Pathogen Characteristics
Top of pageApproximately 20 different species of the genus Pasteurella have been identified using phenotypic and genetic analyses. Of these species, P. multocida is the most prominent of pathogens in domestic animals, causing severe diseases and major economic losses in the cattle, pig, sheep and poultry industries. Isolates are classified into five groups based on capsular antigens namely, A, B, D, E or F and 16 serotypes based on lipopolysaccharide (LPS) antigens. Strains of serogroup A are recognized as the primary cause of fowl cholera and most frequently designated as A:1, A:3 or A:4 (Adler et al., 1999), whereas isolates of serogroups B, D and F are less frequently associated with the disease (Wilson et al., 1993). Atrophic rhinitis of pigs is associated predominantly with capsular type D isolates, bovine and porcine pneumonia are associated mainly with capsular type A strains (Frank, 1989) and haemorrhagic septicaemia of cattle and buffaloes is caused exclusively by capsular type B and E isolates (Carter and Alwis, 1989).
The organisms are small Gram-negative rods or coccobacilli. They are non-motile, non-spore-forming and facultatively anaerobic. Enriched media are most suitable for cultivation of P. multocida. On serum agar, fluorescent, intermediate and blue colonies are produced whereas no growth occurs on MacConkey’s agar. Pasteurella multocida organisms produce oxidase, catalase and indole, and reduce nitrates. They do not produce hydrogen sulfide or urease, and fail to use citrate or liquefy gelatin. Glucose and sucrose are always fermented with the production of acid only. Most strains also ferment sorbitol. Some strains ferment arabinose, xylose and maltose, whereas salicin and lactose are almost invariably not fermented.
The organism is destroyed at 60°C in 10 min, in 0.5% phenol in 15 min, in 1:5000 solution of bichloride mercury and 3.5% cresol in a few minutes. In manure and decomposing carcasses, P. multocida have been observed to be infective for a month and three months, respectively. Avian strains showed some degree of resistance to complement and serum resistance, which is considered as an indicator of virulence of P. multocida for turkeys (Morishita et al., 1990; Ramdani and Adler, 1991). Clinical isolates of P. multocida from cattle exhibited serum resistance while isolates from asymptomatic cattle varied in serum susceptibility.
Recent classification of P. multocida strains of avian, ovine and porcine origin is based on molecular mass heterogeneity of OmpA and OmpH (Davis et al., 2003). Nineteen distinct outer membrane protein (OMP) profiles (OMP-types) of avian strains of P. multocida are identified mainly on molecular mass heterogeneity of the heat-modifiable (OmpA) and porin (OmpH) proteins (Davies et al., 2003). The colorimetric bactericidal assay using XTT may be a valuable assay to differentiate virulent from avirulent strains of avian P. multocida (Choia et al., 1995).
Factors that contribute to virulence of P. multocida strainsCapsule.
P. multocida capsule structure has a pivotal role in the determination of the serogroup type of the bacteria. Five capsular serotypes were identified; A, B, D, E and F. Strains within each serogroup may be further classified by the 16 somatic antigen types. The capsule of type (A) is composed of hyaluronic acid (hyaluronan) and other polysaccharides that could be digested by hyaluronidase. Proteins and lipids might also be tangled within the ‘net’ of polysaccharides. Hyaluronic acid does not exert anti-phagocytic activity as it is non-immunogenic, but the capsular extract of the P. multocida serotype A involves a protein factor of 300 kDa capable of inhibiting bovine phagocytes (Seleim, 1993). The capsules of avian strains provide protection against the action of complement, but have no influence on the association of the organism with phagocytic cells. Removal of hyaluronic acid capsule increases both adhessiveness of the organisms to the animal cell surfaces as well as its susceptibility to phagocytosis.
