ILMU KELAUTAN Desember 2016 Vol 21(4): ISSN

Transcription

1 ISSN Effects of Microencpsulted Syniotic Administrtion t Different Dosges Aginst Hevy Co-infection of White Spot Disese (WSD) Virio hrveyi in Pcific White Shrimp (Litopeneus vnnmei) Yunrty 1, Munti Yuhn 2* Widnrni 2 1 Study Progrm of Aquculture Science, Grdute School, Bogor Agriculturl University, Jl. Agtis, Drmg Cmpus, Bogor, 1668 Ionesi 2 Deprtment of Aquculture, Fculty of Fisheries Mrine Science, Bogor Agriculturl University, Jl. Agtis, Drmg Cmpus, Bogor, 1668 Ionesi E-mil : myhn@gmx.ch Astrct White spot disese (WSD) is one of infectious disese in shrimp cused y white spot syrome virus (WSSV). This study imed to determine the dosge immunologicl effects growth performnces of microencpsulted syniotic (Bcillus NP5 mnnn oligoscchride) t different dosges on Pcific white shrimp. The microencpsulted syniotic ws dministered s feed supplementtion ginst the co-infection of WSSV Virio hrveyi. Syniotic ws encpsulted y spry drying method, further feed supplemented to Pcific white shrimp for 3 dys t dosges of.5% (A), 1% (B), 2% (C) control tretments, i.e. without ny microencpsulted syniotic dministrtion s positive control (D) negtive control (E). The chllenge test ws performed on dy 3 fter feeding supplementtion, then the experimentl shrimps were injected y WSSV intrmusculrly t the infective dosge of 1 4 copies.-ml -1. Afterwrds, 24 hours fter WSSV injection the shrimps were immersed in wter contined cells suspension of V. hrveyi t the cells popultion dosge of 1 6 CFU-.ml -1. All syniotic tretments showed etter results with the vlues of Totl Hemocyte Count (THC), Phenoloxidse (PO) Respirtory Burst (RB), were higher (P<.5) compred to positive control. The specific growth rtes (SGR) of A, B C showed higher thn oth controls of D E. The feed conversion rtio (FCR) vlue of syniotic tretments were lower (P<.5) thn oth controls. However, the dministrtion of microencpsulted syniotic hve not een le to prevent hevy impct of WSSV V. hrveyi co-infection due to lower SR mortlity pttern which continued to increse. Keywords: Syniotic, Litopeneus vnnmei, WSSV, Virio hrveyi, co-infection Introduction Infectious diseses cused y cteri /or viruses is one of fctors cused the decrese production of Pcific white shrimp (Litopeneus vnnmei) (Phuoc et l., 29). One of custive gent of virl disese tht currently widely ttcks the Pcific white shrimp is white spot syrome virus (WSSV). WSSV outrek cused the shrimp mortlity up to 9-1% within 3-1 dys fter exhiiting clinicl signs (Snchez-Mrtinez et l., 27). Beside the virl infections, the cteril infections were lso commonly fou, such s Virio sp. which often fou s the custive gent of secory infection in shrimp (Liu Chen, 24). Severl studies reported tht some pthogens re not lwys ecome single custive gent in the shrimp diseses.there re lso joint infection etween pthogens in the sme host which is clled co-infection (Teixer-Lopes et.l 211). The co-infection diseses etween virus cteri hve een reported y Phuoc et l. (29); either the co-infection y WSSV with V. hrveyi or y WSSV with V. cmpellii. Therefore, the lterntive effective control mesures re needed to prevent co-infection disese y enhncing the immune system of shrimp. One of lterntive methods is y using the syniotic s feed supplementtion. Syniotic ( comintion of proiotic preiotic) pplictionhs een proven to e le in improving immune response, resistnce, growth performnce of shrimp (Li et l., 29) fish (Gerylou et l., 213). The syniotic ppliction in the form s the fresh culture cells suspension hs provided in limited cells lifetime. The technique for improving the cells life time need to e pplied is the microencpsultion method (Anl Singh, 27). Microencpsultion technology is used for coting or lining core mteril with polymeric sustnces *) Correspoing uthor Ilmu Kelutn, UNDIP ijms.uip.c.id DOI: /ik.ijms Received : Accepted :

