Serological types of Pasteurella haemolytica in Kenya

The 12 known serotypes of P. haemolytica and two biotypes A and T were found to occur in Kenya. Biotype T was more common in disease conditions than biotype A, which was more common in the nasal passages of healthy animals. Only biotypes A strains were recovered from cattle and the majority were serotypes 1 and 2, but serotypes 4 and 11 were also isolated. All serotypes were found to occur in sheep and goats, but serotypes 3, 4, 6, 8 and 10 were more commonly associated with pneumonia. It was observed that chickens could harbour both biotypes A and T in pathological conditions. Biotype A serotype 2 was isolated from an adult wildebeest, but the prevalence of P. haemolytica in wild animals needs furter investigation.     

Use of a polymerase chain reaction method to detect the leukotoxin gene lktA in biogroup and biovariant isolates of Pasteurella haemolytica and P trehalosi

OBJECTIVE: To determine whether Pasteurella haemolytica and P trehalosi isolates possess the structural gene for Pasteurella leukotoxin lktA and whether beta-hemolytic activity of these isolates correlated with detection of the lktA gene. SAMPLE POPULATION: 147 P haemolytica isolates from 21 biovariant groups and 101 P trehalosi isolates from 7 biovariant groups. In addition, P multocida and organisms from 7 other genera were tested to establish specificity of the procedure. PROCEDURE: Isolates were observed for beta-hemolysis. A polymerase chain reaction (PCR) procedure was used to amplify the RTX domain of the Pasteurella lktA gene. RESULTS: The lktA gene was detected in 108 (44%) isolates, including 15 associated with respiratory tract disease. All but 2 (98%) of the isolates that had the lktA gene were beta-hemolytic when grown on sheep blood agar. The remaining 140 isolates were negative for the lktA gene and hemolytic activity. CONCLUSIONS AND CLINICAL RELEVANCE: Hemolytic activity of P haemolytica and P trehalosi isolates correlated with detection of the lktA gene for all but 2 isolates. However, 56% of isolates tested were negative for the lktA gene and beta-hemolytic activity. Leukotoxin production and secretion is a major virulence factor when other conditions are favorable for disease development. Therefore, identification of strains that possess the lktA gene may aid in the evaluation of the pathogenic potential of Pasteurella strains carried by wild and domestic animals.     

Susceptibility of Rocky Mountain bighorn sheep and domestic sheep to pneumonia induced by bighorn and domestic livestock strains of Pasteurella haemolytica.

Bighorn sheep were inoculated intratracheally with suspensions of nonhemolytic Pasteurella haemolytica biotype T (10(12) organisms) unique to wild bighorns, with beta-hemolytic P. haemolytica biotype T (10(12) organisms) isolated from clinically normal domestic sheep or intradermally with half a dose of a cattle vaccine containing P. haemolytica biotype A (10(5) organisms). The bighorn strain caused lobar necrotizing bronchopneumonia whereas both domestic livestock strains precipitated fatal septicemia and fibrinous bronchopneumonia. The serotypes given were T3, T4, T15 and A1 and these were recovered from lung lesions and other organs. In three trials, domestic sheep were inoculated intratracheally with suspensions of bighorn sheep pneumonic lungs, and two concentrations of the P. haemolytica bighorn strain (10(4) and 10(12) organisms). One of these sheep was inoculated intrabronchially. The domestic sheep experienced a transient fever and elevated white blood cell counts. After six days, none of the sheep had lung lesions and inoculated organisms could not be recovered. It is suggested that bighorn sheep are very susceptible to P. haemolytica from domestic livestock and should not be allowed in contact with sheep or cattle.     

Polymerase chain reaction techniques for differentiating cytotoxic and noncytotoxic Pasteurella trehalosi from Rocky Mountain bighorn sheep.

