Use of gas chromatography-mass spectrometry versus the limulus amebocyte lysate test for the determination of airborne endotoxin in confined swine buildings

Airborne endotoxin (lipopolysaccharide, LPS) in filter samples collected in three different swine confinement buildings was determined by using the Limulus amebocyte lysate test and by applying gas chromatography-mass spectrometry (GC-MS) to analyze 3-hydroxy fatty acids (3-OH-FAs). The amounts of LPS as shown by GC-MS were 30–50 times larger than the amounts detected by the Limulus test. GC-MS revealed that 21% of the LPS collected on cellulose acetate filters and 26% on polycarbonate filters remained on the filters after buffer extraction. Better correlation with the Limulus test and the 3-OH-FA measurements was achieved when considering the sum of 3-OH C12:0 and 3-OH C14:0 rather than the sum of all of the detected 3-OH-FAs (i.e. those with 12–18-carbon chains), indicating that the bioactivity of the LPS was dependent upon the relative distribution of the 3-OH-FAs. Linear regression analysis between air concentrations of dust and endotoxin/LPS gaveR ²values that varied from 0.407 to 0.739. The air concentrations of LPS were lower in the uninsulated swine house (a green house with an alternative housing system) than in the two insulated buildings (conventional housing systems; one designed as a climate chamber, i.e. climatic parameters could be controlled), whereas the opposite was found for the concentrations of LPS in airborne dust from the three buildings. The numbers of viable bacteria and fungi were highest in the uninsulated swine house. Moreover the relative distribution of 3-OH-FAs in that building differed from the distribution in the insulated buildings, which reflects differences in the microflora, probably mainly due to differences between the housing systems and the design of the buildings.     

Comparison of bioaerosol sampling methods in barns housing swine.

The air in livestock buildings contains bioaerosol levels that are sufficiently high to cause adverse health effects in animals and workers. These bioaerosols are complex mixtures of live and dead microorganisms and their products as well as other aeroallergens. The effectiveness of sampling methods used for quantifying the very high concentrations of microorganisms in these environments has not been well studied. To facilitate an accurate assessment of respiratory hazards from viable organisms in agricultural environments, three bioaerosol sampling methods were investigated: the Andersen microbial sampler method (AMS), the all-glass impinger method (AGI), and the Nuclepore filtration-elution method (NFE). These methods were studied in a parallel fashion in 24 swine confinement buildings. Measurements were taken in two seasons with three types of culture media in duplicate to assess total bacteria, gram-negative enteric bacteria, and total fungi. Methods were analyzed for the proportion of samples yielding data within the limits of detection, intraclass reliability, and correlation between methods. For sampling viable bacteria, the AMS had a poor data yield because of overloading and demonstrated weak correlation with the AGI. Conversely, the AGI and NFE gave sufficient numbers of valid data points (90%), yielded high intraclass reliabilities (alpha greater than or equal to 0.92), and were highly correlated with each other (r = 0.86). The AGI and the NFE were suitable methods for assessing bacteria in this environment, but the AMS was not. The AMS was the only method that consistently recovered enteric bacteria (73% data yield). For sampling fungi, the AGI and AMS both yielded sufficient data and all three methods demonstrated high intraclass reliability.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification and seasonal distribution of airborne fungi in three horse stables in Italy

Fungal agents are responsible for a variety of respiratory diseases both in humans and animals. The nature and seasonal variations of fungi have been investigated in many environments with wide ranging results. The aims of the present report were (i) to evaluate the quality and magnitude of exposure to airborne fungi in three differently structured equine stalls (open air, partially and completely enclosed buildings) during a one-year period, using an air sampling technique and (ii) to compare the distribution and frequency of fungal species, with regards to these different environments. Air samples were collected monthly from December 2001 to November 2002 by means of a surface air sampler (SAS) Super-90, (PBI International, Milan, Italy). Penicillium and Aspergillus spp. were cultured from all the stables in all seasons. Mucoraceae were also recovered in all seasons in stalls 1 and 2, while they were not isolated in spring and fall in stall 3. These fungi were detected in 28.4%, 72.9% and 60.5% of the total number of samples, respectively. Other fungal genera such as Alternaria, Cladosporium, Fusarium, Beauveria and Drechslera were also occasionally recovered.Viable fungal concentrations varied greatly, ranging from below the limit of detection to more than 3000 CFU/m³ for stables 1 and 2, and 1750 CFU/m³ for stable 3. The median fungal concentration was approximately 178 CFU/m³. Total fungal concentration appeared to be highest in summer, winter and spring, and lowest in the fall.     

