Chapter II: Part 2

The ferocity of rats is mainly exercised against members of their own order. The brown rat is undoubtedly the most formidable of the genus in America, and possibly in the world; yet when captured it adapts itself readily to confinement, and in a few days will take food and water whenever offered. The enmity of this species toward other rats and mice is well known. It is supposed to have destroyed the black rat over the greater part of Europe and America, although it is possible that disease carried by the brown rat was a factor in the disappearance of the other species. That the black and the roof rat in tropical countries have not been displaced by the brown rat is probably owing largely to their more arboreal habits. It is not uncommon in the Far East to find two species of rats living side by side in the same locality. An example is _M. imperator_ and _M. rex_ living on one of the Solomon Islands. The first is a burrowing species; the other arboreal. In 1877 two native species of rats, _M. macleari_ and _M. nativitatis_, were found living together in amity on Christmas Island, in the Indian Ocean.[P] About ten years ago the brown rat was accidentally introduced, and it is now thought that both the native species are extinct.

Footnote P:

Proc. Zool. Soc. 1888, pp. 517, 534.

When pressed by hunger rats become cannibals and destroy their weaker fellows. However, when ordinary food is abundant, cannibalism among rats is rare.

PLAGUE INFECTION IN RATS.

By GEORGE W. MCCOY,

_Passed Assistant Surgeon United States Public Health and
Marine-Hospital Service._

The rat is a known or a suspected factor in the transmission of several diseases, yet at present, and perhaps for many years to come, the most immediate and pressing question that concerns us is its relation to the origin and spread of plague among human beings. For this reason a discussion of the reaction of these animals to natural and to artificial infection with _B. pestis_ becomes of prime importance.

Not only are rats believed to be more or less directly responsible for cases of human plague in a community, but in addition, they are believed to be the most frequent medium through which plague is carried from one locality to another, for these animals are good travelers, can live on a very meager ration, and can do without water for a long time if food is available. We have found that on a diet of dry grain alone a rat may live for over a month.

In this connection it may not be amiss to call attention to the importance of the rat as an agent in conveying plague infection to other rodents and especially to ground squirrels. There is every reason for believing that the infection among the squirrels in California was derived originally from rats. Wherry[16] states that more rats than ground squirrels have been trapped in the squirrel burrows in the vicinity of Berkeley, Cal. This shows how easy it might be for rats to infect squirrels and vice versa.

The clinical manifestations of plague in rats are of little importance. It is generally said that the plague-infected rat staggers about with a drunken gait, loses fear of its natural enemies, and is readily captured. Our experience with artificially infected rats indicates that the animals show no marked manifestations of illness until shortly before death when they become quiet, crouch in the corner of the cage, and try to hide.

It is rather surprising to observe that comparatively few plague rats are found dead. In the San Francisco campaign, while no accurate figures are obtainable, certainly not more than 20 per cent of the infected rodents were found dead, the remainder being trapped. This is probably due to the fact that the disease is one of several days’ duration, from two to six most frequently, and during this period there are more chances of catching the sick rodent in a trap than there are of finding the body after death, unless the immediate surroundings are known to harbor infected animals and an especially careful search is made for cadavers in the places, often difficult of access, where rats have their burrows and nests.

As plague is a disease that gives rise to such characteristic gross pathological lesions in man and in laboratory animals, it is but reasonable to expect that equally distinctive lesions would be found in the rat, and this we find to be the case.

Skschivan[1], Kister and Schumacher[2], and other writers have observed and recorded the gross lesions of plague in rats. It remained, however, for the Indian Plague Commission[3], which had the opportunity of examining an enormous number of plague rats in Bombay and elsewhere in India, to crystallize our knowledge of this subject and to point out its field of usefulness.

As to the comparative value of microscopical and macroscopical methods of diagnosis, the Indian Plague Commission[3] states that: “The results of tests carried out for the purpose of comparison make it manifest that the naked eye is markedly superior to the microscopical method as an aid in diagnosis, and as the result of our experience we are prepared to make a diagnosis of plague on the strength of the macroscopical appearances alone, even though the other results of cutaneous inoculation and culture are negative and the animal shows signs of putrefaction.”

Our experience with rat plague, though limited, leads us to the same conclusion as that arrived at by the Indian Commission in regard to the value of the gross lesions of plague in making the diagnosis. To one who is acquainted with them, these lesions are as characteristic as those of any infectious disease in man. It is quite true that occasionally atypical cases are encountered where the majority of the gross lesions are wanting, and in such cases it becomes necessary to resort to the inoculation of animals or to cultural investigations in order to make a diagnosis. Such cases are, however, if anything, rarer than are atypical post-mortem findings in pneumonia or in typhoid fever in man.

MODE OF EXAMINATION.

A brief description of the actual manner of examining rats for plague infection will be given here.

The rats are immersed in any convenient solution for the purpose of killing fleas and other ectoparasites that might be capable of carrying infection from a plague-infected rat.

The following plan of handling rats has been found satisfactory in the federal laboratory at San Francisco. The rats are nailed to a shingle by an attendant. Another attendant reads off the address on the tag attached to the rat, puts a check number on the shingle, and records the address from which the rat was taken and the check number on the card shown on page 48. This card is arranged so as to give the data as to the address from which the rat came, its size, sex, and species. After being checked the rats are dissected and finally, after examination by the medical officer, they are removed from the shingle; any plague-infected rats are burned as soon as the necessary investigation has been made. The dissection is made by reflecting the skin from the whole front of the body and neck so as to expose the cervical, axillary, and inguinal regions. The thoracic and abdominal cavities are then opened with scissors.

In the inspection, careful search for buboes must be made in the regions of the various peripheral lymph glands. The abdominal and thoracic organs must be subjected to a careful scrutiny. It is needless to say that this work should be done in a rat-proof, well-lighted building that is provided with water, gas, and sewer connections. The utmost care should be taken to avoid any undue risk of infection. The wearing of rubber gloves is not necessary. Everyone who has to handle infected animals must be sufficiently alive to the danger of infection.

In the extensive work conducted by the Indian Plague Commission[3], attendants were protected with Haffkine’s prophylactic. This is undoubtedly a wise precaution and should be taken if possible.

For a worktable on which to dissect the rats we use in San Francisco a table which slopes gently from the sides and ends toward the center, where a drain pipe is attached which leads to a vessel containing a disinfectant. The table is covered with sheet lead.

The layman of average intelligence readily learns to recognize the gross lesions of rat plague and it is wise to train the laboratory attendants to do this. Every rat should, however, be subjected to a careful scrutiny by the physician responsible for the work. The great majority of rats may be put aside after a cursory examination as entirely beyond suspicion of infection. Probably 8 or 10 per cent of them will require a very careful examination for the gross lesions of plague. A card which we have found very useful for keeping records of suspected and infected animals is shown on page 34. Probably all of the species of the genus _Mus_ are susceptible to plague infection. I shall, however, confine myself to a consideration of plague in the rats found the world over (_M. norvegicus_, _M. rattus_, _M. alexandrinus_).

In Bombay[18] it has been found that the epizootic among _Mus norvegicus_ appears first and is probably responsible for the diffusion of plague among _Mus rattus_. It precedes the infection among _Mus rattus_ by about ten days, and the opinion is expressed by the Indian Plague Commission that the usual course of the infection is from the _Mus norvegicus_ to the _Mus rattus_, and as the latter rodent is a house dweller in India it is the most frequent source of human infection.

In San Francisco the _Mus rattus_ population is comparatively small, contributing perhaps 2 per cent of the total rat population of the city; but in the section of the city where the large warehouses are found, especially those where oriental goods are stored, about 15 per cent of the rats taken are _Mus rattus_. So far as concerns plague infection about 5 per cent of the rat cases were in _Mus rattus_. It may be of interest to note that the last infection found among rats in San Francisco was among the _Mus rattus_ in a large warehouse near the water front. Two plague-infected rats were found in this building, one October 21, 1908, and the other October 23, 1908. A large number of mummified carcasses, all _Mus rattus_, were found in the building, and it seems not unlikely that a somewhat extensive epizootic had occurred among them. No previous case of rat plague had been found in the city for eighty-five days, though about 25,000 rats had been examined during that period, and none have been found in the six months since, although over 30,000 rats have been examined. Our records show that of 84 infected rats, 79 were _Mus norvegicus_, and the remainder were _Mus rattus_. Some of the latter may have been _Mus alexandrinus_, as the two species (_Mus rattus_ and _Mus alexandrinus_) were not clearly differentiated in the earlier examinations.

