INFECTION AND IMMUNITY, July 1979, p. 345-351 0019-9567/79/07-0345/07$02.00/O
Vol. 25, No. 1
Phenotypic Expression of Genetically Controlled Host Resistance to Listeria monocytogenes E. SKAMENE'* AND P. A. L. KONGSHAVN2 Montreal General Hospital Research Institute' and Department of Physiology,2 McGill University, Montreal, Quebec, H3G 1A4, Canada Received for publication 28 December 1978
Several inbred mouse strains, all of them derived from the C57BL background, have genetically determined increased resistance to infection with Listeria monocytogenes, whereas a variety of other strains are relatively sensitive to this infection. Comparison of the host response to L. monocytogenes in the sensitive A strain and the resistant C57BL/6 (B6) strain revealed that the B6 mice were superior to A mice both in the T-cell-independent and in the T-cell-dependent phase of the response. Although animals of both strains had equal ability to clear their circulation of intravenously administered Listeria and to take up comparable amounts of bacteria in their livers and spleens, already 24 to 48 h after infection the genetic advantage of B6 strain mice to suppress bacterial proliferation was apparent. Both the primary (early and late) and the secondary responses as well as the ability to inactivate the bacterial load after adoptive protection by syngeneic immune lymphocytes were more efficient in the B6 animals, suggesting that the common effector macrophage arm of the antilisterial resistance rather than the lymphocyte arm (mediating the T-cell-dependent phase of response) is genetically controlled.
Genetically determined differences among inbred strains of mice in sensitivity or resistance to infection with Listeria monocytogenes first described by Robson and Vas (20) have been recently confirmed and analyzed in this laboratory (22) and by others (6). The host response and the eventual outcome of an infection with this intracellular facultative parasite would appear to be under the genetic control of a single gene or a group of closely linked genes which, although not yet mapped, is clearly distinct from the classic H-2-linked immune response genes (6, 22). It has become clear, furthermore, that sensitivity and resistance of mice to other infectious agents (BCG, Salmonella typhimurium, Corynebacterium kutscheri, Leishmania donovani, Trypanosoma congolense, Schistosoma mansoni) are also genetically controlled by genes which are not linked to the H-2 locus (1, 3, 13, 17, 18). Since it is possible that in at least some of these infections the protective mechanism involves the action of the same "host resistance" gene, it is of importance to try to elucidate the phenotypic expression of the host resistance gene. For this reason we have analyzed the various facets of host defense against L. monocytogenes in the resistant C57BL/6J mouse strain and in the sensitive A/J strain. MATERILS AND METHODS Mice. Adult female mice, 8 to 12 weeks old, were
used in all experiments. The following inbred strains were purchased from Jackson Laboratories, Bar Harbor, Maine: A/J (A), C57BL/6J (B6), C57BL/6J x A/ J (B6AFI), C3H/H3J, CBA/J, DBA/2J, C57BL/lOSn (B10), B1O.D2Sn (BlO.D2) and BlO.A/SgSn (BlO.A). C3H/HeCr mice originating from Charles River Laboratories, Wilmington, Mass., were purchased from Canadian Breeders, Montreal, Canada.
Bacteria. L. monocytogenes strain EDG, obtained originally from G. B. Mackaness of the Trudeau Institute, Saranac Lake, N.Y., was used throughout the experiments. The organism was kept virulent by passage through mice. A small portion of the stock culture, stored frozen at -70°C, was thawed and used to seed a fresh culture for each inoculation. The culture was grown overnight in Trypticase soy broth, and the number of organisms was determined before injection by spectrophotometry with a nephelometric curve. Infection of mice. In each experiment, six to eight animals per group were infected by injection of Listeria into the lateral tail vein. The dose of infectious inoculum was checked retrospectively by plating out the appropriate dilutions of bacteria in saline on tryptose agar and counting the bacterial colony-forming units (CFU) 24 h later. Determination of LDw. Animals infected with doses of Listeria increasing by 5- or 10-fold concentrations were observed daily for 10 days. The 50% lethal dose (LD5o) was calculated by the method of Reed and Muench (19). Enumeration of Listeria in tis8ues. Mice were assayed for resistance to Listeria infection by determining the numbers of organisms in their livers, spleens, and blood at various times after intravenous 345
346
INFECT. IMMUN.
