JOURNAL OF BACTERIOLOGY Vol. 88, No. 4, p. 1130-1138 October, 1964 Copyright © 1964 Ariierican Society for Microbiology Printed in U.S.A. ELECTRON MICROSCOPIC OBSERVATIONS OF THE DEVELOPMENT OF COXIELLA BURNETII IN THE CHICK YOLK SAC1 R. L. ANACKER, K. FUKUSHI,2 E. G. PICKENS, AND D. B. LACKMAN Rocky Mountain Laboratory, N\ational Institute of Allergy and Infectious Diseases, U.S. Public Health Service, Hamilton, 3Montana Received foI publication 21 May 1964 ABSTRACT doses of penicillin varying from 0 to 4,000 units per egg. ANACKER, R. L. (Rocky Mountain Laboratory, Hamilton, Mont.), K. FUKUSHI, E. G. PICKENS, AND D. B. LACKMAN. Electron microscopic ob- Several reports in the literature indicate that servations of the development of Coxiella burnetii Coxiella burnetii may be morphologically complex. in the chick yolk sac. J. Bacteriol. 88:1130-1138. In their original descriptions of this rickettsia, 1964.-Yolk sac material, obtained daily over a Davis and Cox (1938) and Cox and Bell (1939) period of 1 week from embryos inoculated with described the pleomorphic nature of this or- seed of phase I Coxiella burnctii strain Ohio 314 ganism which passed ordinary bacteriological containing 250 units of penicillin, was examined filters. With the light microscope, they observed by electron microscopy and other techniques for minute coccoid and granular forms, as well as the presence of rickettsiae. The concentration of rickettsiae in the yolk sac, as determined by elec- bacillary forms, of C. burnetii in cells of infected tron microscopy, light microscopy, the comple- guinea pigs and in tissue culture. More recently, ment-fixation test, recovery of organisms, and Kordova (1959a, b; 1960) and Kordova and mouse infectivity, was low for the first 3 days, Rehatcek (1959) reported that they were able to increased rapidly 3 to 5 days after infection, and pass particles of C. burnetii infectious for em- then remained relatively constant. Rickettsiae in bryonated eggs, tissue cultures, ticks, and tick 3- to 7-day cultures, when observed by electron organs through graded collodion membranes of microscopy, had dense fibrillar centers surrounded 60 m,u aveiage pore diameter. by less-dense cytoplasmic material containing With an electron microscope, Rosenberg and granules approximately 15 m,u in diameter. The Kordova (1960, 1962) found in thin sections of whole was enclosed by multiple external layers. Many appeared to be in various stages of binary infected tissue culture cells "intermediate forms" fission, and one form which contained a cross-wall of C. burnetii closely associated with "mature" was observed. These forrms readily combined with ricksettsiac. These forms differed in internal ferritin-labeled specific antibody. In rare in- density and structure, and ranged in size from stances, several kinds of. "atypical" forms which that of the elementary body or mature form, did not combine, with ferritin-labeled antibody approximately 300 to 600 m,u in diameter, to were found in the cytoplasm of yolk-sac cells 4 to forms several microns in diameter. From this 5 days after inoculation; it is not certain whether evidence, Rosenberg and Kordova suggest that these forms are artifacts or normal stages in the the reproductive process of C. burnetii resembles maturation of C. burnetii. These atypical forms that of certain of the larger viruses, as rel)orted were not observed in subsequent experiments in which embryonated eggs were inoculated with by Tajima, Nomura, and Kubota (1957). We attemlted to confirm the electron micro- scopic findings of Rosenberg and Kordova by 1 A preliminary report of this work was pre- studying the development of C. burnetii in the sented at the 63rd Annual Meeting of the Ameri- can Society for Microbiology, Cleveland, Ohio, yolk sac of the embryonated egg. In addition, in May, 1963. an effort to demonstrate an antigenic relationship 2 Present address: The lResearch Institute for between the mature form and intermediate forms, Tuberculosis and Leprosy, Tohoku University, if such existed, we stained yolk sac with ferritin- Sendai, Japan. labeled antibody from a rabbit immunized by in- 1130 l'OL. 88, 1964 ELECTRON MIICROSCOPY OF CONXIELLA BURNETHII 1131 jections of mature rickettsial cells. The results of methacrvlate. As a control of the specificity of our electron microscopic observations and ancil- the ferritin staining, infected yolk-sac material lary studlies are presented in this report. was treated with heterologous antibody con- jugated to ferritin; in no instance was there any 7MATERIALS ANI) MWETHODS nonspecific labeling of C. burnetii. Infection of eggs. In the principal experiment In the later experiments designed to determine to be described below, 398 5-day-old embryonated the effect of penicillin on the morphology of C. eggs were each inoculate(d