The capability of Streptococcus iniae to cause disease in Japanese flounder was investigated by oral and bath (30 min) challenges with low, medium and high inoculation doses. Only with the highest inoculation dose, 9.9 x 10^7 CFU/100g body weight, the oral challenge induced deaths. In the bath challenge deaths were induced even at the lowest inoculation dose, 2.9 x 10^3 CFU/mL water. Hemorrhagic lesions on the fins were observed in dead fish challenged by both methods. It is suspected that S. iniae entered through the body surface such as abrasive sites of the fins to cause disease

Kanai Kinya

Nguyen Huu Thinh

Yoshikoshi Kazuma

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~,mfiJrJE Fish Pathology, 36 (1), 40-41, 2001. 3Experimental Streptococcus iniaeInfection in Japanese FlounderParalichthys olivaceusHuu Thinh Nguyen, Kinya Kanai*and Kazuma YoshikoshiGraduate School of Marine Science and Engineering,Nagasaki University, 1-14 Bunkyo Machi,Nagasaki 852-8521, Japan(Received December 13, 2000)ABSTRACT--The capability of Streptococcus iniae tocause disease in Japanese flounder was investigated byoral and bath (30 min) challenges with low, medium andhigh inoculation doses. Only with the highest inocula-tion dose, 9.9 x 107 CFU/1 00 g body weight, the oral chal-lenge induced deaths. In the bath challenge deathswere induced even at the lowest inoculation dose2.9 x 103 CFU/mL water. Hemorrhagic lesions on th~fins were observed in dead fish challenged by bothmethods. It is suspected that S. iniae entered throughthe body surface such as abrasive sites of the fins tocause disease.Key words: Streptococcus iniae, Paralichthysolivaceus, oral challenge, bath challenge,Japanese flounderStreptococcus iniae is one of the principle causativeagents of streptococcosis in many cultured fish. Theinfection routes of S. iniae have been suggested throughexperimental infections. Through the digestive tract aswell as directly from the water S. iniae can cause diseasein blue tilapia Oreochromis aureus1). The olfactoryorgan has been suggested as a possible route of S. iniaeinfection for hybrid striped bass Morone chrysops x M.saxatilis and nile tilapia O. niloticus2 ). Lactococcusgarvieae, a pathogen of fish streptococcosis, has beensuggested to infect yellowtail Seriola quinqueradiatathrough the digestive trace).Streptococcosis in Japanese flounder Paralichthysolivaceus, an important flatfish species in mariculture inJapan, often occurs during the period of high watertemperature4). Infection routes of S. iniae in flounderare not clear. Elucidation of possible routes of theinfection will be helpful to establish more effective fishhealth management. The purpose of the present work isto investigate the capability of S. iniae to cause disease byoral and bath challenge with three inoculation doses.* Corresponding authorE-mail: kanai@net.nagasaki-u.ac.jp© 2001 The Japanese Society of Fish PathologyMaterials and MethodsFlounder (average weight 101 ± 16 g) were stockedinto 200-L tanks for challenge groups of 20 fish each and30-L tanks for control groups of 5 fish each one daybefore challenge. Each tank was supplied with constantaeration and continuous water flow at 6 Umin. Watertemperature ranged from 25 to 26°C and fish receivedno food during the experiment.S. iniae NUF631, originally isolated from a diseasedflounder, was cultured on Todd-Hewitt (TH) agar platesat 28°C, harvested 18 h after incubation by washing offthe plates with 0.1 M phosphate-buffered saline (PBS),pH 7.2, to make a bacterial suspension. The experi-ment was carried out with low, medium and high inocula-tion doses as described below.For oral challenge, viable count of the bacterial sus-pension was confirmed by TH agar plate count (dupli-cate). Commercial pellet feed ground thoroughly by agrinder was mixed with an appropriate volume of the bac-terial suspension at a ratio of 1(wt) : 2.5(v) to produce aninjectable slurry. Each fish was anesthetized withtricaine methane sulfonate and received the slurry at avolume of 2% body weight by intubation. The intubationwas performed with a 10-mL syringe attached with aplastic catheter that was inserted to the stomach ofexperimental fish. Inoculation doses were 9.9 x 103(low), x105 (medium) and x107 (high) colony formingunits (CFU)/100 g body weight. The fish in controlgroup received the luxury made with PBS only.For bath challenge, water in the tanks was draineduntil the remaining was 10 L and then the bacterial sus-pension was poured in. Viable count of S. iniae in thewater was confirmed by TAOA agar plate count (dupli-cate). TAOA agar, a selective medium for S. iniae, wasprepared according to the descriptions in our publishedwork5). Fish were exposed to the bacterium for 30min. At the end of exposing time, the tanks were filledin with water to reach the initial water level for 30 min,and then water change was commenced again. Inocu-lation doses were 2.9 x 103 (low), x105 (medium) andx107 (high) CFU/mL water. No bacteria were pouredinto the tank of control group.Number of survivors was monitored daily for a 14day period after challenge. Dead fish were taken offand examined for gross lesions both externally andinternally. Their brain and kidney were cultured on THagar and then the grown colonies, if present, were sub-jected to agglutination tests with rabbit anti-So iniaeNUF631 serum to confirm S. iniae infection. Survivorsat the end of the experiment were also examined in thesame way as dead fish.Results and DiscussionNo deaths were observed in the control groups andExperimental Streptococcus iniae infection in Japanese Flounder Paralichthys olivaceus 41o 1 2 3 4 5 6 7 8 9 10 11 12 13 14Days after challengeFig. 