さけ・ます資源管理センター
研究報告 第 5 号 1-6頁
2002年12月

Bulletin of the National Salmon
Resources Center, No. 5: 1-6
December 2002

Investigation of the Transmission Stage ofthe Microsporidian Kabatana takedai in Salmonids
Isao Fujiyama*1, Shigehiko Urawa*2#,
Hiroshi Yokoyama*1, and Kazuo Ogawa*1*1

Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences,
The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan*2
Research Division, National Salmon Resources Center,
2-2 Nakanoshima, Toyohira-ku, Sapporo 062-0922, Japan

Abstract. − In order to examine the transmission route of the microsporidian Kabatana takedai tosalmonid fish, experimental challenges to rainbow trout (Oncorhynchus mykiss) was tried by threemethods (oral intubation, intraperitoneal injection, and immersion) using K. takedai mature sporesisolated from infected fish. However, the experimental infection was not successful by any methods.
To estimate the size of the transmission stage, masu salmon (O. masou) were exposed to infectious
river water filtered by three sets of nylon meshes with openings of 300μm, 100μm and 40μm. As aresult, K. takedai infections occurred among all fish groups. These results suggest that the transmission of K. takedai to fish is not due to the direct spore transmission, but might due to the unknown
infectious stage smaller than 40μm.
Key words : Microsporidia, Kabatana takedai, transmission, plankton net

Introduction
The microsporidian parasite Kabatana takedai(Awakura 1974) is known as an important andendemic pathogen of wild and cultured salmonid
fish. This disease has been reported only in the Chitose River (Takeda 1933), Lake Akan and Tokitonuma of Hokkaido, Japan (Awakura et al. 1966;
Awakura 1978) and in the Taranay and Bryanka
Rivers of Sakhalin, Russia (Vyalova and Voronin
1987; Vyalova 1999).
The parasite infecting the heart and trunk muscle
was first described by Takeda (1933) from rainbow
trout (Oncorhynchus mykiss) in the Chitose Hatchery. Since it was evident that the disease occurred
when river water temperature exceeded 15゜
C fromJuly to September (Awakura 1974; Urawa 1989), thedisease had been controlled by keeping water temperature under 13 ゜
C adding spring water in thehatchery. However, Urawa (2001) found that K.
＃

Corresponding author (urawa@salmon.affrc.go.jp)
Contribution A No. 27 from the National Salmon
Resources Center.
© 2002 National Salmon Resources Center

takedai could form cysts at 13゜
C, and the result ofother experiment in 2001 indicated that the cyst formation occurred even at 11゜
C (Fujiyama, unpublished data).
Generally, it is a common concept that the infection of microsporidian is due to direct transmissionof spores released into the water from dead hostorganisms (Lom and Dyková 1992), which supported by some experimental evidences. Glugea
plecoglossi established the infection by intubation,
injection and immersion with spores (Takahashi
1981), and Loma salmonae formed xenoma by theingestion of infected tissue (Kent et al. 1995). Similarly, K. takedai was reported to be successful inexperimental transmission by ingestion of the feedmixed spores or immersion with spores, although theprevalence was only 10% and 22%, respectively(Awakura 1974).
Awakura (1974) also examined whether
salmonid fish could be infected with K. takedai
through intermediate hosts. Juvenile sockeye salmon(O. nerka) was exposed to the Chitose River water
filtered by four kinds of plankton nets with openingsof 2.0 × 2.0 mm, 0.65-0.80 × 0.70-0.80 mm, 0.200.33 × 0.23-0.30 mm and 0.12-0.18 × 0.04-0.12mm for 75 days in the infective season. As a result,2

さけ・ます資源管理センター研報 Bull. Natl. Salmon Resources Center, No. 5, 2002

only the finest net prohibited the infection with K.
takedai. He suspected that fish were not infectedwith spores directly, but through the involvement ofsmall aquatic organisms as a vector, which weretrapped on 0.12-0.18 × 0.04-0.12 mm-net, butpassed through 0.20-0.33 × 0.23-0.30 mm-net.
We performed infection experiments similar to
those of Awakura (1974) with some improvement inorder to determine the transmission stage of K.
takedai to fish.

