Post on 26-Aug-2020
www.vims.edu
Usage of chemotherapeutants to mitigate
disease pressure in shellfish: a remedy or
a disaster?
Fu-Lin E. Chu and Eric Lund,
Virginia Institute of Marine Science,
College of William and Mary,
Glouscester Point, VA 23062;
Philippe Soudant, LEMAR,
Technopole Brest-Iroise, France
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Presentation outlines
�Diseases in shellfish (bivalves & shrimps)
�Chemotherapeutic treatments and problems
�Alternative approaches: Probiotics, phage-displayed peptides
�Drug/antibiotic: target lipid metabolism and metabolic pathways of the oyster pathogen, Perkinsus marinusDo n
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Problem of diseases
�Various disease agents (e.g., parasitic protozoans, bacteria, virus, fungus…..etc) have been reported frequently to be associated with the outbreak of diseases and subsequent mortality in many cultivated and feral populations of shellfish species.
� Intensive aquaculture practice: high density production and connectivity of systems have lead to large scale disease outbreaks, especially in penaeus shrimp culture.
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Important diseases: marine bivalves
�Economically important species:- Oyster species:Bonamia ostreae Marteilia refingens Marteilia refingensPerkinsus marinus (Dermo); Haplosporidium nelsoni(MSX)… etc. Vibrio spp. Bucephalus sp (Trematode).
- Hard clam, Mercenaria mercenaria (QPX)
- Abalone, Haliptis spp (H. cracherodii, and H. rufescens).: Withering syndrome, a fatal disease caused by theRickettsiales-like bacterium, Candidatus Xenohaliotis californiensis
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Important diseases: Shrimp
�Infectious viral, bacterial, parasitic and
fungal diseases have caused high mortality
in shrimp aquaculture operations: Vibrio
spp.. (e.g., V. harveyi, V. vulnificus, V.
parahaemolyticus, V. anguillarum.) and
virus (e.g., white spot syndrome virus) are
particularly fatal.
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Strategies
� (1) Produce disease resistant strains
� (2) Vaccination?
� (3) Chemotherapeutants/probiotics to
mitigate disease pressure
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Chemotherpeutants to mitigate
disease pressure
�Various antibiotics and chemotherapeutantshave been tested and used in controlling diseases in bivalve and shrimp aquacultures:oxytetracycline (OTC), oxolinic acid (OXA), chloramphenicol, cycloheximide,
enrofloxacin, and streptomycin ….etc.
�Cycloheximide (classified as “mutagen” by EPA, USA); chloramphenicol (a possible/potential precursor of aplastic anemia)
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Chemotherapeutic treatments: The
scenario
�Improper and extensive usage-
(1) Development of antibiotic-resistant pathogens:
- Development of OTC and OXA resistant Vibrioand other bacteria spp. in water, sediment and shrimps sampled from ponds treated with the 2
antibiotics was noted (Tendencia and delaPena, 2002).
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Chemotherapeutic treatments: The
scenario
� Improper and extensive usage-
(2) Potential human hazard due to persistence of
antibiotic resides in targeted organism tissues
(detection of chloramphenicol residues in shrimps resulted
in “0” tolerance of use of ELISA for residue detection and
a ban of shrimp imported from China in 2002).
(3) Ecological impacts: excreted residues and
unused drugs in environment particularly utilized
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Chemotherapeutic treatments: Actions
� (1) Chloramphenicol (a precursor of aplasticanemia) and nitrofurans are banned to use in EU and USA
� (2) For countries allow usage: residues in shrimps not exceed limits set by imported nations (e.g., <1ppb chloramphenicol)
� (3) Required records of usage: (e.g., date, compound used, reason(s) for use and dose).Do n
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Alternative approaches
� I. Development of effective Probiotics -
(1) Development of probiotic bacterial strains based on
competitive exclusion and host imunostimulation (non-
pathogenic live bacteria to inhibit pathogens: bacteriocin-like
inhibitory substance, BLIS).
