Pasteurella multocida pathogenesis: 125years after Pasteur
Marina Harper, John D. Boyce & Ben Adler Australian Research Council Centre of Excellence in Structural and Functional Microbial Genomics, Department of Microbiology, Monash University, Melbourne, Australia Correspondence: Ben Adler, Department of Microbiology, Monash University, Melbourne 3800, Australia. Tel.: 16 139 905 4815; fax: 16 139 905 4811; e-mail: ben.adler@med.monash.edu.au Received 20 July 2006; revised 15 August 2006; accepted 15 August 2006. First published online 11 September 2006. DOI:10.1111/j.1574-6968.2006.00442.x Editor: Ian Henderson Keywords Pasteurella multocida ; pathogenesis; fowl cholera; virulence factors. Abstract Pasteurella multocida was rst shown to be the causative agent of fowl cholera by Louis Pasteur in 1881. Since then, this Gram-negative bacterium has been identied as the causative agent of many other economically important diseases in a wide range of hosts. The mechanisms by which these bacteria can invade the mucosa, evade innate immunity and cause systemic disease are slowly being elucidated. Key virulence factors identied to date include capsule and lipopoly- saccharide. The capsule is clearly involved in bacterial avoidance of phagocytosis and resistance to complement, while complete lipopolysaccharide is critical for bacterial survival in the host. A number of other virulence factors have been identied by both directed and random mutagenesis, including Pasteurella multocida toxin (PMT), putative surface adhesins and iron acquisition proteins. However, it is likely that many key virulence factors are yet to be identied, including those required for initial attachment and invasion of host cells and for persistence in a relatively nutrient poor and hostile environment. Introduction It has been over 125 years since Louis Pasteur rst identied that a bacterium was the causative agent of fowl cholera. In seminal experiments, he also showed that repeated passage of the bacteria produced an attenuated strain incapable of causing disease, but the inoculation of birds with this strain could elicit a protective immune response (Pasteur, 1880, 1881). In this review, we aim to outline the progress that has been made in understanding this highly versatile pathogen. Indeed, many of the molecular determinants for virulence are now identied and understood and we can begin to piece together many of the steps required for successful infection. The species Pasteurella multocida is subdivided into four subspecies that include the type strain, multocida, and three others, gallicida, septica and the recently described tigris (Capitini et al., 2002). This review will consider only the type strain, subspecies multocida. Pasteurella multocida can cause disease in a wide range of animal species and is the causative agent of numerous, economically important dis- eases, including avian fowl cholera, bovine haemorrhagic septicaemia, enzoonotic pneumonia and swine atrophic rhinitis (De Alwis, 1992). Pasteurella multocida strains are classied into serogroups (A, B, D, E and F) based on capsule antigens and further classied into 16 serotypes (116) based primarily on lipopolysaccharide antigens using the Heddleston scheme (Carter, 1955; Heddleston et al., 1972). Fowl cholera is a serious disease of poultry and can present in either acute or chronic forms. The majority of acute fowl cholera cases are caused by serogroup A strains of P. multocida. Obvious clinical signs of acute fowl cholera may not occur until very late in the infection and include depression, rufed feathers, fever, anorexia, mucous dis- charge from the mouth, diarrhoea and an increased respira- tory rate (Rhoades & Rimler, 1989). Haemorrhagic septicaemia, predominantly caused by serotypes B and E strains of P. multocida, is an acute disease that affects cattle and buffalo and is characterized by oedematous swelling of the head and neck, and swollen haemorrhagic lymph nodes (Carter & De Alwis, 1989). Atrophic rhinitis in pigs is caused by strains of P. multocida that express P. multocida toxin (PMT), and typically belong to serogroup D. In pigs, the most obvious symptoms include the shortening and twisting of the snout, dark tear staining and pneumonia (Chanter & Rutter, 1989). Atrophic rhinitis rarely causes death, but it is economically important as it signicantly reduces the growth rate of the infected pigs. FEMS Microbiol Lett 265 (2006) 110 c 2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved Pasteurella multocida can be a primary or secondary agent involved in pneumonia in cattle (predominantly caused by serotype A:1), pigs and, occasionally, sheep (Gilmour, 1978; Chanter & Rutter, 1989; Frank, 1989). Pasteurella multocida can also infect rabbits resulting in rhinitis (snufes) and pneumonia (Manning et al., 1989). Although relatively un- common, human infections have been observed in a range of sites, commonly following cat or dog bites (Weber et al., 1984). Of all the diseases caused by P. multocida, fowl cholera is probably best understood. In birds, it is widely believed that P. multocida enters the host through tissues of the respira- tory tract. Adhesion of P. multocida to turkey air sac macrophages has been demonstrated, and virulent P. multo- cida inoculated into the upper respiratory tract or trachea of turkeys can be subsequently detected in internal organs between 6 and 12 h postinoculation (Rhoades & Rimler, 1990; Matsumoto et al., 1991; Pruimboom et al., 1996, 1999). The mechanisms by which P. multocida invades from the lungs into the bloodstream are poorly understood, although a study using heterophil-depleted chickens experi- mentally infected with P. multocida showed that heterophils may play a dual role. Initially, recruitment of heterophils into the lungs led to tissue damage and invasion, but as the infection progressed, heterophils limited the infection by promoting clearance of the bacteria from the lungs and spleen (Bojesen et al., 2004). Although it has been generally accepted that fowl cholera is a septicaemic infection, bacteria can only be isolated in large numbers from the blood of birds very late in infection, and it has been proposed that this late re-emergence of blood-borne bacteria is due to the rupture of liver and spleen phagocytes (Pabs-Garnon & Soltys, 1971). In the terminal stages of fowl cholera, death is probably caused by massive bacteraemia and endotoxic shock (Carter, 1967; Rhoades & Rimler, 1984). The rst systemic host defence used against most invad- ing bacteria involves the innate immune system and includes phagocytosis and bactericidal properties