An unexpected case of a dog from Poland co-infected with Dirofilaria repens and Dirofilaria Immitis.

BACKGROUND: Dirofilariasis is a vector-borne disease caused by parasitic nematodes of the genus Dirofilaria spp., considered an emerging concern in both veterinary and human medicine. Climate changes and human activities, such as pet travel, contribute to the spread of diseases to new non-endemic regions. Poland is dominated by subcutaneous dirofilariasis caused by D. repens infections. Cardiopulmonary dirofilariasis, also known as a heartworm disease is much more rare with only single autochthonous cases reported so far. Also, imported infections are observed sporadically in dogs traveling to endemic countries. In this study, we report the first case of a dog in Poland, never having traveled abroad, co-infected with Dirofilaria repens and Dirofilaria immitis. CASE PRESENTATION: A 14-year-old mixed breed, an intact male dog with fever, lightly pale mucosal membranes, moderate abdominal pain, and a mild cough was presented in a veterinary clinic in Warsaw, Poland. The examination of the blood sample collected for complete morphology and biochemistry revealed the presence of live microfilariae. Presence of the DNA of both microfilariae species was detected using Real-Time PCR with species-specific primers. CONCLUSIONS: Since the remaining diagnostic methods like Knott's test, antigen test or echocardiography did not reveal the presence of D. immitis, we discussed the impact of microfilariae periodicity and low worm burden infections on the limited efficiency of these techniques. We strongly recommend using a mixed diagnostic approach for the most sensitive and specific diagnosis since the ideal diagnostic method does not exist, and several factors may contribute to misdiagnosis. Furthermore, we considered factors that contribute to the uncontrolled spread of dirofilariasis such as climate changes, introduction of new species of mosquitoes competent for the transmission of the disease, and wildlife animals as an important reservoir of this parasitosis. Given that Poland shares borders with countries classified as endemic and pre-endemic for D. immitis, such as Slovakia and Ukraine, it is reasonable to anticipate a rise in autochthonous heartworm infections and shifts in the epidemiological pattern of dirofilariasis in the coming years.

Dirofilaria immitis conquering the regions in Slovakia previously endemic for D. repens.

The present study was focused on the current state of Dirofilaria species distribution in the territory of the Slovak Danubian Lowland, a region previously identified endemic for Dirofilaria repens. For the research, blood samples of 330 dogs tested positive for dirofilariosis using concentration tests or "rapid heartworm tests" were sent by private veterinary practitioners for further DNA analyses and species determination. The results revealed an unquestionable change in the pattern of Dirofilaria species distribution with Dirofilaria immitis, diagnosed as the agent of mono- and co-infections with D. repens, responsible for 60.00% of all cases. The results showed that in the course of the last 5 years, D. immitis has spread significantly in Slovakia and has become the dominant causal agent of dirofilariosis in the former endemic areas of D. repens distribution, which increases infection risk for both dogs and humans.

Dirofilaria immitis Pulmonary Dirofilariasis, Slovakia.

Dirofilaria immitis is a parasite related to pulmonary dirofilariasis in humans, its accidental hosts. We detected an autochthonous case of D. immitis infection in a woman from Slovakia. The emergence and spread of this parasite in Europe indicates a critical need for proper diagnosis of infection.

Mosquito surveillance of West Nile and Usutu viruses in four territorial units of Slovakia and description of a confirmed autochthonous human case of West Nile fever, 2018 to 2019.

