Investigation of Serum Markers of Hepatic Fibrosis in Equids.

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.

Diagnosis of equine pituitary pars intermedia dysfunction.

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.

Development of a Web App to Convert Blood Insulin Concentrations among Various Immunoassays Used in Horses.

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.

Association between insulin dysregulation and adrenocorticotropic hormone in aged horses and ponies with no clinical signs of pituitary pars intermedia dysfunction.

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.

Carbohydrate pellets to assess insulin dysregulation in horses.

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.

Expression of Cell-Cycle Regulatory Proteins pRb, Cyclin D1, and p53 Is Not Associated with Recurrence Rates of Equine Sarcoids.

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.

Association between forage mycotoxins and liver disease in horses.

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.

Fibroblast-associated protein-α expression and BPV nucleic acid distribution in equine sarcoids.

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.

Clinically and temporally specific diagnostic thresholds for plasma ACTH in the horse.

BACKGROUND: Pituitary pars intermedia dysfunction (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 subgroups 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 interweek differences in diagnostic thresholds. However, from mid-June to early-December diagnostic thresholds showed greater interweek 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.

Failure of serological testing for antigens A and C of Streptococcus equi subspecies equi to identify guttural pouch carriers.

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 S. equi Only one of the nine carriers (11%) was seropositive using a cut-off of OD ≥ 0.5, and only three of nine (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.

Antimicrobial susceptibility of bacterial isolates from ambulatory practice and from a referral hospital.

BACKGROUND: Responsible use of antimicrobials in equine practice relies on knowledge of common bacterial isolates and their antimicrobial sensitivities. OBJECTIVES: To assess the frequency of bacterial resistance to a combination of parenteral penicillin and gentamicin and to trimethoprim and sulfamethoxazole for PO use in a selection of clinical isolates, and subsequently to determine the prevalence of resistance to antimicrobials that might then be used as alternatives to first-line antimicrobials for the same isolates. METHODS: Retrospective analysis of minimal inhibitory concentrations (MICs) of antimicrobials for 6354 bacterial isolates from 365 ambulatory practices and 519 isolates from a referral hospital. The MICs were used to indicate sensitivity or resistance to commonly used antimicrobials and the prevalences of resistance were compared between origin of the isolates, and among antimicrobial drugs. RESULTS: Isolates from the referral hospital were significantly (P < .05) more likely to be resistant to the antimicrobials tested than those derived from ambulatory practice. Overall, 91% of the ambulatory isolates and 64% of the hospital isolates were sensitive to penicillin-gentamicin. For trimethoprim-sulfamethoxazole combination, 82% of the ambulatory practice isolates and 56% of the referral hospital isolates were sensitive. CONCLUSIONS AND CLINICAL IMPORTANCE: Most isolates were sensitive to penicillin and gentamicin as well as trimethoprim-sulfamethoxazole. No predictable efficacious second choice antimicrobial was identified for those isolates resistant to the first-line antimicrobials. The likelihood of isolates being sensitive to second choice antimicrobials was variable but generally higher for ambulatory isolates compared to referral isolates. Bacterial identification and measurement of MIC are essential to make the appropriate antimicrobial choice.

ECEIM consensus statement on equine metabolic syndrome.

Equine metabolic syndrome (EMS) is a widely recognized collection of risk factors for endocrinopathic laminitis. The most important of these risk factors is insulin dysregulation (ID). Clinicians and horse owners must recognize the presence of these risk factors so that they can be targeted and controlled to reduce the risk of laminitis attacks. Diagnosis of EMS is based partly on the horse's history and clinical examination findings, and partly on laboratory testing. Several choices of test exist which examine different facets of ID and other related metabolic disturbances. EMS is controlled mainly by dietary strategies and exercise programs that aim to improve insulin regulation and decrease obesity where present. In some cases, pharmacologic aids might be useful. Management of an EMS case is a long-term strategy requiring diligence and discipline by the horse's carer and support and guidance from their veterinarians.

An evaluation of serum gentamicin concentrations and bacterial susceptibility to gentamicin in equine practice.

