FULL PAPER Pathology

Experimental in Japanese Flounder (Paralichthys olivaceus) Infected with : Pathological Study on the Possible Neural Routes of Invasion and Dissemination of the inside the Body

Eman Moustafa Moustafa MOUSTAFA1,2,4), Nahoko TANGE3), Akinori SHIMADA1)* and Takehito MORITA1)

1)Department of Veterinary Pathology, Tottori University, 4–101 Minami, Koyama-cho, Tottori 680–8553, 2)The United Graduate School of Veterinary Science, Yamaguchi University, Yamaguchi 753–8515, 3)Tottori Prefectural Fisheries Experimental Station, 1166 Ishiwaki, Yurihama, Tohaku-gun, Tottori 689–0602, Japan and 4)Department of Fish Diseases and Management, Faculty of Veterinary Medicine, Kafr El-Sheikh University, Kafr El-Sheikh 33511, Egypt

(Received 19 May 2010/Accepted 13 July 2010/Published online in J-STAGE 27 July 2010)

ABSTRACT. Japanese flounder (Paralichthys olivaceus) were experimentally infected with the highly pathogenic scuticociliate Miamiensis avidus (syn. dicentrarachi) using the immersion method to clarify/identify the possible neural routes of entry and possible ways of dissemination of the scuticociliate in the fish body. were observed on the skin and gills right from day 0–1 post- infection, muscle tissue on day 2 post-infection, reached the brain, and spinal cord on day 3 post-infection, and systemic infection was prominent afterwards. Brain lesions were observed in most of the examined fish from days 3 and 4 post-infection and considered to be the cause of the sudden increase in mortality. Affected fish showed varying degrees of tissue damage including severe epidermal and dermal necrotic lesions, necrotic myositis, encephalitis and myelitis. Whereas, scuticociliates were frequently observed along the optic and/or olfactory nerve in the fish which were accompanied by severe brain lesions but by minimum lesions in the gills and skin, sug- gesting that in addition to skin and/or gills, neural routes including periorbital and nasal routes may play a role in scuticociliate invasion to the brain. Scuticociliates were also observed in the peripheral nerve fibers in the muscle tissue, cranial and spinal nerves, cranial cavity and in the vertebral canal, suggesting that nerve fibers and/or cerebrospinal fluid circulation may be involved in the spread of the scuti- cociliate throughout the body in addition to the blood circulation and connective tissue. KEY WORDS: experimental infection, Japanese flounder, Miamiensis avidus, neural routes, scuticociliatosis. J. Vet. Med. Sci. 72(12): 1557–1563, 2010

