British Journal of Nutrition

Transcription

1 British Journl of Nutrition (2015), 113, doi: /s q The Authors This is n Open Access rticle, distriuted under the terms of the Cretive Commons Attriution licence ( commons.org/licenses/y/3.0/), which permits unrestricted re-use, distriution, nd reproduction in ny medium, provided the originl work is properly cited. Effect of ovine colostrum feeding in comprison with milk replcer nd nturl feeding on the immune responses nd colonistion of enterotoxigenic Escherichi coli in the intestinl tissue of piglets Sugihrto Sugihrto 1,2, Ann-Sofie Riis Poulsen 1, Nuri Cnie 1 nd Chrlotte Luridsen 1 * 1 Deprtment of Animl Science, Fculty of Science nd Technology, University of Arhus, 8830 Tjele, Denmrk 2 Fculty of Animl nd Agriculturl Sciences, Diponegoro University, Semrng, Centrl Jv 50275, Indonesi (Sumitted 4 April 2014 Finl revision received 12 August 2014 Accepted 8 Septemer 2014 First pulished online 6 Mrch 2015) British Journl of Nutrition Astrct The present study investigted the effect of feeding ovine colostrum (BC) to piglets in comprison with feeding milk replcer (MR) nd conventionl rering y the sow on the intestinl immune system nd numer of enterotoxigenic Escherichi coli (ETEC) colonising the intestinl tissue. Piglets (23-d-old) were llocted to one of the following four groups: (1) killed t the eginning of the experiment (Bse); (2) seprted from the sow nd fed BC (BC-fed); (3) seprted from the sow nd fed MR (MR-fed); (4) kept with the sow (Sow-Milk). Blood ws smpled on dys 1 nd 8, nd fecl smples were collected on dys 1, 3, 5 nd 8. On dy 8, piglets were killed nd gstrointestinl digest nd intestinl segments were collected. The frequency of dirrhoe ws found to e higher (P# 0 019) in MR-fed piglets thn in BC-fed nd Sow-Milk piglets. Piglets from the MR-fed group hd the lowest lctic cid cteri:hemolytic E. coli rtio (P tret ¼ 0 064) in the feces. The numer of E. coli colonising the intestinl tissue ws higher (P,0 001) in piglets from the MR-fed group thn in those from the BC-fed nd Sow-Milk groups. Piglets from the Sow-Milk group hd higher (P¼0 020) mucosl IgG concentrtion thn those from the MR-fed group, ut did not exhiit ny difference when compred with piglets from the Bse nd BC-fed groups. Piglets from the BC-fed group exhiited reduced (P#0 037) expression level of Toll-like receptor-4 in the intestinl mucos when compred with those from the MR-fed nd Sow-Milk groups. The expression level of IL-2 ws higher (P# 0 051) in piglets from the MR-fed group thn in those from the other tretment groups. In conclusion, feeding BC rther thn MR to the piglets reduced the colonistion of intestine y ETEC nd modulted the intestinl immune system, wheres no differences were oserved in piglets fed BC nd conventionlly rered y the sows. Key words: Bovine colostrum: Dirrhoe: Enterotoxigenic Escherichi coli: Intestinl immunity: Wek piglets Incresing the litter size hs long een gol mong pig producers, s the numer of offspring orn is n importnt economic trit. Initilly, improvements mde in mngement nd nutrition were imed t incresing the litter size, nd more recently, genetic selection for litter size hs een implemented (1). It hs een reported tht n incresed litter size is geneticlly ssocited with decresed men irth weight nd n incresed within-litter irth weight vrition (2). Therefore, the use of hyperprolific sows in the pig industry not only results in lrge litters, ut lso leds to n increse in the numer of supernumerry nd underprivileged piglets (underweight t irth, referred here s wek piglets) (3). Aprt from its vlue s complete nutrient source, milk contins wide rnge of fctors (e.g. ntiodies nd cytokines) tht cn modulte the development nd mturtion of the immune system nd susequently the immune response of piglets (4,5). During lcttion, lower milk supply per piglet is often oserved in lrge litters. This prticulrly occurs fter the mximl milk production cpcity is ttined (7 15 d) (6). Given tht wek piglets hve lower ility to compete for the udder (7) nd re often less vigorous in suckling (6), their milk intke my e insufficient (7). Hence, it is not surprising tht wek piglets hve poor pre-wening growth performnce nd often fil to thrive, or even die, y the time of wening (8). It hs een suggested tht low ody weight nd immunoincompetence t wening dversely ffect the growth performnce nd viility of piglets fter wening (7,9,10). Thus, efforts to enhnce the growth nd survivl of wek piglets fter wening should e imed t improving not only the wening weight, ut lso the helth nd immunity (11). Arevitions: Bse, piglets killed t the eginning of the experiment; BC, ovine colostrum; BC-fed, piglets seprted from the sow nd fed ovine colostrum; cfu, colony-forming units; DMEM, Dulecco s modified Egle s medium; GIT, gstrointestinl trct; LAB, lctic cid cteri; MR, milk replcer; MR-fed, piglets seprted from the sow nd fed milk replcer; PIOC, porcine intestinl orgn culture; Sow-Milk, piglets kept with the sow; TGF-1, trnsforming growth fctor-1. * Corresponding uthor: C. Luridsen, fx þ , emil chrlotte.luridsen@nis.u.dk

