Antagonistic activity of cellular components of Pseudomonas species against Aeromonas hydrophila

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1 Aquultur 253 (2006) Antgonisti tivity of llulr omponnts of Psuomons spis ginst Aromons hyrophil Bsnt Kumr DsT, Sury Knt Sml, Bisw Rnjn Smntry, Stynryn Sthi, Phlguni Pttnik, Bihunu Kumr Mishr Aquti Animl Hlth Division, Cntrl Institut of Frshwtr Aquultur (CIFA), P. O. Kuslygng, Bhunswr , Oriss, Ini Riv 5 Jnury 2004; riv in rvis form 15 Jnury 2005; pt 31 Jnury 2005 Astrt Antgonisti ffts of Psuomons fluorsns, P. ruginos n P. puti wr stui ginst 12 strins of Aromons hyrophil (Ah1 Ah12). Four iffrnt frtions of llulr omponnt (i.. whol ll prout, ht kill whol ll prout, intr llulr prout n xtr llulr prout) of ll Psuomons spis wr qully fftiv in ruing growth of A. hyrophil strins, s msur y th zon of inhiition in n in vitro snsitivity tst n hv potntil tion ginst A. hyrophil inftion in fishs. D 2005 Elsvir B.V. All rights rsrv. Kywors: Bioontrol; Psuomons spp.; Aromons hyrophil 1. Introution Control of miroil ommunitis with high spis ivrsity in ntur hs n rgr s iffiult (M, 1994). Suh typs of miroil ommunitis n isprs th fft us y th invrsion or ition of rtin xtrinsi pthogni orgnisms. In ition, miroorgnisms n sours of vrity of iotiv nturl prouts of si T Corrsponing uthor. Tl.: * 228/235 (O); (R); fx: E-mil rss: snts@yhoo.om (B.K. Ds). rsrh n ommril intrst tht hv inhiitory fft on miroil growth (Rihlt n Borowitsk, 1984; Cnnll t l., 1988). In quultur prtis tril inftious isss rsult in losss worlwi n to prvnt thm ntimiroil ompouns r us intnsivly. In th pross of ntiioti trtmnt, rug rsistn pttrns n vlop within th pthogni miroil ommunity. Thrfor, ltrntivs to th us of ntiiotis r gining importn in mny ountris (Nogmi n M, 1992; Sugit t l., 1998; Gtsoup, 1999; Bl Ry, 2001). Aoringly, th pplition of miroil ommunitis in quultur for ontrolling pthogni tri shows promis /$ - s front mttr D 2005 Elsvir B.V. All rights rsrv. oi: /j.quultur

2 18 B.K. Ds t l. / Aquultur 253 (2006) Most of th ntiiotis us toy r of miroil origin. In miin, prolm with pthogni miros oming inrsingly rsistnt ginst most ommonly us ntiiotis is ing xprin (Cown, 2001; Lipsith, 2001). Thn som rivtivs of th ntiiotis irumvnt or vn prvnt rpi muttion to rsistn of th pthogns (Hughs, 2003). Th Psuomons spis s fungii to prott th foo from toxi fungi suh s pniillium n otrytis. Th protin pttrns of th iffrnt strins of Stphylous urus n Ltoillus snfrnisnsis r rsistnt to spifi ntiiotis (D Anglis t l., 2001; Corwll t l., 2002; Hkr t l., 2003). A similr pproh ws lso unrtkn in th ultur of Phyllosom lrv of spiny lostr Jsus wrsii (Igrshi t l., 1990). It hs n ror tht ntimiroil sustns prou y som tri sm to ply n importnt rol in ntgonising othr tri in quti osystms (Dopzo t l., 1988; Sughr t l., 1988). It hs lso n rport tht intstinl tri from turot show n inhiitory fft on fish pthogni tri (Wstrhl t l., 1991; Olsson t l., 1992; Sugit t l., 1997). As ths tri r nturl inhitnts of th quti systm, thy oul prsist for rltivly longr prio n might inhiit stlishmnt of fish pthogns in th quti nvironmnt s wll s in th fish host. Thrfor, this prliminry in vitro stuy ws unrtkn to xmin th ntitril