874^ International Journal of Leprosy^ 1989

There are a number of reports thought tobe related to our study However, wesee no direct relation between these findingsand ours, nor are there reports suggestingthat macrophages in leprous plasma reactwith something during cultivation. The na-ture of this cell aggregation is such that wethink the macrophages could be reactingwith antigens in the plasma of leprosy pa-tients, and forming a granuloma in vitro.These results indicate that cell aggregationcan be used as an indicator of the progres-sive stages of leprosy.

—A. Yamagami, M.D.

Chief Bacteriological LaboratoryNational Institzue for Leprosy ResearchAoba-cho, 11igashi-Mitrai,aniaTokyo 189, Japan

—M. Koseki, M.D.

Chief Derinatology Section—S. Kim, M.D.

S. Hazama, M.D.K. Sanada, M.D.

DernzatologistsNational Leprosarium Tania-Zensyo-EnAoba-cho, Iligashi-AlurayamaTokyo 189, Japan

REFERENCES1. AZULAY, R. D. Chemotaxis of monocytes in han-

seniasis. Int. J. Lepr. 50 (1982) 215-216.2. FUQUAY, J. I., Loo, D. T. and BARNES, D. W. Bind-

ing of Staphylococcus aureus by human serumspreading factor in an in vitroassay. Infect. lmmun.52 (1986) 714-717.

3. JONES, G. E., PIZZEY, J. A. and WITKOWSKI, A. Theeffect of monensin on cell aggregation of normaland dystrophic human skin fibroblasts. Exp. CellRes. 159 (1985) 540-545.

4. LAAL, S., BHUTANI, L. K. and NATH, I. Naturalemergence of antigen-reactive T cells in leproma-tous leprosy patients during erythema nodosum le-prosum. Infect. Immun. 50 (1985) 887-892.

5. MASUDA, A. and SCHEINI3ERG, M. A. Peripheralblood monocyte function in leprosy patients. Int. J.Lepr. 48 (1980) 254-259.

6. WEISBART, R. H., GOLDE, D. W., SPOLTER, L.,EGGENA, P. and RINDERKNECHT, H. Neutrophil mi-gration inhibition factor from T lymphocytes (NIF-T): a new lymphokine. Clin. Immunol. Immuno-pathol. 14 (1979) 441-448.

Cultivation of Mycobacterium leprae:A New Approach

To THE EDITOR:We wish to report our recent findings on

our Mycobacterium leprae culture isolates-ICRC strains which may throw new lighton the problem of cultivation of AI. lepraein litro and on the origin ofleprosy-derivedmycobacteria (LDM). In the course of ourinvestigation on antigenic differences among

leprae culture isolates and on the de-velopment of an improved vaccine, we dis-covered that the ICRC bacilli are not purecultures, but consist of tvvo mycobacteriawith distinctive properties.

Since 1958, in our attempts to grow M.leprae in vitro, we have isolated severalstrains of mycobacteria designated as ICRCbacilli (7'8). These culturcs have been grownexclusively on liquid media, e.g., tissue cul-ture conditioned medium and enriched Du-bos' medium (8'2') (The Table). On the oth-

er hand, the majority of LDM are grown onsolid media, and are found to belong tothe AI. avium-intracellulare-scrofilaceum(MAIS) complex. Since some of the ICRCstrains isolated during 1958-1961 hadshown lepromin-like reactivity, the onlymarker of Aí. leprae identification availableat the time, we continued the work on newisolates with detailed studies on some of thestrains (The Table). They were found toshare some AI. /eprae-specific characteris-tics, e.g., lepromin-like activity (6), growthin the mouse foot pad (5), DOPA-oxidaseactivity (22), and antigenic behavior like thatof Aí. leprae in mice (l. However, theyexpress a biochemical profile like that ofavium-intracellulare (8' 21 and Kato, L., per-sonal communication, 1979 ). A candidateantileprosy vaccine prepared from one ofthe strains was found to induce lepromin

57,4^ Correspondence^ 875

THE TABLE. Lsolation and cultivation of ICRC and GMC strains.

l'eriod^Medium Suc-Attempts cesses Strains Authors (Ref.)

