Weaning, particularly the widespread practice of abrupt separation of the mare and foal, has been shown to be a stressful event for horses. Physiological changes in foals measured after weaning include increased blood cortisol concentrations and a subsequent decrease in cell-mediated immune responses. In the randomized, double-blinded, placebo-controlled trial reported here, we assessed the effect of an equine appeasing pheromone (EAP; Modipher EQ, E.A.P. Mist [Pherosynthese s.n.c., LeRieu Neuf, Saint-Saturnin-les-Apt, France]) as an aid for reducing the behavioral and physiological signs of stress during weaning. Fourteen quarter horse foals were separated from their dam (equid mother) between 105 and 146 days of age, in age-matched pairs, and placed in 3.66×3.66m stalls (one treated and one control foal in each stall). Treated foals received the synthetic analogue of the EAP by intranasal wipe 30 minutes before separation and twice daily thereafter for 48 hours. Control foals received placebo by intranasal wipe on the same schedule. The foals were continuously videotaped for 48 hours postweaning to monitor behavioral responses. Blood samples were drawn 24 hours pretreatment and 24 and 48 hours postweaning for evaluation of physiological indicators of stress (serum cortisol) and cytokines as stress-related and immune-mediated response parameters. Interestingly, although behavioral and serum cortisol measures were similar between groups, treatment with EAP had a significant (P<0.05) effect on interleukin 6 and transforming growth factor β, whereas monocyte opiate receptor 1 was significantly upregulated in both groups independent of treatment when compared with baseline values. Although the link between EAP treatment and activation of the measured cytokines remains unexplained, our findings suggest immune-related gene transcription focused on the acute causes of stress in a time frame shortly after weaning. © 2013 Elsevier Inc.

Background

Imported horses that have undergone recent long distance transport might represent a serious risk for spreading infectious respiratory pathogens into populations of horses.

Objective

To investigate the frequency of shedding of respiratory pathogens in recently imported horses.

Animals

All imported horses with signed owner consent (n = 167) entering a USDA quarantine for contagious equine metritis from October 2014 to June 2016 were enrolled in the study.

Methods

Prospective observational study. Enrolled horses had a physical examination performed and nasal secretions collected at the time of entry and subsequently if any horse developed signs of respiratory disease during quarantine. Samples were assayed for equine influenza virus (EIV), equine herpesvirus type-1, -2, -4, and -5 (EHV-1, -2, -4, -5), equine rhinitis virus A (ERAV), and B (ERBV) and Streptococcus equi subspecies equi (S. equi) using quantitative PCR (qPCR).

Results

Equine herpesviruses were detected by qPCR in 52% of the study horses including EHV-2 (28.7%), EHV-5 (40.7%), EHV-1 (1.2%), and EHV-4 (3.0%). Clinical signs were not correlated with being qPCR-positive for EHV-4, EHV-2, or EHV-5. None of the samples were qPCR-positive for EIV, ERAV, ERBV, and S. equi. The qPCR assay failed quality control for RNA viruses in 25% (46/167) of samples.

Conclusions and clinical importance

Clinical signs of respiratory disease were poorly correlated with qPCR positive status for EHV-2, -4, and -5. The importance of γ-herpesviruses (EHV-2 and 5) in respiratory disease is poorly understood. Equine herpesvirus type-1 or 4 (EHV-1 or EHV-4) were detected in 4.2% of horses, which could have serious consequences if shedding animals entered a population of susceptible horses. Biosecurity measures are important when introducing recently imported horses into resident US populations of horses.

The genomes of four strains (MB11, MB14, MB30, and MB66) of the species Corynebacterium pseudotuberculosis biovar equi were sequenced on the Ion Torrent PGM platform, completely assembled, and their gene content and structure were analyzed. The strains were isolated from horses with distinct signs of infection, including ulcerative lymphangitis, external abscesses on the chest, or internal abscesses on the liver, kidneys, and lungs. The average size of the genomes was 2.3 Mbp, with 2169 (Strain MB11) to 2235 (Strain MB14) predicted coding sequences (CDSs). An optical map of the MB11 strain generated using the KpnI restriction enzyme showed that the approach used to assemble the genome was satisfactory, producing good alignment between the sequence observed in vitro and that obtained in silico. In the resulting Neighbor-Joining dendrogram, the C. pseudotuberculosis strains sequenced in this study were clustered into a single clade supported by a high bootstrap value. The structural analysis showed that the genomes of the MB11 and MB14 strains were very similar, while the MB30 and MB66 strains had several inverted regions. The observed genomic characteristics were similar to those described for other strains of the same species, despite the number of inversions found. These genomes will serve as a basis for determining the relationship between the genotype of the pathogen and the type of infection that it causes.

Seven genomes of Corynebacterium pseudotuberculosis biovar equi were sequenced on the Ion Torrent PGM platform, generating high-quality scaffolds over 2.35 Mbp. This bacterium is the causative agent of disease known as "pigeon fever" which commonly affects horses worldwide. The pangenome of biovar equi was calculated and two phylogenomic approaches were used to identify clustering patterns within Corynebacterium genus. Furthermore, other comparative analyses were performed including the prediction of genomic islands and prophages, and SNP-based phylogeny. In the phylogenomic tree, C. pseudotuberculosis was divided into two distinct clades, one formed by nitrate non-reducing species (biovar ovis) and another formed by nitrate-reducing species (biovar equi). In the latter group, the strains isolated from California were more related to each other, while the strains CIP 52.97 and 1/06-A formed the outermost clade of the biovar equi. A total of 1,355 core genes were identified, corresponding to 42.5% of the pangenome. This pangenome has one of the smallest core genomes described in the literature, suggesting a high genetic variability of biovar equi of C. pseudotuberculosis. The analysis of the similarity between the resistance islands identified a higher proximity between the strains that caused more severe infectious conditions (infection in the internal organs). Pathogenicity islands were largely conserved between strains. Several genes that modulate the pathogenicity of C. pseudotuberculosis were described including peptidases, recombination enzymes, micoside synthesis enzymes, bacteriocins with antimicrobial activity and several others. Finally, no genotypic differences were observed between the strains that caused the three different types of infection (external abscess formation, infection with abscess formation in the internal organs, and ulcerative lymphangitis). Instead, it was noted that there is a higher phenetic correlation between strains isolated at California compared to the other strains. Additionally, high variability of resistance islands suggests gene acquisition through several events of horizontal gene transfer.

The genome of Corynebacterium pseudotuberculosis MB20 bv. equi was sequenced using the Ion Personal Genome Machine (PGM) platform, and showed a size of 2,363,089 bp, with 2,365 coding sequences and a GC content of 52.1%. These results will serve as a basis for further studies on the pathogenicity of C. pseudotuberculosis bv. equi.