A rapid simultaneous detection of duck hepatitis A virus 3 and novel duck reovirus based on RPA CRISPR Cas12a/Cas13a.

The mixed infection of duck hepatitis A virus 3 (DHAV-3) and novel duck reovirus (NDRV) has caused significant losses to the global duck farming industry. On-site point-of-care testing of viruses plays a crucial role in the early diagnosis, prevention, and disease control. Here, we proposed an RPA-CRISPR Cas12a/Cas13a one-pot strategy (DRCFS) for rapid and simultaneous detection of DHAV-3 and NDRV. This method integrated the reaction of RPA and CRISPR Cas12a/Cas13a in a single tube, eliminating the need to open the lid during the intermediate processes and thereby avoiding aerosol contamination. On this basis, we proposed a dual RPA-CRISPR strategy coupled with a lateral flow analysis platform (DRC-LFA). This circumvented the necessity for complex instruments, enabling direct visual interpretation of results, making the test more accessible and user-friendly. Our findings demonstrated that the DRCFS method could detect DHAV-3 and NDRV at concentrations as low as 100 copy/µL, while DRC-LFA achieved limit of 101 copies/µL within 35 min. Furthermore, when DRCFS, DRC-LFA, and qPCR were employed collectively for clinical samples analysis, all three methods yielded consistent results. The specificity, sensitivity, and user-friendly of these methods rendered them invaluable for on-site virus detection.

Unveiling the airborne microbial menace: Novel insights into pathogenic bacteria and fungi in bioaerosols from nursery schools to universities.

Bacterial and fungal aerosol pollution is widespread in indoor school environments, and poses potential health risks to students and staff. Understanding the distribution and diversity of microbial communities within aerosols is crucial to mitigate their adverse effects. Existing knowledge regarding the composition of bacterial and fungal aerosols, particularly the presence of potential pathogenic microorganisms in fine particulate matter (PM2.5) from nursery schools to universities, is limited. To bridge this knowledge gap, in the present study, we collected PM2.5 samples from five types of schools (i.e., nursery schools, primary schools, junior schools, and high schools and universities) in China. We used advanced single-molecule real-time sequencing to analyze the species-level diversity of bacterial and fungal components in PM2.5 samples based on 16S and ITS ribosomal genes, respectively. We found significant differences in microbial diversity and community composition among the samples obtained from different educational institutions and pollution levels. In particularly, junior schools exhibited higher PM2.5 concentrations (62.2-86.6 µg/m3) than other schools (14.4-48.4 µg/m3). Moreover, microbial variations in PM2.5 samples were associated with institution type. Notably, the prevailing pathogenic microorganisms included Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Streptococcus pneumoniae, and Schizophyllum commune, all of which were identified as Class II Pathogenic Microorganisms in school settings. Four potentially novel strains of S. commune were identified in PM2.5 samples collected from the university; the four strains showed 92.4 %-94.1 % ITS sequence similarity to known Schizophyllum isolates. To the best of our knowledge, this is the first study to explore bacterial and fungal diversity within PM2.5 samples from nursery schools to universities. Overall, these findings contribute to the existing knowledge of school environmental microbiology to ensure the health and safety of students and staff and impacting public health.

Pathogenic bacteria and fungi in bioaerosols from specialized hospitals in Shandong province, East China.