BackgroundDespite the known circulation of West Nile virus (WNV) and Usutu virus (USUV) in Slovakia, no formal entomological surveillance programme has been established there thus far.AimTo conduct contemporaneous surveillance of WNV and USUV in different areas of Slovakia and to assess the geographical spread of these viruses through mosquito vectors. The first autochthonous human WNV infection in the country is also described.MethodsMosquitoes were trapped in four Slovak territorial units in 2018 and 2019. Species were characterised morphologically and mosquito pools screened for WNV and USUV by real-time reverse-transcription PCRs. In pools with any of the two viruses detected, presence of pipiens complex group mosquitoes was verified using molecular approaches.ResultsAltogether, 421 pools containing in total 4,508 mosquitoes were screened. Three pools tested positive for WNV and 16 for USUV. USUV was more prevalent than WNV, with a broader spectrum of vectors and was detected over a longer period (June-October vs August for WNV). The main vectors of both viruses were Culex pipiens sensu lato. Importantly, WNV and USUV were identified in a highly urbanised area of Bratislava city, Slovakias' capital city. Moreover, in early September 2019, a patient, who had been bitten by mosquitoes in south-western Slovakia and who had not travelled abroad, was laboratory-confirmed with WNV infection.ConclusionThe entomological survey results and case report increase current understanding of the WNV and USUV situation in Slovakia. They underline the importance of vector surveillance to assess public health risks posed by these viruses.

Bartonella species in medically important mosquitoes, Central Europe.

Here, we provide the first mass molecular screening of medically important mosquitoes for Bartonella species using multiple genetic markers. We examined a total of 72,115 mosquito specimens, morphologically attributed to Aedes vexans (61,050 individuals), Culex pipiens (10,484 individuals) and species of the Anopheles maculipennis complex (581 individuals) for Bartonella spp. The initial screening yielded 63 Bartonella-positive A. vexans mosquitoes (mean prevalence 0.1%), 34 Bartonella-positive C. pipiens mosquitoes (mean prevalence 0.3%) and 158 Bartonella-positive A. maculipennis group mosquitoes (mean prevalence 27.2%). Several different Bartonella ITS sequences were recovered. This study highlights the need for molecular screening of mosquitoes, the most important vectors of arthropod-borne pathogens, for potential bacterial agents.

Heartworm on the rise-new insights into Dirofilaria immitis epidemiology.

Until recently Dirofilaria immitis, the causative agent of serious canine heartworm disease, has been detected relatively infrequently in Central Europe in comparison with the predominant D. repens species. In the present study, the elevated number of heartworm cases among dogs from a breeding establishment in south-western Slovakia is described. Out of 25 dogs examined, dirofilariasis was detected by single or several diagnostic approaches in 18 animals, which represents a mean prevalence of 72.0%. D. immitis was confirmed in 16 (64.0%) of the infected dogs and D. repens in 8 dogs (32.0%). All cases of D. immitis infection were detected in areas regarded as D. repens-endemic to date. Following the presented results and discussed circumstances, the question of whether the real prevalence of canine heartworm disease in Slovakia, or even in Central Europe as a whole, has been underestimated, or if D. immitis is currently becoming endemic in this area.

GIS-based environmental analysis of fox and canine lungworm distribution: an epidemiological study of Angiostrongylus vasorum and Crenosoma vulpis in red foxes from Slovakia.

Over a period of intervening years, the distribution of two canine cardiopulmonary metastrongylid nematodes, Angiostrongylus vasorum and Crenosoma vulpis, has been recognised in Central Europe. Here, we report the first epidemiological research conducted in red foxes from Slovakia and the potential influence of selected environmental variables on the parasites' occurrence, quantified by logistic regression. The environmental models revealed that distribution of C. vulpis is not significantly influenced by any environmental variables, and the parasite is present in the whole area under study. Models for A. vasorum revealed some weak influence of environmental variables, as it tends to occur in drier areas with lower proportion of forest. Moreover, A. vasorum shows a typical spatial clustering and occurs in endemic foci identified mainly in the eastern part of Slovakia. A cluster of A. vasorum infection foci was also found in the north-eastern region, where the average winter air temperature regularly falls below - 10 °C.

Cryptic species Anopheles daciae (Diptera: Culicidae) found in the Czech Republic and Slovakia.

