Walter D. James

Growth of Eaton PPLO in Broth and Preparation of Complement Fixing Antigen

Summary Eaton PPLO grew in broth medium supplemented with 20% unheated horse serum and 2.5% yeast extract. Growth was slow and maximal levels were attained on the 9th to 16th day of incubation. A specific CF antigen was prepared from infected broth suspension by concentration (50 X) and treatment with 0.5% phenol. CF antigen was estimated to be 80% as sensitive for serodiagnoses of Eaton infection as immunofluorescence with chick embryo lung sections.

EPIDEMIOLOGY OF M. PNEUMONIAE INFECTION IN MILITARY RECRUITS

M . pneurnoniae (Eaton agent) was originally recovered from filtered throat washings of patients with cold-agglutinin-positive primary atypical pneumonia. The initial isolations were made in embryonated hen’s eggs, but the agent was only demonstrable when infected chick embryo suspensions were subinoculated into rodents which subsequently developed pneumonitis.’ The original studies and later investigations in which the immunofluorescence technique was used to visualize antigen in infected chick embryo lung clearly indicated that the organism was of human origin.I3 Furthermore, these studies suggested that the agent was associated with lower respiratory tract disease in man.2*3 This association was firmly established during later controlled epidemiologic field studies in Marine recruits.4 Final proof that the organism could cause respiratory tract disease in man was provided during a series of studies in vol~nteers.”~ During the past few years M. pneurnoniae has emerged as an important human respiratory tract pathogen wherever appropriate studies have been performed.*~~ The agent described by Eaton was originally thought to be a virus; however, a number of later findings were inconsistent with this view. The organism. was found to be sensitive to chlortetracycline and an organic gold salt, and the morphology of organisms in infected chick embryo lung and tissue culture was clearly unlike that of any known virus.’O-12 The true nature of the agent was finally established when it was grown on a cell-free agar medium and shown to possess all the properties required for classification as a myc~plasma.’~ Much of our understanding of M. pneurnoniae and its ecology have come from studies in military recruits. During World War 11, volunteer studies in recruits performed by the Commission on Acute Respiratory Disease established that the etiologic agent of cold-agglutinin-positive atypical pneumonia was a filterable organism.14 One of the first controlled epidemiologic field investigations in which the organism was firmly associated with naturally occurring lower respiratory tract disease was carried out in Marine recruits in 1959-1960.4 68% of 238 recruits with pneumonia developed fluorescent stainable antibody for M. pneumoniae, whereas only 6% of 262 comparable control individuals without respiratory tract disease had a similar serologic response. Information concerning efficacy of tetracycline therapy has also come primarily from studies in the 1ni1itary.I~ The controversy concerning the effect of tetracycline

Recovery of PPLO of atypical pneumonia on artificial agar medium.

Summary Naturally occurring Eaton PPLO was shown to propagate on an agaryeast extract-horse serum medium. The agent was recovered directly on agar from 12 of 13 serologically positive patients with pneumonia. Eaton PPLO was not recovered from 14 serologically negative pneumonia patients. All Eaton PPLO isolates produced small homogenously granular colonies which could be easily differentiated from the usual “fried egg” or “nippled” colonies produced by non-Eaton oral PPLO. The distinctive colonial morphology of Eaton PPLO may ultimately prove useful for its presumptive identification.

