Transcriptomic Analysis of the Spleen in Diarrheic Piglet Caused by Clostridium perfringens Type C

doi:10.7668/hbnxb.20194102

Abstract

Abstract:

The aim of this study was to obtain the serum inflammatory factor content,spleen pathological changes and transcriptome expression profile characteristics of diarrheic piglets with Clostridium perfringens type C,and to provide a reference for the diarrhea prevention and treatment.Six 7-day-old binary piglets with good health and similar body weight were randomly selected and divided into two groups,namely control group (CS) and treatment group (TS).Each piglet in the TS group was fed with 1 mL bacterial solution (1×10⁹ cfu/mL) every day for 5 consecutive days.Then,piglets in the CS group were fed with culture medium without the bacterial solution.Blood samples were collected and serum was separated on 0,1,3 and 5 days after infection.The serum IL-8,IFN-α and IFN-γ concentration were detected by ELISA.After the experiment,piglets were slaughtered to collect the spleen.The partial spleen was collected for HE staining to observe the pathological changes.Additionally,differentially expressed (DE) genes in the spleen were obtained using RNA-Seq.GO terms and KEGG signaling pathways enrichment analysis were performed.The key spleen genes during the infection of piglets with C.perfringens type C were verified by RT-qPCR.The spleen of piglets in the TS group displayed obvious inflammation,neutrophil infiltration,accompanied by degeneration and necrosis of parenchymal cells;on the 3rd and 5th day,the levels of IL-8,IFN-α,and IFN-γ in the CS group serum were extremely significantly lower than those in the TS group (P<0.01);a total of 1 079 DG genes was screened between TS group and CS group,including 583 up-regulated genes and 496 down-regulated genes.DG genes were mainly enriched in GO terms such as cell response to interferon-β,cell response to DNA damage stimulation,and negative regulation of inflammatory response.Differentially expressed gene were mainly enriched in KEGG signaling pathways such as tuberculosis,hepatitis B,and B cell receptors.These GO terms and KEGG signaling pathways were involved in piglet resistance to C.perfringens type C infection;the RNA-Seq results of the selected candidate genes (such as JAK3,TLR3,ILF2,CXXC1) resistant to C.perfringens type C infection were consistent with the RT-qPCR results.The above results indicated that JAK3,TLR3 and other genes played important roles in the process of piglet spleen resistance to C.perfringens type C infection through key signaling pathways.

Key words: Piglet, Diarrhea, Spleen, Clostridium perfringens type C, Transcriptome

YAN Zunqiang, WANG Pengfei, YANG Qiaoli, HUANG Xiaoyu, GAO Xiaoli, GUN Shuangbao. Transcriptomic Analysis of the Spleen in Diarrheic Piglet Caused by Clostridium perfringens Type C[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(5): 188-196. doi: 10.7668/hbnxb.20194102.

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Figures/Tables 7

Tab.1 Primer information

基因 Gene	获取号 Accession number	序列(5'-3') Sequence(5'-3')	产物大小/bp Product size
JAK3	XM_005654907.3	F:TTCACAAGATCCCTGCCGAC	150
		R:TGTGCTTGGCATCCATCACT
TLR3	NM_001097444.1	F:CAACTGATGCTCCGAAGGGT	204
		R:AGGGTTTGCGTGTTTCCAGA
ILF2	XM_005663409.3	F:GCCGTTTGTGCCACATATCC	207
		R:CCTGGAGCCACGATCAAGTT
ILF3	XM_021083793.1	F:GAACGTAAAACAGCAGGGGC	151
		R:ATCCACCTCGACCTCCATGA
CXXC1	XM_005654427.3	F:ACAAATCCTCTCCACAGCCC	250
		R:GTCACTGGCGAGAGCCG
CSF1	XM_021088653.1	F:GGCCTGTGTCCGAACTTTCT	174
		R:TTGCAATCAGGCTTGGTCAC
AKT2	NM_001256779.1	F:CTGCTGAAGAGTGACGGCTC	196
		R:GGGAGTCCACATGGAAGGTC
CDK8	XM_021065497.1	F:AAAGTGAAGCTGAGCAGCGA	175
		R:ATGCCGACATAGAGATCCCG
β-actin	XM_003124280.5	F:AGGCGGACTGTTAGTTGCAT	187
		R:TGTCACCTTCACCGTTCCAG

Fig.1 Histological result of piglet spleen Red arrows.Infiltration with neutrophilic granulocytes;Black arrows.Parenchymal cells with degeneration and necrosis; Yellow arrows.Spleen trabecular widened and increased;A-C.TS group;D-F.CS group.

Fig.2 The serum content of the inflammatory cytokines IL-8,IFN-α and IFN-γ Different uppercase letter superscripts mean extremely significant difference (P< 0.01);different lowercase letter superscripts mean significant difference (P<0.05).The same as Fig.6.

Fig.3 Screening and cluster analysis of DE gene A.Transcripts expression level;B.The number of DE gene;C.Heat map of DE gene(Rows indicate genes,columns represent samples).

Fig.4 GO term enrichment analyses of differentially expressed gene

Fig.5 KEGG signaling pathway enrichment analysis of differentially expressed gene

Fig.6 Validation of sequencing result by RT-qPCR

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