THE RELATIONSHIP OF YELLOW FEVER OF THE WESTERN HE~IISPHERE TO THAT OF AFRICA AND TO LEX'TOSPIRAL JAUNDICE* BY w. A. SAWYER, M.D., S. I;. T- x11!- of the l);tcteriologic:~l methods used; tllilt it ~VOllltl [l:lSS lL U('YliClCf(l LiltCr C;l[):ll)lC Of hol~ling b:Lc'l; Stap/Iyloc*occlls ~yogl'lrl5~ tZlil'(`llS,' :Llltl that it CONIt bc rcnclil!v tr:lIls;fcrrc[l from ;t Sic]< person t0 ;t we]1 one !vith tllc production of infection, 1)~. tllc mosquito .,~C~CS (I~gj~~/i. The bite of e\`crl 0tlc nl<)squito IVns sufiicicnt to cnusc the (lisease. ITrom 101 S to 102-1 ;L lc>;Itospir;i \\.a5 isolatetl !I!* Noguchi nntl other investigntors (2.1 frOIl1 pticnts in oLltl>rcnks OF >~cllo\v fc\-cr in Ccu:lt]or ((;u:~>~aqui]), iIlfxico (JIcrith ancl \ycrn Crux), l'cru (~IorropGn), and IJrazil (I':ilmeiras). Like the spccilic :lgCIlt stuclictl l)\. the corllrnissior~ (*rlc]cr \i';Llter l:cc(l t]lis organism wns pwsmt in the l?lootl e:~rl>- in the disease, it I?`;~s not Culti\r:ljj]c in the ortlin:lry 494 YELLOTV FEVER culture media for bacteria, and it was able to pass Berkefeld filters (V and N). It seemed to differ, however, in not being easily transferable by AEdes aegypli. Noguchi (3) found that he could infect gui?ca pigs with this leptospira, which he named Lcp/ospira ic/c~oidcs, by means of the bites ol A~rIcs acgy#li which had fed previously on a patient or on infected guinea pigs, but that transmission was obtained infrequently considering the number of mosquitoes employed. Another dificulty in the way of accepting the organism as the causative agent of yellow fever was the uncertainty of the diagnosis in the cnscs in which leptospirae were isolated. In the individual case, infectious jaundice, caused by Lcp/ospirn iclcvo- lzc~erttorrhagiac, is ordinarily indistinguishable from yellow fever on the basis of symptoms. Elliott (4), who was associated with Noguchi in 1918 as clinician of the commission which studictl yellow fever in Gun?xquil, held the following opinion: "Cl inicall\r \~cllow fever is similar to infectious jaundice. The differences _ _ existing bctwccn the Live tlisc:Lscs :ll'pc:lr to l)c chictl>r those of dcgrcc. There is more m;~rl;e(l j:Llinclicc ant1 lass lic~niorrhn~c in >~cllow fcvcr than in infectious jaundice." In the tlifl~*rcnti:tl diagnosis lxt\vccn these tlisc:iscs the nature of tlic prevailing cpitlcmic was of ncccssitjr frcqucntl>~ ;~llo~ed to determine the decision with rcgarcl to tllc in(li\~i~lual cnsc. 7`0 meet this dillicult:7 Koguclii matle com- parative stutlic`s of L. I'cI~:).oi~l~`s and I,. ic.(c'r.c,lftzl'l~fc~~~/~~~~i~~~. an(l decided (2) that they \vcrc tlistinct morl~l~olo~icnll~~ nncl scro!ogicnll!~, and that the evidence nppenrfxl to warrant the conclusion that I*. ir.lt'l'cjiilft'.T is the cause of yellow fever. This \ricw I~ccnrnc gcricr:llI~~ ncccptc~(l. SAWYER, KITCHEN, FROBISHER, AND LLOYD 495 the Americas, as represented by the disease studied by the commission under Reed. Sellards (7) was unable to demonstrate by serological methods any relationship between a yellow fever outbreak in Parahyba, Brazil, and L. icleroides or L. ictero- haentovulwgiae. Theiler and Sellards (8) and Schiiffner and Mochtar (9) com- pared L. ictcyoidcs and L. ictc~dl~acnzorrlLnSiac and obtained strong evidence of their serological identity. Attempts to transfer L. ic`ieroidcs by means of AZJCS acg@; failed in the hands of a number of investigators (Kliglcr (lo), Gay and Scllards (1 l), Schiiffner and Mochtar (9), and Sawyer and Bauer (12)) and they became convinced that this mosquito is not suited to act as intermediate host for L. icfc~oides. When our experiments were undertaken there seemed to be riced for further investigation of the relationships of the "yellow fever" of West Africa; th6 "yellow fever" of South Americcz not due to ;1 leptospira, the "yellow fever" of the Americas apparently causccl by a Ieptospira, and a hypothetical "yellow fcvcr" in which the ctiological factors of the two lnst mcntionecl disenses czre both present. Collection nf Strilills of Ycllo~ FCW~ ?!i;r~s the strains of virus use<1 in our cslxrin~cnts \vcrc the French and the Asibi from West r1frica mcl the 1;. IV. from IZrazil. The French strain was given us b>* Dr. A. \V. Scllards and Dr. Max Thciler of the T~larvnrtl hlctlical School. It had been obtainecl originally from a yellow fcvcr patient in Senegal by* ?rl;dhis, Sellnrds, and Lnigrct (13). During our espcrinlents this strain was indistinguishable from the Asibi strain in its effects on monkeys. `1'11~ r\sibi strain wx sent us from Lagos, Nigeria, by Dr. Henry Beeuwkes, Dirwtor of the \Vcst African J'ello~~ Fever Commission of the Rockefeller Founds- tion. It lind been obtnincd in the Gold Coast from an African native during an attzck of ~cllow fever, and hat1 lien used 1~)~ Stokes, Uauer, and Hudson (5) in most of their cspcrimcnts. The estnblishment in our laboratory of a strain of yellow fever virus from South Arncrica proved to be difficult. IVe are indebted to Dr. 1:. I,. Sopcr nnrl Dr. H. Jfucnch for obtaining for us a large number of specimens of blood from yellow fever patients in r\io de Janeiro. WC? ivish to nckno~~Ict?gc also our special obligation to Dr. Clementin Fraga? Difector of the X;ntionnl Dcpartment of Hedth, and to Dr. Sinval Lins, Chief of the Communicable Disease Section of Hospital !%o SelxstiZo, for their courtesy in granting Dr. Soper and Dr. Muench 496 YELLOW FEVER free access to the patients. Specimens of blood and tissues from monkeys experimentally infected with yellow fever were received through the kindness of Dr. I-I. de 13. AragZo of the Oswald0 Cruz Institute in Rio dc Janeiro and of Dr. N. C. Davis and Dr. J. H. Bauer of the Ucllow Fc~.*er Laboratory of The Iiockcfellcr Foundation at Bahia. The specimens of hl:!nan blood from Rio de Janeiro were taken from 103 patients having yellow fever or suspected of having it. A titrated specimen from each patient was received and also dried blood from three and clotted blood from four. Among the patients suryl>~ing the blood wcrc at lcnst two from whom yellow fever was transmitted to mc;nl;cys in Rio de Janeiro. This lens accomplished by blood inoculation by I):1 Cur~hn ;lnd illunix (1.1) in the case of I;.\\`., and through the bite.5 of mosquitoes 1~)~ r'\r;lgAo (15) in the c;lsc of N-31. `J'hc blootl was drawn on the first day of the tliwasc from 11 patients, on the seconcl from 37, on the third from 5.3, and on the fourth from enc. In 011~ instance the day of disease was not recorded. As soon as rcccivul, the specimens \vc'rc injected into AI. I*JICSIG monkcys,in vnqying amounts, usu:IlI~~ intrnI'cr-itoncall?., rnrclj- sulxxtnncously. Of the 110 spccimcnt;, including il1ll~lic:~tcs;. 