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. earlier by Ormsbee, Lackman, and Pickens (1951) for chick embryos infected with C. burnetii.

TABLE 1. Growth iOf Coxiella burnetii Quantities of ricksettsiae in yolk sacs. Quantities sa chitck yolk sac of rickettsiae in yolk sacs were estimated directly Macchia- in three ways: (i) numbers of rickettsiae in vello- FU/g of Day Cdolk sac* Rickettsiae recoveredi Macchiavello-stained yolk sac smears, (ii) titra- smears tion of rickettsial antigen in ether-extracted yolk-

1 0t 0 Not tested sac suspensions by the CF test, and (iii) the 2 1+ 0 0 recovery of rickettsiae in purified form from the 3 1+ 0 0 yolk sacs. The results of these assays are pre- 4 3+ 300 0.05 sented in Table 1. Yolk-sac smears either were 5 4+ 600 0.30 negative or had only a few rickettsiae for the first 6 4+ 800 0.75 3 days. There was a considerable increase in num- 7 4+ 1,600 0.90 bers on the fourth day, and high levels were found * = nt-fixing on the last 3 days. C. burnetii was not detected by CFU complemeint-fixing units. either of the other two assay l)rocedures for the t Results expressed first 3 days, and then there was a relatively per gram of yolk sac. IIaickettsiae were purified by the glycerol-gradient nnethod. raplid increase in numbers of ricksettsiae. It may t Estimate based on scale of 0 to 4+. be noted that there is, as one would expect, an inverse relationship between the increase in de- .. rickettsiae with time and the TABLE 2. Titration Coxiellbutectable (Table 1) of decrease in the numbers of surviving embryos yolk s,ac in mitce (Fig. 1).

HIighest dilution of yolk sac which Infectivity of the yolk-sac material for mice. To in duced specific antibody response* Day correlate the quantity of rickettsiae present in 'Ft (Phase II) CATT yolk sac at various times after inoculation, as described above, with the number of infectious 10- 10- units at these same intervals, mice were inocu- 2 10-7 10-7 lated intraperitoneally with graded dilutions of 3 10-7 10-9 the yolk-sac material. The highest dilutions of the 4 10-8 10-1 yolk-sac material which induced antibody de-

5 10-9 10'1 tectable 6 weeks after infection are listed in 6 10-1 10-10 Table 2. It may be noted that the increase in 7 10-9 10-10 infectious units is coml)arable to the increase in

* Titers are those ofEpools of five sera obtained rickettsiae determined by the other assay pro- 6 weeks after infection cedures. t CF = complementt-fixation test. Electron microscopic observations. Figure 2, an t CAT = capillary .agglutination test. electron micrograph of a lead hydroxide-stained l'OL. 88, 1964 ELECTRON MICROSCOPY OF COXIELLA BUtRNETII 1133

