destroy rodents (Danysz’s virus and similar preparations) often present
lesions in the liver resembling those due to plague infection. The
granules are, however, larger and more distinct. In these cases the
spleen is enlarged and generally granular, but rarely dark and friable
as in plague infection.
THE SPLEEN.
The size of the spleen of healthy rats of the same weight varies so
greatly that often one can not be sure as to what constitutes an
enlargement of this organ.
In plague rats this organ is markedly enlarged, firm, friable, rather
dark in color, and occasionally presents small granules under the
capsule. As Skschivan[1] pointed out, these granules are not encountered
as often as are granules in the liver. At times the organ presents a
very distinctly mottled appearance. This latter appearance is much more
frequently seen in artificially inoculated rats than in those found
infected in nature. We have seen the organ distinctly slate-colored on
several occasions.
PLEURAL EFFUSION.
The last sign of rat plague is one of great importance when associated
with other suspicious lesions. The effusion is bilateral, and is serous
in character, usually clear, though it is occasionally blood stained.
Pleural effusion is rarely found in rats other than those that are
plague infected. The following table shows in percentage the frequency
of the various macroscopical lesions of acute natural rat plague, as
observed in Bombay and in San Francisco:
────────────────────────┬────────────┬──────┬────────┬───────┬─────────
│Subcutaneous│Bubo. │Granular│ Large │ Pleural
│ injection. │ │ liver. │ dark │effusion.
│ │ │ │spleen.│
────────────────────────┼────────────┼──────┼────────┼───────┼─────────
│_Per cent._ │ _Per │ _Per │ _Per │ _Per
│ │cent._│ cent._ │cent._ │ cent._
Indian Plague │ 69 │ 85 │ 58 │ │ 72
Commission, │ │ │ │ │
Bombay—4,000 rats │ │ │ │ │
Wherry, Walker, and │ 59 │ 14 │ 14 │ 68 │ 71
Howell, San │ │ │ │ │
Francisco—88 rats │ │ │ │ │
Federal laboratory, San │ 85 │ 57 │ 87 │ 74 │ 59
Francisco—62 rats │ │ │ │ │
────────────────────────┴────────────┴──────┴────────┴───────┴─────────
It is recognized that the data from the San Francisco records is so much
smaller than that from the Indian report that perhaps no just comparison
is to be made. However, the figures are quite similar, except for the
small percentage of buboes and of liver lesions in the work of Wherry,
Walker, and Howell. The work of these observers was done in the early
part of the epizootic in San Francisco while the other figures from that
city are drawn from records later in the campaign.
No single sign is pathognomonic, though only once have we been deceived
by what was regarded as a typical plague bubo. This was in a rat that
presented no other suspicious lesions and the inoculation test resulted
negatively.
It is a combination of two or more of the signs that is of moment. The
subcutaneous injection with a typical liver or these signs associated
with a typical spleen afford good grounds for a diagnosis. A rat showing
a typical liver associated with a pleural effusion will usually prove to
be plague infected, and if a large, dark, firm spleen is also found a
diagnosis may be considered as practically established.
As has been pointed out by several writers gross lesions of plague may
be distinguished even in rats that are badly decomposed.
CHRONIC PLAGUE.
No case of natural chronic plague has been encountered in San Francisco.
Only one case was found among the many hundreds of plague rats examined
by the Indian Plague Commission[3] (p. 457) in Bombay. However, this
commission encountered a considerable number of cases among _Mus rattus_
in the Punjab villages of Kasel and Dhand. The lesions were purulent, or
caseous foci. They classify these cases as follows: Chronic plague of
the visceral type, which is further subdivided into splenic nodules and
abscesses, and mesenteric abscesses; chronic plague of the peripheral
type in which abscesses are situated in the regions of the peripheral
lymph glands.
Plague bacilli were either absent or very scanty upon microscopical
examination. They were, however, quite frequently recovered by cultural
methods, and in the great majority of the cases the organisms were fully
virulent. No evidence was forthcoming to show that this chronic rat
plague had anything to do with the recurrence of acute plague among the
rats.
