Polarisation of Major Histocompatibility Complex II Host Genotype with Pathogenesis of European Brown Hare Syndrome Virus.

A study was conducted in order to determine the occurrence of European Brown Hare Syndrome virus (EBHSV) in Denmark and possible relation between disease pathogenesis and Major Histocompatibility Complex (MHC) host genotype. Liver samples were examined from 170 brown hares (hunted, found sick or dead), collected between 2004 and 2009. Macroscopical and histopathological findings consistent with EBHS were detected in 24 (14.1%) hares; 35 (20.6%) had liver lesions not typical of the syndrome, 50 (29.4%) had lesions in other tissues and 61 (35.9%) had no lesions. Sixty five (38.2%) of 170 samples were found to be EBHSV-positive (RT-PCR, VP60 gene). In order to investigate associations between viral pathogenesis and host genotype, variation within the exon 2 DQA gene of MHC was assessed. DQA exon 2 analysis revealed the occurrence of seven different alleles in Denmark. Consistent with other populations examined so far in Europe, observed heterozygosity of DQA (H o = 0.1180) was lower than expected (H e = 0.5835). The overall variation for both nucleotide and amino acid differences (2.9% and 14.9%, respectively) were lower in Denmark than those assessed in other European countries (8.3% and 16.9%, respectively). Within the peptide binding region codons the number of nonsynonymous substitutions (dN) was much higher than synonymous substitutions (dS), which would be expected for MHC alleles under balancing selection. Allele frequencies did not significantly differ between EBHSV-positive and -negative hares. However, allele Leeu-DQA*30 was detected in significantly higher (P = 0.000006) frequency among the positive hares found dead with severe histopathological lesions than among those found sick or apparently healthy. In contrast, the latter group was characterized by a higher frequency of the allele Leeu-DQA*14 as well as the proportion of heterozygous individuals (P = 0.000006 and P = 0.027). These data reveal a polarisation between EBHSV pathogenesis and MHC class II genotype within the European brown hare in Denmark

Prevalence, Patterns, Association with Biofilm Formation, Effects on Milk Quality and Risk Factors for Antibiotic Resistance of Staphylococci from Bulk-Tank Milk of Goat Herds

The objectives of this work were to study the prevalence and the patterns of antibiotic resistance of staphylococcal isolates from bulk-tank milk of goat herds across Greece, to assess possible associations of the presence of antibiotic resistance with the quality of milk in these herds and to evaluate herd-related factors potentially associated with the presence of antibiotic resistance among these staphylococcal isolates. A cross-sectional study was performed on 119 goat herds in Greece. Bulk-tank milk samples were collected for bacteriological examination; staphylococcal isolates were evaluated for resistance to 20 antibiotics. Oxacillin-resistant, resistant to at least one antibiotic, and multi-resistant staphylococcal isolates were recovered from 5.0%, 30.3%, and 16.0% of herds, respectively. Of 80 isolates, 7.5% were resistant to oxacillin, 50.0% were resistant to at least one antibiotic and 27.5% were multi-resistant. Resistance was seen more frequently among coagulase-negative staphylococci (59.3%) than among Staphylococcus aureus (23.8%). Resistance was more frequent against penicillin and ampicillin (41.3% of isolates) and fosfomycin (27.5%). No association was found with biofilm formation by staphylococci. For recovery of oxacillin-resistant isolates, the presence of working staff in the herds emerged as a significant factor; respective factors for the isolation of staphylococci resistant to at least one antibiotic were part-time farming and high (>10) number of systemic disinfections in the farm annually. The same three factors concurrently were also identified to be significant for the recovery of multi-resistant isolates

MLST-Based Analysis and Antimicrobial Resistance of Staphylococcus epidermidis from Cases of Sheep Mastitis in Greece

