POLLÁKOVÁ, J., CSANK, T., PILIPČINCOVÁ, I., PISTL, J.:
Comparative study of various cell lines susceptibility to cytopathic and non-cytopathic strains of Bovine viral diarrhea virus 1 and 2. Acta Virol., 53, 287-289, 2009.

Bovine viral diarrhea virus 1 and 2 (BVDV 1 and 2) (the genus Pestivirus, the family Flaviviridae) is distributed worldwide and causes severe economical losses due to the decreased fertility, abortions, diarrhea, respiratory symptoms, and persistent infection in intrauterinary infected calves. The aim of this study was to compare the susceptibility of different cell lines derived from various tissues to non-cp and cp strains of BVDV. We examined five cell lines: Bovine embryonic lungs (BEL), bovine turbinated (BT), Madin-Darby bovine kidney (MDBK), calf oesopharyngeal (KOP-R), and sheep fetal thymus (SFT-R), which were inoculated with two cp BVDV strains (NADL and Oregon C24V) and two non-cp BVDV strains (PT810 and CS8644). According to titer of virus, the KOP-R cell line was the most susceptible for the isolation of cp BVDV strains and SFT-R and BEL cell lines for non-cp BVDV strains. Any of the cell cultures tested was universal for all BVDV strains. Paradoxically, the lowest harvest of all BVDV strains tested was found in the cell line MDBK, which is one of the most frequently used cell line for the isolation of BVDV.

PISTL, J., NOVÁČKOVÁ, M., JACKOVÁ, A., POLLÁKOVÁ, J., LEVKUT, M., VILČEK, Š.:
First evidence of porcine circovirus 2 (PCV2) in Slovakia, Deutsche Tierärztliche Wochenschrift, 116, 19-23, 2009.

Circoviruses belong to the family of small DNA viruses, which infect a number of animal species. The most important circovirus in swine is porcine circovirus type 2 (PCV2). The most frequent clinical form of circovirus infection in swine is the post-weaning multisystemic wasting syndrome (PMWS). Because Slovakia is one of the countries with unknown prevalence of PMWS, we focused on the detection of Slovak PCV2 isolates from pigs with PMWS-like symptoms (clinical symptoms, pathology) based on generally accepted scientific criteria (biological and molecular-genetic methods). The affected weaned pigs showed general symptoms characteristic for PMWS with pathological-anatomical changes particularly in lungs, lymph nodes and kidneys. Immunohistochemistry and PCR allowed us to prove PCV2 in cryosections of organs from infected animals. Immunoperoxidase test (IPMA) was used to confirm PCV2 in cells of cell culture PK-15 with the highest titre of the virus found in the lymph node tissue. Sequencing proved the specifity of PCR products. BLAST analysis indicated that nucleotide sequences of PCV2 amplified fragments were the most similar to the Austrian isolate of PCV2. This paper presents the first detection and isolation of PCV2 in Slovakia, which was confirmed by laboratory methods at antigenic and genetic level.

VILČEK, Š., VLASÁKOVÁ, M., JACKOVÁ, A.:
LUX real-time PCR assay for the detection of porcine circovirus type 2. J. Virol. Methods 165, 216-221, 2010.

Light Upon eXtension real time PCR (LUX real time PCR) assay was developed for the detection of porcine circovirus type 2 (PCV2). The primers flanking a 114 bp fragment were selected from ORF1. The optimized assay could detect 20 viral copies of pBluescript SK+ plasmid containing inserted PCV2 DNA. The dynamic range of quantitative analysis covered a 7-order interval ranging from 20 to 2.108 genome equivalents per assay with the best result in the range from 2.102 to 2.107 viral copies. The LUX real time PCR assay had a high specificity since it detected PCV2 but not PCV1, CSFV, PRRSV or negative samples. There was good agreement between LUX real time PCR and conventional PCR when lymph nodes from PCV2 infected animals were tested. A comparison of LUX real time PCR with TaqMan PCR and SYBR Green PCR indicated that the amount of viral copies determined using linear calibration curve differed from assay to assay but not more than an order. LUX real time PCR, similar as TaqMan PCR, was more specific in generation of fluorogenic signal than SYBR Green PCR.

