Laboratory assays reveal diverse phenotypes among microfilariae of Dirofilaria immitis isolates with known macrocyclic lactone susceptibility status.

Jeba R J Jesudoss Chelladurai,Pablo D Jimenez Castro,Krystal Chinchilla-Vargas,Matthew T Brewer,Katy A Martin,Ray M Kaplan,Adler R Dillman

doi:10.1371/journal.pone.0237150

Jeba R J Jesudoss Chelladurai, Pablo D Jimenez Castro + Show 5 more

Open Access

https://doi.org/10.1371/journal.pone.0237150

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Abstract

Prevention of canine heartworm disease caused by Dirofilaria immitis relies on chemoprophylaxis with macrocyclic lactone anthelmintics. Alarmingly, there are increased reports of D. immitis isolates with resistance to macrocyclic lactones and the ability to break through prophylaxis. Yet, there is not a well-established laboratory assay that can utilize biochemical phenotypes of microfilariae to predict drug resistance status. In this study we evaluated laboratory assays measuring cell permeability, metabolism, and P-glycoprotein-mediated efflux. Our assays revealed that trypan blue, propidium iodide staining, and resazurin metabolism could detect differences among D. immitis isolates but none of these approaches could accurately predict drug susceptibility status for all resistant isolates tested. P-glycoprotein assays suggested that the repertoire of P-gp expression is likely to vary among isolates, and investigation of pharmacological differences among different P-gp genes is warranted. Further research is needed to investigate and optimize laboratory assays for D. immitis microfilariae, and caution should be applied when adapting cell death assays to drug screening studies for nematode parasites.

Highlights

Dirofilaria immitis is the causative agent of canine heartworm disease, which is capable of causing debilitating cardiovascular and respiratory signs in domestic dogs, cats, wild canids, and mustelids including ferrets
In the presence of macrocyclic lactone (ML), the susceptible Missouri isolate had higher absorbance values when compared to ML-resistant isolates JYD-27, Metairie and Yazoo (p < 0.05, Fig 1)
A similar trend was observed in untreated microfilariae, the difference between drug susceptible and resistant isolates was not as large as that observed in the presence of MLs

Summary

Introduction

Dirofilaria immitis is the causative agent of canine heartworm disease, which is capable of causing debilitating cardiovascular and respiratory signs in domestic dogs, cats, wild canids, and mustelids including ferrets. Control and prevention of D. immitis largely relies on macrocyclic lactone (ML) chemoprophylaxis. This strategy prevents the development of adults from 3rd stage larvae of D. immitis following infection by L3s from mosquito intermediate hosts. This approach has been largely successful for decades in the United States. Over the past ten years, numerous lack of efficacy (LOE) reports have been made by veterinary.

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