INFECTION AND IMMUNITY, May 1975, p. 1122-1132 Copyright © 1975 American Society for Microbiology
Vol. 11, No. 5 Printed in U.S.A.
Susceptibility of Thymectomized and Irradiated Mice to Challenge with Several Organisms and the Effect of Dapsone on Infection with Mycobacterium leprae LOUIS LEVY,* HERMAN NG, MICHAEL J. EVANS,
AND
JAMES L. KRAHENBUHL
Public Health Service Hospital, San Francisco, California 94118*; Stanford Research Institute, Menlo Park, California 94025; and Palo Alto Medical Research Foundation, Palo Alto, California 94301 Received for publication 26 December 1974
B6C3F, mice that had been thymectomized at 8 to 12 weeks of age, subjected to 950 R of whole-body X irradiation, and transfused with syngeneic bone marrow challenged in a footpad with Mycobacterium leprae or M. marinum, or intravenously or intraperitoneally with Listeria monocytogenes. Also, mice inoculated with M. leprae in a hind footpad were administered dapsone in the mouse chow. The thymectomized-irradiated (T + R) mice did not survive as well as non-thymectomized mice when housed in the vivarium with no special precautions, but survived sufficiently well to permit the completion of some long-term experiments. M. leprae multiplied to a higher "ceiling" and survived longer in the T + R mice than in the non-thymectomized controls. But a ceiling to multiplication of M. leprae was imposed, and finally the organisms were killed. The histopathological appearance of the footpad tissues, studied by electron microscopy, was consistent with the measurements of bacterial numbers and viability. Swelling of the footpad after local inoculation with M. marinum was greater in T + R mice than in non-thymectomized controls. Similarly, the number of L. monocytogenes following intravenous challenge was greater in the spleens of T + R than of non-thymectomized mice, and the survival of the T + R mice was impaired after intraperitoneal challenge with L. monocytogenes, compared to the survival of non-thymectomized mice. None of these differences was striking, suggesting that these T + R mice had retained or regained some immune competence. The effects of dapsone treatment of T + R mice inoculated with M. leprae were much the same as those of treatment of non-thymectomized mice. Because these T + R mice were not greatly immunosuppressed, they would not have provided a model of human lepromatous leprosy suitable for chemotherapeutic studies.
were
When a small number (103 7 to 104) of Mycobacterium leprae are inoculated into the footpads of immunologically normal mice of a number of strains, the organisms multiply to a maximum of 106 to 106.3 per footpad. Thereafter, although the number of acid-fast bacilli (AFB) changes only to a very limited degree, the number of viable organisms decreases rapidly, sometimes to an undetectable level (3, 13, 17). When mice are given a large inoculum (105 or more), the M. leprae do not multiply, and bacterial killing begins within a few days after inoculation (2, 12, 13). Inoculation of the footpads of normal mice with M. leprae does not produce lepromatous leprosy, but the limited multiplication of the organism serves as the basis of a very useful laboratory technique (16). In an effort to remove the barrier to multiplication of M. leprae, Rees (11) performed thy1122
mectomy of adult mice followed by lethal wholebody X irradiation and syngeneic bone marrow transfusion. In these mice, the organisms multiplied to a much higher limit, and gross evidence of disease as well as dissemination of the process were apparent in some of the mice so treated (11). However, no investigator working in North America has enjoyed Rees' success. Shepard and Congdon (18) reported multiplication of M. leprae to a level of almost 108 per footpad in 101/C3H hybrid mice that had been thymectomized at 3 months of age, irradiated, and transfused with bone marrow. Evidence of dissemination of the process was found, but no gross disease was encountered. The work to be reported is one result of an effort by the U.S. Leprosy Panel to supply thymectomized-irradiated (T + R) mice to leprosy workers with which to attempt to produce unlimited
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1123
