The use of phage FCL-2 as an alternative to chemotherapy against columnaris disease in aquaculture

This is an elec onic ep in o he o iginal a icle. This ep in may di e om he o iginal in pagina ion and ypog aphic de ail. Au ho (s): Ti le: Yea : Ve sion: Please ci e he o iginal e sion: All ma e ial supplied ia JYX is p o ec ed by copy igh and o he in ellec ual p ope y igh s, and duplica ion o sale o all o pa o any o he eposi o y collec ions is no pe mi ed, excep ha ma e ial may be duplica ed by you o you esea ch use o educa ional pu poses in elec onic o p in o m. You mus ob ain pe mission o any o he use. Elec onic o p in copies may no be o e ed, whe he o sale o o he wise o anyone who is no an au ho ised use . The use o phage FCL-2 as an al e na i e o chemo he apy agains columna is disease in aquacul u e Laan o, Elina; Bam o d, Jaana; Ra an i, Janne; Sundbe g, Lo a-Riina Laan o, E., Bam o d, J., Ra an i, J., & Sundbe g, L.-R. (2015). The use o phage FCL-2 as an al e na i e o chemo he apy agains columna is disease in aquacul u e. F on ie s in Mic obiology, 6, A icle 829. h ps://doi.o g/10.3389/ micb.2015.00829 2015 ORIGINAL RESEARCH published: 19 Augus 2015 doi: 10.3389/ micb.2015.00829 Edi ed by: Hongyue Dang, Xiamen Uni e si y, China Re iewed by: Robe o Bas ías, Pon i ical Ca holic Uni e si y o Valpa aíso, Chile Da en Lee Smi h, Uni e si y o No humb ia a Newcas le, UK *Co espondence: Elina Laan o, Cen e o Excellence in Biological In e ac ions, Depa men o Biological and En i onmen al Science, Uni e si y o Jy askyla, P.O. Box 35, FI-40014 Jy askyla, Finland elina.laan o@jyu. i Special y sec ion: This a icle was submi ed o Aqua ic Mic obiology, asec iono hejou nal F on ie s in Mic obiology Recei ed: 17 June 2015 Accep ed: 28 July 2015 Published: 19 Augus 2015 Ci a ion: Laan o E, Bam o d JKH, Ra an i JJ and Sundbe g L-R (2015) The use o phage FCL-2 as an al e na i e o chemo he apy agains columna is disease in aquacul u e. F on . Mic obiol. 6:829. doi: 10.3389/ micb.2015.00829 The use o phage FCL-2 as an al e na i e o chemo he apy agains columna is disease in aquacul u e Elina Laan o1*,JaanaK.H.Bam o d 1, Janne J. Ra an i 1,2 and Lo a-Riina Sundbe g1 1Cen e o Excellence in Biological In e ac ions, Depa men o Biological and En i onmen al Science, Uni e si y o Jy askyla, Jy askyla, Finland, 2Depa men o Biosciences and Ins i u e o Bio echnology, Uni e si y o Helsinki, Helsinki, Finland Fla obac e ium columna e, hecausa i eagen o columna isdiseasein ish,causes millions o dolla s o losses in he US channel ca ish indus y alone, no o men ion aquacul u e indus y wo ldwide. No el me hods a e needed o he con ol and ea men o bac e ial diseases in aquacul u e o eplace adi ionally used chemo he apies. A po en ial solu ion could be he use o phages, i.e., bac e ial i uses, hos -speci ic and sel -en iching pa icles ha can be can easily dis ibu ed ia wa e low. We examined he e icacy o phages o comba columna is disease. A p e iously isola ed phage, FCL-2, in ec ing F. columna e,wascha ac e izedbysequencing.The47142bp genome o he phage had G +Ccon en o 30.2%,and hecloses simila i ies ega ding he s uc u al p o eins we e ound in Cellulophaga phage phiSM. Unde con olled expe imen al condi ions, wo hos ish species, ainbow ou (Onco hynchus mykiss) and zeb a ish (Danio e io), we e used o s udy he success o phage he apy o p e en F. colu mna e in ec ions. The su i al o bo h ish species was signi ican ly highe in he p esence o he phage. Hund ed pe cen o he zeb a ish and 50% o he ainbow ou su i ed in he phage ea men (su i al wi hou phage 0 and 8.3%, espec i ely). Mos impo an ly, he ainbow ou popula ion was escued om in ec ion by a single addi ion o he phage in o he wa e in a low- h ough ish ank sys em. Thus, F. column a e could be used as a model sys em o es he bene i s and isks o phage he apy on a la ge scale. Keywo ds: aquacul u e, disease, ish, Fla obac e ium columna e,phage he apy In oduc ion In 2014, he WHO (2014) epo edhigh a eso an imic obial esis anceincommondisease- causing bac e ia in all egions o he wo ld. Conce ns o an ibio ic esis ance ha e also b ough a en ion o he la ge amoun s o an ibio ics used in animal p oduc ion, also in aquacul u e (Buschmann e al., 2012;Hollis and Ahmed, 2013). An ibio ics used in aquacul u e may dissol e om he ish eed o he su ounding wa e (Cabello e al., 2013), hus enabling he de elopmen o an ibio ic esis ance in en i onmen al mic obes (Buschmann e al., 2012;Di Cesa e e al., 2013). Indeed, concen a ions o ypically used an imic obials (e.g., e acycline) in ma ine sedimen s can be highe han he minimal inhibi o y concen a ions o mos bac e ia ( e iewed in Cabello e al., 2013). F on ie s in Mic obiology | www. on ie sin.o g 1Augus 2015 | Volume 6 | A icle 829 Laan o e al. Phage he apy agains a ish pa hogen As aquacul u e is he as es g owing indus y o animal p o ein p oduc ion in he wo ld (Bos ock e al., 2010), no el me hods a e needed o managemen o bac e ial diseases. One op ion is phage he apy, which is al eady conside ed a easible subs i u e o an ibio ics in heal hca e, li es ock, and c op p oduc ion (Smi h and Huggins, 1983;Be chie i e al., 1991;Ba ow e al., 1998;Nakai and Pa k, 2002;Jamalludeen e al., 2009;San os e al., 2010;Abedon e al., 2011;Jones e al., 2012). Al