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Journal of Experimental Medicine

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BACTERIOLOGICAL AND CLINICAL STUDIES OF AN
EPIDEMIC OF KOCH-WEEKS BACILLUS CONJUNCTIVITIS ASSOCIATED WITH CELLINCLUSION CONJUNCTIVITIS. 1
BY H I D E Y O N O G U C H I , M.D., aI~D M A R T I N C O H E N , M.D.

(From the Laboratories of The Rockefeller Institute for Medical Research andthe New York Post-Graduate Medical School and Hospital, New York.)
PLATES 41 TO 43(Received for publication, June 21, i915. )While engaged in the study of conjunctivitis associated withepithelial cell inclusions, the occurrence of an epidemic 2 of conjunctivitis due to hemoglobinophilic bacilli enabled us to inves'tigate therelationship between this organism and the epithelial cell inclusions.
Since the discovery of ,the inclusions by yon Prowazek and Halberstaedter, who considered ,them to be the etiological agents of trachoma, an a~bundant literature has arisen. Opinions are still greatly
divided as to the nature of the bo4ies, although the majority of investigators regard them as an independent organism, as claimed by
von Prowazek and Halberstaedter. They do not, however, commit
themselves to the statement as to wh'ether the organism represents
the causative agent of trachoma or not. W e were inclined to thebelief ,that it constitutes an independent organism which produces atrue conjunctivitis of a more or less characteristic clinical course.
Herzog a considered them to be mutation forms of the gonococcus,
while Williams 4 views them as the cell inclusions of various organisms, such as the hemoglobinophilic bacillus and allied species.
1 Read before the Ophthalmological Section of the New York Academy ofMedicine, May 17, I9I 5.
2 Service of Dr. Martin Cohen at the Randall's Island Hospital, New York.
Herzog, H., Arch. f. Ophth., IgIO, lxxiv, 520.
Williams, A. W., Collected Studies from the Bureau of Laboratories, Department of Health, City of New York, 1912-13, vii, 159-247; Jour. Infect. Dis.,
1914, xiv, 261.

304

Hideyo Noguchi and Martin Cohen.

305

Although by means of a special method an organism strikingly
similar to the epithelial cell inclusions has :been made to grow inpure culture, a their pathogenic properties, nevertheless, have not yet
been established.
As a result of our previous studies a tentative conclusion had beenreached that infection of ,the conjunctiva with these organisms produces an independent conjunctival disease which may properly ,be described as cell inclusion conjunctivitis. This organism may be present alone, or it may be associated with other pathogenic organisms.

Outbreak and Course of the Present Epidemic of Koch-Weeks
Bacillus Conjunctivitis.
Prior to the outbreak of the Koch-Weeks bacillus epidemic, 6 oneof us (Cohen) had under observation for about five months IO casesof inclusion conjunctivitis and 4 cases at the outdoor department of
the Post-Gradua'te Hospital. In these cases, smears (and in a few
cases, cultures) were taken weekly and examined (Noguchi), but
no Koch-Weeks bacilli were 'found. On October 3, 1914, one of
the inclusion cases under observation at the Island developed anacute conjunctival inflammation. In the conjunctival smears numerous Koch-Weeks bacilli, as well as epithelial inclusions, could bedemonstrated. The acute conjunctival inflammation spread to theremaining nine inclusion cases. The smears from all showed inclusions as well as numerous Koch-Weeks bacilli.
In an adjoining ward there were under treatment 15 cases of follicular conjunctivitis, 3 cases of interstitial keratitis, and I case ofchronic dacryocystitis. 13 of these contracted the Koch-Weeks bacillus infection. During the entire course, which lasted from two
weeks to seven mon.ths, no pathogenic organisms but the KochWeeks bacilli could be demonstrated in the smears and cultures.
There were 7 patients in the trachoma ward. 2 of these showed
simultaneous presence in the conjunctiva of inclusion and of KochNoguchi, H., and Cohen, M., Arch. of Ophth., 1914, xliii, 117; Your. Exper.

Med., 1913, xviii, 572.
6 In the present article the hemoglobinophilic bacilli found in these cases were
designated as Koch-Weeks bacilli, in order to conform with the usage of thisterm in ophthalmology and bacteriology when speaking of this class of organisms.

Epidemic of Koch-Weeks Bacillus Conjunctivitis.

306

Weeks bacilli. The la'tter organisms were present only in cultures,as there were possibly too few to be recognized in the smears. Thiscondition was present for a short time during this epidemic. The
infection with the Koch-Weeks bacillus apparently produced nochange in the clinical picture of these trachoma cases. The 4 inclusion cases not treated on the Island showed at no time during thepast year the Koch-Weeks bacillus, either in smears or cultures; noteven during a relapse or reinfection, of which one of .these cases had
two, could they be demons.trated. Table I shows the ~bacteriological findings in the cases on which the present report is based.
TABLE

I.

