Trypanosoma evansi: Paraflagellar rod protein 1 and 2 are similar but lack common B cell epitopes.

In an attempt to identify invariant proteins with vaccine potential against African trypanosomes, we investigated the existence of PFR1 protein in Trypanosoma evansi and compared its B cell epitope with that of PFR2 protein of T. evansi using Western blotting and immuno-precipitation assays. The PFR1 gene of T. evansi was amplified by RT-PCR using primers designed based on the open reading frame of PFR1 gene of Trypanosoma brucei. The cloned PFR1 gene of T.evansi was similar to PFR1 genes of T. brucei and Trypanosoma cruzi. The expressed protein from the PFR1 gene was 68.4% homologous to the PFR2 protein of T. evansi, and showed 99.8%, 87%, 77.9% and 77.5% homologous to the PFR1 protein of T. brucei, T. cruzi, Leishmania mexicana and Leishmania major, respectively. Western blot and immuno-precipitation assays showed that antibodies raised against PFR1 and 2 proteins in BALB/c mice recognized the PFR1 and 2 proteins, respectively, with no cross-reactivity. Immuno-agglutination assay showed trypanolytic properties of the anti-PFR1, anti-PFR2 and anti-native PFR sera. These results suggest that PFR1 and PFR2 proteins are components of native PFR antigen and do not share common B cell epitopes.

Immunoprotection of chickens against Eimeria acervulina by recombinant alpha-tubulin protein.

Tubulins are heterodimeric molecules responsible for the polymerization of microtubules in apicomplexan parasites. The alpha-tubulin, a subcellular structural protein of Eimeria acervulina, was cloned and expressed in Escherichia coli as an alpha-tubulin-GST fusion protein. Immunogenicity of the recombinant protein was studied in chickens by subcutaneous injection of 50, 100, or 150 microg of the protein with or without Freund's adjuvant. Immunization with 150 microg alpha-tubulin-GST protein in combination with Freund's adjuvant conferred partial protection against E. acervulina oocyst challenge, as shown by a 36% reduction in oocyst shedding, a marked decrease in intestinal lesion score and a significant increase in body weight gain in comparison with the nonimmunized controls. The results suggest that alpha-tubulin protein may be used as an effective vaccine antigen for the control of Eimeria infection.

Transfected Eimeria tenella could complete its endogenous development in vitro.

Live attenuated coccidiosis vaccines could be used as powerful carriers, expressing exogenous viral and bacterial antigens, to induce protective immunity against pathogenic organisms. We investigated the ability of Eimeria tenella to express an exogenous gene in vitro. Eimeria tenella sporozoites were transfected with the plasmid pH4-2EYFP-Actin3 containing the yellow fluorescent protein gene (yfp) and inoculated into primary chicken kidney cells (PCKCs), followed by incubation at 41 C in a 5% CO2 chamber. Fluorescent sporozoites were observed as early as 15-20 hr post-inoculation (PI). Fluorescence displayed by the expressed YFP protein was visible throughout the schizogony and gametogony stages of the tranfected E. tenella. Fluorescent oocysts were found between 200-327 hr PI. Higher fluorescence intensity was observed in the nucleus than in other compartments of the transfectants, while little or no fluorescence was seen in the refractile globule. The diversity of schizonts, particularly of the first generation, was presented by fluorescent nuclei arranged in different patterns. Our results demonstrated the ability of E. tenella to express an exogenous gene throughout the endogenous development in vitro. Completion of the endogenous development of transfected E. tenella in cell cultures will facilitate the study of foreign antigen expression in Eimeria spp., paving the way for the development of an Eimeria spp. vector vaccine that also carries and delivers other vaccines by oral administration.