Cholera: Death by Diarrhoea

2 CHOLERA: DEATH BY DIARRHOEA

Cholera-anoverviewCholeraisaninfectiousdisease,causedbythebacteriumVibrio cholerae,whichaffectsthetransportofwaterinthesmallintestine.Thebacteriumsecretesatoxin,choleratoxin(CT),whichcausesseverefluidlossfromthebodyintothedigestivetract,leadingtodehydrationandultimatelydeathbydiarrhoea.

V. choleraehascausedsevenworldwidediseaseoutbreaksorpandemicssince1817,killingmillionsofpeopleandinfectingmillionsmore-includingfourepidemicsintheUKaccountingforapproximately55,000deaths.

SoutheastAsiawitnessedthefirstcholeraepidemicin1817,thoughtheconditionof‘dehydratingdiarrhoea’wasmentionedaroundthetimeofHippocrates(460-377BC).Today,choleraisprevalentinCentralandSouthAmerica,AfricaandAsia,thoughnotintheUKasitisusuallyconfinedtocountrieswithapoorsanitationinfrastructure.Worldwide,100,000-300,000casesofthediseasearereportedeachyearwithmorethan94%ofcasesinAfrica.However,thisislikelytobeamassiveunderestimateasmanycountriesinthe

Cholera-ahistoryPriorto1850,itwasthoughtthatcholerawascausedbybreathinginbadair-miasma-andthatprotectionwasofferedbystrongsmellingsubstancessuchasherbsandcamphor.JohnSnow,aprominentLondonphysician,washeraldedastheFather of Cholerawhenhequestionedthemiasmatheoryaftertreatinganumberofcholerapatients

IndiansubcontinentandSoutheastAsiadonotreporttheincidenceofcholera;forexample,nocholeracaseshavebeenreportedinBangladesh,althoughestimatessuggestthattheremaybeasmanyas1millioncasesperyear.Realisticworld-wideestimatessuggest120,000annualdeathswith3-5millionpeoplesufferingfromthedisease.

Haitiangirlsufferingfromcholera

Cartoonofdoctorsvisitingapatientwithcholera

3CHOLERA: DEATH BY DIARRHOEA

butnotsuccumbingtotheconditionhimself.Snowarguedthatdiseasemusthaveenteredthebodythroughthemouthoninfectedfoodordrinkandnotviathelungs,afterobservingthatpatientsbecameillwithsymptomsoriginatinginthegutsuchasvomitingandstomachache.Hehypothesizedthata‘poison’wasingestedwhichreproducedinthedigestivetractand,in1849,hepublishedhisideas,athisownexpense,inapamphletcalledOn the Modes of Communication of Cholera.Snowwasabletoprovehistheoryin1854whenhecarefullymappedtheincidenceofcholeraduringtheLondon1853-1854outbreak.BelievingthatthesourcewasawaterpumpinBroadStreet,Soho,heexaminedwaterfromthepumpunderthemicroscopeanddescribedtheoccurrenceof‘whiteflocculentparticles’.Convincedthattheseparticleswerethesourceofthediseasehepersuadedtheauthoritiestoremovethepumphandle;atthistime,thenumberofcholeracaseslocallywasalreadyindecline,thoughitisthoughtthatSnow’sconclusionsprovidedthebasisforfurtherpreventativemeasures.JohnSnowneveridentifiedthebacterialsourceofinfection;however,throughdatagatheringandstatisticalanalyses,hesuggestedthatthewaterwasthereservoirofinfectionandthemodeoftransmission.Hisrecommendationsofimprovedhygieneandboilingofdrinkingwatermostlywentunheededbythosewhodidnotsupporthisclaimsasthe‘germ’responsibleforcholerahadnotyetbeenobservedmicroscopically.

Whatcausescholera?Choleraiscausedbythecurved,rod-shapedbacteriumVibrio cholerae.Therearemorethan100speciesofVibrio,onlyafewofwhicharepathogenictohumans.Theyarefree-livingbacteriathatarecommonlyfoundinbrackishwaters.Vibriocellsaremotile,propelledbyasinglepolarflagellumand,astheynormallyliveinneutralor

InthesameyearthatSnowmadehisobservationsanItaliananatomist,FillipoPacini,publishedapaperentitledMicroscopical Observations and Pathological Deductions on Choleradescribingthecausativeagentofcholeraandsuggestingtreatmentoptions.However,thepreferredtheoryinItalyatthattimewasthatofmiasmaandassuchPacini’sworkwentunnoticed.

