Persistence of Wolbachia DNA in Dirofilaria immitis-infected canines after doxycycline-only treatment in Rio de Janeiro State, Brazil

Abstract Background Wolbachia are obligatory bacterial endosymbionts of Dirofilaria immitis heartworms, necessary for worm development, reproductive capability, and viability. We quantitatively examined microfilariae and Wolbachia in doxycycline-treated, heartworm-positive dogs. Methods Microfilaremic heartworm-positive dogs were identified in municipalities within the target areas of Rio de Janeiro and enrolled in this study. Dog owners agreed to allow blood draws from their dogs on day 0 and days 60 and 90 (± 5 days), and to administer doxycycline 10 mg/kg BID for 28 days, with weekly phone follow-up to help ensure compliance. Modified Knott’s tests were performed on each experimental day; a portion of each sample was retained and frozen at − 20 °C, and the remaining quantities were submitted for digital PCR (dPCR) of microfilarial and Wolbachia burdens. None of the dogs enrolled in the study received any macrocyclic lactone during the study. Results Thirty-one microfilaremic dogs completed the study, with 27 remaining microfilaremic throughout. Wolbachia were cleared from 25 of the dogs by day 90, demonstrating that the doxycycline dose was fully effective in those cases. Interestingly, 6 dogs remained dPCR positive on the quantitative assessment, and the proportions of Wolbachia / D. immitis on days 60 and 90 were significantly ( p < .0001) lower than on day 0. Results of the comparisons of proportions for day 60 vs. day 90 were not statistically significant ( p = 0.3127). Conclusions This study demonstrates that Wolbachia DNA may persist in some dogs for 90 days after doxycycline-only therapy. These data may reflect the lack of macrocyclic lactone use or higher adult burdens in some dogs; however, it is not possible to confidently conclude these factors entirely underpin our observations. We suggest that further studies should investigate whether this is related to lack of efficacy of doxycycline in certain Wolbachia subpopulations or to other factors.