SKAMENE AND KONGSHAVN
injection. For the blood count, the animals were anesthetized with ether and bled from the retro-orbital plexus with disposable micropipettes (Drummond microcaps). A portion of 0.1 ,ul of the blood from each mouse underwent serial 10-fold dilutions, and each was plated on tryptose agar. The total bacteremia was then calculated assuming the blood volume of each mouse to be 2 ml. The numbers of viable organisms in the spleens and livers were established by plating serial 10-fold dilutions of organ homogenates in saline on tryptose agar. The colony counts were always performed 18 to 24 h later. Adoptive transfer of antilisterial resistance. Spleen cell suspensions from noninfected and 6-dayinfected donors were prepared as described previously (5). Since there were significant differences between the A and B6 mice in the numbers of bacteria remaining in the spleens on day 6 of infection, antibiotic treatment of donors, adopted from the method of North (16), was instituted 24 h before harvest of the spleen cells to reduce or eliminate the bacterial load. The treatment consisted of a single subcutaneous injection of 5,000 U of penicillin G and the addition of ampicillin to the drinking water at 0.5 mg/ml for 12 h. This treatment reduced the numbers of Listeria in the donor spleens to 0 to 103 CFU per spleen, quantities of no significance since the challenge inoculum given to the recipients was 5 x 104 CFU of Listeria. Preliminary experiments established that no transfer of antibiotic activity that might have influenced the outcome of the infection in the adoptively protected recipients occurred under these conditions. Donor cells were routinely injected intravenously 2 to 4 h after inoculating the recipients with the challenge dose of Listeria to allow effective blood clearance of bacteria and lodging of the latter in the tissues. The number of bacteria in the recipients' organs was routinely determined 48 h after infection.
RESULTS Susceptibility of inbred strains of mice to infection with L nwnocytogenes. Nine inbred mouse strains that were tested fell into two sharply defined groups. For five strains (A, C3H/ HeJ, C3H/HeCr, CBA, DBA/2) the LDso was at or below 104 CFU of intravenously administered Listeria, whereas for four strains, all of them with the C57BL background (B6, B10, B1O.A, B1O.D2), the LD5o was at or above 5 X 105 CFU of Listeria. The B6AF1 hybrids exhibited intermediate sensitivity with an LD5o of 2 X 104 CFU of Listeria (Table 1). A lethal dose of infection for each particular strain led to the death of animals in 3 to 6 days. The mice that survived 6 days after a sublethal dose of Listeria always recovered fully from the infection. On the basis of this strain survey for resistance and susceptibility to Listeria, the A mice were employed as representative of the sensitive strains, and the B6 were chosen as representative of the resistant strains in further experiments designed to analyze the host response to Listeria infection.
Kinetics of primary response to infection with Listeria. Mice of the A and B6 strains were infected with graded doses of Listeria (5 X 102 to 5 x 104), and the bacterial colony counts in the spleens and livers were determined 2, 4, and 6 days after infection (Fig. 1). In the mice receiving the lowest dose (5 X 102 CFU of Listeria) that was sublethal for both strains, the classical pattern of response (11) was observed. It was characterized by a peaking of the bacterial burden on day 4 of infection with a subsequent decrease in bacterial counts leading eventually to complete elimination of Listeria. No differences in the responses ofthe sensitive and resistant strains were detected at this dose. However, when higher doses of infectious inoculum were employed, leading to a bacterial burden at or exceeding the level of 105 to 106 CFU TABLE 1. LD50 of mouse strains after intravenous infection with Listeria Mouse strain LD5o A 5x 103 9 x 103 C3H/HeCr 1 x 104 C3H/HeJ CBA 6 x 103 5 x 103 DBA/2
< 0
B6AF1
2 x 104
B6 B10 B10.A B10.D2
6x 5x 5x 5x
105 105 105 105
3tt
24
6i-
5I-
0 Cc
/1
2 8
X
--X
EC6 -+^/ :1 4 52
4 6 2 4 DAYS after INFECTION
6
FIG. 1. Growth curves of L. monocytogenes during the primary response of A (0-----0) and B6 (-O*) mice infected with various doses of Listeria (arrows) on day 0. Mean of 6 to 8 mice per group + standard deviation. except * and t. t Four of six mice died by day 4; * Four of eight mice died by day 6.
