CLOSTRIDIUM WELCHII FOO POISONINB BETT C HOBBS MURIE E SMITH C L OAKLE - PDF document

CLOSTRIDIUM WELCHII FOO POISONINB BETT C HOBBS MURIE E SMITH C L OAKLEY G HARRIEWARRAC AN J C CRUICKSHAN The Food Hygiene Laboratory,Central Public Health Laboratory (Medical Research Council), Colindale, TheWellcome Research Laboratories (Biological Division), Beckenham, Kent, andThe London School of Hygiene and Tropical MedicineCONTENTPAG KWWSVGRLRUJ6 3XEOLVKHGRQOLQHE\&DPEULGJH8QLYHUVLW\3UHVV 7 BETT C HOBB AN OTHER243 outbreak o foo poisonin recorde fo 194 show tha i n les tha 3 %n adequat caus wa foundI thi pape evidenc i brough forwar tha a larg proportio o foopoisonin incident hithert unclassifie ma b du t heat-resistan Clostridiumwelchii. I i suggeste tha th occurrenc o thi typ o foo poisonin i linkewit change i th eatin habit o th community an method fo th preventio o suc foo poisonin ar considered(2 Previous records of food poisoning due to ClostridiLittl attentio wa pai b th earlie worker t th possibilit o foopoisonin du t anaerobi bacteri othe tha Cl. botulinum. Cl. welchii iubiquitous Typ A strain ar frequentl isolate fro norma stool an froa wid variet o foodstuffs I doe no spor readily an Headle (1931 reportetha it spore wer kille i 3 min a 90 C an 5 min o les a 100 C Thesfact hav weighe heavil agains seriou consideratio o Cl. welchii a a caus ofoo poisoning I som instances however failur t isolat organism othe thaCl. welchii i significan number ha le author t incriminat it Klei (1895isolate Cl. welchii fro th stool o patient affecte i tw epidemic o diarrhoeinvolvin 5 an 14 case i a hospital abdomina pai wa a constan symptomvomitin relativel uncommon al patient recovere withi 1 hr Andrewe(1899 isolate Cl. welchii fro th stool o patient sufferin fro mil bu chronidiarrhoe i th sam hospital Th investigation mad i thes tw outbreakwere however no sufficien t establis wit certaint th causativ rol o thorganismAttentio wa agai draw t th possibilit tha anaerobi spore-bearinbacteri migh b responsibl fo foo poisonin i a repor b Kno & MacDonal(1943) wh describe illnesse amon schoo childre afte meal prepare aa centra kitchen Grav mad o th previou da wa foun o severa occasion b heavil contaminate wit anaerobi spore-bearin bacilli Dunca (1944als describe outbreak o diarrhoe i resident o a wartim cam afte thconsumptio o mea an potat pi containin larg number o Cl. bifertnentans.McClun (1945 reporte a serie o fou outbreak o foo poisonin frochicke dishe cooke th da befor the wer served Th symptom appeare8-1 hr afte th mea an include nausea intestina cramp an profusdiarrhoe lastin fo

abou 1 hr. vomitin wa rare Th chicken ha bee a lo pressur fo 3 hr. the wer the remove fro th broth whicwa save fo furthe use Direc microscopi examinatio o al sample o brotshowe larg number o Gram-positiv rods prove b cultur t b Cl. welchii.Attempt t demonstrat enterotoxi productio i mic an guinea-pig werunsuccessful on huma voluntee showe typica symptom afte eatin a samplknow t b contaminate wit Cl. welchii.Fro 194 t 194 a larg numbe o case o haemorrhagi enteriti (enteritinecroticans ' Darmbrand' occurre i nort Germany—th patients afte eatintinne meat rabbi o fish paste develope som hour later sever loweabdomina pai an diarrhoea I th wors case th stool containe bloo an KWWSVGRLRUJ6 3XEOLVKHGRQOLQHE\&DPEULGJH8QLYHUVLW\3UHVV Clostridiu welchi food poisoning 11sloughe mucosa an th patient die fro dehydratio an circulator failuror wher th oedem o th intestina mucos wa sufficientl severe fro intestinaobstruction Cl. welchii wa presen i enormou number h th stool an i thlesions roug colonie wer numerou i culture Thes strain o Cl. welchii wermarkedl heat-resistant survivin 100 C fo 1- hr (Zeissle & Rassfeld 1949) Severa strain examine b on o u (Oakley 1949 produceCl. welchii /?-toxin Thoug i th latte respec the resemble Cl. welchii Typ COakley fo variou reasons regarde the a mor closel relate t Typ B ivie o thei strikin heat-resistanc compare wit thes types however i iconvenien fo th presen t cal the Typ F Hai (1949 examine 10 stoolfro person no sufferin fro enteriti necrotican an showe tha abou one o the containe heat-resistan Cl. welchii. H believe tha th stoolwer a fai sampl o thos o th populatio fro whic the wer obtained andeduce tha one-sixt o th inhabitant o Hambur durin th winte o 1947- hav carrie thi organism I wa no certain however tha th strainisolate fro norma person wer th sam h al respect a thos fro case oenteriti necroticans The wer heat-resistant bu fa les pathogeni fo animals(3 The outbreaks(a General observationsOu ow observation o foo poisonin suspecte t b du t Cl. welchiibegu i Jun 1946 whe on o th author at fo lunc a portio o home-madpor pi prepare a fe day befor an kep i th larder Abdomina pai andiarrhoe occurre durin th night A almos pur cultur o Cl. welchii waisolate fro th fe remainin scrap o pie I Jun 194 a outbrea o gastroenteriti h a factor cantee wa investigated Th illnesse wer shor andalthoug milde tha thos usuall see wit staphylococca foo poisoningresemble th bacteria toxi type Soo afterward a numbe o simila outbreakwer studied an i becam possibl t r

ecogniz a clinica an epidemiologicapattern whic ma b briefl describedClinical. Afte a incubatio perio o 8-2 hr. patient develo acutabdomina pai an diarrhoea nause an vomitin ar rare Pyrexia shiveringheadach an othe sign o infectio ar seldo present Th illnes i o shorduration—on da o less excep i elderl debilitate patient amongs who ii suspecte tha fata case ma hav occurredMedium of infection. Thi i almos invariabl a col o warmed-u mea dismad fro meat boiled braised steame o stewe fo 2- hr o th da befor i required an allowe t coo slowl overnight Roas mea i seldoincriminated Thos wh ea th contaminate mea dishe sa tha the arappetizin an mak n complain abou thei smel o taste Whe receive foexamination th mea varie h appearanc an smell som sample appeanormal other smel sou an sho sign o ga formationCausal organism. Direc smear o suspecte meat sho Gram-positiv bacillresemblin Cl. welchii. Sometime the ar presen h smal number only bu iothe organism ar no numerous thi finding i considere suspicious Direc KWWSVGRLRUJ6 3XEOLVKHGRQOLQHE\&DPEULGJH8QLYHUVLW\3UHVV 7 BETT C HOBB AN OTHEBanaerobi culture o bloo aga usuall sho a profus growt o non-haemolytiCl. welchii o typica colonia appearance aerobi plate ofte sho scant growto saprophyti organism only I Cl. welchii i isolate onl fro enrichmen anno fro direc culture som doub i cas o th diagnosi o Cl. welchii foopoisoningHeat-resistan Cl. welchii wit colonia appearanc an serologica reactionsimila t thos o th strain obtaine fro foo ar isolate fro a hig pro o faece fro patient an other a risk I contro group o person noa ris th proportio o faece yieldin heat-resistan Cl. welchii i low(b) A typical outbreakI Februar 1950 mil foo poisonin occurre i a schoo canteen affectinabou 27 o 47 children 1 o 4 schoo staf an 1 o 1 cantee staff Thshortes incubatio perio recorde wa 6 hr. an th longes 2 hr Mos o thpatient complaine o sudde diarrhoe an abdomina pain som fel nauseabu fe vomited n on complaine o headache pyrexi wa no recordedRecover wa rapi an mos o th childre wer bac a schoo nex day n case occurred Boile sal bee eate col wa though t b responsibleTh mea (Argentine) delivere i smal joint eac weighin 4- lb. wa kep ith larde til midday whe i wa place i lukewar water whic reacheboiling-poin i hal a hour an boile fo 3 hr Th joint wer the removefro th boiler pu o ename trays covere wit a tea-clot an place i th overnight nex da the wer slice an serve wit freshl mad gravyTw othe school ha bee supplie wit th sam batc o sal beef a on thbee wa eate freshl

