Prolonged hyperinsulinemia increases the production of inflammatory cytokines in equine digital lamellae but not in striated muscle.

Hyperinsulinemia is the key feature of equine metabolic syndrome (EMS) which leads to debilitating sequelae. Hyperinsulinemia-associated laminitis (HAL) is one of the major sequelae of EMS, although the pathophysiological mechanisms are not well elucidated. Using an equine model, we hypothesized that expression of inflammatory markers would be increased in digital lamellae and striated muscle following prolonged hyperinsulinemia. Healthy Standardbred horses (5.4 ± 1.9 years) were alternately assigned to a prolonged euglycemic-hyperinsulinemic clamp (pEHC) or control group (n = 4 per group). Following a 48 h pEHC or a 48 h infusion of a balanced electrolyte solution (controls), biopsies were collected from digital lamellar tissue, skeletal muscle and cardiac muscle were obtained. All hyperinsulinemic horses developed laminitis regardless of previous health status at enrollment. Protein expression was quantified via Western blotting. A significant (P < 0.05) upregulation of the protein expression of heat shock protein 90 (HSP90), alpha 2 macroglobulin (A2M) and fibrinogen (α, ß isoforms), as well as inflammatory cytokines including interleukin-1ß were detected in digital lamellae following prolonged hyperinsulinemia. In contrast, protein expression of cytokines and acute phase proteins in heart and skeletal muscle was unchanged following hyperinsulinemia. Upregulation of inflammatory cytokines and acute phase proteins in digital lamellae during prolonged hyperinsulinemia may reveal potential biomarkers and novel therapeutic targets for equine endocrinopathic laminitis. Further, the lack of increase of inflammatory proteins and acute phase proteins in striated muscle following prolonged hyperinsulinemia may highlight potential anti-inflammatory and cardioprotective mechanisms in these insulin-sensitive tissues.

Equine Protozoal Myeloencephalitis: An Updated Consensus Statement with a Focus on Parasite Biology, Diagnosis, Treatment, and Prevention.

Equine protozoal myeloencephalitis (EPM) remains an important neurologic disease of horses. There are no pathognomonic clinical signs for the disease. Affected horses can have focal or multifocal central nervous system (CNS) disease. EPM can be difficult to diagnose antemortem. It is caused by either of 2 parasites, Sarcocystis neurona and Neospora hughesi, with much less known about N. hughesi. Although risk factors such as transport stress and breed and age correlations have been identified, biologic factors such as genetic predispositions of individual animals, and parasite-specific factors such as strain differences in virulence, remain largely undetermined. This consensus statement update presents current published knowledge of the parasite biology, host immune response, disease pathogenesis, epidemiology, and risk factors. Importantly, the statement provides recommendations for EPM diagnosis, treatment, and prevention.

Glucocorticoid receptor density and binding affinity in healthy horses and horses with systemic inflammatory response syndrome.

BACKGROUND: Dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis occurs in horses with systemic inflammatory response syndrome (SIRS). Peripheral resistance to glucocorticoids has not been investigated in horses. OBJECTIVE: To determine if glucocorticoid receptor (GR) function in horses can be measured using flow cytometry, and to use this information to evaluate HPA axis dynamics. ANIMALS: Eleven healthy adult horses in parts 1 and 2. Ten horses with SIRS and 10 age and sex matched controls in part 3. METHODS: Flow cytometry was used to evaluate GR density and binding affinity (BA) in 3 healthy horses in part 1. In part 2, exogenous ACTH was administered to eight healthy horses. Their cortisol response and GR properties were measured. In part 3, CBC, serum biochemistry, cortisol and ACTH, and GR properties were compared between controls without SIRS (n = 10) and horses with SIRS (n = 10), and between survivors and nonsurvivors (n = 4 and n = 6 respectively). RESULTS: Flow cytometry can be used to measure GR properties in equine PBMCs. No correlation was observed between plasma cortisol concentration and GR density or BA in healthy horses (r = -0.145, P = .428 and r = 0.046, P = .802 respectively). Nonsurvivors with SIRS had significantly decreased GR BA (P = .008). Horses with triglyceride concentration > 28.5 mg/dL had increased odds of nonsurvival (OR=117; 95% CI, 1.94-7,060). GR BA <35.79% was associated with nonsurvival (OR = 30.33; 95% CI, 0.96-960.5). CONCLUSIONS AND CLINICAL IMPORTANCE: Tissue resistance to glucocorticoids contributes to HPA axis dysfunction in adult horses with SIRS. These horses might benefit from treatment with exogenous glucocorticoids.

