克氏原螯虾细菌性病原分离鉴定及血清免疫因子变化

Isolation and identification of bacterial pathogens from Procambarus clarkii and study on variations in serum immune factors

克氏原螯虾是当今中国人工养殖最广的淡水螯虾品种。近几年来,稻田养虾面积呈暴发式增长,但随着养殖规模的扩大,养殖环境的恶化,各种病害频发。为探明稻田养殖克氏原螯虾死亡的原因,实验取濒死病虾胃和肠组织进行病原菌分离,用生理生化实验、16S rDNA基因序列测定与系统进化分析鉴定优势菌;用回归感染和药敏实验评价优势菌毒力和药物敏感性,通过血清免疫相关酶活性变化探究优势菌的致病机理。结果显示,分离获得1株优势菌PCW-01,回归感染健康克氏原螯虾,出现与自然发病虾相同的病理症状,活力下降,胃部有积液,半致死量达0.60×10^(5)CFU/g,经理化分析和分子鉴定为维氏气单胞菌,对呋喃妥因、妥布霉素、头孢他啶等7种药物敏感。人工感染后的克氏原螯虾,体内血清谷丙转氨酶(ALT)和溶菌酶(LZM)活性极显著上升,总抗氧化能力(T-AOC)、总超氧化物歧化酶(T-SOD)和谷胱甘肽过氧化物酶(GSH-Px)活性极显著下降,丙二醛(MDA)含量略有上升。研究表明,优势菌PCW-01为致病菌,可失活抗氧化因子,破坏细胞膜结构,导致疾病发生。本研究结果揭示出了克氏原螯虾细菌性疾病的免疫应答规律,为其细菌性疾病的防控提供了科学依据。

In China,Procambarus clarkii is the most widely cultivated freshwater crayfish now,the breeding area and output are increasing every year and the culture models have also been constantly innovated.In recent years,the area of rice-P.clarkii co-culture has been increasing explosively,and its output has accounted for more than 80%of the national total output,so the rice-P.clarkii co-culture has become the basis for the rapid development of crayfish industry.With the expansion of aquaculture scale and deterioration of aquaculture environment,various diseases occur frequently which seriously affect the yield and quality of P.clarkii.In order to find out the death cause of P.clarkii cultured in paddy field,the pathogenic bacteria were isolated from stomach and intestine of these dying crayfish with obvious pathological characteristics,and were identified through physiological and biochemical testing,sequence alignment of 16S rDNA gene and phylogenetic analysis.At the same time the virulence and drug sensitivity of pathogenic bacteria were evaluated by artificial infection and drug sensitivity test,the pathogenic mechanism of pathogenic bacteria was studied by analyzing the variations of serum immune related enzymes activities of P.clarkii injected with pathogenic bacteria.Research results showed that a dominant strain named PCW-01 was obtained and identified as Aeromonas veronii which could cause the healthy P.clarkii to have these same pathological symptoms as the natural disease,such as less exercise,effusion in stomach,excessive intestinal mucus in the regression experiment,and its median lethal dose was 0.60×10^(5) CFU/g to P.clarkii.The analysis of antimicrobial susceptibility test showed that A.veronii(PCW-01)was highly sensitive to furantoin,tobramycin,ceftazidime and other seven antibiotics.The results of serum immune enzyme test showed that in the body of P.clarkii injected with pathogenic bacteria,the activity of lysozyme(LZM)and alanine arninotransferase(ALT)increased considerably(P<0.01),the activity of total antioxidant capacity(T-AOC),total superoxide dismutase(T-SOD)and glutathione peroxidase(GSH-Px)decreased notably(P<0.01),and the concentration of MDA increased slightly(P>0.05).The conclusion was that the strain PCW-01 was the pathogen of the death of P.clarkii,which could inactivate antioxidant factors,destroy cell membrane structure and function,subsequently the pathological phenomena of tissues and organs or death appeared.In the study,these results revealed immune response law of P.clarkii to pathogenic bacteria and will provide scientific support for the prevention and control of the bacterial diseases of P.clarkii in future.