| Acute hepatopancreas necrosis syndrome and Early mortality syndrome,have recently caused serious losses in shrimp farming industry in Southeast As ia and Latin America.Vibrio parahaemolyticus the major pathogen of these syndromes which is a member of Vibrionaceae Veron 1965,Vibrio Pacini 1854,can not only cause various aquatic animal diseases,but also cause human acute diarrhea.In our study,we focused on finding a highly effective broad-spectrum antimicrobial agent,and identifying its biological and chemical properties and toxicity,and explored the mechanisms of its sterilization.(1)We evaluated the sterilization capacity against Vibrio parahaemolyticus of 22kinds of disinfectants They were from halogen disinfectants,quaternary ammonium disinfectants,guanidines disinfectants,aldehyde disinfectants and other types of disinfectants,and these disinfectants were commonly used in shrimp culture under laboratory conditions.In our study,we used quantitative suspension test for the evaluation of these disinfectants and the activity of antiseptics was evaluated by reduction factor tha t was calculated as the difference between logarithms of cfu/mL before and after exposure to tested concentrations of antiseptics.The results showed that iodine bromohydantoin is most effective in halogen disinfectants and the killing logarithm value is 5.6,the bound iodine in quaternary ammonium disinfectant is 5.52.The best effect of antimicrobial activity of polyhexamethylene guanidine chloride(PHMGH)is 6.0.Aldehyde,Bronopol,Potassium monopersulphate triple salt killing logarithm value is 1.0.Thus it can be seen that the antimicrobial activities showed significant differences among these disinfectants.(2)Toxicology test of the disinfectant.According to above tests,we choosed PHMGH as experimental object.In order to promote rational use of disinfectants,we test the toxicology of PHMGH.The results showed that the LC50(lethal concentration of 50%)values at 24h and 48h were 87.49mg/L and 27mg/L,respectively,and the safe concentration is 2.7mg/L.(3)The disinfectant PHMGH protected shrimp of artificial from death.Artificial challenge test of Vibrio parahaemolyticus was performed on healthy shrimps.The Bacterial suspension concentration is 1×10~3,1×10~4,1×10~5,1×10~6 and 1×10~7cfu/mL,respectively.The results showed that the effective concentration of Vibrio parahaemolyticus is 1×10~7cfu/mL.Furthermore,different concentration gradients of PHMGH were set to evalue the effect of PHMGH on lothe shrimp after artificial challenge Vibrio parahaemolyticus.When the concentration of PHMGH exceed 0.2mg/L,it have a significant protected effect of shrimp.(4)Study on the mechanism of sterilization.The mechanism of sterilization of PHMGH against V.p is supposed that when it hydrolysised in water and the bacterial surface is negatively charged,Blocked bacteria breathing passages,caused the bacteria dead.Using he quantitative suspension method to against Gram-positive bacteria,Gram-negative bacteria,to explore the activity of antiseptics of the disinfectant.Then we used the fluorescent dyes SYTO X Green staining the bacteria.The results shows PHMGH caused the bacteria cell in which the envelope cracked damaged the outer membrane layer in cell envelope.(5)The disinfectant applied in Shrimp farming pond.In which,there are a large number of The added PHMGH into the pond make the concentration 0.3mg/L.Continuous detected the number of V.P.The results shown that the number of V.P in shrimp decrease significantly,and in water the V.P were always decreased.In conclusion,PHMGH is a disinfectant that can kill the V.P efficiently,also has low toxicity to the shrimp.It can protect shrimp which infected by V.P from death.The PHMGH has the ability that sterilize a lot of pathogenic bacteria in aquaculture.To a kind of probiotics Bacillus coagulans,it seems safe.The mechanism of sterilization of PHMGH against V.p is the bacteria cytomembrane was attacked at first.Our findings provide theoretical bases for popularization of PHMGH in aquaculture. |
