Particle size distribution of the major Alternaria alternata allergen,Alt a 1, derived from airborne spores and subspore fragments

Fungal fragments are abundant immunoreactive bioaerosols that may outnumber the concentrations of intact spores in the air. To investigate the importance of Alternaria fragments as sources of allergens compared to Alternaria spores, we determined the levels of Alternaria spores and Alt a 1 (the major allergen in Alternaria alternata spores) collected on filters within three fractions of particulate matter (PM) of different aerodynamic diameter: (1) PM_>10, (diameter>10 μm); (2) PM_2.5-10 (2.5–10μm); (3) PM_2.5 (0.12–2.5 μm). The airborne particles were collected using a three stage high-volume ChemVol cascade impactor during the Alternaria sporulation season in Poznań, Poland (30 d between 6 July and 22 September 2016). The quantification of Alt a 1 was performed using the enzyme-linked immunosorbent assay. High concentrations of Alt a 1 were recorded during warm and dry d characterized by high sunshine duration, lack of clouds and high dew point values. Atmospheric concentrations of Alternaria spores correlated significantly (r = 0.930, p < 0.001) with Alt a 1 levels. The highest Alt a 1 was recorded in PM_2.5-10 (66.8 % of total Alt a 1), while the lowest in PM_2.5 (<1.0 %). Significantly more Alt a 1 per spore (>30 %) was observed in PM_2.5-10 than in PM_>10. This Alt a 1 excess may be derived from sources other than spores, e.g. hyphal fragments. Overall, in outdoor air the major source of Alt a 1 are intact Alternaria spores, but the impact of other fungal fragments (hyphal parts, broken spores, conidiophores) cannot be neglected, as they may increase the total atmospheric Alt a 1 concentration.     

Backbone resonance assignment of Alt a 1, a unique β-barrel protein and the major allergen of Alternaria alternata

Alt a 1 is the major allergen of the fungus Alternaria alternata and can be found in the cell wall of its spores. It is a cysteine linked homodimeric protein with a unique β-barrel fold as recently revealed by X-ray crystallography. Despite the elucidation of its structure, its biological function remains unknown. For Alternaria-sensitized patients, contact leads to respiratory allergy and in severe cases to asthma-related death. Here we report the sequence-specific Alt a 1 backbone ¹H, ¹⁵N and ¹³C chemical shift assignment.     

Long-term trends and regional variation in the aeroallergen Alternaria in Cardiff and Derby UK – are differences in climate and cereal production having an effect?

Alternaria is a known aeroallergen, beinga risk factor in childhood and adult asthma.This study compared 1970–1996 daily records ofAlternaria spores in Cardiff and Derby,two very different regions of the UK. There hasbeen a dramatic upward trend in the seasonaltotal for Derby Alternariaspores whereas in Cardiff the trend isdownwards. On certain days in recent yearsDerby Alternaria spore counts haveexceeded 1000 spores per cubic metre of air,but in Cardiff such very high counts have notoccurred since 1970. Derby Alternariaspore levels were most positively correlatedwith wind from the SE (over large stretches ofarable land), whereas in Cardiff the mostpositive correlation was with wind from the north(over grassland). In Derby the increase incereal production, together with highermidsummer temperatures, could account for therising Alternaria counts. This upwardtrend in Derby is expected to continue whereasin Cardiff the coastal position together withthe small amounts of arable production ensureAlternaria spore levels will remain low.The comparison between these two siteshighlights regional differences in the numbersof airborne Alternaria spores andconfirms the importance of maintaining longestablished sites. In Derby these results haveimplications for Alternaria sensitivepatients as very high counts could trigger anasthma attack.     

