Background: Combination doxycycline/macrocyclic lactone (ML) protocols have been shown to provide a more rapid adulticidal and microfilaricidal effect than either MLs or doxycycline alone, although female worms were reported to have a higher tolerance to treatments compared to male worms. The present study aimed to evaluate how ABC transporters may be involved in the synergic effect of the combination treatment. Adult worms of D. immitis were treated in vitro for 24 hours with doxycycline (DOXY), ivermectin (IVM) and a combination of both, and changes in the modulation of ABC transporter genes were measured. Levels of doxycycline inside different treatment media, post-treatment, were determined through HPLC analysis. Results: Quantitative RT-PCR analysis showed the presence of changes in the modulation of ABC transporter genes evaluated in this study. In particular, in female worms, the combination treatment induced a substantial increase in gene expressions, especially of Dim-pgp-10 and Dim-haf-4; whereas in male worms, the greatest increase in gene expression was observed for Dim-pgp-10 and Dim-pgp-11 when treated with DMSO + IVM and DMSO + DOXY/IVM. HPLC analysis of the DOXY concentrations in the media after in vitro treatments of male worms showed a slight difference between the DMSO + DOXY samples and the combination (DMSO + DOXY + IVM), while no difference was observed among females. Conclusions: Further studies are required to explain whether the modulation of cellular efflux plays a role, even partially, in the adulticide effect of doxycycline/macrocyclic lactone combinations in heartworm-infected dogs. To the authors' knowledge, this is the first study to evaluate P-gp expression in adult D. immitis.
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