THE GROSS LESIONS OF NATURAL RAT PLAGUE—ACUTE PLAGUE.

SUBCUTANEOUS INJECTION.

This is the sign which usually first attracts attention. White[4], in discussing plague in rats, states that “the most noticeable post-mortem appearance of the plague rat is the engorgement of the subcutaneous blood vessels, together with a diffuse pink color of the subcutaneous muscles, which have a peculiar dry, waxy translucency.” It has been our experience frequently to have an attendant who is dissecting rats remark that he had found an infected rat after the first incision was made in reflecting the skin. The injection is dark red, and upon close inspection one sees that the small vessels are uniformly distended with blood. It is usually distributed over the whole surface of the body, but on two occasions we have seen it confined to the side of the body on which the primary bubo was found. A bright pink injection is a rather common finding among rats in San Francisco. It is not likely to be mistaken for the injection of plague infection. Subcutaneous œdema, confined to the vicinity of the bubo, is occasionally encountered.

In our experience in San Francisco an injection identical in appearance with that found in plague infection was found only twice, and in each case there was associated with it a small discharging subcutaneous abscess. There were no other lesions in either case and the pus from these abscesses failed to produce plague in guinea pigs.

In a series of 61 consecutive plague rats in San Francisco, injection was present fifty-two times, it was confined to the region of the bubo twice, it was unilateral twice, and was general in distribution forty-eight times. It was slight thirteen times, moderate fifteen times, marked sixteen times, intense eight times.

THE BUBO.

This is the most reliable single sign of plague infection, and when present in typical form is enough on which to base a diagnosis which rarely proves erroneous.

The gland involved is usually surrounded by a more marked injection than is present elsewhere, and an infiltration which at times is hemorrhagic. This surrounding hemorrhage which was common in the plague rats described by the Indian Plague Commission was met with very rarely in San Francisco. The gland proper is usually caseous. The contents may be shelled out very readily, though prior to section the gland feels very firm. In the cases seen at the federal laboratory in San Francisco, the contents of the buboes were recorded as being hemorrhagic four times and as caseous twenty-nine times. Pest-like bacilli were noted as present in 18 cases, in 6 of which the “coccoid” form predominated. They were recorded as absent five times.

Indolent enlargement of the lymph glands is very commonly encountered in rats that are not infected with plague. Among old rats probably 15 per cent will show this. Such glands, however, are tough, elastic, and not surrounded by infiltration. They are not likely to be mistaken for the plague buboes. In the leprosy-like disease of rats, the glands may reach an enormous size.

Observers differ as to the location of the primary bubo. Skschivan[1] states definitely the location of five primary buboes in plague rats seen in Odessa in 1901. Two were in the axilla, two in the inguinal region, and one in the neck. Kitasato[5] says: “To judge from the experience of the past it can be suggested that in examining rats particular attention should be paid to their submaxillary and cervical glands and to the spleen. These organs in most cases show the evidence of infection, if there be any.” From this it would appear that he regarded the neck glands as the most frequent seat of the bubo. It may be remarked here that his experience was derived from plague rats seen in Asia.

We find a marked difference between the experience in San Francisco and that in Bombay. This is demonstrated in the following table, which shows the location in percentage of single buboes in each situation:

───────────────────────────────────────┬───────┬───────┬───────┬───────
│ Neck.│Axilla.│ Groin.│Pelvis.
───────────────────────────────────────┼───────┼───────┼───────┼───────
│ _Per│ _Per│ _Per│ _Per
│ cent._│ cent._│ cent._│ cent._
Indian Plague Commission, Bombay—2,923 │ 75│ 15│ 6│ 4
rats[3] │ │ │ │
Wherry, Walker, and Howell, San │ 12│ 12│ 75│
Francisco[6]—8 rats │ │ │ │
Federal laboratory, San Francisco—32 │ │ 22│ 72│ 6
rats │ │ │ │
───────────────────────────────────────┴───────┴───────┴───────┴───────

The American figures are too small to be of much significance, but one is struck with the fact that in Bombay three-fourths of the buboes are in the neck, while in San Francisco three-fourths of all found are in the inguinal region. We have records of only three multiple buboes found in rats in San Francisco, and in no case was either of the buboes in the neck; while in Bombay, to quote from the report[3], “Of the rats with multiple buboes 54.5 per cent had a bubo in the neck.” Striking as these figures are, we have collected further evidence that the inguinal region is the commonest location of the bubo in plague rats in this vicinity.

