Coxiella burnetii (Cb) in phase I (Ph I) multiplies very poorly in L-cells as shown by light and electron microscopy and infectivity titrations; Cb antigens, however, are detectable by immunofluorescence in most cells. Phase II (Ph II) organisms, in contrast, multiply well and produce typical particles in well-defined cytoplasmic vacuoles. Density gradient centrifugation has been applied to test the heterogeneity of the animal-maintained Nine Mile strain in Ph I and the corresponding egg-grown strain in Ph II. The evidence indicates that both strains are mixtures of Ph I and Ph II organisms, although they reacted as Ph I or Ph II antigens in the complement fixation test. The interaction of Ph I and Ph II organisms was studied in mixed infections in L-cells; suppression, or a less marked enhancement, of the growth of Coxiella burnetii was observed, depending on the conditions. It is suggested that Ph I and Ph II rickettsiae represent genetically different but closely related organisms, Ph I being the "parent" strain and Ph II the "mutant" strain; "phase variation" of Coxiella burnetii results from a selection of the mutant in the population under the prevailing environmental conditions.

Paretsky , D. (University of Kansas, Lawrence), C. M. Downs, and C. W. Salmon . Some biochemical changes in the guinea pig during infection with Coxiella burnetii . J. Bacteriol. 88: 137–142. 1964.—Guinea pigs infected with Coxiella burnetii , the rickettsial agent of Q fever, were studied for 11 days postinfection. Maximal changes in liver lipids, liver phosphorylase, and uridine diphosphate glucose (UDPG)-glycogen glucosyl-transferase activities occurred 3 to 4 days post-infection. In this period, total liver lipids increased from 1.26 to 5.46 mg/mg of N, with the largest increment in the glyceride fraction. Liver glycogen virtually disappeared by the second day, with no chemically detectable restoration until the eleventh day. A pattern of altered phosphorylase and UDPG-glycogen transglucosylase activities was observed, with maximal phosphorylase and minimal glucosyltransferase activities at the third and fourth days. Histochemical observations confirmed chemical analyses for lipids and glycogen.

Paretsky , D. (University of Kansas, Lawrence), R. A. Consigli, and C. M. Downs . Studies on the physiology of rickettsiae. III. Glucose phosphorylation and hexokinase activity in Coxiella burnetii . J. Bacteriol. 83: 538–543. 1962.—Disrupted Coxiella burnetii preparations phosphorylate glucose by a hexokinase system. Glucose-6-phosphate-P 32 , synthesized by the rickettsiae in the presence of P 32 -labeled carbamyl phosphate and glucose, has been isolated as the barium salt and by ion exchange and paper chromatography. C. burnetii hexokinase activity is inhibited by high concentrations of adenosine mono-, di-, and triphosphates, suggesting similarities to hexokinase from other sources.

Journal Article Growth of Pasteurella Tularensis in Cultured Cells Get access John Merriott, John Merriott From the Department of Bacteriology, University of Kansas, Lawrence, Kansas Search for other works by this author on: Oxford Academic PubMed Google Scholar Alice Shoemaker, Alice Shoemaker From the Department of Bacteriology, University of Kansas, Lawrence, Kansas Search for other works by this author on: Oxford Academic PubMed Google Scholar Cora M. Downs Cora M. Downs From the Department of Bacteriology, University of Kansas, Lawrence, Kansas Search for other works by this author on: Oxford Academic PubMed Google Scholar The Journal of Infectious Diseases, Volume 108, Issue 2, March 1961, Pages 136–150, https://doi.org/10.1093/infdis/108.2.136 Published: 01 March 1961 Article history Received: 26 July 1960 Published: 01 March 1961

