Mannheimia haemolytica and the pathogenesis of enzootic bronchopneumonia

Mannheimia (M.) haemolytica (formerly Pasteurella [P.] haemolytica) is the primary aetiological agent of pneumonic pasteurellosis--one of the most important respiratory diseases in cattle and sheep. While bovine pneumonic pasteurellosis is regarded to be mainly caused by M. haemolytica serotype A1, and in Germany during the last years also by serotype A6, sheep can be infected by all serotypes although there is an increased prevalence of serotypes A2 and A5-7. The obligate pathogenicity of M. haemolytica is proven by isolation of pure cultures from pneumonic lungs as well as by infection studies. Knowledge about the virulence mechanisms of M. haemolytica and their molecular basis are fragmentary, most probably due to the complex gene regulation of virulence associated factors in lung tissues. This review summarizes the current literature covering virulence factors to substantiate a model of pathogenesis. After serotype A1 strains have colonized the bovine upper respiratory tract they replace other serotypes by mechanisms unknown to date. After fulminant proliferation in the upper respiratory tract the microorganisms colonize the lower respiratory tract, finally entering alveolar spaces. An inflammatory cascade is initiated by M. haemolytica LPS and Leukotoxin, causing activation of the complement system and release of cytokines. Pathognomonic for bovine pneumonic pasteurellosis is the strong influx of neutrophiles accompanied by accumulation of fibrin, finally causing necrosis of alveolar spaces. Depending on lesion size this fibronecrotizing pneumonia can result in death of the animals. In addition, possible protective antigens are discussed. There is still a great effort in the development of efficacious vaccines against pneumonic pasteurellosis in cattle and sheep caused by various M. haemolytica serotypes worldwide. The scarce knowledge concerning presence and distribution of virulence associated factors in M. haemolytica strains and their role in pathogenesis made it difficult to determine a suitable vaccine candidate in the past. In addition, there is lack of knowledge concerning the variability of virulence factors in individual isolates. Genome sequence analysis of M. haemolytica, enabling proteomics and transciptomics, hopefully will give new insight into the pathogenesis of pneumonic pasteurellosis.     

Comparison of single strains of four serotypes of Pasteurella haemolytica biotype A in experimental pneumonia of sheep

Single strains of serotypes A1, A2, A7 and A9 of Pasteurella haemolytica were separately used in combination with Mycoplasma ovipneumoniae to reproduce pneumonia. Macroscopically and microscopically the pneumonias associated with individual serotypes were similar and it is concluded that serotypes of P haemolytica isolated with low frequency in field disease may be equally virulent to common serotypes.     

Occurrence and diversity of plasmids in ovine isolates of Pasteurella haemolytica.

160 ovine isolates of Pasteurella haemolytica, representing each of the 16 serotypes and also untypable strains, were examined for plasmid content. Plasmid DNA was identified in, and prepared from, strains of serotypes A2, T3, A14 and A16 and also from an untypable strain. The relationship between the plasmids present in the different strains was examined both by restriction fragment profile analysis and by DNA/DNA hybridisation. Both methods gave broadly similar results and showed that each serotype tended to contain either a single plasmid species, or a limited range of species, and that structural similarities could traverse serotype boundaries. None of the plasmid-bearing strains showed any significant level of resistance to a range of antibiotics.     

Genomic distribution of a serotype 1-specific antigen-coding DNA fragment of Pasteurella haemolytica.

