preparation of anti-aeromonas hydrophila
TRANSCRIPT
PREPARATION OF ANTI-AEROMONAS HYDROPHILA
RABBIT SERUM
A THESIS
BY
S. M. RAKIB
EXAMINATION ROLL NO. 10 Fish Aqua JD-18M
SEMESTER: JULY-DECEMBER 2011
REGISTRATION NO. 32625
SESSION: 2005-2006
MASTER OF SCIENCE (M. S.)
IN
AQUACULTURE
DEPARTMENT OF AQUACULTURE
BANGLADESH AGRICULTURAL UNIVERSITY
MYMENSINGH
NOVEMBER, 2011
PREPARATION OF ANTI-AEROMONAS HYDROPHILA
RABBIT SERUM
A THESIS
BY
S. M. RAKIB
EXAMINATION ROLL NO. 10 Fish Aqua JD-18M
SEMESTER: JULY-DECEMBER 2011
REGISTRATION NO. 32625
SESSION: 2005-2006
Submitted to the
Department of Aquaculture
Bangladesh Agricultural University, Mymensingh
in partial fulfillment of the requirements
for the degree of
MASTER OF SCIENCE (M. S.)
IN
AQUACULTURE
NOVEMBER, 2011
PREPARATION OF ANTI-AEROMONAS HYDROPHILA
RABBIT SERUM
A THESIS
BY
S. M. RAKIB
EXAMINATION ROLL NO. 10 FishAquaJD-18M
SEMESTER: JULY-DECEMBER 2011
REGISTRATION NO. 32625
SESSION: 2005-2006
Approved as to style and content by:
Prof. Dr. M. Mamnur Rashid
Supervisor
Prof. Dr. Md. Gias Uddin Ahmed
Co-supervisor
Prof. Dr Md. Ali Reza Faruk
Chairman, Examination Committee
And
Head, Department of Aquaculture
Bangladesh Agricultural University
Mymensingh
NOVEMBER, 2011
DEDICATED TO MY BELOVED PARENTS
ABSTRACT
An experiment was conducted to prepare anti-Aeromonas hydrophila serum in two rabbits
(Oryctolagus cuniculus). Formalin killed cell (FKC) and heat killed cell (HKC) were
prepared by adding 0.5% formalin and 2.5 h heat treatment at 60°C of freshly cultured
Aeromonas hydrophila (AQC810) isolated from naturally infected koi fish kidney
respectively. Slide agglutination tests were performed for the determination of the presence
of specific antibody against Aeromonas hydrophila in the serum collected from the blood of
pre-immune rabbits or post immune rabbits. No agglutination of pre-immune serum with the
homologous antigen proved that the rabbits had no previous Aeromonas contamination.
Rabbits were injected subcutaneously with the FKC added with same amount of Freund’s
complete adjuvant. Three booster doses were given without the adjuvant at weekly intervals
after second week of first injection. Agglutination titration of anti-Aeromonas hydrophila
rabbit serum against HKC and FKC of homologous bacteria showed that the titers increased
with the post inoculation days which rose at their peak during the fifth week of immunization
and after three consecutive booster doses at weekly intervals starting from second week of
first immunization. Rabbits were sacrificed after six weeks of immunization, whole blood,
collected and centrifuged to accumulate the serum that were complement-inactivated by
heating to 58º C for 30 min and stored at -20°C in appendorph tubes, with the aim of rapid
diagnosis of Aeromonas infections in fishes. Result of this study would initiate the way of
vaccine development against MAS (motile Aeromonas septicemia) in the aquaculture fishes
of Bangladesh.
ACKNOWLEDGEMENTS
The author expresses his heartiest and sincere praises to the “Almighty Allah” the omnipotent,
omnipresent, omniscient, the Supreme Authority of the Universe who has created us to explore the
hidden fact of the nature for the benefit of mankind and enabled him to complete this piece of
research work successfully in time.
The author sincerely expresses his deep sense of gratefulness, indebtedness and profound respect to
his honourable teacher and supervisor, Professor Dr. M. Mamnur Rashid Department of Aquaculture,
Bangladesh Agricultural University, Mymensingh for his scholastic supervision, valuable suggestions,
sympathetic co-operation, helpful advice, constructive criticism, painstaking correction of the
manuscript and affectionate feeling at all stages of this study period, research work and preparation of
this thesis.
The Author also finds great pleasure to express his sincere appreciation and immense indebtedness to
his co-supervisor, Professor Dr. Gias Uddin Ahmed., Department of Aquaculture, Bangladesh
Agricultural University, Mymensingh for his sympathetic consideration, valuable suggestions and
constructive criticism throughout the research period and the thesis work.
The author is very interested to express his gratitude, indebtedness and profound respect to his
honourable teachers, Prof. Dr. Kirtunia Juran Chandra, Prof. Dr. Md. Mohsin Ali, Prof. Md. Jahir
Uddin Miah, Prof. Dr Monoronjan Das, Prof. Dr. Gias Uddin Ahmed, Prof. Dr. S.M. Rahmatulla,
Prof. Dr Md. Ruhul Amin, Prof. Dr. Ahsan Bin Habib, Prof. Dr. M.A. Salam and Dr. Mohammad
Mahfujul Haque Associate Professor for their valuable suggestions and sympathetic co-operation
throughout the research period.
The author expresses his gratefulness to his senior researcher Forhad Hossain for their valuable
suggestions, sympathetic co-operation, and helpful advice throughout the research period.