GOLDOVÁ, M., TÓTH, Š., LETKOVÁ, V., MOJŽIŠOVÁ, J., KOŽAROVÁ, I., POMFY, M.:
Comparison of the histological methods in the diagnostic of deer cysticercosis. Helmintologia, 45, 121 – 125, 2008.

Histochemical methods for the detection and diagnosis of the developmental stages of the canine tapeworm, from the genus Taenia found in the heart and lungs of red deer (Cervus elaphus) and roe deer (Capreolus capreolus) hunted in Eastern Slovakia, is presented here. Detailed morphology of cysticerci (Cysticercus spp.), based on microscopic and histochemical analysis is described. For confirmation and demonstration of PAS-positive substances in the body of parasitic tissue (tegument and mesenchyme) the McManus - PAS method was used. The histochemical method according to Van Kossa was very effective for confirmation of calcareous corpuscles, which are one of the most important histological markers of cestode tissues (larva or adult).

JACKOVÁ, A., NOVÁČKOVÁ, M., PELLETIER, C., AUDEVAL, C., GUENEAU, E., HAFFAR, A., PETIT, E., REHBY, L., VILČEK, Š.:
The extended genetic diversity of BVDV-1: Typing of BVDV isolates from France. Vet. Res. Commun., 32, 7-11, 2008.

Of 47 BVDV-1 isolates grouped in the 5′-UTR phylogenetic tree, 9 isolates fall into BVDV-1b, 2 isolates into BVDV-1d and 33 isolates into the BVDV-1e group. Three isolates formed a new phylogenetic group indicating new, the twelfth BVDV subtype “l” - BVDV-1l. The typing of viruses in 5′-UTR was also confirmed in Npro region with 14 selected isolates, including all 3 isolates grouped in BVDV-1l cluster. Our study demonstrates that the analysis of broader collection of BVDV isolates may result in the higher genetic diversity of BVDV-1 and new viral subtypes could be identified. The study of genetic diversity of BVDV isolates is useful for the understanding of pestivirus evolution as well as for molecular epidemiology.

VLASÁKOVÁ, M., JACKOVÁ, A., VILČEK, Š.:
Genetic typing of porcine circovirus type 1 (PCV-2) isolates from Slovakia. Res. Vet. Sci., 90, 168-173, 2011.

Of 120 clinical specimens obtained from pigs breed on 28 PMWS-affected farms in Slovakia, PCV-2 DNA was detected by single PCR in 77 samples. A short 224 bp fragment of ORF2 was used for preliminary grouping of isolates by phylogenetic analysis. Nucleotide sequences of the entire ORF2 region provided more precise genetic typing and segregation of preselected isolates (n=10) into two known PCV-2a (n=1) and PCV-2b (n=9) genotypes. Recognising of the complete genome sequences of three selected isolates was useful for their definitive grouping into genotypes PCV-2b, cluster 1A and PCV2a, cluster 2D. No correlation between the mutations and geographic origin of isolates was observed.

LEVKUT, M., REVAJOVÁ, V., LEVKUTOVÁ, M., ŠEVÍČKOVÁ, Z., HERICH, R., BORUTOVÁ,
R., LENG, L.:
Leukocytic responses of broilers following dietary contamination with deoxynivalenol and/or treatment by dietary selenium supplementation. Brit. Poult. Sci., 50, 181-187, 2009.

1. This experiment was to investigate the effects of natural dietary contamination with a mycotoxin product (deoxynivalenol: DON) and/or with dietary selenised yeast (Se-yeast), on respiratory burst and phagocytic activity of granulocytes and the frequency of B- and T-lymphocytes in peripheral blood of broilers.

2. Sixty one-day-old chicks of both sexes were divided into 4 groups, each of 15 birds, fed on a control diet that contained 0.2mgDON/kg and 0.4mg Se/kg (CON group), a diet supplemented with 1mg Se-yeast/kg (Se-yeast group), a diet contaminated with 3mgDON/kg (DON group) or a diet contaminated with DON and supplemented with Se-yeast (DON plus Se-yeast group).

