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Hog production has been substantially intensified in Eastern Canada. Hogs are now fattened in swine confinement buildings with controlled ventilation systems and high animal densities. Newly designed buildings are equipped with conventional manure handling and management systems, shallow or deep litter systems, or source separation systems to manage the large volumes of waste. However, the impacts of those alternative production systems on bioaerosol concentrations within the barns have never been evaluated. Bioaerosols were characterized in 18 modern swine confinement buildings, and the differences in bioaerosol composition in the three different production systems were evaluated. Total dust, endotoxins, culturable actinomycetes, fungi, and bacteria were collected with various apparatuses. The total DNA of the air samples was extracted, and quantitative polymerase chain reaction (PCR) was used to assess the total number of bacterial genomes, as a total (culturable and nonculturable) bacterial assessment. The measured total dust and endotoxin concentrations were not statistically different in the three studied production systems. In buildings with sawdust beds, actinomycetes and molds were found in higher concentrations than in the conventional barns. Aspergillus, Cladosporium, Penicillium, and Scopulariopsis species were identified in all the studied swine confinement buildings. A. flavus, A. terreus, and A. versicolor were abundantly present in the facilities with sawdust beds. Thermotolerant A. fumigatus and Mucor were usually found in all the buildings. The culturable bacteria concentrations were higher in the barns with litters than in the conventional buildings, while real-time PCR revealed nonstatistically different concentrations of total bacteria in all the studied swine confinement buildings. In terms of workers' respiratory health, barns equipped with a solid/liquid separation system may offer better air quality than conventional buildings or barns with sawdust beds. The impact of ventilation rates, air distribution, or building design still has to be explored.

Numerous residents in agricultural areas are concerned about the impact that the swine industry may have on the ambient air quality. They assume there is a risk because there is limited information on the airborne contaminant that may originate from these facilities. The objective of the project was to assess the impact of swine production on ambient air quality related to public health in farming communities. Of the six chosen communities, three were considered not to be in a swine production area, whereas the three others were considered to be within a swine production area. Data were collected during three periods in spring and summer 2006. Ammonia and hydrogen sulfide concentrations were monitored on a continuous basis whereas odor concentrations and intensities were monitored twice a week. Odor concentrations were measured by dynamic olfactometry and odor intensities were determined by trained odor assessors. Public health was evaluated by survey questionnaires sent to a sample of residents in each of the six communities. Average NH(3) concentrations ranged from 6.9 to 12.6 ppb for nonexposed communities and from 8.9 to 18.3 ppb for exposed communities. Average H(2)S concentrations ranged from 1.1 to 1.5 ppb for nonexposed communities and from 1.1 to 1.6 ppb for exposed communities. For a community in a swine production area, ambient NH(3) and H(2)S concentrations were found to be higher than those communities not in a swine production area; however, that difference was not significant and they were within air quality standards for public health and safety. Odor concentrations showed no significant difference between the nonexposed and exposed communities and between evening and morning periods. Odor intensities were found to be significantly higher in the communities within swine production areas. More research will be required to fully understand the correlation between specific physical symptoms from residents and the presence of odors from swine production.

The Canadian poultry production industry contributes nearly $10 billion to the Canadian economy and employs nearly 50,000 workers. However, modern poultry facilities are highly contaminated with airborne dust. Although there are many bioaerosols in the poultry barn environment, endotoxin is typically attributed with the negative respiratory symptoms observed in workers. These adverse respiratory symptoms have a higher prevalence in poultry workers compared to workers from other animal confinement buildings. Workers in cage-housed operations compared to floor-housed facilities report a higher prevalence of some respiratory symptoms. We review the current state of knowledge on airborne dust in poultry barns and respiratory dysfunction in poultry workers while highlighting the areas that need further investigation. Our review focuses on the aerobiological pathway of poultry dust including the source and aerosolization of dust and worker exposure and response. Further understanding of the source and aerosolization of dust in poultry operations will aid in the development of management practices to reduce worker exposure and response.

