399 TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE
EXPERIMENTAL
AND HYGIENE,
Vol. 69. No. 4. 1975.
MADUROMYCOSIS
II. EXPERIMENTAL MYCETOMA IN MICE CAUSED BY REPEATED INJECTIONS OF PHIALOPHORA JEANSELMEI V. KAMESWARA
RAO AND C. RAM PRASAD
Department of Pathology, Guntur Medical College, Guntur-522004,
A.P. India
Introduction Animal models of human maduromycosis (MACKINNON, 1954) facilitate the assayof antibiotics (MURRAY and COLICHON, 1962) and promote the understanding of pathogenesis. Hitherto, such lesions have been produced by repeated injections of Mycetama mycetomi (LAVERAN) in saline,a single injection along with killed tubercle bacilli (MURRAY et al, 1960) or a single dose ofZ%ialophora jeanselmei with cortisone (KAMESWARA RAO and PFUSAD, 1973). A single dose was found to be ineffective. Even the successful lesion failed to progress, unlike the human lesion, possibly because of the host susceptibility or the mode of infection. We suggested, on histological evidence, that hypersensitivity may play a decisive role in establishing the lesion, even after a single injection along with cortisone (KAMESWARA RAO and PRASAD, 1973), since the effects of cortisone on hypersensitivity have been demonstrably unpredictable (THOMAS, 1953; VOGEL et al, 1955; FRENKEL, 1962). However, in order to establish the role of hypersensitivity, it is imperative that cortisone is avoided and positive serological evidence demonstrated. The present experiment has been designed with this objective.
Material
and methods
Animals
52 albino mice were divided into 4 groups. One group of 22 animals received 16 or fewer weekly subcutaneous injections at different sites of I’. jeanselmei in FREUND’S complete adjuvant. The other 3 groups of 10 animals each were given, intraperitoneally, a single dose, respectively of the fungus in saline, the fungus in adjuvant and the adjuvant only. Sensitization
Using the techniques described by us (KAMESWARA RAO and PFUSAD, 1973) P. jeanselmei was cultured on Sabouraud’s glucose agar, harvested, suspended in saline to contain 5 mg. in 0.25 ml. and thoroughly mixed with 0.25 ml. of adjuvant under sterile conditions by the modification of the technique described by HERBERT (1967). 10 animals survived 7, and 5 animals 16, weekly injections of 0.5 ml. of this suspension at different subcutaneous sites in groins, axillae and back. 7 others died (5 after the 1st or 2nd injection and 2 after the 4th). All the animals were examined weekly for palpable nodules or sinuses. Intraperitoneal
injection
Each of the 15 survivors of sensitization received 5 mg. of live I’. jeanselmei in 0.5 ml. sterile physiological saline intraperitoneally on the 14th day after the last sensitizing injection, (the 7th in 10 and the 16th in 5 animals). The animals were killed 7, 1I and 15 weeks after the intraperitoneal injection, 5 each time, and the lesions studied. All the control animals were killed at the same time as they were exsanguinated, 60 days after the single injection. Serological study 10 days following the 7th injection, the sera obtained from the 10 animals were tested for antibodies by tube precipitation as well as by immunodiffusion in agar on slides (HARTMANN and TOLLIEZ, 1957) against the soluble products of the whole fungus extracted in cold saline for 24 hours (MURRAY, 1961). The pooled sera of the animals in 3 control groups were similarly tested. Cutaneous sensitivity was tested by intradermal injection of 0.01 ml. of 1“,Aby weight of the fungus in saline as well as of the soluble extract into the shaved skin of the anterior abdominal wall of the sensitized animals. 5 normal controls received the same material and 5 others saline only.