Isolation and identification of Rhizoctonia-like fungi from roots of three orchid genera, Paphiopedilum, Dendrobium, and Cymbidium, collected in Chiang Rai and Chiang Mai provinces of Thailand

Three orchid genera, Paphiopedilum, Cymbidium, and Dendrobium, are among the most heavily traded ornamental plants in Thailand. In this study, 27 isolates of Rhizoctonia-like fungi were isolated from root sections of mature orchids in the three orchid genera, collected from diverse horticultural settings in Chiang Mai and Chiang Rai provinces of Thailand. Fungal identification was done by the morphological characterization, the comparison of the internal transcribed spacer and 5.8S ribosomal DNA sequences, and the phylogenetic analysis. Epulorhiza repens was found to be the most common species found in the roots of various species of all three orchid genera, whereas Epulorhiza calendulina-like isolates were strictly found in the roots of Paphiopedilum species. We have also isolated and described an anamorph of Tulasnella irregularis, four new anamorphic species in the genus Tulasnella, and a new anamorphic species in the family Tulasnellaceae. Our study provides information on diversity of root-associated fungi of the orchid genera and at the sampling sites that were rarely addressed in the previous studies.     

Mould biodiversity in homes I. Air and surface analysis of 130 dwellings

Summary Air and surface sampling to quantify and identify fungi were conducted over a 10-year period in 130 Belgian homes, including flats and houses. Homes were selected based on the medical files of allergic, mostly asthmatic patients to assess possible mould responsibility in the aetiology of the disease. Air sampling was done with Reuter centrifugal air sampler using different sampling times and incubation temperatures to detect mesophilic and thermotolerant or thermophilic fungi. More than 50 genera were detected, withCladosporium, Penicillium andAspergillus being the most common. The main species detected wereAspergillus versicolor, several species ofPenicillium, Cladosporium sphaerospermum andCladosporium herbarum. Surface sampling was conducted concomitant with air sampling. Surfaces with and without visible mould growth were tested equally by Rodac contact plates or swabs. AlthoughCladosporium herbarum was shown to be a common species in indoor environments by air sampling, it was not detected by surface sampling from visible mould growth. This finding suggests that its presence in dwellings is from an outdoor origin and not indicative of indoor proliferation.Aspergillus versicolor, Cladosporium sphaerospermun, andPenicillium chrysogenum were the most common species isolated from surfaces showing visible mould growth.     

Contribution of leaf surface fungi to the air spora

High concentrations of airborne fungal spores frequently occur from spring through fall in temperate areas of the world. Although it is generally assumed that fungi on leaf surfaces are contributors to the air spora, little data are available comparing the types of fungi found on leaf surfaces with those in the atmosphere. Air sampling was carried out with a Burkard Spore Trap located on the roof of a building on the University of Tulsa campus using standard methods. Leaf samples were aseptically collected from Ulmus americana and Quercus palustris trees on campus, placed in sterile plastic bags, and brought to the lab. For each leaf, 4 cm² areas of both upper and lower leaf surfaces were swabbed and plated on malt extract agar with streptomycin. Cultures were incubated at room temperature for 5–7 days and then examined microscopically. Results were expressed as colony forming units (CFU)/cm². Twenty-one fungal taxa were identified from the air samples. The most abundant taxa were Cladosporium, ascospores, basidiospores, and Alternaria; together these four spore types comprised over 90% of the yearly total. Yeasts were the most abundant fungi isolated from both leaf types. Among the mycelial fungi were Phoma species, followed by Cladosporium and Alternaria. Overall twenty genera of filamentous fungi were identified. Yeasts and Phoma are normally splash dispersed and were not identified in the Burkard air samples. However, 10 taxa isolated from leaf surfaces were registered in air samples. Crude estimates of the leaf surface area of each tree suggest that the total fungal load was approximately 5.04×10⁸ CFU for Ulmus and 2.71×10⁸ CFU for Quercus. Of these levels, 19% were from fungi also detected in air samples. The data suggest that some leaf-surface fungi are major contributors to the air spora.     

