Microbiological Analysis of Striped Bass (Marone saxatilis) Grown in a Recirculating System t

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1 948 Jurnl f Fd Prtectin, Vl. 60, N.8, 1997, Pges Cpyright, Interntinl Asscitin f Milk, Fd nd Envirnmentl Snitrins Micrbilgicl Anlysis f Striped Bss (Mrne sxtilis) Grwn in Recirculting System t PATTI C. NEDOLUHA nd DENNIS WESTHOFF* Deprtment f Animl nd Avin Sciences, University f Mrylnd, Cllege Prk, MD 20742, USA (MS# : Received 29 August 1996/Accepted 24 December 1996) ABSTRACT The skin, gills, intestines, tnk wter, nd diet f striped bss grwn in recirculting tnks were exmined fr quntity nd qulity f micr flr. Plte cunts fr the fish nd wter smples were similr t cunts reprted in ther systems. The bcteri grups represented mst frequently in isltes frm the fish were Aermns (14%), Mrxellcee (15%), the Flvbcterium- Chrysebcterium-Cytphg-Sphingbcterium grup (II %), Bcillus (8%), Grup I Pseudmns (6%), the Shewnell- Altermns grup (6%), nd Cmmndcee (5%). The fdbrne pthgens Stphylcccus ureus, mtile Aermns spp., nd Vibri mimicus were islted repetedly. V.prhemlyticus nd V.chler were ech islted nce, nd n Listeri, Plesimns, Slmnell, r Yersini enterclitic were islted. Clse txnmic reltinships were bserved mng sme f the bcteri fund n the fish nd in the wter; bcteri frm the skin nd gills were mre likely t be relted t ech ther nd t the wter isltes thn t the intestinl isltes. Sme bcteri in the intestines riginted in the diet, but these strins did nt significntly ffect the verll micrflr f the fish nd wter. Key wrds: Finfish, quculture, micrbilgy Interest in the prductin f sefds thrugh quculture hs incresed with the decresing vilbility f wild stcks. The future cnsumer demnd fr sme species f finfish my eventully be met lmst entirely by quculture prducts (9). Amng the limiting fctrs fr the expnsin f quculture grwut fcilities re the cst nd vilbility f lnd nd wter; in mny regins, wter requirements re the gretest brrier t incresing quculture prductin (4). Intensive culture using recirculting tnks, in which wter is clened by filtrtin nd reused, requires less lnd nd wter thn ther systems; nther dvntge is tht less wste wter is dischrged int the envirnment. The high stcking densities used in recircultin systems put fish in clse cntct with ech ther nd subjects them t incresed * Authr fr crrespndence. Tel: ; Fx: ; E-mil: dw26@umi1.umd.edu. t Mrylnd Agriculturl Experiment Sttin Scientific Article N. A79l0, Cntributin N physilgicl stress. This crwding nd stress culd enhnce the spred f bcteril pthgens mng the fish, nd the verll bcteril lds my be higher thn in fish frm less intensive quculture systems r wild stcks. As result, the micrbilgicl sfety f these fish fr humn cnsumptin culd be cmprmised. Striped bss (Mrne sxtilis) nd hybrid striped bss (Mrne chrysps X Mrne sxtilis) re the stte f Mrylnd's tp quculture prduct (2). Hybrid striped bss grwn in erthen pnds (23) nd striped bss grwn in indr flw-thrugh tnks (24) hve been shwn t crry bcteril lds similr t thse f wild fish. The purpse f this study ws t exmine the numbers nd types f bcteri fund n striped bss grwn in recirculting system, with emphsis n detecting fdbrne pthgens. The txnmic reltinships mng bcteri frm the diet, fish, nd wter were ls exmined. MATERIALS AND METHODS Fish culture Striped bss (Mrne sxtilis) were cultured t the Crne Aquculture Fcility in Bltimre, Mrylnd. Fish were grwn in tw identicl but independent clsed systems; ech system cntined six 6-ft (1.8-m) 1,600-liter tnks nd ne 12-ft (3.6-m) 8,000-liter tnk cnnected in series. Tnks were stcked with tw-yer-ld