Isolation And Identification Of Bacterial Pathogen Of Trionyx Sinensis Big Neck Disease

Trionyx sinensis is the name of our country important freshwater aquaculture species, with the feature of its rich nutrition, delicious taste and strong nourishment. There is rare disease in wild Chinese turtle, but with the high density and intensive development of artificial breeding production, all kinds of diseases frequently occur, thus the disease problem of Chinese turtle becomes one of the main obstacles restricting the development of artificial breeding.There are two strains separated from diseased turtle neck and mouth in this study, which are labeled No.1 and No.2, respectively. To test and verify the pathogenicity of No.1 and No. 2 strains, we will conduct artificial infection experiment.The experimental results show that the pathogenic strength of No.1 and No.2 mixed infection is greater than the pathogenicity of No.1 or No.2 infection alone, and intraperitoneal injection is easier to make healthy Chinese turtle pathogenic than soaking method. The main organs of the diseased Chinese turtle are used to observe pathological morphology by tissue slice technique, we discover that intestine, liver, lung, kidneys, trachea, spleen and gonad organ have appeared obvious pathological changes, especially, the mutation of lung tissue is the worst, which may cause breathing difficulties, and then suffocation to death.No.1 and No.2 strains are analyzed by physiological and biochemical identification, joining with their morphological characteristics. We can determine primarily that No.1 bacteria is Aeromonas hydrophila; No.2 bacteria is Frau Coli citrate or folic Coli citrate, two of which belong to Citrobacter freundii, but exactly which kind of bacteria has no conclusive evidences. No.1 and No.2 strains are used to determine 16s rRNA sequences amplification and sequencing. The sequencing results will be inputed to NCBI, with BLAST tools for sequence homology comparison analysis. Then we find that No.1 strain has a closer relative with Aeromonas hydrophila; No.2 strain has a closer relative with Citrobacter freundii. Meanwhile,combining with the morphological characteristics and the result of physiological and biochemical identification, we can confirm that No.1 bacteria is Aeromonas hydrophila, and No.2 bacteria is Citrobacter freundii; The results of drug susceptibility test show that No. 1 and No.2 bacteria are highly sensitive to ceftazidime, cefepime, ciprofloxacin, amikacin, ciprofloxacin, meropenem antibiotics, but which have drug resistance for ampicillin and piperacillin/tazobactam.This study carried out by collecting a case infected a new bacterial disease of "Chinese turtle neck disease", the new bacteria was founded in the multiple Chinese turtles farms of hunan, which is used pathogen separation, purification and 16s rRNA sequence analysis, then infection testing and histopathological study after artificial regression to determine the pathogen pathogenicity, and drug sensitivity experiment preliminarily to confirm therapy of the disease. Therefore, this study could provide a theoretical basis for the therapy of the disease in practical production.