The main species of pathogenic aerobic actinomycetes causing mycetomas

31 TRANSACTIONSOF THE ROYAL SOCIETYOF TROPICAL MEDICINE AND HYGIENE. Vol. 50. No. 1. January, 1956. COMMUNICATIONS THE

MAIN

SPECIES

OF

PATHOGENIC

CAUSING

AEROBIC

ACTINOMYCETES

MYCETOMAS BY

JUAN E. MACKINNON AND RICARDO C. ARTAGAVEYTIA-ALLENDE* Instituto de Higiene, Montevideo, Uruguay. One of us has already published (1954, 1955) the results of studies on the causal moulds of maduromycosis ; we now propose to give our experience on the identification of the aerobic actinomycetes causing similar conditions. We have studied 40 strains of aerobic actinomycetesl using methods similar to those used in the study of the causal organisms of maduromycosis. Our results emphasize the importance of the classical papers by KANTHACK (1893), VINCENT (1894) and BRUMPT (1906), a s w e l l as the views of some modern authors like CONANT and ROSEBURY (1952). Nevertheless the brevity of WAKSMAN and HENRICI (1948), proper to some types of papers, and the multiplicity of species described in the recent monographs of LACAZ (1945) and NEGRONI (1954), produce some confusion in the minds of non-specialized microbiologists. It is advisable to study both cultures and tissue sections with the parasitic grains. These have some very useful specific characteristics which will allow the species to be identified when cultures are not available for study.

METHODS The morphology of the cultures was studied on glucose peptone (l per cent. Difco bactopeptone) agar, on Krainsky medium (glucose 10 g., asparagine 0.5 g., bipotassic phosphate 0.5 g., agar 15 g., and water l litre) and sometimes also in Czapek agar (sodium nitrate 2 g., bipotassic phosphate 1 g., magnesium sulphate 0.5 g., potassium chloride 0.5 g., ferrous sulphate 0.01 g., glucose 30 g., agar 20 g. and water 1 litre). The semi-acidfastness was studied, as recommended by WILSON and MILES (1945), by decolouration for 5 minutes in 1 per cent. sulphuric acid. The tests to find the optima] temperature for growth were made at 20 - 25°, at 30° and at 37°C. T h e proteolytic activity was studied by direct action of the growing strain on milk, gelatine and inspissated serum over a period of 2 months at 30°C. T h e amylolitic activity by culture on nutritive agar with 1 per cent. soluble starch for 1 week. * We are specially indebted to Dr. J. T. Duncan and to Dr. Jacqueline Walker (London School of Hygiene and Tropical Medicine), to Professor H. Galliard (Institut de Parasitologie de la Facult6 de M6decine de Paris), and to the following research workers who provided cultures and tissue sections : P. H. Abbott, F. P. de Almeida, O. da Fonseca, A. Gonzalez-Ochoa, C. S. Lacaz, A. E. Ar~a Le~o, L. Montemayor, Dr. Panja and H. Vaccaro.

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PATHOGENIC AEROBIC ACTINOMYCETES CAUSING MYCETOMAS

The utilization of sugars was studied by the assimilation test, as recommended by

MACKINNON,FERRADA-URzI:IAand MONTEMAYER(1949) and also by studying the acidification of the basal medium recommended by PRIDHAM and GOTTLIEB (1948). The tests were carried out with glucose, maltose, sucrose, lactose and galactose. The results were appreciable after 10 to 15 days at 30°C, but in the case of S. pelletieri a period of 3 to 4 weeks was necessary. A pH meter was used to indicate acidification, and only acidification below 6.4 is noted as positive. The assimilation test gave some constant results from studies made in 1947, 1951 and 1954, but doubtful results were frequent. Some factors, such as the softness of the agar, are important in order to obtain clear results. The utilization of nitrogenous compounds was studied in our basal medium AN (1949). As urea and potassium nitrate were toxic for some strains in the concentrations used in our previous work, more dilute concentrations were also employed. The assimilation test is not very reliable, and besides common necessary precautions such as careful cleaning of glassware, purity of the assayed compounds, etc., we studied several strains at the same time in the same batch of culture medium. We recommend that new strains should be studied at the same time as the strains previously examined. The parasitic form was studied mainly in tissue sections coloured with haemalumeosin. Only in a few cases could we study both the culture and the parasitic form. The inoculation of mice and guinea-pigs with Nocardia brasiliensis allowed us to obtain grains similar to those observed in man.

