Genome-wide association study of equine herpesvirus type 1-induced myeloencephalopathy identifies a significant single nucleotide polymorphism in a platelet-related gene.

Equine herpesvirus type 1 (EHV-1)-induced myeloencephalopathy (EHM) is a neurologic disease of horses that represents one outcome of infection. The neurologic form of disease occurs in a subset of infected horses when virus-induced endothelial cell damage triggers vasculitis and subsequent ischemic insult to the central nervous system. EHM causes considerable animal suffering and economic loss for the horse industry. Virus polymorphisms have been previously associated with disease outcome but cannot fully explain why only some horses develop EHM. This study investigated the role of host genetics in EHM. DNA samples were collected from 129 horses infected with EHV-1 (61 that developed EHM and 68 in which disease resolved without the development of neurologic signs) during natural outbreaks or experimental infections. A genome-wide association study (GWAS) was performed to investigate host genetic variations associated with EHM. Genotyping was performed using the Illumina SNP50 and SNP70 arrays and a custom Sequenom array. Mixed linear model (MLM) analysis using a recessive model identified one marker that surpassed the threshold for genome-wide significance (P<0.001) after Bonferroni correction. The marker (BIEC2_946397) is in an intron of the tetraspanin 9 (TSPAN9) gene, which is expressed in endothelial cells and platelets. The GWAS identified a region in the horse genome that is associated with EHM in the sample population and thus warrants further exploration. Understanding the contribution of host genetic variation to the development of EHM will enhance our knowledge of disease pathophysiology, and lead to improved strategies for treating individual cases and managing outbreaks.

The equine endometrial cup reaction: a fetomaternal signal of significance.

A remarkable feature of equine pregnancy is the development of the invasive trophoblast of the chorionic girdle and its formation of the gonadotrophin-secreting endometrial cup cells in early gestation. The details of this process have been revealed only slowly over the past century, since the first description of the endometrial cups in 1912. This centennial presents an opportunity to review the characteristics of the cells and molecules involved in this early, critical phase of placentation in the mare. The invasiveness of the chorionic girdle trophoblast appears to represent an atavistic attribute more commonly associated with the hemochorial placentae of primates and rodents but not with the more recently derived epitheliochorial placentae of the odd-toed ungulates. The nature of and raison d'être for the strong fetal signals transmitted to the mare by the endometrial cup reaction, and her responses to these messages, are the subject of the present review.

T-cell tolerance to the developing equine conceptus.

One of the most intriguing and dramatic examples of immunological tolerance is displayed by the mammalian foetal-placental unit, which thrives as a semi-allograft in the mother's uterus during pregnancy. The success of the so-called foetal allograft stands in stark contrast to the failure of most tissue and organ grafts to survive without genetic matching of donor and recipient or drastic immunosuppression of the recipient's immune system. Experiments conducted over the past 60 years have revealed multiple mechanisms that enable the conceptus to avoid immunological detection or destruction. Many of these mechanisms are directed towards evading immune-mediated damage by maternal T lymphocytes, and they can be grouped into three classes: (i) downregulation of major histocompatibility complex (MHC) gene expression in placental trophoblast cells; (ii) local and systemic alterations in maternal immune reactivity; and (iii) innate defence mechanisms of the trophoblast cells that comprise the barrier between foetal and maternal tissues. The redundancy in these protective mechanisms helps ensure the transmission of life from generation to generation and provides a rich field of study of ways in which functional immunological tolerance can be manifest. The variation in placental forms and function among mammalian species present opportunities to discover and understand novel tolerogenic mechanisms that may have broad application in biology, medicine and animal husbandry. This review focuses on the evidence obtained from studies of pregnancy in the mare that support the case for selective T-cell tolerance to the mammalian conceptus.

Molecular evidence for natural killer-like cells in equine endometrial cups.