2 slyer or coting mteril, so tht it ecomes smll prticles within microscopic sizes. The lyer of the microcpsule grnules will protect the proiotic cteril cells from the dmging effects of extreme environmentl coition (Widodo et l., 23; Weinreck et l., 21). The common method pplied for microil cells microencpsultion is y spry drying method. This method hs een reported to e le to mintin the cells viility of microencpsulted Bifidocteri cells oligofructose enriched with inulin (Freire et l., 212). This study ws crried out to evlute the effects of the comintion of proiotic Bcillus NP5 commercil preiotic of mnnn oligoscchride (MOS). Proiotic Bcillus NP5 ws isolted from the digestive trct of tilpi (Oreochromis niloticus) (Putr Widnrni, 215). Preiotic MOS ws derived from Scchromyces cerevisie cell wll produced commercilly ws n dditive preiotic for niml feed. The ddition of preiotic mnnn oligoscchride cn improve survivl rte, helth sttus immunity of loster (Cherx tenuimnus) (Sng et l., 211). In the previous studies (Zuidh et l., 215; Muneni et l., 214) hve reported tht the comintion of proiotic cells of Bcillus NP5 Rf R preiotic from sweet potto extrct produced in the form of microencpsulted syniotic, could improve the growth performnce of Pcific white shrimp chllenged y V. hrveyi. On the other h, the efficcy of microencpsulted syniotic, comintion of Bcillus NP5 MOS on Pcific white shrimp co-infected y WSSV V. hrveyi, hs not een known. Bsed on this pproch, this study ws coucted to determine the est dosge of microencpsulted syniotic dministrtion through feed on growth performnce immune responses of Pcific white shrimp co-infected y WSSV V. hrveyi. Mterils Methods Experimentl shrimps used were Pcific white shrimp post-lrve with verge weight of 5.44 ±.44 g iividu -1, otined from Shrimp Production Unit Gelung, Brckish Wter Aquculture Center Situoo, Est Jv. The shrimp were regurlrly PCR checked s specific-pthogen free (SPF) for WSSV IMNV. Proiotic used ws proiotic Bcillus NP5, which hs een mrked s ntiiotic rifmpicin resistnt t dosge of 5 μg ml -1. Preiotic used ws commercil preiotic MOS. Isolte of WSSV V. hrveyi were otined from collection of Brckish Wter Aquculture Center Situoo, Est Jv. Rering Coition Experimentl Design Pcific white shrimp were rered in tnks with size of 6 cm x 35 cm x 3 cm with density of 15 iividu per tnk. Pcific white shrimp were cclimtized for two weeks efore feeding tril. Feed used ws commercil feed with protein content of 28%. The feeding were coucted four times dy, with feeding rte (FR) ws 6%. The feed ws mixed with microencpsulted syniotic powder. The dosges were tken ccording to tretment ws coted y egg lumine s the ier. The test feed ws ir-dried for 5 to 1 minutes or cn e stored in the refrigertor prior to e used. The feeding tril ws coucted for 3 dys. The chllenge test with WSSV filtrte ws performed on dy 31, y injecting the virus filtrte, s much s 1 μl iividu -1 (1 4 copies ml -1 ; it ws mesured using rel time PCR) negtive control were injected with PBS (phosphte uffer sline) in the sme concentrtion. Artificil co-infection of V. hrveyi ws crried out 24 hours fter virus injection y immersing the shrimp in 1 6 CFU ml -1 of the V. hrveyi cells suspension (Phuoc et l., 29), except for the shrimps of negtive control, which were immersed in PBS. At feeding tril period (dy until dy 3) fter the chllenge test, there were intestinl cteril smplings to determine totl cteril count totl hemolymph count oservtion for immune prmeters oservtion. Experimentl design used ws completely romized design (CRD), with five tretments three replictions; the dministrton of microencpsulted syniotic t dosge of.5% (A), 1% (B), 2% (C) controls (without the dministrtion of microencpsulted syniotic) including positive control (D) negtive control (E). Wter qulity ws mintined in optimum coition (temperture t 29 ± C; dissolved oxygen t ±.63 mg L -1 ; TAN t ±.96 mg L -1 ; slinity t 33 ± ppt; ph t ±.239; nitrite t.129 ±.136 mg L -1 ; nitrte t ± 2.17 mg L -1 ). Siphoning ws done every dy to remove feces unconsumed feed. Syniotic Microencpsultion The composition used in the production of microencpsulted syniotic consisted of proiotic Bcillus NP5 RfR with cells density of CFU g -1 of cells wet weight (Wng, 27) preiotic MOS.4% (w/w) (Zhng et l., 212). Proiotic iomss production ws done y suculture technique in SWC-roth medium using 1% inoculnt with density of 1 8 CFU ml -1 (v/v) (Muneni et l., 214; Zuidh et l., 215). 17 Microencpsulted Syniotic Aginst Hevy Co-infection of White Spot Disese V. hrveyi (Yunrty et l.)

