Newly identified variability of the antigen binding site coding sequences of the equine major histocompatibility complex class I and class II genes.

The major histocompatibility complex (MHC) with its class I and II genes plays a crucial role in the immune response to pathogens by presenting oligopeptide antigens to various immune response effector cells. In order to counteract the vast variability of infectious agents, MHC class I and II genes usually retain high levels of SNPs mainly concentrated in the exons encoding the antigen binding sites. The aim of the study was to reveal new variability of selected MHC genes with a special focus on MHC class I physical haplotypes. Long-range NGS to was used to identify exon 2-exon 3 alleles in three genetically distinct horse breeds. A total of 116 allelic variants were found in the MHC class I genes Eqca-1, Eqca-2, Eqca-7 and Eqca-Ψ, 112 of which were novel. The MHC class II DRA locus was confirmed to comprise five exon 2 alleles, and no new sequences were observed. Additional variability in terms of 15 novel exon 2 alleles was identified in the DQA1 locus. Extensive overall variability across the entire MHC region was confirmed by an analysis of MHC-linked microsatellite loci. Both diversifying and purifying selection were detected within the MHC class I and II loci analyzed.

Complex variation in the KLRA (LY49) immunity-related genomic region in horses.

Natural killer (NK) cells play important roles in innate and adaptive immunity, as well as in the reproduction of placental mammals. Ly49 (KLRA) molecules represent a lectin-like type of NK cell receptor encoded within a complex genomic region, the NK cell complex. In rodents and horses, an expansion of the genes encoding Ly49 receptors leading to the formation of a gene family was observed. High sequence similarities and frequent high polymorphism of multiple family members represent an obstacle both for their individual identification and for annotation in the reference genomes of their respective species. Here, we focused on resolving complex variation of the KLRA gene family observed in domestic and Przewalski's horses. The KLRA (LY49) genomic region contains six genes (KLRA2-KLRA7) and one putative pseudogene, KLRA1. Two types of polymorphism were observed in the horses analyzed. Copy number variation between haplotypes was documented for the gene KLRA7 by polymerase chain reaction. As expected, the major source of variation of all KLRA genes, including KLRA7, is because of single nucleotide polymorphisms, many of them being nonsynonymous substitutions. Extensive allelic variability of the expanded KLRA (LY49) genes was observed. For four out of the six functional KLRA, high numbers of novel allelic amino acid sequence variants were identified in the genes studied, suggesting that this variation might be of functional importance, especially in the context of high polymorphism of their presumed ligands encoded by major histocompatibility complex class I genes. In fact, polymorphic amino acid sites were mostly found in the ligand-binding C-type lectin-like domain of the putative receptor molecule.

Fur color change and hormonal development in captive females of northern white-cheeked (Nomascus leucogenys) and buff-cheeked (Nomascus gabriellae) gibbons.

Gibbons of the genus Nomascus exhibit strong sexual dichromatism in fur color. Change of fur color in sub-adult wild Nomascus females is associated with the onset of puberty and the time of their dispersal. The variability in fur change may be influenced by social factors. In this study, we determined whether in captive females of crested gibbons begin reproductive maturity prior to dispersing and with association to their fur color. We collected 287 fecal extracts to analyze pregnandiol -3- glucuronide and 17ß estradiol profiles of 4 sub-adult females (Nomascus leucogenys and Nomascus gabriellae) and 183 samples from their mothers, using enzyme immunoassays. The sub-adult females were monitored from 4 years of age. Their hormone profiles showed the onset of ovulatory cycling between 4.6 and 5.8 years. Based on the information about the estrogen influence to the secondary sex characteristic (fur color of female) the positive link between estrogen concentration and age of the sub-adult females was found. However, the amount of the estrogen can apparently be influenced by the presence of mother. If the mother was presented, the level of estrogen was higher than if the mother was missing. Our findings suggest that the probability of changing to beige fur color by the sub-adult females increased with increased age and if they were without mother. This initial study presents the maternal influence as a possible social factor affecting the fur color change of female offspring.

Emerging and threatening vector-borne zoonoses in the world and in Europe: a brief update.

