Vector-borne transmission of Trypanosoma cruzi among captive Neotropical primates in a Brazilian zoo.

BACKGROUND: Neotropical primates are important sylvatic hosts of Trypanosoma cruzi, the etiological agent of Chagas disease. Infection is often subclinical, but severe disease has been described in both free-ranging and captive primates. Panstrongylus megistus, a major T. cruzi vector, was found infesting a small-primate unit at Brasília zoo (ZooB), Brazil. ZooB lies close to a gallery-forest patch where T. cruzi circulates naturally. Here, we combine parasitological and molecular methods to investigate a focus of T. cruzi infection involving triatomine bugs and Neotropical primates at a zoo located in the Brazilian Savannah. METHODS: We assessed T. cruzi infection in vectors using optical microscopy (n = 34) and nested PCR (n = 50). We used quantitative PCR (qPCR) to examine blood samples from 26 primates and necropsy samples from two primates that died during the study. We determined parasite lineages in five vectors and two primates by comparing glucose-6-phosphate isomerase (G6pi) gene sequences. RESULTS: Trypanosoma cruzi was found in 44 vectors and 17 primates (six genera and eight species); one Mico chrysoleucus and one Saguinus niger had high parasitaemias. Trypanosoma cruzi DNA was detected in three primates born to qPCR-negative mothers at ZooB and in the two dead specimens. One Callithrix geoffroyi became qPCR-positive over a two-year follow-up. All G6pi sequences matched T. cruzi lineage TcI. CONCLUSIONS: Our findings strongly suggest vector-borne T. cruzi transmission within a small-primate unit at ZooB - with vectors, and perhaps also parasites, presumably coming from nearby gallery forest. Periodic checks for vectors and parasites would help eliminate T. cruzi transmission foci in captive-animal facilities. This should be of special importance for captive-breeding programs involving endangered mammals, and would reduce the risk of accidental T. cruzi transmission to keepers and veterinarians.

Infestation of Mauritia flexuosa palms by triatomines (Hemiptera: Reduviidae), vectors of Trypanosoma cruzi and Trypanosoma rangeli in the Brazilian savanna.

To determine the infestation and trypanosome infection of triatomines captured in Mauritia flexuosa palm trees across its geographic distribution in the Brazilian savanna (Cerrado), we sampled 42 localities in eight states and in the Federal District, Brazil, between July 2005 and January 2010. Overall, 2154 specimens of the species Rhodnius neglectus, Psammolestes tertius, Triatoma sordida, and Microtriatoma borbai, were collected. Among the 341 palms sampled, 182 (53.3%) were infested with R. neglectus, which resulted in the capture of 1639 specimens (9.0 insects per infested palm). P. tertius occurred in 26 palms (8%), which resulted in the capture of 484 specimens (19 insects per infested palm). T. sordida (n=30) and M. borbai (n=1) occurred in only one location. From 537 R. neglectus examined, 44 were infected (8%) with Trypanosoma rangeli and/or Trypanosoma cruzi (Tc Id). M. flexuosa was previously recognized as a suitable breeding ecotope for R. neglectus in the Brazilian states of Minas Gerais, Goiás, Tocantins and the Federal District. Our results expand this distribution to other states (São Paulo, Bahia, Mato Grosso, Maranhão and Piauí), and also show that this particular palm tree harbors other triatomine species. Finally, we show that R. neglectus plays an important role in maintaining the enzootic circulation of T. cruzi and T. rangeli in the Brazilian savanna.

Trypanosoma cruzi I genotypes in different geographical regions and transmission cycles based on a microsatellite motif of the intergenic spacer of spliced-leader genes.