We have diligently sought for chronic plague among the rats in San
Francisco, but, as we said above, without success, although a
considerable number of lesions that correspond perfectly to the
description of chronic plague have been submitted to the guinea-pig
inoculation test, but invariably with a negative result. An account of
the lesions of chronic plague as observed among inoculated rats is given
in another part of this paper.
Pound[7] believes that recovery from plague in rats is shown by the
presence of pigmented lymphatic glands. Kister and Schumacher[2] mention
pigment deposits in the inguinal region, but remarked that they are not
characteristic of plague, a view which I believe is correct, as we have
frequently seen them in San Francisco among the older rats, in which
there was no reason to suspect previous plague infection, and they have
been almost uniformly absent in the case of rats that have been
experimentally infected with plague but have recovered.
RAT PLAGUE WITHOUT GROSS LESIONS.
Plague infection may be present in a rat without bringing about any
recognizable gross lesions. For example: Dunbar and Kister[8] mention a
rat, which came from a ship on which plague rats had been found, that
had no lesions, and cultures were negative; but a guinea pig cutaneously
inoculated died of plague.
Among a considerable number of inoculated rats we have very rarely,
perhaps once or twice in a hundred cases, found nothing at the
post-mortem examination that would suggest plague infection, yet
cultures or inoculation of guinea pigs would demonstrate the presence of
_B. pestis_. Such cases are very infrequent, but it should be kept in
mind that they do occur. When a large number of rats are to be examined
it would be impracticable to inoculate a guinea pig from each rat; and
even if one did this the occasionally resistent guinea pig would
introduce a larger error than exists by placing dependence upon the
gross lesions for a diagnosis.
MICROSCOPICAL EXAMINATION.
The exact weight to be given to the morphology of the organisms found in
smears from the organs of a rat suspected of being plague infected is a
matter of individual judgment. Smears from a bubo and from the spleen
may show no organisms at all, or none even remotely resembling _B.
pestis_, and yet by culture and inoculation methods we may be able to
demonstrate that the animal is plague infected. Attention has been
called to this point by several observers, and every worker in this
field has the experience sooner or later.
In other cases the smears will show such numbers of perfectly typical
bipolar bacilli and “involution” (coccoid) forms as to leave scarcely
any doubt as to the nature of the organism. But even here cases that are
not plague are encountered that will deceive even the most experienced.
We have been accustomed to put great dependence on the “coccoid” forms
of the organism, but late in the San Francisco experience, smears from a
splenic nodule that was not regarded as due to plague showed perfectly
typical “involution” (“coccoid”) forms. Animal inoculations and cultures
showed that the tissues contained no plague bacilli.
In addition to these two classes of cases we have a third, where smears
show a few typically shaped bacilli, or where a considerable number of
typical-looking bacilli are found along with many other bacterial forms.
There is no safe rule for reaching a conclusion in these cases, and one
must resort to culture or to inoculation methods, or both. In any such
case it is always a good plan to let the macroscopical findings have
more weight than the microscopical.
The bipolar appearance of _B. pestis_ is so largely dependent upon the
technique of staining, fixing, length of time the stain is allowed to
act, and the length of the washing, that it should never be given great
weight. Here, as elsewhere in bacteriology, many errors are to be
avoided by not depending too much upon the morphology of the organism
under investigation.
BACTERIOLOGICAL DIAGNOSIS OF RAT PLAGUE.
While the gross lesions of rat plague are often sufficiently
characteristic to justify a positive diagnosis, and the gross lesions in
conjunction with the microscopical examination will in other cases
enable us to say definitely that a rat is plague infected, still a
certain number of cases occur in which it is necessary to resort to
other methods, and there are circumstances, such as the first case in a
community, that make a complete bacteriological confirmation of a
diagnosis necessary.
This is not the proper place in which to discuss fully the bacteriology
of plague. However, a brief outline of what is necessary to establish
beyond question the existence of plague infection in an animal will be
given.
_B. pestis_ may often be isolated in culture from the tissues (bubo,
liver, spleen, or heart’s blood) of an infected rat. Unless the tissues
are badly contaminated with other organisms, plate or stroke culture
will yield a growth of _B. pestis_ in pure culture, or isolated
pest-like colonies may be transferred to other media.