Scuticociliates are free-living organisms in sea-water, gested that P. dicentrarachi is a strong pathogen that can feeding on suspended particulate matters (bacteria, microal- cause a primary infection in flounder by penetrating the gills gae, protozoa). Under certain circumstances, however, and skin, followed by its travel via the blood stream to the these may behave as opportunistic histophagous par- other parts of the body [9]. Jung et al. [11] demonstrated asites, and actively feed on cells and tissue residues of cer- that M. avidus successfully invades the host directly from tain mollusks, and , and continue to live seawater, and causes high mortality. The ciliates rapidly and reproduce within the host tissues [5]. Several scutico- invade and proliferate in the skin and gills, and spread to the have been reported as agents to cause scutic- internal organs in the absence of any additional pathogens ociliatosis in farmed marine fish; species of Philasterides such as secondary bacterial invaders. These reports demon- dicentrarachi infects sea-bass [3] and population of sea strated abraded skin and/or gills as the main routes of entry dragons [19], nigricans species infect bluefin tuna and blood vessels as the dissemination route [9, 11]. Based [15] and Uronema marinum infects many species of aquar- on the results from the experimental infection study by ium fishes [2]. intracoelomic injection of the ciliates to the turbot, Puig et In Korea, scuticociliatosis in cultured Japanese flounder al. [17] suggested the connective tissue as a possible quick (Paralichthys olivaceus) was reported to be due to Uronema way of dissemination of the scuticociliate to the body. marinum [8], Philasterides dicentrarachi [12], Munday et al. [15] proposed that the scuticociliate Pseudocohnilembus persalinus [13] and Miamiensis avidus Uronema nigricans entered its host blue fin tuna by the [10]. It was suggested that Miamiensis avidus is the main nasal route; the starting point of infection was traced to the cause of scuticociliatosis in Japanese flounder in a series of olfactory rosettes, from which the parasite moved up the reports [10, 11, 20]. olfactory nerve to the brain, leading to locomotor dysfunc- Recently, a successful experimental immersion infection tion and final death. Experimental infections by Philas- of Japanese flounder (Paralichthys olivaceus) by Philas- terides dicentrarachi using different routes were terides dicentrarachi was performed in Korea. It was sug- successfully attempted in turbot by Parama et al. [16]; after periorbital inoculation of P. dicentrarchi, all fish died *CORRESPONDENCE TO: SHIMADA, A., Department of Veterinary Pathology, Tottori University, 4–101 Minami, Koyama-cho, Tot- within 5–13 days post-inoculation and the infection lesions tori 680–8553, Japan. were largely localized in brain (100% of the fish). Brain e-mail: [email protected] infection was observed in all the fish together with the pres- 1558 E. M. M. MOUSTAFA, N. TANGE, A. SHIMADA AND T. MORITA ence of ciliates in the optic nerve microscopically, suggest- Japan. Compound fish feeds were fed to the fish fries. Two ing that P. dicentrarchi is able to reach the brain not only via to three days before initiation of the experiment, fish were the blood stream but also via nervous tissues [16]. stopped feeding. Fifteen randomly selected fish were exam- In Japan, though the cause of the outbreak of scuticocili- ined under a stereo microscope before necropsy to ensure atosis in Japanese flounder (Paralichthys olivaceus) was that they were free from the parasites in the brain, gills, mus- considered to be unidentified ciliate [23] for many years, it cle and epidermal skin mucus. Fish were divided into 11 has recently become clear that the main cause of the scutic- experimental groups and each group was reared in a sepa- ociliatosis is Miamiensis avidus as demonstrated in our pre- rate tank. The first 10 groups contained 13 (Tank 1), 10 (2), vious study [6]. Exophthalmia, protrusion of eye ball, was 10 (3), 10 (4), 10 (5), 15 (6), 15 (7), 15 (8), 15 (9) and 20 detected in many affected fish accompanied by brain (10) fish, respectively and the 11th group contained 10 fish lesions; the findings enabled us to focus on the significance as the negative control. The fish in each tank were sampled of the neural route for entry and dissemination of the scutic- in the manner to ensure the minimum number for the inves- ociliate. tigation and to avoid an intentional selection of weakened The aim of this study is to clarify/identify the possible individuals. Because of the predicted increased risk of mor- neural routes of entry and possible