title: Combination of reverse transcription real-time polymerase chain reaction and antigen capture enzyme-linked immunosorbent assay for the detection of animals persistently infected with Bovine viral diarrhea virus.
abstract: Bovine viral diarrhea virus (BVDV) is an economically important pathogen of cattle. A successful control program requires early detection and removal of persistently infected (PI) animals. The objective of the current study was to develop, validate, and apply a cost-effective testing scheme for the detection of BVDV PI animals in exposed herds. Pooled samples were screened by using a real-time reverse transcription polymerase chain reaction (real-time RT-PCR), and individual positives were identified with an antigen capture enzyme-linked immunosorbent assay (ACE). The detection limits of the optimized real-time RT-PCR were 10 and 100 RNA copies per reaction for BVDV-1 and BVDV-2, respectively. The semiquantitative results of real-time RT-PCR and ACE or real-time RT-PCR and immunohistochemistry were moderately correlated. The threshold cycle of real-time RT-PCR performed on pooled samples was significantly correlated with the pool size (R(2)  =  0.993). The least-cost pool sizes were 50 at a prevalence of 0.25-0.5% and 25 at a prevalence of 0.75-2.0%. By using the combined real-time RT-PCR and ACE procedure, 111 of 27,932 samples (0.4%) tested positive for BVDV. At this prevalence, cost reduction associated with the application of real-time RT-PCR and ACE ranged from 61% to 94%, compared with testing individual samples by ACE, immunohistochemistry, or real-time RT-PCR. Real-time RT-PCR screening also indicated that 92.94% of PI animals were infected with BVDV-1, 3.53% with BVDV-2, and 3.53% with both BVDV-1 and BVDV-2. Analysis of the 5'-untranslated region of 22 isolates revealed the predominance of BVDV-1b followed by BVDV-2a.
authors.affiliation: Mississippi Veterinary Research and Diagnostic Laboratory, the Department of Pathobiology and Population Medicine, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS, USA.
authors.forename: Lifang
authors.initials: L
authors.lastname: Yan
authors.forename: Shuping
authors.initials: S
authors.lastname: Zhang
authors.forename: Lanny
authors.initials: L
authors.lastname: Pace
authors.forename: Floyd
authors.initials: F
authors.lastname: Wilson
authors.forename: Henry
authors.initials: H
authors.lastname: Wan
authors.forename: Michael
authors.initials: M
authors.lastname: Zhang
mesh_terms: D000818:Animals; D000956:Antigens, Viral / Q000097:blood; D001912:Bovine Virus Diarrhea-Mucosal Disease / Q000097:blood / Q000453:epidemiology / Q000821:virology*; D002353:Carrier State / Q000276:immunology / Q000662:veterinary* / Q000821:virology; D002417:Cattle; D030241:Diarrhea Virus 1, Bovine Viral / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D030242:Diarrhea Virus 2, Bovine Viral / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000662:veterinary*; D016014:Linear Models; D015995:Prevalence; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012680:Sensitivity and Specificity
chemical_list: D000956:Antigens, Viral; D012367:RNA, Viral