Yumei Cai

Source identification of airborne Escherichia coli of swine house surroundings using ERIC-PCR and REP-PCR.

Abstract Evidence is mounting that microorganisms originating from livestock impact the air quality of the animal houses themselves and the public in the surrounding neighborhoods. The aim of this study was to develop efficient bacterial source tracking capabilities to identify sources of Escherichia coli aerosol pollution caused by pigs. Airborne E. coli were isolated from indoor air, upwind air (10 and 50m away) and downwind air samples (10, 50, 100, 200 and 400m away) for five swine houses using six-stage Andersen microbial samplers and Reuter-Centrifugal samplers (RCS). E. coli strains from pig fecal samples were also collected simultaneously. The enterobacterial repetitive intergenic consensus polymerize chain reaction (ERIC-PCR) and the repetitive extragenic palindromic (REP-PCR) approaches were used to study the genetic variability and to determine the strain relationships among E. coli isolated from different sites in each swine house. Results showed that 35.1% (20/57) of the bacterial DNA fingerprints from the fecal isolates matched with the corresponding strains isolated from indoor and downwind air samples (similarity ⩾90%). E. coli strains from the indoor and downwind air samples were closely related to the E. coli strains isolated from feces, while those isolated from upwind air samples (swine house C) had low similarity (61–69%). Our results suggest that some strains isolated from downwind and indoor air originated in the swine feces. Effective hygienic measures should be taken in animal farms to prevent or minimize the downwind spread of microorganism aerosol.

Immune responses of ducks infected with duck Tembusu virus

Duck Tembusu virus (DTMUV) can cause serious disease in ducks, characterized by reduced egg production. Although the virus has been isolated and detection methods developed, the host immune responses to DTMUV infection are unclear. Therefore, we systematically examined the expression of immune-related genes and the viral distribution in DTMUV-infected ducks, using quantitative real-time PCR. Our results show that DTMUV replicates quickly in many tissues early in infection, with the highest viral titers in the spleen 1 day after infection. Rig-1, Mda5, and Tlr3 are involved in the host immune response to DTMUV, and the expression of proinflammatory cytokines (Il-1β, –2, –6, Cxcl8) and antiviral proteins (Mx, Oas, etc.) are also upregulated early in infection. The expression of Il-6 increased most significantly in the tissues tested. The upregulation of Mhc-I was observed in the brain and spleen, but the expression of Mhc-II was upregulated in the brain and downregulated in the spleen. The expression of the interferons was also upregulated to different degrees in the spleen but that of the brain was various. Our study suggests that DTMUV replicates rapidly in various tissues and that the host immune responses are activated early in infection. However, the overexpression of cytokines may damage the host. These results extend our understanding of the immune responses of ducks to DTMUV infection, and provide insight into the pathogenesis of DTMUV attributable to host factors.

Experimental transmission in guinea pigs of H9N2 avian influenza viruses from indoor air of chicken houses

This study aimed to determine the transmission characteristics of H9N2 avian influenza viruses (AIVs) derived from the air. Eight H9N2 AIVs were isolated from chicken houses between 2009 and 2010. We analyzed the phylogenic and pathogenic traits of these isolates. What is more, transmission characteristics in guinea pigs of two airborne isolates were determined in experimental conditions. Phylogenetic analyses indicated that the homologies of HA and NA genes of eight isolates were 95.4-99.7% and 86.6-99.8% respectively. They were able to duplicate in lung tissues of guinea pigs without prior adaptation. Two airborne isolates could both transmit among guinea pigs by direct contact. No infection was detected in aerosol contact animals while H9N2 AIV aerosols were detected in the air of isolators. Aerosol infection dose experiment showed that aerosol median infective dose (ID(50)) of H9N2 AIV to guinea pigs was 3.58×10(6)copies, demonstrating that the aerosols could infect guinea pigs at certain concentrations in experimental condition. In conclusion, H9N2 AIV aerosols were infectious to mammals, suggesting that urgent attention will need to be paid to its transmission.

Cherry Valley Ducks Mitochondrial Antiviral-Signaling Protein-Mediated Signaling Pathway and Antiviral Activity Research.

Mitochondrial antiviral-signaling protein (MAVS), an adaptor protein of retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs)-mediated signal pathway, is involved in innate immunity. In this study, Cherry Valley duck MAVS (duMAVS) was cloned from the spleen and analyzed. duMAVS was determined to have a caspase activation and recruitment domain at N-terminal, followed by a proline-rich domain and a transmembrane domain at C-terminal. Quantitative real-time PCR indicated that duMAVS was expressed in all tissues tested across a broad expression spectrum. The expression of duMAVS was significantly upregulated after infection with duck Tembusu virus (DTMUV). Overexpression of duMAVS could drive the activation of interferon (IFN)-β, nuclear factor-κB, interferon regulatory factor 7, and many downstream factors (such as Mx, PKR, OAS, and IL-8) in duck embryo fibroblast cells. What is more, RNA interference further confirmed that duMAVS was an important adaptor for IFN-β activation. The antiviral assay showed that duMAVS could suppress the various viral replications (DTMUV, novel reovirus, and duck plague virus) at early stages of infection. Overall, these results showed that the main signal pathway mediated by duMAVS and it had a broad-spectrum antiviral ability. This research will be helpful to better understanding the innate immune system of ducks.

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.