This project uses function-based detection via a fundamental understanding of the genetic markers of AR to distinguish harmful organisms from innocuous ones. This approach circumvents complex analyses to unravel the taxonomic details of 1399 pathogen species, enormously simplifying detection requirements. Laval Hospital's fast permeabilization strategy enables AR revelation in

Dynamic dental instruments generate abundant aerosols in the work environment. Dental unit waterlines (DUWL) support a large microbial population and can be a significant source of bioaerosols generated during dental treatments. This study was conducted to characterize bioaerosol generation during dental treatments performed in standardized conditions. Culture-based method (R2A, and blood agar with and without O2) and fluorescence microscopy were used. Dental cleaning procedures were performed in an isolated treatment room with controlled ventilation rate. Andersen microbial samplers were used to collect culturable bioaerosols generated before (baseline), during, and after 2 hr of dental treatments. Inhalable dust samplers were used to measure total bioaerosols content in dental hygienist's and patients' breathing zones. AGI-30 were used for the collection of the endotoxin. The use of fluorescence microscopy in combination with culture demonstrated that dental staff and patients were exposed to up to 1.86 E+05 bacteria/m(3) generated during treatments. Fortunately, bioaerosols returned to baseline within 2 hr after the dental procedures. The small diameter of the aerosols generated (1 microm) suggests that the risk of contact between the aerosolized bacteria and the respiratory system of exposed individuals is likely to occur.

Rapid detection of Bacillus spores is a challenging task in food and defense industries. In situ labeling of spores would be advantageous for detection by automated systems based on single-cell analysis. Determination of antibiotic-resistance genes in bacterial spores using in situ labeling has never been developed. Most of the in situ detection schemes employ techniques such as fluorescence in situ hybridization (FISH) that target the naturally amplified ribosomal RNA (rRNA). However, the majority of antibiotic-resistance genes has a plasmidic or chromosomal origin and is present in low copy numbers in the cell. The main challenge in the development of low-target in situ detection in spores is the permeabilization procedure and the signal amplification required for detection. This study presents permeabilization and in situ signal amplification protocols, using Bacillus cereus spores as a model, in order to detect antibiotic-resistance genes. The permeabilization protocol was designed based on the different layers of the Bacillus spore. Catalyzed reporter deposition (CARD)-FISH and in situ polymerase chain reaction (PCR) were used as signal amplification techniques. B. cereus was transformed with the high copy number pC194 and low copy number pMTL500Eres plasmids in order to induce resistance to chloramphenicol and erythromycin, respectively. In addition, a rifampicin-resistant B. cereus strain, conferred by a single-nucleotide polymorphism (SNP) in the chromosome, was used. Using CARD-FISH, only the high copy number plasmid pC194 was detected. On the other hand, in situ PCR gave positive results in all tested instances. This study demonstrated that it was feasible to detect antibiotic-resistance genes in Bacillus spores using in situ techniques. In addition, in situ PCR has been shown to be more sensitive and more applicable than CARD-FISH in detecting low copy targets.

Rapid detection and quantification of Mycobacterium immunogenum in field samples of metalworking fluids (MWFs) is important for factory fluid surveillance programs. The applicability of the developed DNA extraction and quantitative real-time PCR (qPCR) methods to detect and quantify M. immunogenum in used MWFs was evaluated. Total DNA from these samples was extracted, and M. immunogenum measured by qPCR by comparison with a standard curve derived from plasmid vectors. PCR counts were compared with bacterial culture counts. PCR counts of M. immunogenum varied from 1.42 x 10(3) to 3.68 x 10(6) cells/mL of MWFs. Recovery of M. immunogenum by bacterial culture varied from 2.5% to 70% of qPCR count in corresponding samples. Quantitative PCR could be used to measure M. immunogenum load in MWF samples with greater sensitivity and shorter processing time than the classic bacterial culture-based approach. The proposed qPCR approach could be routinely used in real-time PCR-equipped laboratories to provide early detection of M. immunogenum and to control proliferation that probably leads to hypersensitivity pneumonitis in exposed workers.

The bacterial bioaerosol community of eight swine confinement buildings (SCB) was monitored during two visits in the winter, and one during the summer. To our knowledge, culture-independent approaches and molecular biology tools such as biomass quantification and biodiversity analyses have never been applied to swine building bioaerosol analyses. Total DNA of each sample was extracted and analysed by quantitative real-time polymerase chain reaction, denaturing gradient gel electrophoresis (DGGE) and phylogenetic analysis using primers targeting the bacterial 16S rRNA gene. Even though the total bacterial concentration was higher in winter than in summer, the total bacterial concentration for both seasons was 100 to1000 times higher than the total cultural bacteria. The concentration of bioaerosol was influenced by the temperature indoors, which was regulated with an electronic fan system driving warm air and particles outside of the SCB. Comparison of the DGGE profiles showed the same biodiversity in each SCB during both seasons. The phylogenetic analysis revealed a large number of sequences (93.8%) related to Gram-positive anaerobic bacteria, such as Clostridia, and dominated by the Clostridia cluster I (C. disporicum) and the Clostridia cluster XI (C. glycolycum). The bioaerosol diversity also contained also a low proportion of Bacteroidetes and Lactobacillales-Streptococcales sequences. Analyses of the global community and phylotype diversity showed that the main source of bioaerosols could come from the pig manure slurry.

