Ivermectin performance in horses diagnosed with equine endocrine disorders.

Anthelmintic performance against equine cyathostomins can be evaluated by two different non-terminal measures; the Fecal Egg Count Reduction Test (FECRT) and the Egg Reappearance Period (ERP). Most available FECRT and ERP data have been determined in populations of young horses, and very little information is available from mature and senior horses. Furthermore, it is unknown how commonly occurring equine endocrine disorders such as Insulin dysregulation (ID) and Pituitary pars intermedia dysfunction (PPID) may interfere with these measurements, but it has been suggested that horses with these conditions could be more susceptible to parasitic infections. A research population of senior horses and horses with or without PPID, ID, or both were enrolled in this study. All strongylid egg count positive horses were included in an ivermectin (200 µg/kg) efficacy study. These were distributed among the following groups: ID: six, PPID: three, PPID and ID: seven, and healthy controls: three. Strongylid fecal egg counts were determined on the day of ivermectin administration, at two weeks post deworming, and on weekly intervals until eight weeks post treatment. Determination of FECRT and ERP were carried out following World Association for the Advancement of Veterinary Parasitology guidelines. Results revealed high ivermectin efficacy with mean egg count reduction at 99.7% or above in all groups at two weeks post treatment. Egg reappearance was documented at six and seven weeks in the ID and PPID/ID groups, respectively, whereas the PPID and healthy control groups both had ERP at 8 weeks. Statistical analysis found no significant differences in egg count levels between groups during the study. The expected ERP for ivermectin is 8-10 weeks, meaning that two of the groups displayed shortened ERPs. However, due to the small group sizes, these data should be interpreted with caution. Nonetheless, results do indicate a need for further investigation of the possible influence of endocrine disorders on anthelmintic performance in horses.

Evaluation of seasonal influences on adrenocorticotropic hormone response to the thyrotropin-releasing hormone stimulation test and its accuracy for diagnosis of pituitary pars intermedia dysfunction.

Pituitary pars intermedia dysfunction (PPID) is an age-related neurodegenerative disorder, affecting >20 % of older horses. There is a need for improved endocrine tests for early disease detection, and the thyrotropin-releasing hormone (TRH) stimulation test has been recommended for diagnosis of early or mild cases. However, it is currently not recommended for year-round use due to marked seasonal variability. The aims of this cohort study were to evaluate effects of month and season on adrenocorticotropic hormone (ACTH) responses to TRH stimulation and to derive monthly cut-offs for PPID diagnosis. Sixty-three horses were assigned to control (n = 17), subclinical PPID (n = 21) and clinical PPID (n = 25) groups, based on a composite reference standard that combined clinical history and examination findings with endocrine test results. TRH stimulation tests were performed monthly for a 12-month period. Circannual changes were evaluated with one- and two-way repeated-measures analysis of variance and receiver operating characteristic curve analysis was used to derive cut-off values for basal and TRH-stimulated ACTH. TRH-stimulated ACTH concentrations were lowest in February-May and highest in August-October. Specificity of both basal and 30 min post-TRH ACTH was generally higher than sensitivity, and TRH stimulation had improved diagnostic accuracy compared to basal ACTH, although its sensitivity was not significantly greater year-round. TRH stimulation tests yielded considerably more positive results than basal ACTH in the subclinical group, but few additional positive results in clinical PPID cases. There were large differences between cut-offs that maximised sensitivity or specificity for TRH-stimulated ACTH, highlighting the importance of considering clinical presentation alongside test results in diagnostic decision-making.

Effect of aging on monocyte phagocytic and inflammatory functions, and on the ex vivo inflammatory responses to lipopolysaccharide, in horses.

