[A case of farmer's lung associated with allergic bronchopulmonary aspergillosis]. / L'association inhabituelle de deux maladies pulmonaires immunoallergiques : un cas de pneumopathie d'hypersensibilité associée à une aspergillose bronchopulmonaire allergique.

Hypersensitivity pneumonitis and allergic bronchopulmonary aspergillosis are two forms of lung disease with presumed distinct immunoallergic mechanisms. We report the observation of a 38-year-old French farmer who, for one month, had fever and dyspnoea resistant to antibiotic therapy. A diagnosis of farmer's lung, and allergic bronchopulmonary aspergillosis was made on clinical, biological, functional and radiological evidence and according to the criteria established. The evolution was favorable with antigenic eviction and corticosteroid therapy. This observation is to our knowledge the 5th case that describes the association of hypersensitivity pneumonitis and allergic bronchopulmonary aspergillosis. It suggests the existence of risk factors and immunoallergic mechanisms common to both diseases and discusses the hypothesis that the same antigen(s) is (are) responsible for them.

Identification of Antigenic Proteins from Lichtheimia corymbifera for Farmer's Lung Disease Diagnosis.

The use of recombinant antigens has been shown to improve both the sensitivity and the standardization of the serological diagnosis of Farmer's lung disease (FLD). The aim of this study was to complete the panel of recombinant antigens available for FLD serodiagnosis with antigens of Lichtheimia corymbifera, known to be involved in FLD. L. corymbifera proteins were thus separated by 2D electrophoresis and subjected to western blotting with sera from 7 patients with FLD and 9 healthy exposed controls (HEC). FLD-associated immunoreactive proteins were identified by mass spectrometry based on a protein database specifically created for this study and subsequently produced as recombinant antigens. The ability of recombinant antigens to discriminate patients with FLD from controls was assessed by ELISA performed with sera from FLD patients (n = 41) and controls (n = 43) recruited from five university hospital pneumology departments of France and Switzerland. Forty-one FLD-associated immunoreactive proteins from L. corymbifera were identified. Six of them were produced as recombinant antigens. With a sensitivity and specificity of 81.4 and 77.3% respectively, dihydrolipoyl dehydrogenase was the most effective antigen for discriminating FLD patients from HEC. ELISA performed with the putative proteasome subunit alpha type as an antigen was especially specific (88.6%) and could thus be used for FLD confirmation. The production of recombinant antigens from L. corymbifera represents an additional step towards the development of a standardized ELISA kit for FLD diagnosis.

Immunoreactive proteins of Saccharopolyspora rectivirgula for farmer's lung serodiagnosis.

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.

[Farmer's lung antigens in Germany]. / Farmerlungen-Antigene in Deutschland.

Recent studies suggest that besides the long-known farmer's lung antigen sources Saccharopolyspora rectivirgula (Micropolyspora faeni), Thermoactinomyces vulgaris, and Aspergillus fumigatus, additionally the mold Absidia (Lichtheimia) corymbifera as well as the bacteria Erwinia herbicola (Pantoea agglomerans) and Streptomyces albus may cause farmer's lung in Germany. In this study the sera of 64 farmers with a suspicion of farmer's lung were examined for the following further antigens: Wallemia sebi, Cladosporium herbarum, Aspergillus versicolor, and Eurotium amstelodami. Our results indicate that these molds are not frequent causes of farmer's lung in Germany.

Detection of Saccharopolyspora rectivirgula by quantitative real-time PCR.

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.

New evidence of the involvement of Lichtheimia corymbifera in farmer's lung disease.

Farmer's lung disease (FLD) is a form of hypersensitivity pneumonitis resulting from recurrent exposure to moldy plant materials. We investigated and compared the initial response of respiratory epithelium after exposure to extracts of Sacharopolyspora rectivirgula, Lichtheimia corymbifera (formerly Absidia corymbifera), Eurotium amstelodami and Wallemia sebi. The two criteria for selection of these species were their high prevalence in the hay handled by FLD patients and the presence of high levels of specific precipitins to these molds in FLD patients’ sera. Hydrosoluble extracts were prepared from spores and hyphae grown in culture under optimal conditions for each of the four species. Confluent A549 cells were inoculated with one of the four calibrated soluble extracts. Two mediators, one inflammatory (Interleukin (IL)-8) and one allergic (IL-13), were quantified using real-time PCR and ELISA assay, after four exposure periods (30 min, 2 h, 4 h and 8 h). S. rectivirgula and L. corymbifera extracts were the only ones which induced a marked upregulation of IL-8, as shown by both real-time PCR and ELISA assay 8 h after the initial contact. This study adds to the growing body of evidence that L. corymbifera should be recognized as an etiologic agent of FLD along with S. rectivirgula.

