| Summary: | The canine heartworm, <i>Dirofilaria immitis</i>, 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 <i>D. immitis</i> 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 <i>D. immitis</i> stages and one of them was found in the blood of <i>D. immitis</i>-infected dogs. These miRNAs have not been found in the secretions of other nematodes and could be <i>D. immitis</i>-specific diagnostic biomarkers, which could allow for the early detection of infection.
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