Studies investigating age-related changes in the function of monocytes are currently limited for horses. Thus, the main goal of this study was to determine the effect of aging on monocyte phagocytic capacity and pro-inflammatory cytokine responses to bacterial lipopolysaccharide (LPS). A second goal of this work was to examine the effect of aging on the inflammatory cytokine responses to LPS in a whole blood ex vivo model. Seven healthy young adult (4-6 years of age) and seven healthy senior horses (>20 years of age) were enrolled. Phagocytosis of E. coli, and pro-inflammatory cytokine (TNFα) responses to LPS, were measured in monocytes by flow cytometry. Gene expressions of pro-inflammatory cytokines (TNFα, IL-1ß, IL-6, IL-8, IL-18, CCL-5, CCL-2) were measured in peripheral blood mononuclear cells (PBMCs) and whole blood by RT-qPCR post incubation for 2 h or 6 h with a low (0.01 µg/mL) or a high (1 µg/mL) dose of LPS. Two sets of statistical models were applied to compare the age groups, one adjusted, and one unadjusted for the horses' body condition scores (BCS). The percentage of monocytes that phagocytosed E. coli after 2 h of incubation was significantly lower in senior compared to young adult horses in the BCS-adjusted model. In the senior group, the expression of IL-1ß in 2 h-0.01 µg/mL LPS-stimulated PBMCs was significantly higher than in the young adult group (BCS-adjusted and unadjusted models). In senior horses, expressions of IL-8 and IL-6 in whole blood samples stimulated for 6 h with 0.01 µg/mL LPS and for 2 h with 1 µg/mL LPS, respectively, were significantly lower than in young adult horses (BCS-adjusted models). The results of this study suggest that the phagocytic function of monocytes, as well as their IL-1ß response to LPS may be altered in senior horses. In addition, the whole blood IL-8 and IL-6 gene expression responses to LPS may be insufficient in senior horses. While investigation of the effect of BCS on monocyte functions and whole blood pro-inflammatory LPS-responses was not a major goal of this work, it appears that adiposity may play a role in innate immune cell function, as significant differences between the age groups were often not apparent until the models were adjusted for BCS.

Impact of blue light therapy on plasma adrenocorticotropic hormone (ACTH) and hypertrichosis in horses with pituitary pars intermedia dysfunction.

Blue light therapy can be used in horses to alter the natural photoperiod and inhibit winter hair coat growth. Seasonal increases in ACTH occur in the fall season but are exaggerated in horses with pituitary pars intermedia dysfunction (PPID). Additionally, PPID horses frequently present with hypertrichosis. Thus, blue light therapy was proposed as a potential management tool for hypertrichosis and for investigating the impact of photoperiod manipulation on ACTH. Eighteen PPID horses, aged 18 to 31 yr, from a university-owned research herd were selected and assigned to either the control group (n = 10) or the treatment (blue light therapy) group (n = 8) based on age and clinical history, which included the results of multiple endocrine tests. Consistent daylength of approximately 14.5 h was maintained for the treated horses from July 15 through approximately late October via the extension of natural daylength using wearable masks that provided short wavelength blue light (465 nm) to 1 eye. The control group was exposed to only the natural photoperiod during this time. All horses were housed on the same farm and remained on pasture for the duration of the study. On Day 0, thyrotropin-releasing hormone (TRH) stimulation tests were performed to confirm PPID status; there were no differences between the 2 groups in resting plasma ACTH or plasma ACTH at 10 min after TRH administration. To determine an effect of treatment on ACTH, blood was collected via jugular venipuncture for measurement of ACTH at sequential timepoints over a 16-h period in mid-October. Hair weights were also assessed throughout the study. No differences in resting plasma ACTH were observed between the 2 groups across the seasonal analysis (July and October) or during the 16-h testing. The PPID horses receiving blue light therapy had lighter hair weights compared to the PPID control horses. These results suggest that blue light therapy does not alter ACTH concentrations but could potentially be used as an additional management tool for hypertrichosis in PPID horses. Manipulation of the photoperiod using blue light therapy did not affect seasonal changes in ACTH in this study.

Evaluation of camera trap-based abundance estimators for unmarked populations.

