Br. J. exp. Path. (1976) 57, 296
DEPRESSION OF BURSAL FOLLICLE FORMATION BY CANDIDA ALBICANS INFECTIONS W. H. WAIN* AND R. A. CAWSONt From the Department of Oral Medicine and Pathology, Guy'8 Hospital Medical School, London Bridge, SEI 9RT Received for publication December 3, 1975
Summary.-Sub-lethal i.v. inoculations of Candida albicans into 12-day-old chick embryos have been shown to cause a depression of follicle formation in the bursa of Fabricius. This depression occurred in the absence of any infection in the bursa, although candidal encephalitis was a frequent manifestation of the disseminated infection. A similar depression of bursal follicle formation was caused by the administration of viable C. albicans on to the embryonic chorio-allantoic membrane, in the absence of any disseminated infection. Non-viable extracts of disrupted C. albicans administered on to the chorioallantoic membrane also caused a depression of bursal follicle formation. This immunodeficiency produced by an endotoxin-like principle at a susceptible stage in the development of the chick has been considered in terms of human immunological development and the neonatal occurrence of C. albicans.
HYPOPLASIA of the bursal follicles in the embryonic chick caused by i.v. inoculations of Candida albicans has recently been reported by Lydyard and Ivanyi (1975). Pathological changes in the thymus of mice chronically infected with C. albicans have previously been described (Mankowski, 1968). Administration of allogeneic lymphocytes on to the chorio-allantoic membrane (CAM) of chick embryos has also been shown to cause depression of bursal follicle formation (Walker, Schoefl and Lafferty, 1972). The chick CAM has also been used as a host tissue for model infections by C. albi cans (Cawson, 1973). This plaqueforming response by the CAM to C. albicans (Partridge, Athar and Winner, 1971) is however variable, but has been shown to be due to clumping of the inoculum (Wain, Price and Cawson, 1974). The degree of depression of bursal follicle *
formation caused by C. albicans has therefore been investigated both as an index of pathogenicity and for its possible role in immunodeficiency. The depression of bursal follicle formation by sub-lethal inocula of viable C. albicans and by non-viable extracts of disrupted fungal cells has been examined. Foci of infection resulting from i.v. dissemination of viable C. albicans have been located and similar locations have been examined after inoculation on to the CAM. Dissemination from CAM inoculations has been tested for by viability assays from those infected embryos. MATERIALS AND METHODS
1. Inoculation of embryos.-Fertile eggs from White Leghorn x Rhode Island fowls (Appleby Farm, Ashford, Kent) were maintained in a humidified egg incubator. The viable 12-day embryos were injected with 0-1 ml of inoculum
Present address: Homograft Department, National Heart Hospital, Westmoreland Street, London W.1.
t To whom reprint requests should be addressed.
DEPRESSION OF BURSAL FOLLICLE FORMATION BY CANDIDA ALBICANS either i.v. (as described by Lydyard and Ivanyi, 1975), or on to the CAM as described by Partridge (1968). Yeast cell suspensions were diluted in day-old chick red blood cell diluent (Gibb Biocult) and cell fractions were resuspended in Yeast Nitrogen base (YNB) without amino acids and ammonium sulphate (Difco). Viability of the inoculated eggs was checked daily by candling and the surviving embryos were killed on Day 19. 2. Culture and disruption of Candida albicans.