54 ABSTRACTS AND REPORT.

laboratory, and the reaction must be complete within twenty minutes. The serum must be capable in quantities of . I ce. of giving a precipitate with 2 cc. of dilutions of the suspected material in the proportions of 1-1000, 1-10,000, and 1-20,000, the reaction occurring instantaneously in the first case, within three minutes in the second, and within five minutes in the third. The material under examination must be greatly diluted, approximately 1-1000 of the proteid. Such a dilution is reached when a sample of the liquid gives a slight turbidity when boiled or when a drop of nitric acid is added. This procedure excludes the possibility of getting the so-called" heterologous turbidity," which is occasionally seen with strong concentrations of heterologous blood.

If only minute traces of blood are available for examination, the capillary method advised by Hauser may be employed. A very small quantity of the dilute material is drawn into a fine capillary tube, the lower end of which is then dipped into the specific serum. The serum runs up the tube by capillary attraction, and a positive reaction is indicated by the appearance of a distinct white or grey ring at the junction of the liquids.

By means of the test applied in this way it was found by the author that the anopheles mosquito frequently sucks blood from cattle, and he suggests that the method might be applied to the examination of the blood found in the stomach of the glossina palpalis, the transmitting fly of sleeping sickness, with the idea of discovering from what animal it sucks blood.

The serum for the test must be absolutely clear, and the rabbit appears to be the best animal for the production of the specific sera. The potency of a serum depends upon the individuality of the animal producing it, and not upon the method of production. Only one or two rabbits out of ten treated may yield good sera.

The serum is filtered through a Berkefeld filter, and, as a rule, has good keeping qualities, remaining serviceable for months or even years. No preservative must be added, and in carrying out a test controls must be made.

Reactions may be obtained with dried blood, and also with blood that is in a condition of putridity, provided that the putridity is not so far advanced as to have caused the decomposition of all the proteid. The author was able to get positive reactions with blood that had been kept in tubes for two years and was in a stinking condition. He was also able to get positive results with dried blood-stains that were sixty-six years old, but with material obtained from a mummy 300, years old the result was negative.

Brief details are given by the author of nine cases in which the biological method was applied to the investigation of blood-stains upon the clothes, etc., of criminals, with positive results, these being in some cases confirmed by confession of the person accused of the crime. (Uhlenhuth, Journal of the Royal Institute of Public Health, Vol. XIX., No.2, November 191 I,

pp. 641 - 662.)

AN UNDESCRIBED PATHOGENIC BACTERIUM IN MILK.

THE lesions caused by this bacterium were first met with some years ago in guinea-pigs that were used for experimental inoculation with the object of ascertaining whether tubercle bacilli were present in samples of milk. The lesions occurred in animals that were kept alive somewhat longer than the

ABSTRACTS AND REPORT. 55

usual period, and, although they might be easily mistaken for tuberculous lesions, no acid-fast bacilli could be detected by the most careful search.

It was found that the disease was transmissible from guinea-pig to guinea­pig by subcutaneous inoculation, but all efforts to cultivate the organism c.ausing it were unsuccessful, as were attempts to find it with the micro­scope.

Interest in the disease was revived owing to the discovery that the milk from a tuberculous cow at the Experimental Station at Bethesda caused the disease in guinea-pigs. Milk was collected from this cow on a number of occasions with all possible precautions, and it was found to be fully as virulent as milk taken with less strict precautions.

All attempts to cultivate the causal organism on or~inary media failed, but the severity of the disease affecting the liver suggested the possibility that the addition of bile to the media might solve the difficulty.

Agar containing 6 per cent. glycerin and varying proportions of ox gall was prepared, and it was found that the organism would grow on this medium. growth being more vigorous on agar containing 5 per cent. or more of bile.

