| Chinese soft-shelled turtle(Pelodiscus sinensis)is a unique freshwater species in China.It has great edible and medicinal value,in recent years,with the expansion of artificial cultivation of Chinese soft-shelled turtle,various problems of cultivation have followed.In 2020,there was an explosive death of Chinese soft-shelled turtle farms in Nanfeng County,Jiangxi Province,through the preliminary work of the research group,the pathogen of explosive disease has been identified as Aeromonas veronii,and the Chinese herbal medicine Ampelopsis japonica extract has an inhibitory effect on this bacterium through in vitro antibacterial experiments.In this study,transcriptome sequencing was performed on spleen tissue of Chinese softshelled turtle infected with Aeromonas veronii to screen immune-related genes and pathways,explore the pathogenic mechanism of Aeromonas veronii on Chinese softshelled turtle.Further,the Ampelopsis japonica extract of different concentrations was used to conduct in vitro antibacterial and germicidal tests on Aeromonas veronii,and the optimal concentration was selected to explore its effect on the growth of Aeromonas veronii and its inhibitory effect on virulence genes.The histopathological changes of spleen,non-specific immune function,secretion of immune factors,and expression of immune-related genes were detected by adding Ampelopsis japonica extract to the diet of Chinese soft-shelled turtle,in order to provide a theoretical basis for the pharmacological mechanism of the Ampelopsis japonica extract in alleviating the infection of Aeromonas veronii.Specific research results are as follows:1.Transcriptome analysis of spleen infected by Aeromonas veronii.in Chinese softshelled turtleIn this study,transcriptome sequencing was performed on spleen tissues of Chinese soft-shelled turtle infected with Aeromonas veronii,The results showed that28.6 Gb of Clean Data was obtained by spleen sequencing,and the percentage of Q20 bases was greater than 97.78%.Using the genome of Chinese soft-shelled turtle as the reference genome,the comparison efficiency of each sample was more than 80%,ranging from 83.37% to 86.14%.A total of 1603 nonredundant differential genes(DEGs)were obtained from spleens of the experimental group and control group,in which 902 DEGs were up-regulated and 701 DEGs were down-regulated.The functional enrichment analysis of GO data source database showed that the differential genes were distributed in biological processes(BP)molecular function(MF)and cell components(CC),including ATPase activity(GO:0016887),extracellular exosomes(GO:0070062),and components of plasma membrane(GO:0005887);chemokine receptor 3(CXCR3),interleukin 6(IL6),and tumor factor receptor superfamily member 6B(TNFRSF6B)were identified in secondary nodes with functional enrichment and related to immune stimulation differential genes.According to KEGG database enrichment analysis,there were six signaling pathways closely related to immunity,including phagosome,cytokine-cytokine receptor interaction and Toll-like receptor signaling pathway,among the top 20 signaling pathways enriched.2.Inhibitory effect of the Chinese herbal medicine Ampelopsis japonica extract on Aeromonas veronii in vitroThe effect of the extract of Ampelopsis japonica extract on the inhibitory cycle,MIC(minimum inhibitory concentration),MBC(minimum bactericidal concentration),and growth curve of Aeromonas veronii was tested by the Oxford cup method and the double dilution method,as well as the inhibitory effect of MIC on virulence genes.The results showed that the Ampelopsis japonica extract was highly sensitive to 300 mg/m L,the diameter of inhibition zone was:13.66±0.14 mm,the MIC was:75 mg/m L,and the MBC was:100 mg/Ml.The Ampelopsis japonica extract with MIC 75 mg/m L was used to inhibit the growth of Aeromonas veronii.Compared with the control group with "S" type growth,the bacteria in the experimental group did not grow before 5 h,but grew slowly after 5 h,and the growth curve was smaller than that of the control group,showing an obvious antibacterial effect.Eight virulence factors,including exu(ribozyme),ast(heat stable enterotoxin),ser(serine protease),aer(aerolysin),act(toxic enterotoxin),alt(heat unstable enterotoxin),fla(flagellin)and lip(lipase),were detected by PCR using the c DNA of the control bacteria as templates,the results showed that Aeromonas veronii contained exu,ast,aer,act and fla.Quantitative expression analysis of virulence genes in the experimental group and the control group was conducted by q RT-PCR,the results showed that compared with the control group,exu and ast were not expressed in the experimental group,while other virulence genes aer,act and fla were significantly down-regulated(P<0.01),indicating that Ampelopsis japonica extract can greatly reduce the toxicity of Aeromonas veronii,and it has good bacteriostatic effect.3.Study on Ampelopsis japonica extract against Aeromonas veronii infection of Chinese soft-shelled turtleIn order to further understand the effect of Ampelopsis japonica extract