BAc2rzaiowOGIcAL Rzvizws, Sept. 1976, p. 633-451Copyright 0 1976 American Society for Microbiology

Vol. 40, No. 3Printed in U.S.A.

Diagnosis of Plague: an Analysis of the Yersin-KitasatoControversy

DAVID J. BIBEL* Arm T. H. CHENDepartment ofDermatology Research, Letterman Army Institute ofResearch, Presidio ofSan Francisco,

California 94129,* and Medical Microbiology and Immunology Laboratory, School ofPublic Health,University of California, Berkeley, California 94720

INTRODUCTION .......................................... 633THE INVESTIGATORS ............. ............................ 634Shibasaburo Kitasato ............ ............................. 634Alexandre Yersin ........... ............................... 634

THE DISCOVERY OF THE PLAGUE BACILLUS .............. ................ 635The Arrival of Yersin and Kitasato at Hong-Kong ............. ................ 635Yersin's Report .......................................... 636Kitasato's Reports ........... .............................. 636A Comparison of Papers ............... .......................... 638

OPPOSITION .......................................... 638CONFIRMATION ........... ................................ 639THE PROBLEM .......................................... 640AN ANALYSIS OF THE BACTERIOLOGICAL DATA .......... ................ 642The Question of Streptococcus pneumoniae ................................... 642Bacteriology of Plague .............. ........................... 643

CONTAMINATION ........... ............................... 646KITASATO'S REEVALUATION.......................................... 647CLINICAL APPLICATIONS .................. ....................... 648CONCLUSIONS .......................................... 648LITERATURE CITED ............ ............................. 648

INTRODUCTION

From time to time certain bacteriologicalcontroversies have interested medical histo-rians. Often such disputes concern the priorityof discovery, but sometimes the content of thediscovery is at question. The case in point is theindependent observation of the plague bacillusby Alexandre Yersin and Shibasaburo Kitasatoin 1894. The essence ofthe argument is not whofirst found the microorganism but whether Ki-tasato did in fact observe and isolate the agentof plague. Secondary to this is the technicalquestion of how Kitasato could have erred. Thescientific origins of this dispute are probablymore important than the nature of the contro-versy itself. There is no question that Yersincorrectly described and cultured the bacterium.This fact is reflected in the new name of thebacillus: Yersinia pestis (15, 59). Formerlyknown as Pasteurella pestis, the bacterium hasbeen demonstrated, along with P. pseudotuber-culosis, to be sufficiently unique to form a newgenus (82).Joseph McFarland's classic Textbook Upon

the Pathogenic Bacteria (60), published 2 yearsafter the discovery, asserted that the "bacillusof bubonic plague seems to have met an inde-pendent discovery at the hands of Yersin and

Kitasato in the summer of 1894, during theactivity of the plague then raging at Hong-Kong. There seems to be not the slightest doubtthat the micro-organisms described by the twoobservers are identical." However, recent Eng-lish language textbooks and histories of micro-biology have been totally inconsistent in credit-ing Yersin or Kitasato as the discoverer. Onementions only Kitasato (33), many cite onlyYersin (26, 28, 34), a few declare both investiga-tors as independent co-discoverers (16, 27, 92),two are noncommital (48, 58), but most ignorethe problem by eliminating historical introduc-tions. Furthermore, in the first edition of onemicrobiology textbook (27) both Yersin and Ki-tasato were credited with the discovery, but inthe second edition (28), which appeared just afew years later, only Yersin was cited.

In our analysis of the dispute we have alsonoted a confusion in the literature regardingcertain aspects of diagnostic microbiology, in-cluding morphology, growth, and pathology ofthe plague bacillus. It seems that many of thebacteriological problems that faced Yersin andKitasato in the diagnosis of plague are stillwith us. Using the controversy as a vehicle ofreview, we will trace the origins ofthe varianceand demonstrate some difficulties that clinicalmicrobiologists might encounter with this dis-

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634 BIBEL AND CHEN

ease. Plague is far from being eliminated, ascommonly believed, and even in the UnitedStates, where plague is epizootic in the westernregion (49), human disease and death still occur(18-21). The presumptive identification of Y.pestis is deceptively simple. As much of itsclassification is still based upon morphologicalfeatures, the early bacteriological history, towhich these aspects were central, bears recon-sideration.

THE INVESTIGATORSShibasaburo Kitasato

Shibasaburo Kitasato, born in 1852 in amountainous village of the southern island ofKyusho, was one of the first Japanese micro-biologists (65). His medical education began inKumamoto, where the German professor Mans-feld influenced his medical interests and taughthim German, and ended at the Tokyo MedicalSchool in 1883.Hejoined the Central Hygienic Bureau ofthe

Department of Interior and in 1885 was chosento travel to Germany for training under RobertKoch. Kitasato was the first of many foreignstudents in Koch's laboratory. His first studieswere on dysentery and cholera, outgrowths of

FIG. 1. Shibasaburo Kitasato (1852-1931). Pho-tograph was taken in Koch's laboratory circa 1890.

his experience in Japan. After instructing himin the latest techniques of bacteriology, Kochassigned him to investigate tetanus, not expect-ing much success. The anaerobic techniquesdeveloped by Kitasato and the heat resistanceofthe spores allowed him to isolate Clostridiumtetani in pure culture for the first time. Whilestudying the pathogenesis of the organism, hediscovered and characterized its exotoxin, andin 1890 with Emil von Behring was first todemonstrate the neutralization of toxins in ani-mal blood. When Kitasato's term for foreignstudy was about to expire, Koch appealed to theJapanese minister in Berlin for an extension.The Emperor of Japan granted a special fellow-ship through the Minister of the ImperialHousehold, and Kitasato was able to assistKoch in the investigation of tuberculin as acure for tuberculosis.

In 1892 Kitasato returned to Japan and theGerman government awarded him the title ofProfessor, the first such honor given to a for-eign researcher. He was offered positions byEnglish and American universities, but he feltthat his duty was to Japan. On arriving inJapan, Kitasato had some second thoughts. Hehad hoped that the government would establisha laboratory for him. Disappointed, he statedhis intention to go to the United States, but oneof Japan's great intellectuals, Yukichi Fuku-zawa, persuaded Kitasato to remain by fundinga small private laboratory.The government took over the laboratory in

1899, placing it under the control of the Minis-ter of Interior. Next the government estab-lished a serum institute and in 1905 both labo-ratories evolved into the Imperial Institute forInfectious Disease. In 1914 the institute wastransferred to the Minister of Education andplaced under the authority of the President ofthe competing Tokyo Imperial University. Ki-tasato resigned in protest, and the entire staffdeparted with him to establish the KitasatoInstitute of Infectious Diseases.

Later Kitasato was invited by the Keio-Gi-jiku University to organize their medical fac-ulty. He entered politics in 1917 when the Em-peror nominated him to the House of Peers. Forhis long efforts and noteworthy achievementsin developing Japanese medical science, Kita-sato was given the title of Baron in 1924. Kita-sato, the direct and forceful chief of Japanesemicrobiology, died in 1931.

Alexandre YersinThe second figure in this inquiry is Alex-

andre Smile John Yersin, who was born inAubonne, Switzerland in 1863 (9). After earn-

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ANALYSIS OF THE YERSIN-KITASATO CONTROVERSY 635

FIG. 2. Alexandre Yersin (1863-1943), as photo-graphed around 1900.

ing his Bachelor of Arts degree in 1882, hetraveled to Lausanne, Marburg, and Paris formedical training. At the H6tel-Dieu Hospital inParis, Yersin's interest turned to pathology andinfectious disease. Realizing that he lacked thetemperament for personal patient care, he fledto the quiet isolation of the pathology labora-tory, which had its own disadvantages. A cutincurred during the autopsy of a rabies victimled him to Louis Pasteur for prophylactic treat-ment.

Yersin was so impressed with the research atthe 9cole Normal Sup6rieur that he enteredEmil Roux's laboratory to complete his thesison experimental tuberculosis. In June 1888,after receiving his medical degree, Yersin wentto Berlin to enroll in Koch's course in bacteriol-ogy (then taught by Richard Petri and CarlFrankel) in order to compare the program withthat of the French group. Upon his return,Yersin was able to assist Roux in developingthe Pasteur Institute's first course in bacteriol-

90

ogy. Soon the two scientists took up the investi-gation of Bacillus (Corynebacterium) diphthe-riae, recently discovered by Friedrich Loefflerand Edwin Klebs. In 1888 Roux and Yersinannounced their own discovery, diphtheriatoxin, but after publishing three papers on thesubject Yersin suddenly left the institute.

Intrigued with the idea of travel, adventure,and exploration of the newly united Frenchcolony of Indo-China, Yersin took a position asship physician of the Messageries Maritimesthat sailed between Manila and Saigon. To se-cure official sanction and supplies for explora-tions, he accepted Albert Calmette's offer tojoin the Colonial Health Corps. Calmette hadestablished a Pasteur Institute in Saigon in1891. Yersin led three expeditions into the inte-rior which was not without risk, for besidestropical diseases, tigers, cobras, and unruly ele-phants, the people of what is now Vietnamhave always resisted colonialists.

In 1895 Yersin opened a second Pasteur Insti-tute in Nhatrang, which merged with the Sai-gon facility in 1904 with Yersin as director.Later the laboratories at Saigon, Nhatrang,Hue, Dalat, Pnom-Penh, Vientiane, and Hanoiwere consolidated under a common administra-tion. Yersin was also founder and director ofthe Medical School of Hanoi. He continued thestudy of tropical diseases and the production ofvaccines, but shifted his emphasis to the eco-nomic interests of Indo-China, supporting ex-pansion and improvement of agriculture. Thiscomplex man, who never used his first name,who preferred to remain alone with his hobbiesof astronomy, radio, and photography, and whoavoided whenever possible the many meetingsof medical and scientific societies, died in 1943in his home in Nhatrang.

