Vol. 24, No. 3
INFECTION AND IMMUNITY, June 1979, p. 843-850 0019-9567/79/06-0843/08$02.00/0
Humoral Response to Experimental Petriellidiosis DAVID M. LUPAN'* AND JOHN CAZIN, JR.2 Department of Microbiology, School of Medical Sciences, University of Nevada, Reno, Nevada 89557' and Department of Microbiology, University of Iowa, Iowa City, Iowa 522422
Received for publication 26 March 1979
The humoral antibody response elicited by experimental infection with Petriellidium boydii and Monosporium apiospermum was studied. The variables of fungus strain, route of inoculation, and concentration of inoculum were examined. Viable spores from three strains of P. boydii and from three strains of M. apiospermum were inoculated intravenously into mice. Serum specimens were tested for antibody by the microtiter indirect hemagglutination assay. At 4 weeks after infection, the percentage of positive specimens among survivors varied from 39 to 62%. Variation in fungus strain virulence was not statistically related either to the incidence of positive specimens or to the magnitude of the humoral response. Mice inoculated by either the subcutaneous or the intramuscular route with viable or killed spores from M. apiospermum 813 developed antibody. The mean antibody titer was greater in those animals receiving viable spores, and the magnitude of the humoral response was dose dependent. Animals inoculated intravenously with comparable inocula of the same strain developed similar levels of antibody. However, cultural studies of infected mice showed chronic infection only after intravenous inoculation. The kidney was the organ most consistently involved. Serious infections in humans caused by Petriellidium boydii and its imperfect form Monosporium apiospermum are well documented. Therefore, isolation of these fungi from clinical specimens can be quite important. For example, three recent reports (12, 18, 23) discuss the significance of diagnosis and clinical management of patients with petriellidiosis. Because petriellidioses occur with some frequency, additional studies of these organisms are warranted. The full significance of the pathogenic potential of P. boydii and M. apiospermum for experimental animals may not be fully realized. Few studies on the virulence of P. boydii and M. apiospermnum for experimental animals have been conducted. Most investigations have been primarily oriented toward fulfilling Koch's postulates by inoculating organisms isolated from clinical specimens into eyes, lungs, footpads, or peritonea of a variety of experimental animals (6, 8, 10, 11). Since these studies have been relatively unsuccessful, investigators have concluded that these fungi have little virulence for laboratory animals. On the other hand, our investigations of several isolates of P. boydii and M. apiospermum have shown that individual isolates are virulent for mice, although at considerably different 50% lethal doses (LD5o) (15). Mice were found consistently to be susceptible
to a fulminating petriellidiosis when the intravenous route of infection was utilized. Conversely, lethal infections were difficult to establish when other routes of inoculation were employed unless the natural resistance was sup-
pressed by corticosteroids. Our findings were consistent with those of other investigations (22, 24, 26). Previous immunological studies on P. boydii and M. apiospermum have been limited to the characterization of antigenic relationships among agents causing mycetoma (19, 25) or to a comparison of methods for serological diagnosis of mycetoma (1). These studies have not considered the immune response during experimental infection, nor have they correlated the humoral antibody response with the progress of the disease, presumably due to the lack of a suitable animal model. Inasmuch as the murine model of petriellidiosis appeared to be successful in our laboratory, we decided to investigate the humoral immune response in infected mice. Specifically, our experiments were designed to determine how the humoral response was influenced (i) by strain of invading fungus, (ii) by local versus systemic infection, and (iii) by the inoculum concentration. This information would be useful for consideration of an animal model for studies on the mechanisms of host resistance
843
844
LUPAN AND CAZIN
INFECT. IMMUN.