cooke o th da o deliver withou il effect n informatio wa availabl fro th othe schoolAfte a earlie outbrea a th sam school th kitche staf ha bee instructet kee smal sample o eac prepare foodstuff a smal specime mea wa therefor availabl fo bacteriologica examination A direc microscopi smea o th bee showe occasiona Gram-positiv bacilli A Yeastre milk-aga pou platincubate aerobicall gav a colon coun o abou 150 pe gram. a surfac couno bloo aga b th Mile an Misr techniqu showe anaerobicall 130,00colonie pe gram Direc aerobi culture wer almos sterile wherea direc blooaga anaerobi culture showe a pur growt o Cl. welchii. Smal number o thiorganis wer als isolate fro enrichmen culture o a specime o haricobean i contac wit th mea o th plate Th brin use t pickl th meayielde a strai o Cl. welchii closel resemblin tha fro th meat Th resulto th bacteriologica examinatio o th person concerne ar show i Tabl 1Th detail o th investigatio ar give i Par II Ninetee sample o faecereceive fro patient connecte wit th outbrea al containe heat-resistanCl. welchii. O te repea sample take afte 1 days si wer stil positive ofou take afte 2 day on wa positive o si furthe repea sample take afte3 day on wa positive Th strain isolate fro mea an faece resembleeac othe closel i thei morphology cultura an serologica character an ithei toxi production Positiv stool wer als obtaine fro thre person wh KWWSVGRLRUJ6 3XEOLVKHGRQOLQHE\&DPEULGJH8QLYHUVLW\3UHVV Clostridiu welchi food poisoning 7ha eate th foo withou showin an symptom (a risk no ill) Fiv sampleo faece fro schoo childre wh ha no eate th foo wer negativ foCl. welchii, an fiv sample fro childre fro anothe schoo wer als negativ(person no a risk)Tabl 1 Results of examination for heat-resistant Clostridiu welchi of samplesof food and faeces connected with school canteen outbreak of food poisoningPropertie o Cl. welchiiNo Solubl antigenNo o positiv Provisionasample fo serologica /? S, e 9Descriptio o sampl examine Cl. welchii type a an i K A fi vCol boile bee 1 1 2 tr - -- + + +Harico bean 1 1 .....Brin 1 I .....FaecesPatient 1- day 1 1 2 tr o — — - o tr — + o + + + tr orRepea sample 1 day 1 6 2 tr — — — + + + tr orRepea sample 2 day 4 1 2 tr — — — + +Repea sample 3 day 6 1 . ..... a ris no il 3 3 2 tr — — — + o + + + tr o —Person no a ris 1 — . .....* Tub agglutinatio t titre t Strai died tr = traceI Octobe 194 a outbrea wit th sam character ha occurre a thischool du t sal bee tha ha bee allowe

t coo overnigh i th liqui iwhic i ha bee cooked A th sam tim joint fro th sam picklin tankrefrigerate afte cooking wer eate a tw othe school withou il effect Thioutbrea wa doubtfull ascribe t Proteus vulgaris, thoug th strai isolatefaile t affec tw volunteers(c Survey of other outbreaksFro Septembe 194 t Februar 195 twenty-thre outbreak o foo poisonini th Londo are wer investigated i whic th suspecte agen wa heatresistan Cl. welchii. Th mediu wa usuall pre-cooke mea an th histor oit preparatio followe a unifor pattern Th incubatio periods symptomsduratio o illness an bacteriolog resemble thos alread described Tabl 2give a lis o som o thes outbreaks includin a brie descriptio o th meadis an wher possibl th provisiona serologica typ o th Cl. welchii strainisolate fro foo an faecesAttentio i draw t specia feature o som o thes outbreaks I outbrea 2i a schoo canteen th hig proportio o victim amon th person a ris i tb noted I outbrea 4 i a smal factory th foo concerne wa a pasty Minc vegetables separatel cooke th previou day wer mixed covere witpastr an bake onl sufficientl t coo th pastry A coun o 4 millioCl. welchii pe gra wa obtaine fro th remain o th mea an vegetablfilling Th aerobi bacteria coun wa approximatel 2 millio pe gram thorganism bein mainl colifor bacilli I outbreak 1 2 3 an 1 n faece werreceived bu th epidemiologica pictur an th bacteriolog o th foo lef littldoub tha Cl. welchii wa th causativ agent I outbreak 7 8 1 an 1 nfoo wa received bu th epidemiologica picture an th hig percentag o KWWSVGRLRUJ6 3XEOLVKHGRQOLQHE\&DPEULGJH8QLYHUVLW\3UHVV GOTabl 2 Outbreaks of suspected Clostridiu welchi food poisoning occurring in the London Area, September 194 to January 1952showing meat dishes responsible and some results of serological typing of strains from food and faecesFoo Faece1234567891011121314151617Plac o outbreaArm camSchoo canteeFactor cantee canteeFactor canteeSchoo canteeSchoo canteeMunicipa canteeFactor canteeMunicipa canteeSchoo canteeSchoo canteeMunicipa canteeR.A.F canteeR.A.F canteeFamilMunicipa canteeYea194194194194194195195195195195195195195195195195195No aris8367252401412733014149410Noaffecte73134230541022173394318 Schoo cantee195 205Natur o samplReheate lamb boile previou daReheate steak stewe previou daLamb' tongues boile 3 hr beforBee an vegetabl pasty Bee received mince an boile previoudaReheate beef stewe previou daCol sal beef boile previou daPresume stoc fro mea cookepreviou day No receiveStea pi mea stewe previou dayNo receiveCol sal beef boile pre

viou daMutto pie mea boile previou daReheate beef steame previou dayNo receiveCol sal beef boile previou daStea pudding stewe previou dayNo receiveCol sal beef steame thre daybeforReheate steak stewe previou daReheate beef braise previou daMea pudding minc stewe previouday No receiveBee an por rissole mea roaste + No 1—tw day befor. =No receive o no examinedHeatresistanCl. welchii++++-1++++Provisionaserologicatyp3113No 1-213383'Noexamine1111361782421PatientNopositivfoCl. welchii1118361681421Provisionaserologicatyp2 (18)' 3(13No 1-13No 1- (125(6No 1-6(13(336Control(no a risk1 al negativ3 al negativ4 al negativ8 al negativ2 al negativexcep 1 (no1-6wHpw0Wtc0WWNo 1- KWWSVGRLRUJ6 3XEOLVKHGRQOLQHE\&DPEULGJH8QLYHUVLW\3UHVV Clostridiu welchi food poisoning 8positiv faece fro patient whe compare wit contro group no a ris let a fairl confiden diagnosi o Cl. welchii foo poisoning Tabl 2 show a compariso o th result obtaine fro th faece o patient wit thos o smalcontro group o person no a ris becaus the ha no eate th contaminatefood I schoo outbrea no 6 whe al o ninetee specimen o faece fropatient wer positive al te contro faece (fiv fro childre i a neighbourinschool wer negative I schoo outbrea no 7 wher eigh o te faece fropatient wer positive thre control wer negative I schoo outbrea no 11o seve specimen o faece fro patient si wer positive al eigh control wernegative Thes outbreaks 6 7 an 1 wit tw other ( an 13) yielde fifty-ninesample fro individua patients an o thes fifty-four (91-6% wer positivewherea fro forty-fiv control onl on positiv sampl wa obtaine (2- %)Durin th investigatio o variou outbreaks ou o 12 sample o faece fropatients 11 (89-9% wer positiv fo heat-resistan Cl. welchii; o 38 samplefro person no a ris onl twent (5- % wer positiv (se Tabl 3) O thirtsample o faece fro person wh ha eate foo contaminate wit Cl. welchiibu wh ha no bee il sixtee (53-3% wer positiveTabl 3 Heat-resistant Clostridiu welchi from stool samplesof various groups of personsGrou Descriptio1 Norma peopl2 Miscellaneou case o diarrhoe3 Dysenter (Sonn outbreak4 Foo poisonin othe tha Cl. welchii5 Ol peopl i hospita (health an witdiarrhoeaTota 38 2 5-6 Cl. welchii foo poisonin 12 11 89-I i clea tha heat-resistan strain o Cl. welchii ar foun muc mor frequentli th faece o person wh hav eate th contaminate food speciall i thehav symptoms tha i person no a risk Th bacteriolog o th organism idiscusse full i Par I i whic supportin evidenc fo th causativ rol o thorganis i produced includin th result o volunte

e experiments O threvolunteer wh swallowe cooke mea culture o strain isolate fro incriminate food on ha a shor acut illnes resemblin tha describe b victim oth outbreaks anothe ha a simila mil illnes an th thir ha triviasymptom tha almos escape noticeToward th en o 195 ther wer tw outbreaks th genera character owhic wer simila t thos o Cl. welchii foo poisoning Th predominanorganis isolate fro foo .an faece was however a strai o Cl. welchiiproducin /?-haemolysi o bloo agar Thi wa a characteristi Typ A straini resiste boilin fo 5 bu no 1 min. an faile t agglutinat wit seragains provisiona Type 1- o heat-resistan Cl. welchii (se Par II)I on outbrea col roas bee cooke th da befor i wa eate gav aJ Hygien 6Noexamine41485No13358PositivAo/2-23-7-15 KWWSVGRLRUJ6 3XEOLVKHGRQOLQHE\&DPEULGJH8QLYHUVLW\3UHVV 8 BETT C HOBB AN OTHEBanaerobi surfac colon coun o 4- millio /Miaemolyti Cl. welchii pe gramth aerobi colon coun wa 190,00 pe gram A simila strai o Cl. welchii waisolate i larg number b direc bloo aga cultur fro al o eleye patientsfaeces Simila culture fro fiftee person no a ris yielde fou strain o/?-haemolyti Cl. welchii. I th secon outbrea braise bee cooke 3 day befori wa use gav a anaerobi surfac colon coun o 1 millio /?-haemolytiCl. welchii pe gra an a aerobi colon coun o 35,00 pe gram A similastrai o Cl. welchii wa obtaine fro al o eigh faeca sample fro patientsA th strain wer onl feebl heat-resistant—i n instanc wer th organismisolate fro faece steame fo 1 hr.—i i though likel tha th mea wacontaminate afte cooking Foo an faece fro bot outbreak faile t yielan o th usua food-poisonin organisms(4 The source of infectionTh reservoir o heat-resistan Cl. welchii ar no ye full known Mea mab contaminate befor cookin fro a leas tw differen sources (1 humanfro th hand o cooks o butcher an other handlin carcase i th slaughterhouse th mea marke an th retai shop (2 animal fro th invasio omuscle b intestina organism durin lif an afte death fro faeca matte oth floor o th slaughter-house o fro rats mic o othe animals Wheopportunitie arose therefore faece wer examine fro group o person an variou animals A fe batche o flies wer als examinedHuman. Th carrie rat o heat-resistan Cl. welchii amongs th normapopulation an amongs thos wh ar sufferin fro diarrhoea disease no wit Cl. welchii appear t var slightl wit differen groupsTabl 3 give a summar o th result obtaine fro th examinatio fo heatresistan Cl. welchii o stoo sample fro person presume no a ris froCl. welchii foo poisoningTh first gro

