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 (: ) in Hatchery-produced 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 , 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 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., 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 amami ensis 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 We lfare, 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,l O 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. Two cohorts (hatched in February 2009 and February 2011) of olive flounder raised at hatchery A Fish-to-fish transmission experiments were used to investigate the seasonal pattern of K. Transmission experiments were conducted in a septempunctata infection. Adult flounder (originating land-based facility at the NRIA, Kamiura. For cohabita- in the 2009 cohort) cultured at the private fish farm tion transmission, donor olive flounder (n = 160; mean caused an outbreak of K. septempunctata food poison- body weight = 710 g; prevalence of infection = 54%; ing in the autumn of 2010. Thus, the remaining fish mean intensity of infection = 7.4 × 106 spores/g) were stock at the farm was subsequently kept for our divided equally in number into two 8,000-L tanks and research and reared in non-filtered seawater. From reared at either 16°C or 24°C with 20 uninfected recipi- this infected stock of 2-year-old fish, 10 fish at each ent fish (mean body weight = 555 g). The recipient sampling (with exception of seven fish in the December fish were labeled with anchor tags to distinguish them 2011 sampling) were collected once a month between from the donor fish. The tanks were supplied with elec- April and December 2011, except in August, October trolyzed seawater after dechlorination by activated and November. carbon. The flow rate was maintained at 100 L/h. Fish of the 2011 cohort (hatched in February 2011) After 97 days, all tagged fish (recipient fish) were killed, were raised in a land-based tank supplied with sand-fil- and the muscle tissues were subjected to PCR assay. tered seawater until May 2011, and thereafter with non- For oral transmission, 52 uninfected olive flounder filtered seawater. Sixty fish (less than 1 year old) were (mean body weight = 10 g) were divided equally in num- sampled in June, July and October 2011. In December ber into two 1,000-L tanks in which the water tempera- 2011, approximately 1,000 fish from this stock were ture was kept at 20°C. We then mixed 140 g of transferred to the National Research Institute of Aquac- minced muscle tissue (3.3 × 106 spores/g) from fish ulture (NRIA), Kamiura Branch, Oita Prefecture, where infected with K. septempunctata with 60 g of a commer- K. septempunctata had never been detected. The cial diet (EP crumble, Nisshin Marubeni Shiryou, Inc.). infected fish stock was then maintained with flow- The mixture was extruded through a 2.44-mm opening through sand-filtered seawater. To avoid dispersion of of a 3-mL syringe, cut to approximately 2 mm in length, the parasite out of the facility, effluent water was steril- and prepared as infectious material. The fish in one ized with chlorine. From January to October 2012, 20 tank were fed with the infectious material for 3 days (60 fish (0 to 1 year old) were sampled every 3 months. g/day), whereas the fish in the other tank were fed a All sampled fish were transported on ice to the control diet. After 14 days of the experiment, all of the Laboratory of Fish Diseases at The University of Tokyo surviving fish were dissected, and the muscle tissues for parasite examination. For fish samples from the were checked for K. septempunctata by PCR as 2009 cohort, the body length and weight of the individ- described above. To confirm the presence of orally ual fish were measured; the somatic muscle was inoculated K. septempuntata spores in the digestive removed from the eyed-side dorsal trunk and analyzed tract, the intestinal tissues were also investigated by microscopically for K. septempunctata spores. To PCR assay. determine the intensity of infection (spore density), the muscle tissue (0.5 g) was minced and passed first Exposure of sentinel fish to seawater contaminated with through a 380-m m steel mesh (40 mesh screen, Cell K. septempunctata Dissociation Sieve Tissue Grinder Kit, Sigma) and then Uninfected olive flounder juveniles (approximately through a 100-m m nylon mesh (Cell Strainer, BD 5 cm in body length, n = 40 to 70) were transported Falcon) with PBS. After the spore suspensions were monthly to hatchery A where K. septempunctata infec- centrifuged at 330 ×g for 15 min, the supernatant was tion was enzootic and reared for 2 weeks in a 1,000-L discarded and the pellet was resuspended in 2.0 mL of tank supplied with flow-through non-filtered seawater PBS. The spores were counted with a hemocytometer between June and December 2013, except for to calculate the spore density (number of spores per g November. To standardize the water volume, the of muscle tissue). An infection intensity higher than inflow rate was maintained at 500 L/h. The fish were Kudoa septempunctata infection in olive flounder 63 fed daily with a commercial pellet diet (Otohime EP1, the duration of 3-month rearing in the NRIA was 22.8°C, Nisshin Marubeni). After two weeks, 10 fish were sam- 23.1°C, 21.7°C, 19.4°C, 16.6°C and 14.9°C for groups pled to detect the early stages of infection by quantita- exposed for 2 weeks in June, July, August, September, tive PCR (qPCR) assay as described below. Blood October and December, respectively. To exclude the was taken from the severed caudal vessel using capil- possibility that latent infection at a low water tempera- lary tubes, fixed in 70% ethanol and stored at 4°C until ture led to false-negative results, a subsample (n = 30) use. Before the qPCR assay, ethanol-fixed blood was of the group exposed in December was reared sepa- centrifuged at 330 ×g for 10 min, and the resulting pel- rately in a tank at 20°C for 3 months. let was subjected to DNA extraction. The heart and The qPCR assays were performed according to the somatic muscle were also collected, fixed in 70% etha- slightly modified protocol of Harada et al. (2012). nol and preserved at 4°C until subsequent molecular Briefly, DNA was extracted from 25 mg of each tissue analysis. as mentioned above. The following primers and probe The remaining exposed fish were carried live to an were used: forward (5¢-CGG TCA TAT CAG CCA TGG indoor facility in the NRIA (Kamiura Branch) and kept in ATA AC -3¢), reverse (5¢-CTA TCG ACA AAT TAA TGT sand-filtered seawater for 3 months. After the subse- TCG ATA TGC -3¢) and the Prime Time qPCR probe (6- quent rearing, all surviving fish (n = approximately 30) FAM)-TCA CCA TGT AAA TGG TGG GAG CAT TT – were dissected and examined for K. septempunctata (Iowa BlackFQ) (Medical and Biological Laboratories). infection in the somatic muscle by qPCR and PCR reactions were carried out in a 25-m L reaction microscopy. Uninfected fish (n = 30) for each of the mixture. Each PCR mixture contained 2.0 m L of exposure groups were held in tanks under equivalent extracted DNA, 12.5 m L of PreMix Ex Taq (Takara), and conditions at the NRIA for 3 months as the negative 0.5 m L of each primer and probe. The thermal cycling control and examined for K. septempunctata infection conditions were as follows: 95°C for 30 s, followed by at the end of each experimental period. For groups 45 cycles of 95°C for 5 s and 60°C for 31 s. All qPCR exposed for 2 weeks in July and August, additional sam- reactions were performed in the MiniOpticon Real-Time ples (n = 10) were collected 1 week, 1 month, and 2 PCR System (Bio-Rad). Plasmid DNA containing K. months post exposure. Mean water temperatures for septempunctata 18S rDNA was 10-fold serially diluted

2011 cohort 2009 cohort 100 100 (10)

80 80 (7) (20) 60 60 (10) (60) (20) (10) (10) 40 (20) 40 (10) (20)

Prevalence (%) 20 20 (60) (60) ND ND ND 0 0 Jun Jul Oct Jan Apr Jul Oct Apr May Jun Jul Aug Sep Oct Nov Dec 2011 2012 2011 108 1000 108 1000

107 800 107 800

106 600 106 600

105 400 105 400

104 200 104 200 Mean body weight (g)