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FIG. 6-8. Thin sections of Coxiella burnetii in yolk sac: (6) obtained 7 days after infection and stained with ferritin-labeled antibody and lead hydroxide; (7-8) obtained 5 days after infection and stained with lead hydroxide. ferritin-stained rickettsiae, two dark lines are identical morphology have been found in yolk-sac characteristically visible at the periphery of the cells 3 to 7 days after infection (Fig. 3 to 6). cell. The outermost layer of these rickettsiae con- The principal difference noted at the various sists of the ferritin-antibody conjugate. Forms of intervals after infection was the difference in l' OL. 88, 1964 ELECTRON MICROSCOPY OF COXIELLA BURNETII 1135 numbers of intracellular rickettsiae. No organisms with lesser doses of penicillin (Fig. 13 and 14), were found in sections 2 days after infection. In- atypical forms like those illustrated in Fig. 9 to dividual organisms or groups of two or three 12 were not found. rickettsiae were found rarely in sections of y-olk sac obtained 3 days after infection. Occasion- DIscussIoN ally, medium-sized clusters of organisms were Results of the various approaches employed in found in cells 4 after days infection, but usually this study of the growth of C. burnetii in the chick rickettsiae were difficult to find at this stage. By embryo are in basic agreement. Deaths of the rickettsiae were and day 5, readily found, on days embryos were rare during the first few days of 6 and rickettsiae were often in 7 found large, infection; during this same period, relatively few closely packed clusters. rickettsiae were detected by light and electron Numerous examples of apparent binary fission microscopy, purification or by the of C. been in ma- lprocedures, burnetii have observed yolk-sac CF test. However, after the fourth the mor- terial 4 to days after day, 7 infection. Probably the tality rate of the infected embryos increased sig- sparsity of organisms has prevented our observa- nificantly, and increasing numbers of rickettsiae tion of division to the fourth prior day. Elongated were detected by all of the assay techniques used. forms containing one or more dense central areas From the third to the last day of the major sometimes bearing and equatorial constrictions experiment, C. burnetii of characteristic may be seen in Fig. 3 to 5. In addition, on one morphol- ogy was observed. Electron microscopy at high a transverse septum was in occasion, found C. revealed that C. burnetii in thin in a with magnification burnetii section stained lead hydroxide sections stained with lead hydroxide l)ossessed only (Fig. 7). external This finding is in con- which have been stained multiple lavers. Rickettsiae with lead trast to that of Wissig et al. (1956), who failed to but not with ferritin are in Fig. hydroxide shown demonstrate external limiting membranes around Four distinct lines which a 8. delineate structure most of the cells of Rickettsia mlooseri in thin sec- or structures 200 A thick can be approximately tions of yolk-sac tissue. As we are not in of the cell y-et, certain, seen ceitain regions periphery. in terms of how the external Figures 9 to 12 illustrate in morphology, layers forms, found rarely of C. burnetii compare with the cell wall and yolk sac obtained 4 to 5 days after infection, membrane which differ from the plasma layers of bacteria. From the "typical" forms described of Allison and above. these forms standpoint chemical comp)osition, Although resemble microor- Perkins (1960) showed that the cell walls of C. ganisms in size and shape, they lack a cell wall burnetii, like bacterial cell walls, contain muramic and a well-defined internal structural organiza- acid. or sometimes dense fibrillar tion. these did not One, several, Also, forms react with ferritin- structures are located in the rickettsiae. labeled antibody. centrallv this dense structure corresl)onds to the has to Probably Since penicillin been reported affect the deoxyribonucleic acid-containing body found in morphology of C. biurnetii (Kravehenko, Gudima, R. prowazekii by Ris and Fox (1949). In and M\Iilyutin, and and other an- general, 1961), penicillin our morphological findings are in agreement with are to the growth of tibiotics known affect other those of Stoker, Smith, and Fiset (1956), who rickettsiae and rielated et organisms (Bozeman al., examined thin sections of purified C. burnetii. 1956; Tajima, Samejima, and Nomura, 1959; In addition to the tyl)ical forms of C. burnetii, we to determine whether Suitor, 1963), attempted we found in a few rare instances atypical forms or not was resl)onsible for the penicillin atypical which bear some to the inter- and the forms may relationshil) forms described above intermediate mediate forms reported by Rosenberg and reported by Rosenberg and Kordova (1960). Kordova (1960). We have not been able to induce Thin sections of yolk-sac fragments obtained from the development of the atypical forms by the chick embryos 3, 4, 5, and 6 days after inoculation administration of l)enicillin nor to exl)lain satis- with a 10%O suslension of C. burnetii and 0, 1,000, factorily the origin of these forms. However, the or 4,000 units of lenicillin were examined with typical and atypical forms are serologically dis- the electron microscope. Although the cell walls tinct; ferritin-labeled antibody which readily com- of some of the rickettsiae from eggs inoculated bines with the typical forms of C. burnetii in with 4,000 units of penicillin appeared to be less y-olk-sac tissue fails to react with the atypical rigidl than the cell walls of rickettsiae inoculated forms. This result does not necessarily invalidate 1136 ANACKER ET AL. J. BACTERIOL.