Estimates of species abundance are critical to understand population processes and to assess and select management actions. However, capturing and marking individuals for abundance estimation, while providing robust information, can be economically and logistically prohibitive, particularly for species with cryptic behavior. Camera traps can be used to collect data at temporal and spatial scales necessary for estimating abundance, but the use of camera traps comes with limitations when target species are not uniquely identifiable (i.e., "unmarked"). Abundance estimation is particularly useful in the management of invasive species, with herpetofauna being recognized as some of the most pervasive and detrimental invasive vertebrate species. However, the use of camera traps for these taxa presents additional challenges with relevancy across multiple taxa. It is often necessary to use lures to attract animals in order to obtain sufficient observations, yet lure attraction can influence species' landscape use and potentially induce bias in abundance estimators. We investigated these challenges and assessed the feasibility of obtaining reliable abundance estimates using camera-trapping data on a population of invasive brown treesnakes (Boiga irregularis) in Guam. Data were collected using camera traps in an enclosed area where snakes were subject to high-intensity capture-recapture effort, resulting in presumed abundance of 116 snakes (density = 23/ha). We then applied spatial count, random encounter and staying time, space to event, and instantaneous sampling estimators to photo-capture data to estimate abundance and compared estimates to our presumed abundance. We found that all estimators for unmarked populations performed poorly, with inaccurate or imprecise abundance estimates that limit their usefulness for management in this system. We further investigated the sensitivity of these estimators to the use of lures (i.e., violating the assumption that animal behavior is unchanged by sampling) and camera density in a simulation study. Increasing the effective distances of a lure (i.e., lure attraction) and camera density both resulted in biased abundance estimates. Each estimator rarely recovered truth or suffered from convergence issues. Our results indicate that, when limited to unmarked estimators and the use of lures, camera traps alone are unlikely to produce abundance estimates with utility for brown treesnake management.

Effects of pituitary pars intermedia dysfunction and Prascend (pergolide tablets) treatment on endocrine and immune function in horses.

It remains unclear how pituitary pars intermedia dysfunction (PPID) and pergolide treatment (Prascend [pergolide tablets]) affect endocrine and immune function in horses. To evaluate these effects, blood was collected regularly from 28 university-owned horses (10 Non-PPID, 9 PPID control [PC], and 9 PPID treatment [PT]) over approximately 15 mo. Pergolide treatment was initiated after Day 0 collections. Analyses included ACTH, insulin, total cortisol, free cortisol, complete blood counts, plasma myeloperoxidase, and cytokine/receptor gene expression in basal whole blood and in vitro stimulations (PMA/ionomycin, heat-inactivated Rhodococcus equi, and heat-inactivated Escherichia coli) of whole blood and peripheral blood mononuclear cells (PBMCs). The results were analyzed using a linear mixed model (SAS 9.4) with significance set at P < 0.05. Significant group (P = 0.0014) and group-by-time (P = 0.0004) effects were observed in resting ACTH such that PT horses differed from Non-PPID horses only at Day 0. PT horses had significantly lower changes in ACTH responses to thyrotropin-releasing hormone stimulation tests than PC horses at non-fall time points only, mid-late February 2018 (P = 0.016) and early April 2018 (P = 0.0172). When PT and PC horses did not differ, they were combined before comparison to Non-PPID horses. No significant group or group-by-time effects were seen in resting insulin, total cortisol, or free cortisol; however, significant time effects were observed in these measures. PPID horses had lower absolute lymphocyte (P = 0.028) and red blood cell (P = 0.0203) counts than Non-PPID horses. In unstimulated whole blood, PPID horses had increased IL-8 expression compared with Non-PPID horses (P = 0.0102). In addition, PPID horses had decreased interferon γ production from PBMCs after stimulation with R. equi (P = 0.0063) and E. coli (P = 0.0057) and showed increased transforming growth factor ß expression after E. coli stimulation (P = 0.0399). The main limitations of this study were a limited sample size and an inability to truly randomize the PPID horses into treatment groups. Resting ACTH is likely the best choice for determining successful responses to pergolide. Neither PPID nor pergolide appears to influence insulin, total cortisol, and free cortisol. As measured, systemic immune function was altered in PPID horses, and it is likely that these horses are indeed at increased risk of opportunistic infection. Despite reducing ACTH, pergolide treatment did not appear to influence immune function.

Effects of advanced age and pituitary pars intermedia dysfunction on components of the acute phase reaction in horses.

