Research Note: The structure and diversity of antibiotic resistance genes in animal house environment.

In recent years, a series of public health issues caused by the spread of antibiotic resistance have been widely concerned. The indoor air of livestock and poultry houses is considered to be one of the main sources of environmental contamination of ARGs. This study characterized the micro-organisms and ARGs in the air particulate matter of chicken houses using metagenomics. The study successfully detected 761 different subtypes of resistance genes including aminoglycosides, tetracyclines, MLSB etc., 4 types of mobile genetic elements, and various pathogenic microorganisms from the aerosols in the chicken coop environment. The results showed that the abundance of ARGs in the air of the chicken coop was at a relatively high level, correlation network analysis showed that multiple types of ARGs could promote the emergence of antibiotic-resistant bacteria.

Maternal exposure to particulate matter from duck houses restricts fetal growth due to inflammatory damage and oxidative stress.

The composition of particulate matter (PM) in poultry farms differs significantly from that of atmospheric PM as there is a higher concentration of microbes on farms. To assess the health effects of PM from poultry farms on pregnant animals, we collected PM from duck houses using a particulate sampler, processed it via centrifugation and vacuum concentration, and subsequently exposed the mice to airborne PM at 0.48 mg/m3 (i.e., low concentration group) and 1.92 mg/m3 (i.e., high concentration group) on the fifth day of pregnancy. After exposure until the twentieth day of pregnancy or spontaneous delivery, mice were euthanized for sampling. The effects of PM from duck houses on the pregnancy toxicity of mice were analyzed using histopathological analysis, enzyme-linked immunosorbent assay, and quantitative real-time polymerase chain reaction (qPCR). The results showed that exposure to PM had adverse effects on pregnant mice that reduced their feed intake in both groups. Microscopic lesions were observed in the lungs and placentas of pregnant mice, and the lesions worsened with increased PM concentrations, as shown by alveolar wall thickening, the infiltration of inflammatory cells in pulmonary interstitium, congestion, edema, and cellular degeneration of placenta. In pregnant mice in the high concentration group, exposure to PM significantly increased the expression of inflammatory cytokines in the lungs and placentas, caused oxidative stress, and decreased estrogen level in the blood. Exposure to PM also resulted in the reduced litter sizes of pregnant mice and shorter body and tail lengths in the fetuses delivered. Beyond that, exposure to PM significantly downregulated the levels of antioxidant factor superoxide dismutase and neurotrophic factor Ngf in the brains of fetuses. Collectively, exposure to a high concentration of PM by inhalation among pregnant mice caused significant pregnancy toxicity that led to abnormal fetal development due to inflammatory damage and oxidative stress. These findings established a foundation for future studies on the underlying mechanisms of pregnancy toxicity induced by exposure to PM.

Studies on the airborne bacterial communities and antimicrobial resistance genes in duck houses based on metagenome and PCR analysis.

The threat of antimicrobial resistance (AMR) is on the rise globally, especially with the development of animal husbandry and the increased demand for antibiotics. Livestock and poultry farms, as key sites for prevalence of antibiotic-resistant bacteria (ARB), can spread antimicrobial resistance genes (ARGs) through microbial aerosols and affect public health. In this study, total suspended particulate matter (TSP) and airborne culturable microorganisms were collected from duck houses in Tai'an, Shandong Province, and the bacterial communities and airborne ARGs were analyzed using metagenomics and PCR methods. The results showed that the bacterial communities in the air of duck houses were mainly Actinobacteria, Firmicutes, Proteobactria, Chlamydia, and Bcateroidetes at the phylum level. At the genus level, the air was dominated by Corynebacterium, Jeotgalicoccus, Staphylococcus, Brevibacterium, and Megacoccus, and contained some pathogenic bacteria such as Staphylococcus aureus, Corynebacterium diphtheriae, Klebsiella oxytoca, Acinetobacter baumannii, and Pseudomonas aeruginosa, which were also potential hosts for ARGs. The airborne ARGs were mainly macrolides (10.97%), penicillins (10.73%), cephalosporins (8.91%), streptozotocin (8.91%), and aminoglycosides (8.02%). PCR detected 27 ARGs in airborne culturable microorganisms, and comparative analysis between PCR and the metagenomic data revealed that a total of 9 ARGs were found to the same, including macrolides ErmA, ErmF, tetracyclines tetG, tetX, methicarbamazepines dfrA12, dfrA15, aminoglycosides APH3-VI, ANT2-Ⅰ, and sulfonamides sul2. Moreover, inhalation exposure modeling showed that the workers in duck houses inhaled higher concentrations of ARB, human pathogenic bacteria (HPB) and human pathogenic antibiotic-resistant bacteria (HPARB) than hospital workers. These results provide new insights into airborne microorganisms and ARGs in animal farms and lay the foundation for further study.

