Vol. 11, No. 5 Printed in U.S.A.
INFECTION AND IMMUNITY, May 1975, p. 1094-1099 Copyright © 1975 American Society for Microbiology
Superinfection in Mice Previously Infected with Mycobacterium leprae LOUIS LEVY Leprosy Research Unit, Public Health Service Hospital, San Francisco, California 94118 Received for publication 30 December 1974
Previous studies of the protection of mice by prior infection with Mycobacterium leprae in one hind footpad against challenge with M. leprae in the opposite hind footpad had produced conflicting results; therefore, the problem was restudied. In several experiments, BALB/c mice were inoculated first in the right hind footpad with 5,000 M. leprae and then challenged in the left hind footpad with 5,000 M. leprae of the same strain at intervals after primary infection, at the same time that uninfected mice were inoculated. Multiplication of the M. leprae of the secondary challenge inoculum occurred at the same rate and to the same level as multiplication in uninfected mice when.challenges were made soon after primary infection. Multiplication was slowed but proceeded to the same level in previously infected as in uninfected mice when the challenges were administered between 76 and 106 days after primary infection (47 to 17 days before the M. leprae of the primary inoculum had multiplied to the level of 106 organisms per footpad). Finally, the M. leprae of a secondary challenge administered at the time that the organisms of the primary inoculum had multiplied to 106 per footpad or later not only multiplied more slowly in previously infected than in control animals, but multiplication in the previously infected animals reached a lower maximum. These results are similar to those observed when mice previously infected with M. bovis (BCG), M. marinum, Toxoplasma gondii, or Besnoitia jellisoni were challenged with M. leprae. When a small number of viable Mycobacterium leprae are inoculated into the hind footpads of immunologically competent mice, the organisms undergo limited multiplication, achieving a "ceiling" of about 106 to 106.3 organisms per footpad during the first 100 to 150 days after inoculation (10). Much evidence suggests that the limit to further multiplication is imposed by a cell-mediated immune response mounted by the host. Killing of M. leprae has been shown to proceed rapidly, beginning at the time that multiplication ceases (6, 14). Also at this time, the footpad macrophages in which the organisms reside begin to assume an activated appearance (1). Additional evidence for a cellmediated immune response is the protection of mice against challenge with M. leprae by prior infection with the related organisms M. bovis (BCG) (2, 10-12; Y. Matsuo, Proc. Conf. Biol. Mycobacterioses, New York, Abstr. 1, p. 1, 1967) and M. marinum (8), and with the protozoa Toxoplasma gondii and Besnoitia jellisoni (5), the protection of mice against challenge with M. marinum by prior infection with M. leprae (8), and the protection of mice against challenge with M. leprae by a graft -ver-
sus-host reaction (C. C. Shepard, Int. J. Leprosy, in press). Finally, multiplication of M. leprae proceeds beyond the usual ceiling in mice treated with cortisone (16), and in mice subjected to adult thymectomy and subsequently treated with lethal whole-body X irradiation (9) or antilymphocyte globulin (3). If the mechanism by which mice limit the multiplication of M. leprae is indeed a cellmediated immune response, mice challenged with M. leprae before the onset of the immune response to a prior infection with M. leprae should not be protected against the secondary challenge, whereas mice challenged after killing of previously inoculated M. leprae had begun should be protected against the challenge. Such is the case in the analogous disease caused by the footpad infection of mice with M. marinum (8), and both Kawaguchi (4) and Shepard (C. C. Shepard, Int. J. Leprosy, in press) have successfully demonstrated protection of mice against homologous challenge by prior infection with M. leprae, although Shepard was unable to show protection by prior M. leprae infection in an earlier study (11). The work to be reported was undertaken in an attempt to confirm the
