942 resultados para nested-PCR_


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Sarcoidosis is a rare equine skin disease characterized primarily by an exfoliative and granulomatous dermatitis but also presenting granulomatous inflammation of multiple systems. The current report presents the clinical and histopathological findings of sarcoidosis in a 16-year-old American Quarter Horse gelding with nested polymerase chain reaction Mycobacterium spp. DNA detection within hepatic and skin samples. Mycobacterium spp. may play a role in the pathogenesis of equine sarcoidosis as has been proposed for human sarcoidosis.

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To produce an epidemiological map of neosporosis in Brazil and identify the types of transmission of this disease, the present study evaluated the occurrence of Neospora caninum in Nelore cattle (Bos indicus) in Presidente Prudent, west region of São Paulo state; its vertical transmission; and the early stage in which fetuses are infected. To achieve this, serum samples from 518 slaughtered pregnant heifers and their fetuses were tested by ELISA technique and fetal brain tissues subjected to PCR. One hundred and three heifers (19.88%) had antibodies to N. caninum, as well as 38 (36.8%) of fetuses from 4 months of gestation. The conventional PCR failed to detect N. caninum DNA. These findings show that neosporosis occurs in the area studied and that it may be transmitted the transplacental route, althought N. caninum had not detected in brain tissue from non-aborted fetuses. The use of nested PCR it would be applied to increase the sensitivy of test.

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

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Pigeons (Columba livia) cohabit with humans in urban and rural areas, representing a public health problem since microorganisms are transmitted through the inhalation of dust from their dry feces (chlamydiosis) and through ingestion of their undercooked or poorly refrigerated meat (toxoplasmosis). This study aimed to evaluate the presence of Chlamydophila psittaci and Toxoplasma gondii in pigeons from four cities in São Paulo State, Brazil. C psittaci was evaluated through hemi-nested polymerase chain reaction (hnPCR) using cloacal and tracheal swabs, whereas T. gondii specific antibodies were assessed by means of modified agglutination test (MAT), mouse brain and muscle bioassay, and polymerase chain reaction (PCR). To confirm the infection in mice, T. gondii antibodies were assayed by using indirect fluorescent antibody test (IFAT). Considering C. psittaci, 40/238 (16.8%; 95%CI 12.6-22.1%) samples were positive according to hnPCR, especially for the cities of São Paulo (42.5%) and Bauru (35%). As regards T. gondii, 12/238 (5%; 95%CI 2.9-8.6%) serum samples were positive according to MAT. of these, five samples had titer equal to 1:8; six samples, 1:16; and one sample, 1:32. Bioassay, IFAT and PCR were negative for mouse toxoplasmosis. The absence of T. gondii antibodies suggests that pigeons may be infected with a low concentration of the agent, not detected by the antigen test. Thus, C. psittaci represents an actual problem concerning bird health. (C) 2010 Elsevier B.V. All rights reserved.

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Babesia spp. infections were investigated in Bos taurus x Bos indicus dairy cows and calves and in Boophilus microplus engorged female ticks and eggs. Blood samples and engorged female ticks were collected from 25 cows and 27 calves. Babesia spp. was detected in ticks by microscopic examination of hemolymph of engorged female and by squashes of egg samples. Cattle infection was investigated in blood thin smears and by DNA amplification methods (PCR and nested PCR), using specific primers for Babesia bovis and Babesia bigemina. Merozoites of B. bovis (3 animals) and B. bigemina (12 animals) were detected exclusively in blood smears of calves. DNA amplification methods revealed that the frequency of B. bigemina infection in calves (92.6%) and in cows (84%) and of B. bovis in calves (85.2%) and in cows (100%) did not differ significantly (P > 0.05). Babesia spp. infection was more frequent in female ticks and eggs collected from calves (P < 0.01) than from cows, especially in those which had patent parasitemia. Hatching rates of B. microplus larvae were assessed according to the origin of engorged females, parasiternia of the vertebrate host, frequency and intensity of infection in engorged female tick, and frequency of egg infection. Hatching rate was lower in samples collected from calves (P < 0.01) than from cows, and in those in which Babesia spp. was detected in egg samples (P < 0.01). Published by Elsevier B.V.

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Babesia bigemina infections were investigated in four genetic groups of beef cattle and in Rhipicephalus (Boophilus) microplus engorged female ticks. Blood samples and engorged female ticks were collected from 15 cows and 15 calves from each of the following genetic groups: Nelore, Angus x Nelore, Canchim x Nelore, and Simmental x Nelore. Microscopic examination of blood smears and tick hemolymph revealed that merozoites of B. bigemina (6/60) as well as kinetes of Babesia spp. (9/549) were only detected in samples (blood and ticks, respectively) originated from calves. PCR-based methods using primers for specific detection of B. bigemina revealed 100% infection in both calves and cows, regardless the genetic group. Tick infection was detected by nested-PCR amplifications showing that the frequency of B. bigemina was higher (P 0.01) in female ticks collected from calves (134/549) than in those collected from cows (52/553). The frequency of B. bigemina was similar in ticks collected from animals, either cows or calves, of the four genetic groups (P > 0.05). (C) 2008 Elsevier B.V. All rights reserved.

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

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The objective of the present study was to evaluate different techniques for the detection of Paracoccidioides brasiliensis in soil, e.g., culture, animal inoculation and specific DNA amplification by Nested PCR. We designed species-specific inner primers derived from rDNA regions (ITS, 5.8S gene) and found their sensitivity to be higher than culture and animal inoculation. In addition, the sensitivity of these primers was higher than p27-gene primers developed for detection of P brasiliensis in soil in a previous study. DNA from P brasiliensis was detected in soil artificially seeded with the fungus (positive soil control) and from environmental samples collected in an armadillo burrow.

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Paracoccidioides brasiliensis infections have been little studied in wild and/or domestic animals, which may represent an important indicator of the presence of the pathogen in nature. Road-killed wild animals have been used for surveillance of vectors of zoonotic pathogens and may offer new opportunities for eco-epidemiological studies of paracoccidiodomycosis (PCM). The presence of P. brasiliensis infection was evaluated by Nested-PCR in tissue samples collected from 19 road-killed animals; 3 Cavia aperea (guinea pig), 5 Cerdocyon thous (crab-eating-fox), 1 Dasypus novemcinctus (nine-banded armadillo), 1 Dasypus septemcinctus (seven-banded armadillo), 2 Didelphis albiventris (white-eared opossum), 1 Eira barbara (tayra), 2 Gallictis vittata (grison), 2 Procyon cancrivorus (raccoon) and 2 Sphiggurus spinosus (porcupine). Specific P. brasiliensis amplicons were detected in (a) several organs of the two armadillos and one guinea pig, (b) the lung and liver of the porcupine, and (c) the lungs of raccoons and grisons. P. brasiliensis infection in wild animals from endemic areas might be more common than initially postulated. Molecular techniques can be used for detecting new hosts and mapping 'hot spot' areas of PCM.

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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

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