Katia Cappelli

Genome-Wide Analysis Reveals Selection for Important Traits in Domestic Horse Breeds

Intense selective pressures applied over short evolutionary time have resulted in homogeneity within, but substantial variation among, horse breeds. Utilizing this population structure, 744 individuals from 33 breeds, and a 54,000 SNP genotyping array, breed-specific targets of selection were identified using an FST-based statistic calculated in 500-kb windows across the genome. A 5.5-Mb region of ECA18, in which the myostatin (MSTN) gene was centered, contained the highest signature of selection in both the Paint and Quarter Horse. Gene sequencing and histological analysis of gluteal muscle biopsies showed a promoter variant and intronic SNP of MSTN were each significantly associated with higher Type 2B and lower Type 1 muscle fiber proportions in the Quarter Horse, demonstrating a functional consequence of selection at this locus. Signatures of selection on ECA23 in all gaited breeds in the sample led to the identification of a shared, 186-kb haplotype including two doublesex related mab transcription factor genes (DMRT2 and 3). The recent identification of a DMRT3 mutation within this haplotype, which appears necessary for the ability to perform alternative gaits, provides further evidence for selection at this locus. Finally, putative loci for the determination of size were identified in the draft breeds and the Miniature horse on ECA11, as well as when signatures of selection surrounding candidate genes at other loci were examined. This work provides further evidence of the importance of MSTN in racing breeds, provides strong evidence for selection upon gait and size, and illustrates the potential for population-based techniques to find genomic regions driving important phenotypes in the modern horse.

Molecular Detection, Epidemiology, and Genetic Characterization of Novel European Field Isolates of Equine Infectious Anemia Virus

ABSTRACT The application of molecular diagnostic techniques along with nucleotide sequence determination to permit contemporary phylogenetic analysis of European field isolates of equine infectious anemia virus (EIAV) has not been widely reported. As a result, of extensive testing instigated following the 2006 outbreak of equine infectious anemia in Italy, 24 farms with a history of exposure to this disease were included in this study. New PCR-based methods were developed, which, especially in the case of DNA preparations from peripheral blood cells, showed excellent correlation with OIE-approved agar gel immunodiffusion (AGID) tests for identifying EIAV-infected animals. In contrast, the OIE-recommended oligonucleotide primers for EIAV failed to react with any of the Italian isolates. Similar results were also obtained with samples from four Romanian farms. In addition, for the first time complete characterization of gag genes from five Italian isolates and one Romanian isolate has been achieved, along with acquisition of extensive sequence information (86% of the total gag gene) from four additional EIAV isolates (one Italian and three Romanian). Furthermore, in another 23 cases we accomplished partial characterization of gag gene sequences in the region encoding the viral matrix protein. Analysis of this information suggested that most Italian isolates were geographically restricted, somewhat reminiscent of the “clades” described for human immunodeficiency virus type 1 (HIV-1). Collectively this represents the most comprehensive genetic study of European EIAV isolates conducted to date.

RNA Sequencing of the Exercise Transcriptome in Equine Athletes

The horse is an optimal model organism for studying the genomic response to exercise-induced stress, due to its natural aptitude for athletic performance and the relative homogeneity of its genetic and environmental backgrounds. Here, we applied RNA-sequencing analysis through the use of SOLiD technology in an experimental framework centered on exercise-induced stress during endurance races in equine athletes. We monitored the transcriptional landscape by comparing gene expression levels between animals at rest and after competition. Overall, we observed a shift from coding to non-coding regions, suggesting that the stress response involves the differential expression of not annotated regions. Notably, we observed significant post-race increases of reads that correspond to repeats, especially the intergenic and intronic L1 and L2 transposable elements. We also observed increased expression of the antisense strands compared to the sense strands in intronic and regulatory regions (1 kb up- and downstream) of the genes, suggesting that antisense transcription could be one of the main mechanisms for transposon regulation in the horse under stress conditions. We identified a large number of transcripts corresponding to intergenic and intronic regions putatively associated with new transcriptional elements. Gene expression and pathway analysis allowed us to identify several biological processes and molecular functions that may be involved with exercise-induced stress. Ontology clustering reflected mechanisms that are already known to be stress activated (e.g., chemokine-type cytokines, Toll-like receptors, and kinases), as well as “nucleic acid binding” and “signal transduction activity” functions. There was also a general and transient decrease in the global rates of protein synthesis, which would be expected after strenuous global stress. In sum, our network analysis points toward the involvement of specific gene clusters in equine exercise-induced stress, including those involved in inflammation, cell signaling, and immune interactions.

