Polymerase chain reaction techniques for differentiating cytotoxic and noncytotoxic Pasteurella trehalosi from Rocky Mountain bighorn sheep

Objective-To evaluate 2 polymerase chain reaction (PCR)-based methods for d ifferentiating cytotoxic and noncytotoxic Pasteurella trehalosi from Rocky Mountain bighorn sheep (Ovis canadensis canadensis). Sample Population-23 isolates of P trehalosi from bighorn sheep in Colorado , including 18 from free-ranging herds and 5 from a captive herd. Procedure-Using a sequence of the leukotoxin gene region of P haemolytica s erotype 1, 7 PCR primers were designed. A PCR amplification was performed o n a sample of bacterial cell suspensions from pure cultures of P trehalosi with known in vitro cytotoxic effects. The 2 most promising primer pairs we re used in a study of 23 P trehalosi isolates. Results were analyzed for as sociation with cytotoxicity and 3 distinct ribotypes (E-CO, A(CO), and B-CO ). Results-Significant associations were observed between in vitro cytotoxicit y and PCR results for coding region, between ribotype E-CO classification a nd PCR results for coding region, and between ribotype E-CO classification and PCR results for promoter region. There was a negative association betwe en ribotype P, classification and PCR results for coding and promoter regio ns. Conclusions and Clinical Relevance-The PCR for the leukotoxin A coding regi on may be useful in differentiating cytotoxic from noncytotoxic P trehalosi isolates recovered from bighorn sheep. it may be useful for studying epide miologic features of pasteurellosis in bighorn sheep and for designing vacc ines to protect wild sheep against pneumonia caused by P trehalosi and P ha emolytica.