Combined 16S and Internal Transcribed Spacer analysis revealed the effect of time on microbial community in animal house.

The outbreak of COVID-19 reminds people that aerosols have an important impact on health. The concentration and composition of microbial aerosol in livestock and poultry houses are closely related to the environmental conditions of livestock and poultry houses, and also related to the healthy growth of livestock and poultry. In our study, 16S and ITS sequencing techniques were used to analyze the relation and difference of bacteria and fungi in the air samples of a chicken house. At the age of 7 to 42 d, the operation classification unit (OTU) numbers of bacteria and fungi identified in our results were 2,398 and 986, respectively, of which the shared OTU numbers were 410 and 141, respectively. At the phylum level, Firmicutes, Proteobacteria, and Actinomycetes were the 3 most abundant bacterial phyla, and Ascomycetes and Basidiomycetes were the top 2 phyla in fungi. At the genus level, 7 differential fungal genera were identified, including Debaryomyces, Trichosporon, Wallemia, Aspergillus, Nigrospora, Fusarium, and Vishniacozyma. Compared with other bacterial genera, Lactobacillus, Cetobacterium, and Romboutsia had the highest abundance (more than 5%). The result showed that the Alpha diversity and Beta diversity of fungi were significantly different in different growing periods. However, only Beta diversity showed significant differences among bacteria. In general, the bacterial and fungal diversity of microbial aerosols in the chicken house increased significantly at the age of 7 to 42 d. And the evenness and richness of airborne fungal communities also increased obviously. In a word, we must pay attention to the complex community composition in the chicken house, this is closely related to animal health and the health of surrounding residents. The cooperation and communication between bacteria and fungi in PM2.5 samples provides a new reference to analyze the influence of microbial aerosol.

Understanding the Effect of Compound Probiotics on the Health of Rabbits and Its Mechanisms Through Metagenomics.

In this study, we investigated the effects of probiotics on growth performance, immunity, intestinal flora, and antioxidant capacity of rabbits. Three hundred New Zealand white rabbits were randomly divided into four groups. Groups A, B, C, and D were the lactobacillus group, compound probiotic group, control group, and antibiotic group, respectively. The results showed compared with the control group, the average weight of groups A, B, and D increased by 14.88%, 12.33%, and 11.97%, respectively. Moreover, the index of immune organs and the IgG and IgM in serum of group B were significantly increased (P < 0.05). Meanwhile, the activities of superoxide dismutase (SOD) in group B and catalase (CAT) in group A were significantly increased (P < 0.05). At week 5, the contents of rabbit cecum were taken for metagenome sequencing, and the results showed probiotics increased the relative abundance of Akkermansia, and decreased the relative abundance of Bacteroides. According to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, we found probiotics could enrich metabolic pathways such as carbohydrates, amino acids, and lipids. According to the Comprehensive Antibiotic Resistance Database (CARD), we found antibiotic resistance ontology (ARO) in cecum mainly included ß-lactamases, macrolide 2'-phosphotransferase II, and plasmid-mediated quinolone resistance protein. Among them, there were 1964, 2105, and 1982 types of ARO in group B, group D, and groups A and C, respectively. These results showed probiotics played a beneficial role in maintaining or enhancing the health and growth of rabbits and could replace antibiotics under certain feeding conditions.