A genomic fragment of Pasteurella haemolytica biotype A coding for a serotype 1-specific agglutinating antigen was used as a probe in a series of hybridization experiments to determine distribution of the fragment in various P. haemolytica serotypes as well as other bacteria. Results showed presence of the fragment in seven out of the 12 serotypes tested, all of which belonged to biotype A. Two other serotypes belonging to biotype A, all three serotypes belonging to biotype T, two Pasteurella multocida isolates and Escherichia coli did not have the fragment in their genome. Thus the expression of the P. haemolytica biotype A serotype 1-specific agglutinating antigen (PHA1SAA) seems to be due to serotype-specific regulation of protein expression rather than to genetic deletion. Differences in methylation of the PHA1SAA-coding fragment was also noted in DpnI and Sau3AI genomic DNA digests from the various serotypes analyzed by Southern blot. However, no apparent correlation was observed between methylation and PHA1SAA expression. E. coli with a recombinant plasmid containing a homologous genomic fragment derived from P. haemolytica serotype 2 also expressed PHA1SAA.     

Pasteurella species isolated from the bovine respiratory tract and their antimicrobial sensitivity patterns

Pasteurella haemolytica biotype A, serotype 1 (P haemolytica A1) was the most commonly isolated Pasteurella species from 80 calves examined at necropsy from 40 outbreaks of respiratory disease, the majority of which were pathologically confirmed as bovine pneumonic pasteurellosis (transit fever; shipping fever). Similarly, nasopharyngeal swabs from in-contact and apparently healthy calves indicated the widespread presence of P haemolytica A1. Pasteurella multocida and other serotypes of P haemolytica A1 were found including six isolations of P haemolytica T10, a fairly common pathogen in sheep. Approximately two-thirds of the isolates were tested for their antimicrobial sensitivity patterns and the degree of sensitivity for P haemolytica A1, the most frequently isolated serotype, was chloramphenicol (100 per cent), sulphamethoxazole trimethoprim (98 per cent), oxytetracycline (80 per cent), ampicillin (85 per cent), penicillin (82 per cent), streptomycin (3 per cent) and lincomycin (1 per cent).     

Protein electrophoretic pattern of Pasteurella haemolytica.

Electrophoresis in polyacrylamide gels of the constituent proteins of the 12 serotypes and an untypable strain of Pasteurella haemolytica showed a pattern of bands that divided the group into two. This division conformed to the A and T biotype groupings of Smith (1959) although the serotype A9 showed only minor band difference from the three T serotypes 3, 4 and 10. It was not possible by this method to separate all the type strains from each other by the specific recognition of the patterns of protein mobilities produced.     

Pneumonic pasteurellosis: examination of typable and untypable Pasteurella haemolytica strains for leukotoxin production, plasmid content, and antimicrobial susceptibility

Plasmid DNA screening experiments were conducted to determine whether a relationship existed between the presence of plasmids and antibiotic resistance in Pasteurella haemolytica or the capability to produce hemolysin or leukotoxin (cytotoxin). Regardless of plasmid content, all P haemolytica isolates produced characteristic hemolysis on blood agar plates. Similarly, standardized suspensions of living bacteria and sterile concentrated (approx 200:1) culture supernatant from strains representing each of the 15 recognized P haemolytica serotypes and 7 field strains of P haemolytica (biotype A, serotype 1) produced leukotoxin, which was detected by their capability to cause inhibition of the luminol-dependent chemiluminescence response of bovine neutrophils. However, neither living bacterial suspensions nor concentrated culture supernatant from 4 untypable P haemolytica strains or a P multocida strain caused an inhibition of the luminol-dependent chemiluminescence response. The production of neither hemolysin nor leukotoxin by P haemolytica seemed to be plasmid mediated. Leukotoxin production is apparently a stable phenotypic characteristic of pathogenic P haemolytica strains, and the gene(s) coding for this activity is probably located on the bacterial host chromosome. Antibiotic susceptibility profiles were determined for the different bacterial strains. Studies of ampicillin and penicillin resistance in 8 P haemolytica (biotype A, serotype 1) strains provided evidence that the plasmid, with size of approximately 5,200 base pairs, may code for their resistance to these compounds.     

Characterization and comparison of antimicrobial susceptibilities and outer membrane protein and plasmid DNA profiles of Pasteurella haemolytica and certain other members of the genus Pasteurella.