sensitize erythrocytes. The production and purification of this preparation has been described elsewhere (16). Sheep erythrocytes (Colorado Serum Co., Denver, Colo.) stored in Alsever solution were centrifuged and washed three times with 0.15 M NaCl. SensitizaMATERIALS AND METHODS tion of erythrocytes was accomplished as follows. Fungus strains. Strains of P. boydii and M. apio- Physiological saline (1.0 ml) containing 0.25 mg of spermum were obtained from stock cultures main- freshly dissolved chromium chloride (CrCl3. 6H20) tained at the Department of Microbiology, University was added with constant stirring to a mixture containof Iowa. Partial characterization of some antigens has ing 1.0 ml of antigen solution (1.0 mg of antigen 1 from been described (16). The LD50 values for P. boydii M apiospermum 813 in saline) and 0.5 ml of packed strains 801, 807, and 809 and M. apiospermum strains erythrocytes. After 8 to 10 min at 37°C, the suspension 806, 813, and 815 for intravenously inoculated mice was centrifuged at 500 x g for 10 min, and the superwere 2.95 X 104, 19.1 X 104, 10.0 X 104, 1.29 x 104, 0.95 natant fluid was decanted. The cells were washed four times with 15-ml volumes of saline and suspended in X 104, and 3.72 x 104 spores, respectively (15). Mice. A Webster strain of Swiss mice maintained saline to a final concentration of 0.5%. Since some by the Department of Microbiology, University of hemolysis occurs on addition of chromium chloride, Iowa, was used throughout this study. Male mice were the concentration of sensitized erythrocytes was standapproximately 4 to 5 weeks old and weighed 16 to 20 ardized spectrophotometrically against a known concentration of untreated erythrocytes. g. Food and water were supplied ad libitum. As required by the experimental protocol, mice were inoculated with heat-killed or viable fungus spores in RESULTS 0.2 ml of saline (0.15 M NaCl) by either the intramusInfluence of fungus strain on humoral cular, intravenous, or subcutaneous route. During the inoculation process care was taken to avoid spore response. Previous studies established that insettling in the syringe by rotating the syringe between dividual strains of P. boydii vary in their viruinoculations. At regular intervals after infection, mice lence for mice. For this reason, an initial experwere anesthetized with ether, and blood specimens iment was designed to investigate the relation(0.1 to 0.5 ml) were taken from the retroorbital venous ship of fungus strain to the magnitude of the plexus by using a Pasteur pipette. The serum was humoral antibody response. Six groups of mice collected and stored at -20'C for further study. Fungus spore suspensions. Fungus spores were were infected with a single strain of either P. obtained from cultures grown on Czapek dextrose agar boydii (strain 801, 807, or 809) or M. apiosperat room temperature. Techniques for harvest and sep- mum (strain 806, 813, or 815). All mice were aration of spores from mycelial fragments have been inoculated intravenously via the lateral tail vein described previously (15). Viability of spores was de- since mice are uniformly susceptible to all strains termined by using pour plates of potato dextrose agar of P. boydii when infected in this manner (15). and incubation at 370C. When killed cells were re- The inoculum for each fungus strain was adquired, the appropriate concentration of fungus spores justed to contain approximately 1.0 LD50. was placed in a boiling water bath for 30 min. Spore Viability studies on the inocula revealed that death was confirmed by culture on potato dextrose the quantity of infectious spores varied from a agar. 0.8 LD50 (strain 807) to a high of 1.8 LD50 of low Antibody assay. Antibody to Petriellidium antigens was measured by the indirect hemagglutination (strain 813). Each inoculated spore concentratechnique. Indirect (passive) hemagglutination assays tion was within the calculated confidence inter(IHA) on serum specimens were done by the microtiter val for the LD5o reported in a previous study. method in V Lucite plates (Cooke Engineering Co., The data revealed, however, that any incidental Alexandria, Va.). Serial twofold dilutions of serum increase in spore concentration above the LD50 were made in 25-jl volumes of saline