Exploring the Microbial Community Structure in the Chicken House Environment by Metagenomic Analysis.

The environmental conditions of chicken houses play an important role in the growth and development of these animals. The chicken house is an essential place for the formation of microbial aerosols. Microbial aerosol pollution and transmission can affect human and animal health. In this work, we continuously monitored fine particulate matter (PM2.5) in the chicken house environment for four weeks and studied the microbial community structure in the aerosols of the chicken house environment through metagenomic sequencing. Our results found that bacteria, fungi, viruses, and archaea were the main components of PM2.5 in the chicken house environment, accounting for 89.80%, 1.08%, 2.06%, and 0.49%, respectively. Conditional pathogens are a type of bacteria that poses significant harm to animals themselves and to farm workers. We screened ten common conditional pathogens and found that Staphylococcus had the highest relative abundance, while Clostridium contained the most microbial species, up to 456. Basidiomycetes and Ascomycota in fungi showed dramatic changes in relative abundance, and other indexes showed no significant difference. Virulence factors (VF) are also a class of molecules produced by pathogenic microbes that can cause host diseases. The top five virulence factors were found in four groups: FbpABC, HitABC, colibactin, acinetobactin, and capsule, many of which are used for the iron uptake system. In the PM2.5 samples, eight avian viruses were the most significant discoveries, namely Fowl aviadovirus E, Fowl aviadovirus D, Avian leukosis virus, Avian endogenous retrovirus EAV-HP, Avian dependent parvovirus 1, Fowl adenovus, Fowl aviadovirus B, and Avian sarcoma virus. The above results significantly improve our understanding of the microbial composition of PM2.5 in chicken houses, filling a gap on virus composition; they also indicate a potential threat to poultry and to human health. This work provides an important theoretical basis for animal house environmental monitoring and protection.

Research progress on distribution and exposure risk of microbial aerosols in animal houses.

Environmental aerosols in animal houses are closely related to the productive performance and health level of animals living in the houses. Preferable housing environments can improve animal welfare and production efficiency, so it is necessary to monitor and study these environments. In recent years, there have been many large-scale outbreaks of respiratory diseases related to biological aerosols, especially the novel coronavirus that has been sweeping the world. This has attracted much attention to the mode of aerosol transmission. With the rapid development of large-scale and intensive breeding, microbial aerosols have gradually become the main factor of environmental pollution in animal houses. They not only lead to a large-scale outbreak of infectious diseases, but they also have a certain impact on the health of animals and employees in the houses and increase the difficulty of prevention and control of animal-borne diseases. This paper reviews the distribution, harm, and control measures of microbial aerosols in animal house environments in order to improve people's understanding of them.

Complete Genome Sequence of Bacillus subtilis BYS2, a Strain with a Broad Inhibitory Spectrum against Pathogenic Bacteria.

Bacillus subtilis BYS2 is a strain with a broad inhibitory spectrum against pathogenic bacteria. In the current study, we report the complete genome sequence of Bacillus subtilis BYS2. The chromosome of BYS2 (4,030,791 bp; G+C content, 43.88%) contained 3,914 protein-encoding genes, with 86 tRNAs, 30 rRNAs, and 5 noncoding RNAs (ncRNAs).

Aerosol Transmission of Coronavirus and Influenza Virus of Animal Origin.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused great harm to global public health, resulting in a large number of infections among the population. However, the epidemiology of coronavirus has not been fully understood, especially the mechanism of aerosol transmission. Many respiratory viruses can spread via contact and droplet transmission, but increasing epidemiological data have shown that viral aerosol is an essential transmission route of coronavirus and influenza virus due to its ability to spread rapidly and high infectiousness. Aerosols have the characteristics of small particle size, long-time suspension and long-distance transmission, and easy access to the deep respiratory tract, leading to a high infection risk and posing a great threat to public health. In this review, the characteristics of viral aerosol generation, transmission, and infection as well as the current advances in the aerosol transmission of zoonotic coronavirus and influenza virus are summarized. The aim of the review is to strengthen the understanding of viral aerosol transmission and provide a scientific basis for the prevention and control of these diseases.

