| Avian coccidiosis is caused by members of the genus Eimeria parasitizing the epithelial cells of the intestinal mucosa of chickens.There are seven Eimeria species of chicken,among which E.necatrix is one of the most pathogenic coccidia,mainly damages eight to eighteen-week-old chickens,and causes severe morbidity and mortality.In recent years,with the mass rearing of yellow feather chickens for long feeding period,as well as the use of live vaccines for controlling chicken coccidiosis,acute intestinal coccidiosis caused by E.necatrix become increasingly prevalent.Currently,the control options mainly rely on anticoccidial drugs.However,with the emergence of resistance,drug residues and environment pollution,it urgently needs effective and safe measures to replace anticoccidial drugs.Eimeria is a single-host parasite,and its development undergoes three stages of sporogony,schizogony and gametogony.The sporogony takes place in the external environment.The schizogony and gametogony occur in the epithelial cells of the chicken intestine and then gametes form oocysts discharging with chicken feces.The unsporulated oocysts develop into infective sporulated oocysts.When oocysts are eaten by a chicken,the oocyst wall is crushed in the gizzard,and sporozoites are released from sporocysts by the action of chymotrypsin and bile salts in the small intestine.Sporozoites enter epithelial cells or are taken into intraepithelial lymphocytes,where develop into merozoites.Subsequently,the merozoites invade the epithelial cells again and started a new round of schizogony.After two to three rounds of schizogony,the merozoites proceed sexual replication known as gametogony.Therefore,the sporozoites and merozoites are two key developmental stages to invade and develop in intestinal epithelial cells.Thus,the identification of key molecules related to cellular invasion may help develop effectively measures to control coccidiosis.To date,the key molecules and mechanisms of apicomplexan invasion of host cells are mainly derived from the researches on Plasmodium and Toxoplasma gondii,but there is little research on Eimeria.Here,we have conducted the transcriptomic and proteomic analysis of E.necatrix unsporulated oocysts(UO),sporozoites(SZ)and second-generation merozoites(MZ-2).We have compared the distribution and abundance of differentially expressed genes and proteins and correlate these with possible invasive mechanisms.Furthermore,three differentially expressed genes were selected for prokaryotic expression and functional analysis.Therefore,these results may help elucidate the molecular mechanism of coccidia invasion and find vaccine candidates and drug targets against coccidiosis.1.Transcriptome analysis of the unsporulated oocysts,sporozoites and second-generation merozoites of E.necatrixTotal RNA of per samples were isolated from UO,SZ and MZ-2,respectively.The sample libraries were constructed for the high throughput sequencing.Based on the PacBio platform,the 9 305 110,8 423 031,6 756 870 subreads,34 932,23 040,21 923 transcripts and 4949,4254,4007 genes were obtained from UO,SZ and MZ-2 libraries,respectively.The analysis of AS events indicated that the majority of AS events were 3'splice sites(27.5%)in UO,5'splice sites in SZ(19.09%)and MZ-2(23.93%).In the APA analysis,a total of 955,797 and 840 genes with a single poly-A site were detected in UO,SZ and MZ-2,respectively.After transcripts mapped to the reference genome,a total of 1958(UO),1743(SZ)and 1416(MZ-2)novel genes,and 26810(UO),17804(SZ)and17151(MZ-2)novel transcripts were identified,respectively.In the transcription factors analysis,a total of 194(UO),159(SZ)and 84(MZ-2)transcripts were mapped to 10(UO),11(SZ)and 8(MZ-2)transcription factors families.A total of 5223(UO),3581(SZ)and 2039(MZ-2)lncRNAs,and 3835(UO),4019(SZ)and 2588(MZ-2)fusion transcripts were identified by the third-generation single-molecule real-time(SMRT)sequencing.Based on the Illumina platform,a total of 71 298 596,73 222 160,70 203 690 raw reads were obtained from UO,SZ and MZ-2,respectively.Gene expression analysis showed that a total of 8376,6905 and 7902 differentially expressed genes(DEGs)were detected in SZ vs UO,SZ vs MZ-2 and MZ-2 vs UO,respectively.GO enrichment analysis showed that 6237,5076 and 5833 DEGs were successfully annotated by 113,9 and 102 GO terms in SZ vs UO,SZ vs MZ-2 and MZ-2 vs UO,respectively.KEGG pathway analysis indicated that 664,1324 and 987 DEGs were successfully annotated by 13,31 and 14 signaling pathways in SZ vs UO,SZ vs MZ-2 and MZ-2 vs UO,respectively.A total of 123 DEGs involving in cellular invasion were obtained,among these 50 genes were