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.