Vet. Pathol. 12: 446-459 (1975)
The Ultrastructure of Macrophages in Granulomatous Colitis of Boxer Dogs H. J. VAN KRUININGEN Department of Pathobiology, University of Connecticut, Storrs, Conn.
Abstract. Thirteen cases of granulomatous colitis of Boxer dogs were studied by electron microscopy to define the contents of macrophages and to seek infectious agents. Macrophages were of three types. The most numerous were distended with residual bodies composed of membranes and parallel pairs of membranes, many arranged in circular patterns. A second common form had heterogeneous cytoplasm distended with phagosomes containing numerous small granular spheres of various sizes and electron densities. The least common were ‘young’ macrophages containing phagocytic vacuoles. Rare macrophages in four of 13 dogs contained bacteria. Macrophages in five of the dogs contained abundant coccoid, coccobacillary or lobulated granular structures, 100-500 nm in diameter, resembling chlamydia. Transition from phagocytic particle to phagosome to residual body was apparent. Granulomatous colitis of Boxer dogs is probably caused by a lipid-rich, ribosome-rich, coccoid to coccobacillary organism that possesses a cell membrane and sometimes a nucleoid, and ranges from 100 to 500 nm in size.
Granulomatous colitis of Boxer dogs was first described in 1965 [18], and has since been reported from several different locations in the United States [2, 7-9, 11, 12, 171, and from Holland [14]. Morphologically, the disease is characterized by a diffuse granulomatous inflammation of the mucosa and submucosa of affected cecum, colon, and rectum. Lamina propria and the stroma of the submucosa are infiltrated, distended, and thickened by abundant macrophages on a background of plasma cells, lymphocytes, reticulum cells, fibroblasts, mast cells, and lesser granulocytes. Colonic glands in affected mucosa are splayed laterally or raised luminally by a mantle of leukocytes in the basal lamina propria [ 17, 181. With progression of the lesion, the luminal surface of diseased segments becomes irregular, then corrugated, then ulcerated [15,16]. Affected areas become eccentrically thickened; lumen diameter is eccentrically reduced; there is loss of elasticity and motility; and
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the segment becomes progressively shortened [15]. The lymph nodes that drain colon, cecum, and rectum, and those which receive afferent lymphatics from colic nodes are diseased [15]. In advanced cases generalized lymphadenopathy occurs, and characteristic macrophages can be found in many peripheral lymph nodes. The macrophages that so strikingly dominate the lesion in this disease are distinctive in size and uniform staining. They are 11-17 pm in diameter and round to oval, with abundant homogeneously globular eosinophilic cytoplasm and prominent eccentric nuclei [ 181. Macrophage cytoplasm is strongly periodic acid-Schiff (PAS) positive and remains positive after diastase treatment. Hale’s colloidal iron stain for acid mucopolysaccharides is positive [12]. The cytoplasm shows a lack of reaction for mucus, amyloid, metachromasia, and acid-fastness [15]. The fat stains, oil-red-0 and Sudan black B, stain the cytoplasm, and frozen sections processed by the Schultz method indicate the presence of cholesterol [15]. In formalin-fixed tissues the macrophage layer is orange, suggesting that the lipid-phospholipid-cholesterol contents impart a color that is lost during processing and embedding for light microscopy. Macrophage cytoplasm shows yellow-green autofluorescence, is isotropic to polarized light, and stains positive by Schmorl’s lipoid-lipochrome procedure [12]. Occasional macrophages contain bacteria [15, 181. Electron microscopy was used to learn more about the macrophage contents and to seek infectious agents.
Materials and Methods Diseased colonic mucosa-submucosa was obtained surgically or a t autopsy from 13 Boxer dogs with granulomatous colitis. Sections 1 mm* were immediately minced and fixed in cold 4% glutaraldehyde. After refrigerated fixation for 2 h, the minced tissues were washed and stored refrigerated in 0.2 M sucrose phosphate buffer at pH 7.4. Some time later the tissues were refixed in 1-2% osmium tetroxide, immersed for 1 h in 2% aqueous uranyl nitrate, dehydrated in graded ethyl alcohols, and embedded in epon 812 or epon-araldite. Thin sections were cut with glass and diamond knives and stained with lead hydroxide. Preparations were examined with an RCA EMU-3G or JEM-T6S electron microscope.
