Jun 16, 1975 - siding in the foothills of the SacramentoValley of Calif. (4, 14). The clinical signs included fever, anorexia, depression, edema of the legs,.
INFECTION AND IMMUNITY, Jan. 1976, p. 273-280 Copyright 0 1976 American Society for Microbiology
Vol. 13, No. 1 Printed in U.S.A.
Ultrastructural Observations on Ehrlichia equi Organisms in Equine Granulocytes DONALD M. SELLS,* PAUL K. HILDEBRANDT, GEORGE E. LEWIS, JR., M. B. A. NYINDO, AND MIODRAG RISTIC Department of Veterinary Pathology and Hygiene, College of Veterinary Medicine, University of Illinois,
Urbana-Champaign, Illinois 61801,* and Department of Experimental Pathology and Division of Veterinary Medicine, Walter Reed Army Institute of Research, Washington, D.C. 20012 Received for publication 16 June 1975
The ultrastructure of the etiological agent of equine ehrlichiosis, Ehrlichia equi, was studied in equine peripheral leukocytes. The organisms occurred within membrane-lined cytoplasmic vacuoles of neutrophils and eosinophils. Ovoid, round, and rod-shaped profiles were observed. From 1 to 33 organisms were present in a thin-section profile of a cytoplasmic vacuole. Many cells contained multiple organism-containing vacuoles. The organisms had a cell wall and plasma membrane, and internally they consisted of electron-dense and lucid areas. A great variation in size was observed. The morphological features were most consistent with agents of the genus Ehrlichia.
Equine ehrlichiosis was reported in 1969 as a distinct disease of low mortality in horses residing in the foothills of the Sacramento Valley of Calif. (4, 14). The clinical signs included fever, anorexia, depression, edema of the legs, and ataxia. The hematological changes consisted of thrombocytopenia, leukopenia, and mild anemia. Inclusion bodies, representing the intracellular localization of the causal agent, occurred within the cytoplasm of neutrophils and eosinophils. The pathological alterations included edema, petechiae, and ecchymoses in the subcutaneous tissues and fascia of the legs and vasculitis (4). The taxomic position of the causal agent of equine ehrlichiosis has not been clearly defined. However, it has been suggested that the organism should be grouped with the agents of the genus Ehrlichia (14). The organism has been referred to as Ehrlichia equi (8), and the same designation will be used in this paper. Based on the presence of inclusion bodies in peripheral blood smears, goats, sheep, dogs, cats, rhesus macaques (Macaca mulatta), and baboons (Papio anubis) have been found susceptible to experimental infection with E. equi (4, 8, 14). The disease was clinically mild or inapparent in most of these animals. Dogs infected with E. equi were not protected against challenge with E. canis (8). Definition of ultrastructural features of E. equi was deemed necessary to further characterize it and compare it morphologically with other rickettsial agents.
MATERIALS AND METHODS Two horses were each experimentally inoculated intravenously with 20 ml of E. equi-infected equine blood (provided by David H. Gribble, University of California, Davis) which had been maintained in liquid nitrogen. At day 7 postinoculation, during the acute phase of disease, heparinized blood samples were withdrawn from the horses and centrifuged to layer the cells. The plasma overlying the buffy coat was removed so as not to disturb the cells, and the buffy coat was overlayed with a 2.5% solution of glutaraldehyde in cacodylate buffer. After 30 min the disk of fixed leukocytes embedded in solidified plasma was removed, cut into 1-mm cubes, and fixed an additional hour in 2.5% glutaraldehyde. The tissues were postfixed in osmic acid for 1 h, dehydrated in graded alcohols, and embedded in araldite. Thin sections were cut from the blocks, stained with uranyl acetate and lead citrate, and examined with a Hitachi 11A electron microscope. Buffy coat smears were air dried, fixed in methanol for 10 min, and stained with Giemsa stain for light microscopic examination.
RESULTS Both horses experimentally inoculated with E. equi-infected blood developed signs of acute equine ehrlichiosis characterized by pyrexia, anorexia, thrombocytopenia, and ataxia. The organisms occurring freely or as components of inclusion bodies were readily detected in the cytoplasm of granulocytes of Giemsastained buffy coat smears examined by light microscopy. They were pleomorphic, ranging from oval to rod-like forms, and stained dark blue to purple (Fig. 1-3).
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Ultrastructurally, the E. equi organisms were detected within membrane-lined vacuoles in the cytoplasm of thin-section profiles of neutrophils (Fig. 4) and eosinophils (Fig. 5). Ovoid, round, rod-shaped, and irregular profiles of the organisms were observed. From 1 to 33 thinsection profiles of the organisms were evident within individual vacuoles, and single leuko-
cytes contained multiple vacuoles with variable numbers of loosely arranged organisms (Fig. 6). The organisms lined the periphery of some vacuoles (Fig. 4). In some instances, one or more organisms were found tightly integrated within a cytoplasmic vacuole, lacking any clear space around or between the organisms and wall of the vacuole (Fig. 6). Vesicles were
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was indicated by the presence of dumbbellshaped organisms and organisms in a state of almost complete division (Fig. 11). Although the organisms varied considerably in size, the smallest about 0.18 am in diameter and the largest about 1.40 ,um along the greatest dimension, the internal structure remained constant.
