INFECTION
AND
IMMUNITY, May 1976,
p.
1301-1306
Vol. 13, No. 5 Printed in U.SA.
Copyright C 1976 American Society for Microbiology
Enhancement Activity of Anti-Mycobacterial Sera in Experimental Mycobacterium bovis (BCG) Infection in Mice A. FORGET,* J. C. BENOIT, R. TURCOTTE, AND N. GUSEW-CHARTRAND Department of Microbiology and Immunology, Faculty of Medicine, University of Montreal, Montreal, Quebec, Canada,* and Armand Frappier Institute, Laval, Quebec, Canada'
Received for publication 11 July 1975
The passive transfer of rabbit anti-mycobacterial immunoglobulins was directed against either living or soluble extracts of Mycobacterium tuberculosis strain H37Rv, which promotes the multiplication of the BCG strain of M. tuberculosis in the spleen of mice infected with low doses of this latter strain. This enhancing effect was reduced significantly when antisera were absorbed with living BCG. Moreover, such treatment led to the removal of all hemagglutinating antibodies when antisera were tested against either BCG or H37Rv soluble extracts. Therefore, it is most probable that the enhancing effect is related to the presence of mycobacterial antibodies. Kaliss (14) first introduced the term immunological enhancement to describe the phenomenon in which humoral antibodies were found to facilitate the growth of homotransplanted tumors, whereas tumor rejection normally occurred in the absence of these antibodies. This phenomenon has since been observed in allografts of normal tissue, in graft-versus-host reactions, and in autoimmune diseases (27). Voisin (27) has postulated that nontissue antigens such as bacteria could also be affected by the enhancement phenomenon, though in this particular case only a depression of the immune reaction has been described. Thus, the synthesis of antibodies can be inhibited or delayed when an antigen with its corresponding antibody is injected into animals (18, 26). Forget et al. (8) have observed that the passive transfer of homologous anti-staphylococcal sera favored the onset of the experimental staphylococcal synovitis in chickens. This observation would indicate that an immune enhancing phenomenon is involved in this chronic type of bacterial infection. Moreover, they have shown that the enhancement activity of the antistaphylococcal sera was associated with a relatively high level of immune adherence antibodies, whereas it seemed not to be related with their agglutinin titers (9). To demonstrate that an enhancement phenomenon might be involved during the development of a bacterial infection, one has to establish that specific humoral antibodies protect, in some way, microorganisms against the defense reactions of the host.
In the present report, the enhancement activity of anti-mycobacterial sera was investigated in an experimental BCG infection in mice. The results strongly suggest that rabbit anti-H37Rv sera facilitate the multiplication of BCG in the spleens of mice. MATERIALS AND METHODS Animals. Female CF-1 mice, weighing 12 to 14 g, were distributed at random in glass cages, five per cage, and were fed Purina Laboratory Chow with water ad libitum. Bacterial strains. Mycobacterium tuberculosis var. hominis strain H37Rv was originally received from the Trudeau Institute Inc., Saranac Lake, N.Y. This strain was grown as a pellicle on Sauton medium and was 14 days old when harvested. Suspensions containing 10 mg (moist weight) per ml in Sauton (1:4) were prepared and kept frozen at -55 C until the immunization time. Soluble extracts from H37Rv were prepared according to a method described previously (25). Mycobacterium tuberculosis var. bovis, strain BCG, kindly provided by the BCG Laboratory of the Armand Frappier Institute, Laval, Quebec, Canada, was used for infecting mice. For the first experiment, BCG was grown as a pellicle on Sauton medium at 37 C for 7 days. Then the bacillary mass was ground with stainless-steel balls. The BCG suspension thus obtained was diluted (1 mg/ml) in saline containing 0.5% of calf serum and filtered through a membrane filter (pore size, 3 ,um) to eliminate bacterial clumps and to infect mice with a dispersed suspension ofbacilli (2). For the second experiment, lyophilized BCG vaccine was twice subcultured in Dubos Tween liquid medium at 7-day intervals, and a dispersed suspension of bacilli was prepared as above. The viable cell counts in both preparations were determined by plating serial dilutions of dispersed bacterial suspensions in triplicate onto Dubos solid medium
I Formerly the Institute of Microbiology and Hygiene annexed to the Faculty of Medicine, University of Montreal, Montreal, Quebec, Canada. 1301
1302
INFECT. IMMUN.
