APPENDIX EXPERIMENTAL INOCULATION OF MONKEYS WITH BACILLUS

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APPENDIX EXPERIMENTAL INOCULATION OF MONKEYS WITH BACILLUS INFLUENZAE AND MICROORGANISMS ISOLATED FROM THE PNEUMONIAS OF INFLUENZA

Eugene L. Opie, M.D.; Allen W. Freeman, M.D.; Francis G. Blake, M.D.; James C. Small, M.D.; and Thomas M. Rivers, M.D.

Experiments were undertaken at Camp Pike in December, 1918, to determine whether bacteria freshly isolated from patients suffering with influenza and pneumonia during the outbreak of influenza and its associated pneumonias were capable of producing similar diseases when introduced into the respiratory passages of monkeys. The number of animals available for the study was limited. The attempt was made (a) to determine if B. influenzÆ produces in monkeys a disease comparable to influenza of human beings, and (b) to determine so far as possible, with the limited opportunity, the character of the lesions produced by combinations of pneumococcus or S. hemolyticus with B. influenzÆ and to compare these lesions with lesions produced by pneumococcus or by hemolytic streptococcus alone.

Pfeiffer[107] found monkeys alone susceptible to invasion by B. influenzÆ and obtained no evidence of multiplication of the microorganism within the body of any other animal. A suspension containing mucus from the sputum of a patient with influenza was injected into a monkey. There was elevation of temperature and the animal died after seven days. Lobular patches of atelectasis occurred along the sharp edges of the lungs and the adjacent bronchial branches contained mucus. Cultures on agar from the bronchi remained sterile. Microscopic examination showed the presence of bacilli resembling B. influenzÆ. Death was caused, the author states, by an abscess at the site of inoculation and not by the process in the lungs. Three monkeys received each 0.5 c.c. of bouillon containing a blood agar culture injected into the lung through the chest wall. There was elevation of temperature lasting from three to five days with return to normal every morning. There was cough but little evidence of illness. B. influenzÆ was introduced by a platinum loop into the nose of a monkey. Febrile reaction is recorded lasting four or five days. Pfeiffer found that guinea pigs and mice were resistant to the microorganism. Large doses injected intravenously caused in rabbits intoxication with dyspnea and evidence of profound muscular weakness.

Kamen[108] used a culture of B. influenzÆ which was nonpathogenic for mice, but when it was inoculated into the peritoneal cavity with streptococcus both influenza bacilli and streptococci appeared in the blood. Jacobson[109] found that B. influenzÆ appeared in the blood and viscera of mice killed by intraperitoneal inoculation of B. influenzÆ mixed with cultures of streptococcus either living or killed by heat. B. influenzÆ which had successively passed through mice, simultaneously inoculated with killed streptococci, acquired such virulence that it was capable of producing septicemia when inoculated alone.

Richie[110] introduced by lumbar puncture a suspension of two blood agar cultures of B. influenzÆ obtained from the meninges of a patient with influenzal meningitis into the subdural space of a rhesus monkey. Death occurred in eighteen hours and there was beginning meningitis. B. influenzÆ was present in the exudate in abundance.

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.

Young cultures of B. influenzÆ, subcultured only once after isolation from early cases of influenza, were used. The microorganism was recovered in abundance by throat swab two days later and again from the bronchus at autopsy three days after inoculation. Tuberculosis of mesenteric lymph nodes, of intestine and of liver and several small tuberculous nodules in the lung were found at autopsy. A secondary invasion of the lung by staphylococci had occurred. There was bronchitis with an inflammatory infiltration of the subepithelial tissue of the bronchi by lymphoid and plasma cells. Bronchopneumonia was present, and the bronchi and many of the alveoli contained blood. These changes do not differ essentially from the changes found in many instances of pneumonia following influenza.

In three instances cultures of B. influenzÆ were injected into the trachea by means of a hypodermic syringe.

In one of these experiments (Experiment 4) intratracheal injection of 2 c.c. salt solution suspension of B. influenzÆ (isolated at autopsy from bronchus of the monkey used in Experiment 3), representing growth on 1½ blood agar plates, was made with a needle inserted into trachea just above the suprasternal notch. On the following day a throat culture contained B. influenzÆ in abundance. Three days after inoculation the monkey appeared to be very sick and there was profuse nasal discharge. The animal coughed and sibilant rÂles were heard over the chest. There was no leucocytosis. A throat culture contained B. influenzÆ. Four days after inoculation the monkey was still sick and weak, but appeared much improved and was killed. The trachea and large bronchi contained thick viscid mucus. In the middle lobe of the right lung was a patch of grayish red, airless tissue, firmer than the lung substance elsewhere. Cultures from the trachea, bronchus and lung contained a variety of microorganisms, but B. influenzÆ was not recovered.