AGermanbacteriologist,RobertKoch,suggestedin1882thatcholerawascausedbyabacteriumandthatthisbacteriumsecretedatoxinwhichcausedrapidwaterloss.Kochobservedfaecalsamplesandidentifiedacomma-shapedbacillus,whichhecalledVibrio choleraeafteritsvibratingwiggles.Havingalreadymadehismarkonbacteriologywithhisworkonanthraxandtuberculosis,andhispostulates(seepage11),whichgenerateaformalproofthatacertainmicro-organismcausesadisease,hisideasandobservationsoncholerawerewellreceived.PaciniwasrecognizedforhisworkposthumouslyastheorganismwasofficiallynamedVibrio cholerae Pacini1854.

Artworkofthespreadofcholerainthe1830s

RobertKoch

4 CHOLERA: DEATH BY DIARRHOEA

alkalineconditions,theyaresensitivetoacidicpH.Thebacterialcellsaretiny,aboutonefivehundredthofamillimetrelong,whichmeansthat20ormorecellswouldfitacrossthediameterofahumanhair.

Getting there…Cholerainfectionresultsfromtheingestionofcontaminatedfoodorwater.Tosurvivethejourneythroughthedigestivetract,thebacteriumhastobewelladapted.

Passagethroughthedigestivetract:Entrytothebody:V. choleraecellsenterthebodyandmaketheirwaythroughthedigestivesystem.Approximatelytwo-thirdssurvivetheacidicconditionsofthestomach;survivorsconserveenergyuntiltheyenterthesmallintestinewheretheybeginproductionoftheirflagella.Theflagellum,whichisalongtail-likestructure,propelseachVibriocellforwardthroughthemucuslayertotheintestinalwall.V. choleraealsoproducesenzymesthatdigestthelayersofmucus,whichhelpswithaccesstotheepithelialcellsliningtheintestine,aswellasdetachmentfromcellsthatarebeinglostduetothebody’sdefencemechanisms.Toconserveenergy,onreachingtheintestinewall,thebacterialcellsstopflagellumproduction,as

propulsionisnolongerrequired,andenergyisrefocusedontheproductionofhairlikeappendagescalledfrimbriaeorpiliwhichareformedonthebacterialcellsurface.Thesestructuresaremadeofproteinandallowthebacteriatoattachtotheliningoftheintestine.

Growth:Forsymptomstopersist,thebacteriamustcontinuetomultiplyintheintestine.

Colonisationandinvasion:Whentheyreachthesmallintestine,thebacteriamust‘holdon’andresistthenormaltransitmotionsofthisregion.Theydothisusingtheirfimbriaeorpili.Onceattached,thebacteriainvadethehostcell.ProductionofCTisthefinalstageinpathogenesis.

Causing trouble…Theperiodbetweeningestionandapatientshowingsymptoms(incubationperiod)isusuallyveryshortandcanbeaslittleas2hours,althoughitcantakeupto5days.Mostpeople(75%)infectedwithV. choleraedon’thaveanysymptoms(asymptomatic);however,theyremaincarriersofthedisease,excretingthebacteriaintheirfaeces,usuallyfortwoweeksbutoccasionallyforseveralyearsasinthecaseof‘CholeraDolores’.

Illustrationofthebacterialcellsthatcausescholera

5CHOLERA: DEATH BY DIARRHOEA

CholeraDoloresAsymptomaticcholerainfectionisunlikelytolastmorethantwoweeks;however,long-termcarriershavebeenreported,thoughtheyareextremelyrare.DuringthePhilippineepidemicof1962,Dolores,ahousewife,sufferedamildattackofcholerawhilepregnantwithhersixthchild,andwashospitalisedthoughnottreatedwithantibiotics.ItisthoughtthatshecontractedcholerafromeatingcontaminatedseafoodwhichherhusbandbroughtfromnearbyBacolodcity.SeveralmembersofDolores’familyshowedsymptomsofcholera,thoughnoneneededhospitalization.Doloresintermittentlyexcretedthebacteriumover11years,sohadthepotentialtocausefurtherinfectionalthoughthiswasneverobservedinherlocality.Thisispossiblyduetothefactthatshelivedinanareawherecholerawasendemic.LittlemoreisknownofthiscaseexceptthatV. cholerae wasresidentinherbiliarytract;itisunknownwhatfactorscontributedtoDoloresbeingacarrierforsuchalongperiodoftime.In1973,Dolores’carrierstatewasresolvedspontaneously.

