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    Review of diagnostic techniques for detection of salmonid fish infected with Myxobolus cerebralis

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    Review of diagnostic techniques for detection of salmonid fish infected with Myxobolus cerebralis

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    © All Rights Reserved
    Download as PDF or read online from Scribd
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    97 views16 pages

    Andree2002 - M-Cerebralis DiagReview

    Uploaded by

    Karl Andree
    Review of diagnostic techniques for detection of salmonid fish infected with Myxobolus cerebralis

    Copyright:

    © All Rights Reserved
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    ‘Ameri abs Soy Sypram 2197-21, 2002 {©1003 y the Amen Phe Sly A Review of the Approaches to Detect Myxobolus cerebralis, the Cause of Salmonid Whirling Disease Kaa B.ANousE* ano RongtyP Hesncx Department of Meese nd Epfemicogy, Seve of Verity Mecne ‘Univer of Caldera af Oars Divi alia 95616, USA Fuzanem MacConseu. Montana Fh Wide and Pas 1400 9th Se, Bema, Montana 59715, USA Asta. The mut of whing dase on the aqvaculueof out ove the ast 89 yeas hts ‘ele inthe development of single to more complex mtn to detect the easutve eat Myf cobals. With the absence of tetve extent fine deste es pce ‘crcl I or galt ve aster perspective to thee developers wile dining the relative mene ofthe ferent techies ‘The cument widespread dstrbution of M. oe bral sin pat de to the mvernents of batchery- ‘aie tout for tocking for spore sing that were ‘fen infected but showing no signs of diene Enploying more sensitive diame rocedes dae the pathogen ingly infected ih sone importane means to prevent this snadvertent spread of M, cereal. The detction of sores of the comet se and shape and in the known eget us the basis the currendy acepted dingnonis of M. cera infection. Unfortunatly, many -sysospotean sores share simular morphology, ad improved proaches to discriminating M. ceeba from other Moanbols sp. fund in salmon sues ate needed. There have fon prgiesive improve ments in che detetion and dcrination of M. cavers spowes in fh tse, inching the ino Aiton of mechanial and enyinatie means to release spores fom skeletal clement, analyses of Pooled oe batch samples, andthe we of labeled muds for che specie entiation of spores. More sensitive and spect detection liso the Fansite in fh sue and olgochaetes have een sbeainel using polymerise chan reaction (PCR) nl insu hybedeation, The Benefits thee ew molecular approaches are already cent ae research tol and thet vilaton for we as owe Broaily applied diagnostic reagents should be an immediate pel Mpanbls erebralls, the etiological agent of hing disease isthe most intensively stad of the mysosporean parasite of fish ye elective treatments for this dese have heen developed Experimental nal administcions of faagilin have heen shown to eect poverty inf ‘on, but the parasite noe slime fom the host (ELMathoul and Hofmann 1991). Avoid ance ofthe parasite, to prevent inetions in ep. tive fish, Is dherefore a pisiry management pyoach, Awidance relies on effective dn tim of water o sediments By the we of Vato ‘chemicals (Hofmann and Honan 1972), le violet (UV) ight (Hotimann 1973; Hesick ea 2000), and ene (M. ELMathoul, University cf Murch, unpublished des), Allo these methods «an beelctive n preventing contact beeen the Paste and smeeptibe out ‘Accurate detection of the parsite provides « messre ofthe success of management prcedres simed ne contulling whirling dese. Due tothe ‘impact of whiting dseae on wid sinbow tout In cereain iver ofthe Intermountsin Wet ofthe United Seanes, testing to detect the pathogen is routinely conducted bork’ on wild aad captive: seared stock of tou to 1) understand the curent Aisibution of M. cabal, 2) adenfy point sources and monitor the il of infection, and 3) decemnine prevalence a severity of infection in wild trout populations Surveillance programs can alo target acer hosts and cations where the prste might be presen, nciing worms, water, snd bird oes 7 TT v8 ANDRE AND HEDRICK Whiting diese was int described by Hofer (1903) and came eo be rudd as 2 condition fend primoniyatnong batchery fish (Hally 1976). The dea spread to the United Sores Fron Europe