Lactobacillus acidophilus and Bacillus subtilis significantly change the growth performance, serum immunity and cecal microbiota of Cherry Valley ducks during the fattening period.

This study explored the effects of a Bacillus subtilis and Lactobacillus acidophilus mixture containing the co-fermented products of the two probiotics on growth performance, serum immunity and cecal microbiota of Cherry Valley ducks. This study included 480 one-day-old Cherry Valley ducks divided into four feeding groups: basal diet (control group) and basal diet supplemented with 300, 500, or 700 mg/kg of the probiotic powder; the ducks were raised for 42 days. Compared with the control group, body weight on day 42 and the average daily gain on days 15-42 significantly increased (p < 0.05), and the feed conversion rate significantly decreased (p < 0.05) in the experimental groups. Furthermore, the serum immunoglobulin (Ig) A, IgG, IgM, and interleukin (IL)-4 levels increased significantly (p < 0.05), and IL-1ß, IL-2, and tumor necrosis factor-α decreased significantly (p < 0.05) in the experimental groups. Finally, Sellimonas, Prevotellaceae NK3B31 group, Lachnospiraceae NK4A136 group and Butyricoccus played an important role in the cecal microbiota of the experimental group. Thus, the probiotic powder has impacts on the growth performance, serum immunity and cecal microbiota of Cherry Valley Ducks.

Molecular characterization and functional analysis of duck CCCH-type zinc finger antiviral protein (ZAP).

This is the first study to clone duck CCCH-type zinc finger antiviral protein (duZAP) from Jingjiang duck (Anas platyrhynchos). Full-length duZAP cDNA was 2154 bp and encoded a 717-amino acid polypeptide containing four highly conserved CCCH-type finger motifs, a WWE domain and a poly (ADP-ribose) polymerase (PARP) domain. duZAP was expressed in multiple duck tissues, with the highest mRNA expression in the spleen. Overexpression of duZAP in duck embryo fibroblast cells (DEFs) led to activation of the transcription factors IRF1 and NF-κB, and induction of IFN-ß. Analysis of deletion mutants revealed that both the WWE and PARP domains of duZAP were essential for activating the IFN-ß promoter. Knockdown of duZAP in DEFs significantly reduced poly (I:C)- and duck Tembusu virus (DTMUV)-induced IFN-ß activation. Our findings further the understanding of the role of duZAP in the duck innate immune response.

Identification of a linear epitope within domain I of Duck Tembusu virus envelope protein using a novel neutralizing monoclonal antibody.

Duck Tembusu virus (DTMUV) is a newly emerging pathogenic flavivirus that caused severe egg drop syndrome in laying ducks in China since 2010, leading to massive economic losses to the duck industry. Although the DTMUV E protein is considered to be critical in inducing the protective immune response, the functional epitopes within this protein remain largely unknown. In the present study, we isolated a DTMUV neutralizing monoclonal antibody (mAb) 3B8 from DTMUV E-immunized mice. Epitope mapping showed that mAb 3B8 recognized a novel linear epitope FSCLGMQNR located on the extreme N-terminal of the domain I (EDI) of E protein. Sequence alignment and Western blot analyses showed that the epitope is greatly conserved with high DTMUV-specificity. Moreover, upon cloning the heavy and light chain variable region sequences of mAb 3B8, we prepared the single-chain variable antibody fragment (scFv) 3B8 by connecting the two chains via a flexible peptide linker. The recombinant scFv 3B8 exhibited antiviral activity against DTMUV infection in vitro and in vivo. Our results provide valuable implications for the development of DTMUV vaccines and therapeutics.

Effects of glucose oxidase on growth performance, immune function, and intestinal barrier of ducks infected with Escherichia coli O88.

