Fungal contamination of poultry litter: a public health problem

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.

Fungal contamination in swine: a potential occupational health threat

Poor air quality in a pig-confinement building may potentially place farmers at higher health risk than other workers for exposure to airborne pollutants that may reach infectious levels. The aim of this study was to assess worker exposure to fungi in indoor environments in Portuguese swine buildings. Air samples from 7 swine farms were collected at a flow rate of 140 L/min, at 1 m height, onto malt extract agar supplemented with chloramphenicol (MEA). Surfaces samples of the same indoor sites were obtained by swabbing the surfaces. Samples from the floor covering were also collected from four of seven swine farms. All collected samples were incubated at 27°C for 5-7 days. After lab processing and incubation of obtained samples, quantitative colony-forming units (CFU)/m(3), CFU/cm(2), and CFU/g and qualitative results were determined with identification of isolated fungal species. Aspergillus versicolor was the most frequent species found in air (21%), followed by Scopulariopsis brevicaulis (17%) and Penicillium sp. (14%). Aspergillus versicolor was also the most frequent species noted on surfaces (26.6%), followed by Cladosporium sp. (22.4%) and Scopulariopsis brevicaulis (17.5%). Chrysosporium was the most frequently found genera in the new floor covering (38.5%), while Mucor was the most prevalent genera (25.1%) in used floor covering. Our findings corroborate a potential occupational health threat due to fungi exposure and suggest the need for a preventive strategy.

Exposure to fungi in dockers

Several activities are ensured by dockers increase occupational exposure to several risk factors. being one of them the fungal burden from the load. In this study we aim at characterizing fungal contamination in one warehouse that storage sugar cane from a ship, and also in one crane cabinet that unload the same sugar cane from the ship. Air samples were collected from the warehouse and from inside the crane cabinet. An outdoor sample was also collected, from each sampling site, and regarding as reference. Sampling volume was selected depending in the contamination expected and the air samples were collect through an impaction method in a flow rate of 140 L/min onto malt extract agar (MEA) supplemented with chloramphenicol (0.05%), using the Millipore air Tester (Millipore). Surfaces samples from the warehouse were collected by swabbing the surfaces of the same indoor sites, using a 10 by 10cm square stencil according to the International Standard ISO 18593 (2004). The obtained swabs were then plated onto MEA. All the collected samples were incubated at 27ºC for 5 to 7 days. After laboratory processing and incubation of the collected samples, quantitative (colony-forming units - CFU/m3 and CFU/m2) and qualitative results were obtained with identification of the isolated fungal species. Aspergillus fumigatus present the highest fungal load and WHO guideline was overcome in both indoor sampling sites. The results obtained in this study highlight the need to know better the exposure burden from dockers, and specifically to fungi contamination.

Fungal contamination assessment in Portuguese elderly care centers

Individuals spend 80-90% of their day indoors and elderly subjects are likely to spend even a greater amount of time indoors. Thus, indoor air pollutants such as bioaerosols may exert a significant impact on this age group. The aim of this study was to characterize fungal contamination within Portuguese elderly care centers. Fungi were measured using conventional as well as molecular methods in bedrooms, living rooms, canteens, storage areas, and outdoors. Bioaerosols were evaluated before and after the microenvironments' occupancy in order to understand the role played by occupancy in fungal contamination. Fungal load results varied from 32 colony-forming units CFU m(-3) in bedrooms to 228 CFU m(-3) in storage areas. Penicillium sp. was the most frequently isolated (38.1%), followed by Aspergillus sp. (16.3%) and Chrysonilia sp. (4.2%). With respect to Aspergillus genus, three different fungal species in indoor air were detected, with A. candidus (62.5%) the most prevalent. On surfaces, 40 different fungal species were isolated and the most frequent was Penicillium sp. (22.2%), followed by Aspergillus sp. (17.3%). Real-time polymerase chain reaction did not detect the presence of A. fumigatus complex. Species from Penicillium and Aspergillus genera were the most abundant in air and surfaces. The species A. fumigatus was present in 12.5% of all indoor microenvironments assessed. The living room was the indoor microenvironment with lowest fungal concentration and the storage area was highest.

