Re-assessment of direct fluorescent antibody negative brain tissues with a real-time PCR assay to detect the presence of raccoon rabies virus RNA

The first report of the raccoon variant of rabies virus was in Ontario, Canada in 1999. As part of the control of this outbreak a Point Infection Control (PIC) strategy of trapping and euthanizing vector species was implemented. To evaluate whether this strategy was indeed removing diseased animals, rabies diagnosis was performed on these specimens. During a PIC program conducted in 2003, 721 animals (raccoons, striped skunks and red foxes) were collected and euthanized and brain material from each specimen was divided into two halves; one half was submitted for rabies diagnosis by a direct fluorescent antibody (DFA) test while the other was tested using a sensitive real-time reverse-transcriptase polymerase chain reaction (RT-qPCR), to detect raccoon rabies virus (RRV) RNA. This latter assay can detect less than ten viral copies in 200ng of total cellular RNA. All 721 PIC brain samples were negative by the DFA test but ten of them (5 raccoons, 5 skunks) tested positive for raccoon rabies virus by the RT-qPCR assay albeit at low levels. Three of these samples were confirmed by sequencing of the PCR products. Little correlation was observed between clinical rabies DFA positive scoring categories and viral copy number as determined by RT-qPCR.