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Construction and virulence of a Pasteurella multocida fhaB2 mutant in turkeys.

Pasteurella multocida is the causative agent of fowl cholera. The organism can occur as a commensally in the naso-pharyngeal region of apparently healthy animals and it can be a primary or secondary pathogen in the disease process of birds. The complete genome of an avian strain of P. multocida has been sequenced and was shown to possess two filamentous hemagglutinin genes designated fhaB1 and fhaB2. Filamentous hemagglutinin transposon mutants of a bovine strain of P. multocida are attenuated in mice. Here, we report the construction of an fhaB2 P. multocida mutant in an avian strain P-1059 (A:3). The fhaB2 mutant and the parent were assessed for virulence in turkeys by intranasal and intravenous challenge. Inactivation of fhaB2 resulted in a high degree of attenuation when turkeys were challenged intranasally and to a lesser degree when intravenously administered. Resistance of the fhaB2 mutant and parent strain to killing by serum complement was similar.

Animals↗

Characterization of avian strains of Pasteurella multocida by restriction endonuclease and amplified fragment length polymorphism.

Avian strains of Pasteurella multocida were typed by employing restriction endonuclease analysis (REA) and single enzyme-amplified fragment length polymorphism (AFLP) to evaluate their applicability for epidemiological studies of fowl cholera outbreaks. A total of 72 strains isolated from different avian species (chicken, duck, turkey, quail and goose) belonging to various geographical regions of India were characterized. REA using two different enzymes HhaI and HpaII produced 9 and 18 clusters respectively, whereas Single enzyme-AFLP recognized 32 patterns out of 72 strains typed. The study indicated that REA using HpaII is a simple and resource efficient method, however, further typing with more stringent and rapid method like Single enzyme-AFLP, could drastically enhance investigation in epidemiological studies of fowl cholera outbreaks.

Animals↗

Identification of avian strains of Pasteurella multocida in India by conventional and PCR assays.

The prevalence of capsular and somatic serotypes were studied among 123 Pasteurella multocida strains isolated from chickens (n=94), ducks (22), quails (4), turkeys (2) and geese (1) from different geographical regions of India. All strains exhibited similar cultural and morphological characteristics. Ninety-two of the isolates belonged to serotype A:1, the most prevalent serotype, with serotypes A:3, A:1,3, D:3 and F:3 having two isolates each. Only one isolate was positive for serotypes A:4 and D:1. Twenty isolates were untyped. A multiplex capsular PCR assay generated amplicons of sizes approximately 460, approximately 1044, approximately 657 and approximately 854 bp in 106 isolates identified as capsular serotype-A, 15 in serotype D and two in serotype F. Capsular types B and E were not detected in any of the avian isolates studied. The present findings suggest that a multiplex capsular PCR assay may be suitable for the rapid initial identification serotypes P. multocida during epidemiological studies of fowl cholera.

Animals↗

A 39 kDa protein mediates adhesion of avian Pasteurella multocida to chicken embryo fibroblast cells.

To clarify the role of avian Pasteurella multocida capsule in pathogenesis, adhesion of capsulated strains P-1059, X-73 and Pm-18, and noncapsulated strains P-1059B, Pm-1 and Pm-3 to chicken embryo fibroblast (CEF) cells was compared. Number of adherent organisms of the capsulated strains to CEF cells were approximately three times as much as noncapsulated strains indicating that adhesive properties were enhanced by the presence of bacterial capsule. Pretreatments of the bacterial cells with heat, trypsin, or with antiserum caused a marked decrease in adhesion of capsulated strain P-1059 and its noncapsulated variant P-1059B. However, depolymerization of capsular hyaluronic acid with high dose of hyaluronidase enhanced adhesion of these strains. Combined treatments of the bacterial cells with both hyaluronidase and trypsin significantly (P < 0.05) inhibited the adherence of strain P-1059 as compared to the treatment only with trypsin, but strain P-1059B was not affected. SDS-PAGE profiles of crude capsular extract (CCE) prepared from capsulated strain P-1059 and its noncapsulated variant P-1059B grown on dextrose starch agar (DSA) plates by heating at 56 degrees C in a 2.5% NaCl solution demonstrated eight protein bands of 28, 34, 36, 39, 52, 56, 63 and 93 kDa. The 28, 34 and 36 kDa proteins were commonly major for both strains, and the 39 kDa protein was major only for strain P-1059 but poor in strain P-1059B. Outer membrane protein (OMP) profiles were identical with a major protein at 34 kDa and four minor proteins between the two strains. The adhesion of strain P-1059 and strain P-1059B to CEF cells was inhibited significantly (P < 0.01) by treatment with rabbit antisera against P-1059, P-1059B, CCE or 39 kDa protein of strain P-1059 as compared to the treatment with either PBS or with normal rabbit serum. These results indicated that an antigenic 39 kDa protein in the capsule may be responsible for adhesion of avian P. multocida type A strains to CEF cells as a virulence factor.

Animals↗

Capsule thickness and amounts of a 39 kDa capsular protein of avian Pasteurella multocida type A strains correlate with their pathogenicity for chickens.

