Volume 47. Number 4Printed in the U.S.A.

ISSN 11I48-916N

IN . FERNATIONAI. JOURNAL OF LEPROSY DUPLICATA

INTERNATIONALJOURNAL OF LEPROSY

And Other Mycobacterial Diseases

Ceatro—de EVoEumE 47, NUMBER 4 DECEMBER 1979r. Rehia!da

f3LIGTNeonatally Thymectomized Lewis Rats Infected with

Mycobacterium leprae. 2. Histopathologic andElectron Microscopic Observations'

Peter J. Dawson, Julius C. Ringus, and A. Howard Fieldsteel -

The use of the neonatally thymectomizedLewis rat (NTLR) as a model for the studyof leprosy has distinct advantages overboth other marine hosts and the nine-hand-ed armadillo (Da.s'ypiis novem•in•tits). Theintact mouse develops a self-limiting infec-tion after footpad inoculation with a ceilingof about 10" organisms ( 17 ). A generalizedinfection does not occur after intravenousinfection until 19 to 24 months after inoc-ulation. and even then only comparativelysmall numbers of bacilli are found ( 2 ').

Rees, et a/. ( ' 2 • ' 5 ) have reported that foot-pad inoculation of M. leprue in thymecto-mized. lethally irradiated mice (reconstitut-ed with syngeneic bone marrow) was fol-lowed by disseminated disease within 12months. Other workers have not been en-tirely successful in reproducing these re-

' Received for publication on 2 July 1979: revisedmanuscript received on 16 September 1979.

P. J. Dawson. NI.D.. F.R.C. Path.. Professor andDirector: Julius C'. Ringus. M.D., Assistant Professorof Pathology. Laboratory of Surgical Pathology. De-partment of Pathology. University of Chicago. Chi-cago. Illinois 60637. U.S.A.: A. Howard Fieldsteel.Ph.D., Nlanager, Infectious Diseases Program, LifeSciences Division, SRI International, Menlo Park.California 94025, U.S.A.

sults. largely because of their inability tokeep the mice alive long enough to finishthe experiment (". 15 ). Rees himself has nowmodified his procedure and gives 5 treat-ments of 200 rad at two week intervals ('').Although these mice survive well, theyshow only a tenfold increase in sensitivitycompared to intact animals. While there ispreliminary evidence that M. /eprae repli-cate well in athymic (nu/nu) mice ( 1 ). theseanimals must he maintained under eithergerm-free or specific pathogen-free condi-tions ( 5 ). In contrast, M. /eprae replicatesreadily in the armadillo ( 7 . 2 "). but armadil-los suffer from the disadvantage that theycannot be bred in captivity and have. insonic areas, been reported to harbor othercultivable myobacteria ''). On the otherhand, none of these objections appears toapply to the NTLR. One of us (A1-11-7) haspreviously reported that the animals arelong-lived. relatively resistant to intercur-rent infection, and support the replicationof large numbers of M. leprac followingeither intravenous or footpad inoculation(''). We are now reporting the histopath-ologic and electron microscopic findings inthese rats following intravenous inoculationwith M. leprue.

561

562^ International Journal of Leprosy^ 1979

'FABLE I. LipCriti(Ciati/ details . and bacterial counts on ears of NTLR inoculated withM. leprae.

Animal # SexAge

(days)

Inoculation Results of bacterial count"Age atdeath(days)Route

No. ofM. /eprae

Day afterinoc.

No. ofM. leprae

1,1,1711,146.1 I' 20 i.v. .23 x^10 7 962 4.10 x^10 7 9681,1,17111.145.2 M 30 i.v .23 x^10'

33 II'' .00 x^10' 795' 2.85 x^108 7971. 1 .341.315.3 1 48 .17 x^10' 545 2.68 x^10' 558I„361,337.1 M 29 i.v. .10 x^10 7 376 2.36 x^10" 770I,^.361,339.3 M 27 i.v. .10 x^10 7 376 1.86 x^10" 7701.1.361.331.3 M 30 i.v. .10 x^10' 381 3.02 x^10" 770I„361,332.2 M 30 i.v. .10 x^10; 384 4.41^x^10" 7701_331.308.2 I: 36 i.v. .03 x^10" 571 2.03 x^10' 573

" These were made on the left ear and are expressed per ear.''^intratesticularly.

After i.v. inoculation.