2 924 S. Sugihrto et l. British Journl of Nutrition Bovine colostrum (BC), which is commercilly ville co-product from the diry industry, is known to contin severl growth fctors s well s ntimicroil nd immunomodultory components essentil for host development nd defence functions (4). Of the ioctive components, Ig, especilly IgG, pper to e the most importnt immune components present in BC s they represent % of the totl protein content in the colostrum (4). Bovine colostrl Ig provide the host with pssive immunity (12,13) nd, in concert with lctoferrin, lctoperoxidse nd lysozyme, my ct s ntimicroil gents (4).BC lso contins high mounts of cytokines tht cn modulte the development nd mturtion of the immune system (4,5). Besides the provision of supplementl milk during suckling (11),rtificil rering on milk replcers (MR) (3) hs een reported s good lterntive for rising low-irth-weight piglets efore wening. Tking this into considertion, rering wek piglets rtificilly on BC could e expected to improve the growth performnce s well s to oost the immune system efore wening. The im of the present study ws to investigte the effect of feeding commercil BC product to piglets in comprison with feeding common MR nd conventionl rering y the sow on the intestinl immune system nd colonistion of ETEC in the intestinl tissue. Our hypothesis ws tht BC would e etter source thn MR nd s good s conventionl rering y the sow in terms of optimistion of the immune defence nd intestinl helth of wek piglets. Mterils nd methods Experimentl design The present experiment ws conducted ccording to the licence otined from the Dnish Animl Experiments Inspectorte, Ministry of Food, Agriculture nd Fisheries, Dnish Veterinry nd Food Administrtion. A totl of forty piglets (8 30 (SD 1 45) kg) from four litters otined from the herd t the Fculty of Science nd Technology, Arhus University, Foulum (Denmrk), were used in the present experiment. All piglets were from sows tested homozygote crriers of the dominnt gene encoding for the intestinl F18 fimrie receptors ( DNA mrker genotyping-sed test ws performed on DNA extrcted from hir smples y vn Heringen lortorium.v., Wgeningen, The Netherlnds); thus, piglets used in the present study were susceptile to Escherichi coli F18. Piglets were provided neither dditionl milk nor creep feed from irth to wening (seprtion from the sow). Of the forty piglets, four piglets, one from ech litter, were killed t the eginning of the experiment (Bse) nd thirty-six piglets, nine per litter, were kept live nd rndomly llocted to one of the following three tretment groups: (1) seprted from the sow nd fed commercil BC product (BC-fed); (2) seprted from the sow nd fed commercil MR (MR-fed); (3) kept with the sow (Sow-Milk). Piglets from the BC-fed nd MR-fed groups were seprted from the sows t 23 d of ge, trnsferred to the experimentl stle nd provided either skimmed stndrdised BC (Europen Colostrum Industry S. A.) or commercil MR (Vitfoss) d liitum Tle 1. Chemicl composition of the ovine colostrum (BC), milk replcer (MR) nd sow milk Items BC* MR* Sow milk DM (%) Crude protein (% DM) Crude ft (% DM) Ash (% DM) Ig (% DM) IgG IgA IgM 2 52 ND 0 56 ND, not detected (detection level ¼ 78 ng/ml, equivlent to %). * Chemicl nlyses of BC nd MR were performed y Eurofins Steins Lortorium A/S, Odense, Denmrk. The chemicl compositions of sow milk, especilly DM, protein nd ft, re dopted from Luridsen & Dnielsen (38), while tht of sh is dopted from Aguing et l. (39). The concentrtions of Ig were determined in our lortory. Vlues of Ig for sow milk re the verge vlues from four milk smples (otined from four sows). for 8 d. The chemicl composition of the sow milk, BC nd MR is given in Tle 1. Liquid BC nd MR were prepred using n utomted wet feeder (Mmo Automix 25; Wit-Mmo, Inc.), in which BC nd MR powders were utomticlly mixed with wrm wter (458C; pproximtely 150 g of powder in 1 litre of wter) nd given to the piglets on regulr sis. To help the piglets ecome fmilir with the utomted wet feeder nd stimulte the consumption of liquid BC or MR, sow milk collected from the corresponding sow ws fed to the piglets using the feeder upon rrivl to the experimentl stle. Piglets from the Sow-Milk group were kept with their dms in the frrowing unit until the end of the experiment without ny supplementry feed or milk. To minimise the vrition mong the tretment groups in terms of stress cused y seprtion from the sow, piglets from the Sow-Milk group were trnsported in similr mnner to those in the other groups nd returned to the sows gin. A totl of three littermtes of similr weight were housed together in m 2 pens with ruer mt nd swdust edding. Ech pen ws equipped with n utomted wet feeder nd wter nipple giving permnent free ccess to feed nd wter to ll the three piglets. Temperture in the experimentl stle ws mintined t 248C throughout the experiment nd the pens were equipped with het lmp. To reduce the impct of moving BC-fed nd MR-fed piglets from the sows when compred with Sow-Milk piglets regrding microil contmintion from the environment, mteril ( mixture of edding nd feces) from the floor of pens where the sows were housed ws collected dily nd spred in the corresponding pens where BC-fed nd MR-fed piglets were housed. In this wy, ll piglets were continuously exposed to the microiot of sow fecl origin. Smpling nd dt collection The ody weight of ech piglet ws recorded t the eginning (dy 1) nd t the end (dy 8) of the experiment. BC nd MR consumption ws mesured y recording the weight of the