Dental unit waterlines (DUWL) support growth of a dense microbial population that includes pathogens and hypersensitivity-inducing bacteria, such as Legionella spp. and non-tuberculous mycobacteria (NTM). Dynamic dental instruments connected to DUWL generate aerosols in the work environment, which could allow waterborne pathogens to be aerosolized. The use of the real-time quantitative polymerase chain reaction (qPCR) provides a more accurate estimation of exposure levels compared with the traditional culture approach. Bioaerosol sampling was performed 13 times in an isolated dental treatment room according to a standardized protocol that included four dental prophylaxis treatments. Inhalable dust samples were taken at the breathing zone of both the hygienist and patient and outside the treatment room (control). Total bacteria as well as Legionella spp. and NTM were quantified by qPCR in bioaerosol and DUWL water samples. Dental staff and patients are exposed to bacteria generated during dental treatments (up to 4.3 E + 05 bacteria per m(3) of air). Because DUWL water studied was weakly contaminated by Legionella spp. and NTM, their aerosolization during dental treatment was not significant. As a result, infectious and sensitization risks associated with legionellae and NTM should be minimal.

Aerated organic biofilters treating pig manure exhibit partial nitrogen removal. In order to optimize this process, a better comprehension of its colonization by denitrifiers was needed. Three pilot aerated biofilters, fed with variable Biological Oxygen Demand after five days: Total Kjeldahl Nitrogen (BOD(5):TKN) ratios, were constructed and monitored during 180 days. Nitrogen was analyzed in the gaseous and liquid flows, at different depths in the systems. Denitrifying biomass was characterized by evaluating its observed (nitrogen mass balances) and potential (adapted acetylene inhibition technique) activities and its quantity (real-time PCR on nirS), at different heights inside the biofilters. Denitrification was observed as soon as nitrate was produced by nitrifiers, after approximately 40 days of operation, but the potential to denitrify increased from the beginning of the monitoring period. Biofilter fed with the highest BOD(5):TKN ratio showed significant differences with the others, particularly after 80 days of operation, as its potential activity was lower with a higher observed nitrate removal. Data showed that denitrifiers were mainly localized near the surface of the filter and that a microbiological gradient was present from top to bottom. The potential denitrifying activities were always higher than what was being observed inside the sections studied, suggesting that the biomass could have reduced more nitrate and that conditions found inside the filter did not allow denitrification to completely occur.

The goal of this study was to evaluate the incidence of sensitization to the major molds found in peat dust in workers exposed to stored peat moss and the health impact of this sensitization. Air samples from each plant were obtained to measure the levels of airborne molds, bacteria, and dust. There were 189 workers from 14 peat moss processing plants (3 all-year mixing plants and 11 seasonal plants) recruited for the study. The subjects completed a symptoms questionnaire, underwent spirometric measurements and skin-prick tests, and gave venous blood samples. Blood samples from 43 nonexposed control subjects were also taken. A similar percentage of smokers from both plant types was observed. Twenty-eight percent of the workers tested had a positive serum reaction to at least one of the tested molds. The percentage of positive workers varied from plant to plant, going from none in 4 plants to 14 out of 21 for 1 plant. This variability was not correlated with the airborne levels of molds. FEV tended to be lower in the workers with positive antibodies compared with seronegative workers. IgG positive frequency was higher for those workers employed in the all-year plants, and workers from those plants had lower FEV/FVC than seasonal plant workers. Seasonal plants were more contaminated with molds than all-year mixing plants, suggesting that the duration of exposure may trigger more sensitization than the level of exposure. We conclude that there is a high incidence of mold sensitization in peat moss factory workers and that this sensitization may have a negative respiratory health impact.

