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Bronchial hyperresponsiveness (BHR) is a key feature of asthma, but it may also appear in allergic rhinitis. The type of allergen, as well as regional characteristics, play an important role in the development of BHR. The aim of our study was to analyze allergen sensitization patterns and the factors that affect BHR in allergic rhinitis patients living in temperate continental climate zone.This study retrospectively analyzed allergic rhinitis patients from Eastern Slovakia who underwent skin-prick tests to aeroallergens, spirometry, histamine and methacholine bronchial provocation tests for evaluation of lower airway symptoms. We analyzed the associations between BHR and the pattern of aeroallergen sensitization, lung function parameters, and the total IgE and eosinophil levels.Out of 365 allergic rhinitis patients (age range 16-64 years), 114 showed BHR. Sensitization to house dust mites (HDMs) and grass were the most common. BHR was significantly associated with sensitization to dogs (odds ratio, OR = 2.15, 95% confidence interval, CI: 1.13-4.11) and Alternaria (OR = 2.15, 95% CI: 1.06-4.35); however, BHR did not show a relationship with HDMs sensitization. The levels of total IgE and eosinophils were higher in the BHR-positive group. Sensitization to more than six allergens significantly increased the probability of BHR (p 0.01).Dogs and Alternaria, but not HDMs, were the sensitizing agents most closely associated with BHR. High-grade sensitization and increased total IgE and eosinophil levels were characteristic clinical signs in BHR-positive allergic rhinitis patients in the temperate continental climatic zone.

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.

Objectives: Ticks, as a vector of various infections occur every year in Liptov, a popular tourist location in Slovakia, and their occurrence generally has an increasing trend. As up to now several rickettsiae from the spotted fever group were confirmed in tick vectors from different areas of Slovakia. Therefore, identification of previously unknown areas with endemic occurrence of Rickettsia spp. is of great importance. Material and methods: Questing ixodid ticks, small mammals, birds and wild boars from recreational areas of Liptov valley in northern Slovakia were tested for the presence of Rickettsia spp. and obtained sequences were further analyzed. Results: Overall, 8 out of 55 ixodid ticks (14.5%) tested positive for Rickettsia helvetica and one male tick (1.8%) was infected with Rickettsia monacensis. The identification of this species was based on the sequencing of 23S-5S internal transcribed spacer (ITS), gltA and ompA genes. Rickettsia spp. was detected in 7 rodents (5.8%) and 5 wild boars (5.9%) originated from Liptov valley, using gltA gene primer set. Conclusion: Our results confirm the presence of spotted fever group rickettsiae in tick vectors and hosts from northern part of Slovakia. Moreover, the evidence of the presence of Rickettsia spp. in vectors and in reservoirs indicate the circulation of the pathogen in nature and the need to monitor the situation in this popular location in Slovakia.

Herein we report a case of a 29-year-old man, suffering from a disease of unknown etiology and indicated a history of a bite by the unknown flying insect to both ear lobes in his anamnesis. The patient developed several nonspecific symptoms, such as fever, tension headache, and bilateral swelling of cervical lymph nodes (lymphadenitis). Moreover, he described perspiration, shivering, chills, epistaxis, several collapses, myalgia, and bone pains. Successive medical examinations of the patient confirmed several diagnoses. In addition, a blood sample was examined for the presence of tick-borne pathogens. PCR assay targeting gltA gene fragment of Rickettsia spp. brought a positive result. DNA of Rickettsia–like organism was confirmed by PCR and further sequencing. The rickettsial infection was successfully treated with doxycycline with a significant improvement of his health. This study brings a report of the molecular evidence of Rickettsia-like infection in a human patient from Slovakia, who suffered from several health problems accompanied by nonspecific febrile clinical symptoms. Although the patient did not report the tick bite before the development of clinical symptoms, he reported that he was bitten by an unknown flying insect. Thus, the possibility of rickettsial infection should be considered in similar nonspecific febrile illnesses when the bite of a flying insect is reported in personal anamnesis.

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.

This survey is aimed at investigation of species composition of fleas removed from small terrestrial mammals captured in rural, suburban, and urban types of habitat and molecular screening of the presence and diversity of Rickettsia species in collected ectoparasites. In total, 279 fleas (Siphonaptera) belonging to 9 species of 2 families, Ceratophyllidae and Hystrichopsyllidae, were collected from 115 (46%) out of 250 trapped small mammals of eight species (Apodemus agrarius, Apodemus flavicollis, Apodemus uralensis, Myodes glareolus, Microtus arvalis, Microtus subterraneus, Crocidura leucodon, and Sorex minutus). Rickettsia spp. were found in 2.5% (7/279) of tested fleas, namely in Ctenophthalmus agyrtes, Ctenophthalmus solutus, Ctenophthalmus uncinatus, Megabothris turbidus, and Amalareus penicilliger. Rickettsia felis, Rickettsia helvetica, and unidentified Rickettsia species were detected in fleas infesting small mammals in Eastern Slovakia. The results of the study suggest that some species of rickettsiae have a different range of arthropod vectors.

