Different expression of Defensin-B gene in the endometrium of mares of different age during the breeding season.

BACKGROUND: Despite being one of the major causes of infertility in mares, the mechanisms responsible for equine endometrosis are still unclear and controversial. In the last few years, many investigations focused on local immune response modulation. Since it is generally accepted that endometrial fibrosis increases with age, we hypothesize that older mares could show altered local immune modulation, initiating a pro-inflammatory and tissue remodeling cascade of events that could lead to endometrosis. The aim of this study, indeed, is to evaluate and describe the local gene expression of genes involved in acute inflammatory response and fibrosis (COL1A1, COL3A1, TNFA, MMP9, IL6, TGFB1 and TGFBR1), together with others associated to immune modulation (DEFB4B, IDO1 and FOXP3), in uterine specimens from mares of different age. RESULTS: Twenty-five Standardbred mares were involved in the study with age ranging from 7 to 19 years (mean 10.40 ± 4.42). They were divided by age into two groups: G1 (n = 15, less than 10 years old) and G2 (N = 10, greater than 11 years old). Specimens from the uterus' right horn-body junction were collected and processed for histology evaluation and RT-qPCR assay.Gene expression of DEFB4B, MMP9 and TNFA was higher in younger mares, suggesting a balance in immune modulation and tissue remodeling. Interleukin-6 and COL3A1 gene expressions were greater in older animals, probably indicating inflammatory pathways activation and fibrosis increase. Although no differences in fibrosis and inflammation distribution could be found with histological examination among G1 and G2, our results suggest a possible involvement of DEF4BB in regulating the local immune response in younger mare's uterus (G1); age may contribute to the dis-regulation of DEFB4B transcription and, indirectly, influence the extracellular matrix homeostasis. Transcription of IDO1 and FOXP3 genes, instead, does not seem to be age related, or to be involved in local immune-response and tissue remodeling functions. CONCLUSIONS: Further investigations are needed in order to clarify the interactions between the expression of DEFB4B, IL6, TNFA, COL3A1 and MMP9 and other local signals of immune-modulation and tissue remodeling, in mares in a prospective study design.

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.

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.

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.

Deep sequencing and variant analysis of an Italian pathogenic field strain of equine infectious anaemia virus.

Equine infectious anaemia virus (EIAV) is a lentivirus with an almost worldwide distribution that causes persistent infections in equids. Technical limitations have restricted genetic analysis of EIAV field isolates predominantly to gag sequences resulting in very little published information concerning the extent of inter-strain variation in pol, env and the three ancillary open reading frames (ORFs). Here, we describe the use of long-range PCR in conjunction with next-generation sequencing (NGS) for rapid molecular characterization of all viral ORFs and known transcription factor binding motifs within the long terminal repeat of two EIAV isolates from the 2006 Italian outbreak. These isolates were from foals believed to have been exposed to the same source material but with different clinical histories: one died 53 days post-infection (SA) while the other (DE) survived 5 months despite experiencing multiple febrile episodes. Nucleotide sequence identity between the isolates was 99.358% confirming infection with the same EIAV strain with most differences comprising single nucleotide polymorphisms in env and the second exon of rev. Although the synonymous:non-synonymous nucleotide substitution ratio was approximately 2:1 in gag and pol, the situation is reversed in env and ORF3 suggesting these sequences are subjected to host-mediated selective pressure. EIAV proviral quasispecies complexity in vivo has not been extensively investigated; however, analysis suggests it was relatively low in SA at the time of death. These results highlight advantages of NGS for molecular characterization of EIAV namely it avoids potential artefacts generated by traditional composite sequencing strategies and can provide information about viral quasispecies complexity.

Stored Canine Whole Blood Units: What is the Real Risk of Bacterial Contamination?

BACKGROUND: Bacterial contamination of whole blood (WB) units can result in transfusion-transmitted infection, but the extent of the risk has not been established and may be underestimated in veterinary medicine. OBJECTIVES: To detect, quantify, and identify bacterial microorganisms in 49 canine WB units during their shelf life. ANIMALS: Forty-nine healthy adult dogs. METHODS: Forty-nine WB units were included in the study. Immediately after collection, 8 sterile samples from the tube segment line of each unit were aseptically collected and tested for bacterial contamination on days 0, 1, 7, 14, 21, 28, 35, and 42 of storage. A qPCR assay was performed on days 0, 21, and 35 to identify and quantify any bacterial DNA. RESULTS: On bacterial culture, 47/49 blood units were negative at all time points tested, 1 unit was positive for Enterococcus spp. on days 0 and 1, and 1 was positive for Escherichia coli on day 35. On qPCR assay, 26 of 49 blood units were positive on at least 1 time point and the bacterial loads of the sequences detected (Propionobacterium spp., Corynebacterium spp., Caulobacter spp., Pseudomonas spp., Enterococcus spp., Serratia spp., and Leucobacter spp.) were <80 genome equivalents (GE)/µL. CONCLUSIONS AND CLINICAL IMPORTANCE: Most of the organisms detected were common bacteria, not usually implicated in septic transfusion reactions. The very low number of GE detected constitutes an acceptable risk of bacterial contamination, indicating that WB units have a good sanitary shelf life during commercial storage.

cDNA AFLP-based techniques for studying transcript profiles in horses.

The identification of differentially expressed genes is a fundamental prerequisite for understanding the molecular regulation of most physiological and pathological processes. Among the procedures employed to compare mRNA populations, those that are gel-based appear to hold great promise and are considered excellent tools for studying gene expression in species, such as the equine one, for which little genomic information is available. In the present study, we evaluated two techniques for studying mRNA profiles in horse tissue, one referred to the cDNA-amplified fragment length polymorphism (AFLP) that we called C-AFLP (classical cDNA-AFLP) protocol and the other to ordered differential display (ODD) with some modifications that we named S-AFLP (systematic cDNA-AFLP). Both techniques can be applied in live animals because of the small amount of sample required. We applied the S-AFLP to investigate horse transcript profile modifications during physical exercise. We found two transcripts that are mostly expressed during exercise and immediately after the end of it.

Effect of training status on immune defence related gene expression in Thoroughbred: are genes ready for the sprint?

Athletic performance is both a stress factor and an adaptive response to exercise that may be modulated by training, reduce inflammation and help prevent disease. Studies on the endocrinology of exercise and training have demonstrated the existence of an integrated metabolic network of hormone and cytokine regulation. Subsequent molecular studies have shown that repeated bouts of exercise may establish new basal levels of gene expression at rest. The Thoroughbred horse may be a useful 'exercise model' for inter-individual comparisons between subjects with homogeneous genetic and environmental backgrounds and similar exercise management practices. In this study, the effects of training and acute effort on gene expression were evaluated with a real time PCR approach in athletic (n=10) and sedentary horses (n=9), using a previously characterised panel of genes known to be highly modulated during effort (CXCL2, TLR4, IL1ß, IL8, IL1RII, IL18, IL6 and CEBPß). A 'rest comparison' was performed to evaluate a training effect in both groups while a 'race comparison' was performed in athletic horses only (before, immediately after, and 12h after racing) to determine the effect of acute effort. The results indicated that many of the investigated genes (TLR4, IL1ß, IL1RII, IL18, IL6 and CEBPß) were expressed to a greater extent in athletic horses compared to sedentary animals when both were at rest. However, a time-course comparison in the athletic horses revealed that genes exhibiting the highest levels of expression at rest did not show significant changes after the race. The findings suggested that training may exert a conditioning on gene expression at rest leading to a more prompt response to exercise-induced stress in Thoroughbreds.