Children growing up on farms in Central Europe were protected from asthma and atopy. These children were exposed to a greater variety of environmental fungi and bacteria as compared with children in the reference group who lived in the same regions. The greater diversity of environmental microbial exposure was inversely related to asthma, but not to atopy, independently of farming. These data support the idea that the greater diversity of microbial exposure among children who live on farms is associated with protection from the development of asthma. Within the spectrum of microbial diversity detected, several focal zones were associated with a greater protective potential than others. On a species level, however, the identification of protective microorganisms was not possible.

The transport of environmental microorganisms from animal sheds and barns to the indoor environment has been reported.13 The relationship between microbial exposures and health outcomes has been assessed in school-age children, although exposures may be more relevant when children are younger.2 However, dairy farming, a known source of microbial exposures, has been constant over time, indicating that our findings probably reflect the effects of both current and long-term exposures with considerable accuracy.

Even when indoors, children living on farms were exposed to a greater variety of microbes than children who did not live on farms, as indicated by the distribution of the frequencies of detectable bands or taxa and the summation scores for microbial diversity. The central finding of this analysis was the inverse association of the diversity scores with asthma, which was not confounded by living on a farm. Moreover, the diversity scores explained a substantial proportion of the effect of the farming environment on asthma. Obviously, summation scores are imperfect measures of diversity, since they do not account for cross-correlation patterns and might be influenced by specific effects. In the PARSIFAL data set, however, it was possible to conduct a sensitivity analysis with the use of the results from the factor analysis before rotation. By definition, the first unrotated factor explained most of the variance of all bacterial bands. Because 76% of all variables loaded substantially on this factor (loadings >0.3), this factor can be interpreted as a diversity score that accounts for the cross-correlation matrix of all bacterial bands. This modified diversity score showed the same strong association with asthma and farm residence as did the summation score. It was not driven by specific effects, since the loadings of all band variables were below 0.6.

We can speculate about how the diversity of microbial stimuli may be protective against asthma. Microorganisms trigger the innate immune system through pattern-recognition receptors, such as the toll-like receptors. Activation of several toll-like receptors has been found in children exposed to farming environments.14,15 Combinations of microbial exposures may activate several signaling pathways downstream of these receptors, with subsequent induction of regulatory T cells.16 Type 1 helper T cells may be activated and may counterbalance the predominance of type 2 helper T cells that is characteristic of asthma.17 Mucosal immunity may also play a specific role in the abrogation of response by type 2 helper T cells.18

An alternative interpretation of diversity may be found in the counterbalancing of specific detrimental exposures. Environmental exposure to a broad range of microorganisms may prevent colonization of the lower airways with harmful bacteria, which has been associated with an increased risk of asthma among children and adults.19,20 Balanced colonization of the airways may parallel the beneficial effects of a diverse microbiome at other surfaces, such as the gut and skin.21,22

The notion of diversity as a summation of stimuli, however, is constrained by the facts that the number of pattern-recognition receptors is quite limited and the pathways of the innate immune system are redundant. Consequently, diversity in itself may not explain the protective effects against asthma mediated by the innate immune system, since small numbers of microbial exposures may be sufficient to stimulate all pattern-recognition receptors. Furthermore, overgrowth of harmful bacteria might be achieved by a limited number of bacterial species. Consequently, we sought to narrow the spectrum of microbial exposures to several focal zones. This approach led to the identification of two factors in the PARSIFAL study, each of them accounting for a number of correlated bands in our SSCP analysis, with each band representing several bacterial species with synonymous 16S rRNA sequences. The bacteria covered by factor 4 belong to the staphylococcaceae family, which has been reported to be the predominant bacterial family in samples of house dust from the Karelian region that has the lowest prevalences of atopic diseases.23 One of the bacterial species related to factor 5, L. monocytogenes, had preventive effects on airway hyperresponsiveness and inflammation in a murine model of asthma.24

The analysis of microbial diversity and its relation to asthma in GABRIELA brought two fungal genera into focus: eurotium and penicillium. At first glance, these findings may challenge previous observations suggesting that molds may account for the increased risk of asthma ascribed to dampness.25 However, molds are very heterogeneous, and different genera or species within very large taxa, such as penicillium species, may exert diverse effects. Eurotium species are the sexual form of certain aspergillus species. The identification of penicillium and eurotium as protective factors against asthma is supported by previously published data from the PARSIFAL study in which exposure to extracellular polysaccharides, a generic marker for exposure to aspergillus and penicillium species, was inversely related to the risk of childhood asthma.15

The assessment of bacterial diversity in GABRIELA was based mainly on Gram's staining and morphologic assessment. These broad categories did not allow a further refinement of the bacterial exposure. However, gram-negative rods were found to be protective against atopy. This finding parallels the inverse association between atopy and endotoxin, a cell-wall component of gram-negative bacteria, which has been reported in many previous studies.3,26 An association of atopy with the diversity scores, however, was not found. This contrast between the findings for asthma and those for atopy may indicate that microbial exposures affect the risks of asthma and atopy through different mechanisms; the contrast parallels that between the different genetic determinants of the two conditions.27

In conclusion, the results of both the PARSIFAL study and GABRIELA showed that children living on farms had a wider range of microbial exposures than children in the reference groups, which largely explained the protective effect of the farming environment on the development of asthma in children. Our methods do not allow us to identify specific microbes that may confer protection, but they have allowed us to identify broad families of species within microbial taxa that could be responsible for the effect of the farming environment. The challenge will be to identify these species with the precision needed to allow specific tests of the relationship between microbial exposure and protection against asthma.