Live Spores Research Articles

On the Origin of Live Spores in γ-Irradiated Spore Preparations: a Perfect Example of Poisson Distribution?

The recent revelation of the shipment of γ irradiation-inactivated Bacillus anthracis spore reference materials containing a small number of live spores (Department of Defense [DoD] Laboratory Review. DoD Launches Review of Lab Procedures Involving Anthrax. http://www.defense.gov/news/newsarticle.aspx?id=128939, May 29, 2015) has raised concerns about the safety and security of these materials and doubts on the validity of the protocols and procedures used to prepare these materials. Such inactivated spore materials have historically fulfilled critical needs: as positive controls in assays used to detect these pathogens in suspected samples (live agents cannot be shipped and used in field settings), and are also used in improvement of currently deployed detection methods or development of new methods/platforms for detection of these agents; in other words, to develop and validate detection assays, and for quality assurance activities such as proficiency testing. The committee for comprehensive review of DoD Laboratory procedures, processes, and protocols associated with inactivating B. anthracis spores has found inherent deficiencies in protocols in three phases in the production of inactive spores that could lead to nonsterile products: (1) radiation dosing, (2) viability testing, and (3) aseptic operations (contamination prevention). These deficiencies and other factors contributed to the establishment of protocols that do not completely or permanently sterilize these samples (http://archive.defense.gov/home/features/2015/0615_lab-stats/docs/Review-Committee-Report-Final.pdf, July 13, 2015). The review committee made a number of recommendations, including initiating studies to understand the science of irradiating spores and establishment of standardized protocols across the labs engaged in inactivated spore production. In addition, a clear understanding of the nature of the surviving spores would aid in understanding the phase(s) in which the failure occurred and fixing the problem. Here we consider a few hypotheses.

Whole genome protein microarrays for serum profiling of immunodominant antigens of Bacillus anthracis.

A commercial Bacillus anthracis (Anthrax) whole genome protein microarray has been used to identify immunogenic Anthrax proteins (IAP) using sera from groups of donors with (a) confirmed B. anthracis naturally acquired cutaneous infection, (b) confirmed B. anthracis intravenous drug use-acquired infection, (c) occupational exposure in a wool-sorters factory, (d) humans and rabbits vaccinated with the UK Anthrax protein vaccine and compared to naïve unexposed controls. Anti-IAP responses were observed for both IgG and IgA in the challenged groups; however the anti-IAP IgG response was more evident in the vaccinated group and the anti-IAP IgA response more evident in the B. anthracis-infected groups. Infected individuals appeared somewhat suppressed for their general IgG response, compared with other challenged groups. Immunogenic protein antigens were identified in all groups, some of which were shared between groups whilst others were specific for individual groups. The toxin proteins were immunodominant in all vaccinated, infected or other challenged groups. However, a number of other chromosomally-located and plasmid encoded open reading frame proteins were also recognized by infected or exposed groups in comparison to controls. Some of these antigens e.g., BA4182 are not recognized by vaccinated individuals, suggesting that there are proteins more specifically expressed by live Anthrax spores in vivo that are not currently found in the UK licensed Anthrax Vaccine (AVP). These may perhaps be preferentially expressed during infection and represent expression of alternative pathways in the B. anthracis “infectome.” These may make highly attractive candidates for diagnostic and vaccine biomarker development as they may be more specifically associated with the infectious phase of the pathogen. A number of B. anthracis small hypothetical protein targets have been synthesized, tested in mouse immunogenicity studies and validated in parallel using human sera from the same study.

Recent Developments in Anti-dotes Against Anthrax.

The etiologic agent of disease anthrax, Bacillus anthracis, causes recurrent outbreaks among the livestock and intermittent infections in humans across the world. Controlling animal infections by vaccination can minimize the incidence of disease in humans. Prevention of anthrax in occupationally exposed personnel is achieved through vaccination with either live spores or precipitates of culture supernatants from attenuated strains of B. anthracis. However, anthrax vaccination of the large human population is impractical as well as inappropriate. Broad-range antibiotics like amoxicillin, ciprofloxacin, clindamycin, streptomycin, and penicillin G are recommended for the treatment of human anthrax infections, but the threat of antibiotic resistant strains always remains. Moreover, in the absence of any specific symptom (s) during early infection, the diagnosis of anthrax is delayed causing elevated levels of anthrax toxin component which could be fatal. For these reasons, there is a need to develop new antimicrobial agents against virulent B. anthracis to effectively combat this fatal pathogen. Over the last two decades, extensive studies have been carried out to develop specific inhibitors against virulence factors of B. anthracis such as capsule, protective antigen, lethal factor and edema factor. Research has also been focused in developing inhibitors of anthrax toxin receptors (including the use of receptor decoys) and host furin endoproteases which are required for activation of toxin. This review highlights the recent progress made in developing the diverse countermeasures for anthrax infections targeting B. anthracis virulence factors and their counterparts in host.

