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Daqu and wheat Qu are saccharification and fermenting agents in Chinese huangjiu and baijiu production. This study aimed to investigate the difference between Daqu and wheat Qu in physicochemical indices, microbial communities, functional genes, and the metabolic network of key microbes responsible for flavor synthesis by whole-metagenome sequencing and metabolite analysis. Herein, physicochemical indices indicated that compared with wheat Qu, Daqu exhibited higher protease and cellulase activity and acidity, and lower glucoamylase and amylase enzyme activity. Metagenomic sequencing reveals that although Daqu and wheat Qu community composition have significant differences at species level, they have similar functional genes. Daqu were enriched in Pediococcus pentosaceus, Weissella paramesenteroides, Rasamsonia emersonii and Byssochlamys spectabilis (22.48% of the total abundance), while wheat Qu harbored greater abundances of Saccharopolyspora (54.78%, Saccharopolyspora rectivirgula, Saccharopolyspora shandongensis, Saccharopolyspora hirsuta, Saccharopolyspora spinose, and Saccharopolyspora erythraea). From a functional perspective, the important functions of Daqu and wheat Qu are both amino acid metabolism and carbohydrate metabolism. Meanwhile, a combined analysis among microbiota, functional genes, and dominant flavors indicated S. shandongensis, S. rectivirgula, and S. spinose might be the main contributor to the synthesis of flavor compounds in wheat Qu, while R. emersonii, W. paramesenteroides, Leuconostoc citreum, Leuconostoc mesenteroides, Weissella cibaria and P. pentosaceus may make the greatest contribution to flavor compounds synthesis in Daqu. This study reveals the microbial and functional dissimilarities of Daqu and wheat Qu, and helps elucidating different metabolic roles of microbes during flavor formation.

International audience; Dairy farming is a risk factor for chronic obstructive pulmonary disease (COPD). The aim was to determine predictive markers either in blood samples or in dwelling dust samples by comparing COPD and healthy controls with or without farming activity. Dust was collected and analyzed by real-time quantitative PCR. ELISA and DELFIA® were performed to assay the level of specific IgG and IgE of 10 targeted microorganisms. The dwelling exposure of farmers was higher than in the non-farmers (Especially Eurotium amstelodami and Lichtheimia corymbifera). The IgG response against Wallemia sebi and Saccharopolyspora rectivirgula was more often higher in the farmers than the non-farmers. However, exposure and sensitization to the microorganisms tested cannot explain the occurrence of COPD in the dairy farmers' population. COPD development is probably caused by multiple factors associated with exposure over a period of several years.

Hypersensitivity pneumonitis (HP) is an immune-mediated interstitial lung disease that develops following repeated exposure to inhaled environmental antigens. The disease results in alveolitis and granuloma formation and may progress to a chronic form associated with fibrosis; a greater understanding of the immunopathogenic mechanisms leading to chronic HP is needed. We used the Saccharopolyspora rectivirgula (SR) mouse model of HP to determine the extent to which a switch to a Th2-type immune response is associated with chronic HP. Exposure of wild-type (WT) and tlr2/9−/− mice to SR for 14 wk resulted in neutrophilic and lymphocytic alveolitis that was not dependent on Toll-like receptors (TLRs) 2 and 9. Long-term exposure of WT mice to SR resulted in a significant increase in collagen deposition, protein leakage, and IL-1α accompanied by a decrease in quasistatic compliance and total lung capacity compared with unexposed mice. This was associated with an increase in IL-17 but not IL-4 production or recruitment of Th2 cells. tlr2/9−/− mice exhibited an increase in protein leakage but less IL-1α and collagen deposition in the lungs compared with WT mice, yet they still displayed a decrease in quasistatic compliance, although total lung capacity was not affected. These mice exhibited an increase in both IL-13 and IL-17, which suggests that IL-13 may ameliorate some of the lung damage caused by long-term SR exposure. Our results suggest that lung pathology following long-term SR exposure in WT mice is associated with the IL-17 response and that TLRs 2 and 9 may inhibit the development of the IL-13/Th2 response.

Rationale: Although recent work has shown that CD34 plays an important role in the trafficking of inflammatory cells during Th2-biased inflammatory responses, its role in Th1/Th17-biased disease as well as dendritic cell (DC) trafficking is unknown. Objectives: We used CD34-deficient mice (Cd34−/−) to investigate the role of CD34 in the Th1/Th17-biased lung inflammatory disease, hypersensitivity pneumonitis (HP). Methods: HP was induced in wild-type (wt) and Cd34−/− mice by repeated intranasal administration of Saccharopolyspora rectivirgula antigen. Lung inflammation was assessed by histology and analysis of bronchoalveolar lavage cells. Primary and secondary immune responses were evaluated by cytokine recall responses of pulmonary inflammatory cells as well as draining lymph node cells. Measurements and Main Results: Cd34−/− mice were highly resistant to the development of HP and exhibited an inflammatory pattern more reflective of a primary response to S. rectivirgula rather than the chronic lymphocytosis that is typical of this disease. Cytokine recall responses from Cd34−/− lymph node cells were dampened and consistent with a failure of antigen-loaded Cd34−/− DCs to deliver antigen and prime T cells in the draining lymph nodes. In agreement with this interpretation, adoptive transfer of wt DCs into Cd34−/− mice was sufficient to restore normal sensitivity to HP. CD34 was found to be expressed by wt DCs, and Cd34−/− DCs exhibited an impaired ability to chemotax toward a subset of chemokines in vitro. Finally, expression of human CD34 in Cd34−/− mice restored normal susceptibility to HP. Conclusions: We conclude that CD34 is expressed by mucosal DCs and plays an important role in their trafficking through the lung and to the lymph nodes. Our data also suggest that CD34 may play a selective role in the efficient migration of these cells to a subset of chemokines.

It is considered that hypersensitivity pneumonitis (HP) occurs with a Th1 cell dominance; however, the role of Th1/Th2 balance is still unclear. C57BL/6 (Th1-biased), BALB/c wt (Th2-biased) and BALB/c Stat6 −/− (Th2 deficient) mice were treated with Saccharopolyspora rectivirgula (SR) or saline during 3 weeks, and sacrificed 1 and 4 days (early and late response) after the last administration. Lung isolated T cell subpopulations were analyzed and lung damage extent was quantified. C57BL/6 wt mice exhibited a significant increase in the extent of lung damage when sacrificed at 4 days compared with those sacrificed 1 day after the last SR administration. In contrast, BALB/c wt mice showed a progressive decrease in the extent of lung damage. A significant increase of NKT CD4+ subset was found in C57BL/6 mice while NKT DN cells were increased in BALBc wt mice. Also, NK and γδ T cells were increased in BALB/c mice at 1 and 4 days. Stat6−/− mice behave similar to the C57BL/6 mice, showing a progressive increase in the extent of lung damage. A significant increase in the levels of Th1 and Th2 cytokines was observed in bronchoalveolar lavage from the SR-treated mice. These results confirm a predominant role of the Th1 response in HP and suggest that the control of inflammation by Th2 biased mice may be related with the increase of NKT DN cells and regulatory NK and γδ T cells.