OBJECTIVE: To evaluate 2 polymerase chain reaction (PCR)-based methods for differentiating cytotoxic and noncytotoxic Pasteurella trehalosi from Rocky Mountain bighorn sheep (Ovis canadensis canadensis). SAMPLE POPULATION: 23 isolates of P. trehalosi from bighorn sheep in Colorado, including 18 from free-ranging herds and 5 from a captive herd. PROCEDURE: Using a sequence of the leukotoxin gene region of P. haemolytica serotype 1, 7 PCR primers were designed. A PCR amplification was performed on a sample of bacterial cell suspensions from pure cultures of P. trehalosi with known in vitro cytotoxic effects. The 2 most promising primer pairs were used in a study of 23 P. trehalosi isolates. Results were analyzed for association with cytotoxicity and 3 distinct ribotypes (Eco, Aco, and Bco). RESULTS: Significant associations were observed between in vitro cytotoxicity and PCR results for coding region, between ribotype Eco classification and PCR results for coding region, and between ribotype Eco classification and PCR results for promoter region. There was a negative association between ribotype Aco classification and PCR results for coding and promoter regions. CONCLUSIONS AND CLINICAL RELEVANCE: The PCR for the leukotoxin A coding region may be useful in differentiating cytotoxic from noncytotoxic P. trehalosi isolates recovered from bighorn sheep. It may be useful for studying epidemiologic features of pasteurellosis in bighorn sheep and for designing vaccines to protect wild sheep against pneumonia caused by P. trehalosi and P. haemolytica.     

Characterization of Pasteurella spp isolated from healthy domestic pack goats and evaluation of the effects of a commercial Pasteurella vaccine

OBJECTIVE: To characterize Pasteurella spp isolated from healthy pack goats and evaluate the effects of administration of a commercial Pasteurella vaccine. ANIMALS: 45 goats. PROCEDURE: Pharyngeal swab specimens and blood samples were collected on day 0 before vaccination with a Pasteurella (Mannheimia) haemolytica serotype A1 bacterin. Samples were also collected from 17 goats on days 21 and 35. Isolated Pasteurella spp were assigned to biovariant groups on the basis of results of biochemical utilization tests and serotyped. Serum antibody titers were determined. RESULTS: Multiple strains of Pasteurella spp were isolated from swab specimens and assigned to 30 nonhemolytic and 14 beta-hemolytic biovariant groups. The most common biovariant isolated was nonhemolytic P trehalosi belonging to group 2. This strain was isolated from 41 goats. Nonhemolytic P haemolytica strains were isolated from 31 goats, whereas beta-hemolytic strains of P trehalosi and P haemolytica were isolated from 8 and 35 goats, respectively. Vaccination with the A1 serotype did not affect the proportion of goats from which we isolated each biovariant group or the number of biovariant groups isolated. CONCLUSIONS AND CLINICAL RELEVANCE: Multiple strains of P haemolytica and P trehalosi belonging to nonhemolytic and beta-hemolytic biovariant groups were isolated from the pharynx of healthy domestic pack goats. Because hemolytic activity correlates with leukotoxin production, beta-hemolytic strains may have a greater potential to cause disease in naive populations of wild ruminants. However, vaccination with an A1 serotype bacterin did not decrease the proportion of culture-positive goats.     

Serotypes and electrophoretic protein profiles of Pasteurella haemolytica isolated from pneumonic ovine lungs.

Serotypes and SDS-PAGE protein profiles of P. haemolytica isolated from pneumonic ovine lungs were investigated. Of 268 P. haemolytica isolates, 232 (86.6%) were serotypable. A total of 12 serotypes were recognized in 20 different geographic origins of central Turkey. The most common serotype was A2, followed by A7, A1 and T4. Serotypes A13, A14, A16 and T15 could not be detected. In SDS-PAGE, marked differences between major bands of biotype A and T strains were found. In numerical analysis of protein profiles, biotype A and T strains were separated at 58% similarity level. Biotype A isolates produced a cluster at 80% similarity level, and biotype T isolates at 92% similarity level. No single cut off level was able to discriminate between each serotype studied and isolates could not be clustered on the basis of their geographic origins.     

Serological and molecular comparison of Mannheimia haemolytica and Pasteurella trehalosi strains isolated from wild and domestic ruminants in the French Alps

Over a period of 17 years, 84 bacterial isolates identified as Mannheimia haemolytica or M. glucosida, and 52 isolates identified as Pasteurella trehalosi were detected in the lungs of domestic and wild ruminants in the French Alps. The isolates were serotyped according to their surface capsular antigens, and those sharing common antigens were further characterized by pulsed field gel electrophoresis. The results showed that the bacterial isolates included in the study clustered according to the host species from which they were isolated. These findings indicate that the transmission of serotypes of M. haemolytica, M. glucosida or P. trehalosi from an animal host in which they are common to another species sharing the same geographical space may be a rare epidemiological event.     

