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Granulomatous sub-dermal lesions in sheep inoculated with Dermatophilus congolensis
INTRODUCTION
Dermatophilosis, caused by Dermatophifus congolensis, is a skin diseaseof domestic, animals (Roberts, 1967; Stewart, 1972a, 1972b; Bida and Dennis, 1976) and man (Deans, Gordon, Severinghaus, Droll and Reilly, 196 1; Albrecht, Horowitz. Gilbert, Richard and Connor, 1974) that occurs in all parts of the world, especially the tropics. It damages hide and wool, and causesthe death of many cattle. Bovine dermatophilosis has been reported (Kinjo, Motonaga, Matayoshi, Kudo, Watanabe, Hara, Tabuchi, Momotani and Azuma, 1981) in Okinawa prefecture, Japan, a subtropical region. The disease produces an cxudativc inflammation of the epidermis (Chodnik, 1956; Roberts, 1965a; Oduyc, 1975: Bida and Dennis, 1976, 1977) and has been reproduced experimentally (,4buSamra and Imbabi, 1976; Bida and Dennis, 1977). A different kind of lesion --granulomatous inflammation -~-has been seen on occasion in cattle (Abu-Samra and Imbabi, 1976; Abu-Samra, Imbabi and Magoub, 1976; Gupta and Sinha, 1978; Oduye, 1976)) rabbits (Abu-Samra and Imbabi, 1976), cats (Baker, Breeze and Dawson, 1972; Jones, 1976; O’Hara antI Cordes, 1963; Smith and Cordes, 1972)) and man (Albrecht et al.. 1974). Another atypical lesion -chronic lymphadenitis -was reported in goats by Singh and Murty (1978). Atypical lesions have not been studied very well and the present paper is intended to make good this deficiency. MATERIALS
AND
METHODS
.4nimaL.r Sis adult Corriedale were used.
ewes which
were free from any clinically
dctcctablc
ahnormalitici;
Organisms and Inoculuni A strain of D. congolensis (14637) kept in this institute anaerobically at 4 C was used. Organisms were cultured on brain heart infusion agar (Difco) for 7 days in 10 per cent CO, at 37°C.. Colonies were removed from the solid medium, crushed in a glass homogenizer and then centrifuged and washed 3 times with sterile 0.9 per cent saline and finally made into a cell suspension with a turbidity of Mcfarland No. 4. The inoculum was stored at 4IC and used within 3 h.
34
E.
MOMOTANI
etal.
Experimental Procedure Skin sites for inoculation were prepared on both sides of the back ofthe ewes by clipping off the hair and then washing and disinfecting the sites. Each ewe was inoculated at 4 sites subcutaneously with 2 ml of the suspension mentioned above. The lesions were observed daily. One animal was necropsied at 1, 3, 7, and 14 days after inoculation and the other 2 animals were necropsied 30 days after infection. After the macroscopic examination, the subcutaneous lesions and other organs were collected. A piece of the subcutaneous lesion was divided into 3 parts; one was used aseptically for culture and the other 2 were used for light and electron microscopic examination, respectively. In the abscess lesions, the abscess wall showing granulation or a granulomatous appearance was selected. Tissues for routine histopathology were fixed in 10 per cent neutral buffered formalin and araffin sections were stained with haematoxylin and eosin (HE), periodic acid-Schiff PPAS), MacCallum-Goodpasture and Giemsa stains. For electron microscopy the tissues were fixed with glutaraldehyde and osmium tetroxide, dehydrated and embedded in Epon. Ultrathin sections were cut and stained with both uranyl acetate and lead nitrate and then examined by electron microscope (HS-9, Hitachi). Cultural Examination The tissue blocks were crushed and placed in an anaerobic rolled tube culture with modified VL medium using the CO, gas jet method. The colonies were subcultured on a blood agar plate in 10 per cent CO, at 37’C. The colonies and films of them were examined morphologically. RESULTS
Gross Pathology One day after inoculation, a small amount of purulent material was noted subcutaneously at the injected sites and the subcutaneous connective tissue adjacent to the exudate was oedematous and the fascia near the injected sites was sometimes mildly hyperaemic. subcutaneous micro-abscesses less than Three days after the inoculation, 1.5 cm in diameter were observed in the subcutaneous tissue. They contained an odourless, custard cream-like pus enveloped by thin connective tissue and the surrounding subcutaneous tissue showed hyperaemia and extensive collagenous infiltration. On the 7th day of the infection the subcutaneous nodular lesions were still about 1.5 cm in diameter. The cut surface of most lesions showed purulent contents and the reaction of the surrounding tissues was weak. Fourteen days after inoculation various sizes of nodular lesions, 0.7 to 2.5 cm in diameter, were found. Most of the smaller nodules were granulomatous and did not contain pus, while the larger ones contained pus within fibrous capsules. Thirty days after inoculation the subcutaneous lesions were almost the same as at 14 days. There were small nodules suggesting the onset ofhealing of the lesions, whereas some relatively large nodules, more than 1.5 cm in diameter, showed poor absorption of the abscesses. The pus in the abscesses of all the ewes showed almost uniform properties regardless of the duration of infection. It was yellowish and gelatinous and had a custard cream-like appearance. It contained a number of minute yellowish white granules suggestive of “sulphur granules”. No
ATYPICAL
disseminated skin showed
35
DERMATOPHILOSIS
or metastatic lesions were found. The uninjected no visible lesions.
control
areas of the
Light Microscojy One-day lesions showed moderate or severe infiltration of neutrophils, a few monocytes and macrophages around the inoculated organisms with fine mycelia and clumps of cocci (Fig. 1). In addition, a number of PAS positive granular objects suggesting degenerated organisms were contained in the neutrophils and macrophages. In the 3-day lesions the small abscesses were mostly composed of a central accumulation of neutrophils, cellular debris, bacterial clumps and macrophages in a so-called “starry sky” arrangement, surrounded by a band of fibroblasts and macrophages. Seven-day lesions resembled those seen at 3 days after inoculation but neutrophils and debris had increased in amount, and the peripheral zone composed of histiocytic macrophages and fibroblasts had become more predominant. Inside the peripheral zone, macrophages were joined to each other at their cytoplasmic projections and syncytial multinucleated giant cells were seen (Fig. 2). These cell types usually contained coccoid or filamentous microorganisms. Some bacterial colonies were closely surrounded by macrophages. Macrophages containing the organisms were also scattered in the peripheral connective tissue.
36
E.
Fig. 3. Multiloculargranuloma Fig. 4. Granuloma proliferation
with various
MOMOTANI
sizes ofcentral
et U/.
bacterial
colonies (arrow).
14-Day
lesion. HE x 56.
composed ofepithelioid cells and a central bacterial colony. Giant cell granuloma of peripheral connective tissue are seen. 30-Day lesion. HE x 157.
