Evaluation of urinalysis as an aid in the diagnosis of equine grass sickness

To determine whether urinalysis can aid the diagnosis of equine grass sickness, samples of urine from 15 horses with acute grass sickness, eight horses with subacute grass sickness, 17 co-grazing horses and 17 stabled control horses were analysed. The samples from all of the horses with grass sickness had a significantly higher specific gravity, higher protein and creatinine concentrations and a significantly lower pH; the samples from the horses with acute grass sickness also had significantly higher glucose concentrations. These differences may support a diagnosis of grass sickness but they are not pathognomonic for the disease.     

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.     

Plasma neurofilament pNF-H concentration is not increased in acute equine grass sickness

Although a presumptive diagnosis of acute grass sickness (AGS) can be made on the basis of clinical signs, a definitive ante mortem diagnosis currently requires histological examination of enteric ganglia. Development of an accurate noninvasive ante mortem diagnostic test is therefore warranted. The objective of this study was to determine whether quantification of the plasma concentrations of the heavily phosphorylated form of major neurofilament subunit NF-H (pNF-H), which mirror the degree of axonal degeneration in some human and animal neurodegenerative disorders, could distinguish AGS-affected and control horses. The pNF-H was quantified in plasma from 20 AGS cases and 20 control horses using a commercial enzyme-linked immunosorbent assay kit. Five AGS and 4 control samples had detectable pNF-H concentrations (>0.0759 ng/ml). There was no significant intergroup difference in pNF-H concentrations. It was concluded that plasma pNF-H is not a useful biomarker for the diagnosis of AGS.     

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.     

The sympatho-adrenal system and plasma levels of adrenocorticotropic hormone, cortisol and catecholamines in equine grass sickness

Plasma levels of adrenocorticotropic hormone (ACTH), cortisol and catecholamines were used to study the role of the sympatho-adrenal system in equine grass sickness. Statistical evaluation determined differences of hormone levels between seven horses with grass sickness (one acute, five subacute and one chronic), six horses with colic (one with laminitis) and 16 control horses before and after mild stress. Plasma levels of the hormones were higher in horses with acute and subacute grass sickness than in the other groups. No differences were detected between horses with colic and stressed control horses but some hormone levels differed between control and colic horses and control horses before and after stress. It is possible that hyperactivation of the sympatho-adrenal system is caused by stress but it is uncertain whether the stress is only a result of the severity of the disease or also plays a role in its aetiology.     

Systemic concentrations of antioxidants and biomarkers of macromolecular oxidative damage in horses with grass sickness

REASONS FOR PERFORMING STUDY: The aetiopathogenesis of equine grass sickness (EGS) is unknown. The role of free radical-mediated neuronal damage has not previously been investigated in this condition. OBJECTIVES: To investigate the potential contribution of oxidative damage and antioxidant status to neurodegeneration in EGS. METHODS: Systemic levels of surrogate biomarkers were determined in 10 horses with acute EGS and in 2 control populations; 10 healthy horses co-grazing with the 10 EGS horses at the onset of clinical disease, and 10 healthy mares grazing where EGS has not been reported. RESULTS: EGS horses had alterations in levels of several antioxidants, consistent with oxidative stress, the acute phase response and/or the secondary metabolic complications of EGS. EGS horses had elevated plasma dihydroxyphenylalanine (DOPA) levels. CONCLUSIONS: The elevated DOPA levels probably reflected a generalised disturbance of catecholamine metabolism rather than increased DOPA production via free radical-mediated oxidation of tyrosine. However, there was no evidence of systemic macromolecular oxidative damage. POTENTIAL CLINICAL RELEVANCE: Further work is required to determine whether macromolecular oxidative damage occurring at the neuronal level contributes to EGS.     

Outbreaks of equine grass sickness in Hungary

Equine grass sickness (EGS) occurs mainly in Great Britain, but has once been reported in Hungary. The stud which was affected by EGS in 2001 had no new cases until 2009/10, when 11 of 60 and five of 12 one- to three-year-old colts died or were euthanased due to EGS. Following a few hours in the high-risk field during the winter of 2010/11 further four cases of acute EGS were noted among these horses. The affected horses showed somewhat different clinical signs compared with the cases reported in Great Britain. Histopathological findings in these horses were consistent with EGS. In most examined cases carbofuran, a carbamate was found in the liver by toxicological examination, and it is postulated that carbofuran may influence the immune system and therefore predispose the horses to develop EGS. Carbamates are thought to cause a delayed neurotoxicity in human beings. Further studies are needed to clarify the potential role of carbamates in EGS.     

