Vaccine testing of a recombinant activation-associated secreted protein (ASP1) from Ostertagia ostertagi.

Previous vaccination trials against the economically important cattle parasite Ostertagia ostertagi have indicated the protective capacity of activation-associated secreted proteins (ASPs). The further development of these antigens into a commercial vaccine will require their recombinant expression. The aim of the current study was to clone and express Oo-asp1 in a baculovirus expression system and to evaluate the protective capacity of the recombinant protein against an O. ostertagi challenge infection in cattle. The full coding sequence of Oo-asp1 was cloned in a baculovirus expression vector in frame with a carboxy-terminal Histidine tag and recombinant virus was used to infect an insect cell culture. Western blot analysis with anti-His and anti-Oo-ASP1 antibodies showed the production of recombinant Oo-ASP1. The cell pellet containing the recombinant was subsequently used to immunize seven calves three times intramuscularly with QuilA as adjuvant. Control animals were solely injected with the QuilA adjuvant. The challenge infection with O. ostertagi consisted of 30,000 L3 larvae per animal given over 30 days (1000 larvae/day, 5 days/week) and started the same day as the final immunization. Immunization with the recombinant Oo-ASP1 did not result in any level of protection against the challenge infection. There was no reduction in faecal egg output or in worm burdens. Moreover, Western blot analyses and ELISA indicated that, although the animals raised an antibody response against the recombinant Oo-ASP1, there was hardly a response against the native Oo-ASP1, suggesting that the baculovirus expressed recombinant was wrongly folded or lacked essential secondary modifications. Further analysis of the structure of the native ASPs and their glycosylations is being done.

Gender-enriched transcription of activation associated secreted proteins in Ostertagia ostertagi.

Activation associated secreted proteins (ASP) are members of a nematode-specific protein family belonging to the SCP/Tpx-1/Ag5/PR-1/Sc7 family. Three different types of molecules have been identified in this family: two-domain ASPs and single-domain ASPs showing homology to either the C-terminal or N-terminal domain of the two-domain ASP. The function of these proteins is still unclear, but a role in transition to parasitism and a role as allergen are often suggested. Here we report that the abomasal cattle parasite Ostertagia ostertagi produces at least 15 ASPs, including two-domain and C- and N-type single-domain ASPs. Ten of these are highly transcribed in the L4 stage, whereas others are highly enriched in adult male worms. The latter was especially the case for the N-type single-domain ASPs Oo-ASP1 and Oo-ASP2 and also for Oo-ASP3, which is homologous with the Haemonchus contortus and Ancylostoma caninum C-type single-domain ASPs. Immunohistochemistry showed that Oo-ASP3 was localised in the oesophagus. Oo-ASP1 and Oo-ASP2 on the other hand were localised in the reproductive tract of both male and female worms, suggesting a role in reproduction or in the development of the reproductive tract.

Vaccination against Ostertagia ostertagi with subfractions of the protective ES-thiol fraction.

Previous vaccination trials against Ostertagia ostertagi in cattle have demonstrated the protective capacity of a protein fraction termed ES-thiol, which is enriched for activation-associated secreted proteins (ASPs) and cysteine proteases. In this study, ES-thiol was subfractionated through Q-Sepharose anion exchange chromatography to determine whether the ASPs and/or the cysteine proteases are responsible for the induced protection. Calves (seven/group) were immunized three times intramuscularly with 100 microg of ES-thiol or equivalent amounts of an ASP-enriched fraction, a cysteine protease-enriched fraction or a rest fraction, with QuilA adjuvant. A negative control group only received QuilA. After the final immunization the animals were challenged with a trickle infection of 25,000 infectious L3 larvae (1000 L3/day; 5 days/week). During a 2-month period the geometric mean cumulative faecal egg count (FEC) of the ES-thiol group was reduced by 62% compared to the QuilA control group (P<0.05). Groups injected with the ASP-enriched, the cysteine protease-enriched and the rest fraction demonstrated a reduction in cumulative FEC of 74, 80 and 70%, respectively (P<0.01). Although no significant reductions in worm burdens were observed, adult male and female worms were significantly smaller in all vaccinated groups (P<0.05), except for male worms from the ES-thiol group. These results suggest the protective capacity of ASPs and the presence of other protective antigens in the ES-thiol fraction.