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Journal of Experimental Medicine

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ETIOLOGY OF OROYA FEVER.
VIII. EXPE]~r~W.NTS ON CROSS-IMMUNITY BETWEEN OROYA FEVER
AND VERRUGA PERUANA.
BY HIDEYO NOGUCHI, M.D.

(From tke Laboratories of The Rockefeller Institute for Medical Research.)(Received for publication, December 30, 1926.)

A common etiologic origin of Oroya fever and verruga peruana was
first indicated experimentally by the production of verrucous lesionsin monkeys (Macacus rhesus) by inoculation of cultures of Bartonella
bacilliformis isolated from the blood in a fatal case of Oroya fever?
Subsequent experiments with the same strain ~,3 gave evidence that thetype of disease induced by the parasite was dependent upon two factors, the virulence of the strain, and the degree of susceptibility of theindividual animal, various grades of infection being induced in theexperimental animals, from a moderate or extensive local skin affection to a severe systemic disease terminating in fatal anemia. Morerecently, * there has been isolated from the skin lesion of verruga
peruana a microorganism morphologically and serologically identicalwith Bartonella bacilliformis and having similar pathogenic properties.
It remained to be proven that animals which had recovered from infection with the Oroya strain of Bartonella bacilliformis would be able
to resist infection with the verruga strain.
The disease induced by Bartonetla badIliformis in M. rhesus ischronic in nature, but complete recovery may take place and isevident from (1) the subsidence of the febrile reaction, ~2) the disappearance of the skin lesions, (3) the sterility of the blood andlymphatic glands, as shown by cultivation experiments, and finally(4) immunity to the homologous strain. All of the ten monkeys
1 Noguchi, H., and Battistini, T. S., J. Exp. Med., 1926, xliii, 851.
Noguchi, H., J. Exp. Med., 1926, xliv, 697.
a Noguchi, H., ar. Exp. Ivied., 1926, xliv, 715.
4 Noguchi H., J. Exp. ]Wed., 1927, xlv, 175.
781

782

ETIOLOGY O1• OROYA FEVER.

VIII(nine Macacus rhesus and one chimpanzee) selected for the present
investigation had been shown b y cultivation of blood and l y m p h a t i cgland tissue to be free from organisms, and five of them had been
p r o v e d immune to the homologous (Oroya) strain b y one or more
previous i m m u n i t y tests. T h e final test of i m m u n i t y against b o t hstrains was m a d e with suspensions of nodular tissue from M . rhesus
41 (verruga strain, second passage) and M . rhesus 36 (Oroya strain,fifth passage), the Oroya strain being inoculated on the right eyebrowand abdominal skin, intradermally and b y scarification, the verruga
strain similarly on the left side. B o t h suspensions induced welldeveloped lesions in a control monkey, the verruga strain being decidedly the more virulent locally, as shown in the protocol of thisanimal. In the ten animals which had previously passed through acourse of infection with the Oroya strain no signs of infection could beinduced.
M. rhesus I had been inoculated on Nov. 16, 1925, intradermally and intravenously, with the first generation cultures of the blood of Case S. A. 153 BaT.
tondla badlliforrMs was cultivated from the blood several times during the coarseof disease, and nodules arose at the site of intradermal inoculation. The lesions
had disappeared within 2 months, and on Feb. 22, 1926, cultures of blood andlymphatic gland tissue were negative. Mar. 9, 1926, the animal received intravenously and intradermally a mixture of cultures and a suspension of nodular
tissue from M. rhesus 18.3 Blood taken on Mar. 31, and Apr. 13 proved sterile.
On Apr. 29 the animal was inoculated again, intradermally and by scarification,with a mixture of cultures with a suspension of nodular tissue from M. rl~sus 25.3
On May 19, minute lesions appeared to be developing on the eyebrows, but theslight indurations disappeared, and blood and lymphatic gland tissue taken on
June 1 yielded no cultures. The final test with both Oroya and verruga strains
was made on June 3, the inoculation being made intradermaUy and by scarification. No lesions developed.
M. rlmus 2 was first inoculated Dec. 8, 1925, and had passed through a protracted course of infection with the Oroya strain. 1 The blood had yidded cultures
on several occasions, and the nodules at the sites of intradermal injection on theeyebrows were still present on Mar. 27. They had disappeared by Apr. 27, andthe bbod and lymphatic gland tissue taken on that date were negative by culture
test. On Apr. 29 the animal was reinoculated with a mixture of cultures with asuspension of nodular tissue from M. rhesus 25.3 No signs of loeal or systemic
infection appeared, and blood and lymphatic gland tissue proved sterile on Jane
1. On June 3 the animal was inoculated intradermally and by scarification withboth the Oroya and the verruga strains. Blood and lymph node cultures madeJune 22 and July 13 proved sterile. No local lesions developed.

