Enhanced rabies surveillance in roadkill specimens by real-time RT-PCR

Abstract

Roadkill specimens are an important source of samples for enhanced rabies surveillance (ERS) in areas where other methods of sample collection may not be practi-cal. However, the physical condition of roadkill specimens is unpredictable and, in many circumstances, unsatisfactory for rabies diagnostic testing by antigen detection methods. The high sensitivity of real-time reverse transcriptase PCR (RT-PCR) holds promise for rabies diagnostic testing of poor-quality samples. We conducted an evaluation of real-time RT-PCR to detect rabies virus RNA in roadkill samples. A total of 299 specimens were collected from raccoons (n = 232), skunks (n = 46), foxes (n = 17), coyotes (n = 2), a bobcat (n = 1), and a domestic cat (n = 1) across ten states during 2018 – 2021 in the United States. Eight samples (2.7%) were positive using the LN34 pan-lyssavirus real-time RT-PCR assay. These eight rabid animals in areas of high interest for wildlife rabies management would likely not have been identified other-wise. These findings support the use of real-time RT-PCR for samples that would typically be unsuitable for testing by widely used antigenic-based detection methods such as the direct fluorescent antibody test (DFA or FAT) and direct rapid immunohistochemistry test (DRIT).