Fas/Fas ligand (FasL)-mediated apoptosis has been implicated in various lung diseases, but whether Fas/FasL-mediated apoptosis in the lungs plays a critical role in the development of hypersensitivity pneumonitis (HP) is unclear.To explore the functional roles of Fas/FasL-mediated apoptosis in HP.Fas-deficient (lpr/lpr), FasL-deficient (gld/gld), and B6 mice were challenged with Saccharopolyspora rectivirgula (SR) antigen intranasally.lpr/lpr and gld/gld mice exhibited attenuation of HP in terms of histological alterations, influx of immune cells in bronchoalveolar lavage fluid (BALF), and SR-specific immune responses compared with B6 mice, similar to the effects of SR in B6 mice given a caspase inhibitor. The lungs of lpr/lpr and gld/gld mice showed high IL-4 production and low IFN-gamma, IL-8, macrophage inflammatory protein-2, IL-1beta, and tumor necrosis factor-alpha production compared with those of B6 mice. Moreover, mice with chimeric B6 and lpr/lpr bone marrow revealed that apoptosis of nonhematopoietic and BALF immune cells of the lungs enhanced immune responses against SR antigen. Gr-1(+) granulocytes in BALF expressed annexin V and their depletion in B6 mice attenuated HP. Apoptosis of nonhematopoietic cells and Gr-1(+) granulocytes in the lungs enhanced the maturation of pulmonary CD11c(+) dendritic cells and their production of macrophage inflammatory protein-1alpha and monocyte chemoattractant protein-1, resulting in recruitment of immune cells into the lungs during HP.These results suggest that apoptosis in nonhematopoietic cells and Gr-1(+) granulocytes of the lungs promotes HP by enhancing maturation and chemokine production of CD11c(+) dendritic cells.

Hypersensitivity pneumonitis is an interstitial lung disease that results from repeated pulmonary exposure to various organic Ags, including Saccharopolyspora rectivirgula, the causative agent of farmer’s lung disease. Although the contributions of proinflammatory mediators to the disease pathogenesis are relatively well documented, the mechanism(s) involved in the initiation of proinflammatory responses against the causative microorganisms and the contribution of signaling molecules involved in the host immune defense have not been fully elucidated. In the current study, we found that S. rectivirgula induces the activation of protein kinase D (PKD)1 in lung cells in vitro and in vivo. Activation of PKD1 by S. rectivirgula was dependent on MyD88. Inhibition of PKD by pharmacological PKD inhibitor Gö6976 and silencing of PKD1 expression by small interfering RNA revealed that PKD1 is indispensable for S. rectivirgula-mediated activation of MAPKs and NF-κB and the expression of various proinflammatory cytokines and chemokines. In addition, compared with controls, mice pretreated with Gö6976 showed significantly suppressed alveolitis and neutrophil influx in bronchial alveolar lavage fluid and interstitial lung tissue, as well as substantially decreased myeloperoxidase activity in the lung after pulmonary exposure to S. rectivirgula. These results demonstrate that PKD1 is essential for S. rectivirgula-mediated proinflammatory immune responses and neutrophil influx in the lung. Our findings also imply the possibility that PKD1 is one of the critical factors that play a regulatory role in the development of hypersensitivity pneumonitis caused by microbial Ags and that inhibition of PKD1 activation could be an effective way to control microbial Ag-induced hypersensitivity pneumonitis.

CD4+CD25+ Treg cells play a protective role in hypersensitivity pneumonitis by inhibiting IFN-γ-producing T cells. HP results from the repeated inhalation of environmental antigens; however, the roles of CD4+CD25+ Treg cells in HP are unknown. Therefore, we investigated the functions of CD4+CD25+ Treg cells in SR-induced murine HP. More severe HP was observed in CD4+CD25+ Treg cell-depleted mice than in control mice in terms of histological alterations, inflammatory cell numbers in BALF, and the serum level of SR-specific IgG, which were restored by the adoptive transfer of CD4+CD25+ Treg cells. The CD4+CD25+ Treg cell-depleted mice also showed elevated levels of IFN-γ, TGF-β, and reduced IL-4 production in the lungs. Moreover, IL-10 production of CD4+CD25+ Treg cells and direct contact between CD4+CD25+ Treg cells and CD4+ or CD8+ T cells in BALF resulted in reduced IFN-γ production. Taken together, CD4+CD25+ Treg cells play a protective role in SR-induced HP by suppressing IFN-γ production by T cells.

The present study verified the hypothesis that enhanced maturation of antigen-presenting CD11c + cells could explain the viral-induced exacerbated immune response to Saccharopolyspora rectivirgula (SR), the main antigen responsible for farmer’s lung, a classic form of hypersensitivity pneumonitis (HP). Four groups of mice were studied: group 1 received intranasal instillations of saline; group 2 received instillations of SR for 12 weeks; group 3 received instillations of saline and a single infection with Sendai virus on week 3; and group 4 received instillations of SR for 12 weeks with a single administration of Sendai virus on week 3. On week 13, mice were sacrificed and bronchoalveolar lavage was performed. Lungs were harvested, digested with enzymes, and CD11c + cells were analysed in flow cytometry with anti-CD11c, anti-CD86 and anti-major histocompatibility complex class II markers. Immunofluorescence studies were also performed with the same cell surface markers. Both flow cytometry and immunofluorescence results demonstrate that mature CD11c + cells are significantly enhanced in SR-challenged mice simultaneously infected with Sendai virus, compared with other groups. These CD11c + cells persist in the lung for 9 weeks after the virus infection. Maturation of CD11c + cells could explain, at least in part, the virus-induced increased immune response to SR antigens in this model of HP, but mechanisms have still to be elucidated.

Hypersensitivity pneumonitis (HP) is mediated by a Th1 immune response. Transcription factor GATA binding protein-3 (GATA-3) is believed to be a key regulator of Th2 differentiation and thus might play regulatory roles in the pathogenesis of hypersensitivity pneumonitis (HP).We examined the effect of GATA-3 overexpression on the development of HP in mice.Wild-type C57BL/6 mice and GATA-3-overexpressing mice of the same background were used in this study. HP was induced by repeated exposure to Saccharopolyspora rectivirgula, the causative antigen of farmer's lung.Antigen exposure resulted in a marked inflammatory response with enhanced pulmonary expression of T-bet and the Th1 cytokine interferon (IFN)-gamma in wild-type mice. The degree of pulmonary inflammation was much less severe in GATA-3-overexpressing mice. The induction of T-bet and IFN-gamma genes was suppressed, but a significant induction of Th2 cytokines, including IL-5 and IL-13, was observed in the lungs of GATA-3-overexpressing mice after antigen exposure. Supplementation with recombinant IFN-gamma enhanced lung inflammatory responses in GATA-3-overexpressing mice to the level of wild-type mice. Because antigen-induced IFN-gamma production predominantly occurred in CD4+ T cells, nude mice were transferred with CD4+ T cells from either wild-type or GATA-3-overexpressing mice and subsequently exposed to antigen. Lung inflammatory responses were significantly lower in nude mice transferred with CD4+ T cells from GATA-3-overexpressing mice than in those with wild-type CD4+ T cells, with a reduction of lung IFN-gamma level.These results indicate that overexpression of GATA-3 attenuates the development of HP by correcting the Th1-polarizing condition.