Bacteria and fungi are abundant and ubiquitous in bioaerosols in hospital environments. Understanding the distribution and diversity of microbial communities within bioaerosols is critical for mitigating their detrimental effects. Our knowledge on the composition of bacteria or fungi in bioaerosols is limited, especially the potential pathogens present in fine particulate matter (PM2.5) from specialized hospitals. Thirty p.m.2.5 filter samples were collected from five hospitals (i.e., oral, dermatology, chest, eye, and general hospitals) in Shandong Province, East China. The diversity of bacteria and fungi was analyzed at the species level using single-molecule real-time sequencing of the 16 S and internal transcribed spacer 1 (ITS) ribosomal genes, respectively. Significant differences were detected across sampling sites in terms of microbial diversity and community composition in PM2.5 as well as pollution concentrations. The range of PM2.5 concentrations observed in hospital halls was higher, ranging from 39.0 to 46.2 µg/m3, compared to the wards where the concentrations ranged from 10.7 to 25.2 µg/m3. Furthermore, microbial variations in PM2.5 bioaerosols were associated with hospital type. The most dominant pathogens identified were Vibrio metschnikovii, Staphylococcus epidermidis, Staphylococcus haemolyticus, Fusarium pseudensiforme, and Aspergillus ruber. Among these, A. ruber was identified as an opportunistic fungus in a hospital setting for the first time. Nine potentially novel strains of F. pseudensiforme, showing 84.5%-92.0% ITS sequence similarity to known Fusarium isolates, were identified in PM2.5 samples from all hospitals (excluding an eye hospital). This study highlights the importance of hospital environments in shaping microbial aerosol communities. To the best of our knowledge, this is the first study to provide insights into the bacterial and fungal biodiversity of PM2.5 in specialized hospitals, enriching research in healthcare environmental microbiology and carrying significant public health implications.

Common carp intelectin 3 (cITLN3) plays a role in the innate immune response.

Intelectin is a lectin with the capacity to recognize and bind to carbohydrates. In this study, we successfully cloned cITLN3 from common carp, which consists of a signal peptide domain, a FReD domain, and an intelectin domain. The expression levels of cITLN3 were detected in various organs of common carp, including the liver, head kidney, spleen, foregut, midgut, and hindgut, with the highest expression observed in the liver. Following infection with Staphylococcus aureus (S. aureus) or Aeromonas hydrophila (A. hydrophila), the expression level of cITLN3 was significantly upregulated in all organs of common carp. Subsequently, we expressed and purified the recombinant cITLN3 protein using an E. coli expression system. The cITLN3 could aggregate both gram-positive and gram-negative bacteria in the presence of Ca2+, with a stronger affinity for gram-positive bacteria. Moreover, our study demonstrated that cITLN3 displayed a higher binding affinity towards PGN compared to LPS. Furthermore, we observed that cITLN3 had the potential to inhibit bacterial proliferation in common carp and safeguard intestinal integrity during bacterial stimulation. And the results also indicated that cITLN3 might played a role in the Toll-like receptors (TLRs) signaling pathway activation.

One-tube RPA-CRISPR Cas12a/Cas13a rapid detection of methicillin-resistant Staphylococcus aureus.

At present, methicillin-resistant Staphylococcus aureus (MRSA) has caused a serious impact on a global scale. The infection and carrier rate of MRSA in the community is increasing year by year, but there is still no convenient detection system for on-site rapid detection. It is very important to select a rapid detection system to accurately and quickly detect patients infected with MRSA. We have developed a high-efficient single-tube detection platform based on RPA and CRISPR reaction system to detect the genes of mecA and clfA of MRSA. Using this detection platform, visual MRSA detection could be achieved in 30 min. It was observed that this detection platform was capable to successfully detect the target genomic as low as 5 copies µL-1, and the reaction was completed in one step without opening the lid. This detection platform could only detect MRSA, but not other common clinical pathogenic bacteria, such as Salmonella, Pseudomonas aeruginosa, Staphylococcus xylosus, Aeromonas hydrophila, Escherichia coli and Staphylococcus warneri, indicated its satisfactory selectivity for MRSA without interference from other bacteria. The results of clinical samples show that the platform has outstanding advantages in sensitivity, specificity and identification of methicillin resistance. The entire reaction can be completed in one step in the handheld instrument without opening the cover, avoiding aerosol pollution during the reaction. The detection platform combined with handheld instruments will have great application potential in point-of-care testing.

Fungal aerosols in rabbit breeding environment: Metagenetic insight into PM2.5 based on third-generation sequencing technology.