OBJECTIVES: To identify equine orthologs of major NK cell marker genes and utilize them to determine whether NK cells are present among the dense infiltration of lymphocytes that surround the endometrial cup structures of the horse placenta during early pregnancy. STUDY DESIGN: PCR primers were developed to detect the equine orthologs of NKP46, CD16, CD56, and CD94; gene expression was detected in RNA isolated from lymphocytes using standard 2-step reverse transcriptase (RT) PCR and products were cloned and sequenced. Absolute real-time RT-PCR was used to quantitate gene expression in total, CD3+, and CD3- peripheral lymphocytes, and invasive trophoblast. Lymphocytes surrounding the endometrial cups (ECL) of five mares in early pregnancy were isolated and NK marker gene expression levels were assayed by quantitative RT-PCR. MAIN OUTCOME MEASURES: Absolute mRNA transcript numbers were determined by performing quantitative RT-PCR and comparing values to plasmid standards of known quantities. RESULTS: NKP46 gene expression in peripheral CD3- lymphocytes was higher than in CD3+ lymphocytes, CD16 levels were higher in the CD3+ population, and no significant differences were detected for CD56 and CD94 between the two groups. Expression of all four NK cell markers was significantly higher in lymphocytes isolated from the endometrial cups of pregnant mares compared to PBMC isolated from the same animal on the same day (NKP46, 14-fold higher; CD94, 8-fold higher; CD16, 20-fold higher; CD56, 44-fold higher). CONCLUSIONS: These data provide the first evidence for the expression of major NK cell markers by horse cells and an enrichment of NK-like cells in the equine endometrium during pregnancy.

IFPA Meeting 2010 Workshop Report I: Immunology; ion transport; epigenetics; vascular reactivity; epitheliochorial placentation; proteomics.

Workshops are an important part of the IFPA annual meeting. At IFPA Meeting 2010 there were twelve themed workshops, six of which are summarized in this report. 1. The immunology workshop focused on normal and pathological functions of the maternal immune system in pregnancy. 2. The transport workshop dealt with regulation of ion and water transport across the syncytiotrophoblast of human placenta. 3. The epigenetics workshop covered DNA methylation and its potential role in regulating gene expression in placental development and disease. 4. The vascular reactivity workshop concentrated on methodological approaches used to study placental vascular function. 5. The workshop on epitheliochorial placentation covered current advances from in vivo and in vitro studies of different domestic species. 6. The proteomics workshop focused on a variety of techniques and procedures necessary for proteomic analysis and how they may be implemented for placental research.

Identification of equine major histocompatibility complex haplotypes using polymorphic microsatellites.

A system for identifying equine major histocompatibility complex (MHC) haplotypes was developed based on five polymorphic microsatellites located within the MHC region on ECA 20. Molecular signatures for 50 microsatellite haplotypes were recognized from typing 353 horses. Of these, 23 microsatellite haplotypes were associated with 12 established equine leucocyte antigen (ELA) haplotypes in Thoroughbreds and Standardbreds. Five ELA serotypes were associated with multiple microsatellite subhaplotypes, expanding the estimates of diversity in the equine MHC. The strong correlations between serological and microsatellite typing demonstrated a linkage to known MHC class I protein polymorphisms and validated this assay as a useful supplement to ELA serotyping, and in some applications, a feasible alternative method for MHC genotyping in horse families and in population studies.

Equine clinical genomics: A clinician's primer.

