Recurrence of Stachybotrys chartarum during mycological and toxicological study of bioaerosols collected in a dairy cattle shed Caroline Lanier, Veronique Andre, Virginie Seguin, Natacha Heutte, Anne El Kaddoumi, Valérie Bouchart, Rachel Picquet, David Garon To cite this version: Caroline Lanier, Veronique Andre, Virginie Seguin, Natacha Heutte, Anne El Kaddoumi, et al.. Recur- rence of Stachybotrys chartarum during mycological and toxicological study of bioaerosols collected in a dairy cattle shed. Annals of Agricultural and Environmental Medicine (AAEM), Institute of Agricultural Medicine in Lublin, 2012, 19 (1), pp.61-67. hal-02186772 HAL Id: hal-02186772 https://hal-normandie-univ.archives-ouvertes.fr/hal-02186772 Submitted on 13 Nov 2019 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Distributed under a Creative Commons Attribution - NonCommercial| 4.0 International License Annals of Agricultural and Environmental Medicine 2012, Vol 19, No 1, 61-67 ORIGINAL ARTICLE www.aaem.pl Recurrence of Stachybotrys chartarum during mycological and toxicological study of bioaerosols collected in a dairy cattle shed Caroline Lanier1, Véronique André1, Virginie Séguin1, Natacha Heutte1, Anne El Kaddoumi1, Valérie Bouchart2, Rachel Picquet2, David Garon1 1 Université de Caen Basse Normandie, Caen, France 2 Laboratoire Départemental Frank Duncombe, Conseil Général du Calvados, Saint Contest, France Lanier C, André V, Séguin V, Heutte N, Kaddoumi A, Bouchart V, Picquet R, Garon D. Recurrence of Stachybotrys chartarum during mycological and toxicological study of bioaerosols collected in a dairy cattle shed. Ann Agric Environ Med 2012; 19(1): 61-67. Abstract Agricultural occupations associated with animal breeding and the processing of animal materials in confinement systems could potentially lead to bioaerosol exposures. Moulds and mycotoxins could be constituents of bioaerosols and should be studied because of their possible involvement in respiratory diseases and cancers. In order to characterize the fungal contamination of the indoor air in a dairy barn, bioaerosols were collected during 20 days in a cattle farm located in Normandy (France). Mycobiota, mycotoxins and the mutagenicity of bioaerosols were studied. The toxigenic ability of Aspergillus flavus group and Aspergillus fumigatus isolates was also evaluated in vitro. The prevalent airborne moulds were from the following potentially toxigenic species: Aspergillus flavus group, Aspergillus fumigatus, Penicillium chrysogenum, Stachybotrys chartarum, and the allergenic species Ulocladium chartarum, Cladosporium cladosporioides. In comparison with harvesting, grain handling or broiler breeding, the concentrations of viable moulds were lower in the cattle shed. Seasonal variations in levels of several species were also observed. This study revealed that aflatoxins were detected in bioaerosols and, for the first time, showed that farmers are possibly exposed to Stachybotrys chartarum during routine barn work. Moreover, the finding of mutagenicity from bioaerosols needs further investigations on bioaerosol composition. Key words airborne moulds, cattle shed, dairy farm, mutagenicity, mycotoxins INTRODUCTION moulds in vegetal matrices, and could be potentially detected in bioaerosols because of their adsorption on spores and dust Normandy (France) is an agricultural area characterized particles [6, 7]. by bovine breeding for the production of milk and meat [1]. Exposure to bioaerosols in the occupational environment On these farms, cattle are kept on loose straw in sheds for is associated with a wide range of health effects including several months, and fed with corn silage and oilseed cakes. infectious diseases, acute toxic effects, allergies and cancer. Farmers could be exposed to dust generated during daily Various constituents could be aerosolized during activities tasks, such as cattle feeding (preparation and distribution of associated with animal breeding, including those derived from feed), animal care and mulching. This dust is composed of bioaerosols, such as fungi, bacteria, endotoxins, viruses and organic and inorganic particles, gases, chemicals, biological parasites. Exposure to them may result in respiratory diseases agents and other toxicants [2]. The concept of bioaerosols [8]. Respiratory symptoms and lung function impairments: can be applied to airborne agricultural dust. Bioaerosols are asthma, farmer’s lung disease (FLD) or rhinitis, are probably usually defined as aerosols or particulate matter of