The intergenic region of spliced-leader (SL-IR) genes from 105 Trypanosoma cruzi I (Tc I) infected biological samples, culture isolates and stocks from 11 endemic countries, from Argentina to the USA were characterised, allowing identification of 76 genotypes with 54 polymorphic sites from 123 aligned sequences. On the basis of the microsatellite motif proposed by Herrera et al. (2007) to define four haplotypes in Colombia, we could classify these genotypes into four distinct Tc I SL-IR groups, three corresponding to the former haplotypes Ia (11 genotypes), Ib (11 genotypes) and Id (35 genotypes); and one novel group, Ie (19 genotypes). Genotypes harbouring the Tc Ic motif were not detected in our study. Tc Ia was associated with domestic cycles in southern and northern South America and sylvatic cycles in Central and North America. Tc Ib was found in all transmission cycles from Colombia. Tc Id was identified in all transmission cycles from Argentina and Colombia, including Chagas cardiomyopathy patients, sylvatic Brazilian samples and human cases from French Guiana, Panama and Venezuela. Tc Ie gathered five samples from domestic Triatoma infestans from northern Argentina, nine samples from wild Mepraia spinolai and Mepraia gajardoi and two chagasic patients from Chile and one from a Bolivian patient with chagasic reactivation. Mixed infections by Tc Ia+Tc Id, Tc Ia+Tc Ie and Tc Id+Tc Ie were detected in vector faeces and isolates from human and vector samples. In addition, Tc Ia and Tc Id were identified in different tissues from a heart transplanted Chagas cardiomyopathy patient with reactivation, denoting histotropism. Trypanosoma cruzi I SL-IR genotypes from parasites infecting Triatoma gerstaeckeri and Didelphis virginiana from USA, T. infestans from Paraguay, Rhodnius nasutus and Rhodnius neglectus from Brazil and M. spinolai and M. gajardoi from Chile are to our knowledge described for the first time.

Is Rhodnius prolixus (Triatominae) invading houses in central Brazil?

Sylvatic triatomines of the genus Rhodnius commonly fly into houses in Latin America, maintaining the risk of Chagas disease transmission in spite of control efforts. In the recent past, adult bugs collected inside houses in central Brazil were identified as R. prolixus, a primary disease vector whose natural geographical range excludes this region. Three nearly sibling species (R. neglectus, R. nasutus, and R. robustus), secondary vectors with limited epidemiological significance, occur naturally south of the Brazilian Amazon. The specific status of Rhodnius specimens found inside houses in central Brazil is therefore an epidemiologically important (and still debated) issue. We used wing and head geometric morphometrics to investigate the taxonomic status of 230 adult specimens representing all four 'R. prolixus group' species (19 populations from palm trees, domiciles, and reference laboratory colonies). Discriminant analyses of shape variation allowed for an almost perfect reclassification of individuals to their putative species. Shape patterning revealed no consistent differences between most specimens collected inside houses in central Brazil and R. neglectus, and showed that R. robustus and R. neglectus occur sympatrically (and fly into houses) in southern Amazonia. Furthermore, all Brazilian specimens clearly differed from our reference R. prolixus population. Using geometric morphometrics, we confidently ascribed individual triatomines to their species within the problematic 'R. prolixus group', illustrating the potential value of this approach in entomological surveillance. Our results strongly support the idea that R. neglectus, and not R. prolixus, is the species invading houses in central Brazil.

The triatomines of northern Peru, with emphasis on the ecology and infection by trypanosomes of Rhodnius ecuadoriensis (Triatominae).

Information on the distribution and synanthropic behaviour of triatomines is essential for Chagas disease vector control. This work summarises such information from northern Peru, and presents new data on Rhodnius ecuadoriensis - an important local vector infesting 10-35% of dwellings in some zones. Three species are strongly synanthropic and may be suitable targets for chemical control of domestic/peridomestic bug populations. Panstrongylus herreri, the main domestic vector in the area, is probably present in sylvatic ecotopes in the Marañón river system. R. ecuadoriensis and Triatoma dimidiata seem exclusively domestic; biogeographical and ecological data suggest they might have spread in association with humans in northern Peru. Confirmation of this hypothesis would result in a local eradication strategy being recommended. Presence of trypanosome natural infection was assessed in 257 R. ecuadoriensis; Trypanosoma rangeli was detected in 4% of bugs. Six further triatomine species are potential disease vectors in the region (T. carrioni, P. chinai, P. rufotuberculatus, P. geniculatus, R. pictipes, and R. robustus), whilst Eratyrus mucronatus, E. cuspidatus, Cavernicola pilosa, Hermanlentia matsunoi, and Belminus peruvianus have little or no epidemiological significance. A strong community-based entomological surveillance system and collaboration with Ecuadorian public health authorities and researchers are recommended.

Experimental infection with Leishmania (Viannia) braziliensis, and Leishmania (Leishmania) amazonensis in the marmoset, Callithrix penicillata (Primates: Callithricidae)

