Identificação de Plasmodium spp. em primatas neotropicais e em anofelinos em municípios da região de São Luís, estado do Maranhão, Brasil

Malaria is a disease of greater impact on Public Health in tropical countries because of the high morbidity and mortality. Considering malaria as a zoonosis in which primates can act as reservoirs of species of Plasmodium that can infect humans lead us to conduct malaria studies in primates with unquestionable relevance. It is generally know that the distribution of human and primate malaria following the same distribution of Anopheles mosquitoes vectors, in this study we investigated the presence of Plasmodium spp. in neotropical primates and Anopheles in São Luís Island, Maranhão State, Brazil. Samples were blood neotropical primates CETAS-São Luís (n = 141) and wild life (n = 20) Private Reserve Sítio Aguahy, São José de Ribamar. Anopheles mosquitoes were captured in the same reserve (n = 380) and Mangalho site (n = 36), located in the Environmental Protection Area of the Maracanã, São Luís, totaling 54 pools. The primate blood samples were subjected to morphological, serological (IFA and ELISA-test) and molecular (qPCR and conventional PCR) to identify Plasmodium. The pools of mosquitoes were assayed by testing for molecular identification of Plasmodium. Five primates had positive slides (3.10%) with observation trofozoíticas forms. In IFA four primate serum samples sororreagiram against the antigen of P. malariae. Amplified Plasmodium sp. qPCR at 34.16% (55/161) of samples of primates. In conventional PCR 30.43% (49/161) were positive, 47 (47/49) for P. brasilianum/P. malariae and 2 (2/49) to P.simium/P. vivax. Were sequenced DNA samples from four primates which showed identity with P. malariae (n = 2), Plasmodium ZOOBH (n = 1) and P. falciparum (n = 1). Three pools of Anopheles were positive for Plasmodium in qPCR (3/54) and conventional PCR. The sequencing samples showed identity to P. falciparum (n = 1), P. vivax (n = 1) and Plasmodium ZOOBH (EF090276). Due to the continued and increasing encroachment to forests by ...A malária é a endemia de maior impacto na Saúde Pública de países tropicais, devido à alta morbidade e mortalidade. Considerando a malária como uma zoonose, nos quais primatas podem funcionar como reservatórios de espécies de Plasmodium que podem infectar seres humanos nos levam a realizar estudos de malária em primatas com inquestionável relevância. Sabendo-se que geralmente a distribuição da malária humana e de primatas segue a mesma distribuição dos anofelinos, mosquitos vetores, neste trabalho investigaram-se a presença de Plasmodium spp. em primatas neotropicais e em anofelinos na Ilha de São Luís, Estado do Maranhão, Brasil. Colheram-se amostras de sangue de primatas neotropicais do CETAS-São Luís (n=141) e de vida livre (n=20) da Reserva Particular Sítio Aguahy, município de São José de Ribamar. Mosquitos anofelinos foram capturados nesta mesma reserva (n=380) e no Sítio Mangalho (n=36), localizado na Área de Proteção Ambiental do Maracanã, município de São Luís, totalizando em 54 ―pools‖. As amostras de sangue de primatas foram submetidas a testes morfológicos, sorológicos (RIFI e ELISA-teste) e moleculares (qPCR e PCR convencional) para identificação de Plasmodium. Os ―pools‖ de mosquitos foram ensaiados por testes moleculares para identificação de Plasmodium. Cinco primatas tiveram lâminas positivas (3,10%) com observação de formas trofozoíticas. Na RIFI quatro amostras de soro de primatas sororreagiram frente ao antígeno de P. malariae. Amplificaram para Plasmodium sp. na qPCR 34,16% (55/161) das amostras de primatas. Na PCR convencional 30,43% (49/161) foram positivas, sendo 47 (47/49) para P. brasilianum/P. malariae e 2 (2/49) para P. simium/P. vivax. Foram sequenciadas quatro amostras de DNA de primatas, que apresentaram identidade com P. malariae (n=2),com Plasmodium ZOOBH (n=1) e com P. falciparum (n=1). Três ―pools‖ de anofelinos foram positivos para Plasmodium na..

Figure 3. Photomicrographs of the indirect immunofluorescence reaction for IgG antibodies against P. malariae.

<p>A – Positive sample at a dilution of 1:20 from a <i>Sapajus</i> specimen (male) at CETAS in São Luís; B – Negative human control serum.</p

Fig 1. Map of South America highlighting Brazil and the state of Maranhão.

<p>Map of the island of São Luís showing the locations of CETAS, in the municipality of São Luís (light blue) and the Sítio Aguahy, municipality of São José de Ribamar (dark blue). Satellite image of CETAS and Sítio Aguahy, which were the capture and collection sites for blood samples from Neotropical primates (Source: Google Earth^®).</p

Fig 2. Photomicrographs of Plasmodium sp. viewed via light microscopy on thin blood smears from Neotropical primates sampled at CETAS, in São Luís.