The author would like to express special thanks to his friends Istique Haider, Robiul islam, Nazmul,
Momin, Masum, Mahbub, Iqbal, Foysal, Tusar, Sabuj, Sazib, Saiful , Khaja, Khadem, Khokon, Misu
for their helpful suggestions and sympathetic co-operation throughout the research period.
The author specially memorizes departed soul of his father Abdul Jalil Mian and is ever grateful to his
beloved mother Selina Begum, brothers Salehin, Rasel for their everlasting love, patience, moral and
constant blessings, valuable advice, cooperation and countless sacrifice in all aspect throughout his
academic life. The author owes special debts of gratitude to all his family members for their constant
blessings and encouragements in all activities of his University life.
The author also offers thanks to Islam Ali, staff of Animal House for his help during the study period.
The author
CONTENTS
CHAPTER TITLE PAGE
ABSTRACT iv
ACKNOWLEDGEMENTS v-vi
CONTENTS vii-viii
LIST OF TABLES ix
LIST OF FIGURES x
LIST OF PLATES xi
1 INTRODUCTION 1-2
2 REVIEW OF LITERATURE 3-7
3 MATERIALS AND METHODS 8-21
3.1 Collection and acclimatization of experimental rabbits 8
3.2 Cleaning and sterilization of required glass wares and plastic wares
8
3.3 Preparation of physiological saline 10
3.4 Preparation of phosphate buffered saline 10
3.5 TSA (triptic soya agar) plate preparation 10
3.6 Culture of Aeromonas hydrophila bacteria 10-12
3.7 Confirmatory test of A. hydrophila 12
3.7.1 Biochemical test 12
CONTENTS (Continued)
CHAPTER TITLE PAGE
3.7.2 Sensitivity to 0129 test 12
3.7.3 Esculin hydrolysis test 12
3.8 Preparation of FKC (formalin killed cell) 12
3.9 Preparation of HKC (heat killed cell) 13
3.10 Antigenic preparation 13
3.11 Preparing the rabbits for immunization 13
3.12 Slide agglutination test 13-14
3.13 Immunization of rabbit 14-15
3.14 Agglutination titration 15-17
3.15 Collection and preservation of the rabbit anti-serum 19
4
RESULTS
22-27
4.1 Characteristics of Aeromonas hydrophila 22
4.2 Status of pre-immune serum 22
4.3 Slide agglutination results of immune sera 22
4.4 Agglutination titer of immune rabbit serum 26
4.5 Anti A. hydrophila rabbit serum 27
5
DISCUSSION
28-29
6 SUMMARY AND CONCLUSION 30-31
REFERENCES 32-35
LIST OF TABLES
Table
NO. DESCRIPTION PAGE
1 Characteristics of Aeromonas hydrophila bacteria in comparison to Popoff
(1984) and Sabur (2006)
23
2 Slide agglutination test results of pre-immune rabbit serum with FKC and
HKC of Aeromonas hydrophila
24
3 Results of slide agglutination tests of immune serum after different days of
post immunization with FKC and HKC of Aeromonas hydrophila bacteria
25
4 Distribution of agglutination titer of the anti- Aeromonas hydrophila rabbit
serum against HKC and FKC of homologous bacteria
26
LIST OF FIGURES
FIG.
NO. DESCRIPTION PAGE
NO.
1 Slide Agglutination 14
2 Agglutination titration. A diagrammatic representation of 96 well 17
LIST OF PLATES
PLATE
NO. DESCRIPTION PAGE
NO.
1 Acclimatization of rabbits under controlled condition in the experimental
animal house of the Department of Microbiology and Hygiene (A= White
rabbit and B= White rabbit)
9
2 Sterilization of inoculating loop to inoculate bacteria into culture media 11
3 Booster doses injected in the shoulder of the rabbit 15
4 Diluter was touched with the upper meniscus of the 20% diluted antiserum
with PBS
16
5 Antigen drops were poured into the microtitre plate 18
6 Sterilization of diluter to dilute the antibody in PBS in microtitre plate 18
7 Dilution of antibody in PBS in microtitre plate with the help of diluters 18
8 The rabbit was anaeshetized 19
9 The rabbit was sacrificed 20
10 Blood were collected from the heart of rabbit by sterilized plastic syringe 20
11 Blood were kept in slanted position for an hour at room temperature 21
12 Prepared anti-Aeromonas hydrophila rabbit serum 21
Chapter 1
INTRODUCTION
CHAPTER 1
INTRODUCTION
Motile Aeromonas septicemia (MAS) is an important problem now-a-days in Bangladesh Aquaculture
caused by Aeromonas hydrophila. It is a pathogen that causes disease in various species of farm and
wild freshwater fishes in different locations of Bangladesh. Rahman and Chowdhury (1996) isolated it
from an ulcer diseased farmed carp fishes of Mymensingh. Iqbal et al. (1998) detected A. hydrophila,
A. veronii, A. sobria, and A. jandaei as pathogenic bacteria from EUS affected mrigals. Sarker et al.
(2000) studied effect of water temperature on the infectivity of A. hydrophila isolates. Sabur (2006)
isolated five different species of A. hydrophila from six polyculture species of exotic and indigenous
carps. Mamnur Rashid et al. (2008) isolated A. hydrophila from EUS affected shing H. fossilis from a
fish farm of Mymensingh. Alam (2009) detected the pathogen from naturally infected Thai pangas
Pangasius hypophthalmus. Roshid (2009) experimentally infected Thai pangas with A. hydrophila
successively.
