With each group was placed one of the nonvaccinated control heifers, each group of three occupying an isolated stall. To test the resistance conferred by the vaccine, virulent bovine tubercle bacilli from cultures were weighed in a fresh state and inoculated into 19 5 ' of the heifers, intravenously, in 5-mg doses with the following results: Group I. Vaccinated heifers 1 and 2 and control No. 13 were inoculated 1 month after vaccination. Control No. 13, killed 60 days after inoculation, showed advanced miliary tuberculosis of the lungs. Vaccinated heifer No.2 died of verminous bronchitis 5 months and 7 days after inoculation. No lesions resembling tuberculosis were found. Guinea pigs, inoculated with portions of, the apparently normal left bronchial lymph node, contracted tuberculosis. Vaccinated heifer No.1 remained in good condition and was butchered 12 months after inoculation. No lesions of tuberculosis could be found. Portions of the left bronchial lymph node were injected into 4 guinea pigs. These were killed but no indication of tuberculosis could be found in them. Group II. Vaccinated heifers 3 and 4 and control No. 14 were inoculated 3 months after vaccination. Control No. 14 died of miliary tuberculosis 44 days after inoculation. Vaccinated heifers 3 and 4 remained in good condition. They were slaughtered 11 months after the test inoculation and no tuberculous lesions could be found. Portions of their bronchial lymph nodes were inoculated into guinea pigs. These pigs were killed 45 days after inoculation. Those that had received tissue from heifer No.4 were tuberculous, but those inoculated from No.3 were free from tuberculosis. Calmette(12) in the latest edition of his book L'Infection Baeillaire ei lao tuberculose chez l.'Homme et chez les Animau.x attributes great importance to the observation that the guinea pigs inoculated from heifers 1 and 3 remained free from tuberculosis. In previous experiments he had observed that calves vaccinated by the intravenous route and later inoculated by the same route with virulent bacilli, remained free from caseous lesions, but their apparently normal lymph-node tissue would retain the bacilli virulent for guinea pigs for at least 18 months. The fact that guinea pigs did not contract tuberculosis when injected with tissues from heifers 1 and 3 was interpreted by Calmette to indicate that the subcutaneous injection of BCG is more efficacious than the intravenous method of vaccination. Group III. Vaccinated heifers Nos. 5 and 6 and control No. 15 were inoculated 6 months after vaccination. Control No. 15 became .very emaciated, was killed 60 days after inoculation and was found to 5 Only 19 heifers are accounted for in their descriptions.
Blood and milk samples were collected on the same day at intervals of approximately 90 days, sometimes oftener. The blood and milk serums were then subjected to the agglutination test in the same dilutions with the same antigens. The greater part of the approximately 400 cc of the milk not used for the agglutination test ,vas centrifuged and the sediment injected intraperitoneally into two guinea pigs. Whenever an abortion or a normal calving occurred, appropriate material was cultured for Brucella abortus on glycerine glucose gentian-violet agar, and two guinea pigs injected intraperitoneally with a salt-solution suspension of the material. Guinea pigs were killed at from five to six weeks, their blood secured for the agglutination test, and spleen, lungs, and liver cultured for Br. abortus. Blood Agglutination Test.-Four dilutions of blood serum were made as a routine procedure by directly pipetting 0.020, 0.010, 0.005, and 0.002 cc of serum, to ,vhich 1.0 cc of antigen ,vas added, thus making dilutions of 1-50, 1-100, 1-200, and 1-500. The antigen was prepared from 48 to 72-hour growths of several strains of bovine and porcine Brucella abortus by the addition of 0.5 per cent physiological sodium chloride and after thorough shaking by hand was filtered through coarse filter paper or a thin layer of cotton. Addition of carbol-saline was then made to secure a density of 3.4 Gates reading. No attempt was made to adjust the reaction to any p~rticular pH concentration. This suspension ,vas then stored in an ice box and used when the organisms proved to be dead. The serum-antigen tubes were incubated overnight, removed to room t.emperature, and readings made at 24 and 48 hours. Collection and Treatment of the Milk Sa1nples.-Approximately 400 cc of milk constitllted the usual sample, which was collected in approximately equal amounts from all milking quarters. Ordinary market-milk pint bottles were covered with a layer of fine cheesecloth, the paper cap applied, and the whole top part of the bottle covered with medium heavy wrapping paper which ,vas tied around the neck of the bottle with string. These bottles were then sterilized in the autoclave. Ten to 20 cc of each sample was withdrawn from the bottle in a sterile manner to provide a sanlple for the whey-agglutination test. The rest of the milk was distributed in large sterile test tubes and centrifuged for Nov.,1935] Hayes-Barger: Bang's Disease in Naturally Infected Herd
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