Dirofilaria immitis is a zoonotic parasitic nematode that infects domestic and wild canids, among its vertebrate hosts. The genetic analysis of D. immitis nowadays transcends the need for genetic taxonomy of nematodes, such as the study of resistance to macrocyclic lactone. We expanded the use of long-read nanopore-based sequencing technology on nematodes by performing genomic de novo assembly of a D. immitis specimen retrieved from a canine cardiopulmonary dirofilariasis case using the ONT MinION platform, followed by the study of macrocyclic lactone resistance. The assembled genome of D. immitis consists of 110 contigs with an N50 of 3687191. The genome size is 87899012 and contains a total of 9741 proteins; 6 ribosomal RNAs, with three belonging to the small subunit (18S) and three to the large subunit (28S); and 73 tRNAs. Subsequent analysis of six loci previously characterized as being associated to macrocyclic lactone resistance selection pressure showed that four have a genotype associated with either some loss of efficacy or the resistance phenotype. Considering the zoonotic potential of D. immitis, the identification of a resistant parasite alerts for the overuse of macrocyclic lactone in the region, which poses a potential risk to both veterinary and human public health.

Background Dirofilaria immitis is a parasitic nematode endemic in the Mediterranean countries, which causes cardiopulmonary dirofilariosis in wild and domestic animals. Despite being recognized hosts of D. immitis, wild carnivores such as wolves and foxes are frequently disregarded when considering a potential role in the transmission of these zoonotic nematodes. In Portugal, studies available regarding D. immitis circulation are scarce, likely underestimating its relevance. To add knowledge on this, we sought to assess Iberian wolves (Canis lupus signatus) and red foxes (Vulpes vulpes) from northern Portugal for D. immitis antigenemia and microfilaremia. Methods Blood samples from 42 Iberian wolves and 19 red foxes were collected, during 2010–2012, in Peneda-Gerês National Park. Antigenemia was searched for by rapid antigen detection test kits (Uranotest Dirofilaria ®). Microfilaremia was assessed by polymerase chain reaction (PCR). Nucleic acids were extracted from blood using QIAamp® DNA Mini Kit (Qiagen), and DNA was screened for the presence of microfilaria using a conventional PCR targeting the 5.8S-internal transcribed spacer 2–28S regions, followed by bidirectional sequencing, Basic Local Alignment Search Tool analysis and phylogenetic analysis. Results Three red foxes had antigenemia, with an occurrence of 15.8% (95% confidence interval [CI] 3.4–39.6), while showing no evidence for the presence of microfilaremia. No wolf samples presented evidence for D. immitis antigenemia. Nevertheless, two wolves were positive for D. immitis microfilaremia (4.8%; 95% CI 0.6–16.2%) as revealed by PCR and confirmed by bidirectional sequencing. Conclusions Although Dirofilaria microfilaremia in wolves does not necessarily correlate to an endangerment of the infected animal's health, positive individuals can act as a reservoir for further infection if the intermediate mosquito hosts are present. To the best of our knowledge, one single study had reported that wolves were suitable Dirofilaria hosts, but microfilaremia have never been reported. Graphical Abstract

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