| Chinese soft-shelled turtle（Pelodiscus sinensis）is an important amphibian with edible,officinal and researchful value.As one of famous freshwater aquaculture species in China,soft-shelled turtle has high economic benefits and market demands.In recent years,due to the rapid development of high density and temperature control of artificial farming industry,bacterial turtle diseases have made severe economic losses to turtle breeding industry.Mass death-off occurred for unsure reasons in Eco-farm cultured turtles in Xiantao,Hubei province in 2015,and the mortality of peak period of incidence is 100%.In this study,an epidemiological investigation was carried out aiming at main breeding areas in Hubei province.After that,the bacterial pathogen was identified and investigated.In addition,pathogenicity experiment and drug-resistance characteristics of different isolates were investigated.The research results were summarized as follows:1.The investigation of epidemic situation about fulminant death of Pelodiscus sinensis in Hubei provinceBy the means of the epidemiological survey of Pelodiscus sinensis in some main breeding areas of Hubei province from Aug.to Oct.in 2015,we finded out that fulminant death disease mainly outbreaked in high temperature seasons,especially from June to August.And the disease centralizingly occurred in adult turtles more than 200g,which attacked healthy turtle cultured in greenhouse for consecutive years.The sick turtles were floating or lying with less food and movement,which body were gray and covered by bleeding spots,even with small ulcer pit on the back.Fulminant death of Chinese turtle was difficult to control,while it has a long incubation period and high mortality.What’s more,parasites and virus were not detected,and the highest detection rate of bacterial pathogens were Citrobacter freundii,Morganella morganii and Klebsiella pneumoniae.Factors such as temperature fluctuation,deterioration of water quality,genetical characterization decline and misgovern of breeding were very likely to cause the infection of soft shelled turtle.2.The study of identification and pathogenicity of isolated strainsThe research got 3 strains of gram-negative bacillus from the lesion site of the turtles.By observing the phenotypic characteristics of colonies,using traditional physiological and biochemical identification methods to identify what category the isolated bacteria belong to,combining with molecular biology method.Phylogenetic analysis was used to explore the evolutionary position by sequencing 16S rRNA gene.It indicated that both the results were the same,16S rRNA gene of isolates enjoyed high homology with corresponding genes of other similar bacteria.The results showed that isolated pathogen were Citrobacter freundii,Morganella morganii and Klebsiella pneumoniae.Artificial injection with isolated bacteria caused death with different rate,and the symptom were similar to the natural infected diseased turtles in a large scale.After re-isolating,the same pathogenic bacteria were isolated from artificial infected turtles.M.morganii had the lowest LD50,which was 2.3×107CFU/mL,while the mix bacteria solution had highest fatality rate.The above results showed that the strains Citrobacter freundii,Morganella morganii and Klebsiella pneumoniae were the pathogen of fulminant death,infecting healthy turtles with 3 kinds of bacteria.3.The drug-resistance characteristics of three isolated bacteriaTo research the drug-resistance characteristics of 3 isolated strains,23 kinds of antibiotic were chosen to test by disk diffusion method.The results showed that the sensitivity of 3isolates to 23 antibiotics was different:Citrobacter freundii were highly sensitive to Ceftazidime,Ceftriaxone and Levofloxacin while extremely resistant to 17 kinds of drugs such as Vancomycin.;Morganella morganii were very sensitive to Ceftriaxone,Ceftriaxone and highly resistant to 17 kinds of drugs such as Clindamycin;Klebsiella pneumoniae were hypersensitive to 13 kinds of drugs such as Enrofloxacin and extremely resistant to 6 kinds of drugs such as Penicillin.A comparative analysis showed that 3 isolates were absolutely resistant to vancomycin,clindamycin,penicillin,rifampicin,imipenem and ampicillin and completely sensitive to cephalosporin such as ceftazidime and ceftriaxone,and certain extent resistance to other kinds of drug.Different strains were different in drug resistance,which may mainly related to characteristics of the drug,the culture conditions and the different mechanisms of drug resistance.In production,the third and fourth generation cephalosporins can be used to disease treatment.Otherwise,it’s effective to achieve the purpose of preventing and controlling fulminant death of P.sinensis by strengthening the management of aquaculture,sterilizing the pond regularly,controlling the water temperature and quality. |
