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DETECÇÃO E IDENTIFICAÇÃO DE FILÁRIAS EM SAGUINUS BICOLOR E ANÁLISE DE PAISAGEM DA PREVALÊNCIA FILARIAL
Dias, Cindy Alves | Data do documento:
2022
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DIAS, Cindy Alves. Detecção e identificação de filárias em Saguinus bicolor e análise de paisagem da prevalência filarial. 2022. 94 f. Dissertação (Mestrado em Biologia da Interação Patógeno-Hospedeiro) - Instituto Leônidas e Maria Deane, Fundação Oswaldo Cruz, Manaus, 2022.
Resumo
O Saguinus bicolor é um calitriquídeo que habita fragmentos florestais e arredores dos municípios amazônicos de Manaus, Rio Preto da Eva e Itacoatiara. Ele sofre risco de extinção, sendo a limitação de seu território de ocorrência e fragmentação do seu habitat duas das maiores ameaças. Poucos estudos são realizados sobre as doenças infecciosas e parasitos que podem acometer esse primata, tais como as filárias. As filárias são vermes nematódeos que podem parasitar uma grande variedade de animais, incluindo primatas não humanos, humanos e animais domésticos. Além disso, algumas espécies podem causar doenças médicas e veterinárias Este trabalho visou identificar parasitos de filária em Saguinus bicolor através de necropsia, microscopia e ferramentas moleculares, bem como compreender a influência da paisagem sobre a prevalência das filárias nesses primatas. Um total de 71 indivíduos foram amostrados, dentre os quais 62 foram submetidos à necrópsia, com inspeção das cavidades torácicas, abdominais e do espaço subcutâneo, e 10 animais vivos tendo o sangue coletado (onde 1 dos animais foi coletado tanto em vida quanto em morte). Durante as necrópsias, também foi possível coletar sangue de 46 carcaças, totalizando 56 amostras. Destas, 47 foram submetidas à microscopia em busca de microfilárias e todas foram submetidas ao diagnóstico molecular em Nested PCR com o alvo ITS-1, sequenciamento de Sanger e métodos de Next Generation Sequencing. As filárias adultas encontradas foram submetidas à análise morfológica e molecular. Adicionalmente, foi possível georreferenciar 50 animais e coletar dados para desenvolver variáveis demográficas, meteorológicas e geográficas para 49 deles. Esses dados foram usados para calcular a matriz da paisagem em buffers de 500 e 1000 metros e desenvolver modelos lineares generalizados sobre a influência dessas variáveis a prevalência. Um total de 6,45% (4/62) das carcaças foram positivas para a presença de filária adulta. Das 24 filárias adultas encontradas nas 4 carcaças, 14 puderam ser identificadas morfologicamente como Dipetalonema sp., 8 foram identificadas como da família Onchocercidae no Sequenciamento de Sanger e 1 como Dipetalonema gracile, utilizando os métodos de NGS. 6,38% (3/47) das amostras de sangue foram positivas para microfilária na microscopia. 28,57% (16/56) das amostras puderam ser amplificadas na análise molecular e, destas, 75% (12/16) eram de Mansonella mariae, 6,25% (1/16) Dipetalonema sp., 6,25% (1/16) da família Onchocercidae e 12,5% (2/16) não foram identificadas. Para a análise de paisagem, 47% (8/17) dos positivos foram oriundos do Bairro Coroado, em Manaus, especificamente do fragmento florestal da Universidade Federal do Amazonas (UFAM); 12% (2/17) do bairro Tarumã; 12% (2/17) da rodovia AM-010; 5,8% (1/17) do bairro Tarumã-açu; 5,8% (1/17) da rodovia BR-174; 5,8% (1/17) da junção dos bairros Distrito Industrial II/Puraquequara; 5,8% (1/17) no bairro Ponta Negra e 5,8% (1/17) sem localização encontrada. Para os negativos, 56,41% (22/39) foram do bairro Coroado, em Manaus, também do fragmento florestal da Universidade Federal do Amazonas; 5,12% (2/39) do bairro Aleixo; 5,12% (2/39) do bairro Tarumã; 5,12% (2/39) do bairro Jorge Teixeira; 2,56 (1/39) para cada uma das seguintes localidades: cidade de Rio Preto da Eva, para a avenida Governador José Lindoso, os bairros Zumbi dos Palmares II, Parque 10 de Novembro, Santo Agostinho e Nova Cidade, em Manaus, e para as rodovias BR-174 e AM-010. No buffer de 500m, observou-se matriz florestal 60% (9/15) dos positivos, com a urbana sendo a segunda mais ocorrida, em 26,7% (4/15); para os negativos, houve 76% (19/25) de matriz florestal e 20% (5/25) de matriz urbana. No buffer de 1000 metros, percebe-se esse padrão praticamente idêntico. O modelo linear generalizado demonstrou probabilidade maior de positivos conforme a temperatura média mensal aumenta e probabilidade maior de negativos conforme densidade demográfica humana aumenta. A identificação dos parasitos coincide com a literatura, que já descreveu Mansonella mariae e Dipetalonema sp. em primatas e vetores amazônicos. As filárias identificadas não têm relato de serem zoonóticas, porém a família Onchocercidae e o gênero Mansonella abrangem espécies de filária de interesse para saúde pública. A análise de paisagem também coincide com o georreferenciamento organizado nesse trabalho, no qual se observa maior ocorrência dos positivos em áreas periurbanas de Manaus, com características de pouca densidade demográfica e com matriz florestal. Esses aspectos indicam a necessidade de continuidade nos esforços para identificar parasitos filariais em outros municípios amazônicos e compreender as causas ambientais por trás do risco de infecção.
