Volume 52, Number 2Printed in the U.S.A.

INTERNATIONAL. JOURNAL or LEPROSY

Ultrastructural Features of Macrophages ofArmadillos Infected with Actively

Multiplying Mycobacterium lepraelYukiko Fukunishi, Wayne M. Meyers, Gerald P. Walsh, Frank B. Johnson,

Chapman H. Binford, Seitaro Okada, and Mitsugu Nishiura 2

In 1974, Yoshizumi, et al. ( 13) reportedthe electron-microscopic findings of lesionsin the nerves of an armadillo (Dasypus no-vemcinctus) inoculated with Mycobacte-rium leprae and, in ultrathin sections, dem-onstrated intracytoplasmic foamy structuresin the macrophages and Schwann cells ofthe peripheral nerves. This confirmation offoamy structures in the lesions of experi-mental leprosy in the armadillo is importantfor the identification of the etiologic agentas M. leprae; however, direct observationof the three-dimensional ultrastructure ofthe bacilli in the experimental lesions is notpossible in ultrathin sections. Employing thefreeze-etching technique, we have studiedthe three-dimensional ultrastructure of le-sions in armadillos experimentally infectedwith M. leprae. This freeze-etching tech-nique is highly useful because it reveals boththe surface structure of the walls of the ba-cilli and the details of the intracytoplasmicfoamy structures. We compared the findingsin experimental leprosy in armadillos withthose in lepra cells in humans and in mac-rophages of nude mice infected with Al. lep-rae ( 3 . 9).

MATERIALS AND METHODSBy ultrathin sectioning and freeze-etch-

ing, we examined the ultrastructure of ex-perimental lesions in lepromas, lymph

' Received for publication on 7 July 1983; acceptedfor publication on 5 October 1983.

2 Y. Fukunishi, M.D., Ph.D.; W. M. Meyers, M.D.,Ph.D.; G. P. Walsh, Ph.D.; F. B. Johnson, M.D., andC. H. Binford, M.D., Armed Forces Institute of Pa-thology, Washington D.C. 20306, U.S.A. (Dr. Fuku-nishi on leave from Kyoto University). S. Okada, M.D.,and M. Nishiura, M.D., Ph.D., Leprosy ResearchLaboratory, Kyoto University School of Medicine,Sakyo-Ku, Kyoto 606, Japan. Reprint requests to Dr.Fukunishi, National Sanatorium, Oshima Seisho-en,6034-1, Aji-cho, Kita-gun, Kagawa Prefecture, Japan.

nodes, spleens, livers, and lungs of 7 nine-banded armadillos (Dasypus novemcinctus)inoculated with Al. leprac. One armadillowas inoculated directly with 3.4 x 10 8 Al.leprae isolated from a human leproma. Allof the other six animals were inoculated with2 x 10 10 organisms isolated from lesions ofarmadillos infected with M. leprae of hu-man origin that had been passaged once inarmadillos. Specimens were obtained 18months after inoculation from the arma-dillo inoculated with bacilli of human originand 11-13 months after inoculation in thesix animals which received bacilli from ar-madillo passage.

All tissues were from M. leprae-infectedarmadillos that had been killed with anoverdose of ketamine hydrochloride. Spec-imens for ultrathin sectioning were fixedwith 3% glutaraldehyde in 0.06 M phos-phate buffer (pH 7.4) for 24-48 hr, and thenwith 2% 0s0, in distilled water for 14-24hr at 4°C. The tissues were embedded inmethacrylate, cut by ultramicrotome, andthen stained with uranium acetate and leadcitrate. Tissues for study by the freeze-etch-ing technique were fixed with 3% glutaral-dehyde under the same conditions as forultrathin sectioning, and then immersed in20-40% glycerol for 1-2 days at 4°C. Otherprocedures were the same as described pre-viously by Nishiura ( 9 ).

RESULTSIn 6 of the 7 armadillos, ultrathin sections

demonstrated typical intracytoplasmicfoamy structures (Fig. 1). In all of the ar-madillos the freeze-etched preparations re-vealed intracytoplasmic foamy structures inphagolysosomes. These structures con-tained small spherical droplets that ap-peared to have accumulated around the lep-rosy bacilli (Figs. 2-4).

