STREPTOCOCCUS FAECALIS AS A CAUSE OF MORTALITIES AMONG CULTURED MONOSEX-TILAPIA

Assiut Vet. Med. J. Vol. 52 No. 109 April 2006

National Institute of Oceanography and Fisheries

STREPTOCOCCUS FAECALIS AS A CAUSE OF MORTALITIES AMONG CULTURED

MONOSEX-TILAPIA (With One Table and 12 Figures)

- By

SAFINAZ G.M. (Received at 29/2/2006)

البکتريا السبحية من نوع ستربتوکاس فيکالز کسبب للموت في أسماک البلطى

وحيد الجنس المستزرع

صافيناز جمعة محمد تم تسجيل وفيات عالية بنسبة ۲۰% في أحدى مزارع البلطى وحيد الجنس في مدينة الإسکندرية خلال الربيع الماضي. وتم عزل وتصنيف البکترية السبحية من نوع فيکالز کسبب للوفاه. تم عمل العدوى الصناعية بنجاح باستخدام البکتريا المعزولة في أسماک البلطى وحيد الجنس والمبروک الفضي عن طريق الحقن بالتجويف البريتوني. وکانت الأعراض الأکلينيکية عبارة عن خمول في حرکة الأسماک وأعراض تنفسية وتحول لون الجلد للون الغامق مع تساقط القشور وحجوظ في أحدى العينين أو کلاهما ووجود أنزفة على الجلد وخاصة عند قواعد الزعانف واستسقاء بالبطن. وتمثلت الأعراض المرضية الداخلية في أحتقان بالأعضاء الداخلية. بينما کانت التغيرات الهستوباثولوجية تحلل وموت خلايا الکبد وأضمحلال بأنسجة تخليق الدم وتنقرز وموت خلايا الکلى. وقد وجد أن البکتريا السبحية من نوع فيکالز حساسة للعديد من المضادات الحيوية وأن کلا من الأوکسى تتراسيکلين والأموکس سيللين کانا أکثر المضادات الحيوية تأثير على البکتريا.

SUMMARY During last spring (2006) severe mortalities among cultured monosex tilapia were recorded in a private fish farm at Alexandria governorate and caused up to 20% mortality. The isolated bacteria was identified as Streptococcus faecelis. Experimental infection was successful through intraperitoneal injection (i.p.). The clinical signs were sluggish movement, respiratory disorder, darkening, loss of scales, uni-or bilateral exophthalmia, haemorrhages of the skin especially in the base of fins and abdominal distention. The common postmortem lesions were congestion of internal organs. The histopathological changes were hydropic degeneration of hepatocytes, deplation of haemopoietic

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elements and necrosis of kidney tissues. The isolated S. faecalis found to be sensitive to a wide range of antibiotic and oxytetracycline and amoxicillin were the drugs of choice.

Key words: Fish, Tilapia, Strept faecalis.

INTRODUCTION

Streptococcal infection of fish which were rearly reported before 1970, (Robinson and Meyer, 1966) become a major problem worldwide with the intensification of aquaculture (Baya et al., 1990 and Carson et al., 1993).

Now streptococcus sp. has recently created a major disease problem in cultured tilapia and considered of high importance in recent years due to increased reports of outbreaks and the high economic losses caused by gram-positive bacteria in both wild and culture fish (Domenech et al., 1996).

Moreover, streptococcal disease has been documented in both wild and cultured fish with distribution being worldwide (Kitao 1993), specially in Oreochromis niloticus and common carp and caused heavy losses (Eldar and Ghittino (1999).

Also Zlotkin et al., (2003) recorded that streptococcus iniae was capable of causing disease in human who had recently handled infected fish from farms.

In Egypt, massive mortalities from streptococcosis has been recorded in both wild and cultured freshwater fish (Badran, 1994, Khalil, 2002, Ebtesam, 2002 and Refaee, 2005).

Vaccination to streptococcosis has been largely disappointing but antimicrobial compound have met with considerable success (Kitao, 1982).

