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AquacultureMicrobiology

CentreforNanobiotechnology,VelloreInstituteofTechnology,Vellore,TamilNadu,India

NatarajanAmaresan

C.G.BhaktaInstituteofBiotechnology,UkaTarsadiaUniversity,Surat,Gujarat,India

ISSN 1949-2448

Springer Protocols Handbooks

ISSN 1949-2456 (electronic)

ISBN 978-1-0716-3031-0 ISBN 978-1-0716-3032-7 (eBook) https://doi org/10 1007/978-1-0716-3032-7

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Preface

Thismanualconsistsofseveralchaptersthatdealwiththetechniquesinvolvedinthestudy ofaquaticpathogensthatcauseinfections,especiallyinfish.Itcoversawiderangeofbasic andadvancedtechniquesassociatedwithresearchontheisolationandidentificationof bacterial,viral,andfungalpathogens,andprobioticbacteria.Inaddition,itaddressesthe treatmentofpathogensusingseaweedextracts,medicinalplantextracts,andactinomycetes. Theknowledgeandinformationsharedinthismanualprovidesinformationonthe variousprotocolstobefollowedwhileperformingtheexperiments.Theeditorsare extremelygratefultoeachauthororteamofauthorswhofoundtimetowriteacomprehensivechapterbasedontheirexpertiseinvariousprotocols.Theseprotocolscoveredinthis manualarewidelyfollowedbyresearchersand,therefore,willbeextremelyuseful.Postgraduatestudents,researchscholars,postdoctoralfellows,andteachersbelongingtodifferentdisciplinesinmicrobiology,biotechnology,andmarinesciencearethetargetedreaders ofthismanual.Readingthismanualwillkindlefurtherdiscussionsamongresearchers workinginaquaculture,biotechnology,microbiology,andotherrelatedsubjects,thus wideningitsbroaderscope.Researcherswillgainmoreknowledgeoftheinformationshared hereonvariousprotocols.

Vellore,TamilNadu,India

Surat,Gujarat,India October2022 NatarajanAmaresan

JohnThomas

MirunaliniGanesan,RaviMani,andSakthinarenderanSai 2 IsolationandIdentificationofEdwardsiellosis-CausingMicroorganism

SakthinarenderanSai,RaviMani,andMirunaliniGanesan

MethodsforCharacterizing Flavobacterium inFish

YaminiGopi,SakthinarenderanSai,MirunaliniGanesan, andRaviMani 4 IsolationandIdentificationof Citrobacter

SakthinarenderanSai,RaviMani,andMirunaliniGanesan

5 IsolationandIdentificationof InfectiousSalmonAnemiaVirus fromShrimp

HaimantiMondal,JohnThomas,NatrajanChandrasekaran, andAmitavaMukherjee

6 IsolationandIdentificationof Betanodavirus fromShrimp

HaimantiMondal,JohnThomas,NatrajanChandrasekaran, AmitavaMukherjee,andNatarajanAmaresan

7 IsolationandIdentificationof HemorrhagicSepticemiaVirus fromShrimp

HaimantiMondal,JohnThomas,NatrajanChandrasekaran, AmitavaMukherjee,andNatarajanAmaresan

8 IsolationandIdentificationofPathogensfromFish: TilapiaLakeVirus(TiLV)

S.R.Saranya,VernitaPriya,A.T.Manishkumar, andR.Sudhakaran

9 IsolationandIdentificationofPathogensfromShrimp:IHHNV

V.M.Amrutha,R.Bharath,K.Karthikeyan,R.Vidya, andR.Sudhakaran

IsolationandIdentificationof Ichthyophonushoferi fromFishes

HaimantiMondal,JohnThomas,NatrajanChandrasekaran, andAmitavaMukherjee

11 IsolationandIdentificationof Branchiomycesdemigrans fromFishes 73 HaimantiMondal,JohnThomas,NatrajanChandrasekaran, andAmitavaMukherjee

12 IsolationandIdentificationofMicrosporidianParasites, Enterocytozoon hepatopenaei InfectionofPenaeidShrimp

K.Karthikeyan,V.M.Amrutha,S.R.Saranya, andR.Sudhakaran

PART IIISOLATIONAND IDENTIFICATIONOF PROBIOTIC BACTERIA IN AQUACULTURE

13 MethodsforIsolatingandIdentifyingProbioticBacteriafromFishes

MahalakshmiS.Patil,RaghuRamAchar, andAnnCatherineArcher

14 IsolationofProbioticBacteriafromGutoftheAquaticAnimals

S.VidhyaHinduandJohnThomas

15 CharacterizationofProbioticPropertiesofIsolatedBacteria

MahalakshmiS.Patil,RaghuRamAchar, andAnnCatherineArcher

16 AntibacterialActivityofProbioticBacteriafromAquaculture

MahalakshmiS.Patil,AnaghaSudhamaJahgirdar, AnnCatherineArcher,andRaghuRamAchar

PART IIIMETHODSFOR TREATING INFECTIONSIN FISHESAND SHRIMPS

17 PreparationofMarineAlgal(Seaweed)ExtractsandQuantification ofPhytocompounds

S.Thanigaivel,JohnThomas,NatrajanChandrasekaran, andAmitavaMukherjee

18 TreatingBacterialInfectionsinFishesandShrimps UsingSeaweedExtracts

S.Thanigaivel,JohnThomas,NatrajanChandrasekaran, andAmitavaMukherjee

19 PreparationandTreatmentofSeaweedEncapsulatedPelletFeedinFisheries Aquaculture

S.Thanigaivel,JohnThomas,NatrajanChandrasekaran, andAmitavaMukherjee

20 TreatmentUsingSeaweedsinFishesandShrimpbyInVivoMethod

R.Bharath,K.Karthikeyan,R.Vidya,andR.Sudhakaran PART IVTREATMENT USING MEDICINAL PLANTS