3 Bovine colostrum nd pig gut helth 925 British Journl of Nutrition powder provided to the piglets nd ny unconsumed residue on dily sis. For Ig nlyses, lood smples were collected in heprinised Vcutiner tues y puncture of the jugulr vein of ech piglet on dys 1 nd 8. Plsm ws otined fter centrifugtion of lood smples t 2000 g for 10 min nd stored t 2208C until nlysis. Fecl smples were collected from the rectum of ech piglet on dys 1, 3, 5 nd 8 of the experiment. Rectl temperture ws recorded with digitl thermometer (Kruuse) efore fecl smple collection. The consistency of fecl smples (1 ¼ hrd, dry nd cloddy; 2 ¼ firm; 3 ¼ soft with shpe; 4 ¼ soft nd liquid; 5 ¼ wtery nd drk; 6 ¼ wtery nd yellow; nd 7 ¼ fomy nd yellow), used s n indictor of the occurrence of dirrhoe, ws recorded immeditely fter smple collection. A fecl consistency score.3 ws defined s clinicl signs of dirrhoe (14). The frequency of dirrhoe ws clculted y counting pig dys with dirrhoe score. 3. The lertness score (0 ¼ norml nd 1 ¼ depressed or listless) of ech piglet ws lso ssessed visully throughout the experiment. One piglet per litter (four in totl) ws killed using cptive olt gun t the eginning of the experiment (dy 1) nd one piglet per litter nd tretment (12 in totl) ws killed t the end of the experiment (dy 8). The domen ws incised nd gll ldder (for ile collection) ws otined immeditely fter leeding the niml. The gstrointestinl trct (GIT) ws immeditely removed nd divided into five segments (stomch, proximl nd distl smll intestine, cecum nd mid-colon), nd digest from ech segment ws collected for cteril enumertion. The length of the smll intestine, from the pyloric sphincter to the ileocolonic junction, ws mesured efore digest collection. Approximtely 15 cm segments from 10 % (representing duodenum), 50 % (jejunum) nd 90 % (ileum) of the length of the smll intestine mesured from the duodenum were removed, opened lengthwys nd wshed with ice-cold PBS. Mucos ws collected from these segments y gently scrping with glss microscope slide nd immeditely stored t 2208C until Ig nlyses. For gene expression nlyses, mucos (pproximtely 100 mg) collected from the sme sites of the smll intestine (jejunum nd ileum) ws preserved in the RNAlter (1 ml; Sigm-Aldrich) nd stored t 48C for 1 d efore storge t 2208C. Intestinl segments (jejunum nd ileum) were lso collected from piglets killed t the end of the experiment nd used in the porcine intestinl orgn culture (PIOC) experiment. Porcine intestinl orgn culture experiment A totl of twelve piglets, one per litter nd tretment, were used in the PIOC experiment. Immeditely fter incising the domen nd leeding the niml, three jejunl segments nd three ilel segments (15 cm ech) were collected septiclly, immersed in Dulecco s modified Egle s medium (DMEM), kept on ice nd immeditely used in the PIOC experiment. E. coli F18 inoculum ws prepred y retrieving the cteril culture stored t 2808C, streking on lood gr (Oxoid, Deutschlnd GmH) nd culturing t 378C for 18 h. A loopful of E. coli F18 colony ws tken from lood gr nd suspended in 4 ml of PBS. The suspension (0 1 ml) ws poured onto Iso-Sensitest gr (Oxoid) nd incuted t 378C overnight. PBS (10 ml) ws poured into the incuted plte, nd the gr surfce ws gently rued with sterile Driglski sptul to remove the cteril colonies from the gr plte. The cteril suspension ws trnsferred into sterile tue nd diluted 1:20 (v/v) with DMEM to otin n inoculum of colony-forming units (cfu)/ml efore eing used for inoculting the intestinl segments of piglets. The E. coli F18 strin ws isolted t the Dnish Veterinry Institute (Frederikserg, Copenhgen, Denmrk) from the intestinl content of pig with post-wening dirrhoe. It ws found to hrour genes for enterotoxins het stle toxin (ST), het lile toxin (LT), Enteroggregtive Escherichi coli het-stle enterotoxin 1 (EAST1), Shig toxin 2e nd fimrie F18. The cteri were found to e hemolytic when grown on lood gr (Oxoid) (15). Of the collected jejunl nd ilel segments, two pieces were inoculted with E. coli F18 nd one piece ws used s control (not inoculted). The PIOC experiment ws conducted sed on the model descried y Nughton et l. (16) nd modified y Sugihrto et l. (17). In rief, polyethylene tuing (Siltue; Eurphrm; 6 mm in dimeter) ws inserted into either end of the segment nd tied with suture to keep the tuing in plce. The tissue ws wshed with 50 ml of PBS (ph 7 2) using Fill-Mster pump (Type 311; Delt Scientific Medicl; flow rte of 7 7 cm/s). The other end of the segment ws tied, 10 ml of DMEM lone (control) or DMEM contining E. coli F18 ws inoculted, nd the segment ws seled with Teflon plug (5 mm in dimeter). The segment ws immersed in DMEM in 300 ml infusion ottle in shking wter-th t 378C, removed fter 1 5 h nd wshed with 50 ml of PBS fter removing the content. Mucos ws collected from piece of the segment nd preserved in the RNAlter for gene expression nlyses. The remining segment ws weighed nd homogenised (Jnke-Kunkel Ultr-Turrx T25 homogeniser; Bie&Bentsen /s) for 20 s in PBS. E. coli were enumerted on McConkey gr (Merck KGA) fter eroic incution t 378C overnight. Microiologicl nlyses of the fecl nd digest smples Microiologicl nlyses were performed on fecl smples (collected dily from one piglet per pen) nd digest smples collected from piglets tht were killed t the end of the experiment. Approximtely 1 g of feces nd 10 g of digest were suspended in pre-reduced slt medium (18) nd homogenised in lortory pddle lender (Sewrd Stomchers w 80 Biomster; L System) for 2 min. Seril 10-fold dilutions were then prepred in the sme medium nd smples (0 1 ml) were plted on selective medi. Hemolytic E. coli nd coliform cteri were enumerted on lood gr (Oxoid) nd McConkey gr (Merck), respectively, fter eroic incution t 378C for 1 d, wheres lctic cid cteri (LAB) were enumerted on de Mn Rogos Shrpe gr (Merck) fter neroic incution t 378C for 3 d.