multiplication of M. leprae. In this report, the Stroudsburg, Pa.); the drug was added as a crystalline results of inoculation of T + R B6C3F, mice solid to make the largest concentration (0.1%) and with M. leprae, M. marinum, and Listeria dissolved in ethanol for the smaller concentrations monocytogenes are described. The results of the (0.0001, 0.001, and 0.01%). administration of dapsone (4,4'-diaminodipheRESULTS nylsulfone; DDS) to T + R mice inoculated with The mortality of the B6C3F, T + R mice was M. leprae are also reported. greater than that of non-thymectomized mice. MATERIALS AND METHODS The mortality varied among the subgroups of T + R B6C3Fl ([C57BI/6 9 x C3H/AnF 8]Fl) T + R mice; on the average, 50% of the mice hybrid mice were supplied by C. C. Congdon, Oak were dead within 10 months after inoculation Ridge National Laboratory, Oak Ridge, Tenn., under (see Fig. 1). By contrast, only 5% of a large the terms of an interagency agreement between the group of BALB/c mice were dead 10 months National Institute of Allergy and Infectious Diseases after inoculation, and none of a small group of and the Atomic Energy Commission. Mice were nonthymectomized B6C3F, mice were dead 6 thymectomized at 8 to 12 weeks of age; 8 or 9 days months after inoculation, when the experiment later, the mice were subjected to 950 R of whole-body X irradiation and transfused on the same day with was terminated. Congdon has reported (C. C. 106.7 syngeneic bone marrow cells obtained from Congdon, personal communication) nearly 25% 16-week-old donors. Mice were shipped within a few mortality 1 year after thymectomy of B6C3F, weeks of irradiation. Upon arrival in San Francisco, mice, and a little more than 50% mortality after the mice were housed with no special precautions in 14 to 15 months. That the mortality among the vivarium of the Public Health Service Hospital. these mice appears to be greater than that Non-thymectomized B6C3F, mice, furnished by reported by Congdon may merely reflect the Congdon, and locally bred BALB/c mice were also fact that, in this laboratory, mortality was used in this research. calculated from the time of inoculation with M. Mice were inoculated with a strain of M. leprae leprae; the mice were shipped by Congdon carried in mouse passage; harvests of M. leprae from pooled mouse footpad tissues were performed by several weeks after thymectomy and were somemethods that have previously been described (13, 20). times held for several months before inoculaFootpads were fixed for electron microscopy in either tion. The multiplication of M. leprae was studied 2% glutaraldehyde buffered with cacodylate to pH 7.2, followed by postflxation in 1% OSO4 containing in three types of experiments. Both non7.5% sucrose buffered with veronal acetate to pH 7.2, thymectomized and T + R B6C3F, mice were or in the OSO4 fixative alone. After fixation, the inoculated with 103 7organisms per footpad. tissues were dehydrated in a graded alcohol series and T + R mice were inoculated with larger numembedded flat in BEEM capsules in Araldite or Spurr low-viscosity embedding media. Specimens for light bers of organisms. The results of harvests of M. microscopy were sectioned with a glass knife on a leprae from the footpads of these mice at Porter Blum MT2 ultramicrotome at 1.0 im, placed intervals after inoculation are shown in Fig. 2. on glass slides, and stained with toluidine blue. Curves A and B, characterized by a peak near Specimens for electron microscopy were sectioned 106 organisms per footpad between 100 and 150 with a diamond knife, placed on collodion-coated days after inoculation, represent the growth nickel grids, stained with uranyl acetate and lead curves of M. leprae in non-thymectomized citrate, and observed with a Philips 200 electron microscope. Mice were inoculated into the hind footpads with a standard inoculum of M. marinum that had been quick-frozen and stored at -70 C (10). The challenge of mice with L. monocytogenes (type 4a) was carried out by using the appropriate dilution of a stock suspension of bacteria frozen at -90 C in Trypticase soy broth (Difco) containing 1% gelatin. Resistance to Listeria was measured by recording the time to death after intraperitoneal (i.p.) challenge or by counting the number of viable Listeria in the spleens of groups of mice 24 and 48 h after intravenous (i.v.) challenge with Listeria. Spleens were removed and homogenized, and 10-fold serial dilutions were plated on Trypticase soy agar (Difco). Male mice were used for the study of the effects of DDS; female mice were used for all of the other studies. M. leprae multiplied identically in mice of both sexes. DDS was mixed in the mouse meal by means of a liquid-solid blender (Patterson-Kelly Co., East
100
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NO. MONTHS AFTER INOCULATION
FIG. 1. Percentage of survival of B6C3F, T + R mice as a function of the time after inoculation with M. leprae. Symbols: ( ), B6C3F1 mice; (- -), J3ALB/c mice; (-), non-thymectomized mice; (0), T + R mice
