Characterisation of Haemonchus contortus-derived cell populations propagated in vitro in a tissue culture environment and their potential to induce protective immunity in sheep

Cell populations derived from viable Haemonchus contortus Li larvae were pr opagated in vitro in a tissue culture environment for a prolonged period (> 48 months). Microscopic evaluation of H. contortus-derived cell populations revealed gross morphological characteristics highly analogous to those des cribed for cell types originating from species of plant nematodes propagate d in vitro in a tissue culture environment for a briefer period of time (<6 months). The characterisation of extracts harvested from tissue culture po pulations of H. contortus-derived cells by SDS-PAGE analysis detected molec ular fractions of approximately 29, 45, 55, and 200-kDa that closely correl ated with reports for preparations obtained from intact/viable H. contortus larvae. Complementary investigations detected the dual biochemical express ion of phosphohydrolase and aminopeptidase-M activities based on the hydrol ysis of the synthetic enzyme-specific substrates, para-nitrophenylphosphate and leucine-para-nitroanaline, respectively. The identification of phospho hydrolase and aminopeptidase-M-like biochemical activity in fractions harve sted from H. contortus-derived cell populations and propagated in vitro in tissue culture served as evidence validating their parasitic-origin. Furthe r validation of H. contortus-derived cell populations propagated in tissue culture entailed the formulation of Triton X-100 extracts containing potent ial immunoprotective antigene with SEAM adjuvant and its administration by intramuscular injection (100 <mu>g total protein) to healthy sheep (n = 8) on day 0 (left rear-limb) and day +14 (right rear-limb). Animals on day 28 subsequently received a single oral challenge of 10,000 infective L-3-stage H. contortus larvae. Applying ELISA methodologies. increases in antigen-sp ecific IgM and IgG were detected in ovine serum samples. Interpretation of experimental findings revealed that sheep with the greatest antigen-specifi c humoral immune responses (IgG titre 1/3125) also demonstrated a degree of reduced abomasal H. contortuslarvae burdens (60% reduction). Polyclonal an tibody from immunoprotected sheep was subsequently found to recognise both the: (i), digestive tract; and (ii), antigen extracts associated with intac t/viable H. contortus larvae. These experimental findings reveal the potent ial feasibility of propagating parasite-derived cell populations in an in v itro tissue culture environment in a manner that retains their ability to e xpress immunoprotective antigenic fractions. (C) 2001 Australian Society fo r Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.