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.     

Hemorrhagic Gastritis Associated with Renal Failure, Hemoglobinuria, and Isolation of Clostridium perfringens in a Horse

A 3-year-old Quarter Horse mare was presented for acute colic with bright red-black foul-smelling gastric reflux containing long rod-shaped bacteria consistent with Clostridium sp. and red-black urine. The serum creatinine concentration was 5.5 mg/dL (N = 0.9–1.7), and blood urea nitrogen was 41 mg/dL (N = 9–20). At necropsy, the stomach wall was diffusely thickened, hemorrhagic, and edematous. Histopathologically, hemorrhagic necrosis was evident, with numerous colonies of spore-forming rods within the submucosa. Clostridium perfringens was cultured from the stomach contents. Polymerase chain reaction (PCR) genotyping was consistent with type A C. perfringens. Bilaterally, the kidneys were grossly enlarged, diffusely dark red-brown, and congested. The renal tubular epithelium was diffusely, acutely necrotic, with interstitial hemorrhage and massive accumulation of intratubular granular and proteinaceous casts. A diagnosis of massive hemolysis with hemoglobinuria and renal failure due to C. perfringens, type A infection was made. Alpha-toxin–induced intravascular hemolysis occurs rarely in humans and sheep. To our knowledge, this has not been described in horses with clostridial enterocolitis nor in equine clostridial gastritis.     

Antimicrobial‐associated diarrhoea in three equine referral practices

Reasons for performing study: Although antimicrobial‐associated diarrhoea (AAD) is the most frequently observed adverse effect of antimicrobial therapy in horses, few multicentred studies on the prevalence of AAD have been performed. Objectives: To determine the prevalence of AAD in horses that developed diarrhoea after antimicrobial treatment for nondiarrhoeic conditions and identify the antimicrobials used. Methods: The 2009 database of 3 referral hospitals was searched to identify nonhospitalised horses (weanling age or older) treated with antimicrobials for nongastrointestinal conditions. Horses with these criteria that presented with diarrhoea during 2009 were included in the study. Additional information, including antimicrobial administered and results of faecal pathogen testing, was gathered on each hospitalised case. Results: Of the 5251 horses treated with antimicrobials for nongastrointestinal signs, 32 were diagnosed with probable AAD, a prevalence of 0.6% (95% confidence interval: 0.43–0.86%). The AAD‐diagnosed horses had an 18.8% (6/32) mortality rate. Horses with AAD had been treated for an average of 4.2 days. The most frequently used antimicrobials in horses with AAD were gentamicin in combination with penicillin (n = 7), enrofloxacin (n = 7) and doxycycline (n = 4). Clostridium difficile was identified in faecal samples from 4 horses, 2 of which died and Salmonella from 3 horses. Conclusions: Results indicated that the prevalence of AAD is low. Any antimicrobial class commonly used in equine practice is a potential cause of equine AAD. Other risk factors, such as opportunistic enteropathogens, may play a part in the development of diarrhoea secondary to antimicrobial usage. Potential relevance: Although the risk of equine AAD is low, this sequela of antimicrobial treatment is possible especially when opportunistic enteropathogens or other risk factors are present. Because drugs from any antimicrobial class can be potentially involved in AAD, clinicians have additional incentive to ensure the judicious use of antimicrobial agents.     

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     

Quantitative real‐time PCR monitoring of Escherichia coli and Clostridium perfringens with oral administration of Lactobacillus plantarum strain Lq80 to weaning piglets

