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Fish Pathology 15 (3/4) 301-307, 1981. 3 Epizootic caused by β-Haemoltytic Streptococcus Species in Cultured Freshwater Fish Tadatoshi KITAO, Takashi AOKI and Ryomei SAKOH Department of Fisheries, Faculty of Agriculture, Miyazaki University, Miyazaki 880, Japan Streptococcal infections have been frequently observed in cultured freshwater fish, tilapia (Tilapia nilotica), rainbow trout (Salmo gairdneri) and ayu (Plecoglossus altivelis) at farms in various districts of Japan. The causative agents isolated from diseased tilapia, rainbow trout and ayu had the same morphological as well as biochemical characteristics. All the strains were also serological homogeneous. These strains were found to be pathogenic to freshwater fish after intraperitoneal injection. The autoclaved and hot-HCl treated cells did not react with any of the group specific sera used: Lancefield A, B, C, D, E, F, G, H, K, L, M, N, 0 and MG. This or ganism was not identical to any strains of Streptcoccus previously reported. Streptococcal infections have been reported in a wide variety of fish species in marine, brackish and freshwater in the U.S.A. and South Africa (ROBIN SON and MEYER, 1966; WILKINSON et al., 1973; PLUMB at al., 1974; BARHAM at al., 1979). In Japan, the first description of outbreaks of strep tococcal disease occurring in cultured rainbow trout was given by HOSHINA et al., (1958). During the past few years, the incidence of the disease has been increasing in cultured marine fish, especi ally yellowtail (Seriola quinqueradiata) (KUSUDA et al., 1976). Similar infections were also observed in cultured eel (KUSUDA et al., 1978). MINAMI e t al. (1979) also reported epizootics in cultured yellowtail caused by different species of Strepto coccus. Since 1979, there have been large scale epizootics of streptococcal disease in cultured freshwater fish, tilapia (Tilapia nilotica), rainbow trout (Salmo gairdneri) and ayu (Plecoglossus altivelis) on farms in various districts of Japan. These epizootics caused serious economic losses. This new strep tococcal disease is a chronic infection. The in fected fish loose appetite gradually and swim slowly. Moribund animals turn laterally and final ly die. The typical symptoms of this disease were exophthalmus, petechiae on the inside of the oper cule and congestion of caudal fin and mouth. Internal examination of the infected fish showed petechiae and heamorrhage of the intestinal tract, liver and pyloric caeca. The causative agent was isolated from brain tissue in high frequency as well as from kidney, heart and spleen. This organ ism seemed to be different from the other patho genic Streptococcus strains mentioned above. The purpose of the present study is to clarify the morphological as well as biochemical characteri stics of this organism which was isolated from cul tured freshwater fish and to discuss the differences between this organism and the other Streptococ cus which are pathogenic for fish. Materials and Methods Sources of strains Twenty five strains of this new organism were studied. Seven strains were isolated from cultured tilapia, eight strains from cultured rainbow trout and ten strains from cultured ayu. All starins were isolated in Japan from 1979 to 1980. Sources of the strains and details of their isolation are given in Table 1. Cell morphology and motility After 48 hours incubation at 30•Ž in Todd Hewitt (TH) broth (Difco), the organisms were stained by the modified Hucker's Gram stain. The cultures were also examined microscopically to determine cell shape and motility. The cells of KST-2 strain which was isolated from cultured tilapia were observed by transmission as well as scanning elcetron microscopy.

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Text of Department of Fisheries, Faculty of Agriculture, Miyazaki

Cultured Freshwater Fish
Department of Fisheries, Faculty of Agriculture, Miyazaki University, Miyazaki 880, Japan
Streptococcal infections have been frequently observed in cultured freshwater fish, tilapia
(Tilapia nilotica), rainbow trout (Salmo gairdneri) and ayu (Plecoglossus altivelis) at farms in various districts of Japan. The causative agents isolated from diseased tilapia, rainbow trout and ayu had the same morphological as well as biochemical characteristics. All the strains were also serological homogeneous. These strains were found to be pathogenic to freshwater fish after intraperitoneal injection. The autoclaved and hot-HCl treated cells did not react with any of the
group specific sera used: Lancefield A, B, C, D, E, F, G, H, K, L, M, N, 0 and MG. This or ganism was not identical to any strains of Streptcoccus previously reported.