(C) and
ATYPICAL
DERMATOPHILOSIS
:i 7
Typical 14-day lesions consisted of many granulomatous nodules which showed multiloculation (Fig. 3). The granulomas contained a bacterial colon!, surrounded by a narrow band of neutrophils. Epithelioid cells or mature macrophages in sheets and packed closely together were seen around the colon). and the neutrophils. They were surrounded by connective tissue septa. The cells of the monocyte-macrophage system (MMS) with their vesicular nuclei wart’ larger and their cytoplasm was more eosinophilic. Macrophages, monocytrs, lymphocytes and plasma cells were also present in the granuloma. Nests composed of numerous foreign body type and Langhans type giant cells wcrc present among the macrophages, mainly in the peripheral zone of the lesions and between the loculi. Some of the epithelioid cells and giant cells contained numerous micro-organisms in their cytoplasm. The outer zone or wall of the granulomas consisted of fibroblasts, histiocytic macrophages, capillaries, somt collagen and some inflammatory cells. Some abscesses persisted in all the animals. At 30 days the granulomatous lesions in the subcutaneous nod&s closeI!. resembled those seen at 14 days (Fig. 4). Small granulomatous units consisting of giant cells were found. Mild diffuse infiltrations of neutrophils and 1ymphocytt.s were found around the granulomas. One ewe revealed poorly organized ahscesscs in which the granulomatous reaction was not predominant. None of the infected ewes revealed any distinct histopathological changes other than the lesions in the areas injected, except that some of the lymph nodes adjacent to the lesions showed some reactive changes. Electron iMicroscop_~ By the first day after inoculation D. congolensis had caused neutrophil accumulation. The organisms were of various shapes and had intact or degenerated and fragmented coccoid and filamentous elements with parallel rows of beading and were surrounded by neutrophils. A small number of macrophages and a few monocytes were observed. The macrophages had ovoid or irregular cell outlines, cytoplasmic pseudopodia, a few mitochondria, a rough endoplasmic reticulum (RER) and many free ribosomes. These macrophages and neutrophils contained intact bacteria and some bacterial fragments. At 3 days after inoculation the findings were basically similar to those of the one-da! lesions. Macrophages were seen around the bacteria. Some sections showed areas with accumulated macrophages containing numerous coccoid elements of II. congo1ensi.r. Th ese macrophages usually adhered together and sometimes degenerated to varying degrees. Some coccoids were present in the phagosomcs along with some digestive changes. At 7 days the lesions were composed predominantly of macrophages and neutrophils. There were no free organisms apparent. At 14 days the lesions were composed of sheets and knots of epithelioid cells with large euchromatic nuclei and macrophages (Fig. 5) and some giant cells with many large euchromatic nuclei. Many neutrophils were also found in the granuloma. The epithelioid cells showed a variety of cytoplasmic organelles; some of the cells contained much RER and others contained predominantly mitochondria or golgi apparatus. Some mature and immature epithelioid cells which closely approximated each other were apparent. Various degrees of‘
38
E.
MOMOTANI
et
d.
interdigitation of the blunt pseudopodia of adjoining epithelioid cells (Fig. 6 inset), giant cells and some macrophages were found. The lymphocytes and plasma cells were more numerous than in the 7-day lesions. Fibroblasts and fibrocytes were also present. At 30 days after infection the findings were basically similar to those at 14 days. Epithelioid cells were tightly aggregated together, with giant cells (Fig. 6), lymphocytes and plasma cells. Cultural Examination Dermatophilus congolensis was recovered from the subcutaneous lesions of the ewes killed, 3, 7, 14 and 30 days after inoculation. Very small white or greyishwhite colonies developed 2 to 3 days after inoculation in anaerobic roll-tube cultures with the CO, gas jet method. At 7 to 10 days after inoculation small rough colonies were found. The isolates were subcultured from anaerobic modified VL medium on a 10 per cent CO, blood agar plate at 37°C. The colonies showed a clear zone of complete haemolysis. Films made from the colonies stained with Gram and Giemsa confirmed the characteristic morphology of the organisms. DISCUSSION
The results of this experiment confirmed that D. congolensis causes granulomas and abscesses in sheep and that the pathological changes develop with the passage of time. The initial macroscopic lesions were small suppurative foci, and palpable subcutaneous nodular lesions were formed at 7 days after inoculation. At 14 to 30 days after inoculation, most of the nodules were enlarged but some showed various degrees of involution. The involuted nodules showed granulomatous characteristics, though some pus was contained in their centre. In contrast, the enlarged nodules turned into well developed abscesses. Histopathologically, early-stage lesions usually consisted of a central necrotic area, pus and an outer granulomatous area. The granulomatous lesions consisted of bacterial colonies in the centre surrounded by neutrophils, macrophages and epithelioid cells. These lesions usually showed multiloculation, with lobular areas of varying size. A granulomatous reaction predominated over the suppurative inflammatory reaction with the passage of time. No detectable lesions were found in any organs except the lymph nodes draining the inoculated sites. The granulomas found in the present study were similar to those of Actinomyces rown, 1973), N ocardia (Destombes, Mariat, Nazimoff and Satre, 1961)) (B Pseudomonas (Kubo, Osada and Konno, 198 1) and true fungal infection (Robboy and Vickery, 1970; Verghes and Klokke, 1966; Winslow and Steen, 1964). These types of granulomas were classified as complex granulomas (Adams, 1976). On the other hand, another type of granuloma, the pure (epithelioid) granuloma (Adams, 1976)) resulting from the inoculation of Mycobacteria (Adams, 1974, 1976; Browett, Simpson and Blennenhassette, 1979) or muramyl dipeptide (Nagao, Ota, Emori, Inoue, and Tanaka, 198 1; Tanaka and Emori, 1980) differs in some respects from the complex granuloma. In the latter, micro-organisms are characteristically present in the centre and some granulocytes are included among the component cells. The colonies or clumps of micro-organisms in the granulomas are called “sulphur granules” or “grains”. In this type of granuloma,
ATYPICAL
stucdies on erg.anisms
DERMATOPHILOSIS
39
the morphology or staining characteristics of the above mention led have been made (Brown, 1973; Kubo, Osada and Konno, 1980; Ku bo et a,i., 1981; Destombes et al., 1961; Robboy and Vickery, 1970; Verghese and Klc ,kkr, 1966; Winslow and Steen, 1964), but the ultrastructural or function IAl
40
E.
