REVIEW: MAJOR PATHOGENIC COMPONENTS OF PASTEURELLA MULTOCIDA AND MANNHEIMIA (PASTEURELLA) HAEMOLYTICA ISOLATED FROM ANIMAL ORIGIN

By

Ragy S. Seleim

Bacteriology Department, Animal Health Research Institute, Nadi El-Seed St. Dokki, 12311 Cairo, Egypt.

E-mail: Seleim_ragy@hotmail.com.

Keywords

Pasteurella multocida, Mannheimia haemolytica, Pathogenic components

ABSTRACT

Pasteurella multocida and Mannheimia (Pasteurella) haemolytica are causitive agents of several economically significant veterinary diseases occuring in numerous species viz: cattle, buffaloes, sheep, goats, poultry, turkeys, rabbits, horses and camels.  Serious infectious diseases as fowl cholera, bovine hemorrhagic septicemia, and porcine atrophic rhinitis were caused by P. multocida, whereas  M. haemolytica is the causitive agent of shipping fever or pneumonic pasteurellosis. Despite the application of advanced investigation and diagnostic techniques on both the organism and the affected animal species, still Pasteurella infections continue to contribute to heavy losses in the animal production as well as a hazardous threat to human health world wide. The application of the advanced diagnostic techniques as electron microscope investigation or DNA analysis of the microorganisms has helped a great deal in the elucidation of the virulence factors of the organism and their encounter in  pathogenesis, which helped in the development of potential candidate vaccines as well as new-targetted generations of antibiotics. Among the major factors encountered in the pathogenesis of Pasteurella are the polysaccharide capsule, endotoxines or lipopolysaccharede (LPS), Outermembrane proteins (OMP), fimbria and adhesins, Exotoxines, extracellular enzymes and other factors that still to be investigated and elucidated.

In this brief review, the major pathogenic potential components of  P. multocida and M. haemolytica were highlighted as well as their role in the pathogenesis process was discussed in different animal hosts.

P.  MULTOCIDA

Capsule

P. multocida capsule structure has a pivotal role in the determination of the serogroup type of the bacteria. Polysaccharides constituted the major part of the capsule and in some cases may be associated with lipoproteins. Five capsular serotypes were identified namely, 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) was composed of hyaluronic acid (hyaluronan) and other polysaccharides that could be digested by hyaluronidase enzyme. Proteins and lipids might also be intangled among the net of polysaccharides. Hyaluonic acid doesnot exert antiphagocytic activity as it was non immunogenic, but the capsular extract of the P. multocida serotype A involved a protein factor (300kDa) capable of inhibiting the bovine phagocytes (Seleim, 1993). The capsules of avian strains provides protection against the action of complement, but has no influence on the association of the organisms 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. Different results were obtained by other authors who reported that removal of hyaluronic acid capsule decreased the adhesion on cell surfaces and attributed the adherence capabilities of the bacteria to the polysaccharide capsule (Seleim 1993, Esslinger et al., 1994 and Seleim, 1997). The production of capsular material of P. multocida is affected by sub-minimal inhibitory concentration of antibiotics and the  capsular anigens can be detected in porcine tonsils colonised by type (D) strains which was mainly constituted chemically of unmodified heparin, that might have the same function of heparin. The capsular serotype (F) was chemically identified as chondroitin and was reported to infect turkeys (Champlin et al., 2002; De-Angelis et al., 2002).

Fimbriae

Electron microscope studies has elucidated the presence of fimbriae on P. multocida isolated from rabbit pharyngeal cells, pigs tonsilar and nasal cells and calves nasal and tracheal cells. Strains belonging to other serotypes (B, D and E) showed much less adhesive capacity on different cell models  viz:  bovine tracheal epithelial cells, porcine nasal and tracheal epithelial cells, rabbits nasal and tracheal epithelial cells as well as on HeLa cells. The attachment of P. multocida was found to be inhibited by N-acetyl-D-glucoseamine, which suggested that this amino-sugar could be the animal cell receptor to which the fimbriae attached. Different adhessiveness results were obtained following treatment of the organism with pronase, heat, acidity or homogenisation. Results confirmed the involvement of the fimbriae and/or the capsular material in the attachment process as the antifimbrial antibodies specifically inhibited the adherence capabilities of the organism (Seleim 1993; Esslinger et al. 1994). Porcine type A strains adhered more strongly than type D strains to porcine tracheal epithelial cells but the organism seemed to lack the ability to colonise the normal porcine nasal mucosa. There was poor correlation between fimbriae and experimental production of turbinate atrophy in pigs when different isolates were tested as induction of experimental pathogenesis was influenced by many bacterial-host factors. Toxigenic strains of P. multocida may have no detectable fimbriae or flagella, yet can colonise the porcine tonsils and respiratory tract, either alone or with Streptococcus suis inducing different pathogenesis mechanismms Esslinger, et al. 1994).

