Veterinary Parasitology 168 (2010) 19–24 Contents lists available at ScienceDirect Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar Effectiveness of chemotherapeutants against scuticociliate Philasterides dicentrarchi, a parasite of olive flounder Chang-Nam Jin a,1, Ramasamy Harikrishnan b,1, Young-Gun Moon b, Man-Chul Kim b, Ju-Sang Kim b, Chellam Balasundaram c, Moon-Soo Heo b,* a Jeollanamdo Fisheries Technology Office, Jindo, Jeonnan 539 802, South Korea b Marine Applied Microbes and Aquatic Organism Disease Control Lab, Aquatic Biomedical Sciences, School of Marine Biomedicals Sciences, College of Ocean Science & Marine and Enviromental Research Institute, Jeju National University, Jeju 690 756, South Korea c Department of Animal Science, Bharathidasan University, Tiruchirapalli 620 024, Tamil Nadu, India ARTICLE INFO ABSTRACT Article history: The histophagous scuticociliate Philasterides dicentrarchi causes fatal scuticociliatosis in Received 8 May 2009 farmed olive flounder Paralichthys olivaceus. The average monthly prevalence of Received in revised form 11 September 2009 scuticociliatosis with P. dicentrarchi infections was very high from May to July (40 Æ 3 Accepted 1 October 2009 to 79 Æ 2%) in olive flounder at farms of Jeju Island, South Korea, from 2000 to 2004. The prevalence of mixed infection along with Vibrio spp. infection was higher (49 Æ 8%) than that Keywords: of scuticociliatosis alone. To date no effective control measure for P. dicentrarchi infection has Formalin been described and large economic losses continue. In the present study 3 chemotheraputants Jenoclean Hydrogen peroxide (formalin, hydrogen peroxide and Jenoclean) were used. Among these, bath treatment with Philasterides dicentrarchi Jenoclean at a low concentration of 50 ppm proved effective; the results were confirmed with Paralichthys olivaceus in vitro motility assessments and morphological changes in P. dicentrarchi. A similar trend was noted following hydrogen peroxide treatment. However, formalin was only moderately effective at this concentration. Therefore, hydrogen peroxide and Jenoclean are the promising compounds effective at low concentrations with short application time. ß 2009 Elsevier B.V. All rights reserved. 1. Introduction Scuticociliatosis was first reported in olive flounder in 1990, and quickly became a challenging disease with no The olive flounder Paralichthys olivaceus is an economic- effective control (Chun, 2000). After 2000 the disease ally important food fish in Korea with 98% of the domestic inflicted severe mass mortality in many commercial farms demand met through indigenous farming (Jung et al., in Jeju Island (Oh et al., 2006). Recently, 3 species 2001); farmed production commenced in the 1990s when (Pseudocohnilembus persalinus, Uronema marinum and wild stocks of P. olivaceus were exhausted. Currently olive Philasterides dicentrarchi) have been identified as respon- flounder culture is a major activity with about 300 sible for scuticociliatosis in Korean olive flounder farms commercial farms situated in Jeju Island, South Korea. (Kim et al., 2004a,b) with P. dicentrarchi as the main species Reported annual production was 21,368 and 20,000 to involved (Iglesias et al., 2001; Kim et al., 2004a; Jung et al., 25,000 mt in 1998 and 2000, respectively (Statistical Year 2005). Book of Maritime Affairs and Fisheries, 2000; Korea Flounder culture mainly begins in March and ends in National Statistical Office, 2001). December in a land-based raceway culture system with an average mortality of 15–20% (Lee, 2007). The incidence of the disease begins in May (Jin, 2006) with a marked * Corresponding author. Tel.: +82 64 754 3473; fax: +82 64 756 3493. withering of scales followed by imbalanced swimming E-mail addresses: [email protected] (R. Harikrishnan), behavior, anemia, weight loss, brownish skin patches, [email protected] (M.-S. Heo). 1 These authors contributed equally to this work. necrotic lesions, enteritis, excessive body mucus and 0304-4017/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2009.10.001 20 C.-N. Jin et al. / Veterinary Parasitology 168 (2010) 19–24 yellowish intestinal mucus. The infected fishes are dip with yeast extract and 10% fetal calf serum (Alvarez-Pellitero treated with Jenoclean, hydrogen peroxide or formalin et al., 2004). Active ciliates were washed and concentrated by (100–500 ppm) for 15–20 min per day for 3–5 days with dilution with Hanks balanced salt solution (HBSS) containing partial success. Indeed only about 50% of the farmers an antibiotic/antimycotic mixture (PSA) at 1Â to 3Â concen- attempt this and others go for final harvest though the final tration (1Â PSA = 100 U mlÀ1 penicillin, 100 mgmlÀ1 strepto- size is less (220 Æ 40 g) in August to October than in mycin and 0.25 mgmlÀ1 amphotericin B), followed by December (440 Æ 100 g). Farmers at present partially manage centrifugation at 500 Â g for 15 min. This process was repeated scuticociliatosis in the farm with crude application of as needed to obtain clear inocula with viable parasites. antibiotics such as