野口英世著

Journal of Experimental Medicine

所収論文

こ の PDF は Journal of Experimental Medicine に 掲 載 さ れ た 論 文 を Rockefeller
University Press の許可（2020 年 3 月 18 日付）を得てアップロードしています。

P U R E C U L T I V A T I O N IN V I V O O F V A C C I N E V I R U S
F R E E F R O M BACTERIA.*
BY HIDEYO NOGUCHI, M.D.

(From the Laboratories of The Rockefeller Institute for Medical Research.)
PLATES39 TO5~
In spite of much effort n,o method has, up to the present time, been
perfected by which vaccine virus can be propagated in a pure state,
free from con'taminating bacteria. The method of propagating vaccine virus universally practiced today consists in transmitting thevirus from the skin of one calf to that of ano~ther. Although the
inoculation of the virus is carried out under precautions as strtictly
aseptic as possible, the fresh product nevertheless yielded by theskin contains a not inconsiderable number of different bacteria derived from the skin surfaces, the air, etc.
The employment of glycerin as an elective germicide against thenon-spore-bearing bacteria contained in fresh, or so called "green,"
vaccine pulp results in a great reducti'on both in number and varietyof the contaminating micro6rganisms, without at the same time
seriously impairing the activity of the vaccine virus ( I ) . After contact with concentrated glycerin, in a refrigerator, ranging from one
to three months, the virus usually is freed from most of the bacteria
and becomes "ripe " for practical use in vaccina,tion on humanbeings.
Different samples of the ripe vaccine preparations, as issued fromvarious authorized sources, vary in their activities as well as in theirgerm contents. Among t~he organisms which may be encounteredin such preparations are the following: streptococci, staphylococci,
Bacillus coli, Bacillus welchii, Bacillus xerosis, Bacillus subtilis, andsome other aerobic and anaerobic bacteria (2). The action of glycerin on bacterial spores, which may also be present in the pulp, is* Received for publication, March 15, 1915.
589

540

Cultivation of Vaccine Virus.

ahnost nil. Moreover, the prolonged action of glycerin tends gradually to reduce the activity of the vaccine virus itself, so that the
fresh vaccine pulp forfeits some of its efficacy while undergoing the"ripening" process. At the end of the ripening process, which consumes from one to three months, the virus usually still con'tains acertain number of bacteria; bacteria-free virus is practically neverproduced by glycerination. But the residue of living bacteria, fortunately, is to all inten'ts and purposes quite harmless and practicallyto be disregarded. And yet it is apparent that it is not only desirable
to eliminate all bacteria from vaccine virus, but the saving of thetime lost in "ripening" and the preservation unimpaired by glycerinof the full strength of the virus are also highly important considerations.
:HISTORICAL.
The observations of BSraud (3), Weigert (4), Chiari (5, 6), Councilman,
Magrath, and Brinkerhoff (7), and others (8) indicated that the variola virus
localizes in different viscera and organs; the vaccine virus, on the other hand,according to Calmette and Gu6rln (9), and von P r o w a z e k and Yamamoto (IO),
exhibits a high degree of affinity for pavement epithelium. W h e n the vaccine
virus is introduced directly into the blood or peritoneal cavity, it lodges in theepithelia of the skin and mucous surfaces, provided they are injured within a few
hours of the inoculation, while in a few hours more it has completely disappeared
from the circulation or the peritoneum. Should the epithelium not take it up, it
is entirely lost.
I n order to test the question whether still other epithelial cells take up thevirus, Henseval and Convent ( I I ) conducted in 191o a series of experiments on
the testes of rabbits. The virus that they employed contained staphylococci and
Bacillus subtilis. 1 T h e effect was to produce some swelling, but no indurationof the organ. Five to seven days after inoculation the testes were extirpated,emulsified in 66 per cent. glycerin, and tested on the shaved skin of rabbits. The
activity was less than that of the original material. F r o m the first testicularemulsion a second set of rabbits was injected; the swelling and induration produced was greater, but the activity of the emulsion was no stronger. No attempt
was made either to free the vaccine of bacteria or to continue the testicularpassages. Later, Paschen (12) injected vaccine (its bacterial content is notstated) into the testicle of a rabbit and noted the production of necrosis andcellular infiltration. As early as 19o4 Tyzzer (I3) attempted to obtain characteristic vaccinia lesions by inoculation of the testis of rabbits and a calf; theresult was regarded as an acute inflammatory reaction. Von Prowazek andMiyaji (14) state that vaccine virus injected into the testicle of the rabbit can
1 The n u m b e r of contaminating bacteria in the three samples employed was
4, IO, and 25,000 per o.oI c.c., respectively.

Hideyo NoguchL

541

still be detected two days later, and that local necrosis of the tissue appears at
the inoculation site. Nothing is said of the bacterial content.
That the testicle of the rabbit affords a favorable site for the multiplicationof parasitic micro6rganisms can be inferred from the experiments of Parodi(15) and later of Uhlenhuth and Mulzer (16) with Treponema pallidum, of
Nichols (17) with Treponema pertenue, and of a host of later workers with
Treponema pallidum ( 1 8 ) . I therefore turned my attention to the cultivationof pure strains of vaccine virus in the rabbit's testicle.
PURIFICATION OF VACCINE VIRUS PRIOR TO CULTIVATION.

At the outset and before describing the details of the experimentson the cultivation of vaccine virus ~t is necessary to point out amaterial difference between the employment of the rabbit's testicle
for Treponema paUidum and for vaccine virus. In the case of theformer the multiplication of the spirochzet~e takes place slowly, sothat the contaminating 'bacteria which are carried into the testicle
with the inoculated fragments of syphilitic tissue may in time be
suppressed by the bactericidal action of the vigorous tissues. On
that account pure strains of the pallidum may be thus developed
after a few passages. Vaccine virus, on the other hand, multiplies
quickly, and the inflammatory reaction and necrosis of tissue which
ensue quickly suppress the bactericidal process of the testicular tissues and place them in a fav.orable state for bacterial development.
On that account the virus must first be freed from the bacterial contamination by other means.
Preliminary Purification.--Several antiseptic or disinfecting substances may be employed f'or reducing or removing contaminating
bacteria. Glycerin is universally employed in practice to reduce thenumber, because of the small effect which it exerts on the virus itself.
At low temperatures the antiseptic action is too weak to eliminate
all bacteria, but at 37 ° C. in 60 per cent. strength all bacteria except
spore-bearing species may be destroyed in a few days. Hence it issuitable for the purification when the virus is devoid of sporogenous
bacteria. Addition of I per cent. phenol alone or I per cent. phenol
and 6o per cent. glycerin both to the emulsion brings about a quicker
elimination of bacteria, but .this is accompanied by a more rapid deterioration of the virus itself. The same is true with the additionof one part per thousand of oil of cloves to 60 per cent. glycerin.

542

Cultivation of Vaccine Virus.

Recently Fornet ( I 9 ) , who has experimented with cultivation invitro of vaccine virus, has recommended ether for removing the bacteria which do not possess spores. An emulsion of the virus can be
freed of all bacteria except sporogenous ones by being shaken atroom temperature for f~rty-eight hours with an excess .of ether.
The activity of the virus is, however, cons~iderably diminished byether treatment. By the ether method a vaccine pulp devoid ofspore-bearing bacteria obtained as follows was secured free of all
bacteria, and hence was suitable for cultivation in a pure statein vivo.
A sample 2 of glycerinated virus free from sporogenous bacteria was incubatedat 37 ° C. for two days or longer, in order to destroy practically all the bacteria
still present in it. The skin on the dorsal side of a rabbit was shaved and thoroughly cleansed with soap and rinsed with sterile distilled water. It was again
shaved closely, after which the glycerinated virus was applied, in such strength
as to produce separate eruptions. The vaccinated surface is protected from contamination by means of a sterile bandage. On the fourth or fifth day the
bandage was removed and the surface washed first with absolute alcohol and thenwith ether. Several vesicles were selected and each was cleansed with 5 per cent.
lysol solution and washed alternately with absolute alcohol and ether on sterilegauze. The eruptions were scraped with the edge of a sharp scalpel and thescrapings emulsified in several cubic centimeters of sterile saline solution. The
emulsion was mixed with several volumes of ether and shaken in a sealed vessel
for varying periods of time at room temperature. Samples were removed at tke
expiration of I, 2, 4, 8, 12, 24, and 48 hours, from each of which cultures wereprepared. W h e n spores are absent sterility is usually obtained in twelve hours.
The vaccinal activity falls at times to one-fifth of the original strength, but thecharacteristic properties remain unaltered.
TESTICULAR CULTIVATION IN RABBITS.

The vaccine enmlsion so prepared is employed for the intra'tes~tic utar inoculation of ra%bits. Rabbits wit5 well developed testicles
should be chosen. After ether anesthesia an assistant holds theanimal and fixes l~he testicles to prevent their withdrawal in,to theperitoneal cavity. The scrotal skin which is tightly stretched over
the testicle is sterilized with 5 per cent. lysol solution and then painted
with tincture of iodine. The operator next inserts the needle of a
2 Several samples of the vaccine virus employed in the present investigation
were furnished me by Dr. F. S. Fielder, Assistant Director of the Vaccine Laboratory of the Department of Health of the City of New York, to whom I wish
to express my gratitude.