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findings of Kawaguchi and Shepard and also to 105, 120, 134, 148, and 178 days after primary ascertain the time of onset of the protection inoculation. The linear regression of the log,0 against homologous challenge relative to the number of AFB per footpad on the time after level of multiplication of the M. leprae of the inoculation for the primary infection reached primary inoculum at the time of secondary the plateau level of 106 AFB per footpad 109 days after inoculation. The secondary challenge challenge. inocula multiplied at strikingly similar rates, MATERIALS AND METHODS yielding times to plateau of 99 to 112 days. At Locally bred BALB/c mice were used. The strain of each interval, the multiplication of these secM. leprae had been originally isolated from a patient ondary inocula in the footpads of previously with lepromatous leprosy by C. C. Shepard (Center infected mice was much retarded, and in no for Disease Control, Atlanta, Ga.) and had been case did multiplication achieve the plateau carried in mouse passage both in Atlanta and in this laboratory. This strain has been used for virtually all level during 364 to 388 days of observation. A of the studies using the mouse footpad infection with close examination of the data reveals, however, that some multiplication of the secondary chalM. leprae in this laboratory. Mice were inoculated with 103-7 M. leprae in the lenge occurred in previously infected mice; in right hind footpad (RHF), and challenged with a the case of the 105-day challenge, for example, similar inoculum in the left hind footpad (LHF) at multiplication to the level of 105-5 AFB per some interval after the primary infection. Additional footpad (a 62-fold increase) occurred. (Increases mice, matched for age and sex, were inoculated only than twofold appear to be significant at with the primary inoculum or with a challenge inocu- greater lum. Harvests of M. leprae were performed at inter- the 95% level of confidence [unpublished vals from the pooled tissues of three to eight footpads data ]). In experiment m 4-10-73 (Fig. 2a) mice were from all groups of mice, by methods that have been described previously (10, 17). The results of the challenged in the LHF 14 days after primary harvests were used to construct bacterial growth infection in the RHF. The time-to-plateau of curves, from which the time required for multiplica- the M. Ieprae in the primary inoculum was 128 tion to 106 acid-fast bacilli (AFB) per footpad (the days, suggesting that the proportion of viable time to plateau) was determined for each inoculum. organisms in this inoculum was somewhat smaller than that in the inocula of the previous RESULTS experiment. The organisms in the secondary In experiment m 6-27-72 (Fig. 1), mice previ- challenge inoculum multiplied at the same rate ously inoculated in the RHF and uninfected in previously infected as in uninfected mice. control mice were challenged in the LHF 90, In a third experiment, experiment m 10-2-73
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FIG. 1. Challenge with M. Ieprae of mice previously infected with M. leprae. Mice were primarily infected in the RHF with 5 x 103 M. leprae, and challenged in the LHF with 5 x 103 M. leprae at intervals of 90 to 178 days after the primary infection. Previously uninfected mice were also inoculated with each challenge inoculum. Symbols: (0), primary infection and challenges of previously uninfected animals; (0), challenges of previously infected mice. The downward-extending arrows indicate that no AFB were seen at the time of harvest; the points are calculated as if one organism had been seen.
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FIG. 2. Challenge with M. leprae of mice previously infected with M. leprae. (a) Mice were challenged with 5 x 103 M. leprae in the LHF 15 days after primary infection in the RHF with 5 x 103 M. leprae. (b) Mice were challenged similarly at intervals of 48 to 106 days after primary infection. Symbols: (0) primary infection and challenges of previously uninfected mice; (0) challenges of previously infected mice.
(Fig. 2b), challenge of the LHF was carried out at 48, 62, 76, 92, and 106 days after primary infection of mice in the RHF. Multiplication of the challenge inocula was not so uniform as in experiment m 6-27-72; one achieved plateau level only 98 days after inoculation, whereas another required 134 days to reach this level. Despite the variability among challenge inocula, a comparison of the rate of multiplication in previously infected mice with that in uninfected mice may readily be made. Multiplication appears to have occurred at the same rate in previously infected as in uninfected mice for both the 48- and 62-day challenges. The difference in times to plateau for the 76-day challenge is significant at the 95% level of confidence, and multiplication is obviously slower in previously infected than in uninfected mice for the 92- and
reached the plateau level was slowed. Protection to homologous challenge appeared to be more solid when the secondary challenge was administered between 31 and 4 days before the primary infection had reached the plateau level of 106 per footpad, and was most evident when mice were challenged after the M. leprae of the primary inoculum had multiplied to plateau.
