Desenvolvimento de métodos imunoquímicos e moleculares para o diagnóstico da neosporose

Abstract

Neospora caninum is a protozoan phylogenetically related to several coccidia with importance in veterinary medicine. This obligate intracellular parasite causes abortions in cattle and neoromuscular paralysis in dogs. Interaction ligand/receiver are required that allow the parasite explore your ability invasive, being the initial contact mediated for immunodominant surface proteins as Ncp43 and Nc-p29. The study of antigens and recombinant proteins of N. caninum have generated a serie of informations aiming the improvement of diagnosis and differentiation of this protozoan and other agents related to it, especially Toxoplasma gondii.In the present work was carried out a literature review focusing in the proteins involved in host-parasite interaction used as vaccine targets and application in diagnostic tests due to its specificity. Among the described proteins, antigens of surface Nc-p43 and Nc-p29 are widely studied due to their specifity and ability to induzer a protective immunity, and not reported cross reaction with other Apicomplexa parasite when used in trials diagnostics. Furthermore, the expression of specific proteins of N. caninum, Ncp43 and Nc-p29, in prokaryote system, are here described, followed by their evaluating concerning their antigenicity, using sera from animals naturally infected with N. caninum; and their specificity through reaction with sera from animals infected with T. gondii. The rNc-p43 protein was used for polyclonal antibody production in rabbit, purified and conjugated to peroxidase (HPR) and fluorescein isothiocyanate (FITC), in order to detect the recombinant and native Nc-p43, respectivelly. pAb and pAb/HRP were able to recognize rNc-p43, which was evaluated by Dot blot and ELISA assays, while pAb/FITC was able to mark the apical complex of tachyzoites. A blocking enzyme-linked immunosorbent assay (b-ELISA) was performed to evaluate the performance of pAb/HRP as diagnostic tool. The average percentage of inhibition for the positive sera pool and the negative sera pool of cattle neosporosis was significantly different (p <0.0001). These results suggest that pAb may bind to the same epitopes of Ncp43 and anti-N. caninum antibodies positive samples tested. b-ELISA using pAb/HRP is an interesting option for diagnostic tests that are available for neosporosis since fewer steps are involved in their implementation and their shape prevents cross-reactivity with anti-species-specific antibodies. In short, this work describes the cloning and expression of Nc-p43 and Nc-p29 proteins N. caninum in prokaryotic system, to produce a monnoespecific polyclonal antibody against the recombinant protein Nc-p43 and evaluation of its suitability as a tool in diagnosis of neosporosis.