(2) Marine secondary metabolites (MSMs), and materials of
biological origins
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Probiotics: trials in shrimp aquaculture
� Rengpipat et al. 2000
(1) They treated (inoculated) the shrimp culture (the blacktiger shrimp, Penaeus monodon), a probiotic bacteria : Bacillus S11 (BS11, isolated from the gastrointestinal tract of P. monodon) for 90 days, measured several immune parameters (e.g., total hemocytecounts, phagocytic activity, and phenoloxidase etc.) before and after V. harveyi challenge. (2) Overall probiotic-treated (BS11) group had greater mean immunity indexes compared with controls(3) Cumulative shrimp mortality in 10 days after V. harveyi challenge: probiotic-treated (BS11) group was 45.7% compared to 64.5% in untreated group.(3) So, treated with probiotic bacteria: stimulate host immune response and improve survival
*Shrimps were cultured in an aerated, biofiltered-recirculating system and fed with PF (1:3 BS11 to feed)
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Rengpipat et al. 2003: probiotic treatment enhanced growth
PF: 1 kg wet wt (≈100g DW) of BS11 (≈1010 CFG g-1). 80 shrimps/cage (2 m2). Cages
Were placed in one 500 m2 pond. P-treatment enhanced significantly the growthDo n
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Mean survival of the two trials:
Generally survival was better in shrimps fed PF
Rengpipat et al. 2003
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Gullian et al. 2004: immunomodulation
The probiotic Vibrio P62 and Bacillus P64 were used, V. alginolyticus (ili strain)
is a positive control. Shrimp (P. vannamei, 1.5 g) were randomly distributed in
aquariums (50L, 10 shrimp/aquarium, fed with commercial pellets. Bacteria were
inoculated to aquarium every 2 days from day 15 to 25 with 50% water exchange
after 20h exposure). Do not
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Immunity index of shrimp treated with different probiotic bacteria
Mean wt of shrimps treated with different probiotic bacteria
Gullian et al. 2004
Mean immune index:
Higher in P-treated groups (except
the P62 group) than the control.
Mean wt of shrimps:
Higher in P-treated groups than
the control
a a a
b
a ab b
Results: Stimulated immune
response and enhanced growth
(1) Competitive exclusion
(2) ImmunostimulationDo not
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Probiotics: trials in marine
bivalves
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Probiotics: trials in marine bivalves
� Gibson et al. 1998
Treated C. gigas larvae with P-Aeromons media A199:
Enhanced survival (≈100% as good as the control) in the
P-treated larval culture culture after challenge with V.
tubiashii compared to the non-treated, V. tubiashii
challenged control (100% mortality at 120 hrs)
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Effect of Ovoglobulins on Bacillary Necrosis Caused by V. tubiashii in larval survival
Takahashi et al.
2000:
Addition of ovoGs:
Improved survival of C. gigas
larvae. The ovoGs-treated
groups have a survival as good
as the control (non-challenge
and no addition of ovoGs)
after challenge with the
pathogenic V. tubiashii.
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Takahashi et al.
2000:
Increased ovoGs concentration
resulted in increased survival
of C. gigas larvae
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Marine secondary metabolites
• Marine alga, Ulva fasciata and sponge
Dendrilla nigra: incorporated into the feed
(1000 and 500 mg/kg feed respectively):
enhanced shrimp’s immune parameters
(e.g., total hemocyte counts, % of
hyalinocytes, agglutination in Ulva
treatment, … Selvin et al. 2004)
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Alternative new approaches
� II. Usage of phage-displayed peptides: molecular
blocking of pathogen’s active sites
III. Develop effective disease control dose while
minimizing residues in target organisms in the
environment
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Blocking active sites of key proteins of white spot syndrome
virus of shrimp (WSSV) by phage-displayed peptides (Yi et al. 2003)
Four peptides were constructed and three of them were tested
against WSSV in fresh water crayfish for a period of 20 days (n=15).
Peptide 2E6 produced lowest mortality and the longest LT50
(>20 days).
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Alternative new approaches
�Biological controls:
II. Molecular blocking of pathogen’s active sites
III. Develop effective disease control dose while
minimizing residues in target organisms in the
environment: using triclosan to target lipid
metabolism and metabolic pathways
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Geographic Distribution of Dermo (Perkinsus marinus), MSX
(Haplosporidium nelsoni) and SSO (H. costale)
Dark shading=diseases are epizootic or enzootic
Light shading=parasites have been reported, but not causing
recognizable mortality (modified from Ford & Tripp 1996)
* * *
Presently Dermo is the most prevalent and destructive disease for eastern and
Gulf Coast oysters, particularly in the mid-Atlantic regions (e.g, , the Chesapeake
Bay). It has caused significant mortality in eastern oyster populations along the
east and coast of US. Do n
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Three lipid metabolic pathways of interest for
drug targeting in protozoan parasites
�Phosphatidylcholine synthesis
�Glycosylphosphatidylinositol (GPI) anchor
synthesis
�Fatty acid synthesis
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Triclosan inhibits the enoyl-ACP reductase in
type II fatty acid synthases
�Type I fatty acid synthases:� found in animals, fungi and some mycobacteria
�complete pathway is associated with a single protein complex.
�cytosolic
�Type II fatty acid synthases:� found in plants, many bacteria and some protozoans
�separate, dissociated proteins catalyze each reaction in the pathway
�activity is associated with the presence of a plastid
The ability of de novo fatty acid synthesis and the presence of a non-photosynthetic plastid in the biflagellate zoospore stage of P. marinus suggest this parasite using Type II pathway.