striped bss (verge weight 680 g); the 6-ft tnks cntined 30 fish ech (12.8 g fish per liter), nd the 12-ft tnks hd 18 fish ech (1.5 g/liter). Ech system cntined n zne twer with n Aqu-Flw mdel CD wtt zne genertr, 24-in. (61-cm) rpid snd filter, nd 21-ft 3 (0.59-m 3 ) trickle-dwn bifilter inculted with Bct-Pur (Aqutic Ec-Systems, Inc., Appk, FL), mixture f Nitrsmns nd Nitrbcter spp. The wter turnver rte thrugh the bifilter ws 0.5 time per hur (8,000 liters/h), nd the wter turnver rte thrugh the zne twer ws 1.5 times per hur (24,000 liters/h). Fish were mved int the clsed systems tw weeks befre smpling begn; feed ws withheld fr 48 h befre mving. The fish were ffered cmmercil diet (38% crude prtein, 10% crude ft, 4% crude fiber) d libitum 2 t 3 times dily, but cnsumed little f the ffered feed during the tw weeks befre smpling begn. The weight f the fish smpled incresed frm 810 g n the first dy f smpling t 1,083 g n the lst dy (Tble 1). The wter slinity ws mintined t 3.0 t 6.0 ppt, temperture rnged frm 19.9 t 22.4 C, nd the ph ws held between 7.8 nd 8.1.

2 MICROBIOLOGY OF AQUACULTURED STRIPED BASS 949 TABLE 1. Weight f smpled fish nd intestinl cntents Smpling tril Weight f fish, men:!: SD (g) 810 ± ± 99 1,088 ± 215 1,017 ± 115 1,113 ± 313 1,083 ± 89 Weight f intestinl cntents (g) Cllectin f smples Fish, wter, nd feed smples were tken n ech f six smpling dys seprted by tw-week intervls. On ech smpling dy, three fish were remved frm ech f the tw 12-ft tnks using dip nets, trnsferred t sterile plstic bgs, nd immeditely pcked in ice. All hndling ws dne with sterile glves. Wter smples were cllected frm ech tnk frm pprximtely 1 ft (30 cm) belw the surfce (16) using sterile 100-ml bttles nd immeditely plced in ice. Feed smples were remved frm the current supply using sterile scps nd held in sterile bgs n ice until exmined. Exmintin f smples begn within 2 h f cllectin. Micrbilgicl nlyses On ech smpling dy, the six fish cllected were divided int tw grups f three fish ech bsed n tnk fr the micrbilgicl nlyses. Skin surfce smples were btined by swbbing n re 10 by 2 cm n the side f ech fish using clcium lginte swb (Clgiswb, Spectrum Lbrtries, Ls Angeles, CA). The swbs frm ech tnk grup were pled in sterile Stmcher bgs (Tekmr-Dhrmn, Cincinnti, OH) cntining 10 ml sterile 0.1 % (wt/vl) peptne wter with 1% (wt/vl) sdium hexmetphsphte nd hmgenized fr 2 min in Stmcher 80 Lb Blender (Sewrd Medicl, Lndn). The gills were excised nd pled by grup in sterile Preplt Bgs (Integrted BiSlutins, Inc., Mnmuth Junctin, NJ); the peritnel cvity ws pened septiclly nd the intestinl cntents fr ech grup pled in sterile Stmcher bg (Sewrd Medicl, Lndn). Feed smples (25 g) were plced int sterile Preplt bgs. All smple bgs were weighed nd enugh sterile 0.1 % peptne wter dded t mke 1:10 dilutin. Smples were then hmgenized fr 2 min in Stmcher 80 Lb Blender r Stmcher 400 Lb Blender (Sewrd Medicl, Lndn), depending n smple mss. All fish, feed, nd wter smples were serilly diluted in 0.1 % peptne wter nd spred plted nt plte cunt gr (PCA, Difc Lbrtries, Detrit, MI) with 0.5% (wt/vl) NCl. Replicte pltes were incubted erbiclly r nerbiclly t 35, 22, r 70C fr 2, 4, nd 7 dys, respectively. Clnies frm ech set f erbic plte cunts with 25 t 250 clnies were numbered, nd eight clnies were selected frm ech set using rndm number tble t eliminte bis. Selected clnies were streked t purity n Trypticse sy brth gr (TSBA) prepred frm 30 g Trypticse sy brth nd 15 g gr (BBL, Clumbi, MD). Enrichments were used t exmine the fish nd wter smples fr the presence f the fdbme pthgens Aermns spp. (26), Plesimns shigellides (20), Vibri spp. (7), Listeri mncytgenes (10), Slmnell spp. (21), Stphylcccus ureus (3), nd Yersini enterclitic (41). Presumptive psitive isltes were streked t purity n TSBA. A MIDI Micrbil Identifictin System (MicrbillD, Inc., Newrk, DE) ws used t identify isltes bsed n their ftty cid methyl ester (FAME) prfiles. The MIDI system hrdwre nd sftwre, smple prcessing prtcls nd chrmtgrphic prmeters were s described previusly (29, 32). Islte prfiles were cmpred t the Aerbe (TSBA) Dtbse (versin 3.9) t determine the mst likely mtch. The Librry Genertin Sftwre (versin 4.15) ws used t generte cluster nlyses nd dendrgrms t seprte the isltes int distinct grups bsed n their FAME prfiles. Isltes nt identified by the MIDI system r identified with very lw similrity cefficients were nlysed using crbhydrte utiliztin prfiles with BIOLOG system (Bilg, Inc., Hywrd, CA) (32); the metblic fingerprints were cmpred t either the grm psitive r grm negtive dtbse (relese 3.50, versin DE). Sttisticl nlyses The lgrithms f the plte cunts frm the skin, gills, intestines, wter, nd feed were exmined using nlyses f vrince (ANOVA) (33); clcultins were perfrmed using generl liner mdel n Sttisticl Anlysis Systems sftwre (SAS Institute, Cry, NC). Fisher's (prtected) lest significnt difference test (33) ws used t seprte mens. The experimentl design ws rndmized cmplete blck, nd the tretment design fr ll smples ws 2 X 3 fctril; incubtin temperture nd tmsphere were the min effects, nd smpling dy ws the blck effect. The interctin between incubtin temperture nd tmsphere ws ls exmined. Cunts belw the limit f detectin were represented with number equl t tw-thirds the limit f detectin fr the sttisticl nlyses nd clcultin f mens (Dr. Lrry Duglss, University f Mrylnd, Deprtment f Animl Sciences, persnl cmmunictin). '" E u.. U 2.5 cl2.q 1.5 bbb RESULTS Plte cunts Differences between mens were cnsidered significnt when P < The min effects (incubtin temperture nd tmsphere) fr the skin, gills, wter, nd diet were significnt (Figures I nd 2), but nly temperture ws significnt fr the intestines; there were n interctins between the min effects. Fr ll smples, the 35 e nd 22 e cunts were significntly higher thn the crrespnding 7 e cunts. Fr the skin, gills, nd wter cunts, the bbc bbbc ~rc 35 C ~35 C 22 C ~22 C rc erbic~ nerbic erbic~ nerbic erbic~nerbic FIGURE 1. Plte cunts (men ± SEM) frm the skin, gills, intestinl cntents, nd culture wter f striped bss grwn in recirculting system. Mens with the sme letter re nt significntly different (P > 0.05). gills

3 950 NEDOLUHA AND WESTHOFF 6.Q>5 rr )3 2 1 II 35 C erbic ~ 22 C 1:;2;221 nerbic Aermns Mrxellcee FCCS Bcillus Pseudmns Grup 1 Shewnell/Altermns Cmmndcee Brevundimns Crynefrms Misc. pseudmnds Micrccccee Vibrincee Methyfbcterlum/Rhdbcter Brevibcterium cetylicum CDC Grup DF-3 Xnthmns unknwn Ncrdifrms Misc. Grm psitives Misc. Grm negtives ~ 35 C ~ nerbic D 7 C erbic ~7 C C 22 C erbic ~ nerbic FIGURE 2. Plte cunts (men ± SEM)frm the dietfed t striped bss grwn in recirculting system. Mens with the sme letter re nt significntly different (P > 0.05). erbic cunts were cnsistently higher thn the nerbic cunts in ech rund, but the differences were nt sttisticlly significnt fr ll cmprisns (Figure 1); the 35 C nd 22 C cunts were significntly different fr skin nd gills under nerbic cnditins but nt fr skin, gills, nd wter under erbic cnditins. N trends were bserved ver time fr the cunts frm the skin, gills, wter, r diet; the lg cunts fr the intestinl cntents styed between 2 nd 4 lg CFU/ml fr the first five smpling runds, then incresed t between 5 nd 6 lg CFU/ml