21 TreatmentUsingMedicinalPlantsinFishandShrimp.

VernitaPriya,A.T.Manishkumar,K.Karthikeyan, andR.Sudhakaran

22 PreparationofFeedandCharacterizationofFeedSupplemented withPhytocompounds

N. Chandra Mohana, A. M. Nethravathi, Raghu Ram Achar, K. M. Anil Kumar, and Jalahalli M. Siddesha

23 IsolationandIdentificationof Actinomycetes .

HaimantiMondal,JohnThomas,andNatarajanAmaresan

24 AssayofHemolyticActivity

HaimantiMondal,JohnThomas,andNatarajanAmaresan

25 CytotoxicityAssay

HaimantiMondal,JohnThomas,andNatarajanAmaresan

26 AntibacterialActivityandExtractionofBioactiveCompound fromActinomycetes

HaimantiMondal,JohnThomas,andNatarajanAmaresan

27 IsolationandIdentificationofHarpacticoidCopepod

M.F.YasmeenandA.Saboor

Contributors

RAGHU RAM ACHAR • DivisionofBiochemistry,SchoolofLifeSciences,JSSAcademyofHigher Education&Research,Mysuru,Karnataka,India

NATARAJAN AMARESAN • C.G.BhaktaInstituteofBiotechnology,UkaTarsadiaUniversity, Bardoli,Surat,Gujarat,India

V.M.AMRUTHA • AquacultureBiotechnologyLaboratory,SchoolofBioSciencesand Technology,VelloreInstituteofTechnology,Vellore,TamilNadu,India

K.M.ANIL KUMAR • DepartmentofEnvironmentalScience,JSSAcademyofHigher Education&Research,Mysuru,India

ANN CATHERINE ARCHER • DepartmentofMicrobiology,JSSAcademyofHigherEducation &Research,Mysuru,Karnataka,India

R.BHARATH • VITSchoolofAgriculturalInnovationsandAdvancedLearning(VAIAL), VelloreInstituteofTechnology,Vellore,TamilNadu,India;AquacultureBiotechnology Laboratory,SchoolofBioSciencesandTechnology,VelloreInstituteofTechnology,Vellore, TamilNadu,India

N.CHANDRA MOHANA • DepartmentofMicrobiology,JSSAcademyofHigherEducation& Research,Mysuru,India

NATRAJAN CHANDRASEKARAN • CentreforNanobiotechnology,VelloreInstituteofTechnology, Vellore,TamilNadu,India

MIRUNALINI GANESAN • CentreforOceanResearch,Col.Dr.JeppiaarOceanResearchField Facility,SathyabamaInstituteofScienceandTechnology(DeemedtobeUniversity), Chennai,TamilNadu,India

YAMINI GOPI • CentreforOceanResearch,Col.Dr.JeppiaarOceanResearchFieldFacility, SathyabamaInstituteofScienceandTechnology(DeemedtobeUniversity),Chennai, TamilNadu,India

K.KARTHIKEYAN • AquacultureBiotechnologyLaboratory,SchoolofBioSciencesand Technology,VelloreInstituteofTechnology,Vellore,TamilNadu,India

RAVI MANI • CentreforOceanResearch,Col.Dr.JeppiaarOceanResearchFieldFacility, SathyabamaInstituteofScienceandTechnology(DeemedtobeUniversity),Chennai, TamilNadu,India

A.T.MANISHKUMAR • AquacultureBiotechnologyLaboratory,SchoolofBioSciencesand Technology,VelloreInstituteofTechnology,Vellore,TamilNadu,India

HAIMANTI MONDAL • CentreforNanobiotechnology,VelloreInstituteofTechnology,Vellore, TamilNadu,India

AMITAVA MUKHERJEE • CentreforNanobiotechnology,VelloreInstituteofTechnology,Vellore, TamilNadu,India

A.M.NETHRAVATHI • PostGraduateDepartmentofStudiesandResearchinBiotechnology, SahyadriScienceCollege(Autonomous),KuvempuUniversity,Shivamogga,India

MAHALAKSHMI S.PATIL • DepartmentofMicrobiology,JSSAcademyofHigherEducation& Research,Mysuru,Karnataka,India

VERNITA PRIYA • AquacultureBiotechnologyLaboratory,SchoolofBioSciencesand Technology,VelloreInstituteofTechnology,Vellore,TamilNadu,India

A.SABOOR • P.G.&ResearchDepartmentofZoology,TheNewCollege(Autonomous), Chennai,TamilNadu,India

SAKTHINARENDERAN SAI • CentreforOceanResearch,Col.Dr.JeppiaarOceanResearchField Facility,SathyabamaInstituteofScienceandTechnology(DeemedtobeUniversity), Chennai,TamilNadu,India

S.R.SARANYA • AquacultureBiotechnologyLaboratory,SchoolofBioSciencesandTechnology, VelloreInstituteofTechnology,Vellore,TamilNadu,India

JALAHALLI M.SIDDESHA • DivisionofBiochemistry,SchoolofLifeSciences,JSSAcademyof HigherEducation&Research,Mysuru,India

R.SUDHAKARAN • AquacultureBiotechnologyLaboratory,SchoolofBioSciencesand Technology,VelloreInstituteofTechnology,Vellore,TamilNadu,India

ANAGHA SUDHAMA JAHGIRDAR • DepartmentofStudiesinZoology,UniversityofMysore, Mysuru,Karnataka,India

S.THANIGAIVEL • DepartmentofBiotechnology,FacultyofScience&Humanities,SRM InstituteofScienceandTechnology,Kattankulathur,TamilNadu,India

JOHN THOMAS • CentreforNanobiotechnology,VelloreInstituteofTechnology,Vellore,Tamil Nadu,India

S.VIDHYA HINDU • JayaCollegeofArtsandScience,Thiruvallur,TamilNadu,India

R.VIDYA • VITSchoolofAgriculturalInnovationsandAdvancedLearning(VAIAL), VelloreInstituteofTechnology,Vellore,TamilNadu,India

M F YASMEEN • P G & Research Depar tment of Zoology, Justice Basheer Ahmed Sayeed College for Women (Autonomous), Chennai, Tamil Nadu, India

Isolation and Identification of Pathogens from Fishes

IsolationandIdentificationof Aeromonas sp.fromFishes

MirunaliniGanesan,RaviMani,andSakthinarenderanSai

Abstract

Aeromonasis animportantbacterialpathogenthatisfrequentlyisolatedfromdiseasedfishthroughoutthe world.Bacterialfishdiseases,especiallybacterialhemorrhagicsepticemiaandmotile Aeromonas septicemia infreshwaterfish,causedgreatlosses.Theresearchersobservedhistopathologicalchangesinintestines,the liver,andkidneysoftheaffectedfish.Manyresearchershaveisolatedandidentified Aeromonashydrophila fromcarp,catfishes,perches,andeelsandfoundthattheyarehighlypathogenic.Here,wedescribethe methodsusedtoisolateandidentify Aeromonas spp.infishbybiochemicaltestsandothermeans.Ourgoals aretoprovidethereaderwithguidelinesonhowtoidentify Aeromonas infectionandisolatethebacteria.