An update on Sarcocystis neurona infections in animals and equine protozoal myeloencephalitis (EPM).

Equine protozoal myeloencephalitis (EPM) is a serious disease of horses, and its management continues to be a challenge for veterinarians. The protozoan Sarcocystis neurona is most commonly associated with EPM. S. neurona has emerged as a common cause of mortality in marine mammals, especially sea otters (Enhydra lutris). EPM-like illness has also been recorded in several other mammals, including domestic dogs and cats. This paper updates S. neurona and EPM information from the last 15 years on the advances regarding life cycle, molecular biology, epidemiology, clinical signs, diagnosis, treatment and control.

Accurate antemortem diagnosis of equine protozoal myeloencephalitis (EPM) based on detecting intrathecal antibodies against Sarcocystis neurona using the SnSAG2 and SnSAG4/3 ELISAs.

BACKGROUND: Recent work demonstrated the value of antigen-specific antibody indices (AI and C-value) to detect intrathecal antibody production against Sarcocystis neurona for antemortem diagnosis of equine protozoal myeloencephalitis (EPM). OBJECTIVES: The study was conducted to assess whether the antigen-specific antibody indices can be reduced to a simple serum : cerebrospinal fluid (CSF) titer ratio to achieve accurate EPM diagnosis. ANIMALS: Paired serum and CSF samples from 128 horses diagnosed by postmortem examination. The sample set included 44 EPM cases, 35 cervical-vertebral malformation (CVM) cases, 39 neurologic cases other than EPM or CVM, and 10 non-neurologic cases. METHODS: Antibodies against S. neurona were measured in serum and CSF pairs using the SnSAG2 and SnSAG4/3 (SnSAG2, 4/3) ELISAs, and the ratio of each respective serum titer to CSF titer was determined. Likelihood ratios and diagnostic sensitivity and specificity were calculated based on serum titers, CSF titers, and serum : CSF titer ratios. RESULTS: Excellent diagnostic sensitivity and specificity was obtained from the SnSAG2, 4/3 serum : CSF titer ratio. Sensitivity and specificity of 93.2 and 81.1%, respectively, were achieved using a ratio cutoff of ≤100, whereas sensitivity and specificity were 86.4 and 95.9%, respectively, if a more rigorous cutoff of ≤50 was used. Antibody titers in CSF also provided good diagnostic accuracy. Serum antibody titers alone yielded much lower sensitivity and specificity. CONCLUSIONS AND CLINICAL IMPORTANCE: The study confirms the value of detecting intrathecal antibody production for antemortem diagnosis of EPM, and they further show that the antigen-specific antibody indices can be reduced in practice to a simple serum : CSF titer ratio.

Treatment with histamine-type 2 receptor antagonists and omeprazole increase the risk of diarrhoea in neonatal foals treated in intensive care units.

REASONS FOR PERFORMING STUDY: The use of anti-ulcer medication in the neonatal intensive care unit (ICU) is common due to the concern for development of catastrophic gastric ulcerdisease. In man, however, the use of acid-suppressive medication has been shown in some studies to be a substantial riskfactorfor the development of Clostridium difficile-associated diarrhoea (CDAD), bacteraemia and neonatal sepsis. OBJECTIVE: The purpose of the study reported herein is to evaluate the influence of anti-ulcer medications on the development of diarrhoea in the neonatalfoal. HYPOTHESIS: The use of anti-ulcer medication does not alter the incidence of diarrhoea in foals treated in an ICU. METHODS: The records of 1710 foals from 6 different equine hospitals were examined and the use of anti-ulcer drugs was recorded. The presence of in-hospital acquired diarrhoea, CDAD, Clostridium perfringens-associated diarrhoea, neonatal sepsis and salmonellosis were documented. In addition, the presence of gastric ulceration, duration of hospital stay and short-term outcome were examined. RESULTS: The use of anti-ulcer medications increased the odds of in-hospital diarrhoea by 2.0 (95% CI 1.4-2.9; P < 0.0001), relative to the use of no anti-ulcer medication. There was no significant association of anti-ulcer medication with CDAD (P = 0.3189) (OR 2.0; 95% CI 0.4-9.5). Further, results indicated that decreased prevalence of gastric ulceration was not associated with use of anti-ulcer drugs among foals in the study for which these data were known (P = 0.5522). CONCLUSIONS: Use of anti-ulcer drugs increases the odds of developing diarrhoea, and may not reduce the incidence of gastric ulceration in hospitalised equine neonates. POTENTIAL RELEVANCE: The use of anti-ulcer drugs in neonatal foals being treated in a hospital setting should be carefully evaluated on an individual basis to determine if such use is warranted.