Results The predominating macrophages appeared the same from case to case, and were generally of one of three morphologic types. Most frequently seen
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was a macrophage distended with numerous round, membrane-enclosed residual bodies (aged phagosomes) (fig. 1,2). The residual bodies varied from 0.75 to 3.2 pm in diameter, but the majority were similar in size, approximately 1.3 pm in diameter. They were enclosed in a unit membrane and had a consistency of intermediate electron density, that is, gray. The mass of the residual body was composed of membranes and parallel pairs of membranes, many of which were arranged in circular patterns. Many of the residual bodies contained one or more round electron-dense particles varying from 100 to 500 nm in diameter. The sizes and shapes of the dense particles varied considerably from one residual body to the next. In many residual bodies, less distinct and less dense particles had the appearance of dense particles fading into residual body substance. Often two or more residual bodies appeared coalesced to form a larger body. A second type of macrophage that occurred commonly had a heterogeneous cytoplasm composed of small electron-dense spheres, small granular electron-dense spheres, various sized granular gray spheres, small spherical vesicles, and odd-shaped membranous forms (fig. 3-5). Electron-dense myelin figures occurred occasionally. The smallest electron-dense and granulardense spheres were approximately 90-100 nm in diameter. In most macrophages the heterogeneous agglomerate was either partially or completely packeted within unit membranes. The size of these packets or phagosomes corresponded to the size of the residual bodies, suggesting that the former became the latter after digestion by the macrophage. Some macrophages contained both packeted agglomerate and residual bodies. A third form of macrophage, which occurred less frequently, was smaller and less filled, and contained few sphere-rich phagosomes, phagocytic vacuoles holding bacteria or structures suggestive of smaller microorganisms (fig. 6-8). In four cases few scattered macrophages contained one to 12 rodshaped bacteria approximately 1.6-2.2 pm long and 0.6-0.7 pm in diameter (fig. 6). In five cases small numbers of macrophages contained granular electron-dense structures suggestive of chlamydia (fig. 6-14). These were
Fig. 1. Parts of two adjacent macrophages containing residual bodies. The smaller residual bodies in the cell on the right occurred less frequently than the larger ones, 1 .O pm in diameter, in the cell on the left. The residual bodies are composed of membranes and parallel pairs of membranes, many arranged in circular patterns. Fig. 2. Part of a macrophage from the lamina propria of diseased colon. Cytoplasm is filled with residual bodies, 1.3 pm in diameter and slightly larger. Few granular dense particles occur among the membranes of the residual bodies.
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Fig.4. Parts of three adjacent macrophages, each distended with phagosomes composed of granular spheres of various sizes and electron densities. Note the electron-lucent nucleoid in 250-nm granular sphere at arrow. Compare with figures 2 and 5 [18].
Fig.3. Macrophage of the heterogeneous type, filled with phagosomes containing various sized, small granular spheres of various electron densities. Two bacteria are present,
the larger of which is 1.9 pm long.
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100-500 nm in diameter, encompassed by a limiting membrane, were rich in ribosomes, and had either an electron-dense or electron-lucent nucleoid. Coccoid, coccobacillary, ring-form, and lobulated ‘mature forms’ were seen. Most were within phagocytic vacuoles, few seemed to be free within the cytoplasm, and others were incorporated in phagosomes. In one case, three extracellular yeasts were identified, and in one other an extracellular structure resembling a mycoplasma was seen.
Discussion
Material from all 13 dogs was not examined with the same degree of intensity. All had macrophages filled with residual bodies, but not all were scrutinized for younger macrophages containing microorganism-like structures. Macrophages of the heterogeneous type were seen in most cases. Bacteria were seen by electron microscopy in four cases, but there is little doubt that repeated attempts would reveal few bacteria-filled macrophages in all cases. The bacteria that were seen were of such a size that they should have been readily apparent by light microscopy, and in fact they are [18, fig. 61. The bacteria were too few to be regarded as likely causative agents. In similar diseases caused by bacteria, such as Whipple’s disease and Johne’s disease, macrophage upon macrophage is crammed with bacilli. Furthermore, the composition of the residual bodies and phagosomes of active and mature macrophages in granulomatous colitis of Boxers is inconsistent with the size and composition of the bacteria present. The smaller electron-dense coccoid, coccobacillary, and lobulated structures are more likely etiologic agents, since a transition from phagocytic particle to phagosomes to residual body was apparent. The small tight circular arrays of membranes within the residual bodies suggested coccoid precursors of the granular electron-dense type and size seen in the heterogeneous macrophages and in phagocytic vacuoles. The relationship between the small, Fig. 5. Vesicular membranous structures within macrophage cytoplasm. The 450-nm structure at arrow appears to have a unit membrane and granular ribosome-like contents within which are more dense areas and one pale zone. This structure is similar to a chlamydia1 elementary body, a lysosome or a degenerate mitochondrion. Compare with figure 2 [lo]. Fig. 6. Macrophage containing six bacteria within phagocytic vacuoles. Note that the cytoplasm also contains several phagosomes composed of small granular electron-dense spheres. Bacteria are 2.2 x 0.7 pm in size.
