Surveillance of single nucleotide polymorphisms correlated to macrocyclic lactone resistance in Dirofilaria immitis from client-owned dogs across the United States

Abstract

Dirofilaria immitis is a parasitic filarial nematode and the causative agent of heartworm disease in canids and other species. Heartworm disease is predominantly managed via macrocyclic lactone (ML) - based chemoprophylactics. Through opportunistic sampling, genotypically and phenotypically confirmed ML-resistant D. immitis isolates have been isolated in the Lower Mississippi River Valley region (LMRV); however, the pervasiveness of resistant isolates in the USA has not been evaluated. This study aimed to evaluate the geographic distribution and prevalence of genotypically ML-resistant heartworms in client-owned dogs across the USA over a 3-year period. Owner consent was obtained to collect microfilaremic blood samples from heartworm-positive dogs from participating clinics. Veterinarians completed a questionnaire on the known history of each dog, including treatment and travel history. A total of 310 microfilaremic blood samples were collected from 45 geographically diverse veterinary clinics located in 22 states. Microfilariae were filtered from blood, DNA extracted utilizing the QIAGEN QIAamp DNA Micro Kit and samples sequenced by the Génome Québec Innovation Centre to determine allele frequencies at nine SNP sites previously correlated with ML resistance. The highly predictive 2-SNP model was used to identify genotypically susceptible, mixed, and resistant populations. Computational analysis indicated 111 (35.8 %) were genotypically susceptible, 96 (31.0 %) were genotypically resistant, and 103 (33.2 %) were genotypically mixed. The genotypically mixed and ML-resistant infections were located within and outside of the endemic LMRV, as far north as Michigan, which indicates canine populations outside of the LMRV are at increased risk for transmission of potentially ML-resistant heartworm infections than previously hypothesized. Veterinary practitioners across the USA need to be aware of the potential risks of ML resistance heartworm infections and ensure patient compliance with recommended prevention protocols.

Keywords: Drug resistance; Genotype; Heartworms; Macrocyclic lactones; Molecular markers; Surveillance.

Graphical abstract

Fig. 1

Alternate allele frequency of the 310 USA clinical samples at the nine SNP molecular markers of interest. The red circles represent the reference base for each SNP molecular marker, and the blue circles represent the alternate base for each SNP molecular marker. The alternative allele frequencies were prepared in comparison to the D. immitis reference genome nDi.2.2.

Fig. 2

Predictive correlation of the top 2 SNP markers (SNP1: Scaffold nDi.2.2scaf00046 Positions 76278, and SNP2: Scaffold nDi.2.2scaf00046; 22857) of the 310 USA clinical samples. The green squares represent genotypically ML-susceptible samples (SUS), the red triangles represent genotypically ML-resistant samples (RES), and the blue circles represent genotypically mixed samples (MIX).

Fig. 3

Distribution of the 310 USA clinical samples collected between 2021 and 2023, with pie sizes proportional to the number of observations per clinic. Clinics with less than three observations did not meet the inclusion cut-off. Clinics with greater than 20 observations are further identified by town name. The predicted genotypic distribution presented is spatially contiguous and encompasses ≥99 % of the data, with clinics from California contributing ≤1 %. The green represents genotypically ML-susceptible samples (SUS), the red represents genotypically ML-resistant samples (RES), and the blue represents genotypically mixed samples (MIX).