of serum compo- nents such as complement. However, P. multocida has mechanisms to evade this system, including the presence of a capsule (Boyce & Adler, 2000; Chung et al., 2001). Active immunity against P. multocida infection is mainly humoral; vaccination of birds with killed P. multocida stimulates protection against homologous challenge, and opsonization studies in mice have shown that bactericidal antibodies are produced (Wijewardana & Sutherland, 1990). Virulence factors Capsule Pasteurella multocida can be classied by serological meth- ods into ve capsule groups designated A, B, D, E and F. The composition and structure of the capsular material found in P. multocida serotypes A, D and F are very similar to mammalian glycosaminoglycans and consist mainly of hya- luronan, heparosan and unsulphated chondroitin, respec- tively (Pandit & Smith, 1993; Rimler, 1994; DeAngelis, 1996; DeAngelis & Padgett-McCue, 2000). The genes required for the synthesis and transport of these capsular types are encoded within a single region on the genome (Townsend et al., 2001). However, recently, in type A, D and F strains, an additional gene encoding a heparosan synthase was identi- ed outside of the known capsule biosynthesis region. The synthase encoded by this gene, renamed hssB (formerly pgla), is transcribed 10-fold less than the synthase within the capsule operon, uses a different acceptor and gives rise to smaller molecular weight polymer products. It was pro- posed that the expression of this gene may give rise to capsular variation (Deangelis & White, 2004). In general, strains that possess a capsule are more virulent than their acapsular variants (Heddleston et al., 1964; Snipes et al., 1987; Tsuji & Matsumoto, 1989). The important role of capsule in the pathogenesis of P. multocida has been clearly demonstrated as genetically dened, acapsular mu- tants constructed from both serogroup A and B strains were strongly attenuated in mice (Boyce & Adler, 2000; Chung et al., 2001). The serogroup A acapsular mutant was also shown to be avirulent in chickens and was unable to establish growth in chicken muscle (Chung et al., 2001). Interestingly, despite its apparent lack of persistence, vacci- nation of chickens with high doses of this acapsular mutant stimulated protective immunity (Chung et al., 2005). It is widely accepted that capsule plays a signicant role in resistance to phagocytosis, and this has been demonstrated in vitro by Harmon et al. (1991) and others, who have correlated sensitivity to phagocytosis with the presence and thickness of the bacterial capsule (Truscott & Hirsh, 1988; Harmon et al., 1991; Pruimboom et al., 1996). Moreover, studies using murine macrophages clearly demonstrated that a genetically dened acapsular serotype B mutant was more susceptible to uptake than its wild-type parent (Boyce & Adler, 2000). Resistance to complement-mediated lysis is clearly important for virulence, and experiments on P. multocida type A strains have shown that serum resistance correlates with the possession of a capsule (Snipes & Hirsh, 1986; Hansen & Hirsh, 1989). A genetically dened acapsu- lar mutant was no longer serum resistant in normal avian serum compared with the serotype A wild-type parent and complemented mutant (Chung et al., 2001). In contrast, a genetically dened, acapsular mutant of a serotype B strain showed no reduction in serum resistance in either bovine or murine serum compared with the parent strain (Boyce & Adler, 2000). The basis for this difference between capsular types remains unknown. However, despite this evidence suggesting no loss of serum resistance, the serotype B FEMS Microbiol Lett 265 (2006) 110 c 2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved 2 M. Harper et al. acapsular mutant was strongly attenuated in mice and had increased sensitivity to phagocytosis by murine macro- phages (Boyce & Adler, 2000). Lipopolysaccharide Pasteurella multocida lipopolysaccharide plays a critical role in the pathogenesis of disease. It stimulates humoral im- munity and is considered to be a protective antigen. Mono- clonal antibodies raised against the lipopolysaccharide from a serotype A strain were bactericidal and protected mice against homologous challenge (Wijewardana et al., 1990). In addition, an opsonic monoclonal antibody against lipopo- lysaccharide from a serotype B strain of P. multocida partially protected mice against P. multocida infection (Ramdani & Adler, 1991). There are conicting reports as to the endotoxic proper- ties of lipopolysaccharide isolated from P. multocida. Intra- venous inoculation of lipopolysaccharide from serotype B:2 stains could reproduce clinical signs of haemorrhagic septi- caemia in buffalo (Horadagoda et al., 2002), but the endotoxic properties of lipopolysaccharide from serotype A strains is less clear. Studies have indicated that chicken embryos and mice are highly susceptible, but that turkey poults are relatively resistant (Ganeld et al., 1976; Rhoades & Rimler, 1987; Mendes et al., 1994). It is clear that P. multocida requires a complete lipopoly- saccharide structure in order to replicate in vivo and cause disease. In a recent study using signature-tagged mutagen- esis, a mutant attenuated in mice and chickens had an insertional inactivation of the gene waaQ PM (encoding a putative heptosyl transferase) required for the addition of heptose to lipopolysaccharide (Harper et al., 2004). Using mass spectrometry and nuclear magnetic resonance, it was demonstrated that the predominant lipopolysaccharide gly- coforms extracted from this mutant were severely truncated. Complementation experiments demonstrated that provid- ing a functional waaQ PM gene in trans restored both the lipopolysaccharide structure and growth in mice to wild- type levels (Harper et al., 2004). A galE mutant of P. multocida had signicantly reduced viability in mice (Fernandez de Henestrosa et al., 1997). Included in the role of galE in bacteria is the epimerization of UDP-glucose to UDP-galactose before lipopolysaccharide assembly, and this mutant probably expressed an altered lipopolysaccharide, although the structural analysis of the lipopolysaccharide was not reported (Fernandez de Henes- trosa et al., 1997). The complete lipopolysaccharide structure of strains VP161 and X73 belonging to Heddleston serotype 1 and PM70, a Heddleston serotype 3 strain, have been deter- mined and all possess a structure similar to the lipopolysac- charide or lipo-oligosaccharide (LOS) of Neisseria spp. and Haemophilus spp. The lipopolysaccharide from the P. mul- tocida strains had highly conserved inner cores with a