Avaliação da exposição a fungos e partículas em explorações avícolas e suinícolas: estudo

O projeto “Avaliação da Exposição a Fungos e Partículas em Explorações Avícolas e Suinícolas” contemplou um elevado número de colheitas ambientais e biológicas e respectivo processamento laboratorial, sendo apenas possível a sua concretização graças ao financiamento disponibilizado pela Autoridade para as Condições de Trabalho. Foi realizado um estudo transversal para avaliar a contaminação causada por fungos e partículas em 7 explorações avícolas e 7 explorações suinícolas. No que concerne à monitorização biológica, foram medidos os parâmetros espirométricos, utilizando o espirómetro MK8 Microlab, avaliada a existência de sintomas clínicos associados com a asma e outras doenças alérgicas, através de questionário adaptado European Community Respiratory Health Survey e, ainda, avaliada a sensibilização aos agentes fúngicos (IgE). Foram ainda adicionados dois objetivos ao estudo, designadamente: aferir a existência de três espécies/estirpes potencialmente patogénicas/toxinogénicas com recurso à biologia molecular e avaliar a exposição dos trabalhadores à micotoxina aflatoxina B1 por recurso a indicador biológico de exposição. Foram colhidas 27 amostras de ar de 25 litros nas explorações avícolas e 56 de 50 litros nas explorações suinícolas através do método de impacto. As colheitas de ar e a medição da concentração das partículas foram realizadas no interior e no exterior dos pavilhões, sendo este último considerado como local de referência. Simultaneamente, a temperatura e a humidade relativa também foram registadas. As colheitas das superfícies foram realizadas através da técnica de zaragatoa, tendo sido utilizado um quadrado de metal inoxidável de 10 cm de lado, de acordo com a International Standard ISO 18593 – 2004. As zaragatoas obtidas (20 das explorações avícolas e 48 das explorações suinícolas) foram inoculadas em malte de extract agar (2%) com cloranfenicol (0,05 g/L). Além das colheitas de ar e de superfícies, foram também obtidas colheitas da cama das explorações avícolas (7 novas e 14 usadas) e da cobertura do pavimento das explorações suinícolas (3 novas e 4 usadas) e embaladas em sacos esterilizados. Cada amostra foi diluída e inoculada em placas contendo malte extract agar. Todas as amostras foram incubadas a 27,5ºC durante 5 a 7 dias e obtidos resultados quantitativos (UFC/m3; UFC/m2; UFC/g) e qualitativos com a identificação das espécies fúngicas. Para a aplicação dos métodos de biologia molecular foram realizadas colheitas de ar de 300 litros utilizando o método de impinger com a velocidade de recolha de 300 L/min. A identificação molecular de três espécies potencialmente patogénicas e/ou toxinogénicas (Aspergillus flavus, Aspergillus fumigatus e Stachybotrys chartarum) foram obtidas por PCR em tempo real (PCR TR) utilizando o Rotor-Gene 6000 qPCR Detection System. As medições de partículas foram realizadas por recurso a equipamento de leitura direta (modelo Lighthouse, 2016 IAQ). Este recurso permitiu medir a concentração (mg/m3) de partículas em 5 dimensões distintas (PM 0.5; PM 1.0; PM 2.5; PM 5.0; PM10). Nas explorações avícolas, 28 espécies/géneros de fungos foram isolados no ar, tendo Aspergillus versicolor sido a espécie mais frequente (20.9%), seguida por Scopulariopsis brevicaulis (17.0%) e Penicillium sp. (14.1%). Entre o género Aspergillus, Aspergillus flavus apresentou o maior número de esporos (>2000 UFC/m3). Em relação às superfícies, A. versicolor foi detetada em maior número (>3 × 10−2 UFC/m2). Na cama nova, Penicillium foi o género mais frequente (59,9%), seguido por Alternaria (17,8%), Cladosporium (7,1%) e Aspergillus (5,7%). Na cama usada, Penicillium sp. foi o mais frequente (42,3%), seguido por Scopulariopsis sp. (38,3%), Trichosporon sp. (8,8%) e Aspergillus sp. (5,5%). Em relação à contaminação por partículas, as partículas com maior dimensão foram detectadas em maiores concentrações, designadamente as PM5.0 (partículas com a dimensão de 5.0 bm ou menos) e PM10 (partículas com a dimensão de 10 bm ou menos). Neste setting a prevalência da alteração ventilatória obstrutiva foi superior nos indivíduos com maior tempo de exposição (31,7%) independentemente de serem fumadores (17,1%) ou não fumadores (14,6%). Relativamente à avaliação do IgE específico, foi apenas realizado em trabalhadores das explorações avícolas (14 mulheres e 33 homens), não tendo sido encontrada associação positiva (p<0.05%) entre a contaminação fúngica e a sensibilização a antigénios fúngicos. No caso das explorações suinícolas, Aspergillus versicolor foi a espécie mais frequente (20,9%), seguida por Scopulariopsis brevicaulis (17,0%) e Penicillium sp. (14,1%). No género Aspergillus, A. versicolor apresentou o maior isolamento no ar (>2000 UFC/m3) e a maior prevalência (41,9%), seguida por A. flavus e A. fumigatus (8,1%). Em relação às superfícies analisadas, A. versicolor foi detetada em maior número (>3 ×10−2 UFC/m2). No caso da cobertura do pavimento das explorações suinícolas, o género Thicoderma foi o mais frequente na cobertura nova (28,0%) seguida por A. versicolor e Acremonium sp. (14,0%). O género Mucor foi o mais frequente na cobertura usada (25,1%), seguido por Trichoderma sp. (18,3%) e Acremonium sp. (11,2%). Relativamente às partículas, foram evidenciados também valores mais elevados na dimensão PM5 e, predominantes nas PM10. Neste contexto, apenas 4 participantes (22,2%) apresentaram uma alteração ventilatória obstrutiva. Destes, as obstruções mais graves encontraram-se nos que também apresentavam maior tempo de exposição. A prevalência de asma na amostra de trabalhadores em estudo, pertencentes aos 2 contextos em estudo, foi de 8,75%, tendo-se verificado também uma prevalência elevada de sintomatologia respiratória em profissionais não asmáticos. Em relação à utilização complementar dos métodos convencionais e moleculares, é recomendável que a avaliação da contaminação fúngica nestes settings, e, consequentemente, a exposição profissional a fungos, seja suportada pelas duas metodologias e, ainda, que ocorre exposição ocupacional à micotoxina aflatoxina B1 em ambos os contextos profissionais. Face aos resultados obtidos, é importante salientar que os settings alvo de estudo carecem de uma intervenção integrada em Saúde Ocupacional no âmbito da vigilância ambiental e da vigilância da saúde, com o objetivo de diminuir a exposição aos dois factores de risco estudados (fungos e partículas).

