http://doc.rero.ch View metadata,citationandsimilarpapersatcore.ac.uk hc hudb ie orfrt thiswork. which shouldbecitedtorefer Published in"Parasitology133:11–18,2006" H45 ae,Switzerland. Basel, CH-4051 H10 Fribourg CH-1700 e Universita der Tel: # Switzerland. + Basel, 4051 Unite REFARDT D. from example in an microsporidians three : their in quantify parasites and intracellular discriminate detect, to PCR Quantitative orsodn uhr olgshsIsiu der Institut Zoologisches author: via Universita Corresponding parasites * of discrimination life-cycle all allow of and DNA parasite stages detect they because analysis. routine for approach feasible an not Species is microscopy, that electron species. transmission morphology by ultrastructural on parasite dis- done is for or identification between sufficient of parasites not analysis microsporidian crimination is so of microscopy and identification light characters by distinct samples morphology few a offers Additionally, only stages life invisible. represent other and remain ongoing an because of fraction missed intensity easily low of light is infection spores, An using extracellular stage. transmission of detected the presence the typically by are microscopy they intracellular are parasites, they although example: detection, are prominent a their on quantification. and depends identification parasites of study The INTRODUCTION distinct few offer they and parasite the of form microsporidia the visible quantify to chemistry microscopically Green SYBR light on based intestinalis only assay Glugoides (qPCR) microscopy the PCR light quantitative a using are developed quantification We spores diagnostics. and characters. and discrimination because studies Their host-parasite difficult respect. for this is important in problematic are are parasites of Microsporidial quantification and discrimination detection, Reliable SUMMARY epooeuigtertoo aaiet otDAa h esr fifcinitniy eso htti esr is measure this that show unequal We of words: samples intensity. Key compare infection to of applied be measure in can the method variance as this residual Additionally, DNA qPCR. the host volume. of of to resolution improves parasite required, 90% greatly is of than resolution and ratio higher more robust more Where the magnitude qPCR. for of of using resolution order account propose the an impairs extraction we than considerably variance DNA more this is and and and data Sampling intensity animals infection whole microscopy. in light intensities than infection of sensitive quantification the allows assay The unicto,diagnostics. quantification, urn drs fbt uhr:Zooice Institut Zoologisches authors: both of address Current 11606.Emi:[email protected] E-mail: 41612670362. C-ae ehd vroeteedrawbacks these overcome methods PCR-based clgee Evolution et Ecologie ´ ¨ ae,Eouinbooi,Vslas ,CH- 1, Vesalgasse Evolutionsbiologie, Basel, t Glugoides ¨ ae,Eouinbooi,Vslas 1, Vesalgasse Evolutionsbiologie, Basel, t # , * Switzerland , and coprabayeri Octosporea , Octosporea .EBERT D. , De preetd Biologie de ´partement , + Ordospora 11707.Fax: 41612760375. and # ropr colligata Ordospora Microsporidia, , , Universite 1 eFribourg de ´ hti a aiyapidt h td fother of study in the adopted to easily applied (b) and easily infections (a) microparasitic is that magna 2003 Mouton (Cheesman evolution interactions and host-parasite ecology the of on studies in applied often nldn unicto fhmnpathogenic human of Hester (e.g. microsporidia biology, quantification molecular (Higuchi in use question including general in its Despite template 1993). DNA number copy the every initial infer of to at used reaction be on measured then data can kinetics and is techniques fluorescent DNA by cycle amplified the of Here, (qPCR). amount PCR quantitative can via infections obtained be on data Quantitative primers. specific gata in ftemcoprda parasites microsporidian the intestinalis infec- of quantify and