Effect of airborne <Emphasis Type="Italic">Alternaria</Emphasis> conidia,ozone exposure,PM<Subscript>10</Subscript> and weather on emergency visits for asthma in school-age children in Kolkata city,India

Alternaria is one of the important allergenic fungal spores having special role in childhood asthma. Aerodynamic particle of <10 μm diameter (PM₁₀) and ozone exacerbate respiratory conditions including asthma. There is no documentation determining the role of airborne concentration of Alternaria conidia, pollutants like PM₁₀ and ozone and weather on school-age (5–18 years) children causing life-threatening episodes requiring emergency hospitalization for asthma in the megacity of Kolkata, Eastern India. We examined the relationship of daily hospitalization for asthma in school-age children and daily concentration of outdoor Alternaria conidia, ozone, PM₁₀ and weather parameters in the atmosphere of Kolkata during 2010 using nonparametric generalized additive model (GAM). After a baseline survey (2008–2009), asthma hospitalization data for school-age children (based on case history and symptom-medication) were collected from two important city hospitals near study area in 2010. Daily Alternaria concentration was recorded using Burkard volumetric sampler. Its allergenic potential was confirmed by skin reactivity. Ozone and PM₁₀ concentration data were collected from West Bengal Pollution Control Board. The daily time series analyses of data were performed using GAM. There are two peak seasons of asthma hospitalization, one in spring-early summer (end of March to mid April) and other in autumn (mid September–October). In GAM analyses, Alternaria conidia, ozone and PM₁₀ were found to be significant both in spline and LOcal regrESSion smoothing method. Airborne Alternaria conidia, ozone and PM₁₀ in the city of Kolkata are found to have effect on school-age children with asthma and appear responsible for severe attacks leading to hospitalization.     

<Emphasis Type="Italic">Alternaria</Emphasis> spores in the atmosphere of Sydney,Australia, and relationships with meteorological factors

Alternaria spores are found in the atmosphere in many locations around the world. They are significant from a human health perspective because they have been known to trigger allergic respiratory disease such as asthma and hay-fever. The presence of Alternaria spores in the atmosphere has been related to meteorological factors in past studies, but this has not been done previously in Sydney, Australia. This paper reports the results of such a study in Sydney. Alternaria spore concentration data for the period 19 August 1992 to 31 December 1995 were examined with meteorological data for the same period. The daily Alternaria spore concentration was compared to the meteorological data for the same day and for up to 3 days previously. The analysis methods were Spearmans rank correlation and multiple regression. Alternaria spores appear in the atmosphere of Sydney year-round, although they peak over spring, summer, and autumn. A number of meteorological factors, including mean, minimum, and maximum, temperature, dew point temperature, and air pressure, are significantly correlated with the atmospheric concentration of Alternaria spores. Some of these meteorological variables (temperature and dew point temperature) show significant correlations with a 1, 2, and 3 day lag, as well as for the same day. Regression models indicate that up to 31.1% of the variation in Alternaria spore concentration can be explained by meteorological factors. There is potential for the results of this study to be used by public health authorities in the prediction of Alternaria spore concentrations in Sydney.     

A new Alternaria species from grapevine in China

Twelve Alternaria strains were isolated from pedicels and rachis from different parts of grapevines in China. In a phylogenetic analysis, nine showed a close relationship with Alternaria longipes, and are described as a new species, A. viniferae sp. nov., but the other four clustered with A. alternata and A. arborescens within the alternata species-group. The new species can clearly be separated from other related small-spored Alternaria species based on sequences of portions of the glyceraldehyde-3-phosphate dehydrogenase (gpd) and Alternaria major allergen (Alt a 1) genes as well as by distinctive morphology.     

The influence of meteorological parameters on <Emphasis Type="Italic">Alternaria</Emphasis> and <Emphasis Type="Italic">Cladosporium</Emphasis> fungal spore concentrations in Beja (Southern Portugal): preliminary results