THE DISCOVERY OF THE PLAGUEBACILLUS

The Arrival of Kitasato and Yersin atHong Kong

Both investigators had come to Hong Kong inJune 1894 to study the epidemic of bubonicplague which, spreading through southernChina, had claimed over 40,000 lives (41). Ki-tasato was accompanied by the pathologist Ta-nemichi Aoyama, several medical students,and assistants. Arriving June 12, members ofthe Japanese Commission were afforded gra-cious hospitality by the acting superintendentof the Government Civil Hospital, James A.Lowson, and were given a room at the KennedyTown Hospital (52).

Yersin, on the other hand, with only a serv-ant and some laboratory equipment, including

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a microscope and autoclave, found himselfwithout facilities, quarters, or government sup-port. Yersin had used the epidemic as a pretextfor exploring the Yunnan province of China (9).He had been granted funds by the Minister ofPublic Instruction through the efforts of Pas-teur and Roux, but the Governor-General ofIndo-China turned down the project. Calmetteused his influence to obtain a favorable deci-sion, and Yersin departed Hanoi on June 12,landing in Hong Kong 3 days later.

Yersin was upset more by his lack of access topatients than by his poor reception. He used hisexperience in impromptu field studies duringhis explorations to build a straw hut for hislaboratory and to obtain the necessary speci-mens. He bribed the English sailors whose taskit was to carry the dead to the cemeteries (57).Later, after an appeal to the Governor, Yersinwas permitted patient contact and pathologicalmaterial.

Kitasato and Yersin met only once, but withGerman as their common language, communi-cation was poor. Cooperation was certainlyminimal. Kitasato, however, did present Yersina stained preparation of a supposed pure cul-ture of plague bacillus. Upon vie*ing the slide30 years afterward, Lagrange, a former assist-ant of Yersin, stated that he was unable todecide whether the bacteria were truly plaguebacilli or pneumococci (57). Yersin noted in hisdiary that Kitasato seemed to ignore the buboin his pathological examination (9, 57), a mis-take which was immediately corrected.

Yersin's ReportYersin made his discovery on June 20. As our

focus is on the bacteriology of plague, it wouldbe helpful to recall his classic description of thebacillus (97):The pulp of the bubo, in every case, was filled

with a thick puree of short, thick bacilli withrounded ends, easily colored with aniline dyes butnot by the method of Gram. The ends of the bacilliare more stained than the center. Often the bacilliappear to be covered by a capsule. One can recover agreat amount from the buboes and lymph nodes ofthe diseased. The blood also contains them but notin such great numbers and only in very grave anddeadly cases.The pulp of the bubo, when inoculated on agar,

gives rise to white transparent colonies whose edgesseem iridescent when examined under reflectedlight. The culture does even better on glycerin agar.The bacilli also grow in coagulated serum.

In broth, the bacilli show a very characteristicaspect similar to erysipelas: clear liquid with flockedparticles along the length and bottom of thetube....

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These cultures examined under the microscopeshow true chains of short bacilli, some appearinglike a ball. On agar, if you examine these cultureswith great care and high magnification, one can seebacilli among normal forms that sometimes are thinand sometimes fat chains of rods joined laterally.These swollen and abnormal forms become more andmore numerous in old cultures and stain poorly.

Yersin noted that mice, rats, and guineapigs, but not pigeons, succumbed within 2 to 5days to infection when inoculated with diseasedtissues or cultures. The spleen was enlargedwith eruptions which, to him, resembled mili-ary tubercles, and the microorganism could beisolated from blood, lymph nodes, spleen, liver,and other organs. Of interest was his remarkthat the organism, when found in blood, wasmore elongated than in lymph nodes. Photo-graphs ofthe plague bacilli in pulp and in brothwere included in his report.

Kitasato's ReportsIn comparison, there are two notes on Kita-

sato's studies. The first appeared as an editorialaccount in The Lancet of 11 August 1894 (30).Kitasato's host, Lowson, had sent to the jour-nal's editors some slide preparations and illus-trations made by Kitasato and him. Presum-ably the editors, but possibly Lowson, statedthat "the organism-which is a bacterium re-sembling the bacilli found in the hemorrhagicsepticemias, except that the ends are somewhatmore rounded- when stained lightly appearsalmost like an encapsulated diplococcus, butwhen more deeply stained it has the appear-ance of an ovoid bacillus, with a somewhatlighter center, especially when not accuratelyfocussed. When, however, it is focussed moreaccurately it is still possible to make out thediplococcus form."One of Kitasato's drawings seems to depict a

pure culture in broth, although Lowson apolo-gized for not having been able to tell about theappearance of the microorganisms when culti-vated in vitro. The figure is ambiguous at best.Drawn from medium magnification (aboutx530), bacteria are small with occasionalchains. Some thick, elongated structures arediscerned, but typical round involution formsare not illustrated. In comparison to Yersin'sdescription, the meager evidence tips towardthe discredit of Kitasato, but the hastily drawnfigures should not be taken too seriously.Two weeks later the same journal published

Kitasato's preliminary but only bacteriologicalstudy, which was sent from Hong Kong on July7 (52). Because Kitasato knew little English(80), the article probably was translated from

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ANALYSIS OF THE YERSIN-KITASATO CONTROVERSY 637

the German or Japanese. This point is impor-tant because Japanese is an imprecise lan-guage, tending toward descriptions in generalterms. Furthermore, it is not known how fluentKitasato was in German. Although languagemay have significantly contributed to the con-troversy, we lack additional information andmust set this matter aside.On June 14, Kitasato, examining some 11-h-

old postmortem material, found bacilli in thebubo, but refrained from judging the signifi-cance due to the age of the tissue and the oppor-tunity for contamination. From the blood prep-aration of a patient with severe disease hemade his first careful observations (52):Under the microscope I found bacilli with cap-

sules, the poles of which were stained much deeperwith aniline dyes than the middle part; this gavethem a great likeness to the bacilli of chicken chol-era (bacillus cholerae gallinarum). On the next dayall the serum cultivations which were prepared inthe incubator from the different organs of the bodyand of blood from the finger tips showed a growth ofmicroorganisms, which, under the microscope, werenot to be distinguished from those which we found inthe blood and in the interior of the bubo at the firstpost-mortem examination. The bacilli differed onlyby being a little longer and staining more easily inthe middle than those from the blood.

In another part of the paper Kitasato reiter-ates that "the bacilli are rods with roundedends, which are readily stained by the ordinaryaniline dyes, the poles being stained darkerthan the middle part, especially in blood prepa-rations, and presenting a capsule sometimeswell marked, sometimes indistinct. The bacillifound in the spleen are best stained by a solu-tion of methyl blue."Then discrepancies with Yersin's observa-

tions emerge: "I am at present unable to saywhether or no [sic] 'Gram's double stainingmethod' can be employed...The bacilli showvery little movement and those grown in theincubator, in beef-tea, make the medium some-what cloudy."

Kitasato's description of the morphology ofcolonies is more extensive than that of Yersin'spaper.The different colonies are of a whitish-grey colour

and by a reflected light have a bluish appearance;under the microscope they appear everywhere as ifpiled up with 'glass-wool', later as if having dense,large centres. If a cover-glass preparation is madefrom a cultivation on agar-agar, and having beenstained, is observed under the microscope longthreads of bacilli are seen, which might, by carelessinspection, be mistaken for a coccus chain, but arerecognized with certainty as 'threads of bacilli' un-der closer observations.

Kitasato found Hong Kong's temperature toohigh to properly test gelatin media. An agar-gelatin stab formed a fine dustlike growthalong the puncture and very little on the sur-face. The question of optimal temperature wasdiscussed by Kitasato, but his statements werecontradictory. He first declared that "thegrowth ofthe bacilli is strongest on blood serumat the normal temperature of the human being(34 C.)," but later stated that "as mentionedbefore the bacilli grow best at a temperature offrom 36 C. to 39 C." He apologized for not yetbeing able to determine the minimal growthtemperature. On potato there was no growthafter 10 days when incubated from 28 to 300C,but at 37TC after a few days a small amount ofgrowth was detected. No spores were found inany preparation.

In studying plague victims Kitasato exam-ined their blood daily and observed the samebacillus as found in buboes and internal organsobtained from 15 autopsies. The detection ofthebacillus in blood was not consistent and oftenrequired the preparation of many slides. Be-cause the appearance of the bacillus differedslightly than those from buboes and internalorgans (the latter stained more easily in themiddle), Kitasato was careful to study bothisolates grown under identical conditions. Hedetermined that serum cultures produced thesame form of bacillus. "In any where cultiva-tions are prepared from parts of any internalorgans or from the blood taken from the finger-tips, with careful observation of all due precau-tions, pure cultivations of one and the samebacillus are always obtained."

Kitasato's engrossment with blood stemmedfrom a desire to use the septicemic stage as aneasier means of obtaining specimens for labora-tory diagnosis rather than piercing the verypainful bubo. His report suggested that plaguewas generally a septicemic disease. Kitasato'squestion of whether it was possible to make adiagnosis of bubonic plague from examinationof blood smears was answered in the affirma-tive for many cases. He warned, however, that"a good deal of bacteriological practice is re-quired, or such diagnosis is impossible," andfurther stated that for safety the blood shouldbe cultured as well.The injection into laboratory animals ofblood

or tissue from plague victims or of cultures gaveessentially the same results as Yersin's trials.In the area of inoculation Kitasato observed ablack and red edematous zone that was infil-trated with a gelatinous exudation. The spleenwas enlarged and the lymph nodes were some-times swollen. Animals also died when fed in-

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fected tissue or pure cultures. Kitasato, likeYersin, noted the deaths ofnumerous mice andrats in Hong Kong and also examined the car-

casses. The same bacilli were found in a mouse.

He tested the ability of his isolated plaguebacillus to withstand the effects of desiccation,heat, phenol, and calcium hydroxide, and con-

cluded with a variation of Koch's postulates:

(1) This bacillus occurs in the blood, in buboes,and in the internal organs of the plague-strickenonly; (2) This bacillus is not to be found in any otherinfectious disease; (3) With this bacillus it is possibleto produce in animals the identical symptoms whichthe disease presents in human beings. From thisevidence we must come to the conclusion that thisbacillus is the cause of the disease known as thebubonic plague; therefore, the bubonic plague is aninfectious disease produced by a specific bacillus.