MATERIALS AND METHODS

Pregnant inbred rats of the Wistar/Lewisstrain were obtained from Charles RiverBreeding Laboratories, Inc., Wilmington,Massachusetts, U.S.A. Although specificpathogen-free when obtained, neither theynor their offspring were maintained in thiscondition. Thymectomy was carried out inall instances between 5 and 16 hours afterbirth, using the method previously de-scribed ( 1 ). The strain of M. leprae used inthese experiments was originally isolatedfrom a leprosy patient by C. C. Shepard ofthe Center for Disease Control in Atlanta.Georgia. It has been maintained in mice andrats in the laboratory of one of us (AFIF)for the past ten years and retains all of itsoriginal growth patterns and characteris-tics.

Thymectomized rats were inoculated in-travenously with from 1.10 to 3.17 x 10 7M. leprae at 20 to 28 days of age. One ratreceived, in addition. 10' M. /eprae in-tratesticularly 20 days after i.v. inoculation.The rats were killed 573 to 968 days afterinoculation when bacillary counts, per-formed on one ear by the method of Shep-ard and McRae 01, indicated disseminatedinfection.

Complete autopsies were performed oneight rats. Tissues for histological exami-nation were fixed in 10% formal in andstained with hematoxylin and eosin andFite's stain ("). One mm cubes of tissuefrom footpads, snout, tail, sciatic nerve,and testes were fixed in 2% glutaraldehyde.

post-fixed in osmium tetroxide, and embed-ded in either Epon 812 or Araldite.

RESULTS

The number of M. leprae in one ear offour animals was determined just beforedeath and in the remaining animals approx-imately one year before they were killed,i.e., at about the midpoint of the experi-ment. The results (Table 1) indicated dis-seminated infection with massive replica-tion of M. leprae. In general, there was acorrelation between the numbers of organ-isms counted in the ear and those found inthe various organs microscopically (Table2).

Gross autopsies revealed no significantfindings except in the animal inoculated in-tratesticularly where there was a small yel-low area at the apparent site of inoculation.By light microscopy, significant histologicalchanges were seen only in the nonhair bear-ing, distal parts of the body, namely. thefootpads, snout, ears, tail, and testes. In allof these organs there was a rather widevariation in the degree of histologic change.In the footpads the principal change wasedema (which was not seen grossly) andmacrophage infiltration. This was mostmarked in the dermis and subcutaneous tis-sues where the number of macrophagesvaried greatly from animal to animal. Insome footpads there were large granuloma-toils masses of cells with foamy cytoplasm,in others, the infiltration of macrophageswas diffuse (Fig. I). Occasional giant cells

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47, 4^ Dawson, et al.: Leprosy in Rats^ 563

TABLE 2. Average distribution of M. le-prae in various tissues following inocula-tion.

Tissue"^Average (range) 5

Snout^

5 plus (4-5)Footpads^

5 plus (4-5)Tail^

4 plus (2-5)Testes^

3 plus (0-5)Ears^

3 plus (2-4)Spleen^

2 plus (0-3)Lymph nodes^

2 phis (0-2)Bone marrow^

2 plus (0-2)Lu ng^ I plus (0-2)I.iver^

I plus (0-3)

" Organisms were not identified in other tissues.'' Because M. Icprae were localized to only certain

areas of the tissue it seemed preferable not to quantifythem in terms of the number of fields examined. They,were therefore quantitated on the following I to S plusscale:

I plus = a single organism identified in the section;2 plus = several organisms or clusters of organims

seen:3 plus = organisms easy to find;4 and 5 plus = large and very large numbers pres-

ent.

were present. Generally, the immediatesubepidermal zone of the dermis wasspared. The muscles of the footpads werefrequently edematous and infiltrated byfoamy macrophages. Individual muscle fi-bers were often fragmented. Small nerveswere also swollen and vacuolated. In oneanimal, macrophages were seen infiltratingthe synovial membranes of the joints of thefoot. Although mast cells were fairly nu-merous and occasional plasma cells wereseen, lymphocytes and polymorphonuclearleukocytes were conspicuous. by their ab-sence. Fite stains revealed enormous num-bers of M. leprae within macrophages,which in some instances could be seen inH and E sections as pale, amorphous, ba-sophilic masses within the cytoplasm.These were equivalent to the numerous glo-bi seen with the Fite stain. Large numbersof bacilli could also be seen in fibroblast-like cells between the muscle bundles whilesmaller numbers were present within theadventitia of blood vessels, the perineuriumof nerves, and the sarcoplasm of musclecells. Bacilli were not seen within the epi-dermis, which appeared normal microscop-ically. Many organisms appeared to belying free in the tissues.

Flo. I. Edema and histiocytic infiltration of dermisand underlying muscle of the footpad (H&E x40).

