Immunological and biological characterization of Coxiella burnetii, phases I and II, separated from host components

Abstract

Coxiella burnetii, phase I and II, cells cultivated in the yolk sac of chicken embryos were separated from host cell components by two cycles of isopycnic Renografin gradient centrifugation. Initial steps in the purification of viable C. burnetii involved differential centrifugation and sedimentation through an aqueous solution of 30% sucrose and 7.6% Renografin. After the first, but not the second, cycle of Renografin gradient centrifugation, the cells were passed through microfilter glass filters which facilitated the removal of host components. The integrity of morphologically different cell variants was maintained during purification procedures by suspending highly purified C. burnetii in phosphate-buffered saline-sucrose solutions. C. burnetii, phases I and II, obtained by these methods appeared to be free from host cell components by serological methods while retaining morphological integrity and infectivity for yolk sacs and experimental animals. Average yields of C. burnetii were 2.83, 1.5, and 0.84 mg (dry weight) per yolk sac of the Ohio strain (phase I), 9 Mile strain (phase I), and 9 Mile strain (phase II), respectively. Recovery of phase I cells averaged about 70%, whereas the recovery of phage II cells was approximately 40%. The temporal sequence of phase I and II antibody response was demonstrated in infected and vaccinated animals. Also, no antibody response in mice and guinea pigs to yolk sac antigens was detectable after two injections of vaccine or viable cells. Importantly, this is the first report of the separation of viable phase II cells of C. burnetii free of host components.

Biochemical and immunological properties of Coxiella burnetii cell wall and peptidoglycan-protein complex fractions

“…Organism and growth conditions. The cultivation and separation of phase I C. burnetii (Ohio strain [CBOI]) from host components were previously described (26).…”

“…C. burnetii cell variants were separated according to susceptibility and resistance to osmotic shock, ultrasonication, and pressure treatment followed by lowand high-speed centrifugations. Purified C. burnetii variants were stored at -20°C in phosphate-buffered saline-sucrose (26). The cells were thawed at room temperature, and subfractions were prepared as follows.…”

Identification ofCoxiella burnetiisurface-exposed and cell envelope associated proteins using a combined bioinformatics plus proteomics strategy

“…Handling of live bacteria was in a Biosafety Level 3 laboratory. Bacteria were inactivated with 0.5% phenol and purified by rate‐zonal sedimentation in gradients of Renografin (Ultravist 370; 0.769 g/mL) at 112 500 × g and 4°C for 90 min . Gimenez staining was used to evaluate the cell purity.…”

Expression of β-lactamase in Coxiella burnetii transformants

“…PII: S 0 3 7 8 -1 0 9 7 ( 0 0 ) 0 0 0 6 4 -1 lated and partially puri¢ed from host cells as previously described [8]. Organisms were further puri¢ed by centrifugation in celite [9] or in 30% sucrose (w/v), 7.6% renogra¢n in PBS [10]. Cell preparations used for electroporation were stored at 320³C or used immediately and DNA was introduced into C. burnetii cells by electroporation using conditions previously described [6].…”