We report the distribution of mosquitoes of the maculipennis complex in two distinct areas of the Czech Republic (Bohemia and South Moravia) and in one locality of neighbouring Slovakia with emphasis on the detection of the newly described cryptic species Anopheles daciae (Linton, Nicolescu & Harbach, 2004). A total of 691 mosquitoes were analysed using a species-specific multiplex PCR assay to differentiate between the members of the maculipennis complex. In the Czech Republic, we found Anopheles maculipennis (with a prevalence rate of 1.4%), Anopheles messeae (49.0%) and Anopheles daciae (49.6%). In Slovakia, only An. messeae (52.1%) and An. daciae (47.9%) were detected. In this study, An. daciae was documented for the first time in the two countries where it represented a markedly higher proportion of maculipennis complex species (with an overall prevalence almost reaching 50%) in comparison to previous reports from Germany, Romania and Poland. The determination of the differential distribution of maculipennis complex species will contribute to assessing risks of mosquito-borne diseases such as malaria or dirofilariasis.

Canine dirofilariosis endemic in Central Europe-10 years of epidemiological study in Slovakia.

The study presents the comprehensive results of a detailed epidemiological study on canine dirofilariosis in Slovakia, Central Europe. More than 4000 dogs were investigated, and several epidemiological factors were considered. The mean prevalence in individual regions ranged from 2.0 % in northern Slovakia to more than 25.0 % in the south-western part of the country, with the nematode Dirofilaria repens confirmed as the dominant causative agent. Canine dirofilariosis occurred more often in animals more than 3 years old and in dogs of large and giant breed sizes. Short-haired animals were infected more often than dogs with a long coat. Also, the infection was significantly more prevalent in animals kept in rural areas in comparison with urban environments.Counts of microfilariae (mf) in peripheral blood reached their highest levels in May and August and corresponded to activity peaks and population maximums of potential vectors, the mosquito species Aedes vexans and Culex pipiens, the two most prevalent species in Slovakia. Moreover, two dogs naturally infested with D. repens were included in the experiment in order to monitor daily microfilarial periodicity. This fluctuation showed the same tendency in both animals, with a peak of circulating mf recorded at 4 a.m. and minimal mf counts at 4 p.m.

Case of human Dirofilaria repens infection manifested by cutaneous larva migrans syndrome.

Dirofilaria repens, parasite of subcutaneous tissues of dogs and other carnivores, represents high infection risk for animals and humans in Europe. In men, infection usually presents as nodule in subcutaneous tissues or, less often, the lesions are localised around the eyes. The work presents first confirmed clinical case of human D. repens infection connected with cutaneous larva migrans syndrome. In patient, the migration of the worm caused true signs of creeping eruption, elevated sinuous track under the skin. It was connected with severe pain, burning and erythema of adjacent skin. Symptoms appeared at least three times, approximately once a month, always in the evening or night and lasted from several minutes to several days. In December 2014, during the scratching of residual pruritic lesion, patient removed 6-cm long, whitish worm from the wound. Morphological features (longitudinal ridges) and PCR amplification of cytochrome oxidase subunit 1 (CO1) confirmed Dirofilaria repens as etiological agent of infection. Herein, presented case confirmed that D. repens infection in humans can be associated with real creeping eruption, clinical sign of cutaneous larva migrans and should be included in its differential diagnosis.

Contribution to host diversity, genetic diversion, and epidemiology of Trichinella pseudospiralis in Slovakia, central Europe.