10 \vcrc` injcctccl into separate animals. The rem:1ining 9-k \vcrc tli\.i(lv(l into 1-f gr~`u~>s anti ~wc~lcd, :~nd-lk~rt of cadi of the 14 niisturcs \v;is injcctc(i into 011~ or t\vo nionl;c~~s. In sonic instances animals \vhich sho\vd no s~7nl~t oms \vcrc inoculatctl Inter \vith another specimen as the numlxr of a\-ailal)lc n!0nl;c!x \~a5 linnitcd. Test injections with a known virus were not gi\rcn at the c>nti of the cjbscr\-ation period to cictcrminc whether immunit} to >~cllo\v fever hat1 \)con csta!~lishecl as \vc 1x1~1 no J-ellow fever virus during the early Iurt of tlic \\,orl;. The rect;ll tcnlpcraturc of the monkq~s under observa- tion was tnkcn t\\,icc ca;lcll ~l;\>~. The results from th*h inoculation of the specimens of human blood sent from Rio de Janeiro \vcrc I!niiornll~~ ncgatii-c. Alost of the monl;e)5 dcvelopcd no fever. Six had iwlatctl ri5c5 of tcnnI)craturc \\hich sccmcd of no significance. jVe conclutiul tll;it it \\`:15 not I~r;~ctic:ll,lc to i5olatc strains of yellow fever from the blood of patients in Soli tli :\nncricn \\.hilc \vorl;ing at so great a distance from the source of nin t uri;ll. SAWYER, KITCHEN, PROBISHER, AND LLOYD 497 intraperi toneally or subcutaneously. Six of the animals died from causes other than yellow fever, The other animals remained normal except one. A monkey which had been inoculated with a specimen of frozen liver, F.W. strain, tenth passage in monkeys, showed fever on the seventh day and recovered. After an observation period of 28,days from the date of inoculation this monkey was given a test injection of African yellow fcvcr virus, Asibi strain, and was found to be immune. The strain obtained by bleeding this monkey at the beginning of its fever was carried through many passngcs in this lal)orntory, and the F.\V. virus used in our experiments was derived from this succession. Like the African strains, the F.W. strain thus procured could withstand long storage under suitable conditions. At one time the strain was apparently lost, but was reestablished by inoculating a monkey with stored liver tissue. The specimen of liver had been kept continually frozen for 67 days. Between cxperi- mcnts this strain nras successfully prcscrvctf in storage for varying intervals up to 92 days in monkey blootl dried in the frozen state by the methods ~mxl by Sawyer, Lloyd, and Kitchen (16) in preserving IIfricnn strains of yellow fever virus.* We have not yet tested oltlcr dried blood spccimcns of the F.11'. strain by inocula- tion of monkeys. The material from Bahia, Brazil, corkted of 2s specimens of blood and liver from 20 monkeys experimentall>~ infected with ~~cllow fever of the 13.13. strain (27 specimens) and the S.R. strain (1 specimen). These strains had been obtained from yellow fcvcr patients in fkkhia, Brazil, 1~17 Da\G ant1 But-kc (17). This material was injcctcd into monl3 :~ntl tlic 13.13. strain was established in our laboratory, but the virulence of this strain for ~nonl;c~~s was so row that it secmcd unsuitable for our projcctctl cross-inlnluriit?. csperimcnts. In the hands of Davis and I- has been tested after one >.enr in storage and found to be highllr virulent for rhcslrs n1onkq.s. 498 YELLOW FEVER with African or South American yellow fever virus and of the liver tissue of 10 monkeys infected with African virus. Cultures were made in a wide variety of media, including the Noguchi lepto- medium, and guinea pigs were inoculated. The results were essen- tially negative. Of the specimens of citrnted human blood sent us from Rio de J,znciro, 66 were cxnmincd also by I-I. R. Mullcr and IX. ID. Tilden (20) at The Rockefeller Institute for Medical Research. Two of these specimens yielded cultures of a spiral organism agreeing with the cultures of L. i&roidcs isolated in yellow fever epidemics by Dr. Noguchi. Both of the specimens containing lcptospirae came from severe cases with the symptoms of yellow fever; one of the cases was fatal. XIotll spccimor~s were :tmong the first 36 examif&, which group was included in the 103 specimens inoculated by us into monkeys. In 0rrIcr to show the rclat ionship of ihmcricnn yellow fever to the African, monkeys were immunized with :Imcrican yellow fever virus and afterward tested for susccptibi1it.y to iIfricnn virus, and vicll 2W.W. In Table I arc given the results of the tests of clcvcn monkeys which had exhibited fever after intrnpcritoncnl inoculation with the F.W. strain of American yellow fever virus. Alonke~~s which had not shown fever after inoculation with this strain were somSncs found to be immune to African virus and sometimes not. They were all excluded from this experiment as some had probably not been infected. Three of the monkc~~s were inoculated n second time with the I;.Ii'. strain frown 5 to 7 w.xks after the first inoculation, in order to m:d:e certain that they had become immune to :Imcricnn ~7ellow fever virus. To obtain additional eviclencc regarding the immunit\- produced nnd a better idea of its clegrec, blood w:ls taken from six of the animals lxtivccn the 16th and 4lst days after the last injection of American \Gus, and the scrutn ~~3s tcstecl ior protective power against Africnn virus. fill elcvcn of the monl;c~-s were tested 1)~ intrnperitoneal injection of African virus when nt Icast 30 clan-s hntl elapsed after their last inoculation with Americnn virus, and the high virulence of the IIfrican \Grus used was shown by its effect on control 3nim:d5. I:scept when otherwise stated in the t&le, the period SAWYER, KITCHEN, FROBISIIER, AND LLOYD 499 of observation after the test inoculation was 30 days or longer. Only fernpcra- tures of 4OOC. or over were accepted as fever unless otherwise stated. AI1 of the eleven monkeys immunized against American yellow fever virus survived inoculation with q highly virulent strain of African yellow fever virus, and only one showed fever within the period of TABLE I Immunity to African Yello~w Fear Virus in Monkeys after Infection with m/i m&can Mon- key First inoculation with Americxn virus - St&n F. IV. l".\V. F.W. I:.