虹鳟源嗜冷黄杆菌的分离鉴定及致病性研究

为确定患病虹鳟的病原，本实验从患病鱼溃烂肌肉中分离到2株细菌，分别命名为CH06和CH07，经回归感染证实分离菌株为导致此次虹鳟患病的病原菌，并进一步对其形态特征、理化特性、分子特征、血清型及耐药性进行分析。结果显示，CH06和CH07株在TYES琼脂平板上呈煎蛋状外观，产黄色素，氧化酶和过氧化氢酶呈阳性，能水解明胶和酪蛋白，不能水解淀粉，不能利用果糖、半乳糖和七叶苷等。16S rRNA比对结果显示，CH06和CH07株与嗜冷黄杆菌模式株NBRC 15942的同源性分别为99.35%和99.42%。综合菌株理化和分子特性确定CH06和CH07株为嗜冷黄杆菌。利用多重PCR方法鉴定CH06和CH07株的血清型均为1型(Fd型)；多位点序列分型(MLST)分析表明，CH06和CH07株的基因型分别为ST-12和ST-78型，且均属于CC-ST10克隆型。人工感染结果显示，CH06和CH07株对虹鳟幼鱼具有较高致病性，其半致死浓度(LD₅₀)分别为7.1×10⁵和1.1×10⁵ CFU/mL，攻毒剂量与临床病症出现时间呈反比，从人工感染实验鱼的肌肉、脾脏等组织中可重新分离到嗜冷黄杆菌。组织病理变化显示，病鱼肝细胞肿胀，空泡变性，部分肝细胞溶解坏死，细胞核溶解消失；脾脏充血、出血，淋巴细胞减少，红细胞和含铁血黄素增多；肌纤维间隙增宽、断裂、弯曲不齐，部分肌细胞肌浆溶解呈蜂窝状。CH06和CH07株对10种抗菌药物的耐药谱略有不同，均对氨苄西林和甲氧苄啶-磺胺甲噁唑敏感；CH06株对恩诺沙星、氟苯尼考等耐药，而CH07株对恩诺沙星和氟苯尼考中度敏感。本研究首次报道了我国虹鳟源嗜冷黄杆菌的分离鉴定及生物学特性，以期为虹鳟细菌性冷水病的防控提供科学依据。

Isolation, identification and pathogenicity of Flavobacterium psychrophilum from Rainbow trout(Oncorhynchus mykiss)

As the causative agent of bacterial cold-water disease (BCWD), Flavobacterium psychrophilum mainly infects juvenile salmonids and causes great economic loss in salmonid aquaculture worldwide. F. psychrophilum is characterized by a broad geographical distributions and hosts, inducing typical clinical signs of caudal peduncle erosion, skin ulcerations, pale gills, enlarged spleen, ascites and spiral swimming behavior in diseased fish. Although studies have been carried out on the epidemiology and pathogenicity of this bacterium in many countries, there is still no report on F. psychrophilum strains isolated from cultured salmonids in China. In the present study, two pathogenic bacterial strains (CH06 and CH07) were isolated from the muscle lesions of diseased rainbow trout (Oncorhynchus mykiss) using a polyphasic approach. The results indicated that CH06 and CH07 isolates had an egg-like appearance on TYES agar, producing flexirubin-like pigments, and positive for oxidase and catalase. It can hydrolyze gelatin and casein, but cannot hydrolyze starch or use fructose, galactose and esculin. The results of 16S rRNA alignment suggested that the homology of CH06 and CH07 isolates with F. psychrophilum type strain NBRC 15942 strain were 99.35% and 99.42%, respectively. Based on the biochemical and molecular characteristics results, CH06 and CH07 isolates were identified as F. psychrophilum. The multiplex PCR method was used to identify the serotypes of CH06 and CH07 isolates as type 1 (Fd type). MLST analysis indicated that the genotypes of CH06 and CH07 isolates were ST-12 and ST-78, respectively, both belonging to CC-ST10. The artificial infection results indicated that CH06 and CH07 isolates were highly pathogenic to juvenile O. mykiss with the LD₅₀ values of 7.1 × 10⁵ and 1.1 × 10⁵ CFU/mL, respectively. The dosage of the challenge was inversely proportional to the time when the clinical signs appeared. F. psychrophilum was successfully re-isolated from muscle, spleen of artificially infected fish. Histopathological changes showed that the hepatocytes of the diseased fish were swollen and vacuoles degenerated. Some hepatocytes were lysed and necrotic and the nucleus was dissolved. The spleen was congested and hemorrhagic with reduced amounts of the lymphocytes and increased amounts of the red blood cells and the hemosiderin. The muscle fiber gap was widened and broken, with uneven bending. Parts of the muscle cell sarcoplasmic dissolution was honeycomb-like. CH06 and CH07 isolates exhibited slightly different resistance profiles to 10 antibiotics. They were both highly susceptible to ampicillin and trimethoprim-sulfamethoxazole, while CH06 isolate was resistant to enrofloxacin and florfenicol and CH07 isolate was moderately susceptible to enrofloxacin and florfenicol. This is the first report on the biological characteristics of F. psychrophilum isolates recovered from cultured O.mykiss in China, and it is expected to provide scientific support for the prevention and control of BCWD in salmonid aquaculture.