ways of dissemination of tality of the fish associated with progression of the disease, the ciliates in the fish body through the pathological study of the initial numbers of fish in latter tanks were increased so Japanese flounder (Paralichthys olivaceus) experimentally that the total number of the fish examined in each tank infected by immersion method with a scuticociliate, would be equal (Miwa and Nakayasu [14]). Miamiensis avidus. All tanks were placed in a big water bath provided with a titanium heater to maintain a steady temperature of 20°C. MATERIALS AND METHODS Water in the fish tanks was slightly aerated constantly dur- ing the experiment. Ciliates isolation and cultivation: Scuticociliates were Experimental infection: On the first day of infection isolated from the brain of naturally infected Japanese floun- experiment, each tank was filled with 10 l of filtered seawa- ders reared at Tottori Prefectural Fisheries Experimental ter and fish were released into the tanks in numbers stated Station in Japan during an outbreak of the disease in 2005. above. A small piece of the brain of Japanese flounder containing Ten milliliters of the culture medium holding 106 cells of active ciliates was inoculated into Epithelioma Papillosum the scuticociliate was centrifuged at 3,500 rpm for 10 min at Cyprini (EPC) cells previously cultured in MEM3 [Mini- room temperature. For preparation of concentrate of the cil- mum Essential Medium (Invitrogen, Tokyo, Japan) supple- iates, the supernatant was discarded and the sediment was mented with 3% fetal bovine serum and 1% Antibiotic- suspended with 1 ml of filtered seawater. Ten equal concen- Antimycotic, liquid (Invitrogen)]. Scuticociliates multi- trates of the scuticociliates were prepared for immersion. plied with EPC were then kept at 25ºC for a period of over- These concentrates were added to each of tanks 1 to 10. The night to 3 days. For subculture, 1 l of the culture concentration of the ciliate in the immersion tanks was supernatant including the scuticociliates was added to 10 ml approximately 100 cells/ml. At 24 hr later, a further 20 l of of YEHS [Yeast Extract Horse Serum liquid medium: 2% filtered seawater were added to each tank. From the next ‘Lab-lemco’ powder (Oxoid, Tokyo, Japan), 0.5% BactoTM day onwards, half of the water in each tank was removed Yeast Extract (Difco 0.5% glucose, 0.8% sodium chloride and replaced by 15 l of filtered seawater on a daily basis. and 5% inactivated horse serum)], and incubated at 18ºC. The fish were observed for maximally ten days after the The scuticociliates were sub-cultured and maintained in the infection without feeding. medium, and the concentration of the scuticociliate in the Just 30 min after the start of immersion (infection), five culture medium was approximately 105 cells/ml. live fish were sampled from tank 1. From the first day to the The isolates were morphologically and genetically identi- tenth day after the infection, five live fish were sampled fied as Miamiensis avidus, and classified serologically, from each of tanks 1 to 10 on each day and examined for the using immobilization assays and Western blotting, into possible neural routes of entry and ways of dissemination of “serotype I” and it was named JF05To [21]. the ciliates (Table 1). The fish were checked from the sur- Aquarium: In the present study, 11 acrylic tanks of 30 l face to the interior of the body using stereo microscope capacity containing seawater, filtered with cartridge filter before necropsy in order to examine the evidence of infec- (0.5 µm, Millipore, Tokyo, Japan), were employed. Each of tion. Fish were euthanatized with an exposure of eugenol these tanks contained one perforation at the upper part of a [Fish/Crustacea anesthetic FA100 (Tanabe Seiyauku, Co., side wall for water drainage. A net was attached to the per- Ltd., Osaka, Japan)]. All dead fish of each tank were sam- foration in the tank to prevent the outflow of the young fish. pled upon confirmation of the death without consideration Fish: All fish used in the experiment (143 young fry of about the duration after the infection. All the fish, live and Japanese flounder of total length: 27.7 mm) were reared dead, were fixed in Davidson’s solution (330 ml of 95% eth- until the 94th day after hatchering in the institution con- anol, 220 ml commercial formaldehyde solution containing trolled under the same conditions as those in the laboratory 35% formaldehyde and 8% methanol, 115 ml glacial acetic in Tottori Prefectural Fisheries Experimental Station in acid and 335 ml distilled water). After one week, David- EXPERIMENTAL FISH SCUTICOCILITOSIS IN JAPAN 1559