Observer agreement study of cervical-vertebral ratios in horses.

REASON FOR PERFORMING STUDY: Different examiners or the same examiner were observed at different times producing slightly different results when obtaining cervical-vertebral ratios. If the difference is substantial, then measurement variability would have an important impact of radiographic interpretation. OBJECTIVE: To quantify agreement and repeatability of these measurements. METHODS: An observer agreement study was performed using 75 horses. Measurements were made at C3-4 and C6-7 by a board-certified radiologist and an imaging resident. Intra- and interobserver agreement was quantified using Bland-Altman plots. Repeatability was assessed as the percentage of differences between duplicate measurements by the radiologist that were within ± 2 s.d. of the differences. RESULTS: At C3-4, the limits of agreement for the intra-vertebral ratio were between -5 and 4% for the intra- and -5 and 6% for interobserver comparison. For the intervertebral ratio, they were between -9 and 8% for the intra- and -10 and 10% for interobserver comparison. At C6-7, the limits of agreement for the intra-vertebral ratio were between -6 and 5% for the intra- and -6 and 8% for interobserver comparison. For the intervertebral ratio, they were between -7 and 7% for the intra- and -6 and 13% for interobserver comparison. At C3-4, all measurements were 95% repeatable (differences typically ≤ 4% and always ≤ 8%) for the intra-vertebral ratio and 96% repeatable (differences typically ≤ 8% and always ≤ 11%) for the intervertebral ratio. At C6-7, all measurements were 98% repeatable (differences typically ≤ 6% and always ≤ 7%) for the intravertebral ratio and 92% repeatable (differences typically ≤ 6% and always ≤ 10%) for the intervertebral ratio. CONCLUSIONS: Cervical-vertebral ratios typically varied by 5-10% within and between examiners. POTENTIAL RELEVANCE: When using cervical-vertebral ratios in practice, the impact of measurement agreement should be factored into the interpretation of the test result because measurement variability may lead to misdiagnosis and limit the clinical usefulness of these tests.

Antibody coefficients for the diagnosis of equine protozoal myeloencephalitis.

BACKGROUND: Diagnosis of equine protozoal myeloencephalitis (EPM) remains a challenge for equine practitioners. Current utilized methods have inadequate sensitivity and specificity, because of a high number of false positive results. HYPOTHESIS/OBJECTIVE: Evaluation of antibody indices to Sarcocystis neurona should provide high sensitivity and specificity for diagnosis of EPM. ANIMALS: Archived samples from 29 clinical patients. METHODS: Archived serum and cerebrospinal fluid (CSF) samples from clinical patients with either EPM (14) or cervical vertebral compressive myelopathy (CVM) (15) were examined and tested for anti-S. neurona antibodies by the SnSAG2 ELISA. The results were used to calculate the antibody index (AI) and C-value. Sensitivity and specificity were calculated, and the AI, C-value, immunoglobulin G (IgG) concentrations, and anti-S. neurona titers compared. In addition, negative CSF was spiked in varying concentrations with blood from a horse with a high anti-S. neurona titer, and the tests repeated. RESULTS: Results demonstrated that the IgG concentration, anti-S. neurona titer, AI, and C-value were significantly higher (P < .05) in horses with EPM than in those with CVM. Sensitivity and specificity of the AI was 71 and 100%, respectively, and that of the C-value was 86 and 100%, respectively. In addition, the AI and C-value from the samples spiked with S. neurona positive blood remained below 1 (eg, negative) in CSF with a red blood cell (RBC) count up to 10(5) RBC/µL. CONCLUSIONS/CLINICAL IMPORTANCE: Results of the study demonstrate the value of calculating the AI and C-value in the diagnosis of EPM in horses. In addition, the test is robust in the presence of blood contamination.