Capsule thickness of avian Pasteurella multocida type A strains was determined by transmission electron microscopy after labeling with polycationic ferritin and compared with their pathogenicity for chickens. The capsule thickness of P. multocida strains Pm-18 and X-73 was 81.4 and 50.1 nm on average, respectively. These strains were highly virulent for chicken, whereas the less virulent strains Pm-1 and Pm-3 had a thin and irregular capsule, 21.0 and 29.8 nm on average, respectively. However, the thickest capsule was observed in strain P-1059, 101.2 nm on average, and the strain revealed moderate virulence. The noncapsulated variant P-1059B, which was derived from strain P-1059, revealed low virulence. The six P. multocida strains were examined with regard to protein content on the capsule of organisms. Amounts of total proteins of crude capsular extract (CCE) from capsulated strains were approximately twice those of the noncapsulated strains. The amount of an antigenic 39 kDa protein in the CCE were found to correlate with the capsule thickness, since heavily capsulated strains exhibited the greatest amount, whereas noncapsulated strains including noncapsulated and low virulent variant P-1059B possessed little 39 kDa protein. The results demonstrated that the capsule thickness and the quantity of a 39 kDa capsular protein of avian P. multocida type A strains correlated with their pathogenicity for chickens.

Animals↗

Characterization of a 39kDa capsular protein of avian Pasteurella multocida using monoclonal antibodies.

The role of a 39kDa protein of avian Pasteurella multocida in pathogenesis of fowl cholera was investigated using monoclonal antibodies (Mabs). Mabs were prepared by immunization of BALB/c mice with a crude capsular extract (CCE) of P. multocida strain P-1059 (serovar A:3). Totally eight hybridomas producing Mab were obtained. Immunoblot analysis of the hybridomas revealed that all the Mabs recognized a 39kDa protein of CCE. Treatment of CCE antigen with proteinase K or periodic acid indicated that the epitope recognized was proteinaceous. The Mabs reacted with a major 39kDa protein of CCE from encapsulated strains but not with any protein of non-capsulated strains indicating that a direct correlation between encapsulation and the 39kDa protein. Immunoelectron microscopy on strain P-1059 and the non-capsulated derivative P-1059B (serovar -:3) reacting with the Mabs and gold-labeled anti-mouse IgG indicated that the protein is associated with the capsule. The Mabs significantly inhibited the adherence of encapsulated P. multocida strains to chicken embryo fibroblast cells, but only slightly that of non-capsulated strains. Mice passively immunized with the Mabs were protected from lethal challenge with virulent strains P-1059 and X-73 (serovar A:1). Thus the capsular 39kDa protein was determined to be an adherence factor and a cross-protective antigen of avian P. multocida type A strains.

Animals↗

Differentiation of Pasteurella multocida isolates from cases of atrophic rhinitis in pigs from Zimbabwe by RAPD and ribotyping.

Atrophic rhinitis in pigs is rarely reported in Southern Africa. To determine the relationship between Pasteurella multocida clones from clinical cases of atrophic rhinitis, twenty-one strains were characterised by selected phenotypic and genotypic methods. Biochemical analysis classified 18 strains as P. multocida subspecies multocida, whilst the remainder were grouped into separate unassigned biotypes. Capsular groups A (16/21) and D (l/21) were found among the isolates by PCR. Four ribotype patterns were obtained following HpaII ribotyping, whilst random amplification of polymorphic DNA (RAPD) revealed three main clusters. However, subclusters were also noted for each RAPD cluster. Our results indicate that RAPD offers a better discrimination of strains than ribotyping and that none of the phenotypic characters were directly related to the genotypic clusters.

Animals↗

Invasion of chicken embryo fibroblast cells by avian Pasteurella multocida.

Invasion of chicken embryo fibroblast (CEF) cells by the virulent encapsulated Pasteurella multocida strains P-1059 (serovar A:3) and X-73 (serovar A:1) and an avirulent noncapsulated derivative P-1059B (serovar -:3) was investigated. The number of intracellular bacteria increased for all the strains after 2, 4 and 6 h post-inoculation to CEF cells. By 6 h post-inoculation, the number of invaded bacteria of encapsulated strains was significantly higher than noncapsulated strain and reached 150- and 112-fold for strains P-1059 and X-73, respectively, while it was 9-fold for strain P-1059B as compared to the number of invaded bacteria recovered after 2 h post-inoculation. Electron microscopy of invasion by encapsulated strains showed that the bacteria were adhering to CEF cells membrane after 1 h of inoculation. By 4-h, one or two bacteria were detected within membrane-bound vacuoles of the intracellular space. The number of intracellular bacteria markedly increased at 14 h post-inoculation. Invasion of all strains was inhibited significantly when the monolayers were treated with periodic acid (P<0.001) or trypsin (P<0.05). The treatment of bacteria with hyaluronidase did not affect invasion. The present results indicate that avian P. multocida capsular type A strains are invasive and that the receptor on CEF cell surface might be glycoprotein.