Table 1. Histological detection of experimentally infected fish with Miamiensis avidus by immersion method Days post-immersion Tissue 0 1234 567 8910 Scuticociliate + + + + + + + + + + + Skin No. of fish/55 5555 555 44 3 Scuticociliate + + + + + + + + + + + Gills No. of fish/53 3223 355 33 2

Muscle Scuticociliate – – + + + + + + + + + No. of fish/50 0455 555 55 5

Eye Scuticociliate – – – + + + + + + + + No. of fish/50 0033333232 Scuticociliate – – – + + + + + + + + Brain No. of fish/50 0044455443 Scuticociliate – – – – + + + + + + + Nasal cavity No. of fish/50 0001355332 Scuticociliate – – – + + + + + + + + Spinal cord No. of fish/50 0044455443 Gastro- Scuticociliate – – – – + + + + + + + intestinal tract No. of fish/5 0 0 0 0 3 3 3 3 3 2 2 Scuticociliate – – – – – + + + + + + Liver No. of fish/50 0000333221 Scuticociliate – – – – – + + + + + + Kidney No. of fish/50 0000443322 son’s solution was switched to 75% ethanol solution. observed in the epidermis and gill mucosal epithelium. On Histopathology: After gross examination, all fish stored day 1 post-infection, the number of fish with evidence of finally in 75% ethanol were transported to the laboratory of infection and scuticociliates densities within the affected Veterinary Pathology of Tottori University, cut into trans- areas remained unchanged. There was an increase in sever- verse sections, dehydrated and embedded in paraffin wax. ity of the lesions as demonstrated by the extensive epider- Subsequently, sections 2–5 m thick were stained with mal damage associated with epidermal ulcer. Then, the hematoxylin & eosin (HE) for light microscopy. scuticociliates gradually disseminated within the entire body and appeared in subcutaneous connective tissue, mus- RESULTS cle tissue and peripheral nerve endings on day 2 post-infec- tion, in the periorbital cavity surrounding the eye, eye, optic Clinical symptoms: Bleached spots on the skin and der- nerve, brain, spinal nerve and spinal cord on day 3 post- mal necrotic lesions were the first observable clinical symp- infection, in the nasal cavity disseminating into the olfactory toms, which appeared just 30 min after the infection. nerve towards the brain from day 4 post-infection and in the Scuticociliates were observed on the skin and fins from day gastrointestinal tract, liver and kidney from days 4 and 5 1 post-infection, and detected to spread to the organs as fol- post-infection onwards. Since then, the infection was gener- lows; gills, eyes, brain and spinal cord from day 3 post- alized and systemic; brain infection was observed in most of infection; nostrils from day 4 post-infection; lips and mouth the examined fish. The scuticociliates distribution became from day 8 post-infection (data not shown). Few fish in the more evident by both ciliate density and numbers of the control group were found dead during the course of the lesions in the affected organs and tissues. study, with detecting the infection of ciliates in some of Histopathology: Histopathological examination on day 1 them. However, PCR examination was negative for post-infection revealed extensive epidermal destruction, Miamiensis avidus (data not shown). subsequent dermal degeneration with lymphocytic, mono- Scuticociliates dissemination inside the body: Results of cytic and eosinophilic granular cell infiltration associated the histological detection of Miamiensis avidus organism in with the presence of a high number of the scuticociliates. the experimentally infected fish are summarized in Table 1. On day 2 post-infection, there was necrotic degeneration in The first ciliate infection was observed in the skin and gills the muscles together with the scuticociliates invading in individuals of the group sampled on day 0 (30 min after underneath the epidermal layer and in the subcutaneous con- the immersion). High densities of scuticociliates were nective tissue. The scuticociliates were also observed in the 1560 E. M. M. MOUSTAFA, N. TANGE, A. SHIMADA AND T. MORITA nerve bundles (Fig. 1A) and nerve fiber endings (Fig. 1B) in 6 onwards, many scuticociliates containing red blood cells the muscle tissues affected. On day 3 post-infection, severe in the cytoplasm were observed in the gills, skeletal mus- infection of the scuticociliates was observed in the perior- cles, skin and brains of the fish. bital cavity (Fig. 2A), eye, optic nerve accompanied by inflammatory cells infiltration (Fig. 2B). The scuticociliates DISCUSSION were also observed in the brain, spinal cord, spinal nerve (Fig. 3) and in the vertebral canal (Fig. 4). From day 4 Miamiensis avidus is a highly invasive and destructive onwards, the scuticociliates were observed in the blood ves- histophagous endoparasitic scuticociliate infecting Japanese sels of a variety of tissues and/or organs (Fig. 5), nasal cav- flounder. In the present study, an experimental infection of ity, along the olfactory nerve and in the olfactory lobe, Japanese flounder was successfully achieved by immersion where severe inflammatory changes were detected (Fig. 6A method to clarify/identify the possible neural routes of entry and 6B) and third ventricle. Since then, the infection and ways of dissemination of the scuticociliate in the fish became more systemic and scuticociliates were observed in body. the lamina propria and lamina muscularis of the gastrointes- Although there was a slight difference in the density of tinal tract and in the abdominal cavity. Periglomerular, per- the scuticociliates between the five alive fish samples from itubular and perivascular mononuclear inflammatory cells each tank which may give an impact on the course of the infiltration along with the presence of the scuticociliates pathogenesis, much attention was paid to clarify/identify the inside blood vessels were observed in the kidney. From day possible neural routes of entry and ways of dissemination of

