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Memórias do Instituto Oswaldo Cruz

Print version ISSN 0074-0276On-line version ISSN 1678-8060

Mem. Inst. Oswaldo Cruz vol.33 no.4 Rio de Janeiro  1938

http://dx.doi.org/10.1590/S0074-02761938000400008 

Infecções experimentaes na Leishmaniose visceral americana

Aristides Marques da Cunha

RESUMO

Passando em revista as experiencias de infecção que effectuamos e que acabamos de relatar, vemos que, as culturas isoladas de casos de Leishmaniose visceral americana quer do homem quer do cão, são capazes de infectar hamsters, rhesus e cães de maneira identica ao que acontece com as outras formas de Kala-Azar. Notamos ainda que a evolução da doença e as lesões observadas nos animaes assim infectados, se assemelham ao que tem sido observado nessa enfermidade aproximando-a de maneira singular do Kala-Azar mediterraneo. É sobretudo no cão que a semelhança se torna mais patente. A infecção da pelle e as lesões por ella provocadas que observamos, não differem em nada das que tem sido descriptas na infecção natural do cão e já assignaladas tambem na infecção experimental desse animal, embora de maneira menos completa do que fazemos agora aqui. As lesões oculares com formação de placas de keratite, a infeccção massiça do intestino nos casos fataes de infecção e até mesmo as lesões do figado descriptas por Adler como peculiares á infecção experimental, (infiltração em torno da veia central do lobulo) constituem outros tantos caracteres que mostram a completa analogia entre as infecções do cão que obtivemos e as já observadas no Kala-Azar mediterraneo. Além disso, a presença de Leishmanias na pelle, as vezes em grande numero e a constancia dessa localisação parasitaria, vem mostrar que o cão apresenta as condições necessarias para desempenhar o papel de depositario de virus tal como acontece no Kala-Azar mediterraneo, tanto mais que é elle encontrado naturalmente infectado, nas regiões em que grassa a doença. Mas não é só a infecção do cão que mostra essa semelhança; nos outros animaes tambem se verifica o mesmo facto e até pequenos signaes, taes como a tumefacção das patas assignalada nos hamsters infectados com Leishmania infantum, foram tambem observados aqui. Por outro lado, a reacção de sôro-agglutinação, conforme mostramos em trabalho anterior, não permite a separação das especies do genero Leishmania, pois todas ellas, quando recentemente isoladas, possuem identica constituição antigenica, que se modifica depois, pela conservação longo tempo em cultura. É esse facto, que deu logar ás conclusões contradictorias a que chegaram os autores que se ocuparam do assumpto bem como os primeiros resultados que obtivemos. Deante de todos esses factos, nos julgamos autorizados a concluir como já fizemos anteriormente, que o agente da Leishmaniose visceral americana é identico á Leishmania infantum. Ao terminar, queremos deixar consignados nossos agradecimentos ao Dr. E. chagas, por ter posto a nossa disposição as culturas de Leishmania por elle isoladas, tornando possivel deste modo, a execução do presente trabalho.

