Accurate detection of Bovine Leukemia Virus using recombinant p24-ELISA and PCR

Abstract

ACCURATE DETECTION OF BOVINE LEUKEMIA VIRUS USING RECOMBINANT P24-ELISA AND PCR Gutierrez, Geronimo; Dus Santos, Maria Jose; Alvarez, Irene; Lomonaco, Marina; Politzki, Romina; Rodríguez, Sabrina; Trono, Karina Introduction: Diagnosis of Bovine leukemia virus (BLV) infection is currently carried out by agar gel immunodiffusion (AGID) and ELISA (1). Even when both tests are approved by the OIE for trade purposes, several factors can contribute to yield conflicting results that need to be confirmed. In this work, ELISA and PCR were used as complementary tools to confirm the real sanitary status for specific situations as breeding or commercialization. The performance characteristics of an in-house developed recombinant ELISA (rp24-ELISA) were evaluated according to international guidelines (2,3). Material & methods: The BLV p24 major capsid protein was expressed as a recombinant thioredoxin-6xHis fusion protein in Escherichia coli in our laboratory. The purified rp24 was used as antigen for the ELISA test (rp24-ELISA) to detect antibodies to BLV. To assess the performance characteristics of this test 710 field serum samples were analyzed by rp24-ELISA and a commercial AGID test (UNLP AGID). Amplification of the envelope gene by nested PCR (nPCR) (4) and an in-house Western Blot assay were used to confirm discordant results on a well-characterized herd. Results: rp24-ELISA and AGID showed a 90.5% of concordance when the 710 field serum samples were analyzed on the accuracy trial. rp24-ELISA could detect 269 positives samples while AGID only detected 210. When nPCR was carried out 18 out of 22 samples that were declared as positive by rp24-ELISA were positive by nPCR. In addition, the rp24-ELISA was able to detect 5 out of 21 infected animals that were not detected by AGID. Five out of 8 samples that were not declared as positives by AGID were detected by WB and rp24-ELISA. Complementary, the rp24-ELISA demonstrated to be a precise assay with excellent repeatability, reproducibility and better analytical sensitivity than AGID Discussions & conclusions Taking these results into account, the rp24-ELISA could be adopted as an official test method for diagnosis and control of BLV in Argentina, and together with the nested-PCR assay could be considered as complementary tools for confirmatory situations. References 1- World Organization for Animal Health (OIE). 2004. Manual of Diagnostic Test and Vaccines for Terrestrial Animals (mammals, birds and bees). Chapter 2.3.4. Enzootic bovine leukosis 2- World Organization for Animal Health (OIE). 2004. Manual of Diagnostic Test and Vaccines for Terrestrial Animals (mammals, birds and bees). Chapter 1.1.3. Principles of validation and of diagnostic assays for infectious diseases. 3- International Committee of Harmonisation. Tripartite Guidelina Q2A: Text on Validation of Analytical Procedures, 1994, and Tripartite Guideline Q2B: Validation of Analytical Procedures: Methodology, 1996. 4-Lew, AE; Bock, RE; Molloy, JB; Minchin, CM; Robinson, SJ; Steer, P. Sensitive and specific detection of proviral bovine leukemia virus by 5´Taq nuclease PCR using a 3´ minor groove binder fluorogenic probe. J Virol Methods, 2004 Feb;115(2):167-7