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FIG. 2. Log number of M. leprae per footpad as a function of time after inoculation of B6C3F1 mic. Symbols: (@), non-thymectomized mice; (0), T + R mice. Bacterial growth curves are labeled as follows: (A) experiment m 11-30-70 controls; (B) experiment m 4-19-71 controls; (C) experiment m 11-30-70; (D) experiment m 4-19-71; (E) experiment m 5-19-71, small inoculum; (F) experiment m 6-1-71; (G) experiment m 5-19-71, large inoculum; (H) experiment m 12-6-72. The small numbers indicate the times to plateau of organisms passaged to BALBIc mice from the harvest indicated. contrast to these curves are C to F, which represent the multiplication of M. leprae in B6C3F, T + R mice. These growth curves increase initially at the same rate as do those for multiplication in non-thymectomized mice and continue to increase, although with a diminishing rate, reaching a plateau between 107 and 107-7 AFB per footpad after about 200 days. The same plateau is reached when larger inocula are used (curve G, 105-4, and curve H, 104.8 per footpad). In the cases of curves C and D, a decrease in the number of organisms was observed in harvests performed after 350 days; the 399-day harvest in the
B6C3F, mice. In
curves
experiment represented by curve C yielded 106-7
AFB, and the 437-day harvest in the experiment represented by curve D, not shown in Fig. 2, yielded 106.5 AFB per footpad. M. leprae harvested 169 days and 231 days after inoculation of the mice of curve C and 180 days after inoculation of the mice of curve H multiplied rapidly in passage, reaching the level of 106 per footpad 106 to 112 days after passage. On the other hand, the organisms harvested 322 days after inoculation of the mice of curve C
required 138 days to achieve the same level, and those harvested after 399 days required 374 days to reach the plateau level of 106 M. leprae per footpad in passage mice. With given strains of mice and of M. leprae, the time from inoculation to multiplication to the level of 106 AFB per footpad should be directly proportional to the proportion of viable organisms in the inoculum (4). If the doubling time of this strain of organisms is about 2 weeks and the duration of the lag phase about 30 days, one viable organism will multiply to 106 per footpad (20 doublings) in approximately 370 days. Thus, the proportion of viable M. leprae in the 399-day harvest from the mice of curve C was no larger than 5 per 5,000. (Because about 80% of an inoculum is lost from the footpad within the first hour after inoculation, an inoculum of 5,000 organisms must contain at least five viable M. leprae in order to provide the single viable necessary for infection [8].) On the other hand, multiplication to 106 organisms per footpad within 106 to 112 days suggests that virtually all of the organisms in these inocula were viable.
VOL. 11, 1975
M. LEPRAE CHALLENGE OF T + R MICE
Histological study of the footpad tissues was carried out in animals sacrificed at various phases of the growth curves described in the previous section (Fig. 2). The infection involved primarily cells of the connective tissue and to a lesser degree nerve and muscle tissues. An example of the area of tissue studied is presented in Fig. 3. During multiplication, organisms were found only in connective tissue cells, primarily in macrophages (Fig. 4a and 5a). Early in bacterial multiplication, macrophages containing organisms were observed in small groups (Fig. 5a), and most organisms were found free in the cytoplasm of the cells (Fig. 5b). As multiplication progressed, the number of macrophages containing organisms increased (Fig. 4a). Late in multiplication, however, many macrophages appeared activated and organisms were observed in phagosomes, often associated with lysosomes (Fig. 6). At this time, organisms were also found in other types of connective tissue cells. Organisms were found in the lipid portion of fat cells and free in the cytoplasm of fibroblasts in tendons (Fig. 4b, 5c,
1125
and d). In both cell types, the organisms appeared solid. After the plateau had been reached at about 200 days, organisms were found in muscle and nerve in addition to the previously mentioned cell types (Fig. 4c and d). For the most part, macrophages appeared activated and the organisms were found in phagosomes associated with lysosomes. Later after plateau, many organisms appeared to be degenerating, and many globi were observed. These clusters of organisms were found both free in the cytoplasm and surrounded by phagosomes. Globi in phagosomes often contained many degenerate organisms. The organisms in fat cells were frequently solid, lying free in the lipid portion of the cell. Organisms in muscle also appeared solid and were found free in the sarcoplasm of the cell, both singly and in clusters. Organisms in nerve bundles were found in macrophages between individual fibers and occasionally free in the cytoplasm of Schwann cells. At no time was a large mononuclear cell infiltrate noted. However, the number of tissue macrophages
FIG. 3. Cross-section of a mouse footpad. x 125.