tri- heptose unit linked via a Kdo residue to lipid A, but there were signicant variations in the outer core between the three different strains (St Michael et al., 2005a, b, c). Inter- estingly P. multocida lipopolysaccharide isolated from the two Heddleston type I strains contains terminal phospho- choline residues, which in other bacteria plays a key role in bacterial adhesion to, and invasion of, host cells by binding directly to the platelet-activating factor (PAF) receptor (Cundell et al., 1995; Schenkein et al., 2000; Swords et al., 2000; Serino & Virji, 2002). Although the role of phospho- choline residues on the lipopolysaccharide of P. multocida remains unknown, it has been shown in the bovine model that lipopolysaccharide from P. multocida assists in adhesion to neutrophils and transmigration through endothelial cells (Galdiero et al., 2000). In addition to phosphocholine residues, all the P. multo- cida strains studied to date have phosphoethaolamine residues attached to various sites on their lipopolysacchar- ide. Notably, in addition to phosphocholine residues, the X73 strain has phosphoethaolamine residues attached to each of the terminal galactose sugars (St Michael et al., 2005a). In other Gram-negative bacteria, phosphoethaola- mine is added to a number of sites within the inner core of lipopolysaccharide by specic transferases, and in Neisseria meningitidis the expression and position of these residues affects the ability of the bacteria to resist the innate immune response (Ram et al., 2003). A clear role for phosphoethao- lamine in P. multocida has not been dened. Fimbriae and adhesins There are many genes, including ptfA, mA, p1, p2, hsf_1 and hsf_2 on the P. multocida genome that encode proteins similar to mbriae or brils in other bacteria. It is likely that mbriae play a role in the surface adhesion, as mbriae have been observed on some P. multocida serotype A strains that were able to adhere to mucosal epithelium, but not on the surface of those strains unable to adhere (Glorioso et al., 1982; Rebers et al., 1988; Isaacson & Trigo, 1995; Ruffolo et al., 1997). Type IV mbriae (pili) have been isolated and characterized from P. multocida serotypes A, B and D (Ruffolo et al., 1997) and are often associated with virulence in other bacteria because of their role in attachment to host cell surfaces. The subunit gene, ptfA, has been isolated and sequenced from a number of strains and the predicted protein sequences showed signi- cant variation between strains (Doughty et al., 2000). However, the role of mbrial structures in P. multocida virulence is still unproven. The P. multocida Pm70 genome contains a region encod- ing proteins with signicant similarity to the Flp pilin locus FEMS Microbiol Lett 265 (2006) 110 c 2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved 3 Pasteurella multocida pathogenesis in Actinobacillus actinomycetemcomitans, encoding a re- cently described type IV mbrial subfamily (Kachlany et al., 2001b; May et al., 2001). This locus encodes proteins predicted to be the Flp pilin subunits and the proteins required for the pilin assembly. Despite the lack of physical evidence for Flp pili in P. multocida, there is evidence through STM studies in mice that the products of these genes are required for virulence. Two genes, p1, encoding a Flp pilin subunit and tadD, predicted to encode a compo- nent of the secretion apparatus required for Flp pilin assembly, have been inactivated in two independent STM studies using transposon mutagenesis, and both mutants were signicantly attenuated in mice (Fuller et al., 2000; May et al., 2001; Kachlany et al., 2001a, b; Harper et al., 2003). Two P. multocida genes, pfhaB1 and pfhaB2, share sig- nicant similarity with a class of genes that encode lamen- tous haemagglutinins, which in Bordetella pertussis play a major role in the colonization of the upper respiratory tract (Kimura et al., 1990; Mooi et al., 1992). Mutation of these genes in P. multocida resulted in signicantly reduced virulence in mice (Fuller et al., 2000). More recently, a pfhaB2 mutant was constructed in a fowl cholera strain, P1059, and shown to be highly attenuated in turkeys when administered intranasally, but only moderately attenuated when given intravenously, suggesting that pfhaB2 has a signicant role in initial colonization or invasion (Tatum et al., 2005). Toxins In general, most P. multocida strains that cause fowl cholera, haemorrhagic septicaemia or pneumonia are not known to express any toxins. The dermonecrotic toxin, PMT, ex- pressed mainly by serogroup D strains, is the only toxin identied to date and is responsible for the clinical and pathological signs of atrophic rhinitis (Foged et al., 1987; Rimler & Rhoades, 1989). Both puried native and recom- binant PMT toxin can be used to experimentally induce clinical signs of disease (Foged et al., 1987; Lax & Chanter, 1990). PMT acts intracellularly by modulating the Ga q subunit in the phospholipase C signal transduction pathway (Mur- phy & Rozengurt, 1992; Wilson et al., 1997). In pigs, this leads to atrophy of nasal turbinates where bone resorption occurs due to uncontrolled proliferation of osteoclasts, and regeneration of bone is prevented by the inhibition of osteoblasts (Sterner-Kock et al., 1995; Mullan & Lax, 1998). PMT is also a potent mitogen, inducing many cellular effects including rearrangements in the actin cytoskeleton (Zywietz et al., 2001). Like cholera and pertussis toxins, PMTactivates dendritic cells to mature cells but, unlike the other toxins, it is a poor adjuvant and appears to suppress the antibody response (Bagley et al., 2005). It has been proposed that PMT blocks chemotaxis-induced migration of dendritic cells to regional lymph nodes and might, therefore, in a natural infection, limit the development of an adaptive immune response (Blocker et al., 2006). The PMT toxin gene resides on a lysogenic bacteriophage belonging to the Siphoviridae family. As the PMT toxin has no signal sequence and no known mechanism of export, it has been suggested that the lytic phase of the bacteriophage may mediate the release of the toxin (Pullinger et al., 2004). The toxin is an effective immunogen; a toxoid developed by deletion mutagenesis of the cloned PMT toxin gene was found to protect mice and their offspring against challenge with puried PMT (Petersen et al., 1991). Similarly, a genetically modied PMT toxin, where there were two key amino acid substitutions, led to a nontoxigenic protein that protected pigs against experimental challenge with the wild- type strain (To et al., 2005). Iron regulated and iron acquisition