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The putative protective antigen, p26/23, from adult Haemonchus contortus was isolated. A soluble extract from adult helminths obtained from the abomasa of hyperinfected (12,000 infective larvae) female Manchego lambs and treated with a mixture of protease inhibitors was subjected to affinity chromatography (hexylgluthatione) to eliminate the enzyme gluthatione S-transferase. The eluate was analyzed by electrophoresis under denaturing and reducing conditions (sodium dodecyl sulfate polyacrylamide gel electrophoresis), electrotransferred to nylon membranes, and assayed by Western blot with sera from immunized lambs. The bands recognized by the lambs' sera corresponding to proteins with a molecular weight of 23-26 kDa (p26/23) were excised, eluted, and separated by reverse-phase chromatography. This allowed the isolation of a single protein, which was expressed in both infective larvae (L3) and the adult stage of the parasite. The first 20 amino acids of the N-terminal end of the purified protein were determined. The partial amino acid sequence revealed a 100% identity with the N terminus of one of the peptides present in the p26/23 immunoprotective fraction previously tested by us against sheep haemonchosis. No significant homology with any reported sequence was found except for the deduced sequence of a hypothetic H.contortus protein (HCC00515). Immunolocalization studies showed that the protein was expressed in the hypodermic chords of the nematode.

Haemonchosis, caused by the abomasal nematode Haemonchus contortus, is a common parasitic disease of sheep. Our previous results showed that a soluble fraction from adult stages of the nematode (p26/23) induced partial protection against challenge. Recombinant DNA technology was applied to obtain a synthetic protein (rHcp26/23). Immunological assays (ELISA, Western blotting, and immunolocalization), using sera from lambs immunized with p26/23, confirmed the identity of the recombinant protein and demonstrated that the synthetic protein is equivalent to the purified protein employed in the previous immunoprophylaxis studies. Vaccination of lambs with 300 μg of rHcp26/23 and Freund's adjuvant elicited a notable specific antibody response. Immunization did not induce any significant protection after challenge with 16000 infective larvae of H. contortus, and comparable values for parasite faecal egg output, packed cell volume, and abomasal parasite burdens were found in vaccinated and control animals.

Individual bands (15) from electroblotted soluble extracts of adult Haemonchus contortus were excised and three peptides of molecular weight ca. 56 (F4), 39 (F8) and 18.5 kDa (F14) used to vaccinate 4-4.5-months-old lambs against the nematode. Immunizing doses from each peptide were administered in 1 ml Freund complete adjuvant (first 50 microg injection) and 1 ml Freund incomplete adjuvant (second and third 50 microg injections) to six lambs. Two weeks after last immunization, animals were challenged with 300 L-3/kg live weight (LW). Lambs were slaughtered 34 days after challenge. Immunization induced a strong antibody response estimated by enzyme-linked immunosorbent assay, whereas no peripheral lymphoproliferative response was observed. Lambs in the F8-vaccinated group showed on average delayed pre-patent period, lower faecal egg counts and reduction of abomasal worm burdens, although the differences were not statistically significant.