The Establishment Of DF-1 Cell Culture Model For Eimeria Tenella And Preliminary Founctional Studies Of EtSerpin And EtAMA1

Avian coccidiosis causes severe economic losses on the poultry industry, which is infected by Eimeria spp. The normal control strategies have relied on prophylactic medication and immunization with live vaccines now. But due to the danger of live vaccines' transmission and the drug resistant emergence of the parasites, Novel approaches are urgently wanted to study on the coccidiosis control. The cell culturing procedure of coccidia has facilitated the detailed study of the mechanism(s) underlying anticoccidial drug effects and the development of coccidia.1. The establishment DF-1 cell culture model for E. tenellaIn order to search a novel cell culture system for the development of coccidia, DF-1 was firstly used in vitro culture of coccidia. The sporozoites could invade the DF-1 cells within 24 h p.i. and develop into mature first generation schizonts at 60 h p.i. . After incubated for 72 h p.i. , the free merozoites could be observed in the cell culture. And the sporozoites developed better and faster in DF-1 cells at 41℃in 5% CO₂ than at 37℃in 5% CO₂. Sporozoites of E. necatrix, E. maxima and second generation merozoites of E. tenella purified from chicken caecum were separately inoculated into DF-1 cell, only sporozoites of E. necatrix developed into first generation merozoites at 72 h p.i. . Sporozoites of E. maxima could invade the DF-1 cell but not develop. No second generation merozoites could be observed in DF-1 cells and develop into oocysts.2.Application of DF-1 cell culture for the invasion activity assay of sporozoites.DF-1 cell line was used for analyzing the invasion activity of sporozoites. Purified sporozoites of E. tenella were irreversibly labeled by the dye carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE) and infected DF-1 cells, then cells were collected and analyzed by flow cytometry. The infected cells, non-infected cells, and free sporozoites were gated using the software RXP for subsequent delineation and counting of the infected (containing the labeled sporozoites) and non-infected (fluorescence-free) cells. thus a reliable detection method was established. Assessed the ability of DF-1, MDBK, VERO, BHK, MDCK to support sporozoites invasion in vitro at 37℃and 41℃, the DF-1 cell line supported the highest degree of invasion. To obtain the best invasion rates of sporozoites in DF-1cells, the culture conditions were optimized as the following: 1×105 DF-1 cells per well were seeded into 24 well plates in DMEM medium containing 10% fetal calf serum and incubated at 37℃, in 5% CO₂ for 24 h. 3×10⁵ labeled sporozoites were used to infect in DMEM medium containing 5% fetal calf serum. After 8¹2 h of culture at 41℃, in 5% CO₂, the infected cells were collected for assay.3. Bioinformatics and cloning of Serpin and AMA1 gene from E. tenella Serpin and AMA1 were identi?ed from the common poultry parasite E. tenella by expressed sequence tag (EST) analysis and the rapid ampli?cation of cDNA ends (RACE) technique. The full-length cDNA of EtSerpin was 1 918 bp (GenBank accession number: HQ830031), and had an open reading frame (ORF) of 1 248 bp encoding a polypeptide of 414 amino acids with the theoretical isoelectric point of 5.26 and predicted molecular weight of 45.5 kDa. SignalP program analysis revealed that the protein most likely contains a signal peptide of 28 amino acids. A Serpin domain was identified in the amino acid sequence. Selected Serpin genes of 8 species to construct phylogenetic tree with the EtSerpin and the result showed that EtSerpin was closer to the Serpin of T. gondii in evolution. The cDNA of EtAMA1 was 2 349 bp (GenBank accession number: JN032081), and had an open reading frame (ORF) of 1 608 bp encoding a polypeptide of 535 amino acids with the theoretical isoelectric point of 5.79 and predicted molecular weight of 54.59 kDa. SignalP program analysis revealed that the protein most likely contains a signal peptide of 23 amino acids. The protein with a AMA1 domain was composed with a extracellular region(1⁴46 aa), a transmembrane region(447⁴69 aa) and a intracellular region (470⁵35 aa). Selected AMA1 genes of 8 apicomplexan parasites to construct phylogenetic tree with the EtAMA1 and the result showed that EtAMA1 was closer with the AMA1 of E. maxima, T. gondii and N. caninum in evolution. Real time quantitative PCR analysis revealed that the EtSerpin and EtAMA1 genes were all expressed at higher levels in sporozoites than in the other developmental stages (unsporulated