bacteria from fish and other aquatic animals - n. buller (2004) ww
TRANSCRIPT
Bacteria from Fish and Other Aquatic Animals
A Practical Identification Manual
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Bacteria from Fish and OtherAquatic Animals
A Practical Identification Manual
Nicky B. Buller
Senior MicrobiologistDepartment of Agriculture
South PerthWestern Australia
CABI Publishing
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CABI Publishing is a division of CAB International
CABI PublishingCAB InternationalWallingfordOxfordshire OX10 8DEUK
Tel: +44 (0)1491 832111Fax: +44 (0)1491 833508E-mail: [email protected] site: www.cabi-publishing.org
CABI Publishing875 Massachusetts Avenue
7th FloorCambridge, MA 02139
USA
Tel: +1 617 395 4056Fax: +1 617 354 6875
E-mail: [email protected]
©N.B. Buller 2004. All rights reserved. No part of this publication may be reproducedin any form or by any means, electronically, mechanically, by photocopying, recordingor otherwise, without the prior permission of the copyright owners.
A catalogue record for this book is available from the British Library, London, UK.
Library of Congress Cataloging-in-Publication DataBuller, Nicky B.
Bacteria from fish and other aquatic animals : a practicalidentification manual / Nicky B. Buller.
p. cm.Includes bibliographical references and index.
ISBN 0-85199-738-41. Aquatic animals--Microbiology. I. Title.
QR106.B85 2004579.3′176--dc21 2003009624
ISBN 0 85199 738 4
Typeset by AMA DataSet, UK.Printed and bound in the UK by Biddles Ltd, King’s Lynn.
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Contents
List of Tables and Figures vii
Foreword: J.A. Plumb ix
Acknowledgements x
Introduction xii
1 Aquatic Animal Species and Organism Relationship 11.1 Host Species, Bacteria and Disease 11.2 Bacterial Diseases 11.3 Bacteria and Relationship to Host 371.4 Taxonomy and Disease Status of Bacteria 75
2 Bacteriological Culture Techniques: Microscopy, Culture and Identification 832.1 Specimen Collection and Submission 842.2 Culture and Incubation 852.3 Examination of Culture Plates 852.4 Biochemical Identification Tests 1142.5 Inoculation of Biochemical Identification Sets 1152.6 API Identification Systems 116
3 Interpretation of Biochemical Identification Tests and Sets 1173.1 Conventional Media: ‘Biochem Set’ 1173.2 Identification Tests and their Interpretation 1173.3 Using the Biochemical Identification Tables 1233.4 Interpretation and Identification of Genera and Species 1233.5 Antisera Available 136
4 Biochemical Identification Tables 1374.1 Results for Conventional Biochemical Tests – ‘Biochem Set’ 1374.2 Results for API Kits 138
5 Technical Methods 2225.1 Total Bacterial Count (TBC) 222
v
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5.2 Microscopy 2245.3 Storage of Isolates 224
6 Techniques for the Molecular Identification of Bacteria 2256.1 Molecular Identification by PCR Using Specific Primers 2256.2 PCR Protocols 2346.3 Molecular Identification by 16S rDNA Sequencing 2366.4 Fluorescence in Situ Hybridization (FISH) 242
7 Preparation of Media for Culture and Identification 2447.1 General Isolation and Selective Media 2447.2 Biochemical Test Media 261
Further Reading and Other Information Sources 278
Appendix: Common Name and Scientific Name of Aquatic Animals 281
Glossary of Terms 287
References 292
Index 329
vi Contents
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List of Tables and Figures
Tables
Table 1.1. Host species and organism relationship.Table 1.2. Bacterial pathogens and saprophytes of fish and other aquatic animals.
Table 2.1. Outline of steps for culture and identification.Table 2.2. General culture.Table 2.3. Specific culture requirements of organisms.Table 2.4. Microscopic and cultural characteristics.
Table 3.1. Interpretation of tests for ‘biochem set’.Table 3.2. Carnobacterium, Lactobacillus, Vagococcus and Renibacterium differentiation.Table 3.3. Differential tests for some non-fastidious fish pathogenic Gram-positive cocci and rods.Table 3.4. Differential characteristics of the genera within the family Flavobacteriaceae.Table 3.5. Further differential characteristics for the genera in the family Flavobacteriaceae.Table 3.6. Additional tests for differentiation of biotypes of P. damselae.Table 3.7. Differentiation of V. alginolyticus and V. harveyi.
Table 4.1. Aeromonas salmonicida (non-motile Aeromonas species).Table 4.2. Aeromonas spp. Phenotypic tests according to DNA hydridization groups.Table 4.3. Aeromonas spp. – motile.Table 4.4. Anaerobes.Table 4.5. Brucella spp.Table 4.6. Cryptococcus.Table 4.7. Cytophaga–Flavobacteriaceae–Bacteroides group – pathogenic.Table 4.8. Cytophaga–Flavobacteriaceae–Bacteroides group – environmental.Table 4.9. Gram-negative coccobacilli and cocci.Table 4.10. Gram-negative, oxidase-negative rods.Table 4.11. Gram-negative, oxidase-positive rods.Table 4.12. Helicobacter spp.Table 4.13. Gram-positive cocci.Table 4.14. Gram-positive rods.Table 4.15. Mycobacterium and Nocardia spp.Table 4.16. Mycobacterium spp. – additional tests.Table 4.17. Mycoplasma spp.Table 4.18. Listonella spp.
vii
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Table 4.19. Moritella spp.Table 4.20. Photobacterium spp.Table 4.21. Vibrio – pathogenic species.Table 4.22. Vibrio – environmental species.Table 4.23. API 20E database biochemical results.Table 4.24. API 20E database numbers (organisms listed alphabetically).Table 4.25. API 20E database numbers (numbers in ascending order).Table 4.26. API 20NE database biochemical results.Table 4.27. API 50CH database biochemical results.Table 4.28. API Coryne database biochemical results.Table 4.29. API 20 Strep database biochemical results.Table 4.30. API Rapid ID32 Strep database biochemical results.Table 4.31. API Zym database results.Table 4.32. Strains quoted in references.
Table 5.1. Total bacterial counts at sites in oyster hatcheries: indication of healthy and diseasedstates.
Table 6.1. List of specific primers available for PCR detection of aquatic organisms.Table 6.2. Standard PCR protocol.Table 6.3. Universal eubacterial primers for 16S rDNA sequencing.Table 6.4. Suggested primer pairs.Table 6.5. Universal primers for amplification of 16S rDNA from Vibrio spp.Table 6.6. Sequencing primers for the 16S rDNA from Vibrio spp.Table 6.7. Sequencing PCR master mix.
Figures
Figure 4.1. Schematic for using the biochemical identification tables.
Photographic section after p. xiv
viii List of Tables and Figures
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Foreword
While aquatic animal diseases have been a concern for centuries, a profusion of marine and fresh-water aquaculture and environmental concerns across the globe in the last 75 years has promptedincreased interest in these diseases, particularly those caused by bacteria. As aquaculture continues tobecome more intensive and expands into new frontiers, fish health problems are likely to becomemore significant. No group of marine or freshwater animals, including mammals, fish, birds, molluscs,shellfish, reptiles and amphibians, have escaped the effects of bacterial diseases. Hundreds of bacterialspecies can be either pathogenic to wild and cultured aquatic animals or pose a potential disease threatunder favourable conditions. Furthermore, the costs incurred by governments, private aquacultureand the public, due to bacterial-related diseases and attempts to control them, totals millions of dollarsannually as a result of lost aquatic resources. In order to successfully cope with these disease-producingmicrobes in a cost-effective manner, prompt and accurate identification is essential.
Until now there has been no single source available for use in identifying bacterial microbes from somany diverse marine and freshwater animals. However, Bacteria from Fish and Other Aquatic Animals:a Practical Identification Manual now provides just such a source with global application. This practical,user-friendly identification manual will be of great value to inexperienced and experienced bacteriolo-gists, microbiology teachers and/or students, aquatic animal health researchers or diagnosticians, as wellas to workers in public health facilities or medical laboratories who work with marine and freshwater fish,birds, mammals, molluscs, shellfish, reptiles or amphibians. Aided by numerous tables and colourfigures the author discusses conventional bacterial identification procedures, commercially availabledata-based identification kits, molecularly based PCR and 16S rDNA sequencing, thus providing utilityto a broad scientific sector. In this single volume one can find biochemical, biophysical and molecularcharacteristics of nearly 400 species of aquatic bacteria, media on which they are cultured and a briefdiscussion of many diseases with which they are associated. Assembling this manual was a monumentaltask and its author, Nicky B. Buller, is to be highly commended for providing this invaluable addition toaquatic microbiology.
John A. PlumbDepartment of Fisheries and Allied Aquacultures
Auburn UniversityAlabama, USA
ix
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Acknowledgements
I would like to acknowledge Dr Jeremy Carson (Department of Primary Industries, Water and Environ-ment, Tasmania), and Nick Gudkovs (Australian Animal Health Laboratories, Geelong, Victoria)for allowing me to visit their respective laboratories, particularly in the early part of my career, fordiscussions on fish bacterial diseases and isolation techniques. In addition, to Dr Jeremy Carson andhis laboratory who, over the years, have confirmed the identification of some of our Vibrio andFlavobacterium strains isolated from diagnostic cases. In particular, for confirmation of the identityof Animal Health Laboratory Department of Agriculture (AHLDA) diagnostic strains mentioned inthis manual (Flavobacterium columnare, Listonella anguillarum, Vibrio agarivorans, Vibrio halioticoli,and Vibrio mediterranei). To Dr Annette Thomas (Department of Primary Industries, Queensland),thank you for the gift of cultures of Vibrio alginolyticus, Vibrio (carchariae) harveyi, and Streptococcusiniae and for our many discussions over the years on veterinary and fish pathogens. Thank you toDr Bruno Gomez-Gil (CIAD/Mazatlán Unit for Aquaculture, Mexico) for test results on V. rotiferianusnot listed in the literature, and for supplying the paper on Vibrio pacinii, before publication, forinclusion in this manual. To Dr Fabiano Thompson (Laboratory for Microbiology, Ghent University,Belgium) for providing further API 20E reactions for Vibrio brasiliensis, Vibrio neptunius, Vibrio xuiinot listed in the journal article. To the fish pathologists who I have worked with, in particular Dr BrianJones and in memorandum Dr Jeremy Langdon, who have helped me to a better understanding offish diseases. I am also grateful to the Fisheries Research and Development Council for fundingto enable me to attend workshops on fish diseases held at the University of Tasmania in 1996 andattendance at the fish bacteriologists’ workshops held at the Australian Animal Health Laboratories,Geelong, in 2000 and 2001.
The photograph of Renibacterium salmoninarum was obtained from the Animal HealthLaboratory (AHL) archives. There was no documentation as to the source of this photograph andthus I am unable to acknowledge the origin. The rest of the photographs were taken by the authorand were of cultures obtained as diagnostic submissions at AHL, type strains, or from Dr A. Thomas. Thephotographs of Flavobacterium columnare adhering to gill tissue are courtesy of Dr Brian Jones.
A great many microbiologists and other scientists have been responsible over the years for develop-ing and refining the media and techniques used for isolation and identification of bacteria. The mediaquoted from the literature in this book are referenced to include the originator and refiners of thosemedia. My apologies if I am remiss in leaving anybody out.
Due to the expense of producing the photographic plates, assistance towards their costwas achieved through sponsorship from the following organizations. Their assistance is gratefullyacknowledged.
x
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Principal sponsor:Agriculture, Fisheries and Forestry – Australia (AFFA), Aquatic Animal Health Subprogram, fundedthrough Fisheries Research and Development Corporation (FRDC).
Other sponsors:Department of Fisheries, Western AustraliaOxoid AustraliaAnimal Health Laboratories, Department of Agriculture, Western Australia.
The contribution by the Animal Health Laboratories, Department of Agriculture, Western Australia,is also gratefully acknowledged for the use of digital photographic equipment, culture media andbacterial cultures used for the photographic section.
I would also like to acknowledge Mr Tim Hardwick at CAB International, for his assistance inbringing this manuscript into publication.
Acknowledgements xi
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Introduction
This manual attempts to provide a source that enables the identification of bacteria that may be foundin animals that inhabit the aquatic environment. The emphasis is on bacteria from farmed aquaticanimals. In the words of Louis Pasteur, ‘chance favours the prepared mind’; therefore, an informedmicrobiologist will have a better chance of identifying those bacteria.
Our knowledge about the isolation and identification of bacteria from aquatic animals and theaquatic environment is expanding at a rapid rate. New organisms, be they pathogens, environmental,normal flora or potential probiotics, are being described and reported each month. This has happeneddue to an increase in aquaculture research, an increase in intensive fish farming systems, an increase inthe international trade of live aquatic animals and products, and the emergence of new diseases. Moreand more laboratories are becoming involved in the isolation and identification of these bacteria ineither a diagnostic or research capacity. In this manual there is an emphasis on bacteria of interest to theaquaculture industry either as pathogens, normal flora or strains that may be used as probiotics. Somebacteria that have been isolated from diverse habitats are also included. This manual attempts to providethese laboratories with an up-to-date and standardized database of methods and biochemical identifica-tion tables that can be used to isolate and identify bacteria from aquatic sources. Molecular diagnostics isbecoming more routine in many laboratories and a section on molecular identification of bacteria usingthe PCR and 16S rDNA sequencing is also included in this manual.
Many laboratories receive samples not only from veterinary sources, but also samples from zooanimals such as penguins, seals, seabirds, and aquatic mammals both captive and wild. From otheraquatic sources, samples for analysis may come from fish, both wild and cultured, freshwater andmarine, aquarium fish, tropical fish, and cultured aquatic animals such as abalone, pearl oysters,seahorses, lobster, crayfish, yabbies, marron and prawns. All these hosts have their own microflora andpotential bacterial pathogens and are found in a diverse range of habitats from tropical to cool temperateclimates. This manual includes as many of those bacteria as possible that may be found during examina-tion of samples from this diverse range of host and habitat. Not only pathogens, but also environmentaland saprophytic organisms, are included to aid in the understanding of the microflora that may be foundin such samples. Many bacteria from the more extreme environments have also been included as, withthe increase in aquaculture throughout the world, and the increasing knowledge about the microflora ofsuch habitats, these organisms may find their way into some laboratories via the samples submitted.Therefore, those isolates that are capable of growing on the isolation media recommended for aquaticorganisms such as ZoBell’s or Marine agar 2216 (Difco) are included in this manual. In addition, I haveincluded some of the Antarctic organisms that have been suggested as a low-cost food source for somemarine finfish because they are rich in omega-3 polyunsaturated fatty acids significant in the diet (Nicolset al., 1996) and thus they may be cultured from samples that come into a laboratory.
xii
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Medical laboratories are also required to identify an increasing number of bacteria from aquatichabitats that may be involved in clinical infections. This book may also assist in the identification of suchbacteria that are not normally listed in the commercial databases such as API (bioMérieux).
Results of phenotypic test results reported in the literature can be confusing. It is important toperform tests by the methods that have been used by the reporting literature. In this book, the majority ofbiochemical tests have been performed according to West and Colwell (1984) and Cowan and Steel(1970), and these methods are listed. Results are also included from the commercial identification kitsavailable, namely, API 20E, API 50CH and API-ZYM from bioMérieux. Results from these tests are listedin the appropriate tables. Phenotypic tests that may produce different results between biochemical tubemedia and commercial identification kits include citrate reaction, decarboxylases, indole, and somecarbohydrates. Where different strains have been used in the literature and different phenotypic resultsreported, the results of these organisms have been listed separately. This is an attempt to enablemicrobiologists to provide the best possible identification of an organism isolated from a diagnosticor research sample.
A clearly defined set of biochemical tests is used as much as possible in this manual and from this themajority of pathogens and non-pathogens encountered from aquatic sources can be identified to genuslevel and, and for the more commonly known bacteria, to species level. The aim of having a defined set isso that laboratories that make in-house media can prepare media that will enable as many bacteria aspossible to be cultured and identified in the routine laboratory without having to prepare an excessivenumber of test and growth media. Some of the problems with variations in biochemical reactionsreported in the literature are due to different methods being used. This manual provides a standardizedset of biochemical identification methods for aquatic organisms and the reactions reported here arebased on this defined set.
Bacteria that require specialized media and identification tests are included here to assistlaboratories in identification of these organisms. In the case of Brucella, Mycoplasma and Mycobacteria,these methods are intended only as a guide, as these organisms should always be sent to a laboratorythat specializes in their identification. Other media, such as alternative methods for the detection ofcarbohydrate fermentation by Flavobacterium species for example, are also included.
The terms fermentation and utilization are often confused in the literature, and in some cases ithas been difficult to assess by which method the test was done. It is important to distinguish betweenfermentation and utilization as they are separate methodologies and a bacterium may show a positivetest for fermentation of a carbohydrate, yet negative for utilization of that same carbohydrate whentested as a sole carbon growth source. Basically, fermentation refers to the fermentation or breakdownof a carbohydrate, commonly called a ‘sugar’. The breakdown products are detected by a pH change inthe medium indicated by a colour change in the pH indicator, usually phenol red. Utilization refers to atest where a carbon source is assessed as a sole growth source for that bacterium. There are no othernutrients in the medium and growth is observed macroscopically, seen as an increase in the turbidity ofthe test medium. There are no pH indicators in utilization tests. The exception to this is citrate utilizationusing the Simmons method.
Cryptococcus (a yeast) is also included in this manual, because although it is not a bacterium, it isa zoonotic hazard to fish pathologists, veterinary pathologists, microbiologists and other laboratorypersonnel who may be dealing with samples from aquatic mammals that are susceptible to thisorganism. It is therefore included so as to alert staff to the biological hazards of some samples. Otherzoonotic organisms include Brucella, Mycobacteria and Nocardia. Many of the bacteria from aquaticsources may cause infections in humans and these are listed in Table 1.1.
Layout of the Manual
This manual is divided into sections basically according to the steps taken to isolate and identify anunknown bacterium. Experienced microbiologists may find some sections basic, but these are includedfor the benefit of students and newly graduated microbiologists or for researchers who are unfamiliarwith the techniques of bacteriology, particularly those used in a diagnostic laboratory.
Introduction xiii
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Thus the sections encompass the host and the microbe, isolation techniques, phenotypic(biochemical) identification techniques, molecular identification and a media section.
In the phenotypic identification section is a flow chart (see Fig. 4.1, p. 138) that directs themicrobiologist to the appropriate biochemical identification table required for the identification ofthe unknown organism. The biochemical identification tables are named according to genus such asAeromonas and Vibrio, or to Gram reaction and cell shape or oxidase reaction.
In the conventional identification tables, the organisms are listed in alphabetical order (with theexception of the Vibrio tables, 4.21 and 4.22) under headings of Pathogen or Environmental. ThePathogen and Environmental differences refer to the pathogenicity for fish and aquatic animals, andgenerally not to humans or terrestrial animals. The inclusion of biochemical reactions for saprophyticand other species is intended to assist with the identification and ensure that the correct identification ismade where species have similar results. In the Vibrio tables, the organisms are listed according to theirgroupings based on ODC, LDC and ADH reactions. The intention is to use these groupings as the start-ing point for identification, similar to a flow chart. Tables for the API databases have the organisms listedalphabetically.
Significance
Fish and other aquatic animals (farmed and wild) are prone to bacterial infections in the same way asland animals, especially when they are stressed. Disease may occur systemically or be confined toexternal surfaces such as the skin or gills. In many instances, the pathogenic bacteria are ubiquitousin the environment, or may form part of the normal internal bacterial flora of an aquatic animal.One study suggested that up to 28 different Vibrio species may be found in the hepatopancreas(104 CFU/g), intestine and stomach (106 CFU/g) of healthy shrimp. The Vibrio species identifiedincluded V. alginolyticus, V. parahaemolyticus, V. cholerae and P. damselae. In diseased states onlyone or two Vibrio species are found (Gomez-Gil et al., 1998). Therefore, many factors need to beconsidered in making a disease diagnosis, such as clinical signs and symptoms, pathology, amount ofgrowth of the cultured bacteria, the numbers of different species cultured, the tissue site from which itwas isolated, and sterility of the specimen collection (Lightner and Redman, 1998).
Bacterial microflora on the surface of fish are heterogeneous in their salt requirement for growth.This requirement for salt (halophilic) is usually retained after serial subculture. Likewise the gut of fishcan be composed almost exclusively of halophilic vibrios (Liston, 1957; Simidu and Hasuo, 1968).Therefore, when attempting culture for pathogens, their salt requirement needs to be taken into account.This also applies to the biochemical identification tests.
Bacterial diseases affecting aquatic animals are detailed in texts such as Austin and Austin (1999),and Woo and Bruno (1999) (see Chapter 8 ‘Further Reading and Other Information Sources’). In thismanual, the diseases and the bacterial cause are presented as a quick reference tabular format only.
xiv Introduction
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Photographs of Culture and MicroscopicAppearance of Organisms
The microscopic and cultural appearance of 31 species of bacteria and some of the biochemical testresults are detailed here. Obviously not all bacteria can be presented, however, genera and specieshave been photographed on commonly used media to show their cultural appearance on thatmedium. Some species such as Vibrio mimicus and Vibrio cholerae are photographed to indicate howsimilar they can be by cultural appearance and how similar they may appear to motile Aeromonasspecies. Likewise the motile Aeromonas species all appear similar on blood agar media, whereas thenon-motile Aeromonas salmonicida has a distinctive colony appearance, slow-growing, with pigmentproduction after a few days incubation.
Reactions of some biochemical tests are included for those not familiar with these reactions.
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Fig
. 1.A
eromonas hydrophila
on BA
, 24 h.
Fig
. 2.A
eromonas hydrophila on M
CA
, 24 h.
Fig
. 3.A
eromonas hydrophila on T
CB
S,
24 h.F
ig. 4.
Aerom
onas hydrophila,G
ram stain.
Fig
. 5.A
eromonas janadaeion B
A,
48 h.F
ig. 6.
Aerom
onas janadaeion MC
A,
3 days.F
ig. 7.
Aerom
onas janadaei,G
ramstain.
Fig
. 8.A
eromonas veroniissp. sobria
onB
A, 48 h, 25
oC.
Fig
. 9.A
eromonas veroniissp. sobria
onM
CA
, 24 h, 25oC
.F
ig. 10.A
eromonas veroniissp. sobria
on TC
BS
, 24 h, 25oC
.F
ig. 11.
Aerom
onas veronii ssp.sobria, G
ram stain.
Fig
. 12.Atypical A
eromonas salm
onicida(A
ustralian strain) on BA
, 3 days.F
ig. 13.A
typicalAerom
onas salmonicida
(Australian strain) on B
A, 7 days.
Fig
. 14.Atypical A
eromonas salm
onicida(A
ustralian strain) showing pigm
ent on NB
agar.
Fig
. 15.AtypicalA
eromonas
salmonicida
(Australian strain),
Gram
stain.
Fig
. 16.C
ryptococcus neoformans
var. gattiisubculture on BA
, 3 days.F
ig. 17.
Cryptococcus. O
riginal isolationplate (S
trep selective agar), 7 days.F
ig. 18.
Cryptococcus.
Gram
smear of tissue/culture, and w
ithin tissue/culture.
Fig
. 19.E
dwardsiella hoshinae
on BA
, 2days, 25
oC.
Fig
. 20.E
dwardsiella hoshinae
on MC
A,
24 h, 25oC
.F
ig. 21.
Edw
ardsiella hoshinae, G
ram stain.
Fig
. 22.E
dwardsiella tarda
on BA
, 24 h,25
oC.
Fig
. 23.E
dwardsiella tarda
on MC
A,
24h.
Fig
. 24.E
dwardsiella tarda, G
ram stain.
Fig
. 25.E
rysipelothrix rhusiopathiaeon B
A, 24 h, 25
oC.
Fig
. 26.E
rysipelothrix rhusiopathiae,G
ram stain.
Fig
. 27.F
lavobacterium johnsoniae
on BA
.F
ig. 28.
Flavobacterium
johnsoniaeon N
A, 48 h, 25
oC.
Fig
. 29.F
lavobacterium johnsoniae
on AO
, 48 h, 25oC
.
Fig
. 32.F
lavobacterium colum
naresubculture on A
O, 5 days.
Fig
. 33.F
lavobacterium colum
naresubculture on A
O, 5 days.
Fig
. 34.F
lavobacterium colum
narem
agnified colonies.F
igs 35 an
d 36.
Flavobacterium
columnare. H
E section. C
ellsattached end-on to skin tissue. G
ramsm
ear of cells from culture.
Fig
s 30 and
31.F
lavobacterium
johnsoniae.G
ram sm
ear of gill tissueand cells from
pure culture.
Fig
. 37.H
afnia alveion BA
, 48 h, 25oC
. F
ig. 38.
Hafnia alveion M
CA
, 48 h,25
oC.
Fig
. 39.H
afnia alvei, Gram
stain.
Fig
. 40.Listonella anguillarum
on BA
, 3days, 25
oC.
Fig
. 41.Listonella anguillarum
on M
SA
-B, 24 h, 25
oC.
Fig
. 42.Listonella anguillarum
on TC
BS
,48 h, 25
oC.
Fig
. 43.Listonella anguillarum
,G
ram stain.
Fig
. 44.M
ycobacterium m
arinumon
BA
, 5 days. F
ig. 45.
Mycobacterium
marinum
introut kidney, Z
N stain.
Fig
. 46.M
ycobacterium m
arinumin
trout kidney, ZN
stain. F
ig. 47.
Nocardia, G
ram stain.
Fig
. 48.P
hotobacterium dam
selaessp.
damselae
on MS
A-B
, 2 days, 25oC
.F
ig. 49.
Photobacterium
damselae
ssp. damselae
on BA
, 48 h.F
ig. 50.
Photobacterium
damselae
ssp. damselae
on TC
BS
, 2 days, 25oC
.F
ig. 51.
Photobacterium
damselae
ssp. damselae, G
ram stain.
Fig
. 52.P
hotobacterium dam
selaessp. piscicida, 13 days.
Fig
. 53.P
lesiomonas shigelloides
onB
A, 24 h, 25
oC.
Fig
. 54.P
lesiomonas shigelloides
onM
CA
, 24 h, 25oC
.F
ig. 55.
Plesiom
onas shigelloides,G
ram stain.
Fig
. 56.P
seudomonas fluorescens
onB
A, 24 h.
Fig
. 57.P
seudomonas fluorescens
onM
CA
, 2 days.F
ig. 58.
Pseudom
onas fluorescens,G
ram stain.
Fig
. 59.R
enibacterium salm
oninarum,
2–3 weeks.
Fig
. 60.S
treptococcus (difficile)agalactiae
Group B
on BA
, 3 days.F
ig. 61.
Streptococcus (difficile)
agalactiaeG
roup B, G
ram stain.
Fig
. 62.S
train of Streptococcus iniae
on BA
, 2 days with w
eak β-haemolysis.
Fig
. 63.S
train of Streptococcus iniae
on BA
, 2 days with stronger
β-haemolysis.
Fig
. 64.S
treptococcus iniae. Gram
smear.
Fig
. 65.V
ibrio agarivoranson M
SA
-B,
7 days, 25oC
.
Fig
. 66.V
ibrio alginolyticus on MS
A-B
,24 h. G
rowth has sw
armed across plate.
Fig
. 67.V
ibrio alginolyticus on BA
, 24 h. F
ig. 68.
Vibrio alginolyticus
on TC
BS
, 24 h.
Fig
. 69.V
ibrio alginolyticus,G
ram stain.
Fig
. 70.V
ibrio choleraenon-01 on B
A,
24 h. F
ig. 71.
Vibrio cholerae
non-01 on MC
A.
Fig
. 72.V
ibrio choleraenon-01 on
TC
BS
.F
ig. 73.
Vibrio cholerae, G
ramstain.
Fig
. 74.V
ibrio fluvialison B
A, 24 h,
25oC
.F
ig. 75.
Vibrio fluvialis
on MS
A-B
, 48 h,25
oC.
Fig
. 76.V
ibrio fluvialison M
CA
, 24 h,25
oC.
Fig
. 77.V
ibrio fluvialison T
CB
S, 24 h,
25oC
.
Fig
. 78.V
ibrio furnissiion BA
, 48 h. F
ig. 79.
Vibrio furnissiion M
CA
, 48 h.F
ig. 80.
Vibrio furnissiion T
CB
S, 48 h.
Fig
. 81.V
ibrio fluvialis, Gram
stain.
Fig
. 82.V
ibrio harveyion MS
A-B
, 48 h.F
ig. 83.
Vibrio harveyion B
A, 48 h.
Fig
. 84.V
ibrio harveyion TC
BS
, 48 h.F
ig. 85.
Vibrio harveyi, G
ramstain.
Fig
. 86.V
ibrio mim
icuson B
A, 24 h.
Fig
. 87.V
ibrio mim
icuson B
A, 48 h.
Fig
. 88.V
ibrio mim
icuson M
CA
.F
ig. 89.
Vibrio m
imicus
on TC
BS
.
Fig
. 90.V
ibrio mim
icus, Gram
stain.F
ig. 91.
Vibrio ordaliion M
SA
-B, 2 days.
Fig
. 92.V
ibrio ordaliion BA
, 3 days,25
oC. N
o growth on T
CB
S.
Fig
. 93.V
ibrio ordalii, Gram
stain.
Fig
. 94.V
ibrio parahaemolyticus
onM
SA
-B, 48 h. G
rowth has sw
armed
across plate.
Fig
. 95.V
ibrio parahaemolyticus
on BA
,24 h, 25
oC.
Fig
. 96.V
ibrio parahaemolyticus
onT
CB
S, 24 h.
Fig
. 97.V
ibrioparahaem
olyticus. Gram
smear.
Fig
. 98.V
ibrio proteolyticuson B
A, 24 h,
25oC
. Show
ing swarm
ing colonies.F
ig. 99.
Vibrio proteolyticus com
pletelycovers on M
SA
-B, 24
h.F
ig. 100.
Vibrio proteolyticus
on TC
BS
,24 h.
Fig
. 101.V
ibrio tubiashiion MS
A-B
, 2days, 25
oC.
Fig
. 102.V
ibrio tubiashiion TC
BS
, 3days, 25
oC.
Fig
. 103.V
ibrio tubiashii, Gram
stain.
Fig
. 104.V
ibrio vulnificuson M
SA
-B, 2
days, 25oC
. F
ig. 105.
Vibrio vulnificus
on TC
BS
, 2days, 25
oC.
Fig
. 106.V
ibrio vulnificus, Gram
stain.
Fig
. 107.Y
ersinia ruckerion BA
, 24 h,25
oC.
Fig
. 108.Y
ersinia ruckerion MC
A, 24 h,
25oC
.F
ig. 109.
Vibrio identification
discs. Top = 150 µg disc (sensi-
tive). Bottom
= 10 µg disc
(resistant).
Fig
. 110.M
otility positive at 25oC
(left), but negative at 37oC
(right)for Y
ersinia ruckeri.
Fig
. 114.D
ecarboxylase reactions. Left toright: arginine dihydrolase (positive); lysinedecarboxylase (positive); ornithine decar-boxylase (negative); and control tube(negative).
Fig
. 115.D
Nase reaction. N
egativereaction on left and positive reaction onright.
Fig
. 116.Indole reaction.
Negative on the left and posi-
tive on the right. Vibrio para-
haemolyticus
with 0.85%
NaC
l,and 2%
NaC
l, respectively.
Fig
. 117.P
late showing gelatin hydroly-
sis and growth on 3%
and 0% N
aCl.
Fig
. 118.M
ethyl Red reaction.
Positive.
Fig
. 111.C
arbohydrate fermen-
tation reaction. Sucrose-positive
(yellow) and sucrose-negative
(red), 24 h, 25oC
.
Fig
. 112.C
itrate test.Y
ersinia ruckeri, citrate-posi-tive (blue) at 24
oC, but cit-
rate-negative (green) at37
oC.
Fig
. 113.D
ecarboxylase reactions. Left toright: arginine dihydrolase (negative); lysinedecarboxylase (positive); ornithine decar-boxylase (positive); and control tube (nega-tive).
1Aquatic Animal Species and Organism Relationship
1.1 Host Species, Bacteria and Disease
This chapter deals with the relationship betweenthe host species and the bacterial flora thatmay be either part of the normal flora of thathost, or pathogenic for that host. This informationis presented in two formats. Table 1.1 liststhe aquatic animal hosts in alphabetical orderunder their common name. The scientific nameis in parentheses. Some hosts are groupedunder their Family name, which is in capitals.For example, trout and salmon are listedunder SALMONIDS; dolphin, porpoises, sealsand whales are listed under SEA MAMMALS;aquarium fish are listed under ORNAMENTALS.The adjacent columns in the table list the bacteriathat have been reported to be either pathogensof the host or that are considered part ofthe normal flora, the tissue site of infection,or the pathology presented and the diseasestate. Some organisms are considered to beopportunistic pathogens and in a healthy hostmay be part of the normal flora. In a stressedanimal, these same bacteria may overcomehost defence mechanisms and cause morbidityor infection in the animal. There are some organ-isms that have been identified and isolated from ahost, but the pathogenicity of the organism isunknown, as virulence studies were not carriedout.
In the second format, in Table 1.2, theinformation is presented by listing the bacteriain alphabetical order, with adjacent columnslisting the name of the disease, the tissuesite where the organism may be found, theaquatic animals where the organism has been
reported, and the geographical location of thedisease.
1.2 Bacterial Diseases
The following section provides more detail thanin the tables on some of the more commonlyrecognized fish bacterial diseases. The diseasesare described in a brief form, as there are othertexts available that provide more comprehensivedetail on diseases of fish. See recommendedtexts in Chapter 8, ‘Further Reading and OtherInformation Sources’.
Bacillary necrosis of Pangasius
This is a recently reported disease found infreshwater catfish (Pangasius hypophthalmusSauvage) in Thailand. The causative organismhas been identified as Edwardsiella ictaluri,which in catfish in America causes the diseaseknown as enteric septicaemia of catfish. Thedisease in Pangasius presents as multifocal, whitelesions, irregular in size and found in internalorgans, predominantly the kidney, liver andspleen. In histology the lesions appear as areasof necrosis and pyogranulomatous inflammation.The causative bacterium was identified as E.ictaluri biochemically; however, when examinedmicroscopically the bacterial cells showed agreater pleomorphism in length and size thannormally seen with other strains of E. ictaluri(Crumlish et al., 2002).
©N.B. Buller 2004. Bacteria from Fish and Other Aquatic Animals:a Practical Identification Manual (N.B. Buller) 1
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2 Chapter 1
Abal
one
Pseu
doal
tero
mon
assp
p.,
Shew
anel
lasp
p.Vi
brio
agar
ivor
ans
(pat
hoge
nici
tyno
tdet
erm
ined
)Vi
brio
med
iterra
nei
Tiss
ueM
orta
lity,
lesi
ons
135
Hal
iotis
disc
usha
nnai
Vibr
ioha
liotic
oli
Gut
Nor
mal
mic
roflo
ra67
8
Red
abal
one
( Hal
iotis
rufe
scen
s)Vi
brio
algi
noly
ticus
Sick
larv
aear
eun
able
tosw
iman
dre
mai
nat
the
botto
mof
the
pond
Mor
talit
yin
larv
ae30
Hal
iotis
tube
rcul
ata
Vibr
io(c
arch
aria
e)ha
rvey
iW
hite
pust
ules
onfo
otM
orta
lity
576
Japa
nese
abal
one
( Sul
culu
sdi
vers
icol
orsu
prat
exta
)Vi
brio
harv
eyi(
stra
inw
asno
n-lu
min
esce
nt,O
DC
-neg
ativ
e,ur
ease
-neg
ativ
e)
Whi
tesp
ots
onfo
ot,t
issu
ele
sion
s,ne
crot
icde
gene
ratio
nin
mus
cle
fibre
sM
ass
mor
talit
y,lo
seab
ility
toad
here
581
Smal
laba
lone
( Hal
iotis
dive
rsic
olor
supe
rtext
a )Vi
brio
para
haem
olyt
icus
Org
anis
min
haem
olym
phW
ither
ing
synd
rom
e,m
ass
mor
talit
y49
9
Alga
Mar
ine
alga
( Ulv
ala
ctuc
a)Ps
eudo
alte
rom
onas
ulva
eH
asan
ti-fo
ulin
gpr
oper
ties
231
Re d
a lg a
( Del
esse
riasa
ngui
nea)
Zobe
lliaga
lact
anov
oran
s61
Allig
ator
Allig
ator
mis
siss
ippi
ensi
s1.
Edw
ards
iella
tard
a2.
Myc
opla
sma
allig
ator
is3.
Past
eure
llam
ulto
cida
4.St
aphy
loco
ccus
5.Ed
war
dsie
llata
rda,
Salm
onel
lam
iam
i ,S.
java
,S.h
artfo
rd1,
3.La
rge
inte
stin
e,cl
oaca
.Con
gest
ion
inki
dney
sw
ithne
crot
icfo
ci,p
erito
nitis
,ul
cers
inga
stric
muc
osa
2.Pu
lmon
ary
oede
ma,
inte
rstit
ial
pneu
mon
ia,p
eric
ardi
tis,m
yoca
rditi
s,m
enin
gitis
,syn
oviti
s3,
4.Lu
ngs
1,5.
Nep
hriti
s.Pa
thog
enic
ityno
tcon
clus
ive
2.Ac
ute
mul
tisys
tem
icin
flam
mat
ory
dise
ase
3,4.
Pneu
mon
ia
128
129
520
804
823
Ambe
rjack
.Jap
anes
eam
berja
ck.
See
Yello
wta
il
Anch
ovy
( Eng
raul
ism
orda
x)Te
naci
bacu
lum
mar
itim
umH
aem
orrh
agic
lesi
ons
onsn
out,
eye
and
mid
sect
ion
ofth
ebo
dyD
isea
se15
4
Arct
icCh
arr(
Salv
elin
usal
pinu
sLi
nnae
us).
See
unde
rSAL
MO
NID
S
Host
Path
ogen
Norm
alflo
raTi
ssue
site
Dise
ase
stat
usRe
f
Tab
le1.
1.H
osts
peci
esan
dor
gani
smre
latio
nshi
p.
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Color profile: DisabledComposite 150 lpi at 45 degrees
Aquatic Animal Species and Organism Relationship 3
Arte
mia
spp.
See
unde
rshr
imp
–br
ine
shrim
p
Ayu
( Ple
cogl
ossu
sal
tivel
isTe
mm
inck
and
Schl
egel
)1.
Flav
obac
teriu
mps
ychr
ophi
lum
2.Li
ston
ella
angu
illaru
m01
and
02(E
urop
ean
desi
gnat
ion)
3.Ps
eudo
mon
asan
guilli
sept
ica
4.Ps
eudo
mon
aspl
ecog
loss
icid
a5.
Ren
ibac
teriu
msa
lmon
inar
um6.
Stre
ptoc
occu
sin
iae
7.Vi
brio
chol
era
non-
01(n
egat
ive
for
orni
thin
ede
carb
oxyl
ase)
4.H
aem
orrh
agic
asci
tes
5.W
hite
nodu
les
inki
dney
,abd
omen
swol
len
with
fluid
,exo
phth
alm
ia
1.C
old
wat
erdi
seas
e2.
Vibr
iosi
s3.
Dis
ease
4.M
orta
lity,
bact
eria
lha
emor
rhag
icas
cite
s(B
HA)
5.Ba
cter
ialk
idne
ydi
seas
e(B
KD)
6.M
orta
lity,
stre
ptoc
occo
sis
7.M
ass
mor
talit
y
434
442
561
564
568
582
712
722
757
803
Baitf
ish.
Amer
ican
baitf
ish
( Pim
epha
les
prom
elas
)Se
eM
inno
ws
Barr
amun
di( L
ates
calc
arife
rBl
och)
See
Bass
Bass
Euro
pean
seab
ass
( Dic
entra
rchu
sla
brax
Linn
aeus
)1.
Aero
mon
ashy
drop
hila
2.Ph
otob
acte
rium
dam
sela
ess
p.pi
scic
ida
3.Ps
eudo
mon
asan
guilli
sept
ica
4.M
ycob
acte
rium
mar
inum
and
Myc
obac
teriu
msp
p.5.
Stre
ptoc
occu
sin
iae
6.Te
naci
bacu
lum
mar
itim
um
1.En
larg
emen
tofs
plee
n,er
ythe
ma
and
swel
ling
ofan
us2.
No
obvi
ous
gros
spa
thol
ogy,
enla
rged
sple
enw
ithw
hite
nodu
les
3.O
rgan
ism
isol
ated
from
head
–kid
ney
and
sple
en4.
Deg
ener
atio
nin
eye,
exop
htha
lmia
,sk
inle
sion
s,ne
crot
icar
eas
ingi
lls4,
5.O
rgan
ism
sin
hear
tand
sple
en,
extre
me
sple
nom
egal
y6.
Pale
skin
zone
sw
ithye
llow
edge
s,le
adin
gto
necr
otic
lesi
ons
onbo
dyar
ound
fins,
oral
cavi
ty,e
yes
and
gills
1.M
orta
litie
s2.
Fish
past
eure
llosi
s3.
Hae
mor
rhag
icse
ptic
aem
ia4.
Myc
obac
terio
sis
5.Ex
udat
ive
men
ingi
tisan
dpa
noph
thal
miti
s6.
Der
mal
necr
osis
.Fin
gerli
ngs
very
susc
eptib
le.S
tress
-rela
ted
inol
derf
ish
60 91 96 140
183
209
227
Larg
emou
thba
ss( M
icro
pter
ussa
lmoi
des
Lace
pède
)Ed
war
dsie
llata
rda
Edw
ards
iella
icta
luri
Inte
stin
e,liv
er,s
plee
nIn
tern
alor
gans
pale
and
anae
mic
,blo
odw
ater
y,ha
emor
rhag
icno
dule
sin
stom
ach
wal
land
sero
saof
inte
stin
e
627
823
cont
inue
d
19Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:17 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
4 Chapter 1
Sea
bass
(Lat
esca
lcar
iferB
loch
)Al
sokn
own
asba
rram
undi
orba
rram
undi
perc
h
1.Fl
avob
acte
rium
john
soni
ae2.
Phot
obac
teriu
mda
mse
lae
ssp.
dam
sela
e3.
Phot
obac
teriu
mda
mse
lae
ssp.
pisc
icid
a4.
Stre
ptoc
occu
sin
iae
5.Vi
brio
harv
eyi
1.Su
perfi
cial
skin
eros
ion
onpo
ster
ior
flank
s,pe
ctor
alfin
san
doc
casi
onal
lyth
elo
wer
jaw
3.Ex
opht
halm
ia,h
aem
orrh
agic
skin
lesi
ons,
sept
icae
mia
4.O
rgan
ism
isol
ated
from
brai
n
1.D
isea
sein
juve
nile
s2,
3,4,
5.M
orta
litie
s12
713
514
573
4
Sea
bass
( Lat
eola
brax
japo
nicu
sC
uvie
r),Ja
pane
sese
aper
chN
ocar
dia
serio
lae
Whi
te-y
ello
wno
dule
sin
gill,
hear
t,ki
dney
,liv
er,s
plee
nN
ocar
dios
is15
5
Sea
bass
( Pun
tazz
opu
ntaz
zoC
uvie
r)Ae
rom
onas
hydr
ophi
laEn
larg
emen
tofs
plee
n,er
ythe
ma
and
swel
ling
ofan
usM
orta
litie
s22
7
Strip
edba
ss( M
oron
esa
xatil
isW
alba
um),
(Roc
cus
saxa
tilis
)1.
Car
noba
cter
ium
pisc
icol
a2.
Cor
yneb
acte
rium
aqua
ticum
3.M
ycob
acte
rium
mar
inum
and
Myc
obac
teriu
msp
p.4.
Phot
obac
teriu
mda
mse
lae
ssp.
pisc
icid
a5.
Serra
tiam
arce
scen
s6.
Stre
ptoc
occu
sin
iae
7.Vi
brio
chol
erae
non-
01an
dVi
brio
mim
icus
Did
notc
ause
deat
hsin
avi
rule
nce
assa
y;ho
wev
er,m
aybe
oppo
rtuni
stic
path
ogen
inst
ress
edfis
h
1.H
yper
aem
iaan
dha
emor
rhag
ein
liver
,ki
dney
,spl
een
and
brai
n2.
Org
anis
min
brai
ntis
sue,
exop
htha
lmia
3.N
odul
arle
sion
sin
allo
rgan
s4.
Org
anis
mis
olat
edfro
min
tern
alor
gans
,enl
arge
dki
dney
and
sple
en.
Abno
rmal
skin
pigm
enta
tion
5.N
ecro
sis
ofm
uscu
lart
issu
es
1.M
orta
lity
2.D
isea
se3.
Mor
talit
y4.
Fish
past
eure
llosi
s5.
Mor
talit
yin
finge
rling
s6.
Stre
ptoc
occo
sis
7.Pr
esen
tin
appa
rent
lyhe
alth
yw
ildfis
h.M
ayca
use
mor
talit
yin
stre
ssed
farm
edfis
h
73 75 76 235
333
337
339
474
507
708
Whi
tese
aba
ss( A
tract
osci
onno
bilis
A yre
s ).A
lso
k no w
na s
Whi
tew
eakf
ish.
Fam
ilySe
rrani
dae
Tena
ciba
culu
mm
ariti
mum
Lesi
ons
iney
e,op
ercu
lum
.Les
ions
onbo
dyra
ngin
gfro
msc
ale
loss
tode
epul
cers
inm
uscu
latu
re
Dis
ease
inju
veni
les
154
Gro
uper
Ora
nge-
spot
ted
grou
per
( Epi
neph
elus
coio
ides
Ham
ilton
,an
dE.
tauv
ina
Fors
skål
).Fa
mily
Serra
nida
e
1.St
rept
ococ
cus
inia
e2.
Vibr
ioha
rvey
i1.
Susc
eptib
leto
infe
ctio
n1.
Stre
ptoc
occo
sis
847
848
Blen
ny,V
ivip
arou
sbl
enny
( Zoa
rces
vivi
paru
sLi
nnae
us)
Atyp
ical
Aero
mon
assa
lmon
icid
aR
edul
cers
with
whi
tem
argi
ns.O
rgan
ism
also
isol
ated
from
inte
rnal
orga
nsSk
inul
cers
,sep
ticae
mia
832
Host
Path
ogen
Norm
alflo
raTi
ssue
site
Dise
ase
stat
usRe
f
Tab
le1.
1.C
ontin
ued.
20Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpTuesday, January 20, 2004 1:53:34 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
Aquatic Animal Species and Organism Relationship 5
Brea
m
Blac
kse
abr
eam
( Aca
ntho
pagr
usla
tus
Hou
ttuyn
).Kn
own
asYe
llow
finse
abre
am
1.Ps
eudo
mon
asan
guilli
sept
ica
1.H
aem
orrh
age
and
ulce
rativ
ele
sion
sN
otsu
scep
tible
toin
fect
ion
with
Stre
ptoc
occu
sin
iae
1.As
soci
ated
with
‘win
ter
dise
ase’
.Sep
ticae
mia
225
569
848
Com
mon
brea
m,C
arp
brea
m( A
bram
isbr
ama
Linn
aeus
)Ae
rom
onas
salm
onic
ida
ssp.
achr
omog
enes
Larg
eop
enle
sion
s,ar
eas
ofde
scal
atio
n.Ty
pica
l‘fu
runc
les’
nots
een
Mor
talit
ies
534
One
-spo
tsea
brea
m( D
iplo
dus
sarg
usko
tsch
yiSt
eind
achn
er)
Not
susc
eptib
leto
infe
ctio
nw
ithSt
rept
ococ
cus
inia
e84
8
Red
sea
brea
m,J
apan
ese
seab
ream
( Pag
rus
maj
orTe
mm
inck
and
Schl
egel
).Fa
mily
Spar
idae
1.Ed
war
dsie
llata
rda
2.Li
ston
ella
angu
illaru
m3.
Tena
ciba
culu
mm
ariti
mum
1.Se
ptic
aem
ia,f
ocal
supp
urat
ive
orgr
anul
omat
ous
lesi
ons,
cuta
neou
sul
cera
tions
1.Ed
war
dsie
llosi
s2,
3.In
fect
ion
Sea
brea
m( S
paru
sau
ratu
sLi
nnae
us).
Also
know
nas
Gilt
-hea
dse
abr
eam
.Fa
mily
Spar
idae
1.Li
ston
ella
angu
illaru
m2.
Phot
obac
teriu
mda
mse
lae
ssp.
dam
sela
e3.
Phot
obac
teriu
mda
mse
lae
ssp.
pisc
icid
a4.
Pseu
dom
onas
angu
illise
ptic
a5.
Stre
ptoc
occu
sag
alac
tiae
6.St
rept
ococ
cus
inia
e7.
Vibr
ioal
gino
lytic
us8.
Vibr
ioha
rvey
i9.
Vibr
iosp
lend
idus
2.Le
thar
gy,d
iste
nded
abdo
men
,ha
emor
rhag
eson
fins
and
tail,
pale
liver
3.N
oex
tern
alch
ange
sex
cept
anus
red
and
prot
rude
d,ab
dom
endi
sten
ded,
fluid
inab
dom
inal
cavi
ty,c
onge
sted
sple
en,
pete
chia
lhae
mor
rhag
eson
liver
,gr
anul
omat
ous
lesi
ons
insp
leen
and
kidn
ey,m
ultif
ocal
tissu
ene
cros
is4.
Erra
ticsw
imm
ing
atw
ater
surfa
ce,
sink
toth
ebo
ttom
ofth
eca
gean
ddi
e.As
cite
s,re
nalh
aem
orrh
age
5.H
aem
orrh
agic
area
son
body
,m
outh
,eye
,ope
rcul
uman
dfin
s1,
7,8,
9.U
lcer
s,ha
emor
rhag
es,
exop
htha
lmia
1,2.
Mor
talit
y3.
Fish
past
eure
llosi
s4.
Asso
ciat
edw
ith‘w
inte
rdi
seas
e’,h
aem
orrh
agic
sept
icae
mia
asso
ciat
edw
ithke
ratit
is5.
Stre
ptoc
occo
sis
–ep
izoo
ticw
ith10
0%m
orta
lity
6.Ex
udat
ive
men
ingi
tisan
dpa
noph
thal
miti
s1,
3,7,
8.9.
Prim
ary
path
ogen
sin
viru
lenc
est
udie
s
57 58 60 96 225
242
751
786
853
Silv
erbr
eam
,Whi
tebr
eam
( Blic
cabj
oerk
naLi
nnae
us)
Aero
mon
assa
lmon
icid
ass
p.ac
hrom
ogen
esLa
rge
open
skin
lesi
ons
surro
unde
dby
area
sof
desc
alat
ion.
Typi
cal‘
furu
ncle
s’no
tsee
n
Furu
ncul
osis
,mor
talit
y53
4
Carp
Bigh
ead
carp
( Aris
ticht
hys
nobi
lis)
Edw
ards
iella
icta
luri
627
cont
inue
d
21Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpTuesday, January 20, 2004 1:55:38 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
6 Chapter 1
Com
mon
carp
,Koi
carp
( Cyp
rinus
carp
ioca
rpio
Linn
aeus
)
1.Ae
rom
onas
best
iaru
m2.
Atyp
ical
Aero
mon
assa
lmon
icid
a3.
A.ve
roni
issp
.ver
onii
4.C
itrob
acte
rfre
undi
i5.
Flav
obac
teriu
mco
lum
nare
6.Ye
rsin
iaru
cker
i
7.Ae
rom
onas
eucr
enop
hila
8.A.
sobr
iaw
ere
non-
path
ogen
icto
carp
invi
rule
nce
stud
ies
(452
)9.
Stre
ptoc
occu
sin
iae
and
S.ag
alac
tiae
(S.d
iffic
ile) w
ere
non-
path
ogen
icin
viru
lenc
est
udie
s(2
34)
1,3.
Hae
mor
rhag
e,ne
cros
is,u
lcer
s2.
Ulc
ers,
lesi
ons
3.Al
sobl
oody
asci
tes
fluid
4.In
finge
rling
s5.
Gills
6.C
anbe
infe
cted
with
orw
ithou
tclin
ical
sign
s7.
Asci
tes
1,3.
Path
ogen
icon
viru
lenc
est
udie
s2.
Ulc
erat
ing
derm
alle
sion
s4.
Hea
vym
orta
litie
s,se
ptic
aem
ia,o
ppor
tuni
stic
infe
ctio
n5.
Dis
ease
6.Ye
rsin
iosi
s7.
Non
-pat
hoge
nic
135
209
234
271
379
425
452
473
760
Cau
casi
anca
rp,C
ruci
anca
rp( C
aras
sius
cara
ssiu
sLi
nnae
us)
Esch
eric
hia
vuln
eris
Hae
mor
rhag
ein
eyes
,sw
olle
n,da
rkco
lour
edab
dom
en,y
ello
wliv
er,y
ello
wflu
idin
inte
stin
e
Mor
talit
y51
Iber
ian
toot
hcar
p,Sp
anis
hto
othc
arp
( Aph
aniu
sib
erus
Vale
ncie
nnes
)
Vibr
iopa
raha
emol
ytic
usEx
tern
alha
emor
rhag
e,ta
ilro
tM
orta
lity
10
Silv
erca
rp( H
ypop
htha
lmic
hthy
sm
olitr
ixVa
lenc
ienn
es)
1.Pr
ovid
enci
a(P
rote
us)r
ettg
eri
2.St
aphy
loco
ccus
aure
us1.
Larg
ere
dul
cera
tive
lesi
ons
onth
eab
dom
en,b
ase
ofth
epe
ctor
alfin
and
onth
ehe
ad.O
rgan
ism
isol
ated
from
inte
rnal
orga
ns.P
ond
had
been
ferti
lized
with
poul
tryfa
eces
from
whi
chP.
rettg
eri
was
isol
ated
2.R
edco
rnea
beco
min
gop
aque
Deg
ener
atio
nof
eye
tissu
e
1.M
ass
mor
talit
y2.
Eye
dise
ase
79 688
Catfi
sh
Cat
fish
spec
ies
( Icta
luru
ssp
p.R
afin
esqu
e)Fl
avob
acte
rium
colu
mna
reO
rgan
ism
inki
dney
88
Blac
kbu
llhea
d( A
mei
urus
mel
as–
valid
nam
e),(
Icta
luru
sm
elas
Raf
ines
que
–sc
ient
ific
nam
e)
1.Ed
war
dsie
llaic
talu
ri2.
Flav
obac
teriu
mco
lum
nare
2.Ye
llow
-edg
edsk
iner
osio
non
head
1.En
teric
sept
icae
mia
ofca
tfish
(ESC
),ed
war
dsie
llosi
s2.
Col
umna
risdi
seas
e
88
Blue
catfi
sh( Ic
talu
rus
furc
atus
Vale
ncie
nnes
)Ed
war
dsie
llaic
talu
riES
C33
4
Host
Path
ogen
Norm
alflo
raTi
ssue
site
Dise
ase
stat
usRe
f
Tab
le1.
1.C
ontin
ued.
22Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:17 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
Aquatic Animal Species and Organism Relationship 7
Brow
nbu
llhea
d(Ic
talu
rus
nebu
losu
s )1.
Edw
ards
iella
icta
luri
2.Ed
war
dsie
llata
rda
1.In
fect
ion
inth
ebr
ain,
syst
emic
diss
emin
atio
nan
dlo
caliz
atio
nof
the
orga
nism
inth
evi
scer
alor
gans
and
mus
cula
ture
and
cuta
neou
sul
cers
2.Se
ptic
aem
ia,f
ocal
supp
urat
ive
orgr
anul
omat
ous
lesi
ons,
cuta
neou
sul
cera
tions
1.ES
C,e
dwar
dsie
llosi
s2.
Edw
ards
iello
sis,
oppo
rtuni
stic
infe
ctio
n
334
Cha
nnel
catfi
sh( Ic
talu
rus
punc
tatu
sR
afin
esqu
e)1.
Aero
mon
ashy
drop
hila
2.Ba
cillu
sm
ycoi
des
3.C
arno
bact
eriu
mpi
scic
ola
4.Ed
war
dsie
llaic
talu
ri,an
aero
bic
stra
ins
also
isol
ated
5.Ed
war
dsie
llata
rda
6.Ye
rsin
iaru
cker
i
2.U
lcer
ativ
esk
inle
sion
san
dfo
cal
necr
osis
ofep
axia
lmus
cle
3.H
yper
aem
iaan
dsl
ight
haem
orrh
age
inliv
er,k
idne
y,sp
leen
and
brai
n5.
Bact
eria
isol
ated
from
lesi
ons
onsk
inan
dsu
perfi
cial
mus
cle,
necr
osis
inor
gans
6.H
aem
orrh
agic
rings
arou
ndth
eey
esan
dfro
ntal
fora
men
s
1,2,
3.In
fect
ion
and
mor
talit
y4.
ESC
5.Ed
war
dsie
llosi
s,en
teric
sept
icae
mia
,opp
ortu
nist
icin
fect
ion
Yers
inio
sis
73 203
307
334
547
783
Fres
hwat
erca
tfish
,Sut
chic
atfis
h( P
anga
sius
hypo
phth
alm
usSa
uvag
e)
Edw
ards
iella
icta
luri
Mor
talit
y,m
ultif
ocal
irreg
ular
,whi
tele
sion
son
inte
rnal
orga
ns.N
ecro
sis
and
pyog
ranu
lom
atou
sin
flam
mat
ion
Baci
llary
necr
osis
ofPa
ngas
ius
194
Wal
king
catfi
sh( C
laria
sba
trach
usLi
nnae
us)
( Cla
rias
garie
pinu
s)
1.Ae
rom
onas
hydr
ophi
la2.
Edw
ards
iella
icta
luri
1.U
lcer
ativ
edi
seas
e,m
orta
lity
2.ES
C29 42
6
Whi
teca
tfish
( Am
eiur
usca
tus
Linn
aeus
)Ed
war
dsie
llaic
talu
riES
C33
4
Chub
,Eur
opea
nch
ub( L
euci
scus
ceph
alus
Linn
aeus
)At
ypic
alAe
rom
onas
salm
onic
ida
Skin
ulce
rsan
dfin
rot
Mor
talit
y83
7
Coal
fish
Phot
obac
teriu
milio
pisc
ariu
mIn
test
ine
Non
-pat
hoge
nic
599
767
Cod,
Atla
ntic
cod
( Gad
usm
orhu
aLi
nnae
us)
1.At
ypic
alAe
rom
onas
salm
onic
ida
2.Li
ston
ella
angu
illaru
mse
roty
pe02
3.Vi
brio
salm
onic
ida
4.C
arno
bact
eriu
msp
p.5.
Phot
obac
teriu
milio
pisc
ariu
m2.
Lesi
ons
4,5.
Inte
stin
e1.
Skin
ulce
rs2.
Infe
ctio
n,vi
brio
sis
4.N
on-p
atho
geni
c
186
232
599
712
767
cont
inue
d
23Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:18 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
8 Chapter 1
Cora
l
Ocu
lina
pata
goni
caVi
brio
shilo
nii(
sai d
t obe
al a
t er
subj
ectiv
esy
nony
mof
V.m
edite
rrane
i)Bi
nds
toco
ral
Cor
albl
each
ing
59 458
742
Poci
llopo
rada
mic
orni
sVi
brio
cora
lliily
ticus
Whi
tesp
ots
seen
at3–
5da
ysan
dco
mpl
ete
tissu
ede
stru
ctio
naf
ter2
wee
ksTi
ssue
lysi
san
dde
ath
83 84
Crab
Blue
crab
( Cal
linec
tes
sapi
dus)
Vibr
ioch
oler
ae-li
ke(2
%of
crab
s)Vi
brio
para
haem
olyt
icus
(23%
ofcr
abs)
Vibr
iovu
lnifi
cus
(7%
ofcr
abs)
Hae
mol
ymph
,dig
estiv
etra
ctPr
esen
tin
appa
rent
lyhe
alth
ycr
abs
212
Swim
min
gcr
ab( P
ortu
nus
tritu
berc
ulat
us)
Vibr
ioha
rvey
i(in
itial
lyca
lled
Vibr
iosp
.zo
ea)
Mas
sm
orta
lity
inzo
eall
arva
e39
0
Craw
fish
Amer
ican
fresh
wat
ercr
ayfis
hR
edsw
amp
craw
fish
( Pro
cam
baru
scl
arki
i)
Vibr
ioch
oler
aeVi
brio
mim
icus
Mor
talit
ies
507
Cray
fish
Aust
ralia
nfre
shw
ater
cray
fish
Yabb
y( C
hera
xal
bidu
s)R
edcl
aw( C
hera
xqu
adric
arin
atus
)
Vibr
iom
imic
usH
aem
olym
phM
orta
lity,
vibr
iosi
s13
523
084
0
Crev
alle
,Tre
vally
,Jac
kcr
eval
le( C
aran
xhi
ppos
Linn
aeus
)Vi
brio
harv
eyi
Der
mal
lesi
ons
Infe
ctio
n45
3
Croc
odile
Edw
ards
iella
tard
aPa
thog
enic
ityno
tkno
wn
Cro
codi
lus
acut
usEr
ysip
elot
hrix
rhus
iopa
thia
eBl
acke
ned
plaq
ues
onsc
ales
Cut
aneo
usle
sion
s40
8
Cai
man
croc
odile
( Cai
man
croc
odilu
s )Er
ysip
elot
hrix
rhus
iopa
thia
eN
ecro
ticfo
ciun
dert
hesc
ales
ofth
eab
dom
enan
dth
eja
wSe
ptic
aem
ia40
8
Cro
cody
lus
nilo
ticus
Myc
opla
sma
croc
odyl
iSw
olle
njo
ints
.Org
anis
mal
sofo
und
inlu
ngs
Exud
ativ
epo
lyar
thrit
is44
1
Host
Path
ogen
Norm
alflo
raTi
ssue
site
Dise
ase
stat
usRe
f
Tab
le1.
1.C
ontin
ued.
24Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:18 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
Aquatic Animal Species and Organism Relationship 9
Dab
(Lim
anda
liman
daLi
nnae
us)
Atyp
ical
Aero
mon
assa
lmon
icid
aR
ound
,red
ulce
rsw
ithw
hite
mar
gin
ofne
crot
ictis
sue
Skin
ulce
rs83
2
Dace
,Com
mon
dace
( Leu
cisc
usle
ucis
cus
Linn
aeus
)1.
Aero
mon
assa
lmon
icid
ass
p.sa
lmon
icid
a2.
Atyp
ical
Aero
mon
assa
lmon
icid
a
1,2.
Skin
ulce
rs1.
Furu
ncul
osis
2.In
fect
ion
323
352
Dam
selfi
sh(F
amily
Pom
acen
trida
e)
Blac
ksm
ith(C
hrom
ispu
nctip
inni
sC
oope
r)Ph
otob
acte
rium
dam
sela
ess
p.da
mse
lae
Skin
ulce
rsFa
tali
nfec
tion
due
topr
oduc
tion
ofa
cyto
lysi
n50
4
Stag
horn
dam
selfi
sh,Y
ello
wta
ilcl
ownf
ish
( Am
blyg
lyph
idod
oncu
raca
oBl
och,
Amph
iprio
ncl
arki
iBe
nnet
t)
Pseu
doal
tero
mon
aspi
scic
ida
List
onel
laan
guilla
rum
and
Vibr
iopa
raha
emol
ytic
usdi
dno
tapp
ear
tobe
invo
lved
indi
seas
ein
expe
rimen
tali
nfec
tions
Dis
ease
deg
gs,s
een
asw
hite
brai
nan
dsp
inal
cord
,sun
ken
irreg
ular
eyes
,hea
rtre
cede
din
toyo
lksa
c
Infe
ctio
n,m
orta
lity
572
Dani
oSe
eun
derO
RN
AMEN
TAL
Disc
usfis
hSe
eun
derO
RN
AMEN
TAL
Dolp
hin
See
unde
rSEA
MAM
MAL
S
Eel
Amer
ican
eel(
Angu
illaro
stra
taLe
sueu
r)1.
Aero
mon
assa
lmon
icid
a1.
Foca
lles
ions
prog
ress
tola
rge
de-p
igm
ente
dne
crot
icpa
tche
sth
ende
tach
atde
rmo-
epid
erm
alju
nctio
nto
form
larg
eul
cers
that
expo
seun
derly
ing
mus
cle
1.U
lcer
ativ
esk
indi
seas
e,m
orbi
dity
584
Euro
pean
eel(
Angu
illaan
guilla
Linn
aeus
)1.
Atyp
ical
Aero
mon
assa
lmon
icid
a2.
List
onel
laan
guilla
rum
sero
type
053.
Pseu
dom
onas
angu
illise
ptic
a4.
Vibr
iofu
rnis
sii
5.Vi
brio
vuln
ificu
sse
rova
r04
6.Ye
rsin
iaru
cker
i
7.Ae
rom
onas
ench
elei
a1.
Skin
lesi
ons
2.Le
sion
s3.
Subc
utan
eous
haem
orrh
ages
.Ab
dom
inal
dist
ensi
on,o
rgan
ism
inin
tern
alor
gans
4.H
aem
orrh
ages
inin
test
inal
tract
5.O
rgan
ism
cultu
red
from
gills
,int
estin
alco
nten
ts,k
idne
y,sp
leen
6.C
anbe
infe
cted
with
orw
ithou
tclin
ical
sign
s
1.In
fect
ion
2.Vi
brio
sis
3.Se
ptic
aem
ia4.
Mor
talit
y.Vi
rule
ntin
path
ogen
icity
stud
ies
5.D
isea
se6.
Yers
inio
sis
7.N
on-p
atho
geni
c
201
209
240
241
271
323
356
541
712
cont
inue
d
25Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:18 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
10 Chapter 1
Japa
nese
eel(
Angu
illaja
poni
caTe
mm
inck
and
Schl
egel
)1.
Atyp
ical
Aero
mon
assa
lmon
icid
a2.
Edw
ards
iella
tard
a3.
Flav
obac
teriu
mco
lum
nare
4.Ps
eudo
mon
asan
guilli
sept
ica
5.Vi
brio
vuln
ificu
sbi
ogro
up2,
sero
var
Eco
ntai
nsvi
rule
ntan
dav
irule
ntst
rain
s
Oth
erbi
otyp
esof
V.vu
lnifi
cus
are
non-
path
ogen
icfo
reel
s2.
Sept
icae
mia
,foc
alsu
ppur
ativ
eor
gran
ulom
atou
sle
sion
s,cu
tane
ous
ulce
ratio
ns,a
bsce
ssed
orul
cera
tive
lesi
ons
inki
dney
orliv
er4.
Hae
mor
rhag
ican
dul
cera
tive
lesi
ons.
Lesi
ons
inm
outh
,ope
rcul
um,b
rain
,liv
eran
dki
dney
5.Le
sion
s
2.Ed
war
dsie
llosi
s,‘p
arac
olon
dise
ase’
4.‘S
ekite
n-by
o’(re
dsp
otdi
seas
e)5.
Vibr
iosi
s
26 506
746
757
799
800
830
Eel(
Angu
illare
inha
rdtii
)Ph
otob
acte
rium
dam
sela
ess
p.da
mse
lae
429
Eel
Lact
ococ
cus
garv
ieae
Eel(
Hyp
erop
lus
lanc
eola
tus
LeSa
uveg
e)At
ypic
alAe
rom
onas
salm
onic
ida
Hae
mor
rhag
eson
snou
t,ca
udal
finan
dta
il.Ty
pica
lum
bona
tefu
runc
les
onfla
nkSk
inul
cers
197
Elve
rsAe
rom
onas
allo
sacc
haro
phila
Dis
ease
527
Gre
enm
oray
eel(
Gym
noth
orax
fune
bris
)Sp
otte
dm
oray
eel(
G.m
orin
ga)
Myc
obac
teriu
m-tr
iple
x-lik
eFl
orid
skin
nodu
les
arou
ndth
ehe
adan
dtru
nkth
atar
eso
ftgr
ey,g
elat
inou
san
dta
n-co
lour
ed
Prol
ifera
tive
skin
dise
ase
345
Sand
eel(
Amm
odyt
esla
ncea
Cuv
ier)
Atyp
ical
Aero
mon
assa
lmon
icid
aH
aem
orrh
ages
onsn
out,
caud
alfin
and
tail.
Typi
calu
mbo
nate
furu
ncle
son
flank
Skin
ulce
rs19
7
Flou
nder
Flou
nder
( Pla
ticht
hys
flesu
sLi
nnae
us)
Atyp
ical
Aero
mon
assa
lmon
icid
aan
dox
idas
e-ne
gativ
est
rain
sEp
ider
mal
ulce
ratio
ns–
dark
,red
wou
nds
usua
llyro
und
buts
ome
irreg
ular
form
sse
en
Skin
ulce
rdis
ease
323
829
831
832
Gre
enba
ckflo
unde
r( R
hom
boso
lea
tapi
rina
Gün
ther
)At
ypic
alAe
rom
onas
salm
onic
ida
Ulc
erat
ive
derm
alle
sion
s82
6
Host
Path
ogen
Norm
alflo
raTi
ssue
site
Dise
ase
stat
usRe
f
Tab
le1.
1.C
ontin
ued.
26Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:18 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
Aquatic Animal Species and Organism Relationship 11
Japa
nese
floun
der,
also
know
nas
Oliv
eflo
unde
r(Pa
ralic
hthy
sol
ivac
eus
Tem
min
ckan
dSc
hleg
el)
1 .At
ypic
alAe
rom
onas
salm
onic
ida
2.Ed
war
dsie
llata
rda
3.La
ctoc
occu
sga
rvie
ae4.
Noc
ardi
ase
riola
e5.
Phot
obac
teriu
mda
mse
lae
ssp.
pisc
icid
a6.
Stre
ptoc
occu
sin
iae
7.Te
naci
bacu
lum
mar
itim
um8.
Vibr
ioic
hthy
oent
eri
9.W
eiss
ella
helle
nica
stra
inD
S-12
2.Se
ptic
aem
ia,f
ocal
supp
urat
ive
orgr
anul
omat
ous
lesi
ons,
cuta
neou
sul
cera
tions
4.Tu
berc
ules
inki
dney
,gills
and
sple
en.
Absc
esse
son
epid
erm
is5.
Hae
mor
rhag
icse
ptic
aem
iaan
dw
hite
area
sof
gran
ulom
ain
the
kidn
ey,l
iver
,sp
leen
8.In
test
inal
necr
osis
,opa
que
inte
stin
es9.
Pres
enti
nin
test
ine
1.In
fect
ion
2.Ed
war
dsie
llosi
s3.
Stre
ptoc
occo
sis
4.N
ocar
dios
is5.
Fish
past
eure
llosi
s6,
7,8.
Mor
talit
y9.
Prob
iotic
pote
ntia
l
139
273
385
389
442
455
567
570
Sum
mer
floun
der(
Para
licht
hys
dent
atus
Linn
aeus
)1.
Myc
obac
teriu
msp
p.w
ithho
mol
ogy
toM
.mar
inum
and
M.u
lcer
ans
2.Vi
brio
(car
char
iae)
harv
eyi
1.G
ranu
lom
asin
the
kidn
ey,l
arge
red-
tan
colo
ured
mul
tilob
ulat
edm
ass
inki
dney
2.R
edde
ning
arou
ndan
alar
ea,
dist
ende
dab
dom
enfil
led
with
fluid
,en
terit
isan
dne
cros
is
1.M
ycob
acte
riosi
s2.
Flou
nder
necr
otiz
ing
ente
ritis
(FIN
E)
373
710
Gra
ylin
g( T
hym
allu
sth
ymal
lus
Linn
aeus
)1.
Aero
mon
assa
lmon
icid
ass
p.sa
lmon
icid
a2.
Atyp
ical
Aero
mon
assa
lmon
icid
a3.
Yers
inia
ruck
eri
1,2.
Skin
ulce
rs1.
Furu
ncul
osis
2,3.
Infe
ctio
n20
932
335
2
Gre
enkn
ifefis
h( E
igem
anni
avi
resc
ens
Vale
ncie
nnes
)Ed
war
dsie
llaic
talu
riEn
teric
sept
icae
mia
426
Gre
enlin
g–
ma r
ine
fish
( Hex
agra
mm
osot
akii)
Atyp
ical
Aero
mon
assa
lmon
icid
aIn
fect
ion
385
Gro
uper
See
Sea
bass
Hadd
ock
( Mel
anog
ram
mus
aegl
efin
usLi
nnae
us).
Fam
ilyG
adid
ae–
cod
and
hadd
ock
Atyp
ical
Aero
mon
assa
lmon
icid
aSk
inul
cers
Infe
ctio
n32
3
Halib
ut
Atla
ntic
halib
ut( H
ippo
glos
sus
hipp
oglo
ssus
Linn
aeus
)1.
Atyp
ical
Aero
mon
assa
lmon
icid
a2.
Tena
ciba
culu
mov
olyt
icum
1.Sk
inul
cers
2.D
isso
lves
chor
ion
and
zona
radi
ata
ofth
eeg
gsh
ells
1.In
fect
ion
2.O
ppor
tuni
stic
path
ogen
ofeg
gsan
dla
rvae
323
324
Gre
enla
ndha
libut
( Rei
nhar
dtiu
shi
ppog
loss
oide
sW
alba
um)
Arth
roba
cter
rhom
biO
rgan
ism
isol
ated
from
sple
enan
din
tern
alor
gans
Path
ogen
icity
notd
eter
min
ed60
0
cont
inue
d
27Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:18 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
12 Chapter 1
Herr
ing
Balti
che
rring
( Clu
pea
hare
ngus
mem
bras
Linn
aeus
)
1.Ps
eudo
mon
asan
guilli
sept
ica
2.Ph
otob
acte
rium
iliopi
scar
ium
1.H
aem
orrh
ages
inth
eey
e2.
Inte
stin
e1.
Dis
ease
2.N
on-p
atho
geni
c50
359
976
7
Hum
an( H
omo
sapi
ens)
1.A.
allo
sacc
haro
phila
2.Ae
rom
onas
cavi
ae3.
Aero
mon
ashy
drop
hila
ssp.
dhak
ensi
s4.
Aero
mon
ashy
drop
hila
5.A.
jana
daei
6.A.
schu
berti
i7.
A.so
bria
8.A.
trota
9.A.
vero
niis
sp.s
obria
10.A
erom
onas
vero
niis
sp.v
eron
ii11
.Bru
cella
abor
tus
12.B
ruce
llasp
ecie
s13
. Bur
khol
deria
(Pse
udom
onas
)ps
eudo
mal
lei
14.C
hrom
obac
teriu
mvi
olac
eum
15.C
rypt
ococ
cus
neof
orm
ans
16.E
dwar
dsie
llata
rda
17.E
rysi
pelo
thrix
rhus
iopa
thia
e18
.Gra
nulic
atel
lael
egan
s19
.Hal
omon
asve
nust
a20
.Myc
obac
teriu
mm
arin
um21
.Pho
toba
cter
ium
dam
sela
ess
p.da
mse
lae
22.R
ahne
llaaq
uatil
is23
.Rao
ulte
llapl
antic
ola
24.S
hew
anel
laal
gae
25.S
trept
ococ
cus
inia
e26
.Vag
ococ
cus
fluvi
alis
27.V
ibrio
chol
erae
01&
0139
28. V
ibrio
chol
erae
non-
01
1,2.
Faec
es3,
4.Fa
eces
5.W
ound
infe
ctio
n,st
ool,
bloo
d6.
Wou
nd,a
bsce
ss,b
lood
,ple
ural
fluid
7. 9,10
.Fae
ces
11.R
espi
rato
ryin
fect
ion,
abor
tion
12.H
eada
ches
,las
situ
de,s
inus
itis
13.W
ound
infe
ctio
ns,p
neum
onia
and
sept
icae
mia
14.W
ound
infe
ctio
ns15
.Cer
ebra
lspi
nalf
luid
16.I
ntes
tine
17.S
uppu
ratin
gsk
inle
sion
s18
.End
ocar
ditis
19.W
ound
with
wat
ery
disc
harg
efo
llow
ing
fish
bite
20.S
kin
lesi
ons
22.C
onta
min
ated
intra
veno
usflu
id24
.Leg
ulce
rs,s
eptic
aem
ia,o
titis
med
ia,
faec
es25
.Wou
nds
follo
win
gha
ndlin
gfis
h26
.Bite
-wou
nd,b
lood
cultu
reis
olat
esan
dfro
mpe
riton
ealf
luid
27.F
aece
s28
.Blo
odcu
lture
,fae
ces
29.B
lood
and
spin
alflu
id30
,31,
32,3
3.Fa
eces
34.W
ound
s,fa
eces
1.D
iarrh
oea,
rena
lfai
lure
inin
fant
2,3,
4,8.
Dia
rrhoe
a5.
Wou
ndin
fect
ion,
diar
rhoe
a6,
8.In
fect
ion
7.Ac
ute
rena
lfai
lure
inan
infa
nt9,
10.D
iarrh
oea
11,1
2.Br
ucel
losi
s13
.Mel
ioid
osis
14.I
nfec
tion
15.Z
oono
sis,
mor
talit
y17
.Ery
sipe
loid
18.E
ndoc
ardi
tis19
.Wou
ndin
fect
ion
20.I
nfec
tion
21.W
ound
infe
ctio
n22
.Sep
sis.
Also
infe
ctio
nin
imm
unoc
ompr
omis
edho
sts
23.S
eptic
aem
iain
new
born
s24
.Inf
ectio
n25
.Cel
lulit
is26
.Clin
ical
infe
ctio
ns27
.Cho
lera
28.C
ellu
litis
,m
enin
goen
ceph
aliti
s,se
ptic
aem
ia,d
iarrh
oea
29.M
enin
gitis
30,3
1,32
.Dia
rrhoe
a33
.Gas
troen
terit
is,f
ood
pois
onin
g34
.Inf
ectio
n,fo
odpo
ison
ing
120
123
125
142
143
151
177
196
255
259
276
310
346
347
348
378
383
392
482
527
555
588
653
732
792
816
822
Host
Path
ogen
Norm
alflo
raTi
ssue
site
Dise
ase
stat
usRe
f
Tab
le1.
1.C
ontin
ued.
28Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:19 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
Aquatic Animal Species and Organism Relationship 13
29.V
ibrio
cinc
inna
tiens
is30
.Vib
rioflu
vial
is31
.Vib
riofu
rnis
sii
32.V
ibrio
hollis
ae33
.Vib
riopa
raha
emol
ytic
us34
.Vib
riovu
lnifi
cus
Japa
nese
med
aka
(Ory
zias
latip
esTe
mm
inck
and
Schl
egel
)M
ycob
acte
rium
absc
essu
sM
ycob
acte
riosi
s73
6
Lam
prey
Aero
mon
assa
lmon
icid
ass
p.sa
lmon
icid
aM
aybe
carri
ers
ofth
edi
seas
e.O
rgan
ism
foun
din
kidn
eySu
ccum
bto
infe
ctio
nfo
llow
ing
stre
ssin
stre
ss-te
stex
perim
ents
352
Lobs
ter.
Amer
ican
lobs
ter
( Hom
arus
amer
ican
us,H
.ga
mm
arus
L)
1.Ae
roco
ccus
virid
ans
var.
hom
ari
2.Li
ston
ella
angu
illaru
m1.
Org
anis
min
haem
olym
ph1.
Gaf
fkem
ia,h
igh
mor
talit
y2.
Vibr
iosi
s11
429
982
7
Mac
kere
l
Mac
kere
l(Sc
ombe
rsco
mbr
usLi
nnae
us)
Myc
obac
teriu
msp
p.G
rey-
whi
teno
dule
sin
sple
enan
dvi
scer
alor
gans
Myc
obac
terio
sis
515
Japa
nese
jack
mac
kere
l( T
rach
urus
japo
nicu
sTe
mm
inck
and
Schl
egel
)
1.La
ctoc
occu
sga
rvie
ae2.
List
onel
laan
guilla
rum
3.Vi
brio
trach
uri(
late
stin
form
atio
nsu
gges
tsth
isis
aju
nior
syno
nym
ofV.
harv
eyi)
3.H
aem
orrh
ages
inor
gans
and
exop
htha
lmia
3.D
isea
se40
074
3
Mah
i-mah
i(C
oryp
haen
ahi
ppur
usLi
nnae
us)a
lso
know
nas
Dol
phin
fish
Pant
oea
(Ent
erob
acte
r)ag
glom
eran
sH
aem
orrh
ages
iney
e,an
ddo
rsal
and
late
ralm
uscu
latu
reM
orta
lity,
oppo
rtuni
stic
path
ogen
325
Mak
onbu
( Lam
inar
iaja
poni
ca)
1.Ps
eudo
alte
rom
onas
bact
erio
lytic
a2.
Pseu
doal
tero
mon
asel
yako
vii
1.Pr
oduc
esa
red
pigm
ento
nth
eLa
min
aria
and
caus
esda
mag
eto
the
seed
supp
ly2.
Deg
rade
sfro
nds
1.R
edsp
otdi
seas
e2.
Spot
dise
ase
677
679
Milk
fish
( Cha
nos
chan
osFo
rssk
ål)
Vibr
ioha
rvey
iEy
ele
sion
s,ex
opht
halm
ia,o
paqu
enes
s,ha
emor
rhag
esin
eyes
Eye
dise
ase
and
mor
talit
y39
0
cont
inue
d
29Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:19 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
14 Chapter 1
Min
now
Baitf
ish
Amer
ican
baitf
ish,
Fath
ead
min
now
( Pim
epha
les
prom
elas
Raf
ines
que)
Yers
inia
ruck
eri
Yers
inio
sis
Bullm
inno
ws
(Fun
dulu
sgr
andi
sBa
ird)
Stre
ptoc
occu
ssp
p.N
on-h
aem
olyt
icgr
oup
BEx
opht
halm
ia,p
etec
hial
haem
orrh
age
inab
dom
en,o
edem
ain
liver
Mor
talit
y,st
rept
ococ
cosi
s63
7
Eura
sian
min
now
( Pho
xinu
sph
oxin
usLi
nnae
us)
Atyp
ical
Aero
mon
assa
lmon
icid
aEx
tens
ive
haem
orrh
agic
skin
lesi
ons
Mas
sm
orta
lity
331
Min
now
List
onel
laan
guilla
rum
Fish
kill
135
Men
hade
n( B
revo
ortia
patro
nus
Goo
de)
Stre
ptoc
occu
sag
alac
tiae
Stre
ptoc
occo
sis
MO
LLUS
CS
Biva
lve
( Nod
ipec
ten
nodo
sus)
Vibr
iobr
asilie
nsis
,V.n
eptu
nius
V.ne
ptun
ius
isol
ated
from
dise
ased
and
heal
thy
anim
als
Path
ogen
icity
notk
now
n74
0
Clam
Man
ilacl
am( R
udita
pes
philip
pina
rum
)and
(R.d
ecus
satu
s)Vi
brio
tape
tisIn
vasi
onof
pallia
l(m
antle
)cav
ityan
dad
here
nce
toth
epe
riost
raca
llam
ina
atth
egr
owin
ged
geof
the
shel
l.M
ain
char
acte
ristic
feat
ure
isa
brow
nco
nchi
olin
depo
sito
nth
ein
ners
urfa
ceof
the
shel
l
Brow
nrin
gdi
seas
e(B
RD
)14 10
814
661
061
1
Cla
ms
( Mer
cena
riam
erce
naria
),Bi
valv
em
ollu
scs
1.Vi
brio
tubi
ashi
i1.
Larv
aece
ase
swim
min
g,di
gest
ion
oftis
sue
1.Ba
cter
ialn
ecro
sis
and
syst
emic
dise
ase
321
762
Mus
sel(
Prot
otha
caje
doen
sis
Lisc
hke)
Shew
anel
laja
poni
ca39
7
Oys
ter
East
ern
oyst
er( C
rass
ostre
avi
rgin
ica )
1.R
oseo
bact
ersp
p.st
rain
CVS
P2.
Vibr
iotu
bias
hii
2.Sh
ewan
ella
colw
ellia
na1.
Lesi
ons,
man
tlere
tract
ion,
conc
hiol
inde
posi
tsin
side
shel
l2.
Prom
otes
settl
emen
tofo
yste
rlar
vae
1.Ju
veni
leoy
ster
dise
ase
(JO
D)
2.N
orm
alflo
ra10
410
581
481
5
Host
Path
ogen
Norm
alflo
raTi
ssue
site
Dise
ase
stat
usRe
f
Tab
le1.
1.C
ontin
ued.
30Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpTuesday, January 20, 2004 1:57:04 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
Aquatic Animal Species and Organism Relationship 15
Japa
nese
oyst
er(C
rass
ostre
agi
gas )
Paci
ficoy
ster
(Cra
ssos
trea
giga
s)
1.Al
tero
mon
assp
ecie
s(n
otsp
ecia
ted)
2.Li
ston
ella
angu
illaru
m3.
Noc
ardi
acr
asso
stre
ae4.
Phot
obac
teriu
mda
mse
lae
ssp.
dam
sela
e5.
Vibr
iosp
lend
idus
biov
arII
6.Vi
brio
sple
ndid
us7.
Vibr
iotu
bias
hii
8.Vi
brio
vuln
ificu
sse
rova
rE(s
ome
stra
ins
are
aviru
lent
forT
aiw
anes
eee
ls)
9.Ae
rom
onas
med
ia10
.Aer
omon
assp
p.11
. Alte
rom
onas
spp.
12. P
seud
omon
assp
p.13
. Vib
riosp
p.To
talb
acte
rialc
ount
inso
fttis
sue
=2.
9×
104
colo
nies
per
gram
,and
inha
emol
ymph
=2.
6×
104
colo
nies
perm
l(R
ef59
6)
1.La
rval
deat
hs2.
Failu
reof
velig
erla
rvae
tom
aint
ain
them
selv
esin
the
wat
erco
lum
n3.
Foca
lare
asof
brow
ndi
scol
orat
ion
onth
em
antle
,org
reen
-yel
low
nodu
les
onab
duct
orm
uscl
e,gi
lls,h
eart
and
man
tle5.
Org
anis
mis
olat
edfro
mgo
nads
ofbr
oods
tock
.Hig
hba
cter
iall
oad
inla
rvae
,ba
cter
iain
shel
lmar
gin,
loss
ofci
liaan
dve
lum
.7.
Larv
aece
ase
swim
min
g,di
gest
ion
oftis
sue
1,2,
4.M
orta
lity
3.N
ocar
dios
is5.
Mas
sm
orta
lity.
Dis
ease
rese
mbl
esba
cilla
ryne
cros
is6.
Mor
talit
y.D
isea
seis
stre
ss-a
ssoc
iate
d7.
Bact
eria
lnec
rosi
san
dsy
stem
icdi
seas
e9.
Prob
iotic
agai
nst V
.tub
iash
ii
26 222
270
280
294
321
466
467
596
721
762
798
Med
iterra
nean
oyst
ers
Vibr
iole
ntus
513
Scal
lop
Argo
pect
enpu
rpur
atus
Aero
mon
ashy
drop
hila
Vibr
ioal
gino
lytic
usLa
rval
deat
hs65
0
Pect
enm
axim
us1.
Vibr
iope
cten
icid
a2.
Ros
eoba
cter
galla
ecie
nsis
1.Af
fect
sla
rvae
1.Vi
brio
sis
662
470
Pike
,Nor
ther
npi
ke( E
sox
luci
usLi
nnae
us)
Yers
inia
ruck
eri
209
Mud
skip
per
List
onel
laan
guilla
rum
Fish
kill
135
Mul
let
Blac
km
ulle
t,G
rey
mul
let,
Flat
head
mul
let(
Mug
ilcep
halu
sLi
nnae
us)
1.Ed
war
dsie
llata
rda
2.Eu
bact
eriu
mta
rant
ella
e3.
Lact
ococ
cus
garv
ieae
(tent
ativ
eid
entif
icat
ion)
1.Se
ptic
aem
ia,f
ocal
supp
urat
ive
orgr
anul
omat
ous
lesi
ons,
cuta
neou
sul
cera
tions
2.O
rgan
ism
reco
vere
dfro
mbr
ain,
liver
,ki
dney
,blo
od3.
Fish
leth
argi
c,ex
opht
halm
ia,c
onge
stio
nan
dha
emor
rhag
ein
sple
enan
dki
dney
,w
hite
spot
s,as
cite
s,gr
anul
omas
,m
acro
phag
esan
dba
cter
iain
orga
ns
1.Ed
war
dsie
llosi
s2.
Mor
talit
y3.
Sept
icae
mia
,chr
onic
men
ingi
tis
157
343
764
cont
inue
d
31Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:19 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
16 Chapter 1
Born
eom
ulle
t,La
rges
cale
mul
let
( Liz
am
acro
lepi
sSm
ith)
Stre
ptoc
occu
sin
iae
Susc
eptib
leto
infe
ctio
nSt
rept
ococ
cosi
s84
8
Silv
erm
ulle
t(M
ugilc
urem
aVa
lenc
ienn
es)
Vibr
ioha
rvey
iO
rgan
ism
isol
ated
inpu
recu
lture
Hae
mor
rhag
icse
ptic
aem
ia23
Strip
edm
ulle
tPh
otob
acte
rium
dam
sela
ess
p.pi
scic
ida
Fish
past
eure
llosi
s
Wild
mul
let(
Liza
klun
zing
eriD
ay)
Stre
ptoc
occu
sag
alac
tiae
Hae
mor
rhag
eson
body
espe
cial
lyin
eye,
mou
th,o
perc
ulum
and
fins.
Org
anis
mcu
lture
dfro
mbr
ain,
eye
and
bloo
d
Stre
ptoc
occo
sis,
epiz
ootic
242
ORN
AMEN
TAL
FISH
Blac
kac
ara
( Cic
hlas
oma
bim
acul
atum
Linn
aeus
)M
ycob
acte
rium
chel
onae
absc
essu
sM
.for
tuitu
m,M
.sim
iae
Myc
obac
terio
sis
474
Com
ets
( Cal
lopl
esio
psal
tivel
isSt
eind
achn
er)
Flav
obac
teriu
mco
lum
nare
Ulc
ers
Mor
talit
y13
5
Dani
o,Si
ndda
nio
( Dan
iode
vario
Ham
ilton
)Ed
war
dsie
llaic
talu
riEn
teric
sept
icae
mia
Blue
disc
usfis
h( S
ymph
ysod
onae
quifa
scia
tus
Pelle
grin
)1.
Aero
mon
ashy
drop
hila
2.A.
jana
daei
3.M
ycob
acte
rium
fortu
itum
2.O
rgan
ism
inliv
er3.
Fin
lesi
ons
1,2.
Dea
ths
3.M
ycob
acte
riosi
s11
613
5
Elec
tric
blue
hap
( Sci
aeno
chro
mis
ahli T
rew
avas
)Ae
rom
onas
jana
daei
Dea
ths
135
Fire
mou
thci
chlid
( Tho
richt
hys
mee
ki,C
ichl
asom
am
eeki
Brin
d)M
ycob
acte
rium
chel
onae
absc
essu
sM
ycob
acte
riosi
s47
4
Flyi
ngfo
x( E
palz
eorh
ynch
oska
lopt
erus
Blee
ker)
Stre
ptoc
occu
sin
iae
Isol
ated
inpu
regr
owth
Dea
ths
135
Gol
den
shin
er( N
otem
igon
uscr
ysol
euca
sM
itchi
ll)1.
Stre
ptoc
occu
sag
alac
tiae
grou
pB
2.Ed
war
dsie
llaic
talu
ri1.
Stre
ptoc
occo
sis
627
Host
Path
ogen
Norm
alflo
raTi
ssue
site
Dise
ase
stat
usRe
f
Tab
le1.
1.C
ontin
ued.
32Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:19 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
Aquatic Animal Species and Organism Relationship 17
Gol
dfis
h( C
aras
sius
aura
tus
Linn
aeus
)1.
Atyp
ical
Aero
mon
assa
lmon
icid
a2.
Aero
mon
assa
lmon
icid
ass
p.no
va3.
Flav
obac
teriu
mbr
anch
ioph
ilum
4.M
ycob
acte
rium
chel
onae
absc
essu
s5.
Vibr
ioch
oler
a(n
on-0
1)
1,2.
Ulc
erat
ive
skin
lesi
ons,
haem
orrh
ages
3.Ba
cter
iaon
gill
surfa
ce,f
lare
dop
ercu
la,
hype
raem
ic,s
wol
len
gill
tissu
e,ex
cess
muc
uspr
oduc
tion
5.Se
ptic
aem
ia
1,2.
Gol
dfis
hul
cerd
isea
se(G
UD
)3.
Bact
eria
lgill
dise
ase
4.M
ycob
acte
riosi
s5.
Mor
talit
ies,
oppo
rtuni
stic
infe
ctio
n,st
ress
-rela
ted
40 474
602
639
695
825
Gup
py( P
oeci
liare
ticul
ata
Pete
rs,
Lebi
stes
retic
ulat
us)
Myc
obac
teriu
mfo
rtuitu
mFi
nle
sion
s,em
acia
ted,
swam
inci
rcle
sM
ycob
acte
riosi
s11
6
Mol
ly
Ballo
onm
olly
( Poe
cilia
spp.
)Es
cher
ichi
avu
lner
isEx
opht
halm
iain
eyes
,pal
egi
lls,e
mpt
ydi
gest
ive
tract
,ope
ned
oper
culu
m,y
ello
wliv
er
Mor
talit
y51
Blac
km
olly
( Poe
cilia
sphe
nops
Vale
ncie
nnes
)Fl
avob
acte
rium
colu
mna
reW
hite
spot
son
the
back
,hea
dan
dsk
inul
cers
Mor
talit
y21
4
Silv
erm
olly
( Poe
cilia
spp.
)Es
cher
ichi
avu
lner
isPa
leliv
er,b
ento
rcur
ved
body
,thi
nnes
sM
orta
lity
51
Osc
ar( A
stro
notu
soc
ella
tus
Agas
siz,
Apis
togr
amm
aoc
ella
tus)
Myc
obac
teriu
mfo
rtuitu
mSk
inle
sion
son
head
Myc
obac
terio
sis
Ram
sSt
rept
ococ
cus
agal
actia
e(S
.diff
icile
)gr
oup
BO
rgan
ism
inpu
regr
owth
inliv
erD
eath
s13
5
Ros
yba
rbs
( Pun
tius
conc
honi
usH
amilt
on)
Edw
ards
iella
icta
luri
Mor
ibun
d,or
gani
smin
inte
rnal
orga
nsM
orta
lity
of40
%37
4
Siam
ese
fight
ing
fish
( Bet
tasp
lend
ens
Reg
an)
1.Ed
war
dsie
llata
rda
2.M
ycob
acte
rium
fortu
itum
1.M
orib
und,
sing
leor
mul
tiple
cuta
neou
sul
cers
of1
mm
diam
eter
.Org
anis
min
inte
rnal
orga
ns2.
Lesi
ons
1.M
orta
lity
of70
%2.
Myc
obac
terio
sis
374
633
Tetra
Blac
ksk
irted
tetra
( Hyp
hess
obry
con
spp.
)Ed
war
dsie
llata
rda
Sept
icae
mia
,foc
alsu
ppur
ativ
eor
gran
ulom
atou
sle
sion
s,cu
tane
ous
ulce
ratio
ns
Edw
ards
iello
sis
374
548
cont
inue
d
33Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:20 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
18 Chapter 1
Neo
nte
tra(H
yphe
ssob
ryco
nin
nesi
Mye
rs–
scie
ntifi
cna
me,
Para
chei
rodo
nin
nesi
Mye
rs–
valid
nam
e)
1.Fl
avob
acte
rium
colu
mna
re2.
Myc
obac
teriu
mfo
rtuitu
m1.
Skin
disc
olor
atio
n,w
hite
area
sof
necr
osis
.Org
anis
min
skin
,gills
,mus
cle
and
inne
rsur
face
ofsc
ales
2.Le
sion
s
1.M
uscl
ein
fect
ions
2.M
ycob
acte
riosi
s11
654
365
6
Serp
aete
traSt
rept
ococ
cus
agal
actia
e(S
.diff
icile
)gr
oup
BIs
olat
edin
pure
heav
ygr
owth
Dea
ths
135
Thre
e-sp
otgo
uram
i(Tr
icho
gast
ertri
chop
teru
sPa
llas)
Myc
obac
teria
spec
ies
Myc
obac
terio
sis
672
Otte
r,Eu
rope
anot
ter(
Lutra
lutra
)1.
Bruc
ella
pinn
iped
iae
2.St
aphy
loco
ccus
lutra
e3.
Vago
cocc
uslu
trae
1.O
rgan
ism
isol
ated
from
tissu
es2.
Org
anis
min
liver
,spl
een,
lym
phno
de3.
Org
anis
mfo
und
inbl
ood,
liver
,lun
gs,
sple
en
1.Br
ucel
losi
s2.
3.Pa
thog
enic
ityno
tkno
wn
262
264
267
477
Paci
ficst
agho
rnsc
ulpi
n( L
epto
cottu
sar
mat
usG
irard
)M
ycob
acte
rium
scro
fula
ceum
Live
rsw
hite
and
friab
leM
ycob
acte
riosi
s47
4
Perc
h
Euro
pean
perc
h( P
erca
fluvi
atilis
Linn
aeus
)1.
Aero
mon
assa
lmon
icid
ass
p.ac
hrom
ogen
es2.
Yers
inia
ruck
eri
1.La
rge
open
lesi
ons,
desc
alat
ion.
No
typi
cal‘
furu
ncle
s’se
en2.
Dis
ease
isas
soci
ated
with
poor
envi
ronm
enta
lcon
ditio
ns
1.M
orta
lity
2.Ye
rsin
iosi
s53
477
2
Silv
erpe
rch
(Bid
yan
perc
h)( B
idya
nus
bidy
anus
Mitc
hell)
Atyp
ical
Aero
mon
assa
lmon
icid
aC
utan
eous
ulce
rsSk
indi
seas
e,ul
cera
tive
derm
atiti
s82
5
Whi
tepe
rch
( Mor
one
amer
ican
a,G
mel
in, R
occu
sam
eric
anus
)Ph
otob
acte
rium
dam
sela
ess
p.pi
scic
ida
Org
anis
mis
olat
edfro
min
tern
alor
gans
Fish
past
eure
llosi
s,m
assi
vem
orta
lity
708
Yello
wpe
rch
( Per
cafla
vesc
ens
Mitc
hill)
Myc
obac
teriu
mch
elon
aeG
ranu
lom
atou
spe
riton
itis
and
hepa
titis
Myc
obac
terio
sis
204
Pike
No r
the r
np i
k e( E
sox
luci
usLi
nnae
us)
Atyp
ical
Aero
mon
assa
lmon
icid
aSk
inul
cers
Infe
ctio
n32
3
Host
Path
ogen
Norm
alflo
raTi
ssue
site
Dise
ase
stat
usRe
f
Tab
le1.
1.C
ontin
ued.
34Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:20 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
Aquatic Animal Species and Organism Relationship 19
Pira
rucu
( Ara
paim
agi
gas
Cuv
ier),
agi
gant
ictro
pica
lfre
shw
ater
fish
Salm
onel
laar
izon
aeO
rgan
ism
isol
ated
from
liver
,spl
een,
hear
t,ki
dney
,blo
ody
exud
ate
inbo
dyca
vity
,cor
neal
opac
ity
Sept
icae
mia
447
Plai
ce,E
urop
ean
plai
ce( P
leur
onec
tes
plat
essa
Linn
aeus
)Li
ston
ella
angu
illaru
mse
roty
pe07
Lesi
ons
Vibr
iosi
s71
2
Plat
ies,
Sout
hern
plat
yfis
h( X
ipho
phor
usm
acul
atus
Gnt
her)
Flav
obac
teriu
mco
lum
nare
Whi
tesp
ots
onth
eba
ckan
dhe
ad,a
ndsk
inul
cers
Mor
talit
y21
4
Porp
oise
See
unde
rSEA
MAM
MAL
S
Praw
nSe
eun
derS
hrim
p
Rabb
itfis
h
Mar
bled
spin
efoo
t(Si
ganu
sriv
ulat
usFo
rssk
ål)
1.M
ycob
acte
rium
mar
inum
2.Ps
eudo
mon
aspu
trefa
cien
s3.
Stre
ptoc
occu
sin
iae
1.W
hite
-yel
low
nodu
les
inth
esp
leen
2.Sk
indi
scol
orat
ion,
and
foca
lnec
rosi
sbe
com
ing
haem
orrh
agic
,abd
omin
alas
cite
s,ex
opht
halm
ia,o
edem
aan
dne
cros
isin
kidn
eys
3.Sy
stem
icdi
seas
ew
ithdi
ffuse
visc
eral
haem
orrh
ages
1.M
ycob
acte
riosi
s–
infe
ctio
nsp
read
from
cage
fish
tow
ildra
bbitf
ish
2,3.
Mor
talit
y
218
260
666
853
Whi
te-s
potte
dsp
inef
oot(
Siga
nus
cana
licul
atus
Park
)St
rept
ococ
cus
inia
eAs
cite
s,he
pato
meg
aly,
sple
nom
egal
ySt
rept
ococ
cosi
s,m
ass
mor
talit
y84
8
Red
Sea
fish,
Wild
fish,
Strip
edpi
ggy
( Pom
adas
ysst
riden
sFo
rssk
ål),
Varie
gate
dliz
ardf
ish
( Syn
odus
varie
gatu
sLa
cepè
de)
Stre
ptoc
occu
sin
iae
Org
anis
mcu
lture
dfro
mbl
ood.
No
mar
ked
gros
ssi
gns
ofdi
seas
eM
orbi
dity
183
Redf
ish ,
Red
drum
( Sci
aeno
psoc
ella
tus
Linn
aeus
)1.
Euba
cter
ium
spec
ies
(tent
ativ
eid
entif
icat
ion)
2.St
rept
ococ
cus
inia
e
1.O
rgan
ism
isol
ated
from
brai
n,liv
er,
kidn
eyan
dbl
ood
2.Sk
inle
sion
s,ex
opht
halm
ia,e
yede
gene
ratio
n,ne
crot
icar
eas
ofgi
llro
t.O
rgan
ism
inhe
arta
ndsp
leen
1,2.
Mor
talit
y34
318
3
REPT
ILES
Snak
esEd
war
dsie
llata
rda
Isol
ated
from
faec
esC
arrie
rsta
tus
orpa
rtof
the
norm
alflo
ra39
9
Torto
ise
Cor
yneb
acte
rium
test
udin
oris
Mou
thle
sion
s18
0
cont
inue
d
35Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:20 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
20 Chapter 1
Turtl
es(C
aspi
ante
rrapi
n–
Mau
rem
ysca
spic
a,Ea
ster
nbo
xtu
rtle
–Te
rrape
neca
rolin
aca
rolin
a,M
issi
ssip
pim
aptu
rtle
–M
alac
lem
ysko
hni,
Nor
ther
ndi
amon
dbac
kte
rrapi
n–
Mal
acle
mys
terra
pin
terra
pin,
Pain
ted
turtl
e–
Chr
ysem
yspi
cta,
Red
-ear
edtu
rtle
–C
hrys
emys
scrip
tael
egan
s ,St
inkp
ottu
rtle
–St
erno
ther
usod
orat
us,T
rava
ncor
ecr
owne
dtu
rtle
–M
elan
oche
lys
triju
gaco
rona
ta)
Edw
ards
iella
tard
a,Sa
lmon
ella
durh
amC
loac
aeC
arrie
rsta
tus
606
Che
loni
am
ydas
,C.c
aret
ta,
Eret
moc
hely
sim
bric
ata
1.Ae
rom
onas
hydr
ophi
la2.
Der
mat
ophi
lus
chel
onae
3.Fl
avob
acte
rium
spp.
4.M
ycob
acte
rium
spp.
5.Ps
eudo
mon
assp
p.6.
Vibr
ioal
gino
lytic
us
7.Ac
inet
obac
terc
alco
acet
icus
8.Ba
cillu
ssp
p.,M
icro
cocc
ussp
p.,
Mor
axel
lasp
p.,P
rote
ussp
p.
2.Sk
in4.
Lung
lesi
ons
7.O
ralc
avity
8.Pa
rtof
norm
alflo
raon
skin
,ora
lcav
ityan
dtra
chea
1.As
soci
ated
with
bron
chop
neum
onia
,ulc
erat
ive
stom
atiti
s,an
dtra
umat
icul
cera
tive
derm
atiti
sca
used
bybi
ting
2.Sk
inle
sion
s,sk
inab
sces
s,sc
abs
3.As
soci
ated
with
traum
atic
ulce
rativ
ede
rmat
itis
caus
edby
bitin
g,ul
cera
tive
stom
atiti
s,br
onch
opne
umon
iaan
dke
rato
conj
unct
iviti
s–ul
cera
tive
blep
harit
is5.
Asso
ciat
edw
ithtra
umat
icul
cera
tive
derm
atiti
sca
used
bybi
ting,
ulce
rativ
est
omat
itis,
aden
itis,
perit
oniti
s,br
onch
opne
umon
iaan
dke
rato
conj
unct
iviti
s–ul
cera
tive
blep
harit
is
300
301
529
Host
Path
ogen
Norm
alflo
raTi
ssue
site
Dise
ase
stat
usRe
f
Tab
le1.
1.C
ontin
ued.
36Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:20 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
Aquatic Animal Species and Organism Relationship 21
6.As
soci
ated
with
traum
atic
ulce
rativ
ede
rmat
itis
caus
edby
bitin
g,br
onch
opne
umon
iaan
dos
teom
yelit
is
Turtl
es( P
seud
emis
scrip
ta)
Aero
mon
ashy
drop
hila
Infe
ctio
n61
4
Haw
aiia
ngr
een
turtl
eC
itrob
acte
rfre
undi
i,H
afni
aal
vei,
Kleb
siel
laox
ytoc
a,Ph
otob
acte
rium
dam
sela
e ,Ps
eudo
mon
asflu
ores
cens
,Pse
udom
onas
putre
faci
ens ,
Pseu
dom
onas
stut
zeri ,
non-
haem
olyt
icSt
rept
ococ
cus
spp.
,Vib
rioal
gino
lytic
us,V
ibrio
fluvi
alis
Nas
alsw
abs
and
cloa
cals
wab
sfro
map
pare
ntly
heal
thy
turtl
esPa
thog
enic
ityno
tdet
erm
ined
.M
ostl
ikel
yno
rmal
flora
from
thes
esi
tes.
5
Leat
herb
ack
turtl
e( D
erm
oche
lys
coria
cea )
Phot
obac
teriu
mda
mse
lae
ssp.
dam
sela
eFl
uid
inbo
dyca
vity
,cal
care
ous
nodu
les
inlu
ngpa
renc
hym
a,lu
ngs
cong
este
dVa
lvul
aren
doca
rditi
san
dse
ptic
aem
ia59
0
Logg
erhe
adse
atu
rtle
( Car
etta
care
tta)
Aero
cocc
usvi
ridan
sG
asin
dive
rticu
lum
,with
mul
tifoc
algr
anul
omat
ous
sero
sitis
.Gre
enm
ucoi
dflu
idan
dfib
rinon
ecro
ticm
embr
ane
onm
ucos
a.
Oes
opha
geal
dive
rticu
lum
755
Rid
ley
sea
turtl
e( L
epid
oche
lys
oliv
acea
)Vi
brio
mim
icus
Red
uced
viab
ility
ofeg
gsC
ause
offo
odpo
ison
ing
inhu
man
s4
Rhyn
chop
elat
esox
yrhy
nchu
sTe
mm
inck
and
Schl
egel
,Coa
stal
fish
–Ja
pan
( The
rapo
nox
yrhy
nchu
s )
Vibr
ioho
llisae
Inte
stin
alco
nten
tsN
on-p
atho
geni
c58
0
Roac
h( R
utilu
sru
tilus
Linn
aeus
)1.
Aero
mon
assa
lmon
icid
ass
p.ac
hrom
ogen
es2.
Atyp
ical
Aero
mon
assa
lmon
icid
a3.
Yers
inia
ruck
eri
1.La
rge
open
lesi
ons,
area
sof
desc
alat
ion.
No
typi
cal‘
furu
ncle
s’se
en2.
Cut
aneo
usul
cers
3.D
isea
seis
asso
ciat
edw
ithpo
oren
viro
nmen
talc
ondi
tions
1.M
orta
litie
s2.
Ulc
erat
ive
derm
atiti
s3.
Yers
inio
sis
352
534
772
825
826
Rock
fish,
Schl
egel
’sbl
ack
rock
fish
( Seb
aste
ssc
hleg
eliH
ilden
dorf)
Atyp
ical
Aero
mon
assa
lmon
icid
aU
lcer
onth
etru
nk.O
rgan
ism
isol
ated
from
the
kidn
eyan
dbr
ain
Mor
talit
y38
540
3
cont
inue
d
37Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:20 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
22 Chapter 1
Rock
ling
Four
bear
dro
cklin
g(E
nche
lyop
usci
mbr
ius
Linn
aeus
)
Atyp
ical
Aero
mon
assa
lmon
icid
aSk
inul
cers
832
Rotif
er( B
rach
ionu
spl
icat
ilis)
1.Vi
brio
rotif
eria
nus
2.V.
nept
uniu
s1,
2.Is
olat
edfro
mro
tifer
flow
-thro
ugh
cultu
resy
stem
.Pat
hoge
nici
tyno
tkno
wn
305
740
Rudd
( Sca
rdin
ius
eryt
hrop
htha
lmus
Linn
aeus
)1.
Aero
mon
assa
lmon
icid
ass
p.ac
hrom
ogen
es2.
Yers
inia
ruck
eri
209
318
SALM
ONI
DS
Arct
icCh
ar( S
alve
linus
alpi
nus
Linn
aeus
)1.
Aero
mon
assa
lmon
icid
ass
p.sa
lmon
icid
a2.
Atyp
ical
Aero
mon
assa
lmon
icid
a3.
Serra
tialiq
uefa
cien
s
4.C
arno
bact
eriu
msp
p.1,
2.Sk
inul
cers
3.R
edne
ssan
dsw
ellin
gar
ound
anus
,as
cite
s,ha
emor
rhag
icin
tern
altis
sues
1.Fu
runc
ulos
is2.
Infe
ctio
n3.
Mor
talit
y4.
Non
-pat
hoge
nic
323
352
648
715
Salm
on
Atla
ntic
salm
on( S
alm
osa
lar
Linn
aeus
)1.
Aero
mon
assa
lmon
icid
ass
p.sa
lmon
icid
a2.
Atyp
ical
Aero
mon
assa
lmon
icid
a3.
Flav
obac
teriu
mps
ychr
ophi
lum
4.Li
ston
ella
angu
illaru
m5.
Mor
itella
visc
osa
6.M
ycob
acte
rium
chel
onae
7.N
ocar
dia
serio
lae
8.Pa
steu
rella
skye
nsis
9.Ph
otob
acte
rium
dam
sela
ess
p.da
mse
lae
10.P
seud
omon
asan
guilli
sept
ica
11.R
enib
acte
rium
salm
onin
arum
12.S
erra
tialiq
uefa
cien
s13
.Stre
ptob
acillu
sm
onilif
orm
is-li
keor
gani
sm14
. Ten
acib
acul
umm
ariti
mum
15.V
agoc
occu
ssa
lmon
inar
um
22.C
arno
bact
eriu
min
hibe
ns23
.Car
noba
cter
ium
spp.
24. L
acto
baci
llus
spp.
25. P
hoto
bact
eriu
milio
pisc
ariu
m26
.Vib
riopa
cini
i
1.Sk
inul
cers
2.O
rgan
ism
from
head
kidn
ey,c
utan
eous
lesi
ons
3.Er
osio
nof
fins
5.Sk
inul
cers
,int
erna
ldar
k-br
own
pete
chia
eor
ecch
ymot
icha
emor
rhag
e6.
Gra
nulo
ma-
like
nodu
les
intis
sues
7.G
ranu
lom
atou
sle
sion
son
body
8.M
ultif
ocal
area
sof
coag
ulat
ive
necr
osis
inki
dney
,liv
er,s
plee
n10
.Pet
echi
alha
emor
rhag
eson
skin
,m
outh
and
anus
,per
itone
uman
dliv
er11
.Whi
teno
dule
sin
kidn
ey12
.Sw
ellin
gof
kidn
eys,
blee
ding
atth
ean
usan
din
test
ine,
gast
roen
terit
is13
.Org
anis
min
trace
llula
rin
tissu
es,
endo
thel
ialc
ells
ofki
dney
glom
erul
ien
larg
ed
1.Fu
runc
ulos
is2.
Sim
ilart
ofu
runc
ulos
is3.
Mor
bidi
tyra
ther
than
mor
talit
y4.
Vibr
iosi
s5.
Win
teru
lcer
s6.
Myc
obac
terio
sis
7.N
ocar
dios
is8.
Mor
talit
y9.
Path
ogen
icin
chal
leng
eex
perim
ents
10.H
aem
orrh
agic
dise
ase
11.B
acte
rialk
idne
ydi
seas
e12
,13.
Mor
talit
y14
.Bac
teria
lsto
mat
itis
(mou
thro
t)in
smol
ts15
.Mor
talit
y16
.Blin
dnes
s17
.Pos
sibl
epa
thog
en
49 100
107
117
132
133
137
144
232
306
330
352
411
412
416
450
506
519
Host
Path
ogen
Norm
alflo
raTi
ssue
site
Dise
ase
stat
usRe
f
Tab
le1.
1.C
ontin
ued.
38Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:21 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
Aquatic Animal Species and Organism Relationship 23
16.V
arra
calb
mi
17.V
ibrio
loge
i18
.Vib
rioor
dalii
19.V
ibrio
salm
onic
ida
20.V
ibrio
wod
anis
21.Y
ersi
nia
ruck
eris
erot
ype
I
14. Y
ello
w-c
olou
red
plaq
ueon
teet
han
dor
alca
vity
15.P
erito
nitis
,hae
mor
rhag
icas
cite
s,re
tain
edeg
gs,e
ngor
ged
test
es,l
angu
idsw
imm
ing
16.D
eep
skin
lesi
ons,
eye
lesi
ons,
haem
orrh
agic
and
pyog
ranu
lom
atou
sle
sion
sin
gills
,kid
ney,
liver
and
pseu
dobr
anch
17.S
kin
lesi
ons
21.H
aem
orrh
agic
mus
cula
ture
25.I
ntes
tine
18. M
orta
lity
20.O
ppor
tuni
stic
infe
ctio
nin
‘win
teru
lcer
’dis
ease
21.E
nter
icre
dmou
th(E
RM
)22
.Inh
ibits
grow
thof
L.an
guilla
rum
and
A.sa
lmon
icid
a23
.Pot
entia
lpro
biot
ic24
.Nor
mal
mic
roflo
ra25
.Non
-pat
hoge
nic
538
564
599
605
611
622
648
682
683
712
767
771
824
828
Chi
nook
salm
on( O
ncor
hync
hus
tsch
awyt
scha
Wal
baum
)1.
Edw
ards
iella
tard
a2.
Flav
obac
teriu
mbr
anch
ioph
ilum
3.F.
colu
mna
re4.
Myc
obac
teriu
mne
oaur
um5.
Myc
obac
teriu
msp
p.6.
Ren
ibac
teriu
msa
lmon
inar
um7.
Tena
ciba
culu
mm
ariti
mum
8.Ye
rsin
iaru
cker
i
2.Ba
cter
iaat
tach
edto
gill
epith
eliu
m3.
Gill
lesi
ons
4.O
rgan
ism
pres
enti
ney
es,
exop
htha
lmia
,and
ocul
arle
sion
s6.
Gill
lesi
ons
1.Ed
war
dsie
llosi
s2.
Bact
eria
lgill
dise
ase
(BG
D)
3.M
orta
lity
4.Pa
noph
thal
miti
s5.
Myc
obac
terio
sis
6.Ba
cter
ialk
idne
ydi
seas
e(B
KD)
7.M
orta
lity
8.ER
M
36 53 88 137
154
209
245
802
Paci
ficsa
lmon
,Coh
osa
lmon
( O.
kisu
tch
Wal
baum
)1.
Aero
mon
assa
lmon
icid
a(o
xida
se-n
egat
ive
stra
in)
2.Fl
avob
acte
rium
colu
mna
re3.
F.ps
ychr
ophi
lum
4.Li
ston
ella
angu
illaru
m01
5.R
enib
acte
rium
salm
onin
arum
1.Fi
nger
lings
affe
cted
.Sof
tkid
neys
,oc
casi
onal
fish
with
haem
orrh
age
onfin
,ot
herw
ise
noex
tern
alsi
gns
1.Fu
runc
ulos
is3.
Col
d-w
ater
dise
ase
4.Vi
brio
sis
5.BK
D
168
712
748
749
757
765
Che
rrysa
lmon
( Onc
orhy
nchu
sm
asou
mas
ouBr
evoo
rt).K
now
nas
Yam
ame
inJa
pan
Flav
obac
teriu
mbr
anch
ioph
ilum
BGD
802
Sock
eye
salm
on( O
.ner
kaW
alba
um)
1.Fl
avob
acte
rium
bran
chio
philu
m2.
Yers
inia
ruck
eri
1.Ba
cter
ium
atta
ches
togi
llep
ithel
ium
1.BG
D2.
ERM
137
802
cont
inue
d
39Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:21 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
24 Chapter 1
Salm
onid
s–
Trou
t
Broo
ktro
ut(S
alve
linus
font
inal
isM
itchi
ll)1.
Atyp
ical
Aero
mon
assa
lmon
icid
a2.
Edw
ards
iella
tard
a3.
Flav
obac
teriu
mbr
anch
ioph
ilum
4.N
ocar
dia
spp.
5.Ye
rsin
iaru
cker
i
1.Sk
inul
cers
3.Ba
cter
iaat
tach
togi
llep
ithel
ium
4.N
ecro
sis,
thro
mbo
sis
inki
dney
,spl
een,
gills
1.Sk
inul
cers
and
sept
icae
mia
2.Ac
ute
bact
eria
lsep
ticae
mia
3.BG
D4.
Noc
ardi
osis
5.ER
M
137
604
765
824
Brow
ntro
ut,S
eatro
ut,S
teel
head
trout
( Sal
mo
trutta
trutta
Linn
aeus
)1.
Aero
mon
assa
lmon
icid
ass
p.sa
lmon
icid
a2.
Atyp
ical
Aero
mon
assa
lmon
icid
a3.
Flav
obac
teriu
mbr
anch
ioph
ilum
4.Fl
avob
acte
rium
colu
mna
re5.
Haf
nia
alve
i6.
List
onel
laan
guilla
rum
027.
Pseu
dom
onas
angu
illise
ptic
a
1,2.
Skin
ulce
rs3.
Skin
arou
nddo
rsal
finbl
anch
ed,b
utno
ulce
ratio
nse
en5.
Org
anis
man
dle
sion
sin
the
kidn
ey6.
Lesi
ons
7.Pe
tech
ialh
aem
orrh
ages
onsk
in,m
outh
and
anus
,per
itone
uman
dliv
eran
dat
base
offin
s
1.Fu
runc
ulos
is2.
Skin
ulce
rsan
dse
ptic
aem
ia3.
Col
umna
risdi
seas
e,ch
roni
cm
orta
lity
4.BG
D5.
Opp
ortu
nist
icpa
thog
en6.
Vibr
iosi
s7.
Hae
mor
rhag
icdi
seas
e
88 352
652
712
802
824
828
Rai
nbow
trout
( Onc
orhy
nchu
sm
ykis
sW
alba
um)
Prev
ious
nam
e( S
alm
oga
irdne
riR
icha
rdso
n)
1.Ae
rom
onas
salm
onic
ida
ssp.
salm
onic
ida
2.At
ypic
alAe
rom
onas
salm
onic
ida
3.At
ypic
alAe
rom
onas
salm
onic
ida
(gro
wth
at37
°C)
4.Ae
rom
onas
sobr
ia5.
Car
noba
cter
ium
pisc
icol
a6.
Clo
strid
ium
botu
linum
7.Es
cher
ichi
avu
lner
is8.
Edw
ards
iella
tard
a9.
Flav
obac
teriu
mbr
anch
ioph
ilum
10.F
.psy
chro
philu
m11
.Jan
thin
obac
teriu
mliv
idum
12.K
lebs
iella
pneu
mon
iae
13.L
acto
cocc
usga
rvie
ae14
.Lac
toco
ccus
pisc
ium
15.L
isto
nella
angu
illaru
m16
.Mic
roco
ccus
lute
us17
.Mor
itella
visc
osa
31.C
itrob
acte
rfre
undi
i1,
2,3.
Skin
ulce
rs4.
Path
ogen
icfo
rfin
gerli
ngs
invi
rule
nce
assa
y(7
50)
5.Bi
late
rale
xoph
thal
mia
,per
iocu
lar
haem
orrh
ages
,asc
ites
fluid
and
haem
orrh
ages
inliv
er,s
wim
blad
der,
mus
cle
and
inte
stin
e6.
Toxi
nin
seru
man
din
test
inal
cont
ents
7.H
aem
orrh
agic
lesi
ons
onsk
in,b
lood
yex
udat
ein
dige
stiv
etra
ct,l
iver
yello
wan
dhy
pera
emic
,gon
ads
haem
orrh
agic
8. 9.Ba
cter
iaat
tach
togi
llep
ithel
ium
12.F
inan
dta
il15
.Les
ions
16.O
rgan
ism
isol
ated
from
kidn
ey,s
plee
nan
das
cite
sflu
id17
.Ski
nle
sion
s,pe
tech
ialh
aem
orrh
age
inliv
eran
dpe
riton
ealm
embr
anes
1.Fu
runc
ulos
is2,
3Sk
inul
cers
,sep
ticae
mia
4.H
aem
orrh
agic
sept
icae
mia
5.C
hron
icdi
seas
ew
ithlo
wle
vel
mor
talit
y6.
Botu
lism
7.M
orta
lity
8.Ed
war
dsie
llosi
s9.
BGD
10.
11.M
orta
lity
infry
,pos
sibl
eop
portu
nist
icin
fect
ion
12.F
inan
dta
ildi
seas
e13
.Mor
talit
y14
.Pse
udok
idne
ydi
seas
e,La
ctob
acillo
sis
15.V
ibrio
sis,
sept
icae
mia
16.I
sola
ted
from
mor
ibun
dfis
h17
.Mor
talit
y
296 40 43 48 51 73 76 81 82 107
135
137
141
195
205
233
234
352
268
Host
Path
ogen
Norm
alflo
raTi
ssue
site
Dise
ase
stat
usRe
f
Tab
le1.
1.C
ontin
ued.
40Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:21 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
Aquatic Animal Species and Organism Relationship 25
18.M
ycob
acte
rium
mar
inum
19.N
ocar
dia
aste
roid
es20
.Pho
toba
cter
ium
dam
sela
ess
p.da
mse
lae
21.P
lesi
omon
assh
igel
loid
es22
.Pse
udom
onas
angu
illise
ptic
a23
.Ren
ibac
teriu
msa
lmon
inar
um01
,02
,03,
0424
. Ser
ratia
mar
cesc
ens,
S.pl
ymut
hica
25.S
taph
yloc
occu
sw
arne
ri26
Stre
ptoc
occu
sag
alac
tiae
(S.
diffi
cile
)27
Stre
ptoc
occu
sin
iae
28Va
goco
ccus
salm
onin
arum
29.V
ibrio
orda
lii30
.Yer
sini
aru
cker
i
18. L
esio
nsin
kidn
ey19
.Les
ions
inki
dney
20.O
rgan
ism
pres
enti
nhe
adki
dney
21.P
etec
hial
haem
orrh
ages
inin
test
ine
22.P
etec
hial
haem
orrh
ages
onsk
in,
mou
than
dan
usan
dba
seof
fins,
perit
oneu
man
dliv
er23
.Whi
teno
dule
sin
kidn
ey25
.Exo
phth
alm
ia,a
scite
sflu
id,l
esio
nson
fins,
orga
nism
inki
dney
and
liver
26,2
7.O
rgan
ism
sin
brai
nan
dey
e28
.Los
sof
equi
libriu
m,h
aem
orrh
age
arou
ndey
ean
dgi
ll,le
sion
son
body
,co
nges
tion
insp
leen
and
liver
.Per
itoni
tis,
haem
orrh
agic
asci
tes,
reta
ined
eggs
,en
gorg
edte
stes
,lan
guid
swim
min
g30
.Hae
mor
rhag
ear
ound
mou
than
din
test
ines
18. M
orta
lity,
myc
obac
terio
sis
19.N
ocar
dios
is20
.Pat
hoge
nic
inch
alle
nge
expe
rimen
tsan
dna
tura
lin
fect
ion
21.P
ossi
ble
oppo
rtuni
stic
path
ogen
22.H
aem
orrh
agic
dise
ase
23.B
KD24
.Mor
talit
yin
finge
rling
s25
.Dis
ease
dan
ddy
ing
trout
–op
portu
nist
icin
fect
ion
26,2
7.M
enin
goen
ceph
aliti
s,se
ptic
aem
ia28
.Chr
onic
dise
ase
with
mor
talit
y29
.Vib
riosi
s30
.ER
M31
.Opp
ortu
nist
icpa
thog
en
542
564
579
604
618
640
682
707
712
716
748
750
752
753
757
802
824
828
835
853
Amag
otro
utPs
eudo
mon
asch
loro
raph
isH
aem
orrh
ages
,inc
reas
edas
cite
sIn
fect
ion
332
Sard
ine
Paci
ficsa
rdin
e,So
uth
Amer
ican
pilc
hard
( Sar
dino
pssa
gax
Jeny
ns)
Tena
ciba
culu
mm
ariti
mum
Glid
ing
bact
eria
seen
asa
tan-
colo
ured
pseu
dom
embr
ane
over
the
body
Dis
ease
154
SEA
BIRD
S
Cran
e(S
andh
illcr
ane
–G
rus
cana
dens
is)
Edw
ards
iella
tard
a,Sa
lmon
ella
hartf
ord ,
S.ja
vaLa
rge
inte
stin
e82
3
Eagl
e,Ba
ldea
gle
( Hal
iaee
tus
leuc
ocep
halu
s )Ed
war
dsie
llata
rda
Clo
acal
swab
Bird
was
sick
butn
oot
her
evid
ence
ofin
fect
ion
823
Gul
l,R
ing-
bille
dgu
ll( L
arus
dele
war
ensi
s )Ed
war
dsie
llata
rda
Larg
ein
test
ine
823
Hero
n(G
reat
blue
hero
n–
Arde
ahe
rodi
as)
Edw
ards
iella
tard
aLa
rge
inte
stin
e82
3
Loon
(Com
mon
loon
–G
avia
imm
er)
Edw
ards
iella
tard
aIn
test
inal
cont
ent
Hae
mor
rhag
icen
terit
is,i
ntes
tinal
cont
entd
ark
and
tarry
823
cont
inue
d
41Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:21 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
26 Chapter 1
Pelic
anBr
own
pelic
an(P
elec
annu
soc
cide
ntal
isca
rolin
ensi
s )
Edw
ards
iella
tard
aIn
test
inal
cont
ent,
lung
,liv
erH
aem
orrh
agic
ente
ritis
,int
estin
alco
nten
tdar
kan
dta
rry82
3
Peng
uin
Gen
too
peng
uin
from
sub-
Anta
rctic
regi
onSa
lmon
ella
ente
ritid
is,S
.hav
ana,
S.ty
phim
uriu
mC
arrie
rsta
tus
612
Mac
aron
ipen
guin
( Eud
ypte
sch
ryso
loph
us)
Burk
hold
eria
(Pse
udom
onas
)ps
eudo
mal
lei
Pin-
poin
t,w
hite
lesi
ons
inliv
eran
dlu
ng,
fluid
inai
rsac
s,or
gani
smin
sple
en,l
iver
,he
artb
lood
Mel
ioid
osis
516
Peng
uins
( Apt
enod
ytes
pata
goni
ca,
Eudy
ptes
cres
tatu
s,Py
osce
lispa
pua ,
Sphe
nisc
usde
mer
sus,
Sphe
nisc
ushu
mbo
ldti)
Prov
iden
cia
fried
eric
iana
Isol
ated
from
faec
esof
capt
ive
peng
uins
559
Shad
Giz
zard
shad
( Dor
osom
ace
pedi
anum
Lesu
eur)
Aero
mon
asso
bria
Isol
ated
inpu
recu
lture
from
kidn
ey,l
iver
,sp
leen
Isol
ates
path
ogen
icfo
rfin
gerli
ngtro
utin
viru
lenc
eas
say
Epiz
ootic
insp
awni
ngfe
mal
es75
0
Sea
brea
mSe
eun
derB
ream
Sea
gras
sVi
brio
aero
gene
sVi
brio
diaz
otro
phic
usPr
esen
tin
sedi
men
t31
969
2
Seah
orse
( Hip
poca
mpu
sku
daan
dH
ippo
cam
pus
spec
ies)
Vibr
ioha
rvey
iEx
tern
alha
emor
rhag
es,h
aem
orrh
agic
liver
,asc
ites
fluid
Mor
talit
y11 13
5
Sea
drag
on,L
eafy
sea
drag
onPh
ycod
urus
equi
sVi
brio
harv
eyi
Isol
ated
inpu
recu
lture
from
hear
tblo
od,
liver
Dea
th.P
atho
geni
city
ofth
eor
gani
smno
tdet
erm
ined
135
Seal
.See
unde
rSEA
MAM
MAL
S
Sea
Lion
.See
unde
rSEA
MAM
MAL
S
SEA
MAM
MAL
S(O
rder
Cet
acea
)Se
am
amm
als
incl
ude
the
thre
em
amm
alia
nor
ders
cons
ider
edto
betru
eaq
uatic
mam
mal
s.Th
ese
are
Cet
acea
(wha
les,
dolp
hins
and
porp
oise
s),
Pinn
iped
ia(s
eals
,sea
-lion
san
dw
alru
ses)
,and
Sire
nian
s(s
eaco
ws)
(Fos
tere
tal.,
2002
).
Dolp
hin
Dol
phin
fish
–se
eM
ahi-m
ahi
Host
Path
ogen
Norm
alflo
raTi
ssue
site
Dise
ase
stat
usRe
f
Tab
le1.
1.C
ontin
ued.
42Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:22 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
Aquatic Animal Species and Organism Relationship 27
Atla
ntic
whi
te-s
ided
dolp
hin
( Lag
enor
hync
hus
acut
us)
1.Br
ucel
lace
tace
ae2.
Hel
icob
acte
rcet
orum
1.O
esop
hage
alul
cera
tion,
abor
tion,
hepa
tican
dsp
leni
cco
agul
ativ
ene
cros
is2.
Org
anis
mfo
und
ingl
andu
larm
ucos
aof
mai
nst
omac
h
1.Br
ucel
losi
s2.
Gas
tric
ulce
ratio
n26
232
726
732
732
8
Amaz
onfre
shw
ater
dolp
hin
( Inia
geof
frens
is)
Stre
ptoc
occu
sin
iae
Subc
utan
eous
absc
esse
sG
olfb
alld
isea
se62
5
Turs
iops
adun
cas
Past
eure
llam
ulto
cida
Inte
stin
alha
emor
rhag
ean
dba
cter
aem
ia.
Sour
ceof
infe
ctio
nw
asco
ntam
inat
ion
from
loca
lbird
rook
ery
Ente
ritis
,mor
talit
y72
6
Turs
iops
geph
yreu
sH
elic
obac
ters
pp.
Pres
enti
nde
ntal
plaq
ueM
ayac
tas
are
serv
oirf
orga
stric
infe
ctio
nan
dga
stric
ulce
rs30
3
Atla
ntic
bottl
enos
edo
lphi
n( T
ursi
ops
trunc
atus
)1.
Bruc
ella
spec
ies
2.Bu
rkho
lder
iaps
eudo
mal
lei
3.C
lost
ridiu
mpe
rfrin
gens
4.Ed
war
dsie
llata
rda
5.Er
ysip
elot
hrix
rhus
iopa
thia
e6.
Hel
icob
acte
rcet
orum
6.M
annh
eim
iaha
emol
ytic
a8.
Phot
obac
teriu
mda
mse
lae
ssp.
dam
sela
e9.
Phot
obac
teriu
mda
mse
lae
ssp.
pisc
icid
a
1.Ab
ortio
n2.
Oed
ema,
haem
orrh
age
and
nodu
les
inlu
ngs
3.Ab
sces
sin
dors
alm
uscl
e4.
Puru
lent
mas
titis
5.U
lcer
son
skin
seen
asrh
ombo
idpl
aque
s,lu
ngs
cong
este
dan
doe
dem
atou
s.O
rgan
ism
inal
lorg
ans
6.O
rgan
ism
cultu
red
from
faec
es7.
Hae
mor
rhag
ictra
chei
tis
1.Br
ucel
losi
s2.
Mel
ioid
osis
3.C
lost
ridia
lmyo
sitis
4.M
astit
is5.
Erys
ipel
as,s
eptic
aem
ia6.
Ulc
ers
inoe
soph
agus
and
fore
stom
ach
–ga
strit
is7.
Sept
icae
mia
8.W
ound
infe
ctio
n9.
Past
eure
llosi
s
349
247
312
292
328
726
Com
mon
dolp
hin
( Del
phin
usde
lphi
s )1.
Bruc
ella
ceta
ceae
2.H
elic
obac
ters
pp.
3.Pa
steu
rella
mul
toci
da4.
Stap
hylo
cocc
usde
lphi
ni
1.Su
bcut
aneo
usle
sion
2.O
rgan
ism
sfo
und
ingl
andu
larm
ucos
aof
mai
nst
omac
h3.
Inte
stin
alha
emor
rhag
e4.
Puru
lent
skin
lesi
ons
1.Br
ucel
losi
s2.
Gas
tric
ulce
ratio
n3.
Ente
ritis
4.Sk
inin
fect
ion
327
267
404
658
726
778
Paci
ficw
hite
-sid
eddo
lphi
n( L
agen
orhy
nchu
sob
liqui
dens
)1.
Burk
hold
eria
pseu
dom
alle
i2.
Hel
icob
acte
rcet
orum
1.O
edem
a,ha
emor
rhag
ean
dno
dule
sin
lung
s2.
Org
anis
mcu
lture
dfro
mfa
eces
1.M
elio
idos
is2.
Ulc
ers
inoe
soph
agus
and
fore
stom
ach
–ga
strit
is
349
328
Strip
eddo
lphi
n( S
tene
llaco
erul
eoal
ba)
1.Br
ucel
lace
tace
ae2.
Cry
ptoc
occu
sne
ofor
man
s(y
east
)3.
Actin
obac
illus
delp
hini
cola
1.M
enin
gitis
2.Pu
lmon
ary
infe
ctio
n3.
Isol
ated
from
vario
ustis
sues
1.Br
ucel
losi
s2.
Pulm
onar
ycr
ypto
cocc
osis
3.Pa
thog
enic
ityno
tdet
erm
ined
278
262
263
267
cont
inue
d
43Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:22 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
28 Chapter 1
Porp
oise
Har
bour
porp
oise
(Pho
coen
aph
ocoe
na)
1.Br
ucel
lace
tace
ae2.
Erys
ipel
othr
ixrh
usio
path
iae
3.St
rept
ococ
cus
dysg
alac
tiae
ssp.
dysg
alac
tiae
Lanc
efie
ldG
roup
L4.
Vago
cocc
usfe
ssus
5.Ac
tinob
acillu
sde
lphi
nico
la6.
Actin
omyc
esm
arim
amm
aliu
m7.
Actin
obac
illus
scot
iae
8.Ar
cano
bact
eriu
mpl
uran
imal
ium
9.Ph
ocoe
noba
cter
uter
i
1.Ly
mph
node
s2.
Cut
aneo
usle
sion
s3.
Inte
stin
e,ki
dney
,lun
g,sp
leen
4,5.
Isol
ated
from
vario
ustis
sues
6.Lu
ng7.
Live
r,lu
ng,b
rain
and
sple
enof
stra
nded
porp
oise
9.U
teru
s
1.Br
ucel
losi
s2.
Erys
ipel
as3.
Sept
icae
mia
,br
onch
opne
umon
ia,m
yoca
rditi
s,py
elon
ephr
itis
4,5,
6,7,
8,9.
Path
ogen
icity
not
dete
rmin
ed
262
263
265
266
267
369
370
404
480
658
686
727
Sea
lion
Sea
lion
( Zal
ophu
sca
lifor
nian
us)
1 .Bu
rkho
lder
iaps
eudo
mal
lei
2.C
lost
ridiu
mpe
rfrin
gens
3.Ed
war
dsie
llata
rda
4.Es
cher
ichi
aco
li5.
Past
eure
llam
ulto
cida
6.Sa
lmon
ella
heid
elbe
rg,S
.ne
wpo
rt ,S.
oran
ienb
urg
1.O
edem
a,ha
emor
rhag
ean
dno
dule
sin
lung
s2.
Infe
ctio
nin
mus
cle
with
gas
and
pus
3.Ab
sces
ses
inlu
ngs,
sang
uino
-muc
opur
ulen
texu
date
intra
chea
and
term
inal
bron
chi
4.Ve
rruco
us,g
rey-
red
lesi
ons
inat
riove
ntric
ular
valv
es5.
Yello
w,p
us-fi
lled
fluid
inpl
eura
lcav
ity6.
Rec
over
edfro
mhe
alth
yan
imal
s
1.M
elio
idos
is2.
Clo
strid
ialm
yosi
tis3.
Bact
eria
lpne
umon
ia,
resp
irato
ryin
fect
ion
Path
ogen
icity
notc
oncl
usiv
e4.
Endo
card
itis
5.M
orta
lity
6.N
on-p
atho
geni
cor
carri
erst
atus
298
312
349
430
435
804
Seal
s( P
inni
pedi
a)
Com
mon
seal
orH
arbo
urse
al( P
hoca
vitu
lina)
1.Br
ucel
lapi
nnip
edia
e2.
Myc
opla
sma
phoc
icer
ebra
le3.
Myc
opla
sma
phoc
irhin
is4.
Stre
ptoc
occu
sph
ocae
5.Va
goco
ccus
fess
us
6.Ar
cano
bact
eriu
m(C
oryn
ebac
teriu
m)p
hoca
e7.
Arth
roba
cter
nasi
phoc
ae8.
Atop
obac
terp
hoca
e9.
Myc
opla
sma
phoc
idae
1.G
astri
cly
mph
node
,spl
een
2.Is
olat
edfro
mbr
ain,
hear
t,lu
ng,n
ose,
thro
at3.
Isol
ated
from
pus
inlu
ng4.
Lesi
ons
inlu
ng,e
xuda
tein
bron
chi
5.O
rgan
ism
isol
ated
from
liver
and
kidn
eyin
pure
grow
th6.
Org
anis
min
nasa
lpas
sage
1.Br
ucel
losi
s2,
3.As
soci
ated
with
resp
irato
rydi
seas
e4.
Pneu
mon
ia–
oppo
rtuni
stic
infe
ctio
nin
seal
mor
billiv
irus
infe
ctio
n5,
6,7,
8.Pa
thog
enic
ityno
tkn
own
404
182
295
449
267
369
479
613
Host
Path
ogen
Norm
alflo
raTi
ssue
site
Dise
ase
stat
usRe
f
Tab
le1.
1.C
ontin
ued.
44Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpTuesday, January 20, 2004 1:57:58 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
Aquatic Animal Species and Organism Relationship 29
7.O
rgan
ism
isol
ated
from
inte
stin
ean
dly
mph
node
s.Th
ean
imal
had
lym
phad
enop
athy
and
pulm
onar
yha
emor
rhag
e9.
Isol
ated
from
resp
irato
rytra
ct
9.Av
irule
nt65
866
070
0
Gre
yse
al( H
alic
hoer
usgr
ypus
)1.
Aero
mon
ashy
drop
hila
2.Br
ucel
lapi
nnip
edia
e3.
Burk
hold
eria
pseu
dom
alle
i4.
Stre
ptoc
occu
sph
ocae
5.Ac
tinom
yces
mar
imam
mal
ium
6.Ar
cano
bact
eriu
mph
ocae
1.O
rgan
ism
inlu
ngan
dliv
er.I
sola
tepo
sitiv
efo
raer
olys
inge
ne,c
ytot
oxin
and
haem
olys
in2.
Isol
ated
from
lung
3.O
edem
a,ha
emor
rhag
ean
dno
dule
sin
lung
s4.
Lesi
ons
inlu
ng,e
xuda
tein
bron
chi
5.Is
olat
edfro
min
test
ine
1.Se
ptic
aem
ia2.
Bruc
ello
sis
3.M
elio
idos
is4.
Pneu
mon
ia–
oppo
rtuni
stic
infe
ctio
nin
seal
mor
billiv
irus
infe
ctio
n5,
6.Pa
thog
enic
ityno
tcle
ar.
Isol
ated
from
seal
sw
ithse
ptic
aem
iaan
dpn
eum
onia
454
349
262
370
267
613
700
Har
pse
al( P
hoca
groe
nlan
dica
)Br
ucel
lasp
p.O
rgan
ism
inly
mph
node
sBr
ucel
losi
s26
1
Hoo
ded
seal
( Cys
toph
ara
cris
tata
)1.
Bruc
ella
pinn
iped
iae
2.Ac
tinom
yces
mar
imam
mal
ium
2.Is
olat
edfro
mlu
ng1.
Bruc
ello
sis,
stre
ss-re
late
d2.
Path
ogen
icity
notd
eter
min
ed26
237
026
7
Nor
ther
nfu
rsea
l(C
allo
rhin
usur
sinu
s )1.
Salm
onel
laad
elai
de,S
.he
idel
berg
,S.n
ewpo
rt,S.
oran
ienb
urg
2.Ac
inet
obac
terc
alco
acet
icus
,Ac
tinob
acillu
ssp
p.,A
erom
onas
eucr
enop
hila
,Alc
alig
enes
faec
alis
,E.
coli,
Ente
roba
cter
spp.
,Kl
ebsi
ella
spp.
,Mor
axel
lasp
p.,
Pseu
dom
onas
fluor
esce
ns,
Pseu
dom
onas
spp.
,S.e
pide
rmid
is,
Stre
ptoc
occu
ssp
p.3.
Baci
llus
spp.
,Cor
yneb
acte
rium
spp.
,E.c
oli,
List
eria
spp.
,M
orax
ella
spp.
,Nei
sser
iacu
nicu
li,Pr
oteu
sm
irabi
lis,S
taph
yloc
occu
sep
ider
mid
is,S
trept
ococ
cus
spp.
1.R
ecov
ered
from
heal
thy
anim
als
2.R
ectu
m3.
Oro
phar
ynx
1.N
on-p
atho
geni
cor
carri
erst
atus
2,3.
Nor
mal
flora
298
779
Rin
ged
seal
( Pho
cahi
spid
a)Br
ucel
lasp
ecie
sO
rgan
ism
inly
mph
node
sBr
ucel
losi
s26
1
cont
inue
d
45Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:22 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
30 Chapter 1
Sout
hG
eorg
ian
Anta
rctic
furs
eal
Salm
onel
laen
terit
idis
,S.h
avan
a,S.
new
port,
S.ty
phim
uriu
mC
arrie
rsta
tus
612
Seal
Myc
opla
sma
Seco
ndar
yin
fect
ion
due
tose
alm
orbi
lliviru
s
Sout
hern
elep
hant
seal
( Miro
unga
leon
ina )
Fack
lam
iam
iroun
gae
Nas
alsw
abIs
olat
edfro
map
pare
ntly
heal
thy
juve
nile
anim
al36
8
Wha
le
Belu
gaw
hale
,Whi
tew
hale
( Del
phin
apte
rus
leuc
as)
1.H
elic
obac
terc
etor
um2.
Myc
obac
teriu
mm
arin
um1.
Inap
pete
nce
and
leth
argy
.Oes
opha
geal
and
fore
stom
ach
ulce
rs2.
Pyog
ranu
lom
atou
sde
rmat
itis
and
pann
icul
itis
1.G
astri
tis2.
Myc
obac
terio
sis
329
327
328
111
Fals
eki
llerw
hale
( Pse
udor
cacr
assi
dens
)Bu
rkho
lder
iaps
eudo
mal
lei
Oed
ema,
haem
orrh
age
and
nodu
les
inlu
ngs
Mel
ioid
osis
349
Kille
rwha
le( O
rcin
usor
ca)
1.Bu
rkho
lder
iaps
eudo
mal
lei
2.C
andi
dasp
p.3.
Clo
strid
ium
perfr
inge
ns
1.O
edem
a,ha
emor
rhag
ean
dno
dule
sin
lung
s2.
Nec
rotic
skin
lesi
ons,
blow
hole
lesi
ons,
can
beco
me
syst
emic
3.To
xaem
ia,o
edem
atou
sly
mph
node
s,liq
uefie
dm
uscl
e
1.M
elio
idos
is2.
Infe
ctio
n3.
Clo
strid
ialm
yosi
tis
349
312
726
Min
kew
hale
( Bal
aeno
pter
aac
utor
ostra
ta)
1.Br
ucel
lasp
ecie
s2.
Gra
nulic
atel
laba
laen
opte
rae
1.Is
olat
edfro
mliv
eran
dsp
leen
2.Is
olat
edfro
mpu
regr
owth
from
liver
and
kidn
eyof
beac
hed
wha
le
1.D
isea
sest
atus
notk
now
n.W
hale
caug
htdu
ring
com
mer
cial
fishi
ng2.
Path
ogen
icity
notd
eter
min
ed
171
179
478
Paci
ficpi
lotw
hale
( Glo
bice
phal
asc
amm
oni )
Can
dida
spp.
Infe
ctio
nof
nare
sus
ually
asa
seco
ndar
yin
fect
ion
Infe
ctio
n72
6
Sow
erby
’sbe
aked
wha
leAc
tinob
acillu
sde
lphi
nico
laIs
olat
edfro
mva
rious
orga
nsPa
thog
enic
ityno
tdet
erm
ined
263
Sea
Urch
inVi
brio
diaz
otro
phic
usPa
rtof
gutf
lora
319
Shar
ks
Brow
nsh
ark
( Car
char
hinu
spl
umbe
us)
Phot
obac
teriu
mda
mse
lae
ssp.
dam
sela
e31
4
Host
Path
ogen
Norm
alflo
raTi
ssue
site
Dise
ase
stat
usRe
f
Tab
le1.
1.C
ontin
ued.
46Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpTuesday, January 20, 2004 1:58:52 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
Aquatic Animal Species and Organism Relationship 31
Nur
sesh
ark
(Ore
ctol
obus
orna
tus)
.An
imal
held
inca
ptiv
ityPh
otob
acte
rium
dam
sela
ess
p.da
mse
lae
Org
anis
mis
olat
edfro
mor
gans
Mor
talit
y61
8
Sand
bars
hark
Vibr
io(c
arch
aria
e)ha
rvey
iIs
olat
edfro
mki
dney
316
Smoo
thdo
gfis
h( M
uste
lus
cani
sM
itchi
ll)an
dSp
iny
dogf
ish
( Squ
alus
acan
thia
sL)
Alte
rom
onas
spp.