Peat moss is organic matter colonized by large numbers of microorganisms. Storage prior to its processing may result in massive microbial growth. These biological contaminants can become airborne during processing. Our goals were (a) to evaluate concentrations of bioaerosols (inhalable dust, molds, bacteria) in peat moss processing plants that used dust removing systems, and (b) to evaluate the presence of these microorganisms in peat moss. Fourteen plants from Eastern Canada were visited; 3 plants operated all year (all-year mixing plants), and 11 plants functioned only during summer months (seasonal). Air samples were taken throughout the day at different work sites using IOM cassettes for inhalable dust and All-Glass Impinger-30 samplers and Andersen six-stage impactors for microorganisms. Samples of nonprocessed and bagged peat moss (solid material) were also taken and analyzed. A total of 25 work sites for air sampling and 33 solid material samples were analyzed. Air samples contained up to 441.7 mg/m3 of inhalable dust and up to 1.0 x 10(8) CFU/m3 mesophilic molds and 3.3 x 10(5) CFU/m3 bacteria. Seasonal plants were more contaminated with molds and dust than all-year mixing plants. Sieving sites were the most highly contaminated work sites. Airborne dust concentration was significantly correlated with molds and bacteria. Up to 3.8 x 10(7) CFU/g (dry weight) and 4.8 x 10(7) CFU/g (dry weight) molds and bacteria, respectively, were found in the solid material samples. Airborne contaminants did not correlate with solid material content. Despite the use of dust removing systems, peat moss processing plants contain very large amounts of microbially contaminated bioaerosols that do not correlate with the quality of the processed peat. Efficiency of dust removing systems could influence the contamination levels.

The ultraviolet (UV) Fluorescent Aerodynamic Particle Sizer (FLAPS), a flow cytometer-like apparatus was developed by the Canadian Department of National Defence for real-time detection of autofluorescence of biological aerosol particles such as bacterial spores. The direct relation between autofluorescence intensity and viability has recently been reported and viable spore are more autofluorescent in UV (Laflamme, Frontiers in Bioscience). The goal of this manuscript is to describe a flow cytometry sorting protocol based on UV-induced autofluorescence. An EPICS® ELITE ESP flow cytometer equipped with a UV laser and cell sorter was used to mimic the optical properties of FLAPS and to study the two extremes of a spore population according to its autofluorescence (lower level of autofluorescence (LLA) and higher level of autofluorescence (HLA) spores). Bacillus subtilis var niger was used as a surrogate for Bacillus anthracis spores and sorted using autofluorescence intensity as the main criterion. The protocol developed in our laboratory to sort Bacillus spores according to their autofluorescence properties is described. Purity of each sorted population was greater than 95%. Using autofluorescence as the main criterion, we demonstrate that it is possible to separate two distinct spore populations.

Two workers employed in a hardwood floor plant presented symptoms suggestive of hypersensitivity pneumonitis (HP). At that plant, kiln-dried wood often shows moldy growth and is subsequently brought inside for processing. This study evaluated the environment in attempt to identify the causative antigen and verify whether other workers of this and similar plants had or were at risk of developing HP. Dust from dust-removing systems and molds on the surface of wood planks were collected and air samples taken from a sister plant. Blood samples, spirometry, and symptoms' questionnaires were obtained from 11 co-workers. Dense Paecilomyces growth was observed on the surface of the dried processed wood in the index plant. This fungal genus was not detected in the sister plant. An additional worker had symptoms suggestive of HP, and his bronchoalveolar lavage revealed a lymphocytic alveolitis. The 3 confirmed cases of HP and the other 10 workers had positive specific IgG antibodies to Paecilomyces. We report 3 cases of HP out of 13 workers and a 100% sensitization to molds in workers of a hardwood processing plant. This rate is much higher than what is commonly seen in other environments associated with HP. The drying process is suspected of being responsible for the massive Paecilomyces contamination likely responsible for the HP.

Background: Human cases of hypersensitivity pneumonitis (HP) have been reported among machinists for over 10 yr. Although mycobacteria have been implicated as causal agents, this has not been established in experimental studies and the mechanisms remain unclear. Other constituents of in-use metalworking fluids (MWFs) may also contribute to the development of lung disease. We investigated the potential for Mycobacterium immunogenum (MI) in MWFs to induce HP. Methods: Mice were exposed intranasally for 3 wk to MI (isolated from MWFs), Saccharopolyspora rectivirgula (positive control), saline, endotoxin, MWFs spiked with endotoxin and/or MI, used MWFs, and particulate-fortified used MWFs. Responses were assessed 96 h after the last exposure. Results: Mice exposed to MI in MWFs developed lung pathology consistent with HP along with significantly more monocytes and neutrophils in lung lavage, increased CD4+/CD8+ T-lymphocyte ratio, and marked pulmonary lymphocytosis on histologic examination when compared with saline-treated control mice. Mice with Grade 2 or higher pathology (0–4 point scale) exhibited significantly elevated macrophage inflammatory protein–1α and IL-10 and a trend toward higher RANTES 96 h after the final dose. Endotoxin coexposure augmented lung pathology. Conclusion: MWFs containing mycobacteria induced granulomatous lung lesions, peribronchiolar lymphocytosis, increased cell concentrations in lavage, and up-regulation of several cytokines. These findings are consistent with HP.

Recent biological terrorism events have indicated that bacterial spores such as Bacillus anthracis are real threat agents. Real time detection of biological agents is possible with the use of an ultraviolet Fluorescent Aerodynamic Particle Sizer (FLAPS) that measures particles' intrinsic fluorescence. It is important to know whether intrinsic fluorescence could be used to estimate agents' viability. Two categories of Bacillus spore populations can be differentiated by the intensity of intrinsic fluorescence emitted by ultraviolet (UV) stimulation : autofluorescent and non-autofluorescent. This study was performed to determine whether intensity of autofluorescence correlates with spore viability. Spores were analyzed using flow cytometer (equipped with a cell sorter) to mimic optical properties of FLAPS. Autofluorescent and non-autofluorescent spores were sorted according to the intensity of autofluorescence emitted following UV stimulation. Culturability, membrane integrity, membrane potential and dipicolinic acid (DPA) content were assessed. Autofluorescent spores were 1.7 times more culturable than the corresponding non-autofluorescent population. Moreover, a small proportion of autofluorescent spores exhibited extracellular membrane damages. Autofluorescent spores also showed higher membrane potential activity and contained higher levels of DPA. In conclusion, this study documents that the overall viability potential of bacterial spores can be assessed by UV flow cytometry used in the FLAPS technology.

This Working Group was assembled to review and evaluate methods currently used to estimate work site exposures to biological agents and to present recommendations on suitable measurement strategies. The current state of exposure assessment was evaluated for environments with organic dust including agriculture, composting, sewage and waste treatment processing, peat moss harvesting and handling, cotton and textile processing, greenhouse work, and grass seed processing. Methods for measurement of microbial contaminants in indoor environments were also considered. Important methods are emerging that use quantitative PCR for assessment of microbial agents. Difficulties exist with optimization of extraction to yield contaminant-free DNA without significant DNA loss. Advances in assays for microbial agents (e.g., endotoxin and glucans) as well as allergens have increased the utility of exposure assessment for these agents. A crucial area for further development is international harmonization of methodologies to reduce interlaboratory variability and to facilitate establishment of exposure guidelines. Endotoxin exposure assessment using the Limulus amebocyte lysate method is a high priority for harmonization because of its importance as a pulmonary inflammatory agent in many occupational settings.

Large volumes of metalworking fluids (MWFs) are used in manufacturing industries for cooling and lubrication of metal pieces and tools during machining. MWFs accumulate microbial growth through continuous recirculation and reuse. We studied the progression of microbial contamination for 6 months after dumping, cleaning and recharging (DCR) of a large semi-synthetic MWF system managed with several biocides. Fresh, uncontaminated fluid was added to the system after extensive cleaning. The following samples were collected and analyzed: pre-DCR fluid (before system cleaning); neat fluid diluted to 6% with water; in use MWF 12 h and 1, 3 and 6 months post-DCR. Samples were analyzed for total microorganism concentrations by direct counting using fluorescence microscopy and by plate counting on various media (R2A, BHI, Middlebrooks and rose bengal under aerobic conditions). In addition, PCR was performed for the detection of mycobacteria. There was a rapid progression in the total bacterial counts as determined by fluorescence microscopy: 5.7 x 10(7) cells/ml in the pre-DCR used fluid, no measurable bacteria in the neat fluid, 6.9 x 10(6) cells/ml after 12 h and 2.2 x 10(6), 3.6 x 10(8) and 6.1 x 10(8) cells/ml after 1, 3 and 6 months, respectively. On average, only 0.2% of the direct count organisms were quantified on R2A cultures. PCR showed the presence of mycobacteria in the used MWF at 3 and 6 months. Mycobacteria were also identified from cultures on Middlebrooks and R2A. This study demonstrates that standard methods for cleaning MWF systems are inadequate since residual bacteria in the system can rapidly repopulate the newly charged MWF.

Germination of Bacillus anthracis spores is necessary for the transcription of plasmidic genes essential to the infection. Assessing germination potential is crucial to predict the risk associated with pathogenic Bacillus exposure. The aim of this study was to set up a viability assay based on membrane potential in order to predict the earliest germination event of spores. B. cereus and two strains of B. subtilis were used. The spores were isolated with a sodium bromide gradient. Approximately 10(7) spores were incubated at 37 degrees C in tryptic soy broth (TSB). Aliquots were harvested at predetermined times and stained with 3,3'-dihexyloxacarbocyanine iodide [DiOC(6)(3)] or with bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC(4)(3)]. Fluorescence characteristics were obtained using flow cytometry. The earliest detectable activation of membrane potential occurred after 15 min of incubation in TSB using DiOC(6)(3). Using DiBAC(4)(3), the earliest detectable signal was after 4 h of incubation. Control experiments using carbonyl cyanide m-chlorophenylhydrazone (CCCP)-treated spores did not show any change in the fluorescence intensity over time. Since no membrane potential and no germination were detected in CCCP-treated spores, the activation of membrane potential seems to be associated with germination. DiOC(6)(3) can be used as an early membrane potential indicator for spores. DiBAC(4)(3), by contrast, is not a early membrane potential marker.

