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BACKGROUND The influence of Plasmodium spp. infection in the health of Southern brown howler monkey, Alouatta guariba clamitans, the main reservoir of malaria in the Atlantic Forest, is still unknown. OBJECTIVES The aim of this study was to investigate the positivity rate of Plasmodium infection in free-living howler monkeys in an Atlantic Forest fragment in Joinville/SC and to associate the infection with clinical, morphometrical, haematological and biochemical alterations. METHODS Molecular diagnosis of Plasmodium infection in the captured monkeys was performed by Nested-polymerase chain reaction (PCR) (18S rRNA and coxI). Haematological and biochemical parameters were compared among infected and uninfected monkeys; clinical and morphometrical parameters were also compared. FINDINGS The positivity rate of Plasmodium infection was 70% among forty captured animals, the highest reported for neotropical primates. None statistical differences were detected in the clinical parameters, and morphometric measures comparing infected and uninfected groups. The main significant alteration was the higher alanine aminotransferase (ALT) levels in infected compared to uninfected monkeys. MAIN CONCLUSIONS Therefore, Plasmodium infection in howler monkeys may causes haematological/biochemical alterations which might suggest hepatic impairment. Moreover, infection must be monitored for the eco-epidemiological surveillance of malaria in the Atlantic Forest and during primate conservation program that involves the animal movement, such as translocations.

The aim of this study was to identify Plasmodium spp. in blood samples from nonhuman primates (NHPs) in the state of Maranhão, using classical and alternative techniques for examination of human malaria. A total of 161 blood samples from NHPs were analyzed: 141 from captive animals at a Wildlife Screening Center (CETAS) and 20 from free-living animals in a private reserve. The techniques used were microscopy, rapid diagnostic test (RDT), Indirect fluorescent antibody test (IFAT) and molecular techniques (semi-nested PCR, quantitative real-time PCR and LAMP). Two serological methods (dot-ELISA and indirect ELISA) were also standardized with rhoptry protein-soluble antigen of P. falciparum and P. berghei. Trophozoite forms of Plasmodium sp. were identified on slides from five different animals. No samples were positive through RDT and LAMP. Four samples were seropositive for P. malariae through IFAT. The samples showed low reactivity to ELISA. Plasmodium sp. was detected in 34.16% (55/161) of the samples using qPCR based on the 18S rRNA gene. After sequencing, two samples showed 100% identityl to P. malariae, one showed 97% identity to Plasmodium sp. ZOOBH and one showed 99% identity to P. falciparum . PCR was shown to be the most sensitive technique for diagnosing Plasmodium in NHP samples. Resumo Neste estudo objetivamos identificar Plasmodium spp. em amostras sangue de primatas não humanos (PNH) do estado do Maranhão, utilizando técnicas clássicas e alternativas para o exame da malária humana. Foram analisadas 161 amostras de sangue de PNH, sendo 141 de CETAS (cativeiro) e 20 de reserva particular (vida livre), utilizando microscopia, teste de diagnóstico rápido (RDT), imunofluorescência indireta (IFI) e técnicas moleculares (semi-nested PCR, PCR em tempo real quantitativo e LAMP). Dois métodos sorológicos (dot-ELISA e ELISA indireto) também foram padronizados com antígenos solúveis de roptrias de P. falciparum e P. berghei. Formas trofozoíticas de Plasmodium sp. foram identificadas em lâminas de cinco animais diferentes. Nenhuma amostra foi positiva em TDR e LAMP. Quatro amostras foram soropositivas para P. malariae na IFI. Os soros de PNH mostraram baixa reatividade pelo ELISA indireto. Plasmodium sp. foi detectado em 34,16% (55/161) das amostras utilizando a qPCR baseada no gene 18S rRNA. No sequenciamento, duas amostras mostraram identidade com P. malariae (100%), uma com Plasmodium sp. ZOOBH (97%) e uma com P. falciparum (99%). A PCR mostrou ser a técnica mais sensível para diagnósticos de Plasmodium em amostras de PNH.

