Exploration of the sensitivity to macrocyclic lactones in the canine heartworm (Dirofilaria immitis) in Australia using phenotypic and genotypic approaches

Abstract

AbstractCanine heartworm disease is a potentially deadly cardiopulmonary disease caused by the mosquito-borne filarial nematodeDirofilaria immitis. In Australia, the administration of macrocyclic lactone (ML) drugs has successfully reduced the prevalence ofD. immitisinfection. However, the recent re-emergence ofD. immitisin 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 approaches to examine the sensitivity to ML drugs inD. immitisin Australia. To do this, we surveyed 45 dogs from Queensland and New South Wales across 3 years (2019-2022) for the presence ofD. immitisinfection using an antigen test, quantitative Modified Knott’s test, and qPCR targeting bothD. immitisand theD. immitissymbiontWolbachia. A phenotype observed by utilising sequential quantification of microfilariae 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 forD. immitisinfection despite reportedly receiving ‘rigorous’ heartworm prevention for 12 months prior to the study, according to the owners’ assessment. The phenotype and genotypic assays in this study did not unequivocally demonstrate the presence of ML-resistantD. immitisin Australia. Although the failure of 16 dogs to reduce microfilaremia by >90% after ML treatment was considered a suspect phenotype of ML-resistance, no genotypic evidence was discovered using the genetic SNP analysis. The traditional quantitative Modified Knott’s test can be substituted by qPCR targetingD. immitisor associatedWolbachiaendosymbiont DNA for a more rapid measurement of microfilariae levels. More definitive phenotypic evidence of resistance is critically needed before the usefulness of SNPs for the detection of ML-resistance in Australia can be properly assessed.