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Herpesviruses establish a well-adapted balance with their host's immune system. Despite this co-evolutionary balance, infections can lead to severe disease including neurological disorders in their natural host. In horses, equine herpesvirus 1 (EHV-1) causes respiratory disease, abortions, neonatal foal death and myeloencephalopathy (EHM) in ~10 % of acute infections worldwide. Many aspects of EHM pathogenesis and protection from EHM are still poorly understood. However, it has been shown that the incidence of EHM increases to >70 % in female horses >20 years of age. In this study we used old mares as an experimental equine EHV-1 model of EHM to identify host-specific factors contributing to EHM. Following experimental infection with the neuropathogenic strain EHV-1 Ab4, old mares and yearling horses were studied for 21 days post-infection. Nasal viral shedding and cell-associated viremia were assessed by quantitative PCR. Cytokine/chemokine responses were evaluated in nasal secretions and cerebrospinal fluid (CSF) by Luminex assay and in whole blood by quantitative real-time PCR. EHV-1-specific IgG sub-isotype responses were measured by ELISA. All young horses developed respiratory disease and a bi-phasic fever post-infection, but only 1/9 horses exhibited ataxia. In contrast, respiratory disease was absent in old mares, but all old mares developed EHM that resulted in euthanasia in 6/9 old mares. Old mares also presented significantly decreased nasal viral shedding but higher viremia coinciding with a single fever peak at the onset of viremia. According to clinical disease manifestation, horses were sorted into an EHM group (nine old horses and one young horse) and a non-EHM group (eight young horses) for assessment of host immune responses. Non-EHM horses showed an early upregulation of IFN-α (nasal secretions), IRF7/IRF9, IL-1β, CXCL10 and TBET (blood) in addition to an IFN-γ upregulation during viremia (blood). In contrast, IFN-α levels in nasal secretions of EHM horses were low and peak levels of IRF7, IRF9, CXCL10 and TGF-β (blood) coincided with viremia. Moreover, EHM horses showed significantly higher IL-10 levels in nasal secretions, peripheral blood mononuclear cells and CSF and higher serum IgG3/5 antibody titres compared to non-EHM horses. These results suggest that protection from EHM depends on timely induction of type 1 IFN and upregulation cytokines and chemokines that are representative of cellular immunity. In contrast, induction of regulatory or TH-2 type immunity appeared to correlate with an increased risk for EHM. It is likely that future vaccine development for protection from EHM must target shifting this 'at-risk' immunophenotype.

Objectives: Diagnosis of equine septic arthritis is not straightforward, and increasing time between onset, diagnosis, and treatment can have serious consequences for quality of life. Defensins are used in diagnosis of human joint infection. The presence of beta defensins (BDs) in equine synovial fluid and their utility as a biomarker of sepsis has not been investigated; therefore, our objectives were to (1) compare in vitro gene expression of lipopolysaccharide (LPS)-stimulated equine neutrophils to unstimulated neutrophils and (2) compare BD protein expression from normal, aseptically inflamed, and septic equine joints., Animals: 5 horses for isolated neutrophil BD expression and 21 synovial fluid samples from 14 horses., Procedures: RT-qPCR analysis was performed for BD gene expression of stimulated and unstimulated equine peripheral neutrophils. BD protein expression was evaluated from equine joints with no disease, aseptic inflammation, and septic inflammation using a commercial ELISA designed for horses and analyzed with a Kruskal-Wallis test (significant at P < .05)., Results: A significant increase was noted in expression of BD-3 in LPS stimulated as compared to unstimulated neutrophils. There were no significant differences in BD expression noted between joints with no disease, aseptic inflammation, and septic inflammation. Low case numbers and different types of cases in the aseptic inflammation group were main limitations. BD expression patterns in samples from stimulated equine peripheral neutrophils and synovial fluid were identified., Clinical Relevance: BDs are detectable in equine synovial fluid and can be stimulated from peripheral neutrophils. Further examination is needed to define their role as biomarkers of joint disease.

