| Aerosol is a dispersed system which is formed by solid or liquid particles suspended in a gas medium. Microbiological aerosol is the biological aerosol formed in the air, which is connected with the dry solid particles and liquid particles in the suspended state of a single cell. Microbial aerosols can be damaged by skin, mucous membranes, respiratory tract and digestive tract, The presence of some pathogenic microorganisms in the microbial aerosols（e.g., Acinetobacter, Staphylococcus aureus, Aspergillus, avian influenza virus, etc.） is an important cause of air pollution, causing the disease of animals and humans. People usually take the particle aerodynamic diameter ≤ 2.5 μm called PM2.5. Microbiological aerosol that aerodynamic diameter ≤ 2.5 μm is an important part of PM2.5. A large number of epidemiological and toxicological studies show that PM2.5 can cause the body’s respiratory system, immune system, circulatory system damage, causing respiratory system,cardiovascular system diseases, which increased mortality. Although there are a lot of relevant researches about PM2.5 at home and abroad, the mechanism of the damage of respiratory system caused by PM2.5 has not been fully clarified. People tend to focus on the study of the physical and chemical composition of PM2.5 on the pathogenesis of the body, it is rare to study the microbial composition and its mechanism in PM2.5.The live bird markets（LBM） are often located in densely populated areas. The airborne PM of LBM contains a large number of harmful biological components（e.g. gram-negative bacteria, fungi, endotoxin and influenza virus） especially in PM2.5. Thus, short term exposure or long-term exposure to airborne PM of the LBM may have potential health risks. Based on this, we take the live bird market as the research object, the test cycle is one year, to monitor the microbiological aerosols in PM2.5, and based on this, in order to reveal the mechanism of its harm to human beings and animals, to assess the risk, we performed different particle size and microbial composition of aerosol induced mouse lung injury and cause pulmonary inflammatory response and proteome research.There are five parts of this study.1 The diversity and dynamic changes of bacteria in particulate matter(PM2.5) collected from the live bird marketThrough the detection of bacteria in PM2.5 by the 16 S rDNA high throughput sequencing technology, we found that the samples of PM2.5 contains a variety of bacteria（accounted for42.29% of the biomass）. Among them, the highest value appeared in August 2014, reaching1295 kinds of bacteria in PM2.5 and the lowest value appeared in October 2014, reaching 614 kinds of bacteria. The bacterial species in PM2.5 is between 6141295 and the pathogenic or pathogenic bacteria accounts for about 9.45%18.34% of the total bacterial species. For example, Acinetobacter were able to detect in April（7.90%）, June（4.80%）, July（28.62%）,August（26.08%）, September（20.71%）, October（13.83%） and November（1.41%）. Its relative abundance accounts for 1.41%28.62% of the total bacterial count in PM2.5 samples.Corynebacterium were able to detected throughout the year in PM2.5. Its relative abundance accounts for 2.93%17.59% of the total bacterial count in PM2.5 samples, and the highest proportion reached to 17.59% of the total bacterial species in July 2014.2 The diversity and dynamic changes of fungi in particulate matter(PM2.5) collected from the live bird marketThrough the detection of fungi in PM2.5 by the ITS high throughput sequencing technology, we found that the samples of PM2.5 also contains a variety of fungi（accounted for 53.68% of the biomass）. Among them, the highest value appeared in August 2014,reaching 1437 kinds of fungi species in PM2.5 and the lowest value appeared in April 2015,reaching 777 kinds of fungi species. The fungal species in PM2.5 is between 7771437 and the pathogenic or pathogenic fungi accounts for about 6.85%10.31% of the total fungal species.For example, Aspergillus were able to detected throughout the year. Its relative abundance accounts for 2.09%10.02% of the total fungal species in PM2.5 samples and and the highest proportion reached to 10.02% of the total fungal species in April 2015. Trichosporon can also be detected throughout the year. Its relative abundance accounts for 0.05%13.11% of the total fungal species in PM2.5 samples and accounts for a large proportion of the total fungal species in PM2.5 in July（4.57%）, August（13.11%）, September（6.29%） and October（7.09%）.3 Development of the real-time RT-PCR method for the rapid detection of H5 avian influenza virus in the airIn order to rapidly monitor the airborne H5 AIVs in particulate matter(PM2.5) collected from the live bird market and quantify its concentration, the specific primers and probe were designed according to the H5 conserved regions, and then the real-time RT-PCR method was established and used to detect air samples from the live bird market.In the research, the mean concentrations ranged from 01.47×104 copies/m3 air in PM2.5throughout the year. The concentration of H5 AIVs was higher in winter and spring, the highest concentrations were reached to 1.47×104 copies/m3 in December 2014 and the positive rate was 33%. The average concentrations in winter and spring were6.85×1011.47×104 copies/m3 air and the positive rate were 16.67%50%. However, the concentration of H5 AIVs was lower in summer and autumn, and the positive rate was small.The H5 AIVs in