Age, neurodegenerative disorders, and dysfunction of insulin secretion may be correlated with increased systemic concentrations of acute phase markers. Thus, the study aimed to determine the effect of age, pituitary pars intermedia dysfunction (PPID), and insulin dysregulation (ID) associated with PPID, on markers of the acute phase reaction. Twenty-nine mix-breed horses of both sexes were classified into groups: (1) healthy adult controls, (2) healthy non-PPID geriatric horses, (3) PPID ID+ horses, and (4) PPID ID- horses. Whole blood proinflammatory cytokine gene expression and serum concentrations of pro- and anti-inflammatory cytokines and acute phase proteins were measured. The data were analyzed using the Mann-Whitney U-test, and correlations between groups of data were assessed using Spearman's correlation coefficient. The tests were statistically significant if P < 0.05. No differences in the whole blood cytokine gene expression, serum cytokine concentrations, or acute phase proteins were noted between the groups. In the PPID ID group, there was a strong correlation between the ACTH concentration after the administration of thyrotropin-releasing hormone and the expression of IL-8 (r = 0.941; P = 0.0321). In the PPID ID+ group, there was a strong correlation between basal insulin concentrations and serum amyloid A (SAA; r = 0.936; P = 0.0083) as well as between postprandial insulin concentrations and SAA (r = 0.965; P = 0.001). These data suggest that neurodegeneration in horses moderately affects circulating markers of inflammation and that ID in horses with PPID influences acute phase inflammatory markers.

Effects of equine metabolic syndrome on inflammation and acute-phase markers in horses.

Obesity and metabolic disorders are associated with systemic low-grade chronic inflammation, both in humans and animals. The aim of the study is to assess the effects of obesity and hyperinsulinemia on individual components of the acute-phase reaction in equine metabolic syndrome (EMS) horses. Eight mixed-breed EMS and six control, age-matched horses of both sexes were included in the study. Animals were classified as EMS or control based on the assessment of BCS, cresty neck score, and basal insulin >50 µU/mL and/or insulin responses to the oral sugar test (OST) >60 µU/mL. Peripheral venous blood was collected. The expression of proinflammatory cytokines, the concentration of circulating cytokines, and acute-phase proteins (serum amyloid A, C-reactive protein, haptoglobin, activin A, and procalcitonin) were measured. The data were analyzed using the Mann-Whitney test, whereas correlations were examined using Spearman's correlation coefficient. The tests were statistically significant if P ≤ 0.05. There were no differences in cytokine gene expression, circulating cytokine concentrations, or concentrations of acute-phase proteins between the EMS and the control groups. There was a strong correlation between the basal concentration of insulin and the serum concentrations of IL-6 (r = 0.71, P < 0.05). Activin A was positively correlated with post-OST insulin concentrations (r = 0.707, P = 0.05), indicating that this marker of inflammation could warrant further investigation in horses with EMS.

Alterations in T cell-related transcripts at the feto-maternal interface throughout equine gestation.

INTRODUCTION: The tolerance of pregnancy by the maternal immune system is balanced between recognition and protection. In the human this is controlled by balancing helper T cell populations (Th1, Th2) in addition to immune suppression from the regulatory arm (Tregs), but this has not been evaluated in the horse. METHODS: RNA sequencing was performed on chorioallantois and endometrium of mares at 120, 180, 300 and 330 days of gestation (n = 4/stage), as well as 45-day chorioallantois (n = 4) and diestrus endometrium (n = 3). Transcripts were selected for relativity to Th1, Th2, or Treg-associated. qPCR and immunohistochemistry were used to confirm the results of select differentially expressed genes. RESULTS: In the endometrium, Th1 transcripts were highest in the diestrus mare and decreased as gestational length progressed. In contrast, Th2 transcripts were upregulated in comparison to the diestrus mare and highest in mid gestation. Treg transcripts were found increased in comparison to the diestrus mare, but decreased prepartum. In the chorioallantois no Th1 transcripts changed. The majority of Th2 transcripts increased from 45 to 300 days gestation, and then decreased prepartum. Treg-related transcripts trended down in the chorioallantois from 45 days to 120 days gestation, followed by an upregulation to 300 days and a secondary decline prepartum. DISCUSSION: The mare experiences a complex and evolving immune profile within the tissues of the feto-maternal interface. This consists of a balance between the Th1 and Th2 response, and a dynamic Treg response that is hypothesized to regulate overall events within the immune system.

A high protein meal affects plasma insulin concentrations and amino acid metabolism in horses with equine metabolic syndrome.