containing bovine can result in greatly elevated mortality rates serum albumin. Antigen-sensitized erythrocytes (25 (Table 1). For example, the inoculation of 1.74 added to each dilution, tl of a 0.5% suspension) were after strain 2.5 h of incubation x 104 viable spores of M. apiospermum and the results were recorded than at room temperature (22 to 250C). The reaction end- 813, which is approximately 1.8-fold greater level a mortality produced LD50, determined the causof serum dilution the as highest was taken point ing complete agglutination of sensitized erythrocytes. of 76.4%. Similar comparisons can be made for A negative control containing 25 MuI of sensitized cells other isolates. The frequency of deaths after and 25 yl of bovine serum albumin-saline was included petriellidiosis was greatest 3 to 6 days post-inwith each assay to determine nonspecific aggregation oculation, after which time the mortality rates of sensitized erythrocytes. All serum specimens were declined rapidly, so that deaths rarely occurred inactivated before titration by incubation at 560C for after 21 days postinfection. Survivors were 30 min and subsequently adsorbed for 1 h at room tested for humoral antibody at 28 days postintemperature with an equal volume of packed sheep erythrocytes to remove anti-erythrocyte agglutinins. fection. Each group of mice responded to the individA semipurified antigen preparation from culture filtrates of M. apiospermum strain 813 was used to ual fungus strains by producing antibody (Table
to petriellidiosis or for examining the parameters that could influence the usefulness of a serological test for petriellidiosis.
HUMORAL RESPONSE TO PETRIELLIDIOSIS
VOL. 24, 1979
845
TABLE 1. IHA antibody titers of intravenously inoculated mice % of
Strain
No. of mice Viable spore nclm in moculu
mf
No. of survivors with the following titers:'
~~~~~surviwi VorM %
Mr
tality
ihGMTc
positive sera'
200 4.4 x 104 83.5 48.5 P. boydii 801 100 15.2 x 104 49.0 39.2 P. boydii 807 9.0 x 104 73.2 46.8 175 P. boydii 809 180 1.7 x 10' 64.5 53.1 M. apiospermum 806 300 1.7 x 104 76.4 62.0 M. apiospermum 813 3.0 x 104 56.7 43.6 180 M. apiospermum 815 a Percentage of survivors at 28 days with positive sera. b Reciprocal of IHA titer (log2). e Geometric mean titer of survivors.
1); however, some survivors within each group lacked measurable levels of antibody. The range of IHA-positive sera among survivors varied from a low of 39.2% (strain 807) to a high of 62% (strain 813). There was no consistent correlation between strain virulence, i.e., percentage of mortality, and ability of a strain to induce a humoral response. Furthermore, there did not appear to be any significant difference by chi-square analysis or analysis of variance between strains in terms of the percentage of surviving animals with positive sera or in the levels (titer) of antibody produced. The geometric mean IHA titer (all survivors considered) was greater for M. apiospermum strain 813 than for any other fungus strain. For this reason additional studies were conducted with strain 813 alone. A control group of 20 mice inoculated only with diluent was negative for antibody to Petriellidium antigens. Humoral response to chronic petriellidiosis. Most human petriellidioses are chronic infections. Therefore, we investigated the effects of inoculation route and size of inoculum on antibody response during a chronic murine petriellidiosis. Initially, two routes of inoculation and three concentrations of infectious spores were chosen. Groups of 15 mice were inoculated with either 3.2 x 10', 3.2 x i05, or 3.2 x 106 viable or nonviable spores of strain 813 by the intramuscular or subcutaneous route. An additional group of uninoculated mice was retained as a control. After inoculation the groups of animals were bled at 4-week intervals, and the sera were tested for antibody to M. apiospermum. In contrast to animals inoculated by the intravenous route (Table 1), none of the animals inoculated intramuscularly or subcutaneously died of infection before the first serum sampling period (28 days). Regardless of route of inocula-
1
2
3
4
5
6
7
5 12 9 10 15
3 2 7 13 13 13
5
1 3 1 4 3 1
1
1 2
1
10
4 7
9 10
1
2
2
1.24 0.92 0.94 1.14 1.44 0.90