DISCUSSION The purpose of this study was twofold: (i) to confirm the results of earlier work by Shepard, who showed that prior M. leprae infection did not protect mice against homologous challenge, or, on the other hand, to confirm more recent reports by Kawaguchi and by Shepard that prior M. leprae infection did indeed confer protection; and (ii) to time the onset of protec106-day challenges. tion against homologous challenge conferred by Interpretation of the results of the work prior M. leprae infection. Shepard (11) had reported here is facilitated by relating the found that multiplication of M. leprae inocuadministration of the secondary challenge to the lated into the LHF of CFW mice infected with time to plateau of the primary inoculum rather M. leprae in the RHF 8 months earlier was not than to the time of primary inoculation. Thus, different from that in the LHF of uninfected there was no protection when the secondary control mice. Kawaguchi (4) found that challenge was administered 114, 75, and 61 days C57BL/6 mice inoculated into the RHF with before the primary inoculum had multiplied to 103 M. leprae were protected against challenge the plateau level, whereas the multiplication of in the LHF with 104-3 organisms 25 weeks later, a secondary challenge inoculum administered whereas no protection was demonstrated when 47 days before the primary inoculum had the secondary challenge was administered only
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5 weeks after primary infection. More recently, Shepard (Int. J. Leprosy, in press) reported that prior inoculation of CFW mice into the RHF with 103- M. Ieprae, which had multiplied to the level of 106 AFB per footpad by 225 days later, were protected against challenge with 103-7 organisms in the LHF 254 days after the primary infection. In the same experiment, Shepard showed that inoculation of mice in one hind footpad with 106 3 M. Ieprae, an inoculum too large to permit multiplication (10), protected mice against challenge in the opposite hind footpad with 103 7 organisms 4 weeks later. The results of the experiments reported here confirm that prior M. Ieprae infection confers protection against homologous challenge and appear to suggest two important qualifications. First, as Kawaguchi (4) suggested, timing of the secondary challenge is critical. Protection was evident only to challenges administered no earlier than 76 days after primary infection and appeared to become more solid the later the challenge was administered relative to the multiplication of the primary inoculum to the plateau level. Second, the protection was at no time absolute, in the sense that the M. Ieprae of the secondary challenge inoculum failed entirely to multiply. Challenge inocula administered between 47 and 17 days before the primary infection had reached the plateau level all multiplied to that level, but multiplication was delayed relative to multiplication in uninfected animals. The failure of Shepard's original study (11) to demonstrate protection may have resulted simply from his having performed too few harvests during logarithmic multiplication of the challenge inoculum. The challenge inocula of experiment m 6.27.72, administered between 19 days before to 69 days after the primary inoculum had reached plateau, all failed to achieve the plateau level, but multiplication to some level lower than 106 per footpad occurred in the case of all but the 148-day challenge. It appears easy to explain the timing of the onset of protection. If the lag phase of multiplication of M. Ieprae in mice may be assumed to be of the order of 30 days, then a challenge inoculum, administered 30 days before multiplication of the primary infection to the level of 106 organisms per footpad, would have begun to multiply only at the time that killing of the organisms of the primary infection was well established. Killing of the M. Ieprae at the site of the primary infection begins only after multiplication of the organisms has reached the level of 106 or so per footpad, and is undoubtedly the mechanism by which a limit to multiplication is imposed. Because killing begins only after mul-