4 926 S. Sugihrto et l. British Journl of Nutrition The numer of E. coli nd LAB is presented s the LAB:hemolytic E. coli rtio or the LAB:totl coliform cteri rtio. Immunogloulin nlyses The concentrtions of totl IgG, IgA nd IgM in the mucos, plsm nd sow milk nd those of IgA nd IgM in ile were mesured using the pig Ig ELISA quntifiction kit (Bethyl Lortories). The sme Ig types were mesured in the skimmed stndrdised BC nd commercil MR using the ovine Ig ELISA quntifiction kit (Bethyl Lortories). All ssys were performed ccording to the mnufcturer s instructions nd run in duplictes. The intr-ssy (withinplte) CV for the ELISA ws % nd the inter-ssy (etween-plte) CV ws %. Before the ssys, the mucos smples were vortexed in PBS (1:10, w/w) for 60 s nd centrifuged t 2000 g for 10 min nd the superntnt ws otined nd used for the determintion of Ig concentrtions nd totl protein content. The concentrtion of IgA in the intestinl mucos is expressed s the reltive mount of IgA to tht of totl protein. Totl protein content in the mucos ws quntified using the Advi 1650 utonlyser (Siemens Medicl Solutions). Gene expression nlyses The mucos ws homogenised y trnsferring it into lysis uffer (Mcherey-Ngel GmH & Co. KG) nd treted with rotor sttor homogeniser (TissueLyser LT; Qigen GmH) until it ws uniformly homogeneous. Totl RNA ws isolted using the NucleoSpin RNA kits (Mcherey-Ngel) ccording to the mnufcturer s protocols. The qulity nd quntity of RNA were ssessed using grose gel electrophoresis nd spectrophotometer (NnoDrop ND-1000; Sveen Werner), respectively. All smples were djusted to 200 ng/ml nd converted into complementry DNA using the High Cpcity cdna Reverse Trnscription Kit (Invitrogen) ccording to the mnufcturer s instructions. All PCR were run in 384-well pltes y mixing 2 ml of complementry DNA with 8 ml of the mix contining the primers, the proe nd 2 TqMn Mster Mix (ctlogue no ; Applied Biosystems) (Tle 2). The rections were run in duplicte for the trget genes nd triplicte for the housekeeping gene under stndrd mplifiction conditions determined for the Applied Biosystem ViiA 7 Rel-Time PCR system (Life Technologies). Gene expression cycle threshold (C t ) vlues were recorded using the Applied Biosystem ViiA 7 softwre. Glycerldehyde-3-phosphte dehydrogense (GAPDH) ws used s the housekeeping gene. The C t vlue for the trget genes of ech smple ws corrected y sutrcting the C t vlue of the GAPDH gene from the C t vlue of the corresponding gene (DC t ). The smples collected from the jejunl mucos of Bse piglets were used s the reference smples, nd the DC t vlue of ll the smples ws sutrcted from the verge DC t vlue of the reference smples (DDC t ). The fold chnge in the expression of ech trget gene ws clculted using the following formul: 2 2DDCt. Clcultions nd sttisticl nlyses Dt on weight gin, frequency of dirrhoe, rectl temperture, lertness score nd plsm Ig concentrtions were otined from ech piglet, while dt on microil counts, mucosl nd iliry Ig concentrtions, nd gene expression levels were otined from one piglet per pen. Bcteril popultion counts (cfu) were normlised using log trnsformtion, nd dt on weight gin of ech piglet were collected per pen efore nlysis. Dt on cteril counts otined from ETEC F18-inoculted jejunl or ilel smples (PIOC experiment) were verged per piglet efore nlysis. The frequency of dirrhoe nd lertness scores were nlysed using the GLIMMIX procedure of SAS (SAS Institute) Tle 2. Sequences of primers nd proes used in rel-time PCR Trgets Primer sequences Accession no. GAPDH Forwrd: 5 0 -GTCGGAGTGAACGGATTTGG-3 0 AF Reverse: 5 0 -CAATGTCCACTTTGCCAGAGTTAA-3 0 Proe: 5 0 -CGCCTGGTCACCAGGGCTGCT-3 0 IL-10 Forwrd: 5 0 -GAGGAGGTGAAGAGTGCCTTTA-3 0 L20001 Reverse: 5 0 -CTCACCCATGGCTTTGTAGACA-3 0 Proe: FAM-CCTCTCTTGGAGCTTGC-MGB TNF- Forwrd: 5 0 -AACCCTCTGGCCCAAGGA-3 0 X57321 Reverse: 5 0 -GGCGACGGGCTTATCTGA-3 0 Proe: FAM-TCAGATCATCGTCTCAAAC-MGB IL-2 Forwrd: 5 0 -GATCTCTCCAGGATGCTCACATTT-3 0 X56750 Reverse: 5 0 -CTCCAGAGCTTTGAGTTCTTCTACT-3 0 Proe: FAM-CCCAAGCAGGCTACAGAA-MGB COX-2 Forwrd: 5 0 -GGGACGATGAACGGCTGTT-3 0 NM_ Reverse: 5 0 -CACAATCTTAATCGTTTCTCCTATCAGT-3 0 Proe: 5 0 -AGACGAGCAGGCTGA-3 0 TLR-4 20 TgMn w Gene Expression Assy, ctlogue no (Applied Biosystems) TGF-1 20 TgMn w Gene Expression Assy, ctlogue no (Applied Biosystems) GAPDH, glycerldehyde-3-phosphte dehydrogense; COX-2, cyclo-oxygense-2; TLR-4, Toll-like receptor-4; TGF-1, trnsforming growth fctor-1.

5 Bovine colostrum nd pig gut helth 927 British Journl of Nutrition with tretment s fixed effect nd litter nd pen s the rndom effects to ccount for the mesurement eing mde in piglets from different litters nd different pens. Dt on performnce nd iliry Ig concentrtions were nlysed with mixed model using the MIXED procedure of SAS with tretment nd litter s the fixed nd rndom effects, respectively. The remining dt tht were otined on vrious dys (rectl temperture, LAB:E. coli rtio in feces nd plsm Ig concentrtions) or from vrious segments of the gut (LAB: E. coli rtio in the digest, numer of E. coli colonising the intestine, mucosl Ig concentrtions nd gene expression levels) of the sme piglet were nlysed using the MIXED procedure with tretment nd smpling dy or gut/intestinl site s the fixed effects nd litter s rndom effect nd tking into ccount tht smples originted from the sme niml. For dt on rectl temperture nd plsm Ig concentrtions, which were otined from piglets from different litters nd pens, the rndom effects of litter nd pen were imposed. The interction etween tretment nd smpling dy or gut/intestinl site ws initilly included in the model, ut excluded when it ws not