Organisms present.

No. of cases.

I. Epithelial cell i n c l u s i o n s a l o n e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6
2. Epithelial cell i n c l u s i o n s at first, w i t h s u b s e q u e n t K o c h - W e e k s i n f e c t i o n 17
3. K o c h - W e e k s bacillus a l o n e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13
4. K o c h - W e e k s bacillus at first, w i t h s u b s e q u e n t a p p e a r a n c e o f i n c l u s i o n s 2
5. P n e u m o c o c c u s w i t h i n c l u s i o n s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
ITotal..........................................................

39

In acute catarrhal conjunctivitis it is at times difficult, or impossible, to determine the causative organism from the clinical manifestations alone. This is due to the great variability in virulence of
the usual infecting organisms, and to the resisting qualities of theindividual patient. Yet in many of the acute infections of the conjunctiva, the etiological diagnosis as made by the clinical appearance could be confirmed subsequently by the bacteriological diagnosis, especially in the subacute state. In mixed infections, such adiagnosis is much more difficult.
In a few instances, even smears and cultures of the conjunctival
scrapings fail to clear up the diagnosis, as no pathogenic organisms
can be found. That the microscope alone is not sufficient to exclude
the presence of an organism is shown by the fact that the scrapings from cases of trachoma which showed neither inclusions nor
the Koch-Weeks bacilli (as proved similarly by cultures), when
inoculated on the conjunctiva of the higher apes (baboons) produced
symptoms similar to those which are seen in inclusion cases in man.
They were, however, of a milder type and lasted only about ten
days. The conjunctival scrapings from the inoculated animals re-Hideyo Noguchi and Martin Cohen.
vealedthe presenceofbacilli or allied organisms
Weepithelial
appearedinclusions,

but

no

Koch-Weeks

in t h e s m e a r s o r in t h e c u l t u r e s .

shall now discuss the clinical manifestations

cases mentioned

307

in t h e g r o u p o f

in T a b l e I.

The clinical course of inclusion conjunctivitis has already been described by
CohenY In the early stage, the clinical manifestations resemble those seen in
the Koch-Weeks bacillus infection, and it is only by the examination of smears
or cultures that the diagnosis can be positively made. Briefly, they are as follows: In the beginning there is moderate edema of the lids, with mucopurulent
secretion. Somewhat later isolated reddish translucent follicles appear in thelower palpebral conjunctiva and upper folds, these two sites becoming involved
simultaneously. At a later stage, the upper palpebral conjunctiva takes on abrick red color and assumes the characteristic granular appearance. These regular and progressive manifestations o f the disease retrogress after about twomonths by absorption of the contents of the follicles and papules. These disa p p e a r first from the upper portions of the conjunctiva, when the conditions
resemble those of cases of follicular conjunctivitis; and later, in about threemonths, they begin to disappear from the lower conjunctiva. At the end of fourmonths, the conjunctiva is again in a normal condition.
This is the usual course, but at times there are deviations, due to externalor unknown conditions, or to a previous affection of the conjunctiva; as, whengrafted on a follicular conjunctivitis, the original follicles become larger andnew ones make their appearance. In the stage of retrogression the papules become absorbed and the original follicles, owing to relaxation of surrounding
pressure, again become prominent, the course being then one of a follicular conjunctivitis. In 2 cases of inclusion conjunctivitis, there remains solely, at theend of nine months, a fine papillary condition of the upper tarsal conjunctiva;
one of these two cases has had two relapses or infections.
It may be mentioned here in passing, that of the 75 cases of inclusion conjunctivitis studied by us during the past five years, 58 cases are still under observation of Dr. Cohen. 2 of these still show a diffuse and linear cicatrization ofpractically the entire palpebral conjunctiva, but no corneal or other involvement
is present. 2 other cases of this group still show a fine papillary hypertrophy ofboth tarsal conjunctivae, with no corneal or other complications. The remaining
54 cases have remained normal for the past four and a half years. 17 cases
were observed for six months, and were normal when last seen.
4 patients with follicular conjunctivitis became infected with inclusions. The
follicles previously present became enlarged and congested, and new papules andfollicles appeared on the upper tarsal conjunctiva and the conjunctiva of thelower lid. These cases had the appearance of a severe type of inclusion conj unctivitis.
For several weeks after the beginning of the disease, the smears in all cases
showed an abundance of epithelial cell inclusions. Toward the end of the disease, the number of inclusions gradually diminished. Yet after three months'
duration, although no inclusions were demonstrable in the smears, the clinicalaspect of the disease was usually still evident.
7 Cohen, 1V[., Arch. of Ophth., 1913, xlii, 29.

