The molecular biology of Pasteurella multocida

Veterinary Microbiology 72 (2000) 3±25

The molecular biology of Pasteurella multocida Meredith L. Hunta,*, Ben Adlera, Kirsty M. Townsendb 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-®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 *

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

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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 epidemiology, cloning and characterisation of individual genes, genome mapping, extrachromosomal 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 characterisation 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.

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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 speciesspeci®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.

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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 HScausing 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

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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 characterisation 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-

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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

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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 discrimination 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

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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;

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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 Cterminal 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,

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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 subsequently 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.

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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

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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

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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 nonconjugative 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

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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).

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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

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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/ResearchProject/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. References Ackermann, H.W., Karaivanov, L., 1984. Morphology of Pasteurella multocida bacteriophages. Can. J. Microbiol. 30, 1141±1148. Amigot, J.A., Torremorell, M., Pijoan, C., 1998. Evaluation of techniques for the detection of toxigenic Pasteurella multocida strains from pigs. J. Vet. Diagn. Invest. 10, 169±173. Azad, A.K., Coote, J.G., Parton, R., 1994. Construction of conjugative shuttle and suicide vectors for Pasteurella haemolytica and P. multocida. Gene 145, 81±85. Bills, M.M., Medd, J.M., Chappel, R.J., Adler, B., 1993. Construction of a shuttle vector for use between Pasteurella multocida and Escherichia coli. Plasmid 30, 268±273. Blackall, P., Pahoff, J., Fegan, N., Marr, G., 1996. Application of molecular ®ngerprinting to studies on outbreaks of porcine pasteurellosis and pleuropneumoniae. Agri. Practice 17, 23±27. Blackall, P.J., Fegan, N., Chew, G.T., Hampson, D.J., 1998. Population structure and diversity of avian isolates of Pasteurella multocida from Australia. Microbiology 144, 279±289. Blackall, P.J., Pahoff, J.L., Marks, D., Fegan, N., Morrow, C.J., 1995. Characterisation of Pasteurella multocida isolated from fowl cholera outbreaks on turkey farms. Aust. Vet. J. 72, 135±138. Blackwood, R.A., Rode, C.K., Read, J.S., Law, I.H., Bloch, C.A., 1996. Genomic ®ngerprinting by pulsed ®eld gel electrophoresis to identify the source of Pasteurella multocida sepsis. Pediatr. Infect. Dis. J. 15, 831±833. Boulnois, G.J., Roberts, I.S., 1990. Genetics of capsular polysaccharide production in bacteria. Curr. Top. Microbiol. Immunol. 150, 1±18.