Equine metabolic syndrome (EMS) is characterized by an abnormal insulin response to a glycemic challenge but despite the known insulinotropic effects of certain amino acids, there is a paucity of data evaluating the impact of dietary protein on insulin dynamics in these horses. The objective was therefore to assess insulin and amino acid responses following intake of a high protein meal in healthy horses and those with EMS. Six mature horses diagnosed with EMS and six age-matched control horses without EMS were used. Horses were fed 2g/kg body mass (BM) of a high protein pellet (31% crude protein) at time 0 and 30min, for a total of 4g/kg BM, following an overnight fast. Blood samples collected during a 4h period were analysed for plasma glucose, insulin, amino acids and urea concentrations. Glucose concentrations were not different between groups (P=0.2). Horses with EMS had a 9-fold greater insulinemic response to the consumption of a high protein meal compared with controls (P=0.046). Post-prandial levels of histidine, citrulline, tyrosine, valine, methionine, isoleucine, leucine and ornithine were higher in horses with EMS (P<0.05). Baseline urea nitrogen concentrations were not significantly different between groups (P=0.1). Knowing that certain amino acids are insulin secretagogues, these results illustrate that consumption of a high protein meal caused a hyperinsulinemic response and affected amino acid dynamics in horses with EMS. These findings suggest that dietary protein content should be taken into consideration in the management of horses with insulin dysregulation.

Metabolic and inflammatory responses to the common sweetener stevioside and a glycemic challenge in horses with equine metabolic syndrome.

Extracts derived from the leaves of the stevia plant (stevioside) are commonly used as sweeteners for humans and horses. Stevioside appears to be safe for human consumption, including for individuals with insulin dysregulation. In the horse, the safety or metabolic effects of stevioside on normal animals or on those with metabolic dysfunction are unknown. Furthermore, the inflammatory response to a glycemic challenge or to stevioside in horses is not well defined. Therefore, the objective of this study was to measure the effects of stevioside and a glycemic challenge on insulin, glucose, and inflammatory responses in horses with a common metabolic dysfunction (equine metabolic syndrome or EMS) compared with non-EMS controls. To accomplish this, 15 horses were selected; 8 EMS and 7 age-matched controls. An oral sugar test was performed using Karo corn syrup (karo) or stevioside in a random crossover design. Horses were given 0.15 mL/kg body weight of karo or its equivalent grams of sugar in stevia dissolved in water. Blood samples were collected by jugular venipuncture before administration of either stevia or karo and at 60 and 240 min after administration. Serum was used for glucose and insulin determination and plasma for isolation of peripheral blood mononuclear cells (PBMCs) for inflammatory cytokine analysis via flow cytometry and reverse transcription PCR (RT-PCR). Stevia appeared to stimulate lower glycemic and insulinemic responses when compared to karo, in particular in EMS horses. EMS and control horses had inverse inflammatory responses to administration of either stevia or karo with EMS horses having a proinflammatory response (P ≤ 0.05). These data provide evidence as to why horses with EMS may be predisposed to developing laminitis, potentially as a result of an exaggerated inflammatory response to glycemic and insulinemic responses. Furthermore, the data provide new avenues for exploring mechanisms behind the syndrome, in particular when using a glycemic challenge.

Pituitary pars intermedia dysfunction does not necessarily impair insulin sensitivity in old horses.

Equine pituitary pars intermedia dysfunction (PPID) has been associated with reduced insulin sensitivity in comparison with younger adult horses; however, the difference in insulin sensitivity between horses with PPID and aged-matched controls has not been well characterized. The objective of the study was to determine if aged horses with PPID had reduced insulin sensitivity and alterations in the insulin-mediated signaling pathways in the skeletal muscle when compared with healthy aged horses. Isoglycemic hyperinsulinemic clamp procedures were conducted in 12 horses that were classified as either PPID (n = 6; age: 25.0 ± 2.5 yr; mean ± standard deviation) or non-PPID, aged-matched controls (control) (n = 6; age: 25.7 ± 2.0 yr). Blood samples were taken before and during the clamp procedures to measure plasma glucose, insulin, and amino acid concentrations, and 2 muscle biopsies were collected from the gluteus medius muscle, one in the basal state and the second at the end of the clamp procedure (insulin-stimulated state). Plasma insulin concentrations increased ∼9-fold during the clamp compared with basal conditions (P < 0.001) in both groups. During the last 30 min of the clamp, the rate of glucose infusion required to maintain isoglycemia in horses with PPID was similar to that in the control horses (P = 0.67). The plasma concentrations of most indispensible amino acids were lower in the insulin-stimulated state than the basal state (P < 0.05). PPID status did not have an effect on the activation of factors associated with protein synthesis and breakdown; however, factors associated with protein synthesis had increased phosphorylation in the insulin-stimulated state, compared with basal. The results from this study provide evidence that PPID is not always associated with impairments in insulin sensitivity.