hough he isk o de elopmen o phage esis ance in he a ge bac e ia needs o be conside ed, by e.g., using mul iphage cock ails, phage he apy has g ea po en ial in he ea men o aqua ic o ganisms. In con as o an ibio ics, phages a e hos -speci ic and sel -en iching pa icles ha can be can easily dis ibu ed ia wa e low wi hin ea ing uni s. Columna is disease (caused by Fla obac e ium columna e, Bac e oide es) is a signi ican p oblem in eshwa e ish a ming wo ldwide (Pulkkinen e al., 2010;Decle cq e al., 2013). Cu en ly, ish wi h columna is disease a e ea ed wi h an ibio ics. Columna is disease is a good candida e a ge o phage he apy since he disease is mainly ex e nal (on he ish gills, ins, and skin; Be na de , 1997;T ipa hi e al., 2005)and hebac e ia ansmi h oughwa e (Welke e al., 2005;Kun u e al., 2009). The p esen s udy cha ac e izes a phage, FCL-2 (Laan o e al., 2011), in ec ing F. columna e and examines how he phage ea men affec s columna is disease in ainbow ou (Onco hynchus mykiss)andzeb a ish(Danio e io)unde expe imen alcondi ions.Weshow ha hephage can signi ican ly al e he ou come o in ec ion by F. columna e. In addi ion o p o ing i s unc ionali y in a speci ic sys em, his s udy highligh s he use o F. columna e and i s phage as a model o he implemen a ion o phages as a ea men , namely phage he apy, in a eal disease con ex in a eal hos . This sys em could also be used o es ima e he possible (en i onmen al) side effec s o he me hod in p ac ice, such as he effec o he p esence o ish hos and an ibio ic ea men s on e olu ion o phage esis ance, o he effec o phage addi ions on na u al bac e ial communi y. Ma e ials and Me hods Bac e ia and Phage Fla obac e ium columna e s ain B185 and phage FCL-2 we e bo h isola ed om a ish a m ea ing mainly salmonid inge lings du ing a columna is ou b eak in 2008 (Laan o e al., 2011). Bac e ia and phage we e cul u ed in Shieh medium (Decos e e e al., 1997), wi hou ob amycin (24◦C, 110 pm) and s o ed in 10% glyce ol and 10% e al cal se um a –80◦C. The op ical densi y o B185 was measu ed a 570 nm o de e mine he colony o ming uni s (CFU) pe mL based on ou unpublished analyses. Fo plaque assays he “double laye aga ” –me hod (Adams, 1959) was used as ollows: mel ed op aga (0.7%) including 300 µLo hos bac e ium and 100 µLo phagedilu ionwaspou edonShieh aga and g own o 48 h a oom empe a u e. Phage s ocks we e p epa ed om Shieh aga pla es wi h con luen lysis by adding 5mLo Shieh-mediumon opo apla eandincuba eda 6 ◦C o 6h.Thelysa ewascollec ed, il e ed, and s o ed a +4◦Cand o longe pe iods a –80◦Cwi h10%glyce ol.Phagemo phology was s udied p e iously (Laan o e al., 2011), bu a highe quali y ansmission elec on mic oscope was ob ained o his s udy (Jeol JEM-1400 a 80 kV). Fo ansmission elec on mic oscopy (TEM) analysis, he phage was pelle ed (Beckman coul e L-90K, 70 Ti- o o , 25 000 ×g,2h,+4◦C) and washed wice wi h 0.1 M ammonium ace a e, pelle ed as abo e and suspended in 0.02 M po assium phospha e. The phage was spo ed on a coppe - coa ed g id o 2 min and excess suspension was d ied wi h il e pape . Ten mic oli e s o 1% phospho ungs a e, a pH 6.5, we e applied on he g id o 1 min and he g id was d ied wi h il e pape . Phage Adso p ion and S abili y The phage adso p ion a e was measu ed in h ee eplica es in 1 mL o Shieh medium wi h loga i hmic phase B185 cells (1.4 ×108CFU), wi h a doubling ime o app oxima ely 10 h a RT (Zhang e al., 2014). Phage (2.2 ×104PFU, esul ing o a MOI o 1.6 ×10−4)wasadded oeach ubeand ubeswe eshakenand hen le o s and a RT; he con ol con ained only he medium wi hou cells. The bac e ium-phage mix u e was pelle ed a e 1, 5, 10, 20, 40, and 60 min by cen i uga ion (15 000 ×g)and he PFU mL−1o he ee phage pa icles in he supe na an a each ime poin was de e mined o calcula e he co esponding numbe o adso bed phage pa icles. The adso p ion a e was de e mined as he exponen ial dec ease o ee phage pa icles du ing he ime o incuba ion. The phage abili y o emain in ec i e in diffe en buffe s was es ed by dilu ing FCL-2 lysa e (o iginal 1.2 ×1011 PFU mL−1) a a a io o 1:100 in Shieh medium, in o 20 mM T is-HCl (pH 6.1, 7.6, and 8.1) and 20 mM po assium phospha e (pH 6.0, 7.2, and 8.0). All dilu ions we e kep a 6◦C(andone dilu ion in Shieh medium a oom empe a u e) o 6 weeks and PFU mL−1was de e mined a e 1 and 6 weeks as desc ibed abo e. Phage Genome Sequencing The p o ocol de eloped by San os (San os, 1991), wi h sligh modi ica ions, was used o isola e phage DNA om he phage lysa e. B ie ly, phage pa icles we e p ecipi a ed by adding 40 mM ZnCl2and incuba ing o 5 min, ollowed by pelle ing o he phage p ecipi a e (15 000 ×g, 5 min). DNA was pu i ied using aGeneJET TM Genomic DNA isola ion ki column (Fe men as). The phage genome was sequenced in wo pla o ms (Ion To en PGM wi h 100 bp ki and comme cially wi h Roche 454 a LGC Genomics, Ge many) and he da a we e combined because nei he o he me hods esul ed in whole genome sequence. Ini ially, in-house Ion To en un was used o de no o assembly. Since he assembly did no yield a single con ig, we decided o y o imp o e he assembly by using also comme cial pai ed-end 454 sequencing. All analyses we e done using GS De No o Assemble e sion 2.9 (454 Li e Sciences; Roche) which uses O e lap Layou Consensus (OLC) me