Pathogenicity of Allescheria boydii for Mice

Transcription

1 INFECTION AND IMMUNITY, Nov. 1973, p Copyright American Society for Microbiology Vol. 8, No. 5 Printed in U.S.A. Pathogenicity of Allescheria boydii for Mice DAVID M. LUPAN AND JOHN CAZIN, JR. Department of Microbiology, University of Iowa, Iowa City, Iowa Received for publication 21 May 1973 Allescheria boydii and its imperfect state, Monosporium apiospermum, were studied to determine whether asexual or sexual strains might exhibit different pathogenic potentials for mice. Six different strains of the fungus were inoculated into mice by the intravenous, intracerebral, intraperitoneal, and intranasal routes. Cortisone-treated mice regularly developed infections after inoculation by any of the routes tested. Mice that had not been treated with cortisone were most susceptible to infection by the intravenous route and least susceptible to infection by the intranasal or intraperitoneal route; nevertheless, all animals that did not receive cortisone were considerably more resistant to infection by the fungus than were comparable groups of cortisone-treated animals. Pathogenicity of the fungus appears to be strain dependent and entirely unrelated to its sexual or asexual form. Studies made to determine accurate viable spore counts of the fungus revealed that the highest viable spore count was generally observed using Sabouraud dextrose agar or potato dextrose agar at an incubation temperature of 37 C for a period of 5 days. The ascomycete isolated from a mycetoma by Boyd and Crutchfield in 1921 was considered by Shear (32) to be a new species which he named Allescheria boydii. In 1909, Tarozzi isolated an imperfect fungus that was subsequently named Monosporium apiospermum by Saccardo in 1911 (as cited by Ajello [1]). Emmons (7) established the true relationship between the two fungi when he observed cleistothecia similar to those of A. boydii in a strain of M. apiospermum isolated by Shaw and Macgregor (31). Epidemiological studies by Ajello (1) established that A. boydii is found widely in our environment. One might expect, therefore, that sporadic infections by this fungus are likely to occur. Although A. boydii normally is considered one of the etiological agents of maduromycosis, considerable evidence is available to show that the fungus may also produce other types of infection. Benham and Georg (3) demonstrated A. boydii as the etiological agent of a case of meningitis, and the organism was isolated from prostatic secretions of a patient suffering from chronic prostatitis (22). In an article on pulmonary allescheriasis, Travis et al. (36) cited numerous references in which A. boydii was implicated in otomycosis, septicemia, and pulmonary infections. M. apiospermum has also been isolated from a corneal ulcer (23). Attempts to produce experimental infection in mice with A. boydii generally have been 743 unrewarding. Although several investigators (1, 9, 12) were successful in producing intraperitoneal infection in mice, all failed to show evidence of pathology in the animals. Evidence for infection was based largely on the fact that the fungus could be reisolated several weeks after the initial inoculation. Even though Ajello (1) reported that spores combined with 5% hog gastric mucin would produce death in mice within a period of 1 week, Travis et al. (36), Schmitt et al. (29), and Reyes (25) were unable to confirm his findings. Conant et al. (6) feel that laboratory animals are generally unsuitable for duplicating a disease similar to that occurring in man. Since it has been reported that A. boydii, when isolated from clinical maduromycosis, is usually in the asexual form (15), the reproductive form of the fungus may reflect some difference in its pathogenicity. An attempt was made, therefore, to determine whether asexual or sexual strains might exhibit more pathogenic potential for mice. MATERIALS AND METHODS Fungal strains. Strains of A. boydii and M. apiospermum used in this study came from stock cultures maintained in the Department of Microbiology, University of Iowa. A number of the strains have been previously described (4). Strain 813 (M. apiospermum) was isolated in January 1968 from a cerebral abscess, and strain 815 (M. apiospermum)