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.     

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).     

Survey of restriction-modification systems and transformation in Mannheimia haemolytica and Pasteurella trehalosi

A significant obstacle to molecular studies of Mannheimia (Pasteurella) haemolytica, has been its resistance to genetic transformation. The lack of competence of many M. haemolytica strains has been attributed to the presence of restriction modification systems. In this study, representative strains of 12 M. haemolytica serotypes and four Pasteurella trehalosi serotypes were successfully transformed by electroporation using a recombinant vector derived from the native M. haemolytica A1 serotype plasmid pNSF2176. Transformation was achieved despite PCR-based evidence for the presence of genes encoding a type I restriction enzyme, phaI, and a type II restriction enzyme hsdM, in each of the M. haemolytica strains.     

Novel protease produced by a Pasteurella trehalosi serotype 10 isolate from a pneumonic bighorn sheep: characteristics and potential relevance to protection

A strain of Pasteurella trehalosi serotype 10, E(CO)-100, isolated from a bighorn sheep that had succumbed to pneumonic pasteurellosis during an epizootic, was compared to well-characterized strains of P. trehalosi serotype 10 and Mannheimia haemolytica serotype 1. The gene for leukotoxin A (lktA) from E(CO)-100 was sequenced and found to be identical on an amino acid basis to a published sequence for lktA from P. trehalosi serotype 10. However, the toxic activity in culture supernatant measured over time for E(CO)-100 was quite different from reference strains. Typically, the ability of the supernatant to lyse target cells increases over time corresponding to the logarithmic growth of the organism, peaks at mid to late phase, then declines gradually. Supernatant from E(CO)-100 exhibited a sharp decline in toxicity after mid-logarithmic growth to undetectable levels. Investigation of this anomaly using a commercial kit with a porcine gelatin/bovine albumin substrate matrix revealed high protease activity in the supernatant of this strain compared to another P. trehalosi serotype 10 and to a M. haemolytica serotype 1. Protease activity was also visualized using gelatin based zymogram gels. This protease was not substrate specific as it was shown to degrade leukotoxin. Activity was neutralized by bighorn sera in a titratable manner. There was an association between the ability to neutralize protease and low pneumonic lung scores in bighorn sheep experimentally challenged with E(CO)-100 (r=0.5, P=0.1). This previously unidentified protease may be an important protective antigen in vaccines designed to prevent pneumonic pasteurellosis resulting from P. trehalosi in bighorn sheep.     

Domestic sheep (Ovis aries) Pasteurellaceae isolates from diagnostic submissions to the Caine Veterinary Teaching Center (1990-2004)

A retrospective study of Pasteurellaceae isolated from domestic sheep (Ovis aries) was conducted. The aim was to identify Pasteurellaceae present in animals that were clinically healthy and others with evidence of respiratory disease. The bacteria had been isolated from samples submitted to the University of Idaho Caine Veterinary Teaching Center as part of disease diagnostic testing. The 844 isolates identified mainly three species of Pasteurellaceae: Mannheimia haemolytica, Pasteurella multocida, and Pasteurella (Bibersteinia) trehalosi. A total of 114 biovariants were identified among these three species. Individual biovariants were identified 1-180 times. Two of those (M. haemolytica 1 and P. (B.) trehalosi 2) constituted 36% of the isolates, and were the only biovariants sufficiently numerous to account for >7% of the total isolates. Samples were primarily submitted from sheep with signs of respiratory disease. Eighty percent of biovariants were identified most often in animals with signs of respiratory disease, but 26% of biovariants were isolated from both sheep with respiratory disease and apparently healthy sheep. P. multocida constituted 4.7% of isolates, and were exclusively associated with animals with respiratory disease. The ability of isolates to produce beta-hemolysis on culture media was not associated with animals with respiratory disease (odds ratio 0.77, 95% CI 0.50-1.19). The inference of this study is limited due to the retrospective study design. However, it is the first study that provides an extensive baseline list of biovariants associated with respiratory disease in domestic sheep.     