u o sample wa collecte fro forty-fiv health childre anadult no a ris i school an factorie wher outbreak o suspecte Cl. welchiifoo poisonin ha occurred 2- % o sample wer positiveTh 14 sample o faece i th secon group o whic 2- % wer positivecam fro a publi healt routin laboratory The ha bee submitte foinvestigatio fro case o diarrhoe diagnose a dysentery ' pyrexi o unknoworigin' o on o th enteri diseases bu no suspecte foo poisoning Th eightsample i th thir group showin 3-8 o positives wer al fro case oSonn dysentery Th fourt grou o sixty-fiv samples i whic 7- % werpositive wer relate t person sufferin fro foo poisonin no though t bdu t Cl. welchii. I vie o th highe percentag o positive i thi groucompare wit th othe thre group o person no a ris i seem likel tha fe o th case o foo poisonin ma hav i fac bee cause b Cl. welchii.Afte a outbrea o suspecte Cl. welchii foo poisonin amon ol peopl ia hospita th fifth grou o faece wer examine fro fifty-three ol peopl ineighbourin hospital sai t b fre fro foo poisoning Fro on hospita two fourtee sample fro health person an fro th othe thre o twenty-on KWWSVGRLRUJ6 3XEOLVKHGRQOLQHE\&DPEULGJH8QLYHUVLW\3UHVV Clostridiu welchi food poisoning 8sample fro health person an thre o eightee sample fro thos witdiarrhoe wer foun t b positive Therefor eigh ou o fifty-three (15- %sample o faece i thi grou wer positive Thi figure, highe tha tha o thothe groups ma indicat tha heat-resistan Cl. welchii i particularl prevaleni elderly person i hospital speciall thos wit diarrhoea I wa no possiblt sa whethe th presence o th organis wa relate t th onse o diarrhoeaTh excretio o Cl. welchii usuall stop 1- week afte th contaminate fooha bee eaten i i known however tha th organis ma persis fo considerabl longe periods I on instanc a coo produce positiv stool fo sommonths Furthe investigation ar neede alon thes linesSewage. Sewag swabs a designe b Moor (19486) eac samplin th sewago sevent houses wer examine fo Cl. welchii. Thes swabs take primaril apar o anothe investigation wer obtaine fro variou part o th countryTh result ar show i Tabl 4Tabl 4 Results of examination for heat-resistant Clostridiu welchi ofbatches of sewage swabsSewag swabBatc12345678OrigiColne WiltWallingford BerkWantage BerkBodmin CornwalMarlow Berk anEastern BerkBeccles SuffolHoole CheshirPrudhoe NorthumberlantNoexamine12211168PositiviNo61117068fo Cl. welchii— */40-62-51-76-46-0100-100-ProvisionaserologicatypNo 1-3- (5No 1- (132(12 an 3(13(1No 1-6(121(26(4No 1- (4No 1-.—6(1No 1- (53(

16(2No 1- (5Tota 12 7 56-Figure i bracket indicat numbe o strainsAnimals. Th carrie rat amon liv animals includin rat an mice an i livan recentl slaughtere cattl wa investigated Th result ar give i Tabl 5Cattl faece wer obtaine fro a far an tw slaughter-houses O thseventy-si sample o pi faeces fifty-two wer obtaine fro th feedin passageservin pen unde condition i whic i wa no possibl t b sur tha contaminatio ha no sprea fro pe t pen O twelv sample take fro slaughterhous pen fou wer positiv an o twelv sample fro individua pig non wapositive6- KWWSVGRLRUJ6 3XEOLVKHGRQOLQHE\&DPEULGJH8QLYHUVLW\3UHVV 8 BETT C HOBB AN OTHERTh ra faece wer collecte b loca authorit officer durin th proces oexterminationTabl 5 Heat-resistant Clostridiu welchi from animalsSampleFaecePigRat an micCattlRa meaPorBeeVeaLamNoexamine74115571iNo1621110PositivAo/18-14-1-20-2414-0T investigat th occurrenc o heat-resistan Cl. welchii i ra meat smalsample wer collecte fro variou sources Afte direc anaerobi culture wermad o bloo agar th portion o mea wer shake wit brot i screw-cappejar an steame fo 1 hr Th lid tightene an th jar coole an incubateaerobicall overnight nex da thes mea broth wer plate ou aerobicallan anaerobically Th result wer groupe accordin t th origi o th samplan the ar show i Tabl 5 Som o th group wer small unfortunatelybecaus i wa difficul t obtai ratione meatI wil b see tha th highes percentag o positive wa foun i sample obee an pork Th fourtee sample o lam wer al negativeFlies. Heat-resistan Cl. welchii wa isolate fro batche o blowflie collectefro differen sources a hospital a butcher' shop a fried-fis shop a slaughterhous an a refus sortin depot Th majorit o flies wer o th genu Lucilia(greenbottles bu som Calliphora an Phormia (bluebottles wer als includedthe wer remove fro th trap som hour befor examinationTw collection wer mad fro eac site th flies bein attracte t th trapb live bait I th first collectio eac batc consiste o severa hundre fliessom o whic ha touche th live bai whic migh possibl hav bee thsourc o infection Th secon collectio wa therefor mad usin sterilize trapi suc a wa tha th flies coul no com i contac wit th bait Onl smalcatche wer obtained rangin fro tw t fifteen flies, but nevertheless Cl.welchii wa isolate fro ever batch Non wa isolate fro a sampl o livebai an i seem reasonabl t assum tha th organis wa carrie b th fliesi th wil stateTh strain o Cl. welchii wer isolate b treatin a suspensio o grounflies i Ringe solutio b th metho use fo faeces Som strain faile tagglutinat wi

t th serologica typin ser 1-8 other appeare t belon tth provisiona type 3 an 7I seem likel tha th blowfl ma pla a importan rol i th sprea oCl. welchii especiall a i breed an feed o mea an othe anima products KWWSVGRLRUJ6 3XEOLVKHGRQOLQHE\&DPEULGJH8QLYHUVLW\3UHVV Clostridiu welchi food poisoning 8(5 CommentTh outbreak o foo poisonin describe abov wer almos al cause b meatha ha bee cooke an allowe t coo slowl overnight condition verfavourabl fo th multiplicatio o survivin anaerobes Fro 8 t 2 hr afteth foo wa eaten symptom appeare an a larg proportio o th person aris wer usuall affected Th sam mil brie typ o illness characterize bcoli an diarrhoe withou vomiting wa observe i al th outbreaks Heatresistan strain o Cl. welchii predominate i th foo an wer frequentl preseni larg numbers A organis wit th sam character wa isolate fro thfaece o abou 9 % o patient an othe person a ris an wa foun ver muc frequentl (averag 5-2% i th faece o norma person o person sufferinfro diarrhoea illnesse o differen origin Salmonell organism wer no isolatean th othe recognize bacteria cause o foo poisonin wer virtuall excludedHeat-resistan Cl. welchii occur i th faece o pig an cattle an o rat anmice i wa als foun i variou marke sample o pork bee an veal an iflies. Thes ar possibl source o infection an i i clea tha th method opreparatio o th food afforde ampl opportunit fo bacteria multiplicationonc contaminatio ha occurred Th epidemiologica evidenc i thu compatibl th vie tha thes outbreak o foo poisonin wer cause b heat-resistanCl. welchii.PAR I BACTERIOLOG(1 General characters of the heat-resistant strains of Clostridiu welchi(a) MorphologyTh morpholog o th organis i direc smears apar fro sligh variation isize i typica o Cl. welchii a describe i an text-book Spore hav rarel beesee i smear fro foodstuffs althoug structure resemblin spore ar seeoccasionall i direc smear fro colonies Furthe wor o th sporulatio o welchii wil b describe later(b) Colonial appearanceTh colonie o bloo aga diffe i appearanc an haemolysi fro thos usuallgive b Cl. welchii Typ A O primar isolatio the ar non-haemolytic aftea fe day a fain haemolysi ma develop Th colonie ar approximatel2- mm i diamete afte 1 hr The ar usuall smoot wit a entir edgebu the ma b opaqu an slightl raise o umbonat i th centr wit a flattranslucen peripher showin radia striations O first isolatio the ma bsmall rough an flat wit a irregula edg resemblin a vin leafTh organism isolate fro th suspecte foodstuff i th first outbrea t binvestigate wer al o th roug

