Differential ABC transporter gene expression in adult Dirofilaria immitis males and females following in vitro treatment with ivermectin, doxycycline or a combination of both

Chiara Lucchetti,Marco Genchi,Chiara Bazzocchi,Laura Helen Kramer,Alice Vismarra,Paolo Serventi,Luigi Venco,Alessandro Menozzi,Simone Bertini

doi:10.1186/s13071-019-3645-y

Chiara Lucchetti, Marco Genchi + Show 7 more

Open Access

https://doi.org/10.1186/s13071-019-3645-y

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Abstract

BackgroundCombination 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.ResultsQuantitative 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.ConclusionsFurther 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.

Highlights

Combination doxycycline/macrocyclic lactone (ML) protocols have been shown to provide a more rapid adulticidal and microfilaricidal effect than either Macrocyclic lactones (MLs) or doxycycline alone, female worms were reported to have a higher tolerance to treatments compared to male worms
Collection and maintenance of D. immitis adults Seven privately-owned dogs that had never received macrocyclic lactones or doxycycline were diagnosed with patent D. immitis infection
Determination of gene expression profile after treatment Quantitative RT-PCR analysis of Dim-pgp-10, Dimpgp-11 and Dim-haf-4 gene expression showed very little variation between NI controls and medium with dimethyl sulfoxide (DMSO) alone

Summary

Introduction

Combination doxycycline/macrocyclic lactone (ML) protocols have been shown to provide a more rapid adulticidal and microfilaricidal effect than either MLs or doxycycline alone, female worms were reported to have a higher tolerance to treatments compared to male worms. Protocols combining doxycycline, which targets the bacterial endosymbiont Wolbachia, together with a macrocyclic lactone, are currently recommended either as pre-adulticide preparation (resulting in less severe post-melarsomine complications) [7, 8] or as an alternative to melarsomine when this is unavailable or contraindicated [6, 9]. Doxycycline has various detrimental effects on developing and adult heartworms, including degeneration of oocytes and early embryonic stages, but its adulticide efficacy is low and slow [10]. The use of macrocyclic lactones as adulticides has been implicated in the development of drug resistance and is no longer recommended [11, 14]

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