Use of pressor therapy in 34 hypotensive critically ill neonatal foals.

BACKGROUND: Arginine vasopressin (AVP) is used in human medicine in the management of vasodilatory shock and cardiac arrest, but it is not widely used in equine neonatal intensive care because of concerns about potential side effects and suboptimal efficacy. This retrospective study reports the clinical use of AVP and norepinephrine (NE) in foals with refractory hypotension. OBJECTIVES: To report the cardiovascular responses and fluid balance in critically ill, hypotensive foals receiving either NE or AVP. DESIGN: The medical records of neonatal foals (<7 days of age) from 2000 to 2007 admitted to the Marion duPont Scott Equine Medical Center were reviewed. RESULTS: The use of exogenous AVP infusion was associated with a significant increase in mean arterial pressure (MAP) and urinary output, and a significant decrease in heart rate. NE administration was also associated with a significant increase in MAP. CONCLUSIONS: The findings of this first report of the clinical treatment of foals with refractory hypotension support the use of AVP and NE.

Blood glucose concentrations in critically ill neonatal foals.

REASONS FOR PERFORMING STUDY: Critical illness is associated with hyperglycemia in humans, and a greater degree and duration of hyperglycemia is associated with nonsurvival. Hypoglycemia is also seen in critically ill humans, and is associated with nonsurvival. This might also be true in the critically ill foal. OBJECTIVES: To investigate the association of blood glucose concentrations with survival, sepsis, and the systemic inflammatory response syndrome (SIRS). METHODS: Blood glucose concentrations at admission (515 foals) and 24 hours (159 foals), 36 hours (95), 48 hours (82), and 60 hours (45) after admission were analyzed. Logistic regression analyses were performed to investigate the association of glucose concentrations with survival, sepsis, a positive blood culture, or SIRS. RESULTS: 29.1% of foals had blood glucose concentrations within the reference range (76-131 mg/dL) at admission, 36.5% were hyperglycemic, and 34.4% were hypoglycaemic. Foals that did not survive to hospital discharge had lower mean blood glucose concentrations at admission, as well as higher maximum and lower minimum blood glucose concentrations in the 1st 24 hours of hospitalization, and higher blood glucose at 24 and 36 hours. Foals with blood glucose concentrations <2.8 mmol/L (50 mg/dL) or >10 mmol/L (180 mg/dL) at admission were less likely to survive. Hypoglycemia at admission was associated with sepsis, a positive blood culture, and SIRS. CONCLUSIONS AND POTENTIAL RELEVANCE: Derangements of blood glucose concentration are common in critically ill foals. Controlling blood glucose concentrations may therefore be beneficial in the critically ill neonatal foal, and this warrants further investigation.

Prophylactic administration of ponazuril reduces clinical signs and delays seroconversion in horses challenged with Sarcocystis neurona.

The ability of ponazuril to prevent or limit clinical signs of equine protozoal myeloencephalitis (EPM) after infection with Sarcocystis neurona was evaluated. Eighteen horses were assigned to 1 of 3 groups: no treatment, 2.5 mg/kg ponazuril, or 5.0 mg/kg ponazuril. Horses were administered ponazuril, once per day, beginning 7 days before infection (study day 0) and continuing for 28 days postinfection. On day 0, horses were stressed by transport and challenged with 1 million S. neurona sporocysts per horse. Sequential neurologic examinations were performed, and serum and cerebrospinal fluid were collected and assayed for antibodies to S. neurona. All horses in the control group developed neurologic signs, whereas only 71 and 40% of horses in the 2.5 and 5.0 mg/kg ponazuril groups, respectively, developed neurologic abnormalities. This was significant at P = 0.034 by using Fisher exact test. In addition, seroconversion was decreased in the 5.0 mg/kg group compared with the control horses (100 vs. 40%; P = 0.028). Horses with neurologic signs were killed, and a post-mortem examination was performed. Mild-to-moderate, multifocal signs of neuroinflammation were observed. These results confirm that treatment with ponazuril at 5.0 mg/kg minimizes, but does not eliminate, infection and clinical signs of EPM in horses.

Equine endotoxaemia--a state-of-the-art review of therapy.

