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Short Term in Vitro Culture of Cryptocaryon Irritans, a Protozoan Parasite of Marine Fishes

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Short Term in Vitro Culture of Cryptocaryon Irritans, a Protozoan Parasite of Marine Fishes 魚 病 研 究 Fish Pathology,39(4),175-181,2004.12 2004 The Japanese Society of Fish Pathology Short Term in vitro Culture of Cryptocaryon irritans, a Protozoan Parasite of Marine Fishes Apolinario V. Yambot1,3 and Yen-Ling Song1,2* 1Institute of Zoology, National Taiwan University, Taipei 106, Taiwan, ROC 2Department of Life Science , National Taiwan University, Taipei 106, Taiwan, ROC3 Present address: College of Fisheries-Freshwater Aquaculture Center , Central Luzon State University, Philippines (Received March 19, 2004) ABSTRACT--Attempts were made to cultivate Cryptocaryon irritans in vitro at 23-25℃. Attachment of theronts and subsequent enlargement into trophonts were achieved in two experi ments using strips of trypticase soy agar (TSA, supplemented with 3% NaCl) as an attachment substrate in filtered seawater. In the third experiment, transformation of theronts into trophonts was achieved in an enriched liquid medium composed of 50% filtered seawater, 30% Leibovitz L-15 and 20% fetal calf serum without attachment onto the TSA. Sizes (mean ±SD) of the trophonts, 114.6 ± 57.9 μm to 295.9 ± 130 μm, were from a recorded size range (50 to 700 μm) of the parasite in vivo. Although only limited numbers of theronts (0.28-1.71%) transformed into trophonts, these results showed that the in vitro culture of C. irritans is potentially feasible as evidenced by the enlargement of the trophonts within the in vivo size range using either a solid medium as an attach ment substrate or a liquid medium without attachment. There is a need, however, to determine essential factors that influence the transformation of the trophonts into viable tomonts capable of producing theronts. Key words: Cryptocaryonirritans, in vitro culture, theront, trophont, culture media The white spot disease in marine fishes caused by Matthews, 1995; Dickerson and Clark, 1996; Yoshinaga the ciliated protozoan parasite, Cryptocaryon irritans, is and Nakazoe, 1997). Manifestation of acquired protec posing a problem to marine aquaculture. The parasite tive immunity in the different fish species against C. is considered one of the most devastating parasites of irritans suggests a potential role of vaccine as a prophy cultured marine fishes. Ornamental, wild and cultured lactic measure. fish species in marine cages and pens are included in Under laboratory conditions, immunization of fish the list of hosts of C. irritans. The parasite is widely dis against the parasite looked promising (Burgess and tributed in various marine waters worldwide (Colorni and Matthews, 1995; Yoshinaga and Nakazoe, 1997; Bryant Burgess, 1997). Diversified isolates of C. irritans, et al., 1999). However, difficulty in obtaining a sufficient including a low salinity variant infecting pond-reared sea amount of parasite organisms for large-scale production bream, were characterized recently by Yambot et al. of the vaccine is a major problem. Continuous propa (2003). gation of the parasite in vivo requires considerable Earlier works demonstrated that freshwater fish ac space, effort and resources. Moreover, maintaining a quire immunity against lchthyophthirius multifiliis, a constant supply of healthy host fish to produce large freshwater counterpart of C. irritans (Clark et al., 1987, amounts of parasites for vaccine development is costly 1988; Burkart et al., 1990). Similarly, resistance and impractical. against C. irritans has been observed in fish surviving In vitro cultures of other protozoan parasites of from the infection of the parasite (Burgess and fishes had been performed by various researchers (Uzmann and Hayduk, 1963; Noga, 1987; Noga and * Corresponding author Bower, 1987; Woo and Li, 1990; Wang and Belosevic, E-mail: song @ccms.ntu.edu.tw 1994). The in vitro techniques reduce space and other 176 A. V. Yambot and Y.-L. Song resources and eliminate the use and maintenance of liv Taipei, Taiwan. Infected samples were chosen based ing host fishes. The in vitro culture aiming for a sus on the appearance of white spots on the body of the tained growth of I. multifiliis has been previously fish. Healthy naive grouper fingerlings (each 4-6 cm in attempted (Noe and Dickerson, 1995; Ekless and length) were procured from a hatchery in southern Matthews, 1993; Nielsen and Buchmann, 2000). The in Taiwan. The fingerlings which were reared in tanks and vitro propagation appears a promising alternative for pro fed with fresh small shrimp at least one week prior to ducing the parasite in sufficient quantity to immunize the infection were used for the in vivo propagation of the fish against white spot disease. parasite. The present study reports the results of the first After their exit from the original host, 10 to 20 phase of experiments on the screening of different tomonts of C. irritans were