Diagnosis of PPID in horses relies on measurement of plasma adrenocorticotrophic hormone (ACTH) concentrations. Because measured ACTH values vary between analytical platforms, and published diagnostic thresholds apply almost exclusively to the Immulite 1000 chemiluminescent assay (CLA), there is a need for validated, assay-specific thresholds for contemporary analysers. This study aimed to transfer and validate established Immulite 1000 diagnostic thresholds for plasma ACTH to three contemporary systems (Immulite 2000XPi, Tosoh AIA-900, and Tosoh AIA-360) using the Clinical Laboratory Standards Institute (CLSI) guidelines. Surplus clinical EDTA plasma samples were analysed across pairs of analysers to enable method comparison and reference interval transference. The Immulite 2000XPi showed close agreement with the Immulite 1000 (r=0.962, P<0.0001; bias -3.9 pg/mL (95% CI: -12.2-4.4)), enabling reliable linear transference of thresholds. Comparison of Immulite 2000XPi with the AIA-900 revealed moderate correlation and non-linear, concentration-dependent bias (r=0.770, P<0.0001; bias 30.8 pg/mL (95% CI: -110-171)), necessitating threshold-specific regression. The AIA-360 and AIA-900 showed excellent agreement (r=0.993, P<0.0001; bias 4.023 pg/mL (95% CI: -1.4-9.4)). Platform-specific low (90% sensitivity) and high (90% specificity) diagnostic thresholds were generated for four seasonal periods for all analysers. The proposed analyser-specific thresholds provide clinicians and laboratories with more validated guidance for interpreting plasma ACTH results across contemporary analysers, supporting more accurate diagnosis and management of PPID in horses.

Prognostic information in equine liver disease can be gleaned from historical, clinical, clinicopathological, ultrasonographic, and histopathological features. However, given the multiple causes and subtypes of liver disease affecting horses worldwide, prognostic data might not be suitable for universal application across all cases. Most evidence has been established for horses with chronic hepatopathies and extrapolation to acute cases should be avoided. In chronic liver disease the presence and severity of clinical signs and serum markers including total globulins, albumin, and bile acids are most helpful. The strongest prognostic associations are seen with histopathologic changes in biopsies, especially fibrosis and biliary hyperplasia.

This study compared the in vitro stability of equine plasma adrenocorticotrophic hormone (ACTH) and serum insulin measured by chemiluminescent assay (CLA; Siemens Immulite 2000XPi) and immunofluorescent assay (IFA; Tosoh AIA-900) after storage at 4°C or room temperature (approximately 21°C) for clinically relevant periods. Fresh equine EDTA blood for ACTH and coagulated blood for insulin were aliquoted and analysed immediately (T0) and after storage. ACTH was assessed at 24, 48 and 72 hours; insulin was assessed at 24 hours, 72 hours and 7 days. Percentage recovery relative to T0 was analysed by repeated-measures ANOVA and confirmed by linear mixed-effects modelling. Agreement between assays at T0 was assessed using Bland-Altman analysis, with additional evaluation of normality of inter-assay differences, proportional bias and log-transformed ratio-based agreement. ACTH concentrations declined significantly over time at both temperatures with both assay methods. Under refrigeration, ACTH recovery was significantly greater with IFA than CLA at all time points, whereas no significant inter-assay difference was detected at room temperature. Bland-Altman analyses showed that baseline ACTH agreement varied with concentration magnitude, indicating proportional bias on the original scale; log-transformed analysis reduced this concern for refrigerated ACTH, although some residual concentration-dependent disagreement remained at room temperature. In contrast, insulin remained comparatively stable, with no meaningful assay-by-time interaction and no clear evidence of proportional bias on the original scale. These findings support prompt chilled handling of ACTH samples and suggest that, when immediate analysis is not possible, the IFA method provides results closer to the initial ACTH concentration than the CLA method, whereas insulin appears relatively robust to short-term storage conditions.