Anti-pathogenic rabbit serum is widely used in fish disease laboratories for identification of endemic
fish disease and other serological research related in fish immunology. The only method to prevent
disease in aquaculture is strengthening the defense mechanism of fish through prior administration of
an antigen prepared for the homologous pathogen (Robertsen 1999). Mamnur Rashid (1997) prepared
anti-Edwardsiella tarda rabbit serum. Swain et al. (2007) prepared Rabbit anti-rohu globulin
conjugate from healthy adult rohu. Maji et al. (2005) produced hyperimmune serum against the crude
antigen of A. hydrophila from two New Zealand White male rabbits.
The disease MAS is not only reducing fish production but also degrading the quality of affected fishes
causing loss of huge amount of money. So, effective rapid diagnosis method should be developed in
order to rapidly identify the pathogen. Immunodiagnosis is a rapid diagnosis measure which is done
by in-vitro antigen antibody reaction. Proper identification of any bacteria is only possible by the
immunodiagnostic method for which antibody against a type strain of the bacteria must be raised.
Prepared antibody would be used to identify the A. hydrophila bacteria of any suspective sample by a
rapid diagnostic method called “slide agglutination method” which will need only one minute. This
antibody would also be used to serotype all A. hydrophila isolates in order to understand intra specific
relationship/variation of the bacteria all over Bangladesh. This will also be helpful for the preparation
of vaccine against the pathogen A. hydrophila. Thus, appropriate remedial measures can be taken
against MAS within a very short time.
So the present study was performed to attain the following objectives:
• To prepare anti-Aeromonas hydrophila rabbit serum;
• To test the efficacy of the different booster doses in the production of the antiserum;
• To determine the titer of the antiserum against Aeromonas hydrophila; and
• To establish rapid diagnosis process of Aeromonas infection.
Chapter 2
REVIEW OF LITERATURE
CHAPTER 2
REVIEW OF LITERATURE
The following literatures were reviewed in favour of the present study.
Lamers (1986) showed that the number of antibody forming cells in the head kidney peaked
at day 9 and the serum antibody response followed with a peak at day 15. When the antigen
was emulsified in adjuvant, the antigen processing and the antibody responses were effected
in carp (Cyprinus carpio) by immunization of Aeromonas hydrophilla.
Toranzo et al. (1987) found that the usefulness of the slide agglutination assay for a rapid
diagnosis of fish diseases was evaluated using a total of 80 pathogenic bacteria and
environmental isolates belonging to the genera Vibrio (28), Pasteurella (5), Aeromonas (26),
Yersinia (6), Edwardsiella (8), Pseudomonas(6) and Lactobacillus(1). Selected strains from
each bacterial group were used as antigens for rabbit immunization.
Ada (1993) observed that water soluble proteins were closely related in structure to the
homologous rabbit serum. When such proteins are monomeric and not aggregated, they may
evoke feeble responses unless adjuvants are used. For soluble antigens, Freund’s complete
adjuvant would be the best choice. It remained the gold standard by which other adjuvants. It
was used to increase the antigenic growth.
Gupta et al. (1993) used adjuvant to prepare antigen for immunization. They stated that an
adjuvant would be a substance, which augmented immune response in a non-specific manner. Goding and James (1993) showed that subsequent injections might be either soluble or
aggregated. Therefore, when antigen would be water soluble protein, the use of an adjuvant
would usually be essential. He stated that if the antigen would be of very low molecular
weight or be poorly immunogenic for any other reason, strong responses to immunogenic
carrier molecule might be possible by using adjuvant.
Stewart-Tull, (1995) showed that Freund’s complete adjuvant was a water-in-oil emulsion in
which killed and dried Mycobacterium bovie bacteria were suspended in the oil phase.
Freund’s incomplete adjuvant did not contain bacteria. Other adjuvant included aluminium
compounds for instance aluminium hydroxide gel and potassium alum.
Rangdale (1995) evaluated the potential of a number of serodiagnostic techniques of rapid
detection of Aeromonas hydrophila in diseased fish and reported that agglutination and
ELISA technique might be useful for the identification of A. hydrophila from bath plate/broth
grown cultures and directly from infected fish.
Mamnur Rashid (1997) produced Anti-Edwardsiella tarda serum in a New Zealand white
rabbit. The author used Freund’s complete adjuvant with FKC at a ratio of 1:1 and injected 1
ml of the mixture subcutaneously at multiple sites. The author inoculated three successive
boaster doses at weekly interval after the second week of the first injection without adjuvant.
Titer rose was 1:1280 for FKC and 1:640 for HKC respectively. Guz (2001) showed that the inhibitory activity of the mucus and serum of carp
experimentally infected with A. hydrophila F6/95 bacteria was much higher in relation to the
F6
/95 proteases than the control group. In the experimentally infected fish, the inhibitory
activity of serum increased faster (after 3 days) compared to that of the mucus (after 7 days).
The inhibitory activity of the mucus and serum depended not only on clastase inhibitors but
also on humoral immunity.
Jha (2001) suggested that pre-xylose ampicillin agar was the best medium for the recovery of
A. hydrophila from fishes and for differentiation by simple criteria of that genus from other
common water born bacteria.
Rahman et al. (2001) observed that temperature affected both of the cell membrane structure
of A. hydrophila and phagocytic activity of gold fish macrophage.