Fungal aerosols are a vital environmental hazard factor impeding the development of the rabbit breeding industry and threatening public health. This study aimed to determine fungal abundance, diversity, composition, diffusion, and variability in aerosols in rabbit breeding environments. Twenty PM2.5 filter samples were collected from five sampling sites (i.e. En5, In, Ex5, Ex15, and Ex45) in a modern rabbit farm in Linyi City, China. Fungal component diversity at the species level was analyzed in all samples using third-generation sequencing technology. Results revealed that fungal diversity and community composition in PM2.5 significantly differed across different sampling sites, and different pollution levels. The highest concentrations of PM2.5 and fungal aerosols (i.e., 102.5 µg/m3 and 18.8 × 103 CFU/m3, respectively) were found at Ex5, and these concentrations were found to decrease as the distance from the exit increased. However, no significant correlation was observed between the internal transcribed spacer (ITS) gene abundance and overall PM2.5 levels, except for Aspergillus ruber and Alternaria eichhorniae. Although most fungi are not pathogenic to humans, zoonotic pathogenic microorganisms that cause pulmonary aspergillosis (e.g., Aspergillus ruber) and invasive fusariosis (e.g., Fusarium pseudensiforme) were observed. The relative abundance of A. ruber was higher at Ex5 than that at In, Ex15, and Ex45 (p < 0.01), and the relative abundance of the fungal species decreased with an increase in distance from the rabbit houses. Moreover, four potential novel strains of Aspergillus ruber were discovered, with 82.9%-90.3% of the nucleotide and amino acid sequences similar to those of reference strains. This study highlights the importance of rabbit environments as a source in shaping fungal aerosol microbial communities. To the best of our knowledge, this is the first study to uncover the initial characteristics of fungal biodiversity and dispersion of PM2.5 in rabbit breeding environments, contributing to infectious disease control and prevention in rabbits.

Common carp Peptidoglycan Recognition Protein 2 (CcPGRP2) plays a role in innate immunity for defense against bacterial infections.

PGRP is a family of pattern recognition molecules of the innate immune system. PGRPs are conserved from insects to mammals and have diverse functions in antimicrobial defense. Here we cloned a common carp PGRP ortholog, CcPGRP2 containing a conserved C-terminal PGRP domain. We tested the expression levels of CcPGRP2 in the liver, spleen, kidney, foregut, midgut, and hindgut of the highest level in the liver. The expression of CcPGRP2 upregulated in common carp infected with Aeromonas hydrophila (A. hydrophila) or Staphylococcus aureus (S. aureus). Recombinant CcPGRP2 protein expressed in Escherichia coli (E. coli) system and the purified CcPGRP2 could maintain the integrity of intestinal mucosa of common carp infected with A. hydrophila. In addition, CcPGRP2 could agglutinate or bind both gram-positive and gram-negative bacteria in a Zn2+-dependent manner. CcPGRP2 has a stronger agglutination and bacterial binding ability in gram-positive bacteria than in gram-negative bacteria. It is perhaps because CcPGRP2 could bind peptidoglycan (PGN) with a higher degree to lipopolysaccharide (LPS). And CcPGRP2 shows antimicrobial activities in the presence of Zn2+. Our results of CcPGRP2 provided new insight into the function of PGRP in the innate immunity of the common carp.

Nitrogen-Doped Multiwalled Carbon Nanotubes Enhance Bone Remodeling through Immunomodulatory Functions.

