| Fasciola gigantica(F.gigantica)is a common parasitic helminth.It is widely distributed in tropical Asia,Africa and parts of Europe and is also regarded as a ‘tropical fluke’.In our country,the epidemic of F.gigantica mainly occurs in the southern provinces or regions of Huaihe river,and its high prevalence has caused considerable economic losses to the animal husbandry industry,especially in the buffalo breeding industry.Water buffalo is an important breed of cattle with economic value,but it is susceptible to F.gigantica infection,and such infection can casue pathological liver damage,hepatomegaly,splenomegaly,anemia,jaundice and other symptoms,and in severe cases,death may occur.However,no fluke vaccine is available.Currently,although drugs are the most common method for the treatment of F.gigantica infection,the resistance of broad-spectrum antihelminthic drugs is rapidly increasing.In the present study,a total of 18 swamp water buffaloes were used as the animal model of F.gigantica infection,and buffaloes were randomly divided into 3 infection groups and 3 uninfected groups(control groups).At 3-,42-and 70-days post infection(3,42 and 70 dpi),the liver,hepatica lymph nodes(h LNs),peripheral blood lymphocytes(p BLs)and spleen tissues of buffaloes were collected for sequencing.The main methods and results of this study are in the following:(1)Using RNA-seq technology,we performed transcriptome sequencing of three lymphoid tissue samples(h LNs,p BLs and spleen).After the processing of raw data using software,the high-quality transcriptome data was aligned to the reference genome,so that we can identify the number of transcripts and quantitatively analyze the expression profiles,as well as characterize the differential expression patterns between infected and control groups.Bioinformatics analysis showed that,at 3 dpi,the differentially expressed transcripts(DETs;P-value < 0.05 and Log2 Fold Change ≥ 1.5)of h LNs had the most significant changes(> 2,500 DETs),but at all infection time points the DETs of p BLs and spleen were relatively lower than the changes of 3 dpi(< 1,000 DETs).GO(Gene Ontology)function enrichment analysis indicated that the upregulation of DETs in h LNs at all infection time points mainly involved Th1/Th2 type innate and adaptive immunity.At 3and 42 dpi,the upregulation of DETs in the infected p BLs was capable of activating the host’s innate immunity and producing inflammatory responses.However,at 70 dpi,the immune responses of p BLs and spleen were both suppressed,especially for Th1 type innate immunity.In addition,WGCNA(weighted gene co-expression network analysis)and ROC curve(receiver operating characteristic curve)analysis revealed potential host genes that were related to F.gigantica infection,such as immunoglobulin(IGL-λ and IGL-κ),cytokines(IL-4,IL12 and IL-21),and antimicrobial peptides(CATHL6 and CATHL7).(2)Using i TRAQ technology,we peformed protein quantification and differential expression analysis of the obtained liver,h LNs and spleen.The results showed that there were > 100 differentially expressed proteins(DEPs;P-value < 0.05 and Log2 Fold Change≥ 1.2)in each affected tissue,but the maximum number of changes(> 1,800 DEPs)was detected at 3 dpi.Functional enrichment of DEPs showed that the upregulation of DEPs by h LNs at 3 dpi was mainly involved in biological processes related to immune response,such as transendothelial migration of leukocytes,B cell receptor signaling pathways,and chemokine signaling pathways.At 70 dpi,DEPs were not significantly enriched in any immune-related biological process.In this study,we also found that,in the infected liver,the drug-related metabolic pathway was downregulated significantly,such as drug metabolism-cytochrome P450.Moreover,subcellular localization analysis showed that DEPs related to the extracellular space and cell membrane played a major role in mediating the host immune response.(3)Using small RNA sequencing technology,we performed micro RNA(mi RNA)analysis from the obtained liver and spleen tissues.By referring to the available swamp buffalo reference genome,this study identified 428 mi RNA members from 202 conserved families,and also identified 2 novel bovine-specific mi RNAs.The quantitative and differential expression analysis of liver and spleen mi RNAs showed that there were 23 differentially expressed mi RNAs(DEMs;P-value < 0.05 and Log2 Fold Change ≥ 1.5)in the infected liver.At 3 dpi,the number of DEMs was downregulated significantly(n = 7),while the upregulation of DEMs mainly occurred at 42(n = 10)and 70(n = 5)dpi;there were 14 DEMs in the infected spleen,and the upregulation of DEMs was more significant at 42 dpi(n = 7).GO function enrichment indicated that the downregulation of DEMs in all affected tissues is mainly involved in several biological processes related to cytokines and immunity,while DEMs are mainly involved in biological processes such as metabolism and transcription of the body.In addition,based on the existing F.gigantica mi RNA data sets,we detected 20 mi RNAs excreted by F.gigantica in the infected tissues,among which Fgi-Mir-87＿3p and Fgi-Mir-10-P1＿5p were the two mi RNAs with the highest expression(each > 100 reads)in the infected liver and spleen,respectively.Interaction study between F.gigantica and buffalo showed that F.gigantica mi RNAs were able to regulate host immune-related pathways,such as chemokine signaling pathway,PI3K-Akt signaling pathway,IL-17 signaling pathway,and T-B cell signaling pathway.In summary,using mutil-omics data and integrated bioinformatics analysis,this study initially clarified buffalo genes,proteins and mi RNAs closely related to F.gigantica infection,and revealed that the effects of F.gigantica infection to liver and several lymphoid tissues at different infection stages.The results of this study not only provide a useful reference for clarifying the infection mechanism of F.gigantica and demonstrate the molecular mechanism of buffalo susceptibility,but also it lays the foundation for the screening of new diagnostic targets and drug development. |
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