Levels of fecal or intestinal lactobacilli, Escherichia coli and Clostridium perfringens, and the prevalence of clostridial alpha toxin gene and heat‐stable toxin (ST) gene of enterotoxigenic E. coli (ETEC) were monitored in weaned piglets before (day 0) and during (days 7, 14, and 21) the administration of Lactobacillus plantarum strain Lq80. Lactobacilli were enumerated in a culture‐dependent method. The remainders were determined by quantitative real‐time PCR. In this quantitative real‐time PCR method, the detection limit was proved to be as low as 10³ cells/g feces or intestinal contents. Number of lactobacilli increased from day 0 to day 7 (P < 0.05), to day 14 (P < 0.05), and to day 21 (P = 0.07) in the Lq80‐administered group. L. plantarum contributed to as low as 10% of the lactobacillal population in the Lq80‐administered group. The number of E. coli and C. perfringens, and the prevalence of alpha toxin gene in feces or intestinal contents of the Lq80‐administered group decreased, at least in the first week of the postweaning period. Oral administration of L. plantarum strain Lq80 can stimulate the growth of indigenous lactobacilli and decrease ST‐producing ETEC and C. perfringens in the intestine of postweaning piglets.     

An acute outbreak of equine dysautonomia (equine grass sickness) in a group of eight Przewalski's horses (Equus ferus [caballus] przewalskii)

Equine grass sickness (EGS) is a neurodegenerative disease affecting grazing equids of which a single case of the chronic clinical presentation has previously been reported in a Przewalski's horse (Equus ferus [caballus] przewalskii). A group of 8 Przewalski's horses were moved to a new enclosure, recently vacated by a group of 4 Eastern kiang (Equus kiang holdereri) that showed no evidence of disease. After 23 days the first Przewalski's horse showed clinical signs of acute EGS including flank sweating, belly kicking, rapid loss of body condition, cessation of faecal passage, nasogastric reflux and mouthing water. It was subjected to euthanasia within 48 h due to lack of therapeutic response. Within 24 h of this first case developing clinical signs, a further 5 Przewalski's horses showed similar clinical signs of acute EGS and were subjected to euthanasia. Post mortem examinations confirmed acute EGS, with all animals demonstrating typical chromatolysis, cytoplasmic hypereosinophilia, cellular swelling, vacuolation, pyknosis and loss of nuclei in approximately 90% of neurones in the cranial cervical and cranial mesenteric ganglia and myenteric and submucosal plexi of the ileum. Two Przewalski's horses within the group showed no clinical signs of disease. No single pathogen was identified as the causal agent, but the epidemiological pattern of the outbreak was typical for that previously reported for acute EGS in domestic equids. All affected animals and the 2 surviving Przewalski's horses had low antibody titres to Clostridium botulinum type C. This is the first report of acute EGS in a herd of Przewalski's horses.     

Equine coronavirus: An emerging enteric virus of adult horses

Equine coronavirus (ECoV) is an emerging virus associated clinically and epidemiologically with fever, depression, anorexia and less frequently colic and diarrhoea in adult horses. Sporadic cases and outbreaks have been reported with increased frequency since 2010 from Japan, the USA and more recently from Europe. A faeco‐oral transmission route is suspected and clinical or asymptomatic infected horses appear to be responsible for direct and indirect transmission of ECoV. A presumptive clinical diagnosis of ECoV infection may be suggested by clinical presentation, haematological abnormalities such as leucopenia due to lymphopenia and/or neutropenia. Confirmation of ECoV infection is provided by specific ECoV nucleic acid detection in faeces by quantitative PCR (qPCR) or demonstration of coronavirus antigen by immunohistochemistry or electron microscopy in intestinal biopsy material obtained ante or post mortem. The disease is generally self‐limiting and horses typically recover with symptomatic supportive care. Complications associated with disruption of the gastrointestinal barrier have been reported in some infected horses and include endotoxaemia, septicaemia and hyperammonaemia‐associated encephalopathy. Although specific immunoprophylactic measures have been shown to be effective in disease prevention for closely‐related coronaviruses such as bovine coronavirus (BCoV), such strategies have yet not been investigated for horses and disease prevention is limited to basic biosecurity protocols. This article reviews current knowledge concerning the aetiology, epidemiology, clinical signs, diagnosis, pathology, treatment and prevention of ECoV infection in adult horses.     