The snout was always involved and oftencontained large numbers of macrophagesreminiscent of lepromatous leprosy. Thesewere found beneath the mucous membraneof the nasal passages, as well as beneaththe skin (Fig. 2), and often were presentaround sebaceous glands. Moderate orlarge numbers of acid-fast bacilli werefound within the macrophages (Fig. 3) withthe formation of globi. Small numbers ofbacilli were also seen in perichondrial fi-broblasts as well as in striated muscle cells,sebaceous cells, and small nerves. In theears the changes were mild by comparison.There was a scanty focal infiltrate of foamymacrophages again often in relation to se-baceous glands. Moderate numbers of M.leprae were present within macrophages aswell as perichondrial fibroblasts. The fewsmall nerves seen in the ears were not in-

FiG. 2. Heavy infiltration by macrophages of thesnout (H&E x40).

4

564^ International Journal of Lepro.sy^ 1979

Fici. 3. M. leprae Within macrophages in the snout(Fite x400).

volved. The tail also showed a focal histio-cytic infiltrate. The cells were lying individ-ually beneath the epidermis and in the deepdermis and subcutaneous tissue. Organismswere generally numerous, particularly nearthe base of the tail around sebaceousglands. Organisms were also present insmall nerves.

The testis was a major site of involve-

FIG. 4. Severely reduced spermatogenesis and fo-

cal macrophage infiltration of the interstitium of the

testes (H&E x40).

FiG. 5. M. leprae in the interstitium of testes. In-

sert. M. leprae within a Kupfler cell in the liver (Fite

x 1.0001.

ment in animals injected intravenously aswell as in the rat inoculated intratesticular-ly. There was generally a complete absenceof spermatogenesis. and the tubules werelined only by Sertoli cells. The interstitiumwas edematous and contained numeroussmall collections of foamy macrophages(Figs. 4 and 5). In some testes, these tendedto be located beneath the tunica albuginea.In the rat inoculated intratesticularly therewas focal fibrosis with dystrophic calcifi-cation and a scanty infiltrate of plasma cellswith an occasional polymorphonuclear leu-kocyte, which were interpreted as beingsecondary to the inoculation. The testescontained large numbers of M. leprae, themajority of which were within macro-phages, but some were present in the ad-ventitia of blood vessels and an occasionalorganism was present intracellularly withinthe tubules, but it was not clear whetherthese were Sertoli cells or macrophages.Occasional M. leprae were found in retic-uloendothelial cells in the liver (Fig. 5),spleen, bone marrow, lymph nodes, andlung. generally unaccompanied by other

FIG. 6. Degenerating M. /eprae lying s ithin double

membrane bound vacuoles in the cytoplasm of amacrophage in the snout (x10.000).

Flo. 7. M. liTrae lying free in the sarcoplasm ofa muscle cell in the snout (x20,000).

47, 4^ Dawson, et al.: Leprosy in Rats^ 565

histopathological changes. A single mac-rophage containing organisms was seen inthe medulla of one kidney. The remainderof the tissues was essentially unremark-able, and no organisms were seen in them.These included hair-hearing skin, sciaticnerve, leg muscles, diaphragm, intestinaltract, seminal vesicles, female genital tract,and brain. However, occasional foci ofbronchopneumonia and chronic pyelone-phritis were found in some animals.

Electron microscopy confirmed that themajority of organisms were within macro-phages. Most of the fibroblast-like cellsseen with the light microscope were foundto have the ultrastructural characteristics ofmacrophages. The intracellular appearanceof the bacilli was similar irrespective of thelocation of the cells. Both intact and frag-mented M. leprae were contained withindouble membrane bound vacuoles (Fig. 6)while the cytoplasm was remarkable for theincreased numbers of organelles, particu-larly heterogeneous lysosomes. An occa-sional bacillus was seen apparently lyingfree in the cytoplasm.

Electron microscopy of the footpads andsnout showed intact bacilli in clusters offrom 3 to 200 organisms lying free in thecytoplasm of striated muscle, i.e., not con-tained within an organelle (Fig. 7). Themyofibrils adjacent to the organisms showeddegeneration with the formation of numer-ous myelin figures. Bacilli were present insmall numbers within the Schwann cellsand perineural cells of nonmyelinated andmyelinated nerve fibers (Fig. 8a, 1). Severalvenules and lymphatic channels containedbacilli lying free within their lumens andwithin endothelial cells (Fig. 9). By lightmicroscopy numerous organisms appearedto he extracellular, possibly because of thevery heavy infiltration. However, by elec-tron microscopy virtually all of these bacillishowed degenerative changes and weresurrounded by fragments of degeneratedcytoplasm. suggesting that they had beenliberated by death of the macrophageswhich originally had phagocytosed them.Examination of the muscles proximal to thefootpad showed involvement of the musclewith the presence of a small number of or-ganisms. The sciatic nerve and thigh mus-

566^ International Journal of Leprosy^ 1979

FIG. R. M. leproe lying free in the cytoplasm of

Schwann cells surrounding a) nonmyelinated and h)

myelinated axons in the rat snout (x12,000).

ties appeared normal, and bacilli could notbe demonstrated within them.

