An Immunological Purification of Typhus Rickettsiae

Abstract

The demonstration of independent metabolism in obligate intracellular parasites is of importance for an understanding of their activity in the host cell. The observation of Bovarnick and Snyder (1949) that purified suspensions of typhus rickettsiae prepared from infected yolk sacs oxidized glutamate, and to a leser extent pyruvate and succinate, has stimulated the search for other enzymes in these organisms. Bovarnick and Miller (1950) reported a glutamate-aspartate transammnase, and Wisseman et al. (1952) concluded on the basis of their observations that the enzymes active against a-ketoglutarate, fumarate, malate, and oxalacetate were also present in rickettsiae. In this laboratory it was noted that marked esterase activity was present in the suspensions prepared in a manner similar to that of Bovarnick and of Wisseman. These observations raised the question whether the enzyme activities should be attributed to the microparasite or to traces of host material which might not be eliminated in the purification procedure. This paper reports experiments designed to solve this problem. MATERIALS AND METHODS RickleUsio2 suspension. The Wilmington strain

METABOLIC ACTIVITY INCOXIELLA BURNETII

“…This final preparation was a dense, white suspension of C. burnetii. Contaminating material could not be detected in these suspensions by direct microscopic examination, by the sucrose density-gradient technique (Ribi and Hoyer, 1960), by complement-fixation (CF) tests with antiyolk-sac serum, or by the formation of a visible precipitate upon the addition to the suspension of antiserum containing antibodies to normal yolk-sac tissue (Karp, 1954). The yield, which was estimated turbidimetrically (Ormsbee, 1964), was 200 to 600 ,ig of rickettsial cells per gram of yolk sac.…”

Some characteristics of heavy and light bands of Rickettsia prowazekii on Renografin gradients

“…Rapidly thawed yolk sac suspensions were mixed with 1.67 volumes of 50% (wt/vol) sucrose in brain heart infusion broth (BHI; 3.7%; BBL Microbiology Systems, Cockeysville, Md.). After centrifugation at 16,300 x g (Sorvall GSA rotor), the rickettsial pellet was suspended in 50 to 100 ml of a 1/10 dilution of rabbit anti-normal yolk sac serum (10) in 6% bovine serum albumin (BSA; Armour fraction V, Reheis Chemical Co., Phoenix, Ariz.) (3) in 122 mM KCl + 23 mM NaCl (pH 7.0). The suspension was held in an ice bath for 60 to 90 min, and the flocculated host material was removed by centrifugation at 500 x g for 10 min.…”

Effects of Cortisone on Experimental Murine Typhus I

“…A modification of the Poltz ether extracted rickettsial antigen was used (Craigie, 1945). To remove the yolk sac material which remained after ether extraction and washing in buffered saline the rickettsiae were treated with antiyolk sac serum as described by Karp (1954). After the last centrifugation, the rickettsiae were resuspended in sterile saline and made up to a standard suspension giving a turbidity reading of 20 in the Coleman spectrophotometer at a wave length of 600 and 1:10,000 merthiolate was added.…”