inthe 1950 with shipments fot for human consumption and now foul 23 teats Tota 1990, Andee ec al 1999). Since the ely 1990, the parasite has become eos “certo wit smonkd populations in theres (Fhe Incemounean West (Nehring ond Waller 1996, Vincent 1996) The unprtance of deer the paras therefore gten with commercial toca federal rearing wu (onan 1990) ved wih the potential detamental impacts the rat con have on il and native stout pul tions (Nefwing au Walker 1996) The mame wing disease” is deriv fom the eating swimaing bdhavior exhibited dt the the ace phe of he deve, Concur wih {he aera simingbohasion the fo wally ‘ahi caudal melons dae CO presure 70 eres this cpm by éhe fast inlay reponse tothe pre. The inflamma mpinaes tn oe gna along the spine reves onto the mca of es, engin een tn he cam tid ofthe body (EPMatbook a 1995) The abet swimming Pehaior bas Been fecetly aed ope ttor enrol Be the porate and the ft flammatory reps ‘aetretng the uper ena cd and Tower bu Ste (Ren a. 2000) ano det he disupin A te auttory onzans was previo thot ‘Developmental sages of ML cera each the cet trl pen sytem via peer neve tes pene Tato he skin ‘The kn, al the russ pores CGpevilli isthe main ste of attachment for she ‘esos tractinomyon (TAM) stage (ELM Tl st al 1995). Howeve, che sin and ental aru tse are ites fr proliferation ofthe para ‘ean no sporgenes (EL-Mboul et 1995). Tar reaching skeletal clement, the pasasite “deseo carage by de prssion of te ese. Destron ofthe crag, featensve, can eal pment clea deformities of various seventy ‘hema ck coll oss ano sais thalgned jr, foesoingof the sou xc thal, an foshorening ofthe operelian es the dessucton of the cniginows elements that Jenks to developenentof permanent skeletal efor tae which chrscrerie che eran pase of the {seme (Hedrick eal 1998). These skeletal abnor ‘nals wil pers even afer cual melanosis sub side. Sporgenesis begins after the parte has ntl the eaeoge: Through seen divisions Ticrcpic tulle spores wih protective Tse ate formic then can ie dormant Ue delet. cases where skeletal aeration te Irma the fch can be conser agymptoati Carin, harboregspore seats in the bones “alae tha arlene afer death ofthe fo "The development i the fs host repress cy half ofthe lifecycle of M. cereals. The at She ab eis an oligochacre wor Tier Snes im which a asc sequential proileatve nd apuropic development takes pice. The ei ‘Reanomyaon of aetinporean stage that fms “fon the speogenest nthe worms the thoeatge tt teaches othe is (Figure 1A Wolf NY Mak 1984, Wolf eta. 198, Maki aod ‘Wolf 1983). The degree which the ese devel sinh fa st dependent on te species a ee the mond (Hesck eal 1998) and the ‘hse ofthe nective sag. The ir of nfctive Sens that might Be found nthe wate is depend fhe om the ype sin, and abundance of the ‘Shstsete hot (Besichamp et als 2001) and Savvenmenral variable sany tha remais t0 be toon fly eso “The inal management approaches to conta sisting dee in Bore andthe United Stes Were cid and sever, with desrction of fh Monks led by hatchery dsiaecton (Horch 1981 Hofmann 1990). Fish were also destroyed in thse and sea by employing toxic cheias in ‘in fio wo contain the paste (nah 1970). AS the life eycle ofthe porate became better wns rood, lisonal contol measies were posible Reducing the olgoccte absar by replacing feather ote pons with concrete ces ae ‘Rang you wll water ror to expose “perareer uplies fr ctl prevent he Sharaf whirling diseae root that were ed in ‘ese conto howevet, werectten infected. The ict hee our fr grein one mens by which the pathogen fas spel to ne ==> topic regions Testing acm war with UY Fhe orang srt wel ater forall tages of eat ting were means that completly prevented M ‘eal ncn in ror rased in cers fais {Fofimann 1975; Hedck eal. 200) The eaent management of whiting dosase pers upon senatve and specific detection as foe M, ena Several mehoss bare teen devrte for this pups, nciing relate ne) co Figut 1. Scanning eectronmicogrephs and ight photomicrographs showing acinosporean and Imjosporeon stage spores ot M cereals 1k and show the actiosporean tage of certs which evel in the olgochete erm hast 1A show the atinaporenn spore stage inmedstay her {tachment via the polar flan tothe skin of Yalow out 18 ad IDshow the myreanrean stage Spore, which develops the slmonid fh host. Using seanning eeconmirorcony