Paliy_02_2022.indd UDC 591.619(477.54) MONITORING OF ANIMAL DIROFILARIOSIS INCIDENCE IN KHARKIV REGION OF UKRAINE A. P. Paliy1,2*, N. V. Sumakova1, O. V. Pavlichenko2, A. P. Palii2, O. I. Reshetylo3, L. M. Kovalenko3, N. P. Grebenik3, L. V. Bula3 1National Scientifi c Center "Institute of Experimental and Clinical Veterinary Medicine", 83, Pushkinska st., Kharkiv, 61023 Ukraine 2State Biotechnological University, 44, Alchevskyh st., Kharkiv, 61002 Ukraine 3Sumy National Agrarian University, 160, Herasym Kondratiev st., Sumy, 40021 Ukraine *Corresponding author E-mail: paliy.dok@gmail.com A. P. Paliy (http://orcid.org/0000-0002-9193-3548) N. V. Sumakova (http://orcid.org/0000-0002-6092-6054) O. V. Pavlichenko (http://orcid.org/0000-0002-6577-6577) A. P. Palii (http://orcid.org/0000-0001-9525-3462) O. I. Reshetylo (https://orcid.org/0000-0002-5167-3622) L. M. Kovalenko (https://orcid.org/0000-0002-4350-2284) N. P. Grebenik (https://orcid.org/0000-0002-1254-3374) L. V. Bula (https://orcid.org/0000-0002-4698-9307) Monitoring of Animal Dirofi lariosis Incidence in Kharkiv Region of Ukraine. Paliy, A.  P., Sumakova, N. V., Pavlichenko, O. V., Palii, A. P., Reshetylo, O. I., Kovalenko, L. M., Grebenik, N. P., Bula, L. V. — A total of 38 species of mosquitoes of the Culicidae family of two subfamilies Anophelinae and Culicinae belonging to 6 genera, were identifi ed on the territory of Kharkiv Region from 2009 to 2019. Dirofi laria larvae were found in 932 insects, which was 4.46 % of the surveyed mosquitoes. Th e prevalence of the infection of female mosquitoes by dirofi laria was 4.46 ± 0.24 %, while the mean intensity of dirofi lariae was 5.4 ± 2.1. Infection of mosquitoes with dirofi lariae is currently detected in 11 districts of Kharkiv Region and the City of Kharkiv. Th e infection rate of mosquitoes of the genus Aedes was 3.83 ± 0.63 %, of the genus Culex — 0.75 ± 0.34 %, of the genus Anopheles — 0.12 ± 0.39 %, whereas females of the genera Culiseta, Coquillettidia, and Uranotaenia were uninfected. It was found that in Kharkiv Re- gion there were 2 types of canine dirofi lariae — Dirofi laria immitis (Leidy, 1856) and Dirofi laria repens (Railliet & Henry, 1911), with a predominance of Dirofi laria repens. For the period from 2009 to 2019, we examined 378 blood samples from dogs (190 males and 188 females) aged from 8 months to 13 years with suspected dirofi lariasis. According to the results of the studies, 140 samples were positive, the prevalence of infection by the larvae of Dirofi laria spp. in blood samples from dogs with suspected dirofi lariasis was 37.03 ± 0.12 %, while infection with Dirofi laria immitis was 2.86 ± 0.45 %, and Dirofi laria repens — 97.35 ± 0.24 %. Th e number of positive samples from mongrel dogs was 42 (30 %). It was found that cats are more oft en amicrofi lariaemic than dogs. K e y w o r d s: dirofi lariasis, dirofi lariae, vectors, dogs, blood, mosquitoes. Zoodiversity, 56(2):153–164, 2022 DOI 10.15407/zoo2022.02.153 154 A. P. Paliy, N. V. Sumakova, O. V. Pavlichenko, A. P. Palii, O. I. Reshetylo et al. Introduction Parasitic diseases of animals bring signifi cant economic losses, and many diseases are socially signifi cant parasitocenoses (Pisarski, 2019; Saichenko et al., 2021). Despite the progress made in studying and solving the problems of parasitic diseases, many issues of their distribution, clinic, pathogenesis, specifi c prevention and treatment remain relevant (Bogach et al., 2020; Rzayev, 2021; Maurizio et al., 2021). Dirofi lariasis, being a transmissible natural focal zooanthroponosis, is an actual problem of both veteri- nary and medical parasitology. Th is disease is common in many countries of the world. Th e largest number of human cases of dirofi lariasis was registered in the Mediterranean countries, Asia (Tarello, 2008; Anvari et al., 2019), including China (Hou et al., 2011), and in the last two decades — in some European countries (Landum et al., 2014; Genchi & Kramer, 2017), America (Dantas-Torres & Otranto, 2013), Brazil (Furtado et al., 2009), Malaysia (Shekhar et al., 1996). In four provinces of the Republic of Serbia, microfi lariae Dirofi laria repens (Leidy, 1856), D. immitis (Rail- liet & Henry 1911), and Acanthocheilonema reconditum were