Maejo Int. J. Sci. Technol. 2015, 9(03), ; doi: /mijst Maejo International Journal of Science and Technology

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1 Mejo Int. J. Si. Tehnol. 215, 9(3), ; doi: /mijst Mejo Interntionl Journl of Siene nd Tehnology Full Pper ISSN Aville online t Effets of Mus (ABB group) preioti nd Billus sutilis S11 proioti on growth nd disese resistne of ultivted Pifi white shrimp (Litopeneus vnnmei) Rthn Boonmee 1 nd Sirirt Rengpipt 1, 2, * 1 Progrmme in Biotehnology, Fulty of Siene, Chullongkorn University, Bngkok 133, Thilnd 2 Deprtment of Miroiology, Fulty of Siene, Chullongkorn University, Bngkok 133, Thilnd * Corresponding uthor. E-mil: srengpipt@gmil.om Reeived: 26 June 214/ Aepted: 14 Novemer 215/ Pulished: 17 Novemer 215 Astrt: Extrt of ripe nn (BN) with preioti tivity sore of 1.77 were demonstrted to support the growth of proioti Billus sutilis S11 (BS11). BN nd BS11, seprte nd omined, were then fed to the Pifi white shrimp (Litopeneus vnnmei) over 9 dys in the riny seson nd in the dry (hot) seson. In oth sesons, the highest prodution ws found in shrimp fed with BS11 plus 1% BN. After hllenging with Virio hrveyi 639, the lowest umultive mortlity ws found in shrimp fed with BS11 nd 1% BN in the riny seson, inditing higher shrimp prodution following the synioti use of the BS11 proioti omined with the BN preioti. Keywords: Pifi white shrimp, Litopeneus vnnmei, proioti, Billus sutilis S11, preioti, Mus (ABB group), nn INTRODUCTION The frming of Pifi white shrimp (Litopeneus vnnmei) hs reently eome mjor eonomi industry in Thilnd. However, from 213 onwrds the industry hs fed severe low due to outreks of ute heptopnreti nerosis disese [1]. Billus sutilis S11 (BS11) hs een proposed s proioti terium for the lk tiger shrimp (Peneus monodon) [2, 3] nd lso for the Pifi white shrimp [4] due to the terium s ility to survive the pssge through the gstrointestinl trt of these shrimp nd enhne the growth nd defense ginst pthogens inluding Virio hrveyi. The inlusion of inulin-derived fruto-oligoshride (FOS) into regulr niml feed hs lso een shown to hve rod ntiteril effet ginst suh pthogens s Esherihi oli, Slmonell spp., Clostridium spp., enteroteri, euteri nd oliforms [5]. Inulin nd

2 Mejo Int. J. Si. Tehnol. 215, 9(3), ; doi: /mijst oligofrutose hve een reported to our in severl fruits inluding the nn, in whih inulin ontent of.3-.7% of fresh weight were reported [6]. Conversion of inulin to oligofrutose normlly ours through ontrolled prtil enzymti hydrolysis whih resemles tht of the proessing of hiory roots [7]. The most populr nn ultivr in Thilnd, Mus (ABB group) or Klui Nmw in Thi, is hyrid of Mus umint Coll nd M. lisin Coll [8]. Both of the prentl plnts typilly ontin onsiderle mount of dietry fire [8], whih hs een limed to hve preioti tivity tht promotes the growth of teri within the smll intestine leding to the olon nd provides potentil helth enefits [9,1]. In this study the use of proioti oupled with preioti, n pproh known s syniotis, is undertken in order to evlute the effets of the BS11 proioti nd Mus (ABB group) preioti supplemented to the regulr feed on the growth nd disese resistne of the Pifi white shrimp MATERIALS AND METHODS Bteril Strins BS11 ws isolted from the gstrointestinl trt of Peneus monodon ught from the Gulf of Thilnd s previously desried [2, 3] nd shown to e good proioti for Litopeneus vnnmei [4]. Virio hrveyi 639, pthogeni terium, lso isolted from P. monodon tht hd died from luminesent disese, ws kindly provided y the Shrimp Culture Reserh Centre, Chroen Pokphnd Feedmill, Smutskorn, Thilnd. The terium ws ultured in trypti soy gr (TSA), trypti soy roth (TSB) with 1.5% (w/v) gr supplemented with 2% (w/v) NCl, or in thiosulphte-itrte-ile-slt-surose gr (TCBS) nd ws used for induing V. hrveyi