The effect of month and breed on plasma adrenocorticotropic hormone concentrations in equids.

Use of plasma adrenocorticotropic hormone (ACTH) concentrations for the diagnosis of PPID in equids requires awareness of other factors that might influence ACTH concentrations, including breed and time of year. This study was designed to investigate effects of breed on plasma ACTH concentrations and potential interactions between breed and time of year. Ten breeds were selected from a laboratory database containing data on plasma ACTH concentrations. Breeds represented were Arabians, Cobs, Connemara ponies, donkeys, Irish Sports horses, New Forest ponies, Shetland ponies, Thoroughbreds, Warmbloods and Welsh breeds. Multivariable analysis was used to determine the effects of month and breed on plasma ACTH concentrations. A circannual pattern of ACTH concentration was documented, with a nadir in April and a peak in September. Arabian horses and donkeys had significantly higher ACTH concentrations than some other breeds, primarily from May to November, whereas in Shetland ponies and Welsh breeds, relatively higher ACTH concentrations only occurred from July to November. The annual increase in plasma ACTH appeared to commence in late April/early May in response to increasing daylight and decreased again promptly after the late September equinox as daylength fell below 12 h per day. Inter-breed differences in plasma ACTH were substantial and indicated that breed and time of year should be considered when interpreting plasma ACTH concentrations in equids suspected for PPID.

The effect of pergolide mesylate on adrenocorticotrophic hormone responses to exogenous thyrotropin releasing hormone in horses.

Thyrotropin releasing hormone (TRH) stimulation testing is often used to support a diagnosis of pituitary pars intermedia dysfunction (PPID) in horses although it is unclear whether or not repeat TRH stimulation testing post-treatment is a valid means of assessing response to medical therapy. Laboratory submissions from 64 suspected equine PPID cases were examined including the initial pre-treatment TRH stimulation test and a follow up test within 100 days of starting medical therapy with pergolide. In a subset of cases, further follow-up tests were examined beyond 100 days of starting treatment. Results from tests conducted between 1 July and 30 November were excluded. Significant improvements were seen in both the baseline and TRH-stimulated adrenocorticotrophic hormone (ACTH) concentrations within 100 days with no further improvements seen in the subset of cases examined thereafter. Although 88% (n = 56/64) of all cases showed a decreased response to TRH post-treatment, only 24% (n = 9/38) of horses with positive pre-treatment TRH stimulation tests normalised following treatment, with a further 34% (n = 13/38) improving into an equivocal test outcome category. Most commonly (42%; n = 16/38), horses with positive pre-treatment TRH stimulation tests remained positive following treatment, although 75% (n = 12/16) of these showed a numerically lower post-treatment response to TRH. These results will help inform practitioners of expected changes in TRH stimulation test results when assessing response of horses with PPID to medical therapy with pergolide.

A study of the environmental survival of Streptococcus equi subspecies equi.

BACKGROUND: Streptococcus equi represents a common hazard to equids worldwide. Environmental contamination with bacteria shed from an infected horse may represent a significant source of contagion and further knowledge of ex vivo bacterial survival under different conditions is important for disinfection and isolation protocols. OBJECTIVES: To determine the potential duration of survival and vigour of growth of S. equi inoculated onto surfaces relevant to equine veterinary practice and stabling in summer and winter. STUDY DESIGN: Repeat sampling of environmental inocula of S. equi. METHODS: Cultures of S. equi were inoculated onto wood, a shoe sole, cotton overalls, inside a nasogastric tube, inside a dental rasp, in a wet plastic bucket and onto a fence post both in the summer and winter seasons. Frequent resampling and culture from the inoculated sites was conducted until no viable bacteria were found. Bacterial viability was determined by both duration (time to first negative culture) and vigour of growth (growth score over the first 3 days of culture) and compared between inoculated sites and times of year. RESULTS: Bacterial viability was enhanced by a wet local environment and by the winter season. Survival tended to be short in the summer (up to 9 days in wet sites and up to 2 days in dry sites) but much longer in the winter (up to 34 days in wet sites and up to 13 days in dry sites). Vigour of bacterial growth was also greater in the winter than in the summer as judged by 3-day-growth scores. MAIN LIMITATIONS: Direct comparison with the variable size and nature of naturally shed infectious material is difficult. CONCLUSIONS: Veterinarians and personnel handling horses should be aware that S. equi may survive in an equine environment for longer than previously found, especially when protected by wet and cold conditions.