found in 49.2 %, 7.2 % and 2.1 % of dogs, respec- tively (Tasić et al., 2008). Th e prevalence of dirofi lariasis among Australian hunting dogs ranges from 12.5 to 21.0 % (Orr et al., 2020). Over the past 10 years, the prevalence of dirofi lariasis among dogs has increased in Taiwan (Wu & Fan, 2003). Dirofi lariae were found in 118 (20.0 %) police dogs and 10 (8.4 %) military dogs located in Slovakia (Miterpáková et al., 2010). In Poland, foci of D. repens infection in dogs were noted in 2009 with a prevalence of 37.5 % (Cielecka et al., 2012). In Austria, more than 50 cases of dirofi lariasis among dogs caused by both D. immitis and D. repens were reported (Fuehrer et al., 2016). In Ukraine, the registration of cases of dirofi lariasis has become mandatory since 1975, and this disease has was included in the national epidemiological surveillance system. Until December 31, 2012, 1533 cases were registered, of which 1465 cases were in the last 16 years. Most cases of dirofi lariasis were registered in six regions: Kyiv, Donetsk, Zaporizhzhay, Dnepropetrovsk, Kherson and Chernyhiv. In 1997–2002 the highest in- cidence rate was noted in Kherson Region in the south of the country (9.79 per 100 000 people), and the lowest in Western Ukraine (0.07–1.68 per 100 000 people) (Sałamatin et al., 2013). In Kharkiv Region of Ukraine from 2002 to 2004, 13 cases of human disease with dirofi lariasis were registered. In 2011, 10 people fell ill with dirofi - lariasis in Kharkiv Region, 7 of them in the city of Kharkiv. Th us, in the Kharkiv Region, there is a tendency to an increase in this disease cases among people (Filipcova et al., 2016). Th e causative agent of the disease — dirofi laria (literally from the Latin “evil thread”) parasitizes ca- nines, less oft en cats and other carnivores. Today, three types of dirofi laria are distinguished: Dirofi laria im- mitis, Dirofi laria repens, Acanthocheilonnema grassi (imaginal stage) (Magnis et al., 2013). Th e causative agent D. immitis causes a cardiac form of the disease, as it parasitizes in the heart cavity, D. repens parasitizes in the subcutaneous tissue, causes the cutaneous form of dirofi lariasis, which is oft en found in dogs, cats and people in Ukraine. Th e imaginal stage of A. grassi can also inhabit body cavities, muscles, and subcutaneous tissue (Ionică et al., 2014). Infection of humans and animals occurs through the bites of blood-sucking mosquitoes of the Culicidae family (genera Anopheles, Ochlerotatus, Aedes, Stegomyia, Culex, Culiseta, Coquillettidia, etc.) (Bocková et al., 2015; Trari et al., 2017). Mosquitoes are infested by domestic dogs, wild carnivores (wolves, foxes, bears, etc.), and more rarely by cats (Todorovic & McKay, 2020). In a study of insects in Slovakia, D. repens was identifi ed in Anopheles messeae and An. maculipennis mosquitoes and the Cx. pipiens complex (Miterpáková et al., 2010). According to the data of the Kharkiv Laboratory Center, the number of mosquitoes of the genus Aedes in 2013 compared to 2010 decreased by 0.6% and amounted to 20.5%, of the genus Anopheles — by 0.7% and amounted to 17.7%, of the genus Culex — by 1.5% and amounted to 18.2% (Filipcova et al., 2016). An increase in the number of stray animals, their mass migration in nature and in settlements, and the process of urbanization contribute to an increase in the transmission of dirofi lariasis from wild carnivores to domestic animals and humans (Diaz, 2015). Since dirofi lariasis is a vector-borne disease, its spread and infec- tion rates have undergone a signifi cant transformation under the infl uence of global climate change (Simón et al., 2010). Material and methods Th e seasonal dynamics of the mosquito population was monitored regularly in the city of Kharkiv and Kharkiv Region. Th e studies began in the last ten days of April — the fi rst ten days of May aft er the emergence of the fi rst generation mosquitoes, and then the studies were carried out once a decade until the end of the insect activity season. We regularly counted and caught mosquitoes during their mass fl ight (from April to September) in the basements of multi-storey buildings, in the territories of summer cottages, private households, near water bod- ies, on dogs, people in diff erent