Fig. 1. A. Scuticociliates (arrows) inside the nerve bundles (N. B.) in the muscle tissue (M). B. Scuticociliates (arrows) in the nerve end- ings (N. End.) in the muscle tissue (M). Muscle tissue from infected fish taken on day 2 post-infection. Bar=100 μm.

Fig. 2. A. Eye (E) showing severe infection of the scuticociliates (arrows) in the periorbital cavity (Per. C.). Bar=50 μm. B. Optic nerve (Opt. N.) showing scuticociliates (arrows) with inflammatory changes. Optic nerve from infected fish taken on day 3 post-infection. Bar=50 μm. EXPERIMENTAL FISH SCUTICOCILITOSIS IN JAPAN 1561

Fig. 3. Scuticociliates (arrows) in the spinal cord (Sp. C.) and in Fig. 5. Scuticociliates (arrows) inside the blood vessels of semi- the spinal nerve (Sp. N.) with severe inflammatory changes. Spi- circular canal in the cranium. Cranium from infected fish taken nal cord from infected fish taken on day 3 post-infection, on day 5 post-infection, Bar=100 μm. Bar=100 μm.

Whereas, the scuticociliates were frequently observed along the optic and/or olfactory nerve in the fish which were accompanied by severe brain lesions but by minimum lesions in the gills and skin. The findings might suggest that in addition to skin and/or gills, neural routes including the periorbital and nasal routes may play a role in the current scuticociliate invasion to the brain [15, 16]. Extensive epidermal damage, dermal degeneration, hyperplasia of branchial epithelium, necrotic myositis, encephalitis and myelitis were observed in the present study; these changes are in agreement with previous studies on the pathology of the outbreaks and experimental cases of scuticociliatosis in fish [1, 7, 9–11]. Brain infection was observed in most of the fish examined, suggesting that the brain is one of the most susceptible organs. Pathological examination of the dead fish taken from day 6 post-infection onwards, when a sudden leap in mortality detected, showed Fig. 4. Scuticociliates (arrows) in the spinal cord (Sp. C.) accom- panied by minimum inflammatory changes and in the vertebral extensive damage in the brain without severe lesions in the canal. Spinal cord from infected fish taken on day 4 post-infec- other tissues and organs. These findings suggest the signif- tion, Bar=100 μm. icance of the brain lesions as the cause of death of the infected fish. Blood stream has been regarded as a major way of dis- the scuticociliate in the host tissue. The same experimental semination into the other tissues [9, 11, 16, 18, 22]. In addi- design was used by Miwa and Nakayasu [14]. tion, the connective tissue was previously suggested as a Few mortality cases were observed in the control group possible quick way of dissemination of the scuticociliate to till day 10 post-infection. This might be due to some stress the body [17]. In the present study, the scuticociliates were factors, lack of nutrients, and/or some infectious agents observed in and along the peripheral, spinal and cranial either bacteria or parasite. Some fish were found to be nerve fibers, cranial cavity and in the vertebral canal, sug- infected individually with ciliates which were PCR negative gesting that the ciliates could spread from the entry sites not for Miamiensis avidus. However, the scuticociliate infected only through blood stream and connective tissue but also groups showed higher mortalities than the control group. through nerve fibers and/or cerebrospinal fluid circulation. In the present study, the scuticociliates were firstly observed in the skin and/or gills from day 0 post-infection ACKNOWLEDGMENTS. The author would like to thank and then disseminate to different body tissues, suggesting Dr. Tarek Balabel, associate professor of Animal Behaviour that the first possible invasion route may be the dermal and/ and Management, Kafr El-Sheikh University, Egypt for or branchial epithelium (skin and/or gill) [4, 9, 11, 16]. continuous encouragement and help in editing the manu- 1562 E. M. M. MOUSTAFA, N. TANGE, A. SHIMADA AND T. MORITA

