Serologic Investigation on Tick-Borne Encephalitis Virus, Kemerovo Virus and Tribec Virus Infections in Wild Birds.

The present study reports on serosurvey on the tick-borne encephalitis virus European subtype (TBEV; genus Flavivirus), and the tick-borne Kemerovo (KEMV) and Tribec (TRBV) orbivirus (genus Orbivirus) infections in tick-infested and non-infested birds. No virus RNA was detected in the blood clots. Birds were infested mostly by Ixodes ricinus, but Haemaphysalis concinna and I. frontalis were observed too. TBEV, KEMV and TRBV neutralising antibodies (NAb) were detected in the screening microtitration neutralisation test (µVNT). Seropositive samples were further examined in simultaneous µVNT to distinguish TBEV infection from WNV and USUV. KEMV and TRBV infections were also further examined by µVNT against each other. The demonstrated results point to increased TBEV and TRBV seroprevalence in birds over the past several years. This is the first study on KEMV infection in the Slovak bird population, and seropositive juvenile birds suggest its occurrence in a new geographic area. The results indicate the significance of tick infestation rates, seropositivity and specific NAb titre. The reservoir role of birds for TBEV, KEMV and TRBV remains unclear. However, targeted monitoring of birds and vectors is an effective measure of surveillance of arbovirus introduction into new geographic areas.

Opportunities and Limitations of Molecular Methods for Studying Bat-Associated Pathogens.

Bats have been identified as reservoirs of zoonotic and potentially zoonotic pathogens. Significant progress was made in the field of molecular biology with regard to infectious diseases, especially those that infect more than one species. Molecular methods, sequencing and bioinformatics have recently become irreplaceable tools in emerging infectious diseases research and even outbreak prediction. Modern methods in the molecular biology field have shed more light on the unique relationship between bats and viruses. Here we provide readers with a concise summary of the potential and limitations of molecular methods for studying the ecology of bats and bat-related pathogens and microorganisms.

Serological survey of lyssaviruses in synanthropic bats and human exposure to bats in Slovakia.

INTRODUCTION: Bats are considered natural reservoirs for lyssaviruses. A total of 17 out of 19 known lyssaviruses circulate in bat populations. Lyssaviruses cause rabies in animals and humans. The transmission of lyssaviruses from European bats to terrestrial animals and humans is rare, but the risk of infection still exists even in developed countries. Slovakia is currently a rabies-free country. OBJECTIVE: The aim of the study was to assess the potential circulation of EBLV-1 in synanthropic bats present in human inhabited buildings, and to give an overview of human exposure to bats. MATERIAL AND METHODS: A passive serological survey targeted the prevalence of antibodies to bat lyssaviruses in synanthropic bats between 2009 - 2019. A total of 598 bats of the species Pipistrellus pipistrellus, Pipistrellus pygmaeus, Eptesicus serotinus, Nyctalus noctula and Vespertilio murinus were captured in buildings mainly in Eastern Slovakia, and examined by the rapid fluorescent focus inhibition test (RFFIT). RESULTS: Lyssavirus-specific antibodies were detected in 2 (0.3%) of the 598 examined bats. Additionally, brain tissues of bats found dead were examined using the standard fluorescent antibody test (FAT) with negative results. An overview of available data on human exposure to bats recorded in Slovakia from 2007 - 2019 is also included. CONCLUSIONS: The study confirmed the presence of lyssavirus antibodies in synanthropic bats in Slovakia, suggesting the active circulation of bat lyssaviruses in bat populations exploiting human buildings. Although the seroprevalence was found to be extremely low, the results show that any case of human exposure to bats must be treated with caution in order to protect public health.

Genetic Characterization of a Neurovirulent West Nile Virus Variant Associated with a Fatal Great Grey Owl Infection.

This study reports on a fatal case of a captive great grey owl infected with the West Nile virus (WNV) in the zoological garden Kosice, eastern Slovakia (Central Europe). The tissue samples of the dead owl were used for virus isolation and genetic characterization. The novel isolate is genetically closer to Hungarian, Greek, and Bulgarian strains from the central/southern European clade of lineage 2 than to the strains previously isolated in Slovakia. Interestingly, it carries NS3-249P, a molecular virulence determinant associated with higher neurovirulence, which has not previously been observed in Slovakia. Subsequent serological investigation of the captive owls revealed additional seropositive animals, indicating local WNV transmission. Although no WNV-positive mosquitoes were found, the presence of the WNV principal vector Culex pipiens complex together with the described fatal case and further serological findings indicate an endemic focus of bird-neurovirulent WNV variant in the area.

The first serological evidence of Anaplasma phagocytophilum in horses in Slovakia.

Anaplasma phagocytophilum is the causative agent of granulocytic anaplasmosis. It affects humans and several wild and domesticated mammals, including horses. The aim of our study was a preliminary survey of the occurrence of these re-emerging pathogens in horses in Slovakia. The sera from 200 animals of different ages and both sexes were tested for the presence of A. phagocytophilum antibodies by indirect immunofluorescence assay. Subsequently, detection of the 16S rRNA gene fragment of A. phagocytophilum was attempted by polymerase chain reaction (PCR) in each blood sample. Our results confirmed the presence of specific antibodies in 85 out of 200 individuals (42.5%), but no significant changes were found between the animals of different ages and sexes. However, the PCR analysis did not detect any positive animals. Our data represent one of the highest values of seropositivity to A. phagocytophilum in horses in Central Europe. These results may contribute to a better understanding of the circulation of A. phagocytophilum in this region, thus indicating a potential risk to other susceptible species.

A Serosurvey of Flavivirus Infection in Horses and Birds in Slovakia.