1957-19631966-19721973-19771978-19801980-19831984-1989

TCCM'TCCM/DubosMod. DubosbMod. DubosEnriched DubosMod. Dubos plus processed

human plasma

C-1,5,10C-44C-49—G-75G-86

Bapat 1961 (8)Bapat & Khanolkar 1972Bapat & Modak, 1977 (3.6)BapatKale & Bapat, 1985 (2.2l.2)Bapat, et al. GMC strains

13^

411^

58^

67

10^

76

TCCM tissue culture conditioned medium.b Mod. Dubos = modified Dubos' medium.

conversion and reversa! reactions in LL pa-tients, and has the efficacy and potencyequivalent to that of BCG plus M. leprae

(10,12,13vaccine and Bapat, et al., personalcommunication, 1980). Experimentally, theirradiated ICRC bacilli were found to in-duce strain-specific sensitization in mice (20).A similar pattern of sensitization, i.e., dif-ferential sensitization to different ICRCstrains, was observed in both ICRC-vacci-nated LL patients and unvaccinated lepro-matous patients (4). The observations sum-marized above strongly indicated theapparent presence of "M. leprae" activityin the ICRC cultures.

The nature of AI. leprae culture isolates-ICRC bacilli has remained cnigmatic for along time, particularly because of theexpression of dual characteristics ofboth M.leprae and AI. avium - intracellulare. A pos-sibility of "recombination" or "fusion" oftwo mycobacteria as a mechanism for thephenomenon has been proposed (2). How-ever, the fact that the two seis of cnzymecharacteristics are mutually exclusive in-dicates that the mechanism may be remote.An alternate hypothesis of the existence oftwo mycobacteria in the ICRC culture waspresented in 1986 (3). This concept of twopopulations appeared to bc a better and morepractical approach to explore the issue.

We already know that an inoculum ofICRC bacilli on L6wenstein-Jensen (LJ)medium yields M. avium - intracellulare (8and Kato, L., personal communication,1979). On this background, we preferredand adopted a physical method—densitygradient centrifugation ('9)—to search forthe other component, if any, in the ICRCculture harvest. The results of the experi-ments revealed that the long-term 111. lepraeculture isolates-ICRC bacilli did, indeed,

contamn just two distinct organisms. Thisdiscovery, that the ICRC culture is not apure strain but consists of two mycobacter-ia, thus supports the hypothesis (3). By con-notation, the discovery has considerablesignificance and importance for the culti-vation problem and the vaccine design.Therefore, we examined the two fractionsfor M. /eprae-specific markers. The datacollected are presented in this communi-cation, mentioning a few immcdiate impli-cations.

MATERIAIS AND METHODSCultures. The ICRC strains (recalled

from Dr. L. Kato, Catherine Booth HospitalCentre, Quebec, Canada) and the GrantMedical Collcge (GMC) strains—new iso-lates —were grown in modified Dubos' me-dium with human serum (21). A type-strainAt aviam was grown in Sauton's medium.A few samples of fresh AI. leprae were pre-pared from human LL skin biopsies col-lected aseptically.

Gradient system. A discontinuous su-crose density gradient (5%-30%) was madeand approximately 10'" organisms wereloaded immediately on it. The tubes werecentrifuged at 1100 x g x 20 min. Theresults showed a clear-cut dissociation ofthe original suspension into two compo-nents, the "light" band of 1.07 g/m1 densityand the pellet of "heavy" organisms of >1.12 g/m1 density. Further enrichment wasdone routinely. Under similar conditions,on a discontinuous Perco!! (19.27) gradient,the "light" population separated at the den-sity of 1.06 g/m1; the "heavy" populationformed a sharp band ai the density of 1.09g/ml. The separation was slow (1 hr 20 min),but the bands were very sharp and stable(The Figure). The results of separation were

A

876^ International fournal of Leprosy^ 1989

THE FIGURE. Separation of "light" and "heavy" populations on density gradients at 1100 x g. A = Sucrose,20 min; B = Percoll, 80 min.

reproducible with ali the strains currentlymaintained as well as with stored samples.

Identifieation tests. The primary aim wasto establish that they were two differentmycobacteria. The studies were conductedon two strains, grown in large quantity, andthe fractions were subjected to the testsadopted for AI. /eprae-specifie markers.

