ABSTRACTS A~D REPORT.

the remainder being I '9. Of these animals 3 were purchased. In 19 herds comprising 492 alllmais there were no reactors, as was also the case in 1908•

Dairy 2.-15 herds comprising 419 animals. 33 reactors and 3 doubtful. I n I herd there were 16 reactors out of 24 animals. In 8 herds containing 218 animals there were none.

Dairy 3.-Animals tested 957. Reactors 66, doubtful reactions 18. I

herd of 45 animals contained 16 reactors . . Dairy 4.-Animals tested 1233. Reactors 61, doubtful reactions 19. 2

herds comprising 39 and 67 animals contained respectively 12 and 10 reactors.

Of 481 animals belonging to private owners 35 reacted. In 1 herd 25 reacted out of 50. Apart from this herd the percentage of reactors was only 2 '4. The owners were requested to dispose of all reacting animals in order to eradicate the disease, provided they were not of great value for breed­ing purposes. The test is repeated on the reacting animals after the lapse of three months. A second clinical examination of badly infected herds is made in order that animals affected with open tuberculosis may be immediately disposed of.

The following is a summary of the results, excluding the figures for West Prussia, which are not comparable with the others. Total number of animals examined 183,102; tuberculosis oftbe lungs, 1"285 per cem.; udder, '212 per cent.; uteru<, '082 per cent.; other forms, '005 per cent. (Nevermann, Bed. Tier. f¥ochens., NO.3, 18th January 19 12, pp. 54-57·)

JOHNE'S DISEASE: CULTIVATION OF THE BACILLUS.

SOME of the experiments made by the author were carried out with a culture received from Twort, and some with material derived from natural cases of the disease. .

Tbe first case was one which had been obtained from an infected herd, and which had been at the laboratory for some time. The animal was clinically affected, and a reaction had been obtained with avian tuberculin. At the post-mortem no tuberculous lesions could be discovered, and the charactenstic lesions of J ohne's disease in the intestine were not very pronounced. Acid-fast bacilli were not especially numerous in either the glands or tbe intestine.

Seed material was sown out on the following media :-1. Blood serum (borse) with 4 per cent. glycerin added. 2. Blood serum with the addition of about i the volume of peptone

liver broth and 4 per cent. glycerin. 3. Egg (yolk and white) with 4 per cent. glycerin. 4. Blood serum with 4 per cent. glycerin and 2 per cent. dead tubercle

bacilli. The latter were obtained from a two-months-old broth culture, and before being added to the medium were mixed with a small quantity of salt solution, and heated to 1000 C. for one and a half hours. The bacilli were of the human type.

5. Blood serum with i volume of hver broth, 2 per cent. dead tubercle bacilli, and 4 per cent. glycerin.

These media were all solidified in the slanting position by heating to 80 0 to 85° C. in a water bath.

6. Serum agar with 2 per cent. dead tubercle bacilli, and about I per cent. glycerin.

ABSTRACTS AND REPORT.

7· Medium prepared according to Twort's directions. The tubes were sealed with paraffin and kept at 37 0 C.

149

No growth occurred on media 1 to 3 in six weeks, while on the others multiplication of the bacilli took place.

The most luxuriant growth was obtained on NO.5. At six weeks the whole surface of the medium .was covered with colonies that were visible to the naked eye, and which later attained a size varying from t to 1 mm.

By reflected light the colonies were greyish-white in colour, and by transmitted light yellowish-brown. They were circular in outline, and under a low magnification the surface was observed to present a slightly wrinkled appearance. The colonies were tough. In older cultures there was observed the formation of a thin delicate membrane of wrinkled growth extending round the colonies. Growth on the other media presented similar appearances, but developed more slowly. Subcultures on the same medium yielded quite abundant growths in about four weeks.

Since the presence of such large quantities of tuberc1e bacilli in the medium rendered further investigations regarding the conditions of growth more difficult, an attempt was made to extract from the tubercle bacilli the substance which rendered the cultivation of the bacllJus possible.

Glycerin was found to be of great value for this purpose, and the extract was prepaJ ed in the following way :-

The bacilli were derived from a two-months-old broth culture. The liquid (about 200 cc.) was decanted and the growth mixed with about 40 cc. of glycerin. After thorough shaking the mixture was heated to 100

0

C. for about an hour, and subsequently allowed to stand at room temperature for several days. The pale yellowish-brown liquid so obtained contained only a few tubercle bacilli. The addition of 5 per cent. of this liquid to one of the above-mentioned media was found to completely replace the tu.bercle bacilli.

