| The present study aims was to investigate the similarities and differences in pathogenicity between precocious strains(PTsx)and virulent strains(Tsx)of Eimeria tenella(E.tenella),and investigate the pathways of pathogen-related genes damage host cells.The model of E.tenella infection in chickens and the model of primary chicken embryo caecal epithelial cells infected with E.tenella in vitro was established.RT-q PCR,Hoechst 33342 / Annexin V-FITC / PI fluorescent labeling,spectrophotometry,Western blot and yeast to express foreign proteins were used to detect the infection rate,apoptosis and autophagy of host cells infected with Tsx and PTsx.RNA-Seq was used to detect and compare the merozoite stage transcriptomes of PTsx and Tsx,and screening out the pathogenicity-related genes of E.tenella.Yeast expression of foreign proteins was used to express the pathogen-related genes of E.tenella,and the effects of recombinant protein on the infection rate,apoptosis and autophagy of E.tenella host cells were examined.The results showed that:(1)After E.tenella infection,the average weight gain of chickens inoculated with the same dose of Tsx group was significantly lower than PTsx group(p < 0.01),and the average pathological scores were significantly higher than PTsx group(p < 0.01).Chickens in the Tsx infection groups all discharged blood stools to different degrees.Among them,40 % of chickens in the high dose Tsx group(45 000 per bird)died,but the chickens in the PTsx infection groups did not showed blood stools and death.In addition,the average weight gain of the low dose groups(4 500 per bird)was significantly higher than the same strain high dose group(p < 0.01),while the average lesion score was significantly lower than same strain high dose group(p < 0.01).Microscopic examination revealed that capillary hemorrhage and villous shedding of chicken cecum mucosa in the same dose Tsx inoculation group were more severe than in the PTsx group.It shows that E.tenella virulent strain is more pathogenic than the precocious strain,and the pathogenicity is related to the inoculation dose.(2)There was no significant difference between the infection rates,the apoptotic rates and Caspase-3absorbance within the same dose inoculation groups of the host cells after inoculation of E.tenella 4 h-72 h in vitro(p > 0.05),but the infection rates,the apoptotic rates and Caspase-3 absorbance of Tsx inoculation groups were extremely significantly higher than PTsx group at 120 h(p < 0.01).The apoptosis rates of cecal and glandular epithelial cells in PTsx groups was extremely significantly lower than that in the same dose inoculated Tsx groups after inoculated E.tenella 5 days in vivo(p < 0.01).Both in vivo and in vitro,the apoptosis rates and Caspase-3 absorption value of the high dose groups(400,000 per well or 45,000 per bird)inoculated with the same type of strains were higher than those of the low dose groups(200,000 per well or 4,500 per bird)(p < 0.01).These observations indicate that E.tenella precocious strains and virulent strains have the same effect on host cell apoptosis in the early and middle stages of their development.However,the effect of precocious strains in inducing apoptosis of host cells in the later stages of E.tenella development is significantly lower than that of virulent strains.With the increase of infection dose,the induction of apoptosis of host cells by precocious strains and virulent strains increased.(3)The relative expression of Beclin-1 m RNA and the ratios of LC3?/? in the PTsx inoculation groups were significantly lower than the Tsx groups with the same inoculation dose after inoculation of E.tenella 4 h-120 h in vitro(p < 0.05).The relative expression of Beclin-1 m RNA and the ratios of LC3?/? in the inoculated groups were significantly higher than that in the non-inoculated group at 4-120 h after inoculation of Tsx(p < 0.05).However,for the PTsx groups,these indicators were significantly higher in the high dose group than those in the non-inoculated group at 120 h after inoculation(p < 0.05).On the 5th day after inoculation of E.tenella in vivo,when the inoculation dose were same,the relative expression level of Beclin-1 m RNA and the LC3?