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.