Potential role of multiple rectal biopsies in the diagnosis of equine grass sickness

Two samples were taken postmortem from the rectum of each of 14 horses with grass sickness and 10 control horses, and four sections stained with haematoxylin and eosin were examined. By using as a criterion of grass sickness the presence of three chromatolytic neurons, 10 of the 14 cases were positive and none of the control horses was positive, giving a sensitivity of 71 per cent and a specificity of 100 per cent. No other histological features appeared to be of diagnostic value, and staining for Nissl substance in neurons with cresyl fast violet and methyl green-pyronin did not improve the sensitivity of the test.     

Sodium nitroprusside, a convenient source of dietary cyanide for the study of chronic cyanide toxicity

1. The suitability of sodium nitroprusside (SNP) as a source of dietary cyanide has been tested with growing chicks and with hens by measuring plasma or urinary thiocyanate; the major detoxication product of cyanide. 2. Growth and food intake were depressed and plasma thiocyanate concentration was increased in chicks in a progressive manner as the dietary concentration of SNP was increased from 0 to 0.5 g/kg. 3. In laying hens, urinary excretion of thiocyanate was increased fivefold by including SNP in the diet at 0.3 g/kg. 4. It is concluded that SNP is a suitable and convenient source of dietary cyanide for the investigation of chronic cyanide toxicity.     

Evaluation of three ancillary treatments in the management of equine grass sickness

Brotizolam, acetylcysteine and aloe vera gel were evaluated as ancillary treatments for 29 cases of equine grass sickness. None of the treatments had any significant beneficial effect on the survival of the horses. However, 11 of 13 horses with mild chronic grass sickness survived solely with intensive nursing care.     

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.     

Analysis of peritoneal fluid as a diagnostic aid in grass sickness (equine dysautonomia)

The analysis of peritoneal fluid is of value in the differential diagnosis of equine colic but its characteristics have not been evaluated in grass sickness. Peritoneal fluid was collected from 15 normal horses and from 11 cases of medical colic, 11 cases of surgical colic, 20 cases of acute grass sickness and 13 cases of subacute grass sickness. The fluid was analysed for its appearance, total and differential white cell count, specific gravity, total protein concentration and total and intestinal alkaline phosphatase activity. Fluid from cases of medical colic was normal in these respects. Surgical cases were unique in having bloodstained fluid with a high alkaline phosphatase activity. Grass sickness cases had a higher specific gravity and protein content than the cases of medical colic although the appearance of the fluid was similar. Grass sickness cases were distinguishable from cases of surgical colic on the basis of the appearance of the fluid and its lower alkaline phosphatase activity.     

Cholinergic activity of intestinal musclein vitro taken from horses with and without equine grass sickness

Equine grass sickness (EGS) is a pan-dysautonomia of horses that involves central and peripheral neuronal degeneration and ultimately depletion. This is the first reported functional study on the motility of equine intestine taken immediatelypost mortem from horses with EGS. Strips of smooth muscle from the small intestine of healthy and EGS-affected horses were suspended in an organ bath and their motility was measured isometrically. The activity of the cholinergic system was studied. Physostigmine enhanced the motility of all muscle strips. Tissues taken from horses suffering from acute grass sickness (AGS) had the longest latency before a measurable response could be obtained (p<0.05). The ileum appeared to be damaged by EGS to a greater extent than the duodenum. For the duodenal strips the enhanced rate of spontaneous contractions was significant (p<0.05) for both normal tissue and that affected by grass sickness but this was not the case for the ileal strips. Muscarinic receptor sensitivity investigation using bethanecol suggested a hypersensitivity of receptors with AGS material,Abbreviations AGS acute grass sickness - CGS chronic grass sickness - ED₅₀ median effective dose - EGS equine grass sickness - VIP vasoactive intestinal peptide     

Histopathological changes in the coeliaco-mesenteric ganglia of horses with 'mal seco', a grass sickness-like syndrome, in Argentina.

'Mal seco' is a grass sickness-like syndrome of horses in Argentina. A histopathological study was made of the coeliaco-mesenteric ganglia of four horses with 'mal seco' and of four horses that died from other causes. The severity and extent of the lesions found in the horses with 'mal seco' was greatest in the two with the shortest clinical course. Degenerative changes consisted mainly in the loss of Nissl substance, cytoplasmic vacuoles, neuronophagia, intercellular and intracytoplasmic eosinophilic bodies, and pyknotic and eccentric nuclei. The coeliaco-mesenteric ganglia of the control horses had no histological lesions. The histological lesions in the horses with 'mal seco' were very similar to those described in the coeliaco-mesenteric ganglia of horses with grass sickness in Europe and it is suggested that 'mal seco' and grass sickness may be the same disease.     

