Associations between the presence of specific antibodies to the West Nile Virus infection and candidate genes in Romanian horses from the Danube delta.

The West Nile virus (WNV) is a mosquito-borne flavivirus causing meningoencephalitis in humans and animals. Due to their particular susceptibility to WNV infection, horses serve as a sentinel species. In a population of Romanian semi-feral horses living in the Danube delta region, we have analyzed the distribution of candidate polymorphic genetic markers between anti WNV-IgG seropositive and seronegative horses. Thirty-six SNPs located in 28 immunity-related genes and 26 microsatellites located in the MHC and LY49 complex genomic regions were genotyped in 57 seropositive and 32 seronegative horses. The most significant association (pcorr < 0.0002) was found for genotypes composed of markers of the SLC11A1 and TLR4 genes. Markers of five other candidate genes (ADAM17, CXCR3, IL12A, MAVS, TNFA), along with 5 MHC class I and LY49-linked microsatellites were also associated with the WNV antibody status in this model horse population. The OAS1 gene, previously associated with WNV-induced clinical disease, was not associated with the presence of anti-WNV antibodies.

Worldwide frequency distribution of the 'Gait keeper' mutation in the DMRT3 gene.

For centuries, domestic horses have represented an important means of transport and served as working and companion animals. Although their role in transportation is less important today, many horse breeds are still subject to intense selection based on their pattern of locomotion. A striking example of such a selected trait is the ability of a horse to perform additional gaits other than the common walk, trot and gallop. Those could be four-beat ambling gaits, which are particularly smooth and comfortable for the rider, or pace, used mainly in racing. Gaited horse breeds occur around the globe, suggesting that gaitedness is an old trait, selected for in many breeds. A recent study discovered that a nonsense mutation in DMRT3 has a major impact on gaitedness in horses and is present at a high frequency in gaited breeds and in horses bred for harness racing. Here, we report a study of the worldwide distribution of this mutation. We genotyped 4396 horses representing 141 horse breeds for the DMRT3 stop mutation. More than half (2749) of these horses also were genotyped for a SNP situated 32 kb upstream of the DMRT3 nonsense mutation because these two SNPs are in very strong linkage disequilibrium. We show that the DMRT3 mutation is present in 68 of the 141 genotyped horse breeds at a frequency ranging from 1% to 100%. We also show that the mutation is not limited to a geographical area, but is found worldwide. The breeds with a high frequency of the stop mutation (>50%) are either classified as gaited or bred for harness racing.

Twelve toll-like receptor (TLR) genes in the family Equidae - comparative genomics, selection and evolution.

Toll-like receptors (TLRs) represent an important part of the innate immune system. While human and murine TLRs have been intensively studied, little is known about TLRs in non-model species. The order Perissodactyla comprises a variety of free-living and domesticated species exposed to different pathogens in different habitats and is therefore suitable for analyzing the diversity and evolution of immunity-related genes. We analyzed TLR genes in the order Perissodactyla with a focus on the family Equidae. Twelve TLRs were identified by bioinformatic analyses of online genomic resources; their sequences were confirmed in equids by genomic DNA re-sequencing of a panel of nine species. The expression of TLR11 and TLR12 was confirmed in the domestic horse by cDNA sequencing. Phylogenetic reconstruction of the TLR gene family in Perissodactyla identified six sub-families. TLR4 clustered together with TLR5; the TLR1-6-10 subfamily showed a high degree of sequence identity. The average estimated evolutionary divergence of all twelve TLRs studied was 0.3% among the Equidae; the most divergent CDS were those of Equus caballus and Equus hemionus kulan (1.34%) in the TLR3, and Equus africanus somaliensis and Equus quagga antiquorum (2.1%) in the TLR1 protein. In each TLR gene, there were haplotypes shared between equid species, most extensively in TLR3 and TLR9 CDS, and TLR6 amino acid sequence. All twelve TLR genes were under strong negative overall selection. Signatures of diversifying selection in specific codon sites were detected in all TLRs except TLR8. Differences in the selection patterns between virus-sensing and non-viral TLRs were observed.

Effect of genetic variation in the MHC Class II DRB region on resistance and susceptibility to Johne's disease in endangered Indian Jamunapari goats.