DifferenttypesofV. choleraeinfectionOver100serogroups(organismsgroupedonthebasisoftheircellsurfaceantigens)ofV. cholerae exist;however,onlytwocauseepidemiccholera.TheyareserogroupO1andserogroupO139.SerogroupO1canbeclassifiedfurtherbasedonphenotypeintoElTorandClassical;thesearereferredtoasbiotypes.ElToristheinfectiveagentresponsibleforthecurrentpandemic;Classicalhasnotbeenidentifiedsincethemid-1990s.V. choleraeO1biotypescanbefurtherclassifiedintoserotypesbasedontheresultsofaserumagglutinationtest;therearethreeO1serotypes,Inaba,OgawaandHikojima.V. choleraeO139,arelativelynewserogroup,hasnotreachedpandemicpotentialsofar,thoughitsoccurrenceisbeingcloselymonitoredasitspatternofinfectionandcapacitytosurviveinwater,suggestthatitmaybebothmoreinfectiousandmorevirulentthanV. choleraeO1.

ItisworthnotingthatthegeneforCTiscarriedonabacteriophage(avirusthatinfectsbacteria)insideV. cholerae.So,intheory,ifthisvirusweremoremobileitmightbepossibleforanewserotypetoemergeagaininthefuture,justasO139hasdonequiterecently.

Bacteria V. cholerae

Serogroup O1 O139

Biotype ElTor Classical

Serotype Inaba Hikojima Ogawa Inaba Hikojima Ogawa

Molecularmodelofthesecondarystructureofcholeraenterotoxin

6 CHOLERA: DEATH BY DIARRHOEA

LaboratorymethodsfordiagnosisofV. choleraeManydiseasescausediarrhoea;however,theproductionoffrequentwaterystoolsrequiresfastertreatmentduetothehighriskofdehydration.Lookingatastoolsampleunderthemicroscopecanhelpthemedicalpractitionermakeadiagnosis,however,thisisoflittleusetopatientsasthosewithanydiarrhoealdiseasereceivethesametreatment,i.e.fluidandelectrolyte(saltsandsugars)replacement.

Conventionalculturemethodsarethe‘goldstandard’fordiagnosisofV. cholerae.StoolsamplesorrectalswabsareplatedonTCBS(thiosulphatecitratebilesalts)agar(V. cholerae coloniesappearyellowastheyfermentglucose).Suspectedcoloniesareselectedforfurtheranalysiswithbiochemicalandserologicaltestssuchslideagglutination.

Incholeraendemicareas,wherelaboratoryfacilitiesareoftenlimited,rapidimmunochromatographicdipsticktestcanbeusedtodiagnosecholera.Itisverysimpletouse,thedipstickisdippedintoastoolsample.Iftworedlinesappearonthedipstick,thenthepatienthascholera,ifonlyoneredlineappears,thetestisnegative.Ittakesbetween2-15minutesforthetesttodevelop.

SymptomsMostpeoplewithcholeraareasymptomaticand,ofthosethatdodevelopsymptoms,80%havemilddiarrhoeawhichisdifficulttodistinguishfromthatcausedbyotherpathogenssuchasviruses.However,allofthesepeoplecarryV. cholerae,excreteitandcanspreadittoothers.Severecholera(choleragravis),whichaccountsforfewerthan10%ofcases,ischaracterizedbythe uRapidonsetofviolentwaterydiarrhoea.Thediarrhoeais‘strawcoloured’withflecksofmucus andisoftendescribedasresemblingricewaterVomitingExtensivedehydrationLegcrampsAsfluidislost,thebloodthickensandtheskinbecomesblue-greyincolour;patientsalsobegintosufferlethargy,alackofconsciousness,confusionandoccasionallyfever.Itisthisrapiddehydration-asmuchas1litreoffluidcanbelosteveryhour-whichcanprovefatalwithin24hoursofdevelopingthedisease.Inchildren,therapidlossoffluidcanleadtoseveresaltimbalance,whichcancauseconvulsionsandcardiacarrestinadditiontothesymptomsdescribed.Thefatalityrateinuntreatedcasescanbeashighas30-50%.Ifpatientsaregivenrehydrationtherapy,thenthedeathrateisbelow1%.