Introduction Fungal spores constitute an important fraction of bioaerosols in the atmosphere. Objectives To analyse the content of Alternaria and Cladosporium spores in the atmosphere of Beja and the effect of meteorological conditions on their concentrations. Methodology The daily and hourly data of Alternaria and Cladosporium fungal spores concentration in the atmosphere of Beja were monitored from April 12, 2012 to July 30, 2014, based on the Portuguese Aerobiology Network methodology. The influence of meteorological conditions on the studied types of fungal spore concentrations was assessed through Spearman’s correlation analysis. Results During the study period, 20,741 Alternaria spores and 320,862 Cladosporium spores were counted. In 2013, there were 5,822 Alternaria spores and 123,864 Cladosporium spores. The absolute maximum concentrations of Alternaria and Cladosporium spores were recorded on November 8, 2013, with 211 and 1301 spores/m³, respectively. Temperature, insolation and wind direction parameters showed a positive correlation with Alternaria and Cladosporium spore levels, while relative humidity and precipitation presented a negative correlation, which is statistically significant. Wind speed only showed a statistically significant positive correlation in terms of Alternaria spore levels. Conclusion Alternaria and Cladosporium spores are present in the atmospheric air of Beja throughout the year, with the highest concentration period occurring during spring and autumn. There was a clear effect of meteorological parameters on airborne concentrations of these fungal spores.     

Atmospheric concentrations and intradiurnal pattern of <Emphasis Type="Italic">Alternaria</Emphasis> and <Emphasis Type="Italic">Cladosporium</Emphasis> conidia in Tétouan (NW of Morocco)

Fungal spores of Alternaria and Cladosporium are ubiquitous components of both indoor and outdoor air samples and are the main causes of human respiratory allergies. Monitoring these airborne fungal spores during 2009–2014 was carried out by means of Hirst-type spore trap to investigate their airborne spore concentrations with respect to annual load, seasonality and overall intradiurnal pattern. Alternaria and Cladosporium spores are present throughout the year in the atmosphere of Tétouan, although they show seasonal variations. Despite important differences between years, their highest levels presented a first peak during spring and a higher second peak in summer or autumn depending on the year. The spore concentrations were homogeneously distributed throughout the day with slight increase of 7.6 and 3.7% on average between 12–14 and 14–16 h for Alternaria and Cladosporium, respectively. The borderline of 3000 sp/m³ of Cladosporium linked to the occurrence of allergic diseases was exceeded between 13 and 31 days. Airborne spores of Alternaria overcame the threshold value of 100 sp/m³ up to 95 days, suggesting that Cladosporium and Alternaria could be clinically significant aeroallergens for atopic patients.     

Atmospheric concentrations of selected allergenic fungal spores in relation to some meteorological factors,in Timişoara (Romania)

Present investigation was undertaken to study the dynamics of relationships between atmospheric fungal spores and meteorological factors in western Romania. The airborne spore sampling was carried out by employing volumetric sampling. A total of nine meteorological parameters were selected for this investigation. During 2008–2010, it was found the same pattern of behaviour in the atmosphere for selected spore types (Alternaria, Cladosporium, Pithomyces, Epicoccum and Torula). The spores occurred in the air throughout the whole year, but maximum concentrations were reached in summer. Cladosporium and Alternaria peak levels were observed in June. Epicoccum peak value was found in September. The relationships between airborne spore concentrations and environmental factors were assessed using the analysis of Spearman’s rank correlations and multiple linear regressions. Spearman’s rank correlation analysis revealed that maximum, minimum and mean temperature, and number of sunshine hours were strongly (p < 0.01) and directly proportional to the concentration of all analysed fungal spores. Negative and significant correlations were with daily mean relative humidity. The variance explained percentage by regression analyses varied between 30.6 and 39.6 % for Alternaria and Cladosporium airborne spores. Statistical methods used in this study are complementary and confirmed stable dependence of Alternaria and Cladosporium spore concentrations on meteorological factors. The climate change parameters either increased temperatures, changed precipitation regimes or a combination of both affected allergenic fungal spore concentrations in western Romania. This study demonstrates the need for investigations throughout the year, from month to month, regarding the correct interpretation of airborne spore relationships with meteorological parameters.     

