The Australian paralysis tick, Ixodes holocyclus causes a potentially fatal paralysis in domestic animals, livestock and humans with companion animals (mainly dogs) most commonly affected. Current treatment regimes include administration of a commercial tick anti-serum (TAS), prepared as hyperimmune serum in dogs, to neutralise the effects of the toxin. However, each new batch must be standardised using an expensive and highly subjective bioassay performed in neonatal mice. There is currently an urgent need for a more cost effective and rapid in vitro assay that can be more objectively and accurately quantified. Further understanding of the composition of the toxin molecule is also required to develop toxin-specific reagents necessary for these assays. One of the main objectives of this study was to develop a suitable immunoassay to replace the existing mouse bioassay for assessing batches of tick anti-sera for use in tick paralysis therapy in dogs. Initially an enzyme-linked immunosorbent assay (ELISA) was established to detect and quantify antibody specific for I. holocyclus toxin in dog sera. Using a partially purified antigen extracted from I. holocyclus salivary glands, good discrimination was achieved between reactive (hyperimmune) and non-reactive (naïve) sera. The hyperimmune dog sera reacted very strongly with the antigen compared to negligible reactions of serum from dogs not exposed to I. holocyclus. The reactions of hyperimmune sera were also significantly weaker to a non-toxin antigen control extracted from the salivary glands of the non-toxic tick Rhipicephalus microplus, indicating the assay was detecting toxin-specific responses. Furthermore, each of the hyperimmune sera that reacted strongly and specifically with the I. holocyclus antigen in the ELISA also strongly neutralised toxin in the mouse bioassay. Together these findings support the suitability of this ELISA for assessing the potency of batches of commercial dog hyperimmune sera for use as therapy for tick paralysis in dogs. Sera from dogs that were experimentally infested with ticks and sera from patient dogs, presenting at veterinary clinics with signs of tick paralysis, were also screened for antibodies to I. holocyclus antigen using the ELISA. Twenty-eight out of 29 sera from animals with single or multiple exposures to ticks failed to recognise the I. holocyclus antigen indicating the ELISA is not suitable as a diagnostic test to detect toxin-specific antibodies in animals with limited exposure to I. holocyclus infestation. A panel of toxin-specific monoclonal antibodies (mAbs) was produced as research tools to analyse and purify tick toxin components. Rats were successfully immunised against tick toxin using a combination of inoculation of partially purified salivary gland antigen and exposure to tick infestation. The latter approach preserved the native confirmation of the toxin using a natural route of immunisation and rats were chosen due to their high tolerance of multiple tick infestations over several days. While fusion of rat spleen cells with mouse myeloma cells has been reported several times in the literature, the resulting hybridomas are unstable with fastidious culture requirements. Optimisation of the culture conditions revealed that most rat-mouse hybridoma lines grew best in serum-free medium supplemented with 5% foetal bovine serum. Of 600 hybridomas produced, only 12 were shown to be specific for the Ixodes antigen, as determined by ELISA. A selection of these hybridomas representing various patterns of affinity and/or antigen specificity were further analysed for toxin-neutralising ability in a mouse bioassay. Notably, the most potent toxin-neutralising mAb in mice, showed a specific but relatively moderate reaction to Ixodes antigen in the ELISA. The most potent toxin-neutralising mAbs inactivated toxin as strongly as the commercial TAS used for immunotherapy in dogs with tick paralysis. This suggests that mAbs may present an alternative source of immunotherapy, providing a potentially endless supply of a highly consistent reagent and negating the need to use live animals for both the production of tick antiserum and the continual testing of reagent batches. The toxin-neutralising mAbs were also used to analyse I. holocyclus toxin in polyacrylamide gel electrophoresis (PAGE) and Western blot to identify specific toxin proteins. The most potent neutralising mAbs consistently recognised high MW proteins (100-200 kDa) in a smeared pattern. Although this was contrary to previous reports of low molecular weight components (3-5 kDa) in holocyclotoxin, this study was the first to use mAbs prepared to native toxin. The large molecular weight structures likely represent presucursors to, or complexes of the smaller peptides, previously identified. When the Toxin-neutralising mAbs were assessed as ligands to affinity purify toxin components from crude Ixodes SG extracts, toxin components of 110 and 32 kDa were consistently identified. These purified proteins represent good candidates for N-terminal sequencing to further identify the toxin components in I.holocyclus salivary glands.
| Identifer | oai:union.ndltd.org:ADTP/282532 |
| Creators | Sonja Hall-Mendelin |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
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