Sodium-glucose co-transporter-2 antagonist (SGLT2i) drugs are used to control hyperinsulinemia in horses but are associated with development of hypertriglyceridemia. Clinical laboratory submissions were examined from 985 horses sampled within 60 days of starting SGLT2i therapy. Signalment data, drug identity and time of year were examined for association with serum triglyceride (TG) concentration post-treatment with the SGLT2i drugs canagliflozin and ertugliflozin. The study also examined associations between TG and other post-treatment markers including insulin, glucose, adrenocorticotropin (ACTH), total adiponectin and gamma-glutamyl transferase (GGT). Variables associated with higher post-treatment TG included Shetland ponies, Dartmoor ponies and canagliflozin (Shetland mean ratio 1.87, 95% CI 1.37-2.56, P<0.001; Dartmoor mean ratio 2.21, 95% CI 1.33-3.67 P=0.002; Canagliflozin mean ratio 1.23, 95% CI 1.04-1.46 P=0.018). Warmbloods had lower post-treatment TG (mean ratio 0.67, 95% CI 0.45-1.00, P=0.048). Horses with moderate TG increases (2.5-5.0 mmol/L) were more likely to be Shetland ponies (OR 2.32, 95% CI 1.21-4.45, P=0.011) and to be older (OR 1.03 95% CI 1.01-1.06, P=0.017) and less likely to be Connemara ponies (OR 0.23, 95% CI 0.06-0.84, P=0.026), Warmbloods (OR 0.12, 95% CI 0.02-1.00, P=0.049) or receiving ertugliflozin (OR 0.57, 95% CI 0.37-0.87, P=0.010). Post-treatment insulin was negatively associated with post-treatment TG (P=0.001). Shetland and Dartmoor ponies were more likely to suffer hypertriglyceridemia when treated with SGLT2i drugs, and Warmbloods appeared less likely. Some effect of older age and use of canagliflozin were also seen leading to recommendations to consider alternative dosing strategies with this drug, especially when used in high-risk breeds.

BACKGROUND:Limited published data exist regarding the diagnostic utility of beta-endorphin (β-END) for pituitary pars intermedia dysfunction (PPID), either compared with or in combination with adrenocorticotropic hormone (ACTH). HYPOTHESIS/OBJECTIVES:To evaluate β-END as a potential diagnostic test for PPID in comparison with ACTH and to explore the diagnostic performance of combined tests. ANIMALS:Ninety-eight equids aged 19-32 years, owned by the foundation "Stiftung für das Pferd," were included. METHODS:A cross-sectional study was conducted in January 2025. Haircoat abnormalities were scored as absent, mild, or pronounced, and plasma β-END and ACTH concentrations were measured. Horses were classified based on haircoat abnormalities, with pronounced haircoat abnormalities defining the PPID group (n = 39) and absence of haircoat abnormalities defining the control group (n = 59). Receiver-operating characteristic (ROC) analyses were performed for individual and combined diagnostic tests. Previously established ACTH cut-offs were combined with study-derived maximum Youden-Index cut-offs. RESULTS:Beta-endorphin showed an area under the ROC curve (AUROC) of 0.88 for PPID diagnosis, with a sensitivity of 76.9% and specificity of 93.2%. Corresponding AUROCs, sensitivities, and specificities were 0.87, 71.8%, and 88.1% for ACTH measured by chemiluminescent assay (CLA), and 0.83, 79.5%, and 69.5% for ACTH measured by immunofluorescent assay (IFA). Combining β-END at 115 pg/mL with ACTH (CLA) at 24.3 pg/mL yielded 88% sensitivity and 89% specificity. CONCLUSIONS AND CLINICAL IMPORTANCE:These preliminary findings support further evaluation of β-END as an adjunct diagnostic test for PPID.

This study aimed to further define and quantify possible cross-reactive peptides when measuring plasma adrenocorticotropic hormone (ACTH) concentration in equids. Equine plasma samples were spiked with known concentrations of exogenous manufactured peptides comprising human ACTH1-39, ACTH18-39 (corticotropin-like intermediate lobe peptide, CLIP) and ACTH7-38 (corticotropin inhibiting peptide, CIP). All samples were assayed in duplicate using Siemens Immulite 2000xpi chemiluminescent assay (CLA) and Tosoh AIA-900 immunoflurorescent assay (IFA). As expected, ACTH1-39 was measured by both assays although higher values were reported using IFA (mean 132 % of actual concentrations) than CLA (mean 104 % of actual concentrations). ACTH18-39 was detected by the CLA but not the IFA (mean 29 % actual concentration) whereas ACTH7-38 was detected by the IFA, but not the CLA (mean 65 % actual concentration). The study further clarifies that these ACTH immunoassays are likely to report higher measured ACTH1-39 concentrations than are actually present in the sample although additional work is needed to elucidate the diagnostic and pathophysiologic implications of these findings.

