Indian Journal of Experimental Biology
Vol. 39, October 200 1 , pp. 962-983
Review Article
Leprosy bacillus - possibly the first chemoautotrophic human pathogen
cultivated in vitro and characterised
l
A N Chakrabarty, Sujata G Dastidar*, Aninda Sen, Parthajit Banerjee & Raktim Roy*
Department of Medical Microbiology and Parasitology, Calcutta University College of Medicine, Calcutta 700020, India
Fax: 248-6604/1620; email: [email protected]
*Division of Microbiology, Department of Pharmaceutical Technology, Jadavpur University, Calcutta 700032, India
Leprosy bacillus (LB) and leprosy derived in vitro culture forms, the chemoautotrophic nocardioform (CAN) bacteria,
showed an extremely close homology and identity with each other as regards a chemoautotrophic nutritional pattern, a no­
cardioform morphology, a weak acid-fastness coupled with Gram and Gomori's stain positivity, an exclusive mycolate and
lipid profile, a phenolic glycolipid (PGL-I) and a highly sequestrated DNA characteristic, namely, a unique small size, a low
GtC % mole, an exceptionally high y and UV radiation resistance, and a high thermal resistance. LB/CAN bacteria (CANb)
gave positive signals for 36 kDa protein PCR, as well as, for 65 kDa epitope, and hybridisation with two or more probes and
also by RFLP-analysis. Both LB/and CAN bacteria exhibited bacillary multiplication in the mouse footpads (MFP), nerve
infiltration and evidences for local pathogenicity associated with pronounced systernic invasion. A highly reproducible mu­
tilation model could be established which enabled a successful application of the postulates of Koch. The proof of their total
identity was their anergic reactions in LL cases counterpoised against Mitsuda type strong nodular responses, mirroring the
reactions of leprosy bacilli in IT cases, in accordance with the dictum of XIth International Leprosy Congress ( 1 978). Thus,
the chemoautotrophic nutritional requirements of LB, entirely unsuspected for a medically important pathogenic bacterium,
having dimorphic (both bacillary and mycelial) characters with spores, mycelia and granules and unique pathogenicity of
multilation manifested through the virulence factor, the enzyme collagenase, made LB or M leprae the highly enigmatic
bacterium for so long.
Since the discovery of leprosy bacillus (LB) as the
causative agent of leprosy in 1 873, all attempts to
cultivate it in vitro have proved unsuccessful. In view
of this, the International Leprosy Congress held in
1978 concluded that, there is no proof that a genuine
leprosy bacillus has been obtained. Thus, it had re­
mained a general belief, though disappointingly, that
leprosy bacillus had not been cultivated in vitro so
far l .
However, progress in other fields of microbiology
had led to magnificent discoveries of many newer
types of bacteria, viruses, viroids, prions etc, as well
as, understanding of genetic codes, synthesis of pro­
teins, discovery of enzymes and metabolic pathways.
In this background, failure to cultivate the leprosy
bacillus by discovering the mysteries of its nutritional
pathways, had doubtless become an exciting but also
challenging task for scientists.
Ever since the discovery of causative agent of lep­
rosy by Hansen2 , attempts had been made to cultivate
it in vitro, and try to establish its pathogenic role in
compliance with the postulates of Koch3• Excellent
I present address - Department of Microbiology, Calcutta Medical
College, Calcutta 70007 3, India
records of such attempts are provided by Wilson and
6
s
Miles4 , Dharmendra and Rees and Young • Table 1
also presents a short summary of some more recent
7
contributions in this direction • 1 2• Despite some frag­
mentary interesting evidences, the concensus had re­
mained that the leprosy bacillus had still not been
cultivated.
To begin with, one finds that the leprosy bacillus
has survived in nature, as well as, in human/and ani­
mal hosts, possibly for many millennia. Thus, it cre­
ated hopes that its metabolic/nutritional enigmas may
possibly be solved in future by approaching these in a
systematic but an unorthodox manner.
Firstly, it may be appreciated that each of the dif­
ferent groups of microbes (e.g. bacteria, rickettsiae,
chlamydiae and viruses) is characterised by different
metabolic/nutritional peculiarities3 , and the leprosy
bacillus may belong to yet another nutritionally dis­
tinctive but hitherto unidentified group. Therefore, the
task of cultivating the leprosy bacillus by solving its
nutritional puzzle may begin by examining the princi­
pal metabolic pathways that are known today and po­
tentially possible in the microbial world.
CHAKRABARTY et at. : LEPROSY BACILLUS-POSSIBLY THE RRST CHEMOAUTOTROPIC HUMAN PATHOGEN
1. Nutritional pathways and types known among
bacteria
3
The studies on bacterial nutrition 1 . 1 4 have sug­
gested 2 main groups of microbes, viz. autotrophs and
heterotrophs. The former requiring only simple inor­
ganic compounds to support life, obtaining the neces­
sary energy either from sunlight or oxidation of vari­
ous inorganic substances (chemoautotrophs); organic
compounds are not utilised by chemoautotrophs. Such
autotrophic organisms are mostly environmental and
free-living.
Heterotrophs on the other hand are described as
organotrophs. Studies by various workers have re­
vealed that the differences between chemoautotrophs
and heterotrophs may not be absolute, because there
7
are many facultative autotrophs4. 1 5- 1 . Thus, the classi­
l8
fication of Knight , specially the first two stages, re­
late to autotrophs and may explain the nutritional re­
quirements of the leprosy bacilli, and so these two
stages have been described in the following paras.
Stage l-Carbon is assimilated as CO2 and nitrogen
from inorganic sources, specially NH3 . Energy re­
quired for reduction of CO2 and synthesis of proto­
plasm is derived either by oxidation of simple inor­
ganic compounds (chemoautotrophs), or by use of
radiant energy (photosynthetic autotrophs).
Stage 2-Energy and carbon compounds for as­
similation are derived primarily by utilisation of com­
pounds already more reduced than CO2 , with assimi­
lation of nitrogen from simple sources (N2 , NH3 ,
N03 ). Protoplasm is thereby synthesised. Compounds
more reduced than CO2 are CO, C�, C2HS in n­
alkane series, including liquid paraffins, tetradecane
etc, and benzene (CJI6) and its derivatives; and on the
other hand are simple sources of N namely, NH 0H,
4
�N03, (N�hS0 ' urea etc. These would constitute
4
the nutritional basis of some organisms belonging to
I
this category 8 .1 9 .
Organisms that grow by metabolising simple nutri­
tional sources of carbon and nitrogen constitute che­
moautotrophs that can adapt to various ecosystems.
Most of these compounds can be derived from fossil
fuels (FF) or their derivatives. FFs are - benzene,
naphthalene, toluene, xylene, aniline and diphenyla­
mine, which are coal tar derivatives, while methane,
ethane and their higher analogues, (tetradecane, hexa­
decane and liquid paraffins) are FFs derived from pe­
l
troleum 9 . These compounds, of C and N, in a mineral
salt minimal medium, constitute the complete growth
medium for chemoautotrophs I 8. 1 9, including the soil
963
chemoautotrophs, where primarily most FF deposits
- 1
are located (Table 3) 1 9 2 .
Chemoautotrophs are widely distributed in the soil
and marine environments22, many of them exist with­
6 7
out FF, but survive on other elemental sources 1 . 1 ;
however, these fall beyond the scopes of our present
study.
It may now be worthwhile to examine the question
of nutritional pathways of leprosy bacillus in the light
of above possibilties. Since leprosy bacillus had re­
vealed its inability to grow on any complex/conven­
tional media e.g. on those composed of blood/serum
or tissue culture medium, it could be safely concluded
l
that it is not a heterotroph 8, despite its paradoxical
growth in human or animal hosts (for explanation, see
section 4). The only probability left was that it could
basically be a chemoautotroph, belonging to Stage 2
,
l
of the nutritional category described above 8 .
2. Leprosy bacillus probably as the first chemoauto­
trophic pathogen isolated.
While the leprosy bacillus failed to metabolise
animal proteinslhormones/tissue culture medium 1 99
etc. provided in the test tube,surprisingly, ,when all
Table I-Some media used for in vitro cultivation of M. leprae
Growth medium
°C
Enriched. complex
+ mycobactin. eschelin
Kato et al. 7
Propagation
Conclusions
34;
1 5-22
±
MIN product
Enriched. complex
Bapat et al. 8
37;
?
Symbiont
with MAIS
Enriched. complex.
tetradecane etc.
Chatterjee et al.9
32;
8-10; -20
±
Coccoid
bodies
Enriched. complex.
repeated changes
10
Bhatia et al.
37; 35;
15; 4; -20
?
Numerous
forms
Sauton's enriched
with foetal serum
II
Veeraraghavan
5 - 10
?
Low temp.
growth
Chemically defined
and
supplemented
12
Dhople et al.
34
±
dependent
Critical O2
requirement
* Vide list of References; MIN - mycobacterial or nocardial;
MAIS - Mycobacterium avium - intracellulare - scrofulaceum complex8 ; ? doubtful or difficult; ± minimal
-
-
INDIAN J EXP BIOL, OCTOBER 2001
964
these heterotrophic nutritional factors were replaced
by simple C and N sources (see above), excellent
growth of the presumptive leprosy bacilli from lep­
23
rosy tissues of man . 24 and animals (mouse footpads,
25 26
armadillos, nude mice) . , occurred without any dif­
ficulty which could also be propagated indefinitely in
vitro (Table 2) on various chemoautotrophic media
3
(Table 3 l •24 • These are described under sections 3, 4
and 5. Nutritional requirements in chemoautotrophic
media indicated that C and N substrates are possibly
available to the leprosy bacillus from fossil fuels (FF)
19
for its life support , and these therefore, appear as
chemoautotrophs par excellence.