proprtis of Psuomons fluorsns, P. ruginos n P. puti ginst th fish pthogni tri Aromons hyrophil for th first tim. 2. Mtrils n mthos 2.1. Isoltion of tri Diss fishs showing vrious lsions on th skin n in th musultur, n hlthy fishs wr us for isoltion of tri. Psuomons isoltion gr (Hi mi, Ini) ws us for isoltion of pur ulturs of Psuomons spis. P. fluorsns (PF1) ws isolt from th livr of golfish (Crssius urtus), P. ruginos (PA1) ws isolt from th intstin of liming prh (Ans tstuinus) n P. puti (PP1) ws isolt from skin lsions of rohu (Lo rohit). Th 12 strins of A. hyrophil isolt from skin lsion, livr, kiny n intstin of mrigl, t fish, golfish n murrls wr tkn to stuy th ntgonisti tivity ginst iffrnt llulr omponnts of Psuomons spis. Th iffrnt spis of Psuomons n A. hyroplhil strins wr slt n hrtriz s on thir iohmil proprtis (Holt n Krig, 1984). Dominnt isolts wr purifi n intifi using onvntionl iohmil tsts sri y M Fn (1976) n Wst n Colwll (1984). A. hyrophil (Ah1 Ah12) strins isolt from iffrnt orgns suh s livr n kiny, s wll s sws of skin lsions of vrious frshwtr fishs mintin in th lortory wr lso us for th prsnt stuy. A. hyrophil ftr 24 h of inution in nutrint roth ws strk on Rimlr-Shott s mium (Hi mi, Ini) for purity. Twlv strins of A. hyrophil wr us for th ntgonisti stuy ginst ht kill whol ll prout (HK WCP), whol ll prout (WCP), intr llulr prout (ICP) n xtr llulr prout (ECP) of th Psuomons spis Prprtion of iffrnt llulr omponnts Four typs of ntigni omponnts,.g. ht kill whol ll prout (HK WCP), whol ll prout (WCP), intr llulr prout (ICP) n xtr llulr prout (ECP) wr prpr from P. fluorsns, P. ruginos n P. puti. Brifly pur ulturs of ll th tri (PF1, PA1 n PP1) wr grown sprtly in stril onitions in 250 ml of rin hrt infusion roth (Hi mi, Ini) t 30 8C for 24 h. Pur ulturs of h trium wr ivi into fiv qul volums of 50 ml n h 50 ml ws tkn for prprtion of HK WCP, WCP, ECP n ICP sprtly. Th optil nsity (OD) of 24 h ol ulturs ws tkn for h trium n simultnous plting ws rri out in triplit n th olony forming unit/ml ws lult. Th optil nsity t 546 nm of P. fluorsns ws whih orrspon to fu/ml, whrs n OD of P. ruginos orrspon to fu/ml n n OD of P. puti, orrspon to fu/ml rsptivly. Th protin stimtion of ll th protin frtion of Psuomons spis ws oring to Brfor (1976). Th minimum mount of protin ontnt (70 Al) of th lowst protin

3 B.K. Ds t l. / Aquultur 253 (2006) onntrtion of Psuomons spis ws tkn s stnr n oringly th othr protin frtions of othr Psuomons spis wr lult n hrg in nutrint gr plts Whol ll prout (WCP) All th tri (P. fluorsns, P. ruginos n P. puti) grown sprtly in rin hrt infusion roth wr hrvst ftr 24 h inution n ntrifug t 10,000 g for 10 min t 4 8C. Th tril pllt ws wsh twi n rsuspn in phospht uffr slin (PBS) (ph 7.2) n us for th ntgonisti stuy Extr llulr prout (ECP) Th suprntnts otin ftr ntrifuging 24 h ol ulturs of tri in rin hrt infusion roth wr filtr (0.22 Al). Thy wr furthr onntrt with 20% PEG 6000, ilys ginst PBS (ph 7.2) n us s ECP Ht kill whol ll prout Psuomons ulturs grown in ulk wr hrvst y ntrifugtion t 10,000g for 10 min t 4 8C. Th tril pllt ws wsh twi n rsuspn in phospht uffr slin (ph 7.2). Thy wr ht kill t 60 8C for 1 h in wtr th n finlly stor t 20 8C Intr llulr prout (ICP) All th tri (P. fluorsns, P. ruginos n P. puti) wr grown sprtly in rin hrt infusion roth n ntrifug t 10,000g for 10 min t 4 8C. Th tril pllt ws wsh twi n r-suspn in phospht uffr slin (ph 7.2), to 2% of th initil volum. Th ll pllts wr thn sonit t 50 Hz for 5 min (Artlk Soni Dismmrtor mol 150) filtr through syring with 0.45 Al filtr n finlly stor t 20 8C until furthr us Bioontrol stuy Ovrnight growth ulturs of iffrnt strins of A. hyrophil wr inut on Trypton Soy gr (Hi mi, Ini) plts sprtly y th lwn ultur mtho. Thn 6 mm imtr wlls wr m in h plt with th hlp of wll puntur (Hi mi, Ini) n 70 Al of whol ll prout (WCP) orrsponing to fu/ml, xtrllulr prout (ECP) orrsponing to fu/ ml, ht kill WCP orrsponing to fu/ ml n intr llulr prout (ICP) orrsponing to fu/ml wr hrg in th rsptiv wlls of iffrnt A. hyrophil plts n inut t 37 8C for 24 h. Th zon of inhiition roun th hrg wll ws ror ftr inution. Simultnously, ontrol smpls wr mintin with stril phospht uffr slin (ph 7.2) pl into th rsptiv wlls prpr s mntion ov. In ll th ss, th triplit plts wr mintin long with th PBS ontrol for ioontrol stuy. Slt ntiiotis, Chlormphniol (30 mg), nliixi i (30 mg), furzolion (50 mg), norfloxin (30 mg), oxyttrylin (30 mg), iprofloxin (15 mg), gntmyin (15 mg) n nitrofurntoin (10 mg) wr tst in ignosti snsitivity mium (Hi mi, Ini) ginst iffrnt A. hyrophil strins to ompr th fftivnss of iffrnt llulr omponnts of Psuomons spis. A rquir mount of ignosti snsitivity mium (DST) (Hi mi, Ini) ws prpr. Stril ptriplts (98 mm imtr) wr pour with 20 ml DST mium n llow to soliify t room tmprtur. A. hyrophil ulturs wr inoult y th lwn ultur mtho. Thn th ov ntiiotis wr pl in iffrnt ultur plts n inut t 37 8C for 24 h. Aftr inution th osrvtions wr not. Th zons of inhiition of h prout wr ompr with th rommn zon of inhiition of th ntiiotis ginst th ulturs. Th zons of inhiition of th ntiiotis ginst th A. hyrophil strins wr trt s positiv ontrol Sttistil nlysis All th t wr nlyz y running th gnrl linr mol progrm vill in SAS softwr. Th mns wr ompr using Dunn s multipl rng tst (Dunn, 1955) to fin th iffrn t 5% ( P 0.05) lvl.

4 20 B.K. Ds t l. / Aquultur 253 (2006) Rsults 3.1. Protin stimtion of iffrnt llulr omponnts of Psuomons spis Th protin ontnts of ICP, WCP, HK WCP n ECP of P. fluorsns wr 5.13, 4.91, 4.85 n 4.75 mg/ml rsptivly. Similrly, th protin ontnts of ICP, WCP, HK WCP n ECP of P. ruginos n P. puti wr 6.21, 5.97, 5.73, 5.63, 5.72, 5.31, 4.85 n 4.72 mg/ml rsptivly. Th ICP of P. ruginos ontin highst protin onntrtion (6.21 mg/ ml) n lowst protin ontnt in ECP of P. puti (4.72 mg/ml). Th minimum quntity of protin frtion (70 Al) hrg on nutrint gr plt ginst A. hyrophil strins of P. puti ECP ws foun to 340 Ag. Equl protin onntrtion pr mililitr of h llulr omponnts of th tri ws just to 340 Ag orrspon to 70 Al Bioontrol stuy using P. fluorsns (PF1) Rsults otin from th WCP, HK WCP, ECP n ICP of PF1 ginst A. hyrophil r shown in Fig. 1. It ws rvl tht th ECP of PF1 ws foun to highly fftiv n prou zon rnging from 60 to 85 mm ginst Ah1 to Ah12 strins (Fig. 1). Th highst ror zon ws osrv with Ah10 isolt from skin lsions of Chnn mrulius follow y Ah6 (81.33 F 2.60 mm). ECP of PF1 ginst Ah11 prou mn zon siz of F 0.88 