Foureen marmosets (Callithrix penicillata) were inoculated intradermally with promastigotes and/or amastigotes of Leishmania (Viannia) brazilensis (L. (V) b.) strains MHOM/BR/83/LTB-300MHOM/BR/85/LTB-12 MHOM/BR/81/LTB-179 and MHOM/BR/82/LTB-250. The evolution of subsequent lesions was studied for 15 to 75 weeks post-inoculation (PI). All but of the L. (V) b. injected marmosets developed a cutaneous lesion at the point of inoculation after 3 to 9 weeks, characterized by the appearance of subcutaneous nodules containing parasites. parasites were isolated by culture (Difco Blood Agar) from all 11 positive animals. The maximum size of the lesions was variable and ranged between 37 mm² to 107 mm². Ulceration of primary nodules became evident after 3 to 12 weeks in all infected marmosets, but was faster and larger in 5 of the 11 animals. The active lesions persisted in 9 out of 11 Callithrix until the en of the observation period, which varied from 15-75 weeks. In 3 animals spontaneous healing of their lesions (13 to 25 weeks, PI) was observed buth with cryptic parasitism. In another 2 infected animals there was regression followed by reactivation of the cutaneous lesions. The appearance of smaller satellite lesions adjacent to primary ones, as well as metastatic lesions to the ear lobes, were documented in 2 animals. Promastigotes of L. (Leishmania) amazonensis (L.(L)a.) MHOM/BR/77/LTB-16 were inoculated in 1 marmoset. This animal remained chronically infected for 6 months and the lesions developed in a similar manner to L.(V)b. infected marmosets. No significant differences in clinical and parasitological behaviour were observed between promastigote or amastigote derived infections of the 2 species. Both produced chronic, long lasting lesions which eventually healed. The same was true for parameters of size and ulceration. Skin tests converted to parasite in 11 of 15 inected masmosets and in 1-0 of 12 parasites positive animnals. Moderate levels of circulating antibodies we also observed by IFAT/IgG assays. In spite of the failure to rep[roduce the mucosal form of the disease, an important aspect of the Callithrix model in experimental cutaneous leishmaniasis lies in the reproduction of 2 clinical events that are common in humans, namely, the chronic ulceration and spontaneous healing of the lesions

Histopatologia de Leishmaniose tegumentar por Leishmania braziliensis braziliensis: 4. Classificaçäo histopatológica / Histopatology of tegumentary leishmaniasis caused by Leishmania braziliensis braziliensis: 4. Histopathological classification

Analisaram-se as alteraçöes histológicas encontradas em 162 casos de Leishmaniose Tegumentar da localidade de Três Braços, Estado da Bahia, dos quais 131 (80,9%) eram de portadores de lesöes cutâneas e 31 (19,1%) de portadores de lesöes mucosas. Analisaram-se, também, o comportamento clínico dos cinco padröes histológicos, já antes descritos, em relaçäo à terapêutica. O melhor prognóstico esteve sempre ligado ao padräo de Reaçäo Exsudativa e Granulomatosa, ou seja, a uma fase na qual o organismo, tendo lançado mäo de um mecanismo endógeno de lise parasitária, já circunscreveu a área de necrose por uma reaçäo granulomatosa, e esta é agora apenas o elemento residual. A açäo terapéutica nessa fase somente acelera a resoluçäo natural do caso. O grupo seguinte é amplo, e compreende os casos em que a lesäo pertence aos padröes de Reaçäo Exsudativa Celular (formas cutâneas), Reaçäo Exsudativa e Necrótica e Reaçäo Exsudativa e Necrótico-Granulomatosa. Nesses casos, o mecanismo de auto-controle da lesäo encontra-se ainda em curso, e a açäo terapêutica encurta o período de evoluçäo natural. Os casos do padräo de Reaçäo Exsudativa e Tuberculóide tiveram um prognóstico variável. Houve boa resposta à terapêutica quando o granuloma tuberculóide característico desse padräo surgiu em pacientes jovens, com curto tempo de evoluçäo da doença e intradermorreaçäo näo exacerbada. Nos demais casos tuberculóides - principalmente em pacientes adultos com longo tempo de evoluçäo da doença e intradermorreaçäo exarcebada -, a resposta foi menos satisfatória

Histopatologia da leishmaniose tegumentar por Leishmania braziliensis braziliensis: 2. Resposta humoral tissular / Histopathology of tegumentary leishmaniasis caused by Leishmania braziliensis braziliensis: 2. Tissue humoral response

