昇温処理に伴うクリノストマム属吸虫のメタセルカリア寄生キンギョの大量死

誌名誌名 魚病研究

ISSNISSN 0388788X

巻/号巻/号 531

掲載ページ掲載ページ p. 44-47

発行年月発行年月 2018年3月

農林水産省 農林水産技術会議事務局筑波産学連携支援センターTsukuba Business-Academia Cooperation Support Center, Agriculture, Forestry and Fisheries Research CouncilSecretariat

;@.frHiJfo/E Fish Pathology, 53 (1), 44-47, 2018. 3

Short communication

Mass Mortalities of Goldfish Carassius auratus Infected with

Clinostomum Metacercariae, Associated with Elevated

Water Temperature

Shinya Yasumoto*, Tsukasa Kabayama, Masakazu Kondo and Yukinori Takahashi

Department of Applied Aquabiology, National Fisheries University, Japan Fisheries Research and Education

Agency, Yamaguchi 759-6595, Japan

(Received October 2, 2017)

ABSTRACT-Mass mortalities of goldfish Carassius auratus occurred at some farms and wholesalers in Japan, where fish were held at around 33°C to control herpesviral hematopoietic necrosis. All dead fish showed light to mod­erate Clinostomum infection. Cysts containing metacer­cariae were accompanied by hemorrhage and congestion, and metacercariae were activated and excysted during the high temperature water treatment. When infected fish were experimentally treated at 20°c, 31 °c, 33°C and 35°C, mortality rates reached to 0%, 5%, 40% and 100%, respec­tively. The experimental results indicated that the eleva­tion of water temperature against the virus infection caused the mass mortalities in C/inostomum-infected goldfish.

Key words: goldfish, C/inostomum, mass mortality, her­pesviral hematopoietic necrosis, high tem­perature water treatment

Clinostomids are common parasites worldwide. Snails are the first intermediate host, fishes are the sec­ond intermediate host, and herons are the final host of these trematodes (Bullard and Overstreet, 2008). In Japan, several fish species serve as a second intermedi­ate host of Clinostomum complanatum, including gold­fish Carassius auratus, common carp Cyprinus carpio, leach Misgurnus anguillicaudatus, and ayu Plecoglossus altivelis (Aohagi et al., 1992; Aohagi et al., 1995). In fish, the cercariae of these parasites develop into metacercariae within a cyst. Generally, Clinostomum does not cause great harm to infected fish except in cases of heavy infection (Lo et al., 1981; Lo et al., 1985).

* Corresponding author E-mail: yasumotos@fish-u.ac.jp

© 2018 The Japanese Society of Fish Pathology

From September to November 2015, mass mortali­ties of cultured goldfish occurred at some Japanese farms and wholesalers. In all cases, fish had been held at around 33°C with a boiler heating system to control herpesviral hematopoietic necrosis of goldfish (HHNG), which is caused by cyprinid herpesvirus 2 (CyHV-2; Tanaka, 2005). All dead or moribund fish were infected with Clinostomum metacercariae. We examined the cause of the mass mortalities by observing gross and histopathological features, and we performed a repro­ducibility test to evaluate elevated water temperature as the potential cause.

Materials and Methods

Spontaneous diseased goldfish Diseased goldfish were obtained from two fish

farms in Nagano and Aichi prefectures, where the mor­tality rates at these farms were about 90% and 70%, respectively. Ryukin goldfish from farm N in Nagano (body weight 8.9-12.7 g; 5 dead, 13 moribund, and 6 surviving fish) and ranchu goldfish from Aichi (body weight 10.0-16.9 g; 2 dead, 7 moribund and 5 surviving fish) were used for observation of gross and histopatho­logical features. Samples from the trunk kidney, spleen, and cysts were subjected to bacterial isolation on BHI agar plates at 28°C.

Goldfish was reported as a host of clinostomids (Kim and Nagasawa, 1996). Removed metacercariae from cysts were large (body length 1-4 mm) and yellow­ish with the acetabulum in forebody, and the cysts were in the muscle close to the skin. From these features, the parasite was identified as a trematode of the genus Clinostomum.

