INTERNATIONAL JOURNAI. OF Lo izn isv Volume 70, Number 4Printed in the U.S.A.

(ISSN 0145-916X)

INTERNATIONALJOURNAL OF LEPROSY

and Other Mycobzcicric l Diseases

VOLUME 70, NUMBER 4 DECEMBER 2002

Dosage and Site of Entry Influence Growth andDissemination of Mycobacterium leprae in T900r Mice'

Gigi J. Ebenezer, Shantha Arumugam, and Charles K. Job'

Many studies have reasonably docu-mented that a large number of bacilli areshed and dispersed from the nose. mouthand ulcerating skin lesions of untreated lep-romatous patients (2'4'

5. I"), but the route of

entry of M. leprue, evolution of the type ofdisease. dissemination of the disease, andits transmission in the community is stillnot very clear. Several carefully controlledexperimental studies on animal modelshave been done and these experiments innude mice have shown that M. leprae canbe transmitted through broken nasal mu-cosa ( 3 .") and abraded skin C). Our prelimi-nary study to infect intact skin on the flanksof T90(lr mice, an area with a temperaturerelatively higher than that of feet. was notsuccessful ( 5 ). To further substantiate thisfinding and to determine whether there wasa difference in the growth and dissemina-

' Received for publication on 3 July 2002. Acceptedfor publication on 7 October 2002.

' G. J. Ebenezer. M.D.. I-lead. Department ofHislopathology and Experimental Pathology: ShanthaAruinueam. B.Sc.. Senior Laboratory Technician. GradeII. Department of Experimental Pathology: and Prof.Charles K. Job. M.D.. FRC Path. DAMS. Emeritus Sci-entist, Schieffelin Leprosy Research and Training Cen-ter, Karigi, Vellore, District. Munilna du, India 632106.

Reprint requests to: I)r. Gigi Ebenezer, Head. De-partment of I listopatholo_y and Experimental Pathol-ogy, SchicMelin Leprosy Research & Training Center,Karigiri. Vellore District. Tantilnadu. India 632106.

tion of organisms when M. leprae were in-oculated into two different sites (footpadand flank), a comprehensive study wasdone and the outcomes are presented.

MATERIALS AND METHODSA group of sixty-eight CBA mice were

thymectomized at six to seven weeks ofage. Three weeks later they were irradiatedwith 900 rad and a syngeneic bone-marrowcell transfusion as administered intraperi-toneally ("). These immunodeficient mice(T900r mice) were now ready for inocula-tion with M. leprue. M. hp/we suspensionswere obtained from the lepromatous nod-ules that developed on the footpads ofT900r mouse previously inoculated with M.lepruc. The mice were randomized. 36 wereinoculated intradermaiiv in both the flanksand the other 32 in both the footpads. Ineach of these two groups four different con-centrations of M. leprae inoculations wereused. Inocula of M. leprue containing 10 7 ,

1 0°. 10' and I W in 0.1 ml were used in theClank group of animals. In the footpadgroup of animals. the different concentra-tions were diluted in 0.03 nil for inocula-tion. The animals were fed with standardfood pellets and unlimited water.

One mouse from each M. leprue concen-tration group was sacrificed at the 6th. 'thand 12th month. All the remaining animalswere harvested at the 15th month. The skin

245

2002246 International Journal o/ Leprosy

along with the subcutaneous tissue and themuscle at the injected area on both sideswere dissected. Tissues from the left flankswere harvested and the AFB were counted.Tissues from the right flanks were immedi-ately fixed in 10% buffered formal i n andsent for histopathological studies. Sectionsof 4 pm thickness were made and stainedwith hematoxylin and eosin (H&E) andmodified Fite Faraco stain ( 7 ). Autopsieswere done on mice sacrificed at the end ofthe 15th illonth and clippings from the earlobes and internal organs, such as the liver,lung, kidney, heart, and spleen, were sub-jected to histopathological examination.Similar techniques were followed for ani-mals inoculated in the footpad. It was notpossible to do an autopsy on one animal inthe 104 mouse footpad group because thesacrificed animal was inadvertently dis-carded.

RESULTSThe M. leprae smear counts comparing

both groups at different periods of time isgiven in Table 1. The harvested countshowed that 104 in 0.1 ml inoculum did notpromote growth in the mice injected in theflank, but growth was seen in all of the micethat were inoculated in the footpad with thisconcentration of M. leprae. In all other M.leprae concentration groups, there wasgrowth of M. leprae, but it was quantita-tively more in the footpad-inoculated ani-mals than in the flank-inoculated animals.In the footpad group of animals, the multi-plication was almost double that of theflank group of animals when the inoculationcount was 10 5 and l06 , but with the highestconcentration of 10 7 , this was not so.