MOMOTANI
et
cd.
properties of the component cells, such as the granulocytes and the monocytemacrophage system (MMS) have been only incompletely understood. In the present study, monocytes, macrophages, epithelioid cells and giant cells in the granulomas were observed with the electron microscope. The monocytes, which at first were very simple cells with poor cytoplasmic organelles, changed into cells with complex and abundant organelles. Large euchromatic nuclei and abundant cytoplasm, filled with many lysosomes, mitochondria, golgi profiles and RER, were seen in the epithelioid and giant cells. Projecting finger-like processes were also seen in the present study and they interdigitated with equivalent processes of neighbouring cells. They have been described by many workers as one of the characteristics of epithelioid cells and giant cells (Adams, 1974, 1976; Browett et al., 1979). Electron microscopical findings of the component cells of the MMS in the present granulomas were basically similar to those in other kinds of granulomas found in animals or man caused by infectious agents or certain chemicals (mycobacteria (Adams, 1974; Browett et al., 1979), muramyl dipeptide (Tanaka and Emori, 1980), Schistosoma (Epstein, Fukuyama, Danno and Kwan-Wong, 1979) and beryllium and zirconium (Elias and Epstein, 1968)). Varying numbers of D. congolensis in the cytoplasma of cells of the MMS were observed by light and electron microscopy. The macrophages had a number of phagosomes or phagolysosomes containing organisms which showed either a degraded or an intact appearance. They were in the peripheral area of the lesions The well developed epithelioid cells and at 3 to 14 days after inoculation. Langhans and foreign body giant cells sometimes also contained the organisms. It has been reported that these cells in the MMS do not exhibit phagocytic activity has been found activity (Browett et al., 1979) but weak phagocytic (Adams, 1976). From this viewpoint, the present findings suggest that the microorganisms either remained intact in the cells throughout the maturing process from macrophage to more advanced cells or they remained within the structure without undergoing visible degradation. Although in some mycobacteria it has been reported that killed bacteria continued to be demonstrated in cultured macrophages for a long period following inoculation, it is possible that, as in experiments with living bacteria (Bendixen, Black and Jorgensen, 1981; Barbieri and Correa, 1967), Dermatophilus congolensis may well survive as an intracellular organism under some conditions. The abscesses or granulomatous lesions caused by D. congolensis in the present study were ofsomewhat different structure from those in the usual spontaneous or experimental dermatophilosis. Dermatophilosis is usually regarded as a superficial exudative dermatitis which is limited to the epidermis (Bida and Dennis, 1976, 1977; Chodnik, 1956; Oduye, 1975; Roberts, 1965a). The main inflammatory cells in the lesions are neutrophils (Roberts, 1965a, 1966). A few atypical dermatophilus infections which show small or large differences from the typical dermatophilosis have been reported. Abu-Samra et al. (1976) and Oduye (1976) described a bovine dermatophilosis with granulomatous inflammation in the dermis; the lesions found in the very severe cases were composed of macrophages, epithelioid and giant cells and fibroblasts. Experimental infection with D. congolensis has revealed similar findings (Abu-Samra and Imbabi, 1976)
ATYPICAL
DERMATOPHILaOSIS
41
but Dermatophilus organisms were not demonstrated in the granulomatous reaction in these reports. In Japan, Kono (1965) described a bovine granulomatous dermatitis with Langhans type giant cells in the dermis and of unknown aetiology. The histological specimens of the dermatitis were re-examined by the present authors and it was ascertained that the lesions were very similar to those reported by Oduye ( 1976). Recently Kinjo et al. ( 1981) reported the existence of bovine dermatophilosis in Okinawa prefecture, Japan, in which Kono (1965) had Further detailed histopathological discovered a granulomatous dermatitis. examination of spontaneous dermatophilosis might well reveal granulomatous changes. There have been a few reports of atypical dermatophilosis occurring in tissues other than the skin in animals. Four feline cases have been reported. O’Hara and Cordes (1963) reported 2 cases of granuloma caused by Dermatophilus: one lesion involved the glossal muscle, and the other the serous surface of the urinary bladder. Baker et al. (1972) reported the condition on the tongue of a cat. In these studies the distinct morphology of the Dermatophilus organisms was seen in the histological specimens but the authors did not isolate the organism. Finally, (1972) described Dermatophilus-like micro-organisms in Smith and Cordes granulomas in feline lymph nodes. However, Roberts (1967) denied that 11. congnlensis was responsible for the feline chronic inflammatory lesions in the popliteal lymph node of a cat from which he isolated D. congolensis. In the goat, Singh and Murty (1978) isolated the organism from an intractable suppurativt lymphadenitis. Albrecht et al. (1974) reported a case of unusual human dermatophilosis in an 8-year-old boy with a deficiency of B lymphocyte-bearing FC receptors and lymphokine production. In this chronic nodular disease, epithelioid cells and giant cells were found around the central necrotic area. The organisms were demonstrated by the fluorescent antibody technique. Th( chronic or granulomatous lesions mentioned above were similar to those found in the present study. It should be recognized that D. congolensis can cause a suppurative or a granulomatous inflammation, but the role of the granulomatous lesions in natural field dermatophilosis is not yet known precisely. We should at least recognize that D. congolensis can produce types of lesion other thau ;I superficial dermatitis.
SUMMARY
Dermatophilus congolensis was inoculated subcutaneously into 6 ewes and the resulting lesions were examined by light and electron microscopy. The organism was recovered from the subcutaneous lesions. The lesions were suppurative in the early stages and granulomatous in the advanced stage, but abscesses were the usual lesion observed. The granulomas were composed of several layers; central bacterial colony, neutrophil layer, layer of macrophages, epithelioid cells and Langhans and foreign body type giant cells with a periphery of connective tissue. Multiloculated granulomas were observed. Electron microscopic examination confirmed the cell types of the lesions. Epithelioid and giant cells were characterized by the shape Znd number of the nuclei and complex and abundant
42
E.
MOMOTANI
t?t d.
organelles. Interdigitations of the blunt pseudopodia of adjacent cells were also found. It is concluded that the organism can produce both granulomatous lesions and abscesses which differ from those of orthodox dermatophilosis but are similar to those described in “atypical dermatophilosis”.