Analisaram-se a resposta humoral nas lesöes de 90 pacientes de Leishmaniose Tegumentar - causada por Leishmania braziliensis brasiliensis -, utilizando o método da imunoperoxidase para identificar nos tecidos a presença de IgA, IgG, IgM, fraçäo C3 do complemento e fibrina. Constataram-se a presença de IgA, IgC e IgM nos plasmócitos tissulares, com predominio de IgG. Admitiram-se que a passagem dessas imunoglobulinas para os tecidos possibilitando a opsonizaçäo do parasitos e/ou de seus antígenos, permitiria a ocorrência de fenômenos necróticos que representam um dos mecanismos eficazes de reduçäo da carga parasitária. Efetivamente, nas áreas de necrose e nas paredes dos vasos inflamados identificaram depósito de imunoglobulinas, fraçäo C3 do complemento e fibrina - elementos do hospedeiro que fazem parte dos imunocomplexos. Interpretaram essa necrose tissular como o resultado da açäo de imunocomplexos na regiäo de eqüivalência ou com discreto excesso de antígenos (tipos ARTHUS). A presença de antígenos parasitários, expressos nas membranas dos macrófagos quando em contato com imunoglobulinas tissulares, na fase inicial da lesäo, possibilitaria a instalaçäo de uma reaçäo antígeno-anticorpo, a qual explicaria o aparecimento da necrose na Leishmaniose Tegumentar

Histopatologia na leishmaniose tegumentar por leishmania braziliensis braziliensis: 3. Reaçäo celular nos tecidos / Histopathology of tegumentary leishmaniasis caused by Leishmania braziliensis braziliensis: 3. Cellular reactions in tissues

Analisam-se as alteraçöes histopatológicas observadas em 378 casos de Leishmaniose Tegumentar da localidade de Três Braços Estado da Bahia, dos quais 307 eram de portadores de lesöes exclusivamente cutâneas, 54 de portadores de lesöes exclusivamente mucosas e 17 de portadores de lesöes cutâneo-mucosas. A infiltraçäo histiolinfoplasmocitária, na maioria dos casos, parece desempenhar o papel de resposta celular inespecífica à presença de um irritante tecidual, porém, nos casos de forma mucosa, näo se pode afastar a possibilidade de que esse infiltrado esteja participando de uma reaçäo de tipo autoagressivo. O plasmócito constitui um elemento quase constante nas lesöes desenvolvidas, mas näo tem sido observado nas lesöes residuais, quer em via de cura ou já cicatrizadas; sua presença nestes casos denota, quase sempre, tendência à recidiva. Os mastócitos foram observados em lesöes tanto da forma cutânea como da forma mucosa, mas predominavam nas primeiras. Seu número foi significantemente maior no padräo de Reaçäo Exsudativa e Necrótico-Granulomatosa, onde os fenômenos necróticos säo bem desenvolvidos. Os eosinófilos apresentaram associaçäo significativa com os mastócitos, confirmando a existência de um eixo bidirecional entre estas duas células, o qual deve participar da modulaçäo inflamatória, na Leishmaniose Tegumentar. Dois tipos de reaçäo granulomatosa foram observados: um desorganizado, em relaçäo, muitas vezes, com a necrose tissular, e outro organizado, mais raro, do tipo tuberculóide ...

El cultivo "in vitro" como instrumento práctico para el diagnóstico y aislamiento primario de Leishmania braziliensis braziliensis: 2. Estudios en pacientes de áreas endémicas / In vitro cultivation as a practical tool for the diagnosis and primary isolation of Leishmania braziliensis braziliensis: 2. Studies in patients of endemic areas

El cultivo "in vitro" de Leishmania braziliensis braziliensis constituye un método útil en el trabajo de campo, para el aislamiento primario de ésta subespécie de Leishmania. Cultivos en dos medios difásicos de agar sangre (DAB y EVANS) y dos medios líquidos (SCHNEIDER'S y AR-103) realizados en pacientes con lesiones cutáneas de Leishmaniasis Tegumentaria Americana (LTA) demostraron: 1) Similar sensibilidad de los medios DAB y SCHNEIDER'S cuando utilizamos el procedimiento de aspiración de las muestas con aguja. 2) Rendimiento sensible y reproducible, con el medio DAB, cuando comparado, en repetidas ocasiones, con el medio EVANS. 3) Incremento significativo en el aislamiento primario de Leishmania braziliensis braziliensis mediante la ejecución, en la misma lesión de cada paciente, de tres aspiraciones consecutivas en sitios diferentes de la úlcera activa (50% de positividad, con el medio DAB)

Características epidemiológicas da leishmaniose tegumentar americana em uma regiäo endêmica do Estado da Bahia: III. Fauna flebotomínica / Epidemiological characteristics of American tegumentary leishmaniasis in an endemic region of the State of Bahia: III. Phlebotomine fauna