DECREASED VIRULENCE AND PROTECTIVE EFFECT OF GENETICALLY STABLE TEMPERATURE‐SENSITIVE MUTANTS OF MYCOPLASMA PNEUMONIAE

Mycoplasrna pneirmoniae is an important cause of pneumonia in children and young adults.1.2 The best estimate of the impact of this organism comes from the surveillance studies of Grayston and Foy in Seattle. They estimated that M. pneirrnoniae caused one pneumonia illness per 1,000 persons per year.2 In addition, several high-risk populations with a considerably higher incidence of disease, such as military recruits and college students, have been identified by us and other investigators.”s Although the organism is sensitive to tetracycline and erythromycin and the severity of disease can be diminished by treatment, these antibiotics do not eradicate M . pneumoniae from the patient’s respiratory tract.G Furthermore, the onset of disease often may be insidious, and considerable debility may occur before the illness is identified correctly. For these reasons it is clear that an effective vaccine is needed. Several inactivated M . pneumoniae vaccines have been developed that were antigenic in Fernald and Clyde, however, have shown that prior infection of the respiratory tract induced significantly more resistance to experimental pulmonary disease in the Syrian hamster than did parenteral inoculation of organisms, although the latter form of immunization stimulated a higher level of serum a n t i b ~ d y . ~ . ~ Hence, it was not surprising that inactivated vaccines that were reasonably effective in stimulating the development of serum metabolisminhibiting antibody in man were found to be less effective in inducing resistance to naturally occurring M . pneumoniae. At most, a 50% protective effect was observed in field trials in which two different vaccines were eval~ated.~,7 In addition, the possibility of sensitization was raised when it was found that vaccinees who failed to develop serum antibody following vaccination experienced more serious disease after experimental challenge than did unvaccinated controls.1° During experimental infection of the hamster with M . pneumoniae, the site of localization of the organism appears to be superficial, involving only the epithelial layer of the respiratory passages.ll Similarly, the organism remains localized to the epithelial surface following infection of organ cultures of human fetal trachea.12 The superficial nature of M . pneumoniae infection in the lower respiratory tract suggests that local immune mechanisms (secretory IgA and/or cell-mediated immunity) in the respiratory tract should be of greater importance than serum antibody in protection from disease.11J2 This inference can also be drawn from the findings of Fernald and Clyde, who observed that resistance to experimental challenge in the hamster was stimulated more effectively by infection of the respiratory tract than by parenteral inoculation of organisms.*~9 In recent studies

Growth of Laboratory and Naturally Occurring Strains of Eaton Agent in Monkey Kidney Tissue Culture.

Summary An egg adapted strain of Eaton agent multiplied in rhesus monkey kidney tissue culture. Antigen concentration within the cells was insufficient to permit visualization by immunofluorescence. An eclipse phase was demonstrated during replication in monkey kidney cells. In a simultaneous test monkey kidney culture was found to be as sensitive as embryonated eggs for recovery of naturally occurring strains of Eaton agent. Fourteen strains were recovered in tissue culture from 17 patients with pneumonia.

SEROLOGICAL AND IMMUNOGENIC ACTIVITIES OF DIFFERENT FRACTIONS OF MYCOPLASMA PNEUMONIAE

Several serological tests have been recently developed for the study of M . pneumoniae. The test methods involve complement-fixation, indirect-hemagglutination, gel-diffusion and growth-inhibition (tetrazolium reduction-inhibition) . The aim of this study was to determine the nature of various antigens of M . pneumoniae which react in these different serological procedures. In addition, we were interested in defining the nature of the antigens of this organism which stimulate antibodies measurable by the different serological techniques. Such information would have relevance to our basic understanding of this important respiratory tract pathogen and to the future preparation of purified vaccines containing only those antigens which stimulate protective antibodies. It should be emphasized that our major interest was in recovering serologically active fractions and not in describing the chemical composition of the organism. M . pneumoniae, strain FH, grown in broth medium was fractionated by chemical procedures and by chromatography on Sephadex G 2 5 and GlOO and on thin-layer silica gel. After sonication at lOKC for one hour, the sediment of the M . pneumoniae suspension was fractionated by chemical methods while the supernatant fluid was fractionated by Sephadex G25 chromatography. All fractions were lyophilized and tested for serological and immunogenic activity as 1 % solutions or suspensions in phosphate buffered saline (PBS) . Complement-fixation (CFT) ,l indirect-hemagglutination (IHA) ,2 growth-inhibition (tetrazolium reduction-inhibition [TRI]3) and gel-diffusion tests4 were performed as described previously. In addition, various fractions of M . pneumoniae were tested for their capacity to block the effect of indirect-hemagglutinating and growth-inhibiting antibody. A convalescent human serum and a hyperimmune rabbit serum were used to measure the serologic activity of the various fractions of M . pneumoniae. These sera contained high titers of specific antibodies and lacked both antibodies against growth medium constituents and antibodies against other human mycoplasma species, except for a low titer of complement-king and indirect-hemagglutinating antibodies for M . salivarium in the rabbit antiserum. The rabbit serum was used at a dilution at which heterotypic reactivity could not be demonstrated. To study the immunogenicity of different fractions of M . pneumoniae, rabbits and guinea pigs were inoculated twice intradermally with a mixture of fraction and adjuvant. The method used for chemical fractionation of the sediment of the sonicated organism suspension is shown in TABLE 1. This procedure yielded one lipid fraction, three protein fractions and four polysaccharide fractions. The supernatant fluid from the sonicated organism suspension yielded four fractions following chromatography on Sephadex G-25 (TABLE 2 ) . The first SG-25 fraction was rechromatographed on Sephadex GlOO and one major frac-