Table 1. Combination of three kinds of nets to filtrate riverwater in four test groups for infection experiments with K.
takedai.
Openig size of net (μm)
Group
300

100

40

Total
numberof nets
0

A
B

○

C

○

○

D

○

○

1
2
○

3

Materials and Methods
Experimental fish
Juvenile rainbow trout used in the experimental
infection trial (trial 1) were obtained from
Yamanashi Prefectural Fisheries Technology Centerand maintained in dechlorinated tap water at 16゜Cbefore the beginning of the trial. Underyearling masu
salmon in the plankton net trial (trial 2) were provided from the Chitose Hatchery, where the fish had
been reared in the pond supplied with spring water,which were not contaminated with K. takedai. The
fish were held in flow-through tanks with well waterat 11゜C before the beginning of the trial. These fish
were fed commercial dry pellets.
Spore preparation
Mature spores used in the trial 1 were preparedby the following methods. Cysts of K. takedai wereisolated from masu salmon reared in the Chitose
Hatchery, squashed and passed through a steel mesh
(Cell Dissociation Sieve-Tissue Grinder Kit, Sigma,
U.S.A.) and a nylon mesh series (100 μm, 75 μm,
50 μm, 25 μm, 10 μm, and 5 μm) to remove tissuedebris. The filtrate was layered onto equal volume of
50% Percoll and centrifuged at 750×g for 15 min.
The pellet was resuspended in distilled water andstored at 4゜
C with antibiotics (100 U/ml penicillin,
100 μg/ml streptomycin and 0.25 μg/ml amphotericin B, Gibco. BRL, U.S.A.) until used. Spore viability defined as the extrusion rate of polar tube stimulated by 30% hydrogen peroxide was about 60%.
Spore infection experiment (trial 1)
Juvenile rainbow trout (mean body length, 6.9cm) were challenged with K. takedai spores by thefollowing methods.
Intubation: Twenty three fish were anesthetizedwith 50 ppm tricainemethanesulphonate (MS222,

Sigma, U.S.A.), and then intubated orally into thestomach with K. takedai spores (1×107 spores/fish)using a 1 ml tipped with a 5 cm sterilized silicon
tube (1 mm inner diameter and 2 mm outer diameter).
Injection: Twenty three fish were anesthetized
and injected with spore suspensions (1 × 10 7
spores/fish) intraperitoneally.
Immersion: Twenty three fish were exposed to 2 lof spore suspension (2.6×103 spores/fish) in a 3-lplastic container at 18゜
C for 24 hours with a gentle
aeration.
Unexposed control : Twenty three fish weremaintained in the 3-l plastic container with 2 l ofdechlorinated tap water at 18゜
C for 24 hours with agentle aeration.
The experimental fish were transferred to each of
three 60-l glass tanks with a circulated filtration unit
C, and fed with dry pellets.
maintained at 18±2゜Plankton net experiment (trial 2)
From July 29 to August 12 in 2002, 30 juvenile
masu salmon were kept in each of the four 120-l
flow-through tanks supplied with Chitose River
water filtrated by combination of three sets of nylon
mesh with openings of 300 μm, 100 μm and 40 μm
at the mean flow rate of 6.4 (5.2-9.0 in range) l/min(Table 1). As a positive control group, 30 fish were
kept in a tank supplied with unfiltered river water.
The river water temperature during the exposure was
16.0-17.4゜
C. Plankton nets were washed once in twoor three days in order to prevent the nets from clogging. Then the experimental fish were stocked inflow-through tanks supplied with parasite-free well
water at 18゜C, and fed with commercial dry pellets
for 5 weeks.

Fujiyama et al. − Transmission stage of K. takedai in salmonids

Sampling and parasitological examination
Trial 1: Half of the surviving fish in each tankwere removed and sacrificed with MS222 overdosed
at 22 days post exposure (PE). The heart and trunk
muscle were examined to detect K. takedai cysts
using a dissection microscope. At 33 days PE, allremaining surviving fish were sacrificed and examined as described above.
Trial 2: After maintained under spring water for
5 weeks, the fish were weighed, measured and examined immediately for K. takedai cysts as describedabove. The number of parasite cysts was recorded
for infected fish.