Aerobiological investigations inside repositories of the National Archive of the Republic of Cuba

The objectives of this study were to evaluate the microbial prevalence inside six repositories of the National Archive of the Republic of Cuba in 2?months of the year and to examine some of the physiological features of fungi isolated in order to evaluate their potential for biodeterioration. The microbiological sampling was conducted in February and September using a slit impactor as air sampler. Appropriate selective culture media were used to isolate fungi and bacteria. Temperature and relative humidity were measured during the samplings. The cellulolytic activity and the production of acids and pigments of the fungi isolated were qualitatively determined. Total viable microbiota and bacteria concentrations were greater in February while the fungal concentration was higher in September. Aspergillus, Cladosporium, Penicillium, Curvularia and Alternaria were the predominant fungal genera in February while Cladosporium prevailed in September, although Fusarium, Mucor and Neurospora genera were also isolated in this month. The fungi isolated were capable of degrading cellulose and excreting pigments and acids. The Gram-positive bacteria group prevailed in the air and Corynebacterium, Streptomyces, Bacillus, Streptococcus, Staphylococcus, Enterobacter and Serratia were some of the genera identified.     

Caracterización de la micobiota aérea en dos depósitos del Archivo Nacional de la República de Cuba

BackgroundThe high relative humidity and temperatures in tropical countries create favorable conditions for development of fungi that are not only a risk to human health but they can also colonize documentary support.AimsTo study the concentration of the airborne fungi in two repositories of the National Archives of the Republic of Cuba, the mycobiota deposited on different photographic supports and maps preserved in these repositories, and to determine the taxonomic characterization of the fungi isolated.MethodsThe air sampling was performed using a sedimentation method, and the supports (6 pictures and 7 maps) were analyzed using moistened sterile swabs.ResultsThe Cladosporium genus was predominant, followed by Aspergillus and Penicillium genera. Filamentous fungi were isolated in all the photos and maps, and yeasts were only isolated from a photographic supports and a map. We identified several species of Aspergillus and Penicillium genera, but Aspergillus niger and Aspergillus flavus predominated. Candida and Rhodotorula were the yeast genera isolated.ConclusionsThe fungal concentration of the air demonstrated that the environments were not contaminated. From the 26 species of filamentous fungi isolated only 5 were detected in the indoor air of the repositories and on one or more of the document supports analyzed (representing a 19.3%). This shows that there is a low ecological relationship between the fungi detected in the indoor air and those that were isolated from the supports studied.     

小蓬竹根际土壤微生物及内生真菌多样性分析

为了解喀斯特典型物种-小蓬竹根际土壤微生物及不同部位内生真菌多样性,采用沿等高线等距离取样法采集小蓬竹根际土壤及健康植株,通过可培养对根际土微生物及内生菌进行分离,利用分子技术对其进行鉴定,根据鉴定结果构建系统发育树,并计算小蓬竹根际土壤微生物和根茎叶内生真菌多样性。结果如下：（1）共从根际土壤、根、茎、叶分离得到139个真菌菌株,隶属于27属,其中根际土壤分离得到34个真菌菌株隶属于12属,根部分离得到的63个内生真菌菌株隶属于17个属,茎部分离得到的14个内生真菌菌株隶属于8个属,叶部分离得到28个内生真菌菌株隶属于9个属;（2）根际土壤共分离得到41株细菌菌株,隶属于7个属26个种,20株放线菌菌株,隶属于1属15种;从Shannon-Wiener多样性指数、均匀度指数、Simpson指数排序来看,真菌主要表现为根 > 根际土壤 > 茎 > 叶,细菌和放线菌多样性均较低。（3）按层次聚类分析可分别将真菌、细菌、放线菌聚为3支。小蓬竹根际土壤、根、茎和叶具有丰富的微生物多样性,不同部位菌群组成存在差异性（P<0.05）,且存在以假单胞菌属、芽孢杆菌属等为优势属的抗盐耐旱菌群,这有助于揭示小蓬竹对喀斯特生境的适应性,以及为微生物-植物群落之间相互关系提供一定基础数据,为后期寻找小蓬竹相关耐性功能菌奠定基础。     