OBSERVATIONSProperties of "light" and"heavy" organisms

The yield. The proportionate yield of the"light" and the "heavy" organisms was 30:70 on a wet weight basis of the total of 1 g/1at 3 weeks.

Morphology and staining characteristics.13oth of the fractions contained acid-fastrods, the majority of which were solidlystained. The "light" acid-fast bacilli (AFB)were slender with uniform spreading; the"heavy" AFB were thicker with few clumps.

Pyridine extraction (") and Ziehl-Neel-sen staining. After a 2-hr extraction withfresh pyridine, a slide preparation of the"light" and the "heavy" organisms, alongwith a fresh AI. leprae smear as the control,indicated that the "light" AFB lost acid-fastness (also .11. /eprae); the "heavy" AFBdid not do so and showed pink rods.

D-DOPA oxidase activity (2 )• The re-sults were reproducible in ali of the testswith standardized suspensions, showing thatthe "light" organisms readily oxidizedD-DOPA. They even showed pigment for-mation on the sediment. The experimentsshowed AOD (480 nm) = 0.16-0.2; the ac-tivity of the "heavy" AFB was ,A0D 0.03—0.06 as that of the heat-killed bacilli.

Catalase aetivity. Catalase activity wasnegative for the "light" AFB and positivefor the "heavy" AFB.

Aryl-sulfatase. The results were positivefor the "light" AFB and negative for the"heavy" AFB.

Tellurite reduction. Tellurite reduction

57,4^ Correspondence^ 877

was negative for the "light" organisms; pos-itive for the "heavy" bacilli.

Detection of PGL-I antigen (16). AnELISA with polyclonal rabbit antibody tophenolic glycolipid-I (PGL-I) showed ahigher concentration of PGL-I on "light"AFI3 than on the "heavy" AFB.

Growth on LJ medium. The inoculum of"light" organisms did not show growth ai4 weeks; in the same period, the "heavy"AFI3 produced small colonies of AFB.

Further studies on purified "light" and"heavy" AFB for their lepromin activity,extraction of mycoside, genome size, G +Ccontent, DNA homology, protcin profiles ofsonicates by SDS-PAGE and by the Amibsprotein method are in progress.

Type strain M. aviamAl. avidin bacilli grown in Sauton's me-

dium and placed on the sucrose gradientwere found to contam n only the "heavy" or-ganisms (D = 1.12 g/m1) and no diffuse dis-tribution of any lighter organisms.

Study of fresh M. leprae suspension fromhuman skin hiopsy. The reproducibility offractionation with small samples of A/. lep-rae culture isolates prompted us to examinethe fresh, human AI. leprae suspension. A3-ml suspcnsion of minced tissue \'as fil-tered through thin cotton mesh, and lhetrate was loaded on the gradient. The or-ganisms separated as "light" AFB (D = 1.07)and "heavy" AFB. The "light" AFB in thesmear contained many AFB, the majoritywere beaded and a few solidly stained (orig-inal bacterial index 5 +), and showed totalloss of acid-fastness on the pyridine extrac-tion (PE) test. The "heavy" AFB were fewin number and were found to be PE rcsis-tant. The paueity of the material and scar-eity of the LL biopsies prevented any othertests, except for the inoculation onto mod-ified Dubos' medium.

COMMENTSThe abovc data show that A/. Icprae iso-

lates-ICRC strains contain two distinct my-cobacteria, and this appears to be the char-acteristic of ali (ICRC and GMC) strainsgrown in liquid media. The evidence sup-ports the concept (ff two populations as pre-sented in 1986 (3). The properties of the"light" and the "heavy" AFB examined sofar establish that they are two distinct my-cobacteria, both physically and bacteriolog-

ically. Thcy grow as an established co-cul-ture system, and the dissociation of the twomycobacteria has been demonstrated for thefirst time.

The "heavy" AFB, the major componentfrom available data, corrcspond to M.avium-intracellidare. The "light" AFBsharing M. leprae characteristics, particu-larly the D-DOPA-oxidase activity at theculture levei, may correspond to the "AI.leprae activity" shovvn by the ICRC bacilli(22). The final identity status of the "light"AFB will bc established when DNA ho-mology data are available.