198

52, 2^Fukunishi, et al.: Ultrastructure—Armadillo Leprosy^199

FIG. 1. Ultrathin section of leproma from an ar-madillo with experimental leprosy. Note the largenumber of leprosy bacilli in a phagolysosome of a mac-rophage. Arrows indicate an electron-transparent zonein the intracytoplasmic foamy structure ( x 10,000).

B = leprosy bacilli; E = electron-transparent zone;R = red blood cell; S = spherical droplet; scale = 1 pm.

FIG. 2. Freeze etching of leproma from an arma-dillo with experimental leprosy. Arrows indicate spher-ical droplets in an intracytoplasmic foamy structure ina macrophage ( x 26,000). (See Fig. I for symbol leg-end.)

FIG. 3. Freeze etching of a lepra cell from an ar-madillo with experimental leprosy. A large amount ofspherical droplets are observed around leprosy bacilli.Arrows indicate spherical droplets ( x 59,500). (See Fig.1 for symbol legend.)

Fic. 4. Freeze etching of lepra cell from an arma-dillo with experimental leprosy. Observe the leprosybacilli and spherical droplets surrounded by phagoly-sosomal membrane ( x 44,000). (See Fig. 1 for symbollegend.)

The organic solvents employed in tissueprocessing for ultrathin sections extract lip-ids from the foamy structures, and this ex-traction is believed to be responsible for theelectron-transparent zones in the intracy-toplasmic foamy structures in macrophagesin the ultrathin sections. In freeze-etchingreplication, however, the fine details of theintracytoplasmic foamy structures are pre-served as aggregations of small sphericaldroplets around bacilli in phagolysosomes.

Almost all of the bacilli in these arma-dillos were long and slender, with bandstructures visible on the smooth cell wallsurfaces (Figs. 5-7). Each band is composedof two circumferential fine lines on the sur-face of the cell wall (Fig. 7). All of thesecharacteristics of the cell wall and band

200^ International Journal of Leprosy^ 1984

FIG. 5. Ultrathin section of leprosy bacilli in a lepracell from an armadillo with experimental leprosy( x 37,000). (See Fig. I for symbol legend.)

FIG. 6. Freeze etching of leprosy bacilli in a lepracell from an armadillo with experimental leprosy. Ba-cilli are long and slender with band structures on thecell wall of the bacillus (arrow) ( x 47,000). (See Fig. Ifor symbol legend.)

structures of the bacilli are typical of thoseof M. leprae as seen in tissues from leprosypatients. The ultrastructural findings in le-sions of armadillos infected with M. lepraeobtained directly from patients and fromfirst passage M. leprae were the same.

In one of the armadillos receiving pas-saged bacilli, there were early lesions onlyin the spleen. We believe that the M. lepraehad not multiplied sufficiently in this ani-mal to produce typical intracytoplasmicfoamy structures; however, the bacillishowed the characteristic features describedabove.

DISCUSSIONThe intracytoplasmic foamy structures in

experimental armadillo leprosy have beenconfirmed by ultrathin sectioning (Yoshi-zumi, et al.'') and freeze etching (present

Fu;. 7. Freeze etching of leprosy bacilli in a lepracell from an armadillo with experimental leprosy. Ar-rows indicate band structures on the cell wall surface.Each band structure is composed of two thin lines( x 100,000). (See Fig. I for symbol legend.)

study). Since similar intracytoplasmic loamystructures are consistently observed in hu-man lepra cells and in nude mice lesionscaused by inoculation with M. /ePraC, thisfoamy substance seems to have a close re-lationship to the multiplication of M. lepraein host cells ( 3 ).

However, similar foamy structures werealso found in naturally acquired leprosy-likedisease in armadillos by Marchiondo, et al.( 7) and Walsh, et al. (personal communi-cation). According to the studies alreadypublished, the pathogens of this natural lep-rosy-like disease in armadillos have biolog-ical characteristics very similar to those ofhuman leprosy bacilli ( 8 . 12).

Although morphological criteria includ-ing ultrastructural features are very usefulfor the detection of differences among dif-ferent mycobacteria, they are not the deci-sive criteria for the identification of bacillifrom experimental models. All of the ultra-structural, immunological, and biologicaldata must be taken into consideration.