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FIG. 9-12. Thin sections of "atypical" forms found in yolk sacs infected with Coxiella burnetii: (9) ob- tained 4 days after infection and stained with lead hydroxide; (10-11) obtained 5 days after infection and stained with ferritin-labeled antibody and lead hydroxide; (12) obtained 5 days after infection and stained with ferritin-labeled antibody. the hypothesis that these forms are intermediates From the results of our experiments, it appears in a complex life cycle, because forms which differ that the growth of C. burnetii is very much like structurally conceivably could also differ anti- that of bacteria, rather than like that of agents genically. of the psittacosis group. After an initial lag phase, VOL. 88, 1964 ELECTRON MICROSCOPY OF COXIELLA BURNETII 1137

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FIG. 13-14. Thin sections of Coxiella burnetii with irregular contoursfrom eggs inoculated with 4,000 untits of penicillin; obtained 5 days after infection and stained with fe rritin-labeled antibody and lead citrate. growth of the rickettsiae, as measured by a amino acids, sugars, and proteins on the sta- variety of parameters, proceeded at a rapid rate; bility of rickettsiae. J. Bacteriol. 59:509-522. this period of rapid growth was followed by a BOZEMAN, F. M., H. E. Hopps, J. X. I)ANAUSKAS, stationary phase, during which the number of E. B. JACKSON, AND J. E. SMADEL. 1956. on the of rickettsiae. I. A remained constant. Study growth tissue organisms approximately culture system for quantitative estimations Electron micrographs of rickettsiae examined of Rickettsia tsutsugani ushi. J. Immunol. during the phase of rapid growth revealed numer- 76:475-488. ous examples of apparent binary fission. Binary CAULFIELD, J. B. 1957. Effects of varyiig the fission of another rickettsia, R. rickettsii, was vehicle for OS04 in tissue fixation. J. Biophys. previously observed in l)hase microscopic studies Biochem. Cytol. 3:827-830. by Schaecter, Bozeman, and Smadel (1957). Al- Cox, H. R., AND E. J. BELL. 1939. The cultivation though our evidence does not exclude the possi- of Rickettsia diaporica in tissue culture and bility of a complex growth cycle, binary fission in the tissues of developing chick embryos. appears to be an important mode, if not the only Public Health Rept. (U.S.) 54:2171-2178. C. burnetii. DAVIS, G. E., AND H. R. Cox. 1938. A filter-passing one, of replication of infectious agent isolated froin ticks. I. Isola- LITERATUJRE CITED tion fromi Dernuacentor andersoni, reactions in aniials, and filtration experiments. Public ALLISON, A. C., AND H. R. PERKINS. 1960. Pres- Health Rept. (U.S.) 53:2259-2267. ence of cell walls like those of bacteria in FREEMAN, J. A., AND B. 0. SPURLOCK. 1962. A new rickettsiae. Nature 180:796-798. epoxy embedment for electron microscopy. ANDRES, C. A., C. MORGAN, K. C. Hsu, R. A. J. Cell Biol. 13:437-443. RIFKIND, AND B. C. SEEGAL. 1962. Electron KORDOVA, N. 1959a. Filterable particles of Coxiella microscopic studies of experimental nephritis burneti. Acta Virol. 3:25-36. with ferritin-conjugated antibody. J. Exptl. KORDOVA, N. 1959b. Microscopic study of tissue Med. 115:929-936. cultures infected with filtrable Coxiella burneti BOVARNICK, M. R., J. C. MIILLER, AND J. C. SNY- particles. Folia Microbiol. (Prague) 4:237-239. DER. 1950. The influence of certain salts, KORDOVA. N. 1960. Study of antigenicity and im- 1138 ANACKER ET AL. J. BACTERIOL.

