R-Plasmids in Pasteurella multocida.

Multiple drug-resistant strains of Pasteurella multocida were associated with a high incidence of fatal pneumonia in feedlot cattle. A representative strain, CAH160, resistant to tetracycline (Tc), streptomycin (Sm), and sulfonamide (Su) was studied. The minimal inhibitory concentration (MIC) of Tc was 32 μg/ml while Sm had an MIC of 256 μg/ml. Plasmid DNA was isolated from CAH160 by cesium chloride-ethidium bromide centrifugation. Agarose gel electrophoresis showed that at least three distinct species of plasmid DNA were present. DNA isolated from CAH160 was used to transform Escherichia coli K12 strain C600 r_k^?m_k^?. Transformants resistant to Tc; to Sm, Su; and to Tc, Sm, Su were obtained. Contour length measurements of plasmid DNA isolated from transformant cells showed that Tc resistance was associated with a 3-Mdal plasmid (pSR10), while Sm, Su resistance resided on a 2.7-Mdal molecule (pSR11). More than 20% of the transformants were resistant to Tc, Sm, Su and contained both plasmid species. In E. coli the MIC of Tc was 256 μg/ml and that of Sm was 64 μg/ml. The buoyant density of pSR10 was 1.699 g/cm³, while the density of pSR11 was 1.709 g/cm³.     

Cloning and expression of the dermonecrotic toxin gene of Pasteurella multocida ssp. multocida in Echerichia coli

A DNA library of Pasteurella multocida ssp. multocida strain CVI 47459 was constructed in the Lambda GEM-11 vector. Recombinant clones that encoded dermonecrotic toxin (DNT) were identified immunologically with antiserum raised against purified DNT. By comparing the DNA restriction maps of the immunoreactive recombinants, we located the DNT gene. Hybridization studies with 10 strains of P. multocida ssp. multocida suggested that strains that do not produce the DNT do not contain sequences homologous to the DNT gene.     

Naturally acquired Pasteurella multocida subsp. multocida infection in a closed colony of rabbits: characteristics of isolates

Twelve litters, comprising 41 rabbits aged 35 to 60 days old, in a closed university colony, were monitored for acquisition of nasal Pasteurella multocida subsp. multocida infection. Isolates from 11 infected rabbits were characterized by colonial morphology, capsular type, biotype and antibiotic resistance. Selected isolates were further characterized by somatic antigen typing. Two major strains of P. multocida subsp. multocida were detected in the colony. One strain had mucoid colonies, fermented few carbohydrates and was serotype A:5, whereas, the other strain had smooth iridescent colonies, non-typeable capsular antigen, type 3 somatic antigen and fermented more than twice as many carbohydrates.     

Heat-labile toxin-producing isolates of Pasteurella multocida from rabbits.

Five of one hundred forty seven isolates of Pasteurella multocida from rabbits were found to produce heat-labile toxin. Each isolate was assayed for the ability of potassium thiocyanate (KSCN) extracts to cause dermonecrosis in guinea pig skin, ability of bacteria or filtrates to cause cytotoxicity in cell cultures, and reactivity with monoclonal antibodies to heat-labile P. multocida toxin. Five capsular type D isolates produced dermonecrosis and reacted with monoclonal antibodies to toxin. Filtrates of all five of these isolates were cytotoxic for cell cultures. Potassium thiocyanate extracts of all five isolates caused pleuritis and pneumonia in rabbits after intranasal inoculation. Turbinate atrophy was seen in 5 of 19 rabbits inoculated intranasally with toxic extracts. Heat-labile toxin was not produced by 109 capsular type A isolates or 19 nontypable isolates.     

Candidate vaccine antigens and genes in Pasteurella multocida.

