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.     

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.     

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.     

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.     

Population-based study of fecal shedding of Clostridium perfringens in broodmares and foals

OBJECTIVE: To determine the percentage of broodmares and foals that shed Clostridium perfringens in their feces and classify the genotypes of those isolates. DESIGN: Prospective cross-sectional study. ANIMALS: 128 broodmares and their foals on 6 equine premises. PROCEDURES: Anaerobic and aerobic bacteriologic cultures were performed on feces collected 3 times from broodmares and foals. All isolates of C. perfringens were genotyped. RESULTS: Clostridium perfringens was isolated from the feces of 90% of 3-day-old foals and 64% of foals at 8 to 12 hours of age. A lower percentage of broodmares and 1- to 2-month-old foals shed C. perfringens in their feces, compared with neonatal foals. Among samples with positive results, C. perfringens type A was the most common genotype identified (85%); C. perfringens type A with the beta2 toxin gene was identified in 12% of samples, C. perfringens type A with the enterotoxin gene was identified in 2.1% of samples, and C. perfringens type C was identified in < 1% of samples. CONCLUSIONS AND CLINICAL RELEVANCE: Clostridium perfringens was identified from the feces of all but 6 foals by 3 days of age and is likely part of the normal microflora of neonatal foals. Most isolates from broodmares and foals are C. perfringens type A; thus, the clinical relevance of culture results alone is questionable. Clostridium perfringens type C, which has been associated with neonatal enterocolitis, is rarely found in the feces of horses.     

A prospective study of the roles of clostridium difficile and enterotoxigenic Clostridium perfringens in equine diarrhoea

Faecal samples from adult horses and from foals with diarrhoea or with normal faeces were evaluated for the presence of Clostridium difficile, C. difficile toxins, C. perfringens enterotoxin (CPE) and C. perfringens spore counts. Clostridium difficile was isolated from 7/55 horses (12.7%) and 11/31 foals (35.5%) with colitis, but from 1/255 normal adults (0.4%) and 0/47 normal foals (P<0.001). Clostridium difficile toxins A and/or B were detected in 12/55 diarrhoeic adults (21.8%) and 5/30 diarrhoeic foals (16.7%) but in only 1/83 adults (1.2%) and 0/21 foals with normal faeces (P<0.001 and P<0.05, respectively). Clostridium perfringens enterotoxin was detected in 9/47 diarrhoeic adults (19%) and 8/28 diarrhoeic foals (28.6%), but was not detected in 47 adult horses (P<0.002) or 4 foals (P = 0.22) with normal faeces. The positive predictive value of isolation of C. perfringens with respect to the presence of CPE was only 60% in adult horses and 64% in foals. There was no association between total C. perfringens spore count and CPE in the faeces. The overall mortality rate from colitis was 22% for adult horses and 18% for foals. Clostridium difficile toxin-positive adult horses with colitis were less likely to survive than C. difficile-negative horses with colitis (P = 0.03). This study provides further evidence that C. difficile and enterotoxigenic C. perfringens are associated with equine enterocolitis.     

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.     

Comparison of IgG antibody levels to Clostridium botulinum antigens between euthanased and surviving cases of chronic grass sickness

Serum from 12 horses suffering from chronic grass sickness (CGS) were assayed for IgG antibodies against botulinum neurotoxins C and D (BoNT/C and BoNT/D) and to a surface antigen extract of a neurotoxin negative strain of Clostridium botulinum type C. Collectively, the six surviving CGS cases demonstrated significantly higher initial IgG levels (P=0.05) against surface antigens than the six that were subsequently euthanased. The surviving animals also demonstrated higher initial IgG levels against the BoNT/C but not reaching significance (P=0.06). The two groups demonstrated no difference between IgG levels against BoNT/D. This study supports existing evidence of the involvement of C. botulinum type C in the aetiology of grass sickness.     

Systemic antibodies to Clostridium botulinum type C: do they protect horses from grass sickness (dysautonomia)?

The aetiology of equine grass sickness (EGS) is still unknown. There is increasing evidence that toxicoinfection with Clostridium botulinum type C is involved. Epidemiological evidence shows that resistance to EGS can occur in older horses and those that have been on a particular pasture for longer or have been in prior contact with the disease. This resistance may be in the form of an immune response to the aetiological agent. Levels of systemic antibodies to the surface antigens of C. botulinum type C (using the closely related and safe C. novyi type A as a phenotypic marker) and to the botulinum type C neurotoxin (BoNT/C) were investigated in horses with and without EGS. Horses with grass sickness were found to have significantly lower levels of systemic IgG to both surface antigens and BoNT/C. Horses with low levels of systemic immunity to these antigens may be more susceptible to developing EGS. There were no significant differences in antibody levels between the different categories of EGS, suggesting systemic immunity to C. botulinum type C does not play a significant role in influencing the severity of the disease. However, horses that had been in contact with EGS or that were grazing land where it had occurred frequently in the past had significantly higher antibody levels to these antigens. These horses may have been exposed to subclinical doses of C. botulinum type C and BoNT/C, resulting in the production of a protective immune response against the putative aetiological agent. This finding is of potential significance for the prospect of prevention of EGS by vaccination against C. botulinum type C.     

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 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.     

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     

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.     

Antibodies against equine herpesvirus 1 in the cerebrospinal fluid in the horse

Neutralizing antibodies against equine herpesvirus 1 were measured in serum and cerebrospinal fluid of 16 horses and ponies from a closed herd both before and after vaccination with modified live equine herpesvirus 1. These titers were also measured in 22 neurologically normal and 15 neurologically abnormal horses at a teaching hospital. Animals from the closed herd had prevaccination serum titers up to 1:8 and postvaccination serum titers up to 1:128. Horses from the teaching hospital had serum titers up to 1:64. Cerebrospinal fluid titers were not detected in the vaccinated horses or the neurologically normal horses but a low titer (1:8) was noted in one neurologically abnormal horse. This titer probably resulted from hemorrhage into the cerebrospinal fluid following trauma.     

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.     

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.     

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.     

Bacteriologic examination of equine fecal flora as a diagnostic tool for equine intestinal clostridiosis

The fecal flora of 56 clinically healthy and 23 sick horses were examined bacteriologically for counts of Clostridium perfringens, molds, coliforms, alpha- and beta-hemolytic streptococci, and microbes belonging to genus Bacillus, as well as for the presence of Salmonella spp. Of the healthy horses, 85.7% had a C perfringens count less than 10(1) colony-forming units/g of feces. Of the healthy horses, lowest counts were found in race-horses. Of the sick horses, equine intestinal clostridiosis was diagnosed in 2 horses with large C perfringens counts (10(4) to 10(7) colony-forming units/g) and with acute diarrhea. The 7 isolates of C perfringens were identified as serotype A. Salmonella spp were not detected from any of the horses. The study indicated that diagnosing equine intestinal clostridiosis based on the determination of the fecal C perfringens count was suitable.     

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.     

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.