The outer membrane protein (OMP), plasmid, and antimicrobial resistance profiles of Pasteurella haemolytica serotypes 1 through 12, a bovine isolate of P multocida, a chicken isolate of P multocida, and an unidentified Pasteurella species of bovine origin were examined. Isolates of P haemolytica serotypes belonging to the same biotype possessed similar OMP profiles. Biotype A isolates contained 2 prominent OMP of 43 kilodaltons (kD) and 29 kD, whereas biotype-T serotypes contained 3 major OMP of 43, 36, and 25 kD. The major OMP profiles of the 2 P multocida isolates and the unidentified Pasteurella species were different from each other and from P haemolytica isolates. Plasmid DNA screening indicated both plasmid-containing and plasmid-free P haemolytica and P multocida isolates. Multiple drug resistance was found in pasteurellae isolates with and without plasmids. However, a relationship between drug resistance and plasmid isolation was found in 3 of 4 haemolytica serotype 1 field isolates, all of which contained a 2.51-megadalton plasmid and had multiple drug resistance for benzylpenicillin, ampicillin, streptomycin, and tetracycline.     

Comparison of antibody responses in cattle to outer membrane proteins from Pasteurella haemolytica serotype 1 and from eight untypeable strains.

Membrane associated proteins from 8 untypeable Pasteurella haemolytica strains were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and compared with those of P haemolytica serotypes 1 and 2. Cattle antisera obtained from P haemolytica serotype 1 vaccine trials were used in immunoblotting assays to compare the membrane proteins from the 8 untypeable strains with those from P haemolytica serotypes 1 and 2. Densitometry was used to identify bands, and using linear regression analyses, the peak area optical densities (measuring antibody response) were correlated to lesion scores from the vaccinated calves. Significant antibody responses to proteins of 99, 69, 60, 55, 47, 45, 39, 33, 30, 16, and 14.5 kDa were detected for 4 or more of the 8 P haemolytica untypeable strains. Serotypes 1 and 2 of P haemolytica contained a comigrating 30-kDa protein. Antibody responses to proteins of 39, 33, and 32.5 kDa were significant for 3 of the untypeable strains and had significant correlation to lesion scores. Antibody responses to various other proteins were significant for 2 untypeable strains each.     

Serotyping of Mannheimia (Pasteurella) haemolytica isolates from the upper Midwest United States.

Mannheimia (Pasteurella) haemolytica biotype A serotype1 (A1) is the primary bacterial agent responsible for the clinical signs and pathophysiologic events in bovine pneumonic pasteurellosis. The goal of this study was to determine the prevalence of other serotypes of M. haemolytica biotype A organisms obtained from the upper Midwest diagnostic laboratories. A total of 147 M. haemolytica isolates were collected from Minnesota, South Dakota, and Michigan. Isolates were tested against M. haemolytica antisera obtained from the National Animal Disease Center, Ames, Iowa. Results indicated that M. haemolytica serotype 1 represented approximately 60%, serotype 6 represented 26%, and serotype 2 represented 7% of the total examined isolates. In addition, 7% of the isolates were serotype 9, 11, or untypable. This finding suggests that M. haemolytica serotypes other than serotype 1 can be isolated from the lung lesions of diseased cattle and seem to be capable of causing the pathologic changes observed in the lung with pneumonic pasteurellosis.     

Purification and partial characterization of a macrophage cytotoxin from Pasteurella haemolytica

A protein from Pasteurella haemolytica that was highly immunogenic and toxic toward bovine alveolar macrophages was partially purified. When isolated from culture supernatants of P haemolytica serotype 1 or serotype 6, the protein reacted on Ouchterlony immunodiffusion tests with antisera from 12 serotypes of P haemolytica, but did not cross-react with antisera to serotypes of P multocida. This indicated that the protein may be specific for P haemolytica. Bacteria were grown in dialysis culture in a brain-heart infusion and calf-serum growth medium. The protein was isolated from the medium by ultrafiltration and size-exclusion chromatography and has a molecular weight of approximately 150,000 daltons. The protein, which is highly immunogenic and has the characteristics of a virulence factor, is common to all serotypes of P haemolytica, and may be an effective agent for immunization against P haemolytica in cattle.     

