Purification and characterization of the vascular permeability factor produced by Bacillus cereus

Purification of an extracellular protein exhibiting the vascular permeability activity produced by Bacillus cereus was performed by ammonium sulfate precipitation followed by chromatography on DE-32 cellulose, Sephadex G-100, and Sephadex G-75. The purified protein was found to be electrophoretically and antigenically almost homogeneous although it contained a trace of contaminant. The molecular weight of the protein was calculated to be 45,000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The purified protein showed vascular permeability activity and mouse lethal toxicity, and caused fluid accumulation in ligated mouse intestinal loops, whereas it did not show any hemolytic and lecithinase activities. From these findings, the purified protein is suggested to be an enterotoxin (or a diarrheagenic toxin) responsible for diarrhea caused by B. cereus in a diarrheal-type food poisoning.     

变质鸡蛋中蜡样芽孢杆菌的分离与鉴定

从山东地区保存超过14 d大范围出现变质鸡蛋中分离到一株细菌,经过培养特性、生理生化鉴定和16S rDNA鉴定,鉴定为蜡样芽孢杆菌(Bacillus cereus).分别将变质鸡蛋、煮熟的变质蛋、健康鸡蛋进行厌氧培养和细菌计数,发现健康鸡蛋和煮熟的变质蛋无蜡样芽孢杆菌,而变质鸡蛋中的细菌数量达到1.3×108CFU/g.本试验表明,蛋黄中过量的蜡样芽孢杆菌可能与鸡蛋变质有关,应注意过量添加蜡样芽孢杆菌可能影响蛋品的保鲜和食用安全性.     

蜡状芽孢杆菌的分离培养与鉴定

从牛奶中分离到一株蜡状芽孢杆菌CO4-1,经生长特性培养、生化试验、灌服动物试验,证明该菌株具有营养要求低、生长速度快、合成蛋白质能力强、耐高温、耐酸碱等特点,菌体及其产物对动物无任何不良反应,对肠道病原菌具有一定的抑制作用。     

Fluid accumulation in mouse ligated intestine inoculated with the vascular permeability factor produced by Bacillus cereus.

Partially purified vascular permeability (VP) factor (VPF) of Bacillus cereus induced fluid accumulation in the ligated intestinal loops of mouse (MIL) and rabbit (RIL), suggesting that the VP activity may correlate with fluid accumulation in ligated intestinal loops of these animals. Fluid accumulation was observed at 6-8 hr in 55-67% of mouse intestinal loops inoculated with 40-50 immunodiffusion units (IDU) of partially purified VPF, whereas it was found at 2 hr in all loops with 400-600 IDU of partially purified VPF. In rabbit intestinal loops with 120-190 IDU of partially purified VPF, fluid accumulation was observed at 6 hr. From these findings, VPF produced by B. cereus can be easily detected in both MIL and RIL. The intestinal tissue of mouse intestinal loops was histopathologically damaged at different concentrations of the VPF to induce fluid accumulation. With 50 IDU of partially purified VPF, severe edema was found in the laminia proprial layer and submucosa. With 600 IDU of partially purified VPF, on the other hand, severe necrosis in the surface epithelium of villus and laminia proprial layer, and hyperemia in the submucosa were observed, suggesting that partially purified VPF may be cytotoxic and/or intestinecrotic.     

Bacillus cereus from the environment is genetically related to the highly pathogenic B. cereus in Zambia

To follow-up anthrax in Zambia since the outbreak in 2011, we have collected samples from the environment and the carcasses of anthrax-suspected animals, and have tried to isolate Bacillus anthracis. In the process of identification of B. anthracis, we collected two isolates, of which colonies were similar to B. anthracis; however, from the results of identification using the molecular-based methods, two isolates were genetically related to the highly pathogenic B. cereus, of which clinical manifestation is severe and fatal (e.g., pneumonia). In this study, we showed the existence of bacteria suspected to be highly pathogenic B. cereus in Zambia, indicating the possibility of an outbreak caused by highly pathogenic B. cereus.     

Bacillus cereus var. toyoi enhanced systemic immune response in piglets

Probiotic bacteria have been suggested to stimulate the host immune system. In this study we evaluated the immunomodulatory effects of probiotic Bacillus cereus var. toyoi on the systemic immunity of piglets. A pool of 70 piglets was divided into a probiotic or control group. We determined the ratios of peripheral blood mononuclear cell (PBMC) subsets and measured proliferative responses and cytokine production of PBMCs and effects on vaccination responses. Blood samples of probiotic-treated piglets showed a significantly lower frequency of CD8(high)/CD3+ T cells and CD8(low)/CD3+ T cells and a significant higher CD4+/CD8+ ratio. IL-4 and IFN-gamma production of polyclonally stimulated PBMCs was on average higher in the probiotic group. Specific proliferative responses of PBMCs to Influenza vaccination antigens were significantly higher and antibody titers against H3N2 Influenza and Mycoplasma vaccination antigens were on average higher in the probiotic group. In conclusion, B. cereus var. toyoi therefore alters the immune status of piglets as indicated by changes in the ratios as well as functionalities of systemic immune cell populations.     