The pathogenesis of Johne's disease (JD), caused by Mycobacterium avium subsp. paratuberculosis (MAP), is complex and has not been completely understood yet. In the present study, we analysed the polymorphism in the exon-2 of the caprine major histocompatibility complex (MHC) Class II DRB region and its association with resistance or susceptibility to JD. A total of 203 Jamunapari goats, which is an Indian endangered breed highly susceptible to JD, kept at a single farm were studied. On the basis of clinical signs, microscopic examination, faecal culture, ELISA and diagnostic PCR, 60 and 143 goats were classified as resistant and susceptible to JD, respectively. PCR-based restriction fragment length polymorphism (PCR-RFLP) with two enzymes, PstI and TaqI, was used to assess variation in the DRB gene(s) in all 203 goats studied. Two di-allelic single nucleotide polymorphisms (SNPs), here referred as 'P' and 'T', were tested. In each of them, three genotypes were found in the group analysed. The minimum allele frequencies (MAFs) were 0.233 and 0.486 for the P and T SNPs, respectively. Statistically significant associations between alleles, individual genotypes and composed genotypes of both SNPs were found. The frequency of p and t alleles, of individual pp and tt and of composed pptt genotypes were significantly higher (P(corr) < 0.001) in the 'resistant' group as compared to the 'susceptible' group, while the P and T alleles were associated with susceptibility (P(corr) < 0.001). In heterozygous genotypes, susceptibility was dominant over resistance. The effects of both SNP on resistance and susceptibility were comparable and composed heterozygous genotypes showed intermediate levels of susceptibility in terms of the odds ratio and P-values calculated.

Immunity-related gene single nucleotide polymorphisms associated with Rhodococcus equi infection in foals.

In previous work, we found significant associations of horse polymorphic microsatellite and immunity-related (IR) gene markers with Rhodococcus equi infection of foals. Here, a statistically significant association between a single nucleotide polymorphism (SNP) within the interleukin 7 receptor-encoding gene (IL7R) with high R. equi burden in transtracheal aspirates was found (Fisher's F = 0.043, odds ratio: 8.00, 95% confidence interval: 1.127-56.795). Further positional and/or functional candidate genes investigated TLR2, IL13, IL17A, IL28R, TACE/ADAM 17 and GBP1, were not associated with infection in this study. SNPs analysed were found by sequencing and appropriate restriction fragment length polymorphism markers were developed. Their associations with R. equi infection were tested by genotyping thoroughbred foals from the original study. The association was confirmed by analysing genotypes composed with genes previously reported to be associated with R. equi infection in the same group.

Genetic susceptibility to West Nile virus infection in Camargue horses.

West Nile virus (WNV) is a mosquito-borne zoonotic neurotropic virus capable to cause lethal meningoencephalitis (WNE) in infected hosts such as birds, horses, and humans. Due to their sensitivity, horses serve as sentinel species in areas at risk. We studied a population of Camargue horses living in Southern France in two zones with endemic WNV circulation where WNV outbreaks were recorded in 2000 and 2003-4. Two sets of microsatellite markers located in MHC and Ly49 genomic regions were genotyped as well as multiple SNPs in ten immunity-related candidate gene regions. Associations between genetic polymorphisms and resistance/susceptibility to WNE were tested. While single marker associations were weak, compound two-gene genotypes of SNPs located within the MAVS, NCR2 and IL-10 genes and microsatellites HMS082 and CZM013 were associated with susceptibility to WNE. Combinations of microsatellite markers CZM009, ABGe17402 and ABGe9019 were associated with simple seroconversion without clinical signs of WNE (resistance). In addition, a distribution of polymorphic markers between WNV-IgG seropositive horses and a control group of WNV-IgG seronegative horses was tested. One SNP in the OAS1 gene (NC_009151.3:g.21961328A>G) was significantly associated with the seropositive phenotype (pcorr = 0.023; OR = 40.5 CI (4.28; 383.26); RR = 8.18 CI (1.27; 52.89) in the Camargue breed. In compound genotypes, SNP markers for SLC11A1, MAVS, OAS1, TLR4, ADAM17 and NCR2 genes and ten microsatellites showed non-random distribution between seropositive and seronegative groups of horses. Further analysis of associated markers could contribute to our understanding of anti-WNV defense mechanisms in horses.

Report of the 3rd Havemeyer workshop on allergic diseases of the Horse, Hólar, Iceland, June 2007.

