Isolation And Identification Of Nocardia Seriolae And Establishment Of Its Fluorescent Quantitative PCR Detection Method

Nocardia seriolae is a common pathogen in aquaculture production,which can cause chronic systemic granuloma in fish.Due to the lack of effective prevention methods of the disease,the mortality rate of infected animals was 30%-60%,which seriously jeopardized the healthy and sustainable development of aquaculture industry.Therefore,it is of great theoretical significance to study the pathogenicity of Nocardia seriolae and to establish a rapid diagnostic method for Nocardia seriolae.In April 2020,a disease characterized by bleeding skin,ulceration and nodules of internal organs occurred in Micropterus salmoides raised in a fishery in Linquan County,Anhui Province,which was suspected to be Nocardia disease.This study isolated and identified the pathogen of the disease that is Nocardia seriolae.On this basis,the pathogenicity of the Nocardia seriolae was further studied,and Fluorescent quantitive Polymerase Chain Reaction（q PCR）was established for the rapid diagnosis of Nocardia seriolae disease.The main research contents and results are summarized as follow:1.Isolation and identification of pathogenic bacteriaThe tissue nodules of diseased fish were inoculated TSB medium to isolate bacteria at first.The dominant strain was obtained after purification,which was named as isolates NI.Then,on the basis of screening out the best culture method for NI isolates,the morphological,physicochemical characteristics and drug sensitivity of the isolates were preliminarily identified.Finally,by amplifying and sequencing the 16S r RNA gene of the NI isolates,and amplifying the 16S-23S r RNA transcriptional spacer of Nocardia seriolae by PCR specifically.The NI isolates was further identified as Nocardia seriolae.2.Study on the pathogenicity of Nocardia seriolae NI isolatesFour different concentrations of NI isolates（5.00×10⁸CFU/m L,6.25×10⁷ CFU/m L,7.8×10⁶ CFU/m L and 9.75×10⁵ CFU/m L）were used to artificially infect experimental fish.Its LD₅₀ was 1.29×10⁷CFU/m L.Then,the gills,head kidney,heart,stomach,liver,spleen,body kidney,intestine and muscle tissues of artificially infected fish were collected for pathological section and HE staining.The main pathological changes were degeneration and necrosis in the cell of tissues,granulomatous lesions in internal organs.3.Establishment of a fluorescent quantitative PCR method for detection of Nocardia seriolaeThe highly conserved Gluconate Operon Transcriptional repressor（Gnt R）of Nocardia seriolae was selected as the test target gene.Firstly,the recombinant clone plasmid p MD19-T-Gnt R was constructed as the positive standard.Then,the positive standard was used to optimize the reaction conditions of q PCR,and the standard curve was established and the linear regression equation was deduced.Lastly,the specificity,sensitivity and stability of the established method were evaluated.The sensitivity of q PCR was 8.98×10³ copies/μL,100 times higher than that of conventional PCR.The Coefficients of variation between intra-group and inter-group were less than 10%.Using the method to measure the organization microbial load of artificial infection fish for the experiment.The results showed the microbial load from high to low is head kidney,heart,liver,stomach,spleen,intestine,muscle,body kidney,and gill tissue,respectively.At the same time,Nocardia seriolae was isolated from the detected tissues（except gills）.To sum up,this study isolated and identified a strain with strong pathogenicity from diseased Micropterus salmoides in Linquan County,Anhui Province.With Gnt R gene as the detection target,a fluorescence quantitative PCR method for detection of Nocardia seriolae was successfully established.The method has strong specificity,high sensitivity and good stability.And it can be applied to the diagnosis of Nocardia seriolae disease.