3. Silicon chemoautotrophy
Besides FFs mentioned above, silicon (Si) from
diatomaceous fossil earth can be used as an additional
source of energy and for protein synthesis as it occurs
22 31
in humans, bacteria, fungi and diatoms . . 32 and can
21
be obtained by scavenging . Our studies showed that
the acid-fast chemoautotrophic bacteria from leprosy
tissues, as well as, several reference strains of myco-
bacteria and nocardiae tested, could be trained to
grow on a Si minimal medium initially having a small
quantity of C supplied as asparagine and gradually
32
replace C in their biosynthetic processes . Electron
probe microanalyser data on these bacteria confirmed
31
a significant Si uptake , and availability of different
types of FF in both the human and soil ecosystems 19.33
(See section 4 for further information and Fig Ic).
4. Special metabolic substrates for chemoautotrophs
It has been mentioned earlier (Section 1 ) that Stage
2 chemoautotrophs may show some evolutionary or
adaptive digressions from typical behaviour, as the
differences between nutritional stages are not abso­
lute l 8 • Thus, we find that leprosy bacillus or CAN
bacteria as chemoautotrophs are able to utilise
urea/asparagine (carbamide/carboxylamide) 19 , both
being more reduced than CO2 • Gelatin is a substrate
readily utilised by the leprosy bacillus, like most
members of Nocardia spp and seems necessary for its
human fathogenicity and survival in human/animal
3
hosts24. ; thus, collagenase present in leprosy bacillus
Table 2-Details of the sources of leprosy bacillus (LB) strains included in the study
Strain and donor(s)
(Leprosy bacillus)
t
B2647
B2439
B263 1
B. Maestre (P 30) (BM)
BH2
MLCD 68
MLCD 7 1 (R7 1 )
MLA 699
Nodule of 'nude' mice
Biopsy or slit skin smear/fluid
from human cases; School of
Tropical Medicine, Calcutta
In vitro culture of Nocardia
brasiliensis
Source of specimen
MFP* work-up :
38th Passage
MFP work-up :
42nd Passage
MFP work-up :
35th Passage
MFP work-up :
30th Passage
Human biopsy
Homogenate
Armadillo spleen
(Freeze dried)
Armadillo spleen
(Suspension)
Armadillo spleen
harvests
Nude mice LB/CANb
harvested
LL case # 106 (total)
TT case # I (total)
Case history of human/animal
biopsy
Untreated, Female (49),
Unknown Leprosy type.
Untreated, Male (20),
Lepromatous.
Untreated, Female (40),
. Borderline (Dimorphous)
Untreated, Female (26)
Lepromatous
Untreated, Male ( 1 4) Borderline, (Dimorphous)
Prototype of 22 similar isolates
3
Surviving ( J 0 ) M. lep raelCANb
after y-irradiation
4
Surviving ( J 0 ) M. lep ra elCANb
after y-irradiation
10 9
Not irradiated; contained ca. 1 0 / 1 0
AFB/CANb per ml
'° 9
AFBILB/CANb ca. 1 0 1 1 0 1 ml
Dr. Shepard's
strains from
CDC, USA
Maximum CFU/ml AFBILB varied between
10
8
J 0 to 1 0
8
S.abouraud's (ca. J 0
CFU/ml) Dextrose Agar
and Gelatin minimal agar
t Strains B2647, B2439, B263 1 , B Maestre were donated by Dr. Van Landingham, USA; Strain BH2 from authors; strains MLCD68,
MLCD7 1 (R7 1 ) , MLA 699 from Dr R J W Rees, IMMLEP, London; nodule, 'nude' mice from Dr. Md Ishaque, Quebec, Canada;
biopsy 1 slit-skin smears from Dr S K Chaudhuri, Calcutta School of Tropical Medicine; N brasiliensis from Dr M Sanyal, Calcutta
School of Tropical Medicine
* MFP - mouse foot pad; CANb - Chemoautotrophic nocardioform bacteria
CHAKRABARTY et al. : LEPROSY BACILLUS-POSSIBLY THE FIRST CHEMOAUTOTROPIC HUMAN PATHOGEN
can cause a breakdown of collagen and release of
gelatin, which is further broken down by the gelati­
nase also elaborated by it. However, it needs to be
mentioned that
in vivo,
even in absence of gelatin, the
leprosy bacillus can also survive by making use of
22
other substrates like NHt salts, urea, asparagine, Si ,
waxeslliquid paraffins etc . from commensal myco­
bacteria, and from
in vitro environments.
The nucleic
965
However, the speed of growth of different leprosy
bacilli in the mouse footpad (MFP) varied (Table 2)
significantly 36 (for further information, see sections
17, 18).
However, Knight' s description of nutritional types
did not lead to discovery of pathogenic chemoauto­
trophs, which continued to be known only among soil,
22
marine or deep oceanic bacteria • In vitro cultivated
acid bases are available in plenty in the hosts; xan­
forms of LB provided an opportunity to examine their
thine or hypoxanthine, as well as, guanine, are the
relationship with
nucleic acid bases that are needed by leprosy bacilli as
2
auxotrophs, lacking ability to synthesise these 6, 33 36.
tissues, or clarify several other controversial concepts
(see later section
in vivo LB
from human infectious
24).
Table 3-Minimal medium (MM) and complete media
Basic Chemicals
g%
Trace elements
g%
KH P04
2
K HPO.
2
(NH.hS04
C - source(s)
(More reduced than CO )
2
(see below)
0.3
0. 1
0.8
0. 1-1 .0
MgS04
MnS04
ZnS04
FeS04
CaCh
0.0005
Gelatin + MM
Paraffin + gelatin + MM
Paraffin + urea + MM
Gelatin + MM + agar/agarose
0. 1
Complete media for chemoautotrophy·
Designated
Designated
Designated
Designated
GM medium
(1)
PGM medium (2)
PUM medium (3)
GMA medium (4)
* Addition of guanine at a concentration of 40 IlglmL is essential to satisfy the auxotrophic requirements of LB/CANb33
Table 4--Correlation of bacterial structures observed between in vivo and in vitro cultural forms
Features
Existing description and
name(s) (in vivo)
Probable identity and
suggested nomenclature (in vitro)
Bacillary
Bodiess. 6
Solidly staining AFB
Weakly staining AFB
Pink/violet, blue staining (non AFB)
AFB with polar or beaded bodies
Granularlbroken forms
Irregular staining forms
Non-solid staining forms
Cuneiform AFB
AFB in clumpslclusterslbundles
Spore-like bodies8 and
4
Coccoid bodies9• 23. 2
Granules in chains2?*
Cysts rings with dots2?*
Globi smaillIarge28
Granules2?
Mycetoma granules2?*
FilamentslSpider legs2?"
Ascospore-like2?*
Thallus Iike2?"
Dark masses2?*
4
Vegetative bacilli (young)2
Vegetative bacilli (aging)23
Vegetative bacilli (0Id)))23
Sporulation (early» 26
* Arthrospores (early»29. 30
Arthrospores (early)
Arthrospores (late)
Branching mycelial fragments
Mycelial aggregates
Free arthrospores30
Free blastospores29.30
* Blastospores in hyphae23
Hyphal rings with blastospores
Globular clumps of fragmented myceliaS
Macroglobi (Nocardioform granule)30
Nocardial granule2?
Nocardial hyphae2?
Nocardial granule2?
Nocardial granule2?
Aggregates of blastopores
Coccoid bodies
Globi
'Fungal' bodies
* Blasto�res, arthrospores and mycelia; hyphae and granules pertain to nocardioform characteristics with N. brasiliensis as the type
species;2 * includes references by Bhatia and his colleagues
966
INDIAN J EXP B IOL, OCTOBER 2001
5. Evolution ofthe concept of chemoautotrophic
nocardioform bacteria
A search for a group that would satisfactorily ac�
commodate the leprosy derived chemoautotrophic
bacteria revealed that it would probably fit best within
21
the group described as nocardioform by Prauser , 37 ,
The nocardioforms exhibited (Figs l c, 2b) fungacious
mycelia that broke up into rod shape or coccoid ele­
ments. On broad morphological grounds, it is not one
which can be defined sharply, and individual strains
967
CHAKRABARTY et al. : LEPROSY BACILLUS-POSSIBLY THE FIRST CHEMOAUTOTROPIC HUMAN PATHOGEN
Fig. l-{a) Growth ( 1 0 days old) of LB/CAN bacteria in gelatin minimal (liquid medium) with a top up paraffin layer; magnified (3 X);
(b) Growth of colonies' (45 days old) of LB/CAN bacteria on gelatin minimal agarose medium slant; magnified (4 X); and (c) Growth
1
(2 /2 year old) of LB/CAN bacteria on gelatin minimal agarose slant, submerged nocardioform mycelia, thickened by intertwining and
bear "conidial" chains. Possibly silicon was scavenged from the glass waIl; magnified 6 X.
Fig. 2-{a) Growth (lO days old) of LB/CAN bacteria in slide culture in GM medium. Coccoid bodies, arthrospores are seen in early
stage, and sprouting out of coccoid bodies into rods seen ( 'rod-coccus' transformation); some hyphae show a row of blastospores (B);
scattered arthrospores (A) are seen. (800 X in oj.f. Z-N stain); (b) Slender wavy mycelia fragmenting into acid-fast "lepra bacilli"; 7 d
old growth from GMA. Some precursors of arthrospores (A) and blastospores (B) and cluster (C) of arthrospores can be seen (800
. X in
oj.f. Z-N stain). LB/CAN bacteria.
Fig. 3-{a) Mycelia apprearing thickened by intertwining with each other, with pockets formed at intersections with slender mycelia
which had produced numerous "lepra bacilli" within and around; both arthrospores (A) and blastospores (B) are seen. (800 X in oj.f. Z-N
stain). LB/CAN bacterium; (b) Hyphae thickened by duplication/interwining, showing prominent arthrospores (A) and blastospores (B)
(800 x in oj.f. Z-N stain).