The negative effects of dietary antibiotics have become a widespread concern. It is imperative to search for a new type of green, safe, and efficient feed additive that can replace antibiotics. This study was to investigate the effects of glucose oxidase (GOD) on growth performance, immune function, and intestinal barrier in ducks infected with Escherichia coli O88. First, we established the E. coli challenge model of ducks through a preliminary experiment and then carried out the formal experiment by using 144 1-day-old male lean Peking ducklings (50 ± 2.75 g). All ducks were randomly assigned to 1 of 3 dietary treatment groups of basal diet (control), 30 mg/kg virginiamycin (antibiotic), and 200 U/kg GOD (1,000 U/g). Each group consisted of 6 replications with 8 birds per replicate. At day 7, all ducks were orally administered 0.2 mL E coli O88 (3 × 109 cfu/mL) twice, 8 h apart based on the preliminary experiment. The experiment lasted for 28 d. Dietary supplementation with GOD improved growth performance of ducks infected with E. coli. The GOD increased contents of Ig in plasma and secreted Ig A in jejunal mucosa. The GOD group had lower concentrations of inflammatory cytokines (tumor necrosis factor-α, IL-1ß, and IL-6) and their upstream regulator Toll-like receptor 4 in the jejunum of ducks than the control group. Supplementation with GOD increased villus height and decreased crypt depth in the jejunum. The gene expression of tight junction proteins (zonula occludens-1, claudin-1 and claudin-2) was enhanced by adding GOD. The GOD decreased intestinal permeability by reducing the concentrations of diamine oxidase and D-lactic in plasma of ducks. There were no significant differences in almost all the indices tested between the GOD and the antibiotic groups. In conclusion, supplementation of GOD improved growth performance, immune function, and intestinal barrier of ducks infected with E. coli O88. Glucose oxidase may serve as a promising alternative therapy to antibiotics to relieve or prevent colibacillosis in ducks.

Effects of Achyranthes bidentata polysaccharides on performance, immunity, antioxidant capacity, and meat quality in Pekin ducks.

This study was conducted to evaluate the effects of Achyranthes bidentata polysaccharide (ABP) on growth performance, antioxidant capacity, immune function, relative organ weight, ileal microflora, and meat quality in Pekin ducks. A total of 1,200 female 1-day-old Pekin ducklings (51.2 ± 0.2 g) were blocked based on body weight (BW) and randomly allocated into 3 treatments with 10 replicates of 40 birds each. The experiment lasted for 6 wk, and dietary treatments included corn-soybean meal-based diet supplemented with 0, 0.02, and 0.04% ABP. The supplementation of ABP increased (P < 0.05) body weight gain (BWG) and final BW linearly during day 22 to 42 and day 1 to 42, respectively, but decreased (P < 0.05) feed-to-gain ratio (F/G) linearly during day 22 to 42 and day 1 to 42. The inclusion of ABP increased (P < 0.05) serum superoxide dismutase, glutathione peroxidase, total antioxidative capacity, catalase, complement3, complement4, immunoglobin A, immunoglobin G, interleukin-2, interferon-γ, and tumor necrosis factor-α linearly. The relative weight of breast meat was increased (P < 0.05) linearly, but the relative weight of abdominal fat was decreased (P < 0.05) linearly with the increasing dietary ABP supplementation. The supplementation of ABP increased (P < 0.05) ileal Lactobacilli counts linearly, whereas decreased (P < 0.05) Escherichia coli counts linearly. Taken together, the inclusion of ABP promoted BWG and final BW during day 22 to 42 and the entire experiment, decreased F/G during day 22 to 42 and day 1 to 42, and partially improved antioxidant activities, immunity, and gut microflora in Pekin ducks.

Effects of pantothenic acid on growth performance and antioxidant status of growing male white Pekin ducks.

