K. M. Townsend

Genetic Organization of Pasteurella multocida cap Loci and Development of a Multiplex Capsular PCR Typing System

ABSTRACT Current serotyping methods classify Pasteurella multocida into five capsular serogroups (serogroups A, B, D, E, and F) and 16 somatic serotypes (serotypes 1 to 16). In the present study, we have developed a multiplex PCR assay as a rapid alternative to the conventional capsular serotyping system. The serogroup-specific primers used in this assay were designed following identification, sequence determination, and analysis of the capsular biosynthetic loci of each capsular serogroup. The entire capsular biosynthetic loci ofP. multocida A:1 (X-73) and B:2 (M1404) have been cloned and sequenced previously (J. Y. Chung, Y. M. Zhang, and B. Adler, FEMS Microbiol. Lett. 166:289–296, 1998; J. D. Boyce, J. Y. Chung, and B. Adler, Vet. Microbiol. 72:121–134, 2000). Nucleotide sequence analysis of the biosynthetic region (region 2) from each of the remaining three serogroups, serogroups D, E, and F, identified serogroup-specific regions and gave an indication of the capsular polysaccharide composition. The multiplex capsular PCR assay was highly specific, and its results, with the exception of those for some serogroup F strains, correlated well with conventional serotyping results. Sequence analysis of the strains that gave conflicting results confirmed the validity of the multiplex PCR and indicated that these strains were in fact capsular serogroup A. The multiplex PCR will clarify the distinction between closely related serogroups A and F and constitutes a rapid assay for the definitive classification of P. multocida capsular types.

The molecular biology of Pasteurella multocida

Pasteurella multocida is an important veterinary and opportunistic human pathogen. The species is diverse and complex with respect to antigenic variation, host predeliction and pathogenesis. Certain serological types are the aetiologic agents of severe pasteurellosis, such as fowl cholera in domestic and wild birds, bovine haemorrhagic septicaemia and porcine atrophic rhinitis. The recent application of molecular methods such as the polymerase chain reaction, restriction endonuclease analysis, ribotyping, pulsed-field gel electrophoresis, gene cloning, characterisation and recombinant protein expression, mutagenesis, plasmid and bacteriophage analysis and genomic mapping, have greatly increased our understanding of P. multocida and has provided researchers with a number of molecular tools to study pathogenesis and epidemiology at a molecular level.

Role of Capsule in the Pathogenesis of Fowl Cholera Caused by Pasteurella multocida Serogroup A

ABSTRACT We have constructed a defined acapsular mutant inPasteurella multocida X-73 (serogroup A:1) by disrupting the hexA gene through the insertion of a tetracycline resistance cassette. The genotype of thehexA::tet(M) strain was confirmed by PCR and Southern hybridization, and the acapsular phenotype of this strain was confirmed by electron microscopy. ThehexA::tet(M) strain was attenuated in both mice and chickens. Complementation of the mutant with an intact hexAB fragment restored lethality in mice but not in chickens. In contrast to the results described previously for P. multocida serogroup B (J. D. Boyce and B. Adler, Infect. Immun. 68:3463–3468, 2000), thehexA::tet(M) strain was sensitive to the bactericidal action of chicken serum, whereas the wild-type and complemented strains were both resistant. Following inoculation into chicken muscle, the bacterial count of thehexA::tet(M) strain decreased significantly, while the wild-type and complemented strains both grew rapidly over 4 h. The capsule is thus an essential virulence determinant in the pathogenesis of fowl cholera.

Comparative susceptibility of planktonic and 3-day-old Salmonella Typhimurium biofilms to disinfectants

Aims:  To compare the susceptibility of a 3‐day‐old biofilm and planktonic Salmonella to disinfectants at different exposure times. We hypothesize that Salmonella biofilms are more resilient to disinfectants compared to planktonic Salmonella.

Molecular characterisation of avian Pasteurella multocida isolates from Australia and Vietnam by REP-PCR and PFGE

A total of 95 isolates of Pasteurella multocida were analysed by pulsed field gel electrophoresis (PFGE) using the enzyme ApaI, including 73 avian isolates from Australia and 22 from Vietnam. The majority of field isolates were capsular Type A, with the predominant somatic serovars of 1, 3, 4 and 3,4. Twenty-one distinct profiles were evident among the Australian isolates, with only 3 profiles observed among the 22 P. multocida strains isolated from Vietnam. Within the Australian isolates, related and unrelated outbreaks could be identified by PFGE. These results correlated well with previously published studies, with greater discrimination shown by PFGE. Repetitive extragenic palindromic sequence PCR (REP-PCR) analysis of representative isolates from PFGE classifications yielded 21 profiles, with most of the subgroups in accordance with PFGE analysis. While REP-PCR was shown to be less discriminating than PFGE, the epidemiological relatedness of strains compared favourably between the techniques. Thus, the ease and rapidity of REP-PCR while maintaining a high level of differentiation, supports the use of REP-PCR as a competent alternative to the more labour-intensive PFGE system for strain identification and epidemiological studies of avian P. multocida.

