Isolation And Identification Of Pathogens From Trionyx Sinensis And Protective Effect Of Scutellaria

Bacterial disease is a common disease in Trionyx sinensis breeding,which seriously threatens the healthy breeding of T.sinensis.In this study,pathogenic strains were obtained from two disease outbreaks in a T.sinensis farm in Xiantao,Hubei.The isolates were identified,and it was verified that they were the pathogenic bacteria that caused the infection of T.sinensis through regression infection experiments,and the phenotype and whole genome of the isolates were analyzed.The protective effect of traditional Chinese medicine extract Baicalin on Aeromonas hydrophila infection of T.sinensis was further explored,in order to lay a foundation for the healthy breeding and disease prevention and control of T.sinensis.The main results are as follows:(1)Isolation and identification of P.seudomonas aeruginosa and A.hydrophila from T.sinensisBacterial infection can cause outbreaks of various diseases in T.sinensis,bacterial disease is one of the important factors that endanger the healthy breeding of T.sinensis.In this study,the first illness of diseased T.sinensis manifested as loss of appetite and fed hardly in the early stage of the onset,followed by hemorrhage on the body surface,rotting of the back,and occasional blood in the stool.After dissecting,it was found that the liver was enlarged and there was a large amount of ascites in the abdominal cavity.The second disease manifested as loss decreased mobility,trauma on the back shell accompanied by inflammation and decay,redness of the bottom plate,and a large number of bleeding spots.After dissecting,the liver was whitish and fibrotic,and the outer wall of intestinal tract was wrapped in fat.In this study,the pathogenics of T.sinensis were isolated.They were identified as A.hydrophila and P.aeruginosa based on 16 S r RNA gene sequence,scanning electron microscope observation and biochemical characteristics.Artificial infection tests confirmed that the isolates were the pathogen causing the disease of T.sinensis.Drug sensitivity tests demonstrated that the three P.aeruginosa were sensitive to eight antibiotics(such as ceftazidime)and resistant to six antibiotics(such as florfenicol,doxycycline and sulfaisoxazole).According to the results of MLST(Multilocus Sequence Typing),the three isolates all belonged to sequence type 252(ST252).The further e BURST analysis showed that ST252 formed a clonal complex CC252,of which ST252 was the founder ST.In addition,drug sensitivity tests demonstrated that A.hydrophila from T.sinensis was sensitive to nine antibiotics(such as florfenicol,doxycycline,sulfagan,aminoglycosides and carbapenems).The results for aquaculture antibiotics demonstrated that the isolate was sensitive to neomycin,enrofloxacin,floxacin,and florfenicol,resistant to sulfaisoxazole.(2)Whole-genome analysis of P.aeruginosa FG10 and A.hydrophila B3The whole genome of FG10 had a size of 4.90 Mb,the average G+C content of65.3%,and 5956 coding sequences.The whole genome has been deposited at Gen Bank under the accession number JAJGXC000000000.In comparison with the Virulence Factor Database(VFDB),873 virulence-related genes were predicted,which were mainly associated with adhesion,secretion systems,and toxin.In comparison with the Comprehensive Antibiotic Resistance Database(CARD),some drug(such as fluoroquinolone,carbapenem,and peptide)resistance related-genes were predicted.The whole genome of B3 had a size of 4.90 Mb,the average G+C content of60.79%,and 4600 coding sequences.In comparison with the Virulence Factor Database(VFDB),648 virulence-related genes were predicted,which were mainly associated with adhesion,secretion systems,and toxin.In comparison with the Comprehensive Antibiotic Resistance Database(CARD),the main drug resistance methods are via changing the antibiotic targets and antibiotic efflux.The main drug resistance antibiotics are aminoglycosides,quinolones,and peptides.(3)Protective effect of traditional Chinese medicine additives on T.sinensis infected with Aeromonas hydrophilaIn this study,we determined that Scutellaria and Baicalin had obvious bacteriostatic effect on A.hydrophila B3 by inhibition zone method,and Baicalin is the effective component of Scutellaria.After feeding T.sinensis with feed Scutellaria additives for three months,it was found that the mortality rate was reduced after B3 infection,and the expression of immune-related genes myd88,il6,c3 a,and dusp7 was also significantly higher than those of control(p<0.001).The Scutellaria additives could significantly promote the immunity of T.sinensis.At the same time,it was found that the Scutellaria additives could not promote the growth of T.sinensis(p<0.001).Through the sequencing of gut microbial diversity,it was found that the diversity of gut microbial composition of T.sinensis fed with Scutellaria was relatively simple and not enriched.