2. Changes in number of survivors after bath challengewith S. iniae NUF631. Inoculation dose (CFU/mLwater); 2.9x 107 (0), 2.9x 105 (v), 2.9x 103 (6).o 1 2 3 4 5 6 7 8 9 10 11 12 13 14Days after challengeFig. 1. Changes in number of survivors after oral challengewith S. iniae NUF631. Inoculation dose (CFU/100 gbody wewight); 9.9 x 107 (0), 9.9 x 105 (v), 9.9 x 103(6).Referencespathological lesions were more obvious. Hemorrhagesof fin were severe and extended to the proximalmargin. Congestion of the liver, spleen and kidneyresulting in severe swelling and darkness in color ofthese organs were observed. Some dead fish showedtypical lesions of the disease such as clouding cornea,internal hemorrhagic and exophthalmic eyes, acitesresulting in abdominal swelling, and pale liver, spleenand kidney. Gastric and intestinal lesions were notobserved in any dead fish. These observations werecommon in the fish challenged by either challengemethod.Although the oral challenge was reported to produceS. iniae infection 1), the capability of the bacterium toenter through the mucosal epithelia of the stomach and/or intestine has not been proved. If S. iniae possessesthis capability indeed, it requires, as the present resulthas shown, a high number of viable cells to causedisease. It may be such a case in practical culture offlounder that flounder ingests decayed visceral organs orbody of infected fish. Otherwise deaths in the oral chal-lenge group employed with the high inoculation dosemay due to the infection of S. iniae excreted to the tankalong with feces, because hemorrhagic lesions on thefins of dead fish were observed like bath-challenged fishand relatively low number of viable cells was enough tocause disease in the bath challenge groups.In the bath challenge experiment deaths wereinduced even at the lowest inoculation dose. This resultimplies even though fish may ingest S. iniae-containingwater during the exposing period, it is unlikely to occurthe oral route infection. It has been suggested thattransmission of streptococcosis caused by S. iniae fromfish to fish could occur via direct contact and abrasion6).Basing on the observation of hemorrhagic lesions on thefins of dead fish, it seems reasonable to suspect that S.iniae entered through the body surface such as abrasivesites of the fins.The result of this work showed that S. iniae infectioncould be induced by oral and bath challenge, thoughbath challenge induced disease more easily than oralchallenge. Bacteriological and histopathological studiesare needed to clarify the exact sites of entry and thecourse of infection.1) Perera, R. P., S. K. Johnson and D. H. Lewis (1997): Aquac-ulture, 152, 25-33. 2) Evans, J. J., C. A. Shoemaker and P.H. Klesius (2000): Aquaculture, 189, 197-210. 3) Taniguchi,M. (1982): Nippon Suisan Gakkaishi, 48, 1717-1720. 4)Nakatsugawa, T. (1983): Fish Pathol., 17, 281-285. 5)Nguyen, H. T. and K. Kanai (1999): J. Appl. Microbiol., 86, 769-776. 6) Shoemaker, C. A., J. J. Evans and P. H. Klesius(2000): Aquaculture, 188, 229-235.20....,....(}-{}-~---f?---r~~-------------,18(/) 16~~ 14~ 12~(/) 104-e 8~(1)~ 6~z 42o--+---.,...---,.-.,....-...,-......,....---,-0( l--r-or---~~---r-----r-~--r--.......2018C/) 16~~ 14~ 12~(/) 104-e 8~(1)~ 6~z 42o-t--r-~--r----r----r----,..--.,-r--~-,......--r----,.-~--,....---fthe oral challenge groups with the low and mediuminoculation doses. In the other challenge groups,deaths were observed. Death in these groups began atday 2 and the majority of deaths occurred between 2 and7 days after challenge except for those observed in thebath challenge group with the low inoculation dose. Theinitiation of deaths in this group occurred later, at day 5(Figs. 1 and 2). S. iniae was recovered from all deadfish and one survivor in the bath challenge group with thelow inoculation dose.Gross examinations of the early deaths showed,externally, small hemorrhagic lesions on the fins, particu-larly the dorsal and pectoral fins, and, less frequently, onthe skin. Internally, mild swelling of the liver, spleen andkidney was observed. For those of the later deaths,

ヒラメレンサ球菌症の実験感染

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ヒラメレンサ球菌症の実験感染

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