Molecular detection and quantification of bluetongue virus in small ruminant and Culicoides vectors in Gujarat, India

Abstract

The present study was carried out to detect bluetongue virus (BTV) in Culicoides vectors and the biological samples of small ruminants with the use of PCR based molecular methods. The vectors were collected using the UV LED CDC traps, placed near animal sheds and then identified by morphological and PCR based methods. Primers specific to genus Culicoides and species Culicoides oxystoma were used in PCR reaction. The end point RT-PCR protocols were used for tentative and confirmatory diagnosis of BTV and its two serotypes viz. BTV-1 and BTV-16, whereas, the real time RT-PCR was used for confirmation of BTV and quantization of viral RNA load. Out of a total of 132 blood samples and 55 tissue samples viz., spleen, liver, kidney, thymus and heart blood of aborted fetuses and 09 pooled samples of Culicoides, total 21 (6.93%) samples i.e., 11 blood samples, 08 spleens and 02 pooled Culicoides samples were detected positive for BTV by NS1 gene specific RT-PCR. In nested PCR, 8 additional samples were found positive for BTV. Among these 29 positive samples, 6 were collected from spleens of aborted fetuses which was indicative of transplacental transmission of BTV among small ruminants. All the positive samples were of either of BTV-1 and BTV-16 serotypes as confirmed by serotype specific PCR. In real time PCR, plasmid vector based positive control was prepared, which confirmed the 21 samples to be BTV positive, whereas, quantization of viral RNA, showed that out of all clinical samples tested, highest and lowest RNA copy numbers i.e. 3566.41 and 81.79 were found in blood sample and spleen of sheep, respectively.