Effects Of Stress From Three Water Environmental Factors Onvirulence And Pathogenicity Of Bacillus Cereus

1 Identification and toxicity detection of Bacillus cereusA strain pathogenic bacterium from disease Cynoglossus semilaevis Günther was identified by 16 Sr DNA,6 kinds of virulence gene were detected by RT-PCR as well as extracellulase activity,hemolytic activity and median lethal dosage were determined of Danio rerio and drug susceptibility was tested by Kirby-Bauer method.The purpose of this study was to evaluate the potential pathogenicity of Bacillus cereus by virulence genes and exotoxin;the drug susceptibility was conducted to provide reference for aquatic animal disease prevention.The results showed that the strain of bacterium was identified as Bacillus cereus which carried with virulence genes of hemolysin(hbl C),enterotoxin(bce T),cytotoxin(Cyt K),pleiotropic regulator(plc R),non hemolytic toxin(nhe A)and without cereulide(ces).Its extracellulase activity(hemolysin,proteinase,diastase,lipase and urease)had been detected.LD50 for challenged zebra fish was 3.26×108 cfu/m L.Drug susceptibility testing showed that the strain of bacterium was sensitive to 15 kinds of drugs.2 Effects of water environmental stress factor on virulence gene's expression of Bacillus cereusThe effects of single water environmental stress factor on the relative expression levels of virulence genes of Bacillus cereus were studied by semi-quantitative PCR.This study was carried out to investigate the effect of wanter environmental stress factors on the relative expression levels of virulence genes in Bacillus cereus and understand the pathogenicity of Bacillus cereus as well as provide reference for disease prevention of aquatic animal.The results showed that temperature had no significant effect on the m RNA expression levels of Cyt K and plc R(P>0.05),whereas temperature had significant effect on that of bec T,hbl C and nhe A(P<0.05).The relative expression levels of Cyt K,plc R,hbl C and nhe A all reached the peak value at 35?,whlie that of bec T reached the peak value at 25?.p H had no significant effect on the relative expression levels of Cyt K and nhe A(P>0.05),whereas p H had significant effect on those of bec T,hbl C and nhe A(P<0.05).The relative expression levels of Cyt K,bec T,plc R and hbl C all reached the peak value at p H 8.5,while that of nhe A reached the peak value at p H 7.2.The relative expression levels of 5 kinds of virulence gene wereaffectd by sodium nitrite concentration(P<0.05),and all reached the peak at the concentration of 3.0 mg/L sodium nitrite.3 Effects of water environmental stress factor on the activities of extracellular products of Bacillus cereusIn order to explore the pathogenicity of Bacillus cereus for fish from the angle of the extracellular products,the effects of single water environmental stress factors on the activities of extracellular products of Bacillus cereus were studied.The results showed that temperature had significant effects on the activities of extracellular products(P<0.05),and all of the activities of extracellular products reached the highest at the temperature of 35?.p H had significant effect on the activities of extracellular products(P<0.05).Except for the activities of amylase reached the highest at p H 7.2,whlie the activities of the other 4extracellular products reached the highest at p H 8.5.The sodium nitrite concentration had significant effect on the activities of other 4 extracellular products(P<0.05).The hightest activities of amylase appeared in 0.75 mg/L of sodium nitrite,And the activities of 4 kinds of extracellular products reached the highest at the sodium nitrite concentration of 3.00 mg/L.This trial was conducted to study the virulence of Bacillus cereus4 Effect of combined water environmental stress factors on virulence and pathogenicity of Bacillus cereusThe orthogonal experiment was designed to investigate the relationship among environmental stress factors,fish immunity and pathogenicity of Bacillus cereus.The results showed that combination of 3 kinds of environmental stress factor's had no significant effects on the relative expression levels of 5 kinds of virulence gene(P>0.05).And the relative expression level of Cyt K reached the peak value at the 4th group(temperature: 25?;p H: 8.5;sodium nitrite concentration: 1.50 mg/L),whereas that of bec T reached the peak value at the1 st group(temperature: 35?.p H: 8.5;sodium nitrite concentration: 0.75 mg/L),while the relative expression level of hbl C,plc R,nhe A reched the peak value at the 9th group(temperature: 25 ?;p H: 6.0;sodium nitrite concentration:1.50 mg/L).3 kinds of environmental stress factors had no significant effect on the activities of 5 kinds of extracellular products(P>0.05).The activities of lipase and amylase reached the highest at the1 st group(temperature: 35?;p H: 8.5;sodium nitrite concentration: 0.75 mg/L),the activity of hemolysin reached the highest at the 1st group(temperature: 25?;p H: 6.0;sodium nitriteconcentration:1.50 mg/L),while the activities of protease and urease reached the highest at the 8th group(temperature: 30 ?;p H: 6.0;sodium nitrite concentration: 1.50 mg/L).The order of factors affecting the mortality of zebra fish caused by Bacillus cereus was temperature>sodium nitrite>p H.But the combination of 3 kinds of environmental stress factors had no significant effect on the mortality of zebra fish caused by Bacillus cereus.And the mortality of zebra fish caused by Bacillus cereus reached the peak value at the 9th group(temperature: 25?;p H: 6.0;sodium nitrite concentration:1.5 mg/L).The changes of the mortality of zebra fish caused by Bacillus cereus was consistent with the changes of the relative expression levels of 5 kinds of virulence gene and the activities of 5 kinds of extracellular products of Bacillus cereus.