Separation of Viable Rickettsia typhi from Yolk Sac and L Cell Host Components by Renografin Density Gradient Centrifugation

Abstract

Rickettsia typhi cultivated in the yolk sac of chicken embryos or in L cells irradiated 7 days previously was separated from host cell components by two cycles of Renografin density gradient centrifugation. Preliminary steps involved differential centrifugation and centrifugation over a layer of 10% bovine plasma albumin of infected yolk sac suspensions, or trypsinization and passage through filters of wide porosity of infected L cell suspensions. Rickettsial preparations obtained by these methods appeared to be free from host cell components while retaining high levels of hemolytic activity, egg infectivity, and capacity to catabolize glutamate. Average yields were 3.3 mg of rickettsial protein per yolk sac or 0.44 mg per 16-oz (ca. 475-ml) L cell culture. Extracts from these two preparations displayed malate dehydrogenase activity of electrophoretic mobility identical to each other but quite different in migration patterns from the corresponding host cell enzymes. This method of separation of rickettsiae from host cell constituents appears to be particularly well suited for the study of rickettsial enzymatic activity.

A Fluorescent Antibody Test for Detection of the Rickettsia Causing Disease in Chilean Salmonids

“…The antigen preparation was partially purified with a modification of the method of Weiss et al (1975). Briefly, spent medium was collected from five 150-cm 2 flasks of fully lysed CHSE-214 cells, and rickettsiae with accompanying host cells and debris were concentrated by centrifugation at 17,300 x gravity for 15 min.…”

Phylogenetic position of Rickettsia tsutsugamushi and the relationship among its antigenic variants by analyses of 16S rRNA gene sequences

“…Rickettsia tsutsugamushi, a causative agent of scrub typhus, is unique among Rickettsia species because of its lack Rickettsia sibirica, ATCC VR151T (R = reference strains, T = type strain), were used. All strains were propagated in suspension culture of L929 cells, and R. tsutsugamushi strains and R. sibirica were purified by Percoll and Renografin density gradient centrifugation, respectively [13,14]. from R. tsutsugumushi were prepared as described previously [151.…”

Purification of Cell Envelopes of Rickettsia tsutsugamushi

“…Differences between these rickettsial envelopes were recognized by morphological observations (5). Purification procedures for the other rickettsiae by Renografin density gradient centrifugation ( 1,8) were not applicable to R. tsutsugamushi due to its surface properties difficult to separate from host cell components, this suggesting also that the adhesive nature of the envelope of this rickettsia is different from that of the others. On the other hand, Eisemann and Osterman (2) described antigens in partially purified fractions of the envelopes of R. tsutsugamushi prepared by differential centrifugation from the rickettsial homogenate, and Hanson (3) mentioned some proteins localized in the outer membrane by monitoring the constituents before and after treatment of rickettsia with detergents.…”