Experimental infection of Aphanomyces invadans and susceptibility in seven species of tropical fish

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1 Veterinry Worl, EISSN: Aville t RESEARCH ARTICLE Open Aess Experimentl infetion of Aphnomyes invns n suseptiility in seven speies of tropil fish Seyeeh F. Afzli 1, Hssn Hj. Moh Du 1, Iss Shrifpour 2, Mohmm Afshrns 2 n Shiv Shnkr 3 1. Deprtment of Veterinry Clinil Stuies, Fulty of Veterinry Meiine, Universiti Putr Mlysi, UPM, Serng, Selngor, Mlysi; 2. Irnin Fisheries Reserh Orgniztion, Tehrn, Irn; 3. Deprtment of Foo Engineering, Mokpo Ntionl University, Kore. Corresponing uthor: Hssn Hj. Moh Du, e-mil: hssnm@upm.eu.my, SFA: s.f..845@gmil.om, IS: ishrifpour@yhoo.om, MA: mfshrns@yhoo.om, SS: shivioteh@gmil.om Reeive: , Revise: , Aepte: , Pulishe online: oi: /vetworl How to ite this rtile: Afzli S.F, Hssn M.D, Shrifpour I, Afshrns M, Shnkr S (2015) Experimentl Infetion of Aphnomyes invns n suseptiility in seven speies of Tropil fish, Veterinry Worl 8(9): Astrt Aim: Epizooti ulertive synrome (EUS) uses y quti oomyete fungus, Aphnomyes invns is ngerous fish isese of wie rnge of fresh n rkish wter, wil n frme fish throughout the worl. The ojetive of the present stuy ws to etermine the suseptiility of numer of tropil fish speies to the EUS n ompre the severity of infetion etween experimentl groups. Mterils n Methos: Snkehe, Chnn strit (Bloh, 1793); snkeskin gourmi, Trihopous petorlis (Regn, 1910); koi rp, Cyprinus rpio (Linneus, 1758); rohe tfish, Clris mroephlus (Günther, 1864); golfish, Crssius urtus (Linneus, 1758); liming perh, Ans testuineus (Bloh, 1792); n Nile tilpi, Oreohromis nilotius (Linneus, 1758) were hllenge y intrmusulr injetion using zoospores of Aphnomyes invns (NJM9701). The infete fish skins n musles were exmine for EUS histopthologil hrteristis, n the results on the severity of lesions n mortlity were nlyze using SPSS progrm. Results: All zoospore-injete fish were shown to e suseptile to the EUS infetion exept Nile tilpi. Although, the generl histopthologil pttern ws similr in the zoospore-injete group, ut there were some vrition in grnulomtous retion, tht is the presene or sene of gint ells, n time of mortlity were etete. The result of sttistil nlysis showe tht there ws signifint ifferene etween speies, ( 2 = n p<0.01). Conlusion: Gourmi, koi rp, n tfish were emonstrte to e highly suseptile while golfish n liming perh were foun to e moertely suseptile to the EUS infetion. These finings suggeste tht the ellulr response of fish to myoti infetion n grnulomtous retion vrie in ifferent fish speies, whih oul not e n initor of suseptiility or resistnt to the EUS itself, lthough it ws shown tht the grnultion rte n the level of mturity or soliifition (onsolition of grnuloms) were higher in resistnt fish. Keywors: Aphnomyes invns, epizooti ulertive synrome, freshwter fish, histopthology. Introution Epizooti ulertive synrome (EUS) is linil fish onition tht rguly h the most serious soio-eonomi impt ffeting the fresh n rkish wter fish over the pst 40 yers n hs een liste y the Aquti Animl Coe sine 1995 [1]. The isese propgtes through the ountries of the Asi-Pifi region with ire onsequenes for fish resoures n the livelihoo of fishermen [2]. EUS is isese tht mnifests in ute skin n musle ulertion with the onsequene of signifint mortlity in freshwter fish. The quti oomyete fungus, Aphnomyes invns, hs een ientifie s the ustive gent of EUS [3,4] whih releses proteolyti enzyme filitting penetrtion of fish tissue n preipitting shllow to eep ulers, leing to high mortlity in fish popultion [5]. Until to te, no effetive prophylti mesures n/or vines re ville