Antibodies to Pseudogymnoascus destructans are not sufficient for protection against white-nose syndrome.

White-nose syndrome (WNS) is a fungal disease caused by Pseudogymnoascus destructans (Pd) that affects bats during hibernation. Although millions of bats have died from WNS in North America, mass mortality has not been observed among European bats infected by the fungus, leading to the suggestion that bats in Europe are immune. We tested the hypothesis that an antibody-mediated immune response can provide protection against WNS by quantifying antibodies reactive to Pd in blood samples from seven species of free-ranging bats in North America and two free-ranging species in Europe. We also quantified antibodies in blood samples from little brown myotis (Myotis lucifugus) that were part of a captive colony that we injected with live Pd spores mixed with adjuvant, as well as individuals surviving a captive Pd infection trial. Seroprevalence of antibodies against Pd, as well as antibody titers, was greater among little brown myotis than among four other species of cave-hibernating bats in North America, including species with markedly lower WNS mortality rates. Among little brown myotis, the greatest titers occurred in populations occupying regions with longer histories of WNS, where bats lacked secondary symptoms of WNS. We detected antibodies cross-reactive with Pd among little brown myotis naïve to the fungus. We observed high titers among captive little brown myotis injected with Pd. We did not detect antibodies against Pd in Pd-infected European bats during winter, and titers during the active season were lower than among little brown myotis. These results show that antibody-mediated immunity cannot explain survival of European bats infected with Pd and that little brown myotis respond differently to Pd than species with higher WNS survival rates. Although it appears that some species of bats in North America may be developing resistance to WNS, an antibody-mediated immune response does not provide an explanation for these remnant populations.

Human pDCs express the C-type Lectin receptor Dectin-1 and uptake and kill Aspergillus fumigatus spores in vitro (MPF4P.734)

Abstract Plasmacytoid dendritic cells (pDCs) are the body’s most potent producers of type-1 IFNs, and are key responders to viruses like HIV, serving as an important link between the innate and adaptive immune responses. Although they are mainly anti-viral, evidence suggests pDCs also play an integral part in anti-fungal immunity. We demonstrated that human pDCs express the C-type Lectin receptor (CLR), Dectin-1, and that it is further upregulated after stimulation with HSV, Influenza, and the synthetic DNA ODN, CpG-A. In contrast, we did not find expression of Dectin-2, another CLR, on pDCs. Dectin-1 recognizes β-linked glucans, a component of the cell wall of opportunistic fungi like Aspergillus, Pneumocystis, and Candida, and is involved in immune functions including the uptake of fungi, induction of cytokine production, and direction of T-helper cell differentiation. Using Fluorescent Aspergillus Reporter (FLARE)-labelled A. fumigatus spores, which allow us to detect and differentiate between live and dead spores using flow cytometry, we showed that human pDCs can phagocytose and kill Aspergillus spores in vitro within a six-hour time frame. We hypothesize that Dectin-1 is the receptor responsible for this uptake. Although pDCs are classically considered IFN producers, stimulation with spores did not result in IFN-α production by pDC. Results from this ongoing study will shed further light on the role of human pDCs in anti-fungal immunity.

Hormesis and trade-offs: a comment.

Hormesis and trade-offs: a comment.