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increased. These results are similar to those reported previously for immunologically intact mice (1). During logarithmic multiplication, most macrophages containing organisms do not appear activated, whereas after multiplication ceases and a stationary phase is reached, most macrophages appear activated. Cessation of multiplication of the organisms associated with actiwas
vation of macrophages suggests that the T + R mice were not totally depressed immunologically. The main differences noted between this and the previous study were the lack of a large mononuclear cell infiltrate during the stationary phase and the presence of organisms in cell types other than macrophages and muscle. The first difference probably reflects the depressed immune response of these animals; the second
1127
M. LEPRAE CHALLENGE OF T + R MICE
VOL. 11, 1975
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FIG. 6. Organisms in macrophages during the late stages of multiplication. (A) Organisms free in the cytoplasm, and (B) those associated with phagosomes and lysosomes. x23,600.
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M. LEPRAE CHALLENGE OF T + R MICE
may 1 )e associated with the longer multiplication p]hase and the greater number of organisms presenlt. The results of treatment with DDS of B6C3F1 T + R mice inoculated with M. leprae are summ arized in Fig. 7, which is a semilogarithmic p ilot of the number of AFB harvested per footpaid as a function of time after inoculation. The ciurve representing the multiplication of M. leprae? in untreated B6C3F, T + R mice (curve A), thoe same curve as curve D in Fig, 2, reveals a doul bling time during logarithmic multiplication (1between 91 and 147 days) of about 12 days. Extra] polation of this curve back to day 70 shows that t: he organisms had multiplied to a level of 101.5 Vvhen treatment was begun. The first two harvessts of the mice treated with the smallest conceintration of DDS (curve B), both performe d during administration of the drug, yielde d approximately 105 5 AFB per footpad. This souggests that multiplication had continued for at )out 45 days, a little more than three doubliings, after DDS treatment was begun. Multi]plication of M. Ieprae appears to have resumled immediately after drug administration was sttopped, at the same rate as in the control mice. The harvests made during drug administratiorn to the mice receiving larger concentrations of DDS all yielded 104 6 or fewer AFB,
1129
indicating that multiplication had ceased immediately when drug administration was begun. Multiplication of the organisms resumed immediately after treatment with 0.001% DDS was stopped (curve C), also at the same rate as in the untreated mice. After the administration of 0.01% DDS was stopped (curve D), there was a delay of about 40 or 50 days before multiplication of M. leprae resumed. The results of later harvests of the mice administered 0.1% DDS (curve E) are more difficult to interpret. Because of a shortage of mice, no harvests were performed between 205 and 387 days; simply connecting the points representing these two harvests suggests that the organisms multiplied slowly during this interval. However, the yield of 104-7 AFB per footpad from the 205-day harvest may not represent multiplication; it appears likely that additional harvests between 205 and 300 days would have yielded similarly small number of organisms. In summary, multiplication of M. leprae continued for about three generations after administration of 0.0001% DDS was begun to B6C3F, T + R mice, whereas it ceased immediately when larger concentrations of the drug were administered. Multiplication of M. leprae resumed immediately after administration of DDS in concentrations of 0.0001 and 0.001% was stopped, whereas larger concentrations produced a delay of resumption of multiplication characteristic of bacterial killing or prolonged bacteriostasis (4, 5, 15). 7.0B6C3F1 control mice, two groups of B6C3F1 / */ / T + R mice, and BALB/c mice were inoculated each in both hind footpads with 103 9 M. mari0 num, and the development of the pathological /C /D A^ /a 6.0process was followed by repeated measurements of footpad thickness. (Footpad thickness was measured with a Schnelltaster [H. C. Kroplin, 0 Schiichtern, Germany; available as Quicktest 0 zi from The Dyer Co., Inc., Lancaster, Pa.]. The 5.0ci precision of this measurement was ±0.10 mm.) The results of this experiment are presented in Fig. 8. Footpad thickness was maximal in all four experimental groups between 12 and 22 days after inoculation. BALB/c mice are 4.0smaller than B6C3Fl mice; swelling of the DoS footpads of these mice occurred generally to a 7 100 200 3X smaller degree than was the case for the non4 TIME AFTER INOCULATION (day.) thymectomized B6C3F, mice. Footpad thickening was generally greater in the T + R mice than FiG. 7. Log number of M. Leprae per footpad as a functio n of time after inoculation of B6C3F, T + R m the non-thymectomized controls. There was mice. S ymbols: (-),untreated mice; (0), mice treated no significant difference in the degree of thickwith DiDS during the period indicated by the shaded ening of the footpads between young (prepared bar bel ow. Curve A, untreated controls; B, 0.0001% about 10 weeks prior to inoculation) and old DDS; C 0.001% DDS; D, 0.01% DDS; E, 0.1% DDS. (prepared a year earlier) T + R mice. The 95% 0
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LEVY ET AL.
spontaneously or after some intervention that impairs immune function, is much sought after, D,
Vol. 11, No. 5 Printed in U.S.A.
