Dr. Gary Richards

Bacteriophage-based Interventions forPathogenic Vibrios in ShellfishGary P. Richards, Ph.D.USDA, Agricultural Research ServiceDelaware State UniversityDover, Delaware

Problem and ARS Response• In April 2008, attended NSA conference and first learned of aproblem on the West Coast with Vibrio tubiashii (Vt)• Caused 70% mortalities in larval shellfish in hatcheries for 3 years• Important West Coast stakeholders requested ARS help with theirVibrio problems• Our lab has expertise in both vibrio and virus research• I developed plan to combat Vt using phages• Secured permission from NPL (Lindsay) and Area Director(Swietlik) to modify research objectives to include phage studies

Vibrio tubiashii• Naturally occurring marine bacterium• Infects and kills larval and juvenileshellfish• Particularly problematic in hatcherysettings• May be an opportunistic pathogenwhen shellfish are stressed (acidity,temperature, salinity, etc.)Vibrio tubiashii

How Big is the Problem?• Reduced Whiskey Creek hatchery production from10 billion larvae per year to 3 billion per year• Leads to shortage of larvae for commercial planting• Reduces native species from natural settings• Will likely affect water quality and the naturalbalance of the coastal environment• Problem here and in other countries• How serious is it in other countries???

What are Phages?• Phages are bacterial viruses• Play key role in maintaining bacterial balance• Most prevalent life form on earth• Levels approach 28 million/oz. seawater• Many different kinds of phages• Bacterial species and strain specific• No known phages for V. tubiashii

Use of Phages in Food Processing• Phage cocktail against Listeria wasapproved for use in the meat industry in2006• Phages against E. coli developed for meatand produce• Phages against Salmonella proposed forthe treatment of eggs

Research Plan to Inactivate Vibrio tubiashiiin Shellfish Hatcheries using Phages• Develop methods to detect phages against Vt• Screen seawater for phages• Characterize the phages and grow them up in lab• Perform pilot studies on the effectiveness of phages toprevent larval mortalities from Vt• Test mixtures of phages (cocktails) in a commercialhatchery

Vibrio tubiashii Stock CulturesObtained and grew Vt strains implicated in hatchery dieoffsStrain Source TCBS Growth API CodeX00-12-1 Netarts Bay, OR GG- yellow 104402400-90-6 Kona, Hawaii GG- yellow 004402400-90-7 Kona, Hawaii GG- green 104400400-90-9 Kona, Hawaii GG- yellow 104403407-52-1 Quilcene, WA NG 000000408-52-1 Netarts Bay, OR GG- green 004000408-99-1 Bayside, CA GG- yellow 1044134Dr. Evans Fish (Dr. Evans) GG- yellow 1044125

Developed Phage IsolationTechniques• Seawater Filtration• Liquid Enrichment Culture• Quantitative Plaque/Phage Assay

• Plan: Screen for Vt phages• Obtained 200 seawatersamples from CA, OR,WA, NJ, HI, and DE• Tested by enrichment orplaque assay, or both• Picked potential phagesfor further ID

100%90%80%70%60%PercentSurvival50%40%30%20%NCVT 10^6 Control10%Phage 10^7 Control1235DaysLarval oyster survival up to 5 days0%Phage 10^6 Control10^6 VT / 2 Hr / 10^7 Phage10^6 VT / 2 Hr / 10^6 Phage10^7 Phage / 2 Hr / 10^6 VT10^6 Phage / 2 Hr / 10^6 VT10^6 VT + 10^7 Phage10^6 VT + 10^6 Phage

Summary of ResultsTreatmentPercent Larval SurvivalAfter 3 DaysLarvae only 100%Larvae + Vibrio 0%Larvae + Phage 100%Larvae + Vibrio + Phage (2 h later) 40%Larvae + Phage + Vibrio (2 h later) 70%Larvae + Phage + Vibrio (added at same time) 60%

Current and Future Plans• Currently performing DNA sequencing of the phages (Ag. & FoodDevelopment Authority, Ireland)• Continuing to screen seawater for phages against other strains of Vt(Kona Coast Shellfish in Hawaii)• Continuing lab-based pilot studies (AquaTechnics, WA)• Will continue to isolate and characterize new phages (ARS)• Demonstrate effectiveness of phage cocktails in commercial hatcheries(Taylor Shellfish and Whiskey Creek)• Next: Develop phage-based methods to eliminate V. parahaemolyticusand V. vulnificus from shellfish

Phage interventions have the potential to lessenVibrio problems here and abroadGary Richards302 857-6419gary.richards@ars.usda.gov