Effect of storage time and temperature on the results of analysis of synovial and mesothelial fluids.

REASONS FOR PERFORMING STUDY: Delays between collection and laboratory analysis of equine body fluid samples are common in practice; however, the effects of delays on the accuracy of results and diagnostic interpretation are unknown. OBJECTIVES: To assess the effects of storage time and temperature combination on protein and cell parameters of equine synovial and mesothelial cavity fluids and determine whether any changes affect clinicopathological interpretation. STUDY DESIGN: In vitro experiment. METHODS: Body fluid samples obtained from horses during diagnostic investigation were divided into 7 aliquots and total protein concentration (TP), total nucleated cell count (TNCC) and neutrophil morphology were analysed immediately (T0 ) and at 24 (T24 ), 48 (T48 ) and 72 h (T72 ) after storage at 4 or 22°C. Linear mixed models were used to analyse effects of fluid type and storage conditions on TP, TNCC and neutrophil morphology grade. Changes in interpretation of samples over time and diagnostic performance at each analysis point were recorded. RESULTS: A total of 32 samples were collected from 23 horses. Storage had no effect on TP. Cell count was influenced by fluid type and was significantly reduced at T72 for storage at 4°C and T24 , T48 and T72 for 22°C (P<0.001). Neutrophil morphology grade was significantly greater at T24 , T48 and T72 than at T0 for both 4 and 22°C (P<0.001). For 9 samples, the diagnostic interpretation changed over time. Specificity and positive predictive value at each analysis point was 100%; however, sensitivity and negative predictive value decreased with greater storage duration and temperature. CONCLUSIONS: Alterations in the TNCC and neutrophil morphology of body fluid samples occur when analysis is delayed, especially with higher storage temperatures, and may influence interpretation and clinical decision-making. Body fluid samples should be analysed as soon as possible after collection to minimise preanalytical errors due to storage.

Investigation of single and paired measurements of adrenocorticotropic hormone for the diagnosis of pituitary pars intermedia dysfunction in horses.

BACKGROUND: Paired measurement of ACTH concentration may be more reliable than a single measurement. HYPOTHESIS/OBJECTIVES: To determine whether the mean of 2 measurements of ACTH concentration is more reliable in assessing pituitary pars intermedia dysfunction (PPID) than a single measurement. ANIMALS: Paired ACTH measurements were performed on (1) 148 occasions from 124 horses being investigated for PPID, (2) 90 occasions from 76 horses with PPID that were receiving treatment with pergolide, and (3) 63 occasions from 50 horses in which there was no clinical suspicion of PPID. Histologic examination of the pars intermedia was performed in 67 of the untreated horses. METHODS: Outcome of testing using single and the mean of paired samples was compared directly and both methods were compared against histology, which was considered the gold standard. RESULTS: Paired ACTH measurement altered binary classification as healthy or diseased in 6 of 211 cases, all off which had equivocal initial ACTH concentrations between 20 and 39 pg/mL. Using histology as the gold standard, optimal sensitivity and specificity for diagnosing PPID were 69.4 and 80.9%, respectively, for a single measurement and 72.2 and 76.2%, respectively, for paired measurements. The area under the receiver operating characteristic curve was 0.72 and 0.73 for single and paired measurements compared with histopathologic diagnosis, respectively. CONCLUSIONS AND CLINICAL IMPORTANCE: Paired measurement of ACTH concentration offers no advantage over a single measurement.