VOL. 25, 1979
IMMUNOGENETICS OF LISTERIOSIS
of Listeria in the livers and spleens by day 2 or 3 of infection, the response of the A strain mice was clearly deficient, compared with that of the B6 strain mice. Whereas the B6 mice showed the characteristic pattern of response after the dose of 5 x 103 to 5 x 104 CFU of Listeria, the A mice were not able to control the bacterial proliferation in their organs and eventually succumbed to infection. Thus, 68% of A mice died 5 to 6 days after infection with 5 x 103 CFU of Listeria and 100% of A mice died 4 to 6 days after infection with 5 X 104 CFU of Listeria. Development of splenomegaly. The spleen weights of animals infected with graded doses of Listeria (5 x 10' to 5 x 104 CFU) 6 days previously were compared in the sensitive and resistant strains of mice. In animals infected with the lower doses of Listeria, the spleen weight was not significantly different in the two strains, but at the higher dose of 5 x 103 the spleen weight was significantly greater in the B6 than in the A strain of mice (Fig. 2). Early events in Listeria infection. It was apparent when analyzing the kinetics of the primary response to Listeria that the differences in resistance between the sensitive and the resistant strains were already developing early in the course of infection, during the nonspecific phase of the response. The early events in Listeria infection, namely the blood clearance and organ uptake of bacteria, were studied in the following set of experiments. (i) Blood clearance and organ uptake of bacteria. Six mice of each strain were injected intravenously with doses of Listeria ranging from 5.1 x 104 to 4.6 x 107, and the bacterial counts in the peripheral blood and in the liver and spleen homogenates were performed 5 min
347
and 1 h after infection. It had been established in preliminary experiments that there was no detectable uptake of bacteria to other organs such as lungs, bone marrow, or brain. No substantial differences were found between the blood clearance and organ uptake values at 5 min and 1 h, and therefore only the 1-h data are shown (Fig. 3). There was no difference between the Listeria-sensitive (A) and the Listeria-resistant (B6) mice in blood clearance and organ uptake of Listeria over the wide range of bacterial dosage employed. For each dose, about 90% of the inoculum lodged in the liver, and the remainder lodged in the spleen. The numbers of Listeria in the peripheral blood in 1 h constituted less than 1% of the inoculum at high bacterial doses, whereas at lower doses no bacteremia was detected. The higher doses used in this experiment would, of course, have proven lethal to the mice of both strains if the animals had not been sacrificed. The use of these doses was, however, necessary when testing the clearance and uptake mechanisms to recover significant numbers of bacteria from the blood and organs within the first hour of infection, before any substantial bacterial multiplication had occurred. For example, the blood clearance could not be assessed with a dose of less than 4.6 X 106 CFU of Listeria. (ii) Early kinetics of bacterial proliferation. In the first experiment, 6 to 8 mice of each strain were infected with graded doses of Listeria ranging from 5 x 104 to 5 x 107 CFU and the bacterial colony counts were determined in the spleens and livers at 1 and 24 h after infection (Fig. 4). Although there were no significant differences in the control of bacterial proliferation in A and B6 mice on day 1 of infection at the lower doses, differences became clearly evident 0.26 with the higher doses of the original inoculum ----o A (e.g., 5 x 107 CFU), the A mice being less well --* B6 'a 0.22 able to control the bacterial proliferation. This point is also illustrated in a second experiment in which mice of both strains were infected with 0.18 graded doses of Listeria ranging from 102 to 5 x 104 CFU, and the bacterial colony count in the U 0.14 was determined 3 days after the infection. spleen -i Again, no difference between the sensitive and co-,4 ena. u.1u resistant strains was apparent at the lower doses of inoculum. However, as the dose of Listeria was increased, the difference in the response to 0.06 5x102 5x103 Listeria between the two strains increased, as DOSE of LISTERIA (CFU) shown by the difference in the slopes of the two curves (Fig. 5). Data obtained from the bacterial 6 B6 mice in A and 2. FIG. Spleen weight infected days previously with different doses of L. monocyto- counts recovered from the liver were similar. Table 2 summarizes the kinetics of bacterial eight mice per group + standard genes. Mean of six deviation except group t where four of eight mice proliferation during the first 24 to 72 h after infection and clearly shows that the genetic addied by day 6.
0
z
to
8I
INFECT. IMMUN.
SKAMENE AND KONGSHAVN
348
B
A
86
B6 A
A
86
A
7
I-.
'
I s " A l fi 5as?
.s 1-
vno3
4.6 x 107
§>~
^
* g
~
*
;
Secondary responses to infection with Listeria. The development of secondary responses to Listeria infection were compared in sensitive and resistant strains of mice in the following series of experiments. In the first experiment, mice of the A and B6 strains were infected with graded doses of Listeria (5 x 10 to 5 X 103 CFU) and 6 weeks later were challenged with a secondary infection of 105 CFU of Listeria. Proliferation of the secondary| bacterial inoculum in the spleens and livers twasdetermined 48 h after the secondary infection. Similar results were obtained for the spleen and liver counts, and only the splenic bacterial 8
Wf 5.lxO 1
Lw 5.1x105 DOSE of LISTERIA (CFU) 4.6x 10
FIG. 3. Blood clearance (B) and early tissue uptake of L. monocytogenes in the livers (L) and spleens (S) of the A (empty bars) and B6 (hatched bars) mice infected with various doses of bacteria 1 h previously. Means of six mice per group ± standard error (vertical lines). LIVER SPLEEN
7
*-. B6
z uJ Co 0
6
0-
B6 8 -_ 7 C+ fI
0L
5 .1
.I
.1
.1
'IO
.1
z 6 < 5
Vol. 25, No. 1
Phenotypic Expression of Genetically Controlled Host Resistance to Listeria monocytogenes E. SKAMENE'* AND P. A. L. KONGSHAVN2 Montreal General Hospital Research Institute' and Department of Physiology,2 McGill University, Montreal, Quebec, H3G 1A4, Canada Received for publication 28 December 1978
Several inbred mouse strains, all of them derived from the C57BL background, have genetically determined increased resistance to infection with Listeria monocytogenes, whereas a variety of other strains are relatively sensitive to this infection. Comparison of the host response to L. monocytogenes in the sensitive A strain and the resistant C57BL/6 (B6) strain revealed that the B6 mice were superior to A mice both in the T-cell-independent and in the T-cell-dependent phase of the response. Although animals of both strains had equal ability to clear their circulation of intravenously administered Listeria and to take up comparable amounts of bacteria in their livers and spleens, already 24 to 48 h after infection the genetic advantage of B6 strain mice to suppress bacterial proliferation was apparent. Both the primary (early and late) and the secondary responses as well as the ability to inactivate the bacterial load after adoptive protection by syngeneic immune lymphocytes were more efficient in the B6 animals, suggesting that the common effector macrophage arm of the antilisterial resistance rather than the lymphocyte arm (mediating the T-cell-dependent phase of response) is genetically controlled.
Genetically determined differences among inbred strains of mice in sensitivity or resistance to infection with Listeria monocytogenes first described by Robson and Vas (20) have been recently confirmed and analyzed in this laboratory (22) and by others (6). The host response and the eventual outcome of an infection with this intracellular facultative parasite would appear to be under the genetic control of a single gene or a group of closely linked genes which, although not yet mapped, is clearly distinct from the classic H-2-linked immune response genes (6, 22). It has become clear, furthermore, that sensitivity and resistance of mice to other infectious agents (BCG, Salmonella typhimurium, Corynebacterium kutscheri, Leishmania donovani, Trypanosoma congolense, Schistosoma mansoni) are also genetically controlled by genes which are not linked to the H-2 locus (1, 3, 13, 17, 18). Since it is possible that in at least some of these infections the protective mechanism involves the action of the same "host resistance" gene, it is of importance to try to elucidate the phenotypic expression of the host resistance gene. For this reason we have analyzed the various facets of host defense against L. monocytogenes in the resistant C57BL/6J mouse strain and in the sensitive A/J strain. MATERILS AND METHODS Mice. Adult female mice, 8 to 12 weeks old, were
used in all experiments. The following inbred strains were purchased from Jackson Laboratories, Bar Harbor, Maine: A/J (A), C57BL/6J (B6), C57BL/6J x A/ J (B6AFI), C3H/H3J, CBA/J, DBA/2J, C57BL/lOSn (B10), B1O.D2Sn (BlO.D2) and BlO.A/SgSn (BlO.A). C3H/HeCr mice originating from Charles River Laboratories, Wilmington, Mass., were purchased from Canadian Breeders, Montreal, Canada.
Bacteria. L. monocytogenes strain EDG, obtained originally from G. B. Mackaness of the Trudeau Institute, Saranac Lake, N.Y., was used throughout the experiments. The organism was kept virulent by passage through mice. A small portion of the stock culture, stored frozen at -70°C, was thawed and used to seed a fresh culture for each inoculation. The culture was grown overnight in Trypticase soy broth, and the number of organisms was determined before injection by spectrophotometry with a nephelometric curve. Infection of mice. In each experiment, six to eight animals per group were infected by injection of Listeria into the lateral tail vein. The dose of infectious inoculum was checked retrospectively by plating out the appropriate dilutions of bacteria in saline on tryptose agar and counting the bacterial colony-forming units (CFU) 24 h later. Determination of LDw. Animals infected with doses of Listeria increasing by 5- or 10-fold concentrations were observed daily for 10 days. The 50% lethal dose (LD5o) was calculated by the method of Reed and Muench (19). Enumeration of Listeria in tis8ues. Mice were assayed for resistance to Listeria infection by determining the numbers of organisms in their livers, spleens, and blood at various times after intravenous 345
346
INFECT. IMMUN.
SKAMENE AND KONGSHAVN
injection. For the blood count, the animals were anesthetized with ether and bled from the retro-orbital plexus with disposable micropipettes (Drummond microcaps). A portion of 0.1 ,ul of the blood from each mouse underwent serial 10-fold dilutions, and each was plated on tryptose agar. The total bacteremia was then calculated assuming the blood volume of each mouse to be 2 ml. The numbers of viable organisms in the spleens and livers were established by plating serial 10-fold dilutions of organ homogenates in saline on tryptose agar. The colony counts were always performed 18 to 24 h later. Adoptive transfer of antilisterial resistance. Spleen cell suspensions from noninfected and 6-dayinfected donors were prepared as described previously (5). Since there were significant differences between the A and B6 mice in the numbers of bacteria remaining in the spleens on day 6 of infection, antibiotic treatment of donors, adopted from the method of North (16), was instituted 24 h before harvest of the spleen cells to reduce or eliminate the bacterial load. The treatment consisted of a single subcutaneous injection of 5,000 U of penicillin G and the addition of ampicillin to the drinking water at 0.5 mg/ml for 12 h. This treatment reduced the numbers of Listeria in the donor spleens to 0 to 103 CFU per spleen, quantities of no significance since the challenge inoculum given to the recipients was 5 x 104 CFU of Listeria. Preliminary experiments established that no transfer of antibiotic activity that might have influenced the outcome of the infection in the adoptively protected recipients occurred under these conditions. Donor cells were routinely injected intravenously 2 to 4 h after inoculating the recipients with the challenge dose of Listeria to allow effective blood clearance of bacteria and lodging of the latter in the tissues. The number of bacteria in the recipients' organs was routinely determined 48 h after infection.
