James H. Rust

SUSCEPTIBILITY OF RODENTS TO ORAL PLAGUE INFECTION: A MECHANISM FOR THE PERSISTENCE OF PLAGUE IN INTER-EPIDEMIC PERIODS

Oral infection of rodents with Pasteurella pestis has been demonstrated with both fully virulent and avirulent strains. Sustained rodent plague epizootics have been initiated and maintained in the absence of the classical flea vector. Transmission was due to cannibalism of the dying rodents by their healthy cagemates. Oral infection is considered to provide a plausible mechanism for the persistence of plague in an area where conditions are temporarily unsuitable for flea transmission.

Plague Toxin: its Effect in vitro and in vivo.

The murine toxin of Pasteurella pestis inhibited the respiration of heart mitochondria from the rat and the mouse but had little or no effect on the respiration of mitochondria from the rabbit, chimpanzee, dog, and monkey. Alterations occurred in tile S-T segments of the electrocardiogramus recorded corded from rats injected with � to 10 LD50 of toxin, but not in those from rats dying of hemorrhagic shock, hypoxia, intoxication with glucose, or Escherichia coli endotoxin. No abnormalities were observed in electrocardiograms from rabbits injected with large amounts of toxin.

THE BIOCHEMISTRY AND PHYSIOLOGY OF THE PLAGUE MURINE TOXIN

Our work on microbial toxins started in 1952 when the United States Army was interested in plague because of the possibility, during the Korean conflict, that United States troops might be exposed to it in Manchuria, which has been the historical breeding place of pandemics of plague. Investigations on plague toxin have been prompted by the observations of McCrumb el aZ.,l in Madagascar, that antibiotics administered 36 to 48 hours after onset of the disease failed to save patients in spite of the fact that on autopsy the organs and blood were sterile. Furthermore, such plague victims a t autopsy showed typical symptoms of so-called toxic deaths. Our primary objective from the start of this work has been to isolate and purify this toxin and to study the mechanism of its action at an enzymatic level. At the time this work was started, the plague bacillus was known to contain several distinct and readily separable antigenic components? Two of these, the envelope antigen and the toxin, have been studied in some detail, particularly in regard to their role in immunity. The recent work of Burrows and his co-workers3 and Lawton and his co-workers4 revealed a number of additional significant antigenic components, but our interests have remained in the toxin that Pasfewella pestis produces : the toxin responsible for the death of mice or rats and consequently referred to as the “murine” toxin. Our initial studies dealt with the purification of the toxin by chemical means.6 These involved extraction of the toxin from acetone-dried cells with 2.5 per cent sodium chloride, followed by ammonium sulfate and isoelectric precipitations, treatment with magnesium chloride and alcohol precipitation, calcium phosphate gel absorption and elution, and chloroform extraction. The product thus obtained behaved as a homogenous protein in the ultracentrifuge and electrophoresis cells and was found to be free of nucleic acids, carbohydrates, and capsular material. However, the purity of this material, when ascertained by the very sensitive immunological gel diffusion precipitation reactions of Oudin and Ouchterlony, showed a t least two and, often, three or more individual zones of precipitation. Since our major objective had been to determine the mechanism of action of the toxin a t an enzymatic level, it became imperative to obtain preparations that were “pure.” The high resolving power for fractionating protein mixtures afforded by continuous paper electrophoresis led to the employment of this technique for the final purification of the toxin.0 The continuous-flow, hanging-curtain electrophoresis apparatus developed by Karler’ has been utilized in the isolation from crude lysed cell cultures of small quantities of toxin that exhibited one band in the very sensitive gel diffusion precipitation reactions.

Serological Response of Rhesus Monkeys (Macaca mulatta) to Immunization and Infection with Pasteurella pestis

Summary Living attenuated Pasteurella pestis vaccine EV76(51f) was an effective immunizing agent for Macaca mulatta by either the oral or parenteral route. As determined by serological tests and challenge experiments, a killed plague vaccine was less effective.