| Bdellovibrio and like organisms(BALOs) is a group of highly motile delta-proteobacteria that prey on other gram-negative bacteria. They hunt other pathogenic and/or potentially pathogenic bacteria in water and biofilms, and showed a very non-specific predatory spectrum of their host bacteria. Besides, BALOs are shown to be unable to prey on eukaryotic cells and as such pose no direct risk to human or animal health. These features make BALOs a possible candidate as a bio-control agent. In aquaculture or related fields, BALOs are attracting attention as a possible alternative bio-agent for farmed animals. In this thesis, two BALOs strains were isolated from marine sediment, and the efficacy of strain BDHSH06 in imporving aquatic animals’ survival and growth, controlling potentially harmful and/or pathogenic bacteria/number and altering bacterial community structures in rearing waters was investigated. Results of these studies would indicate if BALOs strain BDHSH06 could be used as bio-control agent in mariculture.1. BALOs strains BDH12 and BDHSH06 were isolated from marine sediment, with Shewanella putrefaciens strain 12 and Vibrio parahaemolyticus strain SH06 as preys, respectively, when using double layer agar technique. They were identified as BALOs morphologically by transmission electron microscopy, while partial 16 S r DNA sequencing analysis revealed that they showed no close relationships with members of the known genera Bacteriovorax, Vampirovibrio, Bdellovibrio or Peredibacter. Prey range analysis revealed that individually, BDH12 and BDHSH06 lysed 47 strains(82.5%) and 48 strains(84.2%) of the total 57 preys tested respectively. In combination, they lysed 98.2%(56 of 57) strains. All strains of V. parahaemolyticus, Vibrio cholerae and Vibrio alginolyticus tested could be lysed by both strains. Biological characterizations revealed that both strains had the optimal p H, salinity and temperature at 7.2, 3% and 30°C, correspondingly. They could not utilize autoclaved, dead cells as hosts.2. An 85-day Penaeus monodon rearing experiment was performed using BDHSH06 as predatory bacterium. Shrimp survival rate, body length and weight in test tanks were 48.1 ± 1.2%, 99.8 ± 1.00 mm and 6.36 ± 1.50 g respectively, significantly(P<0.05) higher than those in control, viz. 31.0 ± 2.1%, 86.0 ± 1.11 mm and 4.21 ± 1.56 g. With the addition ofBDHSH06, the total number of bacteria(TNB) and vibrio(TNV) were significantly reduced(P<0.05) by 1.3- 4.5 log cfu·ml-1/ cfu·g-1, in both water and shrimp intestines as compared to control. Concentration of BALOs in rearing water in test were 0- 24 pfu·ml-1. No BALOs detected in intestines. The effect of BDHSH06 on bacterial community structures in rearing water was also examined using PCR amplified 16 S r DNA-denaturing gradient gel electrophoresis(PCR-DGGE). DGGE profiles on rearing water samples from control and test revealed that BDHSH06 reduced approximately 44% bacterial species over the 85-day rearing period, among which approximately 57.1% were nonculturable.3. In this study, strain BDHSH06 was applied during a 63-days rearing of Haliotis diversicolor aquatilis combining interval water exchanged. The results showed the TNB and TNV in BDHSH06 added groups in water significant(P<0.05) decreased from 4.28 ± 0.12 log cfu·ml-1 and 2.55 ± 0.16 log cfu·ml-1 to 3.56 ± 0.16 log cfu·ml-1和 1.90 ± 0.15 log cfu·ml-1 respectively, and in intestines they reduced from 7.22 ± 0.13 log cfu·g-1 and 5.33 ± 0.22 log cfu·g-1 to 5.96 ± 0.02 log cfu·g-1 and 4.21 ± 0.21 log cfu·g-1. Concentration of BALOs in rearing water in test were 1.78- 228.89 pfu·ml-1. No BALOs detected in intestines. PCR-DGGE profiles from different periods of control and test revealed the richness value of bacterial community, Shannon index and Pielou’s evenness index were 0.54- 0.97, 1.38- 2.82, 0.35- 0.61 and 0.50- 0.75, 1.30- 2.31, 0.34- 0.56 respectively. When the numbers of bacterial community less than 21, the BDHSH06 reduced the relative concertration of dominant bacteria, and increased the relative concertration of non-dominant bacteria; when the numbers of bacterial community increased quickly, the BDHSH06 efficiently reduced the numbers of bacterial community and the relative concertration of bacteria.4. With the addition of BDHSH06, the TNB in water in test from 4 hours after BDHSH06 added significantly(P<0.05) reduced by 4.4%- 23.6%, the TNB before rearing water exchanged were lower than that in control but no significant(P>0.05) difference. The TNV in water both in 4 hours after BDHSH06 added and before rearing water exchanged were significantly(P<0.05) reduced by 2.6- 44.7%. The TNV in algae in test significantly(P<0.05) reduced but the TNB was no significant(P>0.05) difference. The TNB in intestines significantly(P<0.05) reduced by 2.2%- 35.1%, the TNV significantly(P<0.05) reduced by5.0%- 39.1% as compared to control in Haliotis discus hannai Ino rearing experiment.Concentration of BALOs in rearing water in test were 1.85 ± 0.05 log pfu·ml-1- 2.57 ± 0.02 log pfu·ml-1. No BALOs detected in intestines and in algae. Richness value of bacterial community, Shannon index and Pielou’s evenness index from control and text were 0.60-0.80, 2.40- 2.67, 0.57- 0.64 and 0.48- 0.80, 2.23- 2.68, 0.54- 0.67 respectively. Richness value(Rs) and Shannon diversity index(H’) of bacterial community in test reduced as compared to control. The BDHSH06 reduced the relative concertration of bacteria.5 With the addition of BDHSH06, Richness value(Rs) of bacterial community in Scophthalmus maximus rearing water in control was 0.64- 0.88, an average of 0.76; Shannon diversity index(H’) of bacterial community was 2.34- 2.96, an average of 2.56; Pielou’s evenness index was 0.58- 0.66, an average of 0.61. Richness value(Rs) of bacterial community in test was 0.56- 0.92, an average of 0.72; Shannon diversity index(H’) of bacterial community in test was 2.32- 2.86, an average of 2.54. Pielou’s evenness index was 0.56-0.66, an average of 0.61. BDHSH06 had little effect on the number of bands, Richness value, Shannon diversity index and Pielou’s evenness index in Scophthalmus maximus rearing water. BDHSH06 couldn’t reduce the relative concertration of bacteria.BDHSH06 could significantly reduce the TNB and TNV both in water and intestines in Penaeus monodon, Haliotis diversicolor aquatilis and Haliotis discus hannai Ino rearing experiment. BDHSH06 had a siginificant role for preventing aquatic animals’ bacterial disease, especially Vibriosis. The concentration of BALOs in rearing water in test were less than 2.6 log pfu·ml-1. BDHSH06 could improve the bacterial community structures in rearing waters, and has broad application prospect. The application effect of BDHSH06 is better when p H, salinity and temperature of the rearing water was at 6.8- 7.2, 30‰ and 25- 30°C correspondingly, and the TNB and TNV in water exceeded approximately 104 cfu·ml-1 and 103 cfu·ml-1, and in intestines exceeded approximately 104 cfu·g-1 and 103 cfu·g-1. |
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