proteins Iron is an essential element which must be acquired by bacteria in order to survive. Because of its inherent toxicity, the level of free iron available in vivo is very limited and P. multocida, like other bacterial species, has developed multiple mechanisms for iron uptake. Sequence analysis of P. multocida PM70 revealed that a relatively large proportion of the genome (over 2.5%) encodes 53 proteins with similarity to proteins involved in iron uptake or acquisition (May et al., 2001). Comparisons of P. multocida grown in iron-rich, iron- depleted media or in vivo has demonstrated that many high molecular weight outer membrane proteins are regulated by iron levels and have therefore been called iron-regulated outer membrane proteins (IROMPs) (Snipes et al., 1988; Choi-Kim et al., 1991). Pasteurella multocida grown under iron-limited conditions also induces a stronger protective response in mice compared with the same strain grown under iron-replete conditions (Kennett et al., 1993), and it is thought that IROMPs may, therefore, play a signicant role in cross-protective immunity (Glisson et al., 1993; Ruffolo et al., 1998). However, analysis of IROMPs using a proteo- mics approach identied only one protein, PM0805, that was up-regulated and only one, OmpW, that was down- regulated under low-iron conditions (Boyce et al., 2006). Transferrin receptors utilized by bacterial species in the Pasteurellaceae and Neisseriaceae families usually consist of two iron binding receptors TbpA and TbpB (Gray-Owen & Schryvers, 1996), but recent evidence suggests that the transferrin receptor in bovine strains of P. multocida is composed of only a single protein TbpA (Ogunnariwo & Schryvers, 2001). However, this receptor may not be present in all P. multocida strains; a recent PCR and DNA FEMS Microbiol Lett 265 (2006) 110 c 2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved 4 M. Harper et al. hybridization study found that the tbpA gene is present only in some bovine and ovine clinical isolates (Ewers et al., 2006). Iron acquisition proteins are believed to play a role in the disease process; serogroup A mutants with inactivated ExbB, ExbD, TonB, or HgbA proteins are attenuated in mice (Fuller et al., 2000; Bosch et al., 2002a). ExbB, ExbD and TonB are part of the TonB transport complex, required to transport and provide energy for iron sequestration, and HgbA is predicted to be a haemoglobin-binding protein required for the acquisition of iron from host proteins (Bosch et al., 2002b). Sialic acid metabolism Sialidases are produced by some bacterial species and act to remove sialic acid from host glycosylated proteins and lipids for use as a carbon source. These enzymes may also enhance bacterial virulence by unmasking key host receptors and/or reducing the effectiveness of host defences such as mucin. Most P. multocida strains produce sialidase and both cell bound and extracellular sialidases have been reported in P. multocida (Scharmann et al., 1970; Drzeniek et al., 1972; White et al., 1995). Bacterial sialidase is produced in vivo in goats after transthoracic challenge with either P. multocida or M. haemolytica (Straus & Purdy, 1994; Straus et al., 1996). Passive protection of mice against P. multocida A:3 homo- logous challenge has been demonstrated using rabbit anti- sera raised against partially puried sialidase, although there was some difculty in removing anti-LPS antibodies from the serum before administration into mice (Ifeanyi & Bailie, 1992), thus raising the question about the specicity of the protective antibodies. Two sialidases, NanH and NanB, have been cloned and characterized from a fowl cholera isolate of P. multocida (Mizan et al., 2000). These sialidases differed in their specicity, with both able to utilize 2,3 0 sialyl lactose, but only NanB able to fully utilize 2,6 0 sialyl lactose. It was proposed that the presence of two sialidases with slightly different specicities would enhance the metabolic capacity of P. multocida in the host (Mizan et al., 2000). In addition, there is increasing evidence to suggest that P. multocida is capable of scavenging sialic acid from the environment for both the sialylation of cell components and for nutrients via a catabolic breakdown pathway (Steenber- gen et al., 2005). Furthermore, the uptake, but not catabo- lism, of sialic acid was shown to be essential for virulence in mice (Steenbergen et al., 2005). Two genes, pm0188 and pm0508, encoding sialic acid transferases, have been identi- ed in the P. multocida genome. Interestingly, the product of pm0188 has been shown to be a multifunctional enzyme capable of four functions. It exhibits transferase activity linking sialic acid to galactose with either 2,3 or 2,6 linkages with varying activity. In lower, more acidic pH conditions, it can function both as a sialidase capable of cleaving 2,3 sialyl linkages, but not 2,6 linkages, and as a trans sialidase, transferring 2,3 linked sialyl groups to another galactose residue (Yu et al., 2005). An unequivocal role in pathogen- esis has not yet been demonstrated. Hyaluronidase Although the role of hyaluronidase in pathogenesis has not been determined, it is present in many of the serotype B strains of P. multocida that cause bovine haemorrhagic septicaemia. A study of 74 P. multocida strains representing all capsular serotypes found that only the type B strains, isolated from haemorrhagic septicaemia infections, pro- duced hyaluronidase (Carter & Chengappa, 1980). Another study of 176 strains of P. multocida representing different serotypes also found hyaluronidase activity conned to serotype B, but more specically B:2, and it was suggested that a test for hyaluronidase activity could be used to presumptively identify B:2 strains (Rimler & Rhoades, 1994). Outer membrane proteins Early studies on the P. multocida outer membrane showed that a 37 kDa protein was among ve identied as possible protective immunogens based rstly on radioimmunopre- cipitation results using protective immune rabbit sera, and secondly on their location in the outer membrane (Lu et al., 1988). Monoclonal antibodies raised against the 37 kDa protein were able to passively protect rabbits and mice against infection with P. multocida with strong protection afforded against homologous strains, and some limited protection against heterologous strains (Lu et al., 1991). A protein of similar molecular mass (39 kDa) was identi- ed in the P. multocida A:3 strain P1059; its expression was shown to correlate with the presence and amount of capsule present on the cell (Borrathybay et al., 2003b; Ali et al., 2004a). Pasteurella