Streptococcal infections have been reported in a wide variety of fish species in marine, brackish and freshwater in the U.S.A. and South Africa (ROBIN SON and MEYER, 1966; WILKINSON et al., 1973; PLUMB at al., 1974; BARHAM at al., 1979). In Japan, the first description of outbreaks of strep tococcal disease occurring in cultured rainbow trout was given by HOSHINA et al., (1958). During the past few years, the incidence of the disease has been increasing in cultured marine fish, especi ally yellowtail (Seriola quinqueradiata) (KUSUDA et al., 1976). Similar infections were also observed in cultured eel (KUSUDA et al., 1978). MINAMI
et al. (1979) also reported epizootics in cultured
yellowtail caused by different species of Strepto coccus. Since 1979, there have been large scale epizootics
of streptococcal disease in cultured freshwater fish, tilapia (Tilapia nilotica), rainbow trout (Salmo
gairdneri) and ayu (Plecoglossus altivelis) on farms in various districts of Japan. These epizootics caused serious economic losses. This new strep tococcal disease is a chronic infection. The in fected fish loose appetite gradually and swim slowly. Moribund animals turn laterally and final ly die. The typical symptoms of this disease were exophthalmus, petechiae on the inside of the oper cule and congestion of caudal fin and mouth. Internal examination of the infected fish showed
petechiae and heamorrhage of the intestinal tract, liver and pyloric caeca. The causative agent was
isolated from brain tissue in high frequency as
well as from kidney, heart and spleen. This organ
ism seemed to be different from the other patho
genic Streptococcus strains mentioned above.
The purpose of the present study is to clarify the
morphological as well as biochemical characteri
stics of this organism which was isolated from cul
tured freshwater fish and to discuss the differences
between this organism and the other Streptococ
cus which are pathogenic for fish.
Materials and Methods
Sources of strains Twenty five strains of this new organism were
studied. Seven strains were isolated from cultured tilapia, eight strains from cultured rainbow trout and ten strains from cultured ayu. All starins were isolated in Japan from 1979 to 1980. Sources of the strains and details of their isolation are given in Table 1.
Cell morphology and motility
Hewitt (TH) broth (Difco), the organisms were
stained by the modified Hucker's Gram stain.
The cultures were also examined microscopically
to determine cell shape and motility. The cells of
KST-2 strain which was isolated from cultured
tilapia were observed by transmission as well as
scanning elcetron microscopy.
302 T. KITAO, T. AOKI, and R. SAKOH
Table 1. Sources of strains of the new organism isolated from culture freshwater fish
Growth under various environmental conditions
Whether or not the strains grow on various
media and culture conditions, such as on 1/4,000
tellurite agar, on 10 and 40 % bile agar, in 0.1%
methylene blue milk, at pH 9.6 at 10• and 45•
and in 6.5 % NaCl broth was examined. The
strains were streaked on the agar plates containing
rabbit blood cells and then incubated anaerobi
cally jars. The plates were examined macroscopi
cally for cell growth after 2 days incubation.
Haemolysis tests were performed on the blood agar
plates made from TH agar medium containing 10 % sheep or rabbit blood. Acid production after carbohydrate fermentations was determined in bromcresol purple semisolid agar base (Eiken) supplemented with 1 % of the appropriate sugar . The following carbohydrates were used : arabinose,
glucose, glycerol, inulin, lactose, maltose, mannitol raffinose, salicin, sorbitol, sucrose, trehalose and xylose. Acid production was determined after 7 days incubation.
Biochemical characteristics
wise indicated. The following tests were carried out :
catalase production, cytochrome oxidase, oxidation
and fermentation of glucose, Voges-Proskauer
and methyl-red reactions, production of H2S,
reduction of nitrate, reaction in litumus milk, bile
dissolation, production of indol, susceptibility
to bacitracin, hydrolysis of gelatin, starch, hip
purate, esculin and arginine, and decarboxylation
of lysine, arginine and ornithine (COWAN, 1974).