hodology. Open eading ames (ORFs) we e p edic ed using Glimme and GeneMa kS, and simila sequences we e sea ched om da abases using BLAST (Al schul e al., 1990)wi hGeneious e sion7.1(c ea edbyBioma e s L d). F on ie s in Mic obiology | www. on ie sin.o g 2Augus 2015 | Volume 6 | A icle 829 Laan o e al. Phage he apy agains a ish pa hogen Fish Expe imen s wi h Zeb a ish (Danio e io) and Rainbow T ou (Onco hynchus mykiss) Fish expe imen s we e conduc ed acco ding o he Finnish Ac on Use o Animals o Expe imen al Pu poses, unde pe mission EASVI-2010-05569/Ym-23 g an ed o L-RS by he Na ional Animal Expe imen Boa d a he Regional S a e Adminis a i e Agency o Sou he n Finland. Unsexed, adul , disease- ee zeb a ish (D. e io)we eob ained om heco e acili iesand esea ch se ices o Tampe e (Tampe e Uni e si y, Finland). P io o he expe imen s, he zeb a ish we e main ained in 250-L aqua ia con aining ae a ed g ound wa e a 25◦C. Rainbow ou (O. mykiss) ywi hnop e iouscon ac wi hF. columna e we e ob ained om a ish a m in cen al Finland (app oxima e weigh in he expe imen s 0.57 g). P io o he expe imen s, he ainbow ou we e main ained in ae a ed g ound wa e a 17◦Cin250-L low- h ough aqua ia. Fo he in ec ion expe imen s, he wa e empe a u e o ainbow ou was g adually ele a ed o 24◦C o e 7 days. Two in ec ion me hods we e used. Zeb a ish we e in ec ed by applying F. columna e di ec ly o he expe imen al aqua ia, esul ing in con inuous in ec ion. The bac e ial le els used o in ec ion we e chosen based on ou p e ious s udies on ainbow ou (see, e.g., Kun u e al., 2009)andonzeb a ish (Laan o e al., 2012;Zhang e al., 2014). Rainbow ou popula ions we e imme sed in wa e con aining he bac e ium o 2 h, a e which ish we e ans e ed o expe imen al aqua ia. The ish we e moni o ed in 2-hou in e als in he zeb a ish expe imen and in 12-hou in e als in he ainbow ou popula ion expe imen . Fish aqua ia had unning numbe s and we e mixed a e addi ion o phage. Mo bid ish ha did no espond o s imuli we e conside ed dead and emo ed om he expe imen . In all expe imen s, in cul i a ions on Shieh aga supplemen ed wi h ob amycin we e aken om mo ibund ish o de e mine he p esence/absence o F. columna e on he ish. Measu ing he E ec o Phage Addi ion o a Flow-Th ough Sys em wi h Rainbow T ou Popula ion In ec ed wi h F. col umna e Fo in ec ion, he ainbow ou y (n=260) we e di ided in o 13 g oups o 20 ish, and placed in 3-L aqua ia con aining 2Lo g oundwa e .Nine ishg pupswe eexposed o 3×106CFU mL−1o F. columna e (calcula ed om op ical densi y a 570 nm based on he da a om unpublished expe imen s) and ou con ol g oups o s e ile Shieh medium o 2 h unde ae a ion a 24.2◦C. A e he challenge, ish popula ions we e ans e ed o expe imen al aqua ia con aining 2Lo g oundwa e wi hacons an in lowo eshwa e (∼0.6 L min−1, esul ing in an app oxima ely 3.3 min u no e ime o he whole wa e body) and ae a ion, a a empe a u e anging om 23.7 o 24.4◦Cdu ing heexpe imen .Thephage was added o expe imen al aqua ia a e ish ans e wi h phage- o-bac e ium a ios o 1:1 ( h ee aqua ia) and 10:1 ( h ee aqua ia) compa ed o in ec ion dose, and he highes amoun was added o wo con ol aqua ia wi h ish exposed o Shieh medium. Du ing phage addi ion, he wa e low was s opped o 60 min o all o he aqua ia. Fish we e moni o ed o 7 days in 12-hou in e als. Du ing he expe imen , wa e sampleswe e akena e 24 and 48 h om he s a o he expe imen o de e mine he numbe o F. columna e cells (CFU mL−1)andFCL-2phages (PFU mL−1)in hewa e .Sampleswe e aken om woaqua ia om he ollowing ea men s: phage- o-bac e ium a ios 1:1 and 10:1, phage con ol, and bac e ial con ol. Wa e samples we e dilu ed and pla ed on a Shieh aga pla e supplemen ed wi h ob amycin (Decos e e e al., 1997)andF. columna e colonies we e coun ed a e a 48-hou incuba ion a oom empe a u e. PFU we e de e mined om he same dilu ion se ies by cul u ing 300 µLo hos bac e iumandphagesampledilu ionsinShieh so aga , as desc ibed abo e. E ec o Phage Addi ion in Expe imen al F. columna e In ec ion o Indi idual Zeb a ish Zeb a ish (n=60) we e placed indi idually in 1-L aqua ia con aining 500 mL o g ound wa e (24.4◦C). Fish (n=40) we e in ec ed in con inuous exposu e wi h 5 ×104CFU mL−1o F. columna e. The phage was added o 20 o hese aqua ia di ec ly a e in ec ion a a phage- o-bac e ia cell a io o 1:1. Phage- o-bac e ia a io om 0,1:1 o 100:1 was es ed in p elimina y s udies. The e was no clea diffe ence be ween a ios o 1:1 and 1:100, he e o e he lowes a io ha had an effec o ish su i al was chosen. Con ol ish ecei ed s e ile Shieh medium (n=10) and phage only (n=10). The o al olume addi ion o each aqua ium was 500 µL. S a is ical Analysis Fish su i al in expe imen al in ec ions was analyzed using IBM- SPSS s a is ics 20. The ish su i al was analyzed wi h Kaplan– Meie su i al analysis. Phage Genome Accession Numbe The nucleo ide sequence o he FCL-2 phage genome has been deposi ed o GenBank (accession numbe KM873719). Resul s Phage Cha ac e iza ion and Genome Analysis Phage FCL-2 has been p e iously de e mined as a membe o Myo i idae (Laan o e al., 2011). TEM analysis e ealed he diame e o he icosahed al capsid o be 55–60 nm. The ail was app oxima ely 85 nm long. A p ominen neck was isible and ail ibe s could be seen (Figu e 1). En ichmen o he phage