Hypersensitivity pneumonitis (HP) is mediated by Th1 immune response. NKT cells regulate immune responses by modulating the Th1/Th2 balance. Therefore, we postulated that NKT cells play a critical role in the development of the HP by modulating the Th1/Th2 response. To address this issue, we explored the functional roles of NKT cells in Saccharopolyspora rectivirgula (SR)-induced HP. In CD1d−/− mice, the HP was worse in terms of histological changes, hydroxyproline levels, the CD4:CD8 ratio in bronchoalveolar lavage fluid, and SR-specific immune responses than in control mice. CD1d−/− mice showed elevated IFN-γ production in the lung during the HP, and this was produced mainly by Gr-1+ neutrophils. The blockade of IFN-γ in CD1d−/− mice attenuated the HP, whereas the injection of rIFN-γ aggravated it. Moreover, the depletion of Gr-1+ neutrophils reduced CD8+ T cell numbers in bronchoalveolar lavage fluid during the HP. The adoptive transfer of IL-4−/− mouse NKT cells did not attenuate the HP, whereas wild-type or IFN-γ−/− mouse NKT cells suppressed the HP. In conclusion, NKT cells producing IL-4 play a protective role in SR-induced HP by suppressing IFN-γ-producing neutrophils, which induce the activation and proliferation of CD8+ T cells in the lung.

Saccharopolyspora rectivirgula is the main cause of farmer's lung disease. The development of recombinant antigens to standardize the serodiagnosis of the disease requires knowledge of the S. rectivirgula genome. We sequenced the genome of an environmental strain, S. rectivirgula DSM 43113. A total of 3,221 proteins were found to be encoded in a short 3.9-Mb genome.

PURPOSE: Saccharopolyspora rectivirgula is the principal cause of farmer's lung disease (FLD). Serodiagnosis is based on immunoprecipitation techniques or enzyme immunoassays with homemade crude antigens and is not standardized. We aimed to produce specific recombinant antigens for the development of a standardized ELISA. EXPERIMENTAL DESIGN: We recruited 41 patients and 43 healthy exposed controls from five university hospital pneumology departments in France and Switzerland. S. rectivirgula proteins were extracted, separated by 2D electrophoresis, and subjected to Western blotting, with sera from FLD patients or controls. FLD-specific proteins were identified by MS and were produced as recombinant antigens. The diagnostic performance of ELISA tests using the recombinant antigens was assessed with all the sera from FLD patients and controls. RESULTS: We identified 25 FLD-specific proteins, some of which play important roles in transport, nutrition, or virulence. We produced 17 of these proteins as recombinant antigens and assessed their suitability for inclusion in the ELISA test. A combination of three of these proteins (SR1FA, SR17, and SR22) proved remarkably effective at discriminating between patients and controls, with a sensitivity of 83% and a specificity of 77%. CONCLUSIONS AND CLINICAL RELEVANCE: The recombinant antigens produced in this study constitute a major step toward the improvement of diagnostic performance and the standardization of FLD serodiagnosis.

Background and Objectives: In immunocytochemical preparations of bronchoalveolar lavage (BAL) cells from patients with extrinsic allergic alveolitis (EAA), we observed the presence of alveolar macrophages with cockade-like structures in their cytoplasm (cockade+ alveolar macrophages). These cockade+ alveolar macrophages may reflect a subpopulation of alveolar macrophages which may show a different predominance in various interstitial lung diseases. In this study we aimed to compare the frequency of cockade+ alveolar macrophages in patients with EAA (n = 14) with the results obtained in patients with sarcoidosis (n = 11), idiopathic interstitial pneumonia (IIP; n = 10) and control subjects (n = 8). We also investigated the expression of the transferrin receptor CD71 on cockade+ alveolar macrophages. Methods: In BAL fluid, the total number of cells and differential counts were determined, and immunocytologic examinations of macrophages and lymphocytes were done using monoclonal antibodies. The percentage of cockade+ alveolar macrophages was determined by counting 300 macrophages in the CD20 field of an immunocytochemical slide. Results: The percentage of cockade+ alveolar macrophages was significantly higher in the EAA group (36 ± 9%) compared to patients with sarcoidosis (12 ± 5%) or IIP (11 ± 10%) and control subjects (3 ± 1%; p < 0.001). The proportion of CD71+ alveolar macrophages was significantly lower in EAA than in the other groups (p < 0.01), and the CD71 antigen was expressed on a significantly lower proportion of cockade+ alveolar macrophages compared to cockade– alveolar macrophages in EAA (p < 0.001). Conclusion: We conclude that cockade+ alveolar macrophages could play a role in the pathogenesis and differential diagnosis EAA.

Hypersensitivity pneumonitis (HP) is an interstitial lung disease that develops following repeated exposure to inhaled particulate antigens. Individuals with HP develop lymphocytic alveolitis,granuloma formation, and fibrosis. HP is categorized as a Th1 disease, and granuloma formation is dependent on T cells and the Th1 cytokine IFN-gamma. We therefore hypothesized that the IFN-gamma-inducible chemokines IP-10, Mig, and I-TAC, which are frequently associated with Th1 diseases, would play an important role in the pathogenesis of disease. We analyzed the expression of multiple chemokines in the lungs of wild-type (WT) and IFN-gamma-knockout (GKO) mice exposed to the particulate antigen Saccharopolyspora rectivirgula (SR). Our results demonstrate the production of IP-10, Mig, and I-TAC in WT mice during the development of HP, whereas GKO mice have reduced levels of IP-10 and no Mig or I-TAC mRNA in the lungs in response to SR exposure. The production of these chemokines is associated with an influx of CXCR3+/CD4+ T cells into lungs of WT mice, which is reduced in GKO mice. These results suggest that IFN-gamma mediates the recruitment of CXCR3+/CD4+ T cells into the lung via production of the chemokines IP-10, Mig, and I-TAC, resulting in granuloma formation.

