Comparison of different PCR tests to detect bovine lentivirus in cell culture and experimentally and naturally infected cattle.

Abstract

Genes targeted Reaction volume DNA Primers dNTPs Taq polymerase MgCl2 Cycles Hot start Misc. env and pol 50 ml 0.5 mg 20 pmol 200 mM 1.25 units 3 mM nested—62 cycles no pol and gag 50 ml 2.0 mg 25 pmol 200 mM 2.5 units 1.5 mM 40 cycles yes 0.01% gelatin 0.1% Triton X-100 pol 100 ml 5,000 cells 10 pmol 200 mM 5 units 1.5 mM 35 cycles yes The bovine lentivirus, also known as the bovine immunodeficiency-like virus (BIV), causes a persistent viral infection in cattle, but at present, it is unknown if it causes clinical disease or clinical immunodeficiency in cattle. Only a single epidemiological study has been reported with BIV, and this study associated a decreased milk yield in cows with BIV.4 In order to test whether BIV causes disease in cattle, a reliable diagnostic test to determine if cattle are infected with BIV is critical. A good diagnostic test should be specific, sensitive, and inexpensive and provide results quickly. However, none of the currently described BIV tests fit all of these categories. Serological tests have been the most commonly used diagnostic tests for BIV. However, loss of antibody to BIV proteins in experimentally infected cattle that are persistently infected with BIV has been reported.3,7,9,10,14 The loss of antibody to the p26 gag antigen, the most important antigen for detection of BIV in western blot tests and in some ELISA tests, can result in an animal considered to be uninfected with this virus even when virus can be demonstrated by both PCR and virus isolation.8,10,15 Virus isolation of BIV from naturally infected cattle remains difficult, with only 3 reported virus isolations.2,11,13 Viral diagnosis by PCR has been shown to be a sensitive diagnostic test in other viral systems, and although 5 PCR tests have been published for the detection of BIV, their sensitivity and specificity have not been compared.5,6,10 The purpose of this paper is to provide a comparison among the currently described PCR tests with standardization of some of the PCR conditions. Three different reports describe a total of 5 different PCR tests (Table 1).5,6,10 For all 5 tests, a total of 39 different templates were used with 0.5 mg of DNA per reaction (Table