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. A PREl;JIMIN ARY REPORT- 'ON AN INVESTIGATnn~, O]:'THE IMMUNITY REACTIONS IN EGYPTIAN. BILHARZIASIS,

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By MAJOR N.H~MILTON 'FAIRJ,EY; , .) , . > ./

Austra,lian:Army; Medical 'Corps. :Pathologist AuslraUan . Genf!ral Hospital. ,\

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SECTION .. r:-Introduotiorr.;~einarks.ontb,e cellular.humoral theory of im:ip.unity. 'I. SECTION I!;-Obncerning clinical' aspects of 'Bilharziasis:' ....

.. SECTIO~ III;,~The eellularresponse foll~wing Bilharzial hlfection:':"" ' • " i

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,(1) Observ'atiOnson 'the blqod picture in man.>

(a) The cellular response during thll toxremic;stage of Bilh'arzias~s: (b) The cellular response dliringthe stages of localized Bilharzi:tsis.

r(i) Blood count~ in vesical B~lha~Ziasis(S.hal11zatobium) ma<j,e from the sixth to the eighteenth month aftednfection. . , '\ ' ," ,. '. / , \>

(ii) Blood counts in mixed: Bi,lharzialjnfeptions'(S. mansoJ1,i a~d . S. hcematobium) ma.de from the/sixth tq the eighteenth . . month aftel' infection. .' . . . . "

. (2) ObSllfvations o~ ilie blood picture, etd .• in monl>eys eiperimentally inflicted with Egyptian:Bilharzi~sis. . .

: (a)Obser~ations on the,blood plcturesinmonkeys spontaneously rt\hO~eri:hg frpm, the disease'. .

(b) Obse~vation~b~ the blood picture, ~tc., in m6rikeys.dying:earlyfroll1/ ' ! the disease, . \ '

(c) ~he cellular response observed in the focal Bilharziallesions of the organs of infected monkeys. ,/ \,

t3)'Resume- .. . .

Table I. ~Data ,co!le6teq on the.imm,vn1ty response inin'£e6te~xhonk'eys. " I" :' \ ~., '. ( ! . " ,. . . :,," \' ,

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244, ,\ The Immunity' Redctions,'in Egyptian, Bilharzi~~is ""

, SECTION ri.-The cokplementfiiation J;ea6tion in Bilh~r~iasis: /

(1) 'The phenomenon o~complem~nt fixation." • , ~ /, , (2) , Teohnique emploj'edin"the o6mplementfi'xl}ti~n test forBilhar~iasis .

• (3) SJgnifi~anoe of theoompleme~tfixatlon test in Bilharziasis and its pra.~J;ioal ' , ; I ~ applicati<?n. ___ ll', ~

',,, ' " (4) AIJ. irive~tigation of the~,sera of 311 hospItal patients llMiig all antlge,n a ," , ) , ;, sal~t~e extract ofjnfect~d Planorbis"s:q,ail's' livers (antigen '~AI,,,). '/" (:' ,~.'

! :' "; ," ," . I, ,-{1\) An investigation, of the sera 'of' 280, cases, 'using as antigen 8011 alcoholic

, extrlj.ct,oI infected1livers (antigen ":8"). ,,';, , ',(6)R~latidrlship betviillluthe gr~de of eosinophilia and the,complement fixation~' , :,,: reaction in man. " "> ' " ' ",

',(7) Pt~otioal vaJ:ue of tn'e oom'Pl~mentifixation ~eactiO,n. (a) The di~gnostic value of the' c<implem~tfixation'reaotion' iri~ :an: •

, harziasis,l' , ' , " • -:. "', \' . " \ / ' ' \" , ",,' I )

; j .(b) The complem~~tnxation/Jeaction as 'an index of tla~ ,therapeutic' , " a'ct!i>n of drugs in th?tr~atment qfthe"disease.' , '

(8) Deductions whiph oan be legitimately derived from the work'reQorded, abovE!' ,

SEd;lON V.-':'-':PreJiininary note on 'the investig~ti;n f<;>t, the ,existenoe of' a, precipitin te~t ill "Bilharziasis. ' ' j

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" ,'I SECTION'I.-:--INTRODUC,:!;ION. / , " • "t

,';THE modern'investigation of imtnunity by sucn observers as Leishman ~nd Wiight I hasdetinitely sbownt1;le in'~dequac{ of eitber'.of the two

, ,previous 8c11001s. Qfib,(mght on t1;lis subject. comprising as they did :;the I :': cellular theqry" q'fMetchnikoff" and, the hl:un9raldoct;i~e" of, ,Nuttall.

The-body' e1ll,ploysb'oth the ,cellular andthe humoral lines 6fvdefence. , ' " Under th~influeilCe of bacterial invasion certain cellular elaments 6f

" t4.eblo~qareincrea'Se9. in 'quanti,tYby the chem~()ta~tic action of a ~toxin • on,thebo:tle marrow an!! hrer;riopojetio organs,'wbile the "humor~ or fluids " 'are similar1y~einforcedwith, immune 'bodies antagonIstic to the'Invading par~l'iteand, it's t,oxin. " ",' I, ", " " ' " '~' 'I' " '

/, The cellu.~arr.eaction to p~rasitic'invasiop, appears to follow certain " ,constant< lines., ,'Wher~as I the pyogen;ic group 6fbacteria,.constantly ~ produce a.p61Yq:lorphonuclear, neutrophile leucocytosis; helminthic infec:

,tions arel fo~16wed 'by ,a' relative and absolute increase of the eosinopbil~ , I~ucbeyt\ls" while in tbe Iprotozoa~ difl~ases, \ 1.~., . :iha}arias, ,leishmani!1sis,', 'tr.ypaposomi\1$is,and less c9rrimonly, recQgnized relapsing fever and aIDoobic, dysentery, etC.,: a rela.ti\reinc~ease of' the large monopuclear, elements 'of the ,blood~ is appaterit~ ,'i'" ,/ ' "',, "/".

, '"The humoral rea.ction i~evidenced, by tHe presence of,antibop,ies in the sera ofpl;1tieIitssuffering .from certain bacterialdiseasesand isquite~asilYI ' demonstrated. Thusdn typlioid fever.agglutinin'can be demonstrl;1tedin the ,blood by the Widal ieaction,anilimnni,nebodyis also "fbund to he

:'Ipresentbyutilizing' thecdmplementfix~tion reaction' of Bdrdet arid , Gengou{l]i,e~ployingas~antigen a ,saline suspep.sionof Bacil,ius typhosus.'

SiIilihtrly i:n the, ease 'of certain 'protozoaJ disel;1ses the productiqn' of both ,,\ ~,

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Jlsggl11tinin ~'. and: immune, bodY"has 'beeil:derriQh~ttate({' ;rhus,Wasser'-' Ilhanrt [2] 'using a)~ antige~ saline .ext~ap~ 9(syphiIitic liver"has~hdjvn fixation of the co~plementwith thlL seJ,'a of.syp\liliticpatients, arid. / Nogucb.i after successfully, growing . Trepont;ma i palZidumde.monstrated

.' ~their 'agglutinatibn' insalirte suspe;r:uiion wh~n miiedwith lUetlcs'eta. '" / Much less' is. known of the hu'mmariij3spiHlie ~f thebod~-toinivasion by:

the par/1siticmetazoa.·· . "1 "/ '. . ...• '. . \:, -; I '. '. ,;

' .. In tbe cysticstageo£, hydatid disease' precipitfrt an~ijmrtiun:ebody Jlave bee:(lde'litonsttated in the serum o~ man; who' acts. as the intermediate h6st'.

, for ,T(lJn'iaechi?J;ococc;;'s..In 190,7 Fleig and' Lisboririe[3] aemo'zl'sttated ,',~Ii specific precipitin in the . sera:.of i patients' " suffl;1ring .' fr,6m "hydat{d -·.disease. Welch" and Cb,apman [4] .. have/·corifirmedait~ exteuJi.ed.these ooservatiQJ7l,s.Gti~dini [~], .Welhherg 'and 1?arvu, [6]; E'ckenstein and· '.' others: uSlng\as antigen hydatid fluid, demonsttate'a:the presence· ~£ anti::', bbdy.in the hloodof'patlentssufferingfromthi;s disease,wi~p atechniq~e:'

, similar' to (the Wassermann' reaction. ,'. . ,', ' , '.' f· In t~e presentpaper;i~by means ,of ~"~pe~ificeomplefuent fixatioiF,

_ reaction; defirii~eevidence:lsaddupeilofanimtpune, bo~Y ih, the Ileraof patients ~ffected either,wiith'tbe vef:lic'al (Schistosoma, ,?l-almatobiuni) .. Qr.tl1e ,intestinal(S:,man~oni) type' of Egyptia;n,l;>ilharziasis. This is;tpenrst. · .reco;rdof'a specific'substanqe having qeenisolated from the . body of a.n .' · in~ertebrate in~ermediary (Bullinus and. Planorbis) infected 'withan:uma.h \

parasite and.which will reactwjthhumalliseru~\ (i.e.,jtlia~:Qf ,the definitive h,ost) .. Investigatiol1,o£ tl1e sera 9fthese patients'for an ~gglutinl'\iti&rior' a,.

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prec~pitin reactlQnhas YIelded nEjlgatIve resultsop to thE:) time of .writing ....•. ' J • TJ:?e incidence Ofimfnune body,:as' sbo~n by .~ .. speoifi,cc'ompleII;lent. fixation reaction, in a disease due t,oso ltighly,organized 'a Jiarasiteas a,..

. ,trematode 'worm, is. of. 'col1siaerableimpdrtance.While ,there .is much ", , ' , , ' ~ , ,;,,'. " \ , ' I .. ' ' , ",)'"

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analogy between the. precipitin' and~ complementfixatibn'reaction obtained . ' ' , . ,'from hydatidfltiid and similar reactionsobtained:by'tne mere iptr,~dllcti6n', \ '""

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':9£ a fOJ,'eign protei~intoan'drganismi such a,~reaction jn,billnir~iasis can ' c'. ;, -only pe,:}ntyrpretedas a. true' irrimllllity' r~s'pop.se. '. This being 'the -ca~~~,i~ .. i~;~ot uJ;llikel~ that. certajn other h,elminthic infeqtiplls'are:SimiIaHy i

accompanied, with the formation of immune. body, specific)forth~ invaq;ipg "i

parasite and its to:xins. ,This seems especially likely ,wh~re.theparasi:te inhabit~ the vascula,r' system, an~ fSives rise tq an eo~ip.9philia. Bahr [7], / .. /i

· bas.alr~ady drawn att~Iltion to tbe possible tQxic {actdr,i,n the pr~~uction\ ,.'. , '. of the pat.b()logicallesio:qs in filar~aJ:;is. ,Con~iderhlg' the similari~yi? ,these ( , ,

lesions to ,those ,seen jn bilharziasis? one ,<ca'nwithcertaintyprediGt 'a,. ':.

Sl?e~ific .comp~e,mep.t >fix~tion reaction,'fp.v the' former :dise~>se.·: following", the ,I,

; preparation Qfa ;suitab!e antigen; sUGhas an extractofinfecteCl lymphl1tip '." " : glan!ls~ .Tbe demonstration of such a ·i.eacti6n in filariasis. wouldrbe of I.

considetabl~ '; v~Jlle.

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SECTIONIL~CO~H3ER:NiNG CE;RTAINEARLY CLiNICAL ASPECTS OF, "', .' 'BILHARZIASlfL " \ ..

• ,As, adetailed,aecouIit' of. the cllriidalaspects' of. ea,rly EgYPti~nbi1haliziasis 'wllLbe publishediat,aiateidate, only abriefoutUne oLthem.w,illbe given: .

