4. RESULTADOS Y DISCUSIÓN
4.1. Métodos de detección, recuperación e identificación de Aeromonas
4.1.1. The improved PCR of the fstA (ferric siderophore receptor) gene
differentiates the fish pathogen Aeromonas salmonicida from
other Aeromonas species.Beaz-Hidalgo R, Latif-Eugenín F, Figueras MJ.Vet. Microbiol. (2013) 166:659-563.
Resumen
Bacterias del género Aeromonas son microorganismos autóctonos del medio acuático y varias especies son capaces de producir septicemia y la enfermedad hemorrágica ulcerativa en peces salvajes y de piscifactoría causando una alta mortalidad. La especie más patógena en peces es Aeromonas salmonicida, siendo responsable de brotes epidémicos de furunculosis en salmónidos, así como en otros peces cultivados como el rodaballo, la dorada, el halibut y el pez dorado. Nuevas especies como Aeromonas aquariorum, Aeromonas tecta y Aeromonas piscicola también han sido aisladas recientemente a partir de peces enfermos. Además se ha demostrado que la identificación fenotípica y genética en base a la secuenciación del gen 16S ARNr no permite diferenciar entre las especies A. piscicola, A. bestiarum y A. salmonicida asociadas a la patología de peces. El objetivo del presente estudio es reevaluar dos protocolos de PCR previamente descritos, basados en la detección de los genes fstA y gyrB, para la detección específica del patógeno A. salmonicida ensayando todas las cepas tipo del género Aeromonas así como varias cepas de las especies A. piscicola y A. bestiarum. A diferencia de resultados previamente publicados, se demostró que la PCR del gen gyrB no es específica para la detección de A. salmonicida ya que presenta una reacción cruzada con otras especies. Sin embargo, corroborando resultados previos se demuestra que la PCR del gen fstA es específica para la detección de A. salmonicida y se propone un protocolo más optimizado para su detección.
Shortcommunication
The
improved
PCR
of
the
fstA
(ferric
siderophore
receptor)
gene
differentiates
the
fish
pathogen
Aeromonas
salmonicida
from
other
Aeromonas
species
RoxanaBeaz-Hidalgo,Fadua Latif-Eugenı´n, Marı´a Jose´ Figueras*
UnitatdeMicrobiologia,DepartamentdeCie`ncesMe´diquesBa`siques,FacultatdeMedicinaiCie`nciesdelaSalut,IISPV,UniversitatRovirai Virgili,Reus,Spain
1. Introduction
ThespeciesAeromonassalmonicidaisthecausalagentof furunculosis, a disease known to affect salmonids and otherfishspeciessuchasAtlanticcod(GadusmorhuaL.), halibut(Hyppoglossushyppoglossus(L.)),turbot(Scophtha- musmaximus(L.))orseabream(SparusaurataL.)among others (Bernoth et al., 1997; Austin and Austin, 2007; Goldschmidt-Clermont et al., 2009; Godoy et al., 2010; Noga, 2010; Figueras et al., 2011; Beaz-Hidalgo and Figueras,2012).DifferentiationofA.salmonicidafromits
closelyrelatedspeciesAeromonasbestiarumorAeromonas piscicola (also associated with fish) on the basis of phenotypicandgeneticmethodsisverydifficult(Martı´- nez-Murcia et al., 2005; Beaz-Hidalgo et al., 2010). For instancethesespeciesshare99.8–100%similarityamong their16SrRNAgenesequencesmakingthemnotusefulfor their identification, a problem solved by using house- keeping genes like the rpoD or gyrB that show more interspecificdifferences(Soleretal.,2004;Beaz-Hidalgo etal.,2009,2010;Figuerasetal.,2011;Beaz-Hidalgoand Figueras,2012).
SeveralmolecularPCRmethodshavebeendescribedfor
ARTICLE INFO Articlehistory:
Received22November2012 Receivedinrevisedform8May2013 Accepted19June2013 Keywords: Aeromonassalmonicida Fishpathogen Furunculosis PCR fstA gyrB ABSTRACT
Themembersofthegenus Aeromonas areautochthonous ofaquatic ecosystemsand severalspecieshavebeenassociatedtosepticaemia,ulcerativeandhaemorrhagicdiseases infish,causingsignificantmortalityinbothwildandfarmed,freshwaterandmarinefish species.ThespeciesAeromonassalmonicidaisgenerallyrecognizedasthemostimportant fishpathogenresponsibleforepidemicoutbreaksoffurunculosisinsalmonids,alsobeing abletoproduceinfectionsinotherculturedfishsuchasturbot,halibut,seabreamor goldfish.Newspecies,i.e.Aeromonasaquariorum,AeromonastectaandAeromonaspiscicola, haverecentlybeendiscoveredandisolatedfromdiseasedfish.ThespeciesA.piscicolaand Aeromonas bestiarum are practically impossible to differentiate phenotypically and genetically(whenusingthe16SrRNAgene)fromeachotherandfromA.salmonicida.Inthe presentstudy,twopreviouslydescribedPCRprotocols,basedonthefstAandgyrBgenes, forthespecificdetectionofA.salmonicidawerere-evaluatedwiththetypestrainsofall AeromonasspeciesandwithasetofA.piscicolaandA.bestiarumstrains.Contrarytowhat hadbeenpublishedpreviouslyitwasdemonstratedthatthegyrB-PCRisnotspecificforA. salmonicida because of cross-reactions with other Aeromonas species. However, in agreementwithpreviousresults,A.salmonicidawasdetectedonthebasisofthefstA-PCR, forwhichanimprovedprotocolwasproposed.
