EXPERIMENTAL STUDIES OF THE NASOPHARYNGEAL SECRETIONS FROM INFLUENZA PATIENTS : VI. IMMUNITY REACTIONS.

The experiments described furnish additional evidence of the pathogenic character and the virtual identity of the various strains of the active agent derived from the nasopharyngeal secretions of influenzal patients with which the transmission experiments in rabbits have been carried out. The active material has been shown to be of antigenic nature, so that rabbits are protected from the effects of a second inoculation. The experiments indicate also the antigenic identity of the various strains of the active agent with each other and with Bacterium pneumosintes. Finally, the experiments show that the protection may persist for 14 months which is the longest period yet tested

EXPERIMENTAL STUDIES OF THE NASOPHARYNGEAL SECRETIONS FROM INFLUENZA PATIENTS : IX. THE RECURRENCE OF 1922.

From the nasopharyngeal secretions of patients in the early hours of uncomplicated epidemic influenza during the recurrence in New York City in January and February, 1922, we have again obtained an active agent, pathogenic for rabbits, and have identified this active agent as Bacterium pneumosintes. Four new strains of this micro-organism have been isolated in pure culture and identified with the 1918-19 and 1920 strains on morphological, cultural, and serological grounds. All of the significant characteristics of the old strains, including their effect upon the resistance of the lungs of rabbits to secondary invasion with other bacteria, have been noted in the new strains, which thus have served to confirm and extend our original observations

EXPERIMENTAL STUDIES OF THE NASOPHARYNGEAL SECRETIONS FROM INFLUENZA PATIENTS : IV. ANAEROBIC CULTIVATION.

From the filtered nasopharyngeal washings of patients in the first 36 hours of uncomplicated epidemic influenza and rarely in later stages of the disease, we have cultivated a minute bacilloid body, Bacterium pneumosintes, 0.15 to 0.3 microns in length, of constant cultural characters and capable of indefinite propagation on artificial media. This organism, not of the nature of ordinary bacteria, was also recovered in pure culture from the unfiltered and filtered lung tissue of rabbits and guinea pigs inoculated with unfiltered and filtered nasopharyngeal washings of early influenza cases, both from the first epidemic of 1918–19 and from the second one of 1920. The organism grows only under strictly anaerobic conditions, passes Berkefeld V and N filters, and withstands the action of sterile 50 percent glycerol for a period of months. It has been recovered from cultures contaminated with a variety of ordinary bacteria such as Bacillus pfeifferi, pneumococci, streptococci, and staphylococci, and has been experimentally cultivated in symbiosis with them. Similar cultivation of control materials uniformly failed to yield growths of this organism. The materials tested consisted of the unfiltered and filtered nasopharyngeal washings of persons free from influenza, some of whom were suffering from acute coryza, the lung tissue of normal rabbits and of rabbits with bacterial respiratory infections, and the uninoculated media. The intratracheal injection in rabbits and guinea pigs of mass cultures of this organism has induced effects on the blood and lungs of these animals which are not to be distinguished from those obtained with the nasopharyngeal secretions of patients in the early hours of epidemic influenza. From the pulmonary lesions thus induced the same organism has been recovered in pure culture, and has been found to cause similar lesions on subsequent animal passage. Its pathogenicity is not lost by prolonged artificial cultivation. Our experiments indicate that the cultivable bodies obtained directly from human nasopharyngeal washings and from affected rabbit lungs are strains of the same organism. This organism appears to be the source of the reactions which occur in experimental animals—rabbits and guinea pigs—as a result of the intratracheal injection of nasopharyngeal washings obtained during the early hours of uncomplicated epidemic influenza in man

EXPERIMENTAL STUDIES OF THE NASOPHARYNGEAL SECRETIONS FROM INFLUENZA PATIENTS : XII. THE EFFECTS OF SUBCUTANEOUS INJECTIONS OF VACCINES OF BACTERIUM PNEUMOSINTES IN MAN.