It is unwise, however, to trust to cultural methods alone. In the
majority of doubtful cases it is advisable to inoculate guinea pigs or
white rats. The lesions of plague in these animals are quite
characteristic, and _B. pestis_ may readily be recovered from their
tissues if cultures are made at once after death.
A pure culture of the organism under suspicion is obtained from the
naturally infected animal or from a laboratory animal inoculated from
the one under suspicion. This culture is studied in regard to its
morphology; first, on agar, where it grows as a short rod, or often in
the shape of a coccus; second, in broth, where it often grows in
streptococcus-like chains; third, on agar containing 3 per cent sodium
chloride, where most extraordinary alterations in morphology occur,
giving large balloon-shaped bodies, objects resembling gigantic cocci
and enormous trypanosome-shaped forms, the so-called “involution” forms.
These involution forms must not be confused with the so-called
“involution” (coccoid) forms of the organism found in smears from animal
tissues.
We think it worth while to call special attention to the great
diagnostic value of involution forms developed when _Bacillus pestis_ is
grown on salt agar. No other organism that we have had the opportunity
of working with gives forms that are at all likely to be mistaken for
those of _Bacillus pestis_, except _B. mallei_, and of course the other
points of difference would at once serve to distinguish the latter
organism.
_B. pestis_ is Gram negative, though this point is of no great value
except to distinguish the “coccoid” forms from pus cocci.
The appearance and character of the culture should be as follows:
_Agar._—Smooth, glistening, round whitish colonies which are found to be
sticky when touched with an inoculating needle.
_Broth._—A scanty surface growth which falls, often in globular masses,
when the tube is gently agitated; and a fine flocculent precipitate.
_Litmus milk._—Generally rendered slightly acid.
_Glucose broth._—Rendered slightly acid. Gas is not formed.
_Lactose broth._—Unchanged in reaction. Gas is not formed.
The other cultural reactions are of no material assistance in the
identification of the organism. Indeed, in routine work the appearance
of the growth on agar and in broth, together with the involution forms
on salt agar, are sufficient for identifying the organism.
The plague bacillus is a nonmotile organism, a point worth bearing in
mind.
A culture answering the above description when rubbed into the shaven
skin of a guinea pig or a white rat should cause the death of either of
these animals of plague within ten days, and an organism must be
isolated from their tissues after death corresponding to the one
inoculated.
If one wishes to be doubly certain, one may inoculate a series of
laboratory animals, giving to half of them a sufficient dose of antipest
serum. The protected animals should recover, or markedly outlive the
controls, which should die in the usual time.
As to the virulence of cultures of the bacillus from cases of rat plague
Klein[17] states “that _B. pestis_ bred in the rat is of decidedly less
virulence than that bred in the human subject; moreover, the former is
liable, outside the animal body, to a much greater extent to rapidly
lose its virulence.” It is evident that in any given epidemic it will be
very difficult to say just which strain, rat or human, one is dealing
with.
In the case of the strains of _B. pestis_ recovered from rats in San
Francisco we have seen nothing to justify such an opinion as Klein
expresses. The cultures are all highly virulent and retain their
virulence under artificial cultivation.
The value of inoculation by the cutaneous method to demonstrate the
presence of plague infection in putrefying tissue is well known. We have
had one example in which the value of inoculation by this method was
proven in the case of a rat that was so badly decomposed as not to admit
of any opinion being formed as to whether the animal was infected or
not. A rat was brought from a warehouse where a typical plague rat had
been taken a few days previously. The specimen was so badly decomposed
that the abdominal organs could not be distinguished with any degree of
certainty. Smears from tissue that was thought to represent spleen were
negative so far as pest-like organisms were concerned. A guinea pig
vaccinated from this splenic material died in seven days of typical
plague, and a pure culture of _B. pestis_ was obtained from its organs.
Kolle and Martini [9] compare the cutaneous method of inoculation to the
use of an agar plate in separating plague bacilli from other organisms,
and so regularly does _B. pestis_ penetrate the skin and infect the
animal, and so rarely do other organisms do this, that it offers a
certain and accurate method of “filtering out” _B. pestis_ from any
badly decomposed tissue.