FORGET ET AL.
without Tween followed by an incubation period of 18 days at 37 C. Preparation of immune sera to M. tuberculosis H37Rv. Two types of antisera directed against two different preparations of M. tuberculosis strain H37Rv were produced in New Zealand rabbits. For the first experiment, three rabbits were injected intravenously, twice weekly for 5 weeks, with 1.5 mg of living bacilli mixed with 1% alum (13). For the second experiment, three rabbits were injected intravenously, twice weekly for 6 weeks, with 1.3 mg of soluble extracts of H37Rv mixed with alum as above. In both schedules of immunization, rabbits were bled 1 week later and the corresponding sera were pooled. These sera will be thereafter referred to as antiserum 1 and antiserum 2, respectively. Pooled sera from three noninjected rabbits were used as normal serum. All sera were first heat inactivated for 30 min at 56 C, and the gamma globulin fractions were precipitated with ammonium sulfate. After precipitation, the protein concentration, as measured by biuret reaction, was adjusted to 3.3%, which may be considered as equivalent to a twofold concentration serum as far as immunoglobulins are concerned. Bis-diazotized-benzidine hemagglutination test. The bis-diazotized-benzidine hemagglutination test as adopted previously by one of us (24) was used for the detection of mycobacterial hemagglutinating antibodies in antisera 1 and 2. For the sensitization of rabbit erythrocytes, 7.5 mg of the soluble H37Rv and BCG extracts was first diluted in 3 ml of phosphate buffer. To these solutions were added 0.1 ml of a 50% suspension of washed erythrocytes and 0.5 ml of a bis-diazotized-benzidine-phosphate buffer solution (10). After an incubation of 15 min at room temperature, the mixture was centrifuged, and the cells were washed and suspended in 2.5 ml of 1% rabbit normal serum. Finally, 0.05 ml of sensitized erythrocytes were suspended in 0.5 ml of a serial twofold dilutions of aliquots of the antisera 1 and 2. Absorption of antisera. One volume of the antisera 1 and 2 or of the normal serum was absorbed with one-half volume of washed, packed BCG obtained from a 14-day-old culture on Sauton medium. These absorptions were carried out at room temperature for 1 h and at 4 C for 16 h. Bacilli were removed first by centrifugation and then by filtration on membrane filter (0.45 ,um). Infection of mice with BCG. The present experiments were performed with low doses of BCG because with large doses (i.e., 105 or more) the organisms grew little if at all in the spleens of mice (4, 5, 17, 21, 22), whereas with low doses (i.e., 101 to 104) the rate of multiplication was dose dependent (1, 20). In the research of Benoit and Panisset (1) in which they used doses of about 500 to 25,000 bacilli, the BCG reached the highest level of multiplication after 4 weeks. Mice were infected according to a technique currently in use by one of us (19). Under nembutal anesthesia (1 mg per 10 g of body weight intraperitoneally) the mice were infected intravenously into the right jugular vein with appropriate small doses ofthe BCG suspension (0.25-ml volume).
Treatment of BCG-infected mice with antisera. In the two experiments performed, 0.1 ml of antisera nonabsorbed or absorbed with BCG and normal serum was injected intraperitoneally to the mice 6 h before and 24 h after the intravenous inoculation of BCG suspensions. The mice were sacrificed 4 weeks later. In the first experiment, the spleens were frozen and stored at -70 C before the viable cell counts were determined. However such treatment was found to reduce the number of colony-forming units (CFU) recovered from the spleens by comparison with those recovered from unfrozen spleens. Thus, in the second experiment all the viable cell counts were determined with unfrozen spleens. Determination of spleen CFU. Spleens from sacrificed mice were removed aseptically and ground with 90-mesh alundum in 5 ml of saline containing 2% of calf serum in a small sterile mortar. The suspension obtained was further diluted 1:10 with saline containing 0.2% of calf serum, and a volume of 0.1 ml was plated in triplicate onto Dubos solid medium without Tween. The petri dishes (50 by 12 mm) were read 18 days after incubation at 37 C, and the total spleen CFU were calculated.