was done on pla es because despi e o op imiza ion o cul u e condi ions and pa ame e s, he cells in ec ed wi h phage ha e no lysed unde liquid cul u e. Sequence o he FCL-2 genome was ob ained by combining Ion To en and 454 da a. Using bo h da a se s ( o al numbe o sequences: 34680; o al numbe o bases: 6155764) we we e able o de no o assemble a single 47 142 bp long con ig wi h co e age o 125.9 and G +Ccon en o 30.2%.Se en y- ou ORFs all in o wa d di ec ion we e p edic ed wi h Glimme and GeneMa kS. Fo 18 o hese, a unc ion was p edic ed by BLAST sea ch and 32 we e assigned as hypo he ical p o eins, lea ing 24 ORFs (32.4%) wi hou any signi ican homologs in BLAST sea ch (as o Feb ua y 11, 2015). ORFs wi h p edic ed F on ie s in Mic obiology | www. on ie sin.o g 3Augus 2015 | Volume 6 | A icle 829 Laan o e al. Phage he apy agains a ish pa hogen FIGURE 1 | (A) Phage FCL-2 iewed unde ansmission elec on mic oscopy (TEM; scale ba 40 nm). (B) G aphic ep esen a ion o he genome o ganiza ion o FCL-2 wi h colo s indica ing he pu a i e unc ions o open eading ames (ORFs) as shown in he igu e. unc ions included p o eins in ol ed in packaging ( e minase and phage po al p o ein; Figu e 1 and Supplemen a y Table S1) and a p o ease, a DNA-me hylase, and an ssDNA-binding p o ein. No di ec lysis genes we e de ec ed al hough a ma ch o ahyd olasegenewas ecei ed:Thiscouldpossiblyha ea ole in he lysis o he cell. Mos o he p edic ions co esponded o s uc u al p o eins (e.g., ail and ail shea h p o ein) loca ed om 13 338 o 28 169 bp in he genome. These s uc u al p o eins we e ound o ma ch he Cellulophaga phage phiSM and ma ches o se en o he Cellulophaga phages we e also ob ained om hese genes. Fu he , om he p edic ed hypo he ical p o eins, one ma ched he Fla obac e ium phage 11 b and wo hi s we e linked o he genome o p ophage 6H (Cas illo e al., 2013). These wo p ophage 6H- ela ed p o eins, Ib A and Ib B, a e also ound in F. psych ophilum ( he hos o 6H) and in F. columna e genomes. Addi ionally, wo ORFs ups eam om he Ib AB complex we e iden ical o genes ound in F. columna e ATTC 49512 genome (accession numbe in NCBI: NC_016510.2, FCOL_05310 (WP_014165166.1), and FCOL_05310 (WP_014165168.1). Phage Adso p ion Tes and S abili y In he adso p ion es , 27% o phage FCL-2 had adso bed on he cells o F. columna e B185 a e 1 min and 41% a e 10 min. A e 1h,50%o headdedphageshadadso bed, esul ingin oan es ima ed a e o adso p ion being 0.5 h−1.Thes abili yo FCL-2 i e s was no affec ed by s o age empe a u e (Table 1). A e a 6-week pe iod, he i e o he lysa e s o ed a oom empe a u e was less han ha o he lysa e s o ed a 6◦C. The o e all i e o e 6 weeks dec eased 10- old when s o ed in Shieh medium o T is-HCl buffe , and 100- old when s o ed in he p esence o po assium phospha e. E ec o Phage Addi ion on Su i al o he Rainbow T ou Popula ion in a Flow-Th ough Sys em To mimic eal li e condi ions a a ish a m, he phage efficiency was es ed in p e-in ec ed popula ions o ainbow ou inge lings unde con inuous wa e low. The ish we e gi en phage immedia ely a e bac e ial exposu e (2-hou imme sion in bac e ia). A signi ican inc ease in su i al o ish ollowing expe imen al phage ea men s was obse ed (Figu e 2,Table 2). A he end o he expe imen (7 days pos in ec ion), he mean su i al o he ish in ec ed only wi h F. columna e (in ec ion con ol) was 8.3%. Phage ea men p o ec ed he ish om in ec ion and esul ed in a 41.7% ( a io 10:1) and 50% ( a io 1:1) mean su i al (P<0.001 o all pai wise compa isons agains phage- ea ed g oups and in ec ion con ol; Table 2). Fish in he nega i e con ol g oup (no in ec ed) su i ed 80–100 h pos in ec ion, a e which backg ound mo ali y was obse ed due o he expe imen al condi ions, a he han he ea men s. Ne e heless, in pai wise compa isons, su i al o ish in he nega i e con ol g oup (no bac e ia, no phage) did no diffe om hose ecei ing phage ea men s a e bac e ial in ec ion. In he ainbow ou ial, all diseased ish ea ed wi h bac e ia only we e posi i e o F. columna e hizoid mo phology acco ding o in samples. The cul i a ions om he phage- ea ed ish showed mainly a ough colony mo phology (possibly due o he p esence o phage in he sample, see Laan o e al., 2012)wi h occasional hizoid colonies. All ish no exposed o bac e ia we e ee o F. columna e. E ec o Phage Addi ion o he Su i al o Zeb a ish in a Con inuous In ec ion Model To es i zeb a ish can po en ially be used as a model sys em o s udy phage–bac e ium in e ac ions, including he hos , ins ead o ainbow ou (due o he poo a ailabili y o disease- ee inge lings and sensi i i y o labo a o y condi ions), an in ec ion expe imen was pe o med wi h indi idual con inuous exposu e o bac e ium and phage. The su i al o he non-in ec ed con ols in he expe imen was 100% e sus 0% in he in ec ion con ol g oup ea ed only wi h bac e ia. Phage addi ion signi ican ly inc eased ish su i al, esul ing in 60% o su i al (Figu e 3, Table 2). In bac e ial cul u e samples aken om zeb a ish, all ish ea ed wi h bac e ia only we e posi i e o he i ulen hizoid colony mo phology o F. columna e.Th eeo hesixdead ish ha we e ea ed wi h phage we e posi i e o F. columna e non- i ulen ough ype (possibly due o he p esence o phage in he sample, see Laan o e al., 2012). Fu he , all he ish in bac e ia- ee con