A detection assay forCampylobacter fetus in bovine semen by restriction analysis of PCR amplified DNA |
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Authors: | M D Eaglesome M I Sampath M M Garcia |
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Institution: | (1) Agriculture-Agrifood Canada, Animal Diseases Research Institute, Station H, PO Box 11300, K2H 8P9 Nepean, Ontario, Canada |
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Abstract: | A rapid screening assay forCampylobacter 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 ofC. 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 threeC. 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 ofC. fetus, were amplified, as were some otherCampylobacter species. Restricting the amplified products byAluI differentiatedC. fetus from the other organisms. There was no visible product generated by PCR fromC. 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 detectingC. fetus subsp.venerealis. PCR amplification followed by REA could be used to screen bovine semen rapidly forC. fetus. In most cases, sequencing of C1/C2 PCR generated products would be preferable for distinguishing between the two subspecies ofC. fetus.Abbreviations AI
artificial insemination
- bp
base pair
- Cff
Campylobacter fetus subsp.fetus
- Cfv
Campylobacter fetus subsp.venerealis
- EYT
egg yolk Tris
- MH
Mueller-Hinton
- PCR
polymerase chain reaction
- REA
restriction endonuclease analysis
- TE
Tris-EDTA |
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Keywords: | bull Campylobacter fetus polymerase chain reaction restriction endonuclease analysis semen |
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