103 103 Intensity (No. of spores/g) of (No. Intensity 0 0 Jun Jul Oct Jan Apr Jul Oct Apr May Jun Jul Aug Sep Oct Nov Dec 2011 2012 2011 0-year 1-year 2-year Fig. 1. Prevalence (upper row) and intensity (lower row) of Kudoa septempunctata infection in the somatic muscle of olive flounder produced in February 2009 (right column) and February 2011 (left column) at hatchery A. In the upper graphs, columns and black parts represent the prevalence and the ratio of heavy infection with > 1 × 106 spores/g, respectively. Numbers in parentheses are the numbers of fish examined in each sampling. Arrowheads on the X-axis of the 2011 cohort graphs indi- cate the time when the fish stock was transferred to the NRIA (non-enzootic area). ND = no data. In the lower graphs, open circles and black squares represent the intensity (spore density) and mean body weight, respectively. The dotted lines indicate the threshold level of a heavy infection potentially causing food poisoning. 64H. Yokoyama, M. Lu, K. Mori, J. Satoh, T. Mekata and T. Yoshinaga with TE buffer to generate a standard curve that was Determination of the infective period used to estimate a reliable endpoint. As a result, a lin- The prevalence of infection in the heart, blood and ear relationship with a correlation of determination (R2) muscles of sentinel fish 2 weeks post exposure (p.e.) of 0.999 and slope of –3.434 were displayed (data not exhibited a marked seasonality, peaking at 80–90% in shown). To determine the prevalence of infection, sam- July (Fig. 2). Only 10–20% infection prevalence was ples were considered positive when > 2.2 × 101 copies recorded in the groups exposed in June or August. of 18S rDNA were detected, which was equivalent to This pattern was similar to that in the muscle at 3 the results of Harada et al. (2012). This was estimated months p.e. (Fig. 2). No infection was observed in to be the lower limit for accurate quantification of the either the increased temperature group or the natural qPCR from plasmid DNA. temperature group in December. No K. septempunc- tata infection was detected in any of the negative controls. Results

Field monitoring of K. septempunctata infection at three Development of K. septempunctata in sentinel fish hatcheries exposed in July and August In hatchery A, K. septempunctata infection was not K. septempunctata was first detected in the heart detected by PCR in May or July, whereas the October as early as 1 week p.e. in both groups exposed for samples were identified as positive for infection by both 2 weeks in July and August (Fig. 3). Thereafter, K. PCR (47%) and microscopy (35%) (Table 1). In the septempunctata was also found in the blood and October samples, K. septempunctata was also somatic muscle at two weeks p.e. The prevalence detected by PCR from the heart (10%). In contrast, no was similar (80–90%) among the three tissues exam- K. septempunctata was found in the muscle of flounder ined two weeks p.e. in July, but the amounts of target from hatcheries B and C. DNA varied greatly, between 101 and 104 copies, among the individual fish (data not shown). However, Periodical investigation of K. septempunctata infection at 1 month p.e., the prevalence of infection sharply in zero- to two-year-old hatchery-produced flounder decreased to as low as 10% and increased again up to In olive flounder juveniles (less than 1 year old) of 73% in the muscle at 3 months p.e. At two and 3 the 2011 cohort, K. septempunctata was not detected until October 2011, when a heavy infection (> 1 × 106 2 weeks p.e. spores/g) was first found in two out of 60 fish (Fig. 1). 100 a Heart 30 After transfer to the NRIA, the prevalence of infection in Blood 80 Muscle 25 0- and 1 year old fish was approximately 30%, except Temp. for in January 2012 (60%). The ratios of heavily 60 20 infected flounder were consistently 15%, except for in 40 April 2012 (0%). The intensity of infection varied 20 15 largely from October 2011 to October 2012 (104–107 Temperature (ºC) Temperature spores/g), although the mean body weight increased 0 10 Jun Jul Aug Sep Oct Dec almost 4-fold (from 155 g to 600 g) during this period. 