ols we e nega i e o F. columna e. Phage and Bac e ial Coun s du ing Expe imen s Wa e samples we e aken om he expe imen al aqua ia du ing he ainbow ou expe imen . Du ing he ainbow ou popula ion es in a low- h ough sys em, he FCL-2 phage was isola ed om he wa e in a leas 1 ×104PFU mL−1e en F on ie s in Mic obiology | www. on ie sin.o g 4Augus 2015 | Volume 6 | A icle 829 Laan o e al. Phage he apy agains a ish pa hogen TABLE 1 | Phage FCL-2 s abili y as plaque o ming uni s (PFU) pe mL du ing 6-week s o age. RT 6◦C T is-HCl Po assiumphospha e Week Shieh Shieh pH 6 pH 7,5 pH 8 pH 6,0 pH 7,2 pH 8,0 19.7 ×1010 1.5 ×1011 1.8 ×1011 2×1011 3.3 ×1011 6.3 ×1010 8.5 ×1010 9.7 ×1010 63.6 ×1091×1010 1.5 ×1010 1.6 ×1010 1.3 ×1091.9 ×1094.3 ×1095×109 The o iginal phage i e a he beginning o he expe imen was 1.2 ×1011 PFU mL−1.Allexpe imen swe epe o meda 6 ◦C, excep o Shieh ea men , which was pe o med a oom empe a u e (indica ed as RT). FIGURE 2 | Fish longe i y in hou s (squa es, le axis; mean ±SE) and su i al pe cen age (do s, igh axis; mean ±SE) o ainbow ou a e exposu e o Fla obac e ium columna e B185 and phage FCL-2 in h ee eplica e ish popula ions (n=20). Fish we e in ec ed by imme sion (2 h) and phage was added in o expe imen al aqua ia a e bac e ial challenge (in phage- o-bac e ium a ios o 1:1 and 1:10). See Table 2 o s a is ical compa isons. a e 24 and 48 h pos in ec ion. This indica es ha he phage was able o eplica e in he sys em and was no washed away by he cons an wa e low. The maximum F. columna e coun s in he popula ion expe imen we e 8.8 ×102CFU mL−1in he bac e ial in ec ion con ol and om he phage- ea ed aqua ia, 2.7 ×102CFU mL−1.Allo hecoloniescoun edwe eo he hizoid mo pho ype. No F. columna e was isola ed om he wa e samples o he bac e ia- ee con ol g oup. Discussion He ein, he i s a emp s o apply phage he apy agains columna is disease, caused by F. columna e,a ep esen ed.The i s genome o a phage in ec ing F. columna e (FCL-2) was sequenced and cha ac e ized o i s abili y o inc ease ish su i al in he p esence o he pa hogen. S uc u al p o eins o FCL-2 we e ela ed o hose o Cellulophaga phages. Cellulophaga,amembe o he Bac e oide es g oup, is a close ela i e o Fla obac e ia (Holm eld e al., 2007). Using FCL-2, i was shown ha phages ha e g ea po en ial in con olling columna is disease in small- scale expe imen al sys ems, bu also in ainbow ou popula ions in low- h ough anks. Tho ough cha ac e iza ion o a phage is essen ial o i s he apeu ic use in o de o a oid unwan ed in e ac ions ha migh inc ease he i ulence o i s bac e ial hos (Gill and Hyman, 2010). Genome sequencing e ealed ha a hi d o he phage ORFs’ had no homologs in he da abase, which is ypical o en i onmen al phage genomes (Hu wi z and Sulli an, 2013). Only ew ORFs o he FCL-2 phage we e ela ed o Fla obac e ium phages, mos p obably due o he low numbe o hese phage genomes a ailable. A gene possibly ela ed o hos lysis (hyd olase) was iden i ied in he FCL-2 genome. Clea signs o possible in eg a ion o hos genome (in eg ases) o ep esso s we e absen , and lysogeny o he hos a e exposu e o he phage has no been de ec ed (Laan o e al., 2012). A ew hos hypo he ical p o eins we e iden i ied, including p o eins o unknown unc ion, Ib A and B (Supplemen a y Table S1). In F on ie s in Mic obiology | www. on ie sin.o g 5Augus 2015 | Volume 6 | A icle 829 Laan o e al. Phage he apy agains a ish pa hogen TABLE 2 | S a is ics o pai wise compa isons in ish su i al in phage he apy expe imen s agains Fla obac e ium columna e in ainbow ou (Onco hynchus mykiss)andzeb a ish(Danio e io)usingKaplan–Meie su i alanalysis,pai wiselog ankMan elCox es . Expe imen and o e all s a is ics T ea men S a is ics o pai wise compa isons In ec ion con ol Phage 1:1 Phage 1:10 Rainbow ou , popula ion Nega i e con ol χ2=49.272, p<0.001 NS NS Phage 1:1 χ2=57.235, p<0.001 Phage 1:10 χ2=31.824, p<0.001 NS Only phage χ2=64.390, p<0.001 χ2=6.741, p=0.009 χ2=12.777, p<0.001 Zeb a ish, con inuous in ec ion Nega i e con ol χ2=26.199, p<0.001 χ2=4.9542, p<0.026 Phage 1:1 χ2=17.853, p<0.001 P- alues g ea e han 0.05 a e designa ed as non-signi ican (NS). FIGURE 3 | Fish longe i y in hou s (squa es, le axis; mean ±SE) and su i al pe cen age (do s, igh axis; mean ±SE) o he zeb a ish a e exposu e o F. c o l u m n a e s ain B185 (in ec ion con ol), B185 and phage FCL-2 (1:1), and o g ow h medium o phage only (nega i e con ol). See Table 2 o s a is ical compa isons. he genome analysis o F. psych ophilum p ophage 6H i was hypo hesized ha hese wo p o eins could be possible i ulence ac o s due o hei ela ion o he Ib AB complex in Esche ichia coli (Cas illo e al., 2013). This complex is possibly in ol ed in hos –pa hogen in e ac ions and ela ed o esis ance o se um and Ig-binding (Sand and Hill, 2000). In e es ingly, Ib A and Ib B and he wo ORFs loca ed ups eam can also be ound in he sequenced genome o F. columna e ATTC 49512, indica ing acommono igin o hese egions.Clea ly,mo es udiesa e needed o unde s and he phage li e cycles and phage–bac e ium in e ac ions in F. columna e. The esul s om phage he apy ials in animal p oduc ion (e.g., in poul y, swine, and ca le) ha e gi en encou aging esul s (Smi h and Huggins, 1983;Be chie i e al., 1991;Ba ow e al., 1998;Jamalludeen e al., 2009;San os