ß2013ElsevierB.V.Allrightsreserved.
Veterinary
Microbiology
independentPCRmethodsthattargetA.salmonicidausing thefstA(encodingtheferricsiderophorereceptor)andgyrB (encodingthe DNA gyrase subunitB) genes respectively (Beaz-Hidalgoetal.,2008)butnothingisknownaboutthe possiblecrossreactionwithotherAeromonasspecies,suchas
associatedwithfish,thatwerenottestedastheywerenot knownatthattime.Thereforetheaimofthepresentstudyis tore-evaluatethespecificityofthetwoPCRmethodsusing thetypestrainsofallAeromonasspeciesandstrainsofthe closelyrelatedspecies,A.bestiarumandA.piscicola.
Table1
ComparativeresultsofthePCRamplificationofthetargetedgyrBandfstAgenesobtainedwithstrainsofthecloselyrelatedspeciesA.salmonicida,A. bestiarumandA.piscicola.
Species Strain Hostspeciesa
PCRamplification
gyrB fstA A.salmonicidab CECT894T Salmon + +
A.salmonicidac CECT895T Trout + + A.salmonicidad CECT896T Masou + + A.salmonicidae 34MELT Pollutedriver + + A.salmonicidae
93MEL Pollutedriver + + A.salmonicidae
85MEL Pollutedriver + + A.salmonicidaf LMG20223T Commonroach + +
A.salmonicida R20 Trout + + A.salmonicida R21 Salmon + + A.salmonicida R23 Turbot + + A.salmonicida R25 Turbot + + A.salmonicida R29 Clam + + A.salmonicida R30 Clam + + A.salmonicida R64 Trout + + A.salmonicida R67 Turbot + + A.salmonicida R103 Salmon + +
A.salmonicida LMG18998 Woundexudate + + A.salmonicida CECT5209 Seawater + + A.salmonicida CECT5220 Seawater + + A.salmonicida CECT5230 Wellwater + + A.salmonicida CECT5231 Pipewater + + A.salmonicida CECT5227 Mussel + + A.salmonicida CECT5218 Cake + + A.salmonicida CECT5221 Cake + + A.salmonicida CECT5225 Mussel + + A.salmonicida LMG19037 Cake + + A.salmonicida CECT5249 Water + + A.bestiarum CECT4227T Unknownfishspecies +
A.bestiarum R3 Lamprey + A.bestiarum R5 Salmon + A.bestiarum R54 Lamprey + A.bestiarum R72 Trout + A.bestiarum R77 Lamprey + A.bestiarum R80 Salmon + A.bestiarum R92 Trout + A.bestiarum R201 Salmon + A.bestiarum R208 Salmon + A.bestiarum R209 Salmon + A.bestiarum R215 Salmon + A.bestiarum CECT5238 Wellwater + A.piscicola CECT7443T Salmon +
A.piscicola R4 Lamprey + A.piscicola R9 Trout + A.piscicola R94 Trout + A.piscicola R200 Salmon + A.piscicola R210 Salmon + A.piscicola R213 Trout + A.piscicola R214 Salmon + A.piscicola R219 Bogue + a
Scientificnamesforfishandshellfishspeciesare:Turbot,Scopthalmusmaximus(L.);trout,Oncorhynchusmykiss(Walbaum)andSalmotruttaL.;masou, Oncorhynchusmasou(Brevoort),salmon,SalmosalarL.;lamprey,PetromyzonmarinusL.,bogue,BoopsboopsL.;commonroach,RutilusrutilusRafinesque;
2. Materialsandmethods
2.1. Bacterialstrainsandgrowthconditions
Seventy-one strainswere used in the present study (Tables1and2),includingallthetypestrains(n=25)and 46referenceandnon-referencestrainsofA.salmonicida,A. bestiarum andA. piscicola, isolatedfrom varioussources (diseasedfish,seawater,water,bivalves,cakeandwound exudate),thathadbeenidentifiedpreviouslybymolecular methods (Beaz-Hidalgo et al., 2010). The isolates were grownfor24hontrypticasesoyagar(TSA,Pronadisa)and wereincubatedat258CfortheA.salmonicidastrainsandat 308CfortheotherAeromonasstrains.