Living bacteria rheology: Population growth, aggregation patterns, and collective behaviour under diferente shear flows

The activity of growing living bacteria was investigated using real-time and in situ rheology-in stationary and oscillatory shear. Two different strains of the human pathogen Staphylococcus aureus-strain COL and its isogenic cell wall autolysis mutant, RUSAL9-were considered in this work. For low bacteria density, strain COL forms small clusters, while the mutant, presenting deficient cell separation, forms irregular larger aggregates. In the early stages of growth, when subjected to a stationary shear, the viscosity of the cultures of both strains increases with the population of cells. As the bacteria reach the exponential phase of growth, the viscosity of the cultures of the two strains follows different and rich behaviors, with no counterpart in the optical density or in the population's colony-forming units measurements. While the viscosity of strain COL culture keeps increasing during the exponential phase and returns close to its initial value for the late phase of growth, where the population stabilizes, the viscosity of the mutant strain culture decreases steeply, still in the exponential phase, remains constant for some time, and increases again, reaching a constant plateau at a maximum value for the late phase of growth. These complex viscoelastic behaviors, which were observed to be shear-stress-dependent, are a consequence of two coupled effects: the cell density continuous increase and its changing interacting properties. The viscous and elastic moduli of strain COL culture, obtained with oscillatory shear, exhibit power-law behaviors whose exponents are dependent on the bacteria growth stage. The viscous and elastic moduli of the mutant culture have complex behaviors, emerging from the different relaxation times that are associated with the large molecules of the medium and the self-organized structures of bacteria. Nevertheless, these behaviors reflect the bacteria growth stage.

