| In recent years, a large amount of vibrio was discovered in the hepatopancreas and muscle of reared Litopenaeus vannamei, and the major epidemic diseases of this shrimp, such as early mortality syndrome, white feces syndrome, luminous bacterial disease were often happened in Hainan. This thesis chosed diseased shrimps from five typical regions of Litopenaeus vannamei aquiculture, included Haikou, Wanning, Dongfang, Changjiang and Lingao, in Hainan Province.514 typical strains were isolated from 48 disease shrimp samples.91 strains of Vibrio harveyi (VH),24 strains of V. alginolyticus (VA) and zero V. parahaemolyticus (VP) were all determined by their reported specific PCR detection methods. Five percent strains of VH and VA determined above were randomly selected to identify again by the physiological and biochemical characteristics and 16S rDNA sequences, which was consistent with the PCR detected results. Thus, except for the three representative Vibrios, some other Vibrios were still in shrimp cultured systems in Hainan. The results of the artifical infected tests of both of the VH and the VA indicated that only VH strain PBVH3311 isolated from Dongfang generated obvious pathogenic and similar symptom with the natural diseased shrimps by feeding and dipping protocols. Therefore, we assumed that most of the vibrios isolated from diseased shrimp were opportunistic pathogens, namely, their virulence was closely depending on environment. Hence, the shrimp vibriosis in Hainan would be controlled by improving reared environment.The molecular serotyping results, depended on the ERIC-PCR technology, indicated that 91 strains of VH were belong to a sole group at 45% of the similarity, and were classified to 14 serotypes at 80% of the similarity. Of the 14 serotypes,36 strains of VH from Dongfang were included an identical group. However, the other strains had no obvious regional relation. The 24 strains of VA were in the same serotype at 45% of the similarity. However, they were classified to 15 serotypes at 80% of the similarity and also had no obvious regional relation. Therefore, it is hard to prevent the shrimp vibriosis caused by VH and VA in Hainan because the two species exhibited a larege number of molecular serotypes.The studies have suggested that WCPW15003, WCPW15004, and HKPW15005 have high hereditary stability and have significant antagonism to PBVH3311 by the agar diffusion method and two layer plating method. The inhibited zone diameters of the three strains to PBVH3311 were 16.93mm,3.54mm and 22.86mm, respectively. These three strains were identified to be Pseudoalteromonas piscicida by the 16S rDNA sequences and the physiological and biochemical characteristics. The three strains showed no exoT?exoU?exoS?exoY?phzH?pilB?phz??phz??apr?lasA? lasB which were common virulence genes of Pseudoalteromonas. During the period of two weeks, Litopenaeus vannamei (body weighted 1.5?2.5 g) had no death or other disnormal living phenomenon when the concentration of antagonistic bacteria was 1.7×108 CFU/ml. The three strains could grow well at 20??40? and pH 5-9; WCPW15003 and WCPW15004 grew well at 0?50‰ of salinity, while optimal salinity of HKPW15005 was 0?100‰. According to the results of the broad-spectrum antibacterial property, the three strains antagonized strongly to the mixture of 91 strains of VH or 24 strains of VA (inhibited zone diameters exceeded> 5mm). Fortunately, they had similar antagonism to other possibly pathogenic vibrios in our laboratory, including 10 strains of VH, two strains of VA and one strain of VP isolated from diseased fish. Finally, we detected the antagonistic ability of the three bacteria to PBVH3311 (final concentration=2.1×107 CFU/ml), compared with an efficient antoganistic product as a positive control. The findings showed that the most obvious antagonism was at 96 hours when the vibrio cells were decreased into 4.6×106 CFU/mL or 4.9×105 CFU/mL,1.08×107 CFU/mL or 1.06×106 CFU/mL,1.05×107 CFU/mL or 4.9×106 CFU/mL,1.5×107 CFU/mL or 3.2×106 CFU/mL and 1.7×107 CFU/mL or 4.8×106 CFU/mL for each of the ten-fold diluted suspension ranged from 107 CFU/mL to 103 CFU/mL of WCPW15003 or HKPW15005, respectively, which meant that the vibrio cells were decreased 78.1% or 97.6%,48.6% or 94.9%,50.0% or 76.7%,38.6% or 84.8% and 19.0% or 77.1%, respectively. WCPW15004 (103 CFU/mL) had the obvious antagonism at 120 hours and the vibrio cells were 1.79×107 CFU/ml and decreased 14.8%. In a word, the development of a security prevention and control module against the vibriosis of L. vannamei based on the probiotics is very possible in the future. |
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