Background Distribution and biology of Pholeoixodes ticks is not very well understood. The goal of the study was to collect new data on the Pholeoixodes tick occurrence in Slovakia. Methods Tick infestation of red foxes in the regions of Košice, Prešov, Bratislava and Žilina was studied during the period 2017–2018. Ticks were collected from the fur of animals using tweezers and identified using appropriate keys. In total, 146 red foxes (Vulpes vulpes) were investigated. Results In total, 39 (26.7%) of animals were found to be infected with ticks from five species. Pholeoixodes ticks were found on 13 (3.4%) of the foxes: Ixodes hexagonus (Leach, 1815) on 5 specimens (3.4%), in the Košice, Prešov and Žilina regions; I. crenulatus (Koch, 1844) on 8 specimens (5.5%) in the Prešov and Bratislava regions; Ixodes ricinus (Linnaeus, 1758) collected from 25 (17.2%) foxes in every locality; Dermacentor reticulatus (Fabricius, 1794) from 5 foxes (3.4%) in the Košice, Prešov and Žilina regions; Haemaphysalis concinna (Koch, 1844), from 4 foxes (2.8%) from the Košice region. Conclusions Ixodes hexagonus has been previously recorded in Slovakia. However, this is the first finding of I. crenulatus in the country. The morphological features of the I. crenulatus specimens found in Slovakia were identical to those of ticks described in Poland and descriptions given in identification keys.

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.

During 2004-2011, we collected green lizards and Ixodes ricinus ticks in Slovak Karst National Park in Slovakia; 90% (36/40) of lizards and 37% of ticks removed from lizards were infected with family Anaplasmataceae bacteria. Only Candidatus Cryptoplasma sp. REP (reptile) was identified in these samples. Green lizards transmit this bacterium.

Background Despite 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. Aim To 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. Methods Mosquitoes 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. Results Altogether, 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. Conclusion The 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.

Ticks will diminish productivity among farm animals and transmit zoonotic diseases. We conducted a study to identify tick species infesting slaughter bulls from Adama City and to screen them for tick-borne pathogens. In 2016, 291 ticks were collected from 37 bulls in Adama, which were ready for slaughter. Ticks were identified morphologically. Total genomic DNA was extracted from ticks and used to test for Rickettsia spp. with real-time PCR. Species identification was done by phylogenetic analysis using sequencing that targeted the 23S-5S intergenic spacer region and ompA genes. Four tick species from two genera, Amblyomma and Rhipicephalus, were identified. Amblyomma cohaerens was the dominant species (n = 241, 82.8%), followed by Amblyomma variegatum (n = 22, 7.5%), Rhipicephalus pulchellus (n = 19, 6.5%), and Rhipicephalus decoloratus (n = 9, 3.0%). Among all ticks, 32 (11%) were positive for Rickettsia spp. and 15 (5.2%) of these were identified as R. africae comprising at least two genetic clades, occurring in A. variegatum (n = 10) and A. cohaerens (n = 5). The remainder of Rickettsia-positive samples could not be amplified due to low DNA yield. Furthermore, another 15 (5.2%) samples carried other pathogenic bacteria: Ehrlichia ruminantium (n = 9; 3.1%) in A. cohaerens, Ehrlichia sp. (n = 3; 1%) in Rh. pulchellus and A. cohaerens, Anaplasma sp. (n = 1; 0.5%) in A. cohaerens, and Neoehrlichia mikurensis (n = 2; 0.7%) in A. cohaerens. All ticks were negative for Bartonella spp., Babesia spp., Theileria spp., and Hepatozoon spp. We reported for the first time E. ruminatium, N. mikurensis, Ehrlichia sp., and Anaplasma sp. in A. cohaerens. Medically and veterinarily important pathogens were mostly detected from A. variegatum and A. cohaerens. These data are relevant for a One-health approach for monitoring and prevention of tick-borne disease transmission.

This survey is aimed at investigation of species composition of fleas removed from small terrestrial mammals captured in rural, suburban, and urban types of habitat and molecular screening of the presence and diversity of

Echinococcus multilocularisis 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 mitochondrialnad1gene analysis. In 2016, 11-years old Siberian husky female suffering from inappetence was presented to the Veterinary Clinic in Žilina. 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 ofE.multilocularis nad1gene 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 ofnad1gene. 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.