Protein kinase D1 is essential for the proinflammatory response induced by hypersensitivity pneumonitis-causing thermophilic actinomycetes Saccharopolyspora rectivirgula.

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-kappaB 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.

[Granulomatous diseases and pathogenic microorganism].

Granuloma formation is a chronic inflammatory reaction where macrophage system and other inflammatory cells are involved. After some antigen exposure and processing, T cells, macrophages, epithelioid cells, and giant cell are activated, and granulomas are formed. Granuloma is considered as a defense mechanism against antigens, which stay in the organs without inactivation. Granulomas including fibroblasts extra-cellular matrix surround and isolate the antigens. Granulomas are classified to noninfectious granulomas and infectious granulomas. However recent studies revealed pathogenic microorganism are suspected to be a cause of granuloma in non-inflammatory diseases. Balance between pathogenic microorganisms and defense mechanisms of the host might be important in the special immunologic reaction. In some cases, it is hard to clearly classify infectious and noninfectious granulomas. Recently, Eishi et al. reported that latent infection of Propionibacterium acnes might be cause of sarcoidosis. Several hypersensitivity pneumonias are considered to be caused by exogenous microorganisms. The symposium was organized to know and clarify the new mechanisms of non-infectious granulomatous lung diseases and pathogenic microorganisms. This report is a summary of a symposium entitled "Granulomatous Diseases and Pathogenic Microorganism", organized in the 82nd Japanese Society for Tuberculosis (president Dr. Mitsunori Sakatani, M.D.). 1. Imaging of Granulomatous Lung Diseases: Masanori AKIRA (Department of Radiology, National Hospital Organization Kinki-chuo Chest Medical Center) High-resolution computed tomography (HRCT) is a useful tool in the evaluation of parenchymal changes in patients with a granulomatous lung disease. In sarcoidosis, the HRCT findings include small, well-defined nodules in relation to lymphatic roots, lymph node enlargement, and middle or upper lobe predominance. The appearances of subacute hypersensitivity pneumonitis include ill-defined centrilobular nodules, ground-glass opacity, and air trapping especially on expiratory CT scan. Those of Langerhans cell histiocytosis include bizarre thin-walled lung cysts, centrilobular nodules and upper lobe predominance. Each of granulomatous lung disease has some characteristic HRCT appearances, but they all are non-specific for diagnosis. HRCT is also useful for grading of parenchymal changes in granulomatous lung diseases. 2. Histopathology of granulomatous lung diseases with special reference to differential diagnosis of infectious disease: Tamiko TAKEMURA (Department of Pathology, Japanese Red Cross Medical Center) The lung is commonly involved by various granulomatous diseases of various etiology. It is difficult to pathologically differentiate these granuloumatous diseases to conduct appropriate therapy, because of morphological similarity of epithelioid cell granuloma, variable etiology, and difficulty of identification of causative agents. Granulomatous diseases generally are divided into infectious and non-infectious ones for treatment. Although infectious granulomas usually reveal necrosis and abscess, non-infectious ones occasionally also reveal necrosis. In cases with granulomas in the lung, it is necessary to explore the etiologic agents including environmental ones. 3. Sarcoidosis and Propionibacterium acnes: Yoshinobu EISHI (Department of Pathology, Tokyo Medical and Dental University) P. acnes can cause latent infection in peripheral lung tissue and the mediastinal lymph nodes and persist intracellularly in a cell-wall-deficient form. This dormant form of P. acnes can be activated endogenously under certain environmental conditions (hormones, stress, living habits, etc.) and proliferate in cells at the sites of latent infection. Granulomatous inflammation occurs in sarcoidosis patients with hypersensitivity to intracellular proliferation of the cell-wall-deficient bacteria, which can infect other cells or organs when spread via the lymphatic or blood streams. The timely use of antibiotics may not only kill the bacteria proliferating at the site of disease activity, but also prevent endogenous activation of P. acnes. If long term administration of antibiotics eradicates dormant forms of the bacteria persistent in organs, it may lead to complete remission of sarcoidosis. 4. Farmer's lung and thermophilic actinomycetes: Takashi MOURI (Pulmonary Division, Iwate Prefectural Kitakami Hospital), Kohei YAMAUCHI, Hiroshi INOUE (Third Department of Internal Medicine, Iwate Medical University, School of Medicine), Kazuki KONISHI (Morioka Tsunagi Onsen Hospital) Farmer's lung is caused by the allergic reaction to inhalation of thermophilic actinomycetes. Acute symptoms are chill, fever, cough and dyspnea. Fine crackles is characteristic. Pathologically, alveolitis with lymphocytes infiltration and epithelioid cell granuloma and Masson's body are characteristics. Bronchoalveolar lavage analysis shows elevated lymphocytes and diverse CD4/8 ratio (high in average). Isolation from the environment improves the symptoms. Sometimes patients need steroid therapy, 0.5 to 1.0 mg/kg of predonisolone. Pulse therapy can be applied for severe cases. SLX analogue can prevent lymphocytes infiltration and granuloma formation in mice model. Some of acute farmer's lung show poor long term prognosis, showing emphysematous, fine granular or small nodules in chest CT. These chronic farmer's lung might be diagnosed as IIPs. 5. Hot tub lung: Takashi OGURA (Kanagawa Cardiovascular and Respiratory Center) Hot Tub Lung (HTL) is a disorder caused by exposure to Mycobacterium avium complex (MAC) organisms contaminating hot tub water. Whether this disease represents true infection or hypersensitivity pneumonitis is contoroversial. Recent reports support the theory that this disease represents a hypersensitivity pneumonitis rather than infection. The physicians should suspect a hypersensitivity pneumonitis reaction to MAC in the investigation of patients with hypersensitivity pneumonitis of unknown cause.