308

Epidemic of Koch-Weeks Bacillus Conju~ctivitis.

On Oct. 23, 1914, one of the inclusion cases on the Island developed aslight conjunctival inflammation, which smears and cultures showed to be dueto the Koch-Weeks bacillus. In the succeeding two weeks, as previously mentioned, additional cases of inclusion conjunctivitis contracted the Koch-Weeks
bacillus infection. Conjunctival smears and cultures examined weekIy for the
past several months showed at first the simultaneous presence of inclusion and
Koch-Weeks bacilli. In the succeeding two months, the findings varied, at times
only one of the two organisms being present, at other times, both. Possibly
this was due to relapse or reinfection. In the next two months, although noorganism was present in smears and cultures, there was still evidence of inflammation. Ultimately the conjunctiva became normal in all but one case. In thiscase there is still at the end of a year evidence of an inclusion conjunctivitis in
the retrogressive stage.
In an adjoining ward, where mild follicular conjunctivitis cases were being
treated, 13 contracted the Koch-Weeks bacillus infection. Previous to the KochWeeks infection, all these cases showed over various areas of the conjunetiva
follicles varying in n u m b e r and size, and showing no inflammatory reaction.
The first case in this ward developed on Nov. I, 1914. Only 3 of the 16 cases
escaped the Koch-Weeks infection. The Koch-Weeks bacillus was present ingreat numbers, but no other pathogenic organisms could be demonstrated throughout the entire course of the epidemic, which lasted six months.
T h e clinical manifestations in these follicular cases were as follows. T h e r ewere slight edema of the llds and conjunctival congestion with mucopurulent
secretion. Both eyes became successively involved. T h e acute infection lasted
six to eight weeks. A t the end of this period, the original follicles were stillpresent.
In one of the typical follicular cases it was difficult to determine whether theacute infection was due to the Koch-Weeks bacillus or to the epithelial inclusions. The symptoms were those of a severe inclusion conjunctivitis. But notwithstanding repeated examinations extending over two months, no inclusions
could be found. Nevertheless, judging from the clinical course and from thefact that cell inclusions are often absent in the subacute stage, this and similarcases may be considered due to epithelial inclusions.
2 cases of interstitial keratitis on the Island Eye W a r d and I at the Dispensary had normal conjunctivae, when they suddenly developed an acute catarrhal
conjunctivitis. In the smears and cultures, which were taken weekly for fourmonths, the Koch-Weeks bacillus was present, even at a time when the conjunctiva was free from inflammation. T h e inflammatory stage lasted from six
to eight weeks. Inclusions could at no time be demonstrated in the smears.
T h e clinical appearance in these cases was moderate edema of the lids, andconjunctival congestion with mucopurulent secretion. In one case there waspresent a srnall scleroconjunctival h e m o r r h a g e with a small phlyctena. Conjunctival furrows or folds also appeared in those cases when the congestion
diminished.
As regards the last group in the table, 2 cases at first showed the Koch-Weeks
bacillus in the conjunctival smears. Later, inclusion cells made their appearance,
associated with their clinical manifestations. In the first ease there was presentHideyo Noguchi and Martin Cohen.

