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The result of inoculation experiments was to show as a general rule that when the plague bacillus was cultivated for successive generations in artificial media it rapidly lost its virulence.
When rats were found dead in houses in which cases of plague had occurred, plague bacilli were often to be found in their lymphatic glands and other organs. These animals sometimes had well-developed buboes, most commonly in the inguinal region.
Mice, rats, guinea-pigs, and rabbits confined in the same cages with infected animals usually died of the disease.
IV.—DIAGNOSIS OF BUBONIC PLAGUE.
The diagnosis of the plague is based upon the clinical characteristics and upon the morbid appearances above described, and upon the demonstration of the presence of the plague bacillus.
In the dead body, if the processes of decomposition are not too far advanced, the existence of the plague can be proved with certainty by demonstrating the presence of the bacillus in the spleen, the buboes, or in other affected organs, either by microscopical examination, or in twenty-four to forty-eight hours by culture experiments. I have never seen a case of plague without glandular swellings.
A monkey, that chewed and sucked a piece of sugar-cane infected by a pure culture of the bacillus, died in five days of the disease. The post-mortem examination showed very slight swelling of the inguinal glands, great congestion of the intestine, and swelling of the mesenteric glands and of the spleen.
In the countries in which the plague is generally met with, if bacteriological methods of diagnosis are not available, the disease is liable to confusion with pernicious malarial fevers, with malignant typhus of rapid course, with splenic fever, and with other varieties of adenitis. The cases in which mistakes may most readily arise are, on the one hand, those in which, with severe general symptoms, no external buboes make their appearance; and on the other hand those in which, with mild general symptoms, buboes appear the true nature of which is not suspected. But in well-marked cases of plague, with pronounced general symptoms and well-developed buboes, there can, even in the absence of bacteriological investigation, be little difficulty in diagnosis.
A pig ate the spleen of a man that had died of plague; the animal lost flesh rapidly, suffered from diarrhea, and died of plague in twenty-two days. The post-mortem examination showed hæmorrhages in the abdominal walls; the inguinal glands were swollen to the size of a hazel-nut and bluish-red in colour, the sub-maxillary glands were swollen to the size of a hazel-nut, the mesenteric glands were swollen to the size of a bean or a hazel-nut and of a bluish-red colour; there were hæmorrhages in the mesentery, with much congestion and swelling of the stomach and intestinal walls, hæmorrhages in the mucous membrane, and swelling of the intestinal follicles; the spleen was enlarged, the kidneys were swollen and congested, and the lungs engorged with blood. The plague bacillus was found in the interior of the organs, in the glands, and in the blood. Another pig, inoculated subcutaneously on the abdomen with a small fragment from a bubo, died of the plague in forty days. The appearances on post-mortem examination were substantially the same as those just described. The site of inoculation showed moderate congestion and swelling.
Two cats that ate portions of a bubo were ill for seven days and became very thin, but recovered. Fowls that swallowed fragments of organs, and matter infected with pure cultures, died as a rule after three or four days. Plague bacilli were found in the blood and organs.
Pigeons were immune to subcutaneous inoculation.
Mice, guinea-pigs, and rabbits, when inoculated with cultures attenuated either by cultivation through several generations or by heat, were gradually rendered immune against inoculation with virulent cultures.
By successive inoculations from animal to animal with fragments of the organs or with blood, the virulence of the bacillus could be increased to a definite degree varying with the species of animal. By microscopical and bacteriological researches bubonic plague is shown to be a disease in which the blood, the organs, the saliva, the urine, and the fæces of those that are affected with or have succumbed to the disease contain a specific bacillus, pure cultures of which, inoculated into various animals, produce in these animals the same disease.
As already stated, plague can usually, in severe cases at any rate, be recognized by the examination of the blood. For this purpose, one of the finger-tips or a portion of the skin of the body must be carefully cleansed with soap and water, 1% sublimate solution, alcohol and ether, and pricked with a needle sterilised by heat. For direct microscopical examination the blood should be received on a cover glass; for cultivation, on a tube of agar or of alkaline gelatin-peptone solution. In stained preparations of blood, the bacilli are usually found in very small numbers only, two or three in a field or even in the whole preparation. In the agar tubes or in the peptone solution, in 24 to 48 hours, the bacilli grow in the manner already described. As before said, staphylococcus pyogenes aureus is frequently found in addition to the plague bacillus. If the bacillus cannot be detected at the first attempt, the blood must be examined day after day, and in this manner the organism will often eventually be discovered.
If the examination of the blood gives negative results, the urine, which nearly always contains albumen and plague bacilli, should then be examined. Cleanse the neighbourhood of the urinary meatus with sublimate solution, and while the patient is urinating suck up some of the urine with a sterilised pipette. The urine should then be examined in hanging-drops and in stained preparations; and with a few drops (about five) plate cultivations should be made. In hanging-drops the bacilli can usually be seen at once, sometimes in chains of three or four links. Stained preparations generally give the same results. On the agar-plates, after twenty-four hours, colonies of plague bacilli make their appearance; these are small, white or greyish-white, with a bluish sheen by reflected light, and with iridescent borders. In addition to the plague bacilli, pus-cocci are sometimes found; these are readily distinguished by their greater luxuriance of growth and by other peculiarities. For confirmation, cultures from the colonies should always be made on agar (coagulated with the tubes oblique), on bouillon, and on gelatine; inoculation of mice, rats, and guinea-pigs should also be undertaken. If no urine is passed, an attempt may be made, in case of need, to draw some off by the catheter.
The blood drawn by puncture from the buboes and from the other glandular swellings always shows the bacillus on microscopical examination and on cultivation.
By these methods the diagnosis of plague can almost always be made with certainty, alike in severe cases with or without buboes, and in milder cases.
The presence of the bacillus in the vomit, the fæces, and the saliva, can often be detected in the following manner.
First make plate cultures, and from these make cultures on alkaline gelatin-peptone solution. In the latter, the bacillus grows along the bottom and the sides of the tubes. After twenty-four hours, these cultures should be examined microscopically, and fresh cultures should be made.