The air spora of an orchid greenhouse

The orchid collection of the ELTE Botanical Garden, Budapest, Hungary was monitored for airborne fungi using viable and non-viable air-sampling methods (Hirst-type and a 3-stage Andersen sampler) with three different culture media. A new culture method was also applied to identify fungal spores from Hirst-type samples. The aim of this study was to determine the diversity, human- and phytopathological potential of the air spora. To find out sources of airborne fungi, samples were collected from the air in an adjacent greenhouse and outdoors, and from necrotic plants. A total of 58 genera were found in the air samples. Cladosporium and Penicillium spp. were common members of the airborne biota. A high proportion (27.5%) of identified genera may be presented as a member of microbial consortium associated with the orchids. Airborne fungi potentially pathogenic to humans were also detected. One species, Zygosporium masonii, was new to Hungary. Statistical analysis indicated that conditions of sampling had significant effects. The principal component analysis elucidated the three principal components representing 75.34% of the total variance; the clusters of variables were related to the three types of culture media. Relative abundance of small-sized spores was high, presumably because of the fungal species composition and accelerated sedimentation of large spores in still air. Apparently, in the studied orchid greenhouse, a specific mycobiota developed due to the climate and hosts (Orchideaceae) grown there.     

Keratinophilic fungi of poultry farm and feather dumping soil in Tamil Nadu,India

Soils of 10 poultry farms from Namakkal and 12 feather dumping sites from Chennai were studied for the presence of keratinophilic fungi. A total of 34 species belonging to 19 genera and one non-sporulating fungus were recovered. Sixteen species of fungi and one non-sporulating fungi were common to both sites, eight species were specific to Namakkal and nine species were specific to Chennai. Dermatophytes and closely related fungi were represented by six species belonging to five genera. Fungal species commonly found in the soil samples included Chrysosporium keratinophilum (73%), Trichophyton mentagrophytes (68.2%), Microsporum gypseum (64%), Myceliopthora vellerea (32%), Chrysosporium state of Arthroderma tuberculatum (27.3%) and Geomyces pannorum (23%). Non-dermatophyte fungi were represented by 28 species belonging to 14 genera and one non-sporulating fungus.     

Detection and identification of moulds in dutch houses and non-industrial working environments

The mycoflora of indoor non-industrial environments is reported from “case” studies in The Netherlands. Both air sampling by a RCS-Reutcr centrifugal air sampler and surface sampling by swabs and cellotape preparations were carried out in homes, archives and libraries, musea, offices and schools. Common species encountered in these indoor environments are Aspergillus versicolor, Penicillium brevicompactum, P. chrysogenum, Cladosporium spp. and the xerophilic fungi Eurotium spp. and Wallemia sebi. Aspergillus fumigatus, Scopulariopsis spp. and Stachybotrys chartarum were occasionally isolated. It is not always possible to detect the mycoflora growing on surfaces by air sampling. Therefore direct microscopical examination and sampling from surfaces in addition to air sampling is strongly recommended for the detection of viable moulds in indoor environments. Selection of the most suitable media for isolation of fungi is discussed.     

Studies on the fungal flora in the lung of human necropsy cases. A critical survey in connection with the pathogenesis of opportunistic fungus infections

An ecological study was carried out on the fungal flora in the human lung of 159 autopsy cases. Fungi were isolated from 129 cases (81.1%). Filamentous fungi consisting of 918 strains were isolated from 113 cases, and the dominant genera were Aspergillus and Penicillium. Yeasts were isolated from 58 cases, and the dominant genus appeared to be Candida.With morbid anatomical study, the incidence of the fungus isolation was examined from various points of view. It was concluded that even a healthy respiratory parenchyma of the human lung cannot be assumed as aseptic. However, only a few numbers of a few genera of air-born fungi were isolated.Special stress was laid on the exposure of the respiratory parenchyma of the human lung to air-born fungi in connection with the pathogenesis of opportunistic fungus infections.     