The objective of this review is to introduce equine clinicians to the rapidly evolving field of clinical genomics with a vision of improving the health and welfare of the domestic horse. For 15 years a consortium of veterinary geneticists and clinicians has worked together under the umbrella of The Horse Genome Project. This group, encompassing 22 laboratories in 12 countries, has made rapid progress, developing several iterations of linkage, physical and comparative gene maps of the horse with increasing levels of detail. In early 2006, the research was greatly facilitated when the US National Human Genome Research Institute of the National Institutes of Health added the horse to the list of mammalian species scheduled for whole genome sequencing. The genome of the domestic horse has now been sequenced and is available to researchers worldwide in publicly accessible databases. This achievement creates the potential for transformative change within the horse industry, particularly in the fields of internal medicine, sports medicine and reproduction. The genome sequence has enabled the development of new genome-wide tools and resources for studying inherited diseases of the horse. To date, researchers have identified 11 mutations causing 10 clinical syndromes in the horse. Testing is commercially available for all but one of these diseases. Future research will probably identify the genetic bases for other equine diseases, produce new diagnostic tests and generate novel therapeutics for some of these conditions. This will enable equine clinicians to play a critical role in ensuring the thoughtful and appropriate application of this knowledge as they assist clients with breeding and clinical decision-making.

Report of the Second Havemeyer EHV-1 Workshop, Steamboat Springs, Colorado, USA, September 2008.

This report summarises the findings of the Second Havemeyer EHV-1 Workshop, which was held in Steamboat Springs, Colorado, USA in September 2008. A total of 38 delegates, consisting of veterinary clinicians and scientists from academia and industry participated in a series of sessions that focused on equine herpesvirus myeloencephalopathy (EHM). Each session consisted of a review, followed by short presentations on current research topics. The sessions included EHM epidemiology, in vivo and in vitro models for studying EHM, EHV-1 virulence determinants, real-time PCR diagnostics, antiviral medications and new vaccination technologies. The report summarises the key advances identified during and since the meeting. Citations are restricted to selected reviews and papers published since the workshop.

Genome sequence, comparative analysis, and population genetics of the domestic horse.

We report a high-quality draft sequence of the genome of the horse (Equus caballus). The genome is relatively repetitive but has little segmental duplication. Chromosomes appear to have undergone few historical rearrangements: 53% of equine chromosomes show conserved synteny to a single human chromosome. Equine chromosome 11 is shown to have an evolutionary new centromere devoid of centromeric satellite DNA, suggesting that centromeric function may arise before satellite repeat accumulation. Linkage disequilibrium, showing the influences of early domestication of large herds of female horses, is intermediate in length between dog and human, and there is long-range haplotype sharing among breeds.

Modeling trophoblast differentiation using equine chorionic girdle vesicles.

The chorionic girdle of the equine conceptus is comprised of specialized trophoblast cells which, at day 36-38 of equine pregnancy, gain an invasive phenotype and invade the endometrium to form endometrial cups. Studies of equine endometrial cups remain difficult to perform because of the invasive techniques required to obtain cup tissue and because sampling requires termination of the pregnancy. In this study we developed a system to model trophoblast differentiation and trophoblast-immune interactions in vitro and in vivo. We utilized a method of culturing chorionic girdle pieces in serum-free medium to promote spontaneous formation of vesicle structures enriched for terminally differentiated binucleate cells that secreted equine chorionic gonadotrophin (eCG). Immunohistochemical staining and scanning electron microscopy showed that the cells of the vesicles closely resembled the outer layers of chorionic girdle immediately prior to invasion. Chorionic girdle vesicles were harvested after 72h in culture and ectopically transplanted via injection into the vulvar mucosa of recipient mares. At 7, 14, 21 and 28days after transplantation, biopsies of the injection sites were obtained. Immunohistochemical labeling of cryostat sections of the biopsies with a panel of monoclonal antibodies to horse trophoblast molecules demonstrated survival, differentiation, and presence of trophoblast cells for at least 21days. Serial sections of the biopsies labeled with antibodies to the equine lymphocyte surface markers CD4 and CD8, together with lymphocyte microcytotoxicity assays, revealed that the recipients mounted both cellular and humoral antibody immune responses to the transplanted trophoblast cells. This new method for culturing equine chorionic girdle trophoblast cells, and for transplanting trophoblast vesicles to ectopic sites, should allow identification of key aspects of trophoblast differentiation and the interactions that occur between invasive trophoblast and the maternal immune system.