Climatic changes, landscape management, massive human, animal and commodity transportation represent important factors which are contributing to the spread of zoonotic diseases. The environmental and socioeconomic factors affecting the incidence of vector-borne zoonoses and possibilities for the reduction of disease impacts are discussed in the article. The most important zoonoses with expanding area of incidence and/or increasing occurrence are summarized, with special emphasis on the European region. While some diseases and their respective pathogens are indigenous to Europe (e.g. Lyme disease), others have been introduced to Europe from tropical areas (e.g. chikungunya or dengue fever). These emerging diseases may represent a serious threat in near future and better understanding of their spreading mechanisms, pathogenesis and consequent treatment is very important.

Genetic diversity and population structure of African village dogs based on microsatellite and immunity-related molecular markers.

The village and street dogs represent a unique model of canine populations. In the absence of selective breeding and veterinary care, they are subject mostly to natural selection. Their analyses contribute to understanding general mechanisms governing the genetic diversity, evolution and adaptation. In this study, we analyzed the genetic diversity and population structure of African village dogs living in villages in three different geographical areas in Northern Kenya. Data obtained for neutral microsatellite molecular markers were compared with those computed for potentially non-neutral markers of candidate immunity-related genes. The neutral genetic diversity was similar to other comparable village dog populations studied so far. The overall genetic diversity in microsatellites was higher than the diversity of European pure breeds, but it was similar to the range of diversity observed in a group composed of many European breeds, indicating that the African population has maintained a large proportion of the genetic diversity of the canine species as a whole. Microsatellite marker diversity indicated that the entire population is subdivided into three genetically distinct, although closely related subpopulations. This genetical partitioning corresponded to their geographical separation and the observed gene flow well correlated with the communication patterns among the three localities. In contrast to neutral microsatellites, the genetic diversity in immunity-related candidate SNP markers was similar across all three subpopulations and to the European group. It seems that the genetic structure of this particular population of Kenyan village dogs is mostly determined by geographical and anthropogenic factors influencing the gene flow between various subpopulations rather than by biological factors, such as genetic contribution of original migrating populations and/or the pathogen-mediated selection. On the other hand, the study of oldest surviving dogs suggested a biological mechanism, i.e. a possible advantage of the overal heterozygosity marked by the the microsatellite loci analyzed.

MYD88 and functionally related genes are associated with multiple infections in a model population of Kenyan village dogs.

The purpose of this study was to seek associations between immunity-related molecular markers and endemic infections in a model population of African village dogs from Northern Kenya with no veterinary care and no selective breeding. A population of village dogs from Northern Kenya composed of three sub-populations from three different areas (84, 50 and 55 dogs) was studied. Canine distemper virus (CDV), Hepatozoon canis, Microfilariae (Acantocheilonema dracunculoides, Acantocheilonema reconditum) and Neospora caninum were the pathogens studied. The presence of antibodies (CDV, Neospora), light microscopy (Hepatozoon) and diagnostic PCR (Microfilariae) were the methods used for diagnosing infection. Genes involved in innate immune mechanisms, NOS3, IL6, TLR1, TLR2, TLR4, TLR7, TLR9, LY96, MYD88, and three major histocompatibility genes class II genes were selected as candidates. Single nucleotide polymorphism (SNP) markers were detected by Sanger sequencing, next generation sequencing and PCR-RFLP. The Fisher´s exact test for additive and non-additive models was used for association analyses. Three SNPs within the MYD88 gene and one TLR4 SNP marker were associated with more than one infection. Combined genotypes and further markers identified by next generation sequencing confirmed associations observed for individual genes. The genes associated with infection and their combinations in specific genotypes match well our knowledge on their biological role and on the role of the relevant biological pathways, respectively. Associations with multiple infections observed between the MYD88 and TLR4 genes suggest their involvement in the mechanisms of anti-infectious defenses in dogs.

Positive selection in the SLC11A1 gene in the family Equidae.

