Rhésus foram infectados com culturas recentemente isoladas de L. brasiliensis. As inoculações foram feitas por via intradérmica nas arcadas superciliares e, excepcionalmente, na superfícia do nariz. O prazo da incubação foi de 1 a 2 mêses. A marcha da infecção varia conforme as lesões se limitam à pele ou se estendem às mucosas. No primeiro caso, a duração da infecção foi de 3 a 8 meses e terminou sempre pela cura espontânea. Quando as lesões se estendem às mucosas elas persistem e se agravam, enquanto as lesões cutâneas observadas no mesmo animal, regridem, tendendo para cicatrização e cura. Cães também foram injetados com resultados positivos. As inoculações foram praticadas por via intradérmica na superfície do nariz. O prazo de incubação foi de cêrca de 2 mêses e as lesões observadas se limitaram à pele e terminaram pela cura. Uma cotia (Dasyprocta agouti) foi inoculada não se mostrando infectada. Um rhésus, curado de uma infecção pela L. brasiliensis, inoculado de novo com outra amostra do mesmo parasito, não se mostrou infectado. A extensão das lesões às mucosas nas infecções experimentais do rhésus pela L. brasiliensis, fato ainda não veificado na L. trópica, constitue mais um argumento em favor da independência daquela espécie.

Rhesus specimens were infected with recently isolated cultures of L. brasiliensis. The inoculations were made intradermically into the superciliary arches and, exceptionally, into the skin of the nose. The incubation period from 1 to 2 months. The course of the infection varies according to the extention of the lesios if limited to the skin of invading the mucous membranes. In the first case, the infection lasted form 3 to 8 months and always termninated in spontaneous cure. When the changes extend to the mucous membranes, they persist and evolute, whilst the skin changes observed on the same animal become regressive and tending towards cicatrization and cure. Also dogs were inoculated with positive results. The inoculation were carried out by intradermic way into the skin of the nose. The incubation period was of about 2 months and the changes observed were confined to the skin and terminated in cure. One agouti (Dayprocta agouti) was inoculated but was not infectes. One Rhesus which recovered from an infection by L. brasiliensis, reinoculated with another strain of the same parasite, was also not infected. The extent of the changes to the mucous membranes, in experimental infections of Rhesus by L. brasiliensis, a fact not yet observed in L. tropica, consitutes one more argument in favour of the independence of the species.

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.

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.

No presente trabalho estudamos o comportamento de alguns ciliados do genero Balantidium nas culturas em meios artificiaes. Utilisamos de preferencia nesses estudos o meio para ameba de Dobell e Laidlaw, enriquecidos com amido de arroz. As especies com que trabalhamos foram: B. coli isolado do homem; Balantidium sp. do porco e B. simile do Macaca mulatta. A especie originaria do homem, foi por nós cultivada com grande facilidade durante um tempo bastante longo, fazendo-se os repiques com o espaço de 24 e 48 horas. Observamos com grande constancia nessa especie o apparecimento do phenomeno de conjugação nas culturas de 24 horas (cultura original ou repiques). Os pares eram constituidos por fórmas pequenas medindo em média 38,6 X 32,1 micra ao contrario das fórmas neutras que mediam em média 75,5 X 57,8 micra, sendo de notar que fórmas pequenas eram tambem encontradas em grande numero isoladas (preconjugantes). Essas fórmas se caracterisavam nos preparados corados, não só pelo seu tamanho como tambem pela grande dimensão do micronucleo que tinha em média 6 micra de diametro. Pelo estudo do material corado verificamos que os elementos depois de se reunirem pela região do peristoma dando origem aos pares, soffrem 2 divisões successivas (mitoses) do micronucleo e dos 4 elementos assim formados, 3 soffrem degeneração, vindo o restante novamente a se dividir para formar os pronucleos em torno dos quaes se processa uma condensação do plasma. Depois da troca dos pronucleos migradores que se vão collocar em cada um dos elementos em conjugação ao lado dos pronucleos estacionarios, dá-se a formação de 2 fuzos de divisão, parallelos (gonomeria) só então processando a fuzão dos 2 pronucleos (estacionario e migrador). Dos 2 novos elementos originados dessa divisão, um vae constituir o novo micronucleo e o outro depois de soffrer nova mitose, vae dar origem a duas placentas que crescendo pouco a pouco vão se fundir mais tarde para dar origem ao novo macronucleo. Os macronucleos dos elementos que entraram em conjugação, permanecem durante quasi todo o phenomeno embora em via de degeneração por picnose, fragmentando-se mais tarde para serem reabsorvidos na pahse em que se processa a fusão dos pronucleos. O Balantidium sp. existente nas fézes do porco, foi tambem por nós cultivado com grande facilidade, occorrendo o phenomeno de conjugação constantemente nas culturas de 24 horas. Com o B. simile parasita do M. mulatta o resultado foi completamente differente. Essa especie é dificil de cultivar e só se obtem resultados usando certos artificios de technica taes como juncção de uma substancia tampão ao meio de cultura conseguindo-se então uma intensa proliferação do ciliado 24 horas depois da sementeira, quer nos tubos iniciaes como nos repiques (4ª a 5ª passagem). Essa especie ao contrario da do homem e da do porco absolutamente não conjunga nas culturas e isso podemos affirmar baseados em longa observação. Nas culturas se passa identico facto ao que já foi por nós observado e descripto em material de fézes de M. mulatta, isto é, a « Endomixia ». Nos preparados corados feitos com o material das primeiras culturas como dos repiques, pudemos observar toda a evolução do phenomeno, isto é, desde as fórmas iniciaes (mitose do micronucleo e inicio de degeneração do macronucleo) até as phases finaes do processo. Nesse material verificamos mais uma vez, de maneira clara que a formação do micronucleo se faz a custa de uma das placentas como já tivemos occasião de assignalar em nosso trabalho acima referido. Nelson em recente trabalho (Observations and Experiments on Conjugation of the Balantidium from the Chimpanzee - Amer, Jour. of Hyg., Vol. 20, nº 1, 1934), põe em duvida o phenomeno de endomixia estudado por nós nessa especie de Balantidium. A affirmação desse autor não merece ser discutida por não ter elle apresentado a unica prova que poderia invalidar a nossa observação e que seria, a demonstração da existencia de pares de conjugantes, facto esse que o proprio autor declara nunca ter observado em material que teve occasião de estudar. Quanto ao nosso material que Nelson diz ter examinado temos a declarar se tratar apenas de algumas laminas nas quaes podiam ser vistas algumas das phases do phenomeno e que foram cedidas por nós ao Professor Hegner quando de passagem pelo Brasil. Baseados em longas pesquisas, concluimos que o Ealantidium simile representa uma especie de ciliado em que a conjugação parece tersido substituida de maneira permanente, pelo phenomeno de endomixia. Facto identico occorre, segundo estudos nossos ainda ineditos, em um grupo de ciliados, ito é, na familia Cyathodiniidae. Para esses ciliados propomos aqui a denominação de ciliados azygoticos ou parthenogeneticos.

