Supplemental dietary selenium enhances immune responses conferred by a vaccine against low pathogenicity avian influenza virus.

Selenium is a trace mineral that has antioxidant activities and can influence the immune system. However, antiviral effects of selenium have not been well studies in chickens. Chickens were therefore fed diets supplemented with two levels of two different sources of selenium (organic: selenium enriched yeast; SEY or inorganic: sodium selenite; SS). Chickens in the control groups did not receive supplemental dietary selenium. At 14 and 21 days of age, chickens were vaccinated with an inactivated low pathogenicity avian influenza virus (AIV, subtype H9N2) vaccine and blood samples were collected to determine the level of antibodies using hemagglutination inhibition (HI) and ELISA. At 30 days of age, chickens were also challenged with the same virus and swab samples were collected to assess the amount of virus shedding. Antibody levels, as measured by HI, increased significantly in the chickens that received higher levels of SEY at 16 days post vaccination. ELISA titers for IgM and IgY were higher in selenium supplemented chickens. Comparing to challenged control, virus shedding was lower in organic as well as inorganic selenium treated groups. Therefore, it may be concluded that supplemental dietary selenium could enhance vaccine conferred immunity thereby impacting protection against viral challenge in chickens.

Characterization of immunogenicity of avian influenza antigens encapsulated in PLGA nanoparticles following mucosal and subcutaneous delivery in chickens.

Mucosal vaccine delivery systems have paramount importance for the induction of mucosal antibody responses. Two studies were conducted to evaluate immunogenicity of inactivated AIV antigens encapsulated in poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles (NPs). In the first study, seven groups of specific pathogen free (SPF) layer-type chickens were immunized subcutaneously at 7-days of age with different vaccine formulations followed by booster vaccinations two weeks later. Immune responses were profiled by measuring antibody (Ab) responses in sera and lachrymal secretions of vaccinated chickens. The results indicated that inactivated AIV and CpG ODN co-encapsulated in PLGA NPs (2x NanoAI+CpG) produced higher amounts of hemagglutination inhibiting antibodies compared to a group vaccinated with non-adjuvanted AIV encapsulated in PLGA NPs (NanoAI). The tested adjuvanted NPs-based vaccine (2x NanoAI+CpG) resulted in higher IgG responses in the sera and lachrymal secretions at weeks 3, 4 and 5 post-vaccination when immunized subcutaneously. The incorporation of CpG ODN led to an increase in Ab-mediated responses and was found useful to be included both in the prime and booster vaccinations. In the second study, the ability of chitosan and mannan coated PLGA NPs that encapsulated AIV and CpG ODN was evaluated for inducing antibody responses when delivered via nasal and ocular routes in one-week-old SPF layer-type chickens. These PLGA NPs-based and surface modified formulations induced robust AIV-specific antibody responses in sera and lachrymal secretions. Chitosan coated PLGA NPs resulted in the production of large quantities of lachrymal IgA and IgG compared to mannan coated NPs, which also induced detectable amounts of IgA in addition to the induction of IgG in lachrymal secretions. In both mucosal and subcutaneous vaccination approaches, although NPs delivery enhanced Ab-mediated immunity, one booster vaccination was required to generate significant amount of Abs. These results highlight the potential of NPs-based AIV antigens for promoting the induction of both systemic and mucosal immune responses against respiratory pathogens.

Influenza A virus subtype H9N2 infection disrupts the composition of intestinal microbiota of chickens.

The impact of low pathogenic influenza viruses such as subtype H9N2, which infect the respiratory and the gastrointestinal tracts of chickens, on microbial composition are not known. Twenty-day-old specific pathogen-free chickens were assigned to two treatment groups, control (uninfected) and H9N2-infected (challenged via the oral-nasal route). Fecal genomic DNA was extracted, and the V3-V4 regions of the 16S rRNA gene were sequenced using the Illumina Miseq® platform. Sequences were curated using Mothur as described in the MiSeq SOP. Infection of chickens with H9N2 resulted in an increase in phylum Proteobacteria, and differential enrichment with the genera Vampirovibrio, Pseudoflavonifractor, Ruminococcus, Clostridium cluster XIVb and Isobaculum while control chickens were differentially enriched with genera Novosphingobium, Sphingomonas, Bradyrhizobium and Bifidobacterium. Analysis of pre- and post-H9N2 infection of the same chickens showed that, before infection, the fecal microbiota was characterized by Lachnospiracea and Ruminococcaceae family and the genera Clostridium sensu stricto, Roseburia and Lachnospiraceae incertae sedis. However, post-H9N2 infection, class Deltaproteobacteria, orders Clostridiales and Bacteroidiales and the genus Alistipes were differentially enriched. Findings from the current study show that influenza virus infection in chickens results in the shift of the gut microbiota, and the disruption of the host-microbial homeostasis in the gut might be one of the mechanisms by which influenza virus infection is established in chickens.