RESULTS Hemagglutinating titers of anti-mycobacterial sera. The antiserum 1 obtained from rabbits immunized with the live H37Rv had a hemagglutinating titer of 1:10,240 when tested with antigenic extracts isolated either from H37Rv or BCG, whereas the titer of the antiserum 2, prepared from rabbits immunized with a soluble extract of H37Rv, was 1:1,280 when tested with the same antigenic preparations (Table 1). Absorptions of aliquots of both antisera with living BCG completely removed the hemagglutinating antibodies directed against either antigenic extracts of BCG or H37Rv. No detectable TABLE 1. Effect of absorption with living BCG on the hemagglutinating titers of H37Rv antisera tested against H37Rv and BCG extracts Hemagglutinating ti-
terd against extract of:
Seruma
Treatment
Antiserum lb
Nonabsorbed Absorbed
H37Rv 10,280 0
BCG 10,280 0
Antiserum 2c
Nonabsorbed Absorbed
1,280 0
1,280 0
Normal serum
0 Nonabsorbed 0 0 Absorbed 0 Sera referred to the gamma globulin fraction isolated by ammonium sulfate precipitation. bAntiserum to living H37Rv bacilli. c Antiserum to H37Rv soluble extracts. d Reciprocal of last dilution showing a positive hemagglutinating pattern.
EXPERIMENTAL BCG INFECTION IN MICE
VOL. 13, 1976
hemagglutinating antibody was found in normal serum. BCG infection of mice treated with antimycobacterial sera. In the first experiment, four groups of mice were treated, respectively, with nondiluted antiserum 1, antiserum 1 diluted 1:4, normal serum, and saline. Each group was further divided in two subgroups which were infected with 7,300 and 14,600 living units of BCG (Table 2). In the group treated with nondiluted antiserum and infected with 14,600 bacilli, 5 out of 11 mice had more than 150 CFU (with a mean number of 230 CFU) per spleen, whereas the 6 others had 0 or less than 150 CFU. In the group of mice treated with the diluted antiserum and infected with either 7,300 or 14,600 mycobacteria, more than 150 CFU per spleen were recovered in 5 of 13 (mean 480) and in 4 of 10 (mean 375) animals, respectively. In the group treated with normal serum no CFU were detectable in the spleens of 26 mice, and only 3 of 26 mice treated with saline had a mean number of 150 CFU per spleen. In the second experiment (Table 3), the mice were treated with antiserum 2 previously absorbed or not with BCG and infected with two different doses of BCG. In the groups of mice infected with 14,000 bacilli and treated with nondiluted and nonabsorbed antiserum, the number of CFU recovered from their spleen was significantly higher than the number of CFU recovery from the spleen of mice treated with the corresponding absorbed antiserum.
