‹›•aŒ¤‹† Fish Pathology,40(4),175-180,2005.12 2005 The Japanese Society of Fish Pathology

Occurrence of the Myxosporean Emaciation Disease Caused by leei in Cultured Japanese Flounder Paralichthys olivaceus

Hiroshi Yasuda1 ,Tsuyoshi Ooyama1, Atsushi Nakamura1, Kazuo Iwata1, Oswaldo Palenzuela2 and Hiroshi Yokoyama3*

1 Miyazaki Prefectural Fisheries Experimental Station,Miyazaki,889-2162,Japan 2Instituto de Acuicultura de Torre de la Sal(CSIC) ,12595 Ribera de Cabanes, Castellon, Spain 3 Department of Aquatic Bioscience , Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657,Japan

(Received July 29,2005)

ABSTRACT-The myxosporean emaciation disease has been recently found in Japanese flounder Paralichthys olivaceus cultured in land-based tanks in Miyazaki Prefecture, Japan. Affected fish showed external signs of distended abdomen, rectal prolapse, sunken eyes and a typical bony ridge on the skull. Parasitological and histopathological examinations revealed that the develop mental stages of Enteromyxum Ieei parasitized the intestinal epithelium of diseased fish. No other enteric myxozoans were observed in the affected fish. A molecular analysis of SSU rDNA con firmed that the causative myxozoan was identified as E. leei. Experimental transmission of E. leei was achieved to naive Japanese flounder and tiger puffer Takifugu rubripes by feeding with gut tissues taken from infected Japanese flounder. The present study indicates that the myxosporean emaciation disease of Japanese flounder is caused by E. leei.

Key words: , , emaciation disease , Paralichthys olivaceus, Japanese flounder, Takifugu rubripes, tiger puffer

Since the mid-1990s, the myxosporean emaciation farm was situated in the vicinity of a tiger puffer farm, has occurred extensively in many farmsdisease of tiger where the myxosporean emaciation disease was puffer Takifugu rubripes in sites located in western enzootic. The first outbreak of this disease was in Japan. Affected fish showed external signs of severe November 2001, when water temperature was 21.5•Ž. emaciation including sunken eyes(enophthalmia),bony A preliminary observation showed that most of the ema ridges on the head and a tapered body(Tin Tun et al., ciated flounder(mean body length,25.8 cm;mean body 2000;Ogawa and Yokoyama,2001).By subsequent weight,150.3 g)were infected with enteric myxozoans. parasitological and molecular studies,three enteric In the present study, parasitological and molecular myxozoans,Enteromyxum fugu(= fugu), analyses were conducted to identify the causative agent

Enteromyxum leei(=Myxidium sp.TP)and Leptotheca (or agents)of flounder emaciation. In addition, fugu, were found from the intestine of diseased fish(Tin attempts of fish-to-fish transmission of the disease Tun et al.,2000;Yanagida et al.,2004; 2005).Subse among tiger puffer and Japanese flounder were carried quently, the latter two were shown to be respon out. sible for the emaciation disease(Tin Tun et al.,2002). In recent years, a similar disease has been noticed Materials and Methods in Japanese flounder Paralichthys olivaceus farmed in Miyazaki Prefecture, Japan. Japanese flounder were Fish and disease diagnosis cultured in concrete tanks at a land-based site supplied Moribund cultured Japanese flounder(0-year-old; with flow-through sea water. The Japanese flounder mean body length,23.0 cm)were obtained from a com mercial fish farm located in Miyazaki Prefecture in Octo * Corresponding author ber2002.Following visual examination of symptoms, E-mail: [email protected] fish were dissected, and then the gut tissues were 176 H. Yasuda, T. Ooyama, A. Nakamura, K. Iwata, O. Palenzuela and H. Yokoyama excised. Fresh smears of intestinal scrapings were Transmission trial among Japanese flounder observed by light microscopy. Imprint slides of the As donor fish, three diseased Japanese flounder intestinal mucosa were prepared and stained with Diff (0-year-old; average body weight,241 g)were provided Quik (International Reagents Corp., Japan)for parasito- by the fish farm on January 6,2005.Myxosporean-free logical examination. For histology, pieces of the intesti juvenile Japanese flounder were purchased from a seed nal tissues were fixed in 10% buffeted formalin and production company and kept in acryl tanks(ca.140L) embedded in paraffin wax. Tissue sections(5ƒÊm) equipped with a closed re-circulating filtration system were stained with hematoxylin-eosin(H.E.)&Uvitex 2B, and a thermo-regulator adjusting the water temperature and observed by light or fluorescent microscopy.In to 23•Ž.Ten recipient Japanese flounder(average addition, the kidney was sampled and BHI agar plates total length,19.8 cm; average body weight,71.5g)were were inoculated for bacterial isolation. fed with pieces of the pooled chopped intestines(12.6g from the donors.While feeding the infected) gut tissue, Molecular analysis of myxozoan the filtration system of the tank was interrupted for one DNA cloning and sequencing of the parasite's small hour to guarantee the exposure.As a negative control, subunit ribosomal DNA gene(SSU rDNA)were done ten uninfected Japanese flounder(average total length, according to Palenzuela et al.(2002)and Yanagida et al. 19.8 cm; average body weight,71.9 g)were kept sepa