Pathogenicity and Immune Responses of Aspergillus fumigatus Infection in Chickens.

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-1ß, 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-1ß, Cxcl-8, and IFN-γ. These data will help further understanding of the pathogenesis and immune responses of A. fumigatus infection in the chicken.

Benefit of Dietary Supplementation with Bacillus subtilis BYS2 on Growth Performance, Immune Response, and Disease Resistance of Broilers.

A strain of Bacillus subtilis (B. subtilis) BYS2 was previously isolated from Mount Tai, which is located in Tai'an City in the Shandong Province of China. The strain was then stored in the Environmental Microbiology Laboratory at Shandong Agricultural University. To evaluate the effect of the bacterium preparation in broiler production, we fed the bacterium (106 CFU/g) to 1-day-old broilers and continued this feeding for 6 weeks to analyze its effect on growth and immune performance. We found that the average weight of the bacterium-fed group increased by 17.19% at weeks 5 compared to the control group (P < 0.05). The height of the villi in the duodenum and jejunum and the ratio of villi to crypt were significantly increased in the bacterium-fed group at weeks 5 (P < 0.05). Also, the IgG in the serum of broilers in the experimental group increased by 31.60% (P < 0.05) and IgM 30.52% (P < 0.05) compared with those in the control group. The expressions of the major pattern recognition receptors (PRRs), antiviral proteins, pro-inflammatory cytokines, and ß-defensins were significantly higher than those in the control group (P < 0.05). Meanwhile, the bursa immune organ indices of broilers in the experimental group were significantly higher than those in the control group (P < 0.05). Also, after 5 weeks of continuous feeding, when infected with Escherichia coli (E. coli) O1K1 and Newcastle disease virus (NDV) F48E8, the content of bacteria and virus in tissues and organs of the experimental group decreased significantly, and the survival rate of infected chickens increased by 31.1% and 17.7%, respectively (P < 0.05). These results show that the anti-infective B. subtilis BYS2 could, to some extent, replace antibiotics to promote growth, improve innate immunity, and enhance disease resistance in broilers.

Detection of microbial aerosols in hospital wards and molecular identification and dissemination of drug resistance of Escherichia coli.

Antibiotic-resistant bacteria (ARB) present a global public health problem. Microorganisms are the main cause of hospital-acquired infections, and the biological contamination of hospital environments can cause the outbreak of a series of infectious diseases. Therefore, it is very important to understand the spread of antibiotic-resistant bacteria in hospital environments. This study examines the concentrations of aerobic bacteria and E. coli in ward environments and the airborne transmission of bacterial drug resistance. The results show that the three wards examined have an average aerobic bacterial concentration of 132 CFU∙m-3 and an average inhalable aerobic bacterial concentration of 73 CFU∙m-3, with no significant difference (P > 0.05) among the three wards. All isolated E. coli showed multi-drug resistance to not only third-generation cephalosporin antibiotics, but also quinolones, aminoglycosides, and sulfonamides. Furthermore, 51 airborne E. coli strains isolated from the air in the three wards and the corridor were screened for ESBLs, and 12 (23.53%) were ESBL-positive. The drug-resistance gene of the 12 ESBL-positive strains was mainly TEM gene, and the detection rate was 66.67% (8/12). According to a homology analysis with PFGE, 100% homologous E. coli from the ward at 5 m and 10 m outside the ward in the corridor shared the same drug-resistance spectrum, which further proves that airborne E. coli carrying a drug-resistance gene spreads out of the ward through gas exchange. This leads to biological pollution inside, outside, and around the ward, which poses a direct threat to the health of patients, healthcare workers, and surrounding residents. It is also the main reason for the antibiotic resistance in the hospital environment. More attention should be paid to comprehensive hygiene management in the surrounding environment of hospitals.