Table 5-Lipid profiles of LB, CAN bacteria and mycobacteria
Bacterium
M. leprae
CAN bacteria*
LL2, LL9, BM, A699, M. gordonae
M. avium, M. simiae, M. malmoense,
M. kansasii
ADMI, ADM2
ADM3
ADM4, ADM5 and other mycobacteria
� or keto
(Type IV)
q'or
methyl
(Type I)
a' or
methoxy
or methoxy
like (Type III)
+
+
+
+
+
+
+
+
+
+
+
Tuberculostearic acid or
10- methyloctadecanoic acid
(lO Methylstearic acid 10
Me-C 1 8 :0)
-*
+
+
+
+
ro or
dicarboxy
(Type VI)
+
+
+
+
+
+
+
* Portaels F and Larsson L (personal communications : 1 99 1 ). ADMI to ADM5, armadillo-derived mycobacteria, groups 1 to 5.
of nocardioforms may not reveal all the basic features
proposed by Prauser 37. Thus, the term nocardioform
was intended to bring together in an informal manner,
a number of bacteria with similar characters, e.g., My­
cobacterium, Nocardia, Rhodococcus, aurantiaca­
21
gordona etc . Rhodococcus often has a rod-coccus
life-cycle, and occupies an intermediate position be­
tween several groups of nocardioforms.
Thus, leprosy bacilli and their in vitro cultivated
counterparts firstly appear not only to belong to the
nocardioforms which further belong to a special group
as chemoautotrophs. and hence, has been called che­
23. 24
moautotrophic nocardioform (CAN) bacteria
.
However, other chemoautotrophic organisms also oc­
cur not infrequently, and have a common soil ecology
although many species of each genus have possibly
learnt to adapt to animal hosts and thereby acquire
21. 33
pathogenicity
6. Micromorphological variation of CAN bacteria
CAN bacteria often have conflicting and confusing
variations as observed by different workers (Table 4).
Nocardia brasiliensis possesses acid-fast, slender,
wavy. branching and fragmenting hyphae which are
(X'
•
1
2
3
•
4
•
5
Fig. 4-Mycolate chromatography (TLC) of CAN bacteria : lanes
1 , 2 CAN R7 1 , CAN BM; biopsy derived LB, lane 3; lane 4, M.
lepraemurium; lane 5, M. gordonae. All except 4 seem identical
in mycolate pattern.
INDIAN J EXP BIOL, OCTOBER 2001
968
1 2 / 03/ 1992
UG
TR10-1 G C- M S
L AB - B T h e
D ata Syst e m
I nstru m e nt : Trio - l
Sample : sensiti vity
BMSCAN
6 · 61
1 00
41 77920
87
#3
(0 )
7ST
"
5· 44
6·61
4177920
74
#2
5· 44
4·17
O ������F=MT��
3 1 5 233088
100
TIC
#1
UG
12 / 03 / 1992
LA B - B
The TRIO - l GC MS Data System
Intrument : Tri o - l
6.62
·I. FS
( b)
1425408
87
#3
7-70
0
6 · 62
100
1802240
74
#2
7-70
·I. FS
5·45
0
100
�
8·12
I
7.78
253421328
T IC
#1
t.FS
0
Min
12·0
13·0
14
Fig. 5(a), (b)--Lipid profile of CAN bacterial strains CAN-BM (vide Table 2) and CAN-LL9 by Gas-chromatography mass-spectrometry
with controls. Prominent constituent was palmitic acid only without any significant presence of fatty acids or tuberculostearic acid
(Courtesy : Professor Lennart Larsson, University of Lund, Sweden). LB/CANb grown on gelatin minimal medium.
,
CHAKRABARTY et al. : LEPROSY BACILLUS-POSSIBLY THE FIRST CHEMOAUTOTROPIC HUMAN PATHOGEN
frequently interspersed with bluish coccoid bodies 7 , 9
3 3 3
(identified as blastospores 29, 0; 4. 9 ; see Fig. 3a, b)
, 3
and irregular staining arthrospores 20. 21 2 . 24 (see also
Sections 7, 8) and serve as a convenient model for
reference description of the nocardioform morphol­
3
ogy 8-40 : the former are quite pleomorphic in size and
shape and seem to be blastospores, commonly seen in
many actinomycetes 4 1 , eumycetes, but not uncom­
monly among nocardiae, typically in N. brasiliensis
3.3
appearing as bulging conidia 29. 0 8 . Under favour­
able conditions, a coccoid body (spore) germinates 40
into a vegetative bacillary filament 21 . 37 which stains
red when stained according to Ziehl-Neelsen method.
The bacterial filaments lengthen, branch out to form
(i) tufts of hyphae or mycelia, and form (ii) entangled
compact masses, called granules or clumping of
3
macrophages 29. 9. 42-44 (Fig. 2a, b).
These granules appear possibly to be the in vitro
counterparts of the globi seen in vivo in the leproma­
tous leprosy (LL) cases (Fig. 3a, b). The globular or
the circumscribed morphology seems . to be due to
their location within the host macrophage cells (Vir­
chow cells; see Refs 5, 6). The granules or globi rep­
3
resent aggregates of CAN bacteria (see Fig. 7) or
, ,3 .3 .
leprosy bacilli and other actinomycetes 21 29 0 9 44 .
7.
Granules and glohi
The long filaments (see above) of LB/CAN bacteria
found in the initial stages, fragment into slender acid­
fast bacilli which may look like lepra bacilli (Fig. 2b).
The unfragmented filaments, as well as, CAN bacteria
differentiate into cells with refractory or granular cy­
toplasm 5 and are characterised as nocardioform ar­
throspores (see below) 20. 21 . 24. 25 ; these do not bulge
the cell-wall, or develop into blastospores which
.3 .3 3
cause swelling of the bacterial cells29 0 8. 9 (Figs 3a,
b); the latter, when detached from the parent cells,
look like coccoid bodies, and may lie singly or in
Table 6---D ifferentiation of Mycobacteria, Nocardia, Corynebac­
teria etc. on the basis of G+C% mole and genome size of DNA
Organisms
Corynebacterium
Mycobacterium
M. tuberculosis
M. leprae
CAN bacteria : LL9, TI33
Rhodococcus
Nocardia
G+C % mole
Genome size
5 1 -59
6 1 -73
6 1 -73
56-57
57-59
59-69
6 1-69
1 . 1 x 1 09
2 x 1 09
1 .9 x 1 09
'
( 1 . 6- 1 .8) x 1 09
9
1 .6 x 1 0
?
?
* Due to probable deletion mutation with a smaller size result­
ing in a loss of capacity of heterotrophism
969
Table 7-Radiation resistance of leprosy bacillus and related ones
Radiation
UV Ray
y Ray
Organisms
Corynebacteria
Mycobacteria
CAN bacteria #
S
S
R : 2.5 M Rad
R : 2.5 M Rad
S
S
R*
R
Rhodococcus (Nocardia related)*
(Micrococcus radiodurans)
Deinococcus radiodurans
R : 2.5 M Rad
R
M. leprae #
S
Sensitive; R
Resistant # From the results of Dr. RJW
Rees, IMMLEP, London on tissue harvests of armadillo which
yielded both M. leprae and CAN bacteria; * some members
only, 2.5M Rad 2500000 RadslUnits of radiation; R* also
resistant to UVRIsunlight as substantial reduction in viability
87
did not occur even by 7 days .
=
=
=
=
Table 8-Collagenase aided mutilation - Effects of different fac­
tors on experimental mutilation of mouse footpads (MFP)
Type of Inoculum
CFUIMFP
in O. 1 ml*
Mouse
Footpads
Inoculated
(No. )
Effects observed
(> 4 months)
Permanent con­
tractures clawing!
deformities! mu­
tilations observed
in MFP (No.)
CAN Bacteria (3)
8
1 07/ 1 0 /109 CFU 1 mouse
footpad*
Le os" bacillus
10 110 1 1 09 BI 1 mouse
footpad*
72
54
52
30
78
o
20
20
o
e
Controls
Uninoculated cage-mates
Litters of infected mice
Forelimbs (uninfected)
Collagenase alone
Heated CAN bacteria ·
alone
Nocardia brasiliensis 102+
1
o
t
*Collagenase : 40 JlgIMFP; Collagenase producer, but not
mutilogenic
Table 9--Gelatin particle agglutination test (GPAT) and HUTRA
test
Antisera
Source of
Gelatin
PGL-I
against
Leprosy
particle
HUTRA
CANbacillus
agglutinatitre
antigen
tion titre
LL2
LL9
R 71
BM
LL HUMAN
LL HUMAN
LL ARMADILLO
MFP (x 30)
LL control serum
KIT control serum
2 048
512
2048
512
5 12
256
512
256
256
256
512
256
LL, lepromatous leprosy; MFP, mouse footpad passaged 30 times
970
INDIAN J EXP BIOL, OCTOBER 2001
2,4
c1usters 5 3 (Figs 2a, 3b), Although the thickness of
hyphae of Actinomyces spp, practically never exceeds
8
1 micron across 29, 30, 3 , a blastospore may measure
several fold its width, but may also be as small as tiny
2
dots 9, 44 . Slide culture of LB (Fig. 2a) shows forma­
tion of these spores from hyphal elements, which may
in turn produce a fresh vegetative growth.