Hideyo Noguchi.

543

sterile syringe contain'ing a I to IO or I to -2o dilution of the emulsion in'to 'the testicular parenchyma along the long axis. The pointof the needle is prevented from passing through the parenchyma to
the tunica vaginalis. The contents are now gently forced out of thesyringe into the organ at different regions by turning the directionof the needle. A~bout one cubic centimeter of emulsion is injected
into a testicle weighing from two to three grams. The organ isgently massaged to distribute the virus throughout the entire organ.
The operation is practically painless.
The method just described is employed for the inoculation of thetesticular strains of the virus, in which case an emulsion of tes'ticle
previously inoculated with the virus is used. The stock emulsion isprepared by grinding up the aseptically removed organs with sterile
saline or 6o per cent. glycerin solution in the proportion of one gramof the tissue to two or three cu~bic centimeters of the fluid and any
dilution of it; about i to IO or I to 20 in saline is prepared for 'thepurpose of the injection.
The testing for bacteria in the enmlsion is an important point. Cultures are set up with plain and glucose bouillon, ascitic fluid with andwithout bouillon, and asciti'c fluid with a piece of fresh sterile rabbit
kidney with and whhout a layer of sterile paraffin oil. The cultures
are incubated at 37 ° C. for three to four days, and then subcultures
on plain and ascitic glucose slant agar, deep layer glucose agar, andascitic tissue deep layer agar are made. 3 Film preparations stainedby Gram or by Giemsa are also examined under the microscope, bothfrom the cultures and from the testicles removed from 'the animal.
In order to avoid occasional bacterial infection it has been foundwell to inoculate both testicles of each animal. The skin may bevaccinated at the same time. It is, moreover, advisable to use atleast two rabbits for each transfer, because sometimes a rabbit reacts
poorly to the vaccinal inoculation of both skin and testicle; but oncethe virulence of the virus has reached a certain height this precaution
is no longer necessary.
In the latter part of the present work initial culture in tissue bouillon andsubcultures in glucose "agar (deep) and plain agar (plate) were found to be
sufficient.

544

Cultivation of Vaccine Virus.
RESULTS

OF EXPERIMENTS

WITI-IRABBITS.

As the accompanying table shows, l~he passage of the testicularvirus from animal to animal offered some difficulty until it had been
carried on for a number of generations. In this experiment success
was not assured until the virus had been 'transferred about ten times.
That this result was due to increase in virulence of the virus in thelater generations may be deduced from the greater activity manifested when i't was tested upon the skin, as well as from the greater
severity of the reactions in the testicles themselves (table I).
TABLE
Dateoftransfer.

Generation. Results.

4 From

this

:

:
~

~

+
+
+
+
+*

I
2
3
4
5
6
7
8
9
io
II
12
13
14
15
16
17
18
19

++
++
++

;;7
++
++
++
++
++
++
++
++
++
++

20

21
22
23
24
25

]
i

material

++
++
++
+ +
+-1-t--I-

26
27
28
29
30
31
two

to comefurtherbacks Fromthis two generations
one out of several

Tworabbits
result.

[ Generation. R e s u l t s .

Sept. 28-Oct. 3.
Oct.
3- 7 .............
Oct.
7-I2 .............

+6

6 Onlyfactory

D a t e of transfer.

i

I914
Jan. 29-Feb. 2 ........
Feb.
2- 9 ...........
Feb.
9-I4 ...........
Feb. I4-22 ...........
Feb. 23-27 ...........
Mar. io-i6 ...........
Apr. 22-25 ...........
May
I- 6 ...........
May 25-29 ...........
May 29-June 3 .......
June
3- 6 ...........
June
8-12 ............
June I2-I5 ...........
June I8-25 ...........
June 25-July 2 .........
July
2- 8 ............
July
8-I3 ............
July 13-16 ...........
July I7-2o ...........
July 20--23 ...........
July 26-3 ° ...........
July 3o-Aug. 3 ........
Aug.
4- 7 ...........
Aug. I1-17 ............
Aug. I7-22 ...........
S e p t . I-- 4 . . . . . . . . . . .
Sept. 7-1o ............
Sept. 12-16 ...........
Sept. I6-2o ...........
Sept. 21-24...........
Sept. 24-28...........

so it was necessary

I.

were

were
rabbits

inoculated

Oct.

I2--I6 .............

Oct.

I6-2o .............

1

36

Oct.
OCt.

20-25 .............
25--29 . . . . . . . . . . . . .

[
[

37

38
39
40
41
42
43
44
45
46
47
48
49
5o
5I
52

29-Nov. 3 .........
3- 7 .............
7-I2 .............
I2-I6 .............
I6-2I .............
21-26 .............
26-3o ............
3o-Dec. 5 ........
5- 9 ............
9-13 .............
I3-I6 ............
16-2o ............
2o-24 ............
27-31 ............
1915
Jan.
4- 8 ............
Jan.
8-12 .............
Jan. 15-19 .............

i
i
]
]
]
i

]
I

53
54
55

Jan.

23-27 .............

i

56

Jan.
Feb.
Feb.
Feb.

3I-Feb. 4 .........
8-12 .............
I6-2o .............
24-28 .............

i

57
58
59
60

generations
carried
gave

with

35

Oct.
Nov.
Nov.
Nov.
Nov.
Nov.
Nov.
Nov.
Dec.
Dec.
Dec.
Dec.
Dec.
Dec.

were

to this generation

thiscarriedand to start

]
I

on

and

+++
+++
+++
+++
+++
+++
+++
+ + +
i

then

lost,

again.

on and lost, necessitating

good

++
++
++
++
++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++

32
33
34

a return.

results.

material,

but

only

one

gave

a satis-Hideyo Noguchi.

545

The testicular strain indicated in the ta'ble has passed throughsixty successive generations within twelve months. It is interesting
to find that considerable resistance to testicular adaptation was exhiSited at the seventh transfer, after which no serious obstacle wasmet in carrying on the passages.
TESTICULAR VACCINAL PROCESSES I N RABBITS.

In order to follow the course of 'the vaccinal processes in the testicular tissue the following experiments were carried out.
Thirteen male rabbits were inoculated into each testicle with I c.c. of a 1:2odilution of the saline testicular emulsion derived f r o m the rabbit inoculated withthe thirtieth generation of the testicular strain and castrated under ether anesthesia on the fourth day.
The testicles of these animals were removed one after the other and tested
successively for activity on the skin, cornea, and testicles of normal rabbits every
twenty-four hours, over a period of eighteen days, and then after 3, 4, 5, and8 weeks.
Table II shows the results obtained.

During the first twenty-four hours the testicle presents li~lechange, except that microscopic foci of infiltration of polynuclear
leucocytes and exudate are observed in the interstitial spaces (figure 32, compare with normal, figures 30 and 3 I). _At the end of
forty-eight hours the swelling and induration of the organ begin to
increase rapidly and the tes'ticle becomes congested and edematous(figure 2). The conten't of the virus, which was almost zero after
twenty-four hours, now reaches about IOO times that found at theend of 'the first day. "When examined in sections an enormous leucocytic infiltration is seen in the interstitial tissues, and some leucocytes are contained in tt~bules (figure 33). The testicular cells arehydropic and fill up the tubular lumen. A't the end of three days
the infiltration has increased in intensity and extent (figures 3 and18, compare with normal figure I7), and the vaccinal activity hasrisen to a't least 3o0 times that present twenty-four hours and about
three times 'that present f~rty-eight hours after the inoculation.
The external changes present in the four day specimen (figures 4
and 34) resemble those occurring in the three day specimen, exceptthat the testicle is more compact and less elastic and the amount of546

CultiVation of Vaccine Virus.
TABLE

Testicles
removedII.
Tests for activity of emulsions on rabbits.

Gross appearanceof specimens.

after

24 hrs.
48 hrs.

Almost no swelling; t r a u m a t i c hemorrhages
injection, edema, and moderate i n d u r a t i o n (figure 2)

Dilution ofemulsion.
I :IO
I

IOO

I

1,000

Skin.

Cornea.

Testicle.

I eruption.5..i..+...

I : I0I

I00
I

3 dys.

M a r k e d induration, great congestion,
greyish mottling, edema (figure 3)

++

++

-I-

.....

i

-

+

4 dys.

Severe congestion, edema, i ndura t i on
and swelling; numerous yellowish
grey spots on the organ (figure 4)

++

++

-

5 dys.

Similar to the last (figure I)

++
++

++
++
......

÷......

?
++

++
++

-t-

......

5 dys.

Similar to the last (figure 5)I

5 dys.

6 dys.

7 dys.

8 dys.

Extensive hemorrhages; otherwise similar to the last

?
++

++
++

+

L. . . . . .

Z

M a r k e d induration, b u t s ome w ha t lesst h a n on the previous day. Less
edema (figure 6)++

I n d u r a t i o n somewha t less t h a n on thepreceding day.
Yellowish grey
specks prominent on the surface(figure 7)
General induration disappearing; organ
flabby; disseminated yellowish grey
spots (figure 8)

++

++

÷

.....

