Immune Response Of Ducks Research Articles

The molecular basis of differential host responses to avian influenza viruses in avian species with differing susceptibility.

Highly pathogenic avian influenza (HPAI) viruses, such as H5N1, continue to pose a serious threat to animal agriculture, wildlife and to public health. Controlling and mitigating this disease in domestic birds requires a better understanding of what makes some species highly susceptible (such as turkey and chicken) while others are highly resistant (such as pigeon and goose). Susceptibility to H5N1 varies both with species and strain; for example, species that are tolerant of most H5N1 strains, such as crows and ducks, have shown high mortality to emerging strains in recent years. Therefore, in this study we aimed to examine and compare the response of these six species, to low pathogenic avian influenza (H9N2) and two strains of H5N1 with differing virulence (clade 2.2 and clade 2.3.2.1) to determine how susceptible and tolerant species respond to HPAI challenge. Birds were challenged in infection trials and samples (brain, ileum and lung) were collected at three time points post infection. The transcriptomic response of birds was examined using a comparative approach, revealing several important discoveries. We found that susceptible birds had high viral loads and strong neuro-inflammatory response in the brain, which may explain the neurological symptoms and high mortality rates exhibited following H5N1 infection. We discovered differential regulation of genes associated with nerve function in the lung and ileum, with stronger differential regulation in resistant species. This has intriguing implications for the transmission of the virus to the central nervous system (CNS) and may also indicate neuro-immune involvement at the mucosal surfaces. Additionally, we identified delayed timing of the immune response in ducks and crows following infection with the more deadly H5N1 strain, which may account for the higher mortality in these species caused by this strain. Lastly, we identified candidate genes with potential roles in susceptibility/resistance which provide excellent targets for future research. This study has helped elucidate the responses underlying susceptibility to H5N1 influenza in avian species, which will be critical in developing sustainable strategies for future control of HPAI in domestic poultry.

DDX1 from Cherry valley duck mediates signaling pathways and anti-NDRV activity

Novel duck reovirus (NDRV) causes severe economic losses to the duck industry, which is characterized by hemorrhagic spots and necrotic foci of the livers and spleens. DEAD-box helicase 1 (DDX1) plays a critical role in the innate immune system against viral infection. However, the role of duck DDX1 (duDDX1) in anti-RNA virus infection, especially in the anti-NDRV infection, has yet to be elucidated. In the present study, the full-length cDNA of duDDX1 (2223 bp encode 740 amino acids) was firstly cloned from the spleen of healthy Cherry valley ducks, and the phylogenetic tree indicated that the duDDX1 has the closest relationship with Anas platyrhynchos in the bird branch. The duDDX1 mRNA was widely distributed in all tested tissues, especially in the duodenum, liver, and spleen. Overexpression of duDDX1 in primary duck embryo fibroblast (DEF) cells triggered the activation of transcription factors IRF-7 and NF-κB, as well as IFN-β expression, and the expression of the Toll-like receptors (TLR2, TLR3, and TLR4) was significantly increased. Importantly, after overexpressing or knocking down duDDX1 and infecting NDRV in DEF cells, duDDX1 inhibits the replication of NDRV virus and also regulates the expression of pattern recognition receptors and cytokines. This indicates that duDDX1 may play an important role in the innate immune response of ducks to NDRV. Collectively, we first cloned DDX1 from ducks and analyzed its biological functions. Secondly, we proved that duck DDX1 participates in anti-NDRV infection, and innovated new ideas for the prevention and control of duck virus infection.