The effect of selenium supplementation on vaccination response and immune function in adult horses.

Selenium status has been reported to affect immune function across many different species. Yet few studies have focused on the effect of Se status on the equine immune system. This study examined the effect of Se supplementation on vaccination response and immune function in mature horses. Twenty-eight horses were blocked by age and sex and were randomly allocated to 1 of 4 dietary treatment groups: low Se (LS), adequate Se (AS), Se-yeast (SP), and sodium selenite (SS). For 35 wk, horses allocated to LS, SP, and SS received a low-Se diet (0.06 mg/kg DM) with the intention to lower Se stores, whereas AS received an adequate Se diet (0.12 mg/kg DM). A 29-wk repletion phase was as follows: LS and AS were kept on the diets fed during the depletion period, whereas SP and SS received the depletion diet plus their respective Se supplements to achieve a dietary Se concentration of 0.3 mg/kg DM. The Se status of the horses was monitored using whole blood Se and glutathione peroxidase (GSH-Px) activity as indicators. At wk 22 and 25 of the repletion phase, horses were vaccinated intramuscularly with 10 mg ovalbumin (OVA). Horses were also vaccinated against equine influenza at wk 25. Blood samples were collected for 7 wk after initial vaccination for serum separation and at 0, 3, and 5 wk postvaccination for peripheral blood mononuclear cell (PBMC) isolation and whole blood cytokine mRNA evaluation. At wk 22 of the repletion phase, both Se and GSH-Px were greater for SP and SS compared with AS and LS (P < 0.001). Serum vitamin E was similar between treatments. Response to OVA vaccination, evaluated as OVA-specific IgG production, cytokine mRNA expression of PBMC stimulated with OVA in vitro, and lymphocyte proliferation, was unaffected by Se status. Similarly, memory response to the influenza vaccine was not affected by Se status. However, decreased mRNA expression of selected cytokines was observed in PBMC stimulated with phorbol 12-myristate 13-acetate for LS compared with AS, SP, and SS (P < 0.05). Whole blood mRNA expression of IL-10 was greater for SS compared with LS, AS, and SP (P = 0.043). Although the OVA and influenza vaccination responses were unaffected by Se status, other measures of immune function did indicate that low Se status could adversely affect cell-mediated immunity.

Granzyme B-mRNA expression by equine lymphokine activated killer (LAK) cells is associated with the induction of apoptosis in target cells.

Lymphokine-activated killer (LAK) cells are a subset of cytotoxic cells capable of lysing freshly isolated tumor cells. While LAK activity is typically measured using the (51)Cr-release assay, here we used a non-radioactive flow cytometric method to demonstrate equine LAK activity. Equine peripheral blood mononuclear cells (PBMC) were stimulated in vitro with recombinant human interleukin 2 (hIL-2) to generate LAK cells. An equine tumor cell line, EqT8888, labeled with carboxyfluorescein succinimidyl ester (CFSE) was used as target cells. Following incubation of the targets with different concentrations of LAK cells, Annexin V was added to identify the early apoptotic cells. With increasing effector to target cell ratios, EqT8888 apoptosis was increased. We also measured interferon-gamma, granzyme B and perforin mRNA expression in the LAK cell cultures as possible surrogate markers for cytotoxic cell activity and found granzyme B mRNA expression correlated best with LAK activity. Also, we found that the reduced LAK activity of young horses was associated with decreased granzyme B mRNA expression. Our results indicate that fluorescence-based detection of LAK cell activity provides a suitable non-radioactive alternative to (51)Cr-release assays and mRNA expression of granzyme B can be used as surrogate marker for these cytotoxic cells.

Characterization of the interferon gamma response to Lawsonia intracellularis using an equine proliferative enteropathy challenge (EPE) model.