The occurrence of Bacillus cereus in Finnish dog food sausages; a microbiological and physiochemical survey.

A bacteriological survey of five brands of commercial dog food sausages showed that two of them were heavily contaminated with B. cereus. One of the two brands had a suspected association with food-poisoning incidents in dogs. In addition to B. cereus the other sausage brand was found to contain a high number of sulphite reducing anaerobes. B. cereus was detected in almost every raw material of the two brands of sausages. The pasteurization during manufacturing did not kill all the sporeforming organisms. The water activity of the sausages were equal to or more than 0.99 and pH in a range from 6.2 to 7.0. Methods of preventing the growth of microbes in the sausages after manufacturing are discussed.     

Effects of Bacillus cereus var. toyoi on immune parameters of pregnant sows

Changing immune parameters during pregnancy have previously been reported in humans and cattle, and have been suggested to contribute to increased susceptibility to infections. However, data regarding immune parameters during pregnancy in sows are rare. In this study, we investigated the peripartal immune status of sows using phenotypical (FACS analysis) as well as functional (proliferation assays, cytokine analysis) parameters of peripheral blood mononuclear cells (PBMCs) in pregnant sows. In previous studies, we reported a modulation of the immune system after feed supplementation of the probiotic Bacillus cereus var. toyoi in piglets [Schierack, P., Wieler, L.H., Taras, D., Herwig, V., Tachu, B., Hlinak, A., Schmidt, M.F., Scharek, L., 2007. Bacillus cereus var. toyoi enhanced systemic immune response in piglets. Vet. Immunol. Immunopathol. 118, 1–11]. Here, we extended these previous studies to include investigations of possible probiotic effects on the peripartal immune status of sows and their reproductivity. We show that immune parameters of sows change during pregnancy, the proliferative response of PBMCs to several bacterial antigens in control animals decreased from days 90 to 30 ante partum. Relative numbers (%) of CD3+CD8+, CD4+, cytotoxic T, CD14+ and CD21+ cells were reduced compared to non-pregnant sows. In contrast, the proliferative response of PBMCs of probiotic-treated sows increased during pregnancy. Bacterial antigens primarily stimulated the proliferation of naïve CD21+ cells and the relative CD21+ cell numbers were elevated in the probiotic group in the absence of effects on other immune cell populations. The clinical and microbial status of both control and probiotic sows was similar, excluding pre-existing health problems or infections as responsible for the immunological changes, and feed supplementation also had no significant effects on reproductivity. The results suggest that the probiotic B. cereus var. toyoi can alter the proliferative response of lymphocytes and affects the immune cell population ratios of pregnant sows. How and to what extent this may affect health and reproductivity should be the focus of further studies.     

鱼粉中蜡状芽胞杆菌的分离鉴定及其致病性研究

本文在国内首次报道了由蜡状芽胞杆菌引起的腹泻病。某鸡场鸡群因饲喂了某批鱼粉而引致发病, 经检查证明鱼粉中含有蜡状芽胞杆菌, 经计数为６０ ×１０６ 个／ｇ , 远远超出鱼粉的卫生标准。文中对该菌的致病性进行了初步研究, 结果可致死小鼠, 药敏试验结果表明该菌对蒽诺沙星、环丙沙星敏感。该研究表明, 对饲料尤为鱼粉的卫生问题, 即饲料源性疾病问题应引起畜牧及兽医工作者的密切注意。     

Research note: effect of Bacillus cereus on performances of growing bulls.

The effect of including Bacillus cereus (BC) in a diet of growing bulls was examined. 64 German Holstein bulls (initial body weight: 187 kg) were allotted to one of two treatments (control and BC). The bulls were fed corn silage ad libitum and a restricted amount of concentrate (without BC or with 100 mg BC/kg). They were slaughtered with a final body weight averaging 555 kg. No significant treatment differences were detected for any feedlot performance or carcass characteristics measured. Bulls fed BC had a daily weight gain of 1472 g, control animals of 1425 g. Based on these findings, it may be concluded that under these conditions the inclusion of BC will not significantly improve performance of growing bulls.     

Development of murine monoclonal antibodies against an enterotoxin produced by Bacillus cereus.

Three murine monoclonal antibodies (MAbs) were prepared against an enterotoxin (ET) produced by Bacillus cereus. Although these MAbs were found to react with the ET, their specificities appeared to be different in competitive binding assays. One of the MAbs (D-8), which was highly reactive with the ET, will be useful in developing immunological methods to detect crude ET and to isolate the ET in high yield.