It has been reported that multiwalled carbon nanotubes (MWCNTs) can reportedly positively affect growth and differentiation of bone-related cells and therefore offer great potential in biomedical applications. To overcome negative immune responses that limit their application, specific doping and functionalization can improve their biocompatibility. Here, we demonstrated that nitrogen-doped carboxylate-functionalized MWCNTs (N-MWCNTs) enhance bone remodeling both in vitro and in vivo with excellent biocompatibility, via stimulation of both bone resorption and formation. We revealed that 0.2 µg/mL N-MWCNTs not only increase the transcription of osteoblastogenic and osteoclastogenic genes but also up-regulate the activities of both TRAP and AKP in the differentiation of bone marrow stromal cells (BMSCs). Additionally, intramuscular administration of N-MWCNTs at a dosage of 1.0 mg/kg body weight enhances bone mineral density and bone mass content in mice, as well as induces potentiated degree of TRAP- and ARS-positive staining in the femur. The positive regulation of N-MWCNTs on bone remodeling is initiated by macrophage phagocytosis, which induces altered production of inflammatory cytokines by immune response pathways, and consequently up-regulates IL1α, IL10, and IL16. These cytokines collectively regulate the central osteoclastogenic transcription factor NFATc1 and osteoblastogenic BMP signaling, the suppression of which confirmed that these factors respectively participate in N-MWCNT-mediated regulation of osteoclastic and osteoblastic bone marrow stem cell activities. These results suggest that N-MWCNTs can be readily generalized for use as biomaterials in bone tissue engineering for metabolic bone disorders.

Prevalence of Fowl Adenovirus Serotype 4 and Co-Infection by Immunosuppressive Viruses in Fowl with Hydropericardium Hepatitis Syndrome in Shandong Province, China.

Fowl adenovirus serotype 4 (FAdV-4) is the pathogenic agent of hydropericardium hepatitis syndrome (HHS) in chickens and ducks, which has caused huge economic losses for the Chinese poultry industry since 2015. In order to objectively determine the prevalence and co-infection status of the virus in Shandong province in China, we analyzed a total of 679 clinical cases of chickens and ducks from 36 farms in the province. The results showed that the FAdV-4 infection rate was 65.2% (443/679), and the rate in breeder ducks was almost two-fold higher than that in breeder chickens (68.57% vs. 34.30%). Notably, co-infection by H9N2 avian influenza virus, infectious bursal disease virus, and/or chicken infectious anemia virus was very common in the 443 FAdV-4-positive cases. Furthermore, phylogenetic analysis of the hexon genes of four Shandong FAdV-4 isolates revealed that these strains clustered into Indian reference strains, indicating that the Shandong FAdV-4 strains might have originated in India. These findings provide the first data on the prevalence and co-infection status of FAdV-4 in Shandong province, which may serve as a foundation for the prevention of FAdV-4 in the field.

Airborne Transmission of a Serotype 4 Fowl Adenovirus in Chickens.

Serotype 4 fowl adenovirus (FAdV-4) is the main pathogen for hydropericardium syndrome (HPS) in chickens. It has caused major economic losses in the global poultry industry. Currently, FAdV-4's transmission routes in chickens remain unclear. Here we investigate the airborne transmission routes of FAdV-4 in chickens. A total of 45 ten-day-old chickens were equally divided into three groups (infected group/isolator A, airborne group/isolator B, and control group/isolator C). Of note, isolators A and B were connected by a leak-free pipe. The results showed that the virus could form a viral aerosol, detected in isolators two days post infection (dpi). The viral aerosol reached a peak at 4 dpi in the infected group. Healthy chickens in the airborne group were infected by the virus at 8 dpi. The chickens of the airborne group demonstrated subclinical symptoms capable of shedding the virus for some time. This finding suggests that FAdV-4 can be efficiently transmitted among chickens by aerosol transmission. These findings have significant implications for developing strategies to control this infectious disease epidemic.

Dissemination of Escherichia coli carrying plasmid-mediated quinolone resistance (PMQR) genes from swine farms to surroundings.