Effects of metformin hydrochloride on blood glucose and insulin responses to oral dextrose in horses.

REASONS FOR PERFORMING STUDY: Metformin is a potential therapeutic agent for the treatment of insulin resistance (IR). In laboratory animals, orally administered metformin reduces intestinal glucose absorption and may therefore affect insulinaemic responses to oral carbohydrate ingestion. OBJECTIVES: To determine whether pretreatment with metformin reduces plasma glucose concentration and insulin responses following consumption of dextrose in horses. STUDY DESIGN: Therapeutic cross-over study. METHODS: Seven healthy Standardbred and Thoroughbred geldings were subjected to an oral dextrose challenge test on 4 occasions: with and without metformin, before and after induction of IR with dexamethasone. Metformin was administered by nasogastric tube at 30 mg/kg bwt 1 h before administration of dextrose. Glucose and insulin concentrations in plasma/serum were measured at regular intervals during each test. Linear mixed models were specified for each predetermined outcome variable, and for each model the 'treatment' was included as a fixed effect with 4 categorical levels (none, metformin, dexamethasone and dexamethasone with metformin) and horse accounted for as a random effect. RESULTS: In healthy horses, the administration of metformin resulted in a statistically significant reduction in peak glucose concentration (P = 0.002), area under the glucose curve (P<0.001) and insulin concentration 120 min after dextrose administration (P = 0.011). Following the induction of IR, administration of metformin was associated with significant differences in peak glucose concentration (P<0.001), the percentage increase in glucose concentration (P = 0.010), the area under the glucose curve (P<0.001) and insulin concentration at 120 min (P = 0.034) and 150 min after dextrose administration (P = 0.014). CONCLUSIONS: Metformin resulted in reduced glycaemic and insulinaemic responses both in healthy horses and in horses with experimentally induced IR. POTENTIAL RELEVANCE: Metformin may benefit horses with naturally acquired IR by reducing glycaemic and insulinaemic responses to dietary nonstructural carbohydrates. Further investigations into the mechanisms of action of metformin in horses and controlled clinical trials are warranted.

In equine grass sickness, serum amyloid A and fibrinogen are elevated, and can aid differential diagnosis from non-inflammatory causes of colic.

Equine grass sickness (EGS) is a debilitating and often fatal neurodegenerative disease. A presumptive diagnosis of EGS may be made on the basis of clinical signs and subjective ancillary tests, but a definitive antemortem diagnosis can only be made following histopathological examination of intestinal biopsies. It has previously been reported that horses with EGS may show clinical and clinicopathological signs of systemic inflammation. The objective of this study was to (a) quantify acute inflammatory markers in blood samples collected from acute, subacute and chronic EGS cases, and (b) compare them with (i) clinically normal horses co-grazing with acute EGS cases (co-grazers), (ii) horses with other causes of colic and (iii) healthy horses. Serum amyloid A (SAA), serum activin A and plasma fibrinogen were quantified. There were marked increases in SAA and fibrinogen in EGS cases compared with healthy horses, co-grazers and non-inflammatory colic cases. The concentrations of SAA and fibrinogen in EGS cases were not significantly different from inflammatory colic cases. When concentrations of SAA, fibrinogen and activin A in each EGS subgroup were compared, no significant differences were detected. Activin A concentrations were significantly elevated in EGS cases and co-grazing horses; this could reflect the presence of subclinical disease in some horses that do not develop clinical signs of EGS, and suggests widespread exposure to the aetiological agent. When faced with sparse antemortem diagnostic techniques, identification of marked increases in acute phase protein concentrations may help to differentiate EGS from other causes of abdominal pain, such as intestinal obstructions; however, there could be diagnostic difficulty in differentiating other inflammatory abdominal conditions, such as peritonitis or enteritis.

Circannual variation in plasma adrenocorticotropic hormone concentrations in the UK in normal horses and ponies, and those with pituitary pars intermedia dysfunction.