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Fig. 7. Young macrophage from the lamina propria of diseased colon. Granular lobulated electron-dense structures in phagocytic vacuoles are suggestive of chlamydia. They vary from 180 to 365 nm in diameter. Compare with figure 20 [I]. Reproduced by permission of Gaines Dog Research Center, and Gas/roenterology.
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Fig. 8. Young macrophage containing at least six phagocytic vacuoles, within which three granular electron-dense structures occur. Note the ring form with apparent nucleoid at upper right.
100-1 50 nm, granular electron-dense spheres in the heterogeneous macrophages and the larger more discrete 300-500 nm chlamydia-like structures in phagocytic vacuoles and phagosomes is not yet clear. Perhaps the smaller spheres were the causative agents (miniature reticulate bodies of chlamydia) [13], or perhaps they were not agents at all. The variety of sizes and forms in the 100-500 nm range would be consistent with chlamydia [l, 3-5, 10, 131. No larger, paler initial bodies have been seen, however, and the electronlucent nucleoid noted has not been described for chlamydia.
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Experimental chlamydia1 infection in calves produces a diffuse granulomatous lesion of colonic lamina propria and submucosa [6], but large, foamy macrophages, such as occur in the Boxer colitis, have not been described. Light microscopy has failed to demonstrate Wolbach-Giemsa-stained chlamydia in epithelial cells or in macrophages in the canine disease, whereas they are readily demonstrable in epithelial cells of infected calves [6]. Granulomatous colitis of Boxer dogs has morphologic similarities to another chlamydia-induced intestinal disease, colonic lymphogranuloma venereum. Mild and moderately affected Boxer dogs respond to long-term treatment with antibiotics with activity against chlamydia, notably tylosin and chloramphenicol [15, 161. It is possible that granular electron-dense forms, 100-500 nm in diameter, represent various stages of mycoplasma. Bacteriologic cultivation, including the use of PPLO broths, failed to recover mycoplasmas from 10 cases studied. Some of the structures seen, particularly those in figures 9 and 10, resemble rickettsia [ 11, but the smaller and odd-shaped forms are inconsistent with rickettsia1 phases. On the basis of these observations, it is suggested that granulomatous colitis of Boxer dogs is caused by a lipid-rich, ribosome-rich, coccoid to coccobacillary organism, which possesses a cell membrane, and often a nucleoid element, and ranges from 100 to 500 nm in size. Acknowledgements Part of this study was conducted at the New York State Veterinary College, Ithaca, N.Y., where it was supported financially by departmental funds provided by Dr. C.G.
Fig. 9. Phagosome containing three coccobacillary ribosome-rich structures suggestive of chlamydia, rickettsia or mycoplasma. Structure at the left is 230 x 460 nm. Compare er al. [ I ] and LEPINAY er a / . [lo]. with ANDERSON Fig. 10. Two coccobacillary ribosome-rich structures suggestive of chlamydia, rickettsia, or mycoplasma. They appear free within the cytoplasma of a macrophage, adjacent t o a phagosome. Note the limiting cell membrane and electron-lucent nucleoid. Smaller structure is 320 nm in diameter. Reproduced by permission of Comparative Pathology Bulletin. Fig. 11. Similar ribosome-rich structure within a phagocytic vacuole. A limiting membrane is discernible, and the nucleoid appears as homogeneous dense material containing an electron-lucent vacuole. Structure is 400 nm in diameter. Compare with figure 19 of ANDERSON er nl. [I]. Fig. 12. Similar coccoid structure, 200 nm in diameter, apparently free in cytoplasm. Fig. 13,14. Coccoid ribosome-rich structures resembling chlamydia, within phagocytic vacuoles in macrophage cytoplasm.
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RICKARD, and in part by National Institutes of Health General Research Support Grant Funds allocated by Dr. G.C. POPPENSIEK. Additional studies were conducted a t the University of Connecticut with support from University of Connecticut Research Foundation Dr. Grant No. 0104-35-096. The author thanks Mrs. B.S. BROWN,Dr. J. D. STRANDBERG, C.N. BURKE,Mrs. LAMIA KHAIRALLAH, and Mrs. ELIZABETH JOHNSON for technical assistance; and Miss PATRrcrA T I M M I N S for manuscript preparation.
