Standing gustatory papillae biopsy procedure for antemortem diagnosis of equine grass sickness.

OBJECTIVE: Diagnosing equine grass sickness (EGS) requires histopathological evidence of chromatolysis and/or neuronal loss in peripheral autonomic ganglia. Previous investigators performed postmortem biopsies of gustatory papillae located on the tongue and found chromatolytic subgemmal neurons in all 13 EGS horses. The present study aimed to design a standardized lingual biopsy sampling method through a transbuccal approach in healthy standing horses and assess the quality of the obtained samples, to allow antemortem diagnosis of EGS in clinical cases. ANIMALS: 6 healthy horses. METHODS: A transbuccal approach was performed bilaterally in 6 healthy standing horses. After having reached a deep level of sedation, horses were placed in stocks and a Günther mouth gag was inserted. Local anesthesia followed by a vertical full thickness incision was performed on both cheeks. Foliate papillae biopsies were carried out using an arthroscopic rongeur inserted through each incision site under oral endoscopic control. Tongue movements were restricted with diazepam. Histological assessment of taste buds and subgemmal plexi neurons was performed using H&E-stained longitudinal sections. RESULTS: The procedure was well tolerated in all horses. Minor complications observed were a transient facial paralysis, some incisional fluid collection, and abscesses. Ten samples (10/12) were suitable for assessment of neuronal perikarya. CLINICAL RELEVANCE: This procedure was safe for subgemmal plexus biopsy in healthy standing horses. The obtained samples were adequate as long as they were neatly cut lengthwise for inclusion. The technique was also used for 2 clinical cases and revealed the complete absence of neuronal perikarya, confirming chronic EGS.

Reparación de lesiones en nervios mediante el implante de prótesis obtenidas de segmentos acelulares de nervio isogénico / Repair of nerve injury by implanting prostheses obtained from isogenic acellular nerve segments

Introducción. Cuando se produce una sección nerviosa con separación significativa de los cabos es necesario utilizar una prótesis, a modo de puente, para suturarlos. La mejor prótesis es un segmento de nervio autógeno, pero presenta importantes inconvenientes. Nuestro objetivo es comparar la eficacia de la sutura simple con la tubulización para el implante de una prótesis de nervio isogénico descelularizado. Material y método. Se utilizan 4 grupos de ratas Wistar. Grupo 0: animales donantes de nervio ciático. Grupo 1: recibió el implante con sutura término-terminal. Grupo 2: recibió el implante dentro de un tubo de ??-caprolactona. Grupo 3: lo recibió en un tubo de poliláctico-co-glicólico. Se evaluó la función motora (índice ciático) y la extensión de la regeneración (estudio histológico) a las 3 semanas del implante. Resultados. La regeneración ha sido irregular en los 3 grupos experimentales. En todos hay implantes en los que las fibras nerviosas regeneran la longitud máxima estudiada (15mm) y otros en los que la regeneración es muy escasa. La longitud media de regeneración es mayor en el grupo de sutura directa (G1), aunque la velocidad es similar en los 3. El grupo 1 muestra el mayor porcentaje de regeneración, aunque la variabilidad de los resultados impide que esta diferencia alcance significación estadística. No hemos hallado diferencias significativas entre los dos grupos con tubos de diferentes polímeros. Conclusión. Para implantar prótesis de nervios isogénicos descelularizados es más eficaz, en nuestras condiciones experimentales, la sutura término-terminal que los tubos de polímeros biocompatibles (AU)

Introduction. When a nerve section with a significant gap occurs, it is necessary to use a prosthesis to suture it. To date an autologous nerve segment graft appears to be the best treatment; but it has several important disadvantages. Our goal is to study the effectiveness of an isogenic acellular nerve prosthesis comparing a simple suture with tubulisation. Material and method. Four groups of Wistar rats were used. The animals in Group 0 served as donors of nerve segments to graft. Group 1 received the implant with an end-to-end suture. In group 2, the implant was sutured inside an ??-caprolactone tube. Group 3 received it in a polylactic-co-glycolic acid tube. We evaluated the motor function (sciatic index and step test in motion), and the regeneration length by histological study of regeneration, after a maximum of 3 weeks. Results. Regeneration was uneven in the three groups. In all groups, there were implants with regenerated nerve fibres at the maximum studied length (15mm) and others where regeneration was scarce. The mean regeneration length was greater in the direct end-to-end suture group (G1), although the regeneration speed was similar in the three groups. Group 1 showed the highest percentage of regeneration, but the variability of results prevents this difference reaching statistical significance. We found no significant differences between the two groups with polymer tubes. Conclusion. For the implantation of isogenic acellular nerve prosthesis, under our experimental conditions, the direct end-to-end suture was more effective than when it isprotected with biopolymer tubes (AU)