,·he.re., it w3rs)he.e",istence of ce~t8;jngroups'ofel),rly'symptoin$-;cc8;lled/ .\ hereafter "'toxffimic symptoms :~":'-that p,rst sugge~ted·the,necesfPi~y fqr'a ," complete ij:tvestigatioll: ofiihe)mmuriity response ill bilharziasis;.. "

,>The initial \toxffimjc\symp~oms .of. pil~arziasis;were)irstdesc:dl;>ed\in cpnnexion:w~thBehistosomaja:ponicum.". E~celleIitaescriptioris,of.,this stage ofltbei?-.fec~ioll:were giv/fn)yEdgar ,['81 in19U,Basse~t;Srpith [9] in 1912, .·~¥i:yagawal1D}j1l.1913, and. Laning Dl] i 111914 ... ~The ,initialstage~8;s" . characterized by a high afternoon pyrexia lasting. from three to, five week's, ,

. ,aSlow pulse,emaciatibn,oodeti:ta"and' urticaria, pain, in the upper abdomen; , ' . paroxysmal Q01lghwit,h'Dccasional',pulmoI).ary ,duIlness,diarrhoo3! or con~, stipatioll. mental. dep~ession/ ,ltnd marked eosinophilia. 'B>ecovery from'

<this, stage'followed; I1nd subseipiently th~ patient, dev~loped abilharzial , dyseptery ~iththepl1!ssage of ova of S. 3dpOnic'tf,m.' ,There 'might l1!lsb occureIl,larg.errie~~ 'of. ,the· spleen,)iver; etc.; and. finally. asqjt.es:a,p.d dl?atb,

, In 191~ Arcthibald [itA] described three cases of o1:Jscur~ pyr~xia witQ.~ out eosinophilia which: at: a.utopsy were found to have intestinal bilharziasis (S.marulOni~) " . ' ", . ' ., , , . " , In.~ 1916,Lawton [12J noticed: sillfilar sympto.ms for) the ,first time'in EgyptiaIh bilharziasis. They occurred am'ongst, troopers, of the Austrl11ian Light Horse, sta.tlolled.at.T~l~el-'Kebir on the, sweet,water canal z~)Deii1th..e~' ' Delta. ~", \' . . _. ,. _ / 'j" "'.' , ,\.,

"',The )ni~iaL sy-mptoms., comprised .abdominal pain, .en~argemerit ,and' tenderneSS ofl tpe liyerapd splee~;also pyre)l:ia~ . u:t;ticaria,and. dian;h.ooa. 'A nH~Q.-grade eosinophilia'Yi1s always present,and the.fffices. contained:.

lateral-spined ova,.. Sub,sequently' these. sYI4Ptoms complefelysubsideq.. ,.', " " From:Septemb(?r, f916" toJ:uiie;l918, I have had abundant <?pportunity"

of confirming~ !);ndexpendingLawton's observations; Dur.ing t.hi,~ period . seventy-Bve. ca.ses.d£ b~fha:riiasisiamoJ;lgst the troops have. been, carefully "

\. investigated. In~ddiiion toshowing,that. the great majority of eases from 'I,

:Tel~el~Kebit, (including those of;biL~ton';s series) weretnixed.irifections. '. CS.> hcematobi"um. andS. rftwnsoni):' a simil~r group of' toxffimic' symptoms ',w:asnO.teain~aseries ofp,qre ~.kaJ~,atobiuminfection. '~,' '"

.,,,) . An. analysi$ of.these ·cl1sessho:w.ed}ha,t thirty;:eight outof8event~~fiye cas~s, 9r50'o' per cent,(prese:tlpeqearly tOXffimic 'symptoms.' In these cases I

:two~stages wereinoted in:thedi~ease.'/ '., \., , .(lJ· A,s £age 'of' toxremia 'occurring from. four to" ten wee,ks.after infection, and follO.wed. by r~c0verY· ...._, . " .'. " '. c "

. (2)Alate.r stl1ge' :oflocali~e"d • Bilharfliasis chaiaetedz.ed by vesical Isymptbms,.in,B.!taJmatobium ·infechbns"'~Iidl)y the, intestinal symptoms'

'iriS., ma,nsoni infection's. ': Symptojtls of the lqcalized dlse~se appeareqat., , f"O,' ". / ><," r ,- • I ,', ,,- ). • \ J ,

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,a vari~bl,e periQd-fr~m>thr~etQ ~i~hteen ,months";""atterprinial!y.iIi~~, ! fection.,

,/The cases showi~gth~lnani{esti1tions of ,a toxroiIlic~tage\were~ivi~ip~e, ,". in~othrei gro~ps:- , '., .:"'" ',' "'. '..'" .. ' ,.'., '. (l)rThose.presentingasyndmme 81milar'tO' thatJlescribed by Lawton, viz. : ,nrticaria, as,sociated with prolonged pyrexia, abdomjnal, and,·perhaps; pulmonary symptoms. These were the common cases. " " ' /' .(2) Thosecharacterized)by urticaria, and pyrexia,pf .lessth~ntendays!

" ,'\ ,dui:a.tion-less.commontban~'group 1: " . . I; , • . '

, (3) , Tq.ose ,presenting urticatia\ only. This, 0cCurred, in 'only a few cases, ,It is thus seen that the toxremie, stage of the disease maY·be entirelyansent;

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maybe evidencea 'by. symptoms ofa"profqun<1 t()xrem,i'a,' or the symptoms' , may bl'l~so'slightasto healrilOstneglecte.d. '. '. '1/. .' . i ' ,.', •

The v:arlal::>lIity"of., these tmi.:ffimic' symptomsJ believet~ be ;d~pendent on the in,terisity ofthEr infection, j;e.,tothe,.mass,actionof the ;tokin libel!ated,bythe~a'turiqg and aduI~wol!msa;ndtliei~.ova:,., ',. ; .' "'(: ,

In the followip:g ,pages ,the immunity l!esponse!.iribilharziasiswillbe cqnsidered iromthefollowing. aspe(;ts :~ .. ~ . . . . . .'. . \',' .

. . c,(i) The cellular response a!'} it is manifesteQ : firstly,by.chatlge.s in the c. circulfl<ting bloOd ; and, sec0I!dly, bytheva;scuhl.raIld .tisstl~cellular{reaCtion in'focaJbilharzial lesions. ~. '.' .; .

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.(2): .Thehumoralrespon~e as jnd~cated ,by ihvestiga.tiOnofthecbmple':-mentfixati0rl ~ild the precipitin.reaetions ... " ..

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SEcTIoN Ut-THE .cELr:ULAR'RE~PONSE FOL~~~:i~GiBltE:ARZIAL ", j. INFECTION .

. Though'a number of ~6bserva,tjons~realreadyreco~de!X on the blood ' pioture'i11 bilha!,ziasisbyDouglas 'and Iiardy; [13J ;iKauts~y . [1~1,' -:',

,Bal£our'. [15J; Day, [16J, Con:Qorahd Bazaret [17],and. others,:futther work is requi:red ,in "view'6f the.·,recerit labours .0f'Leiper U&J An 'aelUon-

. ;stratirig' the existence of' the ,two species of Scbistosomatn.~S.v~{£mato~' bium and S. mansoni).' ".', " . \ "Tbedbject of thepreseht' etudy 'istwofo!d;it is:-'-'

(1) To follow 'the changes jnjthe blood picture'dbring.,the, ~~fferent , , stages of the disease. ' ' '. .'" , ... '. /. ,

'(2) To estiJ;nate whetBer any' ~iff~rence in, the bloo.d pictllre exists in pure infection with !thetwo species ,of/ bilharzia, 'and whether any-feature .is accentuated jn~ixedinfe,ction with bOth specie~. ' . . .

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(1) Obsefvations on the 'Blood' Picture in.Ma1L

" (a). The OellularResponse' during the To{!;amiicStageQj J3i~h~;fziasis.- / Mdstof the previous observ;ations in this st~ge bLbilh-arzil\isishave' ,been,

'. ,made on .inf~cti0n~ withB. japonivurn; Leiper ht:1s ~e~o±geif ~1J,n; 85 ,jJer , "

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" " .,(lent eoSinophili(L tnbne ,of 'hij'vcas!'ls ; and Bassett':Smithni:)te,ii,in. a naval , ", officer an. eosihophil'i'aoL 68 per, cent, ,with "a leucocytosis of '30,000 per" ,I

b}lbic millimetre;" La;wton's.obser"ati9nson the blood picture during 'this, Jltage.6fE~yptian bilha~~ia~is'are, ppe only 'ones recorded in the l~terature. .

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, ',He,found a' constan,'I; l§!UCbcytosisvarying from 13,000 to 22,00()'per cubic : 'mipimet~e, 'the average ,count, ceq~alling '18,900 ,,' per, ", c:llbic miHjn;tetre. Differentillilwhite ,counts yielded a high/eosinophilia-.The lowest ,eosinophilia was 36)er, c~,nt'; ,tbe highest' 7,6, per cent,and the a;;~rage~ount wis about: 50 pe~ cent. In casesexainin'ec).imnredi!).telyf'aJter. the toxre):nic' symptoms had, subsided; 'I have noted different1iaJcounts with<fortfthiee'

•• ' . per,cent'ltnd fottYi~five per, cent of eosin,?phile c~lls. ", ' ",,',,:, '," I,

'(b), Thf( Oellular ~esponser1uringthel Stq,ges df Loca~iiea Bilha:rziixsis.By the,titpe\tbesymptot9s of"localized bilbarziasisybave'become, estab-' lisb~<!l, the' blood pjcture, haS cb:;mged' cQn!riderably ifr0ID tbat observed ill

'th,e i4itiaJtoxreD)ic stages ,of the, diseasE!. BO,th1the totarl~ucocyte'coupt' a..?-dth!'lI>e'rcentageo~, eosiI?ophilecellshave~ecteased. f' " "",'

In experimentally infected mopkeys I have noted ,thatip certain cases .the ,totalleu6ocyte .counp an<f,the9iff~rential eosinophile(count rapidly di~inish,jt ,may, be ~even nprlllal after the, twelfth ,to the fourteerith ,,*eek. " 111 $. jdponi~um infectibns, ~imilar:cha~ges h'ave been observed, b1,1t froll:).- " observations,on, Aq,stralia1:t'troOps it is e,videntthat inEgyptiari)bilhar~ , ~iasis (both S.mansoniand S. : h.mma to bium) this decline is ,a more gradual

. one ~n man." p'roI?the ifollrth:rii~nth omv.3;rd the,re'is I). .£alluntil the; "~st~blis4ment .of a 'moderate,' grade, ,', eo~iriopllile a~d 'leuco,cy,te ' c~unt as;, I

mdICated. below. . ,(i) BloCid/Jounts, inVesicdl BJjlharz~dsifJi(S'ihd3'1natobium)" made jr~in

the SiqJt'l1, to the Eigl£teenth Mdnth /oziQ'Wing'1nfe,ction.. ,', " .. " In thirty-five eases ,t;hea~erageto.talleucocyte count:c was: 10,030 ,per cu,bic millitpetre;the lowest cQunt was 4, 700; ,and ,the ;highest21,870per cubic 'millimetre. ': t" i.,.' .'...,,', "," i' ',,'

dn tbirty~sixC:al3es the qverdgeldifJereritial.cou~t w:i~:as~nderi~ , " '

" {l)Poly~orphanuclears • I' ..:, .. ' .45',1 per oent (2) Lyrophocytes , . . . . . 28'2 ,,'

, (S)Large roono1J.uclears , . 12'6' ". (4) Eosinoplliles.\ . . lS'5 r :

, (5).Basophiles ... /' 0'6 H

'InJotty~~evenca8es the'eosinophiie count.wasas follows,:-'

( In. 7 ca;es th\l~osi~ophiles ~er~ under'5ptir cent· '",'14' ',~, between'.. ,. qand lO'per cent

'~'; '.lS" .',. " \ <:, ',IQ ": 15 "'~ . ";, I '7 \ ',-" i. 15 ,,20

" SI 11" ~,,'\ "", ,,'.,. ,,aQ\,/;,'25' ',' \".":-,t~"1 1 '" \ "I ," 25,,':30 "I

_', ," 2, .", ", ," '", ,,... .r. :3~",,"4q "",', ,

. '. "In:~aEies'adb1ittedt~ liQspit~Iat', tli~'-ulide~n1entibned; periods 'after .initial .infectj~)D.~~~-av:erageeoFiiiIOphile·cqurit,w:asas f61Io'Ws:~ ,: ",

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,Excluding th~ cases ,admitted' eight ri1onth~, ~fte'rinfe~tion"the ge~~ral 'tehd(,)ncy of tbe,eosinophileIGoun,t'w~s, to decrease 'fr9mthefsixth to ~the

"eighte~ntbmoDth. , " " ' , ," i,' /. ,'", ' . \, ,The average' coUnt. sllowed an eosinophile lel1cocytosjs, 'with. also '!t.

. constant increase, in the large mohonuch~ai cells,and, a 'decreaf?e' in ,t~e ' pOlymo;rphonu(jlearelements.' "I , /' • , " "

, . " ,These observations fully c~mfi:rm the ;work of Day ion' the blood picture' " in early J3jg-yptian: bilharziasis.,. ',' . 1 " , ," ", " '

Th~re pay-ebean so fe~ p~re 's .. m(insJ)ni inf~ctions in t~epresentseries \ that it is:q.ot p()ssible ;to give any }ltatistical bI'ood 6ounts1 of them. 1 :"

. '(ii) Blood Counts" in Mixed.}B,ilharzial InjectibiM(S.mansoni and IS. hmmatobium) , ma'd:e' frOrrlrl tlfe Sixth to the, Eighteenth Month after /

I' Injection., ' ' ,,' . " I"~ ' '

,:. ,\ , Qase~!of mixed infectipns with S. mafbwni an.d S.hiem(#obi'/,IJm gavetlYe", fpllowing 'results:~ 'i " ."