The very large numbers of M. lepraeseen in the testes were for the most partcontained within macrophages. Again in-tact and fragmented bacilli were found in-tracellularly within phagolysosomes whilefragmented bacilli were found extracellu-larly. Organisms were also seen in the ad-ventitial cells of some blood vessels. A veryoccasional bacillus was found within theseminiferous tubules, but we could not becertain if these were within macrophages orSertoli cells.

DISCUSSIONOur results show that following intrave-

nous inoculation of M. leprae in the NTLR,the infection localized primarily to the non-hair bearing areas of the body, namely thefootpads, snout, ears, and tail. The mostsevere changes and the largest numbers ofbacilli were found in the footpads althoughthe actual number of bacilli varied widely.The most severely infected pads showed aheavy infiltrate with macrophages with ob-

FIG. 9. M. leprae lying free within the cytoplasm

of an endothelial cell in the snout (x16,000).

vious degenerative changes in muscle andnerve, which did not extend to the thighmuscles and sciatic nerve. In contrast,moderate numbers of bacilli were found insections of the ears with only a slight in-crease in inflammatory cells.

The snout was also remarkable for heavybacillation accompanied by a marked mac-rophage infiltrate in areas covered by bothskin and mucous membrane. While in someareas the lesions bore a superficial resem-blance to lepromatous leprosy, both thecharacter and distribution of the inflam-matory infiltrate were different. One im-portant difference from humans was thevery large numbers of extracellular organ-isms seen in the rat, although the vast ma-jority. if not all, appeared nonviable by thecriteria of Rees and Valentine ("). Origi-nally, it was felt that these were due simplyto rupture of the cells either in vitro or dur-ing sectioning, but since this is not seen inhuman lepromatous leprosy, it is probablynot the correct explanation. Rees and Wed-dell ("') and Esiri, et al. ( 2) have postulatedthat M. leprae replicate in muscle cells andthat this is the source of the continued in-

47, 4^ Dawson, et al.: Leprosy in Rats^ 567

fection since these cells cannot form phago-somes and hence are unable to kill the or-ganisms, which eventually are liberated intothe tissues. However, comparison of thenumbers of bacilli found in muscle cells andmacrophages makes it unlikely that theformer are the source of the extracellularbacilli. Further, such organisms would heexpected to appear nonheaded.

Particularly striking was the ease withwhich organisms were found within retic-uloendothelial cells in the liver, spleen,lungs, and lymph nodes. It is not clearwhether these resulted from clearing of theorganisms from the blood stream or frommigration of macrophages from the primarysites of infection in the nonhair hearing ex-tremities. Localization of disease was alsonoted in the interstitium of the testes evenin animals inoculated intravenously. Here,as elsewhere, organisms were found extra-

Our findings in the NTLR are similar tothose previously reported by Evans andLevy in the footpads of intact BALB/c mice(") and by Rees and colleagues in thymec-tomized and irradiated CBA mice 1").The only major difference in the distribu-tion of the organisms was our inability tofind them within the sciatic nerve, a con-stant site of involvement in the mouse ( 2 ').

The exact role of T-cell depletion on thecellular events leading to disseminated lep-rosy in the NTLR has not been explored.While the rats showed depletion of thethymic dependent regions in the spleen andlymph nodes, there is evidence to suggestthat they retain some 'I'-cell function sincetheir lymphocytes are able to respond invitro to concanavalin A, although to a re-duced extent (Colston and Fieldsteel, un-published). Evans and Levy (") have de-scribed an alteration in the appearance ofthe macrophages which they correlate withthe development of cellular immunity andtermination of the logarithmic phase of bac-terial growth in the intact mouse. Our mod-el is, of course, quite different as the infec-tion is generalized. But it is of interest thatvirtually all the macrophages examined byus with the electron microscope were acti-vated, i.e., possessed numerous organellesand bacilli contained within double mem-brane hound phagolysosomes. It should henoted, however, that all of our animals

were examined one year or more after in-oculation. Rees, et al. ( 15) have shownthat replacement of the lymphoid tissue ofthymectomized irradiated mice will restorethe hosts' ability to limit the infection. Thishas not so far been attempted in the rat.The exact nature of the perturbation in lym-phocyte/macrophage interaction that per-mits the progressive replication of M. le-pro(' has yet to he worked out in eithermodel.