mm. Similrly, WCP, HK WCP n ICP of PF1 wr qully fftiv n h triil fft ginst ll strins of A. hyrophil (Fig. 1). Out of th four prout omponnts of PF1, ECP prou th highst tivity in lmost ll ss, xpt for Ah9. WCP of PF1 ws lso qully fftiv s th ECP n th inhiitory tivity ws highst in Ah1, Ah3, Ah6, Ah7, Ah8, Ah9 whn ompr to ICP n HK WCP (Fig. 1) s ompr to PBS ontrol (0 mm). Signifint iffrns ( P V0.05) of inhiitory ffts wr ror in ll th ss s ompr to positiv ontrol Antgonisti stuy using P. puti (PP1) Th zons of inhiition prou y PP1 ginst Ah1 to Ah12 r shown in Fig. 2. Th zon siz of ht kill WCP of PP1 ws highly fftiv n prou zons rnging from 52 to 71 mm. Th Fig. 1. Zons of inhiition of four ru frtions of P. fluorsns ginst A. hyrophil strins. (Vlus r rprsnt s mnfs.e., mn ring ommon suprsript r not signifint to h othr.)

5 B.K. Ds t l. / Aquultur 253 (2006) Zon of inhiition (mm) Ah1 Ah2 Ah3 Ah4 Ah5 Ah6 Ah7 Ah8 Ah9 Ah10 Ah11 Ah12 Strins Positiv Control Ciprofloxin Positiv Control Gntmiin ECP-Extrllulr Prout ICP-Intrllulr Prout HK WCP- Ht kill Wholll Prout WCP-Wholll Prout Fig. 2. Zons of inhiition of four ru frtions of P. puti ginst A. hyrophil strins. (Vlus r rprsnt s mnfs.e., mn ring ommon suprsript r not signifint to h othr.) ovrll tivity of HK WCP ws th highst mong th four prout frtions tst n foun signifintly iffrnt ( P V0.05) s ompr to th positiv ontrol. Whrs WCP of PP1 prou zon inhiition rnging from 38 to 55 mm; ICP of PP1 prou zons rnging from 46 to 68 mm n ECP of PP1 prou zon siz from 34 to 56 mm. Th lst triil tivity ws ror in th s of ECP of PP1 ginst Ah3 n th highst ror triil tivity of PP1 ginst Ah6 initing th fftivity of WCP of PP1 ginst Ah1 s ompr to PBS ontrol (0 mm). All th four llulr omponnts show signifint ( P V0.05) inhiitory tivity ginst 12 strins of A. hyrophil (Fig. 2) whn ompr to th positiv ontrol Antgonisti stuy of P. ruginos (PA1) Th four llulr omponnts of PA1 wr tst s ntgonisti gnts/omponnts ginst A. hyrophil (Fig. 3). All th four frtions prou ntgonism s msur y th zon of inhiition grtr thn 52 mm n wr foun signifintly iffrnt ( P V0.05) s ompr with th positiv ontrol. WCP of PA1 prou highst ntgonism ginst Ah3 (72.66 F 0.88 mm) follow y HK WCP ginst Ah3 (70.33 F1.20 mm) n Ah2 (71.00 F0.57 mm). WCP of PA1 prou zon of 62.00F0.57 mm ginst Ah6 n ICP of PA1 prou zon of 57.33F0.88 mm ginst th Ah2 strin s ompr to th PBS ontrol (0 mm) A. hyrophil A. hyrophil strins s Ah2, Ah3, Ah4, Ah5, Ah6, Ah10, Ah11 n Ah12 ginst P. fluorsns show signifint ( P 0.05) iffrn in ECP ompr to othr xtrts s wll s ontrol; Ah7 n Ah8 i not show ny signifint iffrn mong th xtrts tst. A. hyrophil strin (Ah9) show signifintly highst zon of inhiition of WCP ompr to ontrol n othr xtrts. Th xtrts otin from P. puti show signifintly ( P 0.05) highst zon of inhiition in HK WCP xtrt ginst A. hyrophil strins ompr to ontrol n othr xtrt tst. Whrs, WCP of P. puti show signifintly highst zon of inhiition in Ah9 strins of A. hyrophil. Th xtrts otin from P. ruginos show signifintly highst zon of inhiition in ECP ginst A. hyrophil strins ompr to ontrol n othr xtrts tst. Whr s, WCP of P. ruginos show signifintly highst zon of inhiition in Ah3, Ah5 n Ah9 strins of A. hyrophil rsptivly.