3 The coting mterils used on syniotic microencpsultion were dentured whey protein (DWP) mltodextrin. DWP process ws otined through enzymtic rection ccording to procedure tht hve een implemented y De Cstro-Cislghi et l. (212). Before microencpsultion process,.4% preiotic MOS (w/w)wet weight ws dissolved in sterile PBS, then dded y 1% mltodextrin (w/v). MOS mltodextrin ws heted t temperture of 8 C for 3 minutes (Freire et l., 212). Formultion of syniotic (proiotic MOS), whey protein mltodextrin ws 1:1 (v/v):1% (w/v) (Muneni et l., 214; Zuidh et l., 215). The mterils were further homogenized using stirrer plte for 3 minutes. Furthermore, the spry drying process ws performed using spry dryer (Mini unchi 19) with temperture rnging from 1 up to 11 C t the inlet C t the outlet. Prmeters Oservtion Prmeters oserved were immune response prmeters including totl hemocyte count, phenoloxidse ctivity (Liu Chen, 24) respirtory urst ctivity (Singh et l., 213), totl cteril count in the intestine, survivl rte (SR) (Sng et l., 211), growth performnce prmeters including specific growth rte (SGR) feed conversion rtio (FCR) (Akrmi et l., 212). Sttisticl Anlysis Dt were tested y nlysis of vrince (ANOVA), if there were significntly differences then continued with Duncn test using SPSS version 16. Result Discussion Totl hemocyte count (THC) t the e of tretment or 3 dys fter the feeding tril showed tht ll syniotic tretments (A, B, C) were significntly different (P<.5) from positive control (D) negtive control (E). The highest THC ws fou in B (1%). Incresing of THC t the e of tretment ws cused y dministrtion of microencpsulted syniotic. The ddition of syniotic to the feed will increse totl hemocyte survivl of Pcific white shrimp (Prtid- Arngure et l., 212). Hemocyte will recognize the foreign prticles which get into the shrimp ody. Hemocyte cells will respo through severl mechnisms such s intrcellulr signling cscdes, phgocytosis, encpsultion nodulr ggregtion (Rodriguez Le Muollc, 2). After the chllenge test, THC decresed except in negtive control tretment (Figure 1). This ws due to the functioning of shrimp immune system. The reduction of hemocytes is the effect of immune system work Tle 1. Totl vile cteril count (TBC), presumptive Virio count (PVC), totl Bcillus NP5 Rf R totl V.hrveyi Rf R Bcteril popultion TBC PVC Totl Bcillus NP5 Rf R Totl V.hrveyi Rf R Tretments A B C D E A B C D E A B C D E A B C D E Before feeding tril (Log CFU g -1 ) After feeding tril (Log CFU g -1 ) 4.77 ± ± ± ± ± ± ± ± ± ± ± ± ±.28 After the chllenge test (Log CFU g -1 ) 5.31 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±.16 Nd Note: different superscript letters in the sme column row iicte significnt different results huruf (P<.5), ; not determined. Microencpsulted Syniotic Aginst Hevy Co-infection of White Spot Disese V. hrveyi (Yunrty et l.) 171

4 due to hemocyte infiltrtion in infected tissues (Cost et l., 29). Phenoloxidse (PO) ctivity (Figure 2) of ll syniotic tretments were significntly different (P<.5) from controls. The highest PO ctivity ws fou in B, followed y C A. At the e of tretment or fter 3 dys of feeding tril, there ws incresing of PO ctivity tht ws suspected due to the ddition of microencpsulted syniotic, in which syniotic is comintion of proiotic preiotic which showed improvement of PO ctivity (Chiu et l., 27). Reduction of PO ctivity occurred fter the chllenge test, except in negtive control. PO ctivity THC were correlted to ech other. The more hemocyte count, the more ProPO production, vice vers (Smith et l., 23). RB ctivity (Figure 3) of