tions discriminate detect, to protocol the of stages different infections in life-cycle. infections multiple hosts’ of of detection analysis or growth, measurement parasite is allow that of There methods novel limited. for need are dire interactions experiments analyse reason host-parasite to techniques one on the that theory: is this behind for lags research empirical Daphnia nterhs,tefehae freshwater the host, their in ee erpr h eeomn faqPCR a of development the report we Here, nterhs,tefehae crustacean freshwater the host, their in , a , u olwst rdc ipeprotocol simple a produce to was goal Our . b eltm uniaiePR neto intensity, infection PCR, quantitative real-time , asnadBry 03,qC snot is qPCR 2003), Barry, and Wasson ; tal et , , hmnd Muse du chemin coprabayeri Octosporea 04 Mucklow 2004; . e10 ´e tal et , 02 Menotti 2002; . and tal et ropr colli- Ordospora 04.Here, 2004). . ahi magna Daphnia tal et provided byRERODOCDigitalLibrary Glugoides Daphnia 2003; . tal et brought toyouby tal et . . . CORE http://doc.rero.ch GnakAcsinn.A698) another AY649786), no. of parasite Accession to identical (GenBank are LSU the bayeri Oc. 2005). Ebert, and Lass Ebert 2003; Ebert, 1994; ecology, Ebert, systems the (e.g. used host-parasite investigate of been evolution that and have epidemiology studies parasites in three repeatedly All 2001). (Ebert, Tva of the metapopulation in a most in the is parasite It common parasites. million several harbour may aaieseis(re,17;SindladEbert, and many Decaestecker Stirnadel to 1997; host 1974; is (Green, and species lakes parasite and ponds of zooplankter magna Daphnia system host-parasite The METHODS AND MATERIALS limitations. its its on as comment well applica- and as method usefulness the correction qPCR demonstrate this of of We bility resolution improved. the greatly samples, total is the of in content variation for DNA correcting By 2004). measure reverse Pfaffl, qPCR, to a in (RT PCR similar used quantitative as real-time methods transcriptase is DNA quantification approach relative host This the of to intensity. use parasite infection the of propose of we ratio cases, are the DNA these affected, host In to primarily affected. parasite is less total of sample amounts The a relative in while extraction. DNA DNA of variation sample amount during additional a and introduced of control on is to volume difficult strongly the be depends times, may At but quality. DNA sample parasite of ments with experience previous little qPCR. with laboratories net a oyadoaisadtasisboth transmits and (Jı ovaries vertically and and body horizontally of fat parasite microsporidian parasites. infects a thousand also few is a and to monotonically harbours usually up increases host single A host number chronic. are a the infections in autoinfection, to parasites Due of faeces. after the and with merogony Once host. undergo their they of y gut cells, the the of half inside upper the transmit in cells epithelial parasites infect and spores unicellular infectious via horizontally These 1997). 1996, of parasites microsporidian mns oysz fa dl nmlrne rm1 from ranges mm. animal 5 adult to exper- same an different of size in the Body repeatedly iments. used therefore be can and genotype parthenogenesis cyclical hr r oDAsqecspbihdfor published sequences DNA no are There uniaiePRalw eypeiemeasure- precise very allows PCR Quantitative asnwsoe r rdcdadreleased and produced are spores new days 3 lgie intestinalis Glugoides ¨ e t SrN,ISad5 and ITS SSUrRNA, its yet , .magna D. mneacieaoi otenFinland southern in archipelago rminne tas(rsae:Caoea sa is Cladocera) (Crustacea: Straus (Larsson tal et and 05.I erdcsby reproduces It 2005). . tal et lbliom magnivora Flabelliforma .magna D. ropr colligata Ordospora tal et ´ oe,13) Hosts 1936). rovec, 00 aaland Capaul 2000; . 98.Currently 1998). . coprabayeri Octosporea .magna D. (Larsson .magna D. k n of end tal et that are . 5 . 