Streptomyces somaliensis

(BRUMPT~

1906)

Seven strains isolated from cases of Madura foot in Sudan were studied. They were received already identified from Dr. J. T. DUNCAN (three strains) and from Dr. P. H. ABBOTT. Macroscopic cultural characteristics. On Krainsky medium a thin, smooth and soft pellicle is formed. Two strains developed a short, light ochreous aerial mycelium. On glucose peptone agar, creamy coloured, inconsistent, non-adherent pellicles are formed which may show delicate furrows. The culture may become brownish and even blackish. Isolated colonies may show radiated furrows or be crateriform. No diffusible pigment. Microscopic characteristics. Non-segmented vegetative mycelium with some swellings (chlamydospores) about 1 ~tin diameter. The aerial mycelium shows chains of conidia 1.25 in diameter (Figure 1) typical of the genus Streptomyces. The species is not semi-acidfast and stains well by Gram's method. Biologicalproperties. Optimal temperature 30°C. On a basal medium with asparagine, glucose and maltose slightly favoured the growth. No acidification was recorded in Pridham and Gottlieb's media. Peptone and asparagine were assimilated. Ammonium sulphate, potassium nitrate and urea were not assimilated. All the strains were proteolytic, and they hydrolysed the starch. The parasitic grains. Grains from four cases were studied and showed the same characteristics. They were yellowish, up to 1.25 ram. in diameter, round or ova land compact. They are composed of a matrix of amorphous material showing some slits which we think were artificially produced. Embedded in this substance, filaments of actinomycetes are easily observed because they stain well by haemalum. The filaments are abundant near to the periphery of the grains (Figure 2) but not at the extreme periphery. Some grains show scarce filaments or sectors without filaments. The matrix may show some affinity for eosin at the periphery but no clubs were observed.

T h e parasitic grains of aerobic actinomycetes producing mycetoma.

FIGURE 1. (X 100). Culture of Streptomyces somaliensis. Chains of conidia typical of the genus Streptomyces. FmURE 2. (X 80). Streptomyces somaliensis. Section of a parasitic grain. A b b o t t ' s case. FIGURE 3. (X 46). Streptomyces madurae. S e c t i o n ' o f a p a r a s i t i c grain. Mazza's case.

FIGURE 4. (X 60). Streptomyces rnadurae. Section of a parasitic grain. A palisade of long clubs is seen in the upper left corner. Dekester's case.

(To face page 32)

FIGURE 5. (x 170). Streptomyces madurae. Section of a parasitic grain showing the mantle of filaments, which stained well by haemalum, and the palisade of clubs, Dekester's case. FmURE 6. (x 170). Streptomyces pelletieri. Section of lobulated parasitic grains.

FIOURE 7.

(x 170).

Nocardia brasiliensis. Section of a parasitic grain in human tissues. berg's original case.

FlOURE 8.

(x 170).

Nocardia brasiliensis, Sections of parasitic grains in tissues of mice inoculated with strain 824 received from F. de Almeida.