An experiment was conducted to investigate the effects of dietary pantothenic acid levels on growth performance, carcass traits, pantothenic acid status, and antioxidant status of male white Pekin ducks from 15 to 42 D of age and to evaluate the requirement of this vitamin for growing ducks. Different levels pantothenic acid (0, 2, 4, 6, 8, and 10 mg/kg) were supplemented to a corn-soy isolate protein basal diet to produce 6 dietary treatments with different analyzed total pantothenic acid levels (4.52, 6.44, 8.37, 9.88, 12.32, and 14.61 mg/kg). A total of 240 15-day-old male white Pekin ducks were allotted to 6 dietary treatments with 8 replicate pens of 5 birds per pen. At 42 D of age, growth performance, carcass traits, tissue pantothenic acid concentrations, and antioxidant status of white Pekin ducks were examined. Significant effects of dietary pantothenic acid on BW, average daily weight gain (ADG), plasma, and liver pantothenic acid concentrations were observed (P < 0.05) but not carcass traits. The growing ducks fed the basal diet without pantothenic acid supplementation had the lowest BW, ADG, plasma, and liver pantothenic acid content among all ducks (P < 0.05). In addition, the ducks fed the basal diet without pantothenic acid supplementation showed the lowest antioxidant capacity indicated by greatest plasma malondialdehyde content and lowest liver total antioxidant capacity (P < 0.05). And, these criteria responded linearly as dietary pantothenic acid levels increased (P < 0.05). These results indicated that dietary pantothenic acid supplementation improved growth performance and antioxidant status of the growing ducks. In accordance with the broken-line model, the pantothenic acid requirements (based on dietary total pantothenic acid) of male white Pekin ducks from 15 to 42 D of age for BW, ADG, and plasma and liver pantothenic acid contents were 10.18, 10.27, 12.06, and 10.79 mg/kg, respectively.

Heterologous prime-boost: an important candidate immunization strategy against Tembusu virus.

BACKGROUND: Tembusu virus (TMUV), a newly emerging pathogenic flavivirus, spreads rapidly between ducks, causing massive economic losses in the Chinese duck industry. Vaccination is the most effective method to prevent TMUV. Therefore, it is urgent to look for an effective vaccine strategy against TMUV. Heterologous prime-boost regimens priming with vaccines and boosting with recombinant adenovirus vaccines have been proven to be successful strategies for protecting against viruses in experimental animal models. METHODS: In this study, heterologous and homologous prime-boost strategies using an attenuated salmonella vaccine and a recombinant adenovirus vaccine expressing prM-E or the E gene of TMUV were evaluated to protect ducks against TMUV infection for the first time, including priming and boosting with the attenuated salmonella vaccine, priming and boosting with the recombinant adenovirus vaccine, and priming with the attenuated salmonella vaccine and boosting with the recombinant adenovirus vaccine. Humoral and cellular immune responses were detected and evaluated. We then challenged the ducks with TMUV at 12 days after boosting to assay for clinical symptoms, mortality, viral loads and histopathological lesions after these different strategies. RESULTS: Compared with the homologous prime-boost strategies, the heterologous prime-boost regimen produced higher levels of neutralizing antibodies and IgG antibodies against TMUV. Additionally, it could induce higher levels of IFN-γ than homologous prime-boost strategies in the later stage. Interestingly, the heterologous prime-boost strategy induced higher levels of IL-4 in the early stage, but the IL-4 levels gradually decreased and were even lower than those induced by the homologous prime-boost strategy in the later stage. Moreover, the heterologous prime-boost strategy could efficiently protect ducks, with low viral titres, no clinical symptoms and histopathological lesions in this experiment after challenge with TMUV, while slight clinical symptoms and histopathological lesions were observed with the homologous prime-boost strategies. CONCLUSIONS: Our results indicated that the heterologous prime-boost strategy induced higher levels of humoral and cellular immune responses and better protection against TMUV infection in ducks than the homologous prime-boost strategies, suggesting that the heterologous prime-boost strategy is an important candidate for the design of a novel vaccine strategy against TMUV.

Effects of grape seed extract on performance, immunity, antioxidant capacity, and meat quality in Pekin ducks.