tion or concentration of spores, the humoral response, as indicated by the geometric mean IHA titer, was greater in groups receiving viable spores than in identical groups inoculated with killed spores (Fig. 1). This difference was greatest in groups receiving the largest inoculum. The peak humoral antibody response to the intramuscular inoculation occurred within 4 weeks; and, although in one instance the mean IHA titer appeared to plateau between 4 and 8 weeks (3.2 x 106 spores), it decreased linearly after 4 weeks at the lower spore concentrations. The maximum response to the subcutaneous inoculation occurred approximately 8 weeks after infection. Local fungus proliferation may have occurred by this route, providing prolonged stimulation of the humoral response by fungus antigen. However, in comparison, the magnitude of the humoral response was greater after an intramuscular than after a subcutaneous inoculation of 3.2 x 106 viable spores. All of the mice inoculated subcutaneously with 3.2 x 106 viable spores developed localized, suppurative lesions at the site of infections, which appeared 5 to 7 days postinfection and were usually resolved by 10 to 14 days postinfection. Only 40% of the mice inoculated similarly with 3.2 x 105 viable spores developed lesions, and none of the animals inoculated with the lowest spore concentrations had macroscopically visible lesions. At the conclusion of the 16-week experiment, mice inoculated with viable spores were sacrificed, and the brain, lung, liver, spleen, and kidney tissues were excised. After the organs were thoroughly minced, a sample of each was subcultured on mycobiotic agar (Difco Laboratories, Detroit, Mich.). All cultures were negative. Uninoculated control mice were also tested at regular intervals throughout the 16-week experiment, and the sera were negative by IHA. An additional experiment was conducted to
846
LUPAN AND CAZIN
INFECT. IMMUN.
INTRAMUSCULAR 3 2 .10 5SPORES
-5
0.98 x
104
1.96 x
104
3.92 x 104
W
1,
7-
0
LAS.6.
,
INFECTION AND IMMUNITY, June 1979, p. 843-850 0019-9567/79/06-0843/08$02.00/0
Humoral Response to Experimental Petriellidiosis DAVID M. LUPAN'* AND JOHN CAZIN, JR.2 Department of Microbiology, School of Medical Sciences, University of Nevada, Reno, Nevada 89557' and Department of Microbiology, University of Iowa, Iowa City, Iowa 522422
Received for publication 26 March 1979
The humoral antibody response elicited by experimental infection with Petriellidium boydii and Monosporium apiospermum was studied. The variables of fungus strain, route of inoculation, and concentration of inoculum were examined. Viable spores from three strains of P. boydii and from three strains of M. apiospermum were inoculated intravenously into mice. Serum specimens were tested for antibody by the microtiter indirect hemagglutination assay. At 4 weeks after infection, the percentage of positive specimens among survivors varied from 39 to 62%. Variation in fungus strain virulence was not statistically related either to the incidence of positive specimens or to the magnitude of the humoral response. Mice inoculated by either the subcutaneous or the intramuscular route with viable or killed spores from M. apiospermum 813 developed antibody. The mean antibody titer was greater in those animals receiving viable spores, and the magnitude of the humoral response was dose dependent. Animals inoculated intravenously with comparable inocula of the same strain developed similar levels of antibody. However, cultural studies of infected mice showed chronic infection only after intravenous inoculation. The kidney was the organ most consistently involved. Serious infections in humans caused by Petriellidium boydii and its imperfect form Monosporium apiospermum are well documented. Therefore, isolation of these fungi from clinical specimens can be quite important. For example, three recent reports (12, 18, 23) discuss the significance of diagnosis and clinical management of patients with petriellidiosis. Because petriellidioses occur with some frequency, additional studies of these organisms are warranted. The full significance of the pathogenic potential of P. boydii and M. apiospermum for experimental animals may not be fully realized. Few studies on the virulence of P. boydii and M. apiospermnum for experimental animals have been