Wild animals, especially canids, are important reservoirs of vector-borne pathogens, that are transmitted by the ticks and other bloodsucking arthropods. In total, 300 red foxes (Vulpes vulpes), shot by the hunters in eastern and northern Slovakia, were screened for the presence of vector-borne pathogens by PCR-based methods Blood samples were obtained from nine red foxes and tissue samples originated from 291 animals (the liver tissue samples from 49 foxes and spleen samples from 242 red foxes). Babesia vulpes and haemotropic Mycoplasma species were identified by amplification and sequencing of 18S rRNA and 16S rRNA gene fragments, respectively. Overall, the presence of these pathogens was recorded in 12.3% of screened DNA samples. Altogether 9.7% (29/300) of investigated foxes carried DNA of Babesia spp. In total, 12 out of 29 Babesia spp. PCR – positive amplicons were further sequenced and identified as B. vulpes (41.4%; 12/29), remaining 17 samples are referred as Babesia sp. (58.6%; 17/29). Overall prevalence of B. vulpes reached 4.0% (n = 300). Thirteen (4.3%) samples tested positive for distinct Mycoplasma species. To the best of our knowledge, this study brings the first information on B. vulpes infection in red foxes in Slovakia, and the first data on the prevalence and diversity of haemotropic Mycoplasma spp. in European red fox population. Moreover, co-infections with B. vulpes and Mycoplasma spp. were confirmed in 1.7% of tested DNA samples. The relatively high rates of blood pathogen' prevalence and species diversity in wild foxes indicate the role of the fox population in the maintenance of the parasites in sylvatic cycles and strengthen the assumption that foxes play an important role in spreading of infectious microorganisms within and outside the natural foci.

This study provides the very first evidence of Hepatozoon canis infection in dogs from Slovakia, a Rhipicephalus sanguineus sensu lato free area. In total, 297 red foxes (Vulpes vulpes) and 293 dogs, from three regions of Slovakia, were screened for the presence of H. canis using a conventional 18S rRNA polymerase chain reaction (PCR). Genomic DNA of this blood parasite was detected in 51 (17.1%) foxes from all sampling regions in Slovakia, whereas the overall prevalence in examined dogs was significantly lower, only 1.0%. Identity between nucleotide sequences from Slovak foxes and dogs ranged from 97.3 to 100%. Phylogenetic analysis showed that H. canis isolates from Slovakia are divided into two clusters, suggesting the coexistence of strain variants with unknown pathogenicity which needs to be further explored.

The distribution and prevalence of zoonotic pathogens infecting ixodid ticks in Western Europe have been extensively examined. However, data on ticks and tick-borne pathogens in Eastern Europe, particularly Ukraine are scarce. The objective of the current study was, therefore, to investigate the prevalence of Anaplasma phagocytophilum, Anaplasmataceae, Rickettsia spp., Babesia spp., Bartonella spp., and Borrelia burgdorferi sensu lato in engorged and questing ixodid ticks collected from five administrative regions (oblasts) of Ukraine, namely Chernivtsi, Khmelnytskyi, Kyiv, Ternopil, and Vinnytsia. The ticks were collected from both wild and domestic animals and from vegetation. Of 524 ixodid ticks collected, 3, 99, and 422 ticks were identified as Ixodes hexagonus, Ixodes ricinus, and Dermacentor reticulatus, respectively. DNA samples individually extracted from 168 questing and 354 engorged adult ticks were subjected to pathogen-specific PCR analyses. The mean prevalence in I. ricinus and D. reticulatus were, respectively: 10 % (10/97) and 3 % (12/422) for A. phagocytophilum; 69 % (67/97) and 52 % (220/422) for members of the Anaplasmataceae family; 25 % (24/97) and 28 % (117/422) for Rickettsia spp.; 3 % (3/97) and 1 % (6/422) for Babesia spp.; and 9 % (9/97) and 5 % (20/422) for Bartonella spp. Overall, between the five cities, there was no significant difference in the prevalence of any of the pathogens for the respective ticks (p > 0.05). The prevalence of B. burgdorferi s. l. in the questing and engorged I. ricinus varied from 0 to 27 % and 14-44%, respectively, with no statistical significance identified between the five cities (p > 0.05). In addition to reporting the updated data for Kyiv and Ternopil, this study is the first to provide the prevalences of the tick-borne pathogens for Chernivtsi, Khmelnytskyi, and Vinnytsia. This investigation is also the first to detect Neoehrlichia mikurensis in ixodid ticks from Ukraine. These new data will be useful for medical and veterinary practitioners as well as public health officials when diagnosing infections and when implementing measures to combat tick-borne diseases in Ukraine.