/? ratios of PTsx inoculation groups were significantly lower than the Tsx group(p < 0.05).Whether in vivo or in vitro,the relative expression of Beclin-1 m RNA and the LC3?/? ratios of the high-dose groups inoculated with the same strains were higher than the low dose groups(p < 0.05).These observations indicate that both virulent strains and precocious strains can promote autophagy in host cells.The effect of the same dose of precocious strains on the autophagy of host cells is significantly lower than that of virulent strains;the induction effect of the same type of E.tenella on the autophagy of host cells increases with the increase of the infection dose.(4)Compared with the transcriptome of Tsx 96 h merozoite,there were 2189 up-regulated genes and740 down-regulated genes in PTsx 96 h,961 up-regulated genes and 3671 down-regulated genes in Tsx 120 h.Differential genes were mostly enriched in GO subsets such as cell and cell membrane components,macromolecular substance metabolism process and transmembrane transport,and KEGG pathways such as Ribosome,Spliceosome and Protein processing in endoplasmic reticulum.MIC3,MIC4,ROP17,ROP21,ROP27,ROP30,ROP35,ROP38,GRA9,GRA10 and GRA11 were all expressed in merozoites of PTsx 96 h,Tsx 96 h and Tsx 120 h,and they were all enriched in the pathogenesis mechanism pathway of Toxoplasmosis.RT-q PCR showed that there was no significant difference in the relative expression of ROP38 in PTsx 96 h,Tsx 96 h and Tsx 120 h merozoites(p > 0.05).The relative expression of MIC3,MIC4,ROP17,ROP27,ROP30,ROP35,GRA10 and GRA11 in PTsx 96 h merozoites were significantly lower than that in Tsx 96 h merozoites(p < 0.05),the relative expression of GRA9 in PTsx 96 h merozoites was significantly higher than that in Tsx 96 h merozoites(p < 0.05).The relative expression of ROP17 in Tsx120 h merozoites was significantly higher than Tsx 96 h merozoites(p <0.05),the relative expression of MIC3,MIC4,ROP21,ROP27,ROP35,GRA9 and GRA10 in Tsx 120 h merozoites were significantly lower than Tsx 96 h merozoites(p <0.05).It shows that MIC3,MIC4,ROP17,ROP21,ROP27,ROP30,ROP35,ROP38,GRA9,GRA10,and GRA11 are E.tenella pathogenicity-related genes.The expression of ROP38 in the merozoites of E.tenella precocious strain 96 h,virulent strain 96 h and 120 h is similar.the expression of MIC3,MIC4,ROP17,ROP27,ROP30,ROP35,GRA10 and GRA11 in the merozoites of precocious strain 96 h are down-regulated,the expression of GRA9 in the merozoites of E.tenella precocious strain 96 h is up-regulated.The relative expression of ROP17 in the merozoites of E.tenella virulent strain 120 h is up-regulated,the expression of MIC3,MIC4,ROP21,ROP27,ROP35,GRA9 and GRA10 are down-regulated in the merozoites of E.tenella virulent strain 120 h compared with the merozoites of E.tenella virulent strain 96 h.(5)The yeast expression system of Et ROP38,Et ROP30 and Et GRA11 proteins was constructed,the recombinant proteins of 14 k D,58 k D and 40 k D were expressed,respectively.The corresponding purified recombinant proteins of Et ROP38,Et ROP30 and Et GRA11 were obtained after purification and concentration.This indicates that the yeast expression systems of Et ROP38,Et ROP30 and Et GRA11 are constructed successfully,and the recombinant proteins of Et ROP38,Et ROP30 and Et GRA11 are obtained.(6)The stimulation index(SI)of the cells in the 1-100 ?g/m L protein-treated groups was significantly higher than the blank control group at 4-48 h after Et ROP38 stimulation(p < 0.05),and within this concentration range,there was no significant difference in SI of cecal epithelial cells of chicken embryos(p > 0.05).At 4-120 h after E.tenella inoculation,the infection rates of Et ROP38 + E.tenella treatment group were significantly higher than that of E.tenella inoculation control group(p < 0.01),the apoptosis rates and Caspase-3 activity of host cells in Et ROP38 treatment group were lower than that of blank control group(p < 0.05),and these indexes in Et ROP38 + E.tenella treatment group were also lower than E.tenella inoculation control group(p < 0.05).The relative expression of Beclin-1 m RNA and the ratios of LC3?