Impact of agricultural practices on microbiology of hay, silage and flour on Finnish and French farms.

Exposure to microorganisms in farm environments may cause respiratory disorders, e.g. asthma, organic dust toxic syndrome and allergic alveolitis. By reducing microbiological deterioration of organic materials, some agricultural practices have a protective effect. Microbiological analyses were carried out on hay, silage and flour samples (n=107) from farms in Finland and France (n=23) that use different methods of haymaking. High concentrations of Absidia corymbifera were found in approximately 35 % of French hay samples and only 10 % of Finnish hay samples. Concentrations of Eurotium spp. were found in 20 % of hay samples from both regions. High concentrations of Wallemia sebi typified Finnish hay (38 %) more than French hay (8 %). Rhodotorula yeast was frequently and abundantly found in Finland, but never in France. The method used to make hay appeared to be the main factor affecting the microbiology of the hay. A. corymbifera and Eurotium spp. concentrations were smaller in low-density square bales than in others. In conclusion, our results emphasize the importance of good agricultural practice in the microbiological quality of fodder.

Farmer's lung disease and microbiological composition of hay: a case-control study.

Previous studies performed in France have suggested that handling hay contaminated with high amounts of moulds, and especially Absidia corymbifera and Eurotium amstelodami, may favour farmer's lung disease. The circumstances favouring farmer's lung disease and the distinctive microbiological composition of hay samples that provoke attacks need to be specified. We present a case-control study which investigates the agricultural practices and the microbiological composition of hay handled in patients with farmer's lung disease as compared to those of a representative control population. Ten cases identified the hay they were handling at the onset of symptoms. The location, type of farm and working conditions were similar to those of the control farms. Conversely, the microbiological composition of hay differed, with significantly higher amounts of E. amstelodami (P < 0.01), A. corymbifera (P = 0.003), mesophilic Streptomyces (P < 0.01), thermophilic Streptomyces (P < 0.01) and Saccharomonospora viridis (P < 0.01) than in the control population. Our results demonstrate that hay identified by patients as having a harmful effect is characterized by a higher total amount of microorganisms, notably five microorganisms that seem discriminative. Mean concentrations are 2- to 115-fold higher in hay suspected to cause symptoms than in hay from a representative panel of farms. Handling hay with high amounts of these five microorganisms constitutes a risk factor for farmer's lung disease that should be considered for the development of prophylactic measures.