Comparative susceptibility of Salmonella Typhimurium biofilms of different ages to disinfectants

There is a general consensus that with increasing age a biofilm shows increased resistance to antimicrobials. In this study the susceptibility of 3-, 5- and 7-day-old Salmonella enterica serovar Typhimurium biofilms to disinfectants was evaluated. It was hypothesized that 7-day-old biofilms would be more resistant to disinfectants compared to 3- and 5-day-old biofilms. Biofilms were formed using the MBEC™ system and treated with six chemical disinfectants for 1 and 5 min. Four disinfectants at the highest concentration available showed 100% reduction in viable cells from all ages of biofilms after exposure for 5 min, and ethanol at 70% v/v was the least effective against biofilms, followed by chlorhexidine gluconate (CG). At the recommended user concentrations, only sodium hypochlorite showed 100% reduction in viable cells from all ages of biofilms. Benzalkonium chloride and CG were the least effective against biofilms, followed by quaternary ammonium compound which only showed 100% reduction in viable cells from 5-day-old biofilms. Overall, the results from this study do not display enhanced resistance in 7-day-old biofilms compared to 3- and 5-day-old biofilms. It is concluded that under the conditions of this study, the age of biofilm did not contribute to resistance towards disinfectants. Rather, the concentration of disinfectant and an increased contact time were both shown to play a role in successful sanitization.

Acute septicaemic pasteurellosis in Vietnamese pigs

Sixteen isolates of Pasteurella multocida were cultured from cases diagnosed as acute septicaemic pasteurellosis in Vietnamese pigs. The HSB-PCR assay provided rapid presumptive determination of 10 isolates of P. multocida identified as haemorrhagic septicaemia (HS) causing type B cultures (B:2, B:5, B:2,5). Serological designation using the Carter and Heddleston typing systems confirmed these findings, and identified the six HSB-PCR negative cultures as either A:1, A:3 or D:3,4. Biochemical fermentation and REP-PCR revealed phenotypic and genotypic identity between P. multocida type A:1 isolated from Vietnamese pigs and poultry. Marked homogeneity was also demonstrated among HSB-PCR positive swine isolates, which were shown to possess genotypic identity with P. multocida type B:2 from buffaloes diagnosed with HS.

PCR detection and analysis of Pasteurella multocida from the tonsils of slaughtered pigs in Vietnam.

A total of 36 tonsil swab samples were collected from healthy swine prior to slaughter at the abattoirs in Can tho and Tien giang provinces of Southern Vietnam. The presence of Pasteurella multocida in these samples was detected by the combination of direct cultivation and isolation, mouse inoculation and the polymerase chain reaction (PM-PCR). P. multocida was detected in 16 samples by PCR, with 17 strains ultimately isolated. All samples were negative for serogroup B by HSB-PCR and conventional serotyping, with isolates identified as A:3, D:1 or D:3. In addition, all samples were determined to be negative for the P. multocida toxin (PMT). Characterisation of isolated P. multocida by REP-PCR and biotyping revealed nine distinct REP profiles and seven biotypes among the 17 isolates. Some correlation was seen with P. multocida isolated from a previous Australian outbreak of acute swine pasteurellosis, and those isolated from fowl cholera outbreaks in Vietnamese poultry.

Application of nox-restriction fragment length polymorphism for the differentiation of Brachyspira intestinal spirochetes isolated from pigs and poultry in Australia.

Sixty-nine intestinal spirochetes isolated from pigs and poultry in eastern Australia were selected to evaluate the effectiveness of a species-specific PCR-based restriction fragment length polymorphism (RFLP) analysis of the Brachyspira nox gene. For comparative purposes, all isolates were subjected to species-specific PCRs for the pathogenic species Brachyspira hyodysenteriae and Brachyspira pilosicoli, and selected isolates were examined further by sequence analysis of the nox and 16S ribosomal RNA genes. Modifications to the original nox-RFLP method included direct inoculation of bacterial cells into the amplification mixture and purification of the PCR product, which further optimized the nox-RFLP for use in a veterinary diagnostic laboratory, producing sufficient product for both species identification and future comparisons. Although some novel profiles that prevented definitive identification were observed, the nox-RFLP method successfully classified 45 of 51 (88%) porcine and 15 of 18 (83%) avian isolates into 5 of the 6 recognized species of Brachyspira. This protocol represents a significant improvement over conventional methods currently used in veterinary diagnostic laboratories for rapid specific identification of Brachyspira spp. isolated from both pigs and poultry.

The demonstration of Pasteurella multocida in the alimentary tract of chickens after experimental oral infection.

A selective medium containing polymyxin B, crystal violet, thallous acetate, bacitracin and cycloheximide in 10% sheep blood dextrose starch agar, and a modified Pasteurella multocida-specific polymerase chain reaction (PCR) assay were developed for the respective isolation and detection of P. multocida from chicken alimentary tract. The selective medium and the PCR assay were highly sensitive, detecting 100 cfu from colon contents. These techniques were used to follow the localisation of an orally administered virulent P. multocida in chickens. Pasteurellae could be isolated from the crop of some birds up to 30 h, occasionally from other sites after 28 h. It was concluded that the crop was a likely site for colonisation and that infection was most likely to occur through the mucosa of the jejunum or ileum.