Characterization of Clostridium perfringens in the feces of adult horses and foals with acute enterocolitis

Up to 60% of cases of equine colitis have no known cause. To improve understanding of the causes of acute colitis in horses, we hypothesized that Clostridium perfringens producing enterotoxin (CPE) and/or beta2 toxin (CPB2) are common and important causes of severe colitis in horses and/or that C. perfringens producing an as-yet-undescribed cytotoxin may also cause colitis in horses. Fecal samples from 55 horses (43 adults, 12 foals) with clinical evidence of colitis were evaluated by culture for the presence of Clostridium difficile, C. perfringens, and Salmonella. Feces were also examined by enzyme-linked immunosorbent assay (ELISA) for C. difficile A/B toxins and C. perfringens alpha toxin (CPA), beta2 toxin (CPB2), and enterotoxin (CPE). Five C. perfringens isolates per sample were genotyped for the following genes: cpa, cpb, cpb2 consensus, cpb2 atypical, cpe (enterotoxin), etx (epsilon toxin), itx (iota toxin), netB (necrotic enteritis toxin B), and tpeL (large C. perfringens cytotoxin). The supernatants of these isolates were also evaluated for toxicity for an equine cell line. All fecal samples were negative for Salmonella. Clostridium perfringens and C. difficile were isolated from 40% and 5.4% of samples, respectively. All fecal samples were negative for CPE. Clostridium perfringens CPA and CPB2 toxins were detected in 14.5% and 7.2% of fecal samples, respectively, all of which were culture-positive for C. perfringens. No isolates were cpe, etx, netB, or tpeL gene-positive. Atypical cpb2 and consensus cpb2 genes were identified in 15 (13.6%) and 4 (3.6%) of 110 isolates, respectively. All equine C. perfringens isolates showed far milder cytotoxicity effects than a CPB-producing positive control, although cpb2-positive isolates were slightly but significantly more cytotoxic than negative isolates. Based on this studied population, we were unable to confirm our hypothesis that CPE and CPB2-producing C. perfringens are common in horses with colitis in Ontario and we failed to identify cytotoxic activity in vitro in the type A isolates recovered.     

Outcome and long‐term follow‐up of 20 horses undergoing surgery for caecal impaction: A retrospective study (2000–2008)

Reasons for performing study: If untreated, caecal impaction may progress to rupture of the caecum and reports of long‐term outcome for horses undergoing surgery for caecal impaction are required. Objectives: To describe short‐ and long‐term complication rates for horses undergoing surgery for caecal impaction in an otherwise life‐threatening gastrointestinal condition. Methods: Case records were reviewed for horses undergoing surgery for caecal impaction. Horses were included in the study if an abnormally large, fluid‐distended or feed‐impacted caecum was the primary diagnosis at surgery and excluded if the caecum had already ruptured upon opening the abdomen, necessitating euthanasia. Short‐term follow‐up data were obtained from clinical records; and included complications, repeat celiotomy and survival to discharge. Long‐term survival was defined as survival for >1 year post operatively. Long‐term follow‐up data were obtained through telephone interviews with owners and referring veterinary surgeons. Survival, occurrence of post operative colic and subsequent use of the horse were recorded. Results: Twenty horses underwent surgery for caecal impaction: 16 horses underwent caecal bypass (typhlotomy and removal of caecal contents, ileal transection followed by ileocolostomy); 3 underwent typhlotomy alone; and one horse underwent typhlotomy followed by a second procedure where caecal bypass was performed. Five horses (25%) were admitted for colic evaluation with primary caecal impactions, in 10/20 (50%) of cases the impaction was secondary to previous orthopaedic surgery and in 5/20 (25%) caecal impaction was identified at repeat celiotomy following a previous colic surgery. Sixty‐five percent (13/20) of horses survived to discharge; 11/13 (85%) of horses discharged survived long term; 100% (3/3) horses that received typhlotomy alone were discharged and survived long term. Conclusions: The prognosis following caecal impaction surgery is fair, if the horse survives to discharge then prognosis for long‐term survival is good. Potential relevance: Surgery for caecal impaction carries a fair prognosis for a potentially life threatening disease, in particular where caecal dysfunction is suspected.     

Cardiorespiratory effects of enoximone in anaesthetised colic horses

Reasons for performing study: No studies have been reported on the effects of enoximone in anaesthetised colic horses. Objective: To examine whether enoximone improves cardiovascular function and reduces dobutamine requirement in anaesthetised colic horses. Methods: Forty‐eight mature colic horses were enrolled in this prospective, randomised clinical trial. After sedation (xylazine 0.7 mg/kg bwt) and induction (midazolam 0.06 mg/kg bwt, ketamine 2.2 mg/kg bwt), anaesthesia was maintained with isoflurane in oxygen and a lidocaine constant rate infusion (1.5 mg/kg bwt, 2 mg/kg/h). Horses were ventilated (PaCO₂<8.00 kPa). If hypotension occurred, dobutamine and/or colloids were administered. Ten minutes after skin incision, horses randomly received an i.v. bolus of enoximone (0.5 mg/kg bwt) or saline. Monitoring included respiratory and arterial blood gases, heart rate (HR), arterial pressure and cardiac index (CI). Systemic vascular resistance (SVR), stroke index (SI) and oxygen delivery index (DO₂I) were calculated. For each variable, changes between baseline and T10 within each treatment group and/or colic type (small intestines, large intestines or mixed) were analysed and compared between treatments in a fixed effects model. Differences between treatments until T30 were investigated using a mixed model (α= 0.05). Results: Ten minutes after enoximone treatment, CI (P = 0.0010), HR (P = 0.0033) and DO₂I (P = 0.0007) were higher and SVR lower (P = 0.0043) than at baseline. The changes in CI, HR and SVR were significantly different from those after saline treatment. During the first 30 min after enoximone treatment, DO₂I (P = 0.0224) and HR (P = 0.0003) were higher than after saline administration. Because the difference in HR between treatments was much clearer in large intestine colic cases, an interaction was detected between treatment and colic type in both analyses (P = 0.0076 and 0.0038, respectively). Conclusions: Enoximone produced significant, but short lasting, cardiovascular effects in colic horses. Potential relevance: Enoximone's cardiovascular effects in colic horses were of shorter duration than in healthy ponies.     

Crib‐biting in US horses: Breed predispositions and owner perceptions of aetiology

Reasons for performing study: Crib‐biting is an equine stereotypy that may result in diseases such as colic. Certain breeds and management factors have been associated. Objectives: To determine: breed prevalence of crib‐biting in US horses; the likelihood that one horse learns to crib‐bite from another; and owner perceptions of causal factors. Methods: An initial postal survey queried the number and breed of crib‐biting horses and if a horse began after being exposed to a horse with this habit. In a follow‐up survey, a volunteer subset of owners was asked the number of affected and nonaffected horses of each breed and the extent of conspecific contact. The likelihood of crib‐biting given breed and extent of contact was quantified using odds ratio (OR) and significance of the association was assessed using the Chi‐squared test. Results: Overall prevalence was 4.4%. Thoroughbreds were the breed most affected (13.3%). Approximately half of owners believed environmental factors predominantly cause the condition (54.4%) and crib‐biting is learned by observation (48.8%). However, only 1.0% of horses became affected after being exposed to a crib‐biter. The majority (86%) of horses was turned out in the same pasture with other horses and extent of contact with conspecifics was not statistically related to risk. Conclusion: This is the first study to report breed prevalence for crib‐biting in US horses. Thoroughbreds were the breed more likely to be affected. More owners believed either environmental conditions were a predominant cause or a combination of genetic and environmental factors contributes to the behaviour. Only a small number of horses reportedly began to crib‐bite after being exposed to an affected individual, but approximately half of owners considered it to be a learned behaviour; most owners did not isolate affected horses. Potential relevance: Genetic predisposition, not just intensive management conditions and surroundings, may be a factor in the high crib‐biting prevalence in some breeds, and warrants further investigation. Little evidence exists to suggest horses learn the behaviour from other horses, and isolation may cause unnecessary stress.     