Table 2 shows organs where Mycobacte-rium leprae had disseminated. A histo-pathological examination of the skin fromthe flank-inoculated group of animals

FIG. I . Photomicrograph to show skin from theflanks with a subcutaneous granuloma composed ofmacrophages (H&E x80).

showed focal granulomas occupying thedermis and subcutaneous tissues. The gran-ulomas consisted of foamy macrophages,some with granular cytoplasm, and fewlymphocytes (Fig. 1). Acid-fast stain showedmacrophages containing bacilli that weremostly fragmented and granular. The ears,liver, spleen, heart, kidney showed no sig-nificant lesions, except small nonspecificmononuclear cellular inflammatory infiltra-tion of the liver, lungs and kidneys. Dis-semination of bacilli was not seen in thisgroup of animals.

A histopathological examination of foot-pad-inoculated nice showed granulomas in

TABLE 1. Growth of M. leprae with varying inoculum dosage of M. leprae.

Inoculum dosage ofM. leprae

Bacterial count

6th month 5th month 12th month 15th month

Flank Footpad Flank Footpad Flank Footpad Flank Footpad

1 0' 1.8x 105 8.4 x 10' 7.7 x 105 I x 10 8 1.5x 105 1.4x 10' I.3x 10' 2.3 x 10 8

10" 4.6x105 1x10' 5.7x10 3.8x10' 5.7x10 5 8.8x10' 3.6x10 5 6.3x108

105 1.1x10` 1.2x106 Nil 1.6x10' 6.9x103 2x10 8 6.6x10 2 4.8x108

104 Nil 2.2x 105 Nil 7.3x 106 Nil 9.9x 10' Nil 1.5x 108

70. 4 Ebenezer, et al.: Growth of M. leprae in T900r Mice 247

Fin. 2. Photomicrograph showing a large subepi-Ihelial granuloma in the footpads composed almost en-tirely of macrophage (H&E x160).

the footpads in all the animals regardless ofthe M. leprae concentration (Fig. 2). Gran-ulomas consisting of macrophages filledwith bacilli were consistently seen at the6th, 8th, 12th and 15th month. Autopsiedtissue revealed extensive dissemination ofacid-fast bacilli (AFB) forming microgran-ulomas in ears (Fig. 3). The bacillary indexof the granuloma (BIG) was 4+ (Fig. 4).The lung and liver showed scattered AFBinside the macrophages.

DISCUSSIONWhen M. leprae, in differing concentra-

tions, were injected intradermally into theflank skin of the mice. they were taken upby macrophages and aggregated at the siteof inoculation (Fig. 1). However, thesebacilli were found to be granulated andfragmented signifying degenerated bacteria.The smear count of bacilli was either lessthan that inoculated or zero when the con-centration of M. leprae in the inoculum wasthe lowest. These features demonstrate thatthere was no multiplication of bacilli at theflank site. This could be due to the relative

TABLE 2. Dissemination of M. lepraeinto various organs.

Mice inoculated Mice inoculated

Organs in footpad in dank

Kr 10' 10" 10' 10' 10' 10' 10'

Ears ND + + —I.ungs ND + + —Liver ND + + —Spleen — — — —Heart — — — —Kidney — — — —

increased warmth of the (lank ( 13 ) comparedto the footpad ( 12). Injection of the M. lep-rae inoculum into the mouse footpadshowed more than a doubling of the organ-ism, and the organisms themselves did notdisplay the fragmentation that was seen atthe flank site. This clearly demonstrates themultiplication of M. lepree in the mousefootpad. However the multiplication wasnot uniform in all of the concentrations. Thelowest (104 ) and the highest (I0 7 ) inoculumdid not demonstrate a high growth rate likethat found with the 104 and 10' inoculum.

t to. 3. Photomicrograph of cross-section of earshowing small collections of macrophages forming mi-crogranulomas situated above the cartilage (H&E x80).

248 International Journal of Leprosy 2002

This again clearly demonstrates that opti-mal levels of inoculum exist that promotegrowth of the organism in the mouse foot-pad and inoculating more bacilli than thisoptimal load does not yield better results.

Dissemination Of M. leprae to the inter-nal organs as seen in the footpad-inoculatedanimals was completely absent in the flank-inoculated mice. Multiplication at the siteof entry and dissemination to the internalorgans, when the inoculum is injected intothe mouse footpad and when no growth ordissemination occurs when injected into theflank, demonstrates that local environmentalchanges, such as relatively cooler tempera-tures and an optimum dose of M. leprae atthe site of entry, are conducive factors forthe development of the disease.

In a recent study, it was found that a ma-jority of the leprosy mono-lesions were lo-cated in the uncovered parts of the bodywith a special predilection for the posterioraspects of the lower extremities ('). In chil-dren, these sites are most vulnerable totrauma. They are also relatively cooler, andour animal study demonstrates that M. lep-rae multiply better in cooler parts, such asthe footpad, and disseminate more to theear lobes. This may explain why the firstand early lesions occur in the extremitiesand face where the temperature is low. Inmice, the entry of M. leprae through a rela-tively cooler entry point (i.e., the footpad)allows better growth of M. leprae locally,needs smaller doses of inoculum to promotegrowth, and allows dissemination of the or-ganism to the internal organs. The site ofentry and the dose of M. leprae may have arole in determining whether the person willbe infected or not.