Summary. Inhalation of Saccharopolyspora rectivirgula (SR) can cause the disease Farmer's Lung, a classic example of hypersensitivity pneumonitis. Th1, but not Th2, cell lines can adoptively transfer experimental hypersensitivity pneumonitis (EHP). Substantial amounts of IL12 appear in bronchoalveolar lavage fluid (BALF) after a single intratracheal (IT) injection of SR, and SR-induced IL12 secretion by both a macrophage cell line and alveolar macrophages. We tested the hypothesis that IL12 is essential for the development of EHP by addition of anti-IL12 to cultured cells, and adoptive transfer of EHP in IL12p40–/– animals. We transferred SR cultured spleen and lung associated lymph node cells from SR sensitized mice (both IL12p40–/– and wild type), to naive recipients (both wild type and IL12p40–/–). The addition of anti-IL12 to cultures of sensitized cells could not ablate the ability of these cells to transfer EHP. Cultured cells from IL12p40–/– animals were fully capable of transferring EHP. In contrast, IL12p40–/– recipients of both wild type and IL12p40–/–-cultured cells were less able to express EHP (lung histology and BALF characteristics) than wild type mice, and had more eosinophils in both lung tissue and BALF. We conclude that IL12 is not necessary for development of cells able to adoptively transfer EHP, but that it is required for full expression of EHP in recipient animals.

Background Resident gut microbiota are now recognized as potent modifiers of host immune responses in various scenarios. Recently, we demonstrated that perinatal exposure to vancomycin, but not streptomycin, profoundly alters gut microbiota and enhances susceptibility to a T H 2 model of allergic asthma. Objective Here we sought to further clarify the etiology of these changes by determining whether perinatal antibiotic treatment has a similar effect on the T H 1/T H 17-mediated lung disease, hypersensitivity pneumonitis. Methods Hypersensitivity pneumonitis was induced in C57BL/6 wild-type or recombination-activating gene 1–deficient mice treated perinatally with vancomycin or streptomycin by repeated intranasal administration of Saccharopolyspora rectivirgula antigen. Disease severity was assessed by measuring lung inflammation, pathology, cytokine responses, and serum antibodies. Microbial community analyses were performed on stool samples via 16S ribosomal RNA pyrosequencing and correlations between disease severity and specific bacterial taxa were identified. Results Surprisingly, in contrast to our findings in an allergic asthma model, we found that the severity of hypersensitivity pneumonitis was unaffected by vancomycin, but increased dramatically after streptomycin treatment. This likely reflects an effect on the adaptive, rather than innate, immune response because the effects of streptomycin were not observed during the early phases of disease and were abrogated in recombination-activating gene 1–deficient mice. Interestingly, Bacteroidetes dominated the intestinal microbiota of streptomycin-treated animals, while vancomycin promoted the expansion of the Firmicutes . Conclusions Perinatal antibiotics exert highly selective effects on resident gut flora, which, in turn, lead to very specific alterations in susceptibility to T H 2- or T H 1/T H 17-driven lung inflammatory disease.

We have sequenced the genome of Saccharopolyspora rectivirgula , the causative agent of farmer's lung disease. The draft genome consists of 182 contigs totaling 3,977,051 bp, with a GC content of 68.9%.

Institut Universitaire de Cardiologie et de Pneumologie de Quebec (IUCPQ), Quebec City, Quebec Correspondence: Dr Yvon Cormier, Institut Universitaire de Cardiologie et de Pneumologie de Quebec (IUCPQ), 2725 Chemin Ste-Foy, Quebec City, Quebec G1V 4G5. Telephone 418-656-8711 ext 95280, e-mail yvon.cormier@med.ulaval.ca Hypersensitivity pneumonitis (HP) is a respiratory disease caused by a delayed immune response to a variety of antigens, primarily consisting of bacteria, fungi and bird proteins. Symptoms include shortness of breath, cough and fever, which occur within hours of exposure to the offending antigen. Continuous contact with the antigen can lead to permanent lung damage. HP was first described in farmers >100 years ago (1), while its cause and immune mechanisms were identified in the early 1960s by Dr Jack Pepys (2). Canadian contributions to the understanding of HP date back to the early 1970s, when Drs Peter C Warren and Freddy Hargreave, both fellows with Dr Pepys, came to Canada from the United Kingdom. While working with Dr Pepys, Dr Hargreave developed an antigen provocation test to help diagnose bird fancier’s lung (3). On arrival to Canada (McMaster University, Hamilton, Ontario), he published an extensive review on HP in the Canadian Medical Association Journal (4). He subsequently switched the focus of his research to asthma to become one of Canada’s leading experts on the subject. Dr Peter C Warren (University of Manitoba, Winnipeg, Manitoba) published an article in 1977 that had a major impact on our understanding of the pathophysiology of HP (5). He reported that cigarette smokers exposed to farmer’s lung antigen, Saccharopolyspora rectivirgula (SR, then called Micropolysporafaeni), had significantly lower levels of serum-specific antibodies to this bacteria. This observation led to numerous questions that will be addressed later. The major Canadian contribution to the understanding of HP came from Laval University in Quebec City, Quebec. Since 1982, investigators have published 84 original and review articles, and 16 book chapters on the subject. Farmers played a major role in the prevention of farmer’s lung. When I started my practice in Quebec City in 1977, we were seeing between 20 and 30 new cases of farmer’s lung per year. Now we may see one every two or three years. The difference? Not scientific research, but farming technology. The solution was to eliminate hay and straw molding. This would prevent farmer’s lung and improve the quality of these stored foliages. Farmers could not change the wet climate in Eastern Canada, which resulted in hay being stored too wet, leading to molding. The solution was in the methods of hay making and storage. The first trials of these new methods of handling hay were not all successful. Investigators added acetic acid to the hay, but that caused corrosion of the bailers. One company sold sachets of lactobacillus, which the farmers inoculated hay with. The concept was that if the lactobacillus grew first, it would prevent SR from growing. One of my graduate students at the time, Dr Caroline Duchaine, now a highly successful aerobiologist at Laval University, challenged that concept. What she did was simple. She took samples of hay at different humidity levels and performed a double-blinded test with or without the lactobacillus. She published the results of that study (6), which, to us, confirmed the futility of the treatment. More successful approaches included crushing freshly cut hay through a type of wringer to squeeze out most of the juice; equipping barns with huge fans that act as hay dryers; and wrapping hay in plastic bags, preventing the entry of oxygen, which is essential for SR growth. The latter solution, although effective, is environmentally questionable and certainly spoils the view of the countryside. Although farmer’s lung is related to humid summer conditions typical in Eastern Canada, Dr James Dosman’s group identified cases in the drier climates of Saskatchewan (7). Most importantly, in Saskatchewan, the disease is probably related to different antigens (penicillium species) rather than SR, which causes HP in more humid environments. This antigen was also found to be responsible for HP in a workplace in Toronto, Ontario (8). Now that farmers have almost eliminated farmer’s lung, do we still see HP in Canada? Yes we do. HP is no longer a farmer’s issue but one of multiple workplaces and the home. The number of settings, environments and causes of HP is continually increasing. Metal workers are exposed to aerosolized metal working fluid that often contains mycobacteria that can cause HP (9). In 2004, Dr Caroline Duchaine and her team studied the microflora of metal fluid used in a major automobile manufacturer in Ontario after they had outbreaks of metal-fluid HP. She found that when mycobacteria are established in the biofilms of the fluid system, it is very difficult, if not impossible, to eradicate them (10). We also observed HP in wood transformation plants (11), peat moss packaging units (12) and in a duck abattoir (unpublished personal observation). HP can be caused by molds that grow in saxophones (13) and clothes dryer vents, or can be associated with raising pigeons, doves or love birds, or having down pillows in the home. Dr Hubert Reynolds (Hershey, Pennsylvania, USA) was the first to describe the accumulation of large number of inflammatory lymphocytes in the lungs of patients with HP (14). The question raised by that report involved the peculiar case of asymptomatic individuals who are exposed to an environment that can cause HP who have serum antibodies (precipitins) to the antigen. In the early 1980s, we studied several asymptomatic dairy farmers with serum antibodies to the SR antigen and found that most of them also had an increased number of lymphocytes in their lungs (15). Although they exhibited an inflammatory reaction in their lungs, they remained asymptomatic, with no lung damage over the 20 years they were followed-up (16). Returning to Dr Warren’s article on cigarette smoking, we tested a model of HP in guinea pigs. A group of animals was exposed to cigarette smoke (provided by Rothman, British American Tobacco, USA) while a control group was not. Cigarette smoke decreased the inflammatory response to the antigen challenge (17). Nicotine was considered to be the likely substance that protected against HP (18), as it does for other diseases such as ulcerative colitis (19). It has often been observed that at the beginning of an HP attack, subjects report flu-like symptoms. We found that a viral infection could destabilize the immune response and trigger HP (20). We also reported the presence of viral antigen in lungs of HP patients (21). In 1990, Dr Marc Lalancette, a medical resident at Laval, made an important observation that was published in the American Journal of Respiratory and Critical Care Medicine (22). He found that, in farmers who experienced permanent lung damage from farmer’s lung disease, it CommentAry