tiplication of a small inoculum to this level but immediately after administration of a large inoculum, it appears that a critical concentration of antigen is required to initiate the chain of events that culminates in the killing of the M. leprae in situ, presumably by activated macrophages. At the time of a secondary challenge, administered after the primary inoculum has reached the plateau level, there should be no shortage of antigen nor of specifically sensitized lymphocytes. The difficulty occurs in trying to understand the two phenomena, perhaps related, that characterize the inhibited multiplication of M. leprae in the secondary challenge inocula, namely, slowing of multiplication during the logarithmic phase and the imposition of a lower ceiling to multiplication. The presence of small numbers of sensitized lymphocytes or of activated macrophages in the LHF of animals, in the RHF of which killing of M. Ieprae had already begun, may result in the killing of some M. Ieprae during logarithmic multiplication, resulting in apparent slowing of the process. The lower ceiling may reflect the presence in the animal, and possibly in the LHF, of a background of antigen, sensitized lymphocytes, or activated macrophages, that serves to decrease the critical concentration of antigen required to initiate the operation of the mechanism by which the M. leprae are prevented from multiplying further. The lower ceiling may also represent an artifact. All of the harvests represented by the data points of Fig. 1 and 2 were performed on the pooled tissues from three to eight footpads. Multiplication of M. leprae in the footpad of one mouse but not in those of the other mice included in the pool would be indistinguishable from the situation in which M. Ieprae multiplied to a lower ceiling in each of the footpads composing a pool. In fact, Shepard has recently shown (Int. J. Leprosy, in press) by harvesting footpads individually that M. leprae multiplied in some previously infected mice and not in others. Unfortunately, no harvests of single footpads were carried out in this study. Navalkar et al. (7) have suggested that some quantities of M. leprae antigen are detectable within a few days of inoculation of mice with 103 M. Ieprae, as demonstrated by the appearance of specific plaque-forming cells in the spleen and measurable titers of hemagglutinating antibody in the serum. The plaque-forming cell response decays rather rapidly, although the antibodies persist. Why a much larger number of organisms is required to initiate the immunological response that produces bacterial killing is unknown at this time. Perhaps a much
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FIG. 3. Challenge with M. leprae of mice previously infected with other mycobacteria. (a) Mice were challenged in both hind footpads with M. leprae 1 week after the last of four weekly subcutaneous injections of 104 to 106 viable BCG into the inguinal areas (2). (b) Mice were challenged in the LHF with M. leprae 27 days after primary infection in the RHF with viable M. marinum (8). (c) Mice inoculated in the LHF with 5 x 103 M. leprae were challenged in the RHF with viable M. marinum 70 days later. Symbols: (0), multiplication of M. leprae in control mice; (0), multiplication of M. leprae in mice previously infected with BCG (a) or M. marinum (b), or challenged with M. marinum during logarithmic multiplication of M. leprae (c). The downward-extending arrow indicates no AFB were seen at the time of harvest. 7he upward-extending arrow indicates the time of M. marinum challenge. ml1-30-70
0 0 Ui.
TIME AFTER INOCULATION (Days) FIG. 4. Challenge With M. leprae of mice previously infected with T. gondii (a) or B. jellisoni (b). Mice were challenged in both hind footpads with M. leprae 40 days after primary infection with T. gondii or B. jellisoni (5). Symbols: (0) multiplication of M. leprae in control mice; (0) multiplication of M. leprae in mice previously infected with Toxoplasma or Besnoitia.