309

a mild catarrhal conjunctivitis, papules being present on the lower tarsal conjunctiva alone. In this respect it differed from other inclusion cases where thepapules are present also on the upper tarsal conjunctiva. It is possible that thelesions at times can only be detected microscopically. A second examinationin this case revealed the epithelial inclusions. Perhaps the inclusions were
missed in making the first examination, as it is likely that in some stages of thedisease only a few epithelial cells contain inclusions.
The second patient originally had a follicular conjunctivitis. H e then contracted an acute catarrhal conjunctivitis, the secretion containing for three
weeks the Koch-Weeks bacillus alone. A t the end of this time, when the conjunctivitis improved, he again developed an acute conjunctival inflammation, andthe smears showed n u m e r o u s inclusions and a few Koch-Weeks bacilli. Thiscase then followed the usual course of an inclusion conjunctivitis, the conjunctiva ultimately r e t u r n i n g to a normal condition.
I n ' a ward assigned to m i n o r eye affections, where there were a few mild
Koch-Weeks cases, a routifle examination of the conjunctivae of all the cases
was made, in order to determine the presence of the Koch-Weeks bacillus. 2cases showed the Koch-Weeks bacilli, in spite of the fact that the conjunctivae
were entirely normal t h r o u g h o u t the whole period. In another case in the sameward degenerated Koch-Weeks bacilli were found in the conjunctival smears.
This case then developed an acute inflammation, showing all the symptoms of an
inclusion conjunctivitis with the added findings of epithelial cell inclusions in thesmear examination.
In our routine examinations made during the past five years Koch-Weeks
bacilli have rarely been found. Recently we encountered eight cases of pneumocoecal conjunctivitis. Clinically these cases resembled acute catarrhal conjunctivitis, due to the Koch-Weeks bacillus, and were so diagnosed before thesmears and cultures showed the presence of numerous pneumococci without any
Koch-Weeks bacillus. The conjunctival secretions in these cases were typicallyserous, being a l s o associated with diffuse congestion. T h e duration was from
one to two weeks. One of the patients, included already in this paper, had originally an inclusion conjunctivitis. Later, he contracted a Koch-Weeks bacillus
infection. A f t e r four weeks, when apparently cured, another acute inflammation
appeared, which was due to the pneumococcus. This infection was followed by
the appearance of inclusion conjunctivitis, the smears showing both pneumococcus and inclusion cells. A t present, the clinical symptoms are those of an
inclusion conjunctivitis in a state of relapse or reinfection.
T h e t r e a t m e n t adopted in all these cases was irrigation of the conjunctival
sac with a saturated solution of boric acid every two hours, and the application
to the conjunctiva, once a day, of a silver nitrate solution, until the acute symptoms had subsided, when irrigation alone was continued. This method of treatment had practically but little effect on the course of the inclusion cases, whereas
in the Koch-Weeks infections decided improvement was observable. T h e disappearance of the organisms from the smears and culture was, however, not at
all influenced by the application of the silver nitrate.

310

Epidemic of Koch-Weeks Bacillus Conju~ctivitis.

Bacteriological and Experimental Studies of the Present Epidemic. s
Our technique for obtaining smears and cultures was as follows.
The upper lid was everted and then the short end of a sterile slide
was gently rubbed over the upper tarsal conjunctiva where there isleast likelihood of contamination. By means of a platinum loop,
a part of the scrapings removed was used for the purpose of cultivation and animal experiments. The remainder of the materials
on the slide was transferred to another slide where a thin spread was
made, and examined by means of the Giemsa or Gram stain.
Since our problem deals with the Koch-Weeks infection, the culture media employed were chiefly blood agar and tissue ascitie fluid.
The cultural findings are summarized below,
In the present epidemic it was noticed that various strains of theso called Koch-Weeks bacilli or hemoglobinophilic organisms in cultures varied in size to such an extent that they may be divided into athin (Figs. 4'to 7), a medium (Figs. 8 to II, 20 to 23), and a coarse
type (Figs. 16 .to 19), according to their morphology. The coarse
variety were bacillary or coccoid (Figs. 12 to I5) and resembled culturally and morphologically some strains of Bacillus influenJe (Figs.
24 and 25) derived from the respiratory organs; while the thinnervariety was much narrower and often shorter. After several days'
cultivation at 37 ° C. on blood agar, some of the organisms became
somewhat granular and unevenly stainable; when found in large
masses they often assumed the appearance of the granular forms of
the so called trachoma bodies (Figs. 5 to 7, 9 to I I, I3 to 15, I7 toI9). But these granules did not break down so as to approach theminuteness of the elementary bodies (Figs. 32, 33, 36, 39, 43, 45)
which are found in the uncomplicated cases of inclusion conjunctivitis or trachoma. The degenerated or involuted bacilli do not havethe sharp contour of the elementary granules. Transplants madefrom these somewhat degenerated granular masses of the hemoglobinophilic organisms give a good growth of typical bacillary or semicoccoid forms. The character of the colonies on the blood agar
is also distinctive, in that they remain minute, sharply elevated witha pointed top, and appear more grayish than the colonies of theSThis part of the work was carried out at The Rockefeller Institute.

311

Hideyo Nogucl~i a~d Martin Cohen.

influenza type varieties, which show a tendency to spread and areof a more dewy aspect. It is quite possible that the minute type
constitutes a group by itself, but further study is required to determine its rela~cion to'the other groups. At all events several s.trains
of influenza bacillus 9 derived from cases of meningitis and pneumonia appear quite different from the conjunctivitis strains in theirmorphological features (Figs. 24 to 28). The frequency withwhich organisms of different type were found in the present epidemic is shown in Table II.
TABLE II.
Types.
Organisms present.
I. E p i t h e l i a l inclusions first, w i t h s u b s e q u e n t K o c h W e e k s i n f e c t i o n (i 7 cases) . . . . . . . . . . . . . . . . . . . .
2. K o c h - W e e k s bacillus a l o n e (13 cases) . . . . . . . . . . . .
3. K o c h - W e e k s bacillus first, w i t h s u b s e q u e n t a p p e a r a n c e of inclusions (2 cases) . . . . . . . . . . . . . . . . . . . . .