Anthrax Outbreak Among Cattle and its Detection by Extractable Antigen 1 (EA1) Based Sandwich ELISA and Immuno PCR

Anthrax, a fatal zoonotic disease caused by spore forming bacteria Bacillus anthracis is predominantly witnessed among herbivorous animals that are highly sus- ceptible to the pathogen. Typically B. anthracis spores existing in soil, initiate the infection during grazing and animal to animal spread has been rarely reported. A sus- pected outbreak of anthrax was observed among cattle in Gundlupet taluk neighboring Bandipur national park and tiger reserve during January 2013. Blood samples collected from domestic cattle in Gundlupet region were examined by EA1 sandwich ELISA and immunocapture PCR. Of the 64 samples examined, 12 recovered during the course of outbreak were found positive by the diagnostic methods and further resulted in recovery of B. anthracis isolates that were confirmed by standard methods indicating prevalence of the pathogen among diseased cattle. The outbreak could be contained by the administration of oxytetracycline and live spore vaccine. The isolates recovered from the cattle were susceptible to the major antibiotics penicillin, ampi- cillin, ciprofloxacin, nalidixic acid, tetracycline, erythro- mycin and chloramphenicol whereas resistance could be seen for the antibiotic neomycin (25 %). The ambiguity for initial symptomatic identification due to the absence of bleeding from natural orifices could be overcome by the EA1 immunoassay kits employed in this study.

English

Egg white curd cheese is a curdled protein concentrate made from egg whites. Its advantages are: high protein content, a taste similar to cow's milk curd cheese, minimum carbohydrates, and no fat or cholesterol. Thus, it offers an attractive substitute for cow's milk curd cheese to many followers of a “modern lifestyle” as well as those living with health problems (sensitivity to milk products). Both the internal and sensory characteristics of egg white curd cheese were studied. In addition, the level of potentially dangerous microbes in the product was calculated. The fresh product was in excellent microbiological condition, and over a storage period of three weeks, no Salmonella or Staphylococcus aureus could be detected in it. The levels of Enterobacteriaceae and all live aerobic spore counts were completely satisfactory. In summary, it can be stated that we have succeeded in developing a microbiologically stable product which is applicable for numerous diets, has a flavor resembling mild cow's mild curd cheese, lacks enhancers and preservatives, and can even be consumed as it is.   Key words: Eggwhite, curdcheese, Salmonella, paleolithic diet.

Comparison of Bacillus atrophaeus spore viability following exposure to detonation of C4 and to deflagration of halogen-containing thermites

Energetic materials are being considered for the neutralization of spore-forming bacteria. In this study, the neutralization effects of a monomolecular explosive were compared to the effects of halogen-containing thermites. Bacillus atrophaeus spores were exposed to the post-detonation environment of a 100 g charge of the military explosive C-4 at a range of 50 cm. These tests were performed in the thermodynamically closed environment of a 506-l barometric calorimeter. Associated temperatures were calculated using a thermodynamic model informed by calculations with the Cheetah thermochemical code. Temperatures in the range of 2300–2800 K were calculated to persist for nearly the full 4 ms pressure observation time. After the detonation event, spores were characterized using optical microscopy and the number of viable spores was assessed. Results showed live spore survival rates in the range of 0.01%–1%. For the thermite tests, a similar, smaller-scale configuration was employed that examined the spore neutralization effects of two thermites: aluminum with iodine pentoxide and aluminum with potassium chlorate. Only the former mixture resulted in spore neutralization. These results indicate that the detonation environment produced by an explosive with no chemical biocides may provide effective spore neutralization similar to a deflagrating thermite containing iodine.

Quantifying spore viability of the honey bee pathogen Nosema apis using flow cytometry

Honey bees are hosts to more than 80 different parasites, some of them being highly virulent and responsible for substantial losses in managed honey bee populations. The study of honey bee pathogens and their interactions with the bees' immune system has therefore become a research area of major interest. Here we developed a fast, accurate and reliable method to quantify the viability of spores of the honey bee gut parasite Nosema apis. To verify this method, a dilution series with 0, 25, 50, 75, and 100% live N. apis was made and SYTO 16 and Propidium Iodide (n = 35) were used to distinguish dead from live spores. The viability of spores in each sample was determined by flow cytometry and compared with the current method based on fluorescence microscopy. Results show that N. apis viability counts using flow cytometry produced very similar results when compared with fluorescence microscopy. However, we found that fluorescence microscopy underestimates N. apis viability in samples with higher percentages of viable spores, the latter typically being what is found in biological samples. A series of experiments were conducted to confirm that flow cytometry allows the use of additional fluorescent dyes such as SYBR 14 and SYTOX Red (used in combination with SYTO 16 or Propidium Iodide) to distinguish dead from live spores. We also show that spore viability quantification with flow cytometry can be undertaken using substantially lower dye concentrations than fluorescence microscopy. In conclusion, our data show flow cytometry to be a fast, reliable method to quantify N. apis spore viabilities, which has a number of advantages compared with existing methods.