Summary An attenuated USSR vaccine culture of P. tularensis was examined for dissociation and found heterogeneous with regard to colony type morphology. Live BV, the more immunogenic of two variants isolated from the culture, proved effective for the protection of the mouse against s.c. challenge with strain SCHU S4; in contrast, killed cultures were not immunogenic. Guinea pigs vaccinated with live BV cells and challenged with strain SCHU S4 exhibited an appreciably longer survival time than animals vaccinated with killed preparations but eventually they succumbed. Although SCHU S1-11, one of several colony type variants of reduced virulence isolated from strain SCHU S1, proved equally as immunogenic for the mouse as BV and afforded the guinea pig greater protection than BV, the relatively high residual virulence of this variant for the guinea pig precluded its use as a vaccine strain. Serial animal passage of BV permitted the isolation of a culture that was ultimately selected for the production of live vaccine. The culture was designated the live vaccine strain (LVS) and is currently being used for the production of lyophilized live vaccine for use in man. Strain SCHU S4 proved superior to strain 503 as a challenge strain for quantitating the protection afforded test animals administered live vaccines. Data on the immunogenicity and storage stability of lyophilized live tularemia vaccine produced from cells harvested from GCHA and more recently from MCPH proved sufficiently encouraging to have warranted an extension of this study to a systematic evaluation of the vaccine in the monkey and subsequently in man.

Journal Article The Phagocytosis of Pasteurella Tularensis by Rat Mononuclear Cells as Influenced by Normal Serums and Various Irritants Get access Helen Mcelree, Helen Mcelree From the Department of Bacteriology, University of Kansas, Lawrence, Kansas Search for other works by this author on: Oxford Academic PubMed Google Scholar Cora M. Downs Cora M. Downs From the Department of Bacteriology, University of Kansas, Lawrence, Kansas Search for other works by this author on: Oxford Academic PubMed Google Scholar The Journal of Infectious Diseases, Volume 109, Issue 1, July 1961, Pages 98–106, https://doi.org/10.1093/infdis/109.1.98 Published: 01 July 1961 Article history Received: 13 January 1961 Published: 01 July 1961

The growth of rickettsiae in tissue culture cells has provided a simplified technique for studying the physiological and morphological properties of intracellular rickettsiae and the effects of rickettsial multiplication on the living host cell. Early workers in this field were Nigg and Landsteiner (1932) and Pinkerton and Haas (1931, 1932a, b) who studied Rickettsia prowazekii in minced guinea pig tunica and cultures of scrotal sac exudate, respectively. Pinkerton and Haas (1932c) also observed extensive nuclear invasion of the spotted fever rickettsia in tissue culture cells. Q fever rickettsiae were cultivated by l3urnet (1938) in minced tissues obtained from 12-day chick embryos. Two series of cultures were carried concurrently in tissue culture for over 4 months. The first appearance of rickettsiae in the infected cells was 4 days after initial penetration, the organisms being distributed in groups throughout the cytoplasm and eventually promoting degenerative changes in the host cells. Weiss and Pietryk (1956) succeeded in growing the California strain of Coxiella burnetii in monolayer cultures of chick entodermal cells. The rickettsiae could not be observed in the cytoplasm of the cells until the 4th day and did not become numerous until the 5th day of infection. Cell to cell infection apparently occurred, for the infection usually remained confined to a few contiguous cells, forming discrete foci of infection, from which the "focus count" method of titration was developed. The MB III strain of cells was used by Bozeman et al. (1956) in studying rickettsial growth. Most of the experiments were concentrated on the Karp strain of Rickettsia tsutsugamushi, whereas other rickettsiae such as the Gilliam strain of R. tsutsugamushi, the Breinl strain of R. prowazekii, the Wilmington strain of Rickettsia mooseri, the Bitterroot strain of Rickettsia rickettsii and the Henzerling strain of C. burnetii were also studied. These rickettsiae grew well in the tissue culture cells but differed in the location of their initial infections, R. tsutsugamushi growing in areas adjacent to the nucleus, whereas R. mooseri, R. prowazekii, R. rickettsii, and C. burnetii multiplied in scattered areas throughout the cytoplasm of the cells. Recent studies on the growth and morphology of rickettsiae in tissue culture have been made by Schaechter et al. (1957) using the 14pf strain of rat fibroblasts. Infected cells of both the eggadapted lines of the Karp strain of R. tsutsugamushi and the B3itterroot strain R. rickettsii were observed using phase microscopy. The size and morphology of the organisms and their mode of escape from the infected cells were described. The division of single cells of R. rickettsii by binary fission was recorded photomicrographically on three separate occasions. Several workers have substantiated the hypothesis that rickettsial organisms divide by transverse fission. Bacot et al. (1921) supported this view using light microscopy techniques as did Wissig and co-workers (1956) who made their observations with the electron microscope. In the present paper, the multiplication of C. burnetii in the L strain mouse fibroblast provided a highly satisfactory system with which to investigate various aspects of this host-parasite relationship. The effects of rickettsial invasion on the host cells, the minimum time necessary for infection to occur, the extent of rickettsial multiplication in the cells over various periods of incubation, the effects of streptomycin on rickettsial invasion and multiplication, and maintenance of the rickettsiae through numerous transfers in the tissue culture cells were studied. The fluorescent antibody technique was employed to substantiate identity of the organism and to demonstrate the specificity of this system.