\v. F.W. xiv. 17. IV. F.W. I'. \v. 1'. IV. I'. w. I:ebrilc period' 5.5 to 12.0 2.5 to 15.0 2.5 to 9.0 7.0 A! 12.0 7.5 to O.S$ 3.0s 2.0 to s.5 6.0 to 7.5 3.0 to 4.5 4.og 4.0 to 12.0 Second inoculation with American virus Strain lCebrilc period' 1:. IV. I1 .o to 13.0 I:.lV. None INo t inoculated Not inoculnted F. 1%`. / Xdnc Not inoculated Not inocuhtcci Sot inoculntccl Sot inocul;~tecI Not iribculatccl Not inoculated Protection test of monkey's serum against African virust ProtectedI/I Protected Protected Pro tee tedf Protected Not tested I'rotectcd~ ISot tested Not testccl Not tested Not tested Test of monkey for immunity by inoculation with African virus (hsibi) Immune Immune Immune Immune Immune Immune Immune Immune 11 Immune 7 Immune Immune * lhpressed in tin!-s after inoculation. t I'erforrnccl with Jhperimcnt IV; sxme methotls, same virus (Asibi), regular amount of serum, nnd snnle controls (Table IV). $ 11x1 one elevation of tempernture; Monkey Ii on 14th day after inoculation, hrol~~c~~~~~t~ 7th, hr0nkc\+I on 8th. $ hlasimum temperature 39.7"C. (JIonkq~ G) or 39.9"C. !Y~fonkcy I;). I/ 0lm3-vecl for 2-I da>3 only. 71 Fc\rer 3.0 to 6.0 day. 0bserved for 12 da:,3 only. observntion. The scrcz of six of these monl;cys taken before the test inoculation were tested for power to protect monkeys against the same African strain. All protected against death, although three permitted fever. 500 YELLOW FEVER Tests of Mod~eys ITnzmmizcd with Ajricalz Yellow Fever Virus for Iv2712 unity to Anzerica72 Virm As it was a rare exception for n monkey to survive inoculation with fresh virus of the African strains which we used (French and Asibi), it was necessary in our cross immunity tests'to use monkeys which TABLE II Monkey __-~-.. _ 3,I N 0 I' Q 1: S r , 1 U Control : cs - _ First inoculation with African virus Strain _-.-.- --_- Asibi Asibi French French French French r1sibi .flsibi French -~- .__ None None-i Norwl Noncf 12.5 to 15.0 6.0 to 7.0 5.0 3.0,3..5 1.5 to 5.0 Sccon(l inoculntion with African virus Protection test of monkey's serum nfiainst American virus CIC.\V.) sot tested Not tcstctl Not tcstcxl sot tested sot tested Sot tcstctl lK.J,t tcstctl sot tcstetl IIoul,tf11l:: . . Test of monkey for immunity by inoculntion with American virus (i'.W.) - ----- Imnune" 11 Immune' Jmmune Immune Immune 11 Immune Immune Immune Immune -- ITcl)riIc period 2.5 to 15.0 * Irat received an injection of the 13. B. strain of Americnn virus but, zs the control monkq~ as ~11 3s the nnimnl tested shonw1 no rcxlion, the monkey wns reinoculated 14 da!-s later with the I;. \V. strain. T I'rotectcd 1,~. an injection of .Africnn immune serum from recovered African nnti\rcs (~Ionl;c~-s S, and I') or from immunized AIonkey A,Tahle IV (XIonkey 0). $ IIacl irrcplar unesplninecl rises of tempcrnturc from thy of inoculation. 5 ~lonkc~- C \\~ns control for the esnmin:ltion of the sern of Monkeys M, N, 0, I`, Q, Il. The other tests wcrc m:ltle \Yith the controls shown in Table V. Ij Observation pcriotl only* 20 ~13~3 (LIonkey AI) or 21 da>5 (hlonkcy Q). had been protcctetI against death 13~7 injections of Afric,zn immune serum or which h;\(l rccci\-etf virus attenuated by ~0111~ method of prcserv,2tion or LJT long storage. Xine animals immunized against African virus wt`rc ~~\~:lil;kl,lc for tcstillg. Particulars with regard to the tests arc sho~vn irl `.i'dh II. SAWYER, KITCHEN, FROBISIIER, AND LT,OYD SO1 Eight of the monkeys tested had been given a second injection of the African virus from 20 to 48 days after the first as a test of immunity to that virus. The monkeys were tested by inoculation with American virus (F.W. strain) after an interval of between 31 and 39 days from the last injection of African virus, except in one instance in which the interval was 143 days. They were kept under ob- servation for 30 days or more, except when otherwise stated in the table. Blood was taken from one of the animals before the test inoculation and the serum was tested for protective power against American virus. As the monkey receiving this swum with the virus hnd irregular fevers not due to the yellow fever virus, it was impossible to know whether an attack of fever has caused by the virus and no conclusion could be drawn, as death does not ordinarily result from the F.W. virus in this laboratory. All nine of the monkeys immunized against African yellow fever wcrc free from fcvcr fnllowirlg inoculntiorl wit11 the F.W. strain of American virus. In the cross immunity tests, monl;q-s immunized against American yellow fever virus rcsistccl African yellow fclvcr virus and, convcrscly, those immunized with the African virus resisted the American virus. An esccptional opportunity to invest igi'ntc tbc relationship of American yellow fc\rcr to Africnn yellow fever and to leptospiral jaun- clicc wins prcsentcd by an epidemic of yellow fever in Rio de Janeirc. Through the courtesy of Dr. I-1. de B. Arag and Dr. F. L. Sopcr we rccei\-cd 1.5 spccimcns 0i serum from l-1- persons who had. recoverccl recently from l-cllow fever in that city. They are listed in Table III under tlic heading "Recent epidemic," 311~1 our findings with regard to these sc`ra arc sunirnarizcd there. In 311 the 1-l cnscs :I definite diagnosis of yellow fcvcr had been mntle on clinical grounrts. In one case (S.>I.) the diagnosis had been proven, for Arag (15) hnd trnnsferrcd the infection to n monkey bar menns of mosquitoes. In eight crises the attack \VX described as severe; in six ns mild. Sis of the scrn ;vcrc di\*ided in Nciv York and a portion of each was sent to the, 502 YELLOW FEVER TABLE III Pro~tx~ion liy Scra of Persom Recovered from American Yellow Fever agaittst African Ycltow Fcvcr Virm ad Lcpiospiva ictcroides or L.. iclcvo~~acntorr~~agiae Serum Recent epidemic: N. M. c. F. D. A. rd. A. (1 st spccinicn) ;* I5 s. G. v. M. R. M. A. (2nd specimen) J. &I. hr. ?\I. M. J. S. R. 13. S. J. 11. c. 11'. G. - Protection of monliey against yellow fever virus' + 4 - (1 .S)i -- i -t I- - 4T + + -- t - (.{.S)$ - (2 4) ** - (4.O)S - (2 .O) + + -( -t + + + + -t + -- i 3.5)) -(-1 5) - (4.0) -t- - (4. 5) Protection of guinea pig against leptospird I zs 55 I 2 2iG P.4 - - - - - - - - - - - -I- - - -- t -2 2, .$! $ E2 PI - - - - - - - - - - - + - 1-t + - V Y 23 >d e$ I& -tS -11 - - - - - +I - + - +'..' Experiment `I I II II II IT IIT III III TTT IV - A A A A A A A n B B C C C C C * `1'1~~ \-cllow fever yirus ~vns of the strnii wrimcnts I and IV and French in Espcrimcnts II nnd ITT. The strains of leptospirg used were Le~lospiru iclcroidc-s, Brazil 49, in Espcrimcnts rl anal 13, and Lrpfospiro icfcrolraenzorrlzagriac, Rat I, in Eqwrimcnt C. t The figure in I,nrcnthesis is the number of tl;~~-s from inoculation to the first observntion of farer in coIumn "I'rcvcntccl Fever," and from inocuIntion to death in column "l'rc\~ntcd rkath.