Allergic diseases occur in most mammals, although some species such as humans, dogs and horses seem to be more prone to develop allergies than others. In horses, insect bite hypersensitivity (IBH), an allergic dermatitis caused by bites of midges, and recurrent airway obstruction (RAO), a hyperreactivity to stable born dust and allergens, are the two most prevalent allergic diseases. Allergic diseases involve the interaction of three major factors: (i) genetic constitution, (ii) exposure to allergens, and (iii) a dysregulation of the immune response determined by (i) and (ii). However, other environmental factors such as infectious diseases, contact with endotoxin and degree of infestation with endoparasites have been shown to influence the prevalence of allergic diseases in humans. How these factors may impact upon allergic disease in the horse is unknown at this time. The 3rd workshop on Allergic Diseases of the Horse, with major sponsorship from the Havemeyer Foundation, was held in Hólar, Iceland, in June 2007 and focussed on immunological and genetic aspects of IBH and RAO. This particular venue was chosen because of the prevalence of IBH in exported Icelandic horses. The incidence of IBH is significantly different between Icelandic horses born in Europe or North America and those born in Iceland and exported as adults. Although the genetic factors and allergens are the same, exported adult horses show a greater incidence of IBH. This suggests that environmental or epigenetic factors may contribute to this response. This report summarizes the present state of knowledge and summarizes important issues discussed at the workshop.

Single nucleotide polymorphisms of interleukin-1 beta related genes and their associations with infection in the horse.

In previous work, we found significant associations of horse chromosome 15 (ECA15) microsatellite markers HMSO1 and HTG06 with two horse infections, Rhodococcus equi and Lawsonia intracellularis, respectively. Interleukin-1 beta subunit and interleukin-1 receptor antagonist encoding genes (IL1B and IL1RN) could be considered as candidate genes underlying the associations reported. Therefore, we identified single nucleotide polymorphisms (SNPs) within three interleukin-1 beta functionally related genes: IL1B, IL1RN and Casp1 (interleukin-1 beta converting enzyme/caspasel encoding gene). Using appropriate restriction fragment length polymorphism (PCR-RFLP) and/or single strand conformation polymorphism (PCR-SSCP) markers, their associations with the two infections by genotyping foals from the original study were tested. In addition, the physical localization of one of the two closely located genes, IL1RN, was re-assessed by fluorescence in-situ hybridization (FISH). A statistically significant association between an intronic SNP of the Casp1 gene with R. equi infection was found. The IL1RN gene was localized to 15q13-q14 in agreement with its originally reported physical position.

Microsatellite markers for evaluating the diversity of the natural killer complex and major histocompatibility complex genomic regions in domestic horses.

Genotyping microsatellite markers represents a standard, relatively easy, and inexpensive method of assessing genetic diversity of complex genomic regions in various animal species, such as the major histocompatibility complex (MHC) and/or natural killer cell receptor (NKR) genes. MHC-linked microsatellite markers have been identified and some of them were used for characterizing MHC polymorphism in various species, including horses. However, most of those were MHC class II markers, while MHC class I and III sub-regions were less well covered. No tools for studying genetic diversity of NKR complex genomic regions are available in horses. Therefore, the aims of this work were to establish a panel of markers suitable for analyzing genetic diversity of the natural killer complex (NKC), and to develop additional microsatellite markers of the MHC class I and class III genomic sub-regions in horses. Nine polymorphic microsatellite loci were newly identified in the equine NKC. Along with two previously reported microsatellites flanking this region, they constituted a panel of 11 loci allowing to characterize genetic variation in this functionally important part of the horse genome. Four newly described MHC class I/III-linked markers were added to 11 known microsatellites to establish a panel of 15 MHC markers with a better coverage of the class I and class III sub-regions. Major characteristics of the two panels produced on a group of 65 horses of 13 breeds and on five Przewalski's horses showed that they do reflect genetic variation within the horse species.

Candidate gene molecular markers as tools for analyzing genetic susceptibility to morbillivirus infection in stranded Cetaceans.

Morbilliviruses, such as Cetacean morbillivirus (CeMV) or Phocine distemper virus (PDV), represent a growing threat for marine mammals on both hemispheres. Because free-ranging animal populations strongly rely on natural resistance mechanisms, innate immunity-related genes and virus cell entry receptor genes may represent key factors involved in susceptibility to CeMV in Cetaceans. Using the next generation sequencing technology, we have sequenced 11 candidate genes in two model species, Stenella coeruleoalba and Phocoena phocoena. Suitable single nucleotide polymorphism markers of potential functional importance, located in genes coding for basigin (BSG, CD147), the signaling lymphocyte activating molecule (SLAMF1), the poliovirus-related receptor-4 (NECTIN4, PVRL4), toll-like receptors 3, 7, 8 (TLR3, TLR7, TLR8), natural resistance-associated macrophage protein (SLC11A1) and natural cytotoxicity triggering receptor 1 (NCR1), were identified in each model species, along with MHC-DQB haplotypes unique for each species. This set of molecular markers represents a potentially useful tool for studying host genetic variation and susceptibility to morbillivirus infection in Cetaceans as well as for studying functionally important genetic diversity of selected Cetacean populations.