Made available in DSpace on 2019-10-06T16:00:21Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-07-01. Added 1 bitstream(s) on 2019-10-09T18:35:04Z : No. of bitstreams: 1 S1984-29612018000300363.pdf: 2770381 bytes, checksum: c15ec391d851104f6d38492a03ad8099 (MD5) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis The aim of this study was to identify Plasmodium spp. in blood samples from nonhuman primates (NHPs) in the state of Maranhão, using classical and alternative techniques for examination of human malaria. A total of 161 blood samples from NHPs were analyzed: 141 from captive animals at a Wildlife Screening Center (CETAS) and 20 from free-living animals in a private reserve. The techniques used were microscopy, rapid diagnostic test (RDT), Indirect fluorescent antibody test (IFAT) and molecular techniques (semi-nested PCR, quantitative real-time PCR and LAMP). Two serological methods (dot-ELISA and indirect ELISA) were also standardized with rhoptry protein-soluble antigen of P. falciparum and P. berghei. Trophozoite forms of Plasmodium sp. were identified on slides from five different animals. No samples were positive through RDT and LAMP. Four samples were seropositive for P. malariae through IFAT. The samples showed low reactivity to ELISA. Plasmodium sp. was detected in 34.16% (55/161) of the samples using qPCR based on the 18S rRNA gene. After sequencing, two samples showed 100% identityl to P. malariae, one showed 97% identity to Plasmodium sp. ZOOBH and one showed 99% identity to P. falciparum. PCR was shown to be the most sensitive technique for diagnosing Plasmodium in NHP samples. Laboratório de Parasitologia Animal Curso de Medicina Veterinária Universidade Federal de Rondônia – UNIR Centro de Estudos da Malária Superintendência de Controle de Endemias – SUCEN Departamento de Saúde do Estado de São Paulo Instituto de Medicina Tropical de São Paulo – IMTSP Universidade de São Paulo – USP Laboratório de Patologia Veterinária Curso de Medicina Veterinária Universidade Federal de Rondônia – UNIR Laboratório de Imunoparasitologia Faculdade de Ciências Agrárias e Veterinárias – FCAV Universidade Estadual Paulista – UNESP Laboratório de Imunoparasitologia Faculdade de Ciências Agrárias e Veterinárias – FCAV Universidade Estadual Paulista – UNESP FAPESP: 10/12820-4 FAPESP: 12/03961-9

Plasmodium malariae is a protozoan parasite that can cause human malaria. The simian parasite Plasmodium brasilianum infects New World monkeys from Latin America and is morphologically indistinguishable from P. malariae . Here, we report the first full draft genome sequence for P. brasilianum .

Background The quartan malaria parasite Plasmodium malariae is the widest spread and best adapted human malaria parasite. The simian Plasmodium brasilianum causes quartan fever in New World monkeys and resembles P. malariae morphologically. Since the genetics of the two parasites are nearly identical, differing only in a range of mutations expected within a species, it has long been speculated that the two are the same. However, no naturally acquired infection with parasites termed as P. brasilianum has been found in humans until now. Methods We investigated malaria cases from remote Yanomami indigenous communities of the Venezuelan Amazon and analyzed the genes coding for the circumsporozoite protein (CSP) and the small subunit of ribosomes (18S) by species-specific PCR and capillary based-DNA sequencing. Findings Based on 18S rRNA gene sequencing, we identified 12 patients harboring malaria parasites which were 100% identical with P. brasilianum isolated from the monkey, Alouatta seniculus. Translated amino acid sequences of the CS protein gene showed identical immunodominant repeat units between quartan malaria parasites isolated from both humans and monkeys. Interpretation This study reports, for the first time, naturally acquired infections in humans with parasites termed as P. brasilianum. We conclude that quartan malaria parasites are easily exchanged between humans and monkeys in Latin America. We hypothesize a lack of host specificity in mammalian hosts and consider quartan malaria to be a true anthropozoonosis. Since the name P. brasilianum suggests a malaria species distinct from P. malariae, we propose that P. brasilianum should have a nomenclatorial revision in case further research confirms our findings. The expansive reservoir of mammalian hosts discriminates quartan malaria from other Plasmodium spp. and requires particular research efforts., Highlights • We found human infections with ‘Plasmodium brasilianum’, a quartan malaria parasite of New World monkeys in South America • We show that in areas of close contact humans and non-human primates are concurrently infected with quartan malaria parasites • We conclude that quartan malaria parasites can transcend host species boundaries with impunity We found naturally acquired infections in humans with Plasmodium brasilianum parasites, a quartan malaria parasite which usually infects more than 35 monkey species in South America. This confirms that malaria parasites, which cause the quartan type of fever (two days without fever between fever peaks), are easily exchanged between humans and monkeys in Latin America. The wide host reservoir of quartan malaria parasites requires particular malaria research efforts.