the mics costing ‘he posterior of he Spore Mh, polar capsule pores for extrusion of pol aments ad prone ‘roaves (6) are evident by mew molecular agproehes. In this review, we ‘eamine che histrcal development thee dee tion methods foe M. carbs nd discuss their stenaths and weaknesses, tales prticlar DIAGNosTIC METHODS A postive dans of when disease depenon “hservation of the characteristic rom clita sis fan demonstration ofthe peeence of M casas spores aociated with eamlage. These signs Include a al-chasing wining behavior a en da melness daring the ace phase, Later in infection, thee signs may be accompanied by Akefoites present throughout the skeleton but ‘sully mos pronounced in dhe cranium an spe ‘These deformities can vary considerably in seve 'y, making light infections difiele wo in Alto, hacer cold-water dete, cectsbock Injuries, adder insufictencies can cane kele- tal deformities andor melas, whi resembles whiting disease in sonide (Margolis etal. 1996). Diagnoses of whiting disease require demonstrating the presence of M. crea spores (pcovidel ample ie hae pssed for shem to devel op) by microncopie examination of extits fom ‘nil sbeleeon and stned esse sections fom the sume region. Although presorogoni SOs an be observed in stxned tse sections (Her ‘ean and Kerting 1985, de uae pore tages thor fo asthe end point of mysexporean so fpaie development ste the accepted standard 10 remy species inthe phy Mca. Du to che fie wopim of M. ceebls,ealy proces Ueveloped for dingoosis of ‘whining disease ihvstvel excision and then caling of keel Inene prog to. miresopic observation (Meh 92d Lucky 1970) However, several Myzobols spp. exhib sar oro (Fare 2) and a ‘Bijou in cranial sues Hesick tal. 191) therefore, insifiient co ase dingroses of ‘thing disease on gros ena sgns or the pre hee of spores in ane eat alone Ulimatly fatinmation of hing dsesse depends on obscr- otom of characteris goss clinical sins and ‘Keceton of spon ofthe seguir sie and shape role with Isto in cree in stained tse ‘Seron rom suspect slmoni sh (gue 3) “The erent ai staal proces rec mended bythe Feh Health Section (FHS) of the American Furies Society (AFS) require chat then of the fu he proces by ether of wo Teprved extaction techniques flowed by mic ‘Eipic examination 0 detenatrate presence of {jores (ce blow). The skeletal clement of the ‘sum A Magn 1 co © ANDREE AND Hf Ick ha ate subjected to ereymatedigeton and cen tration, or the eal © groan crctly and rhe Fomogenae is pased though a plankton cen Tefen both poor the pal st ae fpoee for micripie observation. Adio fel bends are pce in fate and following a thu pean ebing, sections ae prepared and ‘ened with hematin au es and exanined toca thatthe spores or developnental taps of th parse are presen cae (Lore aos Aman {T1994 A moicton of thee guelines sed See laborers to ut fh es al thine sail plane snd proces each bal sp ‘Ely One has sed for spore wolacon and en Teo, a the oe als wed for histo Trl A weakness of this approach isthe ym ‘Rese dbus of pores and. developmental ‘Kage between the wo halves ofthe head, which, Foner a low para onecnations can Teal Peder resus Between hstobgia and ce “umeration aalaes (Andre et al 1998). Spore Description “Texonomie clasieation of members ofthe gens polis othe spss evel ean be dificult and tReand ofan expenence parstologis for destip> tion ofthe sae and morphologic eitera associa ‘Meh the pore may be seeded (Lom 1987 Walt wid Markiw 1976), When spores or gross ns of the disease are mbintrreted, rbianoses of ring dase can occur that may have expe eee) cons figure 2. light potanicoaraps of pres tlt rm rainbow wou ania ie. Fle 2 Cae see prfaton sig the pein yen gest method. mire 28.5 2 pbc eer fours in PTD erator lxalzed by Neoigy in he Bai sem, THs eeedinen shown ater prfiation on Prcall graben THE CAUSE OF SALMONID WHIRLING DISEASE m1 te, Flour 2A section ofan incsion of ‘cerebral spores n eran bone sane th Inetiylene hae sive consequences fr che aquaculuris (Mangos a. 1996). The macsporin stage spre of M cerca as leniclo shape measuring 9.7 in enath and 85 jn n wath with rao aly ied par capsules of 4.25 3.1 um (Lom and ot smans 1970). The polar laments make 5-6 tune within the polar capsules and ae expelled thrsush pores atthe anteroe end ofthe spore (Ege 1B), “The posterie aspect of the M, ermal spre coveted