Neurodegeneration in equine grass sickness is not attributable to niacin deficiency.

BACKGROUND: The aetiology of equine grass sickness (EGS) is currently unknown. We hypothesised that an acute deficiency of niacin (vitamin B3), which plays a key role in neural homeostasis, may contribute to neurodegeneration in EGS. Niacin deficiency can potentially result from ingestion of niacin antagonists produced by pasture mycotoxigenic fungi. OBJECTIVES: To compare the niacin status of EGS and control grazing horses. A secondary objective was to compare blood concentrations of vitamins B1, B2 and B6 in EGS and control grazing horses to determine if the status of these vitamins was altered in EGS. STUDY DESIGN: Case-control study. METHODS: Indices of niacin status, namely the erythrocyte nicotinamide adenine dinucleotide:nicotinamide adenine dinucleotide phosphate ratio (NAD:NADP ratio) and erythrocyte concentrations of NAD and NADP, were compared in blood collected from EGS and healthy control grazing horses. Blood concentrations of vitamins B1, B2 and B6 were also compared. RESULTS: There was no significant intergroup difference in the NAD:NADP ratio, the main index of functional niacin status (control group: median 2.1, interquartile range [IQR] 1.8-2.6; EGS group: median 2.1, IQR 1.9-2.6). EGS horses had significantly higher (median value increased by 25%) concentrations of NADP. There were no intergroup differences in blood concentrations of vitamins B1, B2 and B6. MAIN LIMITATIONS: The interpretation of data was limited by the lack of previously defined equine reference ranges for many of the analytes. Sample size was low. CONCLUSIONS: Niacin deficiency does not contribute to EGS neurodegeneration.

Equine grass sickness in Scotland: A case-control study of environmental geochemical risk factors.

REASONS FOR PERFORMING STUDY: We hypothesised that the apparent geographical distribution of equine grass sickness (EGS) is partly attributable to suboptimal levels of soil macro- and trace elements in fields where EGS occurs. If proven, altering levels of particular elements could be used to reduce the risk of EGS. OBJECTIVES: To determine whether the geographical distribution of EGS cases in eastern Scotland is associated with the presence or absence of particular environmental chemical elements. STUDY DESIGN: Retrospective time-matched case-control study. METHODS: This study used data for 455 geo-referenced EGS cases and 910 time-matched controls in eastern Scotland, and geo-referenced environmental geochemical data from the British Geological Survey Geochemical Baseline Survey of the Environment stream sediment (G-BASE) and the James Hutton Institute, National Soil Inventory of Scotland (NSIS) datasets. RESULTS: Multivariable statistical analyses identified clusters of three main elements associated with cases from (i) the G-BASE dataset - higher environmental Ti and lower Zn, and (ii) the NSIS dataset - higher environmental Ti and lower Cr. There was also some evidence from univariable analyses for lower Al, Cd, Cu, Ni and Pb and higher Ca, K, Mo, Na and Se environmental concentrations being associated with a case. Results were complicated by a high degree of correlation between most geochemical elements. CONCLUSIONS: The work presented here would appear to reflect soil- not horse-level risk factors for EGS, but due to the complexity of the correlations between elements, further work is required to determine whether these associations reflect causality, and consequently whether interventions to alter concentrations of particular elements in soil, or in grazing horses, could potentially reduce the risk of EGS. The effect of chemical elements on the growth of those soil microorganisms implicated in EGS aetiology also warrants further study.

