Differentiation of allergenic fungal spores by image analysis, with application to aerobiological counts

The ability of an image analysis routine to differentiate between spores of eleven allergenic fungal genera was tested using analysis based on seven basic and up to 17 more complex features, extracted from digitised images. Fungal spores of Alternaria, Cladosporium, Fusarium, Aspergillus, Penicillium, Botrytis, Epicoccum, Exserohilum, Ustilago, Coprinus and Psilocybe were examined in a series of experiments designed to differentiate between spores at the genus and species level. Linear and Quadratic Discriminant Analysis of feature measurements, recorded for 100 to 1600 spores per taxon, differentiated between genera and species with a high level of accuracy. Genus comparisons using only seven basic features resulted in 98% accuracy for the recognition of conidia belonging to Cladosporium, Fusarium and Epicoccum. Differentiation between conidia of Aspergillus and Penicillium was the least reliable, with 56% of Aspergillus conidia correctly identified and 41% misidentified as Penicillium. At the species level, conidia of Cladosporium macrocarpum, Fusarium moniliforme (microconidia), F. oxysporum (microconidia), F. solani (macroconidia), Alternaria helianthi and A. brassicae were consistently identified with 86--100% accuracy. Reduced levels of accuracy in the identification of spores by image analysis reflected similarities between species in their spore morphology. The application of image analysis to aerobiological counting methods is discussed in relation to the results obtained.     

Airborne fungal spores in the coastal plain of Israel: A preliminary survey

Airborne spores were monitored during the years 1993–1995 in three cities along the coastal plain of Israel: Ramat Gan, Tel Aviv (Ramat Aviv) and Haifa. Seasonal fluctuations in the concentration of airborne spores were recorded. The following genera of fungi were identified:Alternaria, Cladosporium, Coprinus, Curvularia, Drechslera. Diplococcum, Epicoccum, Fusarium, Leptosphaeria, Pithomyces, Puccinia, Sphacelotheca, Stemphylium andUstilago. Unidentified spores were very rare and in negligible numbers. The dominant airborne fungal spores wereCladosporium andAlternaria. The monthly variations in airborne spores, observed among the three cities, seem to be rather minor. The recorded levels of airborne spores were below the concentrations that are accepted as threshold levels for provocation of clinical responses.     

Airborne fungal spores in the Campus of Anchieta (La Laguna,Tenerife/Canary Is.)

A quantitative and qualitative study was carried out of the atmospheric fungal propagules in two university buildings in the Campus of Anchieta (La Laguna, Tenerife, Canary Islands, Spain) and in the outdoor atmosphere, in order to ascertain the most abundant fungi and the concentration of spores in the air, as possible sources of respiratory allergies caused by this type of aeroallergens. Sampling was carried out using two kinds of traps (Burkard Personal Culture Sampler and Burkard Personal Slide Sampler), as well as adhesive tape. Sample 1, indoor, identified between 650-320 CFU m -3 depending on the culture medium, with outdoor values between 670-440 CFU m -3 ; sample 2, recorded between 630-80 CFU m -3 inside and 580-300 CFUm -3 in the outdoor atmosphere. The most represented genera were Cladosporium and Penicillium , although the following were also identified: Agaricales, Alternaria, Aspergillus, Botrytis, Drechslera, Coprinus, Curvularia, Chaetomium, Exserohilum, Fusarium, Helminthosporium, Leptosphaeria , Myxomycetes, Paecilomyces, Pleospora, Stemphylium, Tetraploa, Torula and Ulocladium .     