We report here on the behaviour of some Ciliates of te genus Balantidium in artificial culture media. For this study we have employed mainly Dobell and Laidlaw's amoeba medium to which rice starch was added. We investigated Balantidium coli, isolated from man; Balantidium sp., from the pig; and Balantidium simile, from Macaca mulatta, the Rhesus monkey. We could cultivate the human parasite very casily for a long time, transplanting it every 24 to 48 hours. Conjugation was very constantly observed in this species in culture about 24 hours old, either original cultures or transplants. The conjugants are found in pairs as small forms about 38,6 X 32,1 micra. The neutral individuals reach an average size of 75,5 X 57,8 micra. Small isolated preconjugant forms were also found in large numbers. These pre-conjugant forms in stained slides could be distinguished not only by their small size but also by the large size of their micronulcei which reached an average of 6 micra in diameter. The study of the stained material shows that the conjugants associate in pairs attaching themselves one to another by their peristomal region. Then their micronuclei divide twice by mitosis. From the four micronuclei thus three degenerate the remaining one dividing again in order to form the pronuclei around which a condensation of the cytoplasm is produced. After the exchange og the migratory pronuclei takes place, they are found in cach of the conjugants side by side with the stationary pronuclei. In each pair of pronuclei two mitotic spindles are formed, paralled one to another (gonomery) the fusion of both pronuclei (stationary and migratory) only then taking place. From both nuclei formed by this mitotic division simultaneous to the nuclear fusion, two new nuclei result one of which will become the new micronucleus. The other new nucleus divide again to form two placentae which will slowly grow and then fuse again to form the new macronucleus. The macronuclei originally found in both conjugants in spite of being found to degenerate by pycnosis are usually seen throughout almost all the conjugating process. Later on they break up in fragments which are reabsorved during the stage of pronuclear fusion. The species of Balantidium occurring in the pig was also very easily cultivated, conjugation being constantly observed in 24 hours old cultures. In Balantidium simile things were quite different. This species is difficult to cultivate, good results being obtained only when special technique is used as, for instance, when a buffer is added to the culture medium. In such a case abundant growth and multiplication of the Ciliate is seen after 24 hours either in the original cultures or in the transplants to the 4th. or 5th. subcultures. On the ground of a prolonged observation we can assert that Balantidium simile, in opposition to what one sees in the human and pig parasites, never conjugate in culture. In the original cultures and transplants of Balantidium simile the same phaenomena occur that we described from the faeces of Macaca mulatta, the Rhesus monkey, that is endomixis. On the stained slides we could observe the entire process, since the appearance of the initial forms (mitosis of the micronucleus and beginning of the degeneration of the macronucleus) until the final stages. Once more in this material we could see in a very clear way that the micronucleus is formed from one of the placentae, as we had the opportunity to show in a previous paper. Nelson in a recent paper (Observations and Experiments on Conjugation of the Balantidium from the Chimpanzee - American Journal of Hygiene, vol 20, n. 1, 1934), express his doubts about the phaenomena of endomixis we described in Balantidium simile. This author's opinion does not deserve discussion since he did not present the only proof that could invalidate our conclusions, that is the demonstration of the occurrence of pairs of conjugants, a fact he never succeeded to observe in the material at his disposal. As for our material that Nelson says to have examined it consists only in a few slides we gave to Professor Hegner when he visited Brazil. In these slides some stages of endomixis could be seen. On the ground of long lasting researches we conclude that Balantidium simile is a Ciliate in which conjugation seems to have disappeared and been relaced entirely and permanently by endomixis. According to our researches a similar fact accurs among another group of Ciliates, the families Cyathodiniidae and Enterophrydae. We propose to name azygotic or parthenogenetic the Ciliates in which this phaenomenon is observed.