Resumo em Inglês
Saguinus bicolor is a callitrichid that inhabits forest fragments and the outskirts of the Amazonian municipalities of Manaus, Rio Preto da Eva and Itacoatiara. It is at risk of extinction, being the limitation of its territory of occurrence and the fragmentation of its habitat two of the biggest threats. Also, few studies are carried out on infectious diseases and parasites that can affect this primate, such as filariae. Filariae are nematode worms that can parasitize a wide variety of animals, including primates, humans and domestic animals. Some can cause diseases of medical and veterinarian interest. This work aims to identify filarial parasites in Saguinus bicolor through necropsy, microscopy and molecular tools, as well as understand the influence of the landscape on the prevalence of filaria in these primates. 71 individuals were sampled, where 62 were submitted to necropsy, with inspection of the thoracic, abdominal and subcutaneous cavities, and 10 were sampled alive (where 1 of the animals was collected both in life and in death). From the necropsies, it was also possible to collect blood from 46 carcasses, totaling 56 blood samples. From these, 47 samples were submitted to microscopy to look for microfilariae and all samples were submitted to molecular analysis in Nested PCR with the ITS-1 target, Sanger sequencing and Next Generation Sequencing methods. The adult filariae found were submitted to morphological and molecular analysis. Additionally, it was possible to georeference 50 animals and collect data to develop demographic, meteorological and geographic variables for 49 of them. These data were used to calculate the landscape matrix in buffers of 500 and 1000 meters and to develop generalized linear models on the influence of these variables on prevalence. 6.45% (4/62) of the carcasses were positive for the presence of adult filaria. Of the 24 adult filariae found in 4 carcasses, 14 could be morphologically identified as Dipetalonema sp., 1 as Dipetalonema sp. with the NGS methods, 8 of the Onchocercidae family in the Sanger Sequencing. 6.38% (3/47) of blood samples were positive for microfilaria. 28.57% (16/56) of the samples could be amplified in the molecular analysis and, of these, 75% (12/16) were from Mansonella mariae, 6.25% (1/16) from Dipetalonema sp., 6.25% (1/16) from the Onchocercidae family and 12.5% (2/16) were not identified. For the landscape analysis, 47% (8/17) of the positives came from Coroado neighborhood in Manaus, in the forest fragment of the Federal University of Amazonas; 12% (2/17) from Tarumã Saguinus bicolor is a callitrichid that inhabits forest fragments and the outskirts of the Amazonian municipalities of Manaus, Rio Preto da Eva and Itacoatiara. It is at risk of extinction, being the limitation of its territory of occurrence and the fragmentation of its habitat two of the biggest threats. Also, few studies are carried out on infectious diseases and parasites that can affect this primate, such as filariae. Filariae are nematode worms that can parasitize a wide variety of animals, including primates, humans and domestic animals. Some can cause diseases of medical and veterinarian interest. This work aims to identify filarial parasites in Saguinus bicolor through necropsy, microscopy and molecular tools, as well as understand the influence of the landscape on the prevalence of filaria in these primates. 71 individuals were sampled, where 62 were submitted to necropsy, with inspection of the thoracic, abdominal and subcutaneous cavities, and 10 were sampled alive (where 1 of the animals was collected both in life and in death). From the necropsies, it was also possible to collect blood from 46 carcasses, totaling 56 blood samples. From these, 47 samples were submitted to microscopy to look for microfilariae and all samples were submitted to molecular analysis in Nested PCR with the ITS-1 target, Sanger sequencing and Next Generation Sequencing methods. The adult filariae found were submitted to morphological and molecular analysis. Additionally, it was possible to georeference 50 animals and collect data to develop demographic, meteorological and geographic variables for 49 of them. These data were used to calculate the landscape matrix in buffers of 500 and 1000 meters and to develop generalized linear models on the influence of these variables on prevalence. 6.45% (4/62) of the carcasses were positive for the presence of adult filaria. Of the 24 adult filariae found in 4 carcasses, 14 could be morphologically identified as Dipetalonema sp., 1 as Dipetalonema sp. with the NGS methods, 8 of the Onchocercidae family in the Sanger Sequencing. 6.38% (3/47) of blood samples were positive for microfilaria. 