1303
Essentially the same results were obtained when the mice, infected with the same dose, were treated with the same antiserum at a 1:8 dilution. In these groups, the ratio between the total recovered spleen CFU and those injected was more than 1 in mice treated with nonabsorbed antiserum, whereas it was less than 1 in those treated with absorbed antiserum. In the groups of mice infected with twice that dose (i.e., 28,000 bacilli), no significant difference in the CFU recovered from the spleens was observed between mice treated with nonabsorbed and absorbed, undiluted antiserum; however, at a 1:8 dilution there was some differences, but this was not significant at a 0.05 level. DISCUSSION The data obtained by different investigators who were using in their experiments the intravenous BCG infection in mice measured by the ratio of CFU recovered from the spleens over CFU injected at different intervals could be divided into three groups according to the range of infective doses. First, with extremely small infective doses (about 101 to 103), the data cited by Pierce et al. (20) showed that the maximum of bacilli recovered from the spleen was proportional to the size of the infective dose; second, Benoit and Panisset (1), using intermediate infective doses (about 500 to 25,000), observed that the smaller the dose the higher the rate of multiplication of the bacilli in the spleens at 4 weeks; and third, using large infective doses
TABLE 2. Effect of treatment with H37Rv antiserum 1 on the recovery of BCG in the spleens of BCG-infected mice No. of mice 4 weeks after infection with: No. of mice
BCG dose injected Passive treatment (2 doses of 0.1 ml given intravenously intraperitoneally)
13 11
7,300 14,600
Antiserumb (nondiluted) Antiserumb (nondiluted)
13 10
7,300 14,600
Antiserum 1:4c Antiserum 1:4e
13 13
7,300 14,600
Normal serum (nondiluted) Normal serum (nondiluted)
More than 150 0 or less than CFU (mean 150 CFUa no. of all groups, 300) 1 12 6 5
8 6
5 4
13 13
0 0
9 Saline 2 7,300 1 14 Saline 14,600 The bacterial culture count technique does not allow a count of less than 150 CFU per spleen. "Immunoglobulin fraction of a pool of antisera prepared from rabbit immunized with living M. tuberculosis. See text for further details. ' Diluted 1:4 in saline. 11 15
1304
INFECT. IMMUN.
FORGET ET AL.
TABLE 3. Effect of treatment with absorbed and nonabsorbed H37Rv antiserum 2 on the recovery of BCG in the spleens of BCG-infected mice BCG CFU recovered from the spleens 4 BCG dose in- Passive treatment (2 doses of Total recovered weeks after infection No. jected intra- 0.1 ml given intraperitone(spleen CFU/inof venously per ally 6 h before and 24 h after jected CFU) Arithmetic means + mouse mice bacterial P values" SEa infection) (CFU)
5 6 7 8
14,000 14,000 28,000 28,000
Nondiluted antiserumn Nonabsorbed Absorbed" Nonabsorbed Absorbed
18,040.0 8,716.7 26,207.1 24,412.5
± ± ± ±
1,570.6 1,786.2 8,520.0 8,145.2
p
AND
IMMUNITY, May 1976,
p.
1301-1306
Vol. 13, No. 5 Printed in U.SA.
Copyright C 1976 American Society for Microbiology
Enhancement Activity of Anti-Mycobacterial Sera in Experimental Mycobacterium bovis (BCG) Infection in Mice A. FORGET,* J. C. BENOIT, R. TURCOTTE, AND N. GUSEW-CHARTRAND Department of Microbiology and Immunology, Faculty of Medicine, University of Montreal, Montreal, Quebec, Canada,* and Armand Frappier Institute, Laval, Quebec, Canada'
Received for publication 11 July 1975
The passive transfer of rabbit anti-mycobacterial immunoglobulins was directed against either living or soluble extracts of Mycobacterium tuberculosis strain H37Rv, which promotes the multiplication of the BCG strain of M. tuberculosis in the spleen of mice infected with low doses of this latter strain. This enhancing effect was reduced significantly when antisera were absorbed with living BCG. Moreover, such treatment led to the removal of all hemagglutinating antibodies when antisera were tested against either BCG or H37Rv soluble extracts. Therefore, it is most probable that the enhancing effect is related to the presence of mycobacterial antibodies. Kaliss (14) first introduced the term immunological enhancement to describe the phenomenon in which humoral antibodies were found to facilitate the growth of homotransplanted tumors, whereas tumor rejection normally occurred in the absence of these antibodies. This phenomenon has since been observed in allografts of normal tissue, in graft-versus-host reactions, and in autoimmune diseases (27). Voisin (27) has postulated that nontissue antigens such as bacteria could also be affected by the enhancement phenomenon, though in this particular case only a depression of the immune reaction has been described. Thus, the synthesis of antibodies can be inhibited or delayed when an antigen with its corresponding antibody is injected into animals (18, 26). Forget et al. (8) have observed that the passive transfer of homologous anti-staphylococcal sera favored the onset of the experimental staphylococcal synovitis in chickens. This observation would indicate that an immune enhancing phenomenon is involved in this chronic type of bacterial infection. Moreover, they have shown that the enhancement activity of the antistaphylococcal sera was associated with a relatively high level of immune adherence antibodies, whereas it seemed not to be related with their agglutinin titers (9). To demonstrate that an enhancement phenomenon might be involved during the development of a bacterial infection, one has to establish that specific humoral antibodies protect, in some way, microorganisms against the defense reactions of the host.
In the present report, the enhancement activity of anti-mycobacterial sera was investigated in an experimental BCG infection in mice. The results strongly suggest that rabbit anti-H37Rv sera facilitate the multiplication of BCG in the spleens of mice. MATERIALS AND METHODS Animals. Female CF-1 mice, weighing 12 to 14 g, were distributed at random in glass cages, five per cage, and were fed Purina Laboratory Chow with water ad libitum. Bacterial strains. Mycobacterium tuberculosis var. hominis strain H37Rv was originally received from the Trudeau Institute Inc., Saranac Lake, N.Y. This strain was grown as a pellicle on Sauton medium and was 14 days old when harvested. Suspensions containing 10 mg (moist weight) per ml in Sauton (1:4) were prepared and kept frozen at -55 C until the immunization time. Soluble extracts from H37Rv were prepared according to a method described previously (25). Mycobacterium tuberculosis var. bovis, strain BCG, kindly provided by the BCG Laboratory of the Armand Frappier Institute, Laval, Quebec, Canada, was used for infecting mice. For the first experiment, BCG was grown as a pellicle on Sauton medium at 37 C for 7 days. Then the bacillary mass was ground with stainless-steel balls. The BCG suspension thus obtained was diluted (1 mg/ml) in saline containing 0.5% of calf serum and filtered through a membrane filter (pore size, 3 ,um) to eliminate bacterial clumps and to infect mice with a dispersed suspension ofbacilli (2). For the second experiment, lyophilized BCG vaccine was twice subcultured in Dubos Tween liquid medium at 7-day intervals, and a dispersed suspension of bacilli was prepared as above. The viable cell counts in both preparations were determined by plating serial dilutions of dispersed bacterial suspensions in triplicate onto Dubos solid medium
I Formerly the Institute of Microbiology and Hygiene annexed to the Faculty of Medicine, University of Montreal, Montreal, Quebec, Canada. 1301
1302
INFECT. IMMUN.
FORGET ET AL.
without Tween followed by an incubation period of 18 days at 37 C. Preparation of immune sera to M. tuberculosis H37Rv. Two types of antisera directed against two different preparations of M. tuberculosis strain H37Rv were produced in New Zealand rabbits. For the first experiment, three rabbits were injected intravenously, twice weekly for 5 weeks, with 1.5 mg of living bacilli mixed with 1% alum (13). For the second experiment, three rabbits were injected intravenously, twice weekly for 6 weeks, with 1.3 mg of soluble extracts of H37Rv mixed with alum as above. In both schedules of immunization, rabbits were bled 1 week later and the corresponding sera were pooled. These sera will be thereafter referred to as antiserum 1 and antiserum 2, respectively. Pooled sera from three noninjected rabbits were used as normal serum. All sera were first heat inactivated for 30 min at 56 C, and the gamma globulin fractions were precipitated with ammonium sulfate. After precipitation, the protein concentration, as measured by biuret reaction, was adjusted to 3.3%, which may be considered as equivalent to a twofold concentration serum as far as immunoglobulins are concerned. Bis-diazotized-benzidine hemagglutination test. The bis-diazotized-benzidine hemagglutination test as adopted previously by one of us (24) was used for the detection of mycobacterial hemagglutinating antibodies in antisera 1 and 2. For the sensitization of rabbit erythrocytes, 7.5 mg of the soluble H37Rv and BCG extracts was first diluted in 3 ml of phosphate buffer. To these solutions were added 0.1 ml of a 50% suspension of washed erythrocytes and 0.5 ml of a bis-diazotized-benzidine-phosphate buffer solution (10). After an incubation of 15 min at room temperature, the mixture was centrifuged, and the cells were washed and suspended in 2.5 ml of 1% rabbit normal serum. Finally, 0.05 ml of sensitized erythrocytes were suspended in 0.5 ml of a serial twofold dilutions of aliquots of the antisera 1 and 2. Absorption of antisera. One volume of the antisera 1 and 2 or of the normal serum was absorbed with one-half volume of washed, packed BCG obtained from a 14-day-old culture on Sauton medium. These absorptions were carried out at room temperature for 1 h and at 4 C for 16 h. Bacilli were removed first by centrifugation and then by filtration on membrane filter (0.45 ,um). Infection of mice with BCG. The present experiments were performed with low doses of BCG because with large doses (i.e., 105 or more) the organisms grew little if at all in the spleens of mice (4, 5, 17, 21, 22), whereas with low doses (i.e., 101 to 104) the rate of multiplication was dose dependent (1, 20). In the research of Benoit and Panisset (1) in which they used doses of about 500 to 25,000 bacilli, the BCG reached the highest level of multiplication after 4 weeks. Mice were infected according to a technique currently in use by one of us (19). Under nembutal anesthesia (1 mg per 10 g of body weight intraperitoneally) the mice were infected intravenously into the right jugular vein with appropriate small doses ofthe BCG suspension (0.25-ml volume).
Treatment of BCG-infected mice with antisera. In the two experiments performed, 0.1 ml of antisera nonabsorbed or absorbed with BCG and normal serum was injected intraperitoneally to the mice 6 h before and 24 h after the intravenous inoculation of BCG suspensions. The mice were sacrificed 4 weeks later. In the first experiment, the spleens were frozen and stored at -70 C before the viable cell counts were determined. However such treatment was found to reduce the number of colony-forming units (CFU) recovered from the spleens by comparison with those recovered from unfrozen spleens. Thus, in the second experiment all the viable cell counts were determined with unfrozen spleens. Determination of spleen CFU. Spleens from sacrificed mice were removed aseptically and ground with 90-mesh alundum in 5 ml of saline containing 2% of calf serum in a small sterile mortar. The suspension obtained was further diluted 1:10 with saline containing 0.2% of calf serum, and a volume of 0.1 ml was plated in triplicate onto Dubos solid medium without Tween. The petri dishes (50 by 12 mm) were read 18 days after incubation at 37 C, and the total spleen CFU were calculated.
RESULTS Hemagglutinating titers of anti-mycobacterial sera. The antiserum 1 obtained from rabbits immunized with the live H37Rv had a hemagglutinating titer of 1:10,240 when tested with antigenic extracts isolated either from H37Rv or BCG, whereas the titer of the antiserum 2, prepared from rabbits immunized with a soluble extract of H37Rv, was 1:1,280 when tested with the same antigenic preparations (Table 1). Absorptions of aliquots of both antisera with living BCG completely removed the hemagglutinating antibodies directed against either antigenic extracts of BCG or H37Rv. No detectable TABLE 1. Effect of absorption with living BCG on the hemagglutinating titers of H37Rv antisera tested against H37Rv and BCG extracts Hemagglutinating ti-
terd against extract of:
Seruma
Treatment
Antiserum lb
Nonabsorbed Absorbed
H37Rv 10,280 0
BCG 10,280 0
Antiserum 2c
Nonabsorbed Absorbed
1,280 0
1,280 0
Normal serum
0 Nonabsorbed 0 0 Absorbed 0 Sera referred to the gamma globulin fraction isolated by ammonium sulfate precipitation. bAntiserum to living H37Rv bacilli. c Antiserum to H37Rv soluble extracts. d Reciprocal of last dilution showing a positive hemagglutinating pattern.