Linden-

J U A N E. M A C K I N N O N AND RICARDO C. ARTAGAVEYTIA-ALLENDE

33

Inoculations of mice and guinea-pigs were not successful ; but S. somaliensis survived up to 14 days in the peritoneum of the inoculated mice. D~SCUSS~ON The same principal characteristics were recorded in the seven strains, and the parasitic form was also characteristic in the four cases which we studied. An interstitial substance was observed and it seems also to be continued at the periphery of the grain by a similar substance forming an envelope or sheath which was well described by BRUMPT. BRUMPT (1906) believed that the grain was due to a mould which he named Indiella somaliensis, but in 1913 he recognized that it was an actinomycete, and proposed the name Indiellopsis somaliensis. CHALMERSand CHRISTOPHERSON(1916) placed BRUMPT'S species in the genus Nocardia, and also described a new species which they named Nocardia convoluta. This species would differ because of the absence of a sheath. Nevertheless the figures of CHALMERS and CHRISTOPHERSONare not very different from those of BRUMPT. The grains of N. conVoluta are said to be soft, and those of AT. sornaliensis hard, but this may be due to dryness. Moreover one of us, during a visit to Paris, was able to study BRUMPT'Spreparations labelled IndieUa somaliensis, and the grains looked like those observed in the four cases studied in this paper (ABBOTT'S cases). The cultures described by CHALMERSand CHRISTOPHERSON are similar to those we have studied and even chains of conidia are described and photographed. Undoubtedly CHALMERSand ARCHIBALDwere the first to cultivate the species I. somaliensis. The colonies are usually yellowish but according to BRUMPT some might be reddish, and orange according to CHALMERSand CHRISTOPHERSON. According to GONZALES-OcHoAand VAZQUEz-Ho¥os (1953) the antigens of Streptomyces somaliensis are different from those of the common contaminant streptomyces. Up to the present the species has been observed in Abyssinia in the original case of BOUFFARD (1902) studied by BRUMPT (1906). FULLEBORN(1911) observed a new case in West Africa. BALFOURfound the species in the Sudan (1911), and also CHALMERS and CHRISTOPHERSON (1916). The three strains received from DUNCAN and the four cases from ABBOTT (1954) show that the species is not rare in the Sudan. Outside Africa a case by YAZBEK(1920) in S~o Paulo, Brazil, seems to have been observed. The case of Madura foot recorded by KOCH and STUTZER(1911) in Egypt, attributed to N. madurae by the authors and to a new species named dctinomyces avadi by DODGE (1935), might be due to S. somaliensis. The case of ONORATO, recorded in Tripolitania, is probably due to S. somaliensis but not sufficient data are available.

Streptomyces pelletieri (LAVERAN, 1906) Three strains were studied. Two strains were received from the Facult6 de M6decine de Paris with the names Actinomyces pelletieri and A. africanus (Pijper and Pullinger). The third strain was cultivated from red grains obtained by Abbott in the Sudan from a case of Madura foot. Macroscopic cultural characteristics. Slow growth in all media. On Krainsky's medium small, hard, red or purple, adherent, colonies. The border of the colonies is paler. No diffusible pigments. On glucose peptone agar the colonies may be pale at first but they rapidly become red. Poor growth on Czapek. No veil in liquid media. Microscopic characteristics. Non-segmented branched vegetative mycelium with some

34

P A T H O G E N I C AEROBIC A C T I N O M Y C E T E S C A U S I N G M Y C E T O M A S

swellings up to 1 [x in diameter. No conidia were observed. The organism is not semiacidfast and stains well by Gram's method. Biological properties. Growth is better at 37 ° than at 30°C. The three strains showed proteolytic activities but they did not hydrolyse the starch. On the basal medium with asparagine only glucose favoured the growth, and on the Pridham and Gottlieb's basal medium acidification was observed only with glucose. Peptone and asparagine were utilized ; urea and potassium nitrate were not ; and doubtful results were recorded with ammonium sulphate. The parasitic grains. We studied tissue sections from a case in Senegal, received from the Facult6 de M6decine de Paris and from Abbott's Sudanese case. The grains are deep red in colour, rather small, and they rarely reach 1 ram. in diameter. They ~re very irregular in shape and have smooth or denticulate edges. Some grains seem to be enveloped by a thin refringent hard pellicle. Usually the grains are formed by several lobules united by rather narrow isthmi or separated. The shape of the lobules is frequently geometrical (Figure 6). The filaments are not easily seen. Inoculations of mice and guinea-pigs were not successful. The fungus survived for 4 days. DISCUSSION The three strains have shown identical morphological and biological properties and the parasitic grains seem to show also specific characteristics. Some authors have affirmed that the grains of S. somaliensis may show some reddish hue, but those of S. peUetieri are always deeply red coloured. They are snaall, lobulated and the lobules are frequently triangular or adopt some other geometrical shape. The generic position of the species is not clear. LAVERAN(1906) named his species Micrococcuspelletieri, THIROUXand PE~LETIER (1912) named it Oosporapelletieri, and in the discussion of this paper PINOY claims that it must be placed in the genus Nocardia. WAI
Streptomyces madurae (VINCENT,1894) Eight cultures were studied. All were isolated from cases of Madura foot. One of these strains was isolated by Ch. NICOLLE in North Africa and preserved at the Instituto O. Cruz, Rio Janeiro. Two strains were isolated in Bello Horizonte, Brazil ; one strain in Silo Paulo, Brazil, by LACAZ ; and another in Santiago, Chile, by FLORES, VACCAROand