3

Results
Spore infection experiment
The cysts of K. takedai were not observed in theheart and trunk muscle of rainbow trout challenged
using any of the three methods (Table 2). While a
few fish died during this experiment, the cause ofdeath was not considered to be K. takedai infection
by gross observation.
Plankton net experiment
The prevalence of K. takedai was 71.4-90.0% in
4 test groups (Table 3). The mean number of cysts in
the heart and trunk muscle of the infected fish was
1.3-2.0 and 14.9-17.5 cysts per fish, respectively.
There were no significant differences in the prevalence nor number of cysts among all test groups. A
few fish were died in Group B, C and D at the beginning of this experiment, but the cause of these deaths
was not considered to be K. takedai infection bygross observation.

Statistical analysis
Statistical comparisons were performed between
the four groups for trial 2. A comparison of theprevalence (percentage) and mean number of cysts in
the heart and trunk muscle of infected fish were performed using chi-square analysis and Kruskal-Wallis
ANOVA, respectively. Significant differences werereported at the α=0.05 level of probability.

Table 2. Prevalence of infection with Kabatana takedai in the heat and trunk muscle of rainbow trout by three experimental
infection methods, 23 and 33days post exposure (PE).
Infection method

23 days PE

33 days PE

Heart

Trunk

Heart

Trunk

Intubation

0/8*

0/8

0/8

0/8

Injection

0/10

0/10

0/10

0/10

Immersion

0/7

0/7

0/8

0/8

Control

0/9

0/9

0/10

0/10

* Number of fish infected / number of fish examined.
Table 3. The prevalence and intensity of infection with Kabatana takedai in the heart and trunk muscle of masu salmon reared in
the river water filtrated by different opening sizes of plankton nets. The opening size and combination of nets used for each test
group were shown in Table 1.
Test group1

Number of fish
examined

Prevalence (%)*1

A

30

90.0

B

27

88.9

C

27

81.5

D

21

71.4

* Number of fish infected with K. takedai / Number of fish examined.
*2 Mean number ± SD (range) of cysts in the infected fish.

Intensity*2
Heart

Trunk

2.0 ± 1.3

16.0 ± 13.4

(1-5)

(1-57)

2.0 ± 1.0

14.9 ± 11.3

(1-4)

(3-45)

1.8 ± 0.7

17.5 ± 10.6

(1-3)

(3-52)

1.3 ± 0.8

15.5 ± 15.0

(1-3)

(3-46)

4

さけ・ます資源管理センター研報 Bull. Natl. Salmon Resources Center, No. 5, 2002

Discussion
Since K. takedai was first described by Takeda(1933), several aspects of studies on this parasite
have been reported; the temperature-dependency ofthe parasite's development, the infection route, thehost response, and the epizootiology in the ChitoseRiver and Chitose Hatchery (Awakura and Kurahashi 1967; Awakura 1974; Urawa 1989, 2001).
However, the prevention method of K. takedai infection in the Chitose Hatchery has not been establishedyet, because the life cycle and critical temperature
requirement in the development of this parasite are
not elucidated yet.
We could not establish the artificial infection by
any exposure methods using K. takedai spores.
Awakura (1974) reported that K. takedai could succeed in the transmission and forming of cysts byingestion of the feed added spores or immersion with
spore suspensions. However, Awakura (1974) alsomentioned that there might be some problems withthe fluctuating water temperature during the experiment and the challenge method. In this study, thewater temperature was fixed at 18゜C and the quantitative method of infection was used. Replicatedexperiments under the similar condition to that of
Awakura (1974) could not reproduce the infection
with K. takedai (Awakura, unpublished data). The
present study also suggests that spores are not theinfectious stage to fish.
Most of the fish were infected with K. takedai
within a short period (2 weeks) exposing to the riverwater. Awakura (1974) presumed that the rotifer
Euchlanis dilatata which flowed from the upstreamof the Chitose River might act as the intermediateorganism in natural transmission through the fieldand experimental studies. The size of E. dilatata is
about 200-270 μm (Mizuno 1991), therefore therotifer had to be trapped on 100 μm or 40 μm-net. If
E. dilatata was the intermediate organism, the fish ofGroup C or D would have been expected to be uninfected with K. takedai. Awakura (1974) reported thatthe fish exposed to the river water filtrated by 0.120.18×0.04-0.12 mm net were uninfected with K.
takedai at all, but also mentioned that the flow rateof the river water through the finest net (about 3l/min) was a quarter of the other groups because of
the clogging of the net. It is suspected that this clogging was one of the reasons why the fish were unin-