Fimbriae
Using electron microscopy, pili with at least two distinct morphologies, rigid and curly, are observed on strains of P. multocida isolated from pigs with atrophic rhinitis. Rigid pili had a strong tendency to lie flat along the side of the outer cell membrane (Isaacsona and Trigob, 1995). Toxigenic strains of P. multocida may have no detectable fimbriae or flagella, yet can colonize the tonsils and respiratory tract, inducing different pathogenesis mechanisms (Esslinger et al., 1994).
Outer membrane proteins (OMP)
A complex OMP profile of more than 40 protein bands was demonstrated in P. multocida isolated from haemorrhagic septicaemia cases. However, no single protein band could be identified as unique to all strains that caused haemorrhagic septicaemia. OMP bands, 27-kDa, 34-kDa and 36k-Da, are common to all isolates regardless of serotype. One of the major virulence proteins of the OMPs is haemoglobin-binding protein, which is considered as a specific receptor for haemoglobin. The gene encoding the haemoglobin binding protein (hgbA) has been identified and sequenced (Johnson et al., 1991; Seleim 1993; Seleim 1996; Bosch et al., 2002). Three different types of OMP patterns were categorized, I, II and III, based on the mobility of the heavy (H) and weak (W) protein bands between 28 and 40 kDa. Protein H, which forms an immunogenic complex with lipopolysaccharides of P. multocida was found to be similar to the pore protein of Enterobacteriaceae. OMP type I strains are highly pathogenic to pigs. OMP patterns in bovine strains of type A appeared analogous, (Seleim, 1996). Serum from vaccinated animals contains antibodies that react only with a few OMP bands. OMP (50-kDa) from avian P. multocida inhibits phagocytic capacity of avian phagocytes.
Lipopolysaccharide ( LPS) endotoxins
LPS from P. multocida, which is similar to LPS of Enterobacteriacae, play an essential role in causing diseases like haemorrhagic septicaemia in buffaloes. Antibody reacting with the LPS of type A strains has given protection against mouse and rabbit infection, whereas the role of LPS appears to play a subordinate role in protection with type B. LPS stimulate tumour necrosis factor (TNF-alpha) release from bovine alveolar macrophages and many other tumour inhibition factors and interleukins are released and triggered by the mitogenic action of the endotoxins (Beinhoff et al, 1992; Horadagoda et al., 2001; Lax and Thomas 2002).
Exotoxins
The production of toxins by P. multocida, in particular those of capsular serotype D, produced a factor designated dermonecrotic toxin (DNT). Purified DNT (112 kDa to 160 kDa) can be recovered from sonic and culture fluids. Dermonecrotizing activity of DNT is of pivotal importance in pathogenesis and is considered a potential candidate for vaccine.
Multocidin or siderophores
Iron is an essential element for P. multocida growth. When grown in conditions of iron deprivation the organism is able to secrete a growth enhancing factor that functions as a siderophore (multocidin). Several types of siderophores are synthesized by P. multocida. The organism is also capable of obtaining iron for growth by a non-siderophore-mediated mechanism. Such acquisition was associated with the production of a number of high molecular weight, iron-regulated OMPs including an 82-kDa receptor protein for transferrin-bound iron. This capacity is observed in bovine strains but not in avian strains (Bosch et al., 2002).
Extracellular enzymes
Lipase is released by many strains of P. multocida and is considered as a potential virulence factor for this organism. Hyaluronidase produced by P. multocida type B is correlated with the virulence of strains isolated from haemorrhagic septicaemia. Whereas magnitude of neuraminidase activity is correlated with higher virulence of strains isolated from different species. Avian strains are lacking extra cellular enzymatic activity (Carter and Chengappa 1980; Fuller et al., 2000; Pratt et al., 2000).
Plasmids
Plasmids recovered from P. multocida isolated from various animal species confer antibiotic resistance (R factor) and encode many toxins. Avian strains contained plasmids correlated with complement resistance. Plasmid profiles serve as virulence markers as well as epidemiological tools for identifying different strains from the similar phenotypes of P. multocida (Jablonski et al., 1992; Rubies et al., 2002).