. ' ' . '. In 29 cases the avera:ge, total leucocyte count waslO,50q ,per c.mril. The minimum count'was.: ' .• 5,600 ThemaximuIl)"count was ',. , ,", " .22,500 '

In, thirty~one ~as~s the average dilfe.renUal count, was::::"":' '\ (1) 'Polymorp!loll-uclears .. . ,'44'4per cent

(2) LymphoQytes .•• , 25'1 ' , '(3} ta,rge InonO;Jlluclea.rs , ,Jo,g "

(4)' EQsinophiles ..' .. ,'" " '18'6' " " ,'(5)Basophiles .. ,..,1'0, "

, ! '" EosinopJiJi,le coun~ , . '

i r. In' 'I' ml.~etheeosinophiles'wer~ under I .. ! ," 5 per cent /, , ,;, 7 c9,se~ between '.. 5 and 10 per ,cent

,,'4 " '~', -I';. IQ" 15 ,,-;, 8 " ,,' ",' 15'" 20 ,,4. i, ' i" 20., ,,(25 " ,,2 ,,,,, ,\, , PI 25 I, 30 ;, 3 \;'.' 1. ':,~ '" 30 /;. 35 'I ,,'.

,,)2:',,(: """,' ~O.".,45 '--,,.,\ ,\

Tbe only difference in the blo~d pict;q.refr6m tha:t' of single pure irlfec~ ,.,,~ tion" with , S. htem:cifobiu;';", is,; thy ,defihjtely' higher. ~o!sinophilia-18'6per

. c.ent, in, mixed illfootion&, as against 13~5 per cent in single infedions. ' '~' / / I' ., I. • '

(2) bbs~r1)ation~,' on the. Blood Picture" etc., in I M mikeys,e'xperimentally , " 'infected with Egyp~ian Bilharzia,sis.," ,

, Th~,tollowing~bser.v:ations / are based op.:fifteenmoukeys artificially ''infectedwith\Egyptia,nbilha,liziasis. " , ". " , ' ,". '

Of eleven monkeys infected with S: mi1rnso'ni. ,two died bfthe ~isease: , " "~ OUour mQnkeys iufectedwitll' S.' hmin(1toqium tbree diedoLthediseas(3.

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At ~,va~iable, time afteriJ1fec~i6n, l;e.,' fromthe:~~cond to' the sixth week, ,some or all of r th(3fpllowing' 'syrllptomsdevel6ped : Emaciation. 'a~orexia, muscularweaKness,tremoral).Q, definite -shivering attacks, :,' If the infec~ed m~nkeys were going tbdie of the disease,these sY'lnptoms i increasedJin intensity; if recovery wast,6 ensue th~y:dimiilish~d. Follow~ ing recovery,;Jocl:\olizingsymptoms supervened at a later date. 'In infectioJ1 :with S: rnapsoni, dysenterlc symptomsllot uncomrp.otily".a'pp~aredauringl the seventh: to tenth week; ill' infection with S. ltcematobium, vesical ones 1 I,

\" ~ " '1,' , . " , .'" - / 'I - • - I, • _' .-

were not,ed from, the tenth to the'twelfth week." ' , ' " , ,! (a)Q,bs.ervati~ns on the, Blood Pi~t~re, etc,!'inMonkeysspo~tan;e~usly

"'reco'Qering.jrom Jthe'Disea~e.---'-InthEnen monkeys that recover~d from the' initial toxjpeff~ets of ra, massivecercarial, inf~cti6na' remarkable ,cellular~ , humoral respoiisewas,presentin every case~,The leucocytosis (luring,the fifthtot~e t.welfthweek variedfr:om 13,000 to ?4;000 per cubic ,millimetre,

Moilkey: K

,,-' -'----'-, '-,--'--,-' -' _'~_, __ ,----"-,- ------'- -, --,---'-, -' ----; -' --'--'-~~~~J~ro~~' ,so ~nsoni S .. ma;nsoni S. mansoni S. mansoni" :S"'.manso1i.i S. mansoni, ·s. mansoni

-'-----'-------~--, -' -, ----' ,-' --, - -,-'-',-. - -' -'-J '---I,

Week, ;\luriI!g - w4ichob~erI ya.tions were 'made'

5th 7.tb: ' 8th

5th 7th

,8th,

, --------------I-------~--'-"~---------'-Oomplement p+++'p ++++ p ++++ p +++'+ ' P ++++ P++++ p ++++ ~fixation* ",', P ++++ P ++++ . "

-------' -' ----' - ---::...----~--'~ -~.......,":"-'-- '""'--:----'~ ---'--~- -----~ Edsiliophilia .. 22'2per'Cent 21'6percent 10'7 percent' 42 per cent, 13:6perc-ent U'4percent '35per-cent'"

, ",' ' ':' 63'2 ;,' 24' '" " ~, ' i, , ' "

.Leucocytes p~r ~5,OOO ~"7 13,'200:-::- -"7J.i.50~ -]:5~600 -io.60o~ --34>400- ~17.000-cubic'milli- ' " ~8,OOO 14,700 metre

, _, _,_' _' __ . __ -.". __ ~ __ -:.-_....!..._ ~ ____ '_, _ ....:.-::..-__ --; ___ , _,_~ -:-~-~:-.- ~_"i_, __ '_'

;Recovery' or, Recovered' ;Recovered Recove,red" Recovered Recovered ,Rec0vel'ed Recovered I

death I

-----.:--"""+....::.....~-- -,-'-"1"-- :--...:.::..,;....:........:..:....--- ------,----"-'- --,-_'--"'" -' -'-'---C Post _ morteni Paired worms Pairedwotms Paired worms Paired worms Paired worms Paire(1 worms Pai.r~dworms, /

examiJ'!,llition and lateral· a-ndLla,teral- and lateral· an,dlateral·, andlateral-, and lateral· and1ateralspined'ova 'spined ova spfped ova spined ova spined.dv'a spined;ova sp~ned ova found "found found' found found ,found, found

--------;Microscdp,ic Psej,ldo -, tu-' 'Pseudo --tu' P~eudo ~ t1i~ Pseudd-tu- 1 Pseudo -,til- ,Pseuao"~ tu- 'Pseudo - tu-

picture . of, 'bercles of ' bercles ' ~of . bercles of bercl-es of bercles. of bercles of bercles of liver Bilharzial ' Bilharzial Bilharzial Bilharzial Bilharzial, ' Bilharzial' Bilharzia!

origin and origi~ arid origin and origin and origin and origi~ and origin and 'El 0 sino - , eOSlno· e'o si no-' 13'0 sin 0- ,e'osino - eOSlno- , ;.eosino: phiIe infil- phiIe infil- phile infil- phile:infil- phileinfll: ' phile infiI- phileinfil-tration in tration in '£ration in trati'on in tration in tratiori in' tration in

. ' periport,al, .. periportal- periporta:l peri portal' periportal periportal periportal' 'zones' oc- .zones; DC· zones; 00- , <zo~es .~ . oc" zon\lS ; 6c-\ zones ;'OO~ zones; / 00-ca si;;nal 'c'asion'al c asional ,.c.asional casiO,njl.1 casioJ;l1» I ca.sio,ti,al ovum pvum ,.OVUlll ovum' - :ovum , ovum ovum

, ,

* : To obtaill blood for, the complement ,fixation 't~S.t.,the ,ventricle of the heart w~~Pllnctured

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IN EXPERIMENTALLY INFElCTED 'MONKEYS.' , ,

',' If',

L 0' I p 8 ,; u -:-'-' -' - --',--, -,-' .-:.- -'---, --' - --' ----0' ~,",:"",,"," __ r ___ --'-' ~--' -'-' -' ,-'~-:-

s. ma"'s,o",i "'.s. mansoni, '$, hwm~tobium S. mansoni S. hwmatobiuin'S.,hwmatobium ,So hcematobium , , , ___ ,_, _____ ,_' -'-,--4-'-'--" __ , _____ ....:...._,~ ..... --,-, _': __ '_' _~..-----1_,_' __ , ____ ,_,

,~th 12th

5th 9tl'r

8th" 9th,

-3rd 2nd 8th

, " , , ' . / ~ _, _____ '_ --,---, -, __ ', ...J.. ____ ' _ ___ , ___ --,-_', _, ___ ,_' --'-r -_..4:--' ':"~--' -' --' ~-, -

P+++ ,Negative 'P ++++ NegatIve '~Negative Negative Negative p++++ ,P +++- ,Negative ---,--'--'---~-- -'----- -' ----- ----' ---' ----------'------'-,--23'4 percent 15'5'per,cent n'3,percent 'O'6p~rcent O'3'percent l:l'9per cent, 30'8, 16'6 -' -' ~--' ---'--"- ,.,----'-"-----'- --"'--- -~-'-' --' -', b,--'-;------' ---,,-+--- -' -' -':"'-' -

13,800' 7500" " 2 560 is; 800 '" ;, 1:250 ,

2,200 2,200, 8,800

~---'-"- --' ~~ -'-~-~- ~-~~-,.- --~-' - -' ---' -'~'---~--~- -":'::-, ' ___ " /' ~ecoveted, Recovered Re()overed " Died Died Died, 'Died' I

Pairedwo-;ms Paired ';"Ohns Paired worms Pai;Mworm~ ---' ---,-, ~-7--' ,.....:... ~---'--"""'I~-ir-e-d,;o-r-ni-s andlateral~ and lateral- arid ,termi- and lateral- " :andlateral~

, spined' ova spined,ov,3. nal-spined spine'd ova' spined ova. , .foup.d , fdund ~va ' found ,found, founa, ----~ -'--' -r ---, --'----' ------ ---, ---- -' ,---'---,-,-' -, ,-,-, -- -,-'--" -,-,.

Pseudo - tu: Pseudo-tu- Pseudo -tu; Pseudo- tU" Cloudy swell- Cloudy' swell~ bercles of bercI'es· of ,bercles Of ' ,bercles of ing of par- ing of par-

i Bilharzial, :j3illiarzial , Bilharzial Bilharzial ench,yma enchymJa origin. and drigin and origil! and ' origin and vasc,uta,r, ' vas c'uT a,r

" eo s i n,o- eos'ino- eOSlno- eo s i n'o- e Ago r,g e- engorge-phileinftl- pliile inlil- phile iuftl- , phile infil- ment; peri- ment ; peri. ,tration; in tration' in tration in tration' in portal inftt portal inftl-periportal ' pe~iport~l periporta,l periportal, ,,' fration with tration with zones; 0C:, zones; oc-, zones;' oc- zones;,oc- m'on"onu- monon n-

, ca s.ional .ca si 0 na I c~sional MS i ona1 clear, cells clear cells, ovum: ' ovUm , o~uU?- ovum

a.~d ,the'tequisite alnOi1Uto£J:)lo~d«() c,g.) aspiraten wftha'10-e,c. syringe. - \ ' /

Intense Pseudo ~,tucloudy' ' bercles" of

s),<:,elli)1g of . Bilharziai' p'arenchyma origin and.

eo si n'o - , phile, infll: , tration in"

_pei:ip~rtal ~ ,,i I zones; OC-,'

cl!osion fll , / ; ovull}.

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2152 " , The Immunit'r/ Reactions ,in' JjJgyptian, Bilhar~iasis, "., ') ( .', \ I'" \ ..

'; , T4us~he ser!l'/of,these fiyemonkeys neveryield!'lda positive:comple~. rD.Emt> fixation, reaction . .Inal'lc,ases there was aprbgr:essivele~c6penia ., a~d!~n elltire absence of eosinophilia w:ith oneexception;,Thisex;ception / occurred in Monkey I, T" who, during the ninth week, showed an ~osihophilia ~f 11'9 'p~r cent. I '.' , ! .!