Passed Asst. Surg. J. D. Long, Public Health and Marine-Hospital Service, who has had an extensive experience with rat plague in Oakland, Cal., tells me that the majority of the buboes were found in the groin, very few in the neck. Acting Assistant Surgeon Wherry, Public Health and Marine-Hospital Service, informs me that in a series of plague rats examined after the report made in association with Walker and Howell[6], the cervical bubo was very rarely encountered.

Particular care was taken to look for cervical buboes, as it seemed rather inconsistent to find the other lesions so fully in accord with those found in India, yet to have the location of the bubo to differ so radically. We have not encountered a mesenteric bubo in our work in San Francisco. The Indian Plague Commission found none in over 5,000 naturally infected plague rats. As mesenteric buboes are very commonly encountered in plague infection brought about by feeding, they conclude that the absence of these buboes in naturally infected rats is strong evidence that the infection does not enter by the alimentary canal.

THE GRANULAR LIVER.

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.

The most striking difference between natural and artificial plague in rats is the presence of a reaction at the site of inoculation in the majority of cases where the organism is introduced subcutaneously, and in about a third of the cases where the infectious material is rubbed on the shaven skin (cutaneous inoculation). The local reaction may exist only as a yellowish-brown crust, overlying a granulating surface, and associated with a trifling thickening of the skin and subcutaneous tissue. It may appear as one or more firm papules 3 or 4 millimeters in diameter. The most frequent appearance is a brawny œdematous and blood-stained reaction which extends over an area perhaps an inch in diameter; at times purulent change may be well advanced. Very rarely one finds so extensive an œdema as to cause the lesion to somewhat resemble the widespread gelatinous reaction seen so commonly in the guinea pig. On one or two occasions we have seen an extensive slough at the site of inoculation.

BUBO.

It is very exceptional that one finds in cases of induced plague the typical, firm, caseous bubo surrounded by an infiltrated area, as is so commonly seen in natural infection in rats. The glands are sometimes enlarged and injected without other changes. The commonest lesion, however, is a markedly enlarged gland which upon close inspection is seen to have a number of yellowish points just under the capsule. These points are especially well seen when a section is made through the gland. The gland may be squeezed out of the capsule and it breaks down readily enough when pressure is made upon it; but the uniform necrotic process that one sees so often in natural rat plague is absent.

LIVER.

Granular lesions precisely like those found in natural infections are very common. If the rat has died on the sixth day or later, the ordinary lesions are apt to be replaced by necrotic foci that may be as much as 2 millimeters in diameter.

SPLEEN.

This organ is found mottled more frequently than in natural plague infection, and large granules are much more common.

The subcutaneous injection is rarely so well marked as it is in natural infections.

Pleural effusion of the same nature as that found in natural plague is common. Hemorrhagic foci are not rare in the lungs, and occasionally the organs are partly consolidated.

CHRONIC PLAGUE DUE TO ARTIFICIAL INOCULATION.

Occasionally a rat that has been inoculated but has survived a week or longer, will show, when killed, only an abscess at the site of the injection. Stained smear preparations may show a large variety of bacterial forms. We have not been able to demonstrate the presence of _B. pestis_ in these lesions, yet there is no doubt but that the lesion is the result of the inoculation.

A lesion more frequently found is a caseous or a purulent lymphatic gland. If the inoculated rat has been killed about ten days after the inoculation, in some cases one or more of the peripheral lymph glands will be found to be surrounded by an infiltration, and the gland itself will be purulent or less frequently caseous. Such lesions are occasionally met with in rats in which there is no suspicion of plague infection; but they are seen so frequently among rats that have survived artificial inoculation with _B. pestis_, there is no doubt but that in these cases they are the result of the inoculation. In several such cases pest-like organisms have been demonstrated in smears, and acute plague has been produced in guinea pigs by inoculation with the pus found in these lesions. Not infrequently in these cases the spleen will be found enlarged and looking very much like the organ in acute plague, but cultures from this organ in such cases have in my experience remained sterile.

