A detection assay for Campylobacter fetus in bovine semen by restriction analysis of PCR amplified DNA.
A rapid screening assay for Campylobacter fetus in bull semen was developed using the polymerase chain reaction (PCR) and restriction endonuclease analysis (REA) to complement isolation by culture. An oligonucleotide primer pair (C1/C2) from the hypervariable region of 16S rRNA of C. fetus was used to amplify a 362 base pair fragment by PCR. The PCR/REA assay, which is completed in 10 hours, detected as few as three C. fetus subsp. venerealis cells in experimentally infected raw bull semen and in semen diluted with milk or egg yolk Tris (EYT). All the strains tested, of both subspecies of C. fetus, were amplified, as were some other Campylobacter species. Restricting the amplified products by AluI differentiated C. fetus from the other organisms. There was no visible product generated by PCR from C. sputorum subsp. bubulus, a saprophytic organism found in the prepuce of bulls, or from seven other species of bacteria found in semen. A modification of the PCR assay, using another primer pair (C3/C2) and two temperature PCR cycling conditions, increased the probability of detecting C. fetus subsp. venerealis. PCR amplification followed by REA could be used to screen bovine semen rapidly for C. fetus. In most cases, sequencing of C1/C2 PCR generated products would be preferable for distinguishing between the two subspecies of C. fetus.
Agriculture-Agrifood Canada, Animal Diseases Research Institute, Nepean, Ontario.
M D
MD
Eaglesome
M I
MI
Sampath
M M
MM
Garcia
D000818:Animals; D001483:Base Sequence; D002169:Campylobacter Infections / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary*; D002168:Campylobacter fetus / Q000235:genetics / Q000302:isolation & purification*; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis* / Q000382:microbiology; D017931:DNA Primers / Q000737:chemistry; D004262:DNA Restriction Enzymes; D004269:DNA, Bacterial / Q000032:analysis* / Q000302:isolation & purification; D008297:Male; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000662:veterinary; D015203:Reproducibility of Results; D015183:Restriction Mapping; D012661:Semen / Q000382:microbiology*; D012680:Sensitivity and Specificity
D017931:DNA Primers; D004269:DNA, Bacterial; D004262:DNA Restriction Enzymes