In central Europe, at least three flaviviruses circulate among vectors and vertebrate hosts. West Nile virus (WNV) and Usutu virus (USUV) are mosquito-borne viruses maintained in the nature by enzootic cycle between mosquitoes and birds. Tick-borne encephalitis virus (TBEV) is a flavivirus causing annual human cases in Slovakia. The aim of this study is the prevalence assessment of flavivirus infections in horses (n = 145) and birds (n = 109) by enzyme-linked immunosorbent assay (ELISA) and confirmation by neutralization test (VNT). WNV antibodies have been detected in 11.7% of tested horses and 11.9% of tested birds and confirmed in 6.9% of horse and 9.2% of bird samples. None of the WNV seropositive or dubious horses had WNV IgM (ELISA), and none of the tested horses had USUV neutralizing antibodies. Autochthonous WNV infections have been confirmed in 16.7% of horses without international travelling history. Most of them were from western Slovakia with known endemic WNV transmission. An autochthonous WNV infection in a horse from highland area of Kremnické vrchy (central Slovakia) with unknown data of WNV circulation and without travelling history was detected. TBEV antibody was detected in 6.2% of horses and in 3.4% has been confirmed. In two horses, WNV and TBEV infection could not be distinguished. Confirmed WNV seropositive were eight raptors showing nonspecific signs or suffering from trauma, one white stork, and one house sparrow. The sparrow was caught in a locality in eastern Slovakia, where WNV RNA had been previously detected in sparrows. USUV neutralizing antibodies were present in pooled sample from four Eurasian great tits. Because of insufficient volume, TBEV VNT was not carried out in birds. Results further prove the endemicity of WNV and other vector-borne flaviviruses in natural and accidental hosts in Slovakia, giving better insight in flavivirus epidemiology in European countries in general.

Concentrations of selected toxic elements in ewe living near an environmentally loaded area of eastern part of Slovakia.

INTRODUCTION AND OBJECTIVE: Research focused on the monitoring of selected heavy metals in ewes' blood. Concentrations of selected toxic elements, lead, cadmium and zinc, in ewes living near an environmentally-loaded area, concerned 15 ewes (aged 3-4 years) in good physical condition, during the spring of 2014 and 2015 in the eastern part of Slovakia. The aim of the research was to determine the concentration of selected heavy metals and state the correlations of selected heavy metals in ewes' blood. MATERIAL AND METHODS: Within the period of 2 years, 15 ewe were evaluated. Ewes' blood samples were collected twice during the spring season from a farm located in area Spis, eastern Slovakia, and then analysed for heavy metal contents. In the area under investigation, contamination with heavy metals was assumed as a result of intensive agricultural development and former mining activities. The level of selected heavy metals in the experimental group of animal blood was determined using an optical spectrophotometry. RESULTS: Statistical analyses were carried out using the Statistica programme. The significant differences between means were calculated by the statistical method of the non-parametric Mann-Whitney´s U test. The statistical test experimental group of ewes in 2014 and 2015 confirmed the presence of selected heavy metals in ewes. The measured values of Cd (P=0.0003), Pb (P=0.0200) and Zn (P=0.0018) showed significant differences when comparing the years 2014 and 2015. CONCLUSIONS: The obtained and analysed blood samples confirmed the presence of selected heavy metals in ewes from area of Spis in eastern Slovakia, which belongs the sub-region or is among the localities environmentally burdened. The conclusions are centred on the population's interest and concern for the environment, as well as on the preoccupation with factors that affect the satisfaction of basic needs, the local agricultural development and former mining activities.

Detection of West Nile virus and tick-borne encephalitis virus in birds in Slovakia, using a universal primer set.

West Nile virus (WNV) is a mosquito-borne neurotropic pathogen that presents a major public health concern. Information on WNV prevalence and circulation in Slovakia is insufficient. Oral and cloacal swabs and bird brain samples were tested for flavivirus RNA by RT-PCR using newly designed generic primers. The species designation was confirmed by sequencing. WNV was detected in swab and brain samples, whereas one brain sample was positive for tick-borne encephalitis virus (TBEV). The WNV sequences clustered with lineages 1 and 2. These results confirm the circulation of WNV in birds in Slovakia and emphasize the risk of infection of humans and horses.

Increasing incidence of Geomyces destructans fungus in bats from the Czech Republic and Slovakia.

BACKGROUND: White-nose syndrome is a disease of hibernating insectivorous bats associated with the fungus Geomyces destructans. It first appeared in North America in 2006, where over a million bats died since then. In Europe, G. destructans was first identified in France in 2009. Its distribution, infection dynamics, and effects on hibernating bats in Europe are largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: We screened hibernacula in the Czech Republic and Slovakia for the presence of the fungus during the winter seasons of 2008/2009 and 2009/2010. In winter 2009/2010, we found infected bats in 76 out of 98 surveyed sites, in which the majority had been previously negative. A photographic record of over 6000 hibernating bats, taken since 1994, revealed bats with fungal growths since 1995; however, the incidence of such bats increased in Myotis myotis from 2% in 2007 to 14% by 2010. Microscopic, cultivation and molecular genetic evaluations confirmed the identity of the recently sampled fungus as G. destructans, and demonstrated its continuous distribution in the studied area. At the end of the hibernation season we recorded pathologic changes in the skin of the affected bats, from which the fungus was isolated. We registered no mass mortality caused by the fungus, and the recorded population decline in the last two years of the most affected species, M. myotis, is within the population trend prediction interval. CONCLUSIONS/SIGNIFICANCE: G. destructans was found to be widespread in the Czech Republic and Slovakia, with an epizootic incidence in bats during the most recent years. Further development of the situation urgently requires a detailed pan-European monitoring scheme.