On the surface of this medium the organism forms small, slightly convex, pale grey colonies. The water of condensation remains clear, but is covered with a very thin, almost imperceptible, layer, which has the appearance of fine white dust. A similar layer forms on the surface of some liquid media. Cover-glass preparations of this layer appear to be composed of the organism in pure culture. In stab cultures growth occurs mainly about the top of the stab. .

No growth is obtained on gelatin. On the surface of potato there is a thin flat glistening layer, very faintly tinged with pink. It is not certain whether any growth takes place in milk. Liquid from the sixth generation of milk tubes appears to be innocuous. Growth is slow on all media.

The optimum temperature appears to be 37-39° c., and a temperature of 60' maintained for fifteen minutes is fatal to the organism.

The organism is Gram-positive. Morphologically it resembles the tubercle bacillus of the bovine type. When stained with Lreffier's methylene blue the bacilli appear very minute, but with aniline gentian violet they appear larger.

The organism has repeatedly been isolated from lesions in the guinea-pig, it has been grown in pure culture, has set up the disease in other guinea-pigs, and has been recovered from the tissues of these.

So far as is known the guinea-pig is the only animal susceptible, although it has been tried on rabbits, swine, sheep, cats, dogs, chickens, and cattle. Disease in the guinea-pig is very chronic. Infection in the guinea-pig may be by inoculation or ingestion of cultures or naturally infected milk, but there are no well-marked lesions until after six weeks or more.

Leszims.-Extreme enlargement and redema of the lymphatic glands gener­ally; in the lungs small glistening nodules, which are apparently enlarged minute lymph glands not ordinarily seen; the conversion of the minute nodules into larger necrotic areas; enormous enlargement of the spleen, with irregular thickening of the capsule; enlargement and degeneration of the liver, with the formation of irregular pale yellow or dirty white areas that are apparently formed by a proliferation of the connective tissue; diffuse paren­chymatous nephritis, causing the formation of fibrous nodules in the cortex. In male guinea-pigs there is degeneration of the testicles, converting them into cavities containing creamy pus.

There is no local lesion after subcutaneous inoculation. In a small proportion of guinea·pigs there were enlargements about some

ABSTRACTS AND REPORT.

of the joints, rendering them stiff. This condition was found to be associated with the presence of a micrococcus, and the fact suggested the possibility that this was an organism that was harmless in itself, but capable of doing harm when associated with another organism. This idea was supported by the fact that the joint disease had never occurred among the guinea-pigs before, and that it made its appearance in one guinea-pig out of six, two of which were inoculated with a culture of the bacillus, two with a pure culture of the micrococcus, and two with mixed cultures. It was one of the latter that became affected. The two that received the pure culture of the micro­coccus remained perfectly healthy, and the other four all developed the characteristic lesions caused by the bacillus. It is admitted that the evidence, though suggestive, is inadequate.

The actual presence of the bacillus in the milk, udder, and supramammary glands was proved in the following way. The bacillus was proved to be pre~ent in numerous cases in milk taken from infected cows with every possible pre­caution against contamination. A cow whose milk had been proved to be infective by inoculation into guinea-pigs was killed. Immediately after death the udder was washed and disinfected and the teats ligatured, the whole udder and supramammary glands being then removed. The skin was dis­sected away, and the whole of the surface exposed was scorched with a bunsen flame. The quarters were then incised with sterile knives, and milk collected from each in sterile pipettes from places well removed from the teats. With equal precautions fragments of udder tissue were taken from one fore quarter and one hind quarter, from the supramammary gland, and from the liver and spleen. This material was used for inoculation as follows :-

One guinea-pig, milk from right fore quarter-positive. One guinea-pig, milk from right hind quarter-negative. One guinea-pig, milk from left fore quarter-positive. Two guinea-pigs, milk from hind quarter-negative. Two guinea-pigs, tissue from fore quarter-positive. Two guinea-pigs, tissue from hind quarter-negative. Two guinea-pigs, tissue from supramammary gland-positive. Two guinea-pigs, tissue from spleen-negative. Two guinea-pigs, tissue from liver-negative. . These results showed that both the milk and the tissue from the fore-

quarters were positive, and from the hind quarters negative; but the exist­ence of the bacillus in the depth of the gland was indicated by the positive result obtained with material obtained from the supramammary gland.