Toxoplasma gondii is hypothesized to manipulate the behavior of warm-blooded hosts to promote trophic transmission into the parasite's definitive feline hosts. A key prediction of this hypothesis is that T. gondii infections of non-feline hosts are associated with costly behavior toward T. gondii's definitive hosts; however, this effect has not been documented in any of the parasite's diverse wild hosts during naturally occurring interactions with felines. Here, three decades of field observations reveal that T. gondii-infected hyena cubs approach lions more closely than uninfected peers and have higher rates of lion mortality. We discuss these results in light of 1) the possibility that hyena boldness represents an extended phenotype of the parasite, and 2) alternative scenarios in which T. gondii has not undergone selection to manipulate behavior in host hyenas. Both cases remain plausible and have important ramifications for T. gondii's impacts on host behavior and fitness in the wild.

Equine herpesvirus-1 is the cause of respiratory disease, abortion, and equine herpesvirus myeloencephalopathy (EHM) in horses worldwide. EHM affects as many as 14% of infected horses and a cell-associated viremia is thought to be central for EHM pathogenesis. While EHM is infrequent in younger horses, up to 70% of aged horses develop EHM. The aging immune system likely contributes to EHM pathogenesis; however, little is known about the host factors associated with clinical EHM. Here, we used the "old mare model" to induce EHM following EHV-1 infection. Peripheral blood mononuclear cells (PBMCs) of horses prior to infection and during viremia were collected and RNA sequencing with differential gene expression was used to compare the transcriptome of horses that did (EHM group) and did not (non-EHM group) develop clinical EHM. Interestingly, horses exhibiting EHM did not show respiratory disease, while non-EHM horses showed significant respiratory disease starting on day 2 post infection. Multiple immune pathways differed in EHM horses in response to EHV-1. These included an upregulation of IL-6 gene expression, a dysregulation of T-cell activation through AP-1 and responses skewed towards a T-helper 2 phenotype. Further, a dysregulation of coagulation and an upregulation of elements in the progesterone response were observed in EHM horses.

Equine herpesvirus 1 (EHV-1) affects horses worldwide and causes respiratory disease, abortions, and equine herpesvirus myeloencephalopathy (EHM). Following infection, a cell-associated viremia is established in the peripheral blood mononuclear cells (PBMCs). This viremia is essential for transport of EHV-1 to secondary infection sites where subsequent immunopathology results in diseases such as abortion or EHM. Because of the central role of PBMCs in EHV-1 pathogenesis, our goal was to establish a gene expression analysis of host and equine herpesvirus genes during EHV-1 viremia using RNA sequencing. When comparing transcriptomes of PBMCs during peak viremia to those prior to EHV-1 infection, we found 51 differentially expressed equine genes (48 upregulated and 3 downregulated). After gene ontology analysis, processes such as the interferon defense response, response to chemokines, the complement protein activation cascade, cell adhesion, and coagulation were overrepresented during viremia. Additionally, transcripts for EHV-1, EHV-2, and EHV-5 were identified in pre- and post-EHV-1-infection samples. Looking at micro RNAs (miRNAs), 278 known equine miRNAs and 855 potentially novel equine miRNAs were identified in addition to 57 and 41 potentially novel miRNAs that mapped to the EHV-2 and EHV-5 genomes, respectively. Of those, 1 EHV-5 and 4 equine miRNAs were differentially expressed in PBMCs during viremia. In conclusion, this work expands our current knowledge about the role of PBMCs during EHV-1 viremia and will inform the focus on future experiments to identify host and viral factors that contribute to clinical EHM.