parts of the region. Since 2015, light traps have been used to catch mosquitoes. All mosquitoes sitting on the surface were counted in 3–4 places on an area of 0.25–0.5–1 m2, depending on their density. With a larger mosquito population, there was a smaller survey area. Th en the number of insects per 1 m2 was counted. Counting the number of mosquitoes in the vegetation surrounding the building was carried out by catching them with a net for 10 minutes at 2–3 points. In suburban conditions, to account for 155Monitoring of Animal Dirofi lariosis Incidence in Kharkiv Region of Ukraine the number of adult mosquitoes, regular surveys were carried out in the same backyards. Th e modern species names and the taxonomic position of mosquitoes are given according to Reinert (2009) and Wilkerson with co-authors (2015). Female mosquitoes, 20 individuals each, were placed in laboratory tubes and delivered to the labora- tory of veterinary sanitation and parasitology of the National Scientifi c Center “Institute of Experimental and Clinical Veterinary Medicine”. Aft er species identifi cation, entomological dissection of insects was carried out, the number of egg-laying was determined, and dirofi lariae were identifi ed. Th e preparation of mosquitoes for microscopic examination for microfi lariae infection was carried out according to modern methods (Manrique- Saide et al., 2010; Silaghi et al., 2017). Females were immobilized with chloroform. Before dissection, the wings and legs of the mosquito were removed and placed on a glass slide under a stereoscopic microscope. Holdin g the abdomen with a dissecting needle, the body of a mosquito was dissected with a spear-shaped needle in the middle of the thoracic region. To extract the malpighian glands and intestines, the chitinous cover of the abdo- men was torn between segments 2 and 3, counting from the posterior end. Th en one needle was stuck into the insect’s chest, the other into the end of the abdomen and moved apart in diff erent directions. Th e preparations prepared from females saturated with blood, aft er opening, were treated with several drops of a 3.0 % solution of acetic acid. Aft er drying in air, all preparations were fi xed for 10–15 minutes, stained with Romanovsky-Giemsa stain for 20–30 minutes and microscoped with a lens magnifi cation × 90–100. To dissect the thoracic region, the head of the mosquito was cut off , the needle was pressed fl at on the insect’s chest and carried forward. Th e preparation was microscoped in saline under a cover glass (×40 objective). Th e rate of feeding insects was determined by microscopic examination: all females with the presence of blood in the midgut were considered to be feeding on blood. From clinics in Kharkiv and the region received blood samples from dogs and cats for parasitological studies. A blood test for the presence of dirofi lariae was performed by direct microscopy of a drop of fresh blood under a low microscope magnifi cation (×10) — the easiest, most convenient, and fastest method for diagnosing dirofi lariasis (Chagas et al., 2020). Th e mobile larvae of the parasite are noticeable by their active movement be- tween erythrocytes. Concentration research methods (modifi ed Knott method) were also used (Weil & Ramzy, 2007; Chungpivat & Taweethavonsawat, 2008). Microscopic identifi cation of L1 dirofi laria larvae was performed in a native blood smear and in blood serum (Furtado et al., 2010; Simsek et al., 2011). Also, for the diagnosis of dirofi lariasis, immunostrips were used — immunochromatographic instrumentless test systems for express analysis of invasion. When screening for cardiopulmonary dirofi lariasis, antigen tests for adult dirofi laria were used (Trancoso et al., 2020). Results Th e studies were carried out in the period from 2009 to 2019 on the territory of Kharkiv Region of Ukraine. During the study period, the average number of mosquitoes in vegetation ranged from 0.62 to 3.96 per m2, and by autumn it increased from 0.67 to 5.53 per m2. Indoors, the number of mosquitoes ranged from 1.22 to 3.71 per m2. According to the monitoring data on the territory of Kharkiv Region, the main groups of blood-sucking mosquitoes were the genera Anopheles, Aedes, Culex, Culiseta, Coquillettidia