W. P. Larson

Temperature Reactions in Mice Infected with Pneumococci.

Summary The body temperature of mice is markedly influenced by the surrounding temperature. Pneumococcic infections in mice are associated with a marked drop in temperature. Infected mice held at incubator temperature succumb more rapidly than those at room temperature. Incubator temperature promotes the infective processes in mice more than the protective action of sulfanilamide.

The effect of sodium ricinoleate upon bacterial toxins, and the value of soap-toxin mixtures as antigens.

The present study is a continuation of the work published by Larson and Nelson 1 on the effect of castor oil soap upon bacterial toxins. It was shown that sodium ricinoleate possessed the property of detoxifying soluble toxins as well as endotoxins. In the present paper an attempt is made to analyze the mechanisms of the detoxifying action of soaps, and further, to study the antigenic properties of toxins which have been thus detoxified. The work was begun by subjecting diphtheritic toxin and tetanus toxin to the action of the sodium salts of a large number of fatty acids as: ricinoleic, oleic, stearic, palmitic, myristic and lauric acids, and two acids with uneven numbered carbon chains, ennenoic and hendecenoic acids. Our procedure was to mix the toxin with an equal volume of a one per cent soap solution except in those cases where a one percent solution of soap gels, in which cases weaker solutions were used. It was found that the soaps of oleic, stearic and palmitic acids had no detoxifying action whatever on bacterial toxins. The soaps of lauric, myristic, ennenoic and hendecenoic acids possess some detoxifying action upon toxins, but do not compare with sodium ricinoleate in detoxifying properties.

The antigenic properties of pneumococci and streptococci treated with sodium ricinoleate.

The property of sodium ricinoleate to neutralize bacterial toxins and destroy the pathogenicity of some of the pathogenic bacteria has been emphasized in a series of papers published from this laboratory. 1 , 2 , 3 The present paper concerns the effect of sodium ricinoleate upon the pathogenic and antigenic properties of the pneumococcus and streptococcus scarlatinæ. If a solution of sodium ricinoleate is added to a virulent culture of the pneumococcus, so that the final dilution of soap is 0.1 per cent, the micro-organism loses its pathogenicity instantly. Ten cc. or more of such a culture may be injected into rabbits intraperitoneally without ill effects. Twenty-four hours after such an injection, large amounts of agglutinins are present in the blood stream. Following such treatment, the animals resist many lethal doses of pneumococci. The serum of rabbits thus immunized protects normal rabbits against intraperitoneal and intravenous infections. We have studied the effect of sodium ricinoleate upon one strain of streptococcus scarlatinæ. It loses its power to grow upon culture media in less than five minutes when treated with 0.5 per cent sodium ricinoleate. Streptococci so treated with soap produce agglutinins in rabbits within twenty-four hours following intraperitoneal injection.

Inhibitory Action of Peptone on Sulfapyridine Adsorption.

The bacteriostatic effect of sulfapyridin on the pneumococcus in vitro and the inhibitory action of peptone have been shown previously. 1 The findings compare with those reported by Lockwood, 2 who studied the effect of sulfanilamide on the streptococcus under similar conditions. The effect of peptone in preventing drug-action suggested the possibility of an interference in adsorption of the drug. To test this, a study was made of the adsorption of sulfapyridine by activated carbon particles. Varying amounts of activated carbon were added to solutions containing 10 mg of sulfapyridine and 0.85 g of sodium chloride per 100 cc. After allowing the reaction to take place for 15 minutes, the carbon was removed by filtration, and the filtrate was tested for the presence of the drug by the method described by Marshall. 3 Adsorption was found to occur, as shown in Fig. 1 (solid line). If 1% peptone (Parke-Davis) is added to the solution containing the drug, and adsorption by carbon allowed to take place, the removal of the drug is retarded. This is shown in Fig. 1 by the broken line. That this adsorption is selective is illustrated by the following experiment: 2 cc of a 1:1000 solution of sulfapyridine were added to 8 cc of saline, and adsorbed with 20 mg of carbon. After 15 minutes, the mixture was divided into 2 parts; to one, 5 cc of 0.85% saline was added, and to the other 0.85% saline containing 1% peptone. These were allowed to stand for another 15 minutes, when they were filtered and tested for sulfapyridine. The portion to which saline had been added showed adsorption of 73% of the drug, as compared with a removal of 55% in the portion to which peptone had been added. This suggests that peptone is able to displace the drug from the surface of the carbon particles.

Protective Action of Sulfapyridine in Rabbits Infected with Pneumococci.

Most of the studies on the protective action of sulfanilamide and sulfapyridine on streptococcic and pneumococcic infections have been made on white mice. In the work here reported rabbits were used as the experimental animals. Intracutaneous inoculations were made in order to permit the observation of differences in the local lesions occurring in the treated and the control groups. Rabbits weighing approximately 2 kilos were given 0.3 cc of a 1-100 dilution of an 8-hour culture of a Type II pneumococcus. The strain used had been transferred alternately through mice and veal broth for more than a year. Intraperitoneal injections of 0.2 cc into mice, in dilutions of 1:10,000,000, kills 50% of the animals. Sulfapyridine, 2-(sulfanilamide)-pyridine, was administered orally by suspending in 10% acacia and permitting the suspension to trickle down the throat from a large syringe. The treated animals were given 0.5 g one hour before inoculation, 0.5 g 3 hours after inoculation, and 0.5 g every 12 hours thereafter for a total of 5 g. In addition to the difference in mortality, there was observed a marked difference in the lesions produced in the treated and control animals. Rhoads and Goodner 1 have reported oedema and a spread of the cutaneous lesion by gravity in rabbits inoculated endermally with pneumococci. These results were evident in our control group. The treated animals showed little or no oedema, and probably because of this, showed little or no spread by gravity. That an infection was present in the skin was evidenced by the area of inflammation in the skin of treated animals. The above authors have also stated that in occasional animals they find signs of hemorrhage in the skin. In the lesions of our control series there were extensive hemorrhages, caused either by the virulence of our strain or by the age of the culture.

