the molecular biology of pasteurella multocida

23
The molecular biology of Pasteurella multocida Meredith L. Hunt a,* , Ben Adler a , Kirsty M. Townsend b a Bacterial Pathogenesis Research Group, Department of Microbiology, Monash University, Clayton Vic. 3168, Australia b Veterinary Pathology and Anatomy, School of Veterinary Science & Animal Production, The University of Queensland, St Lucia Qld 4072, Australia Abstract Pasteurella multocida is an important veterinary and opportunistic human pathogen. The species is diverse and complex with respect to antigenic variation, host predeliction and pathogenesis. Certain serological types are the aetiologic agents of severe pasteurellosis, such as fowl cholera in domestic and wild birds, bovine haemorrhagic septicaemia and porcine atrophic rhinitis. The recent application of molecular methods such as the polymerase chain reaction, restriction endonuclease analysis, ribotyping, pulsed-field gel electrophoresis, gene cloning, characterisation and recombinant protein expression, mutagenesis, plasmid and bacteriophage analysis and genomic mapping, have greatly increased our understanding of P. multocida and has provided researchers with a number of molecular tools to study pathogenesis and epidemiology at a molecular level. # 2000 Elsevier Science B.V. All rights reserved. Keywords: Pasteurella multocida; Molecular biology; Genetics 1. Introduction Over a century has passed since the first attempts by Louis Pasteur at immunisation against infection with the Gram negative facultative bacterium, Pasteurella multocida, the organism which bears his name. During this time, considerable research into the mechanisms of immunity, host predilection, virulence and pathogenesis of P. multocida has resulted in only very small increases in our understanding of the organism. Safe and effective vaccines against pasteurellosis are still lacking, and until recently there had been no extensive characterisation of this organism at the molecular level. The lack of genetic tools for use in P. multocida hindered investigations at a time when great inroads were Veterinary Microbiology 72 (2000) 3–25 * Corresponding author. 0378-1135/00/$ – see front matter # 2000 Elsevier Science B.V. All rights reserved. PII:S0378-1135(99)00183-2

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Page 1: The molecular biology of Pasteurella multocida

The molecular biology of Pasteurella multocida

Meredith L. Hunta,*, Ben Adlera, Kirsty M. Townsendb

aBacterial Pathogenesis Research Group, Department of Microbiology,

Monash University, Clayton Vic. 3168, AustraliabVeterinary Pathology and Anatomy, School of Veterinary Science & Animal Production,

The University of Queensland, St Lucia Qld 4072, Australia

Abstract

Pasteurella multocida is an important veterinary and opportunistic human pathogen. The species

is diverse and complex with respect to antigenic variation, host predeliction and pathogenesis.

Certain serological types are the aetiologic agents of severe pasteurellosis, such as fowl cholera in

domestic and wild birds, bovine haemorrhagic septicaemia and porcine atrophic rhinitis. The recent

application of molecular methods such as the polymerase chain reaction, restriction endonuclease

analysis, ribotyping, pulsed-®eld gel electrophoresis, gene cloning, characterisation and

recombinant protein expression, mutagenesis, plasmid and bacteriophage analysis and genomic

mapping, have greatly increased our understanding of P. multocida and has provided researchers

with a number of molecular tools to study pathogenesis and epidemiology at a molecular level.

# 2000 Elsevier Science B.V. All rights reserved.

Keywords: Pasteurella multocida; Molecular biology; Genetics

1. Introduction

Over a century has passed since the ®rst attempts by Louis Pasteur at immunisation

against infection with the Gram negative facultative bacterium, Pasteurella multocida, the

organism which bears his name. During this time, considerable research into the

mechanisms of immunity, host predilection, virulence and pathogenesis of P. multocida

has resulted in only very small increases in our understanding of the organism. Safe and

effective vaccines against pasteurellosis are still lacking, and until recently there had been

no extensive characterisation of this organism at the molecular level. The lack of genetic

tools for use in P. multocida hindered investigations at a time when great inroads were

Veterinary Microbiology 72 (2000) 3±25

* Corresponding author.

0378-1135/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.

PII: S 0 3 7 8 - 1 1 3 5 ( 9 9 ) 0 0 1 8 3 - 2

Page 2: The molecular biology of Pasteurella multocida

being made into understanding the molecular basis of pathogenesis in many other

bacterial pathogens.

Substantial progress towards a better understanding has been recently made, with a

number of groups studying different aspects of the molecular biology of P. multocida.

This review brings together the current molecular knowledge of P. multocida, including

detection and identi®cation by polymerase chain reaction (PCR), molecular epidemiol-

ogy, cloning and characterisation of individual genes, genome mapping, extrachromo-

somal elements, tools for genetic manipulation and methods for mutagenesis.

2. Molecular identi®cation and differentiation of P. multocida

Since its ®rst isolation in 1881, detection, identi®cation and characterisation of

P. multocida has relied on the ability to cultivate or purify the organism in the laboratory.

The puri®ed organism is subsequently classi®ed according to phenotypic traits such as

morphology, carbohydrate fermentation patterns and serological properties. However,

culture conditions can in¯uence the expression of these attributes thus diminishing the

stability and reliability of phenotypic methods for strain identi®cation (Matsumoto and

Strain, 1993; Jacques et al., 1994). In recent years, identi®cation and characterisation has

favoured analyses that re¯ect one of the most fundamental properties of an organism, its

genetic information. Genotypic characterisation possesses versatility surpassing that of

traditional phenotypic methods, as nucleic acid analyses facilitate identi®cation and rapid

detection of an organism, determination of its taxonomic position, and investigation of

intra-species genetic relationships.

Molecular approaches such as DNA hybridisation and nucleic acid ampli®cation have

allowed bacterial detection directly from clinical samples, dramatically reducing the time

required for identi®cation. While such methods have had the greatest impact on the

detection of organisms that are generally dif®cult or slow to cultivate in the laboratory,

molecular technology has also signi®cantly in¯uenced the identi®cation and character-

isation of P. multocida.

2.1. Detection and identi®cation of P. multocida by speci®c PCR assays

Since the initial development of the PCR in 1985, the basic principle of in vitro nucleic

acid ampli®cation through repetitive cycling has had extensive applications in all aspects

of fundamental and applied clinical science (Rapley et al., 1992). The PCR method is no

longer used simply as a tool for the rapid production of large quantities of a de®ned target

sequence. The technique now plays a critical role in the clinical laboratory, as rapid and

speci®c detection of microorganisms has provided remarkable advances in the diagnosis

of infectious agents, particularly in cases where the presence of an organism has

signi®cance (Relman and Persing, 1996). Modi®cations to sample preparation have

allowed PCR analysis to be performed on clinical specimens, dramatically reducing the

time required for bacterial identi®cation. Furthermore, the incorporation of multiplex

PCRs can yield detailed information with regards to diagnosis, pathogenesis or antibiotic

resistance, if relevant oligonucleotide primers are available.