infetion in the shrimp. All ulture medi were purhsed from Difo (USA). Esherihi oli ATCC nd Bifidoterium nimlis susp. ltis BB12 were provided y the Deprtment of Miroiology nd the Deprtment of Food Siene, Fulty of Siene, Chullongkorn University respetively. Lti id teri (LAB), nmely Ltoillus (L.) idophilus IAM 174, L. sei susp. sei IAM 145, L. fermentum IAM 1148, Ltoous (L.) ltis susp. remoris IAM 115 nd L. ltis susp. ltis IAM 1198 were otined from Riken Bioresoure Centre, Jpn. Preprtion of Mus (ABB group) Extrt The ripe (yellow skin) fruits of Mus (ABB group) were peeled nd slied. The slied flesh (1 g) ws homogenised in 1 ml of hot wter (1ºC) nd left to stnd for 1 min. Suspended prtiles were removed y entrifugtion t 116 g nd 4ºC for 1 min., nd the superntnt ws hrvested [11] nd used s the ripe nn extrt (BN) in susequent experiments. Lrger sles of BN preprtion ws performed s desired nd kept t 4 C efore use. Preioti Ativity Sore Preioti tivity sore (PAS) ws determined s previously reported [12]. In rief, n overnight ulture of B. nimlis susp. ltis BB12, L. idophilus IAM 174, L. sei susp. sei IAM 145, L. fermentum IAM 1148, L. ltis susp. remoris IAM 115 nd L. ltis susp. ltis IAM 1198 were trnsferred t 1% (v/v) in triplite into individul tues ontining mnully prepred ltoillus de Mn, Rogos nd Shrpe roth without dextrose (mmrs-d) (1 ml) supplemented with either 1% (w/v) gluose, 1% (w/v) inulin, or 1% (w/v) BN. The tues were then inuted t 37ºC under n neroi ondition in ndle jrs [13]. An overnight ulture of

3 Mejo Int. J. Si. Tehnol. 215, 9(3), ; doi: /mijst either BS11 or E. oli ATCC (.1 ml) ws trnsferred in triplite into ulture tues ontining mnully prepred TSB without dextrose (mtsb-d) (1 ml) nd lso into the sme roth ut with either 1% (w/v) gluose, 1% (w/v) inulin or 1% (w/v) BN nd inuted eroilly t 37 o C [13]. After 24 hr of inution, smples were hrvested nd enumerted on MRS gr (Difo, USA) for LAB nd on TSA for BS11 nd E. oli. All hemils for mnully prepred ulture medi were otined from Merk (Germny). Inulin ws from Sigm-Aldrih (USA). The PAS ws determined using the following formul [12]: where Pp nd Pg re the proioti log olony forming units (CFU)/mL when grown on the preioti nd gluose respetively; Ep nd Eg re the enteri log CFU/mL when grown on the preioti nd gluose respetively; nd the susript numers represent the ulture time ( nd 24 hr). By definition, sustrtes with high PAS support good growth of the proioti teri, with ell densities (CFU/mL) omprle with BS11 grown on mtsb-d with gluose. Preprtion of Shrimp Feed Commeril feed (Chroen Pokphnd Foods Puli Co., Thilnd) ws used s the regulr feed nd then supplemented with the proioti nd/or preioti efore use. The feed mixed with BN t 1%, 2% nd 3% (v/w) (designted s 1%BN-, 2%BN- nd 3%BN-feeds respetively) were prepred. BS11 ws ultured in TSB t 3 o C with shking t 2 rpm for 24 hr nd then wshed three times with sterile norml sline solution y entrifuging t 116 g nd 4ºC for 1 min. The ell pellet ws hrvested nd mixed into the feed. The wet ells were thoroughly mixed with the regulr feed t rto of 1:3 respetively (~2.5% of teri y weight) nd designted s BS11-feed. In ddition, the BS11 ws o-supplemented into the 1%, 2% nd 3% BN-feeds (designted s BS11&1%BN-, BS11&2%BN- nd BS11&3%BN-feeds respetively). Eh mixture ws spred out, dried in n oven for 1-2 hr t 37 C nd then stored in len plsti g t 4ºC until use. Eh th ws exmined for vile BS11 ells (CFU/g). Cultivtion of Shrimp The post-lrve of the Pifi white shrimp (Litopeneus vnnmei), ged 2 dys fter hthing, were otined from ommeril shrimp frm in Pthumthni provine nd limtised in n erted tnk with losed reirulting wter system (ontining 25 mg/l NCl) t 3 C until they rehed 1-2 g in weight. They were then trnsferred into 2-L high-density polyethylene ulture tnk (.64 x 1.5 