This study was conducted to evaluate the effects of grape seed extract (GSE) on growth performance, immunity, antioxidant capacity, relative organ weight, jejunum morphology, ileal microflora, and meat quality in Pekin ducks. A total of 1,500 female 1-day-old Pekin ducklings (52.0 ± 0.2 g) were blocked based on body weight (BW) and randomly allocated into 3 treatments with 10 replicates of 50 birds each. The experiment lasted for 6 wk, and dietary treatments included corn-soybean meal-based diet supplemented with 0, 0.01, and 0.02% GSE. The supplementation of GSE increased (P < 0.05) body weight gain (BWG) and final BW linearly but decreased (P < 0.05) feed-to-gain ratio (F/G) linearly during day (D) 22 to 42 and the entire experiment. The inclusion of GSE increased (P < 0.05) serum superoxide dismutase, glutathione peroxidase, total antioxidative capacity, catalase, complement4, immunoglobin G, interleukin-2, and interferon-γ linearly but decreased (P < 0.05) serum malondialdehyde linearly. The relative weight of carcass, breast meat, and spleen in GSE treatments was increased (P < 0.05) linearly, whereas the relative weight of abdominal fat was decreased linearly (P < 0.05). Birds fed GSE1 and GSE2 diets had lower (P < 0.05) cook loss, 2-thiobarbituric acid reactive substances, and drip loss on day 3 and 5 linearly but higher (P < 0.05) pH24h and water-holding capacity. The addition of GSE decreased (P < 0.05) jejunum crypt depth and ileal Escherichia coli counts linearly but increased (P < 0.05) jejunum villus height: crypt depth ratio and ileal Lactobacilli linearly. Taken together, the inclusion of GSE increased final BW and BWG, decreased F/G during day 22 to 42 and day 1 to 42, partially improved antioxidant activities, immunity, meat quality, and gut health in Pekin ducks.

Duck PIAS2 negatively regulates RIG-I mediated IFN-ß production by interacting with IRF7.

The protein inhibitor of activated STAT (PIAS) proteins are important signal transduction modulator family and regulate the innate immune signaling pathway induced by certain transcription factors, including NF-κB, IRF3, and JAK/STAT. The PIAS protein mechanism that regulates innate immune response in mammals has been well described in the literature; however, whether the PIAS gene exists in ducks as well as the role of PIAS in duck IFN-ß expression is still unclear. Here, we cloned duck PIAS (duPIAS), finding PIAS2 could repress IFN-ß production. DuPIAS2 contains SAP-PINIT-RLD-S/T characteristic domains, and its overexpression could inhibit virus-induced IFN-ß promoter activation. Moreover, duPIAS2 interacts with duck interferon regulatory factor 7 (IRF7) and inhibits IFN-ß promoter activation induced by duck IRF7. Additionally, its inhibitory function does not rely on its SUMO E3 ligase activity but rather its C-terminal portion. The above results demonstrate that duPIAS2 is a repressor of IFN-ß production induced by duck IRF7.

Effects of methylsulfonylmethane on growth performance, immunity, antioxidant capacity, and meat quality in Pekin ducks.