JUAN E. MACKINNON AND ltICARDO C. ARTAGAVEYTIA-ALLENDI~

35

GASlC (1938). Two strains were isolated in Mexico by GONZALEz-OcHoA,and the last strain in India by PANJA. Macroscopic cultural characteristics. Moderately fast-growing actinomycete. On Krainsky medium, cream-whitish, shiny, consistent colonies adherent to the medium. In some cultures, heaped reddish zones were frequently but irregularly observed, and one strain never showed reddish pigmentation. On glucose peptone agar a more luxuriant growth was observed forming a hard crust, creamy coloured, rather shiny and with thin furrows. No diffusible pigments. Microscopic characteristics. Non-segmented m y c e l i u m with some swellings up to 1~ in diameter. The organism is not semi-acidfast and stains well by Gram's method. Biological properties. All the strains grew much better a t 37°C. than at 30°C. All the strains showed proteolytic activity and hydrolysed the starch. All the strains utilized glucose and did not utilize lactose. Doubtful results were obtained with galactose, and irregular ones with maltose and sucrose. On the Pridham and Gottlieb's medium, results were more clear ; only glucose seemed to be utilized and acidification was recorded only with glucose. All the tested nitrogenous compounds were utilized. The parasitic grains. We prefer to express our opinion on the parasitic form in the following paragraph. No grains were obtained by inoculation of cultures ; nevertheless, the species survived up to 25 days in the peritoneum of the inocolated mice. DISCUSSION

Streptomyces madurae is a rather monomorphic species. The morphology is very constant as well as the biological properties. We have not been able to study cultures and parasitic grains from the same case. Nevertheless, the clear descriptions of cultures and tissue grains by VINCENT (1894) allow us to think that some tissue sections from a case of DEKESTERin Africa (Figures 4 and 5) and from another case of Mazza's in Argentina (Figure 3) are due to S. madurae. The grains of S. madurae may reach a larger size than those of other species. They are white-yellowish but sometimes they may show a pinkish hue. The central part of the grain is made up of scant, loosely and irregularly packed filaments, shows lacunar zones and the grain may even appear as a hollow sphere. The lagoons or the loosely woven zones are surrounded by a more dense network, a mantle according to KANTHACK, which stains very well with haemalum. From thiszone bundles of filaments radiate. Clubs are usually observed but they show some special characters. They are elongated, up to 25~, tape-like, sometimes branched, and their ends may be pointed. They usually stain pale with eosin. Some grains show clubs in only some sectors, and others do not show clubs. Perhaps the best description of the grains of S. madurae is that of KANTHACKin 1893. KANTHACK did not obtain cultures, and named his micro-organism Oospora indica. The description seems to us so perfect that it would be possible to discuss the priority of the name indica published in 1893, over madurae published in 1894. Nevertheless, KANTItACK believed that black grains similar to those produced by Madurella mycetomi were due to the same micro-organism or to a variety. He believed that the black grains were degenerated white grains. The characters described for O. indica correspond to two quite different, species, and this name became a nomen confusum. The synonymy of S. madurae must comprehend the species dctinomyces brumpti isolated in Yugoslavia by BORDJOSKIand MILOC!IEVITCH (1935). We agree also with YAZBEK(1920) who considers that Discomyces bahiensis isolated in Bahia, Brazil, by PIRAJA DA SILVA(1918)

36

PATHOGENIC AEROBIC ACTINOMYCETES CAUSING MYCETOMAS

is identical with or very close to S. madurae. FROES(1930) also found the species in Brazil. On the other hand, the granules described under the name madurae by CotmTOlS, DE LooF, THYS and VANBREUSEGHEM(1954) show some peculiarities which are not of the species. The generic position of the species is far from settled. GONZALEz-OcI~OAand SANDOVAL (1951) state that they have observed chains of conidia, and they placed the species in the genus Streptomyces. VINCENT (1894) also described some rather short chains. The geographical distribution of the species is very wide. It has been found in India, Africa, Europe, North America and South America.