BACKGROUND: Therapeutic drug monitoring and minimum inhibitory concentration (MIC) data allow more informed use of gentamicin. HYPOTHESIS/OBJECTIVES: To measure peak and trough serum gentamicin concentrations in horses after a 6.6 mg/kg dose of gentamicin given IV and the MIC of gentamicin of bacteria for which gentamicin might be selected. METHODS: Retrospective analysis of hospital records. Peak and trough plasma gentamicin concentrations were measured after 6.6 mg/kg gentamicin IV in 339 hospitalized horses. The MIC of gentamicin was measured for 503 isolates from ambulatory practice and 33 from hospital practice. The distribution of gentamicin concentrations and MIC results were compared to current recommendations for MIC breakpoints. RESULTS: The median serum gentamicin concentration at 60 minutes after administration (C60min ) was 21.4 µg/mL with a distribution indicating that bacteria with MIC ≥2 µg/mL were unlikely to be exposed to sufficient gentamicin for effective killing. Approximately 90% of isolates from ambulatory practice and 36% of hospital isolates had MICs at or below breakpoints for susceptibility with most of the remainder unlikely to be responsive, even to higher IV doses. CONCLUSIONS AND CLINICAL IMPORTANCE: Gentamicin at a dosage of 6.6 mg/kg IV is likely to be effective against the majority of infections encountered in ambulatory practice, but less effective in an equine hospital. Because there was a dichotomy of most bacteria as being clearly susceptible or clearly resistant to gentamicin, it appears unlikely that higher doses would have been more efficacious, especially in the hospitalized population in our study.

Therapeutics for Equine Endocrine Disorders.

Equine endocrine disease is commonly encountered by equine practitioners. Pituitary pars intermedia dysfunction (PPID) and equine metabolic syndrome (EMS) predominate. The most logical therapeutic approach in PPID uses dopamine agonists; pergolide mesylate is the most common. Bromocryptine and cabergoline are alternative drugs with similar actions. Drugs from other classes have a poor evidence basis, although cyproheptadine and trilostane might be considered. EMS requires management changes as the primary approach; reasonable justification for use of drugs such as levothyroxine and metformin may apply. Therapeutic options exist in rare cases of diabetes mellitus, diabetes insipidus, hyperthyroidism, and critical illness-related corticosteroid insufficiency.

Endocrine Disease in Aged Horses.

Aging horses may be at particular risk of endocrine disease. Two major equine endocrinopathies, pituitary pars intermedia dysfunction and equine metabolic syndrome, are commonly encountered in an aging population and may present with several recognizable signs, including laminitis. Investigation, treatment, and management of these diseases are discussed. Additionally, aging may be associated with development of rarer endocrinopathic problems, often associated with neoplasia, including diabetes mellitus and other confounders of glucose homeostasis, as well as thyroid, parathyroid, and adrenal diseases. Brief details of the recognition and management of these conditions are presented.

Viraemic frequencies and seroprevalence of non-primate hepacivirus and equine pegiviruses in horses and other mammalian species.

Non-primate hepacivirus (NPHV), equine pegivirus (EPgV) and Theiler's disease associated virus (TDAV) are newly discovered members of two genera in the Flaviviridae family, Hepacivirus and Pegivirus respectively, that include human hepatitis C virus (HCV) and human pegivirus (HPgV). To investigate their epidemiology, persistence and clinical features of infection, large cohorts of horses and other mammalian species were screened for NPHV, EPgV and TDAV viraemia and for past exposure through serological assays for NPHV and EPgV-specific antibodies. NPHV antibodies were detected in 43% of 328 horses screened for antibodies to NS3 and core antibodies, of which three were viraemic by PCR. All five horses that were stablemates of a viraemic horse were seropositive, as was a dog on the same farm. With this single exception, all other species were negative for NPHV antibodies and viraemia: donkeys (n=100), dogs (n=112), cats (n=131), non-human primates (n=164) and humans (n=362). EPgV antibodies to NS3 were detected in 66.5% of horses, including 10 of the 12 horses that had EPgV viraemia. All donkey samples were negative for EPgV antibody and RNA. All horse and donkey samples were negative for TDAV RNA. By comparing viraemia frequencies in horses with and without liver disease, no evidence was obtained that supported an association between active NPHV and EPgV infections with hepatopathy. The study demonstrates that NPHV and EPgV infections are widespread and enzootic in the study horse population and confirms that NPHV and potentially EPgV have higher frequencies of viral clearance than HCV and HPgV infections in humans.