|Knowles Jr, Donald|
Submitted to: Clinical and Vaccine Immunology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/9/2007
Publication Date: 10/15/2007
Citation: Hoesing, L.M., White, S.N., Lewis, G.S., Mousel, M.R., Knowles Jr, D.P. 2007. Development and Validation of an Ovine Progressive Pneumonia Virus Quantitative PCR. Clinical and Vaccine Immunology. 14(10):1274-1278
Interpretive Summary: Molecular diagnostic tests are highly sought for accurate detection of infectious pathogens of sheep. This study describes the development and validation of a new ovine progressive pneumonia virus (OPPV) quantitative (q) PCR test for detection of OPPV infection in sheep. The OPPV qPCR targets a conserved portion of the envelope gene, the tm region. Paired serum and peripheral blood leukocyte samples were obtained from 396 sheep. This OPPV qPCR was compared against a standard serological diagnostic test, competitive (c) ELISA. The OPPV qPCR had a positive concordance of 96.2% and a negative concordance of 97.7% when compared to the cELISA. In addition, cloning and sequencing of the tm from 18 different sheep confirmed the qPCR target. This OPPV qPCR may be used as a confirmatory or supplemental diagnostic tool for OPPV infection.
Technical Abstract: Ovine progressive pneumonia virus (OPPV) infects at least one sheep in eighty-one percent of U.S. sheep flocks as measured by serological diagnostic tests and can cause viral-induced mastitis, arthritis, dypsnea, and cachexia. Diagnostic tests that quantify OPP proviral load in peripheral blood leukocytes (PBL) may aid in the identification of infected and super-spreader OPPV sheep and are highly sought. In this study, we developed primers and a Taqman® probe to the highly conserved transmembrane (tm) region of the OPPV envelope gene and established a real-time quantitative (q) PCR assay. This new OPPV qPCR assay was evaluated against a competitive inhibition enzyme-linked immunosorbent assay (cELISA) using 396 PBL samples and sera from Dubois, Idaho sheep ranging in ages 3 to 6 years of the Columbia, Rambouillet and Polypay breeds. The new OPPV qPCR had a positive concordance of 96.2±2.3% and a negative concordance of 97.7±2.5% as compared to the cELISA with a kappa value of 0.93 indicating excellent agreement between the two tests. In addition, the presence of tm was confirmed from the three OPPV qPCR positive and cELISA negative sheep and from fifteen sheep with different OPP proviral loads by cloning and sequencing. These data indicate that the OPPV qPCR may be used as a confirmatory or supplemental diagnostic tool for OPPV infection.