Immunity-related genes are a suitable model for studying effects of selection at the genomic level. Some of them are highly conserved due to functional constraints and purifying selection, while others are variable and change quickly to cope with the variation of pathogens. The SLC11A1 gene encodes a transporter protein mediating antimicrobial activity of macrophages. Little is known about the patterns of selection shaping this gene during evolution. Although it is a typical evolutionarily conserved gene, functionally important polymorphisms associated with various diseases were identified in humans and other species. We analyzed the genomic organization, genetic variation, and evolution of the SLC11A1 gene in the family Equidae to identify patterns of selection within this important gene. Nucleotide SLC11A1 sequences were shown to be highly conserved in ten equid species, with more than 97 % sequence identity across the family. Single nucleotide polymorphisms (SNPs) were found in the coding and noncoding regions of the gene. Seven codon sites were identified to be under strong purifying selection. Codons located in three regions, including the glycosylated extracellular loop, were shown to be under diversifying selection. A 3-bp indel resulting in a deletion of the amino acid 321 in the predicted protein was observed in all horses, while it has been maintained in all other equid species. This codon comprised in an N-glycosylation site was found to be under positive selection. Interspecific variation in the presence of predicted N-glycosylation sites was observed.

Seroprevalence of Toxoplasma gondii and Encephalitozoon cuniculi in rabbits from different farming systems.

The breeding of domestic rabbit (Oryctolagus cuniculus) for human consumption has a long tradition mainly in European and Asian countries. Infections that can affect the production of meat or even be transmitted from animals to humans are important to monitor, especially for public health reasons as well as for their impact on animals health. This study aimed to collect sera from rabbits bred in different conditions and test the presence of Toxoplasma gondii and Encephalitozoon cuniculi antibodies. Whether infections were active or latent was assessed by determining the occurrence of IgM or IgM together with IgG antibodies which indicated active infection whereas latent infection was characterized by finding IgG antibodies only. An ELISA test was performed with 1883 sera samples collected throughout the Czech and Slovak Republics. The seroprevalence of T. gondii in 902 samples from 6 commercial farms (CF) was very low with only 4 rabbits (0.4%) being positive. In total 99 (10.1%) individuals out of 981 samples from 29 household farms (HF) were positive for T. gondii antibodies. Only 2 (50%) of the T. gondii positive CF rabbits had active infections while the rest were latently infected. The serological results showed that 35 (35.4%) rabbits from the T. gondii positive HF group suffered from active infection. Out of CF samples 185 (20.5%) were positive for E. cuniculi. Antibodies of E. cuniculi were detected in 497 (50.7%) HF rabbits. Active E. cuniculi infections were determined in 85.9% of CF and 56.3% of HF rabbits; respectively. Interestingly, the E. cuniculi positive rabbits were significantly more often positive for anti-T. gondii antibodies in comparison to E. cuniculi negative individuals. Prevalence of T. gondii in CF rabbits was negligible. According to our results meat of HF rabbits still poses a risk of T. gondii infection. Nevertheless, the risk is on its lowest level in 20 years which is apparently caused due to changes in feeding practices. The occurrence of E. cuniculi antibodies was significantly lower in rabbits from commercial farms, apparently because of better hygiene conditions.

Brain is the predilection site of Toxoplasma gondii in experimentally inoculated pigs as revealed by magnetic capture and real-time PCR.

Pigs represent an important source of food in many countries, and undercooked pork containing tissue cysts is one of the most common sources of Toxoplasma gondii infection for humans. A magnetic capture method for the isolation of T. gondii DNA and quantitative real-time PCR targeting the 529 bp TOXO repeat element were used to estimate the parasite burden in different tissues of pigs experimentally infected with T. gondii oocysts, and to determine the predilection sites of T. gondii in this host species. The highest concentration of T. gondii DNA was found in brain tissues, equivalent to [median] 553.7 (range 3857.7-121.9) parasites per gram, followed by lungs, heart and dorsal muscles with median values corresponding to 0.3 (range 61.3-0.02); 2.6 (range 7.34-0.37) and 0.6 (range 2.81-0.31) parasites per gram of tissue, respectively. Skeletal muscles from fore and hindlimb, liver and kidney presented very low infection burdens equivalent to [median] ≤0.2 parasites per gram of tissues, and no parasite DNA could be detected in the spleen. This study contributes to understanding the value of different pig tissues as a source of T. gondii infection for humans and shows that the brain, while not being of major importance as human food source, may represent a first-line selection tissue when performing non-serological surveys (e.g. bioassays, histopathological, immunohistochemical or molecular studies) to detect T. gondii infections in pigs.