e al., 2010), simila o phage he apy s udies agains ish diseases (Nakai and Pa k, 2002; Pa k and Nakai, 2003;Higue a e al., 2013;Khai na e al., 2013). The in e es on efficiency o phages agains F. psych ophilum (S enholm e al., 2008;Cas illo e al., 2012)andF. columna e (Laan o e al., 2012;P asad and Kuma , 2012)hasbeen ecen ly inc easing, e lec ing he global conce n owa d la obac e ial pa hogens in aquacul u e. Gene ally, he efficiency o a phage as a po en ial he apeu ic is measu ed by i s capaci y o des oy bac e ial cells a he si e o bac e ial in ec ion (Le in and Bull, 2004). P e ious s udies ha e adminis e ed he phages ia ish eed and injec ion (Nakai e al., 1999;Pa k and Nakai, 2003;Cas illo e al., 2012;Kim e al., 2015), which could be labo ious when handling la ge numbe s o ish. Howe e , as columna is disease is mainly ex e nal, phages could be also applied di ec ly in he wa e . He ein, a signi ican inc ease was obse ed in he su i al o zeb a ish and ainbow ou when he phage was applied in he wa e sho ly a e bac e ial exposu e. When he phage was added in o aqua ia di ec ly a e bac e ia, zeb a ish su i al inc eased mos likely due o a dec ease in bac e ial in ec ion dose. In con as , in he ainbow ou expe imen , bac e ia we e allowed o colonize he ish o 2 h p io o placing hem in he F on ie s in Mic obiology | www. on ie sin.o g 6Augus 2015 | Volume 6 | A icle 829 Laan o e al. Phage he apy agains a ish pa hogen expe imen al low- h ough anks whe e he phage was added. Hence, he inc ease in ish su i al was likely caused by he associa ion o phage and bac e ia on ish su aces a he han ia di ec effec s on in ec ion dose, al hough he p esence o phage in he wa e is also likely o educe he ansmission o disease be ween indi idual ish. Su p isingly, he phage was isola ed om he ank wa e 2 days pos in ec ion, indica ing ha i can pe sis and eplica e in he anks despi e wa e low when hos bac e ia a e p esen . Fu he mo e, he FCL-2 i e emained s able o e a 6-week pe iod, indica ing p omising pe sis ence and s o age quali ies equi ed o he de elopmen o he apeu ic applica ions in aquacul u e. Bac e ia ha e de eloped mul iple mechanisms agains phage in ec ion (Samson e al., 2013); ne e heless, in he con inuous a ms ace be ween a phage and i s hos , phages co-e ol e o ou compe e hese mechanisms. The e o e, esis ance o a ce ain phage may no be a p oblem in phage he apy (Ö mälä and Jalas uo i, 2013), especially as main aining esis ance can be cos ly o bac e ia (Laan o e al., 2012, e iewedin Koskella and B ockhu s , 2014). Indeed, ou p e ious esul s sugges ha ollowing s ong phage selec ion F. columna e changes colony mo pho ype (Laan o e al., 2012). Rapid de elopmen o phage esis ance may hus no be a p oblem in F. columna e as he esis an cells a e no capable o causing disease in ish (Laan o e al., 2012). Howe e , he phage- esis an ough mo pho ype has no been isola ed om disease ou b eaks, indica ing ha en i onmen al condi ions ou side he labo a o y in luence he e olu ion o pe sis ence o phage esis ance o F. columna e a ish a ms. The e o e, mo e s udies on phage esis ance mechanisms in his ish pa hogen a e needed. The phages isola ed agains F. columna e s ains a e e y hos speci ic when compa ed o Fla obac e ium sp. phages om ish a ms and ou side he ish a ming en i onmen (Laan o e al., 2011). This is p omising as i sugges s ha o he bac e ial species would no be affec ed. As Meaden and Koskella (2013) indica e, he possibili y ha o he species o bac e ia migh be in luenced should be aken in o accoun . Al hough he p esen s udy shows ha phages signi ican ly inc eased ainbow ou su i al in low- h ough wa e , he condi ion mos ele an o ish a ming, he e a e s ill many aspec s ha need o be op imized be o e eaching p oduc ion scale. Fi s , he popula ion s uc u e o F. columna e a a ms is complex and se e al s ains co- occu and may co-in ec ish (Suomalainen e al., 2006;Kun u e al., 2012). The e o e, mul iple phages and hei associa ions wi h he hos bac e ia need o be cha ac e ized and es ed o efficiency in mixed in ec ions and o p e en e olu ion o phage esis ance. Second, al hough phage adminis a ion h ough wa e is ela i ely simple, hewa e masses ha need obemain ained sa u a ed a e la ge and phage willbequicklydilu ed.Sinceou da a sugges ha phages a e able o pe sis in low- h ough anks, his challenge may no be insu moun able, especially i phages a e used o educing he in ec i e bac e ial dose in he anks. Simila ly, he iming o ea men needs o be op imized, as ish in he ea ing uni s a e o en a diffe en s ages o in ec ion du ing an ou b eak. P oblems ela ed o en ichmen o phage FCL-2 in liquid cul u e could be a se e e p oblem conce ning he la ge scale p oduc ion o phages o phage he apy, a leas when using his speci ic phage. Also, F. columna e is a slow- g owing bac e ium, wi h a doubling ime app oxima ely o 10 h (Zhang e al., 2014), which could b ing ex a challenges o p oduc ion scale. Fu he , he op imal dose o phage (phage– bac e ium a io) is c ucial o ea men efficacy and needs o be e alua ed, a ac no ho oughly conside ed in ea ly phage he apy ials (Ca l on, 1999). Despi e hese challenges in he applica ion o phage he apy in aquacul u e, i would be wo hwhile o assess whe he phage he apy could be combined wi h o he ea men s. I