Current laboratory reference values for serum insulin concentrations do not account for seasonal fluctuations and differences associated with breed, sex, and age. We hypothesized that serum insulin concentrations would differ with the season, breed, sex, and age. Laboratory records from 21 236 cases. Cases were included where basal serum insulin concentration (INS) or serum insulin concentration after administration of Karo light syrup (INSpk) was measured, along with plasma glucose, breed, age, and sex. Multivariate analysis was used to investigate a seasonal effect on serum insulin concentrations, alongside the effect of breed, age, and sex. Basal serum insulin concentration in winter was significantly higher than in the other seasons (p < 0.001). Serum insulin concentration following administration of Karo light syrup in winter was significantly higher than in summer and fall (p < 0.001). The breed effect was assessed in the 9 most prevalent breeds and the donkey. Shetland ponies had significantly higher INS and INSpk than all breeds except Welsh ponies(p < 0.01). Welsh ponies had significantly higher INS than all other breeds except Shetland ponies (p < 0.01). Welsh ponies had significantly higher INSpk than all breeds (all p < 0.001), except Arabians, New Forest ponies, and Shetland ponies. Females had significantly higher INS than males (p < 0.001) and there was a positive and significant association between age and INS (est = 0.02; SE = 0.002, p < 0.001). Insulin concentrations are influenced by season, breed, age, and sex. This information is essential for better understanding and management of insulin dysregulation.

BACKGROUND:High concentrations of adrenocorticotropic hormone (ACTH) are used as an indicator of pituitary pars intermedia dysfunction (PPID), but other factors that may influence ACTH need to be understood, if diagnostic reference ranges for ACTH are to be used with confidence. Insulin dysregulation (ID) could be one such factor, as insulin affects pituitary hormones in other species. OBJECTIVES:To test the hypothesis that a relationship exists between high insulin and high ACTH in aged (>15-year-old) animals with no clinical signs of PPID. STUDY DESIGN:A cohort study. METHODS:Thirteen horses and eleven ponies (17-25 years-old; mares and geldings) were clinically examined for signs of PPID in the spring (November 2020) and autumn (April 2021). On the same day, blood samples were taken before and 2 h after an oral glucose test (OGT). Concentrations of insulin, glucose, ACTH and cortisol were measured. RESULTS:There was no association between ACTH and cortisol. However, there was a positive linear correlation between ACTH and post-OGT (insulin in the autumn (r = 0.427, p = 0.04). Two horses and six ponies had ACTH above the cut-off value for PPID diagnosis, and of these eight animals, six also had insulin concentrations above the cut-off value for ID. MAIN LIMITATIONS:The cohort was small and thyrotropin-releasing hormone (TRH) stimulation tests were not performed. CONCLUSIONS:In autumn, high ACTH was associated with ID, when no clinical signs of PPID were present. Because ACTH is used in PPID diagnosis, further work is required to understand this interaction.

Liver disease is common in equine practice, and treatment and prognosis are dependent on histopathologic examination of biopsies. Liver biopsy is invasive and expensive which restricts its use. Serum markers are used to predict hepatic fibrosis in humans. This study aimed to investigate the enhanced liver fibrosis (ELF) test, based on serum Hyaluronic Acid (HA), procollagen III N-terminal peptide (PIIINP), and tissue inhibitor of metalloproteinase 1 (TIMP-1) to detect hepatic fibrosis in equids. Four groups were included; two with increased serum concentrations of liver-derived enzymes and a liver biopsy (group H; 10 horses and ponies and group D; 10 donkeys) and two without any evidence of liver disease (group HC; 10 horses and ponies and group DC; 10 donkeys). All samples were analyzed for concentrations of HA, PIINP, and TIMP-1. Given the failure to detect TIMP-1 in most subjects, a novel eELF (equid ELF) score was calculated, based on HA and PIIINP. HA and PIIINP concentrations and the eELF score, were compared with determined hepatic fibrosis. HA, PIIINP, and eELF were significantly greater in horses and ponies with a histopathologic fibrosis score ≥ 2 compared with those < 2. A similar observation was found with donkeys for HA and eELF. A significant correlation was found between fibrosis score and HA, PIIINP, and eELF for horses and ponies, and between fibrosis score and HA and eELF in donkeys. Serum HA and the eELF score might be useful serum markers to predict and monitor hepatic fibrosis in horses, ponies, and donkeys.

