Evaluation of Synaptophysin as an immunohistochemical marker for Equine Grass Sickness

Bryony Waggett, EGSF Research Assistant, University of Edinburgh, (2010)

The diagnosis of grass sickness (GS) is achieved in most cases by assessment of clinical signs, however in less clear-cut cases, confirmation of diagnosis may require assessment of small intestinal (ileum) biopsies which are collected during surgical exploration of the horse’s abdomen (Scholes et al. 1993). In most cases an experienced pathologist can confirm or refute a diagnosis of GS based on microscopic evaluation of nerves in haematoxylin and eosin stained sections of the small intestine, however, false-negatives (i.e. nerves appear normal so pathologist suggests that the horse does not have GS) can occur with this staining technique. This obviously has welfare implications when the horse is recovered from surgery only to be euthanased shortly thereafter when more obvious clinical signs of GS develop.

Synaptophysinstudy

Neurons showing marked synaptophysin expression in horse ileum (small Intestines)

Synaptophysin is a membrane protein found in synaptic vesicles of nerves in healthy horses, which can be stained using immunohistochemical methods. A previous study (Hilbe et al. 2005) showed that synaptophysin accumulates abnormally within the nerves of GS cases, however this study used only a small sample size and did not compare GS cases with colic cases which are the ones that are most difficult to differentiate from GS. An EGSF funded study performed at the Dick Vet Equine Hospital by Waggett et al., compared synaptophysin staining in ileum sections from the following groups of horses: GS cases, colic cases, control cases and botulism cases. This study confirmed that there was an accumulation of synaptophysin within nerves of the ileum in GS cases and not in cases of colic, botulism or controls. Using the criteria of density of neurons and synaptophysin staining, a statistically generated decision tree based on assessment of synaptophysin staining facilitated correct differentiation of all GS cases and non-GS cases. Additionally, samples from three cases that had been false negatives according to conventional staining methods (frozen sections stained with haematoxylin and eosin), were correctly diagnosed. While this study suggests that this technique can aid the histopathological diagnosis of GS, full evaluation of the decision tree in a prospective study with a larger number of horses is required before it can be used confidently by pathologists diagnosing GS.

Furthermore in contrast to GS, botulism did not alter ileal neuron density or synaptophysin labelling. This indicates that different mechanisms cause neuronal damage and/or dysfunction in GS and botulism, and that botulinum neurotoxins do not cause GS. It does not however rule out a causal role for some of the other toxins produced by Clostridium botulinum.

Full reference: B.E. Waggett, B.C. McGorum, D.J. Shaw, R.S. Pirie, N. MacIntyre, U. Werney and E.M. Milne. (2009) Evaluation of Synaptophysin as an immunohistochemical marker for Equine Grass Sickness. Journal of Comparative Pathology. Vol.142 Pages 284-290.

The other references cited can be found in the EGSF Reference list.