Molecular detection of Setaria tundra (Issaitshikoff & Rajewskaya, 1928) and Setaria cervi (Rudolphi, 1819) in red deer in Slovakia.

Filaroid nematodes Setaria tundra (Issaitshikoff & Rajewskaya, 1928) and Setaria cervi (Rudolphi, 1819) are internal parasites from family Onchocercidae with occurrence in the northern hemisphere. They have a considerably wide range of final host, including many species of family Cervidae. Intermediate hosts and vectors at the same time, are represented by the several mosquito species, mostly of genus Aedes. Infection of Setaria is relatively harmless and especially in wild cervids usually pass unnoticed. Although in some cases it can induce peritonitis which might be a life threatening condition.This study was determined to reveal the presence of helminths Setaria tundra and Setaria cervi in red deer (Cervus elaphus) in Slovakia. The parasites were identified morphologically and genetically, based on the sequences of a fragment of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene. For this purpose we used partial results of our longer parasitological monitoring realized in one particular hunting area located in eastern Slovakia, near the city of Kosice. A total of 60 red deer individuals were tested, of which one was found to be infected with Setaria tundra (prevalence of 1.7%) and four were detected to be infected with Setaria cervi (prevalence 6.7%). The intensity of infection was very low, only one specimen of Setaria spp. in each positive animal.

The first report of Setaria tundra (Issaitshikoff & Rajewskaya, 1928) in Slovakia by using of molecular methods.

Setaria tundra is a filarioid parasite occurring in the northern hemisphere. Adult forms of helminths are located free in the peritoneal cavity of its definitive host - cervids, while microfilariae are presented in the host's bloodstream. Intermediate hosts are represented by several mosquito species, mainly of the genus Aedes.Nematode S. tundra is well adapted to roe deer (Capreolus capreolus) and therefore is the infection usually asymptomatic. In this study we present the first report of S. tundra in Slovakia. During a period 2022 a total of 6 roe deer coming from eastern Slovakia (Trebisov district) were examined. Nematodes were found during the evisceration process in the abdominal cavity of 3 specimens Intensity of infection was in range from 5 to 38 helminths per host. Mean intensity of infection reached 18.3 parasites per host. The helminths were identified as S. tundra by morphological examination and molecular typing of the COI gene. This study is the first report of S. tundra in Slovakia.

Morphological and molecular identification of adult Stichorchis subtriquetrus in beaver in Slovakia.

The European beaver (Castor fiber) was a fully eradicated species in Slovakia. Thanks to year-round protection and several different reintroduction programs the population is now increasing. However, there are limited reports about their health status.A 2-year-old female European beaver, was found dead by road near town Hanusovce nad Toplou in eastern Slovakia in 2021. Necropsy was carried out at the Department of Breeding and Diseases of the Game, Fish and Bees, Ecology and Cynology, University of Veterinary Medicine and Pharmacy in Kosice. Necropsy revealed a total of 13 trematodes, collected from the caecum and colon. Based on morphological and molecular analysis digenean trematode Stichorchis subtriquetrus (Rudolphi, 1814) was identified. This is the first record of adult helminth Stichorchis subtriquetrus in beaver in Slovakia.

Engineering the Single Domain Antibodies Targeting Receptor Binding Motifs Within the Domain III of West Nile Virus Envelope Glycoprotein.

West Nile virus (WNV) is a mosquito-borne neurotrophic flavivirus causing mild febrile illness to severe encephalitis and acute flaccid paralysis with long-term or permanent neurological disorders. Due to the absence of targeted therapy or vaccines, there is a growing need to develop effective anti-WNV therapy. In this study, single-domain antibodies (sdAbs) were developed against the domain III (DIII) of WNV's envelope glycoprotein to interrupt the interaction between DIII and the human brain microvascular endothelial cells (hBMEC). The peripheral blood mononuclear cells of the llama immunized with recombinant DIIIL297-S403 (rDIII) were used to generate a variable heavy chain only (VHH)-Escherichia coli library, and phage display was performed using the M13K07ΔpIII Hyperphages system. Phages displaying sdAbs against rDIII were panned with the synthetic analogs of the DIII receptor binding motifs, DIII-1G299-K307 and DIII-2V371-R388, and the VHH gene from the eluted phages was subcloned into E. coli SHuffle. Soluble sdAbs purified from 96 E. coli SHuffle clones were screened to identify 20 candidates strongly binding to the synthetic analogs of DIII-1G299-K307 and DIII-2V371-R388 on a dot blot assay. Among them, sdAbA1, sdAbA6, sdAbA9, and sdAbA10 blocked the interaction between rDIII and human brain microvascular endothelial cells (hBMECs) on Western blot and cell ELISA. However, optimum stability during the overexpression was noticed only for sdAbA10 and it also neutralized the WNV-like particles (WNV-VLP) in the Luciferase assay with an half maximal effective concentration (EC50) of 1.48 nm. Furthermore, the hemocompatibility and cytotoxicity of sdAbA10 were assessed by a hemolytic assay and XTT-based hBMEC proliferation assay resulting in 0.1% of hemolytic activity and 82% hBMEC viability, respectively. Therefore, the sdAbA10 targeting DIII-2V371-R388 of the WNV envelope glycoprotein is observed to be suitable for in vivo trials as a specific therapy for WNV-induced neuropathogenesis.