A fauna flebotomínica da regiäo de Três Braços, uma área endêmica de leishmaniose cutânea-mucosa localizada no sudeste do Estado da Bahia, na regiäo cacaueira, é muito variada. Foram identificadas 30 espécies de Lutzomyia em 13.535 exemplares coletados entre os anos de 1976 e 1984. Lu. withmani foi a espécie altamente predominante no ambiente peridoméstico e no interior das residências, com percentuais de 99,0 e 97,5, respectivamente. Na floresta, as espécies predominantes foram Lu. ayrozai e Lu. yuilli, aparecendo Lu. whitmani com apenas 1,0% do total de exemplares examinados. Lu. flaviscutellata, vetor comprovado da Leishmania mexicana amazonensis, foi também coletada em baixos índices. Lu. wellcomei, vetor da L. braziliensis braziliensis na Serra dos Carajás, Pará, Brasil, näo foi encontrada na regiäo de Três Braços onde o parasito causando infecçöes humanas é predominantemente L. b. braziliensis. Embora näo se tenha encontrado infecçäo natural por promastigotas em 1.832 fêmeas de diversas espécies examinadas, discute-se a possibilidade de Lu. whitmani ser um vetor da L.b. braziliensis na regiäo, mantendo, provavelmente, a transmissäo entre o cäo e o homem

Composiçäo racial e a avaliaçäo da reaçäo intradérmica de Montenegro em portadores da leishmaniose cutâneo-mucosa / Racial composition and evaluation of Montenegro intradermal reaction in patients with mucocutaneosis leishmaniasis

Em um estudo retrospectivo, indivíduos com a forma mucosa da leishmaniose foram pareados (sexo, idade e ocupaçäo) a outros pacientes com a forma cutânea em atividade, näo demonstrando-se diferença significante da freqüência nos grupos raciais. Do mesmo modo, o teste intradérmico de Montenegro näo se mostrou diferente nos diversos grupos raciais, entre os 96 pares de pacientes estudados. Uma maior enduraçäo foi observada em pacientes com o acometimento mucoso quando comparado aos cutâneos

El método de cultivo "in vitro" como instrumento práctico en el diagnóstico y el aislamiento primário de Leishmania braziliensis braziliensis: I. Observaciones de laboratório / The in vitro culture method as practical instrument in the diagnosis and primary isolation of Leishmania braziliensis braziliensis: I. Laboratory observations

Comparamos diferentes medios artificiales y procedimientos de cultivo in vitro de Leishmania braziliensis braziliensis (Lbb) aislada de lesiones de hamsteres experimentalmente infectados. Observamos que la aspiración de material de la lesión, usando jeringa de 5 ml y aguja 22g (25 x 8mm), mostró resultados satisfactorios siendo un procedimiento conveniente de colecta. El medio de Agar Sangre Difco (DAB) es un medio eficiente para el aislamiento de Lbb. El medio de Evans demostró similares resultados al DAB, mientras que el medio líquido de Schneider y el químicamente definido AR-103 fueron nítidamente inferiores. En la propagación in vitro de Lbb, el medio de Schneider permitió un buen crecimiento del flagelado, alcanzando densidades de 0,21 - 2,57 x 10**8 promastigotes por ml ya, en el 5§ día de cultivo. La adición de 5-fluorocytosine a los tubos de cultivo (150micron grama/ml) para evitar la contaminación por hongos, no inhibió el crecimiento de Lbb

Leishmania mexicana in proechimys ihering denigratus moojen (Rodentia, echimyidae) in a region endenic for American cutaneous Leishmaniasis

Três isolados de Leishmania foram obtidos de cinco entre 27 exemplares do roedor Proechimys iheringi denigratus capturados na regiäo de Três Braços, na mata atlântica do Estado da Bahia, Brasil. O isolamento desse parasito foi feito através de inoculaçäo de triturado de pele, braço e fígado em patas de hamsters. Em pelo menos um dos casos, (MTB-574), o parasito foi isolado da pele. Metástase foi observada nos hamsters inoculaçäos, os parasitos cresceram abundantemente em meios artificiais de cultura e um padräo suprapapilário típico foi obtido em Lutzomyia longipalpis, indicando que o parasito pertence ao complexo L. mexicana. Todos os isolados reagiram positivamente com anticorpos monoclonais de L. m. mexicana e L. m. amazonensis. A análise isoenzimática diferenciou o parasito de isolados padröes de L. m. mexicana, L. m. amazonensis, L. m. aristedesi, L. m. pifanoi, L. m. garnhami e L. m. ssp(Goias-W. Barbosa). O parasito parece ser uma subespécie de L. mexicana muito próxima a L. m. amazonensis, da qual difere pela menor mobilidade eletroforética de GPI, PEP e ALAT. Este é o primeiro registro do isolamento de um parasito do gênero Leishmania em um roedor capturado no Estado da Bahia