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PubMed2006

Analytical verification of a multiplex PCR for identification of Bordetella bronchiseptica and Pasteurella multocida from swine.

Bordetella bronchiseptica and Pasteurella multocida are etiologic agents of progressive atrophic rhinitis (PAR) and bronchopneumonia in swine. Only dermonecrotic toxin-producing strains of P. multocida play a role in atrophic rhinitis while both toxigenic and nontoxigenic strains have been associated with pneumonia. Monitoring and investigation of outbreaks involving these bacteria require sensitive and accurate identification and reliable determination of the toxigenic status of P. multocida isolates. In the present study, we report the development, optimization, and performance characteristics of a multiplex PCR assay for simultaneous amplification of up to three different targets, one common to all P. multocida strains, one found only in toxigenic P. multocida strains, and one common to B. bronchiseptica strains. Based on analysis of 94 P. multocida isolates (31 toxigenic) and 126 B. bronchiseptica isolates assay sensitivity is 100% for all amplicons. Evaluation of 22 isolates of other bacterial genera and species commonly found in the swine respiratory tract demonstrated a specificity of 100% for all gene targets. The limit of detection for simultaneous amplification of all targets is 1-10pg of DNA per target, corresponding to a few hundred genomes or less. Amplicon mobility in agarose gels and sequence analysis indicate the amplicons are highly stable. The data presented establish this multiplex PCR as a reliable method for identification of B. bronchiseptica and both toxigenic and nontoxigenic P. multocida that may greatly simplify investigations of swine PAR and bronchopneumonia.

Animals↗
PubMed2006

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↗
PubMed1989

The effect of titanium dioxide inhalation on the pulmonary clearance of Pasteurella haemolytica in the mouse.

Four inhalation exposure chambers were designed and built in which up to 80 mice per chamber could be accommodated. Exposure for several weeks to an inert inorganic particulate aerosol of titanium dioxide at a respirable aerosol concentration of 20 mg m-3 impaired the clearance of Pasteurella haemolytica in proportion to the duration of exposure. Other groups exposed to a lower respirable concentration of 2 mg m-3 exhibited similar clearance rates relative to mice maintained in clean air. A recovery experiment showed that inhalation of TiO2 at 20 mg m-3 for 10 days impaired bacterial clearance at least 10 days after cessation of exposure to TiO2.

Administration, Inhalation↗
PubMed2003

Septicaemia and arthritis in pigs experimentally infected with Pasteurella multocida capsular serotype A.

Twenty-five caesarean-derived, colostrum-deprived (CDCD) pigs and 18 specific pathogen-free pigs, aged 8 to 14 weeks, were inoculated intranasally or intratracheally with Pasteurella multocida capsular serotype A, isolated from a severe pneumonic lesion in a growing pig. The pigs were killed for necropsy on day 6 or 14 post-inoculation (PI) or, in the case of the only fatally infected animal, examined at the time of death. One CDCD pig, inoculated intratracheally with 5 ml of a bacterial suspension containing 1.7x10(9) colony-forming-units/ml, died of septicaemia on day 1 PI. Histological lesions such as severe pleuropneumonia, thrombi in glomerular capillaries, haemorrhage of the spleen, and abscesses in the tonsillar crypts were observed. The organism was recovered from a number of sites and its antigens were detected immunohistochemically in the pneumonic lesions, blood vessels of the tissues, and tonsillar crypts in the dead pig. Pneumonia, pleural adhesions and suppurative arthritis in the extremital joints were observed grossly in 3/29, 8/29 and 7/29 intratracheally inoculated pigs, respectively. In intranasally inoculated pigs, no macroscopical abnormalities were seen; histologically, however, exudative bronchopneumonia and fibrinous pleurisy were observed in 9/14 and 4/14 pigs, respectively. No significant changes were seen in the tissues of uninfected control pigs. The organism was recovered from the lesions and P. multocida type A antigen was demonstrated immunohistochemically. The organism was rarely recovered from the liver, spleen or lymph nodes (bronchopulmonary or mesenteric). The results suggest that P. multocida capsular serotype A alone can cause not only pneumonia in pigs but also septicaemia or arthritis.