organic the most widely studied among organic dust associated health origin [3]. These organic particles include pollens, plant effects [9, 10]. Among bioaerosols constituents, moulds and fragments, bedding materials, manure and microorganisms. mycotoxins could be responsible for several diseases. Farmers The microbial part may consist in bacteria, endotoxins, exposed to mould dust may exhibit signs of alveolitis [11], moulds and mycotoxins. and severe toxic irritative reactions can occur after a single The agricultural environment is an important source of inhalation of high levels of spores [12]. Studies have suggested exposure to airborne fungi. Moulds belonging to Aspergillus, that inhalation exposure to mould spores is another cause Penicillium, Cladosporium and Alternaria genera were of organic dust toxic syndrome [13]. Larsson et al. [11] have previously identified from bioaerosols generated during grain also shown that asymptomatic dairy farmers exposed to unloading and handling [4], and can also be associated with airborne mould dust may have signs of immunostimulation the handling of feedstuffs, e.g. corn silages and oilseed cakes and inflammation in their alveolar space. [5]. Mycotoxins are toxic fungal metabolites produced by Exposure to mycotoxins like aflatoxins and ochratoxin can also occur by inhalation during peanut or livestock Address for correspondence: David Garon, Université de Caen Basse-Normandie, feed processing and grain handling [6, 14]. Mycotoxins Centre François Baclesse, avenue Général Harris, BP 5026, 14076 Caen Cedex 05, France. represent the only clearly established non-viral biological E-mail: [email protected] occupational carcinogens. Several cases of pulmonary Received: 10 April 2011; accepted: 04 January 2012 cancers were reported in workers exposed to aflatoxins via 62 Annals of Agricultural and Environmental Medicine 2012, Vol 19, No 1 Caroline Lanier, Véronique André, Virginie Séguin, Natacha Heutte, Anne El Kaddoumi, Valérie Bouchart, Rachel Picquet, David Garon. Recurrence of Stachybotrys… the respiratory route [15, 16]. In Denmark, an increase in the Tween 80 (0.05%, w/v) (Sigma-Aldrich, St. Louis, MO, USA). risk of liver cancer has been reported for workers exposed After 30 min of magnetic shaking at 420 rpm, 3 dilutions to aflatoxins in concerns processing livestock feed [17]. of the suspension (10-1, 10-2 and 10-3) were made. 1 mL of Although various toxic effects of mycotoxins (cytotoxicity, each dilution (triplicate) was deposited in a Petri dish (90 neurotoxicity, immunosuppressive effects), as well as the mm diameter), and the culture medium constituted by malt carcinogenicity of mycotoxins standards have been widely extract (1.5%)/agar (1.5%) medium (MEA) complemented with studied [18, 19], little is known about occupational airborne chloramphenicol (0.05 %, w/v) was poured over it, following exposures to mycotoxins and the genotoxicity of bioaerosols the dilution plates method. The plates were incubated at 25°C generated during farming activities. and 30°C and viable moulds were counted each day during the Because the evaluation of fungal contamination and 7 days of incubation. The fungal concentration was expressed genotoxicity of the ambient air in dairy shed has rarely been as colony-forming units per cubic meter of air (cfu/m3). The investigated, the aims of this study were: 1) to characterize quantification limit corresponded to the minimum number fungal contamination (moulds and mycotoxins) of bioaerosols of colonies countable from the filter (80 colonies). Each colony collected in a dairy shed during 20 days, 2) to study the was isolated and purified on MEA. Aspergilli and Penicillia toxigenic abilities of Aspergillus fumigatus and Aspergillus were identified after culture on Czapek yeast autolysate agar flavus group isolates, 3) to evaluate the mutagenic proprieties (CYA) and 25% glycerol nitrate agar (G25N) [20], and Fusaria of bioaerosols samples using the Ames test. on potato dextrose agar medium (PDA) [21]. The identity of each colony was checked through macro- and microscopic examinations [20, 21, 22, 23, 24, 25, 26]. MATERIALS AND METHODS Isolates of Aspergillus fumigatus and Aspergillus flavus group were preserved on agar slants (MEA) at 4°C before Chemicals and reagents. The 17 mycotoxin standards their multi-mycotoxin screening by HPLC-MS/MS. (aflatoxins 1B , B2, G1, G2, M1, citrinin, diacetoxyscirpenol, fumagillin, gliotoxin, HT-2 toxin, mycophenolic acid, Toxigenic ability of Aspergillus flavus and