Thirteen volunteers were injected subcutaneously with a vaccine prepared from two strains of Bacterium pneumosintes. Three doses were given corresponding, by opacity tests, to 1,000, 2,000, and 2,000 million staphylococci. The intervals between the several injections were 5 and 8 days. Local reactions of mild and transitory character only were noted. Constitutional reactions, also mild and transitory, consisting of headache, depression, and generalized muscular pains, were infrequently observed. None of the men was prevented by the vaccine from pursuing his ordinary duties. The vaccinations induce a transitory leucocytosis and lead to the appearance of specific agglutinins in the blood serum

EXPERIMENTAL STUDIES OF THE NASOPHARYNGEAL SECRETIONS FROM INFLUENZA PATIENTS : III. STUDIES OF THE CONCURRENT INFECTIONS.

1. Concurrent infections in the experiments described may be regarded as of accidental nature and are not causally related to the typical effects induced in rabbits by a material wholly free from ordinary bacteria. 2. The influenzal agent exerts an effect on the pulmonary tissue which encourages the invasion of the lung and subsequent multiplication there of ordinary bacteria, such as the pneumococcus, streptococcus, and Bacillus pfeifferi. 3. A similarity is believed to exist between the conditions under which concurrent infections arose in the inoculated rabbits and those which seem to favor the occurrence of concurrent infections during epidemic influenza in man. In no instance did death occur in the rabbits as a result of the uncomplicated effects of the influenzal agent alone. When death occurred in any of the inoculated animals concurrent infection of the lungs by ordinary bacteria was present. The microorganisms most commonly met with under these conditions were Pneumococcus Type IV and atypical Type II, streptococci, and hemoglobinophilic bacilli. Other kinds were encountered less often

EXPERIMENTAL STUDIES OF THE NASOPHARYNGEAL SECRETIONS FROM INFLUENZA PATIENTS : V. BACTERIUM PNEUMOSINTES AND CONCURRENT INFECTIONS.

During the course of animal experiments with the anaerobic filter-passing organisms cultivated from epidemic influenzal sources, certain pulmonary infections with ordinary bacteria have been observed. The experiments also have shown that the lungs of animals infected with Bacterium pneumosintes are less resistant than normal lungs to infection with ordinary bacteria. The demonstration of this fact invites a comparison of the course of these experimental bacterial infections with the sequence of postinfluenzal pneumonias attributable to similar organisms in man. These observations furnish additional proof of the identity of Bacterium pneumosintes and the active agent derived from the nasopharyngeal secretions of patients in the early hours of epidemic influenza

EXPERIMENTAL STUDIES OF THE NASOPHARYNGEAL SECRETIONS FROM INFLUENZA PATIENTS : XI. ANTIBODIES IN THE BLOOD AFTER RECOVERY FROM EPIDEMIC INFLUENZA.

Although Bacterium pneumosintes is a stable organism which agglutinates only in low dilutions of the serum of actively immunized rabbits, the serological reactions of this microorganism have always been found to be consistent and specific. The results of the agglutination and precipitation reactions described in this paper indicate that the serum of normal persons does not contain demonstrable agglutinins or precipitins for Bacterium pneumosintes. By contrast, agglutinins have been demonstrated in the serum of seventeen persons among nineteen who were examined from 10 days to 5 months after recovery from epidemic influenza. The serum of ten persons who had influenza, followed in three instances by pneumonia, 2½ to 3½ years before, proved negative. In one instance the appearance of specific agglutinins against Bacterium pneumosintes was found to be coincident with an attack of uncomplicated influenza. In twelve of fifteen instances in which agglutinins were found, precipitins against Bacterium pneumosintes were demonstrated also. It is noteworthy that these antibodies may persist in the blood at least 5 months after recovery from the disease

EXPERIMENTAL STUDIES OF THE NASOPHARYNGEAL SECRETIONS FROM INFLUENZA PATIENTS : VII. FURTHER OBSERVATIONS ON THE CULTURAL AND MORPHOLOGICAL CHARACTERS OF BACTERIUM PNEUMOSINTES.

