养殖中华鳖蜡样芽孢杆菌的分离、鉴定和致病性研究

2016年夏杭州某中华鳖(Pelodiscus sinensis)养殖场出现大量发病, 病鳖呈厌食、反应迟钝、四肢无力、池边独游、濒死前不停摇头等病症, 死亡率20%以上。用营养琼脂从典型症状濒死中华鳖分离到革兰阳性短杆状, 芽孢近中生或中生的分菌株; API 50 CHB对分离菌株T91-1鉴定结果显示与蜡样芽孢杆菌(Bacillus cereus)参考菌株ATCC 14579 97.4%相似; T91-116S rDNA和gyrB基因序列与ATCC 14579相似度为99%, 确认T91-1为蜡样芽孢杆菌。T91-1在羊血平板呈典型β溶血, 牛奶平板上出现明显透明圈, 表明存在溶血性和胞外蛋白酶类毒素。用T91-1肌肉注射感染健康中华鳖LD₅₀为4.91×105CFU/ind., 死亡鳖出现与自然发病鳖类似症状; 感染发病中华鳖血和肝印/涂片瑞氏-姬姆萨染色可见组织间隙有大量芽孢杆菌。自然发病鳖肾、肝、肺、心病理切片可见大量芽孢杆菌, 病鳖血管扩张充血, 嗜酸性粒细胞浸润。T91-1灌胃ICR乳鼠3d后可全部死亡, 死亡鼠呈皮下出血等症状; T91-1腹腔注射和灌胃ICR 4周龄小鼠12—24h可出现迟钝、厌食等症状, 1周内未出现死亡。上述结果表明蜡样芽孢杆菌对中华鳖有很强毒力, 对小鼠有较强肠毒性, 是引起本次杭州中华鳖暴发性疾病的病原。

ISOLATION,IDENTIFICATION AND PATHOGENICITY ANALYSIS OF BACILLUS CEREUS FROM CHINESE SOFT-SHELLED TURTLES, PELODISCUS SINENSIS

The epizootic outbreaks were recorded in Chinese soft-shelled turtle farm in Hangzhou, Zhejiang province in summer of 2016. The diseased turtles were an orexia, lags in responsive, abnormal or lonely swimming. The moribund turtles would creep and lie on the shore with continuous head-shaking with a 20% mortality. Gram positive rod with single endospore were isolated from liver, kidney, spleen and blood of moribund turtles with typical symptoms. Stain T91-1 was identified as Bacillus cereus with API 50 CHB strip, 97.4% similarity to B. cereus reference strains by BioMérieux APIWEB Plus software V3.2.2. The identification was confirmed by phylogenetic analysis using 16S rDNA and gyrB whole gene, 99% similarity to type strains ATCC14579 of B. cereus T91-1 that showed typical β-hemolytisis on sheep blood agar and large transparent circle on the milk agar, indicating the existence of hemolytic and extracellular protease toxin. The T91-1 challenged turtles died from day 3 to day 7 with the symptoms similar to natural infected turtles, and LD₅₀ of B. cereus T91-1 was 4.91×105 CFU/ind. Large number of Bacillus-like rods were observed in blood and liver smear stained with Wright-Giemsa. A large number of Bacillus-like were found in kidney, liver, lung and heart of native diseased turtles with vascular dilatation and congestion, as well as infiltrated eosinophils. All ICR suckling mice were killed at day 3 after oral administrated with T91-1 at concentration ranged from 1.2×106 CFU/mL to 1.2×108 CFU/mL with hemorrhagic subcutis and cerebrum. Four weeks old mice were injected or oral administrated with T91-1 at 1×107—109 CFU/mL, which showed anorexia, lags in responsive within 12h to 24h, but no death was recorded. These results demonstrated that B. cereus T91-1, with the strong pathogenicity to Chinese soft-shelled turtles and enterotoxigenic to mice, could be the pathogen of outbreaks of Chinese soft-shelled turtles diseases in this farm in Hangzhou.