,Pho
toba
cter
ium
spp.
,Pse
udom
onas
spp.
,Sh
ewan
ella
putre
faci
ens,
Vibr
iosp
p.
Alli
sola
ted
from
lesi
ons
inth
ehe
adki
dney
ofhe
alth
ysh
arks
The
role
ofth
ele
sion
s,fro
mw
hich
thes
eor
gani
sms
wer
ecu
lture
d,in
mor
bidi
tyan
dm
orta
lity
isun
know
n
109
Blac
ktip
shar
k( C
arch
arhi
nus
limba
tus )
,Lem
onsh
ark
(Neg
aprio
nbr
eviro
stris
Poey
),N
urse
shar
k( G
ingl
ymos
tom
aci
rratu
m),
Tige
rsh
ark
( Gal
eoce
rdo
curv
ieri)
1.Ae
rom
onas
salm
onic
ida
Alte
rom
onas
spp.
Mor
axel
lasp
p.N
eiss
eria
spp.
Phot
obac
teriu
mda
mse
lae
ssp.
dam
sela
ePh
otob
acte
rium
dam
sela
ess
p.pi
scic
ida
Ples
iom
onas
shig
ello
ides
Vibr
ioal
gino
lytic
usVi
brio
harv
eyi
2.Vi
brio
harv
eyi
3.Ph
otob
acte
rium
dam
sela
ess
p.da
mse
lae
1.Is
olat
edfro
mva
rious
tissu
esi
tes
2.Ac
tive
sple
nic
and
hepa
ticdi
seas
eon
hist
olog
ical
exam
inat
ion
1.Pa
thog
enic
ityno
tass
esse
d2.
Isol
ated
from
expe
rimen
tally
infe
cted
Lem
onsh
arks
butd
idno
tsho
wcl
inic
aldi
seas
e.St
ress
-rela
ted
3.N
otre
cove
red
from
expe
rimen
tally
inoc
ulat
edLe
mon
shar
ks
315
316
Shea
tfish
,Wel
sca
tfish
( Silu
rus
glan
isLi
nnae
us)
1.Fl
avob
acte
rium
bran
chio
philu
m2.
F.co
lum
nare
1.Ba
cter
ium
atta
ches
togi
llep
ithel
ium
2.O
rgan
ism
isol
ated
from
kidn
ey,s
kin
blan
ched
and
ulce
rate
d
1.BG
D2.
Col
umna
risdi
seas
e88 25
180
2
Shrim
p
Blac
ktig
erpr
awn
( Pen
aeus
mon
odon
)1.
Vibr
ioha
rvey
i2.
Vibr
iopa
raha
emol
ytic
us3.
Vibr
iosp
lend
idus
II
1,2,
3.In
fect
she
pato
panc
reas
whe
reth
ere
isan
infla
mm
ator
yre
spon
sein
the
inte
rtubu
lars
inus
es
1,2,
3.M
orta
litie
s36
341
0
Brin
esh
rimp
( Arte
mia
spec
ies)
Vibr
iopr
oteo
lytic
usAf
fect
sm
icro
villi,
disr
upts
gute
pith
elia
lcel
lju
nctio
ns,d
evas
tate
sce
llsan
dtis
sues
inth
ebo
dyca
vity
Dea
ths
135
788
Chi
nese
shrim
p( P
enae
usch
inen
sis )
1.Vi
brio
algi
noly
ticus
2.Vi
brio
harv
eyi
3.Vi
brio
vuln
ificu
sse
rogr
oup
E
4.Vi
brio
paci
nii
1,2.
Affe
cts
larv
alde
velo
pmen
tfro
mzo
east
age
onw
ards
.Ina
ctiv
ity,a
nore
xia,
opaq
uene
ssan
dla
rvae
settl
eto
the
botto
mof
the
tank
1,2,
3.Vi
brio
sis,
mor
talit
y4.
Isol
ated
from
heal
thy
shrim
p98 30
677
7
cont
inue
d
47Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:23 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
32 Chapter 1
Fairy
shrim
p(B
ranc
hipu
ssc
haef
feri
Fish
er,C
hiro
ceph
alus
diap
hanu
sPr
évos
t,St
rept
ocep
halu
sto
rvic
orni
sW
aga)
Aero
mon
ashy
drop
hila
Blac
kno
dule
son
thor
acic
appe
ndag
es,
the
cerc
opod
san
dan
tenn
aeBl
ack
dise
ase
220
Gia
ntfre
shw
ater
praw
n( M
acro
bran
chiu
mro
senb
ergi
i)1.
Aero
mon
asca
viae
2.A.
vero
niis
sp.v
eron
ii3.
Lact
ococ
cus
garv
ieae
3.Ye
llow
ish-
whi
tesp
ots
inm
uscl
e,w
hitis
hm
uscl
ean
dsw
olle
n,ye
llow
hepa
topa
ncre
as,f
luid
accu
mul
atio
nbe
twee
ncu
ticle
and
mus
cle
tissu
e
1.2.
Path
ogen
icin
viru
lenc
est
udie
s3.
Mor
talit
y
156
723
Kuru
ma
praw
n( P
enae
us[M
arsu
pena
eus]
japo
nicu
s,Pe
naeu
sst
yliro
stris
)
1.Vi
brio
harv
eyi
2.Vi
brio
pena
eici
da3.
Acin
etob
acte
rspp
.,Al
tero
mon
assp
p.,B
acillu
ssp
p.,
Cor
yneb
acte
rium
spp.
,Fl
avob
acte
rium
spp.
,Mic
roco
ccus
spp.
,Mor
axel
lasp
p.,
Pseu
dom
onas
spp.
,St
aphy
loco
ccus
spp.
1.Bl
ack
spot
son
exos
kele
ton
2.Se
ptic
aem
ia3.
Isol
ated
from
appa
rent
lyhe
alth
ysh
rimp,
butn
otfro
mdi
seas
edsh
rimp
1.M
orta
lity
2.Sy
ndro
me
933.
Non
-pat
hoge
nic
187
388
198 23
Whi
tesh
rimp
( Pen
aeus
vann
amei
Boon
e)1.
Myc
obac
teriu
mpe
regr
inum
2.Vi
brio
harv
eyi
Vibr
ioxu
ii1.
Mul
tifoc
al,m
elan
ized
nodu
larl
esio
nson
cara
pace
2.Bl
ack
spot
son
exos
kele
ton
1.O
ppor
tuni
stic
infe
ctio
n2.
Mor
talit
y3.
Path
ogen
icity
notk
now
n
23 551
740
Snak
ehea
dfis
h( C
hann
ast
riatu
sFo
wle
r)M
ycob
acte
rium
porif
erae
,lat
erid
entif
ied
byPC
Rto
beM
.for
tuitu
mIn
tern
alno
dula
rles
ions
Myc
obac
terio
sis
633
756
Snoo
k( C
entro
pom
usun
deci
mal
isBl
och)
Vibr
ioha
rvey
iO
paqu
eco
rnea
Infe
ctio
n45
3
Sole
( Sol
ease
nega
lens
isKa
up)
Phot
obac
teriu
mda
mse
lae
ssp.
pisc
icid
aH
aem
orrh
agic
sept
icae
mia
and
whi
tear
eas
ofgr
anul
oma
inth
eki
dney
,liv
er,
sple
en
Fish
past
eure
llosi
s85
5
Dov
erso
le( S
olea
sole
a)Te
naci
bacu
lum
mar
itim
umBl
iste
ring
ofth
esk
inbe
twee
nth
eca
udal
and
mar
gina
lfin
s,lo
ssof
derm
altis
sues
,w
hich
prog
ress
esto
necr
otic
ulce
rs
Blac
kpa
tch
necr
osis
90 539
Spad
efis
hAt
lant
icsp
adef
ish
( Cha
etod
ipte
rus
fabe
r Bro
usso
net)
Vibr
ioha
rvey
iBi
late
rale
xoph
thal
mia
,hae
mor
rhag
esin
and
arou
ndey
es,c
orne
alop
acity
Mor
talit
y23
Host
Path
ogen
Norm
alflo
raTi
ssue
site
Dise
ase
stat
usRe
f
Tab
le1.
1.C
ontin
ued.
48Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:23 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
Aquatic Animal Species and Organism Relationship 33
Spon
ge,M
arin
esp
onge
( Hal
icho
ndria
bow
erba
nki).
Also
know
nas
Cru
mb-
of-b
read
spon
ge
Myc
obac
teriu
mpo
rifer
ae60
8
Squi
d
Lolig
ope
alei
1.Sh
ewan
ella
peal
eana
2.Vi
brio
loge
i1.
Asso
ciat
edw
ithth
ere
prod
uctiv
eor
gan,
the
acce
ssor
yni
dam
enta
lgla
nd,i
nfe
mal
es2.
Sym
bion
tin
light
orga
n
1.2
Nor
mal
flora
257
492
Haw
aiia
nse
piol
idsq
uid
( Eup
rym
nasc
olop
es)
Vibr
iofis
cher
iLi
ghto
rgan
Ligh
torg
ansy
mbi
ont
257
Sepi
ola
affin
is,S
.rob
usta
Vibr
iofis
cher
iVi
brio
loge
iLi
ghto
rgan
Sym
biot
icba
cter
iain
light
orga
n25
7
Stin
gray
( Das
yatis
past
inac
a)An
imal
she
ldin
capt
ivity
Phot
obac
teriu
mda
mse
lae
ssp.
dam
sela
eO
rgan
ism
isol
ated
from
orga
nsM
orta
lity
618
Strip
edja
ck,W
hite
Trev
ally
( Pse
udoc
aran
xde
ntex
Bloc
han
dSc
hnei
der)
1.Ph
otob
acte
rium
dam
sela
ess
p.pi
scic
ida
2.Ps
eudo
mon
asan
guilli
sept
ica
1.H
aem
orrh
agic
sept
icae
mia
and
whi
tear
eas
ofgr
anul
oma
inth
eki
dney
,liv
er,
sple
en2.
Hae
mor
rhag
esin
mou
th,n
ose,
oper
culu
man
dbr
ain.
Org
anis
min
kidn
ey
1.Fi
shpa
steu
rello
sis
2.M
orta
litie
s46
556
7
Stur
geon
Adria
ticst
urge
on( A
cipe
nser
nacc
arii
Bona
parte
)La
ctoc
occu
sga
rvie
aeIn
appe
tenc
e,irr
egul
arsw
imm
ing,
som
ebi
late
rale
xoph
thal
mia
and
abdo
min
alas
cite
s
Mor
talit
y66
9
Sibe
rian
stur
geon
( Aci
pens
erba
erii
baer
iiBra
ndt)
Yers
inia
ruck
eri
Can
bein
fect
edw
ithor
with
outc
linic
alsi
gns
Yers
inio
sis
797
Tenc
h( T
inca
tinca
L.)
Myc
opla
sma
mob
ileIs
olat
edfro
mgi
llsPa
thog
enic
ityno
tsta
ted
439
440
Tila
pia
Nile
tilap
ia( O
reoc
hrom
issp
.,O
.ni
lotic
usni
lotic
usLi
nnae
us)
Also
know
nas
StPe
ter’s
fish
1.Ed
war
dsie
llata
rda
2.St
rept
ococ
cus
agal
actia
e3.
Stre
ptoc
occu
sin
iae
1.Se
ptic
aem
ia,f
ocal
supp
urat
ive
orgr
anul
omat
ous
lesi
ons,
cuta
neou
sul
cera
tions
3.C
entra
lner
vous
syst
emin
volv
emen
t,le
thar
gy,e
rratic
swim
min
g
1.Ed
war
dsie
llosi
s2,
3.St
rept
ococ
cosi
s23
344
2
cont
inue
d
49Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #M.vpTuesday, January 20, 2004 3:31:34 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
34 Chapter 1
Tila
pia
(Sar
athe
rodo
n(T
ilapi
a)au
reus
)1.
Edw
ards
iella
icta
luri
2.St
rept
ococ
cus
inia
e2.
Loss
ofor
ient
atio
n,ex
opht
halm
ia,
pete
chia
ear
ound
anus
,mou
than
dpe
ctor
alfin
s,flu
idin
perit
onea
lcav
ity,
enla
rged
orga
ns
1.Sl
ight
lysu
scep
tible
2.M
orta
lity
621
627
Tila
pia
( Sar
othe
rodo
nni
lotic
us)
Also
know
nas
Nile
tilap
ia1.
Lact
ococ
cus
garv
ieae
2.St
rept
ococ
cus
inia
e3.
Vibr
ioch
oler
aeno
n-01
1.D
erm
alha
emor
rhag
ean
dex
opht
halm
ia,e
pica
rditi
s,pe
riton
itis,
pale
-col
oure
dliv
er,s
plen
omeg
aly,
nodu
lefo
rmat
ion
ingo
nads
2.Lo
ssof
orie
ntat
ion,
exop
htha
lmia
,pe
tech
iae
arou
ndan
us,m
outh
and
pect
oral
fins,
fluid
inpe
riton
ealc
avity
,en
larg
edor
gans
1,2.
Syst
emic
infe
ctio
n–
stre
ptoc
occo
sis
3.Fa
rmm
orta
lity.
May
beop
portu
nist
507
550
621
Turb
ot( S
coph
thal
mus
max
imus
Linn
aeus
)1.
Atyp
ical
Aero
mon
assa
lmon
icid
a.Al
soan
oxid
ase-
nega
tive
stra
in2.
Chr
yseo
bact
eriu
msc
opht
halm
um3.
List
onel
laan
guilla
rum
01an
d02
b,02
a4.
Myc
obac
teriu
mch
elon
aean
dM
.m
arin
um5.
Phot
obac
teriu
mda
mse
lae
ssp.
dam
sela
e6.
Phot
obac
teriu
mda
mse
lae
ssp.
pisc
icid
a7.
Pseu
dom
onas
angu
illise
ptic
a8.
Serra
tialiq
uefa
cien
s9.
Stre
ptoc
occu
spa
raub
eris
10.V
ibrio
sple
ndid
usbi
otyp
eI
11.E
nter
ovib
riono
rveg
icus
12.V
ibrio
chol
erae
non-
01an
dVi
brio
mim
icus
did
notc
ause
deat
hsin
viru
lenc
eas
say
13. V
ibrio
nept
uniu
s14
.Vib
riosc
opht
halm
i
1.Sk
inul
cers
2.Sw
olle
nin
test
ines
,hae
mor
rhag
esin
eye,
skin
and
jaw
,gill
hype
rpla
sia
4.G
ranu
lom
asin
orga
ns7.
Org
anis
min
head
–kid
ney
and
sple
en8.
Swol
len
kidn
eyan
dsp
leen
,yel
low
nodu
les,
foci
ofliq
uefa
ctio
nne
cros
is9.
Lesi
ons,
haem
orrh
age
inth
ean
alan
dpe
ctor
alfin
san
dpe
tech
iae
onth
eab
dom
en,e
xoph
thal
mia
and
pus
inth
eey
es9.
Hae
mor
rhag
esin
mou
th,a
bdom
inal
dist
ensi
on,r
eddi
shflu
idin
perit
onea
lca
vity
11,1
3,14
.Par
tofg
utflo
ra
1.M
orta
lity
2.G
illdi
seas
e,ha
emor
rhag
icse
ptic
aem
ia3.
Vibr
iosi
s4.
Myc
obac
terio
sis
5.M
orta
lity
6.Fi
shpa
steu
rello
sis
7.H
aem
orrh
agic
sept
icae
mia
8.O
ppor
tuni
stpa
thog
en,
mor
talit
y9.
Stre
ptoc
occo
sis,
hepa
tom
egal
y,m
ucoh
aem
orrh
agic
ente
ritis
10.E
pizo
otic
inju
veni
les
11,1
4.N
orm
alflo
ra13
.Pat
hoge
nici
tyno
tkno
wn
268
281
224
149 31 96 475
507
557
617
673
712
740
741
748
749
751
754
791
832
Host
Path
ogen
Norm
alflo
raTi
ssue
site
Dise
ase
stat
usRe
f
Tab
le1.
1.C
ontin
ued.
50Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:23 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
Aquatic Animal Species and Organism Relationship 35
Turb
ot(C
olis
tium
nudi
pinn
isW
aite
)Br
ill( C
.gun
ther
i).Bo
thsp
ecie
sar
eal
sokn
own
asfla
tfish
Vibr
ioca
mpb
ellii-
like,
Vibr
iosp
lend
idus
IO
rgan
ism
inbr
ain,
kidn
ey,l
iver
,whi
chsh
owed
lesi
ons
and
haem
orrh
ages
Acut
em
orta
lity
inju
veni
les,
oppo
rtuni
stic
infe
ctio
n22
1
Yello
wta
il( S
erio
laqu
inqu
erad
iata
and
S.pu
rpur
asce
nsTe
mm
inck
and
Schl
egel
)Al
soca
lled
Rud
derfi
shan
dJa
pane
seam
berja
ck
1.La
ctoc
occu
sga
rvie
ae2.
List
onel
laan
guilla
rum
3.M
ycob
acte
rium
spp.
4.N
ocar
dia
serio
lae
5.Ph
otob
acte
rium
dam
sela
ess
p.da
mse
lae
6.Ph
otob
acte
rium
dam
sela
ess
p.pi
scic
ida
7.St
rept
ococ
cus
inia
e
1.Er
osio
nof
tail
fin,r
edne
ssof
anal
fin,
pete
chia
ein
side
oper
culu
m,e
xoph
thal
mia
4.Ab
sces
ses
inep
ider
mis
,for
mat
ion
oftu
berc
ules
ingi
lls,k
idne
ys,s
plee
n5.
Org
anis
min
sple
enan
dki
dney
,tox
inpr
oduc
ed6.
Bact
eria
lcol
onie
sof
whi
te-g
rey
colo
urse
enon
sple
enan
dki
dney
7.O
rgan
ism
isol
ated
from
brai
n
1.St
rept
ococ
cosi
s3.
Myc
obac
terio
sis
4.N
ocar
dios
is5.
Mor
talit
y6.
Fish
past
eure
llosi
s,m
orta
lity
7.Se
ptic
aem
ia
459
462
424
455
464
236
233
235
Wha
le.S
eeun
derS
EAM
AMM
ALS
Whi
tefis
h( C
oreg
onus
sp.),
Cis
co( C
oreg
onus
arte
diLe
sueu
r),La
kew
hite
fish
( Cor
egon
uscl
upea
form
isM
itchi
ll),P
eled
(Cor
egon
uspe
led
Gm
elin
)Fa
mily
Salm
onid
ae
1.Ae
rom
onas
salm
onic
ida
ssp.
salm
onic
ida
2.At
ypic
alAe
rom
onas
salm
onic
ida
3.Ps
eudo
mon
asan
guilli
sept
ica
4.Ye
rsin
iaru
cker
i
3.Pe
tech
ialh
aem
orrh
ages
onsk
in,m
outh
and
anus
,per
itone
uman
dliv
er4.
Dis
ease
isas
soci
ated
with
poor
envi
ronm
enta
lcon
ditio
ns
1.Sk
inul
cers
,fur
uncu
losi
s2.
Skin
ulce
rs3.
Hae
mor
rhag
icdi
seas
e4.
Yers
inio
sis
323
352
772
828
Wol
f-fis
h( A
narh
icha
slu
pus
Linn
aeus
),Sp
otte
dw
olf-f
ish
(A.
min
orO
lafs
en)
1.At
ypic
alAe
rom
onas
salm
onic
ida
2.C
arno
bact
eriu
mdi
verg
ens
3.C
arno
bact
eriu
msp
p.1.
Atyp
ical
furu
ncul
osis
2,3.
Part
ofin
test
inal
mic
roflo
ra64
8
Wra
sse
( Lab
ridae
)C
lean
erfis
hAt
ypic
alAe
rom
onas
salm
onic
ida
Hae
mor
rhag
icin
tern
alor
gans
,blo
ody
exud
ate
Furu
ncul
osis
468
51Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:23 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
Bacterial gill disease (BGD)
Bacterial gill disease is caused by the bacteriumFlavobacterium branchiophilum, which are largefilamentous Gram-negative rods. The bacteriumattaches to the epithelial surface of the gill(Snieszko, 1981; Ostland et al., 1994).
Bacterial kidney disease (BKD)
The disease agent is Renibacterium salmoninarum,which affects salmonids. The disease begins in achronic form that develops full expression whenthe fish are 1 year old. Systemic granulomatouslesions are seen in all organs but particularly inthe kidney, where grey, necrotic abscesses occurthroughout the tissue, causing enlargement andnecrosis of the kidney (OIE, 2000a).
Bacterial stomatitis (mouth rot)
This condition is seen as yellow-coloured plaqueon the teeth and the oral cavity in Atlantic salmonsmolts in their first year in seawater. Thecausative organism was identified as Tenaci-baculum maritimum. Some differences werenoted between these strains and the referencestrains. The strains from mouth rot had anoptimum temperature of 18–25°C, which isslightly lower than the reference strains, and theyalso had an optimum for media containing 70%seawater. Many of the strains from mouth rot hada- and b-glucosidase activity, an unusual findingfor T. maritimum isolates (Ostland et al., 1999b).
Black patch necrosis
The disease affects Dover sole and is caused byT. maritimum. Initially there is a slight blisteringof the skin surface, which develops into loss ofepithelium and necrotic ulcers (Bernardet et al.,1990).
Brown ring disease (BRD)
This is a disease caused by Vibrio tapetis, andmay cause mass mortality in Manila clams
(Ruditapes philippinarum). It was first reportedin France in 1987 where it caused highmortality in cultured stocks. The disease ischaracterized by a ring of brown deposit ofseveral layers seen between the pallial lineand the edge of the shell. A shell repair processoccurs and this can be seen as white calcifiedareas that cover the brown deposit (Paillard andMaes, 1994).
Enteric redmouth disease (ERM)
This disease is caused by the Gram-negativebacterium Yersinia ruckeri. It is an economicallyserious disease in the rainbow trout farmingindustry of many countries. Clinical signs arehaemorrhagic areas around the mouth, intestinesand other organs.
There are a number of modes of transmissionof the disease, including birds, wild fish andcarrier fish (Willumsen, 1989). The organism alsoforms biofilms on fish tanks, which is a source ofreinfection (Coquet et al., 2002).
Enteric septicaemia of catfish (ESC)
The disease is caused by the bacteriumEdwardsiella ictaluri, and is a major diseaseproblem for the commercial channel catfishindustry. Ornamental species and salmonids aresusceptible and it has also been reported in seabass Dicentrarchus labrax (Hawke et al., 1981).Disease resistance is variable among channelcatfish species, with blue catfish (Ictalurusfurcatus) and Red River strain showing the mostdisease resistance (Wolters and Johnson, 1994).Outbreaks of the disease occur in the tempera-ture range of 18–28°C, although low-levelmortality and carrier status may be seen attemperatures outside this range.
In the acute form, the disease expressesas acute septicaemia. Petechial haemorrhagesoccur around the mouth, the throat and thefins, and internally in the liver and otherorgans. The organism crosses the intestinalmucosa into the internal organs. A chronic formof the disease occurs as a meningoencephalitiswith behavioural changes, and ulceration or‘hole-in-the-head’ (Hawke et al., 1981; OIE,2000b).
36 Chapter 1
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Furunculosis
The causative organism is Aeromonassalmonicida spp. salmonicida, which causes highrates of mortality in salmonid fish. The disease ischaracterized by boil-like inflammatory lesions,known as furuncles, which can penetrate deepinto the musculature. These clinical signs are notalways present (OIE, 2000a).
Pasteurellosis
The disease known as fish pasteurellosis is causedby Photobacterium damselae spp. piscicida(previously Pasteurella piscicida). It can affectmany cultured fish species throughout Japan,USA and Europe. It usually causes high mortality,with very few external or clinical signs of disease,although a dark body colour may be seen. Thespleen usually shows white nodules or tuberclesfrom which the organism can be cultured(Kusuda and Yamaoka, 1972; Hawke et al.,1987; Toranzo et al., 1991; Baptista et al., 1996;Candan et al., 1996; Fukuda et al., 1996).
Petechiae
Petechiae (pinpoint-sized areas of haemorrhage)on the underside and on the abdomen of fishmay indicate septicaemia and generalizedbacteraemia.
Rainbow trout fry syndrome
This condition has been reported from theUK, where diseased fish have anaemia,exophthalmia, pale gills and increased pig-mentation of the skin. The abdomen is swollenwith ascites fluid in the peritoneal cavity and thekidney is swollen. As yet no definitive organismhas been deemed to be the causative organism.A number of bacteria have been suggestedto be associated with the condition. Theseinclude Flavobacterium columnare (previouslyCytophaga columnaris), Janthinobacterium spp.,Micrococcus luteus and Planococcus spp. (Austinand Stobie, 1992b).
Skin and tail rot
Skin and tail rot may be associated with bacteriasuch as Pseudomonas spp., Aeromonas spp.,Flavobacterium or Flexibacter spp. However, thecondition is usually due to poor water quality andis therefore regarded as primarily a managementproblem.
Skin ulcers
Skin ulcers are seen as boil-like or pimple-likeand convex. They may be caused by a varietyof bacteria including typical Aeromonassalmonicida and the many species of atypicalA. salmonicida. When the disease cause isListonella anguillarum, ulcers may be seenas boils with red putrefying and liquefying flesh.
Streptococcosis
This disease is caused by the Gram-positivecocci, Streptococcus agalactiae, a group B,b-haemolytic streptococcus. Clinical signsexhibited with this infection include abnormalbehaviour such as erratic swimming, whirling onthe surface, and C-shaped curvature of the bodywhile swimming at the surface. The eye may beopaque, exophthalmia may be seen and haemor-rhages may be present. Haemorrhagic areas arealso seen on the head and body, in particulararound the mouth, snout, operculum and fins.There may be a haemorrhagic enteritis.
Streptococcus iniae infection
Clinical signs of the disease may vary accordingto the species of fish infected. Chronic infectionseems to occur at a temperature of 25°C,whereas a more acute form of the disease is seenwhen the temperature ranges from 28 to 32°C(Yuasa et al., 1999).
1.3 Bacteria and Relationship to Host
Table 1.2 lists the bacteria that may be pathogensor saprophytes of fish and other aquatic animals.
Aquatic Animal Species and Organism Relationship 37
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Abio
troph
iaba
laen
opte
rae
spp.
nov.
See
Gra
nulic
atel
laba
laen
opte
rae
com
.nov
.17
9
Abio
troph
iael
egan
s(n
utrit
iona
llyva
riant
Stre
ptoc
occi
)Se
eG
ranu
licat
ella
eleg
ans
Achr
omob
acte
rxyl
osox
idan
sss
p.de
nitri
fican
s(p
revi
ousl
yAl
calig
enes
deni
trific
ans )
659
Acin
etob
acte
rbau
man
nii
Gen
ospe
cies
2H
uman
infe
ctio
nsIs
olat
edfro
mhu
man
patie
nts
and
envi
ronm
ent
110
Acin
etob
acte
rcal
coac
etic
usG
enos
peci
es1
1.En
viro
nmen
talo
rgan
ism
2.O
ralc
avity
flora
1.Is
olat
edfro
mso
il2.
Turtl
es2.
Part
ofno
rmal
flora
110
300
301
Acin
etob
acte
rhae
mol
ytic
usG
enos
peci
es4
Path
ogen
icity
notk
now
nfo
rfis
hLe
sion
sin
orga
nsIs
olat
edfro
mAt
lant
icsa
lmon
,cha
nnel
catfi
sh,e
nviro
nmen
tand
hum
ancl
inic
alsa
mpl
es
Nor
way
,USA
110
Actin
obac
illus
delp
hini
cola
Path
ogen
icity
notd
eter
min
edIs
olat
edfro
mva
rious
tissu
esSe
am
amm
als
–ha
rbou
rpor
pois
e,st
riped
dolp
hin,
Sow
erby
’sbe
aked
wha
leSc
otla
nd26
3
Actin
obac
illus
scot
iae
Path
ogen
icity
notd
eter
min
edO
rgan
ism
sin
liver
,lun
g,br
ain,
sple
enIs
olat
edfro
mst
rand
edpo
rpoi
seSc
otla
nd26
5
Actin
omyc
esm
arim
amm
aliu
mPa
thog
enic
ityno
tdet
erm
ined
Isol
ated
from
vario
ustis
sues
,(lu
ng,l
iver
,spl
een,
inte
stin
e)w
ithot
hero
rgan
ism
s
Dea
dho
oded
seal
,dea
dgr
eyse
al,d
ead
harb
ourp
orpo
ise
UK
370
Aequ
oriv
itaan
tarc
tica,
A.lip
olyt
ica,
A.cr
ocea
,A.s
ublit
hinc
ola
Envi
ronm
enta
liso
late
s(m
embe
rof
Flav
obac
teria
ceae
fam
ily)
Isol
ated
from
mar
ine
envi
ronm
ent–
seaw
ater
,sea
ice
Anta
rctic
a11
3
Aero
cocc
usvi
ridan
sAs
soci
ated
with
infe
ctio
nin
oeso
phag
eald
iver
ticul
umO
rgan
ism
inoe
soph
agea
lles
ions
.Lo
gger
head
sea
turtl
eSp
ain
755
Aero
cocc
usvi
ridan
sva
r.ho
mar
i(h
eavy
grow
th)
Hig
hm
orta
lity,
gaffk
emia
dise
ase,
fata
lsep
ticae
mia
May
see
pink
orre
dco
lora
tion
inth
eha
emol
ymph
.Org
anis
ms
mul
tiply
inth
ehe
pato
panc
reas
,ha
emol
ymph
and
toa
less
erex
tent
inth
ehe
arta
ndsk
elet
alm
uscl
e
Lobs
ter,
seaw
ater
and
sedi
men
tin
lobs
terp
onds
.Exi
stfre
e-liv
ing
inm
arin
ebe
ntho
s.C
rabs
and
othe
rcru
stac
eans
may
acta
sre
serv
oirs
orca
rrier
sof
the
orga
nism
Nor
way
,Can
ada
299
719
827
38 Chapter 1
Bact
eria
Dise
ase
Dise
ase
sign
sHo
st/is
olat
ion
site
Dist
ribut
ion
Ref
Tab
le1.
2.B
acte
rial p
atho
gens
and
sapr
ophy
tes
offis
han
dot
her
aqua
tican
imal
s.
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Aquatic Animal Species and Organism Relationship 39
Aero
mon
asal
losa
ccha
roph
ila(H
G15
)(p
revi
ousl
yH
G14
in19
95)
1.D
isea
sed
elve
rs2.
Dia
rrhoe
icst
ools
1.D
isea
sed
elve
rs2.
Faec
es1.
Elve
rson
eelf
arm
2.H
uman
1.Sp
ain
2.So
uth
Car
olin
a,U
SA52
7
Aero
mon
asbe
stia
rum
(HG
2)(fo
rmer
lyge
nom
ospe
cies
DN
Agr
oup
2,A.
hydr
ophi
la)
Path
ogen
icity
indo
ubt
Fish
,riv
erw
ater
,sea
wat
er,s
hellf
ish,
hum
anfa
eces
USA
13
Aero
mon
asca
viae
(HG
4)1.
Sept
icae
mia
,mor
talit
yw
hen
orga
nism
pres
enti
nhi
ghnu
mbe
rs2.
Infe
ctio
n,ga
stro
ente
ritis
1.D
erm
alul
cera
tion,
pres
enti
nhe
pato
panc
reas
2.In
fect
ion
1.Fr
eshw
ater
orna
men
talf
ish,
Atla
ntic
salm
on,o
ctop
us,g
iant
fresh
wat
erpr
awn,
turb
otla
rvae
2.H
uman
Ubi
quito
usin
the
envi
ronm
ent
Turk
ey,K
enya
,Tai
wan
21 723
Aero
mon
ascu
licic
ola
Pres
ence
inaq
uatic
spec
ies
notk
now
nIs
olat
edfro
mm
osqu
itoIn
dia
624
Aero
mon
asen
chel
eia
(HG
16)
(pre
viou
sly
HG
11)
Non
-pat
hoge
nic
Hea
lthy
eels
,fre
shw
ater
Spai
n42
737
924
1
Aero
mon
aseu
cren
ophi
la(p
revi
ousl
yA.
punc
tata
ssp
punc
tata
)(H
G6 )
Non
-pat
hoge
nic
Asci
tes
ofca
rp,d
rinki
ngw
ater
,wel
lwat
erEu
rope
,Ger
man
y37
9
Aero
mon
asgr
oup
501
(form
erly
ente
ricgr
oup
501)
(HG
12)
See
A.sc
hube
rtii
Aero
mon
ashy
drop
hila
ssp.
hydr
ophi
la(H
G1)
(usu
ally
isol
ated
inhe
avy
reas
onab
lypu
regr
owth
fori
tto
beco
nsid
ered
the
prim
ary
path
ogen
)
1.H
aem
orrh
agic
sept
icae
mia
,pe
riton
itis,
reds
ore
dise
ase,
finro
t,re
d-fin
dise
ase,
mor
talit
y.As
soci
ated
with
the
fung
aldi
seas
eep
izoo
ticul
cera
tive
synd
rom
e,ca
used
byAp
hano
myc
esin
vada
ns,i
nca
tfish
and
snak
ehea
dfis
h(T
haila
nd,P
hilip
pine
s).
Opp
ortu
nist
icin
fect
ion
inse
alm
orbi
lliviru
sin
fect
ion
2.O
ppor
tuni
stic
and
prim
ary
path
ogen
3.Bl
ack
dise
ase
4.Pa
thog
enic
ityun
clea
r5.
Gas
troen
terit
is
1.Er
osiv
eor
ulce
rativ
ede
rmal
lesi
ons,
haem
orrh
age
onfin
san
dtru
nk,s
wel
ling
ofan
us,e
ryth
ema
2.R
ed-le
gfro
gdi
seas
e3.
Blac
kno
dule
son
thor
acic
appe
ndag
es4.
Isol
ated
inca
ses
ofab
ortio
n
1.Fr
eshw
ater
and
orna
men
talf
ish,
ayu,
chan
nelc
atfis
h,w
alki
ngca
tfish
,tila
pia,
trout
,tur
tles,
eels
,rep
tiles
,gre
yse
al,
occa
sion
ally
mar
ine
fish
–cu
lture
dAt
lant
icsa
lmon
,sea
bass
,sca
llop
larv
ae.I
ncre
ase
inor
gani
cm
atte
rand
tem
pera
ture
sab
ove
18°C
aid
prol
ifera
tion
ofA.
hydr
ophi
la.
Foun
din
fresh
,bra
ckis
han
dco
asta
lw
ater
s2.
Frog
s,fa
rm-ra
ised
bullf
rogs
3.Fa
irysh
rimps
4.C
attle
,hor
ses,
pigs
5.H
uman
s
1,2.
Ubi
quito
usin
the
envi
ronm
entw
orld
wid
e3.
Alge
ria,G
erm
any,
Spai
n
227
220 21 454 29 300
301
456
497
530
614
650
783
Aero
mon
ashy
drop
hila
ssp.
dhak
ensi
sgr
oup
BD-2
(HG
1)D
iarrh
oea
Cyt
otox
ican
dha
emol
ytic
prop
ertie
sIs
olat
edfro
mdi
arrh
oeal
child
ren
Bang
lade
sh38
3
cont
inue
d
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Aero
mon
asja
ndae
i(H
G9)
(pre
viou
sly
HG
9A.
sobr
ia)
1.Pa
thog
en2.
Clin
ical
sign
ifica
nce
1.Ti
ssue
s2.
Isol
ated
from
bloo
d,w
ound
,di
arrh
oeal
stoo
ls
1.Ee
l2.
Hum
ans
1.Sp
ain
2.U
SA14
3
Aero
mon
asm
edia
(HG
5)1.
Envi
ronm
enta
lorg
anis
m2.
Clin
ical
,gas
troen
terit
is1.
Riv
erw
ater
.Pro
biot
icpr
oper
ties
agai
nstV
.tub
iash
ii2.
Hum
ans
1.U
K15 29
4
Aero
mon
aspo
poffi
iEn
viro
nmen
talo
rgan
ism
Drin
king
wat
erre
serv
oirs
Finl
and,
Scot
land
380
Aero
mon
assa
lmon
icid
ass
p.sa
lmon
icid
a(H
G3)
‘typi
cal’
A.sa
lmon
icid
a(p
rodu
ces
brow
npi
gmen
t)
Gol
dfis
hul
cerd
isea
se(G
UD
),fu
runc
ulos
isin
trout
and
salm
onD
erm
alul
cera
tion
show
ing
typi
cal
umbo
nate
furu
ncle
s.O
rgan
ism
spe
netra
teto
unde
rlyin
gtis
sues
,ki
dney
,coe
lom
icflu
id,s
plee
n,in
test
ine
Man
yfis
h;go
ldfis
h,ca
rp,s
ilver
perc
h,tro
ut,A
tlant
icsa
lmon
,mar
ine
floun
der,
gree
nbac
kflo
unde
r,ee
l,ca
tfish
,car
p,cl
eane
rfis
hFr
ogs,
afte
rfee
ding
from
cont
amin
ated
trout
Hig
hly
viru
lent
stra
infro
mN
orth
Amer
ica,
Euro
pe,U
K,U
SA.N
otpr
esen
tin
Aust
ralia
468
531
584
Aero
mon
assa
lmon
icid
ass
p.sa
lmon
icid
a(n
on-p
igm
ente
dst
rain
s)
Dis
ease
dsa
lmon
Hea
dki
dney
Farm
edAt
lant
icsa
lmon
Inje
ctio
nex
perim
ents
reve
alth
eno
n-pi
gmen
ted
stra
inpr
oduc
eda
high
erm
orta
lity
than
pigm
ente
dst
rain
s
Nor
way
450
Aero
mon
assa
lmon
icid
ass
p.ac
hrom
ogen
es‘A
typi
cal’
A.sa
lmon
icid
aVa
riety
ofpa
thol
ogie
s,ca
rper
ythr
oder
mat
itis,
gold
fish
ulce
rdi
seas
e,ul
cerd
isea
seof
floun
der
Larg
eop
ensk
inle
sion
ssu
rroun
ded
byar
eas
ofde
scal
atio
nw
ithso
ftene
dan
dha
emor
rhag
icde
rmis
Salm
onid
san
dno
n-sa
lmon
ids,
Atla
ntic
cod,
silv
erbr
eam
,per
ch,r
oach
Foun
din
fresh
wat
er,b
rack
ish
wat
eran
dm
arin
een
viro
nmen
ts
Wor
ldw
ide:
Aust
ralia
,Eng
land
,C
entra
land
Nor
ther
nEu
rope
,Ic
elan
d,Ja
pan,
Nor
thAm
eric
a,So
uth
Afric
a
186
534
830
Aero
mon
assa
lmon
icid
ass
p.m
asou
cida
1.‘A
typi
cal’
A.sa
lmon
icid
aSu
perfi
cial
skin
lesi
ons
1.Sa
lmon
ids
–(s
akur
amas
u–
Onc
orhy
nchu
sm
asou
and
pink
salm
on–
O.g
orbu
scha
)
Japa
n43
883
0
Aero
mon
assa
lmon
icid
ass
p.no
vaG
UD
.Cut
aneo
usul
cera
tive
dise
ase
ingo
ldfis
hC
utan
eous
ulce
rsG
oldf
ish,
salm
onid
s/no
n-sa
lmon
ids,
eel,
carp
,mar
ine
fish
Engl
and,
Japa
n,U
SA,
Wor
ldw
ide.
Aust
ralia
ngo
ldfis
hst
rain
sar
eth
ough
tto
belo
ngto
this
subs
peci
es.A
ustra
lian
salm
onid
sar
esu
scep
tible
144
535
695
761
824
825
Aero
mon
assa
lmon
icid
ass
p.pe
ctin
olyt
ica
Envi
ronm
enta
lorg
anis
mPo
llute
driv
erAr
gent
ina
615
Aero
mon
assa
lmon
icid
ass
p.sm
ithia
‘Aty
pica
l’A.
salm
onic
ida
Supe
rfici
alsk
inle
sion
sN
on-s
alm
onid
sEn
glan
d47 83
0
40 Chapter 1
Bact
eria
Dise
ase
Dise
ase
sign
sHo
st/is
olat
ion
site
Dist
ribut
ion
Ref
Tab
le1.
2.C
ontin
ued.
56Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:25 PM
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Aquatic Animal Species and Organism Relationship 41
Aero
mon
assa
lmon
icid
a(a
typi
cals
train
s)M
orta
lity
Varie
tyof
path
olog
ies,
carp
eryt
hrod
erm
atiti
s,G
UD
,ulc
erdi
seas
eof
floun
der,
ulce
rhea
ddi
seas
eof
eels
,ulc
erat
ive
dise
ase
Skin
lesi
ons,
necr
osis
,ha
emor
rhag
icul
cers
,um
bona
tefu
runc
les
and
swel
ling
onhe
adof
eels
.Som
etim
esun
derly
ing
tissu
esaf
fect
ed,p
artic
ular
lyin
farm
edfis
h
Blen
ny,c
arp,
chub
,cle
aner
fish,
dab,
eels
,san
dee
ls,f
loun
der,
gold
fish,
min
now
,rai
nbow
trout
(sal
twat
er),
roac
h,ro
ckfis
h
Balti
cSe
a,D
enm
ark,
Engl
and,
Finl
and,
Japa
n,N
orw
ay,S
outh
Afric
a,U
SA
17 352
331
107
403
468
584
832
Aero
mon
assa
lmon
icid
aat
ypic
alst
rain
sO
xida
se-n
egat
ive
Dea
ths,
ulce
rativ
edi
seas
eLe
thar
gy,s
kin
ulce
rsTu
rbot
,and
floun
derf
rom
asa
lt-w
ater
fish
farm
,coh
osa
lmon
Den
mar
k,Ba
ltic
Sea,
USA
153
617
832
Aero
mon
assa
lmon
icid
aat
ypic
alst
rain
sG
row
that
37°C
Dea
than
dm
orbi
dity
Skin
ulce
rsC
arp,
gold
fish,
roac
hG
arde
npo
nds,
aqua
ria,r
iver
sEn
glan
d40
Aero
mon
assc
hube
rtii(
HG
12)
(pre
viou
sly
calle
den
teric
grou
p50
1)
1.En
viro
nmen
talo
rgan
ism
2.Se
ptic
aem
ia,g
astro
ente
ritis
,di
arrh
oea
2.Ab
sces
ses,
wou
nds,
pleu
ral
fluid
,blo
od2.
Hum
ans,
ofte
nim
mun
ocom
prom
ised
2.U
SA,P
uerto
Ric
oan
dso
uthe
rnco
asta
lsta
tes
ofU
SA34
8 2
Aero
mon
asso
bria
(HG
7)(n
owal
soca
lled
A.ve
roni
issp
.so
bria
)
1.Pe
riton
itis,
epiz
ootic
ulce
rativ
esy
ndro
me.
Path
ogen
icity
indo
ubt
2.D
iarrh
oea,
rena
lfai
lure
,cel
lulit
is,
necr
otiz
ing
gast
roen
terit
isin
adul
t
1.Pe
riton
itis
2.Fa
eces
.Pro
duct
ion
ofcy
toto
xin
1.Fr
eshw
ater
orna
men
talf
ish,
gizz
ard
shad
.May
befo
und
inth
ein
test
ines
ofhe
alth
yfis
h2.
Infa
nt,a
dult
with
alco
holic
liver
cirrh
osis
Ubi
quito
usin
the
envi
ronm
ent
USA
21 259
393
452
750
Aero
mon
astro
ta(H
G14
)(p
revi
ousl
yca
lled
A.en
tero
pelo
gene
s)
1.N
on-p
atho
geni
cfo
rfis
h2.
Dia
rrhoe
a2.
Stoo
lsam
ples
1.Fo
und
inm
arin
e,es
tuar
ine
and
fresh
wat
eren
viro
nmen
ts2.
Hum
ans
Sout
h-Ea
stAs
ia(B
angl
ades
h,In
dia,
Indo
nesi
a,Th
aila
nd),
wor
ldw
ide
142
178
382
Aero
mon
asve
roni
issp
.sob
ria(H
G8)
1.En
viro
nmen
talo
rgan
ism
2.H
uman
s1.
Ubi
quito
usin
the
envi
ronm
ent
2.H
uman
s–
mos
tpat
hoge
nic
ofAe
rom
onas
taxa
Wor
ldw
ide
259
393
Aero
mon
asve
roni
issp
.ver
onii
form
erly
know
nas
ente
ricgr
oup
7(H
G10
)(p
revi
ousl
yca
lled
A.ic
hthi
osm
ia)
1.M
orta
lity
whe
nor
gani
smpr
esen
tin
high
num
bers
2.D
iarrh
oea,
wou
nds,
chol
ecys
titis
1.H
epat
opan
crea
s2.
Faec
es,w
ound
site
1.G
iant
fresh
wat
erpr
awns
2.H
uman
sFo
und
infre
shw
ater
1.Ta
iwan
347 2
178
381
723
Alca
ligen
esfa
ecal
isho
mar
iSe
eH
alom
onas
aqua
mar
ina
cont
inue
d
57Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:25 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
42 Chapter 1
Allo
mon
asen
teric
aEn
viro
nmen
talo
rgan
ism
Isol
ated
from
cont
amin
ated
river
wat
eran
dhu
man
faec
esR
ussi
a41
8
Alte
rom
onas
spec
ies
Bact
eria
lnec
rosi
san
dse
ptic
aem
iaN
ecro
sis,
sept
icae
mia
Larv
alan
dju
veni
lem
ollu
scs
(oys
ter)
Alte
rom
onas
citre
aSe
ePs
eudo
alte
rom
onas
citre
a
Alte
rom
onas
colw
ellia
naSe
eSh
ewan
ella
colw
ellia
na
Alte
rom
onas
com
mun
isSe
eM
arin
omon
asco
mm
unis
Aqua
spiri
llum
spp.
Rep
orto
fan
asso
ciat
ion
inep
izoo
ticul
cera
tive
synd
rom
e–
caus
edby
the
fung
us, A
phan
omyc
esin
vada
ns
Aqua
spiri
llum
indu
ced
slig
htde
rmom
uscu
larn
ecro
ticle
sion
sC
atfis
h–
mild
viru
lenc
eon
ly.S
nake
head
fish
are
noti
nfec
ted
whe
nch
alle
nged
with
Aqua
spiri
llum
spec
ies
Thai
land
497
Arca
noba
cter
ium
bern
ardi
aeIs
olat
edfro
mcl
inic
also
urce
ses
peci
ally
bloo
dan
dab
sces
ses
Hum
an-d
eriv
edst
rain
s27
463
6
Arca
noba
cter
ium
(Cor
yneb
acte
rium
)pho
cae
Path
ogen
icity
notd
eter
min
edTi
ssue
san
dflu
ids
Seal
s.R
ecov
ered
inm
ixed
grow
thfro
mse
als
with
sept
icae
mia
and
pneu
mon
iaSc
otla
nd61
363
6
Arca
noba
cter
ium
plur
anim
aliu
mPa
thog
enic
ityno
tdet
erm
ined
Isol
atio
nsi
teno
tsta
ted
Dea
dha
rbou
rpor
pois
e,de
adsa
llow
deer
UK
480
Arca
noba
cter
ium
pyog
enes
Asso
ciat
edw
itha
varie
tyof
pyog
enic
cond
ition
sM
ucus
mem
bran
es,t
issu
esO
ccur
sin
hum
ans
and
anim
als
Wor
ldw
ide
636
641
Arth
roba
cter
agilis
Envi
ronm
enta
lorg
anis
mW
ater
,soi
l,hu
man
skin
Arth
roba
cter
nasi
phoc
aePo
ssib
lyno
rmal
flora
Nas
alca
vity
Com
mon
seal
(Pho
cavi
tulin
a)18
2
Arth
roba
cter
rhom
biPa
thog
enic
ityno
tdet
erm
ined
Org
anis
mis
olat
edfro
min
tern
alor
gans
Gre
enla
ndha
libut
(hea
lthy)
Gre
enla
nd60
0
Atop
obac
terp
hoca
ePa
thog
enic
ityno
tdet
erm
ined
Inte
stin
e,ly
mph
node
s,lu
ngIs
olat
edfro
mde
adse
alSc
otla
nd47
9
Baci
llus
cere
usBr
anch
io-n
ecro
sis
Foun
don
necr
otic
gills
ofca
rpC
arp,
strip
edba
ssR
ussi
a,U
SA74 63
4
Baci
llus
myc
oide
sM
orta
lity
Ulc
ers
ondo
rsum
,foc
alne
cros
isof
epax
ialm
uscl
eC
hann
elca
tfish
Org
anis
mis
ubiq
uito
usin
soil
and
has
been
impl
icat
edin
dise
ase
inhu
man
san
dpa
rrots
Pola
nd,U
SA30
7
Baci
llus
subt
ilisPa
rtof
bact
eria
lflo
rain
bran
chio
-nec
rosi
sC
arp
Pola
nd63
4
Bact
eria
Dise
ase
Dise
ase
sign
sHo
st/is
olat
ion
site
Dist
ribut
ion
Ref
Tab
le1.
2.C
ontin
ued.
58Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #M.vpTuesday, January 20, 2004 3:32:59 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
Aquatic Animal Species and Organism Relationship 43
Bene
chea
chiti
novo
ra(n
oton
the
listo
frec
ogni
zed
bact
eria
lnam
es)
prev
ious
lyca
lled
Baci
llus
chiti
novo
rus
Ulc
erat
ive
shel
ldis
ease
,she
llro
t,sp
otdi
seas
e,ru
stdi
seas
e.M
ildly
cont
agio
us,c
hron
icse
lf-lim
iting
dise
ase
Affe
cts
the
chiti
nous
plat
esof
the
cara
pace
(dor
sals
hell)
and
plas
tron
(ven
trals
hell)
.She
llbe
com
espi
tted
and
early
lesi
ons
have
blot
chy
dark
colo
ratio
n
Free
-rang
ing
and
capt
ive
turtl
es(s
piny
soft-
shel
led
turtl
es,r
ed-e
ared
slid
ers,
strip
ed-n
ecke
dm
usk,
Sout
hAm
eric
ansi
de-n
eck
turtl
e,Ea
ster
npa
inte
dtu
rtle)
USA
806
Bord
etel
labr
onch
isep
tica
1.Br
onch
opne
umon
ia,s
econ
dary
path
ogen
inph
ocin
em
orbi
lliviru
s(d
iste
mpe
r)in
fect
ion
Lung
,tra
chea
1.Se
als
2.Br
onch
opne
umon
iain
dogs
,lab
orat
ory
anim
als,
cats
,rab
bits
,hor
ses,
turk
eys,
mon
keys
,hum
ans,
asso
ciat
edw
ithat
roph
icrh
initi
sin
pigs
Euro
pe,S
cotla
nd,D
enm
ark,
UK
642
Brev
undi
mon
as(P
seud
omon
as)
dim
inut
aEn
viro
nmen
talo
rgan
ism
685
Brev
undi
mon
as(P
seud
omon
as)
vesi
cula
risEn
viro
nmen
talo
rgan
ism
Foun
din
stre
ams
685
Bruc
ella
abor
tus
Bruc
ello
sis
Posi
tive
sero
logy
Wed
dell
seal
s(L
epto
nych
otes
wed
dellii
)An
tarc
tica
592
Bruc
ella
abor
tus,
B.m
elite
nsis
,B.
suis
and
rare
lyB.
cani
sBr
ucel
losi
sG
ener
ally
host
spec
ific
–B.
abor
tus
(cat
tle),
B.ca
nis
(dog
s),B
.mel
itens
is(g
oats
),B.
neot
omae
(des
ertw
ood
rats
),B.
ovis
(she
ep),
B.su
is(p
igs,
rein
deer
,ha
res)
Zoon
otic
.Use
biol
ogic
alsa
fety
cabi
net
Wor
ldw
ide
185
Bruc
ella
ceta
ceae
(pre
viou
sly
part
ofBr
ucel
lam
aris
s p.n
o v.b
iov a
rI&
II)
1.Br
ucel
losi
s,ab
ortio
n,in
fect
ion
2.Br
ucel
losi
s1.
Abor
ted
fetu
s,su
bcut
aneo
usle
sion
s,sp
leen
,lun
g2.
Illne
ssan
dpo
sitiv
ebl
ood
cultu
re
1.D
olph
ins
(Atla
ntic
whi
te-s
ided
dolp
hin,
com
mon
dolp
hin,
strip
eddo
lphi
n,bo
ttlen
ose
dolp
hin)
,har
bour
porp
oise
,w
hale
2.H
uman
infe
ctio
nZo
onot
ic.W
ork
with
alls
uspe
cttis
sues
and
Bruc
ella
cultu
res
ina
biol
ogic
alsa
fety
cabi
net
Can
ada,
Euro
pe,S
cotla
nd,
USA
261
404
125
267
172
658
Bruc
ella
pinn
iped
iae
(pre
viou
sly
part
ofBr
ucel
lam
aris
spp.
nov.
biov
arI&
II)
1.Br
ucel
losi
s,ab
ortio
n,in
fect
ion
1.Ab
orte
dfe
tus,
subc
utan
eous
lesi
ons,
sple
en,l
ung
1.Se
als
(com
mon
seal
,gre
yse
al,
hood
edse
al,h
arp
seal
,rin
ged
seal
),Eu
rope
anot
ter
Poss
ibly
zoon
otic
.Wor
kw
ithal
lsu
spec
ttis
sues
and
Bruc
ella
cultu
res
ina
biol
ogic
alsa
fety
cabi
net
Can
ada,
Euro
pe,S
cotla
nd,
USA
261
404
125
267
172
658
cont
inue
d
59Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:25 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
44 Chapter 1
Bruc
ella
spec
ies
Bruc
ello
sis
Org
anis
min
liver
and
sple
enof
wha
le,a
ndin
lym
phno
des
ofse
als
Har
pse
al,r
inge
dse
al,m
inke
wha
leC
anad
a,N
orw
ay26
117
1
Budv
icia
aqua
tica
Envi
ronm
enta
lorg
anis
mIs
olat
edfro
mriv
eran
ddr
inki
ngw
ater
Cze
chos
lova
kia,
Swed
en59
1
Burk
hold
eria
(Pse
udom
onas
)ce
paci
aEn
viro
nmen
talo
rgan
ism
Fres
hwat
er,s
oil
Ubi
quito
us29
8
Burk
hold
eria
(Pse
udom
onas
)ps
eudo
mal
lei
Mel
ioid
osis
Sept
icae
mia
,abs
cess
esin
lung
,sp
inal
colu
mn,
liver
,kid
ney
Cet
acea
nsin
ocea
nariu
m–
(fals
eki
ller
wha
le,b
ottle
nose
dolp
hins
,whi
te-s
ided
dolp
hins
,sea
lion,
grey
seal
),sh
eep,
peng
uin,
goat
s,do
g,ga
lah,
cock
atoo
,hu
man
sFo
und
inso
ilan
dw
ater
Zoon
otic
.Use
biol
ogic
alsa
fety
cabi
net
Hon
gKo
ng.
Dis
ease
oftro
pica
land
subt
ropi
calr
egio
ns(A
ustra
lia,
Sout
h-Ea
stAs
ia)
349
516
Can
dida
spp.
(yea
st)
Opp
ortu
nist
infe
ctio
nC
etac
eans
–pa
rticu
larly
dolp
hins
726
Car
noba
cter
ium
alte
rfund
itum
Envi
ronm
enta
lorg
anis
mLa
kew
ater
Anta
rctic
a41
2
Car
noba
cter
ium
dive
rgen
sN
orm
alin
test
inal
mic
roflo
raof
heal
thy
fish
Inte
stin
ean
ddi
gest
ive
tract
Atla
ntic
salm
onju
veni
les,
Atla
ntic
cod,
Arct
icch
arr,
saith
eFr
ance
,Nor
way
176
Car
noba
cter
ium
dive
rgen
sSt
rain
6251
Pote
ntia
lpro
biot
icag
ains
tA.
salm
onic
ida
ssp.
salm
onic
ida,
L.an
guilla
rum
,M.v
isco
sus
Pres
enti
nin
test
ine
Arct
icch
arr(
Salv
elin
usal
pinu
sL.
)64
9
Car
noba
cter
ium
fund
itum
Envi
ronm
enta
lorg
anis
mLa
kew
ater
Anta
rctic
a41
2
Car
noba
cter
ium
gallin
arum
Envi
ronm
enta
lorg
anis
mIs
olat
edfro
mic
esl
ush
arou
ndch
icke
nca
rcas
ses
176
Car
noba
cter
ium
inhi
bens
Stra
inK1
Nor
mal
inte
stin
alm
icro
flora
ofhe
alth
yfis
hFo
und
indi
gest
ive
tract
Inhi
bits
grow
thof
L.an
guilla
rum
and
A.sa
lmon
icid
ain
Atla
ntic
salm
on41
241
1
Car
noba
cter
ium
mob
ileEn
viro
nmen
talo
rgan
ism
Proc
esse
dch
icke
nm
eat
176
Car
noba
cter
ium
(Lac
toba
cillu
s)pi
scic
ola
Pseu
doki
dney
dise
ase,
Lact
obac
illosi
s.Po
st-s
tripp
ing
perit
oniti
s.Se
enin
fish,
1ye
aror
olde
r,fo
llow
ing
stre
sssu
chas
hand
ling
and
spaw
ning
.Mos
tst
rain
sar
eop
portu
nist
ican
dpo
sses
slo
wvi
rule
nce;
how
ever
,oth
erst
rain
sha
vehi
gher
viru
lenc
ean
dca
use
high
mor
talit
y
Epic
ardi
tis,p
erito
nitis
,vis
cera
lgr
anul
omas
,dis
tens
ion
ofab
dom
en,a
scite
sflu
id,b
lood
orbl
iste
rsun
dert
hesk
in.C
olle
ctsa
mpl
esfro
mki
dney
,spl
een,
swim
blad
der.
Viru
lent
stra
ins
caus
ebi
late
rale
xoph
thal
mia
,pe
riocu
lara
ndliv
erha
emor
rhag
es,
and
asci
tes
fluid
Salm
onid
fish,
cutth
roat
trout
,rai
nbow
trout
,chi
nook
salm
on,c
ultu
red
strip
edba
ss,c
hann
elca
tfish
,bro
wn
bullh
ead
catfi
shLe
ssvi
rule
ntin
strip
edba
ssan
dch
anne
lca
tfish
Aust
ralia
,Bel
gium
,Can
ada,
Fran
ce,U
K,U
SA35
3 73 176
752
Bact
eria
Dise
ase
Dise
ase
sign
sHo
st/is
olat
ion
site
Dist
ribut
ion
Ref
Tab
le1.
2.C
ontin
ued.
60Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:26 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
Aquatic Animal Species and Organism Relationship 45
Cat
enib
acte
rium
spp.
See
Euba
cter
ium
spp.
Cel
lulo
phag
a(C
ytop
haga
)lyt
ica
Envi
ronm
enta
liso
late
Mar
ine,
beac
hm
udC
osta
Ric
a16
3
Chr
omob
acte
rium
viol
aceu
mPi
gmen
ted
and
non-
pigm
ente
dst
rain
s
1.En
viro
nmen
tali
sola
te2.
Wou
ndin
fect
ion,
sept
icae
mia
,ab
sces
ses
1.Fo
und
inso
ilan
dw
ater
2.H
uman
s1.
Trop
ical
and
subt
ropi
cal
regi
ons
2.Au
stra
lia,M
alay
sia,
Sene
gal,
Taiw
an,U
SA,V
ietn
am
482
599
Chr
yseo
bact
eriu
m(F
lavo
bact
eriu
m)b
alus
tinum
Flav
obac
terio
sis
Mar
ine
fish
USA
802
Chr
yseo
bact
eriu
m(F
lavo
bact
eriu
m)g
leum
Non
-pat
hoge
nic
tofis
hFo
und
inhu
man
clin
ical
spec
imen
s36
6
Chr
yseo
bact
eriu
min
dolo
gene
s(p
revi
ousl
ySp
hing
obac
teriu
man
dFl
avob
acte
rium
indo
loge
nes)
1.Sy
stem
icin
fect
ion
2.C
linic
also
urce
s1.
Torti
collis
,gro
ssle
sion
s,gr
anul
omas
,enl
arge
dor
gans
1.Fa
rmed
bullf
rogs
(Ran
aca
stes
beia
na)
2.H
uman
spec
imen
san
dho
spita
len
viro
nmen
t
USA
530
844
Chr
yseo
bact
eriu
m(F
lavo
bact
eriu
m)i
ndol
thet
icum
557
Chr
yseo
bact
eriu
m(F
lavo
bact
eriu
m)
men
ingo
sept
icum
1.N
on-p
atho
gen
forf
ish
2.Pe
ricar
ditis
,sep
ticae
mia
3.Sy
stem
icin
fect
ion
4.M
enin
gitis
inin
fant
s
2.Is
olat
edfro
mpe
ricar
dium
,liv
er,
eye
lesi
on3.
Torti
collis
,gro
ssle
sion
s,gr
anul
omas
,enl
arge
dor
gans
1.R
epor
ted
from
fish
bloo
dan
dm
arin
em
ud2.
Bird
s(c
hick
ens,
pige
on,f
inch
)3.
Farm
edbu
llfro
gs( R
ana
cast
esbe
iana
)4.
Hum
anpa
thog
en
USA
,wor
ldw
ide
530
773
Chr
yseo
bact
eriu
m(F
lavo
bact
eriu
m)s
coph
thal
mum
Gill
dise
ase,
haem
orrh
agic
sept
icae
mia
.100
%m
orta
lity
inju
veni
les
Swol
len
gill
lam
ella
e(h
yper
plas
ia),
haem
orrh
agic
sept
icae
mia
,sw
olle
nin
test
ines
cont
aini
ngye
llow
fluid
,ha
emor
rhag
esin
eye,
skin
,jaw
Hea
lthy
and
dise
ased
turb
otC
oast
alw
ater
sSc
otla
nd55
655
7
Citr
obac
terf
reun
dii
1.O
ppor
tuni
stic
infe
ctio
n,lo
wvi
rule
nce
fort
rout
2.Sy
stem
icin
fect
ion
1.H
aem
orrh
agic
spot
son
skin
,ey
ean
dfin
s.O
rgan
ism
isol
ated
from
kidn
ey,l
iver
,spl
een
2.To
rtico
llis,g
ross
lesi
ons,
gran
ulom
as,e
nlar
ged
orga
ns
1.An
gelf
ish
inaq
uaria
,rai
nbow
trout
,su
nfis
h,ca
rpC
omm
only
isol
ated
from
soil,
wat
er,
sew
age,
food
,and
from
orga
nsof
dise
ased
and
heal
thy
anim
als
incl
udin
gm
amm
als,
bird
s,re
ptile
san
dam
phib
ians
2.Fa
rmed
bullf
rogs
( Ran
aca
stes
beia
na)
Indi
a,Ja
pan,
UK,
USA
,w
orld
wid
e42
553
067
575
3
cont
inue
d
61Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:26 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
46 Chapter 1
Clo
strid
ium
botu
linum
Type
EM
orbi
dity
and
mor
talit
yFa
rmed
trout
,sal
mon
,coh
osa
lmon
C.b
otul
inum
can
bea
com
men
sali
ntro
utin
test
ine
and
ongi
lls.T
oxin
usua
llyno
tpr
oduc
edin
live
fish,
buti
spr
oduc
edin
dead
fish
aspa
rtof
the
deco
mpo
sitio
npr
oces
s.Fo
und
inla
kese
dim
ent.
Bact
eriu
mpr
oduc
esto
xin
inan
anae
robi
cen
viro
nmen
t
Brita
in,C
anad
a,D
enm
ark,
USA
141
Clo
strid
ium
perfr
inge
nsTy
peA
1.En
tero
toxa
emia
2.M
yosi
tisat
inje
ctio
nsi
te1.
Gut
cont
ent
2.Ab
sces
sin
mus
cle
Cap
tive
wha
les,
dolp
hins
,sea
lsU
K31
2
Col
wel
liam
aris
prev
ious
lyVi
brio
stra
inAB
E-1
Envi
ronm
enta
lorg
anis
mPs
ychr
ophi
lic,f
ound
inse
awat
erJa
pan
849
Cor
yneb
acte
rium
aqua
ticum
1.Ex
opht
halm
ia2.
Clin
ical
infe
ctio
n1.
Org
anis
mse
enin
brai
n,ha
emor
rhag
esin
eyes
1.St
riped
bass
,rai
nbow
trout
.Org
anis
mfo
und
inna
tura
lfre
shan
ddi
stille
dw
ater
2.R
epor
ted
toca
use
infe
ctio
nin
imm
unoc
ompr
omis
edpa
tient
s3.
Foun
din
fresh
wat
erso
urce
s
1.U
SA2.
Wor
ldw
ide
73 75
Cor
yneb
acte
rium
phoc
aeSe
eAr
cquo
bact
eriu
mph
ocae
Cor
yneb
acte
rium
test
udin
oris
Asso
ciat
edw
ithne
crot
icm
outh
lesi
ons
Mou
thle
sion
sTo
rtois
eSc
otla
nd18
0
Cry
ptoc
occu
slu
pi(y
east
)En
viro
nmen
talo
rgan
ism
Soil
Anta
rctic
a55
Cry
ptoc
occu
sne
ofor
man
sva
r.ga
ttii
(yea
st)
Dea
th,s
eptic
aem
iaO
rgan
ism
isol
ated
from
lung
,bra
in,
lym
phno
des
Dol
phin
Zoon
otic
orga
nism
Isol
ated
from
batg
uano
and
asso
ciat
edw
itheu
caly
ptus
trees
Aust
ralia
Trop
ics
and
sout
hern
hem
isph
ere
278
135
Cyt
opha
gaaq
uatil
isSe
eFl
avob
acte
rium
hyda
tis
Cyt
opha
gaar
vens
icol
aEn
viro
nmen
tali
sola
teSo
ilJa
pan
89
Cyt
opha
gaau
rant
iaca
Envi
ronm
enta
liso
late
Swam
pyso
ilG
erm
any
92
Cyt
opha
gaco
lum
naris
See
Flav
obac
teriu
mco
lum
nare
Cyt
opha
gafe
rmen
tans
Envi
ronm
enta
liso
late
Mar
ine
mud
Cal
iforn
ia89 16
2
Cyt
opha
gahu
tchi
nson
iiEn
viro
nmen
tali
sola
teSo
il89
Cyt
opha
gajo
hnso
nae
See
Flav
obac
teriu
mjo
hnso
niae
Cyt
opha
gala
terc
ula
Envi
ronm
enta
liso
late
Mar
ine
USA
163
Bact
eria
Dise
ase
Dise
ase
sign
sHo
st/is
olat
ion
site
Dist
ribut
ion
Ref
Tab
le1.
2.C
ontin
ued.
62Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:26 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
Aquatic Animal Species and Organism Relationship 47
Cyt
opha
gam
arin
ofla
vaEn
viro
nmen
tali
sola
teSe
awat
erSc
otla
nd92
Cyt
opha
gaps
ychr
ophi
laSe
eFl
avob
acte
rium
psyc
hrop
hilu
m
Der
mat
ophi
lus
chel
onae
Der
mat
ophi
losi
sSk
inle
sion
s,sk
inab
sces
s,sk
insc
abs
Test
udin
es(C
helo
nian
s)–
turtl
ean
dto
rtois
eAu
stra
lia52
9
Der
mat
ophi
lus
cong
olen
sis
Der
mat
ophi
losi
s(re
ferre
dto
as‘lu
mpy
woo
l’an
dm
ycot
icde
rmat
itis
inin
fect
edsh
eep)
Subc
utan
eous
nodu
les
and
lesi
ons
cont
aini
ngca
seou
sm
ater
ial
1.Aq
uatic
spec
ies
–cr
ocod
iles,
bear
ded
drag
on,p
olar
bear
s,se
als
2.H
uman
s,ho
rses
,she
ep,b
lue-
tong
ueliz
ard,
cat,
cattl
e,go
ats,
deer
,mon
keys
,pi
gs,r
abbi
ts,o
wls
,fox
es,g
iraffe
,gaz
elle
Aust
ralia
419
308
699
Die
tzia
mar
is(p
revi
ousl
yR
hodo
cocc
usm
aris
)M
icro
flora
Skin
and
inte
stin
alflo
raC
arp,
soil
USS
R57
3
Edw
ards
iella
hosh
inae
Part
ofno
rmal
flora
Faec
esBi
rds
(puf
fin,f
lam
ingo
)and
rept
iles
(mon
itor,
lizar
d),w
ater
Wor
ldw
ide
317
Edw
ards
iella
icta
luri
Stra
ins
with
alim
ited
tole
ranc
efo
roxy
gen
have
been
isol
ated
(Mitc
hell
and
Goo
dwin
,200
0)
1.En
teric
sept
icae
mia
ofca
tfish
2.En
viro
nmen
t1.
Initi
alin
fect
ion
inbr
ain.
Pete
chia
lha
emor
rhag
eun
derj
awan
dbe
lly,
head
lesi
on,g
ills,k
idne
yin
fect
ed
1.Fr
eshw
ater
orna
men
talf
ish,
cultu
red
chan
nelc
atfis
h(Ic
talu
rus
punc
tatu
s),
Dan
io,w
hite
catfi
sh,g
reen
knife
fish,
bass
,cy
prin
ids
and
Angu
illida
e.C
atfis
har
eth
em
osts
usce
ptib
lesp
ecie
s2.
Isol
ated
from
orga
nica
llypo
llute
dw
ater
s,ur
ine
and
faec
esof
man
,and
inte
stin
alm
icro
flora
ofsn
akes
1.Th
aila
nd,U
SA,V
ietn
am19
433
437
442
650
054
762
7
Edw
ards
iella
tard
a(E
.ang
uillim
ortif
era
isth
ese
nior
syno
nym
,how
ever
, E.t
arda
isco
nser
ved
foru
se.O
rigin
ally
nam
edPa
raco
loba
ctru
man
guilli
mor
tifer
um)
1.Ed
war
dsie
llosi
s,re
dpes
t,em
phys
emat
ous
putre
fact
ive
dise
ase
ofca
tfish
,fis
hga
ngre
ne2.
Syst
emic
infe
ctio
n3.
Hum
ancl
inic
alsa
mpl
es,d
iarrh
oea
1.Se
ptic
aem
ia,a
ndul
cera
tive
derm
atiti
s,in
test
inal
infe
ctio
n.Le
sion
san
dab
sces
ses
inm
uscl
e.Tr
outm
ayha
vem
ucop
urul
ent
disc
harg
efro
mcl
oaca
,con
gest
edsp
leen
,enl
arge
dliv
er,p
etec
hial
haem
orrh
ages
onse
rosa
lfat
and
gills
.Eel
ssh
owab
sces
sed
orul
cera
tive
lesi
ons
inki
dney
orliv
er2.
Torti
collis
,gro
ssle
sion
s,gr
anul
omas
,enl
arge
dor
gans
1.Al
ligat
ors,
ange
lfish
,bla
ckm
ulle
t,ba
ss,
catfi
sh,e
els,
fresh
wat
eran
dor
nam
enta
lfis
h,flo
unde
r,go
ldfis
h,m
arin
em
amm
als,
ostri
ch,r
ainb
owtro
ut,b
rook
trout
,sea
lion,
snak
es,t
ilapi
a,tu
rtles
.Als
opa
rtof
norm
alflo
rain
man
yaq
uatic
anim
als
(fish
,fro
gs,
amph
ibia
ns,r
eptil
es,m
amm
als,
capt
ive
little
peng
uins
,toa
ds,t
urtle
s),a
ndin
habi
tant
sof
aqua
ticen
viro
nmen
tand
surfa
cew
ater
.Fou
ndin
bile
ofhe
alth
ypi
gs3.
Has
been
isol
ated
from
absc
esse
s,m
enin
gitis
,wou
nds,
urin
e,bl
ood,
faec
es,
spin
alflu
id
Ubi
quito
usin
envi
ronm
ent,
Asia
,Au
stra
lia,C
anad
a,Ja
pan,
USA
374
530
606
640
711
800
804
823
cont
inue
d
63Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:26 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
48 Chapter 1
Empe
doba
cter
brev
is(p
revi
ousl
yFl
avob
acte
rium
brev
e)1.
Envi
ronm
enta
l2.
Clin
ical
2.Ey
e,ur
ine,
bloo
dcu
lture
,br
onch
ials
ecre
tion
1.Fi
sh,c
anal
wat
er.M
aybe
path
ogen
icfo
rlab
orat
ory
anim
als
2.H
uman
s
Engl
and,
Irela
nd,S
witz
erla
nd,
Cze
chos
lova
kia
363
775
Ente
roba
cter
aggl
omer
ans
See
Pant
oea
aggl
omer
ans
Ente
roco
ccus
faec
alis
(repo
rted
asE.
faec
alis
ssp.
lique
faci
ens)
1.Po
ssib
lepa
thog
en.I
dent
ityin
doub
t2.
Syst
emic
infe
ctio
n1.
Bact
eria
inliv
eran
dki
dney
,ul
cers
onfin
s1.
Rai
nbow
trout
,cat
fish,
brow
nbu
llhea
d2.
Cra
bsPa
rtof
norm
alin
test
inal
flora
ofm
anan
dan
imal
s.M
ayca
use
noso
com
iali
nfec
tions
1.Ita
ly,C
roat
ia2.
Fren
chM
edite
rrane
anco
ast.
Wor
ldw
ide
Ente
roco
ccus
faec
ium
Nor
mal
flora
.May
caus
eno
soco
mia
lin
fect
ions
Part
ofno
rmal
inte
stin
alflo
raof
man
and
anim
als
Wor
ldw
ide
Ente
roco
ccus
serio
licid
aSe
eLa
ctoc
occu
sga
rvie
ae73
1
Ente
rovi
brio
norv
egic
usN
orm
alflo
raPr
esen
tin
guto
ftur
botl
arva
eN
orw
ay74
1
Erys
ipel
othr
ixrh
usio
path
iae
1.N
on-p
atho
geni
cfo
rfis
h2.
Erys
ipel
as3.
Erys
ipel
oid,
skin
dise
ase,
poly
arth
ralg
ia,s
eptic
arth
ritis
,ren
alfa
ilure
,per
itoni
tis
2.Sy
stem
icdi
seas
ean
dsk
indi
seas
e3.
Skin
dise
ase,
sept
icae
mia
1.Pa
rasi
ticon
fish,
lobs
ter
2.D
olph
ins,
pigs
,kan
garo
os,e
mus
,cat
tle,
shee
p,do
gs,h
orse
s,av
ian
spec
ies,
croc
odile
3.H
uman
path
ogen
,occ
upat
iona
lex
posu
re
Wor
ldw
ide
292
408
229
Esch
eric
hia
coli
Endo
card
itis
Lesi
ons
inhe
artv
alve
sSe
alio
nKo
rea
435
Esch
eric
hia
vuln
eris
1.Se
ptic
aem
ia,m
orta
lity
2.W
ound
s1.
Abno
rmal
ities
and
haem
orrh
ages
ingi
lls,l
iver
,ki
dney
,spl
een
1.Fr
eshw
ater
fish
–ra
inbo
wtro
ut,b
allo
onm
olly
,silv
erm
olly
,cau
casi
anca
rp2.
Hum
ans,
also
isol
ated
from
faec
es.
Foun
din
wat
erof
fish
pond
san
dfis
hfa
eces
1.Tu
rkey
51
Euba
cter
ium
spp.
(iden
tific
atio
nis
tent
ativ
e(U
dey
etal
.,19
77).
Initi
ally
iden
tifie
das
Cat
enab
acte
rium
(Hen
ley
and
Lew
is,1
976)
Mor
talit
yO
rgan
ism
reco
vere
dfro
mbr
ain,
liver
,kid
ney
and
bloo
dG
rey
mul
let,
redf
ish
USA
343
Euba
cter
ium
tara
ntel
lae
(orig
inal
spel
ling
E.ta
rant
ellu
s)M
enin
gitis
.Pos
sibl
ese
cond
ary
path
ogen
Org
anis
mis
olat
edfro
mbr
ain
tissu
eSt
riped
mul
let
Flor
ida
764
Fack
lam
iam
iroun
gae
Nor
mal
flora
Nas
alca
vity
Sout
hern
elep
hant
seal
UK
368
Flav
obac
teriu
maq
uatil
eEn
viro
nmen
talo
rgan
ism
Dee
pw
ellw
ater
Kent
,UK
92
Bact
eria
Dise
ase
Dise
ase
sign
sHo
st/is
olat
ion
site
Dist
ribut
ion
Ref
Tab
le1.
2.C
ontin
ued.
64Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #M.vpTuesday, January 20, 2004 3:33:39 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
Aquatic Animal Species and Organism Relationship 49
Flav
obac
teriu
mbr
anch
ioph
ilum
(Fla
voba
cter
ium
bran
chio
phila
)Ba
cter
ialg
illdi
seas
e(B
GD
).H
igh
mor
talit
yAn
orex
ia,s
uffo
catio
n.Le
sion
san
dw
hite
-gre
ysp
ots
ongi
lls.
Org
anis
ms
seen
ongi
llsu
rface
Fres
hwat
erfis
h(g
oldf
ish)
,sal
mon
ids
Can
ada,
Euro
pe,H
unga
ry,
Japa
n,Ko
rea,
USA
604
802
Flav
obac
teriu
mca
ulifo
rman
sEn
viro
nmen
talo
rgan
ism
Lake
wat
er53
3
Flav
obac
teriu
mco
lum
nare
(pre
viou
sly
calle
dC
ytop
haga
colu
mna
ris,a
ndFl
exib
acte
rco
lum
naris
)
Col
umna
risdi
seas
e,sa
ddle
back
dise
ase,
syst
emic
dise
ase
inse
vere
case
s
Yello
w/b
row
nle
sion
son
gills
Ulc
ers
and
necr
osis
onbo
dysu
rface
,40%
ofin
fect
ions
even
tual
lype
netra
teto
inte
rnal
tissu
esan
dor
gans
Fres
hwat
erfis
h–
(cha
nnel
catfi
sh,b
lue
catfi
sh,c
arp,
whi
teba
ss,l
arge
-mou
thba
ss,b
arra
mun
di,f
athe
adm
inno
w),
blac
kbu
llhea
d,sa
lmon
ids
(bro
wn
trout
),bl
ack
com
ets,
mol
lies,
eels
,neo
nte
tra,p
latie
s.O
ccur
sw
here
wat
erte
mpe
ratu
reex
ceed
s14
°C,p
artic
ular
ly25
–32°
C
Aust
ralia
,Fra
nce,
Hun
gary
,Ja
pan,
USA
,wor
ldw
ide
89 90 88 135
211
214
543
Flav
obac
teriu
mfle
vens
e(p
revi
ousl
yC
ytop
haga
fleve
nsis
)En
viro
nmen
talo
rgan
ism
Lake
wat
erTh
eN
ethe
rland
s89 53
3
Flav
obac
teriu
mfri
gida
rium
Envi
ronm
enta
lorg
anis
mM
arin
ese
dim
ent
Anta
rctic
a37
6
Flav
obac
teriu
mgi
llisia
eEn
viro
nmen
talo
rgan
ism
Envi
ronm
ent
Anta
rctic
a53
3
Flav
obac
teriu
mhi
bern
umEn
viro
nmen
talo
rgan
ism
Fres
hwat
erla
keAn
tarc
tica
532
533
Flav
obac
teriu
mhy
datis
(Cyt
opha
gaaq
uatil
is)
Gill
dise
ase.
Path
ogen
icity
not
dete
rmin
edIs
olat
edfro
mgi
llsof
dise
ased
salm
onC
ultu
red
salm
onid
Euro
pe,U
SA72
0
Flav
obac
teriu
mjo
hnso
niae
prev
ious
lyC
ytop
haga
john
sona
e(in
clud
espr
evio
usly
nam
edFl
exib
acte
raur
antia
cus)
Fals
eco
lum
naris
dise
ase,
gill
dise
ase,
skin
dise
ase
Der
mal
and
gill
lesi
ons
Barra
mun
di,s
alm
onid
s,ko
iand
othe
rfis
h.Is
olat
edfro
mso
ilan
dm
udAu
stra
lia,E
urop
e,Fr
ance
,UK,
USA
89 145
Flav
obac
teriu
mm
enin
gose
ptic
umSe
eC
hrys
eoba
cter
ium
men
ingo
sept
icum
Flav
obac
teriu
mm
izut
aii,
form
e rly
( Sph
ingo
bact
eriu
mm
izut
ae)
Men
ingi
tis.P
atho
geni
city
not
dete
rmin
edSp
inal
fluid
Prem
atur
ein
fant
.Nat
ural
habi
tatn
otkn
own
Japa
n84
4
Flav
obac
teriu
m(C
ytop
haga
)pe
ctin
ovor
umEn
viro
nmen
talo
rgan
ism
Soil
Engl
and
92
Flav
obac
teriu
mps
ychr
ophi
lum
(pre
viou
sly
Flex
ibac
ter
psyc
hrop
hilu
san
dC
ytop
haga
psyc
hrop
hila
)
Bact
eria
lcol
d-w
ater
dise
ase,
pedu
ncle
dise
ase,
rain
bow
trout
frysy
ndro
me,
finro
t
Eros
ion
ofth
epe
dunc
lear
ea,
ulce
rson
scal
es,l
eads
tope
netra
tion
into
tissu
es
Fish
,esp
ecia
llyfry
and
youn
gfis
h,co
hosa
lmon
,chi
nook
salm
on,r
ainb
owtro
ut,
carp
,eel
s,cy
prin
ids,
Japa
nese
swee
tfish
.O
ccur
sw
here
wat
erte
mpe
ratu
reis
belo
w15
°C
Aust
ralia
,Can
ada,
Chi
le,
Den
mar
k,En
glan
d,Fr
ance
,G
erm
any,
Japa
n,Ko
rea,
Spai
n,no
rther
nU
SA
89 90 168
cont
inue
d
65Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:27 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
50 Chapter 1
Flav
obac
teriu
m(C
ytop
haga
)sa
ccha
roph
ilum
Envi
ronm
enta
lorg
anis
mR
iver
wat
erEn
glan
d53
3
Flav
obac
teriu
msc
opht
halm
umSe
eC
hrys
eoba
cter
ium
scop
htha
lmum
Flav
obac
teriu
m(C
ytop
haga
)su
ccin
ican
sPa
thog
enic
ityno
tcon
firm
edIs
olat
edfro
msu
perfi
cial
lesi
ons
ondi
seas
edfis
hSa
lmon
Foun
din
fresh
wat
erU
SA92 16
2
Flav
obac
teriu
mte
getin
cola
Envi
ronm
enta
lorg
anis
mAs
soci
ated
with
cyan
obac
teria
lmat
sAn
tarc
tica
533
Flav
obac
teriu
m(C
ytop
haga
)xa
nthu
mEn
viro
nmen
talo
rgan
ism
Mud
pool
Anta
rctic
a53
3
Flex
ibac
tera
ggre
gans
Envi
ronm
enta
lorg
anis
mM
arin
een
viro
nmen
t,be
ach
sand
Gha
na16
2
Flex
ibac
terc
anad
ensi
sEn
viro
nmen
talo
rgan
ism
Soil
Can
ada
162
Flex
ibac
terc
olum
naris
See
Flav
obac
teriu
mco
lum
nare
Flex
ibac
tere
lega
nsEn
viro
nmen
talo
rgan
ism
Fres
hwat
er,h
otsp
ring
162
Flex
ibac
terf
lexi
lisss
p.–
alga
voru
m–
iola
nthe
–pe
llicul
osus
Envi
ronm
enta
lorg
anis
mFo
und
infre
shw
ater
,lily
pond
Cos
taR
ica
162
Flex
ibac
terl
itora
lisEn
viro
nmen
talo
rgan
ism
Mar
ine
and
fresh
wat
erC
alifo
rnia
162
Flex
ibac
term
ariti
mus
See
Tena
ciba
culu
mm
ariti
mum
551
Flex
ibac
tero
voly
ticus
See
Tena
ciba
culu
mov
olyt
icum
551
Flex
ibac
terp
olym
orph
usEn
viro
nmen
tali
sola
teFo
und
inm
arin
een
viro
nmen
tM
exic
o,U
SA49
4
Flex
ibac
terp
sych
roph
ilus
See
Flav
obac
teriu
mps
ychr
ophi
lum
Flex
ibac
terr
oseo
lus
Envi
ronm
enta
liso
late
Hot
sprin
gC
osta
Ric
a16
2
Flex
ibac
terr
uber
Envi
ronm
enta
liso
late
Hot
sprin
gIc
elan
d89
Flex
ibac
ters
anct
iEn
viro
nmen
tali
sola
teAr
gent
ina
89
Flex
ibac
tert
ract
uosu
sEn
viro
nmen
talo
rgan
ism
Mar
ine
and
fresh
wat
erVi
etna
m16
2
Gra
nulic
atel
laad
iace
nsan
dG
.ele
gans
(pre
viou
sly
Abio
troph
iaad
iace
nsan
dA.
eleg
ans)
refe
rred
toas
nutri
tiona
llyva
riant
Stre
ptoc
occi
(NVS
)
Clin
ical
isol
ates
. Stre
ptoc
occi
that
show
sate
llite
grow
thar
ound
othe
rcol
onie
sN
orm
alflo
raof
mou
th,i
ntes
tinal
and
urog
enita
ltra
ct.M
ayca
use
endo
card
itis,
conj
unct
iviti
s,ot
itis
med
ia
Hum
ans.
Req
uire
pyrid
oxal
hydr
ochl
orid
ean
alog
(Vita
min
B 6)o
rL-c
yste
ine
HC
l(G
.el
egan
s)fo
rgro
wth
421
179
653
Bact
eria
Dise
ase
Dise
ase
sign
sHo
st/is
olat
ion
site
Dist
ribut
ion
Ref
Tab
le1.
2.C
ontin
ued.
66Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:27 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
Aquatic Animal Species and Organism Relationship 51
Gra
nulic
atel
laba
laen
opte
rae
com
.nov
.(p
revi
ousl
yAb
iotro
phia
bala
enop
tera
e )
Path
ogen
icity
notd
eter
min
edIs
olat
edin
pure
grow
thfro
mliv
eran
dki
dney
Beac
hed
min
kew
hale
Scot
land
179
478
Hae
mop
hilu
spi
sciu
mR
e-cl
assi
fied
as‘a
typi
cal’
A.sa
lmon
icid
a.M
ostc
lose
lyre
late
dto
A.sa
lmon
icid
aac
hrom
ogen
es50
Haf
nia
alve
i1.
Hae
mor
rhag
icse
ptic
aem
ia.
Mor
talit
ies
2.In
test
inal
diso
rder
s,pn
eum
onia
,m
enin
gitis
,abs
cess
esan
dse
ptic
aem
ia
1.C
herry
salm
on,r
ainb
owtro
ut,b
row
ntro
ut2.
Hum
ans
Ubi
quito
usin
the
envi
ronm
ents
ofso
il,se
wag
ean
dw
ater
1.Bu
lgar
ia,E
ngla
nd,J
apan
313
652
Hal
omon
asaq
uam
arin
a(s
ynon
ymou
sw
ithAl
calig
enes
faec
alis
hom
ari ,
Del
eya
aest
a,D
.aq
uam
arin
a ,A.
aqua
mar
inus
.Tr
ansf
erre
dto
genu
sH
alom
onas
asH
.aqu
amar
ina)
Mor
ibun
dSo
ftene
dsh
ells
,opa
que
area
son
cara
pace
.Org
anis
ms
isol
ated
from
haem
olym
ph
Lobs
ters
USA
45 719 8
Hal
omon
ascu
pida
(pre
viou
sly
Alca
ligen
escu
pidu
san
dD
eley
acu
pida
)
Mor
talit
ies
Blac
kse
abr
eam
fryJa
pan
463
Hal
omon
asel
onga
taEn
viro
nmen
talo
rgan
ism
Hyp
ersa
line
envi
ronm
ents
The
Net
herla
nds
795
Hal
omon
asha
lodu
rans
Envi
ronm
enta
lorg
anis
mH
yper
salin
een
viro
nmen
tsU
SA,T
heN
ethe
rland
s,Pa
cific
Oce
an33
6
Hal
omon
asm
arin
a(p
revi
ousl
yPs
eudo
mon
asm
arin
aan
dD
eley
am
arin
a)
Envi
ronm
enta
lorg
anis
mM
arin
een
viro
nmen
t66
Hal
omon
asve
nust
a(p
revi
ousl
yAl
calig
enes
venu
stus
)C
linic
alin
fect
ion
Hum
an–
caus
edby
fish
bite
.Pre
sent
inm
arin
ew
ater
sM
aldi
veIs
land
s66 31
0
Hel
icob
acte
rcet
orum
1.O
rgan
ism
foun
din
dent
alpl
aque
2.G
astri
cul
cera
tion
1.Po
tent
ialr
eser
voir
forg
astri
cin
fect
ions
2.O
rgan
ism
ingl
andu
larm
ucos
aan
dth
em
ain
stom
ach
1.C
aptiv
edo
lphi
ns( T
ursi
ops
geph
yreu
s)2.
Dol
phin
s,be
luga
wha
leU
SA30
332
732
9
Hyd
roge
noph
aga
(Pse
udom
onas
)pa
llero
nii
Envi
ronm
enta
lorg
anis
mPr
esen
tin
wat
erG
erm
any,
Rus
sia
834
cont
inue
d
67Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:27 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
52 Chapter 1
Hyd
roge
noph
aga
(Pse
udom
onas
)ps
eudo
flava
Envi
ronm
enta
lorg
anis
mPr
esen
tin
wat
er,m
ud,s
oil
Ger
man
y39 83
4
Iodo
bact
erflu
viat
ilis(p
revi
ousl
yC
hrom
obac
teriu
mflu
viat
ile)
Envi
ronm
enta
lorg
anis
mFo
und
infre
shw
ater
Anta
rctic
lake
s,En
glan
d,Sc
otla
nd,U
biqu
itous
502
Jant
hino
bact
eriu
mliv
idum
Anae
mia
Exop
htha
lmia
,pal
egi
lls,i
nter
nal
sym
ptom
sR
ainb
owtro
utFo
und
inso
ilan
dsp
ring
wat
erSc
otla
nd48
Kleb
siel
lapn
eum
onia
e1.
Fin
and
tail
dise
ase
2.M
icro
flora
1.R
ainb
owtro
ut2.
Mam
mal
ian
tissu
eU
KW
orld
wid
e20
5
Kleb
siel
lapl
antic
ola
Kleb
siel
latre
visa
nii
See
Rao
ulte
llapl
antic
ola
256
228
Kleb
siel
laor
nith
inol
ytic
aSe
eR
aoul
tella
orni
thin
olyt
ica
228
Kleb
siel
late
rrige
naSe
eR
aoul
tella
terri
gena
228
Kleb
siel
laox
ytoc
a22
8
Lact
obac
illus
spp.
,esp
ecia
llya
Lact
obac
illus
plan
taru
m-li
keis
olat
e
Nor
mal
inte
stin
alm
icro
flora
ofhe
alth
yfis
hIn
test
ine
and
dige
stiv
etra
ctAt
lant
icco
d,At
lant
icsa
l mon
,rai
nbow
trout
,wol
f-fis
h,Ar
ctic
char
rFr
ance
,Nor
way
Lact
obac
illus
pisc
icol
aSe
eC
arno
bact
eriu
mpi
scic
ola
Lact
ococ
cus
garv
ieae
Biot
ypes
1–13
(pre
viou
sly
Ente
roco
ccus
serio
licid
a,St
rept
ococ
cus
type
Ian
dSt
rept
ococ
cus
garv
ieae
)
1.La
ctoc
occo
sis,
haem
orrh
agic
sept
icae
mia
,hae
mor
rhag
icen
terit
is,
men
ingo
ence
phal
itis
2.Su
bclin
ical
mas
titis
3.In
fect
ion,
oste
omye
litis
1.Bi
late
rale
xoph
thal
mia
,da
rken
ing
ofsk
in,c
onge
stio
nof
inte
stin
e,liv
er,k
idne
y,sp
leen
,br
ain,
dist
ende
dab
dom
en,b
lood
yas
cite
sflu
idin
perit
onea
lcav
ity.
Org
anis
mse
enin
hear
t,gi
lls,s
kin,
sple
en,e
yes,
kidn
ey2.
Milk
3.Bl
ood,
skin
,urin
e,fa
eces
1.Fa
rmed
rain
bow
trout
,eel
,yel
low
tail,
praw
ns,t
urbo
t,Ad
riatic
stur
geon
Foun
din
seaw
ater
,mud
,int
estin
esof
wild
fish
e.g.
Span
ish
mac
kere
l,bl
ack
scra
per
(Bio
type
s1,
2,3,
4,5,
6,10
)2.
Cow
s,bu
ffalo
(Bio
type
s4,
7,8,
9)3.
Hum
ans
(Bio
type
s1,
2,10
,11,
12,1
3)
Aust
ralia
,Eur
ope,
Fran
ce,I
taly
,Is
rael
,Jap
an,N
orth
Amer
ica,
Saud
iAra
bia,
Spai
n,So
uth
Afric
a,Ta
iwan
,UK,
USA
236
238
237
156
174
157
464
669
731
780
Lact
ococ
cus
pisc
ium
Lact
obac
illosi
s,ps
eudo
kidn
eydi
seas
eR
ainb
owtro
utN
orth
Amer
ica
835
Bact
eria
Dise
ase
Dise
ase
sign
sHo
st/is
olat
ion
site
Dist
ribut
ion
Ref
Tab
le1.
2.C
ontin
ued.
68Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #M.vpTuesday, January 20, 2004 3:34:13 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
Aquatic Animal Species and Organism Relationship 53
List
onel
laan
guilla
rum
(Ser
ovar
s01
,02,
08,0
9)pr
evio
usly
Vibr
ioan
guilla
rum
biot
ype
I.M
osto
fthe
outb
reak
sar
eca
used
byse
roty
pes
01an
d02
Vibr
iosi
s,sy
stem
icdi
seas
e,ul
cera
tive
dise
ase,
necr
osis
Red
spot
son
vent
rala
ndla
tera
lar
eas
offis
h,ul
cera
tive
skin
lesi
ons.
Org
anis
ms
inbl
ood
and
haem
opoi
etic
tissu
es
1.Fi
sh,m
ollu
scs
–(la
rval
and
juve
nile
),ay
u,fla
tfish
(turb
ot,p
laic
efry
,win
ter
floun
der,
sole
,hal
ibut
),lo
bste
r,ee
l,sa
lmon
ids,
(rain
bow
trout
),se
abr
eam
,oc
topu
s2.
Shrim
ps3.
Cra
bs
1.W
orld
wid
e2.
Indo
-Pac
ific
&Ea
stAs
ia3.
UK
341
222
561
563
620
List
onel
lape
lagi
a(p
revi
ousl
yV.
pela
gia
I&II)
Mor
talit
ies
Eros
ion
onfin
san
dta
ilH
aem
orrh
ages
onfin
san
dor
gans
Juve
nile
farm
edtu
rbot
Spai
n
List
onel
laor
dalii
See
Vibr
ioor
dalii
Man
nhei
mia
haem
olyt
ica
(pre
viou
sly
Past
eure
llaha
emol
ytic
a )
1.U
lcer
ativ
est
omat
itis
2.D
isea
se3.
Hae
mor
rhag
ictra
chei
tis
1.R
eptil
es2.
Shee
p,go
ats,
cattl
e3.
Dol
phin
s
1.U
SA70
972
6
Mar
inila
bilia
salm
onic
olor
biov
arag
arov
oran
s(p
revi
ousl
yC
ytop
haga
salm
onic
olor
and
C.a
garo
vora
ns)
Envi
ronm
enta
lorg
anis
mM
arin
em
udC
alifo
rnia
92 89 162
Mar
inob
acte
rhy
droc
arbo
nocl
astic
us(P
seud
omon
asna
utic
a)
Foun
din
mar
ine
envi
ronm
ents
69
Mes
ophi
loba
cter
mar
inus
Envi
ronm
enta
lorg
anis
mFo
und
inse
awat
erJa
pan,
Indi
anO
cean
583
Mic
roco
ccus
lute
usM
orta
lity
Pale
gills
,asc
ites
fluid
,ga
stro
ente
ritis
,int
erna
lha
emor
rhag
es
Rai
nbow
trout
fryEn
glan
d43
Mor
axel
lasp
p.As
soci
ated
with
mor
talit
ySt
riped
bass
USA
72
Mor
itella
japo
nica
Envi
ronm
enta
lorg
anis
mSe
abed
sedi
men
tJa
pan
585
Mor
itella
mar
ina
(Vib
riom
arin
us)
Skin
lesi
ons
Isol
ate
from
surfa
cesk
inle
sion
sAt
lant
icsa
lmon
,sea
wat
eran
dse
dim
ent
ofno
rthPa
cific
Oce
anIc
elan
d,N
orw
ay,P
acifi
cO
cean
,Sc
otla
nd82 99 76
6
Mor
itella
visc
osa
(pre
viou
sly
Vibr
iovi
scos
us)
Win
teru
lcer
dise
ase
Skin
lesi
ons,
haem
orrh
ages
onin
tern
alor
gans
Atla
ntic
salm
on,l
umps
ucke
r,ra
inbo
wtro
ut.F
ound
inco
ldw
ater
Col
dwat
erin
Icel
and,
Nor
way
,Sc
otla
nd81 82 13
250
6
cont
inue
d
69Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:28 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
54 Chapter 1
Myc
obac
teriu
msp
p.M
ycob
acte
riosi
s,sy
stem
icdi
seas
eLe
sion
son
skin
and
kidn
eys.
Nod
ules
inor
gans
,sof
teni
ngof
sple
en,k
idne
y,liv
erG
ranu
lom
asin
tissu
es
Man
ysp
ecie
sof
fresh
wat
er,s
altw
ater
and
orna
men
talf
ish,
fresh
wat
ersn
ails
,fro
gs,
rept
iles,
turtl
es,P
acifi
cgr
een
sea
turtl
es,
New
Zeal
and
furs
eals
,fre
shw
ater
croc
odile
sin
Aust
ralia
Zoon
otic
Wor
ldw
ide
592
737
Myc
obac
teriu
mab
sces
sus
Gra
nulo
mas
,sys
tem
icdi
seas
eN
oob
viou
scl
inic
alsi
gns
ofpi
scin
em
ycob
acte
riosi
s.O
ccas
iona
lext
erna
lgra
nulo
ma
arou
ndbu
ccal
cavi
tyan
dve
nt,
inte
rnal
gran
ulom
as
Japa
nese
med
aka,
fresh
wat
ertro
pica
lfis
h,bl
ack
acar
as,g
oldf
ish,
firem
outh
cich
lid,o
scar
Zoon
otic
USA
474
736
Myc
obac
teriu
mch
elon
aeM
orta
lity,
gran
ulom
as,e
mac
iatio
n,ex
opht
halm
os,k
erat
itis,
skin
ulce
rs,
abno
rmal
swim
min
gbe
havi
our
Mul
tiple
grey
ish-
whi
tem
iliary
gran
ulom
a-lik
eno
dule
sin
tissu
es,
kidn
ey,l
iver
,spl
een
Atla
ntic
salm
on(S
alm
osa
lar),
yello
wpe
rch,
snak
e,tu
rbot
,tur
tleZo
onot
ic
Aust
ralia
,Can
ada,
Portu
gal,
Shet
land
Isla
nds,
Scot
land
,w
orld
wid
e
133
375
204
673
737
Myc
obac
teriu
mfo
rtuitu
m(p
revi
ousl
yM
.pis
cium
and
M.s
alm
onip
hilu
m)
Sept
icae
mia
,fis
hm
aybe
emac
iate
d,ex
opht
halm
ia,i
nfla
mm
atio
nof
skin
Gra
nulo
mas
onsk
inan
din
tissu
es.S
een
asw
hitis
hsp
ots
onliv
er,k
idne
y,he
art,
sple
en
Orn
amen
talf
infis
h–
(bla
ckac
ara,
com
ets,
disc
usfis
h,go
uram
i,gu
ppy,
neon
tetra
,os
car,
Siam
ese
fight
ing
fish)
,Atla
ntic
salm
onZo
onot
ic–
skin
ulce
rsan
ddi
ffuse
pulm
onar
ydi
seas
ein
hum
ans
Aust
ralia
,Sou
thAf
rica,
Thai
land
,w
orld
wid
e11
637
547
463
3
Myc
obac
teriu
mm
arin
umM
ycob
acte
riosi
s.D
erm
atiti
san
dpa
nnic
uliti
sin
capt
ive
whi
tew
hale
Lesi
ons
inki
dney
and
onsk
in.
Nod
ular
lesi
ons
may
besy
stem
icin
allo
rgan
s.G
ranu
lom
asin
orga
ns
Fres
hwat
ertro
ut,f
resh
wat
eror
nam
enta
lfis
h,m
arin
efis
h,ra
bbitf
ish,
sea
bass
,st
riped
bass
,tur
bot,
capt
ive
whi
tew
hale
.Sk
inle
sion
sin
hum
ans
Aust
ralia
,Isr
ael,
Portu
gal,
USA
,w
orld
wid
e,ub
iqui
tous
35 111
135
218
339
474
673
Myc
obac
teriu
mne
oaur
umPa
noph
thal
miti
sO
cula
rles
ions
,nod
ules
inm
uscl
ean
dor
gans
.Org
anis
mis
olat
edw
itha
Rho
doco
ccus
spec
ies
Atla
ntic
salm
on,c
hino
oksa
lmon
.Zo
onot
icC
anad
a53
Myc
obac
teriu
mpe
regr
inum
Myc
obac
terio
sis.
Shrim
pap
pear
edhe
alth
yap
artf
rom
blac
kle
sion
son
cara
pace
Mul
tifoc
al,m
elan
ized
nodu
lar
lesi
ons
inca
rapa
cePa
cific
whi
tesh
rimp
(Pen
aeus
vann
amei
)C
ause
ssk
inin
fect
ion
inhu
man
s.Fo
und
inw
ater
and
soil
USA
551
Bact
eria
Dise
ase
Dise
ase
sign
sHo
st/is
olat
ion
site
Dist
ribut
ion
Ref
Tab
le1.
2.C
ontin
ued.
70Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:28 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
Aquatic Animal Species and Organism Relationship 55
Myc
obac
teriu
mpo
rifer
aeTh
isis
olat
eha
ssi
nce
been
iden
tifie
dby
PCR
asM
.for
tuitu
m
Myc
obac
terio
sis
Inte
rnal
nodu
larl
esio
nsFr
eshw
ater
snak
ehea
dfis
h(C
hann
ast
riatu
s )Pr
evio
usly
repo
rted
from
am
arin
esp
onge
Thai
land
608
633
756
Myc
obac
teriu
msc
rofu
lace
umM
ycob
acte
riosi
sLe
sion
sin
kidn
eyan
dliv
er.L
iver
whi
tean
dfri
able
Paci
ficst
agho
rnsc
ulpi
nU
SA47
4
Myc
obac
teriu
msi
mia
eM
ycob
acte
riosi
sLe
sion
sin
kidn
eyan
dliv
erBl
ack
acar
aFo
und
inen
viro
nmen
talw
ater
Zoon
otic
USA
474
Myc
obac
teriu
msp
p.no
tide
ntifi
edto
know
nsp
ecie
sM
ycob
acte
riosi
sEx
tern
alde
rmal
ulce
rsan
dno
dule
sin
inte
rnal
lesi
ons
Wild
strip
edba
ssC
hesa
peak
eBa
y(U
SA)
337
Myc
obac
teriu
msp
ecie
sM
ycob
acte
rium
tripl
ex-li
keG
ranu
lom
atou
sde
rmat
itis
Flor
idsk
inno
dule
s–
soft,
gela
tinou
s,gr
ey-a
ndta
n-co
lour
edar
ound
head
and
trunk
Gre
enm
oray
eels
,spo
tted
mor
ayee
lsU
SA34
5
Myc
opla
sma
allig
ator
isEp
izoo
ticpn
eum
onia
,pol
yser
ositi
san
dm
ultif
ocal
arth
ritis
Org
anis
mfo
und
intra
chea
,lun
g,jo
intf
luid
,cer
ebro
spin
alflu
id(C
SF)
Amer
ican
allig
ator
USA
128
Myc
opla
sma
croc
odyl
iEx
udat
ive
poly
arth
ritis
Swol
len
join
ts.A
lso
foun
din
lung
sC
roco
dile
sZi
mba
bwe
441
Myc
opla
sma
mob
ileR
eddi
seas
eG
illsTe
nch
USA
439
440
Myc
opla
sma
phoc
icer
ebra
le(p
revi
ousl
yM
.pho
cace
rebr
ale)
Asso
ciat
edw
ithre
spira
tory
dise
ase
Isol
ated
from
,bra
i n,n
ose,
thro
at,
lung
s,he
art
Seal
sN
orth
Sea
295
449
Myc
opla
sma
phoc
idae
(Myc
opla
sma
phoc
ae–
nam
eco
rrect
ion
isno
tleg
itim
ate)
Aviru
lent
Res
pira
tory
tract
Har
bour
seal
sU
SA44
966
0
Myc
opla
sma
phoc
irhin
is(p
revi
ousl
yM
.pho
carh
inis
)As
soci
ated
with
resp
irato
rydi
seas
eIs
olat
edfro
mno
se,t
hroa
t,tra
chea
,lun
g,he
art
Seal
sN
orth
Sea
295
449
Myc
opla
sma
test
udin
isN
on-p
atho
geni
cC
loac
aof
torto
ise
UK
350
Myr
oide
s(F
lavo
bact
eriu
m)
odor
atim
imus
Clin
ical
spec
imen
s,ho
spita
lenv
ironm
ents
774
Myr
oide
sod
orat
us(p
revi
ousl
yFl
avob
acte
rium
odor
atum
)
Clin
ical
spec
imen
s(u
rine,
wou
ndsw
abs,
leg
ulce
r),ho
spita
lenv
ironm
ents
UK,
Cze
chos
lova
kia
362
774
cont
inue
d
71Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #M.vpTuesday, January 20, 2004 3:34:48 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
56 Chapter 1
Noc
ardi
aas
tero
ides
Noc
ardi
osis
Neo
nte
tra,r
ainb
owtro
ut,l
arge
mou
thba
ss,F
orm
osa
snak
ehea
d.Al
soca
uses
infe
ctio
nsin
cats
,cat
tle,d
ogs,
fish,
goat
s,hu
man
s,m
arin
em
amm
als
Arge
ntin
a,Ta
iwan
155
Noc
ardi
abr
asilie
nsis
and
N.t
rans
vale
nsis
Actin
omyc
ete
myc
etom
a66
1
Noc
ardi
acr
asso
stre
aesp
.nov
.N
ocar
dios
isBr
own
disc
olor
atio
non
man
tle,
gree
nor
yello
wno
dule
son
abdu
ctor
mus
cle,
gills
,hea
rtan
dm
antle
Paci
ficoy
ster
sC
anad
a,U
SA27
0
Noc
ardi
afla
voro
sea
Envi
ronm
enta
lorg
anis
mSo
ilis
olat
eC
hina
165
Noc
ardi
ano
vaH
uman
path
ogen
805
Noc
ardi
asa
lmon
icid
aN
ocar
dios
isBl
ueba
cksa
lmon
391
Noc
ardi
ase
riola
e(p
revi
ousl
yN
.kam
pach
i)N
ocar
dios
isAb
sces
ses
and
light
-yel
low
-co
lour
edno
dule
sin
epid
erm
isan
dtu
berc
les
and
gran
ulom
asin
gills
,ki
dney
,liv
er,h
eart
and
sple
en
Cul
ture
dfis
h–
rudd
erfis
hes,
yello
wta
il,Ja
pane
seflo
unde
r,se
aba
ssJa
pan,
Taiw
an15
542
445
5
Noc
ardi
asp
p.Se
ptic
aem
ia,m
orta
lity
Smal
lwhi
tesp
otle
sion
son
derm
is,m
uscl
e,gi
llsan
dor
gans
.Al
sofo
und
inin
tern
alor
gans
Atla
ntic
salm
on,f
arm
edch
inoo
ksa
lmon
,m
arin
efis
h,fre
shw
ater
orna
men
talf
ish.
Isol
ated
from
soil
and
plan
ts
Wor
ldw
ide,
Aust
ralia
,Can
ada,
Japa
n,In
dia,
Taiw
an,U
SA11
7
Oce
anom
onas
baum
anni
iEn
viro
nmen
talo
rgan
ism
Deg
rade
sph
enol
Estu
ary
ofR
iver
Wea
rU
K13
0
Oce
anom
onas
(Pse
udom
onas
)do
udor
offii
Envi
ronm
enta
lorg
anis
mM
arin
een
viro
nmen
t69 13
0
Pant
oea
(Ent
erob
acte
r)ag
glom
eran
s1.
Path
ogen
icity
indo
ubt–
poss
ible
oppo
rtuni
st2.
Hum
ans
1.H
aem
orrh
ages
iney
es,d
orsa
lm
uscu
latu
re2.
Wou
nds,
bloo
d,ur
ine
1.D
olph
infis
h(m
ahi-m
ahi)
2.H
uman
s.Al
sofo
und
inen
teric
tract
3.Fo
und
onpl
ants
urfa
ces,
seed
s,w
ater
.Al
sore
porte
din
ente
rictra
ctof
deer
with
outd
isea
sesi
gns
USA
,ubi
quito
us32
529
124
9
Pant
oea
disp
ersa
Envi
ronm
enta
lorg
anis
mPl
ants
urfa
ces,
seed
s,so
il,en
viro
nmen
tU
biqu
itous
291
Past
eure
llam
ulto
cida
1.Pn
eum
onia
,dea
th,p
leur
isy
(fow
lch
oler
a)2.
Ente
ritis
(con
tam
inat
ion
from
near
bybi
rdro
oker
y)
1.Ex
udat
ein
lung
s,flu
idin
pleu
ralc
avity
2.In
test
inal
haem
orrh
age
1.Al
ligat
or,C
alifo
rnia
nse
alio
n,pe
ngui
ns2.
Dol
phin
sR
espi
rato
rydi
seas
ein
shee
p,go
ats,
cattl
e,ra
bbits
USA
Wor
ldw
ide
430
520
709
726
Bact
eria
Dise
ase
Dise
ase
sign
sHo
st/is
olat
ion
site
Dist
ribut
ion
Ref
Tab
le1.
2.C
ontin
ued.
72Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:28 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
Aquatic Animal Species and Organism Relationship 57
Past
eure
llapi
scic
ida
See
Phot
obac
teriu
mda
mse
lae
ssp.
pisc
icid
a74
5
Past
eure
llask
yens
isM
orta
lity
(low
viru
lenc
e)Ex
hibi
tsig
nsof
loss
ofap
petit
e,m
orbi
dity
Sea-
farm
edAt
lant
icsa
lmon
(Sal
mo
sala
r L.)
Scot
land
100
416
Past
eure
llate
stud
inis
Mul
tifoc
albr
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Phoc
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67
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aybe
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73Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #M.vpTuesday, January 20, 2004 3:35:16 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
58 Chapter 1
Phot
obac
teriu
mfis
cher
iSe
eVi
brio
fisch
eri–
hom
otyp
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nony
m
Phot
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Phot
obac
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759
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Phot
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brio
iliopi
scar
ius)
Non
-pat
hoge
nic
Inte
stin
eH
errin
g,co
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h,sa
lmon
and
cod
livin
gin
cold
wat
ers
599
767
Phot
obac
teriu
mle
iogn
athi
Non
-pat
hoge
nic
Ligh
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icro
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min
ous
orga
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ase
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h,Le
iogn
athu
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3
Phot
obac
teriu
mlo
gei
See
Vibr
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Phot
obac
teriu
mph
osph
oreu
mEn
viro
nmen
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rgan
ism
May
caus
esp
oila
geof
seaf
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Mar
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Sym
biot
icas
soci
atio
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ithm
arin
ean
imal
sin
light
orga
nsof
tele
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s
67
Phot
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mpr
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dum
Envi
ronm
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liso
late
Isol
ated
from
deep
sea
sedi
men
t58
6
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cus
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otile
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ccus
isol
ated
from
seaw
ater
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and
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iled
shrim
p
326
Plan
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cus
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tali
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Nor
thSe
aco
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ring
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rimp,
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awn
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n32
6
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cus
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pres
umpt
ive
iden
tific
atio
nM
orta
lity
Pale
gills
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ites
fluid
,ga
stro
ente
ritis
,int
erna
lha
emor
rhag
es
Rai
nbow
trout
fryEn
glan
d43
Plan
omic
robi
umok
eano
koite
s(p
revi
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yPl
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eano
koite
san
dFl
avob
acte
rium
okea
noko
ites )
Envi
ronm
enta
lorg
anis
mIs
olat
edfro
mm
arin
em
udJa
pan
566
Ples
iom
onas
shig
ello
ides
(pre
viou
sly
Aero
mon
assh
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loid
es)
Poss
ible
oppo
rtuni
stpa
thog
enEm
acia
tion,
pete
chia
lha
emor
rhag
esin
inte
stin
eAf
rican
catfi
sh,e
el,g
oura
mi,
rain
bow
trout
,stu
rgeo
n.C
aptiv
epe
ngui
n,aq
uatic
rept
iles,
ubiq
uito
usin
envi
ronm
ent
Aust
ralia
,Ger
man
y,Po
rtuga
l19
544
3
Prov
iden
cia
rettg
eri
(als
ok n
o wn
a sPr
oteu
sre
ttger
i)Se
ptic
aem
iaO
rgan
ism
isol
ated
from
inte
rnal
orga
ns,u
lcer
ativ
eex
tern
alle
sion
sSi
lver
carp
Asso
ciat
edw
ithpo
ultry
faec
esIs
rael
79
Bact
eria
Dise
ase
Dise
ase
sign
sHo
st/is
olat
ion
site
Dist
ribut
ion
Ref
Tab
le1.
2.C
ontin
ued.
74Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:29 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
Aquatic Animal Species and Organism Relationship 59
Prov
iden
cia
rust
igia
nii
(pre
viou
sly
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eder
icia
na)
Nor
mal
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Faec
esPe
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pten
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bold
ti )
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man
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9
Pseu
doal
tero
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as(A
ltero
mon
as)
anta
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rgan
ism
Mud
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ilsan
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dim
ents
inco
asta
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eas
Anta
rctic
a11
5
Pseu
doal
tero
mon
asba
cter
ioly
tica
Red
-spo
tdis
ease
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ent
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eLa
min
aria
and
indu
ces
dam
ages
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ese
edsu
pply
Cul
ture
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aria
japo
nica
Japa
n67
7
Pseu
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a1.
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icro
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arin
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Far-e
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ance
2.Se
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ring
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285
396
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deni
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239
Pseu
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as(A
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mon
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dist
inct
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icro
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man
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kie
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394
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(pre
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ase
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icro
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onds
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Bay
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679
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tero
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Envi
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enta
liso
late
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ronm
ent
Cal
iforn
ian
coas
t15
0
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tero
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eudo
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rom
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aura
ntia
)
Envi
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enta
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mSu
rface
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ater
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iterra
nean
,Fra
nce
398
286
Pseu
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as(A
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as)
lute
ovio
lace
aEn
viro
nmen
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rgan
ism
Med
iterra
nean
,Fra
nce
287
Pseu
doal
tero
mon
asm
aric
alor
isEn
viro
nmen
talo
rgan
ism
Mar
ine
spon
ge(F
asca
plys
inop
sis
retic
ulat
a )C
oral
Sea
398
Pseu
doal
tero
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as(A
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as)
nigr
ifaci
ens
Non
-pat
hoge
nic
Isol
ated
from
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ater
and
mus
sels
Japa
n39
5
cont
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d
75Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #M.vpTuesday, January 20, 2004 3:35:58 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
60 Chapter 1
Pseu
doal
tero
mon
aspi
scic
ida
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sly
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onas
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mon
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Dis
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deg
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lity
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omac
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Gre
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n,U
SA28
8 77 134
572
Pseu
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bra
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a)En
viro
nmen
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rgan
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ine
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erM
edite
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ranc
e28
3
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doal
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ronm
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Foun
don
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stra
lia23
1
Pseu
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as(A
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undi
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arin
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alifo
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ast
150
Pseu
dom
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illise
ptic
aR
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seas
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eels
.Ass
ocia
ted
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terd
isea
sein
sea
brea
m.
Sept
icae
mia
Hae
mor
rhag
ican
dul
cera
tive
lesi
ons,
haem
orrh
ages
iney
e,no
se,o
perc
ulum
,bra
in,l
iver
,ki
dney
Eels
,rai
nbow
trout
,mar
ine
fish,
sea
bass
,sea
brea
m,a
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alm
on,h
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g,st
riped
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,tur
bot
Den
mar
k,Fi
nlan
d,Fr
ance
,Ja
pan,
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land
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in,
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Aust
ralia
225 96 465
719
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828
Pseu
dom
onas
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rora
phis
1.M
orta
lity
2.O
ppor
tuni
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thog
enH
aem
orrh
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,inc
reas
edas
cite
sflu
id1.
Amag
otro
ut2.
Cra
yfis
h1.
Japa
n2.
UK
332
Pseu
dom
onas
fluor
esce
ns1.
Mor
talit
ies,
sept
icae
mia
.O
ppor
tuni
stpa
thog
en2.
Asso
ciat
edw
ith‘a
typi
calB
GD
’,w
ater
tem
pera
ture
<10 °
C
1.W
hite
nodu
les
insp
leen
,ab
sces
ses
insw
imbl
adde
r.Fi
nor
tail
rot
2.Ba
cter
iaon
gills
,bac
teria
lcel
lsad
here
tola
mel
lare
pith
eliu
m
1.Fr
eshw
ater
orna
men
talf
ish
–ca
rp,
red
osca
r,til
apia
,gol
dfis
h,se
abr
eam
,ra
inbo
wtro
ut2.
Cul
ture
dsa
lmon
ids
(rain
bow
trout
,ch
inoo
ksa
lmon
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ntic
salm
on)
1.W
orld
wid
e,Ja
pan
2.C
anad
a,C
hile
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way
460
548
604
693
Pseu
dom
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plec
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ssic
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Bact
eria
lhae
mor
rhag
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cite
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heav
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lity
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cite
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ultu
red
ayu
Japa
n58
2
Pseu
dom
onas
pseu
doal
calig
enes
Mor
talit
ies
Skin
ulce
ratio
nR
ainb
owtro
utSc
otla
nd42
Pseu
dom
onas
putid
aH
aem
orrh
agic
asci
tes
Atla
ntic
salm
on,y
ello
wta
ilJa
pan.
Ubi
quito
usin
envi
ronm
ent
461
Pseu
dom
onas
putre
faci
ens
See
Shew
anel
laal
gae,
S.ba
ltica
and
S.pu
trefa
cien
s
Bact
eria
Dise
ase
Dise
ase
sign
sHo
st/is
olat
ion
site
Dist
ribut
ion
Ref
Tab
le1.
2.C
ontin
ued.
76Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:29 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
Aquatic Animal Species and Organism Relationship 61
Pseu
dom
onas
stan
ieri
Envi
ronm
enta
lorg
anis
mFo
und
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arin
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nmen
t69
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,abs
cess
es,l
esio
nsin
orga
ns.G
rey-
whi
teen
larg
edne
crot
icab
sces
ses
seen
inki
dney
Salm
onid
s–
brow
ntro
ut,r
ainb
owtro
ut,
broo
ktro
ut,c
hino
oksa
lmon
,coh
osa
lmon
,At
lant
icsa
lmon
,ayu
Can
ada,
Chi
le,D
enm
ark,
Fran
ce,G
erm
any,
Icel
and,
Japa
n,Sp
ain,
USA
,Yug
osla
via,
noti
nAu
stra
lia
564
671
Rho
doco
ccus
(lute
us)f
asci
ans
Mic
roflo
raSk
inan
din
test
inal
flora
Car
p,so
ilU
SSR
573
Rho
doco
ccus
spp.
Pano
phth
alm
itis,
ocul
aroe
dem
a.Pa
thog
enic
ityno
tdet
erm
ined
Ocu
larl
esio
ns,n
odul
esin
mus
cle
and
orga
ns.F
ound
inas
soci
atio
nw
ithM
ycob
acte
rium
neoa
urum
Chi
nook
salm
onC
anad
a53
Ros
eoba
cter
galla
ecie
nsis
Nor
mal
flora
Bact
eria
lflo
raon
larv
aeof
scal
lop
Scal
lop
Pect
enm
axim
usEu
rope
662
cont
inue
d
77Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:29 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
62 Chapter 1
Ros
eoba
cter
spec
ies
stra
inC
VSP
Juve
nile
oyst
erdi
seas
e(J
OD
)R
educ
edgr
owth
rate
,fra
gile
and
unev
ensh
ellm
argi
ns,c
uppi
ngof
left
valv
eIn
tern
ally
–m
antle
retra
ctio
n,le
sion
s,pr
otei
nace
ous
depo
sits
(con
chio
lin)i
nsid
esh
ell
Hat
cher
yre
ared
oyst
ers
( Cra
ssos
trea
virg
inic
a )U
SA10
410
5
Sale
gent
ibac
ter(
Flav
obac
teriu
m)
sale
gens
Envi
ronm
enta
lorg
anis
mH
yper
salin
ela
keAn
tarc
tica
533
Salin
ivib
rio(V
ibrio
)cos
ticol
ass
p.co
stic
ola
Envi
ronm
enta
lorg
anis
mH
yper
salin
een
viro
nmen
tsC
anar
yIs
land
s,Sp
ain
279
371
Salm
onel
laar
izon
aeSe
ptic
aem
iaO
rgan
ism
isol
ated
from
orga
nsPi
raru
cu(tr
opic
alfre
shw
ater
fish)
inzo
oaq
uariu
mJa
pan
447
Salm
onel
ladu
rham
Car
riers
tate
Clo
acal
swab
sAf
rican
mud
turtl
e,ye
llow
-spo
tted
Amaz
ontu
rtle,
helm
eted
terra
pin,
East
ern
box
turtl
e,N
orth
ern
diam
ondb
ack
terra
pin,
Mis
siss
ippi
map
turtl
e,Tr
avan
core
crow
ned
turtl
e,C
aspi
ante
rrapi
n,pa
inte
dtu
rtle,
red-
eare
dtu
rtle,
stin
kpot
turtl
e
USA
606
Salm
onel
laen
terit
idis
,S.h
avan
a,S.
new
port,
S.ty
phim
uriu
mC
arrie
rsta
teFa
eces
Gen
too
peng
uins
,mac
aron
ipen
guin
s,gr
ey-h
eade
dal
batro
ss,A
ntar
ctic
furs
eals
Bird
Isla
ndSo
uth
Geo
rgia
nar
chip
elag
o–
Anta
rctic
a61
2
Salm
onel
lahe
idel
berg
,S.n
ewpo
rt,S.
oran
ienb
urg
Car
riers
tate
inhe
alth
yan
imal
s,or
mild
gast
roen
terit
isR
ecta
lsw
abs
Cal
iforn
ian
sea
lion
pups
,Nor
ther
nfu
rse
alpu
psU
SA29
8
Salm
onel
lagr
oup
Oty
peB
Dea
than
dilln
ess
Sea
turtl
es(C
helo
nia
myd
as)
Wes
tern
Aust
ralia
592
Serra
tiafo
ntic
ola
Envi
ronm
enta
lorg
anis
mW
ater
,spr
ing-
wat
er,s
oil,
wild
bird
s,gu
tco
nten
tofs
lugs
and
snai
lsU
biqu
itous
290
558
Serra
tialiq
uefa
cien
sM
orta
litie
s,se
ptic
aem
ia.O
ppor
tuni
stic
path
ogen
Kidn
ey,s
plee
n,liv
eraf
fect
ed.
Bloo
dyas
cite
s,ha
emor
rhag
esin
tissu
es.R
edne
ssan
dsw
ellin
gar
ound
anus
inAr
ctic
char
r
Arct
icch
arr,
Atla
ntic
salm
on,t
urbo
t.Fr
ance
,Sco
tland
,USA
(mid
-Atla
ntic
regi
on)
500
538
715
791
Serra
tiam
arce
scen
sPa
thog
enic
ityin
doub
t–op
portu
nist
Kidn
eyBa
ss,t
rout
,whi
tepe
rch
USA
pollu
ted
river
76
Serra
tiapl
ymut
hica
Path
ogen
icity
indo
ubt–
oppo
rtuni
st.
Isol
ated
from
mor
ibun
dtro
utO
rgan
ism
sin
inte
rnal
orga
nsR
ainb
owtro
ut.A
ssoc
iatio
nw
ithpo
llutio
nby
dom
estic
sew
age
Scot
land
,Spa
in42 57
9
Bact
eria
Dise
ase
Dise
ase
sign
sHo
st/is
olat
ion
site
Dist
ribut
ion
Ref
Tab
le1.
2.C
ontin
ued.
78Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:30 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
Aquatic Animal Species and Organism Relationship 63
Shew
anel
laal
gae
prev
ious
lyid
entif
ied
asSh
ewan
ella
(Pse
udom
onas
)pu
trefa
cien
sgr
oup
IV,G
ilard
ibi
ovar
2,C
DC
biot
ype
2
1.En
viro
nmen
tali
sola
te2.
Asso
ciat
edw
ithhu
man
faec
es,s
kin
ulce
rs,o
titis
med
ia,a
ndfo
und
inba
cter
aem
ia
1.Ba
cter
ialf
lora
2.Sk
in,b
lood
1.R
edal
gae,
mar
ine
envi
ronm
ent
2.Pa
thog
enic
tohu
man
s1.
Japa
n2.
Wor
ldw
ide,
Can
ada,
Den
mar
k,Sw
eden
,USA
433
360
588
792
Shew
anel
laba
ltica
prev
ious
lySh
ewan
ella
(Pse
udom
onas
)put
refa
cien
sO
wen
’sgr
oup
II
Envi
ronm
enta
liso
late
Oil
brin
eBa
ltic
Sea
851
Shew
anel
labe
nthi
caIn
test
ine,
holo
thur
ia(P
sych
robo
tes
long
icau
da)
112
Shew
anel
la(A
ltero
mon
as)
colw
ellia
na(o
rgan
ism
desi
gnat
edLS
Tin
orig
inal
refe
renc
e)
Auto
chth
onou
sC
ontri
bute
sto
oyst
erla
rvae
sedi
men
t.Ad
here
sto
surfa
ces
such
asoy
ster
shel
l,gl
ass,
plas
tic
Estu
arin
eoy
ster
wat
er.E
aste
rnoy
ster
,( C
rass
ostre
aevi
rgin
ica)
UK
814
815
Shew
anel
lafri
gidi
mar
ina
Envi
ronm
enta
liso
late
Ice,
ice
alga
lbio
mas
s,cy
anob
acte
rialm
atAn
tarc
tica
112
Shew
anel
lage
lidim
arin
aEn
viro
nmen
tali
sola
teIc
eAn
tarc
tica
112
Shew
anel
laha
neda
i(A
ltero
mon
asha
neda
i)En
viro
nmen
tali
sola
teSe
dim
ent,
mar
ine
envi
ronm
ent
Arct
ic40
9
Shew
anel
laja
poni
caEn
viro
nmen
tali
sola
teFo
und
inse
awat
eran
dth
em
usse
l( P
roto
thac
aje
doen
sis)
.Dig
ests
agar
Coa
stal
area
ofSe
aof
Japa
n39
7
Shew
anel
laon
eide
nsis
(form
erly
Shew
anel
lasp
.MR
-1)
Envi
ronm
enta
liso
late
Lake
wat
erO
neid
aLa
ke,L
ake
Mic
hega
n,U
SAan
dBl
ack
Sea
782
Shew
anel
lape
alea
naN
orm
alflo
ra,s
ymbi
otic
Acce
ssor
yni
dam
enta
lgla
ndSq
uid
(Lol
igo
peal
ei)
492
Shew
anel
lapu
trefa
cien
spr
evio
usly
Pseu
dom
onas
putre
faci
ens
Ow
ens
Gro
upI,
Gila
rdib
iova
r1&
3,C
DC
biot
ype
1
Sept
icae
mia
Hae
mor
rhag
icne
cros
ison
body
.Fr
ayed
fins
and
exop
htha
lmia
Rab
bitf
ish
Foun
din
aqua
ticen
viro
nmen
tsin
clud
ing
mar
ine,
sedi
men
ts,o
ilfie
lds,
spoi
ling
fish
Saud
iAra
bia
Anta
rctic
a,U
K,w
orld
wid
e43
366
566
6
Shew
anel
law
oody
iN
on-p
atho
geni
cSq
uid
ink
and
seaw
ater
Albo
ran
Sea
521
Sphi
ngob
acte
rium
mul
tivor
um(p
revi
ousl
yFl
avob
acte
rium
mul
tivor
uman
dgr
oup
Iik,
biot
ype
2)
Clin
ical
sour
ces
Sple
enH
uman
path
ogen
UK,
USA
364
365
844
cont
inue
d
79Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:30 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
64 Chapter 1
Sphi
ngob
acte
rium
(Fla
voba
cter
ium
)spi
ritiv
orum
F.ya
buuc
hiae
isa
juni
orsy
nony
m
Clin
ical
isol
ates
Hum
ans
365
844
Sphi
ngom
onas
(Pse
udom
onas
)pa
ucim
obilis
Envi
ronm
enta
lorg
anis
mC
linic
al,h
ospi
tale
nviro
nmen
t,w
ater
361
Stap
hylo
cocc
usau
reus
Eye
dise
ase
Cor
nea
red
toop
aque
Silv
erca
rp.A
lso
foun
din
mar
ine
envi
ronm
ents
Indi
a32
068
8
Stap
hylo
cocc
usde
lphi
niSk
inle
sion
sPu
rule
ntsk
inle
sion
sD
olph
ins
inca
ptiv
ityEu
rope
778
Stap
hylo
cocc
usep
ider
mid
is1.
Stap
hylo
cocc
osis
2.Sk
inm
icro
flora
1.U
lcer
atio
nan
dha
emor
rhag
eson
fins
1.R
edse
abr
eam
,yel
low
tail.
Also
foun
din
mar
ine
and
estu
arin
ew
ater
s2.
Hum
ans
1.Ja
pan,
Taiw
an32
0
Stap
hylo
cocc
usho
min
isN
ota
know
naq
uacu
lture
path
ogen
Rep
orte
dfro
mm
arin
ean
des
tuar
ine
envi
ronm
ents
and
hum
anso
urce
s32
044
4
Stap
hylo
cocc
uslu
trae
Path
ogen
icity
notd
eter
min
ed.M
aybe
path
ogen
icLi
ver,
sple
en,l
ymph
node
Isol
ated
from
Euro
pean
otte
rsU
K26
4
Stap
hylo
cocc
ussp
p.–
S.ca
pitis
,S.
cohn
ii,S.
epid
erm
idis
,S.
haem
olyt
icus
,S.s
apro
phyt
icus
,S.
sim
ulan
s,S.
xylo
sus
Mic
roflo
raSk
inH
uman
s32
044
468
1
Stap
hylo
cocc
usw
arne
ri1.
Dis
ease
dan
ddy
ing
trout
.O
ppor
tuni
stic
infe
ctio
n2.
Asso
ciat
edw
ithse
ptic
aem
ia,
endo
card
itis,
conj
unct
iviti
s,ur
inar
ytra
ctan
dw
ound
infe
ctio
ns
1.U
lcer
ated
lesi
ons
onfin
san
dex
opht
halm
ia,a
sciti
cflu
id.
Isol
ated
from
liver
and
kidn
ey
1.R
ainb
owtro
ut2.
Hum
ans
1.Sp
ain
296
320
444
Stap
pia
stel
lula
ta-li
keSt
rain
M1
(pre
viou
sly
Agro
bact
eriu
mst
ellu
latu
m)
Pote
ntia
lpro
biot
icin
JOD
May
prev
entc
olon
izat
ion
ofju
veni
leoy
ster
sby
Ros
eoba
cter
stra
inC
VSP
USA
105
Stre
ptob
acillu
sm
onilif
orm
isor
poss
ibly
from
the
Fuso
bact
eriu
mgr
oup.
Iden
tific
atio
nfo
rthi
sdi
seas
eis
notc
oncl
usiv
e(M
aher
etal
.,19
95)
Bact
eria
ldis
ease
Intra
cellu
laro
rgan
ism
sse
enin
tissu
es.E
nlar
gem
ento
fend
othe
lial
cells
ofki
dney
glom
erul
i
Atla
ntic
salm
on.S
een
inpn
eum
onia
inko
alas
,cer
vica
labs
cess
ingu
inea
pigs
,po
lyar
thrit
isin
mic
e,te
ndon
-she
ath
arth
ritis
intu
rkey
.Cau
sativ
eag
ento
fra
t-bite
feve
rin
hum
ans
Irela
nd,w
orld
wid
e16
951
961
1
Bact
eria
Dise
ase
Dise
ase
sign
sHo
st/is
olat
ion
site
Dist
ribut
ion
Ref
Tab
le1.
2.C
ontin
ued.
80Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:30 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
Aquatic Animal Species and Organism Relationship 65
Stre
ptoc
occu
sty
peI
Now
reco
gniz
edas
Lact
ococ
cus
garv
ieae
Stre
ptoc
occu
sag
alac
tiae
grou
pB
Mor
talit
y,ex
opht
halm
ia,h
aem
orrh
age
Hae
mor
rhag
icar
eas
onbo
dy,
mou
th,f
ins.
Org
anis
min
inte
rnal
orga
ns
Blue
fish,
cultu
red
seab
ream
,wild
mul
let,
strip
edba
ss,s
eatro
ut,b
ullm
inno
ws.
Aqua
rium
fish
–ra
ms.
Cau
ses
mas
titis
inca
ttle,
infe
ctio
nsin
man
yot
hera
nim
alsp
ecie
s,an
dne
onat
alm
enin
gitis
inhu
man
s
Aust
ralia
,Kuw
ait,
Che
sape
ake
Bay,
Alab
ama
USA
71 135
242
637
Stre
ptoc
occu
sag
alac
tiae
grou
pB,
Type
1bca
psul
aran
tigen
( Stre
ptoc
occu
sdi
ffici
le)
Sept
icae
mia
,men
ingo
ence
phal
itis
Org
anis
mis
olat
edfro
mbr
ain
Car
p,ra
inbo
wtro
ut,t
ilapi
a,fre
shw
ater
orna
men
talf
ish,
frogs
,mic
e,bo
vine
s,hu
man
s
Aust
ralia
,Isr
ael
233
776
Stre
ptoc
occu
sdi
ffici
leC
onfir
med
asSt
rept
ococ
cus
agal
actia
egr
oup
B,ty
peIb
caps
ular
antig
en77
6
Stre
ptoc
occu
sdy
sgal
actia
ess
p.dy
sgal
actia
e,gr
oup
CAn
imal
path
ogen
Anim
als
–bo
vine
mas
titis
790
Stre
ptoc
occu
sdy
sgal
actia
ess
p.dy
sgal
actia
ese
rova
rLIn
fect
ions
and
sept
icae
mia
with
resu
lting
bron
chop
neum
onia
,m
yoca
rditi
s,os
teom
yelit
is,
pyel
onep
hriti
s,ab
sces
ses
Isol
ated
inal
mos
tpur
ecu
lture
from
lung
,kid
ney,
inte
stin
e,sp
leen
Isol
ated
from
harb
ourp
orpo
ises
stra
nded
orca
ught
infis
hing
nets
.Inf
ectio
nsin
cattl
e,do
gs,p
igs
and
othe
rani
mal
s
Balti
cSe
a,N
orth
Sea
727
790
Stre
ptoc
occu
sdy
sgal
actia
esu
bsp.
equi
sim
ilis,g
rou p
C,G
o rL
Anim
alan
dhu
man
path
ogen
Anim
als
and
hum
ans
790
Stre
ptoc
occu
sin
iae
(Stre
psh
iloiis
aju
nior
syno
nym
)1.
Dea
ths,
men
ingo
ence
phal
itis
2.G
olfb
alld
isea
sein
dolp
hins
3.Sy
stem
icin
fect
ion
4.W
ound
sw
hen
clea
ning
fish.
Dea
ths
1.C
ultu
reor
gani
smfro
mth
ebr
ain.
Also
kidn
eyan
dliv
er2.
Subc
utan
eous
absc
ess
3.To
rtico
llis,g
ross
lesi
ons,
gran
ulom
as,e
nlar
ged
orga
ns4.
Loca
lized
cellu
litis
,ulc
ers
1.Ba
rram
undi
,Eur
opea
nse
aba
ss,
gilth
ead
sea
brea
m,p
uffe
rfis
h,ra
bbit
fish,
reef
fish,
rain
bow
trout
,stin
gray
,tila
pia,
cultu
red
ayu,
flyin
gfo
xaq
uariu
mfis
h,fre
shw
ater
and
salt-
wat
erfis
h2.
Fres
hwat
erAm
azon
ian
dolp
hin
3.Fa
rmed
bullf
rogs
( Ran
aca
stes
beia
na)
4.H
uman
s–
espe
cial
lyel
derly
peop
lean
dfis
hha
ndle
rsM
arin
ean
dbr
acki
shw
ater
Aust
ralia
,Bah
rain
,Bar
bado
s,C
anad
a,C
hina
,Isr
ael,
Japa
n,So
uth
Afric
a,Th
aila
nd,U
SA
223
233
235
135
127
621
625
626
530
848
Stre
ptoc
occu
sm
illeri
(iden
tific
atio
nno
tcer
tain
.S.m
illeri
nota
reco
gniz
edna
me)
Dis
ease
Ulc
ers
onfla
nkan
dta
ilKo
icar
pU
K41 87
cont
inue
d
81Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #M.vpTuesday, January 20, 2004 3:36:31 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
66 Chapter 1
Stre
ptoc
occu
spa
raub
eris
(pre
viou
sly
Stre
ptoc
occu
sub
eris
geno
type
II)
1.St
rept
ococ
cosi
s,m
ucoh
aem
orrh
agic
ente
ritis
2.M
astit
is
1.H
aem
orrh
ages
inan
alan
dpe
ctor
alfin
s,an
dey
es.O
rgan
ism
sis
olat
edfro
mliv
er,k
idne
y,sp
leen
2.M
ilk
1.C
ultu
red
turb
ot2.
Bovi
neSp
ain
224
754
Stre
ptoc
occu
sph
ocae
spp.
nov.
Opp
ortu
nist
icin
vira
linf
ectio
nsSe
als
Nor
way
700
Stre
ptoc
occu
spo
rcin
us(p
revi
ousl
ySt
rept
ococ
cus
infre
quen
s ,La
ncef
ield
grou
psE,
P,U
,V)
Infe
ctio
nsC
ervi
call
ymph
node
san
dm
ilkPi
gsW
orld
wid
e17
5
Stre
ptom
yces
salm
onis
(Stre
ptov
ertic
illium
salm
onis
baso
nym
)
Stre
ptom
ycos
isSa
lmon
ids
USA
41
Tena
ciba
culu
mm
aritim
um(p
revi
ousl
yC
ytop
haga
mar
ina
and
Flex
ibac
term
ariti
mus
)
Mar
ine
colu
mna
ris,e
rosi
vesk
indi
seas
e,gi
llle
sion
san
dul
cera
tive
derm
atiti
s,bl
ack
patc
hne
cros
is,
bact
eria
lsto
mat
itis
(mou
thro
t)
Erod
edm
outh
and
fins,
ulce
rate
dsk
inle
sion
sM
arin
efis
h,es
peci
ally
Atla
ntic
salm
on,
chin
ook
salm
on,s
eaba
ss,r
edse
abr
eam
,bla
ckbr
eam
,roc
kbr
eam
,nor
ther
nan
chov
y,st
riped
trum
pete
r,D
over
sole
,flo
unde
r,ye
llow
tail
Atla
ntic
,Can
ada,
Euro
pe,
Fran
ce,J
apan
,Sco
tland
89 90 91 154
551
605
725
801
Tena
ciba
culu
m(F
lexi
bact
er)
ovol
ytic
umO
ppor
tuni
stpa
thog
enLa
rvae
and
egg
mor
talit
yD
isso
lves
chor
ion
and
zona
radi
ata
ofth
eeg
gsh
ells
Atla
ntic
halib
uteg
gsan
dla
rvae
Nor
way
324
725
Vago
cocc
usfe
ssus
Path
ogen
icity
notd
eter
min
edO
rgan
ism
isol
ated
from
liver
,ki
dney
Isol
ated
from
ade
adha
rbou
rsea
land
porp
oise
Euro
pe36
9
Vago
cocc
usflu
vial
is1.
Envi
ronm
enta
lorg
anis
m2.
Clin
ical
sam
ples
2.Bl
ood,
perit
onea
lflu
id,w
ound
s1.
Riv
erw
ater
,chi
cken
faec
es,d
omes
tican
imal
s(p
ig,c
attle
,cat
s,ho
rse)
2.H
uman
s
UK
177
629
732
Vago
cocc
uslu
trae
Path
ogen
icity
notd
eter
min
edO
rgan
ism
foun
din
bloo
d,liv
er,
lung
s,sp
leen
Otte
rU
K47
7
Bact
eria
Dise
ase
Dise
ase
sign
sHo
st/is
olat
ion
site
Dist
ribut
ion
Ref
Tab
le1.
2.C
ontin
ued.
82Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:30 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
Aquatic Animal Species and Organism Relationship 67
Vago
cocc
ussa
lmon
inar
umLa
ctob
acillo
sis,
pseu
doki
dney
dise
ase,
perit
oniti
s,se
ptic
aem
iaH
aem
orrh
ages
ongi
llsan
din
ocul
arre
gion
,per
itoni
tis,h
eart
lesi
ons,
enla
rged
sple
en,l
istle
ssbe
havi
our
Atla
ntic
salm
on,b
row
ntro
ut,r
ainb
owtro
utAu
stra
lia,F
ranc
e,N
orth
Amer
ica,
Nor
way
542
682
807
Varra
calb
mis
pp.n
ov.
Eye
lesi
ons
(blo
ody
eye)
and
blin
dnes
sLe
sion
sin
kid,
liver
,gills
,ps
eudo
bran
ch.O
rgan
ism
isol
ated
from
eyes
.Ski
nul
cers
Atla
ntic
salm
onN
orw
ay77
1
Vibr
ioae
roge
nes
Envi
ronm
enta
lorg
anis
mAs
soci
ated
with
sea
gras
sse
dim
ent,
foun
din
shal
low
coas
tala
ndoc
eani
cse
awat
ers
Taiw
an69
2
Vibr
ioae
stua
rianu
sEn
viro
nmen
talo
rgan
ism
Foun
das
soci
ated
with
shel
lfish
(oys
ter,
clam
,cra
b)an
des
tuar
ine
wat
ers
USA
747
Vibr
ioag
ariv
oran
sEn
viro
nmen
talo
rgan
ism
Seaw
ater
Abal
one
–pa
thog
enic
ityno
tkno
wn
Aust
ralia
,Med
iterra
nean
Sea
(Spa
in)
135
514
Vibr
ioal
gino
lytic
us1.
Syst
emic
dise
ase,
ulce
rativ
edi
seas
e,ne
cros
is,e
yedi
seas
e,vi
brio
sis,
scal
lop
larv
alm
orta
lity
2.W
ound
infe
ctio
ns,e
xter
nalo
titis
,ce
llulit
is
1.O
rgan
ism
isol
ated
from
orga
nsan
dey
e,sc
allo
pla
rvae
2.W
ound
son
expo
sure
tose
awat
er
1.Au
stra
lian
nativ
ean
din
trodu
ced
fish.
Mol
lusc
s(la
rval
and
juve
nile
),re
dab
alon
e,So
uth
Afric
anab
alon
e,sc
allo
pla
rvae
,eel
,rai
nbow
trout
,sea
brea
m,
turb
ot,t
urtle
s,se
ahor
se.S
hrim
ps2.
Hum
ans
Aust
ralia
,Chi
le,M
exic
o,w
orld
wid
eIn
do-P
acifi
c&
East
Asia
30 300
301
650
Vibr
ioan
guilla
rum
See
List
onel
laan
guilla
rum
Vibr
iobr
asilie
nsis
Path
ogen
icity
notk
now
n.Li
kely
tobe
norm
alflo
raIs
olat
edfro
mbi
valv
ela
rvae
(Nod
ipec
ten
nodo
sus )
Braz
il74
0
Vibr
ioca
lvie
nsis
Envi
ronm
enta
lorg
anis
mSe
awat
erW
este
rnM
edite
rrane
anSe
a,Fr
ance
216
Vibr
ioca
mpb
ellii
Envi
ronm
enta
lorg
anis
mSe
awat
erFl
orid
a,Pu
erto
Ric
o81
982
0
Vibr
ioca
mpb
ellii-
like
Mor
talit
ies
inju
veni
les
Org
anis
min
brai
n,ki
dney
,liv
er.
Org
ans
had
lesi
ons
and
haem
orrh
age
Hat
cher
y-re
ared
turb
otan
dbr
illN
ewZe
alan
d22
1
Vibr
ioca
rcha
riae
Isa
juni
orsy
nony
mof
V.ha
rvey
iSl
ight
diffe
renc
esof
phen
otyp
ebe
twee
nth
est
rain
sar
ere
porte
din
the
liter
atur
e61
9
cont
inue
d
83Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:31 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
68 Chapter 1
Vibr
ioch
oler
ae(n
on-0
1)H
eibe
rggr
oups
I,II
1.Se
ptic
aem
ia2.
Gas
troen
terit
is1.
Skin
haem
orrh
ages
2.D
iarrh
oea,
vom
iting
1.Ay
u,go
ldfis
h,sh
ellfi
sh.I
sola
ted
from
fresh
and
estu
arin
ew
ater
s2.
Hum
ans
–co
nsum
ptio
nof
raw
shel
lfish
1.Au
stra
lia,J
apan
2.Af
rica,
Asia
nco
ntin
ent,
Euro
pe,U
SA,U
K
196
255
275
434
507
Vibr
ioch
oler
aeEl
Tor
(ser
ogro
ups
01&
0139
)C
hole
raH
uman
sFo
und
inaq
uatic
envi
ronm
ent–
surfa
cew
ater
s
Sero
grou
p01
39in
Bang
lade
sh,
Indi
ansu
bcon
tinen
tonl
y.Pa
ndem
icst
rain
275
392 9
Vibr
ioch
oler
ae-li
keD
eath
Lobs
ters
USA
.(O
rgan
ism
does
n’tg
row
inte
mpe
ratu
res
grea
tert
han
25°C
)
Vibr
ioci
ncin
natie
nsis
1.En
viro
nmen
talo
rgan
ism
2.Ba
cter
aem
ia,m
enin
gitis
1.En
viro
nmen
t2.
