Mass Mortality Caused by Blood Fluke (Paradeontacylix) among Amberjack (Seriola dumerili) Imported to Japan Kazuo Ogawa*1 and Mikio Fukudome*2 *1,Department of Fisheries, Faculty of Agriculture, The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan *2 Kagoshima Prefectural Fisheries Experimental Station, Kinko-cho, Kagoshima 892, Japan (Received July 15, 1994) Sudden outbreak of a disease caused by the blood fluke Paradentacylix occurred in May, 1993 among net-cage cultured juvenile amberjack, Seriola dumerili which had been imported from Hainan, China, a few months before the onset of the disease. The cumulative mortality rate ranged from 50 to more than 80% in one month. Dead fish were characterized by opened mouth and opercula, showing typical sign of suffocation. Parasitological examination revealed that eggs of P. grandispinus and/or P. kampachi were accumulated in the afferent arteries of the gills of all dead fish. Since no other pathogens were detected, it was concluded that the mass mortality was caused by these blood flukes. It could not be specified whether the cercarial invasion had occurred prior to or after the introduction of the fish into the Japanese waters. Yellowtail Seriola quinqueradiata is the most widely cultured marine fish of Japan, mainly in Kyushu and Shikoku areas. Because of recent overproduction, there was a substantial decrease in the price of cultured yellowtail. Many farmers have resorted to culture of other species like amberjack, Seriola dumerili. Amberjack has higher market value, but its culture depended on seedlings caught by angling or netting from wild populations in south-western Japan, the catch being in a much smaller quantity than that of yellowtail. This became a major limiting factor for the expansion of amberjack culture, thus importation of amberjack seedlings from China and Hong Kong started in the late 1980s. The number of imported amberjack is steadily increasing, and in 1993 it ex ceeded 10 millions (Ohno, 1993). Amberjack cul ture has to some extent replaced yellowtail culture. Mass mortality of cultured amberjack due to Para deontacylix infection in its vascular system was first diagnosed in Kochi Prefecture, Shikoku in Decem ber 1983 through March 1984 (Ogawa and Egusa, 1986), where the infection has been enzootic until now (H. Andoh, Kochi Prefectural Fisheries Exper imental Station, personal communication). It is suggested that the infection has an annual cycle; cercarial invasion starts in September, eggs accumu late in the gills and heart from November, and mortality, which occurs in winter from December until March, decreases with increasing water temper ature (Ogawa et al., 1989, 1993). In May 1993, a sudden outbreak of blood fluke infection occurred among amberjack which had been imported from Hainan, China, and cultured at sever al localities in Kagoshima Prefecture, Kyushu. The gills of dead fish were full of parasite eggs. This observation was very similar to the known case of Paradeontacylix infection of domestic stocks of am berjack cultured in Kochi Prefecture. However, a marked difference was noted in the seasonality of the outbreaks: in Kochi, mortality occurred in winter months, while, in Kagoshima, it occurred in early summer. This paper describes a case of mass mortal ity among imported amberjack seedlings, identifies the causative agents and compares the present case with the enzootic blood fluke infection of the same host fish in Kochi Prefecture. Materials and Methods Blood fluke infections of different stocks of im ported amberjack seedlings were monitored in March through May, 1993 before and during the outbreak of the disease at Furue, Tarumizu City,
266 K. Ogawa and M. Fukudome Kagoshima Prefecture (Table 1). One to nine (mostly four and over) fish, 6.4-20.1 cm in body length, from each stock were sampled. They com prised 13 different stocks from six shipments (stock nos. 1 to 6 in Table 1), imported from Hainan Island, China or Hong Kong from February to May, 1993. Records of mortality were collected with the help of the local fisheries cooperation. In the survey of O + amberjack in Kochi Prefec ture conducted in 1985 and 1986, Paradeontacylix infected fish could be diagnosed by confirming eggs accumulated in the gill filaments, and all the gill filaments examined were positive for eggs, although flukes were not always recovered from the vascular system (Ogawa et al., 1993). At present, examina tion of gill filaments for eggs seems to be the most reliable and convenient diagnostical method. In this study, ten gill filaments were removed from each fish and examined microscopically for accumulated eggs. In order to identify the causative organisms, a parasitological survey of juvenile amberjack ran domly collected from culture net cages at Uchinoura and Furue Fisheries Cooperation, Kagoshima Pre fecture was conducted on June 