Animals↗

Antimicrobial activity of cathelicidins BMAP28, SMAP28, SMAP29, and PMAP23 against Pasteurella multocida is more broad-spectrum than host species specific.

The antimicrobial activity of linear, cationic alpha-helical peptides from cattle (BMAP28), sheep (SMAP28 and SMAP29), and pigs (PMAP23) were assessed to determine if activity was selective for Pasteurella multocida from a particular animal species or broad-spectrum against all P. multocida tested. The antimicrobial activities of synthetic peptides were determined for P. multocida isolated from cattle (10 isolates), sheep (10 isolates), and pigs (10 isolates) in a broth microdilution assay. All thirty isolates of P. multocida were susceptible to BMAP28 (MICs and MBCs, 1.0-1.9 microM); SMAP28 and SMAP29 (MICs and MBCs, 0.2-0.7 microM); and PMAP23 (MICs and MBCs, 4.3 to > or = 6.8 microM). Overall, the results of this study suggest that synthesized cathelicidins from cattle, sheep, and pigs had broad-spectrum antimicrobial activity against all P. multocida.

Amino Acid Sequence↗

The effects of tick-borne fever on the susceptibility of ovine polymorphonuclear cells to P. haemolytica cytotoxin.

Tick-borne fever predisposes sheep to secondary infections by Pasteurella haemolytica. Polymorphonuclear (PMN) cells obtained from sheep 2 to 7 days after the onset of parasitaemia due to Cytoecetes phagocytophila, the causative agent of tick-borne fever, were significantly more susceptible in vitro to the cytotoxin of P. haemolytica than PMN obtained from age-matched control sheep.

Animals↗

Passive immune cross-protection in mice produced by rabbit antisera against different serotypes of Pasteurella multocida.

Haemorrhagic septicaemia (HS) and fowl cholera (FC) are specific diseases caused by certain serotypes of Pasteurella multocida. Strains that usually cause HS in cattle and water buffalo do not produce FC in avian species, and strains that cause FC do not produce HS in cattle and water buffalo. A variety of P. multocida serotypes, including unusual strains which can cause HS in wild ruminants, were evaluated in passive immune protection studies to determine the immunological relationship between strains associated with HS and FC. Various degrees of cross-protection were seen among the strains. Antiserum against a serotype B:3,4 strain protected against strains capable of causing HS (serotypes B:1, B:2, B:3,4, B:4 and E:2) and FC (serotypes A:1, A:3 and A:5). Antiserum against an FC strain (serotype A:5) similarly protected against strains capable of causing HS and FC. Antigenic analyses indicated that cross-protection was not necessarily induced by serotype-specific capsular (beta) or somatic (gamma) antigens or major porin protein. SDS-PAGE and immunoblots of whole cell lysates of the different HS and FC strains showed many protein-staining bands with similar mobilities and antigenic activity. These cross-reactive antigenic bands occurred in the 20- to 120-kDa range. Adsorption of antiserum with a heterologous serotype removed its reactivity with most of these bands, as well as its ability to cross-protect.

Animals↗

Cloning of a unique sequence specific to isolates of type B:2 Pasteurella multocida.

Two Carter type B Pasteurella multocida isolates, Izatnagar 52 and 25, isolated from cases of haemorrhagic septicaemia (HS), were used in a modified subtractive hybridisation technique with the specific aim of cloning unique DNA sequences related to the pathogenesis of HS. Biochemical and protein analyses have shown these isolates to be similar, but reports indicate that they have differences in pathogenicity. The subtracted inserts were screened against genomic DNA from a wide range of P multocida isolates, with two distinct fragments demonstrating specific hybridisation with Carter type B isolates that cause HS. No identity was observed with either Carter type E isolates or non-HS type B strains. The clones were sequenced and a search of the GenBank database revealed significant identity of the clone A3b (296 nt) to P haemolytica lipoprotein, whereas there was no significant identity with 6b (956 nt). Both these fragments had a high level of identity (72.8 to 76.9 per cent) to the H influenzae Rd genome.

Animals↗

Beware of dogs licking ears.

A patient with right-sided chronic purulent otorrhoea developed meningitis due to Pasteurella multocida transmitted by a dog that frequently licked his ear. We suggest that patients with a perforated tympanic membrane should avoid being licked on their ears by animals.

Aged↗

Immunogenicity of purified lipopolysaccharide or protein-oligosaccharide conjugates of Pasteurella multocida type 6:B in mice.

Purified lipopolysaccharide (LPS) of Pasteurella multocida type 6:B, while toxic at higher doses, was protective at lower dose levels against experimentally-induced pasteurellosis in mice. However, the observed protection was abrogated if such LPS was digested with proteinase K prior to use in immunisation. The O-antigen polysaccharide side-chain (OS) of LPS did not appear to contribute to the observed protection as judged by the fact that immunisation of mice with purified OS or OS-protein conjugates, all of which were nontoxic, failed to confer protection against challenge with homologous virulent organisms. This was despite generation of significant levels of OS-specific antibodies, predominantly either of the IgM or IgG isotypes, in immunised mice.

Adipates↗