Relationship of Ehrlichia canis-infected Mononuclear Cells to Blood Vessels of Lungs1

Transcription

1 INFECTION AND IMMUNITY, Sept. 1974, p Copyright American Society for Microbiology Vol. 10, No. 3 Printed in U.S.A. Relationship of Ehrlichia canis-infected Mononuclear Cells to Blood Vessels of Lungs1 CHARLES F. SIMPSON Department of Veterinary Science, College of Veterinary Medicine, University of Florida, Gainesville, Florida Received for publication 1 April 1974 The lung tissue of six dogs with ehrlichiosis and two control dogs was examined with the electron microscope. Mononuclear cells containing inclusions (morulae) of Ehrlichia canis were adhered at one or more sites to the luminal surfaces of endothelial cells of arterioles or capillaries by way of interdigitations or areas of adherence, or an endothelial cell-bound mononuclear cell was chained to another parasitized or nonparasitized mononuclear cell in the lumen. The bifurcation of arterioles was the most common site at which mononuclear cells clung to the endothelium. Cell-free bodies (morulae), enclosed by a single membrane, were present in the lumens of arterioles. Such cell-free bodies were swollen and vesiculated as compared with intracytoplasmic inclusions (morulae) in mononuclear cells. Ehrlichia canis is responsible for a severe, hemorrhagic disease of dogs known as canine ehrlichiosis (CE). The etiological agent of the disease is the same parasite that was first described in dogs in Algeria by Donatien and Lestoquard and called Rickettsia canis (1). Canine ehrlichiosis has been diagnosed in the United States (2, 10, 11). The principal means of diagnosing the disease is the observation of intracytoplasmic inclusions (morulae) in circulating lymphocytes, neutrophils or, more commonly, monocytes stained with Romanovsky stains (2). However, such parasitized cells are difficult to find in stained blood smears, and inclusions are more readily observed in stained mononuclear cells in impression smears made from the lung, spleen, liver, and kidney (4-6). By electron microscopy, infected mononuclear cells were seen in the lumina of small vessels in the lung, and E. canis was seen in cells construed to be sloughing endothelial cells of the pulmonary vessels (4). The present paper describes the existence of parasitized mononuclear cells in capillaries and arterioles of the lungs of dogs with E. canis, and the relationship of such infected cells to the endothelial cells of arterioles and capillaries. These observations were based on the examination by electron microscopy of numerous sections of lungs of six artificially infected dogs and two control dogs. I Florida Agricultural Experiment Station Journal Series Article No MATERIALS AND METHODS Six preconditioned (wormed, quarantined for 3 weeks) dogs of mixed breed were infected by the intravenous inoculation of 10 ml of fresh blood which contained an isolate of E. canis collected in Florida in The dogs were killed or died after the development of CE, as indicated by the existence of a serous nasal and ocular discharge, depression, anorexia, elevated body temperature, lowered red and white blood cell counts, and intracytoplasmic inclusions (morulae) in Giemsa-stained monocytes in the peripheral circulation. Two preconditioned, noninfected dogs were maintained as controls. Small pieces of lung tissue from each of these eight dogs were immediately processed after death for light and electron microscopic studies. For light microscopy, these tissues were fixed in 10% neutral formalin, embedded in paraffin, cut to 6 um, and stained with hematoxylin and eosin and/or Giemsa stains. For electron microscopy, lung tissues were fixed in 3.5% glutaraldehyde, postfixed in 1% OSO4, and embedded in Araldite. Thin sections on grids were examined with a Philips EM 200 electron microscope after staining with uranyl acetate and lead citrate (9). RESULTS Giemsa-stained blood smears prepared from all infected dogs shortly before death contained monocytes with intracytoplasmic inclusions, which were bluish and granular. In addition, saclike bodies similar to the inclusions in monocytes were seen free in the plasma (Fig. 1). In paraffin-embedded lung- tissue examined by light microscopy, it was observed in infected dogs that mononuclear cells commonly clung to 590