Neuronal chromatolysis in the subgemmal plexus of gustatory papillae in horses with grass sickness.

REASONS FOR PERFORMING STUDY: Diagnosis of equine grass sickness (EGS) can be challenging. We hypothesised that subgemmal plexus neurons are chromatolytic in EGS. If correct, histopathological examination of gustatory papillae biopsies could aid premortem diagnosis of EGS, and EGS could represent a spontaneous model of subgemmal neuronal chromatolysis to facilitate study of the pathology of structures involved in taste. OBJECTIVE: To compare subgemmal plexi and gustatory papillae in EGS and control horses. STUDY DESIGN: Observational study. METHODS: Conventional histology and immunohistochemistry were used to compare subgemmal plexi and gustatory papillae in post mortem samples from 10 EGS and 13 control horses. RESULTS: Chromatolytic neurons were present in all 57 EGS sections which had identifiable neurons, and in only one of 57 control sections. Blinded examination of all haematoxylin-eosin stained sections from each horse for chromatolysis facilitated accurate differentiation of EGS and control horses, with a sensitivity of 100% (95% confidence interval 93.7-100) and specificity of 98.2% (90.6-100) for diagnosing EGS; however, the presence of chromatolytic neurons in one control section indicated that multiple sections per horse must be analysed to achieve diagnostic accuracy. Equine grass sickness was not associated with alterations in taste bud density or morphology, proportion of taste buds with neurofilament immunopositive intragemmal axons or proportion of taste buds containing cells undergoing apoptosis, suggesting taste buds had adequate neurotrophic support at the time of sampling. Horses with EGS had no detectable alteration in lingual gland morphology, but had increased proportions of apoptotic lingual serous gland cells. CONCLUSIONS: While identification of chromatolytic subgemmal neurons in post mortem samples correctly differentiated EGS and control horses, further study is required to evaluate this technique for premortem EGS diagnosis. Equine grass sickness represents a spontaneous model of subgemmal neuronal chromatolysis that facilitates study of the pathology of structures involved in taste.

Equine grass sickness, but not botulism, causes autonomic and enteric neurodegeneration and increases soluble N-ethylmaleimide-sensitive factor attachment receptor protein expression within neuronal perikarya.

REASONS FOR PERFORMING STUDY: Equine grass sickness (EGS) is of unknown aetiology. Despite some evidence suggesting that it represents a toxico-infection with Clostridium botulinum types C and/or D, the effect of EGS on the functional targets of botulinum neurotoxins, namely the soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins, is unknown. Further, while it is commonly stated that, unlike EGS, equine botulism is not associated with autonomic and enteric neurodegeneration, this has not been definitively assessed. OBJECTIVES: To determine: 1) whether botulism causes autonomic and enteric neurodegeneration; and 2) the effect of EGS on the expression of SNARE proteins within cranial cervical ganglion (CCG) and enteric neuronal perikarya. STUDY DESIGN: Descriptive study. METHODS: Light microscopy was used to compare the morphology of neurons in haematoxylin-eosin stained sections of CCG and ileum from 6 EGS horses, 5 botulism horses and 6 control horses. Immunohistochemistry was used to compare the expression of synaptosomal-associated protein-25, synaptobrevin (Syb) and syntaxin within CCG neurons, and of Syb in enteric neurons, from horses with EGS, horses with botulism and control horses. The concentrations of these SNARE proteins in extracts of CCG from EGS and control horses were compared using quantitative fluorescent western blotting. RESULTS: EGS, but not botulism, was associated with autonomic and enteric neurodegeneration and with increased immunoreactivity for SNARE proteins within neuronal perikarya. Quantitative fluorescent western blotting confirmed increased concentrations of synaptosomal-associated protein-25, Syb and syntaxin within CCG extracts from EGS vs. control horses, with the increases in the latter 2 proteins being statistically significant. CONCLUSIONS: The occurrence of autonomic and enteric neurodegeneration, and increased expression of SNARE proteins within neuronal perikarya, in EGS but not botulism, suggests that EGS may not be caused by botulinum neurotoxins. Further investigation of the aetiology of EGS is therefore warranted.

Bodyweight change aids prediction of survival in chronic equine grass sickness.