Abstract Background A glycemic challenge test is used for the diagnosis of insulin dysregulation (ID) in horses and ponies. Different forms of the test exist where the administrative route and dose of glucose vary, which makes interpretation of results challenging. Hypothesis/Objectives To evaluate the palatability of, and blood glucose and insulin responses to, carbohydrate pellets fed as an oral glucose test (OGT), and to establish the diagnostic threshold for ID when using the pellets. Animals University and privately‐owned horses and ponies (n = 157) comprised of 31 breeds and both sexes. Methods Multicenter cohort study. A custom‐produced glycemic pellet was offered for free intake at 0.5 g/kg BW soluble carbohydrate and serum insulin and blood glucose concentrations measured before and after (60, 120, and 180 minutes) the pellets were offered. Pellet acceptance and intake time (those that finished within 10 minutes) were determined to assess palatability. Results The pellets were palatable to 132/157 animals, and ponies found the pellets more (P = .004) palatable than horses. The median intake time (4 [3‐6] minutes) was positively correlated with acceptance grade (r = .51; P < .0001). Consumption of the pellets elicited peak blood glucose (6.6 [5.8‐7.8] mmol/L) and serum insulin (40.5 [19‐99.8] μIU/mL) responses at 120 minutes. At 120 minutes the optimal cut‐off was 83 μIU/mL (95% CI: 70‐99 μIU/mL) for the IMMULITE 2000XPi assay. Conclusions and Clinical Importance The pellets were palatable and a suitable, novel carbohydrate source for the OGT.

Equine pituitary pars intermedia dysfunction (PPID) is common in aged horses. The majority of horses respond well to treatment, but treatment is lifelong, meaning accurate diagnosis of PPID is important. Similar to any condition, there is no perfect laboratory test to diagnose PPID and accuracy is affected by the characteristics of the population in which the test is being evaluated. This review details the importance of consideration of clinical factors and diagnostic test accuracy. Basal adrenocorticotrophic hormone (ACTH) concentration is used most frequently in practice and has very good diagnostic accuracy when used in combination with clinical judgement and the correct application of diagnostic thresholds. The thyrotropin-releasing hormone stimulation test can be used in horses with equivocal test results following basal ACTH testing, or to evaluate subtle cases due to its improved accuracy.

High concentrations of adrenocorticotropic hormone (ACTH) are used as an indicator of pituitary pars intermedia dysfunction (PPID), but other factors that may influence ACTH need to be understood, if diagnostic reference ranges for ACTH are to be used with confidence. Insulin dysregulation (ID) could be one such factor, as insulin affects pituitary hormones in other species. To test the hypothesis that a relationship exists between high insulin and high ACTH in aged (>15-year-old) animals with no clinical signs of PPID. A cohort study. Thirteen horses and eleven ponies (17–25 years-old; mares and geldings) were clinically examined for signs of PPID in the spring (November 2020) and autumn (April 2021). On the same day, blood samples were taken before and 2 h after an oral glucose test (OGT). Concentrations of insulin, glucose, ACTH and cortisol were measured. There was no association between ACTH and cortisol. However, there was a positive linear correlation between ACTH and post-OGT (insulin in the autumn ( r  = 0.427, p  = 0.04). Two horses and six ponies had ACTH above the cut-off value for PPID diagnosis, and of these eight animals, six also had insulin concentrations above the cut-off value for ID. The cohort was small and thyrotropin-releasing hormone (TRH) stimulation tests were not performed. In autumn, high ACTH was associated with ID, when no clinical signs of PPID were present. Because ACTH is used in PPID diagnosis, further work is required to understand this interaction.