HrD~YO ~OGUCHI

783

M. rhesus 3 had been inoculated with the same material as M. rhesus 2 and had
passed through a similar course of disease. Only traces of the local lesions werevisible on Mar. 8, 1926. On Mar. 9 the animal was inoculated intradermally andby scarification with a suspension of nodular tissue from M. rhesus 183 Bloodtaken on Mar. 22 yielded cultures of Bartonella baczTliforrais, and small but definite
lesions developed at the sites of local inoculation. These had disappeared byApr. 28, however, and the blood was sterile. On Apr. 29 a suspension of nodular
tissue from M. rhesus 25~ and cultures were introduced intradermally and by scarification. No local reactions resulted, and on May 19 and June 1 blood and lymphatic gland tissue were sterile. On June 3 the animal received the inoculation ofboth the Oroya and the verruga strains, intradermally and by scarification. Theblood remained sterile, and no local lesions developed.
M. rhesus 125 had been inoculated on Jan. 19, 1926, intravenously, subcutaneously, and by scarification with cultures derived from Monkeys 4, 6, and 7. The
blood was positive by culture on two occasions, and there was high fever for 1
week, but the local lesions were never large, and they disappeared within a month.
On Mar. 25 blood and lymphatic gland tissue were sterile. On Apr. 22 the animal
was inoculated with a suspension of nodular tissue from M. rhesus 18,2 culturesderived from Monkeys 7 and 20, and citrated blood of M. rhesus 25.2 The blood
was negative by culture on May 27, and lymphatic gland tissue was negative on
June 1. No lesions developed. On June 3 the final test with both the Oroya andthe verruga strains was made. The blood and lymphatic tissue were sterile whentested on June 22, and no lesions developed.
M. rhesus 16 had received two injections (Feb. 10 and Feb. 15, 1926) of killedcultures of Bartonella bacilliforrais. No reaction resulted. On Mar. 6 active
suspensions of the skin lesions from M. rhesus 52 and M. rhesus 18 were injected intradermally and by scarification. By Apr. 5 a large nodule had developed on theabdomen. On Apr. 14 it was larger and deep cherry red. By Apr. 28 the lesion
had practically healed. On Apr. 29 the animal received cultures and a suspensionof nodular tissue of M. rhesus 25* intradermally. Small papules appeared at thesites of injection on May 22, but they disappeared, and on June 1 no lesions wereapparent. Blood and lymphatic gland tissue taken on this date were sterile.
The final test with both the Oroya and the verruga strains was made on June 3.
No lesions developed. The blood was sterile when tested on June 21.
M. rhesus 18~ had been inoculated on Feb. 15, 1926, intradermally and by
scarification with a suspension of nodular tissue from M. rhesus 5. Extraordinarily
extensive lesions had developed by Mar. 17, and blood taken on Mar. 18 yielded
cultures of B~rtonella bacilliformis in dilutions as high as 1 : 100,000. The lesions
persisted for many weeks but had become very small, pale, and fibrous by themiddle of June. Blood and lymphatic gland tissue taken on May 15 yielded nocultures. No reaction followed the inoculation of the Oroya and verruga strains on
June 3, and on June 22 the blood and lymphatic tissue were sterile by culture test.
M. rhesus 22, inoculated Mar. 5, 1926, intradermally and by scarification on
5 Noguchi, H., J. Exp. Med., 1926, xliv, 729.

784

ETIOLOGYO]~ O R O Y AFEVER.