4 M.L. Hunt et al. / Veterinary Microbiology 72 (2000) 3±25

Page 3: The molecular biology of Pasteurella multocida

2.1.1. P. multocida-speci®c PCR assays

To date, only three areas of target speci®city have been addressed through the use of

PCR in P. multocida identi®cation. The most fundamental of these, namely a species-

speci®c PCR assay, was developed only recently for detection of P. multocida in mixed

cultures or clinical samples (Kasten et al., 1997a; Townsend et al., 1998a). Two distinct

approaches were used for the development of a P. multocida-speci®c PCR, one rational

and one fortuitous. Kasten et al. (1997a) described the use of oligonucleotide primers

constructed to amplify the psl gene encoding the P6-like protein (Psl) of P. multocida.

This gene demonstrates signi®cant similarity to the P6 protein of Haemophilus in¯uenzae

(Nelson et al., 1988) and H. parain¯uenzae (GenBank accession No. D28887). As

H. in¯uenzae is normally not isolated from poultry and negative results were obtained

with a wide range of avian pathogens, it was postulated that ampli®cation of this gene

could serve as a basis for P. multocida-speci®c detection (PCR-H). However, the

omission of reference strains from the genus Pasteurella sensu stricto is of some concern,

with unknown detection of all P. multocida subspecies and those species present in

the respiratory tract of healthy birds (P. langaa and P. volantium). With this in mind,

the speci®city of the PCR-H assay for P. multocida detection, in poultry or otherwise,

will remain questionable until ampli®cation in other Pasteurella species has been

examined.

It was also shown that neither mouse inoculation nor the PCR assay accomplished total

detection among infected ¯ocks. However, increased detection by PCR may be possible

with further optimisation of the sample preparation procedure. Another disadvantage of

this technique is that to achieve the maximum sensitivity of 10 organisms, additional

hybridisation with psl is required. While PCR technology is being increasingly

incorporated in laboratories around the world, hybridisation is still usually possible only

in specialised laboratories.

The PM-PCR developed by Townsend et al. (1998a) demonstrated a sensitivity of

less than 10 organisms (Lee et al., 1999) without the need for additional hybridisation.

This assay is based on the ampli®cation of a DNA sequence unique to P. multocida

(KMT1) that was isolated by subtractive hybridisation (Townsend et al., 1998a). While

the sensitivity of this assay is high, the speci®city is decreased slightly by the

ampli®cation of Pasteurella canis biovar 2, once recognised as an atypical P. multocida.

Indeed, 16S rRNA sequencing indicates that the two species are closely related (Dewhirst

et al., 1993). As species within Pasteurella sensu stricto other than P. multocida and

P. avium were not examined by Kasten et al. (1997a), it is not known whether P. canis

biovar 2 will remain positive with the PCR-H assay. DNA:DNA hybridisation

studies support the recognition of P. canis biovar 2 as a distinct species (Mutters et al.,

1985).

While false positives may occur with some isolates from pneumonic cattle and pigs

with the PM-PCR, P. multocida subspecies and P. canis biovar 2 can be distinguished on

the basis of indole and mannitol fermentation. Optimisation of the PM-PCR using regions

surrounding the KMT1 sequence may improve the speci®city of the assay, although this

requires further examination. Although some disadvantages in speci®city are evident in

both assays, the assay derived by (Townsend et al., 1998a) is more versatile as additional

hybridisation is not required for optimal sensitivity.

M.L. Hunt et al. / Veterinary Microbiology 72 (2000) 3±25 5

Page 4: The molecular biology of Pasteurella multocida

2.1.2. PCR identi®cation of haemorrhagic septicaemia (HS)-causing serogroup B P.

multocida

Two independently isolated gene sequences unique to HS-causing seroproup B P.

multocida (Brickell et al., 1998; Townsend et al., 1998a) have been utilised in the

development of serogroup B-speci®c PCR assays. Comparative analysis with the H.

in¯uenzae Rd genome indicates that the DNA regions ampli®ed by Townsend et al.

(1998a) and Brickell et al. (1998) are potentially located within about 1 kb of each other.

However, this association has not been examined. Despite the implied close proximity of

these sequences, slight variation in speci®city is evident between the two assays.

To date, the HSB-PCR developed by Townsend et al. (1998a) remains speci®c for HS-

causing serogroup B P. multocida. Serogroup B cultures with the predominant somatic

antigen being either serotype 2 or 5 are speci®cally identi®ed by the ampli®cation of a

�620 bp fragment by the KTSP61 and KTT72 primers. These primers have recently been

employed for detection of HS-causing P. multocida from swine tonsil swabs with no

evidence of non-speci®c ampli®cation (Townsend et al., 1999). In contrast, while the PCR

assay developed by Brickell et al. (1998) demonstrated reasonably speci®c ampli®cation

of serogroup B isolates with HS signi®cance, ampli®cation was also observed with one of

the two serogroup E P. multocida isolates analysed.

Interestingly, both HS-causing serogroup B-speci®c PCR assays were developed by the

fortuitous isolation of DNA sequences unique to these serotypes. With the description of

the serotype A:1 (Chung et al., 1998) and serotype B:2 (Boyce et al., 1999) capsule

biosynthetic loci, rationally derived serogroup A- and B-speci®c PCR assays may be

developed in the near future. However, it should be noted that any PCR assay constructed

from the capsule biosynthetic locus will in theory, identify all organisms within that

capsular type. The speci®city will not extend to the somatic type until type-speci®c

regions have been identi®ed within the P. multocida homologue of the rfb (O-antigen

biosynthetic) gene cluster.

2.1.3. PCR detection of toxigenic P. multocida

The application of PCR technology for P. multocida identi®cation was ®rst reported in

1994 when primers constructed from the sequence of the toxA gene (encoding the

dermonecrotic toxin implicated in progressive atrophic rhinitis) were used to detect

toxigenic P. multocida strains (Nagai et al., 1994). Subsequent PCR assays have been

developed for the direct analysis of toxigenic P. multocida without additional

hybridisation for increased sensitivity (Kamp et al., 1996; Lichtensteiger et al., 1996;

Hotzel et al., 1997). The assay described by Kamp et al. (1996) appears to be the most

sensitive and effective method for large-scale analysis of nasal and tonsillar swabs.

However, the simplistic approach of Lichtensteiger et al. (1996) is more appealing for

small studies despite the potential for false positive ampli®cation (Townsend et al., 1999).