bya mucous envepe that canbe Saal, isd by negative staining with dyes such as Inia tnk (Lom a Hofinana 1971), Thscharsctes hae Been descsited a arable by some author andes appearance may be dependent cn spore maturity (Michell 1959) There i pss gue vin thle by sinning elecuom miconcopy alg each side of che suture line where the two protective ‘ales joi Figure 1B) (Lom and Hfinann 1971, The myncaprean spore stage hasbeen the historical foal point fo the dg of wing dlsease. Detection ofthe charctersie ste and share of the spores by simple micrnconc pose, lures made this stage ofthe parasite moat secogni: she, patcularly since other stages oud the fish (ex nthe kin and nerves) were unknown or more dificult w obsive or their marhelogy ‘oul not be attbued a single species. In al ton, antigen and DNA-based denen apyach, thar resovedcertinaf these concems hal yt to be developed or appli eo M, ceva detection, Spore Staining Technigues The presence of pores in heavily infested ich can be demonstrated by simply ching diseted ke tal elements and observing. them by. hah imicrosapy. The decction of spores in gy Infected fh inci sh with mo clinical sign whirling dress consdeshy moe dil Vor tow macho have theron been develope to lar an canceneat spores ad to san he sng avatiety of dyes uch at methylene ble (Hue 3 tmlachite gre, or sve ite to ite shew \ewetion. Although the rial methods of Pe (1924 dd nor adbocatestsning, MacLean (971) foun cha the use of malachite geen aed inthe entiation of spore, especially foe detection of Spores ln cade preparations of macerated bones, ‘Wolf and Markiw (1979) compared a vary af stains t find one sutable fr tei conjunction with the pepsin-aygsin digestion methad sod Plankton centration Such stain would need imprernate the sutace of the spores und remain sable through che changes of pH and macho shear Fores that spores experience drng ches po ess Among the sai wd many were ne ving the spore parcaton proces dane spore hy altering their morphology. Silver nine as retained in the sores a left the spenes intact, However, the sve itete staining no aeded 3h to the punfcaton proce, fe wae noe ‘soca fr standard ane hat a tol tad tn smewsuring the eficincy of poe recovery fm the selected skeletal elements (Wolf and Math 1979), Some sans, though no species pectic have been used or the kenfition of the sctinospre an ses of M. eral. Crystal violet haw hese ‘sed sucesfilly for saningstinogorean stages % id in thet identieation inner ieee (Thomson and Nehring 200): More specilond staining methods, such ae fluorescein caceate (FDA) an propidium sce (PD), have been ad to india the vibe of gem cel inthe spores lam of the actinoporean stage of M. eels and oder mysosporeane (Yokoyama et al 1997) ee ‘Mechanical and Enzymatic Isolation of Spores aly deagnoste proces, such a dose ws by Plchn (1924), provided tid preparations of Crue ne, aking spore deifeaton dic ‘Xmure complex meth nelvng sequentially pl ‘eng and ltetng saps, followed by cen asin to remove Pony aati, es propo by Prater etal (1971) A clear atin or uantting (pore sao provi inthis prota Real the extensive nature of infestation of spores thou he al eleton and the shall isp tocel ws modified Gurr to ielae the hones of {he lower jew and gil (Td ea 1971). Yetvamore thong septation of spores ff ony ets Mosse using enti igetion of the bones (Le 1973: Markiw and Wolf 1974; Mankiw vent Wolf 1974). A comparison of spore pica fiom metho found that spe yields were simi Sain laces when the mechanical and noma {Craesment of bone were flowed by stepped ‘hn cengaton (Mork ard Welt 1974), Thee ngely gon the acurcy a bly 0 Inch sample the APSFHS Blue Book (Lore anu ‘Armand 1994) sped the ensmatc pepsin ard trypan digestion (r PTD), as developed by Markie Gn Wolf (19748). This method involves the ‘Rental dgestion ofthe Bony’ elements of the {elton afer removal of oft eisue. The dist of the bones istered an eoncentaed pio pun feign by dextrne grant contation, The ‘ule mater fesupended in sal olume Ferexaminaton by it mersopy forthe presence tf apnes This proce couple to the examina tionefsoined tise sesons om the sae cana thes we n the digest Recent madison £9 {he PTD include replacing te pepsin solution if the inal pH of LS rsesabowe 38, as above this pH the opin activity decays A second modification alee the concentration of