2 744 LUPAN AND CAZIN INFECT. IMMUNITY was isolated in December 1968 from an infected knee joint. Stock strains of fungi were maintained on a Czapek medium modified by substitution of 20 g of dextrose per liter for sucrose. All A. boydii strains used in animal pathogenicity studies produced fertile cleistothecia at the time this study was undertaken. Strains designated as M. apiospermum have never been shown to produce ascocarps. Spore suspensions. Fungus spore suspensions were obtained from cultures grown on Czapek dextrose agar contained in 200-ml tissue culture bottles. Spores were harvested in cold 0.15 M NaCl by dislodging them from hyphae with a bent-wire inoculating needle after the fungi had grown for a period of 14 days at room temperature (26 C). Large mycelial fragments were removed from the suspension by filtration through a sterile gauze sponge. The spore suspension was centrifuged, and the spore pellet was washed three times with cold 0.15 M NaCl. The washed spores were finally resuspended in cold 0.15 M NaCl. Viable spore determinations were made by triplicate plating on potato dextrose agar at room temperature and at 37 C. Mice. Male and female white Swiss mice (Webster strain) maintained by the Department of Microbiology at the University of Iowa were used throughout this study. The mice were approximately 4 weeks of age at the beginning of each experiment, and their average weight was 20 g. Food and water were supplied ad libitum. Steroid treatment. Cortisone acetate (The Upjohn Co., Kalamazoo, Mich.) was administered to the animals by subcutaneous injections. The concentration of steroid, contained in 0.1-ml volumes, varied with the route of experimental infection. Pathogenicity determination. Pathogenicity for mice of individual strains of the fungus was determined on the basis of median lethal dose (LD50) values over a 30-day observation period. Probit transformations were used to calculate LD.0 values, and best-fitting lines were determined by the method of Finney (10). The ET,0 value (time in days required for 50% of the animals to die from a given infectious dose) was used in comparing the pathogenicity of individual isolates of A. boydii for steroid-treated and untreated mice. The ET50 values were calculated by the method of Litchfield (19). An extension of time required to kill control animals was interpreted as an indication of a difference in susceptibility or resistance to infection. The randomized complete block design analysis of variance (34) was used to determine the significance of an apparent difference in animal resistance to various routes of inoculation. RESULTS Accurate determinations of LDso values are based on exact counts of total viable infectious spores. Viable spore plate counts can be inconsistent and inaccurate, due to variables such as temperature, assay medium, and length of incubation. For this reason, conditions for optimal viable spore assay were studied on Sabouraud dextrose agar (SDA), potato dextrose agar (PDA), and Czapek dextrose agar (CDA), using five different incubation temperatures (21, 26, 30, 37, and 41 C) and several strains of both A. boydii and M. apiospermum. The highest viable spore counts were generally observed with SDA or PDA at an incubation temperature of 37 C. Potato dextrose agar was chosen as the viable spore assay medium since it is somewhat less opaque than SDA. Viable plate counts obtained with CDA were similar to those observed with PDA and SDA if a longer period of incubation was used. Variation of incubation times and temperatures revealed that optimal conditions were obtained with 5 days of incubation at 37 C. Extensive mycelial growth made individual colonies difficult to distinguish after 5 days. In most instances, plate counts could be made after 3 days of incubation with little change in total counts occurring in the remaining 2 days. Effect of varied cortisone dosages on intravenously or intraperitoneally infected animals. Six groups of mice (10 mice per group) were inoculated intravenously (i.v.) via the lateral tail vein with 2.8 x 103 viable spores of M. apiospermum strain 813 contained in 0.1 ml. Group A did not receive additional treatment, and group B received ten 0.1-ml doses of physiological saline (0.15 M NaCl). Each of four groups, designated as C, D, E, and F, was treated with 10 doses of 0.5 mg, 1.0 mg, 1.5 mg, and 2.5 mg cortisone per dose, respectively. Cortisone and saline were administered to the animals 1 day prior to experimental infection and repeated at 48-h intervals thereafter. Four groups of control animals received doses of cortisone identical to that of the experimental animals but were not inoculated with the fungus. ET,0 values for groups of animals in this experiment were determined so that comparisons could be made between cortisone control animals and experimentally inoculated treated or untreated groups (Table 1). Cortisone alone proved to be very toxic in the higher doses tested, but those animals receiving the fungus inoculum in addition to the steroid still responded in a significantly shorter period of time than control animals. The animals receiving the fungus inoculation and the three highest cortisone concentrations (groups D, E, and F) died by the 7th day postinfection, and all animals of group C died by day 15. Within the 30-day observation period, 40% of the animals in group B, and 50% in group A died. In all cases, cortisone-treated mice were found to be more susceptible to infection than those mice that did not receive cortisone.

