Tick toxinology : isolation and characterisation of the toxin from the Australian paralysis tick, Ixodes holocyclus

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Several species of ticks have been reported to secrete neurotoxins which can be fatal to mammals, including man. Envenomation by the Australian paralysis tick, I. holocyclus, is a considerable veterinary problem affecting tens of thousands of domestic and native animals each year. Envenomation is characterised by a progressive ascending flaccid paralysis and is due to a neurotoxin present in the salivary secretions of the tick. Purification of the neurotoxic component has been largely unsuccessful mainly due to the insensitivity of the biological assay, the minute quantities present and the reported labile nature of the neurotoxin. This dissertation describes the identification, final purification, partial characterisation and preliminary structural characteristics of the neurotoxins from I. holocyclus. The neurotoxins were identified as having a molecular weight of 5 kD by SDS-PAGE using an innovative technique involving adsorption to synaptosomes. The identification subsequently enabled the final purification of three closely related neurotoxins. The neurotoxins were purified from extracts of engorged adult female I. holocyclus by a series of conventional chromatography techniques (heparin Sepharose, DEAE Affi-gel blue, Alkyl Sepharose) followed by C4 and CB reverse phase HPLC. Purity was demonstrated by Tricine-SDS-PAGE and biological activity confirmed by injection into neonatal mice. Total amino acid analysis of the three neurotoxins showed that the neurotoxins were essentially identical. This was latter confirmed by a non-neutralising monoclonal antibody which was capable of recognising a common epitope shared by all three neurotoxins. Amino acid sequence of the neurotoxins was restricted to peptides generated by proteolytic digestion with trypsin as direct N-terminal sequencing revealed an amino-terminal blockage. An accurate molecular weight of 5,640 (HT-II) and 5,460 (HT-I) Daltons has been determined by mass spectrometry for two of the neurotoxins. The small size is comparable to neurotoxins isolated from other arachnids (spiders and scorpions). However, comparisons of the peptide amino acid sequences derived from the proteolytic digestion of HT-II with known protein sequences failed to display any significant homologies. cDNA libraries were produced from poly(A⁺) mRNA isolated from unengorged I. holocyclus ticks. The libraries were characterised and found to be well-represented with tick-derived cDNA sequences. PCR primers and oligonucleotide probes were designed from the partial amino acid sequence obtained for HT-II and used to screen the cDNA libraries. After extensively screening the libraries, no positive clones were obtained. Northern hybridisation analysis of total RNA prepared from unengorged and engorged ticks with the oligonucleotide probe RHT produced a band of the expected size (220 bps), only with total RNA prepared from engorged ticks. This indicated the original cDNA libraries prepared from unengorged ticks were unlikely to contain the gene of interest. PCR based strategies were unable to isolate neurotoxin gene(s) from mRNA prepared from engorged ticks.
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