Outer membrane proteins (OPM).

Complex OMP profile of more than 40  protein bands was demonstrated in P. multocida isolated from haemorrhgic septicaemia cases. Correlation between the electrophoretic pattern and serotypic properties of isolates were established but no one single protein band could be identified as unique to all strains that caused haemorrhagic septicaemia. Common OMP bands (27kDa, 34kDa and 36kDa)  were common to all isolates regardless to serotype. One of the major virulence proteins in OMP was hemoglobin-binding protein which was considered as specific receptor for hemoglobin. The gene encoding the hemoglobin binding protein (hgbA) was identified and sequenced (Johnson et al., 1991; Seleim 1993; Seleim 1996; Bosch et al., 2002). Three different types of OMP patterns were detected I, II, and III from atrophic rhinitis strains. These different types were categorized based on the mobility of the heavy (H) and weak (W) protein bands between 28 and 40kDa. Protein H of P. multocida was found to be similar to the pore protein of Enterobacteriaceae in their surface location, affinity to peptidoglycane, trypsine resistance and resistance to SDS solubilisation. Moreover, the protein H forms an immunogenic complex with lipopolysaccharides.It was not possible to detect protein in the OMP that correlated absolutely with pathogenic strains, but it was observed that OMP type I strains were  highly pathogenic to pigs causing acute atrophic rhinitis, whereas type II and III were  less pathogenic. Bovine strains of type A examined for OMP patterns appeared analogous, and there was slight differences in their protein band profile (Seleim, 1996 ; Gadliero et al., 1998). Serum from vaccinated animals contained antibodies reacted only with few OMP bands.

OMP preparations from avian P. multocida identified a 50 kDa protein has been found to inhibit phagocytic capacity of avian phagocytes. Differences between isolates of the one serotype from fowl cholera were recognised by the position of one of the major proteins in the 34kDa - 38 kDa region and the 34 kDa Protein reacted with serum from chickens experimentally infected with P. multocida of the same serotype (Truscott and Hirsh 1988, Johnson et al., 1989 Irland et al., 1991).  .

Lipopolysaccharide ( LPS)  endotoxines

LPS is a major virulence factor and  played an essential role in causing diseases as haemorrhagic septicaemia in buffalos. Examination of P. multocida strains from different animals confirmed that LPS from P. multocida were slightly similar to LPS of Enterobacteriacae. It was reported that LPS was responsible for the 1-16 somatic serotypes, and when examined electrophoretically the LPS was of low molecular weight. It was confirmed that LPS of P. multocida was shorter than that of Enterobacteriaceae especially those of E.coli  and S. typhimurium. (Horadagoda et al., 2001). P. multocida strains from atrophic rhinitis (type D) showed at least six electrophoretic types of LPS and these frequently coincided both with a certain cell OMP type and with the presence and absence of the pathogenic character of the strain.