Hum
anpa
thog
en2.
USA
120
Vibr
ioco
rallii
lytic
ussp
p.no
v.(p
revi
ousl
yV.
cora
lytic
usYB
)Pa
thog
enof
cora
lTi
ssue
lysi
san
dde
ath.
Smal
lwhi
tesp
ots
seen
onco
rala
t3–5
days
and
com
plet
ede
stru
ctio
nof
tissu
eaf
ter2
wee
ks
Cor
al( P
ocillo
pora
dam
icor
nis)
.Als
ois
olat
edfro
moy
ster
larv
aefro
mth
eAt
lant
icO
cean
Atla
ntic
Oce
an,I
ndia
nO
cean
and
Red
Sea
83 84
Vibr
iocy
clitr
ophi
cus
(orig
inal
lysp
eltV
.cyc
lotro
phic
us)
Envi
ronm
enta
lorg
anis
mEa
gle
Har
bor,
Was
hing
ton,
USA
338
Vibr
iodi
abol
icus
Envi
ronm
enta
lorg
anis
mIs
olat
edfro
mpo
lych
aete
anne
lid(A
lvin
ella
pom
peja
na)i
nde
ep-s
eave
ntPa
cific
Oce
an63
5
Vibr
iodi
azot
roph
icus
Envi
ronm
enta
lorg
anis
mG
astro
inte
stin
altra
ctof
sea
urch
ins
and
mar
ine
anim
als,
surfa
ceof
reed
s,oc
curri
ngin
estu
arin
een
viro
nmen
ts,
seaw
ater
and
sedi
men
ts
Can
ada,
Scot
land
,Eng
land
319
Vibr
iofis
cher
i(s
ynon
ymou
sw
ithPh
otob
acte
rium
fisch
eri)
1.Pa
thog
enic
ityin
doub
t2.
Sym
bion
t1.
Whi
teno
dule
son
skin
,ha
emor
rhag
icul
cera
tion,
tum
ours
inpa
ncre
asan
dbi
ledu
ct2.
Pres
enti
nlig
htor
gan
ofse
piol
idsq
uid
1.Se
abr
eam
,tur
bot
2.Se
piol
idsq
uid
( Eup
rym
nasc
olop
es)
1.Sp
ain
2.H
awai
i10
626
734
248
6
Bact
eria
Dise
ase
Dise
ase
sign
sHo
st/is
olat
ion
site
Dist
ribut
ion
Ref
Tab
le1.
2.C
ontin
ued.
84Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:31 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
Aquatic Animal Species and Organism Relationship 69
Vibr
ioflu
vial
is(p
revi
ousl
ygr
oup
F(F
urni
sset
al.,
1977
)and
grou
pEF
6(H
uqet
al.,
1980
))
1.M
orta
lity
2.En
viro
nmen
t3.
Acut
ew
ater
y,ch
oler
ae-li
kedi
arrh
oea.
Toxi
npr
oduc
ed
1.O
rgan
s2.
Non
-pat
hoge
nic
3.Fa
eces
1.Pl
aice
fry( P
leur
onec
tes
plat
essa
)2.
Foun
din
aqua
tic,p
artic
ular
lyes
tuar
ine
envi
ronm
ent.
Isol
ated
from
mar
ine
mol
lusc
san
dcr
usta
cea
3.H
uman
s
1.D
enm
ark
2.W
orld
wid
e3.
Bang
lade
sh,I
ndia
,Ken
ya,
Mid
dle
East
,Phi
lippi
nes,
Spai
n,Ta
nzan
ia,T
unis
ia
276
378
485
620
687
Vibr
iofu
rnis
sii
(form
erly
V.flu
vial
isbi
ovar
2(a
erog
enic
biog
roup
),gr
oup
F,gr
oup
EF-6
)
1.Pa
thog
enic
ityin
doub
t2.
Envi
ronm
enta
lorg
anis
m3.
Food
pois
onin
g,ga
stro
ente
ritis
1.H
aem
orrh
ages
inin
test
ine
3.D
iarrh
oea
1.Ee
l2.
Riv
erw
ater
,ani
mal
faec
es,m
arin
em
ollu
scs
and
crus
tace
a3.
Hum
ans
Foun
din
aqua
tic,p
artic
ular
lyes
tuar
ine
envi
ronm
ent
1.Sp
ain
2.U
K,w
orld
wid
e3.
Bahr
ain,
Bang
lade
sh,
Indo
nesi
a,Pe
ru,U
SA
123
276
240
485
687
Vibr
ioga
zoge
nes
Envi
ronm
enta
lorg
anis
m67
Vibr
ioha
liotic
olis
pp.n
ov.
Non
-pat
hoge
nic
Mai
ngu
tmic
roflo
raAb
alon
eJa
pan
678
Vibr
ioha
rvey
i(o
rgan
ism
prev
ious
lyid
entif
ied
asV.
carc
haria
eis
said
tobe
aju
nior
syno
nym
ofV.
harv
eyi)
1.M
orta
lity
insh
arks
and
abal
one,
necr
otiz
ing
ente
ritis
insu
mm
erflo
unde
r,ga
stro
ente
ritis
ingr
oupe
r,sy
stem
icdi
seas
e,ul
cera
tive
dise
ase,
necr
osis
,vib
riosi
s2.
Wou
nd(s
hark
bite
)
1.W
hite
spot
son
foot
ofab
alon
e.Lo
seab
ility
toad
here
.Nec
rotic
dege
nera
tion
and
vacu
olat
ion
inth
ele
sion
.Flo
unde
rhav
eex
tend
edab
dom
enan
dre
dan
us.F
ish
–or
gani
smfro
mki
dney
,eye
Shrim
ps–
blac
ksp
ots
onex
oske
leto
n.O
rgan
ism
inle
sion
s,ey
e,he
pato
panc
reas
1.Ab
alon
e,sh
ark,
floun
der,
grou
per,
milk
fish,
mol
lusc
s,oy
ster
s(la
rval
and
juve
nile
),bl
ack
tiger
praw
ns,p
enae
idpr
awns
and
shrim
ps,s
alm
onid
s,se
aba
ss,
sea
brea
m,s
eaho
rse,
shar
ks,o
ctop
us.
Aust
ralia
nna
tive
and
intro
duce
dfis
h2.
Hum
ans
1.Au
stra
lia,E
urop
e,In
done
sia,
Indo
-Pac
ific
&Ea
stAs
ia,
Japa
n,Ph
ilippi
nes,
Sout
hAm
eric
a,Ta
iwan
,Tha
iland
,Ve
nezu
ela,
USA
,wor
ldw
ide
23 11 410
576
581
619
710
734
735
847
Vibr
ioho
llisae
(pre
viou
sly
Gro
upEF
-13)
1.N
on-p
atho
geni
cfo
rfis
h2.
Food
pois
onin
g,ga
stro
ente
ritis
,w
ound
infe
ctio
n,ba
cter
aem
ia
2.D
iarrh
oea
1.Pr
esen
tin
fish
inte
stin
e2.
Hum
ans
Has
ther
mos
tabl
eha
emol
ysin
sim
ilart
oV.
para
haem
olyt
icus
1.Ja
pan
2.U
SA34
655
558
0
Vibr
ioic
hthy
oent
eri
Mor
talit
ies,
inte
stin
alne
cros
isO
paqu
ein
test
ines
Japa
nese
floun
derl
arva
eJa
pan
389
Vibr
iole
ntus
Path
ogen
icity
nots
tate
dAs
soci
ated
with
Med
iterra
nean
oyst
ers
Span
ish
Med
iterra
nean
coas
t51
3
Vibr
iolo
gei
1.Po
ssib
lepa
thog
en2.
Non
-pat
hoge
nic
1.Sk
inle
sion
s2.
Sym
bion
tin
light
orga
n1.
Atla
ntic
salm
on2.
Squi
d( S
epio
la)
1.Ic
elan
d2.
Wes
tern
Paci
fic25
7
cont
inue
d
85Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:31 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
70 Chapter 1
Vibr
iom
edite
rrane
i1.
Non
-pat
hoge
nic
2.Pr
obio
ticpr
oper
ties
3.C
oral
blea
chin
g–
see
unde
rV.
shilo
nii
Appe
ars
tobe
ahe
tero
gene
ous
spec
ies
1.Pl
ankt
on,s
ease
dim
ents
,sea
wat
er2.
Som
est
rain
sar
ea
prob
iotic
fort
urbo
tla
rvae
3.C
oral
blea
chin
g
Spai
n,M
edite
rrane
anco
ast
631
Vibr
iom
etsc
hnik
ovii
1.N
on-p
atho
geni
cfo
rfis
h2.
Perit
oniti
san
dba
cter
aem
ia1.
Isol
ated
from
fow
lwith
chol
erai
cdi
seas
e1.
Foun
din
mar
ine
and
fresh
wat
eren
viro
nmen
t,es
peci
ally
river
s,es
tuar
ies
and
sew
age.
Isol
ated
from
clam
s,co
ckle
s,oy
ster
s,lo
bste
r2.
Hum
ans
Wor
ldw
ide
483
Vibr
iom
imic
us(p
revi
ousl
yVi
brio
chol
erae
Hei
berg
grou
pV)
1.Sy
stem
icdi
seas
ean
dop
portu
nist
2.C
onta
min
anto
nse
atu
rtle
eggs
3.Fo
odpo
ison
ing,
gast
roen
terit
is,
otiti
sm
edia
1.H
aem
olym
ph,i
nfla
mm
atio
nof
peric
ardi
um3.
Dia
rrhoe
a,ea
rinf
ectio
n
1.C
rust
acea
ns–
pena
eid
praw
ns,
yabb
ies,
mar
ron,
fresh
wat
ercr
ayfis
h.Ba
rram
undi
2.R
educ
edvi
abilit
yof
turtl
eeg
gsan
dhu
man
path
ogen
follo
win
gco
nsum
ptio
nof
eggs
3.H
uman
s–
cons
umpt
ion
ofra
wsh
ellfi
sh.
Foun
din
fresh
and
brac
kish
wat
er
1,3.
Asia
,Aus
tralia
,Ba
ngla
desh
,Mex
ico,
New
Zeal
and,
Gua
m,C
anad
a,w
orld
wid
e2.
Cos
taR
ica
416
113
521
023
050
7
Vibr
iom
ytili
1,2.
Non
-pat
hoge
nic
1.N
orm
alflo
ra1.
Mus
sels
2.H
uman
sSp
ain
632
Vibr
iona
trieg
ens
Envi
ronm
enta
liso
late
Salt
mar
shm
ud,w
ater
,oys
ters
UK,
USA
820
Vibr
iona
varre
nsis
Envi
ronm
enta
liso
late
Envi
ronm
enta
l–se
wag
ean
dsu
rface
wat
erSp
ain
768
Vibr
ione
ptun
ius
Mic
roflo
raD
omin
antm
icro
flora
ina
reci
rcul
atin
gsy
stem
forr
otife
rs.I
sola
ted
from
heal
thy
and
dise
ased
biva
lve
larv
ae( N
odip
ecte
nno
dosu
s ),g
utof
turb
otla
rvae
,rot
ifers
305
Vibr
ione
reis
Envi
ronm
enta
liso
late
Seaw
ater
820
Bact
eria
Dise
ase
Dise
ase
sign
sHo
st/is
olat
ion
site
Dist
ribut
ion
Ref
Tab
le1.
2.C
ontin
ued.
86Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:31 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
Aquatic Animal Species and Organism Relationship 71
Vibr
ioni
grip
ulch
ritud
oEn
viro
nmen
tali
sola
teSe
awat
er81
982
0
Vibr
ioor
dalii
(pre
viou
sly
Vibr
ioan
guilla
rum
biot
ype
2an
dLi
ston
ella
orda
lii)
Vibr
iosi
s,ba
cter
ialn
ecro
sis
and
syst
emic
dise
ase
Org
anis
ms
onm
uscl
e,sk
in,g
ills,
dige
stiv
etra
ct,h
eart,
liver
,kid
neys
,sp
leen
.Nec
rosi
san
dha
emor
rhag
e
Mol
lusc
s–
(larv
alan
dju
veni
le),
salm
onAu
stra
lia,J
apan
,Nor
th-W
est
Paci
fic,U
SA,w
orld
wid
e68
0
Vibr
ioor
ient
alis
Envi
ronm
enta
liso
late
Lum
inou
sm
arin
eba
cter
ium
Chi
na84
6
Vibr
iopa
cini
ispp
.nov
.Pa
thog
enic
ityno
tkno
wn
Isol
ated
from
shrim
pla
rvae
(Pen
aeus
chin
ensi
s )C
hina
306
Vibr
iopa
raha
emol
ytic
us(p
revi
ousl
yBe
neck
eapa
raha
emol
ytic
a )
1.Se
ptic
aem
ia,m
orta
lity
2.W
ither
ing
synd
rom
e3.
Food
pois
onin
g,ga
stro
ente
ritis
,w
ound
infe
ctio
n,ba
cter
aem
ia
1.Ex
tern
alha
emor
rhag
e,ta
ilro
t,or
gani
smin
inte
rnal
orga
ns2.
Org
anis
ms
foun
din
haem
olym
ph
3.D
iarrh
oea,
vom
iting
1.C
rust
acea
ns(s
hrim
ps,m
arro
n),m
ulle
t,oc
topu
s,Ib
eria
nto
othc
arp
2.C
ultu
red
smal
laba
lone
3.H
uman
s
1.In
do-P
acifi
can
dEa
stAs
ia,
Spai
n2.
Taiw
an
10 135
272
417
499
Vibr
iope
cten
icid
aPa
thog
enic
Mor
ibun
dsc
allo
pSc
allo
pla
rvae
,shr
imps
,pra
wn
East
Asia
,Ind
o-Pa
cific
,Fra
nce
470
Vibr
iope
lagi
usSe
eLi
ston
ella
pela
gia
Vibr
iope
naei
cida
(pre
viou
sly
Vibr
ioPJ
)Pr
opos
edca
usat
ive
agen
tof
Synd
rom
e93
1.Vi
brio
sis
ofpr
awns
2.Se
ptic
aem
ia,a
ssoc
iate
dw
ithSy
ndro
me
93
1.Br
own
spot
s(n
odul
es)i
nly
mph
oid
orga
nsan
dgi
lls2.
Hae
mol
ymph
1.Ku
rum
apr
awns
.Als
ois
olat
edfro
mhe
alth
ypr
awns
2.Sh
rimp
( Pen
aeus
styl
irost
ris)
1.Ja
pan
2.N
ewC
aled
onia
388
187
676
Vibr
iopr
oteo
lytic
us,s
train
CW
8T2
Path
ogen
Affe
cts
mic
rovi
lli,gu
tcel
ls,
deva
stat
esce
llsin
body
cavi
tyJu
veni
leAr
tem
iasp
p.(b
rine
shrim
p),
used
asliv
edi
ets
fora
quac
ultu
resp
ecie
sEu
rope
67 788
Vibr
ioro
tifer
ianu
sPa
thog
enic
ityno
tkno
wn
Isol
ated
from
rotif
erflo
w-th
roug
hcu
lture
syst
emBe
lgiu
m30
5
Vibr
ioru
moi
ensi
sEn
viro
nmen
tali
sola
teIs
olat
edfro
mdr
ain
pool
offis
hpr
oduc
tpr
oces
sing
plan
tJa
pan
850
Vibr
iosa
lmon
icid
aC
oldw
ater
vibr
iosi
s,H
itra
dise
ase
Sept
icae
mia
,hae
mor
rhag
esin
the
inte
gum
ents
urro
undi
ngth
eor
gans
.Org
anis
mfo
und
inbl
ood
and
kidn
ey
Atla
ntic
salm
on,d
isea
sed
cod
Can
ada,
Faro
eIs
land
s(D
enm
ark)
,Ice
land
,Nor
way
,Sc
otla
nd
198
232
Vibr
iosc
opht
halm
iN
on-p
atho
geni
cPa
rtof
gutm
icro
flora
Juve
nile
turb
otSp
ain
149
254
cont
inue
d
87Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:32 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
72 Chapter 1
Vibr
iosh
iloni
i(p
revi
ousl
yV.
shilo
i,Vi
brio
spec
ies
AK-1
).La
test
info
rmat
ion
sugg
ests
this
isa
late
rsyn
onym
ofV.
med
iterra
nei
(Tho
mps
onet
al.,
2001
a,b)
Cor
albl
each
ing
Adhe
res
and
pene
trate
sin
toth
eep
ider
mis
ofth
eco
ral,
Ocu
lina
pata
goni
ca.C
ultu
refro
mth
eco
ral
muc
us.T
heor
gani
smis
intra
cellu
lara
ndno
n-cu
ltura
ble
Bact
eriu
mbi
nds
toth
eco
rala
tele
vate
dte
mpe
ratu
res
and
disr
upts
the
sym
biot
icpr
oces
sw
ithth
eph
otos
ynth
etic
mic
ro-a
lgae
endo
sym
bion
ts(z
ooxa
nthe
llae)
.Hea
t-sta
ble
toxi
npr
oduc
ed
Med
iterra
nean
Sea
59 458
742
Vibr
iosp
lend
idus
Mor
talit
yin
stre
ssed
oyst
ers
Oys
terl
arva
e(C
rass
ostre
agi
gas)
Fran
ce46
646
7
1.Vi
brio
sple
ndid
usI.
2.Vi
brio
sple
ndid
us-li
ke1.
Sept
icae
mia
.Mor
talit
ies
inju
veni
les
2.M
orta
litie
s1.
Hae
mor
rhag
esat
mou
th,a
nus
and
fins.
Org
anis
ms
inbr
ain,
kidn
ey,l
iver
2.Sk
inha
emor
rhag
es,f
inan
dta
ilro
tero
sion
,dec
ayof
soft
tissu
ebe
twee
nfin
s.Sa
mpl
efro
mki
dney
and
sple
en
1.G
ilt-h
ead
sea
brea
m,r
ainb
owtro
ut,
turb
otla
rvae
,shr
imps
(pra
wns
).Ju
veni
leha
tche
ry-re
ared
turb
otan
dbr
ill2.
Plai
cefry
1.Fr
ance
,New
Zeal
and,
Scot
land
,Spa
in2.
Den
mar
k
281
254 31 221
620
Vibr
iosp
lend
idus
IIBa
cilla
ryne
cros
is,m
orta
lity
Larv
aebe
com
ein
activ
ean
dse
ttle
toth
ebo
ttom
ofth
eta
nkJa
pane
seoy
ster
larv
ae,c
uppe
doy
ster
(Cra
ssos
trea
giga
s)Ja
pan,
Fran
ce72
179
8
Vibr
iota
petis
(pre
viou
sly
Vibr
ioP1
orVP
1or
VTP)
Brow
nrin
gdi
seas
e,hi
ghm
orta
lity
Brow
nco
nchi
olin
depo
sito
nin
ner
surfa
ceof
shel
lbet
wee
npa
lliall
ine
and
shel
lmar
gins
Cul
ture
dM
anila
clam
sEn
glan
d,Fr
ance
,Por
tuga
l,Sp
ain
108
146 14 609
610
Vibr
iotra
chur
iLa
test
info
rmat
ion
sugg
ests
this
isa
juni
orsy
nony
mof
V.ha
rvey
i(T
hom
pson
etal
.,20
02b)
Dis
ease
Hae
mor
rhag
esin
orga
ns,
exop
htha
lmia
Hor
sem
acke
rel
Japa
n40
074
3
Vibr
iotu
bias
hii
Bact
eria
lnec
rosi
san
dsy
stem
icdi
seas
eLa
rvae
ceas
esw
imm
ing,
tissu
edi
gest
ion
Mol
lusc
s(o
yste
rand
clam
)–la
rval
and
juve
nile
,Pac
ific
oyst
erAu
stra
lia,U
K,U
SA32
129
4
Vibr
iovi
scos
usSe
eM
orite
llavi
scos
a82 50
6
Bact
eria
Dise
ase
Dise
ase
sign
sHo
st/is
olat
ion
site
Dist
ribut
ion
Ref
Tab
le1.
2.C
ontin
ued.
88Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:32 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
Aquatic Animal Species and Organism Relationship 73
Vibr
iovu
lnifi
cus
biot
ype
I1.
Envi
ronm
enta
l2.
Infe
ctio
n,sy
stem
icdi
seas
e,ul
cera
tive
dise
ase,
necr
osis
3.W
ound
infe
ctio
n,se
ptic
aem
iafo
llow
ing
cons
umpt
ion
ofra
wse
afoo
des
peci
ally
oyst
ers.
Sept
icae
mia
has
a50
%fa
talit
yra
te
1.M
icro
flora
ofin
test
ines
orgi
llsof
fish,
mus
sels
,cra
bsan
dse
abird
s3.
Inge
stio
nof
cont
amin
ated
seaf
ood,
dam
age
toth
ein
test
inal
wal
l.W
ound
infe
ctio
n
1.R
eser
voirs
incl
ude
clam
s,cr
abs,
oyst
ers,
mus
sels
,flo
unde
r,pl
ankt
on,
seab
irds,
mar
ine
and
brac
kish
wat
ers
2.C
ultu
red
shrim
ps,m
ollu
scs
–la
rval
and
juve
nile
,fis
h3.
Hum
ans
–pe
ople
with
high
seru
miro
nle
vels
,im
mun
ocom
prom
ised
,orw
ithliv
erdi
seas
ear
epa
rticu
larly
susc
eptib
le
1.At
lant
icco
asta
lreg
ions
,D
enm
ark,
Euro
pe,I
ndo-
Paci
fican
dEa
stAs
ia,A
ustra
lia,M
exic
o2.
Aust
ralia
,Ind
o-Pa
cific
and
East
Asia
,Eur
ope,
Japa
n,U
SA3.
Aust
ralia
,Bel
gium
,Den
mar
k,G
erm
any,
Japa
n,H
olla
nd,
Swed
en,U
SA
358
597
746
Vibr
iovu
lnifi
cus
biot
ype
2se
rova
rElip
opol
ysac
char
ide-
base
dO
sero
grou
p,04
1.Vi
brio
sis,
haem
orrh
agic
sept
icae
mia
2.M
ayca
use
illnes
san
dw
ound
infe
ctio
nin
hum
ans
follo
win
gha
ndlin
gof
eels
1.O
rgan
ism
isol
ated
from
gills
,m
ucus
,int
estin
alco
nten
ts,s
plee
n,an
dki
dney
1.Ee
ls–
viru
lent
and
aviru
lent
stra
ins
are
foun
d.C
ultu
red
shrim
ps.F
ound
inbr
acki
shw
ater
,oys
ters
and
sedi
men
t2.
Hum
ans
Den
mar
k,Eu
rope
,Jap
an,
Nor
way
,Spa
in,S
wed
en,T
aiw
an20
135
6 34 25 597
746
Vibr
iovu
lnifi
cus
biot
ype
3Se
vere
wou
ndin
fect
ion
and
bact
erae
mia
Wou
nds
and
bloo
dH
uman
sIs
rael
101
565
Vibr
iow
odan
isN
on-p
atho
geni
c,m
aybe
anop
portu
nist
inw
inte
rulc
erdi
seas
eAt
lant
icsa
lmon
,rai
nbow
trout
,had
dock
Col
dw
ater
ofIc
elan
d,N
orw
ay82 50
6
Vibr
ioxu
iiPa
thog
enic
ityno
tkno
wn
Isol
ated
from
shrim
pcu
lture
wat
eran
dbi
valv
ela
rvae
(Nod
ipec
ten
nodo
sus)
Chi
na74
0
Wei
ssel
last
rain
DS-
12Po
tent
ialp
robi
otic
Non
-pat
hoge
nic
Part
ofin
test
inal
mic
roflo
raof
floun
der.
Spec
ies
ofW
eiss
ella
are
foun
din
ferm
ente
dfo
ods
such
asfis
han
dsa
usag
es
Kore
a13
9
Yers
inia
aldo
vae
prev
ious
lyY.
ente
roco
litic
agr
oup
X2
Envi
ronm
enta
lorg
anis
mIs
olat
edfro
mdr
inki
ngw
ater
,riv
erw
ater
,so
ilan
dfis
h85
Yers
inia
berc
ovie
ripr
evio
usly
Y.en
tero
colit
ica
biog
roup
3B
Envi
ronm
enta
lorg
anis
mIs
olat
edfro
mfre
shw
ater
and
soil
812
Yers
inia
ente
roco
litic
ass
p.en
tero
colit
ica
Gen
eral
lyno
n-pa
thog
enic
form
amm
als
Sucr
ose
nega
tive
stra
ins
from
smal
lro
dent
s.Fo
und
inte
rrest
riala
ndaq
uatic
habi
tats
Japa
n,Eu
rope
,Can
ada,
USA
422
121
cont
inue
d
89Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:32 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
74 Chapter 1
Yers
inia
frede
rikse
nii
Envi
ronm
enta
lorg
anis
mFo
und
inw
ater
.Fis
h,hu
man
s,m
amm
als
and
bird
sm
aybe
heal
thy
carri
ers
423
Yers
inia
inte
rmed
iaEn
viro
nmen
talo
rgan
ism
Atla
ntic
salm
on.F
ound
inw
ater
.Fis
han
dm
amm
als
may
behe
alth
yca
rrier
sAu
stra
lia42
3
Yers
inia
kris
tens
enii
Envi
ronm
enta
lorg
anis
mFo
und
inw
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The information is presented in tabular form sothat it is readily accessible in a summarized form.
The bacterial names are listed in alphabeticalorder. The disease column indicates whether theorganism is a saprophyte, an environmentalorganism or is a pathogen of fish and other aquaticanimals. The next column indicates the diseasesigns that may be seen with an infection caused bythis organism, and then the aquatic species islisted, as is the geographical distribution of theorganism. The final column lists the referencenumber.
1.4 Taxonomy and Disease Statusof Bacteria
New bacteria from the aquatic world are beingdiscovered, identified and named at an increas-ing rate. Some are then reassigned to a differentor newly described genus, while others changetheir species name as more research and studyon the taxonomy follows. The following sectiongives brief details of the current taxonomic statusof a genus or species. Because some of thisinformation is likely to become out of date,microbiologists are encouraged to keep up withsuch information, which is found at taxonomicwebsites. Some addresses and suggested readingcan be found under Further Reading and OtherInformation Sources, p. 278. Refer to these andother texts for a comprehensive coverage ofdiseases.
Abiotrophia spp.
These bacteria, which are known as nutritionallyvariant streptococci (NVS), satellite aroundother bacterial growth, or require L-cysteineor pyridoxal hydrochloride (vitamin B6) forgrowth. A. elegans requires L-cysteine for growth.A. adiacens, A. defectiva and A. elegans areisolated as part of the normal flora from the oralcavity, intestinal tract and genital tract of humans.They have also been isolated in cases of endo-carditis, otitis media and post-surgery woundinfections. They have been included in this bookbecause an organism found in a minke whalewas originally placed into this genus. It has sincebeen placed into the genus Granulicatella alongwith some other previously named Abiotrophia
species. G. balaenopterae, found in the minkewhale, does not require additional growth factorsand grows in air or 5% CO2.
Aequorivita spp.
Strains from this genus belong to the familyFlavobacteriaceae and the order Cytophagales,and have been isolated from the marine environ-ment in Antarctica. They can grow on MA 2216.
Aeromonas spp.
The taxonomy of Aeromonas spp. (familyAeromonadaceae) is in a continual state of fluxas new species are described and the descriptionsof the existing taxa are refined. There is aphenotypic and a genotypic classification.Genotypic classification is based on DNA–DNAhybridization with 16 DNA HG recognized atpresent. The designation of the genospeciesis dependent on the type strain within the HG.Problems exist for the diagnostic microbiologist inthe phenotypic identification of strains. Pheno-typic diversity of strains occurs within a DNA HGand strains from different HG may be bio-chemically similar. In this book the phenotypictests are recorded for the type strains. A table(Table 4.2) is also presented of the phenotypictests according to HG (Abbott et al., 1992;Kaznowski, 1998). An average of the results fromthese two studies is reported. Results from differ-ent references can be difficult to assess. Abbottet al. (1992) stated that when using 22 strainsof HG1, including the type strain A. hydrophilaATCC 7966, 100% were positive for LDC. Thiswas also supported by research that recognizeda new subspecies within the A. hydrophilagroup, with the recognition of A. hydrophilassp. hydrophila and A. hydrophila ssp. dhakensis(Huys et al., 2002b). This study reported resultsfor A. hydrophila ssp. hydrophila of the typestrain LMG 2844, which is equivalent to ATCC7966. However, Kaznowski (1998), using onestrain only (the type strain ATCC 7966), statedthat 0% of HG1 are positive for LDC. Likewise,other researchers have found that the type strainATCC 7966 is negative for LDC (Nielsen et al.,2001). One of the reasons that there are variablereports for LDC may be due to misinterpretation
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of the test, or the use of different methods todetect LDC. In the conventional tube test, LDC isa pale purple colour, particularly in comparisonwith the ADH tube. Also, a stronger result maybe obtained after 48 h incubation rather than at24 h incubation.
HG8 and HG10 can be separated by bio-chemical tests; they are genotypically identicaland are named A. veronii biogroup sobria and A.veronii biogroup veronii, respectively. The typestrain of the non-motile A. salmonicida belongs inHG3, even though HG3 corresponds mainly withthe phenospecies of A. hydrophila.
Aeromonas allosaccharophila and Aero-monas encheleia: The status of these speciesis controversial (Working group on Aeromonastaxonomy. International Journal of SystematicBacteriology, 1999, 49: 1946).
Aeromonas bestiarum was shown to bevirulent to the common carp in pathogenicitystudies (Kozinska et al., 2002).
A. caviae and A. veronii may cause 100%mortality in giant freshwater prawns when eitherorganism is present in high numbers; in the orderof 3.8 × 106 and 3.7 × 105 cells/g of body weight,respectively (Sung et al., 2000).
A. hydrophila is an opportunistic organismfollowing a primary stressor; however, the patho-genesis and virulence of this organism remainsunclear. A number of toxins are produced,including haemolysins, cytotoxins, enterotoxin, acytolytic haemolysin and aerolysin. Aerolysin isa channel-forming toxin that kills cells, is lethalto mice and possesses haemolytic and cytotoxicactivities. It is the main contributor to pathogenic-ity (Chopra et al., 1993). The aerolysin gene is notfound in non-haemolytic strains of A. hydrophila,non-haemolytic strains of A. caviae, or strains of A.sobria (Pollard et al., 1990).
A dominant A. hydrophila, which was nega-tive for the phenotypic tests of LDC and cellobiosefermentation, was found to be associated with dis-ease outbreaks in aquaculture in China (Nielsenet al., 2001). However, this is in contrast to otherfindings that have suggested that LDC-negativeisolates are less virulent than LDC-positiveisolates, where 78% (n = 23) of isolates fromdiseased mammals and 100% (n = 4) of isolatesfrom moribund fish were LDC-positive (Lallierand Higgins, 1988). Although the number of iso-lates tested from moribund fish was small (n = 4),strains of A. hydrophila in these cases were alsoindole-negative, did not produce enterotoxin,
and were negative for agglutination with 0.2%acriflavine.
Virulence studies in strains isolated fromwalking catfish suggested that there was a scale ofvirulence ranging from avirulent, weakly virulent,to strongly virulent. All virulent strains possessedhaemolysin genes, and were able to lyse red bloodcells of a wide variety of animal species. Themajority of avirulent or weakly virulent strainseither did not possess any of the three haemolysingenes tested (AHH1, AHH5, ASA) or had combi-nations of only one or two of the genes. Thesestrains were generally unable to lyse the redblood cells of as many animal species. Thevirulence strains were able to cause infection at adose rate of 104 CFU/ml, whereas avirulent strainsdid not cause disease at this dose (Angka et al.,1995).
A recent article reports a subgroup withinA. hydrophila HG1, and these are referred to asA. hydrophila ssp. dhakensis ssp. nov. Thesestrains were isolated from cases of diarrhoeain children from Bangladesh. They weredifferentiated from A. hydrophila ssp. hydrophilaby genetic tests and the following phenotypictests. A. hydrophila ssp. dhakensis is negative foracid production from L-arabinose, and negativefor utilization of methyl a-D-mannoside, L-fucose,and L-arabinose, whereas A. hydrophila ssp.hydrophila is positive for acid production fromL-arabinose and positive for utilization of methyla-D-mannoside, L-fucose, and L-arabinose (Huyset al., 2002b).
A. ichthiosmia is a later synonym ofAeromonas veronii (Huys et al., 2001).
Aeromonas sobria is now called Aeromonasveronii ssp. sobria. Some commercial systems stillrefer to it as A. sobria. Both terminologies arestated in the tables here.
Aeromonas trota is a junior synonym ofA. enteropelogenes. A. enteropelogenes waspublished first; however, because the nameA. trota is more widely used, this name hasbeen retained (Huys et al., 2002a).
Aeromonas veronii biovar sobria is beingretained so that there is continuity in the literatureand with biochemical identification schemes. Thisorganism was previously called A. sobria but isactually genetically A. veronii (Working groupon Aeromonas taxonomy, 1999, IJSB, 49:1946). This species has been shown to be non-pathogenic to the common carp when tested invirulence studies (Kozinska et al., 2002).
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Aeromonas salmonicida ssp. salmonicida
These strains are characterized by pigmentproduction and by being non-motile. Theybelong to Aeromonas DNA HG3. These virulentstrains are termed ‘typical’ A. salmonicida,possess an A-protein layer and autoagglutinate indistilled water. However, these phenotypic testsare not reliable indicators of the in vivo virulenceof A. salmonicida (Bernoth, 1990; Olivier, 1990).A. salmonicida ssp. salmonicida causes the dis-ease known as furunculosis, so called becauselesions in the dermis are seen as characteristicumbonate ulcers. The organism penetrates intothe underlying tissues and organs. Furunculosisaffects mainly salmonid fish.
The atypical Aeromonas salmonicida
The term ‘atypical’ is used to describe strains ofA. salmonicida that show variations in biochemi-cal reactions, may have slow growth, be slow toproduce pigment, be negative for oxidase, andthat may produce a variety of extracellular pro-teases. The pathogenicity of atypical strains showsgreat variability, and virulence mechanisms differfor those described for ‘typical’ Aeromonassalmonicida. An extracellular A-layer has beendetected, an extracellular metallo-protease anda different iron utilization mechanism has beendescribed for atypical strains compared to typicalstrains (Wiklund and Dalsgaard, 1998). Currentthought suggests that the skin ulceration seen inthe disease may be due to only one well-definedbiotype of atypical A. salmonicida, yet theseorganisms cannot be isolated consistently. Theorganisms are most commonly isolated in thehealing stage of the ulcer, suggesting thatthe infection is not lethal (Wiklund et al., 1999).
A number of subspecies have been named,including A. salmonicida ssp. masoucida, ssp.smithia, ssp. achromogenes, ssp. nova, the latterisolated from non-salmonid fish. The subspeciesof masoucida and smithia have not been reportedsince their initial isolation by Kimura (1969) andAustin et al. (1989). There are many strains ofA. salmonicida reported in the literature thatare atypical and do not fall into any of the above-mentioned subspecies (Wiklund and Dalsgaard,1998). There is some contention about the validityof retaining A salmonicida ssp. and research
continues in order to understand and enabledefinitive identification of this diverse species. Theatypical strains infect a wider variety of fish speciesthan does A. salmonicida ssp. salmonicida.
Alteromonas genus
The genus Alteromonas was divided intotwo genera: Pseudoalteromonas, containing11 species, and Alteromonas, which containsA. macleodii as the type strain. They grow onmarine agar 2216 (Difco).
Brucella spp.
Brucella species are small cocco-bacilli that areintracellular in the cells of mammals. Traditionallythey are classified according to host, as they aregenerally host-specific in their disease. Studiessuggest that the Brucella genus is monospecificand that only one species, Brucella melitensis,should be recognized in the genus. It is proposedthat the other six species be recognized as biovarsthus: B. melitensis biovar abortus, B. melitensisbiovar canis, B. melitensis biovar melitensis,B. melitensis biovar neotomae, B. melitensisbiovar ovis, and B. melitensis biovar suis (Vergeret al., 1985). However, it is practical to retain thecurrent species delineation to avoid confusion(Wayne et al., 1987).
Recent research into the Brucella speciesisolated from marine mammals indicates thatthe monospecific species theory does not applywhen these isolates are considered (Moreno et al.,2002). The Brucella strains isolated from marinemammals appear to be a distinct species based onIS711-based DNA fingerprinting (Bricker et al.,2000). The name Brucella maris, comprising threebiovars, was suggested for the isolates from seals,porpoises, dolphins, an otter and a whale (Jahanset al., 1997). PCR-restriction fragment lengthpolymorphism of two outer membrane proteingenes, omp2 and omp2b, indicates that the iso-lates from sea mammals are a separate group fromthose isolated from terrestrial mammals. The iso-lates from the sea mammals appear to be a moreheterogeneous group than those from terrestrialmammals and, therefore, instead of naming allaquatic isolates as B. maris, it is proposed that theisolates are named according to their host species
Aquatic Animal Species and Organism Relationship 77
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in a similar manner to terrestrial isolates. As yetthis has not been approved by the InternationalCommittee on Systematic Bacteriology. How-ever, the proposal is that the aquatic strains arenow named B. pinnipediae for strains isolatedfrom seals, and B. cetaceae for strains isolatedfrom cetaceans (whales, dolphins and porpoises)(Cloeckaert et al., 2001; Foster et al., 2002).
Carnobacterium spp.
Carnobacterium species can be differentiatedfrom Lactobacillus, as Carnobacterium speciesfail to grow on acetate agar (Rogosa medium,Oxoid; Rogosa et al., 1951). Discrepancies havebeen reported between conventional tube testsand the API 50CH system with results for inulin,lactose, sorbitol and galactose (Baya et al., 1991).The Carnobacterium species are phylogeneticallycloser to the genera Enterococcus and Vagococ-cus than to members of the genus Lactobacillus.
Carnobacterium (Lactobacillus) piscicola mayhave been reported as Corynebacterium spp.,Lactobacillus spp., Listeria spp. or Vagococcussalmoninarum in earlier reports in the literature.
Cytophaga spp.
This genus now contains only two cellulolyticspecies: C. hutchinsonii, the type species, andC. aurantiaca. Their closest relative is the genusSporocytophaga, containing S. myxococcoides,which is cellulolytic and produces microcysts(Bernardet et al., 1996).
Cytophaga aurantiacus, strain NCIMB 1382described by Lewin and Lounsbery (1969),is thought to be a Flavobacterium johnsoniae(Bernardet et al., 1996).
Edwardsiella tarda
This name is conserved for use, althoughEdwardsiella anguillimortifera is the more seniorsynonym. The organism can be found in theenvironment in pond water, lakes, rivers, soil andfrom the cloacae of crocodiles, lizards, seagulls,snakes, tortoises and waterfowl, and may be partof the normal flora or exist in a carrier status. It isa pathogen of many aquatic species including
catfish, trout, eels, and also causes enteritis inhumans. Because it is widespread in the environ-ment, the isolation of the organism from someaquatic animals may make it difficult to assess itspathogenic status. However, site of isolation, clin-ical signs, disease status and histopathologicalexamination should enable a correct diagnosis(Wallace et al., 1966; Otis and Behler, 1973;White et al., 1973; Miyashita, 1984; Humphreyet al., 1986; Reddacliff et al., 1996; Uhland et al.,2000).
Enterococcus spp.
Enterococcus faecalis was previously knownas Streptococcus liquefaciens and Streptococcusfaecalis.
Enterococcus seriolicida was previouslydescribed as Streptococcus species biotype 1 fromAustralian trout in Victoria and Tasmania, and isnow recognized as Lactococcus garvieae.
Enterovibrio norvegicus
This organism has recently been isolated andidentified to be a member of the Vibrionaceae. Itresembles a Vibrio, however, is resistant to thevibriostatic agent 0/129 but grows slowly after3 days on TCBS. It is part of the intestinal micro-flora of turbot larvae (Thompson et al., 2002a).
Flavobacterium/Flexibacter/Cytophaga
The Flavobacterium genus represents pre-dominantly gliding, yellow-pigmented bacteriathat belong to the Flexibacter–Bacteroides–Flavobacterium phylum. Flavobacterium is thetype genus of the Family Flavobacteriaceae, whichincludes the genera Bergeyella, Capnocytophaga,Chryseobacterium, Empedobacter, Gelidibacter,Myroides, Ornithobacterium, Polaribacter,Psychroflexus, Psychroserpens, Riemerella,Weeksella, and misclassified Cytophaga andFlexibacter species. Members of Flavobacteriumgenus are aerobic, Gram-negative rods, produceyellow-pigmented colonies, are motile by gliding,do not hydrolyse cellulose and are widely distrib-uted in soil and freshwater habitats. Some arepathogenic for fish (Bernardet et al., 1996).
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Flavobacterium columnare: Genetic diver-sity has been reported amongst strains of Flavo-bacterium columnare, which enables divisioninto three groups or genomovars. However, thephenotypic characteristics are identical for thethree genomovars and there is no evidence toclassify the groups as new species or subspecies(Triyanto and Wakabayashi, 1999). A genomovaris defined as phenotypically similar but geneticallydistinct (Ursing et al., 1995).
Flexibacter aurantiacus (Lewin and Louns-bery, 1969) strains NCIMB 1382 (previouslyCytophaga aurantiaca) and NCIMB 1455(Flexibacter psychrophilus) are now thought tobe Flavobacterium johnsoniae strains, and havebeen shown to be different from the bona fidestrains of Cytophaga aurantiaca and Flavobac-terium psychrophilum (Bernardet et al., 1996).
The Flexibacter genus contains one species,F. flexis. The only genuine members of theCytophaga genus are C. hutchinsonii (the typespecies), and C. aurantiaca (excluding strainsNCIMB 1382 and NCIMB 1455).
Hafnia alvei
Hafnia alvei is known to cross-react with Yersiniaruckeri antisera (Stevenson and Airdrie 1984;and personal observation).
Haemophilus piscium
Strains originally identified as H. pisciumhave been included with strains of Aeromonassalmonicida. Phenotypic evidence suggests thatthe strain is synonymous with A. salmonicida ssp.achromogenes, yet molecular studies involvingribotyping, RAPD and PCR grouping did notsupport this (Austin et al., 1998).
Helicobacter spp.
Since Helicobacter pylori was first isolated asa cause of gastric ulcer in humans (Marshalland Warren, 1984), the genus has expandedto include some 30 species isolated from thegastrointestinal tract of many different animals.The organisms are microaerophilic and the cellsare fusiform, curved or spiral, and possess flagella
in different numbers and location according tothe species.
Listonella spp.
There is contention in the scientific communityregarding the validity of placing V. anguillaruminto the genus Listonella as proposed byMacDonell and Colwell (1985). There is,however, agreement of the separation of V.anguillarum biotypes 1 and 2 into two species,as V. anguillarum and V. ordalii, respectively(Austin et al., 1997). In this book, V. anguillarumis referred to as Listonella anguillarum.
Listonella anguillarum
This pathogen causes disease in a wide range offish. It is of particular economic importance to thesalmonid industry, as salmonids are particularlysusceptible to disease when being transferredfrom freshwater to salt water.
Listonella anguillarum isolates can bedivided into ten serotypes (European serotypedesignation) based on the detection of the O anti-gen. Of these, only serotypes 01 and 02 have beenisolated from diseased fish, following outbreaks ata distribution rate of 70% and 15%, respectively.In 75% of vibriosis infections in feral fish, serotype02 is isolated. The other serotypes have onlybeen isolated from the environment (Sørensenand Larsen, 1986). Serotype 01 strains tend tobe arabinose-positive. Isolates from serotype 01are a homogeneous group, whereas isolates fromserotype 02 can be further divided into groups02a and 02b based on differences detected bydouble immunodiffusion, dot blot assay andenzyme-linked immunoabsorbant assay (Toranzoand Barja, 1990). Vibrio ordalii, previously classi-fied as L. anguillarum biotype II, cross-reacts withserotype 02 antiserum (Toranzo et al., 1987).
Listonella pelagia strain NCIMB and NCMB1900: Recent phylogenetic analysis of NCMB1900 has indicated that this strain is more likelyto be Vibrio natriegens, and that the originalstrain deposited at NCIMB may have beenlost. Phenotypic tests for strain NCIMB 1900corresponded to those expected for V. natriegens,which is positive for acid production fromL-arabinose, melibiose, and L-rhamnose, witha negative result for lactose fermentation andalginate degradation (Macián et al., 2000). The
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phenotypic results recorded as L. pelagia NCIMB1900 by Lunder et al. (2000) have been recordedas V. natriegens in this book.
Listonella ordalii = Vibrio ordalii
Both names are used in the literature, althoughVibrio ordalii is officially used in taxonomydatabases.
Moritella marina and Moritella viscosa
Moritella marina and Moritella viscosa are closelyrelated and have a sequence similarity of 99.1%based on 16S rDNA sequencing (Benediktsdóttiret al., 2000).
Mycoplasma spp.
Mycoplasma phocicerebrale is a legitimatecorrection to the previously reported name of M.phocacerebrale. The correction of Mycoplasmaphocae to M. phocidae is not legitimate (de Vosand Truper, 2000). Mycoplasma phocirhinis is alegitimate correction from M. phocarhinis.
Nocardia spp.
Members of the Nocardia produce branchedsubstrate hyphae that fragment in situ into rod-shaped to coccoid non-motile elements. Aerialhyphae are numerous and are always present butsome may only be seen microscopically. Hyphaeon a 3-day-old colony may only be 1 mm inlength.
Photobacterium spp.
The species that should be contained in thisgenus is still in dispute. There is agreementof some species rightfully belonging to the genus(P. angustum, P. phosphoreum, P. leiognathi).However, other species are in dispute, such as V.fischeri, V. logei and V. damselae (Lunder et al.,2000) although placing V. damselae into thegenus Photobacterium is generally agreed upon.
It is agreed that Photobacteriumdamselae ssp. piscicida belongs to the genus
Photobacterium rather than Pasteurella whereit was first placed. However, there is stillcontroversy as to whether P. damselae ssp.piscicida is a subspecies of P. damselae or aspecies in its own right (Gauthier et al., 1995b;Thyssen et al., 1998).
Photobacterium histaminum is a later sub-jective synonym of P. damselae ssp. damselae(Kimura et al., 2000).
Photobacterium fischeri and Vibrio fischeriare homotypic synonyms.
Providencia rettgeri and Proteus rettgeri
Providencia rettgeri and Proteus rettgeri arehomotypic synonyms, which means they sharethe same type strain (Brenner et al., 1978).
Pseudoalteromonas species
See also under Alteromonas. The genus Pseudo-alteromonas was formed to include most of thepreviously named Alteromonas species withthe exception of A. macleodii. The majorityof Pseudoalteromonas are associated witheukaryotic hosts and are frequently isolated frommarine waters. The majority grow on MA 2216and show optimal growth at 23°C.
Shewanella putrefaciens
Strain NCIMB 400 has been reclassified asShewanella frigidimarina.
Sphingobacterium spp.
Members of the Sphingobacterium genus have alow G+C content (39–42 mol %) and containsphingolipids.
Streptococcus spp.
Streptococcus agalactiae is a Lancefield group Bstreptococcus and is non-haemolytic. There arenine serogroups of group B streptococcus basedon their capsular polysaccharide.
80 Chapter 1
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Streptococcus dysgalactiae consists of twospecies. Streptococcus dysgalactiae ssp. dysga-lactiae strains are a- or non-haemolytic andare Lancefield group C. Strains belonging toStreptococcus dysgalactiae ssp. equisimilis areall b-haemolytic and may have the Lancefieldgroup C, G or L antigen (Vieira et al., 1998).
Streptococcus difficile has now beenidentified as Streptococcus agalactiae type Ib(Vandamme et al., 1997).
Vagococcus genus
The genus Vagococcus was proposed to accom-modate Gram-positive cocci that were motile andreacted with Lancefield group N antisera (Collinset al., 1989).
Vibrio spp.
Members of the Family Vibrionaceae areColwellia, Listonella, Moritella, Photobacteriumand Vibrio. Those isolated from fish grow well onordinary plate media.
Vibrio carchariae and V. harveyi: V.carchariae is a junior synonym of V. harveyiand the two organisms were indistinguishableby amplified fragment length polymorphisms,DNA : DNA hybridization, ribotyping (Pedersenet al., 1998) and 16S ribosomal DNA sequencing(Gauger and Gómez-Chiarri, 2002).
Vibrio cholerae: A biotyping scheme wasbased on the fermentation of sucrose, D-mannoseand L-arabinose. These biotypes are known asHeiberg types I–VIII. A classification scheme wassuggested where V. cholerae isolated from cholerapatients was classified as 01, and all otherstrains were classified as non-01. Non-01 strainscause gastroenteritis and septicaemia and otherinfections in humans, and have also been reportedto cause infections in terrestrial and aquaticanimals. There are two biotypes, El Tor andClassical; these cannot be differentiated sero-logically and belong to serovar 01. Serovar 01causes the classical cholera epidemics seen inhumans in some countries. Serotype 0139 Bengalis also an epidemic strain of cholera (Furniss et al.,1978; Albert et al., 1993).
Vibrio anguillarum: See notes on Listonellaanguillarum.
Vibrio natriegens: There are some reports inthe literature identifying strain NCIMB and NCMB1900 as Listonella pelagia. Sequencing has shownthat strain 1900 corresponds to Vibrio natriegens(Macián et al., 2000). See also notes underL. pelagia.
Vibrio trachuri: Isolated from Japanese horsemackerel (Trachurus japonicus) and identified asa new species of Vibrio (Iwamoto et al., 1995a).However, further testing has suggested that thisspecies is a junior synonym of Vibrio harveyi(Thompson et al., 2002b).
Vibrio vulnificus: Biotyping and serotyping ofVibrio vulnificus is a little confusing at present.Biotype 1 isolates are indole-positive and associ-ated with human infection, whereas biotype 2isolates are indole-negative and associated withinfection in eels (Tison et al., 1982). However, inDenmark and Sweden 85% of biotype 2 isolatesshowing pathogenicity for eels are indole-positive(Høi et al., 1998b). Biotype 2 strains are serovar Eand these can be further divided into 03 and 04based on LPS-O antigen. Some biotype 2, serovarE, strains from Taiwan have been found tobe avirulent for eels. These strains are positivefor mannitol fermentation (Amaro et al., 1999).Vibrio vulnificus biotype 3 has been put forwardby the Centre for Disease Control, USA, as apossible identification for Vibrio species isolatedfrom wound infections in humans in Israel(Bisharat et al., 1999). It is different from currentlyrecognized V. vulnificus species because ofnegative reactions in cellobiose, citrate, lactose,salicin and ONPG. It is positive by PCR for theV. vulnificus haemolysin gene (Bisharat et al.,1999; Nair and Holmes, 1999). In the conven-tional biochemical identification tables and theAPI 20E tables, the various V. vulnificus strainsand their different reactions are listed so as to assistthe microbiologist when making identification.
Vibrio wodanis has a sequence similarity of98.8% with Vibrio logei strain CIP 103204 basedon 16S rRNA sequencing (Benediktsdóttir et al.,2000).
Yersinia spp.
Yersinia enterocolitica: Y. enterocolitica wasdivided into the subspecies Y. enterocoliticassp. enterocolitica and Y. enterocolitica ssp. pale-arctica. Yersinia mollaretii and Y. bercovieri were
Aquatic Animal Species and Organism Relationship 81
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formerly Y. enterocolitica biogroups 3A and 3B,respectively (Wauters et al., 1988).
Yersinia ruckeri: An original serotypingscheme identified six serovars (Hagermanserovars). Most naturally occurring outbreaks indiseased fish and rainbow trout are caused byserovar I called the Hagerman strain (McCarthyand Johnson, 1982). Some Australian isolates ofY. ruckeri that cross-reacted with serovar I weretermed serovar I′. Serovar II may be significantwhen it is associated with ERM in chinook salmon(Cipriano et al., 1986). Serovar II and serovar Visolates are sorbitol-positive. Serovar III is foundin Australia. Initially it was suggested that sorbitolfermentation was associated with pathogenicity;however, this is not a reliable indicator of virulence(Stevenson and Airdrie 1984; Cipriano et al.,1986).
More recently it has been shown that Y.ruckeri can be divided into clonal groups based onbiotype, serotype and outer membrane proteins,and that clonal groups may relate to virulenceof the organism. There are two biotypes basedon hydrolysis of Tween 20 and Tween 80, andmotility. Biotype 2 strains are non-motile, and donot hydrolyse Tween 80 (Davies and Frerichs,1989). A serotyping scheme based on heat-stableO-antigens identified serotypes 01, 02, 05, 06 and07 in Europe, North America, Australia and South
Africa (Davies, 1990). All serotypes occur inEurope and North America, whereas in Australiaand South Africa only serotype 01 has beenidentified. This study suggested that the Australianisolate described as serotype III was serotype 01by their scheme. Five outer membrane proteinprofiles were identified, which enabled differentia-tion of isolates within a serotype. Serotype 01consisted of six clonal groups. Clone 2 was foundin disease outbreaks in the UK and clone 5 wasfound in disease outbreaks in mainland Europe,North America and South Africa. Clones 3, 4 and6 do not appear to be associated with diseaseoutbreaks. Clones 1 and 3 were found fromAustralian isolates, with clone 3 also being foundin Finland, France, West Germany and the USA.Clone 4 was from Norwegian isolates, and clone 6from isolates in Finland, France, Norway andCanada (Davies, 1991).
Hafnia alvei is known to cross-react withYersinia ruckeri antisera (Stevenson and Daly,1982; Stevenson and Airdrie 1984; and personalobservation).
ERM outbreaks caused by Yersinia ruckeriare generally a result of poor environmentalconditions, resulting in low oxygen level, highwater temperature and poor water quality, allof which stress the fish, making them moresusceptible to disease.
82 Chapter 1
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2Bacteriological Culture Techniques: Microscopy,
Culture and Identification
Successful isolation and accurate identificationof a suspected pathogen depends upon the useof standardized bacteriological culture methods.It is important to work through these methodsin a precise and logical step-by-step manner.Basically the bacteria are grown on culture
media, then inoculated into biochemical identifi-cation tests and those results are recorded andcompared to standard results. Table 2.1 sets outthe steps for the isolation and identification of anorganism and also directs the reader to the loca-tion of the technique or method in this manual.
©N.B. Buller 2004. Bacteria from Fish and Other Aquatic Animals:a Practical Identification Manual (N.B. Buller) 83
Day Activity Method or technique
Day 1 1. Sample collection and preparation2. Inoculation of sample to primary isolation plate media (or broths whereappropriate)3. Incubate at appropriate temperature and atmosphere
2.12.2, Table 2.2, Table 2.3
2.2, Table 2.2, Table 2.3
Day 2(24 h)
1. Examine culture plates2. Select suspect colonies and subculture to BA or MSA-B to obtain puregrowth (secondary plates)3. Re-incubate primary plates4. Incubate all plates at appropriate temperature and atmosphere as before
2.3Table 2.4 for cultural and microscopicappearance2.2, Table 2.2 and 2.3
Day 3(48 h)
1. Re-examine primary plates for slow-growing pathogens2. Check that subcultures on secondary plates are pure3. Perform primary identification tests4. Inoculate appropriate biochemical identification set5. Re-incubate primary plates as before
2.3
2.4, Chapter 3, and media (Chapter 7)2.5, 2.62.2, Table 2.2 and 2.3
Day 4(72 h)
1. Re-examine primary plates for growth of slow-growing pathogens.Re-incubate if disease suggests a pathogen that requires more than 3 daysfor growth2. Examine biochemical identification set/s and record results at 24 hincubation
2.3
Table 3.1
Day 5(96 h)
1. Examine biochemical identification set/s and record results for 48 hincubation. Add reagents for tests for indole, methyl red, nitrate,Voges-Proskaüer2. Interpret results from appropriate identification table
Table 3.1Chapter 3 and Tables 4.1 to 4.22 forbiochemical results (Biochem set), andTables 4.23 to 4.31 for results for API kits
BA, blood agar; MSA-B, marine salt agar.
Table 2.1. Outline of steps for culture and identification.
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2.1 Specimen Collection and Submission
Samples should be collected from live sickanimals or from recently dead animals. Animalsthat have been dead for more than 6 h usuallyhave grossly contaminated organs due to theovergrowth of post-mortem invading bacteria. Itis very difficult for the laboratory to work withthese samples. Unfortunately, it is not alwayspossible to get the samples to the laboratorywithin the ideal time, due to large distancesbetween some laboratories and the aquaculturefarm.
Ideally all samples for bacteriologicalexamination should be collected before the start ofantimicrobial treatment. Samples collected fromaquatic animal species after commencement oftreatment are unlikely to grow any pathogenicbacteria.
Samples may be submitted as swabs, tissuesor whole fish. The most appropriate samples forthe identification of a systemic disease may bekidney, haemolymph or blood. For skin disease,swabs or samples of skin are appropriate. Referto Tables 1.1 and 1.2 for tissue sites where thebacteria are likely to be located.
Transport medium and transport tothe laboratory
If swabs have been collected, then they mustbe placed into a transport medium to preventthe swab drying out and for the organisms toremain viable. Amies transport medium isthe medium of choice for transport of swabsto the laboratory.
Fish, tissues and swabs in Amies transportmedium should be transported to the laboratoryas soon as possible after collection, and preferablyon ice or at 4°C.
Stuarts medium may be unsuitable for trans-port and survival of Vibrio species. Numbers ofviable cells of Vibrio alginolyticus, Vibrio harveyiand Photobacterium damselae ssp. damselaewere greatly reduced after 24 h and were almostnon-viable at 48 and 72 h when tested fromStuarts media kept at 4°C. However, there was noreduction of viable cells when all three organismswere tested from Amies transport media heldat 4°C for 24, 48 and 72 h (H. McLetchie andN. Buller, unpublished).
Sample preparation
Swabs
Swabs require no sample preparation, and areinoculated directly to the appropriate media.
Tissues
Aseptically remove a piece of infected tissuefrom the fish and place into a McCartney bottle.Macerate the tissue with flame-sterilized scissors,and use a sterile, cotton-tipped swab to inoculateto appropriate media. Tissues that have not beenaseptically removed from the fish need to besurface-sterilized. This can be done either byrinsing the piece of tissue in 3–4 washes of sterilewater or saline, or for larger pieces of tissue(walnut-sized), dip in 70% alcohol and flamebriefly for 3–5 s. The tissue is then placed into asterile McCartney bottle or appropriate containerand macerated with flame-sterilized scissors. Asterile, cotton-tipped swab is used to sample thetissue and inoculate to appropriate agar plates,and to make smears for Gram stain.
Lesions
Always collect material from the edge of thelesion, including some of the immediate tissue,to ensure sampling for true invading bacteria.Sampling from the middle of the lesion mayculture secondary invading bacteria that willconfuse the results. Also, the invading bacteriummay no longer be viable at the centre of thelesion.
Whole fish
For small fish, the whole fish is surface-sterilizedby rinsing the fish in 3–4 changes of steriledistilled water or normal saline. For larger fish,surface-sterilize by either flaming with 70%alcohol or washing the area with 3–4 washesof sterile distilled water or normal saline. Usingflamed-sterilized forceps and scissors, make anincision from behind the gills and to the mid-line.Next cut along the mid-line towards the anus. Liftback the flap of flesh to expose the internalorgans. Select the required organs for analysisand remove with flame-sterilized forceps andscissors. Chop tissues as before and inoculateto plates.
84 Chapter 2
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2.2 Culture and Incubation
Culture media
All specimens should be cultured to a general-purpose medium. For freshwater specimens useBA and MCA. TSA can also be used; however,the addition of blood can improve the isolation ofsome organisms such as Aeromonas salmonicida,Carnobacterium and Streptococci. For marinespecimens, use a medium containing Na ions,BA, and MCA. Marine salt agar (MSA-B), whichis prepared from TSA with 5% horse blood and2% NaCl (see Media section), is a good general-purpose medium for marine organisms. Marine2216, commercially available from Difco, andbased on ZoBell’s (1941) original formula, is alsosuitable. MSA-B may give improved growth,particularly on primary culture with commonlyisolated Vibrio species. MA 2216 is more suitablewhen a complex mixture of electrolytes isrequired by some marine organisms.
Isolation and growth conditions: media,temperature and time of incubation
All specimens should be inoculated to thegeneral-purpose medium. This medium is thenincubated at 25°C for 2–5 days as indicated inTable 2.2 for general culture conditions. How-ever, specimens from a particular environmentsuch as a cold water environment or a marineenvironment may need a particular incubationtemperature or NaCl may be required in themedium. These variations are listed in Table 2.3.
Some organisms have special growth require-ments or may be better detected with the use of aselective and/or enrichment medium if they areavailable for that organism. Such requirements forspecific organisms are detailed in Table 2.3.
The preparation of all media mentioned inTables 2.2 and 2.3 is listed in Chapter 7.
2.3 Examination of Culture Plates
Many bacteria from fish and aquatic animalsneed 48 h before colonies appear, or before theyare of a suitable size for examination and subcul-ture. At 24 h incubation, the colony appearanceof different Vibrio species may not be distinguish-able; however, at 48 h incubation, the differencesin physical appearance between different speciesmay be more apparent. The addition of bloodinto a medium such as MSA-B, for Vibrio species,improves the ability to differentiate between thecolonies of different species, and to determinehaemolysis. Generally, a bacterium that is pres-ent in a moderate to heavy growth or as thepredominant organism will be significant. Bear inmind the age of the samples at the time they werereceived at the laboratory. Specimens older than24 h from the collection date may be overgrownwith commensal bacteria.
Select appropriate colonies for subculture.These may be given an individual number (e.g.#1, #2, etc). For example, selected colonies arecircled and numbered on the underside of thePetri dish using a felt pen, and then picked off andsubcultured to fresh plates. Freshwater samplesare subcultured to BA, MCA and TCBS (the latterfor suspect Vibrio species). Samples from marinesources are subcultured to BA, MCA, MSA-B andTCBS. Organisms that require special media forgrowth are subcultured to the appropriate media(see Table 2.3). Some organisms from marineenvironments may need to be cultured to MA2216 media. Incubate a further 24 h or as requiredfor sufficient growth. Pure subculture growthsare used for the inoculation of biochemicalidentification tests.
Microscopic and cultural characteristics
Table 2.4 details microscopic and colony appear-ance, plus the results of the primary tests of Gram
Bacteriological Culture Techniques 85
Fish, aquatic animals Media – freshwater animals Media – salt water animals Temperature optimum (range)
All specimens, tissues BA, MCA MSA-B, BA, MCA 20–25°C, 2–5 daysCold water BA, MCA MSA-B, BA, MCA 15–22°C, 3–5 daysTropical BA, MCA MSA-B, BA, MCA 25°C, 2–5 days
BA, blood agar; MCA, MacConkey agar; MSA-B, marine salt agar containing blood.
Table 2.2. General culture.
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86 Chapter 2
Abio
troph
iaba
laen
opte
rae
BA37
°C,5
%C
O2
orai
r,24
–48
h
Actin
omyc
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r 5%
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–72
h
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with
eith
er2.
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g/l
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ater
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d
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,3–7
days
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usvi
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mar
iN
A+5%
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d,TS
A,BA
,BH
IBSI
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lect
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4–48
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28°C
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days
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asu
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oved
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gBA
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NA
even
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,24–
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–4da
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25°C
,24–
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etel
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onch
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tica
BA,C
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8–72
h
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ella
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Use
biol
ogic
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net
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BA,F
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M)
Isol
ates
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ther
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ter
14da
ys
37°C
,5–1
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and
otte
rs,a
ndso
me
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ates
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spec
ies
requ
ire10
%C
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pseu
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ebi
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bact
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mpi
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BA,T
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15–2
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,48–
72h
Chr
yseo
bact
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bact
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m)
men
ingo
sept
icum
TSA
30–3
7°C
,48
h
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edia
forf
resh
wat
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imal
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edia
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altw
ater
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als
Tem
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Tab
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Bacteriological Culture Techniques 87
Chr
yseo
bact
eriu
msc
opht
halm
umTS
A,AO
Med
ium
K,M
A22
16,A
O-M
25°C
,48
h
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strid
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Anae
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Apl
ates
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ntifi
edby
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ing
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oxin
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uctio
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teri
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toR
ober
tson
’sco
oked
mea
tbr
oth
Incu
bate
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hsfo
r6da
ysat
30°C
pri o
rto
test
ing
fort
oxic
ity
Cor
yneb
acte
rium
aqua
ticum
BA,T
SA,B
HIA
25°C
,48–
72h
Cyt
opha
gahu
tchi
nson
iiD
ubos
med
ium
supp
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ente
dw
ith30
%(w
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ello
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e22
°C,4
8–72
h
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opha
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ine
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69)
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e25
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mat
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°C,2
–3da
ys
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iella
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Stra
ins
with
limite
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lera
nce
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been
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ated
(Mitc
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Edw
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iella
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25–2
8°C
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days
Empe
doba
cter
(Fla
voba
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ium
)bre
vis
TSA
30°C
,2–3
days
Ente
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ccus
spp.
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SA,B
HIA
25°C
,48–
72h
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brio
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NA
and
TSA
supp
lem
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dw
ith1.
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8h.
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ates
at24
and
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ium
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,TSA
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days
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inue
d
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88 Chapter 2
Flav
obac
teriu
mgi
llisia
e,F .
tege
tinco
la,F
.xan
thum
NA,
TSA,
R2A
MA
2216
20°C
,2–7
days
Flex
ibac
ters
pp.–
mar
ine
orig
inAO
-M(A
0m
ediu
mpr
epar
edw
ithar
tific
ials
eaw
ater
( Si g
ma)
at38
g/l )
25°C
,2–7
days
Flex
ibac
terp
olym
orph
usFl
exib
acte
rmed
ium
ofLe
win
(197
4)30
°C,2
–7da
ys
Flex
ibac
terr
oseo
lus
NA
25°C
,2–7
days
Flex
ibac
terr
uber
NA
25°C
,2–7
days
Hal
omon
asel
onga
taTS
A+8%
salt
30°C
,24–
48h
Hel
icob
acte
rspp
.TS
A+
5%bl
ood,
BA,B
ruce
llaag
ar,S
kirro
w’s
med
ium
(VPT
)H
omog
eniz
est
omac
htis
sue
in1
mlB
ruce
llabr
oth
(Difc
o)co
ntai
ning
5%fa
ecal
calf
seru
m.P
lace
100
mlto
p la t
e san
d/or
filte
rthr
ough
a0.
45or
0.8
mmfil
terb
efor
ein
ocul
atin
gto
plat
es(B
utzl
eret
al.,
1973
;Har
pere
tal.,
2000
)
37°C
mic
roae
roph
ilicat
mos
pher
eof
N2,
H2,
CO
2(8
0:10
:10)
for2
–4w
eeks
Com
mer
cial
syst
ems
are
avai
labl
efo
rge
nera
tion
ofco
rrect
atm
osph
ere
(e.g
.M
GC
Anae
roPa
k ,C
ampy
lofro
mM
itsub
ishi
Gas
Che
mic
alC
ompa
ny)
Lact
obac
illus
spp.
,Lac
toco
ccus
spp.
BA,T
SA,B
HIA
25–3
0°C
,48–
72h
List
onel
laan
guilla
rum
TSA
+1%
NaC
l,M
SA-B
,MA
2216
,VAM
22°C
,24–
48h
Mor
itella
visc
osa
TSA
+2%
NaC
l15
°C,4
–9da
ys
Myc
obac
teriu
mab
sces
sus
Mid
dleb
rook
7H10
-AD
Cm
ediu
m25
°C,7
–28
days
Myc
obac
teriu
mch
elon
aeBH
IA,T
SA,A
OM
SA-B
15–2
2°C
,5w
eeks
.(gr
owth
isus
ually
seen
with
in7
days
)
Myc
obac
teriu
mm
arin
um,
M.s
crof
ulac
eum
,M.i
ntra
cellu
lare
,M
.for
tuitu
m
BA,L
owen
stei
n-Je
nsen
(BBL
)20
–23°
C,7
–14
days
Will
grow
in5–
7da
ysat
room
tem
pera
ture
onbo
vine
BA
Myc
obac
teriu
mne
oaur
umBA
25°C
,3–4
days
Myc
obac
teriu
mpe
regr
inum
BA,M
iddl
ebro
ok7H
11ag
ar(D
ifco)
37°C
inC
O2,
4–7
days
Myc
opla
sma
spec
ies
Myc
opla
sma
agar
and
Myc
opla
sma
brot
h37
°Cin
CO
2.Su
bcul
ture
brot
hto
agar
med
ium
ever
y3–
4da
ysfo
r2w
eeks
.Ex
amin
epl
ates
at3–
4da
yin
terv
als
Myr
oide
s(F
lavo
bact
eriu
m)o
dora
tus
TSA
30°C
,48–
72h
Bact
eriu
mM
edia
forf
resh
wat
eran
imal
sM
edia
fors
altw
ater
anim
als
Tem
pera
ture
,atm
osph
ere,
time
Tab
le2.
3.C
ontin
ued.
104Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:36 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
Bacteriological Culture Techniques 89
Noc
ardi
asp
p.-g
ener
al( N
.ast
eroi
des,
N.b
rasi
liens
is,
N.n
ova,
N.p
seud
obra
silie
nsis
,N
.otit
idis
cavi
arum
,N.s
erio
lae,
N.v
acci
nii)
BA25
–30 °
C,1
4da
ys.C
olon
ies
begi
nto
appe
araf
ter5
days
Noc
ardi
acr
asso
stre
aeBH
IABH
IA+
1%N
aCl
28°C
,14
days
Noc
ardi
ase
riola
eBA
,BH
IA,L
owen
stei
n-Je
nsen
med
ium
25–3
7°C
for7
–30
days
Past
eure
llask
yens
isTS
A+
bloo
d+
1.5%
NaC
l.M
A22
16+
bloo
d(n
ogr
owth
with
outb
lood
or1.
5%N
aCl)
22–3
0°C
,in
air,
48h
Pedo
bact
erhe
parin
us,P
.pis
cium
NA,
PY28
°C,4
8h
Phot
obac
teriu
mda
mse
lae
ssp.
dam
sela
eBA
MSA
-B,T
SA+
2%N
aCl,
MA
2216
22–2
5°C
,24–
48h
Phot
obac
teriu
mda
mse
lae
ssp.
pisc
icid
aN
Bw
ith2–
3%N
aCl,
BHIA
with
2%N
aCl,
MA
2216
,or
MSA
-B22
°C,2
4–48
h
Pseu
doal
tero
mon
assp
p.M
A22
1615
–30°
C,1
–5da
ys
Pseu
doal
tero
mon
asan
tarc
tica
TSA,
TSB
MA
2216
,MSA
-B15
°C,5
days
Pseu
doal
tero
mon
asm
aric
alor
isTS
AM
A22
1625
–35°
C,5
days
Pseu
dom
onas
angu
illise
ptic
aBA
,NA,
TSA,
BHIA
.No
grow
thon
Pseu
dom
onas
isol
atio
nag
ar(D
ifco)
20–2
5°C
,7da
ys
Pseu
dom
onas
fluor
esce
nsBA
,Pse
udom
onas
sele
ctiv
eag
ar(B
ecto
n,D
icki
nson
Co)
10–2
5°C
,2–5
days
Ren
ibac
teriu
msa
lmon
inar
umKD
M2,
KDM
C,S
KDM
15–1
8°C
,20–
30da
ys
Ros
eoba
cter
stra
inC
VSP
SWT,
MA
2216
23°C
,5–7
days
Sale
gent
ibac
ters
aleg
ens
MA
2216
,NA,
TSA,
R2A
20°C
,2–7
days
Shew
anel
labe
nthi
ca,S
.col
wel
liana
,S.
hane
dai,
S.ge
lidim
arin
a,S.
peal
eana
,S.w
oody
i
MA
2216
,MSA
-B25
°C,4
8–72
h
Shew
anel
la(A
ltero
mon
as)h
aned
aiTS
A+
0.05
g/ly
east
extra
ct,M
A22
1615
°C,4
8–72
h
Sphi
ngob
acte
rium
(Fla
voba
cter
ium
)m
ultiv
orum
,S.s
pirit
ivor
umN
A,TS
A,PY
28–3
0°C
,48
h
Stap
hylo
cocc
uslu
trae
BA37
°C,2
4h
Stap
hylo
cocc
usw
arne
riBA
,TSA
22–2
5°C
,48–
72h
cont
inue
d
105Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:36 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
90 Chapter 2
Stap
pia
stel
lula
ta-li
kest
rain
M1
SWT,
MA
2216
23–2
5°C
,2–7
days
Stre
ptob
acillu
sm
onilif
orm
isBA
.Req
uire
sad
ditio
nof
20%
seru
mto
brot
hfo
rgro
wth
25–3
7°C
,5–7
days
Stre
ptoc
occu
sin
iae
BA25
°C,2
4–48
h
Stre
ptoc
occu
spa
raub
eris
BA,T
SA22
–37°
C,2
4h
Tena
ciba
culu
m(F
lexi
bact
er)m
ariti
mum
AOm
edia
cont
aini
ng30
%se
awat
er,A
Oco
ntai
ning
ASW
at38
g/l.
Will
notg
row
onAO
with
NaC
lalo
ne.
NaC
land
KCli
ons
requ
ired
forg
row
th.C
a2+, M
g2+
enha
nce
grow
th.I
sola
tion
ofor
gani
smm
aybe
impr
oved
bydi
lutin
gsa
mpl
em
ater
iali
nar
tific
ial
seaw
ater
and
cultu
ring
dilu
tions
toAO
+AS
Wm
ediu
m(O
stla
ndet
al.,
1999
).TY
G-M
,HSM
25°C
for2
–5da
ys(ra
nge
15–3
4°C
)
Tena
ciba
culu
m(F
lexi
bact
er)o
voly
ticum
Asfo
rT.m
ariti
mum
,MA
2216
19°C
,2–5
days
Vago
cocc
ussa
lmon
inar
umBA
,TSA
22–2
5°C
,48
h
Varra
calb
mi
MSA
-B4–
22°C
(opt
imum
15°C
),48
h
Vibr
ioag
ariv
oran
sM
SA-B
,MA
2216
25°C
,48
h
Vibr
ioco
rallii
lytic
usM
A22
16,T
SA+
2%N
aCl
30°C
,24–
48h
Vibr
ioha
liotic
oli
MA
2216
with
orw
ithou
t0.5
%so
dium
algi
nate
25°C
,24–
48h
Vibr
ioho
llisae
TSA
+1%
NaC
l,M
SA-B
,MA
2216
25°C
,37°
C,2
4–48
h
Vibr
iom
imic
usBA
,TSA
MSA
-B25
°C,3
7°C
,24–
48h
Vibr
ioor
dalii,
Vibr
iopa
raha
emol
ytic
usTS
A+
1%N
aCl,
MSA
-B,M
A22
1622
°C,2
4–48
h
Vibr
iosa
lmon
icid
aN
A+
5%bl
ood,
TSA
+1.
5%N
aCl,
MSA
-B15
°C,3
–5da
ys
Vibr
iovu
lnifi
cus
BAC
ello
bios
e-po
lym
yxin
Bco
listin
agar
(CPC
)se
lect
ive
agar
,CC
A,VV
M,M
A22
16,M
SA-B
,TSA
orN
Aw
ith0.
5%(w
/v)N
aCl.
AV.
vuln
ificu
s-sp
ecifi
cpr
obe
isav
aila
ble
25°C
,24
h
Yers
inia
ruck
eri
TSA,
BA,M
CA,
XLD
(Yer
sini
ase
lect
ive
agar
,(YS
A,O
xoid
)is
nots
uita
ble
for
Y.ru
cker
i)
25°C
,24–
48h
Not
eth
atm
otilit
yan
dci
trate
will
bene
gativ
eat
37°C
butp
ositi
veat
25°C
Bact
eriu
mM
edia
forf
resh
wat
eran
imal
sM
edia
fors
altw
ater
anim
als
Tem
pera
ture
,atm
osph
ere,
time
Tab
le2.
3.C
ontin
ued.
106Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:37 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
Bacteriological Culture Techniques 91
Acin
etob
acte
rspp
.N
egPr
edom
inan
tlydi
ploc
occa
lfor
ms
1.0
×0.
7mm
from
plat
egr
owth
–C
olon
ies
circ
ular
,sm
ooth
,tra
nslu
cent
tosl
ight
lyop
aque
,but
yrou
sto
muc
oid
non-
pigm
ente
d.0.
5–2
mm
at24
hat
30°C
+Bi
oche
mse
t
Acin
etob
acte
rha
emol
ytic
usN
egPr
edom
inan
tlydi
ploc
occa
lfor
ms
1.0
×0.
7mm
from
plat
egr
owth
bHC
olon
ies
onTS
Aar
eci
rcul
ar,c
onve
x,sm
ooth
,slig
htly
opaq
uean
dm
ayha
vea
stic
kyco
nsis
tenc
y
+−
Bioc
hem
set
Actin
obac
illus
delp
hini
cola
Neg
Pleo
mor
phic
rods
–w
NG
Col
onie
son
BAin
10%
CO
2ar
e0.
75–1
mm
at24
h,ro
und,
smoo
thgr
ey.B
lood
orse
rum
enha
nces
grow
th.G
row
that
42bu
tnot
at22
°C
−+
Bioc
hem
set,
API2
0E,A
PI20
NE,
API-Z
YM.U
sea
heav
yin
ocul
um
Actin
obac
illus
scot
iae
Neg
Pleo
mor
phic
rods
–N
GC
olon
ies
0.5
mm
roun
d,gr
ey,o
nBA
at24
h.R
equi
res
10%
CO
2.M
aybe
wea
kly
haem
olyt
icon
shee
pbl
ood
−+
Bioc
hem
set,
API2
0E
Actin
omyc
esm
arim
amm
aliu
mPo
sSt
raig
htor
slig
htly
curv
edro
ds,s
ome
bran
chin
g–
On
BA,c
olon
ies
are
0.5
mm
grey
,ent
ire,
conv
exat
48h.
Gro
wth
at37
°Cin
a irw
ith5 %
CO
2
−AP
ICor
yne,
APIr
apid
ID32
Stre
p
Aequ
oriv
itasp
p.N
egR
ods
0.5–
20×
0.2–
0.3
mmO
nM
A22
16,y
ello
wor
oran
geco
loni
es,
com
pact
,circ
ular
,con
vex,
smoo
thw
ithan
entir
eed
ge,n
on-s
prea
ding
,but
yrou
sco
nsis
tenc
y.G
row
thon
NA
+2.
5%N
aCl
Bioc
hem
set,
API2
0E,
API-Z
YM(s
ome
reac
tions
)
Aero
cocc
usvi
ridan
sva
r.ho
mar
iPo
sTe
trad-
form
ing
cocc
usa
NG
aH
o nB A
.Ex a
min
ew
ithIn
d ia
ink
for
enca
psul
ated
cocc
i+
Bioc
hem
set,
APIr
apid
ID32
Stre
p
Aero
mon
ascu
licic
ola
Neg
Rod
On
BAco
loni
essm
all,
grey
,met
allic
shee
nan
db
haem
olyt
icat
24h
++
Bioc
hem
set,
API2
0E,A
PI50
CH
Aero
mon
asen
chel
eia
Neg
Stra
ight
rods
bHN
GN
on-p
igm
ente
dco
loni
eson
TSA
at24
h+
+Bi
oche
mse
t
Aero
mon
aseu
cren
ophi
laN
egSt
raig
htro
dsN
G+
+Bi
oche
mse
t
Aero
mon
ashy
drop
hila
Neg
Smal
lrod
sbH
Wea
k3–
5m
msh
iny,
crea
m,b
ecom
ing
pale
brow
n/gr
een
with
age
++
Bioc
hem
set,
API5
0CH
,API
20E,
API2
0NE,
API-Z
YM
Bact
eriu
mG
ram
Mor
phol
ogy
bHTC
BSCo
lony
char
acte
ristic
sCa
tO
xId
entif
icat
ion
sets
Tab
le2.
4.M
icro
scop
ican
dcu
ltura
lcha
ract
eris
tics.
cont
inue
d
107Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:37 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
92 Chapter 2
Aero
mon
asm
edia
Neg
Rod
s1
×2
mmN
GO
nTS
A,cr
eam
,shi
ny,s
moo
th,r
ound
,rai
sed,
2m
maf
t er 2
days
at22
°C.A
diffu
sibl
ebr
own
pigm
enti
spr
oduc
ed
++
Bioc
hem
set
Aero
mon
assp
p.N
egR
ods
1–3
mmbH
Wea
kgr
owth
,ye
llow
2–3
mm
,gre
y,cr
eam
,shi
ny,r
ound
onBA
and
TSA
++
Bioc
hem
seto
rAPI
20E
Aero
mon
assa
lmon
icid
ass
p.sa
lmon
icid
a
Neg
Smal
lrod
sto
cocc
o-ba
cilli,
1–2
mmv
NG
Brow
ndi
ffusi
ble
pigm
ento
nFA
orTS
A.R
ough
and
smoo
thph
ase,
colo
nies
,0.
5–3
mm
at72
h.N
ogr
owth
at37
°C.F
Ais
pref
erre
dfo
rpig
men
tpro
duct
ion
++
Bioc
hem
set.
API2
0Egi
ves
varia
ble
resu
ltsat
25°C
,im
prov
edre
sults
at30
°C.
FAT
and
aggl
utin
atio
nte
stav
aila
ble
Aero
mon
assa
lmon
icid
ass
p.no
va
Neg
Coc
co-b
acilli
tosm
allr
ods
bHN
GO
nBA
,slo
wgr
owin
g,fri
able
colo
ny,0
.5m
mre
achi
ng4–
8m
mon
day
8.C
olon
ysl
ides
acro
sspl
ate
whe
npu
shed
with
loop
.Pr
onou
nced
zone
ofha
emol
ysis
,sim
ilar
toa
grou
pC
Stre
p.O
nTS
Aa
brow
n-w
ater
-sol
uble
pigm
enti
sno
tsee
nun
tilda
y8
at25
°C.N
ogr
owth
at37
°C
++ v
Bioc
hem
set,
API2
0E,
API-Z
YM.F
ATav
aila
ble,
may
cros
s-re
actw
ithAe
rom
onas
sobr
ia
Aero
mon
assa
lmon
icid
ass
p.pe
ctin
olyt
ica
Neg
Stra
ight
rods
sing
ly,i
npa
irsor
shor
tch
ains
bHN
DC
olon
ies
deve
lop
in24
hat
35°C
o nB A
a nd
TSA.
Prod
uctio
nof
brow
ndi
ffusi
ble
pigm
ent
++
Bioc
hem
set
Aero
mon
asve
roni
iN
egbH
NG
Col
onie
s2
mm
,bH
onBA
at24
h+
+Bi
oche
mse
t,AP
I20E
Allo
mon
asen
teric
aN
egSt
raig
htor
slig
htly
curv
edro
dsG
row
thon
med
iaco
ntai
ning
3–5%
NaC
l.Li
ght
brow
nno
n-di
ffusi
ngpi
gmen
tafte
r2–5
days
at25
or37
°C
++
Bioc
hem
set
Alte
rom
onas
spp.
Neg
Rod
sN
GG
row
thon
MA
2216
assm
allp
ale
yello
wco
loni
es+
+,v
Bioc
hem
set
Arca
noba
cter
ium
phoc
aePo
sC
occo
-bac
illian
dsh
ortr
ods,
sing
ly,
pairs
and
clus
ters
.Non
-aci
dfa
stbH
Col
onie
son
BAat
24h,
whi
te,t
iny,
roun
d,w
ithla
rge
zone
ofbH
vBi
oche
mse
t,AP
I50C
H,
API-Z
YM
Arca
noba
cter
ium
plur
anim
aliu
mPo
sSt
raig
htto
slig
htly
curv
ed,
non-
bran
chin
g,sl
ende
rrod
sa
Ha
haem
olys
ison
BA,s
mal
lcol
onie
s+
APIC
oryn
e,AP
Irap
idID
32St
rep
Arca
noba
cter
ium
pyog
enes
Pos
Coc
coba
cilli
and
shor
trod
s,si
ngly
,pa
irsan
dpa
lisad
efo
rmat
ion
with
shor
tdip
hthe
roid
form
sw
ithcl
ubs,
0.2–
0.9
×0.
3–2.
5mm
bHPi
npoi
nt,h
aem
olyt
icco
loni
esat
24h
onBA
.At
48h
colo
nies
0.5
mm
,circ
ular
,opa
que
whi
tew
ithzo
neof
haem
olys
is2–
3tim
esth
edi
amet
erof
the
colo
ny
−Bi
oche
mse
t.Li
tmus
milk
reac
tion
=ac
id,c
lota
ndre
duct
ion
at48
hus
ing
heav
yin
ocul
um
Bact
eriu
mG
ram
Mor
phol
ogy
bHTC
BSCo
lony
char
acte
ristic
sCa
tO
xId
entif
icat
ion
sets
Tab
le2.
4.C
ontin
ued.
108Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:37 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
Bacteriological Culture Techniques 93
Arth
roba
cter
agilis
Pos
Coc
ciin
pairs
and
tetra
ds,
0.8–
1.2
mmdi
amet
erN
HC
olon
ies
onag
arar
esm
ooth
, mat
t,en
tire,
with
aro
se-re
d-pi
gmen
ttha
tis
wat
er-in
solu
ble
++
Bioc
hem
set
Arth
roba
cter
nasi
phoc
aePo
sIrr
egul
ar-s
hape
dro
ds,n
on-s
pore
form
ing.
Som
eco
ccoi
dfo
rms
NH
Stric
taer
obe.
Gro
wth
onBA
asci
rcul
ar,
entir
e,co
nvex
,gre
yish
-whi
te,1
mm
at24
han
d37
°C
+Bi
oche
mse
t,AP
ICO
RYN
E,AP
I-ZYM
Arth
roba
cter
rhom
biPo
sSi
ngle
cory
nefo
rmro
ds,s
hort
orov
oid
insh
ape
Yello
w-w
hitis
hco
loni
es,1
mm
48h,
BHIA
with
1%N
aCl
++
Bioc
hem
set,
APIC
oryn
e
Atop
obac
ter
phoc
aePo
sSh
orti
rregu
larr
ods
NH
Col
onie
spi
n-po
int,
grey
,sm
ooth
onBA
at24
hat
37°C
i nC
O2
−−
Bioc
hem
set,
APIr
apid
ID32
Stre
p,AP
I-ZYM
Baci
llus
cere
usPo
sR
ods
bHO
nBA
colo
nies
are
whi
te-g
rey,
grou
nd-g
lass
appe
aran
ce,2
mm
at24
h.Be
com
esl
ight
lyye
llow
with
age
+Bi
oche
mse
t
Baci
llus
myc
oide
sPo
sR
ods
inch
ains
Rhi
zoid
colo
nies
with
coun
terc
lock
wis
efil
amen
tous
swirl
ing
patte
rnon
BA+
Bioc
hem
set
Bord
etel
labr
onch
isep
tica
Neg
Thin
rods
with
tape
red
ends
,sin
gly
and
pairs
.May
have
long
filam
ento
usfo
rms
NH
On
BAan
dC
FPA
colo
nies
are
1m
mat
48h
and
may
beha
emol
ytic
orno
n-ha
emol
ytic
.D
epen
ding
onph
ase
varia
tion,
roug
han
dsm
ooth
colo
nies
occu
rand
may
bero
ugh,
trans
luce
ntw
itha
rais
edce
ntre
and
undu
latin
gou
term
argi
n,or
smoo
th,o
paqu
ean
dpe
arl-l
ike
++
Bioc
hem
set
Brev
undi
mon
asdi
min
uta
Neg
Shor
trod
s1–
4×
0.5
mma
HG
row
thBA
,NA,
TSA,
30–3
7°C
,2da
ys.
Pant
othe
nate
,bio
tin,c
yano
coba
lam
inar
ere
quire
das
grow
thfa
ctor
s
++
Bioc
hem
set,
API5
0CH
,API
20N
E,AP
I-ZYM
Brev
undi
mon
asve
sicu
laris
Neg
aH
Yello
wco
loni
eson
BA,T
SA,N
A+
+Bi
oche
mse
t
Bruc
ella
abor
tus
Neg
Smal
lcoc
coid
rod
Gro
wth
at24
his
scan
t,oc
curri
ngw
here
inoc
ulum
ishe
avie
st+
+Se
ebe
low
Bruc
ella
spp.
Neg
Coc
coba
cilli.
May
stai
nfa
intly
Gro
wth
onpr
imar
ycu
lture
7–14
days
.10
%C
O2
requ
ired
fors
ome
stra
ins.
Gro
wth
onTS
Aor
FM.C
olon
ies
are
rais
ed,
conv
ex,e
ntire
edge
and
shin
ysu
rface
,ho
ney-
colo
ured
and
trans
pare
ntin
trans
mitt
edlig
ht
+Po
sitiv
eby
MAF
stai
n.Ph
enot
ypic
test
s,se
roty
ping
–be
stpe
rform
edby
asp
ecia
listl
abor
ator
yW
ork
with
susp
ectt
issu
esan
dBr
ucel
lacu
lture
sin
abi
olog
ical
safe
tyca
bine
t
cont
inue
d
109Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:37 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
94 Chapter 2
Budv
icia
aqua
tica
Neg
Stra
ight
rods
Smal
lcol
onie
son
NA,
0.5
mm
24h
at30
°C.
Tran
sluc
entw
ithsm
ooth
entir
eed
ges
+−
Bioc
hem
set
Burk
hold
eria
pseu
dom
alle
iN
egO
valt
oro
und
cells
with
bipo
lar
stai
ning
at3–
4da
ys.C
ells
may
bem
ista
ken
fors
pore
sas
only
the
perip
hery
stai
ns
Col
onie
s0.
5–1
mm
at24
h,an
d3–
4m
mat
3–4
days
.Whi
te,c
onve
xan
dsm
ooth
,with
whi
tesh
een,
beco
min
gun
even
and
umbo
nate
and
wrin
kled
.Bro
thcu
lture
istu
rbid
with
wrin
kled
pellic
le
++
Bioc
hem
set,
API2
0EW
ork
ina
biol
ogic
alsa
fety
cabi
net
Car
noba
cter
ium
inhi
bens
Pos
Rod
s.0.
2×
0.5–
1.2
mma
H1–
2m
mof
f-whi
teco
loni
eson
BA−
−Bi
oche
mse
t
Car
noba
cter
ium
(Lac
toba
cillu
s)pi
scic
ola
Pos
Very
smal
lrod
,1.1
–1.4
×0.
5–0.
6mm
.D
iplo
cocc
oid
intis
sues
,cha
ins
seen
inbr
oth
cultu
res
NG
1–1.
5m
m,g
rey/
whi
te,s
trep-
like
at25
°Cf o
r48
h.Ve
rylit
tlegr
eeni
ngof
the
agar
.Gro
wth
onBA
,BH
IA,T
SA
−−
Bioc
hem
set,
API2
0St
rep,
API5
0CH
Chr
omob
acte
rium
viol
aceu
mN
egN
Hor
bHPi
gmen
ted
stra
ins
are
deep
purp
le,r
ound
,sl
ight
lyra
ised
onBA
.Non
-pig
men
ted
stra
ins
show
b-ha
emol
ysis
onBA
++
Bioc
hem
set,
API2
0E,A
PI20
NE
Citr
obac
terf
reun
dii
Neg
Rod
sN
G2
mm
grey
colo
nies
at24
hon
BA+
−Bi
oche
mse
t,AP
I20E
Chr
yseo
bact
eriu
mba
lust
inum
Neg
Rod
s,0.
5×
1.0–
3.0
mm–
NG
Muc
oid,
yello
wco
loni
eson
AO+
+Bi
oche
mse
t,AP
I-ZYM
,API
20N
E
Chr
yseo
bact
eriu
m(F
lavo
bact
eriu
m)
gleu
m
Neg
Non
-spo
ring
rods
with
roun
ded
ends
NH
NG
On
NA
circ
ular
,ent
ire,v
isci
dbe
com
em
ucoi
dan
dtra
nslu
cent
afte
r5da
ys.B
right
yello
wpi
gmen
t
++
Bioc
hem
set,
API-Z
YM,A
PI20
NE
Chr
yseo
bact
eriu
min
dolo
gene
sN
egSt
raig
htro
ds0.
5×
1.3–
2.5
mmC
olon
ies
onhe
arti
nfus
ion
agar
are
1m
mat
2 4h
a t3 0
°C.O
n0.
3%ag
argr
owth
spre
ads
outi
na
flow
er-li
kegr
owth
patte
rn
++
Bioc
hem
set,
API2
0NE,
API-Z
YM
Chr
yseo
bact
eriu
mm
enin
gose
ptic
umN
egN
HC
olon
ies
onBA
are
abou
t1m
min
diam
eter
and
surro
unde
dby
azo
neof
gree
nish
disc
olor
atio
nat
24h
++
Bioc
hem
set,
API-Z
YM,A
PI20
NE
Chr
yseo
bact
eriu
msc
opht
halm
umN
egSh
ortr
ods,
2.0
×0.
8mm
NH
NG
Smoo
th,r
ound
,shi
ny,o
rang
epi
gmen
ted
colo
nies
,2–3
mm
afte
r2da
ysat
25o C
o nM
SA-B
,MA
2216
,TSA
.Fre
shis
olat
essh
owgl
idin
gm
otilit
yon
Anac
ker-O
rdal
med
ium
.G
lidin
gab
ility
lost
afte
rsto
rage
++
Bioc
hem
set,
API-Z
YM,A
PI20
NE
Bact
eriu
mG
ram
Mor
phol
ogy
bHTC
BSCo
lony
char
acte
ristic
sCa
tO
xId
entif
icat
ion
sets
Tab
le2.
4.C
ontin
ued.
110Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:38 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
Bacteriological Culture Techniques 95
Clo
strid
ium
botu
linum
Pos
3.4–
7.5
×0.
7mm
oval
,sub
term
inal
spor
esbH
Sem
i-opa
que
totra
nslu
cent
,mat
tcol
ony,
1–3
mm
,irre
gula
rlob
ate
mar
gin
and
rais
edce
ntre
−AP
I rap
i d32
A
Col
wel
liam
aris
Neg
Cur
ved
rods
,0.6
–1×
2–4
mmM
arin
e22
16ag
ar,o
ptim
algr
owth
at15
°C.
Gro
wth
rang
e0–
22°C
,but
nogr
owth
at25
°C+
+Bi
oche
mse
t
Cor
yneb
acte
rium
aqua
ticum
Pos
Rod
s0.
5–0.
8×
1–3
mm.S
light
pleo
mor
phis
mw
ithso
me
club
-sha
ped
form
san
dan
gula
rarra
ngem
ents
ofce
lls
bHO
nBA
and
TSA
colo
nies
roun
d,ra
ised
,ent
ire,
opaq
ue,s
light
lyvi
scid
with
yello
wno
n-di
ffusi
ble
pigm
enta
fter4
8h
at25
°C
+−
Bioc
hem
set,
APIC
oryn
e
Cor
yneb
acte
rium
phoc
oae
Pos
Cor
ynef
orm
rods
NH
Shin
y,ro
und
colo
nies
1m
mon
BA24
hr,
37°C
+Bi
oche
mse
t,AP
ICor
yne,
API5
0CH
,API
-ZYM
Cor
yneb
acte
rium
test
udin
oris
Pos
Dip
hthe
roid
rods
Col
onie
son
BAar
eye
llow
-pig
men
ted
+AP
ICor
yne,
API-Z
YM
Cry
ptoc
occu
sne
ofor
man
sva
r.ga
ttii
Pos
Budd
ing
yeas
tsN
HSl
owgr
owin
g.C
olon
ies
0.5–
1m
mcr
eam
dull,
3da
yson
BA.C
olon
ies
chan
gefro
mcr
eam
todi
rtyye
llow
,lig
htta
nco
lour
afte
r7da
yson
BA
Indi
ain
kfo
rcap
sule
stai
n,ur
ease
posi
tive.
Gro
wth
onSt
rep
sele
ctiv
em
edia
(Oxo
id),
and
SAB
plat
esUs
ebi
olog
ical
safe
tyca
bine
t
Der
mat
ophi
lus
Pos
Bran
chin
gfil
amen
tsco
ntai
ning
row
sof
cocc
i(zo
ospo
res)
.Zoo
spor
esar
em
otile
bHC
olon
ies
onBA
inC
O2
a re
g re y
-wh i
teto
grey
-yel
low
,adh
eren
tand
pitt
heag
ar.A
t48
h,co
loni
esar
etin
y,ro
ugh,
gran
ular
,ra
ised
.Fur
ther
incu
batio
npr
oduc
esco
loni
esup
to2–
3m
m,u
mbo
nate
,mol
arto
oth
crat
erfo
rms
and
beco
me
muc
oid
+Bi
oche
mse
t.M
ake
impr
essi
onsm
ears
from
unde
rsid
eof
fresh
lyre
mov
edsc
abs.
Stai
nw
ithG
iem
sast
ain.
Fila
men
tsar
ebe
stst
aine
dw
ithG
iem
sara
ther
than
Gra
mst
ain
Edw
ards
iella
hosh
inae
Stra
ight
rods
Gro
wth
onN
A,BA
,MC
A.C
olon
ies
flato
rsl
ight
lyco
nvex
,1–2
mm
at24
–48
h,30
and
37°C
+−
Bioc
hem
set,
API2
0E
Edw
ards
iella
icta
luri
Neg
Rod
toco
ccob
acilli
,0.
75×
1.5–
2.5
mmw
NG
Slow
grow
ing,
1–2
mm
,rou
nd,
non-
pigm
ente
d,pa
legr
ey,4
8h
at28
–30°
C.
May
bea
gree
nish
tinge
and
slig
htha
emol
ysis
unde
rthe
colo
ny(p
ale
lem
onco
lour
ona
loop
).M
usty
smel
l
+−
Bioc
hem
set,
API2
0E cont
inue
d
111Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:38 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
96 Chapter 2
Edw
ards
iella
tard
aN
egR
od.1
×1–
2mm
NG
0.5
mm
roun
d,gr
eyco
loni
esat
24–4
8h
(pal
ele
mon
colo
uron
alo
op).
Gro
wth
onBA
,MC
A(N
LF),
SSag
ar.C
olon
ies
will
besm
alle
rin
size
than
Salm
onel
laon
the
sele
ctiv
em
edia
+−
Bioc
hem
set,
API2
0E
Empe
doba
cter
brev
isN
egR
ods
Yello
wco
loni
eson
NA
and
BA,0
.2–2
.5m
mat
24h
and
30°C
,low
conv
ex,e
ntire
edge
.At
day
7on
BAm
ayse
esl
ight
a-h
aem
olys
is
++
Bioc
hem
set
Ente
rovi
brio
norv
egic
usN
egC
ells
0.8
×1.
0–1.
2mm
GO
nM
A22
16co
loni
esar
ebe
ige,
smoo
th,
roun
d,ra
ised
,1m
mat
48h
at28
°C+
+Bi
oche
mse
t,AP
I20E
,AP
I-ZYM
Erys
ipel
othr
ixrh
usio
path
iae
Pos
Gra
m-v
aria
ble
rods
,ple
omor
phic
,ta
ngle
dfo
rms,
2–5
mmN
Ha
HN
DG
row
thon
BAat
24–4
8h,
0.5
mm
grey
-gre
enco
loni
essm
allz
one
ofa
-hae
mol
ysis
−Bi
oche
mse
t,AP
Irap
idID
32St
rep,
APIC
oryn
e.N
ote:
add
afe
wdr
ops
ofst
erile
seru
mto
inoc
ulat
ing
med
ium
toim
prov
ere
actio
nsin
Bioc
hem
set
Euba
cter
ium
spp.
Pos
Long
chai
nsof
pleo
mor
phic
rods
.No
spor
es.I
nol
derc
ultu
res
cells
may
have
‘ovo
id’b
odie
s
bHO
nBA
unde
rana
erob
icco
nditi
ons,
colo
nies
are
haem
olyt
ic,t
rans
luce
nt,s
low
lysp
read
ing,
flat,
and
cont
oure
dw
ithfil
amen
tous
edge
s.An
aero
bic
atm
osph
ere
requ
ired
−−
Bioc
hem
set
Euba
cter
ium
limos
umPo
s0.
6–0.
9×
1.6–
4.8
mm.R
ods
may
have
swol
len
ends
NH
Punc
tate
,circ
ular
,con
vex,
entir
eco
loni
esth
atar
etra
nslu
cent
tosl
ight
lyop
aque
.Les
sth
an1
mm
at48
h.An
aero
bic
−Bi
oche
mse
t,AP
Irap
id32
A
Euba
cter
ium
tara
ntel
lae
Pos
Very
long
filam
ento
us,u
nbra
nche
dro
ds(1
0mm
)bH
NG
2–5
mm
,tra
nslu
cent
colo
nies
,slig
htly
rhiz
oid
and
muc
oid.
Anae
robi
c−
Bioc
hem
set
Fack
lam
iam
iroun
gae
Pos
Ovo
idce
lls(0
.8–0
.9mm
)in
pairs
and
shor
tcha
ins
NH
ND
Col
onie
son
BA0.
5m
mat
24h,
37°C
.C
ircul
ar,e
ntire
,shi
ny,c
onve
xan
dgr
ey-c
olou
red
−AP
Irap
idID
32,A
PI-Z
YM
Flav
obac
teriu
maq
uatil
eN
egC
olon
ies
onAO
med
ialo
w,c
onve
x,ro
und
++
Bioc
hem
set,
API-Z
YM
Flav
obac
teriu
mbr
anch
ioph
ilum
Neg
Long
thin
rods
0.5–
8×
10mm
.Sm
ears
from
colo
nies
may
show
circ
ular
,slig
htly
refra
ctile
,‘cy
sts’
NH
NG
On
AOm
edia
,lig
htye
llow
,sm
ooth
,rou
nd,
rais
ed,c
olon
ies,
0.5–
1m
m,5
days
at18
°C.
No
glid
ing
mot
ility.
Gro
wth
on20
-fold
dilu
teTS
Abu
tnot
on‘fu
ll-st
reng
th’T
SA
V+
Bioc
hem
set,
API-Z
YM,A
PI20
E
Bact
eriu
mG
ram
Mor
phol
ogy
bHTC
BSCo
lony
char
acte
ristic
sCa
tO
xId
entif
icat
ion
sets
Tab
le2.
4.C
ontin
ued.
112Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:38 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
Bacteriological Culture Techniques 97
Flav
obac
teriu
m(F
lexi
bact
er)
colu
mna
re
Neg
Long
thin
rods
(4–8
mm).
Fila
men
tous
NH
NG
Brig
htye
llow
,fla
t,dr
y,rh
izoi
d,sl
owsp
read
ing
grow
th5
days
20–2
5°C
.Ad
here
sst
rong
lyto
agar
V+
Bioc
hem
set,
API-Z
YM,A
PI20
E,AP
I50C
H.Y
ello
wpi
gmen
tcha
nges
topi
nkin
3%N
aOH
Flav
obac
teriu
mfle
vens
eN
egO
nAO
med
ia,l
owco
nvex
,rou
ndco
loni
essu
nken
into
agar
++
Bioc
hem
set,
API-Z
YM
Flav
obac
teriu
mfri
gida
rium
Neg
Rod
s0.
8–2
×0.
5–0.
7mm
,sin
gly
and
inpa
irsG
row
thon
AO,f
latr
ound
yello
wco
loni
esw
ithen
tire
mar
gins
.Gro
ws
onN
A,TS
Aan
dM
A22
16
++
Bioc
hem
set,
API-Z
YM
Flav
obac
teriu
mgi
llisia
eN
egR
ods
2–5
×0.
4–0.
5mm
Ora
nge-
pigm
ente
dco
loni
es,b
utyr
ous,
circ
ular
and
conv
exw
ithen
tire
edge
.Gro
wth
onM
A22
16,N
A,TS
A,R
2A
+−
Bioc
hem
set,
API2
0E
Flav
obac
teriu
mhi
bern
umN
egR
ods
0.7
×1.
8–13
mmG
row
thon
TSA.
Yello
w,m
ucoi
dco
loni
esat
25°C
,gel
atin
ous
at4°
C+
−Bi
oche
mse
t,AP
I20E
,API
20N
E
Flav
obac
teriu
mhy
datis
(Cyt
opha
gaaq
uatil
is)
Neg
Rod
s,8.
0×
0.5
mmN
HN
GM
ucoi
d,ye
llow
-ora
nge
colo
nies
with
flat
spre
adin
gfil
amen
tous
mar
gins
.Inc
ubat
e14
°Cfo
r14
days
+−
Bioc
hem
set,
API-Z
YM,A
PI20
E
Flav
obac
teriu
m(C
ytop
haga
)jo
hnso
niae
Neg
Long
thin
rods
NH
NG
On
AOm
edia
,pal
eye
llow
,5–1
0m
m,f
lat,
smoo
th,r
hizo
idw
ithsp
read
ing,
filam
ento
usm
argi
ns,5
days
.On
BA,y
ello
w,r
ound
,sm
ooth
,1–2
mm
,2da
ys
V+
Bioc
hem
set,
API-Z
YM,A
PI20
E
Flav
obac
teriu
mpe
ctin
ovor
umN
egO
nAO
med
ia,c
olon
ies
low
roun
dw
ithen
tire
mar
gins
+Bi
oche
mse
t,AP
I-ZYM
Flav
obac
teriu
m(C
ytop
haga
)ps
ychr
ophi
lum
Neg
Slen
der,
flexi
ble
rods
1–7
×0.
5mm
NG
Smoo
th,g
loss
y,br
ight
yello
wco
loni
esw
ithth
insp
read
ing
edge
s,5
days
at5–
25°C
.D
oes
nota
dher
eto
agar
V+
Bioc
hem
set,
API-Z
YM,A
PI50
CH
Flav
obac
teriu
msa
ccha
roph
ilum
Neg
On
AOm
edia
,col
onie
sfla
t,sp
read
ing,
sunk
enin
toag
ar+
−Bi
oche
mse
t,AP
I-ZYM
Flav
obac
teriu
msu
ccin
ican
sN
egO
nAO
med
ia,c
olon
ies
flat,
spre
adin
g,w
ithfil
amen
tous
mar
gins
Bioc
hem
set,
API-Z
YM
Flav
obac
teriu
mte
getin
cola
Neg
Rod
s2–
5×
0.4–
0.5
mmYe
llow
-pig
men
ted
colo
nies
,but
yrou
s,ci
rcul
ar,
conv
ex,e
ntire
.Gro
wth
onM
A22
16,N
A,TS
A,R
2A
+−
Bioc
hem
set,
API2
0E cont
inue
d
113Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:38 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
98 Chapter 2
Flav
obac
teriu
mxa
nthu
mN
egG
row
thon
MA
2216
,NA,
TSA,
R2A
++
Bioc
hem
set,
API2
0E
Flex
ibac
ter
poly
mor
phus
Neg
Fila
men
tsm
ultic
ellu
lar.
Aten
dof
each
cell
isa
refra
ctile
gran
ule
oflip
idm
ater
ial
Mar
ine
agar
.Cob
alam
inre
quire
dfo
rgro
wth
.Pe
ach-
colo
ured
pigm
ent
−+
Bioc
hem
set,
API-Z
YM,A
PI50
CH
Gra
nulic
atel
laad
iace
nsG
ranu
licat
ella
eleg
ans
Pos
Coc
ci,i
nclu
ding
pleo
mor
phic
ovoi
dce
lls,c
occo
-bac
illi,r
od-s
hape
dce
llsa
Stra
ins
are
nutri
tiona
llyde
ficie
nt,a
ndsa
tellit
ear
ound
othe
rbac
teria
lgro
wth
.Gro
won
BAw
ithSt
aph
stre
akor
pyrid
oxal
disc
,orB
Asu
pple
men
ted
with
20mg
pyrid
oxal
HC
lper
ml.
For G
.ele
gans
use
L-cy
stei
neH
Cl
(0.0
1%)
−−
Bioc
hem
set,
API2
0St
rep,
API r
api d
I D32
Str e
p
Gra
nulic
atel
laba
laen
opte
rae
Pos
Coc
cus,
sing
lece
llsan
dsh
ortc
hain
sa
NG
Gro
wth
onBA
,0.2
mm
colo
nyin
airo
rCO
2.Is
notn
utrit
iona
llyde
ficie
ntlik
eot
hers
train
sin
the
genu
s
−Bi
oche
mse
t,AP
I-ZYM
,API
20St
rep,
APIr
apid
ID32
Stre
p
Haf
nia
alve
iN
egSh
ortr
ods
Whi
teto
yello
wno
n-m
ucoi
dco
loni
es.G
row
thon
BA,N
A,M
CA,
DC
A+
−Bi
oche
mse
t,AP
I-ZYM
,API
20E
Hal
omon
asaq
uam
arin
a(p
revi
ousl
yAl
calig
enes
faec
alis
hom
ari )
Neg
Stra
ight
orcu
rved
rods
1.5
mm,
bipo
lars
tain
ing
Col
onie
son
MA
2216
,off-
whi
tetra
nslu
cent
,ra
ised
smoo
th2–
3m
mat
24h
at18
°Can
d37
°C.S
light
tend
ency
tosp
read
++
Bioc
hem
set
Hal
omon
asel
onga
taN
egR
ods
sing
lyor
paire
dC
olon
ies
2m
mat
24h,
smoo
th,g
liste
ning
,op
aque
whi
te.A
fter2
4h
colo
nies
spre
addu
eto
flexu
ous
filam
ents
,but
are
nola
rger
than
4m
m.R
equi
res
8%N
aClf
orgr
owth
Bioc
hem
set.
Add
8%sa
ltto
med
ium
Hal
omon
asha
lodu
rans
Neg
Rod
ssi
ngle
,pai
rsor
chai
nsO
nM
A22
16,c
olon
ies
are
smoo
th,g
liste
ning
,tra
nslu
cent
whi
te,c
onve
x,1–
2m
min
24h
at20
or30
°C
Bioc
hem
set
Hal
omon
asve
nust
aN
egR
odAt
48h
colo
urle
ss,m
ucoi
dco
loni
eson
BA,
MC
A+
+Bi
oche
mse
t,AP
I20N
E
Bact
eriu
mG
ram
Mor
phol
ogy
bHTC
BSCo
lony
char
acte
ristic
sCa
tO
xId
entif
icat
ion
sets
Tab
le2.
4.C
ontin
ued.
114Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:39 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
Bacteriological Culture Techniques 99
Hel
icob
acte
rcet
orum
Neg
Fusi
form
tosl
ight
lysp
iral
On
BA,S
kirro
w’s
med
ia(V
PT),
orTS
A+
bloo
d,co
loni
esar
epi
n-po
inta
ndm
aygr
owas
ath
insp
read
ing
film
acro
ssth
epl
ate,
5–14
days
37°C
++
Bioc
hem
ical
s,AP
ICam
py
Hyd
roge
noph
aga
(Pse
udom
onas
)ps
eudo
flava
Neg
Ovo
idce
lls.R
ods
of2.
5mm
inol
der
cultu
res
NG
On
NB
agar
yello
w-p
igm
ente
dco
loni
es2–
4m
mat
3da
ys,s
light
lyirr
egul
arsh
ape
with
anun
dula
ting
mar
gin
W+
Bioc
hem
set
Iodo
bact
erflu
viat
ileN
egSm
allr
od,0
.7×
3.0–
3.5
mmN
GVi
olet
-col
oure
dco
loni
es,t
hin
spre
adin
gm
argi
non
low
nutri
enta
gars
uch
as¼
stre
ngth
NA.
Non
-spr
eadi
ngon
NA
++
Bioc
hem
set
Lact
ococ
cus
garv
ieae
Pos
Coc
coba
cilli
0.7–
1.4
mm,p
aire
dco
cci,
shor
tcha
ins
aH
NG
1m
mgr
ey/w
hite
,rou
nd,w
ithgr
eeni
ngun
der
colo
ny.S
trep-
like
onBA
.Gro
wth
onN
A,TS
A,BA
−−
Bioc
hem
set,
API2
0Stre
p,AP
Irap
idID
32St
rep,
API
50C
H.S
trep
grou
p=
Dne
g
Lact
ococ
cus
pisc
ium
Pos
Coc
coba
cilli
−−
Bioc
hem
set
List
onel
la(V
ibrio
)an
guilla
rum
Neg
Shor
trod
s,cu
rved
orst
raig
ht,
roun
ded
ends
,occ
urrin
gsi
ngly
and
inpa
irs,p
leom
orph
ic.
0.5–
0.7
×1–
2mm
.Rap
idm
otilit
y
bHY
At2
days
colo
nies
are
2m
m,g
liste
ning
crea
m-c
olou
rin
youn
gco
loni
esan
dgr
eeni
sh-p
ale
brow
nin
olde
rcol
onie
son
MSA
-B.H
aem
olys
isun
derc
olon
y.O
nM
SA-B
,V.
chol
erae
and
V.m
imic
usha
vesi
mila
rap
pear
ance
butg
row
mor
equ
ickl
y.O
nN
Aco
loni
esar
eof
f-whi
teto
buff-
colo
ured
,tra
nslu
cent
orop
aque
,circ
ular
,shi
ny,1
–2m
m
++
Bioc
hem
set,
API-Z
YM,A
PI20
E,AP
I20N
E
List
onel
lape
lagi
aN
egN
HG
rey-
trans
luce
ntco
loni
eson
MSA
-B+
+Bi
oche
mse
t
Man
nhei
mia
haem
olyt
ica
Neg
Pleo
mor
phic
rods
,lon
gan
dsh
ort
form
sbH
Gre
yco
loni
es1–
2m
mon
BAat
24h.
b-ha
emol
ysis
seen
++
Bioc
hem
set,
API-Z
YM
Mar
inila
bilia
salm
onic
olor
Neg
Glid
ing,
yello
wto
pale
pink
-pig
men
ted
colo
nies
++
Bioc
hem
set,
API-Z
YM
Mes
ophi
loba
cter
mar
inus
Neg
Coc
coba
cilli,
pleo
mor
phic
NH
NG
Gro
wth
onM
A22
16an
dN
A.C
olon
ies
circ
ular
som
etim
esirr
egul
ar,c
onve
x,gl
iste
ning
,op
aque
,pal
eye
llow
-bro
wn.
Des
crib
esm
arin
eco
ccob
acilli
that
mor
phol
ogic
ally
rese
mbl
eth
eAc
inet
obac
ter-M
orax
ella
grou
p
++
Bioc
hem
set
Mor
itella
mar
ina
Neg
Cur
ved
orst
raig
htro
dsbH
NG
Gre
yish
-cre
am,t
rans
luce
nt,c
ircul
ar,c
onve
xco
loni
eson
MSA
-B+
+Bi
oche
mse
t,AP
I-ZYM
,API
20E
cont
inue
d
115Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #M.vpMonday, January 26, 2004 11:48:01 AM
Color profile: DisabledComposite 150 lpi at 45 degrees
100 Chapter 2
Mor
itella
visc
osa
Neg
Long
cells
whe
ngr
own
inliq
uid
med
ia.S
hort
orel
onga
ted
curv
edro
dson
solid
med
ium
bHN
GC
ream
y-ye
llow
colo
nies
onM
SA-B
and
TSA+
NaC
l,0.
5m
m24
hat
15–2
2°C
.C
olon
ies
are
visc
ous
and
adhe
reto
the
med
ium
.For
mlo
ngth
read
sw
hen
lifte
dfro
mth
epl
ate.
Ligh
thae
mol
ysis
seen
unde
rnea
thco
lony
++
Bioc
hem
set,
API-Z
YM,A
PI20
E,AP
I50C
H
Myc
obac
teriu
mab
sces
sus
AFB
Gro
wth
onM
iddl
ebro
ok7H
10-A
DC
med
ium
in7
days
.Gro
wth
onM
CA
+Bi
oche
mse
t,or
send
tosp
ecia
listl
abor
ator
y
Myc
obac
teriu
mch
elon
aePo
san
dAF
BPl
eom
orph
icro
ds,2
–7×
0.2–
0.5
mmN
HN
GC
olon
ies
onM
SA-B
are
circ
ular
,sm
ooth
,pa
le-c
ream
at7
days
and
15°C
.G
row
thon
MC
A,TS
A,BH
IA
+−
Bioc
hem
set,
orse
ndto
spec
ialis
tlab
orat
ory.
PCR
prim
ers
avai
labl
e.
Myc
obac
teriu
mm
arin
umPo
san
dAF
BPl
eom
orph
icro
ds,2
–7×
0.2–
0.5
mmN
HN
GC
olon
ies
onM
SA-B
are
circ
ular
opaq
ue,
0.2
mm
at7
days
,22 °
C.W
hen
grow
nin
the
light
,col
onie
sar
eye
llow
Bioc
hem
set,
orse
ndto
spec
ialis
tlab
orat
ory.
PCR
prim
ers
avai
labl
e
Myc
obac
teriu
mne
oaur
umG
ram
stai
n=
neg,
AFB
AFB
3–4
×0.
6mm
NH
Yello
wco
loni
eson
BAat
25°C
in5–
7da
ys.
No
grow
that
37°C
.At8
days
,col
onie
s0.
4m
m,s
moo
th,r
ound
clea
r,w
ithsl
ight
y ello
wc o
lou r
.No
g ro w
tho n
MS A
+Se
ndto
spec
ialis
tlab
orat
ory
Myc
obac
teriu
mpe
regr
inum
AFB
Acid
fast
rods
Buff-
colo
ured
colo
nies
onM
iddl
ebro
ok7H
11m
ediu
min
4da
ysun
derC
O2
Send
tosp
ecia
listl
abor
ator
y
Myc
obac
teriu
mpo
rifer
aeAF
BAc
idfa
stro
dsR
apid
grow
thw
ithsm
ooth
colo
nies
onM
iddl
ebro
ok7H
11m
ediu
mSe
ndto
spec
ialis
tlab
orat
ory
Myc
obac
teriu
msp
p.W
eak
pos
non-
stai
ning
,ac
id-fa
st
Pleo
mor
phic
med
ium
tolo
ngro
ds,
non-
bran
chin
g,1.
5–3.
0mm
NH
NG
0.5
mm
off-w
hite
,5–7
days
gran
ular
+Bi
oche
mse
t,or
send
tosp
ecia
listl
abor
ator
yUs
ebi
olog
ical
safe
tyca
bine
t
Myc
obac
teriu
mtri
plex
-like
AFB
Coc
coid
and
rod
form
s.Be
aded
AFB
rods
aH
Gro
wth
o nB A
a fte
r12
we e
k sa t
2 5°C
prod
uces
roug
h,dr
yco
loni
esw
itha
rais
edce
ntre
and
flatb
orde
r
Nes
ted
PCR
Myc
opla
sma
mob
ileC
ells
glid
ein
wet
prep
arat
ion
asse
enby
dark
grou
ndillu
min
atio
n.C
ells
are
elon
gate
d.O
ptim
umte
mpe
ratu
reis
25°C
Myc
opla
sma
set
Bact
eriu
mG
ram
Mor
phol
ogy
bHTC
BSCo
lony
char
acte
ristic
sCa
tO
xId
entif
icat
ion
sets
Tab
le2.
4.C
ontin
ued.
116Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:39 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
Bacteriological Culture Techniques 101
Myr
oide
s(F
lavo
bact
eriu
m)
odor
atim
imus
Neg
Rod
s0.
5×
1–4
mmN
HYe
llow
-pig
men
ted
colo
nies
.Gro
wth
onM
CA,
NA
and
TSA.
No
glid
ing
orsw
arm
ing.
Frui
tyod
our
++
Bioc
hem
set,
API-Z
YM,A
PI20
NE
Myr
oide
s(F
lavo
bact
eriu
m)
odor
atus
Neg
Rod
s0.
5×
1–4
mmN
HYe
llow
-pig
men
ted
colo
nies
,3–4
mm
onM
CA,
NA
and
TSA
at24
h.N
ogl
idin
gor
swar
min
g.Fr
uity
odou
r
++
Bioc
hem
set,
API-Z
YM,A
PI20
NE
Noc
ardi
acr
asso
stre
aePo
sBr
anch
edhy
phae
,fra
gmen
tint
oro
dsan
dco
cci
Dry
wrin
kled
,wax
yco
loni
es,p
ale
yello
w.N
oae
rialh
ypha
e+
Bioc
hem
set,
acid
fast
Noc
ardi
asa
lmon
icid
aPo
sBr
anch
edor
ange
,sub
stra
tem
ycel
ium
,with
whi
teto
pink
aeria
lgro
wth
.Fila
men
tous
colo
nym
argi
ns
Bioc
hem
set
Noc
ardi
ase
riola
ePo
sBr
anch
ing
vege
tativ
em
ycel
iath
atbr
eak
upin
tono
n-m
otile
rods
.C
occo
idan
dlo
ngsl
ende
rm
ulti-
sept
ate
rod
form
s
Mac
rosc
opic
aeria
lmyc
elia
are
notf
ound
.D
ry,w
axy,
wrin
kled
,col
onie
sth
atar
eye
llow
ish-
oran
geon
yeas
text
ract
–mal
tex
tract
agar
(ISP
no2
Difc
o).A
ppea
ras
whi
teco
loni
eson
BHIA
and
yello
wco
loni
eson
LJM
Bioc
hem
set,
wea
kly
acid
fast
Noc
ardi
asp
p.Po
sw
eak
acid
-fast
Coc
coid
toov
alce
lls,l
ong
slen
der
mul
ti-se
ptat
ero
ds,b
ranc
hing
.5–
50mm
NN
G<1
mm
,whi
te,c
ream
,rou
ghor
with
very
shor
tde
nse
myc
eliu
m.G
row
th3–
7da
ys.B
Aan
dN
A
V,+
Bioc
hem
set
Noc
ardi
asp
p.(A
ustra
lian
stra
in)
GPR
Bran
chin
gro
dsC
olon
ies
appe
arat
5da
ys.D
ry,r
ough
,ta
n/ye
llow
,adh
eren
t,1–
2m
mBi
oche
mse
t.M
odifi
edac
idfa
stst
ain
=po
sitiv
e
Pant
oea
(Ent
erob
acte
r)ag
glom
eran
s
Neg
Non
-enc
apsu
late
d,no
n-sp
ore
form
ing,
broa
d,st
raig
htro
d,0.
5–1.
0×
1.0–
3.0
mm
NH
NG
Col
onie
son
NA
are
smoo
th,c
onve
x,en
tire,
and
trans
luce
ntan
dm
ayha
vea
yello
wpi
gmen
t.G
row
thon
BA,M
SA-B
,and
MA
2216
.Gro
wth
at37
°Cbu
tnot
at44
°C
+−
Bioc
hem
set,
API2
0E,A
PI50
CH
Past
eure
llam
ulto
cida
Neg
Coc
co-b
acilli
NH
Gro
wth
onBA
,1–2
mm
at24
–48
h.C
olon
ysi
zean
dap
pear
ance
vary
from
diffe
rent
anim
alsp
ecie
s.C
olon
ies
may
besm
ooth
orm
ucoi
d,da
rkgr
eyw
ithgr
eeni
shap
pear
ance
.D
istin
ctiv
em
usty
swee
tish
smel
l
++
Bioc
hem
set,
API-Z
YM,A
PI20
E
Past
eure
llask
yens
isN
egN
Hor
wea
kG
row
thTS
A-B+
1.5%
NaC
l,at
48h
at22
°C.
Col
onie
sci
rcul
ar,e
ntire
,low
,con
vex,
grey
,0.
5m
m.N
ogr
owth
with
out1
.5%
NaC
lor
bloo
d.N
ogr
owth
at37
°C
−+
wea
kBi
oche
mse
t,AP
I-ZYM
.Add
1.5–
2%N
aClt
ose
tsan
dus
ehe
avy
inoc
ulum
inBi
oche
mse
t.R
ead
afte
r4da
ys cont
inue
d
117Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:39 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
102 Chapter 2
Past
eure
llate
stud
inis
NPl
eom
orph
icro
d0.
2×
1.5–
2mm
bHBA
whi
te,m
ucoi
d,0.
5–1
mm
24–4
8h,
20–3
7 °C
++
Bioc
hem
set
Pedo
bact
erhe
parin
usN
egN
on-s
porin
gro
ds,
0.4–
0.5
×0.
5–1.
0mm
bHsl
owG
row
thon
NA,
1–3
mm
at48
h,ci
rcul
ar,l
owco
nvex
,sm
ooth
,opa
que.
Aye
llow
orcr
eam
yw
hite
non-
fluor
esce
ntpi
gmen
tis
prod
uced
.C
ream
yw
hite
colo
nies
onPY
++
Bioc
hem
set,
API-Z
YM,A
PI50
CH
Pedo
bact
erpi
sciu
mN
egN
on-s
porin
gro
ds0.
4–0.
5×
0.5–
1.0
mmC
olon
ies
onN
Aar
eci
rcul
ar,e
ntire
,low
conv
exat
2da
ys.A
yello
wor
crea
my
whi
te,
non-
fluor
esce
ntpi
gmen
tis
prod
uced
++
Bioc
hem
set,
API-Z
YM,A
PI50
CH
Phoc
oeno
bact
erut
eri
Neg
Pleo
mor
phic
rods
NH
Gro
wth
onBA
with
and
with
outC
O2.
Col
onie
sen
tire,
circ
ular
,low
conv
ex,s
moo
th,g
rey
0 .5
mm
a t4 8
ha t
3 7°C
−+
Bioc
hem
set,
API2
0E,A
PI20
NE,
API-Z
YM
Phot
obac
teriu
man
gust
umN
egSh
ortr
ods
NH
Whi
teco
loni
eson
MA
2216
,MSA
-B+
+Bi
oche
mse
t,AP
I20E
,API
50C
H
Phot
obac
teriu
mda
mse
lae
ssp.
dam
sela
e
Neg
Rod
s,re
lativ
ely
pleo
mor
phic
bHG
Entir
e,sm
ooth
,gre
yish
-whi
te,t
rans
luce
ntco
loni
es,2
–3m
mon
MSA
-B+
+Bi
oche
mse
t,AP
I-ZYM
,API
20E,
API2
0NE,
API5
0CH
Phot
obac
teriu
mda
mse
lae
ssp.
pisc
icid
a
Neg
Smal
lrod
sto
cocc
obac
illi,1
–1.5
mm,
bipo
lars
tain
ing
bHN
G1–
2m
mgr
ey/y
ello
w,s
hiny
,72
h.G
row
thon
BAan
dN
Aw
ith0.
5%N
aCl,
MSA
-B,M
A22
16+
+Bi
oche
mse
t,AP
I-ZYM
,API
20E,
API2
0NE,
API5
0CH
Phot
obac
teriu
milio
pisc
ariu
mN
egPl
eom
orph
icro
ds,s
traig
htan
dcu
rved
NH
GC
olon
ies
smal
lcol
ourle
ssto
grey
ish
opaq
uew
ithen
tire
edge
.May
take
upto
14da
ysfo
rco
loni
esto
appe
ar
++
Bioc
hem
set,
API2
0E,A
PI50
CH
Phot
obac
teriu
mle
iogn
athi
Neg
Shor
trod
sor
cocc
obac
illi,
1–2.
5×
0.4–
1mm
NH
NG
Col
onie
son
MSA
-B,o
ff-w
hite
,tra
nslu
cent
,ci
rcul
ar,s
moo
th,s
hiny
.Lum
inou
sat
3da
ys+
−Bi
oche
mse
t,AP
I20E
,API
50C
H
Phot
obac
teriu
mph
osph
oreu
mN
egSh
ortr
ods
NH
Whi
teco
loni
eson
MSA
-Ban
dM
A22
16+
−Bi
oche
mse
t,AP
I20E
Plan
ococ
cus
kocu
riiPo
sSp
heric
alce
lls1.
0–1.
2mm
,sin
gly,
pairs
and
tetra
ds.M
otile
Col
onie
son
pept
one
yeas
text
ract
agar
are
circ
ular
,sm
ooth
,con
vex,
and
yello
w-o
rang
epi
gmen
ted
+−
Bioc
hem
set
Plan
omic
robi
umok
eano
koite
sN
eg,v
Rod
s0.
4–0.
8×
1–20
mmC
ells
brig
htor
ange
toye
llow
.Req
uire
s3%
NaC
l+
WBi
oche
mse
t.Ad
dN
aCl(
3%)
Bact
eriu
mG
ram
Mor
phol
ogy
bHTC
BSCo
lony
char
acte
ristic
sCa
tO
xId
entif
icat
ion
sets
Tab
le2.
4.C
ontin
ued.
118Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:40 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
Bacteriological Culture Techniques 103
Ples
iom
onas
shig
ello
ides
Neg
Stra
ight
,var
iabl
ele
ngth
rod
NH
W,G
Gro
wth
onBA
and
MC
A1–
2m
m24
hat
25–3
7 °C
.Pun
gent
smel
l+
+Bi
oche
mse
t,AP
I-ZYM
,API
20E
Prov
iden
cia
rettg
eri
Neg
Rod
0.6
×1.
0–1.
5mm
NH
Col
onie
son
BA,T
SAci
rcul
ar,d
iscr
ete,
conv
ex,g
liste
ning
,cre
amy-
whi
te24
hat
37°C
+−
Bioc
hem
set,
API2
0E
Prov
iden
cia
rust
igia
nii
Neg
Non
-spo
ring
rods
0.5
×1–
3mm
NH
On
BA,c
olon
ies
1–2
mm
at24
h,gl
ossy
,se
mitr
ansl
ucen
t,sm
ooth
.Ora
nge-
red
onM
CA
+−
Bioc
hem
set,
API2
0E
Pseu
doal
tero
mon
asge
nus
Neg
Stra
ight
orcu
rved
,non
-spo
refo
rmin
gro
ds0.
2–1.
5×
1.8–
3mm
.Sin
gle
flage
lla
Stric
taer
obes
,gro
wth
onM
A22
16at
20°C
.N
egat
ive
forA
DH
.All
posi
tive
forg
elat
inan
dD
Nas
e
+,w
,v
+Bi
oche
mse
t
Pseu
doal
tero
mon
asan
tarc
tica
Neg
Rod
s,0.
9×
1–3
mm,b
ecom
ela
rger
and
filam
ento
usw
ithag
e(1
0mm
)G
row
that
4–30
°Con
TSA,
MSA
-B,M
A22
16.
Col
onie
sro
und,
smoo
th,b
eige
,con
vex,
muc
oid,
1–2
mm
in5
days
at15
°C.G
row
thfa
ctor
sno
treq
uire
d.Ae
robi
c
++
Bioc
hem
set,
API2
0NE,
API-Z
YM
Pseu
doal
tero
mon
asfla
vipu
lchr
a(P
seud
oalte
rom
onas
aura
ntia
)
Neg
Stra
ight
rods
0.7–
1.5
×1.
5–4
mmO
nM
A22
16,c
olon
ies
1m
m,b
right
pale
yello
w24
h,23
°C.A
t5da
ys,o
rang
e-br
own
togr
een-
brow
n4
mm
.O
nM
SA-B
,brig
ht,m
ucoi
d,da
rk-b
row
nco
loni
es2
days
,the
nm
ediu
mtu
rns
blac
k,an
dha
emol
ysis
isse
en.P
oorg
row
thon
TSA
and
NA
even
with
2%N
aCl
++
Bioc
hem
set
Pseu
doal
tero
mon
asba
cter
ioly
tica
Neg
Rod
sw
ithro
unde
den
ds,
0.6–
0.9
×1.
9–2.
5mm
Gro
wth
onM
A22
16,r
edpi
gmen
tmay
orm
ayno
tbe
pres
ent
+w+
Bioc
hem
set,
API2
0NE
Pseu
doal
tero
mon
asci
trea
Neg
Stra
ight
rods
0.7–
1.5
×1.
5–4
mmbH sl
owO
nM
A22
16co
loni
es0.
5m
m24
hat
23°C
,br
ight
,whi
tish,
beco
min
g4
mm
and
lem
on-y
ello
wup
to4
days
.On
MSA
-B,
6–7
mm
brig
ht,m
ucoi
d,w
hitis
hco
loni
estu
rnin
gbl
ack
arou
ndth
eco
lony
afte
r2–3
days
.Rin
gof
haem
olys
isse
enaf
ter5
days
.Al
sogr
owth
onN
Aw
ithad
ded
NaC
l
++
Bioc
hem
set,
som
eAP
I-ZYM
reac
tions
Pseu
doal
tero
mon
asde
nitri
fican
sN
egR
ods
2–4
×0.
5–0.
7mm
On
MA
2216
,col
onie
sha
vepi
nkto
red
pigm
ent
−+
Bioc
hem
set
Pseu
doal
tero
mon
asel
yako
vii
Neg
Rod
sw
ithro
unde
den
ds,
0.5–
0.8
×1.
8–4.
0mm
Gro
wth
onM
A22
16as
beig
e-co
lour
edco
loni
es,r
ound
,circ
ular
,sm
ooth
,con
vex
++
Bioc
hem
set
cont
inue
d
119Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #M.vpTuesday, January 20, 2004 3:38:02 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
104 Chapter 2
Pseu
doal
tero
mon
ases
pejia
naN
egSt
raig
htro
ds,0
.2–1
.0×
2.0–
3.5
mmG
row
thon
MA
2216
++
Bioc
hem
set
Pseu
doal
term
onas
flavi
pulc
hra
Neg
Rod
s,si
ngle
cells
,0.5
–1.5
mmG
row
thM
A22
16w
ithor
ange
colo
nies
++
Bioc
hem
set
Pseu
doal
tero
mon
aslu
teov
iola
cea
Neg
Gro
wth
onM
A22
16.C
olon
ies
3–5
mm
at4
days
at25
°C,r
egul
ar,c
onve
x,op
aque
,vi
olet
-col
oure
d
−+
Bioc
hem
set
Pseu
doal
term
onas
mar
ical
oris
Neg
Rod
s,si
ngle
cells
,0.
7–0.
9×
1.0–
1.2
mmG
row
thon
MA
2216
,as
roun
dco
loni
es2–
3m
m,c
ircul
ar,r
egul
ar,c
onve
x,tra
nslu
cent
,sm
ooth
,lem
on-y
ello
wpi
gmen
tat4
8h.
Opt
imum
25–3
5 °C
,0.5
–10%
NaC
l
++
Bioc
hem
set
Pseu
doal
tero
mon
aspi
scic
ida
Neg
Ovo
id,G
ram
-neg
ativ
ero
dsO
nM
A22
16co
loni
esar
e3–
6m
m,a
fter2
days
at28
°C.R
aise
dce
ntre
with
light
oran
geto
whi
tepi
gmen
tbec
omin
gda
rker
oran
geat
the
edge
s.W
ater
-sol
uble
pigm
entd
iffus
esin
toth
eag
ar
++
Bioc
hem
set,
carb
ohyd
rate
sus
ing
MO
F,AP
I50C
H
Pseu
doal
tero
mon
asru
bra
Neg
Stra
ight
orsl
ight
lycu
rved
rods
,2–
4×
0.8–
1.5
mmbH
Gro
wth
onM
A22
16br
ight
pink
ish-
whi
teat
2 4h
a t2 3
°C.C
entre
turn
sre
dan
dso
met
imes
blue
at4
days
,siz
e6–
7m
mw
ith2
or3
conc
entri
crin
gs.W
ithbl
ood
adde
dto
MA
2216
,col
onie
sar
ebr
ight
,muc
oid,
red
alm
ost
blac
kw
ithb-
haem
olys
is.C
olon
ies
prod
uce
adi
ffusi
ble
brow
npi
gmen
twith
anod
ouro
fhy
drog
ency
anid
e
++
Bioc
hem
set,
API-Z
YM
Pseu
doal
tero
mon
asul
vae
Neg
Rod
s1.
75–2
.5×
1–1.
5mm
Gro
wth
onTS
Aw
ith2%
NaC
l,M
SA-B
,MA
2216
.Col
onie
son
TSA
are
whi
te,w
here
ason
MA
2216
med
ium
colo
nies
are
purp
leat
48h,
23°C
++
Bioc
hem
set,
API2
0E
Pseu
doal
tero
mon
asun
dina
Neg
Cur
ved
rods
0.7–
0.9
×1.
8–3.
0mm
Gro
wth
onM
A22
16+
+Bi
oche
mse
t
Pseu
dom
onas
angu
illise
ptic
aN
egLo
ngro
ds,s
light
lycu
rved
,5–1
0mm
NH
NG
On
BA,T
SA,B
HIA
,NA
colo
nies
<1m
m,
roun
d,sh
iny,
pale
-gre
yat
4–7
days
.No
grow
thon
Pseu
dom
onas
isol
atio
nag
ar(D
ifco)
++
Bioc
hem
set
Bact
eriu
mG
ram
Mor
phol
ogy
bHTC
BSCo
lony
char
acte
ristic
sCa
tO
xId
entif
icat
ion
sets
Tab
le2.
4.C
ontin
ued.
120Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:40 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
Bacteriological Culture Techniques 105
Pseu
dom
onas
fluor
esce
nsN
egR
ods
Gro
wth
onBA
, lig
ht-g
rey
colo
nies
1.5
mm
24h
at25
°C.3
–5m
mat
48–7
2h
++
Bioc
hem
set,
API 2
0E
Pseu
dom
onas
plec
oglo
ssic
ida
Neg
Rod
s0.
5–1
×2.
5–4.
5mm
bHG
row
thon
TSA,
BA,M
SA-B
,25°
C+
+Bi
oche
mse
t,AP
I-ZYM
,API
20N
E
Pseu
dom
onas
pseu
doal
calig
enes
Neg
Shor
trod
sC
ream
-col
oure
dco
loni
es,‘
gum
my’
cons
iste
ncy
++
Bioc
hem
set
Pseu
dom
onas
stut
zeri
Neg
Stra
ight
and
slig
htly
curv
edro
ds,
som
ebi
pola
rsta
inin
gN
HN
GTh
ere
may
bero
ugh
and
smoo
thco
loni
es.
Col
onie
s0.
5m
m,g
rey,
wrin
kled
,dry
and
buff-
colo
ured
.May
besl
ight
lyye
llow
inco
lour
++
Bioc
hem
set
Ren
ibac
teriu
msa
lmon
inar
umPo
sSm
allb
acilli
,sin
gly
and
inpa
irs(0
.3–1
.5mm
)bH
NG
Col
onie
s2
mm
at20
days
at15
–18°
C,
smoo
th,c
ream
togr
anul
aron
KDM
2m
ediu
m.
On
cyst
eine
seru
mag
arco
loni
esar
eci
rcul
ar,
conv
ex,w
hite
tocr
eam
yye
llow
and
ofva
ryin
gsi
zes
On
Loef
flerc
oagu
late
dse
rum
acr
eam
ym
att
grow
this
seen
On
Dor
sete
ggm
ediu
m,g
row
thap
pear
sas
ara
ised
shin
yye
llow
laye
r
+−
Bioc
hem
set,
API-Z
YMH
aem
olys
isre
porte
dag
ains
tsa
lmon
idR
BCD
oes
notc
ross
-reac
twith
Lanc
efie
ldgr
oup
Gan
tiser
a.C
yste
ine
requ
ired
forg
row
th
Rho
doco
ccus
(lute
us)
fasc
ians
Pos
Stra
ight
orsl
ight
lycu
rved
rods
0.6–
1×
3–6
mmin
angu
laro
rpar
alle
lar
rang
emen
t
Wea
kgr
owth
onN
Aco
loni
esye
llow
,rai
sed,
glis
teni
ng,s
moo
th.G
row
thon
Low
enst
ein-
Jens
enm
ediu
mis
abun
dant
and
yello
w-o
rang
e
+Bi
oche
mse
t
( Rho
doco
ccus
mar
is)
Die
tzia
mar
isPo
sSh
orto
void
rods
0.6–
1×
1–2
mmW
eak
grow
thon
NA,
colo
nies
yello
w,r
aise
d,gl
iste
ning
,sm
ooth
+Bi
oche
mse
t
Rho
doco
ccus
spp.
Pos
Rod
s,sl
ight
lycl
ub-s
hape
d,2–
3×
0.6
mmG
row
thon
BAan
dM
SA-B
in3–
4da
ysat
25°C
.No
grow
that
37°C
(exc
eptf
orR
.equ
i).At
8da
ysco
loni
essl
ight
lydo
med
,rou
nd,
smoo
th,d
ry,d
eep
crea
my
yello
w
−−
Bioc
hem
set
Ros
eoba
cter
galla
ecie
nsis
Neg
Ovo
idro
d,0.
7–1
×1.
7–2.
5mm
On
MA
2216
at25
°C,c
olon
ies
are
circ
ular
0.5
mm
,sm
ooth
,con
vex,
brow
nish
colo
ur,
with
regu
lare
dges
.At7
days
,col
onie
sar
e2
mm
with
diffu
sibl
ebr
own
pigm
entp
rodu
ced
++
Bioc
hem
set
Ros
eoba
cter
spp.
CVS
PN
egR
ods,
0.25
×1.
0mm
Gro
wth
onSW
T,M
A22
16,1
mm
roun
d,no
n-m
ucoi
dat
5da
ys.A
ppea
ranc
eof
pink
pigm
enta
t7da
ys
w+
+Bi
oche
mse
t
cont
inue
d
121Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:40 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
106 Chapter 2
Sale
gent
ibac
ter
sale
gens
Neg
Rod
-sha
ped
cells
,sin
gly,
pairs
,oc
casi
onal
chai
nsG
row
thon
MA
2216
,NA,
TSA
++
Bioc
hem
set
Salin
ivib
rioco
stic
ola
Neg
Cur
ved
rods
0.5
×1.
5–3.
0mm
bHC
olon
ies
circ
ular
,con
vex,
crea
m-c
olou
red
at2
days
at37
°C.M
edia
with
0.5–
20%
NaC
l,M
A22
16,M
SA-B
++
Bioc
hem
set,
add
NaC
l
Serra
tiafo
ntic
ola
Neg
Rod
s0.
5×
3.0
mmG
r ow
t hon
NA
at30
and
37°C
+−
Bioc
hem
set,
API2
0E
Serra
tialiq
uefa
cien
sN
egR
ods
NH
NG
Gro
wth
onBA
,TSA
,MC
A,in
48h
at21
°C.
Non
-pig
men
ted
+−
Bioc
hem
set,
API2
0E
Serra
tiapl
ymut
hica
Neg
Rod
sN
HR
edco
loni
eson
TSA
24–4
8h
at22
°C+
−Bi
oche
mse
t,AP
I20E
Shew
anel
laal
gae
Neg
Shor
tstra
ight
rods
bHN
GC
olon
ies
yello
w-o
rang
eor
brow
naf
ter2
days
,37
°C.G
row
thon
SSag
ar,M
A22
16+
+Bi
oche
mse
t,AP
I20E
Shew
anel
laco
lwel
liana
Neg
Rod
1–3
mmlo
ng.O
nso
lidm
edia
and
inla
teph
ase
cells
beco
me
helic
al,
filam
ento
us–
20mm
NH
Col
onie
son
MA
2216
are
circ
ular
,1m
m,
conv
exw
ithun
dula
tem
argi
ns.A
t7da
ys,
5m
mw
ithirr
egul
arap
pear
ance
.In
brot
hcu
lture
(221
6)pr
oduc
esre
d-br
own
pigm
ent
++
Bioc
hem
set
Shew
anel
lafri
gidi
mar
ina
Neg
Rod
scu
rved
orst
raig
ht,
1–2.
5×
0.5–
0.8
mmG
row
thon
MA
2216
asta
n-pi
gmen
ted
colo
nies
,3–5
days
at10
°C.M
ucoi
dco
loni
esaf
ter1
0da
ysin
cuba
tion.
No
NaC
lre
quire
men
t.O
ptim
alte
mp
=20
–22 °
C
++
Bioc
hem
set,
API2
0E
Shew
anel
lage
lidim
arin
aN
egR
ods
curv
edor
stra
ight
,1.5
–2.5
mm,
sing
lyan
dpa
irsG
row
thon
MA
2216
asta
n-pi
gmen
ted
colo
nies
,3–5
days
at10
°C.M
ucoi
dco
loni
esaf
ter1
0da
ysin
cuba
tion.
NaC
lreq
uire
men
t.O
ptim
alte
mp
=15
–17 °
C
++
Bioc
hem
set,
API2
0E
Shew
anel
laja
poni
caN
egR
ods
1–2
×0.
6–0.
8mm
bHG
row
thon
MA
2216
asci
rcul
ar,s
moo
than
dsl
ight
lypi
nkco
loni
es,a
ndM
SA-B
at25
°C.
Deg
rade
sag
ar
++
Bioc
hem
set
Shew
anel
laon
eide
nsis
Neg
Rod
2–3
mm×
0.4–
0.7
mmN
HG
row
thon
MA
2216
,MSA
-B.P
ale
pink
tobe
ige-
colo
ured
colo
nies
,1–4
mm
,circ
ular
,sm
ooth
,con
vex
++
Bioc
hem
set
Shew
anel
lape
alea
naN
eg2.
0–3.
0×
0.4–
0.6
mmN
HG
row
thon
MA
2216
in2
days
at25
°C.
Col
onie
sop
aque
salm
on-c
olou
red
with
muc
oid
surfa
ce
++
Bioc
hem
set
Bact
eriu
mG
ram
Mor
phol
ogy
bHTC
BSCo
lony
char
acte
ristic
sCa
tO
xId
entif
icat
ion
sets
Tab
le2.
4.C
ontin
ued.
122Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:40 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
Bacteriological Culture Techniques 107
Shew
anel
lapu
trefa
cien
sN
egR
ods
NH
Are
ddis
h-br
own
orpi
nkpi
gmen
tis
prod
uced
++
Bioc
hem
set,
API-Z
YM,A
PI20
E,AP
I20N
E
Shew
anel
law
oody
iN
egN
on-s
poru
latin
gro
d0.
5–1.
0×
1.4–
2mm
NG
Gro
wth
onM
A22
16,a
spi
nk-o
rang
epi
gmen
tco
loni
esat
20°C
++
Bioc
hem
set,
API2
0E
Sphi
ngob
acte
rium
mul
tivor
umN
egN
on-s
porin
gro
ds1
mm,s
ingl
yor
pairs
Yello
wco
loni
eson
NA,
circ
ular
,low
,con
vex
++
Bioc
hem
set,
API-Z
YM
Sphi
ngob
acte
rium
spiri
tivor
umN
egN
on-s
porin
gro
ds1
mmN
HC
olon
ies
onN
A,ye
llow
,low
conv
ex,s
moo
th30
–37°
C+
+Bi
oche
mse
t,AP
I-ZYM
Sphi
ngom
onas
(Pse
udom
onas
)pa
ucim
obilis
Neg
Rod
s,0.
