Int. J. Exp. Path. (1990), 71, 791-797

Experimental visceral leishmaniasis: sequential eventsof granuloma formation at subcutaneous inoculation site

Marcia Dalastra Laurenti, Mirian Nacagami Sotto,Carlos Eduardo Pereira Corbett, Vania Lucia Ribeiro da Matta

and Maria Irma Seixas DuarteInfectious Disease Pathology Laboratory, Department of Pathology, University of Sdo Paulo Medical School,

Sdo Paulo, SP, Brazil

Received for publication I 3 October I989Accepted for publication 20 June I990

Summary. Hamsters were inoculated with 07 Leishmania (Leishmania) chagasi amastigotes inthe hind footpads and killed at 7, I 5, 30, 45, 6o, 75 and go days after infection. We observedmononuclear inflammatory infiltrates with many parasites on the 7th and I5th days ofinfection. On the 30th day there was early granuloma formation. After 45 days the lesion wascharacterized by well defined epithelioid granuloma with multinuclear giant cells whosecytoplasm showed Schaumann bodies. Non-particulate antigenic material was present in themacrophage cytoplasm and between the lamellae of the Schaumann bodies. Granulomaformation has an important role for the control of infection at the inoculation site. The resultsindicate that dissemination of the infection must occur in the first 45 days, before granulomaformation has taken place.

Keywords: visceral leishmaniasis, Leishmaniaimmunoperoxidase

The histopathology of different organsinvolved in visceral leishmaniasis has beendescribed by many authors (Ritterson 1955;Duarte et al. I983; Duarte & Corbett I984,I987; Gutie'rrez et al. I984) but very little isknown about the cutaneous changes whichoccur after parasite inoculation by mosquitobites.Manson-Bahr (I955) and Sen Gupta and

Das Gupta (I95i) described a leishmania-induced granuloma at the site of subcuta-neous inoculation with promastigotes inhuman volunteers. It was histologicallycharacterized by a granulomatous reaction

(Leishmania) chagasi, pathology, granuloma,

with round cells, lymphocytes, plasma cellsand large macrophages with parasites andlate giant and epithelioid cell formation. Thisreaction persisted even when well definedvisceral leishmaniasis lesions developed(Manson-Bahr I959; Manson-Bahr et al.I963).The early histopathological changes after

subcutaneous inoculation of promastigotesofLeishmania donovani in hamsters (Wilson etal. I987), started with an infiltrate of mainlypolymorphonuclear neutrophils and somemacrophages with phagocytosis of parasites.After 24 h the infiltrate was composed

Correspondence: Mdrcia Dalastra Laurenti, Faculdade de Medicina da USP, Depto. de Patologia, Av. Dr.Amaldo, 455, Io andar, sala 26, CEP: OI246, Sao Paulo, SP, Brasil.

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mainly of mononuclear cells with posteriorgranuloma formation. Dissemination of theinfection was detected after 8 weeks by thepresence of parasites in the mononuclearphagocytic cells of the liver and spleen.

This study was designed to investigate thehistopathological changes which develop atthe site of subcutaneous inoculation of Leish-mania (Leishmania) chagasi in the hind foot-pads of the hamster with special attention toparasite antigen kinetics and to granulomaformation.

Material and methods

Animals

Male 45-60 days old outbred hamsters(Mesocricetus auratus) were used, obtainedfrom University of Sao Paulo Medical SchoolGeneral Colony.

Parasites

Leishmania (Leishmania) chagasi (MHOM/BR/72/strain 46). A hamster with approxima-tely 3 months infection was killed and thespleen removed and weighed. The parasitesin spleen smears stained by Giemsa's methodwere counted. The spleen parasite load wascalculated by determining the number ofamastigotes found per IOOO spleen cell nuc-lei x spleen weight (mg) X 2 x IO' (StauberI958). The spleen was homogenized in aglass-tissue grinder and adjusted with salineto give a concentration of I X IO7 amasti-gotes/o. I ml.

Infection

Seven experimental groups, each with fiveanimals, were inoculated s.c. with L. (L.)chagasi in the hind footpads with IO7 amasti-gotes. There were two control animals in

Fig. i. Lymphocyte and macrophage infiltrate in the deep dermis. Many leishmania are present.H&E x 586.

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each experimental group, inoculated witho. i ml of normal spleen homogenate.The animals were killed at 7, 15, 30, 45,

6o, 75 and go days after infection. Frag-ments from the hind footpads, spleen andliver were fixed in buffered io% formalinsolution, pH 7.2, and processed by usualhistopathological techniques and stainedwith haematoxylin-eosin.

Imprints from liver and spleen were fixedwith methanol and stained by Giemsa'smethod.

Fragments from the hind footpads werestained by the immunoenzymatic peroxi-dase-antiperoxidase (PAP) method usingpolyclonal anti-leishmania serum producedin rabbit for the detection of parasite anti-gens. The same fragments were also reactedwith anti-leishmania serum absorbed with apellet of both amastigotes and promastigotesparasites to confirm the specificity of themethod (Sotto et al. I989).

Livers from experimentally infected hams-ters were used as positive controls.