References 1 ANDERSON, D. R.; HOPPS,H.E.; BARILE, M. F., and BLRNHEIM, B.C.: Comparison of
the ultrastructure of several rickettsiae, ornithosis virus, and Mycoplastnu in tissue culture. J. Bact. 90: 1387-1404 (1965). 2 COCKRELL, B. Y.and KREHBIEL, J. D.: Ultrastructural changes in histiocytic ulcerative colitis in a Boxer. Am. J. vet. Res. 33: 453-459 (1972). 3 CUTLIP, R.C.: Electron microscopy of cell cultures infected with a chlamydial agent causing polyarthritis of lambs. Infec. Immunity I: 499-502 (1970). 4 DOUGHRI, A.M.; ALTERA, K. P.; STORZ,J., and EUGSTER, A.K.: Electron microscopic tracing of pathogenetic events in intestinal chlamydial infections of newborn calves. Expl. molec. Path. 18: 10-17 (1973). 5 DOUGHRI, A.M.; ALTERA,K.P.; STORZ,J., and EUGSTER,A.K.: Ultrastructural changes in the chlamydia-infected ileal mucosa of newborn calves. Vet. Pathol. 10: 114-123 (1973). 6 DOUGHRI, A.M.; YOUNG,S., and STORZ,J.: Pathologic changes in chlamydial infection of newborn calves. Am. J. vet. Res. 35: 939-944 (1974). P.C. and CELLO, R.M.: Colitis of Boxer dogs. Gastroenterology 51: 9267 KENNEDY, 931 (1966). J. F.: Colitis in a dog resembling Whipple’s disease in man. 8 KOCH,S.A. and SKELLEY, J. Am. vet. med. Ass. 150: 22-26 (1967). 9 LAWSON,T.L.; GOMEZ,J.A.; STEWART, E.T.; RAMBO,0.N., and MARGULIS, A. R.: Roentgenographic appearance of canine histiocytic ulcerative colitis. Am. J. Roentg. 110: 377-384 (1 970). 10 LEPINAY, A.; ORFILA, J.; ANTEUNIS, A.; BOUTRY,J. M . ; ORME-ROSELLI, L. et ROBINEAUX, R.: Etude en rnicroscopie ilectronique du diveloppement et d e la morphologie de Chlamydia psiltnci dans les macrophages de souris. Annls Inst. Pasteur, Paris 119: 222-23 I (1 970). 1 1 RUSSELL, S. W.; GOMEZ,J.A., and TROWBRIDGE, J.O.: Canine histiocytic ulcerative colitis. The early lesion and its progression to ulceration. Lab. Invest. 25: 509-515 ( 1971). 12 SANDER, C.H. and LANGHAM, R. F.: Canine histiocytic ulcerative colitis. A condition resembling Whipple’s disease, colonic histiocytosis, and malakoplakia in man. Archs Path. 85: 94-100 (1968). 13 TANAM[, Y.and YAMADA, Y . : Miniature cell formation in Ch/amydiapsif/aci.J. Bact. 114: 408412 (1973).
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W.T.C.: Histiocytic ulcerative 14 GAAG,I. VAN DER;HAPPE,R.P., and WOLVEKAMP, colitis in the Boxer. Proc. Voorjaarsdagen, Neth. Sm. Anim. Vet. Assn. P A 0 No. 6, pp. 19-23 (1975). 15 KRUININGEN, H. J. VAN: Granulomatous colitis of dogs; thesis Cornell University, Ithaca (1966). H. J. VAN: Granulomatous colitis of dogs; in The newer knowledge 16 KRUININGEN, about dogs. 16th Gaines Vet. Symp., pp. 19-26 (Gaines Research Center, New York 1966). 17 KRUININGEN, H. J. VAN:Granulomatous colitis of Boxer dogs: comparative aspects. Gastroenterology 53: 114-122 (1967). 18 KRUININGEN, H. J. VAN;MONTALI, R. J.; STRANDBERG, J.D., and KIRK,R. W.: A granulomatous colitis of dogs with histologic resemblance to Whipple’s disease. Pathol. vet. 2: 521-544 (1965).