REFERENCES
Abu-Samra, M. T., and Imbabi, S. E. (1976). Experimental infection of domesticated animals and the fowl with Dermatophilus congolensis. Journal of Comparative Pathology, 86, 157-172. Abu-Samra, M. T., Imbabi, S. E.,.and Mahgoub, E. S. (1976). Dermatophiluscongolensis. A bacteriological, in vitro antrbrotic sensitivity and histopathological study of natural infection in Sudanese cattle. British Veterinary Journal, 132, 627631. Adams, D. 0. (1974). The structure of mononuclear phagocytes differentiating in viuo I. Sequental fine and histologic studies of the effect of Bacillus Calmette-Guerin (BCG) American Journal of Pathology, 76, 17748. Adams, D. 0. (1976). The granulomatous inflammatory response. A review. American Journal of Pathology, 84, 164-191. Albrecht, R., Horowitz, S., Gilbert, E., Richard, J., and Connor, D. H. (1974). Dermatophilus congolensis chronic nodular disease in man. Pediatrics, 53, 902-912. Baker, G. J., Breeze, R. G., and Dawson, C. 0. (1972). Oral dermatophilosis in a cat. Journal of Small Animal Practice, 13, 6499653. Barbieri, T. A., and Correa, W. M. (1967). H uman macrophage culture. The leprosy prognostic test (LPT) International Journal of Leprosy, 35, 377-381. Bendixen, P. H., Black, B., and Jorgensen, J. B. ( 198 1). Lack of intracellular degradation of Mycobacterium paratuberculosis by bovine macrophages infected in vitro and in vivo: Light and electron microscopic observation. American Journal of Veterinary Research, 42, 109-l 13. Bida, S. A., and Dennis, S. M. ( 1976). D ermatophilosis in Northern Nigeria. Veterinary Bulletin, 46, 471478. Bida, S. A., and Dennis, S. M. (1977). Sequential pathological changes in natural and experimental dermatophilosis in Bunaji cattle. Research in Veterinary Science, 22, 18-22. Browett, P. J., Simpson, L. O., and Blennerhassette, J. B. (1979). Experimental granulomatous inflammation: The ultrastructure of the granuloma induced by injection of tubercle bacilli into Freund adjuvant-sensitised guinea-pigs. Journal of Pathology, 129, 191-201. Brown, J. R. (1973). Human actinomycosis. A study of 181 subjects. Human Pathology, 4, 319-330. Chodnik, K. S. (1956). Mycotic dermatitis of cattle in British West Africa. Journal of Comparative Pathology, 66, 179-186. Deans, D. J., Gordon, M. A., Severinghaus, C. W., Kroll, E. T., and Reilly, J. R. ( 1961). Streptothricosis: A new zoonotic disease. New York State Journal of Medicine, 15, 1283-1287. Destombes, P., Mariat, F., Nazimoff, O., and Satre, J. (1961). Apropos des mycetomes a Nocardia. Sabouraudia, 1, 161-172. Elias, P. M., and Epstein, W. L. (1968). Ultrastructural observations on experimentally induced foreign-body and organized epithelioid-cell granulomas in man. American Journal of Pathology, 52, 1207-1223. Epstein, W. L., Fukuyama, K., Danno, K., and Kwan-Wong, E. (1979). Granulomatous inflammation in normal and athymic mice infected with Schistosoma mansoni: An ultrastructural study. Journal of Pathology, 127, 207-2 15. Gupta, P. P., and Sinha, B. P. (1978). Oral dermatophilosis associated with actinomycosis in cattle. zentralblatt fur VeteriniirmediZin. B, 25, 2 11-215. Jones, R. T. (1976). Subcutaneous infection with Dermatophilus congolensis in a cat. Journal of Comparative Pathology, 86, 415-421.
ATYPICAL
DERMATOPHILOSIS
Kin,jo,
4Ii
E., Motonaga, H., Matayoshi, E., Kudo, S., Watanabe, K., Hara, M., Tabuchi, in K., Momotani, E., and Azuma, R. (1981). 0 ccurrence of bovine dermatophilosis the southernmost islands of Japan. National Institute of‘Anima1 Health Quarterly, 21, 163-174. Kono, I. (1965). Granulomatous dermatitis in cattle. Journal of Veterinary Medicine, 48’7, I 3-l 4 (In Japanese). Kubo, M., Osada, M., and Konno, S. (1980). A histopathological and ultrastructural comparison of the sulphur granule in actinomycosis and actinobacillosis. National Institute of Animal Health Quarterly, 28, 53-59. Kubo, M., Osada, M., and Konno, S. (1981). Morphology ofsulphur granules produced by Pseudomonas aeruginosa in cow. National Institute of rlnimal Health Quarterly, 21, 26-3 1. Nagao, S., Ota, F., Emori, K., Inoue, K., and Tanaka, A. ( 1981). Epithelioid granuloma induced by muramyl dipeptide in the immunologically deficient rat. Infection and Immunity, 34, 993-999. Oduye, 0. 0. (1975). Effect of various local environmental conditions and histopathological studies in experimental Dermatophilus congolensis infection on the bovine skin. Research in Veterinary Science, 19, 245-252. Oduye, 0. 0. (1976). Histological changes in natural and experimental Dermatophilus congolensis infection of the bovine skin. In Dermatophilous Infection in Animals and Man. D. H. Lloyd and K. C. Sellars, Eds, Academic Press, London. O’Hara, P. J., and Cordes, D. C. (1963). Granuloma caused by Dermatophilus in two cats. Nele! Zealand Veterinary Journal, 11, 15 1- 154. Robboy, S. J., and Vickery, A. L., Jr. (1970). Tinctorial and morphologic properties distinguishing actinomycosis and nocardiosis. Nezpl England Journal of Medicine, 282, 593-596. Roberts, D. S. (1965a). The histopathology ofepidermal infection with the Actinomycrte Dermatophilus congolensis. Journal of Pathology and Bacteriology, 90, 2 13-2 16. Roberts, D. S. ( 196513). The role of granulocytes in resistance to Dermatophilus congolensis. British journal of Experimental Pathology, 46, 643448. Roberts, D. S. ( 1966). The influence of delayed hypersensitivity on the course of infection with Dermatophilus congolensis. British Journal of Experimental Pathology, 47, 9-16. Roberts, D. S. ( 1967). Dermatophilus infection. The Veterinary Bulletin, 37, 5 13-52 1. Singh, V. P., and Murty, D. K. (1978). An outbreak of Dermatophilus congolensis infection in goats. Indian Veterinary Journal, 55, 674-676. Smith, C. F., and Cordes, D. 0. (1972). Dermatitis caused by Dermatophilus congolensis infection in polar bears (Tharactos maritimus). British Veterinary Journal, 128,366-37 1. Stewart, G. H. ( 1972a). Dermatophilosis: A skin disease of animals and man. Part I. The J’eterinary Record, 91, 537~-544. Stewart, G. H. ( 1972b). Dermatophilosis: A skin disease ofanimals and man. Part II. Thhr Veterinary Record, 91, 555-561. Tanaka, A., and Emori, K. (1980). Epithelioid granuloma formation by a systemic bacterial cell wall component, Muramyl dipeptide (MDP). American journal of Pathology, 98, 733-748. Verghes, A., and Klokke, A. H. (1966). Histologic diagnosis ofspecies of fungus causing mycetoma. Indian Journal of Medical Research, 54, 524-530. \Yinslow, D. J., and Steen, F. G. (1964). C onsiderations in the histologic diagnosis of mycrtoma. American Journal of Pathology, 42, 164-l 69. [Received for publication,
.Nouember 15th, 19821
Dermatophilosis, caused by Dermatophifus congolensis, is a skin diseaseof domestic, animals (Roberts, 1967; Stewart, 1972a, 1972b; Bida and Dennis, 1976) and man (Deans, Gordon, Severinghaus, Droll and Reilly, 196 1; Albrecht, Horowitz. Gilbert, Richard and Connor, 1974) that occurs in all parts of the world, especially the tropics. It damages hide and wool, and causesthe death of many cattle. Bovine dermatophilosis has been reported (Kinjo, Motonaga, Matayoshi, Kudo, Watanabe, Hara, Tabuchi, Momotani and Azuma, 1981) in Okinawa prefecture, Japan, a subtropical region. The disease produces an cxudativc inflammation of the epidermis (Chodnik, 1956; Roberts, 1965a; Oduyc, 1975: Bida and Dennis, 1976, 1977) and has been reproduced experimentally (,4buSamra and Imbabi, 1976; Bida and Dennis, 1977). A different kind of lesion --granulomatous inflammation -~-has been seen on occasion in cattle (Abu-Samra and Imbabi, 1976; Abu-Samra, Imbabi and Magoub, 1976; Gupta and Sinha, 1978; Oduye, 1976)) rabbits (Abu-Samra and Imbabi, 1976), cats (Baker, Breeze and Dawson, 1972; Jones, 1976; O’Hara antI Cordes, 1963; Smith and Cordes, 1972)) and man (Albrecht et al.. 1974). Another atypical lesion -chronic lymphadenitis -was reported in goats by Singh and Murty (1978). Atypical lesions have not been studied very well and the present paper is intended to make good this deficiency. MATERIALS
AND
METHODS
.4nimaL.r Sis adult Corriedale were used.