3. Blood samples collected from the birds at the age of 4 weeks showed that neither B- and T-cell numbers nor granulocytic respiratory burst were influenced by 3mgDON/kg. Blood granulocyte phagocytic activity was not reduced by DON but numbers of heterophils were increased. In the DON plus Se yeast group phagocytic activity was the same as in the CON group. The Se-yeast and DON plus Se-yeast groups had increased numbers of CD3+, CD4+, and CD8+ T-cells as well as IgM+ B-cells in their blood compared to both CON and DON-groups.

4. The results show there is no significant effect of dietary DON up to 3mg/kg on leukocytes apart from the compromised blood granulocytes phagocytic activity and increased numbers of heterophils. The increased numbers of B- and T-lymphocytes in blood of birds fed on diets with supplementation of organic Se indicates some positive effects of this essential microelement on poultry lymphoid cells.

KRÁĽOVÁ-HROMADOVÁ, I., ŠTEFKA, J., ŠPAKULOVÁ, M., OROSOVÁ, M., BOMBAROVÁ, M., HANZELOVÁ, V., BAZSALOVICSOVÁ, E., SCHOLZ, T.:
Intra-individual internal transcribed spacer 1 (ITS1) and ITS2 ribosomal sequence variation linked with multiple rDNA loci: A case of triploid Atractolytocestus huronensis, the monozoic cestode of common carp. Int. J. Paras., 40, 175-181, 2010.

Complete sequences of the ribosomal internal transcribed spacers (ITS1 and ITS2) and karyological characters of the monozoic (unsegmented) tapeworm Atractolytocestus huronensis Anthony, 1958 (Cestoda: Caryophyllidea) from Slovakia were analysed, revealing considerable intra-genomic variability and triploidy in all analysed specimens. Analysis of 20 sequences of each ITS1 and ITS2 spacer yielded eight and 10 different sequence types, respectively, In individual tapeworms, two to four ITS1 and three to four ITS2 sequence types were found. Divergent intra-genomic ITS copies were mostly induced by nucleotide substitutions and different numbers of short repetitive motifs within the sequence. in addition, triploidy was found to be a common feature of A. huronensis. The karyotype of Slovakian A. huronensis possesses three sets of chromosomes (3n = 24, n = 4m + 3st + 1 minute chromosome), similar to the previously described triploicly in conspecific tapeworms from North America. Fluorescent in situ hybridisation (FISH) with a ssrDNA probe revealed two distinct rDNA clusters for each homologue of the triplet number 2. To date, A. huronensis is the only cestode species in which intra-individual ITS sequence variants were found in parallel with its triploid nature and multiple rDNA loci. Some of these molecular and genetic features were observed in several other species of basal or nearly basal tapeworms of the orders Caryophyllidea and Diphyllobothriidea, which indicates that the phenomena may be characteristic for evolutionarily lower tapeworms and deserve more attention in future studies.

HRČKOVÁ, G., VELEBNÝ, S., SOLÁR, P.:
Dynamics of hepatic stellate cells, collagen types I and III synthesis and gene expression of selected cytokines during hepatic fibrogenesis following Mesocestoides vogae (Cestoda) infection in mice. Int. J. Paras., 40, 163-174, 2010.

In the present Study, the relationship between progression of Mesocestoides vogae infection in the liver of mice, the accumulation rate of collagen types I and III, gene expression of fibrogenic factors and cytokines was examined within 6 weeks p.i. Due to asexual multiplication, the total number of larvae in the liver increased considerably and 63.4% were found in collagen Capsules on day 42 p.i. Intense staining for both collagens was recorded in the activated hepatic stellate cells (HSCs) throughout the period of this study in the inflammatory lesions. With progressing infection, cellular expression of both collagens was confined to the flat cells, myofibroblasts, which were scattered among collagen fibres in parenchymal lesions and capsules. Collagen-positive areas mirrored immunostaining of alpha-smooth muscle actin (alpha-SMA) in HSCs and myofibroblasts. Gene expression of both collagens increased rapidly within 14 days p.i. and their expression pattern resembled that for pro-fibrotic cytokine transforming growth factor (TGF)-beta 1 and alpha-SMA protein. IL-10 cytokine expression was up-regulated following day 14 p.i. and that of IL-13 was up-regulated early p.i., then transcription elevated gradually mirroring the activity of other pro-fibrotic markers. In contrast, transcription activity of TNF-alpha and IFN-gamma was elevated shortly after infection, followed by the partial down-regulation of gene expression, indicating the lack of larval killing, enhanced granulomatous inflammation and the perpetuation of hepatic fibrosis. Histomorphometric analysis of the parenchymal fibrous lesions, surface areas of larvae surrounded with the inflammatory infiltrates and surface areas of developing or mature larva-containing granulomas, correlated with the proportion of free and encapsulated larvae, immunostaining and gene expression patterns of collagens and profibrotic markers. At a later stage of infection (day 28 p.i. onwards) collagen I-positive areas occupied a greater surface area and formed mature larval capsules and scars in the liver. In contrast, collagen III was less abundant and was localised mainly in the fibrous lesions in damaged parenchyma, Suggesting their specific up-regulation as the part of host-protecting and tissue-healing responses.