RUDERAL PLANTS: TEMPORARY HOSTS OF ARBUSCULAR MYCORRHIZAL FUNGI IN TRADITIONAL AGRICULTURAL SYSTEMS?

<p>Ruderal plants may serve as temporary hosts of arbuscular mycorrhizal fungi (AMF), by maintaining the availability of active propagules in the soil, which in turn favors rapid colonization of roots of cultivated species during the agricultural cycle. The goals of this study were to: 1) estimate the richness of ruderal plant species in an agricultural plot and determine their mycorrhizal status, 2) quantify the number of live AMF spores in soil samples, and 3) estimate the infection potential and number of active propagules in soil samples from the agricultural site. The agricultural site used was located in Yucatan, Mexico, and consisted of a monoculture of corn subjected to low-impact agricultural practices during the last five years. A total of 20 species of ruderal plants were found at the experimental site, belonging to 11 families. All the sampled species exhibited associations with AMF, and colonization percentages ranged from 11.7±0.07 to 79.6±0.01 among species. The rhizosphere presented an average of 565±324 spores in50 gof dry soil, of which 58.76% of the spores were alive. The inoculum potential of the soil was 50.4±0.05%, while the number of infective propagules was 193.37 (both in 50 mL of soil). Results from this study show that the presence of ruderal species in agricultural sites may promote the maintenance of AMF communities by acting as temporary hosts of these fungal species. In doing so, ruderal species can favor a higher production of infective AMF propagules and thus stronger mycorrhizal interactions with cultivated species. </p>

Detection of a transient mitochondrial DNA heteroplasmy in the progeny of crossed genetically divergent isolates of arbuscular mycorrhizal fungi

Nonself fusion and nuclear genetic exchange have been documented in arbuscular mycorrhizal fungi (AMF), particularly in Rhizophagus irregularis. However, mitochondrial transmission accompanying nonself fusion of genetically divergent isolates remains unknown. Here, we tested the hypothesis that mitochondrial DNA (mtDNA) heteroplasmy occurs in the progeny of spores, obtained by crossing genetically divergent mtDNAs in R.irregularis isolates. Three isolates of geographically distant locations were used to investigate nonself fusions and mtDNA transmission to the progeny. We sequenced two additional mtDNAs of two R.irregularis isolates and developed isolate-specific size-variable markers in intergenic regions of these isolates and those of DAOM-197198. We achieved three crossing combinations in pre-symbiotic and symbiotic phases. Progeny spores per crossing combination were genotyped using isolate-specific markers. We found evidence that nonself recognition occurs between isolates originating from different continents both in pre-symbiotic and symbiotic phases. Genotyping patterns of individual spores from the progeny clearly showed the presence of markers of the two parental mtDNA haplotypes. Our results demonstrate that mtDNA heteroplasmy occurs in the progeny of the crossed isolates. However, this heteroplasmy appears to be a transient stage because all the live progeny spores that were able to germinate showed only one mtDNA haplotype.

Exotic cheatgrass and loss of soil biota decrease the performance of a native grass

Soil disturbances can alter microbial communities including arbuscular mycorrhizal (AM) fungi, which may in turn, affect plant community structure and the abundance of exotic species. We hypothesized that altered soil microbial populations owing to disturbance would contribute to invasion by cheatgrass (Bromus tectorum), an exotic annual grass, at the expense of the native perennial grass, squirreltail (Elymus elymoides). Using a greenhouse experiment, we compared the responses of conspecific and heterospecific pairs of cheatgrass and squirreltail inoculated with soil (including live AM spores and other organisms) collected from fuel treatments with high, intermediate and no disturbance (pile burns, mastication, and intact woodlands) and a sterile control. Cheatgrass growth was unaffected by type of soil inoculum, whereas squirreltail growth, reproduction and nutrient uptake were higher in plants inoculated with soil from mastication and undisturbed treatments compared to pile burns and sterile controls. Squirreltail shoot biomass was positively correlated with AM colonization when inoculated with mastication and undisturbed soils, but not when inoculated with pile burn soils. In contrast, cheatgrass shoot biomass was negatively correlated with AM colonization, but this effect was less pronounced with pile burn inoculum. Cheatgrass had higher foliar N and P when grown with squirreltail compared to a conspecific, while squirreltail had lower foliar P, AM colonization and flower production when grown with cheatgrass. These results indicate that changes in AM communities resulting from high disturbance may favor exotic plant species that do not depend on mycorrhizal fungi, over native species that depend on particular taxa of AM fungi for growth and reproduction.