Genetic diversity and conservation in a small endangered horse population.

The Old Kladruber horses arose in the 17th century as a breed used for ceremonial purposes. Currently, grey and black coat colour varieties exist as two sub-populations with different recent breeding history. As the population underwent historical bottlenecks and intensive inbreeding, loss of genetic variation is considered as the major threat. Therefore, genetic diversity in neutral and non-neutral molecular markers was examined in the current nucleus population. Fifty microsatellites, 13 single nucleotide polymorphisms (SNPs) in immunity-related genes, three mutations in coat colour genes and one major histocompatibility (MHC-DRA) gene were studied for assessing genetic diversity after 15 years of conservation. The results were compared to values obtained in a similar study 13 years ago. The extent of genetic diversity of the current population was comparable to other breeds, despite its small size and isolation. The comparison between 1997 and 2010 did not show differences in the extent of genetic diversity and no loss of allele richness and/or heterozygosity was observed. Genetic differences identified between the black and grey sub-populations observed 13 years ago persisted. Deviations from the Hardy-Weinberg equilibrium found in 19 microsatellite loci and in five SNP loci are probably due to selective breeding. No differences between neutral and immunity-related markers were found. No changes in the frequencies of markers associated with two diseases, melanoma and insect bite hypersensitivity, were observed, due probably to the short interval of time between comparisons. It, thus, seems that, despite its small size, previous bottlenecks and inbreeding, the molecular variation of Old Kladruber horses is comparable to other horse breeds and that the current breeding policy does not compromise genetic variation of this endangered population.

Detection of shrew-borne hantavirus in Eurasian pygmy shrew (Sorex minutus) in Central Europe.

Recently, it was found that not only rodents but also shrews are reservoir hosts of hantaviruses. In Central Europe, only Seewis virus, associated with the Eurasian common shrew (Sorex araneus), has been recognized until now. In the present report, tissue samples from shrews belonging to Crocidurinae and Soricinae subfamilies, trapped in Czech Republic, Germany, and Slovakia, were screened for the presence of novel hantaviruses. Three new hantavirus partial L-segment sequences were obtained from pygmy shrews (Sorex minutus) trapped in Czech Republic and Germany. Complete nucleocapsid protein- and glycoprotein precursor-coding S- and M-segment sequences were then determined for the newly recognized hantavirus strains, CZ/Beskydy/412/2010/Sm, CZ/Drahany/420/2010/Sm, and DE/Dürrbach/1912/2009/Sm. Phylogenetic analyses showed that they represent strains of Asikkala virus (ASIV), a novel hantavirus also found in pygmy shrews from Finland. Our study reveals a broad geographic distribution of ASIV across Europe and indicates pygmy shrew as the primary reservoir host. Future studies will have to determine the pathogenic relevance of ASIV.

Major histocompatibility complex and other allergy-related candidate genes associated with insect bite hypersensitivity in Icelandic horses.

Insect bite hypersensitivity (IBH) is an allergic dermatitis of horses caused by bites of insects. IBH is a multifactorial disease with contribution of genetic and environmental factors. Candidate gene association analysis of IBH was performed in a group of 89 Icelandic horses all born in Iceland and imported to Europe. Horses were classified in IBH-affected and non-affected based on clinical signs and history of recurrent dermatitis, and on the results of an in vitro sulfidoleukotriene (sLT)-release assay with Culicoides nubeculosus and Simulium vittatum extract. Different genetic markers were tested for association with IBH by the Fisher's exact test. The effect of the major histocompatibility complex (MHC) gene region was studied by genotyping five microsatellites spanning the MHC region (COR112, COR113, COR114, UM011 and UMN-JH34-2), and exon 2 polymorphisms of the class II Eqca-DRA gene. Associations with Eqca-DRA and COR113 were identified (p < 0.05). In addition, a panel of 20 single nucleotide polymorphisms (SNPs) in 17 candidate allergy-related genes was tested. During the initial screen, no marker from the panel was significantly (p < 0.05) associated with IBH. Five SNPs associated with IBH at p < 0.10 were therefore used for analysis of combined genotypes. Out of them, SNPs located in the genes coding for the CD14 receptor (CD14), interleukin 23 receptor (IL23R), thymic stromal lymphopoietin (TSLP) and transforming growth factor beta 3 (TGFB3) molecules were associated with IBH as parts of complex genotypes. These results are supported by similar associations and by expression data from different horse populations and from human studies.