Womanwithcholeragettinghereyesexamined

7CHOLERA: DEATH BY DIARRHOEA

TreatmentTreatmentofthesymptomsofcholerafocusesonfluidreplacementaswateraloneisnoteasilyabsorbedbythebody.TheWorldHealthOrganization(WHO)recommendscleanwaterandrehydrationsaltstorestoreelectrolytebalance.Oralrehydrationtherapy(ORT)employsrehydrationsalts,whicharereadilyavailableinpharmacies;theseareofteninshortsupplyinareasofhighinfectionrate.ORThasbeenrevolutionaryincholeratreatmentand,ifproperlyused,ithugelyreducesboththeneedforhospitalizationandmortality.Somecasesmaybetreatedwithantibiotics,althoughgenerallytheindividual’simmunesystemislefttodestroythepathogen.Inextremecases,Hartmann’ssolution(asolutionofsodium,chloride,lactate,potassiumandcalciumions,whichisisotonicwithblood)isadministeredbyintravenousinjectiontoreplacebodyfluidsandmineralsalts.

TransmissionofcholeraCholerainfectionrequiresalargedoseofbacteriaandistransmittedbythefaecal-oralroute,forexamplebydrinkingfromawatersupplycontaminatedwithinfectedexcrement.Thebacteriacanalsobespreadtofoodifinfectedpeopledon’twashtheirhandsthoroughlyaftergoingtothetoiletandbeforefoodpreparation.Infectionisoftennotedafterthefuneralofacholeravictimwherecontaminationoccursasaresultofpoorhygieneinfoodpreparation.

Theenvironmentcanalsobeareservoirofinfectionasthebacteriaarepresentinbrackishwaters,ofteninassociationwithaquaticorganismsatalowlevel,andsometimesinanun-culturablestate.Shellfishlivingincontaminatedwatercantransmitcholeratoo.Theyarefilterfeedersand,astheystrainthewaterforfood,thebacteriabecomeconcentratedinsidethem.Anyoneconsumingshellfishthatarenotproperlycookedcanbecomeill.

Twogirlswashingdishesinmuddyriverwater

RetrospectivediagnosiscanbeperformedbyanalysisofthebloodforantibodiesagainstV. choleraeandCT.AlthoughidentificationofV. choleraeisnotoftenusedintreatment,thisinformationhelpstopreventfurtheroutbreaks.

8 CHOLERA: DEATH BY DIARRHOEA

Physiologicaldifferencesaffecting infectionCholerainfectioncanbespectralinthatsymptomscanvarywithdose,serogroup,biotypeandserotype,thoughphysiologicalvariationbetweenpeoplecanalsoplayapart.Itisthoughtthatthosewhohavereducedornonexistentstomachacidduetodiseaseorailmentaremoresusceptibletocholera.Inahealthyindividual,thestomachacidactsasafirst-lineofdefenceagainstinfectionandkillsmanyV. choleraecellsbeforetheytravelto

PreventionGoodsanitation,cleandrinkingwaterandimprovedhygienepractices,suchaswashinghandsaftervisitingthetoiletandbeforepreparingfood,areallstrategiesthatneedtobefollowedtopreventtheoutbreakofcholera.Belowaresometipsfortravellersonhowtoavoidinfection,fromfoodanddrink,whenvisitinganareawherecholeraispresent.

FOOD DRINK

CookallfoodsthoroughlyUsebottled,boiled,orchemicallytreatedwatertowashdishes,cleanteethandpreparefood

Rawfruitandvegetablesshouldbeavoidedunlessyouthoroughlywashandpeelthem

yourself

Onlydrinkbottledwaterthathasthesealstillintact

Icecreamfromdoubtfulsourcesmayalsobecontaminatedthereforeshouldbeavoided

Avoidicecubesindrinks

Foodmustbeproperlypreparedandstillhotwhenserved

Boilunpasteurisedmilkbeforeyoudrinkit

Becarefulwheneatingfoodfromstreetstalls

Avoidsaladsastheymayhavebeenwashedincontaminatedwater

Avoidrawfishandshellfish

A simple rule is: Boil it, cook it, peel it or forget it!

VaccinesandimmunityOralvaccinesshowlong-lastingprotectionagainstcholerawithfewside-effects,althoughtheyprovideinsufficientprotectionforchildrenunder2yearsofage.Vaccineshoweverareonlyadvisedforpreventativeuseratherthanasamethodfor

theintestine.AnotherpossibilityforphysiologicalvariationamonginfectedindividualsmaybetheavailabilityofsurfacereceptorsforCTonthehostcellsurface,thoughthishasnotbeenproven.

9CHOLERA: DEATH BY DIARRHOEA

controllingoutbreaksandshouldbeusedintandemwithstandardpreventionandcontrolmeasures.Alsoimmunity,naturalorartificial,isserogroup-specific;forexampleimmunityprovidedagainstV. choleraeO1doesnotprotectfromO139.WHOrecommendsvaccinationforpeopleoccupyingslumsorrefugeecampsduetohighdensitypopulationsandincreasedriskofdiseasespread.