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.     

Surgery: Short‐ and long‐term evaluation of surgical treatment of strangulating obstructions of the small intestine in horses: A review of 224 cases

Summary

A retrospective study was carried out of 224 horses operated for strangulating small intestine obstructions. Fifty‐four horses were euthanized and 5 horses died during surgery which means that 165 (73%) were allowed to recover. Of these, 53 horses were euthanized or died in the clinic and 112 (50%) were discharged from the hospital. Of 90 horses available for follow‐up 1 year postoperatively, 76 (84%) were still alive. The most important causes of death or reasons for euthanasia in the direct post‐operative period were post‐operative paralytic ileus, (adhesive) peritonitis and intra‐abdominal haemorrhage. After discharge from the hospital the reasons were (adhesive) peritonitis and (recurrent) colic. Of the horses which survived for at least 1 year, 16% sometimes suffered from colic, 12% experienced problems with incisional woundhealing and 4% suffered from jugular vein thrombosis. All were in good or reasonable condition and 88% performed at (approximately) the same level as before the operation.

The type of surgical intervention (i.e. enterotomy, enterectomy) did not significantly influence the outcome of surgery, whereas the type of anastomosis did. End‐to‐end jejunojejunostomy had a better prognosis than side‐to‐side jejunocaecostomy.

It was concluded that strangulating obstructions of the small intestine still carry a poor to guarded prognosis. Mortality was highest in the direct peri‐operative period. Once discharged from hospital, prognosis can be considered to be fair to good. Attempts to improve outcome should be directed at a better handling of the ileal stump during surgery and at the prevention of post‐operative ileus and the formation of adhesions.     

Seroprevalence of antibotulinum neurotoxin type C antibodies in horses in Israel

REASONS FOR PERFORMING STUDY: Clostridium botulinum type C is prevalent in Israel and outbreaks recorded in many species, other than horses. Association between levels of anti-BoNT/C antibodies and equine grass sickness (EGS) have been demonstrated but seroprevalence of anti-BoNT/C antibodies in horses has not been reported nor has EGS been reported in Israel. OBJECTIVES: To determine the seroprevalence of specific anti-BoNT/C antibodies in horses in Israel and to determine whether age, breed and gender, or geographical region of farms are potential risk factors for exposure to BoNT/C. HYPOTHESIS: Anti-BoNT/C antibodies are prevalent among horses in Israel and farm and horse-level variables are associated with increased risk for exposure. METHODS: Serum samples from 198 horses were collected and the levels of specific anti-BoNT/C antibodies were determined using enzyme-linked immunosorbent assay (ELISA). For each categorical variable indicator variables were created and the odds ratio (OR) and 95% confidence intervals (95% CI) for the outcome variable were calculated using a univariable and multivariable logistic regression models. RESULTS: A total of 61 (30.8%) horses were ELISA positive for anti-BoNT/C IgG antibodies. The farm and its geographical region were associated significantly with seropositivity, horse-level variables, such as gender and breed, were also associated with seropositivity. Quarter Horse and Warmblood mares placed in the southern region of Israel had the highest odds to be tested positive for anti-BoNT/C IgG antibodies. CONCLUSIONS AND POTENTIAL RELEVANCE: Several farm and various horse-level risk factors for exposure to BoNT/C, found in this study, could be correlated to previously reported risk factors of EGS. Studies are required to determine the predisposing factors that cause EGS, which is apparently not present in Israel.     