Analysis of the predicting variables for daily and weekly fluctuations of two airborne fungal spores: <Emphasis Type="Italic">Alternaria</Emphasis> and <Emphasis Type="Italic">Cladosporium</Emphasis>

Alternaria and Cladosporium are two fungal taxa whose spores (conidia) are included frequently in aerobiological studies of outdoor environments. Both spore types are present in the atmosphere of Malaga (Spain) throughout almost the entire year, although they reach their highest concentrations during spring and autumn. To establish predicting variables for daily and weekly fluctuations, Spearman's correlations and stepwise multiple regressions between spore concentrations (measured using a volumetric 7-day recorder) and meteorological variables were made with results obtained for both spore types in 1996 and 1997. Correlations and regressions were also made between the different taxa and their concentrations in different years. Significant and positive correlation coefficients were always obtained between spore concentrations of both taxa, followed by temperature, their concentrations in different years, sunshine hours and relative humidity (this last in a negative sense). For the two spore types we obtained higher correlation and regression coefficients using weekly data. We showed different regression models using weekly values. From the results and a practical point of view, it was concluded that weekly values of the atmospheric concentration of Alternaria spores can be predicted from the maximum temperature expected and its concentrations in the years sampled. As regards the atmospheric concentration of Cladoposrium spores, the weekly values can be predicted based on the concentration of Alternaria spores, thus saving the time and effort that would otherwise be employed in counting them by optical microscopy.     

Seasonality in Antarctic Airborne Fungal Spores

Airborne fungal spores were monitored over periods of up to 131/2 months at three sites on Signy Island in the maritime Antarctic. Fungal spore concentrations in the air were much lower than in other parts of the world. Concentrations were very low during the austral winter but increased during the austral summer. Chlamydospores were the most abundant fungal spore type found. Spores of Cladosporium spp. were the second most frequently trapped form. All spore types samples were most abundant in the summer months, except for chlamydospores, which were most numerous during the winter. The concentration of Cladosporium spores in the air at Signy Island was compared with the concentrations of this spore type found in the air in other parts of the world. It was evident that Cladosporium loses its dominance as the most abundant component of the air spora with increasingly high latitude. The peak concentration of fungal spores occurred at two sites following the start of the thaw; at the third site, the peak occurred with the arrival of spores by long-distance transport from more northerly regions.     

Micoflora atmosferica de la ciudad de Trujillo (Peru) III. — Generos aislados durante el año de 1971

There were 1762 fungi isolated from the atmosphere of Trujillo city (Peru) during the year 1971. These isolants belonged to 23 genera of fungi. The most frequently isolated were: Cladosporium, Penicillium, Fusarium, Stemphylium, Alternaria, Helminthosporium, Aspergillus and Tilachlidium. Nine genera are reported for first time from the air of the city (Tilachlidium, Cephalosporium, Mucor, Aureobasidium, Hormiscium, Trichoderma, Stachybotrys, Paecilomyces and Fisidium). Six genera may be alergenic for asthmatic patients of Trujillo city.     

Detection of fungal infectous agent of wheat grains in store-pits of Markazi province, Iran

Wheat is an economic and important crop that provides approximately 20% of food calorie in the world. It is first crop in Iran and cultivated in the most areas of this country. Store-pit fungi make undesirable changes in quality and appearance of wheat grains. Even, some fungi produce different mycotoxins which are toxic to human and livestock's that use wheat grains as source of food. In this study, several samples were randomly collected from each of five store-pits located in different areas of Markazi Province including: Arak, Mahallat, Khomein, Saveh and Sarband. Grains were treated on PDA, and blotter, agar and washing test also used for isolating and detection of fungi. At least 100 grains per each sample were randomly used for each test and treatment. The fungi that determined in this study were Cochliobolus australiensis, Cladosporium herbarum, Epicoccum sp., Tilletia leavis, Aspergillus flavus, A. niger, A. fumigatus, Alternaria alternata, Alternaria sp., Penicillium italicum, P. digitatum, Fusarium sp., Rhizopus sp., Ustilago tritici, Scytalidium sp. Among these fungi the most isolates were belonged to Cladosporium, Alternaria, Rhizopus and Fusarium. Cladosporium herbarum was the most common in different sampling areas. Tilletia laevis and Ustilago tritici were just recovered in washing test. This study revealed that different fungi are associated with wheat grains in store-pits in Markazi Province. Some of them like Aspergillus flavus normally produce aflatoxin, a very toxic and carcinogenic mycotoxin that is harmful for human.     