In two additional experiments (Experiments 6 and 7) cultures of B. influenzÆ forty-eight hours old were injected into the trachea of monkeys. The microorganism was recovered in cultures made from the pharynx two days later. These animals were only slightly sick.

Introduction of B. InfluenzÆ and S. Hemolyticus into the Trachea.—In view of the frequent association of B. influenzÆ and S. hemolyticus in the sputum of patients with streptococcus pneumonia following influenza and in the bronchi and lungs of those who have died with this disease, the two microorganisms were injected simultaneously into the trachea of monkeys.

B. influenzÆ and S. hemolyticus in Experiment 7 produced bronchitis and bronchopneumonia. There was acute inflammation of the interstitial tissue of the lung, and acute lymphangitis with numerous polynuclear leucocytes within the lumen of the lymphatics was present. B. influenzÆ and S. hemolyticus were present in the trachea at autopsy four days after inoculation. It is probable that part of the injected culture entered the tissue outside the trachea, for an abscess was formed in this situation. It is noteworthy that acute pericarditis occurred and both S. hemolyticus and B. influenzÆ were found in the pericardial exudate. B. influenzÆ not infrequently exhibits this tendency to penetrate in association with other bacteria localities which it does not invade independently.

In a second experiment (Experiment 8) in which B. influenzÆ and S. hemolyticus were injected into the trachea, both microorganisms were recovered from the throat on the day following inoculation; on the fifth day S. hemolyticus alone was recovered and on the sixth day a throat culture was negative both for S. hemolyticus and B. influenzÆ.

Introduction of B. influenzÆ and of Pneumococcus or of Pneumococcus Alone into the Trachea.—In two experiments B. influenzÆ and Pneumococcus Type III were simultaneously injected into the trachea.

In Experiment 9 a large male monkey was used and intratracheal injection made with syringe and needle of 5 c.c. salt solution suspension of Pneumococcus Type III and B. influenzÆ (growth on 5 blood agar plates of mixed cultures of Pneumococcus III and B. influenzÆ). On the following day the animal was very sick, lying on the floor of its cage, and was dead two days after inoculation.

The dosage of bacteria in this experiment was large. The lesions in gross appearance and microscopically resembled those seen in many instances of pneumonia following influenza. In the trachea there was loss of ciliated epithelium, congestion of the subepithelial tissue, hemorrhage and infiltration with plasma cells. The lungs were consolidated and red and there were hemorrhage and edema. B. influenzÆ, as in human cases, was abundant in the bronchi, less abundant in the consolidated lung, being present though scant in the left lung, and absent in cultures from the right. B. influenzÆ as in Experiment 8 with streptococcus had entered the left pericardial cavity in company in this experiment with Pneumococcus III.

In Experiment 10 a very large monkey received by intratracheal injection, made with syringe and needle, 5 c.c. salt solution suspension of Pneumococcus III and 3 strains of B. influenzÆ, (2 recently isolated from cases of influenza and 1 from autopsy in a case of postinfluenzal pneumonia). The animal died twenty-four hours later.

This simultaneous introduction of B. influenzÆ and Pneumococcus III in large quantity has produced rapidly fatal pneumonia with lobar distribution. Hepatization was homogeneous and red, and outside the consolidated parts of the lung there was hemorrhage and edema. The lesion resembled that found when death has occurred within a few days after the onset of pneumonia following influenza, but had no distinctive characters establishing its relation to pneumonia following influenzÆ.

In Experiment 11 Pneumococcus III alone in small amount was introduced into the trachea of a small monkey. The animal was very sick, but its condition improved and recovery seemed probable. The animal was killed seven days after inoculation, and typical lobar pneumonia with gray hepatization was found at autopsy.

Experiment 11

November 20, 1918.—Small monkey; throat culture: negative for B. influenzÆ, pneumococcus and S. hemolyticus. November 28 and December 6.—Nose and throat cultures again negative for B. influenzÆ. December 9—4:30 P.M.—Intratracheal injection with syringe and needle of 0.33 c.c. of an eighteen hour broth culture of Pneumococcus Type III. December 10.—The animal is sick, huddled up in his cage with head down; there is rapid respiration with expiratory grunt and the mucous membranes are moderately cyanotic. There is frequent cough. Throat culture: Pneumococcus III present in abundance. December 15.—The animal appears to be better. Respirations are still rapid but less labored. December 16.—The animal is improving but very weak and emaciated.

Autopsy.—The pleural cavities contain no fluid. On the right side are several strands of fibrin. The right lower lobe with the exception of a small patch at the summit and the lower part of the middle lobe are voluminous, have a dull gray surface covered by a scant layer of fibrin and are firmly consolidated. On section the consolidated tissue has a gray color and is conspicuously granular, the granulation resembling, on a slightly smaller scale, that seen in human lobar pneumonia. The bronchi contain a small amount of viscid fluid.

