Platelet distribution width and mean platelet volume in the interpretation of thrombocytopenia in dogs.

OBJECTIVE: To investigate the use of platelet volume indices in the interpretation of thrombocytopenia in dogs with systemic disease. METHODS: Case records of 80 control dogs and 159 thrombocytopenic dogs with systemic disease were reviewed retrospectively. The relationships between mean platelet volume, platelet distribution width and platelet count in systemically well dogs were established. Knowledge of these relationships was used to interpret thrombocytopenia in terms of the underlying disease process. RESULTS: There was a positive relationship between platelet distribution width and mean platelet volume. Both platelet distribution width and mean platelet volume varied in a negative relationship with platelet count, which was unaffected by signalment, underlying disease, variations in haemotological parameters or serum proteins. CLINICAL SIGNIFICANCE: The interpretation of mean platelet volume and platelet distribution width in dogs requires simultaneous reference to the platelet count. Platelet volume indices did not aid interpretation of thrombocytopenia in terms of underlying pathological processes in the population studied.

Histological assessment of ß-amyloid precursor protein immunolabelled rectal biopsies aids diagnosis of equine grass sickness.

BACKGROUND: An accurate, minimally invasive, ante-mortem diagnostic test for equine grass sickness (EGS) is currently lacking. Although histological examination of haematoxylin and eosin-stained rectal biopsies for chromatolytic neurons is insensitive as a diagnostic test for EGS, we hypothesised that its diagnostic accuracy could be improved by immunolabelling for ß-amyloid precursor protein (ß-APP), which has increased expression in cranial cervical ganglia (CCG) neuronal perikarya in EGS. OBJECTIVES: To develop a grading scheme for assessing the distribution and intensity of ß-APP immunoreactivity within individual rectal submucosal neurons and subsequently to determine the value of the distribution of different grades of neurons in EGS diagnosis. STUDY DESIGN: Retrospective case-control diagnostic accuracy study. METHODS: Initially, a standardised grading scheme was developed and ß-APP immunoreactivity in individual neuronal perikarya and axons was compared in sections of CCG and ileum from EGS and control horses. The grading scheme was then refined before being blindly applied to submucosal neurons in rectal biopsies derived from 21 EGS and 23 control horses. RESULTS: ß-APP immunoreactivity was increased in neuronal perikarya and axons in sections of CCG, ileum and rectum from EGS horses compared with controls. For rectal biopsies, a mean immunoreactivity grade exceeding 1.1 was 100% specific and sensitive for EGS, and the presence of at least one neuron with diffuse labelling of the entire cytoplasm (grade 3) was 95% sensitive and 100% specific for EGS. MAIN LIMITATIONS: Although the diagnostic criteria facilitated the discrimination of the EGS and control biopsies evaluated in this study, further prospective validation using a larger sample set is required. CONCLUSIONS: Histological assessment of ß-APP immunolabelled rectal biopsies is more sensitive than conventional histological examination in EGS diagnosis. Further validation is required before this technique can be advocated for use in clinical decision making.

Equine grass sickness, but not botulism, causes autonomic and enteric neurodegeneration and increases soluble N-ethylmaleimide-sensitive factor attachment receptor protein expression within neuronal perikarya.

REASONS FOR PERFORMING STUDY: Equine grass sickness (EGS) is of unknown aetiology. Despite some evidence suggesting that it represents a toxico-infection with Clostridium botulinum types C and/or D, the effect of EGS on the functional targets of botulinum neurotoxins, namely the soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins, is unknown. Further, while it is commonly stated that, unlike EGS, equine botulism is not associated with autonomic and enteric neurodegeneration, this has not been definitively assessed. OBJECTIVES: To determine: 1) whether botulism causes autonomic and enteric neurodegeneration; and 2) the effect of EGS on the expression of SNARE proteins within cranial cervical ganglion (CCG) and enteric neuronal perikarya. STUDY DESIGN: Descriptive study. METHODS: Light microscopy was used to compare the morphology of neurons in haematoxylin-eosin stained sections of CCG and ileum from 6 EGS horses, 5 botulism horses and 6 control horses. Immunohistochemistry was used to compare the expression of synaptosomal-associated protein-25, synaptobrevin (Syb) and syntaxin within CCG neurons, and of Syb in enteric neurons, from horses with EGS, horses with botulism and control horses. The concentrations of these SNARE proteins in extracts of CCG from EGS and control horses were compared using quantitative fluorescent western blotting. RESULTS: EGS, but not botulism, was associated with autonomic and enteric neurodegeneration and with increased immunoreactivity for SNARE proteins within neuronal perikarya. Quantitative fluorescent western blotting confirmed increased concentrations of synaptosomal-associated protein-25, Syb and syntaxin within CCG extracts from EGS vs. control horses, with the increases in the latter 2 proteins being statistically significant. CONCLUSIONS: The occurrence of autonomic and enteric neurodegeneration, and increased expression of SNARE proteins within neuronal perikarya, in EGS but not botulism, suggests that EGS may not be caused by botulinum neurotoxins. Further investigation of the aetiology of EGS is therefore warranted.

Observations on the interstitial cells of Cajal and neurons in a recovered case of equine dysautonomia (grass sickness).

This paper describes the histopathological and immunohistochemical changes in the central, autonomic and enteric nervous systems in a well-documented case of equine dysautonomia (ED), after the animal had recovered without significant residual clinical signs. Evidence of neuronal degeneration, such as neuronal chromatolysis, glial scars or a decrease in density of neurons, was not observed in the midbrain, pons, medulla oblongata or spinal cord, including the nuclei of cranial nerves III, V, VII, X and XII. In addition, no evidence of muscle denervation or re-innervation, such as group atrophy of clusters of cells or grouping of cells of similar size in the triceps muscle, was observed. These findings support the hypothesis that the chromatolytic motor neurons may not progress to cell death in ED. The autonomic ganglia showed evidence of previous neuronal loss. In the myenteric and submucosal plexuses of the ileum there was significant loss of enteric neurons. However, in the ileum, a continuous network of interstitial cells of Cajal (ICC) was observed by means of immunohistochemical techniques. It is possible that recovery without significant residual clinical signs was associated with lack of damage to, or more likely recovery of, the ICC, which have a pacemaker function in the intestinal tract.

Heparin, platelet aggregation, neutrophils, and cardiopulmonary bypass.

UNLABELLED: Cardiopulmonary bypass (CPB) is associated with both neutrophil activation and failure of platelets to form large stable aggregates. We aimed to determine the effects of heparin and of neutrophil activation on platelet aggregation in whole blood. Fourteen patients undergoing routine aortocoronary bypass grafting and NSAID-free for over 7 days were studied before and after heparinisation, and at end-CPB. Whole blood, anticoagulated with rHirudin, was stirred for 3 minutes, and macroaggregation in response to collagen (0.6 microg. mL(-1)) or the neutrophil stimulant fMLP (10(-7)M) was determined by whole blood impedance aggregometry. Microaggregation was measured by counting unaggregated single platelets (corrected for haemodilution). The blood of volunteers was studied in vitro. PATIENTS: Before CPB, heparin effectively abolished platelet macroaggregation induced by collagen (20.5 to 1.4 Ohms) or fMLP (3.9 to 0 Ohms (p<0.0001). CPB had no additional effect. Heparinisation also reduced the platelet count from 127 (110-170) to 95 (64-117). The inhibition of macroaggregation could not be reversed by ex vivo heparinase. VOLUNTEERS: In vitro, the same heparin concentration, as measured in vivo (4 micromL(-1)), inhibited collagen-induced macroaggregation (20.3 to 14.7 Omega), but this effect was less than that observed ex vivo and was reversed by heparinase. In vitro heparin promoted fMLP macroaggregation (2.9 to 8.6 Omega). The inhibition of macroaggregation resulted from heparinisation, per se, rather than CPB and was insensitive to heparinase. There was less inhibition by in vitro heparin, which was reversible by heparinase, indicating a direct effect of heparin in vitro. The disparate findings are suggestive of an indirect action by heparin in vivo on macroaggregation, although heparin had a small direct stimulatory action on microaggregation.

Autonomic neurone degeneration in equine dysautonomia (grass sickness).

Histological investigations were undertaken on four sympathetic autonomic ganglia and on the myenteric and sub-mucosal plexuses of the jejunum in healthy animals, in naturally occurring cases of acute, sub-acute and chronic equine dysautonomia and in ponies in which neuronal damage had been induced by the injection of acute grass sickness sera. The degree of neuronal damage is related to the type of dysautonomia. The coeliac-mesenteric ganglion reacts differently from other ganglia and is less severely damaged in cases of short duration. Extensive experimentally induced damage to the coeliac-mesenteric ganglion, even when jejunal damage is also present, is not associated with clinical illness. It is proposed that the rate of autonomic neurone loss and the extent of the damage may both influence the clinical manifestations of grass sickness.

Histopathology of the brain-stem nuclei of horses with "Mal seco", an equine dysautonomia.