The composition of microbial aerosols, PM2.5, and PM10 in a duck house in Shandong province, China.

Poultry-emitted air pollutants, including microbial aerosols and particulate matter, have raised concerns due to their potential negative effects on human health and the environment. High concentrations of microbial aerosols can also significantly affect duck production performance, leading to immunosuppression and increased disease susceptibility. We determined the concentrations, distributions, and biological components of the microbial aerosols and particulate matter in a duck house environment. The concentration ranges of the bacteria, fungi, Gram-negative bacteria, Escherichia coli, and endotoxin in the duck houses were 3.3 to 5.2 × 104 CFU/m3, 3.8 to 11.9 × 103 CFU/m3, 2.1 to 3.6 × 103 CFU/m3, 1.3 to 2.7 × 102 CFU/m3, and 0.65 to 2.2 × 103 EU/m3, respectively. We also found the endotoxin levels were higher than the standard that can cause pneumonia (2,000 EU/m3). The concentration ranges of the PM2.5 and PM10 samples were 1.1 to 1.6 × 102 µg/m3 and 1.2 to 1.9 × 102 µg/m3, respectively. At the phylum level, the top 5 bacteria identified in the PM2.5 fraction were Actinobacteria, Firmicutes, Proteobacteria, Bacteroidetes, and Fusobacteria, with Actinobacteria (50.55%) as the most abundant. At the genus level, 293 bacterial groups were identified. Actinobacteria (39.01%) was the most abundant phylum, followed by Firmicutes (5.44%) and Proteobacteria (4.56%). The bacterial distributions that differed between the PM2.5 and PM10 samples were Lactobacillales, Bacilli, Firmicutes, and Bacteroidetes; the fungi that differed were Microbotryomycetes, Sporidiobolales, Agaricomycetes, and Polyporates. Microbial allergens and pathogens were also identified. Corynebacterium had a relative abundance of more than 30% in the PM2.5 and PM10 distributions. Aspergillus was the main fungal allergen and opportunistic pathogen, with a relative abundance of 10%. In conclusion, our research supports that the microbial composition in the duck house environment poses a potential threat to the health of both the ducks and the duck house workers.

Proteomic analysis of ESBL-producing Escherichia coli under bentonite condition.

The dissemination of extended spectrum beta-lactamases (ESBL) genes through gene transfer attracts wide attention. Bentonite is widely used as a feed additive in an animal-breeding environment. In order to obtain a better understanding of the effect of bentonite on Escherichia coli carrying ESBL gene, proteomic analysis was carried out to screen the key proteins. The results showed that a total of 31 proteins were differentially expressed, including 21 up-regulated proteins and 10 down-regulated proteins. These proteins were involved in biosynthetic process, metabolic process, stress response, transport, anaerobic respiration, proteolysis, hydrolase, protein folding, transcription, salvage, and other. The transcriptional level of four genes (mipA, gntY, tldD, and arcA) was in consensus with proteomic results. This study revealed the differentially expressed proteins involved when E. coli was incubated under bentonite and PBS condition, which implied the possibility that bentonite may promote the transfer of ESBL gene between E. coli.

Galectin-1 induces immune response and antiviral ability in Cherry Valley ducks after duck plague virus infection.