typ bu thes hav rarel bee see since Ii possibl tha som differenc i th bloo aga mediu use a tha tim waresponsibl fo th roug colonies Zeissle & Rassfeld-Sternber (1949 describeth appearanc o roug colonie o Cl. welchii Typ F an note ho the differefro the normal smooth colonies of Type A strains. A further variation in thecolonia appearanc wil b describe late i th sectio dealin wit inducesporulation KWWSVGRLRUJ6 3XEOLVKHGRQOLQHE\&DPEULGJH8QLYHUVLW\3UHVV 8 BETT C HOBB AN OTHER(c Biochemical reactionsTh fermentatio reaction o thes strain ar simila t thos o an othe typo Cl. welchii. Aci an ga ar produce fro glucose maltose lactos ansaccharos bu no fro mannito o salicin Aci an clo ar usuall forme ilitmu milk O man hundred o strain examine al bu tw gav a positivNagle reactio inhibite b Typ A antitoxin(d) Heat-resistance and sporulationI wa know tha som o th foodstuff examine fro suspecte outbreakan fro whic w isolate heat-resistan Cl. welchii ha bee boile fo 2- hro eve longe befor bein lef overnigh i th kitche o larder I i i assumetha th organis wa alread presen o th mea befor i wa cooked the itdevelopmen afte cookin mus depen o th surviva o sporesTh power o resistanc t hea o Cl. welchii var fro 1 min a 98 C(Dunham 1897) l±hr a 100 C (Rodella 1910 vo Hibler 1906) t 1-4hr a100 C (Zeissle & Rassfeld-Sternberg 1949)On o th strain isolate durin th presen wor wa grow i a larg volumo cooke mea fo 4 hr a 37 C the gentl boile t imitat th stewin omea i a canteen I survive thi treatmen fo 5 hr bu no fo 6 hr I anotheexperimen culture seale i ampoule survive 4 hr bu no 5 hr submersio ia boilin -wate bath A overnigh cooke mea cultur o anothe strai survivesteamin fo 1 hr bu no autoclavin a 1 lb (117 C. fo 1 minI wa observe tha som strain retaine thei heat-resistanc throug severasubculture an storag i th refrigerato fo man months Wit other i waimpossibl t demonstrat heat-resistanc eve afte th first subcultur i cookemea mediumI ha bee note tha heat-resistan Cl. welchii coul b readil isolate frosample o faece whic ha bee steame fo 1 hr. wherea i wa rarel possiblt isolat the fro foodstuff b th sam methodWil (1898 observe tha spore i stool wer mor resistan t hea tha thosi cultures Simond (1915 observe tha th sporulatio o Cl. welchii i suspension o faece ha a importan bearin o th relationshi o thi organis intestina disturbances H commente o th variatio i th readines wit differen strain o Cl. welchii forme spores an suggeste tha mosimportan amon th numerou factor tha influence sporulatio wer threactio o t

h mediu i th presenc o absenc o fermentabl carbohydratan th temperature H observe tha sporulatio occurre promptl anabundantl i pur culture an i sterilize alkalin an neutral bu no acidsuspension o faeces Al th evidenc suggest tha heat-resistanc i dependeno th formatio o sporesI orde t find ou th approximat proportio o spore t vegetativ forman th effec o hea o mea brot culture o Cl. welchii, strai 298 (Lab no.)incubate fo 5 2 an 4 hr a 37 C th supernatan fluid wa divide intsevera parts Eac par wa heate fo 1 min a variou temperature ranginfro 5 t 100 C Count wer mad o a unheate contro an o eac heate KWWSVGRLRUJ6 3XEOLVKHGRQOLQHE\&DPEULGJH8QLYHUVLW\3UHVV Clostridiu welchi food poisoning 8sampl immediatel afte remova fro th wate bath Th result ar show iTabl 6 the indicate tha i youn culture o 5 hr th vegetativ cell werkille a 50-60 C an tha n spore wer formed I 2 an 4 hr culture thvegetativ cell wer destroye a 60-70 C O thre 2 hr culture tw showeapproximat proportion o spore t vegetativ form o 1 t 37,00 an 1 t8800 whil i th thir cultur n spore wer demonstrated Th abilit oCl. welchii, strai 2985 t produc spore i cooke mea mediu afte 2 hrTabl 6 Effect of heat on 5 2 and 4 hr. cultures of Clostridiu welchi and theapproximate proportion of spores to vegetative forms in these culturesSurfac plat coun (anaerobic pe ml o cultur Approximatr * , proportio oAg o Afte heatin fo 1 min a variou temperature ( C. spore tcultur Unheate , « , vegetativ(hr. contro 5 6 7 8 9 10 form5 2- xlO6 7xlO6 50 50 50 Ni24(1 40xl06 22xlO6 8,50 50 50 50 50 Ni24(2 13 xlO6 — 3- xlO6 — — — 3,50 1/37,0024(3 10 xlO6 — — — — — 12,00 1/8,8048(1 7 xlO6 5xlO6 — 25,00 15,00 38,00 110,00 1/6448(2 6 xlO6 — 46,00 — — — 15,00 1/4,50appear t b variable I th tw 4 hr cultures spore wer presen i thproportion o 1 t 64 an 1 t 450 vegetativ formsThes fe experiment suggeste th reaso fo variatio i th surviva oheat-resistan Cl. welchii. The als demonstrate th presenc o spore i culturewhic appeare t b fre fro spore whe staine films wer examinedVariou method wer use i a attemp t increas th formatio o sporesmostl withou success N spore coul b see i films fro culture i cookemea mediu wit 0-005 manganes sulphat adde o i Brewer' thioglycollat medium A slid cel techniqu usin a slopp nutrien aga containin0- % thioglycollat an als plai nutrien aga gav promisin bu unreproducibl results Th mediu wa melted allowe t cool an inoculate wita 4 hr cultur o heat-resistan Cl.

welchii; a smal amoun wa pipette o ta steril slid an covere wit a steril cover-slip an th cultur incubateaerobicall i a Petr dish O tw occasions a least numerou spore werdemonstrate whic presente a unforgettabl pictur unde a phase-contrasmicroscope Man o th spore appeare unusuall larg an som wer containei grotesqu an swolle vegetativ cells A ver hig proportio o th bacteriacell wer arrange i palisade an containe larg ova subtermina o almoscentra sporesTh onl metho whic ha s fa consistentl encourage th sporulatio othi organis wa evolve fro th followin procedure 10 ml o a 4 hrnutrien brot cultur o Cl. welchii, strai 2985 wer centrifuge an th deposiwa washe twic an resuspende i distille water Thi suspensio wa lef oth benc fo 6 weeks durin thi tim n spore wer see i staine smear mada weekl intervals A fe ml o th 6-wee suspensio wer incubate i cookemea mediu an faile t gro i 2 hr. 0- % o steril starc solutio waadde an th cultur re-incubated Afte 5 hr th formatio o ga wa noticedan a staine smea o th cultur showe occasiona spores Th followin day KWWSVGRLRUJ6 3XEOLVKHGRQOLQHE\&DPEULGJH8QLYHUVLW\3UHVV 8 BETT C HOBB AN OTHEKa bloo aga plat inoculate fro thi 5 hr cultur an incubate anaerobicallshowe approximatel 4 % o norma colonies th remainde wer eithe smalan 'poache egg o 'pin-point i appearance Film wer mad fro thesthre type o colony Thos wit a typica appearanc showe n spore whil thothers 'poache egg an 'pi point' consiste almos entirel o fre spore ansporin bacilli bacill fro thes atypica colonie wer agglutinate b th antiseru t th paren strain Ther wa a grea variatio i th siz o th centraan subtermina spores som wer large ova an thicke tha th bacilli otherwer small Thes colonie showe involutio forms lon filaments, club-shapean shor coccoi forms an som larg swolle bacill stainin feebly Afte two thre subculture throug cooke mea mediu o o bloo aga n spore werfound th atypica colonie reverte t th norma appearanc an showe nonsporin bacill only Whe th origina starc cooke mea cultur wa incubatefo 2 hr befor subcultur n atypica colonie wer foundThi metho o obtainin spore fro Cl. welchii, strai 2985 wa repeatesevera time wit simila results th metho ha no ye bee use fo othe strainsTh germinatio tim o th spore varie a grea deal Th additio o 0- %starc t th cooke mea mediu speede u th growth whic wa noticeablafte 6-2 hr. withou starc growt di no occu fo 4 hr Onc germinatioha commence th spore gre rapidl int non-sporin bacilli Th cooke meacultur wa therefor examine frequentl an plate ou a soo a growt