7×
1.4
mmN
HG
row
thon
NA
and
BA.Y
ello
w-p
igm
ente
dco
loni
esci
rcul
ar,l
ow,c
onve
xat
2da
ysat
22°C
++
Bioc
hem
set
Stap
hylo
cocc
usde
lphi
niPo
sC
occi
0.8–
1mm
,mos
tlyin
clus
ters
buta
lso
sing
lyan
dpa
ired
bHN
GO
nN
A,co
loni
es5–
7m
mci
rcul
ar,s
moo
th,
opaq
ueto
trans
luce
ntw
ithin
cuba
tion
+−
Bioc
hem
set.
Coa
gula
sete
st.
API-Z
YM
Stap
hylo
cocc
usho
min
isPo
sC
occi
inte
trads
,occ
asio
nally
pairs
NH
Col
onie
s3–
5m
m,s
moo
th,d
ull,
opaq
ue,
slig
htly
umbo
nate
with
beve
lled
edge
.Pi
gmen
tpal
eye
llow
togr
ey-w
hite
+Bi
oche
mse
t,C
oagu
lase
,D
Nas
e
Stap
hylo
cocc
uslu
trae
Pos
Coc
cisi
ngly
,pai
rsan
dcl
uste
rsbH
Col
onie
s1.
5–2
mm
,sm
ooth
,rou
nd24
h,37
°C+
−C
oagu
lase
test
,DN
ase
test
,Bi
oche
mse
t
Stap
hylo
cocc
usw
arne
riPo
sC
occi
0.5–
1.2
mmdi
amet
erin
pairs
and
sing
ly,o
ccas
iona
llyte
trads
NH
Col
ony
grow
thin
24h
at37
°C,3
–5m
m,
smoo
th,r
ound
,stic
ky.M
osts
train
sar
ebr
ight
yello
w-o
rang
eor
with
aye
llow
ring
arou
ndth
eed
geof
the
colo
ny.2
0%of
stra
ins
are
non-
pigm
ente
d
+Bi
oche
mse
t,C
oagu
lase
,D
Nas
e
Stap
pia
stel
lula
ta-
like
stra
inM
1N
egM
otile
rods
Gro
wth
onM
A22
16,S
WT
at23
°C.M
ucoi
dco
loni
esw
ithlig
ht-b
row
npi
gmen
tat3
days
.Po
orgr
owth
unde
rana
erob
icco
nditi
ons.
Form
sst
ar-s
hape
dag
greg
ates
whe
ngr
own
inliq
uid
med
ia
++
Bioc
hem
set,
API2
0NE
Stre
ptob
acillu
sm
onilif
orm
isN
egPl
eom
orph
icro
dsfre
quen
tlyin
chai
ns,t
angl
edfil
amen
tsw
ithbu
lbou
ssw
ellin
gs
NH
Cle
ar,n
on-h
aem
olyt
icco
loni
es,0
.5m
mat
4 8h
o nB A
inC
O2
and
37°C
.In
brot
hcu
lture
prod
uces
disc
rete
fluff-
ball-
like
colo
nies
−−
Bioc
hem
set
cont
inue
d
123Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:41 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
108 Chapter 2
Stre
ptob
acillu
sm
onilif
orm
is-li
keor
gani
sm.I
Dno
tco
nclu
sive
(Mah
eret
al.,
1995
)
Neg
Coc
coba
cilli
0.4–
0.6
mmin
tissu
es.
Old
ercu
lture
ssh
owfil
amen
tous
cells
with
poss
ible
bran
chin
gan
dso
me
term
inal
swel
lings
bHG
row
thon
BHIA
cont
aini
ngfe
talc
alfs
erum
and
NaC
l.C
olon
ies
0.1
mm
at4–
8da
ys,
aero
bica
llyat
15–2
2 °C
.Col
onie
sof
f-whi
te,
friab
le,c
onve
x,gr
anul
ar,‘
brea
d-cr
umb’
appe
aran
ce.O
lder
colo
nies
wer
eco
ncav
e‘m
olar
toot
h’ap
pear
ance
. bH
afte
r7–1
4da
ys
−−
Bioc
hem
set.
Wea
kly
acid
-fast
Stre
ptoc
occu
sag
alac
tiae
grou
pB
Pos
Coc
ciin
smal
lcha
ins
bHG
row
thon
BA.C
olon
ies
1m
m,p
ale
grey
at24
h,zo
neof
b-ha
emol
ysis
Bioc
hem
set,
API2
0St
rep,
APIr
apid
ID32
Stre
p,St
rep
grou
ping
antis
era
(gro
upB)
Stre
ptoc
occu
sag
alac
tiae
grou
pB,
Type
1b( S
.diff
icile
)
Pos
Coc
ciin
smal
lcha
ins
with
varia
ble
diam
eter
NH
Cel
lsad
here
stro
ngly
toag
ar.O
nBH
IAco
loni
es1
mm
at48
h.O
ptim
algr
owth
at30
°C.T
ests
mus
tbe
perfo
rmed
atth
iste
mpe
ratu
re.N
ogr
owth
at37
°Cun
less
grow
nin
mic
roae
roph
ilicat
mos
pher
e(5
%O
2,10
%C
O2,
85%
N2)
−St
rep
grou
ping
(gro
upB)
,AP
I20
Stre
p,AP
Irap
idID
32St
rep,
API5
0CH
,Bio
chem
set
Stre
ptoc
occu
sdy
sgal
actia
ess
p.dy
sgal
actia
ese
rova
rL
Pos
Coc
ciin
chai
nsbH
Col
onie
s1
mm
light
grey
onBA
with
zone
ofha
emol
ysis
at24
h−
APIr
apid
ID32
Stre
p.St
rept
exA-
G=
nega
tive.
Lanc
efie
ldgr
oup
L=
posi
tive,
Baci
traci
n=
sens
itive
Stre
ptoc
occu
ssp
ecie
s(G
roup
B)Po
sC
occi
inch
ains
bHN
G0.
5–1
mm
grey
-whi
te,z
one
ofha
emol
ysis
onBA
+Bi
oche
mse
t,AP
Irap
idID
32St
rep,
Stre
ptex
A-G
Stre
ptoc
occu
sin
iae
Pos
Coc
cus
0.3–
0.5
mm.l
ong
chai
nsbH
Gro
wth
onBA
,NA,
BHIA
,TSA
.Col
onie
son
BA1
mm
whi
te,u
mbo
nate
,opa
que
cent
resp
otat
24h.
b-ha
emol
ysis
surro
unde
dby
diffu
serin
gof
aha
emol
ysis
.Hae
mol
ysis
may
beva
riabl
e
−AP
Irap
idID
32St
rep,
API2
0St
rep,
API5
0CH
,Bio
chem
set.
Rea
ctio
nsat
25°C
a re
slow
erth
anat
37°C
Stre
ptoc
occu
spa
raub
eris
Pos
Shor
trod
sto
cocc
obac
illiin
pairs
orsh
ortc
hain
sa
HC
olon
ies
1.5–
2m
mat
24h,
roun
d,w
hitis
han
dsl
ight
lya
-hae
mol
ytic
−−
Bioc
hem
set,
API5
0CH
,API
rapi
dID
32St
rep.
Incu
bate
at37
°Cfo
r24
h
Stre
ptoc
occu
sph
ocae
Pos
Coc
ci1
mmin
diam
eter
,sin
gly,
pairs
orch
ains
bHO
nBA
colo
nies
circ
ular
,ent
ire,s
moo
th,
glis
teni
ngno
n-pi
gmen
ted.
0.8
mm
at24
h,37
°C
−Bi
oche
mse
t,AP
I20
Stre
p,AP
I50C
H
Stre
ptoc
occu
spo
rcin
usPo
sSp
heric
alto
ovoi
dce
llsin
smal
lch
ains
bHO
nBA
colo
nies
are
smal
l,el
evat
ed,e
ntire
and
haem
olyt
ic−
Bioc
hem
set,
API2
0St
rep,
API5
0CH
Bact
eriu
mG
ram
Mor
phol
ogy
bHTC
BSCo
lony
char
acte
ristic
sCa
tO
xId
entif
icat
ion
sets
Tab
le2.
4.C
ontin
ued.
124Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:41 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
Bacteriological Culture Techniques 109
Stre
ptom
yces
salm
onis
Pos
Myc
elia
ND
Bric
k-re
dto
oran
gesu
bstra
tem
ycel
iaan
dw
hite
topi
nkan
dye
llow
shad
esof
aeria
lm
ycel
ia
Bioc
hem
set
Tena
ciba
culu
m(F
lexi
bact
er)
mar
itim
um
Neg
Slen
der,
flexi
ble
rods
,0.5
×2–
30mm
,oc
casi
onal
lyup
to10
0mm
long
NG
On
AO-M
med
ium
,pal
eye
llow
oror
ange
,fla
t,th
inirr
egul
arco
loni
esw
ithun
even
edge
s.Sl
owsp
read
ing
grow
th,5
days
.Col
onie
sne
verl
arge
rtha
n5
mm
.Col
onie
sad
here
stro
ngly
toth
eag
ar.G
row
thva
riabl
eon
MA
2216
.Bro
wn
pigm
ento
nAO
-Mw
ith0.
5%ty
rosi
ne
V+
Bioc
hem
set,
API-Z
YM,A
PI20
E,AP
I50C
H
Tena
ciba
culu
m(F
lexi
bact
er)
ovol
ytic
um
Neg
Long
slen
derr
ods
0.4
×2–
20mm
NH
NG
Pale
yello
wco
loni
es,g
lidin
gm
otilit
y.O
nM
A22
16co
loni
eslig
htbr
owni
shye
llow
,fla
ttene
dan
del
evat
edw
ithre
gula
redg
es.R
apid
loss
ofvi
abilit
y,th
eref
ore
see
anar
eaof
lysi
sin
the
cent
reof
a5-
day-
old
colo
nyw
ithvi
able
cells
atth
eed
ge
++
Bioc
hem
set,
API-Z
YM
Vago
cocc
usfe
ssus
Pos
Cel
lsco
ccoi
d,el
onga
ted
indi
rect
ion
ofth
ech
ain,
sing
ly,p
airs
,sho
rtch
ains
aH
ND
Gro
wth
onBA
,37°
C,5
%C
O2,
s ma l
laH
colo
nies
.Cel
lsm
otile
−Bi
oche
mse
t,AP
Irap
idID
32St
rep,
API-Z
YM
Vago
cocc
usflu
vial
isPo
sC
ells
ovoi
d,el
onga
ted
indi
rect
ion
ofth
ech
ain,
sing
ly,p
airs
,sho
rtch
ains
aH
ND
On
BA,c
olon
ies
slig
htly
larg
erth
anEn
tero
cocc
ussp
ecie
san
da
H.I
mpr
oved
grow
thin
CO
2
−Bi
oche
mse
t,AP
Irap
idID
32St
rep,
API-Z
YM,L
ance
field
grou
pN
=po
sitiv
e
Vago
cocc
uslu
trae
Pos
Coc
cus,
sing
lean
din
chai
ns.C
ell
slig
htly
elon
gate
da
HSm
all0
.1–0
.2sm
ooth
colo
nies
onBA
afte
r2 4
ha t
3 7°C
and
5%C
O2.
Mot
ile−
Bioc
hem
set,
APIr
apid
ID32
Stre
p,AP
I-ZYM
Vago
cocc
ussa
lmon
inar
umPo
sC
occo
baci
lli.Fo
rms
chai
nsin
liqui
dm
edia
aH
NG
Col
onie
s0.
5–1
mm
,whi
te-g
rey,
glis
teni
ng.
Stre
p-lik
e.Sl
ight
gree
ning
ofag
araf
ter2
–3da
ys
−N
DBi
oche
mse
t,AP
I20
Stre
p,AP
Irap
idID
32St
rep,
API
50C
H,S
trep
grou
ping
Varra
calb
mi
Neg
Slen
der,
stra
ight
rods
1.7–
3.5
mmN
egor
aH
NG
1m
mop
aque
,gre
y,co
nvex
,adh
eren
tco
loni
esw
hich
leav
ean
impr
inti
nth
eag
ar.
aH
seen
afte
r1w
eek
−+
Bioc
hem
set
Vibr
ioae
roge
nes
Neg
Stra
ight
tosl
ight
lycu
rved
rods
0.6–
0.8
×2–
3mm
Col
onie
sfla
t,ci
rcul
ar,o
ff-w
hite
afte
r2da
yson
PYag
ar+
−Bi
oche
mse
t
Vibr
ioae
stua
rianu
sN
egSt
raig
htor
curv
edro
d,0.
5×
1.5–
2mm
bHY
Gro
wth
onM
SA,T
SAco
ntai
ning
0.5%
NaC
lBi
oche
mse
t.Ad
ditio
nof
NaC
lto
set,
optio
nal co
ntin
ued
125Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:41 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
110 Chapter 2
Vibr
ioag
ariv
oran
sN
egR
od2–
4×
0.4–
0.6
mmG
Non
-pig
men
ted
colo
nies
onM
SA-B
and
MA
2216
that
prod
uce
ash
allo
wpi
tin
the
med
ium
++
Bioc
hem
set,
API2
0E,
API-Z
YM.A
ddN
aClt
ose
t
Vibr
ioal
gino
lytic
usN
egN
HY
Gre
yco
loni
es.S
war
mac
ross
MSA
-Ban
dM
A22
16in
24h
at25
°C+
+Bi
oche
mse
t,AP
I20E
,AP
I-ZYM
,API
20N
E.Ad
dN
aClt
ose
ts
Vibr
iobr
asilie
nsis
Neg
Rod
,2.5
–3×
1mm
YG
row
thon
TSA+
2%N
aCl.
Col
onie
sbe
ige,
trans
luce
nt,c
onve
x,ro
und,
smoo
th,2
–3m
maf
t er 4
8h
at28
°C
++
Bioc
hem
set,
API2
0E,A
PIZY
M
Vibr
ioca
lvie
nsis
Neg
Slig
htly
curv
edor
stra
ight
rods
.0.
25–1
.0×
0.75
–2.5
mmG
Gro
wth
onM
A22
16.C
olon
ies
brow
nish
,tra
nslu
cent
,circ
ular
,sm
ooth
,con
vex,
entir
eat
25–3
0°C
.No
grow
that
37°C
++
Bioc
hem
set,
API-Z
YM.A
ddN
aClt
ose
t
Vibr
ioch
oler
aeN
egSl
ight
lycu
rved
rods
W, b
HY
Col
onie
sro
und
smoo
th2–
3m
mgr
eeni
sh-g
rey
onM
SA-B
++
Bioc
hem
set,
API2
0E,
API-Z
YM,A
PI20
NE
Vibr
ioch
oler
ae01
39N
egC
urve
dro
dsbH
YC
olon
ies
grey
ish-
opaq
uew
ithda
rker
cent
res
+Bi
oche
mse
t.R
esis
tant
tovi
brio
stat
icco
mpo
und
0/12
9
Vibr
ioci
ncin
natie
nsis
Neg
Rod
0.7–
2mm
YC
olon
ies
1–2
mm
crea
m,r
ound
,sm
ooth
,gl
ossy
,24
hat
25an
d35
°C+
+Bi
oche
mse
t.Ad
dN
aClt
ose
t
Vibr
ioco
rallii
lytic
usN
egR
ods
1.2–
1.5
×0.
8mm
YO
nM
A22
16,c
olon
ies
are
3m
mat
3da
ys,
crea
m-b
eige
,rou
nd,e
ntire
,sm
ooth
++
Bioc
hem
set,
API2
0NE.
Add
NaC
lto
sets
(3%
)
Vibr
iocy
clitr
ophi
cus
Neg
Rod
s0.
6×
1.5–
2.5
mmC
olon
ies
4m
m,c
ream
-col
oure
d,ci
rcul
ar,f
lat
onM
A22
16+
+Bi
oche
mse
t.Ad
dN
aClt
ose
t
Vibr
iodi
abol
icus
Neg
Stra
ight
rod
0.8
×2
mmY
Non
-pig
men
ted,
2m
mat
3da
yson
MA
2216
and
swar
min
g(9
mm
with
adde
dgl
ucos
e)+
+Bi
oche
mse
t,AP
I20E
,API
20N
E,AP
I50C
H,A
PI-Z
YM.
A dd
Na C
lto
s ets
Vibr
iodi
azot
roph
icus
Neg
Shor
trod
s,0.
5×
1.5–
2mm
NH
YC
olon
ies
onM
A22
16ar
efla
t,ci
rcul
arof
f-whi
te+
+Bi
oche
mse
ts.A
dditi
onof
NaC
lto
set,
optio
nal
Vibr
io(P
hoto
bact
eriu
m)
fisch
eri
Neg
Rod
s0.
5×
1–1.
5mm
.Sin
gly
orpa
irs,
roun
ded
ends
,stra
ight
orcu
rved
bH VN
Gor
GG
rey
orof
f-whi
te,t
rans
luce
ntco
loni
eson
MSA
-B,1
–2m
m.B
ecom
epa
leye
llow
,and
lum
inou
sat
3da
ys
++
Bioc
hem
set,
API-Z
YM,A
PI20
E,AP
I50C
H.A
dditi
onof
NaC
lto
set,
optio
nal
Vibr
ioflu
vial
isN
egSh
ortr
ods,
stra
ight
orcu
rved
,sin
gly
orpa
irs,m
aybe
pleo
mor
phic
VY
Col
onie
son
BHIA
are
opaq
ue,s
hiny
smoo
th,
roun
d,do
omed
,may
bem
ucoi
dan
dar
e2–
3m
mat
24h,
30°C
.Gro
wth
onBH
IA,
MSA
-B
++
Bioc
hem
set,
API2
0E.A
ddN
aClt
ose
ts
Bact
eriu
mG
ram
Mor
phol
ogy
bHTC
BSCo
lony
char
acte
ristic
sCa
tO
xId
entif
icat
ion
sets
Tab
le2.
4.C
ontin
ued.
126Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:41 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
Bacteriological Culture Techniques 111
Vibr
iofu
rnis
sii
Neg
Stra
ight
tolig
htly
curv
edro
dV
YC
olon
ies
onBH
IAar
eop
aque
,shi
nysm
ooth
,ro
und,
doom
ed,m
aybe
muc
oid
and
are
2–3
mm
at24
h,30
°C.G
row
thon
BHIA
,BA,
MSA
-B
++
Bioc
hem
set,
API2
0E,
API-Z
YM.A
ddN
aClt
ose
ts
Vibr
ioha
liotic
oli
Neg
Rod
s,0.
6–0.
8×
1.7–
2.0
mmN
DG
On
MA
2216
,bei
ge,c
ircul
ar,s
moo
th,c
onve
xco
loni
es+
+Bi
oche
mse
t.Ad
dN
aClt
ose
t
Vibr
ioha
rvey
iN
egSh
ortr
od,s
traig
htor
slig
htly
curv
ed,
roun
ded
ends
occu
rring
sing
lyor
inpa
irs
NH
YG
rey-
colo
ured
,off-
whi
te,r
aise
dsh
iny,
slow
spre
adin
ggr
owth
onM
SA-B
,may
bem
ucoi
d.Lu
min
ous
afte
r3da
ys
++
Bioc
hem
set,
API-Z
YM,A
PI20
E,AP
I20N
E,AP
I50C
H.
Add
NaC
l to
set
Vibr
ioho
llisae
Neg
Rod
,som
esl
ight
lycu
rved
W bHN
Gor
wea
kO
nBA
and
MSA
-B,c
olon
ies
1–2
mm
opaq
ue.
Hae
mol
ysis
afte
r7da
ys+
Bioc
hem
set.
Add
NaC
lto
set
Vibr
ioic
hthy
oent
eri
Neg
Shor
trod
s1.
6–2.
5×
0.6–
0.8
mmY,
wN
on-p
igm
ente
dco
loni
es+
+Bi
oche
mse
t.Ad
dN
aClt
ose
t
Vibr
iole
ntus
Neg
1.5–
3×
0.8–
1mm
GC
olon
ies
onM
A22
16at
24h
at22
°Ca r
e0.
3–0.
5m
m,r
ound
,opa
que,
non-
pigm
ente
d+
+Bi
oche
mse
t.Ad
dN
aClt
ose
t
Vibr
io(P
hoto
bact
eriu
m)
loge
i
Neg
NH
Yello
w-o
paqu
eco
loni
eson
MSA
-B+
+Bi
oche
mse
t,AP
I-ZYM
.Ad
ditio
nof
NaC
lto
sets
,op
tiona
l
Vibr
iom
edite
rrane
iN
egR
ods,
1–2
×0.
5mm
NH
YC
olon
ies
onm
arin
eag
arci
rcul
ar,t
rans
luce
nt,
non-
pigm
ente
d.O
nM
SA-B
,col
onie
sar
e2–
3m
mat
48h,
crea
my
and
muc
oid
++
Bioc
hem
set.
Add
NaC
lto
set
Vibr
iom
etsc
hnik
ovii
Neg
Shor
trod
s,cu
rved
orst
raig
ht,s
ingl
y,pa
irsor
shor
tcha
ins,
0.5
×1.
5–2.
5mm
bHY
orN
G+
−Bi
oche
mse
t.Ad
ditio
nof
NaC
lto
sets
,opt
iona
l
Vibr
iom
imic
usN
egC
urve
dro
dsbH
GC
olon
ies
roun
d,sm
ooth
,2–3
mm
gree
nish
-gre
yon
MSA
-Ban
dBA
++
Bioc
hem
set,
API2
0E,A
PI20
NE,
API-Z
YM.A
dditi
onof
NaC
l,op
tiona
l
Vibr
iom
ytili
Neg
Coc
coba
cilli.
YG
row
thM
A22
16,T
SAin
24h.
Col
onie
sro
und
and
non-
pigm
ente
d+
+Bi
oche
mse
t,AP
I-ZYM
,API
20E,
API2
0NE,
API5
0CH
.A d
dN
a Clt
os e
ts
Vibr
iona
varre
nsis
Neg
Rod
s1–
2×
0.8–
1mm
YC
olon
ies
2–3
mm
roun
dop
aque
non-
pigm
ente
d24
hon
NA
with
2%N
aCl
++
Bioc
hem
set,
API2
0NE,
API
20E.
Add
NaC
lto
sets
Vibr
ione
ptun
ius
Neg
Slig
htly
curv
edro
d,2.
3–3
×1
mmY
Gro
wth
onTS
A+
2%N
aCl.
Smoo
th,r
ound
ed,
beig
eco
loni
es3
mm
at48
han
d28
°C+
+Bi
oche
mse
t,AP
I20E
,API
ZYM
.Add
NaC
lto
sets co
ntin
ued
127Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:35:42 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
112 Chapter 2
Vibr
ioor
dalii
Neg
2.5–
3×
1mm
,cur
ved
bHN
GC
ircul
arco
nvex
off-w
hite
togr
eyco
loni
es,
1–2
mm
.4–6
days
,22°
C+
+Bi
oche
mse
t,AP
I-ZYM
,API
20E.
Add
NaC
lto
sets
Vibr
ioor
ient
alis
Neg
NH
YW
hite
-opa
que
colo
nies
onM
SA-B
++
Bioc
hem
set,
API-Z
YM.A
ddN
aClt
ose
ts
Vibr
iopa
cini
iN
egR
ods
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igm
ente
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cent
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oche
mse
t,AP
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iopa
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ytic
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edro
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rey
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nies
onBA
,MSA
-B,M
A22
16.
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mm
at24
h,3–
4m
m48
h.Sw
arm
acro
sspl
ate
++
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hem
set,
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aClt
ose
ts
Vibr
iope
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urve
dro
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ooth
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ted,
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ter4
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A22
16+
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oche
mse
t,AP
I20E
.Add
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lto
sets
Vibr
iope
naei
cida
Neg
Stra
ight
orsl
ight
lycu
rved
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trod
s,1.
5–2
×0.
5–0.
8mm
GR
ound
circ
ular
,low
,con
vex,
crea
m-c
olou
red
onM
A22
16,M
SA-B
++
Bioc
hem
set,
API5
0CH
,API
20N
E,AP
I20E
.Add
NaC
lto
sets
Vibr
iopr
oteo
lytic
usN
egY/
GFl
atsp
read
ing
colo
nies
that
will
com
plet
ely
cove
ran
MSA
-Bpl
ate
in24
h.C
olon
yco
lour
dark
ens
with
age
++
Bioc
hem
set
Vibr
ioro
tifer
ianu
sN
egC
urve
dro
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.8–1
.2×
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Gro
wth
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A22
16.T
rans
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ntno
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ted
colo
nies
+Bi
oche
mse
t,AP
I20E
,API
ZYM
Vibr
ioru
moi
ensi
sN
egR
ods
0.5–
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×0.
7–2.
1mm
.Und
erEM
,ble
bsse
enon
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surfa
ceG
row
thon
MA
2216
and
PYS-
2m
ediu
mC
olon
ies
circ
ular
and
colo
urle
ss,4
8h
at30
°C+
+Bi
oche
mse
t.Ad
d3%
NaC
lto
set
Vibr
iosa
lmon
icid
aN
egC
urve
dro
ds,0
.5×
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mmN
HG
Col
onie
son
MSA
-Bar
esm
all,
grey
,no
n-pi
gmen
ted
++
Bioc
hem
set,
API5
0CH
,API
20E,
API-Z
YM.A
ddN
aClt
ose
ts
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iosc
opht
halm
iN
egSh
ortr
ods
NH
YR
ound
non-
pigm
ente
dco
loni
eson
TSA
with
1.5%
NaC
l,M
SA-B
,MA
2216
++
Bioc
hem
set,
API2
0E.
Addi
tion
ofN
aClo
ptio
nal
Vibr
iosh
iloni
iN
egR
ods
2.4
×1.
6mm
YO
nM
A22
16co
loni
esha
vea
slig
htly
serra
ted
edge
++
Bioc
hem
set,
API2
0NE.
Use
inoc
ulum
of3%
NaC
l
Vibr
iosp
lend
idus
IN
e gR
o dbH
YW
hite
-opa
que
colo
nies
onM
SA-B
++
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hem
set,
API2
0E,A
PI50
CH
,API
-ZYM
Vibr
iosp
lend
idus
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odbH
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aque
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nies
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++
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hem
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ram
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phol
ogy
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tO
xId
entif
icat
ion
sets
Tab
le2.
4.C
ontin
ued.
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Bacteriological Culture Techniques 113
Vibr
iota
petis
Neg
Coc
coba
cilli
1–1.
5×
0.5
mmG
Gro
wth
onM
SA-B
,MA
2216
,TSA
with
2%N
aCl.
At48
h,co
loni
esci
rcul
ar,t
rans
luce
nt,
non-
pigm
ente
d
++
Bioc
hem
set,
API2
0E,A
PI20
NE.
Add
NaC
lto
set
Vibr
iotu
bias
hii
Neg
Shor
trod
sst
raig
htor
curv
ed,
0.5
×1.
5mm
bHY
Whi
te-g
rey,
opaq
ueco
loni
eson
MSA
-B,m
aybe
muc
oid
++
Fish
set,
API5
0CH
,API
20N
E,AP
I20E
.Add
NaC
lto
sets
Vibr
iovu
lnifi
cus
Neg
Cur
ved
rods
2–3
mmin
leng
thbH
GG
row
thon
MSA
-B,M
A22
16.C
olon
ies
2–4
mm
at48
han
d25
°C,l
ight
grey
,slig
htgr
een
colo
urin
heav
ygr
owth
++
Bioc
hem
set,
API2
0E.A
dd3%
NaC
lto
sets
Vibr
iow
odan
isN
egSh
orto
relo
ngat
edro
dsbH
Col
onie
son
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-B,2
–3m
mat
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roun
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aque
,yel
low
with
buty
rous
cons
iste
ncy.
Hae
mol
ysis
afte
r2da
ys
++
Bioc
hem
set,
API-Z
YM.A
ddN
aClt
ose
ts
Vibr
ioxu
iiN
eg2–
3×
1mm
YG
row
thon
TSA+
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Beig
eco
loni
es3–
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m,r
ound
,sm
ooth
,con
vex
at48
han
d28
°C
++
Bioc
hem
set,
API2
0E,A
PIZY
M
Yers
inia
kris
tens
enii
Neg
Rod
sG
row
thon
NA,
BAin
24h
at28
°C,1
–2m
m.
Cul
ture
sm
ayha
vest
rong
,mus
tyor
cabb
age-
like
odou
r
+−
Bioc
hem
set,
API2
0E
Yers
inia
ruck
eri
Neg
Rod
s,1–
3mm
NH
NG
Col
onie
s2–
3m
m,o
ff-w
hite
,gre
y,sh
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rais
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ntire
.Typ
ical
Ente
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cter
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tyPa
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-like
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ark
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48h.
Gro
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ste
ndto
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egul
ared
ges
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r48
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tion
at25
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+−
Bioc
hem
set,
API-Z
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PI20
Em
ayno
tdiff
eren
tiate
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Haf
nia
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Incu
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rbes
tre
sults
Zobe
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ods
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ded
ends
Gro
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read
ing
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nies
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ydro
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++
Bioc
hem
set,
API2
0E,A
PI20
NE,
API-Z
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dditi
onof
NaC
l,op
tiona
l
Zobe
lliaul
igin
osa
Neg
On
MA
2216
agar
colo
nies
are
oran
gew
ithte
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ous
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read
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ar
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hem
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onof
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l
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ess
stat
edot
herw
ise.
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reaction, oxidase and catalase. The ‘Identifica-tion sets’ column lists the identification sets thatcan be performed for identification of thatorganism.
2.4 Biochemical Identification Tests
Biochemical identification of a bacteriumproceeds through a number of steps. A puresubculture of the organism is used to performprimary identification tests and to inoculatebiochemical identification sets (secondaryidentification tests), composed of either in-houseprepared media, or commercial identificationsets such as API, available from bioMérieux.The identification sets are incubated at theappropriate temperature and for the appropriatetime for reactions to occur. The results arerecorded on a laboratory work sheet or, in thecase of the commercial identification kits, onthe supplied recording sheets. See Chapter 3for the interpretation of biochemical identificationsets. The media and reagents used in the bio-chemical identification are detailed in Chapter 7,along with information on growth characteristicsand reagent reactions.
The tests described in this chapter and Table3.1 form the ‘Biochem set’.
Primary tests
The primary tests include microscopicexamination of smears, in particular the Gramstain, catalase, oxidase, presence of haemolysis,motility, and growth on MCA. More informationon the performance of these tests and theirinterpretation is found in Chapter 3, and methodsof media and reagent preparation are describedin Chapter 7.
Secondary tests: biochemicalidentification sets
Biochemical identification is achieved bysecondary identification tests, which are themain tests used to identify an organism to specieslevel.
Tubed media
CARBOHYDRATE FERMENTATION. L-arabinose,glucose, inositol, lactose, maltose, mannitol,mannose, salicin, sorbitol, sucrose, trehalose,xylose. The carbohydrates are commonly called‘sugars’.
DECARBOXYLASES. ADH, LDC, ODC, decar-boxylase control tube.
OTHER BIOCHEMICAL TESTS. Aesculin, motility,MRVP, nitrate, oxidative fermentative tubes,ONPG, TSI for H2S, indole, urea. See next sectionfor inoculation methods.
GROWTH AT DIFFERENT TEMPERATURES. U s eeither a TSB or glucose tube and incubate at 37°C,40°C or desired test temperature.
GROWTH IN 10% NaCL. To test an organism’sability to grow in the presence of 10% NaCl, dis-pense equal volumes of TSB and 20% NaCl into asterile McCartney bottle or bijou bottle to give afinal concentration of 10% NaCl. Inoculate withthe organism to a concentration of McFarlandopacity tube 1. Incubate at 25°C or optimumtemperature and after 24 h incubation observe forevidence of growth as seen by an increase in theturbidity of the broth.
Plate media
DNase plate, Gelatin/salt plate, MCA, TCBS. Usea MSA-B or BA plate for purity check of theinoculum.
Discs
Ampicillin 10 mg, ‘Vibrio discs’ 0129 10 mg and150 mg. The discs are placed on a lawn inoculumof the bacterium on either a BA or MSA-B plateaccording to the organism’s growth requirements.The inoculum should be prepared to a density ofMcFarland tube 1. Use for Listonella, Moritella,Photobacterium, Vibrio and Aeromonas species.The Vibrio discs (vibrio static agent pteridine0/129) differentiates between Vibrio species andAeromonas species, the latter being resistant toboth concentrations. A zone of 9 mm is classifiedas susceptible for the 0/129 150 mg disc(Bernardet and Grimont, 1989). Also seeChapter 7, ‘Preparation of Media for Cultureand Identification’.
114 Chapter 2
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2.5 Inoculation of BiochemicalIdentification Sets
Examine the subculture plate and, if the growth ispure, use to inoculate biochemical identificationsets. For marine samples, NaCl needs to beadded to the set at a final concentration of 2%.Many enzymes from a variety of Vibrio specieswill not be active at lower NaCl concentrationsand false negative results will be obtained. Thismay apply to indole, VP, ADH, ODC and LDCreactions. Some tests need a heavy inoculum.These are the decarboxylases and ADH, ureaand citrate. Incubate the set at the appropriatetemperature for a minimum of 48 h. ODC, LDC,ADH, MR, VP, and indole usually require48 h incubation as a minimum for a positivereaction even if reasonable growth is seen inthe tubes.
To inoculate the tubes, plunge a sterilePasteur pipette three-quarters of the way into thetubed media, releasing 3–4 drops of inoculum asthe pipette is withdrawn.
After inoculating all the tubes, place one dropof the inoculum on to a purity plate and streak outfor isolated colonies. After suitable incubation,check that the growth is pure.
Freshwater isolates
For bacteria isolated from freshwater sources,inoculate bacterium into sterile normal saline orsterile distilled water (usually 10 ml) to opacityMcFarland 3. Add 3–5 drops of inoculum to eachtube. Some media require a heavy inoculum(opacity approx McFarland 6). These areaesculin, ADH, LDC, ODC, the decarboxylasecontrol, MRVP, citrate and urea.
Marine isolates and addition of NaCl
Fish organs are at physiological saline level.Therefore, be aware that some bacteria mayadapt to this NaCl concentration. By subculturingan organism to BA and MSA-B, or culturing to a0% and 3% salt plate, the salt requirement orpreference can be determined. However, manyof these organisms produce more accurate results
of biochemical tests when the NaCl is added tothe media, usually at a final concentration of 2%.Therefore check the optimal NaCl concentrationrange of the suspect organism from the NaClcolumn in the tables for biochemical results(Tables 4.1 to 4.22).
For bacteria isolated from the marineenvironment, the optimal final salt concentrationin the medium is all-important. Insufficient saltconcentration will lead to false negative resultseven though good growth may be seen in the tube.An example of this is V. parahaemolyticus, whichwill give a negative indole result when aninoculum of physiological saline is used, but apositive result when a final concentration of 2%NaCl is achieved in the tryptone water (indole test)medium (See photographic section).
Tubed media can be prepared with orwithout added NaCl. However, a laboratory maywant to keep media preparation to a minimumand prepare all media without additional NaCl. Atthe time of inoculation, 500 ml of a 20% sterileNaCl stock solution is added to all liquid media(5 ml) (aesculin, indole test, ADH, ODC, LDC,nitrate, MRVP). A sterile 20% salt stock solutioncan be prepared in a Schott bottle to which anautoclavable 2 ml volumetric dispenser is added,and set to 500 ml. An example of a volumetricdispenser is a Socorex Calibrex 520, 2 ml with0.05 ml divisions.
Paraffin oil overlay
ADH, LDC, ODC and control and one tubeof the OF media is overlaid with sterile paraffinoil, to approximately 5 mm depth. For easeof use, paraffin oil can be sterilized in a Schottbottle to which a 2 ml volumetric dispenser isadded (Socorex, Calibrex 520, 2 ml with 0.05 mldivisions).
Incubation
Plate and tubed media are incubated at theappropriate temperature and duration for theappropriate bacterium; 25°C and 2–5 daysis used as a general rule. Refer to incubationguidelines in Table 2.3.
Bacteriological Culture Techniques 115
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2.6 API Identification Systems
API 20E bioMérieux, Marcyl’Etoile, France
API 20NE bioMérieux, Marcyl’Etoile, France
API 50CH bioMérieux, Marcyl’Etoile, France
API rapid A bioMérieux, Marcyl’Etoile, France
API rapid ID 32 STREP bioMérieux, Marcyl’Etoile, France
API 20 Strep bioMérieux, Marcyl’Etoile, France
API Coryne bioMérieux, Marcyl’Etoile, France
API ZYM bioMérieux, Marcyl’Etoile, France
Identification using API 20E
There have been a number of reports in theliterature about the failure of the API 20E systemto identify many Aeromonas and Vibrio species(Santos et al., 1993). Some of this is due in partto the lack of such information in the API data-base. However, it is well known that differencesin reactions occur between the API system andconventional biochemical tests. This is especiallytrue of the decarboxylases, citrate, urea, indoleand VP. Where such differences have beenreported in the literature, these have beenindicated in the conventional tables (Biochemset). When using the biochemical identification
tables in this book, make sure the correctdatabase is referred to, i.e. the conventional data-base (Tables 4.1 to 4.22), or the API 20Edatabase (Tables 4.23 to 4.25).
MacDonell et al. (1982) recommends usinga diluent containing 20‰ salt. They usedmarine salts mix (Instant Ocean) purchasedfrom Aquarium Systems, Mentor, Ohio, which iscomposed of marine salts with salinities adjustedto 20‰. Kent (1982) recommended suspendingbacterial cells in 50% sterile artificial seawater forinoculating the API 20E strips. Artificial seawatersalts can also be purchased from Sigma (seeChapter 7, ‘Preparation of Media for CulturalIdentification’). Alternatively, an inoculum with2% sterile NaCl works well for the commonlyisolated Vibrio, Photobacterium and Listonellaspecies. The optimal NaCl concentration isextremely important for tests such as citrate,urea, MR, VP and indole. For example,V. parahaemolyticus gives a negative result forindole when an inoculum of 0.85% NaCl is used,but a positive indole result is achieved when 2%NaCl is used.
Adjust the suspension of cells to an opacityof McFarland number 1 and inoculate the set.It is recommended that the API 20E system isincubated at 25°C for 48 h. The sugars are read at24 h and the remaining tests are read at 48 h. Thedecarboxylases (LDC, ODC) and ADH reactionsmay not be detected until 48 h. As recommendedby the manufacturer, a negative nitrate reactionshould always have zinc dust added to the reactioncupule to determine a true negative result (seeTable 3.1). See Chapter 4 for interpretation andresults from the API kits.
116 Chapter 2
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3Interpretation of Biochemical Identification Tests and Sets
3.1 Conventional Media: ‘Biochem Set’
Positive sugar fermentation results may berecorded at 24 h. All liquid media must be incu-bated for at least 48 h, and sometimes longer, forpositive results to be achieved. This applies inparticular to ADH, LDC, ODC, aesculin, citrateand MRVP. For interpretation see Table 3.1.
3.2 Identification Tests and theirInterpretation
This section contains notes on the interpretationof identification tests and any problems that mayoccur. A description of each test is also found inChapter 7 ‘Preparation of Media for Culture andIdentification’.
Aeromonas salmonicida fluorescent antibodytest (FAT)
A FAT for the Australian strain of Aeromonassalmonicida has been noted to cross-react withAeromonas sobria.
Aesculin
A positive result is recorded when the colouris pitch-black and when half or more of thetube is blackened (MacFaddin, 1980). Shades ofgrey should not be recorded as positive. Resultsshould be read up to 3 days. Some organisms will
not grow in the aesculin medium despite theoptimal salt concentration. These are Listonellaanguillarum, L. pelagia and Vibrio ordalii. Theseorganisms are all negative for aesculin production.
Some bacteria have been reported toproduce melanin (Coyne and Al-Harthi, 1992)and in the aesculin medium will cause blackeningof the medium (Choopun et al., 2002). Therefore,it is recommended that an aesculin test showingblackening of the medium be tested for truehydrolysis of aesculin by determining the loss offluorescence in long-wave UV light at 354 nm.Aesculin will fluoresce and therefore the presenceof fluorescence will indicate that the aesculin hasnot been hydrolysed (MacFaddin, 1980).
Some plastic tubes are UV-opaque; there-fore, test for fluorescence by holding a Wood’s UVlamp over the top of an opened tube, or pour thecontents into a Petri dish to observe presence orabsence of fluorescence. When using UV light,protect the eyes with UV-opaque glasses.
ADH
Some strains of Vibrio may show contradictoryresults for ADH when using Møller’s or Thornley’smedium. These are V. mediterranei, V. mytili,V. orientalis, most strains of V. splendidus I, andsome strains of V. tubiashii. They are all positiveaccording to Thornley’s method, but negative inMøller’s ADH. The glucose in Møller’s mediumappears to inhibit the reaction due to cataboliterepression of the inducible ADH system (Maciánet al., 1996). Photobacterium species were found
©N.B. Buller 2004. Bacteria from Fish and Other Aquatic Animals:a Practical Identification Manual (N.B. Buller) 117
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118 Chapter 3
Test
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dgr
owth
orla
ckof
grow
thon
0%or
3%N
aCl.
Hol
dpl
ate
agai
nsta
dark
back
grou
ndan
dw
ithtra
nsm
itted
light
note
whe
ther
azo
neof
gela
tinhy
drol
ysis
can
bese
enar
ound
the
bact
eria
lcol
ony.
This
can
bese
enas
azo
neof
clea
ring
onth
e0%
NaC
lsid
eor
asa
zone
ofop
acity
onth
e3%
NaC
lsid
e.C
hillin
gth
epl
ate
at4 °
C,o
rflo
odin
gw
itham
mon
ium
sulp
hate
may
impr
ove
read
abilit
yof
zone
sSe
eph
otog
raph
icse
ctio
n
H2S
Nil
Blac
keni
ngof
med
ium
espe
cial
lyal
ong
the
inoc
ulum
line
Indo
leAd
d3–
7dr
ops
ofKo
vács
reag
ent
Form
atio
nof
uppe
rpin
kla
yeri
ntu
be.S
eeph
otog
raph
icse
ctio
nTh
ete
stso
lutio
nm
usth
ave
NaC
l(2%
final
conc
entra
tion)
adde
dto
itif
the
orga
nism
requ
ires
NaC
lfor
grow
th
Mot
ility
–tu
bem
etho
dN
ilM
otile
bact
eria
grow
and
spre
adth
roug
hth
ese
mi-s
olid
gel.
Spre
adin
gis
seen
asdi
ffuse
grow
them
anat
ing
from
the
line
ofin
ocul
atio
n.N
on-m
otile
bact
eria
dono
tmig
rate
from
the
line
ofin
ocul
atio
n.
Tab
le3.
1.In
terp
reta
tion
ofte
sts
for
‘bio
chem
set’.
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Interpretation of Biochemical Identification Tests 119
Mot
ility
–ha
ngin
gdr
opm
etho
dN
ilH
angi
ngdr
op. P
lace
adr
opof
salin
eon
toa
cove
rslip
. Mix
inso
me
orga
nism
sfro
ma
plat
eor
brot
hcu
lture
inlo
garit
hmic
phas
eof
grow
th.I
nver
tthe
cove
rslip
onto
agl
ass
slid
eth
atha
sbe
enpr
epar
edw
ith3–
4m
mdo
bsof
plas
ticin
eor
equi
vale
nt,s
oth
atth
edr
opco
ntai
ning
the
orga
nism
sis
susp
ende
dfro
mth
eco
vers
lip.
The
cove
rslip
may
bepl
aced
dire
ctly
onto
the
mic
rosc
ope
slid
e,bu
tmus
tbe
exam
ined
befo
reth
esl
ide
has
drie
d.C
are
mus
tals
obe
take
nth
atth
eliq
uid
isno
tpul
led
from
the
slid
eif
itco
mes
into
cont
actw
ithth
em
icro
scop
est
age
appa
ratu
s,as
the
appe
aran
ceof
bact
eria
lcel
lsbe
ing
draw
nto
the
side
ofth
eco
vers
lipm
aygi
veth
eap
pear
ance
ofm
otilit
y.Ex
amin
eun
der ×
40or
×100
obje
ctiv
ean
din
spec
tbac
teria
lcel
lsfo
rde
finite
indi
vidu
alm
otio
nth
atca
uses
the
cell
totra
vers
eth
egl
ass
slid
e.D
ono
tmis
take
mot
ility
forB
row
nian
mot
ion,
whi
chis
seen
asm
ovem
ento
fthe
cell
with
ina
smal
lcon
fined
area
,usu
ally
the
diam
eter
ofth
ece
ll
MR
(Met
hylR
ed)
Add
3–5
drop
sof
Met
hylR
edre
agen
tafte
r48
hin
cuba
tion
(test
forV
oges
-Pro
skau
er(V
P)re
actio
nfir
st)
Pers
iste
nce
ofre
dco
lour
The
reac
tion
shou
ldno
tbe
test
edle
ssth
an48
haf
teri
ncub
atio
nan
dpr
efer
ably
3da
ysbe
caus
ea
fals
e-ne
gativ
ere
actio
nm
aybe
obta
ined
.Aliq
uots
can
beta
ken
and
test
edda
ily.A
nega
tive
reac
tion
shou
ldbe
incu
bate
dfo
r3da
ysSe
eph
otog
raph
icse
ctio
n
VPR
emov
e20
0ml
ofm
ediu
mfro
mM
RVP
tube
and
plac
ein
toa
0.6
mlm
icro
fuge
tube
.Add
one
drop
each
ofVP
Ian
dVP
IIre
agen
ts(c
anus
ere
agen
tsfro
mAP
I20E
kit)
Afte
r10–
20m
in,d
evel
opm
ento
fpin
k-re
dco
lour
indi
cat e
sa
posi
tive
reac
tion.
Ifan
aliq
uoti
sne
gativ
eat
24h,
incu
bate
fora
furth
er24
han
dre
peat
test
.The
tem
per a
ture
ofin
cuba
tion
isim
porta
ntfo
rVP
asso
me
orga
nism
sm
aybe
nega
tive
at37
°C,b
utpo
sitiv
eat
25°C
Nitr
ate
Add
5dr
ops
ofea
chre
agen
t,N
itrat
eA
and
Nitr
a te
B.C
anus
ein
-hou
sepr
epar
edre
agen
ts,o
rrea
gent
sfro
mAP
I20E
kit
Form
atio
nof
are
dco
lour
ispo
sitiv
eFo
rneg
ativ
ere
actio
nsad
da
mat
ch-h
ead
size
amou
ntof
zinc
dust
.For
mat
ion
ofa
pink
colo
urco
nfirm
sa
nega
tive
nitra
tere
sult,
whe
reas
nofu
rther
colo
urw
ithZn
i ndi
cate
sa
posi
tive
reac
tion
OF
med
ium
(oxi
dativ
efe
rmen
tativ
e)N
ilR
ecor
dfo
rmat
ion
ofye
llow
colo
ur(g
luco
sefe
rmen
tatio
n)in
tube
s.R
ecor
dgr
owth
seen
inon
eor
both
tube
s.G
row
thin
the
tube
with
outp
araf
finoi
l(op
entu
be)i
ndic
ates
anox
idat
ive
orga
nism
.Gro
wth
inbo
thth
eop
enan
dco
vere
dtu
be,i
ndic
ates
the
orga
nism
isa
facu
ltat iv
ean
aero
bean
dgr
ows
with
and
with
outo
xyge
n
ON
PGN
ilYe
llow
colo
uris
posi
tive.
Cle
aror
noco
lour
isre
cor d
edas
nega
tive
TCBS
(thio
sulp
hate
-citr
ate-
bile
salts
-suc
rose
agar
)
Nil
Rec
ord
grow
thof
yello
w(Y
)org
reen
(G)c
olon
ies,
orre
cor d
‘no-
grow
th’
See
phot
ogra
phic
sect
ion
‘Vib
riodi
scs’
0/12
910
mgan
d15
0mg
Nil
Rec
ord
azo
near
ound
the
disc
as‘s
ensi
tive’
,and
nozo
neas
‘resi
stan
t’.A
zone
of9
mm
iscl
assi
fied
assu
scep
tible
fort
he0/
129
150
mgd i
s c
Not
es:S
ome
orga
nism
sw
illno
tgro
win
the
aesc
ulin
med
ium
desp
iteth
eop
timal
salt
conc
entra
tion.
Thes
ear
eL.
angu
illaru
m,L
.pel
agia
and
V.or
dalii.
Thes
eor
gani
sms
are
alln
egat
ive
fora
escu
linpr
oduc
tion.
Oth
erte
sts:
Fora
desc
riptio
nof
othe
rtes
ts,s
uch
ashi
ppur
ate
hydr
olys
iset
c,se
eun
derM
edia
(Cha
pter
7).
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to produce alkaline products in Thornley’smedium, yet none possessed a constitutive ADHsystem when tested with more sensitive analyticalmethods (Baumann et al., 1971; West andColwell, 1984). Other Vibrio species such asListonella (Vibrio) anguillarum, Photobacterium(Vibrio) damselae ssp. damselae, V. fluvialisand V. furnissii are positive for ADH byboth methods. Thornley’s method is therecommended method for Vibrio species (Maciánet al., 1996).
Carbohydrate fermentation
In some reports in the literature, the termsutilization and fermentation have been usedinterchangeably and it has been difficult toascertain which result was reported from whichtest method. Fermentation should be used todescribe a result from the breakdown of acarbohydrate or ‘sugar’ reaction such as used inthe conventional biochemical media and the API20E. A pH indicator in the medium is able todetect the acid change caused by the breakdownproducts. Utilization should only refer to tests thatdetermine an organism’s ability to use a sub-stance as a sole carbon source. There is usuallyno pH indicator in the medium and growth isobserved as an increase in opacity or turbidityof the media. The exception is Simmons citratemedium, which determines an organism’s abilityto use citrate as a sole carbon source. There areno other nutrients in this medium. On the otherhand, Christensen’s citrate method containsother nutrients and is not a utilization test forcitrate as a sole carbon source (Cowan and Steel,1970; MacFaddin, 1980).
Carotenoid pigment detection
A few drops of 0.01% aqueous CR (Congo Red)is placed on a few isolated colonies growing ona medium such as AO agar. After 2 min thecolonies are rinsed with water. The coloniesdevelop a red colour if the test is positive and thecolour can last for a number of hours. CR detectsthe presence of extracellular galactosamineglycan (Johnson and Chilton, 1966, citing, E.J.Ordal, personal communication).
Catalase
A loopful of bacterial growth is removed from theculture plate and smeared to a glass slide. A dropof 30% hydrogen peroxide is placed on to thebacterial cells and the appearance of bubblesindicates a positive test. When removing cellsfrom an agar medium that contains blood, caremust be taken not to transfer any of the blood-containing medium with the bacterial growth, asthe presence of blood may lead to a false-positiveresult.
CR
Used for the detection of carotenoid pigment.See under carotenoid pigment detection.
Decarboxylases
ADH, ODC and LDC should be inoculated with aheavy inoculum of the organism. Incubate for aminimum of 2 days and maximum of 14 days.Add salt to give a final concentration of 2% formarine organisms. Without NaCl many tests mayproduce a false-negative reaction.
Flexirubin pigment
Flexirubin pigment may be produced by someof the members of the Flavobacteriaceae family.For its detection, growth is taken from a cultureplate (usually AO medium) and smeared toa glass slide, which is placed on a white back-ground such as a piece of paper. The colonymass is flooded with 20% KOH and examined foran immediate colour change to reddish purple orbrown. It may be helpful to place two bacterialmasses on the glass slide, one that is floodedwith KOH and the other to act as a controlfor colour differentiation. The KOH can also beused directly on the colony growth on a cultureplate; however, the colour change may notbe noticeable if there is only a thin layer ofgrowth (Bernardet et al., 2002). A reversion tothe original colour occurs when the bacterial cellsare flooded with an acidic solution (Reichenbachet al., 1989).
120 Chapter 3
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Gliding motility
The demonstration of gliding motility of theCytophaga–Flavobacterium–Bacteroides groupmay be difficult as much by choice of culturetechnique and as by the definition of glidingmotility. The organism needs to be grown ona low-nutrient-concentration agar such as thatof AO, which contains 1.5% w/v agar. Glidingis affected by the amount of surface moistureand best results are obtained with freshly pouredplates and incubation in a humid atmosphere.Gliding is observed by direct microscopic obser-vation of the swarming edge of bacterial growthon a thinly poured plate after overnight incuba-tion. A high-powered dry lens is used. Glidingis defined as ‘movement that is continuous andregularly follows the long axis of the cells thatare predominantly organized in bundles duringmovement’. It should be noted that spreadingcolonies are not necessarily an indication ofgliding motility, as spreading may result fromother forms of surface translocation mechanisms(Henrichsen, 1972). Gliding can usually besuspected if a colony has a rhizoid shape onAO agar; however, this will not be seen if theagar surface is too dry. Nor will gliding motilitybe seen if bacterial growth is suspended insaline. Gliding motility can also be observed ina hanging drop prepared from a liquid culture(Bernardet et al., 2002).
Haemolysis
Haemolysis is observed on BA. A clear zoneof lysis of the red cells around a bacterial colonyis referred to a b-haemolysis (bH). A greenishtinge seen around some colonies such assome of the Streptococci species, is referredto as a-haemolysis (aH). Streptococcus iniaeshows complete b-haemolysis on sheep bloodagar, but only partial haemolysis when themedium is supplemented with human or bovineblood.
Some Vibrio species are haemolytic onBA without salt, but non-haemolytic on salt-containing media such as MSA-B, even thoughtheir preferred growth medium is with Na ions. Itis suggested that haemolysins may be producedwhen the organism is under stress.
Indole
A false-negative result may be recorded forsalt-requiring organisms if there is an insufficientfinal concentration of NaCl in the test medium.Growth may be seen in the test medium;however, the organism may not express theenzyme unless the NaCl concentration is optimal.To achieve a 2% NaCl final concentration that isoptimal for the majority of marine organisms, add500 ml of 20% NaCl stock solution to 5 ml of testmedium. Refer also to section 2.5 (Inoculation ofBiochemical Identification Sets).
KOH
Used for the detection of flexirubin pigment.See under flexirubin pigment.
Luminescence
Luminescence may be detected by growing theorganism on an appropriate growth medium.Nutrient broth No. 2 (Oxoid) (25 g), NaCl(17.5 g), KCl (1.0 g), MgCl2.6H2O (4.0 g), agar(12.0 g), distilled water (1000 ml) (Furniss et al.,1978). However, reliance on luminescence needsto be treated with caution, as expression of lumi-nescence appears to be dependent on a numberof factors, including media, and ideally should bemeasured with a luminometer rather than by eye(J. Carson, Department of Primary Industries,Water and Environment, Tasmania, 2003, per-sonal communication). Luminescence is optimalafter 18–24 h of incubation at 25°C. It is alsodependent upon aerobic conditions, so that brothcultures need to be shaken to aerate the mediumbefore luminescence can be detected. Lumines-cence is detected by observing the plates or brothculture in a dark room and allowing the eyes toadjust to the dark for 5 min. However, if possible,luminescence should be measured from a brothculture using an instrument such as a WallacMicrobeta Plus liquid scintillation counter, whichmeasures relative light units (Manefield et al.,2000).
Organisms that are positive for luminescenceinclude Photobacterium leiognathi, P. phos-phoreum, Vibrio fischeri, V. logei, V. orientalis,
Interpretation of Biochemical Identification Tests 121
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V. splendidus biovar I (Furniss et al., 1978; Lunderet al., 2000).
Organisms that are negative for luminescenceinclude Aeromonas salmonicida, Enterovibrionorvegicus, Listonella anguillarum, L. pelagia,Moritella marina, M. viscosa, Photobacteriumangustum, Photobacterium damselae ssp.damselae, Plesiomonas shigelloides, Vibrioagarivorans, V. alginolyticus, V. brasiliensis,V. calviensis, V. campbellii, V. coralliilyticus, V.diazotrophicus, V. fluvialis, V. furnissii, V.gazogenes, V. halioticoli, V. ichthyoenteri,V. lentus, V. metschnikovii, V. natriegens, V.navarrensis, V. neptunius, V. nereis,V. nigripulchritudo, V. ordalii, V. pacinii, V.parahaemolyticus, V. penaeicida, V. proteolyticus,V. rotiferianus, V. salmonicida, V. splendidusbiovar II, V. tapetis, V. tubiashii, V. vulnificus,V. wodanis and V. xuii (Furniss et al., 1978;Lunder et al., 2000; Gomez-Gil et al., 2003a,b).Vibrio fischeri NCMB 1281T was reported tobe negative for luminescence (Lunder et al.,2000).
Strains of V. cholerae, V. logei, V. salmon-icida and V. harveyi are variable for luminescence(Furniss et al., 1978; Lunder et al., 2000).
MR
The MRVP medium (Difco) should be inoculatedwith a heavy inoculum of the organism. Saltneeds to be added if required as a growth factorby the organism under identification. Incubate ateither 25 or 37°C according to the organism’srequirements (see Tables 2.2, 2.3, 2.4). Incubatefor 48 h, and then detect the presence ofacetylmethylcarbinol by adding 3–5 drops ofMR. Continuation of a red colour indicates apositive result. For a negative reaction the redcolour disappears as the MR reagent is addedto the reaction tube. The success of this testfor a positive result is dependent on incubationtime, not the amount of growth, althoughobviously growth must be seen in the tubeto indicate that the organism has grown in themedium. The test can be carried out at 24 h ifdesired, by removing an aliquot (200 ml) to amicrofuge tube and testing for MR by the additionof 1 drop of MR reagent. A negative reactionmust be incubated for a further 24 h and re-tested after this time before being classified asnegative. MR may take 3 days for a positivereaction.
Oxidase test
Oxidase test strips are available commercially,and these are recommended because ofstandardization of the reagent. For the manualoxidase test use the oxidase reagent tetramethyl-p-phenylenediamine. Prepare a 1% solution indistilled water and in a light-protecting bottle andstore at 4°C. For use, place a few drops of thereagent on to some filter paper. Using a woodenmatchstick such as an orange stick or a platinumloop, smear bacterial growth on to the moistenedfilter paper. Development of a purple colourwithin 10–30 s denotes a positive.
A nichrome loop should not be used, as itcauses false-positive reactions. This test should notbe performed from organisms grown on mediathat contain sucrose or nitrates. Therefore TCBS isan unsuitable medium from which to determinean oxidase reaction (Furniss and Donovan, 1974;Jones, 1981).
TCBS
This medium is used to identify an organism as aVibrio species. However, there are some Vibriospecies that do not grow on TCBS. These areMoritella species, Photobacterium leiognathi,Vibrio hollisae and Vibrio ordalii. Listonella(Vibrio) anguillarum may show slow growth onTCBS. Strains of Vibrio fischeri may show weakor negative growth.
TCBS is not strictly selective and some otherorganisms will grow, although at a reduced colonysize. Species of Aeromonas and Enterococcus willgrow as small (1 mm) yellow colonies on TCBSplates. Proteus species may grow as 1 mm yellow/green colonies on TCBS. Plesiomonas does notgrow well on TCBS. Some yellow colonies ofVibrio species may revert to green colonies after afew days growth as they use up the sucrose in themedium (Oxoid manual).
VP
The MRVP medium (Difco) should be inoculatedwith a heavy inoculum of bacterium. Salt needsto be added if required by the organism. Incubateat either 25 or 37°C according to the organism’srequirements (see incubation Table 2.3). As ageneral rule use an incubation temperature of25°C for all bacteria isolated from aquatic envi-ronments and aquatic animals. Incubate for 48 h,and then detect acetoin production using the
122 Chapter 3
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reagents. VP can be tested at 24 h if there issufficient growth in the test medium. A negativeresult, however, should be re-incubated for48–72 h. Test an aliquot by placing 200 ml of theincubated medium into a 0.6 ml microfuge tube.Add one drop each of VP reagent A and VPreagent B. Commercial reagents from the API20E kit work well with the Difco MRVP medium.Examine for red colorization up to 20 min.Although the two tests can be performed in thesame tube, it is preferable to test the reactions at24, 48 and 72 h. The incubation time influencesthe production of acetoin.
Vibriostatic agent
Resistance to the vibriostatic agent pteridine(0/129) has been noted in some developingcountries. Microbiologists are advised to continueusing 0/129 as a means of differentiating Vibriospecies from Aeromonas species, but cautionmay be required in some countries (Huq et al.,1992; Nair and Holmes, 1999). A zone of 9 mmis classified as susceptible for the 0/129 500 mgdisc for Vibrio species (Bernardet and Grimont,1989). A zone size of 22 mm is considered sensi-tive for Photobacterium damselae ssp. damselae(Love et al., 1981). Vibrio cholerae strain 0139 isresistant to the 0/129 500 mg disc (Albert et al.,1993; Islam et al., 1994).
3.3 Using the BiochemicalIdentification Tables
The following sections are devoted to the identifi-cation of an unknown organism using phenotypicor biochemical tests. The variations in the litera-ture for different strains are also recorded, so thatthe microbiologist will be aware of the difficultiesencountered when identifying particular species.Hopefully this system should not prove tootedious for the user of the manual; it is intendedto give the users more confidence in namingan organism and to alert them to potentialdifficulties.
In some cases, species are relatively newlydescribed and different researchers have obtaineddifferent biochemical reactions. Rather than pre-senting a consensus result, both results are listed.It must be stressed that difficulties arise when
assessing results of some phenotypic tests whentwo different versions of a biochemical test havebeen used. Where differences were noted fromdifferent journal articles, these are either stated inthe tables, or are detailed under the notes on inter-pretation and the problems encountered, section3.4. Other notes are from the author’s experiencewith particular species. Phenotypic tests are stillthe primary identification system, and thereforemicrobiologists must be aware of the difficultiesencountered with some tests for particular organ-isms. Some species have been newly described onone strain only and as more information is gath-ered about this species it may become evident thatthe type strain is not in actual fact representative ofthe species (Janda and Abbott, 2002). Thereforesubsequent strains isolated and identified asbelonging to this species may show slightly differ-ent results in the biochemical tests. Therefore,reporting these differences may assist the user ofthis manual with identification. For example thetype strain of Vibrio lentus is negative for mannitolfermentation, yet all other species reported thusfar are positive. Likewise with Vibrio penaeicida,where the type strain is negative for indole and50% of other strains are positive. The atypicalAeromonas salmonicida group is another exam-ple. In this case, different biochemical results arereported from strains isolated from different fishspecies. For the diagnostic laboratory trying toidentify an unknown isolate, it may be difficultto say for certain that the isolate is an atypicalA. salmonicida. However, with the phenotypicdetails listed of all isolates from different fishspecies, it may enable a more definitiveidentification, or at least an identification can bemade with slightly more confidence.
There are limitations to all phenotypic(and genotypic) identification systems (Janda andAbbott, 2002). By detailing the biochemical varia-tions it is hoped that the user of the manual will beassisted in making a more informed identification.
3.4 Interpretation and Identification ofGenera and Species
In the biochemical identification tables (Tables4.1–4.31), where authors have recorded a differ-ent result for the same type strain, then theseresults are listed separately. Rather than present aconsensus from the literature, listing the differentresults provides an indication of those tests that
Interpretation of Biochemical Identification Tests 123
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are likely to produce disparate results for the indi-vidual organisms. However, for some results a ‘v’or variable reaction is recorded. Variable resultsmay have been reported in the literature or theorganism itself may be variable in its fermentationof the biochemical.
This next section provides informationabout discrepancies found in the literature orinformation that is specific to the identification of acertain organism.
Aeromonas spp.
Aeromonas spp. – motile strains
All strains of motile Aeromonas species arepositive for the fermentation of glucose andmaltose, and ONPG. All strains are negative forurease and fermentation of inositol and xyloseand are resistant to the vibriostatic reagent 0/129.
Aeromonas hydrophila ssp. hydrophila andA. hydrophila ssp. dhakensis
A subspecies of A. hydrophila, A. hydrophila ssp.dhakensis, has been described (Huys et al.,2002b). The subspecies is differentiated fromA. hydrophila ssp. hydrophila by the followingtests. A. hydrophila ssp. dhakensis is negative foracid production from L-arabinose, and negativefor utilization of methyl a-D-mannoside, L-fucoseand L-arabinose, whereas A. hydrophila ssp.hydrophila is positive for acid production fromL-arabinose and positive for utilization of methyla-D-mannoside, L-fucose and L-arabinose (Huyset al., 2002b).
There are many reports in the literature ofvariable results for LDC. Some reports state thatA. hydrophila (the type strain ATCC 7966) isnegative for LDC (Kaznowski, 1998; Nielsen et al.,2001). However, there are other reports of thetype strain ATCC 7966 being positive for LDC(Abbott et al., 1992; Huys et al., 2002b). One ofthe reasons that there are variable reports for LDCmay be due to misinterpretation of the test, orthe use of different methods to detect LDC. Inthe conventional tube test, LDC is a pale purplecolour, particularly in comparison with the ADHtube. Also, a stronger result may be obtained after48 h incubation than after 24 h incubation.
There are also varying reports for utilizationof citrate and reaction for MR. Citrate is said to be
positive for 60% of strains of A. hydrophila, andMR is positive for 53% of strains (Abbott et al.,1992).
Aeromonas popoffii
Type strain LMG 17541 is indole-negative, butother strains are indole-positive (Huys et al.,1997b).
Aeromonas salmonicida: non-motileAeromonas – general
Identification of an isolate as A. salmonicida isbased on Gram-negative rod, negative motility,catalase positive, oxidase positive, production ofacid from glucose, resistance to vibriostatic agent0/129, and lack of growth at 37°C (Shotts et al.,1980). The initial division for A. salmonicida wasbased on production of a brown water-solublepigment on media containing tryptone. However,these criteria are not necessarily reliable as thereare many reports of atypical strains producingpigment, and strains that grow at 37°C (Austin,1993).
Pigment production occurs under aerobicconditions, whereas no pigment production isseen under anaerobic conditions (Donlon et al.,1983). It has also been shown that pigmentproduction can be reduced in the presence ofD-glucose. In the case of atypical A. salmonicidastrains from goldfish in Australia, which arethought to belong to the subspecies nova, pigmentproduction was intense after 3 days on Columbiaagar, and light-brown at 6 days on TSA. Additionof 0.1% w/v of glucose delayed pigment produc-tion, and 0.15% (w/v) of glucose completely inhib-ited pigment production (Altmann et al., 1992).
Differences in biochemical reactions havebeen noted when Aeromonas salmonicida strainsare incubated at different temperatures such as11, 18 and 28°C (Hahnel and Gould, 1982). Forconsistency, an incubation temperature of 22°Cis recommended and was used in the study byKoppang et al. (2000).
The A-protein layer, a major virulence factorfor A. salmonicida ssp. salmonicida, can be detec-ted using TSA supplemented with CBBA wherebacterial colonies containing the A-protein layerare seen as blue-coloured colonies, and A-protein-negative colonies appear white (Evenberg et al.,1985). Autoagglutination in distilled water alsoindicates the presence of the A-protein layer.
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However, these tests are not reliable indicators ofthe in vivo virulence of A. salmonicida (Bernoth,1990; Olivier, 1990).
Aeromonas salmonicida – atypical strains
The term ‘atypical’ is used to describe isolatesthat are slow-growing, have slow or no pigmentproduction, and biochemical characteristicsdifferent from A. salmonicida ssp. salmonicidaand the subspecies achromogenes, masoucida,salmonicida and smithia. The atypical strainsrepresent a diverse group of organisms, and aredifficult to identify to species level because of therange of phenotypes. A recent study highlightedthe need for standardized phenotypic tests toreduce inter-laboratory variation, and alsosuggests that ‘atypical’ isolates should be definedas any isolate that does not fit the existingclassification of A. salmonicida ssp. salmonicida(Dalsgaard et al., 1998). Therefore, to assistthe microbiologist in the identification of theseorganisms, biochemical tests are reported in thismanual from a number of different isolates fromdifferent fish species (although the list is notextensive). Results that have been achievedusing the standardized recommended methodsare indicated in the Table 4.1. See Chapter 7 formedia composition.
Pigment production can vary and maydepend on the media used. In a study of atypicalstrains by Hänninen and Hirvelä-Koski (1997),FA detected the highest number of strains withpigment (100%), followed by BHIA (86%) andTSA (74%). A. salmonicida ssp. achromogenesproduced pigment on FA, TSA and BHIA, but noton nutrient agar. The addition of L-tyrosine to TSAor BHIA leads to an increase in pigment detection;however, pigment is still detected earlier on FA,within 3–7 days at 20°C. TSA, BHIA and FA arenot recommended for primary isolation, as thenumber of organisms detected is not as high aswhen BA is used. They are, however, suitablefor subculture (Bernoth and Artz, 1989; Austin,1993).
Brucella spp.
The Brucella species isolated from marinemammals are zoonotic (Brew et al., 1999) and assuch should be dealt with in a class III biologicalsafety cabinet.
Isolates from seal and an otter, and someof the strains from cetaceans, have an absoluterequirement for 10% CO2. Most of the strains fromcetaceans do not require CO2 atmosphere forgrowth (Foster et al., 2002).
Brucella species from aquatic mammals givea numerical profile of 1200004 in the API 20NE.The manufacturer’s database will identifythis as ‘good identification for Moraxellaphenylpyruvica’ (Foster et al., 1996a).
Carnobacterium spp.
Carnobacterium is distinguished from Lacto-bacillus by its ability to grow at pH 9.0 but noton acetate agar (pH 5.4) or at pH 4.5.
When testing Carnobacterium piscicolafor carbohydrate fermentation, discrepanciesbetween results for some carbohydrates havebeen reported in the literature. In particular, aweak reaction for a positive fermentation of acarbohydrate tested using Phenol Red Broth basehas been reported as negative when Purple Brothbasal medium is used in the test. Sorbitol andlactose may be positive when Phenol Red Brothbase is used, but negative when Purple Brothbasal medium is used (Toranzo et al., 1993).
Carnobacterium, Lactobacillus, S. iniae,Vagococcus and Renibacterium differentiation
The following characteristics in Tables 3.2 and3.3 give a guide to the differentiation of thesegenera and species. Renibacterium salmoninarumis differentiated mainly by its slow growth andspecific growth requirements. Use KDM2 andKDMC media for culture and isolation. Plates areincubated at 15°C for up to 2 months with initialgrowth of pin-point colonies visible between 2and 8 weeks. The other genera all grow within1–3 days of incubation on general-purpose agarsuch as BA.
Table 3.3 was kindly supplied by Dr JeremyCarson (Department of Primary Industries, Waterand Environment, Tasmania).
Citrobacter freundii
Strains of Citrobacter freundii show variationin reactions for ADH, ODC, fermentation of
Interpretation of Biochemical Identification Tests 125
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126 Chapter 3
Test
Lact
obac
illus
spp.
Car
noba
cter
ium
pisc
icol
aC
arno
bact
eriu
mdi
verg
ens
Lact
ococ
cus
garv
ieae
Lact
ococ
cus
pisc
ium
Vago
cocc
usflu
vial
isVa
goco
ccus
lutra
eVa
goco
ccus
salm
onin
arum
Ren
ibac
teriu
msa
lmon
inar
um
Gro
wth
onac
etat
eag
ar(R
AA)
+−
−−
−−
−−
ADH
v+
++
−−
−−
Acid
from
man
nito
lv
+−
++
+−
−−
H2S
−−
−−
−−
ND
+La
ncef
ield
grou
pD
reac
tion
Posi
tive
Neg
ativ
eW
eak,
dela
yed
reac
tion
ND
Wea
k,de
laye
dre
actio
nLa
ncef
ield
grou
pN
reac
tion
Posi
tivea
Posi
tiveb
Posi
tive
ND
Neg
ativ
eSt
rept
ococ
calg
roup
ing
kitg
roup
sA-
G(O
xoid
)D
Neg
ativ
eN
egat
ive
Dw
eak
reac
tion
ND
Dw
eak
reac
tion
Neg
ativ
e
a Elda
reta
l.(1
999)
repo
rted
apo
sitiv
ere
sult.
Teix
eira
etal
.(19
96)r
epor
tsas
nega
tive.
b Schm
idtk
ean
dC
arso
n(1
994)
repo
rted
ane
gativ
ere
sul t.
Tab
le3.
2.C
arno
bact
eriu
m,L
acto
baci
llus,
Vag
ococ
cus
and
Ren
ibac
teriu
mdi
ffere
ntia
tion.
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sucrose, melibiose, amygdalin and salicin(API 20E), regardless of source or geographicallocation (Toranzo et al., 1994).
Cryptococcus spp. (yeasts)
All Cryptococcus species are positivefor urease, whereas Candida species arenegative.
Edwardsiella spp.
Edwardsiella hoshinae may smell like Plesio-monas shigelloides, which has a strong, pungent,sweetish smell. Vibrio (carchariae) harveyi ATCC35084 has a similar pungent smell, but notas strong as P. shigelloides. E. hoshinae wasreported as indole-positive by Grimont et al.(1980), but negative or weak results werereported by Farmer and McWhorter (1984).Likewise for TSI, Farmer and McWhorter (1984)
Interpretation of Biochemical Identification Tests 127
Test L. garvieae* C. piscicola V. salmoninaruma L. piscium S. iniae
Gram + + + + +Cell shape ec sr sr/cb c (sr/cb)b cHaemolysis a − a − ac, bc,d
Catalase − − − − −VP (plate) 89%+ + −# + −H2S − − + − −Bile-aesculin + + 62%+ − −PYR + + + − 95%+e
ADH + + − − 71%+e
Aesculin + + + + +Glucose + + + + +Galactose + 65%+ − + +Lactose − 35–60%+ − + −Maltose 90%+ 94%+ 17%+ + +Mannitol 90%+ 88%+ 4%+ + +Raffinose − 29%+ − + −Salicin 98%+ + 87%+ + +Sucrose 7%+ + 52%+ + +Sorbitol − − 4%+ − −Trehalose + + 87%+ + +Glycerol − 40–100%+ − − −Inulin − 0–100%+ − − −L-arabinose − − − − (+)b −Dulcitol − − − − −Fructose + + + + +Starch − 0–100%+ 13%+ − +Xylose − − − − −Adonitol − − − − −Melibiose − 30%+ − + (−)b −
All reactions determined by miniaturized microtitre tray tests (Schmidtke and Carson, 1994) or conventional tests. #Type strainis positive; boriginal description reaction in brackets; ca on bovine blood, b on sheep blood. ec, Elongated coccus; sr, short rod;cb, cocco-bacilli; c, coccus; PYR, L-Pyrrolidonyl-b-naphthylamide; ADH, arginine dihydrolase. *Lactococcus garvieae (syn.Enterococcus seriolicida).aSchmidtke and Carson, 1994; bEldar et al., 1994; cWeinstein et al., 1997; dVuillaume et al., 1987; eDodson et al., 1999.
Table 3.3. Differential tests for some non-fastidious fish pathogenic Gram-positive cocci and rods.
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reported a negative result, whereas a positiveresult was recorded by Grimont et al. (1980).
Edwardsiella ictaluri will grow on BrilliantGreen agar, and Salmonella–Shigella agar. MRand VP are positive and negative, respectively, atboth 37 and 20°C. There is no growth at 42°C.The organism grows in 0–1.5% NaCl, but not in2% NaCl.
Enterococcus spp.
Enterococcus faecalis and E. faecium are positivefor Lancefield group D antigen, grow at 45°C,and grow in the presence of 6.5% NaCl. Reliabletests for differentiation between the two speciesare acid production from L-arabinose (E. faecalisis negative and E. faecium is positive) andpyruvate utilization (E. faecalis is positive andE. faecium is negative).
Enterococcus seriolicida is now known asLactococcus garvieae.
Family Flavobacteriaceae
Many samples from the aquatic environment, bethey freshwater or marine, will produce yellow-pigmented colonies on culture. It is important toidentify whether these colonies are pathogensor saprophytes. Clinical information will beextremely useful in knowing how much time andeffort to put into their identification. Thus, ifbacterial cells, usually long and thin, are seenadhering to the surface or epithelium of aquaticanimals, then genera of the FlavobacteriaceaeFamily may be suspected. To aid in their identifi-cation, it is important for the microbiologist tounderstand the complexities of this family. Thefollowing information will assist in this regard.Much of this information has been taken from apaper by Bernardet et al. (2002), setting out theirproposed guidelines for describing members ofthe family Flavobacteriaceae.
The Cytophaga–Flavobacterium–Bacteroidesphylum contains the family Flavobacteriaceae,which consists of 18 genera and two unaffiliatedorganisms – Cytophaga latercula and C. marino-flava – that remain genetically misclassified at thisstage. At various times, this group has been knownas the yellow-pigmented rods, Flavobacterium-like, or Cytophaga group or Flexibacter group,
and the genera and species within this grouphave undergone numerous name changes andreclassifications in recent years. The type genus isFlavobacterium.
Genera of the Family Flavobacteriaceae con-sist of a group of halophilic organisms (toleratingsaline conditions), some of which are psychro-philic (tolerating cold temperatures). They aredescribed as having cells that are Gram-negative,1–10 mm long and 0.3–0.6 mm wide, with somespecies forming filamentous flexible cells and oth-ers coiled or helical cells. Some members exhibit agliding motility and the rest are non-motile. Theoptimum temperature for growth for all genera is25–35°C, with some species being psychrophilicor psychrotolerant. Growth is aerobic for most ofthe genera; however, microaerophilic or anaero-bic conditions are required for some genera.
Sphingolipids are absent and this featuredifferentiates the Family Flavobacteriaceae fromthe Family Sphingobacteriaceae (Tables 3.4 and3.5). None of the genera in the Family Flavo-bacteriaceae digests crystalline cellulose whentested with filter paper, and this characteristicdistinguishes them from the genus Cytophaga,which only contains species that digest crystallinecellulose. It is important to differentiate the pres-ence of the enzyme cellulase from other enzymesthat can degrade cellulose derivatives, such ascarboxymethylcellulose or hydroxyethylcellulose,which may be present in some species ofthe Flavobacteriaceae Family. Only cellulase iscapable of degrading crystalline cellulose, whichis tested using filter paper.
A number of other families are con-tained under the Cytophaga–Flavobacterium–Bacteroides phylum. Like the Family Flavo-bacteriaceae these include genera that produceyellow-pigmented colonies that may be culturedfrom samples received from the aquatic environ-ment. Some of these species include Cyclo-bacterium marinum, Cytophaga hutchinsonii,Flexibacter flexilis, Marinilabilia salmonicolor,Pedobacter heparinus, Sphingobacteriumspiritivorum and others.
Most of the members of the Flavo-bacteriaceae Family are aerobic except for Cap-nocytophaga, Coenonia, Ornithobacterium andRiemerella. An appropriate isolation technique isculture on to BA and incubation in a carbon-dioxide enriched atmosphere with 5–10% CO2
or a commercial gas generated atmosphere thatachieves 5% O2, 10% CO2 and 85% N2. Growth is
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either poor or absent under anaerobic conditions(Bernardet et al., 2002).
Agar digestion is positive for Cellulophaga,Zobellia and variable for Flavobacterium. None ofthe other genera in the Family Flavobacteriaceaedigests agarose.
Researchers describing new species in theFamily Flavobacteriaceae are recommended tofollow the guidelines for description and identifica-tion of new taxa and species in Bernardet et al.(2002).
Flavobacterium columnare
Genetic diversity is reported amongst strains;however, phenotypically they are similar.Genomic group I, which contains the type strainIAM 14301T, are 50% positive for the nitrate test.Nitrate reductase is also variable for genomicgroup II, whereas strains in genomic group III arenegative for nitrate. Strains for genomic group II
and III are able to grow at 37°C but not at 15°C,whereas strains of genomic group I are variable,with 85% showing growth at 15°C and only 75%at 37°C. The genomovars are distinguishable byPCR (Triyanto and Wakabayashi, 1999). Seesection 6.1, Molecular Identification by PCRusing Specific Primers.
Tenacibaculum maritimum
Reactions for T. maritimum in the API ZYMare reasonably consistent in the literature. TheAPI ZYM should be incubated at 22–25°C withovernight incubation.
Hafnia alvei
H. alvei may be differentiated from Yersiniaruckeri, as H. alvei is positive for xylosefermentation whereas Y. ruckeri is negative.
Interpretation of Biochemical Identification Tests 129
Genus Host Pigment typeSeawaterrequirement
Glidingmotility Atmosphere
Growthon MCA
Aequorivita Sea water, sea ice Carotenoid Variable Negative Aerobic Not testedBergeyella Human None Negative Negative Aerobic No growthCapnocytophaga Human and dog Flexirubin Negative Positive Microaerophilic No growthCellulophaga Marine alga and beach mud Yes Variable Positive Aerobic Not testedChryseobacterium Fish, marine mud, human,
cow’s milk, soilFlexirubin Negative Negative Aerobic Variable
Coenonia Peking duck Not tested Negative Negative Microaerophilic No growthEmpedobacter Human Flexirubin Negative Negative Aerobic PositiveFlavobacterium Fish, water, sea ice, soil, mud,
marine lake, AntarcticaYes Negative 11 species,
positiveAerobic Not tested
Gelidibacter Sea ice Carotenoid Positive Positive Aerobic NegativeMyroides Human Flexirubin Negative Negative Aerobic PositiveOrnithobacterium Turkey None Negative Negative Microaerophilic NegativePolaribacter Seawater, sea ice, marine lake Carotenoid Positive Negative Aerobic Not testedPsychroflexus Antarctica Carotenoid Variable Variable Aerobic NegativePsychroserpens Antarctica Carotenoid Positive Negative Aerobic NegativeRiemerella Duck, pigeon Variable Negative Negative Microaerophilic NegativeSalegentibacter Antarctica Carotenoid Negative Negative Aerobic Not testedTenacibaculum Marine algae, marine sponge,
fishCarotenoid, orweak reaction
Variable Positive Aerobic Not tested
Weeksella Human None Negative Negative Aerobic PositiveZobellia Red marine alga, marine
sedimentFlexirubin Positive Positive Aerobic Not tested
Some genera only produce one type of pigment, others none, whereas other genera contain species that produce either aflexirubin-type pigment, a carotenoid-type pigment or both. A ‘yes’ in this column indicates that both or one type of pigmentmay be produced. MCA, MacConkey agar.
Table 3.4. Differential characteristics of the genera within the family Flavobacteriaceae.
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Lactococcus spp.
The Lactococcus genus was created to accommo-date ‘lactic’ or group N streptococci. There arecurrently four species, L. garvieae, L. lactis,L. piscium, L. plantarum and L. raffinolactis.There are varying reports in the literature forgroup N results for L. garvieae and L. piscium.
Lactococcus garvieae
Different results have been noted for ribose,depending on the identification system used.Ribose is positive when using the API 50CH sys-tem, but negative with the API Rapid ID 32 Strepsystem (Vela et al., 2000). L. garvieae may be bio-chemically indistinguishable from Streptococcusparauberis (Doménech et al., 1996). Lactococcusgarvieae (Enterococcus seriolicida) is phenotypi-cally similar to L. lactis; however, they can be dif-ferentiated by sensitivity testing to clindamycin.L. garvieae is resistant to clindamycin, whereasL. lactis is sensitive (Elliott and Facklam, 1996).L. lactis is now known as L. delbrueckii ssp. lactis.
L. garvieae was divided into biotypes (Velaet al., 2000); however, these divisions have been
disputed. Further research indicates that L. garvi-eae strains are relatively homogeneous regardlessof geographical location or aquatic host. What isimportant in biochemical identification is that thedensity of the inoculum be standardized whenusing the API Rapid ID 32 Strep system and thatthe bacterial cells to be used for the inoculum aretaken from BA plates. Reliable, repetitive resultswere only obtained when the inoculum densitywas adjusted using a spectrophotometer, toan optical density of 0.8 at wavelength of 580.Also, cells grown on BA produced reliable resultsfor biochemical tests, whereas growth taken fromTSA (Oxoid) produced variability in biochemicaltests. However, slight variability was still observedfor biochemical tests such as b-galactosidase,hippurate, b-mannosidase, acid from melezi-tose, N-acetyl-b-glucosaminidase and acid frompullulan (Ravelo et al., 2001).
Listonella anguillarum
Some strains are negative for citrate in both theconventional tube test and in the API 20E. TheMR tube may show weak growth and a weak
130 Chapter 3
Genus Aesculin Catalase DNase Gelatin Glucose acid Indole ONPG Nitrate Sucrose Urease
Aequorivita + + 75% Neg + Neg NT NT Neg Neg VBergeyella Neg + Neg + Neg + Neg Neg Neg +Capnocytophaga V V NT V + Neg V V + VCellulophaga NT + V V V NT NT V V VChryseobacterium + + + + + + V V V VCoenonia + + NT Neg + Neg + Neg Neg NegEmpedobacter Neg + + + + + Neg Neg Neg VFlavobacterium 10/14 =+ + or weak V 11/14 =+ V Neg V V V VGelidibacter + + + V + Neg Neg Neg Neg NegMyroides Neg + + + Neg Neg Neg Neg Neg +Ornithobacterium Neg Neg Neg Neg V Neg + Neg Neg +Polaribacter V + or weak NT V + Neg V Neg V NegPsychroflexus V + + V V Neg Neg Neg Neg VPsychroserpens Neg + Neg V Neg Neg V Neg Neg NegRiemerella V + NT + + V Neg Neg Neg VSalegentibacter + + + + V NT + + V VTenacibaculum Neg + + + Neg NT NT V NT NTWeeksella Neg + Neg + Neg + Neg Neg Neg NegZobellia + + + + + + + + + Neg
V, variable; NT, not tested; Neg, negative; +, positive; ONPG, o-nitrophenyl b-D-galactopyranoside.10/14 =+ means that 11 of the 14 species in the genus are positive.
Table 3.5. Further differential characteristics for the genera in the family Flavobacteriaceae.
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positive reaction at 48 h after incubation andsome strains may be negative. Growth on TCBStends to be slow and there is substantially lessgrowth when compared with the amount ofgrowth on MSA-B or BA plates. Strains isolatedfrom an estuarine environment may grow betterin 0.85% NaCl, whereas those strains from amarine environment show optimal growth with2–3% NaCl, and this can be seen clearly on a gel-atin-salt plate. Use growth on this plate or growthon a BA plate, as opposed to growth on MSA-B,as a guide to the salt requirement of the organismwhen preparing the NaCl for the inoculating fluidfor either the conventional biochemical set orthe API 20E. Strain NCIMB 2129 is reported tobe negative for fermentation of sorbitol and tre-halose (Benediktsdóttir et al., 1998). Differencesin citrate, indole and MR are reported for NCIMB2129, NCIMB 6 and ATCC 14181 (Myhr et al.,1991; Benediktsdóttir et al., 1998; Lunder et al.,2000). Serotype 01 strains tend to be positive forL-arabinose (Toranzo and Barja, 1990).
Mesophilobacter marinus
Mesophilobacter marinus describes marinecoccobacilli that morphologically resemble theAcinetobacter – Moraxella group.
Moritella spp.
Moritella (Vibrio) viscosus often needs prolongedincubation times for growth in test media. Moststrains require 1% peptone for optimal growth.Results for salt requirements and temperaturegrowth limits are inconsistent in the literature.One study suggests 1–4% NaCl and a tempera-ture of 25°C (Lunder et al., 2000). A secondstudy suggests 2–3% NaCl with no growth at 4%NaCl, and a temperature range of 4–21°C withno growth at 25°C (Benediktsdóttir et al., 2000).
Photobacterium spp.
Photobacterium damselae ssp. damselae
There are some biochemical differences accord-ing to the biochemical method used and theamount of salt in the inoculum. The tube ureaseis positive; however, in the API 20E the ureaseresult seems to depend on the salt concentrationin the inoculum. When an inoculum of 0.85%NaCl is used, then the urea gives a positive result,but at an inoculum concentration of 2% NaCl, anegative urea may be obtained. Time of incuba-tion is important also, as urea may be weak ornegative at 24 h incubation, but a strong positiveat 48 h incubation. P. damselae ssp. damselaehas been grouped into biotypes (Pedersen et al.,1997) and many of these are listed in the con-ventional biochemical table for Photobacterium.Table 3.6 lists other biochemical tests not listed inthe conventional biochemical table 4.20.
Photobacterium damselae ssp. piscicida
The fermentation reactions may be improvedunder anaerobic conditions. This is easilyachieved by overlaying the medium with sterileparaffin oil. The glucose reaction in the API 20Ekit may be very weak or negative. Likewise, theVP reaction may be variable, and research intothe biochemical properties of isolates fromFrance, Greece, Italy and Japan found that allisolates tested were negative for VP in the API20E system (Bakopoulos et al., 1995).
Pseudoalteromonas spp.
Pseudoalteromonas citrea
First described by Gauthier (1977), the typestrain is ATCC 29719. Similar strains have beendescribed by Ivanova et al. (1998). Some pheno-typic tests are slightly different, which is thought
Interpretation of Biochemical Identification Tests 131
Biotypes 1 2 3 4 5 6 7 8 9
TestLipase + − + + + + + + +Cellobiose + + + + − − + + +
Taken from Pedersen et al. (1997).
Table 3.6. Additional tests for differentiation of biotypes of P. damselae
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to be due to the different ecological niches. Thephenotypic reactions described in this manual arethose of the type strain.
Pseudomonas anguilliseptica
Finnish isolates and the type strain are positivefor gelatin, whereas Japanese isolates are nega-tive. Motility is only seen when the organism isgrown below 20°C (Michel et al., 1992). Thereare three serotypes of P. anguilliseptica based ona heat-labile surface K antigen. Serotypes fromeels in Japan are K− and K+; ayu have K+serotype as do the Finnish isolates (Wiklund andBylund, 1990). Antisera against P. anguillisepticamay show cross-reaction against Pseudomonasputida and Listonella anguillarum; however, if itis used diluted at 1:5 to 1:10, this may overcomethe cross-reaction (Toranzo et al., 1987).
Renibacterium salmoninarum
This organism is the causative bacterium of BKD.It is a fastidious and slow-growing organismand the culture medium used for isolation andgrowth, KDM2, may give inconsistent perfor-mance due to lot-to-lot variations in peptone(Evelyn and Prosperi-Porta, 1989). Consistencyin growth can be improved either by using anurse culture technique, or by supplementationof the growth medium with spent culturemedium. The nurse culture technique involvesplacing a 25 ml drop of nurse culture (preparedfrom cells suspended in saline or peptone) intothe centre of a plate inoculated with the testsample. Once the drop is dry the plates areinverted, sealed to prevent drying, and incubatedat 15°C for 21 days. The supplementation withspent culture broth method can be achievedby preparing new KDM2 medium with 1.5%(v/v) of spent culture medium (Evelyn et al.,1990).
Streptococcus spp.
Streptococcus agalactiae
Lancefield group B. Different reactions are repor-ted for S. agalactiae isolated from human, fish andanimal sources, and this may in part be due to theoptimum incubation temperature for enzyme
reactions. For example, hippurate may be positiveat an incubation temperature of 25°C but nega-tive at 37°C for isolates from fish. The conven-tional VP method shows a negative reactionfor S. agalactiae, but it is positive in the APIrapid ID 32 (Vandamme et al., 1997). Strains ofS. agalactiae from mammalian sources are b-haemolytic; however, most of the strains isolatedfrom fish were reported to be non-haemolyticand were initially identified as S. difficile. Theyare now identified to be S. agalactiae group B,capsular type Ib (Vandamme et al., 1997). Theseisolates are slow-growing and adhere strongly tothe agar plate, which makes it difficult to resus-pend the cells in an inoculating fluid. Conven-tional biochemical tests may need to be incu-bated for more than 48 h for sufficient growth tooccur in the tubes. MR and VP reactions may beweak, and poor growth is seen in the tryptonewater (indole test). The ADH test using con-ventional media may take more than 3 days’incubation before a slight colour change is seen.
For the S. agalactiae strains that were isolatedfrom a disease outbreak in seabream and wildmullet in Kuwait, there are some differencesin the biochemical reactions compared with theATCC type strain (Evans et al., 2002). Therefore,the results of the S. agalactiae isolates from theseabream and the wild mullet are listed separatelyin the API rapid ID 32 Table 4.30.
Streptococcus iniae
The organism shows complete b-haemolysis onsheep BA, but only partial haemolysis when themedium is supplemented with human or bovineblood. Variations in haemolysis may be seenbetween strains.
Strains isolated from either fish or humanshave a slightly different biochemical profile.
S. iniae strains isolated from culturedand wild fish in the Red Sea were similar in bio-chemical profile to other S. iniae strains, exceptfor a negative ADH in the majority of strains, andlate fermentation of galactose and amygdalin asdetected in the API 50CH, which was incubatedfor 72 h before results were recorded (Colorniet al., 2002). The biochemical profile achievedwith the API 20 Strep agrees with other resultsrecorded for fish isolates where 30% of strains maybe negative for ADH, and that variation may beseen in the fermentation of mannitol (Dodsonet al., 1999).
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Streptococcus parauberis
This may be biochemically indistinguishablefrom Lactococcus garvieae (Doménech et al.,1996).
Streptococcus uberis
There are different reports for the reactionto pyrrolidonylarylamidase. Bergey’s Manualof Determinative Bacteriology (1994) reports anegative reaction, whereas Collins et al. (1984)and Doménech et al. (1996) report a positivereaction.
Tenacibaculum maritimum
See under Flavobacteriaceae.
Vagococcus salmoninarum
The genus Vagococcus was proposed to describemotile, Lancefield group N, Gram-positivecocci; however, Schmidtke and Carson (1994)described V. salmoninarum as negative for the Nantigen.
Vibrio spp.
Vibrio agarivorans
These strains degrade agar and the effect can beseen as small depressions on an agar plate after3 days’ incubation at 25°C. This effect increasesover time, and by about 7 days the effect isvery marked. This was particularly the case withisolates from abalone in Western Australia. Thecarbohydrate fermentation reactions may be dif-ficult to read and this effect has been noticed withall strains tested. All carbohydrate fermentationtubes should be compared alongside the glucosereaction, as this will be a bright yellow colour.
The positive reactions are easy to determine, butthose that appear as a weak reaction are, in fact,negative reactions. These may appear as a paleyellow colour in the middle of the tube. However,after 48 and 72 h incubation these reactions arequite clearly negative. V. agarivorans strains firstisolated and reported from the MediterraneanSea show a positive reaction for aesculin anda negative reaction for gelatin (Macián et al.,2001b). Strains from Western Australia all showa negative reaction for aesculin using the con-ventional tube test, and a positive reaction forgelatin when the plate method is used. However,the gelatin result in the API 20E is negative after48 h incubation at 25°C.
Vibrio alginolyticus and Vibrio harveyi
These may be difficult to differentiate (Table 3.7).The VP test differentiates them, as does theadditional test for fermentation of D-glucuronate(Baumann et al., 1984). V. alginolyticus swarmsand completely covers an MSA-B plate in 24 hat 25°C, whereas V. harveyi has a slow orspreading type of growth. V. alginolyticus isurease-negative, whereas V. harveyi is usuallyurease-positive (50% reported as positive).
Vibrio cholerae
Strains of Vibrio cholerae are negative foraesculin; however, some strains cause blackeningof the medium due to the production of melanin(Coyne and Al-Harthi, 1992). To determine truehydrolysis as opposed to melanin production,the aesculin tube must be tested for loss of fluo-rescence (Choopun et al., 2002). The aesculinsolution will fluoresce under long-wave UV light(354 nm), therefore a loss of fluorescenceindicates hydrolysis of aesculin and a positive testresult (MacFaddin, 1980). The plastic tubes thatthe aesculin medium is in may be UV opaque.Therefore the test medium can be poured intoa Petri dish and a Wood’s UV light held overthe dish in a darkened room. An uninoculated
Interpretation of Biochemical Identification Tests 133
VP 10% NaCl Growth at 42°C Urease D-Glucuronate
Vibrio alginolyticus + + + − −Vibrio harveyi − − − v +
Table 3.7. Differentiation of V. alginolyticus and V. harveyi.
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control and a negative result will show a ringof fluorescence around the edge of the liquid,whereas a true positive aesculin will not show thefluorescence. Protect the eyes with UV-opaqueglasses.
VIBRIO CHOLERAE NON-01. Strains isolated fromayu in Japan were found to be negative forODC (Kiiyukia et al., 1992). Normally, V. choleraeisolates are positive for ODC.
Vibrio coralliilyticus
Six strains of this newly described Vibrio specieshave been characterized. There is some bio-chemical variation between strains, with two ofthe six strains positive for ADH as detected in theAPI 20NE, and four strains negative (Ben-Haimet al., 2003).
Vibrio fischeri
A positive result is obtained for VP in the API 20Esystem, but VP is negative in MRVP medium(Difco).
Vibrio fluvialis and Vibrio furnissii
Both species are indole-negative when tested forindole production in peptone water containing1% NaCl. When indole is tested using heartinfusion broth, 14% of V. furnissii are positive,and 4% of V. fluvialis are positive. Both speciesare positive for indole in the API 20E system. TheAPI 20E database may identify these speciesas Aeromonas species (Brenner et al., 1983). V.furnissii may show a weak pink indole reactionwhen approximately 12 drops of Kovács reagentis added to a 48 h tryptone water (5 ml). Thecolour disappears after a minute and appearsas a dirty orange-brown colour. V. furnissiiwill show a negative ADH reaction if no NaCl isadded to the reagent tube. ADH will be positivewhen the final salt concentration is 2% (Buller,2003). V. furnissii strains produce gas in theglucose tube, whereas V. fluvialis strains donot produce gas. V. furnissii strains (57%) maybe positive for L-rhamnose, whereas V. fluvialisstrains are negative, and 63% of V. fluvialisare positive for cellobiose, whereas V. furnissiiis negative (Lee et al., 1981; Brenner et al.,1983).
Vibrio (carchariae) harveyi ATCC 35084
Citrate and gelatin (plate method) are negativeat 24 h and need to be read at 48 h or longer.The culture has a pungent smell similar toPlesiomonas shigelloides.
Vibrio harveyi
The strain ATCC 14126 is positive for lumines-cence and negative for urease, whereas ATCC35084 is negative for luminescence and positivefor urease (Alcaide et al., 2001). Strains NCIMB1280, ATCC 14126 and ATCC 14129 arereported to be gelatin-positive (Baumann et al.,1984; Benediktsdóttir et al., 1998). Strains ATCC14126 and ATCC 14129 are reported to benegative for fermentation of sorbitol, whereasstrain ATCC 35084 is positive after 2 days(Alcaide et al., 2001; Buller, 2003). Haemolysisis variable, and is reported to be positive againstsheep red blood cells (Alcaide et al., 2001).See Table 3.7 for further tests to differentiateV. harveyi and V. alginolyticus.
Vibrio neptunius
The type strain is positive for ADH; however,other strains are reported to be negative for ADH(Thompson et al., 2003).
Vibrio parahaemolyticus
In the API 20E, strain ATCC 43996 shows a moreclearly positive reaction in the arabinose testwhen incubated for 48 h and prepared with a 2%NaCl inoculum. However, ADH shows a weakpositive reaction with a 2% NaCl inoculum andincubation at 48 h. With the conventional tests,indole will record a negative result if normalsaline is used as the inoculum. A positive resultis recorded with 1.5% and 2% NaCl as a finalconcentration.
Vibrio mediterranei
At 48 h these are large, creamy, mucoid colonieson MSA-B. After 24 h incubation and aninoculum of 2% NaCl, the ADH in the API 20E ispositive and at 48 h incubation LDC may also bepositive. However, in the conventional tube testsonly LDC is positive after 48 h incubation.
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Vibrio proteolyticus
The growth swarms quickly across an agar plate,within 24 h, and could be mistaken for V.alginolyticus. In the conventional citrate testa positive result may not be seen until 48 h.A positive urea is seen in the conventional tubetest, but is negative in the API 20E at 48 h andwith an inoculum of 2% NaCl.
Vibrio scophthalmi
The type strain in positive for chitin hydrolysis,whereas other strains are negative (Farto et al.,1999).
Vibrio scophthalmi and V. splendidus biovar Imay be difficult to differentiate based on pheno-typic tests and this is compounded due to thevariation of phenotypic test results between strainsin each species (Farto et al., 1999). Indole, ONPGand fermentation of mannitol appear to bethe best differentiating phenotypic tests.V. scophthalmi is negative for all three, whereasV. splendidus biovar I is positive for all three.
Vibrio splendidus
Variations in phenotypic tests are reportedbetween different research groups. One of thetests that seems to be a problem for consistency isADH, although the majority of reports suggestthat ADH is positive. Most V. splendidus I strainswere found to be ADH-positive when tested usingThornley’s method for ADH, yet negative whentested by the more commonly used Møller’sarginine method (Macián et al., 1996). V.splendidus I type strain (NCMB 1, ATCC 33125)is reported to be positive for ADH by bothLunder et al. (2000), and Benediktsdóttiret al. (1998). However, the results vary forV. splendidus biovar 2 type strain NCMB 2251,which is reported to be negative by Lunder, yetpositive by Benediktsdóttir. Both papers reporta negative sucrose, whereas the same type strainis reported positive by Farto et al. (1999). Thestrains are also variable for ONPG. Variationssuch as these make it difficult for the diagnosticlaboratory to identify an unknown.
There are two biovars and both have beenimplicated in disease. Biovar 1 isolates are positivefor luminescence and fermentation of mannose,ribose and melibiose, whereas biovar 2 isolatesare negative for these tests. In addition, biovar 1
is negative for fermentation of glycerol, anddegradation of chitin, with biovar 2 being positive(Benediktsdóttir et al., 1998).
There are reported differences in virulencebetween strains of V. splendidus and it issuggested that valine aminopeptidase as testedby API ZYM is a virulence factor becausepathogenic strains produced the enzyme whereasnon-pathogenic isolates did not (Gatesoupe et al.,1999).
Vibrio vulnificus
The division of Vibrio vulnificus into two mainbiovars is under contention. The original classifi-cation by Tison (1982) was that biovar 1 isolateswere positive for indole and ODC and weremainly isolated from human clinical sources.Biovar 2 strains were isolated mainly fromdiseased eels and were indole- and ornithine-negative. However, there is some overlapbetween these divisions, and therefore the reac-tions for the different strains and the geographicalsite and source of isolation have been included inthe conventional identification tables, and in theAPI 20E tables for profile number. It has beenreported that approximately 20% of V. vulnificusstrains are sucrose-positive (Arias et al., 1998).Note that sucrose medium that is autoclaved forsterility may give false-positive reactions. Filter-sterilized sucrose medium is preferred for moreaccurate fermentation results.
Vibrio wodanis
A range of salt requirements, 0.5–5% NaCl, existswith these strains (Lunder et al., 2000). Onestudy found that 1 of 16 isolates showed growthat 0.5% NaCl, and 16 of 23 isolates showedgrowth at 4% (Benediktsdóttir et al., 2000).
Yersinia spp.
Yersinia frederiksenii
These are rhamnose-positive strains, formerlycalled atypical Y. enterocolitica. Y. inter-media comprises rhamnose-positive, melibiose-positive, raffinose-positive strains, formerlycalled Y. enterocolitica or Y. enterocolitica-like.Y. kristensenii refers to sucrose-negative strains.
Interpretation of Biochemical Identification Tests 135
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Yersinia ruckeri
Strains of Y. ruckeri may be divided into motileand non-motile types, a finding that is geo-graphically biased. Strains from the UK tend tobe non-motile, with occasional non-motile strainsreported from Canada and Norway. Non-motilestrains lack lipolytic activity when tested usingthe Tween 20 and Tween 80 tests. Therefore,Shotts-Waltman medium is inappropriate forthe differentiation isolation of non-motile strainsof Y. ruckeri as it contains Tween 80 (Daviesand Frerichs, 1989). These authors suggestedthat non-motile, Tween 80 hydrolysis-negativestrains should be referred to as Y. ruckeri biotype2. Results between conventional tube tests andthe API 20E system may give variable results.These include citrate utilization, gelatin hydroly-sis, VP and nitrate. The nitrate test in the API 20Esystem may give unreliable results for Y. ruckeri,thus a conventional tube nitrate test is recom-mended. After 24 h incubation at 25°C, citrateand gelatin may be falsely negative, therefore48 h incubation is recommended. Motility andcitrate are negative at 37°C but positive at 25°C.The API 20E may show more VP-positive resultsthan the conventional tube test (Davies andFrerichs, 1989).
Y. ruckeri may be differentiated fromHafnia alvei by xylose fermentation. Y. ruckeri isnegative, whereas H. alvei is positive for xylosefermentation.
3.5 Antisera Available
The following are some of the antisera that areavailable commercially.
Aeromonas salmonicida ssp. salmonicida,specific monoclonal antibody (BIONOR MonoAS, BIONOR Aqua, Skien, Norway).
Brucella abortus antiserum (Difco).Listonella anguillarum, specific monoclonal
antibody for serotypes 01, 02, 03 and 04, 05and 07 for environmental serotypes (BIONORMono-Va, BIONOR Aqua, Skien, Norway).
Photobacterium damselae ssp. piscicida,specific monoclonal antibody (BIONORMono-Pp, BIONOR Aqua, Skien, Norway).
Renibacterium salmoninarum, specificmonoclonal antibody (BIONOR Mono-Rs,BIONOR Aqua, Skien, Norway).
Salmonella O and Salmonella H antiserumand specific antiserum for species (Difco).
Staphylase test for identification of Staphylo-coccus aureus (Oxoid).
Streptococcus group A, B, C antiserum(Difco). Antiserum for groups A, B, C, D, F, G(Oxoid).
Vibrio cholerae. Anti-V. cholerae 01 serum(Denka Seiken). Bacto-Vibrio-Cholerae anti-serum for detecting three serotypes in group 01.These are serotype Ogawa (AB O antigen factors),Inaba (AC O antigen factors) and Hikojima (ABCO antigen factors) (Difco).
Yersinia ruckeri specific monoclonal anti-body (BIONOR Mono-Yr, BIONOR Aqua, Skien,Norway).
The BIONOR Mono-Va kit detected serotypesof 01, 02 and 03 of Listonella anguillarum andalso detected the environmental serotypes of 04,05 and 07. However, non-specific agglutinationagainst strains of V. splendidus and motileAeromonas species occurred. A cellular concen-tration of 108/ml is recommended for positiveagglutination (Romalde et al., 1995).
Strains of Photobacterium damselae ssp.piscicida were detected by the BIONOR Mono-Ppkit, with no cross-reactions detected for Aero-monas salmonicida. However, non-specificagglutination occurred from strains of Actino-bacillus pleuropneumoniae, Haemophilusparasuis, Mannheimia haemolytica andPasteurella multocida (Romalde et al., 1995).
Renibacterium salmoninarum was success-fully detected using the BIONOR Mono-Rs kit;however, strains that lacked the p57 surfaceprotein were not detected (Romalde et al., 1995).No cross-reaction with other Gram-positiveorganisms such as Corynebacterium aquaticum,Carnobacterium piscicola, Enterococcus faecalisand Lactococcus garvieae were detected(Romalde et al., 1995).
The BIONOR Mono-Yr kit detected classicalserotypes 01 and 03 of Yersinia ruckeri. For thedetection of 02 serotypes the test needed tobe performed at either 48 h culture growth orfrom subcultures. The kit is unable to detect theY. ruckeri serotypes 05 and 06 because of thecomposition of the serotypes included in the kit. Acell concentration of 108/ml is needed for positiveagglutination. No cross-reaction with other bacte-rial pathogens was found (Romalde et al., 1995).
136 Chapter 3
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4Biochemical Identification Tables
4.1 Results for Conventional BiochemicalTests – ‘Biochem Set’
The following tables (Tables 4.1 to 4.22) are tobe used for interpreting the results obtainedfrom in-house or conventional biochemicalmedia. The tables are listed in alphabetical orderof the table heading. The organisms in the tablesare grouped into those that are consideredto be either pathogens in aquatic animals orenvironmental and saprophytic organisms. Thesaprophytic, environmental and other speciesof bacteria are included under ‘environmental’so as to assist in differentiating a pathogenfrom a closely related species and thus obtain-ing a definitive identification of the unknownorganism.
In a number of cases, different or variablebiochemical tests were recorded in the literatureby different laboratories for the same type strain.Also, differences in a biochemical were recordedfor different strains. These results are includedhere so that the reader will be aware that some testresults may not be reliable and that different resultsmay be recorded by different laboratories. In thecases reported in this Manual, the tests that gavevariable results appeared to be performed usingthe same method. It is known that differentmethods can give different results for the sameorganism, and therefore it is important to be con-sistent with the methods used. Where a differencebetween the conventional biochemical test andthe API 20E was reported, this is recorded in thetables, so that the reader is aware of variations for
different test methods for that particular organism.The numbers in the tables report the percentagesof positives as reported in the literature whena number of strains were tested. The range ofnumbers in the ‘temp’ and ‘NaCl’ columns referto the temperature range (°C) and range of saltconcentrations (%) at which the organism cangrow. A single number indicates the optimumgrowth temperature or NaCl concentration,respectively.
With the exception of the Vibrio tables(Tables 4.21 and 4.22), all other tables list theorganisms alphabetically within the groupings of‘pathogenic’ or ‘environmental’ organisms.
The species in the Vibrio tables are groupedaccording to their ODC–LDC–ADH reaction.Therefore all species with a ODC+, LDC+, ADH-result are grouped together. Likewise otherODC–LDC–ADH reaction combinations aregrouped together. Use these groupings as astarting point to identify an unknown.
Use the tables in conjunction with thesection on interpreting test results and specificreactions noted for some genera and species (seeChapter 3).
An example of a Laboratory Worksheet forrecording results is included on page 177.
Use the following schematic (Fig. 4.1) asa guide to which table or tables (Conventionalmedia, ‘Biochem set’) to use when identifyingan unknown. Start with the result for the Gramstain, then cell morphology and, for the Gram-negatives, a further division based on oxidaseresult.
©N.B. Buller 2004. Bacteria from Fish and Other Aquatic Animals:a Practical Identification Manual (N.B. Buller) 137
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4.2 Results for API Kits
Interpretation of API 20E profile
Use the profile index provided by the manu-facturer as a guide only. Kent (1982) does notrecommend using the profile index provided withthe kit because of misidentifications, especiallywhen attempting to identify an organism notfound in the commercial database. The API 20Esystem will even misidentify organisms whenthe identification is given at the 99% confidence
level. For example, Dalsgaard et al. (1996)found that the API 20E system misidentifiedV. vulnificus. The system gave a greater than98% confidence level as Aeromonas hydrophila,Flavobacterium meningosepticum, Burkholderiacepacia, and between 90 and 95% confidencelevels as V. alginolyticus and V. parahaemolyti-cus. All these ‘isolates’ were in fact V. vulnificus,which they proved using a species-specific DNAprobe.
It is recommended that the API 20E systembe used with caution. Some additional tests may
138 Chapter 4
Fig. 4.1. Schematic for using the biochemical identification tables.
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be required to give a definitive answer. Thisis particularly so for the identification of speciesfrom the genus Aeromonas. Adding conventionalmedia of aesculin, MR, and salicin in addition tothe API 20E system will improve the likelihood ofthe correct identification.
The API 20E database listed in this book is acollation from the literature. For some organismssuch as Vibrio vulnificus, many profile numberswere obtained, indicating the phenotypic strainvariation amongst species.
The reactions for the API 20E are listed inthree different formats (see Tables 4.23, 4.24 and4.25). The first format, in Table 4.23, indicates thereactions obtained for the different tests in thesame order as the API 20E test strip. The organ-isms are listed in alphabetical order. The secondformat, in Table 4.24, lists all organisms in alpha-betical order and then the corresponding API 20Eprofile number. The third format, in Table 4.25,lists the API 20E profile numbers in numericalorder. Use this table first when identifying anunknown. Then use Table 4.23 to check the posi-tive and negative reactions obtained. Tables 4.24and 4.25 should be checked to assess the similaritywith close profile numbers and to determinewhether additional tests should be done to con-firm this initial identification. In some cases thecomplete 9-digit API 20E profile number was notreported in the journal article, as the tests forgrowth on MCA and OF were not reported.In these cases the API 20E profile number isrecorded in the manual as a 7- or 8-digit profilenumber.
The numbers in the cells of the tables are thepercentage of positive strains recorded with thattest result.
Identification using API ZYM
The API ZYM tables are included because manynewly described species are tested for the
production of enzymes using this kit. It is not gen-erally considered a routine diagnostic kit; how-ever, it is useful for some bacteria from veterinaryisolations such as the species from Pasteurella,Actinobacillus and Histophilus ovis and is able todifferentiate between species when phenotypictests may be in doubt (Cousins and Lloyd, 1988).For these organisms the intensity of the colourreactions is consistent for a species and allowsspecies identification. Therefore, the correctinoculum concentration is important. It can alsobe used to confirm or back-up phenotypic testswhen identifying an unknown bacterium isolatedfrom an aquatic animal species.
Species within a genus give very similarresults for the API ZYM; however, there are oftendifferences with one or two enzymes betweenspecies that can give an indication of theidentification of the species. The intensity of thecolour reaction is important. Thus it is importantto be consistent with the inoculum density.The manufacturer recommends a density ofMcFarland tube 6.
For Flavobacterium, Flexibacter and Cyto-phaga species, an incubation time of 12 h isrecommended when incubating at a temperatureof 18–30°C (Bernardet and Grimont, 1989).For an incubation temperature of 37°C use anincubation time of 4 h as recommended by themanufacturer. In the author’s experience isolatesfrom an aquatic environment give better reactionswhen incubated for 24 h at a temperature of 25°C.However, it must be stressed that if the incubationtime and temperature is stated in the tables thenuse these parameters because the results arereported on these. Some references did not statethe time and temperature of incubation thatwas used, and therefore it is assumed that themanufacturer’s recommendations were used.
Improved results may also be obtainedif ammonium salt sugar broth is used asthe inoculating fluid for the Flavobacterium/Cytophaga group from marine and freshwatersources (Bernardet and Grimont, 1989).
Biochemical Identification Tables 139
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140
Test
Gm
Ox
cat
bHm
otPi
gO
DC
LDC
ADH
Nit
Ind
Cit
urea
mr
vpae
sG
onpg
OF
arab
glu
inos
lac
mal
tm
anm
ano
sal
sor
suc
treXy
lH
2SM
CA
TCBS
DN
ase
tem
pN
aCl
0129 10
KfAm
pR
ef
Org
anis
m
Path
ogen
ic
Typi
cal
A.sa
lmon
icid
ass
p.sa
lmon
icid
aN
CM
B11
02H
G3
−+
++
−+
−+
++
−−
−+
−+
++
F+
+ g+
−−
++
++
−−
+−
−+
−+
4–30
0–3
RS
S32
2,45
0
No
grow
th37
°C,A
PI20
ELD
Cm
aybe
neg
200,
475
A.sa
lmon
icid
ass
p.sa
lmon
icid
aAT
CC
1417
4
−+
−+
−+
+−
−−
+F
−+
−+
+−
−−
−−
186
A.sa
lmon
icid
ass
p.sa
lmon
icid
a−r
++
+−
−*−
−+
+−
−−
++
−F
++ g
+−
++
+−
−+
−−
+−
+4–
300.
5–3
R45
0
Non
-pig
men
ted
stra
in
‘Aty
pica
l’
A.sa
lmon
icid
ass
p.ac
hrom
ogen
esN
CM
B11
10T
−r+
++
−+ft
−−
++
+−
−−
+−
++
F−
+ g−
−−
++
+−
−+
−−
v+
−+
4–30
0–4
RR
R45
0,53
4
No
grow
th37
°C.A
PIIn
dm
aybe
neg
200,
322
A.sa
lmon
icid
ass
p.m
asou
cida
−+
++
−−
−+
++
+−
−+
++
++
F+
+ g+
−+
++
−−
++
−+
+−
+30
0–2
RR
322,
450
No
grow
th37
°C.I
nAP
I20E
LDC
,VP
&H
2Sm
aybe
neg
A.sa
lmon
icid
ass
p.no
va−
++
+−
−−
−−
++
v−
−−
−−
F−
+ g−
−−
++
v−
−+
−−
−18
–25
RR
695
Hae
min
requ
irem
ent
A.sa
lmon
icid
ass
p.sm
ithia
−+
+25
−−
−−
−−
v−
−−
−+
+F
+−
−−v
−v
−+
−−
+5–
200–
2R
R47
,450
No
grow
th37
°CAm
eric
anee
l−
+−
−b−
−v
+−
−−
+v
F−
+−
+−
−v
−−
584
Aust
ralia
ngo
ldfis
h−r
++
−−
+b−
v+
+−
−−
−−
−v
F−
+ g−
−−
++
+−
−+
−−
−0.
5–3
R14
4,82
5
Aust
ralia
ngo
ldfis
h−
++
−−
+bt13
5
Balti
cse
atu
rbot
−r−
++
−−b
−−
v−
−−
vv
+−
F+ g
−−
−−
+R
RS
200,
832
Balti
cse
aflo
unde
r−r
−+
+−
−b−
−v
v−
−+
++
vF
+ g−
++v
++
RS
S20
0,83
2
Balti
cse
a(F
inni
sh)
floun
der
−−
++
−h−
−+
v−
−−
++
+F
++ g
−−
−+
−−
−+
−+
RS
S83
1
Balti
cse
ada
b−r
++
−−
−b−
−−
−+
−+
++
−F
+ g−
−+
++w
RS
S83
2
Balti
cse
abl
enny
−r+
+−
−+b
−−
++
+−
+−
+−
F+ g
−+
−+
+R
RR
832
Can
adia
nAt
lant
icco
d−
+−
+s+
+−
−F
−+
−+
++
Can
adia
nAt
lant
icsa
lmon
Keij
stra
in−
+−
−b−
+−
−F
−+
−+
++
186*
Tab
le4.
1.A
erom
onas
salm
onic
ida
(non
-mot
ileA
erom
onas
spp.
).
156Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:36:04 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
141
Can
adia
nsa
blef
ish
AS2
−+
−+b
++
−−
F−
+−
+−
+18
6*
Dan
ish
cod
−+
−+
−v
+−
−+
F+g
v+
−20
0–2
197
Dan
ish
sand
eel
−+
−+b
−−
−+
+−
F+ g
−+
+20
0–2
197
Dan
ish
turb
ot−
−+
−−
−−
−+
−+
++
F−
+ g−
−+
−−
RR
617
Engl
ish
non-
salm
onid
−+
++
−+t
−−
+−
v−
−−
v+
F+
−v
v+
++
−+
++
4–37
0–3
R40
Nor
way
min
now
−+
−−b
+−
+−
F−
−−
+w+
+w−
−+
−−
−R
S33
1
Sout
hAf
rican
rain
bow
trout
−r+
+−
+b+
−+
+F
++
−+
++
−+
2210
7
pigm
entp
ositi
ve+
−+
−+
+−
63
323
pigm
entn
egat
ive
5672
323
Envi
ronm
enta
l
A.sa
lmon
icid
ape
ctin
olyt
ica
−+
++
−+
−−
−v+
++
−+
−+
+F
++ g
+−
++
−+
++
−−
+35
0R
R61
5
VPat
25°C
.Bro
wn
pigm
ent
Furu
ncul
osis
agar
(FA)
ism
ore
sens
itive
topi
gmen
t pro
duct
ion
than
TSA,
TSA
+t.
P=
pigm
entp
rodu
ced,
b=
Pigm
entt
este
don
BA,f
=pi
gmen
ton
FA,h
=pi
gmen
ton
BHIA
,s=
pigm
entp
rodu
ctio
nsl
ow,t
=Pi
gmen
ttes
ted
onTS
A,*
=Pi
gmen
ttes
ted
onTS
Aco
ntai
ning
L-ty
rosi
ne(T
SA+
t).Th
efo
llow
ing
met
hods
are
reco
mm
ende
dso
asto
stan
dard
ize
test
ing
and
redu
cedi
scre
panc
ies
betw
een
diffe
rent
labo
rato
ries.
Car
bohy
drat
efe
rmen
t atio
nis
perfo
rmed
inph
enol
red
brot
h(D
ifco)
with
1%ca
rboh
ydra
te.H
aem
olys
isis
test
edus
ing
BAco
ntai
ning
hors
ebl
ood,
othe
rmet
hods
are
from
Cow
anan
dSt
eel(
1970
)(an
din
this
Man
ual).
Form
arin
eis
olat
esth
ead
ditio
nof
NaC
lto
afin
alco
ncen
tr atio
nof
1.5%
in‘liq
uid’
tube
ssu
chas
MR
VP,a
escu
lin,n
itrat
e,AD
H,L
DC
,OD
Cm
ayim
prov
eco
nsis
tenc
yof
resu
lts.Z
ones
of20
mm
and
grea
t era
reco
nsid
ered
sens
itive
forA
mp
and
Kf.I
ncub
ate
at20
°Can
dre
adat
7–14
days
,Dal
sgaa
rdet
al.(
1998
)(re
f200
).R
efs
197,
450,
831
and
832
also
use
thes
em
etho
ds.
Test
Gm
Ox
cat
bHm
otpi
gO
DC
LDC
ADH
Nit
Ind
Cit
urea
vpae
sG
OF
arab
glu
inos
lac
mal
tm
anm
ano
sal
sor
suc
treXy
lD
Nas
e40
°C01
29 1001
2915
0Am
p
DNA
hybr
idiz
atio
ngr
oup
HG1
−+
++
+−
−96
++
+−
−91
96+
F87
+ g+
−−
+96
++
−+
+−
++
RR
R
HG2
−+
++
+−
−59
94+
++
−83
++
F+
+ g+
−6 7
++
++
++
+−
+−
RR
R
HG3
−+
+9 2
+−
−55
90+
+46
−90
++
F+
+ g+
−5 4
6 7+
++
++
+−
+−
RR
R
HG4
−+
+1 0
+−
−−
++
9770
−−
97+
F+
+−
87+
+31
+7
++
−+
87R
RR
HG5
−+
+−
−50
−−
9 2+
++
−−
92+
F+
+−
++
++
+−
++
−+
−R
RR
HG6
−+
+−
+−
−−
8 7+
++
−−
++
F8 7
+−
++
++
+−
++
−+
−R
RR
HG8/
10−
++
9 0+
−−
57+
++
91−
9010
+F
14+ g
+−
10+
++
−−
++
−+
+R
RR
Gm
=G
ram
reac
tion,
Ox
=ox
idas
e,ca
t=ca
tala
se,b
H=
bha
emol
ysis
,mot
=m
otilit
y,pi
g=
brow
npi
gmen
t,O
DC
=or
nith
ine
deca
rbox
ylas
e,LD
C=
lysi
nede
carb
oxyl
ase,
ADH
=ar
gini
nedi
hydr
olas
e,N
it=
nitra
tere
duct
ion,
Ind
=in
dole
,Cit
=ci
trate
,ur
ea=
urea
hydr
olys
is,v
p=
Voge
s-Pr
oska
uer,
aes
=ae
scul
inhy
drol
ysis
,G=
gela
tinhy
drol
ysis
,OF
=O
xida
tive
Ferm
enta
tive,
arab
=L-
arab
inos
efe
rmen
tatio
n,gl
u=
gluc
ose
ferm
enta
tion,
inos
=in
osito
lfer
men
tatio
n,la
c=
lact
ose
ferm
ent a
tion,
mal
t=m
alto
sefe
rmen
tatio
n,m
an=
man
nito
lfer
men
tatio
n,m
ano
=m
anno
sefe
rmen
tatio
n,sa
l=sa
licin
ferm
enta
tion,
sor=
sorb
itolf
erm
enta
tion,
suc
=su
cros
efe
rmen
tatio
n,tre
=tre
halo
sefe
rmen
tat io
n,Xy
l=xy
lose
ferm
enta
tion,
Dna
se=
hydr
olys
isof
DN
A,40
°C=
grow
that
40°C
,012
9/10
=se
nsiti
vity
tovi
brio
stat
icag
ent0
129
at10
mgco
ncen
tratio
n,01
29/1
50=
sens
itivi
tyto
vibr
iost
atic
agen
t012
9at
150
mgco
ncen
tatio
n,Am
p=
sens
itivi
tyto
ampi
cillin
.Num
bers
refe
rto
perc
enta
geof
posi
tive
stra
ins,
−=
nega
tive,
+=
posi
tive
reac
tion.
HG
=hy
brid
izat
ion
grou
p.D
ata
from
Abbo
ttet
al.(
1992
),Ka
znow
ski(
1998
).
Tab
le4.
2.A
erom
onas
spp.
Phe
noty
pic
test
sac
cord
ing
toD
NA
hybr
idiz
atio
ngr
oups
.
157Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:36:06 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
142
Test
Gm
Ox
cat
bHm
otSW
OD
CLD
CAD
HN
itIn
dC
itur
eam
rvp
aes
Gon
pgO
Far
abgl
uin
osla
cm
alt
man
man
osa
lso
rsu
ctre
Xyl
H2S
MC
ATC
BSD
Nas
ete
mp
NaC
l01
29 1001
2915
0Am
pR
ef
Org
anis
m
Path
ogen
ic
A.al
losa
ccha
roph
ilaH
G15
−+
++
+−
−+
++
++
−−
++
+F
++ g
+−
−+
++
−−
++
−−
++
4–4
0–3
RR
R52
7
A.be
stia
rum
HG
2−
++
++
−−
++
++
+−
++
++
+F
++ g
v−
++
++
+−
++
−+
+4–
42R
R42
7
A.ca
viae
HG
4−
++
10+
−30
−+
++
+−
+−
++
+F
++ g
−−
87+
+31
+−
++
−−
++
4–37
RR
R21
,14
2
A.hy
drop
hila
dhak
ensi
s−
++
++
−−
++
++
+−
−+
++
+F
−+ g
+−
−+
++
+−
++
−−
+−w
25–4
2R
RR
383
A.hy
drop
hila
LMG
2844
TH
G1
−+
++
+−
−+
++
+6 0
−53
++
++
F+
+ g+
−−
++
++
−+
+−
−+
−w+
4–42
0–2
RR
R1, 383,
427
A.ja
ndae
iH
G9
−+
++
+−
−+
++
+7 5
−+
+−
++
F−
+ g+
−−
++
+−
−−
+−
−+
−w+
4–42
0–3
RR
R13
5,14
2,14
3
A.ve
roni
issp
.ver
onii
HG
10−
++
++
−+
+−
++
+−
++
++
+F
−+ g
+−
−+
++
+−
++
−−
+−
+4–
420–
5R
RR
142,
347
Envi
ronm
enta
l
A.cu
licic
ola
−+
++
+−
−+
++
++
−+
−+
+F
−+ g
+−
−+
++
−−
+−
+4–
37R
RR
624
A.en
chel
eia
HG
16−
++
21+
−−
−+
++
−−
v−
++
+F
−+ g
+−
−+
++
+−
75+
−−
+−
+4–
370–
3R
RR
241,
379
A.eu
cren
ophi
laH
G6
−+
+50
+−
−−
++
+v
−+
−+
++
F+
+ g+
−+
++
++
−+
+−
−+
25–3
70–
2R
RR
1, 379,
420,
427
A.m
edia
HG
5A/5
B−
++
25−
−−
−+
++
5 5−
+−
−+
+F
++ g
−−
++
++
9 4−
++
−−
+−
+4–
370–
3R
RR
15,
294
A.po
poffi
i−
++
−w+
−−
−+
+−
70−
+−
++
F50
+ g+
−−
++
+−
−−
+−
50−
+28
–37
0–2
RR
R38
0
The
type
stra
inis
indo
le-n
egat
ive,
othe
rstra
ins
a re
indo
le-p
ositi
ve
A.so
bria
HG
7−
++
++
−−
++
++
+−
45−
−+
+F
−+ g
v−
++
++
v−
++
−+
+−
30–4
20–
5R
RR
21,
420
88%
VPpo
sin
API2
0E
Tab
le4.
3.A
erom
onas
spp.
–m
otile
.
158Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:36:08 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
143
A.ve
roni
ispp
.sob
riaH
G8
−+
++
+−
−+
++
+50
−+
+−
++
F−
+ g+
−5
++
+−
35+
+−
++
+4–
42R
RR
21,
142,
427,
456
A.sc
hube
rtii
HG
12−
++
++
−−
++
+−
+−
++
−+
+F
−+ g
−−
−+
−+
−−
−+
−−
−+
4–42
0–3
RR
R14
2,34
8
gela
t22 °
C,N
on-h
aem
olyt
icsh
eep
bloo
d
A.tro
taH
G14
−+
++
+−
−+
++
++
−+
−−
++
F−
+ g+
−−
++
+−
−−
+−
−+
−4–
42R
RR
142
Aero
mon
asgr
oup
501
HG
13−
++
+−
++
+−
−+
F−
+ g+
−+
23+
−+
0–3
RR
R
Test
Gm
Ox
cat
bHm
otSW
OD
CLD
CAD
HN
itIn
dC
itur
eam
rvp
aes
Gon
pgO
Far
abgl
uin
osla
cm
alt
man
man
osa
lso
rsu
ctre
Xyl
H2S
MC
ATC
BSD
Nas
ete
mp
NaC
l01
29 1001
2915
0Am
pR
ef
Org
anis
m
Path
ogen
ic
Clo
strid
ium
botu
linum
E+r
−+
+−
−−
−−
−−
+−
−−
−−
141
Rod
sw
ithov
alsu
bter
min
alen
dosp
ores
with
appe
ndag
esan
dex
ospo
ria
Clo
strid
ium
perfr
inge
ns+r
−v
−v
−v
++
−+
−v
+v
−
Edw
ards
iella
icta
luri
oxyg
enin
tole
rant
stra
in
−−
+−
−+
+−
+−
−−
−−
−−
−F
−+ g
−−
++
−+
−−
−−
−−
+25
–37
547
Non
-mot
ileat
37°C
Euba
cter
ium
spp.
841
+r−
−−
−−
++ g
++
+−
−−
+20
0–1
343
Euba
cter
ium
spp.
1065
+r−
−−
−+
++ g
++
+−
−−
−20
0–1
343
Euba
cter
ium
tara
ntel
lae
+r−
+−
−−
−−
−−
++
−−
−−
−−
−−
+25
–37
0–1
764
Long
unbr
anch
edfil
amen
tous
rods
Tab
le4.
4.A
naer
obes
.
159Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:36:09 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
144
Test
Gm
Ox
cat
bHm
otC
O2
Nit
cit
ind
MR
VPH
2Sur
eaBF
SOTH
Ala
Asp
Glu
tAr
gO
rnLy
sG
alR
ibXy
lEr
yU
roA
MR
Ref
Hos
t
Org
anis
mSu
rface
Antig
ens
Path
ogen
ic
Bruc
ella
abor
tus
−+
v−
+−
−−
−v
++
++
+−
−−
++++
+++
+++
++
185,
404
Cat
tle
Bruc
ella
cani
s−
++
−−
−−
−+
−−
−+
+++
++−
+++
−−
−18
5,40
4D
ogs
Bruc
ella
ceta
ceae
−+
−−
−+
++
+−v
−+
−−
−+
++
+−
++
−26
7,40
4U
Kdo
lphi
n,po
rpoi
se
Bruc
ella
mel
itens
is−
++
−−
−−
−+
+++
++++
+−
−−
++
+++
++
+18
5,40
4Sh
eep,
goat
Bruc
ella
neot
omae
−−
++
−−
−−
++
−−
+++
−−
+++
−+
+++
+18
5,40
4D
eser
twoo
dra
t
Bruc
ella
ovis
−−
+−
−−
−−
++
+−
++
−−
−−
−+
−+
185,
404
Shee
p
Bruc
ella
pinn
iped
iae
−+
−+
−+
++
+−v
−+
−−
−−
++
+−
+−
−26
7,40
4Se
al,o
tter
Bruc
ella
spp.
−+
+−
++
++
++
−−
261
Rin
ged
seal
,har
pse
al
Bruc
ella
spp.
−+
+−
−+
+−
++
171
Min
kew
hale
Bruc
ella
suis
1−
+−
−−
−+
+−
−−
−++
++++
++++
+++
+++
+++
++
185,
404
Pigs
Bruc
ella
suis
2−
+−
−−
−−
+−
−−
+++
++
++++
+++
+++
+++
++
185,
404
Pigs
,har
e
Bruc
ella
suis
3−
+−
−−
−−
++
−−
++++
++++
−++
++
+++
+++
+18
5,40
4Pi
gs
Bruc
ella
suis
4−
+−
−−
−−
++
−−
++++
++++
−++
+−
+++
−+
+18
5,40
4R
eind
eer
Bruc
ella
suis
5−
+−
−−
−+
−+
+ +−
−+
−++
+−
+++
++
+40
4
Ala
=L-
alan
ine,
Arg
=L-
argi
nine
,Asp
=L-
aspa
ragi
ne,B
F=
grow
thon
med
iaco
ntai
ning
basi
cfu
chsi
n20
mg/m
l(1/
5000
0),b
H=
bha
emol
ysis
,CO
2=
carb
ondi
oxid
ere
quire
men
t,ca
t=ca
tala
se,c
it=
citra
te,E
ry=
mes
oery
thrit
ol,G
al=
D-g
alac
tose
,G
lut=
L-gl
utam
icac
id,G
m=
Gra
mre
actio
n,H
2S=
hydr
ogen
sulp
hide
,ind
=in
dole
,Lys
=L-
lysi
ne,M
R=
Met
hylR
ed,N
it=
nitra
tere
duct
ion,
Orn
=D
L-or
nith
ine,
Ox
=ox
idas
e,R
ib=
D-ri
bose
,SO
=gr
owth
onm
ediu
mco
ntai
ning
safra
nin
O10
0mg
/ml
(1/1
000
0),T
H=
grow
thon
med
ium
cont
aini
ngth
ioni
n20
mg/m
l(1/
5000
0),U
ro=
uroc
anic
acid
,VP
=Vo
ges
Pros
kaue
r,Xy
l=D-x
ylos
e,Su
rface
antig
ens,
A,M
&R
=ag
glut
inat
ion
with
mon
ospe
cific
antis
era.
Tab
le4.
5.B
ruce
llasp
p.
Test
Gm
Ox
cat
bHm
otC
apO
DC
LDC
ADH
Nit
Ind
Cit
urea
mr
vpae
sG
onpg
OF
arab
glu
inos
lac
mal
tm
anm
ano
sal
sor
suc
treXy
lG
Tte
mp
NaC
l
Org
anis
m
Path
ogen
ic
Cry
ptoc
occu
sne
ofor
man
sva
r.ga
ttii
+−
−+
+−
−+
++
+−
−−
+−
−20
–42
Envi
ronm
enta
l
C.l
upi
++
−−
++
−+
++
++
+4–
250–
2
No
ferm
enta
tion,
only
assi
mila
tion.
Anta
rctic
habi
t at.
Gro
wth
ongl
ucos
epe
pton
eye
aste
xtra
ct
Can
dida
spp.
+−
−
Cap
=ca
psul
e;G
T=
germ
tube
.
Tab
le4.
6.C
rypt
ococ
cus.
160Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:36:11 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
145
Test
Gm
Ox
cat
bHm
otG
lidO
DC
LDC
ADH
Nit
Ind
Cit
urea
mr
vpae
sG
onpg
OF
arab
glu
inos
lac
mal
tm
anm
ano
sal
sor
suc
treXy
lH
2SM
CA
CR
KOH
DN
ase
tem
pN
aCl
0129
150
NA
TSA
Ref
Org
anis
m
Path
ogen
ic
Chr
yseo
bact
eriu
mba
lust
inum
−+
+−
−v−
−−
−+v
+−
−v+
+−v
O−
+−
−−
−−
−−v
−v−
−+
−+
+v15
–35
0–2
++
89,8
02
Prev
ious
lyFl
avob
acte
rium
balu
stin
um.Y
ello
wpi
gmen
t
C.s
coph
thal
mum
−+
++
−v
−−
−−
−−
+−
−+
+F
−+
−−
+−
−−
−−
−−
++
15–2
50–
4+
556,
557
Prev
ious
lyFl
avob
acte
rium
scop
htha
lmum
.Ora
nge
pigm
ent
Flav
obac
teriu
mbr
anch
ioph
ilum
ATC
C35
035
−+
+−
−−
−−
++
O−w
+ g−
−−
+−
−v+v
−+
+−w
−−
−−
−10
–25
0–0.
2S
−−
92,8
02
Yello
wpi
gmen
t,no
agar
hydr
olys
is.G
row
thon
1/20
TSA
603
F.co
lum
nare
IAM
1430
1T
−+
+−
−+
−−
−80
−−
−−
−−
+−
O−
−−
−−
−−
−−
−v
++
++
10–3
70–
0.5
S−
−14
5,60
3
Adhe
res
stro
ngly
toag
ar.H
2Sfro
mle
adac
etat
em
ediu
m(o
ther
refe
renc
es11
1,13
9,29
8,29
9)13
5,75
9
F.hy
datis
−−v
++
−+
−−
−+
−−
−−
−+
++
F+
+−
++
++
−−
+−
+−
−−
++
20–3
00–
1R
++
92,7
20
Prev
ious
lyC
ytop
haga
aqua
tilis
.Muc
oid,
oran
ge-y
ello
wco
loni
es89
F.jo
hnso
niae
AHLD
A17
14−
++
−+
−−
−+
−+
+s−
−+
++
F−
w−
−−
−−
−−
−−
−−
−+
−S
++
135
Fjo
hnso
niae
ATC
C17
061
−+
+−
+−
−−
+−
++
++
O+
++
+−
++
−+
+w−
++
+5–
300–
1S
++
145,
603
F.jo
hnso
niae
DSM
2064
−+
++
−−
−−
−+
++
O+
−−
−+
10–3
00–
2S
+89
F.jo
hnso
niae
DSM
2958
5*−
++
+−
−−
+−
++
+O
+−
−+
+10
–30
0–1
S+
+89
F.ps
ychr
ophi
lum
−+v
+70
−w
−−
2−
−−
−−
−+
−O
−−
−−
−−
−−
−−v
−−
−−
−+
+w4–
200–
0.8
S−
−89
,92
Yello
wco
loni
es.G
row
then
hanc
edin
AOm
edia
with
0.5%
trypt
one
90,1
68
Tena
ciba
culu
m(F
lexi
bact
er)m
ariti
mus
−+
+−
++
−−
−−
−+
−I
−−
−−
−−
−−
−−
−−
−+
−+w
14–3
42+
S−
89,8
01
Pale
yello
wco
loni
es.N
oag
arhy
drol
ysis
.Pre
pare
med
iaw
ithAS
Wba
se90
T.(F
lexi
bact
er)
ovol
ytic
us−
++
−+
−−
−+
−−
−+
−O
−−
−−
−−
−−
−−
+4–
253+
−32
4
Pale
yello
wco
loni
es.N
Gon
TCBS
*Thi
sor
gani
smis
nolo
nger
inth
eD
SMZ
data
base
.N
umbe
rsre
fert
ope
rcen
tage
ofpo
sitiv
est
rain
s.1/
20TS
A=
TSA
prep
ared
ata
1:20
dilu
tion,
+s=
slow
reac
tion,
−w=
nega
tive
orw
eak
reac
tion,
Ov
=O
xida
tive
varia
ble
resu
lts,v
=va
riabl
ere
actio
n,N
G=
nogr
owt h
.
Tab
le4.
7.C
ytop
haga
–Fla
voba
cter
iace
ae–B
acte
roid
esgr
oup
–pa
thog
enic
.
161Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #M.vpTuesday, January 20, 2004 4:09:12 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
146
Org
anis
m
Aequ
oriv
itaan
tarc
tica
−+
−−
−−
−−
−v+
+O
−−
−−
−−
−−
−−
−−
−0–
250.
5–6
113
Ora
nge-
pigm
ente
dco
loni
es
Aequ
oriv
itacr
ocea
−+
−−
−−
−−
−+
+O
−−
−−
−−
−−
−−
−−
+v0–
250.
5–6
113
Yello
w-p
igm
ente
dco
loni
es
Aequ
oriv
italip
olyt
ica
−+
−−
−−
−−
+v+
+O
−−
−−
−−
−−
−−
−−
−0–
250.
5–6
113
Yello
w-p
igm
ente
dco
loni
es
Aequ
oriv
itasu
blith
inco
la−
+−
−−
−−
−+
−+
O−
−−
−−
−−
−−
−−
−−
0–25
1–6
113
Ora
nge-
pigm
ente
dco
loni
es
Cel
lulo
phag
aly
tica
−+
+−
+−
−−
−−
+O
−+
−−
−15
–37
1–2
89
Chr
yseo
bact
eriu
m(F
lavo
bact
eriu
m)
gleu
m
−+
+−
−−
−−
−v
−−
v+
+I
+−
−−
−−
−−
++
+25
–37
+36
6
Indo
lepo
sw
ithEh
rlich
sbu
tneg
ativ
eby
K ová
cs
Chr
yseo
bact
eriu
min
dolo
gene
s−
++
−+
−−
−v
+−
++
vO
+−
++
−−
+−
++
+36
557
Chr
yseo
bact
eriu
min
dolth
etic
um−
−+
−+
+−
++
3655
7
Chr
yseo
bact
eriu
mm
enin
gose
ptic
um−
++
−−
−−
−−
−+
vv
−−
++
+O
−+
−+
+v
+−
−−
v−v
−w
−−
+37
–42
0–2
++
89,8
02
prev
ious
lyFl
avob
acte
rium
men
ingo
sept
icum
Cyt
opha
gaal
lerg
inae
−+
+−
−−
−−
++
O−
−+
15–2
50–
1+
89
Cyt
opha
gaar
vens
icol
a−
++
−−
−−
−+
++
O−
−+
15–3
70–
2+
89
Cyt
opha
gafe
rmen
tans
−+
v−
−−
−−
−+
+F
++
−−
−15
–25
1–2
89,1
62
Cyt
opha
gahu
tchi
nson
ii−
++
−+
−−
−−
−+
v−
O+
−−
+−
15–2
50–
189
pigm
ente
dco
loni
es.D
iges
tsce
llulo
se
Cyt
opha
gala
terc
ula
−+
−−
−−
−+
+−
O+
++
+15
–30
1–2
89,4
94
Brig
htre
dpi
gmen
t
Cyt
opha
ga-li
ke−
++
++
−−
−−
−26
+−
−+
+O
+−
−−
−−
+15
–25
0–4
556
Ora
nge
colo
nies
.No
agar
dige
stio
n
Cyt
opha
gam
arin
ofla
va−
++
+F
++
−16
2
Test
Gm
Ox
cat
bHm
otG
lidO
DC
LDC
ADH
Nit
Ind
Cit
urea
mr
vpae
sG
onpg
OF
arab
glu
inos
lac
mal
tm
anm
ano
sal
sor
suc
treXy
lH
2SM
CA
CR
KOH
Das
ete
mp
NaC
l01
2915
0N
ATS
AR
ef
Tab
le4.
8.C
ytop
haga
–Fla
voba
cter
iace
ae–B
acte
roid
esgr
oup
–en
viro
nmen
tal.
162Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:36:14 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
147
Empe
doba
cter
(Fla
voba
cter
ium
)br
evis
−+
+−
−−
−−
−−
+−
−−
+−
O−
−v−
−+
−−
−−
−−
−−
+−
++
28–3
70–
2+
+89
,363
Pigm
ente
dco
loni
es,f
ruity
odou
r80
2
Flav
obac
teriu
maq
uatil
e−
++
−+
−−
−+v
−−
−+v
+v+v
O−
+−
++
−−
−+
−−
−−
−−
−5–
300–
0.5
R−v
+92
,802
89,6
03
Flav
obac
teriu
mfle
vens
e−
++
−+
−−
−−v
+−
+O
+−
−−
−15
–22
0–2
S+
+89
,533
Hyd
roly
ses
agar
92
Flav
obac
teriu
mfri
gida
rium
−+
+−
−−
−−
−−
−+
+−
O−
−−
−−
−+
−−
15–2
80–
5+
+37
6
Flav
obac
teriu
mgi
llisia
e−
−+
−−
−+
−−
−−
−+
−−
O−
+−
−+
++
−+
+−
−−
−−
0–27
0–5
R+
+53
3
Flav
obac
teriu
mhi
bern
um−
−+
+−
−−
+−
−−
−+
++
F+
+−
−−
+−
−+
+0–
300–
2+
+53
2,53
3
Flav
obac
teriu
mm
izut
aii
−+
+−
−v−
+v−
+−
O+
++
−+
++
−−
30–3
7+
+84
4
Flav
obac
teriu
mpe
ctin
ovor
um−
++
++
++
+v
−+
+25
0–1
S+
+92
,533
Flav
obac
teriu
msa
ccha
roph
ilum
−−
++
++
++
−+
−+
−4–
300–
2S
++
92
Hyd
roly
ses
agar
Flav
obac
teriu
msu
ccin
ican
s−
++
+v
++
+F
+w
−−
−+
250
S+
+92
,162
,53
3
Flav
obac
teriu
mte
getin
cola
−−
++
−−
−−
−−
−−
−−
−O
−+
−−
+−
−−
−−
−−
−0–
271–
5R
++
533
Flav
obac
teriu
mxa
nthu
m−
++
−−
−−
+−
−−
−+
+−
O−
+−
−+
++
−+
+−
+−
−−
0–20
2R
++
533
Flex
ibac
tera
ggre
gans
−+
−−
+−
−−
−−
+O
++
+−
−22
–37
1–2
89
Hyd
roly
ses
agar
Flex
ibac
terc
anad
ensi
s−
++
−−
+−
−−
+−
−−
−+
+F
++
−−
−v−
−−
+10
–40
0–1
+89
Flex
ibac
tere
lega
ns−
+−
−−
+O
−−
−22
–25
0–2
162
Flex
ibac
terf
lexi
lis−
+−
−+
−−
−−
−+
−O
−+
−+
−−
+−
−−
−−
5–30
0–0.
2−
−89
,603
Cel
ls15
–20
mm,O
rang
e-pi
gmen
ted
colo
nies
Flex
ibac
terl
itora
lis−
+−
−−
−−
−+
−O
−−
+−
−15
–25
2–5
89,1
62
Pink
pigm
ent
cont
inue
d
163Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:36:16 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
148
Flex
ibac
ter
poly
mor
phus
−+
−−
+−
−−
+v
+O
−−
−22
–32
289
Cel
ls>1
00mm
.Hyd
roly
ses
agar
.Pea
ch-c
olou
red
pigm
ent
Flex
ibac
terr
oseo
lus
−+
−−
−−
−−
+−
O−
−−
−−
22–3
70–
2+
+89
,162
Cel
ls50
–100
mm.B
right
-red
oran
gepi
gmen
t
Flex
ibac
terr
uber
−+
−−
−−
−−
+−
O+
+−
−−
22–3
70–
2+
+89
,162
Brig
htre
d-or
ange
pigm
ent.