A comparative study of the intestinal microbiota of healthy horses and those suffering from equine grass sickness

This study compares quantitatively the microbiota of the gastrointestinal tract of healthy horses with that of horses with equine grass sickness (EGS). Faecal and ileal samples were cultured quantitatively on selective and non-selective media. Confirmed anaerobes were identified to species level.Overall faecal counts gave a ratio of aerobes:anaerobes of approximately 1:1. However, the mean counts in healthy horses of 4.4x10(8) aerobes:3.7x10(8) anaerobes per gram wet weight were different from counts in EGS (means were 10-100-fold higher), with statistically significant differences for the anaerobes (p=0.04). There were 10-100-fold more anaerobic cocci in EGS samples compared to healthy controls. Most of the seven species of anaerobic cocci were found in both healthy horses and EGS. Differences in clostridia isolated between health and disease were notable: fourteen species were isolated from EGS cases, compared to only one (C. bifermentans) in controls. The mean faecal clostridial counts in chronic disease were higher than in controls (10-fold) and in acute EGS (100-fold). In contrast, mean counts for ileal samples from acute cases, showed a 10-fold increase for clostridia compared to 1000-fold reduction in chronic cases (compared to faecal counts). Results indicate an increase in the bacterial numbers in the GI tract of animals with EGS compared to the controls and clostridia are prominent in EGS. Whether the increase in clostridia is the cause of GI stasis or a consequence remains uncertain.     

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.     

Development of a clinical prediction score for detection of suspected cases of equine grass sickness (dysautonomia) in France

Equine grass sickness (EGS) (equine dysautonomia) is a neurodegenerative condition of grazing equines. Pre-mortem diagnosis of EGS is a challenge for practitioners as definitive diagnosis requires ileal/myenteric lymph node biopsies. This study aimed to develop a clinical score that could be used by practitioners to improve the detection of acute or subacute EGS cases in the field. Suspected EGS cases were declared by veterinary practitioners. A case was classified as confirmed positive if ileal or rectal biopsy samples showed neuronal degeneration typical of EGS. A semi-quantitative scoring system, including epidemiological and clinical data, was created to attempt to classify suspected EGS horses into confirmed positive or negative cases. Each variable was weighted based on a boosted regression trees model, while taking into account its clinical relevance. Twenty-eight EGS cases were confirmed by biopsy during the entire study period. The best cut-off value for the score to have a high sensitivity while maximizing specificity was 8, with a sensitivity of 100% and a specificity of 53%. In our dataset, 77% of animals would be correctly classified with this cut-off value of 8. Highest sensitivity was chosen in order to detect the highest number of potential cases. Our score represents an inexpensive and useful tool to aid in the identification of suspected EGS cases in the field and selection for further diagnostics procedures to confirm or rule out the disease. Application of the score to larger populations of animals would be required to further adapt and refine the score.     

Association of admission plasma D-dimer concentration with diagnosis and outcome in horses with colic

Background: Coagulopathies detected in horses with gastrointestinal problems seem to be associated with poor outcome. Plasma D‐Dimer concentration is a sensitive test for assessing coagulopathies. Hypothesis: Plasma D‐Dimer concentration tested on admission is related to diagnosis and outcome in horses with colic. Animals: Four hundred and ninety three horses referred for evaluation of abdominal pain. Methods: Prospective observational clinical study. Horses were grouped according to diagnosis (medical and surgical intestinal obstructions, ischemic disorders with and without intestinal resection, enteritis, peritonitis), outcome (survivors, nonsurvivors), and number of coagulopathies (normal profile, 1 or 2 coagulopathies, subclinical disseminated intravascular coagulation [DIC]). Blood samples were collected on admission and plasma D‐Dimer concentration, clotting times (PT and aPTT), and antithrombin activity were determined. Positive likelihood ratios (LR+) were calculated for evaluation of D‐Dimer cut‐off values, which were later tested in a logistic regression model. Results: Horses with enteritis or peritonitis had significantly (P < .001) higher plasma D‐Dimer concentrations and more severe coagulopathies on admission than horses with other diagnoses. Nonsurvivors also had significantly (P < .001) higher plasma D‐Dimer concentrations at presentation than did survivors, and those horses with subclinical DIC on presentation had an odds ratio (OR) 8.6 (95% confidence interval [CI], 3.3–22.5, P < .001) for nonsurvival. Finally, D‐Dimer concentrations >4,000 ng/mL had a LR+ of 5.9 and an OR 8.8 (95% CI, 4.5–17.1, P < .001) for nonsurvival. Conclusion and Clinical Importance: Plasma D‐Dimer concentration measured on admission can be used to facilitate diagnosis and outcome prediction in horses with colic. A potential cut‐off value for nonsurvival was found at approximately 4,000 ng/mL.     

