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.

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.

Rodents as Sentinels for Toxoplasma gondii in Rural Ecosystems in Slovakia-Seroprevalence Study.

Toxoplasma gondii is a ubiquitous intracellular parasite with felids as definitive hosts and a broad range of intermediate hosts. Rodents are considered suitable sentinels for prevalence studies of many infections, including toxoplasmosis. This study aimed to estimate the seroprevalence of T. gondii in rodents from different localities of Slovakia and investigate the correlation between the seropositivity and the species, age, sex, and sexual activity of animals. Altogether, 1009 wild rodents belonging to 9 species were trapped in 2015 and 2019, and antibodies to T. gondii were detected in 6.7% of the animals. Seropositivity was detected in seven species, ranging from 0.0% in Micromys minutus and Apodemus sylvaticus to 7.7% in A. flavicollis. The females reached significantly higher seropositivity (9.7%) than the males (3.8%), and the adults were positive significantly more often (9.2%) than the subadults (4.9%). The seropositivity differed also among localities, with significantly higher positivity detected in suburban and touristic areas (12.2%) than in localities with a lower level of human activities (5.5%). This study showed that the occurrence of T. gondii varies significantly in rodent species and habitats with various environmental conditions and different levels of anthropic use. Several biological and ecological factors, e.g., soil contamination, soil conditions, the susceptibility of rodent species etc., may influence this variability.

Molecular identification of Taenia hydatigena from domestic and free-living animals in Slovakia, Central Europe.

Taenia hydatigena is a cosmopolitan tapeworm that uses canids or felines as definitive hosts, while the larval stage (metacestode), formerly referred to as cysticercus tenuicollis, infects a wide variety of intermediate hosts, in particular ruminants. In the present study, we used partial nucleotide sequences of the cox1 and nad1 genes of T. hydatigena from different animal species to analyse the intraspecies genetic diversity of this economically important parasite. Twenty-four samples of metacestodes or adults of T. hydatigena from infected sheep, chamois, roe deer, fallow deer, wild boar, and dogs from Slovakia were collected and further analysed. Several haplotypes of T. hydatigena were identified with unique mutations that have not been previously recorded in Slovakia. Analysis of nucleotide polymorphism revealed the existence of 9 and 13 haplotypes, with relatively low nucleotide pairwise divergence ranging between 0.3-1.3 and 0.2-1.8% for the Hcox and Hnad haplotypes, respectively. In general, low nucleotide and high haplotype diversities in the overall population of T. hydatigena from the study indicate a high number of closely related haplotypes within the explored population; nucleotide diversity per site was low for cox1 (Pi = 0.00540) and slightly higher for nad1 (Pi = 0.00898). A molecular study confirmed the existence of genetic variation within T. hydatigena isolates from Slovakia. However, further investigations with more samples collected from different intermediate and definitive hosts are required in order to investigate the epidemiological significance of the apparent genetic differences observed in this study.

Parasitic and Vector-Borne Infections in HIV-Positive Patients in Slovakia-Evidence of an Unexpectedly High Occurrence of Anaplasma phagocytophilum.

In HIV (human immunodeficiency virus) infected people, the immunodeficiency caused by a reduced level of CD4 (cluster of differentiation 4) T-lymphocytes increases the risk of infectious diseases. Additionally, in individuals with immunologically compromising conditions, tick-borne or some parasitic pathogens may cause chronic, debilitating opportunistic infections and even death. The study aimed at determining the IgG seropositivity of HIV-infected patients to Toxoplasma gondii, Toxocara spp., Echinococcus multilocularis, and E. granulosus s.l. and performing the molecular identification of T. gondii and some tick-borne pathogens, namely, Borrelia spp., Babesia spp., Anaplasma phagocytophilum, Rickettsia spp., and Bartonella spp. Out of 89 HIV-positive patients, specific IgG antibodies to T. gondii were detected in 17 (19.1%) and to Borrelia spp. in 12 (13.5%) individuals. Seropositivity to Toxocara spp., E. multilocularis, and E. granulosus s.l. was not recorded. Molecular approaches showed positivity to T. gondii in two (2.2%) patients, and 11 (12.4%) individuals had positive PCR signal for the msp2 gene of A. phagocytophilum. Relatively high prevalence of A. phagocytophilum in HIV-positive patients suggests that these people are more susceptible to some vector-borne pathogens. The presence of opportunistic infections may pose a health risk for patients with weakened immune systems, and should not be neglected during the regular monitoring of the patient's health status.

A Survey of Intestinal Helminths of Dogs in Slovakia with an Emphasis on Zoonotic Species.