seeded into a 1 L beaker with media for the in vitro culture of the protozoan parasite, C. one fingerling for infection by the excysting theronts. irritans. Some features of the trophont produced in vitro Salinity of the seawater was maintained at 35 ppt. were also discussed. Moderate aeration was given in each beaker. Fish feeding was discontinued while infection was in progress. When necessary, partial water change and Materials and Methods collection of fecal matters and other debris by siphoning Maintenance of the parasite were done with great care in each vessel. When most Samples of C. irritans-infected grouper Epinephelus of the trophonts have exited from the experimentally coioides were sourced out from a live fish markets in infected fish body and transformed to tomonts, the fish Table 1. Description of the different media used in the three experiments and summary of results a Includes those media with trophonts; NTG-no trophont growth b Sizes of trophonts (•} SD) were pooled from T2 and T3 In vitro culture of Cryptocaryon irritans 177 hosts were transferred to a new confinement. The T3 (JF1 epithelial cells from javelin grunter Pomadasys adhering tomonts were detached from the bottom of the kaakan, 95% L-15, 5% FCS). Bleeding was carried out beakers using a fine paintbrush. The tomonts were col in tilapia hybrids, Oreochromis sp. (200 g each), using lected by siphoning, cleaned of debris, and seeded into a a 5-mL syringe punctured through the dorsal aorta of the new beaker to infect another naive host fish. caudal peduncle. The blood was placed separately into Eppendorf tubes and allowed to clot overnight at In vitro culture of parasite 4•Ž. The fish serum was then separated by centrifuga Three in vitro culture experiments were carried tion at 1000•~g for 10 min, heat-inactivated at 45•~ for out. The composition of the different media in each 20 min according to Yano (1992) and immediately stored treatment is presented in Table 1. Seawater used was at-80•~ prior to use. The epithelial cells (JF1 cells, gift filter-sterilized (0.22ƒÊm) and each treatment was repli from Dr. Shau-Chi Chi) were cultured as a monolayer in cated three times. Leibovitz L15 medium (supple cell culture flasks using L-15 medium supplemented with mented with 3% NaCl) and trypticase soya agar (TSA, 5% FCS. supplemented with 3% NaCl) were prepared separately The culture of C. irritans was performed in 12-well and sterilized as recommended by manufacturers. culture plates, each well containing 2 mL liquid media. Fetal calf serum (FCS) was heat-inactivated at 56•Ž for TSA strips were added into T1 and T2 as solid substrate 20 min and immediately stored at-80•Ž prior to for the attachment of the parasites. The monolayer of use. Incubation temperature for the in vitro growth of the JF1 cells was added in T3 as another attachment the parasite in culture flasks and plates was maintained substrate. The tomonts seeded into the wells were pre at 23 to 25•Ž in a cell culture incubator. Monitoring of pared according to the procedure described above. the parasite growth in culture flasks and plates was done Each well was seeded with three tomonts. Since bacte two times daily using an inverted microscope. rial contamination was observed in the culture flasks in Surviving theronts and trophonts were assessed as Experiment 1, ciproxin was used as antibiotics and intro alive based on ciliary beating and intact bodies. duced at a dose of 100ƒÊg mL-1 into each well. Photographic documentation of the in vitro raised trophonts at 5-day post-seeding was done to record their Experiment 3 sizes and other features. The culture of the parasite was carried out in 12-well tissue culture plates using four treatments: T1 (50% sea Experiment 1 water, 30% L15, 20% FCS, TSA strips), T2 (50% seawa Late stage tomonts of C. irritans with moving ter, 30% L15, 20% FCS), T3 (50% seawater, 30% L15, theronts inside the cysts (ready to excyst) were collected 20% grouper serum, TSA strips) and T4 (50% seawater, from the in vivo maintenance of the parasite. The 30% L15, 20% grouper serum). The serum concentra tomonts were further cleaned of debris and washed tion was increased to 20% to provide more essential three times with seawater. The tomonts were then nutrients following that of the primary culture of animal washed and soaked three times in filtered seawater with cells (Chi et al., 1999; Freshney, 2000). Collection of antibiotics consisting of 400 IU mL-1 penicillin, 400ƒÊg blood from the grouper (300 g body weight) and serum mL-1 streptomycin and 1ƒÊg mL-1 fungizone for 5 min preparation were performed as described above. each wash, with a quick dip into seawater without antibi Each well contained 2 mL liquid media. The otics between the washes. tomonts were cleaned as described above. Three Ten tomonts were seeded into each of 50-mL tissue tomonts were introduced into each well. To compare culture flasks. Liquid medium was maintained at 20 mL the sizes of in vitro raised trophonts and in vivo in each flask.
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