munogenicity of filterable particles of Coxiella siae. II. Morphologic observations of living burneti. Acta Virol. 4:56-62. rickettsiae in tissue culture cells. Virology KORDOVA, N., AND REHACEK. 1959. Experimental 3:160-172. infections of ticks in vivo and their organs SIDWN-ELL, R. W., AND L. P. GEBHARDT. 1962. Q in vitro with filterable particles of Coxiella fever antibody response in experimentally burneti. Acta Virol. 3:201-209. infected wild rodents and laboratory animals. KRAVCHENKO, A. T., 0. S. GUIDIMA, AND V. N. J. Immunol. 89:318-322. MILYUTIN. 1961. Study of the action of anti- STOKER, M. G. P., K. M. SMITH, AND P. FISET. biotics and specific sera on the development 1956. Internal structure of Rickettsia burnetii of viruses and rickettsias in tissue culture as shown by electron microscopy of thin sec- with use of microcinematography. I. Effect of tions. J. Gen. Microbiol. 15:632-635. penicillin on psittacosis virus and Rickettsia SUITOR, E. C., JR. 1963. Studies on the cell-enve- burneti in tissue culture. Vopr. Virusol. 6:300- lope of the rickettsia-like microorganism, 306. Wolbachia persica. Bacteriol. Proc., p. 133. LuOTo, L. 1953. A capillary agglutination test for TAJIMA, M., Y. NOMURA, AND Y. KUBOTA. 1957. bovine Q fever. J. Immunol. 71:226-231. Structure and development of viruses of the MILLONIG, G. 1961. A modified procedure for lead psittacosis-lymphogranuloma groups observed staining of thin sections. J. Biophys. Biochem. in the electron microscope. J. Bacteriol. Cytol. 11:736-739. 74:605-620. ORMSBEE, R. A., D. B. LACKMAN, AND E. G. TAJIMA, M., T. SAMEJIMA, AND Y. NOMURA. 1959. PICKENS. 1951. Relationships among death Morphology of meningopneumonitis virus curves, complement-fixing values, and in- exposed to penicillin as observed with the fectious endpoints in experimental Q fever. electron microscope. J. Bacteriol. 77:23-34. J. Immunol. 67:257-263. TAWN-DE, S. S., AND J. S. RAM. 1962. Conjugation of PALADE, G. E. 1952. A study of fixation for elec- antibody to ferritin by means of p,p'-difluoro- tron microscopy. J. Exptl. Med. 95:285-298. m, m'-dinitrodiphenylsulphone. Arch. Bio- REYNOLDS, E. S. 1963. The use of lead citrate at chem. Biophys. 97:430. high pH as an electron-opaque stain in elec- WATSON, M. L. 1958. Staining of tissue sections for tron microscopy. J. Cell Biol. 17:208-212. electron microscopy with heavy metals. II. RIBI, E., AND B. H. HOYER. 1960. Purification of Application of solutions containing lead and Q fever rickettsiae by density-gradient sedi- barium. J. Biophys. Biochem. Cytol. 4:727- mentation. J. Immunol. 85:314-318. 730. RIs, H., AND J. P. Fox. 1949. The cytology of WEILSH, H. H., F. W. JENSEN, AND E. H. LEN- rickettsiae. J. Exptl. Med. 89:681-686. NETTE. 1959. Q fever studies. XX. Comparison ROSENBERG, M., AND N. KORDOVA. 1960. Study of intracellular forms of Coxiella burneti in the of four serologic techniques for the detection electron microscope. Acta Virol. 4:52-55. and measurement of antibody to Coxiella ROSENBERG, M., AND N. KORDOVA. 1962. Multi- burnetii in naturally exposed sheep. Am. J. plication of Coxiella burneti in Detroit-6 Hyg. 70:1-13. cell cultures. An electron microscope study. WISSIG, S. L., L. G. CARO, E. B. JACKSON, AND Acta Virol. 6:176-180. J. E. SMADEL. 1956. Electron microscopic ob- SCHAECTER, M., F. M. BOZEMAN, AND J. E. servations on intracellular rickettsiae. Am. J. SMADEL. 1957. Study on the growth of rickett- Pathol. 32:1117-1133.