Fig. 6. A. Scuticociliates (arrows) in the nasal cavity (N.C.), cranial cavity, and along the olfactory nerve (Olf. N.). Inflamatory changes in the olfactory nerve, meningitis and severe haemorrage in the cranial cavity are shown. Bar=50 μm. B. High power magnification of rectangular area of Fig. A. showing the scuticociliates (arrows) with inflammatory cell infiltration in the olfac- tory lobe of the brain. Olfactory lobe from infected fish taken on day 4 post-infection, Bar=100μm.

phora, Scuticociliatida) as the causative agent of scuticociliato- script. Thanks to Dr. Nachiketa Das for reviewing the sis in farmed turbot Scophthalmus maximus in Galacia (NW English. The authors would also like to appreciate the Spain). Dis. Aquat. Org. 46: 47–55. financial support from the Egyptian Embassy to the first 8. Jee, B. Y., Kim, Y. C. and Park, M. S. 2001. Morphology and author (E. M. M. M.). biology of parasite responsible for scuticociliatosis of cultured olive flounder Paralichthys olivaceus. Dis. Aquat. Org. 47: 49– REFERENCES 55. 9. Jin, C. N., Harikrishnan, R., Moon, Y. G., Kim, M. C., Kim, J. 1. Azad, I. S., Al-Marzouk, A., James, C. M., Almatar, S. and Al- S., Balasundaram, C., Azad, I. S. and Heo, M. S. 2009. Histo- Gharabally, H. 2007. Scuticociliatosis associated mortalities pathological changes of Korea cultured olive flounder, Parali- and histopathology of natural infection in cultured silver pom- chthys olivaceus due to scuticociliatosis caused by fret (Pampus argenteus Euphrasen) in Kuwait. histophagous scuticociliate, Philasterides dicentrarachi. Vet. 262: 202–210. Parasitol. 161: 292–301. 2. Cheung, P. J., Nigrelli, R. F. and Ruggieri, G. D. 1980. Studies 10. Jung, S. J., Kitamura, S. I., Song, J. Y., Joung, I. Y. and Oh, M. on the morphology of Uronema marinum Dujardin (Ciliatea: J. 2005. Complete small subunit rRNA gene sequence of the Uronematidae) with a description of the histopathology of the scuticociliate Miamiensis avidus pathogenic to olive flounder infection in marine fishes. J. Fish Dis. 3: 295–303. Paralichthys olivaceus. Dis. Aquat. Org. 64: 159–162. 3. Dragesco, A., Dragesco, J., Coste, F., Gasc, C., Romestand, B., 11. Jung, S. J., Kitamura, S. I., Song, J. Y. and Oh, M. J. 2007. Raymond, J. and Bouix, G. 1995.Philasterides dicentrarchi, n. Miamiensis avidus (Ciliophora: Scuticociliatida) causes sys- sp. (Ciliophora: scuticociliatida), a histophagous opportunistic temic infection of olive flounder Paralichthys olivaceus and is parasite of Dicentrachus labrax (Linnaeus. 1758) a reared a senior synonym of Philasterides dicentrarachi. Dis. Aquat. marine fish. Eur. J. Protistol. 31: 327–340. Org. 73: 227–234. 4. Dykovà, I. and Figueras, A. 1994. Histopathological changes 12. Kim, S. M., Cho, J. B., Kim, S. K., Nam, Y. K. and Kim, K. H. in turbot Scophthalmus maximus due to a histophagus ciliate. 2004. Occurrence of scuticociliatosis in olive flounder Parali- Dis. Aquat. Org. 18: 5–9. chthys olivaceus by Philasterides dicentrarachi (Ciliophora: 5. Elston, R. A., Cheney, D., Frelier, P. and Lynn, D. 1999. Inva- Scuticociliatida). Dis. Aquat. Org. 62: 233–238. sive orchitophryid ciliate infections in juvenile Pacific and 13. Kim, S. M., Cho, J. B., Lee, E. H., Kwon, S. R., Kim, S. K., Kumomoto oysters, Crassostrea gigas and Crassostrea sika- Nam, Y. K. and Kim, K. H. 2004. Pseudocohnilembus persali- mea. Aquaculture 174: 1–14. nus (Ciliophora: Scuticociliatida) is an additional species caus- 6. Eman-Moustafa, M. M., Naota, M., Morita, T., Tange, N. and ing scuticociliatosis in olive flounder Paralichthys olivaceus. Shimada, A. 2010. Pathological study on the scuticociliatosis Dis. Aquat. Org. 62: 239–244. affecting farmed Japanese flounder (Paralichthys olivaceus) in 14. Miwa, S. and Nakayasu, C. 2005. Pathogenesis of the experi- Japan. J. Vet. Med. Sci. 72: 1359–1362. mentally induced bacterial cold water disease in ayu Plecoglos- 7. Iglesias, R., Parama, A., Alvarez, M. F., Leiro, J., Fernandez, J. sus altivelis. Dis. Aquat. Org. 67: 93–104. and Sanmartin, M. L. 2001. Philasterides dicentrarchi (Cilio- 15. Munday, B. L., O’Donoghue, P. J., Watts, M., Rough, K. and EXPERIMENTAL FISH SCUTICOCILITOSIS IN JAPAN 1563

