| Chinese soft-shelled turtle(Pelodiscus sinensis)is a unique and precious aquatic economic animal in China,which has both edible value and medicinal value.In recent years,with the improvement of people’s living standards,the demand for Chinese soft-shelled turtles has gradually increased,so large-scale intensive artificial breeding of Chinese soft-shelled turtles has appeared.However,due to the artificial temperature control,high-density farming and environmental changes,disease microorganisms invaded and led to disease outbreaks.This circumstance not only caused heavy casualties of Chinese soft-shelled turtles,but also brought huge economic losses to the farmers and seriously restricted the development of sustainable aquaculture industry.In 2020,an outbreak of Chinese soft-shelled turtle mortality occurred again in Nanfeng County,Jiangxi Province,with 80%mortality rate at the peak of the incidence.This study will not only isolate and identify the pathogenic bacteria of this outbreak but also explore its pathogenic mechanism.And then we will analyze the antioxidant capacity and antibacterial effect of Chinese herbal medicine extracts on Chinese soft-shelled turtle through in vitro antibacterial experiments.This study aims to provide a theoretical basis for understanding the disease and to clarify the pathogenic mechanism so as to prevent and control this disease.The main results are as follows:1.Isolation and identification of pathogenic bacteria and detection of virulence genesA dominant strain was isolated from the liver of Chinese soft-shelled turtle with obvious disease symptoms.The isolated strain was identified by phenotypic observation,physiological and biochemical identification combined with molecular biology methods,and its evolutionary status was analyzed by phylogeny.The dominant strain of this outbreak disease was identified as Aeromonas veronii(No.ZF2)by the traditional method consistent with the modern molecular biological method.The results of artificial regression infection showed that ZF2 was highly pathogenic to Chinese soft-shelled turtle,the symptoms were similar to those of the naturally infected individuals,while the same pathogenic bacteria were isolated from the diseased tissues.The symptoms of the infected individuals by ZF2 showed red and swollen in neck,bleeding spots on the abdomen,fester on the skirt and limbs;smelly body cavity with gelatinous ascites,ischemia induced white liver,intestinal hyperemia and red;gill-like tissue is red and erosive.Histopathological observation showed that the isolated strain infection can cause myocardial structure loose,muscle fiber rupture,liver cell ballooning,liver blood sinus congestion,the cell nuclear of kidney dissolution,tissue disintegration,spleen with numerous red blood cells and hemosiderin deposition,and a large number of blood cells of intestinal infiltrated whie the intestinal Villi shed.Virulence gene analysis showed that the strain carried three virulence factors of heat-stable enterotoxin(ast),toxic enterotoxin(act)and aerolysin(aer).Eighteen commonly used antibiotics were selected by the drugsensitive paper diffusion method(K-B).And the results showed that ZF2 was sensitive to 9 kinds of antibiotics,such as Cefradine,neomycin and Minocycline,and was intermediate or resistance to other antibiotics.2.Preliminary investigation of the pathogenic mechanism of ZF2To understand the immune response mechanism during ZF2 infection,the Chinese soft-shelled turtles were treated with ZF2 for 24h in experimental group,and the Chinese soft-shelled turtles were treated with 0.85%normal saline in control group.RNA-seq technology was used for sequencing analysis to select key immune genes and pathways related to immunity,so as to explore the molecular mechanism of antibacterial infection.Total of 39.48 Gb Clean Data,with the percentage of Q20 bases above 97.84%were yielded from liver tissue transcriptome sequencing.The genome of Chinese turtle was compared as the reference genome,and the alignment efficiency of each sample ranged from 87.93%to 89.66%.A total of 2138 nonredundant differentially expressed genes(DEGs)were obtained by comparing the experimental group and the control group,including 1292 up-regulated genes and 846 down-regulated genes.The GO enrichment analysis showed that the differentially expressed genes were distributed in biological processes,molecular functions and cell components,and the significantly different genes were enriched in biological processes,which included the response to stimulus,regulation of biological process,cellular response to stimulus and so on.Molecular function included enzyme binding,G protein-coupled receptor activity and so on.The classification of cell components mainly included membrane,intracellular part,etc..The significantly different genes associated with immune stimulation were identified in the secondary nodes,including TRAF3,CSF1,and so on.KEGG pathway enrichment analysis exhibited six signaling pathways,including p53 signaling pathway,TNF signaling pathway and so on,which were enriched and distributed in TOP20.These pathways were closely related to immune stimulation.3.Research on herbal resistance and prevention and control of pathogenic bacteriaIn order to achieve the goal of precise control of the pathogen infection in the breeding process of Chinese soft-shelled turtle,the effectiveness by Chinese herbal extracts controlling of this disease turtle was conducted to evaluate in this paper.Thirty-five Chinese medicinal herbs were selected for the preparation of aqueous extracts,and in vitro antibacterial experiments were conducted using the double dilution method.15 plant extracts had inhibitory effects on ZF2.Three Chinese herbs,Ampelopsis extract,Aloe extract and Asparagus extract had strong antibacterial effects and the minimum inhibitory concentration(MIC)was 1.5625 mg/mL.Ampelopsis extract was mixed into the non-resistant basal diet with 0%(control group)0.075%0.1%0.125%proportion,to evaluate its antioxidant(MDA,CAT,GSH-PX,SOD)and antibacterial(ZF2)effects on Chinese Soft-shelled turtle.The results showed that:The serum MDA content in the experimental groups was significantly lower than that in the control group(P<0.05);the activities of CAT,SOD and GSH-PX in serum of each experimental group increased significantly over time(P<0.05).After 21 days,eight Chinese soft-shelled turtles were selected from each group and were injected ZF2 intraperitoneally with 6×108 CFU/mL.The results showed that the Chinese soft-shelled turtle fed with 4 different concentrations of Ampelopsis extract began to die at the 3rd day and stopped dying at the 10th day.After ZF2 challenge,the mortality of the Control Group(0%)was 75%.Compared with the control group,the mortality of the Chinese soft-shelled turtle fed with 0.075%,0.1%and 0.125%Ampelopsis extract were 50%,37.5%and 12.5%respectively.The experimental results indicated that,mixed feeding of Ampelopsis extract can strengthen the antioxidant and antibacterial ability of Chinese Soft-shelled turtle.The experiment results also showed that adding 1.25 g/kg Ampelopsis extract is the best.In conclusion,based on the analysis of disease identification,pathology and transcriptomics,this study found that ZF2 infection caused severe tissues impairment of Chinese soft-shelled turtle and activated the immune response.And then we evaluated the preventive and control effects of Chinese herbal extracts on ZF2,which provides a theoretical reference for disease prevention and control in Chinese softshelled turtle culture. |
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