waobservedI orde t determin th growt rat o spore o heat-resistan Cl. welchii adifferen temperature i a mediu o cooke meat thre lot o 10 ml o cookemea wer eac inoculate wit 3 ml o a spor suspensio (strai 2985 idistille wate an steame fo hal a hou t destro an vegetativ formsAfte coolin fo hal a hou th inoculate mea medi wer incubate on eaca 3 an 22 C an th thir wa place i th refrigerator Viabl count obloo aga incubate anaerobicall wer carrie ou afte 2 4 6 2 an 2 hrWit a inoculu o les tha fifty spore pe ml ther wa a colon coun o45,00 pe ml afte 6 hr. an 4 millio pe ml afte 2 hr a 37 C Th couna 22°C wa 25,00 pe ml afte 2 hr I th refrigerato ther wa n growti 4 hr I ma b assumed therefore tha a fe spore survivin i cookemeat wil b sufficien t produc a growt o bacill i th mea i lef overnighi a war room(2 Method of isolationTh organis i strictl anaerobi an grow rapidl i brot a 37 C. o blooagar colonie ar wel develope afte 1 hr I ma b isolate fro foodstuffb direc overnigh anaerobi cultur o bloo agarA profuse almos pure cultur fro food i easil recognizable bu amongsa heav growt o facultativ anaerobi organism th colon o non-haemolytiCl. welchii i easil misse o mistake fo som othe organism Isolatio isimplifie i afte incubatio th anaerobi plat i lef o th benc fo 2 hr. wheth aerobi organism wil develo an th anaerobe ca b picke of mor easilyCl. welchii ma als b isolate afte enrichmen overnigh throug cooke mea KWWSVGRLRUJ6 3XEOLVKHGRQOLQHE\&DPEULGJH8QLYHUVLW\3UHVV Clostridiu welchi food poisoning 8mediu o merel b incubatio o th suspecte mea i quarter-strengt Ringesolution I thi metho i used however aerobi organism ma outgro thanaerobes an moreove th presenc o Cl. welchii i enrichmen culture onl ino regarde a o th sam significanc a growt o th organism o direc platesA anaerobi surfac colon coun o bloo aga carrie ou b mean o th Milean Misr techniqu wil giv a usefu estimatio o th proportio o anaerobepresen i th foodstuff i th foo i heavil contaminate wit aerobi organismth valu o suc a coun ma b doubtful Attempt t mak us o heat fo th isolatio o th organis fro food b steamin i brot fo1 hr followe b anaerobi incubatio an subcultur th nex da hav usuallfailed Thi suggest tha th anaerobe i pre-cooke mea ma b recentlgerminate spores tha is activel multiplyin youn organism whic hav noye forme spores T isolat th organis fro stools however goo us ma bmad o it heat-resistanc (Hain 1949) A smal portio o th stool abou thsiz o a pea i steame i brot fo 1 hr. th heate suspensio i incubateanaerobicall overnigh a

n plate o t bloo agar fro positiv sample aalmos pur growt o th organis ma b obtained Th regularit wit whicheat-resistan Cl. welchii ma b isolate fro stoo sample i thi wa suggesttha th organis spore readil durin it residenc i th intestine Direcplatin o faece o t bloo aga withou preliminar hea treatmen wa nosatisfactor fo th isolatio o non-haemolyti Cl. welchii, a it presenc mab obscure b a heav growt o colifor bacilli Fo th isolatio o th /?-haemolyti Typ A strain fro faece direc platin mus b use becaus the d noappea t surviv heat-treatment O Wilso an Blai mediu a use fo thisolatio o enteri pathogen tw strain o Cl. welchii fro foo faile t growwherea the gre o th sam mediu adapte fo th isolatio o Cl. welchii frowater Proteus an som othe organism als gro o thi medium i wa thereforconsidere t b o littl valu fo th direc cultur o Cl. welchii fro faecesA compariso o th isolatio o heat-resistan Cl. welchii fro stool an frorecta swab ha show tha stoo sample giv a highe percentag o positiv I on hospita outbrea o foo poisonin amon elderl person 7 % otwent faece sample wer positive bu onl 4 % o sixty-on comparabl rectaswabs(3 ToxicologyToxin production. Organisms obtaine i pur cultur wer grow fo 5 hr i1 % glucos brot previousl steame fo \ hr an cooled compariso wit othemedi showe tha th on use wa adequat fo estimatio o toxigenicity Thculture wer the filtered throug Seitz-sterilizin pad an th filtrates examinefor toxin a soo a possibl b a metho publishe elsewher (Oakle & Warrack1953)Results. N ne toxin wer detected I genera th toxigeni powe o foodpoisonin strain o C welchii appeare t b slight Trace o a-toxin wer almosinvariabl produced bu th concentratio i nitrate neve exceede th equivalen o on a-unit an wa usuall obviousl fa less Almos al strain examineproduce som deoxyribonuclease a d almos al strain o Cl. welchii o al type KWWSVGRLRUJ6 3XEOLVKHGRQOLQHE\&DPEULGJH8QLYHUVLW\3UHVV 9 BETT C HOBB AN OTHERs fa examined N /?- S-, e- o t-toxi wa detecte i an filtrates, no di anheat-resistan strain produc 0-toxin eve thoug th mediu speciall favoureit production othe type o Cl. welchii includin strain tha wer no heatresistan readil produce i thi medium Th strain varie greatlamon themselve i K (collagenase an /i (hyaluronidase production A fainumbe produce K i quantitie sufficien t attac collage pape an foscreenin test t b possible n strai produce A-antigen Man strain affecteazocoll bu insufficientl fo screenin tests I a strai produce hyaluronidasa al i usuall produce a grea deal th result showe ei

the hyaluronidasactivit a a dilutio o 1 i 512 o n activit a all Repea filtrates fro th strai showe littl o n variatio i antigeni make-up Th genera pattero toxi productio wa thereforea / S e 6 i K A /i v+ _ _ _ _ _ +or - + + +or +an thi gav som assistanc i comparin organism isolate durin a epidemicThu i on sever epidemi o foo poisonin i a institution stoo sample werobtaine fro man person wh ha eate th suspecte food al yielde heatresistan Gl. welchii producin larg amount o hyaluronidase sample froanothe perso wh ha no eate th foo yielde a heat-resistan Cl. welchii,bu thi strai di no produc hyaluronidase Sometimes a i th epidemireferre t i Tabl 1 organism agreein i al respects cultural toxicologicaan serological wer isolate fro food fro sic person an fro person aris wh wer no ill wherea n suc organis coul b isolate fro thos whha no eate th foodClassification of heat-resistant Cl welchii Wit th exceptio o Cl. welchiiTyp F Gl. welchii type ar differentiate fro on anothe b thei abilit tproduc variou toxin (Wilsdon 1931 1932-3 Glenny Barr Llewellyn-JonesDallin & Ross 1933 Oakley 1949) I practice th letha toxin ar give morweigh tha th other i classification s tha five o th si type ar determineb thei capacit t produc on o mor o these—a /? e i. Thus i a straiproduce a a it onl letha toxi i i groupe i Typ A i i produce onla an /? o / only i Typ C i a an e o e only i Typ D; i i produce a, fian e o / an e only i i place i Typ B Typ E produce a an i. Typ F produce / an trace o a woul b classe a Typ C o thi basis budiffer fro thi typ i th marke heat-resistanc o it spore (Zeissle &Rassfeld-Sternberg 1949) Thi metho work fairl wel o th whole especiallwhe i i applie t recentl isolate strains bu wil clearl brea dow i thstrai undergoe an degre o degradation Los o capacit t produc / o e iknow i Typ B an los o / productio i Typ C strains los o capacit tproduc e i suspecte i Typ D strains Examinatio o strain fo subsidiar e.g 8, K, A /i, ma giv usefu indication o th typ fro whic degradeform hav bee derivedTh strain describe i thi agre ver wel wit Typ A strains frowhic the diffe onl i thei heat-resistance Thei feebl a-toxigenicity thei KWWSVGRLRUJ6 3XEOLVKHGRQOLQHE\&DPEULGJH8QLYHUVLW\3UHVV Clostridiu welchi food poisoning 9failur t produc 6, an thei variabl hyaluronidas productio ar n ba tthei inclusio i Typ A fo Evan (1945 ha show tha typica A strain arhighl variabl i al thes respects Fiv ou o thirt suc strain studie bhi produce onl th equivalen o 0-0 uni a-antitoxi pe ml Te ou o thirtproduce n

detectabl 6; twenty-thre ou o thirt n hyaluronidase I i clearlconvenien t retai th food-poisonin strains excep fo Typ F i Typ A untifurthe researc make i possibl t subdivid Typ A o consisten grouping ocharactersAntitoxins in the sera of convalescents. I on epidemi w wer abl t examinser fro a numbe o patient an staff Thes ser wer examine b conventiona method fo Cl. welchii oc, / an e-antitoxins I n cas wa ft- o e-antitoxidetected bu a surprisingl larg numbe o patient (seventee ou o thirty-twoan staf (si ou o eleven possesse detectabl amount o Cl. welchii a-antitoxi(mor tha 0-00 unit) Th distributio contrast markedl wit th onl othecomparabl se o observations tha o Glenny (unpublished) wh foun onlon perso wit detectabl a-antitoxi i 20 examined (Bower Mengl & Paxso(1938 use a metho whic preclude th estimatio o les tha 1 uni Cl. welchiia-antitoxi pe ml Al o Glenny' samples an al o ours wer fa belo thi A Cl. welchii a-toxi i a poo antigen i seem a leas likel tha thpatient an staf o thi institutio ha ha considerabl experienc o it(4 SerologySinc strain o Cl. welchii fro variou source canno b satisfactoril differentiate fo epidemiologica purpose b thei toxicologica an biochemica propertiesth possibilit o differentiatio b serologica method wa investigatedHenderso (1940 foun a wid diversit i th specificit o th somati antigeno th classica Typ A strain o Cl. welchii whic appeare t b strai specificantigen o Type B C an D however possesse similarities Typ B strain witon exceptio share a identica O-antigen wherea Typ D strain showe i specificit an thirtee strain wer place i seve groups Cros Oreaction betwee th variou type wer negligibl o absent I additio t thheat-stabl O-antige Henderso demonstrate a heat-labil L somati componentH wa no able however t detec L antige i Cl. welchii Typ A o C strainsbu onl i thos type ( an D tha produce e-toxin eve her i wa nopresen i al strainsBacteria suspension wer prepare accordin t th method describe bHenderso (1940) The consiste o culture grow overnigh a 37 C i glucosbrot (steame an coole befor inoculation) Th organism wer twic washe resuspende i distille water Fo use suspension wer steame fo 1 hr o3 successiv day ( suspensions o treate wit 0- % formali fo 2 daysAntiser wer prepare b th intravenou inoculatio o rabbit wit si successiv increasin dose o bacteria suspensio a interval o 2- days Formolizesuspension wer foun t produc a bette agglutini respons tha heate i which moreover som antigeni constituen migh b damaged KWWSVGRLRUJ6 3XEOLVKHGRQOLQHE\&DPEULGJH8QLYHUVL