Cel
ls50
–100
mm
Flex
ibac
ters
anct
i−
++
−−
−+
+O
−+
+15
–37
0–1
+89
Flex
ibac
tert
ract
uosu
s−
++
−−
−−
−+
−O
−/+
−−
22–3
70–
289
,162
Ora
nge
pigm
ent
Mar
inila
bilia
salm
onic
olor
−+
+−
−−
−−
++
F+
++
−−
15–3
71–
289
,162
Salm
on-p
ink
colo
ur
Mar
inob
acte
rhy
droc
arbo
nocl
astic
us−
++
−+
+−
−40
–45
269
Prev
ious
lyPs
eudo
mon
asna
utic
a
Myr
oide
sod
orat
imim
us−
++
−−
−−
−−
−+
−+
−O
−+
18–3
789
,362
Myr
oide
sod
orat
us−
++
−−
−−
−−
−−
−+
−+
−al
k−
−−
−−
−−
−−
−−
−+
−−
+18
–37
0–2
++
89,3
62
Prev
ious
lyFl
avob
acte
rium
odor
atum
,fru
ityod
our
802
Pedo
bact
erhe
parin
us−
++
+s−
−−
−−
v−
−v
−−
+−
+O
−+s
++
−−
++
−+
−−
−−
+5–
370–
3+
+89
,163
,72
8
Pedo
bact
erpi
sciu
m−
++
−−
−−
−+
−O
−−
−+
−−
+−
+5–
28+
+72
8
Sale
gent
ibac
ter
sale
gens
−+
+−
−O
−−
−−
−−
0–30
0–20
R53
3
Sphi
ngob
acte
rium
mul
tivor
um−
++
−−
−−
−−
−−
++
14+
O+
+v−
++
−+
+−
++
+−
+−
−60
15–3
70–
2+
+89
,802
Sphi
ngob
acte
rium
spiri
tivor
um−
++
−−
−−
−−
−−
−+
++
+O
27+v
−+v
++
++
−+
++
−+
−−
+15
–37
0–2
++
89,3
65,
802
Zobe
lliaga
lact
aniv
oran
s−
++
−+
−+
−−
−−
++
+O
++
++
+−
+−
++
13–4
50.
5–6
61
Zobe
lliaul
igin
osa
−+
+−
+−
+−
−+
++
O+
+−
++
+13
–30
0.5–
261
alk
=al
kalin
ere
actio
n,C
R=
Con
goR
ed,G
lid=
glid
ing
mot
ility,
KOH
=po
tass
ium
hydr
oxid
e,N
A=
grow
thon
nutri
enta
gar,
TSA
=gr
owth
ontry
pton
eso
yag
a r.
Test
Gm
Ox
cat
bHm
otG
lidO
DC
LDC
ADH
Nit
Ind
Cit
urea
mr
vpae
sG
onpg
OF
arab
glu
inos
lac
mal
tm
anm
ano
sal
sor
suc
treXy
lH
2SM
CA
CR
KOH
Das
ete
mp
NaC
l01
2915
0N
ATS
AR
ef
Tab
le4.
8.C
ontin
ued.
164Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:36:18 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
149
Test
Gm
Ox
cat
bHm
otSW
OD
CLD
CAD
HN
itIn
dC
itur
eam
rvp
aes
Gon
pgO
Far
abgl
uin
osla
cm
alt
man
man
osa
lso
rsu
ctre
Xyl
H2S
MC
ATC
BSD
Nas
ete
mp
NaC
l01
29 1001
2915
0Am
pR
ef
Org
anis
m
Path
ogen
ic
Acin
etob
acte
rsp
p.−c
r+
++
−−
−−
−−
−−
−−
−F
−−
−−
+−
+−
−−
−41
Not
e:Ac
inet
obac
terd
ono
tusu
ally
prod
uce
oxid
ase
Mor
axel
lasp
p.cb
++
+−
−−
−−
−−
−−
−−
−−
−−
−+w
−−
−−
72
Hae
mol
ysis
test
edon
shee
pbl
ood
Taxo
nom
icpo
sitio
nof
both
thes
eor
gani
sms
isin
doub
t
Envi
ronm
enta
l
Acin
etob
acte
rba
uman
nii
−−
+−
−−
−v
−−
−O
+−
−15
–37
110
Acin
etob
acte
rca
lcoa
cetic
us−
−+
−−
−−
+−
−−
O+
−−
15–3
711
0
Acin
etob
acte
rha
emol
ytic
us−
−+
+−
−−
+−
+−
O52
−−
15–3
711
0
Mes
ophi
loba
cter
mar
inus
−cb
++
−−
−+
v+
v+
−v
O+
+−
5–37
1–6
583
Gro
wth
onN
A,M
A22
16
Tab
le4.
9.G
ram
-neg
ativ
eco
ccob
acill
i and
cocc
i.
Org
anis
m
Path
ogen
ic
Citr
obac
terf
reun
dii
−−
++
−12
−v
+−
+−
+−
−−
+F
++ g
+−
++
+−
+10
++
++
−20
–37
0–2
414,
425
Edw
ards
iella
hosh
inae
−−
+−
+−
++
−+
w+
−−
+−
−−
−F
30+ g
v−
−+
++
7 5−
++
−+
+−
25–4
00–
1.5
S31
7
Mot
ileat
25°C
and
35°C
Edw
ards
iella
icta
luri
−−
+−
+−
++
−+
−−
−+
−−
−−
F−
+ g+
−−
+−
+−
−−
−−
−+
−20
–37
0–1.
533
4,37
4
Wea
kly
mot
ileat
25°C
butn
otat
35°C
.Rea
dM
Rat
48h
808
Edw
ards
iella
icta
luri
oxyg
enin
tole
rant
stra
in
−−
+−
−+
+−
+−
−−
−−
−−
−F
−+ g
−−
++
−+
−−
−−
−−
+25
–37
547
Cul
ture
anae
robi
cally
.Non
-mot
ileat
37°C
Test
Gm
Ox
cat
bHm
otSW
OD
CLD
CAD
HN
itIn
dC
itur
eam
rvp
aes
Gon
pgO
Far
abgl
uin
osla
cm
alt
man
man
osa
lso
rsu
ctre
Xyl
H2S
MC
ATC
BSD
Nas
ete
mp
NaC
l01
29 1001
2915
0Am
pR
ef
Tab
le4.
10.
Gra
m-n
egat
ive,
oxid
ase-
nega
tive
rods
.
cont
inue
d
165Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:36:19 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
150
Edw
ards
iella
tard
a−
−+
−+
−+
+−
++
−−
+−
−−
−F
−+ g
+−
−+
−+
−−
v−
−+
+−
420–
237
4,64
0
Mot
ileat
25°C
and
35°C
Esch
eric
hia
vuln
eris
−−
++
+−
++
+−
−−
+−
+−
F+
+ g+
−+
++
+−
−+
+−
+−
−20
–37
RR
51
Haf
nia
alve
i−s
r−
+−
+−
++
−+
−−
−21
87−
−+
F+
+ g+
−−
++
+−
−−
++
−+
−−
4–40
0–4
RPS
313,
652
Hal
omon
ascu
pida
−−
+−
++
++
+−
−−
I+
++
++
+ w+
−10
–25
S
Kleb
siel
lapn
eum
onia
e−
−+
−−
−−
+v−
+−
++
6040
+−
+F
++ g
++
++
++
++
++
−+
−35
0–5
RR
414
Muc
oid
Pant
oea
aggl
omer
ans
−−
+−
+−
−−
−+
−+
−−
++
++
F+
+ g−
−−
++
++
−+
++
−+
−−
4–37
0–6
249,
325
Opp
ortu
nist
,yel
low
pigm
ent,
MR
+ve
with
2%N
aCl
Prov
iden
cia
(Pro
teus
)ret
tger
i−
−+
+−
−−
−+
++
++
−+
−−
F−
++
−−
++
v−
−−
−−
++
18–3
70–
379
Salm
onel
laar
izon
ae−
−+
−+
−+
+−
+−
+−
−−
−F
−+ g
++
++
−+
++
++
+15
–41
0–6
RR
447
Serra
tialiq
uefa
cien
s−
−+
++
−+
+−
−−
+−
−+
++
+F
++ g
++
−+
++
++
++
+−
+−
+4–
370–
5R
R25
0,29
0
Ref
290
isM
R+8
6%at
37°C
,17%
at30
°C71
5
Serra
tialiq
uefa
cien
s−
−+
++
−+
+−
−−
+−
−+
++
+F
++ g
+−
−+
++
++
++
+−
+−
+4–
370–
5R
R71
5
Arct
icch
aris
olat
esar
ein
dole
nega
tive,
inos
nega
t ive
Serra
tiam
arce
scen
s−
−+
v+
−+
+−
+−
+−
−+
+v
+F
−+ g
++
−+
++
++
++
−−
+4–
450–
8R
RR
76
Red
pigm
ent
Serra
tiapl
ymut
hica
−−
+−
−−
31−
−+
−+
−−
++
+F
++ g
−+
−+
++
+v
++
+−
−+
4–37
0–8
RR
R76
,57
9
Red
pigm
ent.
MR
+ve
at37
°Con
ly
Stre
ptob
acillu
sm
onilif
orm
is−
−−
−−
−−
+−
v−
−−
−−
−F
+−
+−
−−
−v
10–2
51–
451
9,61
1
IDof
orga
nism
was
notc
oncl
usiv
e
Stre
ptob
acillu
sm
onilif
orm
is−
−−
−−
−−
−−
−−
−−
−−
F−
w−
w−
−−
−−
−25
–35
169
Rea
ctio
nsof
type
stra
in.R
equi
res
addi
tion
of20
%se
rum
forg
row
th
Yers
inia
inte
rmed
ia−
−+
++
−−
++
++
++w
+−
+F
++
+−
++
++
++
+50
−−
402
Mot
ileat
25°C
butn
otat
35°C
.MR
,VP,
cit+
veat
35°C
,−ve
at25
°C
Test
Gm
Ox
cat
bHm
otSW
OD
CLD
CAD
HN
itIn
dC
itur
eam
rvp
aes
Gon
pgO
Far
abgl
uin
osla
cm
alt
man
man
osa
lso
rsu
ctre
Xyl
H2S
MC
ATC
BSD
Nas
ete
mp
NaC
l01
29 1001
2915
0Am
pR
ef
Tab
le4.
10.
Con
tinue
d.
166Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:36:21 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
151
Yers
inia
ruck
eri
−−
++
+−
++
−85
−+
−+
−−
52+
F−
+ g−
−−
++
97−
−−
97−
−+
−37
0–3
137,
250
Mot
ileat
25°C
butn
otat
35°C
.Citr
ate
+ve
at25
°Cbu
t−ve
at37
°C50
0,65
7
Y.ru
cker
i−
−+
−+
−+
+−
++
+−
+−
++
F−
+−
−+
++
−+
−+
−+
−0–
550
0
Aust
ralia
nst
rain
repo
rted
byLl
ewel
lyn
(198
0)
Y.ru
cker
iser
ovar
I−
Y.ru
cker
iser
ovar
II+
Y.ru
cker
iser
ovar
III−
Y.ru
cker
iser
ovar
IVTh
ese
are
nots
train
sof
Y.ru
cker
i+
+
Y.ru
cker
iser
ovar
V+
Y.ru
cker
iser
ovar
VI
Fish
isol
ates
that
ferm
enta
rab
&rh
amno
sear
eno
tY.r
ucke
ri,bu
tmay
beH
afni
aal
vei
Envi
ronm
enta
l
Budv
icia
aqua
tica
−−
++
−−
−−
+−
−+
v−
−−w
F+
+−
−+ s
−−
−−
−+
++
−4–
370–
4
Citr
obac
terd
iver
sus
−−
++
−+
−60
++
++
+−
−−
+F
++ g
+−
33+
+10
+17
++
−R
R24
8
Ente
roba
cter
aero
gene
s−
−+
+−
++
−−
−−
++
−+
+ g+
++
+40
++
++
−−
0–5
414
Ente
roba
cter
cloa
cae
−−
+−
+−
+−
++
−+
−−
++
++
F+
+ g+
70+
++
++
++
+−
+−
−0–
5R
R41
4
Ente
roba
cter
lique
faci
ens
−−
++
−+
65−
−−
25+
++
++ g
++
60+
++
++
+−
+0–
541
4
Esch
eric
hia
coli
−−
+v
50−
6050
−+
+−
−+
−40
−+
+ gv
−55
+30
+55
++
−−
25–4
40–
541
4
Hal
omon
asel
onga
ta−
+v+
+−
++
+22
6611
1156
4433
F+
67+
+−
300.
1–20
R79
5
Hal
omon
asha
lodu
rans
−−
+−
++
−−
+−
+−
F+
+−
−+
−22
–37
0.1–
2033
6
Hal
omon
asm
arin
a−
Kleb
siel
laor
nith
inol
ytic
a−
−+
−+
+v
F10
–37
228
Kleb
siel
laox
ytoc
a−
−+
−−
++
++
F10
–41
228
Kleb
siel
lapn
eum
onia
eoz
aena
e−
−+
−−
−−
−+
−+
−+
−+
−+
+ g−
−+
++
+−
++
−+
−25
–37
0–5
414
Kleb
siel
larh
inos
cler
omat
is−
−+
−−
−−
−−
+−
+−
+−
−−
F+
+ g−
+−
++
++
++
−+
−25
–37
0–5
414
Kluy
vera
asco
rbat
a−
−+
+−
++
−+
++
−+
−−
+F
+ g+
+41
+v−
5−Kl
uyve
racr
yocr
esce
ns−
−+
+−
++
−+
++
−+
−−
+F
+ g+
+41
+v−
5+
cont
inue
d
167Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #M.vpMonday, January 26, 2004 11:49:27 AM
Color profile: DisabledComposite 150 lpi at 45 degrees
152
Pant
oea
disp
ersa
−−
+−
10−
−+
−+
−+
−+ s
+F
++ g
−+
−+
++
−−
++
+−
−30
–41
0R
R29
1
Yello
wpi
gmen
t.Ty
pest
rain
isO
DC
+ve
Prov
iden
cia
fried
eric
iana
−−
+−
+rt
−−
−−
++
w−
+−
−−
−F
−+ g
+−
−−
−+
−−
+ s−
−−
+−
10–4
00
559
Mot
ileat
25°C
butn
egat
37°C
Rah
nella
aqua
tilis
−−
+−
−−
−+
−+
−89
++
−+
F+
+ g+
−+
++
++
++
++
−+
−−
4–37
0R
R12
4
Mot
ileat
25°C
butn
otat
35°C
.Man
=−v
eby
API2
0E
R.a
quat
ilisG
enos
peci
es2
−−
31−
−−
−+
−79
−+
++
34+
F+
+ g+
−+
++
++
++
++
−+
−−
124
R.a
quat
ilisG
enos
peci
es3
−−
−−
−−
−+
−−
−52
72+
−+
F+
+ g+
−+
++
++
++
++
−+
−−
124
Rao
ulte
llapl
antic
ola
−−
+−
−−
−+
−+
++
++
++
−+
F+
+ g+
++
++
++
++
++
−+
−−
4–41
022
8
Rao
ulte
llate
rrige
na−
−+
−−
−−
+−
+−
++
++
+−
+F
++ g
++
++
++
++
++
+−
+−
4–35
0R
R22
8,40
2
Sim
ilarm
orph
olog
ical
lyto
K.pn
eum
onia
e
Serra
tiafo
ntic
ola
−−
++
−+
+−
+−
+−
+−
+−
+F
++ g
++
++
++
++
+ s+
70−
+−
4–37
290,
558
Serra
tialiq
uefa
cien
s−
−+
−+
−− v
+−
+−
+v
37+
++
F+
+ g+
6416
++
++
++
++
−+
−+
500
Serra
tiam
arce
scen
s−
−+
−+
−+
++
4018
+73
70F
27−
7717
++
+−
+29
0
Serra
tiaru
bida
ea−
−+
+−
−+
−+
2 4+
++
F+
−35
++
−+
++
250,
290
Yers
inia
aldo
vae
−−
+−
+ v−
+−
−+
−+
++
++
−+
F+
++
− v+
++
−+
++
+−
85
Yers
inia
berc
ovie
ri−
−+
−+
−+
−−
+−
−+
+−
+ s−
+F
++
−−
++
++ s
++
++
−+
−81
2
Yers
inia
ente
roco
litic
a−
−+
−−
−v
−+
v−
++
+15
−+
F+
−−
++
+36
250
Yers
inia
frede
rikse
nii
−−
+−
+−
+−
−+
+−v
++
++
−+
F+
+44
22+
++
++
++
20−
−25
–37
042
3
Mot
ility
&VP
posi
tive
at25
°C,b
utne
gativ
eat
37°C
Yers
inia
kris
tens
enii
−−
+−
+−
+−
−+
43−
++
−−
−+
F+
+ g−
2760
++
+−
+−
++
−−
4–41
038
3,40
2
Mot
ileat
25°C
butn
otat
35°C
.VP
−ve
at25
°CYe
rsin
iam
olla
retii
−−
+−
+−
+−
−+
−−
++
−+ s
−F
++
+ s−
++
++ s
++
++
−+
−81
2
Yers
inia
pseu
dotu
berc
ulos
is−
−+
+−
−−
−+
−−
++
−+
−F
++
−−
++
++
−−
++
−v
121
Yers
inia
rohd
ei−
−+
−+
−+ s
−−
+−
++ s
+−
−−
F+
+−
++
++
−+
++
+−
+−
−25
0R
R12
All Y
ersi
nia
and
Haf
nia
spec
ies
shou
ldbe
incu
bate
dat
25–2
8°C
Test
Gm
Ox
cat
bHm
otSW
OD
CLD
CAD
HN
itIn
dC
itur
eam
rvp
aes
Gon
pgO
Far
abgl
uin
osla
cm
alt
man
man
osa
lso
rsu
ctre
Xyl
H2S
MC
ATC
BSD
Nas
ete
mp
NaC
l01
29 1001
2915
0Am
pR
ef
Tab
le4.
10.
Con
tinue
d.
168Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:36:24 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
Test
Gm
Ox
cat
bHm
otSW
OD
CLD
CAD
HN
itIn
dC
itur
eam
rvp
aes
Gon
pgO
Far
abgl
uin
osla
cm
alt
man
man
osa
lso
rsu
ctre
Xyl
H2S
MC
ATC
BSD
Nas
ete
mp
NaC
l01
29 1001
2915
0Am
pR
ef
Tab
le4.
11.
Gra
m-n
egat
ive,
oxid
ase-
posi
tive
rods
.
153
Org
anis
m
Path
ogen
ic
Bene
chea
chiti
novo
ra−
?+
+−
++
++
−+
F+ g
++
+20
–25
1–4
806
Bord
etel
labr
onch
isep
tica
−+
++
+−
−−
−+
−+
+−
−−
−O
−−
−−
−−
−−
−−
−+
25–3
70–
419
9,64
2
Bruc
ella
abor
tus
−+
+v
−+
−−
+s−
−O
v−
−−
−+
−v
350
169
Burk
hold
eria
pseu
dom
alle
i−
++
v+
−−
−+
+−
v−
−−
+v
−O
++
++
++
+−
+−
++
−+
20–4
238
,62
3
APIr
eact
ions
may
bene
gfo
rure
a&
Nit,
18%
pos
forc
it
Del
eya
aqua
mar
inus
−+
+−
+−
+−
+−
−+
−−
−−
−O
++s
−4–
421–
10S
8,45
Prev
ious
lyAl
calig
enes
faec
alis
hom
ari
Jant
hino
bact
eriu
mliv
idum
−+
++
−+
+−
+−
++
+−
O+s
−+
−+
++
++
+−
+s4–
300–
249
6
Purp
lepi
gmen
t
Past
eure
llam
ulto
cida
−+
+−
−−
+−
−+
+−
−−
−−
−−
Fv
+−
v+
−v
+v
v−
−25
–37
016
9,56
2
Past
eure
llask
yens
is−
+w−
−w−
−+
+−
−+
−−
−F
+−
++
++
−−
−+
−−
14–3
21.
5–2
100
Past
eure
llate
stud
inis
−+
++
−−
−−
−+
+−
−+
+F
v+ g
−+s
−85
+−
−v
+v
+v
709
Ples
iom
onas
shig
ello
ides
−+
+9 0
+−
++
9 5+
5 0−c
−v
−−
−+
F−
+ gv
50+
+50
+−
50−
+−
−+
−G50
10–4
20–
3R
SS
169
Pseu
doal
tero
mon
asba
cter
ioly
tica
−+
+w+
−−
−+
O+
15–3
53
677
Red
pigm
ent
Pseu
doal
tero
mon
aspi
scic
ida
−+
++
−−
+−
+O
+−
+−
+−
+25
–40
5–10
134,
572
Pseu
dom
onas
angu
illise
ptic
a−
++
−+
−−
−−
−−
+−
−v
−I
−−
−−
−−
−−
−−
−+
−v
5–30
0–3
SR
Pen
=R
96,
541
Finn
ish
isol
ates
are
gelp
os,J
apan
ese
isol
ates
are
geln
eg82
8
Pseu
dom
onas
chlo
rora
phis
−+
++
−v
v+
++
+O
+−
+−
++
4–35
Gre
enco
loni
es.5
days
forg
row
th
Ps.f
luor
esce
nsbi
ovar
I−
++
+−
−+
−+
+−
O+
+−
++
+−
++
++
4–37
cont
inue
d
169Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #M.vpTuesday, January 20, 2004 4:11:30 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
154
Test
Gm
Ox
cat
bHm
otSW
OD
CLD
CAD
HN
itIn
dC
itur
eam
rvp
aes
Gon
pgO
Far
abgl
uin
osla
cm
alt
man
man
osa
lso
rsu
ctre
Xyl
H2S
MC
ATC
BSD
Nas
ete
mp
NaC
l01
29 1001
2915
0Am
pR
ef
Tab
le4.
11.
Con
tinue
d.
Ps.f
luor
esce
nsbi
ovar
II−
++
+−
−+
++
+−
O+
+−
++
+−
++
++
4–37
Ps.f
luor
esce
nsbi
ovar
III−
++
+−
−+
++
+−
O+
+−
+v
+−
v−
++
4–37
Ps.f
luor
esce
nsbi
ovar
IV−
++
+−
−+
++
+−
O+
+−
++
+−
++
++
4–37
Ps.f
luor
esce
nsbi
ovar
V−
++
+−
−+
−+
+−
O+
+−
+v
+−
vv
++
4v−3
7
Pseu
dom
onas
fluor
esce
ns−
++
++
−−
+v
−+
v−
−−
+−
O+
+v
−+
++
−v
vv
+−
+4–
30R
R62
3
Pseu
dom
onas
plec
oglo
ssic
ida
−+
++
+−
−−
++
−+
−−
−O
−10
–30
0–5
RR
582
Clo
sely
rela
ted
toPs
eudo
mon
aspu
tida
Pseu
dom
onas
pseu
doal
calig
enes
−+
+−
+−
v−
+v
−+
−−
−v
−O
A+
+−
−14
−−
−−
11−
+−
15–4
10
297
Pseu
dom
onas
putid
a−
++
v+
−−
−+
−−
+44
−−
−O
++
−−
2119
+−
−v9
−+
−+
4–35
0–5
297,
623
Pseu
dom
onas
stut
zeri
−+
+−
+−
−−
−+
−+
14−
v−
Ov
+−v
−+
68+
−−
v−
+−v
+−
25–4
10–
629
7,35
9
Wrin
kled
colo
nies
,slig
htly
yello
w62
3
Ros
eoba
cter
stra
inC
VSP
−+
w+
−I
232
104
Shew
anel
lapu
trefa
cien
s−
++
−+
−+
−−
+−
+−
−−
−+
−O
50+s
−−
70−
9−
−35
−−
++
−+
4–37
0–3
433
Varra
calb
imi
−+
−−a
−−
−−
+−
−−
−−
F−
+ g−
++
++
+4–
222
SS
771
Envi
ronm
enta
l
Actin
obac
illus
delp
hini
cola
−+
−−w
−−
++
−+
−−
−+
−−
F+
−−
−−
+−
−−
−−
−30
–42
263
Stra
infro
mM
esop
lode
nbi
dens
.10%
CO
2re
quire
dfo
rgro
wth
Actin
obac
illus
delp
hini
cola
−+
−−w
−−
70+
21+
−−
−+
−−
F+
−−
−−
+−
−−
−−
−30
–42
263
Stra
ins
from
Phoc
oena
phoc
oena
.10%
CO
2re
quire
dfo
rgro
wth
Actin
obac
illus
delp
hini
cola
−+
−−w
−−
−−
++
−−
−+
−−
F+
−−
−−
+−
−−
−−
−30
–42
263
Stra
ins
from
Sten
ella
coer
uleo
alba
.CO
2re
quire
dfo
rgro
wth
Actin
obac
illus
scot
iae
−+
−−w
−−
70−
−+
−−
++
−+
F−
+−
+−
−+
−−
−−
−−
−25
–37
026
5
10%
CO
2re
quire
dfo
rgro
wth
.Nit
=ne
gus
ing
Ros
cota
blet
s
170Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #M.vpTuesday, January 20, 2004 4:12:51 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
155
Achr
omob
acte
rxy
loso
xida
nsde
nitri
fican
s
−+
+v
+−
−−
−+
−+
−−
−−
−O
−−
−−
−−
−−
+−
10–3
70–
429
7
Prev
ious
lyAl
calig
enes
deni
trific
ans
Allo
mon
asen
teric
a−
++
++v
70−
80+
+−
+−
+F
++ g
−−
++
−+
+−
20–3
73–
5
Alte
rom
onas
mac
leod
ii−
++
+−
−−
−+
O+
++
−−
+−
++
2035
–40
2R
R28
6,81
5
Aqua
spiri
llum
spp.
−+
++
−−
−+
−−
+−
−−
I−
−−
+−
−+
−−
−25
0–2
RR
497
Brev
undi
mon
asdi
min
uta
−+
+a
+−
−−
−−
−−
−−
−−
O−
−v−
−−
−−
−−
−−
−−
++1
430
–37
0–5
Brev
undi
mon
as(P
seud
omon
as)
vesi
cula
ris
−+
+a
+−
−−
−−
−−
−+
5852
O+
−46
−−
−−
−−
16−
20−
250–
529
7
Yello
wco
loni
es
Burk
hold
eria
(Pse
udom
onas
)ce
paci
a
−v+
+−
+−
66+
−−
−+
4228
7379
O+v
++
++
−81
+−
+−
25–4
00
RR
297
Fres
hwat
er,y
ello
wpi
gmen
t
Chr
omob
acte
rium
viol
aceu
m−
++
+ v+
−−
−+
+2 0
+−
37−
−86
−F
−+ g
−−
−−
−+
−−
+−
−25
–37
0R
R48
2
Viol
etpi
gmen
t.So
me
non-
pigm
ente
dst
rain
s
Col
wel
liam
aris
−+
++
−+
−−
−+
O−
+−
−−
+0–
223–
4S
S
Hal
omon
asaq
uam
arin
a−
++
−+
−−
−−
−−
+−
−−
−−
−O
−−
−−
−−
−+
−25
–37
0–4
169,
297
Prev
ious
lyAl
calig
enes
faec
alis
Hal
omon
asve
nust
a−
++
−+
−−
−−
+−
++
+−
O−
++
−v
−−
vv
v−
+−
4–37
0–6.
5R
S66
,31
0
Hyd
roge
noph
aga
(Pse
udom
onas
)fla
va
−+
w−
−−
−−
−O
++ g
−+
++
++
+−
−39
Hyd
roge
noph
aga
(Pse
udom
onas
)pa
llero
nii
−+
w+
−−
−−
−+
−−
O−
−−
−−
−−
−−
−−
+30
39,
834
Yello
wpi
gmen
t
Hyd
roge
noph
aga
pseu
dofla
va−
+w
++
+−
−+
−−
−O
++ g
−−
++
+−
++
++
35–4
139
,83
4
Yello
wpi
gmen
t.N
itrat
epo
sw
ithye
aste
xtra
ct
Iodo
bact
erflu
viat
ilis−
++
+ w+
−−
−+
85−
−+
F−
+ g−
−−
+−
+−
−−
+−
−+
4–25
0–1
RR
R50
2
Viol
eton
MC
A.Sp
read
ing
colo
nies
on1/
4N
A
Man
nhei
mia
haem
olyt
ica
−+
++
−−
−−
−+
−v−
−−
−+
F−
++
+−
+−
++w
−20
–37
cont
inue
d
171Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:36:31 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
156
Test
Gm
Ox
cat
bHm
otSW
OD
CLD
CAD
HN
itIn
dC
itur
eam
rvp
aes
Gon
pgO
Far
abgl
uin
osla
cm
alt
man
man
osa
lso
rsu
ctre
Xyl
H2S
MC
ATC
BSD
Nas
ete
mp
NaC
l01
29 1001
2915
0Am
pR
ef
Tab
le4.
11.
Con
tinue
d.
Mar
inom
onas
com
mun
is−
+−
+−
O+
−+
++
−+
−25
+35
–40
286
Mar
inom
onas
vaga
−−
−−
+−
+−
40+
+−
+−
2060
3528
6
Oce
anim
onas
baum
anni
i−
++
−−
+−
+−
−−
−O
−10
–41
1–7
130
Oce
anim
onas
doud
orof
fii−
++
−−
+−
+−
−−
−O
−10
–41
269
,13
0
Nitr
ate
may
bene
gby
tube
test
,pos
byAP
I20N
E
Phoc
oeno
bact
erut
eri
−+
−−
−−
−−
−+
−−
−+
−+
F−
+−
−−
−−
−−
−−
−−
22–3
726
6
Pseu
doal
tero
mon
asan
tarc
tica
−+
++
−−
−−
−−
+−
+−
I−
+−
++
+−
−−
−+
−4–
300.
1–9
115
Pseu
doal
tero
mon
asci
trea
−+
++
+−
−−
−−
−−
−−
−+
−O
+−
−−
+−
−−
+−
+10
–30
1–10
RR
285
Pseu
doal
tero
mon
asde
nitri
fican
s−
+−
+−
+−
+O
+4–
221.
5–5
RR
239
Red
pigm
ent
Pseu
doal
tero
mon
asdi
stin
cta
−+
+
Pseu
doal
tero
mon
asel
yako
vii
−+
++
−−
++
O+
10–3
72
679
Pseu
doal
tero
mon
ases
pejia
na−
++
−−
+35
215
0
Pseu
doal
tero
mon
asfla
vipu
lchr
a−
++
++
−−
−−
−−
−−
−−
+−
O+
−−
+−
−−
+−
+10
–44
0.5–
10R
R28
6,39
8
Pseu
doal
tero
mon
asha
lopl
ankt
isha
lopl
ankt
is
−+
+w+
−−
−+
++
O+
−+
50+
−−
++
−+
4–15
228
5
Pseu
doal
tero
mon
asha
lopl
ankt
iste
traod
onis
−+
++
−−
+O
4–15
228
5
Pseu
doal
tero
mon
aslu
teov
iola
cea
−+
−−
−−
−−
−+
+−
O+ v
−+
−−
−+
−−
+10
–30
2–4
RR
287
Prod
uces
avi
olet
pigm
ent
Pseu
doal
term
onas
mar
ical
oris
−+
++
−−
−+
O10
–37
0.5–
10R
R39
8
172Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:36:31 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
157
Pseu
doal
tero
mon
asni
grifa
cien
s−
++
−−
−+
+O
4–28
239
5
Prod
uces
blac
kpi
gmen
t
Pseu
doal
tero
mon
asru
bra
−+
++
+−
−−
−−
−−
++
O+
−−
−+
−−
−+
+25
–35
2R
R28
3
Red
pigm
ent
Pseu
doal
tero
mon
asul
vae
−+
++
−−
−−
+−
+−
I−
4–25
1–2
SS
S23
1
Pseu
doal
tero
mon
asun
dina
−+
−−
+O
−−
−−
−+
25–3
52
150
Pseu
dom
onas
acid
ovor
ans
−+
+−v
+−
−−
−+
−+
−−
−v−
I−
−−
+w−
−−
v+
−25
–35
029
7
Pseu
dom
onas
aeru
gino
sa−
++
−+
−−
−+
+−
++v
−+
−O
++v
−−
−+
80−
−−
v83
−+
−25
–41
0–5
RR
623
Gre
enpi
gmen
t.M
ayha
vebr
ick-
red
pigm
ent
Pseu
dom
onas
alca
ligen
es−
++
14+
−−
−+
+−
v−
−−
O−
−−
−−
−−
−−
−−
−+
−25
–41
0–5
169
Rod
orfil
amen
t
Pseu
dom
onas
aure
ofac
iens
−+
++
−+
−+
+v
−+
−v
v4–
37
Ora
nge
pigm
ent
Pseu
dom
onas
men
doci
na−
++
++
−−
−+
+−
+50
−−
−O
+−
−−
+−
−−
−+
++
−20
–41
0–6
297
Yello
wpi
gmen
t
Pseu
dom
onas
mes
ophi
lica
−+
+−
+−
v−
−v
−−
O+ w
−−
−−
−−
v25
–35
0
Fatc
ells
,ple
omor
phic
,vac
uola
ted.
Cor
al-p
ink
pigm
ent
Pseu
dom
onas
perfe
ctom
arin
a−
+−
−+
+−
+40
2
Pseu
dom
onas
stan
ieri
−+
+−
−+
−−
402
Ros
eoba
cter
galla
ecie
nsis
−+
++
−−
−−
−−
−−
O+
++
++
15–3
72
662
Ovo
idro
ds,b
row
ndi
ffusi
ble
pigm
ent
Salin
ivib
rioco
stic
ola
ssp.
cost
icol
a
−cv
++
++
−−
−+
12−
20−
53+
++
−F
−+ g
−−
−+
+50
20−
++
−80
+5–
450.
5–20
RS
S27
9
Prev
ious
lyVi
brio
cost
icol
a.Fo
und
inhy
pers
alin
eha
bita
ts37
1
Salin
ivib
rioco
stic
ola
NC
MB
701T
−cv
++
++
−+
−−
−−
−+
+−
−−
F−
+ g−
−−
++
−+
+−
−+
5–45
0.5–
20R
SS
279,
371
Shew
anel
laal
gae
−+
+7 5
+−
+−
−+
−−
−v−
−−
+−
I−
−−
−−
−−
−−
−+
+−
+25
–42
0–10
433,
588,
851
Shew
anel
laam
azon
ensi
s−
++
++
−−
−−
+−
+−
+O
−+
4–30
0–3
Anta
rctic
mar
ine
orga
nism
cont
inue
d
173Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:36:32 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
158
Test
Gm
Ox
cat
bHm
otSW
OD
CLD
CAD
HN
itIn
dC
itur
eam
rvp
aes
Gon
pgO
Far
abgl
uin
osla
cm
alt
man
man
osa
lso
rsu
ctre
Xyl
H2S
MC
ATC
BSD
Nas
ete
mp
NaC
l01
29 1001
2915
0Am
pR
ef
Tab
le4.
11.
Con
tinue
d.
Shew
anel
laba
ltica
−+
+−
+−
+−
−+
−+
−−
−−
−−
O+
++
+4–
3085
1
Shew
anel
labe
nthi
ca−
++
+−
−−
−+
−−
−O
+4–
150–
2
Shew
anel
laco
lwel
liana
−+
+−
+−
−−
−+
+−
−+
−O
−−
−−
−−
−−
−−
−+
8–30
1–5
RR
112,
814
Adhe
res
tosu
rface
s.Su
gars
goal
kalin
e.O
lder
brot
hcu
lture
sha
vere
d-br
own
pigm
ent
815
Shew
anel
lafri
gidi
mar
ina
−+
++
−−v
−−v
+−
+v−
−v
+−v
O−
+−
−+
+−v
−+
+v−v
+v+
0–28
0–8
112
Anta
rctic
mar
ine
orga
nism
Shew
anel
lage
lidim
arin
a−
++
+−
−−
−+
−−
−−
−+
−O
−−
−−
−−
−−
−−
−+
+v4–
151–
611
2
Anta
rctic
mar
ine
orga
nism
Shew
anel
laha
neda
i−
++
+−
−−
−+
−−
−+
O−
−−
−−
−−
−−
−−
+4–
252
409
Shew
anel
laja
poni
ca−
++
++
−+
F+
+10
–37
0–3
397
Deg
rade
sag
ar
Shew
anel
laon
eide
nsis
−+
+−
+−
−−
−+
−−
−+
F+
+15
–40
0–3
782
Shew
anel
lape
alea
na−
++
−+
−+
−+
−−
O−
+−
−−
−−
−−
+4–
300.
1–0.
749
2
Shew
anel
law
oody
i−
++
+−
−−
−+
−−
−+
O−
−−
−−
−+
4–25
211
2
Sphi
ngom
onas
pauc
imob
ilis−
++
−+
−−
−−
−−
−−
+−
+O
−+
+−
++
+−
−+
30–3
729
7
Yello
wpi
gmen
t
Stap
pia
stel
lula
ta-
like
M1
−+
++
−−
++
++
F25
1.5
105
Sten
otro
phom
onas
(Pse
udom
onas
)m
alto
philia
−−
+a
+−
−+
−−
−+
−vv
++
O−
+w−
+v+
−+
+−
+v+
54+
++
350–
562
3
Yello
wco
loni
es
S ee
a ls o
T ab l
e4 .
5fo
rBru
cella
spec
ies.
−c=
nega
tive
inC
hist
ense
n’s
citra
tem
etho
d;O
A=
Oxi
dativ
e;I=
Iner
trea
ctio
n,m
aysh
owan
alka
line
reac
tion;
−a=
nega
tive
oral
pha
haem
olys
is;n
umbe
rsre
fert
ope
rcen
tage
ofpo
sitiv
est
rain
s;nu
mbe
rsin
‘tem
p’an
d‘N
aCl’
colu
mns
refe
rto
rang
e( °
Cor
%N
aCl)
atw
hich
the
orga
nism
will
grow
.
174Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:36:32 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
159
Test
Gm
Ox
cat
mot
Nal
KfG
lyIA
1.5
NaC
l25
°C37
°C42
°Cur
eaN
ithi
pG
GT
APH
2SR
ef
Org
anis
m
H.a
cino
nych
is−
+R
S−
−−
+−
++
327
H.a
cino
nyx
−+
RS
−−
+−
++
540
H.b
ilis−
++
RR
+−
−−
++
++
+54
0
H.c
anis
−−
SI
++
−−
+54
0
H.c
etor
um−
++
+I
S−
−−
++
+−
+−
327,
328,
329
H.c
hole
cyst
us−
++
IR
+−
−−
++
−+
−−
+−
540
H.c
inna
edi
−+
SI
+−
−−
+−
−+
−−
−−
540
H.f
elis
−+
++
RS
−−
−−
++
++
−+
+−
327,
540
H.f
enne
liae
−+
SS
++
−−
−−
+54
0
H.h
epat
icus
−+
+R
R+
++
−+
−+
+−
+54
0
H.m
urid
arum
−+
+R
R−
+−
−+
−+
−−
++
+54
0
H.m
uste
lae
−+
+S
R−
+−
−+
++
+−
++
−32
7,54
0
H.n
emes
trina
e−
+R
S−
−+
+−
+32
7,54
0
H.p
amet
ensi
s−
++
SS
+−
−+
+−
+−
−+
−54
0
H.p
ullo
rum
−+
SR
−+
−+
−54
0
H.p
ylor
i−
++
RS
−−
−−
+−
+−
−+
+−
327,
540
H.t
rogo
ntum
−+
++
RR
++
+−
+−
+54
0
AP=
alka
line
phos
phat
ase
hydr
olys
is;c
at=
cat a
lase
;GG
T=
gam
mag
luta
myl
trans
pept
idas
eac
tivity
;Gly
=gr
owth
in1%
glyc
ine;
Gm
=G
ram
reac
tion;
Hip
=hi
ppur
ate
hydr
olys
is;H
2S=
prod
uctio
nof
hydr
ogen
sulp
hide
;I=
inte
rmed
iat e
susc
eptib
ility;
IA=
indo
xyla
ceta
tehy
drol
ysis
;Kf=
sens
itivi
tyto
ceph
alot
hin
30mg
;Mot
=m
otilit
y;N
al=
susc
eptib
ility
tona
ladi
xic
acid
30mg
;1.5
%N
aCl=
grow
thin
the
pres
ence
of1.
5%N
aCl;
Nit
=ni
trate
redu
ctio
n;O
x=
oxid
ase;
S=
sens
it ive
;R=
resi
stan
t;U
rea
=ur
ease
activ
ity;2
5 °C
,37°
C,4
2°C
=gr
owth
atth
ese
tem
pera
ture
s.Te
sts
fora
lkal
ine
phos
phat
ase
and
hydr
olys
isof
indo
xyl a
ceta
tew
ere
done
usin
gan
ANI-I
dent
disc
from
bioM
erie
ux,G
GT,
AP,h
ip,N
it,H
2Sw
ere
done
usin
gth
eAP
ICam
pyid
entif
icat
ion
syst
em(b
ioM
erie
ux).
Ure
ahy
drol
ysis
was
test
edus
ing
ara
pid
urea
test
from
Rem
el.G
row
thon
1%gl
ycin
eca
nbe
test
edon
agr
owth
med
ium
such
asbl
ood
agar
(BA)
,con
tain
ing
1%gl
ycin
e.Se
nsiti
vity
toN
aan
dKf
wer
edo
neon
BAus
ing
stan
dard
antib
iotic
sens
itivi
tyte
stm
etho
ds.
Tab
le4.
12.
Hel
icob
acte
rsp
p.
175Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #M.vpTuesday, January 20, 2004 4:14:27 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
160
Org
anis
m
Path
ogen
ic
Aero
cocc
usvi
ridan
s+
−a
−−
−−
+75
5
Aero
cocc
usvi
ridan
sva
r.ho
mar
i+
−w
a−
−−
−−
+−
F50
++
+50
+60
50+
+−
10–3
70–
10+
299,
827
Ente
roco
ccus
faec
alis
var.
lique
faci
ens
+c−
−−
+−
+−
−+
−+
Gra
nulic
atel
laba
laen
opte
rae
+c−
−−
−+
−w
+−
+−
−F
−+
−+
−−
−+
−−
179,
478
Lact
ococ
cus
garv
ieae
+−
−a
−−
−−
+−
−−
−+
++
−−
F−
+−
−+
++
+−
V+
−−
v10
–45
0–6.
5−
−46
4,73
1
Gro
wth
in6.
5%N
aCl,
optim
um0%
NaC
l,37
°C.R
esis
tant
tocl
inda
myc
in63
8,78
0
biot
ype
1,2,
12+
−a
−+
++
−+
++
−+
+−
10–4
50–
6.5
780
b io t
y pe
3 ,4 ,
5 ,6 ,
7 ,10
,11,
13+
−a
−+
++
−+
++
−−
+−
10–4
50–
6.5
780
biot
ype
8,9
+−
a−
++
+−
++
−−
−+
−10
–45
0–6.
578
0
Lact
ococ
cus
pisc
ium
+c−
−−
−−
−−
−+
+F
++
−+
++
++
−+
++
−+
5–30
−−
835
Mic
roco
ccus
lute
us+
++
−−
−−
v−
−−
v−
+−
O−
−−
v−
v−
10–4
50–
1043
Plan
ococ
cus
spec
ies
+−
++
−−
−−
+−
−+
−+
O−
+−
−−
−−
372
43
Stap
hylo
cocc
usau
reus
+−
++
−−
++
++
F−
++
++
++
+−
++
+29
6
Stap
hylo
cocc
usde
lphi
ni+
−+
+−
−+
++
−−
F−
++
++s
++
−−
++w
37–4
50–
15−
264,
778
Stap
hylo
cocc
usep
ider
mid
is+
−+
−−
−+
80+
+F
−+
−+
+−
+−
−+
−−
−−w
25–4
50–
729
6
Stap
hylo
cocc
uslu
trae
+c−
++
−−
−+
+−
+F
++
++
v+
++
++
+ w25
–42
0–10
264
Test
Gm
Ox
cat
bHm
otSW
OD
CLD
CAD
HN
itIn
dC
itur
eam
rvp
aes
Gon
pgO
Far
abgl
uin
osla
cm
alt
man
man
osa
lso
rsu
ctre
Xyl H
2SM
CA
Coa
gD
Nas
ete
mp
NaC
lMan An
RAA
Hip
Ref
Tab
le4.
13.
Gra
m-p
ositi
veco
cci.
176Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:36:36 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
161
Stap
hylo
cocc
usw
arne
ri+
−+
−w−
−+
−+
+F
−+
−−
+ s63
−−
−+
+−
−−
25–4
00–
1029
6
Stre
ptoc
occu
sag
alac
tiae
+c−
−−
−−
−−
+−
−−
++
−−
−Fw
−+
−v
+−
+−
−+
+−
−+
25–3
70–
3+
71,
242
Gro
upB,
No
grow
that
10°C
or45
°C.V
Pm
aybe
nega
tive
Stre
ptoc
occu
sag
alac
tiae
(S.d
iffic
ile)
+−
−−
−−
−−
−−
−−
−+
+−
−−
F−
+−
−+
−+
−−
v−
−−
300–
1+
135,
776
Gro
upB,
Type
Ib.H
ip+v
eat
30°C
,−ve
at37
°CSt
rept
ococ
cus
dysg
alac
tiae
ssp.
dysg
alac
tiae
+−
−+
−−
+−
−v
F−
++
−v
−+
0–4
−72
7,79
0
Posi
tive
toLa
ncef
ield
grou
pL
antis
era.
Baci
traci
n=
S
Stre
ptoc
occu
sdy
sgal
actia
ess
p.dy
sgal
actia
e
+−
−v
−−
+−
−F
++
−−
−+
−0–
4−
135,
776
Gro
upC
Stre
ptoc
occu
sin
iae
ATC
C29
177
+−
−+
−−
−−
+−
−−
+−
−−
F−
+−
++
++
−−
++
−−
−w+
10–3
52–
4−
574
No
grow
that
6.5%
NaC
l,or
10°C
o r4 5
°C.I
sola
tes
from
fish
Stre
ptoc
occu
sin
iae
ATC
C29
178
+−
−+
−−
+−
−−
+−
−F
−+
−−
++
++
−+
+−
−w10
–37
2–4
−22
3,62
5
No
grow
that
6.5%
NaC
l,or
10°C
o r4 5
°C.I
sola
tes
from
fish
Stre
ptoc
occu
sin
iae
+−
−+
−−
−−
−+
−−
F−
+−
−+
++
+−
++
−37
2–4
−22
3
Stra
ins
from
hum
ans
Stre
ptoc
occu
sm
illeri
+−
−+
−−
+−
−+
−−
+−
+−
++
−+
−+
+−
++
+−
+−
87
Stre
ptoc
occu
spa
raub
eris
+c−
−a
−−
+−
++
−F
−+
−+
++
++
++
+−
v10
–37
0–4
v17
5,22
4
No
grow
that
6.5%
NaC
l,or
4 °C
o r4 5
°C.N
egfo
rstre
pgr
oup
D
Stre
ptoc
occu
sph
ocae
+−
−+
−−
−−
F−
+−
−+
−+
−−
−−
−25
–37
0−
700
Sens
itive
toba
citra
cin
disc
,som
est
rain
sre
actw
ithst
rep
grou
pC
antis
era
Vago
cocc
usfe
ssus
+cr
−a
+−
−−
−−
F−
+ g−
−−
−−
−−
−−
369
Vago
cocc
uslu
trae
+cr
−a
+−
−−
−−
+−
+F
−+
+−
++
+−
370
−47
7
Vago
cocc
ussa
lmon
inar
um+c
b−
−a
−−
−−
−−
−−
+−
−F
−+ g
−−
−+
−+
+−
++
−w+
v−
5–30
0–6.
5−
−54
2,68
2
May
grow
onTC
BS,w
eak
dela
yed
reac
tion
wit h
stre
pgr
oup
Dan
tiser
a.H
ip&
VPpo
sw
ithAP
I20
STR
EP73
2,80
7
Vago
cocc
usde
scrib
esm
otile
Lanc
efie
ldgr
oup
N.V
.sal
mon
inar
umha
sbe
enre
porte
das
nega
tive
forL
ance
field
grou
pN
cont
inue
d
177Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:36:38 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
162
Envi
ronm
enta
l
Abio
troph
iade
fect
iva
+−
−a
−−
−−
−+
F+
++
20–4
2−
421
Abio
troph
iapa
ra-a
diac
ens
+−
−a
−−
−−
F+
−42
1
Ente
roco
ccus
faec
alis
+−
−a
−−
−−
+−
−−
++
v−
F−
+−
++
++
+v
++
−+
10–4
50–
6.5
+63
8
Ente
roco
ccus
faec
ium
+−
av
−+
−+
+−
+F
++
−+
++
++
−+
+−
10–5
00–
6.5
+
Fack
lam
iam
iroun
gae
+−
a−
−+
−+
−−
−F
−−
−−
−−
+25
–42
0–5
−36
8
Gra
nulic
atel
laad
iace
ns+
−−
a−
−−
−−
−F
−+
−20
–42
−42
1
Gra
nulic
atel
lael
egan
s+
−−
a−
−+
v−
−F
−+
−27
–37
+65
3
Lact
ococ
cus
lact
isSe
rogr
oup
N+
−−
−+
++
F−
++
−−
−+
10–4
50–
2−
731
Phen
otyp
ical
lysi
mila
rto
E.se
riolic
ida.
Sens
itive
tocl
inda
myc
in
Plan
ococ
cus
citre
us+
−+
+−
−−
−−
−v
−−
−−
+−
+−
−−
−−
−−
−−
−−
+5–
300–
15v
326
Plan
ococ
cus
kocu
rii+
−+
+−
−−
−−
−v
−−
−−
v−
v−
−−
−−
−−
−−
−−
+5–
300–
10v
326
Plan
omic
robi
umok
eano
koite
s−v
w+
+−
++
+−
−−
−−
−+
O−
−−
−−
−−
20–3
73–
556
6
Stap
hylo
cocc
usau
reus
anae
robi
us+
−+
−−
++
264
Anae
robi
cgr
owth
only
Stap
hylo
cocc
usca
pitis
++w
−w−
+v
F−
+−
−−
++
−−
+s−
−−
w30
–45
0–10
Stap
hylo
cocc
usca
rnos
us+
−−
−+
+F
+v
−+
+−
v−
−15
–45
0–15
Stap
hylo
cocc
usch
rom
ogen
es+
v−
Fv
+−
−−
Stap
hylo
cocc
usco
hnii
++
−w−
−+w
F−
+−
+80
8080
−−
−+
−−
2015
–45
0–10
Test
Gm
Ox
cat
bHm
otSW
OD
CLD
CAD
HN
itIn
dC
itur
eam
rvp
aes
Gon
pgO
Far
abgl
uin
osla
cm
alt
man
man
osa
lso
rsu
ctre
Xyl H
2SM
CA
Coa
gD
Nas
ete
mp
NaC
lMan An
RAA
Hip
Ref
Tab
le4.
13.
Con
tinue
d.
178Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:36:39 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
163
Stap
hylo
cocc
usco
hnii
urea
lytic
um+
−+
−−
2815
+25
28+
F−
+−
22+
+−
−+
−−
−
Stap
hylo
cocc
usha
emol
ytic
us+
++w
−+
+F
−+
−50
+50
−−
−+
+−
−−
20–4
50–
10
Stap
hylo
cocc
usho
min
us+
+−
−+
wF
−+
−60
+−
−−
−+
+−
−W
20–4
50–
7
Stap
hylo
cocc
ushy
icus
++
−+
−F
−+
+−
v+
−26
4
Stap
hylo
cocc
usin
term
ediu
s+
++
+−
F−w
++
−+
+−
264
Stap
hylo
cocc
ussa
prop
hytic
us+
+−
−−
+F
−+
−+
++
−−
−+
+−
−−
15–4
00–
15
Stap
hylo
cocc
ussc
hlei
feri
coag
ulan
s+
++
F−
−−
++
264
Stap
hylo
cocc
ussc
hlei
feri
schl
eife
ri+
++
F−
v−
−+
264
Stap
hylo
cocc
ussi
mul
ans
++
−w−
+−
F−
+−
+−
+7 7
−−
+77
−−
v15
–45
0–10
+
Stap
hylo
cocc
usw
arne
ri+
+w−
−−
+F
−+
−−
+s+
−−
−+
+−
−−w
25–4
00–
10
Stap
hylo
cocc
usxy
losu
s+
+−
−80
−F
80+
−80
++
+−
−80
80+
−−
15–4
00–
10
Stre
ptoc
occu
sdy
sgal
actia
eeq
uisi
milis
+−
+−
−+
−F
−−
+−
790
Stre
ptoc
occu
spo
rcin
us+
++
++
−F
−+
−+
++
++
++
+−
10–3
70–
6.5
−17
5,62
5
Stre
ptoc
occu
sub
eris
+−
−−
−+
++
−F
−+
−+
++
++
++
+−
10–3
70–
6.5
+17
5,62
5
Vago
cocc
usflu
vial
isAT
CC
4951
5
+cr
−a
+−
−+
−−
+−
vF
−+ g
−−
−+
++
+−
−+
−−
5–40
0–6.
5−
−17
7,73
2
Wea
kde
laye
dre
actio
nw
ithst
rep
grou
pD
ant is
era
629
The
listo
fStre
ptoc
occi
and
Stap
hylo
cocc
iis
not c
ompr
ehen
sive
,and
read
ers
are
urge
dto
use
ate
xtsu
chas
Berg
ey’s
Man
ual,
ifa
Stre
ptoc
occu
ssp
ecie
sdo
esno
tfit
with
the
spec
ies
liste
dhe
re.
Coa
g=
coag
ulas
ere
actio
n;H
ip=
hydr
olys
isof
hipp
urat
e;M
anAn
=fe
rmen
tatio
nof
man
nito
lund
eran
aero
bic
cond
ition
s;R
AA=
grow
thon
Rog
osa
Acet
ate
agar
.
179Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #M.vpTuesday, January 20, 2004 4:16:21 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
164
Test
Gm
Ox
cat
bHm
otSW
OD
CLD
CAD
HN
itIn
dC
itur
eam
rvp
aes
Gon
pgO
Far
abgl
uin
osla
cm
alt
man
man
osa
lso
rsu
ctre
Xyl
H2S
MC
AD
Nas
ete
mp
NaC
lZN
MAF
RAA
Hip
Ref
Org
anis
m
Path
ogen
ic
Arca
noba
cter
ium
phoc
ae+
10+
−−
−−
−−
F−
+ g−
++
+40
60−
−+
+10
−−
636
Baci
llus
cere
us+
++
+−
++
O+
25–4
5−
−74
,30
7
Baci
llus
myc
oide
s+
++
−−
++
O+
25–3
7−
−30
7
Car
noba
cter
ium
pisc
icol
a+s
r−
−−a
−−
−−
+−
−−
−+
++
−+
F−
+ g−
−40
*+
++
+−*
++
−−
−−
0–37
0–6
−−
−−
73,
176
*W
eak
reac
tions
inph
enol
red
med
ium
,neg
ativ
ein
purp
leba
sem
ediu
m75
2
Car
noba
cter
ium
spp.
+r−
−−
−−
−−
+−
−−
−+
++
−v
F+ g
−+v
++
++
v+
+−
−−
10–3
70–
6−
−−
73
Cor
yneb
acte
rium
aqua
ticum
+−
++
+−
−−
−+
−−
−+
+v
+I
−−
−−
−−
−−
4–42
0–5
75
Cor
yneb
acte
rium
test
udin
oris
++
−−
+−
+−
F+
−+
−+
−37
018
0
Der
mat
ophi
lus
chel
onae
++
++
−+
−−
−−
+F
+−
v−
−−
−−
−25
−52
9
Der
mat
ophi
lus
cong
olen
sis
++
++
−−
−+
−−
+F
+−
+v−
−−
−−
−37
−30
8
Erys
ipel
othr
ixrh
usio
path
iae
+r−
−a
−−
−−
−−
−F
++
−+
+−
−w−
−−
−−w
+5–
3729
2
Ren
ibac
teriu
msa
lmon
inar
um+
−+
+−
−−
−−
−−
I−
−−
−−
−−
−15
–18
0−
671
Rho
doco
ccus
spp.
+r−
−−
+F
−+
−−
−−
−+
250–
5−
53
No
grow
th37
°C.G
row
thin
5%
NaC
l
Stre
ptom
yces
salm
onar
is+
+O
−+
+−
w+
−−
+12
–37
41
Bric
k-re
dto
oran
gepi
gmen
ted
myc
elia
Envi
ronm
enta
l
Actin
omyc
esm
arim
amm
aliu
m+
−−
−−
−−
−F
−+
++
−−
−−
−−
−37
0
Arca
noba
cter
ium
bern
ardi
ae+
−v
−−
−−
F+
+−
−−
636
Arca
noba
cter
ium
haem
olyt
icum
+
Tab
le4.
14.
Gra
m-p
ositi
vero
ds.
180Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:36:41 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
165
Arca
noba
cter
ium
plur
anim
aliu
m+
+a
−−
−−
−−
+w+
F−
+−
v−
−−
−−
+
Arca
noba
cter
ium
pyog
enes
+−
+−
−−
−−
−−
−−
++
F−
+ g−
++
+v−
+−
v+v−
++
20–4
0−
−+,
−63
6
Acid
,clo
t,re
duct
ion
inlit
mus
milk
Arth
roba
cter
agilis
+c+
+−
+v−
−−
−−
−−
−−
++
+O
−−
−−
v20
–30
0–1
Arth
roba
cter
nasi
phoc
ae+
+−
−−
−−
+O
−−
−−
−−
25–4
05
+8
Arth
roba
cter
rhom
bi+c
++
−−
−−
+−
+O
−+
−+
++
++
−+
+−
4–30
1–10
600
Atop
obac
terp
hoca
e+
−−
+−
−−
−−
F−
+v
+−
−v
v−
25−
479
Car
noba
cter
ium
alte
rfund
itum
+r−
−−
+−
−nt
−−
F−
+−
−w
−w
nt−
w−
−nt
0–20
1–6
−−
−41
2
Car
noba
cter
ium
dive
rgen
s+r
−−
−−
−−
+−
−−
++
+F
−+
−−
+−
++
−+
+−
−0–
400–
7−
−−
176
Car
noba
cter
ium
dive
rgen
s62
51+r
−−
−−
−−
−−
F−
0–35
0–6
−64
9
Car
noba
cter
ium
fund
itum
+r−
−−
+−
−nt
−−
F−
+−
−+
++
+−
++
−nt
0–20
1–6
−−
−41
2
Car
noba
cter
ium
gallin
arum
+r−
−−
−−
−−
+−
−−
++
+F
−+ g
−−
++
−+
+−
++
+−
0–35
−17
6
Car
noba
cter
ium
inhi
bens
+r−
−+
−−
−−
−−
−+
−−
F+
+ g−
−w
++
++
−+
+−
−0–
301–
6−
−−
+41
2
Car
noba
cter
ium
mob
ile+r
−−
+−
−−
+−
−−
−+
+F
−+g
−−
+−
++
−+
+−
0–35
−17
6
Cor
yneb
acte
rium
phoc
ae+r
+−
−−
−v
−−
F−
+−
+−
+−
−v
−−
613
Cor
yneb
acte
rium
pseu
dodi
phth
eriti
cum
+r−
++
−+
−−
+−
−−
−I
−−
−−
−−
−20
–42
0–5
133
Cor
yneb
acte
rium
pseu
dotu
berc
ulos
is+r
−+
+−
−−
−−
−−
+−
−−
+F
+ g−
−−
−−
−−
20–4
20–
313
3
Cor
yneb
acte
rium
xero
sis
+r−
++
−−
−−
+−
−−
−−
−−
F+ g
−−
−−
−−
−20
–35
0–3
−−
133
Die
tzia
mar
is+
+−
−+
−−
30−
−−
O−
+−
−−
−−
−−
−−
−25
0–7
−−
573
Rho
doco
ccus
equi
++
−+
−−
−−
++
−+
−−
0–7
−−
573
Rho
doco
ccus
fasc
ians
++
−−
15−
++
−−
36O
++
−−
−+
+−
++
+40
250–
5−
−57
3
Wei
ssel
last
rain
DS-
12+c
r
Car
noba
cter
ium
spec
ies
are
very
sim
ilart
oLa
ctob
acillu
ssp
p.Va
goco
ccus
desc
ribes
mot
ileLa
ncef
ield
grou
pN
stra
ins.
+c=
gram
posi
tive
cocc
us;+
cb=
gram
posi
tive
cocc
o-ba
cillu
s;+c
r=gr
ampo
sitiv
eco
ccoi
dro
d;+r
=gr
ampo
sitiv
ero
d;H
ip=
Hip
pura
tehy
drol
ysis
;MAF
=M
odifi
edAc
idFa
stst
ain
reac
tion;
nt=
nott
est e
d;R
AA=
Rog
osa
Acet
ate
Agar
;v=
varia
ble
reac
tions
repo
rted;
w=
wea
kre
actio
n;ZN
=Zi
ehl-N
eils
on.
Also
see
tabl
esfo
r Noc
ardi
aan
dM
ycob
acte
ria.
181Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #M.vpMonday, January 26, 2004 11:50:28 AM
Color profile: DisabledComposite 150 lpi at 45 degrees
166
Test
Gm
Ox
cat
bHm
otO
DC
LDC
ADH
Nit
Ind
Cit
urea
mr
vpae
sG
onpg
arab
glu
inos
lac
mal
tm
anm
ano
sal
sor
suc
treXy
lH
2Ste
mp
NaC
lN
aCl
450
°CH
ipR
ef
Org
anis
m
Myc
obac
teriu
mab
sces
sus
AFB
+−
+6.
5+
736
Myc
obac
teriu
mav
ium
AFB
+−
+−
37–4
55−
737
Myc
obac
teriu
mch
elon
aeAF
B−
+−
−−
+−
+−
−+
−−
−−
15–2
50–
3−
−13
3
Myc
obac
teriu
mfo
rtuitu
mAF
B+
−+
+28
–37
0–5
+56
9
Myc
obac
teriu
mm
arin
umAF
B+
−−
+28
–30
011
1
Rou
gh&
smoo
thco
loni
es.C
ells
may
belo
ngan
dcr
oss-
band
ed
Myc
obac
teriu
mne
oaur
umAF
B25
5−53
Myc
obac
teriu
mpe
regr
inum
AFB
+−
++
+25
–37
+55
1
Myc
obac
teriu
mpo
rifer
aeAF
B−
+25
–37
5+
756
Myc
obac
teriu
msc
rofu
lace
umAF
B−
+56
9
Myc
obac
teriu
msi
mia
eAF
B−
+56
9
Myc
obac
teriu
msp
ecie
sAF
B−
−−
+28
337
Myc
obac
teriu
mtri
plex
-like
AFB
25–3
034
5
Noc
ardi
aas
tero
ides
+−
+−
+−
++ w
−−
+−
+−
+−
−−
−−
−−
−+
−−
20–3
70–
4+
+v
270,
455,
457
Noc
ardi
abr
asilie
nsis
++
+−
++
−−
++
+−
++
−−
+−
−−
−−
30–4
50–
2+
−−
270,
457
Noc
ardi
abr
evic
aten
a+
−−
−+
−−
−−
−+
165
Noc
ardi
aca
rnea
++
−−
+−
+v
++
+16
5
Noc
ardi
aca
viae
+−
+−
+−
v+ w
−−
+−
+−
++
−−
+−
−−
−v
−0–
4+
v45
7
Noc
ardi
acr
asso
stre
ae+
−+
−−
++
−−
+−
−−
−+
+−
22–3
00–
2−
270
Tab
le4.
15.
Myc
obac
teriu
man
dN
ocar
dia
spp.
182Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #M.vpTuesday, January 20, 2004 4:18:45 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
167
Noc
ardi
afa
rcin
ica
+−
++
−+
+−
−+
−−
v−
−−
v−
20–4
50–
4+
455
Noc
ardi
afla
voro
sea
+−
−−
−−
−−
−−
−−
−−
−−
−−
−+
−16
5
Noc
ardi
ano
va+
+−
++
−+
−−
−−
−−
−−
−20
–40
0–4
−+
455,
805
Noc
ardi
aot
itidi
scav
iaru
m+
+−
++
v+
+v
−v
165
Noc
ardi
aps
eudo
bras
iliens
is+
+−
−+
++
−+
+−
−+
++
−20
–37
165,
661
Noc
ardi
asa
lmon
icid
a+
−+
++
++
−−
+−
−−
−−
−−
−10
–30
0–4
−+
455,
391
Noc
ardi
ase
riola
e+
−+
−+
−+
−+
−−
+−
−−
−v
−−
−−
+20
–30
0–2
+−
455
Noc
ardi
atra
nsva
lens
is+
++
−+
vv
−v
165
Noc
ardi
ava
ccin
ii+
−+
++
+v
+−
v16
5
Noc
ardi
asp
p.(A
ust)
+−
+−
++
−−
117
5−=
nogr
owt h
i n5%
NaC
l .
Test
niac
inN
itrat
eTw
een
80ca
tar
ylur
ease
pyr
Feth
io5%
NaC
lM
CA
Ref
Org
anis
m
Myc
obac
teriu
mab
sces
sus
−−
++
++
+−
++
+73
6
Myc
obac
teriu
mav
ium
−−
−v
−−
++
−73
7
Myc
obac
teriu
mch
elon
aev
−−
++
+−
+13
3,73
7
Myc
obac
teriu
mfo
rtuitu
m−
+v
++
++
+11
6,73
7
Myc
obac
teriu
mm
arin
um−
−+
−−
++
+−
−73
7,11
1
Myc
obac
teriu
mne
oaur
um+
+−
53
Myc
obac
teriu
mpe
regr
inum
−+
−+
++
++
551
Myc
obac
teriu
mpo
rifer
ae−
−+
+−
++
++
−75
6
Myc
obac
teriu
msc
rofu
lace
um−
−−
+−
++
+−
737
Myc
obac
teriu
msi
mia
ev
−−
++
++
+−
737
Myc
obac
teriu
msp
p.(n
ew)
−−
−−
++
−33
7
aryl
=ar
ylsu
lfata
se;p
yr=
pyra
zina
mid
ase;
thio
=2-
thio
phen
ecar
boxy
licac
idhy
draz
ide.
Tab
le4.
16.
Myc
obac
teriu
msp
p.–
addi
tiona
ltes
ts.
183Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #M.vpTuesday, January 20, 2004 4:19:55 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
168
Test
Gm
Ox
cat
bHm
otPO
4O
DC
LDC
ADH
Nit
Ind
Cit
urea
mr
vpae
sG
TTC
OF
arab
glu
inos
lac
mal
tm
anm
ano
sal
CS
treXy
lF&
Ste
mp
NaC
lPe
nR
ef
Org
anis
m
Path
ogen
ic
Myc
opla
sma
allig
ator
is+
−−
−+
++
−30
–34
128
Myc
opla
sma
croc
odyl
i+
+−
−−
++
++
++
−25
–42
R44
1
Myc
opla
sma
mob
ile+
++
++v
−−
−+
++
++
++
+4–
30R
440
Cel
lsad
here
stro
ngly
togl
ass
orpl
astic
.Cel
lsgl
ide.
Wea
kgr
owth
onBA
.F&S
with
hors
ese
rum
,not
bovi
nese
rum
Myc
opla
sma
phoc
icer
ebra
le+
++
−−
−+
+−
3729
5
Myc
opla
sma
phoc
idae
a+
−−
660
Myc
opla
sma
phoc
irhin
is+
+−
−−
−+
++
3729
5
Myc
opla
sma
test
udin
is+
−−
−−
−−
+−
−+
++
−−
−F
350
F&S
=fil
man
dsp
ots;
C=
chol
este
rolr
equi
rem
ent f
orgr
owth
;S=
seru
mre
quire
dfo
rgro
wth
;Pen
=Se
nsiti
vity
tope
nici
llin10
Uni
ts.S
eeC
hapt
er7
fors
p eci
ficbi
oche
mic
alte
sts
forM
ycop
lasm
a.
Tab
le4.
17.
Myc
opla
sma
spp.
Test
Gm
Ox
cat
bHm
otSW
OD
CLD
CAD
HN
itIn
dC
itur
eam
rvp
aes
Gon
pgO
Far
abgl
uin
osla
cm
alt
man
man
osa
lso
rsu
ctre
Xyl
H2S
MC
ATC
BSD
Nas
ete
mp
NaC
l01
29 1001
2915
0Am
pR
ef
Org
anis
m
Path
ogen
ic
List
onel
laan
guilla
rum
ATC
C14
181
−+
++
+−
−−
++
−+
−−
+−
++
F−
+ g−
−−
++
+−
++
+−
−−
Y+
10–3
70.
5–7
SS
R50
6
Prev
ious
lyVi
brio
angu
illaru
m
List
onel
laan
guilla
rum
NC
IMB
6
−+
++
+−
−−
++
+−
−+
+−
++
F+
+ g−
−−
++
+−
++
+−
−−
wG
+4–
300.
5–8
SS
81,
563
MR
may
bene
gativ
eor
wea
k.C
itrat
eis
posi
tive
orne
gativ
e
Sero
type
01st
rain
ste
ndto
bepo
sitiv
efo
rara
bino
se
List
onel
lape
lagi
aN
CM
B22
53
−+
+v
+−
−−
−+
++
−+
−−
++
F−
+ g−
−−
++
+−
−+
+−
−v
−10
–37
0.5–
7S
SS
506,
563
Prev
ious
lyVi
brio
pela
gius
II
wG
=w
eak
grow
thof
gree
n-co
lour
edco
loni
es; v
=va
riabl
ere
actio
n.Al
soch
eck
Tabl
e4.
19( M
orite
llasp
p.),
Tabl
e4.
20( P
hoto
bact
eriu
msp
p.),
Tabl
e4.
21( V
ibrio
–pa
thog
enic
spec
ies)
, Tab
le4.
22(V
ibrio
–en
viro
nmen
tals
peci
es).
Tab
le4.
18.
List
onel
lasp
p.
184Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #M.vpTuesday, January 20, 2004 4:20:40 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
169
Test
Gm
Ox
cat
bHm
otSW
OD
CLD
CAD
HN
itIn
dC
itur
eam
rvp
aes
Gon
pgO
Far
abgl
uin
osla
cm
alt
man
man
osa
lso
rsu
ctre
Xyl
H2S
MC
ATC
BSD
Nas
ete
mp
NaC
l01
29 1001
2915
0Am
pR
ef
Org
anis
m
Path
ogen
ic
Mor
itella
mar
ina
ATC
C15
381,
NC
MB
1144
T
−+
++
+−
−−
−+
−−
+−
−+
+F
−+ g
−−
−−
−−
−−
−−
−−
−+
0–20
3–5
RS
S50
6,76
6
Man
o=
pos,
DN
ase
=ne
g(R
ef50
6)
Mor
itella
mar
ina
ATC
C15
381,
NC
MB
1144
T
−+
−+
−+
−−
+−
+F
−+ g
−−
−−
−−
−−
−−
+4–
203
SS
81,
82
Mor
itella
visc
osa
NC
IMB
1358
4−
++
++
−−
+−
+−
−+
5−
−+
−I
−+ g
−−
−−
−−
−−
−−
−−
+4–
211–
4R
SS
506
Isol
ates
from
Scot
land
,Nor
way
.May
need
1%pe
pton
ead
ded
tom
edia
forg
row
th.N
ogr
owth
at25
°CM
orite
llavi
scos
a−
++
−+
−+
I−
+−
−+
−+
−−
−−
−4–
212–
3R
S82
Isol
ates
from
sout
h-w
estI
cela
ndon
ly.
No
grow
that
25°C
Mor
itella
visc
osa
−+
+−
−−
+I
−+
−−
−−
−−
−−
−−
4–21
2–3
RS
82
Isol
ates
from
Nor
thIc
elan
d
Envi
ronm
enta
l
Mor
itella
japo
nica
−+
+R
R58
5
Also
chec
kTa
ble
4.18
( Lis
tone
llasp
p.),
Tabl
e4.
20( P
hoto
bact
eriu
msp
p.),
Tabl
e4.
21( V
ibrio
–pa
thog
enic
spec
ies)
, Tab
le4.
22(V
ibrio
–en
viro
nmen
tals
peci
es).
Tab
le4.
19.
Mor
itella
spp.
Org
anis
m
Path
ogen
ic
P.da
mse
lae
ssp.
dam
sela
eAT
CC
NC
IMB
2184
T
−+
w+
+ w−
−−
++
−−
++
+−
−−
F−
+ g+
−−
+−
+−
−−
−−
−+
G+
10–3
70.
5–5
SS
S26
8,50
6,61
8
80%
ofP.
dam
sela
est
rain
sm
aybe
posi
tive
fort
reha
lose
(ref7
45)
81,
289,
705
P.da
mse
lae
ssp.
dam
sela
ebi
otyp
es
biot
ype
1−
++
++
−−
−+
+−
++
+−
−−
F−
+ g+
−+
−−
−G
0.5–
7S
S61
8
Test
Gm
Ox
cat
bHm
otSW
OD
CLD
CAD
HN
itIn
dC
itur
eam
rvp
aes
Gon
pgO
Far
abgl
uin
osla
cm
alt
man
man
osa
lso
rsu
ctre
Xyl
H2S
MC
ATC
BSD
Nas
ete
mp
NaC
l01
29 1001
2915
0Am
pR
ef
Tab
le4.
20.
Pho
toba
cter
ium
spp.
cont
inue
d
185Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:36:44 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
170
biot
ype
2−
++
++
−−
−+
+−
++
+−
−−
F−
+ g+
−+
−−
−G
0.5–
7S
S61
8
biot
ype
3−
++
++
−−
−+
+−
++
+−
−−
F−
+ g+
−+
−−
−G
0.5–
5S
S61
8
biot
ype
4−
++
++
−−
−+
+−
++
−−
−−
F−
+ g+
−+
−−
−G
0.5–
7S
S61
8
biot
ype
5−
++
++
−−
++
+−
++
+−
−−
F−
+ g+
−+
−−
−G
0.5–
7S
S61
8
biot
ype
6−
++
++
−−
−+
+−
++
+−
−−
F−
+ g+
−+
−−
−G
0.5–
7S
S61
8
biot
ype
7−
++
++
−−
++
+−
++
+−
+−
F−
+ g+
−+
−−
−G
0.5–
5R
R61
8
biot
ype
8−
++
++
−−
−+
+−
++
+−
+−
F−
+ g+
−+
−−
−G
0.5–
7R
R61
8
biot
ype
9−
++
++
−−
−+
+−
++
+−
+−
F−
+ g+
−+
−−
−G
0.5–
7S
S61
8
Hae
mol
ysis
may
bem
ore
appa
rent
onBA
than
MSA
-B
P.da
mse
lae
ssp.
pisc
icid
aAT
CC
1791
1
−cb
++
−−
−−
−+
−−
−−
w+
−−
−F
−+ g
−−
−−
−+
−−
+w−
−−
−−
7315
–35
0.5–
3S
SS
518,
745
Shor
trod
s,bi
pola
rsta
inin
g.VP
resu
ltsva
riabl
eor
neg
byAP
I20E
289,
459
Envi
ronm
enta
l
P.an
gust
umN
CIM
B18
95−
++
−−
−−
−+
+−
−−
++
++
+F
−+ g
−−
v+
−+
−−
+v
+−
−−
+4–
370.
5–6
SS
S74
5
P.ilio
pisc
ariu
mAT
CC
5176
0−
++
−+
−+
++
−−
−+
+−
F−
+ g+
−−
+−
+−
−v
−G
+4–
250.
5–2
SS
81
P.le
iogn
athi
ATC
C25
521
−−
++
−+
−v
−+
++ s
F−
+ g−
−−
−+
−−
−20
–30
0.5–
534
0,74
5
P.le
iogn
athi
LMG
4228
−−
−−
−−
−−
+−
−−
−−
+−
−+
F−
+ g−
−v
−−
+−
−−
−−
−+
−−
20–3
50.
5–6
SS
S74
5
P.ph
osph
oreu
mN
CIM
B84
4−
−+
−v
−−
++
+−
−−
++
v−
+F
−+ g
+−
−+
++
−−
−−
−−
+4–
300.
5–8
RS
81
P.ph
osph
oreu
mN
CIM
B12
82−
−+
−v
−−
−+
+−
−−
++
v−
+F
−+ g
+−
−+
−+
−−
−−
−−
−4–
211–
8S
S81
P.pr
ofun
dum
−−
−+
+−
+−
F+ g
++
−+
−−
−−
5–20
586
Also
chec
kTa
ble
4.18
( Lis
tone
llasp
p.),
Tabl
e4.
19( M
orite
llasp
p.),
Tabl
e4.
21( V
ibrio
–pa
thog
enic
spec
ies)
, Tab
le4.
22(V
ibrio
–en
viro
nmen
tals
peci
es).
Test
Gm
Ox
cat
bHm
otSW
OD
CLD
CAD
HN
itIn
dC
itur
eam
rvp
aes
Gon
pgO
Far
abgl
uin
osla
cm
alt
man
man
osa
lso
rsu
ctre
Xyl
H2S
MC
ATC
BSD
Nas
ete
mp
NaC
l01
29 1001
2915
0Am
pR
ef
Tab
le4.
20.
Con
tinue
d.
186Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #M.vpTuesday, January 20, 2004 4:22:08 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
171
Org
anis
m
V.al
gino
lytic
us−
++
+v+
+53
+−
++
60−
+83
−+
−F
−+ g
−−
−+
++
33−
++
−−
+Y
+15
–42
1–10
RS
R55
2,82
1
V.al
gino
lytic
usN
CIM
B19
03−
+−
+−
++
++
F−
+ g−
−−
++
−−
−+
−+
15–4
21–
10S
S81
V.ch
oler
ae01
−+
++
+−
++
−+
++v
−−
−−a
++
F−
+ g−
−−
++
80+v
−+
+−
−+
Y+
4–42
0–6v
SS
R50
7,82
1
V.ch
oler
aeno
n01
−+
++
+−
++
−+
++
−−
+−a
++
F−
+ g−
−−
++
70−
−+
+−
−+
Y+
10–4
20–
3S
SSv
507
V.ch
oler
ae01
39−
++
+−
++
−+
−75
+F
−+ g
−−
−+
++
−Y
0–3
RR
S9, 39
2
V.m
imic
us−
++
++
−+
+−
++
9 5−
−−
−+
+F
−+ g
−−
−+
++
−−
−+
−−
+G
+4–
420–
6S
SR
210,
230,
507
V.pa
raha
emol
ytic
us−
++
++
++
+−
62+
63−
+−
−+
−F
80+ g
−−
−+
++
5 3−
−+
−−
+G
+20
–40
3–8
RS
V27
2,55
2
V.pa
raha
emol
ytic
usAT
CC
4399
6−
++
++
++
+−
++
−+s
+−
−+
−F
++ g
−−
−+
++
−−
−+
−−
+G
w20
–40
0–3
RPS
135
V.pa
raha
emol
ytic
usAT
CC
2796
9−
++
++
++
−+
−+
−−
+−
F−
+ g−
−+
+−
−G
S49
9
V.(c
arch
aria
e)ha
rvey
iAT
CC
3508
4
−+
+−
++s
++
−+
++ s
++
−−
v−
F−
+ g−
−−
++
+−
+s+
+−
−+
Y/G
+10
–40
0.5–
8R
S11
,13
5,84
7
Col
onie
sof
V.ha
rvey
islo
wly
spre
adac
ross
the
plat
e.C
itm
ayta
ke3–
5da
ys70
,81
V.ha
rvey
iAT
CC
1412
6,AT
CC
1412
9
−+
+w
+−
++
−+
++ w
−−
+−
F−
+ g−
−+
+−
−+
−Y
12–4
03–
6R
S11
Hae
mol
ysis
agai
nsts
heep
RBC
V.vu
lnifi
cus
ATC
C27
562
biot
ype
1,se
rova
rnon
-E
−+
++
+−
++
−+
++
−+
−−
++
F−
+ g−
−+
+−
−−
−−v
+−
−+
G−
20–4
23–
6S
S98
Hum
anst
rain
,USA
–no
n-vi
rule
ntfo
reel
s.C
it=
neg
inAP
I20E
butp
osin
Sim
mon
stu
beci
trate
V.vu
lnifi
cus
ATC
C27
562
biot
ype
1
−+
++
+−
89+
−+
++
−+
−+
+F
−+ g
−−
++
44+
+44
−+
−−
G37
0.5–
7S
S74
6
Asor
igin
ally
desc
ribed
byTi
son
etal
.(19
82)u
sing
conv
entio
nalm
edia
cont
inue
d
Test
Gm
Ox
cat
bHm
otSW
OD
CLD
CAD
HN
itIn
dC
itur
eam
rvp
aes
Gon
pgO
Far
abgl
uin
osla
cm
alt
man
man
osa
lso
rsu
ctre
Xyl
H2S
MC
ATC
BSD
Nas
ete
mp
NaC
l01
29 1001
2915
0Am
pR
ef
Tab
le4.
21.
Vib
rio–
path
ogen
icsp
ecie
s.
187Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #M.vpTuesday, January 20, 2004 4:23:26 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
172
V.vu
lnifi
cus
ATC
C33
184
biot
ype
1,se
rova
rnon
-E
−+
++
+−
++
−+
++
−+
−−
++
F−
+ g−
−+
++
+−
−+
−−
+G
370.
5–6
SS
S98
Hum
anst
r ai n
–U
SA
V.vu
lnifi
cus
biot
ype
1,se
rova
rnon
-E
−+
+v
201
Isol
ated
from
dise
ased
eels
.Bel
gium
(man
=ne
g),S
wed
en(m
an=
pos)
V.vu
lnifi
cus
ATC
C33
187
biot
ype
2,se
rova
rE
−+
++
+−
++
−+
−+
−+
−−
++
F−
+ g−
−+
+−
−−
−−
+−
−+
G+
20–3
53–
6S
S98
Hum
anst
rain
,USA
–vi
rule
ntfo
reel
s
V.vu
lnifi
cus
ATC
C33
149
biot
ype
2,se
rova
rE,0
4
−+
++
+−
−+
−+
−+
−+
−−
++
F−
+ g−
−+
+−
−−
−−
+−
−+
G+
20–3
53–
6S
S98
Viru
lent
fore
els
–Ja
pane
sest
rain
V.vu
lnifi
cus
biot
ype
2se
rova
rE−
++
++
−20
+−
+−
+−
+−
−+
+F
−+ g
−−
++
+−
−−
−+
−−
+G
+20
–35
3–6
SS
26
Taiw
anes
est
rain
s–
aviru
lent
fore
els
V.vu
lnifi
cus
ATC
C33
148
biot
ype
2
−+
++
+−
−+
−+
−+
−+
−+
+F
−+ g
−−
++
−+
+−
−+
−−
G37
0.5–
7S
S74
6
Asor
igin
ally
desc
ribed
byTi
son
etal
.(19
82)u
sing
conv
entio
nalm
edia
V.vu
lnifi
cus
biog
roup
3−
++
++
−+
+−
++
−−
+−
−+
−sF
−+ g
−−
−+
−−
−−
+−
−−
GS
S10
1,56
5
Hu m
a ns t
rain
–Is
rae l
V.tra
chur
i−
++
−+
−+
+−
−−
F−
+−
++
++
−Y
303–
7S
S40
0
V.sh
iloni
i−
++
++
−+
−+
+−
++
F+ g
++
Y16
–37
2–4
SS
59,
458
V.fis
cher
iAT
CC
7744
−+
+−v
+−
−+
−+
−v
++
−−
−20
F−
+ g−
−−
++
−60
−40
−−
−,G
+10
–30
0.5–
6S
SS
Yello
w-o
rang
epi
gmen
t.W
estd
escr
iptio
n=
man
o+v
e
V.fis
cher
iN
CM
B12
81,
ATC
C77
44
−+
++
−+
−+
−+
−+
F−
+ g−
−−
v+v
++v
−−
+v−
v+
0.5–
5S
S81
,34
2
V.fis
cher
iAT
CC
2591
8−
++
−+
−−
+−
+−
−−
−−
−−
−F
−+
−−
+−
−+
−−
−−
−−
−+
10–3
01–
6R
SR
745
VP+v
ein
API2
0E, −
vein
MR
VPm
ediu
m
Test
Gm
Ox
cat
bHm
otSW
OD
CLD
CAD
HN
itIn
dC
itur
eam
rvp
aes
Gon
pgO
Far
abgl
uin
osla
cm
alt
man
man
osa
lso
rsu
ctre
Xyl
H2S
MC
ATC
BSD
Nas
ete
mp
NaC
l01
29 1001
2915
0Am
pR
ef
Tab
le4.
21.
Con
tinue
d.
188Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:36:48 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
173
V.fis
cher
iN
CM
B12
81−
++
++
−−
+−
−−
−+
+−
F−
+ g−
−−
+−
+−
+−
−v
+10
–30
0.5–
7S
SS
506
Gre
yco
loni
es
V.lo
gei
ATC
C29
985T
−+
+−
+−
−+
−−
−+
−+
−F
−+ g
−−
−+
+−
−−
+−
+4–
220.
5–5
SS
S50
6
V.lo
gei
ATC
C15
382
−+
+−
+−
−−
−−
++
−+
−F
−+ g
−−
+−
+−
−+
+−
+4–
250.
5–5
SS
S50
6
V.lo
gei
NC
MB
1143
−+
+−
+−
−+
−+
−+
v−
+−
F−
+ g−
−v
+−
++
−+
+−
+4–
300.
5–5
SS
S81
,74
5
V.lo
gei
NC
IMB
2252
−+
−+
+−
+−
+−
+F
−+ g
−−
+−
+−
−−
+4–
220.
5–5
S81
V.lo
gei
Yello
w-o
rang
epi
gmen
t.St
rain
sar
eci
trate
nega
tive
V.pr
oteo
lytic
us−
++
++
+−
++
++
+33
++
−+
−F
−+ g
−−
−+
+−
−50
76+
−Y/
G+
20–4
01–
10R
SS
67,
788
V.pr
oteo
lytic
usAH
LDA
1735
−+
++
++
−+
++
++
++
+−
+−
F−
+ g−
−−
++
w−
+−
+−
−w
G−
42−
1–10
PSS
135
V.flu
vial
isAT
CC
3380
9−
++
v+
−−
−+
++
+−
+−
72+
+F
++ g
−−
−+
++
−−
++
−−
+Y
+10
–35
1–6
RS
V81
,62
0
V.flu
vial
isN
CTC
1132
7−
++
v+
−−
−+
+−
+−
+−
−+
+sF
++ g
−−
−+
++
−−
++
−−
+Y
+10
–35
1–6
RS
V12
3,48
5
API2
0E
pos
forV
Pan
din
dole
V.fu
rnis
sii
ATC
C35
016T
−+
+v
+−
−−
++
−+
−+
−−
+−
F+
+ g+
−−
++
+−
−+
+−
−+
Y+
20–3
71–
8R
SR
123,
485
API2
0E
pos
forV
Pan
din
dole
V.fu
rnis
sii
ATC
C11
218
−+
++
+−
−−
++
−+
−+
−−
++
F+
+ g+
−−
++
+−
−+
+−
−+
Yw
20–3
71–
8R
S13
5
ADH
=ne
gat
0%N
aCl.
Pos
ADH
at2%
NaC
l
V.sp
lend
idus
IAT
CC
3312
5−
++
v+
−−
−90
++
43−
+−
++
+F
−+ g
−−
−+
++
−−
−v+
−−
−G
−4–
371–
6S
SS
281,
506,
620
Type
stra
inis
citra
tene
gativ
e81
,25
4
V.sp
lend
idus
IIN
CM
B22
51−
++
++
−−
−v
++
+−
+−
−+
+F
−+ g
−−
−+
+−
−−
−+
−G
+4–
300.
5–7
SS
R81
,50
6,81
9
V.tu
bias
hii
ATC
C19
109
−+
+−
+−
−−
83+
+v
30+
−−
++
F−
+ g−
−−
++
−−
++
−−
Y+
10–3
70.
5–6
SS
S10
8,32
1
cont
inue
d
189Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:36:48 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
174
V.tu
bias
hii
NC
MB
1340
T
−+
+w
+−
−−
+s+
+−
−+
−−
++
F−
+ g−
−−
++
+−
−+
+−
−−
Yw+
10–3
00.
5–7
SS
S13
5,50
6
V.ic
hthy
oent
eri
−+
++
−−
−−
+−
−−
−−
F−
+ g−
−−
+−
+−
−+
+−
−Yw
15–3
01–
6S
S38
9
V.ta
petis
−cb
++
+−
−−
−+
+−
−−
−+
+F
−+ g
−−
−+
−+
−−
+−
−G
4–22
1–5
SS
S10
8,14
6
Aesc
ulin
repo
rted
posi
tive
(ref6
10)
587
V.or
dalii
ATC
C33
934
−cv
++
++
−−
−−
−−
−v−
−−
−+
−F
−+ g
−−
−+
+−
−−
+−
−−
−+
4–30
0.5–
7S
SR
v50
6,68
0,81
9
V.or
dalii
DF-
3K−c
v+
++
+−
−−
−−
−−
−−
−−
+−
F−
+ g−
−−
+−
−−
−+
−−
−−
+4–
300.
5–7
SS
Rv
198,
680
V.or
dalii
NC
IMB
2167
−+
−−
−−
−−
−−
−F
−+ g
−−
−+
+−
−−
++
+21
–37
0.5–
5S
S81
V.pe
cten
icid
a−
++
++
−−
−+
−+
−+
−F
−+ g
−−
+−
−−
−−
+4–
301–
6S
S47
0
V.pe
naei
cida
−+
++
−−
−−
+50
67−
+−
++
F−
+ g−
−50
+−
+−
−−
+−
−G
20–3
01–
3S
S18
7,38
8
Type
stra
in=
indo
lene
g
V.sa
lmon
icid
a−
++
−+
−−
−−
−−
−−
−−
−−
−F
−+ g
v−
−−
++
−−
−+
−−
G+
1–22
0.5–
4S
SS
198,
232,
506
V.sa
lmon
icid
aN
CIM
B22
62−
+−
−−
−−
−−
+F
−+ g
−−
−+
−−
−−
−+
1–22
1–4
SS
81
V.co
rallii
lytic
usYB
1T
−+
++
++
++
−+
+F
++
Y25
–30
1–7
SR
83,8
4
4of
6st
rain
sar
eAD
Hne
gativ
e
−aV.
chol
erae
isne
gativ
efo
raes
culin
,but
som
est
rain
sca
use
blac
keni
ngof
the
med
ium
due
topr
oduc
tion
ofm
elan
in. T
ode
term
ine
atru
ene
gativ
eth
elo
ssof
fluor
esce
nce
mus
tbe
mea
sure
dat
354
nm.S
eeun
der‘
aesc
ulin
’ in
inte
rpre
tatio
nof
test
s.In
this
tabl
e,Vi
brio
sar
egr
oupe
dac
cord
ing
toth
eirO
DC
,LD
C,A
DH
reac
tions
.Use
this
asa
star
ting
poin
tin
iden
tific
atio
ns.4
2 −=
nega
tive
grow
that
42°C
.Al
soch
eck
Tabl
e4.
18( L
isto
nella
spp.
),Ta
ble
4.19
( Mor
itella
spp.
),Ta
ble
4.20
( Pho
toba
cter
ium
spp.
),Ta
ble
4.22
( Vib
rio–
envi
ronm
enta
lspe
cies
).
Test
Gm
Ox
cat
bHm
otSW
OD
CLD
CAD
HN
itIn
dC
itur
eam
rvp
aes
Gon
pgO
Far
abgl
uin
osla
cm
alt
man
man
osa
lso
rsu
ctre
Xyl
H2S
MC
ATC
BSD
Nas
ete
mp
NaC
l01
29 1001
2915
0Am
pR
ef
Tab
le4.
21.
Con
tinue
d.
190Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:36:49 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
175
Org
anis
m
Envi
ronm
enta
l
V.di
abol
icus
−+
++
++
+−
++
−−
−−
+−
F−
+−
++
+−
−+
+−
−20
–45
2–5
SS
635
V.ro
tifer
ianu
s−
++
−+
+−
+−
v−
++
F+
+ g−
−+
−+
−Y
28–4
02–
6S
S30
5
V.ci
ncin
natie
nsis
−+
++
−−
+−
+−
+−
+−
+F
++ g
−+
−−
++
−+
+−
Y+
22–3
71–
6R
SS
120
V.m
edite
rrane
i−
++
−+
−−
+−
++
−−
+−
75−
+F
−+ g
−−
−+
++
25+
++
−−
−Y
+20
–30
3–6
SS
S63
1
V.m
etsc
hnik
ovii
−cr
−+
++
−−
++
−20
30−
++
40+
+F
−+ g
−−,
v59
++
+−
30+
+−
−v
Y,−
+4–
400.
5–7
SS
V48
3,81
9
V.br
asilie
nsis
LMG
2054
6T
−cr
++
+−
−−
++
+−
++
++
F−
+−
+−
+−
Y20
–40
2–6
SS
740
V.ne
ptun
ius
LMG
2053
6T
−cr
++
+−
−−
++
++
−+
++
−F
+−
−−
+−
Y20
–35
2–6
SS
740
V.pa
cini
iLM
G19
999T
−+
++
−−
−+
+−
w−
+66
vF
++
−+
−Y
4–35
1.5–
6S
S14
7
V.xu
iiLM
G21
346T
−cr
++
+−
−−
++
+−
++
−−
F+
+−
+−
+−
Y20
–40
2–8
SS
740
V.or
ient
alis
NC
MB
2195
T
−+
+−
+−
−−
++
++
−+
−−
+F
−+ g
−−
−+
+−
−+
+−
Y+
4–35
0.5–
8S
SS
506
Som
eLD
Cpo
sitiv
est
rain
s
V.ae
roge
nes
−−
++
−−
−+
++
+−
+F
−+ g
++
−+
+−
+−
++
20–3
51–
7R
RS
692
V.ae
stua
rianu
s−
++
++
−−
−+
++
+−
−+
++
F−
+ g−
−+
++
−v+
++
−−
+Y
+4–
370.
5–6
SS
S74
7
V.di
azot
roph
icus
−+
+−
+−
−−
++
++
1 6+
−+
−+
F+
+ g−
−+
++
−50
−+
++
−Y
−10
–35
0.5–
6R
SS
319,
819,
820
V.le
ntus
−+
++
−−
−+
++
+−
+F
−+ g
−−
++
+−
−−
+−
G+
4–30
2–6
RR
513
Thor
nley
’sAD
H=
pos,
Møl
lerA
DH
=ne
g.Ty
pest
rain
=m
anne
g
V.m
ytili
−+
++
−−
−+
+−
+v−
−+
−F
++ g
+−
++
−+
−+
++
−−
Y−
10–3
71–
10R
S63
2
V.ne
reis
−+
−+
+−
−−
++
++
−+
−−
+−
F−
+ g−
−−
+−
+−
−+
+−
−Y
+20
–35
3–6
RS
S81
9
Ente
rovi
brio
norv
egic
us−
++
+−
−−
+−
+−
−−
−−
+F
−+
−−
+−
−−
+−
G−
20–2
81.
5–6
RR
741
Test
Gm
Ox
cat
bHm
otSW
OD
CLD
CAD
HN
itIn
dC
itur
eam
rvp
aes
Gon
pgO
Far
abgl
uin
osla
cm
alt
man
man
osa
lso
rsu
ctre
Xyl
H2S
MC
ATC
BSD
Nas
ete
mp
NaC
l01
29 1001
2915
0Am
pR
ef
Tab
le4.
22.
Vib
rio–
envi
ronm
enta
lspe
cies
.
cont
inue
d
191Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:36:49 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
176
V.sc
opht
halm
i−
++
−+
−−
−90
+−
−−
+−
+−
−F
−+ g
−−
−+
−+
−−
+−
−−
−Y
−22
–35
0.5–
3S
S14
9,25
4
V.cy
clitr
ophi
cus
−+
++
−−
+w−
−−
−+
−F
−+ g
−−
+−
−−
++
+−
4–37
2–10
S33
8
V.ag
ariv
oran
s−
++
−+
−−
−−
+−
−−
+−
−+
+F
−+ g
−−
−+
w−
−−
−−
+−
−G
−20
–37
1–6
SS
135,
514
Hyd
roly
ses
agar
.Gel
atin
neg
inAP
I20E
V.ca
mpb
ellii
−+
+−
+−
−−
−+
+58
−50
−50
+−
F−
+ g−
−−
+48
5082
−−
+−
−G
+20
–35
3–6
RS
R82
0
V.ga
zoge
nes
−cr
−+
−−
−−
−−
+−
−+
F+
+ g+
−−
++
++
++
+Y
20–4
23–
6S
S67
Red
toor
ange
colo
nies
V.ha
liotic
oli
−+
+−
−−
−−
−+
+v−
−+
−−
−+v
F−
+ g−
−−
++
+ v−
−−v
−−
−−
G−
15–3
02–
3S
S67
8
V.ho
llisae
−cv
+w
+w−
−−
−+
+−
−−
−−
−−
F+
+ g−
−−
−−
+−
−−
−−
−−
−−
30–3
71–
6R
SS
346,
580
Hae
mol
ysis
wea
kat
24h,
posi
tive
afte
r7da
ys
V.na
trieg
ens
−+
+−
+−
−−
−+
−+
62−
++
67F
++ g
−60
−+
+3 0
+−
++
−−
−Y+
10–4
03–
6R
SS
44,
820
V.na
trieg
ens
NC
MB
1900
−+
+−
+−
−−
−+
−+
−−
−+
−+
F+
+ g−
−−
++
++
−+
+−
−Y
+4–
370.
5–7
SS
S50
6,56
3
V.na
varre
nsis
−+
++
−−
−−
++
+−
−+
+F
−+ g
−−
++
+−
+−
Y10
–40
0.5–
7S
S76
7
V.ni
grip
ulch
ritud
o−
+−
−−
−+
++
−−
50+
+F
−+ g
−+
++
+−
−v+
−+
−G
+20
–30
3–5
RS
S27
7
Col
onie
sbl
ue-b
lack
V.w
odan
isN
CIM
B13
582
−+
++
+−
−−
−89
20+
−−
+F
−+ g
−−
−+
43+
−−
83+
−30
+4–
251–
4S
SS
506
Col
onie
sbl
ue-b
lack
V.ru
moi
ensi
s−
++
−+
−+
+−
−F
++
−+
++
++
−+
2–34
3–6
SS
850
V.ca
lvie
nsis
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++
−+
−+
++
−+
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4–30
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216
V.na
trieg
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stra
inN
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ble
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toba
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ium
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ble
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rio–
path
ogen
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s).
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Gm
Ox
cat
bHm
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OD
CLD
CAD
HN
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dC
itur
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aes
Gon
pgO
Far
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cm
alt
man
man
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lso
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Xyl
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MC
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Con
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192Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:36:50 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
177
Case
Num
ber.
......
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.
Test
Gm
Ox
cat
bHm
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lso
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ate
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idas
e;ca
t=ca
tala
se;b
H=
bha
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ysis
;mot
=m
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=sw
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OD
C=
orni
thin
ede
carb
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H=
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(b-g
alac
tosi
dase
test
);O
F=
Oxi
dativ
efe
rmen
tativ
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ab=
arab
inos
e;gl
u=
gluc
ose;
inos
=in
osito
l;l a
c=
lact
ose;
mal
t=m
alto
se;m
an=
man
nito
l;m
ano
=m
anno
se;s
al=
salic
in;s
or=
sorb
itol;
suc
=su
cros
e;tre
=tre
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se;x
yl=
xylo
se;H
2S=
hydr
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sulp
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;MC
A=
Mac
Con
key
agar
; TC
BS=
thio
sulp
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ate-
bile
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;DN
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;Am
p=
sens
itivi
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cillin
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.
Lab
ora
tory
Wo
rksh
eet
193Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:36:50 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
178
Acin
etob
acte
r/Mor
axel
la−
−−
−−
−−
−−
−−
−−
−−
−−
−−
−+
41
Actin
obac
illus
delp
hini
cola
−+
−−
−−
−−
−+
−+
−−
−−
−−
−−
++
−−
++
3748
0.85
263
Dol
phin
Actin
obac
illus
delp
hini
cola
−21
+70
−−
−−
−+
−+
−−
−−
−−
−−
++
−−
++
3748
0.85
263
Porp
oise
Actin
obac
illus
delp
hini
cola
−+
−−
−−
−−
−+
−+
−−
−−
−−
−−
++
−−
++
3748
0.85
263
Wha
le
Actin
obac
illus
scot
iae
+−
−+
−−
+−
−+
−+
−−
−−
−−
−−
++
−−
++
3748
0.85
265
Aero
mon
asal
losa
ccha
roph
ila+
v+
vv
−−
−+
−+
++
−−
v+
v−
v+
++
++
+41
Aero
mon
asbe
stia
rum
++
+−
−−
−−
++
++
+−
−+
+−
++
++
++
++
2724
0.85
452
Car
p
Aero
mon
asca
viae
++
−−
10−
−−
+8
80+
+−
−−
+−
−+
++
++
++
21
Aero
mon
asca
viae
++
−−
+−
−−
+−
++
+−
−−
+−
−+
++
++
++
2548
0.85
240
ATC
C15
468T
Aero
mon
ascu
licic
ola
++
+−
+−
−+
++
++
−−
−+
+−
−+
++
++
3024
0.85
624
NC
IM51
47T
Aero
mon
asen
chel
eia
++
−−
−−
−−
+−
++
+−
−75
75−
+−
++
++
++
2724
0.85
241,
452
Aero
mon
aseu
cren
ophi
la+
+−
−−
−−
−+
−+
++
−−
−+
++
++
+
Aero
mon
ashy
drop
hila
++
+−
−−
−−
++
++
+−
−−
+−
−+
++
++
++
2548
0.85
240
ATC
C79
66T
Aero
mon
ashy
drop
hila
++
−−
−−
−−
++
−+
+−
−−
+−
−+
++
++
++
674b
ATC
C79
66T
Aero
mon
ashy
drop
hila
++
−−
v−
−−
++
++
+−
−+
+−
++
++
++
++
322
ATC
C14
715
Aero
mon
ashy
drop
hila
++
−−
+−
−−
+−
++
+−
−−
+−
++
++
++
++
2.85
509
WFM
504
Aero
mon
ashy
drop
hila
++
−−
−−
−−
++
++
+−
+−
+−
++
++
++
+2 3
4 82.
8522
7a
Aero
mon
asja
nada
ei+
++
−+
−−
−+
++
++
−−
−−
50−
−+
++
++
+14
3
Aero
mon
asja
nada
ei+
++
−+
−−
−+
++
+w
−−
−−
+−
−+
++
++
+25
480.
8513
5AH
LDA
1718
Aero
mon
aspo
poffi
i+
+−
−+
5 0−
−+
++
++
−−
−+
++
++
Aero
mon
assa
lmon
icid
a−
+−
−−
−−
−−
−+
++
−−
−−
−−
−+
+−
++
+45
0
A.sa
lmon
icid
asa
lmon
icid
a+
++
−−
−−
−−
−+
++
−−
−−
−+
−+
+−
++
+46
8N
CIM
B11
02
A.sa
lmon
icid
aac
hrom
ogen
es−
−−
−−
−−
−−v
−−
++
−−
−+
−−
−+
+−
++
+32
2
A.sa
lmon
icid
aac
hrom
ogen
es−
−−
−−
−−
−+
+−
++
−−
−+
−−
−+
+−
++
+46
8N
CIM
B11
10
A.sa
lmon
icid
am
asou
cida
++
−−
−−
−−
++ v
−+
+−
−−
+−
+−
++
−+
++
322
A.sa
lmon
icid
asa
lmon
icid
a+
+−
−−
−−
−−
−+
++
−−
−−
−+
−+
+−
++
+32
2
A.sa
lmon
icid
asa
lmon
icid
a−
+−
−−
−−
−−
−+
++
−−
−−
−−
−+
+−
++
+67
4
A.sa
lmon
icid
asa
lmon
icid
a−
++
−−
−−
−−
−+
++
−−
−−
−−
−+
+−
++
+67
4
A.sa
lmon
icid
aat
ypic
al+w
−−
−76
1Au
stra
lian
stra
in
A.sa
lmon
icid
aat
ypic
al−
+w−
−−
−−
−+
−−
+−
−−
−w
−−
−+
+−
ww
2572
0.85
135
AHLD
A13
34
Aero
mon
asso
bria
++
+−
−−
−−
++
++
+−
−−
+−
−−
++
++
++
2548
0.85
240
CIP
74.3
3T
Aero
mon
asso
bria
++
+−
50−
−−
++
++
+−
−−
+−
−28
++
++
++
21
Test
son
pgAD
HLD
CO
DC
cit
H2S
ure
TDA
ind
vpG
glu
man
inos
sor
rha
suc
mel
amy
arab
oxN
O2
N2
mot
MC
AO
FTe
mp
Tim
eIn
ocR
efSt
rain
Org
anis
m%
NaC
l
Tab
le4.
23.
AP
I20E
data
base
bioc
hem
ical
resu
lts.
194Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:36:52 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
179
Aero
mon
astro
ta+
++
−+
−−
−+
−+
++
−−
−−
−+
−+
++
++
+37
240.
8514
2AT
CC
4965
7T
Aero
mon
asve
roni
iver
onii
+−
++
+−
−−
++
++
+−
−−
+−
+−
++
++
++
2724
0.85
347,
2
Allo
mon
assp
p.+
+−
−−
−−
−+
−+
++
−−
−+
−+
−2.
8550
9W
FM40
1
Burk
hold
eria
pseu
dom
alle
i−
+−
−63
−−
−−
−+
++
++
−43
−60
++
++
++
−37
480.
8538
Car
noba
cter
ium
dive
rgen
s+
+−
−−
−−
−+
−C
itrob
acte
rfre
undi
i+
v−
−−
+−
−−
−−
−−
−+
++
+−
+−
41
Edw
ards
iella
icta
luri
−−
++
−−
−−
−−
−+
−−
−−
−−
−−
−+
++
+33
4,41
Edw
ards
iella
tard
a−
−+
w+
+−
−+
−−
+−
−−
−−
−−
−−
++
++
2548
0.85
135
AHLD
A13
5
Edw
ards
iella
tard
a−
−+
+−
+−
−+
−−
+−
−−
−−
−−
−−
41
Edw
ards
iella
tard
a–
atyp
i cal
stra
ins
−−
+−
++
−−
+−
−+
+−
−−
−−
−−
−25
480.
8584
5
Ente
rovi
brio
norv
egic
us+
+−
−−
−−
−+
−−
+−
−−
−−
−−
−+
−+
++
2848
2.85
741
LMG
1983
9T
Flav
obac
teriu
mbr
anch
ioph
ilum
−−
−−
−−
−−
−−
++
−−
−−
++
−−
−41
Flav
obac
teriu
mco
lum
nare
−−
−−
−+
−−
−−
+−
−−
−−
−−
−−
+41
Flav
obac
teriu
mco
lum
nare
+−
−−
+−
−−
−+
−−
−−
−−
−−
−+
+−
−−
−25
480.
568
9
Flav
obac
teriu
mgi
llisia
e−
+−
−−
−−
−−
−−
++
−−
−+
−−
−−
2048
1.5
533
Flav
obac
teriu
mhi
bern
um+
−−
−−
−−
−−
−+
+−
−−
++
−−
+−
+25
481.
553
2
Flav
obac
teriu
mhy
datis
−−
−−
−−
−−
−−
++
+−
−−
+−
−+
−41
Flav
obac
teriu
mjo
hnso
niae
+−
−−
+−
−−
−+
+−
−−
−−
−−
−−
++
2572
0.85
135
AHLD
A17
14
Flav
obac
teriu
mps
ychr
ophi
lum
−−
−−
−−
−−
−−
+−
−−
−−
−−
−−
+41
Flav
obac
teriu
mte
getin
cola
−−
−−
−−
−−
−−
−+
+−
−−
−−
−−
−−
2048
1.5
533
Flav
obac
teriu
mxa
ntha
m−
−−
−−
+−
−−
++
+−
−−
+−
−−
++
2048
1.5
533
Haf
nia
alve
i−
−+
++
−−
−−
−−
++
−−
−−
−−
+−
+41
Haf
nia
alve
i+
−+
+−
−−
−−
−−
++
−−
+−
−−
+−
++
++
+65
2
Haf
nia
alve
i+
−+
++
−−
−−
+−
++
−−
+−
−+
+−
++
++
+25
480.
8513
5AH
LDA
1729
Haf
nia
alve
i+
−+
++
−−
−−
−−
++
−−
+−
−−
+−
++
++
+25
480.
8520
3AT
CC
5187
3
Hal
omon
ascu
pida
−+
++
−−
−−
−−
−−
+−
++
+−
−−
−41
Jant
hino
bact
eriu
mliv
idum
−+
−−
+−
−−
−+
++
+−
−−
−−
−−
+41
Kleb
siel
lapn
eum
onia
e+
−v
−+
−v
−−
+−
++
++
++
++
+−
++
++
+41
List
onel
laan
guilla
rum
++
−−
+−
−−
++
++
+−
+−
+−
−−
++
+v
++
2548
0.85
240
NC
MB
6
List
onel
laan
guilla
rum
++ v
−−
+−
−−
++
++
+−
+−
+−
++v
++
++
428,
41
List
onel
laan
guilla
rum
++
−−
−−
−−
++
++
w−
+−
w−
−−
++
−+
+25
482.
85
List
onel
laan
guilla
rum
++
−−
+−
−−
++
++
+−
+−
+−
−+
++
−+
+25
482.
8513
5AH
LDA
1730 co
ntin
ued
195Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #M.vpTuesday, January 20, 2004 4:24:36 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
180
Mor
itella
mar
ina
−−
−−
−−
−−
−−
++
−−
−−
−−
−−
++
+−
++
766
Mor
itella
visc
osa
−−
+−
−−
−−
−−
v+
−−
−−
−−
−−
++
++
+13
2
Pant
oea
aggl
omer
ans
+−
−−
+−
−−
−+
+s+
+−
−+
+−
−+
−+
++
+29
1
Pant
oea
aggl
omer
ans
+−
−−
+−
−−
−+
++
+−
−−
+−
−−
−41
Pant
oea
disp
ersa
+−
−−
+−
−−
−+
+s+
+−
−+
+v
++
−+
++
+29
1
Past
eure
llam
ulto
cida
−−
−−v
−−
−−
+−
−+
+−
+−
+−
−−
++
−+
+42
8
Past
eure
llate
stud
inis
+−
−−
−−
−−
+−
−+
+70
−60
+30
−30
++
−+
++
709
Phoc
oeno
bact
erut
eri
−−
−−
−−
−−
−+
−+w
−−
−−
−−
−−
++
−−
++
266
Phot
obac
teriu
man
gust
um−
+−
−−
−−
−−
++
+−
−−
−+
−−
−+
−−
−+
+26
721.
574
5N
CIM
B18
95
P.da
mse
lae
ssp.
dam
sela
e−
+−
−−
−+
−−
+−
+−
−−
−−
−−
−+
++
++
+26
721.
574
5AT
CC
3353
9T
P.da
mse
lae
ssp.
dam
sela
e−
++
−−
−−
−−
−−
+−
−−
−−
−−
−+
++
++
+25
480.
8524
0AT
CC
3353
9T
P.da
mse
lae
ssp.
dam
sela
e−
+−
−+
−−
−−
−−
+−
−−
−−
−+
−−
++
++
+26
721.
574
5N
CIM
B21
84
P.da
mse
lae
ssp.
dam
sela
e−
+5 5
−15
−85
−−
+20
+−
−−
−−
−15
−75
++
++
+26
721.
574
5
P.da
mse
lae
ssp.
pisc
icid
a−
+−
−20
−−
−−
+−
+−
−−
−−
−−
−+
−−
46+
+26
721.
574
5AT
CC
1791
1
P.da
mse
lae
ssp.
pisc
icid
a−
−−
−−
−+
−−
+−
−−
−−
−−
−−
−+
−−
++
+26
721.
574
5Ab
erra
ntst
rain
P.da
mse
lae
ssp.
pisc
icid
a−
+−
−−
−−
−−
−−
+w−
−−
−−
−−
−+
−−
++
428
ATC
C17
911,
2968
7
P.da
mse
lae
ssp.
pisc
icid
a−
+−
−−
−−
−−
+−
+−
−−
−−
−−
−+
−−
++
2248
1.5
#AT
CC
2969
0,17
911
Phot
obac
teriu
milio
pisc
ariu
m−
++
−−
−−
−−
−−
+−
−−
−−
−−
−+
++
++
599
Phot
obac
teriu
mle
iogn
athi
−+
−−
−−
−−
−+
−+
−−
−−
−−
−−
−+
−+
++
2672
1.5
745
LMG
4228
Phot
obac
teriu
mph
osph
oreu
m−
+w
−−
−−
−+
−+
+−
−−
−+
−+
−2.
8550
9IB
39
Ples
iom
onas
shig
ello
ides
++
++
−−
−−
+−
−+
−+
−−
−−
−−
+41
Prov
iden
cia
(Pro
teus
)ret
tger
i−
−−
−−
−+
++
−−
++
+−
+−
−−
−−
41
Prov
iden
cia
rust
igia
nii
−−
−−
+w−
−+
+−
−+
−−
−−
+w−
−−
++
++
+55
9
Pseu
doal
tero
mon
asul
vae
−−
−−
+−
−+
+−
++
−−
2348
1.5
231
Pseu
dom
onas
angu
illise
ptic
a−
−−
−−
−−
−−
−+
−−
−−
−−
−−
−+
41
Pseu
dom
onas
fluor
esce
ns−
+−
−+
−−
−−
−+
++
++
−+
−−
++
4 1
Pseu
dom
onas
fluor
esce
ns/p
utid
a−
75−
−75
−−
−−
1027
25−
−−
−−
251
20+
26−
++
+−
*
Pseu
dom
onas
plec
oglo
ssic
ida
−−
−−
−−
+−
+−
−+
−42
8
Pseu
dom
onas
putid
a−
−vR
ahne
llaaq
uatil
is+
−−
−+
−−
−−
+−
+−
−+
++
++
−+
++
++
151
Test
son
pgAD
HLD
CO
DC
cit
H2S
ure
TDA
ind
vpG
glu
man
inos
sor
rha
suc
mel
amy
arab
oxN
O2
N2
mot
MC
AO
FTe
mp
Tim
eIn
ocR
efSt
rain
%N
aCl
Tab
le4.
23.
Con
tinue
d.
196Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:36:56 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
181
Sale
gent
ibac
ters
aleg
ens
−20
481.
553
3
Salm
onel
lach
oler
asui
sar
izon
ae+
−+
−+
+−
−−
−+
+−
+−
+−
−−
−−
41
Serra
tiafo
ntic
ola
+−
++
+−
−−
−+
−+
+−
++
−+
+−
++
++
+55
8
Serra
tiafo
ntic
ola
+−
++
+−
−−
−−
−+
++
++
20+
+−
++
++
+29
0
Serra
tialiq
uefa
cien
s+
−+
++
−v
−−
v+
++
v+
−+
v+
−−
+41
Serra
tiapl
ymut
hica
+−
−−
+−
−−
−+
++
++
−−
+−
−+
−+
41
Shew
anel
laal
gae
−−
−+
−+
−−
−−
+−
−−
−−
−−
−−
++
++
+−
433
Shew
anel
lafri
gidi
mar
ina
−−v
−−
+v+
−−
−−
++
+−
−−
+−
−−
++
++
+11
2
Shew
anel
lage
lidim
arin
a−
−−
−−
+−
−−
−+
−−
−−
−−
−−
−+
++
112
Shew
anel
lapu
trefa
cien
s−
−−
++
+−
−−
−+
−−
−−
−35
−−
49+
++
++
−43
3
Shew
anel
law
oody
i−
−−
−−
−−
−−
−+
−−
−−
−−
−−
−+
++
+−
112
Tena
ciba
culu
mm
ariti
mum
−−
−−
−−
−−
−−
+−
−−
−−
−−
−−
+41
Vibr
ioae
stua
rianu
s+
+v
−+
++
++
++
Vibr
ioag
ariv
oran
s+
−−
−−
−−
−−
−−
++
−−
−−
−+
−+
+−
++
2548
2.85
135
AHLD
A17
32
Vibr
ioal
gino
lytic
us−
−+
5 3v
−−
−+
8 3v
++
−−
−+
−67
−+
++
++
552
Vibr
ioal
gino
lytic
us−
−+
−−
−−
−+
w+
++
−−
−+
−−
−+
++
−+
+13
5
Vibr
ioal
gino
lytic
us−
−+
+−
−−
−+
++
++
−−
−+
vv
−+
++
++
650
Vibr
iobr
asilie
nsis
++
−−
+−
−−
++
++
+−
−−
+−
+−
++
+n t
++
2 54 8
2.85
740
LMG
2054
6T
Vibr
ioch
oler
aeno
n-01
+−
++
v−
−−
++
++
+−
−−
+−
−−
++
++
+
Vibr
ioch
oler
ae01
+−
++
v−
−−
+−
++
+−
−−
+−
−−
++
++
+
Vibr
ioch
oler
ae+
−+
+w
−−
−+
+w
++
−−
−+
−+
−+
++
++
+2.
8550
9,55
2W
F110
r
Vibr
iodi
abol
icus
−−
++
−−
−+
+−
++
+−
−−
+−
+−
++
++
+63
5
Vibr
iofis
cher
i−
−+
−−
−−
−−
+−
+−
−−
−−
−+
−+
++
−+
+26
721.
574
5AT
CC
2591
8
Vibr
ioflu
vial
is+
+−
−+
−−
−+
−+
++
−−
−+
−−
++
++
++
+25
480.
8524
0M
EJ31
1
Vibr
ioflu
vial
is+
+−
−81
−−
−+
5 6+
++
−−
−+
−−
++
++
++
+37
480.
8548
5,68
7
Vibr
iofu
rnis
sii
++
−−
63−
−−
8875
++
+−
−−
+−
−+
++
++
++
3 74 8
0 .8 5
6 87
Vibr
iofu
rnis
sii
++
−−
w−
−−
+w
−+
+−
−−
+−
−+
++
++
++
2548
0.85
135
ATC
C11
218
Vibr
iofu
rnis
sii
++
−−
+−
−−
+−
++
+−
−−
+−
−+
++
++
++
2548
0.85
240
ATC
C35
016T
Vibr
ioha
liotic
oli
−−
−−
−−
−−
−−
−w
+−
−−
−−
−−
++
−w
w25
482.
8513
5AH
LDA
1734
Vibr
ioha
rvey
i−
−+
++ w
−−
−+
−+
++
−−
−+
−+
−+
++
++
2 54 8
2.85
581
ATC
C14
129
Vibr
io(c
arch
aria
e)ha
rvey
i−
−+
++
−+
−+
−+
++
−−
−+
−+
−+
++
++
+25
482.
8513
5,58
1,84
7AT
CC
3508
4
Vibr
ioic
hthy
oent
eri
−−
−−
−−
−−
−−
−+
−−
−−
+w−
−−
++
+38
9
cont
inue
d
197Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:50:45 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
182
Vibr
iom
edite
rrane
i+
++
−−
−−
−+
−+
++
++
−+
−+
−+
++
−+
+25
482.
8513
5AH
LDA
1733
Vibr
iom
imic
us+
−+
++
−−
−+
−+
++
−−
−−
−−
−+
++
++
+16
1,21
0
Vibr
iom
imic
us+
−+
+−
−−
−+
−+
++
−−
−−
−−
−+
++
++
+16
1
Vibr
iom
ytili
+−
−−
−−
−+
+−
−+
+−
−−
+−
+−
++
+63
5
Vibr
iona
varre
nsis
−−
−−
−−
−+
−+
++
−−
−+
−−
++
++
+76
8
Vibr
ione
ptun
ius
−+
−−
+−
−+
++
++
−−
−−
+−
−−
++
+nt
++
2548
2.85
740
LMG
2053
6T
Vibr
ione
resi
s−
−−
−−
−−
++
−−
+−
−−
−+
−−
−+
++
635
Vibr
ioor
dalii
−−
−−
−−
−−
−−
++
+−
−−
+−
−−
+−
428
DF
3K
Vibr
iopa
cini
iv
+−
−w
−−
+−
66v
+−
−−
−+
6 6+
−+
++
++
2848
1.5
306
LMG
1999
T
Vibr
iopa
raha
emol
ytic
us21
−+
+−
−−
−−
++
+−
−−
−+
7 7+
++
+*
,552
Vibr
iopa
raha
emol
ytic
us−
−+
+−
−−
−+
−+
++
−−
−−
−−
w+
++
+w+
+48
1.5
135
ATC
C43
996
Vibr
iopa
raha
emol
ytic
us−
−+
++
−−
−+
−+
++
−−
−−
−−
++
++w
++
428
Vibr
iopa
raha
emol
ytic
us−
−+
+−
−+
−+
−+
++
−−
−−
−−
++
++
++
+10
ATC
C17
802
Vibr
iope
cten
icid
a−
−−
−+
−−
−−
−+
+−
−−
−−
−−
−+
++
n t+
+2.
8547
0
Vibr
iope
naei
cida
+−
−−
+−
nt−
50−
++
−−
−−
−+
+−
++
+nt
++
2538
8
Vibr
iopr
oteo
lytic
us−
++
−+
−−
−+
++
++
−+
−−
−−
−+
+w
++
2 54 8
2.85
135
AHLD
A17
35
Vibr
ioro
tifer
ianu
s+
−+
+−
−83
++
−+
++
−−
−+
++
++
nr+
++
2548
2.85
305
LMG
2146
0T
Vibr
iosa
lmon
icid
a−
−−
−−
−−
−−
−−
+−
−−
−−
−−
−+
41
Vibr
iosc
opht
halm
i−
90−
−−
−−
−−
−−
+−
−−
−+
−−
−+
++
nt+
+25
481.
514
9,25
4C
ECT
4638
Vibr
iosp
lend
idus
biov
arI
++
−−
−v−
−−
+−
++
+−
−−
+−
−−
++
++
+22
480.
8525
4
Vibr
iosp
lend
idus
++
−−
−−
−−
+−
++
+−
−−
v+
+−
++
++
2 52.
8528
1
Vibr
iosp
lend
idus
−−
−−
−−
−−
+−
−+
+−
−−
++
+−
++
−−
++
2548
2.85
466
Vibr
iota
petis
+−
−−
−−
−−
+−
++
−−
−−
−−
+−
++
++
+2 5
2.85
108,
146
Vibr
iotu
bias
hii
+−
−−
−−
−+
+−
++
+−
−−
+−
+−
++
++
+32
1,63
5
Vibr
iotu
bias
hii
++ s
−−
−−
−−
+−
−+
+−
−−
+−
+−
++
++
+2 5
4 82.
8513
5
Vibr
iovu
lnifi
cus
biot
ype
1+
−+
+−
−−
−+
−+
++
−−
−−
−+
−+
+2 6
f
V.vu
lnifi
cus
biot
ype
2+
−+
−+
−−
−−
−+
+−
−−
−−
−+
−+
26
V.vu
lnifi
cus
biot
ype
2+
−+
++
−−
−−
−+
+−
−−
−−
−+
−+
26
Test
son
pgAD
HLD
CO
DC
cit
H2S
ure
TDA
ind
vpG
glu
man
inos
sor
rha
suc
mel
amy
arab
oxN
O2
N2
mot
MC
AO
FTe
mp
Tim
eIn
ocR
efSt
rain
%N
aCl
Tab
le4.
23.
Con
tinue
d.
198Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #M.vpTuesday, January 20, 2004 4:26:24 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
183
V.vu
lnifi
cus
biot
ype
2se
roE
+−
++
+−
−−
−−
++
+−
−−
−−
+−
++
2548
26g
V.vu
lnifi
cus
biot
ype
2se
roE
+−
++
+−
−−
−−
++
−−
−−
−−
+−
++
98AT
CC
3318
7a
V.vu
lnifi
cus
biot
ype
2se
roE
+−
+−
−−
−−
−−
++
−−
−−
−−
+−
++
98AT
CC
3314
9b
V.vu
lnifi
cus
biot
ype
2se
roE
+−
+−
+−
−−
−−
++
−−
−−
−−
+−
++
98c
V.vu
lnifi
cus
biot
ype
2se
roE
+−
+−
−−
−−
−−
++
−−
−−
−−
−−
++
98d
V.vu
lnifi
cus
biot
ype
2se
roE
−−
+−
−−
−−
−−
++
−−
−−
−−
+−
++
98e
V.vu
lnifi
cus
+−
++
+−
−−
+−
++
+−
−−
−−
w+
4850
9W
F8A1
110
V.xu
ii−
+−
−−
−−
++
+−
++
−−
−+
−+
++
++
n t+
+2 5
4 82.
8574
0LM
G21
346T
Yers
inia
inte
rmed
ia+
−−
+−
−+
−+
−−
++
v+
++
++
v−
41
Yers
inia
ruck
eri
+−
++
+−
−−
−−
++
+−
−−
−−
−−
−41
Yers
inia
ruck
eri
+−
−+
−−
−−
−−
−+
+−
−−
−−
−−
−67
4,71
7
Yers
inia
ruck
eri
+−
−+
−−
−−
−−
−+
+−
+−
−−
−−
−71
7
Yers
inia
ruck
eri
+−
−+
−−
−−
−+
−+
+−
+−
−−
−−
−71
7
Yers
inia
ruck
eri
+−
++
−−
−−
−+
−+
+−
−−
−−
−−
−+
++
++
2548
0.85
135,
203
AHLD
A13
13
Yers
inia
ruck
eri
+−
++
−−
−−
−−
−+
+−
−−
−−
−−
−+
++
++
674
Yers
inia
ruck
eri
+−
++
−−
−−
−+
−+
+−
+−
−−
−−
−+
++
++
2548
0.85
184
Yers
inia
ruck
eri
+−
++
−−
−−
−+
++
+−
+−
−−
−−
−+
++
++
2548
0.85
184
Yers
inia
ruck
eri
+−
++
−−
−−
−−
−+
+−
−−
−−
−−
−+
++
++
2548
0.85
184
Zobe
lliaga
lact
aniv
oran
s+
−−
−−
−−
−−
−+
++
−−
++
−−
++
+−
+−
307d
1.5
61
Zobe
lliaul
igin
osa
+−
−−
−−
−−
−+
+−
−−
++
+−
+−
307d
1.5
61
#=
Ref
eren
ces:
149,
289,
518,
674,
751,
855.
onpg
=b-
gala
ctos
idas
e;AD
H=
argi
nine
dihy
rola
se;L
DC
=ly
sine
deca
rbox
ylas
e;O
DC
=or
nith
ine
deca
rbox
ylas
e;ci
t=ci
trate
;H2S
=pr
oduc
tion
ofhy
drog
ensu
lphi
de;u
re=
urea
se; T
DA
=try
ptop
hane
deam
inas
e;in
d=
indo
le;v
p=
Voge
s-Pr
oska
uer;
G=
gela
tin;g
lu=
gluc
ose;
man
=m
anni
tol;
inos
=in
osito
l;so
r=so
rbito
l;rh
a=
rham
nose
;suc
=su
cros
e;m
el=
mel
ibio
se;a
my
=am
ygda
lin;a
rab
=ar
abin
ose;
ox=
oxid
ase;
NO
2=
nitra
tere
duct
ion;
N2
=re
duct
ion
toni
trite
gas;
mot
=m
otilit
y;M
CA
=gr
owth
onM
acC
onke
y;O
=ox
idat
ive
ferm
enta
tion;
F=
ferm
enta
tion;
Tem
p=
tem
pera
ture
ofin
cuba
tion;
Tim
e=
time
ofin
cuba
tion;
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199Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:36:58 PM
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184
Acinetobacter/Moraxella 0000004 1 41
Actinobacillus delphinicola 200500416 2 Dolphin, whale 263
Actinobacillus scotiae NCTC 12922 111500416 1 265
Aeromonas allosaccharophila 724613657 1 41
Aeromonas allosaccharophila 724616657 1 41
Aeromonas allosaccharophila 724617657 1 41
Aeromonas bestiarum 704713757 5 Carp 452
Aeromonas caviae ATCC 15468T 324612657 21,240
Aeromonas encheleia 304613457 241
Aeromonas hydrophila ATCC 7966T 704712657 1 240
Aeromonas hydrophila ATCC 7966T 304512657 1 Important to incubate for 48 h forcorrect LDC result
674
Aeromonas hydrophila ATCC 14715 324713757 1 322
Aeromonas janadaei 724714457 2 135,143
Aeromonas salmonicida 200610417 450
Aeromonas salmonicida AHLDA 1334 204402417 1 ‘Atypical’ strain, imported goldfish(Western Australia)
135
Aeromonas salmonicida achromogenes 004412417 322
Aeromonas salmonicida achromogenes NCIMB 1110 004512417 468
Aeromonas salmonicida masoucida 304512517 322
Aeromonas salmonicida salmonicida 200610417 674
Aeromonas salmonicida salmonicida 300610517 322
Aeromonas salmonicida salmonicida 600610417 674
Aeromonas salmonicida salmonicida NCIMB 1102 700610517 468
Aeromonas sobria CIP 74.33T 704712457 240
Aeromonas sobria 724712457 21
Aeromonas trota ATCC 49657T 724610557 142,560
Aeromonas veronii veronii ATCC 35623 114612557 1 347
Aeromonas veronii veronii ATCC 35604 114712457 1 Human, USA 347
Aeromonas veronii veronii ATCC 35606 134612557 1 347
Aeromonas veronii veronii ATCC 35605 134712557 1 Wound isolate, human, USA 347
Aeromonas veronii veronii ATCC 35622 514712557 1 347
Aeromonas veronii veronii 534712557 12 Various strains 2,347,452
Burkholderia cepacia 0004004 3/10 38
Burkholderia cepacia 0206006 1/10 38
Burkholderia cepacia 4304004 4/10 38
Burkholderia cepacia 5304004 1/10 38
Burkholderia pseudomallei 0006727 1/91 38
Burkholderia pseudomallei 0206706 1/91 38
Burkholderia pseudomallei 2006704 1/91 38
Burkholderia pseudomallei 2006706 4/91 38
Burkholderia pseudomallei NCTC8016 2206707 1/91 38
Burkholderia pseudomallei 2006707 5/91 38
Burkholderia pseudomallei 2006726 1/91 38
Burkholderia pseudomallei 2006727 22/91 38
Burkholderia pseudomallei 2202704 1/91 38
Burkholderia pseudomallei 2202706 3/91 38
Burkholderia pseudomallei 2206704 4/91 38
Burkholderia pseudomallei 2206706 21/91 38
Burkholderia pseudomallei 2206707 12/91 38
Burkholderia pseudomallei 2206727 15/91 38
Edwardsiella hoshinae 454412057 *
Edwardsiella hoshinae 474412057 *
Edwardsiella ictaluri 410400057 41,334
Edwardsiella tarda 454400057 41
Edwardsiella tarda AHLDA 135 474400057 135
Organism Strain API 20 E number No of strains Notes Ref
Table 4.24. API 20E database numbers (organisms listed alphabetically).
200Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:36:58 PM
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185
Edwardsiella tarda – atypical 4644100.. 4 Atypical strains from red sea bream,Japan
845
Enterobacter agglomerans 120712057 41
Enterovibrio norvegicus 30440044. 741
Flavobacterium branchiophilum 0006060.. 41
Flavobacterium columnare 0402004.. 41
Flavobacterium columnare 140200410 689
Flavobacterium gillisiae 200412000 533
Flavobacterium hibernum 100603210 532
Flavobacterium hydatis 000612210 41
Flavobacterium psychrophilum 0002004.. 41
Flavobacterium tegetincola 00041000. 533
Flavobacterium xantham 04061241. 533
Hafnia alvei 430410257 41
Hafnia alvei 510411257 652
Hafnia alvei 530511357 135
Hafnia alvei ATCC 51873 5304112.. 1 203
Halomonas cupida 6100530.. 41
Janthinobacterium lividum 2207104.. 41
Klebsiella pneumoniae 521577357 41
Listonella anguillarum V10 304452456 1 Japan 49
Listonella anguillarum V239 304572557 1 Japan 49
Listonella anguillarum PT-87050 304652456 2 Japan 49
Listonella anguillarum 304752456 1
Listonella anguillarum V244 to V246 304752476 5 Japan 49
Listonella anguillarum HT-77003 304752657 1 Japan 49
Listonella anguillarum V240 324472757 1 Japan 49
Listonella anguillarum V241 324562757 1 Japan 49
Listonella anguillarum ET-78063 324632657 1 Japan 49
Listonella anguillarum UB 4346, 434 324712677 2 Spain 49
Listonella anguillarum NCMB 6 3247524.. 1 240
Listonella anguillarum UB (ET-1) 324752557 1 Japan 49
Listonella anguillarum V318, AHLDA 1730 324752656 2 Japan, Australia 49,135
Listonella anguillarum RH-8101, AVL 324752657 6 France, Japan 49
Listonella anguillarum 3247527.. 41
Listonella anguillarum 324752756 428
Listonella anguillarum 324752756 49
Listonella anguillarum LMG 3347 324752757 4 Japan, Norway, Spain 49
Listonella anguillarum UB A078 324752777 1 Spain 49
Listonella anguillarum V320 324772656 1 Spain 49
Listonella anguillarum UB A054–56 324772657 4 Japan, Spain 49
Listonella pelagia 100412456
Listonella pelagia 104612456
Listonella pelagia 124612456
Moritella marina 000600456 766
Moritella viscosa 400400456 132
Moritella viscosa 400600456 132
Pantoea agglomerans CDC 1429–71 100577357 291
Pantoea agglomerans ATCC 14589 120516357 291
Pantoea agglomerans NCPPB 2285 120517257 291
Pantoea agglomerans DNA HG 14589 120517357 291
Pantoea agglomerans 120713257 291
Pantoea dispersa 120713357 291
Pantoea dispersa 120717357 291
Organism Strain API 20 E number No of strains Notes Ref
Table 4.24. Continued.
continued
201Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:36:59 PM
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186
Pasteurella multocida 014452456 428
Phocoenobacter uteri 000100416 266
Phocoenobacter uteri 000500416 266
Photobacterium angustum NCIMB 1895 200702406 745
Photobacterium damselae damselae LMG 7892 200500457 49
Photobacterium damselae damselae 201500457 41
Photobacterium damselae damselae ATCC 33539T 201500457 745
Photobacterium damselae damselae NCIMB 2184 220400157 745
Photobacterium damselae damselae ATCC 33539T 600400457 240
Photobacterium damselae damselae LMG 13639 600500457 6 Various sources 49
Photobacterium damselae piscicida P90029 001100407 1 Atypical strain 745
Photobacterium damselae piscicida ATCC 17911 200400406 ATCC 29687 428
Photobacterium damselae piscicida ATCC 17911 200500406 ATCC 29690, and other strains.Refs: 149, 518, 855, 751
289,674,745
Photobacterium damselae piscicida ATCC 17911 200500407 Majority NCIMB 2058 745
Photobacterium damselae piscicida 220500407 745
Photobacterium iliopiscarium 600400457 1 599
Photobacterium leiognathi LMG 4228 200500017 745
Photobacterium phosporeum IB39 6046021.. 509
Plesiomonas shigelloides 714420457 41
Providencia rettgeri 007431057 41
Providencia rustigianii 026402057 559
Pseudomonas aeruginosa 0206006 1/18 38
Pseudomonas aeruginosa 2002004 1/18 38
Pseudomonas aeruginosa 2006004 5/18 38
Pseudomonas aeruginosa 2206004 9/18 38
Pseudomonas aeruginosa 2206006 1/18 38
Pseudomonas anguilliseptica 000200440 41
Pseudomonas anguilliseptica 020000440 96,541,828
Pseudomonas anguilliseptica 020200440 96,541,828
Pseudomonas fluorescens 0204004.. 2/8 38
Pseudomonas fluorescens 2000004.. 5/8 38
Pseudomonas fluorescens 220000443 1/8 38
Pseudomonas fluorescens 220000453
Pseudomonas stutzeri 0000004.. 1/9 38
Pseudomonas stutzeri 0004004.. 5/9 38
Pseudomonas stutzeri 0004104.. 3/9 38
Rahnella aquatilis 100557317 *
Rahnella aquatilis 120547257 151
Rahnella aquatilis 120557317 *
Salmonella cholerasuis arizonae 560621057 41
Serratia fonticola 530475257 290
Serratia fonticola 530477257 290
Serratia fonticola 530555257 558
Serratia plymuthica 120732257 41
Shewanella algae 050200453 433
Shewanella frigidimarina 06061245. 112
Shewanella frigidimarina 26061245. 112
Shewanella gelidimarina 04020045. 112
Shewanella putrefaciens 070200453 433
Shewanella putrefaciens 070200653 433
Shewanella putrefaciens 070202653 433
Shewanella woodyi 000200452 112
Tenacibaculum maritimum 000200410 41
Vibrio agarivorans AHLDA 1732 100410556 2 From abalone. Pathogenicityunknown
135
Organism Strain API 20 E number No of strains Notes Ref
Table 4.24. Continued.
202Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:36:59 PM
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187
Vibrio alginolyticus 404712456 135
Vibrio alginolyticus 404712457 135
Vibrio alginolyticus 414712457 650
Vibrio alginolyticus 414712557 552
Vibrio alginolyticus 414716557 650
Vibrio alginolyticus 434712457 552
Vibrio brasiliensis LMG 20546T 32471255. 740
Vibrio cholerae LMG 16741 204612457 1 Shrimp, Thailand 49
Vibrio cholerae LMG 16742 324602557 1 Pe. orientalis, PRC, Thailand 49
Vibrio cholerae 01 514612457
Vibrio cholerae non 01 514712457
Vibrio cholerae PS-7701 524712457 1 Pl. altivelis, Japan 49
Vibrio cholerae PS-7705 524712476 1 Pl. altivelis, Japan 49
Vibrio cholerae 91/1198 534612457 1 Carassius auratus, Australia 49
Vibrio cholerae 01 534612457
Vibrio cholerae non 01 LMG 16743 534712457 1 49
Vibrio cholerae WF 110r 534712557 509,552
Vibrio diabolicus 416612557 635
Vibrio fischeri ATCC 25918 400500556 745
Vibrio fluvialis 304612657 485,687
Vibrio fluvialis 304712657 485,687
Vibrio fluvialis 324412757 485,687
Vibrio fluvialis and MEJ 311 324612657 2 240,485,687
Vibrio fluvialis 324712657 485,687
Vibrio furnissii ATCC 35016T 324612657 240
Vibrio furnissii 304712657 687
Vibrio furnissii 324712657 687
Vibrio furnissii ATCC 11218 324512657 Citrate, indole, VP, weak reaction 135
Vibrio (carchariae) harveyi 415412557 135,581,847
Vibrio (carchariae) harveyi 415612557 135,581,847
Vibrio harveyi ATCC 14129 434612557 581
Vibrio (carchariae) harveyi 435412557 135,581,847
Vibrio (carchariae) harveyi ATCC 35084 435612557 135,581,847
Vibrio halioticoli AHLDA 1734 000410456 1 Abalone 135
Vibrio ichthyoenteri 00040245. 389
Vibrio mediterranei AHLDA 1733 704672556 1 Abalone 135
Vibrio mimicus 514610457 1 161
Vibrio mimicus 534610457 1 161,210
Vibrio mytili 106412556 635
Vibrio neptunius LMG 20536T 22670245. 740
Vibrio nereis 00640245. 635
Vibrio ordalii LMG 13544 000402446 1 49
Vibrio ordalii LMG 10951 000402476 3 Salmon 49
Vibrio ordalii V-306 000402556 1 Amago trout, Japan 49
Vibrio ordalii RF-2, PT-81025 000402576 8 O. mykiss, Pl. altivelis, Japan 49
Vibrio ordalii V-11 000412446 1 O. mykiss, Japan 49
Vibrio ordalii F378, F380, V-250 000602546 4 Salmon, Australia; O. mykiss, Japan 49
Vibrio ordalii F379, F381 000612446 2 Salmon, Australia 49
Vibrio ordalii DF 3K 000612446 1 Denmark 428
Vibrio ordalii V-302 304752476 4 O. mykiss, Japan 49
Vibrio parahaemolyticus 410610657 552
Vibrio parahaemolyticus 410610757 552
Vibrio parahaemolyticus ATCC 43996 414610657 1 135
Vibrio parahaemolyticus 430610657 552
Organism Strain API 20 E number No of strains Notes Ref
Table 4.24. Continued.
continued
203Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:37:00 PM
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188
Vibrio parahaemolyticus 434610657 428
Vibrio parahaemolyticus ATCC 17802 434610657 10
Vibrio pectenicida 020600456 470
Vibrio penaeicida 12060455. 388
Vibrio penaeicida 12460455. 388
Vibrio proteolyticus AHLDA 1735 624750456 1 Artemia 135
Vibrio rotiferianus LMG 21460T 5176167.. 305
Vibrio salmonicida RVAU 890206–1/12 000410040 1 Salmo salar, Faroe Islands 49
Vibrio salmonicida RVAU 881129–1/2 000410440 5 Salmo salar, Faroe Islands 49
Vibrio salmonicida RVAU 881129–1/1 000410446 1 Salmo salar, Faroe Islands 49
Vibrio salmonicida RVAU 890206–1/10 000412640 1 Salmo salar, Faroe Islands 49
Vibrio salmonicida RVAU 890206–1/7 520600556 1 Salmo salar, Faroe Islands 49
Vibrio scophthalmi 20040245. 149
Vibrio splendidus 004416516 466
Vibrio splendidus UB S292 204610456 1 Seawater, Sweden 49
Vibrio splendidus 90–0652 224614446 1 Striped trumpeter, Australia 49
Vibrio splendidus RVAU 88–12–686 304610456 3 Sa. Salar, Norway 49
Vibrio splendidus UB S236 304610556 1 Seawater, Sweden 49
Vibrio splendidus RVAU 88–12–711 304612456 1 Sp. auratus, Norway 49
Vibrio splendidus 89–1638 304612556 1 O. mykiss, Australia 49
Vibrio splendidus 304614556 281
Vibrio splendidus 304616556 281
Vibrio splendidus RVAU 88–12–717 324410556 1 Sc. maximus, Norway 49
Vibrio splendidus LMG 16752 324414557 1 Oyster, Spain 49
Vibrio splendidus RVAU 88–12–712 324610456 1 Solea solea, Norway 49
Vibrio splendidus UB S308, LMG 16747 324610556 2 Seawater, Sweden; oyster, Spain 49
Vibrio splendidus LMG 16745 324610557 1 Sp. auratus, Greece 49
Vibrio splendidus LMG 16744, 16750 324612557 2 Sp. auratus, Greece; oyster, Spain 49
Vibrio splendidus LMG 16748 324614556 1 Oyster, Spain 49
Vibrio splendidus LMG 16749 324616556 1 Oyster, Spain 49
Vibrio splendidus LMG 16746 324712556 1 Oyster, Spain 49
Vibrio tapetis 10460055. 108,146
Vibrio tubiashii 106612556 321,635
Vibrio tubiashii 304412556 321,635
Vibrio vulnificus biovar 1 non-serovar E M06–24 434610557 1 Human blood, USA 98
V. vulnificus biovar 1 non-serovar E 532 504600557 2 Diseased eel, Belgium, avirulent foreels
33,98
V. vulnificus biovar 1 non-serovar E 628 514412757 1 Paguara fish, Venezuela 98
V. vulnificus biovar 1 non-serovar E 530 514602557 7 Diseased eel, Belgium, virulent 33,98
V. vulnificus biovar 1 non-serovar E ATCC 27562 514610557 1 Human strain, USA 26,98
V. vulnificus biovar 1 non-serovar E 514610557 23 Human (USA), eels (Spain,Sweden), shrimp (Thailand)
33,98
V. vulnificus biovar 1 non-serovar E B9629, C7184 514610557 Human wound infection, USA 33,98
V. vulnificus biovar 1 non-serovar E UMH1, 374 514610557 Human wound infection, USA 33,98
V. vulnificus biovar 1 non-serovar E ATCC 27562 534600557 Human strain, USA 33
V. vulnificus biovar 1 non-serovar E E109 534600557 Healthy eel, Spain, avirulent for eels 98
V. vulnificus biovar 1 non-serovar E C7184 534610557 1 Human blood, USA, avirulent foreels
98
V. vulnificus biovar 1 serovar E 534 514410557 2 Diseased eel, Sweden, avirulent foreels
33,98
V. vulnificus biovar 1 serovar E ATCC 33186 514610557 Human blood, USA, avirulent foreels
98
V. vulnificus biovar 1 serovar E 521 534600557 Unknown, Australia, avirulent foreels
98
V. vulnificus biovar 1 LMG 12092 104602557 1 Eel, Belgium 33
Organism Strain API 20 E number No of strains Notes Ref
Table 4.24. Continued.
204Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #M.vpTuesday, January 20, 2004 4:27:03 PM
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189
V. vulnificus biovar 1 169 104610557 1 Eel, Belgium 33
V. vulnificus biovar 1 E109 114000557 1 Eel, Spain 33
V. vulnificus biovar 1 160 114610557 1 Eel, Belgium 33
V. vulnificus biovar 1 M626 410610457 1 Eel, Spain 33
V. vulnificus biovar 1 Vv1 504600557 Human wound infection, USA 33
V. vulnificus biovar 1 UMH1 504610557 1 Human wound infection, USA 33
V. vulnificus biovar 1 M631 510610557 1 Eel, Spain 33
V. vulnificus biovar 1 167 514650557 1 Eel, Belgium 33
V. vulnificus biovar 1 VIB 521 516600557 1 Unknown 33
V. vulnificus biovar 1 524610557 97
V. vulnificus biovar 2 serovar E 171 100600557 1 Diseased eel, Sweden 98
V. vulnificus biovar 2 171 104600557 1 Eel, Belgium 33
V. vulnificus biovar 2 serovar E NCIMB 2136 400600557 1 Diseased eel, Japan 98
V. vulnificus biovar 2 420600557 97
V. vulnificus biovar 2 serovar E 520 500400557 1 Shrimp, Taiwan, virulent for eels 98
V. vulnificus biovar 2 serovar E NCIMB 2137 500600457 1 Diseased eel, Japan 33,98
V. vulnificus biovar 2 serovar E ATCC 33149 500600557 1 Diseased eel, Japan 33,98,356
V. vulnificus biovar 2 NCIMB 2138 500600557 1 Diseased eel, Japan 98
V. vulnificus biovar 2 serovar E 121 510200457 1 Diseased eel, Sweden 98
V. vulnificus biovar 2 serovar E 526 510200557 1 Diseased eel, Sweden 98
V. vulnificus biovar 2 serovar E 524 510600557 Diseased eel, Norway 98
V. vulnificus biovar 2 NCIMB 2136 510600557 1 Diseased eel, Japan 33
V. vulnificus biovar 2 510600557 14 Eels (Japan, Norway – serovar E,Spain, Sweden), shrimp, human
33,98
V. vulnificus biovar 2 serovar E 523 514600557 5 Eels (Belgium – serovar E, Spain,Sweden), sea bream (Spain)
33,98
V. vulnificus biovar 2 serovar E NCIMB 2138 520600557 1 Diseased eel, Japan 98
V. vulnificus biovar 2 serovar E E86 520600557 2 Diseased eel, (Japan, Spain) 26,98
V. vulnificus biovar 2 serovar E 525 530600457 1 Diseased eel, Sweden 98
V. vulnificus biovar 2 serovar E ATCC 33187 530600557 1 Human strain, USA 98
V. vulnificus biovar 2 serovar E E105 530600557 Diseased eel, Spain 26,98
V. vulnificus biovar 2 serovar E 530600557 2 Human (USA), eel (Spain) 26,98
V. vulnificus biovar 2 serovar E 530610557 1 Taiwanese strains, avirulent for eels 26
V. vulnificus 818 414412557 1 Unknown, France 98
V. vulnificus 822 504410557 1 Shrimp, Senegal 98
V. vulnificus WF8A1110 534610357 509
Vibrio xuii LMG 21346T 20651275. 740
Yersinia intermedia 115457157 41
Yersinia intermedia 115457357 41
Yersinia intermedia 115477357 41
Yersinia intermedia 115477357 41
Yersinia ruckeri 110410057 674,717
Yersinia ruckeri 110450057 717
Yersinia ruckeri 110550057 717
Yersinia ruckeri ATCC 29473 510410057 2 184,674
Yersinia ruckeri AHLDA 1313 510510057 1 135
Yersinia ruckeri 5105500 20 Environmental strains 184
Yersinia ruckeri 5107500 1 Environmental strains 184
Yersinia ruckeri 530610057 41
Zobellia galactanovorans 10061361. 61
Zobellia uliginosa 100.0..5. 61
*from API database.To be used in conjunction with the API database supplied by bioMerieux.