Keywords Bacterialfishdiseases, Aeromonas,Isolation,Identification,Characterization

1Introduction

Aeromonas spp.areopportunisticpathogensthatfrequentlycause infectionsasaresultofhostdamageorstress[1].Humanillnesses causedbythesebacteriaincludeendocarditis,gastroenteritis,peritonitis,andsepticemia[2].Theyarealsothemostcommoninfectionsinfarmedfish[3]. Aeromonas spp.,include A.caviae, A.veronii, A.salmonicida [4, 5], A.hydrophila [6], A.sobria,and A.bestiarum.Amongthese, A.hydrophila isthoughttobethemost dangeroustoaquaticanimals,producinghemorrhagicillnessin farmedfishonaregularbasis[7]. A.veronii,ontheotherhand, hasrecentlybeenfoundtoinfectfishwithmanyofthesame symptomsandhistologicalabnormalitiesas A.hydrophila.Virulencefactorsareusedtomeasurevirulenceandtoxicityofbacterial infections.Thepathogenicityof A.veronii ismostlyattributableto virulencefactorsandsynergisticinteractions.Virulencefactors includecytotoxicenterotoxins(act,alt,ast),aerolysin(aer),polar flagella(fla),serineprotease(ser),elastase(ahyB),lipase(lip), DNases(exu),glycerophospholipid:cholesterolacyltransferase (gcaT),andtypeIIIsecretionsystem(ascV).Tobetterunderstand

JohnThomasandNatarajanAmaresan(eds.), AquacultureMicrobiology,SpringerProtocolsHandbooks, https://doi.org/10.1007/978-1-0716-3032-7_1, © TheAuthor(s),underexclusivelicensetoSpringerScience+BusinessMedia,LLC,partofSpringerNature2023

thepathophysiologyandepidemiologyof A.veronii,itiscriticalto explorethevirulence-relatedcharacteristicsofclinicalisolates [8].However,dataandinformationregardingsickfisharescarce.

Toclarifywhatconstitutesafavorabledevelopmentplatform forthispathogen,itisessentialtoanalyzethegrowthpropertiesof thebacterialisolateundervariouscircumstances. Aeromonas isan aquatic-specificenvironmentalbacteriumthatcanbeirregularly transferredtopeople[9, 10].Foodsderivedfromanimals,fish, andvegetableshavelongbeenthoughttobekeycarriersof Aeromonas spp.infections.Gastroenteritisisthemostcommonhuman infectioncausedby Aeromonas spp.;however,otherseriousdiseases,suchassystemicinfections,arelesscommonandareusually associatedwithimmunocompromisedindividuals.Furthermore, thesemicrobesareknowntocausemajorinfectionsinfish. Aeromonas spp.cansurviveandmultiplyatlowtemperaturesinavariety offoodproductsstoredbetween 2and10 C,suchasbeef,roast beef,andpork,andcanevenproducevirulencefactorsattheselow temperatures.Althoughtheincidenceoffoodborneoutbreaks causedby Aeromonas spp.hasbeenrelativelylowinthepast,their presenceinthefoodchainshouldnotbeoverlooked.Little researchhasbeenconductedontheincidenceof Aeromonas spp. infrozenfish,despitethefactthattherehavebeenmanysurveyson theprevalenceof Aeromonas spp.infoodproducts.Furthermore, thesestudiesemployedstrainsthatwereincorrectlyidentifiedusing standardbiochemicalapproaches,whichresultedinunsatisfactory results.

2Materials

2.1Isolationof Aeromonas from InfectedFish

2.2Stainingand BiochemicalMethods

2.3Molecular Identification

• Infectedfish.

• Needles.

• Dissectionkits.

• Petriplates.

• Incubator.

• Refertostandardmicrobiologylaboratorymanual.

• PCRreactionfinechemicalsasperstandardprotocols.

• Primers-F-50 AGAGTTTGATCATGGCTCAG30 ,R-50 GGTTACCTTGTTACGACTT30

• Thermocycler.

• Geldoc/transilluminator.

3Methods

3.1FishSampling

3.2Isolation ofBacteria

• Collectfishwithclinicalsignssuchdarkeningskin,external hemorrhages,andinternalbleedingintheliver,kidneys,and skin,oracombinationofthesesymptoms.

• Sterilizetheequipment,suchasneedles,bysprayingthemwith 70%alcoholandthenexposethemtotheflamesdirectly.

• Sterilizethe Petridishesbyautoclavingthemfor10–15minat 121 Cand1atmpressure.

• Dissectliver,spleen,andkidneysfromthefishaftercleaning theirskinwith75%ethylalcohol.

• Homogenizethedissectedorganswith2mLofPBSandcentrifugeat500rpmfor5min.

• Discardthesupernatantandwashthepelletthreetimes withPBS.

• Finallysuspend thepelletwith1mLofsterilePBS.

• PrepareLuria–Bertani(LB) medium(10g/Ltryptone,5g/L yeastextract,10g/LNaCl,15g/Lagar:pH7)and autoclaveit.

• PourtheautoclavedmediaintoPetriplatesandallowtosolidify.

• Add0.1 μLofthesuspendedpelletandspreadplateit.

• Incubateitat37 Cfor24–48handobservethecolonies.

3.3Identificationof Aeromonas Bacteria

3.4Characterization of Aeromonas Bacteria

• Aeromonas isarod-shaped,nonspore-forming,oxidasepositive,glucose-fermenting,facultativelyanaerobic,and Gram-negativebacterium thatlives inwater.

• Aeromonas spp.coloniesontrypticasesoyagararesmooth, convex,and rounded,andthey aretan/buffincolor.

• PerformGramstainingasperthestandardprotocol.

• Aeromonas spp.coloniesontrypticase soyagar aresmooth, convex,androunded,andtheyaretan/buffincolor.Characterizationresultsof Aeromonas spp.areshowninTable 1.

• Weigh25goffishfleshasepticallyandhomogenizefor2minin stomacherbagscontaining225mLofalkalinepeptonewater.

• After18hofincubationat37 C,inoculateanaliquotofthe enrichmentinbloodagarcontaining30mg/Lampicillinand incubatefor24hat28 C.

