Detecção de Campylobacter fetus em lavados prepuciais de touros pela PCR

Abstract

A PCR based on the 23S rDNA sequence was developed for the detection of C. fetus from preputial washings of bulls. Three extraction methods were evaluated for the extraction of DNA in preputial washings: the guanidiniu, tiocynate, the alkaline lysis and the Chelex 100 methods. To determine the specificity of the test, Camppylobacter sp, Helicobacter sp and Escherichia coli reference strains were submitted to PCR. Preputial washings of 47 bulls from one farm with a positive diagnosis of Bovine Genital Campilobactosis were tested by PCR and direct fluorescent antibody test (DFAT). Only C. fetus subps, fetus and C.fetus subps, venerealis were amplified by PCR and the detection limit was 3,8 x 10² CFU/ml of C. fetus. The specificity of the amplified product was confirmed by restriction analysis with Avall. Eight bulls were considered infected by the two tecniques, but three animals were only positive by PCR and seven only by DFAT. The present PCR assay was suitable to detect C. fetus in preputial washing samples.