with 0.5 ml of a 10% burnetii, yolk-sac material was fixed either in 1 % yolk-sac suspension (500 units of penicillin per buffered osmium tetroxide or in 1 %o buffered ml) of the fifth egg passag( of phase I C. burnetii osmium tetr oxide containing 0.14 M sucose strain Ohio 314. At 24-hr intervals, mortalities (Caulfield, 1957) after treatment with the ferritin- were recorded, and yolk sacs from a portion of antibody conjugate. the remaining live embryos were obtained for the Thin sections cut with a l'orter-Blum micro- various studies to be reported below. Ten volk tome were stained with the lead hydroxide stain sacs wereC taken daily through the sixth day; the of Watson (1958), the lead hydroxide stain of yolk sacs from the last seven live embryos were MTillonig (1961), or the lead citrate stain of Rey- u$sed on the last day. nolds (1963), and were examined with a Siemens In subsequent exl)eriments in which only the Elmiskop I. morphology of C. burnetii was of coneern, eggs Serological tests. Titers of mouse sera were de- were inoculated with the same dose of rickettsiae termined bv the complement-fixation (CF) test as above and graded doses of penicillin. After 3 of Welsh, Jensen, and Lennette (1959) and the to 6 days, yolk sacs from a portion of the living capillary agglutination test (CAT) of Luoto embryos were taken for study. (1953). Rickettsial antigen titers of yolk-sac Lighlt iicroscopy. Estimates of the quantities material [diluted with 9 volumes of sucrose-phos- of rickettsiae present in yolk sac were made from phate-glutamate (SPG) solution (Bovarnick, Macchiavello-stained smears. Mliller, and Snyder, 1950), homogenized for 3 min Electron microscopy. For the major experiment, in a Waring Blendor, and extracted with ether] yolk-sac fragments were obtained from the second were determined by the CF test in which 8 CF through the seventh day and were fixed at 0 C units of standard guinea pig Q-fever antiscrum either in 1 c%O buffered osmium tetroxide (Palade, were used. For this test, 1 complement-fixing unit 1952) for 1 hr or in phosphate-buffered saline (pH (CFU) of antigen was defined as the reciprocal 7.4) containing 5(/% formalin for 1 hr. After fixa- of the highest yolk-sac dilution which fixed com- tion in buffered osmium tetroxide, the yolk-sac plement. material was dehydrated in graded dilutions of Purification of rickettsiae. Purified rickettsiae ethanol and embedded in \Iaraglas (Freeman and were obtained by the glycerol-gradient technique Spurlock, 1962) or in methacrylate. of Ribi and Hoyer (1960) from yolk-sac material Yolk-sac material fixed in phosphate-buffered homogenized with 9 volumes of SPG solution for saline containing formalin wras treated with ferri- 3 min in a Waring Blendor. tin-labeled -y-globulin pleviously absorbed with Infectivity of yolk-sac miaterial. Yolk-sac ma- acetone-powdered egg, according to the method terial, which had been blended for 30 sec with 9 of Andres et al. (1962). The y-globulin was de- volumes of SPG solution and frozen until used, rived from a rabbit inoculated with ether-killed, was diluted decimally in SPG solution and purified, phase I C. burnetii strain Ohio 314. injected intraperitoneally into 4-week-old male Ferritin (Nutritional Biochemicals Corp., Cleve- Swiss mice (Rocky MIountain Laboratory strain) land, Ohio) was conjugated to antibody with in groups of five. After 6 weeks, the mice were p , p'-difluoro-m, m'-dinitrodiphenylsulfone, gen- bled from the heart, and the CF titers of the erously supl)lied by J. S. Ram, by the technique sera pooled from each group) of five mice were of Tawde and Ram (1962). The Ayolk-sac material determined. Recently, Sidwell and Gebhardt was washed in cold Tvrode's solution, fixed in (1962) showed that levels of both phase I and buffered osmium tetroxide for 2 hr at 0 C, dehy- phase II antibody are at or near their peaks 6 drated, and embedded in either 1\Iaraglas or weeks after intraperitoneal injection of living 1132 ANACKER ET AL. J. BA~CTERIOL. 20 - virulent C. burnetii. The infectious titer of the 1.9 1.8 yolk-sac material is defined as the highest dilution .7 which stimulated the production of detectable CF a L 6 - 11 antibody. 1.4 - RESULTS 0 1.3 Death rate of infected embryos. The death curve for the infected embryos, corrected for the live " 0.7 - embryos taken each day for the various studies, -j 0.6- is presented in Fig. 1. Up) to the fourth day of the DA7 experiment, the mortality rate was very low. DAY However, after the fourth day, the embryos died FIG. 1. Death curlve jfor 398 5-day-old chick emii- at an accelerated rate; only 7 embryos of the bryos inoculated with 0.5 ml of a 10c0 yolk-sac original 398 were found alive on day 7. This type suspension of phase I Coxiella burnetii strain of death curve is similar to those rel)orted Ohio 314.