sttisticlly significnt. Differences mong the tretment groups were tested using the post hoc Tukey Krmer test. Differences were considered significnt when P# Results re presented s lest-squres mens nd stndrd errors. Results Growth performnce nd frequency of dirrhoe During the experiment, two piglets (from the BC-fed nd MR-fed groups) died nd so dt otined from these piglets were not included in the nlyses. Piglets tht were kept with the sows, i.e. the Sow-Milk group, exhiited higher (P# 0 029) weight gin compred with those seprted from the sows nd fed BC or MR (Tle 3). The frequency of dirrhoe ws higher (P#0 019) in MR-fed piglets thn in BC-fed Tle 3. Growth performnce nd frequency of dirrhoe in piglets in response to the tretments (Lest-squres mens with their stndrd errors) Tretments Items BC-fed MR-fed Sow-Milk SE P Weight gin (g)* Milk intke (g)* NR Pig dys Frequency of dirrhoe BC-fed, piglets seprted from the sow nd fed ovine colostrum; MR-fed, piglets seprted from the sow nd fed commercil milk replcer; Sow-Milk, piglets kept with the sow; NR, not recorded., Vlues within row with unlike superscript letters were significntly different (P#0 05). * Dt presented re the weight gin nd milk intke recorded in ech pen throughout the experimentl period. BC-fed, n 3 pens; MR-fed, n 3 pens; Sow-Milk, n 4 pens. Dt collected from pens with only two piglets (due to the deth of one piglet) were not included in the sttisticl nlyses. Pig dys ¼ numer of piglets numer of dys of dirrhoe scoring. BC-fed, n 11 piglets; MR-fed, n 11 piglets; Sow-Milk, n 12 piglets. Dirrhoe scoring ws conducted on dys 1, 3, 5 nd 8 of the experiment. Frequency of dirrhoe ¼ numer of pig dys with dirrhoe score.3. nd Sow-Milk piglets (Tle 3). There were no significnt differences with regrd to the lertness of piglets throughout the experiment (dt not shown). The rectl temperture did not differ significntly mong the tretment groups throughout the experiment (rnged from 37 to 408C; dt not shown). Rtios of lctic cid cteri:escherichi coli in the feces nd gstrointestinl digest The rtios of LAB:hemolytic E. coli nd LAB:totl coliform cteri in the feces nd GIT digest of piglets re shown in Figs. 1 nd 2, respectively. The LAB:hemolytic E. coli rtio ws higher (P dy ¼ 0 029) during the first 3 d of the experiment thn in the remining 5 d nd tended (P tret ¼ 0 064) to e lower in the feces of MR-fed piglets thn in tht of BC-fed nd Sow-Milk piglets. The Bse nd Sow-Milk piglets hd higher LAB: coliform cteri rtio in the digest collected from the smll intestine when compred with MR-fed piglets (P# 0 043), ut exhiited no differences when compred with BC-fed piglets. The LAB:hemolytic E. coli rtio in the gstrointestinl digest of MR-fed piglets exhiited decresing tendency (P tret ¼ 0 068) when compred with tht in piglets from the other tretment groups. Numer of Escherichi coli colonising the intestinl tissue The numer of E. coli colonising the jejunl nd ilel tissue nd content is summrised in Tle 4. The numer of E. coli ws higher (P# 0 039) in the jejunl nd ilel tissue nd content in the non-inoculted intestinl smples of MR-fed piglets thn in the smples of BC-fed nd Sow-Milk piglets. The numer of E. coli ws higher (P seg, 0 001) in the ilel tissue nd content thn in the jejunl tissue nd content. The numer of E. coli colonising the intestinl tissue in the ETEC F18-inoculted jejunl nd ilel smples of BC-fed piglets ws lower thn tht in the smples of MR-fed piglets, ut there were no significnt differences in vlues (P tret ¼ 0 320). Ig concentrtions in the intestinl mucos, ile nd plsm The concentrtions of totl Ig in the intestinl mucos nd ile of piglets re given in Tle 5. Piglets from the Sow-Milk group hd higher (P¼0 020) concentrtion of IgG in the mucos when compred with piglets from the MR-fed group, ut did not exhiit ny difference when compred with piglets from the Bse nd BC-fed groups. The concentrtion of mucosl IgA ws found to e influenced y the interction etween tretment nd intestinl segment (P tret seg ¼ 0 087), s evidenced y the higher concentrtions of IgA in the jejunum nd ileum of Bse piglets thn in those of piglets from the other tretment groups. Piglets from the MR-fed group hd lower (P¼0 051) concentrtion of totl IgM in the mucos when compred with those from the Bse group, ut did not exhiit ny difference when compred with piglets from the BC-fed nd Sow-Milk groups. Regrdless of tretment, the concentrtion of totl IgM ws higher (P#0 045) in the ileum thn in the duodenum or jejunum, while tht of IgA ws higher (P¼0 034) in the ileum thn

6 928 S. Sugihrto et l. (A) Rtio (B) c Dy of experiment 1 2 Sow-Milk piglets. The expression level of IL-2 ws higher (P#0 051) in the intestinl mucos of piglets from the MRfed group thn in tht of piglets from the other tretment groups. Compred with tht in piglets from the other tretment groups, the expression level of cyclo-oxygense-2 (COX-2) ws lower (P# 0 047) in the intestinl mucos of Bse piglets nd ws significntly higher in MR-fed piglets. The level of IL-10 expression ws lower (P# 0 036) in the intestinl mucos of Bse piglets thn in tht of MR-fed nd Sow-Milk piglets, ut did not differ from tht in BC-fed piglets. No significnt effect of tretment ws oserved on the expression levels of TNF- nd trnsforming growth fctor1 (TGF-1). Dt on gene expression levels in the jejunl nd ilel mucos smples collected from the PIOC model were found to e invlid in the present study due to the inconsistent gene expression ptterns mesured. Compred with those in the control, gene expression levels in the mucos of ETEC British Journl of Nutrition Rtio Dy of experiment Fig. 