2 and 3, 1993. Three to five fish from 6 stocks were sampled (11.8-20.4 cm in body length). The vascular system of host fish from the sinus venosus, heart through the afferent branchial arteries was examined under a stereo microscope for adult flukes. As for gill blood vessels, two out of eight sets of gills were dissected. Flukes obtained were flattened under a cover-slip, fixed in AFA, stained with alum carmine or Heidenhein's iron hematoxylin, dehydrated and mounted in Canada balsam. Infected fish (11.5-19.0 cm in body length; n = 9) at Furue and Uchinoura were examined histological ly. After necropsy, the gills, heart, kidney, liver, spleen, intestine, pyloric caeca, brain and skeletal muscle were fixed in Bouin's solution. Sectioned tissues were stained with hematoxylin and eosin (H &E), and if necessary, with Giemsa. Results Monitoring of blood fluke infection No egg accumulation was observed, when fish were sampled in March and April; eggs were first detected in the gills on May 7 (Table 1). Stocks positive for eggs were introduced on Feb. 16-Apr. 1 from Hainan, China. The infection became apparent at least two months after the arrival. Mortality due to blood fluke infection started in early May (Fig. 1). About 50-80% of stocks were lost until early June, with the highest daily mortality rate reaching almost 15%. No sign of bacterial infection was observed except for a few cases. Gill monogeneans, Heteraxine heterocerca and Zeuxapta japonica were occasionally found, but it was unlikely Table 1. Results of monitoring of blood fluke infection of imported amberjack at Furue, Kagoshima Prefecture in April and May 1993 * No. of fish positive for eggs in gills/no. of fish examined.
Mortality Fig. 1. Daily ( œ; left) mortality rate dumerili infected and of cumulative imported with of amberjack blood ( ; amberjack fluke due to blood fluke infection Fig. 2. Dead juvenile amberjack with open mouth and opercula. Scale in cm. Fig. Gills right) Seriola 267 3. full of parasite eggs. Scale: 0.5 mm. Paradeonta- cylix. that these monogeneans were responsible for the deaths. Common symptoms noticed on gross and microscopic observations included dead fish with open mouth and opercula (Fig. 2) and the gills full of parasite eggs (Fig. 3). Investigation on viral agents was not conducted. (Table 2). A total of 21 flukes were recovered from the heart, ventral aorta and afferent branchial arteries of 11 amberjack with parasite eggs in the gills. Eight flukes were identified as Paradeontacylix kampachi (4.2-6.1 mm in body length; n=4) and 13 as P. grandispinus (1.5-2.1 mm; n=7). No fluke was found from the vascular system of seven out of 18 fish examined in spite of the presence of eggs in the Identification of the parasites Five out of the six stocks examined were found infected, based on the presence of eggs in the gills gills (Table 2). The actual prevalence and intensity of infection may considerably be higher, since the afferent branchial arteries were examined for flukes Table 2. *k: Paradeontacylix Results of parasitological kampachi surveys on imported, g: P. grandispinus. stocks of amberjack in Kagoshima Prefecture
268 K. Ogawa and M. Fukudome from two out of eight gills of each fish. Histopathology 1. Gills: Parasite eggs were accumulated in the gill filaments. They were encapsulated by epithelioid cells and fibroblasts, forming nodules. Sometimes nodules were held together to contain several eggs within a single encapsulation (Fig. 4). Eggs showed different stages of development, some containing ciliated larvae. Among them, dead eggs decomposed inside the nodule were present. Accumulation of eggs sporadically hindered blood flow from the small afferent branchial arteries to capillaries (Fig. 5), but no necrotic tissues were observed in the gills. There was marked hyperplasia of gill tissues, resulting in the fusion of secondary filaments. The connective tissue of tunica intima of the afferent branchial arteries was proliferated and irregularly projected into the lumen. These papilla-like projections occupied a considerable portion of the lumen 4 5 Figs. 4 g. 6 7 8 Histological sections of the gills of imported amberjack infected with Paradeontacylix spp. 4, Encapsulated eggs. 5, Obstructed blood flow by accumulated eggs in the capillaries (arrow: small afferent branchial artery). 6, A cross section of the afferent branchial artery with no pathological changes in a lightly infected fish. 7, Proliferation of the connective tissue of tunica intima of the afferent branchial artery in a heavily infected fish. An unidentified adult flukeis observed in the lumen. Note hollow spaces (arrows) at the base of the proliferated tissue. 8, A saggital section of the afferent branchial artery, occupied by Paradeontacylix kampachi (arrow). All stained with H & E. Scales: 0.2 mm for Fig. 4, 0.1 mm for Figs. 5-7 and 1 mm for Fig. 8.