EXPERIMENTALMADUROMYCOSIS
400
point to multiple intralesions in- mouse by 7 weekly subinjections of P. jeanFreund’s complete points to subcutaneous left groin of mouse by 7 weekly subinjections of P. jeanFreund’s complete
(4
(b) Results
Lesions
Nonspecific nodular granulomata 2-4 mm. in diameter developed in 2 days at the site of the 1st or 2nd subcutaneous injection of the fungus in adjuvant and disappeared within a week in 5 accidentally dead test animals. Essentially similar nodules were seen in 2 animals each dead within 4 days after a single injection of fungus in saline or adjuvant alone. Fungal granulomata developed both at the subcutaneous sites of sensitizing injections (Fig. lb) and in the peritoneal cavity (Fig. la). 15 animals out of 22 survived at least 7 sensitizing injections. 12 out of these 15 developed intraperitoneal lesions and 9 also developed lesions at the site of one of the sensitizing injections. 5 animals dead within the first 2 weeks showed only the nonspecific lesions. 2 animals dead after the 4th sensitizing injection showed the fimgal granuloma. The average number of grains in the peritoneum was 4, ranging from 1-12. Each grain was discrete, 3-5 mm. in diameter, and dark brown or black. There was no evidence of perinodular fibrosis or diffuse peritonitis. The size or the number of grains bore no relation to the length of sensitization (7, 11 or 16 weeks). The subcutaneous lesions developed at only 1 site in 9 animals but at 2 and 4 different sites in 2 animals. Microscopic appearance
The appearance of the grains was similar to that of the older grains described by us earlier in cortisonized animals. In each case, the identity as well as the viability of the fungus was confirmed by microscopic examination, culture on Sabouraud’s medium and in corn meal agar on slides which demonstrated the characteristic phialides and spores. Serological reaction
Circulating antibodies were demonstrable in 2 sensitized animals only, in low titre (1 in 10 and 1 in loo), by tube precipitation. Diffusion in agar gel showed thin lines of precipitation. Apparently, these animals developed very little antibody even after prolonged and repeated sensitization with this fungus. Skin reactions were uniformly negative. Discussion The pathogenicity of a given species of fungus and the serological response to its infection in experimental animals vary according to the species of animal (BINFORD, 1962) and the superimposed variables such as cortisone administration or sensitization. Unlike some fungi like Histoplasma capsdatum, P. jeanselmei and
V. KAMESWARA RAO AND C. RAM PRASAD
401
M. mycetomi are not pathogenic to normal mice at all, while limited lesions can be produced with certainty in almost all the animals given cortisone or sensitized. Meagre serological response, even after prolonged infection, as in this experiment, was often encountered in hamsters infected with a low virulence strain of H. capsulatum (0’ HERN, 1961). Our failure to demonstrate skin sensitivity, in contrast to MURRAY’S success in guinea pigs infected with M. mycetomi (MURRAY, 1961), a fungus not unlike P. jeanselmei in its pathogenicity to animals and man, is intriguing. There is no record, in the literature, of the nature of serological response to P. jeanselmei in animals or in man. Our negative findings, except in two animals in which the weak response was probably due to the cross reactivity with an unrelated fungus, could have been due to the species of animal used or the lack of sensitivity of our technique. MURRAY (196 1) also failed to demonstrate circulating antibodies by complement fixation, haemagglutination and gel diffusion in guinea pigs infected with