Chandran et al. (2002) suggested that immunization of Indian major carps against A.
hydrophila by intraperitoneal injection by two different polyvalent antigen whole cell and
extracellular product (ECP) was very much effective for better survival. It would be
introduced and available in root level farmers.
Faruk et al. (2002) found that a comparison of the response of fish, rabbits and mice sera to
whole cell antigens indicated differences between the species by Western blot. More bands
were recognized by rabbit the mouse serum than with fish serum. The rabbit antisera and
different monoclonal antibodies (Mabs), produced against Flavobacterium psychrophilum
were not suitable for use as diagnostic tools because they failexd to recognize all the F.
psychrophilum bacteria. The rabbit sera raised against both the virulent and bon-virulent
isolates of Flavobacterium psychrophilum, recognized a number of similar protein and
carbohydrates bands on the whole cell profiles of all the isolates examined. However
differences were seen in the staining of high and low molecular w\eight material in the
Western blot profile of the bacterium with these two antisera. This material was recognized
more strongly in bacteria to which the serum had been raised.
Hua and Lu (2002) used antiserum for extracellular product of virulent strain of A.
hydrophila for the detection of its virulent factor.
Hua et al. (2002) tested vaccination against A. hydrophila and Vibrio fluvialis in Carassius
auratus by intraperitoneal injection with a supplement of polysaccharide. They found the
bivalent vaccines protective against the challenge test with those pathogens in a variety of
farmed fishes in China.
Robertson et al. (2003) reported that formalin-inactivated cells of A. hydrophila A3
Sahoo (2003) suggested inclusion of triiodothyronine 5 mg/kg diet in rohu Labeo rohita to
enhance immunity and resistance to Aeromonas hydrophila infection.
-S1
applied as a feed additive had benefit in controlling infection by atypical A. salmonicida in
gold fish.
Majumder (2006) conducted an experiment to prepare an anti-Aeromonas hydrophila serum
in two rabbits (Oryctolagus cuniculus). The author used Formalin killed cell (FKC) and heat
killed cell (HKC) for the immunization of rabbits. Agglutination test was performed to know
antiserum concentrations in the blood of the immunized rabbits. Agglutination titration of
anti-Aeromonas hydrophila rabbit serum showed that the titers increased with the post
inoculation days. Rabbits were sacrificed after six weeks of immunization, whole blood,
collected and centrifuged to accumulate the serum and stored at -20°C in appendorph tubes.
Hasan (2007) showed that when shing Heteropneustes fossilis were infected with Aeromonas
hydrophila in EUS like diseases a great pathological effects were observed. In the liver of the
naturally infected shing focal necrosis and hemorrhages occurred in the tissue. Renal tubules
were degenerated in most of the kidney tissue. Missing of glomerulus and necrosis
surrounding the bowmsan’s capsule. Pyknotic hematopoietic cells were found in the kidney
tissue. Haemorrhages occurred in the whole kidney tissue with fungal granuloma of an
infected shing fish.
Mali et al. (2007) showed that Aeromonas sobria, along with A. hydrophila, has frequently
been reported as a causative agent of motile Aeromonas septicaemia (MAS) in fish and other
aquatic organisms.
Mamnur Rashid et al. (2008) isolated Aeromonas hydrophila from the suspected EUS-
affected shing fish, Heteropneustes fossilis (Bloch) and Hasan et al. (2008) observed
histopathological changes in intestine, liver and kidney of the affected fish. Aeromonas
hydrophila isolates were identified by a series of morphological, physiological and
biochemical tests.
Alam (2009) isolated and characterized Aeromonas hydrophila from naturally infected Thai
pungas Pangasius hypophthalmus. The isolates gave rise to yellowish opaque colonies on
TSA agar, and blue colonies on AIM medium. These colonies were smooth, raised and round
in shape. Zigzag culture from AIM to TSA provided single colonies for stocking. Growth was
observed at 37̊C but no growth was observed at 40˚C. The bacteria were Gram negative,
motile, fermentative, oxidase positive and catalase positive rods. Acid and gas were
produced from dextrose, maltose and sucrose. In lactose and manitol, the bacteria produced
only acid. The bacteria gave positive results in Voges- Proskauer, H2
S, arginine hydrolysis
esculine hydrolysis.
Benjir (2009) isolated Aeromonas hydrophila from naturally infected or apparently healthy
carps (rui, catla, mrigal) catfishes (shing, magur) and perches (koi, tilapia). It gave positive
result in esculine hydrolysis and negative result in 0129 test.
Chapter 3
MATERIALS AND METHODS
CHAPTER 3
MATERIALS AND METHODS
3.1 Collection and acclimatization of experimental rabbits
Two male rabbits: one, white A of 1.0 Kg and another, white B of 0.90 Kg were bought from
Muktagasa Town Market of Mymensingh district. The animals were apparently healthy with
no detectable signs and symptoms of any disease. They were acclimatized for two weeks
under controlled condition in the experimental animal house of the Department of
Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh, providing
appropriate feed and water and maintaining proper hygienic condition. Acclimatization of
rabbits is shown in plate 1. However they were treated with the antibiotic cotrim at a dose of
0.5 tea spoon/Kg body weight at 12 hours interval for 5 days. Fifteen days after the antibiotic
treatment, rabbits were injected for the experiment.