BRNA, M., REVAJOVÁ, V., ŽITŇAN, R., LEVKUT Jr., M., BARAN, M., LEVKUT, M.:
Changes in integrin-positive cells and T cell subpopulations in the peripheral blood and intestine of calves fed soya protein. J. Anim. Feed Sci., 19, 358-367, 2010.

In order to examine the relation of known intestinal lesions to changes in T-cell phenotypes and integrin expression, 16 male 10-day-old Holstein calves were divided into two groups. For 28 days of the experiment, eight males were fed NutriMilk in which 50% of the crude protein was soya protein, and eight control animals, with NutriMilk containing only milk casein. The animals fed soya protein showed shorter jejunal villi with a corrugated surface and deeper crypts compared with the control calves. A higher density of CD8+ cells in the intestinal mucosa and a decrease of these cells in peripheral blood were found in calves fed soya protein. The number of CD11b-positive cells was decreased in the peripheral blood of calves fed soya protein. Lower expression of integrin could be related to the appearance of non-mature polymorphonuclear cells. It is not clear if the decrease in CD11b expression on blood cells could also be influenced by milk replacer, i.e. soya protein.

OBERHAUSEROVÁ, K., BAZSALOVICSOVÁ, E., KRÁĽOVÁ - HROMADOVÁ, I., MAJOR, P., REBLANOVÁ, M.:
Molecular discrimination of eggs of cervid trematodes using the Teflon (PTFE) technique for eggshell disruption. Helminthologia, 47, 147-151, 2010.

Molecular comparative analysis of eggs of four liver and stomach flukes of cervids and domestic ruminants, Fasciola hepatica, Fascioloides magna, Dicrocoelium dendriticum and Paramphistomum cervi, was performed using a new methodological approach for eggshell disintegration. Eggs of all species were crushed mechanically by the Teflon method (PTFE) without use of chemical reagents and an efficient disruption of eggshell was checked microscopically. The egg suspension was then subjected to DNA isolation and PCR amplification using species-specific primers that annealed to the internal transcribed spacer 2 (ITS2) region of ribosomal DNA. The size of PCR products of individual species corresponded well to the size of amplicons obtained from adult flukes. The results provided evidence that the Teflon method does not destroy the structure of egg DNA, thus making the procedure broadly applicable during coprological examinations. Molecular markers introduced here are particularly important for blanket screening and differentiation of morphologically hardly distinguishable F. hepatica, F. magna and P. cervi eggs.

HREŠKO, S., MOJŽIŠ, M., TKÁČIKOVÁ, Ľ.:
Prion protein gene polymorphism in healthy and BSE-affected Slovak cattle. J. Appl. Genet. 50, 371–374, 2009.

Variation of the PrP gene was examined in healthy and BSE-affected Slovak cattle. According to previous studies, the 23-bp indel polymorphism is supposed to be associated with higher susceptibility to BSE. We investigated 301 samples from healthy cattle of various Slovak breeds and 24 samples obtained from tissues of BSE-affected cattle in Slovakia. We examined the PrP gene for the 23-bp indel polymorphism in the putative promoter region, 12-bp indel polymorphism in the first intron of the PrP gene, variations in number of octapeptide repeat units, and presence of the silent AAC›AAT transition in codon 192 within the protein-coding region of the PrP gene. Altogether we found 23 different genotypes in the group of healthy cattle and only 6 genotypes in the group of BSE-affected cattle. Comparison of homozygotes for the 23-bp insertion and heterozygotes showed significant differences (P < 0.05) in genotype distribution between the examined groups. Thereby the homozygous insertion genotype at the 23-bp indel polymorphism site in the promoter region of the prion protein gene seems to have a protective effect against BSE.