W\3UHVV 9 BETT C HOBB AN OTHERThe were therefore use throughou th work althoug i wa realize tha thful antigeni relatio betwee th strain migh no b revealed Th immediatai wa t obtai a clear-cu metho o typ differentiatio fo epidemiologicapurpose only N difference betwee formolize an heate suspension wershow b absorptio tests Simila sera althoug o lowe titre hav beeproduce b intravenou injectio o strain int horsesTube-agglutinatio test wer mad b th standar techniqu fo Salmonellatyphi V agglutinatio tests Firs reading wer mad afte 4 hr i th wate bata 43—45 C an final reading afte th tube ha bee kep fo 1 hr i th colroo an 2 hr a roo temperatureSlide-agglutinatio test whic prove usefu fo rapi routin work wer donwit ser suitabl diluted usually withi th rang 1/ t 1/25 Positiv strainagglutinate immediately Strain no reactin i slid test wer neve positivi tub tests an non-specifi agglutinatio reaction o th slid wer noobservedTabl 7 Comparison of the serology and toxicology of heat-resistant Clostridiuwelchi isolated from food and faeces connected with different outbreaks of foodpoisoning and used for the production of serological typing seraProvisiona Solubl antigenserologicaLab no Sourc typ a /? 8, e, 6, K A /i van (3702/4 Stewe stea 1 tr - - - + + + sl.tr2985/5 Boile sal bee 1 tr - - - - si tr281/5 Boile sal bee 2 tr - -- + +3958/5 Faece 3 + - + - - +3653/5 Boile sal bee 3 f tr - - - + + tr1690/5 Faece 4 tr - - - + + + si tr4322/4 Mea past 4 tr - + - + + +166/5 Faece 5 tr - tr3756/5 Faece 6 v si tr - - - si tr1826/5 Steame lam 7 v f tr - - - - f tr2204/5 Ste 8 f tr - -- + + +tr = trace f = faint v si = ver slightResults. Table 7 an 8 sho th origi o th heat-resistan strain o Cl. welchiiselecte fo serologica study thei toxicologica propertie an antigeni crossreactions O th eigh type s fa identifie an provisionall designate 1-8Type 1 2 an 6- sho n serologica cross-reaction wit othe types Type 3- a smal grou wit som antigeni overla bu wer show b cross-absorptiotest no t b identicalTh relatio betwee thes heat-resistan strain o Cl. welchii an othe typeo th organis wa investigate b tube-agglutinatio tests O seventy-threstrain o Typ A teste agains antiser o Type 1-6 five wer agglutinate bTyp 2 an tw b Typ 3 al o thos teste agains antiser o Type 7 an 8gav negativ resultsFiv ser prepare fro Typ A strain an kindl supplie b D Hendersodi no agglutinat an o th eigh type strain o heat-resistan Cl. welchii. On KWWSVGRLRUJ6 3XEOLVKHGRQOLQHE\&DPEULGJH8QLYHUVLW\3UHVV Clostridiu welchi food po

isoning 9recentl isolate strain however ha bee show t agglutinat wit on o thfive typ A antisera Thes result indicat tha antigeni constituent ma bshare b heat-resistan an othe Typ A strain o Cl. welchii.O si strain o Cl. welchii Typ F connecte wit th outbrea reporte bZeissle & Rassfeld-Sternber (1949) non agglutinate wit th provisionatypin ser 1-6Tabl 8 Tube agglutination of the provisional serological type strains of heat-resistant Clostridiu welchi (unabsorbed sera)Antiser, *� Suspension 1 3 4 5 6 7 81 1/320 _______2 — 1/160 ______3 — — 1/320 1/40 1/160 — — —4 — — 1/40 1/40 1/10 — — —5 — — 1/80 — 1/160 — — —6 _____ 1/40 — —7 ______ 1/160 —8 _______ 1/160— = N agglutinatio eithe wit a 1/5 dilutio o seru i th tub o wit undiluteseru o th slide— = Tub agglutinatio wit seru dilute 1/20 an agglutinate wit undilute bu no1/ seru o th slideO eleve strain als obtaine fro German an sai t b heat-resistanstrain isolate fro norma person thre agglutinate wit th Typ 3 antiseran tw wit Typ 1 antiserum Non o the produce /?-toxin I i possibltha a leas som o thes strain wer simila t th heat-resistan type oCl. welchii describe i thi paperSingl strain o Cl. welchii Type B- fro th Nationa Collectio o TypCulture an fro th Wellcom Cultur Collectio faile t agglutinat witantiser 1-6Tabl 9 show th serologica typ an toxicologica propertie o som strainisolate fro foo an faece i som o th outbreak o foo poisoning I wilb see tha i an on outbrea th organism isolate fro th foo an froth faece o th victim belonge t th sam serologica an toxicologica typealthoug serologicall identica strain isolate fro differen epidemic variei thei hyaluronidas production(5 Attempts to demonstrate pathogenicity(a) Volunteer experimentsTh strai isolate fro mince bee i th firs outbrea studie i Jun 1947wa grow overnigh i cooke mea medium 3 ml o th supernatan flui wertake i mil b a voluntee withou il effectTh strai (Lab no 3702 isolate fro th stea i outbrea 2 (Tabl 2 wagrow i mea brot an give t tw monkey i thei food On monke ata larg amoun an produce on loos stoo som hour later th othe at littl KWWSVGRLRUJ6 3XEOLVKHGRQOLQHE\&DPEULGJH8QLYHUVLW\3UHVV 9 BETT C HOBB AN OTHERan appeare unaffected A 2 hr cooke mea cultur (2 ml. wa the take ba volunteer wh ha a sligh attac o pai an diarrhoe 1 hr later a furthespas occurre 2\ hr later Thi mil reactio i on subjec migh b regardeb som a a psychologica respons onlyFurthe voluntee e

xperiment wer mad wit th sam strain A a preliminarprecautio larg volume o th fluid portio o cooke mea culture wer mixewit potat an offere t tw monkeys on o whic at a larg amount th othemuc less Th animal showe n sign o illness bu a profus growt o heatresistan Cl. welchii, persistin fo 8 day i th monke tha ha eate mos foodwa obtaine fro recta swabs Swab take fro bot monkey befor thexperimen wer negativ fo Cl. welchii.Tabl 9 Comparison of serology and toxin production of heat-resistant Clostridiuwelchi isolated from six outbreaks of food poisoningHeat-resistan Cl. welchiiSolubl antigenProvisiona , *• ^serologica a /? 8, e 6, K A / vSourc typ an iBoile sal bee 2 tr - -- + +Faece (19/1 positive 2 tr - __ + +Boile sal bee 3 tr — — — + + + —Faece (8/ positive 3 tr — — — + + + —Faece (13/1 positive 1 tr — ——+++Boile sal bee 1 tr — — * — — trFaece (6/ positive 1 tr — — * — — trFaece (12/1 positive 6 tr — — — — trFaece (8/1 positive 3 tr — — — — trtr = trace — * affecte azocoll to wea t testFou persons tw o eac sex volunteere t tak par i th experiment Tw18-2 hr culture i cooke mea (10-1 ml. wer used an tw simila uninoculate tube o th sam medium A independen observe allocate th bottles tha on woma (A) an on ma (C receive th cultures th other (B an D)th contro tubes Th shaken-u tube looke alik an i wa afterward agreetha non o th volunteer kne whethe cultur o steril mediu ha beetaken Al th supernatan brot an mos o th mea wer swallowe a 5 p.mon afternoonVoluntee A, wh ha swallowe a culture wok u betwee 4.3 an 5nex mornin wit pai i th uppe abdomen he temperatur wa normal A5.1 a.m th pai wa ver sever bu intermittent graduall diminishin iintensity Abdomina discomfor wit sligh pain headach an nause withouvomitin continue wit sever diarrhoe fo abou 2 hr Sh go u feelin fairlwel an cam t th laboratory bu bega t fee il agai wit a furthe attac odiarrhoea Sh returne hom a 1 a.m an improve durin th day recoverinb th evening Ther wer n furthe symptom th followin dayVoluntee B wh ha bee mildl affecte i th previou experimen wa givea contro bottl o cooke meat sh experience n symptom whatsoever KWWSVGRLRUJ6 3XEOLVKHGRQOLQHE\&DPEULGJH8QLYHUVLW\3UHVV Clostridiu welchi food poisoning 9Voluntee C wh ha swallowe th secon cultur wa a heav sleeper Hwok u a 7.3 a.m wit sligh pai resemblin colic bu failin t recal thexperimen h though i wa 'wind' an afte drinkin a cu o te