Alternaria capsicicola sp. nov., a new species causing leaf spot of pepper (Capsicum annuum) in Malaysia

A new species of Alternaria causing leaf spot of pepper (Capsicum annuum) obtained from the Cameron highlands, Pahang, Malaysia, was determined based on phylogenetic analyses, morphological characteristics, and pathogenicity assays. Phylogenetic analyses of combined dataset of the glyceraldehyde-3-phosphate dehydrogenase (gpd), Alternaria allergen a 1 (Alt a1) and calmodulin genes revealed that the new isolates clustered into a subclade distinct from the closely related Alternaria species A. tomato and A. burnsii. The solitary or short chains of conidia resemble those of A. burnsii. However, conidia with long beaks are morphologically similar to A. tomato. Hence, the pathogenic fungus is proposed as Alternaria capsicicola sp. nov. Pathogenicity assays indicated that A. capsicicola causes leaf spot on pepper.     

Pollen and Mold Spores—An Atmospheric and Field Survey in Los Angeles

A two-year survey of pollen and mold spores by the gravity slide method revealed that there are no clear-cut tree, grass or weed pollen seasons in California.Pollen counts should be correlated with field studies to distinguish the various plants whose pollen have a similar appearance.Spores of Alternaria and Hormodendrum, whose importance in allergic disease of the respiratory tract has been well established for many years, were found all during the year. More hormodendrum spores were collected than the total of all other pollens combined.     

Barrier Disrupting Effects of Alternaria Alternata Extract on Bronchial Epithelium from Asthmatic Donors

Sensitization and exposure to the allergenic fungus Alternaria alternata has been associated with increased risk of asthma and asthma exacerbations. The first cells to encounter inhaled allergens are epithelial cells at the airway mucosal surface. Epithelial barrier function has previously been reported to be defective in asthma. This study investigated the contribution of proteases from Alternaria alternata on epithelial barrier function and inflammatory responses and compared responses of in vitro cultures of differentiated bronchial epithelial cells derived from severely asthmatic donors with those from non-asthmatic controls. Polarised 16HBE cells or air-liquid interface (ALI) bronchial epithelial cultures from non-asthmatic or severe asthmatic donors were challenged apically with extracts of Alternaria and changes in inflammatory cytokine release and transepithelial electrical resistance (TER) were measured. Protease activity in Alternaria extracts was characterised and the effect of selectively inhibiting protease activity on epithelial responses was examined using protease inhibitors and heat-treatment. In 16HBE cells, Alternaria extracts stimulated release of IL-8 and TNFα, with concomitant reduction in TER; these effects were prevented by heat-treatment of the extracts. Examination of the effects of protease inhibitors suggested that serine proteases were the predominant class of proteases mediating these effects. ALI cultures from asthmatic donors exhibited a reduced IL-8 response to Alternaria relative to those from healthy controls, while neither responded with increased thymic stromal lymphopoietin (TSLP) release. Only cultures from asthmatic donors were susceptible to the barrier-weakening effects of Alternaria. Therefore, the bronchial epithelium of severely asthmatic individuals may be more susceptible to the deleterious effects of Alternaria.     

The long-term trends and seasonal variation of the aeroallergen Alternaria in Derby, UK

Very little is known in the UK about long term trends of theAlternaria spore although it is known to trigger asthma. It hasrecently become apparent that Alternaria spore levels areincreasing in Derby and a detailed study of Alternaria wasundertaken to investigate the increase in numbers, seasonal variationand diurnal periodicity. The seasonal (June—October)Alternaria spore concentrations show a distinct upward trendand there is evidence of an earlier seasonal start and an increase inthe seasonal duration. There has been a dramatic rise in the number ofdays with an Alternaria spore count above 50 spores per cubicmetre, with the peak daily count usually occurring in August butoccasionally in late July or early September. August generally has thehighest monthly total and for 1991–1998 there was a positivecorrelation with monthly rainfall and average temperature. Day to dayspore levels show a positive correlation between Alternariaspore concentrations and maximum temperature but a slight negativecorrelation with daily rainfall. The peak time for spore capture is14.00–22.00, and more than half the daily Alternariacatch is caught between 18.00 and 24.00 hours. The upward trend inAlternaria spore concentrations may be responsible forincreasing levels of respiratory disease, especially during harvesttime.     