100 In the 2009 cohort (2-year-old fish), the prevalence b of infection varied from 30% to 90% from April to 80 3 months p.e. December 2011, but no clear pattern was observed in 60 Prevalence of infection (%) the fluctuation of prevalence. A large variation was 40 detected in the intensity of infection with 104–107 spores/gi, sim lar to that for the 2011 cohort, but the 20 ratios of heavily infected flounder varied from 20% to 0 80%, which was in general higher than those in the Jun Jul Aug Sep Oct Dec 2011 cohort. Exposure month Fig. 2. Prevalence of Kudoa septempunctata infection in the Fish-to-fish transmission experiments heart (white columns), blood (gray columns), and Neither the cohabitation (at both 16°C and 24°C) somatic muscle (black columns) of the sentinel fish nor oral transmission trials resulted in K. septempunc- exposed to infectious water in hatchery A for 2 weeks tata infection in the recipient fish (none of the 20 fish a month between June and December 2013 (except examined in each test were infected). The intestines for November), followed by rearing for 3 months at the NRIA (non-enzootic area). The upper (a) and lower of the recipient fish challenged orally were also negative (b) graphs represent the prevalence at 2 weeks and 3 for K. septempunctata by PCR. months p.e., respectively. Prevalence was determined by qPCR and light microscopy. Kudoa septempunctata infection in olive flounder 65

100 a Exposed in July Heart Blood 80 Muscle 60 40 20 0 1 wk 2 wk 1 mo 2 mo 3 mo 100 80 b Exposed in August 60

Prevalence of infection (%) 40 20 0 1 wk 2 wk 1 mo 2 mo 3 mo Time post-exposure

Fig. 3. Prevalence of Kudoa septempunctata infection in the heart (white columns), blood (gray columns) and somatic muscle (black columns) of the sentinel fish exposed to infectious water in hatchery A for 2 weeks in July (a) and August (b) 2013, followed by rearing for 3 months at the NRIA (non-enzootic area). Prevalence was determined by qPCR assay. months p.e. in the group exposed in July, K. sites pseudocysts and the regeneration of the muscle septempunctata spores were microscopically observed fibers associated with the host inflammatory response in the somatic muscle, varying from 104 to 106 spores/g (Moran et al., 1999c). Histopathological studies of (data not shown). Compared with the group exposed olive flounder infected with K. septempunctata will be in July, the overall prevalence and intensity of infection required to examine the host responses and the para- was considerably lower in the group exposed in August site development in the musculature. (Fig. 3). It should also be noted that fish heavily infected with > 1 × 106 spores/g were found in the October sam- ples of fish less than 1 year old. This suggests that Discussion fish stocks potentially causing food poisoning could be The present study reveals the infection dynamics of detected earlier than previously presumed, based Kudoa septempunctata in olive flounder from juveniles on the inspection protocols by the Fisheries to market-size fish. K. septempunctata predominantly Agency. Thus, we recommend that analysis for K. invaded juvenile flounder in July, migrated through the septempunctata infection should be performed not heart and blood and developed mature spores in the only by qualitative methods currently performed somatic muscle within 2 to 3 months. Thereafter, the (conventional PCR or light microscopy of stained infection persisted for at least 2 years. Infection inten- smears) but also by quantitative methods (qPCR or sity varied greatly during the 2-year period; therefore, it spore counting) during the aquaculture period. Early was not clear whether the parasite continued to prolifer- detection will negate the cost of fruitless culture of ate within individual fish. To track the time course infected flounder. development of K. septempunctata in a fish body, it will The reason for the differences in prevalence of be necessary to develop a nondestructive detection infection