Seroprevalence of Dirofilaria immitis in traveling dogs: implications for transboundary pathogen movement

Abstract Background Dirofilaria spp. are parasitic filarial nematodes endemic worldwide that infect dogs but are also zoonotic. Traveling dogs may serve as carriers of zoonotic parasites, including Dirofilaria immitis, introducing them to new regions. However, the risks of importation from endemic areas are unknown. Methods To study seroprevalence in travelling dogs, we first established the signal-to-positive (S/P) cutoffs for the DiroCHEK ™ ELISA assay (Zoetis) by spectrophotometry at three wavelengths (490, 620, or 650 nm). We also validated the assay for testing serum pools using known D. immitis -positive serum. We tested sera collected from 6699 dogs traveling from 34 countries between September 2022 and December 2023 using the spectrophotometric method. Samples were tested in serum pools initially, with pool sizes ranging from 5 to 15 samples based on reported prevalence. All pools were tested with and without heat treatment to disassociate immune complexes. Where sample numbers were insufficient to form a pool, samples were tested individually without heat treatment. Dogs in pools that tested positive were retested individually without heat treatment. Prevalence was calculated from pooled testing and individual testing results. Results We tested 649 serum pools, of which 50 heat-treated pools and 11 non-heat-treated pools tested positive spectrophotometrically. Of these 61 positive pools, 7 tested positive in non-heat treated and heat-treated conditions, resulting in 54 unique pools. During re-testing of individual dogs from the positive pools, 31 pools contained dogs infected with D. immitis , while 23 pools had no positive individuals identified, indicating the presence of cross-reactive nematodes or antigen–antibody complexes. The prevalence of D. immitis and cross-reactive nematodes based on pooled testing was 0–5.32%. Due to the unavailability of specific serological tests for cross-reactive nematodes, they were not identified further. The prevalence of D. immitis based on individual testing was 0–11.1%, which was lower than the reported prevalence in their countries of origin. Conclusions Our findings indicate that traveling dogs may carry D. immitis and cross-reactive nematodes, increasing the risk of introducing these parasites and/or novel parasite genetics to new areas. While pooled testing allows for screening populations rapidly, this methodology may underestimate true prevalence, particularly in low-prevalence regions.