The measurement of the blood insulin concentration, and comparison to cut-offs, is essential in diagnosing insulin dysregulation, a common equine endocrinopathy. However, different insulin assays provide disparate results. We aimed to ease comparison between assays by compiling original and published data into a web app to convert insulin measurements from one assay to another. Data were available for ADVIA Centaur insulin chemiluminescent immunoassay (CLIA), Beckman Coulter insulin radioimmunoassay (RIA), Immulite 1000 CLIA, Immulite 2000 CLIA, Immulite 2000 XPi CLIA, Mercodia equine insulin enzyme-linked immunosorbent assay (ELISA), and Millipore porcine insulin RIA. Linear models were fitted for 13 assay pairs using non-decreasing splines, and integrated into this app. Assay comparisons including data from several studies showed a lower performance. This indicates technical variation between laboratories, which has not been described before, but is relevant when diagnostic measurements and cut-offs are provided by different laboratories. Nevertheless, the models’ overall high performance (median r2 = 0.94; range 0.57–1.00) supports their use to interpret results from diagnostic insulin measurements when the reference assay is unavailable, and to compare values obtained from different assays.

Abstract Background Outbreaks of liver disease in horses are common but the etiology of most remains unknown. Forage mycotoxins have been suspected to be a cause. Objectives To examine the association between outbreaks of liver disease and the presence of mycotoxins in forage stored on the same premises. Animals Premises were identified where ≥4 horses were contemporaneously affected by liver disease, and a control group was formed from premises where ≥4 horses had been examined and found to have no evidence of liver disease. Methods Forage was collected from 29 case and 12 control premises. The forage was analyzed for mycotoxin content using a liquid chromatography/mass spectrometry method, targeting 54 mycotoxins. The presence and distribution of mycotoxins between case and control samples was compared. Results Mycotoxins were found in 23/29 (79%) case samples and 10/12 (83%) control samples (P > .99; relative risk, 0.93; 95% confidence interval [CI], 0.64‐1.75). Median (interquartile range [IQR]) total mycotoxin concentration was similar in case and control samples (85.8 μg/kg [1.6‐268] vs. 315 μg/kg [6.3‐860]; P = .16). Ten mycotoxins were found exclusively in case premises comprising fumonisin B1, 15‐acetyldeoxynivalenol, deoxynivalenol, zearalenone, aflatoxins B1 and G1, methylergonovine, nivalenol, verruculogen, and wortmannin. The median (IQR) concentration of fumonisin B1 was significantly higher in case versus control samples (0 μg/kg [0‐81.7] vs. 0 μg/kg [0‐0]; P = .04). Conclusions and Clinical Importance Several mycotoxins with known hepatotoxic potential were found, alone or in combination, exclusively at case premises, consistent with the hypothesis that forage‐associated mycotoxicosis may be a cause of outbreaks of liver disease in horses in the United Kingdom.

Sarcoids are among the most common tumors diagnosed in equids; their association with bovine papillomaviruses (BPV) infection has been widely reported, but the mechanism of carcinogenesis has not been fully elucidated. To verify whether BPV infection causes dysregulation of the pRb-Cyclin D1-p16CDKN2A-p53 pathway as reported for human papillomavirus (HPV), the study employed immunohistochemistry to test 55 equine sarcoid biopsies for the expression of pRb, Cyclin D1, and p53 cell cycle regulatory proteins and to evaluate the proliferative rate through Ki67. High Cyclin D1 and pRb expression were observed in 51% and 80% of cases, respectively, while low expression was observed in 49% and 20% of cases, respectively. Significantly higher Ki67 proliferation indexes were observed in fibroblastic, nodular, and mixed sarcoids compared to the occult and verrucous. High proliferation was significantly associated with high Cyclin D1 expression. In contrast with previous studies, p53 positivity was not observed in the cases examined in this study. Moreover, follow-up analysis revealed that fibroblastic, mixed sarcoids were associated with significantly higher local recurrence rates while the verrucous subtype was associated with higher rates of new sarcoid development at distant sites.