Kitasato sent notes and cultures to RobertKoch in Berlin. Yersin mailed his preparationsto the Pasteur Institute in Paris, and emilDuclaux delivered the paper at the Academy ofSciences on July 30, 1894 (98).

A Comparison of PapersOn the whole, Kitasato's description of the

plague bacillus is quite similar to that of Yer-sin's, but on closer inspection one can find fourmajor differences and several minor discrepan-cies. The more important include the type ofgrowth in broth, the Gram stain reaction, thepresence of involution forms, and motility. Oth-ers include subtle variation in colony descrip-tion, involvement of lymph nodes in experi-mental infections, and appearance of diseasedorgans. Yersin would have noted motility if hehad seen such activity; on the other hand, Ki-tasato might have easily ignored or simply nothave observed involution forms. Kitasato didnot commit the bacillus to either category ofGram staining, and it is rare that two investi-gators describe colonies exactly alike.Thus, given these two documents alone, the

scientific community readily accepted both in-vestigators as independent co-discoverers. Thesimilarities far outweighed the minor varia-tion in observations. Kitasato's paper, beinglonger and more detailed, presented a greateropportunity for finding fault. Furthermore, be-cause both were already recognized as accom-plished scientists, there was little reason todoubt their results. Two independent workersseemed to have described the same microorga-nism; the plague bacillus must have beenfound.Of interest were the respective directions

taken once the organism was isolated. Kita-sato, a product of the German school, sought

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the destruction ofthe bacillus through chemicaland physical methods; Yersin, who was a fol-lower of Pasteur, addressed the questions ofvirulence and immunity.

OPPOSITIONNot everyone accepted the identity of Yer-

sin's and Kitasato's isolated bacteria. Thebreach occurred in 1895 with the publication ina Japanese journal of an article by Kitasato'sassociate, Aoyama, in Hong Kong (3). Writingin German, Aoyama mentioned that Kitasato'sorganism was morphologically different thanYersin's, that it was isolated from the blood,and that it was partially gram positive (gramvariable?). Considering the frequency of sec-ondary infections in plague, the pathologistconcluded that Kitasato's isolate was merely astreptococcus.

This somewhat obscure report probably didnot circulate extensively beyond Japan (al-though W. Kolle in Berlin did obtain a copy[56]), but Masanori Ogata, who was to suggestthe flea as a vector in plague, wrote an articlethat appeared in a major German journal (67).He related that during the epidemic of plaguein Formosa in 1896 the military physician Mu-rakami sent a culture of bacilli isolated from abubo to the military medical school in Tokyo.There K. Okada studied the organism and con-cluded that it resembled Yersin's bacillus, notKitasato's. Ogata remarked that Kitasato him-self had declared in the Journal of the MedicalSociety of Tokyo that his plague bacillus wastotally different than the bacterium of Yersin.Ogata then listed the differences which

Okada had observed (67). With the exception ofadding that the Kitasato microorganism wasgram positive, the table clearly described motil-ity, capsule, agar colonies, cellular morphol-ogy, agar stab, and experimental pathology inthe basic terms as reported by Kitasato andYersin. Ogata, in addition, referred to Kita-sato's lecture in which he had recorded that inlymph nodes of experimental animals both Yer-sin's bacillus and his blood bacterium could befound and that in most cases lymph node en-largement occurs. Reporting on his own studieson plague, Ogata asserted the difficulty in ob-taining positive slides or cultures from blood,and how often other microorganisms could beobtained. He mentioned that in two acutely illpatients he found in the blood bacteria thatresembled pneumococci.Howard-Jones (43) reviewed other examples

of second- or third-hand accounts of Kitasato'sbacterium by Japanese workers. Finally, in1900 Tatsusaburo Yabe published a report (96)

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describing his personal inspection of Kitasato'sbacterium. Noting the distinct capsule, the lan-cet-shaped diplococci, and the frequency of sec-ondary pneumococcal infections in plague vic-tims, the naval surgeon concluded that the or-ganism was indeed a pneumococcus. He relatedthat Kitasato in November 1899 at Kobe ad-mitted that Yersin's bacillus was the agent ofplague, but affirmed that his bacterium co-existed in septicemia and might be importantin the pathology of plague.Two bits of supportive evidence to Kitasato's

concession can be found. The first is from Kita-sato himself. 'An Abstract of the Report uponthe Epidemic of Pest in Japan from November,1899 to January, 1900" by Kitasato et al. wastranslated in an American medical journal (55).The note stated that "besides the pest-bacilli,septicemic bacilli like those of chicken-cholera,staining deeply by Gram's method, staphylo-cocci, and streptococci were seen." This peculiarsentence can be interpreted two ways. La-grange (57) differentiated pest bacilli and septi-cemic bacilli, but Howard-Jones (43) saw nodistinction. The latter, after reviewing theabridged German version (54), furthermoreconcluded that "nowhere is there the slightesthint that there was ever any difference of opin-ion as to the identity of the plague bacillus."The second account was by Norio Ogata, the

son of the above-mentioned critic. He stated(68) that in Kobe in 1899 in discussing theetiology of plague with Ogata and Nakahama,Kitasato admitted that Yersin's bacillus wasthe plague organism and his was but a plague-associated bacterium. Despite these private ad-missions, Kitasato apparently never issued apublic statement declaring his error. We willreturn to this aspect later.

In 1901 Kitasato provided additional bacteri-ological characteristics of his bacterium. Thedescription is highly significant as it differsconsiderably in some respects from his first re-port. The article (53), which was contributed toStedman's medical encyclopedia, is itself con-tradictory. Included was obvious paraphrasingfrom his first report.He and coauthor Nakagawa assured the bac-

terium's rodlike character, especially distinctin lymph nodes. They added that "in the lungs,heart, brain, and spinal cord it is not rare tofind them presenting an appearance like that ofstreptococci. Again, in artificial cultures thestreptococcus-like appearance is the rule. Onattentive observation, however, these cultureswill be found to consist of bacillary chains andnot cocci groups. On the other hand, cultiva-tions on solidified serum present the real rod-

like shape." Bipolar staining was deemed re-sponsible for the polymorphism. The organismwas gram positive. They described spheroid,ovoid, dumbbell, and large rodlike involutionforms particularly in fluid media. Motility wasbest detected at 370C, and, although the activitywas slight, we are urged not to confuse it withmolecular movement. The bacterium's capsule,seen distinctly in tissue preparations, was loston artificial media. Colonies on agar and coagu-lated serum were now described as moist, grey-yellow, transparent, circular, of irregular mar-gins, and minute size. The colonies at first ap-peared granular, then developed a denser anddarker center, but disappeared by day 4 ofincubation. Kitasato noted the strong resem-blance to S. pneumoniae in the nature of thecolonies and in the growth of a gelatin stab.Regarding the latter, we find the statementthat no growth was seen on the surface. Growthin broth presented a turbid appearance, fol-lowed by formation of fine flocculi and sedimen-tation. Optimal temperature was 36 to 390C.The bacterium was a facultative anaerobewhose growth was more vigorous in anaerobio-sis. Milk was coagulated by 48 h. Further notesinclude that swelling of lymph nodes was oftenobserved in experimentally infected animals,and, although infection could be achievedthrough the alimentary tract, inhalation exper-iments were without success. A toxin, producedin broth, whose activity was reduced by 90%upon heating for 20 min at 600C, was described.

CONFIRMATIONClearly, Kitasato's first accounts and those

that followed are in variance. To add to theconfusion, during the same period researchersin Germany who had received Kitasato's cul-tures were obtaining somewhat different re-sults. Hugh Zettnow (99) studied and photo-graphed cultures and preparations of plagueorganisms received from Elie Metchnikoff atthe Pasteur Institute (probably Yersin's isolates)and from Kitasato by way of Koch's Institutefor Infectious Diseases. He noted at x 320 andwith weak light that Kitasato's bacteria couldbe taken as small chains of cocci, but at highermagnification and with better light bipolar ba-cilli were observed. A photograph of a 21-daygelatin stab culture of Kitasato's organismsshowed continuous growth along the stab, ex-cept at the bottom where beadlike coloniescould be seen. There was growth at the surface.Zettnow provided an important date. He re-corded that Kitasato's preparations were sentto Koch in January 1895, 6 months after the

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first isolations. The slides displayed heart bloodand bubo exudate taken from plague victims inHong Kong. Zettnow did not see any differencesbetween Yersin's bacillus and those in Kita-sato's exudate slide. However, he did state thatthe groups of bacteria seen in the blood smearresembled streptococci.Wilhelm Kolle at the Koch Institute re-

viewed the bacteriology of plague (56), notingAoyama's dissent and concern with secondarystreptococcal infections. Zettnow providedKolle with photographs of Kitasato's prepara-tions. In a comparison with strains obtainedfrom various world wide sources, Kolle foundno differences in morphology. Again photo-graphs of Kitasato's preparations were pub-lished, and these different presentations alsoseem to be Y. pestis. It is important to note thatthe slides were of exudate and not of blood.

THE PROBLEMWhy does this apparent paradox exist? There

are several possibilities: (i) Kitasato did notobserve or culture the plague bacillus, butrather a pneumococcus or pneumococcal-likebacterium; (ii) Kitasato observed Y. pestis in

slide preparations, but his initial cultures were(a) of pneumococci or (b) mixed; (iii) Kitasato'sfirst paper was correct, but either (a) his sub-cultures were subsequently contaminated or (b)a different organism was described in a laterisolation. The fourth alternative, that Kitasatoalways described Y. pestis, is clearly elimi-nated by his own admission and the many de-scriptions of another microorganism by col-leagues.The first choice was advocated by some of

Kitasato's associates (3, 67, 96), Meyer (64), andHoward-Jones (43). Lagrange (57) indicatedthat Kitasato might have seen the plague bacil-lus, but his slide preparations and cultureswere ofS. pneumoniae. Lechevalier and Soloto-rovsky (58), Wu Lien-Teh (95), and Girard (35)accepted the second hypothesis, giving Kitasatotoken credit in the discovery. The fourth idea,in spite of Kitasato's own remarks, was sup-ported by Hirst (41), Severn (80), and Bulloch(70) among others. Except for a short statementof belief by Wilson (91) and an acceptance of itspossibility by Howard-Jones (44), the third con-cept has not hitherto been considered in detail.It is the compromise view suggested as an alter-native by the authors.