In other cases the only lesions will be found in the spleen. The organ is enlarged and contains a number of caseous nodules. These nodules vary in number from four or five to thirty or forty and in size from the head of a pin to a lesion 0.3 centimeter in diameter. In a number of such cases the nature of the lesion has been demonstrated by animal inoculation. For example, in a series of experiments carried out to determine the susceptibility of San Francisco rats to plague infection a large _Mus rattus_ died on the eleventh day after inoculation. The post-mortem examination showed nothing except an enlarged spleen which contained about a dozen caseous nodules, the largest of which was not over 2 millimeters in diameter. The nodules were very firm and the capsule smooth, so that they were held with difficulty with dressing forceps. Cultures from the liver and the spleen remained sterile, but a piece of the spleen was placed beneath the skin of a guinea pig. This animal died of acute plague, and a pure culture of _B. pestis_ was isolated from its liver. In some of these cases the liver will show large, distinct, whitish caseous foci. In another case a small _Mus norvegicus_ was killed on the twelfth day after a cutaneous inoculation from an artificially infected squirrel. No lesion was found except in the spleen which was not materially enlarged, but which presented two small whitish caseous granules on the surface, neither being over 1 millimeter in diameter. A piece of the spleen containing one of these granules was put under the skin of the belly of a guinea pig. The guinea pig died on the fourth day with the usual lesions of acute plague. Occasionally in these cases of chronic plague punctate hemorrhages or even areas of consolidation are found in the lungs.

THE HISTOLOGY OF RAT PLAGUE.

The most recent and satisfactory work on this subject is that of Ledingham [14], who has studied the lesions of both natural and induced plague in rats. The following is a very brief abstract of his work. The reader is referred to the original for a full study of the subject.

NATURAL RAT PLAGUE.

Two groups of cases are distinguished, first, those in which a large number of _B. pestis_ are found in the liver and in the spleen. In the spleen this is accompanied by hemorrhages and congestion of the pulp sinuses and in the liver with congestion of the capillaries. These are early cases.

In the second group, or the later cases, there are extensive reaction changes in the tissues. In the spleen this leads at times to distinct abscess formation, but more frequently to a walling off of the foci of necrosis. In the liver more or less focal necrosis is found; sometimes the areas of “necrosis” may be so extensive that little healthy liver tissue remains. Bacilli are usually to be demonstrated in these areas of necrosis. Giant cells of the Langhans type may be found in the neighborhood of these foci.

The granular appearance of the liver is attributed to “hemorrhages and the focal necroses, together with the fatty changes in the liver cells. It must be understood, however, that a peculiar honeycomb-like vacuolar degeneration of the liver cell protoplasm was far more frequent than any actual, coarse, fatty infiltration. The granular appearance of the spleen is due partly to endothelial catarrh and partly to subcapsular changes.”

In experimental rat plague Ledingham found the lesions to resemble those of the first group of cases referred to above. There is usually marked bacteraemia; focal necroses of the liver are scanty.

In a chronic case, minute abscesses were found scattered through the spleen. In the center of the abscesses were found clumps of degenerated bacilli. The areas were walled off by epithelioid and spindle cells and numerous giant cells of the tubercular type.

IMMUNITY OF RATS.

Contrary to the general impression the wild rat is not an animal especially susceptible to plague infection. The Indian Plague Commission [19] found that when rats are inoculated by the cutaneous method from the spleen of infected rats 59 per cent are immune to infection. A series of experiments conducted in the federal laboratory in San Francisco showed that when inoculated with highly virulent cultures of _B. pestis_ there is an immunity which is, however, more frequent among the large rats. When inoculated cutaneously with tissue containing large numbers of _B. pestis_ from plague infected human beings, rats, or squirrels, about 15 per cent of small rats and about 50 per cent of large ones were found to be immune. There is no good reason for believing that this immunity of San Francisco rats was due to a previous attack of the disease. Indeed, it was known beyond a doubt that some of the immune rats had never had an opportunity of becoming infected with plague in nature and thereby establishing an acquired immunity. We may mention here the fact that has been observed by many workers, and which we have amply confirmed, that rats are readily immunized by antiplague serum.

