Valérie Létourneau

Culture-independent approach of the bacterial bioaerosol diversity in the standard swine confinement buildings, and assessment of the seasonal effect

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.

Culture-Independent Characterization of Archaeal Biodiversity in Swine Confinement Building Bioaerosols

ABSTRACT It was previously demonstrated that microbial communities of pig manure were composed of both bacteria and archaea. Recent studies have shown that bacteria are aerosolized from pig manure, but none have ever focused on the airborne archaeal burden. We sought here to develop and apply molecular ecology approaches to thoroughly characterize airborne archaea from swine confinement buildings (SCBs). Eight swine operations were visited, twice in winter and once during summer. Institute of Occupational Medicine cassettes loaded with 25-mm gelatin filters were used to capture the inhalable microbial biomass. The total genomic DNA was extracted and used as a template for PCR amplification of the archaeal 16S rRNA gene. High concentrations of archaea were found in SCB bioaerosols, being as high as 108 16S rRNA gene copies per cubic meter of air. Construction and sequencing of 16S rRNA gene libraries revealed that all sequences were closely related to methanogenic archaea, such as Methanosphaera stadtmanae (94.7% of the archaeal biodiversity). Archaeal community profiles were compared by 16S rRNA gene denaturing gradient gel electrophoresis. This analysis showed similar fingerprints in each SCB and confirmed the predominance of methanogenic archaea in the bioaerosols. This study sheds new light on the nature of bioaerosols in SCBs and suggests that archaea are also aerosolized from pig manure.

Human pathogens and tetracycline-resistant bacteria in bioaerosols of swine confinement buildings and in nasal flora of hog producers

Swine confinement buildings in eastern Canada are enclosed and equipped with modern production systems to manage waste. Bioaerosols of these swine confinement buildings could be contaminated by human pathogens and antimicrobial resistant bacteria which could colonize exposed workers. We therefore wanted to analyze bioaerosols of swine confinement buildings and nasal flora of Canadian hog producers to evaluate possible colonization with human pathogens and tetracycline-resistant bacteria. Culturable and non-culturable human pathogens and tet genes were investigated in the bioaerosols of 18 barns. The nasal passages of 35 hog producers were sampled and total DNA was extracted from the calcium-alginate swabs to detect, by PCR, Campylobacter, C. perfringens, Enterococcus, E. coli, Y. enterocolitica, tetA/tetC, tetG and ribosomal protection protein genes. Airborne culturable C. perfringens, Enterococcus, E. coli, and Y. enterocolitica were present in the bioaerosols of 16, 17, 11 and 6 of the 18 facilities. Aerosolized total (culturable/non culturable) Campylobacter, C. perfringens, Enterococcus, E. coli and Y. enterocolitica were detected in 10, 6, 15, 18 and 2 barns, respectively. Tet genes were found in isolates of culturable human pathogens. TetA/tetC, tetG and ribosomal protection protein genes were detected in the bioaerosols of all 18 studied buildings. Campylobacter, C. perfringens, Enterococcus, E. coli, and Y. enterocolitica were found respectively in 4, 9, 17, 14 and one nasal flora of workers. One and 10 workers were positive for tetA/tetC and tetG genes, respectively. In swine confinement buildings, hog producers are exposed to aerosolized human pathogens and tetracycline-resistant bacteria that can contaminate the nasal flora.

Bacterial diversity characterization of bioaerosols from cage-housed and floor-housed poultry operations.

BACKGROUND Although bioaerosols from both cage-housed (CH) and floor-housed (FH) poultry operations are highly concentrated, workers from CH operations have reported a greater prevalence of respiratory symptoms. OBJECTIVE The objective of this study was to directly compare bacteria, both quantitatively and qualitatively, in bioaerosols from CH and FH poultry facilities. METHODS Bioaerosols were collected from fifteen CH and fifteen FH poultry operations, using stationary area samplers as well as personal sampling devices. Dust, endotoxin and bacteria were quantified and bacterial diversity was investigated using PCR followed by denaturing gradient gel electrophoresis (DGGE). RESULTS Dust (p<0.001), endotoxin (p<0.05) and bacteria (p<0.05) were significantly higher in personal bioaerosols of FH poultry operations than CH bioaerosols. Although dust and endotoxin did not differ significantly between area and personal samples within each barn type, clustering analysis of DGGE profiles of bacteria revealed that area and personal samples shared less than 10% similarity. These data suggest that area samples are not representative of personal bacteria exposures, which may be affected by worker movement, bacteria carried on the worker and worker location. Personal DGGE profiles from CH and FH operations shared less than 20% similarity and composite analysis showed that bacteria were more prevalent in personal samples from CH bioaerosols than FH bioaerosols. CONCLUSIONS Bacteria concentration and diversity are significantly different between bioaerosols from CH and FH poultry operations.