FIG. 3. Y. pestis from impression smear of spleen of experimentally infected mouse. Wayson's stain.xl,OOO.

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FIG. 4. Y. pestis from blood of experimentally infected mouse. Wayson's stain. x1000.

FIG. 5. S. pneumoniae from 24-hr colony grown on blood agar Gram stain. Kitasato may have confusedthis organism with the plague bacillus. x1000.

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AN ANALYSIS OF THE BACTERIO-LOGICAL DATA

The Question of Streptococcus pneumoniaeConsidering first the possibility that Kita-

sato had both observed and cultured only S.pneumoniae, let us re-examine his first report.We are told that the organism resembled Bacil-lus cholerae-gallinarum, now known as Pasteu-rella multocida. The following is what CarlFlugge in 1886 wrote about this organism (32):"They have been described by Pasteur as cocci,but when highly magnified there is no doubt asto their rod character. The fully-grown individ-uals ... usually show, when stained, an aggre-gation of the colouring matter at the ends, as inthe rabbit septicemia, the dark poles being sep-arated by an unstained central portion. Thebacilli are usually in a state of active division,and thus many forms are found which are con-stricted in the middle, not unlike a diplococcus,and also numerous young individuals in whichthe length is only very slightly greater than thebreadth."The similarity in morphology of the plague

organism to a diplococcus was pointed out inboth the editorial note (30) and Kitasato's re-port (52) in The Lancet. In the former instancethe microorganism's nature was ambiguous;Kitasato assured us that it was indeed a bacil-lus. Obviously the likelihood that Kitasatocould have been mistaken is significant; how-ever, two major items rule against this conclu-sion. Foremost is the quantity and distributionof Y. pestis in the body of plague victims. Kita-sato might have been in error regarding thebacterium in blood, but it is inconceivable thathe did not observe the massive population ofthe plague bacillus in the buboes and ulti-mately the internal organs. Kitasato's descrip-tion is clear enough, and Aoyama's report (3)eliminates the possibility that all the patientswere misdiagnosed. However, it was soon real-ized that some patients were seen who did notsuffer from plague (52). Secondly, Zettnow (99)and Kolle (56) agree that at least the slidepreparations from tissue exudates show bacilliindistinguishable from Yersin's bacillus.On the other hand, superficial evidence indi-

cating that Kitasato's cultures were contami-nated with, if not entirely consisting of, pneu-mococci is not wanting. Here the characteris-tics to examine are colony and cellular mor-phology, growth on potato, in gelatin, and inbroth, temperature relationships, motility, andpathology. Considering the first report, we noteat most a very atypical pneumococcal colony.The colonies of S. pneumoniae normally are

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characteristic. Young colonies are 1 mm in di-ameter, mucoid, transparent, smooth, round,and dome shaped but soon, especially on mediawith blood or serum, undergo autolysis causingthe central region to collapse. The result is achecker-like or nailhead appearance (5). Al-though type III colonies can become large, theyare quite mucoid, and no colony type has adense center. Rough variants have been ob-served, but these are mycelial-like in nature.Other factors counter the implication that Ki-tasato described pneumococcal colonies. The or-ganism grows quite poorly on ordinary mediaespecially when first isolated. He noted thatglycerine agar enhanced growth, and althoughpneumococci do respond in this way (61), theyproduce lactic acid which causes rapid deathand lysis, features not cited in the first paper.

Kitasato's account, nevertheless, leaves roomfor misidentification. The lancet-shape and cap-sule ofthe pneumococcus with careless observa-tion could easily be confused with the envelopedplague bacillus. Kitasato did not record thepresence of involution forms or clarify his state-ment on Gram staining. We should note herethat as S. pneumoniae grows, it becomes gramnegative, and with the coming of autolysismany involution forms are produced (5, 15, 61).Like the plague bacillus, pneumococci grow

poorly, if at all, on potato, and in a gelatin stabthere is filiform or beaded growth along thetract without liquefaction. Some doubt iscaused by Kitasato's observation that little sur-face growth on gelatin was seen, which con-forms somewhat more to S. pneumoniae thanto Y. pestis. Yet we must remember that Zett-now's photograph showed typical growth at thesurface and along the stab.

Kitasato's remarks on optimal temperatureare confusing, but they characterize pneumo-cocci at 370C better than plague bacilli at about290C (15). Slight motility characterizes neitherorganism, but the host range of laboratory ani-mals tends to fit that of plague bacilli. Fowl areresistant to both, and rabbits are not consist-ently susceptible for Y. pestis. However, guineapigs are usually resistant to pneumococci (90).Interestingly, when susceptible animals are in-oculated subcutaneously, edematous exuda-tions and septicemia are found with both micro-organisms (61). Death is somewhat faster withthe pneumococcus, taking 24 to 72 h. The factthat Kitasato described infrequent involvementof lymph nodes indicates, but does not confirm,a pneumococcal etiology. The route of inocula-tion, which was not described, can influencepathogenesis, including the swelling of lymphnodes. In general, pathological analysis can dif-

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ferentiate the two experimental infections, andKitasato's description is more suggestive of Y.pestis.The best evidence for streptococci is the slight

turbidity of broth. Pneumococci produce brothcultures of low turbidity whereas plague bacillitypically yield flocculent strands. As we shalldiscuss later, this does not rule out Y. pestis.The evidence, particularly morphology and

pathology, is substantial that Kitasato's firstpaper did not describe solely streptococci, inspite of the accusations by his Japanese con-temporaries. Nonetheless, the possibility ofmixed cultures is strong. Deferring for the mo-ment further analysis of the contamination hy-pothesis, we will now attempt to show, througha discussion of the bacteriology of plague, that,except for a few very minor errors, Kitasato'sfirst report may have been essentially accurate.

Bacteriology of PlagueCritics might cite turbidity of Kitasato's

broth cultures as proof that he did not isolatethe plague bacillus. We should bear in mindthat Kitasato's exact words were "somewhatcloudy" which, although nebulous, implies alow turbidity. Y. pestis, as Yersin himself re-ported (97), characteristically produces floccu-lent strands in liquid media, but the literatureindicates that this property is not consistent(13, 29, 63, 64, 86). For instance, in Topley andWilson's first edition of their text (86) we foundthe statement that the plague bacillus causeslittle or no turbidity, and in reference to stalag-tite formation this sentence appeared: "Thisproperty is not peculiar to the plague bacillusnor is it possessed by all strains ofthat species."Also, K. F. Meyer (64) mentioned that smoothcolony types produce uniform turbidity,whereas rough colonies give granular sedi-ments with a completely clear supernatant.Stalgtite and stalagmite formation were saidto be enhanced if sterile oil is placed on thesurface, but that smooth colonies recently iso-lated in vivo'do not readily produce these fea-tures.

Pollitzer (72) took a cautious stance, warningthat "one should not be too dogmatic in exclud-ing the possibility that Pasteurella pestis mayunder peculiar circumstances produce uniformturbidity in broth," but admitted that suchgrowth was rare and indicative of contamina-tion. Petrie (70) never observed turbid growthby Y. pestis, but, nevertheless, wrote that vari-able turbidity might be present and was relatedto growth conditions and bacterial strain.With a search of the literature for the origins

of the turbid broth characteristic, it soon be-

came evident that the bulk of the fundamentalbacteriology was developed between 1900 and1930 and little new information as to growthand morphology had been added since. Thelarge number of early studies was due to thelast great plague epidemic at the turn of thecentury, which eventually fell upon India.Sparked by the Yersin and Kitasato reports,several plague commissions were organized (41,70). The Austrians sent Albrecht, Muller,Ghon, and Poech; the German commission in-cluded Gaffky, Pfeiffer, Sticker, and Dieu-donne; Fraser, Wright, and Ruffer representedthe British; the Russian group consisted ofWyssokowitz and Zabolotny. There were alsocommissions from Egypt, Italy, and Ceylon.Later an advisory committee was appointed bythe Secretary of State for India, the Royal Soci-ety, and the Lister Institute.As was mentioned by Meyer (64), broth char-

acteristics of Y. pestis were correlated with col-ony type. Albrecht and Ghon (1) were first toobserve two colonial forms: one being small,round, sharply defined, raised, grey-white butbluish in transmitted light; the other beinglarger with a central nucleus and delicate pe-riphery with wavy edges. Pirie (71) observedthat broth inoculated with pure and stable,smooth S type cultures was uniformly butthinly turbid. Others who found S types to yieldhomogenous growth in broth include Rachin-sky (74), Bessanova and Lenskaya (90), andWats and Pudaval (88). All plague strainstested by Bhatnagar (11) produced many typesof colonies, including small, sticky, dew-drop,large, flat, smooth, opaque, and sunflower orfried-egg colonies. In 1943 Jawetz and Meyer(46) again confirmed colonial variation, butsuggested that differences in agar thicknessand moisture were probably more importantthan inherent strain characteristics. Both sta-ble S and variable R types were obtained fromhuman buboes by Eisler et al. (31). Periph-erally, Wade (87) reported that the type ofgrowth in broth was influenced by varioussugars and that glycerin media would not pro-duce stalagtites.

Kitasato might have examined a relativelystable S type, and, therefore, his description ofbroth would be quite reasonable. His, of course,was a fresh isolate whose activities may havediffered a little from microorganisms which hadbeen subcultured numerous times. Certainly,the distinct bipolarity of staining is quickly lostin vitro (64, 95). Stalagtites, which might havedeveloped in Kitasato's cultures with longerincubation, possibly may have been prevented,for shaking or even slight vibration will disrupt

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their formation (70, 86). The composition ofbroth (beef-tea) also may have been influentialin production of turbidity. Because there is suf-ficient room for variation in description andbecause variation in growth is recognized in thelaterature, we believe that Kitasato's claimcannot be denied outright.

