Secretory microRNA Profiles of Third- and Fourth-Stage Dirofilaria immitis Larvae with Different Macrocyclic Lactone Susceptibility: In Search of Biomarkers for Early Detection of Infection.

Lucienne Tritten,Guilherme G Verocai,Erica J Burkman,Tobias Clark

doi:10.3390/pathogens10070786

Abstract

The canine heartworm, Dirofilaria immitis, is among the most important parasites of dogs in the United States and worldwide, and may cause severe and potentially fatal disease. Current diagnostic recommendations rely on serological detection of an adult female antigen, and visualization of microfilariae in the blood. Therefore, a reliable diagnosis can be only performed approximately six months post-infection. There is a growing need to characterize novel diagnostic markers that are capable of detecting the early stages of heartworm infection, in special markers associated with third-stage larvae (L3) and fourth-stage larvae (L4). The early detection of infection would guide medical interventions that could impede the development of patent infections and further parasite transmission. We cultured D. immitis L3 and L4 of two laboratorial strains with different susceptibility statuses to macrocyclic lactone drugs in vitro. Excretory/secretory microRNAs were sequenced and analyzed. We identified two miRNA novel candidates secreted abundantly by both L3 and L4 of both strains. These candidates were previously detected in the secretions of other D. immitis stages and one of them was found in the blood of D. immitis-infected dogs. These miRNAs have not been found in the secretions of other nematodes and could be D. immitis-specific diagnostic biomarkers, which could allow for the early detection of infection.

Highlights

Our objective was to assess the consistency of the excretory/secretory miRNA profiles of L3 and L4 of two D. immitis strains in vitro, despite their different macrocylic lactone susceptibility status
We found a good agreement between the current study and previously published data for D. immitis [16], there was generally an even higher agreement between L3 and L4 secretions from the same study, highlighting the impact of the inherent inter-experimental variation on resulting data
Regardless of the macrocyclic lactone susceptibility status of the two strains we have examined, our data suggest that the strain involved will have little impact on the most abundantly secreted miRNAs

Summary

Introduction

The canine heartworm, Dirofilaria immitis, is arguably the most important parasite of dogs in the United States of America (USA) and across many countries worldwide. Heartworm is known to cause severe and often fatal disease in infected dogs, cats, and ferrets [1,2]. In addition to being the most prevalent filarial parasite of dogs in the country, heartworm incidence has been increasing in recent years, in the southeastern. The current guidelines of the American Heartworm Society recommend annual testing of all dogs in order to ensure the achievement and maintenance of successful prophylaxis

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