Browsing by Author "Malta-Vacas, J."
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- Accessing indoor fungal contamination by Aspergillus fumigatus complex using conventional and molecular methods in portuguese poultriesPublication . Viegas, C.; Malta-Vacas, J.; Sabino, R.; Viegas, S.; Veríssimo, C.Epidemiological studies showed increased prevalence of respiratory symptoms and adverse changes in pulmonary function parameters in poultry workers, corroborating the increased exposure to risk factors, such as fungal load and their metabolites. This study aimed to determine the occupational exposure threat due to fungal contamination caused by the toxigenic isolates belonging to the complex of the species of Aspergillus flavus and also isolates from Aspergillus fumigatus species complex. The study was carried out in seven Portuguese poultries, using cultural and molecular methodologies. For conventional/cultural methods, air, surfaces, and litter samples were collected by impaction method using the Millipore Air Sampler. For the molecular analysis, air samples were collected by impinger method using the Coriolis μ air sampler. After DNA extraction, samples were analyzed by real-time PCR using specific primers and probes for toxigenic strains of the Aspergillus flavus complex and for detection of isolates from Aspergillus fumigatus complex. Through conventional methods, and among the Aspergillus genus, different prevalences were detected regarding the presence of Aspergillus flavus and Aspergillus fumigatus species complexes, namely: 74.5 versus 1.0 % in the air samples, 24.0 versus 16.0 % in the surfaces, 0 versus 32.6 % in new litter, and 9.9 versus 15.9 % in used litter. Through molecular biology, we were able to detect the presence of aflatoxigenic strains in pavilions in which Aspergillus flavus did not grow in culture. Aspergillus fumigatus was only found in one indoor air sample by conventional methods. Using molecular methodologies, however, Aspergillus fumigatus complex was detected in seven indoor samples from three different poultry units. The characterization of fungal contamination caused by Aspergillus flavus and Aspergillus fumigatus raises the concern of occupational threat not only due to the detected fungal load but also because of the toxigenic potential of these species.
- Fungal contamination of poultries litter: A public health problemPublication . Viegas, C.; Carolino, E.; Malta-Vacas, J.; Sabino, R.; Viegas, S.; Veríssimo, C.Although numerous studies have been conducted on microbial contaminants associated with various stages related to poultry and meat products processing, only a few reported on fungal contamination of poultry litter. The goals of this study were to (1) characterize litter fungal contamination and (2) report the incidence of keratinophilic and toxigenic fungi presence. Seven fresh and 14 aged litter samples were collected from 7 poultry farms. In addition, 27 air samples of 25 litters were also collected through impaction method, and after laboratory processing and incubation of collected samples, quantitative colony-forming units (CFU/m³) and qualitative results were obtained. Twelve different fungal species were detected in fresh litter and Penicillium was the most frequent genus found (59.9%), followed by Alternaria (17.8%), Cladosporium (7.1%), and Aspergillus (5.7%). With respect to aged litter, 19 different fungal species were detected, with Penicillium sp. the most frequently isolated (42.3%), followed by Scopulariopsis sp. (38.3%), Trichosporon sp. (8.8%), and Aspergillus sp. (5.5%). A significant positive correlation was found between litter fungal contamination (CFU/g) and air fungal contamination (CFU/m³). Litter fungal quantification and species identification have important implications in the evaluation of potential adverse health risks to exposed workers and animals. Spreading of poultry litter in agricultural fields is a potential public health concern, since keratinophilic (Scopulariopsis and Fusarium genus) as well as toxigenic fungi (Aspergillus, Fusarium, and Penicillium genus) were isolated.