2 nasml n hsguetemethod. the parasites gauge of thus number and sample the a to in readings qPCR DNA us relate on allowed to This done life suspensions. were spore all tests from initial quantify extractions parasite, to a applied of be stages can qPCR Although extraction DNA and spores of Purification by publication online the in (2005). available Ebert is study this Tuzetia observed (Mangin been never has transmission horizontal and Vizoso magnivora 2004; verti- Flabelliforma Ebert, and and horizontally (Vizoso both cally transmits and sporous lee hog 25 a through filtered sntrsle steeaen lrsrcua data ultrastructural no are there on as available parasites resolved these not between is relationship taxonomic the na1 lhmgnzr lee hog 25 a through filtered homogenizer, ml 10 a in ude netdadult infected hundred ooeie ihamnpsl na1 a in minipestle min a 15 with for centrifuged homogenized and 5000 tube ml at 50 a into mesh ipaeetppte(pedr,Hamburg, (Eppendorf, pipette displacement sdt uhrmiigtsu rmteminipestle the from OB was tube. tissue but the remaining into homogenate flush the buffer to to lysis used directly Tissue added not extraction. was to 1 Whole prior a DNA. minipestle in less homogenized (Qiagen, were yielded phenol/chloroform/isoamyl Kit animals but Tissue extraction) Germany), alcohol (DNeasy methods tested Hilden, extraction a also Other with were Germany). extracted Peqlab, Kit, Erlangen, DNA be Tissue (E.Z.N.A. conveniently kit commercial can phase-contrast pensions using counting bacterial ruling) 400 a with microscopy with (Thoma assessed chamber was spores of 50 in re-suspended n etiue o 5mna 10 at min 15 for centrifuged and etppte.Teeoe Multipette a Therefore, pipettes. buffer ment elution 200 warmed was of (70 use extraction the DNA requires protocol the all of of volume 30% About of germinated. spores they that indicating fe rtaedgsin eann prsof spores Remaining digestion. intestinalis G. protease after hce ne 400 were suspensions under Spore h. 2–3 checked for lasted digestion aeul vrado lo 5 uo HS-40 10 Ludox 1 at min a 25% 10 for in of centrifuged Switzerland) ml Buchs, 1 100 (Sigma, on in overlaid re-suspended carefully were parasites For in used parasites all on information Detailed N rmwoeaiaso rmsoesus- spore from or animals whole from DNA x )wihaet h ouei i displace- air in volume the affects which C) .intestinalis G. sp.). g c bayeri Oc. tal et For . c bayeri Oc. and 95 hr hsprst snamed is parasite this where 1995, . c bayeri Oc. 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Poes sple ihtekit) the with (supplied -Protease eedgse.Tefia elution final The digested. were r m r fwtradconcentration and water of l m . magnification. ehit 1 a into mesh m Daphnia and coprabayeri Octosporea hs-otatmicrocopy phase-contrast a nyoesoetype spore one only has 4 05 nml were animals 10–50 , g r colligata Or. h pr eltwas pellet spore The . otterrefringency, their lost 4 eehomogenized were g . ltb iha with tube ml 5 elt rmall from Pellets . . ltb and tube ml 5 m tal et fwater, of l . 1 ltube, ml 5 . ltube ml 5 shetero- is several , m positive 2005). . .The l. m m http://doc.rero.ch ie hc a cu ntehbttof magna habitat D. the in ramosa para- occur against Pasteuria may primers which tested we sites addition Decaestecker In (sensu 2005). 2 and 1 Microsporidium obtusa pnaeu neto nasemi-natural and a in population) infection spontaneous a beetle), diving a from lated bryozoa), freshwater from sporidia cristatellae galeata mlf 3 pfamn fteLURAgene LSUrRNA the of fragment to of bp 137 OctoFwd used a amplify were Primers OctoRev ACCAAGCAACCCTACTCTGC AF394525). and of CGAGCGAACAAGAAATAGCC no. downstream of bp a 331 Accession gene SSUrRNA amplify located the to fragment used bp 114 GCTTTGACACT- were GlugoRev GCATTCTTTGAGT and ATCAGTC lgie intestinalis Glugoides ewli schaefernai of parasites in Berwaldia sites microsporidian binding place against different tested to was