Vol. 21, No.

JOURNAL OF CLINICAL MICROBIOLOGY, May 1985, p. 808-814

0095-1137185/050808-07$02.00/0
Copyright 1985, American Society for Microbiology

Intracellular Multiplication of Leprosy-Derived Mycobacteria in

Schwann Cells of Dorsal Root Ganglion Cultures
R. MUKHERJEE AND N. H. ANTIA*
The Foundation for Medical Research, 84-A R. G. Thadani Marg, Worli, Bombay 400 018, India
Received 1 October 1984/Accepted 5 February 1985

Organized nerve cultures of dorsal root ganglia from neonatal mice were infected with Mycobacterium leprae,
the causative agent of leprosy. A significant multiplication of the acid-fast bacilli was observed within the
Schwann cell component of the culture. The growth of these bacilli was sensitive to antileprosy drugs and was
not observed directly in bacteriological media. These organisms were brightly stained with the monoclonal
antibody to phenolic glycolipid-I, a M. leprae-specific marker. The antigenic, pathogenic, and biochemical
characteristics of this mycobacterium are under investigation.

men.
Addition of radioisotopes and autoradiograms. The viability of the Schwann cells in infected cultures was assessed
initially by a dye exclusion test with 0.2% (wt/vol) trypan
blue in phosphate-buffered saline and later on by the incorporation of general metabolic precursors, such as 2 to 4 p.Ci
of ['4C]acetate (specific activity, 56.7 mCi/mmol), 1 ,uCi of
[3H]proline (specific activity, 1,500 mCi/mmol), and 1 ,uCi of
[3H]leucine (specific activity, 6,800 mCi/mmol). Some of the
cultures were pulse-labeled with 1 to 2 ,uCi of [3H]thymidine
per ml (specific activity, 15,200 mCi/mmol) at different times
(1, 2, or 3 weeks postinfection). Isotopically labeled cultures
were coated with Ilford emulsion and processed autoradiographically as described previously (32). Silver grains on the
intracellular bacilli were counted under bright illumination
with red filters or under dark background illumination as

MATERIALS AND METHODS

Organized nerve cultures. The organized nerve cultures of

the sensory ganglia of newborn Swiss white mice were grown
on collagen-coated cover slips or in tissue culture flasks
(25-cm2 culture surface area) as described elsewhere (31).
*

suggested by Rogers (35).

Assessment of bacillary multiplication. (i) Cover-slip cultures. On days 1, 14, and 28 postinfection, the cultures were
fixed in 10% formalinized saline and stained by the ZiehlNeelsen method. These cultures were scanned under oil
immersion with the 63 x objective of a light microscope. The

Corresponding author.
808

Downloaded from jcm.asm.org by on October 31, 2007

Cultures were enriched for Schwann cells with cytosine

arabinoside, an antimitotic agent, and maintained in a growth
medium consisting of 70% Dulbecco modified Eagle minimal
essential medium containing 600 mg of glucose per 100 ml,
20% fetal calf serum (GIBCO Laboratories), 10% chicken
embryo extract, and 100 IU of penicillin per ml.
Infection with M. leprae. Eight- to ten-day-old cultures
were inoculated (per milliliter) with 5 x 106 to 8 x 106 M.
leprae cells isolated from fresh nodules of untreated lepromatous leprosy patients by homogenization and brief treatment
with trypsin (1); the cultures were then incubated at 37C.
Some of the cultures were terminated after 72 h. Others were
washed, fed with growth medium, and maintained for extended periods ranging from 1 to 6 weeks postinfection.
These cultures were viewed regularly under an inverted
microscope (model IM35; Carl Zeiss) to detect any degenerative changes or secondary contamination. The old medium
was drained and replaced with fresh growth medium twice a
week.
Control cultures consisted of either noninfected cultures
of the same age as infected cultures or cultures infected with
autoclaved M. leprae obtained from the same biopsy speci-