fected. During the fish was exposed to river waters
in this experiment, the organisms trapped on each net
were fixed by 10% neutral buffered formalin andobserved. As a result, E. dilatata were found in boththe 100 μm and 40 μm nets. The four test groups
resulted in high prevalence of infection with K.
takedai, which were not significantly different
among 4 groups. Therefore, this study shows that theorganism related to K. takedai infection could passthrough all nets easily.
In the microsporidians Amblyospora and Parathelohania species which are infective to mosquitoes, aspore formed in a copepod interemediate host is
responsible for horizontal transmission to the mosquito host (Micieli et al. 2000). Sweeney et al.
(1985) demonstrated that uninucleate spores of
Amblyospora dyxenoides from the mosquito (Culex
annulirostris) are infective to the copepod (Mesocyclops albicans) and that there is a sequence ofdevelopment and achievement different type ofmature spores infective to mosquitoes. Similarly,
Andreadis (1985) succeeded the transmission of A.
connecticus to the copepod (Acanthocyclops
vernalis). Though the report about the obvious existence of intermediate organisms in the fish
microsporidians have not been published yet, someauthors have suggested that intermediate or carrier
hosts may be involved (McVicar 1975, Olson 1976).
The present results also suggest the presence of
unknown transmission stage of K. takedai to fish,
whose size might be much smaller than 40 μm. Further studies related to the transmission stage to fish
are needed for elucidation of the life cycle of K.
takedai.
In 1999, due to the unusual increase of watertemperature, heavy infection of K. takedai occurredamong juvenile masu salmon in the Chitose Hatchery, and approximately 100,000 infected fish were
disposed to prevent this parasite from spreading(Urawa 2001). Since it is difficult to cure the infected fish by temperature manipulations (Urawa 2001),
the most important things is the prevention of K.
takedai. The findings in the present study show apossibility that the unknown transmission stage of K.
takedai may exists. For the establishment of the prevention method in the Chitose Hatchery, further
studies should be performed to clarify the transmission stage and infective period to fish in the ChitoseRiver.

Fujiyama et al. − Transmission stage of K. takedai in salmonids

Acknowledgements
The authors wish to express our appreciation to
the staffs of National Salmon Resources Center andChitose Hatchery for their kind help to provide fish
and information for this study, and their generous
hospitality. We also thank Yamanashi Prefectural
Fisheries Technology Center for assistance with providing fish. Similarly we are grateful to Dr S.-J. Lee
for her technical help.

References
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さけ・ます資源管理センター研報 Bull. Natl. Salmon Resources Center, No. 5, 2002

サ ケ 科 魚 類 に 寄 生 す る 武 田 微 胞 子 虫 Kabatana
takedai の感染源の推定
藤山勲・浦和茂彦・横山博・小川和夫武田微胞子虫Kabatana takedaiのサケ科魚類への感染経路を調べるため，感染魚から分離した成熟胞子を用いてニジマスに対する経口投与，腹腔内注射，浸漬による人工感染を試みた．その結果，いずれの方法によっても心臓および体側筋肉中にシストの形成はみられず，感染は不成立に終わった．次に，感染源の大きさを推定するため，3種のプランクトンネット（300 μm ，100 μm ，40 μm ）で濾過した千歳川河川水でサクラマスを飼育した
ところ，いずれのネットを通過した河川水によっても感染が起きた．以上の結果より，魚への感染は胞子による直接伝播ではなく，40μm以下の未知の感染ステージが寄与していることが示唆された．