ll syniotic tretments were significntly different (P<.5) from controls, with the highest RB ctivity ws fou in B, followed y C A tretments. Incresing of RB ctivity t the e of tretment iicted tht dministrtion of syniotic t different dosges to Pcific white shrimp could oost the shrimp immune system. Li et l. (29) reported tht feed supplementtion with Bcillus OJ gve significnt influence on the immune response disese resistnce. Reduction of RB ctivity fter the chllenge test, llegedly ssocited with the declining of THC fter chllenge test. RB ctivity occurs in hemocyte cells which perform elimintion ctivity of foreign prticles within phgocytosis process (Rodriguez Le Muollc, 2). The totl 1 Totl Hemocyte Count (x1 6 sel.m -1 ) Tretment Figure 1. Totl hemocyte count (THC) of Pcific white shrimp. Administrtion of microencpsulted syniotic.5% (A), 1% (B), 2% (C), without dministrtion of microencpsulted syniotic s positive control (D) negtive control (E). Different superscript letters on the sme oservtion period iicte significnt different results (P<.5). Note : = efore feeding tril, = After feeding tril, = After the chllenge test.8 Phenoloxidse ctivity (OD 492 nm) Tretment Figure 2. Phenoloxidse (PO) ctivity of Pcific white shrimp. Administrtion of microencpsulted syniotic.5% (A), 1% (B), 2% (C), without dministrtion of microencpsulted syniotic s positive control (D) negtive control (E). Different superscript letters on the sme oservtion period iicte significnt different results (P<.5). Note : = efore feeding tril, = After feeding tril, = After the chllenge test 172 Microencpsulted Syniotic Aginst Hevy Co-infection of White Spot Disese V. hrveyi (Yunrty et l.)

5 Respirtory Brust ctivity (OD 63 nm) Tretment Figure 3. Respirtory urst (RB) ctivity of Pcific white shrimp. Administrtion of microencpsulted syniotic.5% (A), 1% (B), 2% (C), without dministrtion of microencpsulted syniotic s positive control (D) negtive control (E). Different superscript letters on the sme oservtion period iicte significnt different results (P<.5). Note : = efore feeding tril, = After feeding tril, = After the chllenge test Specific growth rte (%) Tretment Figure 4. Specific growth rte (SGR) of Pcific white shrimp. Administrtion of microencpsulted syniotic.5% (A), 1% (B), 2% (C), without dministrtion of microencpsulted syniotic s positive control (D) negtive control (E). Different superscript letters on ech r iicte significnt different results (p<.5). Feed Conversion rtion Tretment Figure 5. Feed conversion rtio (FCR) of Pcific white shrimp. Administrtion of microencpsulted syniotic.5% (A), 1% (B), 2% (C), without dministrtion of microencpsulted syniotic s positive control (D) negtive control (E). Different superscript letters on ech r iicte significnt different results (p<.5). c cteril cells count (TBC) t the e of microencpsulted syniotic tretment showed decline of TBC in ll microencpsulted syniotic tretments tht were significntly different (P<.5) compred to D (positive control) E (negtive control). The lowest TBC ws fou in B the highest ws fou in E; it is likely due to the role of the present of preiotic mterils in the shrimp s digestive trct. According to Ringo et l. (21) the preiotic oligoscchrides showed its ility to Microencpsulted Syniotic Aginst Hevy Co-infection of White Spot Disese V. hrveyi (Yunrty et l.) 173