conducted. Most investigations have been primarily oriented toward fulfilling Koch's postulates by inoculating organisms isolated from clinical specimens into eyes, lungs, footpads, or peritonea of a variety of experimental animals (6, 8, 10, 11). Since these studies have been relatively unsuccessful, investigators have concluded that these fungi have little virulence for laboratory animals. On the other hand, our investigations of several isolates of P. boydii and M. apiospermum have shown that individual isolates are virulent for mice, although at considerably different 50% lethal doses (LD5o) (15). Mice were found consistently to be susceptible
to a fulminating petriellidiosis when the intravenous route of infection was utilized. Conversely, lethal infections were difficult to establish when other routes of inoculation were employed unless the natural resistance was sup-
pressed by corticosteroids. Our findings were consistent with those of other investigations (22, 24, 26). Previous immunological studies on P. boydii and M. apiospermum have been limited to the characterization of antigenic relationships among agents causing mycetoma (19, 25) or to a comparison of methods for serological diagnosis of mycetoma (1). These studies have not considered the immune response during experimental infection, nor have they correlated the humoral antibody response with the progress of the disease, presumably due to the lack of a suitable animal model. Inasmuch as the murine model of petriellidiosis appeared to be successful in our laboratory, we decided to investigate the humoral immune response in infected mice. Specifically, our experiments were designed to determine how the humoral response was influenced (i) by strain of invading fungus, (ii) by local versus systemic infection, and (iii) by the inoculum concentration. This information would be useful for consideration of an animal model for studies on the mechanisms of host resistance
843
844
LUPAN AND CAZIN
INFECT. IMMUN.
sensitize erythrocytes. The production and purification of this preparation has been described elsewhere (16). Sheep erythrocytes (Colorado Serum Co., Denver, Colo.) stored in Alsever solution were centrifuged and washed three times with 0.15 M NaCl. SensitizaMATERIALS AND METHODS tion of erythrocytes was accomplished as follows. Fungus strains. Strains of P. boydii and M. apio- Physiological saline (1.0 ml) containing 0.25 mg of spermum were obtained from stock cultures main- freshly dissolved chromium chloride (CrCl3. 6H20) tained at the Department of Microbiology, University was added with constant stirring to a mixture containof Iowa. Partial characterization of some antigens has ing 1.0 ml of antigen solution (1.0 mg of antigen 1 from been described (16). The LD50 values for P. boydii M apiospermum 813 in saline) and 0.5 ml of packed strains 801, 807, and 809 and M. apiospermum strains erythrocytes. After 8 to 10 min at 37°C, the suspension 806, 813, and 815 for intravenously inoculated mice was centrifuged at 500 x g for 10 min, and the superwere 2.95 X 104, 19.1 X 104, 10.0 X 104, 1.29 x 104, 0.95 natant fluid was decanted. The cells were washed four times with 15-ml volumes of saline and suspended in X 104, and 3.72 x 104 spores, respectively (15). Mice. A Webster strain of Swiss mice maintained saline to a final concentration of 0.5%. Since some by the Department of Microbiology, University of hemolysis occurs on addition of chromium chloride, Iowa, was used throughout this study. Male mice were the concentration of sensitized erythrocytes was standapproximately 4 to 5 weeks old and weighed 16 to 20 ardized spectrophotometrically against a known concentration of untreated erythrocytes. g. Food and water were supplied ad libitum. As required by the experimental protocol, mice were inoculated with heat-killed or viable fungus spores in RESULTS 0.2 ml of saline (0.15 M NaCl) by either the intramusInfluence of fungus strain on humoral cular, intravenous, or subcutaneous route. During the inoculation process care was taken to avoid spore response. Previous studies established that insettling in the syringe by rotating the syringe between dividual strains of P. boydii vary in their viruinoculations. At regular intervals after infection, mice lence for