and Uranotaenia. Th e most numerous genus Aedes in the region consisted of 23 species. In total, according to our monitoring, 38 species of mosquitoes belonging to 6 geners of two subfamilies Anophelinae and Culicinae of the Culicidae family were found in the studied region (table 1). We carried out xenomonitoring of the presence of dirophilaria larvae in mosquitoes collected in the districts of Kharkiv Region and the City of Kharkiv during their activity (from April to September). During the whole period we examined 20,876 specimens of blood-sucking mosquitoes of diff erent genera (table 2). Dirofi laria larvae were found in 932 insects, which was 4.46 % of the surveyed mos- quitoes. Th e prevalence of microfi lariae invasion of female mosquitoes was 4.46 ± 0.24 %, while the mean intensity was 5.4 ± 2.1 dirofi lariae. According to the data of previous years, females infected with dirofi laria were identifi ed in the city of Kharkiv, Balakliia, Zmiiv, Chuhuiv districts of Kharkiv Region. In 2009, infected females were detected in Izium and Kharkiv districts, in 2011 — in Krasnokutsk District, in 2012 — in Kolomak District, in 2013 — in Krasnohrad District, in 2014 — in Zolochiv District, in 2016 — in Sakhnovshchyna and Vovchans’k Districts. Infection of mosquitoes with dirofi lariae has been detected at the present in 11 districts of the Kharkiv Region and the City of Kharkiv. 156 A. P. Paliy, N. V. Sumakova, O. V. Pavlichenko, A. P. Palii, O. I. Reshetylo et al. In the salivary glands of female mosquitoes, dirofi lariae in the amount of 2 to 11 specimens were detected starting from May 24 to September 28, and in some years (2013, 2015) — until October 18 in the amount of 2 to 5 dirofi lariae. In blood clots in the intestine, the largest number of dirofi lariae (15–33) was detected from July to September. Th e morphological characteristics of dirofi lariae in a blood clot from a mosquito intestine revealed that D. repens was 300–360 μm long, 5.8–8.0 μm wide, without sheath. Th e dorsal part of the parasite was rectangular in shape, enlightened, the caudal part was wide, long, the tail end was bent in the form of an umbrella handle. In D. immitis, the body length was 262–349 μm, and the width was 4.8–7.0 μm. Th e dorsal part was dark-colored in the form of a cone, the caudal part was thin and straight. According to our data, D. immitis larvae were detected in mosquitoes for the fi rst time in 2013. It was revealed that 75 % of females produced at least 2 ovipositions, that is, they fed on blood at least once. Diff erent level of infestation of female mosquitoes with species of Dirofi laria spp. larvae was revealed (table 3). T a b l e 1. Species composition of mosquitoes of the Culicidae family on the territory of the Kharkiv Region (2009–2019) Genus Subgenus Species Anopheles Meigen, 1818 Anopheles Meigen, 1818 Anopheles (Anopheles) plumbeus Stephens, 1828 Anopheles (Anopheles) claviger Meigen, 1804 Anopheles (Anopheles) maculipennis Meigen, 1818 Anopheles (Anopheles) messeae Falleroni, 1926 Anopheles (Anopheles) atroparvus Van Th iel, 1927 Anopheles (Anopheles) hyrcanus Pallas, 1771 Aedes Meigen, 1818 Ochlerotatus Lynch Arribalzaga, 1891 Aedes (Ochlerotatus) diantaeus Howard, Dyar et Knab, 1913 Aedes (Ochlerotatus) intrudens Dyar, 1919 Aedes (Ochlerotatus) caspius Pallas, 1771 Aedes (Ochlerotatus) dorsalis Meigen, 1830 Aedes (Ochlerotatus) pulcritarsis Rondani, 1872 Aedes (Ochlerotatus) cantans Meigen, 1818 Aedes (Ochlerotatus) riparius Dyar et Knab, 1907 Aedes (Ochlerotatus) behningi Martini, 1926 Aedes (Ochlerotatus) excrucians Walker, 1856 Aedes (Ochlerotatus) annulipes Meigen, 1830 Aedes (Ochlerotatus) fl avescens Muller, 1764 Aedes (Ochlerotatus) cyprius Ludlow, 1919 Aedes (Ochlerotatus) communis De Geer, 1776 Aedes (Ochlerotatus) sticticus Meigen, 1838 Aedes (Ochlerotatus) cataphylla Dyar, 1916 Aedes (Ochlerotatus) leucomelas Meigen, 1804 Aedes (Ochlerotatus) detritus Haliday, 1833 Aedes (Ochlerotatus) punctor Kirby in Richardson, 1837 Aedes (Ochlerotatus) pullatus Coquillett, 1904 Aedimorphus Th eobald, 1903 Aedes (Aedimorphus) vexans vexans Meigen, 1830 Aedes Meigen, 1818 Aedes (Aedes) cinereus Meigen, 1818 Aedes (Aedes) rossicus Dolbeshkin, Goritzkaja et Mitrofanova, 1930 Finlaya Th eobald, 