A - ring-shaped trophozoite in sample from Sapajus sp. (male); B – schizont in sample from Callithrix jacchus (male). Giemsa staining. Bar = 10 µm

Natural Plasmodium infection in neotropical primates in the island of São Luís, state of Maranhão, Brazil

The states that make up the Legal Amazon Region, which include the state of Maranhão, account for 99% of registered cases of human malaria in Brazil. It is also believed that transmission of malaria from nonhuman primates (NHP) to humans occurs in this region, because of current reports of seroepidemiological results from samples from humans and NHP coexisting in the same areas. This study aimed to make morphological, serological and molecular diagnoses of Plasmodium spp. in neotropical primates on the island of São Luís, state of Maranhão, Brazil. The diagnostic techniques used were optical microscopy, the polymerase chain reaction (PCR) and the indirect immunofluorescence assay (IFA). From June 2009 to April 2010, 70 NHP were sampled: 50 at the Wild Animal Screening Center (CETAS), located in the municipality of São Luís and 20 free-living individuals that were caught in a private reserve located in the municipality of São Jose de Ribamar, state of Maranhão. Under an optical microscope, 140 slides (two from each animal) were evaluated and five animals (7.1%) were found to be positive. IFA did not detect anti-Plasmodium spp. From PCR on the 70 animals sampled, amplified Plasmodium spp. products were observed in 13 samples, of which eight (61.5%) were from free-living animals and five (38.5%) were from animals at CETAS

Felicola subrostratus parasitando gatos domésticos de São Luís, Maranhão, Brasil: relato de caso

http://dx.doi.org/10.5007/2175-7925.2013v26n3p255O Felicola subrostratus tem sido descrito como ectoparasito específico de felinos, mas sua infestação é incomum. Este manuscrito relata a primeira ocorrência do malófago F. subrostratus parasitando gatos domésticos do município de São Luís do Maranhão, Brasil. Relatam-se sete casos da infestação sobre gatos domésticos adultos jovens (uma fêmea da raça Persa, duas fêmeas mestiças de Persa, três machos mestiços de persa e um macho sem raça definida), todos de pelagem negra, provenientes de uma mesma casa com acesso à rua. Os animais foram examinados e, observado a presença de ectoparasitos, que foram coletados e fixados em álcool 70º GL, clarificados e montados entre lâminas e lamínulas para posterior identificação. Os animais foram acompanhados durante um mês sem nenhum tratamento para observar evolução da ectoparasitose. Dentro do período citado, todos os gatos machos apresentaram áreas extensas de alopecia e tricorrexia. Nas fêmeas, apenas a gata de raça Persa manifestou pequenas áreas de alopecia. Depois do período de acompanhamento se instituiu tratamento com amitraz (banho na concentração 5:1000), sem obter nenhuma melhora. Estabeleceu-se então tratamento com fipronil (0,25 g/100 mL) que debelou a infestação com apenas uma aplicação. Seis meses após tratamento os pêlos voltaram ao normal. Este relato constitui o primeiro diagnostico de Felicola subrostratus no Estado do Maranhão com posterior recuperação

Serological and molecular techniques applied for identification of Plasmodium spp. in blood samples from nonhuman primates

Abstract 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

Extraction and recovery technique for myxozoan parasites from the Piaractus mesopotamicus kidney embedded in paraffin

http://dx.doi.org/10.5007/2175-7925.2013v26n4p263Tissues fixed and embedded in paraffin for histopathological studies keep their cell characteristics. There are several protocols for extracting genetic material from tissue embedded in paraffin, but there is no protocol for material aimed at the direct identification of parasites. The lack of techniques which describe the recovery of parasites from tissue embedded in paraffin has led us to test a technique for recovering myxosporean parasites found in Piaractus mesopotamicus kidney fragments embedded in paraffin, for a rapid, direct, and economic identification. Once the excess paraffin was removed from the kidney fragment, this was deparaffinized in xylene, hydrated in 70% alcohol, placed in an Eppendorf tube containing 70% alcohol, and left under vigorous and constant agitation in a vortex until the tissue was disintegrated. The precipitated material was mixed with the 70% alcohol and 20 µL were collected for preparing the smears, which were stained with Giemsa. Myxobolus sp. spores at many developmental stages were observed by light microscopy. The technique has proved to be useful for recovering myxosporean parasites from tissue embedded in paraffin and it constitutes an effective tool for prevalence studies when the myxosporean parasites are not detected in fresh mounts

Identification of Plasmodium spp. in Neotropical primates of Maranhense Amazon in Northeast Brazil.

In the Brazilian Amazon region, malaria caused by Plasmodium malariae is considered to be a zoonosis because of cross-transfer of the parasite between humans and Neotropical primates. To contribute information on this issue, we investigated occurrences of natural infection with Plasmodium sp. among Neotropical primates in the Maranhense Amazon (Amazon region of the state of Maranhão), in the northeastern region of Brazil. Blood samples were collected from 161 Neotropical primates of six species that were caught in an environmental reserve (Sítio Aguahy) and from captive primates (CETAS-Wildlife Screening Center, municipality of São Luís), in Maranhão. Plasmodium sp. was diagnosed based on light microscopy, PCR, qPCR and LAMP for amplification of the 18S rRNA gene. Serum samples were also assayed by means of indirect immunofluorescence for IgG antibodies against P. malariae/P. brasilianum, P. falciparum and P. berghei. Parasites were detected through light microscopy on five slides from captive primates (four Sapajus spp. and one Callithrix jacchus). In the molecular tests, 34.16% (55/161) and 29.81% (48/161) of the animals sampled were positive in the qPCR and PCR assays, respectively. In the PCR, 47/48 animals were positive for P. malariae/P. brasilianum; of these, eight were free-living primates and 39 from CETAS, São Luís. One sample showed a band in the genus-specific reaction, but not in the second PCR reaction. Anti-P. malariae/P. brasilianum IgG antibodies were detected in four serum samples from Sapajus spp. in captivity. In this study, circulation of P. malariae/P. brasilianum in Neotropical primates was confirmed, with low levels of parasitemia and low levels of antibodies. The importance of these animals as reservoirs of human malaria in the region studied is still unknown. This scenario has an impact on control and elimination of malaria in this region