6 1 c Survivl Rte (%) 5 Tretment Figure 6. Survivl rte of Pcific white shrimp. Administrtion of microencpsulted syniotic.5% (A), 1% (B), 2% (C), without dministrtion of microencpsulted syniotic s positive control (D) negtive control (E). Different superscript letters on the sme oservtion period iicte significnt different results (p<.5). Note: : = After feeding tril, = After the chllenge test improve the helth sttus suppresed the presence of other uneneficil intestinl cteril cells inthe host s intestinl system. The presumptive Virio cells count (PVC) decresed fterthe shrimps supplemented y syniotic. The PVC counts of A B tretments were significntly lower; different (P<.5) from C, D E tretments. The lowest PVC vlue ws fou in A the highest PVC vlue ws fou in E. This effect suspected due to the role of competitiveness of proiotic cells, ccording to Cerezuel et l. (211). Proiotic cells provided some enefits y producing nticteril molecules in the form of cteriocin which cn directly inhiit other uneneficil cteril cells or viruses ctively prticipte ginst the infection. The high Bcillus NP5 Rf R cells popultion in B (dosge 1%) iicted tht Bcillus NP5 Rf R could survive in the intestinl trcts reched the trget orgns. It lso showed tht proiotic cells hveresulted eneficil effects on shrimp, ecuse hiering the movement of other cteril cells in the intestinl wll (trnsloction), improving the function of mucosl rrier chrcterized y incresing of non-specific immune responses production or in the modulting the inflmmtion (Cerezuel et l., 211). Moreover, preiotic is non-digested food which is eneficil to the host y selectively stimulting the growth of certin cteri in the intestine (Mhious et l., 26). The totl popultion of V. hrveyi Rf R cells fter the chllenge test incresed, long with the incresing of PVC. Bcteril cells which entered through wter food will influence the intestinl microflor, so tht they result in the fluctution of the intestinl cteril cells in qutic nimls s their micro environments (Austin, 26). The microil popultion fluctution my cused incresing of V. hrveyi Rf R cells popultion fter the chllenge test, especilly in the chllenge test process tht ws done y immersion. The incresing of TBC, PVC V. hrveyi Rf R vlues fter the chllenge test llegedly cused y the dministrtion of microencpsulted syniotic, ccording to Chiu et l. (27) who stted tht the ddition of proiotic cteril cells fter certin period in shrimp incresed cteril popultion in the intestinl trct. The specific growth rte (SGR) the feed conversion rtio (FCR) of shrimp fter the feeding tril re presented in figure 4. In this study, it ppered tht SGR of A, B C tretments showed the etter results compred to those of controls, the lowest FCR ws fou in B (1%). This ws cused y the pressence of proiotic cells preiotic in the shrimp intestinl trct. According to Dniels et l. (21) who used syniotic contining Bcillus spp. cells mnnn oligoscchrides hs lso resulted eneficil performnces; in which it could improve growth, feed conversion, immune response disese resistnce on loster Homrus gmmrus L. In ddition, Zhng et l. (212) stted tht the ddition of.4% MOS could improve growth performnce; incresing the weight (WG) specific growth rte (SGR); i.e y the vlues of 38% y 2.51%, respectivelly. The shrimps chllenged y WSSV through injection V. hrveyi through immersion showed the survivl rte tht continued to decline. According to Phuoc et l. (29) tht co-infection of WSSV V. hrveyi cused mortlity up to 8% this pttern tht continued to rise in their experiment. The co-infected shrimps showed the mortlity pttern susceptiility to disese which continue to rise further wekening the shrimp coitions due to WSSV infection. Furthermore, the wekened 174 Microencpsulted Syniotic Aginst Hevy Co-infection of White Spot Disese V. hrveyi (Yunrty et l.)