larger concentration of antigen is required to stimulate the proliferation of specifically sensitized thymus-directed lymphocytes than that of antibody-producing cells. Or perhaps more antigen is required locally to attract the sensi-
tized lymphocytes or activated macrophages. The ability of M. leprae to multiply in the LHF after killing of M. leprae in the RHF had begun speaks for the second mechanism. Consistent with this hypothesis are the results of inocula-
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tion with M. leprae of mice previously infected with BCG (2, 11-13; Matsuo, Proc. Conf. Biol. Mycobacterioses. Abstr. 1, p. 1, 1967) (Fig. 3a), M. marinum (8) (Fig. 3b, and c), T. gondii and B. jellisoni (5) (Fig. 4). In each case some multiplication of M. leprae occurred. On the other hand, previous mycobacterial infection, which might have produced cross-reacting lymphocytes as well as activated macrophages, appeared to confer more solid protection than did previous protozoal infection, which could have produced only a population of activated macrophages. ACKNOWLEDGMENTS This work was supported in part by the U.S. Leprosy Panel of the U.S.-Japan Cooperative Medical Science Program administered by the Geographic Medicine Branch, National Institute of Allergy and Infectious Diseases. U.S. Public Health Service grant R22 AI-07801. The dedicated assistance of Jeraldine A. Anandan, James E. Lynch, Lydia P. Murray, Nancy M. Ullmann. and Sheila M. O'Neill is gratefully recognized. LITERATURE CITED 1. Evans, M. J., and L. Levy. 1972. Ultrastructural changes
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in cells of the mouse footpad infected with Mycobacterium leprae. Infect. Immun. 5:238-247. Evans, M. J., H. E. Newton, and L. Levy. 1973. Early response of mouse footpads to Mycobacterium leprae. Infect. Immun. 7:76-85. Gaugas. J. M. 1968. Enhancing effect of antilymphocytic globulin on human leprosy infection in thymectomized mice. Nature (London) 220:1246-1248. Kawaguchi, Y. 1972. Superinfection with leprosy bacilli in mice. Int. J. Leprosy 40:91-92. Krahenbuhl, J. L., L. Levy, and J. S. Remington. 1974.
Resistance to Mycobacterum leprae in mice infected with Toxoplasma gondii and Bensnoitia jellisoni. Infect. Immun. 10:1068-1071. 6. Levy, L. 1970. Death of Mycobacterium leprae in mice and the additional effect of dapsone administration. Proc. Soc. Exp. Biol. Med. 135:745-749. 7. Navalkar, R. G., P. H. Patel, R. R. Dalvi, and L. Levy. 1975. Immune response to Mycobacterium leprae: plaque-forming cells in mice. Infect. Immun. 10:13021306. 8. Ng, H., P. L. Jacobsen, and L. Levy. 1973. Analogy of Mycobacterium marinum disease to Mvcobacterium leprae infection in footpads of mice. Infect. Immun. 8:860-867. 9. Rees, R. J. W. 1966. Enhanced susceptibility of thymectomized and irradiated mice to infection with Mycobacterium leprae. Nature (London) 211:657-658. 10. Shepard, C. C. 1960. The experimental disease that follows the injection of human leprosy bacilli into footpads of mice. J. Exp. Med. 112:445-454. 11. Shepard, C. C. 1965. Vaccination against experimental infection with Mycobacterium leprae. Am. J. Epidemiol. 81:150-163. 12. Shepard, C. C. 1966. Vaccination against human leprosy bacillus infections of mice: protection by BCG given during the incubation period. J. Immunol. 96:279-283. 13. Shepard, C. C. 1968. A comparison of the effectiveness of two freeze-dried BCG vaccines against Mycobacterium leprae in mice. Bull. W.H.O. 38:135-140. 14. Shepard, C. C., and Y. T. Chang. 1967. Effect of DDS on established infections with Mycobacterium leprae in mice. Int. J. Leprosy 35:52-57. 15. Shepard, C. C., and C. C. Congdon. 1968. Increased growth of Mycobacterium leprae in thymectomizedirradiated mice after footpad inoculation. Int. J. Leprosy 36:224-227. 16. Shepard, C. C., and D. H. McRae. 1965. Mycobacterium leprae in mice: minimal infectious dose, relationship between staining quality and infectivity, and effect of cortisone. J. Bacteriol. 93:790-796. 17. Shepard, C. C., and D. H. McRae. 1968. A method for counting acid-fast bacteria. Int. J. Leprosy 36:78-82.