Dogs are the most popular pets worldwide; however, close contact with people increases the risk of transmission of different zoonotic parasites. This study aims to determine the prevalence of gastrointestinal helminths in dogs in Slovakia. A total of 495 faecal samples collected from pet, shelter, guard, working (police), and hunting dogs, as well as dogs from segregated Roma settlements between 2016 and 2021, were examined using flotation and molecular methods. Eggs of intestinal helminths were detected in 134 (27.1%) samples. Microscopically, six different species/genera/families, namely, Toxocara canis (14.7%), Toxascaris leonina (1.6%), Trichuris vulpis (6.3%), Capillaria spp. (1.4%), Ancylostoma/Uncinaria spp. (8.3%), and taeniid eggs (4.0%), were recorded. Molecular analyses revealed infection with Echinococcus multilocularis in 2.2% of dogs and 0.4% of the animals were infected with Taenia hydatigena. The results showed a correlation between the occurrence of intestinal helminths and the availability of veterinary care, as dogs from Roma settlements and shelter dogs were the most often infected (66.7% and 39.2%, respectively). On the other hand, working animals were in the best health condition, with only 2.5% being positive. The relatively frequent occurrence of zoonotic species points to the constant need for preventive measures and regular deworming of dogs.

Human Taeniases in Slovakia (2010-2019): Genetic Analysis of Taenia saginata Isolates.

Taenia saginata and Taenia solium, known as beef and pork tapeworm, are foodborne pathogens of global importance having a substantial impact on human health and economy. The study aimed to summarize the occurrence of human Taenia spp. infection in past 10 years in Slovakia based on reports of clinicians and diagnostic laboratories to Public Health Authority of the Slovak Republic. Altogether, 19 human cases were reported in Slovakia in 2010-2019, with the incidence of infection ranging from 0.00 to 0.12/100,000 inhabitants per year. In two patients T. saginata infection was confirmed molecularly. Nucleotide sequences of the analyzed nad1 gene fragments derived from both patients were identical and in a phylogenetic tree clustered together with T. saginata Tsa isolate (AM503345), as well as with a homologous sequence of the completely sequenced mitochondrial genome of T. saginata. Similarly, cox1 nucleotide sequences derived from one of the patients and isolates from Asia and/or Europe were identical. A cluster for cox1 partial gene sequence was placed separately from closely related Taenia asiatica and/or T. solium isolates in the phylogenetic tree.

The dwarf tapeworm Hymenolepis nana in pet rodents in Slovakia-epidemiological survey and genetic analysis.

Rodents are popular companion animals and are often kept as pets for children. However, they can be reservoirs of a variety of zoonotic pathogens. As little attention is being paid to the possibility of acquiring parasitic infections from pet rodents, the occurrence of Hymenolepis nana in rodents from pet shops and breeding clubs of Slovakia was surveyed, with parallel genetic analyses to type isolates from rodent species. In 2016-2018, pooled faecal samples from 119 boxes with 228 mice, 191 rats, 124 hamsters and 25 Mongolian gerbils were collected from 12 pet shops and 3 breeding clubs in five cities of eastern Slovakia. H. nana eggs were detected in 25 (21.0%) boxes. Animals from pet shops were infected more frequently (24.6% positive boxes) than those from breeding clubs (17.2%), without statistical significance. The highest prevalence was recorded in rats from pet shops, where 41.7% of boxes contained parasite eggs. Hamsters and mice in pet shops were also frequently infected; in 23.8% and 25% of boxes, respectively, H. nana eggs were observed. Prevalence in rats and hamsters from breeding clubs was lower, but in mice surpassed 40%. Nine samples with positive PCR products in any of the four DNA regions, mitochondrial cox1 and nuclear pmy, ITS1 and ITS2 targets, gave profiles characteristic of H. nana. The results imply the risk of zoonotic transmission of hymenolepiasis in Slovakia. Particular attention should be given to hygiene level maintained while keeping rodents. Furthermore, rodents intended for sale should be tested for parasites and then dewormed.

Oxyurid nematodes of pet rodents in Slovakia - a neglected zoonotic threat / Nematódeos oxiurídeos de roedores de estimação na Eslováquia - uma ameaça zoonótica negligenciada

Abstract The role of rodents as reservoirs of helminths of public health importance is not well known. The zoonotic potential of Syphacia spp. has been confirmed; therefore, the study aimed to estimate the occurrence of oxyurid nematodes in small rodents from pet shops and breeding clubs in Slovakia. Fecal samples of 586 pet rodents kept in 133 cages were collected between 2016 and 2018 and examined by Faust´s flotation method. Four species of oxyurid nematodes, Syphacia muris, S. obvelata, Aspiculuris tetraptera and Paraspidodera uncinata were detected. A. tetraptera was found in the faecal samples of all rodent species included in this survey. The number of positive boxes varied from 5.4% in hamsters to 70.0% with mice. The prevalence of Syphacia muris was highest in Mongolian gerbils where up to 75.0% boxes were positive; S. obvelata was found in 26.7% of boxes with mice, 25.0% of boxes with Mongolian gerbils and 3.2% of boxes with rats. The high prevalence of Syphacia spp. in all animal species points out the infection risk for humans. Animals offered for sale are often in close contact with human beings; therefore they should be regularly tested for parasites and then effectively dewormed.