We believe that detection ofa small num-ber of PE-resistant "heavy" organisms in afresh M. leprae suspension may be the keyto the question of the origin of LDM. Inalmost ali of the attempts to grow Al. lepraein culture, when growth is seen it is invari-ably ofmycobacteria belonging to the MAIScomplex, irrespective of the medium, thesource of Al. leprae, or the heavy size ofinoculum (15, 26). Their presence in the orig-inal inocula has been generally accepted(IS. 26). From our observations, it is not dif-ficult to imagine a few "trapped" "heavy"organisms in purified Al. lepra(' prepara-tions from armadillo-liver tissue which maybe the seed of LDM. The AFB in the scd-iment obtained during Aí. leprae (A) puri-fication appears to have been ignored. Thesurvival and limited growth of Al. leprae ina variety of special media lias been reportedwith the evidence of growth in the mousefoot pad, but eventually they seem to beswamped by MAIS, and/or eliminatedthrough subcultures (14,24,26).

Kato (23) had proposed that "concomitantmycobacteria play a role of an ctiologicalco-factor by providing mycobactin to pro-mote the growth of Al. leprae in vivo (17.").Based on this principie, he has placed Al.leprae in the category of a microbe-depen-dent microorganism of the AI. Novinho.-culosis type. Wc seem to have empiricallydeveloped a similar system with the ICRCculture, unknowingly using the same con-cept. This constant yield of "light" bacilliwith a growth limit ai 3 weeks suggests amutually controlled growth of the two my-cobacteria (8. '5).

The "light" AFB obtained from AL lepraeculture isolates of the ICRC type, in aliprobability, could be vcry dose to AI. lep-rae. This could have been the component

878^ International Journal of Leprosy^ 1989

capable ofgrowth in the mouse foot pad (5);the source of protein antigcns crossreactingwith M. /eprae-derived proteins (9); and theorganisms with epitopes reacting with hu-man T-cells derived from TT patients (").The responses incluced by the ICRC vaccinein LL patients, particularly the reversal re-action mentioned earlier, indicate that the"light" AFB componcnt may be antigeni-cally closer to ,1/. leprae than any other my-cobacterium. This revelation, that the ICRCbacilli contam n an AFB componcnt sharingM. leprae antigens mixed with M. anium-intracellulare, explains why a single injec-tion of the ICRC vaccine is as effective asConvit's 211. leprae plus BCG vaccine. TheICRC vaccine preparation inherently em-bodies exactly the same rationalc on whichConvit designed his vaccine.

The "light" AFB component appears un-able to grow as a single entity. However, theco-culture method and subsequent separa-tion offers a rapid quantitative assay to mea-sure the growth and to evolve a better me-dium for higher yields of the "light" AFBwith other mycobacteria or growth factorsuch as mycobactin. The practical applica-tions of the system are many: a) a qualitycontrol standard to monitor the "light" AFBcontent of the vaccinc batch and to iricreasepotency; b) isolation ofpure organisms froma single biopsy source against pooled bacillileading to the study ofstrain variations withreference to antigenic epitopes; c) design ofa polyvalent vaccine by combination of the"light" AFB from the different strains, e.g.,derived from drug-resistant AI. leprae; andd) to answer many unsolved questions.

In brief, we believe that through the co-culturc system yielding ICRC/GMC strainsfrom M. lepra(' isolates a new avenue isopened up to have a fresh look at the cul-tivation problem. The culture method hasbecn reproducible for many ycars. It is cx-pected that this will pave the way to solvethe most formidable, century-old puzzle inmicrobiology, namely, the cultivation ofM.leprae ia nitro.

— Dr. C. V. Bapat

Emeritus Medical Scientist (ICMR)% Neuropatholop, (MitGrau! Medical CollegeBomba), 400 008, índia

Acknowledizinents. The project on leprosy has beensponsored by the Maharashtra Council for Science andTechnology. Government of Maharashtra 1trla Smar-ak Kosh (IISK), Bombay Hospital; and the lndianCouncil of Medical Research (ICMR), New Delhi, In-dia. The laboratory facilities were provided by GrantMedical College (GMC), Bombay. The experimentswere conducted by Dr.(Mrs.) Vaijayanti P. Kale andMiss Aarti Trivedi supported by IISK, and by MissSheela Nair. Grateful thanks are duelo Dr. K. B. Sainis,BARC: Dr. P. R. Mahadevan, Foundation for MedicalResearch, and Dr. W. S. Bhatki and Mr. R. O. Chu-lawala of Acworth Leprosy Hospital; Dr. Padhi,T.I.F.R., and Dr. Damayanti Shah, R.M.C. M. leprae(A), lepromin, and soluble .1/. leprae sonicatc wcrekindly supplied by Dr. R. J. W. Rees, Medical ResearchCouncil, London.