Airborne porcine circovirus in Canadian swine confinement buildings

Porcine circovirus type 2 has been linked to many diseases, such as postweaning multisystemic wasting syndrome and can be found in most commercial swine confinement buildings around the world. Although the exact role of the virus in the appearance of disease in animals is not fully understood, the mechanisms responsible for the transmission of the virus are currently believed to happen mostly by contact. Nevertheless, the possibility of airborne transmission cannot be rejected. This study investigated the presence of the virus, total bacteria and total dusts in aerosols. Air samples were taken with gelatin filters in swine confinement buildings and were analyzed by quantitative polymerase chain reaction. Interestingly, concentrations of airborne PCV2 of up to 10(7) genomes per cubic meter of air were detected. Airborne dust concentrations were correlated to airborne concentrations of PCV2 and total bacteria. Although the infectivity potential of the airborne viral loads were not evaluated, it is clear that the virus can become airborne in detectable concentrations in commercial swine confinement building environments. The significance of this finding in an epidemiological point of view will need further investigation.

Presence of zoonotic pathogens in physico-chemically characterized manures from hog finishing houses using different production systems

Hog production has been intensified in Eastern Canada, by 50% over the last 20years. Wastes are now managed with conventional production systems (slatted floor), litter systems or source separation systems. We studied the presence of total and fecal coliforms, Campylobacter, Clostridium perfringens, Enterococcus, Escherichia coli, Salmonella, Yersinia enterocolitica, Giardia and Cryptosporidium in the manure of all of these production systems. The concentrations of the studied zoonotic pathogens did not differ between the conventional and the litter systems investigated. The source separation system yielded separated solid and liquid fractions. Total and fecal coliforms, C. perfringens, Enterococcus, E. coli, and Y. enterocolitica were found in the solids and the liquid fractions of a source separation system.Campylobacter species were not detected in the liquid fractions. Zoonotic pathogens are therefore, found in the manure of Eastern Canadian hog finishing houses equipped either with conventional, sawdust litter or source separation systems. Pathogens inactivation will require changes in manure management practices or the use of manure disinfection methods.

Detection of Streptococcus suis in Bioaerosols of Swine Confinement Buildings

ABSTRACT Streptococcus suis is an important swine pathogen that can cause septicemia, meningitis, and pneumonia. Also recognized as an emerging zoonotic agent, it is responsible for outbreaks of human infections in Asian countries. Serotype 2 is the predominant isolate from diseased animals and humans. The aerosolization of S. suis in the air of swine confinement buildings (SCB) was studied. The presence of S. suis in bioaerosols was monitored in SCB where cases of infection had been reported and in healthy SCB without reported infections. Using a quantitative-PCR (qPCR) method, we determined the total number of bacteria (1 × 108 to 2 × 108 airborne/m3), total number of S. suis bacteria (4 × 105 to 10 × 105 airborne/m3), and number of S. suis serotype 2 and 1/2 bacteria (1 × 103 to 30 × 103 airborne/m3) present in the air. S. suis serotypes 2 and 1/2 were detected in the air of all growing/finishing SCB that had documented cases of S. suis infection and in 50% of healthy SCB. The total number of bacteria and total numbers of S. suis and S. suis serotype 2 and 1/2 bacteria were monitored in one positive SCB during a 5-week period, and it was shown that the aerosolized S. suis serotypes 2 and 1/2 remain airborne for a prolonged period. When the effect of aerosolization on S. suis was observed, the percentage of intact S. suis bacteria (showing cell membrane integrity) in the air might have been up to 13%. Finally S. suis was found in nasal swabs from 14 out of 21 healthy finishing-SCB workers, suggesting significant exposure to the pathogen. This report provides a better understanding of the aerosolization, prevalence, and persistence of S. suis in SCB.

