Biomarkers of exposure to cyanogens in horses with grass sickness

To test the hypothesis that equine grass sickness may be associated with the ingestion of cyanogenic glycosides from white clover (Trifolium repens), the concentrations of whole blood cyanide, and plasma and urinary thiocyanate, the main metabolite of cyanide, were measured in 12 horses with acute grass sickness and 10 horses with subacute grass sickness, and in 43 control horses, of which 21 were co-grazing with cases of acute grass sickness, 12 grazed pastures where grass sickness had not been reported, and 10 were stabled horses. The healthy horses which grazed with cases of acute grass sickness had higher concentrations of blood cyanide, and plasma and urinary thiocyanate than the other control horses, consistent with an increased exposure to cyanogens. The horses with grass sickness had no evidence of a recent intake of cyanogens, but may have been exposed to increased levels of cyanogens before they became anorexic.     

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.     

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.     

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.     

Equine grass sickness: serologic evidence of association with Clostridium perfringens type A enterotoxin.

Clostridium perfringens type A enterotoxin seroneutralization was carried out on sera from 50 horses recovered from grass sickness and from 100 other horses with no record of having had the disease. Of the affected horses, 70% had seroneutralizating titers higher than 1:64, half of these being equal or higher than 1:128. More than 88% of the horses with no record of grass sickness had titers lower than 1:64. These data support the theory of association between C perfringens type A toxins and grass sickness.     

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.     

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.     

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     

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.     

Barium swallow as an aid to the diagnosis of grass sickness

Twenty-five horses, suspected on clinical grounds of having grass sickness, were given a barium swallow. Using image intensification the passage of the barium bolus was followed radiographically from the pharynx to the stomach. All 18 horses in which grass sickness was later confirmed at post mortem examination showed defective oesophageal motility. Although the specificity of the abnormality could not be proven, the technique offers a valuable aid to the ante mortem diagnosis of grass sickness.     

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.     

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.     

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.     

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.     

The association of Clostridium botulinum type C with equine grass sickness: a toxicoinfection?

The cause of grass sickness, an equine dysautonomia, is unknown. The disease usually results in death. Gastrointestinal (GI) dysfunction is a common clinical manifestation in all forms of the disease. It is generally thought that equine grass sickness (EGS) is caused by an ingested or enterically produced neurotoxin which is absorbed through the GI tract. Clostridium botulinum was first implicated as a causative agent when it was isolated from the GI tract of a horse with EGS in 1919. The aim of the present study was to investigate the hypothesis that EGS results from toxicoinfection with C. botulinum type C: growth of the bacterium in the GI tract with production of toxin (BoNT/C). Ileum contents and faeces from horses with EGS were investigated for BoNT/C, and indirectly for the presence of C. botulinum type C, and compared with control samples from horses without EGS. BoNT/C was detected directly by ELISA in the ileum of 45% (13/29) of horses with EGS compared to 4% (1/28) of controls, and in the faeces of 44% (20/45) of horses with EGS compared to 4% (3/77) of controls. Levels of up to 10 Mlg toxin/g wet weight of gut contents were observed. The one control horse with detectable toxin in the ileum had been clinically diagnosed as having acute EGS, but this was not confirmed by histopathology. The organism was detected indirectly by assaying for BoNT/C by ELISA after enrichment in culture medium. C. botulinum type C was shown to be present in 48% (14/29) of ileum samples and 44% (20/45) of faecal samples from horses with EGS, compared with 7% (2/27) of ileum samples and 8% (6/72) of faecal samples from controls. These results support the hypothesis that EGS results from a C. botulinum type C toxicoinfection.     

Functional and histopathological evidence of cardiac parasympathetic dysautonomia in equine grass sickness

The parasympathetic terminal cardiac ganglia were examined in three normal horses and in five horses with grass sickness. Histopathological changes, consistent with those observed in other ganglia of horses with grass sickness, were identified in the terminal cardiac ganglia of the affected horses. A functional analysis of cardiac autonomic control by time domain analysis (TDA) of heart rate variability was applied to eight horses with grass sickness, and double-paired to 16 normal horses on the basis of their age, sex and breed, and the time of day at which the electrocardiographs were obtained. There were statistically significant differences between the normal and the affected horses in terms of the triangular index (P=0.01), a geometric method of TDA, the sNN100 index (P=0.009), a statistical method of TDA, and the R to R interval (P<0.0005). These results indicate that there is a pathological and functional parasympathetic decentralisation to the heart in equine grass sickness.     

Preliminary study of mucosal IgA in the equine small intestine: specific IgA in cases of acute grass sickness and controls

REASONS FOR PERFORMING STUDY: There is much evidence to suggest that group III Clostridium botulinum (types C and D) are involved in the aetiology of equine grass sickness (EGS). Antibodies have been detected previously in the blood and high levels associated with resistance to disease. Specific mucosal antibodies in the gastrointestinal (GI) tract are likely to be important in protection, and this study was performed to ascertain if such antibodies could be detected and if their levels were related to disease state. OBJECTIVES: To develop a method for quantifying IgA antibodies to C. botulinum types C and D in the GI tract of horses and to relate antibody levels to disease status. METHODS: Samples of tissue (n = 25: 6 duodenum, 7 jejunum and 12 ileum) were taken from acute grass sickness (AGS) cases and from control horses (n = 12; 4 samples from each site) at post mortem. They were extracted with the detergent saponin in the presence of protease inhibitors and assayed for total IgA, for specific IgA against botulinum neurotoxins types C and D (BoNT/C or BoNT/D), and against surface antigens of a BoNT/C negative strain of C. botulinum type C (SA) and of Clostridium tetani (TetSA), as a control. Specific IgA was expressed as percentage total IgA. RESULTS: Compared to controls, significantly higher levels of specific IgA against BoNT/C were detected in the jejunum (P = 0.04) and ileum (P = 0.02) of AGS cases. Similarly, higher specific levels against BoNT/D were demonstrated in duodenum (P = 0.01) and jejunum (P = 0.02). Significantly higher levels of IgA against SA were demonstrated only in duodenal samples (P = 0.01). CONCLUSIONS: Levels of IgA antibody to BoNTs in control horses were at near undetectable levels, suggesting no recent exposure to toxins. In AGS cases, significantly higher levels of specific IgA were detected predominantly in jejunum and ileum. POTENTIAL RELEVANCE: If specific IgA is protective then any successful vaccine for EGS should induce a mucosal response.     

Unusual compound of small molecular weight in the serum of horses with acute grass sickness

An unusual compound of small molecular weight has been detected in serum from horses with acute grass sickness by solvent extractions and thin-layer chromatography. The substance has not been detected in the serum of normal horses or cases of equine colic and apparently disappears from grass sickness serum after two to three days of clinical illness. Although this compound is found in sera which are known to possess in vivo neurotoxicity, this property could not be demonstrated in either the total chloroform extract which contains the compound or in the hydrophilic serum components remaining after extraction. These findings suggest that, although the compound may be related to the neurotoxin present in the original serum, the toxicity is inactivated during the extraction procedure or that the emulsion necessary for administration precludes absorption of an active toxin.     

Do hares suffer from grass sickness?

An autopsy study of one dead and two sick hares from an East Anglian estate on which two mares had died of grass sickness revealed that two of the hares were suffering from a polyganglionopathy and alimentary tract changes, remarkably similar to those seen in grass sickness in horses. No such abnormalities were found in two healthy hares from the same locality.