Mycobacterium leprae, the causative agent of leprosy, has

not yet been cultivated in vitro. Hanks (20, 21) described it
as an obligate intracellular parasite requiring a suitable
cellular host for growth and multiplication. Consequently,
attempts were made to cultivate this organism in several
types of cultured cells, with only marginal results (8, 10, 19,
27). However, except for macrophages, the other cells used
in these studies were not natural hosts for M. leprae. The
possibility of cultivating M. leprae in Schwann cells, the
other natural host, has not been explored, although there is
considerable evidence that they are probably the target cells
of M. Ieprae (2, 3, 5, 24, 39, 42). A histopathological study of
the early lesions of both lepromatous leprosy and tuberculoid leprosy revealed the presence of acid-fast organisms
primarily in the Schwann cells of the nerves (5). Dissemination of these acid-fast organisms to other tissues, such as the
liver and spleen, is seen only at a later stage of excessive
bacillary proliferation (2, 5). The bacteriological and morphological indices of the bacilli in nerves and Schwann cells
are generally higher than those in the skin and other tissues
(9). Schwann cells have also been known to harbor "persistor" and "resistor" organisms (4, 41). It can, therefore, be
speculated that Schwann cells in tissue cultures could also
serve as a host for the prolonged survival and multiplication
of M. leprae.
Schwann cells can be cultured in vitro in isolation as a
monolayer (11) or as a component of organized cultures of
dorsal root ganglia (13) and can be easily identified (11, 13).
The advantage of the latter model is that in this system, the
Schwann cells express most of their functional properties,
e.g., they associate with axons, acquire a basement membrane, secrete collagen, and synthesize myelin (12-14).
These cells, therefore, are physiologically analogous to their
in vivo counterparts and may be an appropriate host. Therefore, Schwann cells in organized nerve cultures were infected with M. leprae and maintained for extended periods
to study the fate of the intracellular bacilli.

Vol.. 21. 1985

MULTIPLICATION OF LEPROSY-DERIVED MYCOBACTERIA

:?.Ngwilit

809

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_t
(ibf

A. f-

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ofi&

wi

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FIG. 1. Portions of M. leprae-infected organized nerve cultures showing a gradual increase in the bacterial load (arrows) of Schwann cells
day 1 (A), day 14 (B), and day 28 (C) postinfection. Ziehl-Neelsen stain.

bacillary load of 200 Schwann cells sampled at several parts

of each culture was determined.
(ii) Flask cultures. Cells from flask cultures were harvested
with trypsin (0.25% [wt/vol])-EDTA (0.1% [wt/vol]), centrifuged at 92 x g for 10 min in a Sorvall Rc-5B refrigerated
superspeed centrifuge with an SS-34 rotor, suspended in
phosphate-buffered saline, and homogenized in an all-glass
homogenizer. The homogenate was centrifuged at 92 x g,
and the supernatant thus obtained was centrifuged at 20,800
x g for 10 min. The pellet was suspended in isotonic saline
with 1% (vol/vol) bovine serum albumin and counted by the
method of Hart and Rees (22). Bacteria were counted on
days 1, 7, 14, 21, 28, 35, and 42 postinfection.

Testing for mycobacterial contaminants. Mycobacteria inoculated into organized nerve cultures and harvested from
them were inoculated into nutrient agar and into Lowenstein-Jensen, Dubos, and Sautons bacteriological media.
The extractability of acid fastness with pyridine was tested
by the technique of Convit and Pinaridi (15).
Antileprosy drugs. Infected cultures from day 1 postinfection were incubated in the continuous presence of 1 ,ug of
dapsone (DDS) or 5 ,ug of rifampin per ml. The feeding
medium and the drugs were replaced twice a week.
[3H]thymidine (1 ,Ci/ml) was added to each culture 7 days
before termination. The cultures were terminated on day 7,
15, or 21 postinfection and processed for autoradiography.

100
-J

80

z
z
4

60

0.20

10

20

-DAY-1-

10

20

30

40

10

20

30 40

DDAY-14

50

60

70

80

90

100

DAY- 28

NUMBER OF AFB PER CELL

FIG. 2. Frequency distribution of AFB within Schwann cells of dorsal

from 30 cultures, 10 per group.

root

ganglion

cultures on

days 1, 14,

and 28

postinfection.

Data are

Downloaded from jcm.asm.org by on October 31, 2007

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810

MUKHERJEE AND ANTIA

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J. CLIN. MICROBIOL.