Resumo O papel dos roedores como reservatórios de helmintos de importância para a saúde pública não é bem conhecido. O potencial zoonótico de Syphacia spp. foi confirmado; portanto, o estudo teve como objetivo estimar a ocorrência de nematóides oxiurídeos em pequenos roedores de pet shops e clubes de reprodução na Eslováquia. Amostras fecais de 586 roedores mantidos em 133 gaiolas foram coletadas entre 2016 e 2018 e examinadas pelo método de flotação de Faust. Foram detectadas quatro espécies de nematódeos oxiurídeos, Syphacia muris, S. obvelata, Aspiculuris tetraptera e Paraspidodera uncinata, A. tetraptera foi encontrado nas amostras fecais de todas as espécies de roedores incluídas nesta pesquisa. O número de gaiolas positivas variou de 5,4% em hamsters a 70,0% em camundongos. A prevalência de Syphacia muris foi maior nos gerbilos da Mongólia, onde até 75,0% das gaiolas foram positivas; S. obvelata foi encontrada em 26,7% das gaiolas com camundongos, 25,0% das gaiolas com gerbilos da Mongólia e 3,2% das gaiolas com ratos. A alta prevalência de Syphacia spp. em todas as espécies animais aponta o risco de infecção para os seres humanos. Animais oferecidos para venda estão frequentemente em contato próximo com seres humanos; portanto, eles devem ser regularmente testados quanto a parasitas e, então, efetivamente desparasitados.

Oxyurid nematodes of pet rodents in Slovakia - a neglected zoonotic threat.

The role of rodents as reservoirs of helminths of public health importance is not well known. The zoonotic potential of Syphacia spp. has been confirmed; therefore, the study aimed to estimate the occurrence of oxyurid nematodes in small rodents from pet shops and breeding clubs in Slovakia. Fecal samples of 586 pet rodents kept in 133 cages were collected between 2016 and 2018 and examined by Faust´s flotation method. Four species of oxyurid nematodes, Syphacia muris, S. obvelata, Aspiculuris tetraptera and Paraspidodera uncinata were detected. A. tetraptera was found in the faecal samples of all rodent species included in this survey. The number of positive boxes varied from 5.4% in hamsters to 70.0% with mice. The prevalence of Syphacia muris was highest in Mongolian gerbils where up to 75.0% boxes were positive; S. obvelata was found in 26.7% of boxes with mice, 25.0% of boxes with Mongolian gerbils and 3.2% of boxes with rats. The high prevalence of Syphacia spp. in all animal species points out the infection risk for humans. Animals offered for sale are often in close contact with human beings; therefore they should be regularly tested for parasites and then effectively dewormed.

Alveolar echinococcosis in a dog; analysis of clinical and histological findings and molecular identification of Echinococcus multilocularis.

Echinococcus multilocularis is a zoonotic tapeworm of medical and veterinary importance that occasionally infects accidental intermediate hosts causing severe disease or even death. In dogs, alveolar echinococcosis has been reported mainly in central Europe and Canada. The paper presents clinical, biochemical and histological signs of alveolar echinococcosis in a dog from Slovakia and the results of mitochondrial nad1 gene analysis. In 2016, 11-years old Siberian husky female suffering from inappetence was presented to the Veterinary Clinic in Zilina. Biochemical blood examination and blood count showed only minor changes, but abdominal ultrasonography showed the hepatomegaly and the presence of nonhomogeneous hypodense lobulated formation in the left liver lobe. Histological and molecular examinations of excided tissue confirmed the diagnosis of alveolar echinococcosis of the liver. BLAST analysis of E. multilocularis nad1 gene revealed that the nucleotide sequence did not exactly match the previously identified M1 (AJ237639) and/or M2 genotype (AJ237640). In total, two single nucleotide polymorphisms (SNPs) occurred within overlapping region of nad1 gene. In the discussion, clinical and laboratory findings of the infection in other dog patients are compared and the possibilities of diagnosis and therapy of the disease are discussed.

A Community-Based Study to Estimate the Seroprevalence of Trichinellosis and Echinococcosis in the Roma and Non-Roma Population of Slovakia.

Trichinellosis and cystic and alveolar echinococcosis are serious parasitic diseases transmissible between animals and humans. Moreover, alveolar echinococcosis is considered one of the most dangerous of human helminthoses. Roma communities are particularly numerous in Central and Eastern Europe. They are often concentrated in economically undeveloped regions and live in segregated localities with unsatisfactory housing and sanitary conditions. The study aimed to find out the seroprevalence of Trichinella and Echinococcus infections in the Roma population of segregated settlements and to compare it with the seropositivity of the non-Roma population of eastern Slovakia. Out of 823 samples, three sera showed seropositivity to Trichinella in the ELISA (Enzyme-linked immunosorbent assay) test. Subsequent Western blot reaction (WB) confirmed seropositivity in two Roma women. ELISA seropositivity to E. multilocularis was recorded in six persons (0.73%), and five (0.61%) respondents were seropositive to E. granulosus, but WB confirmed the presence of antibodies to Echinococcus spp. in one Roma participant. Positive persons suffered from unspecific clinical symptoms; Trichinella-positive persons reported headache, cough, fatigue, and muscle pain. The Echinococcus-positive participant suffered from headache and back pain. The study showed that the worse living conditions of the Roma community did not significantly influence the occurrence of Trichinella and Echinococcus infections in this minority.