1903 Aedes (Finlaya) geniculatus Olivier, 1791 Culiseta Felt, 1904 Culiseta Felt, 1904 Culiseta (Culiseta) alaskaensis alaskaensis Ludlow, 1906 Culiseta (Culiseta) annulata Schrank, 1776 Culicella Felt, 1904 Culiseta (Culicella) morsitans Th eobald, 1901 Culex Linnaeus, 1758 Culex Linnaeus, 1758 Culex (Culex) territans Walker, 1856 Culex (Culex) pipiens Linnaeus, 1758 Culex theileri Th eobald, 1903 Barraudius Edwards, 1921 Culex (Barraudius) modestus Ficalbi, 1890 Coquillettidia Dyar, 1905 Coquillettidia Dyar, 1905 Coquillettidia (Coquillettidia) richiardii Ficalbi, 1889 Uranotaenia Lynch Arribálzaga, 1891 Pseudofi calbia Th eobald, 1912 Uranotaenia unguiculata unguiculata Edwards, 1913 157Monitoring of Animal Dirofi lariosis Incidence in Kharkiv Region of Ukraine It was found that females of the genus Aedes were the most infected. Th us, the infection of mosquitoes of the genus Aedes was 3.83 ± 0.63 %, of the genus Culex — 0.75 ± 0.34 %, of the genus Anopheles — 0.12 ± 0.39 %, while in females of the genera Culiseta and Mansonia (Uranotaenia) dirofi lariae were not found. For the period from 2009 to 2019 we examined 378 blood samples from dogs (190 males and 188 females) aged from 8 months to 13 years with suspected dirofi lariasis (table 4). It was found that in Kharkiv Region there were 2 species of canine dirofi lariae — D. immitis and D. repens, with a predominance of D. repens. According to the results of the blood sample studies, dirofi lariasis was detected in 140 (37.04 %) samples out of 378 sam- ples with a prevalence of infection 37.03 ± 0.12. Moreover, two samples were amikrofi lari- emic, but the test system detected antigen to D. immitis. In two samples, dirofi lariae were detected both by the direct blood test method and by the concentration method, and the antigen to D. immitis was detected by the test system. In 136 samples (35.99 %), dirofi lariae were detected by both methods, but antigen to D. immitis was not detected. In 238 cases (62.96 %), the diagnosis of dirofi lariasis was not confi rmed. Th e number of positive samples from mongrel dogs was 30 % (42). Th e prevalence of infection by larvae of Dirofi laria spp. in blood samples from dogs with suspected dirofi lariasis was 37.03 ± 0.12 %, while infection with D. immitis was 2.86 ± 0.45 %, and D. repens — 97.35 ± 0.24 %. T a b l e 2. Results of a study of female mosquitoes for infection with dirofi laria larvae N Year Number of investigated, ex. Number of infected microfi lariae, ex. Prevalence of the infec- tion microfi lariae, % Th e mean intensity of infec- tion microfi lariae, ex. 1 2009 1506 80 5.31 5.5 ± 2.5 2 2010 1520 91 5.98 4.0 ± 1.0 3 2011 1560 79 5.06 5.0 ± 2.0 4 2012 1585 81 5.11 5.5 ± 2.5 5 2013 1900 96 5.04 5.5 ± 2.5 6 2014 1350 87 6.44 4.0 ± 2.0 7 2015 1690 81 4.79 7.0 ± 3.0 8 2016 2195 85 3.87 4.0 ± 1.0 9 2017 2750 88 3.20 5.5 ± 2.5 10 2018 2850 93 3.26 5.5 ± 2.5 11 2019 1850 71 3.84 3.5 ± 1.5 Total 20876 932 4.46 5.4 ± 2.1 T a b l e 3. Infection of mosquitoes with dirofi laria larvae Year Number of investigated, eх. Infection by larvae of Dirofi laria spp. female mosquito Number of infected, ex. Genus Aedes, % Genus Culex, % Genus Anopheles, % 2009 1506 80 4.51 0.8 0 2010 1520 91 5.10 0.92 0 2011 1560 79 3.84 1.22 0.13 2012 1585 81 4.42 0.69 0 2013 1900 96 4.37 0.68 0 2014 1350 87 5.48 0.96 0 2015 1690 81 3.96 0.82 0 2016 2195 85 2.78 0.68 0.41 2017 2750 88 2.50 0.47 0.22 2018 2850 93 2.10 0.52 0.28 2019 1850 71 3.10 0.48 0.27 Total 20876 932 3.83 ± 0.63 0.75 ± 0.34 0.12 ± 0.39 158 A. P. Paliy, N. V. Sumakova, O. V. Pavlichenko, A. P. Palii, O. I. Reshetylo et al. We noted a diff erence in the character of movement in the erythrocytic layer of larvae of D. immitis and D. repens. Th us, D. immitis dirofi lariae are distinguished by directional wave-like movement along the body axis, while D. repens move chaotically, mainly in one place. According to the anamnesis, it was found that the most frequently positive result was found in blood samples of dogs aged 4 to 8 years. Th e maximum number of positive sam- ples was obtained in dogs aged 5 years (27), and the minimum — in dogs aged from 8 months to 2 years and from 11 to 13 years (1) (fi g. 1). A positive test was detected in a puppy of the Alabai breed at the age of 8 months, the puppy was from a local dog kennel. A positive test was found in a mongrel dog at the age of 12 years and in a Dachshund at the age of 13 years. According to the results obtained, it can be stated that the breed has no eff ect on the possibility of infection with dirophilariosis. Infection with dirofi laria larvae was practically independent of sex: the prevalence in males (71) was 50.7 %, and in females (69) — 49.3 %, while the mean intensity was 354.5 ± 183.1 (from 8 to 450) larvae in 1 ml of blood. Until 2019, we did not study blood from cats for dirofi lariasis. In July 2019, 25 blood samples of cats aged from 2 to 5 years were tested for dirofi lariosis. Animals were admit- ted to the animal shelter aft er being caught on the streets of Kharkiv with signs of skin Table 4. Th e number of blood samples tested for infection with Dirofi laria spp. larvae Year Number of of samples analyzed Number of of positive samples Prevalence of infection D. immitis D. repens positive % positive % 2009 32 7 21.8 0 0 7 100 2010 28 11 39.3 0 0 11 100 2011 43 16 37.2 0 0 16 100 2012 35 15 42.9 0 0 15 100 2013 41 14 34.1 0 0 14 100 2014 40 18 45.0 2 11.1 16 88.9 2015 39 12 30.8 1 8.3 11 91.7 2016 25 11 44.0 0 0 11 100 2017 29 12 41.4 0 0 12 100 2018 37 14 37.8 1 7.1 13 92.9 2019 29 10 34.5 0 0 10 100 Total 378 140 37.03 ± 0.12 4 2.86 ± 0.45 136 97.35 ± 0.24 N um be r o f i nf ec te d do gs Age, month 0 0,8 1 2 3 4 5 6 7 8 9 10 11 12 13 5 10 15 20 25 30 Fig. 1. Infection of dogs with dirofi lariae depending on age. 159Monitoring of Animal Dirofi lariosis Incidence in Kharkiv Region of Ukraine lesions (15) and pulmonary syndrome (10). Th e modifi ed Knott method gave a positive result in 17 animals, the antigen test was positive in 14 animals, but this included those with a negative Knott method. According to Knott method, dirofi lariae were present in the blood of 68 % of cats with suspected dirofi lariasis. Th e antibody test was positive in 20 animals. Th at is, 80 % of the examined animals were infected, and 56 % were sick, based on the antigen test. Also, during the year, a study was conducted of 8 blood samples from cats, received from clinics in the city of Kharkiv, with suspected dirofi lariasis. Th e native smear test was negative, the modifi ed Knott method was positive in 2 animals, and antigen testing was positive in one animal. Th at is, infection was registered in 25 % of the examined animals, and 12.5 % turned out to be sick based on the antigen test. Infection by dirofi laria larvae is practically independent of sex; the prevalence of in- fection in male cats (16) was 48.5 %, in female cats (17) — 51.5 %. In this case, the mean intensity averaged 44.3 ± 22.9 (from 1 to 90) larvae in 1 ml of blood. Th e minimum number of positive samples was determined in cats aged from 2 to 4 years and the maximum — at the age of 5 years (16). Among stray animals with suspected dirofi lariasis, the infection rate was 68 %, among domestic animals — 25 %. Th e results showed that cats were more oft en amycrofi lariemic than dogs. Infection with dirofi lariae in cats is less likely to cause dirofi lariasis, but there are also two forms of D. immitis and D. repens. Discussion Th e quality of the resulting livestock products, their safety, as well as the health of animals and humans directly depends on the epizootic and epidemiological situation in the region and the level of contamination of environmental objects with pathogens of certain diseases (Shkromada et al., 2019; Paliy et al., 2019). Recently, more and more attention has begun to be paid to ectoparasites and the determination of their role in the occurrence of outbreaks of infectious and invasive diseases (Paliy et al., 2018 b, 2021). Arthropod vector pathogen surveillance is now an important tool for surveillance programs across Europe (Rudolf et al., 2014). Th e fauna of blood-sucking mosquitoes in Kharkiv Region of Ukraine, according to our collection, includes 38 species of mosquitoes that belong to 6 genera. According to other studies, 30 species of blood-sucking mosquitoes of three genera were found on the territory of the studied region: Anopheles (An. maculipennis, An. messeae); Aedes (Ae. cataphylla, Ae. leucomelas, Ae. caspius