Susceptibility to chemotherapeutics
All strains were tested for their sensitivities to 16 chemotherapeutics : chloramphenicol (Sankyo)
(CM), tetracycline hydrochloride (Lederle) (TC), doxycyline (Pfizer) (DOTC), streptomycin sulphate
(Meiji) (SM), kanamycin sulphate (Meiji) (KM), aminobenzyl penicillin (Fujisawa) (ABP), cefazolin
(Fujisawa) (CEZ), colistin (Niphonkayaku) (CL), josamycin (Yamanouchi) (JM), erythromycin (Dainippon) (EM), spiramycin (Kyowa) (SPM),
Epizootic by Haemolytic Streptococcus sp.
303
iichi) (NA), sulfamonomethoxine (Daiichi) (SA),
trimethoprim (Takeda) (TMP) and ormethoprim
(Daiichi) (OMP). The serial, two-fold agar dilu-
tion method for determination of the minimal
inhibitory concentrations (MICs) of various drugs
has been described (oOKI et al., 1974).
Serological analysis
rabbit injected intravenously with heat killed cells.
A slide agglutination test was used to investigate
serological relationships among the strains.
Antigen extractions were prepared as described
by Lancefield and the group was determined by the
capillaly precipitin techniques (LANCEFIELD, 1928;
MEDREK and BARNES, 1962), using grouping anti-
serum for Lancefield A, C, E, F, G, H, K, L, M,
N, 0, and MG (Difco), and B and D (Wellcome).
Pathogeicity for fish
The KST-2 strain isolated from cultured tilapia
was grown in TH broth at 30• for 24 hr. Fish
used had a mean body weight of 300 g for tilapia
and 10 g for rainbow trout. Fish were held at
18• in 20 liter tanks. Groups of 10 fish were
inoculated with suspensions of 107 and 108 cells by
intraperitoneal injection. Tilapia were given
stress by shifting the temperature down to 13•
2 days after injection. The fish were kept for 12 hr
at the lower temperature, then shifted back to 18•.
Results
fish were all small gram-positive cocci which were
non motile, encapsulated, 0.3•`0.5 ƒÊm in diameter
and most often occurring in long chains in TH
broth (Table 2). Transmission and scanning elec-
tron micrographs of the KST- 2 strain are shown in
Figs. 1 and 2, respectively.
Biochemical characteristics
All of the isolates grew very well on brain heart
infusion agar, nutrient agar containing with rabbit
blood cell and heart infusion agar. All of the or-
ganisms were able to grow on 10 bile agar and
under anaerobic conditions. None of the strains
grew on the media containing 1/4,000 tellurite, on
40%, bile agar, in 0.1 methylene blue milk, at
pH 9.6 or in 6.5% NaCl broth. All of the or-
ganisms grew at the temperature range of 20 to
37• but did not grow at 10 and 45• (Table 3-1).
Table 2. Morphological characteristics of the strains
isolated from cultured freshwater fish
Fig. 1. Electron micrograph of Streptococcus. KST-
2 strain isolated from cultured tilapia. Early
stationary phase of growth. Negative stained.
Magnification•~ 9,000
fication•~9,000.
Table 3-1. Biochemical characteristics of the strains
isolated from cultured freshwater fish
Table 3-2. Biochemical characteristics of the strains
isolated from cultured freshwater fish
ƒÀ-
esculin and arginine was demonstrated by all of
the isolates, but gelatin and hippurate were not
hydrolyzed by the strains (Table 3-1).
Other significant biochemical reactions of these
organisms commonly used to characterize Strep
tococci are shown in Table 3-2.
Acid was produce from glucose, maltose, man nitol, salicin, sucrose and trehalose. Arabinose,
glycerol, inulin, lactose, raffinose, sorbitol and xylose were not fermented (Table 4).
Susceptibility to chemotherapeutics The distribution of MICS of 16 chemotherapeutic
agents against 25 strains are shown in Table 5.