Airborne fungal spores in an industrial area: seasonal and diurnal periodicity

Qualitative and quantitative studies of atmospheric fungal spores at a chloralkali factory, Jayashree Chemicals. were made during 1993 employing culture plate and rotorod methods. A total of 57 sporulating fungal types, including three sterile mycelial forms, were recorded by the culture plate method and 51 spore types, including the hyphal fragments and unidentified spores, were recorded by the rotorod method. As to the seasonal variation, winter was found to be the greatest contributor of fungal spores as compared to the summer and rainy season. Instead, when considering the hour of the day, the peak number of fungal propagules was recorded at noon (12.00 h) followed by evening and morning values, an exception being recorded in winter months, when maximum CFUs ofCladosporium were monitored in the morning. The seasonal variation in fungal concentration and composition was found to be influenced by temperature, rainfall and relative humidity, whereas diurnal incidence was the effect of varying temperature and relative humidity during day time only. Moderate temperature and relative humidity favoured the maximum fungal spore load in the atmosphere.Cladosporium, Nigrospora, Alternaria, Lasiodiplodia, Drechslera, Pestalotia, Curvularia, Epicoccum, Aspergillus, Penicillium andChaetomium were the commonest fungal spores in the factory area.     

Airborne fungi in an Italian rice mill

Summary Studies employing volumetric spore trap (VSP) and gravity settling culture plates (GSC) were conducted in order to analyse the air spora of a rice mill at Pavia, Italy, from October-December 1988. Results revealed a variety of fungal spores belonging to different genera and including recognized rice pathogenic fungi. The most frequent genera by GSC method includedAcremonium, Alternaria, Aspergillus, Aureobasidium, Cladosporium, Epicoccum, Fusarium, Helminthosporium, Mucor, Nigrospora, Penicillium, Rhizopus, Trichoderma, Trichothecium, and some unidentified fungi. Environmental assessment of fungal spores by VSP revealed that the most prevalent fungi were:Alternaria, Cladosporium, Epicoccum, Helminthosporium, Nigrospora, Pyricularia, Tilletia and hyaline, dark and coloured types of ascospores and basidiospores. Airborne fungal spore concentrations were particularly high (5,000–6,000 spores/m³) in the rooms of the rice mill where the initial stages of rough rice transformation take place, and dropped to 2,500 spores/m³ in the last room, where workers are. During a temporary interruption of the working processes, air spora concentration dropped below 1,000 spores/m³.Cladosporium, Epicoccum andNigrospora spores were predominant in all subdivisions of the indoor environments of the rice mill.     

Aerobiological study of Alternaria and Cladosporium conidia in the atmosphere of Almeria (SE Spain)

This study analysed the seasonal and intradiurnal behaviour of fungal spores from Alternaria and Cladosporium on air samples collected in the city of Almeria (SE Spain), as well as the influence that meteorological parameters have on the concentration of these type of spores. Aerobiological sampling was made during four years (1998-2001) using a Hirst-type volumetric spore trap. Spores of Alternaria and Cladosporium were detected throughout the year, reaching the highest concentrations from May to October. The diurnal patterns of these taxons reflected a presence similar of spores during a 24 h period, with values horary close to 4% of total sampling daily. The correlations show a positive association with temperature, hours of sunshine and accumulate rainfall, but negative with daily rainfall.     