Evaluation of salting as a hay preservative against farmer's lung disease agents.

Salting is a traditional, empirical practice used commonly in dairy farming regions to prevent moulding and heating in hay. Our aim was to evaluate the effect of salting hay on the proliferation of microorganisms, particularly thermophilic actinomycetes and moulds involved in farmer's lung disease. Fifty-one pairs of salted and unsalted hay bales from 14 farms were produced during the haymaking season between March and July. Both the salted and the unsalted bales came from the same field, and were packed and stored under identical conditions. Sampling was performed by microbiological analysis including 6 culture media during the winter following salting (January-February). The use of salt did not significantly decrease the amount of Saccharopolyspora rectivirgula , the actinomycetes most commonly involved in farmer's lung disease, or that of Absidia corymbifera, Eurotium amstelodami and Wallemia sebi , three moulds responsible for farmer's lung disease in eastern France. Our results are important in that they can inform farmers and dispel the false sense of security induced by salting, which is reinforced by the misconception that palatable hay is healthy hay.

Microbiological evolution of hay and relapse in patients with farmer's lung.

BACKGROUND: Recent studies in France have shown that Absidia corymbifera and, to a lesser degree Eurotium amstelodami and Wallemia sebi, play a role in farmer's lung disease (FLD), but that Saccharopolyspora rectivirgula, classically incriminated, does not. Little is known about farmers' reactions to these fungi or the circumstances which lead to exposure. AIMS: To investigate the conditions which favour the development of these microorganisms in hay and to analyse the relation between their concentration and the risk of occurrence of FLD. METHODS: Sequential microbiological analyses of each batch of hay stored in 10 farms at risk for FLD and a serological survey of 10 farmers (five with a past history of FLD). RESULTS: Exposure to microorganisms varied widely according to farms and periods. These microorganisms usually reached a peak in January and proliferated when harvesting conditions favoured excessive humidity in hay (rain during harvest, soil in the hay). Three of the five FLD patients presented with FLD respiratory recurrence and positive serology for A corymbifera during the winter (2000-01), after exposure to a significantly higher amount of A corymbifera than other farmers. Similar, but less significant, results were found for E amstelodami exposure, but not with W sebi. CONCLUSIONS: Results contribute to confirming A corymbifera as a major aetiological agent of FLD in Doubs, and encourage further studies with a view to implementing preventive measures.

Experimental hypersensitivity pneumonitis: role of MCP-1.

Inhalation of Saccharopolyspora rectivirgula causes "farmer's lung" disease, a classic example of hypersensitivity pneumonitis (HP). Monocyte chemoattractant protein-1 (MCP-1) is increased in the bronchoalveolar lavage fluid of mice challenged with S rectivirgula, and S rectivirgula induces MCP-1 secretion by alveolar macrophages. We tested the hypothesis that MCP-1 and its receptor CC chemokine receptor-2 (CCR2) are essential to the development of experimental HP by treating mice with MCP-1 antibody and using CCR2(-/-) mice. Administration of anti-MCP-1 did not change the response to intratracheally administered S rectivirgula. CCR2(-/-) animals responded in a fashion similar to that of wild-type animals to intratracheally administered.S rectivirgula. To determine the influence of the MCP-1-CCR2 interaction in vitro, we transferred S rectivirgula-cultured spleen cells from S rectivirgula-sensitized mice, to naïve recipients. Later, challenge of the recipients with intratracheal S rectivirgula and examination of both lung histology and bronchoalveolar lavage fluid characteristics were used to determine whether adoptive transfer had occurred. We found that cultured cells from CCR2(-/-) animals were fully capable of adoptive transfer. We conclude that interaction of MCP-1 with CCR2 is not necessary for the development of pulmonary inflammation in response to intratracheally administered S rectivirgula or cells able to adoptively transfer experimental HP.