dorsalis, Ae. excrucians, Ae. vexans); Culex (C. punctatus, C. obsolеtus) (Gazzavi-Rogozina, 2015). Perhaps this is due to the fact that they were collected mainly in urban areas. Th e researchers note that the species composition and number of mosquitoes of the genus Aedes are changing in Kharkiv region. In recent years, the percentage of Aedes geniculatus mosquitoes in the number of mosquitoes of this genus has been increasing. In addition, in some districts of the region (Zmiiv, Derhachi, Chuhuiv) and the city of Kharkiv, mosquitoes Mansonia richiardii are more and more oft en recorded (Bodnia & Potapova, 2016). According to the data of the Kharkiv Regional Laboratory Center, the population rates of bloodsucking mosquitoes (of the Aedes and Culex genera) of utility rooms and basements are 12.6 % against 12.5 % in 2014. According to our data, the population of basements with bloodsucking mosquitoes averaged 13.2 %, that is, slightly higher According to observations (Filipcova et al., 2016), 563 specimens were examined for the presence of dirofi laria nematodes in Kharkiv Region malaria mosquitoes, while dirofi lariae were not identifi ed. In the study of 463 specimens non-malaria mosquitoes in 3 specimens revealed dirofi laria nematodes (0.6 %). A year later, according to laboratory autopsy data, the incidence of Anopheles mosquitoes with nematode dirofi lariae was 0.8–0.9 % (Bodnia 160 A. P. Paliy, N. V. Sumakova, O. V. Pavlichenko, A. P. Palii, O. I. Reshetylo et al. et al., 2016). In Belarus, out of 467 female mosquitoes collected in the Brest and Minsk regions, Dirofi laria spp. were detected in two pools (5.56 %) (Șuleșco et al., 2016). According to the obtained results obtained, the infection of mosquitoes of the genus Aedes was 3.83 ±0.6 3 %, of the genus Culex — 0.75 ± 0.34 %, of the genus Anopheles — 0.12 ± 0.39 %, and in females of the genus Culiseta (Felt, 1904), Coquillettidia (Dyar, 1905), and Uranotaenia (Lynch Arribálzaga, 1891) dirofi lariae were not found. It was revealed that females of the genus Aedes were the most infected. Perhaps the diff erence in results is due to the fact that the material was collected in diff erent places and at diff erent terms. Today dirofi lariasis of dogs is widespread in the world. In Lithuania, in a study of 2280 blood samples, 61 (2.7 %) were positive for the presence of D. repens, while the infection rate was signifi cantly higher in dogs from shelters (19.0 %; 19/100) than in domestic dogs (1.9 %; 42/2180) (Sabūnas et al., 2019). In studies in Poland, dirofi lariae belonging to the species D. repens were found in blood samples from dogs from Warsaw and 18 districts of the Mazovian Voivodeship. Th e average prevalence of this species in the province was 25.8 % with an average intensity of 9 dirofi lariae. Th e highest prevalence, reaching 52.9 %, was found in the Radom Region, and the lowest prevalence, at 4.2 %, was found in the Grójec Region (Demiaszkiewicz et al., 2014). Our results are consistent with other researchers (Simón et al., 2012), who also indicate the distribution of two main species of dirofi laria — D. immitis and D. repens. According to existing data, in Kharkiv Region, dirofi lariae were found in 10.7 % of the surveyed dogs, while in some settlements the percentage of aff ected dogs reached 19 % and more (Bodnia et al., 2016). According to our data, the prevalence of infection by the larvae of Dirofi laria spp. in dogs with suspected dirofi lariasis was 37.03 ± 0.12 %. Infection with D. immitis was 2.86 ± 0.45 %, and D. repens — 97.35 ± 0.24 %. A large number of dogs infected with D. repens, in our opinion, is a constant source of infection for dipterans, and as a result, people. In cats with suspected dirofi lariasis, infection with dirofi lariae was 66.7 %. Th e infection with D. immitis was 54.6 ± 0.45 %, and D. repens — 46.4 ± 0.24 %. Diro- fi lariae in the blood of dogs and cats were found year-round from January to December, but in June and July there were a bit more such cases. A study of the seasonal dynamics of D. im- mitis infection in stray and indoor dogs showed that the proportion of infected dogs in spring and summer was higher than in colder seasons (autumn and winter) (Khedri et al., 2014). It was found that dirofi lariasis