Isolation and characterization of tick toxins

Abstract

Ticks are the most important ectoparasites of domestic animals in the Republic of South Africa. Annual losses due to ticks amount to several hundred million Rand. They may cause disease since they may serve as vectors of pathogens. Certain tick species cause toxicoses which are generally believed to develop as the result of the transfer of toxins from tick to host. In the present work, attempts were made to isolate and characterize toxins from Rhipicephalus evertsi evertsi and Argas (Persicargas) walkerae ticks. These ticks cause paralysis in sheep (Spring lamb paralysis) and poultry, respectively. Furthermore, tick egg toxins were investigated because they may have a bearing on the paralysis toxins. In addition, methods for the purification and detection of Cowdria ruminantium were investigated as a first step in the study of the assumed toxin produced by this pathogen. C. ruminantium is the causative agent of heartwater and transmitted by Amblyomma ticks. A toxin was isolated by chromatofocusing from the salivary glands of female R. evertsi evertsi in the toxic feeding phase. The toxin was shown to reach a maximum concentration during this phase. The toxin was found to be homogeneous according to isoelectric focusing, sodium dodecyl sulfate polyacrylamide gradient gel electrophoresis and gel permeation chromatography. Toxicity was assayed by means of a Xenopuslaevis nerve muscle preparation. The toxin exhibited a reversible inhibition of muscle contraction when applied directly onto the nerve. The molecular weight was found to be 68 kdal by gel permease chromotography and the pI equal to 6. Attempts were also made to isolate a paralysis toxin from whole A. (P.) walkerae fully engorged larvae. Problems were encountered due to the very high concentrations of host hemoglobin in these ticks. No homogeneous toxin could be obtained. Toxicity was assayed with 1 day old chickens. The egg toxins of R. evertsi evertsi, Boophilus decoloratus, B. microplus and Byalomma truncatum were found to be fast-binding or slow-binding inhibitors of trypsin and chymotrypsin. Fast-binding inhibition of trypsin by R. evertsi evertsi (Ki = 16 nM) and of chymotrypsin by B.decoloratus ( Ki = 36 nM) and by H. truncatum ( Ki = 23nM) was observed. Slow-binding inhibition of trypsin by B. microplus (Ki = 4.8 nM), by -B. decoloratus (Ki= 4.1 nM) and by H. truncatum (Ki= 0.9 nM) was demonstrated. Immunological cross reaction was observed between the toxins from B. microplus and B. decoloratus. The in vivo functions of the toxins need to be further investigated. C. ruminantium organisms were isolated by lectin affinity chromatography and Percell density gradient centrifugation. Isolated fractions were analyzed by intravenous inoculation into sheep, protein determination, electron-microscopy and enzyme-Iinked immunosorbent assay. The purification procedures could be completed in a few hours using either infected sheep brain or nymphae as starting material. The density gradient centrifugation method permitted the recovery of viable populations of the organism possessing different densities. ELISA methods for the detection of C. ruminantium antibodies during the course of heartwater disease in sheep as well as antigen in blood and ticks were developed. By using appropriate controls the assays were rendered specific for C. ruminantium. The antibody assay showed that lgM antibodies reached an maximum on the 4th day after infection and disappeared on the 7th day. lgG antibodies first appeared on the 8th day and continued to increase up to at least the 28th day. The earliest day of C. ruminantium antigen detection in the host was in plasma and serum on day 4 after inoculation. Erythrocytes showed the highest concentration of antigen which reached a maximum two days after the febrile reaction. C. ruminantiwn antigen was detected in various tick tissue and hemolymph. In females, the gut and synganglion and in males the salivary glands and gut showed the highest levels. ELISA also showed that the infective mouse strain (k'umm strain) and infective sheep strain (Ball 3 strain) of C. ruminantium possess common antigens.