Active Immunity to Pneumococci in Rabbits Protected by Sulfapyridine.

The results of experiments on the protection of rabbits, infected with pneumococci, by oral administration of sulfapyridine have been reported previously. 1 The purpose of this communication is to present data obtained in the study of the immunity in animals that had recovered from the infection produced under such circumstances. Rabbits weighing approximately 2 kilos were inoculated intracutaneously with 0.3 cc of a 1/100 dilution of an 8-hour culture of a pneumococcus type II. The inoculum contained an average of 300,000 organisms as determined by plate-count. The virulence of the organism is such that an average of 1.5 organisms are necessary to cause an infection in 66% of a statistically sized group of mice weighing 20 g. This we have taken to be our minimal lethal dose. Its virulence for rabbits is such that 0.3 cc of a 1/1000 dilution of the original culture, containing 30,000 organisms, will kill all of the rabbits inoculated. The drug-treated animals were given sulfapyridine orally in a 10% acacia suspension. Each animal received a total of 5 g of the drug, 0.5 g 1 hour before infection, 0.5 g 3 hours after the initial dose, and 0.5 g every 12 hours thereafter until the full dosage was administered. Daily record was made of the temperature, and the size and nature of the skin-lesion. Rabbits recovering from the primary infection were reinoculated intracutaneously in the same general areas as previously, but not at the site of the original lesion if a scar had persisted. Only normal areas of the skin were used for reinoculation. Type-specific Immunity. Twelve rabbits reinoculated intracutaneously with 0.3 cc of the whole 8-hour culture of the homologous type II pneumococcus 30 days after the original infection, showed no rise in temperature, and no local reaction around the point of inoculation.

Protective Action of Sulfapyridine Against Type II Pneumococcal Infections in Mice.

Summary With subcutaneous inoculations of 4000 to 8000 average lethal doses of a Type II pneumococcus in mice, the survival rates at both 30 and 60 days were (1) with 0.5% sulfapyridine in the food, 44%, and (2) with 1.0% sulfapyridine in the food, 63.4%. It is believed that the slight variations in drug-intake from day to day are more than counterbalanced by a more or less continuous drug-absorption from ingested food plus drug.

The effect of the surface tension of the medium upon bacterial toxins.

Tetanus toxin, diphtheritic toxin, botulinus toxin and endotoxin, obtained from the actinomyces gypsoides, were treated with castor oil soap to determine its effect upon these various toxins. Tetanus toxin and diphtheritic toxin were found to be completely detoxified when sufficient castor oil soap was added to reduce the surface tension below forty dynes. Guinea pigs treated with one hundred lethal doses of the respective toxins, with the soap, failed to develop symptoms. Botulinus toxin appears to be more resistant to soap than the other two soluble toxins. This observation offers a possible explanation of the fact that neither diphtheritic nor tetanus toxins are absorbed in toxic form by way of the digestive tract; while botulinus toxin is readily absorbed by the digestive organs. The contents of the intestinal canal having a low surface tension, because of the bile and soaps present, probably destroy tetanus and diphtheritic toxins, but not botulinus toxin. Nelson and Henrici1 have shown that the actinomyces gypsoides, an organism very pathogenic for guinea pigs, possesses a potent endotoxin which kills rabbits and guinea pigs regularly. When shaken in a two per cent solution of castor oil soap, so as to effect a thorough “wetting”, this organism loses its pathogenicity, and its endotoxin is also completely destroyed. Whether or not the effect of soap on bacterial toxins is entirely a surface effect, or is due, in part, to some other property of the soap, cannot be determined definitely at this time. Control experiments have convinced us that the action of the soap upon the bacterial toxins is not due to the low hydrogen ion. The effect of soap on the antigenic properties of bacterial toxins will1 be discussed in a future paper.

The precipitin test in the diagnosis of tuberculosis

The precipitin test has been found to give reliable results in the diagnosis of active tuberculosis. The antigen is prepared by disrupting tubercle bacilli, preferable an old culture, with carbon dioxide by the method described by Larson, Hartzell and Diehl. 1 The disrupted bacteria are filtered through paper in order to remove the shells. The clear filtrate is layered over the serum to be tested, and the tubes incubated for a period up to two hours. A definite cloudy ring at the interface of the two fluids indicates a positive reaction. The cloud often appears within the first five minutes. In the far advanced cases, however, the reaction develops more slowly, but is usually very definite. Upon standing several hours the ring gradually becomes dispersed. Thus far the blood serum of 190 cases have been examined. Of these, 100 were patients in the University Hospital and Dispensary, but not in the tubercular clinics. Ninety cases, representing all stages of tuberculosis, were from a local sanatorium. From the 100 cases not suspected of having tuberculosis, eleven positive reactions were obtained. Six of these have since been found to have evidence of tuberculosis. One died from hypertension, and at autopsy an active lesion was found in the apex of the right lung. An interesting case was that of a newborn, whose mother was a far advanced case, giving a negative Von Pirquet but a positive precipitin test. The blood of the newborn gave a heavy precipitin reaction. Of the 90 sanatorium cases 85 were positive and 5 negative. The 5 negative cases were either “healed” or “arrested.” We have found that the acid fast actinomycetes, as A. gypsoides and A. asteroides, make a good antigens as the tubercle bacillus.