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

2. “ Introduction ” 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 economy. 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, 1999 and Klesius et al., 2000). Bacterial pathogens are the most serious disease problem in tilapia production causing 80% of fish mortalities (Plumb, 1999; Woo and Bruno, 1999; Clark et al., 2000 and Shoemaker et al., 2000). Historically, Streptococcus sp. are not serious pathogens of fish, but recently, these bacteria have become more prominent in wild and cultured fish (Baya et al., 1990). Now 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 infections and the high economic losses caused by gram-positive bacteria in both wild and cultured fish (Eldar et al., 1995 and Domenech et al., 1996). Moreover, Weinstein et al., (1996) and Zlotkin et al., (2003) recorded that Streptococcus iniae was capable of causing disease in humans who had recently handled infected fish from aquaculture farms. In Egypt, Nile tilapia (Oreochromis niloticus) population facing streptococcosis in several areas, notably in Lake El-Temsah in Ismailia governorate (Badran, 1994) recorded a serious mass mortality among fish due to streptococcal infection, in Kafr El-Sheikh governorate (Khalil, 2000) who recorded massive mortalities from streptococcosis and (Ebtesam, 2002) recorded streptococcosis in Lake El-Ibrahimia in Upper Egypt.

3. Saçilik et al., (2000) analyzed different gram positive cocci by SDS-PAGE in order to obtain better discrimination between different gram positive cocci and found a common 18 and 35 kD characteristics bands of Streptococcus spp. and Enterococcus faecalis. Also Barnes et al., (2003) used it for characterization of Streptococcus iniae. Saçilik et al., (2000) analyzed different gram positive cocci by SDS-PAGE in order to obtain better discrimination between different gram positive cocci and found a common 18 and 35 kD characteristics bands of Streptococcus spp. and Enterococcus faecalis. Also Barnes et al., (2003) used it for characterization of Streptococcus iniae. Enlightenment the previous argument the present study was conducted to fulfill the gap about Streptococcus species infection among cultured freshwater fish in Egypt. Also, availability of use of total bacterial proteins in SDS-PAGE for characterization of the isolated streptococcus. In addition to using of polymerase chain reaction (PCR), as a rapid and sensitive method for identification of streptococcus with potential diagnostic value.

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

6. Prevalence of Streptococcosis among the examined fish. % of the diseased fish from the same species Number of clinically diseased fish Number of examined fish Fish species 17.2103600Oreochromis niloticus 27.5165600Clarias gariepinus

7. Seasonal prevalence of Streptococcosis among Nile tilapia (O. niloticus) niloticus) and African catfish (C. (C. gariepinus). C. gariepinusO. niloticusSeason % from examined fish/season No. of infected fish/season No. of examined fish/season % from examined fish/season No. of infected fish/season No. of examined fish/season 3045150 1827 150Spring 5481150 31.3347 150Summer 16.6725150 13.3320 150Autumn 9.3314150 69 Winter

8. Seasonal prevalence of Streptococcosis among Nile tilapia (O. niloticus).

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

10. Prevalence of Streptococcosis among O. niloticus niloticus and African catfish (C. gariepinus) gariepinus) in relation to locality and the total examined fish. Catfish (C. gariepinus)Tilapia (O. niloticus)Locality *% of infected No. of infected No. of examined *% of infected No. of infected No. of examined 23382003435200Domiata 35.85920026.227200Sharkia 41.86820039.841200Kafr El-Sheikh 100165600100103600Total

11. Clinical signs

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

28. O. niloticus niloticus naturally infected with Streptococcus spp. spp. showing enlarged spleen.

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

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

31. Number of dead Monosex tilapiaNumber of dead O. niloticus No. of injected Dose days Control2X10 8 2X10 7 5X10 6 5X10 5 5X10 4 Control5X10 8 5X10 7 5X10 6 5X10 5 5X10 4 1-----------101 -2---------- 2 -421---22--- 3 -23121-3211- 4 -223---4321- 5 -12-31-11221 6 ---1-1---112 7 --1-1---1-1- 8 ---1----11-- 9 ---------1-- ------------ 11 ---1-------1 10 12 ------------ 10 13 ------------ 10 14 ------------ 10 15 10 %100% 80%60%30%0%100% 80%50%40% % of dead Experimental infection of fish with different concentrations of Streptococcus faecalis:

32. Number of dead Monosex tilapiaNumber of dead O. niloticus No. of injected Dose days Control5X10 8 5X10 7 5X10 6 5X10 5 5X10 4 Control5X10 8 5X10 7 5X10 6 5X10 5 5X10 4 ------------101 ------------ 2 -2---------- 3 -31111-312-- 4 -221---42-1- 5 -23-1--2411- 6 --12---111-- 7 -1-1----1-1- 8 ---1---1---- 9 --1----111-- --1-----1--- 11 ---------1-1 10 12 ---1-------- 10 13 ------------ 10 14 ------------ 10 15 0 %100%90%70%20%10%0%100% 60%30%10% % of dead Experimental injection of fish with different concentrations of Streptococcus faecium:

33. O. niloticus niloticus injected with Streptococcus faecalis faecalis showing slight exophthalmia.

34. O. niloticus niloticus injected with Streptococcus faecalis faecalis showing congestion of the internal organs.

35. Sensitivity testes for isolated Streptococcus spp. RRRRR Optochin SSSSS Nalidexic acid SSSSS Vancomycin SSSRS Trimethoprim +Sulfamethoxazole SRSSI Streptomycin SRSSS Penicillin SRSSR Tetracycline RRSIR Kanamycin SSSSR Erythromycin SISSR Ciprofloxacin SSRSS Chloramephenicol SRIRR Ampicillin SRIIS Amoxicillin S. sp 3.S. sp 2.S. sp 1.S. faeciumS. faecalis Antimicrobial agent

36. M: Marker. 1: Standard Streptococcus faecalis 2: Streptococcus faecalis 3: Streptococcus faecium 4: Streptococcus sp1 5: Streptococcus sp2 6: Streptococcus sp3 SDS-PAGE of bacterial cell protein.

37. 9 35.21 18.21 15 23 118.4122S. sp 3 9 35.21 18.21 15 23 84,7918S. sp 2 9 35.21 18.21 15 23 84,7918S. sp 1 9 35.21 18.21 15 23 96.0214S. faecium 9 35.21 18.21 15 23 118.4122S. faecalis 9 35.21 18.21 15 23 118.4122Standard Number of common bands Molecular weight of diagnostic bands/KD Number of diagnostic bands Highest molecular weight/KD Total number of bands Species The number of protein bands and highest molecular weight, number of diagnostic bands and its molecular weight:

38. 2218 1422 2424.00 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 protein

39. S. sp 2.S. sp 1.S. faeciumS. faecalisStandard Strain 0.83S. faecalis 0.580.50S. faecium 0.580.670.75S. sp 1. 0.830.750.670.75S. sp 2. 0.670.750.580.920.83S. sp 3. Similarity coefficient among species by protein electrophoresis Dendrogram among species by protein electrophoresis.

40. The polymorphic bands, shared and total bands in related to the type of primer used for Streptococcus identification: Total No. of bands.No. of shared bands No. of polymorphic bands Primer code No.Serial number 918 OP.A- 02 1 505 OP.A- 09 2 707 OP.A- 10 3 514 OP.Z-01 4 606 OP.B-02 5 633 OP.C-01 6 716 OP.C-04 7 734 OP.C-06 8 716 OP.A- 11 9 615 OP.O-20 10 65115410Total

41. M: Marker. 1: Standard Streptococcus faecalis 2: Streptococcus faecalis 3: Streptococcus faecium 4: Streptococcus sp1 5: Streptococcus sp2 6: Streptococcus sp3

42. Agarose gel electrophoresis of amplified RAPD-PCR products with primers Z1, B2. M: Marker. 1: Standard Streptococcus faecalis 2: Streptococcus faecalis 3: Streptococcus faecium 4: Streptococcus sp1 5: Streptococcus sp2 6: Streptococcus sp3

43. 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 sp1 5: Streptococcus sp2 6: Streptococcus sp3

44. Agarose gel electrophoresis of amplified RAPD-PCR products with primers A11, O20. M: Marker. 1: Standard Streptococcus faecalis 2: Streptococcus faecalis 3: Streptococcus faecium 4: Streptococcus sp1 5: Streptococcus sp2 6: Streptococcus sp3

45. Similarity coefficient among Streptococcus species with total of primer: S. sp 2.S. sp 1.S. faeciumS. faecalisStandard Strain 0.96S. faecalis 0.750.74S. faecium 0.650.610.60S. sp 1. 0.680.750.620.61S. sp 2. 0.790.640.780.670.64S. sp 3. Dendrogram using average linkage from total of primers data among Streptococcus species.

46. Similarity coefficient between Streptococcus species with total of data (primers and protein). S. sp 2.S. sp 1.S. faeciumS. faecalisStandard Strain 0.94S. faecalis 0.720.69S. faecium 0.630.620.63S. sp 1. 0.720.750.630.64S. sp 2. 0.760.660.730.720.68S. sp 3. Dendrogram using average linkage from total of data (primers and protein) among Streptococcus species.

47. 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.

48. 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.