Isolation and characterization of different promising fungi for biological waste management of polyurethanes

As a highly resistant polymer family, polyurethanes (PU) are responsible for increasing environmental issues. Then, PU biodegradation is a challenging way to develop sustainable waste management processes based on biological recycling. Since the metabolic diversity of fungi is a major asset for polymer degradation, nearly thirty strains were isolated from sampling on six different PU wastes-containing environments. A screening of the fungi on four thermoplastic PU (TPU) with different macromolecular architectures led to the selection of three strains able to use two polyester PU as sole carbon source: Alternaria sp., Penicillium section Lanata-Divaricata and Aspergillus section flavi. Weight loss, FT-IR, Scanning Electron Microscopy and Size Exclusion Chromatography analyses revealed that these three fungi degrade slightly and similarly a fatty acid dimer-based TPU while variability of degradation was noticed on a polycaprolactone-based TPU. On this last TPU, robust analysis of the degraded polymers showed that the Penicillium strain was the best degrading microorganism. Membrane enzymes seemed to be involved in this degradation. It is the first time that a strain of Penicillium of the section Lanata-Divaricata displaying PU biodegradation ability is isolated. These newly discovered fungi are promising for the development of polyester PU waste management process.     

Transmission identification of Escherichia coli aerosol in chicken houses to their environments using ERIC-PCR

In order to study E. coli aerosol spreading from chicken houses to their surrounding air, air samples, including indoor and outdoor air (upwind 10 and 50 m as well as downwind 10, 50, 100, 200 and 400 m away) of 5 chicken houses were collected using six-stage Andersen microbial samplers and Reuter-Centrifugal samplers (RCS). E. coli concentrations (CFU/m3 air) collected from different sampling sites were calculated. E. coli strains from chicken feces samples were also isolated. Furthermore, the enterobacterial repetitive intergenic consensus (ERIC)-PCR method was applied to amplify the isolated E. coli strain DNA samples. Through the genetic similarity analyses of the E. coli obtained from different sampling sites, the spreading of bioaerosol from animal houses to the ambient air was characterized. The results showed that the isolated E. coli concentrations in indoor air (9―63 CFU/m3) in 5 chicken houses were higher than those in upwind and downwind air, but there were no significant differences between the indoor and downwind sites 10 m away from all the 5 houses (P>0.05). The phylogenetic tree indicated that a part of the E. coli (34.1%) isolated from indoor air had 100% similarity with those isolated from feces, and that most of E. coli isolated (54.5%) from downwind at 10, 50, 100 or even 200 m had 100% similarity with those isolated from indoor air or feces too. But those isolated from upwind air had a lower similarity (73%―92%) with corresponding strains isolated from indoor air or feces. Our results suggested that some strains isolated from downwind air and indoor air originated in the chicken feces, but most of isolates obtained from upwind air samples did not come from the chicken feces or indoor air. Effective hygienic measures should be taken in animal farms to prevent or minimize downwind spreading of microorganism aerosol.     

The mycoflora of peanuts in Egypt

Twenty-six genera and Sixty-six species, in addition to two varieties of Aspergillus nidulans and one variety of Humicola grisea were isolated from 80 peanut seed (uncovered or covered within shell at the time of sampling) samples and 40 samples of shells collected from different places in Egypt.At 28 °C, the broadest spectrum of genera and species was recorded in the shells (21 genera and 53 species), followed by the naked seeds (16 genera and 38 species) and the covered seeds (17 genera and 33 species). Aspergillus and Penicillium were regularly the most common genera in the three substrates. A. niger, A. fumigatus, A. flavus A. terreus, A. ochraceus and P. funiculosum were generally the most common fungal species recovered from the three substrates. A. flavus was represented in 80%, 60% and 80% of the samples constituting 16.1%, 8.4% and 27.2% of the total count of fungi of the shells, covered and uncovered seeds respectively.At 45 °C, nine genera and sixteen species were collected of which Aspergillus fumigatus was extremely dominant in the three substrates, followed by A. terreus and A. niger. Penicillium was completely eliminated.     

Concentration and distribution characteristics of airborne fungi in indoor and outdoor air of Tehran subway stations

The concentration and distribution characteristics of airborne fungi were investigated in indoor and outdoor air of two metro stations (Imam Khomeini and Sadeghiyeh stations) in Tehran subway. Samples were taken from indoor air at each station from platform and ticket office area also from adjacent outdoor air of each station. Indoor sampling was conducted for two types of trains, old and new. The concentration of airborne fungi ranged from 21 CFU/m³ at the outdoor air of Imam Khomeini station to 1,402 CFU/m³ in the air samples collected from the platform of this station. Results showed that airborne fungi concentrations at indoor air were higher than the outdoor air (p < 0.05), and fungal levels significantly correlated with the number of passengers (p < 0.05; r = 0.68) and RH % (p < 0.05; r = 0.43). Sixteen genera of fungi were isolated in all sampled environments. The predominant genera identified in indoor and outdoor air were Penicillium spp. (34.88 % of total airborne fungi) and Alternaria spp. (29.33 % of total airborne fungi), respectively. The results of this study showed that the indoor air quality in subway is worse than the outdoor air.     