Lawsonia intracellularis is the etiological agent of infectious intestinal hyperplasia for which several clinical diseases have been described including proliferative enteropathy (PE), intestinal adenomatosis, and ileitis. While initially recognized as the causative agent of PE in pigs, L. intracellularis is now viewed as an emerging cause of intestinal hyperplasia in a wide range of mammalian species, including horses. Equine proliferative enteropathy (EPE) has been reported worldwide though definitive diagnosis is difficult and the epidemiology of the disease remains poorly understood. Weanlings, in particular, appear to be most at risk for infection, though the reasons for their particular susceptibility is unknown. Using an infectious challenge model for EPE, we demonstrate that EPE, like porcine proliferative enteropathy, can exhibit three clinical forms: classical, subclinical and acute. Out of six pony weanlings, one developed signs of classic EPE, one developed acute EPE, and two developed subclinical EPE. Attempts to induce pharmacological stress through the use of dexamethasone failed to have any effect on outcome. Peripheral blood cells collected from those weanlings that developed clinical EPE exhibited decreased expression of interferon-gamma (IFN-γ) following in vitro stimulation with L. intracellularis. By contrast, those weanlings that did not develop clinical disease generated a robust IFN-γ response. These results indicate IFN-γ likely plays a significant role in protection from disease caused by L. intracellularis in the equid.

Exercise-induced increases in inflammatory cytokines in muscle and blood of horses.

REASONS FOR PERFORMING STUDY: Studies have demonstrated increases in mRNA expression for inflammatory cytokines following exercise in horses and have suggested those markers of inflammation may play a role in delayed onset muscle soreness. However, measurement of mRNA expression in white blood cells is an indirect method. No studies to date have documented the cytokine response to exercise directly in muscle in horses. HYPOTHESIS: This study tested the hypothesis that exercise increases cytokine markers of inflammation in blood and muscle. METHODS: Blood and muscle biopsies were obtained from 4 healthy, unfit Standardbred mares (∼ 500 kg). The randomised crossover experiment was performed with the investigators performing the analysis blind to the treatment. Each horse underwent either incremental exercise test (GXT) or standing parallel control with the trials performed one month apart. During the GXT horses ran on a treadmill (1 m/s increases each min until fatigue, 6% grade). Blood and muscle biopsies were obtained 30 min before exercise, immediately after exercise and at 0.5, 1, 2, 6 and 24 h post GXT or at matched time points during the parallel control trials. Samples were analysed using real time-PCR for measurement of mRNA expression of interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and interleukin-1 (IL-1). Data were analysed using t tests with the null hypothesis rejected when P < 0.10. RESULTS: There were no changes (P > 0.10) in IL-1, IL-6, IFN-gamma or TNF-alpha during control. Exercise induced significant increases in IFN-gamma, IL1 and TNF-alpha in blood and significant increases in IFN-gamma, IL-6 and TNF-alpha in muscle. There were no significant changes in mRNA expression of IL-1 in muscle or IL-6 in blood following the GXT. These cytokine markers of inflammation all returned to preGXT levels by 24 h post GXT. CONCLUSION: High intensity exercise results in a transient increase in the expression of inflammatory cytokines in muscle and blood.

Immunosenescence of the equine immune system.

It is widely recognized that advanced age is associated with alterations in immunological responses that likely contribute to increased morbidity and mortality in the elderly population. This decreased efficacy of the immune system with age is referred to as 'immunosenescence' and has been reported for a number of species. Similar age-related changes are seen in horses and are manifested as decreased responsiveness to vaccination in vivo and diminished proliferative responses to mitogens in vitro. The underlying mechanism responsible for these impaired immunological responses remains unknown. This paper reviews some of our recent findings on immunosenescence in horses. Recent results on the immune response of aged horses to vaccination with a novel recombinant influenza vaccine are also discussed.

Advanced age in horses affects divisional history of T cells and inflammatory cytokine production.

