A Recombinant Attenuated Salmonella enterica Serovar Typhimurium Vaccine Encoding Eimeria acervulina Antigen Offers Protection against E. acervulina Challenge

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Abstract
Coccidiosis is a ubiquitous disease caused by intestinal protozoan parasites belonging to several distinct species of the genus Eimeria . Cell-mediated immunity (CMI) is critically important for protection against Eimeria ; thus, our approach utilizes the bacterial type III secretion system (TTSS) to deliver an antigen directly into the cell cytoplasm of the immunized host and into the major histocompatibility complex class I antigen-processing pathway for induction of CMI and antigen-specific cytotoxic T-lymphocyte responses in particular. To accomplish this goal, Eimeria genes encoding the sporozoite antigen EASZ240 and the merozoite antigen EAMZ250 were fused to the Salmonella enterica serovar Typhimurium effector protein gene sptP in the parental pYA3653 vector, yielding pYA3657 and pYA3658, respectively. SptP protein is secreted by the TTSS of Salmonella and translocated into the cytoplasm of immunized host cells. The host strain chromosomal copy of the sptP gene was deleted and replaced by a reporter gene, xylE . The newly constructed vectors pYA3657 and pYA3658 were introduced into host strain χ8879 (Δ phoP233 Δ sptP1033 :: xylE Δ asdA16 ). This strain is an attenuated derivative of the highly virulent strain UK-1. When strain χ8879(pYA3653) as the vector control and strain χ8879 harboring pYA3657 or pYA3658 were used to orally immunize day-of-hatch chicks, colonization of the bursa, spleen, and liver was observed, with peak titers 6 to 9 days postimmunization. In vitro experiments show that the EASZ240 antigen is secreted into the culture supernatant via the TTSS and that it is delivered into the cytoplasm of Int-407 cells by the TTSS. In vivo experiments indicate that both humoral and cell-mediated immune responses are induced in chickens vaccinated with a recombinant attenuated Salmonella serovar Typhimurium vaccine, which leads to significant protection against Eimeria challenge.