Y. pestis is not motile (15, 63). Kitasato'sremark that the bacillus was slightly motile isperplexing, for one usually does not record thedegree of this characteristic. Brownian move-ment is the easy explanation, and one shouldnot be distracted by Kitasato's later denials(53). Pollitzer (72) presents the situation suc-cinctly: "Plague strains may show such markedBrownian movement that great experiencemay be necessary to decide whether a givenbacillus is immotile or not." Kitasato, being thefirst even to see the microorganism, naturallycan be excused for his hasty observation.There are several minor points that would

not be worth discussing, if it were not for Ogataand Okada's list of notable differences (67). Ki-tasato described his organism as producinggrey-white colonies that appeared blue bytransmitted light. Yersin's was white with iri-descent edges. These subjective characteristicscommonly are viewed differently with each ob-server and, thus, are oflow significance. Never-theless, we should mention that other plagueworkers have interpreted the coloration of Y.pestis as did Kitasato (1, 11, 13, 62).The presence of surface growth in agar or

gelatin stab cultures also has been suggested asan important differential feature. Ogata incor-rectly listed Kitasato's culture as having nosurface growth when, in fact, a small amountwas present. More important, but not listed,was Kitasato's description of dustlike pointsalong the stab, which is not quite typical of theplague bacillus. Y. pestis normally gives a thinfiliform growth along the entire stab with con-fluent growth toward the top. Older culturesoften display small feathery projections, espe-cially near the top of the tube (13, 47, 64, 92).Most workers record a thin growth on thesurface, but Petrie (70) asserts that suchgrowth is not always found. Whereas gelatinalone was used by others, Kitasato was forcedby the heat of Hong Kong to incorporate agar.The proportion of gelatin and agar, the temper-ature of incubation, and the size of inoculumcould have influenced the appearance of Kita-sato's cultures. Perhaps even more importantare the variation and lack of uniform consist-ence in agar and gelatin available at this time.Furthermore, Ogata's table suggested that

Kitasato always observed capsules whereas

they were sometimes absent on Yersin's orga-nism. Kitasato reported capsules whose edgeswere sometimes well delineated but were alsosometimes indistinct. We have not been able toascertain why both investigators' observationsof capsules were considered as showing signifi-cant differences between the two isolates. Ac-tually, Kitasato's was the more accurate, for Y.pestis does not normally produce a true capsulebut rather an envelope (2, 77, 95). The existenceand nature of the diffused envelope was a sub-ject of controversy. Rowland (77) examined thismatter as part of the team investigating plaguein India, and found that he could enhance theenvelope by growing the organism in broth con-taining 10% serum, especially at 3600 instead ofat 2000. Capsules were detected occasionally invivo.The Gram stain is the most perplexing aspect

of Kitasato's report. His statement that he wasas yet unable to determine whether or not theprocedure is applicable to the plague bacilluscould mean that he had not performed the test,an unlikely situation, or that he had difficultywith reagents or in interpretation of results. Y.pestis is gram negative, so ifsome cells retainedthe stain, then contamination is strongly indi-cated. Hirst (41) suggested that Kitasato maynot have had the necessary aniline dye or mayhave had problems with the "tricky" method.Kitasato must have had the necessary re-agents, since the diagnostic importance of theGram stain was well recognized by 1894 andKitasato himself had used it to characterizeClostridium tetani (51). However, at this timethe technique was not as simple or reliable astoday, although even in contemporary timesvariable performance and misinterpretation ofthe test have troubled taxonomists (8, 75, 76).Gram's original procedure of 1883 (7) proba-

bly was still in use in 1894. Ehrlich's anilinegentian violet was impure, unstable, and gaveinconsistent results (7). High-quality commer-cial reagents became available only after 1895(25). Acidity of pus, reagents, or the organismsthemselves could cause poor results. An addi-tional difficulty then and now is the decoloriza-tion step. Gram employed absolute alcohol.Water-diluted solutions (which occur whilewashing off of the decolorizer) could increasethe rate of decolorization in many instances,but 50% alcohol was found ineffective in deco-lorizing the plague bacillus (47, 81). Hence,timing is especially important in preventingunder- or over-decolorization, and temperaturemay have some effect as well. Bismark brownwas sometimes used by Gram, but othercounterstains were found as useful. However,

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high concentrations could cause gram-positivemicrobes to appear negative. Gram and otherworkers at times did not use any counterstainat all, but this, too, had a disadvantage: gram-negative cells may appear gram positive. Fi-nally, the plague bacillus does not take theusual counterstains well and other stains areused for routine observations (6, 63, 95). Meth-ylene blue was the most frequently used in the19th and early 20th century. (Incidentally, heatfixation of Y. pestis is not as satisfactory asalcohol fixation for demonstration of bipolarstaining [63, 721.) Goldenberg et al. (37) con-tended that Gram's method was decidedly infe-rior for observing bipolarity.A clue to the question of Gram staining was

provided by Kitasato's assistant Ishigami who,in 1900 (45), noted that unless solutions werefreshly prepared, the ends of the plague bacil-lus (Yersin's) tended to retain weakly the gen-tian violet dye. In brief, neither the reagentsnor the procedure of Gram staining in 1894 wasstandardized, and results were often inconsist-ent. There are many reasons why Kitasatowould not have been able to ascertain the reac-tion of the plague bacillus.We had some difficulty finding a satisfactory

explanation of Kitasato's rather damaging dataon relationships of temperature to growth. Un-less there was a printing error, he was contra-dictory, for 340C and later 36 to 390C were givenas optimal temperatures. Hong Kong was toohot for gelatin media, yet he was able to incu-bate potato at 280C. No growth was detected onthis rather poor medium at 280C, but at 370C athin growth was observed. Y. pestis is said tohave a minimal growth temperature of -20C(84), 00C (48, 63), or 40C (70) and a maximaltemperature of 400C (15), 430C (48, 63, 70), or450C (84), a very wide range for a pathogen.However, optimal growth is definitely belowthat presented by Kitasato. Some figures foundin the literature include 25 to 300C (13), 27 to280C (84), 280C (63), 28 to 290C (48), 30°C (70),and 30 to 350C (86). Sokhey (83) did report thatan inoculum of 500 colony-forming units on nu-trient agar would not grow at 270C, but at37.50C would yield some colonies. However, aninoculum of 5,000 colony-forming units grewbest at 27°C. When the plague bacilli wereplated on blood agar, no differences were foundwith concentration, and optimal growth was at270C. Kitasato did not state how he judged opti-mal growth. We can only assume, for the sakeof argument, that Kitasato's work was limitedand not quantitative and that his subjectiveimpressions were wrong.

Certain aspects of plague pathology have

been controversial. One aspect pertinent to ourdiscussion is septicemia. Kitasato's associatescriticized his emphasis on diagnosis from bloodsmears and cultures rather than concentratingon the bubo. Except for the period near thevictim's death, when the bacillus spread to thespleen and other organs, plague was suppos-edly limited to lymph nodes. The British PlagueResearch Commission sampled the blood of28 patients, taking a total of 74 specimens. Ofthese, 30 were culturally positive but only 6showed the bacilli in smears (81). Teissier et al.(85) had rather different results, finding septi-cemia in nearly every case and often in theearly stages ofthe disease. Kirschner (50), from237 patients, obtained 212 positive blood cul-tures. Ohoto (69) was similarly successful with72.2% positive results, and Schoebl (78) andGonzaga (38) agreed that bacteremia occursearly. With additional investigations it wassoon established that showering of bacilli fromthe bubo into the blood is frequent, early, gen-erally progressive, and does not necessarily in-dicate a fatal outcome. Today blood cultures arerecommended for early diagnosis (33, 63).

Kitasato's report has been poorly understood.Although he claimed to have observed theplague bacillus in blood smears from 25 to 28patients, it often required several slides and pa-tient, careful examination to detect even smallnumbers of the microorganisms. As such, Ki-tasato readily realized the importance of bloodcultures. Kitasato's observations, far frombeing nonsense or indicative of contamination,seem very reasonable. He apparently was thefirst to recognize the early bacteremic state andthe diagnostic use of blood cultures at a timewhen absolutely nothing was known about theinfectious process. It should be noted that, ac-cording to Ishigami (45), most of the patientsseen in Hong Kong were in the final stage ofthe disease.There were a few minor anomalies in Kita-

sato's experimental animal infections. Method-ology was not specified, but subcutaneous inoc-ulations were the common approach at thetime. Dosage was not given either, since quan-titation was not yet appreciated. If a large inoc-ulum or a particularly virulent bacillus wasused, then death might have been caused bytoxemia, with the result that nodules mighthave been indistinct or even absent on the oth-erwise swollen spleen (70, 95). Because inbredstrains had not yet been developed, Kitasato'sresults could have varied markedly with eachlaboratory animal. This might also explain theabsence of swollen lymph nodes in every case.It is interesting to note that plague-infested

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rodents in the wild normally do not developenlarged nodes or spleens (J. D. Marshall, Jr.,personal communication).A discussion of Y. pestis would not be com-

plete without mentioning its pleomorphism.Cellular morphology is very sensitive to envi-ronment, and varieties of coccal bacillary, andeven moldlike forms, have been observed (47,77). Different sugars can influence shape andstaining characteristics (87). Within the samebroth culture one might find coccal forms at thetop and uniformally staining long bacilli to-ward the bottom (89). Low incubation tempera-tures can produce elongated forms (12), andYersin (97) described long bacilli in blood prep-arations. Photographs (14, 97) of such smearsconfirmed Kitasato's note that the central areaof the plague bacillus took up less stain thanthose cells located in buboes. Of course, involu-tion forms display a wide assortment of shapes.Hankin and Leumann (40) were able to en-hance their formation with media containing2.5 to 3.5% salt.We should furthermore point out that Kita-

sato was working in the midst of an epidemic.The atmosphere for the bacteriological investi-gation of plague, whose etiology and means ofspread were yet unknown, was not conducive tocautious, well-detailed, and controlled work.The first few days probably were intense withfirst impressions governing the scope of action.Kitasato's report was dated July 7, only 3weeks after his arrival in Hong Kong. Underthe circumstances, it is not unreasonable foreven an expert bacteriologist to make someerrors. Despite his lack of facilities, Yersin wasprobably at an advantage working alone, apartfrom the frenzy of the hospitals and the dis-tracting formalities and social interactions as-sociated with governmental commissions. Withthese considerations, we feel that Kitasato'sreport stands up fairly well to criticism. Wehave little doubt that he did isolate, study, andreasonably characterize the plague bacillus.Kitasato should not be denied this credit.