x.56 m). Suspended solids were removed with filtering unit onsisting of three lyers: snd t the ottom, oyster-shells in the middle nd fire filler on the top. Wter ws trnsferred into the filter unit y n ir-lift pump nd pssed through the unit y grvity into the ulture tnk. All the ulture tnks were pled outdoors under roof nd sun-shde net over. A totl of eight different feeding regimes, viz. the regulr feed (ontrol) nd the BS11-, 1%BN-, 2%BN-, 3%BN-, BS11&1%BN-, BS11&2%BN- nd BS11&3%BN-feeds were evluted. Eh tretment ws performed in triplite using 3 shrimp in eh tnk. The shrimp were fed three times dily t 9. m, 1. pm nd 6. pm t 1% of their net ody weight. This set of experiment ws performed twie, one in the riny seson nd one in the hot seson. The shrimp ody weight nd survivl, totl teri, BS11 nd Virio spp. ounts from the ultivtion tnk nd

4 Mejo Int. J. Si. Tehnol. 215, 9(3), ; doi: /mijst intestines of moriund shrimp, together with totl hemoyte ount (THC) nd phenoloxidse (PO) tivity of live shrimp were monitored efore nd fter hllenging with V. hrveyi 639. Wter smples (1 ml) were olleted from the entre of eh tnk every 15 dys nd their ph, lklinity, dissolved oxygen, slinity, temperture, nd mmonium nd nitrite ontents were monitored s desried previously [14]. Virio hrveyi Chllenge Virio hrveyi 639 ws ultured in TSB with 2% (w/v) NCl or on TCBS nd used for induing the infetion. After feeding for 9 dys, the shrimp in eh tretment were exposed to pthogeni V. hrveyi 639 t ~1 7 CFU/mL y immersion in tnk wter. Ten shrimp were rndomly trnsferred to eh 4-L wter tnk (three tnks per tretment). The remining shrimp were kept nd fed with the sme regime s efore. No wter ws exhnged etween the tnks therefter for 3 dys. During the tril, the numer of live shrimp ws mesured nd the moriund shrimp were removed every 24 hr, nd from this the umultive mortlity ws reorded. The heptopnres nd intestine were disseted from eh moriund shrimp nd exmined for the presene of V. hrveyi 639 initilly y isoltion on TCBS gr, nd susequent identifition s V. hrveyi ws performed s previously desried [15]. Defense Prmeters from Shrimp Hemolymph Shrimp hemolymph (1 µl) ws olleted from the ventrl-sinus using 26-guge needle nd 1-mL syringe ontining 2 µl of ntiogulnt solution (1% w/v sodium itrte). A 2-µL drop of the mixture ws pled in hemoytometer nd the THC determined under light mirosope t 4 x mgnifition. After olleting the hemolymph nd pelleting the ells y entrifugtion t 8 g nd 4ºC for 1 min., the pellet ws wshed nd olleted in ie-old odylte uffer (Sigm-Aldrih, USA) t ph 7. nd then homogenised with sonitor for 1 se. The homogente ws entrifuged t 16 g nd 4ºC for 2 min. to otin the hemoyte lyste superntnt. The PO tivity of the superntnt ws mesured spetrophotometrilly t 49 nm using L-3,4-dihydroxyphenyllnine (Sigm-Aldrih, USA) s the sustrte s desried previously [4, 16, 17]. One unit of enzyme tivity ws defined s n inrese in sorne per min. per µg protein [18]. The protein ontent in the hemoyte lyste superntnt ws mesured y Brdford s method [19] using ovine serum lumin (Sigm-Aldrih, USA) s stndrd. Sttistil Anlysis The dt on shrimp growth, survivl nd disese resistne re presented s men ± stndrd devition (SD). The signifine of differenes etween mens ws tested y nlysis of vrine nd Dunn s multiple rnge tests with epted signifine t p <.5 level [2]. RESULTS AND DISCUSSION Preioti Ativity Sore BS11 grown in mtsb-d with 1% inulin or 1% BN hs resonly high PAS of 1.42 ±.2 nd 1.77 ±.35 respetively, with 1.25-fold higher growth in BN- thn inulin-supplemented mtsb-d, ut this is not sttistilly signifint (Figure 1). In ontrst, B. nimlis susp. ltis BB12 growth in mmrs-d with 1% inulin is mrkedly lower thn tht in mmrs-d with 1% BN giving low PAS of -.18 ±.8 nd -.36 ±.2 respetively. The inility of B. nimlis susp.