Susceptibility of Thymectomized and Irradiated Mice to Challenge with Several Organisms and the Effect of Dapsone on Infection with Mycobacterium leprae LOUIS LEVY,* HERMAN NG, MICHAEL J. EVANS,
AND
JAMES L. KRAHENBUHL
Public Health Service Hospital, San Francisco, California 94118*; Stanford Research Institute, Menlo Park, California 94025; and Palo Alto Medical Research Foundation, Palo Alto, California 94301 Received for publication 26 December 1974
B6C3F, mice that had been thymectomized at 8 to 12 weeks of age, subjected to 950 R of whole-body X irradiation, and transfused with syngeneic bone marrow challenged in a footpad with Mycobacterium leprae or M. marinum, or intravenously or intraperitoneally with Listeria monocytogenes. Also, mice inoculated with M. leprae in a hind footpad were administered dapsone in the mouse chow. The thymectomized-irradiated (T + R) mice did not survive as well as non-thymectomized mice when housed in the vivarium with no special precautions, but survived sufficiently well to permit the completion of some long-term experiments. M. leprae multiplied to a higher "ceiling" and survived longer in the T + R mice than in the non-thymectomized controls. But a ceiling to multiplication of M. leprae was imposed, and finally the organisms were killed. The histopathological appearance of the footpad tissues, studied by electron microscopy, was consistent with the measurements of bacterial numbers and viability. Swelling of the footpad after local inoculation with M. marinum was greater in T + R mice than in non-thymectomized controls. Similarly, the number of L. monocytogenes following intravenous challenge was greater in the spleens of T + R than of non-thymectomized mice, and the survival of the T + R mice was impaired after intraperitoneal challenge with L. monocytogenes, compared to the survival of non-thymectomized mice. None of these differences was striking, suggesting that these T + R mice had retained or regained some immune competence. The effects of dapsone treatment of T + R mice inoculated with M. leprae were much the same as those of treatment of non-thymectomized mice. Because these T + R mice were not greatly immunosuppressed, they would not have provided a model of human lepromatous leprosy suitable for chemotherapeutic studies.
were
When a small number (103 7 to 104) of Mycobacterium leprae are inoculated into the footpads of immunologically normal mice of a number of strains, the organisms multiply to a maximum of 106 to 106.3 per footpad. Thereafter, although the number of acid-fast bacilli (AFB) changes only to a very limited degree, the number of viable organisms decreases rapidly, sometimes to an undetectable level (3, 13, 17). When mice are given a large inoculum (105 or more), the M. leprae do not multiply, and bacterial killing begins within a few days after inoculation (2, 12, 13). Inoculation of the footpads of normal mice with M. leprae does not produce lepromatous leprosy, but the limited multiplication of the organism serves as the basis of a very useful laboratory technique (16). In an effort to remove the barrier to multiplication of M. leprae, Rees (11) performed thy1122
mectomy of adult mice followed by lethal wholebody X irradiation and syngeneic bone marrow transfusion. In these mice, the organisms multiplied to a much higher limit, and gross evidence of disease as well as dissemination of the process were apparent in some of the mice so treated (11). However, no investigator working in North America has enjoyed Rees' success. Shepard and Congdon (18) reported multiplication of M. leprae to a level of almost 108 per footpad in 101/C3H hybrid mice that had been thymectomized at 3 months of age, irradiated, and transfused with bone marrow. Evidence of dissemination of the process was found, but no gross disease was encountered. The work to be reported is one result of an effort by the U.S. Leprosy Panel to supply thymectomized-irradiated (T + R) mice to leprosy workers with which to attempt to produce unlimited
VOL. 11, 1975
M. LEPRAE CHALLENGE OF T + R MICE
1123