-Clinical isolates of Candida albicans were maintained in Sabouraud liquid medium and periodically passaged through viable chick embryos by inoculation on to the CAM at a concentration of 1 x 106 cells/ml. Early stationary phase cells were used for inocula preparation as described in Wain, Price and Cawson (1975) and dilutions prepared in day-old chick red blood cell diluent as required. Continuous culture of C. albicans in 5% glucose yeast nitrogen base (Difco) (YNB) at pH 5*4, 370, flow rate 1-25 ml/min (dilution rate 0 06), aeration at 800 ml/min air, 400 ml/min C02, oxygen concentration 3-5 ppm was carried out in a Biotec Polyferm FE007 (LKB) for 24 h. The cells in the fermentor were harvested, resuspended to 5 ml in 5% glucose YNB and passed 5 times through a small " X "-press (LKB) at - 250. The frozen, disrupted suspension was thawed, centrifuged for 10 min at 3000 g at 00 and the supernatant removed as " cytoplasm ". The pellet was resuspended to the original volume in YNB, without amino acids and ammonium sulphate, and centrifuged again at 3000 g for 10 min at 00. The supernatant was removed as " cell wall extract " and the pellet resuspended to the original volume in YNB without amino acids and ammonium sulphate to provide a " cell wall suspension ". 0-1 ml of each fraction was plated out in duplicate for viability assays. Further 0-1 ml aliquots of these fractions were used as the inocula for the 12-day-old chick embryos. 3. Histology.-At the 19th day, after 7 days of infection, the viable embryos were killed and the CAM and the pelvic region containing the lower gut and bursa of Fabricius were removed and fixed in a buffered 10% formol saline. After 24 h fixation the bursa was dissected from the cloaca and transected equatorially. The 2 half-bursas were processed and prepared in the usual way for paraffin sections. The examinations for disseminated infections were carried out on brain, liver, heart, kidney and spleen, fixed and processed in an identical manner. Transverse 5-,um sections of bursa, spleen, kidney and liver, and longitudinal 5-,um sections of brain and heart were cut to provide representative sections of each organ. The sections were stained with haematoxylin and
297
eosin and with periodic acid-Schiff for detection of the fungi. The sections of bursa were examined for follicular development and the number of follicles in 5 representative folds of each half of each bursa were counted and expressed as a follicle count per fold. Since the size distribution of the follicles assayed by Lydyard and Ivanyi (1975) provided so little more information than the mean total follicular count per fold, this refinement of their assay was not adopted.
4. Retention of viability by the inoculum.The infected CAM was flooded with buffered 10% formol saline for 60 min in order to kill the inoculum on the CAM, which was then washed twice with day-old chick red cell diluent to remove the fixative. The CAM was removed and the remainder of the egg including the shell was homogenized, diluted and plated out on Sabouraud maltose agar. Alternatively the embryo and the brain were homogenized separately and assayed for viable C. albicans by similar plating procedures. RESULTS
1. Mortality of embryos Inoculation of viable C. albicans at 1 x 106 cells per egg on to the CAM resulted in total mortality, while the LD50 was 1 x 105. Intravenous inoculation also resulted in an LD50 of 1 x 105 cells/ egg, and the mortality at 2 x 106 cells/egg was 77%, which was greater than that described by Lydyard and Ivanyi (1975). 2. Effects of i.v. or CAM inoculation of C. albicans on the bursa of Fabricius Twelve-day old embryos were given sub-lethal inocula of C. albicans at 1 x 103 and 1 x 104 cells per egg. The results from these experiments are presented in Table I and show considerable depression of bursal follicle formation with little mortality. A similar depression has been shown after inoculation of chick embryos on the CAM with allogeneic lymphocytes (Walker et al., 1972). Previous work with candidal infections of the CAM (Cawson, 1973) was extended to examine the effect on the bursa. These results are shown in Table II for a range of sub-lethal concentrations and a number of different isolates. The depression of bursal follicle formation associated with candidal infection of the
298
W. H. WAIN AND R. A. CAWSON
TABLE I.-Mortality and Depression of Follicle Formation in Chick Embryos Following i.v. Inoculation with Candida albicans Surviving embryos Strain of C. albican8 Control 12 12 12 12 12 3 3 3153 3153 1 2 9 *P < 0-005.