1. Lctic cid cteri:hemolytic Escherichi coli rtio (A) nd lctic cid cteri:totl coliform cteri rtio (B) in the feces of piglets. Vlues re lest-squres mens, with their stndrd errors represented y verticl rs.,,c Men vlues with unlike letters were significntly different (P#0 05). Four piglets were nlysed per tretment nd dy of experiment (fecl smpling). BC-fed ( X ), piglets seprted from the sow nd fed ovine colostrum; MR-fed ( W ), piglets seprted from the sow nd fed commercil milk replcer; Sow-Milk ( P ), piglets kept with the sow. (A) P tret ¼ 0 064; P dy ¼ 0 029; P tret dy ¼ (B) P tret ¼ 0 143; P dy ¼ 0 784; P tret dy ¼ (A) Rtio (B) Stomch Proximl SI Distl SI Cecum Colon Gstrointestinl segment in the duodenum, ut did not differ from tht in the jejunum. No significnt differences were oserved with regrd to the concentrtions of IgA (P¼0 236) nd IgM (P¼0 062) in the ile of piglets; however, Bse piglets tended (P# 0 089) to hve lower concentrtion of IgM when compred with piglets from the other tretment groups. There were no significnt differences in the plsm concentrtions of Ig mong the tretment groups. Irrespective of the tretment, the concentrtion of IgG ws lower (P dy, 0 001) on dy 8 thn on dy 1 (Tle 6). Gene expression levels in the intestinl mucos Gene expression levels in the jejunl nd ilel mucos of piglets re shown in Fig. 3. The genes were expressed t higher (P seg # 0 010) levels in the ilel mucos thn in the jejunl mucos, except for the IL-2 gene. The expression level of TLR-4 ws lower (P#0 037) in the jejunl nd ilel mucos of Bse nd BC-fed piglets thn in those of MR-fed nd Rtio ,, Stomch Proximl SI Distl SI Cecum Colon Gstrointestinl segment Fig. 2. Lctic cid cteri:hemolytic E. coli rtio (A) nd lctic cid cteri:totl coliform cteri rtio (B) in the gstrointestinl digest of piglets. Vlues re lest-squres mens, with their stndrd errors represented y verticl rs., Men vlues with unlike letters were significntly different (P#0 05). Four piglets were nlysed per tretment nd gstrointestinl segment. Bse ( ), piglets killed t the eginning of the experiment; BC-fed ( ), piglets seprted from the sow nd fed ovine colostrum; MR-fed ( ), piglets seprted from the sow nd fed commercil milk replcer; Sow-Milk ( ), piglets kept with the sow. SI, smll intestine. (A) P tret ¼ 0 068; P seg, 0 001; P tret seg ¼ (B) P tret ¼ 0 302; P seg, 0 001; P tret seg ¼

7 Bovine colostrum nd pig gut helth 929 Tle 4. Numer of Escherichi coli colonising the jejunl or ilel tissue nd content in the porcine intestinl orgn cultures of smples collected from the treted piglets (Lest-squres mens with their stndrd errors) Tretments* P Items BC-fed MR-fed Sow-Milk SE Tret Seg Tret seg Non-inoculted tissue (log cfu/g),0 001, Jejunum Ileum Non-inoculted content (log cfu/ml) 0 020, Jejunum Ileum E. coli-inoculted tissue (log cfu/g) Jejunum Ileum E. coli-inoculted content (log cfu/ml) Jejunum Ileum British Journl of Nutrition F18-inoculted segments were higher, lower or similr, with no cler ptterns. Therefore, the vlues re not reported or discussed here. Discussion BC-fed, piglets seprted from the sow nd fed ovine colostrum; MR-fed, piglets seprted from the sow nd fed commercil milk replcer; Sow-Milk, piglets kept with the sow; Tret, tretment; Seg, segment of the smll intestine; cfu, colony-forming units., Vlues within row with unlike superscript letters were significntly different (P#0 05). * Four piglets were nlysed per tretment (of ech smll-intestinl site, one segment ws not inoculted nd two segments were inoculted with Escherichi coli F18). Intestinl segments (collected from the treted piglets) were not inoculted with E. coli F18 during the porcine intestinl orgn culture (PIOC) experiment. Intestinl segments (collected from the treted piglets) were inoculted with E. coli F18 (8 96 (SE 0 29) log colony-forming units/ml for ech segment) during the PIOC experiment. Aout 3 weeks of ge, piglets hve the lowest concentrtions of mternl ntiodies cquired from the sow milk nd their immune system is not s well developed s tht of dults (7). Thus, due to similrities, the use of 23-d-old piglets in the present study could provide some informtion regrding wek piglets hving n incompetent intestinl immune system. Indeed, t this ge, piglets re lso highly susceptile to ETEC F18, given tht F18 receptors on porcine enterocytes re functionlly ctive (F18 receptors re not yet fully ctive in piglets under out 3 weeks of ge) (19). Severl studies hve reported positive effects of BC-supplemented diets on the immune responses of piglets (12,20). In generl, BC tretment ws minly used to provide dditionl Ig to wened piglets following the loss of pssive protection provided y the sow milk. However, dt on the efficcy of BC supplements in enhncing ntiody responses in piglets fter Tle 5. Concentrtions of totl Ig in the intestinl mucos nd ile of piglets in response to the tretments (Lest-squres mens with their stndrd errors) Tretments* P Items Bse BC-fed MR-fed Sow-Milk SE Tret Seg Tret seg IgG (mg/g) Duodenum 0 66, 0 85, Jejunum 0 93, 0 50, Ileum 0 60, 0 68, IgA (mg/g) Duodenum Jejunum Ileum IgM (mg/g) Duodenum , , 1 22 Jejunum , , 0 72 Ileum , , 0 94 Biliry IgA (mg/l) Biliry IgM (mg/l) Bse, Piglets killed t the eginning of the experiment; BC-fed, piglets seprted from the sow nd fed ovine colostrum; MR-fed, piglets seprted from the sow nd fed commercil milk replcer; Sow-Milk, piglets kept with the sow; Tret, tretment; Seg, segment of the smll intestine., Vlues within row with unlike superscript letters were significntly different (P#0 05). * Four piglets were nlysed per tretment nd smll-intestinl site.

