BACKGROUND: Parasites employ proteases to evade host immune systems, feed and replicate and are often the target of anti-parasite strategies to disrupt these interactions. Myxozoans are obligate cnidarian parasites, alternating between invertebrate and fish hosts. Their genes are highly divergent from other metazoans, and available genomic and transcriptomic datasets are limited. Some myxozoans are important aquaculture pathogens such as Sphaerospora molnari replicating in the blood of farmed carp before reaching the gills for sporogenesis and transmission. Proliferative stages cause a massive systemic lymphocyte response and the disruption of the gill epithelia by spore-forming stages leads to respiratory problems and mortalities. In the absence of a S. molnari genome, we utilized a de novo approach to assemble the first transcriptome of proliferative myxozoan stages to identify S. molnari proteases that are upregulated during the first stages of infection when the parasite multiplies massively, rather than in late spore-forming plasmodia. Furthermore, a subset of orthologs was used to characterize 3D structures and putative druggable targets. RESULTS: An assembled and host filtered transcriptome containing 9436 proteins, mapping to 29,560 contigs was mined for protease virulence factors and revealed that cysteine proteases were most common (38%), at a higher percentage than other myxozoans or cnidarians (25-30%). Two cathepsin Ls that were found upregulated in spore-forming stages with a presenilin like aspartic protease and a dipeptidyl peptidase. We also identified downregulated proteases in the spore-forming development when compared with proliferative stages including an astacin metallopeptidase and lipases (qPCR). In total, 235 transcripts were identified as putative proteases using a MEROPS database. In silico analysis of highly transcribed cathepsins revealed potential drug targets within this data set that should be prioritised for development. CONCLUSIONS: In silico surveys for proteins are essential in drug discovery and understanding host-parasite interactions in non-model systems. The present study of S. molnari's protease arsenal reveals previously unknown proteases potentially used for host exploitation and immune evasion. The pioneering dataset serves as a model for myxozoan virulence research, which is of particular importance as myxozoan diseases have recently been shown to emerge and expand geographically, due to climate change.

• Klíčová slova
• Aquaculture, Drug targets, In silico screening, Myxozoa, Parasite, Proteases,
• MeSH
• antiparazitární látky farmakologie   terapeutické užití   MeSH
• faktory virulence MeSH
• fylogeneze MeSH
• kapři mikrobiologie   MeSH
• Myxozoa genetika   růst a vývoj   MeSH
• nemoci ryb parazitologie   terapie   MeSH
• objevování léků MeSH
• parazitární nemoci u zvířat parazitologie   terapie   MeSH
• proteasy genetika   MeSH
• transkriptom MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antiparazitární látky MeSH
• faktory virulence MeSH
• proteasy MeSH

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

• Publikační typ
• tisková chyba MeSH

Myxozoans (Cnidaria: Myxozoa) are an extremely diversified group of endoparasites some of which are causative agents of serious diseases in fish. New methods involving gene expression studies have emerged over the last years to better understand and control myxozoan diseases. Quantitative RT-PCR is the most extensively used approach for gene expression studies. However, the accuracy of the results depends on the normalization of the data to reference genes. We studied the expression of eight commonly used reference genes, adenosylhomocysteinase (AHC1), beta actin (ACTB), eukaryotic translation elongation factor 2 (EF2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1), DNA-directed RNA polymerase II (RPB2), 18S ribosomal RNA (18S), 28S ribosomal RNA (28S) across different developmental stages of three myxozoan species, Sphaerospora molnari, Myxobolus cerebralis and Ceratonova shasta, representing the three major myxozoan linages from the largest class Myxosporea. The stable reference genes were identified using four algorithms: geNorm, NormFinder, Bestkeeper and ΔCq method. Additionally, we analyzed transcriptomic data from S. molnari proliferative and spore-forming stages to compare the relative amount of expressed transcripts with the most stable reference genes suggested by RT-qPCR. Our results revealed that GAPDH and EF2 are the most uniformly expressed genes across the different developmental stages of the studied myxozoan species.

• MeSH
• algoritmy MeSH
• messenger RNA genetika   metabolismus   MeSH
• Myxozoa genetika   MeSH
• Oncorhynchus mykiss parazitologie   MeSH
• paraziti genetika   MeSH
• referenční standardy MeSH
• regulace genové exprese * MeSH
• transkriptom genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• messenger RNA MeSH

OBJECTIVE: The aim of the study was to evaluate the seroprevalence of West Nile virus (WNV) among the variable population of Eastern Slovakia. METHODS: A serologic survey was conducted using 464 serum samples. The basic demographic, epidemiologic and clinical information was obtained for each serum sample at the time of specimen collection. The presence of antibodies against WNV was investigated using a commercial enzyme-linked immunosorbent assay (ELISA). All the ELISA positive samples were further analysed by a neutralization test with WNV and Usutu virus. RESULTS: Three serum samples (0.65%) from the participants (N = 464) were considered positive for antibodies to WNV. A 29-year-old female was repeatedly exposed to mosquito bites working as a shepherdess and participating in many outdoor activities. Two other females (61 and 76 years old) were treated at the Department of Neurology due to monoparesis of the upper extremity, vertigo; both had a significant epidemiological history with frequent tick and mosquito bites and stay in an endemic region. CONCLUSIONS: Although there was no evidence of WNV infection in the Slovak Republic, the epidemiological situation in the neighbouring countries warrants vigilance and appropriate measures, including the introduction of specific diagnostic tools into clinical practice. The constant monitoring of birds and mosquitoes also seems necessary.