In1894,soonafterKoch’sidentificationofcholera,aninjectable,whole-cell,killedvaccine,wasdevelopedthatinduced48%protectionforthreemonthsagainstV. cholerae, serotypes InabaandOgawa.Thevaccine,initiallytrialledinIndia,wasneverendorsedbyWHO,althoughitmaystillbeavailableinsomecountries.

Protectiveimmunityinthoseexposedtocholeraisinducedalmostexclusivelybyantibodiesproducedintheintestine.Theseantibodiespreventbacterialcolonisationandmultiplication,andtheyinactivatetheCT.ImmunoglobulinsIgA,IgGandIgMhaveallbeendetected,althoughIgAisthemostimportant.TheantibodiespreventtheCTfrombindingwithreceptorsonthecellsurface.NaturalimmunityisprovidedbyIgMfollowedbyaswitchtoIgG.Thereisa3-yearperiodpostinfectionwherepatientsremainimmunetocholeraasaresultofacquired,naturalimmunity.

InareaswherecholeraisendemicsuchasBangladesh,infectionratesarelowamongadultswhencomparedtothechildreninthesamearea,whereasinareaswherenewepidemicsariseratesarehigherintheadultpopulation.Thisdiscrepancyillustratesaresistancelinkedtothepresenceofcirculatingvibriocidalantibodiestocholera.

Effective oral vaccines available against cholera:

uWC/rBS(Dukoral)–thisisawhole-cell,killedV. cholerae O1vaccinewithpartoftheCT protein.Trialshaveshownhighlevelsofprotection(85-90%)overaperiodof6monthsfor 2dosesofthevaccineinallagegroups,thoughprotectiondeclinesafter6monthsinyoung childrenandremainsatabout60%inolderchildrenandadultsafter2years.Protectionis providedagainstV. choleraeO1serotypesInabaandOgawa,andbiotypesClassicalandEl Tor.Dukoralalsoprovidesshort-termprotectionagainstEscherichia colienterotoxin,whichis ofaddedbenefittotravellers.

uVariantWC/rBS–acheaperversionoftheWC/rBSvaccinewhichdoesnotcontainanyCT; ithasbeenshowntohaveanefficacyof66%after8monthsinallagegroups.Thisvaccineis onlylicensedinVietnam.

uCVD103-HgR(Orochol)–thisvaccineconsistsofanattenuated,live,geneticallymodified V. choleraeO1InabastrainthathasbeenengineeredtoproducepartoftheCT.Itconfers highprotection(95%)againstO1(ClassicalandElTor)invirginvolunteers,i.e.thosenot previouslyexposedtothebacterium.Protectioninendemicregionshasnotbeenshownin aneffectivetrial,thoughretrospectivestudieshaveshownprotectioninanongoingoutbreak inMicronesia,agroupofislandsintheWesternPacific.Orocholistheonlyvaccineavailable asasingledose,which,duetoadministrationlogistics,ismoreviableforpre-emptiveand long-termoutbreakcontrolinongoingfieldconditionsaccordingtoWHO.However,due toalackofevidenceforitseffectiveness,thevaccinewaswithdrawnin2004.

10 CHOLERA: DEATH BY DIARRHOEA

Furtherinformation uCDC-www.cdc.gov/nczved/divisions/dfbmd/diseases/cholera/

uECDC-www.ecdc.europa.eu/en/healthtopics/cholera/

uNetDoctor-www.netdoctor.co.uk/travel/diseases/cholera.htm

uTextbookofbacteriology-www.textbookofbacteriology.net/cholera.html

uWHO-www.who.int/topics/cholera/en/

EducationInordertocontrolandultimatelystopthespreadofacholeraoutbreak,communityeducationisparamount.Manyofthebasichygienemessages,whileperhapssimpletorelate,areoftendifficulttoimplementduetocost.Therefore,alternativesolutionsarerequiredtolimittransmission,forexampleWHOsuggeststheadditionoflimejuicetofoodandwatertoinactivateV. cholerae.Ideallyincommunitiesmostaffectedbycholera,awarenesscampaignscontinuethroughouttheyearincreasinginfrequencyasthecholeraseasonapproaches.Clearinformationispresentedwithmessagesadaptedforcultural,socialandeconomiccircumstances,andtheinformationisdeliveredgraphically,byradiobroadcastorastalksinareaswherepeoplearewaitingorcongregated.