Coarse.

Medium.

Thin.

i
I

7
IO

9
2

i
i
(round type) i

i

As will be seen from Table II, the thin variety occurred veryfrequently in the cases in which the pa.tients had been previously
infected with the inclusions. In the simple Koch-Weeks cases themedium was predominating. It is significant that in the smears
taken from the cases infected with the inclusions and Koch-Weeks
bacilli it is not difficult to differentiate the initial bodies of the former from the bacilli, because the initial bodies are much larger andoval in shape, and take up a deeper blue stain. Fig. 3 shows a typical inclusion near the nucleus and some Koch-Weeks bacilli in thesame cell. The hemoglobinophilic bacilli are seen to be taken up
by the polynuclear leucocytes more frequently than by the epi.thelial
cells (Fig. I ) , while the inclusion bodies are chiefly epithelial andseldom leucocytic in nature (Figs. 29 to 45). So far as could beascertained in stained specimens the bacilli remain well preserved
within the cells and do not seem to disintegrate into granules (Fig.
2). On the other hand, the initial bodies are often ill defined anddo not show any distinct bacillary forms. They are far more
9 We are indebted to Dr. Martha Wollstein for these strains, for which wehere wish to express our thanks.

312

Epidemic of Koch-Weeks Bacillus Conjunctivitis.

pleomorphic than the hemoglobinophilie bacilli (Figs. 29, 34, 37,
40, 4I, 43, 46, and 47)The isolation of the hemoglobinophilic organisms, irrespective ofthe type, was easily accomplished by means of the blood agar; but
in many instances ~he preliminary microscopical search in the smears
failed to demonstrate the presence of a few organisms, until they
could be found by the cultural procedure. I,t must be understoodthat in cases where the inclusions only were found, repeated efforts
to isolate the hemoglobinophilic organism's were made, with, however,invariably negative results. In this connection it may be added thata number of cases clinically diagnosed as trachoma was also studied
in order to see if any hemoglobinophilic organism could be isolated.
The results were uniformly negative. A few cases were also foundin the same wards, which may have been infected through contact
with the inclusion cases and which, although carefully observed
from the earliest stage of the disease, did not at any period show thepresence of any hemoglobinophilic bacilli.
Transmission of the Inclusion Bodies to Animals.--In order tofind ou,t whether the inclusion virus ever exists in the form of ahemoglobinophilic bacillus, scrapings from the conjunctivae of thesecases were inoculated into the conjunctiva of a baboon. Prior to the
inoculation the conjunctiva of :the baboon was examined to ensure
the absence of such a bacillus. ' Within seventy-two hours after
the inoculation the conjunctiva showed moderate congestion, edema,
and a few minute papules; a small quantity of a mucopurulent discharge was present at the inner canthus. The smears and cukure
were made from the conjunctival scrapings. The result showedthat there were numerous cell inclusions, but no hemoglobinophilieorganisms 1° (Figs. 46 to 51). The examinations were continued
regularly for a period of two weeks and the results were unvarying.
As was previously shown by various investigators, the yon Prowazek
bodies are transmissible to higher apes, but the hemoglobinophilic
or Koch-Weeks bacillus is not.
Attempts To Produce K, och- Weeks Bacillus Conjunctivitis in A nimals.--Several attempts were made to transfer the hemoglobinolo Cultures made after z4 and 48 hours were also negative in regard to thehemoglobinophilic bacillus.

Hideyo Noguchi and Marlin Cohen.