A number of model systems have been employed to investigate age-associated changes in immune function. The purpose of the current study was to characterize senescent T cells and to investigate the inflamm-aging phenomenon both in vitro and in vivo using the old horse as a model. We examined whether decreased T cell proliferation induced by Con A is caused by increased apoptosis. We also utilized intracellular CFSE to analyze changes within each round of cell proliferation, in particular cytokine production. Intracellular staining with flow cytometry, RT-PCR, and ELISA were used to measure pro-inflammatory cytokines both in vitro and in vivo. While lymphocytes from old horses exhibit decreased proliferation, this is not the result of increased apoptosis. Instead, a larger percentage of the T cells remain in the parent generation and produce significant amounts of IFNgamma. Likewise, old horses have increased frequency of CD8-IFNgamma+ T cells and TNFalpha producing cells. We also show that old horses have elevated levels of IL-1beta, IL-15, IL-18 and TNFalpha gene expression in peripheral blood and significant levels of TNFalpha protein in serum, all characteristics of inflamm-aging.

Relationships among inflammatory cytokines, obesity, and insulin sensitivity in the horse.

Recent studies associate obesity and insulin resistance in horses with development of abnormal reproductive function and debilitating laminitis. The factors contributing to insulin resistance in obese horses are unknown. However, human studies provide evidence that elevated inflammatory cytokines such as tumor necrosis factor alpha (TNFalpha), IL1, and IL6 play direct roles in development of obesity-associated insulin resistance. Thus, inflammation may be a key link between obesity and insulin resistance in horses. The aim of the current investigation was to examine possible relationships between obesity, inflammatory cytokines, and insulin sensitivity (IS) in the horse. Age was recorded and BCS and percent body fat (% FAT) were determined as measures of obesity in 60 mares. In addition, blood mRNA expression of IL1, IL6, and TNFalpha and circulating concentrations of TNFalpha protein (TNFp) were determined in each mare. Finally, fasted concentrations of insulin were determined, and IS was determined using the hyperinsulinemic, euglycemic clamp. Significant correlations between several variables provided evidence for the design of 4 population regression models to estimate relationships between measures of obesity, inflammatory factors, and IS in the sample population. The results of these analyses revealed that IS decreased as BCS and % FAT increased (P < 0.001) in the sample population. Additionally, increased IL1 (P < 0.05) and TNFp (P < 0.01) were associated with decreased IS. However, increased TNFalpha (P < 0.001) was associated with decreased IS only in mares 20 yr of age and older. Increased BCS and % FAT were associated with increased expression of TNFalpha (P = 0.053) and IL1 (P < 0.05), and increased TNFp (P < 0.05). Surprisingly, increased BCS and % FAT were associated with decreased IL6 expression (P = 0.05) in mares <20 yr of age. Finally, evaluation of the influence of obesity and inflammatory cytokines on IS within the same model suggested that BCS and % FAT (P < 0.001) with TNFalpha [mRNA (P = 0.07) and protein (P < 0.05)] are inversely associated with IS independently of one another. Combined, these results provide the first evidence associating obesity with increased inflammatory factors in the horse. Furthermore, the results suggest that an interrelationship exists among obesity, inflammatory cytokines, and IS in the horse and emphasize the need for further studies to elucidate the nature of these relationships.

Foals are interferon gamma-deficient at birth.

The increased vulnerability of foals to specific pathogens such as Rhodococcus equi is believed to reflect an innate immunodeficiency, the nature of which remains poorly understood. Previous studies have demonstrated that neonates of many species fail to mount potent Th1 responses. The current research investigates the ability of circulating and pulmonary lymphocytes of developing foals to produce interferon gamma (IFNgamma). Peripheral blood mononuclear cells (PBMC) were prepared from up to 10 horse foals at regular intervals throughout the first 6 months of life. Bronchoalveolar lavage (BAL) samples were collected at 1, 3 or 6 months of age from three groups of five foals. The PBMC and BAL cells were stimulated in vitro and IFNgamma production was measured by intracellular staining. In addition, RNA was extracted from freshly isolated and in vitro stimulated PBMC and BAL cells for quantitation of IFNgamma gene expression by real time PCR. Newborn foals exhibited a marked inability to express the IFNgamma gene and produce IFNgamma protein. This deficiency was observed in both circulating and pulmonary lymphocytes. However, IFNgamma gene expression and protein production increased steadily throughout the first 6 months of life, reaching adult levels within the first year of life. These findings suggest that foals are born with an inherent inability to mount a Th1-based cell mediated immune response which may contribute to their susceptibility to intracellular pathogens.