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.
Kennedy
James A
JA
Colorado State University Veterinary Diagnostic Laboratory, Rocky Ford Branch, Rocky Ford 81067, USA. James.Kennedy@colostate.edu
Mortimer
Robert G
RG
Powers
Barbara
B
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