PM2.5 were only detected in June 2014（6.14×101 copies/m3 air, 33.33%positive rate） and August 2014（4.24×101 copies/m3 air, 16.67% positive rate）.4 Study on lung injury induced by PM(PM2.5+, PM2.5, PM10+, PM10) in mice4.1 The diversity and relative abundance of bacteria in PM2.5 and PM10 collected from the Live Bird MarketIn this research, the bacterial aerosols in PM2.5 were detected by the 16 S rDNA high throughput sequencing technology. We found that there are large abundant bacterial species in PM2.5. Among them, Acinetobacter（26.18%）, Corynebacterium（21.07%）, Enterococcus（6.85%）, Aeromonas（1.58%）, and Pseudomonas（0.89%） were considered to be potentially harmful bacteria on human health especially the children, the elderly and the live poultry slaughtering staff.For the PM10, there are also large abundant bacterial species in PM10. Among them,Acinetobacter（24.84%）, Enterococcus（20.65%）, Corynebacterium（7.86%）, Staphylococcus（1.07%）, Vibrio（0.06%） and Neisseria（0.06%） were considered to be potentially harmful bacteria on human health.4.2 The diversity and relative abundance of fungi in PM2.5 and PM10 collected from the Live Bird MarketIn this research, the fungal aerosols in PM2.5 were detected by the ITS high throughput sequencing technology. We found that there are large abundant fungal species in PM2.5.Among them, Cladosporium（8.37%）, Aspergillus（7.18%）, Trichosporon（4.94%）, Fusarium（3.84%）, and Cryptococcus（0.01%） were known potential pathogenic fungi to humans.For the PM10, there are also large abundant fungal species in PM10. Among them,Aspergillus（6.41%）, Fusarium（0.08%）, Trichosporon（0.03%）, and Trichothecium（0.01%）were considered to be harmful potentially harmful fungi on human health.4.3 Study on lung injury induced by single stimulation to aerosols(PM2.5+, PM2.5, PM10+,PM10) in mice（acute phase model）In the acute phase model, a significant increase in the expression of TLR2, TLR3, TLR4 and TLR7 protein in the lung was observed. The concentrations of TLR2 and TLR4 were significantly increased in all 4 PM groups(PM2.5+, PM2.5, PM10+ and PM10). In addition, the expression levels of TLR2 and TLR4 in(PM10+)-treated and(PM2.5+)-treated group were significantly higher than the(PM10)-treated and(PM2.5)-treated group（p<0.05）. The expression levels of TLR3 and TLR7 in(PM10)-treated and(PM2.5)-treated group（p<0.05）exhibited no significant differences compared with the control. However, the expression levels of TLR3 and TLR7 in(PM10+)-treated and(PM2.5+)-treated group increased significantly compared to the control group（p<0.05）. These observations indicate that both,TLR2 and TLR4 play a leading role in causing lung injury in mice, and the role of the biological components of PM cannot be overlooked. A significant increase in the expression of IL-1β, TNF-α, IL-8, IP-10 and MCP-1 protein was observed in the acute phase model.4.4 Study on lung injury induced by repeated stimulation to aerosols(PM2.5+, PM2.5,PM10+, PM10) in mice（chronic phase model）In the chronic phase model, significantly increased expression of TLR2 and TLR4 in the lungs was observed, and these protein concentrations were higher than that of the acute phase model and the trends are the same. For cytokines and chemokines, IL-1β, TNF-α, IL-8, IP-10,IFN-γ, MIP-1α, and MCP-1 are the main effect factors in the chronic infection model of lung injury in mice. Through the histological examination of the chronic phase model, we found that the PM that containing microbial components had a far more damage on lung than other groups. The results showed that the biological components of PM play a vital role in lung injury in the chronic phase model and PM2.5 are more efficiently retained in the alveolar lung portion and cause more severe lung injury than the coarse fraction(PM10).5 Proteomic analysis of the lungs of mice infected with PM2.5In this study, two dimensional electrophoresis analysis was used to investigate the host response against PM2.5 infections after 72 h. Forty-five differentially proteins were identified by mass spectrometry. Among them, there are 30 up-regulated proteins and 15down-regulated proteins. These proteins are related to PM-host interactions and the functions include antioxidant response, stress response, Endocrine disorders, tissue damage and abnormalities, immune response, etc. For the up-regulated proteins, Galectin-1 is involved in the process of anti-inflammation and could inhibit the infection of the organism, activate the NF-kappaB signaling pathway, and make the T cell aggregation and other functions. serum amyloid A is the main protein in the acute stage of infection which is involved in the acute inflammation reaction of the body. Peroxiredoxin-2 participates in the antioxidant response of the body and the defense effect of the phosphate polysaccharide. Solution Structure Of Coactosin-Like Protein played a defense against the fungus, etc. For the down-regulated proteins, Surfactant-associated protein A Pulmonary participates in the exchange process of gas. Beta-2-globin, partial involves in the process of oxygen transport. Apoa1 protein can inhibit the release of cytokines involved in the inflammatory response. Annexin A4 can inhibit the release of IL-8 and the activation of NF-kappaB signal pathway. This study laid a foundation for further understanding of the mechanism of PM2.5 on the lung infection in mammals and development anti-PM2.5 drug targets. |
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