International audience; We propose using the surface of pine trees needles to biomonitor the bioaerosol emissions at a composting plant. Measurements were based on 16S rRNA gene copy numbers of Saccharopolyspora rectivirgula, a bioindicator of composting plant emissions. A sampling plan was established based on 29 samples around the emission source. The abundance of 16S rRNA gene copies of S. rectivirgula per gram of Pinus halepensis needles varied from 104 to 102 as a function of the distance. The signal reached the background level at distances around the composting plant ranging from 2 km to more than 5.4 km, depending on the local topography and average wind directions. From these values, the impacted area around the source of bioaerosols was mapped.

Cultured murine CD4+ cells from Saccharopolyspora rectivirgula sensitized C3H/HeJ (Th1 bias) donors can adoptively transfer murine experimental hypersensitivity pneumonitis (EHP). We sensitized BALB/c mice (Th2 bias) with S. rectivirgula, obtained spleen and lung associated lymph node (LALN) cells, cultured the cells with specific antigen, and attempted adoptive transfer of EHP. We also treated both C3H/HeJ and BALB/c donor mice with IL4 and anti-IFNγ before exposure to S. rectivirgula and then cultured cells from both spleen and LALN before attempted transfer of EHP. We found that cultured spleen and lung associated lymph node cells can adoptively transfer EHP in both C3H/HeJ and BALB/c mice as demonstrated by infiltration of the recipient lungs with CD4+ lymphocytes. Treatment of both mouse strains with IL4 and anti-IFNγ did not change the ability of cultured cells to adoptively transfer EHP. We conclude that EHP induced by S. rectivirgula can occur in animals with either a Th1 or a Th2 bias and is not altered by treatment with IL4 and anti-IFNγ. This suggests that attributes of the antigen and not genetic background or cytokine environment at the site of initial sensitization determines the results of exposure to S. rectivirgula.

Farmer's lung disease (FLD) is common in the east of France. In the absence of the primary recognized FLD agent, Saccharopolyspora rectivirgula, its etiology remains unknown. A prospective case-control study was performed to find the etiology of FLD in this area. Eleven patients were matched with 11 healthy control farmers. Twenty-two urban subjects constituted the nonexposed control group. Microorganisms from cowshed air and fodder were identified and counted. The antigens of the microorganisms most frequently isolated at the 22 farms were used for serological tests. Farms of patients with FLD contained more Absidia corymbifera than those of healthy farmers (p < 0.05 in air, p < 0.01 in fodder). Electrosyneresis, performed with A. corymbifera somatic antigen, differentiated 9 of 11 patients with FLD from control subjects (p < 0.01). Other significant results were obtained with Eurotium amstelodami (p < 0.01) and Wallemia sebi (p < 0.05). In contrast, no significant results were obtained with the other seven antigens tested, including S. rectivirgula. Absidia corymbifera and, to a lesser degree, W. sebi or E. amstelodami are likely to be the main causes of FLD in this area. Modifications in working conditions over time could explain the emergence of these new contributing etiologies.

Study objectives: Serum KL-6 (Krebs von den Lungen-6) has been recognized to be a marker for the activity of diffuse interstitial lung diseases. The purpose of the study is to evaluate serum KL-6 measurement as a marker for farmer’s lung disease (FLD). Design: A cross-sectional survey of a cohort of dairy farmers. Retrospective measurement of KL-6 stored serum samples from those dairy farmers previously screened for FLD. Setting: University hospital screening project for FLD within a dairy-farming community in Japan. Participants: Four hundred seventy-two dairy farmers were invited to attend a local clinic. Measurements and results: We examined serum KL-6 concentrations in 272 farmers. Subjects were classified into three groups: (1) 5 farmers with FLD, (2) 30 farmers with positive serum precipitating antibodies to Saccharopolyspora rectivirgula and/or Thermoactinomyces vulgaris without FLD (Ab 1 ), and (3) 237 farmers without these antibodies (Ab 2 ). Serum KL-6 concentrations in the FLD group were significantly higher than those in the Ab 1 and the Ab 2 groups (1,263 6 288 [SEM], 328 6 57, and 207 6 6 U/mL, respectively, p < 0.001). Serum KL-6 concentrations in those with FLD were significantly higher than KL-6 concentrations from stored screening samples from the same individual when FLD was not diagnosed (1,263 6 288 and 419 6 209 U/mL, respectively, p < 0.05). Serum KL-6 concentrations of the Ab 1 group were significantly higher than those of the Ab 2 group (p < 0.001). In the Ab 1 group, farmers with high serum KL-6 concentrations had lower permeability coefficients than farmers with normal serum KL-6 concentrations (p < 0.05). These results may suggest that subclinical FLD can be detected in farmers with high KL-6 concentrations and precipitating antibodies. Conclusion: Serum KL-6 concentration can be a useful marker for assessing the activity of FLD and may be able to be used to detect subclinical disease. (CHEST 2000; 118:445‐ 450)