[Exposure to Saccharopolyspora rectivirgula among cattle breeders in the province of Reggio Emilia and the risk of extrinsic allergic alveolitis (farmer's lung)]. / Esposizione a Saccharopolyspora rectivirgula degli allevatori di bestiame bovino della provincia di Reggio Emilia e rischio di alveolite allergica estrinseca (Farmer's Lung).

INTRODUCTION: Nearly 2.350 dairy farms (and 137.000 milk cows) are located in the province of Reggio Emilia, Italy, to produce the famous Parmigiano-Reggiano" cheese. Feeding is hay-based both in the cold season and (together with grazing) in the warm season. This requires a large production of hay and frequent handling by the farmers. Hay is packed in large cylindrical bales, "round bales" (nearly 2.41 m3), or, rarely, in traditional small prisms-shaped bales (about 0.15 m3), only used on small farms. We estimated there were 6.000-9.000 the workers exposed to hay dust. The risks for the farmer's health due to the hay dust exposure are well known; in particular Farmer's Lung disease (FL) is rather frequent in this Region (1.5%-3.0% among people exposed). We studied hay and air pollution by Saccharopolyspora Rectivirgula (SR) in relation to these two different hay-packing techniques (hay dried in the open air) both in flat and in hilly areas. METHODS: On 56 cattle-farms, hay and air samples were collected and analyzed using a six-stage Andersen sampler and a sedimentation chamber (SC) for hay samples with plastic Petri dishes containing culture medium. RESULTS: Round bales were richer in SR spores than the small prism-shaped bales (n = 37, mean = 6.20 logn ufc/m3 in SC, ds: 3.87 vs n = 15, mean = 2.40 logn ufc/m3 in SC; ds: 4.16) and they seem to produce higher air pollution (n = 30, mean = 5.30 logn ufc/m3; ds: 3.71 vs n = 15, mean = 2.32 logn ufc/m3; ds: 2.99). In hilly areas the pollution produced by round bales (in hay and air) was higher than in flat areas. On the contrary hay from small bales produced in hilly areas was poorest in SR spores. CONCLUSIONS: An heavy exposure to actinomycetes spores, therefore, comes from "round bales" hay handling, especially when the bales are produced in mountain areas. New drying systems, probably, can reduce this risk and raise hay quality.

Airborne microflora in Quebec dairy farms: lack of effect of bacterial hay preservatives.

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.

Saccharopolyspora rectivirgula from Quebec dairy barns: application of simplified criteria for the identification of an agent responsible for farmer's lung disease.

Saccharopolyspora rectivirgula (Micropolyspora faeni) is one of the major agents responsible for farmer's lung disease, a form of hypersensitivity pneumonitis. It is frequently isolated from the air of contaminated barns. The identification of this actinomycete is difficult because most of its phenotypic characteristics are variable and classical tests are not easy to perform on actinomycetes. Fatty acid analysis is very useful for the identification of these strains, but is not available except in some research or reference laboratories. Morphological (microscopic and macroscopic observations), physiological and biochemical tests (growth properties; macromolecules degraded; citrate utilisation and acid production from carbohydrates; resistance to antibiotics, lysozyme and heat), cell wall and fatty acid analyses and IgG analyses with serum from patients with farmer's lung were performed on 12 environmental isolates presumed to be S. rectivirgula and two control strains of S. rectivirgula. From this, a simple and rapid scheme for the identification of this actinomycete is proposed: optimal growth temperature (55 degrees C); colony appearance based on morphology (filamentous) and colour (beige to orange-brown); microscopic morphology (chains of spores on both aerial and substrate mycelium); growth on NaCl 10%; cell-wall analysis (type IV); and the verification of antibody response with serum from a patient with farmer's lung. This last criterion is important to confirm the immunogenicity of the strains identified as S. rectivirgula. This scheme provides an accurate and efficient way of identifying S. rectivirgula strains and evaluating exposure to this bacterium. The study shows the limited value and the lack of reproducibility of some classical biochemical tests.

Fatal cases of farmer's lung in a Canadian family. Probable new antigens, Penicillium brevicompactum and P olivicolor.

Three Canadian farmers, including a married couple and another close relative, died from progressive pulmonary fibrosis. Their histories and investigations were compatible with chronic farmer's lung (FL). Our environmental and immunologic studies indicate Penicillium brevicompactum and P olivicolor as probable new antigens of FL in a cool and dry climate.

