Genome structure and population genomics of the canine heartworm Dirofilaria immitis.

The heartworm, Dirofilaria immitis, is a filarial parasitic nematode responsible for significant morbidity and mortality in wild and domesticated canids. Resistance to macrocyclic lactone drug prevention represents a significant threat to parasite control and has prompted investigations to understand the genetic determinants of resistance. This study aimed to improve the genomic resources of D. immitis to enable a more precise understanding of how genetic variation is distributed within and between parasite populations worldwide, which will inform the likelihood and rate by which parasites, and in turn, resistant alleles, might spread. We have guided the scaffolding of a recently published genome assembly for D. immitis (ICBAS_JMDir_1.0) using the chromosomal-scale reference genomes of Brugia malayi and Onchocerca volvulus, resulting in an 89.5 Mb assembly composed of four autosomal- and one sex-linked chromosomal-scale scaffolds representing 99.7% of the genome. Publicly available and new whole-genome sequencing data from 32 D. immitis samples from Australia, Italy and the USA were assessed using principal component analysis, nucleotide diversity (Pi) and absolute genetic divergence (Dxy) to characterise the global genetic structure and measure within- and between-population diversity. These population genetic analyses revealed broad-scale genetic structure among globally diverse samples and differences in genetic diversity between populations; however, fine-scale subpopulation analysis was limited and biased by differences between sample types. Finally, we mapped single nucleotide polymorphisms previously associated with macrocyclic lactone resistance in the new genome assembly, revealing the physical linkage of high-priority variants on chromosome 3, and determined their frequency in the studied populations. This new chromosomal assembly for D. immitis now allows for a more precise investigation of selection on genome-wide genetic variation and will enhance our understanding of parasite transmission and the spread of genetic variants responsible for resistance to treatment.

Exploring multiplex qPCR as a diagnostic tool for detecting microfilarial DNA in dogs infected with Dirofilaria immitis: A comparative analysis with the modified Knott's test.

Current recommendations to diagnose cardiopulmonary dirofilariosis in dogs caused by Dirofilaria immitis involves tandem antigen and circulating microfilariae tests. The modified Knott's test is an important tool in heartworm diagnosis, allowing identification of circulating microfilariae. However, the subjective nature of the modified Knott's test affects its accuracy and diagnostic laboratories usually do not provide a quantitative outcome. Quantitative enumeration of microfilariae enables clinicians to track treatment progress and acts as a proxy for detecting emerging macrocyclic lactone resistance. There is a need for better diagnostic tools suitable for routine use to efficiently and accurately quantify the presence of D. immitis microfilaremia. The aim of this study was to determine whether the quantitative modified Knott's test can be substituted by multiplex quantitative polymerase chain reaction (qPCR) targeting D. immitis and associated Wolbachia endosymbiont DNA in canine blood samples. To do this, genomic DNA samples (n = 161) from Australian dogs, collected as part of a previous 2021 study, were assessed in a TaqMan qPCR targeting DNA of D. immitis, Wolbachia sp. and Canis lupus familiaris. Of the 161 genomic DNA samples, eight were considered positive for D. immitis microfilariae. The qPCR assay demonstrated good efficiency (E = 90 to 110%, R2 > 0.94). Considering the performance and efficient use of bench time, this TaqMan qPCR assay is a suitable alternative to the modified Knott's test for quantitative enumeration of microfilariae (Cohen's kappa coefficient [κ]: κ = 1 using D. immitis qPCR marker, κ = 0.93 using Wolbachia qPCR marker). The qPCR data demonstrated a comparable result to that of the quantitative modified Knott's test in a 2022 survey of D. immitis in Australian dogs (n = 23) before and after macrocyclic lactone (ML) administration. Improving the detection and diagnosis of canine heartworm infections will assist veterinarians in better managing and controlling disease outcomes and will be valuable for tracking the spread of ML resistance in Australia.

Whole-genome reference of Dirofilaria immitis from Australia to determine single nucleotide polymorphisms associated with macrocyclic lactone resistance in the USA.

For the past 30 years, chemoprophylaxis with macrocyclic lactone (ML) anthelmintics has been the primary strategy for canine heartworm (Dirofilaria immitis) control in both the USA and Australia. ML-resistant D. immitis isolates have been confirmed to exist in the USA and studies have shown that 42 single nucleotide polymorphisms (SNPs) are associated with phenotypic ML-resistance. Currently, ML-resistance has not been reported in any Australian clinical cases of canine heartworm. The aim of the study is to determine whether the 42 SNPs associated with resistance to MLs in the isolates from the USA are present in adult heartworms from a clinical case in Australia. Five adult D. immitis obtained from a dog at post-mortem (Sydney, Australia) were sequenced using the Illumina sequencing technology. The genomic analyses revealed 6 out of the 42 SNPs associated with ML-resistance to be present in our samples, 3 out of the 6 SNPs identified were nonsynonymous SNPs but not in candidate genes for ML-resistance. ML-susceptibility profile was mixed using the 42-SNP and 10-SNP models, but the 5-SNP, 3-SNP and 2-SNP models demonstrated ML susceptibility for all five individuals. In this study, the first whole-genome reference of D. immitis from Australia establishes a new baseline for comparative studies and will be valuable for tracking ML-resistance emergence.