To examine the role of His452 of the Saccharopolyspora rectivirgula β-galactosidase in the binding of a tightly bound, catalytically important Mn 2+ ( i.e. , class II Mn 2+ ) ion, His452 was replaced with Phe or Glu and the respective site-directed mutants, H452F and H452E, were characterized. Neither mutant contained Mn 2+ in an Mn 2+ -free buffer and both were virtually inactive in the absence of Mn 2+ (their relative activities being less than 0.03% that of the fully activated wild-type enzyme). When Mn 2+ was added, however, the mutants were activated to 3% (for H452F) and 0.8% (for H452E) of the full activity of the wild type. The Mn 2+ concentrations needed for half-maximal activation of H452F and H452E were, respectively, 15,000 and 5000 times higher than the reported dissociation constant (2 nM) of the class II Mn 2+ , suggesting that His452 plays a key role in the binding of this catalytically important Mn 2+ . Activation of the mutants by Mn 2+ , albeit very weak, contrasts with a lack of any such metal activation previously observed with the two corresponding mutants of Escherichia coli lacZ β-galactosidase.

Hypersensitivity pneumonitis (HP) is a granulomatous inflammatory lung disease that is usually triggered by organic antigens. At early time points after inhalation of antigen, neutrophilic inflammation is prominent in the lungs. Interleukin (IL)-8 is a potent chemoattractant for neutrophils and it is known that alveolar macrophages can release IL-8 after exposure to organic antigens. However, the role of respiratory epithelial cells in the production of IL-8 in HP is unknown. We exposed A549 epithelial cells to the thermophilic bacteria Saccharopolyspora rectivirgula (SR), and measured IL-8 release via enzyme-linked immunosorbent assay (ELISA) and IL-8 messenger RNA (mRNA) induction via Northern analysis. We observed a dose- and time-dependent release of IL-8 in response to SR. The maximal release of IL-8 was measured at 24-48 hours after exposure. There was also an increase in release of IL-6 in a time-dependent fashion. SR induced a peak increase in IL-8 mRNA at 12-24 hours. SR also triggered expression of the DNA-binding activity of NF-kappa B, a transcription factor that mediates activation of the IL-8 gene. Both corticosteroids and IL-10 blocked the production of IL-8. The release of IL8 was not mediated through IL-1 beta. These data suggest that SR-induced IL-8 production in airway epithelium may play a role in the initial inflammatory response in HP.

Pediococcus pentosaceus is a lactic-acid producing bacterium inoculated in hay to prevent hay deterioration. This study sought to verify the effect of this treatment on the barn microenvironment. Air samples were obtained from 19 barns using bacterial hay treatment and from 18 control barns with six-stage Andersen samplers and all-glass impingers. Appropriate culture media were used for the recovery and identification of microorganisms. Endotoxins were measured with chromogenic Limulus amoebocyte lysate assay. Median values (respectively for treated and untreated hay barns) were: 5.28 x 10(5) and 3.84 x 10(5) colony-forming units (CFU)/m3 for total bacteria; 3.18 x 10(6) and 4.5 x 10(6) CFU/m3 for molds; 1.36 x 10(3) and 1.74 x 10(3) endotoxin units/m3 for endotoxin levels; and 1.03 x 10(3) and 3.00 x 10(3) CFU/m3 for Saccharopolyspora rectivirgula. No viable P. pentosaceus were recovered. The presence of S. rectivirgula, the causative agent for farmer's lung, was not influenced by the hay treatment. Since no significant difference was observed in any of the airborne contaminants, this type of hay treatment probably does not protect farmers from the respiratory effect of ambient microbial contaminants.

Our study aimed to elucidate the clinical role of Absidia corymbifera as an antigen of farmers lung. Using the ELISA technique, a total of 76 serum samples of patients with suggested farmers lung were investigated for IgG-antibodies against A. corymbifera as well as against Saccharopolyspora rectivirgula, Thermoactinomyces vulgaris, and Aspergillus fumigatus. Only 5 of76 (7%) sera revealed antibodies against A. corymbifera, all of which presenting with at least one other positive antigen. We conclude that in Germany an investigation for IgG-antibodies against A. corymbifera does not improve the sensitivity of the diagnosis of farmers lung. However, this should be a focus of further research.

Hypersensitivity pneumonitis (HP) is an immunoallergic disease characterized by a prominent interstitial infiltrate composed predominantly of lymphocytes secreting inflammatory cytokines. Dendritic cells (DCs) are known to play a pivotal role in the lymphocytic response. However, their cross talk with microorganisms that cause HP has yet to be elucidated. This study aimed to investigate the initial interactions between human monocyte-derived DCs (MoDCs) and four microorganisms that are different in nature ( Saccharopolyspora rectivirgula [actinomycetes], Mycobacterium immunogenum [mycobacteria], and Wallemia sebi and Eurotium amstelodami [filamentous fungi]) and are involved in HP. Our objectives were to determine the cross talk between MoDCs and HP-causative agents and to determine whether the resulting immune response varied according to the microbial extract tested. The phenotypic activation of MoDCs was measured by the increased expression of costimulatory molecules and levels of cytokines in supernatants. The functional activation of MoDCs was measured by the ability of MoDCs to induce lymphocytic proliferation and differentiation in a mixed lymphocytic reaction (MLR). E. amstelodami -exposed (EA) MoDCs expressed higher percentages of costimulatory molecules than did W. sebi -exposed (WS), S. rectivirgula -exposed (SR), or M. immunogenum -exposed (MI) MoDCs ( P < 0.05, Wilcoxon signed-rank test). EA-MoDCs, WS-MoDCs, SR-MoDCs, and MI-MoDCs induced CD4 + T cell proliferation and a Th1-polarized immune response. The present study provides evidence that, although differences were initially observed between MoDCs exposed to filamentous fungi and MoDCs exposed to bacteria, a Th1 response was ultimately promoted by DCs regardless of the microbial extract tested.