3.2 Cleaning and sterilization of required glass wares and plastic wares
Experimental glass wares and plastic wares such as syringe, porcelain mortar, glass bottle
eppendorph tubes, conical flask, measuring cylinder, pipettes, petridish, and screw-capped
glass container were treated with 2% sodium hypochliride solution for cleaning. After
overnight soaking in a household dishwashing detergent solution (Trik, Reckitt and Colman
Bangladesh Ltd.) the glass wares were cleaned by brushing and washed thoroughly in
running tap water and rinsed three times with distilled water. The cleaned glass wares were
then dried in a drier at 70°C overnight and sterilized by a dry sterilizer at 160°C for 1.5 h.
The tips for micropipettes were placed in a tip -dispensing box, autoclaved and dried as
above. The appendorph tubes were placed in a straight neck wide-mouth screw-capped glass
container, autoclaved and dried in the same manner. All sterile glasswares and plastic wares
were placed in a clean place.
Plate 1. Acclimatization of rabbits under controlled condition in the experimental
animal house of the Department of Microbiology and Hygiene (A= White
rabbit and B= White rabbit).
A
B
3.3 Preparation of physiological saline:
Physiological saline (PS) was prepared by dissolving 0.85 g of chemically pure sodium
chloride (NaCl) in 100 ml of distilled water in a conical flask. The PS was then sterilized by
autoclaving at 121°C for 15 minutes. After sterilization, the saline was cooled and kept at 4°C
for future use.
3.4 Preparation of phosphate buffered saline
Phosphate buffered saline (PBS) was prepared by dissolving 8 grams of sodium chloride
(NaCl), 0.2 grams of potassium chloride (KCl), 2.89 grams of disodium hydrogen phosphate
(Na2HPO4, 12H2O) and 0.2 grams of potassium hydrogen phosphate (KH2PO4
) in 1000 ml
of distilled water. The pH of the solution was measured by pH meter and adjusted to 7.0-7.2.
The solution was then sterilized and stored as above.
3.5 TSA (triptic soya agar) plate preparation
Required amount (40 g/L) of triptic soya agar (TSA) was weighed and placed in a clean
conical flask. Required amount of distilled water was poured in the agar medium and mixed
well. The mouth of conical flask was covered with aluminum foil. The conical flask was
heated on hot plate and then sterilized by autoclaving at 121°C for 15 minutes. After
autoclaving it was placed in clean chamber until the temperature cooled down to 60°C and
then poured to sterile petridishes at an amount of 20- 30ml. After solidification, the plates
were put in upside down position for at least over night and then kept at 4°C for future use.
3.6 Culture of Aeromonas hydrophila bacteria
Previously collected Aeromonas hydrophila bacteria from fish disease laboratory of
Department of Aquaculture, Bangladesh Agricultural University, Mymensingh and
stored in TSA slants with paraffin oil, were streaked onto TSA plates and incubated at 25°C
for 48 h to observe characteristic colony appearance. Subsequent subcultures were done from
these plates for getting pure culture of the bacteria. Inoculation of inoculating loop is shown
in plate 2.
Plate 2. Sterilization of inoculating loop to inoculate bacteria into culture media.
Fresh TSA agar slants were then prepared as above for future use. A. hydrophila organisms
were freshly cultured to test their morphological, biochemical and physiological
characteristics for reconfirmation of their specific characters.
3.7 Confirmatory test of A. hydrophila
3.7.1 Biochemical tests
Biochemical tests were performed as described below.
3.7.2 Sensitivity to 0129 test
A fresh culture of test organism was obtained. A sterile swab was prepared from the pure
culture. This swab was streaked onto a non-selective blood agar plate (containing 0.5% NaCl)
in three directions to obtain a heavy, confluent growth. Aseptically one 150 µg 0129 disk was
placed onto the agar surface. The plate was incubated aerobically at 35◦
3.7.3 Esculin hydrolysis test
C for 24 h. The zone
of inhibition was observed.
Bile esculin agar base was added with distilled water to bring the volume 1.0 l and mixed
thoroughly. One g esculin was added with esculin agar base, mixed thoroughly and heated
with frequent agitation until boiling. Autoclaving was done at 115ºC for 15 min, cooled at
45◦-50◦
C and distributed onto sterile Petri dishes. Jig jag culture was given onto the prepared
medium from fresh culture. The plate was incubated aerobically at 35˚C for 24 h. Upper
layer of the medium turned blackish colour if the bacteria hydrolyzed esculin.
3.8 Preparation of FKC (formalin killed cell)
For formalin killed cell preparation 100 mg colonies were collected from a freshly culture
plate of A. hydrophila and suspended into sterile PS. One drop of formalin was added to the
10 ml of bacterial suspension (0.5%) washed twice in physiological saline and centrifuged.
The formalin treated suspension was incubated at 37°C for 2 h and 100 µl was plated onto
TSA plate, incubated at 37°C for 1day to observed no growth any bacteria
3.9 Preparation of HKC (heat killed cell)
For heat killed cell preparation, 10ml of such bacterial suspension was allowed to kill by heat
treatment keeping at 60°C for 2.5h in a water bath .After heat treatment; 100µl was incubated
onto a TSA plate and kept at 37°C for 1day to observe no growth of bacteria.
3.10 Antigenic preparation
Antigen was prepared by mixing with 1.5 ml FKC (formalin killed cell) and 1.5 ml FCA
(freund’s adjuvant, complete) in a sterilized porcelain pot and stirred with a grinder until
complete mixing. After, completion of proper mixing this antigenic preparation was stored
for future use.