Multilocus phylogeny and MALDI-TOF analysis of the plant pathogenic species Alternaria dauci and relatives

The genus Alternaria includes numerous phytopathogenic species, many of which are economically relevant. Traditionally, identification has been based on morphology, but is often hampered by the tendency of some strains to become sterile in culture and by the existence of species-complexes of morphologically similar taxa. This study aimed to assess if strains of four closely-related plant pathogens, i.e., accurately Alternaria dauci (ten strains), Alternaria porri (six), Alternaria solani (ten), and Alternaria tomatophila (ten) could be identified using multilocus phylogenetic analysis and Matrix-Assisted Laser Desorption Ionisation Time of Flight (MALDI-TOF) profiling of proteins. Phylogenetic analyses were performed on three loci, i.e., the internal transcribed spacer (ITS) region of rRNA, and the glyceraldehyde-3-phosphate dehydrogenase (gpd) and Alternaria major antigen (Alt a 1) genes. Phylogenetic trees based on ITS sequences did not differentiate strains of A. solani, A. tomatophila, and A. porri, but these three species formed a clade separate from strains of A. dauci. The resolution improved in trees based on gpd and Alt a 1, which distinguished strains of the four species as separate clades. However, none provided significant bootstrap support for all four species, which could only be achieved when results for the three loci were combined. MALDI-TOF-based dendrograms showed three major clusters. The first comprised all A. dauci strains, the second included five strains of A. porri and one of A. solani, and the third included all strains of A. tomatophila, as well as all but one strain of A. solani, and one strain of A. porri. Thus, this study shows the usefulness of MALDI-TOF mass spectrometry as a promising tool for identification of these four species of Alternaria which are closely-related plant pathogens.     

Phylogenetic,toxigenic and virulence profiles of <Emphasis Type="Italic">Alternaria</Emphasis> species causing leaf blight of tomato in Egypt

Species of Alternaria are serious plant pathogens, causing major losses on a wide range of crops. Leaf blight symptoms were observed on tomato leaves, and samples were collected from various regions. Isolation was done from symptomatic tomato leaves, and 15 representatives were selected from a collection of 65 isolates of Alternaria species. The virulence of Alternaria isolates was investigated on detached leaves (DL) and whole plants of tomato cv. Super strain B. A phylogenetic analysis was performed based on three partial gene regions, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the RNA polymerase second largest subunit (RPB2) and the Alternaria major allergen gene (Alt a 1). The potentiality of Alternaria isolates to produce toxins was also investigated on the basis of thin-layer chromatography (TLC). Our investigations revealed that Alternaria isolates showed different levels of virulence either on tomato plants or DL. Based on the phylogeny of three genes, Alternaria isolates encompassed two species of small-spored morphospecies: A. alternata (14 isolates) and A. arborescens (single isolate). The produced toxins varied among Alternaria isolates with tenuazonic acid (TeA) being the most abundant mycotoxin produced by most isolates. This study highlighted on other Alternaria species in Egypt that might represent a serious concern for tomato producers as causal agents of leaf blight over other species, i.e. A. solani.     

First volumetric record of fungal spores in the atmosphere of Montevideo City,Uruguay: a 2-year survey