Cultures were obtained on slanting serum agar and upon blood serum containing i- the volume of peptone liver broth, to both of which media 5 per cent. glycerin extract had been added, from the mesenteric gland of a cow which had reacted to avian tuberculin, and which on slaughter showed no tuberculous lesions.

The bacilli obtained a~reed in their morphology and staining reactions with those cultivated by Tworl.

Subcultures upon slanting serum agar containing 5 per cent. glycerin extract showed luxuriant greyish-white growths composed of numerous colonies. In stab cultures growth was slower and took the form of a greyish-white growth, which gradually extended along the needle track from above downwards.

Upon the surface of agar containing the extract there was developed either an irregular greyish-yellow mass of colonies, or discrete colonies, each of which consisted of a yellowish-brown centre surrounded by a slightly wrinkled, brittle, greyish-yellow pellicle. The diameter of these colonies after about three months in the incubator was 2 to 4 mm.

In stab cultures a thick irregular zone of growth was observed in the uppermost parts of the needle track.

In liver broth to which blood serum and glycerin extract had been added there was observed after an interval of about four weeks a slight amount of sediment at the bottom of the tubes. This gradually increased in amount until there was a considerable amount of deposit composed of clumps of bacilli of various sizes. In some cases there was a greyish-white pellicle of growth along the side of the tube. The liquid remained limpid. A growth was obtained on the surface of the broth already described with a. 5train which had been grown for several generations on glycerin-extract-

ABSTRACTS AND REPORT.

agar. The growth took the form of a thick greyish-white wrinkled film, which spread very slowly.

No growth was obtained upon suitable media put up under anrerobic conditions.

Subsequently attempts were made to get the bacillus to grow on media which did not contain any glycerin extract. In some cases solidification of the media was effected by using a 3 per cent. watery solution of agar. This was mixed with an equal quantity of serum, together with 1 per cent. of one of the following substances in weak acid solution-somatose, Heyden's medium, Witte's peptone, Liebig's meat peptone, and nutrose. In other experiments ! per cent. dextrose and 3 per cent. glycerin were added. Broth (glycerinised) was also prepared from a tuberculous udder, and tuberculous liver obtained from cattle. Finally, attempts were" made to obtain cultures upon serum·agar, ordinary agar, broth, liver broth, etc. N one of these appeared to be suitable, save that there was possibly a slight amount of growth on the media containing somatose and Heyden's medium.

Inoculation experiments into guinea-pigs failed to set up the disease, and produced at the most a small encapsuled abscess at the seat of inoculation. The yellowish-brown crumbling contents of such abscesses contained a few granular acid-fast bacilli.

Experiments carried out with rabbits had similar results. Up to the present the experiments made upon calves have had the

following results. All the calves used were tested with tuberculin and passed. The

inoculations were made subcutaneously and intravenously, and caused no reaction. Three months later the animals were tested with ordinary tuberculin and did not react, but with avian tuberculin, three weeks later, typical reactions were obtained.

Subsequently tuberculin prepared from cultures described was used, and in the case of one calf gave rise to a typical tuberculin reaction. In the others no rise of temperature was observed.

Finally, experiments were carried out with guinea-pigs with the object of ascertaining whether J ohne's bacillus is capable of conferring any immunity against tuberculosis. The animals received two comparatively large doses of culture, and a month later a dose of bacilli of the bovine type. Control animals were inoculated with the bovine bacilli alone. All the animals were weighed twice weekly, and it was found that the control animals commenced to lose weight sooner than the others. The author concludes that a slight degree of immunity was established. (HaIth, Zeitschr. f. Infekt. tarasi/o Krankh. u. Hyg. d. Haust., Vol. XL, NO.5, 14th May 1912, pp. 378-387.)

. A CAMPAIGN AGAINST THE HYPODERMA BOVIS.

THE losses caused by this parasite fall under three headings :-1. Loss due to perforation of the hide. 2. Decrease in the production

of milk. 3. General loss of condition. 1. Loss due to perforation of the hide. This has been variously estimated

in the countries where the fiy occurs. In I889 it was computed that the annual loss sustained by the United States amounted to 35 to 60 million dollars. In Great Britain it has been variously estimated at from 40 to 200 millions, the latter figure appearing to be greatly exaggerated. In Germany the parasite has a very unequal distribution, and the annua]