Epizootic by β-Haemolytic Streptococcus sp. 305
Table 5. Minimal inhibitory concentrations (ug/ml) of naturally occurring Streptococcus strains isolated from cultured freshwater fish to selected chemotherapeutics
All of the strains tested were highly sensitive to CM,
TC, DOTC, ABP, CEZ, JM, EM, SPM, TMP and
OMP. MICS of these drugs against these strains
were from 0.0125 to 1.6 ,ƒÊg/ml. The strains were
moderately sensitive to SM, KM and NF, where
as none of the strains was sensitive to CL, NA and
SA.
used to test the antigenic relationship among the
organisms. The titer of this antiserum against
KST-2 strain was 1: 640. All of the strains iso
lated from cultured tilapia, rainbow trout and ayu
were strongly agglutinated with this antiserum.
Lancefield extracts of this organism did not re
act with any of the grouping sera used : Lance
field A, B, C, D, E, F, G, H, K, L, M, N, O and MG
Pathogenicity for fish Five of 10 rainbow trout were killed within 2
weeks after injection of 107 cells of the organism. The group injected with 108 cells also had 50% mortality. Deaths occurred in 4 or 10 and 6 of 10 tilapia 2 weeks after injection of 107 and 108 cells, respectively. There was no mortality in control fish. The organism was recovered from fish which died.
Discussion
It is obvious that the isolates from cultured tilapia, rainbow trout and ayu can be classified into Streptococcus by their morphological as well as biochemical characteristics. All strains from these three difference sources had the same morphologi cal and biochemical characteristics. The strains were also found to be serologically homogeneous. The organisms from tilapia, rainbow trout and ayu were considered to be same or at least, very similar to one another.
HOSHINA et al., (1958) reported streptococcal infections of cultured rainbow trout and considered the causative organism to be Streptococcus faecalis. Similar epizootic occurred in golden shiner (Note migonus crysoleucas) in a hatchery in Arkansas and the causative agent was also identificated as Streptococcus (ROBINSON and MEYER, 1966), The organisms isolated in Arkansas and the Strep tococcus strains from 8 species of marine fish along the Alabama and northwest Florida coast of the
gulf of Mexico (PLUMB et al., 1974) were quite similar in their characteristics, that is, they were all nonhaemolytic, type Ib, Lancefield group B Streptococci. In recent years, streptococcal in fections were observed in cultured yellowtail at farms in various areas of Japan. The causative agent was similar to S. faecalis and S. faecium
306 T. KITAO, T. AOKI, and R. SAKOH
Table 6. Comparision in biochemical characteristics between the isolates from cultured fresh
water fish and Streptococcus strains which have been previously reported
(KUSUDA et al., 1976). This organism was also
isolated from an epizootic of cultured eel (KUSVDA
et al., 1978). MINAMI et al. (1979) reported ƒÀ-
haemolytic Streptococcus infection of cultured yel
lowtail and the bacterial isolates had characteristics
similar to S. equismillis. The biochemical chara
cteristics of the organism we isolated were com
pared with those of other Streptococcus strains
which have previously reported to be pathogens of
fish. The differences of biochemical characteri
stics among these strains are shown in Table 6.
There are many biochemical differences between
our organism and the ƒ¿-haemolytic Streptococcus
sp. isolated from cultured yellowtail and eel. The
differences of some biochemical characteristics are
also recognized between our organism and Strepto
coccus sp. strains isolated sea catfish and white
bass in the U.S.A. Our organism was also serolo
gically distinct from other strain.
Some variation of biochemical characteristics
were observed between the streptococcal strain
isolated by MINAMI et al. (1976) and our organism
with regard to decarboxylation of arigine, acid
production from glycerol and lactose, although
the strain supplied by Minami was weakly
agglutinated with antiserum for KST-2 strain. Accordingly, the strains isolated from cultured freshwater fish, tilapia, rainbow trout and ayu
were considered not to be similar to other fish
pathogenic Streptococcus strains which have been previously reported.
There is also no question that biochemical chara
cteristics of our organism were different from any type strains of Streptococcus species which are described in Bergey's Manual, 8th edition (1974).