phage he apy could dec ease he bac e ial in ec ion dose du ing disease ou b eaks and educe he isk o ansmission, he need o an ibio ic ea men could be delayed o a ge ed o smalle g oups o in ec ed indi iduals. In he long un, his would lead o a educ ion in an ibio ics use in aquacul u e. Conclusion The phage he apy has po en ial as an efficien and sus ainable disease managemen me hod in he case o columna is disease and aquacul u e in gene al. I is shown he ein ha F. columna e and i s phage o m a good model o he s udy o phage he apy as he disease and ea men can be s udied in he ele an hos s. This model could be used o s udy he ecological and e olu iona y effec s o phage he apy. Fu he ,aquacul u econdi ionswi h high popula ion densi ies o ish hos s can hus be eplica ed in he labo a o y. Finally, using zeb a ish as a model hos could ex end he bene i s o he expe imen al sys em o include he ish hos immune esponse du ing phage he apy. Au ho Con ibu ions EL, JB, and L-RS designed he s udy. EL pe o med he expe imen s. EL, JR, and L-RS analyzed he da a. EL and L-RS p epa ed he ables and igu es and w o e he manusc ip . All au ho s edi ed he manusc ip . Acknowledgmen s This wo k was suppo ed by he Finnish Cen e o Excellence P og am o he Academy o Finland, he CoE in Biological In e ac ions 2012–2017 (#252411), and by Academy o Finland g an s #251106 (JB) and #272995 (L-RS). The au ho s wish o hank D . Heidi Kun u, Ree a Pen inen, MSc, Hanna Kinnula, MSc, Ka ja Neu onen, MSc, Ms. Heidi Pi inen, Ms. Ka i Mäkelä, and M . Pe i Papponen o assis ance in he labo a o y. Supplemen a y Ma e ial The Supplemen a y Ma e ial o his a icle can be ound online a : h p://jou nal. on ie sin.o g/a icle/10.3389/ micb. 2015.00829 F on ie s in Mic obiology | www. on ie sin.o g 7Augus 2015 | Volume 6 | A icle 829 Laan o e al. Phage he apy agains a ish pa hogen Re e ences Abedon, S. T., Kuhl, S. J., Blasdel, B. G., and Ku e , E. M. (2011). Phage ea men o human in ec ions. Bac e iophage 1, 55–65. doi: 10.4161/bac .1.2.15680 Adams, M. H. (1959). Bac e iophages.NewYo k,NY:In e sciencePublishe s,Inc. Al schul, S. F., Gish, W., Mille , W., Mye s, E. W., and Lipman, D. J. (1990). Basic local alignmen sea ch ool. J. Mol. Biol. 215, 403–410. doi: 10.1016/S0022- 2836(05)80360-2 Ba ow, P., Lo ell, M., and Be chie i, A., J . (1998). Use o ly ic bac e iophage o con ol o expe imen al Esche ichia coli sep icemia and meningi is in chickens and cal es. Clin. Diagn. Lab. Immunol. 5, 294–298. Be chie i, A. J ., Lo ell, M. A., and Ba ow, P. A. (1991). The ac i i y in he chicken alimen a y ac o bac e iophages ly ic o Salmonella yphimu ium. Res. Mic obiol. 142, 541–549. doi: 10.1016/0923-2508(91)90187-F Be na de , J. F. (1997). Immuniza ion wi h bac e ial an igens: Fla obac e ium and Flexibac e in ec ions. De . Biol. S and. 90, 179–188. Bos ock, J., McAnd ew, B., Richa ds, R., Jauncey, K., Tel e , T., Lo enzen, K., e al. (2010). Aquacul u e: global s a us and ends. Philos. T ans. R. Soc. B 365, 2897–2912. doi: 10.1098/ s b.2010.0170 Buschmann, A. H., Tomo a, A., López, A., Maldonado, M. A., Hen íquez, L. A., I ano a, L., e al. (2012).Salmon aquacul u e and an imic obial esis ance in he ma ine en i onmen . PLoS ONE 7:e42724. doi: 10.1371/jou nal.pone.0042724 Cabello, F. C., God ey, H. P., Tomo a, A.,I ano a,L.,Dölz,H.,Millanao,A., e al. (2013). An imic obial use in aquacul u e e-examined: i s ele ance o an imic obial esis ance and o animal and human heal h. En i on. Mic obiol. 15, 1917–1942. doi: 10.1111/1462-2920.12134 Ca l on, R. M. (1999). Phage he apy: pas his o y and u u e p ospec s. A ch. Immunol. The . Exp. 47, 267–274. Cas illo, D., Espejo, R., and Middelboe, M. (2013). Genomic s uc u e o bac e iophage 6H and i s dis ibu ion as p ophage in Fla obac e ium psych ophilum s ains. FEMS Mic obiol. Le . 351, 51–58. doi: 10.1111/1574- 6968.12342 Cas illo, D., Higue a, G., Villa, M., Middelboe, M., Dalsgaa d, I., Madsen, L., e al. (2012). Di e si y o Fla obac e ium psych ophilum and he po en ial use o i s phages o p o ec ion agains bac e ial cold wa e disease in salmonids. J. Fish Dis. 35, 193–201. doi: 10.1111/j.1365-2761.2011.01336.x Decle cq, A. M., Haeseb ouck, F., Van den B oeck, W., Bossie , P., and Decos e e, A. (2013). Columna is disease in ish: a e iew wi h emphasis on bac e ium-hos in e ac ions. Ve . Res.44:27.doi:10.1186/1297-971 6-44-27 Decos e e, A., Haeseb ouck, F., and De iese, L. A. (1997). Shieh medium supplemen ed wi h ob amycin o selec i e isola ion o Fla obac e ium columna e (Flexibac e columna is) om diseased ish. J. Clin. Mic obiol. 35, 322–324. Di Cesa e, A., Luna, G. M., Vigna oli, C., Pasqua oli, S., To a, S., Pa oncini, P., e al. (2013). Aquacul u e can p omo e he p esence and sp ead o an ibio ic- esis an En e ococci in ma ine sedimen s. PLoS ONE 8:e62838. doi: 10.1371/jou nal.pone.0062838 Gill, J. J., and Hyman, P. (2010). Phage choice, isola ion, and p epa a ion o phage he apy. Cu . Pha m. Bio echnol. 