FutureCurrentresearchintocholeraisfocusedonunderstandingtheCTitself.Anotherareaofanti-diarrhoealresearchisdevelopmentofapillthatwillpreventthediarrhoeapost-infection.Twodrugs,chlorpromazineandnicotinicacid,havebeenshowntobeeffectiveinanimalmodels,thoughthemechanismisyettobeunderstood.ResearchcontinuesintovaccinedevelopmentagainstbothO1andO139serogroups;severalpotentialnewvaccinesarecurrentlyinclinicaltrialsinBangladesh,IndiaandThailand.

Choleraresearch

11CHOLERA: DEATH BY DIARRHOEA

Koch’spostulatesRobertKochdevelopedasetofpostulatesforprovingthataspecificmicro-organismcausesaspecificdisease. uPostulate1:Thesuspectedpathogenic micro-organismshouldbepresentin allcasesofthediseaseandabsentfrom healthyindividuals[thispostulateisoften disregardedas,inthecaseofcholerafor example,itispossibletobeacarrierand notshowsymptomsofthedisease (asymptomatic)]. uPostulate2:Thesuspected micro-organismshouldbegrownin pureculture(notethecorrectmedium mustbeselectedforsuccessfulculture). uPostulate3:Cellsfromapurecultureof thesuspectedmicro-organismshould causediseaseinahealthyanimal. uPostulate4:Themicro-organismshould bereisolatedandshowntobethesame astheoriginal.

PaintingofRobertKochworkinginhislaboratory

TheSocietyforGeneralMicrobiology(SGM)isaprofessionalbodyforscientistswhoworkinallareasof

microbiology.AnimportantfunctionoftheSocietyisthepromotionofthepublicunderstandingofmicrobiology.

SGMproducesanddistributesawiderangeofresourcestosupportmicrobiologyteachinginschoolsandcolleges

acrossallkeystagesandpost-16.Italsorunstrainingcoursesinpracticalmicrobiologyforteachersandtechnicians,

andoccasionalworkshops.TheSocietyalsooffersaninformationservicetoteachersandparticipatesinschools

competitionsandotheractivities.

SchoolMembershipoftheSGMoffersmanybenefitsincluding:

Microbiology Today,theaward-winningmagazine

DiscountsonSGMINSETcourses

PrioritymailingsofSGMteachingresources

Contact

SGM,MarlboroughHouse,BasingstokeRoad,SpencersWood,Reading,RG71AG,UK

T: 01189881800

F: 01189885656

E:education@sgm.ac.uk

W:www.microbiologyonline.org.uk

Cholera: Death by DiarrhoeaWrittenbyVicki Symington

DesignedbyDariel Burdass

EditedbyDariel Burdass

ProofreadbySusan Andrews

AcknowledgementsThanksareduetoProfessor Charles Penn (University of Birmingham)forhishelpfulcommentsonthistext.Everycarehasbeentakentoensurethattheinformationiscorrect,buttheauthorwillbepleasedtolearnofanyerrorsthathaveremainedundetected.

PicturecreditsFront cover,EYEOFSCIENCE/SPL*,p.2 top half,THONYBELIZAIRE/GETTYIMAGES,p.2 lower right,NATIONALLIBRARYOF

MEDICINE/SPL,p.3 bottom left,SPL,p.3 upper right,LIBRARYOFCONGRESS/SPL,p.4 top half,JIMDOWDALLS/SPL,p.5

bottom half,DRMARKJ.WINTER/SPL,p.6 middle,KARENKASMAUSKI/GETTYIMAGES,p.7 top half,PETERMENZEL/SPL,

p.8 middle,THINKSTOCK,p.10 top half,MAUROFERMARIELLO/SPL,p.11 upper right,WELLCOMETRUSTIMAGES,p.11 bottom

half,USALIBRARYOFCONGRESS/SPL*SPL,SciencePhotoLibrary

CopyrightCholera: Death by Diarrhoea iscopyright.TheSocietyforGeneralMicrobiologyassertsitsmoralrighttobeidentifiedunderSection

77oftheDesign,PatentsandCopyrightAct,UK(1988).

Educationaluse:Electronicorpapercopiesoftheresourcesorindividualpagesfromitmaybemadeforclassroomuseandbona

fideeducationalresources,providedthatthecopiesaredistributedfreeofchargeoratthecostorreproductionandtheSGMis

creditedandidentifiedascopyrightholder.

© 2011 Society for General Microbiologyy