Tese apresentada ao Programa de Pós-Graduação em Biologia Experimental do Departamento de Medicina da Fundação Universidade Federal de Rondônia, como requisito parcial par obtenção do título de Doutor em Biologia Experimental. Submitted by Rejane Sales (rejane.lima@unir.br) on 2014-09-11T18:51:46Z No. of bitstreams: 1 MAISA S ARAUJO - Tese Doutorado 2013.pdf: 5591726 bytes, checksum: c75a6ba5e25d1e82846af595701b1a87 (MD5) Approved for entry into archive by Rejane Sales (rejane.lima@unir.br) on 2014-09-11T18:54:01Z (GMT) No. of bitstreams: 1 MAISA S ARAUJO - Tese Doutorado 2013.pdf: 5591726 bytes, checksum: c75a6ba5e25d1e82846af595701b1a87 (MD5) Made available in DSpace on 2014-09-17T18:49:21Z (GMT). No. of bitstreams: 1 MAISA S ARAUJO - Tese Doutorado 2013.pdf: 5591726 bytes, checksum: c75a6ba5e25d1e82846af595701b1a87 (MD5) Previous issue date: 2013 Em estudos anteriores sobre a malária em primatas não humanos, nativos de florestas do continente Americano, estão registradas apenas duas espécies de plasmódios, Plasmodium brasilianum e P. simium, parasitas semelhantes aos da malária humana, P. malariae e P. vivax, respectivamente. Estes parasitas infectam naturalmente primatas da família Cebidae e Atelidae. O P. brasilianum foi observado em ampla distribuição geográfica e parasitando um grande número de espécies de primatas, ao contrário do P. simium, restrito a três espécies nas áreas do Sul e Sudeste da Mata Atlântica do Brasil. Todavia, é possível que essa distribuição reflita uma subestimação, pois esses dados foram baseados, em sua maioria, em análises microscópicas e penalizadas pelas dificuldades em obter amostras, limitando-as a um pequeno número de animais. A introdução, no presente trabalho, de metodologias moleculares em adição à microscopia para a detecção e identificação dos parasitas da malária, permite abordar o problema das relações entre malária de primatas não humanos e malária humana, uma vez que interpretações conflitantes sobre a origem humana das malárias de primatas não humanos no continente Americano ou, ao contrário, origem simiana da malária humana, tem sido proposta por diferentes autores. Peculiaridades da região de Rondônia, com áreas florestais de instabilidade devido à intensa intervenção ambiental de origem humana favorecendo o contato entre o homem e animais da floresta, permitiu um estudo preliminar da prevalência da malária de primatas não humanos e sua possível relação com a malária humana local. Foram coletadas 184 amostras de sangue de primatas não humanos e analisadas utilizando-se microscopia e técnicas moleculares. As amostras estudadas foram de animais silvestres e de cativeiro. Os animais silvestres foram de duas origens: (i) áreas de instabilidade ecológica (local de construção de hidrelétricas, extração de madeira, etc) e (ii) áreas atualmente estáveis (antigas áreas de mineração e extração de borracha, atividade agrícola estável, etc.). De forma geral, a frequência de infecções por Plasmodium foi de 10,3% (19/184), sendo maior em animais selvagens (12,6%) do que em animais de cativeiro (4,1%). A maioria das infecções observadas nos animais de diferentes espécies foi por P. brasilianum (18/19). Nenhum caso de infecção por P. simium foi detectado. No entanto, foram observados 3 casos de infecção por P. falciparum, estes em animais silvestres e de cativeiro (1,6%), sendo 2 dos casos infecções mistas com P. brasilianum. Todas as amostras positivas foram de animais pertencentes às famílias Cebidade, Atelidae, Pitheciidae e um dos casos de infecção por P. brasilianum inclui pela primeira vez a família Aotidae. A prevalência de P. brasilianum em primatas não humanos capturados em área próxima a habitações humanas, bem como casos de P. malariae na população humana local e casos positivos de P. falciparum em primatas não humanos constituem evidências adicionais da possível interrelação entre malária humana e de primatas não humanos e que estes podem eventualmente constituir em reservatório natural para a malária humana, o que implicaria em futuras ações de controle da doença na região.