multocida can adhere to, and invade, chicken embryo broblasts, and this adherence was inhib- ited by both monoclonal and polyclonal antibodies raised against the 39 kDa protein (Borrathybay et al., 2003a; Al-haj Ali et al., 2004; Ali et al., 2004a, b). Passive immunization of mice with a monoclonal antibody against the 39 kDa protein or active immunization with afnity puried 39 kDa pro- tein, demonstrated that antibodies raised against this pro- tein were cross-protective against serovars A:1 and A:3 (Ali et al., 2004a, b). The actual identity of this 39 kDa protein was not reported, but recently a 39 kDa protein which can stimulate cross-serotype protection (Rimler, 2001) was iso- lated from outer membrane protein extracts of the same A:3 strain, P1059, used in the above studies. This protein was identied as PlpB (Pasteurella lipoprotein B), using peptide mass ngerprinting (Tabatabai & Zehr, 2004) and is FEMS Microbiol Lett 265 (2006) 110 c 2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved 5 Pasteurella multocida pathogenesis predicted to be an ABC transport protein required for the uptake of methionine into the cell (Merlin et al., 2002). One of the major outer membrane proteins of P. multo- cida is OmpH. Antibodies raised against this protein pro- vide some protection against disease. Monoclonal antibodies specic for OmpH passively protect mice against P. multocida challenge (Marandi & Mittal, 1997) and vaccination with the native, but not recombinant, OmpH protein elicits protective immunity in birds against homo- logous challenge (Luo et al., 1997). In addition, antibodies raised to an OmpH synthetic peptide, Cyclic-L2, provided partial protection in chickens against homologous challenge (Luo et al., 1999). Recent studies examining the ability of P. multocida to bind host extracellular matrix proteins have shown that the bacteria can adhere to bronectin and collagen type IX. Proteins identied as possible adhesins include OmpA, Oma87, Pm1069 and the iron related proteins, Tbp (trans- ferrin binding protein) and the putative TonB receptor HgbA (Dabo et al., 2005). OmpA, a b-barrel, ion channel protein, has been speci- cally identied as having a direct role in adhesion. Homo- logs of this protein are important adhesins in Escherichia coli, Haemophilus inuenzae and other bacteria. Notably, recombinant P. multocida OmpA binds to bovine kidney cells and interacts with host extracellular matrix molecules heparin and bronectin (Dabo et al., 2003). Concluding remarks Pasteurella multocida is capable of causing disease in a wide range of animals and birds. There is a clear correlation between capsular type and the disease, with serotypes A and F typically associated with fowl cholera, serotypes B and E with haemorrhagic septicaemia in cattle, and serotype D strains expressing PMT toxin with atrophic rhinitis in swine. What is unclear is whether the expression of a particular type of lipopolysaccharide or the type and amount of proteins presented on the bacterial surface also contribute to the disease specicity. Other virulence factors such as neuraminidases, iron sequestering proteins and metabolic enzymes play key roles in acquiring and utilizing substrates for growth within the host, often a relatively nutrient poor and hostile environment. There has been little progress in understanding exactly how P. multocida invades mucosal surfaces to gain access to the blood and how the host responds to the infection. However, signicant advances have been made in identify- ing bacterial factors critical to P. multocida pathogenesis. It is known that capsule and lipopolysaccharide are essential for normal disease progression in fowl cholera and there is some progress in understanding the bacterial response to the in vivo environment (Boyce et al., 2002, 2004) and identify- ing bacterial factors required for disease progression (Fuller et al., 2000, Harper et al., 2003). References Al-haj Ali H, Sawada T, Hatakeyama H, Katayama Y, Ohtsuki N & Itoh O (2004) Invasion of chicken embryo broblast cells by avian Pasteurella multocida. Vet Microbiol 104: 5562. Ali HA, Sawada T, Hatakeyama H, Ohtsuki N & Itoh O (2004a) Characterization of a 39 kDa capsular protein of avian Pasteurella multocida using monoclonal antibodies. Vet Microbiol 100: 4353. Ali HA, Sawada T & Noda K (2004b) Protectivity of an immunoafnity-puried 39 kDa capsular protein of avian Pasteurella multocida in mice. J Vet Med Sci 66: 16031604. Bagley KC, Abdelwahab SF, Tuskan RG & Lewis GK (2005) Pasteurella multocida toxin activates human monocyte-derived and murine bone marrow-derived dendritic cells in vitro but suppresses antibody production in vivo. Infect Immun 73: 413421. Blocker D, Berod L, Fluhr JW, Orth J, Idzko M, Aktories K & Norgauer J (2006) Pasteurella multocida toxin (PMT) activates RhoGTPases, induces actin polymerization and inhibits migration of human dendritic cells, but does not inuence macropinocytosis. Int Immunol 18: 459464. Bojesen AM, Petersen KD, Nielsen OL, Christensen JP & Bisgaard M (2004) Pasteurella multocida infection in heterophil- depleted chickens. Avian Dis 48: 463470. Borrathybay E, Sawada T, Kataoka Y, Ohtsu N, Takagi M, Nakamura S & Kawamoto E (2003a) A 39 kDa protein mediates adhesion of avian Pasteurella multocida to chicken embryo broblast cells. Vet Microbiol 97: 229243. Borrathybay E, Sawada T, Kataoka Y, Okiyama E, Kawamoto E & Amao H (2003b) Capsule thickness and amounts of a 39 kDa capsular protein of avian Pasteurella multocida type A strains correlate with their pathogenicity for chickens. Vet Microbiol 97: 215227. Bosch M, Garrido E, Llagostera M, de Rozas AMP, Badiola I & Barbe J (2002a) Pasteurella multocida exbB, exbD and tonB genes are physically linked but independently transcribed. FEMS Microbiol Lett 210: 201208. Bosch M, Garrido ME, Llagostera M, de Rozas AMP, Badiola I & Barbe J (2002b) Characterization of the Pasteurella multocida hgbA gene encoding a hemoglobin-binding protein. Infect Immun 70: 59555964. Boyce JD &Adler B (2000) The capsule is a virulence determinant in the pathogenesis of Pasteurella multocida M1404 (B:2). Infect Immun 68: 34633468. Boyce JD, Wilkie I, Harper M, Paustian ML, Kapur V &Adler B (2002) Genomic scale analysis of Pasteurella multocida gene expression during growth within the natural chicken host. Infect Immun 70: 68716879. Boyce JD, Wilkie I, Harper M, Paustian ML, Kapur V &Adler B (2004) Genomic-scale analysis of Pasteurella multocida gene FEMS Microbiol Lett 265 (2006) 110 c 2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved 6 M. Harper et al. expression during growth within liver tissue