Background The gut microbiota is associated with the modulation of mucosal immunity and the etiology of inflammatory bowel diseases (IBD). Previous studies focused on the impact of bacterial species on IBD but seldom suspected archaea, which can be a major constituent of intestinal microbiota, to be implicated in the diseases. Recent evidence supports that two main archaeal species found in the digestive system of humans, Methanobrevibacter smithii (MBS) and Methanosphaera stadtmanae (MSS) can have differential immunogenic properties in lungs of mice; with MSS but not MBS being a strong inducer of the inflammatory response. We thus aimed at documenting the immunogenic potential of MBS and MSS in humans and to explore their association with IBD. Methods To validate the immunogenicity of MBS and MSS in humans, peripheral blood mononuclear cells from healthy subjects were stimulated with these two microorganisms and the production of inflammatory cytokine TNF was measured by ELISA. To verify MBS and MSS prevalence in IBD, stool samples from 29 healthy control subjects and 29 patients suffering from IBD were collected for DNA extraction. Plasma was also collected from these subjects to measure antigen-specific IgGs by ELISA. Quantitative PCR was used for bacteria, methanogens, MBS and MSS quantification. Results Mononuclear cells stimulated with MSS produced higher concentrations of TNF (39.5 ng/ml) compared to MBS stimulation (9.1 ng/ml). Bacterial concentrations and frequency of MBS-containing stools were similar in both groups. However, the number of stool samples positive for the inflammatory archaea MSS was higher in patients than in controls (47% vs 20%). Importantly, only IBD patients developed a significant anti-MSS IgG response. Conclusion The prevalence of MSS is increased in IBD patients and is associated with an antigen-specific IgG response.

Summary Hypersensitivity pneumonitis (HP) is a pulmonary disease characterised by inflammation that can be caused by, amongst other substances, a subset of 4 thermophilic mycelial bacteria:Saccharopolyspora rectivirgula, Saccharomonospora viridis, Thermoactinomyces sacchari, and Thermoactinomyces vulgaris. Air sampling analyses in highly contaminated environments are often performed to evaluate exposure to these species which are difficult and fastidious to identify by conventional techniques. The aim of this study was to use amplified ribosomal DNA restriction analysis (ARDRA) to develop a method of identification for those thermophilic organisms that would be more rapid and simple. Strains of these 4 species were obtained from the American type culture collection (ATCC) and were characterized using biochemical tests and ARDRA patterns obtained on their partial-lenght amplified 16S rDNAs. To validate this approach, ARDRA with two restriction enzymes, Taq I and Hha I, was applied to 49 thermophilic actinomycete-like strains from environmental samples (sawmills). The results obtained show that combining some cultural characteristics and biochemical tests, such as xanthine or hypoxanthine decomposition, growth in the presence of NaCl, lysozyme or novobiocin, and spore resistance over 100 °C provide a rough identification and selection of the genera of interest. Consequently, target species could be confirmed by digestion of partial-lenght 16S rDNA with the use of Taq I and Hha I restriction enzymes that gave specific restriction patterns. ARDRA analyses on the 49 environmental actinomycete-like organisms revealed the presence of 8Saccharopolyspora rectivirgula, 2Saccharomonospora viridis, and 15Thermoactinomyces vulgaris strains, the other strains had restriction patterns different than those of the species of interest. Results of the present study will be applicable to other potential HP environments such as dairy barns, peat bogs and compost plants.

Environmental assessment data collected in two prior occupational hygiene studies of swine barns and sawmills allowed the comparison of concurrent, triplicate, side-by-side endotoxin measurements using air sampling filters and bioaerosol impingers. Endotoxin concentrations in impinger solutions and filter eluates were assayed using the Limulus amebocyte lysate assay. In sawmills, impinger sampling yielded significantly higher endotoxin concentration measurements and lower variances than filter sampling with IOM inhalable dust samplers. Analysis of variance for repeated measures showed that this association remained after controlling for other factors such as replicate, sawmill, sawmill operation, wood type, and interaction terms. Endotoxin concentrations in the swine barns were 10-fold higher on average than in sawmills. These samples demonstrated comparable endotoxin concentration estimates for impinger and filter methods although the variability was lower using the impinger method. In both occupational settings, side-by-side replicates were more uniform for the impinger samples than for the filter samples. This study demonstrates that impinger sampling is an acceptable method for quantitation of area endotoxin concentrations. Further, when sampling is performed with impingers for airborne microorganism quantitation, these same impinger solutions can yield valid endotoxin exposure estimates, negating the need for additional filter sampling.