Fungal contamination of sandpits from recreational parks and schools: a potential risk for human health

Sandpits used by children are frequently visited by wild life which constitutes a source of fungal pathogens and allergenic fungi. This study aimed to take an unannounced snapshot of the urban levels of fungal contaminants in sands, using for this purpose two public recreational parks, three elementary schools and two kindergartens. All samples were from Lisbon and neighboring municipalities and were tested for fungi of clinical interest. Potentially pathogenic fungi were isolated from all samples besides one. Fusarium dimerum (32.4%) was found to be the dominant species in one park and Chrysonilia spp. in the other (46.6%). Fourteen different species and genera were detected and no dermatophytes were found. Of a total of 14 species and genera, the fungi most isolated from the samples of the elementary schools were Penicillium spp. (74%), Cladophialophora spp. (38%) and Cladosporium spp. (90%). Five dominant species and genera were isolated from the kindergartens. Penicillium spp. was the only genus isolated in one, though with remarkably high counts (32500 colony forming units per gram). In the other kindergarten Penicillium spp. were also the most abundant species, occupying 69% of all the fungi found. All of the samples exceeded the Maximum Recommended Value (MRV) for beach sand defined by Brandão et al. 2011, which are currently the only quantitative guidelines available for the same matrix. The fungi found confirm the potential risk of exposure of children to keratinophilic fungi and demonstrates that regular cleaning or replacing of sand needs to be implemented in order to minimize contamination.

Bibliotecas na saúde… e a saúde nas bibliotecas?

Em arquivos e bibliotecas a presença de fungos é considerada nefasta pelas suas implicações na conservação e leitura de documentos históricos e pela sua associação a problemas de saúde sentidos pelos funcionários e utentes que frequentam estes locais. De acordo com alguns autores, os problemas de saúde mais reportados por funcionários em Bibliotecas e Arquivos são dermatite, rinite, alergias e asma. Embora revestida de inegável importância, existem poucos estudos internacionais sobre a temática e, em Portugal, a contaminação fúngica em ambiente arquivístico e em bibliotecas é ainda muito pouco conhecida. O estudo realizado em quatro Arquivos Portugueses teve como objectivo conhecer a contaminação fúngica, contribuindo para a análise da qualidade do ar interior desses espaços e sua comparação com estudos internacionais. Para isso foram recolhidas amostras de ar e de superfícies e estas foram analisadas por métodos clássicos de cultura e, quando necessário, por métodos de biologia molecular. A avaliação foi feita quantitativa e qualitativamente, considerando os requisitos legais em vigor. No que respeita à análise do ar, o número de unidades formadoras de colónias (UFC)/m3 nunca excedeu as 500 (limite legislado), tendo sido verificada contaminação interior em todos os locais estudados. Comparativamente aos estudos realizados anteriormente em contextos semelhantes foram encontrados níveis elevados de contaminação por leveduras nas amostras de ar analisadas em Arquivos Portugueses. Não foi identificado nenhum fungo patogénico neste estudo, mas em quase todas as amostras estavam presentes fungos potencialmente toxinogénicos. Dentro do grupo dos Aspergillus, o A.versicolor mostrou predominância, tendo este fungo reconhecidas capacidades de emissão de micotoxinas em ambiente de interior. A inclusão de amostras de superfície revelou-se vital para conhecer todo o espectro fúngico existente em cada um dos locais estudados, incluindo a detecção de Stachybotrys chartarum e a do fungo potencialmente queratinofílico, Chrysosporium carmichaelli. Tanto para a saúde como para a conservação, o recente estudo realizado em quatro arquivos permitiu retirar importantes conclusões e reforçar a necessidade de vigilância, sendo também útil para a definição de padrões de qualidade no campo do património cultural.

Scheduling of head-dependent cascaded reservoirs considering discharge ramping constraints and start/stop of units

This paper is on the problem of short-term hydro scheduling (STHS), particularly concerning head-dependent reservoirs under competitive environment. We propose a novel method, based on mixed-integer nonlinear programming (MINLP), for optimising power generation efficiency. This method considers hydroelectric power generation as a nonlinear function of water discharge and of the head. The main contribution of this paper is that discharge ramping constraints and start/stop of units are also considered, in order to obtain more realistic and feasible results. The proposed method has been applied successfully to solve two case studies based on Portuguese cascaded hydro systems, providing a higher profit at an acceptable computation time in comparison with classical optimisation methods based on mixed-integer linear programming (MILP).

