The Ixodes holocyclus tick causes paralysis in up to 10,000 companion and domestic animals each year in Australia. Treatment requires the removal of the parasite and the administration of a commercial tick antiserum that is prepared from hyperimmune dogs. Each batch of this serum is initially tested for toxin-neutralising potency in a mouse bioassay that is expensive, time consuming, and subjective. With the aim of developing a rapid in vitro assay to replace the bioassay, we used a partially purified antigen prepared from I. holocyclus salivary glands to develop an ELISA to detect toxin-reactive antibodies in hyperimmune dog sera. The optimised ELISA reliably detected antibodies reactive to I. holocyclus salivary gland antigens. Parallel testing of sera with a negative control antigen prepared from the salivary glands of the nontoxic tick Rhipicephalus (Boophilus) microplus provided further evidence that we were detecting toxin-specific antibodies in the assay. Using the ELISA, we could also detect antibodies induced in rats after experimental infestation with I. holocyclus. This assay shows promise as an alternative means of assessing the potency of batches of hyperimmune dog serum and to screen for toxin-reactive monoclonal antibodies produced from immunised rodents. 1. Introduction Ixodid or hard ticks cause most toxicoses [1], affecting humans and animals around the world [2, 3]. The most severe form of toxicosis results in the paralysis of the infested host. Globally, just under 70 species of ticks have been described as being capable of inducing paralysis [2], the most important being Ixodes holocyclus in Australia, Dermacentor andersoni, D. variabilis, and Argas (Persicargas) radiatus in North America, I. rubicundus in South Africa, Rhipicephalus evertsi evertsi and A. (P.) walkerae in Ethiopia, and A. (P.) radiatus in the Nearctic region of North America [4]. In Australia, I. holocyclus can cause paralysis in a range of domestic animals and livestock [5], affecting up to 10,000 companion animals and up to 100,000 livestock per year [6]. It is considered highly toxic with one female able to kill a dog [1] or sheep [7]. I. holocyclus is found along the eastern seaboard of Australia and is most abundant from early spring to late summer. Paralysis is induced by a neurotoxin that is transmitted to the host in the saliva of a female I. holocyclus when the tick takes a blood meal. During feeding, toxicity in the salivary glands increases, peaking after 4-5 days of engorgement [8]. The treatment of paralysed hosts requires removal of the parasite and
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