Nocardia brasiliensis (LINDENBERG, 1909) We think that the generic name Nocardia must be used for the semi-acidfast species. The type species Nocardia farcinica Trevisan is indeed a semi-acidfast micro-organism. We distinguish two main species : N. brasiliensis and N. asteroides which are capable of producing mycetomas in man. Eighteen cultures of N. brasiliensis have been studied. Of these cultures, 11 were isolated from cases of Madura foot by GONZALEz-OcHoA in Mexico (10 cases) and by ALMEIDA in S~o Paulo, Brazil (one case) ; two strains were isolated from localized mycetomas of the leg by MONTEMAYORin Venezuela, and GONZALEZ-OcHoAin Mexico. We received, also from GONZALEz-OcHoA, tWO strains isolated from localized mycetoma of the thigh and two strains from thoracic mycetoma. One strain isolated from a cervico-facial mycetoma in S~o Paulo was received from ALMEIDA. Macroscopic cultural characteristics. Rapid growing actinomycete. On Krainsky medium, heaped-up colonies with membranous consistency and marked furrows and filamentous edge. The colour may vary from pale ochre in some strains to orange or red ochre (incarnatus) in other strains. Scarce aerial mycelium in Krainsky medium but more abundant in Czapek's. The cultures with aerial mycelium produce an earthy odour. The more pigmented strains also produce some pigmentation of the medium. On glucose peptone agar the cultures are even more heaped up, furrowed and membranous. From strains with abundant aerial mycelium we could obtain variants with very scarce aerial mycelium, but all the other morphological and biological properties did not change. On liquid media a coarse membranous veil was formed. Microscopic characteristics. A non-fragmented mycelium prevails. The aerial mycelium is not different from the vegetative mycelium. All the strains stained well by Gram's method and were semi-acidfast. Biologicalproperties. Some strains grew better at 30°C than at 37°C, but in the majority it was difficult to decide what was the optimal temperature of growth. All the strains showed proteolytic activities but the starch was not hydrolysed. All the nitrogenous compounds tested were utilized. On the basal medium with asparagine, glucose and galactose were utilized by all the strains ; maltose, sucrose and lactose were not. The same results were obtained in the basal medium of Pridham and Gottlieb, and acidification was recorded only in the media with glucose and galactose. The parasitic grains. We were able to study tissue sections from LINDENBERG'S case preserved at the Institut de Parasitologie de la Facult6 de Mddecine de Paris. Irregular grains of moderate size, built up by lobules without clubs. The filaments do not stain by haemalum.

JUAN E. MACKINNON AND RICARDO C. ARTAGAVEYTIA-ALLENDE

37

Mice were inoculated in the peritoneum. After 3 weeks, abscesses of 1 to 3 rnm. in diameter were observed around the pancreas, in the omentum, between the liver and diaphragm, close to the testicles, etc. Abundant grains were observed (Figure 8). These were soft yellow grains, irregular, formed by lobules with the shape of some characters of the alphabet. In smears we could observe the acidfastness of the filaments. The lesions of the mice are not evolutive, and after 4 months they are sterile and heal. Nevertheless N. brasiliensis survived for more than 3 months. In guinea-pigs inoculated in the testicles we observed the formation of grains with clubs. DISCUSSION

LINDENBERG (1909) gave the name Discomyces brasiliensis to a culture isolated in Silo Paulo, Brazil, from a mycetoma of the knee. He distinguished his new species by its morphological characteristics in culture and by its parasitic grains. The acidfastness of the micro-organism and its proteolytic activity on milk are recorded. Most of the microbiologists believed that N. brasiliensis was a synonym of N. asteroides. YAZBEK(1920) after studying five new strains also isolated in S~o Paulo, and one strain received from LIND~NBERG, affirmed that the species has proteolytic activity over the gelatin but considers that it is identical with Actinomyces boris. ALMEIOAand LACAZ (1941) claim that N. brasiliensis is a good species, and GONZALEZ-OcHoA(1945) emphasizes the importance of the proteolytic activity to distinguish N. brasiliensis from N. asteroides. Since 1947 we have been studying the biological properties of actinomycetes and we have found that the strains with proteolytic activity utilize galactose. This property has remained unchanged up to the present. MARIAT and LAVALLE(1955) have made similar observations. The synonymy of the species is not well known. The strain named Actinomyces mexicana by BO'~D and C~UTCHFIELDis difficult to identify with N. brasiliensis. On the other hand, we suspect that Nocardia pretoriana isolated in South Africa by PIJVER and PULLINCER(1927) may be a synonym of N. brasiliensis. ERIKSON (1935) affirmed that N. pretoriana has proteolytic activity. SARTORY,MEYER and MEYER (1930) described a case of mycetoma in Europe attributed to a new variety of N. asteroides. DODGEthinks it is a new species and named it Actinomyces serratus. The macroscopic characteristics of the cultures are rather similar to those of N. brasiliensis. It is not yet possible to write about the geographical distribution of the species. It seems a frequent cause of nocardiosis in tropical and subtropical countries of South America and in Mexico.