Reproducibility test Based on the observed mass mortalities and the

results of gross and histopathological features, we sus­pected that the cause of mass mortalities was excyst­ment of Clinostomum metacercariae induced by warm water. Thus, we performed a reproducibility test for mass mortality at several temperatures.

Eighty infected ryukin goldfish (body weight 8.5-13.2 g) from farm N, each of which had four to seven cysts on the mandible to ventral abdomen, were used for the reproducibility test. Although these fish were from the same farm mentioned above, they had never been exposed to warm water (33°C) to control HHNG. Instead, they had been held at around 20°c at farm N.

We established four experimental groups (20 fish held at 20°c, 31 °c, 33°C or 35°C), each of which was housed in an aquarium (50 L) with an airlift filter. All fish were kept at 20°c and fed a commercial diet for 1 wk before starting the experiments. After pretreatment, water temperatures were elevated gradually (over 2 to 3 h) to 31 °c, 33°C, or 35°C by using a ceramic .heater,

Mass mortalities of C/inostomum-infected goldfish 45

and mortalities were recorded daily for 1 wk. The 20°C group was kept at that temperature throughout the experimental period and served as the control. The gross features of all fish were observed, and some were used for histopathological examination. Bacterial isola­tion from the kidney and spleen was performed as described above.

Histopathological examination After anesthetization with quinaldin , fish were dis­

sected by using scissors and scalpel and then exam­ined. The head including cysts , gill , abdominal organs and muscle were fixed in 10% formalin or Bouin's solu­tion and processed for histopathological examination . Tissue sections (2-4 µm) were stained with Mayer's hematoxylin and eosin, Azan, or May-Grunwald-Giemsa.

Results

Spontaneous diseased goldfish The C/inostomum infection rate was 85 .8%

(109/127) at farm N. No pathogenic bacteria were iso­lated from any of the fish .

In surviving fish, four out of 11 fish had two to four cysts (without hemorrhage or congestion) on the mandi­ble, and they were able to swim with vigor. Cysts were pale yellow and spherical or elliptical (1 .5-3.0 mm in length), often accompanied by swelling on the body sur­face (Fig. 1 A) . The other surviving fish had no cyst or signs of abnormality. In all surviving fish, the gill , brain and abdominal organs were normal. Histopathological analysis revealed that metacercariae were covered with a cyst wall, which consisted of collagen fiber and con­nective tissue (Fig . 1 B).

All dead or moribund fish had two to eight protrud­ing cysts distributed from the mandible to ventral abdo­men. Most of them were degenerate or accompanied by hemorrhage and congestion (Fig. 2A). Degenerated cysts caused ulcers on the body surface (Fig . 2A and B) . Two to eight metacercariae could be surgically removed from individual fish (Fig. 2C). In some fish , metacercar­iae had migrated to the abdominal cavity and exited the body wall from a degenerated cyst (Fig. 2D). Histologically, the gill, brain and abdominal organs were normal, and no other parasites were found. In dead and moribund fish, histopathological observation of cysts containing metacercariae revealed that the cyst wall had undergone liquefaction necrosis and many inflammatory cells had invaded into the cyst (Fig. 3A). In muscle tis­sue surrounding the cysts, muscle fibers had undergone necrosis and hyalinization, and inflammatory cells were observed (Fig. 3B) . In the trunk kidney, the hematopoi­etic tissue was necrotic (Fig . 3C) . Mild congestion and cloudy hepatocytes were observed in the liver (Fig . 3D) . The other organs had no pathological signs.

Fig. 1. Gross and histopathological features of surviving fish infected with Clinostomum. A: Cysts, pale yellow and spherical or elliptical, accompanied by swelling on the body surface, but not by hemorrhage and congestion (bars= 10 mm). B: Histopathological features of the cyst. The metacercaria (me) was covered with cyst wall (cw) , which consisted of collagen fibers and con­nective tissue (Azan; bar= 500 µm) .

Fig. 2. Gross features of spontaneously dead fish and the metacercariae. A: Degenerate cysts (black arrow­head) and hemorrhaged and congested cysts (white arrowhead). B: Degenerated cysts (closed arrow­heads) causing ulcers on the body surface. C: Microscopic image of metacercariae . D: Migrated metacercariae (arrows) observed in the gill chamber and abdominal cavity. Bars: A and B, 5 mm ; C, 1 mm.