ginst EUS. If Copyright: The uthors. This rtile is n open ess rtile liense uner the terms of the Cretive Commons Attriutin Liense ( retive ommons.org/lienses/y/2.0) whih permits unrestrite use, istriution n reproution in ny meium, provie the work is properly ite. sientifi evelopment nnot solve this miroiologil prolem, it is likely to give signifint negtive impt on the inome of fish frmers who rely on fish of wil origin for their livelihoo. The potentil effets of n introution of the pthogen through interntionl tre of tropil fish re ltently hzrous. In the urrent stuy, snkehe n Nile tilpi were use euse of their importne s EUSsuseptile n resistnt fish, respetively for the omprison of infetion. The other freshwter fish speies were selete for experimentl infetion ue to their signifint mrket emn in the Asin qurium tre n foo inustry in orer to etermine their vulnerility to the ustive gent of EUS n to ompre the severity of infetion in eh speies. Mterils n Methos Ethil pprovl The present stuy ws pprove y Institutionl Animl Ethis Committee of the Universiti Putr Mlysi. Veterinry Worl, EISSN:

2 Aville t Experimentl hllenges For oomyete fungi ulture n sporultion, hemp sees were employe s it. Aphnomyes invns isolte NJM9701 (ourtesy of Dr. Birgit Oitmnn) myeli were enourge to grow on sterile hemp sees plnte on the gluose-peptone (GP) gr. Susequently, 6 mm growing hyphe on the hemp sees were trnsferre into GP-yest roth (GPY) with peniillin-streptomyin (10 ml/l) n inute t 25 C for 5 ys, then wshe three times in utolve pon wter (APW). The myelium mts from the tip of olonies together with sees were loe into 1.5 ml mirotues ontining APW for 24 h [6]. Fish utilize for hllenge experimenttion were snkehe, Chnn strit (80±20 g); snkeskin gourmi, Trihopous petorlis (12.5±2 g); koi, Cyprinus rpio rpio (25.5±4.5g); golfish, Crssius urtus (16.5±1 g); rohe tfish, Clris mroephlus (30±3.5g); liming perh, Ans testuineus (30±2 g); n Nile tilpi, Oreohromis nilotius (25±0.5 g). All fish were linilly helthy n kept in quri using e-hlorinte wter equippe with ir stones n qurium heters mintine t 20 C. Two replite hllenge groups n one ontrol ws set-up for eh fish speies utilizing 22 fish per tnk. Fish were nesthetize using MS-222 (triine methnesulfonte) t 150 ppm prior to injetion with 0.2 ml of 10,000 spore/ml suspension (i.e spores/fish) intrmusulrly into the left sie of the oy elow the orsl fin. The ontrol fish were inoulte with n equl volume of APW, n ll fish were monitore ily for hrteristi EUS linil signs uring the 35-ys tril. Two fish from eh tnk were smple t y 1, 2, 4, 6, 8, 10, 12, 14, 21, 28, 35 post-injetion (p.i.) ut e n/or severely ffete fish were ollete s pproprite. Skin n musle tissues (4-5 mm ues) whih exise from lesion res were fixe in 10% phosphte-uffere formlin solution, proesse through n utomti tissue proessor n emee in prffin. The prffin loks were then setione t 4-5 μm thikness with rotry mirotome n mounte on len glss slies. The slies were then stine with routine hemtoxylin n eosin n perioi i shiff (PAS) [7]. Sttistil nlysis The severity of utneous lesions typil of EUS infetion in fish ws sore reltive EUS lesions in snkehe, eing known n EUS-suseptile speies (Figure-1). Dt on the severity of lesions n mortlity were nlyze using SPSS progrm. To ompre etween the experimentl n ontrol groups, Chisqure test ws pplie, n the severity mong seven fish speies ws ompre using Kruskl Wllis test. Results Experimentl hllenges Gross oservtions Fish in the ontrol group (APW-injete) i not show ny ehviorl or linil signs of isese throughout the experiment. The positive ientifition of A. invns septte hyphe (Figure-2) from the zoospore-injete