*' $ Guincn pig had fcvcr but survival and ~~3s found to Lc immune to a second injection of L. ic-l~~roiiic~s. SAWER, KITCHEN, PROBISHER, AND LLOYD 503 TABLE III-CmcZuded :- Serum Doubtful cases: Car. I;. M. D. H. D. Dj. A. Former epidemics: G. I?. c. IT. II. D. 0. L. (1st test) D. 0. L., double amount I. J. I<., 0.4 of amount P. s. R., double amount Tirn;aafrter brief brief brief brief 9 years 5 years 23 years 83 years 30 years Protection of monkey against yellow fever virus* Prevented fever -(1.5) -(1.5) + + - (5.0) - (2 .O) - (2.0) - (4.0) + - (2.0) - (4.0) - (4.5) + + + - (5 3) - (9.0) + + - (4.5) Protection of guinea pig against leptospirae. a Y cf? pcd ca r& - - - - - = Experiment n .t: ir Y 2 C'S i+ III III III III II II II IV III IV $1 N 0 serum rcmai L icd for test. C C C n r3 u ifrest fUric3n I'cllo~v Fever Commission of the Rockcfcllcr Foundation in Lagos. The results of the tests mndc 11~ the Commission in :1frica were reported by IIud- son, Philip, and Davis (2 1). They found that the wra of N.R,I., C.I;., Rl.R., hI.A., and J.31. protected monkeys against injection of LZfrican yellow fever virus (Cases 16, 15, 12, 14, and 17 in their series). One of the sern, however, that of DA (Case 13 in their series) failed to protect either of two monkeys against the injcc- tion of the virus. Four of these scra, those of N.>I., C.F., MA., and J.M., were tested in guinea pigs against L. ic-fe~olrrlcnrorl,Iragiar, and in every case the Pfeiffer phcnomcnon was absent and the animal died of leptospirosis. The results of the protection tats in monkeys were in agreement with those of the tests we performed independently in New York with the same sera, nnc! their I'feiffer tests gave results consistent with those \ve obtained using different strains of Icptospira. E.vpltr~rutiolr oj Tublr~s III utzd It'. The results of tests with the scrn of persons who had rccovcrcd from American yellow fever for protective power ngninst African yellow fever virus and against L. ic/crnidcs or L. icferolrael}rorrlru.~j~le arc shown in Txblcs III and I\`. The results of the tests of the scrn themselves arc in Table III ant1 those of the corresponding control tests arc in Tnble IV. The tests for protection against ~*ellow fever lrirus were made in four sepnrntc experiments 504 YELLOW FEVER TABLE IV Coutrol Tcsis in iltc Ex/wimcnfs Rccordcd i?t Table III Serum 10 mos. 9 1110s. 2,' 1110s. -' 1.. Protection of monkey against yellow fever virus - Prevcntetl fever -t- - (6.5) -I- - (xi)** - (2.5) - (1 ..i) : -(1.5) -(1.5) `I II Prcventetl clc~ltll -t- + -I- - (4.S) - (6.5) - (1.5) - (4.0) - (3.5) - (6.0) Protection of guinea pig against leptospira - - t - - $` + 9 - - % +z..c $2 2% p1 - -t* - - - f: -I- _ +* - Experiment SAJVYER, KITCHEN, FROIHSIIER, AND LLOYD 505 TABLE Iv-Cmtcludcd = Serum No serum, full amount of virus X0 serum, 0.1 amount of virus No serum, 0.01 amount of virus No serum, full amount of virus No serum, 0.1 amount of virus No xrum, 0.01 amount of virus No serum, 0.1 amount of virus No serum, full amount of virus No serum, 0.01 amount of virus Snl t solution Salt solution Salt solution Protection of monkey against yellow fever virus Prevented fever -(1.5) - (2.0) - (10.0) - (6.0) -(Z.O) II - (3.0)** - (2.5);~1 - (2.5) Prevented death - (5.0) - (4.5) - (11 .o: 4- Ii - (4.0) - (S.5) - (5.0) - (4.5) - (4.0) ZE Protection of guiiieh'pig against leptospira 7 hlonkey survived7 nlthough it had received no serun tj Temperrtture rose only to 39.S"C. $1 Guinea pig died on night following inoculation. Experiment dcsignatecl b\v the Roman numernls I, II, III, and IV, and the tests for protec- tion against icptospir;lc wcrc mntlc in three cspcriments, A, 13, and C. In order to show which controls l~elong to each cspcriment the numbers and letters idcn- tif>-ing them 3x-c given in the column hexled `lEsperiment." The scrn tested were injected intraperitoncnlly into .,11-., rlrcs~us monkeys in amounts of 1.5 cc. per kilogram of 1~0~1~~ 1vcight except where otherwise stated in the column hc:~clccl "&urn." The expression "tlouhlc amount" means that 3.0 cc. per kilogrnm 1~3s given, and "0.3 amount" mc`ans 0.6 cc. per kilogrnm. ThCSe \-nrintions in amount of serum do not apply to the tests with leptospirae. The "time after attack" wns measured from the onset of the disease to the time of bleeding to procure the serum. 506 YELLOW FEVER Inoculations of monkeys with African yellow fever virus were given subcutane- ously six hours after the serum had ken given intraperitoncnlly. The amount of the virus injected was 0.3 cc. per kilogram of bocly weight in expcrimwts I, IT, cud III, and 0.2 cc. per kilogram in Experiment IV. The temperatures of the monkeys were ta$en twice each-,&y. To eliminate the personal equation as far as possible, only tcmperaturcs of 40oC. or above were considered as signifying fever except when otherwise stated in the tables. In the column headed "Prcvcntctl fever" the plus sign (+) signifies that the monkey had no fever and remained well during an observation period of 30 days after inoculation. The minus sign (-.) in this column indicates that fever developed, and was probably due to yellow fever, unless explained in a footnote. In the column "Prevented death" a plus sign (+) shows that the animal did not die. Survival was not necessarily due to the action of the serum, for very rarely rccovcries follow infection with the strains of African virus in use, as for example in the case of one of the controls receiving no serum. A minus sign (-) in this column signiftcs that the rnonlqy died of callow fever. The diagnosis in all fatal cnscs of ~~ellow fever was cletcrmincd 11~ finding characteristic lesions on post- mortem csnminntion 2nd on hi~tologicnl cs3niinntion of the tissues. In the tests of scra for their power to protect guinea pigs against lcptospirae a strain of L. ic/croidcs, nrnzil 49, was used in Esperimcnts A and B, and one of I;. ic-l(,r-niitrc,lllo~),~z[l.si~l~,, Rat I, in IT from n \vilcl rat of Xcw York. In the l'fcilfcr tests 1 cc. of tlic scrxtn to 1x2 tested ~3s mixed with 1 cc. of an active ailturc of leptospirac. 311~1 1 cc. of the misturc was injcct.cd immeclintely into the pcritoncnl cn\rit\r of x guinw pig. Fluicl wns ~vithdrawn from the pcri- toncal cn\.it!- of the guincn pig 30, 60, 00, and 120 minutes after inocul:ltion :tnd esnminetl under the clnrk-ficltl microscope. If the T'feiffur phenomenon was nbscnt, the result w;ts rccorclccl \vith :I minus sign (-) under "T'fciffcr phenomenon." In the cxcs in \vhich the rccorcl ~110~s n plus sign the T'fciffcr phenomenon was prcscnt :\ncl unnii~tnl;:11)1~. The guinc3 p ~ iqi \vhich 112~1 I)vc>n inocul:~tccl in the l'fciffcr tests \vcre kept untlcr olxcr\Tnt iOn to cletcrminc rvhct her the!. \verc protected aginst experimental lcptospirnl jnuntlicc 1,~. the st'ra injcctccl. 