3 VOL. 8, 1973 PATHOGENICITY OF A. BOYDII FOR MICE A similar experiment was performed on six groups of mice inoculated intraperitoneally (i.p.) with 8.3 x 106 viable spores of M. apiospermum strain 813. Results (Table 2) are similar to those in the previous experiment. In general, however, the response to infection in this experiment was delayed. Even though the i.p.-inoculated animals received a 2,900-fold greater viable spore dosage, the i.v.-inoculated animals (Table 1) had significantly lower ET,. values than comparable groups of i.p.- inoculated animals (Table 2). Ten doses of 0.5 mg of cortisone did not significantly reduce resistance to i.p. infection even with high viable fungus spore concentrations. Since 10 doses of 1.0 mg of steroid per dose proved to be somewhat toxic to control animals (Tables 1 and 2), a treatment schedule of five 1.0-mg doses of coritsone was selected for later experimental infections involving the i.p. or intranasal (i.n.) route. Group TABLE 1. Treatment'ET50 Comparisons of strain virulence by route of inoculation. Three strains of A. boydii (801, 807, 809), three strains of M. apiospermum (806, 813, 815), and four routes of inoculation were tested. The animals inoculated i.v. received 0.1 ml containing 10' spores via the lateral tail vein, and groups inoculated intracerebrally (i.c.) received 2 x 10' spores in 0.02 ml. Animals inoculated i.p. were given 107 spores in a 0.5-ml inoculum and the i.n.- inoculated animals received 106 spores in 0.03 ml applied directly into the nares. Animals inoculated by the i.c. or i.n. route were lightly anesthetized with ether before inoculation. Steroid-treated animals infected by the i.v. or i.c. route were given ten 0.5-mg doses of cortisone; whereas, the i.p.- and i.n.-treated groups received 5 doses of 1.0 mg each. Results (Table 3) of these experiments show that cortisone treatment markedly altered the pathogenic potential of each fungus strain for ET50 values for intravenously inoculated animals Infected animalsa Control animals A None B Saline (> 30)e 40 C 0.5 mg of cortisone (> 30) 0 D 1.0 mg of cortisone (> 30) 40 E 1.5 mg of cortisone F 2.5 mg of cortisone a Inoculated with 2.83 x 103 spores of M. apiospermum (strain 813). b Ten 0.1-ml subcutaneous doses. cp= d Observation period of 30 days. e Mortality of 50% did not occur within the observation period. TABLE 2. ETl0 values for intraperitoneally inoculated animals Infected animalsa Control animals Group Treatment".d. Mor- Fiducial Morl ET,, o Flimitac tality' ET,, limits tly A None (> 30)e 20 B Saline (> 30) 7 C 0.5 mg of cortisone (> 30) 33 (> 30) 0 D 1.0 mg of cortisone (> 30) 40 E 1.5 mg of cortisone F 2.5 mg of cortisone a Inoculated with 8.3 x 106 spores of M. apiospermum (strain 813). " Ten 0.1-ml subcutaneous doses. cp= d Observation period of 30 days. Mortality of 50% did not occur within the observation period. ETo ctlimited tality Mor- ET,, limits tality talit 745

4 746 LUPAN AND CAZIN INFECT. IMMUNITY TABLE 3. Effect of cortisone treatment on ET,, values of animals infected by different routes of inoculation Intravenous Intracerebral Intraperitoneal Intranasal Strain Un- Un Untreated Treated Untreated Treated treated Treated treated Treated a 3.9 > > > ( )" ( ) 4/10 ( ) 1/10 ( ) 0/10 ( ) 807 > 30c 8.1 > > > /1od ( ) 2/10 ( ) 0/10 ( ) 0/10 ( ) 809 > > > > /10 ( ) 2/10 ( ) 1/10 ( ) 0/10 ( ) > > ( ) ( ) ( ) ( ) 1/10 ( ) 0/10 ( ) > > > ( ) ( ) 3/10 ( ) 2/10 ( ) 0/10 ( ) > > > ( ) ( ) 4/10 ( ) 1/10 ( ) 0/10 ( ) a ET50 in days. I Fiducial limits (P = 0.05). c Observation period of 30 days. d Number dead per total number. the routes of inoculation employed. None of the untreated mice died when inoculated i.n., whereas, in cortisone-treated animals there was at least a 50% response for each fungus strain. In groups inoculated by the i.p. route, a few of the untreated animals died, but a 50% response did not occur unless the animals were cortisone treated. Only with strain 806 was there a 50% response in untreated animals infected by the i.c. route. The ET5. values for i.n.