La especie productora de malaria en primates, Plasmodium brasilianum, fue encontrada por primera vez en Costa Rica en 6 de 104 ejemplares de monos congo o aulladores (Alouatta palliata). Los animales fueron capturados y anestesiados por medio de dardos que contenían hidrocloruro de tiletamina y zolazepam (Zoletil®) combinados en partes iguales. Para estudiar estos animales por parásitos sanguíneos, se prepararon frotis sanguíneos que luego se tiñeron y se estudiaron en el laboratorio, encontrándose las formas de trofozoitos jóvenes o avanzados así como gametocitos y esquizontes. La morfología característica de algunos estados evolutivos, como por ejemplo, las formas en banda de trofozoitos avanzados y los esquizontes en forma de margarita o "rosetta" permitieron el diagnóstico de la especie. Puesto que se han encontrado casos humanos infectados con este organismo y éste es casi indiferenciable de Plasmodium malariae, una especie parásita del ser humano, se discute el hallazgo de este parásito desde un punto de vista epidemiológico en el área de la salud

An anopheline survey was carried out in two simian malaria areas in the Brazilian Amazon, Balbina and Samuel, to determine the potential vectors of Plasmodium brasilianum. The most abundant and/or acrodendrophilic anophelines in the forest and the most likely vector were Anopheles mediopunctatus, An. nuneztovari, An. oswaldoi, An. triannulatus and An. shannoni. An. darlingi and An. marajoara were captured essentially in anthropic habitats outside the forest and are unlikely to be involved in the transmission of P. brasilianum among monkeys within the forests and from monkeys to man in their surroundings in the Amazon.

An antigen, designated here as the parasitized erythrocyte membrane antigen (PEMA), is present in the erythrocyte membrane surrounding all intraerythrocytic stages of Plasmodium brasilianum. An antibody specific for PEMA appeared in 21 (50%) of 42 antisera from Saimiri sciureus monkeys naturally infected with P. brasilianum. Of these 42 sera, nine (21.4%) contained antibody to the ring-infected erythrocyte membrane antigen (RESA); of these nine sera, six did not react with PEMA. Sera of humans infected with P. malariae reacted with PEMA and RESA in a similar pattern; i.e., of 83 antisera, 71 (85.5%) reacted with PEMA and 30 (36%) reacted with RESA. Only one of these latter 30 sera were not reactive with PEMA. Of 167 sera from humans infected with P. falciparum but not P. malariae, 133 (79.6%) reacted with RESA; of these, 43 (25.7% of the total) reacted with PEMA but not RESA. Although PEMA was demonstrated with P. brasilianum and RESA with P. falciparum, neither PEMA or RESA could be demonstrated with P. malariae. Interactions of PEMA and RESA and the corresponding antibodies offer a method whereby the two morphologically similar quartan species, P. malariae and P. brasilianum, can be readily distinguished from each other and may furnish clues to genetic separation of the two and the mechanisms of interaction of quartan malaria and P. falciparum where they are coendemic.

Since 1963, reported malaria transmission in Haiti has been restricted to Plasmodium falciparum. However, screening of Haitian refugees in Jamaica in 2004, by microscopic examination, identified P. falciparum, P. vivax, and P. malariae. PCR confirmed the P. malariae and P. falciparum but not P. vivax infections. DNA sequencing and rRNA gene sequences showed transmission of P. malariae. This report confirms that P. malariae is still being transmitted in Haiti.

Chronic quartan malarial infection has been established in the common marmoset (Callithrix jacchius). Plasniodium brasilianunm from a douroucouli monkey (Aotus trivirgatus) was used to infect splenectomized twin animals, passed to an intact animal, and then to 4 other intact adults, 2 pairs of twins. In 2 of the 4 latter animals there was continuing patency with parasitaemias of ≤0·5% parasitized erythrocytes for 30 weeks. The other 2 had lower initial levels of paraaitaemia; in 1 of these parasitaemias remained low or subpatent. All marmosets developed lymphocytosis. One animal became ill 30 weeks after infection with anaemia, weight loss and mild proteinurea, the other 3 remained well. Histological examination showed minor changes in the kidneys; spleens of infected animals showed marked follicular hyperplasia and phagocytosis of pigment. The livers showed sinusoidal hyper cellularity and pigment deposition and in splenectomized animals, a marked lymphoid follicular hyperplasia in the portal tracts.

Samples of blood taken from 2.046 primates relocated by the "Curupira project" were examined for the presence of simian malaria parasites. This project aimed to rescuing the animals that lived within the forest that began to be flooded by the Tucurui dam built in the Tocantins river, Amazon basin, Brazil. Malaria parasites similar or identical to Plasmodium brasilianum were found in seven species or subspecies: Alouatta belzebul belzebul, A. belzebul nigerrima, A. seniculus, Chiropotes satanas, Saimiri sciureaus, Callicebus moloch and Saguinus midas niger. This last species was never reported infected with malaria parasites before.

