RESUMO
It is hypothesized the existence of marker antigens capable to define early (acute phase) or late (convalescent phase) infection course with Anaplasma marginale. Five parasitologically free hybrid calves were splenectomized and experimentally infected with a Zulia isolate of Anaplasma marginale. Sera samples were collected before inoculation and during the overall period of the infection. The course of infection was followed through Giemsa stained blood smears and by packed cell volume (PCV). The period of infection was divided into the prepatent phase in which no apparent infection was evident, an acute phase in which a rapid increase of the parasitemia and the hematocrit reduction were conspicuous and a convalescent phase subjectively considered as a marked decrease in parasitemia, after treatment of the infected animals with Oxytetracycline. Western blot assays and immunoplot analyses were performed. The frequency analysis of the detected antigens showed that some of them reacted with normal or prepatent sera, being non-specific cross reaction. Band 63 (p65 kDa) was specific in the acute phase. Bands 188; 68 (p70 kDa) and 58 (p60 kDa) were recognized mainly in the late or convalescent phases. These results also showed that band 18 (p19 or MSP5) was a marker for specific infection with Anaplasma.
Se hipotetiza que existen antígenos marcadores capaces de definir el curso de la infección con Anaplasma marginale, tanto durante la fase temprana (aguda) como la tardía de recuperación (convalecencia) de la infección. Cinco becerros, mestizos, parasitológicamente negativos a A. marginale, fueron esplenectomizados y experimentalmente infectados con un aislado Zulia de A. marginale. Las muestras de suero fueron colectadas antes de la inoculación y durante todo el periodo de la infección. El curso de la infección fue monitoreado a través de la determinación del hematocrito y de la parasitemia usando frotis sanguíneos teñidos con colorante de Giemsa. El periodo de la infección fue dividido en: fase prepatente, en la que la infección no era evidente, fase aguda en la que un rápido incremento de la parasitemia y reducción del hematocrito son conspicuas y la fase convaleciente, después del tratamiento del animal con oxitetraciclina y en la que se evidencia un claro descenso de la parasitemia. Los sueros colectados fueron usados para los ensayos de Inmunotransferencia y el análisis de las frecuencias de reconocimiento por inmunoplot. El reconocimiento de cada molécula mostró que hay antigenos que reaccionaron con sueros normales o prepatente, es decir, los antigenos de reacción cruzada no específica. Los antigenos marcadores específicos son la banda 63 (proteína p65 kDa) para la fase aguda y las bandas 188; 68 (p70 kDa) y 58 (p60 kDa) para la fase convaleciente. Los resultados obtenidos demuestran también que la banda 18 (p19 kDa o MSP5) es un marcador especifico de infección con Anaplasma.
RESUMO
Proteolysis of endogenous proteins may play a key role in the adaptation of T. cruzi to the different host environments to which it is exposed during its complex life cycle. For this reason, we have attempted to study the intracellular pathways of protein degradation in the non infective epimastigotes form (EP strain) of T. cruzi. Following intracellular proteolysis by pulse chase experiments with 35 S methionine, we observed a significant inhibition (50 percent) of the degradation of endogenous proteins in log phase parasites in the presence of inhibitors of lysosomal functions, such as chloroquine and E 64. A significant increase in proteolysis was observed in stationary phase parasites which was reverted to log phase values by supplementing the chase medium with 0.5 percent glucose or 10 percent serum, or in the presence of chloroquine. Under this condition of nutritional stress, we could observe an increase in the activity of acid proteases. A significant increase in the degradation rates was observed when abnormal proteins were induced in the parasite by amino acid analogs and puromycin. This increase was not affected by E 64, suggesting the participation of non lysosomal mechanisms in the degradation of rapidly degradable abnormal proteins. Under these conditions, we could observe an increase in high molecular weight conjugates of ubiquitin with respect to endogenous proteins. These results suggest the importance of lysosomal mechanisms in the degradation of cellular proteins in nutritional optimal conditions and during nutritional deprivation, and the possible involvement of the ubiquitin system in the degradation of high turnover proteins