8 930 S. Sugihrto et l. Tle 6. Concentrtions of totl Ig in the plsm of piglets in response to the tretments (Lest-squres mens with their stndrd errors) Tretments P Items BC-fed (n 11) MR-fed (n 11) Sow-Milk (n 12) SE Tret Dy Tret dy IgG (mg/l) 0 606, Dy Dy IgA (mg/l) Dy Dy IgM (mg/l) Dy Dy BC-fed, piglets seprted from the sow nd fed ovine colostrum; MR-fed, piglets seprted from the sow nd fed commercil milk replcer; Sow-Milk, piglets kept with the sow; Tret, tretment. British Journl of Nutrition wening hve een inconsistent. Boudry et l. (12) reported tht orl supplementtion with BC (piglets received 5 g of BC orlly dily for 3 weeks) incresed serum IgA concentrtions (fter 3 weeks of BC supplementtion, ut not efore), ut trnsiently decresed totl IgG concentrtions nd hd no influence on totl IgM concentrtions. In ddition, totl IgG, IgA nd IgM concentrtions in intestinl fluid smples were not ffected y BC supplementtion. In nother study, Boudry et l. (20) demonstrted tht BC whey supplementtion (20 g/kg of BC whey incorported in the diet during the first 2 weeks fter wening nd 10 g/kg for the next 2 weeks) incresed circulting IgA concentrtions y 25 % (during the 1st week fter wening, ut not during the remining experimentl period) nd hd no impct on totl IgG nd IgM concentrtions. Tking this into considertion, feeding commercil BC preprtion s the only dietry ingredient, rther thn s supplement, could e speculted to hve eneficil effects on the immune responses of rtificilly rered wek piglets efore wening to greter extent. It is known tht the nutritionl composition of diet, prticulrly protein, ffects the immunocompetence of nimls (21). There ws sustntil difference in the protein content of BC nd tht of the other two diets in the present study. Indeed, protein in BC is minly composed of Ig (70 80 % of the totl protein content) (4) nd hence ny influence of BC on the immune system of piglets cn resonly e ssumed to e due to Ig (nd other immune fctors such s cytokines) in BC. In the present study, feeding commercil BC product rther thn MR ws found to hve the cpcity to reduce the frequency of dirrhoe in piglets nd no differences were ctully found in the frequency of dirrhoe in BC-fed piglets nd Sow- Milk piglets. Corresponding results hve een reported y Huguet et l. (22), who found tht feeding BC-supplemented diet reduced dirrhoel episodes in wened piglets. BC hs een reported to contin growth fctors essentil for promoting the growth nd development of neworn piglets (4). Yet, feeding BC could not improve the growth performnce of piglets in the present study, s evidenced y the finding tht piglets stying with their dms gined more weight thn those rtificilly rered on BC. All piglets used in the present study were geneticlly susceptile to ETEC expressing F18 fimrie. Previous studies hve shown tht BC exhiits ntimicroil ctivity through its functionl components such s Ig (4,13). In ccordnce with this, cteril counts in the fecl nd digest smples reveled tht feeding BC resulted in lower counts of hemolytic E. coli (indictor of ETEC) compred with feeding commercil MR. The men hemolytic E. coli count in the feces ws 5 97 (SE 0 72) log cfu/g in BC-fed piglets, 6 58 (SE 1 34) log cfu/g in MR-fed piglets nd 5 87 (SE 0 42) log cfu/g in Sow-Milk piglets. The men hemolytic E. coli count in the digest cross the GIT ws 6 34 (SE 1 63) log cfu/g in BC-fed piglets, 7 05 (SE 1 90) log/cfu in MR-fed piglets nd 5 28 (SE 0 78) log/cfu in Sow-Milk piglets. These vlues re within the norml rnge for hemolytic E. coli enumerted in the feces nd intestinl content of piglets fter wening (23,24). Consistent with the cteril counts in the fecl nd GIT digest smples, jejunl nd ilel tissue smples collected from BC-fed (nd Sow-Milk) piglets exhiited significnt reduction in the recovery of E. coli from the tissue when compred with smples collected from MRfed piglets. Dt from the PIOC experiment lso showed tht, reltively to feeding MR, feeding BC is ssocited with lower colonistion of E. coli in the intestinl tissue fter inocultion with ETEC F18. This is in greement with the results of erlier studies reporting n inhiitory effect of BC on the dherence of enterohemorrhgic E. coli nd enteropthogenic E. coli to the intestinl mucos of mice nd HEp-2 cells, respectively (25,26). In nother in vitro study, pre-tretment with BC ws found to decrese the ttchment of Shig toxin-producing E. coli O111 to colon-derived cell lines (HT-29) (27). Altogether, we infer tht BC is cple of inhiiting the ttchment nd/or colonistion of ETEC in the intestinl mucos of piglets. It hs previously een shown tht BC feeding could preserve the lnced microiot in the intestine of piglets (4,28). Accordingly, the results of the present study reveled tht the LAB:hemolytic E. coli nd LAB:coliform cteri rtios in the fecl nd digest smples collected from BC-fed piglets were higher thn those in smples collected from MR-fed piglets. This further provides n indiction tht BC is cple

9 Bovine colostrum nd pig gut helth 931 (A) 30 (B) Fold chnge Fold chnge Jejunum Ileum 20 0 Jejunum Ileum Smll-intestinl segment Smll-intestinl segment (C) 12 (D) British Journl of Nutrition Fold chnge (E) Jejunum Ileum Smll-intestinl segment Fold chnge (F) Jejunum Ileum Smll-intestinl segment Fold chnge ,, Fold chnge Jejunum Ileum Smll-intestinl segment 0 Jejunum Ileum Smll-intestinl segment Fig. 3. Gene expression levels of Toll-like receptor-4 (A), cyclo-oxygense-2 (B), TNF- (C), IL-2 (D), IL-10 (E) nd trnsforming growth fctor-1 (F) in the intestinl mucos of piglets. Vlues re lest-squres mens with their stndrd errors represented y verticl rs., Men vlues with unlike letters were significntly different (P#0 05). Four piglets were nlysed per tretment nd smll-intestinl segment. Bse ( ), piglets killed t the eginning of the experiment; BC-fed ( ), piglets seprted from the sow nd fed ovine colostrum; MR-fed ( ), piglets seprted from the sow nd fed commercil milk replcer; Sow-Milk ( ), piglets kept with the sow. (A) P tret ¼ 0 011; P seg ¼ 0 010; P tret seg ¼ (B) P tret ¼ 0 046; P seg ¼ 0 007; P tret seg ¼ (C) P tret ¼ 0 100; P seg ¼ 0 009; P tret seg ¼ (D) P tret ¼ 0 009; P seg ¼ 0 171; P tret seg ¼ (E) P tret ¼ 0 022; P seg ¼ 0 005; P tret seg ¼ (F) P tret ¼ 0 161; P seg ¼ 0 002; P tret seg ¼ of inhiiting the growth of coliform cteri nd ETEC in the gut of piglets. The results lso reveled tht the LAB: hemolytic E. coli rtio in the feces decresed with time fter experimentl tretment. However, the decrese ws minly oserved in piglets tht were seprted from the sows, suggesting tht seprtion from the sows ltered the lnced gut microiot in piglets. As hs een oserved in the present study, previous studies hve reported tht the numer of enteric cteril pthogens increse, wheres LAB popultion levels re suppressed fter wening (4,10,29).

