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SummaryDirofilaria immitis, the agent of canine dirofilariosis, is a common parasite of domestic and wild carnivores with zoonotic potential and worldwide distribution, being endemic in many countries. Bulgaria is among European countries recognized as endemic for this heartworm parasite. In the present study, D. immitis adults recovered from pulmonary arteries of domestic dog and golden jackal originating from the Pazardzhik region in southern Bulgaria, and from red fox originating from the Plovdiv region in central-southern Bulgaria, were genetically analyzed in nuclear targets. The first PCR amplification of the internal transcribed region 2 (ITS2) of the ribosomal DNA with previously published D. immitis-specific primers yielded single fragments in size of 302 bp that is characteristic for these heartworms. PCR products of three isolates, resulted from the second amplification of the 5.8S-ITS2 region (235 bp) with pan-filarioid primers, were subjected to direct DNA sequencing. Identical nucleotide composition was detected across the screened target region for these Bulgarian isolates. When the 5.8S-ITS2 sequences were phylogenetically compared to the GenBank-retrieved D. immitis sequences in a worldwide context, the neighbor-joining analysis has shown three discrete clades. The first clade was composed of D. immitis isolates from Europe (including the studied Bulgarian samples), Asia and South America, in the second clade samples from Asia and South America were placed, whereas the third clade was formed by two Brazilian dog isolates originated from the north and southeast part of the country. The purpose of the present study was to verify the taxonomic characterization of D. immitis nematodes from Bulgaria based on morphology and compare their genetic structure with filariae obtained from the different world regions using molecular assays. It also summarizes previous epidemiological and ecological studies on the parasite distribution and prevalences in different hosts and regions undertaken so far in Bulgaria.

A nematode species from the Protostrongylidae family, unknown in the parasite fauna of Bulgaria until now, was found in the bronchi of a European brown hare (Lepus europaeus Pallas, 1778 L.) from a southwestern region of the country. At first the systematic identification of the found specimens was attributed to two possibilities-Protostrongylus terminalis Passerini (1884) Schulz, Orlow & Kutass, 1933 and Protostrongylus cuniculorum (Joyeux & Gaud, 1946) Schulz & Kadenazii, 1949. The autonomy of these two species was discussed based on data available in the literature. Morphological and morphometric data about the male and female specimens are provided in the present materials. After comparing these data with the ones available in the literature, the helminths were related to the species P. cuniculorum. P. cuniculorum is reported for the first time as part of the helminth fauna of the European brown hare from Southeastern Europe and Bulgaria in particular.

During the period 2009–2016, seven hundred and ninety lungs from brown hares (Lepus europeus L.) from different regions of Bulgaria were investigated for presence of parasites. The results showed that 11.14% of the lungs were parasitised. Four parasite species were established: two nematodes – Protostrongylus tauricus and P. cuniculorum (recorded for the first time as a part of the helminth fauna of the country), one cestod – larval form of Taenia pisiformis – Cysticercus pisiformis and one arthropod – larval form of Linguatula serrata – Pentastomum denticulatum. The infected hares came mainly of the south parts of the country. The highest prevalence of infection was observed in the South West region, followed by the South Central and South East regions. From the northern parts of the country parasites were established only in the northeastern districts near to Black Sea and Danube River. The ratio of prevalence of infection in female vs male animals was 1.4:1. Infections with protostrongylids and cysticerci were established at the earliest in hares at the age of 4–6 months and with P. denticulatum: in hares between 7 and 9 months. The prevalence of infection varied in animals from the different age groups. The highest prevalence of infection was observed in the young (4–6 months) and adult hares (over 3 years of age).

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During the period 2009–2016, brown hares (Lepus europeus L.) from different regions of Bulgaria were investigated for infections with liver parasites as well as for seroprevalence of protozoan Toxoplasma gondii. Three helminth species were found in the liver: one trematode – Dicrocoelium dendriticum, one cestode – larval form of Taenia pisiformis – Cysticercus pisiformis and one nema­tode – Micipsella numidica. Dicrocoeliasis was found in all parts of the country with infected hares originating mainly from the western regions. The total prevalence of infection with D. dendriticum was 10.59%. The intensity of infection ranged from 1 to 184, 26.67 on the average. The total prevalence of infection with C. pisiformis was 16.8%. The prevalence of infection was highest among hares from the southern parts of the country, and no cysticercosis was registered in the western parts. The extent of infestation with cysticerci was different – from single cysticerci on the omentum or serosa of some organs in the abdominal cavity to innumerable ones that affected also the organs in the thoracic cavity. However, cases in which the animals were slightly affected, predominated. The total prevalence of infection with M. numidica was 5.31%, and the intensity of infection varied between 1 and 6 parasites in one animal. The nematodiasis was established mainly in hares from southern Bulgaria. The total seroprevalence of T. gondii was 6.15% and the protozoan-positive animals originated from three districts: Stara Zagora, Plovdiv, and Burgas. The current studies also showed that the prevalence of D. dendriticum infection among animals increased with age. No correlation was observed between prevalence of infection with C. pisiformis and M. numidica and host age. No correlation was found between the prevalence of the helminth infections and sex of the animals.