• Select more or less than five presumptive Aeromonas colonies for fur ther identification, but consider only one isolate representing a species from each sample for incidence calculation.

Table1

Thecharacterizationresultforthefollowing Aeromonas spp.[12]

Resultsforthefollowingspecies

Characteristics

Physiologicalcharacteristics

Gramstain

Shape

Motility

Gasfromglucosea

Methylred

Voges–Proskauera

Lysinedecarboxylasea

Ornithinedecarboxylasea

Vibriostatic0/129

Productionof: Indolea

H2SfromL-cysteine

Hydrolysisof:

DNase

Gelatin

Betahemolysis

Alphahemolysis

Prolin

Acidfrom:

(continued)

Resultsforthefollowingspecies

Characteristics

Adonitol

D-Arabitol Nd

L-Arabitol Nd Nd

L-Arabinosea

D-Arabinose

Nd +

Nd Nd

D-Fucose Nd Nd

L-Fucose Nd Nd Galactose

Gluconate

Dulcitol Nd Lactose

Inositol D-Mannose

D-Sorbitola

Saccharose(sucrose)a

Utilizationof: Acetate

Argininedihydrolase

Protease

Hemagglutination

Antibioticsusceptibility

Ampicillin

Nd Nd

Characteristics

Resultsforthefollowingspecies

+showspositiveresult, showsnegativeresult,+/ undeterminable, Nd notdetermined, R resistantagainstthe antibiotic, S susceptibletoantibiotic, M mediumresistance, NA notavailable aMajortestsfor Aeromonas spp.

• Maintainthestockculturesofeachstrainforshortperiodsat roomtemperature onblood agarbaseslantsandforlonger storage,andeitherfreezeat 70 Cin20%(w/v)glycerol–Todd–Hewittbroth(Oxoid,Madrid,Spain)orlyophilizein 7.5%horseglucoseserumasacryoprotector.

3.4.1HemolyticActivity

• On an agar basis (Oxoid) supplemented with 5% sheep er ythrocytes, the strains are evaluated for hemolytic activity.

3.4.2ProteolyticActivity

• Streakeachsuspensionovertheplateswithfivemicrolitersand incubatefor24hat22 Cand37 C.

• Presenceofdistinctcolorlesszonearoundthecoloniesindicates hemolyticactivity[11].

• Determinecaseinhydrolysisbystreakingeachsuspensiononto Mueller–Hintonagar(Oxoid)containing10%(w/v)skimmed milk(Difco,Barcelona,Spain)andincubateat37 Cfor24h.

• Caseinaseactivityisshownbytheappearanceofatranslucent zonearoundthecolonies.Eachsuspensionisputin4-mm-diameterwellscutintoanagarosegelandincubatefor20hat 22 C.

• Precipitateunhydrolyzedgelatinbyimmersingtheplatesina saturatedammoniumsulfatesolutionat70 C.

• Gelatinaseactivityisshownbytheappearanceofatranslucent zonearoundthecolonies.

3.4.3LipolyticActivity

3.4.4NucleaseActivity

3.4.5CongoRedDye Uptake

• Streak5mLsuspensionontoresazurin–butteragarandincubateat37 Cfor24h.

• Presenceofpinkcoloniesindicateslipaseactivity.

• Extracellularnucleases(DNases)aredeterminedonDNaseagar plates(Difco)with0.005%methylgreen.

• Streak5 μL bacterialsuspensionontotheplatesandincubateat 37 Cfor24h.

• Apinkhaloaroundthecoloniesindicatesnucleaseactivity.

• TheabilitytotakeupCongoreddyeisdeterminedonagar platessupplementedwith50 μg/mL/mLofCongored dye.

• Streak5 μLbacterialsuspensionontotheplatesandincubateat 37 Cfor24h.

• Orangecoloniesareconsideredpositiveandexpressdifferent intensitiesinthedyeuptakeas+and++.

3.4.6Molecular Identification

Reidentifyallstrainsonthebasisoftherestrictionfragmentlength polymorphismpatterns(RFLP)obtainedfromthe16SrDNAfollowingthemethoddescribedbyBorrelletal.[9].

Add200 μMeach ofdATP,dTTP,dCTP,anddGTP.

Add5 μL1 PCRbuffer(50mMKCL,10mMTris–HCl, pH8.3).

Add 5 μL 2 5 mM MgCl2

3.4.7Antimicrobial Susceptibility

Acknowledgments

Add2 μLof5pMofeachprimer. Add3 μLof10ngofgenomicDNA.

PerformPCRusingthefollowingconditions:denaturationat 93 Cfor3min,followedby35cyclesat94 Cfor1min,56 Cfor 1min,and72 Cfor2min.Afterthefinalcycle,extensionat72 C isallowedfor10min.

Theresistanceofallstrainstodifferentantimicrobialagentsis determinedbythediskdiffusionmethodasdescribedbyInstitute CALS[12].

TheworkissupportedbytheMinistryofEarthSciencesgrant (MoES)(MoES/36/OOIS/53/2016),Govt.ofIndia,and SathyabamaInstituteofScienceandTechnology.

References

1. JeneyZ,JeneyG(2020)SinceJanuary2020 ElsevierhascreatedaCOVID-19resourcecentrewithfreeinformationinEnglishandMandarinonthenovelcoronavirusCOVID-19. TheCOVID-19 resourcecentre ishostedon ElsevierConnect,thecompany’ spublicnews andinformation

2. JandaJM,AbbottL(2006)Theenterobacteria.ASMPress,Washington,D.C.

3. HossainS,DahanayakePS,DeSilvaBCJetal (2019)MultidrugresistantAeromonasspp. isolatedfromzebrafish(Daniorerio):antibiogram,antimicrobialresistancegenesandclass 1integrongenecassettes.LettApplMicrobiol 68:370–377. https://doi.org/10.1111/lam. 13138

4. ZhuM,WangXR,LiJetal(2016)IdentificationandvirulencepropertiesofAeromonas veroniibv.sobriaisolatescausinganulcerative syndromeofloachMisgurnusanguillicaudatus. JFishDis39:777–781. https://doi.org/10. 1111/jfd.12413

5. LongM,NielsenTK,LeisnerJJetal(2016) Aeromonassalmonicidasubsp.salmonicida strainsisolatedfromChinesefreshwaterfish containanovelgenomicislandandpossible regional-specificmobilegeneticelementsprofiles.FEMSMicrobiolLett363:1–8. https:// doi.org/10.1093/femsle/fnw190