VILČEK, Š., WILLOUGHBY, K., NETTLETON, P., BECHER, P.:
Complete genomic sequence of a border disease virus isolated from Pyrenean chamois. Virus Res., 152, 164-168, 2010.

Pestivirus infections occur in cattle, sheep, pigs and numerous other species within the Artiodactyla. Here we report analysis of the full-length genome sequence of the pestivirus strain H2121 which was recently isolated from Pyrenean chamois and typed as border disease virus (BDV) genotype 4. Comparison with full-length genomic sequences of the approved pestivirus species Bovine viral diarrhea virus-1 (BVDV-1), BVDV-2, BDV, and Classical swine fever virus, as well as pestivirus strains Giraffe-1 and Th_04 confirmed that the chamois pestivirus strain is most similar to BDV. The viral genome of H2121 is 12,305 nucleotides long and contains one large open reading frame. The latter encodes a polyprotein consisting of 3,899 amino acids and is flanked with 376 nucleotides long 5´ untranslated region (UTR) and 229 nt long 3´ UTR. The genome organization of the chamois virus is reminiscent to that of other pestiviruses. Compared to other BDV strains including BDV-1 strain X818 and BDV-2 strain Reindeer-1, the 5´ UTR and ORF of the chamois virus are very similar in length, while the 3´ UTR of H2121 is 31 to 44 nucleotides shorter. In contrast to other BDV strains, the genome of the chamois virus contains a unique four amino acid insertion at the N-terminus of NS2. Apart from these differences, no further insertion/deletion or genetic rearrangement were present in the genome of the chamois pestivirus.

MIKULA, I. Jr., BHIDE, M. R., PASTOREKOVÁ, S., MIKULA, I.:
Characterization of ovine TLR7 and TLR8 protein coding regions, detection of mutations and Maedi Visna virus infection. Vet. Immunol. Immunopathol., 138, 51-59, 2010.

Toll-like receptors (TLRs) 2, 3, 4, 7, 8 and 9 play a crucial role in the recognition of viral entities and modulation of the innate immune system. This work presents sequence analysis of ovine TLR7 and TLR8 genes, depicts novel mutations and describes frequencies of mutations in Maedi Visna infected and healthy sheep. Totally 48 samples of the breed Tsigai were analyzed for the presence of mutations. Within 20 mutations, 14 were silent whereas 6 were missense. The frequencies of missense mutations in the Maedi Visna infected compared to non-infected sheep were: Lys115Glu (P - 0.766, F-test), Asn117 (P - 0.380) and Lys818Arg (P - 0.739). These three mutations were localized in extra LRR (lucine rich repeat) region of TLR7, while mutation Ile73Leu (P - 0.498) was located within LRR2 motif. Both mutations in TLR8, Asn165Lys (P - 1.0) and Tyr349His (P - 0.700), were present in extra LRR region. The secondary structure analysis of ovine TLR7 and TLR8 revealed conserved LRR motif structure, however with some irregularities compared to cattle and human. Transmembrane domains of TLR7 and TLR8 showed 100% homology between sheep and cattle wherein no mutations were found. In both TLRs TIR domains were highly conserved with occurrence of 4 silent mutations. Mutations in TLR7 and TLR8 may play an important role as predisposition factor for Maedi Visna infection. Considering the sequence homology among sheep, cattle and human genes encoding TLR7 and TLR8, we predict their similar function, localization and downstream signaling.

MUCHA, R., BHIDE, M. R., CHAKURKAR, E. B., NOVÁK, M., MIKULA, I.:
Toll-like receptors TLR1, TLR2 and TLR4 gene mutations and natural resistance to Mycobacterium avium subsp. paratuberculosis infection in cattle. Vet. Immunol. Immunopathol., 128, 381-388, 2009.