mice. For this reason, an initial experwere anesthetized with ether, and blood specimens iment was designed to investigate the relation(0.1 to 0.5 ml) were taken from the retroorbital venous ship of fungus strain to the magnitude of the plexus by using a Pasteur pipette. The serum was humoral antibody response. Six groups of mice collected and stored at -20'C for further study. Fungus spore suspensions. Fungus spores were were infected with a single strain of either P. obtained from cultures grown on Czapek dextrose agar boydii (strain 801, 807, or 809) or M. apiosperat room temperature. Techniques for harvest and sep- mum (strain 806, 813, or 815). All mice were aration of spores from mycelial fragments have been inoculated intravenously via the lateral tail vein described previously (15). Viability of spores was de- since mice are uniformly susceptible to all strains termined by using pour plates of potato dextrose agar of P. boydii when infected in this manner (15). and incubation at 370C. When killed cells were re- The inoculum for each fungus strain was adquired, the appropriate concentration of fungus spores justed to contain approximately 1.0 LD50. was placed in a boiling water bath for 30 min. Spore Viability studies on the inocula revealed that death was confirmed by culture on potato dextrose the quantity of infectious spores varied from a agar. 0.8 LD50 (strain 807) to a high of 1.8 LD50 of low Antibody assay. Antibody to Petriellidium antigens was measured by the indirect hemagglutination (strain 813). Each inoculated spore concentratechnique. Indirect (passive) hemagglutination assays tion was within the calculated confidence inter(IHA) on serum specimens were done by the microtiter val for the LD5o reported in a previous study. method in V Lucite plates (Cooke Engineering Co., The data revealed, however, that any incidental Alexandria, Va.). Serial twofold dilutions of serum increase in spore concentration above the LD50 were made in 25-jl volumes of saline containing bovine can result in greatly elevated mortality rates serum albumin. Antigen-sensitized erythrocytes (25 (Table 1). For example, the inoculation of 1.74 added to each dilution, tl of a 0.5% suspension) were after strain 2.5 h of incubation x 104 viable spores of M. apiospermum and the results were recorded than at room temperature (22 to 250C). The reaction end- 813, which is approximately 1.8-fold greater level a mortality produced LD50, determined the causof serum dilution the as highest was taken point ing complete agglutination of sensitized erythrocytes. of 76.4%. Similar comparisons can be made for A negative control containing 25 MuI of sensitized cells other isolates. The frequency of deaths after and 25 yl of bovine serum albumin-saline was included petriellidiosis was greatest 3 to 6 days post-inwith each assay to determine nonspecific aggregation oculation, after which time the mortality rates of sensitized erythrocytes. All serum specimens were declined rapidly, so that deaths rarely occurred inactivated before titration by incubation at 560C for after 21 days postinfection. Survivors were 30 min and subsequently adsorbed for 1 h at room tested for humoral antibody at 28 days postintemperature with an equal volume of packed sheep erythrocytes to remove anti-erythrocyte agglutinins. fection. Each group of mice responded to the individA semipurified antigen preparation from culture filtrates of M. apiospermum strain 813 was used to ual fungus strains by producing antibody (Table
to petriellidiosis or for examining the parameters that could influence the usefulness of a serological test for petriellidiosis.
HUMORAL RESPONSE TO PETRIELLIDIOSIS
VOL. 24, 1979
845
TABLE 1. IHA antibody titers of intravenously inoculated mice % of
Strain
No. of mice Viable spore nclm in moculu
mf
No. of survivors with the following titers:'
~~~~~surviwi VorM %
Mr
tality
ihGMTc
positive sera'
200 4.4 x 104 83.5 48.5 P. boydii 801 100 15.2 x 104 49.0 39.2 P. boydii 807 9.0 x 104 73.2 46.8 175 P. boydii 809 180 1.7 x 10' 64.5 53.1 M. apiospermum 806 300 1.7 x 104 76.4 62.0 M. apiospermum 813 3.0 x 104 56.7 43.6 180 M. apiospermum 815 a Percentage of survivors at 28 days with positive sera. b Reciprocal of IHA titer (log2). e Geometric mean titer of survivors.