2 VOL. 10, 1974 LI '_ J FIG. 1. Intact mononuclear cell contains an Ehrlichia canis inclusion (morula) (L). The cell-free body (morula) (L,) apparently originated from the adjacent, ruptured mononuclear cell. Giemsa stain. x 1,800. the endothelium of arterioles. In control dogs, sticking of mononuclear cells to the endothelium was not observed. An intimate relationship between parasitized mononuclear cells and endothelial cells of arterioles and capillaries was observed by electron microscopy. Mononuclear cells containing inclusions of E. canis were adhered at one or more sites to the luminal surfaces of endothelial cells by way of interdigitations and/or areas of adherence. The extent of adherence of the plasma membranes of apposing mononuclear cells and endothelial cells apparently varied from cell to cell because in some micrographs there were several interrupted, adhered areas, and in other micrographs only one or two isolated zones of attachment of apposed membranes were observed (Fig. 2, 3). Areas of adherence were characterized by either the absence of an intercellular space or a narrow intercellular space (Fig. 4). Sometimes as many as two parasitized mononuclear cells were attached by way of areas of adherence to a small segment of the E. CANIS-INFECTED MONONUCLEAR CELLS I 591 endothelium of an arteriole observed at a magnification of x8,000 (Fig. 5). An E. canis organism was not always seen in both sectioned mononuclear cells. It is entirely possible that an inclusion was present in each cell but was not observed in one of the cells because of the plane through which the cell was sectioned. In many micrographs, it was not unusual to observe a parasitized mononuclear cell chained to a second infected mononuclear cell, or rarely a noninfected mononuclear cell which in turn was adhered to the endothelium (Fig. 5). After examination of numerous sections, it was concluded that the bifurcation of small arteries was the most frequent site at which sticking of parasitized mononuclear cells to endothelial cells occurred (Fig. 6). Mononuclear cells, with inclusions of E. canis, in small capillaries of the lung were extensively adhered to the endothelium by numerous areas of adherence. As a consequence, only a small portion of the lumen of the capillary was patent (Fig. 7). Sticking of leukocytes to the endothelium of arterioles or capillaries was not observed in the lung tissue of control dogs. Inclusions of E. canis in mononuclear cells had a characteristic appearance. Inclusions were surrounded by a single membrane. In places where the membranes were closely related to those of the endoplasmic reticulum, it could be erroneously interpreted that the inclusion was confined by a double membrane (Fig. 8). Usually the inclusion was closely related to the Golgi apparatus. The granular elementary bodies, some in division, in inclusions were confined by a double membrane, each of which was about 7.5 to 9.0 nm thick and of similar electron density. Saclike bodies, enclosed by a single membrane and similar to morulae within cells, were seen free in the lumens of arterioles. These saclike bodies were swollen and vesiculated as compared with intracellular inclusions; they appeared to have been liberated from a mononuclear cell as a result of rupture of the parasitized cell (Fig. 9). Apparently, parasitized mononuclear cells free in the lumens of vessels, as well as parasitized mononuclear cells attached to the endothelium of vessels, were potential sources of saclike bodies free in the lumens. DISCUSSION Several studies of CE have noted that E. canis is more readily found in impression

3 FIG. 2. There are several areas of adherence (arrows) that cause the mononuclear cell (N), with E. canis inclusions, to be attached to the endothelium (E) of an arteriole. The smooth muscle cell (S) in the wall of the arteriole is normal. x 7,000. Downloaded from on July 11, 2018 by guest 'V FiG. 3. A mononuclear cell (N) with an E. canis inclusion (L) is attached to the endothelium (E) by an area of adherence (arrow). x 15,

4 _ X7. \v~~~~~~~~~ ~~fi*2: FIG. 4. Areas of adherence, characterized by absence of an intercellular space (arrows) or a narrow intercellular space (±), adhere mononuclear cells with inclusions (N) and without inclusions (N1) to the endothelium (E). x35,000. Insert: High magnification of the intercellular space (arrow) shown in blocked off area. x70,000...'.a _ FIG. 5. Two parasitized mononuclear cells (N, N1) are adhered to the endothelium (E) of an arteriole. One parasitized mononuclear cell (N1) is chained to another parasitized mononuclear cell (N2) in the lumen by an area of adherence (arrow). Smooth muscle cells (S) in the wall of the arteriole are normal. x 7,