oocysts, sporulated oocysts and second-generation merozoites).4. Expression and and preliminary founctional studies of EtSerpin geneThe EtSerpin gene sequence encoding the mature protein was amplified by PCR, cloned into the pET28(a) vector and expressed in E. coli. The purified recombinant protein (rEtSerpin) showed a good antigenicity and a chymotrypsin activity. Anti-serum of rEtSerpin obtained from rabbits could identify natural Serpin protein (45.5 kDa) in the parasite, which mean the serum could be used to study the function of EtSerpin. The purified rEtSerpin antibody inhibited the invasion capacity of E.tenella sporozoites in DF-1 cells. The antibody was also applied to detect the localization of EtSerpin during ?rst schizogony in DF-1 cells. The results showed EtSerpin concentrated on the apical end when sporozoites invaded the cells and secreted into the host cells after developed, and the EtSerpin was also abundantly expressed on the surface of trophozoites and schizonts. The localization of EtSerpin was also shown by immunofluorescence during the development of sporozoites in the caeca. We found that EtSerpin had increased expression on the surface of trophozoites and schizonts which look like forming a barrier between the host cells and the parasites, and the protein also involved in forming the wall of oocysts. Chickens inmmuned with rEtSerpin were slightly better than the control group when the chickens were infected with sporulated oocysts. Although the lesion score artery score was not more significant than control group, in view of the oocyst production, the immune group had significant difference than control group, and 60μg purified recombinant protein was better than 30μg protein. The level of antibodies was detected by ELISA and showed antibodies of rEtSerpin was produced after chickens inmmued one time and could last a long time.4. Expression and preliminary founctional studies of EtAMA1 geneThe EtAMA1 gene sequence encoding the ectodomain sequence was amplified by PCR, cloned into the pGEX-6P-1 vector and expressed in E. coli. The purified recombinant protein (rEtAMA1) showed a good antigenicity and inmmued rabbits to obtain anti-serum. The purified rEtAMA1 antibodies inhibited the invasion capacity of sporozoites in DF-1 cells. Anti-rEtAMA1 antibodies were applied to detect the localization of EtAMA1 during ?rst schizogony in DF-1 cells. The results showed EtAMA1 concentrated on the apical end when sporozoites invaded the cells and also abundantly expressed on the surface of trophozoites; but when trophozoites developed into immature schizonts, the expression of EtAMA1 was reduced; and when the parasites developed into mature schizonts, EtAMA1 mainly on the surface of merozoites; When merozoites released from schizonts, EtAMA1 mainly concentrated on the apical end of the parasites, and some residual EtAMA1 was also found on the surface of schizont. The localization of EtAMA1 was also shown in the chickens caeca. The results showed that when sporozoites adhered to the caecal epithelial cells, a amount of EtAMA1 expressed on the sporozoites'surface; when sporozoites developed into first generation schizonts, EtAMA1 expressed on the surface of parasites; when first generation merozoites released, the expression of EtAMA1 focused on the apical end of merozoites; when parasites developed into second generation schizonts, EtAMA1 concentrated on the surface of merozoites; when the parasites developed into third generation schizonts (or gametocytes), EtAMA1 exppressed on the membrance of schizonts (or gametocytes) and distributed with a small defined region in schizonts; when parasites entered into sexual reproduction stage, the expression of EtAMA1 was significantly increased in microgametocytes and decreased in macrogametes; At the same time, EtAMA1 concentrated in the cytoplasm of fertilized macrogametes as small defined regions and also highly expressed around the membrance of parasites in the contents of caeca. When parasites developed into unsporulated oocysts, EtAMA1 expression was reduced and only some protein around the oocyst wall. Chickens inmmuned with rEtAMA1 were better than the control group when the chickens were attacked with sporulated oocysts, and 100μg purified recombinant protein was better than 50μg protein. The level of antibodies were detected by ELISA and showed antibodies of rEtAMA1 was produced after chickens inmmued for 7 days and could last a long time. Cellular immunity in the caecum was significantly inhibited by the parasites, but the high dose immunization group (100μg) was better than the control group.