Pasteurella multocida is the causative agent of fowl cholera and other diseases of production animals. Isolates are classified into five groups based on capsular antigens and into 16 serotypes based on LPS antigens. Strains causing fowl cholera are most frequently designated A:1, A:3 or A:4. Whole cell bacterins can provide some degree of protection, but only against the homologous LPS serotype. There is good evidence that cross-protective antigens are expressed only under in vivo conditions. Empirically derived, live, attenuated vaccines can protect against heterologous serotypes, but because the basis for attenuation is undefined, reversion to virulence is not uncommon. Work in our laboratory is aimed at using a variety of approaches to identify potential protective antigens or virulence genes to be used as candidates for attenuating mutations or as the basis for vaccine antigen delivery systems. The gene encoding an outer membrane protein, Oma87, which is a homologue of the D15 protective antigen of Haemophilus influenzae, was cloned and sequenced. Rabbit antiserum prepared against recombinant Oma87 could passively protect mice against infection. Type 4 fimbriae form the basis of vaccines against ovine footrot and bovine keratoconjunctivitis. We have identified type 4 fimbriae on the surface of P. multocida, purified the fimbrial subunit protein, PtfA, and determined its N-terminal amino acid sequence. Subsequent cloning of the ptfA gene and its inactivation will now be used to assess the importance of type 4 fimbriae in virulence. There has long been anecdotal evidence for the importance of capsule in virulence, but unequivocal genetic evidence for such a role is lacking. We have cloned and characterised the capsule biosynthetic locus in P. multocida A:1 and identified four bex genes involved in capsule transport and genes encoding enzymes involved in the biosynthesis and transfer of the N-acetyl glucosamine and glucuronic acid components of the capsule. It has been suggested that the low concentration of available iron in vivo acts as an environmental cue for expression of cross-protective antigens. Accordingly, we have cloned and characterised the gene encoding transferrin binding protein, Tbpl, so that its role in immunity and virulence can be investigated. Although P. multocida is not normally considered haemolytic, we have observed haemolysis under anaerobic conditions. Standard library construction and screening resulted in the identification of the mesA gene which encodes an esterase enzyme resulting in a haemolytic phenotype under anaerobic conditions. Virulence studies with mesA- mutants were performed to assess its role in pathogenesis. Using a promoterless phoA gene vector system, the cloning of proteins homologous to known surface proteins of other species as well as proteins unique to P. multocida, allowing their potential as vaccine components to be assessed.     

Partial characterization of plasmids from rabbit isolates of Pasteurella multocida.

Plasmids have not been reported for isolates of Pasteurella multocida from rabbits. We assayed 28 isolates of rabbit P. multocida for plasmids and sought to determine whether or not plasmid presence correlated with clinical or pathologic findings, serotype, toxin production, possession of pili, or biochemical characteristics. Fourteen isolates bore a single 1.6 Md (covalently closed circular form in 0.7% agarose gels) plasmid. An additional isolate had two plasmids which migrated as a closely-spaced doublet, centered around 1.6 Md. Eleven isolates appeared to have identical plasmids, according to Hae III and Hinf I digests. The apparent linear size of this common plasmid in 2% agarose gels was 2.1 Md, as calculated from the sums of the sizes of Hae III or Hinf I digestion fragments. Linearization of the common plasmid with Msp I produced an apparent size of 2.5 Md in 0.7% agarose gels. No correlations between presence of the common plasmid and somatic serotype, toxigenicity, presence of pili, antimicrobial resistance, selected biochemical characteristics, anatomic site from which the bacteria were cultured, or disease status of the host were found.     

Morphology of Pasteurella multocida bacteriophages

Twenty-one tailed phages with icosahedral heads belong to the Myoviridae, Siphoviridae, and Podoviridae families and to four morphological types. Type AU, with 10 phages, has a contractile tail and is morphologically identical with coliphage P2. Lysates contain contracted tail sheaths assembled end-to-end and abnormal structures with long tails and multiple tail sheaths. Types C-2 and 32, with one and three phages, respectively, have long, noncontractile tails. Type 22 includes seven phages, has a short tail, and resembles coliphage T7. Our results agree with previous biological data and suggest that types AU, C-2, 32, and 22 correspond to four different phage species.     

Survival of toxigenic Pasteurella multocida in aerosols and aqueous liquids.

The survival of toxigenic Pasteurella multocida in air and liquids was studied to identify possible risk factors in the etiology of atrophic rhinitis. In aerosols, at low relative humidity (28%), the viability of toxigenic P. multocida 5 min after aerosolization was at least 22% of its initial value. Viability at low relative humidity declined to 8% after 45 min. Viability at high relative humidity (79%) was 69% after 5 min and declined to 2% after 45 min. Survival of toxigenic P. multocida in liquids depended on storage and constituents in the liquid. Toxigenic P. multocida became nonculturable 1 to 14 days after inoculation in water and artificial seawater, depending on the storage temperature. Toxigenic P. multocida stored at 37 degrees C could be detected for up to 6 days in pig slurry and more than 36 days in Bacto Tryptose broth and nasal lavages. However, in Bacto Tryptose broth and nasal lavages stored at 4 degrees C, P. multocida was detected for up to 14 days whereas at 15 and 37 degrees C it was detected for more than 49 days. These results suggest that aerosols and fomites can play a role in the transmission of atrophic rhinitis.     

Genome sequence of Pasteurella multocida subsp. gallicida Anand1_poultry

We report the finished and annotated genome sequence of Pasteurella multocida gallicida strain Anand1_poultry, which was isolated from the liver of a diseased adult female chicken. The strain causes a disease called "fowl cholera," which is a contagious disease in birds. We compared it with the published genome sequence of Pasteurella multocida Pm70.