5 FIG. 6. Bifurcation of an arteriole. Several parasitized mononuclear cells (N) and a nonparasitized mononuclear cell (N1) are adhered to the endothelium (E). There is also an unattached mononuclear cell (N2) with an E. canis inclusion in the lumen. Smooth muscle cells in the wall of the vessel are normal. x4,000. Downloaded from on July 11, 2018 by guest FIG. 7. In a capillary, a mononuclear cell (N) with an inclusion is adhered extensively to the endothelium by several areas of adherence (arrows). Only a small portion of the vascular lumen (P) is patent. x9,

6 FIG. 8. E. canis inclusion is surrounded by a single membrane (arrows), and this membrane is closely associated with membranes of endoplasmic reticulum (r). Golgi complex (G) is close to the inclusion. Subunits in the inclusion, some in division (U), are surrounded by a double membrane. x23,000. Downloaded from on July 11, 2018 by guest FIG. 9. Swollen, cell-free body (morula) is present (arrow) in the lumen of an arteriole. Apparently the body and free mitochondria (M) were derived from the adjacent ruptured mononuclear cell. (N). The endothelium (E) is intact. x5,

7 596 SIMPSON smears of lung tissue than in monocytes in the peripheral circulation (4-6). Our results agree with this observation; an explanation for this finding was seen in electron microscopic studies of lung tissue. In the lung, it was seen that parasitized mononuclear cells were commonly adhered to the endothelium of arterioles and capillaries, or mononuclear cells were chained to parasitized or possibly nonparasitized mononuclear cells that were adhered to the endothelium. In sections, of course, it was impossible to determine whether cells which apparently lacked inclusions might actually have contained such structures if the cell had been sectioned in a deeper or more superficial plane. In addition, unattached parasitized mononuclear cells, as well as saclike bodies (morulae), were frequently seen in the lumens of arterioles. An explanation for the sticking of parasitized mononuclear cells to the endothelium of arterioles and capillaries, especially at bifurcations, is lacking at this time. However, it is possible that, as in inflammation, such sticking might be due to alteration of electrochemical charges (8) or accumulation of acid mucosubstances (3, 7) between endothelial cells and parasitized mononuclear cells since, as described here, in CE there is a selective attraction between endothelial and mononuclear cells. ACKNOWLEDGMENT The technical assistance of J. W. Carlisle is acknowledged. INFECT. IMMUNTY LITERATURE CITED 1. Donatien, A., and F. Lestoquard Existence in Algerie d'une Rickettsia du chien. Bull. Soc. Pathol. Exot. 28: Ewing, S. A Differentiation of hematozoan parasites of dogs. p In Proc. Livestock Sanitary Ass., 69th Annu. Meet. Braun-Brumfield, Inc., Ann Arbor. 3. Grant, L The sticking and emigration of white blood cells in inflammation, p In B. W. Zweifach, L. Grant, and R. T. McCluskey (ed.), The inflammatory process, 2nd ed. Academic Press Inc., New York. 4. Hildebrandt, P. K., J. D. Conroy, A. E. McKee, M. B. A. Nyindo, and D. L. Huxsoll Ultrastructure of Ehrlichia canis. Infect. Immunity 7: Huxsoll, D. L., P. K. Hildebrandt, R. M. Nims, H. L. Amyx, and J. A. Ferguson Epizootiology of tropical canine pancytopenia. J. Wildl. Dis. 6: Huxsoll, D. L., H. L. Amyx, I. E. Hemelt, P. K. Hildebrandt, R. M. Nims, and W. J. Gochenour, Jr Laboratory studies of tropical canine pancytopenia. Exp. Parasitol. 31: Jones, D. B The morphology of acid mucosubstances in leukocytic sticking to endothelium in acute inflammation. Lab. Invest. 23: Marchesi, V. T Some electron microscopic observations on interactions between leukocytes, platelets, and endothelial cells in acute inflammation. Ann. N.Y. Acad. Sci. 116: Reynolds, E. S The use of lead citrate at high ph as an electron opaque stain in electron microscopy. J. Cell Biol. 17: Simpson, C. F Structure of Ehrlichia canis in blood monocytes of a dog. Amer. J. Vet. Res. 33: Walker, J. S., J. D. Rundquist, R. Taylor, B. L. Wilson, M. R. Andrews, J. Barck, A. L. Hogge, Jr., D. L. Huxsoll, P. K. Hildebrandt, and R. M. Nims Clinical and clinicopathologic findings in tropical canine pancytopenia. J. Amer. Vet. Med. Ass. 157:43-55.