Significance of spores of LB--Since the original
6
observation of Hansen 2, on spores of LB, a wide va­
riety of such forms (conidiospores, arthro­
6 ,
spores 20,21 ,2 ,29 30 and blstospores 29,30) has been reported
among
the
actinomycete
nocardio­
2,8
forms 20,2 1 , 9 3 comprising Corynebacterium, Myco-
bacterium, Nocardia, Rhodococcus
etc.. There are
several reports23 , 24 on occurrence of spores among LB
and LB-derived CAN bacteria and other in vitro cul­
tures 9, 10. The report of Microbiology Workshop of
1 4th ILC (Ref. 34) noted the occurrence of arthro­
spores and blastospores in LB/CAN bacteria. Katoch
et at. could also repeatedly observe the mycelial and
spore-like structures in LB/CAN bacterial culture
maintained by them (see Refs 60-62). Desikan and
Desikan 35 have reported on persistent foci of LL
macrophage granuloma in the tissues of patients de­
spite prolonged chemotherapy and lack of any overt
LB in such lesions , which could presumably be due to
Fig. 6- 1 1-{6) Smear of growth of LB/CAN bacteria from GM liquid medium after 12 days, showing cigar-shaped acid-fast bacilli strik­
ingly similar to cigar-shaped cells within globi in LL cases. Hyphae bearing arthrospores (A) and blastospores (B) can be seen (800 X in
o.i.f. Z-N stain); (7) Smear of a ruptured granule (in a homogeniser) of LB/CAN bacteria from GM agarose (22 days old). Mycelial net­
work, bacilli with clumps of coccoid bodies are seen (800 in o.iJ. Z-N stain); (8) Mouse footpad harvest showing LB into which strain
BM was passaged. (800 X in o.iJ. Z-N stain); (9) Experimental mutilation in hind footpads of mice 6 months after inoculation of 1 07
CAN bacteria in the MFP adjuvanted with collagenase. This shows moderate degree of clawing (side view), with necrotic vascular
8
changes; ( 1 0) Mutilation seen in the hind MFPs 8 months after inoculation of 1 0 CPU of CAN bacteria aided with collagenase. J 2
shows loss of some terminal toes with necrotic vascular granuloma; and ( I I ) Section of mouse liver tissue showing numerous granules of
CAN bacteria throughout the parenchymal cells and the sinusoidal spaces. The granules appear to primarily consist of blastospores or
'blue' bodies. (800 X in o.i.f. Fite-Faraco stain).
CHAKRABARTY et al. : LEPROSY BACILLUS-POSSIBLY THE FIRST CHEMOAUTOTROPIC HUMAN PATHOGEN 97 1
viable spores in these tissues. The viability and infec­
tivity of the so called artI-.rospores (and blastospores)
are difficult to determine and remained controver­
sial 44. 45. Many workers believe that these are the non­
viable forms of LB (Refs 36, 44, 45). The fact that the
arthrospores of many actinomycete or eumycotic or­
ganisms are both viable and infective, it suggests that
those of LB or CAN bacteria may have similar char­
acteristics 6. 27. The coccoid bodies on the other hand,
seem to be the seeds which can withstand many in­
imical conditions e.g. host phagocytosis and antago­
nistic immune factors, higher temperatures (in vitro
90°C for 1 5 min) 43, strong UVR, intense gamma­
radiation (2.5 megarads for 114 hr) and exposure to
other agents like Na-dodecyl sulfate (SDS), lysozyme
etc.23.25 (see also section 8 and Table 7). These seeds
seem to have potential to propagate the leprosy ba­
cillus in nature and human environments under inimi­
cal conditions as well as, during chemotherapy. Under
favourable conditions (see below), these spores ger­
minate into vegetative bacilli4O. The growth cycles
may repeat indefinitely 2 1 .37.40 . For more details see
below (Section 8, Table 7, Fig. 3a, b).
Spores are easily identified by acid-fast stain, Go­
mori's, as well as, Gram stains (partially). The blasto­
spores lack mycolates and do not show the acid-fast
red colour. These take up blue or deep blue stain of
methylene blue, and are called blue coccoid bodies9• 1 O•
8. Problems of morphological variations of leprosy
.
bacilli and CAN bacteria (CANb)
These are described in Table 4 and the following
paragraphs. It is evident that in vitro CAN bacteria
have more hyphaVmycelial elements, as well as, ar-
throspores, blastospores and granules, whereas, the
leprosy bacilli in vivo have a preponderance of bacil­
lary rods (lepra bacilli) or bacilli - packed phagocytic
cells; some showing irregular staining arthrospores,
others coccoid bodies (or blastospores); yet others as
non-sporulating hyphae or mycelia which are en­
countered infrequently in the tissueslblood, but can
occur during massive, overwhelming LL infections;
or in the nostrils in LL cases which somewhat simu­
late in vitro conditions. Thus, these show a distinct
dimorphism between the culture (in vitro) form and
the infectious in vivo bacillary form [cf. dimorphic
fungi 3. 17. 30]. This diversity has produced considerable
complexity.
Apparently,
medical
microbiolo­
gists/leprologists are trained for and familiar with the
recognition of typical lepra bacilli from tissues, but
considerable confusion seems to exist in recognising
all other form variants occurring both in vitro and in
vivo (Table 4). The coccoid bodies, mycelial or hy­
phal forms, mycelial granules encountered repeatedly
(Figs 2a, 3b, 6, 7 ) are either overlooked or not recog­
nised as true form variants of the leprosy bacilli, as
also those bacilli that are weakly or incompletely
acid-fast (see below; section 9).
The blastospores or the seeds are of great impor­
tance in the life cycle of leprosy bacillus/CAN bacte­
ria (Fig. 3b). Adverse biological (nutritional), immu­
nological (host CM!), chemical (drugsi5 and physical
agencies (higher temperature, y and UV [see Table 7]
radiations, particularly in vitro) may lead to a switch­
over of vegetative cells (lepra bacilli) into blasto­
spores and arthrospores. The reverse process i.e. a
germination (Figs 2a, 3a) of spores (Fig. 6) occurs
upon withdrawal of the harmful influences and the
Table 1 0000promin versus CAN-AGS responses in humans
Antigen
AFB
mimi *
No.
cases
Status
Lepromin
Alone
CAN-AGS*
CAN-AGS
160
3
IT
160
40, 80, 160+
24
31
IT
CAN-AGS
CAN-AGS
160
160
CAN-AGS
Alone
160
9
3
Vaccinated
3
LL
BL
LL
Tf
Dennal
Response
Nodular
> l O mm
Anergy (-)
Nodular
> 3 - > l O mm
Weak,
Variable
Nodular*
> lO mm
73/99
* mlmL, 1 million bacteria in each millilitre x thereof
+
Includes 6 cases of sonicated CAN-AGS*; independent of Lepromin
described in Table 10
* CAN - AGs (4) as
Incidence
Lepromin
Control
3/3
OK
92/92
80/81
OK, Except 3
OK, Dhannendra-AG
Variable
Weak,
Variable
6/6
Parallel
to Lepromin
Not used
97 2
INDIAN J EXP BIOL, OCTOBER 2001
triggering effects of CaCI 2 , gelatin, xylene etc. and
availability of guanine 34, 55, similar to gennination of
spores4O as have been observed elsewhere. Additional
factors are also probably involved in different eco­
systems.
Difficulties of identification of CANblstored
LB­
Rees devised protocols to declump leprosy bacilli de­
rived from armadillo splenic harvests26; Shepard et
al. 36 (vide also Ref. 66) have applied a similar treat­
ment procedure on leprosy bacilli from mouse footpad
(MFP) harvests. Despite these, all specimens of lep­
rosy bacilli predominantly showed non-acid fast
clumps/granules or free bacteria after storage and
transhipment. We also noted that many human biopsy
and MFP harvests initially comprising mostly AFBs,
gradually changed over, on storage, to non-AFB
granular/bacillary fonns. On the other hand, CAN
bacteria cultures consisting of a variable mixture of
AFBs/non-AFBs when sent by us to several leprosy
workers (D. Williams, USA; J. van Embden, The
Netherlands; P. Klatser, The Netherlands) could not
be easily recognised by them as cultures containing
any typical LB/AFB. This was obviously because of
fonn variations that occur spontaneously on storage as
already observed by us (Table 4). Contrarily, several
workers who had previous experience of such fonn
variants (D. Pal4 1 , Calcutta; P. Basak, Calcutta; S.
Gokhale, Pune) had no difficulty in recognising such
dimorphic bacteria: van Soolingen (The Netherlands);
F Portael (Belgium); V N Bhatia (Calcutta), because
of their theoretical and practical understanding27 (per­
sonal communication to authors). Katoch et al. on the
other hand could repeatedly verify these as true in
vitro cultural fonns of or derived from leprosy bacilli
as described by US46. 60-62 (also personal communica­
tion to authors; see section 1 5). The spontaneous
clumping has also been described in vitro by Yama­
gami et al. 42 of macrophages.
9. Acid-fastness of leprosy bacillus and CAN
bacteria
Although nocardioform morphology is typical of
this group, CAN bacteria apart from exhibiting such
morphology, possess other distinctive characteristics
too. These are moderately acid-fast when smears are
prepared from leprous biopsy materials and stained
according to Ziehl-Neelsen stain; however, these are
much weakly acid-fast or even non acid-fast when
obtained from stored biopsy homogenates, or from
patients receiving chemotherapy, as well as, from in
vitro cultures and many other sources 23. 27. 45 . For this
reason, use ()f H 2S0 ( 1 %) coupled with a shorter
4
period of decolorisation has been recommended for
acid-fast stain of leprosy bacillus/CAN bacteria 23. 24.
Interestingly, the nocardiae themselves are weakly
acid-fast and are stained, using H 2 S0 (1 %) only.
4
Some mycobacteria are also weakly acid-fast, how­
ever, not as weak as LB/nocardiae are. The weaker
level of acid-fastness of LB is also reflected by its
pyridine extractability in contrast with that of typical
mycobacteria6. Other distinctive staining characteris­
tics of LB/nocardiae are their ready staining with
Gram's23 and Gomori's33 stains, in contradistinction
with that of mycobacteria.
Despite the striking similarities between the leprosy
bacillus and CAN bacteria on morphological grounds,
more studies were carried out to find if further ho­
mology and identity exist between them.