5 Mejo Int. J. Si. Tehnol. 215, 9(3), ; doi: /mijst ltis BB12 to grow well in the presene of inulin or BN is in greement with tht previously reported for different strin of B. ifidum NCI on inulin-s with PAS of ±.8 [12]. Among the other tested LAB, the highest PAS of 1.94 ±.4 nd 1.97 ±.43 in mmrs-d with 1% inulin nd 1% BN respetively ws deteted in L. ltis susp. remoris IAM 115. These re 1.4- nd 1.1-fold higher thn PAS for BS11 on the medi supplemented with inulin nd BN respetively. None of the other tested LAB grew s well in mmrs-d with 1% BN ompred to tht with 1% inulin (PAS < 1), exept for L. ltis susp. remoris IAM 115. However, the LAB evluted in this study seem to grow well in the presene of inulin ut not in the presene of BN (Figure 1). Similr to tht reported previously [12], the PAS is oserved to e dependent on oth the teril strin nd the preioti type/dose. Apprently, BN n reple inulin nd support BS11 growth. Figure 1. PAS of BS11 grown in mtsb-d nd vrious LAB grown in mmrs-d supplemented with either 1% inulin or 1% BN s preioti. Dt re shown s men SD from triplite nlysis, nd mens with different lowerse letters (for inulin) or upperse letters (for BN) re signifintly different (P <.5). Effets of BS11- nd BN-Feeds on Shrimp Weight, Survivl nd Prodution The weights of L. vnnmei mong the eight tretments were not signifintly different fter 3 dys of ulture in the riny seson (Figure 2). The verge wter qulity in ll tretments (4-6 mg/l dissolved oxygen, mg/l slinity, ph , -.5 mg/l mmoni, 8-15 ppm lklinity nd -.5 mg/l nitrite) ws within sfe limits for shrimp ulture [21]. The temperture vried only slightly, rnging etween ºC in the riny seson. The wter qulity in ll tretments in the hot seson ws similr, exept for nrrower ph rnge ( ) nd roder temperture rnge ( ºC), whih inluded mrkedly high limte temperture of up to 41.5ºC etween 11m-1pm for one-week period fter 3 dys of ulture. This elevted temperture ws likely to hve indued signifint het stress in the surviving shrimp. After 9 dys of ulture in the riny seson, the highest verge shrimp weight (15.7 ±.75 g) ws found in those fed with 2%BN-feed, whih ws signifintly higher thn tht in the ontrol

6 Mejo Int. J. Si. Tehnol. 215, 9(3), ; doi: /mijst (Figure 2). In ontrst, the shrimp survivl fter 9 dys of ulturing with feed supplemented with BS11&1%BN ( %) or BS11&2%BN ( %) were signifintly greter thn tht of the ontrol (Figure 2). Thus, there ppers to e mrked synioti funtion of the BS11 proioti nd BN preioti on the shrimps survivl ut not on their weight. The highest verge shrimp prodution ourred in shrimp fed with BS11&1%BN-feed (38.9 ± 36.2 g), whih ws signifintly higher thn tht in the ontrol (Figure 2e). In the trils performed in the hot seson the highest inrese in the verge shrimp weight fter 9 dys of ulture ws oserved in shrimp fed with BS11&2%BN-feed (15.7 ±.74 g) (Figure 2). On the other