Reproducibility test Mortality rates in the reproducibility test are shown

in Fig. 4. All fish in the 35°C group and eight fish (40%) in the 33°C group died within 1 wk. The cysts of dead fish showed hemorrhage and inflammation from the mandible to ventral abdomen. Histopathological analy­sis revealed liquefaction necrosis of cyst wall and muscle fiber as well as inflammation. These gross and histopathological features were the same as those observed in spontaneously dead or moribund fish. On the other hand , only one fish (5%) died in the 31 °C

46 S. Yasumoto, T. Kabayama, M. Kondo and Y. Takahashi

Fig. 3. Histopathological features of spontaneously dead fish (A, Azan ; inset in A, May-Grunwald-Giemsa; B-D , hematoxylin and eosin). A: The cyst wall (cw) sur­rounding the metacercariae (me) causing liquefaction necrosis and invasion of inflammatory cells . Inset:

100

80

i :0 ~ 60 i3 E

! 40 "' ,; E :,

U 20

Details of several kinds of inflammatory cells . B: Muscle fiber necrosis and inflammatory cells (arrow­heads) in th e muscle tissue surrounding cysts . Hematoxylin and eosin. C: Necrotic hematopoietic tissue in the trunk kidney. D: Mild congestion and cloudy hepatocytes in the liver. Bars: A, 500 µm ; inset in A, 20 µm ; B, 50 µm; C and D, 100 µm.

-6-20t:: .....,_3 I t::

-&- 33t::

-+-35"C

G ~ ! ! ! ! ! ! 0 2 4 6

Days post elevating water temperatures

Fig. 4. Cumulative mortality rates during the reproducibility test. Eighty infected goldfish, each of which had four to seven cysts from the mandible to ventral abdomen, were used in this experiment. Four experimental groups (n = 20, respectively ; h. 20°C, .._ 31 °C, 0 33°C, • 35°C} were established. Water tempera­tures were gradually raised from 20°C to 20°c , 31 °c , 33°C or 35°C, and mortalities were recorded daily for 1 wk.

group; it had four cysts on the mandible, but these were not accompanied by hemorrhage or inflammation. Histopathological analysis revealed metacercariae were covered with cyst wall. All fish survived in the 20°c group.

In the surviving fish in the 20°C, 31 °C and 33°C groups, the cysts were not accompanied by hemorrhage or inflammation, and metacercariae were covered with cyst wall. The other organs had no pathological signs. No pathogenic bacteria were isolated from any of the experimental fish .

Discussion

Mass mortalities of goldfish were caused by elevat­ing water temperature (to around 33°C) to control HHNG on fa rms . All spontaneous dead or moribund fish showed light to moderate infection with Clinostomum metacercariae, and the cysts were accompanied by hemorrhage and congestion . The histopathological features included liquefaction necrosis of the cyst wall and muscle fibers accompanied by invading inflamma­tory cells . Although some surviving fish were infected with metacercariae, the cysts were not accompanied by hemorrhage or congestion and metacercariae were cov­ered with cyst wall. Lo et al. (1985) reported that the process of the excystment and migration of metacercar­iae caused the congestion and hemorrhage followed by the serious tissue damage. It is likely that the same process of excystment and migration of the metacercar­iae had occurred in the present samples. Therefore, we concluded that the histopathological features around the cysts of the dead and moribund fish were caused by the activated matacercariae. The relationship between the induced topical tissue damages around the cysts and the histopathological features in the kidney and liver was not clear. More detailed pathological investiga­tions would be needed to reveal the phenomena.

In the reproducibi lity test, all fish in the 35°C group and 40% of fish in the 33°C group died. One fish in the 31 °C group died due to another cause , because histo­pathological analysis indicated that metacercariae were not activated . In Lo et al. (1987) , no metacercariae were activated at 30°C and some were activated at 33°C. Results of the reproducibility test support those findings and well reproduced the mass mortality rates on goldfish farms. About 90% of the fish with the infection rate of 85 .8% at farm N died , whereas only 40% of infected fish in the 33°C group in the reproducibility test died. Lo et al. (1987) reported that the average time necessary for the excystment of metacercariae at > 33°C was 14 min , and the rate of excystment increases as temperature rose . At farm N, the water temperature was controlled by a boiler heating system with . an error of several degrees, which is larger than the temperature variability of ceramic heaters used in an aquarium . It appears that the pond water temperature at farm N tem­porarily exceeded 33°C, which resulted in the extremely high mortality. Consequently, mass mortalities of gold­fish at the farm were caused by elevating the water tem­perature of Clinostomum-infected goldfish.