3.11 Preparing the rabbits for immunization
After collection, 1 ml blood from each rabbit was collected by cutting the ventral peripheral
vein of ear by a new blade and simultaneously adding vaseline as an anti-coagulant and
collecting the blood drops in a test tube. They were then fed with the antibiotic cotrim
suspension at a dose of .5 tea spoon/kg body weight at 12 hours intervals for 5 consecutive
days. After completion of antibiotic dose the rabbits were reared for 15 days to acclimatize
with the animal house condition prior to immunization. The collected blood were centrifuged
at 1000 rpm for 10 minutes, the supernatant sera were separated and subjected to
agglutination tests.
The rabbits were fed with normal diet (grass, vegetables, maize and tube well water).
3.12 Slide agglutination test
Slide agglutination tests were performed for the determination of the presence of specific
antibody against A. hydrophila in the rabbit serum collected from the blood of pre-immune
rabbit or immunized with FKC. One drop of PS and one drop of anti-serum were taken on the
slide and kept at room temperature for two minutes to observe the agglutination reaction.
Clumping of cells on the slide was considered as positive and not clumping of the cells were
negative.
Fig. 1. Slide Agglutination.
3.13 Immunization of rabbit
1.0 ml of adjuvant-mixed-antigen was taken into a sterile syringe for injecting into the white
A rabbit (1.0 Kg body weight). The rabbit was then placed on the table for injecting, furs of
its shoulder were cut out and the skin was disinfected with 70% alcohol cotton. The antigen
was then injected subcutaneously in to five sites in the shoulder of the animal (0.20ml site-1
After 15 days 1 ml blood was collected from ear of each rabbit as above and subjected to
agglutination titration of the serum with the FKC as previously done with pre-immune serum.
Immediately after the collection of blood 1 ml FKC was injected in same way to each rabbit
without mixing with the Freund’s adjuvant, complete as the first booster dose.
).
The White B rabbit (0.9 Kg body weight) was also immunized with 1 ml antigen in the same
way.
One week after the injection of the first booster dose, another 1 ml blood was collected from
each rabbit and second booster doses were given to them. These 1 ml bloods were subjected
to agglutination titration as above to read the efficacy of the sera.
Clotting(Agglutination) one drop of antiserum
one drop of PS
…………..……….
After 1 week of the second booster dose, blood collection, injection of third booster dose and
agglutination titration were done in the same way. Injection of the booster dose is shown in
plate 3.
Plate 3. Booster doses injected in the shoulder of the rabbit.
3.14 Agglutination titration
Ninety six (96) well U-bottomed microtitre plates (Nunck, Japan) were used for the
agglutination titration work to determine the antibody titres of the sera of rabbits. Each well
of the microtiter plate was filled with one drop (10 µl) of sterile PBS. Diluters were heated to
red heat, cooled in air, touched with the upper meniscus of the 20% diluted anti-serum with
PBS and put into the first well of each line (plate 4). They were mixed well with the PBS and
then put in the second well and after mixing well, in the 3rd well. In this way the diluters were
put into the 11th well to make serial dilutions of the 20% serum from the first well up to the
11th well. The 12th well of each line was kept untouched with the serum and designated as the
negative control well. One drop of each of FKC or HKC was then put into each of the 12
wells. Duplicate lines were used for each antigen (FKC or HKC).
Plate 4. Diluter was touched with the upper meniscus of the 20% diluted antiserum with PBS.
The microtiter plate was then wrapped with a plastic wrapper, shaked by a micro shaker for 5
minutes, incubated at 37°C for 2 h and kept at 4°C over night. Next morning the plate was
observed upon a tube light to read the titer optically. The numerical expression of the titer
was as follows. Strength of the agglutination in the first well was expressed as 20, a
numerical expression of the titer of that well and doubling the number for each well up to the
eleventh well (Fig. 2). The right most well showing the agglutination appeared as power of
the antibody (antibody titer). The lower value from the two duplicate lines was accepted.
Agglutination titration is shown in plates 5, 6, and 7.
Titer
20 40 80 160 320 640 1280 2560 5120 12040 24080 Control
well no.
1 2 3 4 5 6 7 8 9 10 11 12
Fig. 2. Agglutination titration. A diagrammatic representation of 96 well
microtiter plate.
Plate 5. Antigen drops were poured into the microtitre plate.
Plate 6. Sterilization of diluter to dilute the antibody in PBS in microtitre plate.
Plate 7. Dilution of antibody in PBS in microtitre plate with the help of diluters.
3.15 Collection and preservation of the rabbit anti-serum After one week of the third booster dose, when the titers rose sufficient enough, the rabbits
were anaesthetized (Plate 8) and sacrificed (Plate 9); total bloods were collected from their
heart into a sterilized plastic syringe (Plate 10) and kept in slanted position for one hour at
room temperature (Plate 11). After the blood became clotted, the antiserum was collected by
a pipetter in centrifuge tubes. After centrifugation at 1000 rpm for 15 minutes, the serum was
separated from the RBC and heated at 58°C for half an hour to inactivate the complements.
Such prepared complement inactivated anti-Aeromonas hydrophila rabbit serum was then
allocated to 1 ml in each sterile eppendorph tube and stored at 20°C for future use.
Prepared anti-Aeromonas hydrophila rabbit serum is shown in plate 12.
Plate 8. The rabbit was anaesthetized.