After artificial cultivation for a period of over 3 years Bacterium pneumosintes has maintained its original morphological and cultural characteristics, when grown in the original medium. Adaptation to a saprophytic existence has been accompanied by a loss of pathogenicity. Our strains now grow readily under strictly anaerobic conditions in a variety of media with peptone broth as a base, enriched with fresh tissue, blood, or by the growth of other bacteria. Surface colonies have been obtained on blood agar plates in. an anaerobic jar. These various methods of cultivation are adapted to special purposes. In broth cultures Bacterium pneumosintes grows in larger forms than in the ascitic fluid-tissue medium, but the identity of the microorganisms is proved by their serological reactions and by reversion to the minute forms on transfer to the original medium

EXPERIMENTAL STUDIES OF THE NASOPHARYNGEAL SECRETIONS FROM INFLUENZA PATIENTS : I. TRANSMISSION EXPERIMENTS WITH NASOPHARYNGEAL WASHINGS.

An active substance has been detected, by the methods described, in five patients in early stages of epidemic influenza during 1918–19 and two patients in early stages of epidemic influenza during 1920 It was not detected in twelve cases of the same disease in which the onset of obvious symptoms occurred more than 36 hours before washing of the nasopharynx was carried out, nor was it found in the secretions of fourteen individuals free from the syndrome of influenza either during the epidemics or the interval between them. With this substance a clinical and pathological condition has been induced in rabbits, affecting the blood and pulmonary structures mainly, which could be maintained and carried through at least fifteen successive animals. For this reason, and also because of the dilution between passages, we are led to believe that we were dealing with the actual transmission of a multiplying agent rather than with a passive transference of an original active substance. In some of the experiments secondary infections by ordinary bacteria were encountered. The relation of these microorganisms to this active substance will be dealt with fully in another communication. However, the essential effects were produced by a substance wholly unrelated to these bacteria. The similarity that exists between the effects produced in rabbits on the blood and the lungs and those occurring in man in epidemic influenza provides a basis for further investigation on the inciting agent of epidemic influenza

EXPERIMENTAL STUDIES OF THE NASOPHARYNGEAL SECRETIONS FROM INFLUENZA PATIENTS : X. THE IMMUNIZING EFFECTS IN RABBITS OF SUBCUTANEOUS INJECTIONS OF KILLED CULTURES OF BACTERIUM PNEUMOSINTES.

A series of rabbits was subcutaneously injected with three measured doses of killed cultures of two strains of Bacterium pneumosintes derived from the nasopharyngeal secretions of influenza patients. These rabbits were subsequently tested for the development of serum antibodies and for the presence of an induced immunity to the living organisms, with the following results. The serum of eleven of fifteen rabbits, tested from 10 to 27 days after the final subcutaneous injection, specifically agglutinated Bacterium pneumosintes, whereas normal rabbit serum did not. Nineteen vaccinated rabbits were subjected to protection experiments. Two of them were unaffected by an intratracheal injection of Bacterium pneumosintes, contained in the lung tissues of previously infected animals, in a dose which typically affected the control rabbits. Fifteen of the other seventeen proved to be completely resistant when tested by intratracheal injections of Bacterium pneumosintes cultures that produced typical infections in the controls. Ten of these fifteen rabbits were injected intravenously with living cultures of pneumococcus, Streptococcus hamolyticus, or Bacillus pfeifferi in doses which were non-infective under normal conditions, but infective, as experience has shown, in the presence of a primary lesion caused by Bacterium pneumosintes. In none of these animals did infection develop. The two remaining rabbits of the seventeen were not protected against Bacterium pneumosintes by the vaccination, and they further developed a secondary pulmonary infection with Bacillus pfeifferi after its intravenous injection. Control rabbits similarly injected intratracheally with Bacterium pneumosintes, and then intravenously with the pneumococcus, streptococcus, or Bacillus pfeifferi in doses that had proved non-infective for normal rabbits, uniformly developed a secondary infection with these organisms. The mildness of the local reactions and the absence of general signs, following vaccination with Bacterium pneumosintes, indicate that similar injections would be well tolerated in man. There is no evidence that the subcutaneous injection of large doses of the heat-killed organisms reduces the resistance of the animal body to infections with other bacteria. In single rabbit experiments the resistance to intravenously injected pneumococci, streptococci, or Bacillus pfeifferi has been found unreduced immediately after vaccination with Bacterium pneumosintes