÷

.....

I

i

++

++

I

9 dys.

I n d u r a t i o n gone; soft and pale, slightly
below original volume; some focal
infiltration (figure 9)

&

I :I0
I : IOO
I : 1,000
I : I0,00o

IO dys.

I n d u r a t i o n gone; general a t r o p h y ; some
focal induration (figure zo)

_+ ÷÷

<_+

......

i .....

<+......

i

I " I0I : IO0
I : 1,000

I

I I dys.

M a r k e d reduction in size; no i n d u r a t i o n(figure I I)

: I0,000

I :I0÷ I÷. . . ÷. . .

2+

I : I00-

I : 1,000
I : I0,000

12 dys.

No induration, soft and pale, muchsmaller t h a n original (figure I2)

_

+

I :I0
I : IO0
I : 1,000
I: IO,OOO]

..

1

i

I

-

.

I.

.

.

.

.

.

.

.

I

.

.

.

Hideyo Noguchi.
TABLE
Testicles
removedafter

13 dys.

547

II.--Concluded.
T e s t s for activity o f e m u l s i o n s o n rabbits.

Gros s a p p e a r a n c e o f s p e c i m e n s .

N o r m a l in size, pale and soft; somegreyish spots

Dilution ofemulsion.

Skin.

Cornea. Testicle.

I ;2
I:I0
I

:20

I : I00

14 d y s .

Similar to the last

I :2
I : I015 d y s .

Small, pale, flabby; few greyish foci

I :20
1:2

I:IO
16 d y s .

17 d y s .

18 d y s .
21 d y s ,28 d y s ,35 d y s .
56 d y s .

I :20
I 2
I
I
M a r k e d atrophy
I
I
I
Similar to the last
I
I
Small, fibrous; few m i n u t e loci of in- I I
filtrationII
Similar to the lastII
M a r k e d atrophy; small area of necrotic I I
tissue present
II
One testicle reduced to a small fibrous I
mass; the other less atrophiedI

Similar to the last<+-

. ....

+
--

--

m

<+

+

+

I eruption--+

exudate in the tunica vaginalis and in the testicular tissue itself ismore copious. The color of the testis has become purplish red,spotted here and there witch irregular yellowish areas of different
dimensions (figures 13 to I6). The organ is easily torn. The
sections show numerous groups of several tubules ea'ch, which havelost the property ,of taking up l~he basic stain, indicating a total necrosis of the structures (figures 19 and 36). These changes are
not universal, since areas exist in which no apparent serious alteration has taken place. At this stage the vaccinal power is nearly
three times as great as it was at the end of three days, and at least
I,OOO times as great as at the end of the twenty-four hour period.
The changes in 'the five day specimen are about ~he same as thoseof the four day specimen. The vaccinal activity seems now to haveattained its maximum height, since almost confluent eruptions on theskin of rabbits are yielded in dilutions of I to I,OOO. The sections
indicate beginning disintegration of the leucocytic elements and of

548

Cultivation of Vaccine Virus.

affected tubular cells (figures 5, 29, 29 a, and 35 ; compare with normal, figure 28).
At the expiration of six days the testicle has become of softer
consistency (figure 6), and the edema and cellular infil'tration havebegun to recede. Microscopically loci of small round cells in theinfiltrated areas and fibrin masses are distinguished (figures 37 and
38). The vaccinal activity is similar to that of the five day specimen. After seven days the resolution of the infiltration proceeds
more rapidly (figures 7 and 20), while the vaccinal activity is lessthan that of the preceding day. From now on the testicles diminish
rapidly in volume, so that the ten day specimen is below normal insize (figures 8, 9, Io, 2I, and 22). The organs are now pale andof soft Xexture. The act.ivity of the virus is much less than it wasat the end of seven days. At the end of eleven days it is still weaker.
From twelve 'to eighteen days the shrinkage of the organ continues
and the sections show loss of testicular cells (figures II, I2, 23 to 27,
and 39 to 44) and collapse of the tubules. The virus has now disappeared wholly or almost wholly, and in some instances no vaccinal
effect could be obtained in any concentration. Hence this period may
be regarded as that of elimination of the virus, while the exact
moment of disappearance probably depends upon the quantity of thevirus originally developed and the degree of immunity displayed.
At later periods, namely after five weeks, n,o virus could be detected,
while the atrophy of the testicular parenchyma may be complete, sothat a fibrous mass containing a few unusually thin tubules, linedwith a single layer of epithelial cells may alone remain (figures 27
and 45).
The tunica vaginalis is often intensely infiltrated with polynuclear
leucocytes (figure 46) and contains a considerable amount of thevirus; apparently the virus multiplies here as ft does in other epithelial cells. The epididymis shows a slight infiltration only during
the early period.
Spermatogenesis ceases quickly and the various sperm cells quickly
degenerate under the influence of the vaccinal process.

Hideyo Noguchi.

549

E F F E C T S OF T H E T E S T I C U L A R V A C C I N A L S T R A I N ON T H E

SKINAND CORNEA.

The testicular Strains of the virus were employed to inocula'te theshaved skin and scarified corneal surfaces of rabbits. The effectsof the vaccinations so carried out may be followed in table III.
They will be recognized as characteristic of the effects produced by
active vaccine virus as usually prepared in the calf. The microscopical features of the process are also typical, including the presence of 'the Guarnieri, or vaccine, bodies so called (figures 49 and50).
TABLE III.
Average course in rabbits.

At theend of
24 hrs.
48 hrs.
3 dys.
4 dys.
5 dys.
6 dys.
7 dys.
8 dys.
9 dys.
IO dys.

Skin.

Cornea.

Temperature. 8

Diffuse reddening
Mottled reddish areas
Fairly defined fiat erythema, in part confluent
In part confluent, mostly discrete raised
papules with induration and areola
Distinct raised round vesicles with areola

Slight swelling
Slight turbidity
Distinct turbidity

38.9 °
39.5 °

Advancing in degree and extent
Sometimes ulceration
Ulceration
No change

40.8 °

Ulcer persists
Ulcer persists

38.9 °
38.7 °

Ulcer persists

38.8 °Development into pustular eruptions
Pustules with scanty content; tendency
to dry up
Inflammatory processes disappearing
Stage of crust formation and desquamation
Stage of crust formation and desquamation

EFFECTS OF VARYINGTHE

CONCENTRATION
SKIN~ CORNEA~

40.0 °

40.20
39.8 °
38.8 °OF T E S T I C U L A R VIRUS U P O NANDTESTICLE.

In order to determine the sensitiveness of the skin, cornea, and testicle to vaccine virus, emulsions of the testicular virus of different
concentrations were prepared and inoculated into the several parts
mentioned.
For this purpose a specimen of testicle representing the t'hirty-firstpassage or generation was employed. The dilutions in sterile saline
were as follows: I to IO, I to IOO, I to 300, I to I,OOO, I to 3,0o0,
I to IO,OOO, I to 3o,ooo, I to IOO,OOO. The skin and cornea weres A v e r a g e f r o m fifteen rabbits.

550

Cultivation of Vaccine Virus.

inoculated in the usual manner, and o. 5 of a cubic centimeter was injected into the testicle.
The result is summarized in table IV and may be stated as follows :Up to a dilution of I to 300 the testicular emulsion causes marked
vaccinal reactions in the skin, cornea, and testis. The skin surface
still reacts slightly at the I to I,OOO dilution, Lbut the cornea doesnot. Beyond the I to I,OOO dilution the skin reacts no longer, while
the testicle continues to react even to dilutions of I to IOO,OOO, thelimit of the experiment. However, dilutions of I to 3,ooo andhigher retarded somewhat the testicular reaction, which in the end
T A B L E IV.
Reactions following the inoculation ofdifferent dilutions of the emulsion
as indicated in the first column,

Dilution ofemulsion.

Cornea.
Testicle.

Skin.

I :IO
I : IO0

I : 300
I: I,O00

I : 3,OOO

I : IO,O00

I : 30,000

I : IO0,O00

+++
++
++
+

+
+
+
-

3 dys. already marked
induration and edema
3 dys. already marked
induration and edema
3 dys. already marked
induration and edema
3 dys. already marked
induration and edema
3 dys. only slight induration; 5 dys.
marked increase
3 dys. only slight induration; 5 dys.
marked increase
3 dys. only slight induration; 5 dys.
marked increase
3 dys. only slight induration;
5 dys.
marked increase

Tests of activity of emulsions prepared f r o mtesticles 9 which had been injectedwith different dilutions.
Dilution o f e m u l sion of eachtesticle.

Skin.

Cornea.

Testicle.

++
++
++
++
++
++
++
++
++
++

I

: IO0

+

+

I

: I,O00-t-......

I

: IO0

++

++

I

: I,O00

I

: IO0

++

++

I

: I,O00

I: IO0
I : 1,000
I: I00

I : 1,000
I " I00
I : 1,000++1++÷i iI : 1,000

++
+

I

: IO0

+÷ ;;;I: I,O00

I : IO0+++

I .....