Nocardia asteroides

(EPPINGER,1891)

Four cultures were studied. Two were type cultures and two were newly isolated. The type cultures are one strain received from the National Collection of Type Cultures, and rePuted to be EPPINGER'S strain, and a strain received from the Facult6 de Mddecine de Paris and isolated by MAcCULLUM(1902)from a case of peritonitis. The new strains are one isolated in Rio de Janeiro by FONSECAfrom a case o f Madura foot and one isolated in Montevideo, Uruguay, from a mycetoma of the leg and foot. There exist some morphological differences between the type strains and the new isolates. Macroscopic cultural characteristics. On Krainsky medium EvvI~c~R's strain produces a soft, inconsistent, creamy growth which little by little acquires some orange and rose hue. On glucose peptone agar some furrows are observed but the culture is always soft. MACCALLUM'Sstrain produces some more consistent growth and its colour is orange. On liquid media an inconsistent veil is formed.

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PATHOGENIC AEROBIC ACTINOMYCTESE CAUSING NIYCETOMAS

The new strains, isolated from mycetomas, produce membranous, consistent, furrowed cultures similar to those of N. brasiliensis. The four strains may produce aerial mycelium. Microscopic characteristics. EPPINGER'Sand MACCALLUM'Sstrains produce a fragmented mycelium. The two newly isolated strains produce a non-segmented mycelium. Biological properties. The two type strains and the Brazilian strain grow as well at 37 ° as at 30°C. The Uruguayan strain grows better at 30 ° than at 37°C. The four strains have not proteolytic activities and did not hydrolyse the starch. The four strains were able to utilize all the nitrogenous compounds tested. On the chemically defined medium of PRIDHAM and GOTTLIEB only glucose favoured the growth, and acidification was recorded only in the medium with glucose. Experimentalpathogenicity. The EPHNGER'S and the Uruguayan strains were inoculated into the peritoneum of mice. Small abscesses were observed after 15 to 20 days around the pancreas and close to the testicles. Some clumps of filaments were observed, but no grains such as occurred in human lesions with the Uruguayan strain. After 1 month the lesions were sterile. On the other hand, the inoculation of the Uruguayan strain into the testicles of guinea-pigs produced lesions with abundant grains with clubs, and Eppinger's strain produced very scarce small grains only occasionally. All the guinea-pigs were cured spontaneously after 3 months. The large abscesses produced by the Uruguayan strain opened spontaneously through the skin. DISCUSSION

The two strains isolated from mycetomas showed the morphological characteristics of The study of these last properties was carried out on several occasions between 1947 and 1954. The Uruguayan strain was studied immediately after being isolated, and we cannot say that it has lost any properties while preserved in our collection of cultures. According to prevailing views we should identify both FONSECA'S strain and our own with N. brasiliensis, although we recognize that these views may appear rather simple. Actinomyces freeri Musgrave and Clegg (1907), isolated in the Philippine Islands from a case of Madura foot, has also been considered a synonym of N. asteroides, especially by HENRICI and GARDNER (1921). This actinomycete had not proteolytic activities, but the morphological appearance of the cultures is much like that of N. brasiliensis. We think that there exist very striking similarities between the Uruguayan and Brazilian strains isolated from cases of mycetoma and the strain isolated in the Philippine Islands from a similar case.