Immunogenomic analysis of insect bite hypersensitivity in a model horse population.

Equine insect bite hypersensitivity (IBH) is a seasonal IgE-mediated dermatosis caused by bites of insects of the genus Culicoides. A familial predisposition for the disease has been shown but, except for the MHC, the genes involved have not been identified so far. An immunogenomic analysis of IBH was performed in a model population of Old Kladruby horses, all living in the same environment. Clinical signs of IBH were used as phenotypic manifestation of IBH. Furthermore, total serum IgE levels were determined in the sera of these horses and used as an independent phenotypic marker for the immunogenetic analysis. Single nucleotide polymorphisms (SNPs) in candidate immunity-related genes were used for association analyses. Genotypes composed of two to five genes encoding interferon gamma -IFNG, transforming growth factor beta 1 -TGFB1, Janus kinase 2 -JAK2, thymic stromal lymphopoietin -TSLP, and involucrin -IVL were associated with IBH, indicating a role of the genes in the pathogenesis of IBH. These findings were supported by analysis of gene expression in skin biopsies of 15 affected and 15 unaffected horses. Two markers associated with IBH, IFNG and TGFB1, showed differences in mRNA expression in skin biopsies from IBH-affected and non-affected horses (p<0.05). Expression of the gene coding for the CD14 receptor molecule -CD14 was different in skin biopsies at p<0.06. When total IgE levels were treated as binary traits, genotypes of IGHE, ELA-DRA, and IL10/b were associated with this trait. When treated as a continuous trait, total IgE levels were associated with genes IGHE, FCER1A, IL4, IL4R, IL10, IL1RA, and JAK2. This first report on non-MHC genes associated with IBH in horses is thus supported by differences in expression of genes known to play a role in allergy and immunity.

Rodent damage to natural and replanted mountain forest regeneration.

Impact of small rodents on mountain forest regeneration was studied in National Nature Reserve in the Beskydy Mountains (Czech Republic). A considerable amount of bark damage was found on young trees (20%) in spring after the peak abundance of field voles (Microtus agrestis) in combination with long winter with heavy snowfall. In contrast, little damage to young trees was noted under high densities of bank voles (Myodes glareolus) with a lower snow cover the following winter. The bark of deciduous trees was more attractive to voles (22% damaged) than conifers (8%). Young trees growing in open and grassy localities suffered more damage from voles than those under canopy of forest stands (χ² = 44.04, P < 0.001). Natural regeneration in Nature Reserve was less damaged compared to planted trees (χ² = 55.89, P < 0.001). The main factors influencing the impact of rodent species on tree regeneration were open, grassy habitat conditions, higher abundance of vole species, tree species preferences- and snow-cover condition. Under these conditions, the impact of rodents on forest regeneration can be predicted. Foresters should prefer natural regeneration to the artificial plantings.

Broad geographical distribution and high genetic diversity of shrew-borne Seewis hantavirus in Central Europe.

For a long time hantaviruses were believed to be exclusively rodent-borne pathogens. Recent findings of numerous shrew- and mole-borne hantaviruses raise important questions on their phylogenetic origin. The objective of our study was to prove the presence and distribution of shrew-associated Seewis virus (SWSV) in different Sorex species in Central Europe. Therefore, a total of 353 Sorex araneus, 59 S. minutus, 27 S. coronatus, and one S. alpinus were collected in Germany, the Czech Republic, and Slovakia. Screening by hantavirus-specific L-segment RT-PCR revealed specific amplification products in tissues of 49 out of 353 S. araneus and four out of 59 S. minutus. S-segment sequences were obtained for 45 of the L-segment positive S. araneus and all four L-segment positive S. minutus. Phylogenetic investigation of these sequences from Germany, the Czech Republic, and Slovakia demonstrated their similarity to SWSV sequences from Hungary, Finland, Austria, and other sites in Germany. The low intra-cluster sequence variability and the high inter-cluster divergence suggest a long-term SWSV evolution in isolated Sorex populations. In 28 of the 49 SWSV S-segment sequences, an additional putative open reading frame (ORF) on the opposite strand to the nucleocapsid protein-encoding ORF was identified. This is the first comprehensive sequence analysis of SWSV strains from Germany, the Czech Republic, and Slovakia, indicating its broad geographical distribution and high genetic divergence. Future studies have to prove whether both S. araneus and S. minutus represent SWSV reservoir hosts or spillover infections are responsible for the parallel molecular detection of SWSV in both species.

