The Study Of E.tenella Perforin-like Proteins And The MRNA Expression Changes For Perforin,granzyme A And Granulysin In The Chicken Gut Upon E.tenella Infection

Avian coccidiosis caused by Genus Eimeria is an economically important disease in intense poultry production,in which Eimeria protozoa parasite in chicken intestinal epithelial cells for their asexual multiplication,then destroy host cells.Eimeria tenella is the most common and pathogenic parasite in the global poultry production.When E.tenella infect chickens,it can synthesize or secret some functional proteins for triggering or evading the host immune responses.Of then,intestinal cytotoxic T lymphocyte cells can kill E.tenella via perforin/granzyme pathway,however,on the other side,E.tenella,and other Eimeria protozoa can also produce perforin-like proteins for their invasion to host cells and/or egress from host cells.Previous research results have shown that perforin-like proteins?PLPs?play important roles in parasitic protozoan life cycle in host cells.Toxoplasma gondii PLP can destroy parasitophorous vacuoles membrane?PVM?and host cell membrane and facilitate the invasion of T.gondii.No reports about Eimeria tenella perforin-like proteins?EtPLPs?and their roles in infection and/or host immune responses have been released.In this study,the full-length EtPLPs gene were cloned and recombinant expressed for immunofluorescence detection.The transcription level of EtPLPs at different developmental stages were also analyzed.Furthermore,the transcription levels of perforin,granzyme A and granulysin in the intestine and caecum of chicken infected with E.tenella were dynamically evaluated with qRT-PCR.The main results of these studies are following:The genes for coding EtPLP₁?1932 bp?and EtPLP₂?4215 bp?were cloned in full length.Then the fragments of these two genes encoding their antigenic epitopes were recombinant expression in E.coli for producing the antibodies.Based on these antibodies,the subcellular localization of EtPLP1were visualized at the apical position.The dynamic expression patterns of EtPLP1 and EtPLP2 during E.tenella life cycle were revealed using qRT-PCR methods.The peak expressions for EtPLP₁ and EtPLP₂ were in sporozoite and first-generation merozoite of E.tenella,and the secondly high expression for EtPLP₁ and EtPLP₂ were sporulated oocyst and second generation merozoite respectively.The expression levels of perforin,granzyme A and granulysin in cecum and small intestine of chickens infected with E.tenella were detected by qRT-PCR.During primary E.tenella infection,granulysin,perforin and granzyme A mRNA expression in caecal tissue was significantly increased at 10days post infection,compared to uninfected birds.In contrast,perforin,granzyme A and granulysin mRNA expression in small intestine tissue was no significantly change E.tenella infection.Our results primarily showed that perforin of E.tenella could play important roles in the pathogenesis of chicken coccidioides,and the perforin-granzyme-granulysin system could take part in the immunoprotection against Eimeria infection of chickens.