ewes which
were free from any clinically
dctcctablc
ahnormalitici;
Organisms and Inoculuni A strain of D. congolensis (14637) kept in this institute anaerobically at 4 C was used. Organisms were cultured on brain heart infusion agar (Difco) for 7 days in 10 per cent CO, at 37°C.. Colonies were removed from the solid medium, crushed in a glass homogenizer and then centrifuged and washed 3 times with sterile 0.9 per cent saline and finally made into a cell suspension with a turbidity of Mcfarland No. 4. The inoculum was stored at 4IC and used within 3 h.
34
E.
MOMOTANI
etal.
Experimental Procedure Skin sites for inoculation were prepared on both sides of the back ofthe ewes by clipping off the hair and then washing and disinfecting the sites. Each ewe was inoculated at 4 sites subcutaneously with 2 ml of the suspension mentioned above. The lesions were observed daily. One animal was necropsied at 1, 3, 7, and 14 days after inoculation and the other 2 animals were necropsied 30 days after infection. After the macroscopic examination, the subcutaneous lesions and other organs were collected. A piece of the subcutaneous lesion was divided into 3 parts; one was used aseptically for culture and the other 2 were used for light and electron microscopic examination, respectively. In the abscess lesions, the abscess wall showing granulation or a granulomatous appearance was selected. Tissues for routine histopathology were fixed in 10 per cent neutral buffered formalin and araffin sections were stained with haematoxylin and eosin (HE), periodic acid-Schiff PPAS), MacCallum-Goodpasture and Giemsa stains. For electron microscopy the tissues were fixed with glutaraldehyde and osmium tetroxide, dehydrated and embedded in Epon. Ultrathin sections were cut and stained with both uranyl acetate and lead nitrate and then examined by electron microscope (HS-9, Hitachi). Cultural Examination The tissue blocks were crushed and placed in an anaerobic rolled tube culture with modified VL medium using the CO, gas jet method. The colonies were subcultured on a blood agar plate in 10 per cent CO, at 37’C. The colonies and films of them were examined morphologically. RESULTS
Gross Pathology One day after inoculation, a small amount of purulent material was noted subcutaneously at the injected sites and the subcutaneous connective tissue adjacent to the exudate was oedematous and the fascia near the injected sites was sometimes mildly hyperaemic. subcutaneous micro-abscesses less than Three days after the inoculation, 1.5 cm in diameter were observed in the subcutaneous tissue. They contained an odourless, custard cream-like pus enveloped by thin connective tissue and the surrounding subcutaneous tissue showed hyperaemia and extensive collagenous infiltration. On the 7th day of the infection the subcutaneous nodular lesions were still about 1.5 cm in diameter. The cut surface of most lesions showed purulent contents and the reaction of the surrounding tissues was weak. Fourteen days after inoculation various sizes of nodular lesions, 0.7 to 2.5 cm in diameter, were found. Most of the smaller nodules were granulomatous and did not contain pus, while the larger ones contained pus within fibrous capsules. Thirty days after inoculation the subcutaneous lesions were almost the same as at 14 days. There were small nodules suggesting the onset ofhealing of the lesions, whereas some relatively large nodules, more than 1.5 cm in diameter, showed poor absorption of the abscesses. The pus in the abscesses of all the ewes showed almost uniform properties regardless of the duration of infection. It was yellowish and gelatinous and had a custard cream-like appearance. It contained a number of minute yellowish white granules suggestive of “sulphur granules”. No
ATYPICAL
disseminated skin showed
35
DERMATOPHILOSIS
or metastatic lesions were found. The uninjected no visible lesions.
control
areas of the
Light Microscojy One-day lesions showed moderate or severe infiltration of neutrophils, a few monocytes and macrophages around the inoculated organisms with fine mycelia and clumps of cocci (Fig. 1). In addition, a number of PAS positive granular objects suggesting degenerated organisms were contained in the neutrophils and macrophages. In the 3-day lesions the small abscesses were mostly composed of a central accumulation of neutrophils, cellular debris, bacterial clumps and macrophages in a so-called “starry sky” arrangement, surrounded by a band of fibroblasts and macrophages. Seven-day lesions resembled those seen at 3 days after inoculation but neutrophils and debris had increased in amount, and the peripheral zone composed of histiocytic macrophages and fibroblasts had become more predominant. Inside the peripheral zone, macrophages were joined to each other at their cytoplasmic projections and syncytial multinucleated giant cells were seen (Fig. 2). These cell types usually contained coccoid or filamentous microorganisms. Some bacterial colonies were closely surrounded by macrophages. Macrophages containing the organisms were also scattered in the peripheral connective tissue.
36
E.
Fig. 3. Multiloculargranuloma Fig. 4. Granuloma proliferation
with various
MOMOTANI
sizes ofcentral
et U/.
bacterial
colonies (arrow).
14-Day
lesion. HE x 56.
composed ofepithelioid cells and a central bacterial colony. Giant cell granuloma of peripheral connective tissue are seen. 30-Day lesion. HE x 157.