The effect of skin allografting on the equine endometrial cup reaction.

This research tested the hypothesis that immunological sensitization of mares by skin allografting, followed by the establishment of pregnancy using semen from the skin-graft donor, would give rise to secondary immune responses to the developing horse conceptus, resulting in an earlier demise of the fetally derived endometrial cups. Maiden mares received skin allografts from a stallion homozygous for Major Histocompatibility Complex (MHC) antigens and/or equivalent autografts and were subsequently mated to the skin-graft donor stallion during the next two breeding seasons. Mares that had been immunologically primed to the foreign MHC class I antigens of the skin-graft donor stallion developed strong secondary antibody responses early in their first pregnancies, whereas autografted mares made weak primary antibody responses in their first pregnancies and strong secondary responses in their second pregnancies. In contrast, histological examination of the endometrial cups after surgical pregnancy termination at Day 60 of gestation revealed no discernible differences between allografted and autografted mares, and there were no significant differences in the concentrations and/or duration of secretion of the endometrial cup-specific hormone, equine chorionic gonadotrophin (eCG), between allografted and autografted mares, nor in either group between first and second pregnancies. The vigorous antibody response observed in the pregnant allografted mares supported the first part of our hypothesis, providing evidence of systemic immunological priming. However, there was a lack of an equivalent heightened cellular response to the endometrial cups. These findings provided strong evidence for an asymmetric immune response to the conceptus, characterized by strong humoral immunity and a dampened cellular response.

A molecular approach to the identification of cytotoxic T-lymphocyte epitopes within equine herpesvirus 1.

Equine herpesvirus 1 (EHV-1) causes respiratory and neurological disease and abortion in horses. Animals with high frequencies of cytotoxic T lymphocytes (CTL) show reduced severity of respiratory disease and frequency of abortion, probably by CTL-mediated control of cell-associated viraemia. This study aimed to identify CTL epitopes restricted by selected major histocompatibility complex (MHC) class I alleles expressed in the equine leukocyte antigen (ELA) A3 haplotype. Effector CTL were induced from EHV-1-primed ponies and thoroughbreds with characterized MHC class I haplotypes and screened against P815 target cells transfected with selected EHV-1 genes and MHC class I genes. Targets that expressed EHV-1 gene 64 and the MHC B2 gene were lysed by effector CTL in a genetically restricted manner. There was no T-cell recognition of targets expressing either the MHC B2 gene and EHV-1 genes 2, 12, 14, 16, 35, 63 or 69, or the MHC C1 gene and EHV-1 genes 12, 14, 16 or 64. A vaccinia virus vector encoding gene 64 (NYVAC-64) was also investigated. Using lymphocytes from ELA-A3 horses, the recombinant NYVAC-64 virus induced effector CTL that lysed EHV-1-infected target cells; the recombinant virus also supplied a functional peptide that was expressed by target cells and recognized in an MHC-restricted fashion by CTL induced with EHV-1. This construct may therefore be used to determine the antigenicity of EHV-1 gene 64 for other MHC haplotypes. These techniques are broadly applicable to the identification of additional CTL target proteins and their presenting MHC alleles, not only for EHV-1, but for other equine viruses.

Report of the equine herpesvirus-1 Havermeyer Workshop, San Gimignano, Tuscany, June 2004.

Amongst the infectious diseases that threaten equine health, herpesviral infections remain a world wide cause of serious morbidity and mortality. Equine herpesvirus-1 infection is the most important pathogen, causing an array of disorders including epidemic respiratory disease abortion, neonatal foal death, myeloencephalopathy and chorioretinopathy. Despite intense scientific investigation, extensive use of vaccination, and established codes of practice for control of disease outbreaks, infection and disease remain common. While equine herpesvirus-1 infection remains a daunting challenge for immunoprophylaxis, many critical advances in equine immunology have resulted in studies of this virus, particularly related to MHC-restricted cytotoxicity in the horse. A workshop was convened in San Gimignano, Tuscany, Italy in June 2004, to bring together clinical and basic researchers in the field of equine herpesvirus-1 study to discuss the latest advances and future prospects for improving our understanding of these diseases, and equine immunity to herpesviral infection. This report highlights the new information that was the focus of this workshop, and is intended to summarize this material and identify the critical questions in the field.