Trichinella pseudospiralis belongs to the non-encapsulated clade of the genus and its epidemiology is influenced by various biotic and abiotic factors. The role of different animal species in the spread and epidemiology of the parasite is still not well understood and further research is needed in the areas where its occurrence has been recorded. In Slovakia, T. pseudospiralis was first documented in 2004 on a poorly maintained industrial pig farm where pigs, rats, and a domestic cat were found to be infected. In the following years, its occurrence was confirmed in foxes, wild boars, and three species of birds of prey. The objective of the present study was to investigate different micromammal and carnivore host species inhabiting the Tatra National Park in the north of Slovakia for the presence of Trichinella parasites. From 2018-2023, 715 small mammals belonging to 19 species and 92 muscle samples from 12 carnivorous species were individually examined for the presence of Trichinella muscle larvae using the artificial digestion method. The research brought new host records and completed the list of sylvatic hosts for T. pseudospiralis in Slovakia - the yellow-necked mouse (Apodemus flavicollis) and raccoon dog (Nyctereutes procyonoides) together with results of the genetic structure study conducted on a fragment of the 28S gene in T. pseudospiralis.

Micromammals as a reservoir for the zoonotic nematode Calodium hepaticum (syn. Capillaria hepatica) in recreational areas of Slovakia.

The hepatic nematode Calodium hepaticum is a zoonotic parasite primarily parasitising small mammals, but it can infect a wide range of mammal species, including humans. Due to its specific life cycle and transmission pattern, it is one of the least studied helminths in the world. The only documented findings of C. hepaticum from Slovakia (former Czechoslovakia) come from the 60s and 70s of the 20th Century, including nine human cases of the infection reported post-mortem. The present study was conducted in the area of these original records in the Tatra National Park (TANAP) and the Kosice Zoo. In TANAP, 484 small mammals of six shrew species (Insectivora: Soricidae) and eight rodent species (Rodentia: Muroidea) were collected. In the Kosice Zoo, 163 rodents from 10 species were sampled. All specimens were examined for the presence of C. hepaticum eggs using the artificial digestion method. The parasite was recorded in two shrew species (Sorex araneus and Neomys fodiens) and five rodent species (Arvicola amphibius, Microtus agrestis, Clethrionomys glareolus, Apodemus flavicollis, and Rattus norvegicus) from TANAP, while in the Kosice Zoo only a single individual of R. norvegicus was found to be infected.

Human alveolar echinococcosis in Slovakia: Epidemiology and genetic diversity of Echinococcus multilocularis, 2000-2023.

Human alveolar echinococcosis (AE) is a serious parasitic disease caused by larval stages of Echinococcus multilocularis. Between January 2000 and October 2023, 137 AE cases were confirmed in Slovakia. The average annual incidence increased from 0.031 per 100,000 inhabitants between 2000 and 2011, to an average of 0.187 since 2012, i.e. about six times. Among patients, 45.3% were men and 54.7% were women; the mean age at the time of diagnosis was 52.8 years. Most cases were diagnosed in the age groups 51-60 years and 61-70 years (33 cases each), and eight patients fell into the age category ≤ 20 years. To better recognize the gene diversity in clinical samples, metacestodes from 21 patients collected between 2013 and 2021 were subjected to DNA sequencing of four mitochondrial genes. Using concatenated sequences of cob (603 bp), nad2 (882 bp) and cox1 (789 bp) gene fragments, 14 isolates (66.7%) were assigned to the European E5 profile of E. multilocularis, two isolates (9.5%) to the E5a subtype, four isolates (19%) to the E4 profile, and one isolate (4.8%) to haplogroup E1/E2. The E5-type profiles and E4 profiles were distributed throughout the country, whereas the E1/E2 profile was found in the patient from western Slovakia. According to the data obtained and GenBank sequences, the E5-type dispersal is so far limited to central-eastern Europe and the variant seems to be indigenous to that region. The admixture with the haplotypes E4 and E1/E2 could have taken place from a historical endemic focus during the fox expansion in the last decades. By employing the nad1 fragment, a typical European haplotype was observed in all 21 resolved Slovak samples. The acceleration in the AE incidence in the last decade suggests the emergence of the disease and the need for further research on human and animal isolates.

Mitochondrial genetic diversity and phylogenetic relationships of Echinococcus multilocularis in Europe.