Mortality of amberjack due to blood fluke infection 269 (Figs. 6 and 7). Sometimes the papillae were fused at the top, leaving hollow spaces at the base of the proliferated tissue (Fig. 7). Host cells, mostly eosin ophils, were infiltrated in the proliferated tissue. Adult flukes were sometimes found in the lumen of the afferent branchial arteries (Figs. 7 and 8). Polyopisthocotylid monogeneans were attached to the gill filaments, but the infection was not common. 2. Heart: Nodular formation of parasite eggs was seen in the inner layer of the ventricle. Some eggs were already dead and degenerated, as in the gills. 3. Other organs: In some cases, unknown myxo sporean infections were detected in the skeletal muscle, brain and lumen of the kidney tubule. No common signs were observed. @ most cases, infected, while those which arrived in May were not infected (data not shown). Further more, some fish had no mortality due to blood fluke infection in May 1993 (stock no. 3 in Table 1; data not shown), when mortality was at its peak period, though they were introduced there approximately at the same time as the stocks with mortality (see Table 1). This implies that imported amberjack were al ready infected before introduction into Japanese waters. However, the life cycle of Paradeontacylix has not yet been elucidated, and no detailed informa tion was available on the amberjack stocks examined in this study before arrival in Japanese waters. Thus, it is still premature to conclude that the infection originated from abroad. Based on the ecological characteristics of these blood flukes, the infection cycle in amberjack persists from September through Discussion Blood fluke Paradeontacylix infection has been known among 0 + amberjack cultured in Kochi early spring in Kochi Prefecture (Ogawa et al., 1993). Alternately, it could be that the fish wee introduced into Japanese waters, when it was still within the period of cercarial invasion. Prefecture (Ogawa and Egusa, 1986). The causative agents of mass mortality in this study were the same, but there are some differences from the previous case of infection. Mortality occurred in winter months (water temperature: 12-17 Ž) in the Kochi case, while it was concentrated mostly in May in this case, when water temperature was increasing from 18.0 to 20.5 Ž. Outbreaks were much more acute than in the previous case in Kochi Pref., where the daily mortality rate was about 1%, but that persisted from December through March, the cumulative mortality rate reaching 50% (M. Yamaguchi, Kochi Prefec tural Fisheries Experimental Station, personal com munication). In this case, the daily mortality rate sometimes exceeded 10%. No marked pathological difference was observed in histological sections be tween the two cases. However, pathological changes tended to be more severe in the Kochi case, where parasite eggs were more heavily loaded in the gills and heart, and the afferent branchial arteries were almost completely occuluded with the papillate growth of tunica initima in some fish. The less severe pathological changes observed in this case may be because of short duration of infection. There was a difference in the occurrence of infec Acknowledgement The authors would like to express their gratitude to the staff of Uchinoura and Furue Fisheries Coop eration, Kagoshima Prefecture for providing them with infected fish specimens. References Ogawa, K. and S. Egusa (1986): Two new species of Paradeontacylix McIntosh, 1934 (Trematoda: Sanguini colidae) from the vascular system of a cultured marine fish Seriola purpurascens. Fish Pathol., 21, 15-19. Ogawa, K., K. Hattori, K. Hatai and S. S. Kubota (1989): Histopathology of cultured marine fish, Seriola purpura scens (Carangidae) infected with Paradeontacylix spp. (Trematoda: Sanguinicolidae) in its vascular system. Fish Pathol., 24, 75-81. Ogawa, K., H. Andoh and M. Yamaguchi (1993): Some biological aspects of Paradeontacylix (Trematoda: San guinicolidae) infection in cultured marine fish Seriola dumerili. Fish Pathol., 28, 177-180. Ohno, J. (1993): Present status and problems in supply and demand of amberjack seedlings. Yoshoku, 35, 57-60. (In Japanese.) tion among stocks. Early introduced stocks were, in