313

philic organisms to the conjunctivae of rabbits (young and adult)
and monkeys (baboon and several Macacus rhesus), 'by in,troducing
several loopfuls of :the twenty-four hour blood agar cultures of .different strains into moderately abraded surfaces of conjunctivae.
The results were completely negative, except in the case of one
rabbit, where the organism was still recoverable after twenty-fourhours. It seems remarkable that such a large quantity of purecultures of freshly isolated strains of ~these organisms from thecases where the inclusions were also present should fail to reproduce
the conjunctivitis, in view of the fact that a comparatively small
number of the inclusions as contained in the scrapings from a patient
can readily reproduce the inclusion conjunctivitis.
From this a conclusion may be warranted that in a conjunctivitis
where the inclusion and the hemoglobinophilic ba'cilli are simultaneously present, two pathogenic factors can be separated by means oftransmission of the material into the conjunctiva of a suitable animal(baboon) in which the inclusion virus alone implants itself upon the
new host, while the bacilli quickly disappear from the inoculated
conjunctiva. For man 'both organisms are pathogenic, but for thebaboon only the inclusion virus is capable of producing infection.
On the other hand, this may not exclude the possibility, as asserted
by Williams and her associates, that a conjunctivitis due to the
Koch-Weeks bacillus may also show some cell inclusions; since
under certain experimental conditions a very suggestive phenomenon, to be related below, has been observed.
Attempts To Produce the Koch-Weeks Bacillus Epithelial Inclusions in Animals.--In order to determine experimentally whether or
not the hemoglobinophilic bacilli when taken up byepithelial cells will
undergo the morphological changes which lead to the formation ofso called inclusion bodies, an intrates,ticular inoculation of the
rabbit with pure cultures of the hemoglobinophilic organisms isolated from the cases already mentioned was resorted to. The local
reaction which follows consists of edema of the scrotum and induration of the testicular parenchyma within twenty-four hours. The
edema gradually disappears within the next few days, while thetesticular induration remains more or less the same for about fivedays, after which it commences to recede. In some instances the314

Epidemic of Koch-Weeks Bacillus Co~@o~ctivitis.

rabbit succumbed to septicemia and probably to intoxication as a result of the introduction of the cultures. The organs were removed atintervals of twenty-four hours, three days, and six days, and thenfixed in sublimate alcohol, and stained by Giemsa's acetone method, 11and, if overstained, treated with a IO per cent solution of Glyceriniithermischung (Grfibler) for a few minutes, as advocated by one ofus. 12 The results show that the injection of the bacilli is followedby an intense leucocytosis, in which the polynuclears invade thetubules in groups. In the twenty-four hour specimens the organisms
are still well distributed along the interstitial spaces ; in the three day
specimens one notices numerous masses of agglutinated bacilli here
and there within the tubular lumina or along the connective tissue.
These masses take on a purplish hue and appear granular in structure and indefinite in outline. They are on the point of disintegration. The granules within and abou,t these bacterial masses are
not so minute as to be mistaken for the elementary bodies of the
inclusion. In the six day specimens some clumped bacteria were
found within the polynuclear leucocytes, but a diligent search failed
to show any typical epithelial cell inclusions. In these six day preparations the number of the bacterial clumps is smaller than at an
earlier period. There were no granules small enough to be regarded
as the typical elementary granules. \Vhile a careful comparison of
the inclusions and the bacterial clumps just referred to will reveal
the .difference between them, this is not always easy to accomplish(Figs. 53 to 55). Fig. 52 shows a mass of granules from a caseof mixed infection of Koch-Weeks bacilli and the yon Prowazek
inclusions, and it appears difficult to determine whether they represent the degenerated bacilli or the inclusion granules. The deep
stain of the mass and the absence of any free bacilli around it seemto indica,te that it belongs to the latter kind. It is also possible tha.tsome of the clumped granules found in the conjunctival smears
from cases of Koch-Weeks infection might have been interpreted as
the cell inclusions and classified with ,the yon Prowazek inclusions.

Attempts To Transmit the Inclusion Bodies to a Parenchymatous
Organ in Animals.--Efforts were also made to transmit the yon
11 Giemsa, G., Deutsch. reed. ~Vchnschr., 19o9, x x x v , 1752.
12-Noguchi and Cohen, Proc. N. Y. Path. Soc., 191o, x, 2o.

Hideyo Noguchi and Martiu Cohen.

315

Prowazek bodies from uncomplicated inclusion cases to the testiclesof rabbits, since this organ offers an excellent medium of growth tovarious highly parasitic organisms which otherwise cannot be easily
cultivatedJ 3 The serapings of conjunctivae from 4 different patients were inoculated into the testicles of 8 rabbits; but in spite of
the large number of the inclusions contained in the conjunctival
scrapings used for this purpose, no success was obtained along thisline. The testicles showed within twenty-four hours some induration and edema, but after a few days resumed ,their normal condition.
Tissues removed after twenty-four hours, three days, and six days
failed to show any cell inclusions when examined in smears andsections. No hemoglobinophilic organism was found in cultures
made from the tissues. This negative finding also tends tostrengthen the view that the cell inclusions found in these cases were
not the Koch-Weeks bacilli, for if they had been it would have resulted in the production of Koch-Weeks orchitis.
CONCLUSIONS.

