Avaliação da vacina de Anaplasma marginale utilizando rMSP1a funcionalizada a nanotubos de carbono, associado ao antígeno inativado produzido in vitro: Parâmetros imunológicos, clínicos e a proteção induzida em bezerros, após desafio experimental

Abstract

Bovine anaplasmosis is an infectious disease caused by intracytocytic rickettsia A. marginale. There is no commercially available inactivated vaccine. Inactivated vaccine produced in IDE8 cells failed due to absence of the MSP1a expression in the rickettsia cultured in this system. Mice immunized with rMSP1a, linked to carbon nanotubes (MWNT), showed significant humoral and cellular responses, indicating that this technology may help in the development of inactivated vaccine. Therefore, the general objective was to evaluate the effect of the immunization of calves with MWNT+rMSP1a, associated with inactivated vaccine of A. marginale produced in vitro, in the induction of immune response, control of rickettsemia and in the development of clinical disease after experimental challenge with heterologous sample A. marginale UFMG1. rMSP1a was expressed in Escherichia coli BL21, purified and covalently linked to MWNTs. Soluble extract of A. marginale (AmUFMG2) was produced by cultivating A. marginale in IDE8 cells. In the Chapter I was evaluated the humoral and cellular response of the calves immunized with the different protocols and possible toxic effects of this immunization. Twenty four holstein calves were divided into four groups and immunized subcutaneously with three doses, 21-21 days, in the following scheme: G1 (control), G2 (AmUFMG2), G3 (MWNT+rMSP1a) and G4 (AmUFMG2 and MWNT+rMSP1a). Blood and plasma samples were collected for total leukocyte counts, evaluation of the immune response and biochemical profile. Immunization with MWNT+rMSP1a induced an increase in the total leukocyte counts, in the number of circulating NK cells, in the T lymphocytes CD4+, CD8+ T, activated T lymphocytes, B cells and high levels of IgG, IgG1 and IgG2 antibodies. Furthermore, MWNTs did not induce changes in the biochemical profile, indicating that the immunization of calves with MWNT+rMSP1a was able to induce significant cellular and humoral response, without generate toxicity. In the Chapter II were evaluated the immunological alterations, clinical and parasitological parameters of calves immunized and experimentally challenged with the heterologous sample A. marginale UFMG1. Fifteen holstein calves were divided into three groups and immunized (via SC) with three doses, 21-21 days, in the following scheme: G1 (control), G2 (MWNT+rMSP1a) and G3 (AmUFMG2 and MWNT+rMSP1a). After immunizations, calves were challenged with 4x107 infected erythrocytes with A. marginale UFMG1. Clinical parameters, such as rectal temperature, packed cell volume, rickettsemia and clinical score were evaluated. Blood and plasma samples were collected for the cellular and humoral response analysis. Calves from G3 showed significant reduction of the levels of rickettsemia, moderate clinical score, better recovery capacity of the packed cell volume and high levels of IgG2. These results suggest that the antigens combination received by the G3 group was able to protect animals from severe score, allowing further approaches to be performed using the inactivated vaccine produced in vitro associated with rMSP1a.