Experimental infection of streptococcus, which may or may not be representative a disease in the natural environment, have been achieved by injection (Cook and Lofton, 1975) and by exposure of fish to streptococcus species (Semino et al., 1996). Monosex tilapia and carp species had been recived considerable attention during the last 10 years in Egyptian aquaculture and sharing in most as polyculture farms.

The aim of this study is to record the isolation and identification of streptococcus species during on outbreak in monosex tilapia farm in Alexandria - Cairo desert road at Alexandria Governorate during spring of 2005. Characterization and pathogenicity were carried out.

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MATERIALS and METHODS

1. Fish:

During an out break in private fish farm heavy mortalities (up to 20%) was occurred in spring of 2005 among monosex-tilapia (average body weight 80 + 5g).

Freshly dead and moribund fish were collected and subjected to clinical, bacteriological, parasitic and mycological examination according to Amlacher (1970). Bacterial isolation was done from blood, kidneys, liver, spleen and ascitic fluid of 50 naturally infected monosex tilapia and striked on tripticase soya agar (TSA), brain heart infusion agar (BHIA), 5% sheep blood agar and Mackonky agar. The plates were incubated at 28C for 48 hours.

The isolated colonies were tested for morphological, culture and biochemical characterization according to Bergey et al., (1994) and Elmer et al., (1997). Also the isolated bacteria were tested biochemically by using APT-20 strep. System (Bio Merieux). The antibiogram of the recovered bacteria was done according to Cruickshank et al., (1975) and Carter and Cole (1990). Estimation of the medial lethal dose (LD50):

The median to that dose (LD50) was calculated for the isolated streptococcus sp. in monosex-tilapia and silver carp according to Reed and Muench (1938). Seventy fish of apparently healthy monosex tilapia (60 + 5g) and 70 silver carp (80 10g) were intraperitoneal (i.p) injected with serial ten fold dilutions (10" - 10') of the isolated bacteria (10 fish/dilution). Ten fish from each fish species were injected (i.p) with 1ml steril saline and served as control. Mortalities were recorded for 8 days. Experimental injection:

A total numbers of 60 apparently healthy (30 monosex tilapia and 30 silver carp) were used in experimental injection. Fish of each type were divided into 3 groups (10 fish/group). The first 2 groups of each species were injected (i.p) with 1ml of sublethal dose of S. faecalis (10 cfu/ml in case of monosex-tilapia and 10 cfu/ml in case of silver carp). The 3rd group of each fish species were injected with Iml sterial saline and served as control. The fish were observed daily for 14 days for clinical signs and mortalities. Specimen from liver, kidney, spleen and gills were collected from injected fish fixed in 10% neutral buffered formalin for histopathological examination according to Roberts (1989).

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Reisolation of injected bacteria was done from freshly dead fish for verification the specificity of death where the injected streptococcus isolate was rei-solated.

RESULTS

Clinical examination:

During the out breaks, mass mortalities (about 20%) was recorded. The naturally infected fish showed loss of appetite, sluggish movement, swimming close to the surface of the water, escape reflex

(-ve), darkening of the skin, detached scales and haemorrhages of skin. Uni or-bilateral exophthalmia and eye turbidity with distented abdomen

and congested vent were observed (Fig. 1).

Internally congestion of the liver, kidneys, presence of fluid in the abdomin and destended of gall bladder were recorded (Fig. 2). Bacteriological examination:

The isolated bacteria was recovered from internal organs and blood of moribund fish during the outbreak. The bacteria was gram positive cocci, arranged in short chain, non motile, pen headed colony, white opaque colour, raised edges and gave a-haemolysis on 5% sheep blood agar. The biochemical characters of the isolated bacteria are illustrated in Table (1). From the morphological, cultures and biochemical characters by traditional method and the manufacturer cretaria of the API-20 streps, we abled to recovered one type of streptococcus, namely streptococcus faecalis.