(C) and
ATYPICAL
DERMATOPHILOSIS
:i 7
Typical 14-day lesions consisted of many granulomatous nodules which showed multiloculation (Fig. 3). The granulomas contained a bacterial colon!, surrounded by a narrow band of neutrophils. Epithelioid cells or mature macrophages in sheets and packed closely together were seen around the colon). and the neutrophils. They were surrounded by connective tissue septa. The cells of the monocyte-macrophage system (MMS) with their vesicular nuclei wart’ larger and their cytoplasm was more eosinophilic. Macrophages, monocytrs, lymphocytes and plasma cells were also present in the granuloma. Nests composed of numerous foreign body type and Langhans type giant cells wcrc present among the macrophages, mainly in the peripheral zone of the lesions and between the loculi. Some of the epithelioid cells and giant cells contained numerous micro-organisms in their cytoplasm. The outer zone or wall of the granulomas consisted of fibroblasts, histiocytic macrophages, capillaries, somt collagen and some inflammatory cells. Some abscesses persisted in all the animals. At 30 days the granulomatous lesions in the subcutaneous nod&s closeI!. resembled those seen at 14 days (Fig. 4). Small granulomatous units consisting of giant cells were found. Mild diffuse infiltrations of neutrophils and 1ymphocytt.s were found around the granulomas. One ewe revealed poorly organized ahscesscs in which the granulomatous reaction was not predominant. None of the infected ewes revealed any distinct histopathological changes other than the lesions in the areas injected, except that some of the lymph nodes adjacent to the lesions showed some reactive changes. Electron iMicroscop_~ By the first day after inoculation D. congolensis had caused neutrophil accumulation. The organisms were of various shapes and had intact or degenerated and fragmented coccoid and filamentous elements with parallel rows of beading and were surrounded by neutrophils. A small number of macrophages and a few monocytes were observed. The macrophages had ovoid or irregular cell outlines, cytoplasmic pseudopodia, a few mitochondria, a rough endoplasmic reticulum (RER) and many free ribosomes. These macrophages and neutrophils contained intact bacteria and some bacterial fragments. At 3 days after inoculation the findings were basically similar to those of the one-da! lesions. Macrophages were seen around the bacteria. Some sections showed areas with accumulated macrophages containing numerous coccoid elements of II. congo1ensi.r. Th ese macrophages usually adhered together and sometimes degenerated to varying degrees. Some coccoids were present in the phagosomcs along with some digestive changes. At 7 days the lesions were composed predominantly of macrophages and neutrophils. There were no free organisms apparent. At 14 days the lesions were composed of sheets and knots of epithelioid cells with large euchromatic nuclei and macrophages (Fig. 5) and some giant cells with many large euchromatic nuclei. Many neutrophils were also found in the granuloma. The epithelioid cells showed a variety of cytoplasmic organelles; some of the cells contained much RER and others contained predominantly mitochondria or golgi apparatus. Some mature and immature epithelioid cells which closely approximated each other were apparent. Various degrees of‘
38
E.
MOMOTANI
et
d.
interdigitation of the blunt pseudopodia of adjoining epithelioid cells (Fig. 6 inset), giant cells and some macrophages were found. The lymphocytes and plasma cells were more numerous than in the 7-day lesions. Fibroblasts and fibrocytes were also present. At 30 days after infection the findings were basically similar to those at 14 days. Epithelioid cells were tightly aggregated together, with giant cells (Fig. 6), lymphocytes and plasma cells. Cultural Examination Dermatophilus congolensis was recovered from the subcutaneous lesions of the ewes killed, 3, 7, 14 and 30 days after inoculation. Very small white or greyishwhite colonies developed 2 to 3 days after inoculation in anaerobic roll-tube cultures with the CO, gas jet method. At 7 to 10 days after inoculation small rough colonies were found. The isolates were subcultured from anaerobic modified VL medium on a 10 per cent CO, blood agar plate at 37°C. The colonies showed a clear zone of complete haemolysis. Films made from the colonies stained with Gram and Giemsa confirmed the characteristic morphology of the organisms. DISCUSSION
The results of this experiment confirmed that D. congolensis causes granulomas and abscesses in sheep and that the pathological changes develop with the passage of time. The initial macroscopic lesions were small suppurative foci, and palpable subcutaneous nodular lesions were formed at 7 days after inoculation. At 14 to 30 days after inoculation, most of the nodules were enlarged but some showed various degrees of involution. The involuted nodules showed granulomatous characteristics, though some pus was contained in their centre. In contrast, the enlarged nodules turned into well developed abscesses. Histopathologically, early-stage lesions usually consisted of a central necrotic area, pus and an outer granulomatous area. The granulomatous lesions consisted of bacterial colonies in the centre surrounded by neutrophils, macrophages and epithelioid cells. These lesions usually showed multiloculation, with lobular areas of varying size. A granulomatous reaction predominated over the suppurative inflammatory reaction with the passage of time. No detectable lesions were found in any organs except the lymph nodes draining the inoculated sites. The granulomas found in the present study were similar to those of Actinomyces rown, 1973), N ocardia (Destombes, Mariat, Nazimoff and Satre, 1961)) (B Pseudomonas (Kubo, Osada and Konno, 198 1) and true fungal infection (Robboy and Vickery, 1970; Verghes and Klokke, 1966; Winslow and Steen, 1964). These types of granulomas were classified as complex granulomas (Adams, 1976). On the other hand, another type of granuloma, the pure (epithelioid) granuloma (Adams, 1976)) resulting from the inoculation of Mycobacteria (Adams, 1974, 1976; Browett, Simpson and Blennenhassette, 1979) or muramyl dipeptide (Nagao, Ota, Emori, Inoue, and Tanaka, 198 1; Tanaka and Emori, 1980) differs in some respects from the complex granuloma. In the latter, micro-organisms are characteristically present in the centre and some granulocytes are included among the component cells. The colonies or clumps of micro-organisms in the granulomas are called “sulphur granules” or “grains”. In this type of granuloma,
ATYPICAL
stucdies on erg.anisms
DERMATOPHILOSIS
39
the morphology or staining characteristics of the above mention led have been made (Brown, 1973; Kubo, Osada and Konno, 1980; Ku bo et a,i., 1981; Destombes et al., 1961; Robboy and Vickery, 1970; Verghese and Klc ,kkr, 1966; Winslow and Steen, 1964), but the ultrastructural or function IAl
40
E.