6. ChandrarathnaHPSU,NikapitiyaC,DananjayaSHSetal(2018)Outcomeof co-infectionwithopportunisticandmultidrug resistantAeromonashydrophilaandA.veronii

inzebrafish:identification,characterization, pathogenicityandimmuneresponses.Fish ShellfishImmunol80:573–581. https://doi. org/10.1016/j.fsi.2018.06.049

7. Marinho-NetoFA,ClaudianoGS,YunisAguinagaJetal(2019)Morphological,microbiologicalandultrastructuralaspectsofsepsis byAeromonashydrophilainPiaractusmesopotamicus.PLoSOne14:1–20. https://doi.org/ 10.1371/journal.pone.0222626

8. SenK,RodgersM(2004)Distributionofsix virulencefactorsinAeromonasspeciesisolated fromUSdrinkingwaterutilities:aPCRidentification.JApplMicrobiol97:1077–1086. https://doi.org/10.1111/j.1365-2672.2004. 02398.x

9. BorrellN,AcinasSG,FiguerasMJ,Martı´nezMurciaAJ(1997)IdentificationofAeromonas clinicalisolatesbyrestrictionfragmentlength polymorphismofPCR-amplified16SrRNA genes.JClinMicrobiol35:1671–1674. https://doi.org/10.1128/jcm.35.7. 1671-1674.1997

10. JandaJM,AbbottSL(2010)ThegenusAeromonas:taxonomy,pathogenicity,andinfection.ClinMicrobiolRev23:35–73. https:// doi.org/10.1128/CMR.00039-09

11. GerhardtP,MurrayRGE,CastilowRN,Nester EW,WoodWA,KriegNR,PhillipsG(1981) Manualofmethodsforgeneralbacteriology. AmericanSocietyforMicrobiology, Washington,DC

12. Institute CALS (2008) Pl Pl Pl Quality 1–6

IsolationandIdentificationofEdwardsiellosis-Causing Microorganism

SakthinarenderanSai,RaviMani,andMirunaliniGanesan

Abstract

Isolationandidentificationofpathogenicbacteriaarecrucialforaquatichealthmanagement.Identifying thecorrectpathogenicbacteriaatthespeciesleveliscrucialinpreparingforprecautionaryanddisease managementpractices.Edwardsiellosisisanepidemiccausedby Edwardsiella sp.Thiscausesgreateconomiclossesforfishcultureinmarineandfreshwaterhabitats.Thischapterdiscussesvariousprotocolsfor theirisolationandidentification.

Keywords Edwardsiella,API20E,Immunoassayblot,Edwardsiellosispolymeraseamplification, PCRassay

1Introduction

Edwardsiellosisisanimportantbacterialoutbreakinmosttropical aquaculturesystems.ThecausativeorganismisGram-negativerod bacteriabelongingtothefamily Enterobacteriaceae andgenus Edwardsiella [1].Itcomprisesfivespecies: E.tarda [2], E.ictaluri [3], E.hoshinae [4],E.piscicida [5],and E.anguillarum [6].Thediseaseoutbreakshavebeenreportedfor 20speciesoffreshwaterandmarinefishworldwide[7, 8].The isolationandidentificationofpathogenicfishspeciesareimportant forthecontrolandpreventionofmajoreconomiclossesinaquaculture.Thischapterdescribestheprotocolforisolatingandidentifyingdifferentidentificationtechniquesusedfor Edwardsiella spp.fromtheinfectedfishspecies.

JohnThomasandNatarajanAmaresan(eds.), AquacultureMicrobiology,SpringerProtocolsHandbooks, https://doi.org/10.1007/978-1-0716-3032-7_2, © TheAuthor(s),underexclusivelicensetoSpringerScience+BusinessMedia,LLC,partofSpringerNature2023

2Materials

2.1Isolationof Edwardsiella from InfectedFish

2.2Stainingand BiochemicalMethods

2.3Molecular Identification

• Infectedfish.

• Scalpelblades.

• Dissectionkits.

• Petriplates.

• Incubator.

• Refertostandardmicrobiologylaboratorymanual.

• PCRreactionfinechemicalsasperstandardprotocols.

• Primers:

1. EtfD-F5′GGTAACCTGATTTGGCGTTC3′ . EttR5′GGATCACCTGGATCTTATCC3′ [9].

2. 16SrRNA-F5′AGAGTTTGATCCTGGCTCAG3′ 16SrRNA-R5′ GGTTACCTTGTTACGACTT3′ [10].

3. EvpP- F 5′ GTGATCAAAGAAAACTGGAGCTCTCTCGACTT3′ REypP-R5′GACCGTCAGGTTTGGAATATAGAA CTGTGT3′ [11].

• Thermocycler.

• Geldoc/transilluminator.

3Methods

3.1Isolationofthe Edwardsiella spp.

• Preparebacterialisolatesfromorganssuchastheliver,intestine, andkidneysfromtheinfestedfish.Usesterilescalpelbladesand takeoutsamplesaseptically.

• Afterdissection,homogenizethetissuesamplesinphysiological saline(0.5%NaClsolution).

• Takehomogenatewithsterileloopandstreakontrypticsoyagar with5%sheepbloodorxyloselysinedeoxycholateagarplateand thenincubateitat37 °Cfor24h.

• Roundorcircularorgrayorgrayish-whitepigmentationof coloniesafter24-or48-hincubationisthecharacteristicfeature of Edwardsiella spp.

• Examinationofthebacterialisolatesbyphenotypic,biochemical,andmoleculartesttoidentifytillspecieslevel.

3.1.1Isolationof E. tarda,E.piscicida, and E. hoshinae Species

IsolationandIdentificationofEdwardsiellosis-CausingMicroorganism13

• Observedforsmallpunctuategrayish-whitecoloniesappearing onxyloselysinedeoxycholateagarafter24hofincubationat 37 °C.

• SubcultureitonMacConkeyagarplatesandincubateat37 °C for24h.

• Subculturealllactosenon-fermentedcoloniesontrypticsoy agarcontaining0.5%NaClandincubateitat37 °Cfor24h.

• Performallidentificationtestsusingovernightcultures intryptic soyagar[12].

3.1.2Isolationof Edwardsiella spp.

