• Presented by

Torky, H.A.; Soliman, M.K. *; El-Ashram, A.M.M. ** and El-Refaee,

A. M. E **

STUDIES ON STREPTOCOCCAL STUDIES ON STREPTOCOCCAL INFECTIONS AMONG CULTURED FRESH INFECTIONS AMONG CULTURED FRESH

WATER FISHES WITH SPECIAL WATER FISHES WITH SPECIAL REFERENCE TO ITS DIAGNOSIS BY REFERENCE TO ITS DIAGNOSIS BY

POLYMERASE CHAIN REACTION TESTPOLYMERASE CHAIN REACTION TEST

“Introduction”Fish is among the most important sources of protein to human consumption, thus the study Fish is among the most important sources of protein to human consumption, thus the study

of the signs and lesions, induced by fish diseases, helps the protection in our national of the signs and lesions, induced by fish diseases, helps the protection in our national economy.economy.

Infectious diseases of cultured fish are among the most notable constraints on the expansion Infectious diseases of cultured fish are among the most notable constraints on the expansion of aquaculture and the realization of its full potential (Plumb, 1999; Woo and Bruno, of aquaculture and the realization of its full potential (Plumb, 1999; Woo and Bruno, 1999 and Klesius 1999 and Klesius et alet al., 2000). Bacterial pathogens are the most serious disease problem in ., 2000). Bacterial pathogens are the most serious disease problem in tilapia production causing 80% of fish mortalities (Plumb, 1999; Woo and Bruno, 1999; tilapia production causing 80% of fish mortalities (Plumb, 1999; Woo and Bruno, 1999; Clark Clark et al.,et al., 2000 and Shoemaker 2000 and Shoemaker et al., et al., 2000).2000).

Historically, Historically, Streptococcus spStreptococcus sp. are not serious pathogens of fish, but recently, these bacteria . are not serious pathogens of fish, but recently, these bacteria have become more prominent in wild and cultured fish (Baya have become more prominent in wild and cultured fish (Baya et al.,et al., 1990). Now 1990). Now Streptococcus sp. has recently created a major disease problem in cultured tilapia and Streptococcus sp. has recently created a major disease problem in cultured tilapia and considered of high importance in recent years because of the increased reports of considered of high importance in recent years because of the increased reports of infections and the high economic losses caused by gram-positive bacteria in both wild and infections and the high economic losses caused by gram-positive bacteria in both wild and cultured fish (Eldar cultured fish (Eldar et al., et al., 1995 and Domenech 1995 and Domenech et al.et al., 1996)., 1996).

Moreover, WeinsteinMoreover, Weinstein et al., et al., (1996) and Zlotkin(1996) and Zlotkin et al., et al., (2003) recorded that (2003) recorded that Streptococcus iniaeStreptococcus iniae was capable of causing disease in humans who had recently handled infected fish from was capable of causing disease in humans who had recently handled infected fish from aquaculture farms. In Egypt, Nile tilapia (aquaculture farms. In Egypt, Nile tilapia (Oreochromis niloticusOreochromis niloticus) population facing ) population facing streptococcosis in several areas, notably in Lake El-Temsah in Ismailia governorate streptococcosis in several areas, notably in Lake El-Temsah in Ismailia governorate (Badran, 1994) recorded a serious mass mortality among fish due to streptococcal (Badran, 1994) recorded a serious mass mortality among fish due to streptococcal infection, in Kafr El-Sheikh governorate (Khalil, 2000) who recorded massive mortalities infection, in Kafr El-Sheikh governorate (Khalil, 2000) who recorded massive mortalities from streptococcosis and (Ebtesam, 2002) recorded streptococcosis in Lake El-Ibrahimia from streptococcosis and (Ebtesam, 2002) recorded streptococcosis in Lake El-Ibrahimia in Upper Egypt.in Upper Egypt.

Saçilik Saçilik et alet al., (2000) analyzed different gram positive cocci ., (2000) analyzed different gram positive cocci by SDS-PAGE in order to obtain better discrimination by SDS-PAGE in order to obtain better discrimination between different gram positive cocci and found a common between different gram positive cocci and found a common 18 and 35 kD characteristics bands of 18 and 35 kD characteristics bands of Streptococcus spp. Streptococcus spp. andand Enterococcus faecalis. Enterococcus faecalis. Also Barnes Also Barnes et al., et al., (2003) used it (2003) used it for characterization of for characterization of StreptococcusStreptococcus iniaeiniae..