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Boyce, J.D., Chung J.Y., Adler, B., 1999. Genetic organisation of the capsule biosynthetic locus of Pasteurella multocida B:2. Vet. Microbiol., In press. Brickell, S.K., Thomas, L.M., Long, K.A., Panaccio, M., Widders, P.R., 1998. Development of a PCR test based on a gene region associated with the pathogenicity of Pasteurella multocida serotype B:2, the causal agent of haemorrhagic septicaemia in Asia. Vet. Microbiol. 59, 295±307. Bukau, B., 1993. Regulation of the Escherichia coli heat-shock response. Mol. Microbiol. 9, 671±680. Buttenschùn, J., Rosendal, S., 1990. Phenotypical and genotypical characteristics of paired isolates of Pasteurella multocida from the lungs and kidneys of slaughtered pigs. Vet. Microbiol. 25, 67±75. Carpenter, T.E., Snipes, K.P., Kasten, R.W., Hird, D.W., Hirsh, D.C., 1991. Molecular epidemiology of Pasteurella multocida in turkeys. Am. J. Vet. Res. 52, 1345±1349. Chaslus-Dancla, E., Lesage-Descauses, M.C., Leroy-SeÂtrin, S., Martel, J.L., Coudert, P., Lafont, J.P., 1996. Validation of random ampli®ed polymorphic DNA assays by ribotyping as tools for epidemiological surveys of Pasteurella from animals. Vet. Microbiol. 52, 91±102. Chaslus-Dancla, E., Lesage-Descauses, M.C., Leroy-Setrin, S., Martel, J.L., Lafont, J.P., 1995. Tetracycline resistance determinants, Tet B and Tet M, detected in Pasteurella haemolytica and Pasteurella multocida from bovine herds. J. Antimicrob. Chemother. 36, 815±819. Chengappa, M.M., Carter, G.R., Chang, T.S., 1979. A streptomycin-dependent live Pasteurella multocida type-3 vaccine for the prevention of fowl cholera in turkeys. Avian Dis. 23, 57±61. Chevalier, G., Duclohier, H., Thomas, D., Shechter, E., Wroblewski, H., 1993. Puri®cation and characterization of protein H, the major porin of Pasteurella multocida. J. Bacteriol. 175, 266±276. Chevallier, B., Dugourd, D., Tarasiuk, K., Harel, J., Gottschalk, M., Kobisch, M., Frey, J., 1998. Chromosome sizes and phylogenetic relationships between serotypes of Actinobacillus pleuropneumoniae. FEMS Microbiol. Lett. 160, 209±216. Chung, J.Y., Zhang, Y.M., Adler, B., 1998. The capsule biosynthetic locus of Pasteurella multocida A1. FEMS Microbiol. Lett. 166, 289±296. Cote, S., Harel, J., Higgins, R., Jacques, M., 1991. Resistance to antimicrobial agents and prevalence of R plasmids in Pasteurella multocida from swine. Am. J. Vet. Res. 52, 1653±1657. Dawkins, H.J.S., 1989. Large DNA separation using ®eld alternation agar gel electrophoresis. J. Chromat. 492, 615±639. DeAngelis, P.L., 1998. Transposon Tn916 insertional mutagenesis of Pasteurella multocida and direct sequencing of disruption site. Microb. Pathog. 24, 203±209. DeAngelis, P.L., Jing, W., Drake, R.R., Achyuthan, A.M., 1998. Identi®cation and molecular cloning of a unique hyaluronan synthase from Pasteurella multocida. J. Biol. Chem. 273, 8454±8458. Delamarche, C., Manoha, F., Behar, G., Houlgatte, R., Hellman, U., Wroblewski, H., 1995. Characterization of the Pasteurella multocida skp and ®rA genes. Gene 161, 39±43. Dewhirst, F.E., Paster, B.J., Olsen, I., Fraser, G.J., 1993. Phylogeny of the Pasteurellaceae as determined by comparison of 16S ribosomal ribonucleic acid sequences. Int. J. Med. Microbiol. Virol. Parasitol. Inf. Dis. 279, 35±44. Diallo, I.S., Bensink, J.C., Frost, A.J., Spradbrow, P.B., 1995. Molecular studies on avian strains of Pasteurella multocida in Australia. Vet. Microbiol. 46, 335±342. Dicker, I.B., Seetharam, S., 1991. Cloning and nucleotide sequence of the ®rA gene and the ®rA200(Ts) allele from Escherichia coli. J. Bacteriol. 173, 334±344. Donnio, P.Y., Allardet-Servent, A., Perrin, M., Escande, F., Avril, J.L., 1999. Characterisation of dermonecrotic toxin-producing strains of Pasteurella multocida subsp. multocida isolated from man and swine. J. Med. Microbiol. 48, 125±131. Donnio, P.Y., Le Goff, C., Avril, J.L., Pouedras, P., Gras-Rouzet, S., 1994. Pasteurella multocida: oropharyngeal carriage and antibody response in breeders. Vet. Res. 25, 8±15. El-Adhami, W., Kyd, J.M., Bastin, D.A., Cripps, A.W., 1999. Characterization of the gene encoding a 26-kDa protein (OMP26) from nontypeable Haemophilus in¯uenzae and immune responses to the recombinant protein. Infect. Immun. 67, 1935±1942. Fernandez de Henestrosa, A.R., Badiola, I., Saco, M., Perez de Rozas, A.M., Campoy, S., Barbe, J., 1997. Importance of the galE gene on the virulence of Pasteurella multocida. FEMS Microbiol. Lett. 154, 311± 316.

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