oxy-tetracycline, gentamycine, tetracy- cline, amoxycilin and cefazolin, and/or chemicals such as 2.2. Subculture and harvesting of ciliates formalin, hydrogen peroxide, sodium chloride and Genoclean at concentrations of 350 Æ 150 ppm; however, no scientific Sterile artificial sea water (20% salinity) prepared using study has yet been conducted to determine relative efficacy Instant Ocean mixture (Aquarium Systems, Sarreborg, of these treatments. France) was used as a basal medium for the initial primary Globally, P. dicentrarchi poses a serious threat to the cultures. Ciliates were maintained in 25-cm2 T-flasks, with aquaculture industry, causing mass mortalities in marine 7 ml media at 20 8C. Subcultures were initiated by fishes such as olive flounder (Yoshinaga and Nakazoe, harvesting cultures in stationary phase, followed by 1993; Jee et al., 2001; Kwon et al., 2003), sea-bass and centrifugation (500–650 Â g, 15 min) and washing with turbot (Dykova´ and Figueras, 1994; Dragesco et al., 1995; HBSS containing 2Â PSA. Parasites were counted in a Iglesias et al., 2001; Sterud et al., 2000). However, detailed Neubauer chamber and inoculated into fresh media identification of its characteristics and possible treatment (Alvarez-Pellitero et al., 2004). measures are yet to be elucidated. For these reasons, it is essential to screen new effective drugs against P. dicen- 2.3. Determination of the efficacy of chemotherapeutants trarchi infection. Recently in vivo (piperazine N-substituted naphthyridines, pyridothienopyrimidines and pyridothie- Effects were observed using 1.5 ml of ciliate suspension notriazines, Quintela et al., 2003) studies report on the (centrifuged and resuspended in 2 mm filtered salt water) therapeutic properties of antiprotozoal compounds on the with 200 ml of suitably diluted therapeutants to give the control of the disease. In finfish culture, formalin baths are desired final concentration. Ciliate densities for each trial widely applied at concentrations of 35 Æ 15 ppm for 90 min ranged from 1.5 Â 104 to 4.3 Â 104 cells mlÀ1. Compounds (Tonguthai and Chanratchakool, 1992; Scott, 1993; Fajer- tested for efficacy in killing the ciliates were formalin Avila et al., 2003) or for a shorter duration of 60 min at (Merck, Germany) at 50, 100, 200, 300, and 400 ppm, concentrations from 167 to 250 ppm (Poupard, 1978)in hydrogen peroxide (Merck, Germany) at 25, 50, 100, 150, treating protozoan ectoparasites. 200, and 300 ppm and Jenoclean at 50, 100, 200, 300, 400, To the best of our knowledge, there is a paucity of and 500 ppm. Jenoclean was prepared by one-kg comprises studies on the chemotherapy of scuticociliatosis in olive Atacama extract 97% (Zeolites) and citric acid 3%. Sterile flounder. However, antibiotics like amprolium, monensin, polystyrene microplates (IWAKI) with 24 wells with 0.5 ml oxy-tetracycline and pyrimethamine + sulphaquinoxaline of medium per well (surface-to-volume ratio 4 cm2 mlÀ1) (Bayley, 1995) and amprolium, formalin and monesin were used. Each trial was performed in triplicate. After (Novotny et al., 1996) have been tested in vitro against inoculating each well with P. dicentrarchi, the plates were Anophryoides haernophil. The aim of this study is to assess incubated at 19 Æ 1 8C(Crosbie and Munday, 1999). Effects of the in vitro efficacy of several chemotherapeutic com- therapeutants on motility were observed periodically by pounds (formalin, hydrogen peroxide and Jenoclean) scanning several fields of view at 100Â and 200Â magnifica- against scuticociliatosis caused by P. dicentrarchi. tion using an inverted microscope (Zeiss Invertoscope ID 02) 2. Materials and methods Table 1 Scoring system for appraising the effects of various compounds on motility and morphology of Philasterides dicentrarchi (Crosbie and Munday, 1999). 2.1. Isolation of ciliates Score Interpretation P. olivaceus (mean weight 20 5 g; mean length Æ Motility 13 Æ 2 cm; n = 150) were collected from 15 local land-based 4 No effect; motility normal flounder farms affected with scuticociliatosis located in 3 Motility slowed in more than 50% of ciliates Sambo, Gujwa-eup, west of Jeju Island (338240600N, 2 Approximately 50% of ciliates stationary, but cilia 12683204600E), South Korea during May to September 2005. still beating 1 More than 50% of ciliates stationary, but cilia still The affected fish showed varying levels of tissue damage beating including severe epidermal and dermal necrotic lesions (Jin, 0 No sign of motility or cilia movement 2006). P. dicentrarchi were isolated from the brain, gill, ascites Morphology or ulcerated skin of the collected samples. We cloned and 4 No change; cells elliptical identified P. dicentrarchi using species-specific primers (Kim 3 Less than 50% of cells round or irregular shape et al., 2004b), ultrastructural and phylogenetic studies 2 Approximately 50% of cells round or irregular shape (Parama et al., 2006). P. dicentrarchi were cultured in 1 More than 50% of cells round and lysis evident 0 Extensive lysis with few cells intact minimum essential medium (MEM, Sigma) supplemented C.-N. Jin et al.