Since the use of fluorescein labeled antibodies for the selective staining of homologous antigens was introduced by Coons and associates,',2 the technique has received wide use and has proved to be a valuable tool in immunologic and microbiologic studies. In addition to the use of fluorescein isocyanate as a labeling agent, as described by Coons, the use of an orange fluorescing isocyanate of rhodamine B has been reported by Silverstein, Eveland and Marshall.8 By using the rhodamine B labeled antibody simultaneously with a different antibody labeled with fluorescein isocyanate, they were able to differentiate between two organisms of unlike antigenic composition in the same smear preparation, one organism appearing red-orange and the other yellow-green when viewed in the ultraviolet microscope. Because of the difficulty in preparation, the danger in the use of phosgene, and the instability of the isocyanates described by Coons and Silverstein, we have undertaken studies to determine if the synthesis of the labeling agents could be simplified and if a chemically active group other than isocyanate could be used in coupling the fluorescent dyes with immune globulin. Recently Goldman and Carver4 overcame the instability of fluorescein isocyanate by absorption to filter paper and have described a simple method of labeling the antiserum. The present report deals with the synthesis of stable, solid isothiocyanates of fluorescein and rhodamine B and the use of these two compounds in "labeling" antibodies for the specific staining of antigens. The dyes have been used in both the direct method of Coons2 and the indirect method of staining as given by Weller and Coons,5 and have shown satisfactory fluorescence when labeled antibody is applied to bacterial smears, bacteria in tissue, and to rickettsiae, bacteria, and viruses in cultured cells.

In previous publications from our laboratory, we have shown that the immunizing ability of Pasteurella tularensis in rabbits, guinea pigs, mice, and rats varies with the species used. Rats may be readily immunized against virulent challenge by killed cultures of virulent or avirulent strains; rabbits and guinea pigs are poorly protected against virulent challenge by either killed cultures or by prior infection with cultures of lowered virulence. Mice, on the other hand, are well protected by infection with certain strains of low virulence. Recently, Bell et al. (1952), by the use of subcutaneous challenge with strains of less than full virulence, have shown that mice may be protected by vaccination with killed culture vaccines. It has been demonstrated in our laboratory and in Eigelsbach's (1951, 1952) that parent strains may dissociate into smooth and nonsmooth colonial types. The smooth variants may or may not show full virulence for mice and other susceptible animals. We have shown that smooth, virulent, immunogenic strains; smooth, moderately virulent, immunogenic strains; and smooth, avirulent, but nonimmunogenic strains may be isolated from various parent cultures. Without exception, nonsmooth strains have been lacking in virulence and immunogenicity. Because of the difficulty in immunizing susceptible animals with killed cultures, it is not clear whether the smooth, nonprotective, avirulent strains lack immunogenicity because they lack a protective antigen or because they fail to multiply in vivo and thus do not produce this protective antigen. We have postulated that the poor immuno-

Summary An antigen which interferes specifically with the agglutination of red cells sensitized with Chang9s ESS has been demonstrated in the tissues of mice infected with murine typhus. The antigen is heat stable, does not sensitize cells, and does not fix complement. The soluble antigen used in the CF test also interferes with the agglutination of sensitized cells, but has not been demonstrated to be present in mouse or guinea pig tissues. The injection of cortisone increases the amount of interfering antigen, probably by enhancing the multiplication of rickettsia in the infected mice. The possible diagnostic use of this antigen is suggested.