The cestode Echinococcus multilocularis is the causative agent of alveolar echinococcosis, a fatal zoonotic parasitic disease of the northern hemisphere. Red foxes are the main reservoir hosts and, likely, the main drivers of the geographic spread of the disease in Europe. Knowledge of genetic relationships among E. multilocularis isolates at a European scale is key to understanding the dispersal characteristics of E. multilocularis. Hence, the present study aimed to describe the genetic diversity of E. multilocularis isolates obtained from different host species in 19 European countries. Based on the analysis of complete nucleotide sequences of the cob, atp6, nad2, nad1 and cox1 mitochondrial genes (4,968 bp), 43 haplotypes were inferred. Four haplotypes represented 62.56 % of the examined isolates (142/227), and one of these four haplotypes was found in each country investigated, except Svalbard, Norway. While the haplotypes from Svalbard were markedly different from all the others, mainland Europe appeared to be dominated by two main clusters, represented by most western, central and eastern European countries, and the Baltic countries and northeastern Poland, respectively. Moreover, one Asian-like haplotype was identified in Latvia and northeastern Poland. To better elucidate the presence of Asian genetic variants of E. multilocularis in Europe, and to obtain a more comprehensive Europe-wide coverage, further studies, including samples from endemic regions not investigated in the present study, especially some eastern European countries, are needed. Further, the present work proposes historical causes that may have contributed to shaping the current genetic variability of E. multilocularis in Europe.

Diversity and geographic distribution of haplotypes of Dirofilaria immitis across European endemic countries.

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.

Pilot research on gastrointestinal parasites of the Tatra chamois (Rupicapra rupicapra tatrica).

INTRODUCTION AND OBJECTIVE: The Tatra chamois (Rupicapra rupicapra tatrica) is a significant representative of the High Tatra Mountains endemic fauna species. In terms of health hazards for these animals, an important role is played by parasitic infections that can lead to a significant depletion of the entire population. OBJECTIVE: The aim of the study was to describe the occurrence of gastrointestinal parasites of Tatra chamois in the current environmental and climatic conditions of the High Tatra Mountains. MATERIAL AND METHODS: During the pilot project in 2014 - 2017, a total of 494 chamois faecal samples were collected from the Slovak High Tatra and 114 samples in the Polish part of the mountain and examined using standard coprological methods. RESULTS: The results revealed that the overall positivity for gastrointestinal parasites in chamois of the Slovak High Tatra reached 74.7%. Most frequent were protozoa - Eimeria spp. (42.7%), helminths were represented by Moniezia spp. tapeworms (23.5%), eggs of GIS-strongylids (7.1%), and sporadically Capillaria spp. (1.4%). The chamois from the Polish Tatra Mts. were infected with Eimeria spp. (43.9%), GIT-strongylids (9.6%), and Moniezia spp. (6.1%). Parasitic infection was determined in 59.6 % of faecal samples from the Polish part of the mountains. Statistical analyses revealed a significant difference in Moniezia spp. occurrence in different Slovak Tatra Mts. Regions, as well as between Slovak and Polish Tatra Mts. CONCLUSIONS: Initial research on the gastrointestinal parasites of the Tatra chamois revealed one indisputable finding - a relatively high prevalence of the genus Moniezia, which is closely linked to the climate and microclimate conditions of the mountains. Further intensive research on parasite composition and distribution in Tatra chamois is needed in broader temporal, ecological, and zoological contexts.

Clinical and Hematologic Findings in Babesia canis Infection in Eastern Slovakia.