This study was conducted to determine the effect of methylsulfonylmethane (MSM) on growth performance, immune function, antioxidant capacity, and meat quality in Pekin ducks. A total of 960 female 1-day-old Pekin ducklings (53.3 ± 0.4 g) were randomly allotted to 3 treatments with 8 replicates of 40 birds, based on their body weight (BW). The experiment lasted 6 wks, and dietary treatments included a corn-soybean meal-based diet supplemented with 0%, 0.15%, and 0.3% MSM, that is, CON, MSM1, and MSM2, respectively. Growth performance, serum profiles, and meat quality were determined. During the period of days 22-42, BW gain (BWG) in MSM2 treatment was higher (P < 0.05) and feed-to-gain ratio (F/G) was lower (P < 0.05) than those of CON and MSM1 treatments. BW gain and final BW in MSM2 treatment were increased (P < 0.05) compared with CON and MSM1 treatments during the period of days 1-42. Serum activities of superoxide dismutase and glutathione peroxidase, total antioxidative capacity, and concentrations of interleukin-2 and interleukin-6 were higher (P < 0.05) in MSM2 than in CON treatment. Ducks in the MSM2 treatment group had lower (P < 0.05) serum malondialdehyde, interferon gamma, and tumor necrosis factor-α levels than those in the CON treatment group. The supplementation of MSM increased (P < 0.05) water-holding capacity and redness (a*) and decreased (P < 0.05) values for 2-thiobarbituric acid and drip loss on day 5. Ducks in the MSM2 treatment group had higher (P < 0.05) pH24h than those in the CON treatment group. Taken together, the inclusion of MSM (0.3%) increased final BW and BWG during periods of days 22-42 and days 1-42, reduced feed-to-gain ratio during the period of days 22-42, and resulted in positive effects on immunity, antioxidant capacity, and meat quality.

Development and application of a fiber2 protein-based indirect ELISA for detection of duck adenovirus 3.

Duck adenovirus 3 (DAdV-3) is a newly identified duck adenovirus that has recently emerged in China. The incidence of duck infection caused by this virus is very high, with very large economic losses to the poultry industry. Thus, there is an urgent need for a serological assay for the specific detection of DAdV-3. To this end, prokaryotic expression of the fiber2 protein of DAdV-3 was used as a coating antigen to establish an indirect enzyme linked immunosorbent assay (ELISA) method for the specific detection of antibodies against DAdV-3. The method was found to be specific, repeatable and more sensitive than the agarose gel precipitation test (AGP). This indirect ELISA method based on the recombinant fiber2 protein may be used for the clinical detection of DAdV-3 infection and for monitoring antibody levels after vaccine immunization and is of great significance for the effective prevention and control of the disease.

Protective role of dryland rearing on netting floors against mortality through gut microbiota-associated immune performance in Shaoxing ducks.

Dryland rearing on netting floors (DRNF) is a new rearing method for ducks, which could prevent duck excreta from polluting water bodies. However, the influence of DRNF on duck production and immune performance remains poorly understood. In this study, 2,280 Shaoxing ducks, an egg-type breed of Sheldrake in China, were chosen and randomly divided into 2 groups to investigate the effects of DRNF on duck farming. During the experimental laying rates, feed-egg ratios, and mortality rates of the 2 groups were calculated and recorded. Serum immune parameters, including thymus index, spleen index, levels of immunoglobulin G (IgG), interleukin 1ß (IL-1ß), and tumor necrosis factor α (TNF-α), were determined. Sequencing of the 16S rRNA gene was used to analyze the variability of gut microbiota in the duck ileum and cecum. The results showed that DRNF significantly reduced the mortality rate of the ducks and increased the thymus index (P < 0.05), compared to the control. No other significant differences were detected in productional and immune indices (P > 0.05). The 16S rRNA sequencing results revealed differentially enriched microbial compositions in the ileum and cecum, which might be responsible for the improved immune function of Shaoxing ducks. For example, an increase in Lactobacillaceae (family), Anaerotruncus (genus), Saccharibacteria (phylum), Flavobacteriaceae (family), and a reduction in Anaerobiospirillum (genus), Lachnospiraceae (family), Blautia (genus) was revealed in the DRNF ducks. In conclusion, DRNF could alter gut microflora, enhance duck immune system, and reduce mortality in Shaoxing ducks.

Molecular cloning and mRNA expression of IFIT5 in tissues of ducklings infected with virulent duck hepatitis A virus type 3.

