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

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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

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Triclosan effect on Perkinsus @ 28C

0

20

40

60

80

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120

0uM 2uM 5uM 10uM

Treatment

% V

iab

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Triclosan effect on Hemocytes @ 26C

0102030405060708090

100110120130

0uM 2uM 5uM 10uM

Treatment

% V

iab

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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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