Evaluation of the SNAP® 4Dx® plus test for the detection of Dirofilaria immitis antigen and characterization of exposure to tick-borne pathogens in wild canids in southern Ontario

Population-level surveys for Dirofilaria immitis in wild canids typically rely on identification of the parasite at necropsy. More recently, some studies have employed the use of the SNAP (R) 4Dx (R) Plus test. However, since the assay was designed for use with domestic dogs it needs to be validated for use with wild canids for accurate interpretation of results. We therefore evaluated the performance of the SNAP (R) 4Dx (R) Plus test for detection of D. immitis in wild canids in southern Ontario. Overall, 199 wild canid carcasses were collected from across the region and assessed for the presence of D. immitis parasites at necropsy; ten were infected. Lung tissue extract (LE) and thoracic fluid filter paper extract (TFE) prepared from each wild canid were tested via the SNAP (R) 4Dx (R) Plus test, which simultaneously tests for the presence of D. immitis antigen and antibodies to Borrelia burgdorferi, Anaplasma spp., and Ehrlichia spp. The prevalence adjusted bias adjusted kappa (PABAK) and Gwet's first -orderagreement coefficient (AC1) were used to assess the level of agreement between sample pairs. The PABAK and AC1 between LF and TFE applied to the SNAP (R) 4Dx (R) Plus test and the necropsy-confirmed D. immitis status indicated a very high level of agreement for all sample pairs. Compared to necropsy-confirmed D. immitis status, the estimated sensitivity and specificity of the SNAP (R) 4Dx (R) Plus test for D. immitis antigen in LE was 80 % (95 % CI 44.4-97.5%) and 98.9 % (95 % CI 96.2-99.9%), respectively. For the TFE, the sensitivity was 70 % (95 % CI 34.8-93.3%) and the specificity was 97.9 % (95 % CI 94.7-99.4%). With respect to the tick-borne pathogen components, 1.5 % (3/199; 95 % CI 0.3-4.5%) of wild canids tested positive for B. burgdorferi antibody; 1.0 % (2/199; 95 % CI 0-3.8%) of LE samples were positive and 0.5 % (1/199; 95 % CI 0-3.1%) of TFE samples were positive. No samples tested positive for antibody to Anaplasma spp. (95 % CI 0-2.3%) or Ehrlichia spp. (95 % CI 0-2.3%). Collectively, the results suggest that the SNAP (R) 4Dx (R) Plus test may be a suitable test for use with LE and TFE for the detection of D. immitis antigen in wild canids from southern Ontario.