CONTAMINATIONThe matter of contamination remains: when

and how did it occur, and why was it ignored?Secondary infections due to streptococci, in-cluding pneumococci, were a common occur-rence in plague victims (3, 35, 67, 78, 81, 96). Itis almost certain that S. pneumoniae was Ki-tasato's contaminant. This was the opinion ofhis Japanese colleagues (3, 67, 96), Lagrange(57), Wu Lien-Teh (95), and Girard (9). EvenZettnow (99) suggested the streptococcal natureof organisms found in Kitasato's blood slides.

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We have previously sketched some similaritiesbetween the plague bacillus and the pneu-mococcus.Many plague investigators have commented

on the organism's superficial resemblance tothe pneumococcus. The ease in which contami-nation can occur is manifest. An importantpoint of the inquiry is the determination of theapproximate time when Kitasato's cultures be-came contaminated and ultimately replaced.The major report was completed July 7, 1894(52); Koch received the cultures January 1895(99); Aoyama's paper (3) was dated June 1895;and Ogata (67) reviewed the work conducted in1896.Although, as we have demonstrated, Kita-

sato's report may not have reflected contamina-tion, the chances are good that the event oc-curred in June 1894 with a blood culture. Sub-sequent growth studies of broth and optimaltemperature would conform to pneumococci. Bythe end of the Hong Kong investigation manycultures would be mixed, including those se-lected for the return trip. Eventually the con-taminant would overwhelm the plague bacil-lus.Even if the supposition that the report was

essentially accurate is indeed correct, contami-nation still would have taken place in July. Yetthe cultures of the following January yieldedonly plague bacilli. Girard (35) was intriguedby this paradox and sought the answer in mi-crobial interactions as influenced by tempera-ture. Primarily concerned with pneumonicplague being masked by pneumococcal pneu-monia, he found that each organism developedindependently and in different lobes of thelung. When cultured together in serum broth at34WC, the pneumococcus developed faster, re-tarding Y. pestis. Incubating at 200C allowedgreater growth ofthe plague bacillus, but for upto 48 h the pneumococcus still was dominant.Nutrient broth without glucose or serum didnot significantly alter the situation. At 20 to260C plague bacilli were always isolated, but at370C they were virtually eliminated on subcul-ture. Girard observed similar effects on solidmedia, but after 5 days the pneumococcal colo-nies lysed permitting Y. pestis to develop. Ofgreat relevance was the result obtained whenmixed cultures were injected intraperitoneallyinto guinea pigs. Only the plague bacillus wasfound in two of four animals. From this workGirard surmised (9) that on transit to Germanythe pneumococcus lysed leaving gram-negativedebris. The still inhibited plague bacilli wouldrecover on transfer as a pure culture.Tantamount with this discussion is the mys-

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tery why Kitasato did not recognize the con-taminant, and if he did, then why he chose notto admit the error. It is understandable how atfirst he could miss the presence ofpneumococci.Surely, once Yersin's results were publishedand Kitasato had a chance to examine his cul-tures leisurely, contamination would have beendetected. The pneumococcus was no stranger tothe German-trained bacteriologist, for duringKitasato's 7 years at Koch's laboratory theetiology of pneumonia was debated fully (4).

KITASATO'S REEVALUATIONWhy then did Kitasato not admit that his

cultures had become contaminated? To explainthis we must leave bacteriology and speculateon other matters involved with the conduct ofscience.

Kitasato was a celebrated figure in Japanesemedical circles. His discoveries and honor ofbeing selected to work under Koch's tutelageestablished him as dean ofJapanese bacteriolo-gists. He was the perfect candidate for theplague commission. But such recognition stilldid not fulfill Kitasato's desire for his own re-search institute. Returning in 1892 to his home-land, he was so disappointed that no laboratorywas provided for him that he almost left Japan.Supposing that he recognized the contamina-tion of his culture, he would have to come to acritical decision. He might have lost confidencein his first report, not knowing which charac-teristic was due to the pneumococcus, yet todeny the data would mean a loss of credit fordiscovery. An admission of subsequent contam-ination or mistaken identity would be a graveloss of personal prestige and honor, shatteringhis dream of a research institute. The competi-tion by envious Japanese co-workers could havebeen seen as a continuing threat to his position.On the positive side was the possibility thatYersin might have been wrong. Perhaps Kita-sato felt that until new investigations were car-ried out he could stand on his present results.In the interim he could expand his small labo-ratory and build an even stronger reputationthat would withstand the eventual publicknowledge ofhis error. Kitasato diminished hisbacteriological research on plague and under-took work in the broader field of public healthand hygiene.

In 1899 plague invaded Japan, and with accu-mulating evidence countering his later descrip-tions of the plague organism, Kitasato wasobliged to admit his error. He did so in privateas part of an investigative team studying theepidemic in Kobe. However, he did not agreethat his cultures were of pneumococci or any

other recognized bacterium. Instead, he men-tioned that his isolate was associated withplague and perhaps important in its pathogene-sis. This is not quite the same as declaring hisorganism to be a contaminant, as some havetranslated or interpreted. The nuance is impor-tant since two independent Japanese critics (68,96) had quoted Kitasato in the same terms.Thus, Kitasato's concession is not complete.Yabe related (96) that Kitasato claimed to haveseen Yersin's bacillus in the bubo but chose thesepticemic organism as the agent of plague.This statement may have served as a cover forcontamination because Kitasato had earlier as-serted that the blood-borne organism was thesame as that found in the bubo. He also mighthave actually believed that the pneumococcuswas integrally related to the disease process,but it is more likely that he was desperatelytrying to preserve some creditability.Howard-Jones (4, 43) concludes that the lat-

ter hypothesis was the more probable. Kita-sato's assistant in Hong Kong, Tohiu Ishigami,published in 1900 a textbook on plague that wasrevised by Kitasato. In the book (45) one finds astatement that Kitasato confirmed that Yer-sin's bacillus was the cause of plague, but be-lieved that on invading the blood it took on asecond appearance, becoming gram positive.

In 1899 the government had incorporated Ki-tasato's private laboratory into the Ministry ofInterior. He obtained his dream of directing amajor institute in 1905. Secure in his position,Kitasato eliminated the controversy by ignor-ing it, or, when necessary, by diffusing it. Thecontroversy was never discussed at any inter-national medical congress nor at the 1911 Inter-national Plague Conference at Mukden, China,where Kitasato presided over several sessionson bacteriology and pathology and was vice-chairman of the meeting (94).

In 1926 Lagrange attempted to discredit Ki-tasato's claim to discovery. This paper was veryeffective, influencing Meyer (64) and Scott (79)among others. Within the article is the follow-ing declaration: "However in 1925, as chairmanof the Congress of the Far Eastern MedicalAssociation, before 400 members, amongstwhom were 250 foreign delegates, Kitasato is tobe honoured for having publicly stated thatYersin alone was the discoverer of the plaguebacillus." This appears to be the first and onlypublic admission of error by Kitasato. It is notknown where Lagrange obtained such informa-tion, but it apparently is not true (43, 44).Our scenario, we believe, is plausible. Oth-

ers, which may be equally reasonable, may beconsidered, but the truth of any probably will

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never be ascertained. What does stand, how-ever, are the various descriptions of the natureof Kitasato's isolate(s).

CLINICAL APPLICATIONSThe controversy serves as a warning to clini-

cal microbiologists who may someday be re-quired to identify the ancient scourge. The lab-oratory diagnosis of plague has its pitfalls eventoday, particularly at the presumptive level.

Standardized Gram stain procedures and re-agents have virtually eliminated any chancefor confusion with pneumococci or other gram-positive bacteria, but some gram-negative ba-cilli can at times resemble the bipolar Y. pes-tis (64). Conversely, plague bacilli can mimicother microorganisms. Direct microscopic ob-servation of bubo aspirate or blood smears, al-though useful, cannot alone render presump-tive identification. Y. pestis grows quite slowlyon agar, requiring 2 days for macroscopic visi-bility especially at 370C, and, although mor-phology may vary, it is this slow grow that isdiagnostic. Hence, a negative Gram stain, bipo-lar staining, slow growth on agar, and optimalgrowth at 27 to 280C are normal prerequisitesfor presumptive identification of Y. pestis.

Confirmation is best achieved by specificlysis with bacteriophage at 20'C (6, 17, 37, 39,63). At 370C the bacteriophage can also lyse Y.pseudotuberculosis; therefore, two sets of cul-tures are prepared for incubation at the twotemperatures.Another suitable approach is serology, of

which hemagglutination is the most specificand sensitive (22-24, 37). In addition, fluores-cent-antibody techniques (66, 93) have been em-ployed successfully, although they are not with-out fault (36, 73). The Center for Disease Con-trol confirms Y. pestis by the following criteria(20): (i) microscopic and colonial morphology,(ii) lysis by specific bacteriophage, (iii) stainingwith fluorescent-antibody conjugate to Y. pestisfraction 1, and (iv) production of characteristiclesions in mice that are positive by fluorescentantibody.

