Analysis of protein expression in the autonomic ganglia of horses with respect to equine grass sickness

Equine grass sickness (EGS) is essentially a fatal disease of the horse with clinical symptoms that indicate autonomic nervous system (ANS) dysfunction. Such symptoms include varying degrees of colic, intestinal stasis, dysphagia, anorexia, weight loss, tachycardia, excessive salivation, patchy sweating, ptosis (drooping of the eye lids), rhinitis sicca and muscle tremor (Hudson et al, 2001). Furthermore, a characteristic loss of stainability (chromatolysis) is commonly seen in neurones of the gut wall plexi, prevertebral and paravertebral ganglia, the intermediolateral tract of the spinal cord and certain brain stem nuclei in EGS cases. The disease is recognised in three loosely defined clinical categories; acute (disease duration 1-2 days), sub-acute (2-7 days) and chronic (>7 days). It is hypothesised that a neurotoxin is ingested and the extent of the nervous damage is dose dependent. It is also considered that massive neuronal loss likely results in the acute form, whilst less neuronal damage results in less severe signs and the chronic form of the disease (Cottrell et al, 1999). The aetiology of EGS is largely unknown. However, the working hypothesis is that it is a toxicoinfection with Clostridium botulinum type C, possibly triggered by dietary factors such as cyanide from white clover (Trifolium repens). In this study protein expression in homogenates of cranial cervical ganglia collected from horses with acute EGS (AGS) and from control horses was analysed using two- dimensional electrophoresis (2D-E). Optimal sample preparation and 2D-E techniques were developed for the first time for equine neural tissue. Variations in the protein expression between samples from different locations within the same autonomic ganglia, different autonomic ganglia from the same horse, ganglia from different horses of the same sex and ganglia from horses of both sexes were investigated using the 2D-E method developed. It was concluded that the variations in protein expression between such samples would not affect further comparisons between AGS and control 2D-E tissues and would not interfere with identifying EGS-related proteins. At this point a 2D-E gel was run with sample from control equine autonomic ganglia and sixty spots were subjected to MALDI-MS analysis to construct a preliminary proteome map of equine autonomic ganglia. This will provide a useful reference for future research. The protein expression on 2D-E gels of AGS and control samples was analysed and any differences were highlighted. Fourteen spots were identified as being up-regulated or only occurring on AGS sample gels and were considered to be AGS related. These proteins were analysed using Matrix-Assisted Laser Desorption Ionisation Mass Spectrometry (MALDI MS) and tandem MS. After MS analysis the majority of these proteins were recognised as neural proteins (e.g. beta tubulin and gamma enolase) or serum proteins. The presence or up-regulation of some of the proteins in AGS, such as alpha 1-antitiypsin, chaperonin/HSP60, beta tubulin and gamma enolase may well, with further investigation, help reveal the aetiology of EGS by clarifying the nature of the damage that occurs during EGS