Experimental immunisation of lambs against pneumonic pasteurellosis

Methods of immunising lambs against pneumonic pasteurellosis, caused by several serotypes of Pasteurella haemolytica, were assessed in specific pathogen free lambs. Lambs were vaccinated intramuscularly with sodium salicylate extract (SSE) of P haemolytica, either alone or in combination with heat-killed organisms (HKO). SSE of P haemolytica type A1 protected vaccinated lambs against pneumonia resulting from challenge with the homologous serotype. SSE of type A2 also provided some protection but this was improved by vaccination with a combination of SSE and HKO.     

Phenotypic and genotypic characterization of Mannheimia (Pasteurella) haemolytica-like strains isolated from diseased animals in Denmark.

Trehalose-negative strains of the Pasteurella haemolytica complex have recently been transferred to a new genus, Mannheimia. This genus presently consists of five named species: M. haemolytica, M. glucosida, M. granulomatis, M. ruminalis and M. varigena. The purpose of this study was to investigate the occurrence of these species and lesions associated with these isolates in Denmark. In all 106 M. haemolytica-like strains isolated from pathological material from cattle, sheep, pigs and hares submitted to the Danish Veterinary Laboratory between 1994 and 1998 were investigated. Phenotypic characterization and ribotyping were used for identification in addition to sequencing of the 16S rRNA genes for selected strains. The species allocation was determined by comparison to results from a previous polyphasic taxonomic study. Seventy-one percent of the strains belonged to M. haemolytica, 18% to M. varigena and 8% to unnamed groups within the genus Mannheimia. Single isolates identified as M. glucosida and P. trehalosi, respectively, were detected. Two isolates belonged to M. granulomatis. Forty-three percent of the strains belonged to serotype 1, 41% were untypeable, while the rest belonged to serotypes 2, 7, 9, and 16. The present investigation also showed that a simplified phenotypic characterization using Diatabs Diagnostic Tablets (Rosco, Denmark) represents a useful method for obtaining a quick and reliable species identification. Finally, the investigation confirmed that serotyping does not represent a reliable method for species identification. The heterogeneity of species associated with bovine "pasteurellosis" should be considered in future studies to improve our understanding of the pathogenesis of pneumonic disease.     

Characterisation and biological activity of monoclonal antibodies specific for Pasteurella haemolytica A1 capsule and lipopolysaccharide.

Monoclonal antibodies (mAb) against both Pasteurella haemolytica A1 capsule and lipopolysaccharide (LPS) were produced. Anti-capsule mAb reacted with the homologous A1 serotype only, whereas mAb against LPS reacted with P. haemolytica serotypes A2, A5, A8, A12, A14 and A16 but not with 33 bacterial species or rough LPS mutant strains tested. Both capsule and LPS antigens were visualised on the surface of bacteria by immunogold electron microscopy. Neither of the mAbs demonstrated antibody-dependent complement-mediated killing in vitro but both facilitated phagocytosis in vitro.     

Investigation of outer membrane proteins of Pasteurella. 2: Iron-regulated outer membrane proteins of Pasteurella multocida and Pasteurella haemolytica

Pasteurella multocida and Pasteurella haemolytica produce specific proteins in the outer membrane under iron-depleted conditions. Pasteurella multocida serovar A expresses these proteins of molecular masses of 76 and 96 kDa as determined by electrophoresis. The analogous serovar D produces a further iron-regulated protein of 85 kDa. The Pasteurella haemolytica strains of serovar A1, A6 and T contain iron-regulated outer membrane proteins of molecular masses of 71, 77 and 100 kDa. These proteins possess binding positions for iron ions. Both Pasteurella multocida and Pasteurella haemolytica strains utilize iron from porcine and bovine transferrin, but not from haemin and haemoglobin.     

