Culex and Aëdes mosquitoes are capable of transmitting fowl-pox from diseased to healthy susceptible chickens. The mosquitoes remain infectious for at least 14 days following a meal on diseased fowls.

A study of the growth requirements of Spirochœta obermeieri resulted in the perfection of a method which enabled us (1) to cultivate the organisms consistently from the blood of infected mice and rats, (2) to maintain the viability of cultures for periods of at least 3 to 7 weeks, and (3) to carry them on in successive subcultures by transplanting at intervals of 2 to 4 weeks. This method is essentially the same as the Noguchi technique for the cultivation of the Leptospira group, but emphasizes control of the physicochemical factors that act to limit and prevent growth and prescribes the conditions necessary to counteract the injurious influences. The main facts may be briefly summarized as follows: ( a ) Ascitic fluid, horse or rabbit serum may be used as culture fluids. ( b ) These fluids become progressively more alkaline on exposure to air. ( c ) Uniformly successful results depend chiefly on the proper adjustment and stabilization of the reaction. ( d ) A balanced reaction can be secured by adding 1.0 per cent peptone broth or egg albumin as buffer, and covering the culture with a layer of oil. ( e ) The reaction limits for growth and survival are between pH 6.8 and 8.2, with the optimum at pH 7.2 to 7.4. ( f ) Spirochœta obermeieri is a strict aerobe, consequently in order to permit adequate aeration, the oil layer should not exceed 1.5 cm. in height.

Fourteen typical cases of yellow fever were studied in northern Peru during an epidemic occurring in 1920, nine in Payta in March and April, and five in Morropon and Piura in April and May. The method of investigation was similar to that previously employed, but as the laboratory facilities were very meager certain changes were required. Although in Payta the work was handicapped by the lack of electric light, the scarcity of water and animal food, the unsuitability of the guinea pigs for inoculation, and the changes in culture media due to age, the results obtained under these adverse conditions were by no means negative. While in no instance was there a typical infection produced in animals, either by direct inoculation of blood or with culture materials, yet certain guinea pigs in each series showed temporary febrile reactions or definite hemorrhagic lesions of the lungs indicative of a mild leptospira infection. Direct search for Leptospira icteroides in the blood of patients or in culture materials was not made because the dark-field microscope could not be used. Subsequently, at Piura, the laboratory facilities were vastly, improved, the use of the dark-field microscope was made possible by means of a storage battery, and a fresh stock of young healthy guinea pigs was received from New York, and fresh rabbit serum obtained in Piura. In the study of the materials obtained from five cases of yellow fever in Morropon all these added facilities were taken advantage of, with the result that the outcome was positive and convincing. Cultures from the five cases were examined after 11, 12, and 13 days, and in those from three cases living leptospiras were found. By inoculation into suitable guinea pigs of culture material from these five cases, irrespective of whether or not leptospiras were detected under the dark-field microscope, a typical Leptospira icteroides infection was produced from four of the five cases. In one of these no leptospira had been detected in the culture tubes. Thus one case only yielded negative results, in that no leptospiras were found under the dark-field microscope and the animal inoculation was negative. The leptospira was demonstrated in the blood or organ emulsions of the infected guinea pigs, and further transmission of each strain to other guinea pigs was obtained and pure cultures were secured. A few points of practical significance appeared in the course of the present investigation. One is the importance of using fresh rabbit serum for culture media. Old rabbit serum, whether in pure form or incorporated with agar, etc., which had been kept for several months in a tropical climate, proved to be unsatisfactory for obtaining a growth of Leptospira icteroides . A second point of interest is the variation in susceptibility of guinea pigs to infection with Leptospira icteroides . In two of four series of positive animal inoculations with the Morropon culture materials only one-half of the guinea pigs inoculated with given materials developed typical symptoms. The other half either suffered from a transient mild infection, as evidenced by a few hemorrhagic foci in the lungs, or escaped infection altogether. From these facts it is highly probable that the lung lesions and febrile reactions observed in certain guinea pigs inoculated with the Payta materials were due to a mild leptospira infection. In a comparative experiment the native guinea pigs procured in Payta were found to be more resistant to the leptospira infection than those recently brought from New York. In fact, only a small portion of the former succumbed to typical infection even when inoculated with a virulent strain of Leptospira icteroides obtained from the Morropon epidemic. In conclusion it may be stated that of fourteen cases of yellow fever studied in Peru, a typical leptospira infection, together with the demonstration of the organism in experimentally infected guinea pigs, was obtained in four, while in the majority of instances indications of a mild, non-fatal leptospira infection were observed. In a few cases only were the results entirely negative. The leptospira isolated from Morropon cases of yellow fever, which is morphologically and culturally identical with the Guayaquil and Merida strains of Leptospira icteroides , was also shown by immunity test to be indistinguishable from the Guayaquil organism.