111 the column "l'rcventcd fever" a plus sign meant; tllnt the guinc:l pig rcm:linetl free from fever and a minus sign tlint it clevclopccl fever. In the column "I'rcvented dcnth" a plus sign indicates survi\*nl. not nl~vn!~s due to the serum, xrltl 3 minus sign means de3t.h from csperi- mcnt:ll lvptospir:11 j:lunclicc. 1700tnotcs csplain the irrcgulnr results. I'he tliagno- sis of Iq>tos~~ir;\l j2unkliw in the guinea Ijig w;vi nxulc 1)~ tlic alxxrvntion of the j:luntlicc. cstrcmc licnlc~rrlla~cs, and other clurnctcristic lesions, and usually b\. the ol)servntion of lcptospirnc in the tissues c)r body fuids. SAWYER, KITCHEN, FROIXSHER, AND LLOkD 507 The tests of the sera of the fourteen persons who had recovered recently from yellow fever in the epidemic in Rio de Janeiro gave the following results : 10 persons-the serum protected against African yellow fever virus, but not against Ieptospirne (tests with L. iclcroi~1cs in 8 instances and I,. icfcro- htcmorrha~iue in 2). 2 persons--the serum protcctecl qninst Icptospirac but nGi"ngninst African yellow fever virus (both tests with L. iclcrol~ncnzorrl~aSiac). 2 persons-the serum protected against neither African yellow fever virus nor leptospirae (tests with L. ideroitlcs in one instnnce and L. icfcrohacmor- rlzagine in the other). The results with the second and third groups of the scra in the above cIassificntion were clear cut and iqulxr. `I'hosc with the first group showed a few irregularities in the tests in guinea pigs, as is explained in the notes accompanying Tnblc III. In two cases, in which the guinc,z pigs died too soon for diagnosis, the definitely negative l'feiffer test may be xccptctl ns indirect evidence of the lack of protec- tive power in the serum, since in the other crises negntive Pfeiffer tests were followed by failure to protect ant1 positive PfcilTc*r tests by protection of the guinea pigs. Twc guinea pigs inoculated with serum and Icptospirae devcIopcd fever and recovcretl. That occasional rcco\rcries nre to lx cspccted after inoculation with the strain of IL. icleroidcs usd, in tllc :tlxxnco of n protective serum, is shown by the lxhnvior of a control pinca Ijig \vhicll rcccivccl the same strain with normal horse scruni anal rccovcrctl (`I'nl)le IV). The two species of leptospirx, L. ic/croitlrs and L. iclerolzilcnzorrJlaSiac, appear to be so closcl>r r&ted that tlic!r ni:ly be uxtl intcrch:lngcrrbly in immunological tests for Icptospirnl jnuntlicc, ns in this invcstigxtion of ours, and we have drendy referred to the work of investigators ~~110 ol)t:lined strong evidence of their sero- logical identity. It will lx noticed in Table IV that anti-icteroidcs serum, even when diluted, gave positive Pieiffer reactions with both species of lcptospira. This serum hnd been prepared b>, `I'he Rockefeller Institute by immunizing 3 horse against n number of strains of L. ir-ft,roides. The Ascncc of protecti\re power against Africnn yellow fever virus in four of the sera from recent cxes of yellow fever in Rio de Jnnciro (I).r1., nf.J.S., R.B.S., and C.1i.G.) is in our opinion strong evidence against the prcscnce of ~~cllow fcvcr in these C;LSCS, in view of the cvi- d dence nlrcady presented showing that :Ifrican yellow fever and Amcri- can yellow fever are immunologically the same. That some of the 508 YELLOW FEVER sera sent from Rio de Janeiro shouId be from cases other than of yellow fever casts no reflection on the ability of the diagnosticians, for in many cases it is impossible to distinguish by symptoms between yellow fever and leptospiral jaundice, and mild yellow fever may simulate a number of infections. , As evidence of the lack of protective power in these sern we have the results following the inoculation of one monkey in each cnsc, and the confirmatory observa- tions, already mentioned, of Hudson, Philip, and Davis (21) in the case of D.A. The evidence from the inoculation of one monkey in each case seemed conclusive, however, as there was no suggestion of even partial protection by amounts of serum varying from 3.2 cc. to 5.9 cc. according to the weights of the animals. Observations which bear on the reliability of such negative protection tests have been tnbulntcd by Hudson (22) and relate to scrn of 23 West African natives who hxcl rccovcrctl wccntly from ~~110~ fever during ol~scrvetl epidemics. Of 28 monkeys, each of which had reccivcd 4 cc. of serum and 0.25 cc. to 1.0 cc. of blood virus (Asibi strain), only one succumbed to yeIlow fever. The possibility of error of difignosis in the msc of the person supplying the one negative serum cannot be complctcl~~ ruled out. Hudson gives the estimated number of spontnne- ous recoveries of rlxsrts monkeys after inoculation with the Asibi strain of virus as about two per cent. TWO of the scra from Rio dc Janeiro (M.J.S. and C.W.G.) had strong protective power against .L. ictcrolracrilorrlrtrgicle, and in one of these cases (C.W.G.) the occurrence of a dcfinitc relapse after dis- charge from the 1losI~ital is against the diagnosis of yellow fever and suggest i\-c of lcptospirnl jaundice, which so commonly has an aftcr- f c\`c'r. I-I. R. Carter (23), after L2. wide experience with yellow fever, said of that disease, "I have newr seen a relapse, but other men have. Tllq~ must bc rare." Although the possibility cannot be entirely escIuclcd that the protcxtijvc suhtances in either serum were due to an earlier attack of lcptospiral jnundice, it is highly probable that in both cases the illnesses taken for yellow fever were in reality lepto- spiral jauntlicc (\\`cil's disensc). SAWYER, KITCIIEN, FROl3ISIIER, AND LLOYD 509 second day, moderate albuminuria from the fourth day, and jaundice from the seventh. During the first four days the symptoms were such that the physicians were of the opinion that the disease was not yellow fever, but on the seventh day a definite diagnosis of yellow fever was made. The patient was allowed to get up on the 13th day and to leave the hospital on the 15th. On reaching home he again developed high fever and hcadachc. On the 17th day he was able to leave his bed. Convalescence was slow, and he was still distinctly jaundiced on the 48th day, when 1~ visited our laborat-ory in New York. In Rio de Janeiro he worked at the water front and docks