First case of macrocyclic lactone-resistant Dirofilaria immitis in Europe - Cause for concern

Abstract

Heartworm disease caused by the nematode Dirofilaria immitis is one of the most important parasitoses of dogs. The treatment of the infection is long, complicated, risky and expensive. Conversely, prevention is easy, safe, and effective and it is achieved by the administration of macrocyclic lactones (MLs). In recent years, D. immitis strains resistant to MLs have been described in Southern USA, raising concerns for possible emergence, or spreading in other areas of the world. The present study describes the first case of ML-resistant D. immitis in a dog in Europe. The dog arrived in Rome, Italy, from USA in 2023. Less than 6 months after its arrival in Italy, the dog tested positive for D. immitis circulating antigen and microfilariae, despite it having received monthly the ML milbemycin oxime (plus an isoxazoline) after arrival. The microfilariae suppression test suggested a resistant strain. Microfilariae DNA was examined by droplet digital PCR-based duplex assays targeting four marker positions at single nucleotide polymorphisms (SNP1, SNP2, SNP3, SNP7) which differentiate resistant from susceptible isolates. The genetic analysis showed that microfilariae had a ML-resistant genotype at SNP1 and SNP7 positions, compatible with a resistant strain. It is unlikely that the dog acquired the infection after its arrival in Europe, while it is biologically and epidemiologically plausible that the dog was already infected when imported from USA to Europe. The present report highlights the realistic risk of ML-resistant D. immitis strains being imported and possibly transmitted in Europe and other areas of the world. Monitoring dogs travelling from one area to another, especially if they originate from regions where ML-resistance is well-documented, is imperative. Scientists, practitioners, and pet owners should be aware of the risk and remain vigilant against ML-resistance, in order to monitor and reduce the spreading of resistant D. immitis.

Keywords: Dirofilaria immitis; Drug resistance; Europe; Heartworm; Macrocyclic lactones; SNP genotyping; ddPCR.

Graphical abstract

Fig. 1

Microfilaria of Dirofilaria immitis, displaying characteristic features of the species (pointed cephalic end, straight tail), found at the Knott's test of the dog imported from Louisiana, USA to Italy.

Fig. 2

Two-dimensional (2D) droplet-digital polymerase chain reaction (PCR) plots illustrate the amplification of wildtype and alternate nucleotide (mutant) targets at respective single nucleotide polymorphism (SNP) positions in D. immitis. The absolute quantification of wildtype and mutant targets across SNP1, SNP2, SNP3, and SNP7 positions is depicted in a well characterized ML resistant isolate of D. immitis, JYD-34 (A), and Italian canine sample (B). For each plot, the amplitude in Channel 1 (y-axis) and Channel 2 (x-axis) represents the relative fluorescence of each droplet detected at the FAM and HEX channel, respectively. Green and blue droplets indicate positive droplets for wildtype and alternate allele targets, respectively, orange droplets indicate droplets having both wildtype and mutant targets and black droplets indicate negative droplets with no target amplification. Targets detected are indicated in number of copies per μl of the PCR reaction volume. This was used to calculate the percentage of alternate allele frequency (AAF). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)