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PubMed1999

Characterisation of dermonecrotic toxin-producing strains of Pasteurella multocida subsp. multocida isolated from man and swine.

Thirty-six isolates, from man or swine, of Pasteurella multocida subsp. multocida producing (n = 13) or not producing (n = 23) the dermonecrotic toxin (DNT) were studied by numerical analysis, capsular typing and ribotyping. Toxigenic strains were also characterised by restriction fragment length polymorphism (RFLP) of the toxA gene and pulsed-field gel electrophoresis (PFGE). Numerical analysis differentiated the Pasteurella species and subspecies, but did not discriminate between toxigenic and nontoxigenic strains. RFLP demonstrated that toxA was located in a conserved part of the chromosome of all toxigenic strains. Ribotyping provided evidence of a close association between DNT production and one of the six EcoRI ribotypes designated as E2. In contrast, PFGE provided evidence for significant DNA polymorphism amongst the toxigenic strains. Results of phenotypic and genotypic studies suggested that toxigenic strains do not form a clone within the subspecies multocida. No difference was found between toxigenic strains of porcine or human origin by biochemical characterisation, capsular serotyping or genomic typing methods.

Animals↗
PubMed1999

Genomic DNA restriction site heterogeneity in bovine Pasteurella multocida serogroup A isolates detected with an rRNA probe.

A total of 81 Pasteurella multocida isolates from healthy and diseased dairy and beef cattle originating from various geographical locations was examined by rRNA gene restriction site polymorphism analysis (ribotyping), restriction endonuclease analysis (REA), SDS-PAGE analysis of whole-cell (WCP) and outer-membrane (OMP) proteins, and capsule and somatic serotyping. Bacterial strains were isolated from nose, lung and in one case testicle, of Holstein and cross-bred beef cattle. The isolates represented for the most part serogroup A3 (88%). Ribotyping was performed on DNA digested with HaeII, electrophoresed and then hybridised with 32P-labelled 16S-23S rRNA from Escherichia coli. Six ribotypes (R1-R6) and 10 REA types were found among the 81 isolates with similar discrimination index (DI) of c. 0.60. Protein profiles revealed reproducibility and high levels of polymorphisms among lung isolates. Isolates were compared according to their geographical habitat, their isolation from dairy or from beef cattle and from nasal cavities or lungs. No correlation was apparent between geographical locations and ribotypes. Overall, isolates obtained from dairy cattle were predominantly R1, whereas those obtained from beef cattle were equally distributed between R1 and R2. R1 was more representative of lung isolates. For some strains, particularly the single isolate ribotypes, good correlation was achieved between WCP analysis, REA types and ribotypes. For others, REA to some extent and WCP profiles were able to discriminate among isolates within ribotypes. The data suggest that a combination of ribotyping, REA and WCP analysis is useful for investigating the epidemiology of bovine P. multocida serogroup A.

Animals↗
PubMed2003

Alteration of DNA adenine methylase (Dam) activity in Pasteurella multocida causes increased spontaneous mutation frequency and attenuation in mice.

Pasteurella multocida is one of the primary bacterial pathogens associated with bovine respiratory disease (BRD) complex. Relatively few virulence factors of P. multocida have been characterized, and there is a need for improved vaccines for prevention of BRD. In other Gram-negative species, DNA adenine methylase (Dam) regulates the expression of virulence genes, and appropriate expression of Dam is required for virulence. In this study, the authors cloned and sequenced the P. multocida A1 dam gene and demonstrated that it is able to restore Dam function in an Escherichia coli dam mutant. When P. multocida dam was placed under the control of a constitutively expressed promoter on a plasmid, it caused an increased spontaneous mutation rate in P. multocida. In addition, the plasmid-mediated alteration of Dam production in P. multocida caused it to be highly attenuated in mice. These findings indicate that appropriate expression of Dam is required for virulence of P. multocida, which is believed to be the first report that Dam is required for virulence of a species in the Pasteurellaceae. Therefore, Dam may function as a virulence gene regulator in the Pasteurellaceae, similar to previously reported findings from other Gram-negative species.

Animals↗
PubMed2004

Revised description and classification of atypical isolates of Pasteurella multocida from bovine lungs based on genotypic characterization to include variants previously classified as biovar 2 of Pasteurella canis and Pasteurella avium.