(2004).Briefly, parasite DNA was extracted from rately in the same condition.All fish were fed daily with heavily infected intestinal mucosa scrapings using a commercial extruded pellet (EP).On 32 days PE,all commercial genomic DNA purification kit(Qiagen Gmbh, surviving fish were sampled.Following measurements Germany). Myxozoan nuclear SSU rDNA was ampli of body length, body weight and liver weight, the gut tis fied by PCR using the primers MM18Sf(ctggttgattctgcc sues were excised to examine for myxozoan infection as agtggtc)and MM18Sr (cggtactagcgacgggcg) (Palenzuela described above.In addition,Diff-Quik stained imprints et al.,2002).The products of two parallel reactions(20 were observed to evaluate semi-quantitatively the

L each)were pooled and cleaned using a ƒÊspin-column progress of myxozoan development.The myxozoan cleanup kit(Qiagen).A subsample of the eluent was developmental stages within five visual fields under a checked for purity and concentration in an ethidium bro light microscope with a 400-fold magnification were mide-stained 1% agarose gel. The myxozoan product enumerated.All myxozoan plasmodia were classified was ligated into a cloning vector(pT7-Blue-T,Takara, into three stages; presporogonic,sporogonic and spore Japan)which was used to transform competent E.coli stages,and then their relative frequencies in appearance cells.Transformants were selected by the blue/white were recorded. screening method on plates containing ampicillin and X-Gal.Both DNA strands from two different clones Results were sequenced. Gross and bacteriological observations Transmission trial from Japanese flounder to tiger puffer Affected Japanese flounder showed external signs One emaciated Japanese flounder(0-year-old,353 of distended abdomen, rectal prolapse, sunken eyes and g)was obtained from the fish farm on May 23,2002,and a typical bony ridge on the skull(Fig. 1A).Ascites, served as a "donor fish".Myxosporean-free juvenile mucous liquid in the digestive tract and a flimsy intestinal tiger puffer(average body weight, 7.1g)were purchased wall were also evident.Occassionally,Edwardsiella from a seed production company and held in fiberglass tarda, the causative bacterium of edwardsiellosis, was reinforced plastics(FRP)tanks(1500 L)supplied with isolated from the kidney(about 20% of examined fish), flow-through filtered seawater in Miyazaki Prefectural but it was not found consistently in emaciated fish. Fisheries Experimental Station.The intestine of the donor fish was removed, weighed(1.6g)and finely Parasitological observation chopped.Fifty recipient tiger puffer were fed with Numerous myxozoans with different developmental this material, and they were subsequently sampled at stages were consistently detected both in fresh smears 23, 30,37and 44 days post-exposure(PE) (10fish and in Diff-Quik stained imprints of gut mucosa from per day except the final sampling when all survivors infected fish.In intestinal scrapings, trophozoites were sacrificed).Fifty tiger puffer from the same stock harboring one or two secondary cells exhibited a slow were kept as controls under equivalent conditions rotating and amoeboid movement on the spot.The Myxozoan infections were examined as described commonest stage in Diff-Quik stained intestinal imprints above. During the experimental period, the water tem was a pre-sporogonic plasmodium with a spherical pri perature ranged from 22 to 24•Ž.All fish were daily fed mary cell(10-20ƒÊm in diameter) having a pink nucleus with commercial pellets. and one or two deep blue secondary cells (Fig. 1B).Spore stages were rarely found and when

present, they were mostly immature(Fig.1C).Spores Enteromyxum leei in Japanese flounder 177