Dr. H. J. VAN KRUININGEN, Department of Pathobiology, University of Connecticut, CT 06268 (USA)
Storrs,
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The Ultrastructure of Macrophages in Granulomatous Colitis of Boxer Dogs H. J. VAN KRUININGEN Department of Pathobiology, University of Connecticut, Storrs, Conn.
Abstract. Thirteen cases of granulomatous colitis of Boxer dogs were studied by electron microscopy to define the contents of macrophages and to seek infectious agents. Macrophages were of three types. The most numerous were distended with residual bodies composed of membranes and parallel pairs of membranes, many arranged in circular patterns. A second common form had heterogeneous cytoplasm distended with phagosomes containing numerous small granular spheres of various sizes and electron densities. The least common were ‘young’ macrophages containing phagocytic vacuoles. Rare macrophages in four of 13 dogs contained bacteria. Macrophages in five of the dogs contained abundant coccoid, coccobacillary or lobulated granular structures, 100-500 nm in diameter, resembling chlamydia. Transition from phagocytic particle to phagosome to residual body was apparent. Granulomatous colitis of Boxer dogs is probably caused by a lipid-rich, ribosome-rich, coccoid to coccobacillary organism that possesses a cell membrane and sometimes a nucleoid, and ranges from 100 to 500 nm in size.
Granulomatous colitis of Boxer dogs was first described in 1965 [18], and has since been reported from several different locations in the United States [2, 7-9, 11, 12, 171, and from Holland [14]. Morphologically, the disease is characterized by a diffuse granulomatous inflammation of the mucosa and submucosa of affected cecum, colon, and rectum. Lamina propria and the stroma of the submucosa are infiltrated, distended, and thickened by abundant macrophages on a background of plasma cells, lymphocytes, reticulum cells, fibroblasts, mast cells, and lesser granulocytes. Colonic glands in affected mucosa are splayed laterally or raised luminally by a mantle of leukocytes in the basal lamina propria [ 17, 181. With progression of the lesion, the luminal surface of diseased segments becomes irregular, then corrugated, then ulcerated [15,16]. Affected areas become eccentrically thickened; lumen diameter is eccentrically reduced; there is loss of elasticity and motility; and
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the segment becomes progressively shortened [15]. The lymph nodes that drain colon, cecum, and rectum, and those which receive afferent lymphatics from colic nodes are diseased [15]. In advanced cases generalized lymphadenopathy occurs, and characteristic macrophages can be found in many peripheral lymph nodes. The macrophages that so strikingly dominate the lesion in this disease are distinctive in size and uniform staining. They are 11-17 pm in diameter and round to oval, with abundant homogeneously globular eosinophilic cytoplasm and prominent eccentric nuclei [ 181. Macrophage cytoplasm is strongly periodic acid-Schiff (PAS) positive and remains positive after diastase treatment. Hale’s colloidal iron stain for acid mucopolysaccharides is positive [12]. The cytoplasm shows a lack of reaction for mucus, amyloid, metachromasia, and acid-fastness [15]. The fat stains, oil-red-0 and Sudan black B, stain the cytoplasm, and frozen sections processed by the Schultz method indicate the presence of cholesterol [15]. In formalin-fixed tissues the macrophage layer is orange, suggesting that the lipid-phospholipid-cholesterol contents impart a color that is lost during processing and embedding for light microscopy. Macrophage cytoplasm shows yellow-green autofluorescence, is isotropic to polarized light, and stains positive by Schmorl’s lipoid-lipochrome procedure [12]. Occasional macrophages contain bacteria [15, 181. Electron microscopy was used to learn more about the macrophage contents and to seek infectious agents.
Materials and Methods Diseased colonic mucosa-submucosa was obtained surgically or a t autopsy from 13 Boxer dogs with granulomatous colitis. Sections 1 mm* were immediately minced and fixed in cold 4% glutaraldehyde. After refrigerated fixation for 2 h, the minced tissues were washed and stored refrigerated in 0.2 M sucrose phosphate buffer at pH 7.4. Some time later the tissues were refixed in 1-2% osmium tetroxide, immersed for 1 h in 2% aqueous uranyl nitrate, dehydrated in graded ethyl alcohols, and embedded in epon 812 or epon-araldite. Thin sections were cut with glass and diamond knives and stained with lead hydroxide. Preparations were examined with an RCA EMU-3G or JEM-T6S electron microscope.