Inhibition of lymphocyte proliferation and activation: a mechanism used by equine invasive trophoblast to escape the maternal immune response.

At days 36-38 of gestation, the equine invasive trophoblast cells migrate into the endometrium of the pregnant mare to form the endometrial cups. During their migration, they become surrounded by maternal CD4+ and CD8+ T lymphocytes, and stimulate a cytotoxic antibody response to the paternal major histocompatibility complex class I antigens that they express. Nevertheless, endometrial cup cells remain viable at the site of uterine invasion up to days 80-100 of gestation, suggesting the participation of immunomodulatory mechanisms to the maternal cellular immune response. To determine the effects of the invasive trophoblast cells on lymphocyte proliferation, an in vitro co-culture system was developed using isolated equine invasive trophoblast cells and peripheral blood lymphocytes. Fetal fibroblast cells from the same conceptuses were used as controls. The presence of invasive trophoblast cells or their pre-conditioned medium inhibited 50% or more of lymphocyte proliferation, while fetal fibroblasts had no effect. The invasive trophoblast cell inhibitory factor needed to be present constantly to affect lymphocyte proliferation, and it was ineffective if lymphocytes had been previously stimulated to proliferate. The lymphoproliferative inhibitory mechanism affected lymphocyte subpopulations similarly. In addition, lymphocyte expression of cytokine mRNA including IFNgamma, IL-2, IL-4, and IL-10 was affected compared to controls. The implication of these observations in vivo may explain, in part, the apparent equine maternal immune acceptance of the presence and development of endometrial cup cells.

The second generation of the International Equine Gene Mapping Workshop half-sibling linkage map.

A low-density, male-based linkage map was constructed as one of the objectives of the International Equine Gene Mapping Workshop. Here we report the second generation map based on testing 503 half-sibling offspring from 13 sire families for 344 informative markers using the CRIMAP program. The multipoint linkage analysis localized 310 markers (90%) with 257 markers being linearly ordered. The map included 34 linkage groups representing all 31 autosomes and spanning 2262 cM with an average interval between loci of 10.1 cM. This map is a milestone in that it is the first map with linkage groups assigned to each of the 31 automosomes and a single linkage group to all but three chromosomes.

An ordered BAC contig map of the equine major histocompatibility complex.

A physical map of ordered bacterial artificial chromosome (BAC) clones was constructed to determine the genetic organization of the horse major histocompatibility complex. Human, cattle, pig, mouse, and rat MHC gene sequences were compared to identify highly conserved regions which served as source templates for the design of overgo primers. Thirty-five overgo probes were designed from 24 genes and used for hybridization screening of the equine USDA CHORI 241 BAC library. Two hundred thirty-eight BAC clones were assembled into two contigs spanning the horse MHC region. The first contig contains the MHC class II region and was reduced to a minimum tiling path of nine BAC clones that span approximately 800 kb and contain at least 20 genes. A minimum tiling path of a second contig containing the class III/I region is comprised of 14 BAC clones that span approximately 1.6 Mb and contain at least 34 genes. Fluorescence in situ hybridization (FISH) using representative clones from each of the three regions of the MHC localized the contigs onto ECA20q21 and oriented the regions relative to one another and the centromere. Dual-colored FISH revealed that the class I region is proximal to the centromere, the class II region is distal, and the class III region is located between class I and II. These data indicate that the equine MHC is a single gene-dense region similar in structure and organization to the human MHC and is not disrupted as in ruminants and pigs.