wen t sleeagain I wa hi fre Saturda mornin an h slep unti midday Whe visitea 2 p.m h wa wel an surprise t lear tha h ha take on o th culturesVoluntee D wh ha bee give th secon steril bottl o cooke meat waunaffectedStool fro al fou volunteer take befor th experimen gav n growt o01. welchii afte steamin fo 1 hr Furthe sample wer take o th da followinth experimen an agai afte 1 o 2-da intervals Heat-resistan Cl. welchii warecovere fro th faece o volunteer A an C. Stool fro voluntee B wernegative n sample wer examine fro voluntee D. Voluntee A continue texcret th organis fo 3 days n furthe specimen wer takenT investigat th possibilit tha symptom wer du t a exotoxin voluntee Bdran a Seit nitrat o a duplicat cultur grow a th sam tim a th previouculture an hel fo 3 day i th refrigerator N il effect wer observedTw month later whe i wa hope tha an acquire immunit woul havwaned furthe experiment wer mad b volunteer A an B wit Cl. welchii,strai 3702 A brot cultur withou meat an a washe suspensio o organismfro a simila culture produce n ill-effects Thi strai ha bee repeatedlsubcultured s anothe strain Lab no 281 freshl isolate fro incriminatemeat wa used Washe cell fro cooke mea culture produce n symptomi on voluntee bu cause sligh coli i th other Th centrifuge supernatanfluid o a cooke mea cultur take b on volunteer an a simila fluid filteredthroug a gradoco membran an take b th other produce sligh nause anabdomina discomfort Th inconclusiv result o thes experiment ma havbee du t th developmen o som immunit or perhaps t th remova omea fro th culturesI genera th voluntee experiment indicate tha Cl. welchii, strai 3702growin activel i cooke mea brot produce i volunteer swallowin thwhol cultur symptom resemblin thos observe i th outbreaks Experiment filtrate wer inconclusive A toxi substanc i th filtrat ma b destroyeb th gastri juice bu th whol organism particularl i enclose i meat masurviv passag throug th stomac an produc toxi i th intestine(b) Animal experimentsI additio t th feedin test o monkey alread described culture werintroduce throug a stomac tub int th stomac o monkey anaesthetizewit ether N sign othe tha a occasiona loos stoo wer noted bu thorganis wa isolate fro th faeces Culture give b th mout t guinea-pig n effectTw frogs A an B, wer give approximatel 0- ml b mout o a overnighcultur o Cl. welchii, strai 281 two C an D, wer give approximatel 0- mlo brot fro uninoculate cooke meat Frog C an D remaine quie al daywherea fro B becam ver restles afte 2 hr. continuall movin abou an KWWSVGRLRUJ6 3XEOLVKHGRQOLQHE\

&DPEULGJH8QLYHUVLW\3UHVV 9BETT C HOBB AN OTHERsittin u o it hin legs i di no vomi an afte a furthe 3 hr becam quiean remaine s fo th res o th dayAttempt wer mad t demonstrat pathogenicit o productio o enterotoxii mic b parenta injectio an b ora administratio o cultures Th straino Gl. welchii use showe typica morphologica an biochemica characters angav a positiv Nagle reaction inhibite b Typ A antitoxin The ha beeisolate fro mea considere t hav bee th caus o th outbreak or frofaece o victims a follows outbreak o Octobe 194 (fou strains) Februar195 (seve strains) Jun 195 (seve strains) an late outbreak i 195 an195 (si strains) Th materia consiste o th fluid portio o culture i Robertson' cooke mea incubate fo 2 hr a 37 C Th result appea i Tabl 10Tabl 10 Tests for enterotoxin production of Clostridiu welchi strains in miceand guinea-pigsLabno3702/43790/43864/43954/4281/5293/5294/5298/5322/5323/5324/51465/51466/51467/51468/51469/51470/51471/51690/52985/53653/53756/5166/5914/5C.N.207C.N.199SourcStewe steaBoile beeBoile tonguBoile lamBoile sal beeFaeceFaeceFaeceFaeceFaeceFaeceFaeceFaeceFaeceFaeceFaeceFaeceFaeceFaeceBoile sal beeBoile sal beeFaeceFaeceFaeceTypeTypeMiceIntramuscularD 8 daySssssssssssssss————————2 hr cultureIntraperitoneaD 1 hr———S—————————————S(2SsD 1 hrD 1 hr S (1SD 4 hrD 6 daOraSSssssssssssssssssssssD 4 dayD 1 dayGuinea-pigsIntraduodena injectioc5 hr cultur 2 hr culturS(3———S————————————sS(38(2S(2S(3S(38(2D 1S———s—————————————ssssss8(2hr (2 D 1 hr (2D = died S = survived — = no testedWher mor tha on anima wa used th numbe i indicate i bracketsO th eightee mic injecte intramuscularl wit 0- ml o culture onl on diedo th eight da afte injection O te mic injecte intraperitoneall wit0- ml. thre died al withi 1 hr O twenty-fou mic give 0- ml o culturthroug a fine cathete directl int th stomac al survived N symptom werobserve i th survivin mice whic wer kep unde observatio fo 1 days KWWSVGRLRUJ6 3XEOLVKHGRQOLQHE\&DPEULGJH8QLYHUVLW\3UHVV Clostridiu welchi food poisoning 9Th test mad i guinea-pig wer base o th observation o previouworker (Thomson persona communication wh ha demonstrate a enterotoxi i lam dysenter (Typ B strain o Cl. welchii b injectio directl int duodenum

A a preliminar step thes observation wer confirme wita Typ B strai (C 1990 obtaine fro M A Thomson Guinea-pig inoculateintraduodenall wit 0- ml o 5 hr cooke mea culture o thi strai die 1 hr wit acut enteritis th lowe hal o th smal intestin wa intenselcongested dee purpl i colou an dilate wit fluid. I wa als confirme thatfo a unknow reason som guine pig receivin th sam materia survive showin an symptoms I i advisable therefore t us severa animalfo eac testTh presen experiment wer mad wit eigh strain o Cl. welchii fro anumbe o differen outbreaks Th source o th strains an th number oguinea-pig injected ar show i Tabl 10 I vie o th possibilit tha a labilenterotoxi ma b produce i youn culture only test wer mad wit cookemea culture grow a 37 C fo 5 an fo 2 hr Youn guinea-pig weranaesthetize b th intraperitonea injectio o hexabarbiton o Cyclonal thabdomina cavit wa opene b a vertica incisio an th duodenu exposed0- ml o cultur wa injecte throug a fine needl an th abdome close wita fe sutures O th twenty-si guinea-pig injecte al survived an non showean symptom afte recover fro th anaesthetic Observation wer continuefo a leas 1 daysTh experiment o mic an guinea-pig thu faile t demonstrat enterotoxinTh occasiona death o mic afte parentera injectio wer probabl du t thtrace o a-toxi know t b produce b thes strains repeate test wit thsam strain gav irregula result s tha neutralizatio test wer no practicable(6 CommentTh strain o Cl. welchii isolate fro suspecte foodstuff an fro th faeceo victim i th reporte outbreak o foo poisonin hav som distinctivcharacters The for non-haemolyti colonies whic ma late sho a fainhaemolyti zone The ar heat-resistant usuall withstandin steamin fo 1 hr.som strain wer show t surviv boilin fo severa hours Althoug spore werno readil demonstrabl unde natura conditions certai strain hav beeinduce t spor freel i th laboratory The produc a Nagle reactio inhibiteb Cl. welchii Typ A antitoxin I thei toxicologica reaction the resemblTyp A strains n ne toxin hav bee identified Eigh serologica type havs fa bee recognize b agglutinatio tests the cross-reac serologicall witsom othe Typ A strains bu n relatio t Type B C D E o F ha beeshown I an on outbreak organism o th sam typ wer isolated Attemptt demonstrat pathogenicit i laborator animal wer unsuccessful bu cookemea culture swallowe b volunteer produce gastro-enteriti resemblin thaobserve i th outbreaks I i considere tha th epidemiologica an bacteriologica findings justif th vie tha heat-resistan strain o Cl. welchii ar thcaus o th reporte outbreak o foo poisoningJ Hygien 7 KWWSVGRLRUJ6

 3XEOLVKHGRQOLQHE\&DPEULGJH8QLYHUVLW\3UHVV 9 BETT C HOBB AN OTHERGENERA DISCUSSIOCl. welchii ha lon bee suspecte a a caus o foo poisoning bu th occurrenc o a particula typ capabl o producin spore whic woul survivprolonge boilin wa no recorde befor th wor o Zeissle an hi colleaguesTh difficultie involve i anaerobi wor an th fac tha thi typ o Cl. welchiii non-haemolyti an no eas t pic ou i th presenc o othe bacteri mapartl explai wh i ha no receive mor attentio i relatio t foo poisoningI seem probable however tha a a resul o change i socia habit th miltyp o foo poisonin du t Cl. welchii i becomin mor commonBefor 193 mos person at a home o i smal restaurant servin freshlcooke food A treatmen o foo a hom wa highl individual outbreak ofoo poisonin woul affec onl a fe person and i the wer a mil a mos othos du t Cl. welchii, woul hav occasione littl bu temporar annoyanceAgai th clientel o restaurants thoug workin i a limite area woul i alprobabilit liv farthe afield illnes occurrin a a resul o suc meals thereforemigh no b associate i th min o th victi wit th restaurant moreoverth restauran woul probabl offe som choic o dishe s tha no al personeatin ther o a particula da woul b affectedTh 1939-4 wa change al this Canteen becam common i factories ischool an a municipa ventures existin canteen wer enlarge t suppl manmor meals o wer use o a large scal withou alteration Communa feedinbecam usual s tha larg number o peopl woul ea th sam meal Thconsequen increas i cantee staf le t th employmen a foo handler oman person untraine i th element o foo hygiene an th shortag oequipment refrigerators washin facilities ho water towel an overall madadequat hygieni precaution difficult Abov all th shortag o mea madeconom i it us essential No onl wer 'left-overs hoarde an re-use ipossible bu th practic o cookin mea o th da befor i wa t b eatebecam widespread mea i carve mor economicall whe cold N doub somcantee staf wer genuinel convince tha mea woul no kee overnigh unles iwer cooked Everythin favoure th developmen o heat-resistan Cl. welchii. Ia stew fo instance al aerobe an non-heat-resistan spore-bearer woul b killeoff th oxyge woul b boile ou o th liquid whil glutathion an othe reducinsubstance wer extracte fro th meat Heat-resistan Cl. welchii i presenwoul the germinat an gro freel a th ste coole slowl i th kitchensuc perfec anaerobi condition coul hardl b improve b laborator techniqueTh frequenc wit whic heat-resistan Cl. welchii wa isolate fro thsuspecte foo an fro th faece o patient an person a risk th generasimilarit o th strain culture from foo an