Plate 9. The rabbit was sacrificed.
Plate 10. Blood were collected from the heart of rabbit by sterilized plastic syringe.
Plate 11. Blood were kept in slanted position for an hour at room temperature.
Plate 12. Prepared anti-Aeromonas hydrophila rabbit serum.
Chapter 4
RESULTS
CHAPTER 4
RESULTS
4.1 Characteristics of Aeromonas hydrophila
Results of the morphological, bio-chemical, and physiological characters of Aeromonas
hydrophila compared with the characters by Popoff et al. (1984) and Sabur (2006) are shown
in Table 1.
4.2 Status of pre-immune serum
Results of slide agglutination tests of the pre-immune rabbit serum with FKC and HKC of
Aeromonas hydrophila showed that no agglutination was occurred (Table 2).
4.3 Slide agglutination results of immune sera
Results of slide agglutination at 15 day-, 21 day-, 28 day- and 36 day- post immunization of
the rabbit serum against FKC and HKC of A. hydrophila by 20 fold, 10 fold, and 100% of the
serums are shown in Table 3. At 15 day post-immunization the undiluted antiserum
agglutinated against both FKC and HKC of A. hydrophila but neither of the 10 fold and 20
fold diluted antisera agglutinated. At 21 day post-immunization and having first booster dose
at 15 day post-immunization the undiluted as well as 10 fold diluted antiserum agglutinated
against FKC as well as HKC. However, 20 fold diluted antiserum did not agglutinate. At 28
day and 36 day of post-immunization and after receiving 2nd and 3rd booster dose
respectively, all the 10 fold and 20 fold diluted antisera agglutinated against both of the FKC
and HKC.
Table 1. Characteristics of Aeromonas hydrophila bacteria in comparison to Popoff
(1984) and Sabur (2006)
Characters Characterization by Popoff et al. (1984)
Characterization by Sabur (2006) Present result
Gram stain _ _ _
Shape Rod Rod Rod Motility + + + Sensitivity to 0129 ND ND - Oxidase + + + Catalase + + OF test F F F Acid and gas production from Glucose
+ + +
Acid production from
Lactose + + + Sucrose + + + Maltose + + + Manitol + + - Inositol - - - Sorbitol - - - Rhamnose - - - Esculin hydrolysis ND ND + Methyl-red test - - - Voges-Proskaur + + + Indole + + + H2 + S production + Arginine decomposition + + +
Lysine decarboxilation - - _
Ornithine decarboxilation - - _
Citrate utilization + + + Growth at: 4ºC - - - 5ºC + + + 37°C + + + 40°C - - -
- : Negative + : Positive F : Fermentative ND : Not done
Table 2. Slide agglutination test results of pre-immune rabbit serum with FKC and
HKC of Aeromonas hydrophila
Antigen Agglutination of Serum
20-fold diluted 10-fold diluted Undiluted serum
FKC - - -
HKC - - -
- : No agglutination
Table 3. Results of slide agglutination tests of immune serum after different days of
postimmunization with FKC and HKC of Aeromonas hydrophila bacteria
Post immunization
days Antiserum
Agglutination
FKC HKC
15
20-fold - -
10-fold - -
100% + +
21
20-fold - -
10-fold + +
100% + +
28
20-fold + +
10-fold + +
100% + +
36
20-fold + +
10-fold + +
100% + +
- : No agglutination
+ : Agglutination was observed
4.4 Agglutination titer of immune rabbit serum
The results of the agglutination titration of immune rabbit serum with FKC and HKC of A.
hydrophila at 15 day-, 21 day-, 28 day-, and 36 day- post-immunization are shown in Table 4.
At 15 day post-immunization the titers of the antiserum from white A rabbit were 80 with
FKC and 40 with HKC of A. hydrophila. From white B rabbit, they were 80 with FKC and 40
with HKC. At 21 day post-immunization and after receiving 1st
At 28 day post immunization and after receiving 2
booster dose at 15 day post-
immunization, the titers of the antiserum from the white A rabbit were 640 with FKC of A.
hydrophila and 80, with HKC. From the white B rabbit, they were 320 with FKC and 160
with HKC.
nd
At 36 day post immunization and after receiving 3
booster dose at 21 day post
immunization the titers of the antiserum from the white A rabbit were 1280 and 160 with
FKC and HKC respectively whereas, the titers from white B rabbit were 1280 with FKC and
320 with HKC.
rd
booster dose at 28 day post
immunization the titers of the antiserum were 1280 and 320 with FKC and HKC of A.
hydrophila respectively; and the titers of the antiserum from the white B rabbit were 1280
and 640 with FKC and respectively. HKC showed lower titer than the FKC.
Table 4. Distribution of agglutination titer of the anti- Aeromonas hydrophila rabbit
serum against HKC and FKC of homologous bacteria
Post-immunization
days of blood
collection from rabbit
Titer
FKC HKC
White A White B White A White B
15 days 80 80 40 40
21 days 640 320 80 160
28 days 1280 1280 160 320
36 days 1280 1280 320 640
FKC = formalin-killed cells HKC = heat-killed cells
4.5 Anti A. hydrophila rabbit serum
In total 38 ml blood could be collected from two rabbits. After centrifugation, 17 ml serum
could be collected from the rabbits.
