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 practical. 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 otherwise. 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).

Fig 1. Location of roadkill samples tested for rabies using the LN34 real-time RT-PCR test (red dots indicate positive results, black dots indicate negative or indeterminate results) relative to the 2018 oral rabies vaccination (ORV) zones (in blue) in the eastern U.S.

Fig 2. Photos of roadkills testing positive for rabies using the LN34 real-time RT-PCR test.

Sample order is the same as Table 2, from top left to bottom right.

Fig 3. Brain condition (A), skull condition (B), and estimated number of days in the field (C) for 267 roadkill samples tested for rabies using the LN34 real-time RT-PCR test.

Percent of positive, negative and indeterminate samples are shown by the size of colored bars and correspond to the y-axis. Number of samples per condition/result is written on bars. Brain condition, skull condition, and days in field were all chosen from a list of limited responses (see Methods). Samples from AZ were excluded (n = 32).

Fig 4. Current (A) and previous day (B) temperature in the field for 267 roadkill samples tested for rabies using the LN34 real-time RT-PCR test.

Temperature was recorded in °F. Boxplots show median plus 25% and 75% quartiles. Whiskers show largest observation less than or equal to 1.5 * interquartile range. Outliers are shown as dots. Samples with no temperature data (n = 12 for A, n = 13 for B) were excluded. Samples from AZ were excluded (n = 32).

Fig 5. Cost-benefit analysis comparing proportion of percent of rabies infected animals predicted to be detected (left axis, black line) and cost per informative result (positive/negative result, right axis, orange graph) at different sample degradation score thresholds (x-axis).