| Poultry coccidiosis is an infectious parasitic disease caused by the genus Eimeria.The use of anticoccidial drugs has caused the drug-resistance and drug-residues,so there is an urgent need for immune prevention for replacing anticoccidial drugs to become the dominant way of controlling coccidiosis.The live attenuated vaccines composed of the oocysts of Eimeria precocious strains are the the most widely used coccidiosis vaccine at present,but it has potential adverse effects on the growth of immunized chickens.Previous studies had shown that immunopotentiators could not only improve the immunized efficacy of anti-coccidial live vaccine,but also reduce the adverse effects of vaccination.Compared with the parent strains,the precocious strains of Eimeria have unique biological characteristics such as significantly shortened prepatent period,reduced pathogenicity,and reduced reproductive capacity,but the molecular mechanism of precocity is unknown.In the present study,on the one hand,the effects of levamisole hydrochloride,astragalus polysaccharides,chito-oligosaccharide,bacillus licheniformis and yeast polysaccharide on immunopotentiation to E.tenella and weight gain of immunized chickens were studied by battery test and floor-pen test to obtain the effective dose of each immunopotentiator,which lay the foundation for screening immunopotentiators for anti-coccidial live vaccine.On the other hand,the differences of transcriptome in sporulated oocysts between the E.tenella precocious strain and its parent strain were screened by RNA-seq technology,and the functions and characteristics of two differentially expressed genes,the rhoptry neck proteins 2 and 3(EtRON2 and EtRON3),were preliminarily studied,which lay the foundation for exploring the molecular mechanism of precociousness.1.Evaluation of five immunopotentiators on the effect of immunopotentiation to E.tenella——battery testThe effects of different doses of levamisole hydrochloride,astragalus polysaccharides,chitooligosaccharide,bacillus licheniformis and yeast polysaccharide on immunopotentiation to precocious strain of E.tenella were evaluated by battery test to screen the effective dose of each immunopotentiator.At the age of 3 days,the chicks were first immunized with precocious strain of E.tenella and given immunopotentiators;at the age of 10 days,only the oocysts were used for the second immunization;at the age of 17 days,the chicks were challenged by using virulent strain of E.tenella.The body weight during the immunization period,the ACI after challenge and the serum antibody levels during experiment were used as the evaluation indexes.The results showed that 0.05 mg ~ 0.1 mg of levamisole hydrochloride,2.5 mg ~ 5 mg of astragalus polysaccharides,2.5 mg ~ 7.5 mg of chito-oligosaccharide,1.25 mg of yeast polysaccharides and 10 mg of bacillus licheniformis had the dual effects of immunopotentiation and growth promotion on the chicken infected by E.tenella.2.Evaluation of five immunopotentiators on the effect of immune enhancement to E.tenella——floor-pen testThe effective dose of five immunopotentiators obtained in the battery test was further verified by the floor-pen test.At the age of 3 days,the chicks were immunized with precocious strain of E.tenella and given immunopotentiators;at the age of 24 days,the chicks were challenged by using virulent strain of E.tenella.The body weight during the immunization period,the ACI after challenge and the IgG,IgM,CD4,CD8,TNF-?,TGF-?1,IL-2,IL-4,IL-5 and IL-10 levels during experiment were used as the evaluation indexes.The results showed that 0.1 mg of levamisole hydrochloride could significantly promote the proliferation of chicken CD4 and CD8,the secretion of TNF-? and IL-2.It could also significantly improve the effect of immunopotentiation to Eimeria tenella and had an effect of growth promotion to chickens.2 mg and 6 mg of bacillus licheniformis,5 mg and 7.5 mg of chito-oligosaccharide,and 2.5 mg of yeast polysaccharides could significantly promot the proliferation of chicken CD8 and the secretion of TNF-?,which had the effect of immunopotentiation and growth promotion.5 mg astragalus polysaccharides had an effect of growth promotion to chickens but the effect of immunopotentiation was not significant.The effective dose of astragalus polysaccharides needs further study.3.Transcriptome analysis of the sporulated oocysts between the precocious strain of E.tenella and its parent strainThe precocious strain of E.tenella obtained in our laboratory was used to further precocious selection.After 11 times of selection,the prepatent period of precocious strain decreased from 126 h to 107 h,which was shorten by 19 h.As compared with its homologous parent strain(141 h),the prepatent period of precocious strain was shortened by 34 h.The transcriptome differences of sporulated oocysts between the precocious strain of E.tenella and its parent strain were compared by RNA-seq technology to screen the differentially expressed genes between them.The results showed that a total of 6602 differentially expressed genes were identified,of which 3888 were up-regulated and 2714 were down-regulated.These differentially expressed genes are mainly related to molecular functions such as cationic binding,unfolded protein binding,phosphoric ester hydrolase activity,and participate in endocytosis,ubiquitin mediated proteolysis,glutathione metabolism,protein processing in endoplasmic reticulum and other pathways.22 differentially expressed genes and 5 non-differentially expressed genes obtained from transcriptome data analysis were verified by qPCR and the results showed that the mRNA transcriptional levels of 27 genes were consistent with the results of RNA-seq.This study laid the foundation for exploring the molecular mechanism of the precocious traits of Eimeria.4.Preliminary analysis on the characteristics and functions of rhoptry neck protein 2 and 3 of E.tenellaAccording to the transcriptome analysis results,the characteristics and functions of rhoptry neck protein 2 and 3 of E.tenella(EtRON2 and EtRON3),which were down-regulated expression in precocious strain of E.tenella,were selected for preliminary studies.The results showed that the mRNA transcripts of EtRON2 were the most abundant in the sporulated oocyst,followed by the unsporulated oocyst and merozoite,and the least abundant in the sporozoite.The EtRON2 protein was mainly distributed in rhoptry of the free sporozoites,intracellular sporozoites and second generation merozoites,as well as in the cytoplasm of immature schizonts.The ability of sporozoites to invade DF-1 cells was significantly decreased after incubation with anti-EtRON2 antibody.The mRNA transcripts of EtRON3 were the most abundant in the sporulated oocyst,followed by the unsporulated oocyst,and the least abundant in the sporozoite and merozoite.The EtRON3 protein was mainly distributed in rhoptry of the free sporozoites,intracellular sporozoites and cytoplasm of second generation merozoites,as well as on the surface of immature schizonts.The anti-EtRON3 antibody have significant inhibitory effects on the ability of sporozoites to invade DF-1 cells.The transcriptional levels of EtRON2 and EtRON3 in the sporulated oocysts of precocious strain were significantly lower than that of the parent strain,which was consistent with the results of transcriptome analysis,suggesting that both EtRON2 and EtRON3 were related to molecular mechanism of precociousness of Eimeria.However,the role of EtRON2 and EtRON3 in the invasion and development regulation of Eimeria remains to be further studied. |
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