REFERENCES1. AMMINIKUTTY, J., BAPAT, C. V. and DEO, M. G.

Adoptive transfer of tolerance induced by ICRCbacilli against Mycobacterium leprae in mice. Int.J. Lepr. 54 (1986) 437-445.

2. BAPAT, C. V. Immunological properties of.1/. /ep-rae culture isolates ICRC bacilli: hypothesis onrelationship between^leprae and ML-cultureisolates. Acta Leprol. (Gèneve) 2 (1984) 175-194.

3. BAPAT, C. V. The ICRC-culture strains derivedfrom^leprae: their significance to cultivation of

leprae and leprosy spectrum. In: Proceedingsof the Indo - U.K. Symposium ou Leprosy, Agra,Apri1 7-10, 1986. Agra: The Coronation Press,1987, pp. 250-253.

4. BAPAT, C. V. and KALE, V. P. Differential sensi-tivity to ICRC strains by LMI tests in vaccinatedand unvaccinated leprosy patients and in mice; itssignificance to antileprosy vaccine. Abstract P-32.4th International Colloquium on Mycobacteria—Structure and Function. Pasteur Institute, Paris,Septcmber 1988.

5. BAPAT, C. V. and MODAK, M. S. Growth of ICRCbacilli in foot-pad of mice. Lepr. India 50 (1978)144-155.

6. BAPAT, C. V., MODAK, M. S., D'SouzA, N. G. A.and CHULAWALA, R. O. Immunological identifi-cation of ICRC-bacilli cultivated "in vitro" from

lepraeisolates by lepromin reactivity in leprosypatients. Lepr. India 49 (1977) 472-484.

7. BAPAT, C. V., RANDIVE, K. J. and KHANOLKAR, V.R. "In vitro" cultivation of an acid-fast myco-bacterium from human lepromatous leprosy. In-dian J. Pathol. Bacteriol. 1 (1958) 156-159.

8. BAPAT, C. V., RANADIVE, K. J. and KHANOLKAR,

V. R. Growth characteristic studies on acid-fastmycobacterium isolated from human lepromatousleprosy. Int. J. Lepr. 29 (1961) 329-342.

9. CHIRMULE, N. B., MULHERKAR, R. and Do, M.G. Antigenic profile of ICRC bacilli with specialreference to isolation of immunogcnic subunit. Int.Arch. Allergy Appl. Immunol. 86 (1988) 19-27.

57, 4^ Correspondence^ 879

10. CONVIT, J., ARANZAZU, N., ULRICH, M., ZUNIGA,

M., DE ARAGON, M. E., ALVARADO, J. and REYES,O. Investigations related to the development ofa leprosy vaccine. Int. J. Lepr. 51 (1983) 531-539.

11. CONVIT, J. and PINARDI, M. E. A simple methodfor differentiation of Mycobacterium leprae fromother mycobacteria through routine staining tech-niques. Int. J. Lepr. 40 (1972) 130-132.

12. DEO, M. G., BAPAT, C. V., BHALERAO, V.,CHATURVEDI, R. M., BHATKI, W. S. and CHULA-WALA, R. G. Antileprosy potential of ICRC vac-eine; a study in patients and healthy volunteers.Int. J. Lepr. 51 (1983) 540-549.

13. DEO. M. G., BAPAT, C. V., CHULAWALA, R. G. andBHATKI, W. S. Potential antileprosy vaccine fromkilled ICRC bacilli: a clinico-pathological study.Indian J. Med. Res. 74 (1981) 164-177.

14. DHOPLE, A. M., GREEN, K. J. and OSBORNE, L. J.Limited "in vitro" multiplication of Mycobacte-num leprae. Int. J. Lepr. 57 Suppl. (1989) 320-321.