Babesiosis is a severe disease in cattle worldwide. In Europe, the main causative agent of bovine babesiosis is Babesia divergens. In some areas, this species is reported to have declined or even disappeared, and its etiological role overtaken by other piroplasmid species. Moreover, co-infection with other tick-transmitted pathogens can be expected to complicate diagnosis in cattle. Hence, molecular identification of the causative agent of babesiosis should be a priority. Therefore, samples from 71 domestic cattle, 39 with clinical signs of babesiosis and 32 without, from southern Sweden were screened for Babesia spp. and Anaplasma spp. using molecular methods Babesia divergens was detected in 38 of the samples, and Anaplasma phagocytophilum in 17. Co-infections with both pathogens were frequent, occurring in 18% of the animals with a B. divergens infection. The possibility of co-infection should be considered in diagnosis and treatment of bovine babesiosis.

Lyme borreliosis and tick-borne encephalitis (TBE) are the most common tick-borne diseases in Germany. We collected Ixodes ricinus ticks from 16 high-risk and four low-risk sites distributed in Lower Bavaria and Upper Palatinate based on the number of human TBE cases recorded at the Robert Koch Institute from 2001 to 2009. A total of 8805 questing ticks (8203 nymphs, 602 adults) were collected in 2010 and examined in pools for the presence of tick-borne encephalitis virus (TBEV) using real-time RT–PCR. Overall TBEV prevalence evaluated as the minimum infection rate (MIR) was 0.26 % (23 positive pools/8805 ticks in 1029 pools). TBEV was detected at seven of the 16 high-risk sites, where MIR ranged from 0.16 to 2.86 %. A total of 3969 ticks were examined by PCR for infection with Borrelia burgdorferi sensu lato (s.l.) targeting the 5 S–23 S rRNA intergenic spacer (IGS) region. IGS nucleotide sequences were used to determine genospecies. Selected positive Borrelia samples were subjected to PCR and sequencing targeting the OspA gene, providing 46 sequences for molecular phylogenetic analysis. Of the 3969 questing ticks, 506 (12.7 %) were positive for B. burgdorferi s.l. Seven B. burgdorferi s.l. genospecies were identified: B. afzelii (41.3 %), B. garinii (19 %), B. valaisiana (13.8 %), B. burgdorferi sensu stricto (11.1 %), B. spielmanii (0.4 %), B. lusitaniae (0.2 %), and Candidatus B. finlandensis (0.6 %). Mixed infections were identified in 13.6 % of the ticks. The rate of infection in questing ticks varied among sites from 5.6 % (72 examined, four positive) to 29.5 % (88 examined, 26 positive). B. burgdorferi s.l. occurred at all 20 sites, whereas TBEV was detected only at the high-risk sites where more human TBE cases were reported compared to low-risk sites.

The present survey aimed to investigate flea and tick fauna parasitizing Slovak red fox populations with special emphasis on canine pathogens they transmit. A total of 407 fleas and 105 ticks were collected from 90 red foxes from two geographically distant regions. Seven flea species (Chaetopsylla globiceps, Pulex irritans, Archaeopsylla erinacei, Chaetopsylla rothschildi, Chaetopsylla trichosa, Ctenocephalides canis, and Ctenopthalmus assimilis) and three species of hard ticks (Ixodes ricinus, Ixodes hexagonus, Haemaphysalis concinna) were recorded on sampled animals. Consequently, the DNA of five different pathogen taxa was confirmed in collected arthropod vectors: Bartonella spp. (in P. irritans, Ch. globiceps, and Ct. assimilis), Rickettsia spp. (in A. erinacei, I. ricinus, I. hexagonus, and H. concinna), Anaplasma phagocytophilum (in I. ricinus), Theileria sp. (in Ch. globiceps and H. concinna), and Hepatozoon canis (in I. ricinus and I. hexagonus). Mycoplasma spp., Dipylidium caninum, and Acanthocheilonema reconditum were not found in fleas or ticks in this study.