The technique of the cutaneous method of inoculation, or “vaccination”
as it is sometimes called, is very simple. An area about an inch square
is shaven on an animal’s belly, taking care to abrade the epithelium
slightly. The culture or suspected tissue is rubbed on this shaven area
with a platinum loop or a dressing forceps. Guinea pigs when inoculated
in this manner generally die before the seventh day; white rats die a
day or two earlier.
Kister [10] uses a drop of juice from an organ rich in bacilli for
agglutination experiments with antipest serum. This would appear in many
cases to be of very material assistance, and the objection that it is
difficult to form a uniform emulsion of the bacteria would be avoided.
The well-known tendency of _B. pestis_ to grow in clumps in culture is
the main reason why agglutination reactions have not been more
extensively used in plague work.
Skschivan [1] makes use of Pfeiffer’s phenomenon in establishing the
identity of a given organism as _B. pestis_.
To assist in the early diagnosis of plague, Dunbar and Kister [8]
practiced intraperitoneal inoculation of laboratory animals and used a
parallel series of immunized animals. As is well known, intraperitoneal
inoculation with plague cultures or infected material leads to the early
death of the inoculated animal, and it is evident that the survival of
the immunized animal would afford considerable evidence that the
material used for inoculation contains _B. pestis_.
PEST-LIKE BACTERIA FOUND IN RATS.
The somewhat general impression that there are a considerable number of
organisms that are readily mistaken for _Bacillus pestis_ is not
justified, provided one gives attention to cultural and inoculation
investigations. It is quite true that there are a considerable number of
organisms which in smears from tissues are scarcely to be distinguished
_morphologically_ from _B. pestis_. The similarity, however, usually
ends there. A few resemble plague somewhat closely in cultural
reactions, and especially _B. pseudo-tuberculosis rodentium_ (Pfeiffer)
should be mentioned here; but these differ in pathogenicity. For
example, the above-named organism is not pathogenic for rats.
Neumans[11] reviews the subject of pest-like organisms pathogenic for
rats, and describes an organism belonging to this group which he
isolated from the body of a rat. His work clearly shows that none of the
organisms that have been described should cause any serious difficulty
in the hands of a careful investigator.
Kister and Schmidt[12] describe an organism closely resembling _B.
pestis_ in many respects, and with which guinea pigs could be
successfully infected by the cutaneous method. This organism, which was
also pathogenic for rats and mice, belongs to the hemorrhagic septicæmic
group. It differed from _B. pestis_ in that it gave no involution forms
when grown upon salt agar and was much more rapidly fatal to laboratory
animals.
Augeszky[13] observed an epidemic among gray rats in his laboratory
which was due to a pest-like organism belonging to the Friedlander
group. The animals died after a couple of days of illness. At the
post-mortem examination the spleen was found large, soft, and congested.
There was a hyperæmia of the intestines, lungs, and liver. In the spleen
were found many, and in the heart’s blood few, capsulated bacilli, some
of which resembled _B. pestis_. The cultural reactions were in nowise
similar to those of _B. pestis_. He found that inoculation of rats with
a pure culture of this organism sometimes killed in as short a time as
twenty-four hours, sometimes as late as two or three weeks, and in some
cases the lesions were not very unlike those sometimes produced by _B.
pestis_. However, this organism by its different cultural reactions, and
the fact that the capsule is usually easily demonstrated, would probably
never be a source of any confusion.
ARTIFICIAL INFECTION OF RATS WITH PLAGUE.
For laboratory purposes in general it is customary to use tame white
rats, and in plague work they are especially satisfactory, as they are
easily handled, rarely harbor fleas, are very susceptible to the
infection, and finally and most important, they frequently die a day or
two earlier than guinea pigs. At times it may be necessary to use wild
rats on account of a failure in the supply of white rats, or for the
sake of economy. This may be done very satisfactorily, if one bears in
mind the fact that a considerable number of wild rats are more or less
immune to plague infection, especially when the infectious material is
introduced by Kölle’s (cutaneous) method. Therefore, it is always
advisable to use three or four wild rats where one white rat would be
sufficient. They should be kept in a container of such design that there
is no possibility of their escaping. The inoculation is best conducted
with the animal under the influence of ether.
MODES OF INFECTION.
Rats may be infected experimentally by the ingestion of contaminated
material, and by the application of virulent plague bacilli to a mucous
or a cutaneous surface, or by subcutaneous injection of the organism.
Practically we may confine our study to inoculation by the cutaneous
method, and to subcutaneous inoculation, when the material is injected
in the ordinary manner. A useful modification of the latter method is to
make a small pocket under the skin of the abdomen and thrust the
suspected material into this pocket. This avoids the necessity of making
an emulsion of infectious matter, such as the organs of an animal. The
time that elapses between the inoculation of a rat with virulent culture
of plague bacilli and its death varies somewhat with the size of the
dose and with the mode of inoculation. The following table, compiled
from work in San Francisco, shows the day of death of a few white rats
and a considerable number of wild rats using the strain of _B. pestis_
that was found in the recent epidemic here. Some were inoculated by the
cutaneous and some by the subcutaneous method:
───────────────────────────────────┬─────────────────┬─────────────────
Day of death. │ White rats. │ Wild rats.
───────────────────────────────────┼─────────────────┼─────────────────
Second │ │ 3
Third │ 5│ 27
Fourth │ 7│ 41
Fifth │ 1│ 30
Sixth │ 1│ 9
Seventh │ │ 8
───────────────────────────────────┼─────────────────┼─────────────────
Total │ 14│ 118
───────────────────────────────────┴─────────────────┴─────────────────
The wild rats were all _Mus norvegicus_.
The _lesions_ found, when an artificially inoculated rat is examined
after death, are in a general way similar to those found in naturally
infected rats with certain differences to be mentioned later.
In order to obtain accurate figures as to the frequency of the various
lesions in inoculated rats, I have compiled the data from the records of
the federal laboratory in San Francisco of a considerable number of wild
rats that have been inoculated in the course of various investigations
and have died of acute plague. The rats were practically all of the
species _Mus norvegicus_.
_Artificially inoculated (subcutaneously) plague rats._
──────────────┬─────────┬────────────┬─────┬────────┬────────┬─────────
│ Local│Subcutaneous│Bubo.│Granular│Enlarged│ Pleural
│reaction.│ injection.│ │ liver.│ dark│effusion.
│ │ │ │ │ spleen.│
──────────────┼─────────┼────────────┼─────┼────────┼────────┼─────────
Present │ 36│ 48│ 19│ 47│ 56│ 18
Very extensive│ 1│ [Q]2│ │ │ │ 8
Slight │ 2│ 6│ │ │ │ 8
──────────────┼─────────┼────────────┼─────┼────────┼────────┼─────────
Total present │ 39│ 56│ 19│ 47│ 56│ 34
Absent │ 10│ 4│ 39│ 15│ 3│ 21
Not recorded │ 13│ 2│ 4│ │ 3│ 7
──────────────┼─────────┼────────────┼─────┼────────┼────────┼─────────
Total │ 62│ 62│ 62│ 62│ 62│ 62
──────────────┴─────────┴────────────┴─────┴────────┴────────┴─────────
Footnote Q:
Intense.
All of the lesions aside from the local reaction were present and well
marked in six cases.
_Artificially inoculated (cutaneously) plague rats._
──────────────┬─────────┬────────────┬─────┬────────┬────────┬─────────
│ Local│Subcutaneous│Bubo.│Granular│Enlarged│ Pleural
│reaction.│ injection.│ │ liver.│ dark│effusion.
│ │ │ │ │ spleen.│
──────────────┼─────────┼────────────┼─────┼────────┼────────┼─────────
Present │ 16│ 34│ 42│ 58│ 58│ 22
Very extensive│ 1│ [R]15│ │ │ │ 6
Slight │ 6│ 13│ │ │ │
──────────────┼─────────┼────────────┼─────┼────────┼────────┼─────────
Total present │ 23│ 62│ 42│ 58│ 58│ 28
Absent │ 37│ 6│ 26│ 11│ 8│ 37
Not recorded │ 9│ 1│ 1│ │ 3│ 4
──────────────┼─────────┼────────────┼─────┼────────┼────────┼─────────
Total │ 69│ 69│ 69│ 69│ 69│ 69
──────────────┴─────────┴────────────┴─────┴────────┴────────┴─────────
Footnote R:
Intense.
All of the lesions aside from the local reaction were present and well
marked in five cases.
LOCAL REACTION.