Organism Strain API 20 E number No of strains Notes Ref
Table 4.24. Continued.
205Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:37:01 PM
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190
0000004.. Acinetobacter/Moraxella 1 41
0000004.. Pseudomonas stutzeri 1/9 38
000100416 Phocoenobacter uteri 266
0002004.. Flavobacterium psychrophilum 41
000200410 Tenacibaculum maritimum 41
000200440 Pseudomonas anguilliseptica 41
000200452 Shewanella woodyi 112
0004004.. Pseudomonas stutzeri 5/9 38
0004004.. Burkholderia cepacia 3/10 38
000410456 Vibrio halioticoli AHLDA 1734 1 Abalone 135
000402446 Vibrio ordalii LMG 13544 49
00040245. Vibrio ichthyoenteri 389
000402476 Vibrio ordalii LMG 10951 Salmon 49
000402576 Vibrio ordalii RF-2, PT-81025 O. mykiss, Pl. altivelis, Japan 49
00041000. Flavobacterium tegetincola 533
000410040 Vibrio salmonicida RVAU 890206–1/12 Salmo salar, Faroe Islands 49
0004104.. Pseudomonas stutzeri 3/9 38
000410440 Vibrio salmonicida RVAU 881129–1/2 Salmo salar, Faroe Islands 49
000410446 Vibrio salmonicida RVAU 881129–1/1 Salmo salar, Faroe Islands 49
000412640 Vibrio salmonicida RVAU 890206–1/10 Salmo salar, Faroe Islands 49
000500416 Phocoenobacter uteri 266
000600456 Moritella marina 766
000602546 Vibrio ordalii F378, F380 Salmon, Australia 49
0006060.. Flavobacterium branchiophilum 41
000612210 Flavobacterium hydatis 41
000612446 Vibrio ordalii F379, F381 Salmon, Australia 49
000612446 Vibrio ordalii DF 3K 428
0006727.. Burkholderia pseudomallei 1/91 38
001100407 Photobacterium damselae piscicida P90029 1 Atypical strain 745
004412417 Aeromonas salmonicida achromogenes 322
004416516 Vibrio splendidus 466
004512417 Aeromonas salmonicida achromogenes NCIMB 1110 468
00640245. Vibrio nereis 635
007431057 Providencia rettgeri 41
014452456 Pasteurella multocida 428
020000440 Pseudomonas anguilliseptica 96,541,828
020200440 Pseudomonas anguilliseptica 96,541,828
0204004.. Pseudomonas fluorescens 2/8 38
020600456 Vibrio pectenicida 470
0206006.. Burkholderia cepacia 1/10 38
0206006.. Pseudomonas aeruginosa 1/18 38
0206706.. Burkholderia pseudomallei 1/91 38
026402057 Providencia rustigianii 559
0402004.. Flavobacterium columnare 41
04020045. Shewanella gelidimarina 112
04061241. Flavobacterium xantham 533
050200453 Shewanella algae 433
06061245. Shewanella frigidimarina 112
070200453 Shewanella putrefaciens 433
070200653 Shewanella putrefaciens 433
API 20 E number Organism Strain No of strains Notes Ref
Table 4.25. API 20E database numbers (numbers in ascending order).
206Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:37:02 PM
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191
070202653 Shewanella putrefaciens 433
100.0..5. Zobellia uliginosa 61
100410556 Vibrio agarivorans AHLDA 1732 2 From abalone, pathogenicity unknown 135
100412456 Listonella pelagia
100577357 Pantoea agglomerans CDC 1429–71 291
100600557 Vibrio vulnificusbiovar 2 serovar E
171 Diseased eel, Sweden 98
100603210 Flavobacterium hibernum 532
10061361. Zobellia galactanovorans 61
10460055. Vibrio tapetis 108,146
104600557 Vibrio vulnificus biovar 2 171 Eel, Belgium 33
104602557 Vibrio vulnificus biovar 1 LMG 12092 Eel, Belgium 33
104610557 Vibrio vulnificus biovar 1 169 Eel, Belgium 33
104612456 Listonella pelagia
106412556 Vibrio mytili 635
106612556 Vibrio tubiashii 321,635
110410057 Yersinia ruckeri 674,717
110450057 Yersinia ruckeri 717
110550057 Yersinia ruckeri 717
111500416 Actinobacillus scotiae NCTC 12922 1 265
114000557 Vibrio vulnificus biovar 1 E109 Eel, Spain 33
114610557 Vibrio vulnificus biovar 1 160 Eel, Belgium 33
114612557 Aeromonas veronii veronii ATCC 35623 1 347
114712457 Aeromonas veronii veronii ATCC 35604 1 Human, USA 347
115457157 Yersinia intermedia 41
115457357 Yersinia intermedia 41
115477357 Yersinia intermedia 41
120516357 Pantoea agglomerans ATCC 14589 291
120517257 Pantoea agglomerans NCPPB 2285 291
120517357 Pantoea agglomerans DNA HG 14589 291
120547257 Rahnella aquatilis 151
12060455. Vibrio penaeicida 388
120712057 Enterobacter agglomerans 41
120713257 Pantoea agglomerans 291
120713357 Pantoea dispersa 291
120717357 Pantoea dispersa 291
120732257 Serratia plymuthica 41
12460455. Vibrio penaeicida 388
124612456 Listonella pelagia
134612557 Aeromonas veronii veronii ATCC 35606 1 347
134712557 Aeromonas veronii veronii ATCC 35605 1 Wound isolate, human, USA 347
140200410 Flavobacterium columnare 689
2000004.. Pseudomonas fluorescens 5/8 38
2002004.. Pseudomonas aeruginosa 1/18 38
200400406 Photobacterium damselae piscicida ATCC 17911 ATCC 29687 428
20040245. Vibrio scophthalmi 149
200412000 Flavobacterium gillisiae 533
200500017 Photobacterium leiognathi LMG 4228 745
API 20 E number Organism Strain No of strains Notes Ref
Table 4.25. Continued.
continued
207Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #M.vpTuesday, January 20, 2004 4:28:01 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
192
200500406 Photobacterium damselae piscicida 149,289,751,855
200500406 Photobacterium damselae piscicida ATCC 17911 ATCC 29690 674,745
200500407 Photobacterium damselae piscicida ATCC 17911 Majority NCIMB 2058 745
200500416 Actinobacillus delphinicola 2 Dolphin, whale 263
200500457 Photobacterium damselae damselae LMG 7892 49
2006004.. Pseudomonas aeruginosa 5/18 38
200610417 Aeromonas salmonicida 450
200610417 Aeromonas salmonicida salmonicida 674
2006704.. Burkholderia pseudomallei 1/91 38
2006706.. Burkholderia pseudomallei 4/91 38
2006707.. Burkholderia pseudomallei 5/91 38
2006726.. Burkholderia pseudomallei 1/91 38
2006727.. Burkholderia pseudomallei 22/91 38
200702406 Photobacterium angustum NCIMB 1895 745
201500457 Photobacterium damselae damselae 41
201500457 Photobacterium damselae damselae ATCC 33539 745
204402417 Aeromonas salmonicida AHLDA 1334 1 ‘Atypical’ strain, goldfish 135
204610456 Vibrio splendidus UB S292 Seawater, Sweden 49
204612457 Vibrio cholerae LMG 16741 Shrimp, Thailand 49
20651275. Vibrio xuii LMG 21346T 740
2200004.. Pseudomonas fluorescens 1/8 38
2202704.. Burkholderia pseudomallei 1/91 38
2202706.. Burkholderia pseudomallei 3/91 38
220400157 Photobacterium damselae damselae NCIMB 2184 745
220500406 Photobacterium damselae piscicida 745
2206004.. Pseudomonas aeruginosa 9/18 38
2206006.. Pseudomonas aeruginosa 1/18 38
2206704.. Burkholderia pseudomallei 4/91 38
2206706.. Burkholderia pseudomallei 21/91 38
2206707.. Burkholderia pseudomallei NCTC8016 1/91 38
2206707.. Burkholderia pseudomallei 12/91 38
2206727.. Burkholderia pseudomallei 15/91 38
2207104.. Janthinobacterium lividum 41
224614446 Vibrio splendidus 90–0652 Striped trumpeter, Australia 49
22670245. Vibrio neptunius LMG 20536T Rotifers, bivalve larvae 740
26061245. Shewanella frigidimarina 112
300610517 Aeromonas salmonicida salmonicida 322
324612657 Aeromonas caviae ATCC 15468T 240
30440044. Enterovibrio norvegicus LMG 19839T 741
304412556 Vibrio tubiashii 321,635
304452456 Listonella anguillarum 49
304512517 Aeromonas salmonicida masoucida 322
304512657 Aeromonas hydrophila ATCC 7966 1 674
304512657 Vibrio furnissii ATCC 11218 1 Citrate, indole, VP weak reaction 135
304572557 Listonella anguillarum 49
304610456 Vibrio splendidus RVAU 88–12–686 Sa. Salar, Norway 49
304610556 Vibrio splendidus UB S236 Seawater, Sweden 49
304612456 Vibrio splendidus RVAU 88–12–711 Sp. auratus, Norway 49
304612556 Vibrio splendidus 89–1638 O. mykiss, Australia 49
API 20 E number Organism Strain No of strains Notes Ref
Table 4.25. Continued.
208Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:37:03 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
193
304612657 Vibrio fluvialis 485,687
304613457 Aeromonas encheleia 241
304614556 Vibrio splendidus 281
304616556 Vibrio splendidus 281
304652456 Listonella anguillarum 49
304712657 Vibrio fluvialis 485,687
304712657 Vibrio furnissii 687
304752476 Listonella anguillarum
304752476 Listonella anguillarum 49
304752657 Listonella anguillarum 49
324410556 Vibrio splendidus RVAU 88–12–717 Sc. maximus, Norway 49
324412757 Vibrio fluvialis 485,687
324414557 Vibrio splendidus LMG 16752 Oyster, Spain 49
324472757 Listonella anguillarum 49
324512657 Vibrio furnissii ATCC 11218 1 Citrate, indole, VP weak reaction 135
324562757 Listonella anguillarum 49
324632657 Listonella anguillarum 49
324602557 Vibrio cholerae LMG 16742 Pe. orientalis, PRC 49
324610456 Vibrio splendidus RVAU 88–12–712 Solea solea, Norway 49
324610556 Vibrio splendidus UB S308 Seawater, Sweden 49
324610557 Vibrio splendidus LMG 16745 Sp. auratus, Greece 49
324612557 Vibrio splendidus LMG 16744 Sp. auratus, Greece 49
324612657 Aeromonas caviae 21
324612657 Vibrio fluvialis and MEJ 311 3 485,687,240
324612657 Vibrio furnissii ATCC 35016T 240
324614556 Vibrio splendidus LMG 16748 Oyster, Spain 49
324616556 Vibrio splendidus LMG 16749 Oyster, Spain 49
32471255. Vibrio brasiliensis LMG 20546T Bivalve larvae 740
324712556 Vibrio splendidus LMG 16746 Oyster, Spain 49
324712657 Vibrio fluvialis 485,687
324712657 Vibrio furnissii ATCC 11218 135,687
324712677 Listonella anguillarum 49
324713757 Aeromonas hydrophila ATCC 14715 1 322
324752557 Listonella anguillarum 49
324752656 Listonella anguillarum and AHLDA 1730 2 Japan, Australia (freshwater fish) 49,135
324752756 Listonella anguillarum 41,49,428
324752757 Listonella anguillarum 49
324752777 Listonella anguillarum 49
324772656 Listonella anguillarum 49
324772657 Listonella anguillarum 49
400400456 Moritella viscosa 132
400500556 Vibrio fischeri ATCC 25918 745
400600456 Moritella viscosa 132
400600557 V. vulnificusbiovar 2 serovar E
NCIMB 2136 Diseased eel, Japan 98
404712456 Vibrio alginolyticus 135
404712457 Vibrio alginolyticus 135
410400057 Edwardsiella ictaluri 41,334
410610457 V. vulnificus biovar 1 M626 Eel, Spain 33
API 20 E number Organism Strain No of strains Notes Ref
Table 4.25. Continued.
continued
209Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:37:03 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
194
410610657 Vibrio parahaemolyticus 552
410610757 Vibrio parahaemolyticus 552
414412557 V. vulnificus 818 Unknown, France 98
414610657 Vibrio parahaemolyticus ATCC 43996 135
414712457 Vibrio alginolyticus 650
414712557 Vibrio alginolyticus 552
414716557 Vibrio alginolyticus 650
415412557 Vibrio (carchariae) harveyi 135,581,847
415612557 Vibrio (carchariae) harveyi 135,581,847
416612557 Vibrio diabolicus 635
420600557 V. vulnificus biovar 2 97
4304004.. Burkholderia cepacia 4/10 38
430410257 Hafnia alvei 41
430610657 Vibrio parahaemolyticus 552
434610557 V. vulnificusbiovar 1 non-serovar E
M06–24 Human blood, USA 98
434610657 Vibrio parahaemolyticus 428
434610657 Vibrio parahaemolyticus ATCC 17802 10
434612557 Vibrio harveyi ATCC 14129 581
434712457 Vibrio alginolyticus 552
435412557 Vibrio (carchariae) harveyi 135,581,847
435612557 Vibrio (carchariae) harveyi ATCC 35084 135,581,847
454400057 Edwardsiella tarda 41
4644100.. Atypical Edwardsiella tarda 4 Atypical strains from red sea bream,Japan
845
474400057 Edwardsiella tarda AHLDA 135 ATCC 15947T 135,845
500400557 V. vulnificusbiovar 2 serovar E
520 Shrimp, Taiwan, virulent for eels 98
500600457 V. vulnificusbiovar 2 serovar E
NCIMB 2137 Diseased eel, Japan 33,98
500600557 V. vulnificusbiovar 2 serovar E
ATCC 33149 Diseased eel, Japan 33,98,356
500600557 V. vulnificus biovar 2 NCIMB 2138 Diseased eel, Japan 98
504410557 V. vulnificus 822 Shrimp, Senegal 98
504600557 V. vulnificusbiovar 1 non-serovar E
532 Diseased eel, Belgium, avirulent foreels
98
504600557 V. vulnificus biovar 1 Vv1 Human wound infection, USA 33
504610557 V. vulnificus biovar 1 UMH1 Human wound infection, USA 33
510200457 V. vulnificusbiovar 2 serovar E
121 Diseased eel, Sweden 98
510200557 V. vulnificusbiovar 2 serovar E
526 Diseased eel, Sweden 98
510410057 Yersinia ruckeri ATCC 29473 184,674
510411257 Hafnia alvei 652
510510057 Yersinia ruckeri AHLDA 1313 2 Channel catfish 135,203
510550057 Yersinia ruckeri 20 Environmental isolates 184
510600557 V. vulnificus biovar 2 NCIMB 2136 Diseased eel, Japan 33
510600557 V. vulnificusbiovar 2 serovar E
524 Diseased eel, Norway 98
510610557 V. vulnificus biovar 1 M631 Eel, Spain 33
510710057 Yersinia ruckeri YR55, YR80 2 Environmental isolates 184
510750057 Yersinia ruckeri 1 Environmental isolates 184
API 20 E number Organism Strain No of strains Notes Ref
Table 4.25. Continued.
210Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:37:04 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
195
514410557 V. vulnificusbiovar 1 serovar E
534 Diseased eel, Sweden, avirulent foreels
98
514412757 V. vulnificusbiovar 1 non-serovar E
628 Paguara fish, Venezuela 98
514600557 V. vulnificusbiovar 2 serovar E
523 Diseased eel 98
514602557 V. vulnificusbiovar 1 non-serovar E
530 Diseased eel, Belgium, virulent 98
514610457 Vibrio mimicus 161
514610557 V. vulnificusbiovar 1 non-serovar E
ATCC 27562 Human strain, USA 26,98
514610557 V. vulnificusbiovar 1 non-serovar E
B9629, C7184 Human wound infection, USA 33,98
514610557 V. vulnificusbiovar 1 non-serovar E
UMH1, 374 Human wound infection, USA 33,98
514610557 V. vulnificusbiovar 1 serovar E
ATCC 33186 Human blood, USA, avirulent for eels 98
514612457 Vibrio cholerae 01
514650557 V. vulnificus biovar 1 167 Eel, Belgium 33
514712457 Vibrio cholerae non 01
514712557 Aeromonas veronii veronii ATCC 35622 1 347
516600557 V. vulnificus biovar 1 VIB 521 Unknown 33
5176167.. Vibrio rotiferianus LMG 21460T Rotifer 305
520600556 Vibrio salmonicida RVAU 890206–1/7 Salmo salar, Faroe Islands 49
520600557 V. vulnificusbiovar 2 serovar E
NCIMB 2138 Diseased eel, Japan 98
520600557 V. vulnificusbiovar 2 serovar E
E86 Diseased eel, Spain 26,98
521577357 Klebsiella pneumoniae 41
524610557 V. vulnificus biovar 1 97
524712457 Vibrio cholerae PS-7701 Pl. altivelis, Japan 49
524712476 Vibrio cholerae PS-7705 Pl. altivelis, Japan 49
5304004.. Burkholderia cepacia 1/10 38
5304112.. Hafnia alvei ATCC 51873 1 203
530475257 Serratia fonticola 290
530477257 Serratia fonticola 290
530510057 Yersinia ruckeri AHLDA 1313 135
530511357 Hafnia alvei AHLDA 1729 135
530555257 Serratia fonticola 558
530600457 V. vulnificusbiovar 2 serovar E
525 Diseased eel, Sweden 98
530600557 V. vulnificusbiovar 2 serovar E
ATCC 33187 Human strain, USA 98
530600557 V. vulnificusbiovar 2 serovar E
E105 Diseased eel, Spain 26,98
530610057 Yersinia ruckeri 41
530610557 V. vulnificusbiovar 2 serovar E
Taiwanese strains, avirulent for eels 26
534600557 V. vulnificusbiovar 1 non-serovar E
ATCC 27562 Human strain, USA 33
534600557 V. vulnificusbiovar 1 non-serovar E
E109 Healthy eel, Spain, avirulent for eels 98
API 20 E number Organism Strain No of strains Notes Ref
Table 4.25. Continued.
continued
211Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:37:04 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
196
534600557 V. vulnificusbiovar 1 serovar E
521 Unknown, Australia, avirulent for eels 98
534610357 V. vulnificus WF8A1110 509
534610457 Vibrio mimicus 161,210
534610557 V. vulnificusbiovar 1 non-serovar E
C7184 Human blood, USA, avirulent for eels 98
534612457 Vibrio cholerae 91/1198 Carassius auratus, Australia 49
534612457 Vibrio cholerae 01
534712457 Vibrio cholerae non 01
534712557 Aeromonas veronii veronii 2,347
534712557 Vibrio cholerae WF 110r 509,552
560621057 Salmonella cholerasuis arizonae 41
600400457 Photobacterium iliopiscarium 599
600400457 Photobacterium damselae damselae ATCC 33539T Usually VP positive 240
600500457 Photobacterium damselae damselae LMG 13639 6 Various sources 49
600610417 Aeromonas salmonicida salmonicida 674
6046021.. Photobacterium phosphoreum IB39 509
6100530.. Halomonas cupida 41
624750456 Vibrio proteolyticus AHLDA 1735 1 Artemia 135
700610517 Aeromonas salmonicida salmonicida NCIMB 1102 468
704672556 Vibrio mediterranei AHLDA 1733 1 Abalone 135
704712457 Aeromonas sobria CIP 74.33T 240
704712657 Aeromonas hydrophila ATCC 7966T 1 240
704713757 Aeromonas bestiarum 5 Carp 452
714420457 Plesiomonas shigelloides 41
724610557 Aeromonas trota ATCC 49657T 1 142,560
724613657 Aeromonas allosaccharophila 1 41
724616657 Aeromonas allosaccharophila 1 41
724617657 Aeromonas allosaccharophila 1 41
724712457 Aeromonas sobria 21
724714457 Aeromonas janadaei 2 135,143
Not all references reported the 9-digit profile number, hence some species only have a 7- or 8-digit profile number with no results for growth on MCA orOF results.
API 20 E number Organism Strain No of strains Notes Ref
Table 4.25. Continued.
212Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:37:04 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
197
Org
anis
m
Acin
etob
acte
rcal
coac
etic
us−
−−
−−
−−
−−
−−
−−
−−
+−
−+
+−
2441
Actin
obac
illus
delp
hini
cola
+−
−−
−−
−−
−−
−−
−−
−−
−−
−−
+26
3D
olph
in
Actin
obac
illus
delp
hini
cola
+−
−+
−−
−−
−−
−−
−−
−−
−−
−−
+26
3Po
rpoi
se
Actin
obac
illus
delp
hini
cola
+−
−−
−−
−−
−−
−−
−−
−−
−−
−−
+26
3W
hale
Aero
mon
ashy
drop
hila
+v
++
−+
++
+v
++
++
++
−+
v−
+41
A.sa
lmon
icid
aac
hrom
ogen
es/m
asou
cida
+v
−−
−v
v−
v−
vv
−−
−−
−−
−−
+41
A.sa
lmon
icid
asa
lmon
icid
a+
vv
v−
++
−+
−v
++
++
−−
+−
−+
41
Aero
mon
asso
bria
+v
++
−−
++
+−
++
++
++
−+
+−
+41
Alte
rom
onas
nigr
ifaci
ens
−−
++
+−
++
++
++
395
Bord
etel
labr
onch
isep
tica
−−
−−
+−
−−
−−
−−
−−
−−
+−
++
+37
0.85
102
Type
stra
in
Bord
etel
labr
onch
isep
tica
+−
−−
+−
−−
−−
−−
−−
−−
+−
++
+37
0.85
102
Type
stra
in
Bord
etel
labr
onch
isep
tica
+−
−−
+−
−−
−−
−−
−−
−−
+−
+−
+37
0.85
102
Type
stra
in
Brev
undi
mon
asdi
min
uta
−−
−−
−−
−−
−−
−−
−−
−−
−−
−−
+68
5
Bruc
ella
spp.
+−
−−
+−
−−
−−
−−
−−
−−
−−
−−
+37
0.85
262
Chr
omob
acte
rium
viol
aceu
m−
−+
+−
−+
−+
−−
−+
−+
+−
++
−+
482
Pigm
ente
d
Chr
omob
acte
rium
viol
aceu
m+
−+
+−
−+
−+
−−
−+
−+
+−
++
−+
482
Non
-pig
men
ted
Chr
yseo
bact
eriu
mba
lust
inum
++
−−
++
−−
−w
−−
w−
−−
−w
−+
3748
0.85
773
Chr
yseo
bact
eriu
mgl
eum
+v
−−
++
++
+w
−−
+−
−−
−+
−+
3748
0.85
773
Chr
yseo
bact
eriu
min
dolo
gene
s−
+−
−+
++
w−
w−
−w
−−
−−
w−
+37
480.
8577
3
Chr
yseo
bact
eriu
min
dolth
etic
um−
+−
−−
++
w−
w−
−w
−−
−−
−w
+37
480.
8577
3
Chr
yseo
bact
eriu
mm
enin
gose
ptic
um−
++
−18
++
+−
++
+−
−−
−w
27−
+37
480.
8577
3
Chr
yseo
bact
eriu
msc
opht
halm
um−
−w
−+
++
+−
−+
−+
557
Flav
obac
teriu
mhi
bern
um+
−+
−−
++
++
+−
++
−−
−−
−−
−53
2
Hal
omon
ascu
pida
++
++
++
+−
69
Hal
mon
asve
nust
a+
−−
−+
+−
++
−−
−+
++
+−
++
++
310
Jant
hino
bact
eriu
mliv
idum
+−
−−
−+
−−
++
++
v+
−v
v+
++
+49
6
NO
3TR
PG
luAD
HU
reEs
cG
elPn
gG
luAr
aM
neM
anN
agM
alG
ntC
apAd
iM
ltC
itPa
cO
xTe
mp
Tim
eIn
ocR
efSt
rain
Tab
le4.
26.
AP
I20N
Eda
taba
sebi
oche
mic
alre
sults
.
cont
inue
d
213Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:37:05 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
198
List
onel
laan
guilla
rum
++
++
−−
++
++
++
++
++
+10
8
Myr
oide
sod
orat
imim
us−
−−
−+
−+
−−
−−
−−
−−
−+w
−−
+77
4
Myr
oide
sod
orat
us−
−−
−+
−+
−−
−−
−−
−−
−+w
−−
+24
774
Oce
anom
onas
baum
anni
i+
−−
−−
−−
−−
−−
−−
−−
+−
++
−+
2548
1.5
130
Oce
anom
onas
doud
orof
fii+
−−
−−
−−
−−
−−
−−
−−
+−
++
−+
2548
1.5
130
Phoc
oeno
bact
erut
eri
+−
+w−
−−
−−
−−
−−
−−
−−
−−
−−
+26
6
Phot
obac
teriu
mda
mse
lae
+−
++
+−
−−
−−
−−
−−
−−
−v
−−
+41
P.da
mse
lada
mse
la+
−+
++
v−
−+
−+
−+
+−
−+
−−
++
289
P.da
mse
lapi
scic
ida
−−
++
−−
−−
+−
+−
+−
−−
+−
−−
+28
9
Ples
iom
onas
shig
ello
ides
++
++
−−
−+
v−
−−
vv
vv
−v
−−
+41
Pseu
doal
tero
mon
asan
tarc
tica
−−
+−
−−
+−
+−
++
++
+−
−−
+−
+5
5d11
5
Pseu
doal
tero
mon
asba
cter
ioly
tica
−+
++
+−
−−
−−
−+
+67
7
Pseu
doal
tero
mon
asha
lopl
ankt
is+
++
−−
−28
8
Pseu
dom
onas
angu
illise
ptic
a−w
−−
−−
−+
−−
−−
−v
+−
−+
−−
−+
541
Pseu
dom
onas
chlo
rora
phis
−−
−−
−v
−−
++
++
−−
+v
−+
+−
v41
Pseu
dom
onas
fluor
esce
nsv
−−
v−
−v
−+
v+
+v
−+
+−
++
−+
41
Pseu
dom
onas
plec
oglo
ssic
ida
+−
−+
−−
−−
+−
−−
−−
++
−+
++
+25
4858
2
Pseu
dom
onas
putid
a+
+−
vv
vv
+58
2
Shew
anel
laal
gae
LMG
2265
+−
−−
−−
+−
−−
−−
+−
−+
−+
−−
+85
1
Shew
anel
laba
ltica
NC
TC10
735
+−
+−
−−
+−
+−
−−
++
+−
−+
+−
+85
1
Shew
anel
lape
alea
na−
−+
−+
+49
2
Shew
anel
lapu
trefa
cien
s+
−+
−−
−+
−−
+−
−+
+−
+−
++
−+
433
Sphi
ngom
onas
suba
rctic
a−
−−
−−
+−
++
+−
−+
+−
−−
++
−
NO
3TR
PG
luAD
HU
reEs
cG
elPn
gG
luAr
aM
neM
anN
agM
alG
ntC
apAd
iM
ltC
itPa
cO
xTe
mp
Tim
eIn
ocR
efSt
rain
Tab
le4.
26.
Con
tinue
d.
214Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:37:06 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
199
Stap
pia
stel
lula
ta- l i
kest
r ai n
M1
+−
++
−+
++
++
++
−+
++
−+
2548
1.5
105
Vago
cocc
ussa
lmon
inar
um−
−+
−−
+−
+−
542
Vibr
ioal
gino
lytic
us+
++
−−
v+
−v
−v
vv
vv
−−
+−
−+
41
Vibr
ioal
gino
lytic
us+
++
−−
−+
+−
++
++
+nt
nt+
+nt
+10
8
Vibr
ioal
gino
lytic
us+
++
−−
−+
++
++
++
++
−−
+−
−+
Vibr
ioco
rallii
lytic
us+
−+
−+
++
−+
83+
++
83+
3048
3.5
83,8
4YB
Vibr
io(c
arch
aria
e)ha
rvey
i+
++
−+
++
++
−+
+−
++
−−
++
−+
11AT
CC
3508
4
Vibr
ioch
oler
ae+
++
−−
−+
++
−v
vv
++
−−
++
−+
41
Vibr
iodi
abol
icus
++
+−
−−
++
−−
−−
−+
−+
−+
+−
+63
5
Vibr
ioha
rvey
i+
++
−−
++
++
++
++
++
−−
++
−+
11AT
CC
1412
6
Vibr
ioha
rvey
i+
++
−−
++
++
−−
+−
−+
−−
++
−+
11
Vibr
ioic
hthy
oent
eri
+−
+−
−33
50−
+38
9
Vibr
iom
imic
us+
++
−−
++
−+
++
+−
+23
0
Vibr
iom
ytili
++
++
−+
+−
−+
+−
635
Vibr
iona
varre
nsis
++
+−
−+
++
−+
++
+−
+60
−+
768
Vibr
ione
reis
++
+−
−−
+−
−+
+−
635
Vibr
iope
naei
cida
+−
+−
++
++
++
388
Vibr
iosh
iloni
i+
++
−−
++
++
+45
8
Vibr
iota
petis
++
−−
−+
+−
+−
+−
−−
−10
8
Vibr
iotu
bias
hii
++
++
−+
+−
−+
−−
635
Zobe
lliaga
lact
aniv
oran
s+
−+
−−
++
++
w+
++
+−
−−
−−
−+
307d
2.5
61
Zobe
lliaul
igin
osa
+−
+−
−+
++
++
++
++
w−
−−
−−
+30
7d2.
561
NO
3=
nitra
te;T
RP
=in
dole
;Glu
=gl
ucos
efe
rmen
tatio
n;AD
H=
argi
nine
dihy
drol
ase;
Ure
=ur
ease
;Esc
=ae
scul
in;G
el=
gela
tinhy
drol
ysis
;Png
=p-
nitro
phen
yl-b
-D-g
alac
topy
rano
side
;Glu
=gl
ucos
eas
sim
ilatio
n;Ar
a=
L-ar
abin
ose
assi
mila
tion;
Mne
=m
anno
seas
sim
ilatio
n;M
an=
man
nito
lass
imila
tion;
Nag
=N
-ace
tylg
luco
sam
ine;
Mal
=m
alto
seas
sim
ilatio
n;G
nt=
gluc
onat
eas
sim
ilatio
n;C
ap=
capr
ate
assi
mila
t ion;
Adi=
adip
ate
assi
mila
tion;
Mlt
=m
alat
eas
sim
ilatio
n;C
it=
citra
teas
sim
ilatio
n;Pa
c=
phen
ylac
etat
e;O
x=
oxid
ase;
*AP
Idat
abas
e.
215Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:37:07 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
200
Org
anis
m
Aero
cocc
usvi
ridan
sva
r.ho
mar
iw
++
++
+v
v+
++
++
++
Aero
mon
asbe
stia
rum
HG
2+
−−
++
−−
−−
++
++
−−
−−
++
−+
+−
++
+67
6767
Aero
mon
asca
viae
HG
4+
−−
++
−−
−−
++
+31
−−
−−
+7
−−
+−
++
+90
+87
Aero
mon
ascu
licic
ola
+−
−−
+−
−−
−+
++
+−
−−
−+
−−
−+
−−
−−
−+
−Ae
rom
onas
ench
elei
a+
−−
−+
++
−−
+−
−+
−+
−Ae
rom
onas
eucr
enop
hila
HG
6+
−−
++
−−
−−
++
++
−−
−−
+−
−−
+−
++
++
++
Aero
mon
ashy
drop
hila
HG
1+
−−
++
−−
−−
++
++
−−
−−
+−
−−
+−
++
+−
+−
Aero
mon
ashy
drop
hila
HG
3+
−−
++
−−
−−
++
++
−−
−−
++
−+
+−
++
+62
+54
Aero
mon
asm
edia
HG
5+
−−
++
−−
−−
++
++
−−
−−
+−
−−
+−
++
++
++
Aero
mon
aspo
poffi
i+
−−
−+
++
−−
−+
−−
−+
−A.
salm
onic
ida
‘aty
pica
l’
A.sa
lmon
icid
aac
hrom
ogen
es+
−+
++
++
−−
−−
−Ba
ltic
sea
turb
ot−
−−
−−
−−
−−
−+
+−
−−
−−
−−
−−
−−
−v
−−
−−
Balti
cse
aflo
unde
r−
−−
−v
−−
−−
−+
+v
−−
−−
v−
−−
−−
−+
v−
+−
Balti
cse
ada
b+
−−
−−
−−
−−
v+
+v
−−
−−
−−
−−
+w−
−+
+−
−−
Balti
cse
abl
enny
+−
−−
+−
−−
−+
++
+−
−−
−+
−−
−+
−−
−−
−+
−‘A
typi
cal’
Pigm
entp
os+
−−
−+
−−
−−
++
+−
−−
−+
−−
17−
‘Aty
pica
l’Pi
gmen
tneg
44−
−−
+−
−−
−44
++
−−
−−
+−
−88
−‘A
typi
cal’
Pigm
entn
eg44
−−
−+
−−
−−
44+
+−
−−
−−
−−
88−
Aust
ralia
nst
rain
−−
−−
−−
−−
−A.
salm
onic
ida
typi
cal
++
++
++
+−
++
++
+−
+
A.sa
lmon
icid
asa
lmon
icid
a+
++
++
−+
++
++
−
12
34
56
78
910
1112
1314
1516
1718
1920
2122
2324
2526
2728
29
Test
Gly
Ery
Dar
aLa
rarib
dXyl
lXyl
ado
mdx
gal
glu
frum
neLs
orrh
adu
lin
om
anso
rm
dmm
dgna
gam
yar
bes
csa
lce
lm
alla
c
Tab
le4.
27.
AP
I 50C
Hda
taba
sebi
oche
mic
alre
sults
.
216Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:37:08 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
201
Org
anis
m
Aero
cocc
usvi
ridan
sva
r.ho
mar
iv
++
++
+v
+v
827
Aero
mon
asbe
stia
rum
HG
2−
++
−−
−+
+−
−−
−−
−−
−−
−30
480.
8542
7C
DC
9533
–76
Aero
mon
asca
viae
HG
4−
++
−−
−+
+−
36−
−−
−−
−−
+−
−30
480.
8542
7,21
ATC
C15
468T
Aero
mon
ascu
licic
ola
++
−−
−−
+−
−−
−−
−−
−−
−−
3024
0.85
624
NC
IMB
5147
T
Aero
mon
asen
chel
eia
+−
−Ae
rom
onas
eucr
enop
hila
HG
6−
++
−−
−+
+−
−−
−−
−−
−−
−30
480.
8542
7AT
CC
2330
9T
Aero
mon
ashy
drop
hila
HG
1−
++
−−
−+
+−
−−
−−
−−
−−
+−
−30
480.
8542
7AT
CC
7966
T
Aero
mon
ashy
drop
hila
HG
3−
++
−−
−+
+−
−−
−−
−−
−−
−30
480.
8542
7C
DC
0434
–84
Aero
mon
asm
edia
HG
5−
++
−−
−+
+−
−−
−−
−−
−−
−30
480.
8542
7C
DC
0862
–83
CD
C90
72–8
3
Aero
mon
aspo
poffi
i−
−+
−−
A.sa
lmon
icid
a‘a
typi
cal’
A.sa
lmon
icid
aac
hrom
ogen
es+
+−
++
−−
+s20
7d0.
583
1
Balti
cse
atu
rbot
−−
−−
−−
−−
−−
−−
−−
−−
−−
−+
2014
d83
2
Balti
cse
aflo
unde
r−
v+
−−
−v
+−
−−
−−
−−
−−
−−
+20
14d
832
Balti
cse
ada
b−
+−
−−
−−
−−
−−
−−
−−
−−
−−
+20
14d
832
Balti
cse
abl
enny
−+
+−
−−
++
−−
−−
−−
−−
−−
−+w
2014
d83
2
‘Aty
pica
l’Pi
gmen
tpos
+68
−−
100
3−
−−
−−
−20
7d32
3,35
2
‘Aty
pica
l’Pi
gmen
tneg
+27
−−
6375
−−
−−
−−
207d
323
‘Aty
pica
l’Pi
gmen
tneg
+−
−63
75−
−−
−−
−20
7d32
3
Aust
ralia
nst
rain
+−
−76
1
A.sa
lmon
icid
aty
pica
l−
−−
++
−+
−20
–22
7d35
2
A.sa
lmon
icid
asa
lmon
icid
a−
−−
++
−+
+s20
7d0.
583
1
3031
3233
3435
3637
3839
4041
4243
4445
4647
4849
Test
mel
suc
trein
um
lzra
fam
dgl
ygxy
ltge
ntu
rly
xta
gD
fuc
Lfuc
Dar
lLa
rlgn
t2k
et5k
etTe
mp
Tim
eIn
ocR
efSt
rain
Stra
in cont
inue
d
217Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:37:08 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
202
Aero
mon
asso
bria
+−
−28
+−
−−
−+
++
+−
−−
−+
−−
24+
−−
16−
52+
16
Aero
mon
astro
ta+
−−
−+
−−
−−
++
++
−−
−−
+−
−−
++
−−
−+
+−
Aero
mon
asve
roni
isob
riaH
G8/
10+
−−
14+
−−
−−
++
++
−−
−−
+−
−29
+−
−10
−57
+10
Aero
mon
asve
roni
iver
onii
−−
−−
−+
++
−−
+−
++
++
+−
Arca
noba
cter
ium
bern
ardi
ae+
vv
−+
++
−−
−−
−+
Arca
noba
cter
ium
phoc
ae+
−−
−+
−−
−−
++
+70
−−
−+
30−
−−
+−
−−
−−
++
Arca
noba
cter
ium
phoc
ae−
−−
−−
−−
−−
v+
−+
−−
−−
−−
−−
+−
−−
−−
+v
Arca
noba
cter
ium
pyog
enes
−+
−+
++
++
++
−+
−+
−−
−+
++
Arth
roba
cter
rhom
bi+
−−
−−
−−
−−
++
++
−v
−−
+−
−−
−+
++
++
++
Brev
undi
mon
asdi
min
uta
−−
−−
−−
−−
−−
−−
−−
−−
−−
−−
−−
−−
−−
−−
−Br
evun
dim
onas
vesi
cula
ris−
−−
−−
+−
−−
−+
−−
−−
−−
−−
−−
−−
−−
−−
+−
Car
noba
cter
ium
dive
rgen
s−
−−
+−
−−
−−
++
+−
−−
−−
−−
−+
++
++
−C
arno
bact
eriu
mga
llinar
um−
−−
++
−−
−+
++
−−
−−
−−
−+
++
++
+
Car
noba
cter
ium
mob
ile−
−−
+−
−−
−+
++
−−
−−
−−
−−
+−
++
+
Car
noba
cter
ium
pisc
icol
a+
−−
−+
−−
−−
+w+
++
−−
−−
+v
++
++
++
++
++w
Chr
yseo
bact
eriu
msc
opht
halm
um−
−−
−−
−En
tero
cocc
usfa
eciu
mv
+−
−+
−−
−Fl
avob
acte
rium
colu
mna
re−
−−
−−
−−
−−
−−
−−
−−
−−
−−
−−
−−
−−
−−
−−
Flav
obac
teriu
mps
ychr
ophi
lum
−−
−−
−−
−−
−−
−−
−−
−−
−−
−−
−−
−−
−−
−−
−Fl
exib
acte
rlito
ralis
−−
−−
−−
−−
−−
−−
−−
−−
−−
−−
−−
−−
−−
−−
−Fl
exib
acte
rpol
ymor
phus
−−
−−
−−
−−
−−
−−
−−
−−
−−
−−
−−
−−
−−
−−
−Fl
exib
acte
rros
eolu
s−
−−
−−
−−
−−
−−
−−
−−
−−
−−
−−
−−
−−
−−
−−
Flex
ibac
terr
uber
−−
−−
−−
−−
−−
−−
−−
−−
−−
−−
−−
−−
−−
−−
−Ja
nthi
noba
cter
ium
livid
um+
−+
++
+−
−−
++
++
−v
−+
++
−−
v−
+−
++
+−
Kleb
siel
lapl
antic
ola
+−
++
++
++
++
++
−+
++
++
++
++
Lact
ococ
cus
garv
ieae
−−
−−
+−
−−
−+
++
+−
−−
−+
−−
−+
++
++
++
−
12
34
56
78
910
1112
1314
1516
1718
1920
2122
2324
2526
2728
29
Test
Gly
Ery
Dar
aLa
rarib
dXyl
lXyl
ado
mdx
gal
glu
frum
neLs
orrh
adu
lin
om
anso
rm
dmm
dgna
gam
yar
bes
csa
lce
lm
alla
c
Tab
le4.
27.
Con
tinue
d.
218Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:37:09 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
203
Aero
mon
asso
bria
12+
+−
−−
++
−−
−−
−−
−−
−+
−−
2948
21
Aero
mon
astro
ta−
−+
−−
−+
+−
−−
−−
−−
−−
−−
−37
240.
8520
ATC
C49
657T
Aero
mon
asve
roni
isob
riaH
G8/
10−
++
−−
−+
+−
−−
−−
−−
−−
−30
480.
8542
7C
DC
0437
–84
Aero
mon
asve
roni
iver
onii
−+
+−
−Ar
cano
bact
eriu
mbe
rnar
diae
−+
v+
+v
−+
3748
274
Arca
noba
cter
ium
phoc
ae−
++
−70
−+
+−
−+
−30
−−
−−
−−
+37
2463
6
Arca
noba
cter
ium
phoc
ae−
v−
−−
v−
−−
−−
−−
−−
−−
−−
−37
4861
3
Arca
noba
cter
ium
pyog
enes
−−
+−
+−
++
3748
641
Arth
roba
cter
rhom
bi+
++
−−
−−
−−
++
−−
−−
+−
+−
−25
4860
0
Brev
undi
mon
asdi
min
uta
−−
−−
−−
−−
−−
−−
−−
−−
−−
−−
284
685
Brev
undi
mon
asve
sicu
laris
−−
−−
−−
−−
−−
−−
−−
−−
−−
−−
284
685
Car
noba
cter
ium
dive
rgen
s−
++
−+
−−
−−
+−
−−
−−
−−
+−
−25
7d17
6
Car
noba
cter
ium
gallin
arum
++
−+
−−
−+
−+
−−
−−
+−
176
Car
noba
cter
ium
mob
ile+
++
−−
−−
−−
−v−
−−
−−
−17
6
Car
noba
cter
ium
pisc
icol
a+
++
++
v−
−−
++w
−−
−−
−−
+−
−25
7217
6,73
Chr
yseo
bact
eriu
msc
opht
halm
um−
−En
tero
cocc
usfa
eciu
mv
v−
−−
−−
−v
Flav
obac
teriu
mco
lum
nare
−−
−−
−−
−−
−−
−−
−−
−−
−−
−−
2212
88,8
9,21
1
Flav
obac
teriu
mps
ychr
ophi
lum
−−
−−
−−
−−
−−
−−
−−
−−
−−
−−
2212
88,8
9,21
1
Flex
ibac
terl
itora
lis−
−−
−−
−−
−−
−−
−−
−−
−−
−−
−89
Flex
ibac
terp
olym
orph
us−
−−
−−
−−
−−
−−
−−
−−
−−
−−
−89
Flex
ibac
terr
oseo
lus
−−
−−
−−
−−
−−
−−
−−
−−
−−
−−
89
Flex
ibac
terr
uber
−−
−−
−−
−−
−−
−−
−−
−−
−−
−−
89
Jant
hino
bact
eriu
mliv
idum
−+
−v
−v
−−
+−
−+
−−
++
−−
+−
496
Kleb
siel
lapl
antic
ola
++
+−
+−
−La
ctoc
occu
sga
rvie
ae−
−+
−−
−−
−−
+−
−−
−−
−−
−−
−28
24–9
617
4,23
6AT
CC
4915
6
3031
3233
3435
3637
3839
4041
4243
4445
4647
4849
Test
mel
suc
trein
um
lzra
fam
dgl
ygxy
ltge
ntu
rly
xta
gD
fuc
Lfuc
Dar
lLa
rlgn
t2k
et5k
etTe
mp
Tim
eIn
ocR
efSt
rain
Stra
in cont
inue
d
219Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:37:09 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
204
Lact
ococ
cus
garv
ieae
−−
−−
+−
−−
−+
++
+−
−−
−+
−−
−+
++
++
++
−
Biot
ypes
1,2,
12+
Biot
ypes
3,7,
11,1
3+
Biot
ypes
4,5,
6,10
,+
Biot
ypes
8,9
−La
ctoc
occu
spi
sciu
m−
−−
++
+−
−−
++
++
−−
−−
+−
−−
++
++
++
++
Mor
itella
visc
osa
−−
−−
+−
−−
−−
++
−−
−−
−−
−−
−+
−−
−−
−+
−Pe
doba
cter
hepa
rinus
−−
−+
−+
−−
−+
++
+−
−−
−−
+−
−−
−−
+−
++
+
Pedo
bact
erpi
sciu
m−
−−
−v
−−
++
++
−−
−−
++
++
++
++
+
Phot
obac
teriu
man
gust
um−
−−
−+
−−
−−
++
++
−−
−−
−−
−−
+−
−−
−−
+−
P.da
mse
lae
dam
sela
e+
−−
−+
−−
−−
++
++
−−
−−
−−
−−
+−
−−
−+
+−
P.da
mse
lae
pisc
icid
a−
−−
−+
−−
−−
+w+
++
−−
−−
−−
−−
+−
−−
−−
−−
P.da
mse
lae
pisc
icid
a−
−−
−+
−−
−−
++
++
−−
−−
−−
−−
+−
−−
−−
++
Phot
obac
teriu
milio
pisc
ariu
mv
−−
−+
−−
−−
++
++
−−
−−
−−
−−
+−
−−
−+
−Ph
otob
acte
rium
leio
gnat
hi+
−−
−+
−−
−−
++
++
−−
−−
−−
−−
+−
−−
−−
−−
Stre
ptoc
occu
s(d
iffic
ile)a
gala
ctia
e−
−−
−+
−−
−−
−+
++
−−
−−
−−
−−
+−
−−
−−
+−
Stre
ptoc
occu
s(d
iffic
ile)a
gala
ctia
e−
−−
−+
−−
−−
−+
++
−−
−−
−−
−+
+−
−−
−−
+−
Stre
ptoc
occu
s(d
iffic
ile)a
gala
ctia
e−
−−
−+
−−
−−
−+
++
−−
−−
−−
−+
+−
−−
−−
+−
Stre
ptoc
occu
sin
iae
−−
−−
+−
−−
−?
++
+−
−−
−+
−+
−+
−+
++
++
−St
rept
ococ
cus
inia
e−
−−
−+
−−
−−
−+
++
−−
−−
+−
−+
+−
++
++
+−
Stre
ptoc
occu
sin
iae
−−
−−
+−
−−
−−
++
+−
−−
−+
−−
−+
−+
++
++
−St
rept
ococ
cus
inia
e−
−−
−+
−−
−−
++
++
−−
−−
+−
−−
++
++
++
+−
Stre
ptoc
occu
spa
raub
eris
−−
−−
+−
−−
−+
++
+−
−v
−+
+−
−+
++
++
++
+
Stre
ptoc
occu
sph
ocae
−−
−−
+−
−−
−−
++
+−
−−
−−
−−
−+
−−
−−
−+
−St
rept
ococ
cus
porc
inus
+−
−−
+−
−−
−+
++
+−
−−
−+
+−
−+
v+
++
+v
Stre
ptoc
occu
sub
eris
−−
−−
+−
−−
−+
++
+−
−v
−+
+−
−+
++
++
++
+
12
34
56
78
910
1112
1314
1516
1718
1920
2122
2324
2526
2728
29
Test
Gly
Ery
Dar
aLa
rarib
dXyl
lXyl
ado
mdx
gal
glu
frum
neLs
orrh
adu
lin
om
anso
rm
dmm
dgna
gam
yar
bes
csa
lce
lm
alla
c
Tab
le4.
27.
Con
tinue
d.
220Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:37:10 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
205
Lact
ococ
cus
garv
ieae
−−
+−
−−
−−
−+
−−
−−
−−
−−
−−
2824
–96
174,
211,
236,
237,
780
ATC
C49
156
Biot
ypes
1,2,
12+
+78
0
Biot
ypes
3,7,
11,1
3−
+78
0
Biot
ypes
4,5,
6,10
,−
−78
0
Biot
ypes
8,9
−−
780
Lact
ococ
cus
pisc
ium
++
+−
++
+w−
−+
+−
−−
−−
−+
−−
2548
0.85
835
NC
FB27
78
Mor
itella
visc
osa
−−
−−
−−
++
−−
−−
−−
−−
−−
+−
1572
132
NC
IMB
1348
4
Pedo
bact
erhe
parin
us−
+−
−−
−+
−−
−+
−−
+−
−−
−+
2848
0.5
728
Pedo
bact
erpi
sciu
m+
+−
+−
++
−−
−−
+−
−−
−28
480.
572
8
Phot
obac
teriu
man
gust
um−
−−
−−
−−
−−
−−
−−
−−
−−
−−
−26
721.
574
5N
CIM
B18
95
P.da
mse
lae
dam
sela
e−
−+
−−
−−
−−
−−
−−
−−
−−
−−
2672
1.5
745
ATC
C35
083
ATC
C33
539
P.da
mse
lae
pisc
icid
a−
+w−
−−
+wv
−−
−−
−−
−−
−−
−−
−26
721.
528
8,74
5N
CIM
B20
58N
CIM
B25
918,
ATC
C17
911
P.da
mse
lae
pisc
icid
a−
−−
−−
−−
−−
−−
−−
−−
−−
−−
2672
1.5
745
P90
029
Atyp
ical
stra
in
Phot
obac
teriu
milio
pisc
ariu
m−
−v
−−
−−
−−
−−
−−
−−
−−
v−
−22
2.85
599
Phot
obac
teriu
mle
iogn
athi
−−
−−
−−
−−
−−
−−
−−
−−
−+
−−
2672
1.5
745
LMG
4228
Stre
ptoc
occu
s(d
iffic
ile)a
gala
ctia
e−
+−
−−
−−
−−
−−
−−
−−
−−
−−
−30
Stre
ptoc
occu
s(d
iffic
ile)a
gala
ctia
e−
+−
−−
−−
−−
−−
−−
−−
−−
−−
−30
7223
3N
D2–
22
Stre
ptoc
occu
s(d
iffic
ile)a
gala
ctia
e−
+−
−−
−−
−−
−−
−−
−−
−−
−−
−30
7223
3N
D2–
22
Stre
ptoc
occu
sin
iae
++
−+
−+
+−
+−
−−
−−
−−
−−
−St
rept
ococ
cus
inia
e−
++
−+
−+
+−
+−
−−
−−
−−
−−
−30
7223
3N
D2–
16
Stre
ptoc
occu
sin
iae
−+
+−
+−
++
−+
−−
−−
−−
−−
−−
3072
233,
235
ND
2–16
Stre
ptoc
occu
sin
iae
−+
+−
+−
++
−+
−−
−−
−−
−−
−−
2472
0.85
183
Stre
ptoc
occu
spa
raub
eris
−+
+v
−−v
−v−
−+
−−
+−
−−
−−
−−
3724
224
Stre
ptoc
occu
sph
ocae
−−
−−
−−
−−
−−
−−
−−
−−
−−
−−
3724
700
Stre
ptoc
occu
spo
rcin
us−
++
−−
v−
−−
−−
−−
−−
−−
−37
24–4
80.
517
5
Stre
ptoc
occu
sub
eris
−+
++
−−
−−
−+
−−
+−
−−
−−
−−
3724
224
3031
3233
3435
3637
3839
4041
4243
4445
4647
4849
Test
mel
suc
trein
um
lzra
fam
dgl
ygxy
ltge
ntu
rly
xta
gD
fuc
Lfuc
Dar
lLa
rlgn
t2k
et5k
etTe
mp
Tim
eIn
ocR
efSt
rain
Stra
in cont
inue
d
221Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:37:10 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
206
Tena
ciba
culu
mm
ariti
mum
−−
−−
−−
−−
−−
−−
−−
−−
−−
−−
−−
−−
−−
−−
−Va
goco
ccus
fluvi
alis
−−
−−
+−
−−
−−
++
+−
−−
−+
+−
++
++
++
++
−Va
goco
ccus
fluvi
alis
−−
−−
+−
−−
−−
++
+−
−−
−+
−−
+−
++
++
++
−Va
goco
ccus
salm
onin
arum
−−
−−
60+w
−−
−−
++
+90
−−
−−
70−
−+
++
++
6070
−Vi
brio
carc
haria
eAT
CC
3508
4−
−−
−+
−−
−−
++
++
−−
−−
+−
−−
+−
−+w
−+
+−
Vibr
iodi
abol
icus
+−
−−
+−
−−
−+
++
+−
−−
−+
−−
−+
+−
−−
−+
−Vi
brio
fisch
eri
−−
−−
+−
−−
−+
++
+−
−−
−−
−−
−+
−−
−+
++
−Vi
brio
harv
eyi
ATC
C14
129
−−
−−
+−
−−
−+
++
+−
−−
−+
−−
−+
−−
+w−
++
−
Vibr
iom
ytili
+−
−+
++
−−
−+
++
+−
−−
−+
−−
−+
++
++
++
−Vi
brio
nere
is−
−−
−+
−−
−−
−+
++
−−
−−
−−
−−
+−
−−
−−
+−
Vibr
iope
naei
cida
−−
−−
−+
++
−−
−−
−−
++
−Vi
brio
salm
onic
ida
+sl
−−
−+
−−
−−
++
+−
−−
−−
+−
−−
+−
−−
−−
+−
Vibr
iosp
lend
idus
v−
−−
+−
−−
−+
++
+−
−−
−+
−−
−+
−−
+−
++
v
Vibr
iotu
bias
hii
−−
−−
+−
−−
−+
++
+−
−−
−+
−−
−+
+−
−−
++
−Ye
rsin
iakr
iste
nsen
ii+
−−
++
+−
−−
++
++
+−
−−
++
−−
+v
+−
−+
++
Zobe
lliaga
lact
anov
oran
s−
++
++
++
+−
++
+
Zobe
lliaul
igin
osa
−+
−−
++
12
34
56
78
910
1112
1314
1516
1718
1920
2122
2324
2526
2728
29
Test
Gly
Ery
Dar
aLa
rarib
dXyl
lXyl
ado
mdx
gal
glu
frum
neLs
orrh
adu
lin
om
anso
rm
dmm
dgna
gam
yar
bes
csa
lce
lm
alla
c
Tab
le4.
27.
Con
tinue
d.
222Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:37:11 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
207
Tena
ciba
culu
mm
ariti
mum
−−
−−
−−
−−
−−
−−
−−
−−
−−
−−
2212
ASW
88,8
9,21
1
Vago
cocc
usflu
vial
is−
++
−−
−−
++
−−
−−
−−
−−
177
Spec
ies
Vago
cocc
usflu
vial
is−
−+
−−
−−
−−
+v
−−
−−
−−
−−
−17
7,49
8N
CD
O24
97
Vago
cocc
ussa
lmon
inar
um−
++
−−
−−
−−
50−
−+
−60
−−
−+w
−25
2–7d
542,
807
NC
FB27
77
Vibr
ioca
rcha
riae
ATC
C35
084
−+
+−
−−
++
−−
−−
−−
−−
−+
−−
581
Vibr
iodi
abol
icus
−+
+−
−−
++
−−
−−
−−
−−
−+
−−
2548
2.85
635
Vibr
iofis
cher
i−
−−
−−
−+
−−
−−
−−
−−
−−
−−
−26
721.
574
5AT
CC
2591
8
Vibr
ioha
rvey
iAT
CC
1412
9−
++
−−
−+
+−
−−
−−
−−
−−
+−
−25
242.
8558
1
Vibr
iom
ytili
−+
+−
+−
++
−+
v−
−−
−−
−+
−−
2548
2.85
635
Vibr
ione
reis
−+
+−
−−
+−
−−
v−
−−
−−
−+
−−
2548
2.85
635
Vibr
iope
naei
cida
−−
+−
+25
7d38
8JM
C91
23
Vibr
iosa
lmon
icid
a−
−+
−−
−−
−−
−−
−−
−−
−−
+−
−23
2
Vibr
iosp
lend
idus
+−
+−
−−
++
−−
−−
−−
10−
−+
−−
252.
8528
1
Vibr
iotu
bias
hii
−+
+−
−−
++
−−
−−
−−
−−
−+
−−
2548
2.85
635
Yers
inia
kris
tens
enii
−−
+−
−−
+−
2848
0.85
86
Zobe
lliaga
lact
anov
oran
s+
−−
−30
7d2.
523
DSM
1280
2T
Zobe
lliaul
igin
osa
+w
++
307d
2.5
61D
SM20
61T
Gly
=gl
ycer
ol;E
ry=
eryt
hrito
l;D
ara
=D-a
rabi
nose
;Lar
a=
L-ar
abin
ose;
rib=
ribos
e;dX
yl=
D-x
ylos
e;ad
o=
adon
itol;
mdx
=b-
met
hyl-D
-xyl
osid
e;ga
l=ga
lact
ose;
glu
=gl
ucos
e;fru
=f ru
ctos
e;m
ne=
man
nose
;Lso
r=L-
sorb
ose;
rha
=rh
amno
se;
dul=
dulc
itol;
ino
=tin
osito
l;m
an=
man
nito
l;so
r =so
rbito
l;m
dm=
a-m
ethy
l-D-m
anno
side
;mdg
=a
-met
hyl-D
-glu
cosi
de;n
ag=
N-a
cety
lglu
cosa
min
e;am
y=
amyg
dalin
;arb
=ar
butin
;esc
=ae
scul
in;s
al=
salic
in;c
el=
cello
bios
e;m
al=
mal
tose
;la
c=
lact
ose;
mel
=m
elib
iose
;suc
=su
cros
e;tre
=tre
halo
se;i
nu=
inul
in;m
lz=
mel
izito
se;r
af=
raf fi
nose
;am
d=
star
ch;g
lyg
=gl
ycog
en;x
ylt=
xylit
ol;g
en=
gent
iobi
ose;
tur=
D-tu
rano
se;l
yx=
D-ly
xose
;tag
=D-ta
gato
se;D
fuc
=D-fu
cose
;Lf
uc=
L-fu
cose
;Dar
l=D-a
rabi
tol;
Larl
=L-
arab
itol;
gnt=
gluc
onat
e;2k
et=
2-ke
togl
ucon
ate;
5ket
=5-
keto
gluc
onat
e.N
umbe
rsin
dica
teth
epe
rcen
t age
ofst
rain
sw
itha
posi
tive
resu
lt.
3031
3233
3435
3637
3839
4041
4243
4445
4647
4849
Test
mel
suc
trein
um
lzra
fam
dgl
ygxy
ltge
ntu
rly
xta
gD
fuc
Lfuc
Dar
lLa
rlgn
t2k
et5k
etTe
mp
Tim
eIn
ocR
efSt
rain
Stra
in
223Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:37:11 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
208
Nit
Pyz
Pyra
Pal
bgu
rb
gal
agl
ubN
AGes
cur
ege
lgl
urib
xyl
man
mal
lac
sac
gly
cat
Ref
Actin
omyc
esm
arim
amm
aliu
m−
−−
v−
++
−−
−+
370
Arca
noba
cter
ium
bern
ardi
ae−
−−
−+
−−
+−
480
Arca
noba
cter
ium
haem
olyt
icum
−−
−−
+−
v48
0
Arca
noba
cter
ium
phoc
ae−
−+
+−
++
−−
−−
+v
30+
v+
+v
480,
636
Arca
noba
cter
ium
phoc
ae−
−+
+−
−+
−−
v−
−−
−+
vv
−+
613
Arca
noba
cter
ium
plur
anim
aliu
m−
−v
−+
−−
−w
+−
++
−−
+−
−−
+48
0
Arth
roba
cter
nasi
phoc
ae−
−+
+−
−+
−−
−+
−−
−−
−−
−−
+18
2
Arth
roba
cter
rhom
bi−
−−
−−
+−
−+
−−
−−
++
++
−60
0
Cor
yneb
acte
rium
aqua
ticum
−+
++
−+
+−
+−
+v−
133
Cor
yneb
acte
rium
pseu
dodi
phth
eriti
cum
++
++
−−
−−
−+
−−
133
Cor
yneb
acte
rium
pseu
dotu
berc
ulos
is−
−−
+−
+−
−−
+−
−13
3
Cor
yneb
acte
rium
test
udin
oris
+−
−−
−−
v−
+−
−+
−−
+−
+−
+18
0
Cor
yneb
acte
rium
xero
sis
+−
++
−−
−−
−−
−−
133
Nit
=ni
trate
;Pyz
=py
rrolid
onyl
acry
lam
idas
e;Py
ra=
pyra
zina
mid
ase;
Pal=
alka
line
phos
phat
ase;
bgu
r=b
gluc
uron
idas
e;b
gal=
bga
lact
osid
ase;
agl
u=
agl
ucos
idas
e;bN
AG=
N-a
cety
l-bgl
ucos
amin
idas
e;es
c=
aesc
ulin
;ure
=ur
ease
;ge
l=ge
latin
;glu
=gl
ucos
e;rib
=rib
ose;
xyl=
xylo
se; m
an=
man
nito
l;m
al=
mal
tose
;lac
=la
ctos
e;sa
c=
sucr
ose;
gly
=gl
ycog
en;c
at=
cata
lase
.Ref
=re
fere
nce;
+=
posi
tive
reac
tion;
−=
nega
tive
reac
tion;
num
bers
indi
cate
perc
enta
geof
posi
tive
stra
ins;
w=
wea
kre
actio
n.
Tab
le4.
28.
AP
ICor
yne
data
base
bioc
hem
ical
resu
lts.
224Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #M.vpTuesday, January 20, 2004 4:30:49 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
209
vphi
pae
spy
raa
-gal
b-gu
rb-
gal
Pal
Lap
ADH
ribar
am
anso
rla
ctre
inu
raf
amd
glyg
Hem
Ref
Abio
troph
iade
fect
iva
−−
−+
+−
+−
+−
−−
−−
++
−+
+−
Car
noba
cter
ium
inhi
bens
−+
+−
−−
−−
−−
+−
+−
+w+
+w−
−−
412
Car
noba
cter
ium
pisc
icol
a+
−+
+nt
++
−+
+v
++
v−
+54
2
Ente
roco
ccus
aviu
m+
−+
+−
−−
−−
−+
++
++
++
++
−a
156
Ente
roco
ccus
dura
ns+
−+
++
−+
−−
++
−−
−+
−−
−+
−a
156
Ente
roco
ccus
faec
alis
+−
++
−−
+−
++
+−
++
++
−−
+−
g15
6
Ente
roco
ccus
faec
ium
+−
−+
++
+−
−−
−v
Gra
nulic
atel
laad
iace
ns−
−−
+−
+−
−+
−−
−−
−−
−+
−−
−G
ranu
licat
ella
bala
enop
tera
e−
−+
−−
−−
−+
+−
−−
−−
+−
−−
−47
8,77
3
Gra
nulic
atel
lael
egan
s−
+−
+−
−−
−+
+−
−−
+−
−a
653
Lact
ococ
cus
garv
ieae
+−
++
−−
−−
+−
+−
++
−+
−−
−−
a15
6
Lact
ococ
cus
garv
ieae
+−
++
−−
−−
++
+−
+−
−+
−−
−−
a23
7
Lact
ococ
cus
lact
isss
p.cr
emor
is+
−+
−−
−+
−+
++
−−
−−
+−
−−
−g
156
Lact
ococ
cus
lact
isss
p.la
ctis
+−
++
−−
−−
++
+−
−−
−+
−−
+−
g15
6
Lact
ococ
cus
raffi
nola
ctis
+−
+−
+−
−−
+−
−−
−−
++
−+
+−
g15
6
Stre
ptoc
occu
sin
iae
Fish
stra
ins
−−
++
−+
−+
+7 0
+−
46−
−+
−−
++
ab
183,
223
Stre
ptoc
occu
sin
iae
Hum
anst
rain
s−
−+
−−
−−
++
−−
−+
−−
+−
−+
−a
b22
3
Stre
ptoc
occu
sin
iae
−−
++
−+
−+
++
+−
+−
−+
−−
++
b23
5,84
8
Stre
ptoc
occu
s(d
iffic
ile)a
gala
ctia
e+
−−
−−
−−
++
++
−−
−−
−−
−−
+23
3
Stre
ptoc
occu
s(d
iffic
ile)a
gala
ctia
e+
+−
−−
+−
++
−+s
−−
−−
−−
−−
+77
6
Stre
ptoc
occu
sph
ocae
−−
−−
−−
−+
−−
+−
−−
−−
−−
−−
700
Stre
ptoc
occu
spo
rcin
us+
−+
−−
+−
++
++
−+
++
−−
v−
b17
5
Vago
cocc
usflu
vial
is+v
−+
+−
−−
−v+v
−+
−+
+−
+−
++
+a
629
Vago
cocc
ussa
lmon
inar
um+
++
+−
−−
++
−70
−−
70−
+−
−−
−a
542
VP=
Voge
s-Pr
oska
uer;
hip
=hi
ppur
ate;
aes
=ae
scul
in;p
yra
=py
rrolid
onyl
aryl
amid
ase;
a-g
al=
aga
lact
osid
ase;
bgur
=b
gluc
uron
idas
e;b-
gal=
bga
lact
osid
ase;
Pal=
alka
line
phos
phat
ase;
Lap
=le
ucin
ear
ylam
idas
e;AD
H=
argi
nine
dihy
drol
ase;
rib=
ribos
e;ar
a=
arab
inos
e;m
an=
man
nito
l;so
r=so
rbito
l;la
c=
lact
ose;
tre=
treha
lose
;inu
=in
ulin
;raf
=ra
ffino
se;a
md
=am
ygda
lin;g
lyg
=gl
ycog
en;H
em=
haem
olys
is;R
ef=
refe
renc
e;nt
=no
tte s
ted.
Num
bers
show
perc
enta
geof
posi
tive
stra
ins.
+=
posi
tive
resu
lt;−
=ne
gativ
ere
sult;
w=
wea
kre
actio
n;s
=sl
owre
act io
n.a
=a
haem
olys
is;b
=b
haem
olys
is;g
=no
haem
olys
is.
Tab
le4.
29.
AP
I20
Str
epda
taba
sebi
oche
mic
alre
sults
.
225Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:37:13 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
210
Org
anis
m
Abio
troph
iade
fect
iva
−−
50−
+−
−−
−50
+50
−50
+75
−−
−−
++
−−
+−
−−
v−
−−
*,4
21
Abio
troph
iapa
ra-a
diac
ens
−+
−50
−−
−50
−+
−42
1
Actin
omyc
esm
arim
amm
aliu
m−
−+
−−
v−
−−
+−
−−
++
−+
+−
v−
+−
−−
−−
−−
−−
−37
0
Aero
cocc
usvi
ridan
s−
703
3060
−28
7525
7991
42−
−10
83−
−92
1010
95−
−+
−−
65−
−−
−*
Arca
noba
cter
ium
bern
ardi
ae−
−−
+−
−−
−+
480
Arca
noba
cter
ium
haem
olyt
icum
−v
−−
+−
v−
−48
0
Arca
noba
cter
ium
phoc
ae+
−+w
+v
+−
−+
480
Arca
noba
cter
ium
plur
anim
aliu
m−
−−
+−
−+
−−
−−
−−
+−
+−
v+
−−
−−
−−
−−
−−
−−
−48
0
Arca
noba
cter
ium
pyog
enes
++
−+
++
−v
v48
0
Atop
obac
terp
hoca
e+
−−
−−
++
−−
vv
−−
w−
−w
−−
−+
++
−−
v−
−−
−−
+−
479
Car
noba
cter
ium
alte
rfund
ium
−−
−−
−−
−+
+18
1
Car
noba
cter
ium
dive
rgen
s+
+−
++
++
++
181
Car
noba
cter
ium
fund
itum
−−
−−
−−
++
+18
1
Car
noba
cter
ium
gallin
arum
++
−+
++
++
+18
1
Car
noba
cter
ium
inhi
bens
−+
−+
−−
++
−+w
+−
−−
−−
−−
+−
−+
−−
++
++
−−
−−
412
Car
noba
cter
ium
mob
ile+
−−
−+
−−
−+
181
Car
noba
cter
ium
pisc
icol
a+
−−
+−
++
v−
+w
++
++
−−
−+
ww
+−
w+
−47
9
Ente
roco
ccus
faec
alis
++
−−
−−
++
−+
+−
++
−+
+−
−−
−+
−−
+−
−+
−+
−63
8
Erys
ipel
othr
ixrh
usio
path
iae
42−
+−
−28
−−
−75
−−
−64
−+
8085
−−
−−
−−
−−
−−
−−
−−
*
Fack
lam
iam
iroun
gae
+−
−−
−−w
nr−
−−
+−
−+
−+
−ww
−−
−−
−−
−−
−−
−−
−+
368
Gra
nulic
atel
laad
iace
ns−
+−
30−
−−
−−
−−
−−
+−
7025
−−
−−
+−
−+
−−
−+
−−
−*
,421
Gra
nulic
atel
laba
laen
opte
rae
+−
−−
−−
−−
−−
+−
−−
−+w
+−
−−
++
−−
−−
−−
−−
−+w
478,
479
Gra
nulic
atel
lael
egan
s+
−−
−−
−−
−+
−−
+−
−+
−−v
421
Lact
ococ
cus
garv
ieae
++
−−
−−
++
−−
+−
++
−+
+−
−−
−+
−−
+−
−+
−+
−63
8
Lact
ococ
cus
garv
ieae
++
−−
−−
3575
−50
+−
++
−74
10−
−−
−75
−−
50−
−85
50−
50−
*
Biot
ype
1+
+−
++
−*
,780
Biot
ype
2+
+−
++
+*
,780
Biot
ype
3+
+−
−+
−*
,780
Biot
ype
4+
−−
−−
−*
,780
Biot
ype
5+
−+
−−
−*
,780
11.
11.
21.
31.
41.
51.
61.
71.
81.
91A
1B0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
0A0B
1C1D
1E0C
0D0E
1F0F
Ref
ADH
bglu
bgar
bgur
aga
lpa
lrib
man
sor
lac
trera
fvp
appa
bgal
pyrA
bnag
gta
hip
glyg
pul
mal
mel
mlz
suc
lara
darl
mbd
gta
gbm
ancd
exur
e
Tab
le4.
30.
AP
IRap
idID
32S
trep
data
base
bioc
hem
ical
resu
lts.
226Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:37:15 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
211
Biot
ype
6+
−+
−−
+*
,780
Biot
ype
7+
+−
−+
−*
,780
Biot
ype
8−
+−
−−
−*
,780
Biot
ype
9−
++
−−
−*
,780
Biot
ype
10+
−−
−−
+*
,780
Biot
ype
11+
+−
−+
+*
,780
Biot
ype
12+
−−
++
−*
,780
Biot
ype
13+
−−
−+
−*
,780
Lact
ococ
cus
lact
isla
ctis
++
5−
−−
9526
−50
75−
74+
26−
50−
26−
−+
−−
26−
−85
350
95−
*
Stre
ptoc
occu
sag
alac
tiae
+−
−50
10+
85−
−30
74−
90+
−−
−−
+4
++
−−
+−
−90
26−
−−
*
Stre
ptoc
occu
sag
alac
tiae
+−
−+
31+
+−
−−
++
+−
−−
−+
−+
+−
−+
−−
+−
−−
−g2
42
Stre
ptoc
occu
sag
alac
tiae
(diff
icile
)+
−−
−−
−−
−−
−−
−+
−−
−−
−−
−−
+−
−−
−−
−−
−−
−13
5
Stre
ptoc
occu
sdy
sgal
actia
ess
p.dy
sgal
actia
e+
−−
+−
++
−−
++
−−
+−
−−
−−
++
+−
−+
−−
−−
−40
−72
7
Lanc
efie
ldgr
oup
L=
posi
tive.
Stre
ptex
grou
psA–
G=
nega
tive
Stre
ptoc
occu
sin
iae
–fis
h6 7
+−
83+
−−
+−
−+
−+
−St
rept
ococ
cus
inia
e–
hum
an−
−−
−−
−+
−−
−−
−−
Stre
ptoc
occu
sin
iae
–fis
h+ s
+−
+−
w−
+−
−+
−−
+−
+−
−−
−−
−−
−−
−−
−−
−−
−13
5s
Stre
ptoc
occu
sin
iae
–fis
h+
+−
+−
−+
+−
−+
−−
+−
+−
−−
+−
+−
−+
−−
−−
−−
−13
5#a
Stre
ptoc
occu
sin
iae
–fis
h+
+−
−−
−−
+−
−+
−−
+−
+−
−−
−−
+−
−+
−−
−−
−−
−13
5#b
Stre
ptoc
occu
spa
raub
eris
++
−−
15−
++
++
+1 0
++
−10
−−
50−
−+
−10
+−
−+
3 03 0
−−
*,2
24,
745
Stre
ptoc
occu
sub
eris
++
30+
155
++
++
+20
++
−30
−−
905
−+
−−
+−
−+
5−
−−
*
Vago
cocc
usfe
ssus
−v
v−
−−
−−
−−
−−
−−
−+
−v
−−
−−
−−
−−
−−
−−
v−
369
Vago
cocc
usflu
vial
is−
+−
−−
−+
++
−+
−−
−−
++
+−
−−
+−
−+
−−
−−
v++
−17
7,36
9,62
9
Vago
cocc
uslu
trae
−+
−w−
+−w
+−
+−
+−
−−
−w+
++
−−
−+
−−
+−
−+
−+
+−
477
Vago
cocc
ussa
lmon
inar
um−
++
+−
+−
−−
+−
−+
−−
+−
682,
732
Vago
cocc
ussa
lmon
inar
um−
++
+−
+−
−+
−+
−−
+−
−+
−68
2,73
2
ADH
=ar
gini
nedi
hydr
olas
e;bg
lu=
bgl
ucos
idas
e;bg
ar=
bga
lact
osid
ase;
bgur
=b
gluc
uron
idas
e;a
gal=
aga
lact
osid
ase;
pal=
alka
line
phos
phat
ase;
rib=
ribos
e;m
an=
man
nito
l;so
r=so
rbito
l;la
c=
lact
ose;
tre=
treha
lose
;raf
=ra
ffino
se;
vp=
acet
oin
prod
uctio
n(V
oges
-Pro
skau
er);
appa
=al
anin
e-ph
enyl
alan
ine-
prol
ine
aryl
amid
ase;
bgal
=b
gala
ctos
idas
e;py
rA=
pyro
glut
amic
acid
aryl
amid
ase;
bnag
=N
-ace
tyl-b
gluc
osam
inid
ase;
gta
=gl
ycyl
-tryp
toph
ane
aryl
amid
ase;
hip
=hi
ppur
ate;
glyg
=gl
ycog
en;p
ul=
pullu
lan;
mal
=m
alto
se;m
el=
mel
ibio
se;m
lz=
mel
ezito
se;s
uc=
sucr
ose;
lara
=L-
arab
inos
e;da
rl=
D-a
rabi
nose
;mbd
g=
met
hyl-b
-D-g
luco
pyra
nosi
de;t
ag=
taga
tose
;bm
an=
bm
anno
sida
se;c
dex
=cy
clod
extri
n;ur
e=
urea
se.
Ref
=re
fere
nce;
w=
wea
kre
actio
n;+
=po
sit iv
ere
actio
n;−
=ne
gativ
ere
actio
n;s
=sl
owre
actio
n.N
umbe
rsin
dica
tepe
rcen
tage
ofpo
sitiv
est
rain
s.*
=fro
mAP
Idat
abas
e;#
=AH
LDA
1722
stra
infr o
mim
porte
daq
uariu
mfis
h(a
=re
sults
at37
°Cin
cuba
tion;
b=
resu
ltsfro
m25
°Cin
cuba
tion)
;s=
stra
infro
mQ
ueen
slan
d;g
=st
rain
sfro
mm
ulle
tand
seab
ream
from
Kuw
ait.
227Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:37:16 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
212
Actin
obac
illus
delp
hini
cola
−5
13
−3
−−
−−
50–
2−
−−
−−
−−
−26
3
Actin
omyc
esvi
scos
us−
−−
+−
++
−−
−+
++
+−
−+
−−
+41
Aequ
oriv
itaan
tarc
tica
−+
+−
2011
3
Aero
cocc
usvi
ridan
sva
r.ho
mar
i−
++
+−
+−
−−
−−
+−
−−
−−
−−
−41
Aero
mon
ashy
drop
hila
−5
−4
24
1−
3−
51
−3
−1
−4
−−
254
0.85
NC
TC78
1013
5
Aero
mon
asja
nada
ei−
12
3−
2w
−−
−2
2−
2−
w−
2−
−25
40.
85AH
LDA
1718
135
Aero
mon
assa
lmon
icid
a−
3−
1−
2w
−1
−5
1−
−−
−−
2−
−25
200.
85AH
LDA
1334
135
Aero
mon
asve
roni
isob
ria−
5−
41
3w
−3
−5
11
4−
1−
5−
−25
40.
85AH
LDA
1684
135
Alte
rom
onas
mac
leod
ii−
4−
2−
22
−4
−4
1−
−−
−−
−−
−28
3
Arca
noba
cter
ium
phoc
ae−
42
2−
3−
1v
−5
−3
1−
5−
−−
−63
6
Arth
roba
cter
aure
scen
s−
−−
+−
+−
++
−+
+−
++
++
−+
−41
Arth
roba
cter
nasi
phoc
ae−
+1
−−
+−
−−
−+
−−
−+
−−
−−
MI
MI
MI
CC
UG
4295
318
2
Atop
obac
terp
hoca
e−
+1
1−
−w−
−−
−+
−−
v−
−−
−−
−47
9
Bord
etel
labr
onch
isep
tica
−w
−11
1−
3–4
w−
−−
2w
−2−
−−
−−
−−
−7
40.
85Va
rious
135
Brev
undi
mon
asdi
min
uta
−+
+−
+−
−+
++
−−
−−
−−
−68
5
Brev
undi
mon
asve
sicu
laris
−+
+−
+v
−+
−+
−−
+−
−−
−68
5
Car
noba
cter
ium
alte
rfund
itum
−+
+−
−−
−−
−−
++
−−
−−
+−
−−
250.
85C
CU
G34
643
649
Car
noba
cter
ium
dive
rgen
s−
++
−−
−−
−−
−w
+−
−−
−−
−−
−25
0.85
CC
UG
3009
464
9
Car
noba
cter
ium
dive
rgen
s62
51−
−+
w−
−−
−−
−−
w−
−+
−−
−−
−25
0.85
6251
649
Car
noba
cter
ium
fund
itum
−+
+−
−−
−−
−−
++
−−
−−
+−
−−
250.
85C
CU
G34
644
649
Car
noba
cter
ium
gallin
arum
−+
+−
−−
−−
−−
−w
−−
−−
−−
−−
250.
85C
CU
G30
095
649
Car
noba
cter
ium
inhi
bens
−−
−−
−−
−−
−−
−−
−−
−−
+−
−−
412
Car
noba
cter
ium
inhi
bens
−−
−−
−−
−−
−−
−+
−−
−−
+−
−−
250.
85C
CU
G31
728
649
Car
noba
cter
ium
mob
ile−
++
−−
−−
−−
−−
w−
−−
−−
−−
−25
0.85
CC
UG
3009
664
9
Car
noba
cter
ium
pisc
icol
a−
−−
+−
479
Cel
lulo
phag
aly
tica
−5
34
15
42
31
55
11
−3
25
2−
2212
ASS
89
Chr
yseo
bact
eriu
mba
lust
inum
−5
12
25
53
21
55
−−
−3
32
−−
2512
ASS
89,5
57
Wel
l1
23
45
67
89
1011
1213
1415
1617
1819
20Te
mp
Tim
eIn
ocSt
rain
Ref
Enzy
me
con
alk
C4
C8
C14
aryl
val
cys
trych
rac
pnp
a-g
alb-
gal
b-gl
ucr
a-g
lub-
glu
N-a
bglu
man
fuc
°CH
%N
aCl
Org
anis
mes
tes
tlip
lip
Tab
le4.
31.
AP
IZY
Mda
taba
sere
sults
.
228Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:37:16 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
213
Chr
yseo
bact
eriu
mgl
eum
−5
23
15
52
22
55
−−
−4
54
−−
2212
557
Chr
yseo
bact
eriu
min
dolo
gene
s−
52
31
54
22
25
5−
−−
5−
4−
−22
1255
7
Chr
yseo
bact
eriu
min
dolth
etic
um−
52
31
54
1−
−4
4−
−−
4−
3−
−22
1255
7
Chr
yseo
bact
eriu
mm
enin
gose
ptic
um−
54
43
55
53
44
53
−−
42
52
237
4AS
S89
Chr
yseo
bact
eriu
msc
opht
halm
um−
53
41
54
24
15
5−
−−
31
2−
−55
7
Cor
yneb
acte
rium
phoc
ae−
+−
+−
v−
−−
−+
−−
−−
+−
−−
−61
3
Cor
yneb
acte
rium
test
udin
oris
−−
ww
−+
−−
−−
+w−
−−
−v
+−
−−
180
Cyt
opha
gaal
lerg
inae
−5
24
15
52
13
55
33
25
54
−−
2212
ASS
89
Cyt
opha
gaar
vens
icol
a−
52
31
55
24
14
43
4−
43
52
322
12AS
S89
Cyt
opha
gafe
rmen
tans
−5
13
−5
2−
2−
55
−5
12
55
−−
2212
ASS
89
Cyt
opha
gahu
tchi
nson
ii−
42
41
44
3−
−3
2−
−−
−−
−−
−22
12AS
S89
Cyt
opha
gala
terc
ula
−5
24
15
53
55
44
−−
−−
−−
−−
2212
ASS
89
Edw
ards
iella
tard
a−
5−
−w
51
−−
−4
2−
−−
−−
3−
−25
200.
85AH
LDA
135
135
Empe
doba
cter
brev
is−
53
4−
55
33
25
5−
−−
4−
−−
−22
12AS
S89
Ente
rovi
brio
norv
egic
us−
++
++
+−
−−
−+
+−
nr−
nrnr
nrnr
nr28
482.
85LM
G19
839T
741
Fack
lam
iam
iroun
gae
−−w
−−
−+
ww
−w
ww
−−
−−
−−w
−−
368
Flav
obac
teriu
maq
uatil
e−
42
41
55
2−
−1
2−
−−
5−
−−
−22
12AS
S89
,92
Flav
obac
teriu
maq
uatil
e−
++
+−
++
++
−+
+−
−−
+−
+−
−18
48AT
CC
1194
760
3
Flav
obac
teriu
mbr
anch
ioph
ilum
−5
23
−5
42
2−
44
−−
−1
−−
−−
2512
ASS
92
Flav
obac
teriu
mbr
anch
ioph
ilum
−+
++
−+
++
+v−
++
v−
−+
+−
−−
1848
0.85
603
Flav
obac
teriu
mbr
anch
ioph
ilum
−+
++
−+
++
−−
++
−−
−−
−−
−−
802
Flav
obac
teriu
mco
lum
nare
−5
23
−4
41
31
33
−−
−−
−−
−−
2512
ASS
88,9
2
Flav
obac
teriu
mco
lum
nare
−5
−2
−5
53
−−
54
−−
−3
−1
−−
376
Flav
obac
teriu
mco
lum
nare
−+
++
−+
++
−−
++
−−
−−
−−
−−
1848
NC
MB
2248
603
Flav
obac
teriu
mco
lum
nare
−5
−2
−5
22
−−
11
−−
−−
−−
−−
254
0.85
AHLD
A14
6813
5
Flav
obac
teriu
mfle
vens
e−
51
21
51
1−
−3
31
5−
3−
4−
−22
12AS
S89
,92
Flav
obac
teriu
mfri
gida
rium
−5
−2
−5
51
−1
55
−−
−−
−−
−−
376
Flav
obac
teriu
mhy
datis
−5
24
14
52
−−
45
−−
−4
−2
−−
2212
ASS
89,9
2
Flav
obac
teriu
mhy
datis
−5
−1
−5
53
−−
55
−−
−5
−5
−−
376
Flav
obac
teriu
mjo
hnso
niae
−5
13
15
52
11
45
−3
−4
14
−−
2512
ASS
89,9
2
Flav
obac
teriu
mjo
hnso
niae
−5
34
14
53
12
54
55
15
25
−−
2512
ASS
89
cont
inue
d
229Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:37:17 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
214
Flav
obac
teriu
mjo
hnso
niae
−5
23
15
53
12
54
33
15
25
−−
2512
ASS
89
Flav
obac
teriu
mjo
hnso
niae
−5
−1
−3
11
−−
43
21
−3
32
−−
2537
6
Flav
obac
teriu
mjo
hnso
niae
−+
++
++
++
++
++
++
−+
++
−−
1848
ATC
C17
061
603
Flav
obac
teriu
mpe
ctin
ovor
um−
53
3−
44
2−
−4
4−
21
45
3−
−22
12N
CIM
B90
5992
Flav
obac
teriu
mps
ychr
ophi
lum
−5
23
15
1−
−−
33
−−
−−
−−
−−
2012
NC
IMB
1947
89,9
2
Flav
obac
teriu
mps
ychr
ophi
lum
−+
−+
−+
+−
−−
++
−−
−−
−−
−−
1848
NC
MB
1947
603
Flav
obac
teriu
msa
ccha
roph
ilum
−5
34
−4
42
−−
53
24
−5
−4
−−
2212
ASS
NC
IMB
2072
92
Flav
obac
teriu
msa
ccha
roph
ilum
−5
−1
−2
1−
−−
52
−1
−1
−1
−−
NC
IMB
2072
376
Flav
obac
teriu
msu
ccin
ican
s−
53
3−
44
21
−5
5−
−−
42
4−
−22
12AS
SN
CIM
B22
7792
Flex
ibac
tera
ggre
gans
−5
34
15
43
3−
55
13
−4
25
33
2212
ASS
NC
MB
1443
89
Flex
ibac
tera
uran
tiacu
s*−
52
41
55
3−
−5
3−
1−
53
3−
−22
12AS
SN
CM
B13
8289
Flex
ibac
tera
uran
tiacu
s*−
52
41
55
3−
−5
4−
−−
53
4−
−22
12AS
SN
CM
B14
5589
Flex
ibac
terc
anad
ensi
s−
52
31
55
34
15
54
1−
53
4−
322
12AS
SAT
CC
2959
189
Flex
ibac
terf
lexi
lis−
53
4−
54
31
23
1−
−−
3−
−−
−22
12AS
SN
CM
B13
7789
Flex
ibac
terf
lexi
lis−
+−
+−
++
+−
++
+−
−−
++
−−
−AT
CC
2307
960
3
Flex
ibac
terl
itora
lis−
52
41
55
35
15
5−
−−
−−
−−
−22
12AS
SN
CM
B13
6689
Flex
ibac
terp
olym
orph
us−
52
31
55
34
12
2−
−−
2−
−−
−30
12AS
SAT
CC
2782
089
Flex
ibac
terr
oseo
lus
−4
23
13
32
13
22
−−
−−
−−
−−
2512
ASS
NC
MB
1433
89
Flex
ibac
terr
uber
−5
34
23
32
13
33
−−
−−
−−
−−
2512
ASS
NC
MB
1436
89
Flex
ibac
ters
anct
i−
5−
2−
41
−4
−4
54
4−
43
4−
222
12AS
SN
CM
B13
7989
Flex
ibac
tert
ract
uosu
s−
52
31
54
31
45
4−
−−
51
−−
−22
12AS
SN
CM
B14
0889
Gra
nulic
atel
laba
laen
opte
rae
−−
−+
−+
−−
−−
−−
−−
−−
−+
−−
478
Haf
nia
alve
i−
5−
w1
32
−w
−4
1−
2−
21
w−
−25
40.
85AH
LDA
1729
135
List
onel
laan
guilla
rum
−+
++
−+
++
−−
++
−−
−−
−+
−−
2224
ATC
C14
181
506
List
onel
laan
guilla
rum
−4
−2
−w1
−−
−−
−−
−−
−−
−−
−−
2524
2.85
NC
MB
681
List
onel
laan
guilla
rum
−3
12
22
11
−−
−w
−w
−−
2−
−−
2524
2.85
AHLD
A17
3013
5
List
onel
lape
lagi
aII
−+
++
−+
−−
+−
++
−−
−−
−−
−−
2224
NC
MB
2253
506
Man
nhei
mia
haem
olyt
ica
−5
11−
w−
1–3
0–1
−−
−5
1–2
−1–
2−
−−
−−
0–2
374
0.85
Vario
us13
5
Wel
l1
23
45
67
89
1011
1213
1415
1617
1819
20Te
mp
Tim
eIn
ocSt
rain
Ref
Enzy
me
con
alk
C4
C8
C14
aryl
val
cys
trych
rac
pnp
a-g
alb-
gal
b-gl
ucr
a-g
lub-
glu
N-a
bglu
man
fuc
°CH
%N
aCl
Org
anis
mes
tes
tlip
lip
Tab
le4.
31.
Con
tinue
d.
230Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:37:18 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
215
Mar
inila
bilia
salm
onic
olor
−5
12
−−
−−
−4
21
3−
15
55
−−
2512
ASS
NC
MB
2216
89
Mor
itella
mar
ina
−+
++
−+
−−
−−
++
−−
−−
−−
−−
2224
NC
MB
1144
506
Mor
itella
visc
osa
−+
++
++
−−
−−
++
−−
−−
−−
−−
2224
NC
MB
1358
450
6
Myr
oide
sod
orat
imim
us−
++
+−w
3−w
−w−
−w+
+−
−−
−−
−−
−N
CTC
1118
077
4
Myr
oide
sod
orat
us−
++
+−w
1−w
−w−
−w+
+−
−−
−−
−−
−N
CTC
1103
677
4
Myr
oide
sod
orat
us−
52
3−
2−
−1
−5
5−
−−
−−
−−
−30
12N
CTC
1103
689
Past
eure
llam
ulto
cida
−5
1−w
1–2
−2
0−w
−−
−5
1−
−−
−−
−−
−37
40.
85Va
rious
135
Past
eure
llask
yens
is−
51
2−
2−
−−
−5
5−
−−
−−
−−
−25
4d1.
5N
CTC
1320
410
0
Pedo
bact
erhe
parin
us−
++
+−
+−
−−
−+
++
+−
++
++
−28
40.
85N
CIB
9290
728
Pedo
bact
erhe
parin
us−
52
4−
42
1−
−4
2−
−−
1−
41
−22
12N
CIB
9290
89
Pedo
bact
erpi
sciu
m−
++
++
++
++
++
−+
−+
++
+−
284
0.85
728
Phoc
oeno
bact
erut
eri
−5
15
−1
−−
−−
55
−−
−−
−−
−−
266
Phot
obac
teriu
mda
mse
lae
−+
++
−+
−−
−−
++
−−
−−
−+
−−
2224
NC
MB
2184
506
Phot
obac
teriu
mda
mse
lae
−5
−2
−4
11
−w
51
−1
−−
−4
−−
2520
2.85
AHLD
A16
8313
5
Phot
obac
teriu
mpi
scic
ida
−+
++
−+
−−
−−
++
−−
−−
−+
−−
855
Ples
iom
onas
shig
ello
ides
−4
−1
w4
1−
−−
51
−w
−2
−5
−−
254
AHLD
A19
213
5
Pseu
doal
tero
mon
asan
tarc
tica
−+
++
−+
++
++
++
−−
−+
−+
+−
115
Pseu
doal
tero
mon
asci
trea
−+
++
−−
−w
−−
285
Pseu
doal
tero
mon
asru
bra
−4
−2
12
1−
41
42
−−
−−
−2
−−
283
Pseu
dom
onas
angu
illise
ptic
a−
+−
++
+−
−−
−+
+−
−−
−−
−−
−N
CIM
B19
49T
96
Pseu
dom
onas
plec
oglo
ssic
ida
−+
−+
−+
+−
+−
++
−−
−−
−−
−−
582
Ren
ibac
teriu
msa
lmon
inar
um−
+−
+−
+−
−+
−+
+−
−−
+−
−+
−41
Shew
anel
laal
gae
−+
−+
−+
13−
++
++
−−
−−
−+
−−
433
Shew
anel
laba
ltica
−−
−−
−−
−−
−w
−−
−−
−−
−+
−−
851
Shew
anel
lapu
trefa
cien
s−
+−
+−
+67
−78
89+
+−
−−
−−
+−
−43
3
Sphi
ngob
acte
rium
mul
tivor
um−
+−
+−
++
−−
−+
−−
+−
+−
+−
−36
4
Sphi
ngob
acte
rium
mul
tivor
um−
54
41
51
1−
−5
52
4−
43
53
−37
4AS
SN
CTC
1134
389
Sphi
ngob
acte
rium
spiri
tivor
um−
+−
−−
−−
−+
−+
1−
1−
+−
+−
−N
CTC
1138
636
5
Sphi
ngob
acte
rium
spiri
tivor
um−
52
4−
51
15
25
52
41
32
53
430
12AS
SN
CTC
1138
689
Stap
hylo
cocc
uslu
trae
−5
55
−−
−−
−−
55
−1
−−
−−
−1
264
Tena
ciba
culu
mm
ariti
mum
−5
13–
42–
55
55
55
55
−−
−0–
1−
−−
−18
7d2.
8560
5
cont
inue
d
231Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:37:19 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
216
Tena
ciba
culu
mm
ariti
mum
−5
34
15
53
12
55
−−
−−
−−
−−
2512
2.85
89
Tena
ciba
culu
mm
ariti
mum
−5
23
14
52
31
55
−−
−−
−−
−−
2212
0.85
ATC
C43
398
743
Tena
ciba
culu
mov
olyt
icum
−+
++
−+
+−
−−
++
−−
−−
−−
−−
324
Vago
cocc
usfe
ssus
−v
+w+w
−+
−v
−+
−−
−v
−−
v−
−−
369
Vago
cocc
uslu
trae
−−w
−+
−+
−−
−+
+−
+−w
−+
++
−−
477
Vibr
ioag
ariv
oran
s−
1w
1−
1−
−−
−−
w−
2−
−−
−−
−25
242.
85AH
LDA
1732
135
Vibr
ioal
gino
lytic
us−
5−
2w
41
−2
−5
1−
−−
−−
1−
−25
413
5
Vibr
iobr
asilie
nsis
−+
++
++
+−
−−
−−
−+
−−
−−
−25
241.
5LM
G20
546T
740
Vibr
ioca
lvie
nsis
−+
++
++
−−
−−
++
−+
−−
DSM
1434
7T21
6
Vibr
ioch
oler
aeno
n-01
−3
−3
12
1w
−−
11
−2
−1
−4
−−
2524
0.85
AHLD
A99
613
5
Vibr
iodi
abol
icus
−3
23
13
−−
22
3−
−−
−−
−−
−−
635
Vibr
iofis
cher
i−
+−
+−
+−
−−
−+
+−
−−
−−
+−
−22
24N
CM
B12
8150
6
Vibr
iofu
rnis
sii
−3
−2
14
22
−−
11
−1
−w
−4
−−
2524
0.85
ATC
C11
218
135
Vibr
ioha
liotic
oli
−1
w1
−1
w−
−−
−1
−−
−−
−3
−−
2524
2.85
AHLD
A17
3413
5
Vibr
ioha
rvey
i−
32
2−
1w
−−
15
1−
−−
−−
1−
−25
40.
85AT
CC
3508
413
5
Vibr
ioha
rvey
i−
42
2−
1w
−−
23
1−
−−
−−
2−
−25
42.
85AT
CC
3508
413
5
Vibr
ioha
rvey
i−
++
+−
−−
−−
−+
+−
−−
−−
−−
−AT
CC
3508
484
7
Vibr
iolo
gei
−+
++
−+
−−
−−
++
−−
−−
−−
−−
2224
ATC
C15
382
506
Vibr
iolo
gei
−+
++
−+
−−
−−
++
++
−−
−−
−−
2224
NC
MB
1143
506
Vibr
iolo
gei
−+
++
−+
−−
−−
++
−−
−−
−−
−−
2224
ATC
C29
985
506
Vibr
iom
edite
rrane
i−
52
22
1−
−−
−4
2−
−−
−−
2−
−25
242.
85AH
LDA
1733
135
Vibr
iom
imic
us−
5−
2−
41
−−
w5
1−
1−
−−
3−
−25
42.
85AH
LDA
1654
135
Vibr
iom
ytili
−−
23
13
−−
1−
2−
−−
−−
−2
−−
CEC
T63
263
5
Vibr
iona
trieg
ens
−+
++
−−
−−
−−
++
−−
−−
−−
−−
2224
NC
MB
1900
506
Vibr
ione
ptun
ius
−+
++
++
+−
+−
−−
−−
−−
−+
−22
241.
5LM
G20
536T
740
Vibr
ione
reis
−−
11
−2
−−
−−
−−
−−
−−
−−
−−
LMG
3895
635
Vibr
ioor
dalii
−+
++
−+
−−
−−
++
−−
−−
−+
−−
2224
NC
MB
2167
506
Wel
l1
23
45
67
89
1011
1213
1415
1617
1819
20Te
mp
Tim
eIn
ocSt
rain
Ref
Enzy
me
con
alk
C4
C8
C14
aryl
val
cys
trych
rac
pnp
a-g
alb-
gal
b-gl
ucr
a-g
lub-
glu
N-a
bglu
man
fuc
°CH
%N
aCl
Org
anis
mes
tes
tlip
lip
Tab
le4.
31.
Con
tinue
d.
232Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:37:19 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
217
Vibr
ioor
dalii
−5
−w
−4
w−
−−
ww
−−
−−
−−
−−
254
2.85
DF
3K42
8
Vibr
ioor
ient
alis
−+
++
++
+−
−−
++
−−
−−
−+
−−
2224
NC
MB
2195
506
Vibr
iopa
cini
i−
++
+−
++
v−
w+
+−
66−
+−
−−
−28
41.
5LM
G19
99T
306
Vibr
iopa
raha
emol
ytic
us−
5−
3w
53
−1
−5
1−
−−
−−
−−
−25
202.
85AT
CC
4399
613
5
Vibr
iopa
raha
emol
ytic
us−
+−
−−
−−
−+
−+
+−
−−
−−
+−
−AT
CC
1780
249
9
Vibr
iopa
raha
emol
ytic
us−
+−
+−
−−
−+
−+
+−
−−
−−
+−
−AT
CC
2796
949
9
Vibr
iopr
oteo
lytic
us−
21
2−
2−
−−
−−
w−
−−
−−
w−
−25
242.
85AH
LDA
1735
135
Vibr
ioro
tifer
ianu
s−
++
+−
++
−+
++
+−
−−
−−
−−
−25
242.
85LM
G21
460T
305
Vibr
iosa
lmon
icid
a−
++v
+−
−−
−−
−+
+−
−−
−−
−−
−22
24N
CM
B22
6250
6
Vibr
iosp
lend
idus
I−
++
++
+−
−+
−+
+−
−−
−−
+−
−22
24N
CM
B1T
506
Vibr
iosp
lend
idus
I−
++
++
+−v
−+
−+
v−
+v−
+−
+−
−28
1
Vibr
iosp
lend
idus
II−
++
+−
++
−+
−+
+−
−−
−−
−−
−22
24N
CM
B22
5150
6
Vibr
iota
petis
−+
++
−+
+−
−−
+−
−−
−−
−+
−−
2218
2.85
B109
0T58
7
Vibr
iotu
bias
hii
−2
13
12
−−
−−
−−
−−
−−
−−
−−
LMG
1093
663
5
Vibr
iotu
bias
hii
−+
++
−+
++
−−
−+
−−
−−
−+
−−
2224
NC
MB
1340
T50
6
Vibr
iotu
bias
hii
−+
++
−+
++
−−
−+
−−
−−
−+
−−
2224
NC
MB
1340
506
Vibr
iovu
lnifi
cus
biov
arI
−5
w1
−1
w−
−−
w1
−−
−−
−−
−−
254
0.85
AHLD
A17
1613
5
Vibr
iovu
lnifi
cus
biov
arI
−5
11
−1
ww
−−
−w
−−
−−
−−
−−
254
2.85
AHLD
A17
1613
5
Vibr
iow
odan
is−
+83
+−
+86
−−
−69
+−
−−
−−
77−
−22
24N
CIM
B13
582
506
Vibr
ioxu
ii−
++
++
+−
−−
−+
+−
−−
−−
−−
2524
1.5
LMG
2134
6T74
0
Yers
inia
ruck
eri
−1
−w
w4
1−
−−
21
−2
−1
−3
−−
2520
AHLD
A13
1313
5
Zobe
lliaga
lact
aniv
oran
s−
33
13
307d
2.5
61
Zobe
lliaul
igin
osa
−1
13
130
7d2.
561
*Is
olat
esN
CM
B13
82an
dN
CM
B14
55pr
evio
usly
iden
tifie
das
Flex
ibac
tera
uran
tiacu
sar
eno
wth
ough
tto
beFl
avob
acte
rium
john
soni
ae.
1=
cont
rolw
ell;
2=
2-na
phth
yl-p
hosp
hate
;3=
2-na
phth
yl-b
utyr
ate;
4=
2-na
phth
yl-c
apry
late
;5=
2-na
phth
yl-m
yris
tate
;6=
L-le
ucyl
-2-n
apht
hyla
mid
e;7
=L-
valy
l-2-n
apht
hyla
mid
e;8
=L-
cyst
yl-2
-nap
hthy
lam
ide;
9=
N-b
enzo
yl-D
L-ar
gini
ne-2
-nap
hthy
lam
ide;
10=
N-g
luta
ryl-p
heny
lala
nine
-2-n
apht
hyla
mid
e;11
=2 -
naph
thyl
-pho
spha
te;1
2=
naph
thol
-AS-
BI-p
hosp
hat e
;13
=6-
Br-2
-nap
hthy
l-a-D
-gal
acto
pyra
nosi
de;1
4=
2-na
phth
yl-b
-D-g
alac
topy
rano
side
;15
=nap
htho
l-AS-
BI- b
-D-g
lucu
roni
de;1
6=
2-na
phth
yl-a
-D-g
luco
pyra
nosi
de;1
7=
6-Br
-2-n
apht
yl-b
-D-g
luco
pyra
nosi
de;1
8=
1-na
phth
yl-N
-ace
tyl-b
-D-g
luco
sam
inid
e;19
=6-
Br-2
-nap
hthy
l-a-D
-man
nopy
rano
side
;20
=2-
naph
thyl
-a-L
-fuco
pyra
nosi
de.
Tem
p=
tem
pera
ture
ofin
cuba
tion;
Tim
e=
time
ofin
cuba
tion;
H=
hour
s;d
=da
ys;I
noc
=in
ocul
um;%
NaC
l ind
icat
esfin
alN
aClc
once
ntra
tion
inth
ein
ocul
um.M
I=M
anuf
actu
rer’s
Inst
ruct
ions
;nr=
notr
epor
ted;
Ref
=re
fere
nce
num
ber.
Ref
603
clas
sifie
da
reac
tion
of2
orle
ssas
nega
t ive.
233Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #M.vpTuesday, January 20, 2004 4:31:33 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
218
Actinobacillus scotiae NCTC 12922 265
Actinomyces marimammalium CCUG 41710T = CIP 106509T 370
Aequorivita antarctica ACAM 640T, DSM 14231T 113
Aequorivita crocea ACAM 642T, DSM 14239T 113
Aequorivita lipolytica ACAM 641T, DSM 14236T 113
Aequorivita sublithincola ACAM 643T, DSM 14238T 113
Aeromonas allosaccharophilaHG15
CECT 4199 427
Aeromonas bestiarumHG2
CDC 9533-76 1,21,427
Aeromonas caviaeHG4
ATCC 15468 427,21
Aeromonas encheleiaHG16
CECT 4342 = LMG 16330 241,379,427
Aeromonas eucrenophilaHG6
ATCC 23309, CDC 0859-83 427,21
Aeromonas eucrenophila LMG 3774 = NCMB 74 379
Aeromonas eucrenophila LMG 13057 379
Aeromonas hydrophila dhakensis P21T = LMG 19562T = CCUG 45377T 383
Aeromonas hydrophila hydrophila ATCC 7966T = LMG 2844T 383
Aeromonas hydrophilaHG1
ATCC 7966T, CDC 9079-79 21,427,818
Aeromonas hydrophilaHG3
CDC 0434-84 427
A. hydrophila anaerogenes = A. caviae ATCC 15468 818
A. hydrophila anaerogenes ATCC 15467 Taxonomy 2000
Aeromonas janadaeiHG 9
CDC 0787-80, ATCC 49568 143,427
Aeromonas mediaHG 5B
CDC 9072-83, CDC 0435-84 427,21
Aeromonas mediaHG 5A
CDC 9072-83, CDC 0862-83 427,21
Aeromonas popoffii LMG 17541T 380
Aeromonas salmonicida achromogenes NCMB 1110 450,475
Aeromonas salmonicida pectinolytica DSM 12609T = 34 mel 615
A. salmonicida salmonicida ATCC 14174 450,818
A. salmonicida salmonicida SVLT -1, -2, -5, -6, Non-pigmented strains 450
A. salmonicida salmonicida NCMB 1102 475
Aeromonas salmonicidaHG3
CDC 0434-84 21
Aeromonas schubertiiHG12
CDC 2446-81, ATCC 43700, formerly Enteric group 501 348,427
Aeromonas sobria HG7 CIP 7433, CDC 9538-76 21,427,818
Aeromonas veronii sobriaHG8/10
CDC 0437-84 427
Aeromonas spp.HG11
CDC 1306-83 427
Aeromonas trota ATCC 49657T = LMG 12223T HG 14 142,427
Aeromonas veronii ATCC 35604T = CDC 1169-83 21,347
Aeromonas veronii HG 8, CDC-0437-84 21
Alteromonas aurantia ATCC 33046, NCMB 2052 286
Alteromonas citrea NCMB 1889 285
Alteromonas rubra NCMB 1890 283
Arcanobacterium phocae DSM 10002T, M1590/94/3T 636
Arcanobacterium pluranimalium CCUG 42575T = CIP 106442 480
Arthrobacter rhombi CCUG 38813T 600
Atopobacter phocae CCUG 42358T = CIP 106392 479
Bordetella bronchiseptica ATCC 19395, ATCC 4617, NCTC 8344 102
Brevundimonas diminuta ATCC 11568, LMG 2089 = CCUG 1427 685
Brevundimonas vesicularis ATCC 11426 = CCUG 2032 = LMG 2350 685
Organism Strain number Reference number
Table 4.32. Strains quoted in references.
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219
Brucella maris biovar I NCTC 12890, 2/94 404
Brucella maris biovar II NCTC 12891, 1/94 404
Brucella maris biovar III 247,404
Carnobacterium inhibens CCUG 31728T, strain K1 411,412
Carnobacterium piscicola ATCC 35586 73,353,682
Chromobacterium violaceum 482
Corynebacterium aquaticum RB 968 BA 73,133
Edwardsiella ictaluri CDC 1976-78, ACC 33202 334
Edwardsiella tarda ATCC 15947T 374,640
Enterococcus faecalis ATCC 19433 638
Enterovibrio norvegicus LMG 19839T, CAIM 430T 741
Flavobacterium aquatile NCIB 8694T, LMG 4008T 89,92
Flavobacterium branchiophilum ATCC 35035, BGD-7721 802
Flavobacterium frigidarium ATCC 700810 = NCIMB 13737 376
Flavobacterium gillisiae ACAM 601T 533
Flavobacterium hibernum ATCC 51468 = ACAM 376T 532
Flavobacterium hydatis (C. aquatilis) ATCC 29551 720
Flavobacterium tegetincola ACAM 602T 533
Flexibacter polymorphus ATCC 27820 494
Granulicatella balaenopterae CCUG 37380T, M1975/96/1 478
Iodobacter fluviatilis NCTC 11159T 502
Janthinobacterium lividum NCIMB 9230, NCIMB 9414, DSM 1522 496
Lactococcus garvieae NCDO 2155 224,638
Lactococcus garvieae ATCC 49156T 236,464,682
Lactococcus lactis ATCC 19435 731
Lactococcus piscium NCFB 2778 835
Listonella anguillarum ATCC 14181 506
Listonella anguillarum NCMB 6 = ATCC 19264 (Bagge and Bagge strain) 341
Listonella anguillarum NCMB 407 = PL 1 341
Listonella anguillarum NCMB 571 (Hoshina) 341
Listonella anguillarum NCMB 828 = ATCC 14181 (strain 4063, Smith) 341
Listonella pelagia I (said to be V. natriegens) NCMB 1900T 506
Listonella pelagia II NCMB 2253 506
Mesophilobacter marinus IAM 13185 583
Moritella marina NCMB 1144T = ATCC 15381 82,506,766
Moritella viscosa NCIMB 13584T = NVI 88/478T 82,506
Mycoplasma crocodyli ATCC 51981 441
Mycoplasma mobile 163K 439
Myroides odoratimimus NCTC 11180, LMG 4029 565
Myroides odoratus NCTC 11036, LMG 1233 565
Nocardia seriolae JCM 3360 455
Nocardia spp., Australian strain 98/1655 117
Pantoea agglomerans ATCC 27155T, NCTC 9381 249,291
Pantoea agglomerans ATCC 27155T, ATCC 12287 325
Pantoea dispersa ATCC 14589T 291
Pasteurella skyensis NCTC 13204T, NCIMB 13593T 100
Phocoenobacter uteri NCTC 12872 266
Photobacterium angustum NCIMB 1895 745
Photobacterium damselae ssp. damselae ATCC 33539 = NCIMB 2184T 289,504,506,705,745
Photobacterium damselae ssp. damselae ATCC 35083 268,745
Photobacterium damselae ssp. piscicida ATCC 17911 & NCIMB 2058 289,745
Photobacterium damselae ssp. piscicida 751
Photobacterium damselae ssp. piscicida ATCC 29690, ATCC 17911 518
Photobacterium fischeri NCMB 1281T = ATCC 7744 506
Photobacterium fischeri ATCC 25918 745
Photobacterium fischeri ATCC 7744 340,818
Photobacterium iliopiscarium ATCC 51760 599,767
Organism Strain number Reference number
Table 4.32. Continued.
continued
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220
Photobacterium leiognathi LMG 4228, NCIMB 1895 745
Photobacterium logei ATCC 15382 = NCMB 1143 = PS 207 506
Photobacterium logei NCMB 1143 506
Photobacterium logei ATCC 29985T 506
Photobacterium logei ATCC 15382 82
Photobacterium logei NCIMB 2252, ATCC 29985 506
Providencia friedericiana DSM 2620 559
Pseudoalteromonas antarctica CECT 4664T, NF3 115
Pseudoalteromonas citrea ATCC 29719T, NCMB 1889 285
Pseudoalteromonas elyakovii ATCC 700519T, KMM 162T 679
Raoultella planticola ATCC 33531 228
Raoultella terrigena ATCC 33257 228
Renibacterium salmoninarum ATCC 33209 671
Serratia fonticola ATCC 29844 290
Shewanella algae LMG 2265, IAM 14159 433,792,851
Shewanella baltica NCTC 10735, DSM 9439, CECT 323, IAM 1477, LMG 2250 851
Shewanella colwelliana ATCC 39565 815
Shewanella frigidimarina ACAM 591T 112
Shewanella gelidimarina ACAM 456T 112
Shewanella (Alteromonas) hanedai ATCC 33224 409
Shewanella japonica KMM 3299, LMG 19691 = CIP 106860 397
Shewanella oneidensis ATCC 700550T 782
Shewanella pealeana ANG-SQ1T 492
Shewanella putrefaciens ATCC 8071 433,792
Shewanella woodyi ATCC 51908T, MS32 112
Sphingobacterium spiritivorum NCTC 11386 365
Staphylococcus delphini DSM 20771T 778
Staphylococcus lutrae DSM 10244, M340/94/1 264
Streptococcus (difficile) agalactiae ND 2-22, CIP 103768 233
Streptococcus (difficile) agalactiae LMG 15977 776
Streptococcus iniae ND 2-16, CIP 103769 233
Streptococcus iniae ATCC 29177 626
Streptococcus parauberis NCDO 2020 224
Streptococcus phocae NCTC 12719, 8399 HI 700
Streptococcus uberis NCDO 2038 224
Tenacibaculum (Flexibacter) maritimum NCMB 2154T 801
Tenacibaculum (Flexibacter) ovolyticum NCIMB 13127 = EKD002 324
Vagococcus fessus CCUG 41755 369
Vagococcus fluvialis NCDO 2497, NCFB 2497 177,629
Vagococcus lutrae CCUG 39187 477
Vagococcus salmoninarum NCFB 2777 682,807
Vibrio aerogenes ATCC 700797 = CCRC 17041, FG1 692
Vibrio aestuarianus ATCC 35048, LMG 7909 149,747
Vibrio agarivorans CECT 5084, CECT 5085T = DSM 13756 514
Vibrio agarivorans AHLDA 1732 135
Vibrio brasiliensis LMG 20546T 740
Vibrio calviensis RE35/F12T = CIP 107077T = DSM 14347T 216
Vibrio (carchariae) harveyi ATCC 35084T 11,135
Vibrio cincinnatiensis ATCC 35912 120
Vibrio diabolicus CNCM I-1629 = HE800 635
Vibrio diazotrophicus ATCC 33466 319
Vibrio fluvialis ATCC 49515, NCDO 2497 = NCFB 2497 177,732
Vibrio fluvialis NCTC 11327 123,485
Vibrio furnissii ATCC 35016 = CDC B3215 123
Vibrio furnissii ATCC 11218 135
Vibrio furnissii Group F 687
Organism Strain number Reference number
Table 4.32. Continued.
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221
Vibrio gazogenes ATCC 29988 818
Vibrio halioticoli IAM 14596T 678
Vibrio harveyi ATCC 14126T 11,818
Vibrio hollisae KUMA871, ATCC 33564T 346,580
Vibrio lentus CECT 5110T = DSM 13757 = 40MA 513
Vibrio mediterranei CECT 621T, LMG11258 149,631
Vibrio metschnikovii NCTC 8563 819
Vibrio metschnikovii NCTC 8443 818
Vibrio mimicus ATCC 33653T 210,507,818
Vibrio mytili CECT 632T 635
Vibrio natriegens ATCC 14048 44
Vibrio navarrensis CIP 1397-6 767,768
Vibrio neptunius LMG 20536T 740
Vibrio nereis LMG 3895T 635
Vibrio ordalii NCMB 2167T, ATCC 33509 = DF3K = Dom F3 506,680
Vibrio orientalis NCMB 2195T 506
Vibrio pacinii LMG 1999T 306
Vibrio pectenicida CIP 105190T, A365 470
Vibrio penaeicida JCM 9123, KH-1, IFO 15640 388
Vibrio proteolyticus AHLDA 1735 135
Vibrio proteolyticus CW8T2 788
Vibrio rotiferianus LMG 21460T 305
Vibrio salmonicida NCMB 2262T 232,506
Vibrio salmonicida NCMB 2245 506
Vibrio salmonicida 90/1667-10c 506
Vibrio scophthalmi A089, CECT 4638T 254,149
Vibrio shilonii (possibily V. mediterranei) ATCC BAA-91T = DSM 13774 = AK-1 59,458
Vibrio splendidus I NCMB 1T (= ATCC 33125T) 149,506
Vibrio splendidus I ATCC 33125 281,620
Vibrio splendidus II NCMB 2251 149,506
Vibrio tapetis B1090T, CECT 4600 108,587
Vibrio tubiashii NCMB 1340T 506
Vibrio tubiashii ATCC 19109T 321
Vibrio tubiashii LMG 10936T 635
Vibrio vulnificus 149
V. vulnificusbiovar I non-serovar E f
ATCC 27562 26,746
V. vulnificusbiovar 2, serovar E a
ATCC 33187 98
V. vulnificusbiovar 2, serovar E b
ATCC 33149 98
V. vulnificusbiovar 2, serovar 04 b
ATCC 33149 356
V. vulnificusbiovar 2, serovar E c
NCIMB 2138 98
V. vulnificusbiovar 2 serovar E d
NCIMB 2137 98
V. vulnificusbiovar 2, serovar E e
NCIMB 2136 98
V. vulnificusbiovar 2, serovar E g
Taiwanese strains 26
V. vulnificusbiovar 2
ATCC 33148 746
Vibrio wodanis NCIMB 13582 = NVI 88/441T 506
Vibrio xuii LMG 21346T
Yersinia rohdei ATCC 43380, CDC 3022-85 12
Yersinia aldovae ATCC 35236, CDC 669-83 85
Organism Strain number Reference number
Table 4.32. Continued.
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5Technical Methods
5.1 Total Bacterial Count (TBC)
Diagnostic, research, or industry-based laborato-ries often monitor pond or tank water for bacte-rial load as an indicator of water quality, andhence as a disease indicator. This is done by atest known as the TBC.
There are a number of methods for TBCsand these may be found in most microbiologytextbooks. A basic method for TBCs is describedhere.
Generally, TBCs are performed using a goodgeneral-purpose medium that will support thegrowth of the majority of the organisms that areexpected to be cultured. A selective medium forVibrio species such as TCBS is often used as aculture medium for TBCs. However, this mediumhas a tendency to produce a lower count thanan equivalent sample inoculated to MSA-B plate(or BA in the case of freshwater samples) evenwhen the majority of bacteria cultured are Vibriospp. Therefore, it is not the best primary culturemedium.
The time between collection of the watersample and testing is important. Bacteria in thewater sample will multiply within a few hours atroom temperature. Thus, if there is a delay of morethan 1–2 h between collection and testing, aninaccurate assessment of the bacterial load of theoriginal water sample will be obtained. The watersample must be stored at 4°C either during trans-port or while waiting to be tested in the laboratory.There is no effect on bacterial count due to thesize of the collection container (Simon andOppenheimer, 1968).
There is no predetermined optimal bacterialload of pond or tank water, and this will varyanyway according to the number of fish in thewater, that is, the stocking density. It is suggestedthat laboratories regularly monitor a particulartank or set of tanks and keep a record of theirresults. The bacterial load or TBC is then relatedback to the health of the fish. If a daily log or graphis kept of the TBC against the health of the fish,then the laboratory can eventually determine theoptimal bacterial load for healthy fish, as opposedto the load seen when the fish appear diseased.
Suggested sites for monitoring bacterial loadare the intake water supply, the filtered water sup-ply, fish tanks and the feed supply. The sedimentat the bottom of the tank will have a greaterbacterial count than the water body, particularlywhen there are dead or diseased larvae or fish.
Research in an oyster hatchery indicated thatfour main genera were involved in the bacterialload; these were Vibrio species, Alteromonas spe-cies, Pseudomonas species and Flavobacteriumspecies. Vibrio species were the predominantorganism. Virulence testing showed that a third ofthese Vibrio species, which included Listonellaanguillarum, and two of the ten Alteromonasspecies were pathogenic. The isolates were notidentified to species level. Infection was not seen inthe larvae when they were dosed with the non-pathogenic bacteria. However, fatal infection wasproduced when the bacterial load of the patho-genic bacteria was 1 × 107–5 × 107. At a lowerdose of 1 × 105–5 × 105, infection still occurred,but took 2–3 days longer to develop and forclinical signs to be seen. Thus, infection rates are
©N.B. Buller 2004. Bacteria from Fish and Other Aquatic Animals:222 a Practical Identification Manual (N.B. Buller)
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related to the bacterial load of pathogenic bacteria(Garland et al., 1983).
Bacteria are part of the diet of marine filterfeeders. In another study on healthy oysters(Crassostrea gigas) and horse mussel (Modiolusmodiolus), the total bacterial load of normal florain the haemolymph and soft tissue was 2.6 × 104
colonies/ml, and 2.9 × 104 colonies/g, respec-tively. The predominant flora and percentage ofthe total flora were Pseudomonas spp. (61.3%),Vibrio spp. (27%) and Aeromonas spp. (11.7%) inthe haemolymph, with the predominant bacteriain the soft tissues being Vibrio spp. (38.5%),Pseudomonas spp. (33%) and Aeromonas spp.(28.5%). When the oysters and horse musselwere challenged in virulence studies using Vibriosalmonicida, the TBC increased to 105 colonies/mlin the haemolymph and 6 × 107 colonies/g in thesoft tissue (Olafsen et al., 1993).
Table 5.1 indicates the bacterial countsobtained in both studies.
The microflora in the intestines of healthyjack mackerel (Trachurus japonicus) was found tobe 4.6 × 106 colonies/g and consisted of Vibriospecies. The total bacterial load in the stomachwas 2.6 × 105 colonies/g, with the predominantflora being Vibrio spp., Achromobacter spp.,with smaller counts of Pseudomonas spp.,Flavobacterium spp., Corynebacterium spp.,Bacillus spp. and Sarcina spp. (Aiso et al., 1968).
Equipment
A pipette or pipettes capable of measuring 100 mlto 1000 ml1.5 ml microfuge tubes
An agar medium that supports the growth ofthe target bacteria, such as BA for freshwater orMSA-B, or MA 2216 for salt waterInoculating loopBunsen burnerIncubator set to 25°C
Water
Collect a volume of waterDo dilutions at neat, 10−1, 10−2, 10−3
Dilutions
Neat = 100 ml to a plate and lawn inoculate(spread evenly over the plate)10−1 = 900 ml of sterile distilled water + 100 ml ofsample10−2 = 900 ml of sterile distilled water + 100 ml ofdilution 10−1
10−3 = 900 ml of sterile distilled water + 100 ml ofdilution 10−2
Method
Place 100 ml (or 10 µl) of each dilution into thecentre of an agar plate. Use a separate plate foreach dilution. Spread each inoculum evenly overeach plate using a sterile bent glass rod, or aflame-sterilized inoculating loop. It is important toensure that the inoculum is spread evenly overthe plate so that individual colonies are obtainedfor counting. Clumps of colonies will lead toerroneous results.
Technical Methods 223
Sample site Total bacterial count (cells/ml)
Seawater (filtered, no larvae)a 103–103 a
Tank watera 104–105 a
Sedimenta 107–108 cells/ga
Haemolymph healthy oysterb 2.6 × 104 b
Soft tissue healthy oysterb 2.9 × 104 b
Load of pathogenic bacteria at which mortality is seen. Rapid onset of infectiona 1 × 107–5 × 107 a
Load of pathogenic bacteria at which mortality is seen. Slower onset of infectiona 1 × 105–5 × 105 a
Haemolymph in diseased oysterb 105 b
Soft tissue in diseased oysterb 6 × 107 b
aData from Garland et al. (1983); bData from Olafsen et al. (1993).
Table 5.1. Total bacterial counts at sites in oyster hatcheries: indication of healthy and diseased states.
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Incubation
Place plates in a sealed plastic container andincubate at room temperature or an incubator setto 25°C.
Counting the bacterial colonies
Examine the plates at 24 and 48 h.Count the number of colonies on a plate: using afelt pen, mark each colony from the back of thePetri dish to assist with countingCount = N
Calculation to obtain CFU/ml
If 100 ml is placed on to a plate and then lawninoculated, this is a plate dilution of 10−1. If 10 mlwere placed on a plate, this would be a platedilution of 10−2.
CFU/ml = N × dilution × plate dilution.
Thus if 268 colonies are counted fromthe 10−2 dilution and 100 ml was inoculatedto the plate, the count = 268 × 102 × 10 =2.68 × 105 CFU/ml.
5.2 Microscopy
Most laboratory staff will be familiar with the useof a light microscope. However, for those whoare new to the use of a microscope, some basicsare explained here.
Before use, a microscope should be set up forKoehler illumination. This ensures that the lightentering the microscope is focused so as to pro-duce an evenly illuminated field.
• Place a stained smear on to the stage. Focususing the ×10 objective.
• Close the field diaphragm – this is thediaphragm at the base of the microscope.
• Close the aperture of the iris diaphragm.This is the aperture just under the stage thatthe glass slide rests on.
• There will be a small circle of light. Centrethis circle of light using the condensercentring screws.
• Next, adjust the height of the condenserunder the stage so that the edge of the circleof light is in sharp focus.
• Now open the field diaphragm so that it justdisappears from view.
• Open the aperture diaphragm to suit thecontrast required.
Staff who are new to the use of a microscopeshould remember that examination of a Gram-stained smear of bacterial cells is best observedusing the oil immersion lens, which together withthe ×10 objective gives a magnification of 1000times. The aperture on the substage condensershould be open to half its setting with the lightintensity set so that it is comfortable for theeyes. For the examination of wet preparations thisaperture will need to be almost closed so that agood contrast is obtained and cellular structuresare defined.
Only use lens tissue paper for cleaning theobjective lens. Always remove the oil from thelens after use, as the oil can damage the structureswithin the objective. If oil does get on to any ofthe lenses, it can be removed by soaking somelens tissue with petroleum spirit and gentlywiping across the lens until the oil has beenremoved.
5.3 Storage of Isolates
Isolates may be stored at −80°C in LabLemco broth supplemented with 10% glycerol.For marine organisms use marine broth 2216supplemented with 30% glycerol (Bowman andNichols, 2002).
There are a number of different mediafor freeze-drying of bacteria. One method is tosuspend organisms in 1 ml of inositol horse serumin Wheaton serum bottles or appropriate con-tainer depending on freeze-drying equipment.Snap freeze in liquid nitrogen and followinstructions for freeze-drying equipment (seeChapter 7).
An analysis of different cryoprotectantstested for Flavobacterium columnare, F. psychro-philum and Tenacibaculum maritimum recom-mends the use of a medium containing two-thirds Brucella broth (Difco) and one-thirdhorse or fetal calf serum (Desolme andBernardet, 1996).
224 Chapter 5
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6Techniques for the Molecular Identification of Bacteria
The polymerase chain reaction (Saiki et al.,1985; Mullis and Faloona, 1987) has wideapplications in both diagnostic and researchlaboratories. It is routinely being used in thediagnosis and identification of bacteria. Likewise,16S rDNA sequencing is being used more widelyand is a useful tool for the identification of bacte-ria that are difficult to identify by biochemicaltests. This chapter covers the basics of both thesetechniques.
6.1 Molecular Identification by PCRUsing Specific Primers
A number of specific primers for the detection ofaquatic organisms by PCR have been reported.However, most, if not all, of these primers willundergo a long period of validation before theyare routine in a diagnostic laboratory (Baderet al., 2003). It is suggested they be used intandem with biochemical identification methods.
The PCRs that have been reported for aquaticorganisms are summarized in Table 6.1. Each for-ward and reverse primer is listed together with therecommended annealing temperature, number ofcycles and expected product length in base pairs.The concentration of reagents is not given herebecause of the wide variety used for the individualPCRs. The reader is advised to either refer to thereference or optimize the particular PCR for theirlaboratory conditions and equipment.
DNA extraction techniques, basic PCR proto-cols and preparation of reagents are covered inthis chapter.
Aeromonas spp. PCR
The primers used for Aeromonas hydrophila(Nielsen et al., 2001) also produce a bandat 685 bp with an Australian strain of atypicalA. salmonicida (AHLDA 1334). Aeromonashydrophila strains that are positive usingthese primers are negative for the phenotypictests of LDC and cellobiose fermentation.This PCR may assist in determining that isolatesare A. hydrophila, as phenotypic tests are notalways reliable (Nielsen et al., 2001). However,phenotypic tests would need to be carried outto differentiate from atypical A. salmonicida.A. hydrophila, ATCC 7810, which is positivefor LDC, does not produce an amplified productby this PCR.
A PCR that detects the presence of theaerolysin gene, detected DNA from all haemolyticstrains of A. hydrophila that were cytotoxicto vero and CHO cells, and produced entero-toxin as detected by suckling-mouse assays.No aerolysin gene was detected in non-haemolytic strains of A. hydrophila, non-haemolytic strains of A. caviae, or strains ofA. sobria. Extraneous bands were found withsome strains of A. caviae and Plesiomonasshigelloides, but not at the correct molecularweight (Pollard et al., 1990). The aerolysingene is considered to be a useful virulencemarker for detecting virulent pathogenicAeromonas species (Kong et al., 2002). Perform-ing PCRs using the primers of Nielsen et al.(2001), and the primers of Pollard et al. (1990),may be useful in identifying virulent strains ofA. hydrophila.
©N.B. Buller 2004. Bacteria from Fish and Other Aquatic Animals:a Practical Identification Manual (N.B. Buller) 225
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226 Chapter 6
Aero
mon
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iges
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uces
fragm
ents
180
&80
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7
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ers
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ific
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.
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ang
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.,19
96
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and
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n
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2868
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iels
enet
al.,
2001
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5560
209
Polla
rdet
al.,
1990
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(spe
cific
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Prod
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GG
7e5-
R78
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CAA
GC
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5535
242
Oak
eyet
al.,
1999
Aero
mon
assa
lmon
icid
ass
p.sa
lmon
icid
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CTC
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GC
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iyat
aet
al.,
1996
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mon
assa
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imer
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C55
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øie
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.,19
97Ta
ylor
and
Win
ton,
2002
Aero
mon
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Det
ects
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GAG
GG
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CC
CG
Fille
reta
l.,20
00
Aero
mon
astro
taAL
1TT
GC
CG
CC
CAG
GC
CG
GTG
CTG
AL2
ACC
ACTG
TGTG
GAC
CAG
GG
TA66
3062
2Kh
anet
al.,
1999
Edw
ards
iella
spp.
Ampl
ifies
sodB
gene
from
allE
dwar
dsie
llasp
p.E1
FAT
GTC
RTT
CG
AATT
ACC
TGC
497R
TCG
ATG
TAR
TAR
GC
GTG
TTC
CC
A42
3545
4Ya
mad
aan
dW
akab
ayas
hi,
1999
Flav
obac
teriu
msp
ecie
s-sp
ecifi
cB a
s ed
o n1 6
S rD
NA
Col
-72F
GAA
GG
AGC
TTG
TTC
CTT
TC
ol-1
260R
GC
CTA
CTT
GC
GTA
GTG
6030
?Tr
iyan
toet
al.,
1999
Flav
obac
teriu
mco
lum
nare
with
in16
SrD
NA
FvpF
GC
CC
AGAG
AAAT
TTG
GAT
FvpR
1TG
CG
ATTA
CTA
GC
GAA
TCC
5925
1192
Bade
reta
l.,20
03
Org
anis
mFo
rwar
dpr
imer
5′–3
′R
ever
sepr
imer
5′–3
′AT
CBp
Ref
eren
ce
Tab
le6.
1.Li
stof
spec
ific
prim
ers
avai
labl
efo
rP
CR
dete
ctio
nof
aqua
ticor
gani
sms.
242Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:37:25 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
Techniques for the Molecular Identification of Bacteria 227
Flav
obac
teriu
mco
lum
nare
Gen
omov
ar1
Sequ
ence
with
in16
SrD
NA
Col
-Ta
TTC
AGAT
GG
CTT
CAT
TTG
Col
-Tb
CC
GTT
TAC
GG
GC
GTT
GG
AATA
CAG
5430
?Tr
iyan
toet
al.,
1999
Flav
obac
teriu
mco
lum
nare
Gen
omov
ar2
Sequ
ence
with
in16
SrD
NA
Col
-T1
ATTA
AATG
GC
ATC
ATTT
AC
ol-T
2TC
GTT
TAC
GG
CG
TGG
ACTA
CC
A52
3062
1Tr
iyan
toet
al.,
1999
Flav
obac
teriu
mco
lum
nare
Gen
omov
ar3
Sequ
ence
with
in16
SrD
NA
Col
-T11
GAT
GTG
GC
CTC
ACAT
TGTG
Col
-Tb
CC
GTT
TAC
GG
GC
GTT
GG
AATA
CAG
5630
?Tr
iyan
toet
al.,
1999
Flav
obac
teriu
mps
ychr
ophi
lum
16S
rRN
APS
Y1G
TTG
GC
ATC
AAC
ACAC
TPS
Y2C
GAT
CC
TAC
TTG
CG
TAG
5730
1089
Wik
lund
etal
.,20
00
Flav
obac
teriu
mps
ychr
ophi
lum
16S
rRN
AFP
1G
TTAG
TTG
GC
ATC
AAC
ACFP
2TC
GAT
CC
TAC
TTG
CG
TAG
5435
1088
Urd
acie
tal.,
1998
Flav
obac
teriu
mps
ychr
ophi
lum
16S
rRN
APs
y1C
GAT
CC
TAC
TTG
CG
TAG
Psy2
GTT
GG
CAT
CAA
CAC
ACT
45 5030 39
1100
Toya
ma
etal
.,19
94
Lact
ococ
cus
garv
ieae
Base
don
the
dihy
drop
tero
ate
synt
hase
gene
SA1B
10-1
-FC
ATTT
TAC
GAT
GG
CG
CAG
SA1B
10-1
-RC
GTC
GTG
TTG
CTG
CAA
CA
5830
709
Aoki
etal
.,20
00
Lact
ococ
cus
garv
ieae
B as e
do n
1 6S
rDN
ApL
G-1
CAT
AAC
AATG
AGAA
TCG
CpL
G-2
GC
ACC
CTC
GC
GG
GTT
G55
3511
00Zl
otki
net
al.,
1998
a
Lact
ococ
cus
garv
ieae
IRL
TTTG
AGAG
TTTG
ATC
CTG
GLg
RAA
GTA
ATTT
TCC
ACTC
TAC
TT45
3548
2Pu
etal
.,20
02
Lact
ococ
cus
pisc
ium
Doe
sno
tdiff
eren
tiate
from
L.pl
anta
rum
,L.
raffi
nola
ctis
IRL
TTTG
AGAG
TTTG
ATC
CTG
GPi
plra
RC
GTC
ACTG
AGG
GC
TGG
AT45
3586
3Pu
etal
.,20
02
List
onel
laan
guilla
rum
Hae
mol
ysin
gene
VAH
-P1
ACC
GAT
GC
CAT
CG
CTC
AAG
AVA
H1-
P2G
GAT
ATTG
ACC
GAA
GAG
TCA
5530
603
Hiro
noet
al.,
1996
List
onel
laan
guilla
rum
toxR
gene
VA-U
2C
ACTT
CG
CAA
CC
CG
AAG
AGAC
AVA
-D1
CTG
CTT
AGG
TGC
CAG
TTC
TCC
A62
2030
7O
kuda
etal
.,20
01
Myc
obac
teriu
mge
nus
T39
GC
GAA
CG
GG
TGAG
TAAC
ACG
T13
TGC
ACAC
AGG
CC
ACAA
GG
GA
5030
924
Tala
atet
al.,
1997
Myc
obac
teriu
mch
elon
aeR
estri
ctw
ithAp
aIto
give
fragm
ents
at81
2an
d11
2bp
Res
trict
with
BanI
togi
vefra
gmen
tsat
562
and
362
bpTa
laat
etal
.,19
97
cont
inue
d
243Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:37:26 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
228 Chapter 6
Myc
obac
teriu
mfo
rtuitu
mR
estri
ctw
ithAp
aIto
give
fragm
ents
at67
7,13
2,11
5bp
Res
trict
with
BanI
togi
vefra
gmen
tsat
562
and
362
bpTa
laat
etal
.,19
97
Myc
obac
teriu
mm
arin
umR
estri
ctw
ithAp
aIto
give
fragm
ents
at67
7,13
2,11
5bp
No
rest
rictio
nw
ithBa
nITa
laat
etal
.,19
97
Myc
obac
teriu
mtri
plex
-like
Nes
ted
PCR
For1
CG
AAAG
CG
TGG
GG
AGC
GAA
CA
For2
GG
TGTG
GG
TTTC
CTT
CC
TT
Rev
1AG
ACC
CC
GAT
CC
GAA
CTG
AGAC
CR
ev2
ACG
GG
CC
ATTG
TAG
CAT
55 55
38 5540
9
Her
bst e
tal.,
2001
Noc
ardi
age
nus
NG
1AC
CG
ACC
ACAA
GG
GG
NG
2G
GTT
GTA
ACC
TCTT
CG
A55
3059
6La
uren
teta
l.,19
99
Phot
obac
teriu
mda
mse
lae
ssp.
dam
sela
ean
dpi
scic
ida
Util
izes
pres
ence
orab
senc
eof
urea
sege
ne
Ure
-5TC
CG
GAA
TAG
GTA
AAG
CG
GG
Car
1G
CTT
GAA
GAG
ATTC
GAG
T
Ure
-3C
TTG
AATA
TCC
ATC
TCAT
CTG
CC
ar2
CAC
CTC
GC
GG
TCTT
GC
TG
60 60
30 30
448
267
Oso
rioet
al.,
2000
Pseu
dom
onas
angu
illise
ptic
aPA
FG
ACC
TCG
CG
CC
ATTA
PAR
CTC
AGC
AGTT
TTG
AAAG
4635
439
Blan
coet
al.,
2002
Pseu
dom
onas
plec
oglo
ssic
ida
Base
don
gyrB
codi
ngre
gion
Use
dTa
qMan
real
time
PCR
GBP
A-F
CC
TGC
TGAA
GG
ACG
AGC
GTT
CG
GBP
A-R
AAC
CAG
GTG
AGTA
CC
ACC
GTC
G68
50Su
kend
aan
dW
akab
ayas
hi,
2000
Ren
ibac
teriu
msa
lmon
inar
umD
etec
tsp5
7ge
neC
AAG
GTG
AAG
GG
AATT
CTT
CC
ACT
GAC
GG
CAA
TGTC
CG
TTC
CC
GG
TTT
Brow
net
al.,
1994
Ren
ibac
teriu
msa
lmon
inar
umD
etec
tsp5
7ge
neIn
tern
alpr
obe
FL7
CG
CAG
GAG
GAC
CAG
TTG
CAG
FL10
GG
TGTA
ACG
ATAA
TGC
GC
CA
RL1
1G
GAG
ACTT
GC
GAT
GC
GC
CG
A
RL1
1
60 60
35 35
349
149
Miri
amet
al.,
1997
Ren
ibac
teriu
msa
lmon
inar
umF G
ATC
GTG
AAAT
ACAT
CAA
GG
R GG
ATC
GTG
TTTT
ATC
CAC
CC
6030
149
Leon
etal
.,19
94
Salin
ivib
rio(V
ibrio
)cos
ticol
aIG
SGlu
(cos
B)-s
peci
ficpr
imer
VCO
S-F
CTG
ACG
CTA
TTC
TTG
CG
AVC
OS-
RG
TAAT
CAC
ATTC
GTA
AATG
C55
3518
6Le
eet
al.,
2002
Stre
ptoc
occu
sag
alac
tiae
(b-h
aem
olyt
ic,g
roup
B).
Sem
i-nes
ted
PCR
.Cro
ss-re
actio
nw
ithS.
porc
inus
DSF
1TG
CTA
GG
TGTT
AGG
CC
CTT
T
DSF
2G
GC
CTA
GAG
ATAG
GC
TTTC
T
DSR
1C
TTG
CG
ACTC
GTT
GTA
CC
AA
DSR
1
67 6730 30
450
265
Ahm
etet
al.,
1999
Org
anis
mFo
rwar
dpr
imer
5′–3
′R
ever
sepr
imer
5′–3
′AT
CBp
Ref
eren
ce
Tab
le6.
1.C
ontin
ued.
244Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #L.vpMonday, January 12, 2004 2:37:27 PM
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Techniques for the Molecular Identification of Bacteria 229
Stre
ptoc
occu
sin
iae
16S
rRN
ASi
n-1
(CTA
GAG
TAC
ACAT
GTA
CT(
AGC
T)AA
G)
Sin-
2G
GAT
TTTC
CAC
TCC
CAT
TAC
5535
300
Zlot
kin
etal
.,19
98b
Tena
ciba
culu
m(F
lexi
bact
er)m
ariti
mum
16S
rRN
AM
ar1
TGTA
GC
TTG
CTA
CAG
ATG
AM
ar2
AAAT
ACC
TAC
TCG
TAG
GTA
CG
5839
400
Bade
rand
Shot
ts,1
998
Cep
eda
etal
.,20
03Te
naci
bacu
lum
(Fle
xiba
cter
)mar
itim
um16
SrD
NA
MAR
1AA
TGG
CAT
CG
TTTT
AAA
MAR
2C
GC
TCTC
TGTT
GC
CAG
A45
30To
yam
aet
al.,
1996
Vibr
ioch
oler
ae01
ctxA
gene
CTX
2C
GG
GC
AGAT
TCTA
GAC
CTC
CTG
CTX
3C
GAT
GAT
CTT
GG
AGC
ATTC
CC
AC60
2556
4Fi
elds
etal
.,19
92
Vibr
ioch
oler
ae01
ElTo
rPo
sitiv
efo
r rtx
A,rtx
Can
dct
xBge
nes
Rtx
A-F
CTG
AATA
TGAG
TGG
GTG
ACTT
ACG
Rtx
A-R
GTG
TATT
GTT
CG
ATAT
CC
GC
TAC
G55
3041
7C
how
etal
.,20
01
Vibr
ioch
oler
aeno
n-01
Posi
tive
for r
txA
and
rtxC
gene
sR
txC
-FC
GAC
GAA
GAT
CAT
TGAC
GAC
rtxC
-RC
ATC
GTC
GTT
ATG
TGG
TTG
C55
3026
3C
how
etal
.,20
01
Vibr
ioch
oler
ae01
clas
sica
lPo
sitiv
efo
r ctx
Bge
neon
ly(n
egat
ive
for r
txA
and
rtxC
gene
s)
ctxB
2G
ATAC
ACAT
AATA
GAA
TTAA
GG
ATct
xB3
GG
TTG
CTT
CTC
ATC
ATC
GAA
CC
AC55
3046
0C
how
etal
.,20
01
Vibr
iodi
azot
roph
icus
IGSG
lu(d
iaA)
-spe
cific
prim
erVD
IA-F
AGAT
TCTC
TTG
ATG
AGTG
CC
VDIA
-RTA
CC
TAC
ATC
TCTA
AGAG
ACAT
AG55
3530
0Le
eet
al.,
2002
Vibr
iofis
cher
iLu
xAge
neLu
xA-F
GTT
CTT
AGTT
GG
ATTA
TTG
GLu
xA-R
TCAG
TTC
CAT
TAG
CTT
CAA
ATC
C40
4042
8Le
ean
dR
uby,
1995
Vibr
ioflu
vial
isIG
SGlu(fl
uA)-s
peci
ficpr
imer
VFLU
-FAT
AAAG
TGAA
GAG
ATTC
GTA
CC
VFLU
-RG
TATT
CC
TGAA
TGG
AATA
CAC
5535
278
Lee
etal
.,20
02
Vibr
ioho
llisae
gyrB
gene
HG
-F1
GC
TCTG
TCG
GAA
AAAC
TTG
AH
G-R
2AT
GC
TCAA
AATG
GAA
CAC
AG55
3036
3Vu
ddha
kule
tal.,
2000
Vibr
ioho
llisae
toxR
gene
HT-
F3C
TGC
CC
AGAC
ACTC
CC
TCTT
CH
T-R
2C
TCTT
TCC
TTAC
CAT
AGAA
ACC
G62
2430
6Vu
ddha
kule
tal.,
2000
Vibr
ioni
grip
ulch
ritud
oIG
SGlu
(nig
A)-s
peci
ficpr
imer
VNIG
-FC
ATTT
CTT
TGAA
ACAG
AAAG
TVN
IG-R
TAG
ATAA
GG
GG
ATTG
TTG
CTA
5535
114
Lee
etal
.,20
02
Vibr
iopa
raha
emol
ytic
usgy
rBge
ne.T
his
gene
may
also
bepr
esen
tin
V.al
gino
lytic
us
VP1
CG
GC
GTG
GG
TGTT
TCG
GTA
GT
VP2r
TCC
GC
TTC
GC
GC
TCAT
CAA
TA60
3028
5Ve
nkat
esw
aran
etal
.,19
98Ki
met
al.,
1999
Vibr
iopa
raha
emol
ytic
ustl
gene
.Thi
sge
nem
ayal
sobe
pres
enti
nV.
algi
noly
ticus
L-tl
AAAG
CG
GAT
TATG
CAG
AAG
CAC
TGR
-tlG
CTA
CTT
TCTA
GC
ATTT
TCTC
TGC
58.6
3045
0Be
jeta
l.,19
99
cont
inue
d
245Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #M.vpTuesday, January 20, 2004 4:58:55 PM
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230 Chapter 6
Vibr
iopa
raha
emol
ytic
usto
xRge
neM
ayge
twea
kno
n-sp
ecifi
cba
nds
with
V.al
gino
lytic
us,
V.vu
lnifi
cus
ToxR
1G
TCTT
CTG
ACG
CAA
TCG
TTG
ToxR
2AT
ACG
AGTG
GTT
GC
TGTC
ATG
6320
368
Kim
etal
.,19
99
Vibr
iopa
raha
emol
ytic
usC
lone
dfra
gmen
tpR
72H
spec
ific
fort
his
spec
ies
VP33
TGC
GAA
TTC
GAT
AGG
GTG
TTAA
CC
VP32
CG
AATC
CTT
GAA
CAT
ACG
CAG
C60
3538
7or
320
Lee
etal
.,19
95R
ober
t-Pillo
teta
l.,20
02Vi
brio
pena
eici
da16
SrD
NA
VpF
GTG
TGAA
GTT
AATA
GC
TTC
ATAT
CVR C
GC
ATC
TGAG
TGTC
AGTA
TCT
6235
310
Saul
nier
etal
.,20
00
Vibr
iopr
oteo
lytic
usIG
SIA(p
roC
)-spe
cific
prim
erVP
RO
-CG
CAT
TCTT
ACG
AGTG
TGVP
RO
-RAT
TAG
TTG
TATT
CAA
ATA
5535
133
Lee
etal
.,20
02
Vibr
iosa
lmon
icid
aIG
S0(s
alA)
-spe
cific
prim
erVS
AM-F
TGC
GAT
TTAT
GAG
TGTT
CA
VSAM
-RAC
TCTT
CAT
TGAG
AGTT
CTG
5535
275
Lee
etal
.,20
02
Vibr
iosp
lend
idus
IGS0
(spn
A;sp
nD)-s
peci
ficpr
imer
VSPN
-FG
ATTT
AGTT
AAAG
CC
AGAG
CVS
PN-R
CC
TGAT
AAC
TGTT
TGC
CG
5535
240,
294
Lee
etal
.,20
02
Vibr
iotra
chur
i(ju
nior
syno
nym
ofV.
harv
eyi)
Pstl-
1aTG
CG
CTG
ACG
TGTC
TGAA
TTPs
tI-1b
AAG
CAG
CG
ATG
ACAA
GC
AGT
6035
417
Iwam
oto
etal
.,19
95b
Vibr
iotu
bias
hii
IGSIA
(tubA
)-spe
cific
prim
erVT
UB-
FTG
GG
TCTT
TCAG
GC
CC
GVT
UB-
RC
GAC
GAA
TGAC
CG
TTG
TC55
3539
4Le
eet
al.,
2002
Vibr
iovu
lnifi
cus
Nes
ted
PCR
P1 GAC
TATC
GC
ATC
AAC
AAC
CG
P3 GC
TATT
TCAC
CG
CC
GC
TCAC
P2 AGG
TAG
CG
AGTA
TTAC
TGC
CP4 C
CG
CAG
AGC
CG
TAAA
CC
GAA
57 59
50 50
704
222
Lee
etal
.,19
98
Vibr
iovu
lnifi
cus
Cyt
olys
in-h
aem
olys
inge
neF C
GC
CG
CTC
ACTG
GG
GC
AGTG
GC
TGR G
CG
GG
TGG
TTC
GG
TTAA
CG
GC
TGG
64.5
30–5
0C
olem
anet
al.,
1996
Yers
inia
ruck
eri
YER
8G
CG
AGG
AGG
AAG
GG
TTAA
GTG
YER
10G
AAG
GC
ACC
AAG
GC
ATC
TCTG
6025
575
Gib
ello
etal
.,19
99
Yers
inia
ruck
eri
16S
rDN
AR
uck1
CAG
CG
GAA
AGTA
GC
TTG
Ruc
k2TG
TTC
AGTG
CTA
TTAA
CAC
TTAA
5530
409
LeJe
une
and
Rur
angi
rwa,
2000
Yers
inia
ruck
eri
yruR
/yru
Iquo
rum
sens
ing
gene
IF2
GAG
CG
CTA
CG
ACAG
TCC
CAG
ATAT
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Techniques for the Molecular Identification of Bacteria 231
Mul
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Edwardsiella spp. PCR
The sodB gene (coding for iron-cofactored super-oxide dismutase) from Edwardsiella spp. wasamplified (454 bp for all species) and sequenced.Differences in nucleotide sequence dividedthe species of Edwardsiella into a pathogenicand non-pathogenic cluster. Cluster I comprisespathogenic strains of E. tarda from Japaneseeel, Japanese flounder (Paralichthys olivaceus),Nile tilapia (Oreochromis niloticus) and ayu(Plecoglossus altivelis), atypical E. tarda from redsea bream, Edwardsiella species from Japaneseeel, and E. ictaluri. Non-pathogenic E. tarda andE. hoshinae were found in cluster II (Yamada andWakabayashi, 1999).