of chickens with fowl cholera. Microbes Infect 6: 290298. Boyce JD, Cullen PA, Nguyen V, Wilkie I & Adler B (2006) Analysis of the Pasteurella multocida outer membrane sub- proteome and its response to the in vivo environment of the natural host. Proteomics 6: 870880. Capitini CM, Herrero IA, Patel R, Ishitani MB & Boyce TG (2002) Wound infection with Neisseria weaveri and a novel subspecies of Pasteurella multocida in a child who sustained a tiger bite. Clin Infect Dis 34: e74e76. Carter GR (1955) Studies on Pasteurella multocida I. A haemagglutination test for the identication of serological types. Am J Vet Res 16: 481484. Carter GR (1967) Pasteurellosis: Pasteurella multocida and Pasteurella haemolytica. Adv Vet Sci 11: 321379. Carter GR & Chengappa MM (1980) Hyaluronidase production by type B Pasteurella multocida from cases of hemorrhagic septicemia. J Clin Microbiol 11: 9496. Carter GR & De Alwis MCL (1989) Haemorrhagic Septicaemia. In: Pasteurella and Pasteurellosis (Adlam C & Rutter JM, eds), pp. 131160. Academic Press Limited, London. Chanter N & Rutter JM (1989) Pasteurellosis in Pigs and the Determinants of Virulence of Toxigenic Pasteurella multocida. In: Pasteurella and Pasteurellosis (Adlam C & Rutter JM, eds), pp. 161195. Academic Press, London. Choi-Kim K, Maheswaran SK, Felice LJ & Molitor TW (1991) Relationship between the iron regulated outer membrane proteins and the outer membrane proteins of in vivo grown Pasteurella multocida. Vet Microbiol 28: 7592. Chung JY, Wilkie I, Boyce JD, Townsend KM, Frost AJ, Ghoddusi M & Adler B (2001) Role of capsule in the pathogenesis of fowl cholera caused by Pasteurella multocida serogroup A. Infect Immun 69: 24872492. Chung JY, Wilkie I, Boyce JD & Adler B (2005) Vaccination against fowl cholera with acapsular Pasteurella multocida A:1. Vaccine 23: 27512755. Cundell DR, Gerard NP, Gerard C, Idanpaan-Heikkila I & Tuomanen EI (1995) Streptococcus pneumoniae anchor to activated human cells by the receptor for platelet-activating factor. Nature 377: 435438. Dabo SM, Confer AW & Quijano-Blas RA (2003) Molecular and immunological characterization of Pasteurella multocida serotype A:3 OmpA: evidence of its role in P. multocida interaction with extracellular matrix molecules. Microb Pathog 35: 147157. Dabo SM, Confer AW & Hartson SD (2005) Adherence of Pasteurella multocida to bronectin. Vet Microbiol 110: 265275. De Alwis MCL (1992) Pasteurellosis in Production Animals: A Review. In: Pasteurellosis in Production Animals, ACIAR Proceedings No. 43 (Patten BE, Spencer TL, Johnson RB, Hoffman D & Lehane L, eds), pp. 1118. ACIAR, Bali, Indonesia. DeAngelis PL (1996) Enzymological characterization of the Pasteurella multocida hyaluronic acid synthase. Biochemistry 35: 97689771. DeAngelis PL & Padgett-McCue AJ (2000) Identication and molecular cloning of a chondroitin synthase from Pasteurella multocida type F. J Biol Chem 275: 2412424129. Deangelis PL &White CL (2004) Identication of a distinct, cryptic heparosan synthase from Pasteurella multocida types A, D, and F. J Bacteriol 186: 85298532. Doughty SW, Ruffolo CG & Adler B (2000) The type 4 mbrial subunit gene of Pasteurella multocida. Vet Microbiol 72: 7990. Drzeniek R, Scharmann W & Balke E (1972) Neuraminidase and N-acetylneuraminate pyruvate-lyase of Pasteurella multocida. J Gen Microbiol 72: 357368. Ewers C, Lubke-Becker A, Bethe A, Kiebling S, Filter M & Wieler LH (2006) Virulence genotype of Pasteurella multocida strains isolated from different hosts with various disease status. Vet Microbiol 114: 304317. Fernandez de Henestrosa AR, Badiola I, Saco M, Perez de Rozas AM, Campoy S & Barbe J (1997) Importance of the galE gene on the virulence of Pasteurella multocida. FEMS Microbiol Lett 154: 311316. Foged NT, Pedersen KB & Elling F (1987) Characterization and biological effects of the Pasteurella multocida toxin. FEMS Microbiol Lett 43: 4551. Frank GH (1989) Pasteurellosis of Cattle. In: Pasteurella and Pasteurellosis (Adlam C & Rutter JM, eds), Academic Press Limited, London. Fuller TE, Kennedy MJ & Lowery DE (2000) Identication of Pasteurella multocida virulence genes in a septicemic mouse model using signature-tagged mutagenesis. Microb Pathog 29: 2538. Galdiero M, Folgore A, Nuzzo I & Galdiero E (2000) Neutrophil adhesion and transmigration through bovine endothelial cells in vitro by protein H and LPS of Pasteurella multocida. Immunobiology 202: 226238. Ganeld DJ, Rebers PA & Heddleston KL (1976) Immunogenic and toxic properties of a puried lipopolysaccharide-protein complex from Pasteurella multocida. Infect Immun 14: 990999. Gilmour NJL (1978) Pasteurellosis in sheep. Vet Rec 102: 100102. Glisson JR, Contreras MD, Cheng IHN & Wang C (1993) Cross- protection studies with Pasteurella multocida bacterins prepared from bacteria propagated in iron-depleted medium. Avian Dis 37: 10741079. Glorioso JC, Jones GW, Rush HG, Pentler LJ, Darif CA & Coward JE (1982) Adhesion of type A Pasteurella multocida to rabbit pharyngeal cells and its possible role in rabbit respiratory tract infections. Infect Immun 35: 11031109. Gray-Owen SD & Schryvers AB (1996) Bacterial transferrin and lactoferrin receptors. Trends Microbiol 4: 185191. Hansen LM & Hirsh DC (1989) Serum resistance is correlated with encapsulation of avian strains of Pasteurella multocida. Vet Microbiol 21: 177184. FEMS Microbiol Lett 265 (2006) 110 c 2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved 7 Pasteurella multocida pathogenesis Harmon BG, Glisson JR, Latimer KS, Steffens WL & Nunnally JC (1991) Resistance of Pasteurella multocida A:3,4 to phagocytosis by turkey macrophages and heterophils. Am J Vet Res 52: 15071511. Harper M, Boyce JD, Wilkie IW &Adler B (2003) Signature- tagged mutagenesis of Pasteurella multocida identies mutants displaying differential virulence characteristics in mice and chickens. Infect Immun 71: 54405446. Harper M, Cox AD, St Michael F, Wilkie IW, Boyce JD & Adler B (2004) A heptosyltransferase mutant of Pasteurella multocida produces a truncated lipopolysaccharide structure and is attenuated in virulence. Infect Immun 72: 34363443. Heddleston KL, Watko LP & Rebers PA (1964) Dissociation of a fowl cholera strain of Pasteurella multocida. Avian Dis 8: 649657. Heddleston KL, Gallagher JE & Rebers PA (1972) Fowl cholera: gel diffusion precipitin test for serotyping Pasteurella multocida from avian species. Avian Dis 16: 925936. Horadagoda NU, Hodgson JC, Moon GM, Wijewardana TG & Eckersall PD (2002) Development of a clinical syndrome resembling haemorrhagic septicaemia in the buffalo following intravenous inoculation of Pasteurella multocida serotype