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Abstract
Branchellion torpedinis is a marine leech that exclusively parasitizes elasmobranchs. The purpose of this project was to explore the pathogenic relationship between this relatively unknown leech species and its elasmobranch host, and to elucidate functional elements of the elasmobranch immune response, induced by parasitism. To examine pathogenesis and extent of disease caused by B. torpedinis, 12 yellow stingrays Urobatis jamaicensis were infected with one or three leeches for 14 days. Leeches induced extensive cutaneous ulceration and were associated with anorexia, decrease in host packed cell volume and serum total solids, and mortality in three rays. Decreases in packed cell volume positively correlated with parasite:host ratios and ulcer size. Decreases in total solids also positively correlated with ulcer size. Host erythrocytes observed microscopically within leech intestine confirmed blood feeding. The salivary gland transcriptome of B. torpedinis was evaluated by extracting mRNA from leech salivary tissue and preparing cDNA for 454 pyrosequencing. Genetic sequences with significant homology to bioactive proteins belonging to the ADAMTS (disintegrin, metalloproteinase and thrombospondin motif) superfamily of proteins, anticoagulants, and immunomodulators, were identified. Putative protein activities correlate with gross and microscopic lesions observed in elasmobranchs at leech feeding sites. Serum IgM responses to leech salivary gland extract were examined retrospectively in captive zebra sharks Stegostoma fasciatum by ELISA and Western blot assays in 20 serum samples from six zebra sharks with a 5 year history of leech infection, and 18 serum samples from 8 captive bred zebra sharks with no history of leech exposure. ELISAs demonstrated significantly higher serum IgM titers to salivary gland extract in exposed zebra sharks compared to the non-exposed population. One-dimensional and two-dimensional Western blot assays revealed IgM targeted specific salivary gland proteins within the 40, 55 and 70 kD range. Antigenic proteins identified by liquid chromatographymass spectrometry and de novo peptide sequencing include an ADAMTS family protein, tubulin, aldehyde dehydrogenase, and two unknown proteins. Results from this study provide much needed information on the pathologic potential and relevance of B. torpedinis in captive elasmobranchs and enhances our understanding of this host-parasite relationship, to facilitate better management of infected animals.