Flavobacterium columnare PCR
Primers FvpF and FvpR2, located within the 16SrDNA are said to be specific for F. columnare(Bader et al., 2003). Three primer sets, alsolocated within the 16S rDNA gene, differentiatethe three genomovars of F. columnare. Threeseparate forward primers are available, withgenomovars 1 and 3 having the same reverseprimer (see Table 6.1). Primers for amplifyinggenomovar 2 produce a product band at 621 bp,but also produce bands at 800 and 1000 bp in allFlavobacterium species (Triyanto et al., 1999).The sensitivity of the PCRs can be increased fromtissue samples by using universal primers (Baderet al., 2003) or universal primers followed by theFlavobacterium species-specific set of Col-72Fand Col-1260F (Table 6.1) (Triyanto et al.,1999).
Pseudomonas plecoglossicida PCR
This PCR was conducted using theTaqMan methodology for quantitative real-time PCR. The internal control and targetDNA probes were TGT-P 5′-(FAM)AGATGGCGTGGGCGTTGAAGTAGCGC(TAMRA)-3′, and ISD-P 5′-(VIC)CCTTCACCACCACGGCCGAGCGTGAG(TAMRA)-3′ (Sukenda and Wakabayashi, 2000).
Tenacibaculum (Flexibacter) maritimum PCR
A nested PCR using universal primers of 20F and1500R in the first PCR reaction, followed by anested PCR using T. maritimum-specific primersMar1 and Mar2 in the nested reaction allows
detection of this organism direct from fish tissue(Cepeda et al., 2003). Ready-to-Go PCR beads(Amersham Pharmacia Biotech) were used, with1 pmol of each primer in a 25 ml reaction. Cyclingconditions for the first PCR reaction used apreheating step of 95°C for 5 min, 30 cyclesof 95°C for 30 s, 57°C for 30 s, 72°C for 60 swith a final cycle that used an extension step at72°C for 5 min. The second PCR reaction used apreheating step of 94°C for 2 min, followed by 40cycles of 94°C for 2 s, 54°C for 2 s, 72°C for 10 swith a final cycle that used an extension step of4 min.
PCR for Vibrio spp.
Many of these PCRs will need to undergo aperiod of validation, and as more information isgathered and research done, the specificity ofthese PCRs will also be assessed. Such a case hasbeen found with PCRs for some of the Vibrio spe-cies. There is some doubt as to the specificity ofsome of the primers for Vibrio parahaemolyticusand V. alginolyticus. The gyrB gene has beensuggested to differentiate V. parahaemolyticusfrom V. alginolyticus (Venkateswaran et al.,1998); however, the primers also detectedthe gene in V. alginolyticus when the suggestedannealing temperature of 58°C was used. Speci-ficity for V. parahaemolyticus was improved at anannealing temperature of 60°C (Kim et al., 1999).The tl gene that encodes for a thermolabilehaemolysin and used in a PCR for detectingV. parahaemolyticus (Bej et al., 1999) was foundto occur not only in V. parahaemolyticus butwas also detected in species of V. alginolyticus(Robert-Pillot et al., 2002). The toxR gene, whichis involved in the regulation of many genes, isconserved amongst the Vibrio species; however,there is a low degree of homology that allows forselection of species-specific primers. A PCR thattargets the toxR gene in V. parahaemolyticus mayamplify non-specific amplicons from V. algino-lyticus and V. vulnificus (Kim et al., 1999). Stud-ies suggested that to date the PCR that appears tobe the most specific for V. parahaemolyticus is aPCR that detects a fragment termed R72H. Thissection of DNA is composed of a non-codingregion and a phosphatidylserine synthetase geneand the primers amplify an amplicon of either387 or 320 bp, both of which are consideredspecific for V. parahaemolyticus (Lee et al., 1995;Robert-Pillot et al., 2002).
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Multiplex PCR
A multiplex PCR enabled simultaneous detectionof the human pathogens E. coli, Salmonellatyphimurium, V. cholerae, V. parahaemolyticusand V. vulnificus from shellfish samples. Anoptimized PCR was used with 2.5 mM MgCl2,and an annealing temperature of 55°C (Brasheret al., 1998).
Nested PCR
Any of the above primers in Table 6.1 that aredesigned within the 16S rRNA can be used ina nested PCR reaction to improve detectionsensitivity. The first PCR reaction is done witheubacterial primers, which amplify the entire 16SrDNA, and the specific primers are then usedfor a nested PCR reaction. The detection ofFlavobacterium columnare was improved intissue samples when specific primers were used ina nested PCR with eubacterial universal primers(Triyanto et al., 1999; Bader et al., 2003). Fordetails on universal primers see section 6.3.
An optimized nested PCR was reportedfor Aeromonas salmonicida, Flavobacteriumpsychrophilum and Yersinia ruckeri. The firstround PCR used universal primers for the 16SrDNA gene and the second round of primers werespecies-specific for the three organisms tested.The PCR conditions were optimized so that thesame conditions could be used for all primers(Taylor and Winton, 2002). The universal primersof Weisburg et al. (1991) with forwardprimer fD2 5′-AGAGTTTGATCATGGCTCAG-3′ and reverse primer rP2 5′-GTTTACCTTGTTACGACTT-3′ were used togenerate a 1500 bp fragment of the 16S rDNAgene. This template was then used in secondround PCR with primers for Aeromonassalmonicida AS1 and AS2 (Høie et al., 1997),Flavobacterium psychrophilum PSY1 and PSY2(Toyama et al., 1994), and primers YER8 andYER10 for Yersinia ruckeri (Gibello et al., 1999).In the PCR reaction mix, the final concentrationof MgCl2 was 2.0 mM, dNTP was 200 mM, with100 pmol final concentration for each primer and1.25 U/50 ml of Taq. Thermocycling conditionswere also standardized with an initial denaturationstep of 95°C for 4 min, followed by 30 cycles of95°C for 45 s, annealing for 45°C for universalprimers and 55°C for specific primers, with anextension of 72°C for 90 s. A final cycle with an
extension step of 72°C for 4 min was used. A finalcycle of 4°C infinity was used as a holding cycle(Taylor and Winton, 2002).
Outline of steps for PCR using specific primers
Step Method1 Extract DNA from bacterial colonies2 Amplify DNA using specific primers3 Visualize DNA on agarose gel
DNA extraction from bacterial cells
There are many methods for the extraction ofDNA from bacterial cells and these include bothmanual methods and commercially availablekits. Such kits include Instagene (Bio-Rad),AquaPure genomic DNA kits (Bio-Rad),Chelex-based resin (Bio-Rad), Puregene (GentraSystems), PrepMan (Applied Biosystems),MasterPure (Astral Scientific – Epicentre),Wizard Genomic (Promega), and the DneasyTissue system and QiAamp system fromQiagen.
MANUAL METHOD 1. Suspend bacterial cells in100 ml of a solution of 1 mM EDTA-0.5% Triton-X-100. Boil in a microwave on HIGH setting for5 min (Lee et al., 1998).
MANUAL METHOD 2. Make a suspension ofbacterial cells to 0.5 McFarland density. Centri-fuge at 13,000 rpm for 5 min. Suspend pellet ofcells in a digestion buffer of 50 mM Tris-HCl(pH 8.5), 1 mM EDTA, 0.5% SDS and 200 mg/mlof proteinase K. Incubate for 3 h at 55°C withagitation. Heat inactivate the proteinase K for10 min at 95°C. Cool to 4°C, then centrifuge at13,000 rpm for 10 min. Use supernatant in thePCR reaction. However, the PCR master mix mustcontain a final concentration of Tween 20 toneutralize the effect of SDS, which is inhibitoryto Taq polymerase at concentrations as low as0.01% (Goldenberger et al., 1995).
This method is also suitable for tissue sampleswith an overnight incubation in digestion bufferfollowed by sonication (Goldenberger et al.,1995).
MANUAL METHOD 2 FOR GRAM-POSITIVE BACTERIA.
For Gram-positive bacteria treat cells withlysozyme (1 mg lysozyme/ml TE, pH 8.0).
Techniques for the Molecular Identification of Bacteria 233
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Extraction of DNA from tissue
As for extraction of DNA from cultured bacterialcells, bacterial DNA can also be extracted fromtissue samples. There are many methods avail-able for both manual and commercially availablemethods. The following are some suggestions.
MANUAL METHOD 1. To 100 ml of homogenizedtissue add 100 ml of Chelex-100 resin (Bio-RadLaboratories or Sigma-Aldrich). Heat at 56°C for10 min. Add 200 ml of 0.1% Triton-X-100 andboil for 10 min. Cool on ice, then centrifuge at12,000 rpm for 3 min. Use 5 ml in PCR reaction(Khan and Cerniglia, 1997).
MANUAL METHOD 2. Culture 10 g of tissue into90 ml of TSBYE medium (30 g tryptic soy brothpowder with dextrose, (Difco); 6 g yeast extract;1 l of water). Incubate at 25°C overnight withshaking. Take 0.5 ml of the upper phase of thesample and mix with 1 ml of sterile phosphate buf-fered saline (PBS, 0.05 mol/l, pH 7.4). Centrifugeat 9000 g for 3 min. Wash the pellet three times inPBS and once with sterile water. Resuspend pelletin 50 ml of water, then dilute 1:10 with 1%Triton-X-100 and place in boiling waterbath for5 min. Place immediately on ice. Use 2 ml in PCRmixture (Wang et al., 1997).
6.2 PCR Protocols
Any PCR that is introduced into the laboratoryneeds to be optimized for the Taq enzyme that isused by that laboratory and the primers that areprepared. Differences may occur between differ-ent batches of primers and this may be seen withthe concentration of the dNTPs. For example,a pair of primers that may be optimal at a final
concentration of 100 mM dNTP mix in the PCRreaction; when re-ordered a concentration of200 mM may be optimal.
Method 1 – standard protocol
Table 6.2 suggests a standard PCR protocolthat may be used to amplify from most targetsequences. It is also a basic starting point fromwhich to do optimizations. When optimizing theconcentrations in the PCR mix, the suggestedranges for testing are: dNTP concentration(100–200 mM), primer concentration (0.1–1 mM),magnesium chloride concentration (1–4 mM)and Taq enzyme (1–2.5 Units). Too much ortoo little of any of these reagents will cause non-specific background, mispriming or insufficientproduct generated. In particular, high concentra-tions of dNTP, primer or MgCl2 will lead tonon-specific product (Saiki, 1989; Innis andGelfand, 1990). A negative and positive controlshould be included with each PCR run.
Sometimes bovine serum albumin, gelatin,Tween 20 or DMSO can be added to the PCRreaction mix to help stabilize the Taq enzyme.DMSO may assist in preventing the inhibition ofthe function of the Taq enzyme caused by inhibi-tors present in tissue or from contamination byagar and agar-containing media such as Stuartstransport medium. However, not more than 2%should be used, as greater than this concentrationis inhibitory to Taq.
It is suggested that the reagent volumesfor the PCR master mix be set up in an Excelspreadsheet with the appropriate formulae ineach cell. When a different number of samplesare tested then the master mix is easilycalculated.
Aliquot PCR master mix into 20 ml volumes.Add 30 ml of sterile paraffin oil to each tube if
234 Chapter 6
Reagent ×1 (ml) ×10 (ml) Final concentration
Water 11.8 = 25 minus the volume of reagentsPCR buffer (x10) 2.5 = 2.5 × 10 1dNTP mix (2 mM of each) 2.5 = 2.5 × 10 200 mM (0.2 mM)MgCl2 (25 mM) 2.2 = 2 × 10 2 mMPrimer forward (20 pmol = 5 mM) 0.5 = 0.5 × 10 0.1 mMPrimer reverse (20 pmol = 5 mM) 0.5 = 0.5 × 10 0.1 mMTaq enzyme (5 U/ml) 0.2 = 0.2 × 10 1 UnitTotal volume including volume of DNA to be added 25.2 = 25 × 10
Table 6.2. Standard PCR protocol.
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thermocycler does not have a hot lid. Tubes canbe stored at −20°C until required.
Add 5 ml of template (DNA) and place tubeson thermocycler.
Method 2 – commercial PCR reaction mixture
Many companies offer master mix that containsbuffer, MgCl2, and dNTPs. The user addsthe DNA template, primers and water. Anexample is Ready-To-Go PCR beads fromAmersham Pharmacia Biotech. This is used as a25 ml reaction mix to which primers and 5 ml ofDNA are added. BIOMIX ready-to-go from AstralScientific (Bioline), PCR Master Mix (Promega),and IQ supermix (Bio-Rad laboratories) are otherexamples of commercially available PCR MasterMix.
Thermocycling conditions
The cycling conditions will depend on the typeof thermocycler, the annealing temperature ofthe primers and the type of Taq enzyme used,whether it is a hot-start enzyme or not.
A standard set of cycling conditions beginswith one cycle at 95°C for 1–5 min. Then, 25–35cycles of denaturation at 95°C for 30 s, annealingat 55–68°C 30 s, extension at 72°C for 30 s.A final cycle is suggested as for the last cyclebut with an extension time of 10 min. Thethermocycler can be set to do a hold cycle at 4°C.
The primer annealing temperature dependsupon the length of the primer, the GC content andconcentration. A recommended annealing tem-perature is 5°C below the true Tm of the primers.The Tm is usually provided on the data sheetsent with the primer. The Tm can be estimated bycalculating 2°C for A or T, and 4°C for G or C inthe primer sequence. The annealing temperatureaffects the specificity of the reaction. A lower thanoptimal annealing temperature may lead tomispriming of non-target sequence or the mis-extension of incorrect nucleotides at the 3′ end ofthe primers. A low extension temperature togetherwith high dNTPs also favours mispriming andnon-specificity (Saiki, 1989; Innis and Gelfand,1990).
The optimal number of cycles is between 25and 35. Increasing the number of cycles may leadto problems with the PCR, as a plateau effectis reached where product is no longer amplifiedand a result is that non-specific product may be
amplified preferentially. This plateau is reachedaccording to the number of target copies initiallypresent in the sample and the amount of DNAsynthesized. Also, reagent exhaustion occurs withan extended number of amplification cycles.The recommended number of cycles per startingmaterial is 25–30 (for 3 × 105 target molecules),30–35 (1.5 × 104 molecules), 35–40 (1 × 103
molecules), 40–45 (50 molecules) (Saiki, 1989;Innis and Gelfand, 1990).
Gel electrophoresis
A standard gel for the detection of amplifiedproduct is as follows. Prepare a 2% agarose gelusing chromosomal grade agarose (Bio-Rad).Load 5 ml of amplification product and 5 ml of2 × loading buffer into the wells. Run gel at5 v/cm for 2 h. On a Bio-Rad PowerPac 300,80 v for 90 min produces well-separated bands.Always include a molecular weight marker suchas a 100 bp marker.
The gel is stained for 1 h in 1 litre of distilledwater containing 50 ml of ethidium bromide.Ethidium bromide is a carcinogen and thereforeappropriate precautions need to be taken, such aswearing lab coats and gloves.
The gel is photographed over a UV trans-illuminator. Safety note: Do not expose the skinor eyes to UV light. Laboratory coats, gloves andface shields must be worn when operating UVtransilluminators that are not housed within aclosed instrument.
Reagent preparation of stock andworking solutions
dNTP concentration for PCR amplification
For dNTPs supplied as 100 mM. The finalconcentration in the PCR is required at either100 mM or 200 mM depending on results inoptimization tests.
Therefore, prepare each dNTP to 10 mM bydiluting 1:10. From this prepare a working mastermix of a dNTP solution by adding equal volumesof each dNTP plus an equal volume of water; thatis, a 1:5 dilution for each dNTP to give a finalconcentration of each dNTP of 2 mM. For a finalconcentration of 100 mM in the PCR master mix,use 1.25 ml. For 200 mM use 2.5 ml in a master mixof 25 ml total volume.
Techniques for the Molecular Identification of Bacteria 235
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The nucleotides can also be purchased asa mixture containing all four dNTPs. An exampleis PCR nucleotide mix (Promega) with eachnucleotide at 10 mM concentration.
Preparation of primers
A data sheet is usually sent with the primer, whichdetails the concentration, melting temperatureetc of the primer. Prepare a stock solution of100 pmol in TE buffer (pH 7.0). Therefore, if thedata sheet states a primer is 3500 pmol, add350 ml of TE buffer to the dehydrated primer toobtain a stock solution of 100 pmol.
Prepare a working solution in distilledwater of the required concentration, usually20 pmol.
Primer quantification by spectrophotometry
An accurate quantification can be done byspectrophotometer. Dilute the stock solution ofprimer 1:20 (i.e. 50 ml + 950 ml of water).
The UV absorbance is read at 260 nm. Theconcentration of the primer or oligonucleotidein mg/ml = absorbance × dilution × weight perOD.
Single-stranded DNA (ssDNA) at a con-centration of 33 mg/ml has an absorbance of 1.Therefore 33 mg of ssDNA = 1 OD unit.
Therefore, in the above example, theconcentration of ssDNA mg/ml = absorbance ×20 × 33.
Double-stranded DNA (dsDNA): A260 =OD260 = 1 for a 50 mg/ml solution.
Oligomer – quantitation
For a 20-mer, a stock solution with A260 = 1,contains 5 nmol5 nmol = 33 mg/(20 × 325)For a 40-mer, a stock solution with A260 = 1contains 2.5 nmol2.5 nmol = 33 mg/(40 × 325)
Oligomer – conversion of pmol of primerto mg of primer
Multiply pmol by (length × 325)/1,000,000Example: 51809.88 pmol of a 20-mer (from datasheet)(51809.88 × 20 × 325)/1,000,000 = 336.7 mgprimer
Conversion of mg of primer to pmol of primer
Multiply by 1,000,000/(length × 325).Example: 365.73 mg of a 20-mer (from datasheet)(365.73 × 1,000,000)/(20 × 325) = 56,266.15pmol of primer
Primer concentration for PCR amplification
Micromolar concentrations of primer =pmol/mlThus 20 pmol of primer in 100 ml PCR mixture =20 micromolar (20 mM)
Equation for estimating volume required froma stock concentration
A basic formula for estimating the required vol-ume from a stock solution is as follows:(initial concentration) × (volume needed) =(final concentration) × (volume of sample)Therefore, if you have a stock solution of 5 mMof primer with a final concentration required of0.1 mM in a 25 ml reaction volume the equationis:5 mM × c = 0.1 mM × 25 mlc = (0.1 mM × 25)/5 mMc = 0.5 ml. Therefore add 0.5 ml of the 5 mMstock (or working solution) to a 25 ml reactionvolume.
Storage of oligonucleotides
Oligonucleotides should be stored at −20°C.Store in aliquots to prevent multiple freeze andthaw of oligonucleotides.
6.3 Molecular Identification by 16SrDNA Sequencing
Sequencing the 16S rRNA has been used exten-sively to study bacterial evolution and phylogeny.With a vast number of 16S rRNA sequencesavailable in the National Centre for Biotech-nology Information (NCBI) and the RibosomalDatabase Project (RDP) databases, sequencingthe 16S rDNA is an essential tool in bacterialsystematics and the identification of new species.
It is also a useful tool when used with bio-chemical tests for bacterial identification in thediagnostic laboratory. Ribosomal RNA containsvariable and highly conserved regions that evolve
236 Chapter 6
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very slowly and, therefore, can be specific to agenus.
The 16S rRNA is found in the ribosomes,which consist of proteins and RNA. In prokaryotesthe ribosomes measure 70S, and consist of twosubunits, 50S and 30S. S is the sedimentationrate, or Svedberg unit, during centrifugation.The 16S rRNA is found in the 30 S subunit,whereas the 50S subunit contains the 5S and23S rRNA molecules. 16S is approximately 1600nucleotides, 23S, 3000 nucleotides and 5S isapproximately 120 nucleotides.
Within the gene encoding for the 16S rRNAare areas of sequence that are conserved acrossthe different genera, and it is these areas thathave been used for the design of the universaleubacterial primers. Not all primers will bind to theDNA of the bacterium of interest; however, if acombination of primers is used then a sectionof DNA will be amplified, which can then besequenced for identification.
Outline of steps for sequencing
Step Method1 Extract DNA from bacterial colonies2 Amplify 16S rDNA using universal
eubacterial primers3 Visualize DNA on agarose gel4 Clean up amplified product5 Quantify DNA concentration6 Perform sequencing PCR reaction7 Sequencing (send to a sequencing facility)8 Analysis of sequence information using
BIOEDIT
9 BLAST search for sequence identification
DNA extraction
DNA is extracted from bacterial colonies asdescribed previously.
Amplification of 16S rDNA using universaleubacterial primers
The rDNA contains a number of sites that areconserved across genera, and as such there are anumber of primers that can be used to amplify apart or all of the 16S rDNA for many genera. Theuniversal primers A, B and C amplify regions thatare universally conserved across prokaryotes andeukaryotes and which were initially proposed by
Lane et al. (1985). Since then, a number ofdifferent variations have been proposed. Someprimers are universal for bacteria only, someamplify the entire 16S rDNA, whereas othersamplify different regions. The convention foridentifying the primers is to name them accordingto the number of the E. coli position at which the3′ end of the primer anneals.
Some of the primers that are available toeither amplify all or part of the 16S rDNA aredetailed in Table 6.3. Not all primer combinationswill be appropriate for all bacteria. Table 6.4indicates primer combinations that have proveduseful. The primers may be ordered from acompany such as Qiagen or Invitrogen.
Primers A, B, and C are universal fororganisms from the three primary kingdoms,archaebacteria, eubacteria and eukaryotes (Laneet al., 1985). Primer EUBB (7–26) of Suzuki andGiovannoni (1996) is the same as primer 27f ofLane (1991), except for a degeneracy (M) thatpossibly makes the primer more universal.
Primer 27f was modified because comple-mentarity at the 3′ ends led to self-priming fol-lowed by primer depletion. The modified primerwas referred to as POmod (Wilson et al., 1990).Complementarity in primers and between primerpairs can lead to primer dimers, which is theproduct that is amplified in preference tothe desired sequence on the DNA template(Watson, 1989).
The suggested pairs of primers of 63f and1387r (Table 6.4) were found to improve uponthe primer pair of 27f and 1392r (Table 6.3)(Lane et al., 1991), particularly with difficult DNAtemplates (Marchesi et al., 1998).
The entire rDNA can be amplified with twosets of primer pairs. The primer pairs POmod andPC3mod amplify 789 base pairs of the rDNA andthe pairs P3mod and PC5 amplify the remainderof the rDNA of 721 base pairs (Wilson et al.,1990). Alternatively, the entire 16S rRNA can beamplified with 27f or EUBB as the forward primerand 1525 or EUBA as the reverse primer(Weisburg et al., 1991).
The universal primers 16/23S-F and16/23S-R were used to amplify the intergenicspacer (IGS) region between the 16S rRNA andthe 23S rRNA genes of the Vibrio species and thisregion was subsequently used to design specificprimers for the detection of eight Vibrio species. Astandard PCR reaction mix, and thermocyclingconditions with an annealing temperature of
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238 Chapter 6
Primer(E. coli numbering) Primer sequence 5¢ to 3¢ Target Reference27f (EUBB) (7–26) AGAGTTTGATCMTGGCTCAG Most Eubacteria Lane, 1991; Weisburg et al.,
1991; Suzuki and Giovannoni,1996
20F AGAGTTTGATCATGGCTCAG Eubacterial Weisburg et al., 1991POmod (8–22)modified from 27f
AGAGTTTGATCMTGG Eubacterial kingdom only Wilson et al., 1990
63f (43–63) CAGGCCTAACACATGCAAGTC Marchesi et al., 1998357r (339–357) CTCCTACGGGAGGCAGCAG Most Eubacteria Lane, 1991; Weisburg et al., 1991530f (515–530) GTGCCAGCMGCCGCGG Most Eubacteria, Eukaryotes,
ArchaebacteriaLane, 1991
P3mod f (787–806) ATTAGATACCCTDTAGTCC Eubacterial kingdom only Wilson et al., 1990519r (519–536) GWATTACCGCGGCKGCTG Primer A. Universal for all kingdoms Lane et al., 1985; Lane, 1991PC3mod r (787–806) GGACTAHAGGGTATCTAAT Eubacterial kingdom only Wilson et al., 1990685r3 TCTRCGCATTYCACCGCTAC Most Gram-positives, cyanobacteria Lane, 1991907r (907–926) CCGTCAATTCMTTTRAGTTT Primer B. Universal for all kingdoms Lane et al., 1985; Lane, 1991926f AAACTYAAAKGAATTGACGG Most Eubacteria, Eukaryotes,
ArchaebacteriaLane, 1991
1100r (1100–1114) GGGTTGCGCTCGTTG Most Eubacteria Lane, 1991; Weisburg et al., 1991
1114f GCAACGAGCGCAACCC Most Eubacteria Lane, 19911387r (1387–1404) GGGCGGWGTGTACAAGGC Marchesi et al., 19981392r (1392–1406) ACGGGCGGTGTGTRC Primer C. Universal for all kingdoms Lane et al., 1985; Lane, 19911406f TGYACACACCTCCCGT Most Eubacteria, Eukaryotes,
ArchaebacteriaLane, 1991
PC5 r (1492–1507)Modified from 1492r
TACCTTGTTACGACTT Eubacterial kingdom only Wilson et al., 1990; Lane, 1991
1492r (1492–1512) TACGGYTACCTTGTTACGACTT Most Eubacteria, Archaebacteria Lane, 19911500R GGTTACCTTGTTACGACTT Eubacterial Weisburg et al., 19911525r (1525–1541) AAGGAGGTGWTCCARCC Universal for all kingdoms Lane, 1991; Weisburg et al., 19911525r EUB Amodified from 1525r
AAGGAGGTGATCCANCCRCA Eubacterial kingdom only Suzuki and Giovannoni, 1996
M = C:A; R = A:G; K = G:T; W = A:T (Lane et al., 1985). Other mixtures of nucleotides (known as degeneracies or wobbles) areS = C:G; Y = C:T; V = A:G:C; H = A:C:T; D = A:G:T; B = C:G:T; N = A:G:C:T. F = same sequence as rRNA. R = means thecomplement of rRNA sequence. The primer positions correspond to the nucleotide numbering system of E. coli 16S rRNA(e.g. 9–27). The C in the name of the primers of Wilson et al. (1990) refers to the complementary sequence.
Table 6.3. Universal eubacterial primers for 16S rDNA sequencing.
Forward primer 5′–3′ Reverse primer 5′–3′ Reference
27f GAGTTTGATCCTGGCTCAG 1392R ACGGGCGGTGTGTRC Lane, 199163f CAGGCCTAACACATGCAAGTC 1387R GGGCGGWGTGTACAAGGC Marchesi et al., 1998530F GTGCCAGCMGCCGCGG 1100R GGGTTGCGCTCGTTG Lane, 1991POmod. AGAGTTTGATCMTGG PC3mod. GGACTAHAGGGTATCTAAT Wilson et al., 1990P3mod. ATTAGATACCCTDTAGTCC PC5. TACCTTGTTACGACTT Wilson et al., 199016/23S-F. (1390–1407)TTGTACACACCGCCCGTC
16/23S-R. (474–456)CCTTTCCCTCACGGTACTG
Lee et al., 2002
Table 6.4. Suggested primer pairs.
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55°C, was used. (Lee et al., 2002). See Table 6.1for specific primers.
Primers for sequencing the 16S rDNA gene forthe identification of Vibrio spp.
There is high sequence homology for the 16SrRNA of all Vibrio species and therefore sequenc-ing part of the 16S rDNA will not be of much usewhen trying to identify to species level. The entire16S rDNA needs to be sequenced for identifica-tion to species level. This can be achieved byamplifying the entire 16S rDNA with universalprimers (Table 6.5) and then using this amplifiedproduct as a template for eight sequencingprimers (Table 6.6) (Thompson et al., 2001a).
For the Universal PCR mix use a standardmaster mix. Use standard cycling parameters withan annealing temperature of 55°C. A productof approximately 1.5 kilo base pairs (kbp) isamplified.
Once the entire 16S rDNA is amplified andpurified, the sequencing primers are used inindividual sequencing PCR reactions to sequencethe 1.5 kbp product. For purification of theamplified product and for quantification of DNAsee page 240. Table 6.7 details the sequencingmaster mix.
PCR master mix for universal primers
Use the standard PCR protocol as a guide.DMSO at no more than a 2% final concentrationcan be added to improve the specificity ofhybridization of the primers (Wilson et al., 1990).Prepare PCR master mix in a single tube. Aliquot20 ml volumes to 0.6 ml or 0.2 ml microfugetubes depending upon the requirements ofthe thermocycler. Add 20–30 ml of paraffin oilto each tube (if hot lid is not available on thethermocycler). Tubes can be labelled and storedat −20°C until required for use.
For the PCR add 5 ml of template DNA to a20 ml reaction tube and place in thermocycler.
Thermocycling conditions for universal primers
The thermocycling conditions are much the sameas for the universal PCR, except that a relaxedannealing temperature is used. Therefore, thecycling conditions begin with one cycle at 95°Cfor 1–5 min. Then, 25–35 cycles of denaturationat 95°C for 30 s, annealing at 45–55°C 30 s,extension at 72°C for 30 s. A final cycle issuggested as for the last cycle but an extensiontime of 10 min. The thermocycler can be set to doa hold cycle at 4°C. The annealing temperaturecan be between 45 and 55°C.
Techniques for the Molecular Identification of Bacteria 239
Forward primer 5′–3′ Reverse primer 5′–3′ Reference
EUBB (7-26)AGAGTTTGATCMTGGCTCAG
EUB-AAAGGAGGTGATCCANCCRCA
Weisburg et al., 1991; Suzuki andGiovannoni, 1996; Thompson et al., 2001a
MH1AGTTTGATCMTGGCTCAG
MH2TACCTTGTTACGACTFCACCCCA
Thompson et al., 2001a
Table 6.5. Universal primers for amplification of 16S rDNA from Vibrio spp.
Primer name E. coli position Sequence 5′–3′ Reference
16F358 339–358 CTCCTACGGGAGGCAGT Modified from primer 357r from Lane, 199116F536 519–536 CAGCAGCCGCGGTAATAC Thompson et al., 2001a16F926 908–926 AACTCAAAGGAATTGACGG Modified from primer 926f from Lane, 199116F1112 1093–1112 AGTCCCGCAACGAGCGCAAC Thompson et al., 2001a16F1241 1222–1241 GCTACACACGTGCTACAATG Thompson et al., 2001a16R339 358–339 ACTGCTGCCTCCCGTAGGAG Modified from primer 342r from Lane, 199116R519 536–519 GTATTACCGCGGCTGCTG Modified from primer 519r from Lane, 199116R1093 1112–1093 GTTGCGCTCGTTGCGGGACT Thompson et al., 2001a. Similar to 1100r from Lane, 1991
Thompson et al., 2001a.
Table 6.6. Sequencing primers for the 16S rDNA from Vibrio spp.
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Visualize DNA on agarose gel
To check that the DNA has been amplified,visualize the product on a 2% agarose gel.
Clean-up amplified product
Before sequencing, the amplified product needsto be purified from the other components in thePCR reaction. Commercial kits such as QIAquickPCR kit or QIAprep Spin kit (Qiagen), PCRKleen spin columns (Bio-Rad), Wizard
PCR Preps (Promega) can be used for post-PCRclean-up.
Quantify DNA concentration
Once the DNA is free of other contaminants itneeds to be quantified for the PCR sequencingreaction. This can be done spectrophoto-metrically by reading the absorbance at 260 nm.The concentration of double-stranded DNA inmg/ml = absorbance × dilution × weight per OD.Weight per OD for dsDNA is 50 mg/ml.
Alternatively, the concentration of DNAcan be estimated by gel electrophoresis in a 2%agarose gel and estimating the amount of DNAagainst a quantitative DNA molecular weightmarker, which has known molecular weightsin nanograms per band. Examples are Hyper-Ladder IV from Bioline, GeneRuler from MBIFermentas.
For the sequencing PCR, the concentrationof DNA needs to be 10–30 ng for a reactionvolume of 10 ml.
Concentrating DNA, ethanol precipitation
If the DNA needs concentrating to obtain thedesired concentration for sequencing, then anethanol precipitation is used. If oil has been usedas an overlay, remove as much as possible beforethe post-PCR clean-up or ethanol precipitation.This method is from Sambrook et al. (1989).
1. Add a 1/10 volume of 3 M sodium acetatepH 4.62. Add an equal volume of 95% ethanol (non-denatured alcohol)3. Centrifuge 13,000 rpm for 15 min at roomtemperature4. Carefully remove the supernatant5. Wash twice with 70% ethanol6. Wash once with 95% ethanol
7. Air dry, or vacuum dry8. Add the desired volume of water or TE bufferto the dried pellet.
3 M sodium acetate = 24.6 gm of sodiumacetate (molecular weight = 82.03) in 100 ml ofdistilled water.
Perform PCR sequencing reaction
The sequencing PCR is done using the BigDyeTerminator v3.0 kit from Applied Biosystems.
There is only one primer per sequencingreaction. Therefore, the universal forward primerused in the amplification PCR is used in onesequencing tube, and the reverse primer in asecond tube. It is advisable to do each primer induplicate or triplicate to check for errors due tomispriming.
The sequencing PCR master mix (Table 6.7)is the recommended protocol from Applied Bio-systems for use with their BigDye V3.0 CycleSequencing kit.
The full reaction volume is that recom-mended by the supplier. The volume of wateris used to adjust for the concentration of DNA.Sequencing results will not be obtained if the DNAis too dilute. For a thermocycler without a hot lid,overlay tubes with 30 ml mineral oil.
Thermocycling conditions for thesequencing PCR
Perform one cycle at 96°C for 2 min. Run 25cycles of 96°C for 30 s, 50°C annealing for 30 s,60°C for 4 min. Hold at 4°C. Times may beshorter, depending upon the thermocycler used.
Product purification before sequencing
The product from the sequencing PCR reactionneeds to be purified to remove salts and unincor-porated dye terminators otherwise the first 100
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Reagent Half reaction (ml) Full reaction (ml)
Water (ultra-pure) 0.5 0Terminator ready mix 4.5 8Primer F 0.5 1DNA (10-30 ng/ml) 5.5 11Volume ml (including DNA) 10.5 20
Table 6.7. Sequencing PCR master mix.
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bases of the sequence will not be readable.Clean-up can be done by using Centrisepcolumns from Applied Biosystems, MicroBio-Spin 30 columns from Bio-Rad or DyeExDye-Terminator Removal system from Qiagen.
A manual protocol also gives good resultsand is based on an ethanol precipitation.
1. Into a 0.6 ml microfuge tube place 25 ml of95% ethanol (non-denatured, absolute alcohol)2. Add 1 µl of 3 M sodium acetate, pH 4.63. Add entire PCR product (10 ml if a halfreaction volume was used)4. Place on ice for 10–20 min. Some methodsrecommend a strict 10 min only5. Centrifuge 13,000 rpm at room temperaturein a table-top centrifuge for 30 min. Keep thehinges of the tube to the outside so that the DNApellet can be more easily located6. Carefully remove supernatant withoutdisturbing the pellet, which may not be visible7. Immediately add 125 ml of 70% ethanol andgently roll the tube8. Centrifuge 13,000 rpm for 5 min9. Carefully remove the supernatant withoutdisturbing the pellet10. Air dry or vacuum dry the pellet11. Send the dried product to a sequencingfacility.
Sequencing
The sequencing reaction is not covered in thismanual. Usually, this reaction is carried out by aspecialized laboratory.
Analysis of sequence information using BIOEDIT
The sequence information can be analysedby a number of different programs. Proprietarysoftware from Applied Biosystems is available forpurchase from the company.
However, there are a number of freewareprograms available and a very comprehensiveone is called BIOEDIT. It is available for downloadfrom http://www.mbio.ncsu.edu/Bioedit/bioedit
Citation: Hall, T.A. (1999) BioEdit: a user-friendly biological sequence alignment editor andanalysis program for Windows 95/98/NT. NucleicAcids Symposium Series 41 95–98.
USING BIOEDIT. A brief explanation of theuse of BIOEDIT for analysing sequence data isexplained here, but the reader is referred to the
help notes under ‘General use of BIOEDIT’contained within the program.
The sequence information is usually e-mailedto the client laboratory. Open the e-mail and savethe attachments into a folder on the hard drive.
Open the BIOEDIT program. Select OPENSEQUENCE SET. This opens an ‘open file’ win-dow. The sequence files will have the ending .ab1.Select the sequence to be examined and doubleclick. This places the title of the sequence inthe left-hand side of the BIOEDIT window, and theDNA sequence in the right-hand side window. Achromatogram window is also seen. Position thetwo windows so that both can be seen and checkthe sequence for inaccuracies. In many cases wherea no base is called (N), checking the chromato-graph can interpret the correct base. Do thesame with the duplicate sequence. Once both arechecked a consensus sequence can be obtained.
First select the duplicate sequence by clickingin the left-hand window to highlight the sequencename. Go to the menu bar and EDIT, COPYSEQUENCE. Then click on the window con-taining the first sequence. Go to the menu barand EDIT, PASTE SEQUENCE. The primaryand duplicate sequences will now be in theone window.
While holding down the shift key, click onboth sequence names in the left-hand windowto highlight both sequences. Go to menu barSEQUENCE, PAIRWISE ALIGNMENT, ALIGNTWO SEQUENCES (optimal global alignment).A new window appears with the alignment resultfor the two sequences. From this window go tothe menu bar and select ALIGNMENT, CREATECONSENSUS SEQUENCE. A window nowappears with the consensus sequence. To savethe consensus sequence, highlight the consensussequence in the left-hand window, go to the menubar EDIT, COPY SEQUENCE. Open a new worddocument and select PASTE in the Word menubar. The consensus sequence will appear thus:
>ConsensusGGTACTGACC etc.
Re-name the sequence as appropriate. Thisformat is known as ‘fasta’ format, and is the formatrequired for a BLAST search.
ALIGNING TWO SEQUENCES USING CLUSTAL W.
BIOEDIT also contains the CLUSTAL program. Touse CLUSTAL within BIOEDIT to align two sequen-ces, select the NEW ALIGNMENT icon. Go to the
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document with the sequence to be aligned. Makesure the sequence is in fasta format. Go back tothe BIOEDIT NEW ALIGNMENT window. UnderFILE, select IMPORT FROM CLIPBOARD. Thesequence name and sequence will appear in thewindow. Do this for the second sequence. UnderACCESSORY APPLICATION, select CLUSTAL.Run CLUSTAL. When it is finished, close theCLUSTAL window to see the alignment in theBIOEDIT window.
If the reverse complement is needed, underSEQUENCE go to NUCLEIC ACID, then toREVERSE COMPLEMENT.
CLUSTAL W citation: Thompson, J.D.,Higgins, D.G. and Gibson, T.J. (1994) Clustal W:improving the sensitivity of progressive multiplesequence alignment through sequence weighting,position-specific gap penalties and weight matrixchoice. Nucleic Acids Research 22, 4673–4680.
WEB SOFTWARE FOR MULTIPLE ALIGNMENTS.
http://dot.imgen.bcm.tmc.edu:9331/multi-align/multi-align.htmlhttp://www.ebi.ac.uk/clustalw/Clustal alignmentprogramhttp://www.ncbi.nlm.nih.gov/blast/bl2seq/bl2.html Blast for alignment of two sequenceshttp://www.mbio.ncsu.edu/Bioedit/bioeditBioEdit program for sequence analysishttp://www.technelysium.com.au/chromas.htmlor http://bioinfo.weizmann.ac.il/pub/software/chromas (Chromas is a sequence editor)
BLAST SEARCH FOR SEQUENCE IDENTIFICATION.
The National Centre for Biotechnology Informa-tion is located at http://www. ncbi.nlm.nih.govSelect the BLAST icon. There is a tutorial there.However, BLAST is very easy to use. SelectNUCLEOTIDE BLAST. In the search windowpaste the sequence in fasta format. Press NOWBLAST IT. Press FORMAT to get the results. Theresults are placed in a queue and it may take 1 or 2minutes for the results to be downloaded to thedesktop.
Citation for BLAST: Altschul, S.F., Madden,T.L., Schäffer, A.A., Zhang, J., Zhang, Z., Miller,W. and Lipman, D.J. (1997) ‘Gapped BLASTand PSI-BLAST: a new generation of proteindatabase search programs’. Nucleic AcidsResearch 25, 3389–3402.
The Ribosomal Database Project containsribosomal sequence information at http://rdp.cme.msu.edu/html/
6.4 Fluorescence in Situ Hybridization(FISH)
FISH is used increasingly in clinical laboratoriesfor the identification of bacteria from clinicalsamples (DeLong et al., 1989; Hogardt et al.,2000; Jansen et al., 2000).
Using the same principle, oligonucleotides orprimers that are species-specific as shown by aBLAST search of experimentation can be used todevelop sensitive FISH assays for aquatic bacte-ria. The primers listed in Table 6.1 would be agood starting point for developing specific FISHassays that could be applied to smears of coloniesgrown on agar plates. Although none of theseprimers has been tested as being suitable forspecific probes, it was suggested that eight prim-ers that were species-specific for V. costicola, V.diazotrophicus, V. fluvialis, V. nigripulchritudo, V.proteolyticus, V. salmonicida, V. splendidus andV. tubiashii would be suitable for use as specificprobes (Lee et al., 2002).
Outline of steps for FISH
Step Method1 Prepare smear from bacterial colonies2 Hybridization3 Washing4 Microscopic examination
Oligonucleotide probes
Primers should be selected from Table 6.1 and aBLAST search conducted to ascertain whether theprimers are species-specific. The single-strandedoligonucleotides are synthesized and covalentlylabelled with fluorescein isothiocyanate at the 5′end.
A universal positive probe is prepared usingEUB primer 5′-GCTGCCTCCCGTAGGAGT-3′.This sequence corresponds to positions 338–355on the E. coli numbering system. A universalnegative probe is prepared using primernon-EUB 5′-ACTCCTACGGGAGGCAGC-3′(Amann et al., 1990; Jansen et al., 2000).Fluorescein isothiocyanate is added to the 5′ endof each primer.
Prior to use, the probes are diluted to aconcentration of 10 ng/ml in hybridization buffer.
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Preparation of smears
Bacterial cells from a colony are emulsifiedin a drop (10–15 ml) of sterile saline or steriledistilled water within a marked area on a glassmicroscope slide. The suspension should not betoo thick, but should allow individual cells to beseen under the microscope. Prepare one dropper probe to be tested plus drops for the positiveand negative controls. Allow to air dry.
Once dry, the cells are fixed to the slide byusing a fixative solution of 4% formaldehyde in96% ethanol. Gram-positive organisms mustbe permeabilized before application of thehybridization buffer. The fixed slide is placed intopermeabilization buffer of 1 mg/ml of lysozymefor 5 min. Gram-negative organisms do notneed the permeabilization step (Jansen et al.,2000).
Hybridization
The fixed slides are hybridized in a hybridizationbuffer of 20 mM Tris-HCl, 0.9 M NaCl, 0.1%sodium dodecyl sulphate, pH 7.2, which containsthe probe at a concentration of 10 ng/ml. Thehybridization buffer (10–15 ml) can be placed ontop of the fixed cells. Gram-negative organismsare hybridized for 45 min, and Gram-positivesfor 2 h at a temperature of 50°C (Jansen et al.,2000).
One hybridization method pretreated theslides by placing them in ascending ethanol stepsof 50, 80 and 96% for 3 min each step. Thehybridization buffer then contained 30–40%formamide with 50 ng of probe. This concen-tration of formamide improved the specificity ofthe probe (Hogardt et al., 2000). However, aconcentration of 20% (vol/vol) of formamide didnot show an increase in probe specificity (Jansenet al., 2000).
Washing
The slides are washed in washing buffer of20 mM Tris-HCl, 0.9 M NaCl, pH 7.2, at atemperature of 50°C for 10 min (Jansen et al.,2000).
Examination of smears
The slides are mounted with VectaShield(Vector Laboratories, Burlingame, California), orCitifluor (Citifluor Ltd, London, UK).
The slides are examined for cells showingfluorescence (positive result), using a fluorescencemicroscope and filter set capable of detectingfluorescein. Fluorescence should be seen with thepositive control, and no fluorescence with thenegative control.
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7Preparation of Media for Culture and Identification
7.1 General Isolation and Selective Media
Acetate agar. See Rogosa medium under ‘Test media’
Alkaline peptone water (APW) (May be used as an enrichment medium for Vibrio species; Furnisset al., 1978)
Reagent Amount Preparation of media Description of growth characteristicsPeptone 10.0 g Dissolve reagents in distilled water and
pH to 8.6. Dispense in 10 ml aliquotsto McCartney bottles and autoclave at121°C for 20 min.
May be used as an enrichment medium to isolateVibrio species from contaminated samples such asfaeces and polluted water. To be effective, the APWcultures should be subcultured at 6 h if incubated at37°C and overnight if incubated at 18–20°C.
Sodium chloride 10.0 gDistilled water 1000 ml
Amies transport medium (Oxoid)This transport medium can be purchased ready-made as swabs in tubes of media, or prepared from apowdered medium available from Oxoid.
Formulae: 10.0 g charcoal (pharmaceutical), 3.0 g sodium chloride, 1.15 g sodium hydrogenphosphate, 0.2 g potassium dihydrogen phosphate, 0.2 g potassium chloride, 1.0 g sodiumthioglycollate, 0.1 g calcium chloride, 0.1 g magnesium chloride, 4.0 g agar, 1000 ml distilled water,pH 7.2.
Anacker-Ordal agar (AO) (Used for growth of freshwater and marine Cytophaga, Flavobacteriumand Flexibacter spp.; Anacker and Ordal, 1955, 1959)
Reagent Amount Preparation of media Description of growth characteristicsBacto-tryptone (Difco) 0.5 g Add all reagents to
1000 ml of distilled water,and adjust pH to 7.2–7.4.Autoclave at 121°C for15 min (15 lb/20 min). Coolto 50°C and pour mediainto Petri dishes. Storeplates in sealed plasticbags at 4°C.
Using a cotton-tipped swab, collect samples from skinlesions and gills of fish and inoculate the plate. Colonies ofF. columnare appear at 2–5 days as yellow-pigmented,rhizoid colonies with a thin spreading growth. Seephotographic section.
Early colonies may be viewed with the aid of astereomicroscope. Subculture by cutting out a block of agarcontaining the colony, and invert on to new plate. A Pasteurpipette prepared in the shape of a ‘hockey stick’ with theend unsealed can be used to pick out a plug of agar.
Yeast extract (Difco) 0.5 gSodium acetate 0.2 gBeef extract (Difco) orLab-Lemco powder (Oxoid)
0.2 g
Agar (Difco or Oxoid AgarNo 1)
9 g/l
Distilled water 1000 ml
©N.B. Buller 2004. Bacteria from Fish and Other Aquatic Animals:244 a Practical Identification Manual (N.B. Buller)
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Anacker-Ordal agar – marine (AO-M) (Used for growth of marine Cytophaga, Flavobacteriumand Flexibacter spp. and Tenacibaculum (Flexibacter) maritimum; Anacker and Ordal, 1955, 1959)Add artificial seawater salts (Sigma) at 38 g/l. If using seawater, use sterile at 50–100% final concentra-tion (Ostland et al., 1999b).
Anaerobe plates (ANA). For growth of anaerobic Gram-positive and Gram-negative organisms(Oxoid manual)
Reagent Amount Preparation of media Description of growth characteristicsOxoid Wilkins-ChalgrenAnaerobe agar
21.50 g Suspend powder in water in a 1000 ml Schottbottle with magnetic flea. Autoclave at 121°C for20 min then cool to 50°C in waterbath. Asepticallyadd blood. Pour into Petri dishes.
Inoculated plates are incubated atthe appropriate temperature in ananaerobic atmosphere.Distilled water 500 ml
Sterile equine blood 30 ml
Gram-negative anaerobe plates (ANA-GN). For growth of anaerobic Gram-negative organisms(Oxoid manual)
Reagent Amount Preparation of media Description of growth characteristicsOxoid Wilkins-ChalgrenAnaerobe agar
21.50 g Dissolve agar in water. Autoclave at 121°Cfor 20 min then cool to 50°C in a waterbath.Reconstitute 1 vial of supplement with10 ml sterile distilled water and add to base.Aseptically add blood. Pour into Petri dishes.
Inoculated plates are incubated atthe appropriate temperature in ananaerobic atmosphere.Distilled water 500 ml
Sterile equine blood 15 mlOxoid G-N selective supplement 10 ml
Artificial seawater – ASW: Sea salts (Sigma product number S 9883)
Reagent Amount Preparation of media Description of growth characteristicsSea salt (Sigma) 38 g Add salts to distilled water and
pH to 7.6. Autoclave at 121°C for15 min.
May be added to media for growth of marine organisms. Addto AO medium for isolation of marine Flavobacterium andTenacibaculum species.
Distilled water 1000 ml
Addition of 18.7 g/l makes a 50% seawater concentration of 17.5‰ salinity.
Artificial seawater medium (Lewin, 1974 – Used for isolation of marine Flexibacter/Flavobacterium group. Baumann et al., 1971, quoted in MacLeod, 1968, is the same medium butwithout the trace element mixture – Used for marine Vibrio species)
Reagent Amount Preparation of media Description of growth characteristicsNaCl 20.0 g Dissolve reagents in distilled water.
Add 1.0 ml of trace element solution.Adjust pH to 7.5 and autoclave at121°C for 20 min.
Can be used as an inoculatingmedium for biochemical identificationsets or as an initial isolation broth formarine organisms. Add agar at 15 g/land pour into Petri dishes if a solidmedium is required.
Lewin used this medium for marineFlavobacterium and Flexibacterspecies. The medium used byMacLeod and Baumann did notcontain the trace element solution.
MgSO4.7H2O 5.0 gKCl 1.0 gCaCl2.2H2O 1.0 gDistilled water 1000 mlB (soluble salt of element) 0.5 mg/ml Prepare a stock solution of trace
elements so that the final concentrationof each element is either 0.5 mg/l or0.01 mg/l.
Fe (soluble salt of element) 0.5 mg/mlMn (soluble salt of element) 0.5 mg/mlCo (soluble salt of element) 0.01 mg/mlCu (soluble salt of element) 0.01 mg/mlMo (soluble salt of element) 0.01 mg/mlZn (soluble salt of element) 0.01 mg/ml
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Blood agar – BA
Reagent Amount Preparation of media Description of growth characteristicsOxoid ColumbiaBA base
19.5 g Suspend agar base in water. Autoclave at 121°C for15 min. Cool in waterbath to 50°C. Add blood to cooledagar, mix well and pour into Petri dishes to depth ofapproximately 3 mm. Store at 4°C in sealed plastic bags.(Plates can be left overnight on the bench and sealed inplastic bags the following day. This prevents too muchmoisture build-up once stored in the plastic bags.)
Plates are inoculated with a swab ofthe specimen, and incubated at theappropriate temperature andatmosphere. Plates are examineddaily for growth and haemolysis.
Distilled water 500 mlSterile equine blood 15 ml
Bordetella bronchiseptica selective agar – CFPA medium (Smith and Baskerville, 1979;Rutter, 1981; Hommez et al., 1983)
Reagent Amount Preparation of media Description of growth characteristicsColumbia agar base (Oxoid) 19.5 g Add agars to distilled water and autoclave at
121°C for 15 min. Cool to 50°C. Add sterileblood to cooled agar mixture. Reconstitute1 vial of Bordetella supplement with 2 ml ofdistilled water and add to mixture. Add 5 mlof antibiotic stock. Pour plates and store at4°C.
Colonies are 1 mm at 48 h and maybe haemolytic or non-haemolytic,opaque, smooth and pearl-like, orrough, translucent, raised in thecentre with an undulating margin,depending on phase variation.
Agar technical No. 3 (Oxoid) 10.0 gDistilled water 500 mlBordetella Pertussis supplement(Oxoid, Code SR082E)
2 ml
Sterile equine blood 15 ml
Penicillin 20 mg Antibiotic stock: Add 20 mg of each antibioticto 10 ml of normal saline. Store in fridge.Handle furaltadone with caution and usemask and gloves.
Furaltadone 20 mgNormal saline 10 ml
Brucella agar (Available from Difco or Oxoid media suppliers. See also under Farrell’s medium)
Burkholderia pseudomallei selective media – glycerol medium (Thomas et al., 1979)
Reagent Amount Preparation of media Description of growth characteristicsAgar No 3 (Oxoid) 2.4 g Add all reagents to distilled water (glycerol may
be warmed to aid pipetting). Autoclave at 121°Cfor 15 min. Cool to 50°C and pour into Petridishes. Store plates at 4°C.
Plates are a mauve colour.Colonies appear at 24 h, smooth,mauve-coloured with slight metallicsheen. As incubation increases,colonies become wrinkled andumbonate. Incubate for 4 days.
Glycerol 6.0 mlCrystal violet stock solution 0.5 mlDistilled water 194 mlCrystal violet – Stock(1/5000 dilution)
0.5 g Add 0.5 g of crystal violet to distilled water andstir until dissolved. Store at room temperature.
Distilled water 100 ml
Burkholderia pseudomallei selective broth (Modified from Thomas et al., 1979)
Reagent Amount Preparation of media Description of growth characteristicsMacConkey broth (purple) (Oxoid) 100 ml Prepare MacConkey broth and add
reagents except antibiotics. Autoclave at121°C for 15 min. Cool to 50°C and addfilter-sterilized antibiotics. Asepticallydispense into sterile McCartney bottles.
Detection of Burkholderia can beimproved by incubating material inbroth for 24 and 48 h, followed bysubculture to plates.
Crystal violet 0.001 gGentamycin 0.8 mgStreptomycin sulphate 5000 units
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Burkholderia pseudomallei selective media – Ashdown’s medium (Ashdown, 1979a)
Reagent Amount Preparation of media Description of growth characteristicsTryptone soy agar 40 g Add all ingredients except gentamycin to distilled water.
Autoclave at 121°C for 15 min. Cool to 50°C. Add filtersterilized gentamycin. Mix well and pour into Petri dishes.
Colonies are flat, rough, wrinkledafter 3 days. Colonies ofBurkholderia pseudomallei absorbthe Neutral red after 3 days whereasPseudomonas species have no dyeuptake at 3 days.
Glycerol 40 mlCrystal violet 5 mgNeutral red 50 mgGentamycin 4 mgDistilled water 1000 ml
Carbon dioxide atmosphere. See under ‘Test media’
Cellobiose-Colistin agar – For selective isolation of Vibrio vulnificus (Massad and Oliver, 1987;Høi et al., 1998a)
Reagent Amount Preparation of media Description of growth characteristicsSolution 1 Adjust pH to 7.6. Autoclave at 121°C for
15 min and cool to 55°C.The medium is olive green to lightbrown. Incubate plates at 40°C for24–48 h. V. vulnificus appears as yellowcolonies surrounded by a yellow zonedue to fermentation of cellobiose.
V. cholerae appears as purple coloniessurrounded by a blue zone.
Bacto-peptone (Difco) 10 gBeef extract (Difco) 5 gNaCl 20 gBromothymol blue 40 mgCresol Red 40 mgAgar 15 gDistilled water 900 mlSolution 2 Filter sterilize. Add to cooled reagents of
solution 1 and mix. Dispense into Petri dishes.Cellobiose 15 gColistin 0.03 mg/ml
(4 × 105 U/l)Distilled water 100 ml
CFPA media. See under Bordetella bronchiseptica medium
Dermatophilus selective medium – polymyxin plates (Abu-Samra and Walton, 1977)
Reagent Amount Preparation of media Description of growth characteristicsColumbia agar base(Oxoid)
19.5 g Add agar base to distilledwater and autoclave at121°C for 15 min. Cool to50°C and aseptically addblood and polymixin.Polymixin B may bedissolved in sterile normalsaline first.
Grind scab material using a sterile pestle and mortar. Transferfinely ground material to a bijou bottle and add 2 parts ofdistilled water. Shake thoroughly then allow to settle for 15 min.Take a loopful of surface material and plate to BA and polymixinplates. Incubate at 25 or 37°C depending on the habitat of theinfected animal. Examine plates at 24 and 48 h for presence ofadherent pitted dry or mucoid colonies. This medium is notcompletely selective and some contaminating bacteria will grow.
Distilled water 500 mlSterile equine blood 50 mlPolymixin B (Use at aratio of 1000 IU/ml ofmedium)
62.5 mg
Dubos medium. For isolation of Cytophaga hutchinsonii (Bernardet and Grimont, 1989)
Reagent Amount Preparation of media Description of growth characteristicsNaNO3 0.5 g Add all reagents except cellobiose, to distilled water.
Adjust pH to 7.2 and autoclave at 121°C for 15 min.Cool to 50°C and add filter-sterilized cellobiose solution.Mix well and pour into Petri dishes.
K2HPO4 1 gMgSO4.7H2O 0.5 gKCl 0.5 gFeSO4.7H2O 0.01 gDistilled water 1000 mlAgar 15 gD-cellobiose 30% w/v Prepare cellobiose solution and filter-sterilize.
Note: For isolation of Cellulomonas species add 0.5 g yeast extract.
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Edwardsiella ictaluri medium (EIM). For isolation of Edwardsiella ictaluri (Shotts and Waltman,1990)
Reagent Amount Preparation of media Description of growth characteristicsBacto-tryptone (Difco) 10 g Dissolve all reagents in distilled
water and adjust the pH to 7.0–7.2.Autoclave at 121°C for 15 min.Cool to 50°C and add the 10 mlfilter-sterilized solution 1 containingmannitol, colistin, bile salts andfungizone. Mix well and pour intoPetri dishes.
This medium is used for the isolation ofEdwardsiella ictaluri. Most Gram-negative bacteriaare inhibited with the exception of Proteus spp.,Serratia marcescens, Aeromonas hydrophila andYersinia ruckeri. Gram-positive bacteria areinhibited with the exception of Enterococci. 90%of E. tarda isolates grow on this medium.
E. ictaluri is seen as 0.5–1.0 mm green,translucent colonies at 48 h. Colonies of E. tardahave a similar size and appearance.Proteus species are 2–3 mm, brownish-green andmay swarm. S. marcescens colonies are 2–3 mmand reddish brown. Y. ruckeri are 1–2 mmyellowish green, A. hydrophila are 2–3 mmyellowish-green opaque colonies and Enterococciappear as 0.5 mm yellowish colonies.
Yeast extract (Difco) 10 gPhenylalanine 1.25 gFerric ammonium citrate 1.2 gSodium chloride 5.0 gBromothymol blue 0.03 gAgar (Difco) 17 gDistilled water 990 mlSolution 1Mannitol 3.5 g Add reagents to 10 ml of distilled
water and filter-sterilize.Colistin 10 mgBile salts 1 gFungizone 0.5 mgDistilled water 10 ml
Electrolyte supplement (Added to biochemical test media to improve growth of organism whenNa+ alone is insufficient; Lee et al., 1979)
Reagent Amount Preparation of media Description of growth characteristicsNaCl 100 g Add all reagents to distilled water and
autoclave at 121°C for 15 min.If addition of NaCl alone to biochemical identificationmedia does not improve the growth of an organism, thiselectrolyte supplement may improve growth. It is addedat a rate of 0.1 ml of supplement per 1.0 ml of medium.
MgCl2.6H2O 40 gKCl 10 gDistilled water 1000 ml
Erysipelothrix selective medium. See ‘Wood’s’ broth, and ‘Packer’s’ plates
Farrell’s medium. For the growth of Brucella spp. (Farrell, 1974)
Reagent Amount Preparation of media Description of growth characteristicsOxoid bloodagar base
20.0 g Add agar to distilled water and autoclave at 121°C for15 min. Cool to 50°C and leave at this temperature whilethe other reagents are prepared.
Incubate plates at 37°C in 10% CO2 for14 days.
Isolates from sea mammals appear after4 days incubation, whereas isolates fromseals do not appear until day 10 ofincubation, or do not grow. Incubation ofplates should continue for 14 days.
It has been suggested that the isolationof strains from seals may be improvedby decreasing the concentration orremoving bacitracin and/or nalidixic acidfrom the medium (Foster et al., 2002).
Always inoculate a non-selectivemedium such as blood agar or serumdextrose medium.
Colonies are 1–2 mm in diameter at 5days incubation, pale yellow,translucent, convex and round withentire edges.
Distilled water 500 mlNormal horseserum
25 ml Inactivate the horse serum at 50°C for 35 min.
25% Dextrose(D-Glucose)
20 ml Add 125 g of D-glucose to 375 ml distilled water anddissolve by gentle heating. Dispense into 20 ml aliquotsinto McCartney bottles and with lids loose, autoclave at121°C for 15 min. Store at 4°C.
Oxoid Brucellaselectivesupplement
10 ml Dissolve Brucella supplement in 5 ml sterile distilled waterand 5 ml methanol. Incubate for 10–15 min at 37°C andshake well to dissolve.
For complete Farrell’s medium aseptically combine allingredients while stirring.
Mix well and pour media into plates to an approximatedepth of 3 mm.
Final mediapreparation
Combine all prepared reagents (serum, dextrose,supplement) into agar. Mix well and pour into Petridishes to a depth of 3 mm.
The original medium uses the following antibiotic concentrations: bacitracin (25 units/ml), vancomycin (20 mg/ml), polymixin B(5 units/ml), nalidixic acid (5 mg/ml), nystatin (100 units/ml) and cycloheximide (100 mg/ml) (Farrell, 1974).
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Farrell’s medium is available from Oxoid as Brucella medium base, to which Brucella selective sup-plement (Oxoid code SR83) is added. The selective supplement is based on the Farrell formulation.
Flavobacterium maritimus media (FMM). May improve primary isolation for T. maritimum(Pazos et al., 1996)
Reagent Amount Preparation of media Description of growth characteristicsPeptone 5.0 g Add reagents and powders to sterile
seawater. Adjust pH to 7.2–7.4 and sterilizeby autoclaving at 121°C for 20 min.
Colonies pale-yellow, flat, irregular withuneven edges. Reduces growth of Vibriospecies and Aeromonas species that mayoutgrow Flexibacter on media such as MSA-Bor MA 2216.
Yeast extract 0.5 gSodium acetate 0.01 gAgar 15 gSterile seawater 1000 ml
Flavobacterium psychrophilum medium (FPM). May improve isolation rate and colony size forF. psychrophilum (Daskalov et al., 1999)
Reagent Amount Preparation of media Description of growth characteristicsTryptone T (Oxoid) 0.5 g Add all reagents (except sugars and
skimmed milk) to distilled water andautoclave at 121°C for 15 min. Cool ina 50°C waterbath.
Prepare supplements (galactose,glucose, rhamnose and skimmed milk)as separate 10% (w/v) solutions, andfilter by sterilization thorough a 0.22 mmpore size Millipore Millex porosity filter.Add filter-sterilized solutions to cooledagar medium. Pour into Petri dishes andstore at 4°C.
Colonies grow after 3–6 days andare an intense yellow colour with aspreading and irregular shape.
Yeast extract (Oxoid) 0.5 gBeef extract (Oxoid) 0.2 gSodium acetate trihydrate (Sigma) 0.2 gD(+) galactose (Sigma) 0.5 gD(+) glucose (BDH) 0.5 gL-rhamnose (Sigma) 0.5 gSkimmed milk (Oxoid) 0.5 gWater 1000 mlAgar – bacteriological agar no. 1(Oxoid)
9.0 g
This medium is based on Anacker Ordal (AO) agar (also known as Cytophaga agar) supplemented with galactose, glucose,rhamnose and skimmed milk. The size and number of colonies of F. psychrophilum isolated is increased compared to AO medium.Broth can be prepared without the addition of agar.
Flexibacter polymorphus media (Lewin, 1974)
Reagent Amount Preparation of media Description of growth characteristicsNaCl 20.0 g Dissolve reagents in distilled water.
Adjust pH to 7.5 and autoclave at121°C for 20 min.
The organism is maintained in 5 ml of the brothmedium and subcultured twice weekly. Filamentsseveral hundred microns in length and 1.5 mm wideare produced. They have rounded ends, areflexuous, unbranched and cylindrical. The growingfilaments may be peach coloured. Cultures in lategrowth phase may show a refractile granule at eachend, which is more easily seen after cell lysis.
Cobalamin is essential for growth.
Agar can be added to the medium for plate media.
The organism also grows on agar medium preparedwith seawater and yeast extract (10 mg/ml). Coloniesmay be peach coloured with a filamentous margin.
MgSO4.7H2O 5.0 gKCl 1.0 gCaCl2.2H2O 1.0 gFe (soluble salt) 0.5 mgB (soluble salt) 0.5 mgMn (soluble salt) 0.5 mgCo (soluble salt) 0.01 mgCu (soluble salt) 0.01 mgMo (soluble salt) 0.01 mgZn (soluble salt) 0.01 mgDistilled water 1000 ml
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Flexibacter maintenance medium (Lewin and Lounsbery, 1969; Lewin, 1974)
Reagent Amount Preparation of media Description of growth characteristicsTryptone (Difco) 1.0 g Add all reagents to filtered seawater
and adjust pH to 7.5. Dispensemedia into tubes. Autoclave at121°C for 20 min.
Optimum cobalamin is 0.3 mg/l.
The organisms are maintained in 5 ml of thebroth medium, incubated between 22 and32°C, and subcultured twice weekly.
The Flexibacteria may show a range ofpigmented filaments ranging from pink,orange or yellow colouration. On solidmedium they may produce fimbriate margins.
Agar can be added to the medium for platemedia.
A semi-solid medium can be produced using0.3% agar, and viable filaments can bemaintained for up to 1 month when grownin this medium at room temperature.
Casamino acids (Difco) 1.0 gMonosodium glutamate 5.0 gSodium glycerophosphate 0.1 gCobalamin 1.0 ugFe (soluble salt) 0.5 mgB (soluble salt) 0.5 mgMn (soluble salt) 0.5 mgCo (soluble salt) 0.01 mgCu (soluble salt) 0.01 mgMo (soluble salt) 0.01 mgZn (soluble salt) 0.01 mgFiltered seawater 1000 ml
Glycerol Lab Lemco broth (Used for storage of cultures at −80°C)
Reagent Amount Preparation of media Description of growth characteristicsLab Lemco broth(Oxoid)
0.64 g Add all reagents together. Pipettingof glycerol may be facilitated bypre-warming. Dispense into 2 mlvolumes into Bijou bottles.Autoclave at 121°C for 15 min.Store media at 4°C.
Used as −80°C storage medium. Using a sterilecotton-tipped swab, scrape bacterial growth from an agarplate and inoculate into 1 ml of Glycerol Lab Lemco mediumin a Nunc tube. Use an inoculum of approximately tube 5McFarland opacity. Place tube in cryobox in −80°C freezer.This medium is also suitable for liquid nitrogen storage.
Glycerol 20 mlDistilled water 80 ml
Helicobacter selective media. Use Skirrow’s medium (also known as VPT media; Skirrow, 1977)
Reagent Amount Preparation of media Description of growth characteristicsBlood agar baseNo. 2 (Oxoid)
20 g The agar is dissolved in waterand autoclaved at 121°C for15 min. After cooling to 50°Cadd 15 ml of sterile equineblood. Reconstitute 1 vial ofsupplement using 2 ml of steriledistilled water and add to cooledmedia. Mix thoroughly. Pourmedia into plates to anapproximate depth of 3 mm.Store plates at 4°C.
Other bases such as Columbia agar base (Oxoid), orBrucella medium base (Oxoid), or tryptone soy agar maybe used to prepare this medium. However, it is suggestedthat blood agar base No. 2 is more nutritionally rich andalso that trimethoprim is more active in this base.
To reduce contamination from other organisms, thesample can be filtered through a 0.65 mm filter and thefiltrate cultured to the plate (Butzler et al., 1973).
The plates are incubated at 37°C in a microaerophilicatmosphere of N2, H2, CO2 (80:10:10) for 2–4 weeks. Gasgenerating packs are available from commercial supplierssuch as MGC Anaero Pak Campylo from Mitsubishi GasChemical Company.
Helicobacter species will grow as pin-point colonies andalso appear as a thin spreading film across the plate.
Distilled water 500 mlSterile equine blood 15 mlOxoid Campylobactersupplement (Skirrow)
2 ml
This medium was developed by M.B. Skirrow (1977) for the isolation of Campylobacter species. It is also suitable for the isolation ofHelicobacter species and is widely quoted in many references. It is also available as ready-made media from a number of mediaproducers. The antibiotics are often supplied as a supplement and, depending on the originators of the medium, the supplementswill be named accordingly. For example, Oxoid produces ‘Campylobacter selective supplement (Skirrow) for additions ofvancomycin, polymyxin and trimethoprim, and Campylobacter selective supplement (Blaser-Wang) for additions of vancomycin,polymyxin, trimethoprim, amphotericin B, and cephalothin’. The additive containing the amphotericin B is recommended whenfungal contamination is suspected. The original concentrations described by Skirrow (1977) are vancomycin (10 mg/l), polymyxin B(2.5 IU/ml), and trimethoprim (5 mg/l). A series of papers describing the isolation and identification of Helicobacter cetorum fromdolphins and whales (Harper et al., 2000, 2002a,b) describes the use of TVP and CVA media from Remel. These are, in essence,Skirrow’s media and Blaser-Wang media, respectively.
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Hsu-Shotts agar – HS (For Flavobacterium columnare, F. psychrophilum; Bullock et al., 1986)
Reagent Amount Preparation of media Description of growth characteristicsTryptone 2 g Add all reagents to distilled water, except neomycin
sulphate. Autoclave at 121°C for 15 min. Cool to50°C. Add filter-sterilized neomycin sulphate, mixwell and pour into Petri dishes.
This is a semi-selective medium forthe isolation of Flavobacteriumcolumnare. At 48 h characteristicyellow, spreading colonies are seen.
Yeast extract 0.5 gGelatin 3 gAgar 15 gDistilled water 1000 mlNeomycin sulphate 4.0 mg/ml Prepare solution and filter-sterilize.
Note: HSM. Addition of 18.7 g/l sea salts (Sigma) to make a 50% seawater concentration (17.5%° salinity), and 200 IU/mlpolymyxin B is suitable for isolation of Tenacibaculum (Flexibacter) maritimum (Chen et al., 1995).
Inositol horse serum (Suspension medium for freeze-drying organisms)
Reagent Amount Preparation of media Description of growth characteristicsInositol 5 g Dissolve inositol in horse serum. Filter through
a 0.45 mm filter followed by further filtrationthrough a 0.22 mm filter for sterilization. Checksterility. Dispense into McCartney bottles andstore at 4°C.
Place 1 ml of sterile inositol horse serum into afreeze-dried vial (Wheaton serum bottle). Using asterile cotton swab or loop emulsify a heavysuspension of bacteria in the medium. Snap-freezein liquid nitrogen and follow instructions forfreeze-drying relevant to the equipment being used.
Horse serum 100 ml
KDM2. For growth and isolation of Renibacterium salmoninarum (Evelyn, 1977)
Reagent Amount Preparation of media Description of growth characteristicsTryptone 1 g Add all reagents to distilled water and
adjust pH to 6.5–6.8 (with NaOH).Dispense into tubes and autoclave at121°C for 20 min.
Store at 4°C for 1 month.
Incubate plates at 15°C for up to 2 months.Initial growth is visible between 2 to 8 weeks.Colonies are pin-point to 2 mm in size. Oldcolonies may have a granular or crystallineappearance. To assist in prevention ofovergrowth by fast growing colonies,examine plates every few days andaseptically remove these colonies.
Yeast extract 0.05 gL-cysteine (chlorhydrate) 0.1 gAgar 1.5 gDistilled water 100 mlFetal calf serum 5–10% For use, heat tubes. Cool to 50°C and
add calf serum. Pour into plates.
KDMC. For Renibacterium salmoninarum (Daly and Stevenson, 1985)
Reagent Amount Preparation of media Description of growth characteristicsKDM2 medium As above. Addition of charcoal acts as a
detoxifying agent.Activated charcoal (Difco) 0.1% For use heat tubes. Cool to 50°C and addcharcoal. Pour into plates.
Marine salt agar with blood (MSA-B) (Used as a general-purpose medium for isolation oforganisms from the marine environment)
Reagent Amount Preparation of media Description of growth characteristicsTryptone soy agar(Oxoid)
20.0 g Dissolve TSA and NaCl in waterand autoclave at 121°C for 15 min.Cool in waterbath to 50°C.
Aseptically add 15 ml blood tocooled agar, mix well and pourinto Petri dishes to depth ofapproximately 3 mm. Store at 4°C.
TSA with added blood has the advantageof providing differentiation betweenhaemolytic and non-haemolytic Vibrio spp.
Some Vibrio species will be haemolytic onblood agar and non-haemolytic on MSA-Bdespite the latter medium providing bettergrowth for most marine Vibrio species.
NaCl 7.5 g(= 2% w/v final conc)
Distilled water 500 mlSterile equine blood 15.0 ml
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Marine 2216 agar (Difco) (ZoBell, 1941)
Reagent Amount Preparation of media Description of growth characteristicsBacto marine agar 2216(Difco – complete medium)
55.1 g Add powder to distilled water and boil to dissolve.Autoclave at 121°C for 15 min. pH 7.6 ± 0.2.
Plates are a light amber colour andslightly opalescent.
Distilled water 1000 ml
MA 2216 can be purchased from Difco as a broth or agar. Composition of MA 2216: 5.0 g Bacto peptone; 1.0 g Bacto yeast extract;0.1 g Fe(III) citrate; 19.45 g NaCl; 5.9 g MgCl2 (dried); 3.24 g NaSO4; 1.80 g CaCl2; 0.55 g KCl; 0.16 g Na2CO3; 0.08 g KBr; 34.0 mgSrCl2; 22.0 mg H3BO3; 4.0 mg Na-silicate; 2.4 mg NaF; 1.6 mg (NH4)NO3; 8.0 mg Na2HPO4; 15.0 g agar; 1000 ml distilled water.
Medium K (Used for isolation of Chryseobacterium (Flavobacterium) scophthalmum; Mudarris et al.,1994)
Reagent Amount Preparation of media Description of growth characteristicsYeast extract (Oxoid) 1 g Add all ingredients to seawater and
adjust to pH 7.2. Autoclave at 121°Cfor 15 minutes.
Orange-pigmented colonies are shiny,smooth, round, raised entire, 5–6 mm at48 h, 25°C.
On primary culture, isolates may showslight gliding motility, but lose this afterstorage.
Beef extract (Oxoid) 5 gCasein (Oxoid) 6 gTryptone (Oxoid) 2 gAnhydrous CaCl2 1 gAgar (Oxoid No. 1) 15 g750 ml seawater (aged 30 days)
Middlebrook 7H10-ADC medium (Used for isolation of Mycobacterium abscessus; Teska et al.,1997)
Reagent Amount Preparation of media Description of growth characteristicsNH4SO4 0.05 g Add chemical reagents to distilled water.
Autoclave at 121°C for 15 min. Cool to 50°C.Aseptically add sterile bovine albumin,nalidixic acid and cycloheximide, pyridoxineand biotin.
Media can be prepared as broths or add1.5 g of agar for preparation of plates.
Incubate at 25°C for 14–28 days.Growth occurs in 7 days. Youngcolonies may exhibit a lightblue-green tint. With age, coloniesare off-white to tan in colour.
KH2PO4 0.15 gNa2HPO4 0.15 gSodium citrate 0.04 gMgSO4 0.0025 gCaCl2 0.00005 gZnSO4 0.0001 gCuSO4 0.0001 gL-glutamic acid 0.05 gFerric ammonium citrate 0.004 gPyridoxine 0.0001 gBiotin 0.00005 gMalachite green 0.025 gGlycerol 0.5 mlNalidixic acid (35 mg/ml) 0.0035 gCycloheximide (400 mg/ml) 0.04 gBovine albumin V 0.5 mlD-glucose 0.2 gCatalase 0.0003%Distilled water 100 ml
This medium is modified from Middlebrook’s medium (Middlebrook et al., 1960) by the addition of ADC (albumin, catalase anddextrose).
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Mycoplasma medium – general purpose medium. Bacto Pleuropneumonia-like organism(PPLO) agar and broth with supplements (Difco). (Used for isolation of Mycoplasmas species. Thiscommercial medium, with the added supplements, is based on Hayflick’s medium)
Reagent Amount Preparation of media Description of growth characteristicsPPLO agar 35 g Agar plates
Add dehydrated media to distilledwater and autoclave at 121°C for15 min. Cool to 50–60°C.Aseptically add 300 ml Mycoplasmasupplement or 300 ml of Mycoplasmasupplement S. Mix well. Dispenseinto small (5 cm) Petri dishes. Storein plastic bags at 4°C.
Mince or grind tissue and place a few loopfulsinto a 3 ml broth. Incubate broth at 25 and37°C. A change in pH results in a colourchange from red to yellow, and may indicategrowth of Mycoplasma. Bacterial contaminationmay also change the pH of the medium. Thebacterial contamination can be filtered off bypassing the broth through a 0.22 mm filter.Place drops of the filtered broth to a fresh brothand to a plate.
Subculture 2 drops from a broth culture to aPPLO agar plate every 3 or 4 days. Incubateplates in air and 5–10% CO2 at 25 and 37°C.Subculturing on a Tuesday and a Friday maybe a convenient time frame for the laboratory.
Examine plates under a stereomicroscope forpresence of typical ‘fried-egg’ type colonies.
To differentiate Mycoplasma colonies fromcontaminating bacterial colonies, stain colonieswith Dienes stain. Mycoplasma colonies stainblue with a distinctly dense blue centre and alighter blue periphery. Mycoplasma coloniesretain the stain for 24 h, whereas bacterialcolonies decolorize after 30 min. See Dienesstain in ‘Tests’ section.
Distilled water 700 ml
PPLO broth 21 g BrothsAdd dehydrated media to distilledwater and autoclave at 121°C for15 min. Cool to 50–60°C.Add either supplement as for agar.
Distilled water 700 mlPhenol red
Mycoplasma supplement 1 vial SupplementRehydrate vial with distilled waterand swirl to dissolve. Asepticallyadd 1 vial per 70 ml of PPLO agaror broth.
Sterile distilled water 30 ml
Mycoplasma supplement S 1 vial Supplement S
Rehydrate vial with distilled waterand swirl to dissolve. Aseptically add1 vial per 70 ml of PPLO agar orbroth.
Sterile distilled water 30 ml
DNA (optional) DNA solutionPrepare 0.2% solution by dissolving0.2 g DNA in 100 ml distilled water.Autoclave at 121°C for 15 min.Add 10 ml to 1000 ml of abovemedium to produce a finalconcentration of 1%.
DNA (optional) (Calbiochempolymerized calf thymusDNA) 0.2% solution
1%
Distilled water 100 ml
Formulations
• PPLO agar = per litre contains 50 g Bacto beef heart infusion, 10 g Bacto peptone, 5 g sodiumchloride, Bacto agar 14 g. The broth does not contain the agar component.
• Mycoplasma supplement = per 30 ml vial is Bacto yeast extract 0.01 g, 1.6 g desiccated horseserum.
• Mycoplasma supplement S = Per 30 ml vial contains Bacto yeast extract 0.01 g, 1.6 g ofdesiccated horse serum, 55,000 units penicillin, 50 mg thallium acetate.
• Bacto heart infusion broth (Difco) contains 500 g beef heart infusion, 10 g bacto-tryptone, 5 gsodium chloride.
• The addition of 0.2% DNA can be added to the above medium. DNA is recommended forprimary isolation of the bovine mycoplasma M. bovigenitalium, and is thought to stimulate thegrowth of other Mycoplasma species as well. Therefore it is recommended as an optional mediacomponent (Freundt, 1983).
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Mycoplasma medium – modified Hayflick medium (Chanock et al., 1962) (Modified and usedfor isolation of Mycoplasma mobile; Kirchhoff and Rosengarten, 1984. Also used for many animalmycoplasma species)
Reagent Amount Preparation of media Description of growth characteristicsBacto PPLO broth (Difco) 16.8 g Dissolve PPLO broth in distilled water and
autoclave at 121°C for 15 min.Cool to 50°C for agar containing media, orroom temperature for broths, and asepticallyadd other filter-sterilized ingedients.Dispense in 3 ml volumes to bijou tubes forbroths, or 5 ml volumes to small Petri dishesfor agar plates.
See above under Mycoplasmamedium – general-purpose medium.Horse or bovine serum 200 ml
Yeast extract (50% w/v) 10 mlDNA 0.02 gPenicillin 2000 IU/mlThallium acetate (1.25% w/v) 10 mlDistilled water 800 ml
For agar plates add 1.0% w/v purified agar (Oxoid) or use PPLO agar. Note: Handle thallous acetate with caution. Use gloves andmask.
Mycoplasma medium (Used for isolation of Mycoplasma from crocodiles; Kirchhoff et al., 1997)
Reagent Amount Preparation of media Description of growth characteristicsBrain heart infusion broth (Oxoid) 37 g Dissolve brain heart infusion, yeast extract
and glycerol in distilled water. Glycerol canbe warmed slightly to facilitate pipetting.Autoclave at 121°C for 15 min. Prepareother reagents and filter sterilize. Addeach reagent to cooled autoclaved media.Dispense into 5 cm Petri dishes. Store inplastic bags at 4°C.
Incubate plates in a candle jar orsimilar CO2 generating atmosphere,at 37°C.
Yeast extract (Oxoid) 2 gGlycerol 8 mlThallium acetate (10% w/v) 2.5 mlAmpicillin (5% w/v) 2 mlNAD (1% w/v) 10 mlDistilled water 700 ml
NAD, nicotinamide adenine dinucleotide; TTC, tetrazolium chloride.Kirchhoff et al. (1997) also used this medium as a base to study the metabolism of glucose, arginine hydrolysis, TTC andphosphatase activity.
Nutrient agar (Used as a general isolation medium)
Reagent Amount Preparation of media Description of growth characteristicsNutrient agar (Oxoid) 14.0 g Add agar powder to distilled water. Autoclave at 121°C
for 15 min. Cool to 50°C and dispense into Petri dishes.Store at 4°C.
Distilled water 500 ml
Organic growth requirements
Some strains of Vibrio, e.g. Vibrio ordalii (previously V. anguillarum type II), Moritella (Vibrio) marinaand some strains of Vibro (Photobacterium) logei need 0.05% yeast extract for growth (Baumannet al., 1980). A 10× stock solution of yeast extract can be prepared and then 250 ml added to 5 ml ofbroth media.
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Packer’s plates (Selective medium for Erysipelothrix; Packer, 1943)
Reagent Amount Preparation of media Description of growth characteristicsColumbia blood agar base(Oxoid)
20.0 g Add agar base to water andautoclave at 121°C for 15 min.Cool to 50°C and asepticallyadd sodium azide, crystal violetand blood. Mix well and pourinto Petri dishes.
Colonies are 0.5–1 mm at 24–48 h. It is suggestedthis medium is used in conjunction with Wood’sbroth. Approximately 1 g of chopped or mincedtissue or sample material is placed into 10 ml ofWood’s broth. Incubate at 25°C and subculturefrom broth to BA and Packer’s plates at 24 and48 h. Examine plates at 24 and 48 h for typicalcolonies. Colonies of Erysipelothrix rhusiopathiaeare 0.5–1 mm, grey-green at 48 h, with slighta-haemolysis around the colony. They resemblea-haemolytic Streptococci.
See photographic section.
Distilled water 500 mlSterile equine blood 25 ml
Crystal violet (stock = 0.25 gin 100 ml of distilled water)
2 ml Prepare stock solutions ofcrystal violet and sodium azide.Autoclave at 121°C for 15 min.Sodium azide (NaH3)
(stock = 1 g in 100 ml ofdistilled water)
12.5 ml
Pasteurella multocida selective media. NB plates (Rutter et al., 1984)
Reagent Amount Preparation of media Description of growth characteristicsColumbia agar base (Oxoid) 7.8 g Add agar to distilled water and autoclave
at 121°C for 15 min. Cool to 50°C andaseptically add blood and 1 ml of antibioticstock. Mix well and pour into Petri dishes.
This is a semi-selective medium.Colonies of Pasteurella multocidaappear as grey non-haemolyticcolonies 1–2 mm and may beslightly mucoid.
Distilled water 200 mlSterile equine blood 12 ml
Neomycin sulphate (2.0 mg/ml) 4.0 mg Prepare antibiotic stock by addingneomycin and bacitracin to normal saline.Add 1 ml to cooled agar and blood mixtureas described above.
Bacitracin (3.5 mg/ml) 7.0 mgNormal saline 10 ml
Peptone yeast medium – PY (For growth of Cytophaga, Flavobacterium and Sphingobacteriumspecies; Takeuchi and Yokota, 1992)
Reagent Amount Preparation of media Description of growth characteristicsPeptone 1 g Add all reagents to distilled water and pH to 7.0. Autoclave
at 121°C for 15 min. Cool to 50°C and dispense into Petridishes.
Can be prepared as a broth by omitting the agar.
For growth of Pedobacter heparinus,Sphingobacterium spiritivorum, S.multivorum and Flavobacteriumspecies. Incubate at 28°C for 2 days.
Yeast extract 0.2 gGlucose 0.2 gNaCl 0.2 gAgar 1.5 gDistilled water 100 ml
Poly plates (See under Dermatophilus congolensis)
PYS-2 Medium (For growth of Vibrio rumoiensis; Yumoto et al., 1999)
Reagent Amount Preparation of media Description of growth characteristicsPolypeptone 8 g Add all reagents to distilled water and pH to 7.5.
Autoclave at 121°C for 15 min. Cool to 50°C andaseptically dispense into Petri dishes.
Colonies of Vibrio rumoiensis are circularand colourless. Growth can occur between2 and 34°C with an optimum at 27–30°C.
Yeast extract 3 gNaCl 5 gAgar 15 gDistilled water 1000 ml
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R2A agar (Oxoid CM 906, or Difco) (Used for isolation of Antarctic Flavobacterium spp.;McCammon and Bowman, 2000)
Reagent Amount Preparation of media Description of growth characteristicsAgar CM906 18.1 g Add powder to distilled water and adjust
pH to 7.2. Boil to dissolve. Sterilize byautoclaving at 121°C for 15 min. Cool to50°C and pour into Petri dishes.
This is a nutritionally reduced medium that enhancesrecovery of heterotrophic bacteria from treatedwaters, and assists in recovery of bacteria that arestressed or chlorine-tolerant. The sodium pyruvateincreases the recovery of stressed cells.
Distilled water 1000 ml
Composition g/l of Oxoid CM 906: yeast extract (0.5), tryptone (0.25), peptone (0.75), dextrose (0.5), starch (0.50), di-potassiumphosphate (0.3), magnesium sulphate (0.024), sodium pyruvate (0.3), agar (15.0).
Rogosa acetate agar (Oxoid). See under ‘Tests’ section
Strontium chloride B enrichment broth (Use for isolation of Salmonella spp. and Edwardsiellatarda; Iveson, 1971)
Reagent Amount Preparation of media Description of growth characteristicsBacto tryptone (Difco) 0.5 g Add all reagents to the
distilled water. Dispenseinto 10 ml volumes inMcCartney bottles andautoclave at 121°C for20 min.
Final concentration ofstrontium chloride is3.4% and pH is 5.0–5.5.
Place macerated or chopped sample (0.5 ml) or swab ofsample into broth. Incubate at 37°C for 24 and 48 h.Subculture from broth on to selective media of MCA or DCA.
On MCA plates the colonies of Edwardsiella tarda andSalmonella spp. appear as non-lactose fermenting colonies.On DCA the colonies appear as pale pink to clear colonieswith or without a black centre (H2S). E. tarda colonies areslightly smaller in size than Salmonella and the black centretakes longer to develop.
Sodium chloride 0.8 gPotassium dihydrogenphosphate
0.1 g
Strontium chloride 60% 6.0 mlDistilled water 100 ml
Seawater – artificial. See under artificial seawater
Serum-dextrose agar (Non-selective medium for Brucella species; Alton and Jones, 1967)
Reagent Amount Preparation of media Description of growth characteristicsNutrient agar 95 ml Prepare nutrient agar and cool to 50°C. Add
5 ml of a stock solution of serum-dextrose per95 ml of nutrient agar. Mix and pour into Petridishes.
After 4 days growth, smoothcolonies of Brucella are small,round, glistening and blue orblue-green in colour whenexamined by obliquely reflectedlight. Rough colonies areyellow-white in colour and have adry granular appearance.
Serum-dextrose 5 ml to 95 ml ofnutrient agar
Stock solution ofserum-dextrose
1 g of dextrose per5 ml of serum
For a stock solution of serum-dextrose, dissolvepure dextrose (glucose) in inactivated serum,at a rate of 5 g of dextrose per 5 ml of serum.Filter-sterilize and store at 4°C or −20°C.
This medium is available from Oxoid as Brucella medium base (Code CM169). Inactivated horse serum (5%) is added to theautoclaved base medium.
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Shieh medium + Tobramycin (SM-T) (Selective medium for Flavobacterium columnare andF. psychrophilum; Decostere et al. 1997; Shieh, 1980)
Reagent Amount Preparation of media Description of growth characteristicsPeptone (Difco) 5 g Suspend all chemicals into
1000 ml of distilled water and pHto 7.2. Autoclave at 121°C for15 min. Cool to 50°C and addedfilter-sterilized solution oftobramycin. Mix and pour mediainto Petri dishes.
Ensure the moisture contentremains in the media by storingplates at 4°C in sealed plasticbags. Growth of F. columnare isoptimal when the plates are fresh,but reduces as the surfacemoisture decreases.
Using a cotton-tipped swab, collect samples fromskin lesions and gills of fish and inoculate theplate. Colonies of F. columnare appear at 2–5days as yellow-pigmented, rhizoid colonies with athin spreading growth. See photographic section.
Early colonies may be viewed with the aid of astereomicroscope and a plug of the colony cut outusing an unsealed Pasteur pipette prepared in theshape of a ‘hockey stick’. Subculture the colonyplug to a fresh plate.
Shieh medium plus tobramycin reduces thegrowth and colony size of Aeromonas hydrophilaand prevents growth of A. salmonicida, S.putrefaciens and Ps. fluorescens.
Yeast extract (Difco) 0.5 gSodium acetate 0.01 gBaCl2 (H2O) 2 0.01 gK2HPO4 0.1 gKH2PO4 0.05 gMgSO47H2O 0.3 gCaCl22H2O 0.0067 gFeSO47H2O 0.001 gNaHCO3 0.05 gTobramycin 0.5 mgNoble agar (Difco) 10 gDistilled water (pH 7.2) 1000 ml
Note: The original method states 1 mg/ml of tobramycin; however, 0.5 mg/ml is recommended for Australian strains of F. columnareas growth may be inhibited or reduced at 1 mg/ml of tobramycin (Dr Annette Thomas, Department of Primary Industries,Queensland, 2000, personal communication).
SKDM. (Selective medium for Renibacterium salmoninarum, Austin et al., 1983)
Reagent Amount Preparation of media Description of growth characteristicsTryptone 1.0 g Add all reagents to distilled water and adjust
pH to 6.8. Autoclave at 121°C for 15 min, thencool to 50°C.
Inoculate plates with infectiousmaterial and incubate in a humidatmosphere at 15°C for 12 weeks.Examine plates regularly for thepresence of pin-point colonies.Maximum size of colonies is 2 mm.Colonies are white or creamy, shiny,smooth, raised and round. Oldcolonies may have a granular orcrystalline appearance.
Yeast extract 0.05 gCycloheximide 0.005 gAgar 1.0 gDistilled water 100 mlFetal calf serum 10.0 g Filter-sterilize L-cysteine hydrochloride,
D-cycloserine, polymyxin B sulphate andoxolinic acid. Add these plus sterile fetal calfserum to autoclaved media. Pour into plates.
L-cysteine hydrochloride 0.1 gD-cycloserine 0.00125 gPolymyxin B sulphate 0.0025 gOxolinic acid 0.00025 g
Shewanella marine agar (SMA) (Used for isolation of Shewanella woodyi; Makemson et al., 1997)
Reagent Amount Preparation of media Description of growth characteristicsBacto peptone (Difco) 5.0 g Add all reagents to distilled water.
Autoclave at 121°C for 15 min.A medium suitable for growth of luminousmarine bacteria, especially Shewanella.Bacto yeast extract (Difco) 1.0 g
Bacto agar (Difco) 15.0 g1x sea salts (see below) 200 mlDistilled water 1000 ml
5× sea salts stock: 2.58 M NaCl, 0.125 M MgCl2, 0.125 M MgSO4, 0.1 M KCl, distilled water 1000 ml, pH to 7.5.
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Siem selective medium (Selective medium for Aerococcus viridans; Stewart, 1972; Gjerde, 1984)
Reagent Amount Preparation of media Description of growth characteristicsGlucose 6.5 g Add powders to distilled water and
dissolve. Adjust pH to 7.4. Autoclave.Growth of Gram-positive cocci is promotedwith a change of medium from purple toyellow because of acid production.Incubate at 25°C for 5 days. Subcultureyellow-coloured broth to BA.
Yeast extract 4.5 gTryptone 15.0 gNaCl 6.4 gPhenyl ethyl alcohol 2.5 gBromocresol purple 0.008 gDistilled water 1000 ml
Skirrow’s medium See under Helicobacter selective medium.
SWT (A seawater-based complex medium for growth of marine organisms; Nealson, 1978; Boettcheret al., 1999)
Reagent Amount Preparation of media Description of growth characteristicsTryptone 0.5 g Add reagents to distilled water and autoclave at 121°C
for 15 min. Cool to 50°C and aseptically addfilter-sterilized seawater. Pour into Petri dishes.
Used for cultivation of Vibrio fischeri,Roseobacter spp., Stappia spp.Yeast extract 0.3 g
Glycerol 0.3 gSeawater 70 mlDistilled water 30 mlAgar 1.2 g
TCBS cholera medium (TCBS) (Semi-selective for growth of Vibrio spp.; Kobayashi et al., 1963;Nicholls et al., 1976)
Reagent Amount Preparation of media Description of growth characteristicsTCBS choleramedium (Oxoid)
44.0 g Add powder to distilled water.Autoclave at 121°C for 15 min.Cool to 50°C and dispenseinto Petri dishes. Store at 4°C.
This selective medium will grow the majority of Vibrio species.Some strains, however, such as Vibrio (Listonella) ordalii do notgrow on this medium. Aeromonas spp. and Pseudomonas willgrow weakly as small colonies approximately 1 mm at 24–48 h.
Vibrio species that ferment sucrose appear as yellow colonieswhereas sucrose-negative isolates appear as green colonies.
Distilled water 500 ml
Notes:
• Some Vibrio species grow poorly on TCBS and a few strains do not grow at all on this medium.Some brands of TCBS are more inhibitory than others. Eiken and Oxoid brands supported thegrowth of a greater number of Vibrio species than BBL or Difco brands, in particular for V.cholerae and non-cholera Vibrio spp. Also, it is recommended that each batch of medium istested for growth of Vibrio spp. (Nicholls et al., 1976).
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• TCBS detects a lower number of Vibrio colonies than MSA-B when performing TBCs on water.Colonies that grow on MSA-B and not on TCBS on primary culture often grow on TCBS whensubcultured to that medium, and identify as Vibrio spp. biochemically.
• After prolonged incubation, sucrose-fermenting colonies that initially appeared yellow may turngreen once the sucrose in the medium has been used up or the pH of the medium changes.
• Formulation: yeast extract (5 g), peptone (10 g), sodium thiosulphate (10 g), sodium chloride(10 g), ox bile (8 g), sucrose (20 g), NaCl (10 g), ferric chloride (1 g), bromothymol blue (0.04 g),thymol blue (0.04 g), agar (1.4 g), water (1000 ml) to pH 8.6.
Tryptone soya agar (TSA) (Used as a general-purpose isolation medium)
Reagent Amount Preparation of media Description of growth characteristicsTSA (Oxoid) 20.0 g Dissolve TSA in water and autoclave at 121°C
for 15 min. Cool in waterbath to 50°C and pourinto Petri dishes to depth of approximately 3 mm.Store at 4°C.
Suitable as a general-purpose medium.Add NaCl (7.5 g) to give a finalconcentration of 2% to isolate bacteriafrom marine sources.
Distilled water 500 ml
Tryptone yeast extract salt medium (TYES) (Used for the growth of Flavobacterium columnare;Triyanto and Wakabayashi, 1999)
Reagent Amount Preparation of media Description of growth characteristicsTryptone 0.4 g Dissolve reagents in water and autoclave at 121°C
for 15 min. Cool in waterbath to 50°C and pour intoPetri dishes to depth of approximately 3 mm. Storeat 4°C.
Used for the growth of F. columnare.Incubate plates at 25°C for 24 h.Colonies appear as yellow-pigmented,rhizoid, mucoid, or honeycomb-shapedcolonies.
Yeast extract 0.04 gMgSO4.7H2O 0.05 gCaCl2.10H2O 0.05 gDistilled water 100 ml
Tryptone yeast extract glucose agar (TYG) (Used for growth of F. psychrophilum, F. columnare,T. maritimum, Cipriano et al., 1996)
Reagent Amount Preparation of media Description of growth characteristicsTryptone 0.2 g Add all reagents, except
neomycin, to distilled water.Autoclave at 121°C for 15 min.Cool to 50°C, and asepticallyadd filter-sterilized neomycin.Pour into Petri dishes.
Colonies are yellow with a thin spreading margin, whichindicates the ability of the organism to exhibit glidingmotility. Optimal temperature for F. psychrophilum is14–20°C, for F. columnare 22–30°C, and 15–34°C forT. maritimum. Plates are examined daily for up to 7 days.Examination for colonies using a stereomicroscope mayassist in early detection of characteristic colonies.
Yeast extract 0.05 gGlucose 0.3 gAgar 1.5 gDistilled water 100 mlNeomycin sulphate 0.4 mg
Addition of 10 IU/ml of polymyxin B sulphate may assist as a selective medium for F. columnare (Shamsudin and Plumb, 1996).
TYG-M. Addition of 4 mg/ml neomycin sulphate, 200 IU/ml polymyxin B and 18.7 g/l ASW can beused for isolation of Tenacibaculum (Flexibacter) maritimum (Chen et al., 1995).
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VVM Selective medium for Vibrio vulnificus (Cerdà-Cuéllar et al., 2001)
Reagent Amount Preparation of media Description of growth characteristicsD-cellobiose 15.0 g Dissolve all reagents in distilled water
and bring to boil. Cool to 50°C andadjust pH to 8.5 using 5 M NaOH. (Thismedia does not require autoclaving.)
VVM plates are violet-blue in colour.Vibrio vulnificus is seen as a brightyellow colony with a yellow halo ofdiffusion due to the fermentation ofcellobiose. Other cellobiose fermentingVibrio spp. such as V. campbellii,V. harveyi and V. navarrensis will growon VVM agar. Some strains ofV. aestuarianus, V. alginolyticus,L. anguillarum may also grow on thismedium.
NaCl 10.0 gYeast extract 4.0 gMgCl2.6H2O 4.0 gKCl 4.0 gCresol red 40.0 mgBromothymol blue 40.0 mgPolymyxin B 105 U/lColistin methanesulfonate 105 U/lAgar 15.0 gDistilled water 1000 ml
A modification of this medium, termed VVMc, has the same reagents except the polymyxin B, and recovery rates are virtually thesame (Cerdà-Cuéllar et al., 2001).
VAM (Presumptive differentiation medium for Listonella (Vibrio) anguillarum; Alsina et al., 1994)
Reagent Amount Preparation of media Description of growth characteristicsSorbitol 15 g Dissolve all reagents (except
ampicillin) in distilled water, andboil. Cool to 50°C. Adjust pH to 8.6with 5 M NaOH. Aseptically addampicillin. Pour into Petri dishes.
Store at 15°C. Plates are viablefor 3 weeks. After this time theampicillin begins to lose activity.
The uninoculated medium is a violet-blue colour. After48 h incubation at 25°C, L. anguillarum colonies areflat, round and bright yellow with a diffuse yellow halodue to the fermentation of sorbitol.
V. fluvialis, V. harveyi and V. metschnikovii are ableto grow as yellow colonies on VAM, and therefore mustbe differentiated biochemically from L. anguillarum.V. alginolyticus grows as a non-swarming blue colony.
Yeast extract 4 gBile salts 5 gNaCl 35 gAmpicillin 10 mgCresol red 40 mgBromothymol blue 40 mgAgar 15 gDistilled water 1000 ml
Wood’s broth (Selective broth for detection of Erysipelothrix rhusiopathiae; Wood, 1965)
Reagent Amount Preparation of media Description of growth characteristicsBacto tryptose (Difco) 7.5 g Add tryptose, Lab Lemco and NaCl
to distilled water, pH to 7.5 andautoclave at 121°C for 15 min.Cool to room temperature.
Place swab of material or choppedpieces of tissue into medium. Incubateat 37°C for 48 h. Subculture to BA at24 and 48 h.
Colonies of Erysipelothrix are 0.5 mmto 1 mm, grey-green at 48 h, with slighta-haemolysis around the colony. Theyresemble a-haemolytic Streptococci.
See photographic section.
Lab Lemco powder (Oxoid) 1.5 gNaCl 2.5 gDistilled water 500 mlSterile horse serum (not inactivated) 25 ml Add antibiotics to 10 ml of sterile
distilled water. Aseptically add horseserum and antibiotic solution to cooledbase. Dispense 10 ml volumes toMcCartney bottles and store at 4°C.
Kanamycin 200 mgNeomycin 25 mgVancomycin 12.5 mg
Note: Wood and Packer (1972) modified this medium by replacing the distilled water with 0.1 M phosphate buffer (12.02 gNa2HPO4, 2.09 g of KH2PO4 and 1000 ml distilled water).
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Yersinia selective agar (Yersinia ruckeri does not grow on this medium)
Reagent Amount Preparation of media Description of growth characteristicsYersinia selectiveagar base (Oxoid)
29.0 g Add Yersinia selective base to distilled water andautoclave at 121°C for 15 min. Cool to 50°C.
Yersinia species grow as pinkcolonies with a darker ‘bulls eye’centre at 24–48 h.Distilled water 500 ml
Oxoid YersiniaSelective Supplement
2 ml Reconstitute 1 vial of supplement by aseptically adding1 ml distilled water and 1 ml ethanol. Mix to dissolveand aseptically add to base. Pour into Petri dishes.
7.2 Biochemical Test Media
Aesculin
Reagent Amount Preparation of media Description of testAesculin 0.2 g Dissolve all chemicals,
except aesculin, by boiling.Cool and add aesculin whilestirring. Dispense 5 ml ofmedia into tubes. Autoclaveat 121°C for 15 min.
Store tubes in the dark.
Bacterial cells are inoculated into aesculin broth and incubated atappropriate temperature for 24–48 h. Development of a black colouris positive. Because some bacteria, especially some Vibrio, causeblackening of the medium due to melanin production, true hydrolysisof aesculin must be tested for loss of fluorescence at 354 nmlongwave UV light (MacFaddin, 1980; Choopun et al., 2002).Fluorescence indicates a negative reaction.
Ferric citrate 0.1 gPeptone water(Oxoid)
3.0 g
Distilled water 200 ml
Arginine dihydrolase (Møller). See under Decarboxylases
Arginine dihydrolase (Thornley). See under Decarboxylases
Brucella: Assay of metabolic activity (Alton and Jones, 1967; Jahans et al., 1997)
Reagent Amount Preparation of media Description of testL-alanine 1.25 g/l Dissolve various metabolic
substrates in sterile PBS,pH 7.2 at a concentrationof 1.25 g/l. Sterilize eachsolution by filtration through0.22 mm filter membrane.Store at 4°C.
Prepare MTT solution inPBS at 1.0 g/l. Sterilize byfiltration through a 0.22 mmfilter.
Substrate mediated metabolic activity is assayedby the stimulation of tetrazolium reduction.
Prepare a cell suspension in PBS and adjust theconcentration to 1010 organisms per ml. In aflat-bottomed microtitre plate, place 100 ml ofeach substrate into each well. Place 50 ml of cellsuspension in each well and incubate at 37°C in10% CO2 for 18 h.
After incubation, place 50 ml of MTT in each well.Incubate 1 h at room temperature and add 50 mlof formaldehyde to each well. After 2–4 h, readthe optical density at 630 nm. The metabolicindex for each substrate is expressed as a ratioof the OD with substrate against the OD withoutsubstrate.
L-asparagine 1.25 g/lL-glutamic acid 1.25 g/lL-arginine 1.25 g/lDL-ornithine 1.25 g/lL-lysine 1.25 g/lD-galactose 1.25 g/lD-ribose 1.25 g/lD-xylose 1.25 g/lMeso-erythritol 1.25 g/lUrocanic acid 1.25 g/l3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)
1.0 g/l
Formaldehyde 40%
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Brucella: growth on substrate media (Alton and Jones, 1967; Jahans et al., 1997)
Reagent Amount Preparation of media Description of testBasic fuchsin 20 mg/ml
(1/50,000)A 0.1% stock solution of each dye is made in distilledwater, and sterilized by being held in boiling water for1 h. Stock solution should be renewed after 3 months.
Each dye is then added to a base media such astryptone-soy agar or serum-dextrose agar. Alton andJones (1967) suggest that a laboratory shoulddetermine the concentration of each dye that givesoptimum results, the range being somewhere between1:25,000 and 1:100,000 (10–40 mg of dye per ml ofmedium), using the FAO/WHO reference strains.The concentrations in the ‘amount’ column aresuggested by Jahans et al. (1997).
The required amount of the dye stock solution is addedto the melted base medium, mixed and poured intoPetri dishes and allowed to set. Label each plate withappropriate dye identification.
Prepare a suspension of bacterial cellsin 0.5 ml of sterile normal saline.
The unknown culture should beinoculated on to the same plate alongwith the reference strains. Thereforemark off an appropriate number ofareas on a plate. Using a loop ofculture suspension, make five separatestreaks without recharging the loop onto each specifically marked area. Alsoinoculate a control plate that containsbasic medium only and no dye.Incubate plates at 37°C with andwithout CO2 for 4 days.
Record results of ‘growth’ or ‘nogrowth’ in the presence of each dye.
Safranin O 100 mg/ml(1/10,000)
Thionin 20 mg/ml(1/50,000)
Brucella selective media. See Farrell’s medium
Carbohydrate fermentation (General-purpose medium for detection of carbohydrate fermenta-tion; Vera, 1948, 1950)
Reagent Amount Preparation of media Description of testCystine tryptic medium (Gibco) 7.13 g Basal medium preparation
Boil distilled water and cysteine tryptic medium.Dispense 5 ml volumes into 10 ml tubes.
Carbohydrate preparationTo prepare the carbohydrates, make a 10% solution(3 g in 30 ml).For salicin use a 4% solution (1.2 g in 30 ml ofdistilled water).
For carbohydrates that can be autoclavedPipette 0.5 ml of carbohydrate solution per 5 ml ofbasal medium. Autoclave at 10 lb per 10 min.
For filter-sterilizationFilter-sterilize the carbohydrate solution through a0.22 mm filter.Autoclave basal medium 5 ml in 10 ml tubes. Cool to50°C and aseptically add the filter-sterilizedcarbohydrate solution, 0.5 ml to 5 ml of basal medium.
Fermentation isdetected by a colourchange from red toyellow.
References may statethat carbohydratesare added at 0.5% to1.0%. However,because reversion ofthe reaction mayoccur when 0.5% isused, using aconcentration of 1%ensures againstdepletion of thecarbohydrate by theorganism.
Distilled water 250 mlCarbohydrate (see below) Sterilization
methodCarbohydrateArabinoseAesculinGlucoseInositolLactoseMaltoseMannitolMannoseSalicinSorbitolSucroseTrehaloseXylose
SterilizationFilterAutoclaveAutoclaveAutoclaveAutoclaveFilterAutoclaveFilterAutoclaveAutoclaveFilterAutoclaveFilter
1% finalconcentration
Cystine tryptic medium contains Bacto tryptose, L-cystine, sodium chloride, sodium sulphite, agar and phenol red. With the additionof yeast extract at 0.01%, this medium is also suitable for testing carbohydrate fermentation of the Cytophaga, Flavobacterium,Flexibacter, Tenacibaculum group, plus certain Vibrio species that require yeast extract, such as V. ordalii, Moritella marina andsome strains of V. logei. Baumann et al. (1980) adds yeast extract at 0.05% for strains of Vibrio and Photobacterium spp. thatrequire organic growth factors.
Notes:
• In the literature, some original recipes used bromothymol blue as the pH indicator; however, itmay be toxic to a number of marine bacteria, and therefore phenol red is the recommended pHindicator in carbohydrate fermentation tests (Leifson, 1963).
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• Peptone-containing media may give misleading results for carbohydrate breakdown becauseweak acid production may be masked by peptone-breakdown products (Vera, 1950).
• Sucrose should be filter-sterilized, as it is thermolabile and will give false positive results if sterilizedby autoclaving (Stanier et al., 1966).
• To prevent colour change of the medium caused by CO2 build-up in screw-capped tubes wheninoculated with organism of interest, ensure that lids are loosened for about an hour before resultsare read. Preparing tubed media to only contain half their volume in media also helps to preventpH reversion caused by CO2 build-up.
• Most carbohydrates can be stored at room temperature for 2–3 weeks, with the main stocks storedat 4°C for longer term storage. However, some carbohydrates should always be stored at 4°C toprevent unwanted colour change in the medium. These are arabinose, mannitol, xylose and alsothe oxidative-fermentative media and ONPG media.
Alternative methods for carbohydrate fermentation for specific bacteria
Carbohydrate fermentation media for Flavobacterium spp.
• Broth medium containing 0.05% tryptone, 0.05% yeast extract, 0.0018% phenol red, and filter-sterilized carbohydrate at a final concentration of 0.2% (Wakabayashi et al., 1986).
• AO medium was used as a basal medium for carbohydrate fermentation; however, it wasunsuitable for this test, as colour changes were seen in the negative control tube (Bernardet andGrimont, 1989).
Carbohydrate fermentation media for marine bacteria (Leifson, 1963, used by Gauthier,1976b)
Reagent Amount Preparation of media Description of testCasitone (Difco) 0.1 g The ingredients are dissolved in half-strength
seawater and the pH is adjusted with HCl to 7.5.Autoclave and re-check the pH, which shouldbe about 8. Adjust with HCl if necessary.Filter-sterilize the carbohydrate and addaseptically to the base medium. Dispense 3 mlinto 13 × 100 mm tubes.
Fermentation is indicated by achange in colour from red to yellow.Yeast extract 0.01 g
Ammonium sulphate 0.05 gTris buffer 0.05 gAgar 0.3 gPhenol red 0.001 gArtificial seawater Half strengthCarbohydrate 1%
Notes: Einar Leifson (1963) modified this recipe from the original medium published in 1953 (Hugh and Leifson, 1953). The pHindicator in the 1953 medium (bromothymol blue) was found to be toxic for a number of marine bacteria and therefore phenol red isrecommended. This media is also used to test for oxidation/fermentation. Add individual carbohydrates to the base medium forfermentation tests. This method is similar to the general-purpose medium.
Carbohydrate fermentation media for Pseudomonas and Flavobacterium spp. (Gilardi,1983)
Reagent Amount Preparation of media Description of testOF basal medium(Difco)
9.4 g Add reagent in distilled water and dissolve by boiling.Dispense into 100 ml aliquots. Autoclave at 121°C for15 min. To each 100 ml aliquot aseptically add 10 ml ofa 10% carbohydrate solution. Aseptically dispense each100 ml of respective carbohydrate into 5 ml aliquots in10 ml tubes.
Colour change to yellow indicates apositive fermentation reaction.
Distilled water 1000 ml
Carbohydrate 10%solution
10 ml Filter-sterilize and add as above.
Composition of Difco OF medium: tryptone (2.0 g), sodium chloride (5.0 g), di-potassium phosphate (0.3 g), agar (2.0 g),bromothymol blue (0.08 g), in 1000 ml distilled water, pH 6.8.
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Carbohydrate fermentation media using acid from ammonium salt sugar (ASS). Used forSphingobacterium, Flavobacterium, Shewanella putrefaciens, Pseudomonas spp. Used by Holmeset al. (1975) for testing Pseudomonas spp. Used by Bernardet and Grimont (1989) as an inoculationmedium for API 50CH when testing Flavobacterium. Quoted in Cowan and Steel (1970) from themethod of Smith et al. (1952).
Reagent Amount Preparation of media Description of test(NH4)2HPO4 1.0 g Add ingredients to distilled water and dissolve
by boiling or steaming.
Add indicator and autoclave at 115°C for20 min.
Allow medium to cool to 60°C and addfilter-sterilized carbohydrate solution.
Dispense into tubes and incline tube so thatmedium sets as a slope.
Inoculate and incubate at theappropriate temperature.Examine over 7 days. A colourchange from purple to yellow isindicative of fermentation ofcarbohydrate.
KCl 0.2 gMgSO4.7H2O 0.2 gYeast extract 0.2 gAgar 20 gDistilled water 1000 mlBromocresol purple 0.2% solution 4 mlCarbohydrate 10% solution 100 ml
Notes:
• Bacteria that do not give a reliable fermentation reaction with peptone-containing medium shouldbe tested in ASS. In general, peptone-containing media give the least positive reactions and ASSthe most positive reactions (Cowan and Steel, 1993 edition).
• Reactions using this medium are not reported in this book. Flavobacterium spp., reactionsreported in this book were the same by both the peptone-containing method (Carbohydrategeneral purpose medium) and ASS.
Carbohydrate fermentation media for Vibrio spp. (The following media were used by Baumannet al. (1971) for Vibrio spp. and Beneckea – adapted from methods of Stanier et al., 1966)
• Basal medium (BM): 50 mM tris (hydroxymethyl)aminomethane (Tris)-hydrochloride (pH 7.5),190 mM NH4Cl, 0.33 mM K2HPO4.3H2O, 0.1 mM FeSO4.7H2O and half strength seawater.
• Basal medium agar (BMA): Mix equal volumes of double-strength BM with 20 g of Ionagar(Oxoid) per litre.
• Yeast extract broth (YEB): Add 5 g/l of yeast extract (Difco) to BM.• For Yeast extract agar (YEA) add 20 g of agar (Difco) per litre of YEB.• Carbohydrate fermentation medium (F-2): To YEB add 100 mM Tris-hydrochloride (pH 7.5),
1 g/l of Ionagar (Oxoid), 1 g/l of sodium thioglycollate, and 10 g/l of filter-sterilized glucose.• Carbohydrate fermentation medium (F-3): To BM add 25 mM Tris hydrochloride (pH 7.5),
0.5 g/l of yeast extract (Difco), 1 g/l of Ionagar (Oxoid), 2 ml/l of a 1.6% (w/v) alcoholic solution ofbromocresol purple, and 10 g/l of filter-sterilized carbohydrate.
Carbohydrate fermentation media for Nocardia spp. Basal inorganic nitrogen medium(Gordon et al., 1974, Quoted from Ayers et al., 1919)
Reagent Amount Preparation of media Description of test(NH4)2HPO4 1.0 g Add all reagents except
carbohydrate to distilled waterand adjust pH to 7.0. Dispense5 ml into tubes and autoclave at121°C for 15 min.
Aseptically add 0.5 ml of a 10%solution of each carbohydrate(autoclaved separately) to tubesand allow to set on a slant.
Production of acid due tofermentation of the carbohydrateis seen as a colour change frompurple to yellow.Incubate at 28°C and read from7–28 days.
With the addition of yeast extract,this medium is the same as ASS.
KCl 0.2 gMgSO4.7H2O 0.2 gAgar 15Distilled water 1000 mlBromocresol purple 0.04% solution 15 mlCarbohydrates to be tested (10% solution) 0.5 mlOptionalYeast extract (Difco)
Note: When testing N. seriolae, add 2% w/v yeast extract (Difco) to medium (Kudo et al., 1988).
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Carbon dioxide atmosphere (Used when incubation conditions require a carbon dioxideatmosphere)For laboratories that do not have access to a carbon-dioxide incubator, the following methods offer acheap alternative.
Method 1: Candle jar method. Place agar plates in an airtight container. Place a lighted candle in thejar and seal the lid. This method produces 2% CO2 (J. Lloyd, Department of Agriculture, Western Aus-tralia, 1985, personal communication) with other reports suggesting 6–8% CO2 (Cottral, 1978). If the lidis plastic, then protect from the heat produced by the candle by using an aluminium foil-covered device.
Method 2: ‘ENO salts’ method (Lloyd, 1985). 7.5 g of ENO salts per 10 l container generates 4%CO2. Use an appropriate container that has a well-sealed lid such as a household food container orMitsubishi Gas Chemical Company anaerobe box. Place 3 g of ENO salts (Sigma) into 20 ml of water fora container that measures 22 × 22 × 8 cm or 10 g of ENO salts into 40 ml of water for a container thatmeasures 30 × 30 × 14 cm. This technique needs to be performed quickly, therefore place salts into asmall piece of tissue paper. Place the water into a 50 ml urine container and place into the incubationcontainer. Quickly add the salts to the water and seal lid. Place box at appropriate temperature. ENO is asparkling antacid that contains per 5 g, sodium bicarbonate (2.32 g), sodium carbonate (0.5 g), citricacid anhydrous (2.18 g) and which can be purchased through Sigma chemicals.
Catalase test (from Cowan and Steel, 1970)Smear a colony on to a glass slide. Place a drop of 30% H2O2 on the smear. The presence of bubblesindicates a positive reaction. When picking growth from a blood agar plate, make sure no blood-containing agar is placed on the slide, as false-positive results may occur.
Cellulose digestion (Wakabayashi et al., 1989)To a broth culture medium appropriate to the growth requirements of the organism being tested, add apiece of cellulose filter paper. Observe for disintegration of the filter paper. A characteristic of theCytophaga species is that they digest cellulose, therefore when testing an organism suspected of beinga Cytophaga species use a broth of Anacker-Ordal medium.
Coomassie Brilliant Blue agar (CBBA). Used to detect the A+ protein layer of Aeromonassalmonicida (Udey, 1982; Evenberg et al., 1985; Cipriano and Bertolini, 1988; Markwardt et al.,1989)
Reagent Amount Preparation of media Description of growth characteristicsTSA 44.0 g Add Coomassie Blue and
TSA to distilled water.Autoclave at 121°C for15 min. Cool to 50°C andpour into Petri dishes.
Coomassie Brilliant Blue is a protein-specific dye that resultsin production of dark blue colonies of bacteria that have theA-layer protein. A-layer positive A. salmonicida stain deepblue. This medium is not selective and other bacteria willproduce blue-coloured colonies. However, it assists in theisolation and detection of A. salmonicida.
Coomassie BrilliantBlue R250 (Bio-Rad)
0.1 g
Distilled water 1000 ml
Citrate: Simmons method (Simmons, 1926)
Reagent Amount Preparation of media Description of testSimmons citrate (Difco) 3.63 g Suspend media in distilled water, and
bring to the boil.
Dispense 3 ml of media into tubes.Autoclave at 121°C for 15 min. Layon a slight incline to create slopes.
Development of a deep blue colour is positive.Simmons citrate (Difco) contains 1% NaCl. Citrateis tested as a sole carbon source. (Christensen’scitrate does not test for citrate as the sole carbonsource, as it contains other nutrients).
Distilled water 150 ml
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Congo Red. Tests for presence of extracellular galactosamine glycan in the Flavobacteriaceae(Johnson and Chilton (1966) who quote personal communication with E.J. Ordal for this test;McCurdy, 1969)
Reagent Amount Preparation of media Description of testCongo Red 10 mg Weigh 10 mg of Congo Red and add
to 100 ml of distilled water. Finalconcentration is 0.01% aqueous. Mix well,label bottle and store at room temperature.
Place 1–2 drops of Congo Red on a few isolatedcolonies growing on AO or Shieh’s medium. A positivereaction is red-colour development of colonies. Thiscolour lasts for a number of hours. This test detectsthe presence of extracellular glucans.
Distilled water 100 ml
Congo Red agar (Used for detection of A protein layer of A. salmonicida; Ishiguro et al., 1985)Prepare tryptic soy agar (Difco) containing 30 g/ml of Congo Red. Incubate at 20°C and examine forred-coloured colonies after 48 h.
Dienes stain (Used to differentiate Mycoplasma from Bacteria; Dienes, 1939; Hayflick, 1965).
Reagent Amount Preparation of media Description of testMethylene blue 2.5 g Dissolve all reagents in
distilled water.
Store in a screw-capbottle.
Method I. Place some stain on to a coverslip and allow to dry. Agarblocks containing colonies are excised and placed on to a glassslide with colonies uppermost. Place coverslip on to agar blockcontaining colonies and press coverslip lightly so that there is goodcontact between the colonies and the stain.
Method II. Using a cotton wool swab directly apply the stain to theMycoplasma colonies.
Mycoplasma colonies stain blue with a distinctly dense blue centreand a lighter blue periphery. Mycoplasma colonies retain the stainfor 24 h, whereas bacterial colonies decolorize after 30 min.
Azure II 1.2 gMaltose 10 gSodium carbonate(anhydrous)
0.25 g
Distilled water 100 ml
Some methods contain 0.2 g of benzoic acid.
Digitonin (Used to differentiate Mycoplasma species from Acholeplasma species)
Reagent Amount Preparation of media Description of testDigitonin(1.5% w/v)
1.5 g Dissolve digitonin in ethanol.Warm to 37°C to dissolve.
Place 20 ml of stock solutionon to a 6 mm sterile filterpaper disc (Oxoid). Dry at37°C for 1 h.
Plate suspect Mycoplasma colony to a 5 cm Mycoplasma agar plate. Thiswill either be from a broth or agar plate. When transferring colonies froman agar plate, use a sterile spatula or open-ended ‘hockey stick’ (madefrom a bent Pasteur pipette), to select a plug of agar containing a colony.Invert this agar plug, colony side down, on to a fresh agar plate and rubover the surface. If inoculating the plate from a broth culture, pipette thebroth culture on to the plate and then remove the excess. Allow the plateto dry for a few hours. Place the digitonin disc in the middle of the plate,and incubate at appropriate temperature under CO2 for 3–4 days.
Mycoplasmas are sensitive to digitonin and so a 4 mm zone of inhibitionof growth is seen from the edge of the disc (zone of 14 mm diameter).
Acholeplasma are resistant to digitonin and a zone of less than 1 mm isseen from the edge of the disc.
Ethanol 100 ml
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DNase (Used by West and Colwell (1984) and reported from all sources used in this manual)
Reagent Amount Preparation of edia Description of testDNase test agar(Oxoid)
19.50 g Suspend agar in water in 1000 ml.Autoclave at 121°C for 15 min thencool to 50°C in waterbath. Mix welland pour media into Petri dishes.
Spot-inoculate or streak as a single line, a loop of bacterialculture on to the plate. Incubate plates for 2–7 days at24°C. Flood plate with 1% HCl to precipitate the DNA.A positive reaction is seen as a clear zone around thebacterial streak. See photographic section.
Distilled water 500 ml
See under HCl for preparation of 1M HCl.
Decarboxylases and arginine dihydrolase (Used in Cowan and Steel, 1970, West and Colwell,1984)
Reagent Amount Preparation of media Description of testDecarboxylase base Møller (Difco)(Møller, 1955)
2.1 g In separate bottles,dissolve each individualamino acid in the 200 mlof distilled water anddecarboxylase base.Dispense 5 ml into 10 mltubes. Autoclave at 121°Cfor 15 min.
A tube of basal medium without amino acid isalways inoculated in parallel with the test media.Inoculate with heavy inoculum. Overlay all tubeswith paraffin oil, and incubate for 7–14 days forsome strains. Most strains can be read at 48 h.
Check the bottom of tube and the cloudiness of thetube for signs of sufficient inoculum and growth.
For salt-requiring strains, add 0.5 ml of 20% NaClto tubes before inoculation with organism.
Distilled water 200 mlL-arginine HCl 2.0 gL-ornithine monohydrochloride 2.0 gL-lysine monohydrochloride 2.0 g
Control – use decarboxylase baseand distilled water only
See under Paraffin oil for sterilization proceduresfor the oil.
Arginine dihydrolase (ADH) (Method of Thornley, 1960. Recommended for marine bacteria)
Reagent Amount Preparation of media Description of testBacto-Peptone(Difco)
0.1 g Add reagents to distilled water and pHto 6.8. Dispense 5 ml into 10 ml tubes.Autoclave at 121°C for 15 min.
Note: the original pH described byThornley was 7.2, however pH 6.8 isrecommended by West and Colwell (1984).
A tube of basal medium without amino acid is alwaysinoculated in parallel with the test media. Inoculatewith heavy inoculum. Overlay all tubes with paraffinoil, and incubate for 7–14 days for some strains.Most strains can be read at 48 h.
Check the bottom of tube and the cloudiness of thetube for signs of sufficient inoculum and growth.
NaCl 0.5 gK2HPO4 0.03 gAgar 0.3 gPhenol red 0.001 gArginine HCl 1.0 gDistilled water
Note: Some Vibrio species are negative in Møller’s method for ADH, but positive by the method of Thornley. These are; Vibriomediterranei, V. mytili, V. orientalis, Vibrio splendidus biogroup I and some strains of V. tubiashii (Macián et al., 1996). The glucosein Møller’s medium appears to inhibit the reaction due to catabolite repression of the inducible ADH system (Macián et al., 1996).Baumann et al. (1971) found that Photobacterium spp. can produce alkaline products in Thornley’s medium, yet none possesses aconstitutive ADH system when tested with more sensitive analytical methods (West and Colwell, 1984). Different brands of peptonewere found to give different results and the Difco bacto-peptone was recommended (Thornley, 1960).
Thornley’s medium was modified by Baumann and Baumann (1981) by using basal medium with-out the Tris-HCl. See under Alternative media for carbohydrate fermentation, media for Vibrio species,for this recipe.
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Furunculosis agar (For detection of pigment production from Aeromonas salmonicida; Bernoth andArtz, 1989)
Reagent Amount Preparation of media Description of testBacto-tryptone (Difco) 10 g Add all ingredients to distilled water.
Adjust pH to 7.3, and autoclave at121°C for 15 min. Cool to 50°C andpour into Petri dishes.
Pigment production is best detected by incubatingat 15–20°C for up to 7 days. At 25°C less pigmentproduction is seen.
Pigment is seen as a diffuse brown colorationaround each colony.
Yeast extract (Difco) 5 gL-tyrosine (Merck) 1 gNaCl (Merck) 2.5 gAgar (Oxoid L11) 15 gDistilled water 1000 ml
Note: Furunculosis agar is recommended as a superior medium for detection of pigment production, as a greater number of strainsshow pigment on FA than on TSA, NA or BHIA (Hirvelä-Koski et al., 1994, Hänninen and Hirvelä-Koski, 1997). However, FA is notthe best medium for primary isolation of Aeromonas salmonicida (Bernoth and Artz, 1989) and BA is recommended. (It may bepossible that by adding blood to FA, the medium would be a suitable primary isolation medium.)
Gelatinase (0% AND 3% NaCl) (Smith and Goodner, 1958. Used by West and Colwell, 1984)
ReagentAmount0% NaCl
Amount3% NaCl Preparation of media Description of test
Difco bacto Peptone 1.0 g 1.0 g Add reagents to distilledwater and autoclave at121°C for 15 min. Cool to50°C.
Use split Petri dishes, andlabel plates 0% and 3%per side of each plate.Pour appropriate mediainto each half of each plate.
Store at 4°C.
Spot inoculate the gelatin plate on both the 0%NaCl and 3% NaCl sides with approximately equalamounts of bacterial growth. Incubate at anappropriate temperature 24–48 h.
Production of gelatinase is seen as either a cloudyor clear zone around the area of bacterial growth.The plate should be held up to the light and readagainst a darkish background. Chilling the platesmay provide a sharper contrast between zonesand the unaffected portions of the media. Floodingthe plate with ammonium sulphate may assist withdefinition of zones of clearing.
Difco yeast extract 0.25 g 0.25 gOxoid gelatin 3.75 g 3.75 gNaCl – 7.50 gOxoid agar No 1 3.75 g 3.75 gDistilled water 250 ml 250 ml
This plate method of Smith and Goodner (1958) is used as it detects a change in the composition of gelatin rather than itsliquefaction and thus is a very sensitive method. Either use a split plate with one side 0% NaCl and the other side containing 3%NaCl. This allows the salt requirement of the organism to be detected at the same time. Alternatively, use small (5 cm) Petri dishesfor each NaCl concentration.
Glucose yeast extract agar (GYEA) (For colonial morphology and survival at 50°C of Nocardiaspecies; Gordon et al., 1974)
Reagent Amount Preparation of media Description of testYeast extract 10 g Add reagents to water and pH to 6.8.
Autoclave at 121°C for 15 min. May bepoured into plates, or prepared as tubedslants.
Plate media is used for observation of colonialmorphology.
Tubed slants are used for testing survival at 50°C.
Glucose 10 gAgar 15 gTap water 1000 ml
HaemolysisRecord from growth on BA or MSA-B within 7 days. For some Vibrio species, haemolysis may bemore pronounced on BA than on MSA-B even though their preferred growth medium is MSA-B.
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HCl (1N) (For detection of hydrolysis of DNA in DNase medium)
Reagent Amount Preparation of media Description of testConcentrated HCl, 32% 9.85 ml Add 9.85 ml of 32% acid to 80 ml
water, then make up to 100 ml.To a 24 or 48 h growth on a DNase plate flood with1 M HCl. Wait 1 min and observe over a black tilefor zone of clearing around bacterial growth.
See photographic section.
Distilled water 80 ml
Safety note: Always add acid to water, not vice versa. Other concentrations of HCl are available. To prepare from 35.4%, add 8.9 mlto 80 ml of water, and then make up to 100 ml. To prepare from 37%, add 8.5 ml of acid to 80 ml of water, and then make up to100 ml.
Hippurate hydrolysis (Hwang and Ederer, 1975)
Reagent Amount Preparation of media Description of testHippuric acidsodium salt
0.15 g Dissolve hippuric acid inwater. Dispense 0.4 mlper sterile, 5 mlyellow-capped plastictube. Store at −20°C.
Inoculate medium with a large loopful of bacterial growth from cultureplate. Incubate at appropriate temperature for 2.5 h. Add 200 ml ofninhydrin reagent and incubate a further 10 min. Development ofdeep purple colour is positive. (Original reference suggests a purplecolour as deep as the colour of Crystal violet used in the Gram stain.)
Distilled water 15 ml
Reagent: Ninhydrin.
Ninhydrin reagent (Used for detection of hippurate hydrolysis; Hwang and Ederer, 1975)
Reagent Amount Preparation of media Description of testNinhydrin 0.35 g Dissolve ninhydrin in the acetone/butanol mixture.
Dispense into 5 ml tubes and store in the dark.
To a mixture of organisms incubated in hippuratesolution for 2.5 h, add 200 ml of ninhydrinreagent. Incubate 10 min and observe formationof purple layer.
Acetone 5 mlButanol 5 ml
Hydrogen sulphide productionMany of the references for the biochemical reactions use a method whereby a lead acetate strip (H2Sindicator) is suspended over a tube of growth medium containing cysteine (sulphur source). This is avery sensitive method. However, the preparation of the lead acetate strips is hazardous, (for methodsee Cowan and Steel, 1970). Alternative methods are available but may not have the same sensitivity.The test can be performed in an API 20E or a triple sugar iron tube. A hydrogen sulphide Biostrip(catalogue number TM343) is available from MedVet Science. When using these media be aware ofthe growth requirements of the test organism and the sensitivity of the test.
Indole (Cowan and Steel method 2, 1970; Colwell and West, 1984; MacFaddin, 1980)
Reagent Amount Preparation of media Description of testTryptone broth(Difco)
2.5 g Combine reagents and pHto 7.5. Dispense 5 ml pertube and autoclave at 121°Cfor 15 min.
Use a heavy inoculum and incubate 48 h at 25 or 37°C dependingon growth requirements of the bacterium. To read, add 6–7 dropsof Kovács reagent and shake tube. A positive result is developmentof a cherry red colour in the upper reagent layer on top of the brothmedium. No colour development indicates a negative result.
For organisms that require salt, add 0.5 ml of 20% NaCl to 5 ml ofmedium. Even though a salt-requiring organism may show growthin the tube at 48 h, if NaCl is not present a false-negative reactioncan still occur. See photographic section.
NaCl 1.25 gDistilled water 250 ml
Results between API 20E and tryptone broth may differ. Add NaCl to a final concentration of 2% for marine isolates (i.e. add 500 mlof 20% NaCl to 5 ml of test medium).
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Indoxyl acetate hydrolysis (Mills and Gherna, 1987)Indoxyl acetate discs may be prepared by making a 10% (wt/vol) solution of indoxyl acetate inacetone. Add 50 ml to a blank disc, 0.64 cm in diameter. Discs are available from Oxoid. Allow discs todry and store at 4°C in an amber-coloured bottle. Shelf life of discs is approximately 6 months. Discsare also available commercially from companies such as Remel.
Test: Bacterial growth from an agar plate is scraped on to an indoxyl acetate disc and a drop ofdistilled water is added. A positive result is the development of a dark blue colour within 5–10 min.Alternatively, colonies from an agar plate can be emulsified in 0.3 ml of distilled water. Add an indoxylacetate disc. A positive result is the development of a dark blue colour within 10–15 min (Mills andGherna, 1987).
Kovács indole reagent (Kovács, 1928; Cowan and Steel, 1970; MacFaddin, 1980)
Reagent Amount Preparation of media Description of testp-dimethylaminobenzaldehyde 5 g Handle p-dimethylaminobenzaldehyde and HCl with caution.
Dissolve p-dimethylaminobenzaldehyde by warming in awaterbath at 50°C. Only leave long enough to dissolve,otherwise it turns pink then dark brown. Cool and slowly addthe HCl to the aldehyde–alcohol mixture. The reagent shouldbe light yellow to light brown in colour. Store reagent in abrown glass-stoppered bottle at 4°C.
See under ‘Indole’.Iso-amyl alcohol 75 mlConcentrated hydrochloric acid 25 ml
KOH (Tests for presence of Flexirubin pigments; Reichenbach et al., 1974, 1981)
Reagent Amount Preparation of media Description of testKOH 20% Place 1–2 drops of KOH on to a few isolated young colonies on AO medium.
Detection of a brown or red pigment is positive. Detects flexirubin pigment(Reichenbach et al., 1981). Some reports indicate a purple colour as positive(Mudarris and Austin 1989).
Note: Pigment production may be influenced by factors in the growth medium such as yeast extract, which promotes pigmentproduction through stimulated growth. Also, pH can affect pigment, where a lower pH may reduce pigment production(Reichenbach et al., 1974). (Reichenbach states 5% KOH in methanol.)
MacConkey agar
Reagent Amount Preparation of media Description of testMacConkey agar (Oxoid) 26.0 g Suspend the agar in the distilled
water and autoclave at 121°C for20 min. Cool to 50°C and pour intoPetri dishes. Store plates at 4°C.
Organisms that ferment lactose are seen as darkpink colonies. Non-lactose fermenting coloniesare seen as yellow-clear colonies. Salmonellaspp. and Edwardsiella tarda will appear asnon-lactose fermenting colonies.
Distilled water 500 ml
Marine oxidative-fermentative medium (MOF) (Leifson, 1963). See under Oxidativefermentative marine.
McFarland (nephelometer) standards preparation
McFarlandTube No.
Sulfuric acid1% aqueous solution (ml)
Barium chloride,1% aqueous solution (ml)
Corresponding densityof bacteria – 106
International Units (IU)of opacity
1 9.9 0.1 300 35 9.5 0.5 1500 15
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Method: Mix the 1% sulphuric acid and the 1% barium chloride according to the table. Use clean, clear glass tubes. Plug or capthe tubes and seal with parafilm. Store tubes upright.Use: When comparing bacterial density against the tubes, ensure that the bacterial suspension is in a similar sized glass tube tothe standard. It may be best to prepare the standards in a tube or bottle that the bacterial suspensions will be normally prepared in.Sensitivity to vibriostatic agent (0/129 discs), prepare lawn inoculum at McFarland tube 1.API 20E, API 20NE, API 50CH. Inoculate with inoculum prepared to turbidity of McFarland tube 1.API ZYM. Inoculate with inoculum prepared to turbidity of McFarland tube 5.
(Taken from Difco manual, referenced in Gradwohl’s Clinical Laboratory Methods and Diagnosis. A.C. Sonnenwirth and L. Jarett(eds). C.V. Mosby Company, 1980, p.1363.)
Motility – Hanging drop method (Recommended in West and Colwell, 1984)Place a drop of suspension from an early stationary phase broth culture on to a coverslip. Place smalldollops of vaseline or plasticine at the corners. Invert coverslip and place on to a glass slide so that thedrop of culture hangs free of the glass slide. Examine under phase contrast, or bright-field with thesubstage condenser aperture closed down to give greater contrast. Weakly motile strains may notbe detected in semi-solid motility medium. For some organisms, motility is temperature dependent;therefore, ensure that growth medium is incubated at the appropriate temperature.
MRVP test medium (Clark and Lubs, 1915; Cowan and Steel, 1970; used by West and Colwell,1984)
Reagent Amount Preparation of media Description of testMRVP medium (Oxoid) 3.75 g Dissolve reagent in distilled water and
autoclave at 121°C for 15 min. Dispense5 ml per tube.
Add NaCl for marine organisms. Mustincubate for 2–3 days before testing.
VP reaction: Red coloration may develop upto 18 h after the addition of reagents.
Distilled water 250 ml
For Voges-Proskauer test, see under VP.
Methyl Red reagent
Reagent Amount Preparation of media Description of testMethyl Red 40 mg Add methyl red reagent to ethanol and heat
to 50°C in a waterbath to assist dissolution.Make up to 100 ml total with distilled water.
More ethanol can be added if a precipitateforms.
After incubation at optimal temperature for at least 2days, add 3–4 drops of Methyl Red reagent.Persistence of a red colour indicates a positive result.
See photographic section.
Ethanol 95% 40 ml
Mycoplasma characterization tests (Aluotto et al., 1970 with modifications)
Reagent Amount Preparation of media Description of testHeart infusion broth – stock Add dehydrated media to distilled water.
Using 5M NaOH adjust pH to 7.6.Inoculate a test and substrate controltube with 1 ml of a 24 h culture grownin broth.
Read tests daily for up to 2 weeks. A pHdrop of 0.5 unit or more in the glucose tubecompared with the appropriate substratecontrol tube is a positive reaction.
A rise of 0.5 pH unit in the arginine or ureatubes compared with the appropriatesubstrate tubes indicates a positivereaction.
The pH values are read by comparison witha set of standards ranging in pH from 5.6 to8.4.
Heart infusion broth 25 gDistilled water 1000 mlBasal medium Prepare yeast extract stock, and phenol
red stocks.
To 74 ml of HIB add horse serum, yeastextract, phenol red and test substrate.Using 5 M NaOH of 5 M HCl, adjust toappropriate pH depending on testsubstrate.
Prepare substrate control tubes, that is,basal medium without test substrate.
Filter sterilize and dispense in 5 mlamounts to sterile bijou or otherscrew-capped bottles.
Heart infusion broth (Difco)stock
74 ml
Horse serum (heated 56°C for30 min)
10 ml
Yeast extract (Oxoid) stocksolution of 10% w/v.Filter-sterilize.
5 ml
Phenol red (0.5% w/v)autoclaved
1 ml
Test substrate
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Glucose (10% w/v) 10 ml pH 7.6Arginine (0.2%) 10 ml pH 7.0Urea (10% w/v) 10 ml pH 7.0OF test Dispense 1 ml of sterile medium into
15 × 45 mm tubes.Inoculate duplicate test and substratecontrol tubes with 0.5 ml of an overnightbroth culture. Overlay one test and onecontrol tube with sterile paraffin oil. Incubateat appropriate temperature and examinedaily for up to 2 weeks for pH changecompared with pH standards.
Fermentative organisms produce acid inboth the aerobic and anaerobic tubes,whereas oxidative organisms produce acidin the aerobic tube only.
Basal medium 90 mlGlucose (10% w/v) 10 ml,
pH 7.6
Tetrazolium (TTC) reduction Aseptically add reagents to HIA stock andpour into 5 cm Petri dishes.
TTC can also be tested in broth ratherthan on agar plates.
Inoculate duplicate plates with agar plugstaken from dense growth on culture plates.Agar plugs can be excised using a scalpelblade, alcohol flame-sterilized spatula, oropen-ended ‘hockey stick’ made from abent Pasteur pipette. Slide the inverted agarplug over the surface of the TTC plates.
Incubate one plate aerobically and the otheranaerobically for up to 2 weeks.
Development of a pink-red colour in 3–4days is positive.
HIA stock 74 mlHorse serum 20 mlYeast extract stock 5 mlTTC (2% w/v) 1 ml
Phosphatase Aseptically add reagents to HIA stock andpour into 5 cm Petri dishes.
Phosphatase can also be tested in brothrather than on agar plates.
Inoculate 3 plates with a drop from a 24 hbroth culture. Incubate plates at theappropriate temperature. On days 3, 7 and14, flood plate with 5 M NaOH. A positivereaction is the appearance of a red colour.
HIA 74 mlHorse serum 20 mlYeast extract stock 5 mlPhenolphthalein diphosphate(sodium salt) (1% w/v)
1 ml
Film and Spots 1 Aseptically remove egg yolks andhomogenize with an equal amount ofsterile distilled water. Add the homogenateto HIA at a final concentration of 10%.Pour into 5 mm Petri dishes.
Inoculate the egg yolk medium with testorganism, and incubate in CO2 at 37°C forup to 14 days. Examine macroscopicallyusing reflected light. An iridescent or pearlyfilm seen over areas of heavy growthindicates a positive reaction.
HIA 90 mlEgg yolk 10 ml
Film and Spots 2 An alternative method is to use 20%horse serum in the medium.
Inoculate plates with test organism andincubate in CO2 at 37°C for up to 14 days.The development of small spots in themedium indicates a positive reaction.
HIA 80 mlHorse serum 20 ml
Catalase Flood a plate containing 24–48 h growthwith 30% hydrogen peroxide. Production ofbubbles indicates a positive reaction.
HIA plateSee also Digitonin and Dienesstain
Note: This is the original medium proposed by Aluotto et al. (1970). Bacto heart infusion broth (Difco) contains 500 g beef heartinfusion, 10 g bacto-tryptone, 5 g sodium chloride. An alternative is to use Difco PPLO broth with Mycoplasma supplement, to whichthe phenol red and test substrate is added. Some Mycoplasmas may be inhibited by 1% arginine, as originally proposed by Aluottoet al. (1970), therefore 0.2% is recommended. TTC = 2, 3, 5-triphenyltetrazolium chloride.
pH standards: Add phenol red to basal medium and dispense 3-ml lots into 5 ml tubes. Adjust pHin tubes to give a pH range of 5.6–8.4.