6 22 B.K. Ds t l. / Aquultur 253 (2006) Zon of inhiition (mm) Ah1 Ah2 Ah3 Ah4 Ah5 Ah6 Ah7 Ah8 Ah9 Ah10 Ah11 Ah12 Strins Positiv Control Ciprofloxin ECP-Extrllulr Prout HK WCP- Ht kill Wholll Prout Positiv Control Gntmiin ICP-Intrllulr Prout WCP-Wholll Prout Fig. 3. Zons of inhiition of four ru frtions of P. ruginos ginst A. hyrophil strins. (Vlus r rprsnt s mnfs.e., mn ring ommon suprsript r not signifint to h othr.) 3.6. Drug snsitivity tst Th tst rugs us in th prsnt xprimnt to ompr th zon of inhiition with tht of th four prouts of PF1, PP1 n PA1; prou zons rnging from 21 to 33 mm ginst Ah1 Ah12, whih ws muh lowr, whn ompr to th four prout frtions of PF1, PP1 n PA1. 4. Disussion Th ntitril fft of tri is gnrlly u to ithr iniviul or joint proution of ntiiotis, trioins, sirophors (Grm n Mlhiorsn, 1996), lysozyms n protss n ltrtion of ph y orgni i proution (Sugit t l., 1998). Th inhiition u to suh ompouns is highly pnnt on th xprimntl onitions, whih r iffrnt in vitro n in vivo (Gtsoup, 1999). Sugit t l. (1998) isolt strin of Billus spis tht ws ntgonisti to 63% of th isolts from fish intstin. Aoring to ths uthors, most pthogni strins of Virio or Aromons hv n trgt in vitro tsts. Othr workrs hv lso ttmpt to us othr pthogns,.g. Ewrsill tr, Entroous sriolii, Psturll pisii, Yrsini rukri (Dopzo t l., 1988; Austin t l., 1995; Ruiz t l., 1996; Sugit t l., 1996; Byun t l., 1997; Gison t l., 1998). Som tri r ntgonisti to viruss (Kmi t l., 1987, 1988; Dirkusrkom t l., 1998) n thy my ffiint for ioontrol of virl isss (M t l., 1997). Antgonism of quti miros to fish n shllfish pthogns hs n rviw y Gtsoup (1999). Th ntgonisti proprtis of P. fluorsns to V. nguillrum in Lts nilotius (Grm t l., 1999) n P. fluorsns to A. slmonii in Slmo trutt (Smith n Dvy, 1993) hv n rport n this inhiition is proly u to omptition for fr iron. In th prsnt stuy, P. fluorsns, P. puti n P. ruginos howvr wr ntgonisti to A. hyrophil strins tst in th prsnt stuy whih wr isolt from vrious iss onitions. Th whol ll kill ntigns, ECP, ICP n liv tri of ll th spis wr qully fftiv, s rvl y th zon of inhiition

7 B.K. Ds t l. / Aquultur 253 (2006) to ll th tst strins of A. hyrophil. It ws foun tht th ntiiotis suh s nliixi i, furzolion, norfloxin, oxyttrylin, iprofloxin, gntmyin n nitrofurntoin prou zon siz, whih ws muh smllr thn tht of four llulr omponnts of h trium tst. Du to th inrs in thrputi rsistn to th mor ommonly us ntiiotis (Hjltns t l., 1987; Aoki, 1992; Nsh t l., 1992) n its potntil ntipthogni tions (y its intr n/or xtr llulr prouts or whol ll prouts), thr pprs to potntil for Psuomons spp. in th ontrol of quultur isss in fishs, prtiulrly romonisis. Th inhiition us y th Psuomon sullulr omponnts is qully fftiv ginst th A. hyrophil strins. Howvr, furthr ssys r nssry spilly in vivo, to forst th onntrtion of ICP, ECP, WCP or liv tri n to ntgoniz