10 932 S. Sugihrto et l. British Journl of Nutrition As hs een discussed ove, BC is n importnt source of Ig tht my provide the host with pssive immunity ginst infectious pthogens (4,13). In the present study, the concentrtions of Ig in the BC were much higher thn those in the sow milk nd MR. Yet, the resulting concentrtions of Ig in the mucos of BC-fed piglets did not differ significntly from those in the mucos of MR-fed or Sow-Milk piglets. Similr results were reported y Boudry et l. (12), s hs een mentioned ove. The inility of porcine Ig receptors on the surfce of enterocytes to efficiently ind to the ovine Ig (30) my explin this finding. The ovine colostrl Ig tht re not sored y the intestine (remining in the intestinl lumen) my, however, still provide pssive locl immunity to piglets y inding to the ntigens nd protecting the intestinl tissue through immune exclusion (13). In the present study, feeding BC to the piglets did not result in differences in terms of circultory Ig concentrtions. The low cpcity of the intestine to sor ovine Ig (30) nd/or gut closure (12,31) my explin the poor trnsfer of ovine Ig from the gut to the loodstrem in piglets during the period fter seprtion from the sows. Irrespective of the tretment, the concentrtion of plsm IgG ws higher on dy 1 thn on dy 8 of the experiment. The decrese in totl plsm IgG concentrtions could e ssocited with the ge effect or decy of pssively cquired mternl IgG (12). In ccordnce with the systemic Ig, the concentrtions of totl IgA were higher in the jejunl nd ilel mucos of piglets from the Bse group, killed t the eginning of the experiment, thn in tht of piglets from the other tretment groups, which were killed 8 d lter. Chnges in the diet nd environment of piglets fter seprtion from the sows hve een known to e ssocited with n incresed numer of pthogenic cteri in the vrious regions of the GIT (10). Through TLR, these cteri re le to produce signls tht up-regulte the expression of pro-inflmmtory fctors nd stimulte host immune responses (32). In the present study, BC ws le to reduce the intestinl colonistion y ETEC nd, ccordingly, the incresed expression of TLR-4 ws not oserved in the intestinl mucos of BC-fed piglets. The relese of cytokines or other inflmmtory fctors upon recognition of Grm-negtive pthogenic cteri is one of the most importnt effects of TLR-4 ctivtion (32). In the cse of BC-fed piglets, low ctivtion of TLR-4 y ETEC my therefore e implicted in the lower expression of pro-inflmmtory meditors, pprently s in the lower expression levels of IL-2 (inflmmtory meditors in response to cteril infections (33) ) in the intestinl mucos. TNF- is n inflmmtory cytokine tht is normlly up-regulted upon ntigen stimultion (27). Unlike those of IL-2, the expression levels of TNF- in the intestinl mucos did not differ mong the tretment groups in the present study. BC is known to contin severl potentil proinflmmtory components, including TNF- nd IL-6 (4). Therefore, pre-tretment with BC increses the production of TNF- nd IL-6 in oth ntigen-stimulted nd unstimulted humn monocytic THP-1 cells (ATCCIIB-202) (27). Mny immunomodultory fctors such s TNF- nd IL-10 re lso present in the sow milk (5). Tken together, the potentil of BC nd sow milk to up-regulte the expression of TNF- my therefore explin the lck of difference in the expression levels of this cytokine mong the tretment groups, lthough the smll intestine of BC-fed nd Sow- Milk piglets ws less colonised y the E. coli compred with tht of MR-fed piglets. IL-10 is n nti-inflmmtory cytokine tht plys criticl immunoregultory role (lnces immune ctivtion) during immune responses. There re diverse stimuli tht cn induce the production of this cytokine, mong which re cteri nd cteril products (enterotoxins) (34). Concomitnt with tht of IL-10, the expression of TGF-1, which is n essentil signl for the genertion of regultory T cells nd T-helper 17 cells, is induced y cteril infections (35). In conjunction with the higher popultion levels of E. coli in the intestine of MR-fed piglets tht might led to higher expression levels of IL-10 nd TGF-1 in the mucos, the presence of IL-10 (5) nd TGF-1 (4,5) in the BC nd sow milk ppered to e responsile for the incresed expression levels of these immune meditors in the intestinl mucos of BCnd sow milk-fed piglets (36). This condition my explin the insignificnt differences in IL-10 nd TGF-1expression levels mong the tretment groups in the present study. An interesting finding from the present study is tht genes (TLR-4, COX-2, TNF-, IL-10 nd TGF-1) were expressed t higher levels in the ilel mucos thn in the jejunl mucos of piglets, irrespective of the tretment. Consistent with this, the concentrtions of totl IgA nd IgM were higher in the ilel mucos thn in the jejunl mucos. Bsed on dt from the PIOC experiment, in which more E. coli were enumerted in ilel tissue thn in jejunl tissue, we infer tht the immune responses my prtly e ffected y the degree of colonistion of cteril pthogens in the gut. This is in ccordnce with the results of studies of Wng et l. (35) nd Dudelin et l. (37), who reported tht the colonistion of E. coli or other cteril pthogens in the intestine is responsile for the stimultion of the host-relted immune responses. In conclusion, feeding BC to the piglets reduced the intestinl colonistion of ETEC nd modulted the intestinl mucosl immune system when compred with feeding MR. Rering piglets on BC ws s good s conventionl rering y sows in terms of optimistion of the immune defence of wek piglets. Hence, future reserch should consider the potentil of BC supplementtion in wek piglets. Acknowledgements The uthors thnk Mlene Cilieorg, KU-Life (Copenhgen University), Denmrk, for providing the E. coli strin O138:F18. They lso thnk Krin Durup nd Inger-Mrie Jepsen for their technicl ssistnce during the experiment nd Mette Lykkegrd Jørgensen nd Krin Johnsen for their ssistnce with the niml cre. Funding for the present study ws received through the NEOMUNE Centre ( Erly milk nd microiot to stimulte lter immunity ) y The Dnish Council for Strtegic Reserch nd the Grdute School of Science nd Technology, Arhus

11 Bovine colostrum nd pig gut helth 933 British Journl of Nutrition University, Denmrk, for PhD scholrship (A.-S. R. P.) nd through PhD scholrship received from the Ministry of Eduction nd Culture, Repulic of Indonesi (S. S.). The funders hd no role in the design or nlysis of the study nd in the writing of this mnuscript. Trde nmes or commercil products hve een mentioned in the rticle simply for the purpose of providing specific informtion nd not for recommendtion or endorsement purposes. The uthors contriutions re s follows: S. S. conducted the niml tril, crried out the smple nlyses, nlysed the dt nd drfted the mnuscript; A.-S. R. P. conducted the niml tril nd revised the drfted mnuscript; N. C. contriuted to the study design, conducted the niml tril, ssisted with the dt nlyses nd revised the drfted mnuscript; C. L. ws the project co-ordintor, designed the experiment nd revised the drfted mnuscript. None of the uthors hs ny conflicts of interest to declre. References 1. 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