Antigen was prepared from Plasmodium brasilianum harvested from an infected spider monkey. This antigen was attached to aldehyde-fixed, human type O cells, and was tested against sera from human cases of P. malaria, P. vivax, P. falciparum, and P. ovale infection, and sera from noninfected persons. At dilutions of 1:16 or greater the antigen failed to react in sera from noninfected persons. It reacted at titers of 16 or above with sera from 85% of the persons with P. malariae infection, 83% with P. vivax infection, 70% with P. falciparum infection, and 70% with P. ovale infection.

Background Non-human primates (NHPs) as a source for Plasmodium infections in humans are a challenge for malaria elimination. In Brazil, two species of Plasmodium have been described infecting NHPs, Plasmodium brasilianum and Plasmodium simium. Both species are infective to man. Plasmodium brasilianum resembles morphologically, genetically and immunologically the human quartan Plasmodium malariae. Plasmodium brasilianum naturally infects species of non-human primates from all New World monkey families from a large geographic area. In the family Callitrichidae only the genus Saguinus has been described infected so far. The present study describes the natural infection of P. brasilianum in tamarins and marmosets of the genera Callithrix, Mico and Leontopithecus in the Atlantic forest. Methods One hundred and twenty-two NHPs of the family Callitrichidae housed in the Primate Centre of Rio de Janeiro (CPRJ) were sampled in June 2015, and January and July 2016. The CPRJ is located in the Atlantic forest in the Guapimirim municipality, in the Rio de Janeiro state, where human autochthonous cases of malaria have been reported. The samples were screened for the presence of Plasmodium using optical microscopy and nested PCR for detection of 18S small subunit rRNA gene. The amplicon was sequenced to confirm the molecular diagnosis. Results The frequency of Plasmodium infections detected by nested PCR in New World monkeys of the family Callitrichidae was 6.6%. For the first time, Callitrichidae primates of genera Callithrix, Mico and Leontopithecus were found naturally infected with P. brasilianum. Infection was confirmed by sequencing a small fragment of 18S rRNA gene, although no parasites were detected in blood smears. Conclusions The reported P. brasilianum infection in NHP species maintained in captivity suggests that infection can be favoured by the presence of vectors and the proximity between known (and unknown) hosts of malaria. Thus, the list of potential malaria reservoirs needs to be further explored. Electronic supplementary material The online version of this article (doi:10.1186/s12936-017-1724-0) contains supplementary material, which is available to authorized users.

Background In Brazil, two species of Plasmodium have been described infecting non-human primates, Plasmodium brasilianum and Plasmodium simium. These species are morphologically, genetically and immunologically indistinguishable from the human Plasmodium malariae and Plasmodium vivax parasites, respectively. Plasmodium simium has been observed naturally infecting monkeys of the genera Alouatta and Brachyteles in a restricted area of the Atlantic Forest in the south and southeast regions of Brazil. However, its reported geographical distribution and the diversity of its vertebrate hosts may be underestimated, since available data were largely based on analyses by microscopic examination of peripheral blood, a method with limited sensitivity, considering the potential sub-patent feature of these infections. The present study describes, for the first time, the natural infection of P. simium in capuchin monkeys from the Brazilian Atlantic Forest. Methods Blood samples from 30 non-human primates belonging to nine species kept in the Primate Centre of Rio de Janeiro were collected. Fragments of spleen and liver from one dead monkey found in the neighborhoods of the Primate Centre were also analysed. Molecular diagnosis was performed by nested PCR (18SSU rRNA) and the amplified fragment was sequenced. Results Thirty per cent of the captive animals were infected with P. simium and/or P. brasilianum. The dead monkey tested positive for DNA of P. simium. For the first time, Cebinae primates (two specimens of genus Cebus and two of genus Sapajos) were found naturally infected by P. simium. The infection was confirmed by sequencing a small fragment of 18SSU rRNA. Conclusion The results highlight the possibility of infection by P. simium in other species of non-human primates whose impact could be significant for the malaria epidemiology among non-human primates and, if it becomes clear that this P. simium is able to infect monkeys and, eventually, man, also for the maintenance of transmission of human malaria in the context of a zoonosis in areas under influence of the Atlantic Forest. Electronic supplementary material The online version of this article (doi:10.1186/s12936-015-0606-6) contains supplementary material, which is available to authorized users.