REASONS FOR PERFORMING STUDY: Pituitary pars intermedia dysfunction (PPID) is a common endocrinopathy, frequently diagnosed via plasma adrenocorticotropic hormone (ACTH) concentrations. Seasonal variation in plasma ACTH concentrations has been described in normal horses prompting caution in diagnosing PPID at certain times of the year. The aims of this study were to determine appropriate reference intervals for equine plasma ACTH throughout the year; and to examine the circannual variation of plasma ACTH concentrations in PPID cases. HYPOTHESIS: Plasma ACTH can be used as a test for PPID throughout the year with the use of appropriate reference intervals. METHODS: Data for reference interval calculations were obtained from samples collected from inpatients of Liphook Equine Hospital (non-PPID group, n=156). Data from PPID cases (n=941) were obtained from samples submitted to the Liphook Equine Hospital Laboratory from horses with a clinical suspicion of PPID found to have plasma ACTH concentrations greater than our upper reference interval for that time of year. RESULTS: Upper limits for reference interval of plasma ACTH were 29 pg/ml between November and July and 47 pg/ml between August and October. Circannual variation in plasma ACTH occurred in both non-PPID and PPID horses with the highest ACTH concentrations found between August and October in both groups (P<0.0001). The greatest difference between the 2 populations also occurred between August and October. CONCLUSIONS: Plasma ACTH can be used for the diagnosis and monitoring of PPID throughout the year with the use of appropriate reference intervals. These findings demonstrate an increase in pituitary gland secretory activity during the late summer and autumn in both normal and PPID cases.

Stability of common biochemistry analytes in equine blood stored at room temperature.

REASONS FOR PERFORMING STUDY: Time delays between collection of blood samples and biochemical analysis of equine blood are unavoidably common in equine practice. The effect that delays may have on the accuracy of results of blood biochemical analyses is not well established. HYPOTHESIS: Delays in processing of blood of up to 72 h results in alterations in measured levels of common biochemical analytes that are of potential clinical relevance. Separation of serum prior to storage is protective against the effects of time delays. METHODS: Samples of clotted blood, separated serum and oxalate fluoride plasma from 20 horses were stored and analysed at 0, 24, 48 and 72 h. Graphical exploration of each analyte was undertaken. General linear models with fixed effects were fitted for the whole blood data. The mean bias and 95% limits of agreement were calculated, using bootstrapped data, to assess agreement between pairs of samples analysed at 0 h and other time points. Bland-Altman plots were used to explore general trends in the data. Paired t tests were used to compare the results from whole blood and separated serum. RESULTS: Delays in processing equine blood resulted in significant increases in measured concentrations of aspartate aminotransferase, creatine kinase, lactate dehydrogenase, total bile acids and magnesium. A significant decrease in concentration was identified for glucose (serum and oxalate fluoride preserved plasma). Separation of serum immediately following clot formation resulted in nonsignificant increases in accuracy for some analytes. CONCLUSIONS AND PRACTICAL SIGNIFICANCE: Delays in processing of blood samples may result in biochemical changes of clinical relevance in individual cases; however, in the majority of cases, where delays are only a few days and a number of analytes are assessed concurrently, delays are unlikely to have an effect on the interpretation of results. Separation of serum following clot formation is of limited benefit. Clinical samples in which a delay in processing has occurred may be interpreted with reference to the data presented.

Type 2 diabetes mellitus with pancreatic beta cell dysfunction in 3 horses confirmed with minimal model analysis.

REASONS FOR PERFORMING STUDY: Type 2 diabetes mellitus (T2DM) is diagnosed rarely in equine practice although it may be under-recognised. A greater awareness of the condition and therapeutic considerations would be to the benefit of such cases presenting in practice. More investigation into the pharmacological management of these cases is needed. OBJECTIVES: Three cases of diabetes mellitus were investigated using a specific test for insulin sensitivity and pancreatic beta cell function in order to define accurately and characterise the existence of T2DM in all 3 subjects. METHODS: The insulin-modified frequently sampled i.v. glucose tolerance test was performed in each case and the data so obtained were subject to minimal model analysis of insulin-glucose dynamics. Cases were then monitored following treatment using a combination of dietary modification, metformin, glibenclamide and pergolide. RESULTS: Marked insulin resistance was identified in each case and, furthermore, severe pancreatic beta cell dysfunction was present therefore classifying each case as end stage T2DM. Treatment was nevertheless associated with restoration of normoglycaemia in all cases. CONCLUSIONS: T2DM in horses may be more common than generally considered.In some cases individuals may respond to therapy aimed at restoring insulin sensitivity and pancreatic function. Drugs used in other species for the treatment of T2DM have not yet been adequately tested in horses. POTENTIAL RELEVANCE: T2DM should be considered as an important differential diagnosis in mature to elderly horses and ponies suffering from weight loss, polydipsia and polyuria. Clinicians should be encouraged to offer treatment and management advice when such cases are encountered.

Long-term management of sabulous cystitis in five horses.

Five horses with sabulous cystitis were managed for up to three years. They were treated by emptying the bladder through a urinary catheter and saline lavage with cytoscopic guidance to remove residual sabulous material. The cystitis was treated with antimicrobial and anti-inflammatory medications, and bethanechol chloride was also administered. Frequent catheterisation and emptying of the bladder was an alternative to regular cystoscopic examination with saline lavage but it resulted in the development of a urethral stricture in one case. Four of the horses returned to work and one was retired owing to persistent incontinence.

The effect of metformin on measurements of insulin sensitivity and beta cell response in 18 horses and ponies with insulin resistance.

REASONS FOR PERFORMING STUDY: Laminitis in equids is a very common debilitating disease, and insulin resistance (IR) and hyperinsulinaemia are increasingly recognised as important predisposing factors. Pharmacological modification of IR and hyperinsulinaemia might reduce the risk of laminitis. HYPOTHESIS: Metformin, a drug commonly prescribed for treatment of human IR, may also decrease IR in equids. METHODS: Eighteen horses and ponies with IR and recurrent laminitis were treated with 15 mg/kg bwt metformin per os q. 12 h. Each animal served as its own control by comparing pre- and post treatment proxies for IR, insulin sensitivity (IS) and pancreatic beta cell function while controlling for possible dietary and managemental influences on IR. RESULTS: Evidence of significantly improved IS and decreased pancreatic beta cell secretion was found following metformin treatment. The magnitude of effect was greater at earlier resampling (6-14 days) than at later times (23-220 days). Apparent subjective clinical benefits were good but less favourable than effects on IR. CONCLUSIONS: Metformin is safe and appears to increase IS in equids. POTENTIAL RELEVANCE: Metformin may be indicated as a treatment for IR in equids. Further studies are required to define appropriate selection of subjects warranting therapy, dosing schedule and pharmacokinetics.

Clinical, immunophenotypic and functional characterisation of T-cell leukaemia in six horses.

REASON FOR PERFORMING STUDY: Lymphoid leukaemia (LL) is rare in equids. In man, immunophenotypic classification identifies distinct leukaemic types with different treatment strategies. Improved understanding and classification of equine LL may allow similar advances. OBJECTIVES: To document the clinical, immunophenotypic and functional characteristics in 6 cases of equine LL of T-cell origin. METHODS: The clinical records and pathological findings from 6 cases of equine LL were analysed. Immunohistochemistry to identify T or B lymphocytes was performed on paraffin embedded tissues in 4 cases. Peripheral blood mononuclear cells (PBMC) were phenotyped for expression of CD4, CD8, MHC class I and II and B-cell antigens in 4 cases using monoclonal antibodies (mAbs) and flow cytometry. Neoplastic lymphocytes from 4 horses were stimulated with mitogens. RESULTS AND CONCLUSIONS: Six horses of various breeds were identified with LL of T-cell origin. The clinical course and presenting signs varied. Neoplastic lymphocytes were identified in peripheral blood samples from all horses and tissue invasion was confirmed at examination post mortem in 4 horses. Immunophenotyping identified a predominance of CD3+ T-cells in lymphoid tissues and CD4+ T-cells in circulating peripheral blood mononuclear cells (PBMC) in the affected horses. Neoplastic lymphocytes from the 4 cases that were tested failed to proliferate in response to mitogens. POTENTIAL RELEVANCE: Characterisation of the clinical, pathological and immunological findings in 6 horses with LL has added to reports of this rare condition, characterised it in greater detail and therefore provides a starting point for further investigations.