REASONS FOR PERFORMING STUDY: Objective criteria for predicting survival of chronic grass sickness cases are currently lacking. OBJECTIVES: To determine whether the rate and/or magnitude of bodyweight change during hospitalisation of chronic grass sickness cases can provide an objective predictor of survival to discharge from hospital. Clinicians' recorded indication(s) for euthanasia were also reviewed. STUDY DESIGN: Single centre retrospective observational study. METHODS: Case records of all horses admitted for management of chronic grass sickness to The Dick Vet Equine Hospital between 1998 and 2013 were analysed. Case background, survival to hospital discharge, indication(s) for euthanasia, disease duration at admission and bodyweight changes during the hospitalisation period were analysed, and data for survivors and nonsurvivors compared. Percentage weight change was calculated for 7 day intervals up to 28 days (0-7, 7-14, 14-21, 21-28 days) and for entire periods from the first weight recorded (0-7, 0-14, 0-21, 0-28 days). These results were used to estimate survival probability conditional on weight change. RESULTS: The study sample comprised 213 horses, with 114 survivors (53.5%) and 99 (46.5%) nonsurvivors. Compared with nonsurvivors, survivors had significantly lower median maximum bodyweight loss as a percentage of first weight (survivors 5.9%, interquartile range 1.8-13.5; nonsurvivors 12.7%, 6.4-17.3). Throughout all time periods analysed, survivors had significantly lower median bodyweight loss than nonsurvivors, but no specific time period was more predictive of survival. Highest percentages of total bodyweight loss for individual horses were comparable for survivors (36%) and nonsurvivors (37%). Survival prediction curves reporting percentage survival rates for all time periods analysed provided data to aid prediction of chronic grass sickness survival. CONCLUSIONS: Overall, nonsurvivors had greater bodyweight loss than survivors. Rapidity and magnitude of bodyweight loss were equally predictive of outcome. Percentage survival prediction curves provide objective data to aid discussion of prognosis, but greater predictive specificity with associated sensitivity is required for clinical decision making in individual cases.

Focus on: vaccination against equine grass sickness.

Jo Ireland of the Animal Health Trust describes a new field trial of a potential vaccine against equine grass sickness.

Equine grass sickness in Scotland: a case-control study of signalment- and meteorology-related risk factors.

REASONS FOR PERFORMING STUDY: Equine grass sickness (EGS) remains a frequently fatal disease of equids in Britain. Since previous investigations of signalment- and meteorology-related risk factors for EGS have yielded some conflicting data, further investigation is warranted. OBJECTIVES: To identify signalment- and meteorology-related risk factors for EGS in Scotland. STUDY DESIGN: Retrospective time-matched case-control study. METHODS: This study was undertaken using data for 455 EGS cases and 910 time-matched controls that were referred to the Royal (Dick) School of Veterinary Studies, and average UK Meteorological Office weather station meteorological values from the month of admission of the animal, from the 3, 6 and 12 months prior to admission, and for the entire 1990-2006 period. RESULTS AND CONCLUSION: Signalment-related risk factors associated with an increased risk of EGS were native Scottish pure breeds compared with crossbreeds (odds ratio [OR] = 3.56, 95% confidence interval [CI] 2.43-5.43) and animals living on premises located further north within the study region (OR = 1.08, 95% CI 1.06-1.10). There was a decreased risk of EGS in animals aged 11-20 years compared with animals 2-10 years (OR = 0.32, 95% CI 0.22-0.45), non-native Scottish pure breeds compared with crossbreeds (OR = 0.71, 95% CI 0.54-0.94), and stallions compared with mares (OR = 0.43, 95% CI 0.22-0.86). Meteorology-related risk factors associated with an increased risk of EGS were (if Ordnance Survey northing is excluded) more sun hours (OR>1.43) and more frost days (OR>1.13), while there was a decreased risk of EGS with higher average maximum temperature (OR<0.83). POTENTIAL RELEVANCE: The signalment-related risk factors will help owners identify high-risk animals, thereby allowing them to prioritise management strategies. The identification of meteorological risk factors may assist studies on the aetiology of EGS.