28.57% (16/56) of the samples could be amplified in the molecular analysis and, of these, 75% (12/16) were from Mansonella mariae, 6.25% (1/16) from Dipetalonema sp., 6.25% (1/16) from the Onchocercidae family and 12.5% (2/16) were not identified. For the landscape analysis, 47% (8/17) of the positives came from Coroado neighborhood in Manaus, in the forest fragment of the Federal University of Amazonas; 12% (2/17) from Tarumã Saguinus bicolor is a callitrichid that inhabits forest fragments and the outskirts of the Amazonian municipalities of Manaus, Rio Preto da Eva and Itacoatiara. It is at risk of extinction, being the limitation of its territory of occurrence and the fragmentation of its habitat two of the biggest threats. Also, few studies are carried out on infectious diseases and parasites that can affect this primate, such as filariae. Filariae are nematode worms that can parasitize a wide variety of animals, including primates, humans and domestic animals. Some can cause diseases of medical and veterinarian interest. This work aims to identify filarial parasites in Saguinus bicolor through necropsy, microscopy and molecular tools, as well as understand the influence of the landscape on the prevalence of filaria in these primates. 71 individuals were sampled, where 62 were submitted to necropsy, with inspection of the thoracic, abdominal and subcutaneous cavities, and 10 were sampled alive (where 1 of the animals was collected both in life and in death). From the necropsies, it was also possible to collect blood from 46 carcasses, totaling 56 blood samples. From these, 47 samples were submitted to microscopy to look for microfilariae and all samples were submitted to molecular analysis in Nested PCR with the ITS-1 target, Sanger sequencing and Next Generation Sequencing methods. The adult filariae found were submitted to morphological and molecular analysis. Additionally, it was possible to georeference 50 animals and collect data to develop demographic, meteorological and geographic variables for 49 of them. These data were used to calculate the landscape matrix in buffers of 500 and 1000 meters and to develop generalized linear models on the influence of these variables on prevalence. 6.45% (4/62) of the carcasses were positive for the presence of adult filaria. Of the 24 adult filariae found in 4 carcasses, 14 could be morphologically identified as Dipetalonema sp., 1 as Dipetalonema sp. with the NGS methods, 8 of the Onchocercidae family in the Sanger Sequencing. 6.38% (3/47) of blood samples were positive for microfilaria. 28.57% (16/56) of the samples could be amplified in the molecular analysis and, of these, 75% (12/16) were from Mansonella mariae, 6.25% (1/16) from Dipetalonema sp., 6.25% (1/16) from the Onchocercidae family and 12.5% (2/16) were not identified. For the landscape analysis, 47% (8/17) of the positives came from Coroado neighborhood in Manaus, in the forest fragment of the Federal University of Amazonas; 12% (2/17) from Tarumã Saguinus bicolor is a callitrichid that inhabits forest fragments and the outskirts of the Amazonian municipalities of Manaus, Rio Preto da Eva and Itacoatiara. It is at risk of extinction, being the limitation of its territory of occurrence and the fragmentation of its habitat two of the biggest threats. Also, few studies are carried out on infectious diseases and parasites that can affect this primate, such as filariae. Filariae are nematode worms that can parasitize a wide variety of animals, including primates, humans and domestic animals. Some can cause diseases of medical and veterinarian interest. This work aims to identify filarial parasites in Saguinus bicolor through necropsy, microscopy and molecular tools, as well as understand the influence of the landscape on the prevalence of filaria in these primates. 71 individuals were sampled, where 62 were submitted to necropsy, with inspection of the thoracic, abdominal and subcutaneous cavities, and 10 were sampled alive (where 1 of the animals was collected both in life and in death). From the necropsies, it was also possible to collect blood from 46 carcasses, totaling 56 blood samples. From these, 47 samples were submitted to microscopy to look for microfilariae and all samples were submitted to molecular analysis in Nested PCR with the ITS-1 target, Sanger sequencing and Next Generation Sequencing methods. The adult filariae found were submitted to morphological and molecular analysis. Additionally, it was possible to georeference 50 animals and collect data to develop demographic, meteorological and geographic variables for 49 of them. These data were used to calculate the landscape matrix in buffers of 500 and 1000 meters and to develop generalized linear models on the influence of these variables on prevalence. 6.45% (4/62) of the carcasses were positive for the presence of adult filaria. Of the 24 adult filariae found in 4 carcasses, 14 could be morphologically identified as Dipetalonema sp., 1 as Dipetalonema sp. with the NGS methods, 8 of the Onchocercidae family in the Sanger Sequencing. 6.38% (3/47) of blood samples were positive for microfilaria. 