1); however, some survivors within each group lacked measurable levels of antibody. The range of IHA-positive sera among survivors varied from a low of 39.2% (strain 807) to a high of 62% (strain 813). There was no consistent correlation between strain virulence, i.e., percentage of mortality, and ability of a strain to induce a humoral response. Furthermore, there did not appear to be any significant difference by chi-square analysis or analysis of variance between strains in terms of the percentage of surviving animals with positive sera or in the levels (titer) of antibody produced. The geometric mean IHA titer (all survivors considered) was greater for M. apiospermum strain 813 than for any other fungus strain. For this reason additional studies were conducted with strain 813 alone. A control group of 20 mice inoculated only with diluent was negative for antibody to Petriellidium antigens. Humoral response to chronic petriellidiosis. Most human petriellidioses are chronic infections. Therefore, we investigated the effects of inoculation route and size of inoculum on antibody response during a chronic murine petriellidiosis. Initially, two routes of inoculation and three concentrations of infectious spores were chosen. Groups of 15 mice were inoculated with either 3.2 x 10', 3.2 x i05, or 3.2 x 106 viable or nonviable spores of strain 813 by the intramuscular or subcutaneous route. An additional group of uninoculated mice was retained as a control. After inoculation the groups of animals were bled at 4-week intervals, and the sera were tested for antibody to M. apiospermum. In contrast to animals inoculated by the intravenous route (Table 1), none of the animals inoculated intramuscularly or subcutaneously died of infection before the first serum sampling period (28 days). Regardless of route of inocula-
1
2
3
4
5
6
7
5 12 9 10 15
3 2 7 13 13 13
5
1 3 1 4 3 1
1
1 2
1
10
4 7
9 10
1
2
2
1.24 0.92 0.94 1.14 1.44 0.90
tion or concentration of spores, the humoral response, as indicated by the geometric mean IHA titer, was greater in groups receiving viable spores than in identical groups inoculated with killed spores (Fig. 1). This difference was greatest in groups receiving the largest inoculum. The peak humoral antibody response to the intramuscular inoculation occurred within 4 weeks; and, although in one instance the mean IHA titer appeared to plateau between 4 and 8 weeks (3.2 x 106 spores), it decreased linearly after 4 weeks at the lower spore concentrations. The maximum response to the subcutaneous inoculation occurred approximately 8 weeks after infection. Local fungus proliferation may have occurred by this route, providing prolonged stimulation of the humoral response by fungus antigen. However, in comparison, the magnitude of the humoral response was greater after an intramuscular than after a subcutaneous inoculation of 3.2 x 106 viable spores. All of the mice inoculated subcutaneously with 3.2 x 106 viable spores developed localized, suppurative lesions at the site of infections, which appeared 5 to 7 days postinfection and were usually resolved by 10 to 14 days postinfection. Only 40% of the mice inoculated similarly with 3.2 x 105 viable spores developed lesions, and none of the animals inoculated with the lowest spore concentrations had macroscopically visible lesions. At the conclusion of the 16-week experiment, mice inoculated with viable spores were sacrificed, and the brain, lung, liver, spleen, and kidney tissues were excised. After the organs were thoroughly minced, a sample of each was subcultured on mycobiotic agar (Difco Laboratories, Detroit, Mich.). All cultures were negative. Uninoculated control mice were also tested at regular intervals throughout the 16-week experiment, and the sera were negative by IHA. An additional experiment was conducted to
846
LUPAN AND CAZIN
INFECT. IMMUN.
INTRAMUSCULAR 3 2 .10 5SPORES
-5
0.98 x
104
1.96 x
104
3.92 x 104
W
1,
7-
0
LAS.6.
,
