Isolation And Characterization Of Bacillus, Aeromonas Hydrophila And Their Phage During Pelodiscus Sinensis’s Farming

At present, the farming methods of Pelodiscus sinensis in China is mainly intensiveculture which bring good economic benefits. While the accumulation of bait and feces had ledto the deterioration of water quality and the disorder of the micro-ecological environment,easy to an outbreak of disease during its farming. People mainly take the drug delivery,antibiotics to the food and the use of probiotics to regulate the water quality and to prevent thediseases, and the use of probiotics is the better one because of its non-polluting andeco-friendly. Bacillus is one of the common bacteria of probiotics which has a good effect onwater purification, while there had been bacteriophage pollutions during the production ofBacillus, causing great losses. In addition, phage therapy is a promising treatment withbacterial diseases, high specificity to pathogens, harmless to beneficial bacterias and noresidual, and some scholars had carried out several relevant research on mice, chickens, pigs,rainbow and other organisms which had achieved good results, while there’s no similar studyof P.sinensis, In this paper, bacillus were isolated from the intestinal tracts of P.sinensis, andthen identification of these Bacillus were in search, while one bacteriophage of one of theseBacillus had been isolated, and it’s nature were studied in this paper, discussing theprevention measures to phage pollution of Bacillus. Another bacteriophage had been isolatedfrom the aquaculture water of P.sinensis, and Aeromonas hydrophila is it’s host bacteria,which is the common pathogens of aquaculture.The nature of phage and the dynamicrelationship between the phage and it’s host were studied in our paper, discussing theapplication of phage therapy in aquaculture. The results are as follows:1Three strains of Bacillus were isolated from the intestinal tracts of Chinese soft-shellturtle P.sinensis. One of them was identified as B. pumilus, the other two were identified as B.safensis, based on the results of morphological, physiological and biochemical characteristicsand the homological analysis of16S rDNA sequence.2Using B. pumilus，one of the three strains, as the host bacteria, one bacteriophage hadbeen isolated from the aquaculture water of P.sinensis, and some of its characteristics were studied. The results showed that the phage has a high thermal stability and wide rangeadaptation of pH, that it can grow and reproduce within pH4~9, can also survive in theenvironment below80℃, but it is sensitive to temperatures above80℃, and more sensitiveto UV that all phage were inactivaty more than6min exposure time. In addition, theadsorption time of this phage to its host is less than5min, high incidence of phagecontamination, and the optimal multiplicity of infection is0.001, high ability to infectBacillus.What’s more, this phage’s incubation period was10min, the time of burst phage was70min, and the burst size was about94.25. In short, this bacteriophage has a relatively shortincubation period and a large burst size, with a greater harm to B. pumilus3Using A.hydrophila as the host bacteria, The phage isolated frome the aquaculturewater of P.sinensis is lytic phage, whose plaque is transparent with tidy edge.The resultsshowed that the phage has a wide range adaptation of pH, but sensitive to low pH that therealmost have no plaque when pH<5, the adsorption time of this phage to it’s host is slightlylonger with20min, and it is very sensitive to UV that all lots of phages were die with20sexposure time and all phage were inactivaty more than30s exposure, and this phage is alsovery sensitive to high temperature that almost all phage lost their activity in the environmentabove50℃.The experiment of optimal multiplicity of infection showed that, the phage hadhighest phage titer when MOI=0.1and lowest phage titer when MOI=0.001, different with theother references.The one-step growth curve showed that, this phage’s incubation period was50min, thetime of burst phage was40min, and the burst size was about92.8, having great crackingcapacity. In addition, The dynamic relationship of the phage and its host bacteria results showthat, adding appropriate concentration of phage can effectively reduce the concentration ofA.hydrophila to a safe level that lowe enough to avoid related diseases, and the concentrationof phage only have a slightly change, having a dynamic equilibrium near the firstconcentration ultimately.In summary, the phage of B. pumilus has a great infection capacity to host, and it issensitive to UV irradiation and heat treatmen, UV and heat can be used to eliminate the phage to avoid the phage pollution.The phage of A.hydrophila has short incubation period and largeburst size when infecting the host, having great effectivity in reducing the concentration ofA.hydrophila, but its growth and reproduction require more suitable conditions, as it issensitive to acidic environment, UV and high temperature, which may limit the developmentof the phage therapy.