Part 4
Two lesions of the liver are encountered in plague rats. The one most frequently observed is spoken of by the Indian Plague Commission as “fatty” change, though it is explained that this term refers to the naked eye appearance as, microscopically, the lesion is found to be due to a necrosis of the liver tissue. When this change is present the organ is found to be rather yellowish in color and is studded with an enormous number of yellowish white granules which are about the size of a pin head. This lesion, which was very common in the San Francisco cases, is very readily recognized.
The other lesion is a marking of the organ with grayish white spots; “they are typically of the size of a pin’s point, and give the surface of the organ a stippled appearance as if dusted over with gray pepper”[3] (p. 331). This appearance, which is less frequently encountered than is the preceding one, is more difficult to recognize; indeed the most careful scrutiny is necessary to avoid overlooking it.
Rats that have been fed with certain biological preparations used to 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.