Animals↗
PubMed1998

Pasteurella multocida infection in the chicken embryo.

Pasteurella multocida infection in embryonated chicken eggs was studied by chorio-allantoic membrane inoculation. Strain differences were demonstrated in terms of lesion severity and time to death, especially during the first 24 h post-inoculation. A strain of low virulence gave a clear dose response but more virulent strains did not. Comparable results were obtained by infecting 6-week-old chickens. The main lesions in inoculated embryos appeared as severe vascular involvement of the entire embryo and feather tracts, thickening of the chorio-allantoic membrane, and enlargement and congestion of the yolk sac. The bacteria were demonstrated by transmission electron microscopy, either extracellularly or multiplying intracellularly in hepatocytes, heart tissue, and in the hyperplastic layer of the chorio-allantoic membrane, with resulting damage to the cellular organelles, and severe tissue changes.

Allantois↗
PubMed1990

Increased susceptibility to Pasteurella haemolytica in lambs infected with bovine respiratory syncytial virus.

Groups of conventionally reared, 6- to 8-week-old lambs were inoculated intranasally and intratracheally with either bovine respiratory syncytial virus (BRSV) or BRSV followed by Pasteurella haemolytica 6 days later or with P. haemolytica alone. Lambs infected with P. haemolytica 6 days after experimental infection with BRSV had significantly higher disease scores, greater magnitudes of fever, and higher mortality rates than those infected with BRSV alone or with P. haemolytica alone (P less than 0.05). Mononuclear cells in the peripheral blood and lung lavage obtained from the BRSV-infected lambs were more susceptible to P. haemolytica cytotoxin than those obtained from control lambs (P less than 0.05). P. haemolytica was isolated in significantly greater numbers from lambs infected with BRSV and P. haemolytica than from those infected with P. haemolytica alone (P less than 0.05).

Animals↗
PubMed1996

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↗
PubMed2002

Experimental induction of pneumonic pasteurellosis in calves by intratracheal infection with Pasteurella multocida biotype A:3.

The study aimed to establish an experimental model to investigate the pathogenesis of lung infection by Pasteurella multocida, an important cause of bovine respiratory disease. An experimental model is required to assist the development of an effective vaccine. Sixteen 8-week-old calves were challenged intratracheally with 10(9) or 10(10) colony forming units of P. multocida in either 60 or 300 ml saline in a 2 x 2 factorial experiment. All animals became dull within 2-6h post-infection (p.i.) and two calves were killed humanely because of suspected endotoxic shock. Remaining animals showed increased respiratory rates by 15-20 h p.i. and, at 23 h p.i., calves given the high dose, high volume challenge showed higher (P < 0.05) rectal temperatures. From 24 to 36 h p.i., clinical signs decreased in a majority of animals. Plasma haptoglobin concentrations increased (P < 0.05) in calves given the high volume challenge irrespective of the number of bacteria. At post-mortem examination (4d p.i.), lung lesions, mainly in the apical lobes, were found in all calves. Histopathological examination showed areas of purulent pneumonia with a tendency to abscessation and inflamed interlobular septa characterised by accumulation of neutrophils and oedema. The clinical and pathological responses described were typical of bovine pneumonic pasteurellosis.

Animals↗
PubMed1996

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↗
PubMed1998

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↗
PubMed1984

Pasteurella multocida infections in West Suffolk.

During the 3-year period 1980-1982, Pasteurella multocida was isolated from 19 patients, each with a history of animal contact. One patient, a slaughterman, whose exposure was occupational, developed meningitis. These case reports illustrate unusual features of human infections with this zoonotic pathogen.

Adult↗
PubMed1995

Meningitis in infancy caused by Pasteurella multocida.

A high proportion of household pets are colonised by Pasteurella multocida. The organism can be transmitted to humans by contact with animal saliva and is a recognised, although rare, cause of meningitis in infancy. Intimate contact between infants and family pets should be discouraged.