Interferon-induced proteins with tetratricopeptide repeats (IFITs) are a family of proteins strongly induced downstream of type I interferon signaling. The function of IFITs has been investigated extensively in mammals. IFIT5 is the sole protein in this family found in birds and little information is available about the function of avian IFIT5. In this study, duck IFIT5 was cloned from peripheral blood mononuclear cells. Multiple amino acid sequence alignment and phylogenetic analysis showed that duck IFIT5 is highly homologous to chicken IFIT5. Tissue specificity analysis demonstrated that duck IFIT5 was ubiquitously expressed in all examined tissues of five-day-old ducklings, with the highest expression levels in heart, followed by thymus, cerebrum, liver, and lung; kidney expressed the lowest. Quantitative real-time PCR (qRT-PCR) analysis revealed that duck IFIT5 expression rapidly increased both in vitro and in vivo after stimulation with polyinosinic:polycytidylic acid [poly (I:C)] and infection with virulent duck hepatitis A virus type 3 (DHAV-3), respectively. Altogether, these results indicate that the expression of duck IFIT5 is positively correlated with viral load and may play an important role in the immune response to DHAV-3 infection. This study lays a foundation for further research into the innate antiviral immune responses of ducklings.

Protection against duck hepatitis a virus type 1 conferred by a recombinant avian adeno-associated virus.

The avian adeno-associated virus (AAAV) has been proved to be an efficient gene transfer vector for human gene therapy and vaccine research. In this experiment, an AAAV-based vaccine was evaluated for the development of a vaccine against duck hepatitis a virus type 1 (DHAV-1). The major capsid VP1 gene was amplified and subcloned into pFBGFP containing the inverted terminal repeats of AAAV, and then the recombinant baculovirus rBac-VP1 was generated. The recombinant AAAV expressing the VP1 protein (rAAAV-VP1) was produced by co-infecting Sf9 cells with rBac-VP1 and the other 2 baculoviruses containing AAAV functional genes and structural genes respectively, and confirmed by electron microscopy, Western blotting and immunofluorescence assays. Quantitative real-time PCR revealed that the titer of rAAAV-VP1 was about 9 × 1012 VG/mL. Immunogenicity was studied in ducklings. One day ducklings were injected intramuscularly once with rAAAV-VP1. Serum from rAAAV-VP1-vaccinated ducklings showed a systemic immune response evidenced by VP1-specific enzyme-linked immunosorbent assay and virus neutralization test. Furthermore, all ducklings inoculated with rAAAV-VP1 were protected against DHAV-1 challenge. The data of quantitative real-time RT-PCR from livers of challenged ducklings also showed that the level of virus copies in rAAAV-VP1 group was significantly lower than that of the PBS group. Collectively, these results demonstrate that the AAAV-based vaccine is a potential vaccine candidate for the control of duck viral hepatitis.

Duck interferon regulatory factor 7 (IRF7) can control duck Tembusu virus (DTMUV) infection by triggering type I interferon production and its signal transduction pathway.

Human interferon regulatory factor 7 (IRF7) plays an important role in the innate antiviral immune response. To date, the characteristics and functions of waterfowl IRF7 have not been clarified. This study reports the cDNA sequence, tissue distribution, and antiviral function of duck IRF7. The duck IRF7 gene has a 1536-bp open read frame (ORF) and encodes a 511-amino acid polypeptide. IRF7 is highly expressed in the blood and pancreas of 5-day-old ducklings and in the small intestine, large intestine and liver of 60-day-old adult ducks. Indirect immunofluorescence assay (IFA) showed that over-expressed duck IRF7 was located in both the cytoplasm and nucleus of transfected duck embryo fibroblasts (DEFs), which was also observed in poly(I:C)-stimulated or duck Tembusu virus (DTMUV)-infected DEFs. Titres and copies of DTMUV were significantly reduced in DEFs overexpressing IRF7. Moreover, overexpression of duck IRF7 significantly induced IFNα/ß, but not IFNγ, mRNA expression, and transcription of downstream interferon-stimulated genes (ISGs), such as MX, OASL and IL-6, which were significantly induced by poly(I:C) co-stimulation, was enhanced. Additionally, duck IRF7 overexpression can significantly activate the IFNß promoter in DEFs. Collectively, duck IRF7 plays an important role in host anti-DTMUV immune regulation, which depends on type I interferons and associated signal transduction pathway(s).