I. There are cases in which epithelial cell inclusions may alone bepresent in the conjunctival smears. In such cases no other pathogenic organisms, such as the Koch-Weeks bacillus or the pneumo-coccus, can be demonstrated in smears or cultures.
2. The conjunctiva can become simultaneously infected with theinclusion bodies and Koch-Weeks bacilli or other organisms.
3. In cases of acute or subacnte conjunctival inflammations due
to mixed infections the clinical features of each infection may bepresent. The course of the inflammation is, however, more prolonged.
4. Within recent years, the Koch-Weeks bacillus has only seldom
been found in our routine examinations.
5. The epidemic studied was of a severe type.
6. Clinically it is practically impossible to distinguish pneumococcal conjunctivitis from the Koch-Weeks conjunctivitis. Bacteriological examination of smears and cultures is the only means bywhich the etiological diagnosis can be definitely established.
7. Conjunctivae of certain species of monkeys are susceptible to
13 Noguchi, H., ]our. Exper. Med., 1915, xxi, 539.

316

Epidemic of Koch-Weeks Bacillus Conjunctivitis.

the von Prowazek inclusion bodies, but not to the hemoglobinophilic bacilli isolated from cases of epidemic conjunctivitis.
8. The injection of conjunctival scrapings containing the vonProwazek cell inclusions into the testicles of rabbits produces nocell inclusions in the latter, while the injection of a pure culture of
the hemoglobinophilic bacilli causes an acute inflammation accompanied by numerous clumps of the organisms, simulating the yon
Prowazek bodies at certain stages of their evolution.
9. There exists an apparent morphological similariiy between thedegenerated forms of this variety of the hemoglobinophilic bacilli
and the cell inclusions, both in cultures and in experimental orchitis
in the rabbit. But, as a rule, the elementary bodies of the latter are
much smaller and more sharply defined than the smallest granulesof the former, while the initial bodies are 6igger, more intensely
stainable, and less definite in their contour than the hemoglobinophilic bacilli found in the infected conjunctivm.
EXPLANATION OF PLATES.
All the photographs were made from film preparations, except where otherwise stated. They were stained with Giemsa. The enlargement is uniformly
I,OOO diameters.
PLATE 41.
FIG. I. This shows a mass of the Koch-Weeks bacilli in a film preparationfrom an uncomplicated case of Koch-Weeks conjunctivitis. The bacilli belong
to the thin type and some scattered examples appear coccoid. By focusing, themass is seen to be composed of numerous well defined bacilli which cannot be
confused with the inclusion granules.
Fro. 2. An epithelial cell from a case of uncomplicated Koch-Weeks conjunctivitis. Around the periphery of the cell and near the nucleus along thelower border numerous bacilli are seen to be attached to, or contained within,
the cell body. There is, however, no difficulty in recognizing the bacilli as suchin this instance.
FIG. 3- A film preparation from a case of mixed infection of the KochWeeks and the inclusion organisms. In the field the epithelial cell is seen tocontain a densely stained mass of the initial bodies near the upper right borderof the nucleus and numerous Koch-Weeks bacilli to the left, especially where
there is a leucocyte.
FIG. 4- This represents a twenty-four hour old pure culture of a thin typeof the Koch-Weeks bacilli on blood agar.
Fins. 5, 6, and 7. The appearance of the same organism as in Fig. 4 after
3 days, 5 days, and 8 days, respectively, on the same medium at 37 ° C.

Hidcyo Noguchi and Martin Cohen.