Spring and late fall grazing can lead to metabolic problems in horses (Equus caballus L.) as a result of elevated nonstructural carbohydrates (NSC) in pastures. Therefore, the objectives were to determine the impact of different forage species on blood glucose and insulin concentrations of horses during the spring and late fall. Research was conducted in May (spring) and October (late fall) in St. Paul, MN. Alfalfa (Medicago sativa L.) and mixed perennial cool-season grasses (CSG) were grazed in spring, and CSG and teff (Eragrostis tef [Zucc.] Trotter) were grazed in late fall by six adult horses randomly assigned to a forage in a cross-over design. Jugular catheters were inserted 1 hour before the start of grazing, and horses had access to pasture from 8 AM to 4 PM in the spring and 8 AM to 12 PM in the late fall. Jugular venous blood samples were collected from each horse before being turned out (0 hours) and then at 2 hours intervals after turnout. Plasma and serum samples were collected and analyzed for glucose and insulin, respectively. Corresponding forage samples were taken by hand harvest. Seasons were analyzed separately, and data were analyzed using the MIXED procedure in SAS with P ≤ .05. Teff had lower NSC compared with CSG in the late fall (P ≤ .05) with subsequently lower average glucose, average insulin, and peak insulin in horses grazing teff compared with CSG (P ≤ .05). These results suggest grazing teff could lower the glucose and insulin response of horses during late fall., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Elevated nonstructural carbohydrate (NSC) values in pasture forages can cause adverse health effects in some horses (Equus caballus L.). The objectives of this study were to determine the impact of different forage species on blood glucose and insulin concentrations of horses throughout the grazing season. Research was conducted in July (summer) and September (fall) in St. Paul, MN. Alfalfa (Medicago sativa L.), mixed perennial cool-season grasses (CSG), and teff (Eragrostis tef [Zucc.] Trotter) pastures were grazed by six horses (24 ± 2 years) that were randomly assigned to one of three forage types in a replicated Latin-square design. Jugular catheters were inserted 1 hour before the start of grazing and horses had access to pasture each day from 08:00 to 16:00 hours. Jugular venous blood samples were collected from each horse before being turned out (0 hours) and then at 2-hour intervals following turnout. Plasma and serum samples were collected and analyzed for glucose and insulin, respectively. Corresponding forage samples were taken by hand harvest. Seasons were analyzed separately and data were analyzed using the MIXED procedure in SAS with P ≤ .05. Teff generally had lower (P ≤ .05) equine digestible energy, crude protein, and NSC compared to the other forages. Differences in peak insulin were observed between horses grazing CSG and teff during the fall grazing (P ≤ .05). These results suggest grazing teff could lower the glucose and insulin response of some horses., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Diagnosis of equine pituitary pars intermedia dysfunction (PPID) remains a challenge as multiple factors (stress, exercise, and time of year) influence ACTH and cortisol concentrations. To assess endocrine status in a study designed to evaluate the effects of age and diet on glucose and insulin dynamics, we performed thyrotropin-releasing hormone (TRH) stimulation tests and overnight dexamethasone suppression tests in March, May, August, and October on 16 healthy Thoroughbred and Standardbred mares and geldings. Horses were grouped by age: adult (mean ± SD; 8.8 ± 2.9 yr; n = 8) and aged (20.6 ± 2.1 yr; n = 8). None of the horses showed clinical signs (hypertrichosis, regional adiposity, skeletal muscle atrophy, lethargy) of pituitary pars intermedia dysfunction. Horses were randomly assigned to groups of 4, blocked for age, and fed grass hay plus 4 isocaloric concentrate diets (control, starch-rich, fiber-rich, and sugar-rich) using a balanced Latin square design. Data were analyzed using a multivariable linear mixed regression model. Baseline ACTH was significantly higher in aged horses (mean ± standard error of the mean; 60.0 ± 10.7 pg/mL) adapted to the starch-rich diet compared to adult horses (15.7 ± 12.0 pg/mL) on the same diet (P = 0.017). After controlling for age and diet, baseline ACTH concentrations were significantly increased in October (57.7 ± 7.1 