Pathogenicity of duck plague and innate immune responses of the Cherry Valley ducks to duck plague virus

Duck plague caused by duck plague virus (DPV) is an acute and contagious disease. To better understand the pathogenic mechanism of duck plague virus in ducklings, an infection experiment was performed. Our results showed that typical symptoms were observed in the infected ducklings. DPV could replicate quickly in many tissues, leading to pathological lesions, especially on the spleen. Real-time quantitative PCR demonstrated that expression of many innate immune-related genes was mostly up-regulated in the brain, and the antiviral innate immune response was established, but not sufficient to restrict viral replication. In contrast, although the expression of many major pattern recognition receptors (PRRs) increased in the spleen, the expression of most cytokines was declined. Our study indicates that DPV is a pantropic virus that can replicate rapidly in tissues, causing serious pathological lesions but the immune responses are different in the spleen and brain. To our knowledge, this is the first report to systematically explore the expression profiles of the immune genes in the DPV-infected ducks. Our data provide a foundation for further study of the pathogenicity of duck plague.

The pathogenicity of novel duck reovirus in Cherry Valley ducks

The novel duck reovirus (NDRV) is an emerging, contagious infection. To better realize the pathogenic mechanism of NDRV in ducks, an infection experiment was conducted. The resulting data demonstrated that typical gross lesions were observed in the infected ducks. NDRV was able to replicate in various tissues, leading to these pathological lesions, especially on the liver and spleen. Real-time quantitative PCR showed that the expression of most innate immune-related genes was up-regulated and the antiviral innate immune response could be established in both the liver and spleen. This study indicates that NDRV is a pantropic virus. To resist viral infection, several pathogen recognition receptors can cooperatively recognize NDRV and initiate innate immunity, but the responses are different between different tissues. As far as we know, this is the first systematic investigation of the pathogenicity of NDRV in Cherry Valley ducks based on the hosts innate immunity, and these data will provide new insights into the further study of the disease.

Seasonal Fluctuations of Microbial Aerosol in Live Poultry Markets and the Detection of Endotoxin

Microbial aerosol whose species and concentrations are closely related to human health is ubiquitous. The effect of microbes on human and animal health and production performance is, in many cases, caused by the spreading of air. Samples in this experiment were from a live poultry market (LPM) in Tai’an, China, collected three times a day (8 am, 14 pm, and 20 pm) over three consecutive days each month for 11 months (Original sampling plan was a year, the government due to the environmental protection, the was LPM closed). The main indicators of the test were concentrations of cultural airborne bacteria, airborne fungi, and Gram-negative bacteria. At the same time the species of Gram-negative bacteria and the concentration of endotoxin were tested. Temperature and humidity were recorded in the process of each sampling. The results showed that the diurnal variation of the concentration of bacteria, fungi, and Gram-negative bacteria is higher in the morning and evening, but lower at noon. The concentrations of airborne bacteria and Gram-negative bacteria increased in earlier months and decreased in later months, with the peak appearing in the autumn. The concentration of fungi showed a decrease first and then tended to stabilize, with the peak occurring in the spring. The concentration peak of endotoxin occurred in the summer. Endotoxin levels were significantly correlated with humidity (r = 0.90, p < 0.01). Most bacteria were distributed at the third and fourth stages (2.1–4.7 μm) in the ANDERSEN-6 sampler. The dominant species of Gram-negative bacteria during the four seasons were Pseudomonas aeruginosa, Acinetobacter, Klebsiella pneumoniae, and Salmonella. In China, people have a habit of eating fresh poultry, LPM distribution is widespread, stream of people and traffic flow are large, easily caused the spread of bacteria and viruses, so the LPM microbial aerosol research have significant public health implications.

Expression of Immune-Related Genes of Ducks Infected with Avian Pathogenic Escherichia coli (APEC)

Avian pathogenic Escherichia coli (APEC) can cause severe disease in ducks, characterized by perihepatitis, pericarditis, and airsacculitis. Although the studies of bacteria isolation and methods of detection have been reported, host immune responses to APEC infection remain unclear. In response, we systemically examined the expression of immune-related genes and bacteria distribution in APEC-infected ducks. Results demonstrated that APEC can quickly replicate in the liver, spleen, and brain, with the highest bacteria content at 2 days post infection. The expression of toll-like receptors (TLRs), avian β-defensins (AvBDs) and major histocompatibility complex (MHC) were tested in the liver, spleen, and brain of infected ducks. TLR2, TLR4, TLR5, and TLR15 showed different expression patterns, which indicated that they all responded to APEC infection. The expression of AvBD2 was upregulated in all tested tissues during the 3 days of testing, whereas the expression of AvBD4, AvBD5, AvBD7, and AvBD9 were downregulated, and though MHC-I was upregulated on all test days, MHC-II was dramatically downregulated. Overall, our results suggest that APEC can replicate in various tissues in a short time, and the activation of host immune responses begins at onset of infection. These findings thus clarify duck immune responses to APEC infection and offer insights into its pathogenesis.

Influence of indoor microbial aerosol on the welfare of meat ducks

Abstract The aim of the study was to evaluate the effects of microbial aerosols on ducks’ welfare and provide information on which to establish 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. Different microbial aerosol concentrations 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, fungi, gram-negative bacteria and an AGI-30 microbial air sampler detected endotoxins. Physiological stress was evaluated in the ducks by adrenocorticotropic hormone (ACTH) values in serum. To assess the effects of bioaerosol factors, welfare indicators including fluctuating asymmetry (FA), appearance and gait as well as the Lactobacillus caecal concentration were evaluated. The data showed group D had already reached the highest limit of concentration of airborne aerobic bacteria, airborne fungi, airborne gram-negative bacteria and airborne endotoxin. The ducks in this group had significantly increased serum ACTH values and significantly decreased caecal lactobacilli concentration. Furthermore, appearance and gait scores, wing length and overall FA and caecal Lactobacillus concentration in this group were significantly increased at 6 and 8 weeks of age. In conclusion, high concentrations of microbial aerosol adversely affected the welfare of meat ducks. The microbial aerosol values in group D suggest a preliminary upper limit concentration of bioaerosols in ambient air for healthy meat ducks.