'TaMe I 'present.s in tabulated form sO,lIle of tbe,pbser'Zatio,ns Dfade. ,It" may here be' noted that most of th,e monkeys weregrivet (Gercojiithee.us)'; , "O";ahi" T" were of the 'spedes yd,cacusrhesus. In the gri~et the

/' ,eosillophileis similar: to tpe: huinan, eosinophile,but the cytoplasm, retains '/ ' ,theba~ic staiQlO1age~ than n~ual. /, Itw~stberef6r~,,fouhd necessary, in

staip-ing"bYlLeisbman's,methbd; In orde~' to demonstrate the,eosinophile nature, ,of tlie~cell, ~o w~sh out longer than for, the usualperiod with 9i~tiHed ' water.IntheMaca~us.rhes1ts this/procedure is ullneGessary. Inevety (',ase " p:t;ior to cercarial infeCt:i.on t4e hlqod of each mo:p:key yielded 'a 'negative,' cOinplemerit fix,ation' ~est, and the normal total leucocyte and differential,' whlteicoutits were' observed. ,.' ,! i ; , '

" "'" (c) 'The; Cellular Response obs~rved:i1/; the",Focal' Bitharzial L.esions'of t"h!eOr,{Jffj~8' hi Infected 'Monkeys.-As, I p~bpose ,to describe at 'a later d~te ing~eater ~etail p~tholpgical chang~~ ~:>f 'awid~sprea~,and toxic,~atJlJ,'8 in '

t mprikeys dying in the, first few weeks of ,the disease, I shall briefly.refer ." '. ' f ~ , ,I' ; {,' . ~: ~ "'

here to the, main features. ' ' " ,In ·the< most acute fo;rh ,no fo~al 'lesions are ,ob;erved. - The m9nkeys, die ofalla,cut~ ~oxffi~ia 'With dijfuse cloudy swelling of all organs (see

-table). . .. '" ",' ," ",I

: :The appearance of pseudo~tubercles on the sm;face of and throughout , . 'c~rt3:in visceritj at ,post·:mortem,was the outstaJ:tding feature)n later stages" "

\Tbeirdist,rihritiondiffered,somewhat in the two species; whereas in S.;hcematoliiu'tninfecti()tl:tnese ~efe 'found in the 'in.testine,' liver~ lungs,' and pelvic organs; jn S. mdJtsoni Infections ,theywere corifineuJo the tW9 , fobner orgaris~ , ,"", '" ;' ' '-'," \ " ,'" /' , • : Such:apsEmdo~t)ibercle on: microscopic" section. presents a pictpre of

',eosinophile infiltration, formation of ,giant cells, and mononuclear infiltra';' tion, 'arid sometimes a centralcasea~ion .in ; addition:. Such findings/are

, comparab~e to thefindingsof'Balir {19] in 'acute filariasis. " \ As the/3e changes are not s,trictly,confined in microscopic Erection to the

vidnity of either the adult wor~E! or their ova, it is reasonable to conclude , on ·pathological grou:t;lds alone thatthE!y result, not from" mere mecha.nicaJ"

, irritation, / but Jr9m a; (v,\idespr~ad!and: diffusible, toxin produced by the ;~dUlt. worms, and possibJyhy disintegr!t~i'ngova. '!'

'/

(3) Resume.

,; (1) Ooservations ! on 'bhharzia~is b6th in man !1udin experimentally. inf~cted' monbys showthat the 9y~ologjcal I:~~ponse to the bilharzial toxips 'is lan eo~ihOphile cell. These cells arep~esent(i:Iliri,creasing tiumb'ef~nQt

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only in the circulatingblood:b~tal~pin the l~cal; l:)ilhar~iallt~sjbhs;whethe~ " " in, the liver" intestine" bhtdder,ll:terus or IUllg!,l:l.ndare init,epeildent of}he'

species of infection. (i.e., S. h03matobium or S . .mansoni.) , ' , , " ' . . " (2) Al1'id~tical' cellular 'response results from ~ither'species"o~ infec~ , ' ·tiotl'-::",whether!d1;leto S.manspni or S. ,h03mqtobiu~. ,The presenceQfa' .

. ,mixed,infection evokesa,higher eo~inophiJiathan does a single ol1e.! ; . . . Similady,a miCrQscopic e.xamination o£the local lesions in, the two different' sp~cieso~' infectionreve~ls n~ ~es$ential difference in the .ceUula~ respohse

i '" , _ ". '. _ _ - ",- \, '\'. '( I·

lo~a:Uy,u()r indeed intne focal pathologic\1L,l?icture. ".', :, . ' " ,~. I

, '(3)F-ollowing invasi6n ofthehost bY,bilharziacercarire an eosirlOphile )eucocytosis of ~arked intensity becomes established a,bout the fifth to the

"i ,ei~hth wedk of the disease. , ;In: experimentiJ,llyinfe'cted', monkeys, within ,'this period 'oftinie; a leuco~ytosis, of 34,OQO; tOgether with an eosin<;>philia '.ofsi:ti;y-£our, per cent,h~~ been observed; , , ',' " " ", . " ,(~) :i:f1,man,afterpe~sistini fori ~, few 'weeks, this abrupt, rise in: the

eosinophile elements ,bf,the blood is followed by 'a, stea.dy decline' for ~everiJ.I : months, until. a Ploderate-grade eqsip.ophilia apd l~ucp6ytol3i~ areel3tablishe,d;

, ,Thrts, in observlttionsfro~ the; sixth totlieeighteenth 'month' 6fthe disea'se,. ,tIle average count was as follows :-r- , , '

. tn pureE .. h03matobium,infect~ons the leucpeyt~sis 'eq,uall~d lO(03q per . cubic m:illime~r~i andth~.average eosinophilia was,13'5 per cent:. ., ,.~.. In 'doubl~'infectiQris(S .. h03mat()bi'l.l;mand IS: nransoni) the leucocytosis" . equ!LlIed,10~500 per cubi.c millimetre; .the ~verage eosinophiJia,was lS;6.per I

.. ·cent'> . \ I , • " • ',~. \ I

", In thechro:tlie stage of biI):Ht~zia~is (i:e. ,of m~ny year~' stan~lng) ,:.the ;.' 'eosinophilia is.a much less constant .featur~of the disease. , Th8'ihcidence

. ofa secondary pyogenic infection eng~afted on. tba bilha:rzia)ulceratlo~~ whether v~sical . or "iritest{IiaI--,-results in rt ma~king .', of, the, eosiil;()phile , ce,11sby', inducing ~ri.' response !to,', it :a' polymo'rphonucl~a,r neutrophile"

I' 'leucocytpsis.. ' " \' ", .'. '... .' ' .. ' ". ' I, >l' "," I • . \

, (6), Inbilharzialdisease .eosinophiliamay he abse:nt :~..: , '. ", " /., / / . . (a)· 'In case!1.of hyper-infecti()n durifg, the' early to*i'BInic'stag~ of

. thedi,sease. (See Table 1.)' , \' j' . . (b) In milder cases of the dis.ease,evon.during ,the early,stages of

, . , J

: " locali~ed bilbarziasis. . ", ' .,'" .•.. ". ",! " ,

(c).Iri chrb:q,ic bilharziasis, e&peciaIfy ~f as~ociat!'ld with asecop:dary , pyc;:>genic infection. ' ." . '

, (.7) From a, 'diagnostic standpoint thip,resenceof" an eosinophilia ~may be a materIal. aid to diagnosis. In the, early stages of the disea~e ~ts yalue'

:r is undoubted,cItjs hot so constantly present during I the ,stages ·ofearly !locll:!i;zea'bilha~1iasis"and in 'chronic. b.ilharz~asis its value:is'less,ceriain .

. stilt. It nevera,ffords so constant 'or .s'o cert~i~ari iriqex .to diagnosis as the .. I , .c?lnplement fi:x:atiibnreac~ion now tob.e described. . 7,1\ '

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25f The'Iinmu,'nity, Reactions iri .1J}gyptian Bilharziasis - . /

, . , ,. " . "\ ",-SECTION I¥ .-THE 'CoMi=n::EMENT ,FXXATIONREACTION IN' BI:E.HARZ'IA:SIS';

: ,," The frequency ,with whIch~urticatia, fever aIldother ~yrnptoms ,we~e observed in ,the eaily$tages;;orbllharzi~sis, al1'lODgst Australia:titro,ops ' strongly suggested,thepresence ofE;ome ex~toxin, the product. of the' maturing or of the adult worms.Seeirigthat th,ese initial symptoms'

, \\

, s~bsidedafter a- variable period and the 'patient completely re~overed "/ u:q.til such a. ti,me ,asth'esigns and symptoms of localized ~ilhaI'ziasis supervene~, it seemed ,to me probable. on a priori grounds' l\1at :recov&ry " should beaccompaniedbythe fOJlmationbfsome specific immune bodyin the hI,bod. " / ' .', ',' . ;:' ,I!' " ":,.

. In order to investig~te th,epresence of this hypoth·~tical imm'une body in ,the blbqdof, bilharzia:!. Pfl!tie:r:tt:;;.' the, Ingenious-method qf fixation 0'£ .. \

! pbmplenient-(lescribedbyBordet andiG:engou [20J jn.1901w,a& utilized. A specjfic 'antigen was prep~red from\ the livers of snails (P/c/norbis !Missyv), infected with B. mansoni; and after' seve~al weeks' preliin.inatyexper.imentation I,was,a15le to report iu May,1917,tlle aiscdvery ofapositivecomple-' mentfixation ,test for.bilh~rziasis' more' s'pecifi~for this! disease than is ,the correspo:tiaiIlg'Wa>}serm~nn test for syphilis. ! ,During the Ipaet twelve months inore than 600 speciniensol sera (from '6ases . of bilh'arziasis, frqm , Gfl!sesqf'other specific'ilieeases, as well as from ,normal healthy peop.1e) have. beeri investigated with remarkably satisfactory results.

, (1) The,Phenomeno;'/, oj,oomplemeit't Fixation.

The complement fixation reactioriis :'dependent pn~:th~ .fact' tllat when antigens inac·tiva.ted serum containing .immune body and complement are wixed togetherilimmunebody: firmly combines with complement'in such a! manner ,tllat comple!llent ca,nnolongerbe demonstrated in the'lllixtlir~;, If sucha,~ixture ,IS incubated at 37° C. for, one hour-or more, and to i£' is added iL ~uspension of sheep's red blood corpuscl,es sen,sitiiedwith it suitable quantity

'"of inactivated hoomolytic serum '(o'btainedby i~jecting iJilcreasinglloses" 'of sheep's corpusolesinlorabbits),n? hoomolysi!) ~iUtake ~lace, since there

I is no £,ree complement; / '!'his ~1>sellceof hoomolYElis cori~titutesa positiv~. "reaction and proves the\pr~sence of specific immune body in the inactivated 'serum. If . the ,conipreni~t is, not :qxed !'thenhOOinOlysis"ens'iies.Thi13 constitut~s ,a negative .rea,ctionand ,demonstrates theahs.e,n.ee of specific imn111nebody in the serum un~erinvestigation. " The essentia,l problem in . applying the complement fi4atiou, test, to anynew.disease iseentred in the I

. production ofanantigell,whicb, will yield a,:high percenti;tge.ofpositive. reactio~sdiaghosti(ll:ofthe iiliness ullder consider8(tion, and at!~he ,same time w'illprove specijicfor that qisease only, ,that is to' say, will not give pseudo j

reactioris with the sera of suhject$ s1Jffeiing from .otherinfe'6tjons. ' ,', \, ' During the present investigation two diff~rent. antigens havebe~n

utilized,OI\ea saline ,extract, the o'theran: alcoholic. ext~~.ct 6f 'tl)e, livers ofi~fected snails.· The latter antigen., ihas 'provesl the 'more ' '

", satisfadpry oft~e two~'

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(2) Technique employed: in th(3 Complement Fixation Test jorIJilharziasis.