Cefotaxime↗
PubMed1997

Pasteurella multocida infection of a total hip arthroplasty following cat scratch.

Pasteurella multocida is a well recognized cause of sepsis following animal contact particularly bites and scratches. Spread to prosthetic joints may occur particularly in immunocompromised patients. Immunocompromised patients with prosthetic joints should be warned that animals are potential sources of serious infection and urgent medical advice should be sought if bitten or scratched.

Animals↗
PubMed2002

Pathological and immunological changes after challenge infection with Pasteurella multocida in naive and immunized calves.

Challenge infections of calves with Pasteurella multocida were established to characterize the local inflammatory response and determine the effect of previous exposure to live bacteria on the post-challenge immune response. Experimental infections were established by intratracheal inoculation of P. multocida in both naive calves and calves that had been previously vaccinated with two subcutaneous (s.c.) injections of live bacteria. Histological, immunohistological and cytokine expression studies were performed on bronchoalveolar lavage (BAL) samples, lung parenchymal tissues and lung lymph nodes (LN). In comparison to uninfected control animals in which no lung lesions were observed, a patchy to confluent bronchopneumonia was observed following infection of naive calves, characterized by abscess formation, haemorrhage, oedema and suppurative consolidation. Cellular analysis following infection of naive animals was characterized by an influx of neutrophils in the BAL, with macrophages and dendritic cells observed in the lesion perimeter. A significant increase in the number of CD8(+) blasts expressing MHC (major histocompatibility) II was also observed in the BAL of infected calves. Decreased expression of interleukin (IL)-1 beta and increased expression of IL-8 compared to naive unchallenged controls was apparent in lung LN. In comparison, a more limited pathology was observed in vaccinated animals post-challenge, indicating partial protection conferred by the s.c. immunization with live bacteria. Studies of vaccinated animals showed the presence of bronchial-associated lymphoid tissue (BALT) in the lung tissue and an increase in the number of B-cells and CD4(+) T-cells expressing MHCII in the lung LN after challenge. In contrast to primary infection, there was no significant influx of neutrophils in the BAL. Instead, a population of newly recruited monocytes/macrophages was observed. Increased IL-2 expression and decreased IL-8 expression was observed in the LN, while IL-1 beta expression was not detected. The reduced neutrophil and increase monocyte response in the vaccinated calves may be associated with significant changes in the gamma delta T lymphocyte population in the BAL.

Animals↗
PubMed1999

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.

Amino Acid Sequence↗
PubMed2006

[An unusual case of pulmonary granulomatous lesion in an immunocompetent patient: infection caused by Pasteurella multocida].

We report a case of lung infection caused by Pasteurella multocida in an immunocompetent patient. Diagnosis was made both on histological analysis of a lung wedge resection, showing pyoepithelioid granulomas, and on bacteriological cultures from lung tissue. Histological diagnosis of pulmonary pasteurellosis is difficult, and other etiological causes of pyoepithelioid granulomas of the lung have to be discussed. Pulmonary pasteurellosis is particularly severe in immunocompromised patients, and should be systematically suspected in patients exposed to pets, eventhough a percutaneous exposure is not usually found for these patients.

Adult↗
PubMed2000

Antibody reactions after aerogenous or subcutaneous immunization of pigs with Pasteurella multocida antigens.

Depending upon the antigens used and the initial titres, subcutaneous immunization of weaner pigs by means of a temperature-sensitive mutant of live Pasteurella multocida (serovar A), resulted in a significant rise of the level of specific IgG antibody already present in blood serum, but not in lung lavage fluid, and a specific stimulation of lung clearance as compared to non-immunized controls. Aerogenous immunization of a total of 108 animals in 18 experiments did not influence serum antibody titres but produced a significant rise in lung antibodies and P. multocida clearance as compared to an identical number of controls. In all 12 immunization experiments using the live mutant, increased specific IgA antibodies and in seven out of 10 experiments, elevated IgG antibodies were measured. Only aerogenous immunization was found to be capable of reducing the severity of pneumonia induced by intrabronchial infection.

Aerosols↗