Investigation of a New Electrostatic Sampler for Concentrating Biological and Non-Biological Aerosol Particles

The detection of airborne pathogens is becoming a subject of great concern in modern day society. Recent studies have shown that electrostatic samplers are suitable for collecting microorganisms as well as preserving their viability. In most of these studies, flow rates were lower than 12.5 L/min or required a concentration stage to increase the flow rate to 100 L/min. In the present study, a single stage electrostatic sampler was developed for an efficient collection of microorganisms at 100 L/min. The design is based on the positive DC corona between coaxial cylinders to continuously create charged particles as they pass through the sampler. The physical collection efficiency of the device was investigated by sampling the ambient air particles. The efficiency in collecting live biological samples was determined by sampling live bacterial spores with the collector situated inside an aerosol chamber. It is shown that particles of 0.3–0.35 μm are captured with an efficiency of about 86%, whereas cultivable spores of Bacillus thuringiensis are collected with an efficiency of about 94%. Results are compared with an analytical predictive model. The experimental results are similar to the efficiencies previously reported in the literature; however, the current sampler design features a higher flow rate which enables the device to be used as an alarm trigger in a shorter period of time. Copyright 2013 American Association for Aerosol Research

Drosophila melanogaster Selection for Survival after Infection with Bacillus cereus Spores: Evolutionary Genetic and Phenotypic Investigations of Respiration and Movement

Laboratory populations of D. melanogaster have been subjected to selection for survival after live spores of B. cereus were introduced as a pathogenic agent. The present study was designed to investigate correlated traits: respiration as a metabolic trait and movement as a behavioral trait. An underlying hypothesis was that the evolution of increased survival after B. cereus infection exerts a metabolic cost associated with elevated immunity and this would be detected by increased respiration rates. There was support for this hypothesis in the male response to selection, but not for selected-line females. Two phenotypic effects were also observed in the study. Females especially showed a marked increase in respiration after mating compared to the other assay stages regardless of whether respiration was measured per fly or adjusted by lean mass or dry weight. Given that mating stimulates egg production, it is feasible that elevated metabolism was needed to provision oocytes with yolk. Females also moved less than males, perhaps due to behaviors related to oviposition whereas elevated male activity might be due to behaviors associated with seeking females and courtship. Relatively low movement of females indicated that their elevated respiration after mating was not due to a change in locomotion.

Identification of the Immunogenic Spore and Vegetative Proteins of Bacillus anthracis Vaccine Strain A16R

Immunoproteomics was used to screen the immunogenic spore and vegetative proteins of Bacillus anthracis vaccine strain A16R. The spore and vegetative proteins were separated by 2D gel electrophoresis and transferred to polyvinylidene difluoride membranes, and then western blotting was performed with rabbit immune serum against B.anthracis live spores. Immunogenic spots were cut and digested by trypsin. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry was performed to identify the proteins. As a result, 11 and 45 immunogenic proteins were identified in the spores and vegetative cells, respectively; 26 of which have not been reported previously. To verify their immunogenicity, 12 of the identified proteins were selected to be expressed, and the immune sera from the mice vaccinated by the 12 expressed proteins, except BA0887, had a specific western blot band with the A16R whole cellular lytic proteins. Some of these immunogenic proteins might be used as novel vaccine candidates themselves or for enhancing the protective efficacy of a protective-antigen-based vaccine.

Surface availability of beta-glucans is critical determinant of host immune response to Cladosporium cladosporioides

It is well accepted that mold exposure is a major contributor to the development of asthma, and beta-glucans are often used as a surrogate for mold exposure in the environment. Beta-glucans are an important component of mold spores and are recognized by the immune system by their receptor, Dectin-1. Cladosporium cladosporioides spores have a high beta-glucan content, but the beta-glucans are not available on the surface of live spores. We sought to determine whether altering the exposure of beta-glucans in C cladosporioides through heat killing could alter the immune response through binding to Dectin-1. In a murine model of mold-induced asthma, mice were repeatedly exposed to either live or heat-killed C cladosporioides and the phenotype was determined by the measurement of airway hyperresponsiveness, airway inflammation, and cytokine production. Pro-inflammatory cytokines from dendritic cells were measured by using quantitative PCR and ELISA. Live C cladosporioides induced robust airway hyperresponsiveness, eosinophilia, and a predominately TH2 response, while heat-killed C cladosporioides induced a strong TH17 response and neutrophilic inflammation, but very mild airway hyperresponsiveness. Heat killing of C cladosporioides spores effectively exposed beta-glucans on the surface of the spores and increased binding to Dectin-1. In the absence of Dectin-1, heat-killed spores induced a predominantly TH2 response analogous to live spores. Furthermore, the production of TH17-skewing IL-6, IL-23, and TNF-α by dendritic cells in response to heat-killed C cladosporioides was dependent on Dectin-1. The host immune response to C cladosporioides is dependent on the surface availability of beta-glucans rather than the total beta-glucan content.