'As'in other 'compl~m~nt fixation reactions,it; must:bB' realiz~dthatthis ' test i~· essentia.llyi aqua1ttitative, tiot a qualitative one. It ~s dependent on, the fact that this specific cercarial antigen" in the, presenc,e of serum ,ftorn a;

/ patient infected with bilharziasis (immune body.), will fix a gr~ater qU,antity , of complement than it will in the presence of an aver~ge pooled 'serum d-erived from non-bilharzial subjects. As in ,the' analogous WassermaIlll

'reaction,accurate st~ndardization of the'various reagents used in ,the test' is, "neqessary. , "

; . In thenrst stage or tb.ereaction a'" known qU~lltity of' stapdardized> antigen is mixed with ~ known quantity of inactivated patierit'sserum:iu

" ,the presence of'gI;adedquantities of compiement of an'asc,ertained strength. , ,This system is incubated for one hour at ,37°C. After'this incubation

is complete a given ,quap.tity'of sensiti:z;ed sheep's corpuscles is itaded.to, the' syst,em, and toe whOle incubated at 37° CL fora~other hour~, 'During this period of onehour:s duration readings are made'at jutervalsof a quarter of an hour, thereadingOs ma}le at the end of the hour'being taken,as final. , A ntigen . .,-The" preparatioll 'of the two < diff~rent ' antigens, used duri'ng this investigation ~s as follows :;- , ' , Antigen (A), Saline Extmct.-A number of infected sri~ils' livers were • macerated'in a solution, compo~ed of 0'S5per cent saline and 0',5 percent phenol. The, amount of fluid added was, dependent upon the ,n,Umber of ~s~ails~ livers utilize(!:' ~he mpst satisfa~toryst,andard,wa,s found to 'be one,cubic'cimtill1etre of isolution to each ;snail liver. Thus, if the e}Ctract , were tone madefroni}wenty infected Ii,v:ers, theI1twen~y cubic centimetres '

',of solution were ,added." This rriixtq.re was sha¥eilth()rojlgh~y OIJlm electric 'shaker, for twenty minutes, . and thenin'cilbated at' 37° C. for twenty~fourho,lirsto permit of extraction; it, was then filtered; and 'the

'fresh filtrate used as antigen. ' . ,,/,' 'Antigen (B)" Alcoholic Extrcwt.-A nuipber ,of snails' livers' were

}itacerated, i~ 'a' quantity p£~bs6lute alcoh61-::-g<;lnerallY· thirty livers ,in" thirty cubic. centiin'etres:,of. alc()hol. Thismixtu,re, after thirty minutes" ,thorough shakirig on an electric sQaker, was then ,incubated fortwenty-fQur hours or loriger at 37~ C., being shaken at 'interv:als. ',The mix:ture was t~en filtered. Next, the filtrate was 'evaporated by means ,of a ,Spre~gel's exhaust~pumv, airbeingdravrn:through the solution which was k'ept at 45° c./~:n a water~batlL ,,\yhen thoroughly dry, the' residue was weighed. This'wasaccomplished by weighing the tube while, empty and af~rward,s

- weighing it again containing the evaporated residue, and then estimating' the difference between thetwp~ ",' - " " . .',

"In ,standardizingfor use, twerity,cubic'centimetres of solution of 0'85 per . cent saJine and 0'5 per cent' phenol were added to 0'05 grm: of this residue.' The, ,resultant' mixture was shaken for tbjrty,minutesornio~,e "oil the' electric sh~ker and filtered: 'This fresh filtrate I was used .as'antigen. <, .,' ,,',I:" -"0, --.

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256 The Im/niunity, Reactions, in".Egyp{ian Bilharziasis'

OIlst~ndi~'g it tended to become OPale~~e~\t;~nd' wa~ of a ~aJ?ary:~e.lIow colop.r. " I' ,'I ' , ' ,

",' Inthe,seriesr~por~ed on la fresh alcoholicext~act wasalway§used, ,since e:o.ougp.i:nfected >snails were always ,'easIly o'btainable f SomE) f.of the

, , Sel'a; however, w~real~o tested against an antigen prepared'from alcoholic extract. of snails' livers that' had been stored on ice for five months. The~ 'fel'ults, were fair.l,y satisfacto~y,.though ,the long:stored antigen provedles~, , sensitive tlianthe freshly,prepared one.", " " ,'_, . f

;' Af;te~ considerable investigatioIl the I~aline extract, (antigen ,}' A~')

/.

was· disparded" and' ,the alcoholic extract ,(antigen "B ':) substituted. ,Thereas,ons for 'regarding the ,alcoholic extract 'as superior', are as "I.,

follows:--'- ,,; , " ' \' ,,' , , , , ,"" . , (I) A higher 'percentage of POFlitiye res~lts was :9htained, with the"

alcoholic extract' in bilharzial infection. " ' ' f ' \ . I .' . 1 I ./ '

(2) , 'llherewa:~ no tendency to pSEmdo"reactions in non-bilharzial sera. I

{3i .Accurate' standa'rdizationof I the alcoholic extract Cbuld. :be niore easily: carried out! ,"',,' I'" "'"', " .

. (Jomplernenb",-The complement was obtained, from well-nourished 'healthy 'gumea-pigs 'and' cqllected, lluder ,sterile co:ndi.tions.:Accurate· 'standardIzation ,of complement was' always carried'put. The M.H.D.

,; (minimum hre'illolyti~ dose) 'was6btained", lrithe final test six ahd twelte M.H.D.s were used.' ' i

" A', positive, diagnosis 'w~s/ n;iade whm;e' an inactivated ,serum 'fixed 'six> M;H~D~sofc<?mplementin thepresence~.of specificlantigen. '"L

A.rraiigement'of System for the FinaZ'Test.-TI!ree,rows of tubes as . I

shown b~low:"-o-, ,.', ( .' , .', f ,..'. , i l. '

T,his series coqtain~d :,-Row No. ~"

Antjgen , Patient:s serum f , Diluted complement (six M.;r.D.s)

ch c.~. }M~de up to 0-5 ~;~,~-.Jit, h 0'1 " .• "P-.1 " ' f 0'85 p~r cent sal~ne ,

. , / ..

" . ' Pipettes of, known calibre, staI'!.dardized .. by ,the lY.l'orse gauge (Donalirs', . method), wer~ used'1;o,6btain the right'volumes ofthe reagents.' '/, ,,'f

. . I' " . -

'/ !low No. 2. ' 1,(

Antigen , •• '0'1 ~~c'}' "" ", ' 'Patientis serUm '.. ' ,..' .,.. ~ .0'1 ' , ,Made up to,O'5 ~,c. with,' Diluted c~mplemimt(twelve\M.H.D.s) '". P'2 ::."" 0'85 'per cent saline "

., I. r- - I

" Row No. 3. - , " Patient1s serum· f •• 0'1 c'.p.' '} Made up'tdO'5 c.c~ with

Dilute\i c'oniplement f .. ' .. ' 0'1 '" 0'85 per ce~t saline, , I /

This row (3) serves as a serum control, . and, any :anti-complementary , t~ndency ,in' individual', sera' examined c~1i easily be demonstrated. "

1 .

, Additiohal cOt;ltrols used in. the test were:~' , :, jt)Antigencontr~l'-antigen, one cubic c~ntitp.~t~e ni~de. up' to O~5 I 'i

" cubic centimetre with,O'S5 per cent saline"., , ' .! ' \,

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~, e2) Pool~d ,negati~e ser~~ : i ,';(3) Sure pQSitivesera., , .. " '

, '" The q1iantityof\antig~n flxeu, by a. pboled negative serum ;wa~ ,also determined by adding varying' amo'U,nts of complement., ' , "

/1

r< -, 'The following is an; jn~erpretcitionofiheSiil}; ,utilized in recording

, the Resul~s. \ ' " \', I ... ~ ) '... \ "

P ++++ 100 percent of complement,:fixe,d' , , 1

p+++ 75 '" "

P++ 50 "

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p+ 25 " " "

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{ C01Dplete fixation of, c~:inpl~mentin :"

, tubes 1 and 2' , ' ,

{ Compiete fixati~n of compl~ment.in, , , tuJ>e 1: Partial hremolysis in tube 2

{Complete '~tionl of ~o~plenlent , ~n, t~be It ,Colllplete hrelllplysis" •

m tube 2' ", '

{

'COlllPlete. fiX, ation ofcomplem'erit' in " tube 1. Complete hremolysis in , t~be ,2 , ' "', I , i

Note.~A P + reaction on theabov~ba~is was r~g~rdea asp6si'tive, evidence 'of bilharzia 1 infection only in the presence of strong clinIcaJ sIgns

, of the disease. - , ,0 ',' ,2 - , ',,' ,\ I . H'cemolytic,Serum.----;-T.his was obtained" from r~bbits by in'traperito:ri~.al'

, injection o(increasing doses of sheep'S corpuscltWwhi<ih had been repeatedly, , washed in saline. \ ,The serum used wa;s, of hjghtitre, active in ]>t04,OOO. diluti,on. '1'os,ensitize_ the;sheep's corpusC1~s, folirM.lI~D.s:·o£' amboceptor

or 'hremolyticserum' were used. ',The', M;H.D;'9f ambocept()r was- taken to' be' that amount of-hffimolytic serum which, ,wm~" fqur ,to fiv~M..H:p.s

},of complemeat, was sufficient to produce in one hou.r,at, 37o.C. comple,te hrerb.olysls' in one cubic ,centimetre ofafive 'per cent suspensIon of sheep;s

',corpuscles; , I " -f "'" " ' ", ," : I '

, Sheep's 'Corpuscles.~Equal quantities, of sheep's blood a.ndtwd per cent sodlumcitrat'e in pliysiologicalsaline' werec'mixed togetq.er.Required I

~mounts ,of this mixture "received "three washings with nine times the I "volume of, physiolqgical saline and were finally made up to a five per"

cent srtspension of sheep' s' blood 'inphysiological saline. "," , Sensitizatio'iJ; ,ofGor~uscles:~After four ~.H.D.s of ambpceptor' hf:td

been' addedto:t~e suspension of sheep's ' ~()rpuscles the mixt,ure was" incuba~~a' 'for' thirty min~tes at 37° C. ,After compl~tion of sellsitization / in-, this ~y, 'the suspension of ,corpuscles was kept, ;in I an ice-chest,till' ;reqU'ired forus~.' ", ' , , , Patients' Sera.-Blood was 'usually obtained on the day pte'ceding the

i test and kept ibthe ice"chestti-ll' required; The SerUIn was' then diluted wi~h fbur/t'ime,s its-~volume _ of physiological saline and heatedto.l?6°,C. in a water-batl;1 for twenty-five minut~s.'· By so doing,' all complement was destroyed. \ _ I " , ,- , '

" ' ,Note.,-:-:It 'is :q.ecessary that sera',shollldbe not more thEm, forty.,eight , hours old. I •

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258 The 11IJmunity React,ions'in Egyptian Bizharziasis I . '/ . .,

/ .'; ..... '" , . - . .

(3) S,ignificance of the Complement Fixation, Reaction in Bi?harziasis . . ' . , • \ \' / '\' i /" • .~.':'",

.I~ a I?igroscopic ste?tion of. a heavily infected snail's 'liver' be examined, it w~ll 'be noted that the 'great. mass of 'the liver substance:is in.reality composed ofsporocysts, and cer9arire: They distend theiriter-acinous

.. spaces, 'and cauSe atrophy.'of the parenchyma. Such being the ~ase" it, \ ' • , • < "\

. follows that any extract of such anorgltnis largely an extract .of sporocysts a,nd 'cercarire. . 1

Oh several occasions antigen, was prepared by making extracts from cercarire wqich had' he~n fioated,6ut from the liver, suhstance, but such extracts. were' no more efficacious than those' of infecte'dliver itself, and were ~mich m01;e 9.ifficult.to prepare. \ . .'.. . :' .... '.' . :

.' ,On another "occasion an antigep was, prepared by making' an aJcoholic '. "extract'of adultw;orms collected post mortem from the portal system of an Egyptian who had .die.d of bilharziasis. The great difficu,ltyof obtaining a:' .sufficient quantity of worms made the 'l,llanqfacture of such an antigen" ,impracticable:, .. ,'. " ' •... , '" " > .' '.' ,

, In ~rderto demon!)trate that the sp~cific a~tionof thisap.tigen depended on the presence of bilharzial cercarire, apd.wa.s ,"independentofanye)dract: derivl;ldfrom' normal snail's liver, or from degenerative ,change& induced by parasitic invasion of that organ, the following'experiments. were :carried' out.

'Expel'iment' I.-A. sari ne extrac.t was prepared ,from thirty normal live,rs of. Planorbisboissyi ~o~ use as an antigen, and the identIcal technique' was used as forithe preparation ofaJiltige!f' ~'A/' Fifteen bilharzia.! sera· .. a~d 3r number of known I.l'egati~ese~a were/te,stedag~inst' this antigen. 4bsotutely no tendency to. 'c6mplementfixation:was shown. by an:y . of the.' bilhar~ial a,era; . '; I .', ".' . ' "

. Expe~imentJ,I;;An alcoholic. ,extract was prepared from livers of. snails of the. species Bulliiu.i.s,dYbowski infected with the (jastro,discusa3gyptius cercariffi; from' this, an. antigen was produced by a te~hnique similar to":, tha£describedfor; antigep..!' B.".. . <>' .' .'

A ri,umber of known bilh~rzial·~era"were .. tested, by the cOp1plement 'fi;xation reaqti6n against this antigen,' and" all' yielded negative results .

. Tlie,point isthatthi~ fj.ntigen Illlust h'!>ve containe~ products of th.e· degeneration of the liver tissue;produce<l during the gro~th of sporocysts . and' cercati~,and also' aconcEmtra,ted' extract of thesiBgle,.tailed pigmented cerca~ire .0£0. a3gyptius, (Cobbald) aswelL > ' . '.' / . ""

. ,It follows, tb,el;efore, -that the antige~icpr6perties of ·the e~tract ,of : snails: livers inf(jc~ed ~ith bilharzia. must, be' dependent upoit speqific suhstance~ extracted· froIl). the cercanre, themselves. ". , '..'

.. T~e . antigen therefore, is quite specific, and in .thIS r~spect. differs entirely from the antigen useq. ,in the Wassermann' reaction forthe detec-

, tiori of 'syphilitic immune body. 'Syphilitic antigeB is non-specific in S0 .

. ' ''far tqat a variety. of l3ubstances! of' a -lipoidal character .. act e'qually ase~c~ci6usly.a;s does,antige~ prepa_red from an extract of Treponemata'

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pallida, or a;s anextract,whe,ther it~be saline or alcoholic; pr~pared from a' .syph~litic liver; ," , . ',' ", ' "/.

o Furthermore, this, reaction 'is mote specific than isthe Wassermann reaction 'in 'ano~~er sense of theterni: Prbloqged experiment,s with a variety of sera ,ha:ve failed to demonstrate/any constant tendency to pseudo- ' reaction ill, any disease or group' of, diseases. ' 'rhe sera tested 'inglnde

'tho!;le ()f relapsing 'fe~er,_, syphilis, ~u'bercnlosis" enterica, hyatid disease;', ,:other, helminthic infection's,pellagia, etc. - , ';, • " , '" , "

In, the case 'of thei Wass~rtnann 'reaction there 'are several 'allied protozoaldise,ases~s~chas y~ws, ~elapsing' fe;er, trypanosomiasis, and perhaps malaria as, well, which give 'quit~, commonlypSEmdo-pOsitive complement fixation, reactions. , ' ,

In the majority of 'my serological investigations an, antigen prepared, . from snails ofthespecies i Planorbis boissyi fnfected with S. mansoni was

utilized., ,This alltigen wasfound to be specific for S. htEmalobium.infec..: tions, as well.lli, a similar fashion, an antigen prepar~d,-fro:m' Bnllinns snails i~fecfed with S. hcemq,tobiumcercarirewasfonnd to be quite e~ca- ." cious iridetectjng either of the tw~ v~rieties ofbilharzi!!,sis! S. jmanl!oni , or' ,8. htEmatobiurn: "The similarity of the Initial toxic ~ymptoms in Ilian ' would suggeilttha~ a ,similar toxin is given off by both worms/ a~d, the above ,experiment!) bear out this contention. It. is probable, then, that ,a similar. testis applicable toS.japonicu;m infections as well.; . ..,' ,

In view ,of 3;ll,th~se observed f~~ts I regardtbis comI?l~m~~tfixation. " ' t~st 'as a. true Bordet:Gepgou reactIOn, dependent on the fixatIOn of the ' 60trlplem~;nt by a' specific immune "body '",hiebexists in the sera of.,

bilh,arzial, patients, in the ,presence' of its specific, cercariat'antigen. "

(4) An Investigation la/the Sera of 311 Haspital Patien:ts usi1igas Antigen, ,a,~ali,~e Extract of/nfected Planprbis Sn,ails' ~ivers (A'ntig~n :'A :'). "

. (a) In' a ,group of cases whose bilharziasis was ,of not more than two years' duration, ,39 ,out of 5.4 (or 72 per cent) yielde-d positive reactions. ", , " (b) In another group of cases whose bilharziasis was' of, more than two "

y~ars' duratio~, 81 out' of 46 (or 67'4 p~r cent) yielded positive reactions .. ,,' (c) Thirty-five cases ,yielding positive~Wasse?;mannsY'phi!itic reactions"

,w~re all / negativ~ to 'my' 'test, . pro~ided the saline antigen was freshly prepared and "the sera not more than forty-eight, hours old,.' ' "

,( d) One hundre~,.and':seventy cases suffering from :various 0~4er diseases '.\, were exaITii,ned. All yielded negative results. '(In: this,series \vereinclude,d ' . cases Infected with ankylostom,iasis,.hydatid disease,Ascaris lumbricoides; peiIa~b:a" typhoid, tuberculosis, gbnorrhooa. and. many other diseases.) , :

(e) In twelve. cases a tendency to' ,pseudo-reacti0-11 was o,bserved in "non·bilharzial sera. , ' .' ~, ',', / ' ' \, .

, Of these twelve c,ases six yield.ed a P + reaction only: in the ahs~nce I

,orthe 'clini~M signs oL'bilharziasls S'Ilch a reading, is rega,rdedas valueless, \ . 'so'that these :cases cau be negl~ct;ed ... , " , , \ " '" ' ' ,"~'

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The Trnl1~unity,' Reaptions' in Eg,yptian Bilharzia/sis ' , . ", 0fthe ~emaining SIX' cases (i.e;; 3'6 ,p~r cent' of~n 'negative sera eX~IP-ined).' ' '" > 1 yielded a P ++++' reaction'

1 " P +++ '''" 4 "P,++'

, In the' case of the~e pseudo-re~ctiom;, ,three occu~red, vvhen' using '~n" antigen seven days old, and might have oeen eliminated by the ~se of fresh antigen; The rerilJlining three cases, ,'Yere, dejinitelye~ses oC pseudo;, reac,tions with fresh saline ;antigen~,' , " ,,:, ',' / '", •

,'Itcannot be top strongly emphasized that antigen prepared as a ,saline extract of infected snail's livers ,must· be fresh when used. 'At times~ such' , e~tracts, even)£ k~pt on ice, "quit,e 'un:a~~ouI)-tably developan'ti~c'qmple~, meritary tendency, andmay'lose their, power of ~xjng, complement in the ' presence of bilharz'ial immune body. A similardeg~nerati'on maybe noted ',' in, 'other saline an~igens' prepared either as bact'erial, suspensions, or as:, .. imple 'extracts of. syphilitjclivers. '.. " '

•. ' 'c ..! ' \

/, (5) An, InvestigaW,ortoj'the S~ra' oj 280' Ca,ses using'-'as Antigeli an, Alcoholic ,Extrdct o/Injected Liv(3'1's (Antigen "B "). "

, " '" /" '

, (a) In' a g~oup l of 21 cas~swhose bilharzia!'lis was 'tmd~~ two, years duration, 19 (,--or 90 per ceq't) :yielded p,ositive re!:l>ctions. .. " .. (b) In a, grottp of 85 cases whosebillj.a,rziasis ,was oyer two yeltrs

duratioLl,65 (or 76'5 per c~nt) yi~lded positIve results. .., ,I,

(c) Thirty eases,yieldirig-PGlsitive\ Wa.sser~nann syphilitic reactions, wer~ "all negative to ;tI~is test. i. " ",,' ' . .. . / ..

(d) 'One llundred ,and thIrty-five othetcases, ,Ill eIther normal'health or suffering froci' various 'disease~, 'yielded I~eg~tive ~e8ults.; " , "

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, ,(e) Eseudo,reactions w'ere, never met with., " In only. onecl1se, was a' positive reaction obtaiz:1ed where bilharzia, ova were hot subsequently demoristrated. This occurred in the case of,a 'soldier' who ,had bath~d ina 8W,eet-'Yater carial known to be i~fective ; he preseq.ted ~e.c£~l ~nd sligp,t I1tinary 'syinptoms.A, single examinatioli of the excreta ,proved negative.

" '

'/ '~hisinvestigationc6uid n9t b,e regarded ~s ,qomplete, so ,that bilhar~iasis " could not wi;t.h ceI;tainty pe:exciudedin this case" . ", '

e ," • ~ ! " ' '~~ " "

'," I"

(6)' Relatipnsh~p b~t!ween. the 'Grade ,oj Eosinophilia'and the, COnJplement I

, Fixa'tion Reaction in Man. ,"

, ,'-, , In fiftycase~ of bilh,a'rziasis joint ob!\e,rvatidns were:~ade on, the .. g~ad\'l of'eosinophiliaarid the cOlllplemenLfixation reaction, with tijy object ,of determining the relationship, if,any, existi:r;rg; between the 6ellular ,and the '

I.

humoral reaction to this parasiticinv;asion:, \ " ' The, observations may be' ~ecorded 'a'~ jollo~ '.:-:.., ~,

, ! GroupI . .2..In 23,case!l oCbi~hlnzia~i'syieiding an:eosiriophiiia of'from, " '.'''~, to 10 percent 16 (or 69'5 per cent) yielded positive complement'fixation

reactions. It may be observed, how,ev~r:l ,that tpe anioun,t of complement ' \ " I , • •

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N; 'Hafniltonjj'airley . ' fixed! wa~Jot as gi'eat~s in th~ 'Gr6upslIa~d;nt, i.e~; th'~ 'eoI:J1ple'ment'

\ ," fixationreactions'werenot$'o matked. ,', ,,'I,"~ 'I

;'G1'OUP ii.:-In 21 cases in, which,th~ ,~cisinophiIe, count was 'between' , \10 per cent and 25 per cent 18 (or 85,'70 petceIit)yiel~edst~ongpositi:v~" ' , complem,ent fixatioh .react~ons:,'. .) I \' ,',,' 'i~'~' ",' .,," , '

,,' GroupIII.--'-In \6 cases in, which: the ,~osi!lophile. cQun~ :was b'et1i'een, ".' 25 perce:'~t dnd 40 pet· cent, 6, cases (or 100 per cent) yielfied strong po~itipe,

C01ripif(men~, fixation' req,ctions. .:,',',,',;:,' "':, " ',," " ','" '. Fro~ these, 'ob~~rvatlons it'wIll h~ seen "th'at,th~l?-igheithe gr:aqe ' of

eosinophilia present; the greater .the tendencyforthe~erum t? give positive', , ,re~ults by the corllplement fixation :.;oea'ctiom , It J;llustbe emph3tsiz~d; how- .'

ev:er,. that po~itive complemen~ ,fixation\ reactions have: ,been obtained in' a',; ,,'

\' , number of eases w~ere the 'diff~rEmtia,l'~dun~ ha~ shown 'the Broportiqn qf ' ' \ eosinophi)es,as low as, five per cent or under~ I ' " ,. " ,

; '-.. . (7) p,ractj,cal Value of,thi~ qompl~ment' FixfJ-t,ion 'Reaction: "

There are, two directiops in ,which this ~est' hasa',ptact'ical appli~~tiQn.:. ' (~) ,As )L, means of~iagriosing, cases of bilharziasis iJe'ry' early in the ' ';

, 1-isease (p1,'ior,to,the pnset 'of localizing' sympt~mls in ,th'e 'bladder 'a~d" , ',intestine), and 'also as ,it method,of diagnosisinlater cases where. localizing ,

, ", . <' , ' , I. I .'.', " i

'., symptoms' are obscure and' ova are,difliculttQ ,demonstrate. " "," , , " (b)Asa ther'apeutic~ ind~i to;the'effect Of~ gi~en drUg on the bilharzia.

,worms, lrithis, respe'ct t~isteststaIl:ds, in, tlie; sam,e r€lat~()llship' ;to ,bilharziasisl1s, does 'the Wasser~aJ?~ test to syphilis. , • " ,

(a)' The 'Diagnostic Value,oj, theOompiement' Fixation Reaction in Bilhariia'si~.~-P~rhaps the ~dctical value of th~ complemetitJixatiqn test> can Q~st be' realized by ireports on, th~ follqWing groups of 'cases':-,- , .',

, Report (l).-For th~~e months,the ,bI'oodsof 'cases pres'enting liri:tuLry '"

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" sy:qlpto~s such as hremat~ria,pain; and freque~cy, ,wet;e sent to me for, , examination 'from anumb~rof British military hospitals ip Cairo: In onlY',' ,', three case~ ~a:s' I ~suppli~d with~ny previous information with re'~erenG~ /, ' ' " '~,J" to the presfmc~'of ,ova in the\ u,rine. ,', , ' , " ' ',' ' " , ,.: , '.