3] 7FIGS. 8, 9, io, and II. These represent the appearance of a medium type strainof the Koch-Weeks bacilli after 24 hours, 3 days, 5 days, and 8 days, respectively,on blood agar at 37 ° C.
FIGS. i2, 13, i4, and I5. These show the appearance of a strain of round or
coccoid type of the Koch-Weeks bacilli in a 24 hour, 3 day, 5 day, and 8 day
growth on blood agar, respectively.
FIGs. i6, I7, I8, and I9. These show the appearance of a coarse strain of the
Koch-Weeks bacilli in a 24 hour, 3 day, 5 day, and 8 day growth on blood agar,
respectively. This strain resembles B. influenz¢ more than the others.
FIGS. 20, 2I, 22, and 23. These show 4 different strains of the Koch-Weeks
bacilli isolated from the mixed infection cases in the present epidemic. They
belong to the medium type.
FIGS. 24, 25, and 26. These show 24 hour growths of 3 different strains of
B. influenzce isolated from cases of pneumonia.
FiGs. 27 and 28. These show z4 hour growths of 2 different strains of B.
influen2x isolated from cases of meningitis. Both strains were kept on artificial
media for several years and tend to form threads more than they originally did(Wollstein).
PLATE 42.
FiGs. 29, 3o, 3I, 32, and 33. These show the inclusions at various stages of
evolution in the film preparations made from a case (Tho.) of inclusion conjunctivitis. This patient had been suffering from the inclusion conjunctivitis
when he was superinfected with the Koch-Weeks bacilli, which, in due course,
disappeared from his conjunctiva leaving the original condition little affected.
These inclusions shown here were found in the conjunetiva long after the KochWeeks bacilli had disappeared in smears or in cultures. T h e minute elementary
bodies in Figs. 32 and 33 bear no resemblance to the degenerated forms of the
Koch-Weeks bacilli.
FIGS. 34, 35, and 36. These represent the inclusion bodies in an uncomplicatedcase (Sh.), that is, without any association with the Koch-Weeks bacilli. The
character of the initial bodies in Fig. 34 would definitely dispose of any suggestion as to their being the Koch-Weeks bacilli. Fig. 36 shows a still unburst
intracellular aggregation of the elementary bodies.
FIGS. 37, 38, and 39. Inclusions from another case (Sat.) of pure inclusion
conjunctivitis.
FIGS. 40, 41, and 42. Inclusions from a case (Bol.) of trachoma. T h e initial
bodies as shown in Fig. 4 ° are exceptionally coarse.
FIGS. 43, 44, and 45. Inclusions from an uncomplicated case (For.) of inclusion conjunctivitis. In Fig. 43 a thick mass of the initial bodies is seen to embrace the left side of the nucleus of a cell on the left, while on the right numerous elementary granules are scattered around the cell below. Figs. 44 and 45
show the steps of evolution of the inclusion bodies.
PLATE 43.
FIGS. 46, 47, 48, 49, 50, and 51. Different stages of the evolution of the inclusion bodies and the conjunctiva of a baboon experimentally infected ~vith thescrapings of the affected conjunctivae of man. The structure of the initial318

Epidemic of Koch-Wccks Bacillus Conju~ctivitis.

bodies in Figs. 46, 47, and 48 is quite peculiar and reveals no definite bacillary
outlines. They are deeply stained, almost amorphous, and show indistinct coarse
granules within the mass. T h e elementary bodies are even more minute thanthose in the h u m a n cases, as will be seen in the cell occupying the lower half ofFig. 48. In the same figure one sees an irregularly shaped mass of the initial
bodies at the upper corner. In Figs. 49, 5o, and 5I are shown the enormously
distended cells in which the inclusion granules are rapidly multiplying.
FIC. 52. This shows a dense mass of deeply stained granules in a film preparation from a case of mixed infection. This mass appears to be more of theyon Prowazek type of body than of the degenerated Koch-Weeks bacilli.
FIGS. 53, 54, and 55. Sections of the rabbits' testicles inoculated with purecultures of Koch-Weeks bacilli isolated from the mixed infection cases during
the present epidemic. Fig. 53 shows that the organisms assume a coccobacillaryform when examined within twenty-four hours after they were injected into thetesticle. In photography they appear somewhat like the initial bodies in the
inclusion cases, yet in actual examination of the preparations they are not difficult
to distinguish as the Koch-Weeks bacilli. They are freely capable of cultivation
when a minute portion of the testicle is t r a n s f e r r e d to the blood agar, while thisis not the case with the initial bodies of the inclusion organism. In Figs. 54 and55 the sections removed after 3 and 6 days, respectively, are shown in whichlarge masses of agglutinated and somewhat ill defined bacilli are found in clear
spaces within the lumen of the testicular tubules, They, too, present an appearance not unlike that of the inclusion organism found in experimental conjunctivitis in monkeys, as well as in an epidemic in man. Here, again, a culture on
blood agar is quickly differentiated from the other kind, as the Koch-Weeks
bacilli at this stage still thrive very well in culture, while the pure inclusionmaterial does not give any growth with the hemoglobinophilie bacilli.

THE

J O U R N A L OF E X P E R I M E N T A L

MEDICINE VOL. XXlI.

PLAT E 41.

(Nogucbi and Cohen: Epidemic of Koch Weeks tlaeillus Conjunctivitis.)

T H E J O U R N A L O F E X P E R I M E N T A L MEDICINE VOL. XXII.

PLATE 42.

(Noguchi and Cohen: Epidemic of Koch-Weeks Bacillus Conjunctivitis.)

T H E JOURNAL OF EXPERIMENTAL MEDICINE VOL. XXll.

PLATE 43.

Experimental Inclusion Conjunctivitis in Baboon (46-51 .

Experimental Koch-Weeks Orchitis in Rabbit (53-55).
(Noguchi and Cohen: Epidemic of Koch-Weeks Bacillus Conjunctivitis.)