upregulated in SZ and 73 genes were upregulated in UO.2.Proteome analysis of the unsporulated oocysts,sporozoites and second-generation merozoites of E.necatrixBased on proteome analysis of UO,SZ and MZ-2 of E.necatrix using iTRAQ technology,we identified 983 835 spectra,26 410 peptides and 3606 proteins.The number of annotated proteins in the four databases of GO,KEGG,IPR and KOG were 1725,2143,2386 and 1724,respectively.A total of 388,300 and 592 differentially expressed proteins(DEPs)were identified from SZ vs UO,SZ vs MZ-2 and MZ-2 vs UO,respectively.GO enrichment analysis showed that 166(UO),105(SZ)and 200(MZ-2)DEPs were successfully annotated by 38,20 and 11 GO terms,respectively.KEGG pathway analysis indicated that 89,52 and 105 DEPs were successfully annotated by 102,72 and 125 signaling pathways in SZ vs UO,SZ vs MZ-2 and MZ-2 vs UO,respectively.With InterPro(IPR)enrichment analysis,a total of 47,32 and 64 DEPs were mainly assigned to 66,43 and 84 IPR in SZ vs UO,SZ vs MZ-2,MZ-2 vs UO,respectively.Protein interaction network analysis of DEPs showed that there were 273 nodes,686 edges,153 nodes,230 edges and 321 nodes,864 edges in the PPI network of SZ vs UO,SZ vs MZ-2 and MZ-2 vs UO,respectively.As a result,a sum of 103 DEPs were detected involving in cellular invasion.3.The combined analysis of the transcriptome and proteome of the unsporulated oocysts,sporozoites and second-generation merozoites of E.necatrixTranscriptome and proteome correlation analysis found that the person correlation coefficients of SZ vs UO,SZ vs MZ-2 and MZ-2 vs UO were 0.086,0.297 and 0.317,respectively.In addition to the consistent expression trend of related genes at the mRN A level and protein level,there are also multiple expression patterns.Totally 292,159 and 391 DEGs were identified in SZ vs UO,SZ vs MZ-2,MZ-2 vs UO,respectively.GO enrichment analysis showed that DEGs were involved in GO terms,such as nucleotide binding,small molecule binding,transferase activity,cofactor binding and oxidoreductase activity.KEGG enrichment analysis showed that DEGs were involved in pathways,such as protein processing in endoplasmic reticulum,aminoacyl-tRNA biosynthesis,glutathione metabolism,propanoate metabolism,carbon metabolism and biosynthesis of amino acids and peroxisomes.As a result,a total of 68 DEGs were identified involving in cellular invasion.4.Prokaryotic expression of the SAG,p25 and MIC13 genes and the analysis of immunofluorescence localizationThree DEGs(EnSAG?Enp25?EnMIC13)were amplified with functions of cellular invasion.Then their sequences were inserted into the prokaryotic expression vector pET28a(+)and successfully expressed in E.coli BL21,respectively.The sizes of three recombinant proteins were 26 kDa,23 kDa and 19 kDa,respectively.The recombinant protein rEnSAG and rEnMIC 13 mainly existed in a form of inclusion bodies,and rEnp25 expressed in supernatant.Western blot analysis showed that all three recombinant proteins could be specifically recognized by anti-6×HIS tag monoclonal antibody and mouse anti-rEnSAG,-rEnp25 and-rEnMIC 13 polyclonal antibody.The extracts of MZ-2 could be specifically recognized by mouse anti-rEnSAG,-rEnp25 and-rEnMIC 13 polyclonal antibodies,respectively.Immunofluorescence results showed that EnSAG and EnMIC13 were mainly distributed on the cytoplasm of SZ and MZ-2,Enp25 distributed at the apical end of SZ and the cytoplasm of MZ-2.The real-time PCR results showed that EnSAG transcription level was higher in SZ,and its protein level was higher both in SZ and MZ-2.Enp25 transcription level was higher in UO,and its protein level was higher both in SZ and MZ-2.EnMIC13 transcription level was higher in MZ-2,but its protein level was expressed only in MZ-2.5.The immune protective effects of rEnSAG,rEnp25 and rEnMIC13Three recombinant proteins were mixed with adjuvant to prepare immunogens,respectively.The chickens were divided into three immune dose groups(200 ?g,100 ?g,50 ?g),respectively.At the same time,the two groups were used as positive(unimmunized and challenged with oocysts)and negative(unimmunized and unchallenged with oocysts)controls,respectively.Seven days after the booster immunization,except negative group,the other groups were challenged with oocysts.The protective efficacy was evaluated based on the survival rate,body weight gain,lesion score and reduction in oocysts.As a result,rEnSAG at 100 ?g/bird,rEnp25 and rEnMIC 13 at 200 ?g/bird had a better protective effect.Compared with positive control,the recombinant proteins could decrease the oocyst production,lesion score and improving the weight gain. |
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