Biological principles of heredity of and resistance to disease.

Understanding the biological principles of disease heredity and resistance to disease is a prerequisite for the incorporation of these factors into multi-trait breeding programmes. Mutations represent an evolutionary aspect of disease. The author reviews patterns of Mendelian inheritance and possible causes of non-Mendelian inheritance, such as multifactorial inheritance, expansion of trinucleotide repeats, mitochondiral inheritance and genomic imprinting, in relation to diseases of domestic animals. Host-pathogen interactions underlie genetic variability in resistance to disease. Infectious pathogens endowed with a high potential for evolutionary change use this potential to evade various host defence mechanisms. This interaction may have a competitive or co-evolutionary character. The host immune system copes with the variability of pathogens by using the potential of genetic diversity of lymphocytes in immunoglobulin, T-cell receptor and major histocompatibility complex genes. Possible mechanisms for maintenance of this diversity are discussed in the context of selection for disease resistance.

[Immunoenzyme detection of enzootic bovine leukemia viruses (BLV) in cell cultures]. / Imunoenzymatický prukaz viru enzootické leukózy skotu (BLV) v bunecných kulturách.

There is a description of the method of immunoenzymic test used for detection of enzootic bovine leukosis virus (BLV) on the cells of foetal lamb spleen (FLS) permanently producing BLV and on the lymphocytes of infected animals. In both cases (in FLS cells and lymphocytes) the presence of BLV was demonstrated by the immunoperoxidase test. Using the defined serums from the animals after experimental and natural infection, BLV was detected by the above-mentioned test in FLS cells in all cases of the use of positive serums with anti-BLV antibodies. The specificity of the antibodies was also demonstrated in this way. The presence of BLV was analogically detected in the lymphocytes of the infected animals where the infection time was precisely defined. The method of immunoperoxidase test on lymphocytes is recommended in indicated cases for the demonstration of the presence of BLV in infected animals.

[Detection of enzootic bovine leukemia virus using the syncytium test]. / Prükaz viru enzootické leukózy skotu (BLV) syncytiálním testem (SyT).

BLV detection by the syncytial test was performed in 27 heifers experimentally and naturally infected by the enzootic bovine leukosis virus (BLV). The presence of BLV was demonstrated in 94.7% of the animals. The bovine foetal spleen cells (FBS) were found to be suitable for the syncytial test. Positive animals not reacting to infection by the production of anti-BLV antibodies were identified during the syncytial-test investigation. The importance of this finding for the programme of controlling enzootic bovine leukosis on farms is discussed. As suggested by the results, temporary occurrence of anti-BLV antibodies followed by their disappearance can be observed together with a negative result of the syncytial test in some circumstances. The discussion deals with the problems of the determination of anti-BLV antibodies in milk, and/or milk secretion, by the ELISA method.

[Experimental and natural infection with the enzootic leukosis virus of cattle]. / Experimentální a prirozená infekce virem enzootické leukózy skotu (BLV).

A trial was performed with heifers at the age of six to seven months. The animals were experimentally infected with the lymphocytes of a virus-productive donor. Infection was produced in all the nine cases, as demonstrated by means of the positive syncytial test. As indicated by the results of the trial, the antibodies to the enzootic bovine leucosis virus (BLV) were produced soon after experimental infection. A high sensitivity of the serum-neutralization test and the ELISA method was demonstrated in this connection: by these methods, the antibodies were identified already two to three weeks after experimental infection whereas by the immunodiffusion test they could be detected only after five weeks. Twenty-four animals were exposed to natural contact infection. Within 270 days of the trial, the disease after contact was recorded only in one heifer out of the four that were in close contact with the experimentally infected animals. In this case, as compared with experimental infection, the antibodies were produced much later--after 85 to 93 days. Leucosis was recorded in none of the remaining animals. The reasons why such a favourable result was obtained were the thorough disinfection of the stables after blood collections and the strict observance of the aseptic conditions. The results of experimental infection in three cows were identical with those obtained in young cattle. In the experimentally infected dairy cows, antibodies in milk were determined by the ELISA method. As found, in milk the antibodies to BLV appear two to three weeks later than they do in serum. The ELISA method of BLV antibody detection can be used for the identification of infected animals in herds where enzootic bovine leucosis occurs.

[Biological principles of chemotherapy: cellular response as a result of its evolutionary memory]. / Biologický princip chemoterapie: reakce bunky jako výsledek její evolucní pameti.