SUMMARYThe role of dosage of Mycobacterium lep-

rae and the environment of the inoculatedsite, in producing leprosy lesions in immu-nologically-suppressed, highly-susceptibleT900r mice, was investigated. Various dosesof M. leprae, i.e., 107 , 106, 105 , 104, were in-oculated into both flanks and footpads oftwo different groups of mice. The sites of in-oculation were biopsied for histopathologi-cal examination and for M. leprae counts atthe end of 6, 8 and 12 months. M. lepraemultiplied at the infected site and dissemi-

FIG. 4. Photomicrograph showing clumps of acid-fast bacilli (AFB) in macrophages shown in Fig. 3(modified Fite Faraco stain x800).

nated to other parts of the body at all con-centrations in the mice that were infected inthe footpad with a temperature of 31°C. Inanimals inoculated at the flanks with a tem-perature of 37°C, multiplication was re-corded only when the dosage of M. lepraewas high and there was no dissemination ofthe organism in any of them. The tempera-ture at the site of entry and the dose of in-fecting M. leprae may play an importantrole in the development of leprosy in sus-ceptible individuals exposed to M. leprae.

RESUMEN

Se investigó el efecto de la dosis de Mycobacteriumleprae y el papei del microambiente en el sitio de inoc-ulación, en la producción de lesiones de la lepra en losratones T900r inmunológicamente suprimidos y alta-mente susceptihles. Se inocularon varias dosis de M.leprae (10', 10`', 105 y 1W) tanto en los flancos como enlas almohadillas plantares de dos grupos diferentes deratones. Los sitios de inoculación fueron inuestreadospara su examen histopatológico y para la cuenta de baci-los al final de 6, 8 y 12 meses de inlección. Los bacilosinoculados en la almohadilla plantar (con una temper-

7O, 4 Ebenezer, et al.: Growth of M. leprae in T900r Mice 249

atura de 30°C) se multiplicaron a todas las dosis 2.probadas en el sitio de inoeulacicín y se diseminaron aouras partes del cuerpo. En contraste, en los animales in- 3.oculados en los fl ancos (con una temperatura de 37°C),la multiplicacicín ocurrió solo con Ias dosis altas de baci-

los y no hubo di seminacieín de los mismos a ninguna 4.concentracicín probada. Se concluye que la temperatura

en el sitio de entrada y la dosis infectante de M. leprae,pueden jugar un papel importante en el desarrollo de la 5.lepra en los individuos susceptibles, expuestos al bacilo.

RÉSUMÉ

Le rôle, pour I'obtention de lésions lépreuses, de ladose d'inoculum ansi que de I'environnement au sited' inoculation de Mycobacterium leprae, rut examinéen utilisant des souris hautement susceptihles, im-munologiquement supprimées dc souchc '1'900r. Desdoses de I0', 10c", I0 5 et 10' furem inoculées, soit dans

les flanes, soit dans la plante des pieds de deux dif-férents groupes de souris. Des biopsies des sitesd' inoculation furent réalisées pour évaluer les lésionshistologiques et pour compter les bactéries après 6,8et 12 mois. M. leprae se soot multipliécs localement etse soot disséminées à d'autres parties du corps à touterles concentrations chez les souris infectées dans laplante des pieds, site à une température de 3I °C. Chezles souris inoculées aux flanes à une température de37°C, une multiplication ne fut enregistrée que lorsqueles doses de M. leprae étaient élevées et aucune dvi-dence de dissémination ne rut détectée dans ce groupe.La température au site d'entrée et Ia dose de M. 'comeintectieuse pourraient jouer un rôle important dans ledéveloppement de Ia lèpre chez les individus sensibles

exposés à M. leprae.

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A. C. Mode of transmission of M. leprae in nudemice. Int. J. Exp. Path. 71 (1990) 689-700.MCDOUGALL, A. C. and REES. R. J. W. Ulceratinglepromatous leprosy in a patient with dapsone re-sistant Mycobacterium leprae. Lepr. Rev. 44(1973) 59-64.SHAN'I'HA, A., .Jon, C. K. and EBENEZER, G. J.Preparation of thy mectomized and irradiatedT900r mice: a modification. Indian J. Lepr. 69(1997) 200-201.SHEPARD. C. C. Temperature optimum of Myco-bacterium leprae in mice. J. Bacteriol. 90 (1965)1271-1275.SHORT, D. J. and WOODNOTT, D. P. Measurementof temperature and humidity. In: The JA7'Manualof Laboratory Animal Practice and Techniques.2nd edn. London: Crosby Lockwood & Son Ltd.1969, pp. 70-71.

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