魚病研究 Fish Pathology, 50 (2), 60–67, 2015. 6 © 2015 The Japanese Society of Fish Pathology Research article Infection Dynamics of Kudoa septempunctata (Myxozoa: Multivalvulida) in Hatchery-produced Olive Flounder Paralichthys olivaceus Hiroshi Yokoyama1*, Meibi Lu1, Koh-ichiro Mori2, Jun Satoh2, Tohru Mekata2 and Tomoyoshi Yoshinaga1 1Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan 2National Research Institute of Aquaculture, Kamiura Branch, Oita 879-2602, Japan (Received January 6, 2015) ABSTRACT—Food poisoning of humans, caused by the ingestion of raw flesh of the olive flounder Paralichthys olivaceus infected with Kudoa septempunctata, has recently become a public health con- cern in Japan. The present study investigated the infection dynamics of K. septempunctata in two cohorts of olive flounder produced in a hatchery, where K. septempunctata infection was enzootic, by PCR assay and light microscopy. In less than 1-year-old juveniles of the 2011 cohort (hatched in February 2011), K. septempunctata was not detected in either June or July 2011 even by conventional PCR, but light microscopy detected a heavy infection (> 1 × 106 spores/g) in October 2011. In 2-year-old fish of the 2009 cohort (hatched in February 2009), the prevalence of infection varied from 30% to 90% from April to December 2011, although no clear pattern was observed in the fluctuation of prevalence and intensity. Fish-to-fish transmission of K. septempunctata was not possible orally or by cohabitation. To investigate the infection period and early development of K. septempunctata, uninfected fish were exposed monthly for 2 weeks to the seawater at the infected hatchery. The results indicated that the peak period of infection was July, and that K. septempunctata was detectable in the heart by quantitative PCR assay as early as 1 week post-exposure, then in the blood and somatic muscle at 2 weeks post- exposure. Key words: Kudoa septempunctata, Myxozoa, Paralichthys olivaceus, infection dynamics, food poisoning, olive flounder Marine myxosporeans have been often recognized some of them were forced to close their business or to as pathogens of cultured fish (China et al., 2013; Özer replace the culture fish species with another due to et al., 2014; Shirakashi et al., 2014; Xu et al., 2014). In these infections. addition, some Kudoa species decrease the marketabil- In June 2011, the Ministry of Health, Labor and ity of the host fish because of either post-mortem myoliq- Welfare of Japan recommended that infected olive floun- uefaction (e.g., Kudoa thyrsites and K. lateolabracis) or der should be frozen at –15°C to –20°C for > 4 h macroscopic cysts in the trunk muscle (e.g., K. or heated at 75°C for > 5 min to prevent foodborne amamiensis and K. iwatai) (Moran et al., 1999a; Eiras et illness due to K. septempunctata (webpage of the al., 2014). In recent years, K. septempunctata infec- Ministry of Health, Labor, and W elfare, Japan: tion of the somatic muscle of the olive flounder http://www.mhlw.go.jp/stf/houdou/2r9852000001fz6e- Paralichthys olivaceus has been implicated as a cause att/2r9852000001fzl8.pdf, “accessed 5 January 2015”). of food poisoning in humans, which has become a However, such treatments are impractical because olive major public health concern in Japan (Kawai et al., flounder are commonly eaten raw (sashimi or sushi) 2012). During a food poisoning outbreak, acute diar- (Yokoyama, 2013). The onset of food poisoning was rhea and vomiting occurred within 2–20 h after inges- probably dependent on the spore dose (Kawai et al., tion of the raw flesh of infected olive flounder, although 2012); the threshold spore dose that induces the dis- patients usually recovered within 24 h. However, floun- ease was estimated to be approximately 7 × 107 spores der farmers have suffered serious economic losses, and (Yahata et al., 2015). Considering the average amount of flounder ingested per person, the permissible spore * Corresponding author density in flounder muscle was determined to be < 1 × 6 E-mail: ayokoh@ mail.ecc.u-tokyo.ac.jp 10 spores/g (webpage of the Ministry of Health, Labor, Kudoa septempunctata infection in olive flounder 61 and Welfare, Japan: http://www.mhlw.go.jp/topics/yunyu/ obtained at intervals from three private hatcheries (A, B other/2012/dl/120607–01.pdf, “accessed 5 January and C) in two prefectures located in western Japan. 