Antibody reacting with the LPS of type A strains has given protection against murine and rabbit infection, whereas the role of LPS appears to play a subordiate role in protection with type B. Avian strains from fowl cholera showed some degree of resistance to complement  and serum resistance which was 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 susccepatability. LPS was found to stimulate TNF-a release from bovine alveolar macrophages and many other tumour inhibition factors and interleukines  were released and trigered by the mitogenic action of the endotoxins. The mitogenic action was explained through the disturbance in the host cellular mechanisms, leading to cellular proliferation and blocking apoptosis (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 was a protein of estimated molecular weight ranging from 112kDa to 160 kDa and can be recovered from sonic and culture fluids. Dermonecrotizing actiovity was considered one of the toxic action which encluded, cytotoxicity for embryonic bovine lung cells, lethality, mucoid diarrhoea or splenic atrophy in mice, turbinate atrophy in a range of animals including pigs, rats, rabbits and goats, vascular endothelial damage in the liver of pigs and rats.  Other authors reported the mitogenic and lethal action of the toxines as well as its inhibition of bone cell differentiation as well as inhibition of osteoblast and stimulation of osteoclast activity. Atrophic rhinitis in pigs was basically due to the action of the DNT on the nasal bones. The DNT gene has been cloned into E. Coli  and the recombinant toxin used to produce atrophic rhinitis in pigs. Examination of the biochemical mechanism of action of the toxin on embryonic bovine lung cells did not reveal changes similar to those induced by other bacterial toxins, e.g. in cell ultra-structure protein or nucleic acid  turn over, intracellular concentrations of ATP (Kamps et al., 1990; Lax and Chanter 1990; Lax and Grigoriadis 2001; Rubies et al., 2002). 

Commercial vaccines includes formaldehyde-treated whole cells or formaldehyde-detoxified crude bacterial extracts of  toxigenic organisms were available. Moreover, the toxin and its gene have been sequenced and recombinant derivatve of the toxin have been assessed for the efficacy as much potent vaccine production. Toxoid prepared from purified toxin is effective against the systemic effect of the toxin in rats and against turbinate atrophy in pigs. Serotype B has been reported as potentially active toxin producing, (Rimler and Rhoades; 1989 Thurston et al. 1991). DNT has been associated with atrophic rhinitis in pigs, other hosts were found also to produce the toxins as poultry, calves, cats, dogs, and rabbits. Few human isolates were toxigenic and were reported from respiratory tract infections (Chrisps and Foged 1991; Lax and Grigoriadis 2001;  Lax and Thomas 2002; Rubies et al., 2002). 

Multocidin or sidophores

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). Siderophores selectively bind ferric iron and were involved in receptor-specific iron transport into the bacterial cell. Several types of siderophores were synthesized by P. multocida and M. haemolytica. The organism was 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 OMP including an 82kDa receptor protein for transferrin-bound iron. This capacity was observed in bovine strains and not in avian strains, and was restricted to acquisition from bovine transferrin .Sidophores might be also related to the hemoglobin binding protein but the protein structure, mechanism of action as well as the gene responsible for expresion were different (Bosch et al., 2002).

Extracellular enzymes

 Lipase was ovserved by many strains of P. multocida isolated from different animals and man. The lipase activity was demonstrated on Tween 20, Tween 40, Tween80 and Tween 85. Several Lipases were separated by sepharose 2B column which were considered a potential virulence factors for this organism. Hyaluronidase is another enzyme produced by P. multocida.  Its production appeared to be limited to type B strains and its production was correlated to the virulence of strains isolated from haemorrhagic septicemia. Whereas neuraminidase activity was detected in most sertogroups from different pathogenic species. High neuraminidase production was correlated to higher virulence of strains. Avian strains were repoted as lacking extracellular enzymatic activity. Yet many genes were identified for secreting extracellular enzymes from different species, that expressed high virulence potentials (Carter and Chengappa 1980; Fuller et al.,2000; Pratt et al., 2000).

Plasmids

Plasmids have been recovered from P. multocida that was isolated from various animal species. These plasmids conferred antibiotic resistance (R factor) as well as encoded many toxins. Avian strains contained plasmids correlated to complement resistance which was particularly conserved to the avian isolates. Plasmid profile was considered as virulence marker of P. multocida as well as an essential epidemiological tool for identifying different strains from the same phenotype. P. multocida can be easily transformed with plasmids by electroporation process which subject the bacterial cell membrane to a short electric shock leading to bacterial transformation (Jablonski et al. 1992 Rubies et al., 2002).