Number of cells in 0- 1 ml inoculum
Inoculation
1X 1X 1x 1X 1X 1X
103 104 105 106 107 104 x 105 X 1 X 104 1 x 105 1 X 104 1 X 104 1 X 104
2
Number of surviving embryos 10 36 13 6 3 6
13th day 26 5
11
11
25 2 7
5 5 5 8 3 9 3 6 6 6
5 3 0 8 3 8 2 6 6 6
1 0 0 5 1 7 0 5 6 6
9
5
1
Mean follicular count follicles/fold
Ts.e.
mean
19 12T0-28 15.67+F0-26t 14-73T0 48t 16- 66+F1 * 12* 17-57:F1- 18NS 18 - 61 +F 1 * 17 NS 15-06 1 *07t
*P < 0 05. tP < 0.005. NS Not significant. § Inocula of 0 1 ml of a suspension of cells/ml of C. albicans were used.
1
x
104
CAM was similar to that produced by i.v. inoculation. of infection To establish whether the infecting C. albicans had a direct effect on the bursal epithelium by invasion of the bursa, a programme of investigative histology using periodic acid-Schiff stain was initiated. The results of the survey are presented in Table III and show that in no case was there any evidence of C. albicans infecting the bursa: 33% of the longitudinal sections of chick brain contained foci of infection after i.v. inoculation (Fig. 4),
3. Dissemination
19th day
12th day 28 5
TABLE II-Depression of Follicle Formation in Chick Embryos Following Inoculation of Candida albicans on to the Chorio-Allantoic Membrane Strain of C. albicans§ Control 12 3 3153
Killed
Mean follicular count follicles/fold Ts.e. mean 23 03T+0 39
16-80T0-47* 16* 12+0 50*
20
17- 12T0 83*
13-85F1-25*
15 50+0 44*
16-60+1-25* 19-03 1- 12*
19-03T0-63*
and other organs were infected to a lesser degree. These results are based on single representative sections of each organ from each embryo and must therefore be regarded as under-estimates. By contrast, inoculation on to the CAM produced no foci of infection apart from one embryo containing C. albicans in the kidney. All embryos showed evidence of C. albicans on the CAM (Fig. 5) and, though a few showed fungal penetration through the CAM, there was no histological evidence of dissemination. 4. Viability estimates of disseminated infection Because this histological survey (Table III) provided an under-estimate of the dissemination of the infection, infected embryos were homogenized for viable count estimations of infection, as described by Wain, Price and Cawson (1976). Table III shows that the primary focus of disseminated infection was the brain, and so estimations of infection were made on brains, whole embryos, and the shell, yolk and membranes without the CAM or embryo. There was no disseminated infection in the brain nor in the embryo. Though viable cells were recovered from the whole egg and from the shell, yolk and membranes, contamination from regions
DEPRESSION OF BURSAL FOLLICLE FORMATION BY CANDIDA ALBICANS
299
FIG. 1.-Equatorial section of bursa of Fabricius from control, uninfected chick embryo. H. and E. x22. FIG. 2.-Equatorial section of bursa of Fabricius from chick embryo infected with 1 x 104 Candida albicans. H. and E. x 22. FIG. 3.-Equatorial section of bursa of Fabricius from chick embryo infected via the chorio-allantoic membrane with the " cell wall extract " fraction from 5 x 108 disrupted Candida albicans. H. and E. x 22. FIG. 4. Longitudinal section of chick embryo brain showing encephalitis after i.v. administration of 1 x 104 Candida albicans. PAS x 375. FIG. 5. Transverse section of chorio-allantoic membrane from chick embryo infected with 1 x 104 Candida albicans. PAS x 75.