Response of sheep and cattle to combined polyvalent Pasteurella haemolytica vaccines

The antibody response to various combined polyvalent Pasteurella haemolytica vaccines was studied in sheep and cattle. In sheep, certain oil adjuvant vaccines gave rise to a better antibody response to P. haemolytica than an A1(OH)3-adsorbed vaccine. This finding, however, was not consistent for all serotypes, and with respect to P. multocida, oil adjuvants had no advantage. Furthermore, it was found that the removal of all the culture supernatant fluid during the production process had no deleterious effect on the antigenicity of the product. In cattle, good responses were obtained with both alum-precipitated and A1(OH)3-adsorbed vaccine where all culture supernatant fluid was not removed during the production process. No advantage was gained with oil emulsion vaccines. The degree of immunity afforded to mice and the antibody response to different serotypes of P. haemolytica varied considerably. Further detailed studies with respect to specific serotypes of P. haemolytica are therefore required.     

Seasonal incidence and antibiotic susceptibility patterns of Pasteurellaceae isolated from American bison (Bison bison).

Ninety pharyngeal tonsils were collected from 2-year-old American bison (Bison bison) bulls and sampled for members of the Pasteurellaceae family. Particular attention was paid to seasonal incidence and antimicrobial resistance in serotypes and biovariants. Multiple strains of Pasteurella haemolytica (39%), P. trehalosi (68%), P. multocida (34%) and Haemophilus somnus (13%) were cultured from 86 out of the 90 (96%) tonsil samples. Pasteurella trehalosi was the most common and evenly distributed of the organisms recovered. Pasteurella haemolytica was found in fewer numbers than P. trehalosi, but showed an increase in number of isolates recovered with each sampling period. Pasteurella multocida, both A and D capsular types, was recovered from all sampling periods. No serotype pattern was observed in any of the animal groups sampled. One hundred twenty-seven of 147 (86%) of the isolates were resistant to at least 1 antibiotic, 95/147 (65%) to at least 2 different antibiotics, and 16/147 (11%) to at least 3 antibiotics. The most common resistance pattern observed was to neomycin and spectinomycin (73/147) (49%).     

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.     

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.     

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.     

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.     

Plasmid profile analysis of bovine isolates of Pasteurella haemolytica

Pasteurella haemolytica isolates, obtained from cattle with respiratory tract disease, were characterized as to serotype, antimicrobial susceptibility, and plasmid content. Strains isolated from 2 groups of cattle were compared. Remarkable similarity was seen in the plasmid profiles of isolates of the same serotype. In contrast, isolates of 2 different serotypes had totally different plasmid profiles.     

Fatal Pasteurella haemolytica pneumonia in bighorn sheep after direct contact with clinically normal domestic sheep

Six Rocky Mountain bighorn sheep were raised in captivity from birth (n = 5) or taken from the wild as a lamb (n = 1). After the bighorn sheep were in captivity for over a year, 6 clinically normal domestic sheep were placed on the 2 ha of pasture on which the bighorn sheep were kept. Nasal swab specimens were obtained from all sheep at the time the domestic sheep were introduced. Pasteurella haemolytica was isolated from swab specimens obtained from 4 of 6 domestic sheep, but not from specimens obtained from the bighorn sheep. All 6 bighorn sheep died of acute hemorrhagic pneumonia after exposure to domestic sheep. Death in the bighorn sheep occurred on days 4, 27, 27, 29, 36, or 71 after initial exposure to domestic sheep. Pasteurella haemolytica was isolated from respiratory tract tissue specimens of all bighorn sheep at the time of death. None of the domestic sheep were clinically ill during the study. At the end of the study, 3 of 6 domestic sheep were euthanatized, and at necropsy, P haemolytica was isolated from 2 of them. The most common serotypes in bighorn and domestic sheep were P haemolytica T-3 and A-2. Other serotypes isolated included P haemolytica A-1, A-9, and A-11 in bighorn sheep and A-1 in domestic sheep. On the basis of results of this study and of other reports, domestic sheep and bighorn sheep should not be managed in proximity to each other because of the potential fatal consequences in bighorn sheep.