Quantification of Equid herpesvirus 5 DNA in clinical and necropsy specimens collected from a horse with equine multinodular pulmonary fibrosis

A 15-year-old Belgian gelding was referred for fever, depression, and respiratory distress. Lung biopsy revealed interstitial fibrosis consistent with chronic interstitial pneumonia. Equid herpesvirus 5 (EHV-5) DNA was detected by polymerase chain reaction (PCR) in bronchoalveolar lavage and biopsy specimens. A presumptive diagnosis of equine multinodular pulmonary fibrosis (EMPF) was made, and the horse was administered a systemic treatment with corticosteroids and antiviral drugs. Despite initial clinical improvement, 4 weeks later, the condition of the horse rapidly deteriorated, and the animal was euthanized. Postmortem examination confirmed the presumptive diagnosis of EMPF. The EHV-5 DNA load in different tissues was estimated using a quantitative real-time PCR. Lung had a remarkable viral load, higher than in other organs, especially within the pulmonary fibrotic nodules, and a linkage between high viral burden and the most severely affected tissues was observed. The results suggest that the quantitative real-time PCR is a useful tool to quantify the EHV-5 load in different organs and to understand the relationship between EHV-5 and EMPF. The bronchoalveolar lavage was determined to be a good clinical sample to estimate the EHV-5 load in lung.

Athletic humans and horses: Comparative analysis of interleukin-6 (IL-6) and IL-6 receptor (IL-6R) expression in peripheral blood mononuclear cells in trained and untrained subjects at rest

BackgroundHorses and humans share a natural proclivity for athletic performance. In this respect, horses can be considered a reference species in studies designed to optimize physical training and disease prevention. In both species, interleukin-6 (IL-6) plays a major role in regulating the inflammatory process induced during exercise as part of an integrated metabolic regulatory network. The aim of this study was to compare IL-6 and IL-6 receptor (IL-6R) mRNA expression in peripheral blood mononuclear cells (PBMCs) in trained and untrained humans and horses.ResultsNine highly trained male swimmers (training volume: 21.6 ± 1.7 h/wk in 10-12 sessions) were compared with two age-matched control groups represented by eight lightly trained runners (training volume: 6.4 ± 2.6 h/wk in 3-5 sessions) and nine untrained subjects. In addition, eight trained horses (training volume: 8.0 ± 2.1 h/wk in 3-4 sessions) were compared with eight age-matched sedentary mares. In humans, IL-6 mRNA levels in PBMCs determined by quantitative reverse transcription-polymerase chain reaction were significantly higher in highly trained subjects, whereas IL-6R expression did not differ among groups. In horses, transcripts of both IL-6 and IL-6R were significantly up-regulated in the trained group.ConclusionsUp-regulation of IL-6R expression in PBMCs in horses could reflect a mechanism that maintains an adequate anti-inflammatory environment at rest through ubiquitous production of anti-inflammatory cytokines throughout the body. These findings suggest that the system that controls the inflammatory response in horses is better adapted to respond to exercise than that in humans.

Geographic structuring of global EIAV isolates: a single origin for New World strains?

Equine infectious anaemia virus (EIAV) is classified within the Retroviridae and, like other lentivirus, has the propensity for considerable antigenic variation. An extensive phylogenetic analysis in Bayesian fashion, with significant amounts of new EIAV gag sequence information, revealed a strong geographic compartmentalization clearly related to the phylogeographic history of modern horses, pointing out that New World EIAV strains form a distinct group with a potentially common origin. This evidence suggests that a single founder event may have occurred during the reintroduction of horses to the Americas by European colonists in the 15th century, a possibility that raises many interesting scenarios with implications for all evolutionary and ecological studies.

