An RT-qPCR Assay from Rectal Swabs for the Detection of Rabbit Hemorrhagic Disease Virus 2 in Natural Cases

Abstract

Rabbit hemorrhagic disease virus 2 (RHDV2 or Lagovirus europaeus GI.2) is spreading across North America. This has enabled submissions of lagomorphs for testing to veterinary diagnostic laboratories (VDLs). The liver is currently the gold-standard sample type for testing by RT-qPCR. VDL clients usually seek alternate diagnostic approaches that permit simpler and faster sample collection with less risk of environmental contamination; there is also a necessity for a sample type that can be collected from live animals. Therefore, the goal of this study was to optimize and evaluate an RT-qPCR assay on rectal swabs collected from a group of carcasses of leporids of different species that were submitted to a VDL during an RHDV2 outbreak. A total of 130 carcasses were tested both by liver tissue and rectal swab RT-qPCR. The results of the liver samples were considered the gold standard, and 73 carcasses tested positive and 57 carcasses tested negative in liver. Out of the 73 liver RT-qPCR-positive carcasses, 64 tested positive and 9 tested negative on the rectal RT-qPCR. All 57 liver RT-qPCR-negative carcasses tested negative on the rectal RT-qPCR. The sensitivity and specificity of the rectal RT-qPCR were 88% and 100%, respectively, most likely due to significantly ( $p<0.001$ ) lower viral loads in the rectal swabs (median Ct: 27.03) compared to the liver samples (median Ct: 12.69). Despite being more than 4 logs less sensitive, RT-qPCRs from rectal swabs can be used to screen leporid carcasses for the presence of RHDV2 RNA.