The results of mycological examination proved to be negative while, the parasitic one showed presence of slight infestation with ciliate protozoa (Trichodina sp.). Antibiogram:

The isolated Streptococcus faecalis were proved to be sensilive to amoxicillin, chloramphenical, penicillin, oxytetracycline, Trimethoprim & sulfamethoxazole, nalidexic acid and colistin sulphate and resistant to ciprofloxacin, erythromycin, kanamycin, Tetracyclin and ampicillin. Median Lethal dose (LD50):

The results of LD50 were proved that the isolated S. faecalis was highly virulent to monosex tilapia than silver carp. LD50 value were 10* and 100 respectively.

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Experimental infection:

The experimental infection was successfully induced by (i.) with streptococcus fecalis with no difference in both fish species except that in silver carp, caudal fin erosion was observed. Reisolation of the injected bacteria was succeeded from all dead fish. The control group showed neither clinical signs nor post mortem (P.M.) changes.

The clinical signs and PM lesions were appeared on injected fish as no escape reflexes, restlessness, swim near the water surface and strong respiratory disorder. Death started after 4 and 6 days post infection with a total mortalities of 30 and 40% incase of monosex tilapia and silver carp respectively.

The fish showed haemorrhage in the base of the fins, uni or bilateral exophthalmia, with haemorrhage of the eye. Slight ascites and detached scales. Congetion of the internal organs was observed and some fish from both species showed yellowish liver (Fig. 3, 4, 5, 6 and 7). Histopathological alteration:

The histopathological lesions of naturally and experimentally infected fish were more or less similar.

The gills showed telangictasis beside edema at the base of secondary lamellae Fig. (8).

Hyperactivation of melanomacrophage centers and marked depletion of white pulp with multifocal hyrmphocytic cells depletion were observed in the spleen Fig. (9 and 10).

The liver showed severe hydropic degeneration and congestion of blood vessels Fig. (11).

Depletion of intertubular haemopiotic tissue, cloudy swelling of renal tubules and necrotic of convoluted tubules were the changes recognized in the kidney Fig. (12).

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Assiut Vet. Med. J. Vol. 52 No. 109 April 2006 Table 1: Morphological and biochemical characters of isolated bacteria.

Test

Result

Test

Result

- Gram stain

- Citrate utilization

- Chain

Short

- a-galactosidase

- Motility

-ve

- Hydrolysis of:

- Growth on:

* Argenin dihydrolase

* Triptic soya agar

pen head.

* Esculin hydrolysis

ed white

* Hippurate hydrolysis

Opque

- Acid produced from:

circular

* Ribose

Colonies

* Arabinose

* Macconkey agar

Pink

* Mannitol

Colour

* Sorbitol

- 5% sheep blood

a-hae

* Lactose

Agar

moloysis

1 * Inulin

- At 6.5% NaCl

* Raffinose

- At 10 and 45C

* Sucrose

- Fermentation metab

* Glucose

olism

* Dulcitol

- Production of

*Urease

*Catalase

* Oxidase

* Indole

* Voges Perskauer

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Fig. 1

Fig.3

Fig.2

Fig. 4

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Fig.5

Fig. 7

Fig.6

Fig. 8

545

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w

AS

WANDOWS

WAND

KA

Wie

WE

WWW

W

Fig. 9

Fig. 11

BAR

Fig. 10

Fig. 12

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LEGEND OF FIGURES

Fig. 1: Naturally infected monosex tilapia showing darking of the skin,

congestion of base of fins, fin erosion and unilateral

exophthalmia. Fig. 2: Naturally infected monosex tilapia showing congestion of

internal organs and distended gall bladder. Fig. 3: Experimentally injected monosex-tilapia with Streptococcus

faecalis showing sever exophthalmia. Fig. 4: Experimentally injected monsex-tilapia with Streptococcus

faecalis showing detached scales and fins errosion. Fig. 5: Experimentally injected monosex-tilapia with Streptococcus

faecalis showing congestion of internal organs. Fig. 6: Experimentally injected silver carp showing darking,

haemorrhagic eye exophthamia. Fig. 7: Experimentally injected silver carp showing congestion of

internal organs and yellowish liver. Fig. 8: Gills of experimentally infected monosex tilapia showing