Sexual Phase of Three Species of Pteris (Pteridaceae)

This work describes and compares morphogenesis of the gametophyte, or sexual phase, of three ferns, Pteris deflexa, P. denticulata and P. tristicula, through a palynological analysis of live spores. This study was conducted under laboratory conditions; observations and pictures were made using light and scanning electron microscopy. The pattern of spore germination corresponds to the Vittaria type and gametophytic development corresponds to the Ceratopteris type. We observed that the polymorphous spores of P. denticulata and P. tristicula are viable. Ultrastructural details of the gametophytes revealed that P. denticulata and P. tristicula form antheridia different from those of the remaining Pteris species; the archegonia have necks formed by 4 rows of 4 cells each. The type of reproduction is sexual and sporophytes arise 90 days after sowing.

The impact of Aspergillus fumigatus viability and sensitization to its allergens on the murine allergic asthma phenotype.

Aspergillus fumigatus is a ubiquitously present respiratory pathogen. The outcome of a pulmonary disease may vary significantly with fungal viability and host immune status. Our objective in this study was (1) to assess the ability of inhaled irradiation-killed or live A. fumigatus spores to induce allergic pulmonary disease and (2) to assess the extent to which inhaled dead or live A. fumigatus spores influence pulmonary symptoms in a previously established allergic state. Our newly developed fungal delivery apparatus allowed us to recapitulate human exposure through repeated inhalation of dry fungal spores in an animal model. We found that live A. fumigatus spore inhalation led to a significantly increased humoral response, pulmonary inflammation, and airway remodeling in naïve mice and is more likely to induce allergic asthma symptoms than the dead spores. In contrast, in allergic mice, inhalation of dead and live conidia recruited neutrophils and induced goblet cell metaplasia. This data suggests that asthma symptoms might be exacerbated by the inhalation of live or dead spores in individuals with established allergy to fungal antigens, although the extent of symptoms was less with dead spores. These results are likely to be important while considering fungal exposure assessment methods and for making informed therapeutic decisions for mold-associated diseases.

Analysis of global gene expression changes in human bronchial epithelial cells exposed to spores of the allergenic fungus, Alternaria alternata

Exposure and sensitivity to ubiquitous airborne fungi such as Alternaria alternata have long been implicated in the development, onset, and exacerbation of chronic allergic airway disorders. This present study is the first to investigate global changes in host gene expression during the interaction of cultured human bronchial epithelial cells and live Alternaria spores. In in vitro experiments human bronchial epithelial cells (BEAS-2B) were exposed to spores or media alone for 24 h. RNA was collected from three biological replicates per treatment and was used to assess changes in gene expression patterns using Affymetrix Human Genome U133 Plus 2.0 Arrays. In cells treated with Alternaria spores compared to controls, 613 probe sets representing 460 individual genes were found differentially expressed (p ≤ 0.05). In this set of 460 statistically significant, differentially expressed genes, 397 genes were found to be up-regulated and 63 were down-regulated. Of these 397 up-regulated genes, 156 genes were found to be up-regulated ≥2 fold. Interestingly, none of the 63 down-regulated genes were found differentially expressed at ≤−2 fold. Differentially expressed genes were identified following statistical analysis and subsequently used for pathway and network evaluation. Interestingly, many cytokine and chemokine immune response genes were up-regulated with a particular emphasis on interferon-inducible genes. Genes involved in cell death, retinoic acid signaling, and TLR3 response pathways were also significantly up-regulated. Many of the differentially up-regulated genes have been shown in other systems to be associated with innate immunity, inflammation and/or allergic airway diseases. This study now provides substantial information for further investigating specific genes and innate immune system pathways activated by Alternaria in the context of allergic airway diseases.