Specificity taken regions. variable was highly care and 2002) eune fteMcoprda(Canning rDNA Microsporidia of be the alignment to of an selected sequences on were based sequences species-specific Primer the run. in reactions same amplification different of tem- 60 realization to Annealing set 2000). was Skaletsky, perature inter- and web (Rozen Primer3 the face with designed were Primers PCR quantitative Real-time GC eeue oapiya12b fragment of bp 182 gene a SSUrRNA amplify the to of used were CGTCC CACTGACTCTTTCAT- OrdoRev GTGATGTTGAGTATTT- and GAGCG OrdoFwd Primers emn)wsue.EtatdDAwsstored was DNA Extracted at used. was Germany) ilb,Isih A S)ad0 and USA) MA, Ipswich, 100 Biolabs, DNA), 2 extracted primer, each of 20 all they yet tested, efficiencies. amplification were suboptimal gene Several had rRNA AJ292544). the in no. actin primers the Accession of fragment (GenBank bp 89 of gene a amplify Primers to chosen AF394529). were no. Accession oSaTqDAplmrs Qae,Hilden, (Qiagen, polymerase DNA HotStarTaq oigwr on oyedotmlrsls 0 n 600 fol- results: optimal the yield and to explored found were Biosystems, were lowing reagents of (Applied and concentrations template Mix Various Switzerland). Master Rotkreuz, SYBR PCR with Australia) Green Sidney, Research, (Corbett rmr lgFdCAGAAGCGGGGAAC- GlugoFwd Primers eltm uniaiePRwspromdin performed was PCR quantitative Real-time x c bayeri Oc. m ecinvlmso oo-ee2000 Rotor-Gene a on volumes reaction l 20 , r x coprabayeri Octosporea C. mendotae ), , rooeaplumatellae Bryonosema rcooeapectinatellae Trichonosema GnakAcsinn.AY649786). no. Accession (GenBank acmo atra aaieof parasite bacterial common (a or Ordospora m ftmlt ie %o the of 1% (i.e. template of l , .pulicaria D. x , m oalwfrsimultaneous for allow to C uly vavrai Gurleya /lBA(e England (New BSA g/ml , lbliom magnivora Flabelliforma .intestinalis G. Microsporidium ropr colligata Ordospora r colligata Or. p (from sp. ). Nosema , Pseudonosema , .magna D. (GenBank (GenBank .magna D. . Larssonia Daphnia 5units 25 p (from sp. Daphnia Daphnia p (iso- sp. (micro- tal et tal et and M : . , . : 3 N qiaet f10 of equivalents adult extracted DNA DNA single of a series dilution from a with tested were prsadwr elctd4tms( ie for animals. times infected in (8 occur that times intensities infection 4 replicated bayeri Oc. were and spores ahse,cntn ursec custo) A acquisition). (2 fluorescence control constant negative step, each e fsoe.Te otie N qiaett 10 to num- equivalent DNA known di- contained a They a spores. from of extracted ber with DNA assessed of series were lution qPCR of reproducibility USA). Sidney, NC, JMP Cary, with Inc., Research, Institute calculated (SAS were (Corbett v5.0.1a statistics Rotor- and software using Australia) quantified 6.0 were Gene Samples run. every curve melting a and (60 acquisition) fluorescence (10 eo a oeaantadlto eiso DNA of series infected dilution adult an a from against extracted done was below Daphnia ohlgtmcocp n PR apevolume Sample qPCR. 25 and was microscopy light both colligata Or. qC)i h hl ape aawr log were equivalents Data spore sample. whole or the number microscopy) in the (qPCR) (light either spores calculate to both of used from were Measurements qPCR. methods by extraction remaining the amplified of the standard was 1% and From the extracted was spores. DNA is sample, counting this for laboratory, method our 400 In ruling) under nification. (Thoma contrast-microscopy haemocytometer phase with a using sample) eauain(5mna 95 at min the (15 in denaturation polymerase the to addition mastermix. in Germany) eehmgnzdi 240 in homogenized were wleadult Twelve qPCR and extraction DNA of Reproducibility regression. linear by analysed and transformed Thirty-one microscopy light to qPCR of Comparison dilution. extractions spores. against pure 25-fold standard from the of a equivalents calibration the spore the and by in contained expressed 5- were and a Measurements times extraction, 4 original replicated were series t94 at eeqatfid4tmswt PR(2 extracted qPCR with was times DNA DNA 4 parasite quantified and which were host sample, from every In aliquots separately. 