aff ects dogs of all age groups, in the age range from 8 months and up to 13 years. Th ere was no statistically signifi cant relationship between the prevalence of D. immitis with age, sex, breed, and use of antiparasitic drugs (Anvari et al., 2019). Th e prevalence of D. immitis infection in stray dogs over 5 years old (53.8 %) was higher than in other age groups, while in domestic dogs the infection rate was higher at the age of 3-5 years (27.3 %) (Khedri et al., 2014). Th ere is evidence that the likelihood of infection was signifi cantly higher in older dogs and dogs in an outdoor shelter compared to younger dogs and those kept in an indoor shelter. Th ere were no signifi cant diff erences in infection between sexes and between purebred and crossbred dogs under the same rearing conditions (Hou et al., 2011). Th ese results also exist in humane medicine, where there was no diff erence in positivity for infection with dirofi lariasis between men and women (Furtado et al., 2009). Predictive models have recently forecast that current summer temperatures are ac- celerating incubation of dirofi lariae. Global warming, predicted by the Intergovernmental Panel on Climate Change, suggests that warm summers suitable for transmission of diro- fi lariae in Europe will become common in the coming decades, and if the actual trend of increasing temperatures continues, dirofi lariasis invasion should spread to previously free areas (Genchi et al., 2011) in a northern direction (Jokelainen et al., 2016; Pietikäinen et al., 2017). Th e constant increase in the incidence of D. repens human dirofi lariasis in Ukraine is indicated. Despite eff orts in the fi eld of medicine, infections have become more frequent, and the territory of the spread of the disease has expanded to cover the whole of Ukraine (Sałamatin et al., 2013). 161Monitoring of Animal Dirofi lariosis Incidence in Kharkiv Region of Ukraine An analysis of the seasonality of invasion in Kharkiv Region showed that dirofi lariae are detected year-round, and the peak is in June and July, the month is also marked by an increased dirofi lariaemia in the summer months, its decrease in the fall and an insignifi cant number of larvae in winter. In our opinion, the variability of indicators largely depends on the climatic and geographic characteristics of the region under study, the size of the stud- ied sample of animals, and various diagnostic techniques that were used by the authors to make a diagnosis. Th e fi ght against parasitic diseases of animals is a priority task for specialists in veteri- nary medicine, and its success directly depends on a comprehensive solution to this prob- lem (Paliy et al., 2018 a; Boyko et al., 2020; Bogach et al., 2020). Conclusions Th e average number of mosquitoes (2009–2019) in vegetation ranges from 0.62 to 3.96 per m2, and by autumn it increases from 0.67 to 5.53 per m2. Indoors, the number of mosquitoes ranged from 1.22 to 3.71 per m2. On the territory of Kharkiv Region of Ukraine, 38 species of blood-sucking mosquitoes were found, which belong to 6 genera and 38 species. Th e prevalence of infection by diro- fi lariae of female mosquitoes is 4.46 ± 0.24 % at a mean intensity is 5.4 ± 2.1 microfi lariae. It has been established that the most infected are female mosquitoes of the genus Aedes. Infection of mosquitoes of the genus Aedes is 3.83 ± 0.63 %, of the genus Culex — 0.75 ± 0.34 %, of the genus Anopheles — 0.12 ± 0.39 %, in females of the genus Culiseta, Coquillet- tidia and Uranotaenia microfi lariae were not found. It was found that in Kharkiv Region there were 2 species of canine dirofi lariae D. im- mitis and D. repens, with a predominance of the parasite D. repens. Th e species D. immitis on the territory of Kharkiv Region was identifi ed by us for the fi rst time in 2014. Dirofi lariae in the blood of dogs and cats were found year-round from January to De- cember, but in June and July a little more oft en. In the blood of dogs, dirofi lariae were detected in 140 out of 378 samples with a prevalence of infection 37.03 ± 0.12 with a mean intensity of 354.5 ± 183.1 (from 8 to 450) larvae in 1 ml of blood. Cats are more likely to be amycrofi lariemic than dogs. 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