hnd, the highest shrimp survivl ws deteted in shrimp fed with BS11- feed (81.1 ± 11.7%) (Figure 2d). A lower survivl ws found in the hot seson thn in the riny seson, pprently due to the high wter temperture flututions in the hot seson, whih is ove the optimum temperture for shrimp growth (28-3 o C). This my lso ount for lrger vrine seen in the verge vlues in the hot seson thn in the riny seson. Likewise, the net verge prodution in the hot seson ws lower thn in the riny seson, with the highest prodution level in the hot seson eing found in shrimp fed with the BS11&1%BN-feed (291.1 ±.44 g) (Figure 2f). However, this is still 1.3-fold lower thn the est prodution level found in the riny seson. The results re in greement with the known effets of temperture stress, whih n inrese the shrimp s suseptiility to pthogens, limit growth nd redue survivl rte nd prodution [22, 23]. The higher net prodution of shrimp fed with BS11&1%BN-feed thn with BS11-feed implies the possiility of synioti effet of the omintion of the BS11 proioti nd the BN preioti on prodution. In oth sesons redued shrimp prodution level ws found mostly when shrimp were fed only with BN-feed nd this is signifintly different from those fed with BS11- nd BS11&BN-feeds (Figures 2e nd 2f). This is in greement with previous reports tht the preioti funtion of FOS did not ffet the weight gin nd survivl of the Pifi white shrimp [24], whilst Bio-Mos nd β-1,3-d-glun did not ffet the growth of the western king prwn P. ltisultus [25]. In ontrst, mnnn oligoshrides t 3. g/kg were found to signifintly enhne the weight nd survivl of P. semisultus PL [26], whilst short hin FOS signifintly enhned the survivl nd weight of juvenile white shrimp in dose-dependent mnner [27]. In ddition, the survivl of Indin white shrimp lrve (M 1 -M 3 ) fter dministrtion of inulin (Rftilline ST) + Esy DHA Selo -enrihed Artemi ws signifintly higher thn tht fed with either Esy DHA Selo - enrihed Artemi or inulin-enrihed Artemi [28]. Without the preioti funtion of BN, the proioti BS11 lone seems to signifintly enhne the shrimp prodution level (Figures 2e nd 2f), onsistent with its previously reported proioti properties [8] inluding growth enhnement, indution of helthy pperne [29, 3] nd inresed survivl [31, 32]. It is prole tht BS11 n produe some nti-miroil sustnes tht negtively ffet other pthogens [2, 3] nd/or it n produe useful enzymes or metolites to inrese the uptke of useful nutrients, provide ompetitive exlusion effet on pthogens nd provide some enefits to indigenous indution [2, 3, 25, 33]. Mny other Billus spp. hve previously een reported to t s good proioti ndidtes for Pifi white shrimp y supplementtion into the diet [34-36] or ulture wter [31, 37]. Here, preioti (BN) feeds re demonstrted to onfer enefits on the BS11 growth nd my improve gstrointestinl miroflor of the host s desried previously [9, 24, 38].