++
++
++
++
++
++

was quite identical with that produced by the stronger concentration. In an experiment made somewhat later and after the testicular strain of the virus had been intensified by repeated passages, askin reaction of small degree but of characteristic kind could beelicited in the I to IOO,OOO dilution.
This experiment serves also to bring out distinctly the fact of
9 T h e testicles w e r e r e m o v e d f r o m the a n i m a l s o n the s i x t h day a f t e r the
inoculation.

Hideyo NogucM.

551

the rapid multiplication of the virus in the testicle of rabbits, andis shown in the second half of table IV. The vaccinal effect upon
the skin, cornea, and testicle became essentially the same in kind anddegree, irrespective of the concentration of the virus emulsion injected originally into the testicle. At the expiration of six days thetesticles inoculated with virus in the I to Ioo,ooo dilution yielded avirus emulsion as active as the original testicular material employedfor preparing the stock emulsion. The rapidity and ease of multiplication of the virus within the testicle is well illustrated by this experiment. The causes of the small variations in effect as indicatedin the table as occurring between emulsions of testicles receiving thehigher and lower dilutions of virus are not at once apparent. Possibly certain rabbits restrain the multiplication of the virus more thanothers; possibly concomitant immunity reactions come into play.
TESTICULAR CULTIVATION I N BULLS.

Experiments were next performed on the testicles of young bulls,
with the view of ascertaining whether 'this species of animal would
be suitable for use, both from the quantitative and qualitative relation of the virus, since it has long been customary to produce vaccine
in the skin of this species.
Bull I . - - W e i g h t , 750 pounds. T h e scrotal skin was shaved, washed with soap,
and sterilized by sublimate alcohol. The vaccine virus employed for the injection
consisted of testicular emulsion derived from the testicles of two rabbits which
had been inoculated with the 3oth generation of the testicular strain a n d castrated at the end of seven days. This emulsion produced a fairly thick eruption
on the skin of rabbits in the dilution of I : Ioo. On Oct. I3, I914, io c.c. of theemulsion diluted i : i o o were injected into the right, and I5 c.c. into the left
testicle under aseptic precautions.
The testicles showed signs of swelling on the third day (forty-eight h o u r s ) ,and increased f u r t h e r in size and density up to the end of six days. The left
testicle was much more swollen than the right. The temperature remained normal(38.7 ° to 38.9 °) until the end of the fifth day, when it rose to 4o.6 °, 4o.I ° (6thday), and remained at 40.4 ° as late as the end of the sixth day. On the morningof the eighth day the temperature was 38.7 ° (text-figure I ) .
T h e animal was slaughtered and the testicles were removed under aseptic
precautions on the eighth day. The subcutaneous tissue and the tunica vaginalis
were edematous and hemorrhagic, especially along the needle track. The testicles themselves were also edematous and showed a mass of coagulated blood at
the site where a blood vessel must have been injured by the needle puncture.

Cultivation of Vaccine Virus.

552

C o m p a r e d with the n o r m a l the vaccinated testicles w e r e firmer and m o r e pinkish
in color, besides s h o w i n g n u m e r o u s ecchymoses. T h e left testicle w a s m o r ealtered than the r i g h t ; the f o r m e r weighed 3Io and the latter 25o gm.

06T.18.
A.M.P.M.A.M

P.M.

F,.M.P.M.A.M.P.M.A.M,

P.M.A.M.

P,M.

A.M.P.M.

OCT.19. OCT20
Ilk. M .

P.M,

A.M.P.M.

44 °

43"

42 °

41 °

Z

F40 °

89 °

::3

c-I

z

J

,1,

<

88°
87°
Tzxx-FIG. i.

Bull i.

Emulsions in sterile saline solution were made with the tissues
immediately about the point of insertion of 'the needle and also of
portions remote from the site of injection. The tests for activity
were made upon the shaved skin, cornea, and testicles of rabbits.
T A B L E V.
.'ests o n r a b b i t s .

Bull ~.
Emulsion of tissue around site of injection . . . . . .

Dilution ofemulsion.

Skin.

I:IO

+++÷ ++÷ ÷÷÷÷I : IOO

Emulsion of tissue remote from site of injection

I : I,O00
I : lO,O00<÷

I : IO÷÷!
+I : IO0
I : 1,O00
I : IO,O00

Exudate in tunica vaginalis . . . . . . . . . . . . . . . . . .

Undiluted
I :IO

--

Cornea.!

Testicle.

÷ ÷I
I.....
[

++

++
-

++
++

!

Hideyo Noguchi,

553

The exudate contained in the tunica vaginalis was likewise tested.
Both exudate and emulsions were found sterile for bacteria. Table
V summarizes the results, which show that 'the virus multiplied to amuch smaller extent in the testicular tissues of the bull than is therule in the testicle of the rabbit. That the virus multiplied in somedegree is highly probable; and that the edematous fluid in the tunica
vaginalis was devoid of virus is an interesting point.
Bull z.--Weight, 680 pounds. Oct. 30, I914. The left testicle was injected atfour different sites in order to distribute the virus widely with a I : xoo dilutionof rabbit testicular virus in the thirty-third generation, the total quantity inoculated amounting to 20 c.e. A t the same time the skin of the ventral surface below
the navel was shaved, scarified, and inoculated with some of the same emulsion.
The inoculated testicle a f t e r forty-eight hours was tense on palpation, distinctly
swollen, and indurated. The swelling and induration increased during the next
three days. T h e skin showed distinct papules three days after the inoculation,
which became typical pustules two days later (figure 53). T h e temperature roseto 40 ° to 40.7 ° on ttre fourth day, and was 40 ° on the sixth day. T h e bull was
slaughtered on the sixth day and the testicles were aseptically removed (textfigure 2).
0CT,2gA.M,

P.M.

ocT.30A,M.

P.M.

0CT.31
A,M.

P-M.

NOV.I
A.M.P.M.

NOV.2,
A,M.

P,M

N 0V.~).
A.M,

N0M 4-.

P.M.A.M,

P,M,

44 a

48 o

41 q

,,0o
890

.J

J
B

/"x..._

88 •

87 °

TEXT-FIG. 2.

Bull 2.

T h e scrotum was found to be edematous and infiltrated, but no adhesion waspresent between the tunica and the testieular surface. T h e moderate amount ofserous and hemorrhagic exudate proved to be sterile for bacteria. Upon section

554

Cultivation of Vaccine Vireos.

serous exudate oozed freely from the testicle. The points of injection could be
detected by the presence of numerous minute hemorrhages within areas whichwere distinctly firmer than the surrounding parts. Coagula of blood occurred
here and there (figures 54 and 55). The uninoculated right testicle showed nochange. The weight of the left testicle was 229 and that of the right I99 gm.
E m u l s i o n s w e r e p r e p a r e d b o t h f r o m the t i s s u e s i n u n e d i a t e l y a b o u t
the sites o f i n j e c t i o n a n d f r o m p a r t s r e m o t e f r o m t h e m .
T h e tests
w e r e m a d e o n the skin, c o r n e a , a n d testicles o f r a b b i t s .
TABLE VI.
Ir
Bull 2.

Tests on rabbits.

i]iEmulsion of tissue around zone of injection.[

Dilutionof emulsion,

Skin.

Cornea I Testicle.
i

I :IO

Confluent i + +I : IO0I : I,O00
I : IO,O00

Emulsion of tissue remote from site of injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I :IO
I : IO0I : I,OOO
I : IO,OOO

Exudate in tunica vaginalis . . . . . . . . . . . . .

Undiluted
I

3°

~-~-

.....

r
++
+
<+

++
+
,. . . . . .

--I ......

<+

i

+

++
++
++
++
++
++
+
+

Table VI, which summarizes the results, indicates that the tissues
a'bout t h e sites o f i n o c u l a t i o n c o n t a i n a n a m o u n t o f t h e v i r u s a b o u t
e q u a l to t h a t o f r a b b i ' t ' s testicles i n o c u l a t e d w i t h s i m i l a r m a t e r i a l ,and that the degree of activity possessed by the two testicular strains
is a b o u t t h e s a m e .
O n t h e o t h e r h a n d , t h e e m u l s i o n m a d e f r o m ther e m o t e p o r t i o n s o f t h e testicle p r o d u c e d f a r less effect, a l t h o u g h it
w a s still a c t i v e u p to t h e d i l u t i o n o f a t l e a s t I to I , o o o .
The exudate
in t h e t u n i c a c o n t a i n e d a t r a c e o f the v i r u s , 'but this m a y h a v e b e e n
d u e to a c o n t a m i n a t i o n o f t h i s e x u d a t e in the o p e n i n g o f t h e testicle.

Bull 3.--Weight, 720 pounds. Nov. iI, I9I 4. Both testicles were inoculatedwith an emulsion of a testicular strain obtained from a rabbit inoculated with thetesticular strain of bull 2. The emulsion was, however, contaminated with axerosis bacillus. The local and general reactions were similar to those describei
in the previous two bulls. In spite of the admixture of xerosis bacilli with thevirus the local reaction was not different from those following the injection ofa pure virus. The animal was slaughtered on the ninth day after injection.
The testicular tissues were edematous, showed several well localized areas ofHideyo Noguchi.

