In two species of monkeys Wollstein[[111]] produced fatal meningitis by injecting suspensions of B. influenzæ into the subdural space by lumbar puncture.
During the course of our investigation of pneumonia and influenza, sputum of approximately 400 normal individuals or patients with influenza was injected into the peritoneal cavity of mice. B. influenzæ was found in approximately 150 instances. In only 4 instances was B. influenzæ found in pure culture in the blood; in all other mice in which B. influenzæ appeared in the blood it accompanied pneumococcus or S. hemolyticus.
Before experiments were performed cultures were made from the throats of all monkeys in order to exclude the presence of B. influenzæ. Blood agar plates inoculated with a swab applied to the nasopharynx failed to show in any instance B. influenzæ, pneumococci, or hemolytic streptococci. Streptococci causing green discoloration of blood agar were usually found.
Inoculation of the Nose and Pharynx with B. Influenzæ.—B. influenzæ was introduced into the nose and pharynx of two healthy monkeys. An actively growing culture of the microorganism made on alkaline blood agar and sixteen hours old was used. The culture was the first subculture from a growth obtained from the nose and throat of a patient with influenza. A cotton swab moistened with broth was applied to the surface of the culture. It was introduced into the nostrils and smeared over the pharynx of the animals. A swab moistened with sterile broth was applied to the nose and pharynx of a third monkey as a control; cultures from this animal kept in a cage removed from those inoculated failed to show B. influenzæ.
Experiment 1
November 21, 1918.—Small female monkey; throat culture: negative. November 23.—10:20 A.M.—White blood corpuscles, 16,700; polynuclear leucocytes, 68 per cent; small lymphocytes, 17.5 per cent; large lymphocytes, 8 per cent; large mononuclears, 1 per cent; eosinophiles, 2.5 per cent; basinophiles, 0.5 per cent. 10:30 A.M.—Mucous membranes of nose and throat were inoculated with B. influenzæ as described above. November 25.—The animal appears sick and is huddled in back of its cage; the nose is running. White blood corpuscles, 13,500; polynuclear leucocytes, 44 per cent; small lymphocytes, 30 per cent; large lymphocytes, 22 per cent; large mononuclears, 3 per cent; eosinophiles, 1 per cent. 3:40 P.M.—Free epistaxis occurred after culturing of nose; the swab was discolored with old brownish blood indicating previous epistaxis. Nose culture: B. influenzæ present in abundance; Gram-positive cocci present. Throat culture: negative for B. influenzæ. November 28.—Monkey is more active and appears to be fairly well. Nose and throat cultures: negative for B. influenzæ. December 4.—Monkey is apparently well.
Experiment 2
November 21, 1918.—Small male monkey. Throat culture: negative. November 23.—10:10 A.M.—White blood corpuscles, 10,900; polynuclear leucocytes, 52 per cent; small lymphocytes, 18 per cent; large lymphocytes, 25 per cent; large mononuclears, 3 per cent; eosinophiles, 2 per cent. 10:15 A.M.—Mucous membranes of nose and throat were inoculated by means of moist swab with 4 strains of B. influenzæ recently isolated from acute cases of influenza. November 24.—Monkey is quiet and takes no interest in surroundings. November 25.—Animal appears sick and remains huddled at back of its cage. Nose culture: B. influenzæ present. Throat culture: B. influenzæ present. Swab applied to nose is stained brown with old blood indicating previous epistaxis. November 26.—Animal is still sick; nose is running. White blood corpuscles, 14,400; polynuclear leucocytes, 61 per cent; small lymphocytes, 23 per cent; large lymphocytes, 15 per cent; large mononuclears, 1 per cent. November 27.—White blood corpuscles, 11,300. November 28.—Nose culture: negative for B. influenzæ. Throat culture: B. influenzæ present. November 29.—Animal is active, but still appears sick. White blood corpuscles, 19,300. December 4.—Monkey appears well. Throat culture: B. influenzæ present.
These animals were sick two and six days following inoculation. There was discharge from the nose. In both instances there was epistaxis. The temperature of the animals was subject to such wide variation in relation to external temperature that it could not be used as an index of the progress of the disease. There was no leucocytosis, but in one animal there was some increase in the numbers of leucocytes during recovery. In one animal B. influenzæ present in the nose after two days was absent after four days. In the other animal the organism was repeatedly found in the nose and throat and was still present in the throat eleven days after inoculation. The two animals suffered with a self-limited disease resembling many cases of influenza.
Introduction of Bacillus Influenzæ into the Trachea.—In the attempt to reproduce the bronchitis which occurs in a considerable proportion of all cases of influenza and is almost invariably associated with B. influenzæ, this organism was introduced into the trachea of monkeys. In Experiment 3 a suspension containing young cultures of freshly isolated B. influenzæ was introduced into the trachea by a silver catheter passed through the glottis and larynx into the trachea.