M. mycetomi. In human infections with M. mycetomi, serum antibodies were demonstrated (MAGHOUB, 1964) but not delayed skin sensitivity either to the carbohydrate antigen or the culture filtrate antigen (MURRAY and MOGHUBY, 1964). In view of the success of the present experiment it has to be assumed, deductively, that the serological response must have aided the establishment as well as the limited growth of the lesion. In general, the presence of circulating antibodies does not prove their role in pathogenesis. The cellular immunity manifesting as the enhanced capacity for phagocytosis and the intracellular digestion of the spore forms of the fungus, e.g. Histoplasma (GOOD et al, 1960), may play a decisive role. However, whether the infection by myceliate fungi is primarily intracellular is doubtful. The role of altered peritoneal cellular response in establishing the lesion in cortisone-treated or sensitized mice is under investigation. Summary
Intraperitoneal and subcutaneous lesions were produced in 17 out of 22 mice, sensitized by repeated subcutaneous injections of P. jeanselmei in FREUND’S complete adjuvant. The lesions were essentially selflimiting as were those described by us earlier in cortisone-treated mice. Precipitating antibodies and cutaneous hypersensitivity could not be demonstrated. REFERENCES BINFORD, C. H. (1962). In: Fungi and Fungous Diseases. p. 220 Springfield 111: Charles C. Thomas. FRENKEL, J. K. (1962). Lab. Invest., 11, 192. GOOD, R. A., BRIDGES, R. A. & CONDY, R. M. (1960). Bact. Rev., 24, 115. HARTMANN, L. & TOLLIEZ, M. (1957). Rev. Franc. Et. clin. biol., 2, 197. HERBERT, W. J. (1967). In: Handbook of experimental immunology. p. 1,208, Oxford. Blackwell Scientific lications. KAMESWARA RAO, V. & PRASAD, C. R. (1973). Mycopath. Mycol. appl., 51, 119. MACKINNON, J. E. (1954). Trans. R. Sot. trop. Med. Hyg., 48, 470. MAGHOUB, E. S. (1964). Ibid., 58, 560. MURRAY, I. G., (1961). Ibid., 55, 209. & COLICHON, H. (1962). Ibid., 56, 156. & MOGHRAEIY, I. (1964). Ibid., 58, 557. SPOONER, E. T. C. & WALKER, J. (1960). Ibid., 54, 335. O’HERN, E. M. (1961). J. Immun., 87, 728. THOMAS, L. (1953). Ann. N. Y. Acud. Sci., 56, 799. VOGEL, R. A., MICHAEL, M. JR. & TIMPE, A. (1955). Am. J. Path., 31, 535.
Pub-
EXPERIMENTAL
AND HYGIENE,
Vol. 69. No. 4. 1975.
MADUROMYCOSIS
II. EXPERIMENTAL MYCETOMA IN MICE CAUSED BY REPEATED INJECTIONS OF PHIALOPHORA JEANSELMEI V. KAMESWARA
RAO AND C. RAM PRASAD
Department of Pathology, Guntur Medical College, Guntur-522004,
A.P. India
Introduction Animal models of human maduromycosis (MACKINNON, 1954) facilitate the assayof antibiotics (MURRAY and COLICHON, 1962) and promote the understanding of pathogenesis. Hitherto, such lesions have been produced by repeated injections of Mycetama mycetomi (LAVERAN) in saline,a single injection along with killed tubercle bacilli (MURRAY et al, 1960) or a single dose ofZ%ialophora jeanselmei with cortisone (KAMESWARA RAO and PFUSAD, 1973). A single dose was found to be ineffective. Even the successful lesion failed to progress, unlike the human lesion, possibly because of the host susceptibility or the mode of infection. We suggested, on histological evidence, that hypersensitivity may play a decisive role in establishing the lesion, even after a single injection along with cortisone (KAMESWARA RAO and PRASAD, 1973), since the effects of cortisone on hypersensitivity have been demonstrably unpredictable (THOMAS, 1953; VOGEL et al, 1955; FRENKEL, 1962). However, in order to establish the role of hypersensitivity, it is imperative that cortisone is avoided and positive serological evidence demonstrated. The present experiment has been designed with this objective.