Means of Increasing the Tolerated Dose of Isoniazid in Mice. III. Certain Vitamins

Summary Certain vitamins have been found effective in increasing the tolerated oral dose of isoniazid (INH) in DBA mice. Four of the group, pyridoxal-HCl, pyridoxine-HCl, p-aminobenzoic acid, and nicotinic acid, permitted 50% or more, mouse survival in acute toxicity tests with 8 mg INH (400 mg/kg). Only pyridoxal-HCl, in 1- or 2-molar equivalent concentration, protected 100% of mice against this dose. This vitamin in appropriate concentration formed a hydrazone with INH and the compound containing as much as 20 mg INH/mouse was tolerated for 8 semi-weekly administrations. Blood plasma levels of INH following administration of the hydrazone were somewhat lower than those after a sublethal dose of INH alone, but were constant and more prolonged, presumably due to gradual in vivo hydrolysis of the hydrazone. Six vitamins, as tested, did not interfere with in vitro bacteriostatic action of INH on one avirulent strain of Mycobacterium, but the pyridoxal isonicotinyl hydrazone per se was as effective as INH alone as a tuberculostatic agent.

Rapid Colorimetric Method for Determination of Isonicotinic Acid Hydrazide in Blood Plasma

Summary The color reaction between isonicotinic acid hydrazide (isoniazid) and glutaconic aldehyde liberated from 4-pyridylpyridinium dichloride as described conforms to Lambert-Beers law and is the basis for a sensitive and rapid method for determination of hydrazides in blood plasma. Blood plasma levels in mice and guinea pigs after oral administration of respectively 5 and 50 mg of isoniazid have been found to reach a maximum within 30 minutes to 2 hours and to fall to low levels within 18 hours.

Mycoplasma pneumoniae phosphatidyl glycerol.

Summary When the lipids of M. pneumoniae were fractionated by silicic acid column chromatography, the major serologically reactive material was usually eluted with chloroform-methanol, 9:1. Initially or after rechromatography the 9:1 eluates contained primarily a phospholipid moiety which on chemical analysis appeared to be phosphatidyl glycerol. Although this phospholipid did not react with M. pneumoniae antibodies, it acted as an auxiliary lipid to enhance the activity of the glycolipid haptens which were present in low concentration in the 9:1 eluates.

A Means of Increasing the Tolerated Dose of Isoniazid in Mice

Summary Attempts have been made to find a means of increasing the amount of isoniazid (INH) which mice will tolerate. In the DBA strain of mice; INH alone was lethal for 45% of the animals when 5 mg/20 g mouse (250 mg/kg) were given orally. With an 8 mg dose of INH, lethal per se, simultaneous administration of as little as 25 mg glycine and 10 mg sodium glucuronate mono-hydrate was sufficient to keep alive all the animals tested. By the use of appropriate amounts of the 2 detoxifying chemicals, all mice survived either a single oral dose of 25 mg INH, or 3 doses of 8 mg daily, or 13 doses of 10 mg each given at 72-hour intervals. Larger amounts were not tolerated. A study of the blood plasma concentrations of INH showed that, whereas 24 hours after a 4-mg dose alone only a negligible amount remained, appreciably higher concentrations, approximately 6-fold, persisted for a longer period following 8 or 10 mg INH in combined treatment. In the proportions tested, sodium glucuronate and glycine had no effect on the bacteriostatic action of INH in vitro on one human strain of tubercle bacilli.