Other aberrant ®ndings when using the primers of Lichtensteiger et al. (1996) have been

reported (Amigot et al., 1998). Faint PCR products of the expected size were observed

from samples that were negative by both ELISA and cell culture. It was suggested that

these bands were a result of either low numbers of positive cells not detectable by

other methods, false ampli®cation or contamination with positive DNA. Additional

primer sets within the toxA gene for use in nested or multiplex PCRs may enhance

6 M.L. Hunt et al. / Veterinary Microbiology 72 (2000) 3±25

Page 5: The molecular biology of Pasteurella multocida

the sensitivity and speci®city of the assay, while eliminating the possibility of false

positive ampli®cation.

2.2. Detection of toxigenic P. multocida by colony hybridisation assays

A colony lift-hybridisation assay using a commercially available multicolour detection

kit was recently developed for rapid, highly sensitive and simultaneous detection of

toxigenic P. multocida and Bordetella bronchiseptica (Register et al., 1998). The major

advantage of this assay is the ability to screen the primary isolation plate for suspect

colonies, removing the need for pure cultures and allowing rapid analysis of large numbers

of samples. However, it was noted that while biotinylated probes are suggested for use

with the Genius Multicolor Detection Kit (Boehringer Mannheim), false positive reactions

have been shown to occur with biotinylated bacterial proteins in colony lift-hybridisation

assays (Register, 1998). It is, therefore, essential that biotin is not substituted for digoxigenin

and ¯uoroscein in the preparation of the labelled probe for this assay.

2.3. Molecular characterisation of P. multocida

Repeated efforts have been made to classify the P. multocida species according to

phenotypic attributes such as serological antigen presentation (capsular and somatic) and

biochemical fermentation pro®les. However, these techniques provide limited character-

isation and insuf®cient information for epidemiologic studies of P. multocida (Wilson

et al., 1992). The ability to differentiate phenotypically similar isolates is critically important

in epidemiology, particularly when establishing the identity of bacterial vaccine strains

(Stull et al., 1988). The development of DNA-based techniques has provided alternative

methods of characterisation that overcome the limitations of phenotyping, while identifying

precisely individual strains of closely related bacteria (Owen, 1989). During the last decade,

genomic characterisation techniques have supplemented or replaced traditional typing

methods for the discrimination of isolates from a wide range of bacterial pathogens (Tenover

et al., 1995). Initially, the equipment and reagents required for molecular characterisation

were expensive, and few laboratories were capable of performing these `complicated'

procedures (Wachsmuth, 1986). In recent years, technique optimisation and the increased

availability of equipment have allowed such methods to be incorporated on a routine basis

in most laboratories throughout the world.

Molecular characterisation, or DNA ®ngerprinting as we know it today, encompasses a

large range of methods with variable speci®city and discriminatory power, most of which

have been used to differentiate phenotypically similar P. multocida isolates. The

application of these techniques to P. multocida epidemiology has generated a large

number of publications, many of which could not be reviewed in detail. However,

selected references are reviewed that illustrate areas in which the application has had a

major impact.

2.3.1. Restriction endonuclease analysis

Restriction endonuclease analysis (REA) has proved to be a valuable component of

bacterial epidemiologic studies, particularly in investigations of outbreaks of pasteur-

M.L. Hunt et al. / Veterinary Microbiology 72 (2000) 3±25 7

Page 6: The molecular biology of Pasteurella multocida

ellosis. This method is a highly reproducible technique that is not in¯uenced by the

inconsistent expression of phenotypic traits that limit the sensitivity and speci®city of

conventional typing methods (Snipes et al., 1989). The use of REA, either solely or in

conjunction with ribotyping (see below), can provide sensitive, distinctive banding

pro®les capable of differentiating isolates of similar serotype. Several restriction enzymes

have been used for DNA ®ngerprinting of P. multocida isolates by REA, with HhaI and

HpaII yielding the most informative and easily distinguished pro®les from a wide range

of serotypes (Wilson et al., 1992, 1993; Blackall et al., 1995, 1996; Diallo et al., 1995).

Wilson et al. (1992) demonstrated a high level of differentiation among the

P. multocida somatic reference serotype strains, with each of the 16 serotypes producing

a unique HhaI ®ngerprint pro®le. Such discrimination was also seen within 71 capsular

serogroup B isolates, with 20 HhaI pro®les evident among eight somatic serotypes.

Thirteen of these were recognised among 54 isolates possessing classic HS-causing

P. multocida serotypes (B:2, B:5 or B:2,5), with several instances where isolates of

similar serotype exhibited distinct DNA ®ngerprints.

However, the discriminatory power of REA with HhaI observed among avian and

serogroup B P. multocida did not extend to serogroup E isolates, in which a single unique

HhaI pro®le was shown among 13 E:2 strains (Wilson et al., 1992). Minor pro®le

differences were demonstrated following HpaII digestion, illustrating the potential

requirement for multiple REA typing to characterise fully some P. multocida isolates.

Isolates from outbreaks of septicaemic pasteurellosis in elks have been characterised

by HhaI and HpaII DNA ®ngerprinting, and compared with pro®les previously

established in HS and septicaemic pasteurellosis strains (Wilson et al., 1995). All 70

isolates from the 1987 and 1993 outbreaks were identi®ed as serotype B:3,4 and shown to

exhibit a single common DNA pro®le, regardless of whether HhaI or HpaII endonuclease

was used. These ®ndings indicated that P. multocida isolated from these outbreaks was

endemic to the National Elk Refuge, and clearly distinct from other HS and septicaemic

pasteurellosis isolates of similar serotype.

Considerable genetic heterogeneity has been observed among swine isolates, allowing

detailed epidemiological studies to be performed (Buttenschùn and Rosendal, 1990; Zhao

et al., 1992; Gardner et al., 1994). DNA pro®ling using BamHI digests of paired isolates

of P. multocida from swine lungs and kidneys indicated that despite the high genetic

diversity among serogroup A isolates associated with bronchopneumonia, genotypic

identity was observed among strains isolated from different sites of the same animal

(Buttenschùn and Rosendal, 1990). It was concluded that P. multocida isolated from

kidney lesions represented blood borne dissemination from primary bronchopneumonic

lesions. SmaI ®ngerprinting results of P. multocida associated with progressive atrophic

rhinitis (PAR) supported the hypothesis that a common infectious source exists in

Australian swine herds (Gardner et al., 1994) and that PAR in Australian herds was

associated with the importation of breeder pigs.

The high level of discrimination exhibited by REA has allowed investigations into the

vaccinal safety and ef®cacy of the two principal live attenuated fowl cholera vaccines

(M9 and CU) currently in use in turkeys in the US (Snipes et al., 1990; Wilson et al.,

1993). REA of somatic serotype 3,4 P. multocida isolates from M9-vaccinated and

unvaccinated turkey ¯ocks using the enzyme SmaI revealed eight distinct pro®les that

8 M.L. Hunt et al. / Veterinary Microbiology 72 (2000) 3±25

Page 7: The molecular biology of Pasteurella multocida

were con®rmed by ribotyping (Snipes et al., 1990). It was shown that the pro®le observed

for the M9 vaccine strain was uncommon among unvaccinated turkeys, yet the majority

of isolates from M9-vaccinated ¯ocks possessed the M9 ®ngerprinting pro®le.

Kim and Nagaraja (1990) were able to demonstrate further subtle differences between

the CU (Clemson University) and M9 (a slow-growing variant of CU) strains following

REA of BglII-digested DNA. Several ®eld isolates were shown to possess DNA pro®les

similar to those of either CU or M9 (Kim and Nagaraja, 1990; Wilson et al., 1993),

although the vaccination history of the birds from which the isolates were obtained was

unknown. Once it is ®rmly established that the DNA pro®le of either M9 or CU is

uncommon among unvaccinated birds, REA using HhaI or BglII could become a valuable

tool to assess the level of mortality in fowl cholera outbreaks due to these vaccine strains.

2.3.2. Ribotyping

The banding patterns produced by REA are often complex, making visual

interpretation of results dif®cult. Ribotyping, like REA, utilises restriction enzyme

digestion of genomic DNA and agarose gel electrophoresis for DNA fragment separation.

The additional use of Southern blotting and hybridisation with a labelled DNA probe

reduces the complexity of the restriction patterns, and highlights restriction fragment

length polymorphisms (RFLPs) within the bacterial genome without the computer

analysis that is sometimes required.

Ribosomal RNA (rRNA) molecules are highly conserved, ubiquitous molecules that

constitute the major proportion of RNA in the bacterial cell (Grimont and Grimont,

1986). As rRNA operons vary in copy number and genomic location between strains and

species, DNA probes speci®c for rRNA gene sequences can be used to identify RFLPs

within and/or around the ribosomal operon, thus providing the basis for bacterial strain

differentiation.

Ribotyping, in conjunction with REA, has been used to characterise successfully and

differentiate avian (Snipes et al., 1989; Blackall et al., 1995) and porcine (Zhao et al.,

1992; Gardner et al., 1994) isolates of P. multocida. Ribotyping of avian P. multocida

isolated from fowl cholera outbreaks in turkeys in Australia (Blackall et al., 1995) and the

US (Carpenter et al., 1991) has demonstrated considerable genomic heterogeneity,

providing suf®cient evidence to discount the relatedness of outbreaks previously

indistinguishable by serotyping and biotyping.

Ribotyping of HS-causing isolates of P. multocida demonstrated increased discrimina-

tion among strains of similar serotype than ®eld alternation gel electrophoresis (FAGE),

but more inconsistencies within classi®cations were observed (Townsend et al., 1997a). It

appeared that the limited view of RFLPs within or around the ribosomal operon

occasionally compounded the confusion of the phenotypic classi®cations. Ribotyping in

combination with other genotypic methods could provide greater discrimination among

HS-causing isolates, although a distorted view of genetic relatedness could occur if

ribotyping alone is used.

2.3.3. Pulsed-®eld gel electrophoresis (PFGE)

Field alternation electrophoretic methods, more commonly known as pulsed-®eld gel

electrophoresis (PFGE), remain the `gold standard' ®ngerprinting method for molecular

M.L. Hunt et al. / Veterinary Microbiology 72 (2000) 3±25 9

Page 8: The molecular biology of Pasteurella multocida

epidemiology (Goering, 1993), as polymorphisms throughout the chromosome are

examined without the complexity of REA patterns and the restricted view of genetic

variation produced by ribotyping. Detailed reviews of the development and application of

all PFGE methods have been published previously (Dawkins, 1989; Townsend and

Dawkins, 1993).

PFGE analysis has consistently shown greater discrimination in identi®cation and

differentiation of bacterial species than ribotyping (Prevost et al., 1992; Kristjansson

et al., 1994; Townsend et al., 1997a). REA provides comparable discrimination among

isolates; however, the complex banding patterns sometimes require computer software

analysis to achieve de®nitive interpretation and accurate typing (Wilson et al., 1993;

Kristjansson et al., 1994).

While PFGE analysis has become an integral component of bacterial genetics and

molecular epidemiology during the last decade, it has had limited application in the

comparative typing of P. multocida isolates (Donnio et al., 1994, 1999; Blackwood et al.,

1996; Townsend et al., 1997a). The initial study by Donnio et al. (1994) demonstrated

heterogeneity among P. multocida serotype D:2 isolated from the oropharynx of pig

breeders whose livestock had suffered from pasteurellosis, although there was no

indication of the genetic relatedness between P. multocida isolated from the breeders and

their pigs. Recently, characterisation by PFGE of dermonecrotic toxin (DNT)-producing

strains of P. multocida isolated from humans and swine revealed signi®cant DNA

polymorphisms, although no correlation with host species was evident (Donnio et al.,

1999). It was suggested that the lack of discrimination between toxigenic isolates of

porcine and human origin might indicate colonisation of people from a porcine reservoir.

Discrimination between isolates of similar serotype was observed following PFGE

analysis of HS-causing isolates of P. multocida (Townsend et al., 1997a), with some

correlation to geographic location. HS-causing serogroup B isolates from North America

were clearly differentiated from Asian serogroup B strains, with the latter exhibiting a

high degree of homogeneity. The ability of PFGE to demonstrate genetic relatedness, yet

maintain discrimination, was particularly evident after analysis of the North American

isolates. Cultures representing the original Yellowstone Park Buffalo `B' strain

(Gochenour, 1924) and isolations made from horse blood during subsequent passages

(Stein et al., 1949) were indistinguishable by serotyping, protein and ribotyping studies

(Johnson et al., 1991; Townsend et al., 1997a). PFGE analysis demonstrated polymorphic

restriction pro®les clearly identifying each isolate, illustrating the value of this technique

in bacterial molecular epidemiology.

Recently, an extensive study by Gunarwardana et al. (1999) con®rmed the standing of

PFGE as the `gold standard' technique for molecular epidemiology, as it was shown that

PFGE was more discriminatory than repetitive extragenic palindromic PCR (REP-PCR)

for the characterisation of avian P. multocida. However, REP-PCR compared favourably

with PFGE classi®cations, and appears to be an extremely useful technique for

laboratories without the specialised PFGE equipment.

2.3.4. PCR ®ngerprinting

Several reports have detailed the use of PCR ®ngerprinting for the differentiation of P.

multocida isolates (Chaslus-Dancla et al., 1996; Zucker et al., 1996; Schuur et al., 1997;

10 M.L. Hunt et al. / Veterinary Microbiology 72 (2000) 3±25

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Townsend et al., 1997b, 1998b, 1999; Hopkins et al., 1998; Gunarwardana et al., 1999).

Arbitrarily primed PCR (AP-PCR) was effective in discriminating P. multocida isolates

from the respiratory tract of pigs (Zucker et al., 1996) and also post-vaccination isolates

from turkeys (Hopkins et al., 1998). However, the use of a radioactive label by Hopkins

et al. (1998) to increase sensitivity may limit the feasibility of these primers in some

laboratories. The 32P-dCTP procedure will enhance detection of ampli®ed fragments in

organisms with a high percentage of cytosine in their genome. This bias will also limit the

sensitivity of A�T-rich amplimers, but this did not seem to affect the level of

discrimination among avian P. multocida.

Repetitive extragenic palindromic PCR (REP-PCR) ®ngerprinting was recently shown

to compare very favourably with PFGE in discrimination of avian and swine P. multocida

isolates, exhibiting a high level of differentiation (Townsend et al., 1997b, 1998b, 1999;

Gunarwardana et al., 1999). Interestingly, P. multocida isolated from outbreaks of fowl

cholera (FC) and HS in Vietnam demonstrated minimal variation, with a single REP

pro®le observed among serotype A:1 and HS-causing serogroup B isolates, respectively

(Townsend et al., 1998b; Gunarwardana et al., 1999). Analysis of P. multocida isolates

from the tonsils of healthy pigs at slaughter (Townsend et al., 1999) exhibited genetic

heterogeneity, with some indication that healthy pigs may act as a reservoir for

FC-associated serotypes.

3. Genetics of P. multocida

3.1. Metabolic genes

There have been surprisingly few metabolic genes cloned or characterised in P.

multocida. Mock et al. (1991) described the cloning and sequencing of a gene encoding

the P. multocida adenylate cyclase. Not surprisingly, it was similar to its E. coli

homologue with the deduced protein sequence indicating N-terminal catalytic and C-

terminal regulatory domains. The gene could express adenylate cyclase activity in E. coli

and was subject to similar regulatory mechanisms.

The beta-subunit of the P. multocida tryptophan synthase was cloned, with sequence

analysis indicating high levels of similarity to homologues from other Gram negative

bacteria (Jablonski et al., 1996). The trpB gene encodes a protein of 43.6 kDa that

contains the GGGSNA motif involved in binding to pyridoxal phosphate. The P.

multocida trpB was able to complement a de®ned E. coli mutant, thus con®rming

functional expression of the gene in E. coli. The gene encoding the alpha-subunit, trpA,

was located adjacent to trpB.

The galE gene of P. multocida could complement a galE mutant of Salmonella

(Fernandez de Henestrosa et al., 1997), while the GalE protein was most closely related

to its H. in¯uenzae homologue, with which it shared 85% identity. Of particular

importance was the ®nding that a galE mutant of P. multocida constructed by allelic

exchange was attenuated in virulence for mice. A similar mutant complementation

strategy was used to clone the aroA gene of P. multocida and construct an aroA mutant,

which was also attenuated for virulence in mice (Homchampa et al., 1992). Moreover,

M.L. Hunt et al. / Veterinary Microbiology 72 (2000) 3±25 11

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the mutant could induce immunity against subsequent lethal challenge. A marker-free

aroA mutant was shown to cross protect against heterologous serogroup A strains in

mice (Homchampa et al., 1997) and showed vaccine potential in chickens (Scott et al.,

1999).

Cloning of the ®rA and skp genes of P. multocida was reported by Delamarche et al.

(1995), but no further characterisation was carried out. ®rA encodes a glucosamine

transferase (Dicker and Seetharam, 1991) which appears to be involved in the

biosynthesis of Lipid A, while the function of the skp gene remains uncertain. However,

the skp homologue from H. in¯uenzae was recently shown to elicit protective immunity

against infection in an infant rat model (El-Adhami et al., 1999), suggesting that further

investigation into the role of skp in immunity to pasteurellosis is warranted.

Although no restriction endonucleases have been isolated from P. multocida, a

restriction/modi®cation system has been reported (Hoskins and Lax, 1997) in which

unmodi®ed DNA was cleaved at or near PstI sites. It was not characterised further.

3.2. Outer membrane protein (OMP) genes

The P6 OMP of H. in¯uenzae has been shown to elicit protective immunity in animal

models of infection. Kasten et al. (1995) cloned a gene encoding the P. multocida

homologue of P6 and showed it to be present in all 16 somatic serotypes. However,

immunisation of turkeys with recombinant P6 failed to protect them against subsequent

challenge (Kasten et al., 1997b).

The ompH porin of P. multocida was puri®ed and its N-terminus sequenced

(Chevalier et al., 1993). OmpH is a homologue of the P2 porin of H. in¯uenzae and

a monoclonal antibody against OmpH could passively protect mice against infection

(Vas® Marandi and Mittal, 1997). Luo et al. (1997) cloned the ompH gene and showed

experimentally that ompH had porin activity. Immunisation of chickens with the

recombinant mature length ompH elicited immunity against homologous challenge,

although heterologous protection was not investigated. Subsequent analysis of ompH

from different serotypes showed a high degree of conservation and predicted the presence

of two large external loops. A cyclic synthetic peptide which mimicked the predicted

structure of loop 2 was able to induce partial homologous protection in chickens (Luo

et al., 1999).

The gene encoding the Oma87 OMP was cloned and characterised (Ruffolo and Adler,

1996). Oma87 showed high similarity to the D15 protective outer membrane protein of H.

in¯uenzae (Loosmore et al., 1997) and rabbit antiserum against Oma87 was able to

passively protect mice against infection. Oma87/D15 homologues have been subse-

quently identi®ed in a number of Gram negative species including Neisseria (Manning et

al., 1998) and Shigella (GenBank accession No. AAD23568). The protective epitope(s) of

D15 were localised to the N-terminus of the protein (Yang et al., 1998). Immunisation of

chickens with a serogroup D GST-Oma87 fusion protein containing the N-terminal 25%

of Oma87, failed to protect chickens against challenge with a serogroup A:1 strain

(Harper et al., 1999) despite the >95% sequence identity between Oma87 from the two

strains. Further work is required to determine unequivocally the role of Oma87 in

immunity to pasteurellosis.

12 M.L. Hunt et al. / Veterinary Microbiology 72 (2000) 3±25

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3.3. Heat shock proteins

P. multocida produced increased amounts of several proteins at 428C (Love and

Hirsh, 1994). A 60 kDa protein was subsequently shown to be the GroEL homologue

encoded by the groESL operon (Love et al., 1995). Although the regulation of GroES

and GroEL was not investigated, analysis of the upstream sequences suggests that

P. multocida utilises a s32-based system (Bukau, 1993) to upregulate its heat shock genes.

The 70 kDa protein upregulated at 428C was almost certainly DnaK, but it was not

identi®ed.

3.4. Capsule biosynthetic genes

The entire capsule biosynthetic locus of P. multocida A:1 was cloned and sequenced

(Chung et al., 1998). Analysis revealed a typical Gram negative type of organisation

(Boulnois and Roberts, 1990) in which a central Region 2 encoding sugar biosynthetic

genes and glycosyl transferases is bounded by two regions (1 and 3) which encode

transport functions. The four Region 1 genes were homologues of the H. in¯uenzae

bexABCD genes which encode proteins involved in exporting the polysaccharide capsule

to the bacterial cell surface, including the principle ABC transporter BexA (designated

HexA in P. multocida A:1). The two Region 3 genes (phyAB) appeared to be involved in

phospholipid substitution to anchor the capsule in the outer membrane. The ®ve genes

comprising Region 2 encode enzymes for the synthesis and assembly of the serogroup A

hyaluronic acid capsule. One of the genes, hyaD, was identi®ed as the hyaluronic acid

synthase gene reported by DeAngelis et al. (1998).

In contrast, the organisation of the serogroup B capsule locus differed signi®cantly

(Boyce et al., 1999). The four Region 1 genes (cexABCD) together with phyA constitute

Region 1, while the single phyB gene makes up Region 3. Of the nine genes in Region 2,

three were similar to sugar biosynthesis or glycosyl transferase genes. However, the

deduced protein products of the remaining six showed no similarity to any sequences in

the databases. Nevertheless, based on their genetic and transcriptional organisation, it was

suggested that they were involved in capsule biosynthesis. The structure of the serogroup

B capsule remains to be determined. Interestingly, there is a relatively low level of

sequence similarity between the serogroup A and serogroup B homologues, but the genes

¯anking the loci exhibit almost 100% nucleotide sequence identity (Boyce et al., 1999),

suggesting a similar chromosomal location. The cap locus has been mapped in serogroup

A (see below) but not in serogroup B.

3.5. Toxin genes

Strains of P. multocida serogroup D which cause atrophic rhinitis produce a

dermonecrotic toxin (PMT, for P. multocida toxin), which is the principle virulence

factor in atrophic rhinitis. PMT induces localised osteolysis in the nasal turbinates,

primarily through increased osteoclastic bone resorption. Recombinant toxin derivatives

have been used as vaccine candidates (Nielsen et al., 1991; Petersen et al., 1991).

Although toxin related sequences have occasionally been found in other serotypes, the

M.L. Hunt et al. / Veterinary Microbiology 72 (2000) 3±25 13

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synthesis of PMT is usually restricted to serogroup D. The toxA gene was cloned and

shown to express functional PMT in E. coli (Petersen and Foged, 1989). Sequence

analysis of toxA revealed a gene of 4381 bp encoding a protein of 146.5 kDa as well as

the presence of an upstream negative regulator gene, txaR (Petersen, 1990). Deletion of

txaR resulted in a 10-fold increase in toxin expression. However, a subsequent study

found no effect on toxin production following deletion of txaR (Hoskins and Lax, 1996).

Growth at 308C or under iron replete conditions caused less than a four-fold decrease in

the expression of toxA and these authors suggested that PMT is essentially expressed

constitutively.

4. Bacteriophages in P. multocida

Various researchers have demonstrated the presence of bacteriophages in P. multocida.

11 temperate bacteriophages were isolated from a variety of avian P. multocida strains

(Kirchner and Eisenstark, 1956). Bacteriophages were also isolated from bovine strains of

P. multocida, three of which were demonstrated to be genus and species speci®c (Rifkind

and Pickett, 1954; Gadberry and Miller, 1977). Studies on 21 P. multocida bacteriophages

showed a number with morphological similarity to the P2 and T7 coliphages (Ackermann

and Karaivanov, 1984). A set of 24 bacteriophages was recovered after mitomycin

treatment of mainly atrophic rhinitis isolates. These were subsequently used in a proposed

typing system to discriminate between the toxigenic and non-toxigenic strains of P.

multocida (Nielsen and Rosdahl, 1990), but the scheme has not gained widespread

acceptance.

More recently, some sequence similarity to bacteriophage Mu has been found. Regions

with identity to the Mu sequence appear to be present only in the HS causing serogroup B

strains, and have been used to develop a B:2 speci®c PCR (Brickell et al., 1998). None of

the bacteriophages recovered to date have been studied in suf®cient detail for their use as

tools for the genetic manipulation of P. multocida.

5. Native plasmids of P. multocida

Several groups have undertaken studies to determine the presence of plasmids in

numerous strains of P. multocida and to investigate the correlation between antibiotic

resistance pro®les, virulence attributes and the presence of plasmids. The rate of plasmid

carriage has been shown to vary considerably between different P. multocida collections.

Plasmid carriage in avian isolates from two studies varied from 24 (Price et al., 1993) to

70.7% (Hirsh et al., 1985) of isolates tested. Studies with bovine and porcine strains

by Schwarz et al. (1989) found that 47% of strains carried plasmids. A similar ®gure of

51% of porcine serogroup D isolates carrying plasmids was found by Cote et al. (1991).

P. multocida isolated from rabbits demonstrated the highest plasmid carriage rate of

92% of the 28 isolates tested (Gunther et al., 1991).

P. multocida has been shown to harbour plasmids from 1.3 kb (Diallo et al., 1995) to

ca. 100 kb (Hirsh et al., 1989) in size. However the majority of plasmids identi®ed have

14 M.L. Hunt et al. / Veterinary Microbiology 72 (2000) 3±25

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been between 2 and 6 kb in size. Many phenotypically cryptic plasmids have been

found in P. multocida isolated from avian (Hirsh et al., 1989; Price et al., 1993) and

mammalian hosts (Haghour et al., 1987; Cote et al., 1991; Gunther et al., 1991). A

number of R-plasmids have been identi®ed, which confer various antibiotic resistances.

Most R-plasmids were non-conjugative, but could be readily transferred to other

P. multocida strains and E. coli by transformation. Conjugal transfer of a small non-

conjugative R-plasmid in P. multocida has been reported previously, but this required

the presence of a helper fertility plasmid (Hirsh et al., 1981). Subsequently, Hirsh et al.

(1989) identi®ed a large conjugative R-plasmid that was capable of transferring multiple

antibiotic resistance to E. coli and P. multocida.

Many of the R-plasmids carried resistance to streptomycin (Stm) and sulphonamides

(Sul). Cote et al. (1991) found an R-plasmid carrying the Stm and Sul determinants to be

highly similar to the salmonella plasmid RSF1010. Tetracycline (Tet) resistance was

sometimes present with stm and sul genes on a single R-plasmid or carried on a separate

R-plasmid (Silver et al., 1979; Hirsh et al., 1985). Similar R-plasmids from human and

bovine isolates of P. multocida were found to carry the ROB-1 û-lactamase gene and

confer signi®cant resistance to penicillins (Livrelli et al., 1988; Rosenau et al., 1991).

Kanamycin resistance was found in conjunction with Stm, Sul and Tet resistance on the

large conjugative plasmid of an avian strain (Hirsh et al., 1989) and on an R-plasmid from

serogroup D which also carried Stm, Sul and chloramphenicol resistance genes

(Yamamoto et al., 1990). The production of chloramphenicol acetyltransferases

conferring chloramphenicol resistance in P. multocida has also been shown to be

mediated by R-plasmids of 5.1, 5.5 and 17 kb (Vassort-Bruneau et al., 1996).

Plasmid analysis from complement resistant P. multocida (Lee and Wooley, 1995)

demonstrated a correlation between plasmid carriage in the P. multocida CU strain and an

increase in complement resistance, invasiveness and virulence. However, other reports

have found that plasmids were unrelated to virulence (Gunther et al., 1991; Price et al.,

1993; Diallo et al., 1995).

In most cases the antibiotic resistance genes identi®ed on plasmids were not

characterised further; their presence was deduced on the basis of antibiotic resistance

phenotypes. However, the Tet(B) and Tet(M) classes of tetracycline resistance

determinants were detected in two strains (Chaslus-Dancla et al., 1995) with the authors

suggesting that the genes were chromosomally borne. Hansen et al. (1993) identi®ed a

novel, plasmid-borne tetracycline resistance determinant, designated Tet(H), in an avian

strain of P. multocida. Tet(H) belongs to the ef¯ux-mediated class of tetracycline

resistance determinants. In a subsequent study (Hansen et al., 1996), the tet(H) gene

was detected in the majority of a set of North American isolates of P. multocida and

P. haemolytica. Based on the fact that tet(H) was found located both on the chromosome

as well as on plasmids, it was hypothesised that it might be borne on a transposable

element. This was con®rmed by the discovery of the transposon Tn5706 (Kehrenberg

et al., 1998) which was shown to carry the tet(H) gene with its regulator tetR.

Interestingly, its 228 amino acid transposase enzyme was more closely related to

transposases found in Gram positive bacteria. Other transposons have been used for

insertional mutagenesis described below, but Tn5706 remains the only P. multocida

transposable element identi®ed to date.

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6. Vectors for genetic manipulation

Some native P. multocida plasmids have been modi®ed for use as cloning vehicles.

Vectors such as pBAC64 (Bills et al., 1993), pAKA16 (Azad et al., 1994) and their

derivatives and pIG112 (Wright et al., 1997) have been engineered largely for use as

shuttle vectors between E. coli, P. multocida and other members of the Haemophilus,

Actinobacillus and Pasteurella (HAP) group. Other shuttle vectors based on the broad

host range plasmid RSF1010, have also been used in P. multocida (Lee and Henk, 1997).

Tools such as these provide a useful resource for research into the molecular aspects of

pathogenesis.

The conjugative plasmids, pJM703.1 and pGP704 are unable to replicate in host strains

lacking the pir gene product, and as such have been used as suicide vectors in other

bacteria (Miller and Mekalanos, 1988; Herrero et al., 1990). However, when introduced

into serotype A:1 (PBA100) or D:12 (PM25) P. multocida strains, respectively, these

vectors failed to act as suicide replicons (Homchampa et al., 1992; Fernandez de

Henestrosa et al., 1997). These ®ndings suggest that a lambda pir homologue may be

present on the chromosome of P. multocida. Both groups found that these plasmids could

be lost from P. multocida by culturing for 60 to 100 generations without selection.

Alternative suicide vectors that may be useful in P. multocida are those carrying ColE1

replication origins (Nnalue and Stocker, 1989; Azad et al., 1994) or temperature sensitive

derivatives of RSF1010.

7. Mutagenesis of P. multocida

7.1. Transposon mutagenesis

Transposon mutagenesis has the advantage of generating large numbers of random

mutants that can then be tested for any alteration of their virulence phenotype.

Several transposons have been examined in P. multocida with varying degrees of

success. Nnalue and Stocker (1989) found that while Tn5 did not transpose in

P. multocida, transposition of Tn7 was successful. However, Tn7 was found to

insert into a single site in the P. multocida genome, and as such was not practical

for use in the generation of random mutants (Nnalue, 1990). Two transposons of

potential use in P. multocida as tools for mutagenesis are Tn10 and Tn916. Prior to

their use in avian strains of P. multocida, these transposons were shown to be useful

in Actinobacillus and Haemophilus, respectively (Kauc and Goodgal, 1989; Tascon

et al., 1993).

Using a mini-Tn10 vector and delivery by conjugation, insertion of Tn10 into the

P. multocida genome was shown to be random and stable by Lee and Henk (1996).

More recently, Tn916 has been shown to insert in a quasi-random fashion throughout

the genome of P-1059, a serotype A:3 strain (DeAngelis, 1998). Mutants in a region

related to capsule production were thus identi®ed. By sequencing the P. multocida

DNA ¯anking the transposon in one such mutant, DeAngelis (1998) was able to identify

the P. multocida hyaluronan synthase gene. Both authors have foreshadowed the

16 M.L. Hunt et al. / Veterinary Microbiology 72 (2000) 3±25

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application of these transposons for the identi®cation of genes involved in virulence and

pathogenesis.

7.2. Chemical mutagenesis

Agents such as N-methyl-N-nitro-N-nitrosoguanidine and acridinium salts have been

employed by a number of groups attempting to generate P. multocida mutants that are

suf®ciently attenuated for use as live vaccines (Wei and Carter, 1978; Kucera et al.,

1981). Attenuated strains, some displaying temperature sensitivity or streptomycin

dependence, have been isolated and have shown varying degrees of success as live

vaccines (Chengappa et al., 1979; Hofacre et al., 1989). The genetic basis of attenuation

in these mutants is not de®ned and some of these empirically derived vaccine strains have

been implicated in outbreaks of fowl cholera, suggesting a reversion to virulence and

indicating the instability of the mutant phenotype (Hofacre and Glisson, 1986).