Chapter 5
DISCUSSION
CHAPTER 5
DISCUSSION
Serology and serodiagnosis of bacterial fish pathogens have contributed a lot to fish health
management in recent years. A wide variety of serological procedures have been increasingly
used to provide diagnostic techniques for fish pathogens, detect antibodies against specific
pathogens in fish sera, evaluate vaccines, and establish serological relatedness of strains of
bacterial fish pathogens.
From the view point of developing an effective vaccine against A. hydrophila infection, the
more bacterial serotype present, both locally and internationally, the less the chance there is
of developing an effective vaccine. Also, a vaccine containing many serotypes will be more
expensive to produce. For the vaccine to be effective in the prevention of motile Aeromonas
septicemia (MAS), more work is needed to clarify the antigenic types of A. hydrophila from
different geographical regions. Most disease outbreaks caused by A. hydrophila are currently
confirmed from bacterial culture after isolation of the pathogen from infected fish. This can
be time consuming and is often unsuccessful since the bacterium is particularly fastidious.
The results of the characterization done by Popoff et al. (1984), Sabur (2006) and during the
present study were same.
No agglutination of pre-immune serum with the A. hydrophila antigen proved that the rabbits
had no previous A. hydrophila contamination. Majumder (2006) also found the similar results
during his immunization work on locally available white and black rabbits.
Slide agglutination test results at different days of post-immunization proved that the
antibody started to be concentrated in the serum of experimental rabbits. At 15 days post-
immunization little antibody production in-vivo in the rabbit’s serum was evident by positive
agglutination of 100% serum but negative results of 10 and 20 fold dilutions. From the 4th
week 20 fold diluted antiserum started showing positive agglutination result explaining its
considerable concentration in the blood of the immunized rabbit. Mamnur Rashid et al.
(1994b) found the similar results during their immunization work on New Zealand white
rabbit with Edwardsiella tarda bacteria. Majumder (2006) also found the similar result
during his immunization work on locally available white and black rabbits with Aeromonas
hydrophila.
Results of the agglutination titration of presently prepared Aeromonas hydrophila rabbit
serum against FKC and HKC cells of homologous bacteria showed that the titers increased
with the post inoculation days which rose at their peaks during the 5th week of immunization
and after three consecutive booster doses at weekly intervals starting from 2nd
Although some interesting serological works have been done for different pathogen in
different countries like Mamnur Rashid et al., (1994a,b), Guz (2001), Hua et al. (2002), Okbi
et al. (2005), Fei et al. (2007) and Mali et al. (2007), before Majumder (2006) no such work
had ever been done with Aeromonas hydrophila isolates in Bangladesh.
week of
immunization. However, HKCs always showed lower titers than the FKCs of both the rabbit
as also shown by Mamnur Rashid (1997) and Majumder (2006) which was in confirmation of
the theory of Brock and Medigan (1994). FKC contained more antigen than HKC because
heat destroyed some of the protein.
For a fruitful contribution towards the vaccination steps against A. hydrophila, serotyping of
different isolates of the homologous bacteria from different environmental condition of
Bangladesh aquaculture including those of Sabur (2006) has become an important step from
the result of the present work. Moreover, simultaneous sero-diagnosis of suspected
Aeromonas fish pathogen should also be done with the present anti Aeromonas hydrophila
rabbit serum.
Thenceforth, vaccination trial can be done with the condition of serological homogeneity of
all suspected isolates of Aeromonas hydrophila from MAS suspected fishes.
Chapter 6
SUMMARY AND CONCLUSION
CHAPTER 6
SUMMARY AND CONCLUSION
The experiment was conducted to prepare antibody against Aeromonas hydrophila bacteria in locally
available rabbits, both are white. The rabbits were bought from Muktagasa, Mymensingh.
Aeromonas hydrophila bacteria were collected from the stock of Fish Disease Laboratory of the
Department of Aquaculture, Bangladesh Agricultural University, Mymensingh. Confirmation was
done for proper identification of A. hydrophila bacteria. After confirmatory test A. hydrophila bacteria
were freshly cultured on to TSA plates and their formalin killed cells (FKC) and heat killed cells
(HKC) were prepared by adding 0.5% formalin and undergoing 2.5 h heat treatment of their
suspension respectively.
Slide agglutination tests were performed for the determination of the presence of specific antibody
against A. hydrophila in the rabbit serum collected from the blood of pre-immune rabbit or
immunized with FKC and HKC. No clumping of cells with pre-immune rabbit serum confirmed that
the rabbits did not have any Aeromonas hydrophila contamination.
Rabbits were injected subcutaneously with the FKC added with same amount of Freund’s complete
adjuvant. Three booster doses were given without the adjuvant at weekly intervals of first injection.
Strength of the anti-serum was tested by slide agglutination test and agglutination titration.
After five weeks, when the antibody titer rose to 1280 to 640 the rabbits were sacrificed, blood was
collected, centrifuged, complement inactivated at 58˚C, stored in appendorph tubes and preserved at -
20˚C for future use.
Disease is one of the most constraining factors in the aquaculture practices in Bangladesh. Rapid
diagnosis of fish disease is prerequisite for successful aquaculture operation. This study was
performed to produce anti-Aeromonas hydrophila rabbit serum. This serum can be used as a rapid
diagnostic tool for the identification of Aeromonas hydrophila which causing motile Aeromonas
septicemia (MAS) disease in Bangladesh Aquaculture. Next important task is to serotype all
Aeromonas hydrophila isolates of Bangladesh to understand their serological relatedness. In the
condition of their serological homogeneity, vaccination trails can be performed against MAS.
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