THE SUBEPIDERMAL FUNGI OF CEREAL GRAINS II. THE NATURE, IDENTITY AND ORIGIN OF THE MYCELIUM IN WHEAT

An internal fungal mycelium between the epidermis and cross-layer cells in normal wheat grains is shown to consist of extracellular septate hyphae forming a network on the inner surface of the epidermis, whose cells are occasionally penetrated. The mycelium, which is first observed at a comparatively late stage of maturation of the grain, usually remains as a loose network, although sclerotia and plate-like structures suggestive of drought forms are occasionally present. These appearances support the view, derived from comparison of the drying rates of grain under various climatic conditions, that the spread of the subepidermal mycelium is finally restricted by desiccation beneath the epidermis during the drying out of the ripening grain.
Cultures from surface-sterilized grains of Bersée wheat have shown that the most common subepidermal fungus is Alternaria tenuis (in 64.4% of the grains). Bacteria (28.8%), Mycogone sp. (?) (7.7%), Cladosporium herbarum (5.8%), Pullularia pullulans (4.8%), Fusarium sp. (1.9%), Botrytis cinerea (1.9%) and Stemphylium botryosum (0.9%) were also isolated.
The subepidermal mycelium apparently arises either from systemic infection of the wheat plant or from fungal spores and hyphae present on the outside of the developing grains and among the dead floral parts.     

The effect of meteorological factors on the daily variation of airborne fungal spores in Granada (southern Spain)

A study was made of the link between climatic factors and the daily content of certain fungal spores in the atmosphere of the city of Granada in 1994. Sampling was carried out with a Burkard 7-day-recording spore trap. The spores analysed corresponded to the taxa Alternaria, Ustilago and Cladosporium, with two morphologically different spore types in the latter genus, cladosporioides and herbarum. These spores were selected both for their allergenic capacity and for the high level of their presence in the atmosphere, particularly during the spring and autumn. The spores of Cladosporium were the most abundant (93.82% of the total spores identified). The Spearman correlation coefficients between the spore concentrations studied and the meteorological parameters show different indices depending on the taxon being analysed. Alternaria and Cladosporium are significantly correlated with temperature and hours of sunlight, while Ustilago shows positive correlation indices with relative humidity and negative indices with wind speed. Received: 16 April 1998 / Revised: 27 September 1999 / Accepted: 27 October 1999     

Correlation of spring spore concentrations and meteorological conditions in Tulsa, Oklahoma

Different spore types are abundant in the atmosphere depending on the weather conditions. Ascospores generally follow precipitation, while spore types such as Alternaria and Cladosporium are abundant in dry conditions. This project attempted to correlate fungal spore concentrations with meteorological data from Tulsa, Oklahoma during May 1998 and May 1999. Air samples were collected and analyzed by the 12-traverse method. The spore types included were Cladosporium, Alternaria, Epicoccum, Curvularia, Pithomyces, Drechslera, smut spores, ascospores, basidiospores, and other spores. Weather variables included precipitation levels, temperature, dew point, air pressure, wind speed, wind direction and wind gusts. There were over 242.57 mm of rainfall in May 1999 and only 64.01 mm in May 1998. The most abundant spore types during May 1998 and May 1999 were Cladosporium, ascospores, and basidiospores. Results showed that there were significant differences in the dry-air spora between May 1998 and May 1999. There were twice as many Cladosporium in May 1998 as in May 1999; both ascospores and basidiospores showed little change. Multiple regression analysis was used to determine which meteorological variables influenced spore concentrations. Results showed that there was no single model for all spore types. Different combinations of factors were predictors of concentration for the various fungi examined; however, temperature and dew point seemed to be the most important meteorological factors. Received: 5 July 2000 / Revised: 20 December 2000 / Accepted: 22 December 2000     

The incidence of fungal spores in the ambient air and inside homes: Evidence from London

Little research has been carried out in London concerning fungal spore prevalence yet this information may help to elucidate geographical patterns of asthma and hay fever. Although many types of spore reach peak concentrations outdoors in late-summer, the incidences in the indoor environment may be more important through the winter because of heating and poor ventilation. Daily average concentrations of fungal spores in the ambient atmosphere were monitored with a Burkard volumetric spore trap on an exposed roof in North London from autumn 1991 until the summer of 1992. Indoor spore measurements were taken in 19 homes in the vicinity through the winter months, both by direct air sampling using a portable Burkard sampler and by dust culture. Trends in the occurrence and concentrations of fungal spores indoors and outdoors were examined. Relationships between the abundance of selected allergenic fungi and features of the houses were analysed including age of dwelling, dampness, cleanliness and presence of pets.Aspergillus andPenicillium were the most frequently occurring spore types in the homes. Overall, high spore incidence was associated with dampness and dust accumulation. The outdoor spore samples revealed generally low concentrations through the winter until March when concentrations of many types includingCladosporium, Epicoccum andAlternaria increased in abundance in response to the warmer weather. Even during the late-spring and early-summer, concentrations of most fungal spores were notably below those reported for rural sites.     

