Dirofilaria Immitis: Genotyping European Clinical Samples and US Laboratory Isolates at Single Nucleotide Polymorphic (SNP) Sites Associated With Macrocyclic Lactone Susceptibility and Resistance

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 susceptible isolates, Berkeley, and Georgia II, one 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 associated with macrocyclic lactone susceptibility and 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 to estimate the probability of identity with known susceptible and resistant isolates.Results: In the three laboratory-maintained US D. immitis isolates Berkeley had a 2-SNP pairwise FST of 0.00, Georgia II had a 2-SNP pairwise FST of 0.07 indicating two ML-susceptible genotypes. WildCat had a 2-SNP pairwise FST of 0.33 indicating a ML-resistant genotype. The genotype analysis of the European clinical samples showed that all eleven had 2-SNP pairwise FST of 0.00, which indicates their genotypes, at these SNP sites, are consistent with ML susceptibility. Conclusions: The results of the current study provides a genotypic analysis of European clinical samples with US SNP molecular markers associated with ML susceptibility and resistance. The eleven clinical samples tested show no genomic evidence of ML-resistance. Vigilance is needed to maintain susceptibility to heartworm preventives in regions of the world so far without resistance. The early adoption of genotyping of clinical D. immitis samples could provide an early indication of the potential development of ML-resistance and aid to distinguish clinical cases of heartworm infection due to ML resistance from those due to a lack of prevention or inadequate compliance, as has been seen in North America.