multiplication of M. leprae. In this report, the Stroudsburg, Pa.); the drug was added as a crystalline results of inoculation of T + R B6C3F, mice solid to make the largest concentration (0.1%) and with M. leprae, M. marinum, and Listeria dissolved in ethanol for the smaller concentrations monocytogenes are described. The results of the (0.0001, 0.001, and 0.01%). administration of dapsone (4,4'-diaminodipheRESULTS nylsulfone; DDS) to T + R mice inoculated with The mortality of the B6C3F, T + R mice was M. leprae are also reported. greater than that of non-thymectomized mice. MATERIALS AND METHODS The mortality varied among the subgroups of T + R B6C3Fl ([C57BI/6 9 x C3H/AnF 8]Fl) T + R mice; on the average, 50% of the mice hybrid mice were supplied by C. C. Congdon, Oak were dead within 10 months after inoculation Ridge National Laboratory, Oak Ridge, Tenn., under (see Fig. 1). By contrast, only 5% of a large the terms of an interagency agreement between the group of BALB/c mice were dead 10 months National Institute of Allergy and Infectious Diseases after inoculation, and none of a small group of and the Atomic Energy Commission. Mice were nonthymectomized B6C3F, mice were dead 6 thymectomized at 8 to 12 weeks of age; 8 or 9 days months after inoculation, when the experiment later, the mice were subjected to 950 R of whole-body X irradiation and transfused on the same day with was terminated. Congdon has reported (C. C. 106.7 syngeneic bone marrow cells obtained from Congdon, personal communication) nearly 25% 16-week-old donors. Mice were shipped within a few mortality 1 year after thymectomy of B6C3F, weeks of irradiation. Upon arrival in San Francisco, mice, and a little more than 50% mortality after the mice were housed with no special precautions in 14 to 15 months. That the mortality among the vivarium of the Public Health Service Hospital. these mice appears to be greater than that Non-thymectomized B6C3F, mice, furnished by reported by Congdon may merely reflect the Congdon, and locally bred BALB/c mice were also fact that, in this laboratory, mortality was used in this research. calculated from the time of inoculation with M. Mice were inoculated with a strain of M. leprae leprae; the mice were shipped by Congdon carried in mouse passage; harvests of M. leprae from pooled mouse footpad tissues were performed by several weeks after thymectomy and were somemethods that have previously been described (13, 20). times held for several months before inoculaFootpads were fixed for electron microscopy in either tion. The multiplication of M. leprae was studied 2% glutaraldehyde buffered with cacodylate to pH 7.2, followed by postflxation in 1% OSO4 containing in three types of experiments. Both non7.5% sucrose buffered with veronal acetate to pH 7.2, thymectomized and T + R B6C3F, mice were or in the OSO4 fixative alone. After fixation, the inoculated with 103 7organisms per footpad. tissues were dehydrated in a graded alcohol series and T + R mice were inoculated with larger numembedded flat in BEEM capsules in Araldite or Spurr low-viscosity embedding media. Specimens for light bers of organisms. The results of harvests of M. microscopy were sectioned with a glass knife on a leprae from the footpads of these mice at Porter Blum MT2 ultramicrotome at 1.0 im, placed intervals after inoculation are shown in Fig. 2. on glass slides, and stained with toluidine blue. Curves A and B, characterized by a peak near Specimens for electron microscopy were sectioned 106 organisms per footpad between 100 and 150 with a diamond knife, placed on collodion-coated days after inoculation, represent the growth nickel grids, stained with uranyl acetate and lead curves of M. leprae in non-thymectomized citrate, and observed with a Philips 200 electron microscope. Mice were inoculated into the hind footpads with a standard inoculum of M. marinum that had been quick-frozen and stored at -70 C (10). The challenge of mice with L. monocytogenes (type 4a) was carried out by using the appropriate dilution of a stock suspension of bacteria frozen at -90 C in Trypticase soy broth (Difco) containing 1% gelatin. Resistance to Listeria was measured by recording the time to death after intraperitoneal (i.p.) challenge or by counting the number of viable Listeria in the spleens of groups of mice 24 and 48 h after intravenous (i.v.) challenge with Listeria. Spleens were removed and homogenized, and 10-fold serial dilutions were plated on Trypticase soy agar (Difco). Male mice were used for the study of the effects of DDS; female mice were used for all of the other studies. M. leprae multiplied identically in mice of both sexes. DDS was mixed in the mouse meal by means of a liquid-solid blender (Patterson-Kelly Co., East
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NO. MONTHS AFTER INOCULATION
FIG. 1. Percentage of survival of B6C3F, T + R mice as a function of the time after inoculation with M. leprae. Symbols: ( ), B6C3F1 mice; (- -), J3ALB/c mice; (-), non-thymectomized mice; (0), T + R mice
