Embed Size (px)
Citation preview
Bacteriophage-based Interventions for Pathogenic Vibrios in Shellfish
Gary P. Richards, Ph.D.USDA, Agricultural Research Service
Delaware State UniversityDover, Delaware
Problem and ARS Response
In April 2008, attended NSA conference and first learned of a
problem 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 their
Vibrio 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 juvenile
shellfish
Particularly problematic in hatchery
settings
May be an opportunistic pathogen
when shellfish are stressed (acidity,
temperature, salinity, etc.) Vibrio tubiashii
How Big is the Problem?
Reduced Whiskey Creek hatchery production from
10 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 natural
balance 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 was
approved for use in the meat industry in
2006
Phages against E. coli developed for meat
and produce
Phages against Salmonella proposed for
the treatment of eggs
Research Plan to Inactivate Vibrio tubiashii
in 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 to
prevent larval mortalities from Vt
Test mixtures of phages (cocktails) in a commercial
hatchery
Vibrio tubiashii Stock Cultures
Obtained and grew Vt strains implicated in hatchery dieoffs
Strain Source TCBS Growth API Code
X00-12-1 Netarts Bay, OR GG- yellow 1044024
00-90-6 Kona, Hawaii GG- yellow 0044024
00-90-7 Kona, Hawaii GG- green 1044004
00-90-9 Kona, Hawaii GG- yellow 1044034
07-52-1 Quilcene, WA NG 0000004
08-52-1 Netarts Bay, OR GG- green 0040004
08-99-1 Bayside, CA GG- yellow 1044134
Dr. Evans Fish (Dr. Evans) GG- yellow 1044125
Developed Phage Isolation
Techniques
Seawater Filtration
Liquid Enrichment Culture
Quantitative Plaque/Phage Assay
Plan: Screen for Vt phages
Obtained 200 seawater samples from CA, OR, WA, NJ, HI, and DE
Tested by enrichment or plaque assay, or both
Picked potential phages for further ID
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
PercentSurvival
Days
NC
VT 10^6 Control
Phage 10^7 Control
Phage 10^6 Control
10^6 VT / 2 Hr / 10^7 Phage
10^6 VT / 2 Hr / 10^6 Phage
10^7 Phage / 2 Hr / 10^6 VT
10^6 Phage / 2 Hr / 10^6 VT
10^6 VT + 10^7 Phage
10^6 VT + 10^6 Phage
2
5
1
3
Larval oyster survival up to 5 days
Summary of Results
Treatment
Percent Larval Survival
After 3 Days
Larvae 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. & Food
Development 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. parahaemolyticus
and V. vulnificus from shellfish
Phage interventions have the potential to lessen
Vibrio problems here and abroad
Gary Richards
302 857-6419