The cell reaction to chemotherapy is based on certain general biological principles. The genetic structure of cell populations, their actual reaction to chemotherapy and the prognosis of their development are not accidental. They are determined by previous exposures to chemotherapeutic agents and thus their evolutional experience on Darwinian principles. Chemotherapy occurs always in a defined environment of the host with his active participation. A genetic variability in the host reaction to chemotherapeutic interference also exists and which in the long-term perspective is also of evolutional nature. The complex of evaluation of therapeutic procedures thus involves evaluation and influencing of the host and his variability (immunotherapy). Understanding of molecular mechanisms of the pathogenesis of disease, definition of specific genes, their expression, the role of their products and definition of possible targets of chemotherapy is at present a realistic goal of application of genomics and proteogenomics in medicine. The new philosophy in medicine is thus based on respecting individuality in diagnosis, treatment and prognosis. We only can wish that the trend of respecting individuality will be applied in the future, and not only in medicine.

Genetics of anti-EHV antibody responses in a horse population.

Individual variation in immune responses to herpesviruses was observed in various species. Here, associations between polymorphic molecular markers and life-long anti-EHV-1/4 antibody immune responses were analyzed in a model EHV-infected population of the Old Kladruber horses. Two-dimensional analysis including overall mean titers and titer dynamics expressed by differences between spring and autumn titers allowed identification of low-responders. 50 randomly selected microsatellites and nine single nucleotide polymorphisms in nine immunity-related candidate genes were genotyped. Due to differences (p<0.001) in antibody titers between two color varieties of Old Kladruber horses, separate association studies were performed in the two sub-populations by using the Fisher's exact test. In black horses, the interleukin 4 receptor and MxA protein coding genes, and the microsatellite TKY325 were associated with the responder status. In the grey population, the microsatellite TKY343 showed significant association with anti-EHV antibody responsiveness after Bonferroni corrections.

Karyotypic relationships among Equus grevyi, Equus burchelli and domestic horse defined using horse chromosome arm-specific probes.

Using laser microdissection we prepared a set of horse chromosome arm-specific probes. Most of the probes were generated from horse chromosomes, some of them were derived from Equus zebra hartmannae. The set of probes were hybridized onto E. grevyi chromosomes in order to establish a genome-wide chromosomal correspondence between this zebra and horse. The use of arm-specific probes provided us with more information on the mutual arrangement of the genomes than we could obtain by means of whole-chromosome paints generated by flow sorting, even if we used reciprocal painting with probe sets from both species. By comparison of our results and results of comparative mapping in E. burchelli, we also established the chromosomal correspondence between E. grevyi and E. burchelli, providing evidence for a very close karyotypic relationship between these two zebra species. Establishment of the comparative map for E. grevyi contributes to the knowledge of the karyotypic phylogeny in the Equidae family.

Single nucleotide polymorphisms in four functionally related immune response genes in the horse: CD14,TLR4, Cepsilon, andFcepsilon R1 alpha.

The objective of this study was to identify single nucleotide polymorphisms (SNPs) within four functionally related immune response genes in the horse, and to develop genotyping techniques that could be useful for future genomic studies of horse infectious and allergic diseases. The genes analysed were: the lipopolysaccharide (LPS) receptor gene CD14, the toll-like receptor 4 gene TLR4, the gene Cepsilon encoding the IgE heavy chain molecule and the gene FcepsilonR1 alpha coding for the alpha subunit of the IgE receptor molecule. Horse-specific primers amplifying selected gene regions were designed and SNPs were searched by selective resequencing and/or by PCR-SSCP (polymerase chain reaction-sequence specific conformational polymorphism) or PCR-RFLP (PCR-restriction fragment length polymorphism). Gene expression was analysed by RT-PCR (reverse transcriptase-PCR) of all four genes examined. For CD14, the cDNA sequence was determined and a novel sequence of the 5'UTR region was identified. The protein-coding sequence was identical to that previously deposited in GenBank. 5'UTR, intronic and both synonymous and non-synonymous exonic SNPs were identified. Three SNPs were found in the CD14 gene, four in the TLR4 gene; two SNPs were identified in the Cepsilon gene, and one SNP was found in the FcepsilonR1 alpha gene. PCR-RFLP was developed for genotyping eight of the SNPs identified. The RT-PCR assay showed that all the SNPs reported here are parts of expressed genes. The results showed that important immunity-related genes in horses are polymorphic and that even non-synonymous SNPs with potential functional impact may occur. The methods developed for genotyping and haplotyping the SNPs identified represent, along with markers described previously, a potentially useful tool for genomic analysis of the function and role of these genes in immunity and in mechanisms of disease.