or t s ioontrol ginst A. hyrophil inftion in fish. Hn rsrh is urgntly rquir on th ntipthogni mtolits xrt y Psuomons prtiulrly P. fluorsns, P. puti n P. ruginos, whih oul potntilly us s sustituts to ntiiotis. Aknowlgmnts Th uthors r vry grtful to th Inin Counil of Agriulturl Rsrh (ICAR), Nw Dlhi for proviing th finnil ssistn n th Dirtor, Cntrl Institut of Frshwtr Aquultur (CIFA), Bhunswr for proviing filitis for th xprimnt. Rfrns Aoki, T., Chmothrpy n rug rsistn in fish frms in Jpn. In: Shriff, M., Suhsingh, R.P., Arthur, J.R. (Es.), Disss in Asin quultur: 1. Proing of th First Symposium on Disss in Asin Aquultur, 1992, Novmr 1990, Bli, Inonsi, pp Austin, B., Stuky, L.F., Rortson, P.A.W., Effni, I., Griffith, D.R.W., A proioti strin of Virio lginolytius fftiv in ruing isss us y Aromons hyrophil, Virio ngullrum n Virio orlii. J. Fish Dis. 18, Bl Ry, B., Biofilm ssoit tril flor of frshwtr hitt. M. F. S. thsis. Cntrl Institut of Fishris Eution, Mumi, Ini, pp Brfor, M.M., A rpi n snsitiv mtho for th quntifition of mirogrm quntitis of protin utilizing th prinipl y ining. Anl. Biohm. 72, Byun, J.W., Prk, S.C., Bnno, Y., Oh, T.K., Proioti fft of Ltoillus sp. DS-12 in flounr (Prlihthys olivus). J. Gn. Appl. Miroiol. 43, Cnnll, R.J.P., Owsink, A.M., Wlkr, J.M., Rsults of lrg sl srning progrmm to tt ntitril tivity from frshwtr lg. Br. Phyol. J. 23, Corwll, S.J., Lrsn, M.R., Col, R.T., Wlsh, B.J., Comprtiv protomis of Stphyloous urus n th rspons of mthiillin-rsistnt n mthiillin-snsitiv strins to Triton X-100. Miroiology 148, Cown, L.E., Priting th mrgn of rsistn to ntifugl rugs. FEMS Miroiol. Ltt. 204, 1 7. D Anglis, M., Bini, L., Pllini, V., Coonlli, P.S., Gotti, M., Th i-strss rspons in Ltoillus snfrnisnsis CBI. Miroiology 147, Dirkusrkom, S., Yoshimizu, M., Ezur, Y., Rungpn, L., Dnyol, Y., Virio spp., th ominnt flor in shrimp hthry ginst som fish pthogni viruss. J. Mr. Biothnol. 6, Dopzo, C.P., Lmos, M.L., Loiros, C., Bolinhs, J., Brj, J.L., Tornzo, A.E., Inhiitory tivity of ntiioti-prouing mrin tri ginst fish pthogns. J. Appl. Btriol. 65, Dunn, D.B., Multipl rng n multipl FT tsts. Biomtris 11, Gtsoup, F.J., Th us of proiotis in quultur. Aquultur 180, Gison, L.F., Wooworth, J., Gorg, A.M., Proioti tivity of Aromons mi on th pifi oystr, Crssostr gigs, whn hllng with Virio tuishii. Aquultur 169, Grm, L., Mlhiorsn, J., Intrtion twn fish spoilg tri Psuomons sp. n Shwnll putrfins in fish xtrts n on fish tissu. J. Appl. Btriol. 80, Grm, L., Mlhiorsn, J., Spnggr, B., Hur, I., Nilsn, T.F., Inhiition of Virio nguillrum y Psuomons fluorsns AH2, possil proioti trtmnt of fish. Appl. Environ. Miroiol. 65, Hkr, M., Englmnn, S., Corwll, S.J., Protomis of Stphyloous urus urrnt stt n futur hllngs. J. Chromt. B. Anlty. Th. Biom. Lif. Si. 787, Hjltns, B., Anrsn, K., Egiius, E., Multipl ntiioti rsistns in Virio slmonii. Bull. Eur. Asso. Fish Pthol. 7, Holt, J.G., Krig, N.R., Brgy s Mnul of Systmti Btriology vol. 1. Willims n Wilkins, Bltimor, USA. Hughs, D., Exploiting gnomis, gntis n hmistry to omt ntiioti rsistn. Nt. Rv., Gnt. 4, Igrshi, M.A., Kittk, J., Kwhr, E., Phyllosom ultur with inoultion of mrin tri. Nippon Suisn Gkkishi 56 (11), Kmi, Y., Yoshimizu, M., Ezur, Y., Kimur, T., Srning of tri with ntivirl tivity ginst inftious hmto-