Influence of urban climate upon distribution of airborne Deuteromycete spore concentrations in Mexico City

 The effect of an urban climate upon the spatial and temporal distribution of Deuteromycete spores was studied during 1991 using Burkard volumetric spore traps in two areas of Mexico City with different degrees of urbanization. Deuteromycete conidia formed the largest component of the total airborne fungal spore load in the atmosphere of Mexico City, contributing 52% of the spores trapped in an urban-residential area (southern area) and 65% of those in an urban-commercial area (central area). Among the most common spore types, Cladosporium and Alternaria showed a marked seasonal periodicity with significant differences in concentration (P<0.05) between the dry and wet seasons. Maximum conidial concentrations were found during the end of the wet season and the beginning of the cool, dry season (October–December). Daily mean concentrations of the predominant airborne spore types did not differ significantly between the southern and central areas. Daily mean spore concentrations were significantly correlated (P<0.05) in southern and central areas with maximum temperature (south, r = –0.35; central, r = –0.40) and relative humidity (south, r = 0.43; central, r = 0.29) from the previous day. Moreover, multiple regression analysis of spore concentrations with several meteorological factors showed significant interactions between fungal spores, relative humidity and maximum temperature in both areas. The diurnal periodicity of Cladosporium conidia characteristically showed two or three peaks in concentration during the day at 0200–0400, ∼ 1400 and 2000–2200 hours, while that of Alternaria showed only one peak (1200 to 2000 hours) in both areas. Maximum concentrations of these spores generally occurred 2–4 h earlier in the southern than in the central area. The lag in reaching maximum concentrations in the central area probably resulted from differences in the local conditions between the study areas, and from spores transported aerially into the city from distant sources. The analysis of maximum hourly concentrations of Cladosporium and Alternaria spores during 1 month of the dry season (February), and another month of the wet season (September) showed significant differences between the two study areas. Environmental factors and sources (green areas) affected diurnal changes in conidial concentration in the southern area (urbanization index, UI, 0.25), but not in the central area (UI 0.97). In general, spore concentrations were greatest in the southern area when relative humidities were low, and temperatures and wind velocities were high. It was difficult to establish effects of climatic factors on the spore concentration in the city centre. This probably results from the large amounts of air pollution, the heat island phenomenon, and from the distant origin of trapped conidia obviating aerial transport. Nevertheless, the seasonal and diurnal distributions of conidia found were similar to those reported for other tropical regions of the world. Received: 13 August 1996 / Accepted: 4 December 1996     

The effect of local cropping activities and weather on the airborne concentration of allergenic Alternaria spores in rural Australia

Atopy to the fungus Alternaria is strongly associated with respiratory disease. The prevalences of asthma and of allergy to Alternaria are high amongst children living in rural towns of south-eastern Australia. In such towns, airborne allergenic spores have been proposed to arise from nearby crops, but this has not been tested and crops are unlikely to be the only sources of Alternaria . We sought to identify sources and factors that influence concentrations of spores of Alternaria detected in rural towns. Over two years, we sampled spores in two towns (Wagga Wagga and Moree, New South Wales, Australia), in nearby wheat and cotton crops during harvesting and control periods, in a cotton gin and a grain shed. Alternaria was present in both towns throughout the study, and above the crops, at the gin and grain shed. Daily and annual concentrations were amongst the highest recorded worldwide and peaks persisted for six months in Wagga Wagga and ten months in Moree. Crop maturation affected the spore load in the air more than the actual days of harvest. Regression analysis showed that the overall spore concentrations above towns correlated with those above crops. Variables of rainfall and maximum temperature correlated with concentrations in both towns, and additionally wind direction in Wagga Wagga. In conclusion, crops and produce handling released spores into the air that reached nearby rural towns, with peaks in spore concentrations following warm temperatures and recent rainfall.     