Riemerella anatipestifer infection affects intestinal barrier structure and immune reactions in the duck caecum

ABSTRACT Riemerella anatipestifer (RA) infection causes high mortality and poor feed conversion, leading to great economic losses to the duck industry. This study investigated the effects of RA on the intestinal morphology and immune response of ducks. Histological examination showed that RA infection caused intestinal injury, including significantly reduced mucosal thickness on days 2, 3 and 5, significantly reduced villus height on days 1, 2, 3 and 5 (P < 0.05) and significantly reduced villus height to crypt depth ratios on days 2, 3, 5 and 9 of RA infection (P < 0.05). The expression of intestinal mucosal layer construction-associated genes and tight junction genes was significantly altered on at least one time point (day 1, 2, 3, 5, 9 or 14) after RA infection. Quantitative real-time polymerase chain reaction revealed that RA infection affected intestinal mucosal immune function. The genes encoding TLR4 (toll like receptor-4), TRAF6 (TNF receptor-associated factor 6), MYD88 (myeloid differentiation factor 88), IFN-γ (interferon-γ), IL (interleukin)-4 and IL-8 were significantly upregulated on day 2 of RA infection. Taken together, these results indicate that RA infection negatively affects intestinal barrier function in ducks due to impaired mucosal and villus-crypt structure and alters the mRNA expression of mucous layer construction-, intestinal tight junction-, and intestinal mucosal immunity-related genes.

Molecular cloning, tissue distribution and function analysis of duck TLR7

Toll-like receptors (TLRs) play an important role in detecting pathogen-associated molecular patterns (PAMPs). Among the TLRs, TLR7 is involved in the recognition of antiviral compounds and single-stranded RNA. This study was designed to explore the structure and function of TLR7 in duck (Anas platyrhynchos), a natural host for avian influenza virus. Firstly, the full-length cDNA of Shaoxing egg-laying duck TLR7 (duTLR7) was obtained using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). It consisted of 38 base pairs (bp) 5′-untranslated region (UTR), 187 bp 3′-UTR, and 3270 bp open reading frame that encodes a single protein of 1089 amino acid residues. DuTLR7 shares high identity with TLR7 genes from other vertebrates. In healthy ducks, duTLR7 transcripts were broadly expressed in different tissues, with higher expression levels in the liver, kidney, and thymus. The highest relative transcript level of duTLR7 could be induced with R848 stimulation. In addition, overexpression of duTLR7 by stimulating with poly(I:C) significantly promoted IFN-β, NF-κB, IRF7, TRIF, Mx, STAT1 and STAT2 expressions. Taken together, these results suggest that TLR7 may play an important role in the innate immune response of ducks.

Immunogenicity and protective efficacy of DHBV DNA vaccines expressing envelope and capsid fusion proteins in ducks delivered by attenuated Salmonella typhimurium

Duck hepatitis B virus (DHBV) shares many basic characteristics with hepatitis B virus (HBV) and is an attractive model for vaccine development. In this study, DHBV DNA vaccines were designed to express envelope and capsid fusion proteins to enhance the breadth of immune response in ducks. Attenuated Salmonella typhimurium (SL7207) was used as a carrier and adjuvant to boost the magnitude of immune response. Based on this strategy, novel DNA vaccines (SL7207-pVAX1-LC and SL7207-pVAX1-SC) were generated. Growth kinetics, genetic stabilities and relative transcription levels of the L, S and C genes introduced by these vaccine strains were measured before inoculation to guarantee safety and efficacy. The relative transcript levels of the CD4 and CD8 T genes and the antibody levels (IgY) in ducks receiving the vaccines were higher than those in single gene delivered groups. Additionally, the copy number of covalently closed circular DNA in hepatocytes after DHBV challenge also provided evidence that our fusion vaccines could enhance the protective efficiency against DHBV infection in ducks.

Optimization of inactivated H5N9 highly pathogenic avian influenza vaccine and inactivated Salmonella enterica serovar Typhimurium vaccine with antigen dose and prime-boost regimen in domestic ducks

Owing to the increase in the number of diseases affecting ducks and the demand for food safety by consumers, vaccination has become one of the factors that influence duck meat productivity. The highly pathogenic avian influenza (HPAI) virus is one of the most prevalent and causes one of the most lethal diseases in domestic ducks, and Salmonella enterica serovar Typhimurium is a food-borne pathogen persistent in the domestic duck population. To better understand the optimal usage of HPAI and S. enterica serovar Typhimurium vaccines, we aimed to determine antigen dose, oil and gel adjuvant usage with prime-boost regimen, and vaccination age, inducing the best immune response in ducks, without an effect on body weight gain. In the case of the inactivated H5N9 vaccine, a single dose of vaccine was inadequate to induce proper antibody titer when administered to day-old ducks, which necessitates boost vaccination. Administration of the oil-adjuvanted H5N9 vaccine administration in day-old and 2-week-old ducks resulted in a lower body weight at the time of slaughtering, compared to that of gel-adjuvanted H5N9 vaccine. However, gel-adjuvanted H5N9 vaccine failed to induce proper immune response to an extent recommend by OIE–World Organization for Animal Health. In the case of the Salmonella enterica serovar Typhimurium vaccine, a moderate or low dose of vaccine was appropriate for day-old ducks receiving the gel prime-oil boost vaccination. Single vaccination with oil adjuvants affects the mean body weight of 7-week-old ducks, suggesting that the gel adjuvant is more suitable for meat production. We expect that the use of adjuvants in a prime-boost regimen and at antigen doses set in this study will be helpful to maximize body weight in the case of domestic duck production at the actual farm site.

Molecular cloning and immune responsive expression of LGP2 gene, a pivotal member of the RLR gene family from Muscovy duck Cairina moschata

Laboratory of genetics and physiology 2 (LGP2) is an important intracellular receptor that recognizes viral RNAs in innate immunity. In this study, a novel LGP2 cDNA was identified from the spleen of a Muscovy duck (Cairina moschata). The deduced amino acid sequence of Muscovy duck LGP2 (MdLGP2) consisted of 675 amino acid residues. The peptide contained two main structure domains: six important motifs, including a DExD/H box for RNA helicase activity in the RNA helicase region located at the N-terminal region, and two Zn2+-binding regions with an RNA-binding loop in the C-terminus regulatory domain (CTD). The MdLGP2 mRNA was ubiquitously expressed in the tested tissues, with high expression levels in glandular stomach, colon, ileum, crop, and caecum tissues, and low expression levels in the brain, skin, and heart. The mRNA expression of MdLGP2 was significantly upregulated in the brain, spleen, and lungs of ducks in the early stages of postinfection with H5N1 highly pathogenic avian influenza virus (HPAIV). These results suggested that MdLGP2 was involved in the early stages of antiviral innate immune response in ducks after infection with H5N1 HPAIV. However, whether it plays a positive or negative regulatory role in the host antiviral response requires further investigation.

The CD8α gene in duck (Anatidae): cloning, characterization, and expression during viral infection.

Cluster of differentiation 8 alpha (CD8α) is critical for cell-mediated immune defense and T-cell development. Although CD8α sequences have been reported for several species, very little is known about CD8α in ducks. To elucidate the mechanisms involved in the innate and adaptive immune responses of ducks, we cloned CD8α coding sequences from domestic, Muscovy, Mallard, and Spotbill ducks using reverse transcription polymerase chain reaction (RT-PCR). Each sequence consisted of 714 nucleotides and encoded a signal peptide, an IgV-like domain, a stalk region, a transmembrane region, and a cytoplasmic tail. We identified 58 nucleotide differences and 37 amino acid differences among the four types of duck; of these, 53 nucleotide and 33 amino acid differences were between Muscovy ducks and the other duck species. The CD8α cDNA sequence from domestic duck consisted of a 61-nucleotide 5' untranslated region (UTR), a 714-nucleotide open reading frame, and an 849-nucleotide 3' UTR. Multiple sequence alignments showed that the amino acid sequence of CD8α is conserved in vertebrates. RT-PCR revealed that expression of CD8α mRNA of domestic ducks was highest in the thymus and very low in the kidney, cerebrum, cerebellum, and muscle. Immunohistochemical analyses detected CD8α on the splenic corpuscle and periarterial lymphatic sheath of the spleen. CD8α mRNA in domestic ducklings was initially up-regulated, and then down-regulated, in the thymus, spleen, and liver after treatment with duck hepatitis virus type I (DHV-1) or the immunostimulant polyriboinosinic polyribocytidylic acid (poly I:C).

The immune response of a recombinant fowlpox virus coexpressing the HA gene of the H5N1 highly pathogenic avian influenza virus and chicken interleukin 6 gene in ducks

Ducks have played an important role in the emergence of H5N1 subtype of highly pathogenic avian influenza (HPAI), and the development of an effective vaccine against HPAI in ducks is a top priority. It has been shown that a recombinant fowlpox virus (FPV)-vectored vaccine can provide protection against HPAI in ducks. In this study, a recombinant fowlpox virus (rFPV-AIH5AIL6) coexpressing the haemagglutinin (HA) gene of the H5N1 subtype of the avian influenza virus (AIV) and chicken interleukin 6 gene was constructed and tested in Gaoyou and cherry valley ducks to evaluate the immune response in ducks. These animal studies demonstrated that rFPV-AIH5AIL6 induced a higher anti-AIV HI antibody response, an enhanced lymphocyte proliferation response, an elevated immune protection, and a reduction in virus shedding compared to a recombinant fowlpox virus expressing the HA gene alone (rFPV-SYHA). These data indicate that rFPV-AIH5AIL6 may be a potential vaccine against the H5 subtype of avian influenza in ducks and chicken interleukin 6 may be an effective adjuvant for increasing the immunogenicity of FPV-vectored AIV vaccines in ducks.

Three novel Anas platyrhynchos avian β-defensins, upregulated by duck hepatitis virus, with antibacterial and antiviral activities

Three novel Anas platyrhynchos avian β-defensins (Apl_AvBDs), Apl_AvBD4, 7 and 12, were identified successfully and characterized in tissues from Peking ducks in the present study. The cDNA fragment of Apl_AvBD4 contained 171bp, and encoded 56 amino acids. The complete nucleotide sequences of Apl_AvBD7 and 12 contained 204bp and 198bp open reading frames, which encoded 67 and 65 amino acids, respectively. Both recombinant and synthetic forms of the three Apl_AvBDs showed antibacterial activity against most of the bacteria investigated, including Gram-negative and Gram-positive bacteria, except for Salmonella choleraesuis. In addition, the antibacterial activity of all the three Apl_AvBDs decreased significantly in 150mM NaCl. Significant antiviral activity of the three Apl_AvBDs was shown against duck hepatitis virus (DHV). However, none of the Apl_AvBDs showed significant hemolytic activity. Additionally, the expressions of the three Apl_AvBDs in response to DHV infection was highly variable, and significant upregulation of Apl_AvBD7 in liver was found in response to infection at different time points. Expression of Apl_AvBD4 in thymus, and of Apl_AvBD7 in bone marrow was induced in a time-dependent fashion by DHV infection. In contrast, expression of Apl_AvBD12 was found to be significantly decreased, and was hard to detect in cecal tonsil, spleen, bursa of Fabricius, and thymus of ducks at some time points after DHV infection. The present results demonstrate that Apl_AvBDs play vital roles in the immune response of ducks against bacterial and viral pathogens.

Association of RIG-I with innate immunity of ducks to influenza.

Ducks and wild waterfowl perpetuate all strains of influenza viruses in nature. In their natural host, influenza viruses typically cause asymptomatic infection and little pathology. Ducks are often resistant to influenza viruses capable of killing chickens. Here, we show that the influenza virus sensor, RIG-I, is present in ducks and plays a role in clearing an influenza infection. We show evidence suggesting that RIG-I may be absent in chickens, providing a plausible explanation for their increased susceptibility to influenza viruses compared with ducks. RIG-I detects RNA ligands derived from uncapped viral transcripts and initiates the IFN response. In this study, we show that the chicken embryonic fibroblast cell line, DF-1, cannot respond to a RIG-I ligand. However, transfection of duck RIG-I into DF-1 cells rescues the detection of ligand and induces IFN-beta promoter activity. Additionally, DF-1 cells expressing duck RIG-I have an augmented IFN response resulting in decreased influenza replication after challenge with either low or highly pathogenic avian influenza virus. Implicating RIG-I in the antiviral response to an infection in vivo, we found that RIG-I expression is induced 200 fold, early in an innate immune response in ducks challenged with the H5N1 virus A/Vietnam/1203/04. Finding this natural disease resistance gene in ducks opens the possibility of increasing influenza resistance through creation of a transgenic chicken.