ABSTRACT

With cultures isolated from cases of american visceral leishmaniasis we succeeded in obtaining experimental infections in hamsters (Cricetus cricetus), rhesus monkeys (Macaca mullata) and dogs. Hamsters were infected with strains obtained from man and dogs, the intraperitoneal way having been always employed. When cultures recently isolated are used, infection is obtained practically in 100% of the animals inoculated. The first negative results obtained by us may be explained by the use of cultures isolated some time before (about 7 months 0 and which had lost already their virulence. In some cases external lesions are observed represented by alterations of the skin and swelling of the paws. The skin lesions are observed on the ventral surface and consist in depilation, erythema and exudation. The skin thus affected shows to be extremely friable, rupturing at the movements of the animal when hold. On post-mortem examination, besides the lesions pointed out, the animals present enlargement of the spleen. The parasites are very numerous in the spleen, liver, bone marrow, etc. The changed skin shows considerable hypertrophy of the epithelium with degeneration of the cells of the superficial layers, intensive infiltration of the derm by mononucleate cells full of parasites, and strong hyperemia. In rhesus monkey, we obtained until now, only one case of infection. The inoculation was made by the intraperitoneal way with cultures in Noeller's medium, isolated from a human case of visceral leishmaniasis, found in Chaco Argentino. About 8 months after the inoculation, the monkey was found in agony and was sacrified; the post-mortem examination showed that it was intensely infected. For infecting dogs, we employed young animals, 1 to 2 months old. The cultures were recently isolated (1 to 3 months) from man or dog. For the inoculation we used thick suspensions of flagellates from plate cultures by Mayer and Ray's method. The inoculations were carried out through the intraperitoneal way and renewed 3 to 4 times with intervals from 4 to 8 days. We observed an incubation period of 3 to 4 months when we used cultures obtained from dogs, and a period of 5 to 7 months, when employed human strains. Formerly, in order to verify the infection it, was used the liver puncture; later on we also examined the bone marrow removed by trepanation. We wish to emphasize the advantages derived from the examination of the bone marrow, as there the parasites are much more numerous than in the liver, thus making the examination easier. The infection shows itself in the animal by fever, anemia and emaciation at times attaining cachexia. Apart from this, skin lesions are observed represented by depilation, seborrhea, and even ulceration. In the dog A. we observed keratitis on both eyes, and in the dog C, diminution of vision nearly attaining complete blindness. What called our particular attention, was the infection of the skin. In this, parasites are always found in any region o fthe body, although presenting some elective sites such as the paws where the parasites first appear and where they generally are more numerous. At the beginning of the skin infection we see in the derm macrophages, containing parasites, generally 3 to 4, and arranged along the vascular tracts. With the exception of these macrophages, the skin looks nomral. The number of parasited macrophages increases gradually and then appear the changes of the skin here described. The main change observed is represented by infiltration of the derm by mononucleate cells. Such infiltration is mainly located around the pilo-sebaceous folliches. In other cases, the infiltation is located in the chorion. Finally, in certain cases of intense infiltration, it extends uniformly over the whole derm. The infiltration is sometimes constituted chiefly by parasited macrophages and this occurs mainly when the infiltration is not very intensive. When the infiltration becomes more intensive, the number of not parasited macrophages increases, and in cases of ulceration the parasited macrophages become very rare or entirely disappear. The four dogs experimentally infected which died either from the infection or from an intercurrent disease were all examined post-mortem. On the post-mortem examination, the macroscopical changes observed in a constant manner are not important and only consist in an enlargement of the spleen. In the dogA, we found keratitis already observed in the same living animal, and the intestine intensely congested with sanguinolent contents and the axillary and inguinal lymphatic glands enlarged and congested. Smears from the organs showed very numerous parasites, chiefly in the spleen, liver, bone marrow and lymphatic glands. In liver sections we observed the infiltration mainly located around or near by the intralobar vein a feature which already has been pointed out by Adler in the experimental infection of dog. Parasites were also found in Kupffer's cells, and a few parasited macrophages were observed in the porta-spaces as well as in the tissue of the lobule. The intestine of the same animal was intensely parasited, the Leishmaniae being located mainly in the mucous membrane, in infiltration cells located amongst Lieberkuehn's glands and some parasited macrophages could also be observed in the sub-mucosa. Mice as well as the rodents Dasyprocta agouti and Proechymis oris when inoculated, did not become infeced. From the exposed we may conclude that the experimental infections obtained, present a complete analogy with those described in the Mediterrean Kala-azar. On the other hand, the sero-agglutination test, as shown by us in a previous paper, allows no distinction of the species of Leishmania, as all the strains when recently isolated, have the same antigenic constitution which later on undergoes modifications when the Leishmaniae are preserved in culture for a long time. Thus, we feel authorized to conclude that the agent of the visceral american leishmaniasis is identical with L. infantum.

 

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