• Klíčová slova
• West Nile virus, mosquitoes, seroprevalence,
• MeSH
• Culicidae MeSH
• ELISA MeSH
• protilátky virové MeSH
• séroepidemiologické studie MeSH
• virus západního Nilu izolace a purifikace   MeSH
• západonilská horečka diagnóza   epidemiologie   MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Slovenská republika epidemiologie   MeSH
• Názvy látek
• protilátky virové MeSH

Myxozoans (Cnidaria: Myxozoa) are almost exclusively endoparasites of aquatic vertebrates and invertebrates, with the notable exception being two species of Soricimyxum Prunescu, Prunescu, Pucek et Lom, 2007 described from terrestrial shrews (Soricidae) in central Europe. Myxospores of the two parasites are morphologically indistinguishable, but have SSU rDNA sequences that differ by about 4%. Herein, we report additional molecular and histology data from Soricimyxum fegati Prunescu, Prunescu, Pucek et Lom, 2007 from common shrew (Sorex araneus Linnaeus) from Hungary, and add a new geographic record for S. fegati in pygmy shrew (Sorex minutus Linnaeus) from Slovakia. A limited survey of shrews from the northern United States, Blarina brevicauda Say and Sorex sp. from New York, and Sorex spp. from Oregon, did not discover any infections, which is in stark contrast to the relatively high infection rates (up to 66%) in European shrew populations. We also provide a summary and discussion of literature records of species of Soricimyxum and a host survey. Given the lack of distinguishing morphological or morphometric characters between Soricimyxum spp., and the overlap in vertebrate hosts and geographic ranges, unambiguous identification of these closely related shrew parasites can presently only be achieved through sequence comparison of one or more variable SSU rDNA regions.

• Klíčová slova
• Central Europe, Eurasian pygmy shrew, Eurasian shrew, bile ducts, liver, mammal hosts, myxozoan infection,
• MeSH
• Myxozoa klasifikace   genetika   fyziologie   MeSH
• rejskovití parazitologie   MeSH
• ribozomální DNA genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Maďarsko MeSH
• Slovenská republika MeSH
• Názvy látek
• ribozomální DNA MeSH

The response of chicken to non-typhoidal Salmonella infection is becoming well characterised but the role of particular cell types in this response is still far from being understood. Therefore, in this study we characterised the response of chicken embryo fibroblasts (CEFs) to infection with two different S. Enteritidis strains by microarray analysis. The expression of chicken genes identified as significantly up- or down-regulated (≥3-fold) by microarray analysis was verified by real-time PCR followed by functional classification of the genes and prediction of interactions between the proteins using Gene Ontology and STRING Database. Finally the expression of the newly identified genes was tested in HD11 macrophages and in vivo in chickens. Altogether 19 genes were induced in CEFs after S. Enteritidis infection. Twelve of them were also induced in HD11 macrophages and thirteen in the caecum of orally infected chickens. The majority of these genes were assigned different functions in the immune response, however five of them (LOC101750351, K123, BU460569, MOBKL2C and G0S2) have not been associated with the response of chicken to Salmonella infection so far. K123 and G0S2 were the only 'non-immune' genes inducible by S. Enteritidis in fibroblasts, HD11 macrophages and in the caecum after oral infection. The function of K123 is unknown but G0S2 is involved in lipid metabolism and in β-oxidation of fatty acids in mitochondria.

• MeSH
• down regulace MeSH
• fibroblasty cytologie   metabolismus   mikrobiologie   MeSH
• kultivované buňky MeSH
• kur domácí genetika   metabolismus   MeSH
• kuřecí embryo MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• makrofágy metabolismus   mikrobiologie   MeSH
• nemoci drůbeže metabolismus   patologie   MeSH
• Salmonella enteritidis fyziologie   MeSH
• salmonelová infekce u zvířat metabolismus   patofyziologie   MeSH
• sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů MeSH
• transkriptom * MeSH
• upregulace MeSH
• zvířata MeSH
• Check Tag
• kuřecí embryo MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

During a regular veterinary inspection of fishes from Lake Balaton, Hungary, echinostomatid metacercariae (Digenea), with collar spines characteristic of species of the genera Petasiger Dietz, 1909 and Paryphostomum Dietz, 1909, were found in the lateral line scales of a roach Rutilus rutilus (Linnaeus), an apparently unique site. In a subsequent examination of 586 fishes from 20 different species, similar infections were found in 11 species. The infection was virtually restricted to the lateral line scales, other scales being infected only incidentally. These encysted metacercariae had 27 collar spines, including eight larger angle spines and 19 smaller dorsal spines arranged in two rows. Two types of metacercarial cyst were found. One type had a cyst diameter of 138-171 µm × 105-120 µm and three central dorsal spines that were larger than the remainder and tended to resemble the angle spines. The second type of metacercarial cyst had a diameter of 128-157 µm × 105-115 µm and all 19 dorsal spines of the metacercaria were of a similar size. ITS sequences of the second type of metacercaria exhibited a 100% similarity to sequences of two adult Petasiger phalacrocoracis (Yamaguti, 1939) specimens collected from the gut of Phalacrocorax carbo (Linnaeus) in Hungary and to P. phalacrocoracis deposited in the GenBank database. Sequences obtained from two metacercariae of the first type showed a 2.8-2.9 % difference from sequences of the second type of metacercaria and from those of adult specimens of P. phalacrocoracis from cormorants. Based on these results, the second type metacercaria is considered to be a larval stage of P. phalacrocoracis, but the identity of the first type is uncertain. The unusual location of these metacercariae in the lateral line scales is discussed in relation to their transmission.

• Klíčová slova
• Cyprinidae, Kis-Balaton Reservoir, Lake Balaton, metacercaria, molecular identification,
• Publikační typ
• časopisecké články MeSH