Preliminary description of a polymerase chain reaction test for bluetongue and epizootic hemorrhagic disease viral RNA in bovine semen.

Abstract

Bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) are arthropod-borne orbiviruses of worldwide agricultural and economical importance. These viruses are transmitted from animal to animal by biting midges of the genus Culicoides. Several viruses, including BTV, can be isolated in bovine semen. 5 This finding is of considerable concern to the animal breeding industry. The potential presence of BTV and EHDV in bovine semen has resulted in time-consuming and expensive processes for certifying bovine semen free of these viruses. 9 BTV isolation from semen using cell cultures is difficult because of cell culture toxicity of the spermatozoa and seminal plasma. 4 This problem has been circumvented for bovine herpesvirus by the use of polymerase chain reaction (PCR) procedures on bovine semen samples. 12,13 Recently, capture nested PCR procedures were developed that utilize a reverse target capture for viral RNA extraction and reverse transcriptase/nested PCR for BTV and EHDV RNA detection. 11,14 These capture nested PCR tests are BTV and EHDV group specific and can detect all US stereotypes of these viruses. In this study, the capture nested PCR procedure was applied to the detection of BTV or EHDV RNA in virus-spiked raw semen samples. Dilutions of cell culture source viruses were added to virus-free bovine semen and incubated overnight at 4 C. Initially, samples were diluted 2-fold with 10 mM Tris-HCl (pH 8) and sonicated using a cup-horn sonicator for 1 minute. The samples were then microfuged at 12,000 3 g for 10 minutes at 4 C. The pellet was resuspended in 100 ml of sterile water, and 200 ml of extraction buffer 11 was added. The target RNA was denatured and captured using the biotinylated outer antisense primer and strepavidin-coated magnetic beads a as described previously. 11 The sonication and centrifugation step were useful for reducing the protein material, which could cause sample handling problems due to protein aggregates. However, this step was unnecessary for these samples but may be necessary for samples with a higher protein content. In the results reported here, the samples were diluted 2-fold directly with the extraction buffer, and the target RNA was captured as described previously. 11 The captured target RNA was then denatured with 33% formamide and heat, reverse transcribed, and amplified as described previously. 11,14 The products were detected using agarose gel electrophoresis. The BTV assay consistently detected viral RNA from 1‐100 CCID50/0.5 ml and with less consistency down to 0.1 CCID50/0.5 ml (Fig. 1). Samples spiked with 1,000 CCID50/0.5 ml of virus were not detected perhaps because of template inhibition of the PCR amplification. The EHDV assay with spiked raw semen was 100-fold less sensitive (100‐1,000 CCID50/0.5 ml) than