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Control organisms:Glucose positive = M. bovirhinis ATCC 19884, and negative = M. arthritidis ATCC 19611.Arginine hydrolysis positive = M. arthritidis ATCC 19611, and negative is M. bovirhinis ATCC19884.Urea positive = T-strain mycoplasma, and negative = M. arthritidis ATCC 19611.OF test organisms: Oxidative = M. pneumoniae ATCC 15531, Fermentative = M. bovirhinisATCC 19884.TTC control organisms: Positive aerobic and anaerobic = M. bovirhinis ATCC 19884. Negativefor both is M. arthritidis ATCC 19611.Phosphatase control organisms: Positive = M. arthritidis ATCC 19611. Negative = M. bovirhinisATCC 19884.Film and spots control organisms: Positive = M. gallinarum ATCC 19708. Negative = M.arthritidis ATCC 19611.Sterol requirement: Inoculate into serum-free basal medium, and basal medium with 5 mg bovineserum albumin per ml, 0.01 mg of palmitic acid per ml, and different concentrations of cholesterol(1.0, 5.0, 10.0, 20 mg/ml). Growth is determined by sedimenting the mycoplasma and testing themedium for protein content by the biuret method. Paper disc inhibition method: Use a 1.5% w/vsolution of digitonin (Sigma), and 5, 10, 20% w/v solutions of sodium polyanetholesulphonate(Koch-Light Labs, UK). The widths of the zones of growth inhibition are measured. Zones ofinhibition are 5–10 mm wide.
Ninhydrin reagent Used for detection of hippurate hydrolysis (from Barrow and Feltham, 1993).See under Hippurate hydrolysis.
Nitrate broth (Quoted in Crosby, 1967; Cowan and Steel, 1970; West and Colwell, 1984)
Reagent Amount Preparation of media Description of testKNO3 0.25 g Add reagents to distilled water and
autoclave at 121°C for 15 min. Dispense5 ml into tubes.
Inoculate media with organisms and incubateat the appropriate temperature.
For organisms that require salt, add 0.5 ml of20% NaCl to 5 ml of test media.
Nutrient broth (Oxoid) 3.25 gDistilled water 250 ml
Reagent Amount Preparation of media Description of testNitrate reagent A Add sulphanilic acid to distilled water,
then dissolve in the acetic acid.After incubation for 24 or 48 h, add 5 dropsof nitrate reagent A, and 5 drops of nitratereagent B to nitrate broth. Appearance of ared colour is indicative of a positive reaction.
Tubes that do not show a red colour shouldbe tested with zinc dust (match headamount). Development of a red colourindicates a true negative result, whereasno colour indicates that nitrate is absent,indicating that it was reduced by theorganism to nitrite, which in turn wasreduced – therefore, a positive result.
Sulphanilic acid 1.28 gDistilled water 110 mlAcetic acid 50 ml
Nitrate reagent B Handle with caution and use mask andgloves.
Add dimethyl-a-naphthylamine todistilled water. Then add acetic acidand dissolve by heating to 50°C in awaterbath.
Dimethyl-a-naphthylamine 0.96 mlDistilled water 110 mlAcetic acid 50 ml
Nocardia spp.: growth medium. See Glucose yeast extract agar
0/129. See under Vibrio discs for description of test.0/129 = 2:4-diamino-6:7-diisopropyl pteridine (0/129) phosphate. Discs are available from Oxoidand other companies. Available at concentrations of 10 mg and 150 mg.
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ONPG – o-nitrophenyl-b-D-galactosidase (Cowan and Steel (1970) using the method of Lowe,1962)
Reagent Amount Preparation of media Description of testOxoid peptone water 0.9 g Dissolve the peptone water in distilled water and
autoclave at 121°C for 20 min.Inoculate a tube of ONPG brothand incubate at the appropriatetemperature for 24–48 h.
A yellow colour indicates a positiveresult and indicates the presenceof the enzyme b-galactosidase.
Distilled water 60 mlONPG 0.15 g Dissolve the ONPG in the phosphate solution at pH
7.5 and filter-sterilize. Aseptically add to the peptonewater, and dispense 2.5 ml into sterile tubes.
Store at 4°C and keep protected from the light.
Na2HPO4 0.035 gDistilled water 25 ml
ONPG discs are available from Oxoid. Discs are recommended when testing Flavobacteriaceae.
Oxidase test (Kovács, 1956. Used in Cowan and Steel, 1970; West and Colwell, 1984)
Reagent Amount Preparation of media Description of testTetramethyl-p-phenylenediamine
1% aqueoussolution
Prepare a 1% solution ofoxidase reagent in water. Useimmediately.
The oxidase reagent must bestored in a stoppered darkglass bottle, protected from thelight and stored in the fridge.It should not be used if it hasbecome deep blue in colour.
Place a piece of filter paper in an empty Petri dish.Dampen with freshly prepared oxidase reagent. Usinga platinum loop or wooden orange stick or toothpick,smear a streak of bacterial growth across the paper.Appearance of a deep purple colour in 10–30 secindicates a positive reaction. A purple colour after 2 minmay be a false positive. Discard once the filter paperbecomes a blue colour.
Do not test for oxidase reaction from media that containscarbohydrate such as TCBS and MCA (Jones, 1981).
Note: Commercial oxidase strips are also available and are recommended, as they offer a standardized test method.
Oxidative-fermentative (O-F) media (Media of Hugh and Leifson, 1953. For marine organisms,use marine oxidative fermentative medium of Leifson, 1963)
Reagent Amount Preparation of media Description of testDifco bacto tryptone 0.8 g Mix all ingredients, except horse serum,
in distilled water and boil for 1 min todissolve. Cool slightly and pH to 7.1using 10 M NaOH. Dispense 5 ml totubes and autoclave at 121°C for 15 min.Cool to 50°C and aseptically add 2drops of sterile horse serum per tube.Store at 4°C.
Fermentative organisms produce an acidreaction (yellow colour) in both the sealed andopen tubes. Oxidative organisms produceacid at the surface of the open tube. Growth isseen at the surface of the medium with little orno growth at the bottom of the open tube, andno growth in the sealed tube. Organisms thatfail to either ferment or oxidize glucose mayproduce an alkaline reaction (purple) at thesurface of the open tube.
NaCl 2.0 gK2HPO4 0.12 gGlucose 4.0 gOxoid agar No 1 0.8 gIndicator (see below) 24 mlSterile horse serumnot inactivated
2 drops per5 ml tube
Distilled water 400 ml
Notes from Hugh and Leifson (1953): Metabolism of carbohydrate involves two different mechanisms. One mechanism is calledfermentation and occurs without oxygen and is therefore an anaerobic process. The other mechanism is called oxidation andoccurs in the presence of oxygen and is therefore an aerobic process. The OF medium from Oxoid is based on Hugh and Leifson(1953).
OF indicator
Reagent Amount Preparation of media Description of testCresol red 0.15 g Dissolve the bromothymol blue in water, 0.3 ml of a
1% solution is added to each 100 ml of medium.Bromothymol blue 0.10 gNaOH 0.20 gDistilled water 500 ml
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Marine oxidative fermentative medium for marine organisms (MOF) (Used for carbohydratemetabolism for marine organisms; Leifson, 1963)
Reagent Amount Preparation of media Description of testCasitone (Difco) 0.1 g Dissolve ingredients in distilled water and adjust
pH to 7.5. Autoclave at 121°C for 15 min.Autoclave artificial seawater separately and addto ingredients in distilled water.
Aseptically add 10 ml filter-sterilized glucoseper 100 ml. Aseptically dispense 5 ml to 10 mltubes.
Inoculate 2 tubes of medium with organismand overlay the medium in one tube with alayer of sterile paraffin oil to approximately1 cm depth, or 0.5 ml. Incubate tubes atappropriate temperature.
Organisms that ferment carbohydrateacidify the medium in both tubes, whereasaerobic organisms acidify the medium inthe ‘open’ tube only.
For fermenting organisms record ‘F’ onresults sheet. For aerobes or oxidizingorganisms, record ‘O’ on results sheet.
Yeast extract 0.01 gAmmonium sulphate 0.05 gTris buffer 0.05 mlAgar 0.3 gDistilled water 50 mlArtificial seawater 50 ml
Phenol red(0.1% stock)
1 ml Phenol red: Used at a 0.001% finalconcentration. (Prepare a 0.1% solution anduse 1 ml of this per 100 ml of medium).
Glucose 1.0% Prepare a 10% solution and filter-sterilize.
Note: The indicator bromothymol blue, which is used in the conventional OF medium, may be toxic to some marine organisms.
Paraffin oil (Used for overlay in decarboxylase tests and OF test)
Reagent Amount Preparation of media Description of testParaffin oil As required Dispense into 100 ml bottles or any
appropriate-sized bottle. Hot-air sterilizeat 160°C for 60 min. Do not autoclave asthe oil goes cloudy.
Used as overlay in ADH, LDC, ODC and OF tests.
For ease of use, oil can be dispensed into a 1 l Schottbottle and capped with a Socorex 2 ml dispenser unit.Dispense 0.5 ml into 10 ml media tubes.
Physiological saline. See saline
Rogosa agar (RA) (Oxoid manual) (Selective medium for Lactobacilli; Rogosa et al., 1951)
Reagent Amount Preparation of media Description of testRogosa agar (Oxoid) 82 g Suspend Rogosa agar in distilled water and
bring to the boil to dissolve. Add glacial aceticacid and mix thoroughly. Heat to 90–100°C for2–3 min with frequent agitation. Distribute intosterile tubes or Petri dishes. Do not autoclave.
May assist to differentiate Lactobacillifrom other Gram-positive bacteria suchas Carnobacterium, Arcanobacteriumand Vagococcus species.
Distilled water 1000 mlGlacial acetic acid 1.32 ml
Ingredients (g/l) tryptone (10.0), yeast extract (5.0), glucose (20.0), sorbitan mono-oleate (1.0), potassium dihydrogen phosphate(6.0), ammonium citrate (2.0), sodium acetate (25.0), magnesium sulphate (0.575), manganese sulphate (0.12), ferric sulphate(0.034), agar (20.0).
Saline: physiological saline
Reagent Amount Preparation of media Description of testNaCl 0.85 g Add salt to distilled water. Dispense 10 ml into
McCartney bottles. Autoclave at 121°C for 15 min.May be used as an inoculum forcommercial kit tests and ‘Biochem Set’.Distilled water 100 ml
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Salt – 20% stock solution
Reagent Amount Preparation of media Description of testNaCl 100 g Add NaCl to distilled water. Autoclave
at 121°C for 15 min.For ease of use, 20% NaCl can be dispensed into a 1 lSchott bottle and capped with a Socorex 2 ml dispenserunit with 0.5 ml graduations. Dispense 0.5 ml per 5 ml ofmedia to produce final NaCl concentration of 2% in test.
Distilled water 400 ml
In most cases, the carbohydrate media tubes do not require NaCl in addition to the 2% NaCl inoculum fluid. However, liquid tubemedia such as aesculin, ADC, ODC, LDC, the decarboxylase control, MRVP, indole (TW) and nitrate do require that 500 ml of a20% NaCl solution is added to the 5 ml of media.
Salt tolerance0%, 3% NaCl. See Gelatin/ NaCl split plate. Using a wire loop, spot inoculate equal amounts of
the bacterium to the 0% NaCl side and the 3% NaCl side. After 1–2 days incubation at the appropriatetemperature, examine for areas of clearing or opacity. See photographic section.
10% NaCl. For tolerance to 10% salt, dispense equal volumes of TSB and 20% NaCl stocksolution into a sterile bijou bottle or 10 ml tube. Add bacteria to turbidity of 0.5 or tube 1 McFarlandstandard. Incubate at appropriate temperature for 24–48 h. Look for obvious increase in growth oforganism as seen by cloudiness in the medium.
Survival at 50∞C (Used for Nocardia species; Gordon et al., 1974)Inoculate slants of glucose yeast extract agar, and incubate in a 50°C waterbath for 8 h. Remove fromwaterbath, cool quickly and incubate at 28°C for 3 weeks. Examine tubes for growth.
Tryptophane deaminase (TDA) reagent (For use in the API 20E kit. Can be purchasedcommercially)
Reagent Amount Preparation of media Description of testFerric chloride 3.4 g Dissolve the ferric chloride in 90 ml of water,
and then make up to 100 ml.Used in the API 20E kit. A positive is a browncolour. Proteus strains tend to be positive.Distilled water 100 ml
Triple sugar iron agar (TSI)
Reagent Amount Preparation of media Description of testTriple sugar ironagar (Oxoid)
9.75 g Dissolve reagent indistilled water and pHto 7.4. Dispense in5 ml aliquots to 10 mltubes. Autoclave at121°C for 15 min. Allowto cool and solidify in aslanted position to givedeep butts.
Inoculate the tube using a straight wire. Stab into medium, and thenzig-zag the slope. H2S producers will turn the medium black.Fermentative organisms will acidify the TSI butt (yellow), whereasnon-fermentative organisms will grow on the slant only and show eitherno pH change or an alkaline reaction and, rarely, an acidic reaction.
For some organisms this test method may not be as sensitive as usinga lead acetate paper strip.
A hydrogen sulphide Biostrip (catalogue number TM343) is availablefrom MedVet Science.
Distilled water 150 ml
Urea (Christensen, 1946)
Part A
Reagent Amount Preparation of media Description of testOxoid agar No 1 3.75 g Add agar to distilled water and autoclave at 121°C for
15 min. Cool to 50°C.Add 1% NaCl for marine organisms.
Distilled water 225 ml
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Preparation of Media for Culture and Identification 277
Part B
Reagent Amount Preparation of media Description of testBBL brand ureaagar base
8.7 g Add reagents to distilled water and filter-sterilize througha 0.22 mm Millipore filter.
Add Part A to 25 ml of Part B. Aseptically dispense 5 mlvolumes per tube. Allow to solidify in a sloped position.
Inoculate the slant heavily over theentire surface with a loopful of bacteriaharvested from an agar medium. Brightpink indicates a positive reaction.
Distilled water 30 ml
Note: Urea slopes should be prepared with a deep butt and a short slant (Gilardi, 1983).
Vibrio discs
0/129 = 2:4-diamino-6:7-diisopropyl pteridine (0/129) phosphate, vibriostatic reagent (mg). TheVibrio spp. (including Listonella spp., Moritella spp. and Photobacterium spp.) are sensitive tothis compound, commonly called vibriostatic agent (Shewan et al., 1954). This test assists in thedifferentiation of Vibrio species from other Gram-negative rods, particularly Aeromonas species, whichare resistant to vibriostatic agent. Almost all Vibrio species are sensitive to 0/129 at 150 mg and someare sensitive to 10 mg. However, Vibrio cholerae 0139 has developed resistance to 0129 at 150 mgconcentration (Albert et al., 1993; Islam et al., 1994).
Treat this test in the same manner as ‘sensitivity testing’ used to test an organism as sensitive orresistant to an antimicrobial reagent. Prepare an inoculum in normal saline to the opacity of McFarlandtube 1, a suspension that is just visible to the eye. Use a sterile cotton-tipped swab to lawn inoculate theplate. Use BA for freshwater organisms and MSA-B for marine organisms. Place the two discs on tothe agar surface so that they are at least 4 cm apart. Invert the plate, and incubate at the appropriatetemperature for 24 h. If there is insufficient growth incubate a further 24 h; however, normally the zonesare recorded at 24 h as further growth, especially when testing Vibrio species that swarm, may show afalse resistant result. Record zone sizes as ‘sensitive’ (S) or ‘resistant’ (R). For the 0/129 500 mg disc, azone of 9 mm is susceptible for Vibrio species (Bernardet and Grimont, 1989). A zone size of 22 mm isconsidered sensitive for Photobacterium damselae ssp. damselae (Love et al. 1981).
The discs are purchased from Oxoid or Rosco Diagnostics. The Oxoid codes for the twoconcentrations are DD14 for 0129 10 mg, and DD 15 for 150 mg.
Voges-Proskauer reaction See MRVP test (Clark and Lubs, 1915; Voges and Proskauer, 1898, asreported in Cowan and Steel, 1970. Used by West and Colwell, 1984)
Use for the detection of acetoin from glucose fermentation. The incubation time and temperaturerather than the method influence the production of acetoin.
MRVP test medium (The commercial medium is based on Clark and Lubs medium, 1915)
Reagent Amount Preparation of media Description of testMRVP medium(Oxoid)
3.75 g Dissolve reagent in distilled water andautoclave at 121°C for 15 min. Dispense5 ml per tube.
Add NaCl for marine organisms. Must incubate for2–3 days before testing.
Add VP reagent A, and VP reagent B. Red colorationmay develop up to 18 h after the addition of reagents.
Distilled water 250 ml
MR = methyl red; VP = Voges-Proskauer.
VP test reagents (Barritt, 1936)
Reagent Amount Preparation of media Description of testReagent A Dissolve a-naphthol in ethanol.
Store at 4°C.After incubation of MRVP medium for 48 hat appropriate temperature, place 1 ml intoa test tube. Add 0.6 ml of reagent A, and0.2 ml of reagent B. Examine for pinkcolour up to 4 h at room temperature.
Tests for acetylmethylcarbinol (acetoin).
a-naphthol 5.0 gAbsolute ethanol 100 mlReagent B Weigh KOH. Make up to 100 ml
final volume with distilled water.Store at 4°C.
KOH 40.0 gDistilled water Add to final 100 ml
Note: VP reagent I and II in the API 20E kit are also suitable. Transfer 250 ml of medium to a microfuge tube. Add 150 ml and 50 mlof reagent I and II respectively, shaking after each addition. Read after 10–20 min.
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Further Reading and Other Information Sources
Books on Fish Diseases
Austin, B. and Austin, D.A. (1999) Bacterial Fish Pathogens: Disease of Farmed and Wild Fish. 3rd revised edn. PraxisPublishing, Chichester, UK.
Woo, P.T.K. and Bruno, D.W. (eds) (1999) Fish Diseases and Disorders. Vol. 3: Viral, Bacterial and Fungal Infections.CAB International, Wallingford, UK.
Bergey’s Manual of Systematic Bacteriology, Vol I and Vol II. (1984) Holt, J.G. (ed.) Lippincott Williams and Wilkins,Baltimore, Maryland.
Diagnostic Manual for Aquatic Animal Diseases, 3rd edn (2000) Office International Des Epizooties (OIE), 12 rue deProny, F-75017, Paris, France.
Plumb, J.A. (1999) Health Maintenance and Principal Microbial Diseases of Cultured Fishes. Iowa State UniversityPress.
The fourth edition of the OIE Diagnostic Manual for Aquatic Animal Diseases will be available in July 2003. Thesemanuals are also available on line. http://www.oie.int/eng/normes/fmanual/A_summry.htm
Books on Biochemical Identification Tests
Cowan, S. and Steel, K. (1970) Manual for the Identification of Medical Bacteria. Cambridge University Press,Cambridge.
Barrow, G.I. and Feltham, R.K.A. (1993) Cowan and Steel’s Manual for the Identification of Medical Bacteria,3rd edn. Cambridge University Press, Cambridge.
MacFaddin, J.F. (1980) Biochemical Tests for Identification of Medical Bacteria, 2nd edn. Williams and Wilkins,Baltimore, Maryland.
MacFaddin, J.F. (2000) Biochemical Tests for Identification of Medical Bacteria, 3rd edn. Williams and Wilkins,Baltimore, Maryland.
Other Biochemical Identification Schemes
Alsina, M. and Blanch, A. (1994) A set of keys for biochemical identification of environmental Vibrio species. Journalof Applied Bacteriology, 76, 79–85.
Alsina, M. and Blanch, A. (1994) Improvement and update of a set of keys for biochemical identification of Vibriospecies. Journal of Applied Bacteriology, 77, 719–721.
Carson, J., Wagner, T., Wilson, T. and Donachie, L. (2001) Miniaturised tests for computer-assisted identification ofmotile Aeromonas species with an improved probability matrix. Journal of Applied Microbiology, 90, 190–200.
Schmidtke, L.M. and Carson, J. (1994) Characteristics of Vagococcus salmoninarum isolated from diseased salmonidfish. Journal of Applied Bacteriology, 77, 229–236.
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Journals
Applied and Environmental Microbiology http://aem.asm.org/Aquaculture http://www.elsevier.com/locate/aquacultureBulletin of the European Association of
Fish PathologistsCurrent Microbiology http://link.springer.de/link/service/journals/00284/Diseases of Aquatic Organisms http://www.int-res.com/journals/dao/Fish PathologyInternational Journal of Systematic and
Evolutionary Microbiology http://ijs.sgmjournals.org/Journal of Applied Ichthyology http://www.blackwell-synergy.com/Journals/issuelist.asp?journal=jaiJournal of Applied Microbiology http://www.blackwell-synergy.com/Journals/issuelist.asp?journal=jamJournal of Aquatic Animal HealthJournal of Clinical Microbiology http://jcm.asm.org/Journal of Fish DiseasesVeterinary Microbiology http://www.elsevier.nl/locate/vetmic
Culture Collections
Ecole Nationale Vétérinaire de Toulouse, 23 chemin des Capelles, F-31076 Toulouse cedex 03, France. http://www.bacterio.cict.fr/collections.html
ACAM: Australian Collection of Antarctic Microorganisms, Antarctic CRC, University of Tasmania, Hobart,Australia.
AHLDA: Animal Health Laboratories, Department of Agriculture. 3 Baron-Hay Court, South Perth, WesternAustralia 6151.
ATCC: American Type Culture Collection. Corporate: ATCC, 10801 University Boulevard, Manassas, VA20110–2209, USA. Products & Services Orders: ATCC, PO Box 1549, Manassas, VA 20108–1549, USA.http://www.atcc.org/
CCUG: Culture Collection, University of Göteborg, Department of Clinical Bacteriology, Institute of ClinicalBacteriology, Immunology, and Virology, Guldhedsgatn 10A s-413, 46 Göteborg, Sweden.
CDC: Center for Disease Control, 1600 Clifton Rd, Atlanta, Georgia 30333, USA.CECT: Coleccion Espanola de Cultivos Tipo, Universidad de Valencia, Burjassot, Spain.CIP: Collection de l’Institut Pasteur, Institut Pasteur, 28 Rue du Docteur Roux, 75724 Paris Cedex 15, France.CNCM: Collection Nationale de Culture de Microorganismes, Institut Pasteur, Paris, France.DSMZ (DSM): Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1B,
D-38124, Braunschweig, Germany. http://www.dsmz.de/dsmzhome.htmIAM: Institute of Molecular and Cellular Biosciences (formerly Institute of Applied Microbiology, Culture Collection –
IAMCC), The University of Tokyo, Yayoi, Bunkyo-Ku, Tokyo, Japan.KMM: Collection of Marine Microorganisms, Pacific Institute of Bioorganic Chemistry, Vladivostok, Russia.NCFB: National Collection of Food Bacteria (previously named NCDO). Transferred from the IFR (Institute of Food
Research), Reading, to National Collections of Industrial, Food and Marine Bacteria, 23 Machar Drive,Aberdeen AB24 3RY, UK.
NCIMB: National Collection of Industrial and Marine Bacteria, National Collections of Industrial, Food and MarineBacteria, 23 Machar Drive, Aberdeen AB24 3RY, UK. http://www.ncimb.co.uk/ncimb.htm
NCTC: National Collection of Type Cultures, Central Public Health Laboratory, Colindale Ave., London NW9 5HT,UK. www.phls.co.uk
RVAU: Royal Veterinary and Agricultural University, Copenhagen, Denmark.UB: University of Barcelona, Barcelona, Spain.
Bacterial Names/Taxonomy/Nomenclature
Bacterial nomenclature is continually being updated at the following websites:DSMZ (DSM): Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1B,
D-38124, Braunschweig, Germany. http://www.dsmz.de/dsmzhome.htm
Further Reading 279
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List of Bacterial Names with Standing in Nomenclature: http://www.bacterio.cict.fr/NCBI: http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html/
Fish Disease Web Sites
http://www.fishdisease.net This website is for aquatic animal health professionals and contains information onleaflets on different diseases, an image library on parasites, notification of conferences, jobs and contacts.
http://www.fishbase.org/home.htm or http://www.fishbase.org/search.html This website contains information onfishes that may be useful to fisheries managers, scientists and others. One of its features is the latest scientificname of the different fishes.
280 Further Reading
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Appendix
Common Name and Scientific Name of Aquatic Animals
281
Common name Scientific nameAbalone Haliotis discus hannaiAdriatic sturgeon Acipenser naccariiAfrican cichlid Nimbochromis venustusAmazon freshwater dolphin Inia geoffrensisAmberjack Seriola dumeriliAmerican alligator Alligator mississippiensisAmerican crayfish Orconectes limosus, Pacifastacus leniusculus, Procambarus
clarkiiAmerican eel Anguilla rostrataAmerican plaice Hippoglossoides platessoidesAntarctic fur seal Arctocephalus gazellaArctic char Salvelinus alpinus L.Atlantic bottlenose dolphin Tursiops truncatusAtlantic cod Gadus morhua L.Atlantic croaker Micropogon undulatusAtlantic menhaden Brevoortia tyrannus LatrobeAtlantic salmon Salmo salar L.Atlantic walrus Odobenus rosmarus rosmarusAtlantic white-sided dolphin Lagenorhynchus acutusAustralian oyster Saccostrea commercialisAyu Plecoglossus altivelisBalloon molly Poecilia spp.Banana prawn Penaeus merguiensisBarramundi Lates calcarifer (Bloch)Beluga whale Delphinapterus leucasBighead carp Aristichthys nobilisBlack acara Cichlasoma bimaculatumBlack mullet Mugil cephalusBlack scraper Novodon modestusBlack skirted tetra Hyphessobrycon spp.Blenny Zoarces viviparusBlue fish Pomatomus saltatrixBlue mackerel Scomber australasicusBlue manna crab Portunus pelagicusBlue shrimp P. (Litopenaeus) stylirostris
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282 Appendix
Common name Scientific nameBottlenose whale Hyperodoon ampullatusBoney bream Nematolosa come (Richardson)Borneo mullet Liza macrolepisBottle-nosed dolphin Tursiops truncatusBowhead whale Balaena mysticetusBream Abramis bramaBrine shrimp ArtemiaBrook salmon Salvelinus fontinalisBrook trout Salvelinus fontinalis (Mitchill)Brown bullhead Ictalurus nebulosus (Lesueur)Brown shark Carcharhinus plumbeusBrown-spotted grouper Epinephelus tauvina, E. coioidesBrown trout Salmo trutta m. fario, Salmo trutta m. lacustris L.Burnett salmon Polydactylus sheridani (Macleay)Californian sea lion Zalophus californianusCanadian shrimp Lismata amboiensCarp Cyprinus carpio L.Catfish Clarius batrachus L.Caucasian carp Carassius carassiusChanchito Chichlasoma facetum (Jenyns)Channel catfish Ictalurus punctatus (Rafinesque)Chinook salmon Oncorhynchus tschawytschaChub Leuciscus cephalisChum salmon Oncorhynchus keta (Walbaum)Cichlid Oreochromis mossambicusClam Tapes philippinarumCod Gadus morhuaCoho salmon Oncorhynchus kisutchCommon carp Cyprinus carpio L.Common dolphin Delphinus delphisCommon seal Phoca vitulinaCommon snook Centropomus undecimalisCommon wolf-fish Anarhichas lupusCoral prawn Metapenaeopsis spp.Crucian carp Carassius carassiusCultured flounder Paralichthys olivaceusCutthroat trout Salmo clarkiCuttle fish Sepia officinalisDab Limanda limandaDace Leuciscus leuciscus L.Damselfish Chromis punctipinnisDamselfish Pomacentridae, Amphiprion clarkii (Bennett),
Amblyglyphidodon curacao (Bloch)Danio Danio devarioDiscus fish Symphysodon discus, S. aequifasciatusDolphin Tursiops truncatus, T. gephyreusDolphin fish Coryphaena hippurus L.Eastern freshwater cod Maccullochella ikeiEastern mosquitofish Gambusia holbrookiEastern painted turtle Chrysemys picta pictaEel Anguilla japonica, A. reinhardtiiElephant seal Mirounga leoninaEuropean crayfish Astacus leptodactylus, A. pachypus, A. torrentium, A. astacus,
Austropotamobius pallipesEuropean eel Anguilla anguillaEuropean sea bass Dicentrarchus labrax Serranidae
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Appendix 283
Common name Scientific nameFairy shrimp Branchipus schaefferi (Fisher),Chirocephalus diaphanus
(Prévost), Streptocephalus torvicornis (Waga)False killer whale Pseudorca crassidensFarmed mussel Perna pernaFathead minnow Pimephales promelasFighting fish Betta splendensFiremouth cichlid Cichlasoma meekiFlat-tailed mullet Liza dussumieri (Valenciennes)Flounder Paralichthys olivaceus, P. flesusFlounder Platichthys flesusFour bearded rockling Enchelyopus cimbrius L.Freshwater cod (Australian native) Maccullochella spp.Freshwater dolphin Inia geoffrensisFreshwater prawn Macrobranchium rosenbergiiFur seal Arctocephalus australisGilthead sea bream Sparus auratusGolden shiner Notemigonus crysoleucas (Mitchell)Goldfish Carassius auratus L.Goldsinny wrasse Ctenolabrus rupestrisGourami (three-spot) Trichogaster trichopterusGrass carp Ctenopharyngodon idellaGrayling Thymallus thymallus L.Greater weever Trachinus dracoGreen knife fish Eigemannia virescensGreen moray eel Gymnothorax funebrisGreen sturgeon Acipenser medirostrisGreenback flounder Rhombosolea tapirina GuntherGrey seal Halichoerus grypusGrouper Epinephelus guaz, E. coioidesGuppy Poecilia reticulata (Peters), Lebistes reticulatusHaddock Melanogrammus aeglefinus L.Halibut Hippoglossus hippoglossus L.Harbour porpoise Phocoena phocoenaHarbour seal Phoca vitulinaHarp seal Phoca groenlandicaHerring Arripis georgianusHooded seal Cystophora cristataHorse mackerel Trachurus trachurusIberian toothcarp Aphanius iberusJapanese abalone Sulculus diversicolor supratextaJapanese eel Anguilla japonicaJapanese flounder Paralichthys olivaceusJapanese medaka Oryzias latipesJewel tetra Hyphessobrycon callistus (Boulenger)Johnston crocodiles (freshwater) Crocodylus johnstoniKiller whale Orcinus orcaKing prawn Penaeus latisulcatusKnife fish Gymnotus carapoLake trout Salmo trutta m. lacustris, Salvelinus namaycush WalbaumLargemouth bass Micropterus salmoidesLemon shark Negaprion brevirostrisLittle penguin Eudyptula minorLiving dace Tribolodon hakonensis GuntherLoach Misgurnus anguillicaudatus CantorLobster Homarus gammarus L.
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284 Appendix
Common name Scientific nameLocal mussel Mutilus edulisLong-tom Tylosurus macleayanus (Ogilby)Mackerel Scomber scombrusManila clam Tapes philippinarum, T. decussatus, Ruditapes philippinarumMasu salmon Oncorhynchus masouMenhaden Brevoortia patronusMinke whale Balaenoptera acutorostrataMinnow Phoxinus phoxinus L.Molly Poecilia velifera (Regan)Mud crab Scylla serrataMullet Mugil cephalusMurray cod Maccullochella peeliMussel (Far-eastern) Crenomytilus grayanus and Patinopecten yessoensisMussel Protothaca jedoensis, Mytilus edulis, Mytilus galloprovincialisNarwahl whale Monodon monocerusNeon tetra Paracheirodon innesi, Hyphessobrycon innesiNew Zealand fur seal Arctocephalus forsteriNew Zealand mussel Perna canaliculusNew Zealand sea lion Phocarctos hookeriNile tilapia Oreochromis niloticusNorth-east Atlantic mackerel Scomber scombrusNorthern elephant seal Mirounga angustirostrisNorthern fur seal Callorhinus ursinusNorthern pike Esox lucius L.Northern right whale dolphin Lissodelphis borealisNurse shark Orectolobus ornatusOctopus Octopus vulgaris, O. joubiniOne-spot bream Diplodus sargusOrnamental fish Pterophyllum scalareOscar Astronotus ocellatusOscar Apistogramma ocellatusOtter Lutra lutraOyster Ostrea edulisOyster (eastern) Crassostreae virginicaPacific herring Clupea harengus pallasiPacific oyster Crassostrea gigasPacific salmon Oncorhynchus spp.Pacific staghorn sculpin Leptocottus armatusPacific white shrimp Penaeus vannameiPacific white-sided dolphin Lagenorhynchus obliquidensParadise fish Macropodus opercularis (L.)Pejerrey Odonthestes bonariensisPenguins Aptenodytes patagonica, Eudyptes crestatus, Pyoscelis papua,
Spheniscus demersus, Spheniscus humboldtiPerch Perca fluviatilisPike Esox luciusPilchard Sardinops neopilchardusPilot whale Globicephala scammoniPink salmon Oncorhynchus gorbuschaPink snapper Chrysophrys unicolorPinkfish Lagodon rhomboidesPirarucu Arapaima gigas CuvierPlaice Pleuronectes platessaPompanos Trachinotus carolinus L.Rabbitfish Siganus rivulatus (Forsskål)Rainbow trout Oncorhynchus mykiss (Walbaum)
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Appendix 285
Common name Scientific nameRainbow and steelhead trout Salmo gairdneriRed abalone Haliotis rufescensRed algae Jainia spp.Red claw crayfish Cherax quadricarinatusRed drum, Redfish Sciaenops ocellatusRed-eared slider turtle Chrysemys scripta elegansRed sea bream Pagrus majorRed swamp crawfish Procambarus clarkiiRedtail catfish Phractocephalus hemiliopterusRinged seal Phoca hispidaRisso dolphin Grampus griseusRoach Rutilus rutilus L.Rohu Labeo rohitaRosy barb Puntius conchoniusRudd Scardinius erythrophthalmusSablefish Anoplopoma fimbria (Pallas)Salmon Oncorhynchus kisutch (Walbaum)Saltwater crocodile Crocodylus porosusSand eel Ammodytes lancea (Cuvier), Hyperoplus lanceolatus
(Lesauvege)Sand lance Ammodytes personatus GirardSand whiting Sillago ciliata CuvierSaratoga Scleropages leichardiiSardine Sardinops melanostictus, Sardinops sagnaxScallop Pecten maximus, Argopecten purpuratusScaly mackerel fish Amblygaster posteraSea bass Dicentrarchus labraxSea bream Pagrus major, Evynnis japonicus, Sparus aurata,
Acanthopagrus latusSea catfish Arius felisSea horse Hippocampus angustus, H. barbouri, H. whitei, H. kudaSea lion Otaria flavescensSea mullet Mugil cephalus L.Sea trout Salmo trutta m. trutta L.Sea turtle Chelonia mydasSea-urchin Paracentrotus lividuSepiolid squid Euprymna scolopesShotted halibut Eopsetta grigorjewiShubunkin Carassius spp.Siamese fighting fish Betta splendens ReganSignal crayfish Pacifastacus leniusculusSilver black porgy Acanthopagrus cuvieriSilver bream Blicca bjoerknaSilver bream Acanthopagrus butcheri, A. australis (Owen)Silver carp Hypophthalmichthys molitrix ValenciennesSilver mullet Mugil curema (Valenciennes), Mugil cephalus (L.)Silver molly Poecilia spp.Silver perch Bidyanus bidyanus (Mitchell)Silver trout Cynoscion nothusSmall abalone Haliotis diversicolor supertextaSmallmouth bass Micropterus dolomieuiSnakehead fish Channa striatusSnakehead fish Ophicephalus punctatus, O. striatusSnub-nose garfish Arrhamphus sclerolepsis (Gunther)
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286 Appendix
Common name Scientific nameSockeye salmon Oncorhynchus nerka (Walbaum)Softshell clam Mya arenariaSole Solea soleaSouth African abalone Haliotis midaeSouth American side-necked turtle Podocnemis unifelisSouthern elephant seal Mirounga leoninaSowerby’s beaked whale Mesoploden bidensSpanish mackerel Scomber japonicusSpanner crab Ranina raninaSpiny soft-shelled turtle Trionyx spiniferSpot Leiostomus xanthurusSpotted dolphin Stenella plagiodonSpotted moray eel Gymnothorax moringaSpotted wolf-fish Anarhichas minorSquid Loligo pealeiSquid SepiolaSquid Teuthoidea speciesStarfish Asterias rubensStingray Dasyatis pastinacaStriped bass Morone saxatilis (Walbaum), M. chrysopsStriped dolphin Stenella coeruleoalbaStriped mullet Mugil cephalusStriped-neck musk turtle Sternotherus minor peltiferSturgeon Acipenser naccariiSunfish Mola molaTasmanian lobster Jasus novaehollandiaeTilapia Oreochromis niloticus, O. aurusTilapia Tilapia nilotica, Tilapia aurea, Tilapia mosambicaTilapia Sarotherodon aureus (Steindachner)Tom cod Gadus microgadusTropical shrimp Stenopus hispidusTrout cod Maccullochella macquariensisTurbot Scophthalmus maximus L.Turtle Dermochelys coriaceaTurtle Pseudemis scriptaViviparous blenny Zoarces viviparusWeddell seal Leptonychotes weddelliiWestern rock lobster Panulirus cygnusWhite catfish Ictalurus catus L.White clawed crayfish Austropotamobius pallipesWhite leg shrimp P. (Litopenaeus) vannameiWhite perch Roccus americanus, Morone americanus (Gremlin)White whale Delphinapterus leucasWhitefish Coregonus spp.Whitespotted rabbitfish Siganus canaliculatusWhiting Merlangius merlangusWolf-fish Anarchichas lupus L.Wrasse Labrus berggyltaYellow bass Morone mississippiensisYellow perch Perca flavescensYellowtail Seriola quinqueradiata, S. lalandiYellowfin bream Acanthopagrus australis (Owen)Zebra danio Brachydanio rerio
This list was taken from the references used in this manual.
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Glossary of Terms
287
a or aH (referring to greening of agar) Alpha haemolysisA Alkaline reactionAAHRL Australian Animal Health Reference LaboratoryACAM Australian Collection of Antarctic MicroorganismsADH Arginine dihydrolaseAes AesculinAFB Acid-fast bacteriaAHL Animal Health LaboratoriesAHLDA Animal Health Laboratories Department of Agriculture Culture CollectionAla L-alanineAmp Ampicillin disc 10 mgANA Plate media for anaerobic bacteriaAO Anacker Ordal agar for FlavobacteriaAO-M Anacker Ordal agar with added NaCl for growth of marine FlavobacteriaAPI 50CH API identification system from bioMérieux. Carbohydrate testsAPI 20E API identification system from bioMérieux. Fermentation and enzyme
testsAPI 20NE API identification system. Utilization testsAPI Rapid ID32 Strep API system for Streptococci and other Gram-positivesAPI 20 Strep API identification system for Streptococci and other Gram-positive
organismsAPI ZYM API identification system. Enzyme testsArab L-arabinoseArg ArginineAsp L-asparagineASW Artificial sea waterAT Annealing temperatureATCC American Type Culture Collection, Rockville, Maryland, USAb Beta (refers to clear zone or b haemolysis)BA Blood agarBGD Bacterial gill diseasebH b-haemolysisBHA Bacterial haemorrhagic ascitesBHIA Brain heart infusion agarBKD Bacterial kidney diseasebp Base pairs (in kilo-bases)BRD Brown ring diseaseBrucella agar Used for isolation of Brucella species
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288 Glossary
C Number of cycles in the PCR reactionCa CalciumCat CatalaseCBBA Coomassie brilliant blue agarCCA Cellobiose Colistin agar for isolation of Vibrio vulnificusCCRC Culture Centre for Research and CollectionCCUG Culture Collection of the University of Göteborg, Department of Clinical
Bacteriology, Göteborg, SwedenCDC Centres for Disease Control and Prevention, Atlanta, Georgia, USACFPA Selective media for isolation of Bordetella bronchisepticaCFU colony forming unitsCit CitrateCNCM Collection Nationale de Culture de Microorganismes (Institut Pasteur,
Paris, France)CO2 Carbon dioxideCPC Cellobiose Polymyxin B Colistin agar for isolation of Vibrio vulnificusCr Coccoid rodsCR Congo RedCSF Cerebrospinal fluidcv Curved rodsd DaysDCA Desoxycholate-citrate agarDmso Dimethyl sulphoxideDNase Test for the detection of hydrolysis of DNADSM Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH,
Braunschweig, GermanyEIM Edwardsiella ictaluri mediumEM Electron microscopyERM Enteric red mouthESC Enteric septicaemia of catfishEry Meso-erythritolF Fermentative (facultative anaerobe)FA Furunculosis agar for Aeromonas salmonicidaFAO Food and Agriculture Organization of the United NationsFINE Flounder necrotizing enteritisFM Farrell’s medium for Brucella speciesFPM Flavobacterium psychrophilum mediumG GelatinG Green-coloured colony on TCBS plateGlid Gliding motilityGal D-galactoseGlu GlucoseGlut Glutamic acidGm Gram stain reaction (blue colour = positive, red = negative)GUD Goldfish ulcer diseaseh HoursH2S Hydrogen sulphideHCl Hydrochloric acidHG (DNA) hybridization groupHip HippurateHS Hsu-Shotts agar for Flavobacterium columnareHSM For the isolation of Tenacibaculum maritimumI Inert reaction (in OF test)ID IdentificationIGS Intergenic spacerInd Indole
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Glossary 289
Inos InositolISP No. 2 Yeast malt extract agar (Difco)JOD Juvenile oyster diseaseKCl Potassium chlorideKDM2 Medium for growth of Renibacterium salmoninarumKDMC Medium for growth of Renibacterium salmoninarumKf Cephalothin 30 mg discKOH Potassium hydroxide. 20%KUMA Kumamoto Prefectural Institute of Public Health (culture collection prefix)Lac LactoseLDC Lysine decarboxylaseLJM Lowenstein-Jensen medium for Mycobacteria speciesLMG Laboratorium Microbiologie Gent Culture Collection, Universiteit Gent,
BelgiumLPS LipopolysaccharideLys LysineMA 2216 Medium for growth of marine organisms. Commercially availableMAF Modified acid fast stainMalt MaltoseMan MannitolMan An Fermentation of mannitol under anaerobic conditionsMano MannoseMCA MacConkey agarMg MagnesiumMiddlebrook’s media For the isolation of Mycobacterium speciesmin MinuteMOF Marine oxidative-fermentative mediumMot MotilityMR Methyl RedMRVP Methyl Red Voges-ProskauerMSA-B Marine salt agar – blood medium for growth of marine organismsN NegativeNA Nutrient agarNaCl SaltNaCl 0/3 Plate media containing either 0% NaCl or 3% NaCl concentrationNB Nutrient brothNCFB National Collection of Food Bacteria, Agricultural and Food Research
Council (AFRC) Institute of Food Research, Reading Laboratory,Reading, Berkshire, UK (formerly National Collection of Dairy Organisms)
NCIM National Collection of Industrial Microorganisms, National ChemicalLaboratory, India
NCIMB National Collection of Industrial and Marine Bacteria, Aberdeen,Scotland, UK
NCTC National Collection of Type Cultures, Central Public Health Laboratory,London, UK
ND Not doneNeg NegativeNG No growthNH Non-haemolyticNit NitrateNK Not knownNLF Non-lactose fermentingnmol NanomolesNVI National Veterinary Institute, Oslo, Norwaynm Nanometrent Not tested
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290 Glossary
O Oxidative (aerobic metabolism)OD Optical densityODC Ornithine decarboxylaseOF Oxidative-fermentative testONPG o-Nitrophenyl b-D-galactopyranosideOrn OrnithineOx Oxidase0%–3% Split plate containing gelatin, and concentration of salt at 0% and 3%0129 Vibriostatic agent – 2:4-diamino- 6:7-diisopropyl pteridine phosphatePacker’s plates Selective media for the isolation of Erysipelothrix speciesPBS Phosphate buffered salinePig Pigment productionpmol PicomolesPoly plates Media for the isolation of Dermatophilus speciesPos PositivePS Partially sensitive to vibriostatic discPY Peptone yeast medium for Pedobacter and Sphingobacterium speciesPYR L-Pyrrolidonyl-b-naphthylamidePYS-2 Peptone yeast medium 2R ResistantR2A Medium for isolation of marine Flavobacterium and other organismsRAA Rogosa acetate agarRBC Red blood cellsRib D-riboseS SensitiveSS Media for the isolation of Salmonella and ShigellaSAB Sabouraud’s medium for fungiSal SalicinShieh medium For the isolation of Flavobacterium columnareSiem agar Selective medium for isolation of Aerococcus viridansSKDM Selective medium for Renibacterium salmoninarumSkirrow’s medium For the isolation of Helicobacter speciesSor SorbitolSuc SucroseSW Swarming growth on agar plateSWT Seawater-based complex mediumTHA Todd-Hewitt agarTB Lab Tuberculosis laboratoryTCBS Thiosulphate–citrate–bile salts–sucrose agarTE Tris EDTA bufferTemp TemperatureTm Melting temperatureTre TrehaloseTSA Tryptone soy agarTSA-B Tryptone soy agar with added bloodTSA+NaCl Tryptone soy agar with added salt (2% final concentration)TSB Tryptic soy brothTSI Triple sugar ironTYG Tryptone yeast extract glucose agarTYG-M Tryptone yeast extract glucose agar with added NaCl for growth of
T. maritimumUro Urocanic acidV Variable reaction in literatureVAM Vibrio anguillarum medium for isolation of Listonella anguillarumVP Voges-Proskauer test
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Glossary 291
VPT Vancomycin, Polymyxin, Trimethoprim – Skirrow’s medium for theisolation of Helicobacter species
Vs Variable or slow reactionUK United KingdomUSA United States of AmericaUV Ultraviolet lightVVM Vibrio vulnificus mediumW WeakWHO World Health OrganizationWood’s Broth Selective broth for the isolation of Erysipelothrix speciesXLD Xylose lysine desoxycholate agar for isolation of Salmonella speciesXyl D-xyloseY Yellow-coloured colony on TCBS plateYSA Yersinia selective agarZN Ziehl–Neilson strain for Mycobacteria− Negative reaction−cr Gram-negative curved rod−a Negative result, but may show a-haemolysis after a week+ Positive reaction+gb Gram positive cocco-bacilli+g+ Glucose fermentation positive, gas produced+g- Glucose fermentation positive, no gas produced+rt Positive at room temperature (25°C) but negative at 37°C+s Slow positive reaction. May take 2–4 days for reaction to occur+sr Gram-positive, short rod+w Weak positive reaction
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References
1 Abbott, S.L., Cheung, W.K.W., Kroske-Bystrom,S., Malekzadeh, T. and Janda, J.M. (1992) Identifi-cation of Aeromonas strains to the genospecieslevel in the clinical laboratory. Journal of ClinicalMicrobiology 30, 1262–1266.
2 Abbott, S.L., Seli, L.S., Catino, M. Jr., Hartley,M.A. and Janda, J.M. (1998) Misidentification ofunusual Aeromonas species as members of thegenus Vibrio: a continuing problem. Journal ofClinical Microbiology 36, 1103–1104.
3 Abu-Samra, M.T. and Walton, G.S. (1977) Modi-fied techniques for the isolation of Dermatophilusspp. from infected material. Sabouraudia 15,23–27.
4 Acuna, M.T., Diaz, G., Bolanos, H., Barquero, C.,Sanchez, O., Sanchez, L.M., Mora, G., Chaves, A.and Campos, E. (1999) Sources of Vibrio mimicuscontamination of turtle eggs. Applied and Environ-mental Microbiology 65, 336–338.
5 Aguirre, A.A., Balazs, G.H., Zimmerman, B. andSpraker, T. (1994) Evaluation of Hawaiian greenturtles (Chelonia mydas) for potential pathogensassociated with fibropapillomas. Journal of WildlifeDiseases 30, 8–15.
6 Ahmet, Z., Stanier, P., Harvey, D. and Holt, D.(1999) New PCR primers for the sensitivedetection and specific identification of Group Bb-haemolytic streptococci in cerebrospinal fluid.Molecular and Cellular Probes 13, 349–357.
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789 Vicente, A., Coelho, A. and Salles, C. (1997)Detection of Vibrio cholerae and V. mimicusheat-stable toxin gene sequence by PCR. Journalof Medical Microbiology 46, 398–402.
790 Vieira, V., Teixeira, L., Zahner, V., Momen, H.,Facklam, R., Steigerwalt, A., Brenner, D. andCastro, A. (1998) Genetic relationships among thedifferent phenotype of Streptococcus dysgalactiaestrains. International Journal of SystematicBacteriology 48, 1231–1243.
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795 Vreeland, R.H., Litchfield, C.D., Martin, E.L. andElliot, E. (1980) Halomonas elongata, a new genusand species of extremely salt-tolerant bacteria.International Journal of Systematic Bacteriology30, 485–495.
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800 Wakabayashi, H. and Egusa, S. (1973)Edwardsiella tarda (Paracolobactrum anguilli-mortiferum) associated with pond-cultured eeldisease. Bulletin of the Japanese Society ofScientific Fisheries 39, 931–936.
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825 Whittington, R., Gudkovs, N., Carrigan, M.,Ashburner, L. and Thurstan, S. (1987) Clinical,microbiological and epidemiological findings inrecent outbreaks of goldfish ulcer disease due toatypical Aeromonas salmonicida in south-easternAustralia. Journal of Fish Diseases 10, 353–362.
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828 Wiklund, T. and Bylund, G. (1990) Pseudomonasanguilliseptica as a pathogen of salmonid fish inFinland. Diseases of Aquatic Organisms 8, 13–19.
829 Wiklund, T. and Bylund, G. (1993) Skin ulcer dis-ease of flounder Platichthys flesus in the northernBaltic Sea. Diseases of Aquatic Organisms 17,165–174.
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835 Williams, A.M., Fryer, J.L. and Collins, M.D.(1990) Lactococcus piscium sp. nov. a newLactococcus species from salmonid fish. FEMSMicrobiology Letters 68, 109–114.
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853 Zlotkin, A., Hershko, H. and Eldar, A. (1998b)Possible transmission of Streptococcus iniaefrom wild fish to cultured marine fish. Applied andEnvironmental Microbiology 64, 4065–4067.
854 ZoBell, C.E. (1941) Studies on marine bacteria. I.The cultural requirements of heterotrophicaerobes. Journal of Marine Research 4, 42–75.
855 Zorilla, I., Balebona, M.C., Moriñigo, M.A.,Sarasquete, C. and Borrego, J.J. (1999) Isolationand characterisation of the causative agent ofpasteurellosis, Photobacterium damselae spp.piscicida, from sole, Solea senegalensis (Kaup).Journal of Fish Diseases 22, 167–172.
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Index
0/129culture appearance see photographic
sectionreagent 273test see Vibrio discs
Abalone 2, 67, 69, 281Haliotis discus hannai 2, 281Haliotis tuberculata 2Japanese abalone (Sulculus diversicolor
supratexta) 2Red abalone (Haliotis rufescens) 2, 67, 285Small abalone (Haliotis diversicolor
supertexta) 2, 71, 286South African abalone 67, 286
Abiotrophia 75A. adiacens see Granulicatella adiacensA. balaenopterae 38
see also Granulicatella balaenopteraeA. defectiva 75, 162, 210A. elegans 38, 75
see also Granulicatella elegansA. para-adiacens 162, 210
Acetate agar see Rogosa acetate agarAchromobacter 223
A. xylosoxidans ssp. denitrificans 38, 155Acinetobacter 32, 131
identification 91, 149, 178, 184, 190A. baumannii 38
identification 149A. calcoaceticus 20, 29, 38
identification 149, 197A. haemolyticus 38
identification 91, 149
Actinobacillus 29A. delphinicola 27, 28, 30, 38
identification 91, 154, 178, 184, 192,197, 212
A. scotiae 28, 38, 218identification 91, 154, 178, 184, 191
ActinomycesA. marimammalium 28, 29, 38, 218
identification 86, 91, 164, 208, 210A. viscosus 212
Aequorivita 75identification 86, 91, 129, 130A. antarctica 38, 218
identification 146, 212A. crocea 38, 218
identification 146A. lipolytica 38, 218
identification 146A. sublithincola 38, 218
identification 146Aerococcus
A. viridans 21, 38identification 160, 210, 258
A. viridans var. homari 13, 38identification 86, 91, 160, 200, 201,
212Aeromonas 75
DNA hybridization groups 75, 141identification 124, 139PCR primers 231A. allosaccharophila 10, 12, 39, 76, 218
identification 142, 178, 184, 196A. bestiarum 6, 39, 76, 218
identification 142, 178, 184, 196, 200,201
329
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Aeromonas continuedA. caviae 218, 225
identification 142, 178, 184, 192, 193,200, 201
hosts and diseases 12, 32, 39, 76PCR primers 226
A. culicicolahosts and diseases 39identification 91, 142, 178, 200, 201
A. encheleia 218hosts and diseases 9, 39identification 86, 91, 142, 178, 184, 193,
200, 201taxonomy 76
A. enteropelogenes 40A. eucrenophila 218
hosts and diseases 6, 29, 39identification 86, 91, 142, 178, 200, 201PCR primers 231
A. hydrophila 218, 225culture appearance see photographic
sectionhosts and diseases 3, 4, 7, 15, 16, 20, 21,
29, 32identification 91, 178, 184, 192, 193,
196, 197, 200, 201, 212PCR primers 225, 226, 231taxonomy 75
A. hydrophila ssp. dhakensis 218hosts and diseases 12, 39, 76identification 76, 124, 142taxonomy 75
A. hydrophila ssp. hydrophila 218hosts and diseases 12, 39, 76identification 76, 124, 142taxonomy 75
A. ichthiosmia see Aeromonas veronii ssp.veronii
A. janadaei 12, 40, 218culture appearance see photographic
sectionhosts and diseases 16identification 142, 178, 184, 196, 212
A. media 218hosts and diseases 15, 40identification 92, 142, 200, 201
A. popoffii 40, 218identification 86, 124, 142, 178, 200,
201PCR primers 231
A. punctata 39A. salmonicida
identification 86, 124, 178, 184, 192, 212
A-protein layer 124detection with Coomassie brilliant blue
agar 265detection with Congo Red agar 266
luminescence 122PCR primers 233pigment detection 86, 124
Furunculosis agar 124, 268A. salmonicida ssp. salmonicida 218
antisera 136hosts and diseases 9, 11, 13, 22, 24, 31,
35, 37, 39, 77identification 92, 86, 140, 178, 184, 192,
196, 197, 200, 201PCR primers 226pigment 86non-pigmented strains 40taxonomy 77
A. shigelloides see Plesiomonasshigelloides
A. schubertii 39, 218hosts and diseases 12, 41identification 143
A. sobria 218, 225hosts and diseases 6, 12, 24, 41identification 142, 178, 184, 196, 197,
202, 203PCR primers 226, 231
Aeromonas species 15, 37, 223Aeromonas species PCR 225A. trota 218
hosts and diseases 12, 41identification 143, 179, 184, 196, 202,
203PCR primers 226, 231taxonomy 76
A. veronii ssp. sobria 218culture appearance see photographic
sectionhosts and diseases 12, 40identification 143, 202, 203, 212taxonomy 76
A. veronii ssp. veronii 218hosts and diseases 6, 12, 32, 41, 76identification 92, 142, 179, 184, 191,
195, 196, 202, 203taxonomy 76
Aeromonas group 501 39enteric group 41
Atypical A. salmonicidaculture appearance see photographic
sectiongrowth at 37°C strain 24, 41
330 Index
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hosts and diseases 4, 6, 7, 9, 10, 11, 14,17, 18, 21, 22, 24, 34, 35, 37, 41, 77
identification 125, 178, 184oxidase-negative strains 10, 23, 34, 41,
140pigment 77, 125A. salmonicida ssp. achromogenes 22,
30, 32, 57, 77, 218identification 178, 184, 190, 200, 201hosts and diseases 5, 18, 21, 22, 40taxonomy 79
A. salmonicida ssp.achromogenes/masoucidaidentification 140, 197
A. salmonicida ssp. masoucida 40, 77hosts and diseases 40identification 140, 178, 184, 192
A. salmonicida ssp. novahosts and diseases 17, 40identification 92, 140
A. salmonicida ssp. pectinolytica 40, 218identification 92, 141
A. salmonicida ssp. smithia 40identification 140
Aesculin 130, 133media preparation 261, 262test interpretation 117, 118, 261
Agar digestion 129Agrobacterium stellulatum see Stappia
stellulata-likeAlbatross (grey headed albatross) 62AlcaligenesAlcaligenes denitrificans see Achromobacter
xylosoxidans subsp. denitrificansA. faecalis
hosts and diseases 29see also Halomonas aquamarina
A. faecalis var. homari 41identification 86, 98taxonomy 51
A. venustus see Halomonas venustaAlga 2
Marine alga 2, 57, 60Red alga (Delesseria sanguinea) 2, 63, 74Ulva lactuca 60
Alkaline peptone water 244Alligator 2, 47, 55, 56, 281Allomonas
A. enterica 42identification 92, 155, 179
Alteromonashosts and diseases 15, 31, 32, 42identification 86, 92
taxonomy 77A. antarctica see Pseudoalteromonas
antarcticaA. aurantia 218
see also Pseudoalteromonas flavipulchraA. citrea 218
see also Pseudoalteromonas citreaA. colwelliana see Shewanella colwellianaA. communis see Marinomonas communisA. denitrificans see Pseudoalteromonas
denitrificansA. distincta see Pseudoalteromonas
distinctaA. elyakovii see Pseudoalteromonas
elyakoviiA. espejiana see Pseudoalteromonas
espejianaA. hanedai see Shewanella hanedaiA. macleodii 77
identification 155, 212A. nigrifaciens see Pseudoalteromonas
nigrifaciensAPI 20NE see Alteromonas
nigrifaciens 197A. piscicida see Pseudoalteromonas
piscicidaA. rubra 218
see also Pseudoalteromonas rubraA. undina see Pseudoalteromonas undina
Alvinella pompejana see polychaete annelidAmblyglyphidodon curacao Bloch see
DamselfishAmeiurus species see CatfishAmies transport medium 84, 244Ammonium salt sugar (ASS) 139, 264Ammodytes lancea Cuvier see EelAmphiprion clarkii see DamselfishAmpicillin disc 114Anacker-Ordal agar (AO) 244, 245Anaerobe plates 245Anarhichas species see Wolf-fishAnchovy 2, 66Angel fish 45, 47Anguilla species see EelAO medium see Anacker-Ordal agarAphanomyces invadans 39, 42API identification systems 116
API 50CH 116, 200–207API Coryne 116, 208API 20E 116, 138, 178–196API 20NE 116, 197–199API 20 Strep 116, 209API rapid A 116
Index 331
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API identification systems continuedAPI rapid ID 32 STREP 116, 210–211API ZYM 116, 139, 212–217
Apistogramma ocellatus see OscarAphanius iberus Valenciennes see CarpAptenodytes patagonica see PenguinAquaspirillum
hosts and diseases 42identification 86, 155
Arabinose see Carbohydrate fermentationArapaima gigas Cuvier see PirarucuArcanobacterium
culture requirements 86A. bernardiae
hosts and diseases 42identification 164, 202, 203, 208, 210
A. haemolyticumidentification 164, 208, 210
A. phocae 218hosts and diseases 28, 29, 42identification 92, 95, 164, 202, 203, 208,
210, 212A. pluranimalium 218
hosts and diseases 28, 42identification 92, 165, 208, 210
A. pyogenes 59hosts and diseases 42identification 92, 165, 202, 203, 210
Arctic Charr see SalmonidsArctocephalus species see SealsArdea herodias see HeronArginine dihydrolase
media preparation 267Møller’s method 117, 267test interpretation 117, 118
variable results in Vibrio species 117,120, 267
see also photographic sectionThornley’s method 117, 267
Argopecten purpuratus see ScallopAristichthys nobilis see CarpArtemia species see ShrimpArthrobacter
culture requirements 86A. agilis 42
identification 93, 165A. aurescens 212A. nasiphocae
hosts and diseases 28, 42identification 93, 165, 208, 212
A. rhombi 218hosts and diseases 11, 42identification 93, 165, 202, 203, 208
Artificial seawater medium 245Ascidians 59Ashdown’s medium see Burkholderia
pseudomallei selective mediaAtractoscion nobilis Ayres see BassASW see Artificial seawater mediumAtopobacter phocae 218
hosts and diseases 28, 42identification 93, 165, 210, 212
Atypical bacterial gill disease 60Atypical furunculosis 35Ayu 3, 39, 53, 60, 61, 65, 68, 281
BA see Blood agarBacillary necrosis 15, 72Bacillary necrosis of Pangasius 1, 7Bacillus 20, 29, 223
B. cereushosts and diseases 42identification 93, 164
B. mycoideshosts and diseases 7, 42identification 93, 164
B. subtilis 42Bacterial cold-water disease 49Bacterial gill disease 17, 23, 24, 31, 36, 49Bacterial haemorrhagic ascites (BHA) 3, 60Bacterial kidney disease (BKD) 3, 22, 23, 25,
36, 132Bacterial load 222Bacterial necrosis 14, 42, 71Bacterial stomatitis (mouth rot) 22, 36, 66Baitfish (Pimephales promelas, American
baitfish) 14Balaenoptera acutorostrata see WhaleBarramundi 4, 49, 57, 65, 70, 281Basal medium 264Basal medium agar 264Bass 3, 42, 47, 49, 57, 62, 285, 286
European seabass (Dicentrarchus labraxLinnaeus) 3, 36, 65, 282
Japanese seaperch 4Largemouth bass (Micropterus
salmoides) 3, 49, 56, 283Sea bass 39, 54, 56, 60, 66, 69, 285Sea bass (Lateolabrax japonicus Cuvier)
4Sea bass (Lates calcarifer Bloch) 4, 281Sea bass (Puntazzo puntazzo Cuvier) 4Striped bass (Morone saxatilis Walbaum,
Roccus saxatilis) 4, 44, 46, 53, 54, 57,65, 286
332 Index
348Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #M.vpMonday, January 26, 2004 12:33:45 PM
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White sea bass (weakfish, Atractoscionnobilis Ayres) 4, 55
Bearded dragon 47Benechea chitinovora
hosts and diseases 42identification 86, 153
Bergeyella 78, 129, 130Betta splendens Regan see Siamese fighting fishBGD see Bacterial gill diseaseBidyanus species see PerchBHA see Bacterial haemorrhagic ascitesBiochemical identification tests 114
discs (Vibrio discs) 114growth at different temperatures 114inoculation of biochemical identification
sets 115freshwater isolates 115marine isolates 115
interpretation of biochemical identificationset (‘biochem set’) 117–123
recording results (Laboratory worksheet)177
using the biochemical identification tables123, 137, 140–176
flowchart 138BKD see Bacterial kidney diseaseBlack acara (Cichlasoma bimaculatum
Linnaeus) 16, 54, 55, 281Black disease 32, 39Black parch necrosis 32, 36, 66Black tiger prawn see ShrimpBlacksmith see DamselfishBLAST search 242Blaser-Wang media see Helicobacter selective
mediaBlenny 4, 41, 281, 286Blood agar (BA) 246Bordetella bronchiseptica 218
culture requirements 86Bordetella bronchiseptica selective agar
(CFPA) 246hosts and diseases 43identification 93, 153, 197, 212
Black scraper 52, 281Blicca bjoerkna Linnaeus see BreamBluefish 65, 281Brachionus plicatilis see RotiferBranchio-necrosis 42Branchipus schaefferi Fisher see ShrimpBRD see Brown ring diseaseBream 5, 53, 57, 282, 286
Black sea bream (Acanthopagrus latusHouttuyn) 5, 51, 66
Common bream 5Gilt-head sea bream (Sparus auratus
Linnaeus) 57, 65, 72, 283One-spot sea bream (Diplodus sargus
kotschyi Steindachner) 5, 284Red sea bream (Pagrus major) 5, 64, 66,
285Rock bream 66Sea bream (Sparus auratus Linnaeus) 60,
65, 67, 68, 69, 285Sliver bream (Blicca bjoerkna Linnaeus) 5,
40, 285Brevoortia patronus Goode see MenhadenBrevundimonas
B. diminuta 218hosts and diseases 43identification 93, 155, 197, 202, 203,
212B. vesicularis 218
hosts and diseases 43identification 93, 155, 202, 203, 212
Bromothymol blue indicator 262, 263, 275Brill see TurbotBrown ring disease (BRD) 14, 36, 72Brucella
culture requirements 86assay of metabolic activity 261Brucella agar 246Farrell’s medium 86, 248growth on substrate media 262serum-dextrose agar 256
hosts and diseases 12, 27, 29, 30, 43identification 93, 125, 144, 197taxonomy 77B. abortus
antisera 136hosts and diseases 12, 43identification 93, 144, 153
B. canishosts and diseases 43identification 144
B. cetaceae 219hosts and diseases 27, 28, 43, 77, 78identification 144, 124
B. maris see B. cetaceaeB. melitensis
hosts and diseases 43, 77identification 144
B. pinnipediaehosts and diseases 18, 28, 29, 43, 78identification 144
B. suis 43identification 144
Index 333
349Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #M.vpTuesday, January 20, 2004 5:35:38 PM
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Brucella agar 246Brucella broth 224Budvicia aquatica
hosts and disease 44identification 94, 151
BurkholderiaB. cepacia 61
hosts and disease 44identification 155, 184, 190, 194, 195
B. pseudomalleiculture requirements 86
selective broth 246selective media (Ashdown’s medium)
247selective media (glycerol medium) 246
hosts and diseases 12, 27, 28, 29, 30, 44identification 94, 153, 179, 184, 190,
192
Callinectes sapidus see CrabCalloplesiops altivelis Steindachner see CometsCallorhinus ursinus see SealsCandida species 30
hosts and diseases 44identification 144
Capnocytophaga 78, 128, 129, 130Caranx hippos Linnaeus see Crevalle, Jack
crevalleCarassius species see Carp, GoldfishCarbohydrate fermentation
alternative method for Vibrio spp. 264carbohydrate fermentation media using
acid from ammonium salt sugar 264general purpose medium 262for Pseudomonas spp., Flavobacterium
spp., Nocardia spp., Shewanella spp.,Sphingobacterium spp. and marinespecies 263–264
test interpretation 118, 120, 262, 263see photographic section
Carbon dioxide atmosphere 247, 265Carcharhinus species see SharkCaretta caretta see TurtleCarnobacterium
hosts and diseases 7, 22, 35identification 78, 125–127taxonomy 78C. alterfunditum
hosts and diseases 44identification 165, 210, 212
C. divergenshosts and diseases 35, 44
identification 126, 165, 179, 202, 203,210, 212
C. divergens strain 6251hosts and diseases 44identification 165, 212
C. funditumhosts and diseases 44identification 165, 210, 212
C. gallinarumhosts and diseases 44identification 165, 202, 203, 210, 212
C. inhibens 219hosts and diseases 22identification 86, 94, 165, 209, 210, 212
C. inhibens strain K1hosts and diseases 44
C. mobilehosts and diseases 44identification 165, 202, 203, 210, 212
C. piscicola 219hosts and diseases 4, 7, 24, 44identification 86, 94, 125, 126, 127, 164,
202, 203, 209, 210, 212taxonomy 78
Carnobacterium speciesidentification 164
Carotenoid pigment detection 120, 129also see Congo Red
Carp 40, 41, 42, 45, 47, 49, 61, 65Bighead carp (Aristichthys nobilis) 5, 281Caucasian carp 6, 48, 282Common carp (Cyprinus carpio carpio
Linnaeus) 6, 76, 282Crucian carp (Carassius carassius Linnaeus)
6, 282Goldfish (Carassius auratus Linnaeus) 17Iberian toothcarp 6, 71, 283Koi carp 6, 49, 65Silver carp (Hypophthalmichthys molitrix
Valenciennes) 6, 58, 64, 285Spanish toothcarp (Aphanius iberus
Valenciennes) 6Carp erythrodermatitis 40, 41Catalase 130
method and test interpretation 120, 265for Mycoplasma spp. see Mycoplasma
characterization testsCatenibacterium species see Eubacterium
speciesCatfish 1, 6, 39, 40, 42, 48, 282, 285
African catfish 58Black bullhead (Ameiurus melas, Ictalurus
melas) 6, 49
334 Index
350Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #M.vpTuesday, January 20, 2004 5:35:38 PM
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Blue catfish (Ictalurus furcatusValenciennes) 6, 36, 49
Brown bullhead (Ictalurus nebulosus) 7,44, 48, 282
Channel catfish 7, 36, 38, 39, 42, 44, 47,49, 74, 282
Freshwater catfish (Pangasiushypophthalmus Sauvage) 1, 7
Walking catfish (Clarias batrachusLinnaeus) 7, 39, 76
White catfish (Ameiurus catus Linnaeus) 7,47, 286
CBBA see Coomassie brilliant blue agarCellobiose-Colistin agar 247Cellulomonas culture media see Dubos mediaCellulophaga 129, 130Cellulophaga lytica
hosts and disease 45identification 146, 212
Cellulose digestion method 128, 265Centropomus undecimalis Bloch see SnookCetacea see Sea mammalsCFPA medium see Bordetella bronchiseptica
selective agarCFU see Colony forming unitsChanos chanos Forsskål see MilkfishChelonia mydas see TurtleCherax albidus see Crayfish, Australian
freshwater crayfishCherax quadricarinatus see Crayfish, red clawChirocephalus diaphanus Prévost see ShrimpCholera 12, 68Chorion 11, 66Chromis punctipinnis see DamselfishChromobacterium violaceum 219
hosts and diseases 12, 45identification 94, 155, 197
Chrysemys species see TurtleChryseobacterium 129, 130
C. balustinumhosts and diseases 45identification 94, 145, 197, 212
C. gleumhosts and diseases 45identification 94, 146, 197, 213
C. indologeneshosts and diseases 45identification 94, 146, 197, 213
C. indoltheticumhosts and diseases 45identification 146, 197, 213
C. meningosepticumculture requirements 86
hosts and diseases 45identification 94, 146, 197, 213
C. scophthalmumculture requirements 87
Medium K 252hosts and diseases 34, 45identification 94, 145, 197, 202, 203, 213
Chrysophrys unicolor see SnapperChub (Leuciscus cephalus Linnaeus) 7, 41, 282Cichlasoma bimaculatum Linnaeus see Black
acaraCichlasoma meeki Brind see Firemouth cichlidCichlid (Oreochromis mossambicus) 282
Firemouth cichlid (Thorichthys meeki,Cichlasoma meeki Brind) 16, 54, 283
CitrateChristensen’s citrate method 120Simmons method 120, 265test interpretation 118, 265
see photographic sectionCitrobacter diversus
identification 151C. freundii
hosts and diseases 6, 21, 24, 45identification 94, 125, 127, 149, 179
Clam 14, 67, 70, 73Manila clam (Ruditapes philippinarum) 14,
36, 72, 282, 284Mercenaria mercenaria 14Softshell clam (Mya arenaria) 286
Clarias species see CatfishCleaner fish see WrasseClostridial myositis 27, 28, 30Clostridium
C. botulinumculture requirements 87hosts and diseases 24, 46identification 95, 143toxin detection 87
C. perfringenshosts and diseases 27, 28, 30, 46identification 143
Clupea harengus membras Linnaeus seeHerring
Coalfish 7, 58Coastal fish 21Cockles 70Cod 58, 71, 282, 283, 284, 286
Atlantic cod (Gadus morhua Linnaeus) 7,40, 44, 52, 281
Coenonia 128, 129, 130Cold water disease 3, 23Colistium species see Turbot
Index 335
351Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #M.vpMonday, January 26, 2004 12:34:14 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
Colony forming units (CFU) 224Columnaris disease 6, 24, 31, 49
marine columnaris 66Colwellia maris
hosts and disease 46identification 95, 155
Comets (Calloplesiops altivelis Steindachner)16, 54
Black comets 49Conchiolin deposit 14, 62, 72Congo Red (CR) 120
media preparation 266for use see Carotenoid pigment detection
120Congo Red agar 265Coomassie brilliant blue agar (CBBA) 265Coral 8, 68
Oculina patagonica 8, 72Pocillopora damicornis 8, 68
Coral bleaching 70, 72Coregonus species see WhitefishCorynebacterium
C. aquaticum 219culture requirements 87hosts and diseases 4, 46identification 95, 164, 208
C. phocae see Arcanobacterium phocaeidentification 165
C. pseudodiphtheriticumidentification 165, 208
C. pseudotuberculosisidentification 165, 208
C. testudinorishosts and diseases 19, 46identification 95, 164, 208, 213
Corynebacterium species 29, 32, 223C. xerosis
identification 165, 208Coryphaena hippurus Linnaeus see Mahi-mahiCrab 8, 38, 48, 53, 67, 73, 281, 284, 286
Blue crab (Callinectes sapidus) 8Swimming crab (Portunus trituberculatus)
8Crane (Sandhill crane (Grus canadensis)) 25Crassostrea species see OysterCrawfish
American freshwater crayfish 8, 281Red swamp crawfish (Procambarus clarkii)
8, 281, 285Crayfish 8, 60, 281, 285
Australian freshwater crayfish 8, 70Marron 70, 71Red claw 8, 285
Yabby 8, 70Crevalle 8Crocodile 8, 47, 48, 54, 55, 283, 285
Caiman crocodile (Caiman crocodilus) 8Crocodilus acutus 8Crocodylus niloticus 8
Crumb-of-bread sponge see SpongeCryptococcosis 27Cryptococcus
culture appearance see photographicsection
differentiation from Candida species 127,144
C. lupihosts and disease 46identification 144
C. neoformans 12, 27C. neoformans var. gattii 46
hosts and disease 46identification 95, 144
CSF see Cerebrospinal fluidCulture and incubation 83, 85
culture medium 85freshwater animals 85incubation time and temperature 85, 115saltwater animals 85, 115specific culture requirements of organisms
86–90Culture collections 279Culture media preparation 244–261Cutaneous ulcerative disease 40Cyclobacterium marinum 128Cyprinus carpio carpio Linnaeus see CarpCystophara cristata see SealCytophaga
agar see Anacker-Ordal agarcarbohydrate fermentation 262cellulose digestion 265culture media
Anacker-Ordal agar 120, 121, 244peptone yeast medium 255
taxonomy 78, 128C. agarovorans see Marinilabilia
salmonicolor biovar agarovoransC. allerginae
identification 146, 213Cytophaga aquatilis see Flavobacterium
hydatisC. arvensicola
hosts and disease 46identification 146, 213
C. aurantiaca strain NCIMB 1382 78hosts and disease 46
336 Index
352Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #M.vpMonday, January 26, 2004 12:35:59 PM
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C. columnaris see Flavobacteriumcolumnare
C. fermentanshosts and disease 46identification 146, 213
C. flevensis see Flavobacterium flevenseC. heparina see Pedobacter heparinusC. heparinum see Pedobacter heparinusC. hutchinsonii 128
culture requirements 87Dubos medium 247
identification 146, 213C. johnsonae see Flavobacterium
johnsoniaeC. latercula
culture requirements 87hosts and disease 46identification 146, 213taxonomy 128
C. lytica see Cellulophaga lyticaC. marina see Tenacibaculum maritimumC. marinoflava
hosts and disease 47identification 146taxonomy 128
C. pectinovorum see Flavobacteriumpectinovorum
C. psychrophila see Flavobacteriumpsychrophilum
C. saccharophilum see Flavobacteriumsaccharophilum
C. salmonicolor see Marinilabiliasalmonicolor biovar agarovorans
C. succinicans see Flavobacteriumsuccinicans
C. uliginosum see Zobellia uliginosaC. xanthum see Flavobacterium xanthum
Dab 9, 41, 282Dace (Leuciscus leuciscus Linnaeus) 9, 282Damselfish (Pomacentrida) 9, 57, 60, 282
Blacksmith (Chromis punctipinnis) 9Staghorn damselfish 9Yellowtail clownfish (Amblyglyphidodon
curacao) 9Yellowtail clownfish (Amphiprion clarkii)
9Danio 16, 47, 282, 286
Danio devario Hamilton 16Sind danio 16
Dasyatis pastinaca see StingrayDelesseria sanguinea see Alga
Delphinapterus leucas see WhaleDelphinus delphis see DolphinDelya aquamarinus
identification 153see also Alcaligenes faecalis homari;
Halomonas aquamarinaDelya marina see Halomonas marinaDermatophilosis 47Dermatophilus
culture requirements 87D. chelonae
hosts and diseases 20, 47identification 164
D. congolensishosts and diseases 47identification 95, 164selective medium (polymyxin plates)
247Dermatophilus selective medium (polymyxin
plates) 247Dermochelys coriacea see TurtleDicentrarchus labrax Linnaeus see BassDienes Stain 253, 266Dietzia maris (Rhodococcus maris)
hosts and diseases 47identification 105, 165
Digitonin 266Diplodus sargus kotschyi Steindachner see
BreamDiscus fish 16, 282
Blue discus fish (Symphysodonaequifasciatus Pellegrin) 16, 54
Discs 114DNA extraction 233, 234DNase plate 130
media preparation 267HCl for detection of hydrolysis 269test interpretation 118
see also photographic sectionDolphin 46, 48, 51, 53, 56, 57, 64, 284, 285,
286Amazon freshwater dolphin (Inia
geoffrensis) 27, 65, 281, 282Atlantic bottlenose dolphin (Tursiops
truncatus) 27, 281, 282Atlantic white-sided dolphin
(Lagenorhynchus acutus) 27, 43, 281Bottlenose dolphin 43, 44Common dolphin (Delphinus delphis) 27,
43, 282Pacific white-sided dolphin
(Lagenorhynchus obliquidens) 27,284
Index 337
353Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #M.vpMonday, January 26, 2004 12:36:24 PM
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Dolphin continuedStriped dolphin (Stenella coeruleoalba) 27,
38, 43, 286Tursiops aduncas 27, 281Tursiops gephyreus 51White-sided dolphin 44
Dolphin fish see Mahi-mahiDorosoma cepedianum Lesueur see ShadDubos medium 247
Eagle (Bald eagle, Haliaeetus leucocephalus) 25Edwardsiella
E. anguillimortifera see Edwardsiella tardaE. hoshinae
culture appearance see photographicsection
hosts and disease 47identification 95, 127–128, 149, 184
E. ictaluri 219culture requirements 87
medium (EIM) 248hosts and diseases 1, 3, 5, 6, 7, 11, 16,
17, 28, 47identification 95, 128, 149, 179, 184,
193E. ictaluri anaerobic strains 7, 47, 87
identification 143, 149E. tarda 219
culture requirements 87, 256culture appearance see photographic
sectionhosts and diseases 2, 3, 5, 7, 8, 10, 11,
12, 15, 17, 19, 20, 23, 24, 25, 27, 33,34, 36, 47, 78
identification 96, 150, 179, 184, 185,194, 213, 270
taxonomy 78Edwardsiella species PCR primers 226,
232Eel 9–10, 39, 40, 41, 47, 49, 52, 53, 57, 58,
60, 67, 69, 73, 74, 281American eel 9, 281Anguilla reinhardtii 9, 282Elvers 10, 39European eel (Anguilla anguilla Linnaeus)
9Green moray eel (Gymnothorax funebris)
10, 55, 283Hyperoplus lanceolatus Lesauvege 10Japanese eel (Anguilla japonica) 10, 282Sand eel (Ammodytes lancea Cuvier) 10,
41, 285
Spotted moray eel (Gymnothorax moringa)10, 55, 286
Taiwanese eels 15Eigemannia virescens Valenciennes see Green
knifefishEIM see Edwardsiella ictaluri MediumElectric blue hap (Sciaenochromis ahli
Trewavas) 16Electrolyte supplement 248Elvers see EelEmpedobacter 129, 130Empedobacter brevis
culture requirements 87hosts and diseases 48identification 96, 147, 213taxonomy 78
Emphysematous putrefactive disease of catfish47
Enchelyopus cimbrius Linnaeus see RocklingEngraulis mordax see AnchovyENO salts 265Enteric redmouth (ERM) 23, 24, 25, 36, 74,
82Enteric septicaemia of catfish (ESC) 1, 6, 7, 36,
47Enterobacter 29, 48, 151
Enterobacter agglomerans see Pantoeaagglomerans
Enterococcus 209culture requirements 87identification notes 128taxonomy 78E. faecalis 219
hosts and diseases 48identification 128, 162, 209, 210
E. faecalis var. liquefaciens 160E. faecium
hosts and diseases 48identification 128, 162, 209, 202, 203
E. seriolicida see Lactococcus garvieaeEnterovibrio norvegicus 219
culture requirements 87hosts and diseases 34, 47identification 96, 175, 179, 185, 192, 213luminescence 122taxonomy 78
Epalzeorhynchos kalopterus Bleeker see Flyingfox
Epinephelus coioides see GrouperEretmochelys imbricata see TurtleErysipelothrix rhusiopathiae
culture appearance see photographicsection
338 Index
354Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #M.vpTuesday, January 20, 2004 5:35:39 PM
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culture requirements 87Packer’s plates 255Wood’s broth 260
hosts and diseases 8, 12, 27, 28, 47identification 96, 164, 210
Erysipelothrix selective medium 248Escherichia
E. colihosts and diseases 28, 29, 47identification 151PCR primers 231, 233
E. vulnerishosts and diseases 6, 17, 24, 47identification 150
Esox lucius Linnaeus see PikeEubacterium
hosts and diseases 19, 48identification 96, 143E. limosum
culture morphology 96E. tarantellae
culture requirements 87hosts and diseases 15, 48identification 96, 143
Eudyptes species see PenguinEudyptula minor see PenguinEuprymna scolopes see SquidExtracellular galactosamine glycan see
Carotenoid pigment detection; CongoRed
Facklamia miroungaeculture requirements 87hosts and diseases 30, 48identification 96, 162, 210, 213
False columnaris disease 49Farrell’s medium 248Fascaplysinopsis reticulata see SpongeFermentation see Carbohydrate fermentationFetal calf serum 224Film and spots see Mycoplasma characterization
testsFINE see Flounder necrotizing enteritisFin rot 39, 49, 60, 72FISH see Fluorescence in situ hybridizationFish bite 12, 51Fish gangrene 47Fish pasteurellosis 3, 4, 5, 11, 16, 18, 32, 33,
34, 35, 37, 57Flamingo 47Flatfish see Turbot
Flavobacteriaceae familycarbohydrate fermentation 262, 264gliding motility 121identification notes 128-130pigment detection 120using the API ZYM 139
Flavobacteriosis 45Flavobacterium species 32, 37, 223
culture mediaAnacker-Ordal agar 244, 245, 249artificial seawater medium 245carbohydrate fermentation 262
alternative methods for carbohydratefermentation 263
Flavobacterium maritimus media (FMM)249
Flavobacterium psychrophilum medium(FPM) 249
peptone yeast medium (PY) 255R2A agar 256
differentiation from Cytophaga species 128hosts and diseases 20PCR primers 226taxonomy 78F. aquatile 48, 219
hosts and diseases 48identification 96, 147, 213
F. balustinum see Chryseobacteriumbalustinum
F. branchiophilum 219culture requirements 87hosts and diseases 17, 23, 24, 31, 36, 49identification 96, 145, 179, 185, 190,
213F. breve see Empedobacter brevisF. cauliformans 48F. columnare 224
culture requirements 87culture appearance see photographic
sectionHsu-Shotts agar 251Shieh medium + tobramycin 257tryptone yeast extract salt medium 259tryptone yeast extract glucose agar 259
genomovars 129hosts and diseases 6, 10, 16, 17, 18, 19,
23, 24, 31, 37identification 97, 129, 145, 179, 185,
190, 191, 202, 203, 213PCR primers 226, 227, 232, 233media for freeze-drying 224taxonomy 79
Index 339
355Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #M.vpMonday, January 26, 2004 12:37:00 PM
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Flavobacterium species continuedF. flevense
hosts and diseases 49identification 97, 147, 213
F. frigidarium 219hosts and diseases 49identification 97, 147, 213
F. gillisiae 219culture requirements 88hosts and diseases 49identification 97, 147, 179, 185, 191
F. gleum see Chryseobacterium gleumF. gondwanensis see Psychroflexus
gondwanensisF. hibernum 219
hosts and diseases 49identification 97, 147, 179, 185, 191,
197F. hydatis 219
hosts and diseases 49identification 97, 145, 179, 185, 190,
213F. indologenes see Chryseobacterium
indologenesF. indoltheticum see Chryseobacterium
indoltheticumF. johnsoniae
culture appearance see photographicsection
hosts and diseases 4, 49identification 97, 145, 179, 213, 214taxonomy 79
F. meningosepticum see Chryseobacteriummeningosepticum
F. mizutaiihosts and diseases 49identification 147
F. multivorum see Sphingobacteriummultivorum
F. okeanokoites see Planomicrobiumokeanokoites
F. pectinovorumhosts and diseases 49identification 97, 147, 214
F. piscicida see Photobacterium damselaessp. piscicida
F. psychrophilum 224culture requirements 87
Flavobacterium psychrophilum medium(FPM) 249
Hsu-Shotts agar 251Shieh medium + tobramycin 257tryptone yeast extract glucose agar 259
hosts and diseases 3, 22, 23, 24, 49identification 97, 145, 179, 185, 190,
202, 203, 214media for freeze-drying 224PCR primers 227, 233
F. saccharophilumhost and diseases 50identification 97, 147, 214
F. salegens see Salegentibacter salegensF. scophthalmum see Chryseobacterium
scophthalmumF. succinicans
host and diseases 50identification 97, 147, 214
F. tegetincola 219culture requirements 88host and diseases 50identification 97, 147, 179, 185, 190
F. uliginosum see Zobellia uliginosaF. xanthum
culture requirements 88host and diseases 50identification 98, 147, 179, 185, 190
F. yabuuchiae 64Flexibacter 37
culture mediaAnacker-Ordal agar 244artificial seawater medium 245Flexibacter maintenance medium 250Flexibacter polymorphus media 249
carbohydrate fermentation 262alternative methods for carbohydrate
fermentation 263culture requirements of marine species
88taxonomy 78, 79F. aggregans
hosts and diseases 50identification 147, 214
F. aurantiacusAPI ZYM 214taxonomy 79
F. canadensishosts and diseases 50identification 147, 214
F. columnaris see Flavobacteriumcolumnare
F. eleganshosts and diseases 50identification 147
F. flexilis 128hosts and diseases 50identification 147, 214
340 Index
356Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #M.vpTuesday, January 20, 2004 5:35:40 PM
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F. litoralishosts and diseases 50identification 147, 202, 203, 214
F. maritimus see Tenacibaculummaritimum
F. odoratimimus see Myroidesodoratimimus
F. odoratum see Myroides odoratusF. ovolyticus see Tenacibaculum
ovolyticumF. polymorphus 219
culture requirements 88media 249
hosts and diseases 50identification 98, 148, 202, 203, 214
F. psychrophilus see Flavobacteriumpsychrophilum
F. roseolusculture requirements 88hosts and diseases 50identification 148, 202, 203, 214
F. ruberculture requirements 88hosts and diseases 50identification 148, 202, 203, 214
F. sanctihosts and diseases 50identification 148, 214
F. tractuosushosts and diseases 50identification 148, 214
Flexirubin pigment 129KOH 121method and test interpretation 120
Flounder 10, 41, 47, 66, 73, 282, 283Greenback flounder (Rhombosolea tapirina
Günther) 10, 40, 283Japanese flounder (Paralichthys olivaceus)
11, 56, 57, 69Marine flounder 40Platichthys flesus Linnaeus 10Summer flounder (Paralichthys dentatus)
11, 69Winter flounder 53
Flounder necrotizing enteritis (FINE) 11, 69Fluorescence in situ hybridization (FISH)
242–243Flying fox (Epalzeorhynchos kalopterus Bleeker)
16, 65FPM see Flavobacterium psychrophilum
mediumFreeze-drying of bacteria 224, 251
Frogs 40, 47, 54, 65Bullfrogs 39, 45, 65Rana castesbeiana 65
Fundulus grandis Baird see MinnowFuruncles 10, 35, 36, 40, 41Furunculosis 5, 9, 11, 22, 23, 24, 35, 37, 40,
77Furunculosis agar (FA) 268
Gadus morhua Linnaeus see CodGaffkemia 13, 38Galactosamine glycan see Carotenoid pigment
detectionGaleocerdo curvieri see SharkGastric ulceration 27, 51Gastritis 27, 30Gavia immer see LoonGel electrophoresis 235Gelatin 130Gelatinase
gelatin/salt plate method 268test interpretation 118, 268
see also photographic sectionGelidibacter 78, 129, 130Giant freshwater prawn see ShrimpGill disease 34, 45, 49Ginglymostoma cirratum see SharkGliding motility 129
method and test interpretation 121Globicephala scammoni see WhaleGlucose 130
see also Carbohydrate fermentationGlucose yeast extract agar (GYEA) 268Glycerol Lab Lemco broth 250Glycerol medium see Burkholderia
pseudomallei, culture requirements,selective media
Golden Shiner (Notemigonus crysoleucasMitchill) 16, 283
Goldfish 17, 40, 41, 47, 49, 54, 60, 68, 74,283
Goldfish ulcer disease (GUD) 17, 40, 41Golf ball disease 27, 65Gourami 54, 58, 283
Three-spot gourami (Trichogastertrichopterus Pallas) 18
Gram-negative anaerobe plates 245Granulicatella
G. adiacenshosts and diseases 50, 75identification 98, 162, 209, 210
Index 341
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Granulicatella continuedG. (Abiotrophia) balaenopterae 219
culture requirements (see Abiotrophiabalaenopterae) 86
hosts and diseases 30, 51identification 98, 160, 209, 210, 214
G. eleganshosts and diseases 12, 50identification 162, 209, 210
Grayling 11, 283Green knifefish 11, 47, 283Greenling 11Grouper 4, 11, 69, 282, 283
Orange-spotted grouper (Epinepheluscoioides) 4
Grus canadensis see CraneGUD see Goldfish ulcer diseaseGull, Ring-billed gull (Larus delewarensis) 25Guppy (Poecilia reticulata Peters) 17, 54, 283
Lebistes reticulatus 17GYEA see Glucose yeast extract agarGymnothorax species see EelGymnotus carapo see Knife fish
Haddock 11, 73, 283Haemolysis
Streptococcus iniae 121test description 121, 251, 268Vibrio species 121, 268
Haemophilus piscium 51, 79Hafnia alvei
culture appearance see photographicsection
hosts and diseases 21, 24, 51identification 98, 150, 179, 185, 194, 194,
195, 214differentiation from Yersinia ruckeri 129,
136taxonomy 79
Haliaeetus leucocephalus see EagleHalibut 11, 42, 53, 283, 285
Atlantic halibut (Hippoglossus hippoglossusLinnaeus) 11, 66
Greenland halibut (Reinhardtiushippoglossoides Walbaum) 11
Halichoerus grypus see SealHalichondria bowerbanki see SpongeHaliotis species see AbaloneHalomonas
H. aquamarinaculture requirements (see Alcaligenes
faecalis homari) 86
hosts and diseases 51identification 98, 153, 155
H. cupidahosts and diseases 51identification 150, 179, 185, 196, 197
H. elongataculture requirements 88hosts and diseases 51identification 98, 151
H. haloduranshosts and diseases 51identification 98, 151
H. marinahosts and diseases 51identification 151
H. venustahosts and diseases 12, 51identification 98, 155, 197
Halophilic 128Hayflick’s medium 253Helicobacter
culture requirements 88selective media 250
hosts and disease 27identification 159taxonomy 79H. cetorum
hosts and diseases 27, 30, 51identification 99, 159
Heron (Great blue heron, Ardea herodias) 25Hexagrammos otakii see GreenlingHerring 58, 60, 283, 284
Baltic herring 12Hippocampus species see SeahorseHippoglossus hippoglossus see HalibutHippurate hydrolysis 269Hitra disease 71Hole-in-the-head 36Holothuria 63Homarus species see LobsterHorse serum 224Hsu-Shotts agar 251Hsu-Shotts marine agar (HSM) 251Hugh and Leifson oxidative-fermentative media
274Human 12–13, 51Hydrogen sulphide production (H2S) 118,
269Hydrogenophaga
identification 155H. palleronii
host and diseases 51identification 155
342 Index
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H. pseudoflavahost and diseases 52identification 99, 155
Hyperoplus lanceolatus Lesauvege see EelHyphessobrycon species see TetraHypophthalmichthys molitrix Valenciennes see
Carp
Ictalurus species see CatfishIndole 130
Kovács indole reagent 270media preparation 269test interpretation 118, 121, 269
see also photographic sectionIndoxyl acetate hydrolysis 270Inia geoffrensis see DolphinInositol see Carbohydrate fermentationInositol horse serum 224, 251Intestinal microflora 223Iodobacter fluviatilis 219
culture morphology 99hosts and diseases 52identification 155
Jack crevalle see CrevalleJanthinobacterium 37
J. lividum 219hosts and diseases 24, 52identification 153, 179, 185, 192, 197,
202, 203Japanese amberjack see YellowtailJapanese medaka 13, 54, 283Japanese sweetfish 49JOD see Juvenile oyster diseaseJuvenile oyster disease (JOD) 14, 62
KDM2 132, 251KDMC 251Klebsiella 29
identification 151K. oxytoca
hosts and diseases 21identification 151
K. planticola (now called Raoultellaplanticola)
identification 152, 202, 203K. pneumoniae
hosts and diseases 24, 52identification 150, 179, 185, 195
K. terrigena see Raoultella terrigena
Kluyvera ascorbata, K. cryocrescensidentification 151
Knife fish 283Koehler illumination 224KOH 121, 270
see also Flexirubin pigmentKoi carp see CarpKovács indole reagent see IndoleKuruma prawn see Shrimp
Labeo rohita see RohuLaboratory worksheet 177Labridae see WrasseLabrus berggylta see WrasseLactobacillosis 24, 44, 67Lactobacillus piscicola see Carnobacterium
piscicolaLactobacillus plantarum-like 52Lactobacillus
culture requirements 88Rogosa acetate agar 275
differentiation from similar genera 125–127hosts and diseases 22
Lactococcosis 52Lactococcus
culture requirements 88Rogosa acetate agar 244, 275
identification notes 130L. garvieae 219
hosts and diseases 10, 11, 13, 15, 24, 32,33, 34, 35, 52
identification 99, 126, 127, 130, 133,160, 204, 205, 209, 210differentiation from similar genera
126-127, 130using the API Rapid ID 32 Strep system
130taxonomy 78PCR primers 227
L. lactis 130, 162, 209, 219L. lactis ssp. cremoris 209, 211L. piscium 219
hosts and diseases 24, 52identification 99, 126, 127, 160, 204,
205differentiation from similar genera
126-127PCR primers 227
L. raffinolactis 209Lactose see Carbohydrate fermentationLagenorhynchus species see DolphinLagodon rhomboides see Pinkfish
Index 343
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Laminaria 59Laminaria japonica see MakonbuLamprey 13Larus delewarensis see GullLates calcarifer Bloch see BarramundiLDC see Lysine decarboxylaseLebistes reticulatus see GuppyLeiognathus 58Leiostomus xanthurus see SpotLepidochelys olivacea see TurtleLeptocottus armatus Girard see Pacific Staghorn
sculpinLeuciscus cephalus Linnaeus see ChubLeuciscus leuciscus Linnaeus see DaceLight organ 33, 58, 68, 69Limanda limanda Linnaeus see DabListeria species 29Listonella
L. anguillarum 219antisera 136culture requirements 88
VAM differentiation medium 260culture appearance see photographic
sectionhosts and diseases 3, 5, 7, 9,13, 14, 15,
19, 22, 23, 24, 34, 35, 37, 53, 79identification 99, 130, 168, 179, 185,
192, 193, 198, 214luminescence 122PCR primers 227serotypes 79taxonomy 79, 81
L. ordalii see V. ordaliiL. pelagia 219
hosts and diseases 53identification 99, 168, 185, 191, 214luminescence 122taxonomy 79
L. ordalii see Vibrio ordaliiLiza species see MulletLizard 47Loach 283Lobster 13, 38, 48, 51, 53, 68, 70, 283, 286Loligo pealei see SquidLoon 25Luminescence 121–122Lumpsucker 53Lutra lutra see European otterLysine decarboxylase
media preparation 267method 120, 267test interpretation 118
see also photographic section
MA 2216 252MacConkey agar (MCA) 270Mackerel 13, 281, 281, 284
Horse mackerel 72, 283Japanese jack mackerel (Trachurus
japonicus) 13, 223Spanish mackerel 52, 286
Macrobranchium rosenbergii see ShrimpMacropodus opercularis see Paradise fishMahi-mahi (Dolphin fish) 14, 56Makonbu 13, 59Malaclemys species see TurtleMaltose see Carbohydrate fermentationMannheimia haemolytica
hosts and diseases 27, 53identification 99, 155, 214
Mannitol see Carbohydrate fermentationMannose see Carbohydrate fermentationMarbled spinefoot see RabbitfishMarine 2216 agar 252Marine columnaris see Columnaris diseaseMarine oxidative fermentative medium (MOF)
275see also Oxidative fermentative media
Marine salt agar with blood (MSA-B) 251Marinilabilia
M. salmonicolor 128M. salmonicolor biovar agarovorans
hosts and diseases 53identification 99, 148, 215
Marinobacter hydrocarbonoclasticushosts and diseases 53identification 148
Marinomonas communis 156M. vaga 156
Mauremys caspica see TurtleMCA see MacConkey agarMcFarland (nephelometer) standards
preparation 270Medium K 252Melanochelys trijuga coronata see TurtleMelanogrammus aeglefinus Linnaeus see
HaddockMenhaden 14, 281, 284Mercenaria mercenaria see ClamMerlangius merlangus see WhitingMesophilobacter marinus 219
hosts and diseases 53identification notes 99, 131, 149
Methyl Red also see MRreagent preparation 271test interpretation 119, 122, 271
see also photographic section
344 Index
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Micrococcus 20, 32M. luteus
hosts and diseases 24, 37, 53identification 160
Micropterus species see BassMicroscopy 224Middlebrook 7H10-ADC medium 252Milkfish (Chanos chanos Forsskål) 13, 69Minnow 14, 41, 284
Bullminnows (Fundulus grandis Baird) 14,65
Eurasian Minnow (Phoxinus phoxinusLinnaeus) 14
Fathead Minnow 49, 283Mirounga species see SealsMisgurnus anguillicaudatus see LoachModified Hayflick medium 254Modiolus modiolus see MusselMola mola see SunfishMolluscs 42, 53, 59, 68, 69, 71, 73
bivalve 14, 67, 70, 73Nodipecten nodosus 73
Molly 17, 49, 284Balloon molly 17, 48, 281Black molly (Poecilia sphenops
Valenciennes) 17Silver molly 17, 48, 285
Monitoring bacterial load see Total bacterialcount
Moraxella specieshost and diseases 20, 29, 31, 32, 53identification 149
MoritellaM. japonica
identification 169M. marina 219
culture morphology 99host and diseases 53identification 99, 169, 180, 185, 190,
215carbohydrate fermentation 262organic growth requirements 254
luminescence 122taxonomy 80
M. viscosa 219culture requirements 88luminescence 122hosts and diseases 22, 24, 53identification 100, 131, 169, 180, 185,
193, 204, 205, 215taxonomy 80
Morone americana, Gmelin see Perch
Motilitygliding motility 129hanging drop method 119, 271tube method 118
MRVP see Methyl red; Voges-ProskauerMSA-B see Marine salt agar with bloodMud skipper 15Mugil species see MulletMullet 71, 283, 284, 285
Black Mullet 15, 47, 281Borneo mullet 16, 282Flathead mullet (Mugil cephalus Linnaeus)
15, 285Grey mullet 15, 48Largescale mullet (Liza macrolepis Smith)
16Silver mullet (Mugil curema Valenciennes)
16, 285Striped mullet 16, 48, 286Wild mullet (Liza klunzingeri Day) 16, 65
Multiplex PCR 231, 233Mussel 14, 70, 73, 283, 284
Far-eastern mussel 59, 284Horse mussel (Modiolus modiolus) 223Protothaca jedoensis Lischke 63
Mustelus canis Mitchill see SharksMya arenaria see ClamMycobacterium
culture morphology 100culture requirements 88hosts and diseases 3, 4, 11, 13, 18, 20, 23,
35, 54, 55identification 100, 166, 167PCR primers 227, 228M. abscessus
hosts and diseases 13, 54Middlebrook 7H10-ADC 252
M. avium 166, 167M. chelonae
hosts and diseases 18, 22, 34, 54M. chelonae abscessus 16, 17M. fortuitum
hosts and diseases 16, 17, 18, 32, 54, 55M. marinum
culture appearance see photographicsection
hosts and diseases 3, 4, 11, 12, 19, 25,30, 34, 54
M. neoaurumhosts and diseases 23, 54, 61
M. peregrinumhosts and diseases 32, 54
Index 345
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Mycobacterium continuedM. poriferae
hosts and diseases 32, 33, 55M. scrofulaceum
hosts and diseases 18M. simiae
hosts and diseases 16, 55Mycobacterium species new strain 55
identification 166M. triplex-like
hosts and diseases 10, 55M. ulcerans
hosts and diseases 11Mycoplasma 30
culture requirements 253characterization tests 271-273medium 253, 254modified Hayflick medium 254
differentiation from Acholeplasma speciessee Digitonin
differentiation from bacteria see Dienesstain
hosts and diseases 28, 55identification 100, 168taxonomy 80M. alligatoris
hosts and diseases 2, 55M. crocodyli 219
hosts and diseases 8, 55M. mobile 219
hosts and diseases 33, 55M. phocacerebrale see M. phocicerebraleM. phocarhinis see M. phocirhinisM. phocicerebrale
hosts and diseases 28, 55M. phocidae
hosts and diseases 28, 55M. phocirhinis
taxonomy 80M. testudinis
hosts and diseases 55identification 168
Myroidesculture requirements 88identification notes 129, 130taxonomy 78M. odoratimimus 219
hosts and diseases 55identification 101, 148, 198, 215
M. odoratus 219culture requirements 88
hosts and diseases 55identification 101, 148, 198, 215
NA see Nutrient agarNB plates see Pasteurella multocida selective
mediaNegaprion brevirostris Poey see SharksNeisseria cuniculi 29Neisseria species 31Nematolosa come see BreamNinhydrin 269, 273Nitrate 130
media preparation 273test interpretation 119, 273
Nocardiaculture requirements 89
carbohydrate fermentation media 264survival at 50°C 268, 276
hosts and diseases 24, 56identification 80, 101, 166, 167
microscopic appearance see photographicsection
PCR primers 228N. asteroides
hosts and diseases 25, 56N. brasiliensis
hosts and diseases 56N. crassostreae
culture requirements 89hosts and diseases 15, 56identification 101, 166
N. flavoroseahosts and diseases 56
N. kampachi see N. seriolaeN. nova
hosts and diseases 56N. salmonicida
hosts and diseases 56identification 101, 167
N. seriolae 219culture requirements 89
carbohydrate fermentation media264
hosts and diseases 4, 11, 22, 35, 56identification 101, 167
Nocardia species, Australian strain 219N. transvalensis
hosts and diseases 56Nodipecten nodosus see MolluscsNovodon modestus see Black scraper
346 Index
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Notemigonus crysoleucas Mitchill see Goldenshiner
Nutrient agar 254Nutritionally variant Streptococci 38, 50, 75
OceanomonasO. baumannii
hosts and diseases 56identification 156, 198
O. doudoroffiihosts and diseases 56identification 156, 198
Octopus 39, 53, 57, 61, 69, 71, 284Oculina patagonica see CoralODC see Ornithine decarboxylaseOdobenus rosmarus rosmarus see WalrusOdonthestes bonariensis see PejerreyOF see Oxidative-fermentative testOncorhynchus species see SalmonidsONPG 130
media preparation 274test interpretation 119
Orcinus orca see WhaleOrectolobus ornatus see SharksOreochromi species see TilapiaOrganic growth requirements 254, 262Ornamental fish 16-18, 36, 39, 41, 47, 54, 56,
60, 65, 74, 284Ornithine decarboxylase (ODC)
media preparation and method 267test interpretation 118
see also photographic sectionOrnithobacterium 78, 128, 129, 130Oryzias latipes Temminck and Schlegel see
Japanese medakaOscar 17, 54, 284
Apistogramma ocellatus 17Red oscar 60
Otter 66, 74, 284European otter 18, 43, 64
Oxidasemethod and test interpretation 122, 274
Oxidative-fermentative testmedia preparation 274, 275test interpretation 119for Mycoplasma spp. see Mycoplasma
characterization testsOyster 63, 64, 67, 68, 69, 72, 73, 281
Crassostrea gigas 72, 223Crassostrea virginica 62Eastern oyster 14, 63, 284Japanese oyster 15, 72
Mediterranean oysters 15, 69Pacific oyster 15, 56, 72, 284
Pacific staghorn sculpin 18, 55, 284Packers plates 255Pagrus major see BreamPangasius hypophthalmus Sauvage see CatfishPantoea
P. agglomerans 219hosts and diseases 13, 56identification 101, 150, 180, 185, 191
P. dispersa 219hosts and diseases 56identification 152, 180, 185, 191
Paracentrotus lividu see Sea urchinParacolobactrum anguillimortiferum see
Edwardsiella tardaParadise fish 284Paraffin oil 115, 275Paralichthys species see FlounderPasteurella
P. haemolytica see Mannheimiahaemolytica
P. multocidahosts and diseases 2, 27, 28, 56identification 101, 153, 180, 186, 190,
215Pasteurella multocida selective media 255P. piscicida see Photobacterium damselae
ssp. piscicidaP. skyensis 219
culture requirements 89hosts and diseases 22, 57identification 101, 153, 215
P. testudinisidentification 102, 153, 180
Pasteurellosis 27, 37, 57PCR (polymerase chain reaction) 225, 234, 235
multiplex PCR 231nested PCR 233primer preparation 236specific primers 225
Pecten maximus see ScallopPedobacter
P. heparinus 128culture requirements 89
peptone yeast medium 255hosts and disease 57identification 102, 148, 204, 205, 215
P. pisciumculture requirements 89identification 102, 148, 204, 205, 215
Index 347
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Peled see WhitefishPelecannus occidentalis carolinensis see PelicanPenaeus species see Prawn; ShrimpPenguin 26, 44, 56, 58, 59
Aptenodytes patagonica 26, 59, 284Eudyptes crestatus 26, 59, 284Gentoo penguin 26, 62Little penguin (Eudyptula minor) 47Macaroni penguin (Eudyptes chrysolophus)
26, 62Pyoscelis papua 26, 59, 284Spheniscus demersus 26, 59, 284Spheniscus humboldti 26, 59, 284
Peptone 131, 132, 134, 244, 263, 264, 267Perca species see PerchPerch 18, 40
European perch (Perca fluviatilis Linnaeus)18, 284
Silver perch (Bidyanus bidyanus Mitchell)18, 40, 285
White perch (Morone americana Gmelin,Roccus americanus) 18, 57, 62, 286
Yellow perch (Perca flavescens Mitchill) 18,54, 286
Phenol red indicator 118, 125, 262, 263, 271,272, 275
Phoca species see SealPhocoena species see PorpoisePhocoenobacter uteri 219
hosts and diseases 28, 57identification 102, 156, 180, 186, 190,
198, 215Phosphatase see Mycoplasma characterization
testsPhotobacterium 31
taxonomy 80transport medium 84P. angustum 219
hosts and disease 57identification 102, 170, 180, 186, 192,
204, 205luminescence 122
P. damselae spp. damselae 219culture appearance see photographic
sectionculture requirements 89hosts and diseases 4, 5, 9, 10, 12, 15, 21,
22, 25, 27, 30, 31, 33, 34, 35, 57identification 102, 131, 169, 170, 131,
180, 186, 192, 196, 198, 204, 205, 215additional tests for biotypes 131
luminescence 122PCR primers 228
taxonomy 80P. damselae ssp. piscicida 219
antisera 136culture appearance see photographic
sectionculture requirements 89hosts and diseases 3, 4, 5, 11, 16, 18, 27,
31, 32, 33, 34, 35, 37, 57identification 102, 131, 170, 180, 186,
190, 191, 192, 198, 204, 205, 215PCR primers 228
P. fischeri see Vibrio fischeritaxonomy 80
P. histaminum see Photobacteriumdamselae ssp. damselae
taxonomy 80P. iliopiscarium 219
hosts and diseases 7, 12, 22, 58identification 102, 170, 180, 186, 196,
204, 205P. leiognathi 220
hosts and diseases 58identification 102, 170, 180, 186, 191,
204, 205luminescence 121
P. logei see Vibrio logeiP. phosphoreum
hosts and diseases 58identification 102, 170, 180, 186, 196luminescence 121
P. profundumhosts and diseases 58identification 170
Phoxinus phoxinus Linnaeus see MinnowPhycodurus equis see Sea dragonPhysiological saline see SalinePike 18
Northern pike 18, 284Pimephales promelas see BaitfishPinnipedia see SealsPirarucu 19, 62, 284 (also spelt Piarucu)Plaice 53, 69, 72, 281
European plaice 19Pleuronectes platessa Linnaeus 69, 284
Planococcushosts and diseases 37, 58identification 160P. citreus
hosts and diseases 58identification 162
P. kocuriihosts and diseases 58identification 102, 162
348 Index
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P. okeanokoites see Planomicrobiumokeanokoites
Planomicrobium okeanokoiteshosts and diseases 58identification 102, 162
Platichthys flesus Linnaeus see FlounderPlaties 19, 49
Southern platyfish (Xiphophorus maculatusGünther) 19
Platyfish see PlatiesPlecoglossus altivelis see AyuPelican 26
Brown pelican (Pelecannus occidentaliscarolinensis) 26
Plesiomonas shigelloides 225culture appearance see photographic
sectionhosts and diseases 25, 31, 58identification 103, 153, 180, 186, 196,
198, 215luminescence 122
Pleuronectes platessa Linnaeus see PlaicePocillopora damicornis see CoralPodocnemis unifelis see TurtlePoecilia species see MollyPolar bear 47Polaribacter 78, 129, 130Poly Plates see Dermatophilus selective mediumPolychaete annelid 68Polydactylus sheridani see SalmonidsPolymerase chain reaction 239Polymyxin plates see Dermatophilus selective
mediumPomacentrida see DamselfishPomadasys stridens Forsskål see Red Sea fishPomatomus saltatrix see Blue fishPorpoise 27, 38
Harbour porpoise (Phocoena phocoena)27, 38, 42, 43, 57, 65, 66, 283
Portunus species see CrabPost-stripping peritonitis 44Potassium hydroxide see Flexirubin pigmentPrawn see ShrimpPreservation of bacteria see storage of isolatesPrimers
degenerate primers 238sequencing primers 238for sequencing Vibrio species 239universal primers 233
Probiotic 11, 15, 23, 40, 44, 64, 70, 73Procambarus clarkii see CrawfishProteus
P. mirabilis 29
P. rettgeri see Providencia rettgeritaxonomy 80
Proteus species 20Protothaca jedoensis Lischke see MusselProvidencia
P. rettgerihosts and diseases 22, 58identification 103, 150, 180, 186, 190taxonomy 80
P. rustigianii 220culture morphology 103hosts and diseases 59identification 150, 180, 186, 190
Pseudemis scripta see TurtlePseudoalteromonas
culture morphology 103culture requirements 89hosts and diseases 2taxonomy 80P. antarctica 220
culture requirements 89hosts and diseases 59identification 103, 156, 198, 215
P. aurantia see Pseudoalteromonasflavipulchra
P. bacteriolyticahosts and diseases 13, 59identification 103, 153, 198
P. citrea 220hosts and diseases 59identification 103, 131, 156, 215
P. denitrificanshosts and diseases 59identification 103, 156
P. distinctahosts and diseases 59identification 156
P. elyakovii 220hosts and diseases 13, 59identification 103, 156
P. espejianahosts and diseases 59identification 103, 156
P. flavipulchrahosts and diseases 59identification 103, 104, 156
P. haloplanktis haloplanktisidentification 156, 198
P. haloplanktis tetraodonisidentification 156
P. luteoviolaceahosts and diseases 59identification 104, 156
Index 349
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Pseudoalteromonas continuedP. maricaloris
hosts and diseases 59identification 89, 104, 156
P. nigrifacienshosts and diseases 59identification 157
P. piscicidahosts and diseases 9, 60identification 104, 153
P. rubrahosts and diseases 60identification 104, 157, 215
P. ulvaehosts and diseases 2, 60identification 104, 157, 180
P. undinahosts and diseases 2, 60identification 104, 157
Pseudocaranx dentex Bloch and Schneider seeWhite Trevally
Pseudomonascarbohydrate fermentation 264hosts and diseases 15, 20, 29, 31, 32,
37P. acidovorans 157P. aeruginosa
identification 157, 180, 186, 191, 192P. anguilliseptica
culture requirements 89hosts and diseases 3, 5, 9, 10, 12, 22, 24,
25, 33, 34, 35, 60identification 104, 132, 153, 180, 186,
190, 198, 215PCR primers 228
P. cepacia see Burkholderia cepaciaP. chlororaphis
hosts and diseases 25, 60identification 153, 198
P. diminuta see Brevundimonas diminutaP. doudoroffii see Oceanomonas
doudoroffiiP. fluorescens
culture appearance see photographicsection
culture requirements 89hosts and diseases 21, 29, 60identification 105, 153, 154, 180, 186,
190, 191, 192, 198P. fluorescens/putida 180P. marina see Halomonas marinaP. mendocina 157P. mesophilica 157
P. nautica see Marinobacterhydrocarbonoclasticus
P. palleronii see Hydrogenophagapalleronii
P. paucimobilis see Sphingomonaspaucimobilis
P. perfectomarina see P. stutzeriP. piscicida see Photobacterium damselae
ssp. piscicidaP. plecoglossicida
hosts and diseases 3, 60identification 105, 154, 180, 198, 215PCR primers 228, 232
P. pseudoalcaligeneshosts and diseases 60identification 105, 154
Pseudomonas pseudomallei seeBurkholderia pseudomallei
P. putidahosts and diseases 60identification 154, 180, 198
P. putrefaciens see Shewanella putrefaciensP. putrefaciens hosts and diseases 19, 21Pseudomonas species 223P. stanieri
hosts and diseases 61identification 157
P. stutzerihosts and diseases 21, 61identification 105, 154, 186, 190
Pseudorca crassidens see WhalePsychrobotes longicauda 63Psychroflexus 129, 130
taxonomy 78P. gondwanensis
hosts and diseases 61Psychrophilic 46, 128Psychroserpens 78, 129, 130Pterophyllum scalare 284
see also Ornamental fishPuffer fish 65Puffin 47Puntius conchonius Hamilton see Rosy barbsPY see Peptone yeast mediumPyoscelis papua see PenguinPYS-2 medium 255
R2A agar 246RAA see Rogosa acetate agarRabbitfish 19, 54, 63, 65, 284
Marbled spinefoot (Siganus rivulatusForsskål) 19
350 Index
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White-spotted spinefoot (Siganuscanaliculatus Park) 19, 286
Rahnella aquatilishosts and diseases 12, 61identification 152, 180, 186, 191
Rainbow trout fry syndrome 37, 49Rams 17, 65Rana castesbeiana see FrogsRaoultella
R. ornithinolyticahosts and diseases 61
R. planticola 220API 50CH see Klebsiella planticola
hosts and diseases 12, 61identification 152
R. terrigena 220hosts and diseases 61identification 152
Red disease 55Red Sea fish 19Red spot disease 13, 59, 60Red spot disease see Sekiten-byo diseaseRedfish 19, 48
Red drum 19, 285Red-leg frog disease 39Redpest 47Redsore disease 39Reef fish 65Reference strains 218–221Renibacterium salmoninarum 220
antisera 136culture requirements 89
KDM2 251KDMC 251SKDM 257culture appearance see photographic
sectionhosts and diseases 3, 22, 23, 25, 36, 61identification 105, 125, 126, 132, 164, 215PCR primers 228
Reinhardtius hippoglossoides Walbaum seeHalibut
Reptiles 19, 39, 45, 53, 54, 58Water monitor 47
Rhodococcushosts and diseases 61identification 105, 164R. equi 165R. fascians
hosts and diseases 61identification 105, 165
R. maris see Dietzia marisRhombosolea tapirina Günther see Flounder
Rhynchopelates oxyrhynchus Temminck andSchlegel 21
Riemerella 78, 128, 129Roach 21, 40, 41, 285Roccus species see Bass and PerchRockfish (Schlegel’s black rockfish) 21, 41Rockling (Fourbeard rockling) 22, 283Rogosa acetate agar 256Roseobacter
culture mediumSWT 258
culture morphology 105R. gallaeciensis
hosts and diseases 15, 61identification 105, 157
Roseobacter strain CVSPculture requirements 89hosts and diseases 14identification 105, 154
Rosy barbs 17, 285Rotifer 22, 70, 71Rudd 22, 285Rudderfish see YellowtailRutilus rutilus Linnaeus see Roach
Saddleback disease 49Salegentibacter 129, 130
S. salegensculture requirements 89hosts and diseases 62identification 106, 148, 181
Salicin see Carbohydrate fermentationSaline, physiological saline 275Salinivibrio costicola ssp. costicola
hosts and diseases 62identification 106, 157PCR primers 228
Salmo species see SalmonidsSalmonella
antisera 136culture media
appearance on MCA 270strontium chloride B enrichment broth
256S. adelaide 29S. arizonae 19, 62
identification 150, 181, 186, 196S. durham 20, 62S. enteritidis 30, 62S. hartford 2, 25S. havana 30, 62S. heidelberg 28, 29, 62
Index 351
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Salmonella continuedS. java 2, 25S. miami 2S. newport 28, 29, 30, 62S. oranienburg 28, 29, 62S. typhimurium 30, 62
PCR primers 233Salmonella group O type B 62
Salmonids 22–25, 36, 44, 49, 61, 66, 69, 74,77
Salmon 22–23, 40, 46, 50, 58, 60, 71,282, 284, 285
Atlantic salmon (Salmo salar Linnaeus)22, 36, 38, 39, 40, 44, 53, 54, 56, 57,60, 61, 62, 64, 66, 67, 69, 71, 73, 74,281
Blueback salmon 56Cherry salmon (Oncorhynchus masou
masou Brevoort, Yamame) 23, 51Chinook salmon (Oncorhynchus
tschawytscha Walbaum) 23, 44, 49, 54,56, 60, 61, 66, 282
Coho salmon (Oncorhynchus kisutchWalbaum) 23, 41, 46, 49, 61, 282
Pacific salmon 23, 284Sockeye salmon (Oncorhynchus nerka
Walbaum) 23, 286Trout 24–25, 39, 40, 46, 54, 57, 64, 283,
285Amago trout 25, 60Arctic charr (Salvelinus alpinus Linnaeus)
22, 44, 52, 62, 281Brook trout (Salvelinus fontinalis Mitchill)
24, 47, 61, 282Brown trout 24, 49, 51, 61, 67, 74, 282Cutthroat trout 44, 282Rainbow trout (Oncorhynchus mykiss
Walbaum) 24, 36, 41, 44, 45, 46, 47,48, 49, 51, 52, 53, 56, 57, 58, 60, 61,62, 64, 65, 67, 72, 73, 284
Sea trout 24, 65, 285Steelhead trout (Salmo trutta trutta
Linnaeus) 23, 284Salmon blood spot 74Salt
adding to biochemical sets 115artificial seawater salts 245marine salts mix 116salt requirement 11520% stock solution 276see also Saline
Salt tolerancemedia for detection 268, 276
Salvelinus species see SalmonidsSample collection 84Sample transport 84Sample preparation 84Sand lance 285Sand whiting 285Sardinops species see SardineSaratherodon species see TilapiaSardine 285
Pacific sardine 25Pilchard (Sardinops neopilchardus) 284South American pilchard (Sardinops sagax
Jenyns) 25Scallop 15, 39, 67, 71
Argopecten purpuratus 15, 285Pecten maximus 15, 61, 285
Sarcina species 223Scardinius erythrophthalmus Linnaeus see RuddSciaenochromis ahli Trewavas see Electric blue
hapSciaenops ocellatus Linnaeus see RedfishScleropages leichardii see SaratogaScomber species see MackerelScophthalmus maximus Linnaeus see TurbotScylla serrata see CrabSea bird 73, 74Sea dragon (Leafy sea dragon, Phycodurus
equis) 26Sea grass 26, 67Sea lion 28, 44, 47, 56, 284, 285
Californian sea lion (Zalophuscalifornianus) 62, 282
Sea mammals 26–31, 38, 47Sea salts see Artificial seawaterSea salts stock solution 257Sea urchin 30, 68, 285Seahorse 26, 57, 67, 69, 285Seals (Pinnipedia) 40, 42, 43, 46, 47, 55, 66,
283, 284Antarctic fur seal 62, 281Common seal (Phoca vitulina) 28, 42, 43,
282Grey seal (Halichoerus grypus) 29, 38, 39,
43, 44, 283Harbour seal 28, 55, 66, 283Harp seal (Phoca groenlandica) 29, 43, 44,
283Hooded seal (Cystophara cristata) 29, 38,
43, 283New Zealand fur seal (Arctocephalus
forsteri) 54, 67, 284Northern fur seal (Callorhinus ursinus) 29,
284
352 Index
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Ringed seal (Phoca hispida) 29, 43, 44,285
South Georgian Antarctic fur seal 29Southern elephant seal (Mirounga leonina)
30, 48, 282, 286Weddell seals 43, 286
Seawater see Artificial seawaterSebastes schlegeli Hildendorf see RockfishSekiten-byo disease (red spot disease) 10Senegal 45Sepia officinalis see Cuttle fishSepiola species see SquidSequencing 236–242
BIOEDIT 241BLAST search 242
Seriola species see YellowtailSerratia
S. fonticola 220hosts and diseases 62identification 106, 152, 181, 186, 195
S. liquefaciensfor Arctic char isolates 150hosts and diseases 22, 34, 62identification 106, 150, 152, 181
S. marcescenshosts and diseases 4, 25, 62identification 150, 152
S. plymuthicahosts and diseases 62identification 106, 150, 181, 186, 191
S. rubidaeaidentification 152
Serum-dextrose agar 256Shad 26
Gizzard shad 26, 41Sharks 30–31, 44, 57, 69
Blacktip shark (Carcharhinus limbatus) 31Brown shark (Carcharhinus plumbeus) 30,
282Lemon shark (Negaprion brevirostris Poey)
31, 283Nurse shark (Ginglymostoma cirratum) 31,
284Sandbar shark 31Smooth dogfish (Mustelus canis Mitchill) 31Spiny dogfish 31Tiger shark (Galeocerdo curvieri) 31
Sheatfish 31Wels catfish 31
Shewanellacarbohydrate fermentation 264hosts and diseases 2S. algae 220
hosts and diseases 12, 63identification 106, 157, 181, 186, 190,
198, 215S. amazonensis
identification 157S. baltica 220
hosts and diseases 63identification 158, 198, 215
S. benthicaculture requirements 89hosts and diseases 63identification 158
S. colwelliana 220culture requirements 89hosts and diseases 14, 63identification 106, 158
S. frigidimarina 220hosts and diseases 63identification 106, 158, 181, 186, 190,
192S. gelidimarina 220
culture requirements 89hosts and diseases 63identification 106, 158, 181, 186, 190
S. hanedai 220culture requirements 89hosts and diseases 63identification 158
S. japonica 220culture morphology 106hosts and diseases 14, 63identification 158
S. oneidensis 220culture morphology 106hosts and diseases 63identification 158
S. pealeana 220culture requirements 89hosts and diseases 33, 63identification 106, 158, 198
S. putrefaciens 220hosts and diseases 19, 21, 31, 63identification 107, 154, 181, 186, 190,
191, 198, 215taxonomy 80
Shewanella sp. MR-1 see Shewanellaoneidensis
S. woodyi 220culture media
Shewanella marine agar 257culture requirements 89hosts and diseases 63identification 107, 158, 181, 186, 190
Index 353
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Shewanella marine agar (SMA) 257Shieh medium + Tobramycin (SM-T) 257Shrimp 53, 57, 67, 69, 73, 281, 282, 286
Black tiger prawn (Penaeus monodon) 31,69
Brine shrimp (Artemia sp.) 31, 71, 282Chinese shrimp (Penaeus chinensis) 31, 71Fairy shrimp (Branchipus schaefferi Fisher)
32, 39, 283Fairy shrimp (Chirocephalus diaphanus
Prévost) 32, 283Fairy shrimp (Streptocephalus torvicornis
Waga) 32, 283Giant freshwater prawn (Macrobranchium
rosenbergii) 32, 39, 41, 283Kuruma prawn (Penaeus stylirostris,
P. japonicus) 32, 71Penaeid prawn 57, 69, 70Pacific white shrimp 54, 284Prawn 72White shrimp (Penaeus vannamei Boone)
32Shubunkin 285Siamese fighting fish (Betta splendens Regan)
17, 54, 283, 285Siberian sturgeon see SturgeonSIEM selective media 86, 258Siganus species see RabbitfishSillago ciliata see Sand whitingSilurus glanis Linnaeus see SheatfishSKDM 257Skin and tail rot 37Skin ulcers 37Skirrow’s medium see Helicobacter selective
mediaSMA see Shewanella marine agarSmooth dogfish see SharksSM-T see Shieh medium + TobramycinSnails 54, 61, 62Snakes 19, 47, 54Snakehead fish 32, 39, 42, 55, 56, 285, 286Snook 32, 282Sole (Solea senegalensis Kaup) 32, 53, 57,
286Dover sole (Solea solea) 32, 36, 66
Sorbitol see Carbohydrate fermentationSouth American pilchard see SardineSouthern platyfish see PlatiesSpadefish 32
Atlantic spadefish 32Sparus auratus Linnaeus see BreamSpecimen collection 84Spheniscus species see Penguin
Spiny dogfish see SharksSphingobacteriaceae 128Sphingobacterium
carbohydrate fermentation 264culture media
peptone yeast medium 255taxonomy 80S. heparinum see Pedobacter heparinusS. indologenes see Chryseobacterium
indologenesS. mizutae see Flavobacterium mizutaiiS. multivorum
culture requirements 89hosts and diseases 63identification 107, 148, 215
S. piscium see Pedobacter pisciumS. spiritivorum 128, 220
culture requirements 89hosts and diseases 64identification 107, 148, 215
Sphingolipids 128Sphingomonas paucimobilis
hosts and diseases 64identification 107, 158S. subarctica 198
Sponge 33, 55, 59Sporocytophaga 78Spot disease 13, 43, 59Squalus acanthias see SharksSquid 63, 69, 286
Hawaiian sepiolid squid (Euprymnascolopes) 33, 68, 285
Loligo pealei 33, 63, 286Sepiola affinis 33Sepiola robusta 33
St Peter’s fish see TilapiaStaphylococcus
hosts and diseases 2, 29, 32, 64identification 162, 163S. aureus 6
hosts and diseases 64identification 160
Staphylase test 136S. aureus anaerobius 162S. capitis 64, 162S. cohnii 64, 162S. delphini 220
hosts and diseases 27, 64identification 107, 160
S. epidermidishosts and diseases 29, 64identification 160
S. haemolyticus 64, 163
354 Index
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S. hominis 64identification 107, 163
S. lutrae 220culture requirements 89hosts and diseases 18, 64identification 107, 160, 215
S. saprophyticus 64, 163S. simulans 64, 163S. warneri 25
culture requirements 89hosts and diseases 64identification 107, 161
S. xylosus 64, 163Stappia stellulata-like
culture requirements 90SWT 258
hosts and diseases 64identification 107, 158, 199
Stenella species see DolphinStenotrophomonas (Pseudomonas) maltophilia
158Sternotherus species see TurtleStingray 33, 57, 65, 286Storage of isolates 224Streptobacillus
S. moniliformisculture requirements 90hosts and diseases 64identification 107, 150
Streptobacillus moniliformis-like organism22
identification 108, 150Streptocephalus torvicornis Waga see ShrimpStreptococcosis 37Streptococcus
antiserum Groups 136S. agalactiae
hosts and diseases 5, 14, 16, 33, 37, 65identification 108, 161, 132, 211
Kuwait strains 132PCR primers 228serogroups 80
S. agalactiae (S. difficile) Group B Type Ib220
culture appearance see photographicsection
hosts and diseases 6, 17, 18, 25, 65identification 108, 132, 161, 209, 204,
205, 211PCR primers 228taxonomy 81
S. difficile see S. agalactiae (S. difficile)Group B Type Ib
S. dysgalactiae ssp. dysgalactiae LancefieldGroup L
hosts and diseases 28, 65identification 108, 161, 211taxonomy 81
S. dysgalactiae ssp. dysgalactiae, Group Chosts and diseases 65identification 161taxonomy 81
S. dysgalactiae ssp. equisimilishosts and diseases 65identification 163taxonomy 81
S. garvieae see Lactococcus garvieaeS. iniae 220
culture appearance see photographicsection
culture requirements 90hosts and diseases 3, 4, 5, 6, 11, 12, 16,
19, 25, 27, 33, 34, 35, 37, 65identification 108, 127, 132, 161, 204,
205, 209, 211differentiation from similar genera 125,
127haemolysis 121, 132
PCR primers 229S. milleri
hosts and diseases 65identification 161
S. parauberis 220culture requirements 90hosts and diseases 34, 66identification 108, 133, 161, 204, 205,
211S. phocae 220
hosts and diseases 28, 29, 66identification 108, 161, 204, 205, 209
S. porcinus 66identification 108, 163, 204, 205, 209PCR primers 228
S. shiloi see S. iniaeStreptococcus ssp. non-haemolytic 21Streptococcus ssp. non-haemolytic Group
B 14S. uberis 220
identification 133, 163, 204, 205, 211Streptomyces salmonis
hosts and diseases 66identification 109, 164
Streptomycosis 66Striped jack 33, 60Striped piggy see Red Sea fishStriped trumpeter 66
Index 355
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Strontium chloride B enrichment broth 256Stuarts medium 84Sturgeon 33, 58, 286
Adriatic sturgeon 33, 52, 281Siberian sturgeon 33
Sucrose 130see also carbohydrate fermentation
Sulculus diversicolor supratexta see AbaloneSurvival at 50°C see Nocardia spp.Sunfish 45, 286Swabs 84SWT 258Syndrome 93 32, 71Symphysodon aequifasciatus Pellegrin see
Discus fishSynodus variegatus Lacepède see Red Sea fish
TBC see Total bacterial countTCBS cholera medium 258
test interpretation 119, 122, 258, 259TDA reagent 276Tenacibaculum 129, 130
carbohydrate fermentation 262alternative methods for carbohydrate
fermentation 263T. maritimum 220, 224
culture requirements 90Flavobacterium maritimus media 249Hsu-Shotts marine agar 251tryptone yeast extract glucose agar 259
media for freeze-drying 224hosts and diseases 2, 3, 4, 5, 11, 22, 23,
25, 32, 36, 66identification 109, 129, 133, 145, 181,
186, 190, 206, 207, 215, 216PCR primers 229, 232
T. ovolyticum 220culture requirements 90hosts and diseases 11, 66identification 109, 145, 216
Tench 33, 55Terrapene carolina carolina see TurtleTetra 17
Black skirted tetra (Hyphessobrycon sp.)17, 281
Neon tetra (Hyphessobrycon innesi Myers)18, 49, 54, 56, 284
Serpae tetra 18Tetramethyl-p-phenylenediamine see Oxidase
testTetrazolium chloride reduction see Mycoplasma
characterization tests
Therapon oxyrhynchus see Coastal fishThorichthys meeki see CichlidThymallus thymallus Linnaeus see GraylingTilapia 33, 39, 60, 65, 286
Nile Tilapia (Oreochromi niloticus niloticusLinnaeus) 33, 284
St Peter’s fish 33Saratherodon (Tilapia) aureus 34, 286Sarotherodon niloticus 34
Tinca tinca see TenchTorticollis 45, 47Tortoise 19, 46, 47, 55, 57Total bacterial count (TBC) 222
Oyster hatcheries 223seawater 223tank water 223
Trachinotus carolinus (Pompanos) 284Trachinus draco (Greater weever) 283Trachurus japonicus see MackerelTransport medium 84
Amies transport medium 84Stuarts transport medium 84transport to the Laboratory 84
Trevally see CrevalleTrehalose see Carbohydrate fermentationTribolodon hakonensis (Living Dace) 283Trichogaster trichopterus Pallas see GouramiTrionyx spinifer see TurtleTriple sugar iron agar (TSI) 276Trout see SalmonidsTryptone soya agar (TSA) 259Tryptone yeast extract glucose agar (TYG)
259Tryptone yeast extract salt medium (TYES)
259TSI see Triple sugar iron agarTTC see Tetrazolium chloride reductionTurbot 34–35, 39, 41, 45, 48, 53, 54, 57, 60,
62, 66, 67, 68, 70, 71, 72, 78, 286Brill (Flatfish) 35, 67, 72Colistium guntheri 35Colistium nudipinnis Waite 35
Tursiops species see DolphinTurtles 19–21, 38, 39, 43, 47, 47, 54, 57, 67,
70, 286African mud turtle 62Caspian terrapin (Mauremys caspica) 20,
62Chelonia mydas 20, 62, 285Eastern box turtle (Terrapene carolina
carolina) 20, 62Eretmochelys imbricata 20Hawaiian green turtle 21
356 Index
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Helmeted terrapin 62Leatherback turtle (Dermochelys coriacea)
21Loggerhead sea turtle (Caretta caretta)
21, 38Mississippi map turtle (Malaclemys kohni)
20, 62Northern diamondback terrapin
(Malaclemys terrapin terrapin) 20, 62Pacific green sea turtle 54Painted turtle (Chrysemys picta) 20, 43,
62Pseudemis scripta 21, 286Red-eared turtle (Chrysemys scripta
elegans) 20, 43, 285Ridley sea turtle (Lepidochelys olivacea)
21South American side-neck turtle
(Podocnemis unifelis) 43, 286Spiny soft-shelled turtles (Trionyx spinifer)
43, 286Stinkpot turtle (Sternotherus odoratus) 20,
62Striped-necked musk (Sternotherus minor
peltifer) 43, 286Travancore crowned turtle (Melanochelys
trijuga coronata) 20, 62Yellow-spotted Amazon turtle 62
TYES see Tryptone yeast extract salt medium(TYES)
TYG see Tryptone yeast extract glucose agar
Ulcer head disease of eels 41Ulva lactuca see AlgaeUrea 130
test method 276, 277Utilization, differentiation from fermentation see
Carbohydrate fermentation
Vagococcusidentification notes 124
differentiation from similar genera 124taxonomy 81V. fessus 220
hosts and diseases 28, 66identification 109, 161, 211, 216
V. fluvialis 220hosts and diseases 12, 66identification 109, 126, 163, 206, 207,
209, 211
V. lutrae 220hosts and diseases 18, 66identification 109, 126, 161, 211, 216
V. salmoninarum 220culture requirements 90hosts and diseases 22, 25, 67identification 109, 126, 126, 133, 161,
199, 206, 207, 209, 211differentiation from similar genera
126–127VAM 260Variegated lizardfish see Red Sea fishVarracalbmi
culture requirements 90hosts and diseases 23, 67identification 109, 154
Vibrioalternative method for carbohydrate
fermentation 264differentiation from Aeromonas species
114haemolysis 121hosts and diseases 15, 31luminescence 121, 122PCR primers for sequencing 239taxonomy 81TCBS selective medium 122, 119transport medium 84Vibrio discs (0/129) 114, 119, 123, 277V. aerogenes 220
hosts and diseases 67identification 109, 175
V. aestuarianus 220hosts and diseases 67identification 109, 175, 181
V. agarivorans 220culture appearance see photographic
sectionculture requirements 90hosts and diseases 2, 67identification 110, 133, 176, 181, 186,
191, 216V. alginolyticus
culture appearance see photographicsection
hosts and diseases 2, 5, 15, 20, 21, 31,67
identification 110, 133, 134, 171, 181,187, 193, 194, 199, 216, 260differentiation from V. harveyi 133
PCR primers 232V. anguillarum biovar II see V. ordalii
Index 357
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Vibrio continuedV. brasiliensis 220
hosts and diseases 14, 67identification 110, 175, 181, 187, 193,
216V. calviensis 220
hosts and diseases 67identification 110, 176, 216
V. campbelliihosts and diseases 67identification 176
V. campbellii-like 35, 67V. carchariae see V. harveyiV. cholerae
antisera 136hosts and diseases 8identification 110, 133, 171, 187, 192,
193, 195, 196,199aesculin test 117, 133, 261
PCR primers 231, 233serotypes 81
V. cholerae 01 81hosts and diseases 12, 68identification 171, 181, 187, 195PCR primers 229
V. cholerae serotype 0139 Bengal 81hosts and diseases 12identification 110, 171resistance to Vibrio static agent 123
V. cholerae non-01 81culture appearance see photographic
sectionhosts and diseases 3, 4, 12, 17, 34, 68identification 134, 171, 181, 187, 195,
216PCR primers 229
V. cholerae-like 8, 68V. cincinnatiensis 220
hosts and diseases 13, 68identification 110, 175
V. coralliilyticusculture requirements 90hosts and diseases 8, 68identification 110, 134, 174, 199
V. costicola see Salinivibrio costicola ssp.costicola
V. cyclitrophicushosts and diseases 68identification 110, 176
V. damselae see Photobacterium damselaessp. damselae
V. diabolicus 220hosts and diseases 68
identification 110, 175, 181, 187, 194,199, 206, 207, 216
V. diazotrophicus 220hosts and diseases 30, 68identification 110, 175PCR primers 229
V. fischeri 219hosts and diseases 33, 68identification 110, 172, 173, 181, 187,
193, 206, 207, 216variable results for VP 134
PCR primers 229V. fluvialis 220
culture appearance see photographicsection
hosts and diseases 13, 21, 69identification 110, 134, 173, 181, 187,
193, 260PCR primers 229
V. furnissii 220culture appearance see photographic
sectionhosts and diseases 9, 13, 69identification 111, 134, 173, 181, 187,
192, 193, 216V. gazogenes 221
hosts and diseases 69identification 176
V. halioticoli 221culture requirements 90hosts and diseases 2, 69identification 111, 176, 181, 187, 190,
216V. harveyi 220, 221
culture appearance see photographicsection
hosts and diseases 2, 4, 5, 8, 11, 13, 16,31, 32, 69
identification 111, 133, 134, 171, 181,187, 194, 199, 206, 207, 216, 260differentiation from V. alginolyticus 133
taxonomy 81V. hollisae 221
culture requirements 90hosts and diseases 13, 21, 69identification 110, 176PCR primers 229
V. ichthyoenterihosts and diseases 11, 69identification 111, 174, 181, 187, 190,
199V. iliopiscarius see Photobacterium
iliopiscarium
358 Index
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V. lentus 221hosts and diseases 15, 69identification 111, 175
V. logei 220carbohydrate fermentation 262hosts and diseases 23, 33, 69identification 111, 173, 216organic growth requirements 254
V. marina see Moritella marinaV. mediterranei 221
hosts and diseases 2, 8, 70identification 111, 134, 175, 182, 187,
196, 216arginine dihydrolase test differences 267
V. metschnikovii 221hosts and diseases 70identification 111, 175, 260
V. mimicus 221culture appearance see photographic
sectionculture requirements 90hosts and diseases 4, 8, 21, 34, 70identification 111, 134, 171, 182, 187,
195, 196, 199, 216V. mytili 221
hosts and diseases 70identification 111, 175, 182, 187, 191,
199, 206, 207, 216arginine dihydrolase test differences 267
V. natriegens 219, 221hosts and diseases 70identification 176, 216taxonomy 79, 81
V. navarrensis 221hosts and diseases 70identification 111, 176, 182, 199
V. neptunius 221hosts and diseases 14, 22, 34, 70identification 111, 175, 182, 187, 192,
216ADH strain variation 134
V. nereis 221hosts and diseases 70identification 175, 182, 187, 190, 199,
206, 207, 216V. nigripulchritudo
hosts and diseases 71identification 176PCR primers 229
V. ordalii 221carbohydrate fermentation 262
culture appearance see photographicsection
culture requirements 90hosts and diseases 23, 25, 71identification 112, 174, 182, 187, 190,
216, 217organic growth requirements 254taxonomy 79, 80
V. orientalis 221hosts and diseases 71identification 112, 175, 217
arginine dihydrolase test differences267
V. pacinii 221identification 112, 175, 182, 217hosts and diseases 22, 31, 71
V. parahaemolyticusculture appearance see photographic
sectionculture requirements 90hosts and diseases 2, 6, 8, 9, 13, 31, 71identification 112, 134, 171, 182, 187,
194, 217PCR primers 229, 230, 231, 232, 233
V. pectenicida 221hosts and diseases 15, 71identification 112, 174, 182, 188, 190
V. pelagius see Listonella pelagiaV. penaeicida 221
hosts and diseases 32, 71identification 112, 174, 182, 188, 191,
199, 206, 207PCR primers 230
V. proteolyticus 221culture appearance see photographic
sectionhosts and diseases 31, 71identification 112, 135, 173, 182, 188,
196, 217PCR primers 230
V. rotiferianus 221hosts and diseases 22, 71identification 112, 175, 182, 188, 195,
217V. rumoiensis
culture mediumPYS-2 medium 255
hosts and diseases 71identification 112, 176
V. salmonicida 221, 223culture requirements 90hosts and diseases 7, 23, 71identification 112, 174, 182, 188, 190,
195, 206, 207, 217PCR primers 230
Index 359
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Vibrio continuedV. scophthalmi 221
hosts and diseases 34, 72identification 112, 135, 176, 182, 188,
191chitin hydrolysis 135differentiation from V. splendidus
biovar I 135V. shilonii 221
hosts and diseases 8, 72identification 112, 172, 199
Vibrio species 223Vibrio species zoea 8
Vibrio species zoea see V. harveyiV. splendidus
hosts and diseases 5, 15, 35, 72identification 135, 173, 182, 188, 190,
192, 193, 206, 207test variations between strains 135
PCR primers 230V. splendidus-like 72V. splendidus I 34, 72, 135, 221
hosts and diseases 72identification 112, 135, 173, 182, 217
arginine dihydrolase test differences 267V. splendidus 221
hosts and diseases 15, 31, 72identification 112, 135, 173, 217
V. tapetis 221hosts and diseases 14, 36, 72identification 113, 174, 182, 188, 191,
199, 217V. trachuri
hosts and diseases 13, 72identification 172PCR primers 230taxonomy 81
V. tubiashii 221culture appearance see photographic
sectionhosts and diseases 14,15, 40, 72identification 113, 173, 174, 182, 188,
191, 192, 199, 206, 207, 217arginine dihydrolase test differences 267
PCR primers 230V. viscous see Moritella viscosaV. vulnificus 221
taxonomy, serotypes and biotypes 81culture requirements 90
cellobiose-colistin agar 247VVM 260
culture appearance see photographicsection
hosts and diseases 8, 9, 10, 13, 15, 31, 73identification 113, 135, 138, 171, 182,
183, 188, 189, 191, 193,194, 195, 196,217
PCR primers 230, 231, 232, 233V. wodanis 221
hosts and diseases 23, 73identification 113, 135, 176, 217salt requirement variation 134taxonomy 81
V. xuii 221hosts and diseases 32, 73identification 113, 175, 183, 189, 192,
217Vibrio discs (vibrio static agent 0/129)
test interpretation 114, 119, 123, 277Voges-Proskauer (VP)
media preparation 277test interpretation 119, 122, 277
VP see Voges-ProskauerVPT media see Skirrow’s mediumVVM selective medium 260VVMc 260
Weakfish see Sea bassWeeksella 78, 129, 130Weissella hellenica strain DS-12
hosts and diseases 11, 73identification 165
Wels catfish see SheatfishWhale 43, 46, 282, 284
Beluga whale 30, 51, 281False killer whale (Pseudorca crassidens)
30, 44, 283Killer whale (Orcinus orca) 30, 283Minke whale (Balaenoptera
acutorostrata) 30, 44, 51, 284Pacific pilot whale (Globicephala
scammoni) 30, 284Sowerby’s beaked whale 30, 38, 286White whale (Delphinapterus leucas) 30,
54, 286White-spotted spinefoot see RabbitfishWhite Trevally 33Whitefish 35, 286
Cisco (Coregonus artedi Lesueur) 35Lake whitefish (Coregonus clupeaformis
Mitchill) 35Peled (Coregonus peled Gmelin) 35
Wild fish see Red Sea fishWinter disease 5, 53, 60Winter ulcers 22, 23
360 Index
376Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #M.vpTuesday, January 20, 2004 5:35:45 PM
Color profile: DisabledComposite 150 lpi at 45 degrees
Winter ulcer disease 53, 73Withering syndrome 2, 71Wolf-fish (Anarhichas lupus Linnaeus, A. minor
Olafsen) 35, 52, 282, 286Spotted wolf-fish 35, 285
Wood’s broth 260Worksheet see Laboratory worksheetWrasse (cleaner fish) 35, 40, 283, 286
Xiphophorus maculatus Günther see PlatiesXylose see Carbohydrate fermentation
Yamame see SalmonidsYeast extract 262Yeast extract broth or agar 264Yellowtail 35, 52, 56, 57, 60, 64, 66, 286
Seriola purpurascens 35Seriola quinqueradiata 35
Yellowtail clownfish see DamselfishYersinia
hosts and diseases 73, 74identification 152taxonomy 81Y. aldovae 221Y. frederiksenii
identification 135, 152Y. intermedia
identification 150, 183, 189, 191Y. kristensenii
identification 113, 152, 206, 207Y. pseudotuberculosis 152
Y. rohdei 221Y. ruckeri
antisera 136culture requirements 90PCR primers 230, 233hosts and diseases 6, 7, 9, 11, 14, 15, 18,
21, 22, 23, 24, 25, 33, 35identification 113, 136, 151, 183, 189,
191, 194, 195, 217differentiation from Hafnia alvei 129,
136motility at different temperatures 90,
113, 136see also photographic section
taxonomy and serotyping 82Yersinia selective agar (YSA) 260
Zalophus californianus see Sea lionZobellia 129, 130
Z. galactanovoranshosts and diseases 2, 74identification 113, 148, 183, 189, 191,
199, 206, 207, 217Z. uliginosa
hosts and diseases 74identification 113, 148, 183, 189, 191,
199, 206, 207, 217Zoarces viviparus Linnaeus see BlennyZoBell’s agar see Marine 2216 agarZona radiata 11, 66Zoonotic 43, 44, 46, 48, 54, 55Zooxanthellae 72
Index 361
377Z:\Customer\CABI\A4636 - Buller\A4657 - Buller - Vouchers #VP10 #M.vpTuesday, January 20, 2004 5:35:45 PM
Color profile: DisabledComposite 150 lpi at 45 degrees