SUMMARYWe report the histologic and electron

microscopic findings following intravenousinoculation of M. /eprae into neonatallythymectomized Lewis rats, which werekilled one to two years later. All organs ap-peared normal grossly. Histologic changeswere confined to the footpads, snout, ears,tail, and testes, all of which were involvedin every rat. The tissues were edematousand infiltrated by varying numbers of foamymacrophages. In the footpads muscle fiberswere vacuolated, and small nerves showeddegenerative changes. Large numbers ofM. leprae were present in macrophages andstriated muscle cells and smaller numbersin perineural cells and pericytes, as well aslying free in the tissues. Occasional intra-cellular bacilli were found throughout thereticuloendothelial system. Electron mi-croscopy confirmed that the majority of or-ganisms were within activated macro-phages. Both intact and fragmented bacilliwere contained within double-membranehound vacuoles. Numerous M. /eprac werelying free within the sarcoplasm of striatedmuscle cells. Virtually all of the extracel-lular organisms were degenerating.

RESUMEN

En este trabajo estudiamos los camhios histoldgicosy los ballazgos at microsc(ipio electrintico clue resul-

Limn de la inocultici(in intravenosa con el M. /cprae

en ratas Lewis timectomizadas al nacimiento, las(males fueron sacrificadas I a 2 linos despuits. 'Dodos

los organos tuvicron aria apariencia normal. Los cam-

hios histol(igicos, en today las ratas. estuvieron con-

linados a los cojinetes plantares, la trompa. las (trellis,la cola y los testiculos. Los tejidos estuvieron ede-

matosos e infiltrados por macrotagos espumosos enminteros variables. En los cojinetes plantares, las It-

bras musculares estuvieron vactioladas v los nervios

pequenos mostraron camhios degenerativos. Se oh-servit tin gran minter() de M. leprue en los macr(Wagos

568^ International .lourmil Of Leprosy^ 1979

y en its celulas del intisculo estriado y tin pequeno

ntimero de bacilos en its celulas permetirales y en lospericitos, asi cones algunos hacilos residiendo libre-

mente en los tejidos. Ocasionalmente sc observaronhacilos intraceltilares en el sisteina reticulo endotelial.

La inicroscopia electrtMica coffin - int') que la mayoria

de los inicroorganismos estuvicron dentro de macre,-

fagos activados. Los bacilos. intactos o fragmentados,

se observaron dentro de vacuolas limitadas por mittduhlc membrana. Tainhien se observaron numerosos

Lyra(' residiendo libremente en el Sarcoplasnm de

Its celtilas del imisculo estriado. Todos los inicroor-

ganismos extraeLdulares estuvieron virtualmente de-

generados.

RESUMEOn relate dans eel article les observations histolo-

gigues et de microscopic electronique pratiqueessuite (le intraveinettse de M. leprac chef

des rats nouveau-nes de Lewis thymectomises, tiles

tine oil deux annees plus lard. Tom, Ics organes sent

apparus normaux a tin examen macroscopique. Les

modifications histologiques etaient limitees aux cous-

sine's plantaires, ;11.1 [miscall, aux males. a It queue,

ainsi yu aux leslicules. ces organes Clain atteints chef

lolls les rats. Fes tissus etaient (ledematies et infiltrespar tin nombre variable de macrophages spunteux. I.CS

fibres musculaires dans les coussinets plantaires

etttient vttcuolisees, et les filets net - veils presentment

tine atteinte degenerative. 1)e tres nombreux M. /epraeont pH etre observes dans les macrophages, de mettle

que dans les cellules musculaires strives: on en a

egalement observes en plus petits nombres dans les

cellides, perineum! et dans les pericytes, et egalementClans les tissus. en dehors des cellides. I)es bacilles

infra-celltilaires out egalement etc releves a roccasion

dans le systeme retictilo-endothelial. La microscopicelectronique a confirme que la majorite des organismes

etaient situes a l'interieur de macrophages actives.

Des bacilles intacts. de mettle que des hacilies Crag-

mettles. etaient heberges dans des vacuoles tapissees

(lune double membrane. On a observe de nombreux

M. /eprae libres a l'interieur du stircolemme des cel-

lules musculaires strives. Presque toes ces organismcs

cellulaires etaient degeneres.

Acknowledgments. This work was supported in part

by the U.S.-Japan Cooperative Medical Science Pro-

gram. National Institute for Allergy and InfectiousDiseases. National Institutes of Health. Department

of Health. Education and Welfare (Grant R22 A 1-08417).

We thank Patricia Tsc and Marjorie James for ex-

cellent technical assistance.

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ith

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