7 shrimps would e susceptile to secory infection cused y pthogenic Virio cells which is commonly fou in the mrine environment (Selvin Lipton, 23). Conclusions Administrtion microencpsulted syniotic to shrimp t dosges of.5%, 1% 2% hve showed improvement in the immune system prmeters growth performnces of shrimp fter feeding tril. Survivl rte (SR) vlues of A B tretments were higher thn positive control. However, the dministrtion of microencpsulted syniotic hs not een le to prevent disese cused y hevy co-infection of WSSV V.hrveyi, which ws shown y its low survivl rte (SR) mortlity pttern which continued to rise. References Akrmi, R., Rzeghi-Mnsour, M., Chitsz, H., Ziee, R, & Ahmdi, R., 212. Effect of Dietry Mnnn-oligoscchride (MOS) on Growth Performnce, Survivl, Body Composition Some Hemtologicl Prmeters of Crp Juvenile (cyprinus crpio). J. Animl Sci Advnces. 2: doi : 1.17/s Anl, A.K & Singh, H. 27. Recent Advnces in Microencpsultion of Proiotics for Iustril Applictions Trgeted Delivery. Tres in Food Sci.Technol. 18: doi : /j.tifs Austin, B. 26. The Bcteril Microflor of Fish. Sci. World J.. 6: doi : 1.11/tsw Cerezuel, R., Meseguer, J. & Esten, M.A Current Knowledge in Syniotic use for Fish Aquculture: A Review. J. Aquculture Res. Dev. 8:1-7. doi: / S1-8 Chiu, C.H., Guu, Y.K., Liu, C.H., Pn, T.M. & Cheng, W. 27. Immune Responses Gene Expression in White Shrimp, Litopeneus vnnmei, Iuced y Lctocillus plntrum. Fish Shellfish Immunol.. 23: doi : 1.116/j.fsi Cost, A.M., Buglione, C.C., Bezerr, F.L., Mrtins, P.C.C. & Brrcco, M.A. 29. Immune Assessement of Frm-Rered Peneus vnnmei Shrimp Nturlly Infected y IMNV in NE Brzil. Aquculture. 291: doi: 1.116/j.quculture Dniels, C.L., Merrifield, D.L., Boothoryd, D.P., Dvies, S.J., Fctor, J.R. & Arnold, K.E. 21. Effect of Dietry Bcillus spp. Mnnn- Oligoscchrides (MOS) on Europen Loster (Homrus gmmrus l) Lrve Growth Performnce, Gut Morphology Gut Microiot. Aquculture 34: doi : 1.116/j.quculture De Cstro-Cislghi, F.P., Silv, C.D.R.E., Fritzen- Freire, C.B., Lourenz, C.G. & Snt Ann, E.S Bifidocterium -12 Microencpsulted y Spry Drying with Whey: Survivl Uer Simulted Gstrointestinl Coitions, Tolernce to Ncl, Viility During Storge. J. Food Engineering. 113: doi : 1.116/j.jfoodeng Freire, C.B.F., Prudencio, S.E., Amoni, R.D.M.C., Pinto, S.S., Murkmi, A.N.N. & Murkmi, F.S Microencpsultion of Bifidocteri y Spry Drying in the Presence of Preiotics. Food Res. Int.. 45: doi : 1.16/j. foodress Gerylou, Z., Souffreu, C., Rurngw, E., Meester, L.D., Courtin, C.M., Delcour, J.A., Buyse, J. & Ollevier, F Effects of Dietry Arinoxyln-Oligoscchrides (AXOS) Eogenous Proiotics on the Growth Performnce, Non-Specific Immunity Gut Microiot of Juvenile Sierin Sturgeon (Acipenser erii). Fish Shellfish Immunol.. 35: doi : 1.16/j.fsi Li, J., Tn, B. & Mi, K. 29. Dietry Proiotic Bcillus OJ Somltooligoscchrides Influence the Intestine Microil Popultions, Immune Responses Resistnce to White Spot Syrome Virus in Shrimp (Litopeneus vnnmei). Aquculture 291: doi: 1. 16/j.quculture Liu, C.H & Chen, C.J. 24. Effect of Ammoni on the Immune Response of White Shrimp Litopeneus Vnnmei its Susceptiility to Virio lginolyticus. Fish Shellfish Immunology 16: doi: S (3)113-X. Mhious, A.S., Gtesoupe., Hervi, M., Metiller, R. & Ollevier. 26. Effect of Dietry Inulin Oligoscchrides s Preiotics for Wening Turot, Psett mxim (Linneu, C. 1758). Aquculture Int. 14: doi: 1.17 /s Muneni, W., Yuhn, M. & Widnrni Effect of Micro-Encpsulted Syniotic t Different Microencpsulted Syniotic Aginst Hevy Co-infection of White Spot Disese V. hrveyi (Yunrty et l.) 175