Different samples were collected from three swine farms in China to investigate the spread of antibiotic-resistant Escherichia coli. A total of 130 E. coli isolates were obtained from feces, air, river water, silt, and soil samples and characterized. The susceptibility of the E. coli isolates to 19 antibiotics was tested. The results revealed that the resistance rates of the E. coli isolates against 9 antibiotics were high. The minimum inhibitory concentration (MIC) values of ciprofloxacin, ofloxacin, and nalidixic acid were mainly in the ranges of 2-64, 8-64, and 8-64 µg/ml. The plasmid-mediated quinolone resistance (PMQR) genes qnr, aac(6')-Ib-cr, qepA, and oqxAB were detected by polymerase chain reaction (PCR), and the similarity of E. coli from different samples was identified by pulsed-field gel electrophoresis (PFGE). The detection rates of the qnrA, qnrB, qnrS, aac(6')-Ib-cr, qepA, and oqxAB genes in the E. coli isolates from three swine farms were in the range of 10.87-23.08%, 13.04-20.51%, 40.00-43.48%, 30.43-38.46%, 6.52-12.82%, and 7.69-17.39%, respectively. The PFGE result showed that 49% (49/100) of isolates originating from air, river water, soil, and silt samples had ≥85% similarity to fecal-obtained isolates, and 40.82% (20/49) of them shared the same PMQR genes with fecal-obtained isolates. This indicated that E. coli carrying PMQR genes and originating from feces in swine farms could spread to the external environment, which could be a potential threat to the public environment and human health.

Pathogenic, Phylogenetic, and Serological Analysis of Group I Fowl Adenovirus Serotype 4 SDSX Isolated From Shandong, China.

Hydropericardium hepatitis syndrome (HHS) caused by group I fowl adenovirus serotype 4 (FAdV-4) is an acute and infectious disease in fowl, particularly in broilers aged 3-5 weeks. In June 2015, a highly pathogenic disease outbroke in 25-40 day-old ducklings in Shandong province, characterized similar symptom to HHS. In order to determine the pathogenic mechanism of FAdV-4 (SDSX strain) in meat ducks. We divided 90 25-day-old Cherry Valley meat ducks into three groups (oral, subcutaneous, and control; 30 ducks in each group) and infected them with the virus. HHS, inclusion body hepatitis, and enlargement and hemorrhage of the spleen, kidney, lung, thymus, and brain were observed in FAdV-4-infected meat ducks. Histopathological changes were mainly characterized by severe fatty degeneration in the liver, basophilic inclusion bodies in hepatocytes, and vacuolation in the bursa. More importantly, viral DNA could be detected by quantitative real-time polymerase chain reaction in several viscera tissues (e.g., heart, liver, spleen) on the third day after infection. Notably, the livers of the two infected groups contained the highest concentration of viral DNA. In addition, immune responses were studied based on titer levels of the virus antibody and the levels of inflammatory cytokines interleukin (IL)-2 and interferon (IFN)-γ, and most levels were significantly upregulated, indicating that the host immune responses were activated early in infection. These findings increase our understanding of the pathogenicity of FAdV-4 (SDSX) in meat ducks and provide the foundation for further in-depth study of the pathogenic mechanism of this virus.

Evolution of Tembusu Virus in Ducks, Chickens, Geese, Sparrows, and Mosquitoes in Northern China.

Tembusu virus (TMUV) is a contagious pathogen from fowl that mainly infects ducks and geese, causing symptoms of high fever, loss of appetite, retarded growth, neurological symptoms, severe duck-drop syndrome, and even death. During an epidemiological investigation of TMUV in Northern China, we isolated 11 TMUV strains from ducks, chickens, geese, sparrows, and mosquitoes (2011⁻2017). Phylogenetic analysis of the open-reading frames of genes revealed that these strains clustered into Chinese strains II. The nucleotide and amino acid homologies of NS1 of the strains ranged between 85.8⁻99.8% and 92.5⁻99.68%, respectively, which were lower than those of E (86.7⁻99.9% and 96.5⁻99.9%, respectively), NS3 (87.6⁻99.9% and 98.2⁻99.8%, respectively), and NS5 (86.5⁻99.9% and 97.8⁻99.9%, respectively). Predictions of the tertiary structure of the viral proteins indicated that NS1 in 4 of 11 strains had a protein structure mutation at 180TAV182 that changed a random crimp into an alpha helix. The protein of 6 of 11 strains had a glycosylation site mutation from NTTD to NITD. Furthermore, epidemiological data suggested that TMUV has been circulating in half of China's provinces (17 of 34). Our findings, for the first time, have identified the NS1 protein as a potential hypervariable region for genetic evolution. Additionally, the territorial scope of the virus has expanded, requiring strict bio-security measures or a multivalent vaccine to control its spread.