Mannheimia (Pasteurella) haemolytica

Capsules

M. haemolytica capsule structure was identified as a polyscharide basic structure produced during the logarithmic phase of growth, and can be visualized in organisms grown in vitro and in vivo. The same material was present in the alveoli of experimentally and naturally infected cattle and sheep. Capsules affected adversely the interaction of the organisms and alveolar macrophages. It was found that acapsule mutant of M. haemolytica was easily phagocytized than the capsuled wild strains. Furthermore, it was reported that, the capsular material may interact with pulmonary surfactant, thereby facilitating local adherence of the organism to different host cells (Brogden et al. 1989, Whiteley et al.1990, Czuprynski et al. 1991;  Highlander, 2001). Serotypic differentiation was based on sugar composition of the capsule as well as the composition of LPS. This same material appears to be a means of attachment to epithelial cells and other surfaces as well as to withstanding the activity of phagocytes and complements. Serotype A2 was composed of sialic acid, commonly found in host membranes, this component is purely nonimmunogenic. On the other hand capsular polysaccharide may be strongly antigenic if it contained surface protein which conctitute the potential immunogenic antigens. Different capsular extraction procedure affected the amount of protein extracted with the capsule. For example,  saline extraction revealed  fewer protein bands in the sodium dodicyl sulphate  polyacrylamide gel electrophoresis analysis (SDS-PAGE) than salicylate extraction which was protein rich extract. Capsular polysaccharide uncontaminated by LPS does not stimulate interleukin-1 and tumour necrosis factor released by bovine phagocytes due to the lower antigenic structure (Adlam et al, 1986; McVey et al., 1990; Chae et al., 1990; Highlander 2001).

Exotoxin

M. haemolyica produces a heat-labile protein cytotoxin, The addition of serum or serum proteins increases the speed and concentration of toxin production. In addition, antigenic profiles, particularly in the higher kilodalton range were found to be affected  by the presence of serum protein in culture media. The age of the culture also influenced the expression of exotoxines, which acted directly on the  host cell surface causing cytolysis (pore forming action). These damage was caused only on the suseptaible cells by formig transmembrane pores. Leukotoxines were specifically toxic only for leucocytes, and in particular ruminant  leukocytes, which accounted for its terminology as  leukotoxin. The most susceptible cells included bovine macrophages, lymphocytes, neutrophils from most ruminant species and cultured lymphoma cells. Additionally the leukotoxin lysed platelets, from different animals especially ruminant species. This species cells susceptibility to leukotoxines were directly correlated to the high susceptibility of ruminants and low susceptibility of non-ruminants to infection by M. haemolytica biotype A (Clinkenbeard et al.1989. Majury and Shewen 1991 Confer and Durham 1992). Another degenerative effect of leukotoxin included stimulation of respirtory burst and degranulation of neutrophils as well as generation of arachidonic acid metabolites with potent chemotactic activity. Leukotoxin was found to be protein about 100 kDa that is heat-labile, oxygen stable, pH stable and water soluble. The structural gene for the cytotoxin shares significant sequence homology with the haemolysin of E. Coli and the two proteins of M. haemolytica leukotoxin, designated LKIA and LKIC were homologous to the E. coli HlyA  and HlyC products respectively. There was also sequence homology with the leukotoxin gene of Morganella morgani, Proteus vulgaris and Proteus mirabilis.  Immunological cross-reactivity were also demonstrated between the leukotoxines and the haemolysin of Actinobacillus pleuropneumoniae. A pure DNA sequence encoding M. haemolytica leukotoxin has now been patented and E. coli transformed by a plasmid vector containing the leukotoxin gene sequence and used for production of recombinant leukotoxin crude leukotoxin which administered through the gut to induce a pulmonary immune response in calves (Chang et al. 1986; Devenish et al. 1989; Bowersock et al. 1992; Cravens, 1996;   Schaller et al,. 2000;   Lo, 2001).

Outer membrane proteins (OMP)

Examination of OMP preparations by SDS-PAGE showed major differences between strains of M. haemolytic isolated from the same or different host species. Therefor this phenomenon was used to differentiate between the isolates. Within M. haemolytica, the two biotypes (A) and (T) were also distinguishable by  their LPS SDS-PAGE profile. Yet the individual serotypes were not accurately identifiable by this method due to the great similarity in their protein band resolution. Protein components of the outer membrane were most likely located on the surface of  and their expression differed according to whether the organisms are grown in vivo or in vitro  Nevertheless, antigens unique to in-vivo growth were recognized by addition of  serum derived from animals vaccinated with killed M. haemolytica. which suggested that these antigens were either precursors or shared the antigenic determinants  of other M. haemolytica proteins (Kaights et al. 1990; Rossmanith et al. 1991; Confer et al.1992).