Material
and methods
Animals
52 albino mice were divided into 4 groups. One group of 22 animals received 16 or fewer weekly subcutaneous injections at different sites of I’. jeanselmei in FREUND’S complete adjuvant. The other 3 groups of 10 animals each were given, intraperitoneally, a single dose, respectively of the fungus in saline, the fungus in adjuvant and the adjuvant only. Sensitization
Using the techniques described by us (KAMESWARA RAO and PFUSAD, 1973) P. jeanselmei was cultured on Sabouraud’s glucose agar, harvested, suspended in saline to contain 5 mg. in 0.25 ml. and thoroughly mixed with 0.25 ml. of adjuvant under sterile conditions by the modification of the technique described by HERBERT (1967). 10 animals survived 7, and 5 animals 16, weekly injections of 0.5 ml. of this suspension at different subcutaneous sites in groins, axillae and back. 7 others died (5 after the 1st or 2nd injection and 2 after the 4th). All the animals were examined weekly for palpable nodules or sinuses. Intraperitoneal
injection
Each of the 15 survivors of sensitization received 5 mg. of live I’. jeanselmei in 0.5 ml. sterile physiological saline intraperitoneally on the 14th day after the last sensitizing injection, (the 7th in 10 and the 16th in 5 animals). The animals were killed 7, 1I and 15 weeks after the intraperitoneal injection, 5 each time, and the lesions studied. All the control animals were killed at the same time as they were exsanguinated, 60 days after the single injection. Serological study 10 days following the 7th injection, the sera obtained from the 10 animals were tested for antibodies by tube precipitation as well as by immunodiffusion in agar on slides (HARTMANN and TOLLIEZ, 1957) against the soluble products of the whole fungus extracted in cold saline for 24 hours (MURRAY, 1961). The pooled sera of the animals in 3 control groups were similarly tested. Cutaneous sensitivity was tested by intradermal injection of 0.01 ml. of 1“,Aby weight of the fungus in saline as well as of the soluble extract into the shaved skin of the anterior abdominal wall of the sensitized animals. 5 normal controls received the same material and 5 others saline only.
EXPERIMENTALMADUROMYCOSIS
400
point to multiple intralesions in- mouse by 7 weekly subinjections of P. jeanFreund’s complete points to subcutaneous left groin of mouse by 7 weekly subinjections of P. jeanFreund’s complete
(4
(b) Results
Lesions
Nonspecific nodular granulomata 2-4 mm. in diameter developed in 2 days at the site of the 1st or 2nd subcutaneous injection of the fungus in adjuvant and disappeared within a week in 5 accidentally dead test animals. Essentially similar nodules were seen in 2 animals each dead within 4 days after a single injection of fungus in saline or adjuvant alone. Fungal granulomata developed both at the subcutaneous sites of sensitizing injections (Fig. lb) and in the peritoneal cavity (Fig. la). 15 animals out of 22 survived at least 7 sensitizing injections. 12 out of these 15 developed intraperitoneal lesions and 9 also developed lesions at the site of one of the sensitizing injections. 5 animals dead within the first 2 weeks showed only the nonspecific lesions. 2 animals dead after the 4th sensitizing injection showed the fimgal granuloma. The average number of grains in the peritoneum was 4, ranging from 1-12. Each grain was discrete, 3-5 mm. in diameter, and dark brown or black. There was no evidence of perinodular fibrosis or diffuse peritonitis. The size or the number of grains bore no relation to the length of sensitization (7, 11 or 16 weeks). The subcutaneous lesions developed at only 1 site in 9 animals but at 2 and 4 different sites in 2 animals. Microscopic appearance
The appearance of the grains was similar to that of the older grains described by us earlier in cortisonized animals. In each case, the identity as well as the viability of the fungus was confirmed by microscopic examination, culture on Sabouraud’s medium and in corn meal agar on slides which demonstrated the characteristic phialides and spores. Serological reaction
Circulating antibodies were demonstrable in 2 sensitized animals only, in low titre (1 in 10 and 1 in loo), by tube precipitation. Diffusion in agar gel showed thin lines of precipitation. Apparently, these animals developed very little antibody even after prolonged and repeated sensitization with this fungus. Skin reactions were uniformly negative. Discussion The pathogenicity of a given species of fungus and the serological response to its infection in experimental animals vary according to the species of animal (BINFORD, 1962) and the superimposed variables such as cortisone administration or sensitization. Unlike some fungi like Histoplasma capsdatum, P. jeanselmei and
V. KAMESWARA RAO AND C. RAM PRASAD
401
M. mycetomi are not pathogenic to normal mice at all, while limited lesions can be produced with certainty in almost all the animals given cortisone or sensitized. Meagre serological response, even after prolonged infection, as in this experiment, was often encountered in hamsters infected with a low virulence strain of H. capsulatum (0’ HERN, 1961). Our failure to demonstrate skin sensitivity, in contrast to MURRAY’S success in guinea pigs infected with M. mycetomi (MURRAY, 1961), a fungus not unlike P. jeanselmei in its pathogenicity to animals and man, is intriguing. There is no record, in the literature, of the nature of serological response to P. jeanselmei in animals or in man. Our negative findings, except in two animals in which the weak response was probably due to the cross reactivity with an unrelated fungus, could have been due to the species of animal used or the lack of sensitivity of our technique. MURRAY (196 1) also failed to demonstrate circulating antibodies by complement fixation, haemagglutination and gel diffusion in guinea pigs infected with M. mycetomi. In human infections with M. mycetomi, serum antibodies were demonstrated (MAGHOUB, 1964) but not delayed skin sensitivity either to the carbohydrate antigen or the culture filtrate antigen (MURRAY and MOGHUBY, 1964). In view of the success of the present experiment it has to be assumed, deductively, that the serological response must have aided the establishment as well as the limited growth of the lesion. In general, the presence of circulating antibodies does not prove their role in pathogenesis. The cellular immunity manifesting as the enhanced capacity for phagocytosis and the intracellular digestion of the spore forms of the fungus, e.g. Histoplasma (GOOD et al, 1960), may play a decisive role. However, whether the infection by myceliate fungi is primarily intracellular is doubtful. The role of altered peritoneal cellular response in establishing the lesion in cortisone-treated or sensitized mice is under investigation. Summary
Intraperitoneal and subcutaneous lesions were produced in 17 out of 22 mice, sensitized by repeated subcutaneous injections of P. jeanselmei in FREUND’S complete adjuvant. The lesions were essentially selflimiting as were those described by us earlier in cortisone-treated mice. Precipitating antibodies and cutaneous hypersensitivity could not be demonstrated. REFERENCES BINFORD, C. H. (1962). In: Fungi and Fungous Diseases. p. 220 Springfield 111: Charles C. Thomas. FRENKEL, J. K. (1962). Lab. Invest., 11, 192. GOOD, R. A., BRIDGES, R. A. & CONDY, R. M. (1960). Bact. Rev., 24, 115. HARTMANN, L. & TOLLIEZ, M. (1957). Rev. Franc. Et. clin. biol., 2, 197. HERBERT, W. J. (1967). In: Handbook of experimental immunology. p. 1,208, Oxford. Blackwell Scientific lications. KAMESWARA RAO, V. & PRASAD, C. R. (1973). Mycopath. Mycol. appl., 51, 119. MACKINNON, J. E. (1954). Trans. R. Sot. trop. Med. Hyg., 48, 470. MAGHOUB, E. S. (1964). Ibid., 58, 560. MURRAY, I. G., (1961). Ibid., 55, 209. & COLICHON, H. (1962). Ibid., 56, 156. & MOGHRAEIY, I. (1964). Ibid., 58, 557. SPOONER, E. T. C. & WALKER, J. (1960). Ibid., 54, 335. O’HERN, E. M. (1961). J. Immun., 87, 728. THOMAS, L. (1953). Ann. N. Y. Acud. Sci., 56, 799. VOGEL, R. A., MICHAEL, M. JR. & TIMPE, A. (1955). Am. J. Path., 31, 535.
Pub-