12 in rmr o h unicto of quantification the for Primers eaieqatfiaino ape ntetests the in samples of quantification Relative mlfiainecec fprst rmr and primers parasite of efficiency Amplification h mlfiainpol osse fa initial an of consisted profile amplification The 5 x C–95 for x ,2 t60 at s 20 C, m .W one prsi 0 in spores counted We l. c bayeri Oc. n eerpiae times. 4 replicated were and x .Dlto eisas eetrne of ranges reflect also series Dilution ). ,rsn yhl ere etn o at s 5 for resting degree, a half by rising C, eesree o h aaieusing parasite the for screened were , Daphnia Daphnia ont 0( for (1 10 to down ) m x Daphnia ,1 t72 at s 15 C, fwtr a nlddin included was water) of l rvosyepsdto exposed previously , netdwith infected m x fdinzdwtr split water, deionized of l 2 ecin contained Reactions . x ) 0cce 2 s (20 cycles 50 C), o10 to . 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[host DNA] = F − .SS 0·7 0 ropr colligata Ordospora 0 . 28 . 5.Ti niae that indicates This 25). 2 = r S 2 . . . . 7 33 877 8 0 085 1 51 614 3 0 039 . =0·78 0 − E 0·6 . ,slope ., 78, rmDNA from P F < − rtor -ratio 0·5 . . = 20 72 90 59 0 . plotted 1 0) The 001). . 06 ¡ 0 2 . . . . 912 088 940 060 . 18 5 aaiebre latsur en contrast, means square contrast, (least means P burden highest square the carried parasite C genotype DNA, (least host to parasite readings DNA by P revealed host contrast, as DNA means of total square less intensity contained (least infection higher C P a genotype had than B genotype host 97% explained while 4). genotypes variation, (Fig. total host of between differences 1% sample only correction, this explained After volume differences content. from DNA this total arising of in variation use the the that reduced found ratio We readings. qPCR the from of 71% also DNA. explained was host in of DNA genotype variance amount total host host total varied, and was the in affected samples As variance in 2). total DNA (Table of DNA Host 79% parasite genotypes. only host explained genotype between intensity infection oreSS DNA Parasite (A) Source between differences where genotypes by (C) explained DNA largely host is to parasite variance of ratio the reduced using is by Variance variance. total of a part substantial explains volume infected Sample three genotypes. from parasite samples host of on (B) data DNA qPCR host and to (A) fitted ANOVA 2. Table epl 2005). Hempel, in detect, para- to microsporidian sites qPCR the quantify on and based discriminate method a developed We DISCUSSION B otDNA Host (B) a eraiyapidt te netbaehost- invertebrate other aegypti to e.g. systems, applied microparasite readily be can crustacean freshwater colligata Ordospora C aaieDAhs DNA DNA/host Parasite (C) < < < otgntp 15 genotype Host ro 0 3 Error volume Sample otgntp 4 genotype Host ro 0 1 Error volume Sample ro 0 0 28 Error volume Sample genotype Host codn oraig fprst N alone, DNA parasite of readings to According DNA host to parasite of ratio the calculated We 0 0 0 rblu castaneum Tribolium . . . 001). of ratio the of application after Thus, 001). C genotype from samples However, 001). lgie intestinalis Glugoides (Bedhomme nterhs,teplanktonic the host, their in tal et ahi magna Daphnia 04 or 2004) . , FshradSchmid- and (Fischer ari culicis Vavraia coprabayeri Octosporea ...... 2347 12 826 70 38 4145 64 288 51 42 241 32 1407 42 30 F oeawhitei Nosema hsmethod This . rtor -ratio ...... 