-, i.p.-, and i.c.-inoculated animals treated with cortisone were significantly less than for untreated animals. Animals inoculated i.v. and treated with cortisone responded in significantly less time than untreated groups for each fungus except strain 806, which proved to be almost equally virulent for either treated or untreated animals. A separate experiment was performed to determine the LD50 dose of inoculum for cortisone-treated and untreated mice inoculated by the four different routes. These results (Table 4) show that LD,0 values for cortisone-treated animals inoculated by the i.v., i.c., or i.p. routes are all significantly less (P = 0.05) than the values obtained for untreated animals for comparable strains. Only one strain (813) produced a 50% response in untreated animals inoculated i.p. The LD.o values for i.n.-inoculated, treated animals could not be calculated, since in untreated animals it was impossible to give large enough challenge doses in an inoculum of 0.03 ml. Deaths did not occur in mice inoculated i.n. at the spore doses used unless the animals were cortisone treated. The LD50 values in the i.v. group for strain 801 are equal to 29,500 spores for untreated animals and 1,070 spores for treated animals; and, for strain 809 the LD50 is equal to 100,000 spores for untreated animals and only 3,700 spores for treated animals. When looking at extremes of differences in susceptibility for treated and untreated animals inoculated i.v., strain 813 and 807 show a 12-fold and 120-fold difference in virulence. Extremes in i.c.- inoculated animals with strains 801 and 806 show 280-fold and 3,200-fold differences. This study suggests that mice are most resistant to infection with A. boydii or M. apiospernum by the i.p. or i.n. routes of inoculation. Although infection by these fungi produced striking neurological symptoms, indicating involvement of the central nervous system, the LDse values for untreated mice are greater for the i.c. route of inoculation than for the i.v. route. This would suggest that spore dispersal to other vital organs via the blood-stream may play an important role in pathogenesis. When the LDo0 values of i.v.- and i.c.-inoculated animals are analyzed using the randomized complete-block design analysis of variance, the i.v. route of inoculation is shown to be significantly different (P = 0.005) than the i.c. route

5 VOL. 8, 1973 PATHOGENICITY OF A. BOYDII FOR MICE TABLE 4. Effect of cortisone treatment on LDs0 values of animals infected by different routes of inoculationa Strain Intravenous Intracerebral Intraperitoneal Intranasal Untreated Treated Untreated Treated Untreated Treated Untreated Treated b > > c / / > > / / > > > /lOd / /10 4/ > > / / > / > / > / a Observation period of 30 days. 6 Log 10. c Fiducial limits (P = 0.05). d Number dead per total number. for producing infections in animals even though several of the LD,0 values appear to be nonsignificant because of overlapping fiducial limits. Infected mice that ultimately die always show considerable hyperirritability during the early period of infection. As infection progresses, the animals run in circles or gyrate wildly by continuous sidewise rolling. Finally, the animals become lethargic and die. All infected animals exhibit a marked weight loss, probably due to dehydration and diminished food intake. The time of onset of neurological symptoms appears to be related to spore concentration, use of steroid treatment, and to the route of inoculation. Such symptoms were not seen in uninfected mice treated only with cortisone, and injection of concentrated culture filtrates or inactivated spore suspensions failed to reproduce the neurological symptoms. Histological sections (Fig. 1 and 2) made from the organs of infected mice reveal actual invasion of lung and brain tissues. DISCUSSION Although considerable research has been done on A. boydii, one can question the value of conclusions made from data accumulated with a single isolate. Wolf et