The pathophysiology of endotoxaemia, a leading cause of death in the horse, is beginning to be understood in greater detail. Endotoxin may be absorbed into the systemic circulation in a number of different ways: most commonly the body's normal defense mechanisms are disrupted or bypassed, or the normal clearance mechanisms overwhelmed. Following this wide-spread effects are observed, although the most significant are seen in the cardiovascular system. Fever, arterial hypoxaemia and signs of abdominal pain are also common. With increased understanding of the disease new therapeutic agents have become available, however, while the newer agents offer some advantages it is important to recognise that supportive care is the mainstay of treatment for endotoxaemia. Supportive care consists of aggressive fluid therapy (crystalloid, colloid and hypertonic), the administration of non-steroidal antiinflammatory drugs and, where appropriate, antimicrobials. The principles of supportive care are discussed in detail. Other therapies such as hyperimmune plasma, polymyxin B, pentoxifylline, dimethyl sulfoxide and heparin are commonly used in the treatment of equine endotoxaemia and their use is reviewed here. Furthermore, newer agents such as anti-tumour necrosis factor antibodies, detergent, activated protein C and insulin, which have yet to gain widespread acceptance but may have an important role in the treatment of endotoxaemia in the future, are examined.

Arterial lactate concentration, hospital survival, sepsis and SIRS in critically ill neonatal foals.

REASONS FOR PERFORMING STUDY: Blood lactate concentration has been shown to be a useful clinical indicator in human patients, but has not been formally investigated in critically ill foals. OBJECTIVE: To investigate the association of blood lactate with hospital survival, markers of cardiovascular status, metabolic acid base status, sepsis and systemic inflammatory response syndrome (SIRS). METHODS: A database containing clinical, haematological, plasma biochemical and hospital outcome data on neonatal foals referred to an intensive care unit in 2000-2001 was analysed. Seventy-two foals for which arterial lactate was measured at admission were included in the study. RESULTS: Sixty-one foals had an admission lactate concentration > 2.5 mmol/l. Admission lactate was statistically associated with hospital survival, mean arterial pressure, blood creatinine concentration, bacteraemia, anion gap, lactate concentration at 18-36 h after admission and evidence of SIRS, but not with packed cell volume or heart rate. Lactate at 18-36 h was also associated with survival and evidence of SIRS. Anion gap, base excess, base excess due to unidentified anions (BEua), simplified strong ion gap or bicarbonate correctly classified foals for presence of hyperlactaemia (> 5 mmol/l) in < or = 80% of animals. CONCLUSIONS: Admission blood lactate gives important prognostic information. Lactate should be measured rather than assumed from the anion gap, base excess, BEua, simplified strong ion gap or bicarbonate. POTENTIAL RELEVANCE: Blood lactate concentrations at admission are clinically relevant in neonatal foals and warrant further investigation. This should include the clinical value of measuring changes in lactate in response to treatment.

Comparison of lithium dilution and thermodilution cardiac output measurements in anaesthetised neonatal foals.

Knowledge of cardiac output is expected to help guide the treatment of hypotension associated with critical illness and/or anaesthesia in neonatal foals. However, a practical and safe method of measuring cardiac output has not been described for the foal. Lithum dilution, a new method of cardiac output determination not requiring cardiac catheterisation, has recently been reported in mature horses. We compared this method to thermodilution in isoflurane-anaesthetised foals age 30-42 h and found good agreement between the 2 methods in a range of cardiac outputs 5.4-20.4 l/min. The lithium dilution technique is a practical and reliable method of measuring cardiac output in anaesthetised neonatal foals, and warrants investigation in critically ill conscious foals.

Pharmacology of INS37217 [P(1)-(uridine 5')-P(4)- (2'-deoxycytidine 5')tetraphosphate, tetrasodium salt], a next-generation P2Y(2) receptor agonist for the treatment of cystic fibrosis.

INS37217 [P(1)-(uridine 5')-P(4)-(2'-deoxycytidine 5')tetraphosphate, tetrasodium salt] is a deoxycytidine-uridine dinucleotide with agonist activity at the P2Y(2) receptor. In primate lung tissues, the P2Y(2) receptor mRNA was located by in situ hybridization predominantly in epithelial cells and not in smooth muscle or stromal tissue. The pharmacologic profile of INS37217 parallels that of UTP, leading to increased chloride and water secretion, increased cilia beat frequency, and increased mucin release. The combined effect of these actions was confirmed in an animal model of tracheal mucus velocity that showed that a single administration of INS37217 significantly enhanced mucus transport for at least 8 h after dosing. This extended duration of action is consistent with the ability of INS37217 to resist metabolism by airway cells and sputum enzymes. The enhanced metabolic stability and resultant increased duration of improved mucociliary clearance may confer significant advantages to INS37217 over other P2Y(2) agonists in the treatment of diseases such as cystic fibrosis.

Early postnatal cardiac changes and premature death in transgenic mice overexpressing a mutant form of serum response factor.

Serum response factor (SRF) is a key regulator of a number of extracellular signal-regulated genes important for cell growth and differentiation. A form of the SRF gene with a double mutation (dmSRF) was generated. This mutation reduced the binding activity of SRF protein to the serum response element and reduced the capability of SRF to activate the atrial natriuretic factor promoter that contains the serum response element. Cardiac-specific overexpression of dmSRF attenuated the total SRF binding activity and resulted in remarkable morphologic changes in the heart of the transgenic mice. These mice had dilated atrial and ventricular chambers, and their ventricular wall thicknesses were only 1/2 to 1/3 the thickness of that of nontransgenic mice. Also these mice had smaller cardiac myocytes and had less myofibrils in their myocytes relative to nontransgenic mice. Altered gene expression and slight interstitial fibrosis were observed in the myocardium of the transgenic mice. All the transgenic mice died within the first 12 days after birth, because of the early onset of severe, dilated cardiomyopathy. These results indicate that dmSRF overexpression in the heart apparently alters cardiac gene expression and blocks normal postnatal cardiac growth and development.

Diagnostic validity of electroencephalography in equine intracranial disorders.

Electroencephalography (EEG) is a valuable diagnostic test to identify functional disturbances in brain activity. The purpose of this study was to assess the validity of EEG as a diagnostic indicator of intracranial diseases in horses. The validity of EEG was estimated by comparing clinical, clinicopathologic, and histopathologic findings to EEG findings in 20 horses examined for seizures. collapse, or abnormal behavior between 1984 and 1997. A bipolar left-to-right, back-to-front montage and a bipolar circular montage were recorded from sedated (4) and anesthetized (16) horses. Visual and semiquantitative masked analysis of EEG recording Ist was validated on 10 horses presented for problems other than intracranial diseases. EEG pattern was normal in 7 of the 20 clinically affected horses. Abnormal EEG patterns included high-voltage slow waves and discrete paroxysmal activity with or without generalized activity in 13 horses. Histopathologic diagnoses in 10 horses included meningoencephalitis, neuronal necrosis, congenital anomalies. cerebral edema. and abscess. All of these horses had abnormal EEG patterns (sensitivity, 100%) with a positive neuroanatomic correlation in 7 animals. Localization of histopathologic and EEG abnormalities did not correlate in 15% of the horses (3/20). The cause of neurologic signs could not be explained at postmortem examination in 10 animals and the EEG pattern was normal in 7 of these horses (specificity, 70%). In conclusion, equine EEG was a sensitive tool in the diagnosis of intracranial disorders.

Lymphocyte phenotype subsets in the cerebrospinal fluid of normal horses and horses with equine protozoal myeloencephalitis.

The percentages of T-lymphocytes, lymphocyte subsets CD4+ and CD8+ T-cells, and lymphocyte adhesion molecule CD11a/CD18 were determined in the peripheral blood and cerebrospinal fluid (CSF) of seven normal horses and four horses with equine protozoal myeloencephalitis (EPM) using flow cytometry. There was a greater percentage of CD5+ cells in the CSF (79.0%) than in peripheral blood (67.0%), although this did not achieve statistical significance. Furthermore, the lymphocyte population in CSF comprises a significantly greater (P = .01) percentage of CD8+ T-cells, resulting in a decrease of the CD4/CD8 ratio. Lymphocyte phenotype subsets in peripheral blood or CSF from horses affected with EPM did not differ from normal horses, although CD5+ T-lymphocytes were seen in significantly greater numbers in the CSF of EPM-affected horses (93.2%) than in normal horses (79.0%).