Atypical Pasteurella haemolytica type A from poultry

Atypical strains of Pasteurella haemolytica that failed to ferment maltose were isolated from nodular necrosis in the liver and heart blood of domestic fowl (Gallus domestica). These strains did not typically behave like either of the two well-known biotypes of P. haemolytica. The strains utilized trehalose and produced hydrogen sulfide (H2S), thus behaving like P. haemolytica type T, and produced acid in xylose but not in salicin, thus behaving like P. haemolytica type A. Most of the properties of the strains, however, conformed closely to those of P. haemolytica type A. Detailed characteristics of the isolates are described and discussed.     

Investigations on the species specificity of Mannheimia (Pasteurella) haemolytica serotyping

Eleven serotypes (1, 2, 5-9, 12-14 and 16) have been demonstrated within Mannheimia haemolytica. Subsequent serotyping of 166 Mannheimia haemolytica-like strains, genetically and phenotyphically distinct from Mannheimia haemolytica, and isolated from ruminants, pigs, hares and rabbits showed that 13.2% were typeable, 19 of which were serotype 11 representing strains now being classified as M. glucosida. In addition, three strains belonged to serotypes 6, 9 and 16, respectively. Additionally, the serotyping results of 98 (P.) haemolytica-like isolates from non-ruminant sources collected by the UK Veterinary Investigation Centres during the period 1982-1996 were investigated. None of these isolates have been kept, making further genetic characterization impossible. Among these isolates, 25.5% were typeable representing serotypes 1, 2, 3, 5, 6, 9, 10, 13 and 15. Substantial evidence has been reported indicating that M. haemolytica-like isolates from non-ruminant sources represent species different from M. haemolytica. The present investigation underlines that serotyping does not represent a reliable method for the identification of M. haemolytica or M. glucosida. These observations emphasize that extended phenotypic and genetic characterization is necessary for the proper identification of these organisms.     

Typing and virulence testing of Pasteurella haemolytica field strains

The greater part of 145 typed Pasteurella haemolytica strains from calf could be attributed to Type A 1. Hence, in the context of virulence testing, this is the most important type at present for calf. Clearly manifest pneumonia was caused in calf by other strains of Types A 2, A 6, A 12, and T 10 which were also tested for their virulence parameters. The same applied to a number of strains which had so far been characterised merely as T or A/T strains.     

Cytotoxic effect of serotypes of Pasteurella haemolytica on sheep bronchoalveolar macrophages

Ovine isolates of the 15 known serotypes found within the A and T biotypes of Pasteurella haemolytica were cytotoxic for sheep bronchoalveolar macrophages (BAM). Weaker toxicity for the same target cells was also expressed by non-serotypable ovine isolates of P. haemolytica. The results suggest that cytotoxicity for sheep BAM is a virulence factor common to both A and T biotypes of P. haemolytica.     

Experimental infection of sheep with Mycoplasma ovipneumoniae and Pasteurella haemolytica

A group of Caesarian-derived, colostrum-deprived lambs was inoculated intranasally and intratracheally with a virulent Mycoplasma ovipneumoniae isolate selected from ovine mammary studies and propagated in an ovine mammary gland. Other groups of lambs were inoculated with M. ovipneumoniae in combination with Pasteurella haemolytica type Al or P. haemolytica alone. The M. ovipneumoniae isolate alone did not induce any specific pneumonic lesions in the lambs and when combined with P. haemolytica type Al did not increase the severity of the P. haemolytica-type lesions. Fifty percent of lambs inoculated with P. haemolytica developed a purulent and exudative bronchopneumonia with pleurisy and high titres of P. haemolytica were recovered from these lesions.