11, 2–14. doi: 10.2174/138920110790725311 Higue a, G., Bas ías, R., Tse s adze, G., Rome o, J., and Espejo, R. T. (2013). Recen ly disco e ed Vib io anguilla um phages can p o ec agains expe imen ally induced ib iosis in A lan ic salmon, Salmo sala . Aquacul u e 392–395, 128–133. doi: 10.1016/j.aquacul u e.2013.02.013 Hollis, A., and Ahmed, Z. (2013). P ese ing an ibio ics, a ionally. New Engl. J. Med.369,2474–2476.doi:10.1056/NEJMp1311479 Holm eld , K., Middelboe, M., Nyb oe, O., and Riemann, L. (2007). La ge a iabili ies in hos s ain suscep ibili y and phage hos ange go e n in e ac ions be ween ly ic ma ine phages and hei Fla obac e ium hos s. Appl. En i on. Mic obiol. 73, 6730–6739. doi: 10.1128/AEM.01399-07 Hu wi z, B. L., and Sulli an, M. B. (2013). The Paci ic Ocean Vi ome (POV): a ma ine i al me agenomic da ase and associa ed p o ein clus e s o quan i a i e i al ecology. PLoS ONE 8:e57355. doi: 10.1371/jou nal.pone.00 57355 Jamalludeen, N., Johnson, R. P., Shewen, P. E., and Gyles, C. L. (2009). E alua ion o bac e iophages o p e en ion and ea men o dia hea due o expe imen al en e o oxigenic Esche ichia coli O149 in ec ion o pigs. Ve . Mic obiol. 136, 135–141. doi: 10.1016/j. e mic.2008.10.021 Jones, J. B., Vallad, G. E., I ia e, F. B., Ob ado ic, A., We nsing, M. H., Jackson, L. E., e al. (2012). Conside a ions o using bac e iophages o plan disease con ol. Bac e iophage 2, 208–214. doi: 10.4161/bac . 23857 Khai na , K., Rau , M. P., Chandeka , R. H., Sanmukh, S. G., and Paunika , W. N. (2013). No el bac e iophage he apy o con olling me allo-be a-lac amase p oducing Pseudomonas ae uginosa in ec ion in ca ish. BMC Ve . Res. 9:264. doi: 10.1186/1746-6148-9-264 Kim, J. H., Cho esca, C. H., Shin, S. P., Han, J. E., Jun, J. W., and Pa k, S. C. (2015). Biological con ol o Ae omonas salmonicida subsp. salmonicida in ec ion in ainbow ou (Onco hynchus mykiss) using ae omonas phage PAS-1. T ansbound Eme g. Dis. 62, 81–86. doi: 10.1111/ bed.12088 Koskella, B., and B ockhu s , M. A. (2014). Bac e ia-phage coe olu ion as a d i e o ecological and e olu iona y p ocesses in mic obial communi ies. FEMS Mic obiol. Re . 38, 916–931. doi: 10.1111/1574-6976.12072 Kun u, H. M. T., Sundbe g, L.-R., Pulkkinen, K., and Val onen, E. T. (2012). En i onmen may be he sou ce o Fla obac e ium columna e ou b eaks a ish a ms. En i on. Mic obiol. Rep. 4, 398–402. doi: 10.1111/j.1758- 2229.2012.00342.x Kun u, H. M. T., Val onen, E. T., Jokinen, E. I., and Suomalainen, L.-R. (2009). Sap ophy ism o a ish pa hogen as a ansmission s a egy. Epidemics 1, 96–100. doi: 10.1016/j.epidem.2009.04.003 Laan o, E., Bam o d, J. K. H., Laakso, J., and Sundbe g, L.-R. (2012). Phage-d i en loss o i ulence in a ish pa hogenic bac e ium. PLoS ONE 7:e53157. doi: 10.1371/jou nal.pone.0053157 Laan o, E., Sundbe g, L.-R., and Bam o d, J. K. H. (2011). Phage speci ici y o he eshwa e ish pa hogen Fla obac e ium columna e.Appl. En i on. Mic obiol. 77, 7868–7872. doi: 10.1128/AEM.0557411 Le in, B. R., and Bull, J. J. (2004). Popula ion and e olu iona y dynamics o phage he apy. Na . Re . Mic obiol. 2, 166–173. doi: 10.1038/n mic o822 Meaden, S., and Koskella, B. (2013). Explo ing he isks o phage applica ion in he en i onmen . F on . Mic obiol. 4:358. doi: 10.3389/ micb.2013.00358 Nakai, T., and Pa k, S. C. (2002). Bac e iophage he apy o in ec ious diseases in aquacul u e. Res. Mic obiol. 153, 13–18. doi: 10.1016/S0923-2508(01) 01280-3 Nakai, T., Suqimo o, R., Pa k, K. H., Ma suoka, S., Mo i, K., Nishioka, T., e al. (1999). P o ec i e effec s o bac e iophage on expe imen al Lac ococcus ga ieae in ec ion in yellow ail. Dis. Aqua . O g. 37, 33–41. doi: 10.3354/dao0 37033 Ö mälä, A.-M., and Jalas uo i, M. (2013). Phage he apy: should bac e ial esis ance o phages be a conce n, e en in he long un? Bac e iophage 3:e24219. doi: 10.4161/bac .24219 Pa k, S. C., and Nakai, T. (2003). Bac e iophage con ol o Pseudomonas plecoglossicida in ec ion in ayu Plecoglossus al i elis.Dis. Aqua . O g. 53, 33–39. doi: 10.3354/dao053033 P asad, Y., and Kuma , A. D. (2012). Isola ion and efficacy e alua ion o i ulen bac e iophages speci ic o ish pa hogenic bac e ium, Fla obac e ium columna e.J. Appl. Anim. Res. 38, 169–172. doi: 10.1080/09712119.2010.105 39505 Pulkkinen, K., Suomalainen, L.-R., Read, A. F., Ebe , D., Rin amäki, P., and Val onen, E. T. (2010). In ensi e ish a ming and he e olu ion o pa hogen i ulence: he case o columna is disease in Finland. P oc. Biol. Sci. 277, 593–600. doi: 10.1098/ spb.2009.1659 Samson, J. E., Magadán, A. H., Sab i, M., and Moineau, S. (2013). Re enge o he phages: de ea ing bac e ial de ences. Na . Re . Mic obiol. 11, 675–687. doi: 10.1038/n mic o3096 Sand , C. H., and Hill, C. W. (2000). Fou diffe en genes esponsible o nonimmune immunoglobulin-binding ac i i ies wi hin a single s ain o Esche ichia coli.In ec . Immun. 68, 2205–2214. doi: 10.1128/IAI.68.4.2205- 2214.2000 San os, M. A. (1991). An imp o ed me hod o he small scale p epa a ion o bac e iophage DNA based on phage p ecipi a ion by zinc chlo ide. Nucl. Acids Res. 19, 5442–5442. doi: 10.1093/na /19.19.5442 San os, T. M. A., Gilbe , R. O., Caixe a, L. S., Machado, V. S., Teixei a, L. M., and Bicalho, R. C. (2010). Suscep ibili y o Esche ichia coli isola ed om u e i o pos pa um dai y cows o an ibio ic and en i onmen al bac e iophages. Pa II: in i o an imic obial ac i i y e alua ion o a bac e iophage cock ail and se e al an ibio ics. J. Dai y Sci.93,105–114.doi:10.3168/jds.2009-2299 F on ie s in Mic obiology | www. on ie sin.o g 8Augus 2015 | Volume 6 | A icle 829