Exposure to bioaerosols in composting plants can lead to negative health effects on compost workers. Health complaints vary between cough, irritation of the eyes and the skin, sinusitis, or dyspnea among others. It is fact that compost materials harbor high concentrations of microorganisms, which were aerosolized during handling compost. Within the present study, total cell numbers between 3.4 × 10(4) and 1.6 × 10(8) cell counts per m(3) air were determined after 4',6-Diamidin-2-phenylindol DAPI staining in 124 samples from German composting plants. Special attention should be paid to some specific microorganisms, which are able to cause health complaints. Saccharopolyspora rectivirgula, known to be one of the major causes of extrinsic allergic alveolitis (EAA, also called hypersensitivity pneumonitis, HP), was often found in environments of agricultural production, where the classical form of EAA ('farmer's lung disease') is common, but also in composting plants. In Germany, cases are known where workers had to terminate their work due to this disease. However, up to now, the relevance of S. rectivirgula at composting plants is unexplained. This study showed that high concentrations of airborne S. rectivirgula were found in composting plants similar to that found in agricultural production. Altogether, in 86.7% of the 124 analyzed samples, S. rectivirgula was detected using quantitative real-time polymerase chain reaction (PCR). Estimated concentrations ranged between 1.24 × 10(2) cell counts of S. rectivirgula per cubic meter air next to the rotted residues and 1.5 × 10(7) cell counts next to a converter. Furthermore, our methodical proceedings were verified. To analyze DNA extraction limits through the amount of cells within one sample, the DNA concentration was compared with total cell counts (TCCs). Altogether, when TCC was 2.0 × 10(6) cells were necessary to measure DNA concentrations in 90% of the analyzed samples, whereas cell counts 3.5 × 10(6) cells must be sampled for one DNA extraction assay. With this study, the real-time PCR approach for the detection of S. rectivirgula at workplaces in compost plants was revised, and the results revealed that this method is suitable for occupational exposure measurements.

To understand the etiology of exposure-related diseases and to establish standards for reducing the risks associated with working in contaminated environments, the exact nature of the bioaerosol components must be defined. Molecular biology tools were used to evaluate airborne bacterial and, for the first time, archaeal content of dairy barns. Three air samplers were tested in each of the 13 barns sampled. Up to 10 6 archaeal and 10 8 bacterial 16S rRNA genes per m 3 of air were detected. Archaeal methanogens, mainly Methanobrevibacter species, were represented. Saccharopolyspora rectivirgula , the causative agent of farmer's lung, was quantified to up to 10 7 16S rRNA genes per m 3 of air. In addition, a wide variety of bacterial agents were present in our air samples within the high airborne bioaerosol concentration range. Despite recommendations regarding hay preservation and baling conditions, farmers still develop an S. rectivirgula -specific humoral immune response, suggesting intense and continuous exposure. Our results demonstrate the complexity of bioaerosol components in dairy barns which could play a role in occupational respiratory diseases.

To understand the roles of metal ions on the catalytic properties and thermostability of the thermostable beta-galactosidase of Saccharopolyspora rectivirgula, a thermophilic actinomycete, we have investigated the binding kinetics and requirements of divalent metal ions by equilibrium dialysis, titration, and metal ion buffer techniques. We found that the monomeric multimetal enzyme (M(r) 136,977) had eight specific binding sites for divalent metal ions. These sites were classified as follows: a very tight (class I) site for Ca2+, three tight (class II) sites consisting of two Ca(2+)-specific sites (class IICa) and one Mn(2+)-specific site (class IIMn; Kd for Mn2+, 2.0 nM), and four loose (class III) sites for Mn2+ (Kd, 1.2 microM) and Mg2+ (Kd, 2 microM). Removal of metal ions bound to class II and III sites of the holoenzyme (Ca3Mn5 species; relative Vmax (Vrel), 100%) by a chelating resin at 4 degrees C yielded a less thermostable Ca1 species (Vrel, 1.7%) with a class I Ca2+ ion, removal of which by a chelating resin at 50 degrees C caused a complete irreversible inactivation of the enzyme. Titration studies revealed that stoichiometric binding of Mn2+ to a class IIMn site of the Ca1 species caused a 33-fold activation whereas binding of Ca2+ to class IICa sites had no effect on enzyme activity. Ca1 species could be also activated 8-fold by heating at 60 degrees C for 20 min, suggesting that the catalytically important class II Mn2+ plays important roles in maintaining the native structure essential for activity. Occupation of class III sites by Mg2+ or Mn2+ was of physiological importance to attain sufficient thermostability by which this extracellular beta-galactosidase remained active for a prolonged time at elevated temperatures as was observed during growth of S. rectivirgula.

Concentrations of microorganisms, dust and bacterial endotoxin were determined in the air of 18 animal houses (four cowsheds, nine horse stables and five piggeries) in eastern Poland. Total mesophilic bacteria numbered 568.7–1480.7 colony forming units (CFU)x103 m-3 in piggeries, 43.9–280.5 CFU×103 m-3 in cowsheds, and 26.2–150.1 CFU×103 m-3 in horse stables. Corynebacteria (Corynebacterium spp., Arthrobacter spp.) predominated. Concentrations of Gram-negative bacteria, thermophilic actinomycetes and fungi were smaller (mostly in the range of 102-104 CFU m-3) except for one cowshed and two stables where concentrations of thermophilic actinomycetes exceeded 100 CFU×103 m-3. Thermoactinomyces vulgaris and Saccharopolyspora rectivirgula, both known to be strongly allergenic, were the predominant actinomycetes. Concentrations of airborne dust were 0.15–14.05 mg m-3. There were large concentrations of bacterial endotoxin in the air of piggeries, in the range 1.88–75.0 μg m-3, but concentrations were m...

The current study was done to look at the long-term enhancing effect of a single viral infection on repeated Saccharopolyspora rectivirgula (SR) antigenic challenges in mice and at inflammatory cytokines in this enhancement. Four groups of C57BI/6 mice were studied: Group 1 received intranasal instillations of saline, 3 days per week; Group 2, intranasal instillations of saline plus one intranasal instillation of 80 hemagglutination units (HAU) of Sendai virus after 3 wk of saline; Group 3, instillations of SR, 3 days per week; and Group 4, instillations of SR, 3 days per week, plus one instillation of Sendai virus after 3 wk of SR. Bronchoalveolar lavages (BAL) were performed 15 and 30 wk after the virus inoculation in the appropriate groups. Each time, a two- to threefold increase in BAL cell counts was obtained from virus-infected animals challenged with SR compared with animals that received the SR alone. Animals infected with virus only showed values similar to those of control animals. A higher percentage of large foamy multinucleated cells were found in the BAL from the SR+Sendai group than the SR alone group (7.92 +/- 0.730% compared with 1.8 +/- 0.296%). These cells were not seen in the other groups. BAL levels of TNF-alpha and IL-1 alpha at 15 wk were much higher in SR+Sendai-treated (1,439 +/- 268 and 96 +/- 9 pg/ml, respectively) than SR alone-treated animals (361 +/- 100 and 23 +/- 4 pg/ml). BAL fluid of control animals and Sendai alone animals contained 64 +/- 24 and 65 +/- 15 pg/ml of TNF-alpha, but no IL-1 alpha was detected.(ABSTRACT TRUNCATED AT 250 WORDS)

