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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.

Department of Fisheries, Faculty of Agriculture, Miyazaki

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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 organism 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 (ROBINSON and MEYER, 1966; WILKINSON et al., 1973; PLUMB at al., 1974; BARHAM at al., 1979). In Japan, the first description of outbreaks of streptococcal 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, especially 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 Streptococcus. 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 streptococcal disease is a chronic infection. The infected fish loose appetite gradually and swim slowly. Moribund animals turn laterally and finally die. The typical symptoms of this disease were exophthalmus, petechiae on the inside of the opercule 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.

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•Ž and 45•Ž

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

All strains were incubated at 30•Ž, unless other

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

furazolidone (Takeda) (NF), nalidixic acid (Dai-

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

Antiserum of KST-2 strain was obtained from a

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•Ž 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•Ž 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•Ž

2 days after injection. The fish were kept for 12 hr

at the lower temperature, then shifted back to 18•Ž.

Results

Cell morphology amd motility

The organisms isoalted from cultured freshwater

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•Ž but did not grow at 10 and 45•Ž (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

Fig. 2. Scanning electron micrograph of Strepto-

coccus KST-2 strain isolated from cultured tila-

pia. Early stationary phase of growth. Magni-

fication•~9,000.

304 T. KITAO, T. AOKI, and R. SAKOH

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-

haemolysis was observed on the sheep and

rabbit blood agar plates. Hydrolysis of starch,

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, mannitol, 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.

Serological analysis

Antiserum prepared against KST-2 strain was

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 morphological 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 (Notemigonus 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 Streptococcus 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 infections 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

We are grateful to Mr. Katsuo Shiomitsu of

Kagoshima Prefectural Fisheries Experimental

Station and Dr. Sei Takahashi of Shiga Prefec

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.

This investigation was supported by a grant from

the Japan Fisheries Resources Conservation As

sociation.

References

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