Rickettsiae of the spotted fever group are considered as emerging pathogens; ticks, fleas and mites are known to be their vectors. However, the prevalence and species diversity of rickettsiae in small mammals and the role of these hosts in the circulation of bacteria are much less studied. During 2014-2016, a total of 250 small mammals (Apodemus agrarius, Apodemus flavicollis, Apodemus uralensis, Myodes glareolus, Crocidura leucodon, Crocidura suaveolens, Micromys minutus, Microtus arvalis, Microtus subterraneus, Neomys fodiens and Sorex minutus) were captured in natural, suburban and urban habitats of eastern Slovakia. Ear biopsies of 245 individuals were examined for the presence of SFG rickettsiae by molecular methods. The overall prevalence of Rickettsia spp. in small mammals was 11%. The predominant species, Rickettsia helvetica was confirmed in the striped field mouse (A. agrarius), the yellow-necked mouse (A. flavicollis), the bank vole (M. glareolus) and the common vole (M. arvalis) in natural and suburban habitats, followed by Rickettsia sp. closely related to R. felis identified in A. flavicollis captured in a suburban habitat. Finally, R. slovaca was found in only one yellow-necked mouse (A. flavicollis) captured in a natural habitat, with the sympatric occurrence of Dermacentor marginatus and Dermacentor reticulatus ticks. We assume the presence of R. slovaca especially in sites with the occurrence of Dermacentor spp. All small mammals captured in the urban habitat tested were negative for the presence of rickettsiae. This study brings the first molecular evidence of R. slovaca in a rodent captured in Slovakia. Rickettsia sp. closely related to R. felis was first time detected in A. flavicollis in suburban site of Slovakia. The highest species diversity of rickettsiae was observed in A. flavicollis, and the highest prevalence of bacteria was recorded in M. glareolus. The highest occurrence of rickettsiae-positive small mammals was recorded during the spring and autumn months, May, June and September, respectively. This may be related with the seasonal activity of the tick vectors. This study confirms the long-term persistence of Rickettsia spp. in small mammals in natural and suburban habitats of Slovakia. Some rodent species that have a wider ecological valency may contribute to the maintenance, circulation and dissemination of rickettsiae within and out the natural foci more significantly than those species that have narrower relation to the certain type of habitat.

A survey was undertaken to assess the prevalence of Theileria equi and Anaplasma phagocytophilum in some regions of Ukraine, Poland and Slovakia. Using a specific PCR assays, blood samples from 215 horses were tested. The prevalence of T. equi and A. phagocytophilum infection was 13.95% and 1.4%, respectively. BLAST analysis showed the isolates closest to the T. equi 18S rRNA and A. phagocytophilum msp4 gene sequences in GenBank with a similarity of ≥99%. No significant association was found between the T. equi PCR positivity and the age or sex of the horses. There was a significant association between the origin of horses and T. equi-PCR positivity. No significant association was found between the A. phagocytophilum-PCR positivity and the age, sex or origin.

Background The spirurid nematode Thelazia callipaeda, also called the “Oriental eyeworm”, is the causative agent of canine and human ocular thelaziosis. In the past few years it has started to spread across central Europe and new endemic areas have been established. The present study reports on the first four autochthonous cases of canine ocular thelaziosis in the territory of Slovakia, Central Europe. Results All cases were recorded in dogs living in eastern Slovakia, near the border with the Ukraine. All worms collected were investigated morphologically and their identification further confirmed at the molecular level by PCR amplification and direct sequencing. Nucleotide sequences of partial T. callipaeda cox1 and 28S rDNA gene fragments isolated from Slovak dogs were submitted to the GenBank database under accession numbers KY476400 and KY476401, respectively. Conclusions Considering that all four cases were diagnosed in animals that had never travelled abroad, there is clear evidence of an autochthonous occurrence and thereby the further spread of T. callipaeda across Europe. Moreover, at latitude of 48°N, these cases might be considered as the northernmost recorded cases of autochthonous in western and Central Europe.

Monitoring West Nile virus (WNV) and Usutu virus (USUV) activity now has the highest priority among mosquito-borne pathogenic viruses circulating in the European Union. This study documents a first time detection and the co-circulation of WNV lineage-2 (with the minimal prevalence of 0.46%) and USUV clade Europe 2 (with the minimal prevalence of 0.25%) in mosquitoes from the same habitat of south-western Slovakia and underlines necessity to perform rigorous surveillance in birds, mosquitoes, horses and humans in that country.

To date, only limited data about the presence of ticks and circulation of tick-borne pathogens in urban parks of Kyiv in northern Ukraine are available. In total, 767 ticks (696 Ixodes ricinus and 69 Dermacentor reticulatus) collected in seven urban parks and one suburban oak wood park in Kyiv were individually analyzed by the PCR assays. Tick-borne pathogens, namely spirochetes from Borrelia burgdorferi sensu lato complex, Anaplasma phagocytophilum, and Babesia microti, were detected in 11.1% of tested I. ricinus ticks. In total, 4% of I. ricinus ticks tested positive for the presence of B. burdorferi s.l. (Borrelia afzelii and Borrelia garinii), 5.2% for A. phagocytophilum, and Ba. microti was confirmed in 1.9% of examined ticks. Mixed infections were recorded in four DNA samples, representing the prevalence of 0.6%. One female and two I. ricinus nymphs were simultaneously infected with B. afzelii and A. phagocytophilum, and one female carried B. afzelii and Ba. microti. In addition, 10.1% of D. reticulatus ticks tested positive for Rickettsia raoultii. Identification of infectious agents and their diversity, assessment of the relative epidemiological importance and determination of the prevalence in questing ticks from central parts of the cities are crucial steps towards the tick-borne diseases surveillance in urban environment.