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FIG. 2. Log number of M. leprae per footpad as a function of time after inoculation of B6C3F1 mic. Symbols: (@), non-thymectomized mice; (0), T + R mice. Bacterial growth curves are labeled as follows: (A) experiment m 11-30-70 controls; (B) experiment m 4-19-71 controls; (C) experiment m 11-30-70; (D) experiment m 4-19-71; (E) experiment m 5-19-71, small inoculum; (F) experiment m 6-1-71; (G) experiment m 5-19-71, large inoculum; (H) experiment m 12-6-72. The small numbers indicate the times to plateau of organisms passaged to BALBIc mice from the harvest indicated. contrast to these curves are C to F, which represent the multiplication of M. leprae in B6C3F, T + R mice. These growth curves increase initially at the same rate as do those for multiplication in non-thymectomized mice and continue to increase, although with a diminishing rate, reaching a plateau between 107 and 107-7 AFB per footpad after about 200 days. The same plateau is reached when larger inocula are used (curve G, 105-4, and curve H, 104.8 per footpad). In the cases of curves C and D, a decrease in the number of organisms was observed in harvests performed after 350 days; the 399-day harvest in the
B6C3F, mice. In
curves
experiment represented by curve C yielded 106-7
AFB, and the 437-day harvest in the experiment represented by curve D, not shown in Fig. 2, yielded 106.5 AFB per footpad. M. leprae harvested 169 days and 231 days after inoculation of the mice of curve C and 180 days after inoculation of the mice of curve H multiplied rapidly in passage, reaching the level of 106 per footpad 106 to 112 days after passage. On the other hand, the organisms harvested 322 days after inoculation of the mice of curve C
required 138 days to achieve the same level, and those harvested after 399 days required 374 days to reach the plateau level of 106 M. leprae per footpad in passage mice. With given strains of mice and of M. leprae, the time from inoculation to multiplication to the level of 106 AFB per footpad should be directly proportional to the proportion of viable organisms in the inoculum (4). If the doubling time of this strain of organisms is about 2 weeks and the duration of the lag phase about 30 days, one viable organism will multiply to 106 per footpad (20 doublings) in approximately 370 days. Thus, the proportion of viable M. leprae in the 399-day harvest from the mice of curve C was no larger than 5 per 5,000. (Because about 80% of an inoculum is lost from the footpad within the first hour after inoculation, an inoculum of 5,000 organisms must contain at least five viable M. leprae in order to provide the single viable necessary for infection [8].) On the other hand, multiplication to 106 organisms per footpad within 106 to 112 days suggests that virtually all of the organisms in these inocula were viable.
VOL. 11, 1975
M. LEPRAE CHALLENGE OF T + R MICE
Histological study of the footpad tissues was carried out in animals sacrificed at various phases of the growth curves described in the previous section (Fig. 2). The infection involved primarily cells of the connective tissue and to a lesser degree nerve and muscle tissues. An example of the area of tissue studied is presented in Fig. 3. During multiplication, organisms were found only in connective tissue cells, primarily in macrophages (Fig. 4a and 5a). Early in bacterial multiplication, macrophages containing organisms were observed in small groups (Fig. 5a), and most organisms were found free in the cytoplasm of the cells (Fig. 5b). As multiplication progressed, the number of macrophages containing organisms increased (Fig. 4a). Late in multiplication, however, many macrophages appeared activated and organisms were observed in phagosomes, often associated with lysosomes (Fig. 6). At this time, organisms were also found in other types of connective tissue cells. Organisms were found in the lipid portion of fat cells and free in the cytoplasm of fibroblasts in tendons (Fig. 4b, 5c,
1125
and d). In both cell types, the organisms appeared solid. After the plateau had been reached at about 200 days, organisms were found in muscle and nerve in addition to the previously mentioned cell types (Fig. 4c and d). For the most part, macrophages appeared activated and the organisms were found in phagosomes associated with lysosomes. Later after plateau, many organisms appeared to be degenerating, and many globi were observed. These clusters of organisms were found both free in the cytoplasm and surrounded by phagosomes. Globi in phagosomes often contained many degenerate organisms. The organisms in fat cells were frequently solid, lying free in the lipid portion of the cell. Organisms in muscle also appeared solid and were found free in the sarcoplasm of the cell, both singly and in clusters. Organisms in nerve bundles were found in macrophages between individual fibers and occasionally free in the cytoplasm of Schwann cells. At no time was a large mononuclear cell infiltrate noted. However, the number of tissue macrophages
FIG. 3. Cross-section of a mouse footpad. x 125.