fr the finl rund (dt nt shwn). Bcteril isltes nd identifictin The 1,052 bcteril isltes selected rndmly frm the plte cunts were divided int grups bsed n cluster nlyses ftheir FAME prfiles (Figures 3 nd 4). Micrcccus luteus ws gruped with Stphylcccus spp. under the heding Micrccccee, but M. kristine ws plced in the crynefrm cluster. Brevibcterium cetylicum ws put int its wn cluster, but ther Brevibcterium spp. were gruped with the crynefrms. Other crynefrms were frm the gener Clvibcter, Aurebcterium, Arthrbcter, Crynebcterium, Rthyibcter, nd Cellulmns. skin Il2lI gills. intestines % f rndm fish isltes (n = 814) FIGURE 3. Micrrgnisms islted frm striped bss grwn in recirculting system. FCCS = Flvbcterium, Cytphg, Chrysebcterium, nd Sphingmns; Misc. = miscellneus. FCCS ShewnelliA/termns Vibrincie Pseudmns Grup 1 mtile Aermns A. s/mnicid Cmmndcee Misc. pseudmnds Enterbctericee Mrxellcee unknwn Misc. Grm negtives Bcillus Brevundimns Crynefrms Bcillus Mrxellcee Pseudmns Grup 1 Crynefrms Enterbctericee unknwn Micrccccee Misc. Grm negtives Misc. Grm psitives B. cety/icum Misc. pseudmnds wier.:35'c ~22'C.:7'C % f rndm wter isltes (n=121) % f rndm diet isltes (n=140) diet 35'C E;22'C.7'C FIGURE 4. Micrrgnisms islted frm the culture wter nd diet f striped bss grwn in recirculting system. FCCS = Flvbcterium, Cytphg, Chrysebcterium, nd Sphingmns. Misc. = miscellneus. The Vibrincee were represented primrily by Vibri spp., but Listnell nd Phtbcterium spp. were ls islted. Mst f the Aermns isltes were mtile species, but A. slmnicid ws ls identified. The tw grups lbeled s "miscellneus" cntined species tht were identified but ccurred t lw frequency; these grups d nt represent clusters f relted rgnisms, but re hetergeneus grups plced tgether fr cnvenience. The miscellneus grm-psitives grup included species f Entercccus, Ncrdides, Rhdcccus, Grdn, Aercccus, nd Deincccus, nd miscellneus grm-negtives grup included species f Erwini, Enterbcter, Yersini frederiksenii, Agrbcterium, Phyllbcterium, Actinbcillus, nd Acetbcter. The Mrxellcee cntined the three gener Acinetbcter, Mrxell, nd Psychrbcter. Mst f the isltes frm the Cmmndcee were Hydrgenphg spp., but few Acidvrx were ls fund. The genus Brevundimns ws represented by tw species, B. diminut nd B. vesiculris; this genus ws previusly knwn s Grup IV Pseudmns (30). A cluster identified by the MIDI s very lw mtch Hemphilus infiuenze ws identified s CDC Grup DF-3 by the Bilg system. CDC Grup DF-3 suppsedly hs cellulr ftty cid prfile similr t tht f Cpncytphg (27), but neither rgnism is in the MIDI TSBA dtbse. The isltes did nt cluster with the Flvbcterium- Chry sebcte rium- Cytphg-S phingbcte rium (FCCS) grup. Mst f the rgnisms in the miscellneus Pseudmns grup were members f very tight cluster which ws

4 MICROBIOLOGY OF AQUACULTURED STRIPED BASS 951 nt identified by the MIDI system nd misidentified by the Bilg system s Acinetbcter rdiresistenslgenspecies 9 with very high similrity cefficients. These grmnegtive rds were mtile nd xidse psitive, wheres Acinetbcter spp. re nnmtile nd xidse negtive (12); ls, in the FAME dendrgrms, this grup clustered very clsely with identified Pseudmns spp., nd shwed n reltedness t the Mrxellcee grup. The Grup I Pseudmns cluster cntined mstly P. lcligenes, P. pseudlcligenes, nd P. stutzeri in the smples frm the fish nd wter, but P. erugins, P. putid, nd P. fiurescens were ls present in very smll numbers; the diet smples cntined primrily P. putid. The grups represented mst frequently in isltes frm the skin were Mrxellcee (33%), the Flvbcterium- Chrysebcterium-Cytphg-Spingbcterium (FCCS) grup (16%), crynefrms (9%), Brevundimns (8%), nd ther Pseudmns (6%). On the gills, species frm Aermns (16%), Cmmndcee (12%), FCCS (10%), Grup I Pseudmns (12%), the Shewnell-Altermns (SA) grup (9%), nd Mrxellcee (7%) were the predminnt isltes, nd in the intestines species frm Aermns (28%), Bcillus (27%), SA (9%), FCCS (6%), nd CDC Grup DF-3 (6%) were the mst cmmn isltes. Isltin temperture influenced the prevlence f the bcteril grups islted; fr exmple, P. immbilis ws islted mst ften t 7 C, but nly tw Bcillus isltes were btined t this temperture. Species frm the Methylbcterium-Rhdbcter nd miscellneus Pseudmns grups were nt islted t 7 C. The predminnt grups represented in the isltes frm the fish t 35 C were Aermns (15%), FCCS (13%), Bcillus (II %), Pseudmns (6%), Cmmndcee (7%), Mrxellcee (7%), nd crynefrms (5%). At 22 C, Aermns (11 %), Bcillus (11 %), FCCS (10%), Brevundimns (7%), Cmmndcee (6%), Mrxellcee (6%), SA (5%), nd crynefrms (4%) were the mst frequently represented grups. The Mrxellcee nd Aermns isltes ccunted fr 35% nd 19% f the fish isltes t 7 C, respectively; the ther dminnt grups were the FCCS grup (II %), SA (8%), Pseudmns Grup I (6%), nd CDC Grup DF-3 (4%). All f the intestinl isltes f Bcillus were btined t 35 C r 22 e. The Bcillus spp. islted frm the diet nd intestines were mstly B. pumilus nd B. lichenifrmis; few instnces f B. sphericus ls ccurred. The micr flr f the intestinl cntents cntined high percentges f Bcillus spp. during the first 10 weeks f smpling; t the 12th week, the intestinl micrflr hd becme dminted by Aermns spp., nd n members f Bcillus were islted. Cluster nlyses suggested tht the Bcillus spp. islted frm the intestines f the fish riginted in the diet; hwever, nly ne f the five Bcillus isltes btined frm the externl surfces f the fish nd ne f the tw btined frm the wter were relted t thse frm the diet nd intestines. Psychrbcter immbilis ws nt islted frm the intestines r the wter, but this species ws significnt cmpnent f bth the diet nd the skin f the fish; few instnces f this species were ls islted frm the gills. Almst ll f the P. immbilis isltes frm the diet nd skin were btined t 7 e. The isltes frm the fish gruped int distinctly seprte clusters frm the diet isltes, but these individul clusters were clsely relted; thus, it is nt cler whether the P. immbilis n the fish riginted in the diet. A few ther strins islted frm the fish nd wter clustered clsely with strins frm the diet. All f the P. fiurescens, sme f the B. cetylicum, nd few Mrxell isltes riginted in the feed. Nne f these grups, hwever, mde significnt cntributins t the ttl number f isltes. The Stphylcccus nd crynefrms islted frm the diet clustered seprtely frm thse islted frm the fish nd wter. The ther txnmic grups demnstrted clse reltinships mng the isltes frm the skin, gills, intestines, nd wter but nt the diet. One hundred thirty-six isltes were btined frm the selective enrichments. N Slmnell, Listeri, Plesimns, r Yersini enterclitic isltes were btined; Y.frederiksenii ws islted frm the wter smples, but nt frm the enrichments. Stphylcccus ureus ws islted frm the diet, skin, nd wter. V mimicus ws islted frequently frm the gills, intestines, skin, nd wter, nd ne islte ech f V chlere nd V vulnijicus were fund n the gills. DISCUSSION Since the wter is reused in recirculting system rther thn replced, ne might expect the bcteri frm the diet nd intestines t ccumulte nd estblish themselves s prt f the micrflr f the wter, skin, nd gills. Hwever, with the pssible exceptin f P. immbilis, the rgnisms in the diet ppered unble t successfully cmpete with the strins present in the wter nd n the fish surfces, nd nly few f the isltes frm the intestines were the sme strins s isltes frm the wter, skin, nd gills. The micrflr f the diet hd strng influence n the micrflr islted frm the intestinl cntents during the first 10 weeks; bth diet nd intestines yielded high numbers f Bcillus isltes. This is cntrry t the results seen in striped bss frm the flw-thrugh tnks, where Aermns nd Plesimns isltes were dminnt in the intestinl micr flr thrughut the study (24). Withhlding feed frm the fish befre the strt f this study my hve reduced r eliminted the resident micrflr in the intestines; the intestines f sme fsting fish hve been fund t cntin diminished numbers f bcteri (39) r t be nerly sterile (19). Als, the fish were stressed frm the mve int the clsed system, s they fed prly during the tw weeks befre smpling begn; this my hve delyed redevelpment f the resident micrflr. The bsence f Bcillus spp. in the 12th week f smpling suggests tht the resident micrflr, chrcteristiclly dminted by Aermns spp., becme reestblished by tht time nd utnumbered the Bcillus spp. This thery is supprted by the sudden rise in the plte cunts f the intestinl cntents t 12 weeks; during the first 10 weeks, these plte cunts hd verged I lg lwer thn the cunts frm the previus studies, but t 12 weeks they reched similr levels. The dilutin fctr

5 952 NEDOLUHA AND WESTHOFF required t enumerte the higher numbers f Aermns clnies wuld hve resulted in few Bcillus clnies being present n cuntble pltes. It is unlikely tht the Bcillus strins pssed thrugh the intestinl trct s reprducing cells. The rtis f B. pumilus t B. lichenifrmis in bth the diet nd intestines were identicl t 2.8. Since B. pumilus cnnt grw nerbiclly (B. lichenifrmis is fculttively nerbic) nd the rti f the tw rgnisms remined cnstnt, it is likely tht neither species ws reprducing in the nerbic envirnment f the intestines. These strins were prbbly pssing thrugh the intestinl trct s spres nd did nt becme prt f the estblished r ntive flr f the intestines. The wter cunts in the recirculting system my hve been reduced by the tretment f the wter with zne, which cn be used s disinfectnt in quculture systems (11). Hwever, in recirculting system where zntin ws nt used, bcteril cunts t 20 C frm the grwing wter f crp (Cyprinus crpi) rnged frm 10 4 t 10 5 per ml (25), similr t the 22 C cunts in the current study. Similr results were ls bserved by Sugit et l. (36) in the grwing wter f puffer (Fugu niphbles) hused in glss quriums. Stcking density my ls hve influenced the wter cunts in the current study. In recircultin system grwing tilpi (35), the erbic plte cunts fr the grwing wter were 6.3 X 10 6 CFU/ml in tnks with stcking density f g fish per glln (l00 g fish per liter) nd 5.1 X 10 5 CFU/ml in tnks with stcking density f 11.4 g fish per glln (3 g fish per liter). The stcking density t the strt f this study ws 1.5 g fish per liter in the tnks frm which fish were smpled, but the wter ws shred with tnks tht hd strting stcking density f 12.8 g fish per liter; the highest wter cunts were less thn 10 5 CFU/ml. Mst f the bcteri islted in this study fell int grups similr t thse fund in the tw previus studies, but there were sme ntble differences. Species f Methylbcterium nd Rhdbcter were nt fund in pnds r flw-thrugh tnks. Bth gener re ften sscited with queus envirnments (12, 13); Rhdbcter spp. re ble t utilize mmni, s they my be invlved with nitrgen cycling in the bifilters. Nerly 15% f the isltes frm the fish surfces in this study were S. putrefciens r P. immbilis. S. putrefciens isltes ccurred in