In equine grass sickness, serum amyloid A and fibrinogen are elevated, and can aid differential diagnosis from non-inflammatory causes of colic.

Equine grass sickness (EGS) is a debilitating and often fatal neurodegenerative disease. A presumptive diagnosis of EGS may be made on the basis of clinical signs and subjective ancillary tests, but a definitive antemortem diagnosis can only be made following histopathological examination of intestinal biopsies. It has previously been reported that horses with EGS may show clinical and clinicopathological signs of systemic inflammation. The objective of this study was to (a) quantify acute inflammatory markers in blood samples collected from acute, subacute and chronic EGS cases, and (b) compare them with (i) clinically normal horses co-grazing with acute EGS cases (co-grazers), (ii) horses with other causes of colic and (iii) healthy horses. Serum amyloid A (SAA), serum activin A and plasma fibrinogen were quantified. There were marked increases in SAA and fibrinogen in EGS cases compared with healthy horses, co-grazers and non-inflammatory colic cases. The concentrations of SAA and fibrinogen in EGS cases were not significantly different from inflammatory colic cases. When concentrations of SAA, fibrinogen and activin A in each EGS subgroup were compared, no significant differences were detected. Activin A concentrations were significantly elevated in EGS cases and co-grazing horses; this could reflect the presence of subclinical disease in some horses that do not develop clinical signs of EGS, and suggests widespread exposure to the aetiological agent. When faced with sparse antemortem diagnostic techniques, identification of marked increases in acute phase protein concentrations may help to differentiate EGS from other causes of abdominal pain, such as intestinal obstructions; however, there could be diagnostic difficulty in differentiating other inflammatory abdominal conditions, such as peritonitis or enteritis.

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.

Outbreaks of equine grass sickness in Hungary.

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

Autonomic dysfunction in a Jack Russell terrier.

A 4-year-old Jack Russell terrier was presented with an array of clinical signs suggestive of autonomic dysfunction. Many of the clinical signs were consistent with a diagnosis of dysautonomia; however, both chronicity and resolution of signs contradicted a diagnosis of this disease.

Effects of equine grass sickness on sympathetic neurons in prevertebral and paravertebral ganglia.

Acute equine grass sickness (EGS) is a fatal disease of horses that is thought to be due to ingestion of a neurotoxic agent causing extensive damage to autonomic neurons. The aim of this study was to compare the effects of EGS on neurons in two sympathetic ganglia, the paravertebral cranial cervical ganglion (CCG) and the prevertebral coeliac/cranial mesenteric ganglion (CG/CMG). Specimens from horses with EGS and controls were obtained post mortem and processed using single and double immunofluorescence labelling for PGP 9.5 and HuC/HuD (pan-neuronal markers), TUNEL and caspase 3 (markers for apoptosis), vasoactive intestinal polypeptide (VIP) and galanin (markers of the cell body response to injury following axotomy or exposure to sympathetic neurotoxins) and tyrosine hydroxylase (TH, marker for noradrenaline synthesis). In control horses, all neurons contained PGP 9.5 and HuC/HuD. There was a significant loss of PGP 9.5 and HuC/HuD expression in samples from horses with EGS that occurred to a greater extent in the CG/CMG than the CCG. The number of caspase 3-positive neurons increased significantly in both ganglia, but TUNEL staining of sympathetic neurons was only significantly increased in the CG/CMG in EGS. No VIP was observed in any ganglia; however, there was a significant increase in galanin-positive neurons in both ganglia in EGS. In the CCG, there was a significant shift towards increased fluorescence intensity for TH, possibly indicating an initial accumulation of TH within the cell body. In contrast, TH fluorescence intensity was significantly reduced in the CG/CMG in EGS correlating with the greater loss of neurons. These results demonstrate that EGS can induce a cell body response that is similar to the response of sympathetic neurons to a chemical neurotoxin. EGS also causes loss of sympathetic neurons, some of which occurs via apoptosis. Changes were more marked in the CG/CMG than the CCG indicating that the prevertebral ganglia were affected earlier than the paravertebral ganglia in the pathological process and had undergone greater neurodegeneration.