Eight swine confinement buildings, selected to cover the widest possible range of cleanliness, were visited twice during winter and once during summer to verify the range, seasonal variations, and correlations between biological and chemical contaminants. Physical aspects were graded for dirtiness (1 = clean, 10 = dirty), ventilation, air temperature, number of animals, building, and room size. Air samples were taken to measure relative humidity, CO2, ammonia, total dust, and microbiological counts and/or identification (bacteria and molds); endotoxin levels also were measured. During winter, average measurements and ranges were: CO2 = 0.304% (0.254 to 0.349%); ammonia = 19.6 ppm (1.9 to 25.9 ppm); dust = 3.54 mg/m3 (2.15 to 5.60 mg/m3). There were 883 cfu/m3 (547 to 2862 cfu/m3) of molds, 4.25 x 10(5) cfu/m3 (1.67 x 10(5) to 9.30 x 10(5) cfu/m3) of total bacteria, 29 cfu/m3 (3 to 94 cfu/m3) of thermophilic actinomycetes). A significant decrease in bacterial levels (p = 0.04), dust (p = 0.0008), ammonia (p = 0.005), and CO2 (p0.0001) was observed during summer sampling when compared with winter levels. Mold counts were positively correlated (p = 0.03) with dirtiness scores, while bacterial counts were negatively correlated with this parameter (p0.002), whereas bacteria and endotoxins were correlated with the number of animals (p0.05). Ambient gases (CO2 and ammonia) correlated with each other (p = 0.006). Bacteria were the most important contaminant in swine confinement buildings, and endotoxin levels found were also very high (mean = 4.9 x 10(3) EU/m3). We conclude that a wide range of air contamination exists in swine confinement buildings of different maintenance. There is a decrease in some of these contaminants during summer. Observed dirtiness of the swine confinement buildings has a poor predictive value concerning air quality.

This study investigated whether clean swine confinement buildings (SCB) are less harmful to the respiratory system than older and dirtier facilities. Eight healthy volunteers were exposed for 4 h, at 1 week intervals, to eight SCB representing the widest possible range of cleanliness. Each volunteer and a technician rated the SCB for cleanliness from 1-10, 1 being the cleanest possible. Airborne dust, bacteria, endotoxin levels, molds, and ammonia were measured. For each volunteer measured, before and after each exposure, forced expiratory flows (forced expiratory volume in one second (FEV1), and forced vital capacity), white cells in nasal wash and venous blood, and nasal lavage levels of interleukin (IL)-8 and serum levels of IL-6. A methacholine challenge was obtained at baseline and following each exposure. Cleanliness scores ranged 1.5-8.25. Mean airborne levels were: dust 3.54 mg x m(-3) bacteria 4.25 x 10(5) CFU x m(-3); endotoxins 404 EU x m(-3); molds 883 CFU x m(-3); ammonia 20.7 parts per million (ppm). Expiratory flows decreased after exposure (FEV1 from 4.8+/-0.7 to 4.4+/-0.7, p

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.

A nonsmoking 54-yr-old man, employed in a peat moss packaging plant, developed dyspnea and recurrent fever. The diagnosis of hypersensitivity pneumonitis (HP) was made. Thirteen of 14 coworkers and 13 nonexposed control subjects were studied. Five workers were nonsmokers, two were minimal smokers, and six were smokers. HP was found in another subject. Monocillium sp. and Penicillium citreonigrum, 4.6 x 10(7) CFU/g, were found in the peat moss. Three nonsmokers, the two minimal smokers (including the subject with HP), and the index case had antibodies to these microorganisms; none of the six heavy smokers had antibodies. Serum TNF-alpha was higher in the workers than in the control subjects (0.930 +/- 0.177 versus 0. 350 +/- 0.076). Three of the four asymptomatic seropositive workers and two seronegative smokers were further evaluated. All three seropositive workers had normal lung functions and CT but they all had a lymphocytic alveolitis (30, 34, and 68% lymphocytes in their bronchoalveolar lavage [BAL]). The smokers had normal lung functions, CT, and percentage of BAL lymphocytes (3 and 13%). This study identified a previously unrecognized work environment that can lead to HP and documented a protective effect of smoking on the response to antigens.