Enlightenment the previous argument the present study was Enlightenment the previous argument the present study was conducted to fulfill the gap about conducted to fulfill the gap about Streptococcus Streptococcus species species infection among cultured freshwater fish in Egypt. Also, infection among cultured freshwater fish in Egypt. Also, availability of use of total bacterial proteins in SDS-PAGE availability of use of total bacterial proteins in SDS-PAGE for characterization of the isolated streptococcus. In for characterization of the isolated streptococcus. In addition to using of polymerase chain reaction (PCR), as a addition to using of polymerase chain reaction (PCR), as a rapid and sensitive method for identification of rapid and sensitive method for identification of streptococcus with potential diagnostic value.streptococcus with potential diagnostic value.

Fig : Fig : Streptococcus spp.Streptococcus spp. isolated from fish occurred as gram isolated from fish occurred as gram positive cocci arranged in chain.positive cocci arranged in chain.

Prevalence of Streptococcosis among the examined fish.Prevalence of Streptococcosis among the examined fish.

Fish speciesNumber of examined fish

Number of clinically diseased fish

% of the diseased fish from the same species

Oreochromis niloticus60010317.2

Clarias gariepinus60016527.5

Seasonal prevalence of Streptococcosis among Nile tilapia (Seasonal prevalence of Streptococcosis among Nile tilapia (O. O. niloticusniloticus) and African catfish () and African catfish (C. gariepinusC. gariepinus).).

Season O. niloticusC. gariepinus

No. of examined

fish/season

No. of infected

fish/season

% from examined

fish/season

No. of examined

fish/season

No. of infected

fish/season

% from examined

fish/season

Spring 1502718

1504530

Summer 1504731.33

1508154

Autumn 1502013.33

1502516.67

Winter 15096

150149.33

SpringSummer

AutumnWinter

020406080

100120140

160

Season

No. of examined No. of infected

Seasonal prevalence of Streptococcosis among Nile tilapia (Seasonal prevalence of Streptococcosis among Nile tilapia (O. O. niloticusniloticus).).

Seasonal prevalence of Streptococcosis among African catfish Seasonal prevalence of Streptococcosis among African catfish ((C. gariepinusC. gariepinus).).

0

20

40

60

80

100

120

140

160

SpringSummerAutumnWinter

Season

Prevalence of Streptococcosis among Prevalence of Streptococcosis among O. niloticusO. niloticus and African catfish ( and African catfish (C. C. gariepinusgariepinus) in relation to locality and the total examined fish.) in relation to locality and the total examined fish.

LocalityLocality

Tilapia (O. niloticus)Catfish (C. gariepinus)

No. of examined

No. of infected

*% of infected

No. of examined

No. of infected

*% of infected

Domiata20035342003823

Sharkia2002726.22005935.8

Kafr El-Sheikh2004139.82006841.8

Total600103100600165100

Clinical Clinical signssigns

O. niloticusO. niloticus naturally infected with naturally infected with Streptococcus spp.Streptococcus spp. showing skin ulceration and tail fin erosion.showing skin ulceration and tail fin erosion.

C. gariepinusC. gariepinus naturally infected with naturally infected with Streptococcus spp.Streptococcus spp. showing haemorrhagic ulcers..showing haemorrhagic ulcers..

C. gariepinusC. gariepinus naturally infected with naturally infected with Streptococcus spp.Streptococcus spp. showing skin ulceration and haemorrhages in the base of the fins..showing skin ulceration and haemorrhages in the base of the fins..

C. gariepinus naturally infected by Streptococcus spp. showing haemorrhagic ulceration.

O. niloticus naturally infected with Streptococcus spp. Showing sever exophthalmia and congestion of the eyes.

O. niloticusO. niloticus naturally infected with naturally infected with Streptococcus sppStreptococcus spp . .showing congestion of the eyes and corneal opacityshowing congestion of the eyes and corneal opacity..

O. niloticus naturally infected with Streptococcus spp .showing corneal opacity.

O. niloticus naturally infected with Streptococcus spp .showing different stages of corneal opacity until complete

destruction of eye.

O. niloticus naturally infected with Streptococcus spp. showing haemorrhage at the base of dorsal fin.

O. niloticusO. niloticus naturally infected with naturally infected with Streptococcus spp.Streptococcus spp. Showing Showing inflammation and congestion of the anal openinginflammation and congestion of the anal opening..

O. niloticus O. niloticus naturally infected with naturally infected with Streptococcus sppStreptococcus spp. . showing vertebral column deformitiesshowing vertebral column deformities

O. niloticus O. niloticus naturally infected with naturally infected with Streptococcus sppStreptococcus spp. showing pale liver . showing pale liver and haemorrhage on the skinand haemorrhage on the skin

O. niloticusO. niloticus naturally infected with naturally infected with Streptococcus spp.Streptococcus spp. showing pale liver showing pale liver and congestion of internal organsand congestion of internal organs

O. niloticusO. niloticus naturally infected with naturally infected with Streptococcus sppStreptococcus spp. showing . showing corneal opacity, brownish liver and distended gall bladdercorneal opacity, brownish liver and distended gall bladder..

