Cultivation of European Type of Typhus Rickets in Presence of Live Tissue

Abstract

The cultivation of Rickettsia presents considerable difficulties. Successful cultivation, with demonstrable rickettsia in cells, has been reported in tissue cultures by Wolbach and Schlessinger, 1 and Zinsser and Batchelder. 2 Pinkerton and Hass, 3 and Nigg and Landsteiner 4 were the first to obtain positive subcultures through several successive generations, the latter in flask or Maitland cultures. The last named authors worked with strains producing scrotal lesions in guinea pigs and used the tunica vaginalis infiltrated with rickettsia as the infective material. Pinkerton and Hass, though presumably working with a European strain, report the production (though irregular) of scrotal lesions in guinea pigs.

Our attempts to cultivate a European strain∗ by the methods used by Pinkerton and Hass or Nigg and Landsteiner failed to give positive cultures. Brain as well as tunica tissue was used as infective material but no rickettsia grew out. We, therefore, changed the procedure and used infected lice intestines for starting the culture.

In the experiments reported below successful initial cultures as well as subcultures were obtained by using infected lice intestines as infective material and following in all other respects the procedure employed by Nigg and Landsteiner. Apparently the positive outcome of these experiments is due to the use of heavily infected lice tissue, containing large numbers of rickettsia, to initiate the culture.

The technique was briefly as follows: Infected lice, 8-10 days after having been infected by the Weigle technique, were sterilized by treatment with alcohol and the guts removed aseptically into a drop of saline in a sterile hollow slide. Normal guinea pig tunica was macerated in the drop together with the infected louse gut and the mixture allowed to stand for 15-30 minutes. Small amounts of this material were then placed in small rubber-stoppered flasks containing 3 cc. tyrode-serum (2.5 cc. tyrode solution and 0.5 cc. fresh guinea pig serum). At first the cultures where placed at 37°C, but subsequently they were kept at 30°C. At the latter temperature cultures as well as transplants were obtained. The following typical protocols give the procedure and the results obtained thus far: