Acta path. microbiol. scand. Sect. B, 84: 265-272, 1976
ACQUIRED RESISTANCE OF RCG-VACCINATED RED MICE TO INFECTION WITH L I S T E R I A MONOCY TOGENES ANDR.JESPERSEN Tuberculosis Department, Statens Seruminstitut, Copenhagen, Denmark
Jespersen, A. Acquired resistance of BCG-vaccinated red mice to infection with Listeria monocytogenes. Acta path. microbiol. scand. Sect. B, 84: 265-272, 1976. Infection experiments have shown that red mice, belonging to the vole family, could be infected with Listeria monocytogenes and that they were almost as susceptible to intravenous infection as CF, mice. Vaccination of red mice with BCG induced a resistance which could be demonstrated by prolongation of the survival time of the animals after challenge with Listeria. T h e resistance was greatest in the second and third week after vaccination and was considerably higher after intravenous injection of BCG vaccine than after intraperitoneal or subcutaneous injection. O n the basis of studies concerning the dose of vaccine, the route of vaccination, and the interval between vaccination and challenge, a method by which to evaluate the potency of a strain of BCG has been elaborated. By this method it was found that there was a significant difference between the resistance produced by a strain of BCG which is weakly virulent and one which is strongly virulent for hamsters. Key words: Listeria rnonocytogenes; red mice; native resistance; BCG-induced resistance; potency of BCG strains. Andr. Jespersen, Tuberculosis Department, Statens Seruminstitut, Amager Boulevard 80, DK-2300 Copenhagen S, Denmark.
Received 8.iv.76
Accepted 8.iv.76
Various species of mice belonging to the murid family have been used in the course of time in infection experiments with Listeria monocytogenes (L.m.), whereas the susceptibility of voles to L.m. has not been examined. Red mice, like rabbits, are strongly resistant to M . tuberculosis but very sensitive to M . bouis (Jespersen 1954). Vaccination of red mice with BCG vaccine induces a resistance to infection with M . bouis, the degree of which is dependent on the BCG strain used (Jespersen & Bentzon 1964 a, b ) . Since rabbits are sensitive to L.m., the same might also apply to red mice.
The aim of the present experiment was to examine whether red mice can be infected with L.m. and whether the resistance induced by BCG is sufficiently high to enable the development of a method for determination of the potency of a strain of BCG. MATERIALS AND METHODS
Animals: T h e red mice and the CF, mice were bred a t the farm belonging to Statens Seruminstitut. T h e animals, 2-5 months old a t the commencement of the experiment, were distributed a t random into earthenware jars, one animal in each jar. T h e individual experimental groups consisted of equal numbers of female and male animals. BCG-vaccine: I n expts. 3 and 4, a freeze-dried
265
TABLE 1. Suruiual Times (in Days) of Groups of R e d Mice Injected Intravenously with Varying Doses of L.m. 10-3
0.1 ml i.v.
0.33 x 10-2 0.1 ml i.v.
vaccine produced at the BCG Department7 Statens Seruminstitut, was used. T h e strains of BCG used in expt. 5 were grown in Dubos fluid Tween medium and dispersed by ultrasonics. T h e strain Copenhagen was received from the BCG Department, Statens Seruminstitut, as a freeze-dried routine culture, batch 60, produced on March 17, 1971. This strain is considerably more virulent for hamsters than the Prague strain, but not as virulent as the strongest BCG strains. T h e strain Prague, received as a freeze-dried culture, batch 725, was prepared from the Prague strain on May 12, 1969, by the BCG Department, Statens Seruminstitut. This strain is weakly virulent for hamsters. Each of the two strains was subcultured twice in Dubos medium, after which the culture was treated with uItrasonics for ten minutes (Jespersen & Bentzon 1964 a ) . T h e number of viable units in the doses used for vaccination was determined by inoculation of suitable dilutions on Lowenstein- Jensen medium. Listeria monocytogenes: T h e strain (SSC 1423), provided by Dr. Jorgen Bennedsen, has been described previously, including details concerning its virulence for C3H mice and its maintenance (Bennedsen & Olesen Larsen 1975). A suspension of L . m . in broth was adjusted to a density of 0.025 in Coleman electrophotometer (100) and tenfold dilutions down to 10-7 were made with redistilled water. Two blood agar plates were inoculated with five drops of 0.05 ml per plate of each of the dilutions 10-5, 10.6 and 10-7. Vaccination: Subcutaneous vaccination was carried out by injection of the vaccine into the right groin and intravenous vaccination by injection into a tail vein. Challenge: Challenge was made by injection into a tail vein of 0.1 ml of a standard suspension diluted 100 times (10-2). This dose corresponds to about 2 x 105 viable organisms. T h e animals were challenged a t random. Measurement of resistance: Survival times after
266
0.33 x 10-1 0.1 ml i.v.
10-2 0.1 ml i.v.
10-1 0.1 ml i.v.
challenge were used for determination of the resistance. T h e mice were observed for ten days after the challenge injection. Statistical methods: Evaluation of the results was based on the combination of the number of surviving animals and the survival times of animals which died spontaneously. I n the infection experiments, LD,, and the slope of the log doseresponse curve were determined by the logit
TABLE 2. Suruiual Tim.es (in Days) of Groups of Red Mice and CF, Mice Injected Intrauenously with Varying Doses of L.m. Red mice 0.25 x O.lmli.v.
0.5 x 10-2 O.lmli.v.
5 5
6
S S
8 8
S
S
S
S
10-2 O.lmli.v.
10-2 0.2mli.v. 2 3
S
S
S
S
4 4 4 4 4 6 6 6
S
S
S
S
S
S
3 4 4 4 4
3 3 3 4 5
3 3 3 3 4
7
S
S
7
3 3 4 4 4 4 5 5
CF, mice 2 2 2 2
2 2
S
S
S
S
S
S
3 3
S
S
S
8
S
S
S
S
method on the basis of the number of surviving animals (Finney 1964). Comparison of the survival times was performed by Wilcoxon’s rank sum test ( Wilcoxon 1945).
RESULTS
Expt. I : Natural Resistance of Red Mice to Listeria Table 1 shows the survival times, in days, of red mice injected with varying doses of L . m . T h e three largest doses killed all the animals in the group and the survival times were prolonged the smaller the dose. Five out of eight animals died in the group injected with the next smallest dose, and two out of eight in the group injected with the smallest dose. Statistical analysis: The course of the infection was clearly dose-dependent. T h e slope of the dose-response curve was 4.59 (1.317.87), LD,, 0.20 x lo-*.
Expt. 2: Comparison of the Natural Resistance of Red Mice and CF, Mice to Listeria Table 2 shows the survival times, in days, of groups of red mice and CF, mice injected intravenously with varying doses of L . m . The susceptibility of the two animal species to Listeria cannot be compared on the basis of LD,, since this value cannot be determined in the case of CF, mice (see below). If the susceptibility is evaluated on the basis of the 3. Survival Times (in Days) of Groups Mice Vaccinated Subcutaneously with 106 Viable Units of Freeze-dried BCG and Challenged Intravenously with 0.1 ml 10-fL.m. I, 2, 3 and 4 Weeks Later
TABLE of Red 1.2 x Vaccine
Interval between vaccination and challenge 2 weeks 3 weeks 4 weeks
1 week
3 3 4 4 6
4 5
S
S
S
S
S
5 9
S
S
S
S
4 4 6 7 8 10
lowest dose that can kill all the mice in a for both group, the dose is 0.2 ml species, but the survival times of the CF, mice are shorter than those of the red mice. This latter finding applies irrespective of the dosage. Statistical analysis: As regards the red mice, the slope of the dose-response curve was 5.52 (2.20-8.84), i.e. similar to that found in Expt. 1. In the case of the CF, mice, the slope was 1.32 (-0.7-3.34). Since the slope is not significantly different from 0, LD,, cannot be determined.
Expt. 3: BCG-induced Resistance-Time Occurrence of Maximum Degree of Resistance
of
In order to examine whether BCG vaccine can produce resistance against Listeria infection, and to ascertain the time at which resistance is at a maximum, groups of red mice were vaccinated subcutaneously with 0.03 mg (1.2 x lo6 viable units) of freeze-dried BCG and challenged intravenously with 0.1 ml lo-? L . m . one, two, three and four weeks after vaccination. Table 3 shows the survival times, in days, of the four groups of mice. One week after vaccination, the survival times were the same as those in the corresponding group of non-vaccinated mice in Expts. 1, 4 and 5. After two and three weeks, a considerable resistance had developed. In both groups, four out of six mice survived and the survival times of the survivors were prolonged. Four weeks after vaccination, the resistance had decreased though it was greater than after the first week. Statistical analysis: Changes in the distribution of the survival times during intervals from the first to the second week and from the third to the fourth week, were in both cases near the 5 per cent significance limit (rank sum 1st week = 28, expected 39; p = 5-10 per cent. Rank sum 4th week = 26, expected 39; p = 2-5 per cent). Thus, it is justifiable to conclude that the resistance of the animals was greatest in the second to third week.
267
6 __ 7
8 -
S
I
S
S
S
S
8 Died during the interval between vaccination and challenge. Underlined figures = Median survival times.
s Survivor.
S S
S S
S
S
9
S
S
S
S
S
S
S
S
5 6
S
S
S
8
8 S
S
-
8
6
S
S
S
S
-
S
S -
S
S
4
7 7
S
S
S
S
S
S
S S
S __
S -
S
-
S
S
S
10
Intravenous vaccination 2.1 x 104 2.1 x 105 2.1 x 106
a
S
S
-
S
9 -
8 8
5 7
2.1 x 103
S
S
4
8 -
5
-
5 -
5
S
-
9
7
7
-
~
4 4 4 4
4 4 4 4
6
5
Intraperitoneal vaccination 2.1 x 104 2.1 x 105 2.1 x 106
2.1 x 103
5 7
6 -
S
5 5 5 -
6 7 -_ 7
5 5 -
4 4
4
4
3
Subcutaneous vaccination 2.1 x 104 2.1 x 105 2.1 x 106
4 4
2.1 x 103
4 -
~
4 4 4
3 3
Nonvaccinated
TABLE 4. Survival Times (in Days) of Non-vaccinated Red Mice and Mice Vaccinated Subcutaneously, Zntrafieritoneally or Intravenously with Graded Doses (Viable Units) of Freeze-dried BCG Vaccine Challenged Intravenously with 0.1 ml 10-0 L.m. Three Weeks after Vaccination
Expt. 4: Effect of the Route of Vaccination on BCG-induced Resistance Table 4 shows the survival times, in days, of groups of mice vaccinated subcutaneously, intraperitoneally or intravenously with graded doses of BCG vaccine. Together with a nonvaccinated group they were challenged intravenously with L.m. three weeks after vaccination. All non-vaccinated mice except one died. The median survival time, expressed as the survival times of the two middle animals in the group, (underlined in the Table), was four days. Among the vaccinated mice, the highest resistance was found in animals injected intravenously. This was particularly evident in the groups given the three highest doses, where the number of survivors after increasing doses was eight, nine and nine. I n the group vaccinated with 2.1 x lo3 viable
units, the median survival time was 6.5 days, and four animals survived as against one in the non-vaccinated group. The resistance was slightly increased in all groups of animals vaccinated subcutaneously and intraperitoneally, and there was no difference in the resistance after subcutaneous and intraperitoneal injection, whether estimated on the basis of median survival times or on the number of surviving animals. Statistical analysis: No difference was found in the effect of the three largest doses regardless of the vaccination route. The total frequency of surviving animals injected with these doses was 10/30, 14/29 and 26/30 after subcutaneous, intraperitoneal and intravenous vaccination, respectively. The frequency after intravenous vaccination was significantly higher than that after intraperitoneal and subcutaneous vaccination ( p = 0.3 per cent
TABLE 5 . Survival Times (in Days)
of Non-vaccinated Red Mice and Mice Vaccinated Intravenously with Graded Doses (Viable Units) of Living BCG Vaccine (Strains Prague and Copenhagen) and a Heat-killed Copenhagen Strain and Challenged Intravenously with 0.1 ml 10-2 L.m. Three Weeks after Vaccination ~
Nonvaccinated
~~~~
Prague (living) BCG Copenhagen (living) BCG Copenhagen (killed) BCG 2.5 x lo3 2.5 x lo4 2.5 x lo5 1.5 x lo3 1.5 x lo4 1.5 x 105 1.5 x lo3 1.5 x lo4 1.5 x 105 ~
3 3 3 3 3 4
3 3 4 4 4 4
4
3 3 4 4 4
4
4
4 4 4 4
4
4
4
4
4 4
4 5 5 -
5 5 5
5 5 5 5 5
6
10
6
9
~
4 4 4 -
4
4 4 4
3 4
4 4 5 5 6 9
9 9 -
3 5 5 5 5 6 S S S S
-
4 5 5 6 6 6
3 3 3 3 3 3
7 7 9 9 -
4 4 -
s -
4 -
4 4
3 3
3 3
4
4
4
4
4 4 4 4 4 4 -
4 4 4
4 4
4
-
4
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
6 10
4 5 5 5 5 5 5 7
S
S
S
S
S
S
S
S
S
S
S
S
S
§
S
§
s
S
S
S
5 __ 5 5 5 5 5 6 6
5 5
s -
S
4 4
5 5 5
-
5 5 5 6 6 6
7
s Survivor.
$ Died during the interval between vaccination and challenge, Underlined figures = Median survival times.
269
and 0.01 per cent, respectively) ; with the last two routes, the mutual difference in frequency was not significant ( p > 10 per cent).
Expt. 5 : B C G Resistance Inducpd by Strains of Varying Virulence Using a method based on the results of the above-mentioned experiments, comparison was made of the effect of two viable vaccines produced from a strain, Prague, which is weakly virulent and another strain, Copenhagen, which is strongly virulent for hamsters. The latter vaccine was also examined in heatof killed form. Dilutions lo-', lo-' and the two strains cultured in Dubos fluid medium and the same dilutions of the Copenhagen strain killed by heat were injected intravenously into groups of 20 mice. Three weeks after vaccination, these animals and a non-vaccinated group were challenged with 0.1 ml L.m. injected intravenously. The survival times after challenge of the animals in the ten groups are shown in Table 5. I n the non-vaccinated control group, all animals except two died, and the survival times of the two middle animals in the group were 4 and 5 days. I n the groups vaccinated with killed BCG vaccine, there was no increased resistance; neither the number of survivors nor the survival times in any of the groups deviated from those of the control animals. T h e two highest doses of the Prague vaccine induced significant resistance. T h e number of surviving animals was 5 and 7, respectively, as against 2 in the control group. O n the other hand, the median survival times were not prolonged. The group vaccinated with the lowest dose did not differ from the control group. All doses of the Copenhagen vaccine induced considerable resistance, whether estimated on the basis of the number of surviving animals, viz. 10, 13 and 10, with the three doses, or on the basis of the median survival times. Statistical analysis: Irrespective of the vaccines used, the effect of dosage was not significant. The frequencies of survival of animals vaccinated with Copenhagen, Prague and heat-killed vaccine were 33/59, 15/59 270
and 6/60, respectively. Using the Copenhagen vaccine, the frequency was significantly higher than that observed after the other two vaccines ( p = 0.15 per cent and p
ACQUIRED RESISTANCE OF RCG-VACCINATED RED MICE TO INFECTION WITH L I S T E R I A MONOCY TOGENES ANDR.JESPERSEN Tuberculosis Department, Statens Seruminstitut, Copenhagen, Denmark
Jespersen, A. Acquired resistance of BCG-vaccinated red mice to infection with Listeria monocytogenes. Acta path. microbiol. scand. Sect. B, 84: 265-272, 1976. Infection experiments have shown that red mice, belonging to the vole family, could be infected with Listeria monocytogenes and that they were almost as susceptible to intravenous infection as CF, mice. Vaccination of red mice with BCG induced a resistance which could be demonstrated by prolongation of the survival time of the animals after challenge with Listeria. T h e resistance was greatest in the second and third week after vaccination and was considerably higher after intravenous injection of BCG vaccine than after intraperitoneal or subcutaneous injection. O n the basis of studies concerning the dose of vaccine, the route of vaccination, and the interval between vaccination and challenge, a method by which to evaluate the potency of a strain of BCG has been elaborated. By this method it was found that there was a significant difference between the resistance produced by a strain of BCG which is weakly virulent and one which is strongly virulent for hamsters. Key words: Listeria rnonocytogenes; red mice; native resistance; BCG-induced resistance; potency of BCG strains. Andr. Jespersen, Tuberculosis Department, Statens Seruminstitut, Amager Boulevard 80, DK-2300 Copenhagen S, Denmark.
Received 8.iv.76
Accepted 8.iv.76
Various species of mice belonging to the murid family have been used in the course of time in infection experiments with Listeria monocytogenes (L.m.), whereas the susceptibility of voles to L.m. has not been examined. Red mice, like rabbits, are strongly resistant to M . tuberculosis but very sensitive to M . bouis (Jespersen 1954). Vaccination of red mice with BCG vaccine induces a resistance to infection with M . bouis, the degree of which is dependent on the BCG strain used (Jespersen & Bentzon 1964 a, b ) . Since rabbits are sensitive to L.m., the same might also apply to red mice.
The aim of the present experiment was to examine whether red mice can be infected with L.m. and whether the resistance induced by BCG is sufficiently high to enable the development of a method for determination of the potency of a strain of BCG. MATERIALS AND METHODS
Animals: T h e red mice and the CF, mice were bred a t the farm belonging to Statens Seruminstitut. T h e animals, 2-5 months old a t the commencement of the experiment, were distributed a t random into earthenware jars, one animal in each jar. T h e individual experimental groups consisted of equal numbers of female and male animals. BCG-vaccine: I n expts. 3 and 4, a freeze-dried
265
TABLE 1. Suruiual Times (in Days) of Groups of R e d Mice Injected Intravenously with Varying Doses of L.m. 10-3
0.1 ml i.v.
0.33 x 10-2 0.1 ml i.v.
vaccine produced at the BCG Department7 Statens Seruminstitut, was used. T h e strains of BCG used in expt. 5 were grown in Dubos fluid Tween medium and dispersed by ultrasonics. T h e strain Copenhagen was received from the BCG Department, Statens Seruminstitut, as a freeze-dried routine culture, batch 60, produced on March 17, 1971. This strain is considerably more virulent for hamsters than the Prague strain, but not as virulent as the strongest BCG strains. T h e strain Prague, received as a freeze-dried culture, batch 725, was prepared from the Prague strain on May 12, 1969, by the BCG Department, Statens Seruminstitut. This strain is weakly virulent for hamsters. Each of the two strains was subcultured twice in Dubos medium, after which the culture was treated with uItrasonics for ten minutes (Jespersen & Bentzon 1964 a ) . T h e number of viable units in the doses used for vaccination was determined by inoculation of suitable dilutions on Lowenstein- Jensen medium. Listeria monocytogenes: T h e strain (SSC 1423), provided by Dr. Jorgen Bennedsen, has been described previously, including details concerning its virulence for C3H mice and its maintenance (Bennedsen & Olesen Larsen 1975). A suspension of L . m . in broth was adjusted to a density of 0.025 in Coleman electrophotometer (100) and tenfold dilutions down to 10-7 were made with redistilled water. Two blood agar plates were inoculated with five drops of 0.05 ml per plate of each of the dilutions 10-5, 10.6 and 10-7. Vaccination: Subcutaneous vaccination was carried out by injection of the vaccine into the right groin and intravenous vaccination by injection into a tail vein. Challenge: Challenge was made by injection into a tail vein of 0.1 ml of a standard suspension diluted 100 times (10-2). This dose corresponds to about 2 x 105 viable organisms. T h e animals were challenged a t random. Measurement of resistance: Survival times after
266
0.33 x 10-1 0.1 ml i.v.
10-2 0.1 ml i.v.
10-1 0.1 ml i.v.
challenge were used for determination of the resistance. T h e mice were observed for ten days after the challenge injection. Statistical methods: Evaluation of the results was based on the combination of the number of surviving animals and the survival times of animals which died spontaneously. I n the infection experiments, LD,, and the slope of the log doseresponse curve were determined by the logit
TABLE 2. Suruiual Tim.es (in Days) of Groups of Red Mice and CF, Mice Injected Intrauenously with Varying Doses of L.m. Red mice 0.25 x O.lmli.v.
0.5 x 10-2 O.lmli.v.
5 5
6
S S
8 8
S
S
S
S
10-2 O.lmli.v.
10-2 0.2mli.v. 2 3
S
S
S
S
4 4 4 4 4 6 6 6
S
S
S
S
S
S
3 4 4 4 4
3 3 3 4 5
3 3 3 3 4
7
S
S
7
3 3 4 4 4 4 5 5
CF, mice 2 2 2 2
2 2
S
S
S
S
S
S
3 3
S
S
S
8
S
S
S
S
method on the basis of the number of surviving animals (Finney 1964). Comparison of the survival times was performed by Wilcoxon’s rank sum test ( Wilcoxon 1945).
RESULTS
Expt. I : Natural Resistance of Red Mice to Listeria Table 1 shows the survival times, in days, of red mice injected with varying doses of L . m . T h e three largest doses killed all the animals in the group and the survival times were prolonged the smaller the dose. Five out of eight animals died in the group injected with the next smallest dose, and two out of eight in the group injected with the smallest dose. Statistical analysis: The course of the infection was clearly dose-dependent. T h e slope of the dose-response curve was 4.59 (1.317.87), LD,, 0.20 x lo-*.
Expt. 2: Comparison of the Natural Resistance of Red Mice and CF, Mice to Listeria Table 2 shows the survival times, in days, of groups of red mice and CF, mice injected intravenously with varying doses of L . m . The susceptibility of the two animal species to Listeria cannot be compared on the basis of LD,, since this value cannot be determined in the case of CF, mice (see below). If the susceptibility is evaluated on the basis of the 3. Survival Times (in Days) of Groups Mice Vaccinated Subcutaneously with 106 Viable Units of Freeze-dried BCG and Challenged Intravenously with 0.1 ml 10-fL.m. I, 2, 3 and 4 Weeks Later
TABLE of Red 1.2 x Vaccine
Interval between vaccination and challenge 2 weeks 3 weeks 4 weeks
1 week
3 3 4 4 6
4 5
S
S
S
S
S
5 9
S
S
S
S
4 4 6 7 8 10
lowest dose that can kill all the mice in a for both group, the dose is 0.2 ml species, but the survival times of the CF, mice are shorter than those of the red mice. This latter finding applies irrespective of the dosage. Statistical analysis: As regards the red mice, the slope of the dose-response curve was 5.52 (2.20-8.84), i.e. similar to that found in Expt. 1. In the case of the CF, mice, the slope was 1.32 (-0.7-3.34). Since the slope is not significantly different from 0, LD,, cannot be determined.
Expt. 3: BCG-induced Resistance-Time Occurrence of Maximum Degree of Resistance
of
In order to examine whether BCG vaccine can produce resistance against Listeria infection, and to ascertain the time at which resistance is at a maximum, groups of red mice were vaccinated subcutaneously with 0.03 mg (1.2 x lo6 viable units) of freeze-dried BCG and challenged intravenously with 0.1 ml lo-? L . m . one, two, three and four weeks after vaccination. Table 3 shows the survival times, in days, of the four groups of mice. One week after vaccination, the survival times were the same as those in the corresponding group of non-vaccinated mice in Expts. 1, 4 and 5. After two and three weeks, a considerable resistance had developed. In both groups, four out of six mice survived and the survival times of the survivors were prolonged. Four weeks after vaccination, the resistance had decreased though it was greater than after the first week. Statistical analysis: Changes in the distribution of the survival times during intervals from the first to the second week and from the third to the fourth week, were in both cases near the 5 per cent significance limit (rank sum 1st week = 28, expected 39; p = 5-10 per cent. Rank sum 4th week = 26, expected 39; p = 2-5 per cent). Thus, it is justifiable to conclude that the resistance of the animals was greatest in the second to third week.
267
6 __ 7
8 -
S
I
S
S
S
S
8 Died during the interval between vaccination and challenge. Underlined figures = Median survival times.
s Survivor.
S S
S S
S
S
9
S
S
S
S
S
S
S
S
5 6
S
S
S
8
8 S
S
-
8
6
S
S
S
S
-
S
S -
S
S
4
7 7
S
S
S
S
S
S
S S
S __
S -
S
-
S
S
S
10
Intravenous vaccination 2.1 x 104 2.1 x 105 2.1 x 106
a
S
S
-
S
9 -
8 8
5 7
2.1 x 103
S
S
4
8 -
5
-
5 -
5
S
-
9
7
7
-
~
4 4 4 4
4 4 4 4
6
5
Intraperitoneal vaccination 2.1 x 104 2.1 x 105 2.1 x 106
2.1 x 103
5 7
6 -
S
5 5 5 -
6 7 -_ 7
5 5 -
4 4
4
4
3
Subcutaneous vaccination 2.1 x 104 2.1 x 105 2.1 x 106
4 4
2.1 x 103
4 -
~
4 4 4
3 3
Nonvaccinated
TABLE 4. Survival Times (in Days) of Non-vaccinated Red Mice and Mice Vaccinated Subcutaneously, Zntrafieritoneally or Intravenously with Graded Doses (Viable Units) of Freeze-dried BCG Vaccine Challenged Intravenously with 0.1 ml 10-0 L.m. Three Weeks after Vaccination
Expt. 4: Effect of the Route of Vaccination on BCG-induced Resistance Table 4 shows the survival times, in days, of groups of mice vaccinated subcutaneously, intraperitoneally or intravenously with graded doses of BCG vaccine. Together with a nonvaccinated group they were challenged intravenously with L.m. three weeks after vaccination. All non-vaccinated mice except one died. The median survival time, expressed as the survival times of the two middle animals in the group, (underlined in the Table), was four days. Among the vaccinated mice, the highest resistance was found in animals injected intravenously. This was particularly evident in the groups given the three highest doses, where the number of survivors after increasing doses was eight, nine and nine. I n the group vaccinated with 2.1 x lo3 viable
units, the median survival time was 6.5 days, and four animals survived as against one in the non-vaccinated group. The resistance was slightly increased in all groups of animals vaccinated subcutaneously and intraperitoneally, and there was no difference in the resistance after subcutaneous and intraperitoneal injection, whether estimated on the basis of median survival times or on the number of surviving animals. Statistical analysis: No difference was found in the effect of the three largest doses regardless of the vaccination route. The total frequency of surviving animals injected with these doses was 10/30, 14/29 and 26/30 after subcutaneous, intraperitoneal and intravenous vaccination, respectively. The frequency after intravenous vaccination was significantly higher than that after intraperitoneal and subcutaneous vaccination ( p = 0.3 per cent
TABLE 5 . Survival Times (in Days)
of Non-vaccinated Red Mice and Mice Vaccinated Intravenously with Graded Doses (Viable Units) of Living BCG Vaccine (Strains Prague and Copenhagen) and a Heat-killed Copenhagen Strain and Challenged Intravenously with 0.1 ml 10-2 L.m. Three Weeks after Vaccination ~
Nonvaccinated
~~~~
Prague (living) BCG Copenhagen (living) BCG Copenhagen (killed) BCG 2.5 x lo3 2.5 x lo4 2.5 x lo5 1.5 x lo3 1.5 x lo4 1.5 x 105 1.5 x lo3 1.5 x lo4 1.5 x 105 ~
3 3 3 3 3 4
3 3 4 4 4 4
4
3 3 4 4 4
4
4
4 4 4 4
4
4
4
4
4 4
4 5 5 -
5 5 5
5 5 5 5 5
6
10
6
9
~
4 4 4 -
4
4 4 4
3 4
4 4 5 5 6 9
9 9 -
3 5 5 5 5 6 S S S S
-
4 5 5 6 6 6
3 3 3 3 3 3
7 7 9 9 -
4 4 -
s -
4 -
4 4
3 3
3 3
4
4
4
4
4 4 4 4 4 4 -
4 4 4
4 4
4
-
4
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
6 10
4 5 5 5 5 5 5 7
S
S
S
S
S
S
S
S
S
S
S
S
S
§
S
§
s
S
S
S
5 __ 5 5 5 5 5 6 6
5 5
s -
S
4 4
5 5 5
-
5 5 5 6 6 6
7
s Survivor.
$ Died during the interval between vaccination and challenge, Underlined figures = Median survival times.
269
and 0.01 per cent, respectively) ; with the last two routes, the mutual difference in frequency was not significant ( p > 10 per cent).
Expt. 5 : B C G Resistance Inducpd by Strains of Varying Virulence Using a method based on the results of the above-mentioned experiments, comparison was made of the effect of two viable vaccines produced from a strain, Prague, which is weakly virulent and another strain, Copenhagen, which is strongly virulent for hamsters. The latter vaccine was also examined in heatof killed form. Dilutions lo-', lo-' and the two strains cultured in Dubos fluid medium and the same dilutions of the Copenhagen strain killed by heat were injected intravenously into groups of 20 mice. Three weeks after vaccination, these animals and a non-vaccinated group were challenged with 0.1 ml L.m. injected intravenously. The survival times after challenge of the animals in the ten groups are shown in Table 5. I n the non-vaccinated control group, all animals except two died, and the survival times of the two middle animals in the group were 4 and 5 days. I n the groups vaccinated with killed BCG vaccine, there was no increased resistance; neither the number of survivors nor the survival times in any of the groups deviated from those of the control animals. T h e two highest doses of the Prague vaccine induced significant resistance. T h e number of surviving animals was 5 and 7, respectively, as against 2 in the control group. O n the other hand, the median survival times were not prolonged. The group vaccinated with the lowest dose did not differ from the control group. All doses of the Copenhagen vaccine induced considerable resistance, whether estimated on the basis of the number of surviving animals, viz. 10, 13 and 10, with the three doses, or on the basis of the median survival times. Statistical analysis: Irrespective of the vaccines used, the effect of dosage was not significant. The frequencies of survival of animals vaccinated with Copenhagen, Prague and heat-killed vaccine were 33/59, 15/59 270
and 6/60, respectively. Using the Copenhagen vaccine, the frequency was significantly higher than that observed after the other two vaccines ( p = 0.15 per cent and p