10. Mycolate and lipid profiles as distinctive markers
Mycolic acids occur only among several patho­
genic members of mycobacteria, nocardiae and cory­
nebacteria. These unique branched-chain lipids vary
considerably in chain length, as well as, in the number
of carbon (C) atoms in the chains in each of the dif­
ferent genera 2 1 . 40. While the chain length of myco­
lates and their C numbers are longest and highest re­
spectively among the mycolates produced by myco­
bacteria (C # 60-90), those produced by the nocardiae
are comparatively shorter with fewer C atoms (C #
40-60). However, in some nocardia-related ones, e.g.,
Gordona-Aurantiaca, mycolates may be close (C #
68-74) to those of mycobacteria, and thus there may
be an overlapping characteristic of mycolates among
these 3 groups. The corynebacteria produce much
shorter chains with fewer C atoms 3 8-40.
Apart from their chain length and carbon (C) num­
ber, mycolates also serve as important tools for taxo­
nomic identification at genus and even species levels.
Interestingly, there are . several components of myco­
lates which occur in some typical combinations
among mycobacteria and are useful in their type
identification (Table 5). Determination of mycolate
and other lipid profiles of leprosy bacillus and CAN
bacteria is thus of much importance for proving the
identity of these 2 with each other. We found that
opinion differs as regards occurrence of a methoxy or
methoxy-like mycolate as a third component in M.
Zeprae apart from ex and keto components46 (Table 5;
see also Ref. 5 1 ). It was not detected on TLC when
extraction was done by alkaline methanolysis process;
this, however, was seen regularly when mycolates
CHAKRABARTY et at. : LEPROSY BACILLUS-POSSIBLY THE ARST CHEMOAUTOTROPIC HUMAN PATHOGEN .
were extracted by the acid methanolysis process
(Datta et aI. 47). This could be due to a fragmentation
(Philip Draper, personal communication, 1988) of
some methyl mycolate component resulting in meth­
oxy or methoxy-like artifacts. However, acid metha­
nolysis of mycolates of M. bovis which has a similar
lipid profile with a methyl mycolate component (Por­
taels et al. sl ) does not generate such fragments. Fur­
8
ther, Datta et a1. 47, and Patil et al. 4 , studying M. lep­
rae from human biopsies found that the methoxy-like
component from M. leprae (along with u and keto)
always co-chromatographed with the reference
u, methoxy and keto components from M. tuberculo­
sis following acid methanolysis. This suggested that if
fragmentation of mycolates indeed occurs, it will in­
volve those of M. tuberculosis as well.
These findings indicate that the third type of my­
colate i.e. the methoxy-like component genuinely ex­
ists in M. leprae, and being somewhat alkali labile,
may escape detection if present in small amounts, as
may haf-pen to some in vivo specimens (cf. Kondo &
Kanai 4 ).
It thus appears that CAN bacteria and LB possess
an identical set of 3 different components of myco­
lates which was supported by the findings of Por­
taels SI (Table 5). This controversy, like several oth­
ers, based on biopsy derived characterisation of the
· leprosy bacillus (e.g catalase; tuberculostearic acid
etc) can be settled with reference to in vitro pure cultures of CAN bacteria. On the other hand, this also
serves to identify the leprosy bacillus with CAN bac­
teria, and distinguish these 2 (and M. gordonae) from
all other mycobacteria (Fig. 4).
A similar controversy also centred around whether
another marker namely, tuberculostearic acid (TSA)
occurs as a true lipid constituent of leprosy bacillus,
or not. The finding so. S I of several workers was that
TSA was detectable in leprosy bacillus derived from
armadillo livers when liver contains TSA, but not
when it lacks TSA. Thus, this could be due to leprosy
bacillus or other mycobacteria efficiently absorbing
many host-derived metabolites, including TSA from
liver 21 . 46, rather than producing it endogenously. TSA
may thus originate from a miscellaneous bag of arma­
dillo derived mycobacteria (ADM) which the arma­
dillo usually hosts, even though no viable ADM could
be always detected. The results showed 46 that TSA
could neither be detected in any of the 4 in vitro CAN
bacteria tested (Table 5), nor in any of the most bacil­
liferous (BI > 5) LL biopsies, in contrast to its pres­
ence being reported from 2 biopsies with poor bacil-
973
lary content (BI < 1 ; not in Table 5); the latter could·
presumably be due to TSA-rich mycobacteria (other
than leprosy bacillus) being present in many LL pa­
tients as superinfection. Although, evidences are
much in favour of LB (and CANb) not having any
TSA (Fig. 5a, b) as a genuine constituent of their lipid
layers, the problem was investigated further by
choosing another yardstick of contamination i.e. an
extraneous (ADM) product that was known not to be
a constituent of the leprosy bacillus. These were the
secondary alcohols produced by ADM. The premise
was that if the secondary alcohols were also absent in
the leprosy bacilli, then a contamination of leprosy
bacillus with extraneous metabolites e.g. secondary
alcohols (and TSA) can be excluded and these may be
genuine endogenous products of LB. This premise,
however, turned out to be fallacious, as absence of
these alcohols could be due to their rapid catabolic
removal and failure to be detected. This explains why
there is a wide fluctuation in the content of TSA in
different armadillo-derived M. leprae dependent on
the tissue concentration of TSA in each animal which
is not compatible with an idea of endogenous bio­
synthesis 46. The most crucial evidence in this regard,
however appears to be that of Kusaka and Izumi
(1983i2 in which leprosy bacilli from two human lep­
romata showed absence of TSA. These findings are
also in conformity with the results reported (Table 5,
Fig. 5a, b; and personal communication) by Dr. L.
Larsson. Thus, all the data and their critical evalua­
tion, led to the conclusion that the mycolate and lipid
profiles of leprosy bacillus despite being shrouded in
the complexities of host-derived contamination, ap­
peared to be as described in Table 5. These observa­
tions have been consistently supported by Wayne and
Kubicas3 • Thus, the pure in vitro cultures of CAN
bacteria because of identical patterns uphold the in
vivo findings i.e. LB was TSA negative and possessed
3 mycolate components.
1 1 . Na-palmitate as a mycolate precursor substance
in CAN bacteria / leprosy bacillus
An analysis of the lipid layer of CAN bacteria re­
vealed further that palmitic acid was the principal
constituent of lipid layer (L. Larsson, 1 992, personal
communication to authors, vide Fig. 5a, 5b). The Mi­
crobiology Workshop of the 14th ILC 34 also reported
on a role of palmitate in biosynthesis of mycolates of
5
leprosy bacillus. Kato et al. 4 reported on growth of
leprosy bacillus in a palmitate medium. We devised a
modification of the basic chemoautotrophic cultiva-
974
INDIAN J EXP BIOL, OCTOBER 2001
tion medium which was enriched with sodium palmi­
tate at a concentration of 1 mg/mL dissolved in di­
methyl sulfoxide (DMSO). Our preliminary studies,
showed that there was a preponderance of acid-fast
bacteria staining red, instead of blue or violet staining
nocardioform granules. coccoid bodies of CAN bacte­
ria, suggesting more mycolate synthesis by the cells.
It is known that leprosy bacilli can often be weak
acid-fast, and even more so in vitro (as CAN bacte­
ria), possibly due to insufficient availability of pal­
mitate for synthesis of mycolates in the minimal me­
dia23 •
Thus, the various characters of the leprosy bacillus
and CAN bacteria studied showed a close identity
between them involving a chemoautotrophic metabo­
lism, a nocardioform morphology, a weak acid­
fastness. and a Gram and Gomori stain positivity. Ex­
clusive mycolate and lipid profiles of these bacteria,
seen now, strengthen the trend witnessed at different
stages.
12. Physical characteristics and molecular biology :
Studies on the DNA relationship of leprosy ba­
cilli and CAN bacteria
We examined relationship with respect to their
DNNnucleic acid make-up, between LB and CAN
bacteria. Firstly. each type of DNA was examined for
its G+C % mol (Ref. 55, 56). and for determination of
genome size by gel electrophoresis. Finally, DNA was
further used to carry out studies on gene(s) for 65 kDa
protein for identification of the genus Mycobacterium
and later for gene translating 36 kDa protein for iden­
tification of M. Zeprae specific sequences. The techni­
cal details are given in the following paragraphs
(Section l 3).
13. Studies on mycobacterial DNA
For tests to characterise DNA of LB/CANb, geno­
mic DNA was extracted both from M. leprae and
CAN bacteria and studies were done on 1 6S rRNA,
specific DNA for 36 kDa heat shock protein and an­
other two mycobacterial specific genes 57 with 383
and 44 1 bp fragments (work done in collaboration of
Dr. P. Khandekar of National Institute of Immunol­
ogy, Delhi).
Extraction of DNA from M. leprae showed that the
cells were extremely resistant to lysis. Fresh LL biop­
sies with high BI count yielded rich M. leprae har­
vests which were easier to lyse for extracting DNA.
However, stored LB suspensions/CAN bacterial cul­
tures offered high degree of resistance to lysis both by
SDS digestion method ( 1 00°C for 8 hr) or by French
pressure cell for cell lysis in guanidium buffer 58.
While sequencing of 1 6S rRNA was difficult and la­
borious, base sequencing of the polymorphic region
of Mycobacterium gene that translates a 65 kDa pro­
tein called heat shock protein (common generic and
species specific) ' has proved useful for determining
the position of M. leprae. Furthermore. 65 kDa DNA
is for identification of genus and 36 kDa is species
specific. PCR amplification of 383 bp and 44 1 bp
genes with purified genomic DNA of M. leprae and
CAN bacteria was done. PCR products were analysed
by agarose gel electrophoresis.