Strains deviating in key phenotypic characters, mainly isolated from cases of bovine pneumonia in five European countries, were genotyped in order to examine their genotypic relationship with Pasteurella multocida. Twenty-two strains of Pasteurella avium biovar 2, including variants in indole, xylose and mannitol, 18 strains of Pasteurella canis biovar 2 and variants of this taxon, five strains of P. multocida subsp. septica showing variations in indole and ornithine decarboxylase, nine strains of P. multocida subsp. multocida showing variation in ornithine decarboxylase and mannitol, and type strains of the subspecies of P. multocida were included. Ribotyping was used to examine the relationship of the strains, and 13 types, each containing between one and 20 isolates, were observed. Identical ribotypes were observed in some cases for P. avium biovar 2 and either P. canis biovar 2 or P. multocida subsp. septica. ITS (16S-23S rRNA internal transcribed spacer) fragment-length profiling showed identity of the majority of strains (47 of 52), representing all four taxa, with only five divergent strains. A 16S rRNA sequence comparison of 11 strains representing the main ribotype clusters showed 99.9 % similarity to the type strain of P. multocida subsp. multocida, but only 97.4 % similarity was obtained to P. canis (biovar 1) and 93.7 % to P. avium (biovar 1). A species-specific PCR test for P. multocida gave a positive result with biovar 2 variants of P. avium and P. canis. DNA-DNA hybridizations between strains of P. multocida, biovar 2 variants of P. avium and P. canis, and P. multocida subsp. septica confirmed similarity at the species level. It is proposed, on the basis of genotypic similarity, that P. multocida be reclassified to include the biovar 2 variants of P. avium and P. canis and that the existence of the biovar 2 variants of P. avium and P. canis is highly questionable. It is concluded that the redefined P. multocida is genotypically homogeneous, although phenotypically diverse lineages exist with respect to ornithine decarboxylase, indole and mannitol, characters that have been regarded as essential for identification to the species level. A formal reclassification of the species is not possible, however, since too few strains have been found to vary in these key characters. Considering the phenotypic diversity of P. multocida, identification will have to depend partly on genotypic methods and the source host also seems important for safe diagnosis.

Animals↗
PubMed2004

Genetic diversity among Pasteurella multocida strains of avian, bovine, ovine and porcine origin from England and Wales by comparative sequence analysis of the 16S rRNA gene.

Genetic diversity among 86 Pasteurella multocida isolates was investigated by comparative sequence analysis of a 1468 bp fragment of the 16S rRNA gene. The strains included 79 field isolates recovered from birds (poultry) (22), cattle (21), pigs (26) and sheep (10) within England and Wales, four Asian isolates associated with bovine haemorrhagic septicaemia, and the type strains of the three subspecies of P. multocida. Dulcitol and sorbitol fermentation patterns were also determined to establish correlations between subspecies status and phylogenetic relatedness. Nineteen 16S rRNA types were identified, but these were clustered into two distinct phylogenetic lineages, A and B. Sequences within lineages A and B had a mean number of nucleotide differences of 21.12+/-3.90. Isolates within lineage A were associated with birds, cattle, pigs and sheep, whereas those belonging to lineage B were recovered from birds and a cat. Eighty-seven per cent of the isolates were classified as P. multocida subsp. multocida by dulcitol and sorbitol fermentation patterns, but these have diverse 16S rRNA gene sequences that were represented in both lineages A and B. Avian P. multocida subsp. septica isolates were associated exclusively with lineage B, but bovine P. multocida subsp. septica isolates were present in lineage A. P. multocida subsp. gallicida isolates of avian, bovine and porcine origin represent a homogeneous group within lineage A, but they have the same 16S rRNA type as certain P. multocida subsp. multocida isolates. These findings provide strong support for the view that dulcitol and sorbitol fermentation patterns are inaccurate indicators of genetic relatedness among P. multocida strains. Avian capsular type B isolates and capsular type B and E isolates associated with haemorrhagic septicaemia of cattle and water buffaloes are closely related and form a distinct cluster within lineage A. The current subspecies nomenclature of P. multocida neither accurately reflects the 16S rRNA-based phylogenetic relationships among isolates nor does it adequately encompass the full range of diversity within the species. The study provides a 16S rRNA-based evolutionary framework that will form the basis of further studies into the genetic diversity of P. multocida and will also help in the reclassification of the species.

Animals↗
PubMed2006

Immunization with outer membrane proteins of Pasteurella multocida (6:B) provides protection in mice.

The immunoprotective efficacy of Pasteurella multocida (6:B) outer membrane proteins (OMPs) was examined in the mouse model. Bacterial OMPs were extracted using sarkosyl method and analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and immunoblotting. Prototype vaccines were prepared using OMPs with adjuvants including dioleoyl phosphatidyl choline-based liposome and Montanide ISA206 water-in oil-in water emulsion. Antibody response to the vaccine was monitored using indirect enzyme linked immunosorbent assay. The results of the study showed that immunized mice had high titre with both the formulations. The vaccinated mice were able to survive a live virulent bacterial challenge. Based on the findings of the study it can be inferred that OMPs are important determinants of immunoprotection hence can serve as vaccine candidates against haemorrhagic septicaemia.