smll numbers in pnd-rised fish but nt in fish frm the flw-thrugh tnks, nd P. immbilis ws nt present in either grup. S. putrefciens, which grws rpidly nd cmpetes well t lw tempertures, is ne f the mst imprtnt cntributrs t the spilge f refrigerted fish (5, 8, 34). Isltes f P. immbilis, which hs ls been sscited with the spilge f refrigerted fish (34), were btined primrily t 7 C, reflectin f the rgnism's bility t grw under refrigertin (15). Lrge ppultins were islted frm bth the feed nd skin, nd the P. immbilis n the skin my hve riginted in the diet. This suggests tht diet culd hve n indirect effect n the refrigerted shelf life f qucultured striped bss by cntributing t the spilge flr. The Vibri ppultins were higher in the fish frm the recirculting tnks thn frm the ther systems. Since mny Vibri spp. require NCl fr grwth (7), this my be result f the higher slinity in the recirculting tnks nd the dditin f 0.5% NCl t the PCA. McFrlne et l. (17) fund higher Vibri ppultins in wild striped bss frm mrine envirnment thn in fish frm n esturine envirnment; similrly, slmn rised in sewter hd higher incidence f Vibri thn slmn rered in freshwter (42). V. mimicus ws the mst frequently islted vibri. V. chlere nd V. vulnijicus ccurred t much lwer incidence thn in the ther quculture systems, nd V.prhemlyticus ws nt islted t ll. Nticebly bsent frm the recirculting system ws Plesimns shigellides, significnt cmpnent f the intestinl micrflr in fish frm pnds nd flw-thrugh tnks. Plesimns ws nt reprted in wild striped bss (17) but hs been reprted in ther qucultured species (14, 18, 28, 37, 38); Plesimns is f interest in quculture prducts becuse it cuses dirrhel illness in humns (40). Plesimns is usully sscited with freshwter envirnments, lthugh the rgnism cn grw in culture brths with up t 5% slinity (40); the slinity in this system ws cnsiderbly higher thn in the thers nd my ccunt fr the bsence f this species. Hwever, it is ls pssible tht Plesimns ws nt detected in the recirculting tnks becuse the intestinl micrflr ws prly estblished; furthermre, becuse Plesimns ws s prevlent in the ther quculture systems, smples were directly plted nt the selective nd differentil medi used insted f being pssed thrugh n enrichment brth. The mst frequently islted pthgens were mtile Aermns spp. nd V. mimicus, which hve been implicted in cses f fdbrne dirrhel illness fllwing cnsumptin f undercked nd nd rw sefds (1, 6, 22, 31). A. hydrphil nd sme vibris re f cncern becuse they re cpble f grwing in fds t refrigertin tempertures. These rgnisms re s widespred in the queus envirnment nd ccur s ften in sscitin with sefds tht ttempts t eliminte them frm the fish grwing envirnment wuld be futile. S. ureus ws the nly ther pthgen f cncern islted in this study. Aqucultured fish my be mre susceptible t precpture cntmintin with S. ureus thn wild fish becuse their envirnment is in frequent cntct with humns; hwever, this is prbbly insignificnt cmpred t the cntmintin frm fd hndlers nd prcessing equipment tht ccurs fter cpture. S. ureus cmpetes prly with ther bcteri, s it is primrily cncern in prcessed fds which hve been recntminted. The verll incidence f rgnisms tht culd cuse serius fdbrne illness in humns ws very lw in fish frm the recirculting system. When prperly hndled nd sufficiently cked, qucultured striped bss d nt present risk f fdbrne illness. ACKNOWLEDGMENT The uthrs thnk Dr. Curry Wds nd the stff f the Crne Aquculture Fcility, Bltimre, MD, fr generusly prviding the fish used in this study nd fr ssisting in the cllectin f the fish frm the

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