It was also reported that the protein surface composition of M. haemolytic was modified by the availability of iron and iron-regulated OMP may be expressed in vivo. Properties of the organism such as surface structure hydrophobicity, adherence and other protein related virulence characteristics were probably affected by the conditions of bacterial growth. The isolation of outer membranes and inner membranes of M. haemolytica Al allowed for identification of their major proteins. Two major proteins. 30kDa and 42kDa were detected as well as several minor proteins were also found. Antibodies against the 30 kDa protein were able to recognise 30kDa and 15 kDa proteins in all serotypes of M. haemolytica.   Many methods of OMP extractios were reported for use as antigen in serological detection  and surveillance of infection as well as in preparation of vaccines. do not release significant amounts of the two major OMP. (Cravens et al. 1991; Morck et al. 1991; Simons et al., 1992;  Highlander 2001)

Lipopolysaccharide (LPS)

The basic structure of LPS from M. haemolytica was reported similar to that of other Gram negative bacteria. LPS components were estimated as  10-25 % of the dries cell wall. Its composition differed between vivo grown bacteria and those grown in vetro, and these difference are associated with differences in relation to opsonophagcytosis and complement dependent killing. Moreover, in vetro grown bacteria showed different LPS structure depending on the composition of the culture media used. The LPS constituted a major surface antigen and the somatic serotype was defined according to the antigenic components in their structure. M. haemolytica serotypes of biotype (A) possessed rough type LPS while biotype (T) contains smooth LPS. It was demonstrated that LPS could directly produce bovine endothelial cell injury in vitro. The mechanism of injury was not clarified but it was observed that this toxic effect can be reduced by netrophils. LPS was also found to stimulate  tumour necrosis factor (TNF) release  from bovine alveolar macrophages as well as other cytokines. (Sutherland et al., 1990; Adlam 1992; Utley et al. 1992, Tsai et al. 1988, Brieder et al. 1990, Bienhoff et al. 1992 and Rimsay et al. 1981,  Cravens 1996; Lo 2001; Highlander 2001)

Adhesins

Two types of fimbriae were demonstrated in M. haemolytica, the large and rigid which measured 12nm width, while the smaller and flexible measured 5nm width. In vivo similar structures were seen especially when the organisms were recovered from lavage fluids from an experimentally or naturally infected calves and in organisms adherent to trachael epithelium in a naturally infected calf. The large rigid fimbriae are composed of 35 kDa subunits these fimbrial proteins were proved to be highly immunogenic and provided high antibody titers in the serum of immunized animals as well as contributed in the blockage of colonization of invading pathogens. These characters facilitated the usage of fimbrial protein in vaccine production Failure to demonstrate the fimbrial protein may reflect failure in the method of preparation rather than a variable capacity of the organisms to produce them (Morck et al. 1987; Potter et al. 1988;  Morck et al., 1989;  Lo 2001; Highlander 2001).   

Extracellular enzymes

The majority of M. haemolytica A serotype, but not T serotype, strains produced neuraminidase (which in some literature was reportedto be  cell-associated and in others in culture supernatants. A neutral proteases were also reported which targeted the immunoglobulins which directly influence the immune response of the host. Little was known about the haemolysin that gaves the species its name. Yet some authors reported greater similarity between the M. haemolytica hemolysines and other Gram negative hemolysines and was not plasmidal mediated. (Frank and Tabatabai 1981  (Otulakowski et al, 1983; Chang et al, 1987; Cravens, 1996; Highlander 2001;  Lo, 2001)

Plasmids

Plasmid-mediated antibiotic resistance has been demonstrated in M. haemolytica). The presence of plasmids was not a uniform phenomenon among all species but they were recorded in certain cohorts of animals (Each strain carried only one plasmid, and plasmids are generally small (less than 3.4 Mda). A relationship between drug resistance and plasmid isolation has been found in a limited number of serotype 1 field isolates. Not all plasmids present were associated with antibiotic resistance, conversely multiple resistance could be found in the absence of plasmids but some plasmids were correlated to toxin or other virulence protein production (Rossmanith et al. 1991; Haghour et al. 1987).; Chang et al. 1987 Highlander 2001).   

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First published January 2005