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.     

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.     

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.     

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.     

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.     

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     

A computer-derived protocol to aid in selecting medical versus surgical treatment of horses with abdominal pain

In order to determine which variables are useful in identifying horses with abdominal pain requiring surgery, data were analysed from 219 horses presented at one veterinary teaching hospital. Using multiple stepwise discriminant analysis with a recursive partitioning algorithm, we obtained a decision tree that identifies surgical and non-surgical patients. The prevalence of surgical patients was 79 per cent in this population. The sensitivity, specificity, and positive and negative predictive values of this decision tree were 99 per cent, 55 per cent, 90 per cent and 99 per cent respectively. Compared to the clinical decision, this decision tree yielded more false positives (11 per cent) but almost eliminated false negatives (1 per cent). This decision tree was validated by the jack-knife method and also by evaluation using a new sample in a second veterinary teaching hospital in which the prevalence of surgical patients was 55 per cent. This led to sensitivity, specificity and positive and negative predictive values of 93 per cent, 73 per cent, 81 per cent and 89 per cent respectively.     

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.     

Helminth control used by trainers of thoroughbreds in England

A telephone survey was conducted of the methods used to control parasitic worms at 106 thoroughbred training yards. Most of the horses were allowed access to grass and were therefore at risk of infection. The control methods relied primarily on the use of anthelmintics and appeared reasonably successful because only 44 per cent of trainers reported observing clinical signs of nematode infections. They dosed their horses frequently, 40 per cent treating every four to six weeks and 25 per cent treating every seven to eight weeks. New arrivals on yards were usually treated with anthelmintic but the strategies used would be unlikely to prevent the introduction of anthelmintic-resistant nematodes on most yards or to deal with encysted cyathostomes. Trainers were responsible for the design of most control schemes, and only 42 per cent of them based their choice of anthelmintic on veterinary advice.     

Field study of the prevalence of lameness in horses with back problems

A population of 805 horses (70 per cent dressage, 20 per cent show jumpers and 10 per cent trotters) with orthopaedic problems was examined for signs of lameness and back problems, irrespective of their original complaints. In the horses with a back problem the prevalence of lameness was 74 per cent, and back problems were diagnosed in 32 per cent of the lame horses. These percentages were significantly higher than those recorded in a control population of 399 horses, of which 20 per cent were lame and 12 per cent had back problems. In the group of horses with orthopaedic problems there was a strong association between lameness and back problems and, in particular, there was a high prevalence of lameness among the horses with back problems.     

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.     

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.     

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.     

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.     

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.     

Epidemiological study of wastage among racehorses 1982 and 1983

An epidemiological study of wastage among racehorses was conducted in 1982 and 1983 among six stables, five of which were in Newmarket. The basis of the survey was the inability of horses to take part in cantering exercise as a result of injury or disease. The greatest number of days lost to training was caused by lameness (67.6 per cent) and respiratory problems (20.5 per cent). Conditions of the foot (19 pe cent), muscle (18 per cent), carpus (14 per cent), fetlock joints (14 per cent), tendons (10 per cent) and sore shins (9 per cent) were the major reasons for training days being lost in 198 cases in which a positive diagnosis of the site of lameness was made. The results confirm a previous investigation by Jeffcott and others (1982) and suggest that the diagnosis, therapy and prophylaxis of lameness merit a high priority in research efforts to reduce wastage among young racehorses.     

Antithrombin III activity in horses with colic: an analysis of 46 cases

Antithrombin-III (AT-III) activity was measured at the time of admission in 46 horses referred with colic. Differences in AT-III activities between animals treated medically or surgically and survivors or non-survivors were compared. The mean AT-III values for the horses treated medically (76.2 per cent), surgical survivors (69.5 per cent) and surgical non-survivors (55.9 per cent) were significantly different from the reference value for healthy adults (92 to 108 per cent). The mean AT-III activity of the survivors was significantly (P less than 0.01) greater than that of the nonsurvivors. The majority of the survivors (11 to 13 horses) had AT-III activities exceeding 60 per cent of normal, whereas that of 10 of the 14 non-survivors was less than 60 per cent of normal. There was an apparent distinction between the survivors and non-survivors at approximately 60 per cent of normal AT-III activity.     

Prevalence of benzimidazole resistance on horse farms in Germany

Faecal egg counts (FECs) were made on samples from 1383 horses on 64 farms in northern Germany between August 2000 and November 2001. There were significant differences between the mean FECs in the two years; in 2000, 59.6 per cent of 369 samples were positive and in 2001, 32.6 per cent of 1014 samples were positive for strongyle eggs. The results of a FEC reduction test indicated that resistance to fenbendazole was present on all 10 farms where it had been used, including in 33 of 60 horses tested. In contrast, treatment with ivermectin resulted in the complete elimination of nematode eggs in all the 77 horses tested. The mean LD50 values of the egg hatch test for thiabendazole indicated resistance on all 20 farms investigated and in 94 of 134 samples (70 per cent).     

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.