title: Characterisation of Haemonchus contortus-derived cell populations propagated in vitro in a tissue culture environment and their potential to induce protective immunity in sheep.
abstract: 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.
authors: Veterinary Pharmacology Research Laboratory, Veterinary Research Program, College of Veterinary Medicine, Mississippi State University 39762, Mississippi, MS, USA. coyne@cvm.msstate.edu
authors: C P
authors: CP
authors: Coyne
authors: D
authors: D
authors: Brake
mesh_terms: D000018:Abomasum / Q000469:parasitology; D000818:Animals; D000909:Antibodies, Helminth / Q000096:biosynthesis / Q000097:blood; D000947:Antigens, Helminth / Q000276:immunology / Q000302:isolation & purification; D018826:CD13 Antigens / Q000032:analysis / Q000378:metabolism; D018929:Cell Culture Techniques; D004591:Electrophoresis, Polyacrylamide Gel / Q000662:veterinary; D004797:Enzyme-Linked Immunosorbent Assay / Q000662:veterinary; D006188:Haemonchiasis / Q000276:immunology / Q000517:prevention & control / Q000662:veterinary*; D006189:Haemonchus / Q000276:immunology*; D007150:Immunohistochemistry / Q000662:veterinary; D010744:Phosphoric Monoester Hydrolases / Q000032:analysis / Q000378:metabolism; D012756:Sheep; D012757:Sheep Diseases / Q000276:immunology* / Q000469:parasitology / Q000517:prevention & control
chemical_list: D000909:Antibodies, Helminth; D000947:Antigens, Helminth; D010744:Phosphoric Monoester Hydrolases; D018826:CD13 Antigens