2.2. DNAextractionandPCRamplifications
DNAwasextractedfrompure bacterialcultureswith theInsta-Genematrix(Bio-Rad),asrecommendedbythe manufacturer. Conditionsandprimersfortheamplifica- tionofthefstA(422bp)andgyrB(760bp)geneswereas previously described (Beaz-Hidalgo et al.,2008), except thatthecommercialReady-to-goPCRbeads(GEHealth- care) that include all the lyophilized preparation was substitutedforconventionalreagents.ThePCRamplifica- tions were carried out in T-gradient Thermocycler (Biometra)in50mlvolumecontaining5mlofDNA,7ml dNTP mix (10mM, Applied Biosystems), 1ml of each primerat15mM,5mlof10buffermix,3mlMgCland 0.5mlofTaqDNApolymerase(Invitrogen).PCRproducts wereelectrophoresedat80Vfor1.5hthrough2%agarose
3. Results
ThegyrBprimerswerefoundtoproduceanampliconof theexpectedsizeforallA.salmonicidastrains,forallthe strainsof A. bestiarum and A. piscicolaand for thetype strainsofAeromonasmedia,Aeromonassobria,Aeromonas allosaccharophila, Aeromonas molluscorum, Aeromonas popoffiiand Aeromonasfluvialis(Fig.1and Tables 1and 2).WhenthefstAgenewasinvestigated,thetypical422bp band was only observed for the A. salmonicida strains, although6A.piscicolaand9A.bestiarumstrainsdidshow somedifferent,unspecificbands(Fig.2)thatdisappeared whenthePCRconditionswereoptimizedbyreducingthe amountof MgClfrom 3mlto 1.5ml and the annealing temperaturewas64.78C.Underthesedifferentconditions, onlytheA.salmonicidastrains(includingallthesubspecies testedsalmonicida,masoucida,achromogenes,smithiaand pectinolytica) produced a specific and unique band of 422bp(Fig.3andTable1).
4. Discussion
Thepresentstudyhasre-evaluatedthePCRprotocols forthefstAandgyrBgenesusingallthesubspeciesofA. salmonicida,allthetypestrainsofthespeciesofthegenus Aeromonasandgeneticallyidentifiedstrainsoftheclosely relatedspeciesA.bestiarumandA.piscicolatocheckforany cross-reactions.Incontrast,intheoriginalstudyofBeaz- Hidalgoetal.(2008)thatproposedthesePCRmethods,the sensitivity and specificity was evaluated with 69 bio- chemicallyidentifiedA.salmonicidastrainsincludingonly
Species Strain Origin PCRamplification
gyrB fstA A.hydrophila CECT839T
Milk A.caviae CECT838T
Guineapig
A.media CECT4232T Aquaculturewater +
A.eucrenophila CECT4224T Freshwaterfish
A.sobria CECT4245T Fish + A.veronii CECT4257T Sputum A.jandaei CECT4257T Humanfaeces A.schubertii CECT4240T Cutaneousabscess A.trota CECT4255T Humanfaeces
A.allosaccharophila CECT4199T Eel +
A.encheleia CECT4342T Eel
A.popoffii CECT5176T Drinkingwater + A.simiae IBSS6874T Monkeyfaeces A.molluscorum CECT5864T Bivalvemolluscs + A.bivalvium CECT7113T Bivalvemolluscs + A.tecta CECT7082T Childfaeces
A.aquariorum CECT7289T Ornamentalfish
A.fluvialis CECT7401T Riverwater A.taiwanensis CECT7403T Woundinfection A.sanarellii CECT7402T Woundinfection A.diversa CECT4254T Woundinfection A.rivuli CECT7518T Riverwater
Fig.1.SpecificgyrB-PCRproductsforA.salmonicidaandforotherAeromonasspecies.Lanes:1,A.salmonicidaCECT894T;2,A.mediaCECT4232T;3,A.sobria CECT4245T ;4,A.trotaCECT4255T ;5,A.allosaccharophilaCECT4199T ;6,A.eucrenophilaCECT4224T ;7,A.bivalviumCECT7113T ;8,A.caviaeCECT838T ;9,A. molluscorumCECT5864T ;10,A.popoffiiCECT5176T ;11,A.fluvialisCECT7401T ;12,A.taiwanensisCECT7403T ;13,A.sanarelliiCECT7402T .MW,molecular weightladder(100–2072bp,Invitrogen).Numbersontherightindicatethepositionofthemolecularsizemarkerinbasepairs(bp).Numbersontheleft indicatethesizeoftheexpectedgyrBamplicon.