N. brasiliensis, but they failed to show proteolytic activities and to utilize galactose.

COMME N TS

The problem of the common anaerobic agent of the classical actinomycosis has been very well discussed by several authors and among them by ERIKSON (1949), but our knowledge of the aerobic species producing mycetomas and nocardiosis is incomplete. Each strain, differing in some minor and sometimes variable characteristics, has usually been considered as a new species. Studies of singly-isolated species have often been misleading, and moreover, contaminations of cultures by bacteria are very frequent and not always very apparent. Critical comparisons of many strains were necessary. After studying 38 strains of aerobic actinomycetes isolated from cases of Madura foot or from other localized mycetomas, we think that the number of pathogenic species is not

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39

great. Our studies lead us to recognize the following species: Streptomyces madurae (Vincent), Streptomyces somaliensis (Brumpt), Streptomyces pelletieri (Laveran), Nocardia asteroides (Eppinger) and Nocardia brasiliensis (Lindenberg). Nevertheless, we think that this list is not complete. We have studied the original strain of Nocardia paraguayensis Almeida (1940) and we found it to be a species of Streptomyces closely related to S. albus and other common species. These conclusions agree with those of GO~ZAL~z-OcHoA and SANDOVAL(1951). After the study of the parasitic grains we think, as in the case of the maduromycosis, that the species causing actinornycotic mycetomas may be identified at least in the majority of cases, by the study of tissue sections with grains. The morphology and structure of the grains, the affinity of the filaments for haemalum and their acidfastness are very important characteristics. The grains of S. madurae and S. somaliensis show filaments well stained by haemalum, but those of S. madurae are serpiginous and usually lacunar or hollow grains, while those of S. somaliensis are compact due to an interstitial substance and a sheath. The grains of S. pelletieri are always red, irregular, with smooth or denticulated edges, hard and built up by lobules v)ith geometrical shapes. The grains produced by the semi-acidfast Nocardia show, moreover, filaments which stain badly with haemalum. The clubs are absent in S. somaliensis and S. pelletieri, and may be present in the grains due to other species. When present they also sometimes show some specific characteristics. We think that the present knowledge of the species producing mycetoma justifies our undertaking researches on medical treatment. A methodical study of the sensitivity to drugs and antibiotics might be useful. Moreover, our knowledge of the parasitic form of each species will be useful when no cultures are available. The geographical distribution is not well known, but the facts recorded seem to indicate a wide distribution for all the species. Nevertheless in some regions some species seem to predominate. S. somaliensis in the Sudan, N. brasiliensis in tropical and subtropical Latin America, etc. SUMMARY

Thirty-eight strains of aerobic actinomycetes producing localized mycetoma (Madura foot, etc.) in several zones of the world have been identified as : Streptomyces madurae (eight strains), Streptomyces somaliensis (seven strains), Streptomyces pelletieri (three strains), Nocardia asteroides (two strains) and Nocardia brasiliensis (eighteen strains). The parasitic grains of the named species show characteristics which would be useful for the identification of the species when cultures are lacking. REFERENCES ABBOTT, P. H., (1954). Mycetoma. Clinical and epidemiological study, M.D. Thesis. Cambridge University. AL~CIEIDA,F. (1940). Mycopathologia, 2, 201. --& LAeAZ,C. (1940). Ann. Fac. IVied. S. Paulo, 16, 207. - &- (1941). Ibid., 17, 577. BnJ~FOUR,A. (1911). Fourth Report of the Wellcome Research Laboratories. Vol. A. 365. BORDJOSKI,M. & MILOCrtEVITeH,S. (1935). Ann. Parasit. hum. comp., 13, 36. BOUFF~mD,(1902). Ann. Hyg. Med. colon., 5, 636. BOYD, M. F. & CaUTeHFIELD,E. D. (1921). Amer. J. trop. Med., 1, 215. BRUNPT,E. (1906). Arch. Parasit., Paris, 10, 489. (1913). Prdcis de Parasitologie, 2nd Ed. Paris : Masson et Cie. -