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DISCUSSION The ultrastructure of E. equi is similar to that of agents in the genera Rickettsia (1) and Ehrlichia (6, 12, 13, 15), and the large particles of the genus Chlamydia (1, 2, 3, 10), as well as to the causal agent of bovine petechial fever
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x41,000. found situated within and around some vacuoles (Fig. 6). The plasma membrane of the cell and membrane of the vacuoles appeared to fuse and occasionally bulge above the surface of some cells (Fig. 7). Organisms of varying sizes were contained in such vacuoles. Individual organisms were bound by two distinct membranes (Fig. 6, 8). The outer membrane, or cell wall, was rippled. The inner or plasma membrane was adherent to the internal constituents of the organism. Internally, the organisms consisted of electron-dense and lucid areas. The dense areas contained granules, apparently ribosomes, and in the lucid areas fine fibrils suggestive of deoxyribonucleic acid strands were observed. Occasionally small bodies with the consistency of organisms were observed beneath the cell wall adjacent to the organism (Fig. 9, 10). Multiplication by fission
E. equi differs morphologically from most of the organisms in the genus Rickettsia in that the latter are usually more rod shaped and do not occur within membrane-lined vacuoles, with the exception of Rickettsia sennetsu, an otganism of uncertain classification (1, 11). Examination of E. equi in blood leukocytes did not reveal a developmental cycle with elementary bodies and intermediate bodies typical of organisms in the genus Chlamydia (1, 3, 5). The only mode of reproduction observed was that of fission of the organism. Condensation of small bodies within larger organisms, as described in bovine petechial fever (7), was not observed. The significance of the small dense bodies beneath the cell wall was not determined. Static evidence of the organism entering the cell was not observed. However, it seems likely that entrance may have been by a phagocytic process. Exit of the organisms from the cell probably occurred as a result of rupture of vacuoles after the membrane of the vacuole and plasma membrane of the cell came in close apposition, as has been indicated with other agents (7, 15). Although actual evidence of rupture of a vacuole was not observed in this study, the presence of organisms of all sizes in a single vacuole would suggest that there is no predilection for the release of a particular size of organism presumably to infect other cells. Vesicles such as those observed around and in the vacuoles have been incriminated in the transfer of cellular material associated with the destruction of the constituents of the vacuole (7, 9, 15). However, degenerative changes were not observed in the organisms, nor were degenerative changes observed in neutrophils or eosinophils due to the presence of the organism. The morphological characteristics of the
VOL. 13, 1976
E. EQUI ORGANISMS IN EQUINE GRANULOCYTES
279
FIG. 10. Neutrophil with an inclusion (I) containing numerous organisms. Dense bodies (arrows) are present beneath the cell wall of some organisms. x30,000.
280
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SELLS ET AL.
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causal agent of equine ehrlichiosis and the host cells parasitized are consistent with other organisms classified in the genus Ehrlichia. ACKNOWLEDGMENTS This study was supported in part by contract DADA 17-70-C-0044 from the U.S. Army Medical Research and Development Command to the College of Veterinary Medicine, University of Illinois, Champaign-Urbana, Ill. LITERATURE CITED 1. Anderson, D. R., H. E. Hopps, M. F. Barile, and B. C. Bernheim. 1965. Comparison of the ultrastructure of several Rickettsiae, ornithosis virus, and Mycoplasma in tissue culture. J. Bacteriol. 90:1387-1404. 2. Armstrong, J. A. 1968. Relation of PLT organisms to the Rickettsiae, with special reference to structure and multiplication. Acta Virol. Engl. Ed. 12:15-17. 3. Cutlip, R. C. 1970. Electron microscopy of cell cultures infected with a chlamydial agent causing polyarthritis of lambs. Infect. Immun. 1:499-502. 4. Gribble, D. H. 1969. Equine ehrlichiosis. J. Am. Vet. Med. Assoc. 155:462-469. 5. Higashi, N. 1965. Electron microscopic studies on the mode of reproduction of trachoma virus and psittacosis virus in cell cultures. Exp. Mol. Pathol. 4:2439. 6. Hildebrandt, P. K., J. D. Conroy, A. E. McKee, M. B. A. Nyindo, and D. L. Huxsoll. 1974. Ultrastructure of Ehrlichia canis. Infect. Immun. 7:265-271. 7. Krauss, H., F. G. Davies, 0. A. Odegaard, and J. E. Cooper. 1972. The morphology of the causal agent of
bovine petechial fever (ondiri disease). J. Comp. Pathol. 82:241-246. 8. Lewis, G. E., Jr., D. L. Huxsoll, M. Ristic, and A. J. Johnson. 1975. Experimentally induced infection of dogs, cats, and nonhuman primates with Ehrlichia equi, etiologic agent of equine ehrlichiosis. Am. J. Vet. Res. 36:85-88. 9. Lockwood, W. R., and F. Allison, Jr. 1964. Electron micrographic studies of phagocytic cells. II. Observations on the changes induced in the cytoplasmic contents of human granulocytes by the ingestion of rough pneumococcus. Br. J. Exp. Pathol. 45:294299. 10. Mitsui, Y., M. Fujimoto, and M. Kajima. 1964. Development and morphology of trachoma agent in yolk sac cell as revealed by electron microscopy. Virology 23:30-45. 11. Moulder, J. W. 1974. The Rickettsias, p. 882-890. In R. E. Buchanan and N. E. Gibbons (ed.), Bergey's manual of determinative bacteriology, 8th ed. The Williams & Wilkins Co., Baltimore. 12. Simpson, C. F. 1972. Structure of Ehrlichia canis in blood monocytes of a dog. Am. J. Vet. Res. 33:24512454. 13. Simpson, C. F. 1974. Relationship of Ehrlichia canisinfected mononuclear cells to blood vessels of lungs. Infect. Immun. 10:590-596. 14. Stannard, A. A., D. H. Gribble, and R. S. Smith. 1969. Equine ehrlichiosis: a disease with similarities to tick-borne fever and bovine petechial fever. Vet. Rec. 84:149-150. 15. Tuomi, J., and C. H. von Bonsdorff. 1966. Electron microscopy of tick-borne fever agent in bovine and ovine phagocytizing leukocytes. J. Bacteriol. 92:14781492.