555

a greyish white color, and some hemorrhages caused by the t r a u m a of the needle.
The tissues were not softened. Cultures made from the emulsions of the testicles
showed the presence of a few xerosis bacilli, but no oti:er ba:teria.
The vaccinal activity as tested on the rabbit showed that it was much weaker
than that of bull 2.

Bull 4.--Weight, 14o pounds. Dec. 24, 1914. 4 c.c. of a 1 : 4 o dilution of anemulsion of the fiftieth generation of testicular rabbit virus were injected into
the left, and 3 c.c. of a I : IO dilution of the testicular virus from bull 2 were
injected into the right testicle. T h e organs were examined every t w e n t y - f o u rhours, but no particular difference was noticed on the two sides. Both showedmarked induration within thirty hours. On the fifth day the swelling appeared
to have reached the maximal stage. The temperature rose a f t e r three days andreached 4o.8 ° on the sixth day. T h e animal was slaughtered at the end of six
days (text-figure 3).
DEC2..4 DEC.2.5 DFC.26/)..M.P.M

DEC.P..7 DFC.£8

DI=C.z9 DEC.30

A.M.P.M..e,.M.P.M.A.M.P.M.A.M.P.M.A.M.P.M.A.M.P.M.

44 °

43 °

42 °
uJ
-J
.J

Z

41 °

o_
I-<[
._l

f._.f._../'-

40 °

-"

.39 ~

j
3~

f

--

I

37~
TExT-Fro. 3.

Bull 4.

The testicles had increased in volume, especially the one on the left side
injected with the bull-rabbit strain; they were markedly edematous and congested.
On section numerous greyish yellow specks were seen to be scattered t h r o u g h o u t
the organ, and were especially numerous along the sites of injection. Similar
greyish yellow areas, it will be recalled, occur almost consta~ntly in the inoculated
rabbit's testicles.

The emulsions were made from both testicles separately and tested
for their vaccinal activity, together with the controls of the standard
glycerinated vaccine issued by the Department of Health of New

556

Cultivation of Vaccine Virus.

York City. The testicular emulsions were free .of bacteria; theHealth Department vaccine, of course, was not.
TABLE VII.

Bull 4.

Emulsion of testicle inoculated with bull-rabbit
testicular strain

Tests on rabbits.

Tests on calves.

I
Coruea.I

Testicle.

Skin.

+

÷
< ÷

+

++
++
++
++

Confluent.
+++<+

I : IOI Confluent

I : I00

Confluent

++
++
+
-

++
++
++
++

Confluent.
Almostconfluent.
++
<+

++

++

-

<<+

Confluent.
Almost confluent.
++<+

Dilution ofemulsion.

Skin.

I : IOi : iooI

Confluent
Confluent1,000

I : I0,0o0

Emulsion of testicle inoculated with bull-rabbit
testicular strain no. 2

i

i,ooo

I : IO,OOO

+

+

÷

" N . Y . D . H . standard i : IO
vaccine Ist samplel i : ioo

Confluent
Confluent++I I : 1,000<+
I I : I0,000~g

N . Y . D . H . standard~ I : io
vaccine 2d sample; I ioo
I I : 1,000
I I0,000

Confluent+++++÷ ++÷
++++++

Confluent.
+++

The above experiments (table V I I ) demonstrate that a strong
bacteria-free vaccine virus can be produced in the testicular tissuesof the young bull by injecting the testicular strain derived from a bullor from a rabbit, and they also show that the vaccinal activity reaches
the same strength as that possessed by a standard virus propagatedon the skin of a calf. Moreover, no differences were noted in thetype and course of the vaccinal processes as produced in the s k ~of rabbits and calves by the vaccines from the several sources. The
only distinctions are quantitative ones, since the skin virus is more
quickly diluted beyond effective strength than the testicular virus.
The next experiment was devised to compare the effect of thetesticular inoculation of a testicular and a skin strain of vaccine, respectively, into the same calf.
Bull 5 . - - W e i g h t , 16o pounds. Jan. 4, 1915• 3 c.c. of a I : I O dilution o f anemulsion o f the testicular strain derived f r o m bull 4 w e r e injected into the right,
and 3 c.c. o f a I : IO dilution of the N e w Y o r k City D e p a r t m e n t o f H e a l t h standa r d vaccine into the left testicle. T h e n e x t day the swelling and injection of theright testicle w e r e marked, while the left was f a r less altered. T h i s differenceHideyo Noguchi.

557

became more pronounced, and on the sixth day only the right side presented thetypical reaction, swelling, and induration previously noted, while the left was but
slightly enlarged. T h e animal was slaughtered at the end of five and one-halfdays (text-figure 4).
JAN3. JAN.4 JANS. JAN6, JAN,7 dAN.5. JAN.9
A,M,

P.M.A.m.P.M.A.M.

P,M

A,m,

P.M.A.M.P.M.A.M.P.M.A.M.P.M.

t4 °

43 ~

62 °

(D

.-I

tO

a,0o

O
Z

¢

J

39"

88°
37°
TExT-FIG. 4.

Bull 5.

Upon exposing the testicles it was found that the left showed infiltration insmall amount and only about the site of the injection, while the right was edematous throughout, greatly enlarged, and dotted with numerous minute foci ofgreyish yellow appearance. While the consistency of the left testicle whichweighed 6.5 gin. was about normal, that of the right, which weighed 8 gm., wasdiminished.

The vaccinal strength of 'both testicles was tested on the r.abbit
TABLE VIII.
Tests on r a b b i t s
Bull 5.

Emulsion of testicle inoculated with calf testicular strain no. 4

Emulsion of testicle inoculated with skin strain(N. Y. D. H.)

Dilution ofemulsion.
I

I0

I

I00

I

1,000

I

I0,000

I
I

I0
I00

I

I,O0 0I

I0,000Skin.

Cornea.

Testicle.

Confluent
++
+

+ +
++

++
++
++
++

+
<+

+

+

Cultivation of Vaccine Virus.

558

As table V I I I indicates, the local reaction and the vaccinal activity
were more marked in the case of the testicular than of the skin,strain, from which it may be inferred that adaptation of the strain
to the testicle is necessary as a prerequisite to the production by thismeans of strong vaccine.
Bull 5.--Weight, 225 pounds. Jan. II, 1915. The left testicle was injectedwith an emulsion of the testicle that had been inoculated with the standard skinstrain of vaccine, as mentioned above, and the right testicle with an emulsion of
the testicle previously inoculated with a testicular strain. T h e local and generalreactions were similar to those observed and described, except that the left testicle,
weighing 9 gm., showed far less change than the right, which weighed 12.5 gin.
d.Z~l.1O
A.M.P.M,,.Io,NI I
A.M,

P.M,

dAN.12~ dANI~
A.M.P.M.A.M.P.M-

i.JAN.14,

d~Nl3

A.M.P.M.A.M.

P-M.

UANI6
A.M.P.M.

dANI"/"
A,M.

P,M.

dANI8
A.M.P.M.

44*

43°
42 °

=,
41"

Z
O

40°

h

39 ~

$

J
.I

J

88o
87 °

TExT-FIG. 5.

Bull 6.

(figures 56 and 57). The bull was slaughtered at the end of five and one-halfdays, and the vaccinal activity of each testicle was tested on the rabbit (table
I X and text-figure 5).

Histological Changes.--The histological changes observed in thetesticles of bulls inoculated with testicular strains of vaccine virus
are similar to those enumerated in these organs of the rabbit. An
acute inflammatory reaction arises, in which many polymorphonuclear leucocytes invade the interstitial tissues together with a serous
exudate; the adjacent tubules are compressed by pressure of the exu-Hideyo NogucM.
TABLE

559

IX.
T e s t s on rabbits.

Bull 6.

E m u l s i o n of l e f t t e s t i c l e i n o c u l a t e d w i t h s k i n s t r a i n .

Dilutionof emulsion.

Skin.

Cornea.

I : io

+
<+

+

+++
++
+

++
+

I : I00
I : 1,000

iEmulsionstrainiof r i g h t t e s t i c l e i n o c u l a t e d w i t h t e s t i c u l a r i

I : IO,OOO

I : zoi : IOO
I : 1,000

I : I0,0OO

date, which also invades ~hem, and the epithelial cells degenerate.
The reaction is multifocal and affects parts remote from the site of
inoculation, although the severer effects occur adjacent to the inoculation. Hemorrhages occur also (figures 57 to 66).
IDENTITY OF THE SKIN AND TESTICULAR STRAINS OF VACCINE VIRUS.
It may now be considered as established that vaccine virus derived
from the specific skin lesions is capable of multiplying indefinitely ina pure state within the testis of the rabbit and the bull. There
would appear to be no necessity for restoring the virus to skin surfaces in order to continue t~he propagation in the testicles. The testicular epithelium, once the virus is adapted to it, seems to provide
as suitable a medium for its multiplication as the skin epithelium.
Moreover, in order that the virus should propagate in the testis noprevious injury of the organ is necessary, save that caused by the
injection, so that in this respect the testicle may be regarded as exceeding the skin surface in susceptibility to its presence. Possibly
anatomical structure accounts for this difference; unless scarified
the virus has no opportunity of adhering to and entering intimately
into relation with the skin surface, while injection into the testicle
brings the virus at once into immediate proximity to the tissues composing that organ.
The question which arises is whether 'file skin and testicular strainof virus remain identical in physiological properties. One has, ofcourse, been derived from the other; but it remains to be considered
whether in the course of adaptation the testicular strain has undergone modification.