Fig. 1. Myxosporean emaciation disease of Japanese flounder Paralichthys olivaceus. A: Gross appearance of affected Japanese flounder. Note a bony ridge on the skull. B: Developmental stages of Enteromyxum leei from Japanese flounder. Arrow and arrowhead show pre-sporogonic and sporogonic stages, respectively. Diff-Quik stain. Bar = 10 ƒÊm. C: An immature spore of E. leei observed in intestinal scraping. Bar = 10 ƒÊm. D & E: Histological sections of the infected intestine showing hyperplasia of the gut epithelium. H. E. stain. Bars = 50 ƒÊm. F: Histological section showing Uvitex 2B-positive E. leei

parasitizing the gut epithelium. Uvitex 2B & H. E. stain. Double exposure with UV and normal light. Bar = 50 ƒÊm.

were slightly spindle to crescent-shaped, measuring lium (Figs. 1D & 1E). Detachment and destruction of 15.6 (14-18) ƒÊm long and 8.8 (7-10) ƒÊm wide. Two the gut epithelia associated with proliferation of parasitic polar capsules with a size of 5.5 (4.5-6.5) ƒÊm long and stages were evident. It was noticed that plasmodia 2.7 (2-3) ƒÊm wide were elongated, tapered to their distal located in the gut epithelium were positive for Uvitex 2B ends and arranged obliquely in frontal view. staining (Fig. 1F).