"Mal seco" is a disease of unknown aetiology affecting horses in Argentina. It is similar to grass sickness, a primary dysautonomia of horses in Europe. A histopathological study of the brain stem nuclei of three horses with "mal seco" was performed. Changes were found that consisted of chromatolysis, cytoplasmic vacuoles, eosinophilic sphaeroids, and pyknotic and eccentric nuclei. These changes were most severe at the oculomotor, vestibular and abducent nuclei. The results provide further evidence to suggest that "mal seco" and grass sickness may be the same disease.

Gamma-glutamyl transpeptidase and its multiple forms in the tissues and sera of normal dogs.

The total gamma-glutamyl transpeptidase activity and the proportions of its multiple forms in canine tissues and serum have been measured. The kidney showed the greatest activity, followed by the pancreas and small intestine. After separation by cellulose acetate electrophoresis, two main areas of activity were observed in tissues, an insoluble fraction which remained at the origin and a soluble fraction which migrated towards the anode. The soluble fraction was divisible into six zones differing in mobility and, with the exception of liver and lung, the tissues differed in the distances migrated by these soluble fractions. Serum gamma glutamyl transpeptidase formed one zone corresponding to the soluble fraction of liver and lung. The separation of the multiple forms is of potential value in determining the source of increased serum activities of the enzyme in the dog.

Lactate dehydrogenase and its isoenzymes in the tissues and sera of clinically normal dogs.

The total activity of lactate dehydrogenase (LDH) and the percentage distribution of its isoenzymes in the tissues and sera of clinically normal adult dogs are presented. Total LDH activity was greatest in skeletal muscle followed by heart muscle, kidney, small intestinal mucosa, liver, lung, pancreas and bone. Each tissue had a unique isoenzyme pattern and the proportions of the isoenzymes in serum suggested that liver is the source of normal serum LDH. The tissue isoenzyme patterns were similar to those obtained by other authors in human beings, horses, cattle, sheep and cats although in liver, differences between ruminants and monogastric animals including dogs were evident. The data presented provide a basis for the interpretation of serum LDH isoenzyme patterns in canine disease.

An improved TLC method for the detection of flunixin in equine serum.

A method for flunixin detection in equine serum extracts involving thin layer chromatography, spraying the chromatogram with alkaline sodium hypochlorite solution and heating with a detection limit of 50 ng ml-1 is described.

Serum lipids and lipoproteins in equine colic and grass sickness.

Serum total lipids, lipoprotein fractions, triglycerides, cholesterol, phospholipids and free fatty acids were measured in horses with acute, subacute and chronic grass sickness (equine dysautonomia) and in colic cases. The values were compared with those of normal grazing and stabled horses. A marked individual variation occurred, but total lipids, triglycerides and free fatty acids were significantly higher than normal in grass sickness and colic cases with cholesterol was significantly higher than normal in grass sickness cases only. Pre-beta lipoprotein was significantly increased in colic and subacute grass sickness although all abnormal groups showed this fraction which was absent from normal horses. The percentage of alpha 2b lipoprotein was significantly higher in colic and grass sickness. The changes described are typical of those occurring in fat mobilisation in the horse and are considered to be due to a number of factors including decreased food intake, cortisol and catecholamine release and insulin resistance.

Acute phase proteins in grass sickness (equine dysautonomia).

Four acute phase proteins were assayed in the serum of normal horses and those with acute, subacute and chronic grass sickness, colic and inflammatory conditions, in order to investigate their diagnostic value in grass sickness. The grass sickness and inflammation group had a significantly increased haptoglobin concentration (P less than 0.01-P less than 0.001). Orosomucoid was elevated in acute, subacute and chronic grass sickness and inflammation (P less than 0.001, P less than 0.001, P less than 0.05 and P less than 0.05, respectively). Highest concentrations of haptoglobin and orosomucoid were recorded in subacute grass sickness. Ceruloplasmin was significantly higher in acute grass sickness cases than all other groups except the colic group (P less than 0.05-P less than 0.01). alpha 2-macroglobulin was significantly higher in acute grass sickness than normal, colic and chronic grass sickness cases (P less than 0.01, P less than 0.05 and P less than 0.05). The time scale of changes suggests that the stimulus to haptoglobin and orosomucoid synthesis occurs at the onset of clinical signs whereas the increase in ceruloplasmin and alpha 2-macroglobulin is more likely to reflect haemoconcentration.

Clinical equine dysautonomia and autonomic neuron damage.

Damage to the neurons of selected autonomic ganglia was quantified in relation to the severity of the clinical signs shown in acute, subacute and chronic cases of dysautonomia (grass sickness). No connection between the clinical severity of acute or subacute dysautonomia and the amount of neuronal damage in the superior cervical, stellate and coeliaco-mesenteric ganglia could be demonstrated. However, a higher proportion of normal neurons were found in chronic cases. Jejunal submucosal neuronal damage was correlated with clinical severity but further work is required to confirm this finding and to establish how widespread the alimentary neuronal lesions are in dysautonomia of different severities.