CONCLUSIONSKitasato's plague studies are as controversial

as the nature of the plague bacillus itself. It isdifficult to compare the 19th and early 20thcentury microbiological literature with Kita-sato's report when several different descriptionsof the organism's growth and colonial morphol-ogy can be found. Contemporary textbook ac-counts are largely based upon the results ofearly, diverse plague commissions and uponmodern investigations of endemic plague

BACTERIOL. REV.

strains. It is a significant possibility that thecharacteristics of the plague bacillus may bealtered in an epidemic affecting man. Althoughspecific means of diagnosis are now available,many presumptive bacteriological proceduresare subject to the same problems that underlinethe historical dispute.From our analysis we are confident that Ki-

tasato had examined the plague bacillus inHong Kong during late June and early July1894. For the most part, his report was an accu-rate description of the bacterium, and the docu-ment alone was sufficient for Western scientificcircles to give Kitasato a share in the discovery.Aside from this purely historical consideration,Kitasato's note served well as a foundation forfurther research and field studies undertakenby many investigative commissions. It is onlybecause of the similarity of the plague bacillusto the pneumococcus under specific but commonconditions that Kitasato was lead to subsequenterror and doubt. Kitasato's face-saving effortsmerely furthered the challenges to his claim ofdiscovery. Nevertheless, the contribution of Ki-tasato to the diagnosis of plague and its historyis significant, and this work will endure.

ACKNOWLEDGMENTSWe thank John D. Marshall, Jr. and Alfred M.

Allen for their helpful criticism. We further ac-knowledge The Lancet and the Annales de l'InstitutPasteur for permission to quote from the reports ofKitasato and Yersin. We give special thanks to Bar-bara Bibel for translation of Yersin's report andliterary support.

T. H. Chen was supported by contract no. DADA17-72-C-2104 of the U. S. Army Medical Researchand Development Command.

LITERATURE CITED1. Albrecht, H., and A. Ghon. 1900. Ober die Beu-

lenpest in Bombay. II. C. BakteriologischeUntersuchungen uber den Peste bacillus.Denkschr. Math. Naturw. Klasse Kais. Akad.Wiss. 66. Wein.

2. Amies, C. R. 1951. The envelope substances ofPasteurella pestis. B. J. Exp. Pathol. 32:259-273.

3. Aoyama, T. 1894-97. tiber die Pestepdiemie inHong Kong in Jahre, 1894-1895. MitteilenMed. Fac. Kais. Jpn. Univ. 3:115-238.

4. Austrian, R. 1960. The Gram stain and the etiol-ogy of lobar pneumonia, an historical note.Bacteriol. Rev. 24:261-265.

5. Austrian, R. 1970. Diplococcus pneumoniae(pneumococcus), p. 69-75. In J. E. Blair, E. H.Lennette, and J. P. Truant (ed.), Manual ofclinical microbiology. American Society forMicrobiology, Bethesda, Md.

6. Baltazard, M., D. H. S. Davis, R. Devignat, G.Girard, M. A. Gohar, L. Kartman, K. F.

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.

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Meyer, M. T. Parker, R. Pollitzer, F. M.Price, S. F. Quant, and P. Wagle. 1956. Rec-ommended laboratory methods for the diag-nosis of plague. Bull. W.H.O. 14:457-509.

7. Bartholomew, J. W., and T. Mittwer. 1952. TheGram stain. Bacteriol. Rev. 16:1-29.

8. Berger, U. 1961. A proposed new genus of gram-negative cocci: Gemella. Int. Bull. Bacteriol.Nomencl. Taxon. 11:17-19.

9. Bernard, N. 1955. Yersin. Pionnier-savant-ex-plorateur 1863-1943. La Colombe, Paris.

10. Bessanova, A., and G. Lenskaya. 1930. Bouil-lontrubende Varetaten des B. pestis Untersu-chungen fiber die Dissoziation des B. pestis.Zentrabl. Bakteriol. Parasitenkd. Infeck-tionskr. Hyg. Abt. 1 Orig. 119:430-443.

11. Bhatnagar, S. S. 1940. Bacteriological studieson Pasteurella pestis and Pasteurella pseudo-tuberculosis. I. The morphology, the growthand the dissociation of Pasteurella pestis. In-dian J. Med. Res. 28:1-15.

12. Bouffard, G., and G. Girard. 1923. Le depistagede la peste par la ponction du foie. Son impor-tance prophylactique. Bull. Soc. Pathol. Exot.16:501-524.

13. Breed, R. S., E. G. D. Murray, and N. R. Smith(ed.). 1957. Bergey's manual of determinativebacteriology, 7th ed. Williams and WilkinsCo., Baltimore.

14. Bryan, A. H., and C. G. Bryan. 1953. Bacteriol-ogy. Principles and practice, 5th ed. Barnesand Noble, Inc., New York.

15. Buchanan, R. E., and N. E. Gibbons (ed.). 1974.Bergey's manual of determinative bacteriol-ogy, 8th ed. Williams and Wilkins Co., Balti-more.

16. Burrows, W. Textbook of microbiology, 20th ed.1973. W. B. Saunders Company, Philadelphia.

17. Cavanaugh, D. C., and S. F. Quan. 1953. Rapididentification ofP. pestis using specific bacte-riophage lyophilized on strips of filter paper.Am. J. Clin. Pathol. 23:619-620.

18. Center for Disease Control. 1975. Fatal bubonicplague-California. Morbid. Mortal. WeeklyRep. 24:211.

19. Center for Disease Control. 1975. Human plague-New Mexico. Morbid. Mortal. Weekly Rep.24:277-278, 283.

20. Center for Disease Control. 1975. Plague in hu-mans-New Mexico. Morbid. Mortal. WeeklyRep. 24:341-342.

21. Center for Disease Control. 1975. Human plague- Colorado. Morbid. Mortal. Weekly Rep.24:390, 395.

22. Chen, T. H. 1972. The immunoserology ofplague, p. 223-251. In J. B. Kwapinski (ed.),Research in immunochemistry and immuno-biology, vol. 1. University Park Press, Balti-more.

23. Chen, T. H., and K. F. Meyer. 1954. Studies onimmunization against plague. VII. A hemag-glutination test with the protein fraction ofPasteurella pestis. J. Immunol. 72:282-298.

24. Chen, T. H., and K. F. Meyer. 1966. An evalua-tion of Pasteurella pestis. Fraction 1-specific

antibody for the confirmation of plague infec-tions. Bull. W.H.O. 34:911-918.

25. Clark, G. 1974. A history of quality assurancein biological dyes. ASM News 40:252-259.

26. Cruickshank, R., J. P. Duguid, B. P. Marmion,and R. H. A. Swain. 1973. Medical microbiol-ogy, 12th ed. Churchill Livingston, Edin-burgh.

27. Davis, B. D., R. Dulbecco, H. N. Eisen, H. S.Ginsburg, and W. B. Wood, Jr. 1967. Microbi-ology. Harper and Row, New York.

28. Davis, B. D., R. Dulbecco, H. N. Eisen, H. S.Ginsburg, and W. B. Wood, Jr. 1973. Microbi-ology, 2nd ed. Harper and Row, Hagerstown,Md.

29. Department of Army Technical Manual, TM 8-227-5. 1963. Laboratory procedures in clinicalbacteriology. Headquarters, Department ofArmy, Washington, D.C.

30. Editorial. 1894. The plague at Hong-Kong. Lan-cet 2:325.

31. Eisler, D. M., G. Kubik, and H. Preston. 1958.Colonial morphology and virulence of Pasteu-rella pestis. J. Bacteriol. 76:41-47.

32. Flugge, C. 1890. Micro-organisms with specialreference to the etiology of the infective dis-eases, p. 315. (W. W. Cheyne, translator).New Sydenham Society, London.

33. Foster, W. D. 1970. A history of medical bacteri-ology and immunology. William HeinemannMedical Books, Ltd., London.

34. Frobisher, M., and R. Fuerst. 1973. Microbiol-ogy in health and disease, 13th ed. W. B.Saunders Co., Philadelphia.

35. Girard, G. 1946. L'association pneumocoque-ba-cille de Yersin. Ann. Inst. Pasteur Paris.72:708-718.

36. Goldenberg, M. I. 1968. Laboratory diagnosis ofplague infection. Health Lab Sci. 5:38-45.

37. Goldenberg, M. I., B. W. Hudson, and L. Kart-man. 1970. Pasteurella infections, p. 422-439.In H. L. Bodily, E. L. Updyke, and J. 0.Mason (ed.), Diagnostic procedures for bacte-rial, mycotic, and parasitic infections, 5th ed.American Public Health Association, Inc.,New York.

38. Gonzaga, A. G. 1922. Cultivating plague bacillifrom blood. Brazil-Med. 1:69-73.

39. Gunnison, J. B., A. Larson, and A. S. Lazarus.1951. Rapid differentiation between Pasteu-rella pestis and Pasteurella pseudotuberculosisby action of bacteriophage. J. Infect. Dis.88:254-255.

40. Hankin, E. A., and B. H. F. Leumann. 1897. Amethod of rapidly identifying the microbe ofbubonic plague. Zentrabl. Bakteriol. Parasi-tenkd. Infecktionskr. Hyg. Abt. 1 Orig.22:438-440.

41. Hirst, L. F. 1953. The conquest of plague. Astudy of the evolution of epidemiology. Clar-endon Press, Oxford.

42. Holvey, D. N. (ed.). 1972. The Merck manual ofdiagnosis and therapy, 12th ed. Merck Sharpand Dohme Research Laboratories, Rahway,N.J.

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.

650 BIBEL AND CHEN

43. Howard-Jones, N. 1973. Was Shibasaburo Ki-tasato the co-discoverer of the plague bacil-lus? Perspect. Biol. Med. 16:292-307.

44. Howard-Jones, N. 1975. Kitasato, Yersin, andthe plague bacillus. Clio Med. 10:23-27.

45. Ishigami, T. 1905. A textbook on plague. Re-vised by S. Kitasato. (D. MacDonald, transla-tor). Pathogenic horticulture. D. MacDonald,Adelaide.