Clinical application of parenteral nutrition in the treatment of five ponies and one donkey with hyperlipaemia.

Five ponies and one donkey with hyperlipaemia that occurred secondarily to a variety of primary clinical conditions were treated with lipid-free partial parenteral nutrition comprising equal volumes of 50 per cent glucose and 15 per cent amino acids. The infusion supplied energy and protein at rates of 2.6 kJ/kg per hour and 34.3 mg/kg per hour, respectively. In all six cases there was a prompt and sustained decrease in serum concentrations of triglyceride. In four of the six cases a good response to treatment of the primary condition was also seen and the subjects were discharged successfully. In the remaining two cases, poor clinical response of the primary condition resulted in euthanasia, although hyperlipaemia was nevertheless resolved. The main complication of parenteral nutrition was hyperglycaemia.

Quantification of immune cell populations in the lamina propria of equine jejunal biopsy specimens.

The histological diagnosis of inflammatory bowel disease (IBD) in horses and other species is subjective, and pathological assessments vary considerably as a result. One important criterion is increased infiltration of the lamina propria by eosinophils, plasma cells, lymphocytes or macrophages, but this is difficult to assess without a knowledge of the normal immune cell populations and potential for individual variation. Retrospective jejunal specimens were analysed from 14 horses aged 13-15 years which had not shown clinical or post-mortem signs of gastrointestinal disease. Populations of plasma cells, T lymphocytes (CD3+), B lymphocytes (CD79a+ cytoplasmic membranes), eosinophils, macrophages and neutrophils were counted in 9000-microm2 areas of the villous lamina propria and intercryptal lamina propria for each horse. There were significantly higher counts of plasma cells, B lymphocytes and eosinophils in the intercryptal than in the villous region, which accords with previous findings in dogs. This information will be used as control data for future quantitative morphometrical analysis of immune cells in small intestinal specimens from horses in which IBD has been diagnosed.

Nutritional and clinicopathological effects of post operative parenteral nutrition following small intestinal resection and anastomosis in the mature horse.

REASONS FOR PERFORMING STUDY: There is an absence of data describing the nutritional requirements and nutritional status of horses following surgery for colic; furthermore, the potential effect of parenteral nutrition (PN) on improving nutritional status in such cases is unknown. HYPOTHESIS: Post operative colic cases suffer from a potentially detrimental negative energy balance and the PN formulation developed in this study would lead to clinicopathologically detectable improvements in the subjects' nutritional status. METHODS: Several clinicopathological variables, some known to be associated with nutritional status, were compared in 2 groups of horses in the post operative period following colic surgery; Group N (n = 15) were treated with PN and Group C (n = 15) were starved routinely. RESULTS: Group N had significantly lower serum concentrations of triglycerides, total bilirubin, albumin and urea and significantly higher serum concentrations of glucose and insulin compared with Group C in the post operative period. CONCLUSIONS: The control group of horses demonstrated significant clinicopathological evidence of starvation and the described PN protocol resulted in a demonstrably improved nutritional status in the treated horses. POTENTIAL RELEVANCE: Further study is required to investigate clinical benefits and possible harmful side effects of post operative parenteral nutrition before the technique can be advocated for widespread use in practice.