among the three hatcheries monitored for K. method for the parasite. septempunctata is unknown. However, the results No remarkable decrease was observed in infection give us some hints in understanding the biology of this intensity in 2-year-old fish, implying that infected parasite. First, the geographical distribution of K. fish do not recover from the infection. In contrast, septempunctata may be limited to hatchery A only. Shirakashi et al. (2012) found that the number of K. Sugiyama et al. (1999) reported the localization of K. yasunagai cysts in the brains of infected cultured yellow- amamiensis infection within the Okinawa islands; the tail Seriola quinqueradiata tended to decline over time. prevalence of infection with K. amamiensis in yellowtail Moran et al. (1999c) also reported that K. thyrsites in reared in Motobu area was 100%, though those in the Salmo salar reached a maximum preva- other areas were 0–9%. Although the life cycle of lence of 64% at 26 weeks p.e., followed by recovery Kudoa myxosporeans has not yet been elucidated, it is from the infection within 1 year. The recovery process likely that an alternate invertebrate host may only exist was illustrated histologically by the breakdown of K. thyr- along the coastal area of hatchery A. If so, the selec- 66H. Yokoyama, M. Lu, K. Mori, J. Satoh, T. Mekata and T. Yoshinaga tion of disease-free sites for hatcheries is worthy of con- The qPCR assay proved that K. septempunctata sideration for avoiding Kudoa infections (Yokoyama, was present in juvenile olive flounder as early as 2003). Second, it is possible that the K. septempunc- 1 week p.e.. This implies that the heart is a candidate tata infectious stage was eliminated by sand-filtration organ for the early detection of K. septempunctata in and/or ultraviolet illumination during the rearing period juvenile flounder. However, it is unknown whether the in hatcheries B and C. If this hypothesis is true, our parasite develops in the cardiac musculature or the next step will be to develop effective control measures blood stage only exists in the ventricle cavity. Moran using physical filtration and/or ultraviolet treatment of et al. (1999c) described the early development of K. the water supply (Cobcroft and Battaglene, 2013; thyrsites plasmodia within the cardiac muscle of Atlantic Shirakashi et al., 2014). Alternatively, the absence of salmon 17 weeks p.e. by histological analysis. Kabata K. septempunctata infection in hatchery C may be attrib- and Whitaker (1989) also found K. thyrsites spores uted to delayed hatching periods (August to October) in microscopically in the cardiac muscle of returning the flounder stock, resulting in avoidance of the peak Pacific salmon adults, but the prevalence and intensity period of infection (July). In this case, the develop- of infection were very low. If K. septempunctata prolif- ment of an aquaculture program management strategy erates to develop numerous mature spores in the car- will be useful for reducing Kudoa infection. diac muscle, the heart may be preferentially selected for The failure of K. septempunctata fish-to-fish trans- microscopic inspection because it is not an edible part mission further supports that myxosporean parasites of flounder. always require alternate invertebrate hosts to complete The pattern of the K. septempunctata infectious their life cycle. Although direct transmission of period is most likely influenced by the actinosporean spp. from fish to fish has been docu- dose to which the fish were exposed. Density of acti- mented (China et al., 2013, 2014), these enteric myxo- nospores in the environment has been possibly regu- sporeans probably transmit to fish by vegetative stages lated by water temperature (Alama-Bermejo et al., 2013). released in the excrement rather than by myxospores. In the present study, actinospore release from the Moran et al. (1999b) reported the successful transmis- invertebrate host may be related to an increase in sea- sion of K. thyrsites in Atlantic salmon by the intracoelo- water temperature to 