Development and testing of a unique strain of Pasteurella haemolytica for use in studies on colonization of the respiratory tract of cattle

Abstract

Objective - To develop a unique strain of Pasteurella haemolytica, selectable from nasopharyngeal respiratory tract secretions, that retains the ability to efficiently colonize the respiratory tract of calves. Animals - 26 calves that each weighed approximately 200 kg. Procedure - Rifampicin- resistant mutants of P haemolytica were developed and tested for in vitro growth rate and leukotoxin production. After instillation into the tonsils of calves, an isolate that was efficient at colonizing was selected and transformed, using electroporation, with a 4.2-kilobase (kb) plasmid encoding for streptomycin resistance. This isolate was instilled into the tonsils of 4 of 14 commingled calves to examine transmission of organisms. Nasal secretion and tonsil wash specimens were collected, cultured, and examined for P. haemolytica. Serum antibody concentration was measured by means of indirect hemagglutination. Results - Selected P haemolytica organisms colonized the tonsils and nasal passages for more than 2 weeks. Exposed calves and contact calves shed the organism, which was recovered from specimens of nasal secretions and tonsil washes. The 4.2-kb plasmid was lost during in vivo colonization. Conclusions and Clinical Relevance - The selected rifampicin- resistant P haemolytica organism colonized tonsils and nasal passages in a manner similar to the wild-type organisms. Selective media suppressed other bacterial flora to the extent that a single colony-forming unit was detectable from 200 μl of specimen, a 100-fold improvement in detection sensitivity. The selectable strain spread rapidly among commingled calves. A 4.2-kb plasmid marker was unstable when P haemolytica replicated in vivo.