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increased. These results are similar to those reported previously for immunologically intact mice (1). During logarithmic multiplication, most macrophages containing organisms do not appear activated, whereas after multiplication ceases and a stationary phase is reached, most macrophages appear activated. Cessation of multiplication of the organisms associated with actiwas
vation of macrophages suggests that the T + R mice were not totally depressed immunologically. The main differences noted between this and the previous study were the lack of a large mononuclear cell infiltrate during the stationary phase and the presence of organisms in cell types other than macrophages and muscle. The first difference probably reflects the depressed immune response of these animals; the second
1127
M. LEPRAE CHALLENGE OF T + R MICE
VOL. 11, 1975
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VOL. 11, 1975
M. LEPRAE CHALLENGE OF T + R MICE
may 1 )e associated with the longer multiplication p]hase and the greater number of organisms presenlt. The results of treatment with DDS of B6C3F1 T + R mice inoculated with M. leprae are summ arized in Fig. 7, which is a semilogarithmic p ilot of the number of AFB harvested per footpaid as a function of time after inoculation. The ciurve representing the multiplication of M. leprae? in untreated B6C3F, T + R mice (curve A), thoe same curve as curve D in Fig, 2, reveals a doul bling time during logarithmic multiplication (1between 91 and 147 days) of about 12 days. Extra] polation of this curve back to day 70 shows that t: he organisms had multiplied to a level of 101.5 Vvhen treatment was begun. The first two harvessts of the mice treated with the smallest conceintration of DDS (curve B), both performe d during administration of the drug, yielde d approximately 105 5 AFB per footpad. This souggests that multiplication had continued for at )out 45 days, a little more than three doubliings, after DDS treatment was begun. Multi]plication of M. Ieprae appears to have resumled immediately after drug administration was sttopped, at the same rate as in the control mice. The harvests made during drug administratiorn to the mice receiving larger concentrations of DDS all yielded 104 6 or fewer AFB,
1129
indicating that multiplication had ceased immediately when drug administration was begun. Multiplication of the organisms resumed immediately after treatment with 0.001% DDS was stopped (curve C), also at the same rate as in the untreated mice. After the administration of 0.01% DDS was stopped (curve D), there was a delay of about 40 or 50 days before multiplication of M. leprae resumed. The results of later harvests of the mice administered 0.1% DDS (curve E) are more difficult to interpret. Because of a shortage of mice, no harvests were performed between 205 and 387 days; simply connecting the points representing these two harvests suggests that the organisms multiplied slowly during this interval. However, the yield of 104-7 AFB per footpad from the 205-day harvest may not represent multiplication; it appears likely that additional harvests between 205 and 300 days would have yielded similarly small number of organisms. In summary, multiplication of M. leprae continued for about three generations after administration of 0.0001% DDS was begun to B6C3F, T + R mice, whereas it ceased immediately when larger concentrations of the drug were administered. Multiplication of M. leprae resumed immediately after administration of DDS in concentrations of 0.0001 and 0.001% was stopped, whereas larger concentrations produced a delay of resumption of multiplication characteristic of bacterial killing or prolonged bacteriostasis (4, 5, 15). 7.0B6C3F1 control mice, two groups of B6C3F1 / */ / T + R mice, and BALB/c mice were inoculated each in both hind footpads with 103 9 M. mari0 num, and the development of the pathological /C /D A^ /a 6.0process was followed by repeated measurements of footpad thickness. (Footpad thickness was measured with a Schnelltaster [H. C. Kroplin, 0 Schiichtern, Germany; available as Quicktest 0 zi from The Dyer Co., Inc., Lancaster, Pa.]. The 5.0ci precision of this measurement was ±0.10 mm.) The results of this experiment are presented in Fig. 8. Footpad thickness was maximal in all four experimental groups between 12 and 22 days after inoculation. BALB/c mice are 4.0smaller than B6C3Fl mice; swelling of the DoS footpads of these mice occurred generally to a 7 100 200 3X smaller degree than was the case for the non4 TIME AFTER INOCULATION (day.) thymectomized B6C3F, mice. Footpad thickening was generally greater in the T + R mice than FiG. 7. Log number of M. Leprae per footpad as a functio n of time after inoculation of B6C3F, T + R m the non-thymectomized controls. There was mice. S ymbols: (-),untreated mice; (0), mice treated no significant difference in the degree of thickwith DiDS during the period indicated by the shaded ening of the footpads between young (prepared bar bel ow. Curve A, untreated controls; B, 0.0001% about 10 weeks prior to inoculation) and old DDS; C 0.001% DDS; D, 0.01% DDS; E, 0.1% DDS. (prepared a year earlier) T + R mice. The 95% 0
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LEVY ET AL.
spontaneously or after some intervention that impairs immune function, is much sought after, D,