25°C. Alternatively, it is likely mic injection of putative blood stages from infected fish. linked to the breeding periods of the invertebrate host. However, this is an artificial means of transmission Rangel et al. (2009) documented that actinospores of achieved only in experimental conditions. The infec- Zschokkella mugilis were released only during the repro- tion routes attempted in the present study seem to ductive season of the polychaete alternate host. A reflect the natural environment where fish likely become similar experiment using sentinel fish was previously infected. The negative results in the oral transmission conducted by Alama-Bermejo et al. (2013), which experiment may have been because the fish were revealed double peaks (spring and autumn) in the C. checked for infection too early (2 weeks p.e.). How- puntazzi infectious period. Additionally, direct quantifi- ever, K. septempunctata was detectable within 2 weeks cation of the infectious stage in marine myxosporeans p.e. in fish naturally exposed to infectious water, as in seawater using qPCR has been documented (Alama- shown in the present study. Thus, fish-to-fish transmis- Bermejo et al., 2013; Ishimaru et al., 2014). Similar tri- sion of K. septempunctata is not likely to occur under als are currently in progress with K. septempunctata. the culture conditions in fish farms or in live fish tanks in Although qPCR cannot distinguish between the actino- restaurants. sporean and myxosporean stages, this technique will be The sentinel fish group exposed in July exhibited useful for choosing culture locations and timing water an obvious temporal fluctuation in the development of treatments to minimize K. septempunctata infection. K. septempunctata. The parasite was detected in the heart, blood, and muscle of 80–90% of the sentinel fish Acknowledgments 2 weeks p.e., but most of the parasites disappeared at 1–2 months p.e. Alama-Bermejo et al. (2013) also We acknowledge several private fish farms and observed a very high prevalence of Ceratomyxa olive flounder hatcheries for providing fish material. puntazzi infection in the blood of sharpsnout seabream We thank Drs. Kengo Oota and Taizo Morioka of the Diplodus puntazzo just after exposure to infectious National Research Institute of Fisheries and Environ- water, although the final prevalence was low in the gall- ment of Inland Sea and Dr. Sho Shirakashi of Kinki bladder, the parasite’s target organ. The loss or clear- University for their help with the fieldwork. We are ance of blood stage C. puntazzi may be caused by the grateful to Dr. Tetsuya Harada of the Osaka Prefectural host’s immunological responses (Alama-Bermejo et al., Institute of Public Health for providing the K. 2013). Further studies are required to clarify the K. septempunctata plasmid DNA control. We are also septempunctata mode of infection and migration route grateful to Naoko Funaguma of The University of Tokyo in olive flounder. for her technical assistance with molecular work. This Kudoa septempunctata infection in olive flounder 67 research was partly supported by the Ministry of Agricul- Dis., 58, 1046–1052. ture, Forestry, and Fisheries of Japan (No. 23064 and Moran, J. D. W., D. J. Whitaker and M. L. Kent (1999a): A 2403). review of the myxosporean genus Kudoa Meglitsch, 1947, and its impact on the international aquaculture industry and commercial fisheries. Aquaculture, 172, 163–196. References Moran, J. D. W., D. J. Whitaker and M. L. Kent (1999b): Natural and laboratory transmission of the marine myxozoan para- Alama-Bermejo, G., R. Šíma, J. A. Raga and A. S. Holzer site Kudoa thyrsites to Atlantic salmon. J. Aquat. Anim. (2013): Understanding myxozoan infection dynamics in the Health, 11, 110–115. sea: seasonality and transmission of Ceratomyxa puntazzi. Moran, J. D. W., L. Margolis, J. M. Webster and M. L. Kent Int. J. Parasitol., 43, 771–780. (1999c): Development of Kudoa thyrsites (Myxozoa: Myxo- China, M., H. Nakamura, K. Hamakawa, E. Tamaki, S. Miwa, F. sporea) in net-pen reared Atlantic salmon determined by Meng