telangictasis an edema of the base of secondary lamellae. H & E

(X 250). Fig. 9: Spleen of naturally infected monosex tilapia showing

hyperactivation of melano macrophages centers H & E (X250). Fig. 10: Spleen experimentally infected silver carp showing marked

deplation of white pulp and multifocal hymphocytic cells

deplation (arrow) H&E (X400). Fig. 11: Liver of experimentally infected monosex-tilapia showing

congestion of hepatic blood vesels with severe hydropic

degeneration of the most hepatic cells. H&E (X400). Fig. 12: Kidneys of silver carp showing cloudy swelling of some

convoluted tubules (arrows), multifocal tubular necrosis (arrows head) and depletion of inter tubular haemopiotic tissue H & E (X400).

DISCUSSION

Streptococcal infection of fish which were reported before 1970, (Robinson and Meyer, 1966) and become a major problem world wide with the intensification of aquaculture (Carson et al., 1993). Now streptococcus sp. had recently created a major disease problem in

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cultured tilapia and reported to cause high economic losses in both wild and cultured fish (Domench et al., 1996).

The isolated bacteria from naturally infected monosex tilapia and comprised gram-positive cocci, non-motile, catalase, oxidase and indole negative, grew on blood agar and gave a-haemolysis. From the results of culture, morphological and biochemical tests, the isolated bacteria could be identified as Streptococcus faecalis as guided by Bergey et al., (1994). Also the results agree with those reported by Baye et al., (1990), El-Bouhy (2002) and Zeid (2004). Also Refaee (2005) isolated S. faecalis from naturally infected Oreochromis niloticus. The isolation of S. faecalis from kidney, spleen and liver may be attributed that, this organs more or less considered being tropism for this bacteria due to the nature of septicemia occurred by the microorganism and proved the pathogenicity of this bacteria to fish (Kimura and Kasuda, 1982). Moreover, presence of isolated bacteria in fish tissue revealed high

concentration of these pathogen in the pond water.

The successful induction of the disease experimentally leaves no doubt about the potential pathogenicity of Streptococcus faecalis to monosex tilapia and silver carp. The fact that infection could occur following the presence in water contaminated with S. faecalis confirm the invasive character of the organism. These substantiate findings reported by Boomker et al.,(1979).

Variation in disease signs, including lethargic, darkening, deteched scales, exophthalmia and distended abdomin, while the common postmortem lesions were acute septicemia in nature as they revealed congestion of the internal organs. These results may be due to the haemolytic effect of the exotoxin produced by bacteria (Rasheed and Plumb, 1984).

The isolation of S. faecalis from diseased and dead fish during the epizootic in spring season indicate the pathogenicity of this organism and seasonal threat to fish industry posed by it. The differences in LD50 values and mortality rate in monosex tilapia and silver carp may be attributed to sensitivity of fish to infection and monosex tilapia proved to be sensitive to S. faecalis infection than silver carp. This differences among fish species had been reported by Khalil (2002) and Refaee (2005).

It is interesting to note that the disease was occurred during spring and among sexually mature fish. This may be due to the stress effect during this time of the year which initiates a series of physiological responses ending with immunosuppression (Peters, 1977).

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The histopathological alterations were gill telangictasis, hydropic degeneration, activation of melanamacrophage centers and depletion of haemopiotic elements.

The possible explanation of these changes could be attributed to the strong action of a-haemolysin of S. faecalis (Minamia et al., 1979 and Zeid, 2004). Some changes also reported by Khalil (2002) and Refaee (2005).

The results of antibiotic sensitivity of the isolated bacteria proved that they were sensitive to a wide range of antibiotics (Franks et al., 1998, Domenech et al., 1996 and Khalil, 2000).

The fact that streptococcus species are a common cause of human infections. The zoonotic importance of this organism specially S. faecalis. This work, raised some questions about the use of chicken manure infarms and dangerous of contamination of water used in farms with human sewage. However, it remain to be necessary to investigate how far is the role of man or fish in transmission of this bacteria.

ACKNOWLEDGEMENT

The author greatly acknowledges Dr. E.M. El-Manakhly, Professor of pathology, Fac. of Veterinary Medicine, Alexandria University for his help in the histopathological study.

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