Airborne fungi monitoring in Santiago, Chile

Fungal airborne spores were studied from September 1996throughout August 1997 in Santiago, Chile. Total concentrationsfluctuated between 308 and 10,334 spores/m³/day withan annual mean of 2,154 per m³, the highest dispersion beingduring April and May. Forty-five percent of total fungal content wasfound in autumn. Thirteen genera and 3 other spore types wereidentified. Cladosporium, the most abundant genera in ouratmosphere, contributed with 70.9% of the total fungi counts andreached an annual mean of 1,527 spores/m³/day, itshighest frequency being in autumn. Alternaria appeared as thesecond most frequent genera, with an annual mean of 40spores/m³/day, representing a 1.9% of theannual fungal catch. Altogether, Stemphylium, Torula, Epicoccum,Ganoderma, Helminthosporium, Chaetomiun, Pleospora and othersreached relative frequencies of 0.5% or less. It is concludedthat fungi are present in Santiago's atmosphere all year round, some ofthem with a clear seasonality.     

Seasonal variation of fungal propagules in a fruit market environment, Nagpur (India)

The concentration of airborne fungal spores in a marketenvironment was examined to provide basic information needed to evaluatethe importance of varying levels and heterogeneity. Sampling has beencarried out by rotorod sampler and exposed Petri plate method to obtainthe quantitative and qualitative estimations respectively.Aspergillus was the most frequent and predominant genusdetected. Cladosporium, Penicillium and Alternariaspores were also fairly abundant which are well known as allergenic andpathogenic. The high concentration of airborne spores was recordedduring December–January. While the maximum concentration ofAspergillus was found during summer months in the marketenvironment.     

Airborne Alternaria spores in SE Spain (1993-98)

The annual, seasonal and hourly distribution of Alternaria spores in the air of Murcia, SE España, was studied on a six-year period. The relationships between Alternaria spore concentrations and meteorological factors were investigated. Alternaria is a late afternoon taxon in the Murcia city, with maximum spore concentrations occurring between 13.00h and 21.00h. Alternaria spores are present in the atmosphere throughout the year, with a main spore season extending from March to October, and showing two peaks as a consequence of the summer drop in concentration. Alternaria spore concentrations correlate well with Poaceae and Chenopodiaceae pollen counts, suggesting these plants could be important hosts, but not the only ones, because many crops are growing just when peaks occur. Low wind velocities favoured high spore counts. Correlation with temperature was positive in five of the six years. Mean temperature is the factor which best explain spore levels. The best prediction model obtained explains 74% of the observed variance in Alternaria levels (in a five steps scale) by using mean temperature alone.     

The variation in spore concentrations of selected fungal taxa associated with weather conditions in Szczecin,Poland, 2004–2006

The investigation of airborne fungal spore concentrations was carried out in Szczecin, Poland between 2004 and 2006. The objective of the studies was to determine a seasonal variation in concentrations of selected fungal spore types due to meteorological parameters. The presence of spores of ten taxa: Cladosporium, Ganoderma, Alternaria, Epicoccum, Didymella, Torula, Dreschlera‐type, Polythrincium, Stemphylium and Pithomyces was recorded in Szczecin using a volumetric method (Hirst type). Fungal spores were present in the air in large numbers in summer. The highest concentrations were noted in June, July and August. The peak period was recorded in August for most of the studied spore types: Ganoderma, Alternaria, Epicoccum, Dreschlera‐type, Polythrincium and Stemphylium. Cladosporium and Didymella spores reached their highest concentrations in July while concentrations of Torula were highest in May and Pithomyces in September. Multiple regression analysis was performed for three fungal seasons: 2004, 2005, and 2006. Spore concentrations were positively correlated with minimum temperature for seven spore types in 2004, for five spore types in 2005, and for eight spore types in 2006 (significance level of α = 0.05). Some spore types are also significantly correlation among their concentrations, pressure, relative humidity and rain. Minimum temperature appeared to be the most influential factor for most spore types.