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Background: Dirofilaria immitis, also known as heartworm, is one of the most important parasitic nematodes of domestic dogs, causing a potentially serious disease, cardiopulmonary dirofilariosis, which can be lethal. This species seems to be less 'expansive' than its sister species Dirofilaria repens, and it is believed that climate change facilitates the spread of this parasite to new non-endemic regions. Methods: In total, 122 heartworm isolates were analysed from nine endemic countries in Europe (Portugal, Spain, Italy, Greece, Hungary, Romania, Slovakia, and Ukraine) and a single isolate from Bangladesh by amplification and sequencing of two mitochondrial (mt) DNA markers: cytochrome c oxidase subunit 1 (COI) and dehydrogenase subunit 1 (NADH). The main aim of the current study was to determine the genetic diversity of D. immitis and compare it with D. repens haplotype diversity and distribution. DNA was extracted from adult heartworms or microfilariae in blood. Most isolates originated from dogs (Canis lupus familiaris) while 10 isolates originated from wildlife species from Romania, including eight isolates from golden jackals (Canis aureus), one isolate from a Eurasian otter (Lutra lutra) and one isolate from a red fox (Vulpes vulpes). Results: Median spanning network analysis was based on the combined sequence (1721 bp) obtained from two mt markers and successfully delineated nine haplotypes (Di1-Di9). Haplotype Di1 was the dominant haplotype encompassing 91 out of the 122 sequences (75%) from all nine countries and four host species. Haplotype Di2 was the second most common haplotype, formed solely by 13 isolates from Italy. The remaining sequences were assigned to Di3-Di9 haplotypes, differing by 1–4 SNPs from the dominant Di1 haplotype. There was evidence for geographical segregation of haplotypes, with three unique haplotypes associated with Italy and four others associated with certain countries (Di4 and Di7 with Slovakia; Di8 with Greece; Di6 with Hungary). Conclusion: Diversity in D. immitis mt haplotypes was lower by half than in D. repens (9 vs. 18 haplotypes in D. immitis and D. repens, respectively), which may be associated with the slower expansion of heartworm in Central and NE Europe. NADH gene appears to be conserved in Dirofilaria sp. by showing lower genetic diversity than the analysed COI gene. [ABSTRACT FROM AUTHOR]

Dirofilariasis is a fast-spreading disease of dogs and humans in Europe. We investigated whether Dirofilaria spp. have spread northwards in Europe, invading the Baltic countries. Altogether, 424 blood samples were collected from eight countries in the period 2017–2019, including 227 samples from sled dogs and 197 samples from other dogs. PCR amplification and sequencing were conducted employing three genetic markers (mitochondrial [mt] 12S rDNA, mt cytochrome c oxidase subunit I [COI] gene and mt dehydrogenase subunit I [NAD1] gene). The SNAP test (IDEXX) for detection of D. immitis infections was also implemented. The DNA of D. repens was detected in 59 of 424 dogs (prevalence 13.9%). D. repens was found in sled dogs from Lithuania, Latvia, Poland and Belarus. Only one dog from Estonia was infected, apparently an imported case. The highest prevalence was recorded in Lithuania (38%). Among pet dogs from the Ukraine, six dogs tested positive (3.8%). Our study has revealed a high prevalence of D. repens infections in Lithuania and Latvia, but no evidence for spread of the heartworm D. immitis. We conclude that sled dog kennels constitute hot spots for D. repens transmission. [ABSTRACT FROM AUTHOR]

Subcutaneous dirofilariosis is a fast-spreading infection of dogs, and occasionally of other carnivores and humans. Several factors contribute to its spread, including climate change, which facilitates development and survival of Dirofilaria repens in the mosquito vector. Movement/relocation of infected definitive hosts (dogs) from endemic regions to non-endemic regions is another possible cause of local emergence and the presence of a wide variety of wild reservoirs of the parasite may also contribute to its spread. The main aim of this study was to evaluate the genetic diversity of D. repens from different regions of Europe and to evaluate the spread of identified haplotypes and their geographic origin. A total of 95 D. repens isolates were obtained from Central and Eastern Europe (Poland, Belarus, Ukraine, Austria, Romania), NE Europe (Lithuania, Latvia, Estonia), Italy and Israel. All but two positive samples were obtained from the blood of dogs while one positive sample was obtained from an adult worm from a human case from the Lublin area in SE Poland and one sample was obtained from Anopheles plumbeus mosquito from Austria. Genetic diversity in D. repens isolates was evaluated by PCR amplification and sequencing of three genetic markers, including two mitochondrial genes (mtDNA): the cytochrome c oxidase subunit I (COI) and dehydrogenase subunit I (NADH). Additionally, the genomic marker, internal transcribed spacer 1 (ITS-1) was amplified and sequenced. Haplotypes were differentiated based on sequence alignments by identifying Single Nucleotide Polymorphism (SNPs) using DnaSP and Mega X. PopArt was used to construct a haplotype network including all identified haplotypes. Both mtDNA sequences (COI and NADH) were combined together for phylogenetic and network analyses. Altogether 18 haplotypes (DR1-DR18) were identified in combined mtDNA markers among 95 analysed samples. Haplotype DR1 was the most common encompassing 66 isolates: 42 isolates from Poland (41 from dogs and one from a human), 13 from Lithuania, 4 from Latvia, 2 from Ukraine and 5 from Romania. All other haplotypes grouped around haplotype DR1 separated by 1–5 SNPs, forming a star-like shape. Haplotype DR2 was the second most common haplotype, formed by six isolates from Romania. Interestingly, haplotype DR3 was represented only by four isolates from Israel. The remaining 15 haplotypes were represented by 1–4 isolates of different origins. Our study showed that only minor genetic diversity was found in D. repens since all isolates appear to have clustered in or branched out from haplotype DR1 with 1–5 SNP differences. The genetic diversity appears to be governed by geographic origin since isolates from neighbouring populations (countries) appear to share unique haplotypes while other populations that are geographically distant from individual haplotypes. • 18 different haplotypes (DR1-DR18) of Dirofilaria repens were observed in 10 different countries. • The most common haplotype encompassed 66/95 (69%) of the isolates. • Genetic diversity is related to geographic origin. • Baltic and Eastern European isolates appear to show genetic similarity. [ABSTRACT FROM AUTHOR]