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Triclosan inhibits the growth and disease
progression of Plasmodium spp. via inhibition
of fatty acid synthesis
Surolia and Surolia in:
Nature Medicine, vol 7, #2, 167-172. Feb 2001
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Triclosan is a widely used and
largely unregulated antimicrobicide
5-chloro-2-(2,4 dichlorophenoxy)phenol
halogenated diphenyl ether
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A sampling of triclosan-containing
consumer products
Soap
hand lotion
shampoo
toothpaste
mouthwash
deodorants
plastic cutting boards
plastic toys
shoe insoles
socks
mens underwear
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Questions
�Does triclosan kill or inhibit the growth of
P. marinus in vitro?
�Does triclosan inhibit fatty acid synthesis in
P. marinus?
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Experiment
�Add cells from 7 day old cultures to fresh media with:
�13C or non-labeled sodium acetate (6 mM)
�0, 2, 5 or 10 µM triclosan in ethanol (n=3).
� Sampled at 3 days after triclosan treatment
�Conduct cells counts, check cell viability using neutral red assay and extract/derivatize lipids for GC/FID and GC/MS analysis.Do n
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0
4
8
12
16
0µM 2µM 5µM 10µM
triclosan concentration
mil
lion
cel
ls/m
l
Effect of triclosan on proliferation (counted by microscope)
Reduced meront cell proliferation at concentrations 2-10 µMDo not
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0
20
40
60
80
100
0µM 2µM 5µM 10µM
triclosan concentration
mic
rog
ram
per
g d
w p
er h
rpalmitic acid
arachidonic acid
Triclosan suppressed the synthesis of both palmitic & arachidonic acid
Effect of triclosan on fatty acid synthesis
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Effect of triclosan: A comparison of Perkinsus and hemocytes
Triclosan Effect on Hemocytes @ 28C
0
20
40
60
80
100
120
140
0um 2um 5um 10um
Treatment
% V
iab
ilit
y
Triclosan effect on Perkinsus @ 28C
0
20
40
60
80
100
120
0uM 2uM 5uM 10uM
Treatment
% V
iab
le
Triclosan effect on Hemocytes @ 26C
0102030405060708090
100110120130
0uM 2uM 5uM 10uM
Treatment
% V
iab
le
Effect of triclosan was tested at 28 C:
(1) P. Marinus were cultured at 28C
(2) Oysters were maintained in a
Flow-through flume (ambient T= 26C)
Results:Reduced P. marinus viability
(MTS/PMS assay) at 2-10 µm, while
hemocyte viability decreased
slightly at 10 µm.
aa a a
a aa
ba
c bc b
N=18 N=9
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Experimental conditions: Treated P. marinus infected oysters with 0, 150, 300
or 600 µg triclosan per oyster per day. After 7 weeks (at 21-22 C), 10 oysters from each
replicate tank were Sampled for body burden assay (# of parasite cells/g of w oyster tissues,
mean Mean ±SE,n=30). Initial infection: 34,000 ± 44,000 cells/g ww (n=15, Mean ±SD).
Results:
significantly slow the disease progression as indicated by lower parasite burdens in the
groups treated 300 and 600 µg triclosan/oyster/day
7 week sampling results
0
100000
200000
300000
400000
Control 150ug 300ug 600ug
Treatment
ce
lls
/g w
et
tis
su
es
a ab
bb
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Summary
Tested dose vs FDA allowed level
� Triclosan, 2-10 µM (0.3, 1.5, and 3 µg/ml) inhibits fatty acid synthesis, growth and reduces cell viability of P. marinus cultured in vitro.
� Oyster hemocytes exhibited 40% mortality after 24 hr exposure to 10 µMtriclosan at 28 C (2C higher than the maintenance ambient T), no mortality occurred after 24 hr exposure to same concentration at 26 C: T stress?
� The in vitro tested concentrations are 1000 times lower than the allowed level by FDA in daily used hygiene products such as toothpaste and mouthwash (3 g/L vs 2.9 mg/L for 10 µM).
� The in vivo tested concentration,150, 300, 600 µg (0.52, 1.04 and 2.07 µmole respectively) is 10-20 times lower than the allowed level by FDA in daily used of toothpaste (7.5 mg/dose)
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Future research
�Determine the most effective in stopping disease
progression in infected oysters
�Determine any health effects of triclosan on oysters
�Conduct triclosan bioaccumulation and depuration
studies on oysters
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Summary: overall� Improper and extensive use of
chemotherapeutants: could be a disaster.
� Probiotics: a wise alterative approach-Gaining momentum and results are quite promising: - Increased survival, enhanced resistance and growth- Increased antibacterial activity and digestibility
- Mechanisms: - Immumodulation and immunostimulation- Competitive exclusion
- Production of BLIS (bacteriocin-like inhibitory substance), proteinaceous “antibiotic” substances…., microbial enzymes….
-Non-pathogenic to human?Do not
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Summary:
� Usage of phage-displayed peptides for blocking active sites of
key proteins of pathogenic microbes - seems working ---
molecular approaches are forthcoming
� Develop methods to target pathogens’ metabolic pathway with effective/low dose seems feasible.
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Acknowledgement
�Funding: NSF and ODRP, Sea Grant,
NOAA
�Technical Support: Jennifer Littell
�VIMS, College of William & Mary
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