EXPERIMENTAL BCG INFECTION IN MICE
VOL. 13, 1976
hemagglutinating antibody was found in normal serum. BCG infection of mice treated with antimycobacterial sera. In the first experiment, four groups of mice were treated, respectively, with nondiluted antiserum 1, antiserum 1 diluted 1:4, normal serum, and saline. Each group was further divided in two subgroups which were infected with 7,300 and 14,600 living units of BCG (Table 2). In the group treated with nondiluted antiserum and infected with 14,600 bacilli, 5 out of 11 mice had more than 150 CFU (with a mean number of 230 CFU) per spleen, whereas the 6 others had 0 or less than 150 CFU. In the group of mice treated with the diluted antiserum and infected with either 7,300 or 14,600 mycobacteria, more than 150 CFU per spleen were recovered in 5 of 13 (mean 480) and in 4 of 10 (mean 375) animals, respectively. In the group treated with normal serum no CFU were detectable in the spleens of 26 mice, and only 3 of 26 mice treated with saline had a mean number of 150 CFU per spleen. In the second experiment (Table 3), the mice were treated with antiserum 2 previously absorbed or not with BCG and infected with two different doses of BCG. In the groups of mice infected with 14,000 bacilli and treated with nondiluted and nonabsorbed antiserum, the number of CFU recovered from their spleen was significantly higher than the number of CFU recovery from the spleen of mice treated with the corresponding absorbed antiserum.
1303
Essentially the same results were obtained when the mice, infected with the same dose, were treated with the same antiserum at a 1:8 dilution. In these groups, the ratio between the total recovered spleen CFU and those injected was more than 1 in mice treated with nonabsorbed antiserum, whereas it was less than 1 in those treated with absorbed antiserum. In the groups of mice infected with twice that dose (i.e., 28,000 bacilli), no significant difference in the CFU recovered from the spleens was observed between mice treated with nonabsorbed and absorbed, undiluted antiserum; however, at a 1:8 dilution there was some differences, but this was not significant at a 0.05 level. DISCUSSION The data obtained by different investigators who were using in their experiments the intravenous BCG infection in mice measured by the ratio of CFU recovered from the spleens over CFU injected at different intervals could be divided into three groups according to the range of infective doses. First, with extremely small infective doses (about 101 to 103), the data cited by Pierce et al. (20) showed that the maximum of bacilli recovered from the spleen was proportional to the size of the infective dose; second, Benoit and Panisset (1), using intermediate infective doses (about 500 to 25,000), observed that the smaller the dose the higher the rate of multiplication of the bacilli in the spleens at 4 weeks; and third, using large infective doses
TABLE 2. Effect of treatment with H37Rv antiserum 1 on the recovery of BCG in the spleens of BCG-infected mice No. of mice 4 weeks after infection with: No. of mice
BCG dose injected Passive treatment (2 doses of 0.1 ml given intravenously intraperitoneally)
13 11
7,300 14,600
Antiserumb (nondiluted) Antiserumb (nondiluted)
13 10
7,300 14,600
Antiserum 1:4c Antiserum 1:4e
13 13
7,300 14,600
Normal serum (nondiluted) Normal serum (nondiluted)
More than 150 0 or less than CFU (mean 150 CFUa no. of all groups, 300) 1 12 6 5
8 6
5 4
13 13
0 0
9 Saline 2 7,300 1 14 Saline 14,600 The bacterial culture count technique does not allow a count of less than 150 CFU per spleen. "Immunoglobulin fraction of a pool of antisera prepared from rabbit immunized with living M. tuberculosis. See text for further details. ' Diluted 1:4 in saline. 11 15
1304
INFECT. IMMUN.
FORGET ET AL.
TABLE 3. Effect of treatment with absorbed and nonabsorbed H37Rv antiserum 2 on the recovery of BCG in the spleens of BCG-infected mice BCG CFU recovered from the spleens 4 BCG dose in- Passive treatment (2 doses of Total recovered weeks after infection No. jected intra- 0.1 ml given intraperitone(spleen CFU/inof venously per ally 6 h before and 24 h after jected CFU) Arithmetic means + mouse mice bacterial P values" SEa infection) (CFU)
5 6 7 8
14,000 14,000 28,000 28,000
Nondiluted antiserumn Nonabsorbed Absorbed" Nonabsorbed Absorbed
18,040.0 8,716.7 26,207.1 24,412.5
± ± ± ±
1,570.6 1,786.2 8,520.0 8,145.2
p
