广西人工养殖牛蛙源嗜水气单胞菌的分离鉴定及致病力分析

Isolation,identification and pathogenicity analysis of Aeromonas hydrophila from farmed bullfrog(Rana catesbeiana)in Guangxi

  • 摘要: 【目的】确定引起广西人工养殖牛蛙(Rana catesbeiana)发病死亡的病原菌,了解其致病性、组织病理学特征、耐药性及耐药基因的携带情况,为该病的临床诊断及合理预防提供科学依据。【方法】采用常规细菌分离纯化方法从患病牛蛙的病灶组织中分离优势菌株,通过观察分离菌株的形态特征、生理生化特征和16S rRNA基因序列分析等对病原菌进行鉴定,以纸片扩散法测定其耐药性,通过PCR扩增分析其耐药基因和毒力基因携带情况,通过人工回归感染试验确定病原菌的致病性,并通过组织病理学探究牛蛙感染菌株后的组织特性。【结果】从患病牛蛙腿部分离得到1株优势菌株(编号NFAH-5),综合其形态学、生理生化特性、溶血特性及分子鉴定结果,确定为嗜水气单胞菌(Aeromonas hydrophila)。菌株NFAH-5对多西环素、多粘菌素B和庆大霉素等6种抗生素敏感,对头孢拉定、恩诺沙星和阿莫西林等12种抗生素已产生耐药性;经耐药基因PCR检测,发现该分离菌携带Ant (3″)-I、Intl1和parC等9种耐药基因,与耐药表型基本一致。毒力基因PCR检测结果显示,菌株NFAH-5携带Ser、aer、Lip、exu、LuxS、actaha等7种毒力基因。人工回归感染试验结果表明,菌株NFAH-5感染牛蛙的半数致死剂量(LD50)为1.55×106 CFU/mL,感染后发病牛蛙表现四肢底侧充血发红,肝肿大呈红褐色或黄色,肠内空虚或有出血,肾充血发红,与自然发病牛蛙的症状相似。患病牛蛙的肝细胞发生变性,脾脏红、白髓质间很难辨出,可见大量血源性色素沉着物,肾小球萎缩,肾小管发生玻璃样变。【结论】嗜水气单胞菌是引起广西南宁人工养殖牛蛙发生红腿病的致病菌,结合农业农村部《水产养殖用药明白纸2022年2号》文件中批准使用的药物目录,建议选用敏感药物多西环素进行防治。

     

    Abstract: 【Objective】This study aimed to identify the pathogenic bacteria causing the morbidity and death of bullfrog(Rana catesbeiana),and to understand their pathogenicity,histopathological characteristics,drug resistance and the carrying of drug resistance genes,it would provide reference for the clinical pathological diagnosis and scientific prevention and treatment of bullfrog.【Method】Conventional bacterial isolation and purification methods were used to isolate dominant strains from the lesion tissues of diseased bullfrog. The pathogenic bacteria were identified by observing the morphological characteristics,physiological and biochemical characteristics and 16S rRNA gene sequence of the isolated strains. The drug resistance was determined by disk diffusion method,the carrying status of drug resistance genes and virulence genes was analyzed by PCR amplification,and the pathogenicity of the pathogenic bacteria was determined by artificial regression infection test. Histopathology was used to study the tissue characteristics of infected bullfrog.【Result】A dominant strain(NFAH-5)was isolated from the leg of the infected bullfrog. Based on the morphological,physiological and biochemical characteristics,hemolysis characteristics and molecular identification results,the isolated strain was identified as Aeromonas hydrophila. The strain NFAH-5 was sensitive to six antimicrobial drugs including doxycycline,polymyxin B and gentamicin,and was resistant to twelve antibiotics such as cporadine,enrofloxacin and amoxicillin. According to PCR test of the resistance genes,the isolate carried nine resistance genes such as Ant(3″)-I,Intl1 and parC,which was basically consistent with the drug resistance phenotype.Virulence gene PCR test revealed that isolate NFAH-5 carried seven virulence genes,including Ser,aer,Lip,exu,LuxS,act,and aha. Strain NFAH-5 infected bullfrog with a median lethal dose(LD50)of 1.55×106 CFU/mL. Infection-affected bullfrog had congestion and redness on the underside of the limbs,reddish-brown or yellow liver enlargement,intestinal emptiness or bleeding,and renal congestion. Symptoms were similar to those of naturally diseased bullfrog. The liver cells of the diseased bullfrog were degenerated;the red and white medulla of the spleen were difficult to distinguish,and a large amount of blood-derived pigmentation,glomerular atrophy,and hyaline degeneration of the renal tubules were observed.【Conclusion】A. hydrophila is the pathogenic bacterium that causes red leg disease in breeding bullfrog in Nanning,Guangxi. Combining the list of drugs approved for use in the document Paper on Aquaculture Drugs No. 2 in 2022 by Ministry of Agriculture and Rural Affairs,the sensitive drug doxycycline can be used to control it.

     

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