Galectin-1, as a typical animal galactose-binding protein, it is found on the cell surface and in the extracellular matrix. Cloning the full-length coding sequence of galectin-1 from the spleens of Cherry Valley ducks revealed that the coding sequence of duck galectin-1 (duGal-1) comprises 405 bp, encoding 134 amino acids. Homologic analysis revealed its amino acid sequence is most identical to that of Anas platyrhynchos (98.8%) followed by Gallus gallus. Quantitative real-time PCR analysis indicated that duGal-1 mRNA is broadly expressed in healthy Cherry Valley duck tissues, primarily in the heart and trachea but minimally in the lung and skin. Meanwhile, the duGal-1 expression is slightly upregulated in the infected liver and spleen. Furthermore, the expression levels of ISGs (Mx, PKR, OAS) and some cytokines such as IFN-α, IL-1ß, IL-2, are up-regulated to varying degrees after overexpression the duGal-1, In contrast, Knockdown of duGal-1 found that the expression levels of ISGs and some inflammatory cytokines were down-regulated. Antiviral assay showed that duGal-1 could inhibit viral replications early during infection. This is the first study of the cloning, tissue distribution, and antiviral immune responses of duGal-1, and findings imply it is involved in the early stages of antiviral innate immune responses to duck plague virus infections in ducks.

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.

The PA Subunit of the Influenza Virus Polymerase Complex Affects Replication and Airborne Transmission of the H9N2 Subtype Avian Influenza Virus.

The polymerase acidic (PA) protein is the third subunit of the influenza A virus polymerase. In recent years, studies have shown that PA plays an important role in overcoming the host species barrier and host adaptation of the avian influenza virus (AIV). The objective of this study was to elucidate the role of the PA subunit on the replication and airborne transmission of the H9N2 subtype AIV. By reverse genetics, a reassortant rSD01-PA was derived from the H9N2 subtype AIV A/Chicken/Shandong/01/2008 (SD01) by introducing the PA gene from the pandemic influenza A H1N1 virus A/swine/Shandong/07/2011 (SD07). Specific pathogen-free (SPF) chickens and guinea pigs were selected as the animal models for replication and aerosol transmission studies. Results show that rSD01-PA lost the ability of airborne transmission among SPF chickens because of the single substitution of the PA gene. However, rSD01-PA could infect guinea pigs through direct contact, while the parental strain SD01 could not, even though the infection of rSD01-PA could not be achieved through aerosol. In summary, our results indicate that the protein encoded by the PA gene plays a key role in replication and airborne transmission of the H9N2 subtype AIV.

Characterization and Roles of Cherry Valley Duck NLRP3 in Innate Immunity During Avian Pathogenic Escherichia coli Infection.

The nucleotide-binding oligomerization domain-like receptor (NLR) pyrin domain containing 3 (NLRP3) is a pattern recognition receptor that is involved in host innate immunity and located in the cytoplasm. In the present study, the full-length cDNA of Cherry Valley duck NLRP3 (duNLRP3) (2,805 bp encode 935 amino acids) was firstly cloned from the spleen of healthy Cherry Valley ducks, and the phylogenetic tree indicated that the duNLRP3 has the closest relationship with Anas platyrhynchos in the bird branch. According to quantitative real-time PCR analysis, the duNLRP3 mRNA has a broad expression spectrum in healthy Cherry Valley duck tissues, and the highest expression is in the pancreas. There was significant up-regulation of duNLRP3 mRNA expression in the liver and down-regulation in the spleen after infection with avian pathogenic Escherichia coli (APEC) O1K1, especially at 3 days after the infection. Ducks hatched from NLRP3-lentiviral vector-injected eggs had significantly higher duNLRP3 mRNA expression in the liver, spleen, brain, and cecum, which are tissues usually with lower background expression. The mRNA expression levels of inflammatory cytokines IL-1ß, IL-18, and TNF-α significantly increased after the APEC infection in those tissues. The bacterial content in the liver and spleen decreased significantly compared with the NC-lentiviral vector-injected ducks. In addition, in the duck embryo fibroblasts, both of the overexpression and knockdown of duNLRP3 can trigger the innate immune response during the E. coli infection. Specifically, overexpression induced antibacterial activation, and knockdown reduced the antibacterial activity of the host cells. The IL-1ß, IL-18, and TNF-α mRNA expressions showed up-regulation or down-regulation. The results demonstrate that duNLRP3 has a certain antibacterial activity during E. coli infection. These findings also contribute to better understanding the importance of duNLRP3 in regulating the inflammatory response and the innate immune system of ducks.

