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 differentiating cytotoxic and noncytotoxic Pasteurella trehalosi from Rocky Mountain bighorn sheep (Ovis canadensis canadensis).


METHODS
23 isolates of P. trehalosi from bighorn sheep in Colorado, including 18 from free-ranging herds and 5 from a captive herd.


METHODS
Using a sequence of the leukotoxin gene region of P. haemolytica serotype 1, 7 PCR primers were designed. A PCR amplification was performed on a sample of bacterial cell suspensions from pure cultures of P. trehalosi with known in vitro cytotoxic effects. The 2 most promising primer pairs were used in a study of 23 P. trehalosi isolates. Results were analyzed for association with cytotoxicity and 3 distinct ribotypes (Eco, Aco, and Bco).


RESULTS
Significant associations were observed between in vitro cytotoxicity and PCR results for coding region, between ribotype Eco classification and PCR results for coding region, and between ribotype Eco classification and PCR results for promoter region. There was a negative association between ribotype Aco classification and PCR results for coding and promoter regions.


CONCLUSIONS
The PCR for the leukotoxin A coding region may be useful in differentiating cytotoxic from noncytotoxic P. trehalosi isolates recovered from bighorn sheep. It may be useful for studying epidemiologic features of pasteurellosis in bighorn sheep and for designing vaccines to protect wild sheep against pneumonia caused by P. trehalosi and P. haemolytica.
D000818:Animals; D001426:Bacterial Proteins; D002470:Cell Survival / Q000187:drug effects; D003120:Colorado; D004275:DNA, Ribosomal / Q000235:genetics; D005098:Exotoxins / Q000235:genetics* / Q000633:toxicity; D006460:Hemolysin Proteins / Q000235:genetics* / Q000633:toxicity; D010325:Pasteurella / Q000145:classification* / Q000302:isolation & purification / Q000472:pathogenicity; D016133:Polymerase Chain Reaction / Q000379:methods; D012703:Serotyping; D012756:Sheep / Q000382:microbiology*; D014774:Virulence