Canine babesiosis was considered an imported tick transmitted disease until the first case of autochthonous canine babesiosis in Slovakia was described in 2002. Since then, the number of cases kept increasing every year. The causative agent of babesiosis in dogs is not yet characterized; therefore, the aim of our study was to determine the agent and the rate of infection in the vector tick D. reticulatus in Slovakia. Babesia canis canis was detected in 80 out of 87 blood samples from dogs with clinical manifestations of babesiosis. Six dogs suspected of babesiosis tested positive for presence of Anaplasma phagocytophilum, and one mixed infection of B. c. canis and A. phagocytophilum was detected. B. c. canis was detected in 35.6% questing adults of D. reticulatus. The obtained sequences from blood samples showed 99.7% and from D. reticulatus, 99.4% similarity with the B. c. canis (AY072926) from dogs infected in Croatia. In our study, we characterized the agent of canine babesiosis from blood samples of naturally infected dogs and D. reticulatus, the vector tick. Further, the presence of A. phagocytophilum, bacterium responsible for the canine granulocytic anaplasmosis, was recorded in dogs for the first time in Slovakia.

Anaplasma phagocytophilum is an obligate intracellular bacterium, circulating in the natural foci in enzootic, vector-host cycle. In Europe, A. phagocytophilum is transmitted by Ixodes ricinus ticks. In Slovakia, cervids which are considered as naturally infected reservoirs of A. phagocytophilum are besides the ticks commonly infested with insects from the family Hippoboscidae. In this study, the presence of A. phagocytophilum was confirmed in deer keds (Lipoptena cervi) removed from deer by using of molecular approach. Detection of A. phagocytophilum in deer keds represents the remains of infected blood meal taken from infected deer host, what underlines the potential role of these blood-sucking insects in the mechanical transmission of pathogenic bacteria within the susceptible population of wild animals. Moreover, it may suggest the risk for the transmission of A. phagocytophilum or related pathogens to humans and healthy animals via the bite of infected hematophagous ectoparasites.

Granulocytic anaplasmoses represent a group of emerging tick-borne infectious diseases caused by the obligate intracellular gram-negative bacterium, Anaplasma phagocytophilum (Rickettsiales) that infects granulocytes. It has been known as a ruminant pathogen in Europe since 1932, however, recently it has emerged as a pathogen of humans and domestic animals such as dogs and horses in the Northern Hemisphere, including United States and Europe. Rodents and game animals (especially deer) are presumed to play a crucial role in the maintenance of A. phagocytophilum in natural foci and serve as competent reservoirs. Up to now, the presence of bacterial DNA has been confirmed by molecular methods in a number of domestic and wildlife animals. Circulation of several genotypes has been confirmed in natural foci but the vector competence and the host spectrum involved in its circulation is still under investigation. Human granulocytic anaplasmosis (HGA) typically occurs in spring or summer and clinical manifestations range from mild or self-limiting to severe disease, especially in elderly patients with up to 50% requiring hospitalization and 7% intensive care. So far, no confirmed A. phagocytophilum infections of humans have been reported in Slovakia despite the fact that the presence of anti-anaplasma antibodies has been detected in investigated patients sera. This fact could be explained by non-specific clinical signs of the infection or lack of information in physicians and underdiagnosed or misdiagnosed cases. The purpose of this review is to present biology, ecology and life cycle of A. phagocytophilum and introduce clinical symptoms, diagnosis and treatment of the infection caused by this pathogenic bacterium.

This study brings the data about the occurrence of bacterial and protozoan pathogens in 32 great cormorants (Phalacrocorax carbo sinensis), representing approximately 20% of the population nesting in the surroundings of water basin Liptovska Mara (northern part of Central Slovakia). A survey revealed the presence of tick-borne bacteria Anaplasma phagocytophilum (6.25%) and parasitic protozoa Toxoplasma gondii (3.1%). These data indicate an infectious status of the great cormorant population nesting in Slovakia; they might suggest a degree of environmental contamination by infectious agents and demonstrate the role of migratory seabirds in the circulation and dispersal of pathogens with zoonotic potential.