In Uruguay, aeromycological studies are restricted to a gravimetric analysis performed from December 1942 to March 1944 in Montevideo where spores of Pucciniaceae, Alternaria and Helminthosporium were the only specimens identified. Daily monitoring of airborne fungal spores was carried out for the first time in Montevideo, from April 2012 to March 2014, using a Rotorod sampler in order to evaluate the seasonal variation and influence of meteorological parameters. A total of 548,309.68 spores/m³ were recorded which belong to anamorphs of Higher Fungi (69.18 %), Phyla Ascomycota (12.62 %), Basidiomycota (8.01 %), Oomycota (0.37 %) and Myxomycota (0.06 %). Airborne spores occurred in Montevideo throughout the whole year. However, a seasonal pattern was revealed, with the highest concentrations recorded in autumn and summer. The most abundant spore types were Cladosporium (53.22 %), Alternaria (6.62 %), Didymella Group (5.86 %), Leptosphaeria Group (4.37 %) and Coprinus (4.3 %). Temperature appeared to be the most influential meteorological factor correlating significantly and positively with total spore, Cladosporium, Alternaria and Didymella Group abundance. Relative humidity influenced positively total spore, Cladosporium and Didymella Group concentrations while a weak negative association was obtained for Alternaria. Wind speed correlated negatively with total spore, Cladosporium, Alternaria and Didymella Group. Precipitation showed a negative influence on Alternaria, while positive correlations were observed for Didymella Group. For the first time, fungal spores considered allergenic were recorded in Montevideo atmosphere and the risk of exposure would have been high from December to June. However, long-term sampling is needed to define seasonal prevalence patterns and the influence of meteorological conditions on spore abundance.     

Low incidence of pneumonia in COPD patients treated with inhaled corticosteroids undergoing pulmonary rehabilitation

Background

Based on meta-analyses results, it is currently acknowledged that there is an increased risk of pneumonia in patients with chronic obstructive pulmonary disease (COPD) undergoing inhaled corticosteroids (ICS) treatment. However, this is not found to be true in those with asthma. No data on this risk are available for COPD patients involved in pulmonary rehabilitation program (PR).

Methods

For 1 year, we prospectively studied 2 cohorts of COPD patients—undergoing PR and not undergoing PR. The first group included 438 patients undergoing PR of which 353 were treated with ICS, and 85 were treated with bronchodilators only. The second group was comprised of 76 COPD patients who were treated with ICS, but not PR. The control group consisted of 49 ICS-treated patients with asthma. The diagnosis of pneumonia, when suspected, had to be confirmed with a chest x-ray.

Results

Overall, 6 cases of pneumonia were diagnosed in the first study group: 5 ICS-treated patients and 1 patient treated only with bronchodilators. This corresponded to a rate of 1.41 and 1.17%, respectively, compared to a rate of 6.6% in COPD patients not treated with PR, which was significantly higher (p?=?0.029) than that in the first study group. No case of pneumonia was registered among patients with asthma.

Conclusions

These findings suggest that a significantly lower incidence of pneumonia is found in COPD patients treated with ICS and PR than in patients treated with ICS but not with PR. This observation deserves to be investigated in large populations of PR-treated COPD patients, possibly in multi-centric cohort studies.

     

A 10-year study of <Emphasis Type="Italic">Alternaria</Emphasis> and <Emphasis Type="Italic">Cladosporium</Emphasis> in two Polish cities (Szczecin and Cracow) and relationship with the meteorological parameters

Alternaria and Cladosporium spores belong to the most frequent and allergenic particles in bioaerosol in the temperate climate. The investigation of Alternaria and Cladosporium spore concentrations was performed in two cities in Poland, Szczecin and Cracow, in 2004–2013. The meteorological parameters taken to assess their impact on fungal spores were average, maximum and minimum temperature, relative humidity and average wind velocity. In order to reveal whether changes in dynamics of spore seasons are driven by meteorological conditions, ordination methods were applied. Canonical correspondence analysis was used to explore redundancy among the predictors (meteorological parameters). Prior to ordination analyses, the data were log(x)-transformed. Concentrations of Alternaria and Cladosporium spores were significantly higher in Szczecin comparing to Cracow, but it was also observed the decreasing trend in the spore concentrations in Szczecin. As regards temperature, it was higher in Cracow and was still increasing in the studied years. Relative humidity and wind velocity were significantly lower in Cracow. In Szczecin meteorological conditions did not explain changes in spore season characteristics (insignificant redundancy analysis models), while in Cracow’s redundancy analysis models indicated that spore season parameters were in over 40 % determined by meteorological conditions, mainly air temperature and wind velocity. If they increase, the peak value, total number of spores and their average concentrations in a season will also increase.