Serum tumor necrosis factor activity in horses with colic attributable to gastrointestinal tract disease.

Over a 24-month period, serum tumor necrosis factor (TNF) activity was determined in 289 horses with colic attributable to gastrointestinal tract disease. Serum TNF activity was quantitated by use of a modified in vitro cytotoxicity bioassay, using WEHI 164 clone-13 murine fibrosarcoma cells. Causes for colic, determined by clinical and laboratory evaluation, exploratory celiotomy, or necropsy included: gastrointestinal tract rupture (GTR); ileal impaction; small intestinal strangulating obstruction (SIO); proximal enteritis (PE); transient small intestinal distention; large-colon displacement; large-colon volvulus; large-colon impaction; colitis; small-colon obstruction; peritonitis; and unknown. Each diagnosis was placed into 1 of 3 lesion categories: inflammatory disorders (GTR, PE, colitis, peritonitis); strangulating intestinal obstruction (SIO, large-colon volvulus); and nonstrangulating intestinal obstruction (ileal impaction, transient small intestinal distension, large-colon displacement, large-colon impaction, small-colon obstruction, unknown). The prevalence of high serum TNF activity and/or mortality were evaluated. Differences were tested at significance level of P less than 0.05. Approximately 20% of the 289 horses has serum TNF activity greater than that found in clinically normal horses (greater than 2.5 U/ml). Twenty-three horses (8%) had marked increase in serum TNF activity (greater than or equal to 10 U/ml) which was more prevalent among horses with SIO and PE than in horses of other diagnostic groups, except those with GTR. Mortality and marked increase in serum TNF activity were greater in horses with intestinal inflammatory disorders or strangulating intestinal obstruction than in horses with nonstrangulating intestinal obstruction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification and cloning of two immunogenic Clostridium perfringens proteins, elongation factor Tu (EF-Tu) and pyruvate:ferredoxin oxidoreductase (PFO) of C. perfringens

Clostridium-related poultry diseases such as necrotic enteritis (NE) and gangrenous dermatitis (GD) cause substantial economic losses on a global scale. Two antigenic Clostridium perfringens proteins, elongation factor Tu (EF-Tu) and pyruvate:ferredoxin oxidoreductase (PFO), were identified by reaction with immune sera from commercial meat-type chickens with clinical outbreak of Clostridium infections. In addition to the genes encoding EF-Tu and PFO, C. perfringens alpha-toxin and necrotic enteritis B-like (NetB) toxin were also expressed in Escherichia coli and their corresponding recombinant proteins were purified. Using the four recombinant proteins as target antigens in ELISA immunoassays, high serum antibody titers were observed not only in chickens with clinical signs of Clostridium infections, but also in apparently healthy animals from the same disease-endemic farm. By contrast, no antibodies against any of the proteins were present in the serum of a specific pathogen-free bird. In ELISA using recombinant proteins of C. perfringens, the levels of anti-bacterial protein antibodies were also higher in chickens which were experimentally induced to show NE clinical signs after co-infection with C. perfringens and Eimeria maxima compared with uninfected controls. These results show that two antigenic C. perfringens proteins, EF-Tu and PFO can be useful detection antigens for C. perfringens-afflicted infections in commercial poultry.