The subject of the transfer of infection directly from rat to rat by cutaneous or subcutaneous inoculation through a series of the animals is one that is evidently intimately associated with the preceding subject, as it is quite evident that an immune rat or several of them might terminate a series without any actual diminution in the virulence of the organism transferred. It is quite plain that the success of such an experiment would depend largely upon the number of rats used in each transfer. The Indian Plague Commission [19] had no difficulty in carrying infection through twenty-six transfers, using from six to fifty rats in each transfer.

Pound [7] in a series of eight experiments, was never able to convey the infection successfully beyond the sixth rat, using but one rat for each transfer. There was no apparent lessening of the virulence of the organism and each series appears to have been terminated abruptly by encountering an immune rat.

Baxter-Tyrie [15] says:

It is probable that under certain natural circumstances a reduction in
the virulence of the organism is effected and a comparative immunity
is conferred on the rats. The infection of immigrant rats is, however,
severe, and their arrival is heralded by a heavy mortality. In the
same manner an infected rat imported into a fresh locality produces a
similar result. This attenuation of virulence is responsible for the
condition known as chronic rat plague.

Several experiments conducted in San Francisco to determine this point have given results that I regard as showing merely the presence of a considerable percentage of immunity among the rats. It was observed that in each case certain of the rats died of acute plague even in the last transfer. It was very evident that had certain combinations of immune rats been encountered the experiment might have terminated at any point. On the other hand, by being especially fortunate in using nonimmune rats, the experiments might have given a much higher percentage of cases of acute plague. Unfortunately it was necessary to terminate these experiments in each instance before they could be regarded as completed.

The reason for the natural subsidence of plague among rats in any community is a point about which much more evidence must be obtained before we can speak with any degree of certainty. It may be due to the lack of susceptible material, possibly to a loss of virulence of the organism; but it seems more probable that it is due to a change in the number or relations of the ectoparasites of the rat.

Adequate measures of rat extermination, while they may never bring about the ideal condition of a community that is free from rats, are, as is shown by the recent experience in San Francisco, of the utmost value in shortening the epizootic.

REFERENCES.

Endnote 1:

Skschivan (Centralblatt für Bacter., etc., 1903, Vol. XXXIII, No. 4,
p. 260).

Endnote 2:

Kister & Schumacher (Zeit. für Hyg. u. inf. Krank., Vol. LI, 1905).

Endnote 3:

Indian Plague Commission (Journal of Hygiene, 1907, Vol. VII, No. 3).

Endnote 4:

White (Medical Record, vol. 67, No. 4, Jan. 28, 1905).

Endnote 5:

Kitasato (Philippine Journal of Science, Vol. I, No. 5, 1906).

Endnote 6:

Wherry, Walker & Howell (Journal Am. Med. Assn., April 11, 1908, Vol.
L, No. 15).

Endnote 7:

Pound (1907, Report on Plague in Queensland, B. B. Ham, p. 134).

Endnote 8:

Dunbar & Kister (1904, Centralblatt für Bact., etc., Vol. XXXVI, No.
1, p. 127).

Endnote 9:

Kolle & Martini (Deut. Med. Woch., Jan. 2, 1902, Vol. XXVIII).

Endnote 10:

Kister (Centralblatt für Bact., etc., July 24, 1906, Vol. XLI, No. 7).

Endnote 11:

Neumans (1903, Zeit. f. Hyg. u. inf. Krank., Vol. XLV, No. 3, p. 451).

Endnote 12:

Kister & Schmidt (1904, Centralblatt für Bact., etc., Vol. XXXVI, No.
3, p. 454).

Endnote 13:

Aujeszky (1904, Centralblatt für Bact., etc., Vol. XXXVI, No. 5, p.
603).

Endnote 14:

Ledingham (Journal of Hygiene, 1907, Vol. VII, No. 3).

Endnote 15:

Baxter-Tyrie (Journ. of Hygiene, Vol. V, 1905, p. 315).

Endnote 16:

Wherry (The Journal of Infect. Diseases, Dec. 18, 1908, Vol. V, No.
5).

Endnote 17:

Klein (The Bacteriology and Etiology of Oriental Plague, MacMillan and
Co., London, 1906).

Endnote 18: