Reverse transcription-polymerase chain reaction on pooled samples to detect bovine viral diarrhea virus by using fresh ear-notch-sample supernatants.
Ear-notch samples from 3,599 yearling heifers were collected to detect persistently infected (PI) animals with suspect bovine viral diarrhea virus (BVDV). Individual immunohistochemistry (IHC), individual antigen-capture enzyme-linked immmunosorbent assay (AC-ELISA), and reverse transcription-polymerase chain reaction (RT-PCR) tests with pooled ear-notch supernatants were compared with samples from 3,016 heifers, whereas RT-PCR ear-notch pools and individual AC-ELISA tests were compared with samples from all 3,599 heifers. Four heifers were identified positive by both IHC and AC-ELISA, whereas the remaining heifers were identified negative by both tests. When supernatant from ear notches from 100 animals was randomly pooled and RT-PCR was accomplished on each pool, RT-PCR identified 2 pools that contained 1 positive AC-ELISA sample and 1 pool that contained 2 positive AC-ELISA samples. Further evaluation of the pooled RT-PCR ear-notch supernatant detected 100% (n = 36) samples spiked with supernatant from a single randomly selected positive AC-ELISA ear notch. Although follow-up confirmatory tests were not completed, all 3 methods correlated 100% in detecting suspect PI animals, with a kappa value of 1. The use of RT-PCR on pooled ear-notch supernatant could provide an initial, rapid, cost-effective method of screening cattle herds for BVDV PI animals. Subsequent serial testing with an AC-ELISA to evaluate individual samples included in the positive pool could minimize the length of time other animals are exposed to the virus.
Colorado State University Veterinary Diagnostic Laboratory, Rocky Ford Branch, Rocky Ford 81067, USA. James.Kennedy@colostate.edu
James A
JA
Kennedy
Robert G
RG
Mortimer
Barbara
B
Powers
D000818:Animals; D000956:Antigens, Viral / Q000032:analysis; D001912:Bovine Virus Diarrhea-Mucosal Disease / Q000175:diagnosis* / Q000821:virology; D002417:Cattle; D001908:Diarrhea Viruses, Bovine Viral / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D004431:Ear, External / Q000821:virology; D004797:Enzyme-Linked Immunosorbent Assay / Q000191:economics / Q000379:methods / Q000662:veterinary; D005260:Female; D007150:Immunohistochemistry / Q000191:economics / Q000662:veterinary; D012367:RNA, Viral / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000191:economics / Q000379:methods / Q000662:veterinary*; D012680:Sensitivity and Specificity
D000956:Antigens, Viral; D012367:RNA, Viral