Protective Role of Rabbit Nucleotide-Binding Oligomerization Domain-2 (NOD2)-Mediated Signaling Pathway in Resistance to Enterohemorrhagic Escherichia coli Infection.

Nucleotide-binding oligomerization domain 2 (NOD2), a member of the NOD-like receptors (NLRs) family that is well-known to play a key role in innate immune responses and is involved in innate antibacterial responses. In this study, rabbit NOD2 (rNOD2) was cloned from rabbit kidney (RK) cells. It was distributed in various tissues, and the highest level of rNod2 was detected in spleen. Moreover, the expression of rNod2 was significantly upregulated in the heart, liver, and spleen induced by enterohemorrhagic Escherichia coli (EHEC). Overexpression of rNOD2 induced the expression of pro-inflammatory cytokine, including Il1ß, Il6, Ifn-γ, and Tnf, as well as defensins, including Defb124, Defb125, and Defb128 through the nuclear factor (NF)-κB signaling pathway. Furthermore, overexpression of rNOD2 inhibited the growth of EHEC, and knockdown of rNOD2 or inhibition of the NF-κB pathway promoted its replication. In addition, our results suggest that rNOD2 can significantly activate NF-κB signaling and trigger antibacterial defenses to increase the expression of pro-inflammatory cytokine and defensins after stimulation by EHEC. These findings are useful to further understanding the innate immune system of rabbits and providing a new perspective for the prevention of bacterial diseases in rabbits.

Fowl Adenovirus Serotype 4 SD0828 Infections Causes High Mortality Rate and Cytokine Levels in Specific Pathogen-Free Chickens Compared to Ducks.

Hydropericardium syndrome and inclusion body hepatitis, together called hydropericardium-hepatitis syndrome, are acute infectious diseases found in chickens. These diseases are caused primarily by fowl adenovirus serotype 4 (FAdV-4) strains. In this study, we isolated a FAdV-4 strain (SD0828) from clinically diseased chickens and phylogenetically analyzed the L1 loops of the hexon protein sequences in 3-week-old specific pathogen-free chickens and ducks infected intramuscularly and orally, determining differences in the pathogenicity by observing clinical signs and gross and histological lesions. We also detected the viral load in tissue samples. Postinfection necropsy showed that all chickens but no ducks exhibited typical necropsy lesions. Additionally, all chickens infected intramuscularly died within 2 days postinfection (dpi), and all those infected orally died within 5 dpi, whereas no infected ducks died before 28 dpi. Quantitative real-time polymerase chain reaction analysis was used to determine the viral load in the tissues of hearts, livers, spleens, lungs, and kidneys and in cloacal cotton swabs from infected chickens and ducks at 1, 2, 3, 5, 7, 14, 21, and 28 dpi. The greatest number of viral DNA copies was found in the livers of infected chickens, yet no virus was found in any samples from infected ducks. In addition, the viral load increased over time in both chicken and duck embryo fibroblasts (CEFs and DEFs, respectively); in the former, replication speed was significantly greater than in the latter. Innate immune responses were also studied, both in vivo and in vitro. In CEFs, DEFs, and chickens infected intramuscularly, but not in infected ducks, mRNA expression levels of proinflammatory cytokines (interleukin-6 and -8) and interferon-stimulated genes (Mx and OAS) were significantly upregulated. Although some cytokines showed significant upregulation in the oral chickens, most did not change significantly. Finally, the duck retinoic acid-inducible gene I and its caspase activation and recruitment domain both had significant antiviral functions in CEFs, particularly after 24 h postinfection. Taken together, this research provides new insights into the interactions between FAdV-4 and the innate immune systems of studied hosts (chickens and ducks).