3.2Preliminary Identificationof Isolates

3.2.1GramStaining

• Dothewetobservationofinfectedfish(E.ictaluri issuspected).

• Streakthehomogenateoftissuesamplesintrypticasesoyagar with5%sheepblood(SBA)orbrain–heartinfusion(BHI)agar platesandincubateat26°Cfor48h.

• Subculturetheisolateagaininthesamemediumusing48-h incubatedcultures.

• Performallidentificationtestsusingovernightculturesintryptic soyagar.

• PerformGramstainingandspecificbiochemicaltestforbasic identification.

• Ascetically,prepareauniformsmearofbacterialisolateinaclean glassslideusingadropofsterilewaterorsalinesolution.

• Allowittoair-dryandmildlyfixitwithheat.

• Thenaddfewdropsofcrystalvioletsolutionandleftundisturbedfor1minandwashusingdistilledwater.

• FloodthesmearwithGram’siodineforaminute.

• Thenwashgentlywithdistilledwaterortapwateruntilthe violetcolordisappears.

• Decolorizeusing95%ethylalcoholuntilitrunsclear andimmediately rinsewithwater(5–10s).

• Afterthataddsafranin(counterstain)for45sandagainrinse withdistilledwater.Air-dry,blot-dry,andobserveunderoptical microscope.

3.2.2Biochemical Tests[12]

CatalaseTest

• Detectcatalaseenzymeusinghydrogenperoxideassubstrate whichresultsinwaterandoxygen.

• Performthistest usingovernight-growncultureinTSAplates.

• Edwardsiella spp. shows catalase positive showing bubble formation in tubes or glass slide.

14SakthinarenderanSaietal.

OxidaseTest

TripleSugarIonTest

IndoleProductionTest

• Dothistesttodetectthepresenceofcytochromeoxidase enzymeinbacterialcell.

• Purplecolorformationconfirmsoxidasepositivewithin10son reactionwith1%aqueoussolutionoftetramethylphenylenediaminedihydrochloride.

• Edwardsiella spp.showsnegativeforoxidasetest.

• Thistestindicatestheabilityofbacteriatofermentlactose, sucrose,andglucosebyproducingH2Sgasproduction.

• Edwardsiella spp.showspositiveforthistest.

• AddKovac’sreagenttoSIM(sulfur,indole,andmotility)media.

• Incubatefor24handobserveforthedeepredcoloration.

• Edwardsiella spp.showspositiveforthistest.

Simmons’CitrateTest

LysineDecarboxylaseTest

• GrowthebacterialisolateinSimmons’citrateslantbystabbing inoculumontheslantsurfaceandincubateat37°Cforaweek.

• Thenobserveforcolorchange. Edwardsiella spp.showsnegativeresultagainstthistestindicatingthatitcannotusecitrateas theonlycarbonsource.

• Inoculatethebacterialisolateinlysinebrothandincubateat37°C for4days.

• Observeafter96hof incubation. Edwardsiella spp.showspositive forthistest.

• Thepurplecolorindicatesthedecarboxylationabilityofisolate andmaintainthealkalinepHofthemedium.

3.2.3RapidIdentification

UsingCommercial

API20EKit

• API20Eisaplasticstripcontainingasetof21biochemicaltests forminganidentificationsystemfor Enterobacteriaceae and othernon-fastidiousGram-negativerods.

• Inoculatethebacterialisolateinmicrotubulesubstrateandincubateforidentifyingit.

• Givenumericalvaluesforthecolorchangesofthesubstrate reaction(i.e.,colorchanges),aspermanufacturerguidelines.

• In total, seven-digit number is obtained for identifying species [13]

3.2.4Molecular

IdentificationofIsolates

DNAIsolationof Edwardsiella sp.

PathogenStrainfrom Fishes

IsolationandIdentificationofEdwardsiellosis-CausingMicroorganism15

• IsolatetheDNAfrombacterialstraineithermanuallyorusing kitmethod.

DNAExtractionDirectly fromSpleen

• Amodifiedmanualmethodusingdirectboiledcellsmethod describedbyRamSavan[14].

• Culture1mLofpathogenicisolatesinthebrothandcentrifuge at5000 × g for3minat25 °Ctoobtainbacterialcellpellet.

• Boilthepelletsat95 °Cfor5minbyresuspendingitin250 μL ofTEbuffer(10mmoL/LTris–HCl,1mmol/LEthylenediaminetetraaceticacid(EDTA),pH8.0).

• Again,centrifugeatsamecondition,andthencollectsupernatantanduseditastemplateDNA.

• Dissectoutasmallpartofspleen(20mg)usingsterilescalpel bladeandasepticallytransferto1.5mLmicrocentrifugetube.

• ExtracttheDNAusingmanualmethodorcommercialkit.

• UseDNeasyBloodandTissuekitfromQiagenandfollowthe manufacturerprotocolforGram-negativebacteria.

• QuantifytheDNAextractusingNanoDropspectrophotometer.

IdentificationUsing ConventionalPCRAssay

• PreparePCRmixturesbyadding1 μLof16SrRNA/EvpP/etfD (forward;10 μM),1 μL EvpP/etfD(reverse; 10 μM)and20 μL steriledistilledwater.

• SetthePCRcycleto -95 °Cfor5min,andthen30cyclesof 30sat95 °C,30sat55 °C,and10sat72 °Candafinal extensionat72 °Cfor2min.

• Determinetheamplifiedproductsby1%(w/v)agarosegel electrophoresis.

• PreparetheagarosegelinTAE(Tris–acetate–EDTA)buffer1× , 0.04mMTris-HCL,1mMethylenediaminetetraaceticacid, pH8.0),and0.06 μg/mLofethidiumbromidefor visualization.

• Observedbandsattherangefrom400to445basepairs.

• Forspecies-levelidentification,purifythePCRproductsorelute usingQiaquickPCRcleanupkit(Qiagen)followingthemanufacturerprotocolandsequenceitthroughIlluminaNextgen sequencer.

• Compare the sequences using BLASTN program from the National Center for Biotechnology Information [15].

16SakthinarenderanSaietal.

IdentificationUsing RecombinasePolymerase Amplification(RPA)Assay

RPAisarapid,specific,andsensitiveassayusedfornucleicacid amplificationbyrecombinase,single-chainbindingproteinand DNApolymeraseunderisothermalcondition.

• Use1ng/μLconcentrationofDNAtemplateandaddtoRPA reactionmixture.

