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 L(3) larvae were propagated 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 described for cell types originating from species of plant nematodes propagated in vitro in a tissue culture environment for a briefer period of time (<6 months). The characterisation of extracts harvested from tissue culture populations of H. contortus-derived cells by SDS-PAGE analysis detected molecular fractions of approximately 29, 45, 55, and 200-kDa that closely correlated with reports for preparations obtained from intact/viable H. contortus larvae. Complementary investigations detected the dual biochemical expression of phosphohydrolase and aminopeptidase-M activities based on the hydrolysis of the synthetic enzyme-specific substrates, para-nitrophenylphosphate and leucine-para-nitroanaline, respectively. The identification of phosphohydrolase and aminopeptidase-M-like biochemical activity in fractions harvested from H. contortus-derived cell populations and propagated in vitro in tissue culture served as evidence validating their parasitic-origin. Further validation of H. contortus-derived cell populations propagated in tissue culture entailed the formulation of Triton X-100 extracts containing potential immunoprotective antigens with SEAM adjuvant and its administration by intramuscular injection (100 microg 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-specific IgM and IgG were detected in ovine serum samples. Interpretation of experimental findings revealed that sheep with the greatest antigen-specific humoral immune responses (IgG titre 1/3125) also demonstrated a degree of reduced abomasal H. contortuslarvae burdens (60% reduction). Polyclonal antibody from immunoprotected sheep was subsequently found to recognise both the: (i), digestive tract; and (ii), antigen extracts associated with intact/viable H. contortus larvae. These experimental findings reveal the potential feasibility of propagating parasite-derived cell populations in an in vitro tissue culture environment in a manner that retains their ability to express immunoprotective antigenic fractions.