Sheep that have been immunized by multiple truncated infections with the parasitic nematode Trichostrongylus colubriformis contain anti-larval activity in their intestinal mucus and high-speed mucus supernatants. This activity induces T. colubriformis L3 to clump in vitro and causes a significant reduction in larval establishment in naive sheep after infusion of larvae and mucus into the intestinal lumen via a duodenal cannula. In this report, we provide evidence that one factor contributing to the anti-larval activity of immune mucus is antibody against a 35-kDa L3-specific cuticular antigen. The anti-larval activity in mucus is > 100 kDa by membrane filtration, is heat labile and sensitive to either protease digestion or reduction with DTT. Immunoblotting showed that mucus and supernatants of ultracentrifuged mucus from immune sheep contained IgG1 and IgA antibodies that recognized predominantly a larval antigen with an estimated molecular weight of 35 kDa on SDS-PAGE. Antibodies eluted from the surface of washed larvae that had been incubated in immune mucus also reacted specifically with the 35 kDa antigen on blots of larval homogenate. Immunofluorescence and immunogold electron microscopy showed that the 35 kDa antigen is present on the epicuticle of L3 and is shed during the moult to L4. The antigen is not present in eggs, L1, L2, L4 or adult worms and is found only in extracts of sheaths and L3 before infection and up to 4 days after infection. We hypothesize that the binding of antibody to the larval surface prevents larvae from establishing at their preferred site, causing them to be eliminated from the intestine. Monoclonal antibody PAB-1 recognizes the 35 kDa T. colubriformis larval antigen and also cross-reacts with antigens of similar molecular weight on blots of L3 extracts of the parasitic nematodes Haemonchus contortus and Ostertagia circumcincta; and with a 22-kDa antigen on blots of L3 extracts from Cooperia curticei and Nematodirus spathiger. This indicates that an antigenically related surface antigen with immunizing potential is present on several nematode species and can be identified by mAb PAB-1. The 35 kDa T. colubriformis larval antigen and related molecules in other nematodes are potential novel targets for stimulating host-protective immunity against nematode infections.

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