Animals↗
PubMed1996

Differentiation of Pasteurella multocida subspecies multocida isolates from the respiratory system of pigs by using polymerase chain reaction fingerprinting technique.

PCR fingerprinting technique was applied to subtype 44 Pasteurella multocida subspecies multocida (P.m.sp.m.) isolates from the respiratory system of pigs. Two single primers were tested for their abilities to generate individual fingerprints by using PCR. Primer 1 (core sequence of the M13 phage) grouped the 44 P.m.sp.m. strains into five distinct fingerprinting profiles, while primer 2 ((GACA)4) grouped them into seven profiles. The results suggest that PCR fingerprinting is an efficient technique to detect DNA polymorphism in the species P.m.sp.m. This technique could be used to differentiate P.m.sp.m. strains of the same capsular serotype.

Animals↗
PubMedDate not supplied

Studies of the influence of ochratoxin A on immune and defense reactions in the mouse model.

In the mouse model, the mycotoxin ochratoxin A has a non-selective suppressive effect on various immune and defence reactions. Apart from weight depression, lymphopenia, neutrophilia and eosinophilia, antibody-producing cells, antibody titres in blood serum and phagocytosis of Escherichia coli by blood phagocytes become suppressed. Moreover, immunized animals show a lower survival rate after experimental infection with Pasteurella multocida as well as an increase in oxygen radicals in blood cells.

Animals↗
PubMed1996

A minimal medium for growth of Pasteurella multocida.

We developed a minimal medium supporting the growth of both toxigenic and nontoxigenic strains of Pasteurella multocida to optical densities of > 0.5 (600 nm). P. multocida P1059 (ATCC 15742), one of a number of strains which can cause fowl cholera, was used as the model strain in this study. The medium was composed of 17 ingredients including cysteine, glutamic acid, leucine, methionine, inorganic salts, nicotinamide, pantothenate, thiamine, and an energy source. Leucine was not required for growth but was stimulatory, and thiamine could be replaced by adenine. An additional 46 strains of P. multocida were tested, and 40 out of 46 (87%) strains grew as well as strain P1059 through a minimum of 10 serial transfers. P. multocida toxin (PMT) was produced when cells of a known toxigenic strain (P4261) were cultivated in the minimal medium. No growth of Pasteurella Haemolytica or Pasteurella trehalosi strains was observed in this minimal medium.

Animals↗
PubMed1997

Protection against haemorrhagic septicaemia induced by vaccination of buffalo calves with an improved oil adjuvant vaccine.

An experimental oil adjuvant vaccine was developed against haemorrhagic septicaemia, a disease of cattle and buffalo caused by Pasteurella multocida serotype B and E. Mineral oil, Mercol 52, was used as adjuvant together with Span 85 and Tween 85 as emulsifiers. The vaccine was evaluated by single dose intramuscular immunisation of 1-2 year old buffalo calves. IgG and IgM class antibodies were determined by ELISA. The group of animals immunised with the experimental oil adjuvant vaccine showed a high titre of the IgG class of antibodies measured at 300 days post vaccination. To compare the protective efficacy of the vaccine with the commonly used broth bacterin, another group of buffalo calves was immunised by broth bacterin. This group showed a low level of IgG antibodies. Protection was assessed by challenge with 10(9) viable bacteria of P. multocida type B:2,5 administered subcutaneously, 250 days post vaccination. Animals vaccinated with the experimental oil adjuvant vaccine were fully protected. The other groups of animals, vaccinated with broth bacterin or used as control (non-vaccinated), developed symptoms of haemorrhagic septicaemia. A strong relationship between IgG but not IgM class antibody level and resistance to challenge was observed. The experiment demonstrated that the experimental oil adjuvant vaccine was superior to broth bacterin in providing protection against experimental haemorrhagic septicaemia in young buffalo calves beyond 250 days.

Adjuvants, Immunologic↗
PubMed2006

Pasteurella multocida pathogenesis: 125 years after Pasteur.

Pasteurella multocida was first shown to be the causative agent of fowl cholera by Louis Pasteur in 1881. Since then, this Gram-negative bacterium has been identified 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 identified to date include capsule and lipopolysaccharide. 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 identified 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 identified, including those required for initial attachment and invasion of host cells and for persistence in a relatively nutrient poor and hostile environment.

Animals↗