MOMOTANI
et
cd.
properties of the component cells, such as the granulocytes and the monocytemacrophage system (MMS) have been only incompletely understood. In the present study, monocytes, macrophages, epithelioid cells and giant cells in the granulomas were observed with the electron microscope. The monocytes, which at first were very simple cells with poor cytoplasmic organelles, changed into cells with complex and abundant organelles. Large euchromatic nuclei and abundant cytoplasm, filled with many lysosomes, mitochondria, golgi profiles and RER, were seen in the epithelioid and giant cells. Projecting finger-like processes were also seen in the present study and they interdigitated with equivalent processes of neighbouring cells. They have been described by many workers as one of the characteristics of epithelioid cells and giant cells (Adams, 1974, 1976; Browett et al., 1979). Electron microscopical findings of the component cells of the MMS in the present granulomas were basically similar to those in other kinds of granulomas found in animals or man caused by infectious agents or certain chemicals (mycobacteria (Adams, 1974; Browett et al., 1979), muramyl dipeptide (Tanaka and Emori, 1980), Schistosoma (Epstein, Fukuyama, Danno and Kwan-Wong, 1979) and beryllium and zirconium (Elias and Epstein, 1968)). Varying numbers of D. congolensis in the cytoplasma of cells of the MMS were observed by light and electron microscopy. The macrophages had a number of phagosomes or phagolysosomes containing organisms which showed either a degraded or an intact appearance. They were in the peripheral area of the lesions The well developed epithelioid cells and at 3 to 14 days after inoculation. Langhans and foreign body giant cells sometimes also contained the organisms. It has been reported that these cells in the MMS do not exhibit phagocytic activity has been found activity (Browett et al., 1979) but weak phagocytic (Adams, 1976). From this viewpoint, the present findings suggest that the microorganisms either remained intact in the cells throughout the maturing process from macrophage to more advanced cells or they remained within the structure without undergoing visible degradation. Although in some mycobacteria it has been reported that killed bacteria continued to be demonstrated in cultured macrophages for a long period following inoculation, it is possible that, as in experiments with living bacteria (Bendixen, Black and Jorgensen, 1981; Barbieri and Correa, 1967), Dermatophilus congolensis may well survive as an intracellular organism under some conditions. The abscesses or granulomatous lesions caused by D. congolensis in the present study were ofsomewhat different structure from those in the usual spontaneous or experimental dermatophilosis. Dermatophilosis is usually regarded as a superficial exudative dermatitis which is limited to the epidermis (Bida and Dennis, 1976, 1977; Chodnik, 1956; Oduye, 1975; Roberts, 1965a). The main inflammatory cells in the lesions are neutrophils (Roberts, 1965a, 1966). A few atypical dermatophilus infections which show small or large differences from the typical dermatophilosis have been reported. Abu-Samra et al. (1976) and Oduye (1976) described a bovine dermatophilosis with granulomatous inflammation in the dermis; the lesions found in the very severe cases were composed of macrophages, epithelioid and giant cells and fibroblasts. Experimental infection with D. congolensis has revealed similar findings (Abu-Samra and Imbabi, 1976)
ATYPICAL
DERMATOPHILaOSIS
41
but Dermatophilus organisms were not demonstrated in the granulomatous reaction in these reports. In Japan, Kono (1965) described a bovine granulomatous dermatitis with Langhans type giant cells in the dermis and of unknown aetiology. The histological specimens of the dermatitis were re-examined by the present authors and it was ascertained that the lesions were very similar to those reported by Oduye ( 1976). Recently Kinjo et al. ( 1981) reported the existence of bovine dermatophilosis in Okinawa prefecture, Japan, in which Kono (1965) had Further detailed histopathological discovered a granulomatous dermatitis. examination of spontaneous dermatophilosis might well reveal granulomatous changes. There have been a few reports of atypical dermatophilosis occurring in tissues other than the skin in animals. Four feline cases have been reported. O’Hara and Cordes (1963) reported 2 cases of granuloma caused by Dermatophilus: one lesion involved the glossal muscle, and the other the serous surface of the urinary bladder. Baker et al. (1972) reported the condition on the tongue of a cat. In these studies the distinct morphology of the Dermatophilus organisms was seen in the histological specimens but the authors did not isolate the organism. Finally, (1972) described Dermatophilus-like micro-organisms in Smith and Cordes granulomas in feline lymph nodes. However, Roberts (1967) denied that 11. congnlensis was responsible for the feline chronic inflammatory lesions in the popliteal lymph node of a cat from which he isolated D. congolensis. In the goat, Singh and Murty (1978) isolated the organism from an intractable suppurativt lymphadenitis. Albrecht et al. (1974) reported a case of unusual human dermatophilosis in an 8-year-old boy with a deficiency of B lymphocyte-bearing FC receptors and lymphokine production. In this chronic nodular disease, epithelioid cells and giant cells were found around the central necrotic area. The organisms were demonstrated by the fluorescent antibody technique. Th( chronic or granulomatous lesions mentioned above were similar to those found in the present study. It should be recognized that D. congolensis can cause a suppurative or a granulomatous inflammation, but the role of the granulomatous lesions in natural field dermatophilosis is not yet known precisely. We should at least recognize that D. congolensis can produce types of lesion other thau ;I superficial dermatitis.
SUMMARY
Dermatophilus congolensis was inoculated subcutaneously into 6 ewes and the resulting lesions were examined by light and electron microscopy. The organism was recovered from the subcutaneous lesions. The lesions were suppurative in the early stages and granulomatous in the advanced stage, but abscesses were the usual lesion observed. The granulomas were composed of several layers; central bacterial colony, neutrophil layer, layer of macrophages, epithelioid cells and Langhans and foreign body type giant cells with a periphery of connective tissue. Multiloculated granulomas were observed. Electron microscopic examination confirmed the cell types of the lesions. Epithelioid and giant cells were characterized by the shape Znd number of the nuclei and complex and abundant
42
E.
MOMOTANI
t?t d.
organelles. Interdigitations of the blunt pseudopodia of adjacent cells were also found. It is concluded that the organism can produce both granulomatous lesions and abscesses which differ from those of orthodox dermatophilosis but are similar to those described in “atypical dermatophilosis”.