Equine dysautonomia (grass sickness) is associated with altered plasma amino acid levels and depletion of plasma sulphur amino acids

To determine whether equine dysautonomia (ED) is associated with alterations in plasma amino acid metabolism, plasma amino acid profiles were determined for horses with acute (n = 10), subacute (n = 6) and chronic (n = 7) ED and for healthy cograzing horses (n = 6) and control horses (n = 10). Horses with acute ED had perturbations in plasma amino acid profiles resembling those of severe protein malnutrition. In addition, horses with ED and cograzing healthy horses had depletion of the plasma sulphur amino acids cyst(e)ine and methionine. As similar plasma amino acid perturbations occur in subacute/chronic cyanide toxicity, the role of cyanogenic glycosides in the aetiology of ED warrants further study. Unfortunately, amino acid analysis cannot be used as a definitive premortem diagnostic test for ED, since there was overlap in the individual amino acid levels of control, cograzing and ED horses.     

Urinary cortisol:creatinine ratios in healthy horses and horses with hyperadrenocorticism and non-adrenal disease

Urinary cortisol and creatinine concentrations, and the cortisol:creatinine ratio were compared between 12 healthy horses (group 1), 13 horses with Cushing's disease (group 2), and eight horses with dysautonomia syndrome (equine grass sickness) (group 3). The mean (sd) urinary cortisol concentrations were 112 (55.7), 250 (357) and 864 (526) nmol/litre in groups 1, 2 and 3, respectively; the mean (sd) urinary creatinine concentrations were 18.9 (7.3), 12.0 (6.7) and 45.2 (26.4) nmol/litre in groups 1, 2 and 3, respectively, and the mean (sd) ratios were 6.1 (2.6), 19.8 (23.8) and 21.3 (14.5) (x 10(-6)) in groups 1, 2 and 3, respectively. The urinary cortisol and creatinine concentrations were significantly greater in group 3 than in groups 1 and 2, but the ratios were not significantly different, although there was a trend (P=0.076) towards higher values in groups 2 and 3. A diagnostic cut-off in the cortisol:creatinine ratio for the confirmation of Cushing's disease of more than 6.9 x 10(-6) was associated with a diagnostic sensitivity and specificity of 92.3 and 75.0 per cent, respectively, when compared with healthy horses. However, when group 3 horses were included, a cut-off of more than 7.4 x 10(-6) was associated with a diagnostic sensitivity and specificity of 84.6 and 54.5 per cent, respectively.     

Light Microscopy of the Enteric Nervous System of Horses with or without Equine Dysautonomia (Grass Sickness): its Correlation with the Motor Effects of Physostigmine

Light microscopy was undertaken on sections from the caudal flexure of the duodenum and the terminal ileum proximal to the ileocaecal fold in 5 control horses, 5 horses with acute grass sickness (AGS), and 5 horses with chronic grass sickness (CGS). With the exception of the ileal submucous plexus of the CGS group, the AGS group had the lowest number of neurons as measured using a subjective scoring scheme. The proportion of abnormal neurons in the AGS group was similar in both plexuses and both regions, whereas the values for the CGS group were much higher in the duodenal region than in the ileal region. The motility of tissue adjacent to that used for histology was measured isometrically in vitro. The increase in the rate of contractions following exposure to physostigmine was greatest for the AGS group, both from the duodenal and from the ileal region. The latency was longest for the AGS group, suggesting that the material from this group had the least number of active cholinergic neurons. The studies with physostigmine thus indicated that the most severe functional damage occurred in cases of AGS. These findings confirm that extensive damage occurs in the enteric neurons in equine grass sickness. There was good correlation between the functional cholinergic responses and the extent of neuronal degeneration.     

