Suspect macrocyclic lactone resistance cases with the canine heartworm (Dirofilaria immitis): mismatch between microfilarial suppression test and SNP results in Australia

Canine heartworm disease is a potentially deadly cardiopulmonary disease caused by the mosquito-borne filarial nematode Dirofilaria immitis. In Australia, the administration of macrocyclic lactone (ML) drugs has successfully reduced the prevalence of D. immitis infection. However, the recent re-emergence of D. immitis in dogs in Queensland, Australia and the identification of ML-resistant isolates in the USA poses an important question of whether ML-resistance has emerged in this parasite in Australia. The aim of this study was to utilise phenotypic and genotypic assays to determine the presence of ML-resistant D. immitis in Australia. To do this, we surveyed 45 (n=45) dogs from Queensland and New South Wales across 3 years (2019-2022) for the presence of D. immitis infection using an antigen test, quantitative Modified Knott's test, and qPCR targeting both D. immitis and the D. immitis symbiont Wolbachia. A phenotypic assay utilising a microfilarial suppression test for 23/45 dogs was coupled with genetic testing of filtered microfilariae for SNPs previously associated with ML-resistance in isolates from the USA. Sixteen (16/45) dogs tested positive for D. immitis infection despite reportedly receiving 'rigorous' heartworm prevention for 12 months prior to the study. The phenotypic and genotypic assays in this study did not unequivocally demonstrate the presence of ML-resistant D. immitis in Australia. Although the failure of 16 dogs to reduce microfilaremia by >90% after ML treatment was considered phenotypic evidence of suspect ML-resistance, no genotypic evidence was discovered using the genetic SNP analysis. It is possible that the presence of ML-resistance in this study was masked by the high initial dose of moxidectin (microspheres, 500 ug/kg s.c.) administered to eighteen (18/23) dogs at the time of diagnosis, which had the potential to clear any ML-resistant D. immitis microfilariae prior to post-ML treatment sampling. ### Competing Interest Statement The authors have declared no competing interest.