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Effects of Triclosan on Growth, Viability and Fatty Acid Synthesis of the Oyster Protozoan Parasite Perkinsus Marinus

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Effects of Triclosan on Growth, Viability and Fatty Acid Synthesis of the Oyster Protozoan Parasite Perkinsus Marinus DISEASES OF AQUATIC ORGANISMS Vol. 67: 217–224, 2005 Published November 28 Dis Aquat Org Effects of triclosan on growth, viability and fatty acid synthesis of the oyster protozoan parasite Perkinsus marinus Eric D. Lund1, Philippe Soudant2, Fu-Lin E. Chu1,*, Ellen Harvey1, Stephanie Bolton3, Adolph Flowers4 1Virginia Institute of Marine Science, College of William and Mary, Gloucester Point, Virginia 23062, USA 2Université de Bretagne Occidentale, Institut Universitaire Européen de la Mer LEMAR–Laboratoire des Sciences de l’Environnement Marin (UMR 6539), Technopole Brest Iroise, Place Nicolas Copernic, 29280 Plouzané, France 3Wake Forest University, 1834 Wake Forest Road, Winston-Salem, North Carolina 27106, USA 4Morehouse College, 830 Westview Drive SW, Atlanta, Georgia 30314, USA ABSTRACT: Perkinsus marinus, a protozoan parasite of the Eastern oyster Crassostrea virginica, has severely impacted oyster populations from the Mid-Atlantic region to the Gulf of Mexico coast of North America for more than 30 yr. Although a chemotherapeutic treatment to reduce or eliminate P. marinus from infected oysters would be useful for research and hatchery operations, an effective and practical drug treatment does not currently exist. In this study, the antimicrobial drug triclosan 5-chloro-2-(2,4 dichlorophenoxy) phenol, a specific inhibitor of Fab1 (enoyl-acyl-carrier-protein reductase), an enzyme in the Type II class of fatty acid synthetases, was tested for its effects on via- bility, proliferation and fatty acid synthesis of in vitro-cultured P. marinus meronts. Treatment of P. marinus meront cell cultures with concentrations of ≥2 µM triclosan at 28°C (a temperature favorable for parasite proliferation) for up to 6 d stopped proliferation of the parasite. Treatment at ≥5 µM at 28°C greatly reduced the viability and fatty acid synthesis of meront cells. Oyster hemocytes treated with ≥20 µM triclosan exhibited no significant (p < 0.05) reduction in viability relative to controls for up to 24 h at 13°C. P. marinus meronts exposed to ≥2 µM triclosan for 24 h at 13°C exhibited signifi- cantly (p < 0.05) lower viability relative to controls. Exposure of P. marinus meronts to triclosan con- centrations of ≥20 µM resulted in >50% mortality of P. marinus cells after 24 h. These results suggest that triclosan may be effective in treating P. marinus-infected oysters. KEY WORDS: Triclosan · Dermo disease · Perkinsus marinus · Eastern oyster · Fatty acid synthesis Resale or republication not permitted without written consent of the publisher INTRODUCTION are infected with P. marinus (Burreson & Ragone- Calvo 1996), which has severe ramifications for The protozoan parasite Perkinsus marinus, cau- restoration efforts, oyster disease research and com- sative agent of Dermo disease in the Eastern oyster mercial oyster aquaculture. Crassostrea virginica, has decimated oyster popula- Potential benefits from the development of a protocol tions along the Atlantic coast of North America and to eliminate Perkinsus marinus from infected oysters the Gulf of Mexico for at least 30 yr. One element include (1) increasing the quantity and quality of critical to both the restoration of wild oyster popu- gametes of oysters conditioned for hatchery spawns; lations and the development of commercial aquacul- (2) a reduction in the frequency of spawning required ture operations is a practical method for reducing or to maintain specific strains of oysters; (3) production of eliminating P. marinus from infected oysters. Cur- disease-free spat; (4) production of disease-free oysters rently, virtually all oysters in the mid-Atlantic region for use in research on host–parasite interactions. *Corresponding author. Email: [email protected] © Inter-Research 2005 · www.int-res.com 218 Dis Aquat Org 67: 217–224, 2005 To be practical for use in controlling Perkinsus mari- Expt 1. Effect of triclosan on growth, viability and nus infection, a drug therapy needs to have several fatty acid synthesis by Perkinsus marinus. The effects important features: (1) high specific action against the of triclosan on the fatty acid synthesis rates of P. mari- parasite; (2) low toxicity to the host; (3) application nus were tested by measuring the incorporation of through direct addition to water in holding containers; exogenous 13C sodium acetate into fatty acids of (4) low/no toxicity to humans; (5) low cost and ready P. marinus meront cells. We added 10 ml aliquots of 13 availability. Several studies evaluating potential fresh media containing 6 mM sodium acetate 1,2 C2 chemotherapeutic agents for treatment of Dermo dis- (Cambridge Isotope Laboratories) to culture flasks and ease have been published, but the protocols