• RPAreactionmixtureconsistsof0.42 μMRPAprimers,1× rehydrationbuffer,andDNase-freewater (commercialkit: Cat. no.T00001,JiangsuQitiangeneBiotechnologyCo.,China). Tothismixture,addtheDNAtemplate.

• Leavethereactionundisturbedfor15minat39 °Canddeterminetheamplifiedproductsconfirmedbyagarosegel electrophoresismethod.

• Forspecies-levelidentification,purifythePCRproductsorelute usingQiaquickPCRcleanupkit(Qiagen)followingthemanufacturerprotocolandsequenceusingIlluminaNextgen sequencer.

• RunthesequenceusingBLASTNprogramfromtheNational CenterforBiotechnologyInformation[11].

3.2.5Identificationof Edwardsiella spp.Isolates byELISA

ExtractionofWholeCell Protein(WCP)

Thewholecellproteinprofilingallowsonetodetectthestrainsvery specificallyusingImmunoassayandELISA.

• Inoculateovernightculturesof Edwardsiella spp.intryptone soybroth(TSB)andincubatefor24hat37 °C.

• Fixthebacterialcultureusing2%formalinandkeptat4 °Cfor 24h.Andthencentrifugeat 2800rpmfor45minat4 °C.

• Discardthesupernatantandresuspendthepelletinphosphate buffersaline(pH7.2).Again,centrifugeat5000rpmforabout 15min.

• Afterpelletingdown,withoutdisturbingthem,keepitforsonicationat45Hzfor10min.

• Aftersonication,againcentrifugeat10,000rpmfor15min.

• Thesupernatantiscollectedandstoredat -20 °Cuntilfurther analysis.ThissupernatantisWCPantigen.

ProductionofPolyclonal AntibodiesinRabbit

• InjecttheWCPantigentoawhiterabbitonthehindlegto producepolyclonalantibodies.

• Inject150 μgof WCPantigenalongwiththeemulsionof Freund’scompleteadjuvant(FCA),intramuscularly.

• Give booster dose to the rabbit on 14th and 28th days of immunization.

SDS–PAGE(Sodium DodecylSulfate–PolyacrylamideGel Electrophoresis)Analysis

IsolationandIdentificationofEdwardsiellosis-CausingMicroorganism17

• Collectthebloodsamplesfromrabbiton42nddaypostimmunizationandallowtoclotatroomtemperaturefor2–3h.

• Centrifugeclottedbloodsamplesat5000rpmfor10minto obtainbloodserumandstoreitat -20 °C.

• MixtheWCPantigenand2× SDSgelloadingbufferin1:2ratio andheatat100 °Cfor5min.

• Dotheelectrophoresisofproteinsampleusing12%separating geland4%stackinggel.

• Allowthegeltorunat120Vuntilbromophenolbluedye reachesthebottomofthegel.

• StainthegelwithCoomassiebrilliantblueR-250followedby destaining.

WesternBlottingofWCP Antigen

AfterSDS–PAGE,checkthereactivityoftherabbitantiserumto theWCPantigenusingWesternblotimmunoassay.

• Dotheelectrotransferofrunninggelontoanitrocellulose membrane(0.45 μm).

• Washthemembraneusingdistilledwater,andblockwith5% skimmedmilkpowdersolutioninphosphatebuffer(PBS)containing0.05%Tween20andincubatewithprimaryantibodyat 37 °Cfor1h.

• Again,washitusingPBS,andthenincubateusingsecondary antibody(HRPconjugatedgoatanti-rabbitIgG)at37 °Cfor 1h.

• Finallywashusingdeionizedwater,anddeterminethebound antibodiesbyreactingitwith3,3′,5,5′-tetramethylbenzidine membraneperoxidasesubstrate.

• Stopthecolorreactionbyrinsingtomembranewithdeionized water.

Dot-ELISAforSpecies Identification

• Coatthenitrocellulosepaperstripwith3 μLofWCPantigenof Edwardsiella spp.anddryitforhoursatroomtemperature.

• ThenblockthestripinPBScontaining5%skimmedmilkpowderat37 °Cfor30min.

• Incubateforanhourat37 °C,withprimaryantibody(1:400 dilutions).

• Again,washthemembranestripforafewtimesandincubate withsecondaryantibodywithsameconditionfor30min.

• Fur ther incubate the nitrocellulose paper with anti-bovine complex reagent for 30 min

• Finally,incubatewith3-amino-9-ethylcarbazole(AEC)reagent for10minindark.

• Washthenitrocellulosemembranestripwithdistilledwater, air-dryitatroomtemperature,anddetecttheblots[16].

Acknowledgments

TheworkissupportedbytheMinistryofEarthSciences(MoES) MoES/36/OOIS/53/2016),Govt.ofIndia,andSathyabama InstituteofScienceandTechnology.

References

1. GriffinMJ,QuiniouSM,CodyT,TabuchiM, WareCetal(2013)Comparativeanalysisof Edwardsiella isolatesfromfishintheeastern UnitedStatesidentifiestwodistinctgenetic taxaamongstorganismsphenotypicallyclassifiedasE.tarda.Vet Microbiol165:358–372

2. Ewing WH,McWhorterAC,LAEscobarMR (1965) Edwardsiella:anewgenusofEnterobacteriaceaebasedonanewspecies,E.tarda. IntBullBacteriolNomenclTaxon15:33–38

3. HawkeJP(1979)Abacteriumassociatedwith diseaseofpondculturedchannelcatfish.JFish ResBoardCanada36:1508–1512

4. GrimontPAD,GrimontF,RichardCSR (1980) Edwardsiellahoshinae,anewspeciesof Enterobacteriaceae.CurrMicrobiol4:347–351

5. AbaynehT,ColquhounDJSH(2013) Edwardsiellapiscicidasp.nov.anovelspecies pathogenictofish.JApplMicrobiol114: 644–654

6. ShaoS,LaiQ,LiuQ,WuH,XiaoJetal(2015) Phylogenomicscharacterizationofahighlyvirulent Edwardsiella strainET080813TencodingtwodistinctT3SSandthreeT6SSgene clusters:proposeanovelspeciesasEdwardsiellaanguillarumSp.SystApplMicrobiol38: 36–47

7. KathariosP,KokkariC,DouralaNSM(2015) Firstreportofedwardsiellosisincage-cultured sharpsnoutseabream,Diploduspuntazzo fromtheMediterranean:casereport.BMC VetRes11:155