of the infected CAM adhering to the shell could not be excluded. Depression of bursal follicle formation is thus produced by C. albicans inoculated on to the CAM in the absence of any disseminated infection and this, together with the inability to grow any viable cells from the culture received from Ivanyi and
Lydyard, suggested that the depression might be due to some cell-free extract or toxin. Candidal cells were disrupted in an " X "-press (LKB) to yield 3 fractions, i.e. cytoplasm, cell wall extract and cell wall suspension. Viable counts on these extracts showed that the cell damage, causing loss in viability, was in excess of
300
W. H. WAIN AND R. A. CAWSON
TABLE III.-Mortality of Chick Embryos and Foci of Infection Following i.v. Inoculation with C. albicans Surviving embryos
Infection seen in:
Strain of C. albicans 12 3 1 3153 2 9 Total
12th day 13th day 19th day 16 16 9 5 5 5 6 6 5 6 6 6 6 6 6 6 6 6 45
Heart
37
45
1
Liver 3
Kidney 1
Spleen 2
-
-
1
-
-
-
-
2
Brain 7 1 1
-
-
-
1
-
-
-
-
1
-
-
-
-
1
3
2
6
5 1 15
Bursa -
-
0
TABLE IV.-Mortality and Depression of Follicle Formation in Chick Embryos Following Administration of Fractions of Disrupted Candida albicans on to the ChorioAllantoic Membrane Surviving embryos Fraction 0- 1 ml/egg Control Cytoplasm Wall Extract Control Cytoplasm Wall Extract 1/100 Extract Control Cytoplasm Wall Extract Control Extract
Viable cells No./0 1 ml 0
Equivalent cell No./0 1 ml 0
0
4 7x107 2 9x107 4-2x 107
0
0
0
2 0 0
Protein ,ug/0 1 ml
47x 107 2-9x 107
0
174 136 70 0
134 ND 53 0-53
0
4 2x 107 4 2x 105
0
0
0
ND ND ND
0
44x 108 86x 107 3-1 x 108 0
0
5. 1 x 108
0
0
110 0
0
ND
.
Inoculation _-A
Killed
12th day 13th day 19th day 4 3 1 4 2 0 5 3 0 1 5 1 10 10 8 10 7 9 10 8 9 10 10 6 10 9 7 6 6 6 4 5 4 6 6 6 7 7 7 10 10 10 10 10 10
Mean follicular count, follicles/fold
Ts.e. mean ND ND
18*31 0F 53 14-69:F103* 12* 880F 81t
14 82+0F 87*
13-52T0* 53t 19* 19F0 40
21 - 12F0- 60 17* 59F0* 40* 14- 29TF0*40t
24*66+0F44 16*40F0*39t
*P
DEPRESSION OF BURSAL FOLLICLE FORMATION BY CANDIDA ALBICANS INFECTIONS W. H. WAIN* AND R. A. CAWSONt From the Department of Oral Medicine and Pathology, Guy'8 Hospital Medical School, London Bridge, SEI 9RT Received for publication December 3, 1975
Summary.-Sub-lethal i.v. inoculations of Candida albicans into 12-day-old chick embryos have been shown to cause a depression of follicle formation in the bursa of Fabricius. This depression occurred in the absence of any infection in the bursa, although candidal encephalitis was a frequent manifestation of the disseminated infection. A similar depression of bursal follicle formation was caused by the administration of viable C. albicans on to the embryonic chorio-allantoic membrane, in the absence of any disseminated infection. Non-viable extracts of disrupted C. albicans administered on to the chorioallantoic membrane also caused a depression of bursal follicle formation. This immunodeficiency produced by an endotoxin-like principle at a susceptible stage in the development of the chick has been considered in terms of human immunological development and the neonatal occurrence of C. albicans.
HYPOPLASIA of the bursal follicles in the embryonic chick caused by i.v. inoculations of Candida albicans has recently been reported by Lydyard and Ivanyi (1975). Pathological changes in the thymus of mice chronically infected with C. albicans have previously been described (Mankowski, 1968). Administration of allogeneic lymphocytes on to the chorio-allantoic membrane (CAM) of chick embryos has also been shown to cause depression of bursal follicle formation (Walker, Schoefl and Lafferty, 1972). The chick CAM has also been used as a host tissue for model infections by C. albi cans (Cawson, 1973). This plaqueforming response by the CAM to C. albicans (Partridge, Athar and Winner, 1971) is however variable, but has been shown to be due to clumping of the inoculum (Wain, Price and Cawson, 1974). The degree of depression of bursal follicle *
formation caused by C. albicans has therefore been investigated both as an index of pathogenicity and for its possible role in immunodeficiency. The depression of bursal follicle formation by sub-lethal inocula of viable C. albicans and by non-viable extracts of disrupted fungal cells has been examined. Foci of infection resulting from i.v. dissemination of viable C. albicans have been located and similar locations have been examined after inoculation on to the CAM. Dissemination from CAM inoculations has been tested for by viability assays from those infected embryos. MATERIALS AND METHODS
1. Inoculation of embryos.-Fertile eggs from White Leghorn x Rhode Island fowls (Appleby Farm, Ashford, Kent) were maintained in a humidified egg incubator. The viable 12-day embryos were injected with 0-1 ml of inoculum
Present address: Homograft Department, National Heart Hospital, Westmoreland Street, London W.1.