Discovery of Anas platyrhynchos avian β-defensin 2 (Apl_AvBD2) with antibacterial and chemotactic functions

The cationic, cysteine-rich peptides called β-defensins play a major role in the innate immune response. Here, we describe the identification and characterization of the duck β-defensin-2 homologue, Anas platyrhynchos avian β-defensin 2 (Apl_AvBD2). The 195 base pair open reading frame (ORF) of Apl_AvBD2 has 83% identity with Gga_AvBD2 (chicken) and 85% identity with Mga_AvBD2 (turkey) at nucleotide level. The gene corresponding to the coding region is comprised of three exons and two introns in both Apl_AvBD2 and Gga_AvBD2. The predicted secondary structure of Apl_AvBD2 has the classical “β-defensin core motif” formed by the β-sheet rich structure. Apart from mild expression in tissues like kidney, lung, brain, bursa of Fabricious and ovary, Apl_AvBD2 mRNA show a very high level constitutive expression in bone marrow and spleen, indicating that it is a myeloid defensin. Purified recombinant Apl_AvBD2 demonstrated in vitro antibacterial activity against both Gram-positive and Gram-negative bacteria, with a minimum bactericidal concentration (MBC) of 3.7μM against Micrococcus luteus NCIM 2871 and Escherichia coli NCIM 2685, and of 2.2μM against Reimerella anatipestifer. The immunomodulatory potential of Apl_AvBD2 was shown by chemotaxis of DT-40 chicken B-lymphocytes. The widespread tissue distribution and the potent bactericidal and chemotactic activity make Apl_AvBD2 an important molecule in the innate immune response in ducks. It may play a vital role in the immune response of these birds against bacterial and viral pathogens.

Cloning, in vitro expression and bioactivity of duck interleukin-18

The encoding sequence for duck IL-18 was obtained, using reverse transcription-polymerase chain reaction, from mRNA harvested from Con A-stimulated Gushi (GS) duck splenic mononuclear cells. Recombinant duck IL-18 (rduIL-18) was produced in a prokaryotic expression system. In vitro bioactivity of rduIL-18 was determined in a lymphocyte proliferation assay and in vivo bioactivity of rduIL-18 was assessed by addition to a vaccine. Monoclonal antibody (mAb) and polyclonal antibodies (pAbs) specific for rduIL-18 were generated and subsequently characterized by ELISA, Western blot and neutralizing assays. Sequence analysis of GS duck IL-18 demonstrated an open reading frame (ORF) of 603 base pairs encoding for a 200 amino acid precursor protein. The duck encoding sequence shares 85.3% similarity to the chicken equivalent, at the nucleotide level. A His-duIL-18 fusion protein was recognized in Western blot by mAbs against duck and chicken IL-18 (chIL-18), but not by mAb against human IL-18. Recombinant duIL-18 induced in vitro proliferation of Con A-stimulated duck splenocytes and enhanced the immune response of ducks vaccinated with an inactivated oil emulsion vaccine against avian influenza virus. PAb and mAb 5B2 against rduIL-18 had neutralizing ability, inhibiting the biological activities of both recombinant duIL-18 and endogenous duIL-18. The results indicate that rduIL-18 has the potential to be used as an immunoadjuvant, and the mAb against rduIL-18 further facilitates basic immunobiological studies of the role of IL-18 in the avian immune system.

Effects of Early Cyclophosphamide Treatment on Lymphoid Organs and its Immune Response in Ducks

Cyclophosphamide treatment of newly hatched White Pekin ducks significantly reduced the weight of their lymphoid organs and almost completely eliminated the formation of bursal lymphoid follicles at one week of age. Thereafter, the thymus and spleen of these ducks completely recovered in weight at 7 weeks of age. The bursa, however, retained its light weight and morphological defects, such as the absence of lymphoid follicles. The specific antibody producing-capability to Salmonella pullorum was also eliminated. The total dosage of cyclophosphamide was smaller than that employed in chickens, which makes effective chemical bursectomy in ducks possible.