560

Cultivation of Vaccine Virus.

Several criteria can be employed to decide this point: (a) typeand course of the eruption of ~the skin of susceptible hosts (rabbit,
calf, m a n ) ; (5) Characteristic lesion of the cornea of the rabbit;(c) histological processes within the lesions caused by the virus;(d) phenomena of immunity arising after vaccination; (e) resistance to the action of certain agencies, such as glycerin, ether, chloroform, phenol, drying, and heating.
It may be assumed that merely completely freeing vaccine virus
from the usual bacteria which contaminate it in practice will notchange its physiological properties. This probability is indicated by
the effect.of glycerination which reduces the bacterial content. The
practical advantage of this reduction is obtained in milder vaccinal
effects in man, which are equally protective with the severer effects
resulting from bacterial coSperation.
Hence it may be hoped that the complete removal of the contaminating bacteria should still further improve the results of vaccination in that the secondary operation of bacteria contained within thevirus will be entirely eliminated. The employment of such a purestrain virus will, at the same time, do away entirely with the potential danger of contamination of the virus with tetanus bacilli or
spores, a subject which has always engaged the attention of producers, and respecting which special precautions are taken.
Skin Reactions.--Both skin and testicular strains of virus were
inoculated simultaneously on different parts of the shaved skin surfaces of ra'bbits and calves, after which the course of the vaccination
was followed. No differences were noted (figures 67, 68, and 69).
Cornea.--The rabbit's cornea inoculated with skin and testicularvirus, respectively, undergoes the same series of changes, and in eachcase Guarnieri bodies are produced. The results are indistinguishable (figures 49 and 51).
Histological Lesions.--'When the testic~alar strains of virus are
reimplanted upon the skin of the rabbit and calf, and the minutehistological changes produced are compared wit'h those caused by theskin strains of virus, no essential differences have been detected(figures 47, 48, 5° , and 52). Moreover, the microscopical changes
in the testicles produced by skin or adapted testicular strains of virus
are essentially identical.

Hideyo Noguchi.

561

I M M U N I T Y PHENOMENA.

Considered from the practical standpoint the most important effectof vaccine virus is the immunity to reinoculation t'hat it confers.
Hence, experiments were conducted with the testicular strains inorder to determine the immunity effects which it produces.
Experiment /.--Rabbit. First vaccination, Oct. 5, 1914, at three separate
places on the shaved skin, with regular, glycerinated stock virus. Confluent vaccinal eruptions were produced.
Second vaccination, Feb. 12, 1915. The three places previously vaccinatedwere now revaccinated as follows: (a) an area with some of the virus used previously; (b) an area with rabbit testicular strain; (c) an area with bull testicular.strain. The three areas reacted in identical manner : each area became congested,
but neither vesicles nor papules appeared. The reddening of the skin was probably allergic in origin (2o).
The control rabbit developed confluent eruptions from each of the three samples of virus.
Experiment //.--Rabbit. First vaccination, Oct. 5, 1914, at three separate
places on the shaved skin, with a testicular strain of vaccine virus from a rabbit.
Confluent vaccinal eruptions were produced.
Second vaccination, Feb. 12, 1915. The three places previously vaccinatedwere revaccinated as follows: (a) an area with some of the virus used previously; (b) an area with bull testicular strain; (c) an area with New York City
Department of Health vaccine. On the following day slight transient scatterederythema was noted, but neither papules nor pustules.
The control rabbit developed typical confluent eruptions from each of thethree samples of virus.
Experiment / / / . - - R a b b i t . First vaccination, Feb. 2, 1915, at three places on
the shaved skin, with testicular strain from a bull calf (No. 4). Confluent vaccinal eruptions were produced.
Second vaccination, Feb. 17, 1915. The three places previously vaccinatedwere revaccinated as follows: (a) an area with some of the virus used in thefirst vaccination; (b) an area with a rabbit testicular strain; (c) an area withthe regular New York City Department of Health vaccine. Only moderateallergic reactions were observed.
Two other rabbits similarly tested gave identical results with the above experiment, except for the presence in one of them of a slight erythema along thescarified lines, which lasted three days.
Controls on a normal rabbit showed that the strength of the vaccine strains
employed for the foregoing experiments was such as to produce confluent
eruptions.

The experiments described are conclusive in demonstrating thatthe skin and testicular strains of vaccine virus yield identical immunity reactions both as regards kind and degree.

562

Cultivation of Vaccine Virus.
T E S T I C U L A R C U L T I V A T I O N AS A M E A N S OF P R O P A G A T I O N OF
P U R E V A C C I N E VIRUS.

It is obvious that a method of propagating the vaccine virus freefrom bacterial contamination would remove all danger of accidental
infection through the introduction at the time of vaccination of an
impure preparation containing certain bacteria. Without stopping
to consider how often severe effects have resulted from bacterial
contamination of the virus it may be urged that the use of a pure
preparation will altogether preclude the possibility of a mixed infection from this source. The indefinite propagation of a highly
potent vaccine virus in the testicular tissues of animals is comparatively a simple procedure, so that by means of a strictly aseptic technique combined with careful bacteriological control ,of the product,it will now become a simple matter to supply the medical profession
with an absolutely pure virus, for the purpose of vaccination. As iswell known, the method hitherto practised for propagating vaccine
consists in utilizing the emulsion of the skin pulp from vaccinatedcalves. The usual fresh pulp contains many and various bacteria,
so that to free the pulp of them it must be left in contact for a periodwith 60 per cent. glycerin or i per cent. phenol, before it is regarded
as ready for use on human subjects. Since the strength of thevirus also deteriorates with time the process of vaccine preparation
has not been perfected from the practical point of view. On theother hand, a pure virus should be ready for use as soon as the usual
bacteriological and potency tests are completed, that is, within about
one week after the preparation of the emulsion of the testicle containing it.
The introduction of a pure vaccine int.o the public health service
would be welcome, even if it were somewhat more expensive to produce than the usual vaccine. As a matter of fact it appears to beeven more economical than the latter, as the following calculationshows.
A m a l e rabbit w e i g h i n g a b o u t 3 kg. yields f r o m 7 to IO gin. of t e s t i c u l a r
m a t e r i a l c o n t a i n i n g t h e p u r e c u l t u r e o f vaccine. W h e n this a m o u n t o f tissue ise m u l s i f i e d in f r o m 15 to 20 c.c. o f 5o per cent. sterile glycerin, it will yield f r o m
2o to 28 c.c. o f a finished p r o d u c t o f h i g h potency. A s a m p l e o f this p r o d u c t is
d i l u t e d I : I,OOO a n d tested on t h e skin of rabbits. I f a c o n f l u e n t e r u p t i o n results,

Hideyo Noguchi.

563

which may reasonably be expected, the stock suspension may be further diluted
with four times its volume of sterile 5o per cent. glycerin, since a sample ofglycerinated virus which produces a confluent eruption in a dilution of I:2oo
is sufficiently strong for use in man. Hence a single rabbit may yield I25 c.c.
of finished glycerinated virus, or even more.
The average yield from the calf, according to Fielder, 1° is put at 4o to Ioo
gin. of fresh pulp, which emulsified with four parts of 5o per cent. glycerin
gives 25o to 3oo c.c. of vaccine virus ready for use within three months.
A comparison of the cost is in f a v o r of the rabbit vaccine. F o r
the preparation of I,OOO cubic centimeters o f the finished vaccine
product about 40 rabbits or 4 calves would be required. T h e initialcost of the f o r m e r would be considerably smaller.
Moreover, the employment of rabbits instead o f calves has advantages of another kind f r o m those which have just been emphasized.
T h e prevalence of f o o t - a n d - m o u t h disease a m o n g cattle has closed
the m a r k e t in m a n y states to calves, so that the propagation o f vaccine virus in the o r d i n a r y way has recently suffered. N o such int e r r u p t i o n is to be feared with respect to the use of rabbits. D u r ing the prevalence of such an epidemic it 'becomes essential also Co
keep the calves intended for vaccination under observation before use
f o r a sufficient period to insure their f r e e d o m f r o m foot-and-mouthdisease infection.
Finally, it m a y be added that the bull m a y also come to be used
for the testicular cultivation of the virus. H o w e v e r , the m e t h o d has
not yet been w o r k e d out for this animal as completely as it has forthe rabbit. H e n c e it must be left to the future to determine whether
the bull is strictly suitable for the purpose. This part o f m y work
suffered interruption because of the prevailing epidemic of foot-andm o u t h disease and the resulting quarantine upon cattle.
STANDARDIZATION OF VACCINE VIRUS.
Several methods are employed for determining the efficacy andpotency of vaccine virus before distri'buting it for general use. T h e
accepted methods agree in using animals before permitting the virus
to be used on man. Thedbald Smith recommends the skin of thecalf, and requires that the eruptions produced there must correspondwith those of the skin of a child. Calmette and Gu6rin (9, 21) andlo On the other hand, Rosenau (I) gives the amount of fresh pulp from acalf as 2o to 40 gm., which will yield 5o to 12o c.c. of the glycerinated emulsion.

564

Cultivatio~, of Vaccine Virus.

Camus (22) employ the skin of the rabbit; while Chaumier (23) andothers 11 first employ animals, and then themselves observe the vaccination effects on a few children before issuing the virus. There isagreement among the authorities that the skin of the rabbit andcalf are reliable indications of the effects produced on human subjects.
Henseval and Convent (24) consider that a preparation which
produces on the skin of the rabbit almost confluent eruptions in adilution of I to .500 should be considered a suitable vaccine. Such asample always causes an uninterrupted eruption along a line (linear
eruption) on the skin of a child. They recommend as still usable
even a weaker specimen provided it produces a continuous eruption
in a I to Ioo dilution.
The testicular vaccine strains which the author prepared in rabbits
either fulfill or exceed the above requirement, since a few samples
produced confluent eruptions in dilutions of I to Io,ooo. On theother hand, the products from the testicles of the bull were less active,
and a few only of the preparations reached the lower standard.
PURE VACCINATION IN M A X .

The tests which were carried out in detail and with minute carein animals with the pure testicular strains of vaccine virus all indicated that the virus would produce typical vaccinal effects in thehuman subject.
Several adults, chiefly physicians familiar with the cultivation experiments, immediately volunteered for vaccination, which was
carried out by the linear and scarification methods. Among thetests, which were all revaccinations, some were positive and others
negative. In the positive results fhe vaccinal eruption was typicaland no secondary infection arose Co complicate or intensify the vaccination.
Among those in whom the vaccination was positive was a physician. After his experience he vaccinated his own child, three years
and two months old, who had not previously been successfully
1~ T h e D e p a r t m e n t of H e a l t h of the City of New York issues only the preparations which have produced on primary vaccination cases fifteen typical takes
on fifteen insertions made on five children, thus requiring IOO per cent. of positive takes for each sample of vaccine.

Hideyo Noguchi.

565

vaccinated. The linear eruption appeared on the fifth day, increased
in intensity during the three succeeding days when it reached the
maximum, and then receded. No pain or itching was complainedof, and the highest temperature was 37.5 ° C., which was noted on
the eighth day. On the fourteenth day scab formation was well advanced. I am indebted to the father of the child for the two ~hotographs (figures 70 and 71) which illustrate the vaccinal effects
on the fifth and sixth days.
SUMMARY.

Vaccine virus freed from all associated bacteria by means of suitable disinfecting agents can be propagated in a pure state in thetesticles of rabbits and bulls. The virus cultivated in this manner
is not only devoid of all bacteria, but appears capable of indefinite
transfer from one animal to another. Sixty passages in rabbits ofa pure strain have been made within one year.
Several transfers from testicle 'to testicle are required to bringabout accurate adaptation of t~he virus to the testicular parenchyma,
so that continued propagation in this way can be certainly secured.
During the first transfers from testicle to testicle the activity of thevirus may be less than the original skin specimen from which thepure strain was derived; but as the transfers proceed the activity
rises until, when the adaptation is complete, the activity of the testicular equals that of the skin strain.
The multiplication of the virus within the testicle is maximum on
the fourth or fifth day after inoculation; the quantity of virus remains about stationary until the eighth day, when diminution begins.
At the expiration of five weeks no more virus could be detected in
the testicle.
The vaccinal processes in the skin, cornea, and tes'ticle of rabbits
are practically identical whether the virus employed for the inoculation has been the original skin strain or the pure testicular strain;
and the skin lesions produced in the calf with the two strains arealso identical.
In conformity with the finding mentioned in the last paragraphit has been found that human beings react to the pure testicularstrain of vaccine virus in an entirely typical manner. In the case566

C u l t i v a t i o n of V a c c i n e V i r u s .

b o t h o f o r i g i n a l v a c c i n a t i o n a n d r e v a c c i n a t i o n t h e v a c c i n a l effects
c a n n o t be d i s t i n g u i s h e d f r o m t h o s e a r i s i n g f r o m u n c o m p l i c a t e d s k i nvirus.
P u r e s t r a i n s o f t e s t i c u l a r v i r u s a r e r e a d i l y p r o d u c e d , a n d o n c e sec u r e d t h e y m a y be p r o p a g a t e d in a p u r e s t a t e b y t h e m e t h o d d e s c r i b e din r a b b i t s o r bulls w i t h o u t d i f f i c u l t y a n d w i t h e c o n o m y .
The purestrains thus obtained should supply an ideal form of virus for emp l o y m e n t in t h e v a c c i n a t i o n o f h u m a n b e i n g s .
BIBLIOGRAPHY.
i. Rosenau, M. J., Preventive Medicine and Hygiene, New York, 1913.
2. Francis, E., Conditions Surrounding Tetanus Spores Artificially Implanted
into Vaccine Virus, Bull. Hyg. Lab., U. S. P. H. and M.-H. S., 1914,
No. 95, 11.
3. BOraud, B. J., Recherches sur l'orchite et l'ovarite varioleuses, Arch. g~n. de
todd., I859, i, 274, 557.
4. Weigert, C., ~3ber pocken~ihnliche Gebilde in parenchymatSsen Organen undderen Beziehung zu Bakteriencolonien, Breslau, 1875.
5. Chiari, H., Ueber Orchitis variolosa, Ztschr. f. Heilk., I886, vii, 385.
6. Chiari, Weitere Beitr~ige zur Lehre von der Orchitis variolosa. Ztschr. f.
Heilk., 1889, x, 340.
7- Councilman, W. T., Magrath, G. B., and Brinkerhoff, W. R., The PathologicalAnatomy and Histology of Variola, Jour. Med. Research, 19o4, xi, 12.
8. A complete literature up to I9O4 is given in the article of Councilman,
Magrath, and Brinkerhoff, loc. cir. Also a review on this subject till I911
is to be found in the work of Paschen, E., ~ber den Erreger der Variolavaccine. Immunit~itsverh~.Itnisse bei Variolavaccine, in Kraus, R., andLevaditi, C., Handbuch der Technik und Methodik der Immunit~itsforschung, Jena, I9II, lte Erg~.nzungsband, 465.
9. Calmette, A., and GuOrin, C., Recherches sur la vaccine expOrimentale, Ann.
de l'Inst. Pasteur, 19Ol, xv, 161.
IO. yon Prowazek, S., and Yamamoto, J., Experimentelle und morphologischeStudien tiber das Vakzinevirus, Mi~nchen. reed. Wchnschr., 19o9, Ivi, 2627.
II. Henseval, M., and Convent, A., Contribution ~ l'Otude expOrimentale: l'injection de vaccin dans le testicule, Bull. de l'Acad, roy. de todd. de Belgique,
Series 4, 191o, xxiv, 635.
12. Paschen, loc. cit., p. 483 (footnote).
13. Tyzzer, E. E., The Etiology and Pathology of Vaccinia, Jour. Med. Research,
19o4, xi, 18o.
14. yon Prowazek, S., and Miyaji, S., Weitere Untersuchungen fiber das Vaccinevirus, Centralbl. f. Bakteriol., zte Abt., Orig., 1914, lxxv, 144.
15. Parodi, U., Ueber die Uebertragung der Syphilis auf den Hoden des Kaninchens, Centralbl. f. Bakteriol., Ire Abt., Orig., 19o7, xliv, 428.
16. Uhlenhuth, P., and Mulzer, P., BeitrSge zur experimentellen Pathologie undTherapie der Syphilis mit besonderer Berficksichtigung der Impf-Syphilis
der Kaninchen, Arb. a. d. k. Gsndhtsamte., 1913, xliv, 307.

Hideyo Noguchi.

567

17. Nichols, H. J., Experimental Yaws in the Monkey and Rabbit, Jour. Exper.
Med., I91O, xii, 616.
18. Noguchi, H., A Method for the Pure Cultivation of Pathogenic Treponema
pallidum (Spirochwta pallida), Jour. Exper. Med., 1911, xiv, 99.
19. Fornet, V~r., Die Reinkultur des Pockenerregers, Berl. klin. 147chnschr., 1913,
1, 1864; Ueber den Pockenerreger, ibid., p. 2325.
2o. yon Pirquet, C., Klinische Studien fiber Vakzination und vakzinale Allergie,
Leipzig, 19o7.
21. Gufrin, C., Contr61e de la valeur des vaccins jennfriens par la numfrationdes 616ments virulents, Ann. de l'Inst. Pasteur, 19o5, xix, 317.
22. Camus, L., Recherches sur l'immunit6 vaccinale. De Faction antiviruleute deshumeurs des animaux vaccinfs. Ses variations, ses relations avec l'action
bact6ricide, Jour. de physiol, et de path. gdn., 19o8, x, 455.
23. Chaumier, E., Le vaccin Z devant la soci6t6 m6dicale des h6pitaux de Lyons,Tours, 19o4; cited by Henseval and Convent, loe. cir., p. 617.
24. Henseval and Convent, Le contr61e de l'activit6 du vaccin variollque. Etude
expfrimentale, loc. cir., p. 616.
25. Calkins, G. N., The Life History of Cytorytes variolae, Guarnieri, ]our. Med.
Research, 19o4, xi, 136.
EXPLANATION OF PLATES.
PLATE 39.
FIG. 1. The right testicle shows swelling due to the injection of pure vaccine
virus made five days previously; the left side is normal.
FIGS. 2 to 12. The conditions of the testicles at various stages in the vaccinalprocesses produced by means of a pure vaccine virus, as follows: 2, 48 hours;
3, 3 days; 4, 4 days; 5, .5 days; 6, 6 days; 7, 7 days; 8, 8 days; 9, 9 days; lO, Io
days; Ii, 11 days; and 12, i2 days after the inoculation. The yellowish grey
mottles of irregular dimension can be seen in the specimens shown in figures 4,5, and 6. The marked atrophy of the organ is easily noticeable in the specimens
shown in figures 9, 11, and 12. All natural size.
PLATE 40.

FIG. I3. A testicle four days after inoculation with pure vaccine virus. It
shows marked vascular injection, edema, and several small yellowish grey spots
visible on the surface.
Flc. 14. The organ on section, in which several lobules can be seen undergoing necrosis, as indicated by the greyish yellow, somewhat indurated areas.
The parenchyma has bulged out of the tunica albuginea on account of the edema.
FIGS. 15 and 16. Two specimens of testicles at the height of the vaccinalprocesses, one (figure I6) with hemorrhagic reactions. In the latter instance thenecrotic changes are much more pronounced, as is indicated by the generalyellowish grey color of the lobules on section.
PLATES 41 AND 4 2.
FIC. I7. The structure of the testicle of a normal rabbit under a low power(magnified I3 diameters).

568

Cultivation of Vaccine Virus.

FIG. I8. Diffuse interstitial infiltration of the vaccinated testis a f t e r threedays. The compression of the tubules through intertubular edema and infiltration is well shown.
FIG. I9. A four day specimen which presents a large, irregularly defined infiltration and the destruction of the tubules.
FIGS. 20 to 24. Gradual absorption of the inflammatory products with distinct
diminution of tubules. There are still many foci of infiltration.
FIGS. 25 and 26. The structure of testicles which had been vaccinated threeand seven weeks previously.
FIG. 27. A longitudinal section of a testicle which shrank as the result ofvaccination fifty-six days previously. There are only a few tubules left, allwithout any spermatogenetic activities. Magnification: X I 3 throughout.
PLATE 43.
FIG. 28. A section of normal rabbit's testicle stained by the Borrel-Calkinmethod (25).
FIGS. 29 and 29 a. The peculiar round or oval bodies of varying size within
the affected cellular elements in the vaccinated testicles of rabbits. These bodies
appear brilliantly red when stained by the Borrel-Calkin method, and they resemble the vaccine bodies so called. These bodies may have been derived from thenuclei of the cells under the influence of the vaccine virus. Magnification : X 34othroughout.

PLATE 44.
Fins. 3o and 3I. The structure of a normal testicle of the rabbit.
FIG. 32. A n early polynuclear infiltration of the interstitial spaces of a vaccinated testicle of the rabbit after twenty-four hours.
FIG. 33- A similar but more advanced stage after three days.
FIG. 34. Extensive involvement of the testicular tubules as well as the interstitial tissues a f t e r four days.
FIG. 35. A focus wherc polynuclears are seen to have begun to break up after
five days.
FIG. 36. A n area of extensive necrosis occurring in a specimen removed after
four days.
FIG. 37. T h e cellular infiltration and fibrin in a specimen after five days
( W e i g e r t stain).
FIG. 38. A n intratubular infiltration occurring in a specimen seven days a f t e r
inoculation. The cells are basophilic leucocytes. Magnification : X I7o throughout.
PLATE 45.
FIG. 39. A tubule completely filled with leucocytes in a specimen eight daysa f t e r inoculation.
FIGs. 4o and 4 I. E n o r m o u s cellular infiltration along the tunica albuginea innine day specimens.
FIG. 42. The detachment of the degenerated testicular cells from the wall ofaffected tubules in a twelve day specimen.
FIG. 43- A focus of infiltration in a twenty-one day specimen. Such a focus
is rather rare in old specimens.

Hideyo Noguchi.

569

FIG. 44. A focus of infiltration which was found in a fifty-six day specimen.
FIG. 45. T h e general changes of the structure of the vaccinated testicle after
fifty-six days.
FIG. 46. T h e infiltration of the tunica vaginalis in a rabbit with polynuclear
leucocytes in a four day specimen.
FIGS. 47 and,48. The vaccinal reactions on the skin of rabbits. Magnificat i o n : X 17o throughout.
PLATE 46.
FIG. 49. The Guarnieri, or vaccine bodies, so called, in the corneal epitheliumof a rabbit inoculated with pure testicular vaccine virus. Three day specimen.
FIG. 50. T h e vaccinal reaction in the skin of a rabbit inoculated with the testicular strain of vaccine virus. Stained with Calkin's modification of Borrel's
method.
Fro. 51. The vaccine bodies in a rabbit's cornea inoculated with regular vaccine strain.
FIG. 52. N o r m a l rabbit skin stained with the Borrel-Calkin method.
PLATE

47.

Fie,. 53. T h e inoculated (left) and normal (right) testicles of bull 2 on thesixth day a f t e r the injection of pure testicular vaccine from a rabbit. T h e left
side is seen to be somewhat larger than the right. The skin shows typical vaccinal pustules also produced with the same material.
FIG. 54- The inoculated testicle and the tunica vaginalis of the same animal,
the latter showing much hemorrhage.
FIG. 55. T h e same organ on section. One notices the hemorrhagic loci andnecrotic area.
FIGS..56 and 57- The testicles of bull 6. The larger one was inoculated withthe testieular, and the smaller with a skin strain of vaccine virus, and both were
removed on the sixth day.
PLATE 4 8 .
FIG. 58. The structure of a normal testicle of a young bull. One recognizes
at once that the spermatogenesis is in full display.
FIGS. 59 to 61. The reactions following the injection of pure testicular vaccine
strain into the organ. They represent changes found on the sixth day.
FIGS. 62 to 66. T h e vaccinal processes in the testicles of bull calves. T h echanges are similar to those found with young bulls and rabbits, except that the
animals were too young to have any spermatogenesis, even in the unaffected areasof the organ. Both in the young bulls and in the bull calves the interstitial infiltration with polynuclear leucocytes is marked. Magnification : X 17o throughout.
PLATE 49.
FIG. 67. T h e typical vaccinal effect on the skin of a rabbit inoculated with apure testicular strain of vaccine virus on the sixth day.
FIG. 68. A confluent eruption on the skin of a rabbit vaccinated with a puretesticular strain.

570

Cultivation of Vaccine Virus.

Fro. 69. T h e typical vaccinal effect on the skin of a calf inoculated with apure testicular strain of vaccine virus obtained from a rabbit, on the sixth day.
Natural size.
PLATE 5° .
FIGS. 70 and 7I. The fifth and the sixth day phases of the first vaccination
in a child three years and two months old. The virus was prepared in the rabbit
according to the method described in this work, and was absolutely free frombacteria. Notice the uninterrupted linear eruption on both sides. In figure 7z
the mottled appearance around the site of vaccination is due to the adhesion of
the vaccine shield.

THE JOURNAL OF EXPERIMENTAL MEDICINE VOL, XXI.

PLATE 39.

(Noguchi: Cultivation of Vaccine Virus.)

T H E JOURNAL OF EXPERIMENTAL

MEDICINE(Noguehi:

VOL.

XXI.

PLATE 40,

Cultivation of Vaccine V~rus.)

THE JOURNAL OF EXPERIMENTAL MEDICINE VOL. XXl.

PLATE 41.

(Noguch[: Cultivatiqn of Vaccine Virus.)

T H E JOURNAL OF EXPERIMENTAL MEDICINE VOL. XXI.

PLATE 42.

(~*oguchi: Cultivation of Vaccine Virus.)

T H E JOURNAL OF EXPERIMENTAL MEDICINE VOL. XXI.

PLATE 43,

( N o g u c h i : Cultivation of Vaccine Virus.)

T H E JOURNAL OF EXPERIMENTAL MEDICINE VOL. XXI.

PLATE 44.

(Noguchi: Cultivation of Vaccine Virus.)

T H E JOURNAL OF'.EXPERIMENTAI2 MEDICINE VOL. XXl.

PLATE 45.

(Noguchi: Cultivation of Vaccine Virus.)

T H E J O U R N A L OF EXPE.RIMENTAL MEDICINE VOL. XXl.

PLATE 46.

(~XToguchi: Cultivation of Vaccine Virus.)

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

PLATE 47.

(Noguchi: Cultivation of Vaccine Virus.)

T H E JOURNAL OF EXPERIMENTAL MEDICINE VOL. XXl,

PLATE 48.

(Noguchi: Cultivation of Vaccine Virus.)

THE

JOURNAL OF E X P E R I M E N T A L

M E D I C I N E VOL.

(Noguchi:XXl.

P L A T E 49.

Cultivation of Vaccine Virus.)

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

PLATE 50.

(Noguchi: CuItivation of Vaccine Virus.)