t To whom reprint requests should be addressed.
DEPRESSION OF BURSAL FOLLICLE FORMATION BY CANDIDA ALBICANS either i.v. (as described by Lydyard and Ivanyi, 1975), or on to the CAM as described by Partridge (1968). Yeast cell suspensions were diluted in day-old chick red blood cell diluent (Gibb Biocult) and cell fractions were resuspended in Yeast Nitrogen base (YNB) without amino acids and ammonium sulphate (Difco). Viability of the inoculated eggs was checked daily by candling and the surviving embryos were killed on Day 19. 2. Culture and disruption of Candida albicans.-Clinical isolates of Candida albicans were maintained in Sabouraud liquid medium and periodically passaged through viable chick embryos by inoculation on to the CAM at a concentration of 1 x 106 cells/ml. Early stationary phase cells were used for inocula preparation as described in Wain, Price and Cawson (1975) and dilutions prepared in day-old chick red blood cell diluent as required. Continuous culture of C. albicans in 5% glucose yeast nitrogen base (Difco) (YNB) at pH 5*4, 370, flow rate 1-25 ml/min (dilution rate 0 06), aeration at 800 ml/min air, 400 ml/min C02, oxygen concentration 3-5 ppm was carried out in a Biotec Polyferm FE007 (LKB) for 24 h. The cells in the fermentor were harvested, resuspended to 5 ml in 5% glucose YNB and passed 5 times through a small " X "-press (LKB) at - 250. The frozen, disrupted suspension was thawed, centrifuged for 10 min at 3000 g at 00 and the supernatant removed as " cytoplasm ". The pellet was resuspended to the original volume in YNB, without amino acids and ammonium sulphate, and centrifuged again at 3000 g for 10 min at 00. The supernatant was removed as " cell wall extract " and the pellet resuspended to the original volume in YNB without amino acids and ammonium sulphate to provide a " cell wall suspension ". 0-1 ml of each fraction was plated out in duplicate for viability assays. Further 0-1 ml aliquots of these fractions were used as the inocula for the 12-day-old chick embryos. 3. Histology.-At the 19th day, after 7 days of infection, the viable embryos were killed and the CAM and the pelvic region containing the lower gut and bursa of Fabricius were removed and fixed in a buffered 10% formol saline. After 24 h fixation the bursa was dissected from the cloaca and transected equatorially. The 2 half-bursas were processed and prepared in the usual way for paraffin sections. The examinations for disseminated infections were carried out on brain, liver, heart, kidney and spleen, fixed and processed in an identical manner. Transverse 5-,um sections of bursa, spleen, kidney and liver, and longitudinal 5-,um sections of brain and heart were cut to provide representative sections of each organ. The sections were stained with haematoxylin and
297
eosin and with periodic acid-Schiff for detection of the fungi. The sections of bursa were examined for follicular development and the number of follicles in 5 representative folds of each half of each bursa were counted and expressed as a follicle count per fold. Since the size distribution of the follicles assayed by Lydyard and Ivanyi (1975) provided so little more information than the mean total follicular count per fold, this refinement of their assay was not adopted.