al. (38) demonstrated that a single strain of M. apiospermum was autotrophic for its vitamins, whereas Villela and Cury (37) studied a strain of A. boydii which was biotin deficient and unable to grow 747 at 37 C. In other studies involving more than a single isolate of A. boydii or M. apiospermum (4, 5, 14), considerable difference has been noted among individual strains with respect to growth and sporulation. Assuming that differences might also exist in the virulence of individual isolates, the pathogenic potential of three strains of A. boydii and three strains of M. apiospermum were examined in this study. Several investigators (9, 12, 16) have attempted to infect the eyes, lungs, footpads, and peritoneum of guinea pigs with A. boydii strains freshly isolated from clinical materials. In general, very little evidence of infection was observed in the animals. Benham and Georg (3) were able to recover A. boydii from guinea pig spleens 3 weeks after i.p. inoculation of fungus spores, although no pathological changes were observed in the tissues. Subcutaneous inoculation of the fungus resulted only in the production of small localized abscesses. Their general conclusion was that the fungus possesses only a low degree of pathogenicity for experimental animals. Ajello (1) has established that A. boydii, when inoculated by the i.p. route, is pathogenic for mice. He was able to obtain consistent mortalities only when the organism was inoculated in combination with gastric mucin. Schmitt et al. (29) and Reyes (25), using similar procedures, reported that they were unable to obtain i.p. infections that produced death.

6 748 LUPAN AND CAZIN INFECT. IMMUNITY *!~:\! V'*'. fsi^ -or iep.pm j..-4 7% FIG. 1. Lung tissue from a cortisone-treated mouse 6 days after intranasal inoculation with M. apiospermum (813). x400. Since these investigators examined only a single isolate of A. boydii or M. apiospermum, one might suspect that the strains tested were similar to other individual strains that have been shown to be of low virulence (3). Schmitt et al. (29) attempted to infect animals with A. boydii by using known numbers of viable cells. In combination with mucin, they used viable spore doses of approximately 1.4 x 10' spores to infect animals i.p. Our study (Table 4) reveals that mortality of i.p.-infected, untreated animals occurred only with doses greater than 10 million viable spores. Even at large spore doses, the percentage mortality was low, i.e., 20% for strains 806 and 809 and 10% for strain 815. Deaths were not observed within 30 days in animals infected i.p. with strain 807 even with an inoculum as large as 3.5 x 107 viable spores. Steroid treatment of animals was necessary to achieve consistent mortalities in i.p.-inoculated mice. Even with steroid treatment, however, a viable spore inoculum greater than 106 spores was required to produce consistent mortality. This infectious inoculum represents a severalfold-higher dosage level than that required by any other route of inoculation. Data obtained in our study on i.p. infections of mice suggest that investigators who attempted to infect mice i.p. elected the route by which the animals appear to be most resistant to infection. Histological studies on experimental infections with A. boydii are extremely limited. Reyes (25), in studying the pathology of i.p. infection by M. apiospermum spores contained in gastric mucin, found fungal spores associated with necrotic granulomatous areas; however, he did not show actual tissue invasion. In this study, histological sections from mice inoculated with M. apiospermum strain 813 revealed numerous mycelial elements in most tissues. The presence of polymorphonuclear infiltrate and lack of hemosiderin-laden macrophages indicate tissue invasion by the fungus rather than fungus proliferation within a site of trauma. A neurological syndrome similar to that observed in our study with Allescheria-infected mice has been observed by other investigators with different fungi. Pore and Larsh (24) attributed the symptomatology, in mice infected with aleuriospores [sic] from a species of Aspergillus, to damage of the eleventh cranial or spinal accessory nerve. Under in vitro conditions, spores of aspergilli have been shown to enlarge approximately two fold in diameter prior to 46~~~~~~~~ R-,-w q 'W e % w * e'u. X FIG. 2. Brain tissue from a cortisone-treated mouse 6 days after intracerebral inoculation with M. apiospermum (813). x250..t S.%-