7.3. Targeted mutagenesis

A targeted or rational approach to mutagenesis has also been applied for the study of

pathogenesis in P. multocida. A speci®c gene is identi®ed and cloned and then inactivated

either by a deletion of a portion of the gene or by the insertion of a cassette often

encoding antibiotic resistance. This inactivated gene construct is then delivered to the

wildtype organism and reintroduced into the genome via allelic replacement. Isolates

carrying the mutated construct are then selected by virtue of an altered phenotype such as

the resistance to an antibiotic and subsequent genetic analysis to con®rm the mutant

genotypic pro®le.

The aroA and galE metabolic genes have been targets for mutation in P. multocida

using this approach. The aroA mutants PMP1 and PMP3 were derived from the strains

X-73 and P-1059, respectively. They have been shown to be signi®cantly attenuated and

studies illustrate their potential for use as stable, non-reverting, live vaccines against

pasteurellosis (Homchampa et al., 1997). The safety and ef®cacy of PMP1 and PMP3 as

vaccines for use in chickens was recently demonstrated (Scott et al., 1999). High doses of

the live attenuated mutants were administered and provided solid cross-protective

immunity against a serotype 4 strain (PM206). No vaccine associated clinical signs or

lesions were detected and neither mutant strain could be reisolated �24 h after

vaccination. The authors have indicated further work is currently being undertaken to

assess the safety of these live vaccine strains in immunosuppressed birds and to determine

the longevity and extent of heterologous protection provided (Scott et al., 1999).

The galE mutant generated by Fernandez de Henestrosa et al. (1997) was constructed

using a system similar to that of the aroA allelic exchange. An inactivated construct was

introduced using a conjugative plasmid which failed to suicide in the P. multocida D:12

recipient strain. The plasmid was then cured by passaging without antibiotics. The galE

mutant showed reduced virulence when tested in the mouse model. However, before this

strain could be considered as a live attenuated vaccine candidate the authors suggest that

additional mutations in other loci are required to ensure its safety (Fernandez de

Henestrosa et al., 1997).

M.L. Hunt et al. / Veterinary Microbiology 72 (2000) 3±25 17

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8. The genome of P. multocida

8.1. Genome mapping and organisation

A physical and genetic map of an Australian serotype A:1 fowl cholera isolate

(PBA100) was generated by Hunt et al. (1998) using the restriction enzymes ApaI, NotI

and CeuI. This study found the chromosome to be a 2.35 Mb single circular molecule,

with no extrachromosomal elements. Similar sized genomes within the Pasteurellaceae

family have been observed in H. in¯uenzae, 1.8 Mb, (Fleischmann et al., 1995) and

Actinobacillus pleuropneumoniae with sizes ranging between 2.3 and 2.4 Mb (Chevallier

et al., 1998; Oswald et al., 1999). The genetic organisation of P. multocida A:1 strain

PBA100 appeared to have many features common to other bacterial species. A putative

location for the origin of replication was suggested and various genes involved in

transcription were also located in the surrounding regions. Several genes involved in

virulence and immunity in P. multocida and other bacterial pathogens were localised onto

the physical map, such as the type IV ®mbrial subunit gene ptfA, capsule biosynthesis

genes, genes involved in iron acquisition, the skp gene and those encoding outer

membrane porin ompH and the oma87 OMP.

8.2. Organisation of rrn genes

The intron encoded endonuclease CeuI which recognises a 26 bp sequence found

exclusively in the 23S rRNA gene (rrl) (Liu et al., 1993; Toda and Itaya, 1995) was used

to determine the number of rrl genes present in P. multocida. Hunt et al. (1998) found the

rRNA genes of P. multocida to exist as ®ve operons with the gene order rrs-rrl-rrf similar

to that of many other eubacteria. The rrn operons were arranged in two groups being

transcribed divergently away from the oriC region. Single ApaI and CeuI sites were found

in all the rrs and rrl genes, respectively. These restriction enzymes may prove useful for

tracking the number and arrangement of rRNA genes in other P. multocida isolates.

8.3. Genomic diversity

Comparison of rare restriction sites and marker genes between genomic maps of

different species or strains provides one measure of genetic variation at the molecular

level. As no other P. multocida genomic maps are currently available, the genetic map of

P. multocida was compared to that of H. in¯uenzae Rd (Fleischmann et al., 1995). Short

regions of gene order conservation, but no long range co-linearity of gene order, were

found. The genome of A. pleuropneumoniae has recently been mapped (Oswald et al.,

1999). However a comparison to the P. multocida map was not possible due to the low

resolution of these genetic maps.

Genomic heterogeneity within P. multocida has long been recognised from the many

methods used for molecular epidemiology discussed earlier. Avian strains in particular

have been found to exhibit considerable diversity (Blackall et al., 1998). PFGE restriction

pro®le differences have been observed between both ApaI, NotI and CeuI genomic

digests of avian isolates (Hunt et al., 1998; Gunarwardana et al., 1999) As CeuI cleaves

18 M.L. Hunt et al. / Veterinary Microbiology 72 (2000) 3±25

Page 17: The molecular biology of Pasteurella multocida

only in the rrl gene of eubacteria, differences in CeuI restriction pro®les indicate

alterations in the rrn backbone of the chromosome between these strains. Genomic

diversity may have arisen through recombination events such as insertions, deletions,

inversions and duplications or may be due to the large number of separate serotypes being

grouped together in the species. Alternatively the restriction polymorphism seen may be

due merely to point mutations in the rare restriction sites indicating an alteration in the

physical map but no signi®cant alteration of the gene order.

The generation of genetic maps of other P. multocida strains and serotypes to compare

the organisation may help to determine if these restriction polymorphisms relate to a

distinct association between the genome architecture and properties such as virulence,

host speci®city and other metabolic or pathogenic phenotypic aspects.

8.4. Genome sequencing

Sequencing of the P. multocida genome has been undertaken by the Advanced Genetic

Analysis Centre at the University of Minnesota, (http://www.cbc.umn.edu/ResearchPro-

ject/AGAC/Pm/index.html). A valuable database is being constructed from the random

sequencing of an A:3,4 turkey strain. Knowing the sequence of a bacterial pathogen's

genome provides much information about the unique biology of the organism. However,

the genome sequence per se will provide only limited information about the role of

individual genes in pathogenesis. The challenge will be to determine which of the 2000 or

so genes being uncovered by the sequencing project are distinctly involved in virulence,

pathogenesis, host predilection or in the induction of a protective immune response.

Indeed, a number of molecular tools such as transposon mutagenesis discussed above and

microarray (gene chip) technology will be critical in the next wave of P. multocida

research and will elucidate the role of many genes.

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