Microarray analysis after strenuous exercise in peripheral blood mononuclear cells of endurance horses

It is known that moderate physical activity may have beneficial effects on health, whereas strenuous effort induces a state resembling inflammation. The molecular mechanisms underlying the cellular response to exercise remain unclear, although it is clear that the immune system plays a key role. It has been hypothesized that the physio-pathological condition that develops in athletes subjected to heavy training is caused by derangement of cellular immune regulation. The purpose of the present study was to obtain information on endurance horse gene transcription under strenuous conditions and to identify candidate genes causing immune system derangement. We performed a wide gene expression scan, using microarray technology, on peripheral blood mononuclear cells of ten horses chosen from high-level participants in national and international endurance races. The use of three different timepoints revealed changes in gene expression when post-effort samples (T1, taken immediately after the race; and T2, taken 24 h after the race) were compared with basal sample (T0, at rest). Statistical analysis showed no differences in gene expression between T0 and T2 samples, indicating complete restoration of homeostasis by 24 h after racing, whereas T1 showed strong modulation of expression, affecting 132 genes (97 upregulated, 35 downregulated). Ingenuity pathway analysis revealed that the main mechanisms and biofunctions involved were significantly associated with immunological and inflammatory responses. Real-time PCR was performed on 26 gene products to validate the array data.

Clinical, serological and molecular investigations of EHV-1 and EHV-4 in 15 unweaned thoroughbred foals

Fifteen unweaned thoroughbred foals, born on a stud farm to vaccinated mares, were clinically monitored during their first six months of life and repeatedly tested for equine herpesvirus type 1 (ehv-1) and equine herpesvirus type 4 (ehv-4). Nasopharyngeal swabs and blood samples were collected and screened respectively by pcr and seroneutralisation to detect the presence of the virus, explore its role as a possible cause of respiratory disease, and to assess the efficiency of the PCR for the diagnosis of this disease. The foals were divided into three groups on the basis of their clinical signs and whether they had seroconverted to ehv-1 and/or ehv-4: first, foals with no clinical signs of disease that had not seroconverted; secondly, foals with clinical signs that had seroconverted, and thirdly, foals with clinical signs that had not seroconverted. The results indicated that the viruses circulated on the stud farm despite stringent vaccination regimens against them, and confirmed their association with respiratory disease. The absence of significantly different pcr results among the three groups of foals showed that the pcr was effective in confirming the circulation of the viruses on the premises without being particularly helpful as a diagnostic tool.

Transcription of LINE-derived sequences in exercise-induced stress in horses

A large proportion of mammalian genomes is represented by transposable elements (TE), most of them being long interspersed nuclear elements 1 (LINE-1 or L1). An increased expression of LINE-1 elements may play an important role in cellular stress-related conditions exerting drastic effects on the mammalian transcriptome. To understand the impact of TE on the known horse transcriptome, we masked the horse EST database, pointing out that the amount is consistent with other major vertebrates. A previously developed transcript-derived fragments (TDFs) dataset, deriving from exercise-stimulated horse peripheral blood mononuclear cells (PBMCs), was found to be enriched with L1 (26.8% in terms of bp). We investigated the involvement of TDFs in exercise-induced stress through bioinformatics and gene expression analysis. Results indicate that LINE-derived sequences are not only highly but also differentially expressed during physical effort, hinting at interesting scenarios in the regulation of gene expression in relation to exercise.

Exercise-induced up-regulation of MMP-1 and IL-8 genes in endurance horses

BackgroundThe stress response is a critical factor in the training of equine athletes; it is important for performance and for protection of the animal against physio-pathological disorders.In this study, the molecular mechanisms involved in the response to acute and strenuous exercise were investigated using peripheral blood mononuclear cells (PBMCs).ResultsQuantitative real-time PCR (qRT-PCR) was used to detect modifications in transcription levels of the genes for matrix metalloproteinase-1 (MMP-1) and interleukin 8 (IL-8), which were derived from previous genome-wide expression analysis. Significant up-regulation of these two genes was found in 10 horses that had completed a race of 90–120 km in a time-course experimental design.ConclusionThese results suggest that MMP-1 and IL-8 are both involved in the exercise-induced stress response, and this represents a starting point from which to understand the adaptive responses to this phenomenon.