Structural studies of the major glycolipid from Saccharopolyspora genus.

A major glycolipid was isolated from the well characterized Saccharopolyspora species, S. hirsuta, S. rectivirgula, S. erythraea and one not completely identified strain (Saccharopolyspora sp.). On the basis of sugar and methylation analysis, specific enzymatic and chemical degradations of the carbohydrate moiety, its FAB mass spectrometry and NMR spectroscopy characterizations, the carbohydrate part was shown to be the glycerol linked dimannoside alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->1/3)Gro. The internal mannose residue is esterified at C-6 by one fatty acid residue, whereas another fatty acyl chain substitutes the primary methylene position of glycerol. The main fatty acyl residues are anteiso-branched heptadecanoic acid and the iso-branched fatty acids iso-17:0, iso-16:0, and iso-18:0, with the former species being predominant. The major glycolipid has potential value for taxonomic and diagnostic purposes, especially in the specific diagnosis of farmer's lung disease.

Proinflammatory effect of Pediococcus pentosaceus, a bacterium used as hay preservative.

Bacterial cultures, such as Pediococcus pentosaceus, are used to treat hay with the objective of preventing hay heating and moulding, and thus, the development of the microbial growth which causes farmer's lung. The aim of this study was to investigate whether such bacterial cultures have the potential to induce a pulmonary inflammatory response. Mice were instilled 3 days week-1 for 3 weeks with either saline or nonviable preparations of P. pentosaceus, Saccharopolyspora rectivirgula, Lactococcus lactis (control bacteria) or with the combinations of S. rectivirgula and P. pentosaceus. P. pentosaceus induced a significant inflammatory response in the lung which was similar to that produced by S. rectivirgula. L. lactis produced a response of a lower intensity. The total number of cells in bronchoalveolar lavage were: S. rectivirgula: 6.4 x 10(5) cells.mL-1; P. pentosaceus: 4.3 x 10(5) cells.mL-1; S. rectivirgula + P. pentosaceus: 5.4 x 10(5) cells.mL-1, L. lactis: 6.8 x 10(5) cells.mL-1 and saline group 3.7 x 10(4) cells.mL-1. The lung index was higher in S. rectivirgula+P. pentosaceus and P. pentosaceus groups than in S. rectivirgula, L. lactis and saline groups. The quantity of specific immunoglobulin G and A (IgG and IgA) to P. pentosaceus and L. lactis levels (in the blood and/or lavage fluid) were similar to those against S. rectivirgula. In mice, P. pentosaceus has the potential to induce a similar inflammatory response in the lung as S. rectivirgula, which is the most common antigen responsible for farmer's lung disease in Quebec. Further studies are needed to verify whether farmers can develop farmer's lung or other lung responses to this new potential antigen.

Farmer's lung: patients' IgG2 antibodies specifically recognize Saccharopolyspora rectivirgula proteins and carbohydrate structures.

Farmer's lung is a frequent form of extrinsic allergic alveolitis. In Europe and Northern America the main source of the antigenic components that induce farmer's lung is the bacterium Saccharopolyspora rectivirgula (Micropolyspora faeni). It remains unclear, however, which S. rectivirgula components are responsible for the disease. We approached these problems by investigating the serologic reaction of patients with farmer's lung and demonstrated specific binding of patients' IgG2 to S. rectivirgula antigens. No such antibodies were found in exposed, unaffected subjects. Thus IgG2 antibodies reacting with S. rectivirgula antigens are useful for the serologic diagnosis of patients with farmer's lung and for the isolation of disease-causing antigens. After separation of S. rectivirgula extract on concanavalin A-Sepharose (Pharmacia, Uppsala, Sweden), we found that approximately one third of the patients' IgG2 reactivity occurred with nonglycosylated proteins. Among these, we characterized two major acidic proteins with molecular weights of 12 and 30 kd, respectively, and with identical N-terminal sequences. Approximately two thirds of the patients' IgG2 reactivity was observed against concanavalin A-binding glycoproteins that contained mainly glucose, mannose, and galactose residues. Deglycosylation of the concanavalin A-bound fraction indicated that most of the IgG2 reactivity occurred with the carbohydrate components.