Studies of oesophageal function

Lesions of the sympathetic nervous system have been associated with grass sickness for many years (Obel, 1955). Dysphagia is also an accepted clinical feature of subacute or chronic cases. Megaoesophagus has been reported in horses with grass sickness, but it was uncertain whether oesophageal dilation was a primary condition, or a sequel to gastric distension (Greet, 1982). Robertson and others, (1948) suggested that dysphagia was alleviated in affected horses after drainage of gastric contents. It is interesting to note that dysphagia was present in less than half the cases in this study although radiological abnormalities of the oesophagus were similar in all cases. Consistent radiological abnormalities of the oesophagus occurred in 12 out of the 14 horses examined (this consisted of incoordination or atony of the thoracic oesophagus). In the two suspected cases of grass sickness which made a clinical recovery, the radiological abnormalities were slightly different. Contrast material was transferred slowly through the cervical oesophagus and pooled at the thoracic inlet as well as at several sites in the cervical oesophagus. Eventually the contrast material passed through the distal oesophagus into the stomach. Although gastric distension was present in many of the cases examined it was not possible to identify this by radiographic means. It is unlikely that the signs of oesophageal incoordination and dilation were related only to gastric distension, as in most of the cases, contrast material pooled in the oesophagus at the thoracic inlet rather than just cranial to the diaphragm. It seems reasonable to suggest that these radiological abnormalities resulted from neurological impairment of the oesophagus. It would be of particular value to examine horses with obstructive lesions of the stomach or duodenum to evaluate the effect of upper gastro-intestinal obstruction on oesophageal motility. A number of horses with other conditions of the upper alimentary tract have been examined radiographically at these clinics. Two horses suspected of suffering from grass sickness were found to have primary oesophageal abnormalities. One had a diverticulum and the other a localized area of dilation; both showed regurgitation of food and weight loss. Barium swallows in both horses defined the sites of their localized lesions but there was no evidence of either Type I or Type II oesophageal malfunction, and at post-mortem examination the ganglionic changes associated with grass sickness were absent. Examination of one horse with ileocaecal intussusception did not show radiological features of the oesophagus typical of those shown by cases of grass sickness. However, contrast material passed slowly through the upper oesophagus of a foal with congenital megaoesophagus in a manner similar to the Type II oesophageal malfunction described above. It also demonstrated dilation and gross inco-ordination of the thoracic oesophagus with pooling of contrast material at the thoracic inlet and oscillation between the thoracic inlet and diaphragm as seen in Type I malfunction in proven grass sickness. It is recognized that the radiological findings of oesophageal dilation and inco-ordination merely demonstrate the presence of neuromuscular impairment of oesophageal movement. Until more cases of upper gastro-intestinal disease can be examined, the specificity of these functional abnormalities for grass sickness cannot be accurately assessed. However the results assume considerable diagnostic significance when demonstrated in an adult horse which is exhibiting signs of grass sickness, particularly those of colonic impaction. The need for sophisticated equipment and thus the necessity of transport to centres so equipped, is clearly a drawback to the technique but this is often outweighed by the advantage of being able to avoid unnecessary laparotomy. As there is at present no cure for grass sickness, the improved diagnostic capability will allow severely ill horses to be destroyed without delay. It may also identify horses which on rare occasions appear to be making a slow recovery.     

A negative serological relationship between cases of grass sickness in Scotland and Clostridium perfringens type A enterotoxin

In an attempt to compare the equine grass sickness as reported in Europe with that described in the Republic of Colombia, sera from horses experiencing grass sickness in Scotland were used in neutralisation tests with Clostridium perfringens type A enterotoxin. The sera, from acute and chronic cases of the disease, failed to neutralise either crude or partially-purified enterotoxin. Neither were precipitin lines formed when the sera were treated against the toxin in immunoelectrophoresis. These results suggest that grass sickness in Europe and the equine disease in Colombia have a different aetiology.     

Effect of withholding feed on concentration and composition of plasma very low density lipoprotein and serum nonesterified fatty acids in horses

OBJECTIVE: To measure and compare the concentration and composition of very low-density lipoprotein (VLDL) in plasma and selected lipids in serum of horses fed mixed grass hay ad libitum or denied feed for 36 hours. ANIMALS: 4 healthy adult mares. PROCEDURE: Mares were either fed mixed grass hay ad libitum or denied feed for 36 hours beginning at 8:00 AM. Blood samples were collected every 2 hours during the study period and analyzed for nonesterified fatty acid (NEFA), triglyceride (TG), VLDL, and glucose concentrations and composition of VLDL. RESULTS: Withholding feed significantly increased mean serum concentrations of NEFA. By 36 hours, a 16-fold increase in mean serum NEFA concentration and 2-fold increase in mean plasma VLDL concentration, compared with baseline values, were detected. Mean plasma TG concentrations significantly increased with time in feed-deprived horses. Significantly lower overall mean plasma glucose concentrations were detected in feed-deprived horses. Mean percentage of protein in VLDL was significantly lower in feed-deprived horses. Plasma VLDL concentrations varied widely among horses in response to withholding feed. Plasma TG and VLDL concentrations remained unaltered in 2 horses. CONCLUSIONS AND CLINICAL RELEVANCE: Withholding feed significantly increases blood lipid concentrations in horses, but individual horses respond differently. Serum NEFA concentrations were increased in all 4 horses denied feed, indicating mobilization of tissue triglyceride stores. Variation in plasma VLDL concentration in response to withholding feed suggests that its metabolism is strongly influenced by other, as yet undetermined, factors in horses. Differences in the plasma VLDL concentrations among horses in response to withholding feed may be used as an indication of susceptibility to the hyperlipemic syndrome of Equidae.