Avaliação da malato desidrogenase citoplasmática para o diagnóstico de Trypanosoma congolense

Abstract

African trypanosomiasis is a hemoparasitosis transmitted by the tsetse fly that affects man and animals, causing great economic losses. This disease is caused by protozoa belonging to the genus Trypanosoma, with T. congolense, T. vivax and T. brucei spp. the most important species. Animal trypanosomiasis in Africa is responsible for high losses in livestock and in the economy in general. The main measure of control is chemotherapy and chemoprophylaxis, although limited by the number of trypanocides available, and the always increasing existence of resistance. Thus, diagnosis becomes an important tool because it allows the design of adequate treatment; however, the existing methods are not satisfactory. Mostly based on the detection of circulating antibodies, they cannot differenciate between active infection and recent but passed infection following treatment or self-cure. In this context, room for the development of a circulating antigen capture ELISA exists, which would detect active infections, with the added advantage to be open to adaptation into a dipstick format. Cytoplasmic malate dehydrogenase (cMDH) was chosen as candidate antigen due to its assumed abundance in host bloodstream after trypanosome lysis, and the possibility of enzymatic detection. In order to express the recombinant form of cMDH, the coding sequence of the protein was amplified by PCR and cloned into the T-vector pTZ57R/T, sequenced, and subcloned into the expression vector pET32. Expression was realized in the bacteria Escherichia coli strain BL21 (DH3), under the inducible promoter T7. After induction by IPTG a 50 kDa soluble protein was produced, fused to a His.Tag. The recombinant protein was purified by affinity chromatography based on Nickel chelation and used to immunize two rabbits and two chickens to raise polyclonal antibodies, which were used to develop a capture ELISA test. Antibodies raised in rabbit and chicken showed on an immunoblot the ability to recognize native T. congolense cMDH present in a trypanosome lysate, indicating a potential to also detect the presence of circulating cMDH in sera from infected cattle. Indeed, a sandwich-ELISA was developed, using the IgY as capture antibodies, and the rabbit serum as revelating antibodies that 6Avaliação do potencial antigénico da enzima malato desidrogenase citoplasmática para uso em testes de captura para o diagnóstico de Trypanosoma congolense not only could capture circulating cMDH in the serum of T. congolense-infected cattle, but also showed a positive correlation between the intensity of the test and the level of parasitaemia. This study demonstrates that the polyclonal antibodies produced are able to capture native cMDH in serum from T. congolense infected animals with different levels of parasitemia. These results suggest that the cMDH capture system that we developed can be validated as a test for the field diagnosis of T. congolense infections