Preliminary Study On The Molecular Mechanisms Of Egress-mediated Clearance Of Eimeria Spp.

Apicomplexan parasites are obligate intracellular pathogens which undergo complex intracellular life cycles consisting of host cell entry, intracellular development and propagation and egress. Among these processes, egress from the host cells is a pivotal step for apicomplexan parasites since it enables parasites shuttle into neighboring cells for further development. Recendy, as researches continue, the study on egress of apicomplexan parasites induced by immune factors has attracted attentions from many research groups. Our previous study in 2011 found that co-culture of Eimeria tenella sporozoites-infected PCKs with lymphocytes from E. tenella infected chickens could trigger egress of the parasites. Based on this study, we hypothesized that some immune factors could induce egress of sporozoites from infected cells and the egressed parasites could be cleared by another immune factors, which may be another way to eliminate eimerian parasites as important as the way of CTL and autophage in host immune system. In this study, we attempted to study on the molecular mechanisms of egress-mediated clearance of Eimeria spp. Firstly we established a system to analyze the egress of sporozoites in vitro, and then explained the mechanism of egress of eimerian parasites preliminarily using E. tenella sporozoites as model. Results showed that the egress of sporozoite was depended on the development stage of the parasite in host cells which provided the reference of choosing detection time in vivo. Further study discovered that calcium played a vital role during egress occurrence which gave a basis for selection immune factors that have the effect on inducing egress of sporozoites. Furthermore, we found that exogenous nitrc oxide could trigger egress of many species of eimerian sporozoites from infected-PCKs. Further results showed that nitric oxide-induced egress of E. tenella sporozoites had no host cells specific and compared to the freshly isolated sporozoites, the re-invading ability and reproductivity of the egressed parasites significantly decreased by 67.2% and 52.4% individually. Then we co-cultured the egressed parasites with chicken peritoneal macrophages and found that the egressed sporozoites could be captured and killed by macrophages.Next, we stepped into in vivo study based on the foundings obtained from in vitro experiments. For conducting in vivo experiment, establishing a reliable test method was the primary issue. We successively tried to introduce several ways to reflect the egress occurrence in vivo. The methods were that detection the gene expression level of egress-specific protein of the parasites and quantification of the number of sporozoites by Real-Time PCR, quantification of the number of the sporozoites by paraffin sections stained with mixture of aniline blue-orange G and obtaining the qualitative data by paraffin sections with immunohistochemical staining. The results suggested that the methods based on Real-Time PCR were not feasible for our study. The orange G staining showed that the sporozoites number in cecum of vaccined and challenged group was significantly lower than unvaccined and challenged group. Meanwhile, the immunohistochemical staining showed that the proportion of the intact parasitophorous vacuoles without parasites in cecum of vaccined and challenged group was significantly more than vaccined and challenged group and the free sporozoites were hardly detected in vaccined and challenged group. These results suggested that there existed the pathway of egress-mediated clearance of sporozoites in vivo posiibly.In conclusion, our findings demonstrated the possibility that the phenomenon of egress-mediated clearance of eimerian sporozoites existed in vivo. In following study, more credible methods should be involved into in vivo experiments to obtain more reliable data, and to confirm the existence of the pathway of egress-mediated clearance of sporozoites in host immune system finally.