Histological observation Molecular analysis Histological examinations revealed that the intesti- A consensus 1588 bp DNA product (including ampli- nal epithelium was affected by developmental stages of fication primers) was obtained (GenBank accession the enteric myxozoan, causing hyperplasia of the epithe- number; DQ127230). The sequence shared •„99% 178 H. Yasuda, T. Ooyama, A. Nakamura, K. Iwata, O. Palenzuela and H. Yokoyama identity to the enteric myxozoan Enteromyxum leer, both uninfected at the end of the experiment. from sharpsnout sea bream Diplodus puntazzo (AF411334) and from tiger puffer (AY520574). Discussion Transmission from Japanese flounder to tiger puffer To date, four enteric myxozoans have been In all the samplings, developmental stages of described in marine fishes; E. leei from many fish spe enteric myxosporean were detected in tiger puffer fed cies, particularly sparids cultured in the Mediterranean with the infected intestine. Prevalence of infection varied and Red Sea (Diamant et aL, 1994) and also from tiger between 10% and 40% (Table 1). All control fish puffer cultured in Japan (Tin Tun et al., 2000); E. remained uninfected throughout the study. Although scophthalmi from Scophthalmus maximus farmed mean body weight was consistently lower in recipient in Spain (Palenzuela et al., 2002); and E. fugu and fish than in control fish (but not significantly different), no Leptotheca fugu from tiger puffer cultured in Japan (Tin typical external signs of emaciation disease were Tun et al., 2000). Among them, E. scophthalmi, E. fugu noticed. and L. fugu were clearly distinguishable from the current myxozoan of Japanese flounder in the larger spore size, Table 1. Transmission of Enteromyxum leer to tiger puffer the different site of infection, and the different morphol Takifugurubripes by feeding withthe gut tissue of E. ogy of polar capsules, respectively. Spore shape and leei-infected Japanese flounder Paralichthys dimensions of the myxozoan from Japanese flounder olivaceus were consistent with those of E. leei. In addition, plas modia of the present myxozoan were shown to be posi tive for Uvitex 2B staining, which was proposed as a complementary criterion for a diagnostic feature for E. leei (Yanagida et al., 2004). These morphological simi larities are consistent with the definitive identification facilitated by the SSUr DNA analysis, showing >99% identity with other E. leei isolates from sharpsnout sea bream and tiger puffer (Palenzuela et al., 2002; Yanagida et al., 2004). The current sequence has the same length as the isolate from tiger puffer (1588 bps.), 1 bp less than the original sequence from sharpsnout Transmission among Japanese flounder sea bream. Despite this difference in length, corre All the recipient Japanese flounder were infected sponding to a consistent deletion of one adenine base in with the enteric myxozoan at the end of the experiment Japanese isolates (position 169 of AF411334), the re (Table 2). One heavily infected fish died 17 days maining mismatches can be attributed to ambiguously PE. Most of recipient fish exhibited anorexia around resolved residues or previously identified polymorphic 25-29 days PE, and five out of nine fish showed typical sites. Thus, the present species parasitizing Japanese signs of the emaciation disease. Frequencies of the flounder was clearly identified as E. leei. The signifi developmental stages in donor fish were 98.6%, 1.4% cance of the single adenine insertion/deletion in and 0% in pre-sporogonic, sporogonic and spore stages, sequences from Mediterranean and Japanese isolates, if respectively. A slight increment in proportions of any, remains to be investigated. sporogonic (4.9%) and spore (2.9%) stages was noted in This is the first report of E. leei infection in Japanese recipient fish (Table 2). All control fish remained flounder, but the broad host range of E. leei has been already described by different authors. Lc Breton and Table 2. Transmission of Enteromyxumleei to Japanese floun Marques (1995) found that cultured sharpsnout sea der Paralichthys olivaceus by feeding withthe gut tis bream and red sea bream Pagrus major were affected sue of E. leei-infected Japanese flounder by E. leei. Diamant (1998) experimentally demonstrated that E. leei could infect red drum Sciaenops ocellatus. Furthermore, Padros et al. (2001) recorded E. leei infections from 25 different fish species (belong ing to 16 genera, ten families and four orders) reared at an exhibition aquarium. This list has significantly lengthened in the last few years by epidemiological studies in other Mediterranean wild fishes (Palenzuela, unpublished data). In Japan, unidentified developmen tal stages resembling E. leei in the intestine of cultured thread-sail filefish Stephanolepis cirrhifer have also been Enteromyxum leei in Japanese flounder 179 detected (Yasuda, unpublished data). Thus, it is of required to compare host-parasite relations among dif great concern that E. leei could spread further to other ferent fish species. important fishes in aquaculture industry in Japan, and In conclusion, the present study demonstrates that worldwide. the myxosporean emaciation disease of Japanese flounder Scarce information is available on factors causing is caused by E. leei. Successful fish-to-fish trans the myxosporean emaciation disease. Our previous mission of this parasite suggests spreading of E. leei work suggested that the development of E. leei was infection from cultured tiger puffer to Japanese flounder. strongly influenced by the ambient temperature (Yasuda Routes of infection (e.g., neighboring fish farms, wild et al., 2002). Redondo et al. (2002) also reported that fishes or culture seedlings) should be clarified in future infections with E. scophthalmi were established earlier at studies. higher water temperature. Our preliminary experiment showed that a poor growth and a lower liver to body Acknowledgement weight ratio were recorded in some affected fish which were reared at a high density in a tank, suggesting that We thank Mr. Tetsuya Yanagida of The University of severe damages of the gut epithelia caused by E. leei Tokyo for his technical assistance including the histol resulted in malnutrition (Yasuda, unpublished data). ogy. This warrants further studies on the effect of the fish stocking density on onset of the myxosporean emacia tion disease. References Fish-to-fish transmission of Enteromyxum spp. Diamant, A. (1997): Fish-to-fish transmission of a marine could explain the rapid spread of the parasites within fish myxosporean. Dis. Aquat. Org., 30, 99-105. farms (Diamant, 1997; 1998; Redondo et al., 2002; Diamant, A. (1998): Red drum Sciaenops ocellatus Yasuda et al., 2002). However, the nature of the infec (Sciaenidae), a recent introduction to Mediterranean mari culture, is susceptible to Myxidium leei (). tive stages still remains to be clarified. In the current Aquaculture, 162, 33-39. study, the semi-quantitative evaluation of the develop Diamant, A., J. Lom and I. Dykova (1994): Myxidium leei n. sp., mental stages of E. leei allowed us to examine the pro a pathogenic myxosporean of cultured sea bream Sparus gression of parasite maturation in fish hosts. The lack aurata. Dis. Aquat. 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