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The bacillary load and the number of silver grains on the

bacilli of the control and drug-treated cultures were determined and compared.
Immunofluorescence. Cultures were washed with BSS,
fixed for 10 min in 4% formalinized saline, rinsed several
times with BSS containing 5% heat-inactivated horse serum
(BSS-HS), and then incubated at room temperature in a
moist chamber with the monoclonal antibody to phenolic
glycolipid-I (supplied by B. R. Bloom) diluted 1:40 in BSS.
After 40 min, the cover slips were washed with BSS-HS and
incubated with fluorescein-conjugated goat anti-mouse immunoglobulin G (Cappel Laboratories) diluted 1:15 in BSS.
After 30 min, the cover slips were rinsed with BSS-HS,
mounted in 50% glycerol-phosphate-buffered saline, and
sealed with nail varnish. These cultures were viewed under
oil immersion with the 63 x objective of a WL Zeiss microscope equipped with both phase-contrast and fluorescence
optics and were photographed.

duration of the infection (Fig. 2). Over the course of the

infection, the arrangement of intracellular bacilli also gradually changed from that of a few scattered AFB early in the
infection (Fig. 1A) to more regular clumps and eventually
globi after prolonged infection (Fig. 1C). Ultrastructural
studies revealed that the organisms within Schwann cells
were intraphagosomal and surrounded by electron-transport
zones (Fig. 3).
The increases in the numbers of intracellular bacilli observed in cover-slip cultures were corroborated by bacillary
counts in flask cultures. The cumulative increases in the
numbers of AFB within Schwann cells was gradual, peaked
on day 28 postinfection, and declined thereafter (Fig. 4 and
Table 1). The optimum increase over a period of 28 days was
9.34- to 16.82-fold, with a mean of 11.72-fold. The decrease

RESULTS
As reported earlier (31), acid-fast bacilli (AFB) were seen
within Schwann cells and fibroblasts but not in neurons and
axons. In this study, because the cultures were enriched for
Schwann cells, the behavior of intracellular M. Ieprae cells
observed was that of bacteria resident chiefly within Schwann
cells.
The microorganisms contained within Schwann cells were
rod-shaped AFB. No other forms of non-AFB or AFB were
detected. Examination of the cover-slip cultures under a
light microscope revealed bacilli in small, countable numbers localized at the polar end of 70% of the Schwann cells
on day 1 postinfection, whereas on days 14 and 28 postinfection, the Schwann cells were loaded with significantly
increased numbers of bacilli (Fig. 1A through C). The
average number of bacilli per Schwann cell therefore showed
a definite and gradual increase with the increase in the

I0

12
0

-.0

./

z
$LJ
04

14

21
42
28
35
DAYS AFTER INFECTION
FIG. 4. Growth curve of AFB cultured in Schwann cells. Each
point on the graph represents the mean counts for 10 strains of
bacilli.

Downloaded from jcm.asm.org by on October 31, 2007

FIG. 3. Electron micrograph of a thin section of a M. leprae-infected dorsal root ganglion culture fixed in 2% glutaraldehyde and 1%
osmium tetroxide, dehydrated, and embedded in araldite. Note the intracellular bacilli (b) enclosed within a pha.gosome (p) and surrounded
by electron-transparent zones (z). Uranyl acetatelead citrate sta

TABLE 2. Counts of intracellular bacilli, between days 1 and 28

postinfection, subcultured in flask cultures of dorsal root ganglia
Bacillary counts (106) on

TABLE 1. Counts of intracellular bacilli maintained in flask

cultures of dorsal root ganglia
Bacillary count (106) on day postinfectiona:
Strain

14

28

21

35

42

11.1
10.6
11.0
11.4
8.9
9.1
6.2
10.6
11.1
7.2
11.5
12.1
9.4
6.1
6.8
5.4
5.9
6.75
4.6
12.1
14.2
9.1
10.9
10.1
8.03
10.5
11.4
7.2

a The mean bacillary counts x 106 the standard deviation on days, 1, 7,

14, 21, 28, 35, and 42 postinfection were 0.90 + 0.23, 1.69 0.6, 4.17 1.4,

2.2
1.9
1.09
1.06
2.1
1.21
0.95
2.3
2.6
1.4

0.92
0.94
0.69
0.91
1.30
0.63
0.73
1.1
1.02
0.71

1
2
3
4
5
6
7
8
9
10

811

MULTIPLICATION OF LEPROSY-DERIVED MYCOBACTERIA

VOL. 21, 1985

5.1
4.1
3.3
3.4
6.1
3.2
2.4
6.8
4.1
3.2

9.8
8.6

11.6
12.01
9.6
11.5
12.3
7.2
7.1
14.2
11.3
11.9

7.55 1.7, 10.87 2.29, 10.48 2.37, and 9.73 2.35, respectively.