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CHALMERS,A. J. & CHRISTOPHERSON,J. B. (1916). Ann. trop. Med. Parasit., 10, 223. CONANT, N. F. & ROSEBURY,T. (1952). In Dubos's Bacterial and Mycotic Infections of Man. 2nd Ed. Philadelphia : Lippincot Co. p. 634. COURTOIS, G., DE LooF, C., THYS, A. & VANBREUSEGHEM,R. (1954). Ann. Soc. belge Med. trop., 31, 371. DODGE, C. W. (1935). Medical Mycology. St. Louis : C. V. Mosby. EPPINGER, ]yI. (1891). Beitr. path. Anat., 9, 287. ERIKSON, D. (1935). Medical Research Council ; Special Report Ser. No. 203. (1949). Ann. Rev. Microbiol., 3, 23. FLORES, VACCARO,H. & GASIC (1938). Bol. Soc. Cirug. Chile, 16, 21. FR6ES, H. P. (1930). Do " Mycetoma pedis " no Brasil. Thesis. Bahia (Brasil). FULLEBORN,F. (1911). Arch. Schiffs-u. Tropenhyg., 15, 131. GRANTHAM-HILL,C. (1931). Trans. R. Soe. trop. Med. Hyg., 25, 39. GONZALEZ-OCHoA,A. (1945). Rev. Inst. Salubr. Enferm. trop. Mex,, 6, 155. ---& SANDOVAL,M. A. (1951). Caracteristicas de los actinomicetos mils comunes. Congreso Cientifico de la Universidad auton6ma de Mexico. & V~zQVEZ-Hovos, A. (1953). Rev. Inst. Salubr. Enferm. trop., Mex., 13, 177. HENRICI, A. T. & GARDNER,E. L. (1921). J. infect. Dis., 28, 232. I~NTHACK, A. A. (1893). J. Path. Bact., 1, 140. KOCH, J. & STUTZER (1911). Z. Hyg. Infekthr., 69, 17. LACAZ, C. S. (1945). ContribuicTzo para o estudo dos actinomicetos produtores de micetomas. S~o Paulo : LANGERON, M. (1936). Les myc&omes in Nouvelle Pratique Dermatologique, Vol. II, Paris : Masson et Cie. LAVERAN, M. (1906). C . R . Soc. Biol., 61, 340. LINDENBERC, A. (1909). Arch. Parasit., Paris, 13, 265. MAcCULLUM, W. G. (1902). Zbt. Bakt., 1, 31, 529. MACKINNON, J. E. (1954). Trans. R. Soc. trop. Med. Hyg., 48, 470. - (1955). 1bid., 49, 287. , FERRADA-UazlRA, L. V. & MONTEMAYOR, L. (1949). Mycopathologia, 4, 384. MARIAT, F. & LAVALLE,P. (1955). C . R . Acad. Sci., Paris, 240, 255. MUSGRAVE, W. E. & CLECC, M. T. (1907). Philipp. J. Sci., B, 2, 477. NEGRONI, P. (1954). Micosis profundas. Vol. I. Los Micetomas. Buenos Aires : E1 Ateneo. ed. ONORATO, R. (1926). Arch. ital. Sci. Med. colon., 7, 1. PIJPER, A. & PULLINGER,B. D. (1927). J. trop. Med. (Hyg.), 30, 153. PIRAJA DA SILVA, P. (1918). Mere. Inst. Butantan, 1, 187. PRIDHAM, T. G. & GOTTLIEB, D. (1948). J. Bact., 56, 107. SARTORY, A., MEYER, M. & MEYER, J. (1930). Ann. Inst. Pasteur, 44, 298. SILVA, F. & AaAUJO, E. (1938). Rd¢, franc. Ddrm. Vdndrdol., 14, 451. SMITH, E. C. (1928). Trans. R. Soc. trop. Med. Hyg., 22, 1928. THIROI,rX, A. & PELLETIER,J. (1912). Bull. Soc. Path. exot., 5, 585. VINCENT, M. H. (1894). Ann. Im't. Pasteur., 8, 129. WAKSMAN, S. A. & HENRICI, A. T. (1948). In Bergey's Manual of Deterndnative Bacteriology, 6th Ed. Baltimore : Williams and Wilkins Co. ---& LECHEVALIER,H. A. (1953). Actinomycetes and their Antibiotics. Baltimore: Williams and Wilkins Co. WILSON, G. S. & MILES, A. A. (1946). Topley and Wilson's Principles of Bacteriology and Immunity, 3rd Ed. London : E. Arnold and Co. YAZBEI(, A. K. (1920). Dos Myeetomes. Thesis, Fac. Med. S. Paulo. -

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