46. Jawetz, E., and K. F. Meyer. 1943. Avirulentstrains of Pasteurella pestis. J. Infect. Dis.73:124-143.

47. Jennings, W. E. 1903. A manual of plague. Reb-man Ltd., London.

48. Joklik, W. K., and D. T. Smith (ed.). 1972. Zins-ser microbiology, 15th ed. Appleton-Century-Crofts, New York.

49. Kartman, L., A. R. Martin, W. T. Hubbert, R.N. Collins, and M. I. Goldenberg. 1967.Plague epidemic in New Mexico, 1965. Epi-demiologic features and results of field stud-ies. Public Health Rep. 82:1084-1094.

50. Kirschner, L. 1934. Gal als voedingsboden bij dediagnose der pest septichaemie. Geneeskd.Tijdschr. Ned. Indie 74:1141-1159.

51. Kitasato, S. 1889. Ueber den Tetanusbacillus. Z.Hyg. Infektionskr. 7:225-233.

52. Kitasato, S. 1894. The bacillus of bubonicplague. Lancet 2:428-430.

53. Kitasato, S., and A. Nakagawa. 1901. Plague, p.

325-352. In T. L. Stedman (ed.), Twentiethcentury practice, vol. 15. W. Wood and Co.,New York.

54. Kitasato, S., T. Takaki, K. Shiga, and G. Mo-riya. 1900. Bericht uber die Pestepidemie inKobe and Osaka von November 1899 bis Jan-uar 1900. Sanitasabteilung im Ministeriumdes Innern, Tokyo.

55. Kitasato, S., T. Takaki, K. Shiga, and G. Mo-riya. 1901. An abstract of the report upon theepidemic of pest in Japan from November,1899 to January, 1900. (M. Ostheimer, trans-lator). Phila. Med. J. 7:94-95.

56. Kolle, W. 1897. Zur Bacteriologie der Beulen-pest. Dtsch. Med. Wocherschr. 23:146-148.

57. Lagrange, E. 1926. Concerning the discovery ofthe plague bacillus. J. Trop. Med. Hyg.29:299-303.

58. Lechevalier, H. A., and M. Solotorovsky. 1965.Three centuries of microbiology. McGraw-HillBook Co., New York.

59. van Loghem, J. J. 1944. The classification ofplague bacillus. Antonie van Leeuwenhoek J.Microbiol. Serol. 10:15-16.

60. McFarland, J. 1896. A text-book upon the patho-genic bacteria for students of medicine andphysicians, p. 318-321. W. B. Saunders Co.,Philadelphia.

61. MacLeod, C. M. 1965. The pneumococci, p. 391-411. In R. J. Dubos and J. G. Hirsch (ed.),Bacterial and mycotic infections of man, 4thed. J. B. Lippincott Co., Philadelphia.

62. Markl, J. B. 1914. Zur Frage der Mutation beiPest Bacillen. Zentrabl. Bakteriol. Parasi-

BACTERIOL. REV.

tenkd. Infectionskr. Hyg. Abt. 1 Orig. 74:529-540.

63. Marshall J. D., Jr., and D. C. Cavanaugh. 1970.Pasteurella, p. 205-209. In J. E. Blair, E. H.Lennete, and J. P. Truant (ed.), Manual ofclinical microbiology. American Society forMicrobiology, Bethesda, Md.

64. Meyer, K. F. 1948. The pasteurella, p. 415-432.In R. J. Dubos (ed.), Bacterial and mycoticinfections of man. J. B. Lippincott Co., Phila-delphia.

65. Miyajima, M. 1931. Robert Koch and Shibasa-buro Kitasato. Sonor, Geneva.

66. Moody, M. D., and C. C. Winter. 1959. Rapididentification of Pasteurella pestis with flu-orescent antibody. III. Staining Pasteurellapestis in tissue impression smears. J. Infect.Dis. 104:288-294.

67. Ogata, M. 1897. Ueber die Pestepdiemie in For-mosa. Zentrabl. Bakteriol. Parasitenkd. In-fectionskr. Hyg. Abt. 1 Orig. 21:769-777.

68. Ogata, N. 1955. Wer hat die Pestbazillen zuerstentoleckt? Kitasato? Yersin? Oder Kitasatound Yersin? Zentrabl. Bakteriol. Parasi-tenkd. Infectionskr. Hyg. Abt. 1 Orig.163:171-172.

69. Ohoto, 0. 1923. On the B. pestis in blood and itscultivation. Jpn. Med. World 3:136-137.

70. Petrie, G. F., and W. Bulloch. 1929. Bacilluspestis (Syn. Pasteurella pestis), p. 137-224. InMedical Research Council, A system of bacte-riology in relation to medicine, vol. 3. HisMajesty's Stationery Office, London.

71. Pirie, J. H. H. 1929. Plague studies. II. Microbicdissociation of B. pestis and its importance inconnection with the preparation of plaguevaccine and serum. Publ. S. African Inst.Med. Res. 4:191-230.

72. Pollitzer, R. 1954. Plague. World Health Orga-nization, Geneva.

73. Quan, S. F., W. Knapp, M. I. Goldenberg, B. W.Hudson, W. D. Lawton, T. H. Chen, and L.Kartman. 1965. Isolation of a strain ofPasteu-rella pseudotuberculosis from Alaska identi-fied as Pasteurella pestis: an immunofluores-cent false positive. Am. J. Trop. Med. Hyg.14:424-432.

74. Rachinsky, B. 1930. Vergleichende Untersu-chungen uber die R-und S-Formen des Bact.pestis und Bact. Pseudotuberculosis roden-tium. Vestn. Mikrobiol. Epidemiol. Parazitol.9:369-376.

75. Reyn, A. 1970. Taxonomic position of Neisseriahaemolysans (Thjotta and Boe 1938). Int. J.Syst. Bacteriol. 20:19-22.

76. Reyn, A., A. Birch-Andersen, and U. Berger.1970. Fine structure and taxonomic position ofNeisseria haemolysans (Thjotta and Boe 1938)or Gamella haemolysans (Berger 1960). ActaPathol. Microbiol. Scand. Sect. B 78:375-389.

77. Rowland, S. 1914. The morphology of the plaguebacillus. J. Hyg., 13(Plague Suppl. III):418-422.

78. Schobl, 0. 1913. Bacteriological observations

Dow

nloa

ded

from

http

s://j

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als.

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.org

/jour

nal/b

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.

ANALYSIS OF THE YERSIN-KITASATO CONTROVERSY 651

made during the outbreak of plague in Manilain 1912. Philipp. J. Sci. Sect. B: 8:409-426.

79. Scott, H. H. 1953. Conquest of plague. Br. Med.J. 2:1327.

80. Severn, A. G. M. 1927. A note concerning thediscovery of the Bacillus pestis. J. Trop. Med.Hyg. 30:208-209.

81. Simpson, W. J. 1905. A treatise on plague deal-ing with the historical, epidemiological, clini-cal, therapeutic and preventive aspects of thedisease. Cambridge University Press, Cam-bridge.

82. Smith, J. E., and E. Thal. 1965. A taxonomicstudy of the genus Pasteurella using a numer-ical technique. Acta Pathol. Microbiol. Scand.64:213-223.

83. Sokhey, S. S. 1939. Experimental studies inplague. The solid medium of choice and opti-mal temperature of incubation for the growthofplague bacillus. Indian J. Med. Res. 27:321-329.

84. Sokhey, S. S., and M. K. Habbu. 1943. Optimumand limiting temperature for the growth ofthe plague bacillus in broth. J. Bacteriol.46:25-32.

85. Tessier, P., L. Tanon, P. Gastinel, and I.Reilly. 1921. Valeur diagnostique del'h6moculture dans la peste bubonique; frequ-ence de la bacillemie pesteuse. Bull. Mem.Soc. Med. Hop. Paris 45:136-138.

86. Topley, W. W. C., and G. S. Wilson. 1931. Theprinciples of bacteriology and immunity, p.482-490. William Wood and Co., New York.

87. Wade, H. W. 1916. Carbohydrate fermentationby Bacillus pestis, comparing certain Ameri-can and Oriental strains with analysis of dis-crepancies of fermentations with Hiss's serumwater, litmus agar, and bouillon. Philipp. J.Sci. Sect. B 11:159-182.

88. Wats, R. C., and T. K. Pudaval. 1940. A study ofsome virulent and avirulent strains ofPasteu-

rella pestis. Indian J. Med. Res. 27:823-831.89. van Westernik, N. 1906. Ueber die bipolare

Farbung der Pestmikroben. Zentrabl. Bakte-riol. Parasitenkd. Infectionskr. Hyg. Abt. 1Orig. 42:181-184, 283-288.

90. White, B., E. S. Robinson, and L. A. Barnes.1938. The biology of pneumococcus. The bacte-riology, biochemical, and immunologicalcharacters and activities of Diplococcus pneu-moniae. The Commonwealth Fund, NewYork.

91. Wilson, G. S., and A. A. Miles. 1964. Topley andWilson's principles of bacteriology and immu-nity, 5th ed. Williams and Wilkins Co., Balti-more.

92. Wilson, G. S., and A. Miles. 1975. Topley andWilson's principles of bacteriology, virology,and immunology, 6th ed. Williams and Wil-kins Co., Baltimore.

93. Winter, C. C., and M. D. Moody. 1959. A rapididentification of Pasteurella pestis with flu-orescent antibody. II. Specific identification ofPasteurella pestis in dried smears. J. Infect.Dis. 104:281-287.

94. Wu Lien-Teh. 1959. Plague fighter. The auto-biography of a modern Chinese physician. W.Heffer and Sons, Ltd., Cambridge.

95. Wu Lien-Teh, J. W. H. Chun, R. Pollitzer, andC. Y. Wu. 1936. Plague. A manual for medicaland public health workers. Weishengshu Na-tional Quarentine Service, Shanghai.

96. Yabe, T. 1900. Sur le microbe de la peste. Arch.Med. Nav. 74:469-472.

97. Yersin, A. 1894. La peste bubonique a Hong-Kong. Ann. Inst. Pasteur Paris 8:662-667.

98. Yersin, A. 1894. La peste bubonique a Hong-Kong. C. R. Acad. Sci. 119:356.

99. Zettnow, H. 1896. Beitrage zur Kenntniss desBacillus der Bubonenpest. Z. Hyg. Infekt.21:165-169.

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