Cases of H9N2 avian influenza virus (AIV) in poultry are increasing throughout many Eurasian countries, and co-infections with other pathogens have resulted in high morbidity and mortality in poultry. Few studies have investigated the genetic factors of virus airborne transmission which determine the scope of this epidemic. In this study, we used specific-pathogen-free chickens housed in isolators to investigate the airborne transmissibility of five recombinant H9N2 AIV rescued by reverse genetic technology. The results show that airborne transmission of A/Chicken/Shandong/01/2008 (SD01) virus was related to the neuraminidase (NA) gene, and four amino acid mutations (D368E, S370L, E313K and G381D) within the head region of the SD01 NA, reduced virus replication in the respiratory tract of chickens, reduced virus NA activity, and resulted in a loss of airborne transmission ability in chickens. Similarly, reverse mutations of these four amino acids in the NA protein of r01/NASS virus, conferred an airborne transmission ability to the recombinant virus. We conclude that these four NA residues may be significant genetic markers for evaluating potential disease outbreak of H9N2 AIV, and propose that immediate attention should be paid to the airborne transmission of this virus.

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 5 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.

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.

Aspergillus fumigatus is a ubiquitous pathogen in poultry farms, causing aspergillosis in chickens. To study the pathogenicity of A. fumigatus, 14-days-old chickens were infected with fungal conidia (2 × 10⁷ CFU/mL) via thoracic intra-air sacs inoculation. The clinical symptoms, gross and histopathological lesions, and fungal load in the lungs were examined. Additionally, the mRNAs of Toll like receptors (TLR) and pro-inflammatory cytokines were evaluated by quantitative PCR to explore the immune responses induced by A. fumigatus. The results showed that overt depression, ruffled feathers, and dyspnea were observed in the infected chickens as early as 3 days post infection (dpi). Eleven out of 25 infected chickens died from 5 to 9 dpi, and A. fumigatus could also be reisolated from the infected lung. Histopathological examination revealed obvious airsacculitis and pneumonia, characterized by inflammatory cell infiltration (heterophils and macrophages), and granulomatous lesions in the lung. The mRNA expressions of TLR1 and TLR2 were upregulated in the lung and spleen, and most pro-inflammatory cytokines including IL-1β, Cxcl-8, TNF-α, IL-12, and IFN-γ were increased in both the lung and spleen during the tested period, suggesting that the innate immune responses were triggered by A. fumigatus infection, and these cytokines participated in the inflammatory responses against A. fumigatus. These results indicate that A. fumigatus infection by thoracic intra-air sacs inoculation can cause severe respiratory damage in chickens, activate TLR1 and TLR2 mediated immune responses, and elicit large expression of pro-inflammatory cytokines such as IL-1β, Cxcl-8, and IFN-γ. These data will help further understanding of the pathogenesis and immune responses of A. fumigatus infection in the chicken.

Pseudomonas aeruginosa is a common opportunistic pathogen. The potential efficacy of phage therapy has attracted the attention of researchers, but efficient gene-editing tools are lacking, limiting the study of their biological properties. Here, we designed a type V CRISPR-Cas12a system for the gene editing of P. aeruginosa phages. We first evaluated the active cutting function of the CRISPR-Cas12a system in vitro and discovered that it had a higher gene-cutting efficiency than the type II CRISPR-Cas9 system in three different P. aeruginosa phages. We also demonstrated the system’s ability to precisely edit genes in Escherichia coli phages, Salmonella phages, and P. aeruginosa phages. Using the aforementioned strategies, non-essential P. aeruginosa phage genes can be efficiently deleted, resulting in a reduction of up to 5,215 bp (7.05%). Our study has provided a rapid, efficient, and time-saving tool that accelerates progress in phage engineering.