Animal shed Bacillus licheniformis spores possess allergy-protective as well as inflammatory properties.

BACKGROUND: Numerous epidemiologic studies have demonstrated an allergy-protective effect of farm life early in childhood. It has been hypothesized that environmental exposure to microbes may contribute to this effect. Because of their small size and thereby their potential for deposition in lower airways of small children, bacterial spores may be candidates for such allergy-protective effects. OBJECTIVE: To investigate immune responses elicited by exposure to Bacillus spores in experimental settings. METHODS: Animal shed and mattress dusts were analyzed for bacteria and fungi by aerobic and anaerobic growth. Bacillus licheniformis, the most prominent microorganism found in these samples, was investigated with respect to spore specific stimulation of pattern recognition receptors, monocyte-derived dendritic cells and T(H)-cell polarization in vitro as well as to the prevention of asthma development in a mouse model of allergic asthma. RESULTS: In vitro, B. licheniformis spores activated a T(H)1 cytokine expression profile. In vivo application of these spores resulted in less spore-specific but long-lasting immune activation preventing eosinophilia and goblet cell hyperplasia; however, they provoked an influx of neutrophils in lung tissue of asthmatic mice. CONCLUSION: Bacterial spores may contribute to the allergy-protective properties of farming environments, but their persistence in the lung causes ongoing immune activation in mouse experiments.

Application of PCR-SSCP for molecular epidemiological studies on the exposure of farm children to bacteria in environmental dust.

The environmental exposure of farm children to microorganisms in dust has become a focus of interest, since microbial exposure on farms has been related to a reduced prevalence of asthma and atopic diseases in children. Previous studies almost exclusively focused on the determination of microbial counts using conventional culturing or the determination of microbial compounds i.e. endotoxins. In this study PCR-SSCP (single-strand conformation polymorphism) was modified for characterising bacterial communities in environmental dusts and their sensitivity and reproducibility was validated. A fivefold repeated PCR-SSCP analyses of a well homogenised mattress dust, cow-shed dust, swine-shed dust, chicken-shed dust and a horse-shed dust sample, respectively, showed similarities, based on Pearson correlations, ranging from 89.7% to 95.2%. The reproducibility of day to day variations (five days) and gel to gel variations (five gels) was also around 90%. The detection limit of Escherichia coli was 7 x 10(1) cfu g(-1) whereas Listeria monocytogenes and Bacillus licheniformis containing 30% spores showed visible bands at 7 x1 0(2) cfu g(-1). Application of this method to dust samples of 37 sheds and 63 children's mattresses showed that distinct farm environment dusts reflected different SSCP profiles. However, digital analysis of the gels showed that some bands in the profiles of shed- and mattress dusts were found at the same position in the gels. By excision, cloning, sequencing and phylogenetic analyses, these bands were identified as Corynebacterium tuberculostearicum, Corynebacterium mucifaciens, Staphylococcus epidermidis, Acinetobacter lwoffii, Brevibacterium iodinum, Brevibacterium linens and Arthrobacter spp, respectively. These results may reflect transfer of microorganisms from animal sheds to mattresses. In conclusion this study demonstrates that PCR-SSCP is a promising method with sensitive detection limits and moderate sample variances to be applied for epidemiological studies characterizing the exposure of farmers using environmental dust.

Occurrence of Listeria spp. in mattress dust of farm children in Bavaria.

Several epidemiological studies have shown that the farm environment impacts allergy protection mechanisms in children. These associations are not well understood, but it is thought that contact to microorganisms may mediate this effect. For example, heat-inactivated Listeria (L.) monocytogenes have been successfully used as an adjuvant in mouse immunotherapy to modulate airway hyperreactivity and inflammation. In investigating the link between farming lifestyle and prevention of childhood allergy, we examined the prevalence of Listeria spp. in dust specimens from the environment of rural children. A total of 26 farms located in Bavaria (South Germany) were examined. Dust samples taken from mattresses (n=63), cow-sheds (n=30) and swine-sheds (n=10) were qualitatively screened for the presence of viable Listeria spp. according to the ISO 11290-1 method and additionally by L. monocytogenes specific iap-based real-time PCR. Isolates were further characterized by biochemical techniques, serology and multiplex PCR. Nineteen of 26 farms tested positive for Listeria spp. and seven were tested negative. The dominant species found by culturing methods were L. innocua (n=12) and L. monocytogenes (n=8). Viable Listeria spp. were detected in 8% of the mattress dust samples, whereas real-time PCR revealed 60% L. monocytogenes positive specimens. Regarding animal sheds, 28% of dust samples showed viable Listeria spp., while using real-time PCR found that 28% of specimens were L. monocytogenes positive. All strains of L. monocytogenes except one (4ab) belonged to the serotype 1/2a. Our data demonstrate that a substantial number of farm children's beds contain L. monocytogenes. The importance of this result regarding the health of children must be evaluated by epidemiological investigations on both the risk of listeriosis and the effects on protection against allergies.