/?in the host cells of Et ROP38 treatment group were higher than that in the blank control group(p <0.05),and these indexes in Et ROP38 + E.tenella treatment group were also higher than E.tenella inoculation control group(p < 0.05).This indicates that Et ROP38 promote the proliferation activity of chicken embryo caecal epithelial cells,promote the infection of host cells by E.tenella,and inhibit the apoptosis of host cells during the development of E.tenella,and promote the autophagy of host cells.(7)The SI of the cells in the 1-100 ?g/m L protein-treated groups was significantly lower than that of the blank control group at 24-48 h after Et ROP30 stimulation(p < 0.05),and within this concentration range,there was no significant difference in chicken cecal epithelial cells SI among the inoculation groups except for 24 h(p > 0.05).There was no significant difference in the infection rates of host cells between the Et ROP30 + E.tenella treatment group and E.tenella inoculation control group(p > 0.05).The apoptosis rates and Caspase-3 activity of host cells in Et ROP30 treatment group were higher than the blank control group(p < 0.05),and these indicators in Et ROP30 + E.tenella treatment group were also higher than the inoculation control group(p < 0.05).The relative expression of Beclin-1 m RNA and the ratios of LC3?/? in Et ROP30 treated group were lower than the blank control group(p < 0.05),and they were lower in Et ROP30 + E.tenella treated group than inoculation control group(p < 0.05).This indicates that Et ROP30 inhibit the proliferation activity of chicken embryo caecum epithelial cells,promot the apoptosis of host cells and inhibit the autophagy of host cells during the development of E.tenella.(8)At 4-48 h after Et GRA11 stimulation,the SI of cells in the protein treatment groups in the concentration range of 10-100 ?g/m L were significantly higher than those in the blank control group and0.1-1 ?g/m L protein treatment groups(p <0.05).At 4-120 h after E.tenella inoculation,there was no significant difference in the infection rates between the Et GRA11 + E.tenella treatment groups and the inoculation control group(p > 0.05).At most sampling time points,there was no significant difference in the apoptosis rates,Caspase-3 activity and the ratios of LC3?/? between the Et GRA11 treatment group and the blank control group(p > 0.05),there was no significant difference in apoptosis rates and Caspase-3activity between Et GRA11 + E.tenella treatment group and the inoculation control group(p > 0.05).However,the relative expression of Beclin-1 m RNA in Et GRA11 treatment group and Et GRA11 +E.tenella treatment group were significantly lower than that in blank control group and inoculated control group(p < 0.05),respectively.The ratios of LC3?/? in Et GRA11 + E.tenella treatment group were significantly lower than that in the inoculated control group(p < 0.05).This indicates that Et GRA11 promotthe proliferation activity of chicken embryo caecal epithelial cells,the effect of Et GRA11 on E.tenella invasion and apoptosis of host cells is not obvious.Moreover,when Et GRA11 acts alone,it does not affect the autophagy of chicken embryo caecal epithelial cells,but it can inhibit the autophagy of host cells when it cooperates with E.tenella.To sum up: there was no difference in the effect of E.tenella precocious strain and virulent strain on the apoptosis of host cells in the early and middle stages of development,but the induction of apoptosis of host cells in the late stage of development was far lower than that of virulent strain.Both precocious strains and virulent strains can induce host cell autophagy,but the induction effect is weaker than the virulent strains.Et ROP38 inhibits E.tenella host cell apoptosis,but promotes host cell autophagy and E.tenella infection.Et ROP30 induces E.tenella host cell apoptosis,but inhibits host cell autophagy.Et GRA11 cooperates with E.tenella to inhibit host cell autophagy. |
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