8 24 B.K. Ds t l. / Aquultur 253 (2006) poiti nrosis virus (IHNV) from sturin n mrin nvironmnts. Bull. Jpn. So. Si. Fish. 53, Kmi, Y., Yoshimizu, M., Ezur, Y., Kimur, T., Srning of tri with ntivirl tivity from frshwtr slmoni hthris. Miroiol. Immunol. 32, Lipsith, M., Th ris n fll of ntimiroil rsistn. Trns Miroiol. 9, M Fn, J.F., Biohmil Tsts for th Intifition of Mil Btri. Willims n Wikns Co, Bltimor. 310 pp. M, M., Bioontrol of th lrvl rring iotop in quultur. Bull. Ntl. Rs. Inst. Aquult., Suppl. 1, M, M., Nogmi, K., Knmtsu, M., Hirym, K., Th onpt of iologil ontrol mthos in quultur. Hyroiologi 358, Nsh, G., Nithimthhok, C., Tungmni, C., Arkrjmorn, A., Viriosis n its ontrol in pon rr Pnus monoon in Thiln. In: Shriff, M., Susingh, R.P., Arthur, J.R. (Es.), Disss in Asin Aquultur: 1. Proing of th First Symposium on Diss in Asin Aquultur, Novmr 1990, Bli. Inonsi, pp Nogmi, K., M, M., Btri s ioontrol gnts for rring lrv of th r, Portunus triturultus. Cn. J. Fish. Aqut. Si. 49, Olsson, J.C., Wstrhl, A., Conwy, P.L., Kjllrg, S., Intstinl oloniztion potntil of turot (Aophthlmus mximus) n (Limn limn) ssoit tri with inhiitory ffts ginst Virio nguillrum. Appl. Environ. Miroiol. 58, Rihlt, J.L., Borowitsk, M.A., Antimiroil tivity from mrin lg: rsults of lrg-sl srning progrmm. Hyroiologi 116, Ruiz, C.M., Romn, G., Snhz, J.L., A mrin tril strin fftiv in prouing ntgonisms of othr tri. Aqu. Int. 4, Smith, P., Dvy, S., Evin for th omptitiv xlusion of Aromons slmonii from fish with strss-inuil furunulosis y fluorsnt psuomon. J. Fish Dis. 16, Sughr, I., Kimur, T., Hyshi, K., Nkjim, I., Distriution n gnri omposition of lyti nzym-prouing tri in ostl n oni ottom simnts. Nippon Suisn Gkkishi 54 (6), Sugit, H., Mtsuo, N., Shiuy, K., Dguhi, Y., Proution of th ntitril sustns y intstinl tri isolt from ostl r n fish spis. J. Mr. Biothnol. 4, Sugit, H., Shiuy, K., Hn, H., Dguhi, Y., Antitril ilitis of intstinl miroflor of th rivr fish. Fish. Si. 63, Sugit, H., Hiros, Y., Mtsuo, N., Dguhi, Y., Proution of th ntitril sustn y Billus sp. strin NM 12, n intstinl trium of Jpns ostl fish. Aquultur 165, Wst, P.A., Colwll, R.R., Intifition of Psuomon: n ovrviw. In: Colwll, R.R. (E.), Psuomonisis in th Environmnt. John Wily, Nw York, USA, pp Wstrhl, A., Olsson, J.C., Kjllrg, S., Conwy, P.L., Isoltion n hrtriztion of turot (Sophthlmus mximus) ssoit tri with inhiitory ffts ginst Virio nguillrum. Appl. Environ. Miroiol. 57,