1
2
3
4

day postinfection:
28
1
5.04
0.47
9.1
0.78
10.2
0.80
6.2
0.51

Fold

increase

10.72
11.66
12.77
12.35

No increases in the numbers of bacilli were observed with

heat-killed organisms. No growth of AFB was observed
directly in the growth medium at the beginning or the end of
the cultivation period. None of the strains of bacilli either
before or after release from Schwann cells grew in Lowenstein-Jensen, Sautons, or Dubos bacteriological media. These
bacilli also showed the property of extractability of acid
fastness with pyridine.
The host Schwann cells showed no evidence of toxicity
even up to 4 weeks postinfection. Good incorporation of
[14C]acetate, [3H]leucine, and [3H]proline was seen as uniformly distributed silver grains over all the cells in autoradiograms of the infected cultures.
In the next set of experiments, M. leprae cells were
passaged within Schwann cells in flask cultures by releasing
the organisms from the host cells, counting them, and
inoculating them at the original concentration of S x 106/ml
into fresh flask cultures. Table 2 shows that the intracellular
growth of AFB within Schwann cells was transferable and
that the growth patterns of AFB in all four passages were
similar, i.e., in each passage, the optimum increase of ca. 10to 12-fold AFB occurred 28 days postinfection. The AFB
obtained after passage 4 in Schwann cells had the same
characteristics as the original AFB with respect to the
extractability of acid fastness with pyridine and the inability

vi

c)

z
c-

cr.

ax
I

,.
..

,.e

-DAY-7-

-DAY-14-

-DAY-21-

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FIG. 5. Representative autoradiogram of infected Schwann cells under bright-field (A) and dark-field (B) illumination showing the
incorporation of [3H]thymidine by the intracellular bacilli (arrows). (C) The proportion of bacilli with silver grains increased from 7 to 14 days,
with the maximum occurring on day 21 postinfection. DDS and rifampin significantly reduced this proportion. No silver grains were detected
on heat-killed bacilli. Data are from three strains of bacilli. Symbols: 0,Schwann cells with bacilli; O, Schwann cells with bacilli plus 1 ,ug
of DDS per ml; E3, Schwann cells with bacilli plus 5 ,ug of rifampicin per ml; *, Schwann cells with heat-killed bacilli.

Downloaded from jcm.asm.org by on October 31, 2007

in bacillary counts in cultures after 28 days postinfection was

mainly caused by heavily infected cells floating on the
substratum, leading to a fall in the cellular density of the
culture.
The growth of AFB was also evidenced by [3H]thymidine
incorporation and hence DNA synthesis by bacilli in autoradiograms of infected cultures (Fig. 5). Silver grains were
seen on individual bacilli and clumps of bacilli under both
bright-field (Fig. 5A) and dark-field (Fig. SB) illumination.
Grain counts on intracellular bacilli were possible because of
nonincorporation of [3H]thymidine by the nuclei of the host
cells, an observation reported earlier (32). With the increase
in the duration of the infection there was an increased
incorporation of [3H]thymidine which paralleled the concomitant increases in the microscopic counts of intracellular
bacilli (Fig. 5C). Both of these parameters were significantly
reduced by the antileprosy drugs DDS and rifampin at doses
of 1 and 5 ,ug/ml, respectively (Fig. 5C).

Subculture

812

MUKHERJEE AND ANTIA

J. CLIN. MICROBIOL.

4i

FIG. 6. Immunohistochemical staining with the monoclonal antibody to phenolic glycolipid-I of bacilli cultured in Schwann cells. (A and
B) Phase-contrast (A) and fluorescence (B) microscopy of bacilli (arrows) after passage 1 in Schwann cells. Note the intracellular bacilli
intensely stained with the antibody. (C) Same strain as in panels A and B but released from the host after passage 4 and brightly stained with
the antibody.

to grow in bacteriological media. From the in vitro growth

curve (Fig. 4), the generation time of M. leprae was calculated to be 7 days (25).
These organisms cultivated within Schwann cells showed
marked fluorescence with the monoclonal antibody to phenolic glycolipid-I. The organisms from passages 1 through 4
were brightly stained (Fig. 6).