Recombinant fiber-2 protein protects Muscovy ducks against duck adenovirus 3 (DAdV-3).

As a novel duck adenovirus 3 (DAdV-3) infection caused significant economic losses to the poultry industry in China, there is an urgent need to develop a safe and effective vaccine. In the research, fiber-1 and fiber-2 proteins were expressed and purified, respectively. To evaluate the immunogenicity of the two recombinant proteins, we investigated the IgY antibodies and virus-neutralizing antibodies in duck sera. The protective efficacy was evaluated by mortality, virus shedding and histopathological examinations after challenged with the DAdV-3. Results showed that the IgY antibody levels of the fiber-2 group was significantly higher than that of the fiber-1 group and inactivated vaccine group. Ducks vaccinated with fiber-2 group provided full protection with no mortality, no virus shedding and no histological lesions, superior to other groups. These results suggest that the fiber-2 protein can be an ideal candidate for subunit vaccine against the disease.

Comparative transcriptome analysis reveals PERP upregulated during Salmonella Enteritidis challenge in laying ducks.

Salmonella Enteritidis (SE) can be transmitted to eggs through cecum or the ovary from infected layers and causes food poisoning in humans. The mechanism of cecal transmission has been extensively studied. However, the mechanism and route of transovarian transmission of SE remain unclear. In this study, the ducks were orally inoculated with SE, and the ovarian follicles and stroma were collected to detect SE infection. The immune responses were triggered and the innate and adaptive immune genes (TLR4, NOD1, AvßD7, and IL-1ß) were upregulated significantly during the SE challenge. Moreover, the ovary tissues (small follicle and stroma) of susceptible and resistant-laying ducks were performed by RNA sequencing. We obtained and identified 23 differentially expressed genes (DEGs) between susceptible and resistant-laying ducks in both small follicle and stroma tissues ( p < 0.05). The DEGs were predominately identified in the p53 signaling pathway. The expression of key genes (p53, MDM2, PERP, caspase-3, and Bcl-2) involved in the signaling pathway was significantly higher in granulosa cells (dGCs) from SE-infected ducks than those from uninfected ducks. Moreover, the overexpression of PERP resulted in further induction of p53, MDM2, caspase-3, and Bcl-2 during SE infection in dGCs. Whereas, an opposite trend was observed with the knockdown of PERP. Besides, it is further revealed that the PERP could enhance cell apoptosis, SE adhesion, and SE invasion in SE-infected dGCs overexpression. Altogether, our results demonstrate the duck PERP involved in the ovarian local immune niche through p53 signaling pathway in dGCs challenged with SE.

A recombinant adenovirus expressing the E protein of duck Tembusu virus induces protective immunity in duck.

Duck Tembusu virus disease, caused by the duck Tembusu virus (DTMUV), can lead to a severe reduction in egg production and growth retardation in laying ducks and ducklings, respectively. In this study, we engineered a novel recombinant adenovirus expressing the E protein of DTMUV (rAd-E) in AAV-293 cells (analyzed by western blot and indirect immunofluorescence assays). Intramuscular immunization of Cherry Valley ducks with rAd-E was performed to evaluate host cellular and humoral immune responses. Compared to the phosphate-buffered saline administered group and the negative control wild-type adenovirus (wtAd) group, the rAd-E vaccinated group showed increased cellular and humoral responses. The results from the cytokine release and lymphocyte proliferation assays showed that rAd-E induced a stronger cellular immune response than the control group (P<0.01), 4 weeks after primary immunization. The results of enzyme-linked immunosorbent and virus neutralization assays showed that rAd-E induced higher titers of specific neutralizing antibodies, 2 weeks after primary immunization. The DTMUV challenge experiment showed a higher survival rate (80%) of ducks in the rAd-E group, when challenged with 0.5 ml (ELD50=10-2.67/0.2 ml) of the DTMUV strain AH-F10. These results indicate that rAd-E effectively protects ducks against DTMUV infection. Therefore, rAd-E could be a vaccine candidate to provide an effective and safe method for prevention and control of DTMUV infection.