so far inoculated those with 2.5 × 107 cells and 0, 2, 5 or developed to successfully treat P. marinus infection in 10 µM triclosan using ethanol (5 µl ml–1) as a carrier oysters fail to meet several of these criteria (Ray (n = 3). After 72 h at 28°C, cultures were harvested and 1966a,b, Calvo & Burreson 1994, Faisal et al. 1999, cell density and percentage of viable cells for each cul- Delaney et al. 2003). ture flask was determined using a neutral red assay Parasitic protozoans must either acquire lipids from (Borenfreund & Puerner 1985), and fatty acid synthesis their host or synthesize lipids de novo to produce the rates were determined by gas chromatography/mass new cell membrane necessary for cell replication. spectroscopy (GC/MS) (Chu et al. 2002). Inhibiting the ability to synthesize new membrane pre- Expt 2. Viability and cell proliferation of Perkinsus vents the parasite from increasing in surface area, marinus cultures exposed to different levels of tri- thereby halting cell proliferation and disease pro- closan. To test a wide range of triclosan concentra- gression. tions, 21 T-10 tissue-culture flasks containing 10 ml The antimicrobial drug triclosan, 5-chloro-2-(2,4 aliquots of medium were inoculated with 1 ml (0.25 × dichlorophenoxy) phenol, has been used in a wide 106 cells) of 4 d old P. marinus culture. Triclosan solu- array of consumer products such as soaps, tooth- tions of 0, 2, 5, 10, 20, 50 and 100 µM were added with pastes and household plastics for the last 30 yr. ethanol (5 µl ml–1) to the culture flasks (3 replicates per Triclosan inhibits fatty acid synthesis and stops the treatment). Aliquots of 1.2 ml from each culture flask proliferation of protozoan parasites including species were collected after 3 h, 24 h, 3 d and 6 d incubation at of the genera Plasmodium, Toxoplasma, and Trypa- 28°C for determination of cell culture density and via- nosoma in vitro and in vivo (Beeson et al. 2001, bility measurements by flow cytometry (FCM). All the McLeod et al. 2001, Surolia & Surolia 2001, Roberts suspensions were filtered through 80 µm mesh into et al. 2003). The purpose of the present study was to FCM tubes and maintained on ice until FCM analysis. determine whether triclosan may be a useful Expt 3. Viability of oyster hemocytes exposed to tri- chemotherapeutic agent to treat oysters infected with closan. Adult oysters, Crassostrea virginica, were sup- Perkinsus marinus. The effectiveness of triclosan plied by Pemaquid Oyster Company (Waldoboro) and against P. marinus was evaluated by measuring the maintained in a flow-through seawater system at effect of the drug on the growth, viability and fatty ambient temperature (13 to 14°C) and salinity (15 to acid synthesis of in vitro parasite cultures. To address 20). They were processed as follows: hemolymph was concerns about potential toxic effect on host cells, withdrawn from the adductor muscle through a notch the effect of triclosan on oyster hemocyte viability previously (24 h) ground in the oyster shell, using a was also assessed. 1 ml plastic syringe with a 25-gauge needle. Hemo- lymph from each oyster was transferred into an indi- vidual Eppendorf tube held on ice. Only individual MATERIALS AND METHODS samples confirmed by microscope observation to be free of contaminating particles were used. Hemolymph Culture conditions. Perkinsus marinus meront cells from 30 individuals was pooled to constitute 3 repli- were cultivated as previously described (Chu et al. cates of 9 ml of fresh hemolymph. Pooled hemolymph 2002) in medium defined by La Peyre et al. (1993). samples were filtered through 80 µm mesh prior to use. Medium was prepared with artificial seawater (ASW) We combined 1 ml of hemolymph with 1 ml of anti- and adjusted to an osmolarity of 590 (equivalent to a aggregant hemocyte solution (AAHS, Auffret & salinity of 20, Lund et al. 2004). The medium was then Oubella 1995) in 21 Falcon vials (7 vials per hemo- sterilized by 0.2 µm filtration and stored at 4°C until lymph pool), and then added 0, 2, 5, 10, 20, 50 and use. Meronts were inoculated at a concentration of 1 × 100 µM of the ethanol (5 µl ml–1) triclosan solutions to 106 ml–1 and cultivated in 10 ml aliquots of medium in the 2 ml hemolymph + AAHS mix (3 replicates per T-10 tissue-culture flasks at 28°C. Meronts in exponen- ttreatment). The hemolymph was continuously main- tial growth phase (7 d old) were harvested and used for tained on ice until initiation of incubation. All the all assays. Falcon tubes were thus incubated at 13°C (the temper- Lund et al.: Effects of triclosan on Perkinsus marinus 219 ature at which, the oyster were maintained prior to positive chemical ionization (CI). The same column as bleeding). After 24 h incubation, 0.6 ml of each cell that used for GC/FID analysis of the FAME samples suspension was collected and filtered into FCM tubes (J & W DB-WAX, 25 m × 0.32 mm; 0.2 µm film thick- for subsequent FCM analysis. ness) was used for GC/MS analysis. Carrier gas Expt 4.
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