First molecular detection of Onchocerca flexuosa (Wedl, 1856) in red deer in Slovakia.

The present paper deals with the post-mortem diagnostics of onchocerciasis and the molecular detection of causative agents of this disease in wild ruminant ungulates (Cervus elaphus, Dama dama and Capreolus capreolus). The animals were shot in hunting seasons 2017 and 2018, in two regions of the Eastern Slovakia. The total number of examined skins was fifty-eight. The presence of subcutaneous nodules was confirmed in 27.59% (95% CI 16-39) of animals. All positive skins belonged to red deer individuals (47.06%; 95% CI 30-64). The nodules were present mainly in the back area and in the lumbar area, and their sizes ranged from 2.9 to 24.1 mm, with the average count of 10 nodules per animal. Thirteen worms, isolated from the nodules collected from 13 animals, were subjected to molecular identification. Applying the standard PCR method, targeting the mitochondrial 12S rRNA, 16S rRNA and NADH-dehydrogenase gene, and subsequent sequencing, all the worms were identified as Onchocerca flexuosa Wedl, 1856. The sequences were submitted to GenBank under specific accession numbers. Two samples were identified as Onchocerca flexuosa haplotype B, in which T176A and A177T were present. Despite the presence of mutations in the 12S rRNA of the Onchocerca flexuosa, the standardized PCR remains to be a very specific and sensitive method that uses this fragment as a selectable marker for the detection of the studied parasite.

Single Domain Antibodies Targeting Receptor Binding Pockets of NadA Restrain Adhesion of Neisseria meningitidis to Human Brain Microvascular Endothelial Cells.

Neisseria adhesin A (NadA), one of the surface adhesins of Neisseria meningitides (NM), interacts with several cell types including human brain microvascular endothelial cells (hBMECs) and play important role in the pathogenesis. Receptor binding pockets of NadA are localized on the globular head domain (A33 to K69) and the first coiled-coil domain (L121 to K158). Here, the phage display was used to develop a variable heavy chain domain (VHH) that can block receptor binding sites of recombinant NadA (rec-NadA). A phage library displaying VHH was panned against synthetic peptides (NadA-gdA33-K69 or NadA-ccL121-K158), gene encoding VHH was amplified from bound phages and re-cloned in the expression vector, and the soluble VHHs containing disulfide bonds were overexpressed in the SHuffle E. coli. From the repertoire of 96 clones, two VHHs (VHHF3-binding NadA-gdA33-K69 and VHHG9-binding NadA-ccL121-K158) were finally selected as they abrogated the interaction between rec-NadA and the cell receptor. Preincubation of NM with VHHF3 and VHHG9 significantly reduced the adhesion of NM on hBMECs in situ and hindered the traversal of NM across the in-vitro BBB model. The work presents a phage display pipeline with a single-round of panning to select receptor blocking VHHs. It also demonstrates the production of soluble and functional VHHs, which blocked the interaction between NadA and its receptor, decreased adhesion of NM on hBMECs, and reduced translocation of NM across BBB in-vitro. The selected NadA blocking VHHs could be promising molecules for therapeutic translation.

Dandelion (Taraxacum officinale) and Agrimony (Agrimonia eupatoria) as Indicators of Geogenic Contamination of Flysch Soils in Eastern Slovakia.

Contents of potentially toxic elements Fe, Mn, Cr, Ni, Co, V, Cu, and Mo were determined in common dandelion (Taraxacum officinale) and agrimony (Agrimonia eupatoria) to show their usefulness as bioindicators of geogenic soil pollution. Both plants were collected on geochemically anomalous soils developed on flysch sedimentary rocks (Paleogene) of Eastern Slovakia, which also are composed of weathered detritus of some ultramafic rocks. Generally, contents of the investigated association of potentially toxic elements are highly increased in these "serpentine"-like soils. Elevated concentrations were detected in both shoots and roots of the plants. The highest values, which exceed world average values for plants, were observed for Ni content. They ranged from 1.7 to 16.3 mg kg(-1) in dandelion and from 1.6 to 22.6 mg kg(-1) in agrimony. Essential elements, such as Mo, Cu, and Mn, were the most concentrated in plants, whereas Co, V, and Cr were the least concentrated. Although the bioindication value of the common dandelion for anthropogenic soil pollution is well known, it is not mentioned for agrimony in literature, and no data exist to indicate the geogenic pollution for both plants. Dandelion and agrimony are widely used as herbal drugs; therefore, our intention also was to point out another fact, namely, possible high uptake of potentially toxic elements by herbal plants growing on similar soils.

Anaplasma infections in ticks and reservoir host from Slovakia.

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.

Sensitivity of Borrelia genospecies to serum complement from different animals and human: a host-pathogen relationship.

Different Borrelia species and serotypes were tested for their sensitivity to serum complement from various animals and human. Complement-mediated Borrelia killing in cattle, European bison and deer was higher irrespective of the Borrelia species whereas in other animals and human it was intermediate and Borrelia species-dependent. Activation of the alternative complement pathway by particular Borrelia strain was in correlation with its sensitivity or resistance. These results support the incompetent reservoir nature of cattle, European bison, red, roe and fallow deer, at the same time present the probable reservoir nature of mouflon, dog, wolf, cat and lynx. In short, this study reviews Borrelia-host relationship and its relevance in reservoir competence nature of animals.