Isolation and Selection of Duck Primary Cells as Pathogenic and Innate Immunologic Cell Models for Duck Plague Virus.

Duck plague virus (DPV) is a representative pathogen transmitted among aquatic animals that causes gross lesions and immune inhibition in geese and ducks. The mechanism of organ tropism and innate immune evasion of DPV has not been completely deciphered due to a lack of cell models to study the innate immune manipulation and pathogenicity of aquatic viruses. In the present study, we isolated five types of duck primary cells [duck embryo fibroblasts (DEFs), neurons, astrocytes, peripheral blood mononuclear cells (PBMCs), and monocytes/macrophages] to identify appropriate cell models for DPV, using tropism infection and innate immunologic assays. Cells responded differently to stimulation with DNA viruses or RNA virus analogs. DPV infection exhibited broad tropism, as the recombinant virulent strain (CHv-GFP) infected DEFs, neurons, astrocytes, and monocytes/macrophages, but not the PBMCs, as the expression of EGFP was negligible. The basal levels of innate immunity molecules were highest in monocytes/macrophages and lower in DEFs and astrocytes. Conversely, the titer and genomic copy number of the attenuated virus strain was higher in DEFs and astrocytes than in neurons and monocytes/macrophages. The titer and genomic copy number of the attenuated virus strain were higher compared with the virulent strain in DEFs, neurons, and astrocytes. The innate immune response was not significantly induced by either DPV strain in DEFs, neurons, or astrocytes. The virulent strain persistently infected monocytes/macrophages, but the attenuated strain did so abortively, and this was accompanied by the phenomenon of innate immune inhibition and activation by the virulent and attenuated strains, respectively. Blockage of IFNAR signaling promoted replication of the attenuated strain. Pre-activation of IFNAR signaling inhibited infection by the virulent strain. The selection assay results indicated that induction of innate immunity plays an essential role in controlling DPV infection, and monocytes/macrophages are an important cell model for further investigations. Our study provided practical methods for isolating and culturing duck primary cells, and our results will facilitate further investigations of organ tropism, innate immune responses, latent infection, and the effectiveness of antiviral drugs for treating DPV and potentially other aerial bird pathogens.

Molecular cloning and functional characterization of duck DDX41.

DEAD (Asp-Glu-Ala-Asp) box polypeptide 41 (DDX41), a receptor belonging to DExD/H-box helicase family, acts as an intracellular DNA sensor and induces type I IFN production in mammals and fish. However, the function of avian DDX41 in innate immune response is still unknown. In this study, the full-length duck DDX41 (duDDX41) cDNA sequence was cloned for the first time and encoded a putative protein of 618 amino acid residues which showed the high sequence similarity with both zebra finch and chicken DDX41s. The duDDX41 mRNA was widely distributed in all tested tissues, especially the cerebrum, cerebellum, and liver. Overexpression of duDDX41 triggered the activation of transcription factors IRF1 and NF-κB, as well as IFN-ß expression in DEFs. The DEADc domain of duDDX41 played an extremely vital role in duck type I IFN signaling pathway. Knockdown of duDDX41 by siRNA silencing dramatically decreased IFN-ß expression stimulated by poly(dA:dT) or duck enteritis virus (DEV). In addition, the replication of DEV was significantly inhibited in duDDX41-expressed DEFs and was enhanced in DDX41 knockdown DEFs. These results suggest that DDX41 is an important cytosolic DNA sensor and plays a crucial role in duck antiviral innate immune response.