Comparative Transcriptomic Analysis of Immune-Related Gene Expression in Duck Embryo Fibroblasts Following Duck Tembusu Virus Infection.

Duck is a major waterfowl species in China, providing high-economic benefit with a population of up to 20⁻30 billion per year. Ducks are commonly affected by severe diseases, including egg-drop syndrome caused by duck Tembusu virus (DTMUV). The immune mechanisms against DTMUV invasion and infection remain poorly understood. In this study, duck embryo fibroblasts (DEFs) were infected with DTMUV and harvested at 12 and 24 h post-infection (hpi), and their genomes were sequenced. In total, 911 (764 upregulated and 147 downregulated genes) and 3008 (1791 upregulated and 1217 downregulated) differentially expressed genes (DEGs) were identified at 12 and 24 hpi, respectively. Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed that DEGs were considerably enriched in immune-relevant pathways, including Toll-like receptor signaling pathway, Cytosolic DNA-sensing pathway, RIG-I-like receptor signaling pathway, Chemokine signaling pathway, NOD-like receptor signaling pathway, and Hematopoietic cell lineage at both time points. The key DEGs in immune system included those of the cytokines (IFN α2, IL-6, IL-8L, IL-12B, CCR7, CCL19, and CCL20), transcription factors or signaling molecules (IRF7, NF-κB, STAT1, TMEM173, and TNFAIP3), pattern recognition receptors (RIG-I and MDA5), and antigen-presenting proteins (CD44 and CD70). This suggests DTMUV infection induces strong proinflammatory/antiviral effects with enormous production of cytokines. However, these cytokines could not protect DEFs against viral attack. Our data revealed valuable transcriptional information regarding DTMUV-infected DEFs, thereby broadening our understanding of the immune response against DTMUV infection; this information might contribute in developing strategies for controlling the prevalence of DTMUV infection.

A Novel Diagnostic Method to Detect Duck Tembusu Virus: A Colloidal Gold-Based Immunochromatographic Assay.

Duck Tembusu virus (DTMUV) is an emerging pathogenic flavivirus that has resulted in large economic losses to the duck-rearing industry in China since 2010. Therefore, an effective diagnostic approach to monitor the spread of DTMUV is necessary. Here, a novel diagnostic immunochromatographic strip (ICS) assay was developed to detect DTMUV. The assay was carried out using colloidal gold coated with purified monoclonal antibody A12D3 against envelope E protein. Purified polyclonal C12D1 antibodies from BALB/c mice against the envelope E protein were used as the capture antibody. Goat anti-mouse IgG was used to detect DTMUV, which was also assembled on the ICS. Results showed that the ICS could specifically detect DTMUV within 10 min. It also could be stored 25 and 4°C for 4 and 6 months, respectively. The sensitivity of the ICS indicated that the dilution multiples of positive allantoic fluid of DTMUV (LD50: 104.33/0.2 ml) was up to 200. Its specificity and sensibility showed no significant change under the above storage situations. Fifty clinical samples were simultaneously detected by ICS and reverse-transcription polymerase chain reaction with a 93.9% coincidence rate between them. It proved that the ICS in the present study was highly specific, sensitive, repeatable, and more convenient to rapidly detect DTMUV in clinical samples.

Vertical Transmission of H9N2 Avian Influenza Virus in Goose.