on immune function of Chinese soft-shelled turtle,0 mg/kg,750 mg/kg,1000 mg/kg and1250 mg/kg Ampelopsis japonica extract was added to the base diet for feeding Chinese soft-shelled turtle,and the experiment of Aeromonas veronii infection was carried out.The results showed that the survival rate of the 1250 mg/kg group was80%.And to experiment further with this concentration,the cultured Chinese softshelled turtle was divided into four groups: group A(control group),group B(Ampelopsis japonica),group C(Aeromonas veronii),and group D(Ampelopsis japonica + Aeromonas veronii),and the following results were obtained:(1)The survival rate of group C after Aeromonas veronii infection is 40%,and that of group D is 80%,the Ampelopsis japonica extract can significantly improve the survival rate of Chinese soft-shelled turtle infected with Aeromonas veronii.The average weight gain rate(WGR)and average specific growth rate(SGR)were show in B>A>C(P<0.01),D>C(P<0.01),there was no significant difference between A and D.The spleen index in group C was significantly higher than that in groups A,B and D(P<0.05),the bacterial load in spleen of group D was significantly lower than that of group C(P<0.05).(2)Histopathological observation showed that the spleen tissue of group A and B had no abnormal health during breeding,and the structure of tissues was normal without pathological changes.However,in group C,there were obvious lesions and necrotizing inflammation on the 1-7 d after the challenge,mainly manifested as massive infiltration of red blood cells,degeneration of blood nuclei,deformation and necrosis,the red pulp and white pulp could not be distinguished,lymphocytopenia,nucleus pyknosis,fragmentation and dissolution,many inflammatory cells and hemosiderosis such as monocytes,neutrophils and plasma cells were found,leading to cell necrosis.The splenic tissue damage of group D recovered somewhat,but there was still inflammatory cell infiltration;granular degeneration and necrosis were also present in all cells at the 1st to 3rd day,the red pulp and white pulp could be clearly differentiated,and there were inflammatory cells and a small amount of hemosiderosis;however,at the 5th to 7th day of feeding,there was only a little hemosiderosis and slightly more red blood cells,and there was a significant recovery compared with the earlier stage and group C.TUNEL method was used to detect apoptosis in spleen tissue of Chinese soft-shelled turtle in each group.The results showed that apoptosis in spleen tissue of Chinese soft-shelled turtle in group C was significantly increased(P<0.01),there were obvious apoptotic signals.Compared with group C,the apoptosis level of spleen cells in group D was significantly decreased(P<0.01),but there were also a few apoptotic signals.(3)The results of enzyme activity showed that after feeding for 22 days,there was no significant change in the activity of myeloperoxidase(MPO)in serum and spleen in groups A and B(P<0.05),C>D>A(P<0.01),changes of total antioxidant capacity(T-AOC)activity in serum and spleen: B>A>D>C(P<0.01),the changes of acid phosphatase(ACP)in serum were as follows: B>A(P<0.05),A>D>C(P<0.01),the changes of ACP in spleen were as follows: B>A>D>C(P<0.01),alkaline phosphatase(AKP)activity in serum and spleen: B>A>D>C(P<0.01).(4)ELISA detected the secretion levels of inflammatory factors in spleen tissue,the expression level of TNF-α in spleen of four groups was as follows: C>D>A>B(P<0.01),the expression level of IL-10 was as follows: B>A>D>C(P<0.01),the expression level of IL-6 was as follows: C>D>A(P<0.01),A>B(P<0.05).(5)Real-time quantitative analysis of the immune genes screened by the transcriptomes showed that compared with group A,the expressions of CXCR3 and IRF7 in group B were significantly up-regulated after feeding at 22 days(P<0.01),IL6 was significantly down-regulated(P<0.01),TNFRSF6 B was significantly downregulated(P<0.05),there was no significant difference in SOCS3;CXCR3 and IRF7 were significantly down-regulated in group C(P<0.01),IL6,TNFRSF6 B and SOCS3 were significantly up-regulated(P<0.01);IRF7 of Chinese soft-shelled turtle in group D was significantly up-regulated(P<0.05),IL6,TNFRSF6 B and SOCS3 were significantly up-regulated(P<0.01),CXCR3 showed no significant difference.Compared with group C,the expression of CXCR3 and IRF7 in group D,which was fed Ampelopsis japonica extract,was significantly up-regulated(P<0.01),the expression levels of IL6,TNFRSF6 B and SOCS3 were significantly down-regulated(P<0.01).In summary,this study found through transcriptomics,histomorphology,molecular biology and other research methods that combined with in vivo and in vitro tests,it was found that feeding 1250 mg/kg Ampelopsis japonica extract can effectively improve the survival rate of Chinese soft-shelled turtle,reduce pathological damage of spleen tissue,activate immune response,and enhance immune function and resistance to Aeromonas veronii infection.It can be used as an antibiotic substitute for the treatment of Aeromonas veronii infection in culture. |