Fig.2.fstA-PCRproductsforA.salmonicidaandforotherAeromonasspecies.Theexpectedspecificamplicon(422bp)wasonlyobtainedfortheA.salmonicida strainswhileunspecificampliconsofothersizeswerecontainedforA.bestiarumandA.piscicolastrains.Lanes:1–2,A.salmonicidastrainsCECT894T
, 34MELT
;3,A.bestiarumCECT4227T
;4–8,A.bestiarumstrainsR72,R201,R3,R5,R208;9–17A.piscicolastrainsCECT7443T
,R200,R9,R210,R214,R213, R219,R94,R4.MW,molecularweightladder(100–2072bp,Invitrogen).Numbersontherightindicatethepositionofthemolecularsizemarkerinbase pairs(bp).NumbersontheleftindicatethesizeoftheexpectedfstAamplicon.
Fig.3.SpecificfstA-PCRproductsforA.salmonicidaandforotherAeromonasspecieswiththemodifiedcondition.WiththenewPCRconditionsonlythe strainsofA.salmonicidarespondedtotheamplificationshowingtheexpectedbandof422bp.Lanes:1–4,A.salmonicidastrainsCECT894T,CECT895T,CECT
896T
,34MELT
;5–7,A.bestiarumstrainsCECT4227T
,R72,R201;8–9;A.piscicolastrainsCECT7443T
,R200;10,A.salmonicidastrainR20;11–13,A.piscicola strainsR9,R210,R214.MW,molecularweightladder(100–2072bp,Invitrogen).Numbersontherightindicatethepositionofthemolecularsizemarkerin basepairs(bp).
species A. bestiarum or with other recently described species,i.e.A.piscicola,A.tectaandA.aquariorum,which had never been testedbefore usingthis PCRdiagnostic assay.FurthermorethefstA-PCRprotocolwasmodifiedto avoidunspecificPCRamplicons,andtoreducethecost(i.e. PCRreagentswereusedinsteadofusingthecommercial Ready-to-goPCRbeads).
TheresultsobtainedinthisstudyforthegyrBandfstA genesareimportantbecauseotherstudiesmayrelyinthe future on these genes for the specific detection of A. salmonicida (Beaz-Hidalgoet al.,2008). In fact,Kulkarni etal.(2009)targetedthegyrBregiondescribedpreviously (Beaz-Hidalgo et al., 2008) in order to develop a loop- mediatedisothermalamplification(LAMP)protocolforthe specific detectionof furunculosisinAtlantic cod (Gadus morhua).AlaterstudybyKulkarnietal.(2010)usedthe samegyrBregionforthedetectionofA.salmonicida ina multiplex-PCR(m-PCR)assaythatalsotargetedtwoother pathogens, Francisella piscicida and Vibrio anguillarum. However,theauthorsdidnottestanystrainsofthespecies A.bestiarum,A.piscicola,A.media,A.sobria,A.allosacchar- ophila,A.molluscorum,A.popoffiiorA.fluvialis,whichhave been shown to producethe same amplicon as the one expected for A. salmonicida. Among them, A. sobria, A. bestiarum and A. piscicolahavebeenisolated previously fromvariousdiseasedfishspecies,includingtrout,salmon, bogue(BoopsboopsL.),carp,lampreyandtilapia,witha prevalencerangingfrom11.7to25%(Kozin´skaetal.,2002; Kozin´ska,2007;Beaz-Hidalgoetal.,2010;Soriano-Vargas etal.,2010;LiandCai,2011;Kozin´skaandPe˛kala,2012). Few records exist for A. media isolated from trout and salmon(7%ofprevalence,Beaz-Hidalgoetal.,2010)orfor A.allosaccharophilaoriginallydiscoveredfromeelsinSpain (Martı´nez-Murciaetal., 1992)and detectedin carpand trout(1.5%ofprevalenceKozin´ska,2007).
It is important to consider that a quick, correct identification of A. salmonicida is importantin orderto treatthediseasewiththecorrectantimicrobials andfor theearlydetectionofcovertlycarrierfishforthecontrolof furunculosisandofepidemicoutbreaks.Thesepreventive strategies for the controlof furunculosis and otherfish diseasesareaconstantchallengeandneedtoberegularly reviewed in order to recognize the emergence of new, potentiallypathogenicspecies,liketherecentlydiscovered A.piscicola.Inconclusion,thepresentstudydemonstrates that the fstA PCR protocol is specific for detecting A. salmonicida, showing no cross-reactionswithits closely relatedspeciesA.bestiarumandA. piscicolaoranyother Aeromonasspecies.
Acknowledgements
The authors thank Catalina Nu´n˜ ez for her technical assistance.Thisworkwassupportedinpartbytheproject withreferenceAGL2011-30461-C02-02bytheMinisterio
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4.1.2. A culture independent method for the detection of Aeromonas sp. from water