Elisabete Carolino

Occupational Exposure to Poultry Dust and Effects on the Respiratory System in Workers

Farmers are occupationally exposed to many respiratory hazards at work and display higher rates of asthma and respiratory symptoms than other workers. Dust is one of the components present in poultry production that increases risk of adverse respiratory disease occurrence. Dust originates from poultry residues, molds, and feathers and is biologically active as it contains microorganisms. Exposure to dust is known to produce a variety of clinical responses, including asthma, chronic bronchitis, chronic airways obstructive disease (COPD), allergic alveolitis, and organic dust toxic syndrome (ODTS). A study was developed to determine particle contamination in seven poultry farms and correlate this with prevalence rate of respiratory defects and record by means of a questionnaire the presence of clinical symptoms associated with asthma and other allergy diseases by European Community Respiratory Health Survey. Poultry farm dust contamination was found to contain higher concentrations of particulate matter (PM) PM5 and PM10. Prevalence rate of obstructive pulmonary disorders was higher in individuals with longer exposure regardless of smoking status. In addition, a high prevalence for asthmatic (42.5%) and nasal (51.1%) symptoms was noted in poultry workers. Data thus show that poultry farm workers are more prone to suffer from respiratory ailments and this may be attributed to higher concentrations of PM found in the dust. Intervention programs aimed at reducing exposure to dust will ameliorate occupational working conditions and enhance the health of workers.

Genotoxicity biomarkers in occupational exposure to formaldehyde: the case of histopathology laboratories

Formaldehyde, classified by the IARC as carcinogenic in humans and experimental animals, is a chemical agent that is widely used in histopathology laboratories. The exposure to this substance is epidemiologically linked to cancer and to nuclear changes detected by the cytokinesis-block micronucleus test (CBMN). This method is extensively used in molecular epidemiology, since it provides information on several biomarkers of genotoxicity, such as micronuclei (MN), which are biomarkers of chromosomes breakage or loss, nucleoplasmic bridges (NPB), common biomarkers of chromosome rearrangement, poor repair and/or telomere fusion, and nuclear buds (NBUD), biomarkers of elimination of amplified DNA. The aim of this study is to compare the frequency of genotoxicity biomarkers, provided by the CBMN assay in peripheral lymphocytes and the MN test in buccal cells, between individuals occupationally exposed and non-exposed to formaldehyde and other environmental factors, namely tobacco and alcohol consumption. The sample comprised two groups: 56 individuals occupationally exposed to formaldehyde (cases) and 85 unexposed individuals (controls), from whom both peripheral blood and exfoliated epithelial cells of the oral mucosa were collected in order to measure the genetic endpoints proposed in this study. The mean level of TWA(8h) was 0.16±0.11 ppm (<detection limit until 0.51 ppm) and the mean of ceiling values was 1.14±0.74 ppm (0.18-2.93 ppm). All genotoxicity biomarkers showed significant increases in exposed workers in comparison with controls (Mann-Whitney test, p<0.002) and the analysis of confounding factors showed that there were no differences between genders. As for age, only the mean MN frequency in lymphocytes was found significantly higher in elderly people among the exposed groups (p=0.006), and there was also evidence of an interaction between age and gender with regards to that biomarker in those exposed. Smoking habits did not influence the frequency of the biomarkers, whereas alcohol consumption only influenced the MN frequency in lymphocytes in controls (p=0.011), with drinkers showing higher mean values. These results provide evidence of the association between occupational exposure to formaldehyde and the presence of genotoxicity biomarkers.

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.

Occupational Exposure to Aflatoxin B1 in Swine Production and Possible Contamination Sources

Although the adverse health consequences of ingestion of food contaminated with aflatoxin B1 (AFB1) are known, relatively few studies are available on the adverse effects of exposure in occupational settings. Taking this into consideration, our study was developed aiming to elucidate the possible effects of occupational exposure to AFB1 in Portuguese swine production facilities using a specific biomarker to assess exposure to AFB1. In total, 28 workers participated in this study, providing blood samples, and a control group (n = 30) was composed of subjects without any type of agricultural activity. Fungal contamination was also studied by conventional methods through air, surfaces, and new and used floor coverage. Twenty-one workers (75%) showed detectable levels of AFB1 with values ranging from <1 ng/ml to 8.94 ng/ml and with a mean value of 1.91 ± 1.68 ng/ml. In the control group, the AFB1 values were all below 1 ng/ml. Twelve different Aspergillus species were identified. Aspergillus versicolor presented the highest airborne spore counts (3210 CFU/m3) and was also detected in higher values in surfaces (>300 CFU/cm2). Data indicate that exposure to AFB1 occurs in swine barns, and this site serves as a contamination source in an occupational setting.