7 Mejo Int. J. Si. Tehnol. 215, 9(3), ; doi: /mijst () Weight (g) 1 5 () Weight (g) Time (dys) Time (dys) () 1. (d) 1. Survivl (%) Survivl (%) d d d d d d d d d d Time (dys) Time (dys) (e) 4 (f) 4 Produtions (g) Produtions (g) Time (dys) Time (dys) Control BS11 1%BN 2%BN 3%BN BS11&1%BN BS11&2%BN BS11&3%BN Figure 2. Averge live (wet) weights (, ), survivl (, d) nd prodution (e, f) of L. vnnmei during 9-dy ulture period when fed with regulr feed (ontrol) or with BS11-, 1%BN-, 2% BN-, 3%BN-, BS11&1%BN-, BS11&2%BN- or BS11&3%BN-supplemented feeds in riny seson (,, e) nd hot seson (, d, f). Dt re shown s men SD from triplite nlysis, nd mens with different letters re signifintly different etween tretments within the respetive time points. The totl teril nd Virio spp. ounts in the ulture wter of ll tretments rnged etween ~4 x ~3 x 1 7 nd ~2 x ~2 x 1 4 CFU/mL respetively in riny seson, nd etween ~3 x ~1 6 nd ~1 x ~2 x 1 4 CFU/mL respetively in hot seson. As expeted,

8 Mejo Int. J. Si. Tehnol. 215, 9(3), ; doi: /mijst BS11 ws only found in the ulture wter from tnks of shrimp fed with BS11-supplemented feeds nd ws t ~7 x ~2 x 1 3 CFU/mL in riny seson nd ~9 x ~3 x 1 3 CFU/mL in hot seson. Chllenge Test nd Defense Prmeters from Shrimp Hemolymph From Figures 3 nd 3, the THCs in shrimp fed with different supplemented feeds efore nd fter eing hllenged y V. hrveyi 639 re numerilly higher thn those in shrimp fed with regulr feed in oth sesons, ut re only signifintly higher in shrimp fed with BS11- nd BS11&1%BN-feeds. The redution in THC fter eing hllenged with V. hrveyi 639 for ll eight tretments rnges etween 36-56% nd 52-64% in the riny nd hot sesons respetively. The lowest THC redution is oserved in shrimp fed with BS11&1%BN-feed in oth sesons, wheres the highest THC redution is found in shrimp fed with regulr feed. The ontrol shrimp give the lowest verge THC fter V. hrveyi 639 hllenge, t 1.6- nd 2.-fold lower thn those from shrimp fed with BS11&1%BN- nd BS11-feeds respetively in the riny seson (Figure 3), nd t 1.4- nd 2.3-fold lower in the hot seson (Figure 3). From Figures 3 nd 3d, the highest redution in hemoyte PO tivity (84% nd 7% in riny nd hot sesons respetively) fter hllenge with V. hrveyi re oserved in shrimp fed with regulr feed, whilst BS11- nd BS11&%BN-feeds give 55-68% nd 51-63% redution in riny nd hot sesons respetively. The lowest verge hemoyte PO tivity fter V. hrveyi 639 hllenge ours in the ontrol shrimp, eing more thn 2.-fold lower thn tht in the BS11&1%BN- nd BS11-feeds in oth sesons. From Figures 3e nd 3f, mortlity is evident from the first dy onwrds in ll the tretments fter hllenge with V. hrveyi 639. However, the shrimp fed with ontrol feed suffer signifintly higher mortlity from dy 2 nd reh 9% mortlity 3 dys fter V. hrveyi exposure in riny seson nd 42% in hot seson. The improved shrimp survivl, tken to imply disese resistne s ompred to ontrol, is lerly evident in the shrimp fed with ll BN- nd BS11-feeds, with the highest survivl of 66.7% in those fed with BS11&1%BN-feed in riny seson, 9.% survivl in those fed with 2%BN- or 3%BN-feeds in hot seson, nd 86.7% survivl in those fed with BS11&1%BN-feed. BS11-, BN- or BS11&BN-feed thus gives n enhned survivl fter n externl V. hrveyi exposure. In greement with these results, deresed shrimp mortlity fter infetion with V. prhemolytius ws previously reported in shrimp tht were fed with feed ontining V. lginolytius UTM 12, B. sutilis UTM126, Roseoter glleiensis SLV3 nd Pseudomons estumrin SLV22 [36]. Overll, lower mortlity ws oserved in the hot seson during the 3-dy exposure period to V. hrveyi thn in the riny seson. This my well reflet the ~1 3 CFU/mL lower vile Virio spp. levels in shrimp wter in the hot seson ompred with those in the riny seson.