14. Details of PCR technique for amplifzcation of
383 and 441 bp genes
Tests were performed using 5 JlL of the purified
DNA as unit volume; 50 J.1L of PCR reaction mixture
(+ dNTPs; each, 200 J.LM, 1 .25 UTAQ DNA polymer­
ase and 0.5 J.LM. pooled). Required amplification:
probes were TB I + TB2 and TB 1 1 + TB 1 2. M. tuber­
culosis DNA was used as reference DNA (see also
Refs. 57, 58).
Determination of presence of 36 kDa epitope by
PCR was done in collaboration with Dr R Hartskeerl
of Royal Tropical Institure of Amsterdam. The strains
used were armadillo-derived M.leprae and a CAN
bacterium, strain BM. The details of the method fol­
lowed have been described by Hartskeerl et ai. 59 ear­
lier.
15. Experiments on nucleic acid hybridisation
Entire work was undertaken independently from
cultivation steps of CAN bacterialleprosy bacilli.
CAN bacteria from leprous human, mouse foo ad
23 ;26
and armadillo tissue specimens were obtained
and purified cultures were propagated serially. Nine
CAN bacterial cultures gave good growth by ATP­
biomass assessment and also by visual matching, and
20 more CAN bacterial cultures could also be grown
subsequently. Independently cultivating/propagating
leprosy bacillus/CAN bacteria in a new laboratory
(CJIL, ICMR, Agra) was successfully achieved 60-62 .
Extraction of nucleic acids was done as described by
Katoch and COXS8, and adequate growth could be ob­
tained. All subsequent works on the nucleic acids and
mycolate characterisation were carried out on the
cultures developed at CJIL, Agra, India (see also Ref.
82).
Studies were also carried out on nucleic acid hy­
bridisation using restriction fragment length polymor-
�
CHAKRABARTY et al. : LEPROSY BACILLUS-POSSmLY THE ARST CHEMOAUTOTROPIC HUMAN PATHOGEN
phism (RFLP) and special PCR tests60-62 . At a physi­
caUchemical level, G+C% mole determination
seemed of value to exclude some organisms from be­
ing considered either as LB/CANb (Tables 6, 7 )6 .
Additional information on y and UV ray resistances
of bacteria were available to evaluate if such heavily
sequestrated markers (conferring such resistances) can
be helpful in taxonomic identification of leprosy ba­
cillus and the suspected ones. (Table 7). These infor­
mation were available from Dr. R J W Rees,
IMMLEP, London, as well- as, from our own work,
presented at 14th International Leprosy Congress at
Orlando. USA, 1 993 (Microbiology Workshop)34 . The
results showed that on the basis of G+C% mole, lep­
rosy bacillus was close to CAN bacteria and Rhodo­
coccus sp. On the other hand, corynebacteria showed
some similarities, but could be excluded from further
considerations of relatedness to leprosy bacillus on
account of widely differing genome size. All other
bacteria in Table 7 needed to be excluded in consid­
eration of both these DNA parameters.
It was found57-62 on the basis of initial hybridisation
experiments that the results of homology studies be­
tween leprosy and CAN bacteria were definitely
promising. Hybridisation occurred with respect to two
M. leprae specific probes 6 1 . Subsequent detailed in­
vestigation on various gene regions showed marked
differences from the known molecular characterisa­
tions based on a single reference strain of M. leprae.
Information on 28 more clinical isolates is awaited
and when available would be most interesting to find
the extent of diversity or homogeneity that exists
among M. leprae strains (see also subsequent sections
17, 1 8).
16. Evaluation based on other DNA parameters and
probes
Molecular probes, if applicable on in vitro CAN
bacteria and in vivo leprosy bacilli may bridge the
information gap on the characteristics of the latter, as
seen on the basis of in vitro studies. On the other
hand, information based on DNA RFLP tests appear
to be of considerable value, but still needs to be fully
evaluated. The polymerase chain reaction based on
amplification of specific gene sequences (translating
specific proteins of 1 0, 12, 1 8, 36 and 65 kOa) has
proved useful in limited ways, although recent infor­
mation on genotypic strain diversity had made its ap­
plication of limited value34. 63.
The initial idea concerning an absolute genetic ho­
mogeneity of all leprosy bacilli based on analysis of
97 5
only few isolates seemed untenable, as gradually a
large number of strains from different geographical
situations and often associated with discrete clinical
states, became available for analysis and comparison.
It showed in unequivocal terms, that CAN bacteria
which had become indistinguishable from leprosy
bacillus on numerous grounds examined so far, now
show similarity of their DNA too by hybridising with
2 M. Zeprae probes61 . 62 , despite the fact that there are
no valid universal probes for all isolates and other
limitations (vide Section 1 8, para 1).
1 7 . The cluster called leprosy bacillus (M. leprae)
Studies on DNA relatedness revealed a diversity
among different LB isolates which could be due to its
being prevalent for thousands of years over widely
separated regions like India, China, Egypt, Africa,
Europe, as well as, the New World with chances of
segregation and divergent evolution, probably compa­
rable to those existing among plague and tubercle ba­
cille. These differences seen in phenotypes or clinical
types of leprosy bacilli possibly depend on genotype
differences. These often may result ,in a geographical
segregation. Thus, M. leprae may form a dense cluster
of human pathogenic strains, possibly genetic sub­
types, as evident from the work of different workers.
The clinical diversities observed may be accountable
in terms of discrete genetic diversities, e.g. multi­
bacillary or gaucibacillary5, clinical LL or IT types;
lucio type64. s, histoid type64.65 , alopecia type64.6S, hy­
perbacillary single nodule type64. 65, pure neurific
type64.65, ulcerative type64.65 and skin f:ustule type, and
xanthenelhypoxanthene utilising type 3,24.26. These had
thrown new light on several distinctly different bio­
logical types too e.g. those with long/short generation
time (slow/fast growers) ; those with low or high
yields in vitro; slow or fast growers in the mouse
footpads66• These distinct but stable biotypes confirm
the wide genetic variability within the cluster called
leprosy bacillus which actually comprises many het­
, erogeneous subtypes.
18. Results of study on CAN bacteria : Could DNA
homology be the yardsticks of their identifica­
tion ?
Contrary to previous beliefs, studies carried out
during last 5 years on DNA relatedness of different
LBs using reference strains for comparison, revealed
that a widespread diversity exists among these instead
of a uniformitylhomogeneity. Studies on our strains
suggested a considerable degree of homology with
976
INDIAN J EXP BIOL. OCTOBER 2001
different LB strains34,57.59 . Similar studies on homol­
ogy among and with respect to other LB isolates, as
well as reference LB strains, revealed a lack of either
genetic homogeneity or existence of a single identi­
fying yardstick67-70 or adequate number of specific
probes. A variability of hybridisation intensities71 •72,
seen could be accounted for due the fact that only
50% of DNA domain had been explored63 so far.
Thus, on the basis of DNA, leprosy bacilli isolated
from different human/animal/or geographical sources
may be greatly heterogenous but sub-divisible into
different types.
In conclusion, identification of LB, based on nu­
merous characters which are shared by all the strains,
like chemoautotrophic nutrition, nocardioform mor­
phology, specific mycolates, PGL-I, animal pathoge­
nicity and lepromin anergyIMitsuda74 responses may
be relied upon as gold standards. DNA characteristics
may be used to identify subtypes within M. leprae
cluster for investigating ecological, epidemiological
and global biovariability arid not for identifying the
leprosy bacillus as a universal single type being im­
possible for the purpose.
19. Experimental mouse pathogenicity of leprosy
bacillus/and CAN bacteria : from multiplication
to mutilation
Since the discovery of leprosy bacillus by Hansen2
(also widely known as Hansen's bacillus), it was not
possible for a long time to establish either successful
infection with this bacterium or reproduction of a
mutilating disease in animals which was necessary for
qualifying this pathogen as causing leprosy and fulfil
the basic tenets of Koch3 . Shepard's discovery of the
mouse footpad model showed the first extra-human
multiplication of the leprosy bacillus rather than being
a pathogenicity model5 • Gradually, however, with the
discovery of a minimal local pathogenicity coupled
with an occasional nerve infiltration, much of the psy­
chological reservations against it were overcome. The
armadillo infection model which came soon after,
proved its pathogenicity with an assured certainty75 ;
still, however, a mutilation model in animal(s) which
would typically duplicate the human leprosy for
which it had been considered so unique and stigma­
tised, had yet to come. Our studies on pathogenicity
of this germ were aimed at producing such a model
advancing sequentially through the various stages of
infective pathology of the disease ending up in muti­
lation.
20. Mouse pathogenicity of leprosy bacillus/CAN
bacteria
Several variants of the pathogenicity test were rec­
ognised76 - (i) In 2-3 week old mice : footpad multi­
plication of bacilli was transferable in series; (ii)
Nerve infiltration in the footpads was observed occa­
sionally; (iii) Organ infiltration was observed regu­
larly; (iv) Minor deformities of toes at a low fre­
quency occurring spontaneously following footpad
inoculation; and (v) In infant mouse : with the adju­
vanting effects of collagenase77 : deformity, clawing,
contractures of toes of the footpads; loss of toes with
necrotic vascular granuloma, at high frequency; and
progressive bacillary infiltration of the internal organs
and muscles.
21. Characteristics of the bacillary multiplication
model in mouse footpads
The model was reproducible using ca. 103 CPU
(mainly bacilli) / MFP inoculum (Fig. 8) · for each
mouse (n = 40 each batch), 2-3 week old, Swiss A
strain. Periodically sacrificed and autopsied mice
were assessed on the basis of microbiological, histo­
pathological and imprint smears of MFP tissues, as
well as, the internal organs; these showed characteris­
·
tics which had been reported earlier76• Additional
features . noted were post-inoculation inflammatory
. responses at 2, 6 and 10- 1 1 weeks which subsided
slowly. All these reactions were considerably milder
compared with those of Nocardia brasiliensis (control
organism). When the inocula consisted of granules of
CAN bacteria, a mild localised inflammation also oc­
curred which lasted for 4-6 weeks.