REFERENCES
Abu-Samra, M. T., and Imbabi, S. E. (1976). Experimental infection of domesticated animals and the fowl with Dermatophilus congolensis. Journal of Comparative Pathology, 86, 157-172. Abu-Samra, M. T., Imbabi, S. E.,.and Mahgoub, E. S. (1976). Dermatophiluscongolensis. A bacteriological, in vitro antrbrotic sensitivity and histopathological study of natural infection in Sudanese cattle. British Veterinary Journal, 132, 627631. Adams, D. 0. (1974). The structure of mononuclear phagocytes differentiating in viuo I. Sequental fine and histologic studies of the effect of Bacillus Calmette-Guerin (BCG) American Journal of Pathology, 76, 17748. Adams, D. 0. (1976). The granulomatous inflammatory response. A review. American Journal of Pathology, 84, 164-191. Albrecht, R., Horowitz, S., Gilbert, E., Richard, J., and Connor, D. H. (1974). Dermatophilus congolensis chronic nodular disease in man. Pediatrics, 53, 902-912. Baker, G. J., Breeze, R. G., and Dawson, C. 0. (1972). Oral dermatophilosis in a cat. Journal of Small Animal Practice, 13, 6499653. Barbieri, T. A., and Correa, W. M. (1967). H uman macrophage culture. The leprosy prognostic test (LPT) International Journal of Leprosy, 35, 377-381. Bendixen, P. H., Black, B., and Jorgensen, J. B. ( 198 1). Lack of intracellular degradation of Mycobacterium paratuberculosis by bovine macrophages infected in vitro and in vivo: Light and electron microscopic observation. American Journal of Veterinary Research, 42, 109-l 13. Bida, S. A., and Dennis, S. M. ( 1976). D ermatophilosis in Northern Nigeria. Veterinary Bulletin, 46, 471478. Bida, S. A., and Dennis, S. M. (1977). Sequential pathological changes in natural and experimental dermatophilosis in Bunaji cattle. Research in Veterinary Science, 22, 18-22. Browett, P. J., Simpson, L. O., and Blennerhassette, J. B. (1979). Experimental granulomatous inflammation: The ultrastructure of the granuloma induced by injection of tubercle bacilli into Freund adjuvant-sensitised guinea-pigs. Journal of Pathology, 129, 191-201. Brown, J. R. (1973). Human actinomycosis. A study of 181 subjects. Human Pathology, 4, 319-330. Chodnik, K. S. (1956). Mycotic dermatitis of cattle in British West Africa. Journal of Comparative Pathology, 66, 179-186. Deans, D. J., Gordon, M. A., Severinghaus, C. W., Kroll, E. T., and Reilly, J. R. ( 1961). Streptothricosis: A new zoonotic disease. New York State Journal of Medicine, 15, 1283-1287. Destombes, P., Mariat, F., Nazimoff, O., and Satre, J. (1961). Apropos des mycetomes a Nocardia. Sabouraudia, 1, 161-172. Elias, P. M., and Epstein, W. L. (1968). Ultrastructural observations on experimentally induced foreign-body and organized epithelioid-cell granulomas in man. American Journal of Pathology, 52, 1207-1223. Epstein, W. L., Fukuyama, K., Danno, K., and Kwan-Wong, E. (1979). Granulomatous inflammation in normal and athymic mice infected with Schistosoma mansoni: An ultrastructural study. Journal of Pathology, 127, 207-2 15. Gupta, P. P., and Sinha, B. P. (1978). Oral dermatophilosis associated with actinomycosis in cattle. zentralblatt fur VeteriniirmediZin. B, 25, 2 11-215. Jones, R. T. (1976). Subcutaneous infection with Dermatophilus congolensis in a cat. Journal of Comparative Pathology, 86, 415-421.
ATYPICAL
DERMATOPHILOSIS
Kin,jo,
4Ii
E., Motonaga, H., Matayoshi, E., Kudo, S., Watanabe, K., Hara, M., Tabuchi, in K., Momotani, E., and Azuma, R. (1981). 0 ccurrence of bovine dermatophilosis the southernmost islands of Japan. National Institute of‘Anima1 Health Quarterly, 21, 163-174. Kono, I. (1965). Granulomatous dermatitis in cattle. Journal of Veterinary Medicine, 48’7, I 3-l 4 (In Japanese). Kubo, M., Osada, M., and Konno, S. (1980). A histopathological and ultrastructural comparison of the sulphur granule in actinomycosis and actinobacillosis. National Institute of Animal Health Quarterly, 28, 53-59. Kubo, M., Osada, M., and Konno, S. (1981). Morphology ofsulphur granules produced by Pseudomonas aeruginosa in cow. National Institute of rlnimal Health Quarterly, 21, 26-3 1. Nagao, S., Ota, F., Emori, K., Inoue, K., and Tanaka, A. ( 1981). Epithelioid granuloma induced by muramyl dipeptide in the immunologically deficient rat. Infection and Immunity, 34, 993-999. Oduye, 0. 0. (1975). Effect of various local environmental conditions and histopathological studies in experimental Dermatophilus congolensis infection on the bovine skin. Research in Veterinary Science, 19, 245-252. Oduye, 0. 0. (1976). Histological changes in natural and experimental Dermatophilus congolensis infection of the bovine skin. In Dermatophilous Infection in Animals and Man. D. H. Lloyd and K. C. Sellars, Eds, Academic Press, London. O’Hara, P. J., and Cordes, D. C. (1963). Granuloma caused by Dermatophilus in two cats. Nele! Zealand Veterinary Journal, 11, 15 1- 154. Robboy, S. J., and Vickery, A. L., Jr. (1970). Tinctorial and morphologic properties distinguishing actinomycosis and nocardiosis. Nezpl England Journal of Medicine, 282, 593-596. Roberts, D. S. (1965a). The histopathology ofepidermal infection with the Actinomycrte Dermatophilus congolensis. Journal of Pathology and Bacteriology, 90, 2 13-2 16. Roberts, D. S. ( 196513). The role of granulocytes in resistance to Dermatophilus congolensis. British journal of Experimental Pathology, 46, 643448. Roberts, D. S. ( 1966). The influence of delayed hypersensitivity on the course of infection with Dermatophilus congolensis. British Journal of Experimental Pathology, 47, 9-16. Roberts, D. S. ( 1967). Dermatophilus infection. The Veterinary Bulletin, 37, 5 13-52 1. Singh, V. P., and Murty, D. K. (1978). An outbreak of Dermatophilus congolensis infection in goats. Indian Veterinary Journal, 55, 674-676. Smith, C. F., and Cordes, D. 0. (1972). Dermatitis caused by Dermatophilus congolensis infection in polar bears (Tharactos maritimus). British Veterinary Journal, 128,366-37 1. Stewart, G. H. ( 1972a). Dermatophilosis: A skin disease of animals and man. Part I. The J’eterinary Record, 91, 537~-544. Stewart, G. H. ( 1972b). Dermatophilosis: A skin disease ofanimals and man. Part II. Thhr Veterinary Record, 91, 555-561. Tanaka, A., and Emori, K. (1980). Epithelioid granuloma formation by a systemic bacterial cell wall component, Muramyl dipeptide (MDP). American journal of Pathology, 98, 733-748. Verghes, A., and Klokke, A. H. (1966). Histologic diagnosis ofspecies of fungus causing mycetoma. Indian Journal of Medical Research, 54, 524-530. \Yinslow, D. J., and Steen, F. G. (1964). C onsiderations in the histologic diagnosis of mycrtoma. American Journal of Pathology, 42, 164-l 69. [Received for publication,
.Nouember 15th, 19821