Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: The results have been disseminated by a presentation at the Pacific Coast Shellfish Growers Association meeting in September 2007. In addition, the results have been incorporated into a U.S. patent application. Additionally, the results have been disseminated by ongoing contact between the investigator and potential users of the technology such as shellfish hatcheries and nurseries on the east and west coasts of the United States and in Hawaii. This contact has included technical advice for improving hatchery productivity and the hathcery improvements that are required and beneficial in order to use the project product (beneficial probiotic bacteria). In addition, results were disseminated at a Washington State Sea Grant conference for shellfish growers in March 2007. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Project modifications were filed and approved in May 2007 to enable collabortor Dr. Claudia Hase of Oregon State University to provide molecular characterization, virulence determination and diagnostic tools to the project.
Impacts
Ten pilot scale commercial trials were conducted in algal, larval tanks and physical systems in commercial hatcheries during the reporting period. During these pilot scale trials, an outbreak of highly virulent Vibrio tubiashii vibriosis continued and required development of a diagnostic method for the strain, determination of its virulence, research to determine the mechanism of virulence and tests of probiotic for effectiveness against the newly re-emerged pathogen. Results showed that hatchery larvae with probiotic added, when virulent pathogen Vibrio tubiashii was not present, grew and metamorphosed at optimal rates and that addition of the probiotic significantly improved the health status of the larvae, in comparison to control groups not treated with probiotic. While pilot scale trials showed probiotic benefit in terms of enhanced growth and survival in some cases, hatchery scale up results were variable due to extreme variability discovered during this project in the sanitation operations and water treatment effectiveness in hatcheries and nurseries. We therefore instituted an effort to upgrade and more highly standardize hatchery and nursery management and sanitation procedures, in order to establish a basis for probiotic usages. Effective concentration of probiotic bacteria were maintained for over two years in stock algal culture and for up to two months in sanitary expanded algal cultures. A method to isolate continuous algal bag cultures in hatcheries was developed and implemented in several hatcheries, as a precursor to establishing conditions favorable for the use of probiotic bacteria. The general mechanism of action of the probiotics was determined in laboratory studies.
Publications
- No publications reported this period
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Progress 10/01/05 to 09/30/06
Outputs
For the laboratory phase of the project we have completed studies on the optimization of production of probiotics, obtained reagents for rapid confirmation of probiotic strain identity and completed further characterization of the isolates. We determined that we could increase probiotic activity of our isolates by "stressing" the bacteria during culture. Further characterization of the isolates included biochemical characterization and DNA sequence alignments with other sequences from GenBank. Brood stock pretreatment with probiotics, the first listed task in commercial pilot scale testing, has been postponed, due to successful commercial-scale treatment of the eggs obtained from the brood stock. We found that probiotic bacteria could be utilized at effective concentrations in egg suspensions, without harm to the unfertilized or fertilized eggs. This successfully prevents carry-over of bacteria from brood stock to the larval culture. Furthermore, the calculated input of
pathogens was determined to be far greater from hatchery water than from brood stock carry-over to gametes. Probiotic larval trials were conducted in static 190 liter tanks and in 30,000 liter production tanks. The trials in the 190 liter static tanks were promising but need to be repeated in replicated flow-through tanks which are now under construction and nearly completed. Algae-probiotic compatibility was tested at a laboratory scale at AquaTechnics for four algae/probiotic combinations which had not been tested for compatibility during Phase I. All four combinations were found to be compatible, with good growth of the algal cultures and probiotic levels of 10E5 to 10E7. Probiotics were tested for compatibility with continuous (chemostat) algal culture. Nine days after establishing the cultures, the bags were placed in harvesting mode and samples plated on marine agar. The results showed that probiotics were not well maintained in these continuous algal cultures. Based on these
results, a second trial was started in which we first established the probiotic with the algae in flasks, and moved this to the bag system. Three strains of algae were tested. Axenic cultures performed very well. Nine days after the initial addition of probiotic XX1to algae cultures, all three cultures had a high density of algae and effective concentrations of probiotic. However, the algal-probiotic cultures did not fare well after transfer to the bag system. Near-term project focus will be on further trials with various probiotic addition regimens to larval cultures in small-scale flow-through tanks. Further trials with probiotic production in larger-scale algae batch cultures (static tanks) may be conducted in the future in facilities that continue to utilize this method of algae production.
Impacts
Although the bivalve shellfish seed production industry, even for the longest cultured species such as oysters, operates on a commercial scale, the consistency of production is still highly variable and a great scope for improvement of efficiency exists. One of the most serious challenges has always been the management of aggressive opportunistic bacterial infections in larval and juvenile cultures. Often pathogens or a population of bacteria that are antagonistic in some way to the cultured animals establish dominance and result in low productivity and high variability in the success of culture batches. We believe that the use of naturally and locally occurring beneficial bacteria with probiotic activity offers a solution for the management and prevention of bacterial diseases in shellfish seed. Our previous research and case reports have shown that such bacteria are not uncommon, but not all isolates with probiotic activity have culture characteristics (e.g. growth
rate and temperature response) that render them suitable for use in marine hatcheries . These candidates were isolated from marine waters in the vicinity of the shellfish hatcheries and nurseries. We believe we can meet the challenge for the design and validation of a stable and cost effective system for delivery and maintenance of the beneficial bacteria in the culture system. Because these bacteria are show growth activity over a wide temperature range, they are expected to be applicable to other type of marine shrimp hatcheries.
Publications
- No publications reported this period
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