8. YiG,KaiyuW,DefangC,FanlingFYH(2010) Isolationandcharacterizationof Edwardsiella ictaluri fromculturedyellowcatfish(Pelteobagrusfulvidraco).IsrJAquac62:105–115

9. SakaiT,IidaT,OsatomiKKK(2007)DetectionofType1fimbrialgenesinfishpathogenic andnon-pathogenic Edwardsiellatarda strains byPCR.FishPathol42:115–117

10. LanJ,ZhangX-H,WangY,ChenJ,HanY (2008)Isolationofanunusualstrainof Edwardsiellatarda fromturbotandestablish aPCRdetectiontechniquewiththegyrBgene. JApplMicrobiol105:644–651

11. JiangJ,FanY,ZhangS,WangQ,ZhangY, LiuQ,ShaoS(2022)Rapidon-the-spotdetectionof Edwardsiellapiscicida usingrecombinasepolymeraseamplificationwithlateralflow. AquacRep22:100945

12. KebedeBHT(2016)Isolationandidentificationof Edwardsiellatarda fromLakeZeway andLangano,SouthernOromia,Ethiopia.Fish AquacJ7:184

13. NatalijaTP,RozelindraC-R,Strunjak-Perovic I(2007)Commercialphenotypictests(API 20E)indiagnosisoffishbacteria:areview. Veterina ´ rnı´medicı´na52:49–53

14. SavanR,IgarashiA,MatsuokaS,SakaiM (2004)Sensitiveandrapiddetectionof edwardsiellosisinfishbyaloop-mediatedisothermalamplificationmethod.ApplEnviron Microbiol70:621–624

15. CastroN,ToranzoAE,Nu ´ n ˜ ezSetal(2010) Evaluationoffourpolymerasechainreaction primerpairsforthedetectionof Edwardsiella tarda inturbot.DisAquatOrg90:55–61. https://doi.org/10.3354/dao02203

16. Das BK, Sahu I, Kumari S et al (2014) Phenotyping and whole cell protein profiling of Edwardsiella tarda strains isolated from infected freshwater fishes Int J Cur r Microbiol App Sci 3:235–247

MethodsforCharacterizing Flavobacterium inFish

YaminiGopi,SakthinarenderanSai,MirunaliniGanesan,andRaviMani

Abstract

Bacterialgilldisease,rainbowtroutfrysyndrome,Columnarisdisease,andbacterialcold-waterdiseaseare causedbybacteriathatarepathogenictofish.Theybelongtothe Flavobacteriaceae family.Theyarespotted withyellowpigmentationinaquaticorganisms.Thesekindsofbacteriaareisolatedandidentifiedusing techniquessuchasGramstaining,motility,glidingmotility,biochemicaltests,andAPI20E.Itcanalsobe identifiedusingtechnicalmethodsbyplatecountandmicroscopy.MolecularcharacterizationsuchasDNA extraction,polymerasechainreaction(PCR),andphylogeneticanalysisisalsodonetoidentifythespecies.

Keywords Flavobacteriaceae,Yellowpigmentation, Gramstaining, Motility,Glidingmotility,Biochemicaltest,API20E,Microscope,Platecount,DNAextraction,PCR,Phylogeny

1Introduction

Flavobacterium spp.inhabitingfreshandmarineenvironmentsmay produceyellow-pigmentedcoloniesonculture.Itisveryimportant toknowtheclinicalinformationaboutbacterialcellstodifferentiatethemintopathogensorsaprophytes.Therefore,cellsthatproduceyellowpigmentation,usuallylongandthin,adheretothe surfaceorepitheliumofaquaticspeciesbelongingtothefamily Flavobacteriaceae [1].Thegenus Flavobacterium comprisesof morethan100species[2].Mostofthe Flavobacterium spp.are Gram-negative,long,slenderglidingrodsof4–10 μmand 0.3–0.5 μmwide,strictlyaerobic[3],andthecolonysizesare round,convex,rhizoid,andflat,whichgrowanoptimaltemperatureof20–30 Cformostofthespecies.Amongthem,manyare pathogenicandcausativeagentsofvariousdiseasesinfish. Flavobacteriumpsychrophilum isthecausativeagentofrainbowtroutfry syndromeandbacterialcold-waterdisease[4].Thistypeofdisease occursincoldwaterat16 C,withopenlesionsontheexternal surfaceofthefish. Flavobacteriumbranchiophilum and Flavobacter-

JohnThomasandNatarajanAmaresan(eds.), AquacultureMicrobiology,SpringerProtocolsHandbooks, https://doi.org/10.1007/978-1-0716-3032-7_3, © TheAuthor(s),underexclusivelicensetoSpringerScience+BusinessMedia,LLC,partofSpringerNature2023

iumsuccinicans arepathogensthatcausebacterialgilldiseasesin fish[5]. F.johnsoniae, F.hydatis, F.chilense, F.araucananum, F.oncorhynchi, F.plurextorum, F.tructae, F.piscis, F.collinsii, F.branchiarum, F.branchiicola,and F.spartansii areother Flavobacterium spp.associatedwithfishdiseases.Flavobacterialdiseases arealsopathogenictoamphibiansandhumans[6].Theisolation andidentificationof Flavobacterium spp.arebasedonmorphological,biochemical,andmolecularcharacterizations.

2Materials

2.1Isolationof Flavobacterium spp. fromFish

2.2Stainingand BiochemicalTest

2.3Molecular Identification

• Infectedfish.

• Sterileswab.

• Shiehmedium.

• Tobramycin.

• Cytophagaagar.

• Neomycin.

• PolymyxinB.

• Petriplates.

• Incubator.

• Generalstaining,motilitytest,andbiochemicalwerecarriedout asperstandardprotocolsAPI20EKit.

• Shiehbroth.

• Tobramycin.

• Incubator.

• Lyophilizer.

• Primers:

27F:50 -AGAGTTTGATCMTGGCTCAG-30 . 1387R:50 GGGCGGWGTGTACAAGGC-30 [4].

27F:50 -AGAGTTTGATCMTGGCTCAG-30 1492R:50 -TACGGYTACCTTGTTACGACTT-30 [7].

27F:50 -GAGTTTGATCMTGGCTCAG-30

• 518R:50 WTTACCGCGGCTGG-30 [7]. Thermocycler.

• Geldoc.

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