Results The predominating macrophages appeared the same from case to case, and were generally of one of three morphologic types. Most frequently seen
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was a macrophage distended with numerous round, membrane-enclosed residual bodies (aged phagosomes) (fig. 1,2). The residual bodies varied from 0.75 to 3.2 pm in diameter, but the majority were similar in size, approximately 1.3 pm in diameter. They were enclosed in a unit membrane and had a consistency of intermediate electron density, that is, gray. The mass of the residual body was composed of membranes and parallel pairs of membranes, many of which were arranged in circular patterns. Many of the residual bodies contained one or more round electron-dense particles varying from 100 to 500 nm in diameter. The sizes and shapes of the dense particles varied considerably from one residual body to the next. In many residual bodies, less distinct and less dense particles had the appearance of dense particles fading into residual body substance. Often two or more residual bodies appeared coalesced to form a larger body. A second type of macrophage that occurred commonly had a heterogeneous cytoplasm composed of small electron-dense spheres, small granular electron-dense spheres, various sized granular gray spheres, small spherical vesicles, and odd-shaped membranous forms (fig. 3-5). Electron-dense myelin figures occurred occasionally. The smallest electron-dense and granulardense spheres were approximately 90-100 nm in diameter. In most macrophages the heterogeneous agglomerate was either partially or completely packeted within unit membranes. The size of these packets or phagosomes corresponded to the size of the residual bodies, suggesting that the former became the latter after digestion by the macrophage. Some macrophages contained both packeted agglomerate and residual bodies. A third form of macrophage, which occurred less frequently, was smaller and less filled, and contained few sphere-rich phagosomes, phagocytic vacuoles holding bacteria or structures suggestive of smaller microorganisms (fig. 6-8). In four cases few scattered macrophages contained one to 12 rodshaped bacteria approximately 1.6-2.2 pm long and 0.6-0.7 pm in diameter (fig. 6). In five cases small numbers of macrophages contained granular electron-dense structures suggestive of chlamydia (fig. 6-14). These were
Fig. 1. Parts of two adjacent macrophages containing residual bodies. The smaller residual bodies in the cell on the right occurred less frequently than the larger ones, 1 .O pm in diameter, in the cell on the left. The residual bodies are composed of membranes and parallel pairs of membranes, many arranged in circular patterns. Fig. 2. Part of a macrophage from the lamina propria of diseased colon. Cytoplasm is filled with residual bodies, 1.3 pm in diameter and slightly larger. Few granular dense particles occur among the membranes of the residual bodies.
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Fig.4. Parts of three adjacent macrophages, each distended with phagosomes composed of granular spheres of various sizes and electron densities. Note the electron-lucent nucleoid in 250-nm granular sphere at arrow. Compare with figures 2 and 5 [18].
Fig.3. Macrophage of the heterogeneous type, filled with phagosomes containing various sized, small granular spheres of various electron densities. Two bacteria are present,
the larger of which is 1.9 pm long.
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100-500 nm in diameter, encompassed by a limiting membrane, were rich in ribosomes, and had either an electron-dense or electron-lucent nucleoid. Coccoid, coccobacillary, ring-form, and lobulated ‘mature forms’ were seen. Most were within phagocytic vacuoles, few seemed to be free within the cytoplasm, and others were incorporated in phagosomes. In one case, three extracellular yeasts were identified, and in one other an extracellular structure resembling a mycoplasma was seen.
Discussion
Material from all 13 dogs was not examined with the same degree of intensity. All had macrophages filled with residual bodies, but not all were scrutinized for younger macrophages containing microorganism-like structures. Macrophages of the heterogeneous type were seen in most cases. Bacteria were seen by electron microscopy in four cases, but there is little doubt that repeated attempts would reveal few bacteria-filled macrophages in all cases. The bacteria that were seen were of such a size that they should have been readily apparent by light microscopy, and in fact they are [18, fig. 61. The bacteria were too few to be regarded as likely causative agents. In similar diseases caused by bacteria, such as Whipple’s disease and Johne’s disease, macrophage upon macrophage is crammed with bacilli. Furthermore, the composition of the residual bodies and phagosomes of active and mature macrophages in granulomatous colitis of Boxers is inconsistent with the size and composition of the bacteria present. The smaller electron-dense coccoid, coccobacillary, and lobulated structures are more likely etiologic agents, since a transition from phagocytic particle to phagosomes to residual body was apparent. The small tight circular arrays of membranes within the residual bodies suggested coccoid precursors of the granular electron-dense type and size seen in the heterogeneous macrophages and in phagocytic vacuoles. The relationship between the small, Fig. 5. Vesicular membranous structures within macrophage cytoplasm. The 450-nm structure at arrow appears to have a unit membrane and granular ribosome-like contents within which are more dense areas and one pale zone. This structure is similar to a chlamydia1 elementary body, a lysosome or a degenerate mitochondrion. Compare with figure 2 [lo]. Fig. 6. Macrophage containing six bacteria within phagocytic vacuoles. Note that the cytoplasm also contains several phagosomes composed of small granular electron-dense spheres. Bacteria are 2.2 x 0.7 pm in size.