DISCUSSION
The inability to cultivate M. leprae, the causative agent of
leprosy, in laboratories has to a great extent restricted
research work on leprosy (21). Earlier attempts to cultivate
it directly in vitro yielded either no growth or the growth of
AFB not related to M. Ieprae, e.g., ICRC, H-57, and
Corynebacterium spp. (7, 16, 38). Attempts to use several
kinds of cultured cells have failed to yield any significant
growth of the organism (19, 30, 33, 36). These findings
suggest that M. leprae is an extremely fastidious organism
requiring very specific conditions for its growth and multiplication, such as the available in vivo conditions within
macrophages or Schwann cells. Attempts to culture M.
leprae within macrophages have yielded limited multiplication (17, 34, 36, 40). The possibility of cultivating M. Ieprae
in Schwann cell cultures, even though speculated by
Lumsden (26), has not yet been explored.
The present study demonstrates that human-derived M.
leprae cells undergo significant multiplication within
Schwann cells in tissue cultures. Multiplication, as assessed
by examining infected cultures under a light microscope as
well as by making actual quantitative measurements, was
consistent in 25 experiments. In flask cultures, although the
original inoculum consisted of 2.5 x 107 organisms, the
actual number of organisms phagocytosed at 72 h was less
than or around 106 (Table 1). The low level of selective
phagocytosis of viable organisms has previously been re-

ported (31). The remaining nonphagocytosed organisms from

the original inoculum were carefully washed, and the growth
medium was renewed twice a week, to ensure minimal
repeated phagocytosis. These organisms exhibited several
properties distinct from those of other leprosy-derived
mycobacteria. The growth was strictly intracellular, inducing the inhibition of host cell proliferation, a feature reported
earlier as being specific to M. Ieprae (32). Growth was
inhibited by the antileprosy drugs DDS and rifampin. No
growth was observed in the bacteriological media tested.
Acid fastness was extractable with pyridine (15). Although
these characteristics are common to M. leprae, there is still
some reservation about their specificity.
Recent studies (23) have revealed that phenolic glycolipidI, a part of the M. leprae capsule, is a highly specific
molecule which differentiates M. Ieprae from all other
mycobacteria and that the monoclonal antibody generated
against phenolic glycolipid-I is a marker specific for M.
Ieprae (28). The organisms cultivated by us in Schwann cells
had the capacity to produce phenolic glycolipid-I. The other
antigenic and pathogenic characteristics and cell wall composition of this cultivable organism are under investigation
and will be reported shortly (manuscript in preparation).
The faster growth rate of the organisms cultivated within
Schwann cells as compared with that in mouse foot pads (37)
can be attributed either to the difference in the milieu and
temperature of these two systems or to the inherent differences in the growth rates of these two types of organisms.
The retention of the growth potential and original characteristics after four passages demonstrates that within
Schwann cells, M. Ieprae can be continuously subcultured
without cross-contamination with any other species of
mycobacteria.
The growth of M. leprae within Schwann cells is not
entirely surprising. This culture system attempts to over-

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..
..
..
.

MULTIPLICATION OF LEPROSY-DERIVED MYCOBACTERIA

VOL. 21, 1985

come deficiencies described as inherent in direct in vitro

culturing systems (21). M. leprae cells within Schwann cells
are intraphagosomal and have capsules around them. Hence,
these organisms are not leaky and are capable of retaining
metabolic pools required for multiplication, as experimentally demonstrated by the DNA synthesis reported in this
paper and the lipid synthesis to be reported later (manuscript
in preparation). The possibility of host Schwann cells supplying metabolites for the growth and multiplication of M.
leprae remains to be explored. At this stage, evidence in this
direction is indirect but persuasive. Lipids are the important
major cell wall components of M. leprae (29), and Schwann
cells have the ability to synthesize in vitro bulk quantities of
myelin components and other lipids that could be utilized by
M. leprae (6, 13, 18, 43).
ACKNOWLEDGMENT
We are grateful to B. R. Bloom, Albert Einstein Institute, New
York, N.Y., for supplying the monoclonal antibody to phenolic
glycolipid-I.

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