BACKGROUND: Wolbachia is present in all life stages of Dirofilaria immitis. Wolbachia surface protein (WSP) can be highly immunogenic and induce acute inflammatory reactions in the host upon worm death. To avoid the abrupt release of Wolbachia and its antigens from deceased parasites, the American Heartworm Society (AHS) has recommended using doxycycline (DOXY) and having a 1-month wait period between the DOXY treatment and the adulticidal process for Wolbachia elimination. Studies have shown that the 28 day, 10 mg/kg twice daily (BID) administration of DOXY can effectively clear Wolbachia in the bloodstream of the host. The 1-month wait period is hypothesized to allow for further reduction of Wolbachia. However, the levels of Wolbachia in adult parasites after the DOXY treatment remain unknown. METHODS: Forty-five purposely bred dogs were intravenously transplanted with 20 Dirofilaria immitis adults, consisting of 12 females and 8 males. The dogs were divided into nine groups of five dogs each. Two groups each received 5, 7.5, or 10 mg/kg DOXY BID orally for 28 days, and ivermectin (IVM) monthly (6 µg/kg.) Three groups remained untreated as controls. Study animals were necropsied on day 0, day 30, and day 60, following the start of treatment. Adult worms were collected at necropsy and preserved for analysis. Quantitative polymerase chain reaction (qPCR) and immunohistochemistry for WSP were performed on worms collected at each time point. The data were analyzed using a linear mixed model (LMM). Multiple comparisons were adjusted using Tukey's test. RESULTS: The qPCR results showed that all treatment doses significantly reduced Wolbachia levels compared with the control groups at 30 and 60 days. The intradose comparison indicated a significant decrease on day 60 compared with day 30. No significant differences were found between different doses on the two examination dates. Immunohistochemistry indicated the markedly reduced presence of Wolbachia in treatment groups. CONCLUSIONS: All DOXY dosages can be considered effective in reducing Wolbachia on both tested dates (30 and 60 days). On the basis of the further reduction of Wolbachia levels in adult D. immitis, the 1-month rest period in the AHS heartworm treatment guidelines is beneficial. Wolbachia can still be detected on day 60 in all dosage groups.