During a study on high mortality cases of goose embryo in Shandong Province, China (2014-2015), we isolated an H9N2 avian influenza virus (AIV) strain (A/goose/Shandong/DP01/2014, DP01), which was supposedly the causative agent for goose embryo death. Sequence analysis revealed that DP01 shared 99.9% homology in the HA gene with a classic immune suppression strain SD06. To study the potential vertical transmission ability of the DP01 strain in breeder goose, a total of 105 Taizhou breeder geese, which were 360 days old, were equally divided into five groups (A, B, C, D, and E) for experimental infection. H9N2 AIV (DP01) was used for inoculating through intravenous (group A), intranasal instillation (group B), and throat inoculation (group C) routes, respectively. The geese in group D were inoculated with phosphate buffer solution (PBS) and those in group E were the non-treated group. At 24 h post inoculation, H9N2 viral RNA could be detected at vitelline membrane, embryos, and allantoic fluid of goose embryos from H9N2 inoculated groups. Furthermore, the HA gene of H9N2 virus from vitelline membrane, embryo, allantoic fluid, and gosling shared almost 100% homology with an H9N2 virus isolated from the ovary of breeder goose, which laid these eggs, indicating that H9N2 AIV can be vertically transmitted in goose. The present research study provides evidence that vertical transmission of H9N2 AIV from breeding goose to goslings is possible.

Proteomic Analysis of Differential Expression of Cellular Proteins in Response to Avian H9N2 Virus Infection of A549 Cells.

In this study, differentially expressed proteins in A549 cells (human lung adenocarcinoma epithelial cell line) infected with H9N2 avian influenza virus (AIV) were investigated by two-dimensional electrophoresis (2-DE). Sixteen different spots between the groups (ratio > 2, p < 0.05) were identified with mass spectrometry identification. Proteins located in the downstream of the NF-κB and IFN transcription factor pathways were identified, e.g., ISG15. Actin and keratin were also identified, suggesting that the cytoskeleton may plays an important role in the AIV infection of mammalian cells. These findings could provide insights into the interaction between host and influenza viruses and might provide valuable information for clarifying the pathogenesis of viral infections as well.

Effects of Microbial Aerosol in Poultry House on Meat Ducks' Immune Function.

The aim of this study was to evaluate effects of microbial aerosols on immune function of ducks and shed light on the establishment of microbial aerosol concentration standards for poultry. A total of 1800 1-d-old cherry valley ducks were randomly divided into five groups (A, B, C, D, and E) with 360 ducks in each. To obtain objective data, each group had three replications. Concentrations of airborne bacteria, fungi, endotoxin in different groups were created by controlling ventilation and bedding cleaning frequency. Group A was the control group and hygienic conditions deteriorated progressively from group B to E. A 6-stage Andersen impactor was used to detect the aerosol concentration of aerobes, gram-negative bacteria, fungi, and AGI-30 microbial air sampler detect the endotoxin, and Composite Gas Detector detect the noxious gas. In order to assess the immune function of meat ducks, immune indicators including H5 AIV antibody titer, IgG, IL-2, T-lymphocyte transformation rate, lysozyme and immune organ indexes were evaluated. Correlation coefficients were also calculated to evaluate the relationships among airborne bacteria, fungi, endotoxin, and immune indicators. The results showed that the concentration of airborne aerobe, gram-negative bacteria, fungi, endotoxin have a strong correlation to H5 AIV antibody titer, IgG, IL-2, T-lymphocyte transformation rate, lysozyme, and immune organ indexes, respectively. In addition, when the concentration of microbial aerosol reach the level of group D, serum IgG (6-8 weeks), lysozyme (4 week) were significantly higher than in group A (P < 0.05); serum IL-2 (7 and 8 weeks), T-lymphocyte transformation rate, lysozyme (7 and 8 weeks), spleen index (6 and 8 weeks), and bursa index (8 week) were significantly lower than in group A (P < 0.05 or P < 0.01). The results indicated that a high level of microbial aerosol adversely affected the immune level of meat ducks. The microbial aerosol values in group D provide a basis for recommending upper limit concentrations of microbial aerosols for healthy meat ducks.