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Fig. 7. Young macrophage from the lamina propria of diseased colon. Granular lobulated electron-dense structures in phagocytic vacuoles are suggestive of chlamydia. They vary from 180 to 365 nm in diameter. Compare with figure 20 [I]. Reproduced by permission of Gaines Dog Research Center, and Gas/roenterology.
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Fig. 8. Young macrophage containing at least six phagocytic vacuoles, within which three granular electron-dense structures occur. Note the ring form with apparent nucleoid at upper right.
100-1 50 nm, granular electron-dense spheres in the heterogeneous macrophages and the larger more discrete 300-500 nm chlamydia-like structures in phagocytic vacuoles and phagosomes is not yet clear. Perhaps the smaller spheres were the causative agents (miniature reticulate bodies of chlamydia) [13], or perhaps they were not agents at all. The variety of sizes and forms in the 100-500 nm range would be consistent with chlamydia [l, 3-5, 10, 131. No larger, paler initial bodies have been seen, however, and the electronlucent nucleoid noted has not been described for chlamydia.
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Experimental chlamydia1 infection in calves produces a diffuse granulomatous lesion of colonic lamina propria and submucosa [6], but large, foamy macrophages, such as occur in the Boxer colitis, have not been described. Light microscopy has failed to demonstrate Wolbach-Giemsa-stained chlamydia in epithelial cells or in macrophages in the canine disease, whereas they are readily demonstrable in epithelial cells of infected calves [6]. Granulomatous colitis of Boxer dogs has morphologic similarities to another chlamydia-induced intestinal disease, colonic lymphogranuloma venereum. Mild and moderately affected Boxer dogs respond to long-term treatment with antibiotics with activity against chlamydia, notably tylosin and chloramphenicol [15, 161. It is possible that granular electron-dense forms, 100-500 nm in diameter, represent various stages of mycoplasma. Bacteriologic cultivation, including the use of PPLO broths, failed to recover mycoplasmas from 10 cases studied. Some of the structures seen, particularly those in figures 9 and 10, resemble rickettsia [ 11, but the smaller and odd-shaped forms are inconsistent with rickettsia1 phases. On the basis of these observations, it is suggested that granulomatous colitis of Boxer dogs is caused by a lipid-rich, ribosome-rich, coccoid to coccobacillary organism, which possesses a cell membrane, and often a nucleoid element, and ranges from 100 to 500 nm in size. Acknowledgements Part of this study was conducted at the New York State Veterinary College, Ithaca, N.Y., where it was supported financially by departmental funds provided by Dr. C.G.
Fig. 9. Phagosome containing three coccobacillary ribosome-rich structures suggestive of chlamydia, rickettsia or mycoplasma. Structure at the left is 230 x 460 nm. Compare er al. [ I ] and LEPINAY er a / . [lo]. with ANDERSON Fig. 10. Two coccobacillary ribosome-rich structures suggestive of chlamydia, rickettsia, or mycoplasma. They appear free within the cytoplasma of a macrophage, adjacent t o a phagosome. Note the limiting cell membrane and electron-lucent nucleoid. Smaller structure is 320 nm in diameter. Reproduced by permission of Comparative Pathology Bulletin. Fig. 11. Similar ribosome-rich structure within a phagocytic vacuole. A limiting membrane is discernible, and the nucleoid appears as homogeneous dense material containing an electron-lucent vacuole. Structure is 400 nm in diameter. Compare with figure 19 of ANDERSON er nl. [I]. Fig. 12. Similar coccoid structure, 200 nm in diameter, apparently free in cytoplasm. Fig. 13,14. Coccoid ribosome-rich structures resembling chlamydia, within phagocytic vacuoles in macrophage cytoplasm.
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RICKARD, and in part by National Institutes of Health General Research Support Grant Funds allocated by Dr. G.C. POPPENSIEK. Additional studies were conducted a t the University of Connecticut with support from University of Connecticut Research Foundation Dr. Grant No. 0104-35-096. The author thanks Mrs. B.S. BROWN,Dr. J. D. STRANDBERG, C.N. BURKE,Mrs. LAMIA KHAIRALLAH, and Mrs. ELIZABETH JOHNSON for technical assistance; and Miss PATRrcrA T I M M I N S for manuscript preparation.