and H. Yokoyama (2013): Occurrence of the myxo- light microscopy and a polymerase chain reaction test. sporean emaciation disease caused by Enteromyxum leei Dis. Aquat. Org., 37, 185–193. in cultured Malabar grouper Epinephelus malabaricus. Özer, A., T. Öztürk, H. Özkan and A. Çam (2014): First report of Fish Pathol., 48, 88–96. Enteromyxum leei (Myxozoa) in the Black Sea in a poten- China, M., H. Nakamura, K. Hamakawa, E. Tamaki, H. tial reservoirh host C romis chromis. Fish Pathol., 49, Yokoyama, S. Masuoka and K. Ogawa (2014): Efficacy of 57–60. high water temperature treatment of myxosporean emacia- Rangel, L. F., M. J. Santos, G. Cech and C. Székely (2009): tion disease caused by Enteromyxum leei (Myxozoa). Morphology, molecular data, and development of Zschok- Fish Pathol., 49, 137–140. kella mugilis (, ) in a polychaete Cobcroft, J. M. and S. C. Battaglene (2013): Ultraviolet irradia- alternate host, Nereis diversicolor. J. Parasitol., 95, tion is an effective alternative to ozonation as a sea water 561–569. treatment to prevent Kudoa neurophila (Myxozoa: Myxo- Shirakashi, S., A. Morita, K. Ishimaru and S. Miyashita (2012): sporea) infection of striped trumpeter, Latris lineata Infection dynamics of Kudoa yasunagai (Myxozoa: Multi- (Forster). J. Fish Dis., 36, 57–65. valvulida) infecting brain of cultured yellowtail Seriola quin- Eiras, J. C., A. Saraiva and C. Cruz (2014): Synopsis of the spe- queradiata in Japan. Dis. Aquat. Org., 101, 123–130. cies of Kudoa Meglitsch, 1947 (Myxozoa: Myxosporea: Shirakashi, S., T. Nishimura, N. Kameshima, H. Yamashita, H. Multivalvulida). Syst. Parasitol., 87, 153–180. Ishitani, K. Ishimaru and H. Yokoyama (2014): Effective- Grabner, D. S., H. Yokoyama, S. Shirakashi and R. Kinami ness of ultraviolet irradiation of seawater for the prevention (2012): Diagnostic PCR assays to detect and differentiate of Kudoa yasunagai and Kudoa amamiensis (Myxozoa: Kudoa septempunctata, K. thyrsites and K. lateolabracis Multivalvulida) infections in Seriola fish. Fish Pathol., 49, (Myxozoa, Multivalvulida) in muscle tissue of olive flounder 141–144. (Paralichthys olivaceus). Aquaculture, 338–341, 36–40. Sugiyama, A., H. Yokoyama and K. Ogawa (1999): Epizooti- Harada, T., T. Kawai, H. Sato, H. Yokoyama and Y. Kumeda ological investigation on kudoosis amami caused by (2012): Development of a quantitative polymerase chain Kudoa amamiensis (Multivalvulida: Myxozoa) in Okinawa reaction assay for detection of Kudoa septempunctata in Prefecture, Japan. Fish Pathol., 34, 39–43. (In Japanese olive flounder (Paralichthys olivaceus). Int. J. Food with English summary) Microbiol., 156, 161–167. Xu, L.-W., J.-Y. Zhang, J. Feng and J.-G. Wang (2014): Mass Ishimaru, K., T. Matsuura, K. Tsunemoto and S. Shirakashi mortality of cage-cultured orange-spotted grouper Epi- (2014): Seasonal monitoring of Kudoa yasunagai from sea nephelus coioides associated with renal sphaerosporosis water and aquaculture water using quantitative PCR. caused by Sphaerospora epinepheli in South China Sea. Dis. Aquat. Org., 108, 45–52. Fish Pathol., 49, 202–205. Kabata, Z. and D. J. Whitaker (1989): Kudoa thyrsites (Gilchrist, Yahata, Y., Y. Sugita-Konishi, T. Ohnishi, T. Toyokawa, N. 1924)y (M xozoa) in the cardiac muscle of Pacific salmon Nakamura, K. Taniguchi and N. Okabe (2015): Kudoa (Oncorhynchus spp.) and steelhead trout (Salmo septempunctata-induced gastroenteritis in humans after gairdneri). Can. J. Zool., 67, 341–342. flounder consumption in Japan: A case-controlled study. Kawai, T., T. Sekizuka, Y. Yahata, M. Kuroda, Y. Kumeda, Y. Jap. J. Infect. Dis., 68, 119–123. Iijima, Y. Kamata, Y. Sugita-Konishi and T. Ohnishi (2012): Yokoyama, H. (2003): A review: Gaps in our knowledge on Identification of Kudoa septempunctata as the causative myxozoan parasites of fishes. Fish Pathol., 38, 125–136. agent of novel food poisoning outbreaks in Japan by con- Yokoyama, H. (2013): Parasitosis caused by ingestion of raw sumption of Paralichthys olivaceus in raw. Clin. Infect. fish. Jpn. J. Food Microbiol., 30, 100–103. (In Japanese)