- Identificação molecular de Aspergillus fumigatus em amostras de ar interiorPublication . Malta-Vacas, J.; Sabino, R.; Brito, M.; Viegas, C.Os bioaerossóis são essencialmente compostos por partículas fúngicas, bactéricas e esporos de plantas, sendo os fungos responsáveis pela produção de compostos orgânicos voláteis e micotoxinas. A sua presença no ar interior está associada a manifestações alérgicas, tóxicas e infecciosas. Tais respostas dependem não só da susceptibilidade do indivíduo como das espécies e concentrações de fungos presentes. Os trabalhadores de aviários e suiniculturas são profissionais com elevada exposição a estes agentes, pelo que têm maior risco de desenvolvimento de patologias associadas. Com este estudo pretende‑se desenvolver um método rápido e de elevada especificidade e sensibilidade que permita identificar as espécies de fungos clinicamente relevantes em contexto de saúde ocupacional. Foi utilizado o Coriolis Air sampler para proceder à recolha de amostras de ar interior (300 Lmin‑1) em seis instalações de criação de aves e suínos. O DNA fúngico foi isolado das amostras utilizando o kit Zymo ZR Fungal/Bacterial DNA e a sua presença foi confirmada utilizando primers universais para fungos. A presença de Aspergillus fumigatus foi pesquisada por PCR em Tempo Real utilizando sondas Taqman e primers específicos. Os resultados obtidos foram comparados com os determinados por métodos convencionais de cultura. Foram obtidas 25 amostras de 300L de ar em seis instalações. Os resultados obtidos confirmam a presença de A. fumigatus em todas as amostras de ar em que esta espécie foi identificada através de culturas (3/25). Adicionalmente, foi também detectada em amostras de ar correspondendo a instalações em que, através de culturas, esta espécie não foi identificada no ar interior (6/25). Os resultados preliminares sugerem maior sensibilidade dos métodos moleculares relativamente aos métodos convencionais de cultura. Futuramente, pretende‑se aplicar a mesma metodologia para identificação de um maior número de espécies e proceder à sua quantificação.
- Molecular biology versus conventional methods – Complementary methodologies to understand occupational exposure to fungiPublication . Viegas, C.; Malta-Vacas, J.; Sabino, R.
- Occupational exposure to aflatoxin (AFB1) in poultry productionPublication . Viegas, S.; Veiga, L.; Malta-Vacas, J.; Sabino, R.; Almeida, A.; Viegas, C.Aflatoxin B1 (AFB1) has been recognized to produce cancer in human liver. In addition, epidemiological and laboratory studies demonstrated that the respiratory system was a target for AFB1. Exposure occurs predominantly through the food chain, but inhalation represents an additional route of exposure. The present study aimed to examine AFB1 exposure among poultry workers in Portugal. Blood samples were collected from a total of 31 poultry workers from six poultry farms. In addition, a control group (n = 30) was included comprised of workers who undertook administrative tasks. Measurement of AFB1 in serum was performed by enzyme-linked immunosorbent assay (ELISA). For examining fungi contamination, air samples were collected through an impaction method. Air sampling was obtained in pavilion interior and outside the premises, since this was the place regarded as the reference location. Using molecular methods, toxicogenic strains (aflatoxin-producing) were investigated within the group of species belonging to Aspergillus flavus complex. Eighteen poultry workers (59%) had detectable levels of AFB1 with values ranging from <1 ng/ml to4.23 ng/ml and with a mean value of 2 ± 0.98ng/ml. AFB1 was not detected in the serum sampled from any of the controls. Aspergillus flavus was the fungal species third most frequently found in the indoor air samples analyzed (7.2%) and was the most frequently isolated species in air samples containing only Aspergillus genus (74.5%). The presence of aflatoxigenic strains was only confirmed in outdoor air samples from one of the units, indicating the presence of a source inside the building in at least one case. Data indicate that AFB1 inhalation represents an additional risk in this occupational setting that needs to be recognized, assessed, and prevented.
- Occupational Exposure to toxigenic fungi from Aspergillus flavus complexPublication . Malta-Vacas, J.; Sabino, R.; Viegas, S.; Viegas, C.Bioaerosols are mainly composed of fungal particles, bacteria and plant spores, being fungi responsible for the release of VOCs and micotoxins into indoor environments. Aspergillus flavus is a common opportunistic pathogen causing human infections and is involved in the production of aflatoxin and other secondary metabolites associated with toxic and allergic reactions. Poultry workers are exposed to high concentrations of fungi and are therefore more prone to develop associated pathologies. To evaluate occupational exposure of the workers to Aspergillus flavus and aflatoxins, six animal production facilities were selected, including 10 buildings, from which indoor air samples and outdoor reference samples were obtained. Twenty-five duplicate samples were collected by two methodologies: impactation onto malt extract agar of 25L air samples using a Millipore Air Tester were used to evaluate quantitative (CFU/m3) and qualitative (species identification, whenever possible) sample composition; 300 L air samples collected with the Coriolis Air Sampler into phosphate–saline buffer were used to isolate DNA, following molecular identification of Aspergillus section flavi using nor-1 specific primers by real-time PCR. Overall, Aspergillus was the most frequent genus detected. Using conventional methodologies, A. flavus species were identified in five indoor samples belonging to three buildings and in two outdoor samples. Using real-time PCR, aflatoxigenic species were detected in two buildings, although only one was coincident with the ones identified by cultures. Using both methodologies we could quantify viable microorganisms and simultaneously identify potentially toxigenic species, resulting in complementary information useful in the adoption of strategies to minimize exposure to micotoxins.