Journal Article Studies on the Pathogenesis and Immunity of Tularemia: I. The Demonstration of a Protective Antibody in Mouse Serum Get access Lolita Pannell, Lolita Pannell From the Department of Bacteriology, University of Kansas, Lawrence, Kansas Search for other works by this author on: Oxford Academic PubMed Google Scholar Cora M. Downs Cora M. Downs From the Department of Bacteriology, University of Kansas, Lawrence, Kansas Search for other works by this author on: Oxford Academic PubMed Google Scholar The Journal of Infectious Diseases, Volume 92, Issue 2, January 1953, Pages 195–204, https://doi.org/10.1093/infdis/92.2.195 Published: 01 January 1953 Article history Received: 09 October 1952 Published: 01 January 1953

Journal Article Streptomycin Studies in Tularemia. I. The Effect of Streptothricin and Streptomycin on Bacterium Tularense in Vitro and in Vivo (Mouse) Get access S. S. Chapman, S. S. Chapman From Camp Detrick, Frederick, Maryland Search for other works by this author on: Oxford Academic PubMed Google Scholar Cora M. Downs, Cora M. Downs From Camp Detrick, Frederick, Maryland Search for other works by this author on: Oxford Academic PubMed Google Scholar Lewis L. Coriell, Lewis L. Coriell From Camp Detrick, Frederick, Maryland Search for other works by this author on: Oxford Academic PubMed Google Scholar S. F. Kowal S. F. Kowal From Camp Detrick, Frederick, Maryland Search for other works by this author on: Oxford Academic PubMed Google Scholar The Journal of Infectious Diseases, Volume 85, Issue 1, July 1949, Pages 25–38, https://doi.org/10.1093/infdis/85.1.25 Published: 01 July 1949 Article history Received: 18 September 1948 Published: 01 July 1949

Summary and Conclusions 1.The invasion and multiplication of Bacterium tularense has been followed quantitatively after inoculation of the virulent organisms in normal, vaccinated and recovered white rats. 2.Virulent Bact. tularense spread by the usual lymphogenic and hematogenic routes in normal, vaccinated or recovered white rats. 3.Rats immunized by vaccination with killed cultures or by recovery from a previous sublethal infection are able to limit the net increase of virulent organisms and to sterilize their tissues. 4.The routes of spread and the increase of organisms in immune animals differ only in a quantitative fashion from those observed in normal animals. 5.Recovered rats are more solidly immune than vaccinated animals, as indicated by the smaller number of organisms recovered from the tissues and by their lower mortality rate after multiple lethal doses of virulent organisms. 6.The behavior of avirulent strains in normal, vaccinated and recovered animals varies in degree but not in kind from virulent strains.

Summary 1.Living organisms of certain strains of Bacterium tularense were found to elicit a high degree of immunity in white mice when inoculated in sublethal concentrations. 2.Killed vaccines gave markedly less protection in proportion to the amount of antigen injected than was obtained with living vaccines. 3.Strains of low virulence multiplied following their inoculation into white mice, but decreased progressively after the fourth or fifth day until none could be demonstrated in spleen, liver and heart blood on or about the 15th day. 4.Mice immunized with a number of strains of lowered virulence were highly resistant to infection with virulent strains and disposed of the organisms very rapidly. 5.As few as 20 living cells of the Jap strain produced a good immunity when inoculated into white mice. 6.Vaccination of white mice with living Jap cells produced a high degree of immunity within three days following inoculation. 7.The Jap strain of Bact. tularense provided white mice with an immunity against more than one virulent strain.

Summary and Conclusions 1.The agglutinin response in rats vaccinated against Bact. tularense is very rapid and the initial rise parallels roughly the time at which immunity to infection develops. 2.The agglutinin titer in vaccinated, recovered and normal rats after infection, rises rapidly and continues at a high level for a longer period than after vaccination. 3.A high degree of active immunity after vaccination of the rat was found to persist for as long as 99 days whereas a high titer of agglutinins persisted for only 11 to 17 days. 4.Rats are more solidly immune after recovery from infection than after vaccination with killed cultures. This immunity also persists at a high level for at least 114 days. 5.Normal, vaccinated and recovered rats were shown to harbor living organisms in the spleen for 31 to 46 days after infection.