RESULTS Susceptibility of inbred strains of mice to infection with L nwnocytogenes. Nine inbred mouse strains that were tested fell into two sharply defined groups. For five strains (A, C3H/ HeJ, C3H/HeCr, CBA, DBA/2) the LDso was at or below 104 CFU of intravenously administered Listeria, whereas for four strains, all of them with the C57BL background (B6, B10, B1O.A, B1O.D2), the LD5o was at or above 5 X 105 CFU of Listeria. The B6AF1 hybrids exhibited intermediate sensitivity with an LD5o of 2 X 104 CFU of Listeria (Table 1). A lethal dose of infection for each particular strain led to the death of animals in 3 to 6 days. The mice that survived 6 days after a sublethal dose of Listeria always recovered fully from the infection. On the basis of this strain survey for resistance and susceptibility to Listeria, the A mice were employed as representative of the sensitive strains, and the B6 were chosen as representative of the resistant strains in further experiments designed to analyze the host response to Listeria infection.
Kinetics of primary response to infection with Listeria. Mice of the A and B6 strains were infected with graded doses of Listeria (5 X 102 to 5 x 104), and the bacterial colony counts in the spleens and livers were determined 2, 4, and 6 days after infection (Fig. 1). In the mice receiving the lowest dose (5 X 102 CFU of Listeria) that was sublethal for both strains, the classical pattern of response (11) was observed. It was characterized by a peaking of the bacterial burden on day 4 of infection with a subsequent decrease in bacterial counts leading eventually to complete elimination of Listeria. No differences in the responses ofthe sensitive and resistant strains were detected at this dose. However, when higher doses of infectious inoculum were employed, leading to a bacterial burden at or exceeding the level of 105 to 106 CFU TABLE 1. LD50 of mouse strains after intravenous infection with Listeria Mouse strain LD5o A 5x 103 9 x 103 C3H/HeCr 1 x 104 C3H/HeJ CBA 6 x 103 5 x 103 DBA/2
< 0
B6AF1
2 x 104
B6 B10 B10.A B10.D2
6x 5x 5x 5x
105 105 105 105
3tt
24
6i-
5I-
0 Cc
/1
2 8
X
--X
EC6 -+^/ :1 4 52
4 6 2 4 DAYS after INFECTION
6
FIG. 1. Growth curves of L. monocytogenes during the primary response of A (0-----0) and B6 (-O*) mice infected with various doses of Listeria (arrows) on day 0. Mean of 6 to 8 mice per group + standard deviation. except * and t. t Four of six mice died by day 4; * Four of eight mice died by day 6.
VOL. 25, 1979
IMMUNOGENETICS OF LISTERIOSIS
of Listeria in the livers and spleens by day 2 or 3 of infection, the response of the A strain mice was clearly deficient, compared with that of the B6 strain mice. Whereas the B6 mice showed the characteristic pattern of response after the dose of 5 x 103 to 5 x 104 CFU of Listeria, the A mice were not able to control the bacterial proliferation in their organs and eventually succumbed to infection. Thus, 68% of A mice died 5 to 6 days after infection with 5 x 103 CFU of Listeria and 100% of A mice died 4 to 6 days after infection with 5 X 104 CFU of Listeria. Development of splenomegaly. The spleen weights of animals infected with graded doses of Listeria (5 x 10' to 5 x 104 CFU) 6 days previously were compared in the sensitive and resistant strains of mice. In animals infected with the lower doses of Listeria, the spleen weight was not significantly different in the two strains, but at the higher dose of 5 x 103 the spleen weight was significantly greater in the B6 than in the A strain of mice (Fig. 2). Early events in Listeria infection. It was apparent when analyzing the kinetics of the primary response to Listeria that the differences in resistance between the sensitive and the resistant strains were already developing early in the course of infection, during the nonspecific phase of the response. The early events in Listeria infection, namely the blood clearance and organ uptake of bacteria, were studied in the following set of experiments. (i) Blood clearance and organ uptake of bacteria. Six mice of each strain were injected intravenously with doses of Listeria ranging from 5.1 x 104 to 4.6 x 107, and the bacterial counts in the peripheral blood and in the liver and spleen homogenates were performed 5 min