B:2 endotoxin and the role of tumour necrosis factor-alpha. Res Vet Sci 72: 194200. Ifeanyi FI & Bailie WE (1992) Passive protection of mice with antiserum to neuraminidase from Pasteurella multocida serotype A:3. Vet Res Commun 16: 97105. Isaacson RE & Trigo E (1995) Pili of Pasteurella multocida of porcine origin. FEMS Microbiol Lett 132: 247251. Kachlany SC, Planet PJ, DeSalle R, Fine DH & Figurski DH (2001a) Genes for tight adherence of Actinobacillus actinomycetemcomitans: from plaque to plague to pond scum. Trends Microbiol 9: 429437. Kachlany SC, Planet PJ, DeSalle R, Fine DH, Figurski DH & Kaplan JB (2001b) Flp-1, the rst representative of a new pilin gene subfamily, is required for non-specic adherence of Actinobacillus actinomycetemcomitans. Mol Microbiol 40: 542554. Kennett L, Muniandy N & Mukkur TKS (1993) Comparative Protective Potential of Non-living Intact Cells and Puried Outer Membrane and Associated Proteins of Pasteurella multocida Type B:6 Grown Under Iron-Regulated Conditions. In: Pasteurellosis in Production Animals, ACIAR Proceedings No. 43 (Patten BEea, ed.), pp. 144149. ACIAR Press, Canberra. Kimura A, Mountzouros KT, Relman DA, Falkow S & Cowell JL (1990) Bordetella pertussis lamentous hemagglutinin: evaluation as a protective antigen and colonization factor in a mouse respiratory infection model. Infect Immun 58: 716. Lax AJ & Chanter N (1990) Cloning of the toxin gene from Pasteurella multocida and its role in atrophic rhinitis. J Gen Microbiol 136: 8187. Lu YS, Afendis SJ & Pakes SP (1988) Identication of immunogenic outer membrane proteins of Pasteurella multocida 3:A in rabbits. Infect Immun 56: 15321537. Lu YS, Lai WC, Pakes SP & Nie LC (1991) A monoclonal antibody against a Pasteurella multocida outer membrane protein protects rabbits and mice against Pasteurellosis. Infect Immun 59: 172180. Luo Y, Glisson JR, Jackwood MW, Hancock RE, Bains M, Cheng IH & Wang C (1997) Cloning and characterization of the major outer membrane protein gene (ompH) of Pasteurella multocida X-73. J Bacteriol 179: 78567864. Luo Y, Zeng Q, Glisson JR, Jackwood MW, Cheng IH & Wang C (1999) Sequence analysis of Pasteurella multocida major outer membrane protein (ompH) and application of synthetic peptides in vaccination of chickens against homologous strain challenge. Vaccine 17: 821831. Manning PJ, DiGiacomo RF & DeLong D (1989) Pasteurellosis in Laboratory Animals. In: Pasteurella and Pasteurellosis (Adlam C & Rutter JM, eds), pp. 263302. Academic Press, London. Marandi MV & Mittal KR (1997) Role of outer membrane protein H (ompH)- and ompA-specic monoclonal antibodies from hybridoma tumors in protection of mice against Pasteurella multocida. Infect Immun 65: 45024508. Matsumoto M, Strain JG & Engel HN (1991) The fate of Pasteurella multocida after intratracheal inoculation into turkeys. Poultry Sci 70: 22592266. May BJ, Zhang Q, Li LL, Paustian ML, Whittam TS & Kapur V (2001) Complete genomic sequence of Pasteurella multocida, Pm70. Proc Natl Acad Sci USA 98: 34603465. Mendes S, Carmichael KP, Nunnally JC, Glisson JR, Cheng IH & Harmon BG (1994) Lesions resulting from attempted Shwartzman reaction in turkey poults inoculated with Pasteurella multocida lipopolysaccharide. Avian Dis 38: 790796. Merlin C, Gardiner G, Durand S & Masters M (2002) The Escherichia coli metD locus encodes an ABC transporter which includes Abc (MetN), YaeE (MetI), and YaeC (MetQ). J Bacteriol 184: 55135517. Mizan S, Henk A, Stallings A, Maier M & Lee MD (2000) Cloning and characterization of sialidases with 2-6 0 and 2-3 0 sialyl lactose specicity from Pasteurella multocida. J Bacteriol 182: 68746883. Mooi FR, Jansen WH, Brunings H, Gielen H, van der Heide HG, Walvoort HC & Guinee PA (1992) Construction and analysis of Bordetella pertussis mutants defective in the production of mbriae. Microb Pathog 12: 127135. Mullan PB & Lax AJ (1998) Pasteurella multocida toxin stimulates bone resorption by osteoclasts via interaction with osteoblasts. Calcif Tissue Int 63: 340345. Murphy AC & Rozengurt E (1992) Pasteurella multocida toxin selectively facilitates phosphatidylinositol 4,5-bisphosphate hydrolysis by bombesin, vasopressin, and endothelin. Requirement for a functional G protein. J Biol Chem 267: 2529625303. Ogunnariwo JA & Schryvers AB (2001) Characterization of a novel transferrin receptor in bovine strains of Pasteurella multocida. J Bacteriol 183: 890896. FEMS Microbiol Lett 265 (2006) 110 c 2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved 8 M. Harper et al. Pabs-Garnon LF & Soltys MA (1971) Multiplication of Pasteurella multocida in the spleen, liver and blood of turkeys inoculated intravenously. Can J Comp Med 35: 147149. Pandit KK & Smith JE (1993) Capsular hyaluronic acid in Pasteurella multocida type A and its counterpart in type D. Res Vet Sci 54: 2024. Pasteur L (1880) De latt enuation du virus du chol era des poules. C R Acad Sci 91: 673680. Pasteur L (1881) Sur les virus-vaccins du chol era des poules et du charbon. C R Trav Congr Int Dir Agronom session de Versailles 151162. Petersen SK, Foged NT, Bording A, Nielsen JP, Riemann HK & Frandsen PL (1991) Recombinant derivatives of Pasteurella multocida toxin: candidates for a vaccine against progressive atrophic rhinitis. Infect Immun 59: 13871393. Pruimboom IM, Rimler RB, Ackermann MR & Brogden KA (1996) Capsular hyaluronic acid-mediated adhesion of Pasteurella multocida to turkey air sac macrophages. Avian Dis 40: 887893. Pruimboom IM, Rimler RB & Ackermann MR (1999) Enhanced adhesion of Pasteurella multocida to cultured turkey peripheral blood monocytes. Infect Immun 67: 12921296. Pullinger GD, Bevir T & Lax AJ (2004) The Pasteurella multocida toxin is encoded within a lysogenic bacteriophage. Mol Microbiol 51: 255269. Ram S, Cox AD, Wright JC et al. (2003) Neisserial lipooligosaccharide is a target for complement component c4b. Inner core phosphoethanolamine residues dene C4b linkage specicity. J Biol Chem 278: 5085350862. Ramdani & Adler B (1991) Opsonic monoclonal antibodies against lipopolysaccharide (LPS) antigens of Pasteurella multocida and the role of LPS in immunity. Vet Microbiol 26: 335347. Rebers PA, Jensen AE & Laird GA (1988) Expression of pili and capsule by the avian strain P-1059 of Pasteurella multocida. Avian Dis 32: 313318. Rhoades KR & Rimler RB (1984) Avian Pasteurellosis. In: Diseases of Poultry 8th edn (Hofstad MS, Barnes HJ, Calnek BW, Reid WM & Yoder HW Jr, eds), pp. 141156. Iowa State University Press, Ames, IA. Rhoades KR & Rimler RB (1987) Effects of Pasteurella multocida endotoxins on turkey poults. Avian Dis 31: 523526. Rhoades KR & Rimler RB (1989) Fowl Cholera. In: Pasteurella and Pasteurellosis (Adlam C & Rutter JM, eds), pp. 95144. Academic Press Limited, London. Rhoades KR & Rimler RB (1990) Pasteurella multocida colonization and invasion in experimentally exposed turkey poults. Avian Dis 34: 381383. Rimler RB (1994) Presumptive identication of Pasteurella multocida serogroups A, D and F by capsule depolymerisation with mucopolysaccharidases. Vet Rec 134: 191192. Rimler RB (2001) Purication of a cross-protective antigen from Pasteurella multocida grown in vitro and in vivo. Avian Dis 45: 572580. Rimler RB & Rhoades KR (1989) Pasteurella multocida. In: Pasteurella and Pasteurellosis (Adlam C & Rutter JM, eds), pp. 3773. Academic Press Limited, London. Rimler RB & Rhoades KR (1994) Hyaluronidase and chondroitinase activity of Pasteurella multocida serotype B:2 involved in hemorrhagic septicaemia. Vet Rec 134: 6768. Ruffolo CG, Tennent JM, Michalski WP &Adler B (1997) Identication, purication, and characterization of the type 4 mbriae of Pasteurella multocida. Infect Immun 65: 339343. Ruffolo CG, Jost BH & Adler B (1998) Iron-regulated outer membrane proteins of Pasteurella multocida and their role in immunity. Vet Microbiol 59: 123137. Scharmann WR, Drzeniek R & Blobel H (1970) Neuraminidase of Pasteurella multocida. Infect Immun 1: 319320. Schenkein HA, Barbour SE, Berry CR, Kipps B & Tew JG (2000) Invasion of human vascular endothelial cells by Actinobacillus actinomycetemcomitans via the receptor for platelet-activating factor. Infect Immun 68: 54165419. Serino L &Virji M (2002) Genetic and functional analysis of the phosphorylcholine moiety of commensal Neisseria lipopolysaccharide. Mol Microbiol 43: 437448. Snipes KP & Hirsh DC (1986) Association of complement sensitivity with virulence of Pasteurella multocida isolated from turkeys. Avian Dis 30: 500504. Snipes KP, Ghazikhanian GY & Hirsh DC (1987) Fate of Pasteurella multocida in the blood vascular system of turkeys following intravenous inoculation: comparison of an encapsulated, virulent strain with its avirulent, acapsular variant. Avian Dis 31: 254259. Snipes KP, Hansen LM & Hirsh DC (1988) Plasma- and iron- regulated expression of high molecular weight outer membrane proteins by Pasteurella multocida. Am J Vet Res 49: 13361338. St Michael F, Li J & Cox AD (2005a) Structural analysis of the core oligosaccharide from Pasteurella multocida strain X73. Carbohydr Res 340: 12531257. St Michael F, Li J, Vinogradov E, Larocque S, Harper M & Cox AD (2005b) Structural analysis of the lipopolysaccharide of Pasteurella multocida strain VP161: identication of both Kdo- P and Kdo-Kdo species in the lipopolysaccharide. Carbohydr Res 340: 5968. St Michael F, Vinogradov E, Li J & Cox AD (2005c) Structural analysis of the lipopolysaccharide from Pasteurella multocida genome strain Pm70 and identication of the putative lipopolysaccharide glycosyltransferases. Glycobiology 15: 323333. Steenbergen SM, Lichtensteiger CA, Caughlan R, Garnkle J, Fuller TE & Vimr ER (2005) Sialic acid metabolism and systemic pasteurellosis. Infect Immun 73: 12841294. Sterner-Kock A, Lanske B, Uberschar S & Atkinson MJ (1995) Effects of the Pasteurella multocida toxin on osteoblastic cells in vitro. Vet Pathol 32: 274279. Straus DC & Purdy CW (1994) In vivo production of neuraminidase by Pasteurella haemolytica A1 in goats after transthoracic challenge. Infect Immun 62: 46754678. FEMS Microbiol Lett 265 (2006) 110 c 2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved 9 Pasteurella multocida pathogenesis Straus DC, Cooley JD & Purdy CW (1996) In vivo production of neuraminidase by Pasteurella multocida A:3 in goats after transthoracic challenge. Curr Microbiol 33: 266269. Swords WE, Buscher BA, Ver Steeg Ii K, Preston A, Nichols WA, Weiser JN, Gibson BW &Apicella MA (2000) Non-typeable Haemophilus inuenzae adhere to and invade human bronchial epithelial cells via an interaction of lipooligosaccharide with the PAF receptor. Mol Microbiol 37: 1327. Tabatabai LB & Zehr ES (2004) Identication of ve outer membrane-associated proteins among cross-protective factor proteins of Pasteurella multocida. Infect Immun 72: 11951198. Tatum FM, Yersin AG & Briggs RE (2005) Construction and virulence of a Pasteurella multocida fhab2 mutant in turkeys. Microb Pathog 39: 917. To H, Someno S & Nagai S (2005) Development of a genetically modied nontoxigenic Pasteurella multocida toxin as a candidate for use in vaccines against progressive atrophic rhinitis in pigs. Am J Vet Res 66: 113118. Townsend KM, Boyce JD, Chung JY, Frost AJ & Adler B (2001) Genetic organization of Pasteurella multocida cap Loci and development of a multiplex capsular PCR typing system. J Clin Microbiol 39: 924929 (Erratum in J. Clin. Microbiol. (2001) 2039, 2378). Truscott WM & Hirsh DC (1988) Demonstration of an outer membrane protein with antiphagocytic activity from Pasteurella multocida of avian origin. Infect Immun 56: 15381544. Tsuji M & Matsumoto M (1989) Pathogenesis of fowl cholera: inuence of encapsulation on the fate of Pasteurella multocida after intravenous inoculation into turkeys. Avian Dis 33: 238247. Weber DJ, Wolfson JS, Swartz MN & Hooper DC (1984) Pasteurella multocida infections. Report of 34 cases and review of the literature. Medicine 62: 133154. White DJ, Jolley WL, Purdy CW & Straus DC (1995) Extracellular neuraminidase production by a Pasteurella multocida A-3 strain associated with bovine pneumonia. Infect Immun 63: 17031709. Wijewardana TG & Sutherland AD (1990) Bactericidal activity in the sera of mice vaccinated with Pasteurella multocida type A. Vet Microbiol 24: 5562. Wijewardana TG, Wilson CF, Gilmour NJ & Poxton IR (1990) Production of mouse monoclonal antibodies to Pasteurella multocida type A and the immunological properties of a protective anti-lipopolysaccharide antibody. J Med Microbiol 33: 217222. Wilson BA, Zhu X, Ho M & Lu L (1997) Pasteurella multocida toxin activates the inositol triphosphate signaling pathway in Xenopus oocytes via G(q)alpha-coupled phospholipase C-beta1. J Biol Chem 272: 12681275. Yu H, Chokhawala H, Karpel R, Wu B, Zhang J, Zhang Y, Jia Q & Chen X (2005) A multifunctional Pasteurella multocida sialyltransferase: a powerful tool for the synthesis of sialoside libraries. J Am Chem Soc 127: 1761817619. Zywietz A, Gohla A, Schmelz M, Schultz G & Offermanns S (2001) Pleiotropic effects of Pasteurella multocida toxin are mediated by Gq-dependent and -independent mechanisms. Involvement of Gq but not G11. J Biol Chem 276: 38403845. FEMS Microbiol Lett 265 (2006) 110 c 2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved 10 M. Harper et al.