patients an th ver lo carrierat i norma person make i fairl certai tha th patient acquire thei heatresistan Cl. welchii fro th contaminate food I i b n mean s eas t shotha th foo poisonin i du t heat-resistan Cl. welchii. Th organism ar onlfeebl toxigenic the d no produc y#-toxi a all an n enterotoxi ha beedemonstrate b anima experiment Experiment o volunteers however sugges KWWSVGRLRUJ6 3XEOLVKHGRQOLQHE\&DPEULGJH8QLYHUVLW\3UHVV Clostridiu welchi food poisoning 9tha a mil for o foo poisonin ca b produce i ma b ingestio o cookemea culture o heat-resistan Cl. welchii obtaine fro foodsTh typ differentiatio o heat-resistan strain o Cl. welchii i importan foth investigatio o foo poisonin outbreaks i i essentia fo th correlatio oorganism isolate fro th suspecte meat cooke an uncooked fro patientsfaece an fro carrier amongs th kitche staff Eve i th suspecte foower no available a hig percentag o stool showin heat-resistan Cl. welchiio th sam serologica typ woul b a stron indicatio tha th outbrea wa i du t Cl. welchii.Mea ma b contaminate wit Cl. welchii i variou ways—b organism idust o b faeca organism o huma o anima origin an i i clea tha thicontaminatio ca occu i slaughter-houses i transi an i canteens T preventh transfe o Cl. welchii fro th huma intestin t foodstuf vi th handdepends a usual o th persona hygien o th foo handler I canno b tostrongl emphasize tha th preventio o thi typ o foo poisonin lie no ith contro o carrier b treatmen wit anti-bacteria substance no b theiremova fro th kitchen bu i attentio t clea hands car i foo manipulatio and mos importan o all correc foo preparatio an storageAlmos al ou outbreak hav bee associate wit th consumptio o meacooke th previou da an allowe t coo fo lon periods I i eviden thamos o the woul hav bee prevente i th mea ha bee eate ho o thda o whic i wa cooked I migh b wort whil i futur t conside thinstallatio o pressur cooker i canteens thes woul enabl th cookin t bdon i ver muc les tim tha i no possible Whateve th futur policyhowever th practica result o eatin mea o th da afte i i cooke ar cleaenough I certai borough wher man o th first outbreak o Cl. welchii foopoisonin wer investigated th principa canteen i school an factorie havcease t coo thei mea a da ahea o requirements preferrin t us i freshlcooked Thi improvemen i technique encourage b th officer o th locaPubli Healt Departments ha resulte i freedo fro Cl. welchii foo poisonini thes areasI thi idea solutio i no practicable cooke meat intende t b eate oth followin da mus b cool

e quickl an refrigerated i an furthe heatini necessary i mus b don jus befor th mea i eaten Furthermore th heapenetratio durin cookin wil b mor effectiv fo smal joint tha fo largone an th subsequen coolin wil b mor rapid Therefore i seem advisablt us a numbe o smal joint i preferenc t on larg one Cooke mea shoulb kep eithe to col o to ho fo bacteria multiplication i shoul neve ballowe t remai war fo mor tha th shortes possibl time Coolin arrangement fo thi purpos undoubtedl rais economi difficultiesI i suggeste i th Repor o th Manufacture Mea Product Workin Parttha th bes arrangemen fo rapi coolin unde hygieni condition i a cooair-conditione room Facilitie ma no allo thi an th woode cabine witshelves t whic i attache a electri fa an a filter, referre t i thRepor ma solv th difficulty7- KWWSVGRLRUJ6 3XEOLVKHGRQOLQHE\&DPEULGJH8QLYHUVLW\3UHVV 10 BETT C HOBB AN OTHERTh househol typ o refrigerato coul b adapte t tak a serie o shallotray s tha stew an simila fluid dishe ca b divide int smalle quantitieo larg surfac area Ther ma b othe simple device whic ar a effectiveEducatio i method o foo preparatio an storag i a essentia facto i thpreventio o foo poisoningSUMMAR1 Outbreak o mil foo poisonin hav bee investigate i whic heatresistan Cl. welchii appeare t b th causativ organism Th outbreak wercharacterize b coli an diarrhoe withou vomiting commencin 8—2 hr afteingestio o th contaminate food2 Th strain o Cl. welchii* concerne ar onl feebl toxigenic an aparfro th heat-resistanc o thei spores an som colonia characters fit wel intCl. welchii Typ A Th toxi productio an th serolog o th strain i uniforwithi a outbreak3 Mil foo poisonin simila t tha see i natura epidemic ha produce i volunteer b ingestio o culture o heat-resistan Cl. welchii isolatefro contaminate meat4 Infectio i almos invariabl du t mea whic ha bee boiled steamedbraised stewe o insufficientl roasted allowe t coo slowly an eate thnex day eithe col o reheated5 Outbreak o thi kin shoul b prevente b cookin mea immediatelbefor consumption or i thi i impossible b coolin th cooke mea rapidlan keepin i refrigerate unti i i require fo useW wis t than man person fo thei help First thos wh hav providefacilitie an material fo th investigatio o outbreak o Cl. welchii foopoisonin i th Londo Area i particular th Medica Officer o Healt andSanitar Inspector o Publi Healt Department a Southgate WembleyUxbridge Edmonton Islington Barnet Potter Bar Ilford Enfield FinsburyS Pancras Shoreditch Brentwoo an Kensington Secondly th director an

staf o laboratorie o th Publi Healt Laborator Servic wh hav providedetail o outbreak an man hundred o cultures i particula thos aEdmonto an Coppett Roa (London) Manchester Newport BrightonLeicester Epsom Newcastl an SalisburyW ar als mos gratefu fo th generosit an kindl co-operatio i thsuppl o mea an faece samples o Messr Apli an Barret Ltd. Yeovil anth Publi Healt Department o Uxbridge Watford Wemble an Birkenheaan als th Liste Institute Elstree Th flies wer provide throug th kindneso M A A Green Pes Infestatio Laboratory SloughOu thank ar als give t D A G Sign an D Edit Strake o S MarAbbot Hospita fo muc informatio an fo specimen connecte wit a par outbreak t M J Smile o th Medica Researc Council Mil Hill foth preparatio an microscop o slid cell showin spore o Cl. welchii, an tth staf o th Immunolog an Experimenta Patholog Department WelcomResearc Laboratories fo invaluabl technica assistance* Thes strain o Cl. welchii ar i th Nationa Collectio o Typ Cultures KWWSVGRLRUJ6 3XEOLVKHGRQOLQHE\&DPEULGJH8QLYHUVLW\3UHVV Clostridiu welchi food poisoning 10REFERENCEANDSBWES F W (1899) Lancet, 1 8BOWER J O. MENGLE H A & PAXSON N F (1938) J. Immunol. 34 185BUCHBINDER L. OSLER A G & STEITEN G I (1948) Pvbl. Hlth Rep., Wash., 63 109DTJNCAN J T (1944) Mon. Bull. Minist. Hlth Lab. Serv. 3 61DUNHAM E K (1897) Bull. Johns Hopk. Hosp. 8 68EVANS D G (1945) J. Path. Bact. 57 75GLENNY A T. BABR M. LLEWELLYN-JONES M. DALLING T & Ross H E (1933)J. Path. Bact. 37 53HAIN E (1949) Brit. med. J. 1 271HATJGB S (1950) Nord. hyg. Tidskr. 31 189HEADLEE M R (1931) J. infect. Dis. 48 468HENDERSON D W (1940) J. Hyg., Camb., 40 501VO HIBLEB E (1906) Zbl. Bakt. (1 Abt. -Be/.) 37 545KLEIN E (1895) Zbl. Bakt. (1 Abt. Orig.), 18 737KNOX R & MACDONALD E K (1943) Med. Offr, 69 21MCCLTTNG L S (1945) J. Bact. 50 229MOORE B (194 a). Mon. Bull. Minist. Hlth Lab. Serv. 7 136MOORE B (194 6) Mon. Bull. Minist. Hlth Lab. Serv. 7 241OAKLEY C L (1949) Brit. med. J. 1 269OAKLEY C L & WARRAOK G H (1953) J. Hyg., Camb., 51 102RODELLA A (1910) Wien. klin. Wschr. 23 1683SIMONDS J P (1915) Monogr. Rockefeller Inst. no 5WILD O (1898) Zbl. Bakt. (1 Abt. Orig.), 23 913WILSDON A J (1931) 2nd Rep. Inst. Animal Path., Camb., p 53WILSDON A J (1932-3) 3rd Rep. Inst. Animal Path., Camb., p 46ZEISSLER J & RASSFELD-STERNBEBG L (1949) Brit. med. J. 1 267(MS. received for publication 30 vn 52. KWWSVGRLRUJ6 3XEOLVKHGRQOLQHE\&DPEULGJH8QL