Inhibition of Aspergillus fumigatus and its biofilm by Pseudomonas aeruginosa is dependent on the source, phenotype and growth conditions of the bacterium

Aspergillus fumigatus (Af) and Pseudomonas aeruginosa (Pa) are leading fungal and bacterial pathogens, respectively, in many clinical situations. Relevant to this, their interface and co-existence has been studied. In some experiments in vitro, Pa products have been defined that are inhibitory to Af. In some clinical situations, both can be biofilm producers, and biofilm could alter their physiology and affect their interaction. That may be most relevant to airways in cystic fibrosis (CF), where both are often prominent residents. We have studied clinical Pa isolates from several sources for their effects on Af, including testing involving their biofilms. We show that the described inhibition of Af is related to the source and phenotype of the Pa isolate. Pa cells inhibited the growth and formation of Af biofilm from conidia, with CF isolates more inhibitory than non-CF isolates, and non-mucoid CF isolates most inhibitory. Inhibition did not require live Pa contact, as culture filtrates were also inhibitory, and again non-mucoid>mucoid CF>non-CF. Preformed Af biofilm was more resistant to Pa, and inhibition that occurred could be reproduced with filtrates. Inhibition of Af biofilm appears also dependent on bacterial growth conditions; filtrates from Pa grown as biofilm were more inhibitory than from Pa grown planktonically. The differences in Pa shown from these different sources are consistent with the extensive evolutionary Pa changes that have been described in association with chronic residence in CF airways, and may reflect adaptive changes to life in a polymicrobial environment.

Occupational Exposure to Aspergillus by Swine and Poultry Farm Workers in Portugal

Aspergillus is among a growing list of allergens that aggravate asthmatic responses. Significant pulmonary pathology is associated with Aspergillus-induced allergic and asthmatic lung disease. Environments with high levels of exposure to fungi are found in animal production facilities such as for swine and poultry, and farmers working with these are at increased risk for occupational respiratory diseases. Seven Portuguese poultry and seven swine farms were analyzed in order to estimate the prevalence, amount, and distribution of Aspergillus species, as well as to determine the presence of clinical symptoms associated with asthma and other allergy diseases in these highly contaminated settings. From the collected fungal isolates (699), an average incidence of 22% Aspergillus was detected in poultry farms, while the prevalence at swine farms was 14%. The most frequently isolated Aspergillus species were A. versicolor, A. flavus, and A. fumigatus. In poultry farms, A. flavus presented the highest level of airborne spores (>2000 CFU/m3), whereas in swine farms the highest was A. versicolor, with an incidence fourfold greater higher than the other mentioned species. Eighty workers in these settings were analyzed, ranging in age from 17 to 93 yr. The potentially hazardous exposure of poultry workers to mold allergens using sensitization markers was evaluated. Although no significant positive association was found between fungal contamination and sensitization to fungal antigens, a high incidence of respiratory symptoms in professionals without asthma was observed, namely, wheezing associated with dyspnea (23.8%) and dyspnea after strenuous activities (12.3%), suggesting underdiagnosed respiratory disturbances. Further, 32.5% of all exposed workers noted an improvement of respiratory ability during resting and holidays. From all the analyzed workers, seven were previously diagnosed with asthma and four reported the first attack after the age of 40 yr, which may be associated with their occupational exposure. Some of the fungi, namely, the Aspergillus species detected in this study, are known to induce hypersensitivity reactions in humans. This study confirmed the presence and distribution of Aspergillus in Portuguese poultry and swine farms, suggesting a possible occupational health problem and raising the need for preventive and protective measures to apply to avoid exposure in both occupational settings.