Antigenic analysis of this organism was carried out in detail for the group specific polysaccharides by
authors and the results of this study will be reported elsewhere.
The initial origin of this Streptococcus is not yet known. Since cultured fish have been routinely
transferred from one fish farm to another farm in
a different area, Streptococcus strains may have also been transferred with these fish and spread
widely in various districts. Recently this organism has been frequently isolated in pure culture from brain tissue and from kidney, heart and spleen of diseased fish. The high pathogenicity, as well as,
Epizootic by β-Haemolytic Streptococcus sp. 307
the high affinity for brain tissue of various fish is
significant for freshwater fish culture. This infec
tion is expected to be controlled by chemothera
peutics after diagnosis by pathological examina
tion.
Acknowledgements
Kagoshima Prefectural Fisheries Experimental
tural Fisheries Experimental Station for supplying
the materials for this investigation. We would
also like to express our appreciation to Miss.
Hiroko Yago of Department of Veterinary Science,
Miyazaki University for preparing the electron
micrograph.
the Japan Fisheries Resources Conservation As
sociation.
References
AOKI T., S. EGUSA and T. ARAI (1974) : Detection of R factors in naturally occurring Vibrio anguillarum
strains. Antimicrob. Agents Chemother., 6, 534 -538.
BARHAM W. T., H. SCHOONBEE and G. L. SMIT (1979) : The occurence of Aeromonas and Streptococcus in
rainbow trout, Salmo gairdneri RICHARDSON. J. Fish Biol., 15, 457-460.
BUCHANAN R. E. and N. E. GIBBONS (1974) : Bergey's Manual of Determinative Bacteriology. 8th ed.
Williams & Wilkins Co., Baltimore. COWAN S. T. (1974) : Cowan and Steel manual for the
identification of Medical bacteria. 2nd ed. Cambridge
Univ. Press, London & N. Y. HOSHINA T., T. SANO and Y. MORIMOTO (1958) : A
Streptococcus pathogenic to fish. J. Tokyo Univ. Fish., 44, 57-68.
KUSUDA R., K. KAWAI, T. TOYOSHIMA and I. KOMATSU
(1976) : A new pathogenic bacterium belonging to the genus Streptococcus, isolated from an epizootic of
cultured yellowtail. Bull. Japan. Soc. Sci. Fish., 42, 1345-1352 (in Japanese).
KUSUDA R. and I. KOMATSU (1978) : A comparative study of fish pathogenic Streptococcus isolated from
saltwater and freshwater fishes. Bull. Japan. Soc. Sci. Fish. 44, 1073-1078 (in Japanese).
KUSUDA R., I. KOMATSU and K. KAWAI (1978) : Stre
ptococcus sp. isolated from an epizootic of cultured eels. Bull. Japan. Soc. Sci. Fish., 44, 295.
LANCEFIELD R. C. (1928) : The antigenic complex of Stretococcus hemolyticus. II. Chemical and im munological properties of the protein fraction. J. Exp. Med., 47, 469-480.
MEDREK T. F. and E. M. BARNES (1962) : The influence of the growth medium on the demonstration of a
group D antigen in faecal Streptococci. J. Gen. Microbiol., 28, 701-709.
MINAMI T., M. NAKAMURA, Y. IKEDA and H. OZAKI
(1976) : A Beta-hemolytic Streptococcus isolated from cultured yellowtail. Fish Path., 14, 33-38 (in
Japanese). PLUMB J. A., J. H. SCHACHTE, J. L. GAINES, W. PELTIER
and B. CARROLL (1974) : Streptococcus sp. from marine fishes along the Alabama and northwest Florida coast of the gulf of Mexico. Trans. Amer.
Fish. Soc., 103, 358-361. ROBINSON J. A. and F. P. MEYER (1966) : Streptococ
cal fish pathogen. J. Bacteriol., 92, 512. WILKINSON H. W., L. G. THACKER and R. R. FACKLAM
(1973) : Nonhemolytic group B Streptococci of human, bovine, and ichthyic origin. Infect. Im
munity, 7, 496-498.