4. Retention of viability by the inoculum.The infected CAM was flooded with buffered 10% formol saline for 60 min in order to kill the inoculum on the CAM, which was then washed twice with day-old chick red cell diluent to remove the fixative. The CAM was removed and the remainder of the egg including the shell was homogenized, diluted and plated out on Sabouraud maltose agar. Alternatively the embryo and the brain were homogenized separately and assayed for viable C. albicans by similar plating procedures. RESULTS
1. Mortality of embryos Inoculation of viable C. albicans at 1 x 106 cells per egg on to the CAM resulted in total mortality, while the LD50 was 1 x 105. Intravenous inoculation also resulted in an LD50 of 1 x 105 cells/ egg, and the mortality at 2 x 106 cells/egg was 77%, which was greater than that described by Lydyard and Ivanyi (1975). 2. Effects of i.v. or CAM inoculation of C. albicans on the bursa of Fabricius Twelve-day old embryos were given sub-lethal inocula of C. albicans at 1 x 103 and 1 x 104 cells per egg. The results from these experiments are presented in Table I and show considerable depression of bursal follicle formation with little mortality. A similar depression has been shown after inoculation of chick embryos on the CAM with allogeneic lymphocytes (Walker et al., 1972). Previous work with candidal infections of the CAM (Cawson, 1973) was extended to examine the effect on the bursa. These results are shown in Table II for a range of sub-lethal concentrations and a number of different isolates. The depression of bursal follicle formation associated with candidal infection of the
298
W. H. WAIN AND R. A. CAWSON
TABLE I.-Mortality and Depression of Follicle Formation in Chick Embryos Following i.v. Inoculation with Candida albicans Surviving embryos Strain of C. albican8 Control 12 12 12 12 12 3 3 3153 3153 1 2 9 *P < 0-005.
Number of cells in 0- 1 ml inoculum
Inoculation
1X 1X 1x 1X 1X 1X
103 104 105 106 107 104 x 105 X 1 X 104 1 x 105 1 X 104 1 X 104 1 X 104
2
Number of surviving embryos 10 36 13 6 3 6
13th day 26 5
11
11
25 2 7
5 5 5 8 3 9 3 6 6 6
5 3 0 8 3 8 2 6 6 6
1 0 0 5 1 7 0 5 6 6
9
5
1
Mean follicular count follicles/fold
Ts.e.
mean
19 12T0-28 15.67+F0-26t 14-73T0 48t 16- 66+F1 * 12* 17-57:F1- 18NS 18 - 61 +F 1 * 17 NS 15-06 1 *07t
*P < 0 05. tP < 0.005. NS Not significant. § Inocula of 0 1 ml of a suspension of cells/ml of C. albicans were used.
1
x
104
CAM was similar to that produced by i.v. inoculation. of infection To establish whether the infecting C. albicans had a direct effect on the bursal epithelium by invasion of the bursa, a programme of investigative histology using periodic acid-Schiff stain was initiated. The results of the survey are presented in Table III and show that in no case was there any evidence of C. albicans infecting the bursa: 33% of the longitudinal sections of chick brain contained foci of infection after i.v. inoculation (Fig. 4),
3. Dissemination
19th day
12th day 28 5
TABLE II-Depression of Follicle Formation in Chick Embryos Following Inoculation of Candida albicans on to the Chorio-Allantoic Membrane Strain of C. albicans§ Control 12 3 3153
Killed
Mean follicular count follicles/fold Ts.e. mean 23 03T+0 39
16-80T0-47* 16* 12+0 50*
20
17- 12T0 83*
13-85F1-25*
15 50+0 44*
16-60+1-25* 19-03 1- 12*
19-03T0-63*