15. DHOPLE, A. M. and OSBORNE, L. J. Presence ofmycobactin-like substance in Alycobacteriunz lep-rae. Indian J. Lepr. 60 (1988) 348-359.

16. DOUGLAS, J. T., NA, S. O. and LEE, S. W. De-velopment of an ELISA for detection of antibodyin leprosy. Int. J. Lepr. 52 (1984) 19-25.

17. DRAPER, P. and WHEELER, P. R. Workshop Onmicrobiology, XIII International Leprosy Con-gress. Int. J. Lepr. 57 Suppl. (1989) 301-302.

18. EMMRICH, F. and KAUFMANN, S. H. E. HumanT-cell clones with reactivity to Mycobacteriumleprae as tools for the charcterization of potentialvaccines against leprosy. Infect. Immun. 51 (1986)879-883.

19. Isolation of bacteria from yoghurt on gradients ofPercolls generated in sim. Standard Course Ex-perimem. Uppsala: Pharmacia, 1982, p. 44.

20. KALE, V. P. and 13ApAT, C. V. Antigenic cross-reactivity between ICRC bacilli and M. leprae: an"in vitro" evaluation. Indian J. Lepr. 56 (1984)219-231.

21. KALE, V. P., BHAT, A. V. and BAPAT, C. V. Com-parison of biochemical characters of ICRC bacilliwith A/. leprae; effect of substrate alteration in themedium. Indian J. Lcpr. 56 (1984) 212-218.

22. KALE, V. P.. BilAT, A. V. and BAPAr, C. V. DOPA-Oxidase activity of ICRC bacilli. Indian J. Lepr.56 (1984) 58-62.

23. KATO, L. Absence of mycobactin in Mycobacte-rium hprae; probably microbe dependem micro-organism: implications. lndian J. Lepr. 57 (1985)58-70.

24. KIIERA, V., SAWANT, S. and MAHADEVAN, P. R."In vitro" grown M. /eprae-cultivable bacteria withconditioned phenotypic expression. Indian J. Lepr.56 Special Issue (1984) abstract no. 223.

25. PRABHAKARAN, K., HARRIS, E. B. and KIRCHHEI-MER, W. F. O-Diphenoloxidase of Mycobacte-rium lepraeseparated from infected armadillo tis-sues. lnfect. Immun. 12 (1975) 267-269.

26. WHEELER, P. R. Metabolism in Mycobacterium/eprae: its relation to other research on M. lepraeand to aspects of metabolism in other mycobac-teria and intracellular parasites. (Editorial) Int. J.Lepr. 51 (1984) 208-230.

27. WORLD HEALTH ORGANIZATION. Purification ofleprae. WHO/TDR/IMMLEP/SWG(5)/80.3

(1980) p. 26.

Glomerular lmmunoglobulin Deposition in theAbsence of Tissue Damage in Murine Leprosy

To THE EDITOR:A study was undertaken to gain insight

into the immunopathology of the kidneysin murine leprosy, a very neglected subject.Despite the fact that murine leprosy is avisceral, disseminated disease, the kidneysare usually spared. In advanced murine lep-rosy the kidneys appear, at most, pale andenlarged, and when lesions are found theyare limited to the outer aspect of the capsule.In rare cases, bacilli and leprous lesions arefound in the glomeruli, Bowmann's capsule,and ducti COntrOill of the renal parcnchyma('). In our study, a group of 10 albino, adult,NIH mice were inoculated intraperitoneallywith 108 Mycobucterium lepracinurium

(MLM) Hawaiian strain harvested frompreviously infected mice. Six months afterinoculation, the animais were sacrificed andexamined for pathological externai and in-ternai changes. Their kidneys were excisedand divided in half: one half was collectedin 10% Formalin in 0.01 M phosphate-0.15M NaCl (PBS), pH 7.4; the other wasembedded in OCT compound (Miles, Elk-hart, Illinois, U.S.A.), frozen on dry ice, andstored at -70°C until used.

Histological examination of the Forma-kidneys was performed following

standard paraffm sectioning. Two-micron-thick sections were stained with the he-matoxylin-eosin, Masson's trichrome,