The north-eastern part of Poland is considered an area of high risk for infection with tick-borne diseases, including with human granulocytic ehrlichiosis (HGE) agents. The etiological agent of HGE is Anaplasma phagocytophilum. As the animal reservoir for A. phagocytophilum in the environment serve the species from Cervidae and Bovidae families. European bison (Bison bonasus) and elk (Alces alces) are the big ruminant species, reintroduced to the forests of Middle Europe after many decades of absence. In the foci of zoonotic diseases they are able to play a role as natural reservoir to pathogens, however, their status as protected animals means their study has been rare and fragmentary. The studies of B. bonasus were conducted in Białowieża Primeval Forest and A. alces in Biebrza National Park. PCR amplifications were performed using primers amplifing the end of the groES gene, the intergenic spacer and approximately two-thirds of the groEL gene in the first round, and primers that span a 395-bp region of the groEL gene were used in the second round. The positive results were obtained in B. bonasus and A. alces, the prevalence of infection was 66.7 and 20.0%, respectively. Randomly selected samples were sequenced, sequences were compared with GenBank entries using Blast N2.2.13 and determined as A. phagocytophilum. The results presented herein are the first record of the presence of Anaplasma phagocytophilum in A. alces, and at the same time confirm the previous observations regarding the infection of B. bonasus with A. phagocytophilum.

Canine babesiosis is a severe and potentially life threatening infection. In Europe, Babesia canis is considered to be the most common species responsible for the disease. We report two cases of babesiosis caused by Babesia gibsoni. The polymerase chain reaction, restriction fragment length polymorphism analysis and further sequencing of 18S rRNA gene fragments from blood samples of both dogs revealed the identity of isolates with B. gibsoni genotypes from other dogs worldwide. This species was previously not known to infect dogs in Slovakia. It is resistant to traditional anti-babesial therapy. Therefore, correct diagnosis is crucial for the successful treatment, especially in dogs with hemolytic anemia and febrile conditions.

Rodents are important reservoir hosts of tick-borne pathogens. Anaplasma phagocytophilum is the causative agent of granulocytic anaplasmosis of both medical and veterinary importance. In Europe, this pathogen is primarily transmitted by the Ixodes ricinus tick among a wide range of vertebrate hosts. However, to what degree A. phagocytophilum exhibits host specificity and vector association is poorly understood. To assess the extent of vector association of this pathogen and to clarify its ecology in Central Europe we have analyzed and compared the genetic variability of A. phagocytophilum strains from questing and feeding I. ricinus and Ixodes trianguliceps ticks, as well as from rodent’ tissue samples. Tick collection and rodent trapping were performed during a 2-year study (2011–2012) in ecologically contrasting setting at four sites in Eastern Slovakia. Genetic variability of this pathogen was studied from the collected samples by DNA amplification and sequencing of four loci followed by Bayesian phylogenetic analyses. A. phagocytophilum was detected in questing I. ricinus ticks (0.7%) from all studied sites and in host feeding I. trianguliceps ticks (15.2%), as well as in rodent biopsies (ear – 1.6%, spleen – 2.2%), whereas A. phagocytophilum was not detected in rodents from those sites where I. trianguliceps ticks were absent. Moreover, Bayesian phylogenetic analyses have shown the presence of two distinct clades, and tree topologies were concordant for all four investigated loci. Importantly, the first clade contained A. phagocytophilum genotypes from questing I. ricinus and feeding I. ricinus from a broad array of hosts (i.e.,: humans, ungulates, birds and dogs). The second clade comprised solely genotypes found in rodents and feeding I. trianguliceps. In this study we have confirmed that A. phagocytophilum strains display specific host and vector associations also in Central Europe similarly to A. phagocytophilum’ molecular ecology in United Kingdom. This study suggests that A. phagocytophilum genotypes associated with rodents are probably transmitted solely by I. trianguliceps ticks, thus implying that rodent-associated A. phagocytophilum strains may not pose a risk for humans.

In Europe, Anaplasma phagocytophilum circulates in natural foci in a tick-host cycle. Up to now, antibodies against A. phagocytophilum as well as pathogen's DNA were recorded in several domestic and wild animals. Nevertheless, the reservoir host range is still under investigation. Tissue samples from European brown bears (Ursus arctos) were tested for the presence of A. phagocytophilum DNA by a PCR amplification of 16S rRNA gene. The results of our study provides the evidence, that the range of animals involved in the circulation and maintenance of A. phagocytophilum in natural foci, is extended of another ursine carnivore, European brown bear (Ursus arctos).