VIIIeyebrows and abdomen with nodular tissue of M. rhesus 5. Apr. 26, blood diluted
1:1,000 yielded cultures of Bartonella bacilliformis. A nodule had developed on
the right eyebrow, and by May 6 it was very large and pedunculated. The lesion
had disappeared by June 1, and blood and lymphatic gland tissue taken on thatdate were sterile by culture test. The final test with the Oroya and verruga
strains was made on June 3. No lesions developed during 4 weeks of observation,and the blood was sterile by culture test on June 22.
M. rhesus 265 had been infected by the bites of four ticks previously fed on M.
rh~us 18, the ticks having been transferred from Monkey 18 to Monkey 26 on
Mar. 29, 1926. Lymphatic gland tissue (inguinM) taken on Apr. 14 yielded cultures of Bartonella bacilliformis, as did also blood taken on Apr. 24. On May 28
blood and lymphatic tissue were both sterile. The animal was inoculated onJune 3 with both the Oroya and the verruga strains. No lesions developed during
6 weeks of observation. The blood was negative by culture test on June 22.
M. rhesus 31 was inoculated intradermally Apr. 5, 1926, with a suspension of
testicular tissue of Rabbit 1744s and nodular tissue of Dog 1.8 Blood taken onApr. 28 yielded cultures in a dilution as high as 1 : 10,000. The nodules were welldeveloped at this time. May 22, the nodules were reduced in size. May 28, blood
culture sterile. June 1, lesions had disappeared, blood and lymphatic tissue failed
to yield cultures. June 3, final test with Oroya and verruga strains. No reaction.
Blood sterile on June 22.
Chimpanzee (Pan leucoprymnus), had been inoculated Jan. 29, 1926, intraderreally, subcutaneously, and by scarification, with mixture of cultures and suspensionof nodule of M. rhesus 3. Blood positive in dilution of 1 : 10,000 Feb. 23, 1 : 100Mar. 12. Large nodules developed on eyebrows and abdomen. The animal
had recovered completely by May 1, and neither blood nor lymphatic gland yielded
cultures of Bartonella bacilliformis. On June 3 the animal was tested for immunity
against the Oroya and verruga strains in the same way as the nine rhesus monkeys.
No lesions developed, and blood culture was negative on June 28.
T h e following protocol illustrates the effects p r o d u c e d in a noni m m u n e control b y inoculation of the materials used on J u n e 3 forthe final test against the O r o y a and verruga strains of Bartonella

bacilliformis.
M. rhesus 45 was inoculated June 3, 1926, with the same materials and in thesame manner as the recovered monkeys, the Oroya strain being inoculated on theright eyebrow and abdominal skin, the verruga strain at corresponding sites on theleft side. Several minute nodules were recognizable at the sites of inoculation on
Both the testicular tissue of the rabbit and the nodule of the dog yielded cultures of Bartonella bacilliformis; the report of these and other experiments will
be published later.

HIDEYO NOGUCHI

785

the left side within 10 days and had attained considerablesize (8 × 20 ram.) 32
days after the inoculation. The reactions at the sites of inoculation with theOroya strain did not become noticeable until 3 weeks after inoculation. The
nodules reached their maximum size (4 >( 8 ram.) by the middle of July, neverattaining the extent of the lesions on the left side. All the lesions were similar incharacter, however, being of the usual subcutaneous, mular type. Culturesmadewith blood withdrawn on June 28, 1926, 25 days after inoculation, yielded growthof Bartonella baciltiforrnisin a dilution of 1 : 100,000. All the lesions had healedby the middle of August.
The experiments demonstrate that rhesus monkeys which haverecovered from infection with the Oroya strain of Bartonella bacilliformis are completely immune to the verruga strain. The results
warrant the conclusion that the strains from Oroya fever and verruga
peruana are identical, and that both conditions are manifestations ofinfection vdth Bartonella bacilliformis.
The duration of the infection in the present series of animals varied
from 2 to 5 months. The cultural test of lymphatic gland tissue
appears to be extremely important in determining whether or not theanimal is free from infection, as has already been shown in anotherseries of e x p e r i m e n t s / i n which it was found that two animals of agroup of four which had apparently recovered still harbored Bartonella
bacilliformis in certain organs 48 and 58 days, respectively, after inoculation. In this earlier series of animals convalescence was establishedas early as 30 days in some instances, while in others the infection wasstill active 68 days after inoculation. The duration of a fatal infection varies from 25 to 57 daysY
SUMMARY.

Nine monkeys (Macacus rhesus) and a chimpanzee which had recently recovered from an infection with the Oroya strain of BartonelIa
bacilliformis were tested for immunity against the verruga strain of
Bartonella bacilliformis as well as against the homologous strain.
Complete immunity to both strains was demonstrated. The result
establishes the identity of the strains and is in agreement with theresult of comparative serological study.
Noguchi, H., J. Exp. Med., 1927, xlv, 437.

786

ETIOLOGY OF OROYA F E V E R .

VIIIThe criteria of recovery include not only the subsidence of febrilereactions and local lesions but also a negative result of cultural tests ofblood and lymphatic gland tissue.
Recovery may occur as early as 1 month after inoculation, but inmost instances a period of 2 to 5 months is required for the completionof convalescence.