The granules of CAN bacteria evoked typical
granulomatous response in the subcutaneous tissues
and showed their gradual disintegration. Infiltration of
muscles, connective tissues and epithelial cells by
bacillary or mycelial masses was seen frequently, and
that of the nerve bundles only occasionally. Plenty of
mycelial tufts emerged from many granule - laden
macrophages (macrophage globi). By 6-8 months, the
granules of CANb disintegrated nearly completely.
releasing a large number of free acid-fast bacilli
(AFB), single layered rings of AFB , small globi and
some residual mycelia. These AFB . harvested from
mouse foot pad (MFP), were similar to or indistin­
guishable from the bacillary preparations from in vitro
cultures23.24 • and those from the human biopsy derived
leprosy bacillus. or those after passages into MFP; the
identity of these MFP isolates with leprosy bacillus
CHAKRABARTY et al. : LEPROSY BACILLUS-POSSIBLY THE FIRST CHEMOAUTOTROPIC HUMAN PATHOGEN 977
/
was established on the basis of several criteria studied
including 36 kDa gene positivity59. 25 ; other important
21
criteria studied were a typical mycolate profile ,
PGL-I positivity and immunological identity with
lepromin, and mutilation (Figs 9, 1 0).
The histopathological pictures seen in the sections
through liver spleen, kidney, muscles and other tis­
sues revealed a closely similar appearance. Myce­
lia/hyphae were seen running through the tissue layers
beyond tissue boundaries, or parenchymal cells and
collagen layers etc. Kidney showed maximum bacil­
lary masses, as granules or mycelial tufts invading
and disrupting the nonnal architecture. Section of an
infected liver has been shown in Figure 1 1 .
Conversion of human, MFP or armadillo leprosy­
derived AFB into nocardioform granules in vitro, and
their conversion to bacillary forms in vivo (in MFP)
by host immunity mechanisms revealed their dimor­
phic character (Fig. 3a,b) described earlier. Thus, LL
globi can be recognised as rudimentary granules
within the host macrophages in immunodeficient LL
3
cases • 5.
Our studies on multiplication of leprosy bacillus
from different sources as well as of CAN bacteria in
the mouse footpads with higher infecting inoculum
9
showed a multiplication potential up to 10 in course
of 6-8 post-inoculation months. Invasion of kidney
tissue by M. leprae in experimentally infected mice
had been reported78• Such tissue invasion and morbid
pathology as a result of MFP infection with leprosy or
CAN, bacilli had not been often observed or reported
by earlier workers, possibly because of lack of a fa­
miliarity with the dimorphic variations as occur in
vitro or in the animal systems. However, it thus re­
vealed that the immunity mechanisms in mice could
not contain or keep confined the infection within MFP
only, which may get widely disseminated in the
mouse, possibly due to an alteration of immunological
44 18
status comparable to the human LL cases5. • •
22. Studies on experimental mutilation model of lep­
rosy
Strains used were - human biopsy derived leprosy
bacillus (LL l , LL2, BM), and corresponding CAN
bacteria (3 strains), and a reference pathogenic strain
of Nocardia brasiliensis. The mice, as the same strain
used originally, comprised 6- 10 day old ones in each
batch (n=20), were similarly inoculated in both the
hind foot-pads subcutaneously; uninfected mice
served as controls (n=20). Each inoculum comprised
ca. 101 or 108 or 1 09 CFU of AFB of leprosy bacilli or
CAN bacteria in 0. 1 mL of liquid culture medium
(GM) without, or mixed with, 40 J.1g (for each MFP)
of collagenase (type VII lyophilised, Sigma laborato­
ries, USA), injections were given under stringent ste­
rility precautions. The experimental protocol followed
is described in Table 8.
A composite picture of the various deformities cli­
maxing into mutilations and loss of toes was recorded
in Table 8. Figures 9, 1 0 show mutilations in different
stages and that these occurred in significantly high
frequencies when aided with collagenase in the start­
ing inoculum. Earliest deformities manifested around
9
10- 1 2 weeks with the largest inoculum (10 CFU) in
some mice only. Gradually, and with the passage of
time (> 4 months), the earliest seen deformities pro­
gressed to clear-cut clawing, contractures, loss of one
or more toes, and necrotic vascular granuloma (only
with high inoculum supported by collagenase) ending
up in extensive mutilations at high frequency in ani­
mals after 7 -8 months. Animals with inoculum sizes
of lower magnitude showed milder and slower devel­
opment and lesser permanent deformities/mutilations.
In animals where the inocula did not contain any col­
lagenase, these could occasionally develop any clear­
cut clawing, and definite mutilations were never seen.
Biopsy sections from the florid mutilation lesions
showed infiltration of host tissues by leprosy bacil­
lus/and CAN bacterial1 , which could be repeatedly
isolated from such lesions and their identity was veri­
fied19 • Histologically, the sections showed extensive
loss of tissue structure and collagen architecture, with
disintegration of muscle fibres and tissues studded
with acid-fast granules associated with perivascular
infiltration of blood vessels by mononuclear cells and
lymphocytes. Nerve infiltration was frequent.
We discovered that collagenase produced endoge­
nously by the leprosy bacillus or given exogenously
provided in tum gelatin, serving a ready source of
nutrition for the leprosy bacillus or CAN bacteria,
thereby acting as a key metabolic factor responsible
for virulence. Once the growth of these bacteria was
initiated they could produce their own collagenase
and keep the cycle going. N. brasiliensis, though a
good collagenase producer30 and could produce
granulomatous pathology in man and mouse, was un­
able to cause mutilation, possibly due to differences in
the specificity of its collagenase action, or other cru­
cial virulence factors which it lacked. In the context
of our observations, the large number of other evi­
dences on the leproma juice-derived growth factors
81
(Dhople and Hanksso, Dhople and Ibanez ) and gela-
978
INDIAN J EXP BIOL, OCTOBER 2001
nerves and produced clawing, loss of toes in high fre­
quency, and mutilation as in the humans, when adju­
vanted with collagenase; and (4) MFP or armadillo
passaged LB or CAN bacteria yielded identical CAN
bacteria.
Presence of identical mycolate and lipid profile,
lepromin anergy and Mitsuda response, PGL-I and 36
kDa protein specificity, typical genome size and G+C
% mole were in further conformity with the postulates
of Koch.
tin as an important metabolic substrate (Katoch et
al. 82) for the leprosy bacillus (LB/CANb) thus became
explainable. Urea which is another metabolisable sub­
strate of leprosy bacillus (Chakrabarty et al. 23.24) is
also present abundantly in the host systems and may
adequately supplement the nutritional requirements of
the leprosy bacillus (see also Section 4).
In florid lepromatous leprosy cases, a known bio­
chemical process is an extensive destruction of colla­
gen tissues in the body and an excretion of hydroxy­
proline in urine (Dharmendrai. Although mutilation
can frequently occur due to trauma to, and infection
of, the insensitive limbs, possibly even without de­
struction of collagen tissues5 •64,65 , a common under­
lying pathology in many LL cases seems to be an ex­
tension of lepromatous granulomatous process into
connective tissue of bones and joints, resulting in a
loss of collagen materials, leading to their dislocation
and sublaxation. This may result in a shortening of
fingers/toes and other more severe types of mutila­
tions, even in absence of injury and infection5 , as may
also occur when the joints become swollen, and the
digits angulate and shorten65 . The destruction of col­
lagen tissues in the fingers and toes in such cases pos­
sibly accounts for deformities/mutilations 5.34.
Mutilations seen in humans seem to be uniquely
symbolic of leprosy. This is because, leprosy infec­
tions are known to occur naturally or experimentally
in a number of animals. In primates, granulomatous
tissue hyperplasia had been noted in chimpanzees and
sooty mangabey monkeys in the face and ears in some
instances, mimicking the human features of deform­
ity. Yet loss of toes/fingers etc. is not seen commonly.
Armadillos which present a clinical state like human
LL, also do not manifest mutilationlloss of toes etc.
The mouse, possibly used extensively including the
different immunocompromised ones (nude, skid etc.),
also do not commonly develop mutilation, or loss of
toes or other anatomical deformities. Mutilation there­
fore naturally develops only in human LL cases rather
late, and experimentally in the mouse model as de­
scribed.
We have noted that the principles or postulates laid
down to establish the aetiological role of an organism
as a pathogen had met with difficulties with respect to
the leprosy bacilli. The present studies showed that
Koch' s postulates could possibly be fulfilled as ( 1 )
AFBs regularly occur as LB/CAN bacteria in leprosy
cases; (2) these could be easily cultivated and main­
tained in appropriate media as CAN bacteria in vitro ;
(3) CAN bacteriaILB multiplied in MFP, infiltrated
23. PGL - I, lepromin and CAN bacterial antigens as
test parameters
J
The specific anergy to leprosy bacillus appears
largely due to the presence of unique phenolic gly­
colipid (PGL-I). In studying, therefore, anergy to
CAN bacteria (as well as leprosy bacilli), it is neces­
sary to show firstly that all CAN bacteria tested as
antigens (CAN-Ag) do indeed have PGL-I as their
constituent. We, therefore, undertook in vitro tests
(Table 9) to detect the presence of PGL-I; secondly by
intradermal tests for detecting anergy in LL cases,
side by side, with nodular reactions in IT cases with
these antigens. It had been noted earlier (Table 2) that
a large number of CAN bacteria were isolated by
2 .25.27 .36
US 3
; four representative cultures of these CAN
bacteria obtained by in vitro cultivation of LBs (2
from LL cases and 1 each from an armadillo and a
mouse footpad) were used as antigens for being tested
in parallel with the standard human biopsy derived
lepromin comprising LB (Table 9).
Tests were done in 2 different ways - detecting
PGL-I specific antibodies in various positive sera, by
using known PGL-I-antigen (PGL-Ag), and detecting
PGL-Ag in CAN bacteria by using such sera con­
taining anti-PGL-Ab. Antibody detection tests were
based on the reference sera from the manufacturers
(Serodia®, Fujirebio, Japan), and those procured by us
from clinically and bacteriologically confirmed un­
treated LL cases. PGL-I-Ags were provided (i) by the
manufacturers (Fujirebio) as a triglyceride fragment
incorporated within gelatin particles, this was called
gelatin particle agglutination or GPA test; or (ii) as
prepared by us by coating tanned human RBC (0
group, Rh +) with standard (reference) PGL-I ob­
tained from Dr. J.Colston (IMMLEP, MRC, London,
Courtesy WHO); and additionally (iii) by using boiled
antigen (presumably PGL-I, see below, Table : 9) ex­
tracted from CAN bacteria to coat RBC as in (ii) and
perform tests called Human Tanned RBC Agglutina­
tion (HUTRA) test. Preparation of hyper-immune
CHAKRABARTY et al. : LEPROSY BACILLUS-POSSIBLY THE FIRST CHEMOAUTOTROPIC HUMAN PATHOGEN 979
anti-PGL-I rabbit sera was done by us which required
prolonged immunisation
extending over
(10- 1 2 injections i.c/s.c/i.m)
2-2 1/2 months. Noteworthy features
sought an immunological answer to the question of
identity of CANb with LB. We found that anergy, or
contrarily, Mitsuda-type responses towards
4 chemo­
rabbits,
autotrophic nocardioform antigens (CAN-Ags) and a
disappeared in the middle order range and higher di­
and borderline cases of leprosy. The antigens injected
ticles or on RBC as carrier particles, reference (manu­
plete anergy to CAN-Ags was seen in
were immunological paralysis
in
severai
while marked prozone effects at lower dilutions which
lutions. Using known PGL-I,Ag either in gelatin par­
control standard lepromin were tested in 73 LL,
per patient varied from
5
IT
to a minimum of
2. Com­
92/92 instances
facturer's) antiserum, known LL patients' sera, and
tested on
were run in parallel to determine cross-reactivity with
been vaccinated before. Concurrent studies with the
antisera prepared against CANb antigens (see below)
24 LL cases. The anergy was weakly modi­
fied or unmodified in 3 other LL cases which had
each other. The results showed that mutual cross­
same antigens tested on 33 IT cases showed clear­
that PGL-I synthetic Ag-fragment, PGL-I complete
instances. CAN bacteria, therefore, despite their ori­
reactivity of a high order was present which meant
Ag (MRC, WHO) and that extracted from CAN­
bacteria, were closely related or possibly even identi­
cut, dose-dependent, Mitsuda-type responses in
80/8 1
gin from different unrelated leprous human, mouse
footpad (MFP) and armadillo tissues (Table
2), ap-
cal. Once the identity of Ag (PGL-I) was established,
. peared to be immunologically and pathogenetically
dilutions (11500) for slide/tube agglutination tests to
bacillus, on the basis of these and other parameters74
detect PGL-I in CANb.
as described earlier (Table
several hyper-immune rabbit sera were used in high
Results of both the gelatin particle agglutination
test (GPAT) and HUTRA (vide Table
9) tests for de­
tection of PGL-I Ag in various CANb are described
in Table
9. It could be seen that these results tallied
fairly close with each other (both the systems), how­
ever, confirming the presence of PGL-I in all CANb
tested. The higher titres of Ab obtained with HUTRA
identical with each other and also with the leprosy
10).
24. Further parameters of study for distinguishing
or identifying LB with CANb and how these are
applicable in practice
Additional parameters studied were the antibiotic
3
sensitivity8 and electron microscopic morphology84,
as well as, an assessment of taxonomic relatedness
82 or more characters33. These studies .-fur­
tests suggested that the presentation of PGL�I com­
based on
than GPAT whose suitability for the purpose is not
ceedingly close homology of the leprosy bacilli with
pOnse
lates by leprosy bacilli and CAN bacteria with the aid
plete antigen on human RBC was of higher sensitivity
fully understood. Another reason for this observed
difference could be due to differences in res
between humans and rabbits with respect to PGL-1.
The interesting point to note was that PGL-I was
formed in large amount in GM medium45, as evident
from unstained hallow around CANb in acid-fast
stain. Problems observed while immunising rabbits
were the failure to produce antibodies initially in sev­
eral rabbits and prozone effects in others, resembling
immune - complex phenomenon in LL cases.
These antigens thus provided an opportunity to de­
ther illustrated their similarity and showed an ex­
different CAN bacteria. Fulfilment of Koch' s postu­
of collagenase appears to be a strongly sugges­
tive I corroborative point too (vide section
Thus,
nity spectrum (LL to
IT).
Moreover, the responses of
LB at LL and IT poles seem to be respectively, either
one of complete anergy (in LL cases), or one of late
nodular reaction (in IT cases). On the other hand, it is
believed that when a suspect bacillus produces anergy
in LL cases, but a Mitsuda type response in IT
cases74; it is most likely to be a leprosy bacillus 1 • Us­
ing CANb-Ag side by side with lepromin-Ag, we
in vitro culture forms or in vitro cultures of
leprosy bacilli apart from revealing LB in a new light,
had offered solution to some unsolved practical prob­
lems, - the questions of non-viable forms of LB etc.
were evaluated earlier (vide section
questions of practical
solved.
termine the responses of anergy or late nodular reac­
tion among leprosy patients across the leprosy immu­
22).
7). Several other
significance
may
now
Generation time (GT) of LB based on clinical or
be
in
vivo assessments suggested this to vary from 1 2 to 1 3
days6 or even much longer. Determination of GT on
in vitro isolates of CANb showed it to be from ca. 40
to
44 hr (Ref. 85).
earlier
This was significantly shorter than
(in vivo)' assessments, although it was still
much longer than that of most slow-growing bacteria.
One reason f�r such errors seems to be due to the fact
that GT for all bacteria has been determined with re­
spect to
in vitro cultures, and the in vivo values com-
980
INDIAN J EXP B IOL, OCTOBER 200 1
puted for LB represented multiplicity of unanalysed
factors, e.g. host immune processes, opportunities for
, -2
germination of spores (as in the case of LB 9, 1 0 20 4 ;
vide section 7), as well as, other factors like chemo­
therapy. As it was possible to objectively determine
GT by in vitro parameters (instead of indirect in vivo
methods), GT became an important tool, firstly to
suspect genuine phenotypic differences between lep­
rosy bacillus strains, and secondly to find out the ge­
23 25 66
netic basis of such variations5 , - , (vide also section
1 8).
Prior to in vitro cultivation of LB , controversy ex­
isted on almost all the characteristics described for it.
It was presumably so because such descriptions were
based on host-derived and host-modified LB, often
contaminated with host-products. Similar controversy
existed whether the catalase enzyme occasionally re­
ported in biopsy-derived LBs, truly belonged to LB or
was just a host derived contaminant. Surprisingly,
even with the actual detection of a katG gene in LB 86
(for catalase-peroxidase), its non functional nature
remained unexplained, possibly because spores in
Mycobacterium could not be thought of; this, how­
ever, could be easily explained in terms of LB/CAN
bacterial spores (Tables 4, 6), which like all spores do
not store vegetative enzymes3 , and appear non func­
tional (see also Section 7).
It was clear that genesis of most of these problems
was due to failure to appreciate that LB could belong
to a genus other than Mycobacterium, although the
LB had been constantly compared to mycobacteria
and M. tuberculosis for understanding it, too often
41
with misleading results • Because both LB and tuber­
cle bacillus shared similar characters of acid-fastness,
these were included within the genus Mycobacterium
during the later half of 1 9th century when the princi­
ples of nomenclature of bacteria and their appropriate
taxonomic allocation were not well developed, and
often done prematurely and arbitrarily. In such a
background Hansen' s bacillus was allocated to Myco­
bacterium, and as it caused leprosy, it was called My­
cobacterium leprae. However, it was not known until
1 888 when Nocard discovered another group, . later
called Nocardia which was closely related to the my­
cobacteria and many related groups, which were also
acid-fast and often pathogenic. Chakrabarty and Das­
tidar33 on the basis of a taxonomic study of 82 char­
acters initially, suggested that LB could be closer to
Nocttrdia than to Mycobacterium. Pal et al. 4 1 corrobo­
rated these observations concentrating on its human
45
pathogenic aspects. Chakrabarty and Dastidar re-
viewing the data obtained by various workers on the
relationship of LB with mycobacteria and related gen­
era based on molecular biology and taxonomy, felt
that inclusion of LB within Mycobacterium could be
seriously questioned for a re-examination.
In this background, in vitro CAN bacterial cultures
had generated an accurate picture of leprosy bacillus
which had helped redraw I reconstruct with fidelity
the often indistinct/incomplete characters of in vivo
leprosy bacillus. This pertains to nearly all the char­
acters studied. Thus, a vast array of evidences seem to
have finally established the identity of leprosy bacil­
lus with in vitro cultivable form of human pathogenic
chemoautotrophs derived from leprosy tissues.
It is evident that LB is fundamentally a soil chemo­
1
autotroph 9,3 1.32 which has adapted itself to a human
pathogenic career in course of evolution like most of
the nocardioforms or actinomycetes. It is plausible to
hypothesise that LB or soil CAN bacteria are trans­
mitted primarily as soil-to-human infections, and then
as man-to-man one33•86. These involve epidemiologi­
cal and containment strategies for leprosy different
from the one being followed at present which is
mainly based on eradication by use of multidrug ther­
apy (MDT).
Acknowledgement
The authors are grateful to late Dr. S K Chaudhuri,
former Professor & Head, Deptt of Leprology, School
of Tropical Medicine, Calcutta. We also acknowledge
with gratitude the help obtained from Profs F. Por­
taels, L. Larsson and RJW Rees for our work.
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