Serum from yellow fever convalescents from Payta, Piura, and Morropon gave a positive Pfeiffer reaction with the strains of Leptospira icteroides isolated in Guayaquil and Merida. The serum also protected the guinea pigs from these strains in the majority of instances. The Pfeiffer reaction was complete with all recent convalescents (7 to 36 days) but slight or partial in some instances with serum derived from individuals who had had the attack of yellow fever 10 months previously. The virulence of the Morropon strains was found to be approximately the same as that of the Guayaquil or Merida strains. With one strain the minimum lethal dose for the guinea pig was less than 0.00001 cc. of a kidney emulsion from an infected guinea pig. Suitable quantities of the anti- icteroides serum administered to guinea pigs inoculated with 2,000 to 20,000 minimum lethal doses of infective material prevented the development of the infection, or a fatal outcome, according as the serum was given during the incubation period or after fever had appeared. The earlier the administration of the serum the smaller was the quantity needed; during the incubation period 0.0001 to 0.001 cc. was sufficient, during the febrile period 0.01 to 0.1 cc. was required to check the progress of the disease, and even at the time when jaundice had already appeared, the injection of 0.1 to 1 cc. saved three out of four animals inoculated with Strain 3 and one out of three inoculated with Strain 1. The native guinea pigs secured in Payta proved to be unusually refractory to infection with Leptospira icteroides as compared with normal guinea pigs recently imported from New York. Fresh rabbit serum is recommended for culture work with Leptospira icteroides .

Identification of the leptospira isolated from a case of yellow fever in Merida was accomplished by means of an anti-icteroides immune serum prepared in a horse with several Guayaquil strains of Leptospira icteroides . The immune serum showed a protective action of high titer against the Merida strain, thus establishing its efficacy as a therapeutic agent against this strain. Polyvalent anti-icteroides immune serum prepared in the horse or monovalent anti-icteroides immune serums prepared in the rabbit had a definite devitalizing action upon the Merida strain, while immune serums similarly prepared with strains of icterohœmorrhagiœ had no perceptible effect upon the Merida strain. Serums from yellow fever convalescents in Merida gave a positive Pfeiffer reaction with the Merida strain of Leptospira icteroides . The reactions between the Guayaquil strains (Nos. 1 and 5) and two of these serums from convalescents varied from definitely positive to doubtful, owing probably to the diminution of active immune principles in the serums during the prolonged and unfavorable conditions of their transportation.

Injections into guinea pigs of the blood and the emulsions of liver and kidney obtained at autopsy from a fatal case of yellow fever in Merida induced in some of these animals, after a period of several days incubation, a rise of temperature which lasted 1, 2, or more days. When killed for examination at this febrile stage the animals invariably showed hemorrhagic areas of various size, sometimes few and sometimes numerous, in the lungs, and also, though less constantly, in the gastrointestinal mucosa, together with general hyperemia of the liver and kidneys. In a guinea pig (No. 6) inoculated with the liver emulsion of Case 1 there was a trace of jaundice on the 9th day. Injections of the blood or liver and kidney emulsions from such animals into normal guinea pigs reproduced the febrile reactions and the visceral lesions. The majority of the animals which were allowed to live and complete the course of the infection rapidly returned to normal (within several days). Examinations of these surviving guinea pigs after 2 weeks revealed the presence of rather old hemorrhagic foci in the lungs. In the course of further attempts to transfer the passage strain, a secondary infection by a bacillus of the paratyphoid group caused many deaths among the guinea pigs and resulted finally in the loss of the strain from Case 1. Most of the cultures made with the heart's blood taken at autopsy from Case 1 proved to be contaminated with a bacillus of the coli group. The contents of the apparently uncontaminated tubes were inoculated into guinea pigs, but the results were for the most part negative or vitiated by a secondary infection. Dark-field search for the leptospira with the autopsy materials was negative, although prolonged and thorough examination was not practicable at the time of these experiments. Our efforts were concentrated on obtaining positive animal transmission rather than on the time-consuming demonstration of the leptospira, which when unsuccessful does not necessarily exclude the presence of the organism in small numbers. Likewise, the dark-field work with the material from guinea pigs was confined to a brief examination and was omitted in many instances. Under these circumstances no leptospira was encountered in any of the material from Case 1. On the other hand, the results obtained with the specimens of blood from Case 2 were definitely positive, not only in the transmission of the disease directly, or indirectly by means of cultures, into guinea pigs, but also in the demonstration of the leptospira in the primary cultures and in the blood and organ emulsions of guinea pigs experimentally infected with such cultures. Definite positive direct transmissions were obtained with the specimens of blood drawn on the 2nd and 3rd days. No blood was taken on the 4th or 6th days. There were indications of abortive or mild leptospira infection in the guinea pigs inoculated with the blood taken on the 5th day. Regarding the inoculation of cultures from Case 2, it may be stated that only the cultures (leptospira +) made with the blood drawn on the 2nd day caused a definite fatal infection in guinea pigs. From this series a continuous passage in the guinea pig has been successfully accomplished. One of the guinea pigs (No. 48) inoculated with the culture 5 days old (leptospira +) made from the blood taken on the 3rd day presented typical symptoms, and a positive transfer from this to another animal (No. 98) was also made. Cultures of the blood drawn on the 5th and 7th days gave unsatisfactory results, owing to a secondary contamination. Leptospiras were detected in some of the culture tubes containing 2nd and 3rd day specimens of blood from Case 2; they were few in number and for the most part immotile, owing perhaps to some unfavorable cultural condition such as a fungus contamination. Charts 17, 18, and 19 give a summary of the experiments. See PDF for Structure