pg/mL) compared to March (13.2 ± 7.1 pg/mL; P < 0.001), May (12.4 ± 7.1 pg/mL; P < 0.001), and August (24.2 ± 7.1 pg/mL; P < 0.001), whereas post-TRH ACTH was higher in August (376.6 ± 57.6 pg/mL) and October (370.9 ± 57.5 pg/mL) compared to March (101.9 ± 57.3 pg/mL; P < 0.001) and May (74.5 ± 57.1 pg/mL; P < 0.001). Aged horses had significantly higher post-dexamethasone cortisol on the starch-rich diet (0.6 ± 0.1 μg/dL) compared to the sugar-rich diet (0.2 ± 0.1 μg/dL; P = 0.021). Post-dexamethasone cortisol was significantly higher in October (0.6 ± 0.1 μg/dL) compared to March (0.3 ± 0.1 μg/dL; P = 0.005), May (0.2 ± 0.1 μg/dL; P < 0.001), and August (0.3 ± 0.1 μg/dL; P = 0.004). Breed did not influence ACTH or cortisol measurements. In conclusion, in addition to age and time of year, diet is a potential confounder as animals on a starch-rich diet may be incorrectly diagnosed with pituitary pars intermedia dysfunction., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Glucose and insulin dynamics may be different in adult and aged horses., Objectives: To determine the effects of age and dietary carbohydrates on glucose and insulin dynamics in healthy horses., Study Design: Balanced Latin square with four isocaloric diets: CONTROL (hay plus restricted-starch-and-sugar fortified pellets), STARCH (control plus kibbled corn), FIBER (control plus unmolassed sugar beet pulp/soybean hull pellets) and SUGAR (control plus dextrose powder)., Methods: A total of 16 healthy Thoroughbreds and Standardbreds divided into two age groups: ADULT (8.8 ± 2.9 years; n = 8) and AGED (20.6 ± 2.1 years; n = 8). Following dietary adaptation, horses underwent an insulin-modified frequently sampled intravenous glucose tolerance test (FSIGTT), modified oral sugar test (OST) and dietary meal challenge. Outcome variables included: insulin sensitivity (SI), disposition index (DI), glucose effectiveness (Sg) and acute insulin response to glucose (AIRg) from the FSIGTT; peak glucose, peak insulin, time to peak, area under the curve for glucose (AUCg) and insulin (AUCi) from the OST and dietary meal challenge. Data were analyzed using multivariable linear mixed regression modelling., Results: AIRg was higher in AGED (mean [95% confidence interval]; 582.0 [455.0-709.0]) vs. ADULT (358.0 [224.0-491.0]; P = 0.03). ADULT and AGED horses had a higher SI on STARCH (adult: 3.3 [2.3-4.2]; aged: 2.8 [1.9-3.7]) and SUGAR (adult: 3.4 [2.5-4.3]; aged: 4.0 [3.1-4.9]) diets compared with CONTROL (adult: 2.0 [1.1-2.9], P = 0.029 (starch), P = 0.009 (sugar); aged: 1.4 [0.5-2.2], P = 0.009 (starch), P < 0.001 (sugar)). Feeding a STARCH (adult: 21581.0 [15029.0-28133.0]; aged: 35205.0 [29194.0-41216.0]) or SUGAR (adult: 26050.0 [19885.0-32215.0]; aged: 25720.0 [19770.0-31670.0]) meal resulted in postprandial hyperinsulinaemia (AUCi)., Main Limitations: Study cohort contained two insulin-sensitive breeds and no insulin-resistant breeds., Conclusions: Age and diet should be considered when evaluating glucose and insulin dynamics., (© 2017 EVJ Ltd.)

Objective: The purpose of this study was to determine whether there was a correlation between circulating and intra-synovial Dkk-1 and radiographic signs of equine osteoarthritis., Methods: Circulating and intra-synovial Dkk-1 levels were measured in clinical cases using a commercially available human Dkk-1 ELISA. Radiographs were performed of the joints from which fluid was collected and these were assessed and scored by a boarded radiologist for joint narrowing, subchondral bone sclerosis, subchondral bone lysis, and periarticular modelling. Comparisons were made between radiographic scores and the concentrations of Dkk-1 using a Kruskal-Wallis one-way ANOVA. Correlations were calculated using Kendall's statistic., Results: A total of 42 synovial fluid samples from 21 horses were collected and used in the analysis. No significant correlation was identified between Dkk-1 concentrations and radiographic signs of osteoarthritis. Intra-synovial Dkk-1 concentrations were significantly greater (p <0.001) in low motion joints (mean concentration, 232.68 pg/mL; range, 109.07-317.17) when compared to high-motion joints (28.78 pg/mL; 0.05-186.44 pg/mL) (p <0.001)., Clinical Significance: Low motion joints have significantly higher concentrations of Dkk-1 compared to high motion joints. Further research is needed to establish the importance of this finding and whether potential diagnostic or therapeutic applications of Dkk-1 exist in the horse.