28.57% (16/56) of the samples could be amplified in the molecular analysis and, of these, 75% (12/16) were from Mansonella mariae, 6.25% (1/16) from Dipetalonema sp., 6.25% (1/16) from the Onchocercidae family and 12.5% (2/16) were not identified. For the landscape analysis, 47% (8/17) of the positives came from Coroado neighborhood in Manaus, in the forest fragment of the Federal University of Amazonas; 12% (2/17) from Tarumã Saguinus bicolor is a callitrichid that inhabits forest fragments and the outskirts of the Amazonian municipalities of Manaus, Rio Preto da Eva and Itacoatiara. It is at risk of extinction, being the limitation of its territory of occurrence and the fragmentation of its habitat two of the biggest threats. Also, few studies are carried out on infectious diseases and parasites that can affect this primate, such as filariae. Filariae are nematode worms that can parasitize a wide variety of animals, including primates, humans and domestic animals. Some can cause diseases of medical and veterinarian interest. This work aims to identify filarial parasites in Saguinus bicolor through necropsy, microscopy and molecular tools, as well as understand the influence of the landscape on the prevalence of filaria in these primates. 71 individuals were sampled, where 62 were submitted to necropsy, with inspection of the thoracic, abdominal and subcutaneous cavities, and 10 were sampled alive (where 1 of the animals was collected both in life and in death). From the necropsies, it was also possible to collect blood from 46 carcasses, totaling 56 blood samples. From these, 47 samples were submitted to microscopy to look for microfilariae and all samples were submitted to molecular analysis in Nested PCR with the ITS-1 target, Sanger sequencing and Next Generation Sequencing methods. The adult filariae found were submitted to morphological and molecular analysis. Additionally, it was possible to georeference 50 animals and collect data to develop demographic, meteorological and geographic variables for 49 of them. These data were used to calculate the landscape matrix in buffers of 500 and 1000 meters and to develop generalized linear models on the influence of these variables on prevalence. 6.45% (4/62) of the carcasses were positive for the presence of adult filaria. Of the 24 adult filariae found in 4 carcasses, 14 could be morphologically identified as Dipetalonema sp., 1 as Dipetalonema sp. with the NGS methods, 8 of the Onchocercidae family in the Sanger Sequencing. 6.38% (3/47) of blood samples were positive for microfilaria. 28.57% (16/56) of the samples could be amplified in the molecular analysis and, of these, 75% (12/16) were from Mansonella mariae, 6.25% (1/16) from Dipetalonema sp., 6.25% (1/16) from the Onchocercidae family and 12.5% (2/16) were not identified. For the landscape analysis, 47 (8/17) of the positives came from Coroado neighborhood in Manaus, in the forest fragment of the Federal University of Amazonas; 12% (2/17) from Tarumã neighborhood; 12% (2/17) from the AM-010 highway; 5,8% (1/17) from Tarumã-açu neighborhood; 5,8% (1/17) from the BR-174 highway; 5,8% (1/17) from the junction area between the Industrial District II/Puraquequara neighborhood; 5,8% (1/17) from Ponta Negra neighborhood and 5,8% (1/17) with its location not found. For the negatives, 56.46% (22/39) were from Coroado neighborhood in Manaus, also in the forest fragment of the Federal University of Amazonas; 5,12% (2/39) from Aleixo neigborhood; 5,12% (2/39) from Tarumã neighborhood; 5,12% (2/39) from Jorge Teixeira neighborhood; 2,56% (1/39) for each one the following places: city of Rio Preto da Eva; Governador José Lindoso avenue; Zumbi dos Palmares II neighborhood; Parque 10 de Novembro neighborhood; Santo Agostinho neighborhood and Nova Cidade neighborhood, in Manaus; and for BR-174 and AM-010 highways. In the 500m buffer, 60% (9/15) of the positives were observed, with the urban matrix being the second most frequent, at 26.7% (4/15); for the negatives, there was 76% (19/25) of the forest matrix and 20% of the urban matrix (5/25). In the buffer of 1000 meters, this pattern is practically identical. The generalized linear model showed a greater probability of positives as the average monthly temperature increases and a greater probability of negatives as the human population density increases. The identification of the parasites coincides with the literature, which has already described Mansonella mariae and Dipetalonema sp. in primates and Amazonian vectors. None of the filariae detected cause zoonoses, but the Onchocercidae family and the Mansonella genus cover filarial species that are of public health interest. The landscape analysis also coincides with the georeferencing organized in this work, where a higher occurrence of positives is observed in peri-urban areas of Manaus, with characteristics of low population density and with a forest matrix. These aspects indicate the need for continuity in the efforts to identify filarial parasites in other Amazonian municipalities and to understand the environmental causes behind the risk of infection.
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