and other organs were infected to a lesser degree. These results are based on single representative sections of each organ from each embryo and must therefore be regarded as under-estimates. By contrast, inoculation on to the CAM produced no foci of infection apart from one embryo containing C. albicans in the kidney. All embryos showed evidence of C. albicans on the CAM (Fig. 5) and, though a few showed fungal penetration through the CAM, there was no histological evidence of dissemination. 4. Viability estimates of disseminated infection Because this histological survey (Table III) provided an under-estimate of the dissemination of the infection, infected embryos were homogenized for viable count estimations of infection, as described by Wain, Price and Cawson (1976). Table III shows that the primary focus of disseminated infection was the brain, and so estimations of infection were made on brains, whole embryos, and the shell, yolk and membranes without the CAM or embryo. There was no disseminated infection in the brain nor in the embryo. Though viable cells were recovered from the whole egg and from the shell, yolk and membranes, contamination from regions
DEPRESSION OF BURSAL FOLLICLE FORMATION BY CANDIDA ALBICANS
299
FIG. 1.-Equatorial section of bursa of Fabricius from control, uninfected chick embryo. H. and E. x22. FIG. 2.-Equatorial section of bursa of Fabricius from chick embryo infected with 1 x 104 Candida albicans. H. and E. x 22. FIG. 3.-Equatorial section of bursa of Fabricius from chick embryo infected via the chorio-allantoic membrane with the " cell wall extract " fraction from 5 x 108 disrupted Candida albicans. H. and E. x 22. FIG. 4. Longitudinal section of chick embryo brain showing encephalitis after i.v. administration of 1 x 104 Candida albicans. PAS x 375. FIG. 5. Transverse section of chorio-allantoic membrane from chick embryo infected with 1 x 104 Candida albicans. PAS x 75.
of the infected CAM adhering to the shell could not be excluded. Depression of bursal follicle formation is thus produced by C. albicans inoculated on to the CAM in the absence of any disseminated infection and this, together with the inability to grow any viable cells from the culture received from Ivanyi and
Lydyard, suggested that the depression might be due to some cell-free extract or toxin. Candidal cells were disrupted in an " X "-press (LKB) to yield 3 fractions, i.e. cytoplasm, cell wall extract and cell wall suspension. Viable counts on these extracts showed that the cell damage, causing loss in viability, was in excess of
300
W. H. WAIN AND R. A. CAWSON
TABLE III.-Mortality of Chick Embryos and Foci of Infection Following i.v. Inoculation with C. albicans Surviving embryos
Infection seen in:
Strain of C. albicans 12 3 1 3153 2 9 Total
12th day 13th day 19th day 16 16 9 5 5 5 6 6 5 6 6 6 6 6 6 6 6 6 45
Heart
37
45
1
Liver 3
Kidney 1
Spleen 2
-
-
1
-
-
-
-
2
Brain 7 1 1
-
-
-
1
-
-
-
-
1
-
-
-
-
1
3
2
6
5 1 15
Bursa -
-
0
TABLE IV.-Mortality and Depression of Follicle Formation in Chick Embryos Following Administration of Fractions of Disrupted Candida albicans on to the ChorioAllantoic Membrane Surviving embryos Fraction 0- 1 ml/egg Control Cytoplasm Wall Extract Control Cytoplasm Wall Extract 1/100 Extract Control Cytoplasm Wall Extract Control Extract
Viable cells No./0 1 ml 0
Equivalent cell No./0 1 ml 0
0
4 7x107 2 9x107 4-2x 107
0
0
0
2 0 0
Protein ,ug/0 1 ml
47x 107 2-9x 107
0
174 136 70 0
134 ND 53 0-53
0
4 2x 107 4 2x 105
0
0
0
ND ND ND
0
44x 108 86x 107 3-1 x 108 0
0
5. 1 x 108
0
0
110 0
0
ND
.
Inoculation _-A
Killed
12th day 13th day 19th day 4 3 1 4 2 0 5 3 0 1 5 1 10 10 8 10 7 9 10 8 9 10 10 6 10 9 7 6 6 6 4 5 4 6 6 6 7 7 7 10 10 10 10 10 10
Mean follicular count, follicles/fold
Ts.e. mean ND ND
18*31 0F 53 14-69:F103* 12* 880F 81t
14 82+0F 87*
13-52T0* 53t 19* 19F0 40
21 - 12F0- 60 17* 59F0* 40* 14- 29TF0*40t
24*66+0F44 16*40F0*39t
*P