At the post-mortem examination there were no lesions to explain the per­sistent passage of the bacilli with the milk save the presence of a few small areas of slight induration.

A second cow that passed bacilli with the milk was also killed and exam­ined. No lesions other than those found in the first cow were encountered. Experimental animals inoculated from this case are not yet ready for exam­ination. Blood and urine from several infected cows always yielded negative results to inoculation.

It has not yet been possible to transmit the disease to healthy cows either by rubbing a pure culture on the udder and teats, by ingestion, or by sub­cutaneous inoculation.

The bacillus was found in thirty samples of market milk out of 217 exam­ined (nearly 14 per cent.). In a large herd of 140 cows that is probably free from tuberculosis the bacillus was found in the milk from nineteen animals, and in another herd at the Experimental Station at Bethesda the organism was being passed by eleven cows out of thirty-six. Of these eleven animals

ABSTRACTS AND REPORT. 57

eight were extensively tuberculous, one was affected with tuberculosis, and two apparently healthy. One of the tuberculous 'cows had tuberculosis of the udder, and in the guinea-pigs inoculated with the milk both diseases developed, and each seemed to increase the pernicious potency of the other. In stained preparations from guinea-pigs affected with both diseases tubercle bacilli could be easily found, while the other could not be seen, but it could be cultivated on media unsuitable for the tubercle bacillus.

The discovery of this organism, which appears to inhabit the udders of normal cows, and which cannot be certainly excluded from milk, is, in the opinion of the authors, a fresh fact indicating the advisability of the pasteur­isation of all milk before using it for food. (Schroeder and Cotton, American Veterinary Rez1iez£l, Vol. XL., No.2, November 19II, pp. 195-206.)

G.lERTNER'S BACILLUS IN THE CARCASES OF CALVES.

THE author's experience in connection with the occurrence of organisms of this group in meat is at variance with the scanty published references, in that within four months he has been able to isolate from the carcases of eight calves organisms which on morphological, cultural, and serological grounds undoubtedly belong to the Grertner group.

The author is unable to say whether this is due to the material at his disposal or to the methods of investigation employed, but he presumes' that his method of examining the prescapular gland and spleen is more likely to give positive results than examination of the muscular tissue. In simul­taneous examinations of the muscles and glands immediately after slaughter the Petri dishes that had been smeared with muscle frequently remained sterile, while cultures made from the glands showed numerous colonies. In none of the many cases examined during the last three years were cultures from the glands negative if even isolated colonies developed in cultures prepared from the muscles. In everyone of the cases examined in which cultures made direct from the muscle remained sterile the same organism was isolated when the muscle was incubated for twenty-four hours before plate cultures were made. The author could detect no difference between the various glands save in cases in which there was localised disease of some kind, such as slight sero·fibrinous peritonitis resulting from puncture of the rumen in cattle, in which case there were cocci in the precrural gland, the other glands being sterile. The prescapular glands were selected by the author on account of their size and their richness in lymph.

The following was the method adopted :- . A gland or piece of spleen was removed immediately after slaughter and

taken to the laboratory. Any fatty tissue was removed, and the gland, held with sterile forceps, was scorched in the flame of a bunsen burner and then divided across with scissors that had just been heated in the flame. Pressure was exerted upon the piece of tissue still held in the forceps, causing the lymph to well up on to the cut surface. Smears were made with this moist surface on Petri dishes containing agar, endofuchsin agar, and Konrad i­Drigalski agar medium.

The same procedure was adopted with the spleen. The result was visible within fourteen hours. If colonies develop, speci­

mens may be tested by staining with Gram's method, and the motility of the