in l~lnccs whcrc thcrc wcrc many rats. An investigation of this case in Brazil has been published by R. A. Warner (25). . . I As the sera from most of the 14 persons in Brazil who had recently had symptoms like those of yellow fever possessed strong protective power against African yellow fever virus, the conclusion seems justified that the yellow fever of America is the same disease as that of Africa. -The same conclusion has beers reached by Theiler and Sellards (26), Hudson, Baucr, and Philip (27) and Hudson, Philip, and Davis (21) 8s the result of their protection tests with Arnericm serLz. B. Sera, Ttdw Soou. afiev Suqmfcd Yellow Fever Four of the scrn sent LIS came from a town in southern Brazil, and were from cases in which j-ellonr fcvcr was onI\, suspected. In each case the illness was mild and a diagnosis could not bc made from the symptoms. These specimens were tested with the results wcorded under the heading "Doubtful cases" in Table III Two of the scra protcc`tccl complctcl>~ agninst yellow fever and two did not. protect. at all. One of the scra \vhich protected :lgainl;t the virus and the two others were tested for power to protect against II. ii!t~~~oidt~s, and none protected. We were evidcntl~~ dealing with t\vo cnscs of ~rcllo~v fever, ant1 two cnscs of infection other than yellow fever or leptospird jaundice. The testing of the sera from rcccnt cases of yellow fever at Rio de Janeiro indicated that the I3rnzilian disease was the same as that of flfrica. The nssumption ws dso that it w'czs iclenticd with the disensc studied by- Reed in Cub and suppressed by Gorgx3 in Pmanm. The fact, howcvcr, that the ocean passage between Senegal and Drazil rnrt~- be ;1s short xs six dn~5 permits of the possibility of the transport of the i-Ifric:In >-~llon- fc\m= to IBrxil? dthough thcrc is no cvidcnce of this at tllc I>rcxnt tinlc. Fort un;ltc~lJ~ the relationship of the present yellow fcwr Of Xricn nnd I3ruil to the historic ycllo~v fever of America 510 YELLOW FEVER need not be left to specuIation. Direct evidence has been obtained by the examination of the scra of persons who passed through attacks of American yellow fever many years ago. Serological evidence of the unity of the hrqerican yellow fever of the past and the African yellow fever of the present has been secured by Bauer and Hudson (28). Two sera obtained 23 years after attacks of yellow fever in the Panama Canal Zone and one taken 26 years after an attack in Tampico, Mexico, protected monkeys against African yellow fever. A fourth serum taken 26 years after yellow fever in New Orleans failed to protect. The tests were made in duplicate, The results of the tests of five sera from persons who had had yellow fever in the Americas many years ago are given under the heading "Former epidemics" in Table III. The particulars regarding the sources of thcsc scra arc as follows: G.E. had yellow fever in La Union, SnIvador, in July, 1919, soon after arriving from Ilonduras. The attack was x-cry severe and lasted 14 days. It was char- acterized by much vomiting, heavy albuminuria, and jaundice. According to the patient there were other cases of yellow fever in La Union and in Honduras at the time. Blood was obtained for us by Dr. J. E. Elmcndorf, Jr., and informa- tion regarding the case was supplied by Dr. Pcralta Lagos. The interval between the attack and the bleeding was 9 years and 3 months. C.H.I-I. had yellow fever in :jrncnjil in the State of Scrgipe, Brazil, in May, 1923, according lo his statement. 11\`c arc indebted to Dr. 1.3. I<. Washburn and Dr. IInrgrcnvcs of Kingston, Jamaica, for obtaining blood. The blood was rlrawn 5 years after the attack. D.O.T,. had a very severe attack of ~*ellow fever in the city of Panama in June 1905. ITe was nttendcd b>v several doctors who had had a large experience with yellow fever, including Colonel I!`. C. Gorgas, and Dr. I-I. R. Carter. IVe are indebted to Dr. \V. YE. Dceks and Dr. R. C. Connor for putting 71s in touch with D.O.L., and to Captain J. \\1. Smith of the Afctlical Corps of the U. S. Army for dra\ving a specimen of his blood. The intcrvd between the attack of yellow fever 3rd the tlrnwing of blood \v;~s 2,: yc:lrs ant1 5 months. I.J.K. contracted yellow ftlvcr in r\Iorropcin, Peru, in April, 1920, during an epidemic, and had a mild attack. TTc spent a few months in IVest Africa in 1926. In his case the interval ktwccn the tliscnsc and the bleeding was 8 years and 7 months. .A different spc'cimcn of xrurn of 1.J.K. was tcstccl by Tludson, 16aucr, and Philip (37> and found to protect against ;\fricnn virus, I\sil)i strain. P.S.R. had !Vcllolv fc\vcr in II;I\Y~I~I, Cub, in 11ay, lS99, Xl >-ears before the spccimcn Of SCTUl71 \{`A5 tLlliCt1. SAWYER, KITCHEN, FROIlISIIER, AND LLOYD 511 The results obtained in these tests were as follows: Serum taken S+ years after an attack of yellow fever in Peru pro- tected a monkey completely against African yellow fever. Serum taken 9 years after yellow fever in Salvador permitted the appearance of fever in the monkey but prevented death. A specimen obtained 23 years after an undoubted attack of yellow fever in Panama failed to protect when inject-cd in. the usual amount, but prcvcntcd death, while permitting fever, when used in double quantity. The same lamount of normal human serum failed to prevent the death of a control animal in this experiment and in each of three other cxperi- ments (Table IV). A serum taken five years after, yellow fever in Northern Brazil failed to protect. The evidence with regard to the diagnosis in this case was meager. Serum taken 30 years after yellow fever in Cuba did not protect. The results obtained in these tests and those of the other in- vestigators cited show the historic yellow fever of America to have been the same as the present African yellow fever. If we were right in our conclusion, derived from observation of the protective power of American serlt ng,zinst African virus, that the yellow fever of America is the same as that of Africa, then the sera of persons who have reco\rcrcd from yellow fever in Africa should protect likewise against American yellow fever virus. To demonstrate this by experiment proved difficult owing to the low virulence of the F.W. strain of .4mericnn yellow fever virus for monkeys, 3s the strain exists in this laboratory. Only twice in our experience has death resulted from the inocu- lation of a monkey with the I;.\\`. strain. In using this strain in protection tests it wz necessnry, therefore, to base our conclusions on the presence or absence of fever in the esperimentnl animnls after inoculation. Under these circumstances the results of the tests would of newssit>. be inconclusive in relntion to the indi- vidunl specimen of serum, but they should permit conclusions with reg:nrd to 3 group of scrn when comp3rcd with ,zn ndcquatc series of control tests. In Table V are shown the results of tests of the scra of six natives of the Gold Const of \Vest Africa. -411 of these persons had had yellow fever in observed yellow fever epidemics. 11-e arc indebted to Dr. Henry Bceuwkes, Director of the IVest African Yellow Fever Commission of the Rocl;efellcr Foundntion, for these ser;L. 512 YELLOW FEVER TABLE V PyO/ec/ioft by Scra of Pcrsom Recoccrcdjro~~t AfricaIt YcIZozv I;czw against A werican Serum -~ Africnn natives. K. ot. K. n. IT. Owir. J. 0. k'. N. T. C. Controls: Arncrian, recent rc- covery, AI. A. ~Imerican, rcccnt rc- covcry, c. IV. G., tlolll)lc n11101111 t = - Timr from attack t 0 blcc~ling 22 rnos. 23 I` `l " `6 " ;1\`s ,` I Protected monkey from fever after inoculation \A t 11 Americnn ycl!ow fever virus, I:.\\:. strain* I Result of Inter test inoculation OI monkey with Afric;tn virus, Asibi strain No rcnction . ,X0 reactIon Survivcd$ X0 rraction X0 re:lction So reaction I - (4 .o j :: 1 So rcnction I I = Results of other tests of the sera Did not protect agninst L. ic~tcroides (Table IV) Protected against African fever virus . . yellow (Asil,i) Protcctctl yellow against African fever virus (`I'al,le III) T'rotectccl against L. icfero- /rcnrnrrifn~i~~P:, hut not :\fric:ln yellow fever virus (`l'ahl~ ITT) I'rotectctd xpinst I,. if-b- oides and L. ic~lerohrtlor- rhgi~e, hut not African yellow fever wrus (`I.`nlhz IV> Similar specimens in Table IV I/I I Iad brief fc\-cr rcnching 40oC. but recovered. $ 1Ixd no fever but died on 25th dn~v of observation from cnuse other thnn yel- Ion- fc\.cr. /I Fez\-cr indeilnite or nhent. The highest tcmpcrature of the monlicy in ?!I(- case of c. I\`. G. \vas 39.S"C.; in the case of the anti-icteroides serum, 30.7"C.; in the case of the normnl humnn serum, 39.9"C.; in the cxc of the smnll &XC O! virus. 39.SoC. SAWYER, KITCIIEN, Z'ROBISI-IER, AND LLOYD 513 TABLE V-Comluded Serum Controls-Co7zfi?lzrcd: Normal rlzews mon- key, double amount No serum, full amount of virus No serum, 0.01 amount of virus Time from attack b*efZing I Protected monkey frorr fever after inoculation with American yellow fever virus, F.W. strain' - (4.0) - (4.0) T - (~.W Result of Inter test inoculation of monkey with African 1 virus Asibi strain No reaction No reaction Results of other tests of the sera Similar specimens in Table IV . _ 7 Died from dysentery on 2Sth day after inoculation. The methods of performing these tests and tabulating the results are in general the same as in the previous csperimcnts (Tables III and IV). The regular amount of serum injected into the monkc>ys ~~n.s 1.5 cc. per kilogram of body weight, and the double amount was 3.0 cc. The amount of virus-bearing blood injected was uniformly 0.3 cc. per l;ilogrnm, nnd it w:ns injected subcutaneously six hours after the serum had been riven intrnperitoncally. The results of the tests, though clearly unreliable in the individual case on nccount of thrt low virulcncc of the F.W. strain of virus used, show that the rIfrican sern as n group possessed protective power against American yellow fc\ycr virus. In no case did fever appear in a monkey which had received one of these six sera. The three control animals which received the full amount of virus without serum or after nor.mnl human or monkey serum all showed rises of tempera- ture to 39.9"C. or over at the time when fever would bc expected 8s the result of the inocuhtion. `I'hcsc observntions are in agrecmen t with the stronger evidence obtained IJJ~ testing American sera against African virus. The two African strains we usccI in our experiments (French and Asibi) were sinhr tc> cnch other in virulence for monkeys, but they 514 YELLOW FEVfi;R differed markedly in this respect from the one American strain (F.W.). To facilitate comparison, we have summarized our experience with the inoculation of rhesus monkeys with the Asibi and F.W. strains. In doing so we have considered only those cases in which the inoculum was blood taken on the first day of fever from monkeys experimentally infected with yellow fever in this laboratory. The amount of blood was not less than 0.1 cc., expressed as undiluted whole blood. If titrated blood was injected, the specimen was not over 24 hours old, and, if dried blood was used, it was prepared by drying in vacuum in the frozen state and was not over 100 days old, In fatal cases the animals were allowed to die or were killed when moribund. Of 24 monkeys inoculated with African virus of the Asibi strain, all came down with yellow fever and only one survived. Of 20 monkeys receiving American virus of the F.W. strain, 15 developed fcvcr and two of these died. These tivo, Monkeys V and W, had received dried blood 94 and 63 days old, respectively. In the cases of those animals that showed temperatures of at least 4O"C., the incubation periods for the Asibi strain were, minimum 1.5 days, maximum 6, and average 2.7; for the F.W. strain, minimum 1.5 days, maximum 12, and average 4.3. The intervals between inoculation and death were, for the animals inoculated with the Asibi strain, minimum 2.5 days, maximum 10.5, and average 5. The two animals which died after receiving the F.W. strain did so 4.5 and 8 days after inoculation. The Asibi strain of African virus proved to h;lve a much higher virulence for monlqs than the F.W. strain of American virus. The former caused death in 23 of 24 monkeys inoculated, and the latter in only 2 of 20. It dots not follow, however that there is a similar difference between these strains in their virulence for man, nor that these strains arc representative in virulence of the African and American strains in ~~ncrnl. Dr. iu. Paul Ihdson tells us that the fipres of the laboratoqr of the West African Wlow Fever Commis- sion of the Rockefeller Foundation showed, up to the end of 1928, that there 1vas a marked \ynrintion in the \rirulence of African strains for N~ctrc~rs ~Jzcst~s. ~1 strain (I-II'.) obtained from a fatal case in a European killed only one-third of the monl;c~~s inoculated with blood drawn from infected monkeys at the time of fever. The mortality ~3s low also ivhen this strain leas transmitted from animal to animal by rnosqui toes. SAWYER, KITCHEN, FROBISHER, AND LLOYD 515 Comparison of the Lesions Produced by African and American Strains of Yellow F euer Virus The gross and microscopic lesions produced by the Asibi and French strains of African yellow fever virus in o'ur monkeys were in general those described by Hudson (29) for experimental yellow fever as pro- duced in M. ~JZCSZIS by the Asibi strain. The two animals (Monkeys V and W) which died as the result of inoculation with the F.W. strain of American virus showed gross and microscopic lesions such as are produced by the African strains. One animal (Monkey X) killed during a mild attack following inoculation with the F.W. strain showed only very slight changes. Da Cunha and Muniz (30) speak of the varying extent of the liver lesions in monkeys after inoculation w+ith a Brazilian strain of yellow fever virus. In some animals they found extensive necrosis of the hepatic cells and none in others. Although they f6ctid that their virus possessed less virulence than an African strain, it was sufficient at times to cause early death with extensive lesions like those following inoculation with the African strain, CONCLUSlONS 1. The yellow fever now in South America, the present yellow fever of Africa and the historic yellow fever of Panama and other American countries arc the same disease. This conclusion is based on cross immunity tests in monkeys with strains of yellow fever virus from Africa and Brazil and on tests of sera from 25 persons, who had recovered from yellow fever in lrarious places and at various times, for the power to protect monkeys against African or Brazilian virus strains. 2. Cases of leptospiral jaundice (Weil's disease) were present among those diagnosed as yellow fever in the recent epidemic in Rio de J aneiro. This is shown by the isolation of cultures of leptospirae from the blood of two patients by H. R. Muller and E. 13. Tilden of The Rockefeller Institute, and by the demonstration by us of protective power against leptospirae and absence of protective power against yellow fever virus in the sera from two persons after recovery. The isolation of lcptospirae by Koguchi and other investigators from the 516 YELLOW FEVER blood of occasional patients in past epidemics of yellow fever in a number of American countries indicates that Ieptospiral jaundice was present then as well and was diagnosed clinically as yellow fever. 3. The absence of protective power (against leptospirae shown by the Brazilian sera which protected against yellow fever virus and the absence of protective power against yellow fever virus in the scra that protected against lcptospirae point to the probability that Amcri- can yellow fever is not the combined effect of leptospirae and yellow fever virus. The position of L. ictcroidcs, isolated by Noguchi during yellow fever epidemics, now appears to be not that of a secondary invading microorganism in casts of virus yellow fever, but that of the incitant of a form of infectious jaundice, sometimes fatal, often coin- cident in its appcarancc with typical yellow lcvcr ant1 apparently inclis- tinguishablc from it clinically. This lcptospiral disease has not hitherto been separated from true yellow fever. Noguchi's discoveries become: therefore, of the greatest significance in respect to the epidemiology and causation of yellow fever and of infectious jaundice, previousIy ,. . . confusctl one with the other. In all outbrcnks of supposed yellow fcvcr hcrcaftcr tlic csistcncc of the two kinds of jaundice, one due to yellow fcvcr virus and the other to lcptospirae will have to be taken into account. Only the former probably is spread by mosquitoes and rcquircs anti-mosquito rncnsurcs for its control. 4. The only difference obscrvcd by us between the American and UXcx~ strains of ~~cllow fever virus was a pronounced difference in \+rulcncc for monkeys. The virulence of the two African strains studied was very high while that of the one American strain was highly variable and usually low. 61, No. S22, \\`:lshington. I>. C., 101 1. 2. Noguchi, 11. .I. TIV~L ,Ifc,tl. t1)1t1 I/>l,g., 192.5, 28, 1 S.5. 3. Soguchi, II. .I. Ihp. Ilfd., 1919, 30, -101. 4. Elliott, C. :1. :ivr-lr. I,rI. ilId., 1920, 25, 17-l. 5. Stokes, t\., Hnucr, J. II., and IIudson, S. I'. 11 vz. J. Tro&. Med., 1928, 8, 103. 6. :\rngZo, II. dc 13. 1lcmoirs of Os~vddo Cruz Institute, 1925, Supplement 2, 35. 7. Sellnrds, A. \v. rl~)r. 3. TIT/L Med., 1927, 7, 71. SAWYER, KITCIIEN, FROBISIIER, AND LLOYD 517 . 8. Theiler, RI., and Sell&s, A. W. Ant. J. Trap. Med., 1926, 6, 383. 9. Schtiffner, W., and Mochtar, A. r-lrclziv. f. Sclzifls-md Tropenhyg., 1927, 31, 149. 10. Kligler, I. J. Am. J. Trap. Med., 1928, 8, 283. 11. Gay, D. hf., and Sellsrds, A. 1%`. Ann. of Trap. Med., 1927, 21, 321. 12. Sawyer, W. A., and Baler, J. I-1. Ant. J. Trap. Med., 1928, 8, 17. l.?. Mathis, C., Sellat-&, A. W., and Lnigrct, J. Compt. rcm~. st?lrrccs A cad. sci., 1928, 186, 604. 14. Da Cunha, A. M., and Murk, J. Memoirs of Oswaldo Cruz Institute, 1928, Supplement 2, 51. 15. Arq$o, H. de B. J. Am. Med. Assoc., 1929, 92, 550. 16. Sawyer, W. A., Lloyd, W. D. M., and Kitchen, S. I?. J. hp. Med., 1929, 50, 1. 17. Davis, N. C., and Burke, A. W. J. fixI,. Illal., 1929, 49, 975. 18. Davis, N. C. J. &-p. Med., 1929, 49, 985. 19, Frobisher,M., Jr. Properties of yellow fever virus. fitn. J. IIyg., in press. 20. Muller, I-I. R., and Tilclcn, E. B. Lcpiospirn icicroidcs and Brazilian yellow fever, in preparn t ion. 21. Hudson, N. I'., Philip, C. B., and Da\%, G. E. ri~lt. J. Trap. Med., 1929, 9, 223. 22. Hudson, N. P. T,aborntory report for 192S, West African Yellow Fever Commission of Rockcfcllcr Foundntion. Unpublished" $port to Inter- national Henlth Division of the Rockefeller Foundation. 23. Carter, 1-E. R. Yellow Fever. The Practice of Medicine in the Tropics, eclitcd by I3!xn, IV., and Archibald, R. G., Henry Frowde and Hoddcr and Stoughton, London, 1922, 2, 12-M. 24. Bnrrcto, J. de 13. .+f rc-lri~. cz'c 11~fg., Dr~fwrtomeuto Nacioual de Sam/e Publica (iirdsil), 3d !vcnr. X0. 1, 93. 25. M'arncr, R. A. U. S. Ntrwl Med. Bdl., 1929, 27, 7S6. 26. Theiler, >I., and Sellards, .+I. \V. .,lu~. Trap. Ill~d. and Parasit., 1928, 22, 449. 27. Hudson, X. I'., Baucr, J. I-I., and PhiIip, C. B. ..l~z. J. Trap. Med., 1929, 9,l. 28. Baucr. J. H., and Hudson, X. 1). The duration of immunity in human yellow fever, ns shown by the protective power of the serum. (To bc published.) 29. Hudson, S. I'. .-im. J. Path. 192S, 4, 395, 407, 419. 30. Da Cunhx, 11. SI., and JIuniz, J. >Iemoirs of 0sw;tIdo Cruz Institute, 1929, Supplement 5, 17.