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  • Author or Editor: S. Mady Dabo x
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Abstract

Objective—To evaluate a rapid polymerase chain reaction (PCR) fingerprinting technique for discriminating among Pasteurella multocida isolates from laboratory rabbits.

Sample Population—33 P multocida isolates from rabbits with clinical pasteurellosis.

Procedure—PCR assays were conducted with 2 minisatellites (core sequence and modified core sequence of phage M13) and 2 microsatellites ([GTG]5 and [GACA]4). Each bacterium was assigned to a PCR type for each of the primers used. Boiled bacterial extracts and purified genomic DNA were compared by use of PCR assays for phage M13 and (GACA)4. Plasmids were isolated from each bacterium, and their influence on PCR fingerprint was determined, using boiled extracts as a DNA source.

Results—M13 core sequence and M13 modified core sequence yielded 5 and 8 PCR types, respectively. The microsatellites (GTG)5 and (GACA)4 yielded 4 and 9 PCR fingerprint types, respectively. Fingerprint patterns obtained by use of isolated DNA differed from those obtained by use of boiled extracts, although discrimination among P multocida isolates was similar. The presence or absence of plasmids did not affect PCR fingerprints.

Conclusion—Single primer PCR fingerprinting with minisatellite and microsatellite primers is an efficient and reproducible method for the discrimination of P multocida isolates from rabbits and can be performed directly, using boiled bacterial extracts as a source of template, although more bands were obtained from pure genomic DNA. (Am J Vet Res 2000;61:305–309)

Full access
in American Journal of Veterinary Research

Abstract

Objective—To determine efficacy of intranasal vaccination of rabbits with Pasteurella multocida A:3 outer membrane proteins (OMP) expressing iron-regulated OMP (IROMP) in conferring protection against experimental challenge exposure.

Animals—52 male New Zealand White rabbits.

Procedure— Rabbits were vaccinated intranasally on days 0, 7, and 14; some vaccines included cholera toxin (CT) as an adjuvant. Concentrations of intranasal IgA and serum IgG antibodies against P multocida OMP were determined. In experiment A, rabbits were vaccinated with either phospate-buffered saline solution (PBSS), PBSS-CT, OMP-CT, or IROMP-CT, challenge-exposed intranasally on day 16, and euthanatized and necropsied on day 28. Rabbits were also vaccinated with OMP or IROMP without CT and were not challenge-exposed. In experiment B, rabbits were vaccinated with PBSS, PBSS-CT, IROMP, or IROMP-CT. On day 17, rabbits were challenge-exposed intranasally .Nasal bacteria and antibodies were determined on day 24.

Results—In experiment A, OMP-CT vaccination stimulated mucosal and systemic antibody responses to the bacterium and enhanced resistance against challenge exposure. Intranasal bacterial counts were not significantly reduced. Vaccination with IROMP-CT stimulated mucosal and systemic antibodies, enhanced resistance to challenge exposure, and significantly reduced nasal bacterial counts. In experiment B, natural infection was detected in several rabbits at challenge exposure; however, IROMP-CT-vaccinated rabbits had significantly higher serum and nasal antibody responses, compared with other rabbitsIROMP-CT-vaccinated rabbits had significantly lower nasal bacterial counts compared to control rabbits.

Conclusions and Clinical Relevance—Intranasal vaccination of rabbits with P multocida outer membranes containing IROMP and CT stimulated immunity against experimental pneumonic pasteurellosis. (Am J Vet Res 2001;62:697–703)

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in American Journal of Veterinary Research

Abstract

Objective

To determine whether a Pasteurella haemolytica A1 mutant that is unable to produce membrane lipoproteins has reduced susceptibility to complement-mediated killing, and to characterize the mutant strain.

Sample Population

12 sera from cattle resistant to P haemolytica challenge exposure after vaccination with P haemolytica or its antigens, or after natural exposure.

Procedures

Complement-mediated killing assays were performed, using wild-type and mutant strains and, as antibody source, various immune sera from cattle that were resistant to P haemolytica challenge exposure. Antibody response to whole-cell antigens produced by mutant and wild-type strains, production of outer membrane proteins and iron-regulated outer membrane proteins by the 2 strains, and growth of the 2 strains in various media were analyzed.

Results

Compared with wild-type P haemolytica, the lipoprotein mutant strain had increased susceptibility to bovine complement-mediated killing. Aside from the lipoproteins that are not produced by the mutant, immunoblot analysis did not reveal differences between immunoreactive antigens that are produced by the 2 strains. Some iron-regulated, outer membrane proteins, which usually are only produced by P haemolytica under iron-deficient conditions, were produced constitutively by the mutant. The mutant grew to a lower final cell density and at a lower rate under conditions likely to reflect those encountered in vivo.

Conclusions

Lack of 3 membrane lipoproteins resulted in enhanced susceptibility to bovine complement-mediated killing. Site-specific mutagenesis of genes encoding P haemolytica membrane lipoproteins alters production of iron-regulated outer membrane proteins by P haemolytica. Growth characteristics of the mutant suggested that it may have reduced capacity for survival in vivo. (Am J Vet Res 1998;59:1275-1280)

Free access
in American Journal of Veterinary Research