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.

Poly(ADP-ribose) catabolism is a complex situation involving many proteins and DNA. We have developed an in vitro turnover system where poly(ADP-ribose) metabolism is monitored in presence of different relative amounts of two principal enzymes poly(ADP-ribose) transferase and poly(ADP-ribose) glycohydrolase along with other proteins and DNA. Our current results reviewed here show that the quality of polymer, i.e. chain length and complexity, as well as preference for the nuclear substrate varies depending upon the availability of poly(ADP-ribose) glycohydrolase. These results are interpreted in the light of the recent data implicating poly(ADP-ribose) metabolism in DNA-repair.

We have evaluated the influence of growth media and washing on the laser-induced fluorescence spectra of bacteria. Three different bacterial simulants were cultured in three types of growth media. Three kinds of samples were generated from each culture: the culture itself, the growth medium alone, and a triple-washed sample. The materials were injected as aerosols in a lab-sized lidar aerosol chamber to obtain their spectra. Using two different analysis approaches, signature variations were observed between the three kinds of samples for most combinations of growth media/bacteria. This study concludes that the culture media used influences the spectral signatures.

Mycobacteria in metalworking fluids (MWF) are associated with hypersensitivity pneumonitis but are difficult to recover using culture. Quantitative PCR is a promising approach to quantify mycobacteria, but three challenges exist: mycobacterial cell lysis, high-yield DNA extraction, and removal of PCR inhibitors. We used Mycobacterium spp. primers to amplify polymorphic regions of 16S-rDNA flanked with highly conserved regions. A standard curve was constructed by cloning M. immunogenum amplification product. We developed single tube DNA extraction employing mixer mill cell disruption, enzymatic digestions (lysozyme, proteinase K) followed by a mechanical disruption, and column purification. MWF was spiked with M. immunogenum, and DNA was successfully extracted. Mycobacterial 16S-RNA genes were quantified by comparing PCR amplification detection (Cycle Threshold) from our samples with that obtained from the standard curve. Recovery and quantification of mycobacterial DNA from spiked samples approached 100 %. A rapid method for quantification of mycobacteria in MWF was demonstrated.

Background The increasing consumption of sugar worldwide seems to lead to several health problems, including some types of cancer. While some studies reported a positive association between sweet foods intake and breast cancer risk, little is known about their relation to mammographic density (MD), a strong breast cancer risk factor. This study examined the association of sweet foods and drinks intake with MD among 776 premenopausal and 779 postmenopausal women recruited at mammography. Methods A food-frequency questionnaire was used to assess intake of sweet foods, sugar-sweetened beverages and spoonsful of sugar added. Percent and absolute breast density were estimated using a computer-assisted method. Multivariate generalized linear models were used to evaluate associations. All models were adjusted for potential confounders, including age and body mass index. Results For increasing quartiles of sugar-sweetened beverages intake, adjusted-mean absolute density was respectively 32, 34, 32 and 36 cm2 among all women (Ptrend = 0.040) and 43, 46, 44 and 51 cm2 among premenopausal women (Ptrend = 0.007). For increasing quartiles of sweet foods intake, adjusted-mean percent density was respectively 16, 16, 17 and 19% among postmenopausal women (Ptrend = 0.036). No association was shown between intake of spoonsful of sugar added and MD. Conclusion Our results suggest that an increase in sweet foods or sugar-sweetened beverage intake is associated with higher MD.

The aim of the project was to investigate whether there were diminished health effects in swine farm workers during summer compared with winter, as seasonal differences in concentrations of bioaerosols have been reported. Twenty-four workers were visited once during each season. Before and after a work shift, they underwent lung function testing and blood sampling. During work, they wore personal air sampling equipment. The mean endotoxin exposure of the workers was highest during winter (25,690 vs. 6,553 EU/m(3); p = 0.004). Although exposures to endotoxin and CO(2) varied between the seasons, no differences in lung function were found between them. White blood cell concentration increased over the work shift from 5.74-6.82 in winter (p0.0001) and from 5.80-6.38 in summer (p = 0.014). These increases differed between the two seasons (p = 0.032). Plasma tumour necrosis factor concentrations fell over the work shift only during winter (1.34-1.24 pg/ml (p = 0.03) (p = 0.014 for the difference between seasons). Plasma interleukin-6 increased over the work shift independently of season (p = 0.0006). The study supported our hypothesis of adverse effects on lung function and immune system, but less so during summer than during winter among Quebec swine farm workers.