Hawkesford, T. 1997. Fatal encephalitis due to the scuticocili- tus). Vet. Pathol. 45: 546–550. ate Uronema nigricans in sea-caged, southern bluefin tuna 20. Song, J. Y., Kitamura, S. I., Oh, M. J., Kang, H. S., Lee, J. H., Thannus maccoyii. Dis. Aquat. Org. 30: 17–25. Tanaka, S. J. and Jung, S. J. 2009. Pathogenicity of Miamiensis 16. Paramá, A., Iglesias, R., Álvarez, M. F., Leiro, J., Aja, C. and avidus (syn. Philasterides dicentrarachi), Pseudocohnilembus Sanmartín, M. L. 2003. Philasterides dicentrarachi (Cilio- persalinus, Pseudocohnilembus hargisi and Uronema marinum phora, Scuticociliatida): experimental infection and possible (Ciliophora, Scuticociliatida). Dis. Aquat. Org. 83: 133–143. routes of entry in farmed turbot (Scophthalmus maximus). 21. Song, J. Y., Sasaki, K., Okada, T., Sakashita, M., Kawakami, Aquaculture 217: 73–80. H., Matsuoka, S., Kang, H. S., Nakayama, K., Jung, S. J., Oh, 17. Puig, L., Traveset, R., Palenzuela, O. and Padros, F. 2007. His- M. J. and Kitamura, S. I. 2009. Antigenic differences of the topathology of experimental scuticociliatosis in turbot Scoph- scuticociliate Miamiensis avidus from Japan. J. Fish Dis. 32: thalmus maximus. Dis. Aquat. Org. 76: 131–140. 1027–1034. 18. Ramos, M. F., Costa, A. R., Barandela, T., Saraiva, A. and 22. Sterud, E., Hansen, M. K. and Mo, T. A. 2000. Systemic infec- Rodrigues, P. N. 2007. Scuticociliate infection and pathology tion with Uronema-like ciliates in farmed turbot Scophthalmus in cultured turbot Scophthalmus maximus from the north of maximus (L.). J. Fish Dis. 23: 33–37. Portugal. Dis. Aquat. Org. 74: 249–253. 23. Yoshinaga, T. and Nakazoe, J. 1993. Isolation and in vitro cul- 19. Rossteuscher, S., Wenker, C., Jermann, T., Wahli, T., tivation of an unidentified ciliate causing scuticociliatosis in Oldenberg, E. and Schmidt-Posthaus, H. 2008. Severe scutico- Japanese flounder (Paralichthys olivaceus). Fish Pathol. 28: ciliate (Philasterides dicentrarachi) infection in a population 131–134. of sea dragons (Phycodurus eques and Phyllopteryx taeniola-