, /

, These' cases were tested by the'90mplement fixation re~ction, using,' 'as, ,tiritigen the" saline ,extract ,of: infected li~~rs ofPlano~bis snails'· (antigen" A ~').' , , ' "

In fift~en cases a positive diagposisoL bilharziasi's was made on, the' , bloodfindi'llg!Jalone;oLthese • 'I: ' ',: . 1

,J" ," ' )7 c~ses 'gll-V!l P ++++'reactioI)," " ,,1 ",,, p ,+++', ' , " "

. .', ~" 5 ,,/ "p++ "," 2', p+, ' , ,',\,

... Fifteencases'~were teported negative; in. eleven cases the~ cause ,of 'the \ : 'llrinary' trouble was ,trlJ,ced ,to other ccinditiQJls ,such asnephrit~s, renal

calculus, etc. In the f01n! remaining cases bilharzia ova,wered~moristrated~ r:h only: ,three of !the. fifteeni'yielding'a,positiv~ coniplementfIxati9n

reaction ova were not found in' the urIne at~th~t time; : rut· cystoscOpic, (

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T.hiJmm.u/(l,ify peactio.ns, i1J, Egyptian ,Bilharziasis, ~. " I ' .. "

examination rev;e'aled 'a. typi~ar biihar~ial 'lesion, arid, sub~eqU:e~tly ,the OV:1 were demonstrated in ea~hcase. ' , ' , v /', ( ,

,', ,N opseudo-l;'e,actions ,~ere'rE)ported.',Thus ,Qutof,'i9 c~ses of biUlat-" , z'i'asis 15, (or ,79 'perc~ri:t)wete:diagnosedas ~positiveJ arid this,:t, would " emphasize;' using. the irif~rior:salineantigen . .' """, '" ' _ " " ,!

r,

, ~, 'Repor~ (2).~~n' oRtbreali: of' bil~arziasis occurred in Palestine in,' the Mounted Ye.om(l.n'l'Y, who,had;:pe,enl:!athing in sweet-water can!l,ls'in the Fay'oum during the previpus sumIIier: • Systematic' .grinary examination Of' this unit h!l,d been, m~de" andltll the cases'passing ova six wee~f;lprior to my arrival in.:th~ vicinity had beim'diagnosed. ,">' ,: , ' , ,

, : When the complement fixation reactien .was investigated (which' was performed ip. ~'. field raborat~ry se~~t~n" te fift~Em.rriiles bebihd the' :firing line), six'men 'wh<?,ha~l previously b~en diagn,osed stilL remahied with their "

,squadron ; the remainjng:caf:les'in wb,om,no"ova had, previously been found were'e~amined." " " , " " '

. ',", \ "RESULT OF EkAI,[1N'ATtoN:

No. ';Urin~ry:8ymptom~; if any: , Eosinophilia, Cerebrospinal fluid reaction

-----I~-.,.---':-----,--.~-"-~-, ---' -:---~-'-~-, ,--'-'-' -,- ------

Penile pain' , Positiv~' 8;2 per c~nt .. P +- + + +-1 2 3 4 5 6 7 8 9

10 11 12 13 14

Hrematuria , ", ,.." 2'9" P +++ , ," '; ,; • .-,; .,. ./ 2'5 ,,' , P'+++ Urethral burnin'g, pail1~"j "" '," 2'6' P ++ No symptoms.. • .. ' ,,, 6'6 ;. ,p+, Hrematuria' . " ; 6'6' Negative Nil - ", '" Neg~tive' 19:6 / .• P ++++ Urethral pain ", '., '17'5, P ++++

';" bu~n~ng,.. 10:9 F++++ .. '8'2 ... P ++++,

\ Hreni'aturia

Nil ,," Frequency

.. " : ..

..;" 7'2:" ,P +\+++ " •. 2'8,. ," P'++++ " .. ,1'2, (" "P++++ " •• I ,6'2 "" p++

I ' '

The first; ~i:x; case~ wer~ knownt6 be suffering from urinary bilha:rziasis ~hen the,y ~ere exam'ined; .one .o{the~e !was ne&,ative. ,"The last: eight cases had no ova in their urine prior to the performance of the 'coinplen;lBJ1t fixation reaction. SubsequentlY'five oftheseeightc;ases .showed'ovain their 'lirine; unfertunatE)ly:;r was net able to follow up/the oth~r thnie cases

, 'owing to military exigencies~", ',,' ,'; , ' ,'" "," " ,", \' '." , ' Report (3)'7"Thi~ty~tll1,'eecases' of urinary and, rectal 9ilharziasis vyere

e;'aminedfrom th~ As~lum oLthelnsan'e, Abbassia;through'"thecoprte:sy of Dr. W. ,W.' Warnock, Q.1\I.G. Of tliesethirty-thre~ cases:""':':'

-',. .:;.... ';, .,' '. " '"

19 yieldedPf+++' reaction. (1 ," P +++ rj:laction. 9,,, ' P++ rea~tion.

/, .

1, " ,P + reaction:'-' , 3 cases were negative, ' \'

, "An,alcoholi~,antige~ (atiti,gen," B ~'): was,used. Con~id~ring .of these cases were cases of chronic bilb!,l.rziasis.with mental

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tbae'm,any' symptoms, /

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,there~ultsof the ,complement: fixation test'(i.e."nhH~typer .cent positive finding) are extraordinarily satisfa~tory;';, ' " ,

", ' ,Report (4);-:-Thirty cases of adva~ced chronic ,bilh1Lrzi~sis' from/ the ':Kasr-el-Ainy Hdspital wereexamiri'ed'thro:ug:h ~he courtesy, of Professor "F. Cole Madden, :B'.R.C.s.' '. '

" ' .. '

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, ,'Twimtv-three out. ~f the thirty cas~s, 'yielded definitely positive results, (i.,e:, P+ -+ ++;,p+ + +, orP++) ieaclfibns. ,T4e serum' of' two'(,lases I ,

.shbwed delayed hremolysis, and five were negative.rrbree of tll~ negative' , ',' cases w~re moribuIJ.d as a result. of~~condary septIc c'omplications (i.e.; ,(. ,', septic cystitis and pyonephr9si.s) , engrafted on,tq aehroljlic bilharziallrifec-:" '.,

\ '_, ti6n~, One case was 'suffering from urremia at the tim.e his bloodwastested~ , , ; Note'.-At autops~Y' ina ce,rtai.q. propottiort'qfthese a;dV:~Jieed chronic cases bilharzia worms carinot.'be deinonstrated.:It, isquit.e conceivable', .

"that; owing to secon~ary' infectio~, and repeated deposition of ova ;inthe, ' ' '. wal~s' of ,such 9rgans',~s the' })lad~erand co~o~,these focal·' donditions may.

, persist, and even cause ,death to the host (i.e., ,~an) l()ng' after ,tpe'primary , cause (l.e., the wo~ms themselves) nave diM oft, NaturallyinsucJ?,cases, , though beingprim'~r~tyof abllhariia!: origin"orie~ould e~pectto oqt~ina , :p'egative'complement fixation reaction. " " , " " , ,

. (b)'Go:mple'rq.ent Fix.at'ionReac.tiori ,a,s an 'Index. to,the POs~ibleTh~ra~' peuticrActionof Drug.~'inthe Treatme~t of the Di,sease. -:~All 9bservatioris r' hay"e ~a.de oti ~he .~e~()logic~l-t~st :~ep.d ,to 'sho.~· .th~t l"~t, stands)n ~·*c~ . .-!~ ", the . sam~ r~lati9nslJip"to bHharziasis as does the .Waf~sepnann \ test to sypb,.ilis;. It ~houlda:fford ariaccurate index of theactivityoCthe'b~lharzial , . ~worins'in, thebodyin'lliuch tl'le same manner as does'theWassermann test<' in the case 'of 'T: :'iallid'lfm. r;rbat'such is tlle.,case.<is,· indicated'bythe , higher perceritage<;>f positiv~ ,reactions .obtained in early bilharziasis as' I

CoIpp8;red :Wi,th those obtained In:tlieqb,ropic sM;ges ,of the 'disease.' ; '", ' . \ ' In the pa~t it has' been cnstoIIlarytoes,~imate, the ~herapelltic valu~ of any4.rug,in bilharziasis a~cording to the folJowing cri~ei'iai:- , ' '. ~

(1) TheaIIleliol'ation of vesical, ()r fntestinaY&ymptolll;!. "I ~'" \

" (2) .The~ diJ:I?inution intlle~umb~r' 'of ova"passed {n the ~xcreta(Le., ',' urine and freces); " " "i" " j ",',' " /' ,

'Snch "dat~"how~ver" a!ct', only as an 'index 'to /the an1~lioration: o( \' symptoms; 'but 'not to'the',cure' of, the"disease,

, ·Fp.~therm~r~, o~a may b~ deposited in,the walis~oChonowviscer~ . ,(irit~stine, and bladder), and thus'im.prisonedt,hey,may 'not, be :paEis~dout

• 'iri the excreta for, .luany months, it may beev~p Joryearsafterwards.' . What ,is ' really required ,is !!t reaction.' 't,c)" test the' .activityof,~he , ~orms

in the veins; ',for ,Imcb an' index we.now' can look to the : complement. " fixation reaction ,and toa.1esserde'gree'to tb,.e eosinophile,count,. - :'

:' The constant ,association of ova I with local, vesical and intestinal

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bilharziall~sions bas in the':p~st misdirected the though~9f ,the e1irii,cian " '.

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, as w;eU as the pathologif:;t toregar~the' ova, hut not.theparasitesthemselve~" as being the,dominating factor in bilharziasis." .' , '

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, \.' 264 The 1m;munityRe~cti01i8 ·in Egyptian j3ilharziasis. '.,

~.. )' ".': f' •. ' J 1'1 ,_ ,\., , ...• ", " .. ". . • I 1 \ \ " • ' .' ., .-

, ' ," N6Jess~nauthority. tqanLboss, taught 'that"tIiE(e6sedtialthe~~peutic. "

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,problem in bilharziasis ,resolved it~~H into' the, dis~overfof a drug' which 'i'

would· dissolve , the'/~hitinous ,'envelope ,of 'the; 0Yl1m " 'and destroy the mfracidium without at thesametiIrie injuring the humanhosk ,'" ' " In co~traaistinction to sll,ch :aview', I ,bold that an forms oUopal treatmentare"useless Nom the sta~d'point of an ultim~te l'cure; though symptoms~ nl!LY be teinporai-ily r~H,eved thereby. ' . ":;, '., , " ,:' The essential ,therapeutic, problem tl;len is to fir;td some specific drug 'so ' lethal to the ad.ultwotms that itcari.be ,introduced intravepously in sufficient

".'", dosageto kill these/parasites inhabitingthevenolis, system, arid yet not be , ; , 'ex6essively' harmfuJ to man himself." ' ,",. " ," 'I I ..

I,'

, • • \" .'.. • - > ' • ' ,/ .. '. • , ' " • •

',Furthe,rmore, itisJobeiemelllbered that'theproblem is to exterminate , .theparasjtE)s; not' ,bythediscc.n;'E)rY 'of ,a drug producing' ,its letqal ~ffect, by . ,.', dissolving or'perme!).ting the. :toughouter coat; but by thediscQvery of one l(' '

which during \it,s ingestion along with tli'elluman blood ,(which is after, all 'the. ~op:llal food) 'wil~ exert its specific para~iticid~l actiotiaf~er absoFption '

;', I;, through the more delicate lining".of the~limen~ary tract of the worms. . '. \ ," . ,'From the stan~'point' of exp~ri1pental th~rapeutics, there can b~' few diseas~s affording such p:rospect of ultimate cu~e, thus :- , '. ,

.' (1) Th~ rethalaction of various drugs'on 'worms and gercarire, kept alive ' in ~aline'or other suitable solutions,ca:nbe\estimated. ' .

. • ',1.,. .!', \' • I'" ", " "" . " \

., ' , (2) Monkeys cain readily b~. infected. (heavpy or ,otherwise, :as may be.,' " ' "desired);. arid' the 'effect 'cif the intravenous injecti,ort'of ,any,drug~ wI:lich'have' 'beenJound to exerta'Jetbal action on wormElin,vitrocan,thenbe.tested.

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"(3) Th.~ complement fixationte~,t,~ontrqll~dulti'Inat~Iyby, post~n:iortem, exaniinayi6non monkeys, ~eadily afford,san index. of the,effe6t of the drug ,

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uIlder' investigation,~)lithe patasi~es; , .'/ . '. ',' .,.. . ,! Time liIl?itations, thepre,ssure of routine work; and the" absence of.

suitable :ph~rmacologicltl;co~oper.ti~i'0n, h,ave prevented this line o{researc'Jl. . being followed' .out ,in det~il. "When one' considers .the' widespreaclravages 'of ::bilh'arzia~isamong:st the Egyp.tian~;the urgeht desirabil,ityfor sueh an~

", '. iuyestigation, especialIy if instituted in c9njiulctiOll with a synthetic chemist,: must 'be obvious to all. ' .. ' , ,( '," ">,, '"" " ; ,

. Iinvestigated.the:e:li'~~t ~f int;aVimous eusoliri' four' cases ofbil~arzi~sis r s~me/ twelve' months ~g,o, b'ut~ all the ':cases. are'stiiI 8ufferlngfrom,-the .

\,' dIsease. / , ' , I ' , "

I ' ,.' Op 'a "prioJ:igr:oun~s; there was ,no -r9a;Sp,l;l'to hope~ for', satisfa~torY/" : results from ,this form of medication,for'contrary to,the v~e'rs of certaiJ?, " ob~ervers [21J iav~ilable chlorine was f?und t?po~sessno§peciay lethal '"

, effects on cer,carire. 'Thus, t,wo and J1 :ha1£, hours, after ill,lmersion in water" , ,,'! • containing {,patts perl;OCO!OQO, of: available e~lorihe; I Ifo,und ,cerqarire,.

", 'ali~e and· vei;ym0til~/wher~~s as i a, bacte'~icide 1; part p~r \ 1,000.100 is i

'/ universally consideredeffic,ient.", \ '. ", .... , ' , ' . , 'The averagehumberof injections in, these cases was three; glyen at '

, 'i~tervalsoUive daysiji.~·qthesllm'..:t6tal o,f tHe amount inj~ctedineachcase . . , , ' '.~

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was 200 cubic' ce~timet~~s of eusol. ':N'ocon~titutio~ai disturbanc~s fol-" lowed exc~pt slight \,rise ~f; temperature and ' heaaacl).e;' and in o~e ~ase~ ,I,

loirtpain. No change w~s noted in the differen~ili,l eosinophill:lc9un~ nor "

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, . .in the compleinent fixation reaction in any of' the fourcases up to ,the,,' , "'thirdwe'ek' after 'the intravenous i~J'ection~ of eusol. Tbi~ tre~tmen't was I

, I " ,therefore abandoned, " ,I ',' ,

I' '." ",'

",(8) iJed~ctions whichcaldegiti1~ately be'd,.erived jn/m Exjjerimenta,l, , , , -Work reC'orded q,bQve. '/ " ,

i .

(l)Thatfroin,the cerc~rI(l3 of the'twospecies; S. mansoni or S.}~cerna, ," tobium, a specific antigen can b~ produced which 'reacts with bilharzia~ infected' sera.. ' ' ',' ,: ,",' , ' '~ " ',1 " \

, ", -I.' ~., ./ .' '. . '. \

(2) rrhaf this is the first record of a spedfic' substan,ce having been : isolated from tlie, body:of an invertebrate intermediary (BulIinus !Iond' Plariorbish infecte,d witha( human p~rasite,andwhich ,will' react with

" " \ 'I" I· " .' j

humall ~erum '(i.e., that of the, definitive, host). ' , , ", ,(3) Tp,at,' , provided' all the ~pnditions /which I have "ej'iumer~ted be fulfilled, Uleteat is s'till cbmpatable in its results, and is a more specific :one, ' than is, t,he Wass~rlllan~ reaction for syphilis; inasInllch ~s the tetidencyto " _ ,pseudo-reactions appears to be lessmal'ked., /,","':, " '_

(4) That it has.:a 'practical beaiidg)s( shOw,n by,the' high per,centage of " positive, results maintained throughol1t the ,disease, 'but especially in the ",' e'arly,stages, before the appearance of 'Qvi-ln the'-urine orthe exqeta. In', i

-, this :conriexion it is 'applicablefor the examination of large' bodies" of men, : J': for' installce, :on entraJice .to a publi~:"service; undet~coDditions :in 'w,hi-ch it is"

I' . - " ,~reasonable to 'suppose tha't ,a larger number: of rectal, infections ,would "reina'iriundetect~d by the microscopical ,eIi~min~tion of ,the falces. "

',~

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, ", (5)' By' its practical' application' the' discovery of :the'lethal action ,of 'a' , ,parasicitida1.drug may be facilitated. I " :, ,'I'

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SECTIO~, V.~:PRELiMINARY ,NOTE ON. THE IN:V~STIGATION FClR THE

EXISTENCE OF A PRECIPITIN 'I'EST-INBILHARZIASIS. ' 'I ,~, ,

\ Tpe demon~t:r~tion of the presence of ~ posItive complement fixlj.tiorl. ;,' ,'; '\ r~action in bilharziasis led to the h6pe t,hat a 'precipitin test might also be

obtained in ,the serum of this disease. \ ",' " • ' I" In ordert~' investig~te thisp6ssibilitY the followi~g exp~ri~ents~ere"

-' carried out:..:.. ,,' '; '\. " \ :Thelivers, 6fthirty 'flanorbis s~~ils infected .with-Schistosoma ,manso1:lri

were teased oU,t in, saline .and in'cubated witht,hirtycubiccent'i'm'et:res. of ", , ',soIut,ion (composer , of' 0'~5 pet Gent salin,e arid 0'5 per cen:t phenol) 'for,

, 'forty~eight hours, the 'mixtur~ being shaken' on an 'electric shaker. Subsequently it \vas, filtered through a Chamberlain' fitter, and~he filtrate (Af

" used for experiments;, " ", ',' . " '" ", I

''.I,'h,e, sera, of, two cases of human ,bilha~~ia:sis' (mixe,d infections, i.e.;

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266," The Immunity Reactions in 'Egyptian Bilharziasis'

$. mansoni ~nd'S. h(Bmotobiu~)and~f two'iilfp-cted infections) w~l'e utilized;'" , ". ....

monkeys (S. mansoni, . ' ,

'TbrElerows oItubes 'Yere put up in each ca~e~-' .' ,- I '"

In the 1st was filtrate (a), 0'5 c.c.'+ Bilharzial serum, 0·25·c.o. , 2nd' (A); 0'15.. +.. ,;, 0'5." .

3rd ,,' (AI, 0'5" : + physiological saline', 0'5

. . Additio,nal control~ were' u'sed,' in.knownnegati~e! sera ~nd"--filtrate , (Which I. have termed" A ") in similar quantities as 4escribed abo~e. All .results .were entirely negative"even after being left for forty-eight'hours at room tempeiature. Further observations are'. being uowconducted with higher grade' dilutions' oJ bi~hariial sera. ", " '. .. .' "', '. !,.'

" Note.-N 0 agglutination or parasiticidal action of, bil~arzial .setI1lIiWas'· nbtedon live cercariffi \v hen the latter were' suspende'd ill ',bilharzial ~erum'

'in coverslip prepar~tions andobset~ed .underthe, micros(jope~ The loss of 'o4ygen isth6 main factor in immobiliziIlg bilharzialcercarire in coverslip. preparations. as iRcle~rly ind~c~ted by the saline 'or negative serum controls: and/by the fact that 8o'm~ cc;Jrcari~ placed in open~est~tl1besl in .identical, solutions8urvivemuch longe:!;. than silllilar cercarire placed in sealed ·coyer~ sli~ preparations. ' . ., , , .

ACK~O'WLE:oGM:ENTS .

. To my former' COn:lrpa~djngdffice~s,Lie,~tellant"Colonel A., H:'Thw~i£es, .~. D.B.O.,' A:A.M.C., and' Co]oneIW., S'ummons,A.A.M.C.; 'to Oolonel R. Fowler; A.A.M.C., Commanding Officer of Australia~ General :;Hospital,

,and to Colonel R. M. Dow,nes, C:M.G., D.D~M:S., A.LF.,myhest thanks _ are due· forthe'faciIities afforded during this research. . -, , ; " To myfr{eild, Captain'P. :a:. ,Bahr, D'.-S.O.; RA.M.C.,I ,am tinder 'the deepest obligation,as .. throughorit tllEi investigation, he has· 'ponstantly' ren'dElr~d the most valuable'assistance. . . , '. ..., .;' '.'

. . .To the Laboratory Staff of .~ Australian .GeI}eral Hospital,' especially' , 'to,Staff-Serjean't C.F. Sullivan,B.Sc,;who has prepared the microscopic .

. sE(ctions,and' ~ls'o to Corporal R. C. Stephens, and Private, R'V: Panelli, who h,ave rendered much "assistance and. have freque,ntlY"w'orked ov~rtilJ1e to facilitate theinv;estig'ii.tioll, my thanks,are due. .'. 1,:".' ,,", , ,:The Au.straliali'Red Cross havegerier'<;mslyassisted me fina~ciaUyduring ,

, , this research, paying forty perqentof the total expenditure, . '

. LIST OF REFERENOES ..

[1] 'BoimETand GENGOU. ~Ann.·de l'inst. Pasteur, xv,190l;'" .. :" [2]. WA~SERMANN and BilucK. Med.'klin.; 55, 'L905'; and Deutseh: ~d.,W~henschr., xii, 1906. [3J FLEIG and LrsBoNNE. Oomp. rend. de la 800: de Bioi., lxiii, No~ 23, '1907, p.1196.

, [4}.WELCH;CHAP:M:AN, and STOREY. Lancet,April17,1909. ,... ", , [51' GUEIlINI..'~ Recherche suI siero di Sangue, etc.," Gaze .. do! Ospedali et deUeCliniche;',

, No: 153, )906 ; and Nos. (; and 45,,1907. . '.' .',' '-, . . . '. [6J WEINBERG and PARVU: ," Reaction. de Bordet.Gengou dans les, Helminthiasis," Comp.·

. rend. de la Soc. de Bio/;, lxv, No. 28, October 17; 1998, p. 298. " " , .' .. '

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''En BARB. " Filariasis and 'Elephantiasis.in Fiji," Witherby and Co:; Londqn; 19i2. ',Report , to the\London.school,ofTropical Medicine. ,". ' .: ", , " ' ,"'".

[S] EOGAR.' Journ. of State Metf,icine, September, 1913, xxi; No. 9, p: 542; and Statistical" Report of lIealth of !:'l"a~yfor Year 1911, pp.' 167-69, (H. M. Stat.Offlce, 1912). ' ,

[9]' BASSETT-SMITH', Brit. ¥ed. Journ., '1~12,ii;p. 1208: " ' " " , [10] , MIYAGAWA"Oentralbl. f. Bakt" Abt.Orig., May 23, 1913,-lxix, No. S"pp. 132-42. , [11]" LANNING. '" Scnist?somiaflis oq the 'Y',ang~tze R~veJ, with Repoft,of Cases," U.S.faval.

Med, Bull., 'January, 1914,:viii, No.:1, pp. 16c36 (with 17 figs.). ".,' ":, [l1AJARCIiIBALD .. 'Brit. 'Med,. Journ." 1914, xi,' p. 297.' '/ '

.[12} , LAWTON.~' The EarlySymptomsiollowing rnfeci;ion -bY' 'Schistosomum mansoni," by' • . F; B. Lawton,M.:B.~ B.Cp., MedwalJourn. of Australia, September 22, 1917:

[13] DOUGLAS and HARDY. Lanc~t, 1903, p.1009" ' , "', " ' ',' [143 KAUTSK~ .• ' "Die Bilharz!aerkrank," Wien. klin. Refndsch"INo.. 36, 1903: '

,[15] BAlfFOU'R. Lancet, 1903, ii, p. 1649; ",' " ,'" . '.,., ' '/ [163 ,\ DAY" "The Blood Changes in Bii~arziasis;" Lancet, H, No~ember 1i,1911. ,',

[17] CONNOB and BENAZET.·' "Formule leucocytairecl.'image d'Arnej;h dans la Bilharziose," BuJl. Soc.PcithoCexot" June 5:1912, 'p. 396. ",,' , " , " ... "

'[lSf LEIPER(R>T.)."":Report on the , R,esultsof the Bi~ha:rzia ~i[isslon inEgypt,'~JouRNAL , ' OF THE ROYAL ARM~ MEDICAL CORPEi, July, 1915,xxy, No. ,1, pp. 1·55 ; ,August, 1915,

xxv;'NQ: 2"pp. 147-92, with 17 figs.; Sl,lptember, 1915, xxv, No. 3,pp,,253.67,'vi~h figs: 4L55. 0 ' '

[19] BARB. Ibia, ref. [7].' [20] :BORDET et GENGOU. 'Ibid" ret' [1], , ", , [~1] "Memoranda on some, Medical 'Diseases in the ,M~dite'rranea.n "War .. ,Arear.'Yi~h some '

,'Sanitary Notes;" ,Reprintedv.:itll Amendments; 1917..; ,cbap. ~" B,ilharziasis,'p. S. ,- , " . " \ ,.)

POST SCRIPTUM.'(

, ,Sin~e the',nnal'comp}eti~n of' thisarticl~ r:b'ave learned for the fi1;st " time of tgeexperimental' work ofLeiper on infecte<lanlmals, reported on intbeJ=tOYAL ARMY MEDICAL ~C()RPl?J6U:RNAL; Marcb, 19~8i:"p, ,239, ,which has just, come' to hand,', " J' ' ' " '!" '

.. , His conch:isiops !1re .that 'none of thesubst.ances 'of known anthelminthic orcerc,arlacidal val 11e could'be.introduced into the p'ortal aY,stem in, doses lethal to adult parasites., I still tbink, how€ver,that with It c;lomplement ' fixation reaction' now available ,and, 'with the'potentialities of' syntbetic,c:tJ,emistry 'as yet' unexplored, a rational optimism is, justifiable and that' fUrther researyh'aLong th,e line,enunciated,above is ~tiIlurgEmtlyindicatea.:

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