With the methods which have been described we have separated an exotoxin and an endotoxin from cultures of the Shiga dysenteric bacillus. The study of the nature and effect of the poison of this microorganism is thus simplified. The two toxins are physically and biologically distinct. The exotoxin is relatively heat-labile, arises in the early period of growth, and yields an antiexotoxic immune serum. The endotoxin, on the other hand, is heat-stable, is formed in the later period of growth, and is not neutralized by the antiexotoxic serum. The exotoxin exhibits a specific affinity for the central nervous organs in the rabbit, giving rise to a characteristic lesion—mainly, hemorrhages, necroses, and possibly a perivascular infiltration in the gray matter of the upper spinal cord and medulla. The endotoxin exerts a typical action on the intestinal tract, producing edema, hemorrhages, necroses, and ulcerations, especially in the large intestine. In dysentery in man the intestinal lesions predominate, but in severe epidemics paralysis and neuritis have been observed (Osler 17 ). These facts become specially significant from the standpoint of the serum therapy of bacillary dysentery. A potent antidysenteric serum should contain antibodies against the exotoxin as well as the endotoxin. That such a serum can be produced in horses has been experimentally demonstrated.

The growth of all the pathogenic bacteria studied was favorably influenced by the addition of small amounts of tissue extracts. Beef heart, rabbit and cat tissues, and human nasal secretions contain substances favorable to the growth of the organisms tested. The mucosa of different organs, spleen, liver, and kidney, are relatively rich in these substances, while muscle is relatively poor. The favorable effect of the extracts is manifested by an enhancement of growth and a reduction of lag. The water-soluble substances are apparently the ones essential for bacterial development; the ether extract has no effect on growth. Experiments are reported which indicate that the substances in question belong to the class of so called vitamines.

The study of the cultural and agglutinating reactions of seventeen strains of Morgan bacilli is reported. The cultures were obtained from different sources. Culturally all the strains were identical. Antigenically they were highly diversified. The sera produced against some strains have marked agglutinating power for other strains, but absorption tests showed that the cross-agglutinations were often due to group agglutinins. While no conclusion can be drawn regarding the pathogenic significance of this bacillus, the wide diversities of antigenic properties raise the question as to the specific relationship of the various cultures met with as well as their relation to a definite class of pathological processes in man.

The three media as modified—Endo medium, neutral red-brilliant green, and the lead acetate Russell double sugar—have proved in our hands the best combination for the isolation and rapid differentiation of the various organisms belonging to the typhoid-dysentery group of bacilli.

This study of the inhibitive effect of aniline and some of its derivatives and of the triphenylmethane dyes on certain bacteria warrants the following tentative conclusions: 1. The composition and reaction of the medium exert a marked influence on the behavior of the antiseptic. The higher the concentration of organic nitrogenous compounds (peptone) in the medium, the lower is the effective concentration of the dye. The reaction of the medium modifies the specific action of the antiseptic, owing probably to an alteration in the bacterial cell. 2. The germicidal action of the compounds is a function of the benzene nucleus, the added elements or radicals, their number, and, in the case of the dyes, probably the quinoid structure of the nucleus. 3. As far as tested, the increase in the number of alkyl radicals increases the antiseptic power. Methyl green is an interesting exception to this rule, for the change of one of the nitrogens to the quaternary salt is accompanied by an almost complete loss in inhibitive action. 4. The antiseptic power is enhanced to a greater extent by an ethyl than a methyl group, and the second alkyl produces a proportionately greater increase than the first. It appears that the relative position of the introduced group may be a factor in determining the relative improvement in the effectiveness of the compound. 5. The introduction of a methyl group in the nucleus consistently enhances the inhibitive action of the compound and its alkyl derivatives. This is evident from a comparison of the action of aniline and its derivatives with that of toluidine and its corresponding derivatives. 6. The simple aniline derivatives, as well as the dyes, are more toxic for the Gram-positive than the Gram-negative bacteria. Of the former, Bacillus subtilis is more sensitive to the dyes than Staphylococcus aureus , while the reverse is true in the case of the aniline compounds. 7. The most marked specific selective effect is manifested by the triphenylmethane dyes. Bacillus aerogenes and Bacillus typhosus possess a higher resistance to these substances than Bacillus coli or Bacfflus dysenterice . The last is exceedingly sensitive. This partial specificity is apparently a function of the molecule as a whole.