7 VOL. 8, 1973 PATHOGENICITY OF A. BOYDII FOR MICE germination (11). Although it is not known whether spores of Allesch'eria also enlarge prior to germination, such a phenomenon might result in physical blockage of capillaries, and possibly the onset of torticollis. In general, concentrations of steroid used therapeutically in human beings have not produced enhancement of experimental mycotic infections in lower animals. The recommended dosage for a patient with tuberculosis is 100 mg cortisone per day, in combination with antibiotics. Comparable dosage for a 20-g mouse, over a period of 20 days, would total 0.57 mg cortisone. This quantity of cortisone is nearly ninefold less than that required to produce significant effects in Allescheria-infected animals. Even though therapeutic doses of cortisone may not produce a diminished resistance in animals, consideration must be given to an underlying illness in combination with steroid therapy that may result in a much greater overall depression of resistance than steroid therapy alone. In addition, antibiotic therapy in combination with steroids is also known to have an additive effect in increasing susceptibility to infection (30). Studies designed to measure the effects of steroids on experimental infections in animals have generally supported the concept of enhanced susceptibility to mycotic infections in animals receiving steroid treatment. Scherr (28) produced more severe infections and higher mortalities with Candida albicans in animals treated with cortisone postinfection as compared to untreated animals. The quantity of steroid administered to animals was found by Roth et al. (26) to result in marked differences in percentage mortality of infected animals. Moreover, the time interval between steroid administration and fungal infection was found to be important. Enhanced susceptibility to infection was observed between 2 and 120 h after steroid treatment was begun, with maximum susceptibility at 48 h. Louria (20) reported that, in mice infected with Candida albicans, a 90% mortality was observed when 0.5 mg of cortisone per dose was administered 2 days preinfection, but only 70% was observed in mice receiving the identical dose when initiated 24 h postinfection. He stated that significant differences occurred both in mortalities and tissue population as a result of delayed steroid administration. A decrease in animal resistance to fungal infection by steroid treatment is not limited to yeasts but also has been reported for mycelial opportunistic fungi (27, 33). In our study, cortisone treatment was found to alter significantly the resistance of normal mice to experimental 749 allescheriosis. Moreover, the steroid dosage and route of experimental inoculation played a significant role in the mortality of Allescheria-infected mice. The steroid enhanced susceptibility of Allescheria-infected animals was related to the concentration of cortisone and was dependent also on the route of inoculation of the fungus. For example, groups C (Table 1 and 2) were given the same steroid dosage; nevertheless, the ET60 value of i.v.-inoculated animals is significantly less (P = 0.05) than the ET50 value of i.p.