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.

Amino acid substitutions in the neuraminidase protein of an H9N2 avian influenza virus affect its airborne transmission in chickens.

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.

Rapid emergence of a PB2-E627K substitution confers a virulent phenotype to an H9N2 avian influenza virus during adoption in mice.

The worldwide circulation of H9N2 avian influenza virus in poultry, the greater than 2.3 % positive rate for anti-H9 antibodies in poultry-exposed workers, and several reports of human infection indicate that H9N2 virus is a potential threat to human health. Here, we found three mutations that conferred high virulence to H9N2 virus in mice after four passages. The PB2-E627K substitution rapidly appeared at the second passage and played a decisive role in virulence. Polymerase complexes possessing PB2-E627K displayed 16.1-fold higher viral polymerase activity when compared to the wild-type virus, which may account for enhanced virulence of this virus. The other two substitutions (HA-N313D and HA-N496S) enhanced binding to both α2,3-linked and α2,6-linked sialic acid receptors; however, the HA-N313D and N496S substitutions alone decreased the virulence of mouse-adapted virus. Furthermore, this mouse-adapted virus was still not transmissible among guinea pigs by direct contact (0/3 pairs). Our findings show that adaption in mice enhanced the viral polymerase activity and receptor-binding ability, which resulted in a virulent phenotype in mice but not a transmissible phenotype in guinea pigs, indicating that host factors play an important role in adaptive evolution of influenza in new hosts.

First detection of severe fever with thrombocytopenia syndrome virus in the tick species Haemaphysalis concinna in Shandong Province, China.

The aim of this study was to detect severe fever with thrombocytopenia syndrome virus (SFTSV) infection using polymerase chain reaction (PCR) amplification in adult Haemaphysalis concinna ticks. A total of 72 adult H. concinna ticks were obtained from 35 goats, three adult H. concinna ticks (4.17 %) collected from two goats were found to be infected with SFTSV via PCR assay. Sequence analysis showed that the partial segment M glycoprotein gene of SFTSV was about 500 bases long by polymerase chain reaction (PCR) amplification and that the PCR products from the samples had an identical sequence (KP714259). With regard to the phylogenetic analysis, the Nei-Gojobri (Kimura 2-parameter) method was used to construct the phylogenetic trees. Phylogenetic analysis indicated that the obtained sequence closely resembled SFTSV strain from Zhejiang Province (KC189856) and belonged to the same clade. The similarity of these strains was up to 96.62 % (only differing by 17 bases). In addition, phylogenetic analysis also indicated that the sequence obtained from adult H. concinna ticks was most closely related to the sequence isolated from Haemaphysalis longicornis (KF781498) with 97.22 % similarity (differing only by 4 bases) and belonged to the same clade.

Response profiles of cytokines and chemokines against avian H9N2 influenza virus within the mouse lung.

The circulation of H9N2 viruses throughout the world, along with their expanded host range, poses a potential health risk to the public, but the host responses to H9N2 virus in mammals were little known. To obtain insight into the host immune responses to the avian H9N2 virus, the expressions of both cytokines and chemokines in the lungs of infected mice were examined by real-time polymerase chain reaction and enzyme-linked immunosorbent assay. We found that interferon gamma (IFN-γ) was the dominant antiviral component, and IFN-γ-induced protein 10 kDa, interleukin 6, chemokine (C-C motif) ligand 5 and macrophage inflammatory protein-1 alpha all played a role in pro-inflammatory responses to H9N2 viruses. In conclusion, this research can make us further understand the infection characteristics of H9N2 virus in mammalian host by providing the data on mice lung immune responses to the avian H9N2 virus.

Determination of infective dose of H9N2 Avian Influenza virus in different routes: aerosol, intranasal, and gastrointestinal.