Results

Histopathological changes at the inoculationsite, hind footpads, on the 7th day werecharacterized by a mild inflammatory infil-trate of macrophages, plasma cells and lym-phocytes and intra and extra-cellular para-sites into the deep dermis (Fig. i). From theI sth day there was spread of infection to themuscular layer with many parasites. On the30th day there was a diffuse mononuclearinflammatory infiltrate and an initial granu-loma formation and reduction in the numberof parasites. At the 45th day the lesion wascharacterized by well defined epithelioid gra-nulomas, a few polymorphonuclear neutro-phils among the macrophages and very fewremaining parasites (Fig. 2). At this time themultinuclear giant cell cytoplasm contained

Fig. 2. Epithelioid granulomas near to a nerve. H&E x 293.

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round, lamellar, concentric and basophilicstructures known as Schaumann bodies (Fig.3). Similar structures were also seen as smallaggregates within macrophages. This pat-tern, seen at the 45th day, was also presentin the 60-, 75- and go-day infection groups.

Visceral leishmania were demonstrated45 days after infection. Amastigotes of L. (L.)chagasi were found in the mononuclearphagocytic system in the liver and spleen byhistopathological and imprint observations(Fig. 4a, b).Leishmania antigen was detected with

specific antibodies using the peroxidase-antiperoxidase immunoenzymatic method(PAP). Numerous amastigotes were identi-fied after 7, I5, and 30 days infection butwere uncommon at later times after infec-tion.

Non-particulate antigenic material wasalso present in the cytoplasm of macro-

phages and multinuclear giant cells withingranulomas (Fig. 5) after 45, 6o, 75 and godays of infection. The same antigenic mater-ial was seen between the lamellae of theSchaumann bodies within giant cell cyto-plasm (Fig. 6a).

Fusiform subepidermal cells with non-particulate antigenic material within thecytoplasm were found go days after infection(Fig. 6b).The PAP reaction, using antiserum pre-

viously absorbed with parasites, did notreveal leishmania antigen in any group.

Discussion

Inoculation of L. (L.) chagasi in the hindfootpads of hamsters produced an inflamma-tory infiltrate, mainly mononuclear, rich inparasites 7 and i5 days after infection.Macrophages at the 30th day had a tendency

Fig. 3. Schaumann bodies within a multinuclear giant cell and in the centre of a small granuloma.H&E x 586.

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Experimental visceral leishmaniasis

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Fig. 4. Parasitized macrophages in a, Ithe liver and b, the spleen. H&E x I465.

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Fig. 5. Leishmania antigen within a, macrophage cytoplasm (arrow) and b, in multinuclear giant cell.PAPx 586.

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~~~~~~~

Fig. 6. Non-particulate antigenic material a, between the lamellae of Schaumann bodies (arrow) and b,within the cytoplasm of a fusiform subepidermal cell (arrow). PAP x 586.

to develop a nodular arrangement, as iftrying to circumscribe the parasites. Fromthe 45th day onward, well defined epithe-lioid granulomas were present, containinggiant cells with lamellated, concentric andbasophilic structures known as Schaumannbodies (Williams I960). When granulomaformation was present, few parasites couldbe detected by PAP but non-particulateantigenic material was present withinmacrophages, giant cells and between thelamellae of the Schaumann bodies.The Schaumann bodies were frequently

seen in hamster macrophages and are consi-dered in these animals to be indicative of afragile mononuclear phagocytic system(Dumont & Sheldon i965). Schaumannbodies originate from residual bodies, whichare end-products of activated lysosomes(Williams & Williams i968).Granulomas observed at the 45th day of

infection were characterized by numerous

Schaumann bodies such as are seen inexperimental tuberculosis in the hamster(Dumont & Sheldon I965). In this model,these structures were observed aroundmycobacteria or their antigens, which weresurrounded by activated macrophageswhich help to confine the infection. Inexperimental visceral leishmaniasis, therewas a comparable development of Schau-mann bodies at the site of inoculation; at thissite the numbers of parasites decrease butantigenic material persists in the macro-phage cytoplasm as residual material whichis revealed by PAP staining.

Ridley and Ridley (I986) have alreadysuggested that granuloma formation withmacrophage activation is a mechanism forlysis and destruction of the parasite.The finding of degraded antigen within

macrophages and giant cells both in thegranuloma and in subepidermal fusiformcells (Langerhans cells) at the goth day

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indicates the existence of a mechanism ofparasite killing and control of infection at theinoculation site. However, degraded anti-gens are not completely destroyed and willpersist for a long time at this site thusproviding a persistent stimulation for granu-loma formation even after the parasite isdestroyed. It also indicates that the dissemi-nated parasites, found in viscera, must haveleft the inoculation site before granulomaformation, that is, the parasite could spreadonly in the first 45 days of infection while thedefence mechanism was still non-specific. Itis not clear why granuloma formation takesso much time to develop after infection ofLeishmania when after other infections, suchas tuberculosis, it takes only 2-3 weeks.

Acknowledgements

This work was supported by FINEP and LIM-72/HC-FMUSP. We thank Roselaine A.P.Cardoso for technical assistance, Mfircio C.Baptista for photographic technical assist-ance, and Maria Daisi Moraes for secretarialhelp.

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