8 Frequencies for Luminous Viriosis Control in White Shrimp (Litopeneus vnmei). Microiol. Ionesi 8: doi: /mi Prtid-Arngure, B.O., Lun-González, A., Fierro- Corondo, J.A., Flores-Mir, M.C. & González-Ocmpo, H.A Effect of Inulin Proiotic Bcteri on Growth, Survivl, Immune Response, Prevlence of White Spot Syrome Virus (WSSV) in Litopeneus vnnmei Cultured Uer Lortory Coitions. Africn J. Biotechnol. 12 : doi: /AJB Phuoc L.H., Corteel, M., Thnh, N.C., Nuwynck, H., Pensert, M., Aldy-Snz, V., den Broeck, W.Y., Sorgeloos, P. & Bossier, P. 29. Effect of Dosge Chllenge Routes of Virio spp. on Co-Infection with White Spot Syrome Virus in Peneus vnnmei. Aquculture 29: doi: 1.16/j.quculture Putr, A.N & Widnrni Screening of Amylolitic Bcteri s Cidte of Proiotics in Tilpi (Oreochromis sp). Res. J. Microiol.. 1:1 13. doi: /jm Ringo, E., Olsen, R.E., Gifstd, T.O., Dlmo, R.A., Amlu, H. & Hemre, G.I. 21. Preiotics in Aquculture: A Review. Aquculture Nutrition. 16: doi : /j x Rodriguez L. & Le Moullc, G. 2. Stte of the Art Immunologicl Tools Helth Control of Peneid Shrimp. Aquculture 191: doi: S ()421-X. Snchez-Mrtinez, J.G., Guzmn, G.A. & Ruiz, H.M. 27. White Spot Syrome in Cultured Shrimp : A Review. Aquculture Res. 38: doi: /j x Sng, F.M., Fotedr, R. & Filer, K Effect of Dietry Mnnn-Oligoscchride on The Survivl, Growth, Immunity Digestive Enzyme Activity of Freshwter Cryfish, Cherx destructor Clrk (1936). Aquculture Nutrition 17: doi: /j x. Selvin, J. & Lipton, A.P. 23. Virio lginolyticus Associted with White Spot Disese of Peneus monodon. Disese of Aqutic Orgnisms. 57: doi: Singh S.K., Tiwri, V.K., Chdh, N.K., Prksh, C., Sukhm, M., Ds, P., Ml, S.C. & Chnu, T.I Effect of Bcillus circulns Fructooligoscchride Supplementtion on Growth Hemto-Immunologicl Function of Leo rohit (Hmilton, 1822) Fingerlings Exposed To Su-Lethl Nitrite Stress. The Isreli J. Aquculture Bmidgeh. 64:1-11. Smith, V.J., Brown, J.H. & Huton, C. 23. Immunostimultion in Crustcen: does it Relly Aginst Infection. Fish Shellfish Immunol. 15: doi: 1.116/S (2)14-7. Teixeir-Lopes, M.A., Vieir-Giro, P.R.N., Freire, J.E.C., Roch, I.R.C.B., Cost, F.H.F. & Rdis- Bptist, G Nturl Co-Infectious Hypoderml Hemtopietic Necrosis Virus (IHHNV) Infectious Mynecrosis Virus (IMNV) in Litopeneus vnnmei in Brzil. Aquculture 312: doi: 1.116/j.quculture Wng, Y.B. 27. Effect of Proiotics on Growth Performnce Digestive Enzyme Activity of The Shrimp Peneus vnnmei. Aquculture. 269: Doi : 1.116/j.quculture Weinreck, F., Bodnr, I. & Mrco, M.L. 21. Cn Encpsultion Lengthen the Shelf-Life of Proiotic Bcteri in Dry Products. Int. J. Food Microiol. 136: doi: 1.116/j.ijfood micro Widodo, Soeprno & Whyuni, E. 23. Bioenkpsulsi Proiotik (Lctocillus csei) dengn Pollrd dn Tepung Terigu sert Pengruhny terhdp Viilits dn Lju Pengsmn [Bioencpsultion of Preiotics (Lctocillus csei) with Pollrd Whet Flour Its Roles for the Acidifiction Rte]. 14: Zhng, J., Yongjin, L.A., Lixi, T., Huijun, Y., Guiying, L. & Donghui, X Effects of Dietry Mnnn-Oligoscchride on Growth Performnce, Gut Morphology Stress Tolernce of Juvenile Pcific White Shrimp, Litopeneus vnnmei. Fish Shellfish Immunol. 33: doi:1.116/j.fsi Zuidh, A., Yuhn, M. & Widnrni Encpsulted Syniotic Dietry Supplementtion t Different Dosges to Prevent Viriosis in White Shrimp, Litopeneus vnnmei. Hyti Journl of Biosciences. 22: doi : 1.116/j.hj Microencpsulted Syniotic Aginst Hevy Co-infection of White Spot Disese V. hrveyi (Yunrty et l.)