60 16 20 52 60 76 10 01 90 0 79 41 in Aedes 2 ...... and 176 787 216 706 014 974 http://doc.rero.ch hehl o3 to we threshold detection, 0 microscopic examine typically light Using sequences. N gis otDNA. parasite host of against Ratio DNA (B) DNA. parasite (A) of genotypes. Measurements host between differences obscure to with infected samples colligata genotypes in host qPCR different by 3 measured from intensity Infection 4. Fig. ihterslso h w ehd,weewe an than where more methods, was two qPCR of the sensitivity of that results found the with o eibedtcinle t3 threshold at DNA the lies the qPCR, detection of to reliable 1% subjected for If sequences is reliably. successful target extraction detected for 3 than be probability less cannot required, 95% Poisson is is a detection PCR the the If that in distributed. sequences expect target we of quantification Based sample. number considerations, a any of theoretical part a of on only analyses shortcoming that method is expected this Furthermore, a an extraction. low or at parasite DNA this a not efficient in genes less are but rRNA the spores reasons of number 10 Possible copy as concentrations. low detected lower We as spore. equivalents single equiv- DNA a DNA to parasite alent unambiguously detected we conventional In to microscopy. light compared qPCR using by increased hosts freshwater limited. is in knowl- both parasites microsporidian because of of specificity edge parasites complete ensure different cannot of than range rather Daphnia speci- a tested known, with we ficity is Although where surveys. community environmental experiments parasite from individual analyse the samples to population developed and was method This rmnn irsoiinprstsof parasites microsporidian prominent log [parasite b) DNA] a) u blt odtc aaie a strongly was parasites detect to ability Our sn hsapoc,w iciiae between discriminated we approach, this Using 10 − − − − − 1·5 1·4 1·3 1·2 1·1 otn fsmlswsdlbrtl varied deliberately was samples of Content . Host genotype swl sohrc-curn ot,this hosts, co-occurring other as well as ABC r 1250 . 8 fasml hc esthe sets which sample a of 08‰ .intestinalis G. = 70soe.Ti conforms This spores. 3750 log10[parasite DNA/host DNA] 0·5 1·0 1·5 2·0 2·5 0 Host genotype ABC r and 100 c bayeri Oc. = r colligata Or. 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Zbinden by Comments infection of measure the as ratio host DNA intensity. the host 2 to employing parasite after of of reversed intensities were infection genotypes Moreover, accen- were tuated. genotypes host decreased between was differences genotype and host as same the DNA between from host variation samples to intensity, infection parasite of of measure ratio the the intensity. used infection we measure When to quantified Differences was genotypes DNA parasite volume. when host obscured were their genotypes different host between varied 3 deliberately from and each assembled samples We volume. 12 sample in differences for rect destroyed. samples, been has aged DNA or the of degraded part a e.g. where differences quality, de- for sample correct be also in may cannot method populations The tected. between a screening differences prevalence that the is in in drawback without One individual hosts. assessed individual of be per comparison. can DNA their intensity population parasite allows infection to host and Average size of them ratio and amounts The standardizes different age DNA. contain total in of will individual. differ samples ‘averaged’ will structure, an quantifi- populations with and done Because then extraction is DNA cation sampled, and be and can homogenized population Pulkkinen every of fractions (e.g. hosts, of case populations the In 2004). host intensities studies Ebert, infection several epidemiological of assessment in content; the require DNA may total for ttsia die .Gn oihdteEgih Both English. the polished Ganz H. advice. statistical lgie intestinalis Glugoides cor- to method our of applicability the tested We samples correct to imperative is it cases, some In tal et 2004). . and .magna D. r colligata Or. n te small other and ¨ lkrgave lliker establish 7 etr .D,Vra . os,A . ae .W., M. 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