Mass mortalities of Clinostomum-infected goldfish 47

Cultured fish are not usually exposed to elevated water temperature. At most goldfish farms, however, treatment at 33°C for more than 4 days is recommended to control HHNG {Tanaka, 2005). The treatment is nec­essary in goldfish culture because they are usually infected with CyHV-2 during the course of distribution and farming. Pesticide use does not prevent Clinostomum infection because it induces activation and excystment of metacercariae {Lo et al., 1985). Attempting to prevent the invasion of farms by herons is not a realistic option. Therefore, the best option for pre­venting Clinostomum infection is to remove all snails, which serve as the first intermediary host, from the ponds.

Acknowledgments

We thank the goldfish farms in Nagano and Aichi prefectures that provide the samples and information. This study supported by Japan pet design Co., Ltd.

References

Aohagi, Y., T. Shibahara, N. Machida, Y. Yamaga and K. Kagota (1992): Clinostomum complanatum (Trematoda:

Clinostomatidae) in five new fish hosts in Japan. J. Wild/. Dis., 28, 467-469.

Aohagi, Y., T. Shibahara and K. Kagota (1995): Metacercariae of Clinostomum complanatum found from new fish hosts, Lateolabrax japonicus and Leuciscus hakonensis. Jpn. J. Parasitol., 44, 340-342.

Bullard, S. A. and R. M. Overstreet (2008): Digeneans as ene­mies of fishes, In "Fish diseases, Vol., 2" (ed. by J. E. Eiras, H. Segner, T. Wahli and B. G. Kapoor). Science Publishers, Enfield, pp. 817-976.

Kim Y. G. and K. Nagasawa (1996): Infection of Clinostomum complanatum (Rudolphi, 1814) (Trematoda: Digenia) metacercaria in goldfish (Carassius auratus) cultured in Korea. J. Fish Pathol., 9, 1-9.

Lo, C. F., F. Huber, G. H. Kou and C. J. Lo (1981 ): The study of Clinostomum complanatum (Rud. 1819). Fish Pathol., 15, 219-227.

Lo, C. F., S. C. Chen and C.H. Wang (1985): The study of Clinostomum complanatum (Rud. 1814). V. The influ­ences of metacercaria of Clinostomum comp/anatum on fish. Fish Pathol., 20, 305-312.

Lo, C. F., S. C. Chen, C. H. Wang, K. Ogawa and H. Wakabayashi (1987): The mechanism of the metacercarial excystment of Clinostomum complanatum (Rud. 1814). Fish Pathol., 22, 227-235.

Tanaka, M. (2005): Elevating water temperature for control of herpesviral haematopoietic necrosis of goldfish, Carassius auratus. Bull. Saitama Pref. Agr. Forest. Res. Cent., 5, 37-43. (In Japanese)

昇温処理に伴うクリノストマム属吸虫のメタセルカリア

寄生キンギョの大量死

安本信哉・樺山つかさ・近藤昌和・高橋幸則

2015年 9~11月にかけてヘルペスウイルス性造血器壊

死症の昇温治療中にキンギョが大量死した。すべての死

亡魚にはクリノストマム属吸虫のメタセルカリアが軽度

から中度に感染しており，下顎部から腹部にかけて虫体

の被嚢には出血や炎症をともなっていた。病理組織観察

の結果，メタセルカリアの活性化による脱嚢や周辺組織

の壊死・崩壊および炎症性細胞の浸潤が認められた。大

量死を再現するためにクリノストマム感染キンギョを

20°c. 31°c. 33°cおよび35°cで飼育したところ，それ

ぞれ0%, 5%, 40%および100%の死亡がみられた。以

上の結果から，クリノストマム感染キンギョの昇温処理

が大量死を引き起こしたと判断した。

魚病研究， 53(1), 44-47 (2018)