-inoculated mice even though animals infected i.p. received a much larger infectious inoculum. The effect of delayed steroid administration on experimental allescheriosis was not determined. Various mechanisms have been proposed to account for the altered resistance of animals receiving steroid therapy. Experimentally, large doses of corticosteroids are capable of suppressing the inflammatory response irregardless of the irritant (13, 24). Epstein et al. (8) in working with pulmonary aspergillosis have reported that cortisone acts to stabilize lysosomal membranes so that release of factors which contribute to an inflammatory stimulus are inhibited. The primary focus in blocking the inflammatory process appears to be centered around vascular responsibility (18) by maintaining vascular tone, reducing endothelial injury, and by decreasing capillary permeability. The outflow of cells and fluid into the injured area are minimized, thereby decreasing injury and diminishing the normal reparative function. The phagocytic function of leukocytes also may be decreased. Engulfment appears normal, but the polymorphonuclear and mononuclear leukocytes are impaired in their ability to digest engulfed material (2), probably due to a corticosteroid-induced derangement of the metabolic activity of the phagocytes. We have shown that deaths were easier to produce in animals treated with cortisone. Therefore, in normal animals, the alveolar and peritoneal macrophages may be able to prevent overt infections by spores of A. boydii. Large doses of corticosteroids diminish antibody response (18) and alter reticuloendothelial function (17). It is unlikely that a diminished antibody response plays a significant role in the mortality of mice inoculated i.v. or i.c. with A. boydii since death usually occurs before there is a significant stimulation of humoral antibody. It is difficult to differentiate A. boydii from other mycelial pathogens by histological techniques. Consequently, the actual number of Allescheria infections that occur may be greater

8 750 LUPAN AND CAZIN than generally recognized. Louria (21) described an instance in which a pulmonary infection caused by A. boydii would have been misdiagnosed had not positive lung cultures been obtained. Our results confirm that mice are susceptible to experimental allescheriosis. Further study might prove that other animal species thought to be resistant to infection by A. boydii might also be susceptible when cortisone treatment is combined with an experimental infection by the i.v. or i.c. route. The use of ET50 and LD50 values has given accurate quantitative indexes for comparing virulence of these fungi for mice. The virulence of Allescheria or Monosporium spp. for mice is difficult to compare with virulence of other opportunistic fungi since most investigators have not quantitatively measured response to an accurate viable infectious dose. A clear pattern has not evolved in this study to indicate any relationship between sexual and asexual forms of these fungi and their pathogenicity for mice. Pathogenicity of A. boydii or its anascigerous form M. apiospermum appears to be entirely strain dependent. LITERATURE CITED 1. Ajello, L The isolation of Allescheria boydii Shear, an etiologic agent of mycetomas, from soil. Amer. J. Trop. Med. 1: Allison, F., and M. H. Adcock The influence of hydrocortisone and certain electrolyte solutions upon the phagocytic and bactericidal capacity of leukocytes obtained from peritoneal exudate or rats. J. Immunol. 92: Benham, R. W., and L. K. Georg Allescheria boydii, causative agent in a case of meningitis. J. Invest. Dermatol. 10: Cafin, J., Jr., and D. W. Decker Carbohydrate nutrition and sporulation of Allescheria boydii. J. Bacteriol. 88: Cazin, J., Jr., and D. W. Decker Growth of Allescheria boydii in antibiotic-containing media. J. Bacteriol. 90: Conant, N. F., D. T. Smith, R. D. 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9 VOL. 8, 1973 PATHOGENICITY OF A. BOYDII FOR MICE 751 procedures of statistics. McGraw-Hill Book Co., Inc., New York. 35. Thomas, L Cortisone, ACTH and infections. Bull. N.Y. Acad. Med. 31: Travis, R. E., E. W. Ulrich, and S. Phillips Pulmonary allescheriasis. Ann. Intern. Med. 54: Villela, G. G., and A. Cury Studies on the vitamin nutrition of Allescheria boydii shear. J. Bacteriol. 59: Wolf, E. T., R. R. Bryden, and J. A. MacLaren The nutrition of Monosporium apiospermum. Mycologia 42: