Purification and characterisation of tick toxins

Abstract

Ectoparasites can serve as vectors of numerous disease agents i.e. they are involved in maintaining and transmitting for example viral and rickettsial organisms pathogenic to man and animals (Sl). An understanding of these diseases inevitably must involve a study of the vector, pathogen, possible toxins involved and the host. The present study was designed to investigate the origin and characteristics of the toxins. Tick eggs were selected as starting material since they are known to be toxic (S2) and they can be collected in fairly large numbers. Furthermore, these toxins may have a bearing on toxins associated with tick paralysis and tick-symbiont association. Rhipieephalus evertsi evertsi, Hyalomma trunaatum, Boophilus deaoloratus and Boophilus miaroplus were used to prepare crude egg extracts. Toxin purification procedures involved centrifugation, gel permeation chromatography and chrornatofocusing. They were shown to be pure by isoelectric focusing and SDS-PAGE. Protease inhibitory activity was analysed on pancreatic trypsin with N-benzoyl-D-L-arginine 4-nitroanilideHCl as substrate, and pancreatic chymotrypsin with N-succinyl-L-phenylalanine-p-nitroanilide as substrate. The toxins differed with respect to amino acid composition, isoelectric point, molecular mass and inhibitory action on trypsin and chymotrypsin. Molecular mass was determined with SOS-gradient polyacrylamide gel electrophoresis and sedimentation equilibrium centrifugation and the isoelectric points with analytical isoelectric focusing. Some characteristics of the toxins are summarised in Table Sl. The relatively well studied organisms, Riakettsia prowazekii, R. typhi and R. aonorii were selected as test organisms. These studies were later extended to Cowdria ruminantium and organisms from ticks producing sweating sickness. The motivation was to investigate possible toxins produced by C. ruminantium and to investigate the possibility that organisms from sweating sickness producing ticks are involved as agents of the disease. The isolation of rickettsia-like organisms involved high and low speed centrifugation as well as Percell density gradient centrifugation. Electron microscopic studies of the organisms isolated from heartwater infected Amblyomma hebraeum ticks, showed morphological similarities to those found in the choroid plexus of an infected animal. The rickettsia-like organisms isolated from sweating sickness positive and negative H. truncatum ticks and eggs showed a similar distribution of organisms for both strains in Percell density gradients. The above results show that rickettsia-like organisms from ticks may be harvested in a pure, infective form and in large numbers.