Comparison of fungal contamination between hospitals and companies food units

A descriptive study was developed to compare air and surfaces fungal contamination in ten hospitals’ food units and two food units from companies. Fifty air samples of 250 litres through impaction method were collected from hospitals’ food units and 41 swab samples from surfaces were also collected, using a 10 by 10 cm square stencil. Regarding the two companies, ten air samples and eight surface samples were collected. Air and surface samples were collected in food storage facilities, kitchen, food plating and canteen. Outdoor air was also collected since this is the place regarded as a reference. Simultaneously, temperature, relative humidity and meal numbers were registered. Concerning air from hospitals’ food units, 32 fungal species were identified, being the two most commonly isolated genera Penicillium sp.

Air fungal contamination in ten hospitals’ food units from Lisbon

A descriptive study was developed to monitor air fungal contamination in ten food units from hospitals. Fifty air samples of 250 litres were collected through impaction method. Samples were collected in food storage facilities, kitchen, food plating, canteen and also, outside premises, since this is the place regarded as reference. Simultaneously, environmental parameters were also monitored, including temperature and relative humidity through the equipment Babouc, LSI Sistems and according to the International Standard ISO 7726.

Comparison of indoor and outdoor fungi and particles in poultry units

A descriptive study was developed in order to compare indoor and outdoor air contamination caused by fungi and particles in seven poultry units. Twenty eight air samples of 25 litters were collected through the impaction method on malt extract agar. Air sampling and particles concentration measurement were done in the interior and also outside premises of the poultries’ pavilions. Regarding the fungal load in the air, indoor concentration of mold was higher than outside air in six poultry units. Twenty eight species / genera of fungi were identified indoor, being Scopulariopsis brevicaulis (40.5%) the most commonly isolated species and Rhizopus sp. (30.0%) the most commonly isolated genus. Concerning outdoor, eighteen species/genera of fungi were isolated, being Scopulariopsis brevicaulis (62.6%) also the most isolated. All the poultry farms analyzed presented indoor fungi different from the ones identified outdoors. Regarding particles’ contamination, PM2.5, PM5.0 and PM10 had a statistically significant difference (Mann-Whitney U test) between the inside and outside of the pavilions, with the inside more contaminated (p=.006; p=.005; p=.005, respectively). The analyzed poultry units are potential reservoirs of substantial amounts of fungi and particles and could therefore free them in the atmospheric air. The developed study showed that indoor air was more contaminated than outdoors, and this can result in emission of potentially pathogenic fungi and particles via aerosols from poultry units to the environment, which may post a considerable risk to public health and contribute to environmental pollution.

Occupational exposure to aflatoxin (AFB1) in poultry production

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.

Development of a biosensor for urea assay based on amidase inhibition, using an ion-selective electrode

A biosensor for urea has been developed based on the observation that urea is a powerful active-site inhibitor of amidase, which catalyzes the hydrolysis of amides such as acetamide to produce ammonia and the corresponding organic acid. Cell-free extract from Pseudomonas aeruginosa was the source of amidase (acylamide hydrolase, EC 3.5.1.4) which was immobilized on a polyethersulfone membrane in the presence of glutaraldehyde; anion-selective electrode for ammonium ions was used for biosensor development. Analysis of variance was used for optimization of the biosensorresponse and showed that 30 mu L of cell-free extract containing 7.47 mg protein mL(-1), 2 mu L of glutaraldehyde (5%, v/v) and 10 mu L of gelatin (15%, w/v) exhibited the highest response. Optimization of other parameters showed that pH 7.2 and 30 min incubation time were optimum for incubation ofmembranes in urea. The biosensor exhibited a linear response in the range of 4.0-10.0 mu M urea, a detection limit of 2.0 mu M for urea, a response timeof 20 s, a sensitivity of 58.245 % per mu M urea and a storage stability of over 4 months. It was successfully used for quantification of urea in samples such as wine and milk; recovery experiments were carried out which revealed an average substrate recovery of 94.9%. The urea analogs hydroxyurea, methylurea and thiourea inhibited amidase activity by about 90%, 10% and 0%, respectively, compared with urea inhibition.

Sensing Cloud Optimization to Solve ED of Units with Valve-Point Effects and Multi-fuels

In this paper a solution to an highly constrained and non-convex economical dispatch (ED) problem with a meta-heuristic technique named Sensing Cloud Optimization (SCO) is presented. The proposed meta-heuristic is based on a cloud of particles whose central point represents the objective function value and the remaining particles act as sensors "to fill" the search space and "guide" the central particle so it moves into the best direction. To demonstrate its performance, a case study with multi-fuel units and valve- point effects is presented.