PURPOSE: Babesia canis infection occurs in many locations throughout Europe. However, various studies report different clinicopathological findings in affected dogs. This study was focused on changes in clinical and hematologic parameters in dogs with B. canis infection from eastern Slovakia. METHODS: The study was prospective and included 45 dogs with suspected babesiosis. Babesia canis infection was confirmed by PCR in 34 cases and by blood smear microscopy in 24 (70.6%) of them. Hematology results, clinical examination from these dogs, and possible co-infection with other tick-borne pathogens by PCR were subsequently evaluated. RESULTS: The major clinical signs found included lethargy (91%), fever (59%), anorexia (59%), pigmenturia (47%) and icterus (18%). Mortality rate was 6%. Thrombocytopenia was the most common hematologic change, observed in 100% of the dogs with B. canis infection. Other frequent findings were lymphopenia (82%) and anemia (68%). No co-infections were detected. Anaplasma phagocytophilum infection was diagnosed by PCR only in one dog, which was not infected with B. canis. CONCLUSIONS: This study showed that B. canis infection in eastern Slovakia should be diagnosed by PCR when there is clinical suspicion of the disease, as almost 30% of the infected sick dogs did not have demonstrable parasites in their blood smear by microscopy. Lymphopenia is a frequent hematologic finding in B. canis infection and observed even more often than anemia. However, in agreement with previous studies, thrombocytopenia remains the most common hematologic finding associated with B. canis infection.

Thelazia callipaeda in Slovakia - From sporadic cases to endemic areas.

Spirurid nematode Thelazia callipaeda, transmitted by the fruit fly Phortica variegata, is a causative agent of an ocular parasitic disease called also canine thelaziosis. Dogs, cats, and wild canids are considered the primary definitive hosts for the parasite, but humans may also serve as aberrant definitive hosts. For long decades the geographic range of T. callipaeda was strictly limited to the territory of Asia, but after the year 2000, the parasite began to spread rapidly through Europe. The first autochthonous infections of dogs and foxes in Slovakia were recorded in 2016. In the present study, the results of a whole-area surveillance for canine thelaziosis are reported. Altogether, 142 cases of infection caused by T. callipaeda were diagnosed by veterinarians in dogs between 2016 and the first quarter of 2021, and two cases of feline thelaziosis were recorded. The majority of the dogs showed mild ocular signs manifested by conjunctivitis; 8.5% of them suffered from more serious mucopurulent discharge, and in two dogs corneal ulceration was recorded. The screening revealed increasing trends in the occurrence of canine thelaziosis from both a temporal and spatial point of view and unambiguously confirms the endemic status of T. callipaeda in Slovakia with the prospect of its further expansion.

Molecular Study of Echinococcus granulosus Cestodes in Ukraine and the First Genetic Identification of Echinococcus granulosus Sensu Stricto (G1 Genotype) in the Country.

INTRODUCTION: Cystic echinococcosis is a globally distributed zoonotic disease of great medical and veterinary importance, which is caused by the cestode Echinococcus granulosus sensu lato. In Ukraine, two areas of the prominent circulation of the parasite are established, the southern steppe zone with sheep as the main transmitter, and the northern forest-steppe zone and Polissia, where pigs are mainly responsible for maintaining the E. granulosus transmission. METHODS: Given that only a few studies have so far addressed the genetic diversity of the parasite in Ukraine, we have sequenced partial mitochondrial genes of cytochrome c oxidase 1 (789 bp), NADH dehydrogenase 1 (602 bp) and 12S rRNA (333-334 bp) in pig metacestodes from the Sumy region (farms close to Sumy, northeastern Ukraine) and the Kyiv region (a farm in Bila Tserkva, central Ukraine). RESULTS: Four isolates from four pigs in the Sumy region were identified as E. canadensis (G7 genotype), the major E. granulosus s.l. species circulating in Eastern Europe, including the three microvariants (G7A, G7B, G7C). Three isolates from the two pigs in the Kyiv region were classified as E. granulosus s.s. (G1 genotype), including one microvariant (G1A). CONCLUSION: To our knowledge, this is the first genetic record of E. granulosus s.s. with the presumed highest infectivity and virulence among the E. granulosus s.l. species in Ukraine. The finding has implications for public health as local control programmes should take into consideration different development rate of this parasite in dogs and the greater risk of the species for human infection.