Potentially Pathogenic Bacteria and Antimicrobial Resistance in Bioaerosols from Cage-Housed and Floor-Housed Poultry Operations

BACKGROUND Antibiotics are used in animal confinement buildings, such as cage-housed (CH) and floor-housed (FH) poultry operations, to lower the likeliness of disease transmission. In FH facilities, antibiotics may also be used at sub-therapeutic levels for growth promotion. Low levels of antibiotic create a selective pressure toward antimicrobial resistance (AMR) in chicken fecal bacteria. OBJECTIVE The objective of this study was to compare bacteria and AMR genes in bioaerosols from CH and FH poultry facilities. METHODS Bioaerosols were collected from 15 CH and 15 FH poultry operations, using stationary area samplers as well as personal sampling devices. Bacteria concentrations were determined by genus- or species-specific quantitative polymerase chain reaction (PCR) and AMR genes were detected using endpoint PCR. RESULTS Enterococcus spp., Escherichia coli, and Staphylococcus spp. were significantly higher in bioaerosols of FH poultry operations than CH bioaerosols (P < 0.001) while Clostridium perfringens was significantly higher in area bioaerosols of CH operations than FH area bioaerosols (P < 0.05). Campylobacter spp. were detected only in bioaerosols of FH facilities. Zinc bacitracin resistance gene, bcrR, erythromycin resistance gene, ermA, and tetracycline resistance gene, tetA/C, were more prevalent in bioaerosols of FH facilities than CH bioaerosols (P < 0.01, P < 0.01, and P < 0.05, respectively). CONCLUSIONS Most bacteria are more concentrated and most AMR genes are more prevalent in bioaerosols of FH poultry operations, where growth-promoting antibiotics may be used.

Organic components of airborne dust influence the magnitude and kinetics of dendritic cell activation

Bioaerosol exposure in highly contaminated occupational settings is associated with an increased risk of disease. Yet, few determinants allow for accurate prediction of the immunopathogenic potential of complex bioaerosols. Since dendritic cells are instrumental to the initiation of immunopathological reactions, we studied how dendritic cell activation was modified in response to individual agents, combined microbial agents, or air sample eluates from highly contaminated environmental settings. We found that combinations of agents accelerated and enhanced the activation of in vitro-generated murine bone marrow-derived dendritic cell cultures, when compared to individual agents. We also determined that endotoxins are not sufficient to predict the potential of air samples to induce bone marrow-derived dendritic cell activation, especially when endotoxin levels are low. Importantly, bone marrow-derived dendritic cell activation stratified samples from three environmental settings (swine barns, dairy barns, and wastewater treatment plants) according to their air quality status. As a whole, these results support the notion that the interplay between bioaerosol components impacts on their ability to activate dendritic cells and that bone marrow-derived dendritic cell cultures are promising tools to study the immunomodulatory impact of air samples and their components.

Improving Air Quality in Swine Buildings by Using a Combination of Technologies - Pilot-Scale Tests

Abstract. The environmental impact of swine production is still a great concern for the industry as well as for rural populations. Substantial amounts of aerial contaminants such as odors, gases and dust can be emitted by livestock buildings and affect both pig and worker health as well as the nearby environment. Different strategies have successfully reduced some of these airborne contaminants over the years, but few studies looked at their combined effect. Thus, the main objective of this study is to evaluate the reduction of airborne contaminants by using a combination of best practices as compared to conventional management in swine industry at a pilot-scale. Previously in the project, a series of experiments was conducted at a laboratory-scale (4-5 grower-finisher pigs per room) to evaluate the performance of some strategies alone or combined. Results have shown that in order to protect both worker health and reduce environmental emissions, all three tested technologies should be used together: manure separation with a v-shaped scraper, oil sprinkling and an air treatment unit. Following the laboratory-scale results, it was necessary to validate the performance of the technologies at a pilot-scale using two rooms with 16 pigs each. Ammonia emissions for both rooms were quite similar. Unfortunately, the v-shaped scraper didn‘t provide the expected reduction in ammonia emissions. The air treatment unit removed up to 66% of the ammonia on average with peaks up to 80%. For dust, oil sprinkling reduced the dust load by 70% on average.