Immunisation of cattle with cysteine proteinases of Trypanosoma congolense: targetting the disease rather than the parasite.

In order to test the hypothesis that trypanosome cysteine proteinases (CPs) contribute to pathology of trypanosomosis, cattle were immunised with CP1 and/or CP2, the major CPs of Trypanosoma congolense, and subsequently challenged with T. congolense. Immunisation had no effect on the establishment of infection and the development of acute anaemia. However, immunised cattle, unlike control cattle, maintained or gained weight during infection. Their haematocrit and leukocyte counts showed a tendency to recovery after 2-3 months of infection. Cattle immunised with CP2 mounted early and prominent IgG responses to CPs and to the variable surface glycoprotein following challenge. Thus trypanosome CPs may play a role in anaemia and immunosuppression; conversely, anti-CP antibody may modulate the trypanosome-induced pathology.

Antibody responses to invariant antigens of Trypanosoma congolense in cattle of differing susceptibility to trypanosomiasis.

Five trypanotolerant N'Dama (Bos taurus) and five susceptible Boran (Bos indicus) cattle were challenged by tsetse flies infected with Trypanosoma congolense IL 13-E3. These animals had experienced five previous infections with T. congolense, each terminated by drug therapy. Immunoblotting and ELISA were used to determine isotype and specificity of antibody responses to trypanosome invariant antigens. Both IgM and IgG1 were elicited, but the IgG1 responses were directed against a greater diversity of antigens. A 69 kD antigen was the major invariant antigen which elicited IgM antibodies in both breeds, but the N'Damas also responded with high levels of specific IgG1. Analysis of isotypic responses to whole trypanosome extract also revealed lower levels of IgG1 and higher levels of IgM in the Borans than in the N'Damas, suggesting that a dysfunction in the switch from IgM to IgG might occur in infected Boran cattle. A 33 kD antigen appeared to elicit only IgG1. Sera from all five N'Damas and the two Borans which were most resistant to the disease reacted with this antigen prior to and following re-infection. Furthermore, during the primary T. congolense infection in the same animals, anti-33 kD antibodies were detectable in all five trypanotolerant N'Damas, but in none of the five susceptible Borans. Thus, the presence of antibodies to the 33 kD antigen of T. congolense appeared to be associated with a capacity to control the disease.

Identification of a 33-kilodalton immunodominant antigen of Trypanosoma congolense as a cysteine protease.

A 33-kDa protein of Trypanosoma congolense is a major antigen in infected cattle and the production of antibody to this antigen appeared to correlate with enhanced resistance to trypanosomiasis [4]. Immunoelectron microscopy using a monoclonal antibody (mAb 4C5) raised against the 33-kDa antigen showed a lysosomal localisation, similar to that of a previously described 32-kDa cysteine protease of T. congolense. Both mAb 4C5 and anti-33 kDa antibody from infected cattle bound on Western blots to the cysteine protease that had been purified by affinity chromatography on cystatin-Sepharose. Sepharose-coupled mAb 4C5 was used to affinity purify the antigen from bloodstream forms of T. congolense. On sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), the affinity-purified antigen had a molecular mass of 33 kDa under non-reducing conditions, and 40 kDa under reducing conditions. Anti-33-kDa antibody from infected cattle bound to both non-reduced and reduced affinity-purified antigen on Western blots. Serum from a rabbit immunised with the biochemically purified enzyme also bound the affinity-purified antigen. The affinity-purified antigen displayed proteolytic activity in fibrinogen-containing SDS-PAGE and against Azocoll. It hydrolysed benzyloxycarbonyl-Phe-Arg-7-amino-methyl coumarin (Z-Phe-Arg-NHMec) with a Km similar to that of the biochemically purified enzyme. Proteolytic and peptidolytic activities of the antigen were inhibited by the inhibitors of cysteine proteases, cystatin and trans-epoxysuccinyl-L-leucyl-amido (4-guanidino)butane (E-64). On two-dimensional gel electrophoresis, the antigen displayed similar characteristics to those of the biochemically purified enzyme. We conclude that the 33-kDa antigen of T. congolense and the cysteine protease are the same molecule.