Toll like receptors (TLRs) are a class of pattern recognition receptors belonging to the innate immune system. Mutations in the protein coding region of TLRs are associated with altered responsiveness to pathogen-associated molecular patterns (PAMPs). A search was performed for novel mutations in bovine TLR1, TLR2 and TLR4 genes associated with the Mycobacterium avium subsp. paratuberculosis (MAP) infection. The work was also focused on the assessment of linkage between well known mutations in TLR genes (TLR2: Arg677Trp, Pro681His and Arg753Gln; TLR4: Asp299Gly and Thr399Ile), and the susceptibility of cattle to MAP infection. Detection of MAP infection in cattle population (n = 711) was based on IS900 PCR, which revealed 22.50% (n = 160) MAP positivity. Known mutations in TLR2 and TLR4 genes were not found in cattle population. A novel mutation Val220Met was associated (Odd's ratio, OR-3.459) with increased susceptibility to MAP infection. Toll/interleukin-1 receptor (TIR) domain of TLR2 was screened for the presence of mutations, wherein a novel Ile680Val mutation was linked with MAP infection. In silico analysis of the bovine TLR4 ectodomain (ECD) revealed the polymorphic nature of the central ECD and irregularities in the central LRR motifs. LRR11 of the TLR4 showed five missense mutations possibly linked with the increased susceptibility to MAP infection. The most critical position that may alter the pathogen recognition of TLR molecule was 4th residue downstream to LRR domain. Two such missense mutations in TLR4 (Asp299Asn downstream to LRR11, and Gly389Ser downstream to LRR15) were associated with MAP infection. Briefly, the work describes novel mutations in the bovine TLRs and presents their association with the MAP infection.

BHIDE, M. R., MUCHA, R., MIKULA Jr., I., KIŠOVÁ, L., ŠKRABANA, R., NOVÁK, M., MIKULA, I.:
Novel mutations in TLR genes cause hyporesponsiveness to Mycobacterium avium subsp. paratuberculosis infection. BMC Genetics, 10, art. no. 21, 2009.

Background: Toll like receptors (TLR) play the central role in the recognition of pathogen associated molecular patterns (PAMPs). Mutations in the TLR1, TLR2 and TLR4 genes may change the ability to recognize PAMPs and cause altered responsiveness to the bacterial pathogens. Results: The study presents association between TLR gene mutations and increased susceptibility to Mycobacterium avium subsp. paratuberculosis (MAP) infection. Novel mutations in TLR genes (TLR1- Ser150Gly and Val220Met; TLR2 - Phe670Leu) were statistically correlated with the hindrance in recognition of MAP legends. This correlation was confirmed subsequently by measuring the expression levels of cytokines (IL-4, IL-8, IL-10, IL-12 and IFN-γ) in the mutant and wild type moDCs (mocyte derived dendritic cells) after challenge with MAP cell lysate or LPS. Further in silico analysis of the TLR1 and TLR4 ectodomains (ECD) revealed the polymorphic nature of the central ECD and irregularities in the central LRR (leucine rich repeat) motifs. Conclusion: The most critical positions that may alter the pathogen recognition ability of TLR were: The 9th amino acid position in LRR motif (TLR1-LRR10) and 4th residue downstream to LRR domain (exta-LRR region of TLR4). The study describes novel mutations in the TLRs and presents their association with the MAP infection.

PULZOVÁ, L., BHIDE, M. R., ANDREJ, K.:
Pathogen translocation across the blood-brain barrier. FEMS Immunol. Med. Microbiol., 57, 203-213, 2009.

Neurological manifestations caused by neuroinvading pathogens are typically attributed to penetration of the blood-brain barrier (BBB) and invasion of the central nervous system. However, the mechanisms used by many pathogens (such as Borrelia) to traverse the BBB are still unclear. Recent studies revealed that microbial translocation across the BBB must involve a repertoire of microbial-host interactions (receptor-ligand interactions). However, the array of interacting molecules responsible for the borrelial translocation is not yet clearly known. Pathogens bind several host molecules (plasminogen, glycosaminoglycans, factor H, etc.) that might mediate endothelial interactions in vivo. This review summarizes our current understanding of the pathogenic mechanisms involved in the translocation of the BBB by neuroinvasive pathogens.