2015”). This density has been adopted as the permissi- Hatching periods and sampling designs are summa- ble upper limit by the Japanese Food Hygiene Law rized in Table 1. Water treatment methods in the three (Yokoyama, 2013). hatcheries were as follows. Fish in hatchery A were The Fisheries Agency of the Ministry of Agriculture, reared in sand-filtered (specified capture particle size Forestry, and Fisheries of Japan has recommended that > 10 m m) and ultraviolet (UV)-irradiated seawater from fish farmers remove infected fish lots by careful inspec- February to May 2011 and thereafter in non-filtered tion for K. septempunctata prior to shipment to the fish seawater. For fish in hatchery B, culture water was market (webpage of the Fisheries Agency, Japan: sand- and cartridge-filtered, and additionally treated http://www.jfa.maff.go.jp/test/saibai/pdf/kudoa2.pdf, with UV throughout the rearing period. In hatchery C, “accessed 5 January 2015”). Briefly, a polymerase rearing water was sand-, 200-m m and 50-m m cartridge chain reaction (PCR) assay based on Grabner et al. filtered and treated with UV (61 mJ/cm2) for the initial 1 (2012) (or a modified protocol) is performed when juve- month. Thereafter, the fish were reared in sand-fil- nile flounder are introduced into the fish farms from tered seawater. hatcheries, and light microscopic examination of methyl- The muscle tissues (and the heart in several sam- ene blue-stained smears of the muscle tissue is con- plings) were removed from the flounder’s eyed-side dor- ducted before commercial-size fish are shipped to sal trunk and examined for K. septempunctata infection market. by light microscopy and/or PCR assay according to the These control measures seem to have greatly con- slightly modified protocol of Grabner et al. (2012). A tributed to a decrease in food poisoning cases in recent wet mount preparation was made by adding one drop of years. However, little is known about the infection phosphate-buffered saline (PBS) onto the minced dynamics of K. septempunctata, although this informa- muscle tissue and K. septempunctata spores were tion is of considerable importance for the design of observed under a light microscope (CX41, lO ympus). inspection programs. In the present study, we per- For PCR diagnosis, the DNA was extracted from the formed a periodical investigation of K. septempunctata muscle and heart using a QIAamp® DNA Mini Kit infection in hatchery-produced olive flounder and the (Qiagen) following the manufacturer’s protocol. To parasite’s development in sentinel fish monthly exposed amplify the K. septempunctata 28S rDNA, the primer to infectious water. pairs KSf (5¢-GTG TGT GAT CAG ACT TGA TAT G-3¢) and KSr (5¢-AAG CCA AAA CTG CTG GCC ATT T-3¢) were used. PCR reactions were carried out in a 20-m L Materials and Methods total volume mixture. Each PCR mixture contained 0.1 Field monitoring of K. septempunctata infection at three m L of Ex Taq HS (Takara), 2 m L of 10 × Ex Taq Buffer, hatcheries 1.6 m L of dNTP mixture (2.5 mM of each), 0.4 m L of In 2011, hatchery-reared olive flounder juveniles each primer, and 1.0 m L of extracted DNA suspension. (less than 1 year old, n = 60 for each sampling) were All PCR reactions were performed in an iCycler Table 1. Field monitoring of Kudoa septempunctata infection in juvenile olive flounder Paralichthys olivaceus (n = 60) hatched at three private hatcheries in 2011 Prevalence of infection (%) Hatchery Month of hatching Tissue examined Detection method May Jul. Sep. Oct. Nov. Dec. PCR 00 * 47NE NE NE Muscle Microscopy NE NE 35NE NE NE A February 2011 Heart PCR 10NE0NE NE NE Mean body length (cm) 8.0 13.2 NE 21.6 NE NE Muscle PCR NE NE NE0NE NE B February 2011 Mean body length (cm) NE NE NE 15.3 NE NE PCR ** 0000–– Muscle August 2011 Microscopy NE–– 00NE Mean body length (cm) 2.0–– 5.2 10.4 13.6 C Muscle PCR 00NE–– NE September 2011 Mean body length (cm) NE–– 2.0 2.1 NE Muscle PCR 0NE––– NE October 2011 Mean body length (cm) NE––– 1.7 NE *Not examined, **Before hatching. 62H. Yokoyama, M. Lu, K. Mori, J. Satoh, T. Mekata and T. Yoshinaga (Bio-Rad). Denaturation of DNA (95°C for 4 min) was 1 × 106 spores/g was defined as a heavy infection followed by 35 cycles of amplification consisting of 30 s because it was considered as a threshold that causes a for denaturation at 95°C, 30 s for annealing at 53°C and food poisoning (webpage of the Ministry of Health, 30 s for extension at 72°C, ending with a 5 min exten- Labor, and Welfare, Japan: http://www.mhlw.go.jp/topics/ sion at 72°C. PCR products were analyzed by 1.5% yunyu/other/2012/dl/120607–01.pdf, “accessed 5 January agarose gel electrophoresis with TAE (40 mM Tris-ace- 2015”). Fish samples of the 2011 cohort were investi- tate, 1 mM EDTA) running buffer. gated by light microscopy, and the intensity of infection was determined. Additionally, for the first two sam- Periodical investigation of K. septempunctata infection plings (June and July 2011), the muscle samples were in zero- to two-year-old hatchery-produced flounder also analyzed by PCR as describe above.