Bacteriology.—Direct smears from the trachea and the lower lobe of the left lung contain Gram-positive diplococci. Cultures from the trachea and from the blood of the heart contain Pneumococcus III. Cultures from the left lower lobe, from the liver and from the spleen remain sterile.

Microscopical Examination.—There is abundant infiltration of the subepithelial tissue of the trachea with plasma cells. Superficial ciliated epithelium is in places lost. At one point is a small focus of hemorrhage. Alveoli in the consolidated part of the lungs contain polynuclear leucocytes and fibrin and exhibit the appearance seen in lobar pneumonia in man.

Fig. 33.—Experimental lobar pneumonia in the stage of gray hepatization produced by injection of Pneumococcus III into the trachea of a monkey (Experiment 11). The alveoli are uniformly filled with plugs of fibrinous exudate.

In Experiment 12 B. influenzÆ was injected into the trachea and two days later identified in a culture made from the pharynx; four days after inoculation Pneumococcus IV was injected into the trachea. The animal was killed seven days after the first inoculation, and three days after inoculation with pneumococcus. The lower half of the upper lobe of the right lung and the greater part of the lower and middle lobes were consolidated. The pleural surface of the consolidated areas was dull red and covered by a small amount of fibrin. The lower lobe, with the exception of a small part at the summit, was very firmly consolidated, on section pinkish gray in the anterior part and deep red in a small zone at the posterior border. The cut section was conspicuously granular. The trachea and bronchi contained mucus. Cultures from the trachea, the right lung and the right pleural cavity contained Pneumococcus IV in pure culture. Alveoli in the consolidated part of the lung were filled with polynuclear leucocytes and fibrin.

Lobar pneumonia has been produced by the introduction of Pneumococcus IV into the trachea. It is doubtful if preceding inoculation of B. influenzÆ has influenced the course of the disease.

The foregoing experiments have shown that B. influenzÆ introduced into the nasopharynx or into the trachea of monkeys is capable of causing lesions of the mucosa of these structures; the microorganism persists within the nasopharynx or trachea and is recoverable during a variable period of from two to eleven days after inoculation. Spontaneous infection of monkeys with B. influenzÆ has not been observed. The animals infected with the microorganism are ill during several days, but the experimental disease like most instances of human influenza is self limited. Following inoculation of the nose and throat of monkeys with B. influenzÆ there is discharge from the nose, tendency to epistaxis and absence of leucocytosis.

Bronchitis was produced by the introduction of B. influenzÆ into the trachea of monkeys, and the microorganism was recovered from the nasopharynx two and three days following inoculation. There was no leucocytosis. In two experiments death occurred following inoculation, and in both instances it was found that the animal suffered with tuberculosis which had produced only trivial lesions of the lungs. In both animals staphylococci were obtained from the internal organs. There was bronchitis with changes in the bronchi which, although not characteristic, resembled those found in association with B. influenzÆ in man. It is noteworthy that B. influenzÆ is usually found mixed with other bacteria in the bronchi of those who have died with bronchitis and pneumonia following influenza. In the experimental animals there was in places superficial loss of ciliated epithelium, exudation of polynuclear leucocytes, infiltration of the subepithelial tissue with plasma cells and hemorrhage into this tissue.

In one instance simultaneous injection of B. influenzÆ and S. hemolyticus, freshly obtained from autopsy upon a man dying with pneumonia following influenza, caused bronchitis and bronchopneumonia; there were acute lymphangitis and infiltration of the interstitial tissue of the lung with polynuclear leucocytes such as occurs in human cases, but the lesion had not proceeded to suppuration.

In man B. influenzÆ is usually found in greatest abundance upon the mucosa of the respiratory passages, less frequently it invades the alveoli of the lungs and is almost invariably found in association with other microorganisms. In company with other microorganisms B. influenzÆ penetrates into tissues outside the lungs. In Experiment 7 it has entered the pericardium, with streptococcus, and in Experiment 9 with pneumococcus. When B. influenzÆ and streptococcus are injected into the peritoneal cavity of a mouse both organisms appear in the blood, whereas in the absence of streptococcus, B. influenzÆ seldom leaves the peritoneal cavity.

Typical lobar pneumonia has been produced for the first time in monkeys by injecting pneumococci (in quantity as small as 0.33 c.c. of suspension) into the trachea. With the animals available it has not been possible to adjust the dosage of the two microorganisms so that the influence of one upon the other might be determined. Pneumococcus III, in small quantity, introduced into the trachea has produced typical acute lobar pneumonia in the stage of gray hepatization. A similar lesion has been produced with Pneumococcus IV obtained from the lung of a man dead with pneumonia.

                                                                                                                                                                                                                                                                                                           

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