psi or fen Fie fom a inal concent of 05% to 005%, Thereby preventing swan degradation of the {pore (Linda Chisum, Ura Divison of Wife ‘Resures, personal communication). Inu bore tory, acon diction the elacement tf dextrse with peal (Sigs Chemial Compa ny oc he gratin cengtion, “The plankton centstion method ws evoleped os an alemate, time-saving method or Sore bolation from fob diss, (O'Grodnick TTS). In this procedure, fa heads ae homoge |ANDAEE AND HEDRICK ed, and the homogenate i flere eroush une. This crde filtrate then passed toush a Fankoon cenfge and the reating pellet exam Tred microscopically, While the preparation for Ibscronapic cretion i ore eb his alte Tatve fas been accep by the AFSIFHS 2s an Prmoved method for M, cerebral decton (Loe SE Amand 1994). Obert of sores fom the aceon cesnifgatin method can be impeoved bythe abltion of eygsin treatment (0.25%) to Tony he material costed from the plankton enee (Mask and Wolf 1974 “The FID and plankion ces meade swell sted to batch sampling and ae eflectve penne for extating aad concentrating the spores own ah ues Both methods provide he ability to (Rauf the numbers of spores pe sample. Since Cfese mets do pide qc rel pe ‘Gal care abou be take to clean the equipment erween tach simples, to remove all spores thot fap adhere to the apparatus and conta be {june samples The PTD method softer peered ‘Recanttiveaols since dis method has been {Kesomurted wallow detection of rete proper ‘om pres han pe hyseal separatory eth Be hc torekave of the poe fom Pony nelson hes the creymatic dgesion (Mw and Wall {ta 1974), By compari, the plankton cen trig pore reparations are considerably moet tian ove fom the PTD method, making pee entiation more fica, Weakneses of Ba Proves inca signe investment in ae nor ard he abit dec onl the bade ‘pore sage ofthe arse. The length of tne om ‘Bhd expsute ode prs ntl hee specs ate Faly famed depends of nameous factors olin seater emperate (Ely 1979). Therefore, pet fom 3 to TT months pest exponsre to the pane See ay he reed before fu ate suitable or sae lig ung these methods. Lt these procedure “rene designe oe do they detest respon ‘Me tags inthe sh sto any of the develope ‘olor speogonic ages in the ligochsete hos Histological Assessment of Disease in amination of mounted stained tise section ese todenontate the presence fo erie in pepsi tryin digest ox plkton tre exacts, which ae asco with cl Tete in onlrto confirm » dag of whi dnewe: While histological sessment i mor Iya labor intensive nak i les cond ates “THE CAUSE OF SALMONID WHIRLING DISEASE » buch esting ti tl equi to prevent midiag- roses when Mysaolu pp tht are ot M. cee bras ate found in cranial tes other than cari lag. Tisuts are xed and sides pepe following the methods deserted by the AFSIFHS Ble Book (Core and) Amat 1994). Une seceny there Ihave been no uidelines or sessing the degree ox severe of nection ascites with the parse: A more cbjecive, quantitative measure of lesions developed by one ofthe authors (E. MacConnel ann Tom Pk (Wesgton State University), ‘srw available and has been widely sod to eval: tame the impact of M. cal on exposed ish (Baldwin eal. 2000). The hisoloieal assent tesa graded scale hase onthe number and si of catlage lesion, asia snflammation, and involement of sutounding tees All cag rows tue in each section should he examined Best rests are obtained when the godess on to head or whole body sections pr fh (Table 1 and Figure 4). This vcle provides 4 oumneical standard for more objective eomparicos between tnd within diagostic aborts. The grading scale has aso been dnetly beneficial to esearch that eques eration ofthe responses to ine” ‘on sang eld and laboratory tal Immunologic Methods Laced antibodies could theory provide a oe species pectic, objective dercton method. The soecesfl we of eologie methods, such a direct (DFAT) of indirect Maorescent antibody ets (IFAT), requis dat che iil antigens wed or Table 1. MacConnelalawin numeri sale for soning leslon severity. Geode Desrpon ancitody peetaction be well pifisd (Goldman 1968) Unfortunately, preparations of spores pt fed by mechanical andlor enzymatic means usl- Jy contain considerble amounts of fish tinue These spore preparations canbe athe lens of bony debris by tworphave separations ce gradient

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