Staphylococcus epidermidis is an important causal agent of ovine mastitis. A literature search indicated a lack of systematic studies of causal agents of the infection by using multi-locus sequence typing (MLST). The objectives were to analyse MLST-based data and evaluate the antimicrobial resistance of S. epidermidis isolates from ovine mastitis in Greece. The database included 1593 isolates from 46 countries: 1215 of human, 195 of environmental and 134 of animal origin, distributed into 949 sequence types (STs) and cumulatively with 450 alleles therein. Among mastitis isolates, bovine isolates were distributed into 36 different STs and ovine ones into 15 STs. The 33 isolates from ovine mastitis in Greece were in 15 different STs, 6 of these (ST677, ST678, ST700, ST 709, ST710, ST711) assigned for the first time; in addition, 5 alleles (65 for arcC, 59 for aroE, 56 and 57 for gtr and 48 for tpiA) were identified for the first time. The spanning tree of these isolates included 15 nodes and 14 edges (i.e., branches). Among these isolates, 19 showed resistance to antimicrobial agents (tetracycline, penicillin, fucidic adic, erythromycin, clindamycin, cefoxitin). Resistance-related genes (tetK, tetT, msrA, tetM, tetS, ermC, mecA) were detected. There was no association between STs and resistance to antimicrobial agents. Isolates with antimicrobial resistance were recovered more often from flocks where hand-milking was practised

DQA alleles (residues 8–76) of <i>Lepus europaeus.</i>

<p>DQA alleles (residues 8–76) of <i>Lepus europaeus.</i> Polymorphic residues are in bold. Residues contributing to the formation of binding pockets P1, P6 and P9 are indicated. Shaded residues are putative TCR contacts. Crosses indicate residues with hydrogen bonds to the peptide. The boxed area determines αβ pairing.</p

Groups of Danish brown hares individuals according to the presence of EBHSV and the variation in exon 2 DQA gene.

<p>Numbers (N) and percentages of Danish brown hares assessed for the four groups defined in this study: EBHSV negative (EBHSV⁻), EBHSV positive (EBHSV⁺), susceptible (EBHSV⁺ Dead), resistant (EBHSV⁺ not Dead).</p

Phylogenetic tree of the EBHSV isolates.

<p>Phylogenetic tree resulting from the Bayesian analysis, clustering the seven Danish isolates identified in this study combined with sequences available from the EMBL database. The topology of the clusters was similar for the NJ tree. Numbers on branches at the internodes of the clusters correspond to posterior probabilities from the Bayesian analysis. At the end of the branches the designation and origin of the EBHS viruses studied. Probabilities below 50% are omitted.</p

UPGMA phylogenetic tree of the eight <i>Lepus europaeus</i> DQA exon 2 alleles.

<p>UPGMA phylogenetic tree resulting from the analysis of the eight <i>Lepus europaeus</i> DQA exon 2 alleles identified in this study, together with sequences assessed in the study of Koutsogiannouli et al. 2009 available from GenBank. Numbers indicate the percentage bootstrap support (10000 replicates). The evolutionary distances were computed using the maximum composite likelihood method and are in the units of the number of base substitutions per site. The populations in which each allele was present are given abbreviated in parentheses. (The neighbour-joining tree produced similar results with comparable bootstrap values).</p

Percentages indicating the occurrence of the homogygous and heterozygous <i>DQA</i> genotypes.

<p>Percentages indicating the occurrence of the homogygous and heterozygous <i>DQA</i> genotypes in the four groups of <i>Lepus europaeus</i> sampled in Denmark. Bars indicate the occurrence of each genotype in non affected or affected animals. Pies above bars indicate the occurrence of each genotype among the affected <i>L. europeaus</i> individuals in susceptible animals found dead with lesions typical to the EBHS in various organs or resistant not dead animals.</p

Location map of EBHSV positive <i>Lepus europaeus</i> individuals.

<p>Location map of the 65 <i>Lepus europaeus</i> individuals sampled throughout Denmark that were affected by the virus of the European Brown Hare Syndrome (EBHSV).</p