Isolation And Identification Of A Pathogen Causing "Head-shaking Syndrome" Of Pelodiscus Sinensis And Its Effect On Four Immune-related Genes Of Pelodiscus Sinensis

The outbreak of"head-shaking syndrome"disease caused a large scaledeath of Chinese soft-shelled turtle?Pelodiscus sinensis?and serious economic losses to Chinese soft-shelled turtle breeding industry.In order to determine the pathogen,understand the pathogenicity of the pathogen,damage to the organism and propose reasonable control measures,this study designed related experiments.First,pathogenic bacteria were isolated and cultured,and then the pathogenic bacteria were identified by observing colony morphology,Gram staining,determining 16S rDNA,gyrB and rpoB sequenceand the physiological and biochemical indexes of the bacteria.Antimicrobial susceptibility of the bacteria were also tested.Second,the virulence genes included in pathogenic bacteria were measured by PCR method,and visceral tissue sections were used to observe the damage of internal organ.Third,transcriptome sequencing was used to analyze the effect of the bacteria on transcriptome of Pelodiscus sinensis.Finally,to determine the effect of pathogens on Chinese soft-shelled turtle,fluorescent quantitative PCR was used to determine the expression levels of immune genes MyD88,TLR2,TLR3,and TLR4 in various tissues of the organism.The results are as follows:1.Identification results of the pathogens and drug sensitivity results?1?The isolated pathogenic bacteriaare creamy white waxy bulges,positive for Gram staining and have spores.The physiological and biochemical indicators are basically consistent with the description of Bacillus cereus in the system identification manual of common bacteria.The sequence analysis of 16S rDNA gene,gyrB gene and rpoB gene as well as the phylogenetic tree results show that the strain is a new species in Bacillus cereus group,named Bacillus cereus XS0724.The infection by artificial injection can reproduce the symptoms of the disease,presenting as floating on the surface of the water,slow response,weakness of the limbs,shaking head and death.The LD₅₀ is 2.42×10⁵ CFU/kg.?2?Drug susceptibility test shows that this strain is highly sensitive to cephalexin,florfenicol,doxycycline,enrofloxacin,ofloxacin,moderately sensitive to medicamycin,nitrofurantoin,tobramycin,kanamycin,furazolidone,gentamicin,vancomycin,and resistant to rifampicin and ceftazidime2.Virulence genes of pathogenic bacteria and their toxicity to tissues?1?PCR analysis of 10 common virulence genes of Bacillus cereus shows that Bacillus cereus XS0724 contains 7 virulence genes,which were non-hemolytic enterotoxin genes?nheA,nheB,nheC?,hemolytic enterotoxin genes?hblA,hblC,hblD?and intestines Toxin gene?entFM?.It is further shown that this strain is a toxic strain and has high toxicity.?2?Visceral sections showthat liver,spleen,and intestinal are severely damaged,the kidney andlungare lighter,but some lesions are visible.A large number of pathogenic bacteria are found in the liver and spleen.3.Effects of pathogenic bacteria on spleen transcriptome of Chinese soft-shelled turtleTranscriptome analysis resulted in Clean Data of about 42.63 Gb and the Data volume of each sample is more than 6.07 Gb.After Data splicing,a total of 348,665Unigene are obtained.Statistics of differentially expressed genes show that the experimental group?Case?has a total of 18,593 genes,including 9,678 up-regulated genes and 8,915 down-regulated genes,compared with the control group?Control?.KEGG pathway enrichment is mainly concentrated in toll-like receptor signaling pathway,antigen processing and presentation pathway,IL-17 signaling pathway,Th1 and Th2 cell differentiation pathway,autoimmune deficiency,parasitic disease,tuberculosis,human tumor and Staphylococcus aureus infection and other immune-related pathways.4.Expression of immune genes and results in visceral sections of Chinese soft-shelled turtlesinfected by pathogensExcept for the brain,the expression of MyD88 mRNA is extremely significant different from that of the control group?P<0.01?,and the relative expression levels are spleen>muscle>heart>kidney>lung>blood>liver>intestine>brain.Compared with the control group,the expression of TLR2 mRNA is significant differences in the brain?P<0.05?,and extremely significant differences in the other tissues?P<0.01?,but no different in intestinal and blood?P>0.05?.The relative expression levels are muscle>spleen>heart>blood>lung>kidney>liver>brain>intestine.Compared with the control group,the expression of TLR3 mRNA is significant differences in the lungs?P<0.05?,and extremely significant differences in the remaining tissues?P<0.01?.The relative expression levels are muscle>spleen>blood>heart>liver>lung>kidney>intestine>brain.Compared with the control group,the expression of TLR4 mRNA is not different in the intestine?P>0.05?,the lung and blood phases are significantly different?P<0.05?,and the remaining tissues are extremely significant different?P<0.01?.The relative expression levels are muscle>spleen>heart>kidney>liver>lung>blood>brain>intestine.In summary,Bacillus cereus strain XS0724 is pathogenic to Chinese soft-shelled turtles and should be distinguished when selecting Bacillus cereus as a probiotic.This toxic strain is highly toxic and destructive to the liver,spleen and intestines,and drugs that are beneficial to the liver,spleen,and intestinal tract can be given priority during treatment and prevention;cephalexin,florfenicol,and doxycycline can be used for disease treatment.