The Use of Molecular Markers to Investigate Possible Resistance to Heartworm Preventives in Dirofilaria Immitis Samples from Heartworm Positive Dogs in Europe

Background: Dirofilaria immitis is a parasitic filarial nematode. It is the causative agent of dirofilariosis, a potentially fatal pulmonary infection which primarily infects canids and felines. dirofilariosis infections are primarily controlled with a prophylactic macrocyclic lactone (ML) regimen. Recent evidence has confirmed the development of ML-resistant isolates in the US which are genetically distinct from wild-type populations. Previous research clinically validated 9 single nucleotide polymorphism (SNP) molecular markers associated with these ML resistance phenotypes isolated from the USA. Methods: In this study, three D. immitis US laboratory-maintained isolates: two putative susceptible isolates, Berkeley, and Georgia II, one putative resistant isolate, WildCat; and eleven European D. immitis clinical samples, from Italy, Spain, and Hungary were analyzed. The samples tested were fresh microfilaria (mf) in blood or adult female worms shipped in ethanol and rehydrated in phosphate buffered saline (PBS). After DNA extraction, each sample underwent MiSeq sequencing of regions encompassing the 9 SNP sites previously correlated with ML resistance. The nucleotide frequencies of the 9 SNP sites were analyzed and the pairwise fixation index (FST) of the top 2 SNP molecular markers were calculated in order to estimate the probability of identity with known resistant isolates.Results: In the three laboratory-maintained US D. immitis isolates Berkeley had a 2-SNP pairwise FST of 0.00, indicating a ML-susceptible genotype, WildCat had a 2-SNP pairwise FST of 0.33 indicating a ML-resistant genotype, and Georgia II had a 2-SNP pairwise FST of 0.07, which may indicate early selection for ML resistance. The genotype analysis of the European clinical samples showed that all eleven European samples had 2-SNP pairwise FST of 0.00, which indicates their genotypes are consistent with ML susceptibility. Conclusions: Prior to genotyping the European samples, it was possible that the positive heartworm infections could have arisen because of the development of ML-resistance or due to lack of owner compliance or incomplete use of heartworm preventives. Our results indicate no genomic evidence of ML-resistance and suggests that resistance has not developed, so far, in Europe, or been introduced via movement of infected dogs. However, vigilance is needed to maintain susceptibility to heartworm preventives in regions of the world so far without resistance.