BACKGROUND Serology is commonly used as a means of identifying horses that might be chronic and silent carriers of S. equi but its sensitivity is rarely examined. OBJECTIVES The study was designed to investigate the sensitivity of serological testing for antibodies against S. equi antigens A and C to detect guttural pouch carriers of S. equi. STUDY DESIGN Retrospective clinical study. METHODS As part of routine surveillance and quarantine procedures horses arriving at a welfare charity quarantine unit were subject to both microbiological sampling of guttural pouches and also serological testing for antibodies directed at S. equi antigens A and C. Laboratory results and endoscopic findings were examined to determine associations between serological results and guttural pouch carriage of S. equi. RESULTS Of 287 included horses, 9 (3.1%) were found to be guttural pouch carriers. There was no significant association between serological status and guttural pouch carriage of Strep. equi. Only one of the 9 carriers (11%) was seropositive using a cut-off of OD ≥0.5, and only 3 of 9 (33%) using a cut-off of OD ≥0.3. MAIN LIMITATIONS Horses examined in this study were new arrivals at a welfare centre rather than from a general, well-managed, equid population. As a retrospective clinical study, the laboratory test results could not be repeated for further confirmation. CONCLUSIONS Caution is advised when relying on seronegativity to antigens A and C in order to discount the possibility of chronic carriage of S. equi in guttural pouches.

BACKGROUND PPID is commonly investigated using plasma ACTH concentrations but problems exist with currently available diagnostic thresholds. OBJECTIVES To derive temporally specific diagnostic thresholds for equine plasma ACTH concentration to be used alongside clinical judgement in each individual week of the year and appropriate for the degree of clinical suspicion in any given case. Furthermore, to apply these thresholds to compare the prevalence of high and low ACTH in two sub-groups of animals with high and low clinical suspicion of PPID. STUDY DESIGN A retrospective population study examining a large laboratory database of equine plasma ACTH concentrations using an indirect approach to calculate diagnostic thresholds. METHODS Logs of plasma ACTH concentrations from 75,892 individual horses were examined using robust L2 estimation of mixtures of two normal distributions in categories of each week and month of the year. Thresholds dividing the two populations of high-ACTH and low-ACTH horses were then established at different levels of sensitivity and specificity and compared with clinical subgroups of horses divided based on reported clinical signs, as having high (n = 4036) or low (n = 3022) clinical suspicion of PPID. RESULTS For most of the year there were small inter-week differences in diagnostic thresholds. However, from mid-June to early-December diagnostic thresholds showed greater inter-week variability, reaching a maximum in late September and early October. Grouping of high- and low-ACTH compared favourably with grouping based on clinical signs. MAIN LIMITATIONS Given the multiple sources of diagnostic samples, pre-analytical data could not be fully verified. CONCLUSIONS Diagnostic thresholds for equine plasma ACTH vary through the year. It is especially important to consider the temporally specific threshold between June and December. Different clinical thresholds can be used depending on the case circumstances and whether a false positive or false negative diagnosis is deemed least desirable.

Sarcoids are the most common cutaneous tumor of equids and are caused by bovine papillomavirus (BPV). Different clinical subtypes of sarcoids are well characterized clinically but not histologically, and it is not known whether viral activity influences the clinical or histological appearance of the tumors. The aim of this study was to verify whether the development of different clinical types of sarcoids or the presence of certain histological features were associated with BPV distribution within the tumor. The presence of BPV was assessed by polymerase chain reaction (PCR) and visualized in histological sections by chromogenic in situ hybridization (CISH) in 74 equine sarcoids. Furthermore, to better characterize the molecular features of neoplastic cells, immunohistochemistry for S100, smooth muscle actin-α (αSMA), and fibroblast-associated protein-α (FAPα) was performed. The presence of BPV was confirmed in all tissues examined by either or both PCR and CISH (72/74, 97% each). Of 70/74 CISH-positive cases, signal distribution appeared as either diffuse (61/70, 87%) or subepithelial (9/70, 13%); the latter was more frequently observed in the verrucous subtype. However, no statistically significant association was found between clinical subtypes and specific histological features or hybridization pattern. Moreover, CISH signal for BPV was not detected in the epidermis overlying sarcoids nor in the tissue surrounding the neoplasms. By immunohistochemistry, αSMA confirmed the myofibroblastic differentiation of neoplastic cells in 28/74 (38%) sarcoids. Using tissue microarrays, FAPα labelling was observed in neoplastic fibroblasts of all sarcoids, suggesting this marker as a potential candidate for the immunohistochemical diagnosis of sarcoids.