9 Mejo Int. J. Si. Tehnol. 215, 9(3), ; doi: /mijst () 16 Before After () 16 Before After Totl hemoytes (1x1 6 ells ml -1 ) Totl hemoytes (1x1 6 ells ml -1 ) d d d d Tretment Tretment () 7 Before After (d) 7 Before After PO tivity (U min -1 mg protein -1 ) PO tivity (U min -1 mg protein -1 ) (e) Cumultive mortlity (%) Tretment (f) Cumultive mortlity (%) Tretment Control 1%BN 2%BN 3%BN Time (dys) BS11 BS11&1%BN BS11&2%BN BS11&3%BN Control 1%BN 2%BN 3%BN Time (dys) BS11 BS11&1%BN BS11&2%BN BS11&3%BN Figure 3. Men shrimp THCs (, ) nd PO tivities (, d) efore nd fter hllenge for 3 dys y Virio hrveyi 639, nd shrimp umultive mortlity (e, f) during 3-dy hllenge for shrimp fed with regulr feed (ontrol) or BS11-, 1%BN-, 2%BN-, 3%BN-, BS11&1%BN-, BS11&2%BN- or BS11&3%BN-supplemented feeds in riny seson (,, e) nd hot seson (, d, f). Dt re shown s men SD, derived from nine shrimp. Different letters represent signifint differene mong tretments efore or fter hllenge y Virio hrveyi 639.

10 Mejo Int. J. Si. Tehnol. 215, 9(3), ; doi: /mijst The Virio ounts in the intestines of the moriund shrimp from ll the tretments fter hllenge with V. hrveyi 639 for 3 dys rnged etween x 1 7 CFU/g in the riny seson nd 3 x x 1 7 CFU/g in the hot seson, whih were signifintly higher thn those efore V. hrveyi hllenge. Lower Virio ounts from the intestines of shrimp fed with BS11-feed thn with norml or BN-feeds were noted. The derese in totl Virio ounts in the intestines of BS11- fed shrimp in this study grees with previous results on shrimp fed with proioti Hlomons sp. B12 [39], lthough it does not extly mth the shrimp mortlity rtes oserved here. CONCLUSIONS A synioti effet using Billus sutilis S11 proioti teri nd nn preioti on enhning the prodution nd luminous viriosis resistne of the Pifi white shrimp, Litopeneus vnnmei, hs een demonstrted. The results from this study support the possiility of using Billus sutilis S11 nd/or nn preioti s lterntives to ntiiotis in the shrimp frming proess, whih ould led to more sustinle nd environmentlly friendly industry s well s helthier food produt of higher eonomi vlue. ACKNOWLEDGEMENTS The uthors sinerely thnk Roert Buther for revising the mnusript nd providing mny vlule suggestions nd omments. This reserh ws supported y the Thi Government Stimulus Pkge 2 (TKK 2555) under the Projet for the Estlishment of Comprehensive Centre for Edution Reserh Promotion nd y the Ntionl Reserh University Projet of Thilnd, Offie of the Higher Edution Commission (FW643A) nd prtilly y Chullongkorn University 9th Anniversry fund (Rthdphisek Somphot Endowment Fund). REFERENCES 1. FAO, Report of the FAO/MARD Tehnil Workshop on Erly Mortlity Syndrome (EMS) or Aute Heptopnreti Nerosis Syndrome (AHPENS) of Cultured Shrimp (under TCP/VIE/334), Hnoi, Viet Nm, 213, FAO, Rome, S. Rengpipt, W. Phinphk, S. Piytirtitivorkul nd P. Mensvet, Effet of proioti terium on lk tiger shrimp Peneus monodon survivl nd growth, Aquulture, 1998, 167, S. Rengpipt, S. Rukprtnporn, S. Piytirtitivorkul nd P. Mensvet, Immunity enhnement in lk tiger shrimp (Peneus monodon) y proiont terium (Billus S11), Aquulture, 2, 191, P. Sphroen nd S. Rengpipt, Effets of the proioti Billus sutilis (BP11 nd BS11) on the growth nd survivl of Pifi white shrimp, Litopeneus vnnmei, Aquult. Nutr., 213, 19, L. S. Boekner, M. I. Shnepf nd B. C. Tunglnd, Inulin: A review of nutritionl nd helth implitions, Adv. Food Nutr. Res., 21, 43, J. vn loo, P. Coussement, L. de Leenheer, H. Hoeregs nd G. Smits, On the presene of inulin nd oligofrutose s nturl ingredients in the western diet, Crit. Rev. Food Si. Nutr., 1995, 35, E. J. Vndmme nd D. G. Deryke, Miroil inulinses: Fermenttion proess, properties, nd pplitions, Adv. Appl. Miroiol., 1983, 29,

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