Molecular screening of 246 Portuguese Aspergillus isolates among different clinical and environmental sources

Clinical and environmental samples from Portugal were screened for the presence of Aspergillus and the distributions of the species complexes were determined in order to understand how their distributions differ based on their source. Fifty-seven Aspergillus isolates from clinical samples were collected from 10 health institutions. Six species complexes were detected by internal transcribed spacer sequencing; Fumigati, Flavi, and Nigri were found most frequently (50.9%, 21.0%, and 15.8%, respectively). β-tubulin and calmodulin sequencing resulted in seven cryptic species (A. awamorii, A. brasiliensis, A. fructus, A. lentulus, A. sydowii, A. tubingensis, Emericella echinulata) being identified among the 57 isolates. Thirty-nine isolates of Aspergillus were recovered from beach sand and poultry farms, 31 from swine farms, and 80 from hospital environments, for a total 189 isolates. Eleven species complexes were found in these 189 isolates, and those belonging to the Versicolores species complex were found most frequently (23.8%). There was a significant association between the different environmental sources and distribution of the species complexes; the hospital environment had greater variability of species complexes than other environmental locations. A high prevalence of cryptic species within the Circumdati complex was detected in several environments; from the isolates analyzed, at least four cryptic species were identified, most of them growing at 37ºC. Because Aspergillus species complexes have different susceptibilities to antifungals, knowing the species-complex epidemiology for each setting, as well as the identification of cryptic species among the collected clinical isolates, is important. This may allow preventive and corrective measures to be taken, which may result in decreased exposure to those organisms and a better prognosis.

Assessment of Workers’ Exposure to Aflatoxin B1 in a Portuguese Waste Industry

Aflatoxin B1 (AFB1) is considered by different International Agencies as a genotoxic and potent hepatocarcinogen. However, despite the fact that the fungi producing this compound are detected in some work environments, AFB1 is rarely monitored in occupational settings. The aim of the present investigation was to assess exposure to AFB1 of workers from one Portuguese waste company located in the outskirt of Lisbon. Occupational exposure assessment to AFB1 was done with a biomarker of internal dose that measures AFB1 in the serum by enzyme-linked immunosorbent assay. Forty-one workers from the waste company were enrolled in this study (26 from sorting; 9 from composting; 6 from incineration). A control group (n = 30) was also considered in order to know the AFB1 background levels for the Portuguese population. All the workers showed detectable levels of AFB1 with values ranging from 2.5ng ml(-1) to 25.9ng ml(-1) with a median value of 9.9±5.4ng ml(-1). All of the controls showed values below the methods detection limit. Results obtained showed much higher (8-fold higher) values when compared with other Portuguese settings already studied, such as poultry and swine production. Besides this mycotoxin, other mycotoxins are probably present in this occupational setting and this aspect should be taken into consideration for the risk assessment process due to possible synergistic reactions. The data obtained suggests that exposure to AFB1 occurs in a waste management setting and claims attention for the need of appliance of preventive and protective safety measures.

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.

Molecular epidemiology of Aspergillus collected from cystic fibrosis patients

BACKGROUND Aspergillus respiratory infection is a common complication in cystic fibrosis (CF) and is associated with loss of pulmonary function and allergic disease. METHODS Fifty-three Aspergillus isolates recovered from CF patients were identified to species by Internal Transcribed Spacer Region (ITS), β-tubulin, and calmodulin sequencing. RESULTS Three species complexes (Terrei, Nigri, and Fumigati) were found. Identification to species level gave a single Aspergillus terreus sensu stricto, one Aspergillus niger sensu stricto and 51 Aspergillus fumigatus sensu stricto isolates. No cryptic species were found. CONCLUSIONS To our knowledge, this is the first prospective study of Aspergillus species in CF using molecular methods. The paucity of non-A. fumigatus and of cryptic species of A. fumigatus suggests a special association of A. fumigatus sensu stricto with CF airways, indicating it likely displays unique characteristics making it suitable for chronic residence in that milieu. These findings could refine an epidemiologic and therapeutic approach geared to this pathogen.