O. niloticusO. niloticus naturally infected with naturally infected with Streptococcus spp.Streptococcus spp. showing showing bloody ascitis and inflammation of intestinebloody ascitis and inflammation of intestine..

O. niloticusO. niloticus naturally infected with naturally infected with Streptococcus spp.Streptococcus spp. showing showing enlarged spleenenlarged spleen..

O. niloticusO. niloticus naturally infected with naturally infected with Streptococcus spp.Streptococcus spp. showing showing enlarged and congested kidneyenlarged and congested kidney....

C. gariepinusC. gariepinus naturally infected with naturally infected with Streptococcus spp.Streptococcus spp. showing empty intestine and enlarged kidneyshowing empty intestine and enlarged kidney..

Dosedays

No. of injected

Number of dead O. niloticusNumber of dead Monosex tilapia

5X1045X1055X1065X1075X108Control 5X1045X1055X1062X1072X108Control

110-----------1

210----------2-

310---22---124-

410-1123-12132-

510-1234---322-

61012211-13-21-

710211---1-1---

810-1-1---1-1--

910--11----1---

1010--1---------

1110------------

12101-------1---

1310------------

1410------------

1510------------

% of dead40%50%80%100%100%0%30%60%80%100%100%10 %

Experimental infection of fish with different concentrations of Streptococcus faecalis:

Dosedays

No. of injected

Number of dead O. niloticusNumber of dead Monosex tilapia

5X1045X1055X1065X1075X108Control5X1045X1055X1065X1075X108Control

110------------

210------------

310----------2-

410--213-11113-

510-1-24---122-

610-1142--1-32-

710--111---21--

810-1-1----1-1-

910----1---1---

1010--111----1--

1110---1-----1--

12101-1---------

1310--------1---

1410------------

1510------------

% of dead10%30%60%100%100%0%10%20%70%90%100%0 %

Experimental injection of fish with different concentrations of Streptococcus faecium:

O. niloticusO. niloticus injected with injected with Streptococcus faecalisStreptococcus faecalis showing slight showing slight exophthalmiaexophthalmia..

O. niloticusO. niloticus injected with injected with Streptococcus faecalisStreptococcus faecalis showing showing congestion of the internal organscongestion of the internal organs..

Sensitivity testes for isolated Sensitivity testes for isolated Streptococcus sppStreptococcus spp..

RRRRROptochin

SSSSSNalidexic acid

SSSSSVancomycin

SSSRSTrimethoprim

+Sulfamethoxazole

SRSSIStreptomycin

SRSSSPenicillin

SRSSRTetracycline

RRSIRKanamycin

SSSSRErythromycin

SISSRCiprofloxacin

SSRSSChloramephenicol

SRIRRAmpicillin

SRIISAmoxicillin

S. sp3.S. sp2.S. sp1.S. faeciumS. faecalisAntimicrobial agent

M: Marker.1: Standard Streptococcus

faecalis2: Streptococcus faecalis3: Streptococcus faecium4: Streptococcus sp15: Streptococcus sp26: Streptococcus sp3

SDS-PAGE of bacterial cell proteinSDS-PAGE of bacterial cell protein..

935.2118.21

1523118.4122S. sp3

935.2118.21

152384,7918S. sp2

935.2118.21

152384,7918S. sp1

935.2118.21

152396.0214S. faecium

935.2118.21

1523118.4122S. faecalis

935.2118.21

1523118.4122Standard

Number of common

bands

Molecular weight of diagnostic bands/KD

Number of diagnostic

bands

Highest molecular weight/KD

Total number of

bandsSpecies

The number of protein bands and highest molecular weight, number of diagnostic bands and its molecular weight:

2218181422222424.00 

78.296105.845115.67269.189112.4489.57817.6030.9524

28.75791.28785.50621.48977.01275.48418.210.9323

32.46744.36541.3418.97337.84842.7620.2770.8722

10.2616.51914.768 16.86114.08322.4760.8221

4.392 17.877 9.6225.92125.1720.7620

 21.652 5.09510.51712.95325.8560.7419

 25.64331.991  25.00227.2710.7118

14.47536.9543.4548.83221.43840.64529.9390.6617

43.28629.2234.3712.87239.077 32.0440.6316

42.56349.56350.17411.86449.72155.94435.2080.5915

42.053 34.91 61.51666.91638.3810.5614

28.32329.78226.41110.59866.46669.48738.9520.5513

39.94322.043 11.1281.56584.43844.0350.4912

76.90319.37225.24617.48988.8992.74548.9510.4411

54.08439.00341.688 94.47795.30254.720.3910

47.56932.33634.74921.46591.14290.50857.6940.389

33.61321.65327.19815.994 70.94863.5870.358

29.97622.53127.07817.58558.45654.93773.0290.317

36.87920.08526.769 69.54473.02478.8070.296

32.1314.99319.957 67.26159.50184.790.275

22.175   63.30271.0591.3780.254

22.677  12.96666.317 96.0210.243

16.645   30.79341.123109.060.202

14.669   24.57719.077118.4080.181

S. sp3S. sp2S. sp1S. faeciumS. faecalisStandardM. W. KDRelative FrontBand Number

Table (16): Densitometeric analysis of bacterial cell proteinTable (16): Densitometeric analysis of bacterial cell protein

 StrainStandardS. faecalisS. faeciumS. sp1.S. sp2.

S. faecalis0.83    

S. faecium0.500.58   

S. sp1.0.750.670.58  

S. sp2.0.750.670.750.83 

S. sp3.0.830.920.580.750.67

Similarity coefficient among species by protein electrophoresisSimilarity coefficient among species by protein electrophoresis

Dendrogram among species by protein electrophoresis.

The polymorphic bands, shared and total bands in related to the type of primer used for Streptococcus identification:

Serial numberPrimer code No.No. of polymorphic bandsNo. of shared bandsTotal No. of bands.

1OP.A- 02819

2OP.A- 09505

3OP.A- 10707

4OP.Z-01415

5OP.B-02606

6OP.C-01336

7OP.C-04617

8OP.C-06437

9OP.A- 11617

10OP.O-20516

Total10541165

M: Marker.M: Marker.1: Standard1: Standard

StreptococcuStreptococcus faecaliss faecalis

2: 2: Streptococcus Streptococcus faecalisfaecalis

3:3: Streptococcus Streptococcus faeciumfaecium

4: 4: StreptococcusStreptococcus sp1sp1

5: 5: StreptococcusStreptococcus sp2sp2

6: 6: StreptococcusStreptococcus sp3sp3

Agarose gel electrophoresis of amplified RAPD-PCR products with primers Z1, B2.

M: Marker.1: Standard Streptococcus

faecalis2: Streptococcus faecalis3: Streptococcus faecium4: Streptococcus sp15: Streptococcus sp26: Streptococcus sp3

Agarose gel electrophoresis of amplified RAPD-PCR products with primers C1, C4, C6.

M: Marker.1: Standard

Streptococcus faecalis

2: Streptococcus faecalis

3: Streptococcus faecium

4: Streptococcus sp15: Streptococcus sp26: Streptococcus sp3

Agarose gel electrophoresis of amplified RAPD-PCR products with primers A11, O20.

M: Marker.M: Marker.1: Standard1: Standard

Streptococcus Streptococcus faecalisfaecalis

2: 2: Streptococcus Streptococcus faecalisfaecalis

3:3: Streptococcus Streptococcus faeciumfaecium

4: 4: StreptococcusStreptococcus sp1sp15: 5: StreptococcusStreptococcus sp2sp26: 6: StreptococcusStreptococcus sp3sp3

Similarity coefficient among Streptococcus species with total of primer:

Strain StandardS. faecalisS. faeciumS. sp1.S. sp2.

S. faecalis0.96    

S. faecium0.740.75   

S. sp1.0.600.610.65  

S. sp2.0.610.620.750.68 

S. sp3.0.640.670.780.640.79

Dendrogram using average linkage from total of primers data among Streptococcus species.

Similarity coefficient between Streptococcus species with total of data (primers and protein).

 StrainStandar

dS. faecalisS. faeciumS. sp1.S. sp2.

S. faecalis0.94    

S. faecium0.690.72   

S. sp1.0.630.620.63  

S. sp2.0.640.630.750.72 

S. sp3.0.680.720.730.660.76

Dendrogram using average linkage from total of data (primers and protein) among Streptococcus species.

Conclusion

It could be concluded that streptococcosis was detected in high prevalence among cultured fresh water fish in Egypt, especially during summer season. The most common signs of streptococcosis in fish was septicaemia, ulcer formation, uni- or bilateral exophthalmia, haemorrhage of the eye, in some cases change cloudy and destruct of eye (pop-eye) and haemorrhages on the skin especially in the base of fins and tail.

Using of molecular diagnostic technology in diagnosis of bacterial fish pathogens, considered as good tool due to:

1. Highly specific, quick and sensitive. 2. Give accurate diagnosis and accurate treatment. 3. Help in detection of non culturable bacteria. Results of SDS-PAGE and RAPD PCR considered

first record in Egypt and can be used for rapid diagnosis of Streptococcus species.