Recently, several arthropod-borne infections have been introduced into previously non-endemic regions in Europe as the result of various global changes. At the same time, endemic regions are expanding and the risk of co-infections is rising, due to climate change that allows vectors to move and spread infectious diseases into new areas. The aim of the current study was to confirm simultaneous infections with Anaplasma phagocytophilum and/or Babesia canis canis in Dirofilaria-infected dogs from Slovakia, central Europe. Genomic DNA was isolated from 366 blood samples of microfilaraemic dogs without clinical signs of infection. Samples were further screened for the presence of canine tick-borne pathogens using PCR and sequencing. This survey revealed co-infection with four arthropod-borne pathogens, in particular, Dirofilaria repens, Dirofilaria immitis, A. phagocytophilum, and B. canis canis. While D. repens, responsible for canine subcutaneous dirofilariosis, is scattered through the whole territory of the country, D. immitis occurs only in endemic areas of southeastern and southwestern Slovakia in mixed infection with D. repens. Co-infection with A. phagocytophilum was reported in 3.27% of the dogs positive for D. repens; mixed infection with D. repens and B. canis canis was detected in 3.55% of the tested blood samples. Eastern Slovak Lowland represents a natural focus of B. canis canis and is a highly endemic area for canine dirofilariosis. The presence of triple infection with D. repens, A. phagocytophilum, and B. canis canis was detected in one dog originating from the eastern lowland region of Slovakia. This study highlights the importance of co-infected, clinically healthy dogs in the spreading of several different arthropod-borne pathogens and the necessity for detailed epidemiological surveys, especially in newly infested areas.

Anaplasma phagocytophilum is a worldwide distributed bacterium with a significant medical and veterinary importance. It grows within the phagosome of infected neutrophils and is responsible for human granulocytic anaplasmosis (HGA), tick-borne fever (TBF) of small ruminants and cattle, canine and equine granulocytic anaplasmosis, but infects also a great variety of wildlife species. Wild ungulates and rodents are considered reservoirs of infection in natural foci. The objective of this study was to determine the spectrum of animal species involved in the circulation of A. phagocytophilum in Slovakia and to analyze the variability of obtained nucleotide sequences, in order to determine whether genotypes from Slovakia cluster according to host-species or geographical location. Several animal species and vector ticks were screened for the presence of members of the family Anaplasmataceae using PCR based methods. Additional data on the molecular evidence of Anaplasma ovis and Candidatus Neoehrlichia mikurensis are presented. These pathogens were detected in tested sheep flocks and rodents with the mean infection rates of 8.16% and 10.75%, respectively. A. phagocytophilum was genotyped by 16S rRNA and groEL gene sequencing. Bacterial DNA was confirmed in questing ixodid ticks, in domesticated canine, wild rodents and several species of wild ungulates. In European isolates, 16S rRNA gene does not seem to be an appropriate locus for the analyses of heterogeneity as it is too conservative. Similarly, 16S rRNA isolates from our study did not reveal any polymorphisms. All isolates were identical in overlapped region and showed identity with sequences from ticks, horses or ruminants previously isolated elsewhere in the world. On the other hand, the groESL heat shock operon is widely used for determination of diversity and the analyses have already revealed considerable degree of heterogeneity. Tested ungulates were infected with A. phagocytophilum to a considerable extent. High proportions of red and roe deer tested positive and the rates of infection reached over 60.0%. GroEL sequences from canine, wild ungulates and ticks from Slovakia clustered within a clade together with isolates from horses, humans, wild ungulates and ticks from Slovakia or elsewhere in the world. Sequences from rodents clustered apart from those obtained from wild ungulates, ticks and humans. These results suggest that European rodents do not harbour A. phagocytophilum strains with strong zoonotic potential such as those from United States.

TOC summary line: The green lizard is implicated in the transmission cycle of B. lusitaniae., In Europe, spirochetes within the Borrelia burgdorferi sensu lato complex are transmitted by Ixodes ricinus ticks. Specific associations are described between reservoir hosts and individual genospecies. We focused on green lizard (Lacerta viridis) as a host for ticks and potential host for borreliae. In 2004 and 2005, a total of 146 green lizards infested by ticks were captured, and 469 I. ricinus ticks were removed. Borrelial infection was detected in 16.6% of ticks from lizards. Of 102 skin biopsy specimens collected from lizards, 18.6% tested positive. The most frequently detected genospecies was B. lusitaniae (77.9%–94.7%). More than 19% of questing I. ricinus collected in areas where lizards were sampled tested positive for borreliae. B. garinii was the dominant species, and B. lusitaniae represented 11.1%. The presence of B. lusitaniae in skin biopsy specimens and in ticks that had fed on green lizards implicates this species in the transmission cycle of B. lusitaniae.