tissues ws rrie out y the wet mount preprtion metho. EUS ssoite lesions were oserve in ll fishes inoulte with A. invns, exept in Nile tilpi (Figure-3). From 24 h fter inoultion, the min linil oservtions in zoospore-injete fish inlue lolize swelling on the oy n reening. Sle loss n re ulertion unerlying musulture were oserve from y 4 of p.i. The re ulertion further evelope into eep ulertion tht ws overli with fungl olonies from y 6 of p.i. Swimming ehviors were lso ffete, n most fish were oserve swimming with teethering movement, inluing unnturl kwr n forwr movement from y 8 of p.i. In some Figure-1: Visul esription of skin lesion soring system of exmine fish is shown using epizooti ulertive synrome - ffete snkehe lesions. () Sore 1: Skin lnhing lost of sle n epithelil ells. () Sore 2: Re spot n mrke swelling. () Sore 3: Ulertive lesion. () Sore 4: Deep ulers involving unerlying musles. Figure-2: Smer preprtion of epizooti ulertive synrome oomyete fungus Aphnomyes invns re-isolte from rtifiilly infete snkehe () n Snkeskin gourmi (). Typil Aphnomyes invns non-septte hyphe re shown with luster of enyste primry zoospores (rrows) ( 100). Veterinry Worl, EISSN:

3 Aville t fish, the oy olor ws ffete, n it ws oserve tht these fish showe pllor t the site of injetion on y 10 of p.i. All moriun fish showe wekness with loss of ppetite n remine t the ottom of the tnk efore eth. In ontrst to the other speies whih were use in this stuy, no visile inflmmtory response ws foun in Nile tilpi, whih ws use s EUS-resistnt fish uring the ourse of the stuy. ws well in progress, n epithelioi ells were ompnie y ense firolsts lyers. At y 21 p.i., grnulomt of vrying sizes, mostly smll sizes, were fuse together in ifferent res, forming lrger grnulomt surroune y lyers of firous tissue. Degenerte fungl hyphe were oserve in some Histopthology In oth ontrol n tretment groups, few egenertive hnges ue to the neele penetrtion were oserve in ll smples in the erly stges of the isese, however, it ws ontinue with inflmmtory ell infiltrtion, espeilly mrophge response n severe invsive myonerosis in A. invns infete fish. In snkehe, there ws some hemorrhge in the epiermis n hyperemi loo vessels with firolst tivity n myophgi t 24 h fter injetion. At y 2 of p.i., umultions of mononuler inflmmtory ells in the ermis, seprtion of loose onnetive tissue of the ermis, n rupture of pillries leing to hemorrhge were evient. The prominent feture s oserve in infete fish ws the egenertion of musle tissues, loo vessels, n the presene of epithelioi ells. By the 4th y, mrophges were present in irulr pttern of epithelioi ells irumsriing the lesion re n roun the fungl hyphe, with some vrine epenent upon fish speies. At y 6-8 p.i., the regenertion of musle fiers ws tive; the presene of firolst n grnultion tissue were onspiuous in this perio lnspe the entire efetive re. From y p.i., well evelope smll n lrge grnulomt were ominnt, ompose of few lyers of epithelioi ells surroune y vrile thiknesses of firous enpsultions with entrl re of the neroti mteril. Generlly, in other fish speies exept Nile tilpi, the histopthologil pttern ws similr, however with vrition in grnulomtous retion, presene or lk of gint ells, n time of mortlity. These were onsiere to reflet suseptiility (Tle-1). In gourmi (Figure-4), koi rp (Figure-4), n rohe tfish (Figure-4), the grnuloms formtion ws wek n the oomyete fungi hyphe were oserve s eing istriute in most res of the impirment with no grnulom. Wheres, in snkehe (Figure-4), golfish, n liming perh (Figure-5), the grnultion proess e f Figure-3: Epizooti ulertive synrome skin lesions (rrows) of fish injete rtifiilly with Aphnomyes invns spores: () Snkehe, () Snkeskin gourmi, () Koi rp, () Brohe tfish, (e) Golfish, (f) Climing perh. Figure-4: Histopthologil hrteristi of epizooti ulertive synrome-ffete fish intrmusulrly injete with Aphnomyes invns NJM9701 zoospores. () Mture grnulom with neroti enter surroune y firolst lyers (F) in Snkehe (12 pi). Note the eposition of melnin pigments, H & E, 400. () Formtion of grnulomt (rrow) hrterize y thik firolst lyers in Snkeskin gourmi (10 pi), H & E, 200. () A grnulom (rrow) surroune y epithelioi ells in Koi rp (10 pi), H & E, 200. () Foreign oy type gint ells (irle) with surrouning onnetive tissues in rohe tfish (14 pi), H & E, 400. Tle-1: Chrteristi histopthologil finings ompre mong seven fish speies infete with Aphnomyes invns. Histopthology finings fish speies Cellulr infiltrtion Grnuloms Gint ells Free hyphe Woun heling Snkehe Snkeskin gourmi Koi rp Brohe tfish Golfish Climing perh Nile tilpi Develope Immture - Immture - Immture - Develope Develope Well evelope Note: Severity oe: -=Asent, =Mil, =Moerte, =Severe Veterinry Worl, EISSN:

4 Aville t ( 2 =145.11, p<0.01). Exmine fish inluing snkeskin gourmi, koi rp, n rohe tfish showe signifintly higher thn snkehe while lower signifint were reore in golfish n liming perh. Figure-5: Histopthologil hrteristi of epizooti ulertive synrome-ffete fish intrmusulrly injete with Aphnomyes invns NJM9701 zoospores. () Presene of Lnghns type multinulete gint ells (irles) in Golfish (6 pi), H & E, 400. () A numer of grnulomt (rrow) surroune y firolst lyers in liming perh (10 pi), H & E, 400. () Enpsultion fungi y Foreign oy type gint ells (irle) n grnuloms (rrow) surrouning y thik firolst lyers (4 pi), H & E, 400. () Non-psulte hyphe (rrow) in neroti res in Koi rp (18 pi), P.A.S, 200. grnulomt. However, few free fungi hyphe were evient in the infete res whih were onfirme in PAS stining (Figure-5). Formtion of multinulete gint ells ws lerly evient in the infete re in tfish (Figure-4), golfish (Figure-5), n Nile tilpi (Figure-5), ut not in other speies. In Nile tilpi, fully mture grnulomt of vrying sizes together with multinulete gint ells were evient n fille the whole ffete re in very erly stges of infetion. Most grnulomt in vrying res fuse together forming lrge grnulom (fusion) surroune with ense firous lyers n melnin pigments, n no free fungi hyphe were etete in infete tissues. At y 35 p.i., the tilpi s lesion ws hele, n regrless of the presene of grnulomt, the mirorhiteture of the tissue ws lmost ientil to the norml tissue smple. First mortlity ourre in gourmi n koi rp t y 7 p.i., whih ll fish ie t y 14 p.i., n 18 p.i., respetively, showing lethrgy n norexi efore eth. Afterwr, mortlity ws oserve in tfish t y 8 p.i., in golfish t y 10 p.i., in snkehe t 14 p.i., n ll moriun fish were e t y 21 p.i. The first liming perh ie t 15 p.i., n ll the rest were foun e t y 28 p.i while Nile tilpi n fish of the ontrol group i not show ny mortlity. Sttistil nlysis The results of Chi-squre test showe tht there were signifint linil n pthologil ifferenes etween ontrol n experimentl groups for ll seven speies of the exmine fish (Tle-2). The result of Kruskl Wllis test showe tht there ws signifint ifferene in suseptiility etween speies, Disussion In the urrent stuy, erml ulertion n grnulomtous retion were suessfully inue in the experimentlly infete fish vi i.m. injetion of A. invns live zoospores, the egree of whih ws similr to tht woul e expete ue to nturl EUS infetion. It shoul e note tht the infetion moel use in the urrent stuy ws not influene y ny iologil or physiologil ftors, n the environmentl ftors were kept onstnt. The experimentl fish reeive very similr oses of zoospores. Therefore, the fish s immune response n ntomil ftors were the min ftors influening the evelopment of the isese. Although, the responses of the helthy fish uner lortory onitions my not e ientil to tht of fish infete nturlly, it is nonetheless n importnt omponent of unerstning the si responses s evient in suseptile fish. The injete zoospores of A. invns were le to grow in the musles of experimentlly infete fish n germinte into hyphe n proliferte extensively, using nerotizing grnulomtous ermtitis with the inflmmtory response within very short perio fter injetion. However, of more thn 100 fish speies tht hve een reporte to e ffete y EUS, only few of them h een vlite y emonstrting the presene of myoti grnuloms in histologil setions or y isoltion of the pthogeni oomyete fungus, A. invns from the tissues unerlying the ulers [2]. Experimentl infetion, s exeute in USA with A. invns whih were rrie out in four speies of esturine fish, le to similr lesions, n severe pthologies in Atlnti menhen, Brevoorti tyrnnus, n Killifish, Funulus heterolitus were oserve [8]. These historil results supporte the results of the urrent stuy whih prove tht some speies of fish were more sensitive thn others, n fish hving soft epierml skin lyers suh s gourmi, ornmentl rps, n tfish were more vulnerle to infetion y A. invns. Johnson et l. [8] stte tht Atlnti menhen hve n oily flesh, thin n frgile epierml lyer, n reily lose their eiuous sles tht my filitte the ttrtion of n infetion y zoospores of A. invns. Menhen tissue hs lso een reporte to support the inrese growth of hyphe of A. invns s ompre with gr [9]. Thus, the preiletion of A. invns for Atlnti menhen my lso reflet the suseptiility of the host to stress, whih filittes entry, n y nture of the oily flesh, whih serves s signifint supportive nutritionl soure for the oomyete [8]. Prhn et l. [10] investigte the suseptiility of three speies of Inin mjor rp (tl, rohu, n mrigl) n ommon rp to EUS n foun tht Veterinry Worl, EISSN:

5 Aville t Tle-2: Skin lesion sore n signifine of isese in A. invns experimentlly hllenge fish. Speies/ tretment Skin lesion sore Mortlity χ 2 p vlue 0 1 χ Snkehe Exp (%) 0/44 (0) 0/44 (0) 0/44 (0) 24/44 (54.5) 20/44 (45.5) 44/44 (100) <0.001 Snkeskin gourmi Exp (%) 0/44 (0) 0/44 (0) 0/44 (0) 21/44 (47.7) 23/44 (52.3) 44/44 (100) <0.001 Koi rp Exp (%) 0/44 (0) 0/44 (0) 0/44 (0) 23/44 (52.3) 21/44 (47.7) 44/44 (100) <0.001 Brohe tfish Exp (%) 0/44 (0) 0/44 (0) 0/44 (0) 22/44 (50) 22/44 (50) 44/44 (100) <0.001 Golfish Exp (%) 0/44 (0) 0/44 (0) 6/44 (13.6) 22/44 (50) 16/44 (36.4) 44/44 (100) <0.001 Climing perh Exp (%) 0/44 (0) 0/44 (0) 9/44 (25.5) 20/44 (45.5) 15/44 (34.1) 44/44 (100) <0.001 Nile tilpi Exp (%) 39/44 (88.6) 5/44 (11.4) 0/44 (0) 0/44 (0) 0/44 (0) 0/44 (0) 22.5 <0.001 Cntl=Control, Exp=Experimentl, Note: Figures in prentheses inite perentge of eh sore n mortlity, A. invns=aphnomyes invns within perio of 12 ys, there ws 100% mortlity with severe gross lesion evelopment n myoti grnulomtous lesions in Inin mjor rp, wheres, in ommon rp, neither mortlity nor gross visile lesions were oserve. Their finings, s well s histopthologil exmintions y Bruh et l. [11], inferre tht vne fingerlings of Inin mjor rp re highly suseptile to EUS. Oitmnn et l. [12] h onute n experimentl infetion utilizing similr A. invns strin NJM9701 n emonstrte tht Europen tfish n Silurus glnis proue typil EUS ulertive skin lesions. Three-spot gourmi n Trihogster trihopterus were use in their stuy s positive ontrol n emonstrte to e more vulnerle to A. invns infetion s ompre to Europen tfish. Notwithstning injete gourmies inite the hnge swimming ehvior (forwr n kwr teethering movements) t y 6 p.i., however, in the urrent stuy similr swimming ehvior were seen in gourmies t y 7 p.i. In the urrent stuy, oservtions of the inflmmtory hnges in fish fter i.m. inoultion of A. invns inite the ourrene of the hrteristi fetures of n ute to hroni inflmmtion. The min fetures of the hroni inflmmtion were ellulr infiltrtion, hemorrhge, firosis, n vsulriztion in the lesion re t the erly stges of the infetion use y the pssge of the neele inserting the inoulums, n the zoospore tivity whih trnsmute to omplete egenertion of the musle fiers. The min omponents of the inflmmtory response were the inflmmtory ells omprising mononulete ells, gint ells, n firolsts. In this stuy, euse of the presene of the pthogen in the tissue, inflmmtory ells egn to infiltrte into the lesion re resulting in the initition of myophgi t 24 h fter inoultion. Mrophges were oserve in the lesion re from the erly stges while their numer n tivity inrese, ut only the Nile tilpi sueee to initite the heling proess, wheres, in the other exmine fish, the numer of inflmmtory ells erese t the finl stges of smpling. The longterm tivity n inresing numer of mrophges oul e n immunologil retion relte to mrophges responsiility for the evelopment of fish immunity [13]. However, suh fish efense mehnisms eploye ginst the inving fungus were not effetive enough to limit growth of the fungus insie the tissue of fish exmine in this stuy exept in respet to Nile tilpi whih ws known to e resistnt to the EUS. In the urrent stuy, the presene of multinulete gint ells with engulfe hyphe were oserve in the lesion re in rohe tfish, golfish, n Nile tilpi t y 2, 6, n 4 p.i., respetively. Furthermore gint ells with no engulfe fungus were oserve either in the lesion re or in the grnulomt. The numer n tive gint ells signifintly inrese in Nile tilpi y the time, leing to enpsultion of fungi mnifeste in the infete re, however, this retion in rohe tfish n golfish oul not prevent fungl growth in the lesion. Other stuies lso reporte the presene of gint ells in Europen tfish, Silurus glnis [12], golfish, n other fish speies suh s Mirror rp [13], Ayu n Common rp [14], Atlnti menhen, n stripe killifish [6], ut it ws not reporte in Snkehe s experimentlly infete with A. invns [15], wrf gourmi infete y Aphnomyes sp. [16], n Veterinry Worl, EISSN:

6 Aville t in gry mullet or ny other fish speies. Thompson et l. [17] suggeste tht gint ell formtion i not seem to e n initor of resistne to EUS, sine some of the highly suseptile speies (Ayu, tfish, golfish, n puntius) were ple of forming suh, while, the tul impt of this type of response in respet to fungl infetion is still unler. Myoti grnulomtous retion whih is n importnt efense mehnism of fish ginst EUS were oserve in this stuy in ll the experimentlly infete fish, resulting in vrying size n thikness inite y epithelioi ells surrouning the fungl hyphe t ys 6-8 p.i. Previous stuies estlishe tht firous prolifertion n intense grnulomtous retions in response to A. invns my e n importnt ftor inhiiting fungl growth. The results of this stuy emonstrte tht grnulomtous retion oul prevent n hlt fungl growth only in resistnt fish suh s Nile tilpi, ut in suseptile fish, for exmple snkehe, gourmies, koi rp, tfish, golfish, n liming perh, the rte of fungl growth ws more rpi thn grnulomtous retion n free fungl hyphe were etete in the infete re even t the time of mortlity. In the urrent stuy, mortlity in EUS suseptile fish ws oserve from y 7-14 p.i., however, therefter life ws prolonge s reltive to mximum of y 28 p.i. It ws suggeste tht this ely in the time of mortlity ws ue to some egree of resistne offere y the mrophges in the initil stges of infetion [18]. During this perio, the host my e ttempting to prevent the invsive spre of the hyphe in tissue, however, in suseptile fish, the hyphe were le to inve neighoring tissues, proliferte [17] n use extensive myonerosis ue to the relese of proteolysins in lrge res of myotome resulting in moriity or eth. Although in Nile tilpi, the grnulomtous retion ws well evelope with high level of soliifition from very erly stge of isese (y 2 p.i.) leing to enpsultion of ll fungi in infetion re. From the finings of the urrent stuy, it oul e suggeste tht ellulr response of fish to myoti infetion n grnulomtous retion vrie in ifferent fish speies whih oul not e n initor of suseptiility or resistne to the EUS, however, the rte n the level of mturity foun to e higher in resistnt fish. These finings were similr to W et l. [14] who oserve tht EUS resistnt ommon rp, Cyprinus rpio, evelope grnulom fr more rpily thn suseptile Ayu, Pleoglossus ltivelis, without pprent gross mnifesttion. In ontrst, Oitmnn et l. [12] i not oserve ny myoti grnuloms in rtifiilly injete Europen tfish, Silurus glnis, with A. invns isolte NJM 9701, whih ws the sme fungl strin use in the urrent stuy, lthough the formtion of multinulete gint ells n grnulomtous retion in gourmies were reore in their stuy. The present stuy n Phee et l. [19] hve sueee in inuing A. invns infetion in golfish, lthough Oitmnn et l. [12] file to infet golfish vi intrmusulr injetion of A. invns (NJM9701) zoospores. These results my suggest ifferenes in pthogeniity of A. invns strins on ifferent host speies, or the presene of ifferenes in strin suseptiility mong golfish. Conlusion From the finings of the urrent stuy, it ws onlue tht, ompre to the EUS-suseptile snkehe n EUS-resistnt tilpi, tropil snkeskin gourmi, koi rp, n rohe tfish were emonstrte to e highly suseptile, whilst golfish n liming perh were foun to e moertely suseptile to the EUS infetion. Histopthology exmintion ws emonstrte to e prtil n pproprite for etetion of EUS infetion within the 1 st y of the isese. Erly etetion n the pplition of suitle prophylti methos n therefore irumvent erly trnsmission, whih if not ught n mnge n le to serious outrek of the isese n lso the introution of the pthogen into unffete ountries. Authors Contriutions SFA esigne n rrie out the experiment n rfte the finl mnusript. HMD n IS nlyze the histologil results. MA rrie out the sttistil nlysis. SS ws responsile for finl eiting n English proofreing of the mnusript rft. All uthors re n pprove the finl mnusript. Aknowlegments The uthors wish to reor their sinere thnks to the Deprtment of Veterinry Clinil Stuies, Fulty of Veterinry Meiine, Universiti Putr Mlysi for finnil ssistne n proviing ll the neessry filities to rry out the present work. The uthors re inete to Dr. Birgit Oitmnn (CEFAS, Unite Kingom) for her support in proviing A. invns strin (NJM9701). Competing Interests The uthors elre tht they hve no onflits of interests. Referenes 1. OIE. (2014). Infetion with Aphnomyes invns (Epizooti Ulertive Synrome). Ch Aquti Animl Helth Coe - 07/08/2014. Aville from: oie.int/filemin/home/eng/helth_stnrs/h/2010/ hpitre_phnomyes_invns.pf. Aesste on: Lilley, J., Cllinn, R.B., Chinut, S. n Knhnkhn, S. (1998) Epizooti Ulertive Synrome (EUS) Tehnil Hnook. The Aquti Animl Helth Reserh Institute, Bngkok. p Blok, F.C., Blzer, V., Cllinn, R., Hti, K., Krunsgr, I., Mohn, C.V. n Bon-Rentso, M.G. (2005) Outomes of short expert onsulttion on epizooti ulertive synrome (EUS): Re-exmintion of usl ftors, se efinition n nomenlture. In: Wlker, P., Lster, R. n Bon-Rentso, M.G., eitors. Asin Veterinry Worl, EISSN:

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