Study of airborne fungus spores by viable and non-viable methods in Havana,Cuba

The Havana aeromycobiota diversity was studied from November 2010 to October 2011 using two complementary volumetric methods. A total of 35 fungal genera were characterised, 26 of them were recognised only by non-viable methods, six with viable methodology and the other three with both sampling methods. Furthermore, 47 species were identified by cultivation and the spores collected with the non-viable methodology. These could not be included in a specific genus, and thus, were categorised into five fungal types. In general, the main, spread worldwide, mitosporic fungi also predominated the Havana atmosphere. The predominant species were Cladosporium cladosporioides, Aspergillus flavus and Penicillium citrinum. Moreover, several Zygomycetes (Syncephalastrum racemosum, Rhizopus stolonifer and Rhizopus oryzae), Ascomycetes (Chaetomium globosum) and Basidiomycetes such as Coprinus or Ganoderma were isolated. In the present paper, the review of the airborne fungi conducted in previous studies in Cuba was completed by the detection of two new genera and the first isolation of ten new records in the Cuban atmosphere. Most of the fungi detected showed a diurnal pattern with high spore peak at 11.00–12.00.     

Biodeterioration of Mayan buildings at uxmal and tulum,Mexico

Uxmal and Tulum are two important Mayan sites in the Yucatan peninsula. The buildings are mainly composed of limestone and grey/black discoloration is seen on exposed walls and copious greenish biofilms on inner walls. The principal microorganisms detected on interior walls at both Uxmal and Tulum were cyanobacteria; heterotrophic bacteria and filamentous fungi were also present. A dark‐pigmented mitosporic fungus and Bacillus cereus, both isolated from Uxmal, were shown to be acidogenic in laboratory cultures. Cyanobacteria belonging to rock‐degrading genera Synechocystis and Gloeocapsa were identified at both sites. Surface analysis previously showed that calcium ions were present in the biofilms on buildings at Uxmal and Tulum, suggesting the deposition of biosolubilized stone. Apart from their potential to degrade the substrate, the coccoid cyanobacteria supply organic nutrients for bacteria and fungi, which can produce organic acids, further increasing stone degradation.     

A procedure for evaluation of the indoor environment

Exposure to substances found in the indoor environment is known totrigger respiratory illnesses such as asthma. Despite this knowledge,information on how to identify and remove these exposures is scarce. Toaddress this issue, we describe a program of environmental assessmentdeveloped at Children's Mercy Hospital in Kansas City, Missouri. Weevaluated buildings using a standardized assessment protocol. Thisprotocol included a combination of background information, visualinspection, volumetric air sampling, bulk sampling of suspicious areas,antigen measurement of housedust, and general measurements of the indoorclimate. We inspected a total of 63 buildings including 49 single unitstructures, 4 apartments, 4 town homes, 3 schools, 2 office buildings,and 1 mobile home. Most of these locations were identified because ofrespiratory ailments reported by the occupants. A screeningquestionnaire revealed factors that are known to be associated withfungal contamination such as leaks or flooding in 78% of thebuildings. Visual inspection confirmed these sources of contamination inthe majority of structures. Volumetric air sampling identified a highproportion of buildings with airborneAspergillus/Penicillium species. Stachybotrys wasfound in nearly 30% of these structures both in air and onsurface samples, and surprisingly, Stachybotrys spores werefound only in air in nearly 20%. Climactic factors were onlyweakly associated with fungal contamination. The homes of patients withrespiratory diseases often have significant fungal contamination thatcan be identified with a questionnaire. Using this standardizedapproach, factors associated with respiratory illnesses, usually fungalcontamination, frequently can be identified. Whether removal of theoffending exposure will lead to improve health remains to bedetermined.