Kauai, et a!. (") studied the ultrastructuralfeatures of M. bons, Ravenel R-KM strain,and reported that the electron-transparentzone between the phagosomal membraneand the bacillary cell wall is mostly artifact.They suggested that in the artifact-free con-dition the gap between the phagosomalmembrane and the bacillary cell wall is verynarrow and it might actually be a tightphagosome in which a bacillus is wrappedtightly by a phagosomal membrane. Theydid not report the presence of foamy struc-

52, 2^Fukunishi, et al.: Ultrastructure— Armadillo Leprosy^201

tures around M. bovis in this study. In thecase of Al. leprae infections, leprosy bacilliare observed in tight phagosomcs in the ear-ly stage of development of leprae cells, butas the leprosy bacilli multiply in the phago-somes of macrophages, lipidic material isaccumulated and this always has the ap-pearance of spherical droplets in the phago-lysosomes of the lepra cells.

The ultrastructural features of the lipidicmaterial which accumulates around .11. lep-raeniurium are very much different fromthose of the spherical droplets of M. leprae.In the case of Al. lepraeniurium, the peri-bacillary substance is chiefly made up ofcrystalline material attached to the myco-bacterial cell wall when observed by thefreeze-etching technique. Chemically, thiscrystalline material is principally mycosideC according to Draper, et al. ( 42). Takeo,et al. (I I) studied 19 species of mycobacteriagrown in vivo by the freeze-etching tech-nique, and spherical droplet-like structureswere never observed in any of the myco-bacteria which they examined.

Recent studies by Brennan, et al. ( -4 5 ) elu-cidated the biochemical structures of theperibacillary substances of Al. leprac grownin armadillo tissue. The immunological datausing these lipid materials from infected ar-madillo tissue, which are available now,suggest the identity of the peribacillary sub-stance of bacilli in an experimental arma-dillo lesion and the peribacillary substanceof Al. leprae in human lepromatous tissue

1 ,1 , .) From the available data, it seemsmost probable that the peribacillary sub-stance found in the armadillo lesion by freezeetching in this study corresponds to the li-pidic substance which Brennan's groupstudied.

As to the forms of mycobacteria found inexperimental armadillo leprosy, the bacilliwere usually slender and smooth surfacedwith band structures located on the surfaceof the cell wall just like human leprosy ba-cilli.

SUMMARYExperimental leprosy lesions in the ar-

madillo (Dasypus novemcinctus) were stud-ied by freeze etching and ultrathin section-ing. Infected macrophages have distinctintracytoplasmic foamy structures in theform of spherical droplets accumulated

around multiplying bacilli. This finding isthe same as those observed in human lepracells and nude mice macrophages infectedwith M. leprae.

RESUMENEl estudio al microscOpio electrOnico de las lesiones

experimentales en el armadillo (Dasypus novemcinc-tus) por la tecnica de impresiOn por congelamiento yen secciones ultradelgadas, mostrO la presencia de es-tructuras espumosas peculiares en los macrOfagos delas lesioncs. Las caracteristicas de tales estructuras es-pumosas son las mismas que las de las "celulas de lalepra" en los humanos y que las de los macrofagos deratones desnudos infectados con Mycobacterium lep-rae.

RESUMEUne etude menee par microscopic èlectronique sur

les lesions de lepre cxperimentale chez le tatou (Das-rpus novemcinctus) par cryodecapage et des coupes ul-tra minces a revel& des structures intracytoplasmiqucspumeuses distinctes dans les macrophages des lesions.Les caracteristiques de ces structures spumcuses etaientles mémes que celles que l'on relêvent dans des cellulesde lepre humaine et dans des macrophages de sourisglabres infectees par Mycobacterium leprae.

Acknowledgments. This study was supported in partby the Sasakawa Memorial Health Foundation, theImmunology of Leprosy (IMMLEP) component of theUNDP/World Bank/WHO Special Programme for Re-search and Training in Tropical Diseases, the Ameri-can Leprosy Missions, Inc., and the Damien-DuttonSociety for Leprosy Aid, Inc.

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