Thermophilic actinomycetes were isolated from 163 (48.95%) of 333 samples of vegetable substrates and soil from different sites in Anambra and Enugu States in Nigeria. Thermoactinomyces (Tha.) vulgaris was the most common, occurring in 32.4% of samples while Tha. thalpophilus was isolated from 20.1%. Tha. sacchari, Saccharomonospora (Sam.) viridis and Saccharopolyspora (Sap.) rectivirgula were isolated from 3-10.5% of the samples examined. Streptomyces (Stm.) thermovulgaris occurred in 2.7% and Thermomonospora spp. in 0.6% of samples. Except for Stm. thermovulgaris and, perhaps, Thermomonospora spp., all the species isolated are known to be allergenic in man. The five known allergenic species were abundant in hay and maize silage. T. vulgaris and T. thalpophilus were also frequently isolated from rice straw. Tha. vulgaris, Tha. thalpophilus and Sap. rectivirgula were all most numerous in hay while Tha. sacchari was most numerous in maize silage. The widespread occurrence of thermophilic actinomycetes in Nigeria emphasizes the need for investigation of their role in allergic respiratory disorders.

We purified an extracellular thermostable β-galactosidase of Saccharopolyspora rectivirgula strain V2-2, a thermophilic actinomycete, to homogeneity and characterized it to be a monomeric enzyme with a relative molecular mass of 145 000 and s°20,w of 7.1 s. In addition to the hydrolytic activity of 1-O-substituted β-d-galactopyranosides such as lactose [a Michaelis constant K m=0.75 mm and molecular activity (k cat)= 63.1 s−1 at pH 7.2 and 55° C] and p-nitrophenyl β-d-galactopyranoside (K m=0.04 mm k cat= 55.8 s−1), the enzyme had a high transgalactosylation activity. The enzyme reacted with 1.75 m lactose at 70°C and pH 7.0 for 22 h to yield oligosaccharides in a maximum yield (other than lactose) of 41% (w/w). A general structure for the major transgalactosylic products could be expressed as (Gal)c-Glc, where n is 1, 2, 3, and 4 with a glucose at a reducing terminal. These oligosaccharides could selectively promote the growth of the genus Bifidobacterium found in human intestines. S. rectivirgula β-galactosidase was stable at pH 7.2 up to 60°C (for 4 h in the presence of 10 μm MnCl2) or 70°C (for 22 h in the presence of 1.75 m lactose and 10 μm MnCl2). Thus the enzyme is applicable to an immobilized enzyme system at high temperatures (60°C

The thermophilic actinomycete species Saccharopolyspora rectivirgula has been associated with the exogen allergic alveolitis (EAA). EAA is caused by the inhalation of high amounts of airborne spores that can be found for example in environments of agricultural production, compost facilities, mushroom cultivation rooms, or rooms with technical air moistening. Because of the medical relevance of S. rectivirgula, a reliable detection system is needed. Therefore, a quantitative real-time polymerase chain reaction (qPCR) primer system was designed, targeting the 16S rRNA gene of the type strain S. rectivirgula DSM 43747(T) and six other S. rectivirgula reference strains. Our investigation showed that S. rectivirgula presumably own four operons of the 16S rRNA gene, which has to be considered for estimation of cell equivalents. Furthermore, the DNA recovery efficiency from these strains was tested in combination with bioaerosol or material sample as well as the influence of non-target DNA to the recovery rate. Results showed a recovery DNA efficiency of 7-55%. The recovery rate of DNA in a mixture with non-target DNA resulted in ∼87%. In summary, a high amplification efficiency using real-time PCR was found, for which estimated concentrations revealed cell numbers of 2.7 × 10(5) cells m(-3) in bioaerosol and 2.8 × 10(6) cells g(-1) fw(-1) in material samples from a duck house. The specificity of the new developed quantification system was shown by generation of two clone libraries from bioarosol samples, from a duck house, and from a composting plant. Totally, the results clearly show the specificity and practicability of the established qPCR assay for detection of S. rectivirgula.

The effects of daily intranasal instillation of liposomal dexamethasone and free dexamethasone phosphate were compared in a murine model of hypersensitivity pneumonitis induced by Saccharopolyspora rectivirgula (formally known as Micropolyspora faeni). After 3 weeks of antigen and liposome instillations, lung response was evaluated by bronchoalveolar lavage cell counts, lung index and histopathology. Systemic absorption was evaluated by measuring plasma adrenocorticotropic hormone (ACTH) level. Free dexamethasone phosphate induced a dose-dependent response with the maximal effect reached at 1 mg kg-1. At 0.1 mg kg-1, liposomal dexamethasone had a greater effect than free dexamethasone phosphate on bronchoalveolar cells ml-1: 3.01 x 10(5) +/- 0.35 x 10(5) compared to 4.70 x 10(5) +/- 0.34 x 10(5), and lung index: 1.22 +/- 0.10 compared to 1.86 +/- 0.07. Effect on histopathology was similar. Plasma ACTH levels (pg ml-1) were: 75.1 +/- 14.0 for animals receiving antigen and free dexamethasone phosphate (0.2 mg kg-1), and 149.7 +/- 12.0 for animals receiving antigen and liposomal dexamethasone (0.2 mg kg-1). In conclusion, liposome-incorporated dexamethasone is efficient in the treatment of experimental hypersensitivity pneumonitis and, contrarily to free dexamethasone phosphate, does not inhibit ACTH secretion.

Hypersensitivity pneumonitis (HP) is a complex syndrome caused by an exaggerated immune response to the inhalation of a large variety of organic particles. The most frequent antigens that cause HP worldwide are bird proteins (pigeon breeders' disease) and bacteria (Saccharopolyspora rectivirgula). However, fungi are also implicated in many cases, including occupational and nonoccupational outbreaks. The clinical course of the disease is highly variable and its diagnosis clinically challenging since no specific test or biomarker allows a consistent diagnosis. Therefore, a combination of symptoms, bronchoalveolar lavage findings, chest imaging, lab tests, and often biopsies are needed for an accurate diagnosis. Regardless of the cause or the responsible environment, the histopathology is similar and usually consists of a granulomatous interstitial bronchiolocentric pneumonitis characterized by the presence of poorly formed granulomas and a prominent interstitial infiltrate composed of lymphocytes, plasma cells, and macrophages. However, some patients may show a "nonspecific interstitial pneumonia" pattern, or even a usual interstitial pneumonia-like pattern. Importantly, patients with chronic HP may evolve to interstitial fibrosis or develop emphysematous changes, although the reason(s) for these different pathological responses are presently unclear. This review provides a general overview of HP, emphasizing its fungal etiologies, and also examines the currently used clinical criteria for diagnosis and proposes an alternative classification. Challenges for future research include identification of biomarkers that may predict outcome and progression (primarily of chronic HP), and the need for a better understanding of the underlying molecular and genetic mechanisms of the disease.