ETIOLOGY OF YELLOW FEVER
Authors: Noguchi Hideyo 
Published: June 2004

In several series of experiments guinea pigs were variously infected with different amounts of Leptospira icteroides, either in the form of culture, organ emulsion from infected guinea pigs, or a mixture of both. The infecting materials were of different grades of virulence; in some series the amount given was near a single lethal dose, in others a subminimum lethal dose was given, i.e. causing mild infection with recovery in the majority of animals, and in still others the animals were injected with at least 50 minimum lethal doses of a mixture of a culture and a highly virulent organ emulsion from a guinea pig. The animals were inoculated intraperitoneally, and within about 30 minutes each was injected subcutaneously with a different amount of salvarsan or neosalvarsan. The amounts injected were in most series 0.0005, 0.001, 0.002, 0.005, 0.01, 0.02, and 0.03 gm. per 350 to 450 gm. of body weight, and in one series, in addition to this dosage, 0.00005,0.0001, and 0.0002 gm. were also tried.
Among the guinea pigs treated either with salvarsan or with neosalvarsan there were more recoveries than among the controls, but they were not in strict proportion to the amounts of the drugs injected. In the experiments with 50 minimum lethal doses of the infecting material there were several recoveries among those which received 0.001 to 0.002 to 0.003 gm., but all passed through a typical infection with all its symptoms. It is extremely doubtful, therefore, whether salvarsan or neosalvarsan mitigated the severity of the infection. The fact is noteworthy that in the same series of experiments the guinea pigs receiving 0.00005 and 0.0001 gm., or thereabout, of salvarsan died 1 to 2 days sooner than the controls, which died in 6 to 7 days. This suggests a possible earlier injury of the kidneys by the drugs, giving the leptospiras an easier and earlier access to, and localization in this organ. The inefficacy or dubious therapeutic value of salvarsan and neosalvarsan against the experimental icteroides infection of guinea pigs presents a close analogy to the observations already made by several investigators with Leptospira icterohæmorrhagiæ.
Several series of test-tube experiments were also made to determine the direct effect of salvarsan and neosalvarsan on Leptospira icteroides cultures. It was found, the injurious effect of alkalinity being eliminated, that the leptospiras remain motile for at least I hour in a concentration weaker than 1:10,000 of salvarsan or 1:1,000 of neosalvarsan. But they become gradually sluggish and succumb to the effect of the drugs at the end of 18 to 24 hours. The highest dilution which killed the leptospira in 18 hours was somewhere near 1:200,000.
When added to a culture medium, salvarsan and neosalvarsan both suppressed the growth of icteroides when their concentration in the medium was 1:200,000. Hence these two drugs are highly poisonous for Leptospira icteroides.
The serums derived from rabbits which received 0.05 gm. of salvarsan or neosalvarsan per kilo of body weight 1 hour before bleeding proved to be very different from a normal rabbit serum in their behavior toward Leptospira icteroides. In the salvarsanized or neosalvarsanized serums the leptospiras remained active for at least 1 hour but appeared somewhat sluggish at the end of 18 hours, and were all dead and degenerated when examined after 48 hours. On the other hand, the leptospiras mixed with normal rabbit serum lived well and multiplied during the same period of time and under otherwise identical conditions (at 28°C.) To these tubes another portion of culture was added to determine whether or not a rapidly detrimental toxic substance had appeared in the drugged serum while standing for 72 hours, but the organisms remained still active at the end of 1 hour, 24 hours being required to kill them. In another experiment the salvarsanized and neosalvarsanized serums, together with normal serum as a control, were first left standing for 72 hours, after which period a rich culture of icteroides was introduced. The organisms remained uninfluenced for 1 hour in all the serums, but at the end of 24 hours many of those in the drugged serums were dead, and none was left alive at the end of 48 hours. In normal serum they steadily increased in numbers and were all active.
It is evident, then, that salvarsan or neosalvarsan introduced intravenously into the body of the rabbit is present in some form in the blood serum drawn at the end of 1 hour. The substance present in such serum has a slowly operating injurious effect upon Leptospira icteroides. The action of the drugs seems to be slower after passage through the animal body than before. If this phenomenon were to take place also in the infected body injected with these drugs, it is obvious that in a rapidly evolving infectious disease like yellow fever the progress of the infection will be too rapid to allow the drugs to exert their beneficial effect upon the course of the disease.
In direct contrast to the behavior of salvarsan and neosalvarsan in vivo and in vitro, anti-icteroides immune horse serum in a dose of 0.0001 cc., or 1 cc. of a 1:10,000 dilution, protected guinea pigs from an infection with at least 5,000 minimum lethal doses of icteroides when injected simultaneously, but the same serum failed to exert any injurious effect upon the organism when mixed in vitro in a concentration weaker than 1:2,000. A rapid disintegration resulted with a concentration of 1:20 and almost complete agglutination and degeneration in 1:200.
The contrast between chemotherapy, as carried out with salvarsan and neosalvarsan, and serotherapy demonstrated with an immune serum is apparently of considerable practical significance.