Quantitation of alpha-1 proteinase inhibitor in the pulmonary epithelial lining fluid of horses with chronic obstructive pulmonary disease.

The concentration of alpha-1 proteinase inhibitor (API) was measured in the pulmonary epithelial lining fluid (PELF) of horses with chronic obstructive pulmonary disease (COPD) while they had clinical signs and while they had none. The concentrations of total protein, albumin and API were significantly higher in the PELF of animals with clinical signs of COPD. The correlation between albumin and API in the PELF suggested that most of the API was derived from the serum.

The relationship between meteorological features and equine grass sickness (dysautonomia).

Local weather patterns associated with 15 outbreaks of equine grass sickness in eastern Scotland were studied. The majority showed a trend toward cooler drier weather associated with irregular ground frosts. This would not preclude the hypothesis that fungi might be connected with the aetiology of grass sickness.

A comparative study of normal equine populations and those with grass sickness (dysautonomia) in eastern Scotland.

A retrospective survey was made of premises in eastern Scotland on which at least two cases of grass sickness had occurred between 1970 and 1987. For comparison, a further survey of 49 equine establishments, on which no grass sickness had been recorded, was conducted from 1986 to 1988. The results indicated that younger animals are more susceptible, especially those in good physical condition grazing full-time in the spring or early summer. Movement to new grazing increases the risk of grass sickness and identifiable stress may contribute. The nature of the establishment governed the animals' condition during the summer, but it did not appear to influence the prevalence of grass sickness. However, riding schools and livery establishments which experienced the disease kept, on average, significantly more animals than unaffected premises of the same type. No relationship was found between supplementary feeding or stage of pasture growth and grass sickness. The results of the survey support the hypothesis that the causal agent of grass sickness is associated with grazing but multiple factors may influence the expression of illness.

A case-control study of grass sickness (equine dysautonomia) in the United Kingdom.

A case-control study was performed to investigate the epidemiology of grass sickness in the United Kingdom from 1992 to 1995. Data were collected by means of postal questionnaire when cases of grass sickness were identified. Sets of three questionnaires were posted to owners of, or veterinary surgeons attending, cases of grass sickness, with a request to provide information on the case, on one healthy animal on the same premises as the case and on another healthy animal on other premises. Controls were matched to cases by date of onset. After univariate analyses, the probability of grass sickness in horses was modelled using conditional logistic regression techniques. Young animals were found to be at increased rick of grass sickness and females were less likely to become affected, as were animals that had a history of contact with previous cases of the disease. The probability of grass sickness was higher in animals that were on premises where grass sickness had previously occurred, particularly if this was recent. Animals were at particular risk of disease if they changed fields within the previous 2 weeks; the risk thereafter reduced with time. The disease had a seasonal pattern, with a peak from April to June. More than 95% of cases had access to grazing, and 66% occurred after 2 week periods of predominantly dry weather.

Clinical and biochemical features of grass sickness (equine dysautonomia).

An attempt has been made to assess the diagnostic value of clinical features seen at initial examination of horses with grass sickness, colic cases and cases submitted as possible grass sickness but diagnosed subsequently as some other condition. There appears to be no single pathognomonic sign for grass sickness. A range of signs has been associated with grass sickness but these are of value only when related to the length of illness and the history. Biochemical tests related to intestinal tissue damage, stress and dehydration were evaluated and most were found to be of value in diagnosing acute grass sickness. No evidence was found relating selected mineral or vitamin deficiencies to grass sickness, nor were protein changes specific. A marked fall in glutathione peroxidase values was associated with grazing rather than disease.

Decrease in the alpha 1-proteinase inhibitor Spi3 in equine bronchoalveolar lavage fluid.

The alpha 1-proteinase inhibitors of trypsin, Spi1, Spi3A, and Spi3B, in bronchoalveolar lavage fluid (BALF) and serum of horses were separated by electrophoresis, and their proportions were quantified in 12 control horses and 12 with chronic obstructive pulmonary disease (COPD). A significantly lower proportion of Spi3B (P < 0.05) and higher proportion of Spi1 (P < 0.02 to P < 0.01) were detected in BALF, compared with serum, in control and COPD-affected horses and appeared to be attributable to reduced Spi3 activity in BALF. There was no significant difference between the control and COPD groups in this respect, indicating that the decrease in Spi3 may be a physiologic phenomenon. The differences observed may be associated with proteolytic damage to or preferential complex formation by Spi3.