347
and 1 h after infection. It had been established in preliminary experiments that there was no detectable uptake of bacteria to other organs such as lungs, bone marrow, or brain. No substantial differences were found between the blood clearance and organ uptake values at 5 min and 1 h, and therefore only the 1-h data are shown (Fig. 3). There was no difference between the Listeria-sensitive (A) and the Listeria-resistant (B6) mice in blood clearance and organ uptake of Listeria over the wide range of bacterial dosage employed. For each dose, about 90% of the inoculum lodged in the liver, and the remainder lodged in the spleen. The numbers of Listeria in the peripheral blood in 1 h constituted less than 1% of the inoculum at high bacterial doses, whereas at lower doses no bacteremia was detected. The higher doses used in this experiment would, of course, have proven lethal to the mice of both strains if the animals had not been sacrificed. The use of these doses was, however, necessary when testing the clearance and uptake mechanisms to recover significant numbers of bacteria from the blood and organs within the first hour of infection, before any substantial bacterial multiplication had occurred. For example, the blood clearance could not be assessed with a dose of less than 4.6 X 106 CFU of Listeria. (ii) Early kinetics of bacterial proliferation. In the first experiment, 6 to 8 mice of each strain were infected with graded doses of Listeria ranging from 5 x 104 to 5 x 107 CFU and the bacterial colony counts were determined in the spleens and livers at 1 and 24 h after infection (Fig. 4). Although there were no significant differences in the control of bacterial proliferation in A and B6 mice on day 1 of infection at the lower doses, differences became clearly evident 0.26 with the higher doses of the original inoculum ----o A (e.g., 5 x 107 CFU), the A mice being less well --* B6 'a 0.22 able to control the bacterial proliferation. This point is also illustrated in a second experiment in which mice of both strains were infected with 0.18 graded doses of Listeria ranging from 102 to 5 x 104 CFU, and the bacterial colony count in the U 0.14 was determined 3 days after the infection. spleen -i Again, no difference between the sensitive and co-,4 ena. u.1u resistant strains was apparent at the lower doses of inoculum. However, as the dose of Listeria was increased, the difference in the response to 0.06 5x102 5x103 Listeria between the two strains increased, as DOSE of LISTERIA (CFU) shown by the difference in the slopes of the two curves (Fig. 5). Data obtained from the bacterial 6 B6 mice in A and 2. FIG. Spleen weight infected days previously with different doses of L. monocyto- counts recovered from the liver were similar. Table 2 summarizes the kinetics of bacterial eight mice per group + standard genes. Mean of six deviation except group t where four of eight mice proliferation during the first 24 to 72 h after infection and clearly shows that the genetic addied by day 6.
0
z
to
8I
INFECT. IMMUN.
SKAMENE AND KONGSHAVN
348
B
A
86
B6 A
A
86
A
7
I-.
'
I s " A l fi 5as?
.s 1-
vno3
4.6 x 107
§>~
^
* g
~
*
;
Secondary responses to infection with Listeria. The development of secondary responses to Listeria infection were compared in sensitive and resistant strains of mice in the following series of experiments. In the first experiment, mice of the A and B6 strains were infected with graded doses of Listeria (5 x 10 to 5 X 103 CFU) and 6 weeks later were challenged with a secondary infection of 105 CFU of Listeria. Proliferation of the secondary| bacterial inoculum in the spleens and livers twasdetermined 48 h after the secondary infection. Similar results were obtained for the spleen and liver counts, and only the splenic bacterial 8
Wf 5.lxO 1
Lw 5.1x105 DOSE of LISTERIA (CFU) 4.6x 10
FIG. 3. Blood clearance (B) and early tissue uptake of L. monocytogenes in the livers (L) and spleens (S) of the A (empty bars) and B6 (hatched bars) mice infected with various doses of bacteria 1 h previously. Means of six mice per group ± standard error (vertical lines). LIVER SPLEEN
7
*-. B6
z uJ Co 0
6
0-
B6 8 -_ 7 C+ fI
0L
5 .1
.I
.1
.1
'IO
.1
z 6 < 5