BACKGROUND: Low pathogenic H9N2 avian influenza virus (AIV) has been spreading worldwide, leading to huge economic losses to poultry husbandry, but few studies were concerned about its aerosol infection. METHODS: This study compared the infective doses of H9N2 AIV to chickens by three different routes, aerosol infection, intranasal and gastrointestinal infection, and determination of the results was conducted by detecting virus shedding and seroconversion of chickens. RESULTS: The results indicated that chickens were susceptible to H9N2 AIV with a different infection rate which depended on the route of inoculation. H9N2 AIV media aerosol-infective dose (aID50) to chickens was about 491 TCID50, intranasal infection was 398 TCID50, and gastrointestinal infection was 19,952 TCID50. CONCLUSION: The infection ability of H9N2 AIV to chickens was related to its way of invading. The respiratory infection ability was about 40 times more effective than gastrointestinal infection, which suggested that urgent attention should be paid to environmental disinfection to block airborne transmission of influenza virus.

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.

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.

Complete genome sequence of porcine circovirus 2b strain Shandong.

Shandong is a porcine circovirus 2b (PCV2b) strain that was isolated and purified from tissue samples from pigs with postweaning multisystemic wasting syndrome (PMWS) in the Shandong Province of China. Here, we report the complete genome sequence of strain Shandong, which may aid in understanding the molecular characteristics of this strain.

Airborne spread and infection of a novel swine-origin influenza A (H1N1) virus.

BACKGROUND: The novel swine-origin influenza A (H1N1) virus (S-O 2009 IV) can cause respiratory infectious diseases in humans and pigs, but there are few studies investigating the airborne spread of the virus. In January 2011, a swine-origin H1N1 epidemic emerged in eastern China that rapidly spread to neighboring farms, likely by aerosols carried by the wind. METHODS: In this study, quantitative reverse transcription polymerase chain reaction (RT-PCR) was used to detect viruses in air samples from pig farms. Based on two aerosol infection models (Pig and guinea pig), we evaluated aerosol transmission and infection of the novel S-O 2009 IV isolate. RESULTS: Three novel S-O 2009 IV were isolated from the diseased pig. The positive rate and viral loads of air samples were 26.1% and 3.14-5.72 log10copies/m³ air, respectively. In both pig and guinea pig infection models, the isolate (A/swine/Shandong/07/2011) was capable of forming aerosols and infected experimental animals at a range of 2.0-4.2 m by aerosols, but aerosol route was less efficient than direct contact. CONCLUSIONS: The results indicated that S-O 2009 IV is able to be aerosolized by infected animals and to be transmitted to susceptible animals by airborne routes.

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.

Development of a real-time RT-PCR method for rapid detection of H9 avian influenza virus in the air.

Avian influenza virus (AIV) has caused serious epidemics all over the world. Notably, the low-pathogenic AIV H9N2 has been spreading widely, leading to enormous economic losses to the poultry industry. To rapidly monitor airborne H9 AIVs in chicken houses, a real-time RT-PCR method was established and used to detect virus in air samples, and it was also compared with the traditional RT-PCR. The results showed that the real-time RT-PCR possessed high specificity and sensitivity for H9 AIVs, and the sensitivity reached 100 copies/reaction, much higher than the traditional RT-PCR; airborne H9 AIVs were found in the six chicken houses by real-time RT-PCR, and their mean concentrations ranged from 1.25×10(4) to 6.92×10(4) copies/m(3) air. Overall, the real-time PCR is a valuable tool for detecting airborne H9 AIVs.

Detection of Babesia divergens using molecular methods in anemic patients in Shandong Province, China.

Babesiosis (piroplasmosis) is a zoonotic disease caused by an intraerythrocytic protozoan transmitted by Ixodes ticks. The aim of this study was to detect Babesia spp. infection using molecular methods in 377 blood samples from anemic patients. Sequence analysis showed that the 18S rRNA gene was 439 bases long by polymerase chain reaction (PCR) amplification and that the PCR products from the samples had an identical sequence (named Taian China, HM355854). BLAST search showed that the sequence was identical to the 18S rRNA sequences of Babesia divergens. The 18S rRNA sequence for Toxoplasma gondii was included as the outlier for phylogenetic analysis by using the program MEGA4.0 software. The results showed that the 18S rRNA gene sequences obtained from the present study was most closely related to B. divergens Switzerland (DQ312439) with 98.4% similarity (differing only by seven bases). The phylogenetic analysis also revealed that this sequence closely resembled B. divergens strains from other countries and belonged to the same clade. This is the first report of a human being infected by B. divergens in China.

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.