References 1 ANDERSON, D. R.; HOPPS,H.E.; BARILE, M. F., and BLRNHEIM, B.C.: Comparison of
the ultrastructure of several rickettsiae, ornithosis virus, and Mycoplastnu in tissue culture. J. Bact. 90: 1387-1404 (1965). 2 COCKRELL, B. Y.and KREHBIEL, J. D.: Ultrastructural changes in histiocytic ulcerative colitis in a Boxer. Am. J. vet. Res. 33: 453-459 (1972). 3 CUTLIP, R.C.: Electron microscopy of cell cultures infected with a chlamydial agent causing polyarthritis of lambs. Infec. Immunity I: 499-502 (1970). 4 DOUGHRI, A.M.; ALTERA, K. P.; STORZ,J., and EUGSTER, A.K.: Electron microscopic tracing of pathogenetic events in intestinal chlamydial infections of newborn calves. Expl. molec. Path. 18: 10-17 (1973). 5 DOUGHRI, A.M.; ALTERA,K.P.; STORZ,J., and EUGSTER,A.K.: Ultrastructural changes in the chlamydia-infected ileal mucosa of newborn calves. Vet. Pathol. 10: 114-123 (1973). 6 DOUGHRI, A.M.; YOUNG,S., and STORZ,J.: Pathologic changes in chlamydial infection of newborn calves. Am. J. vet. Res. 35: 939-944 (1974). P.C. and CELLO, R.M.: Colitis of Boxer dogs. Gastroenterology 51: 9267 KENNEDY, 931 (1966). J. F.: Colitis in a dog resembling Whipple’s disease in man. 8 KOCH,S.A. and SKELLEY, J. Am. vet. med. Ass. 150: 22-26 (1967). 9 LAWSON,T.L.; GOMEZ,J.A.; STEWART, E.T.; RAMBO,0.N., and MARGULIS, A. R.: Roentgenographic appearance of canine histiocytic ulcerative colitis. Am. J. Roentg. 110: 377-384 (1 970). 10 LEPINAY, A.; ORFILA, J.; ANTEUNIS, A.; BOUTRY,J. M . ; ORME-ROSELLI, L. et ROBINEAUX, R.: Etude en rnicroscopie ilectronique du diveloppement et d e la morphologie de Chlamydia psiltnci dans les macrophages de souris. Annls Inst. Pasteur, Paris 119: 222-23 I (1 970). 1 1 RUSSELL, S. W.; GOMEZ,J.A., and TROWBRIDGE, J.O.: Canine histiocytic ulcerative colitis. The early lesion and its progression to ulceration. Lab. Invest. 25: 509-515 ( 1971). 12 SANDER, C.H. and LANGHAM, R. F.: Canine histiocytic ulcerative colitis. A condition resembling Whipple’s disease, colonic histiocytosis, and malakoplakia in man. Archs Path. 85: 94-100 (1968). 13 TANAM[, Y.and YAMADA, Y . : Miniature cell formation in Ch/amydiapsif/aci.J. Bact. 114: 408412 (1973).
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W.T.C.: Histiocytic ulcerative 14 GAAG,I. VAN DER;HAPPE,R.P., and WOLVEKAMP, colitis in the Boxer. Proc. Voorjaarsdagen, Neth. Sm. Anim. Vet. Assn. P A 0 No. 6, pp. 19-23 (1975). 15 KRUININGEN, H. J. VAN: Granulomatous colitis of dogs; thesis Cornell University, Ithaca (1966). H. J. VAN: Granulomatous colitis of dogs; in The newer knowledge 16 KRUININGEN, about dogs. 16th Gaines Vet. Symp., pp. 19-26 (Gaines Research Center, New York 1966). 17 KRUININGEN, H. J. VAN:Granulomatous colitis of Boxer dogs: comparative aspects. Gastroenterology 53: 114-122 (1967). 18 KRUININGEN, H. J. VAN;MONTALI, R. J.; STRANDBERG, J.D., and KIRK,R. W.: A granulomatous colitis of dogs with histologic resemblance to Whipple’s disease. Pathol. vet. 2: 521-544 (1965).
Dr. H. J. VAN KRUININGEN, Department of Pathobiology, University of Connecticut, CT 06268 (USA)
Storrs,
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