Tula virus in populations of small terrestrial mammals in a rural landscape.

Over 5 years (2000-2004), populations of small mammals from a rural landscape in southern Moravia (Czech Republic) were investigated for the presence of Tula virus (TULV) antigen using the ELISA set Hantagnost. In total, 1566 individuals from 10 species were examined. The prevalence in the common vole (Microtus arvalis Pallas 1778), the main reservoir of TULV, was 10% (n = 871). The prevalence of TULV antigen increases with its population numbers. The highest number of TULV antigen-positive common voles was found in set-aside plots and winter crops, such as rape and winter wheat. All these habitats are important for common vole overwintering. Older and heavier individuals were more often hantavirus antigen positive. From the other small mammal species, 186 pygmy field mice (Apodemus uralensis Pallas, 1811) were examined, of which 3 were positive, which represents the first hantavirus antigen positive record for this species, and of 195 wood mice (Apodemus sylvaticus Linnaeus, 1758) only 1 was positive. The remaining five rodent species (Apodemus flavicollis Melchior, 1834, Mus musculus Linnaeus, 1758, Micromys minutus Pallas, 1771, Myodes glareolus Schreber, 1780, Microtus subterraneus de Sélys-Longchamps, 1836) and two Soricomorpha (Sorex araneus Linnaeus, 1758, Sorex minutus Linnaeus, 1766) were hantavirus antigen negative.

Polymorphism and selection in the major histocompatibility complex DRA and DQA genes in the family Equidae.

The major histocompatibility complex genes coding for antigen binding and presenting molecules are the most polymorphic genes in the vertebrate genome. We studied the DRA and DQA gene polymorphism of the family Equidae. In addition to 11 previously reported DRA and 24 DQA alleles, six new DRA sequences and 13 new DQA alleles were identified in the genus Equus. Phylogenetic analysis of both DRA and DQA sequences provided evidence for trans-species polymorphism in the family Equidae. The phylogenetic trees differed from species relationships defined by standard taxonomy of Equidae and from trees based on mitochondrial or neutral gene sequence data. Analysis of selection showed differences between the less variable DRA and more variable DQA genes. DRA alleles were more often shared by more species. The DQA sequences analysed showed strong amongst-species positive selection; the selected amino acid positions mostly corresponded to selected positions in rodent and human DQA genes.

Age variation in a fluctuating population of the common vole.

We analysed variation in age in a fluctuating population of the common vole ( Microtus arvalis) in southern Moravia, Czech Republic, to test the assumption of the senescence hypothesis that the age of voles increases with increasing population density. Between 1996 and 1998, we monitored the demographic changes by snap-trapping and live-trapping in a field population passing through the increase, peak and decline phase of the population cycle. We used the eye lens mass method to determine the age of snap-trapped animals and those that died in live-traps. The average age of winter males was clearly higher after the peak phase breeding season than before it. No such phase-dependent shift in age, however, was observed in the female component. Male age continued to increase from autumn to spring over the pre-peak winter, and the highest age was in spring of the peak phase year. However, after the peak phase breeding season the highest age was achieved in winter, with the decline phase males during the next spring tending to be younger. The average age of females in spring populations was always lower than in winter populations. The average age of voles from live-traps was always higher than voles from snap-traps, particularly in winter and spring populations, suggesting the presence of senescent animals. Although the density-dependent changes in age are consistent with those observed for other voles, they provide only weak evidence that population cycles in the common vole are accompanied by pronounced shifts in individual age, particularly in female voles.