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STUDIES ON STREPTOCOCCAL INFECTIONS AMONG CULTURED FRESH WATER FISHES WITH SPECIAL REFERENCE TO ITS DIAGNOSIS BY POLYMERASE CHAIN REACTION TEST. Presented by Torky, H.A.; Soliman, M.K. *; El-Ashram, A.M.M. ** and El-Refaee, A. M. E **. “ Introduction ”. - PowerPoint PPT Presentation
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• 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.