Effects of early feeding and dietary interventions on development of lymphoid organs and immune competence in neonatal chickens: A review.

With the ongoing intensification of the poultry industry and the continuous need to control pathogens, there is a critical need to extend our understanding of the avian immune system and the role of nutritional interventions on development of immune competence in neonatal chicks. In this review, we will focus on the ontogeny of the lymphoid organs during embryonic life and the first 2 weeks post-hatch, and how early feeding practices improve heath and modulate the development and function of the immune system in young chicks. The evidence for the positive impact of the nutrition of breeder hens on embryonic development and on the survival and immunity of their chicks will also be outlined. Additionally, we will discuss the vital role of supplemental feeding either in ovo or immediately post-hatch in chick health and immunity and the importance of these approaches in ameliorating immune system functions of heat-stressed chicks. To conclude, we provide some perspectives on a number of key issues, concerning the mechanisms of nutritional modulation of immunity, that need to be addressed. A thorough investigation of these mechanisms may assist in the formulation of diets to improve the immunity and general health status.

PLGA-encapsulated CpG ODN and Campylobacter jejuni lysate modulate cecal microbiota composition in broiler chickens experimentally challenged with C. jejuni.

Campylobacter jejuni is a leading bacterial cause of human gastroenteritis. Reducing Campylobacter numbers in the intestinal tract of chickens will minimize transmission to humans, thereby reducing the incidence of infection. We have previously shown that oral pre-treatment of chickens with C. jejuni lysate and Poly D, L-lactide-co-glycolide polymer nanoparticles (PLGA NPs) containing CpG oligodeoxynucleotide (ODN) can reduce the number of C. jejuni in infected chickens. In the current study, the effects of these pre-treatments on the composition and functional diversity of the cecal microbiota, in chickens experimentally infected with C. jejuni, were investigated using next-generation sequencing. The taxonomic composition analysis revealed a reduction in cecal microbial diversity and considerable changes in the taxonomic profiles of the microbial communities of C. jejuni-challenged chickens. On the other hand, irrespective of the dose, the microbiota of PLGA-encapsulated CpG ODN- and C. jejuni lysate-treated chickens exhibited higher microbial diversity associated with high abundance of members of Firmicutes and Bacteroidetes and lower numbers of Campylobacter than untreated-chickens. These findings suggest that oral administration of encapsulated CpG ODN and C. jejuni lysate can reduce colonization by C. jejuni by enhancing the proliferation of specific microbial groups. The mechanisms that mediate these changes remain, however, to be elucidated.

Oral administration of PLGA-encapsulated CpG ODN and Campylobacter jejuni lysate reduces cecal colonization by Campylobacter jejuni in chickens.

Campylobacter jejuni (C. jejuni) is a major cause of bacterial food-borne illness in humans. It is considered a commensal organism of the chicken gut and infected chickens serve as a reservoir and shed bacteria throughout their lifespan. Contaminated poultry products are considered the major source of infection in humans. Therefore, to reduce the risk of human campylobacteriosis, it is essential to reduce the bacterial load in poultry products. The present study aimed to evaluate the protective effects of soluble and PLGA-encapsulated oligodeoxynucleotides (ODN) containing unmethylated CpG motifs (E-CpG ODN) as well as C. jejuni lysate as a multi-antigen vaccine against colonization with C. jejuni. The results revealed that oral administration of a low (5 µg) or high (50 µg) dose of CpG resulted in a significant reduction in cecal C. jejuni colonization by 1.23 and 1.32 log10 (P < .05) in layer chickens, respectively, whereas E-CpG significantly reduced cecal C. jejuni colonization by 1.89 and 1.46 log10 in layer and broiler chickens at day 22 post-infection (slaughter age in broilers), respectively. Similar patterns were observed for C. jejuni lysate; oral administration of C. jejuni lysate reduced the intestinal burden of C. jejuni in layer and broiler chickens by 2.24 and 2.14 log10 at day 22 post-infection, respectively. Moreover, the combination of E-CpG and C. jejuni lysate reduced bacterial counts in cecal contents by 2.42 log10 at day 22 post-infection in broiler chickens. Anti-C. jejuni IgG antibody (Ab) titers were significantly higher for broiler chickens receiving a low or high dose of E-CpG or a low dose of C. jejuni lysate than for chickens receiving the placebo. Furthermore, a positive correlation was observed between serum IgG Ab titers and cecal counts of C. jejuni in these groups. These findings suggest that PLGA-encapsulated CpG or C. jejuni lysate could be a promising strategy for control of C. jejuni in chickens.

Molecular Characterization of Hemorrhagic Enteritis Viruses (HEV) Detected in HEV-Vaccinated Commercial Turkey Flocks in Germany.

Despite the application of live hemorrhagic enteritis virus (HEV) vaccines, HEV field outbreaks are suspected to still occur in turkey flocks in Germany. Increasing secondary bacterial infections in HEV-vaccinated flocks suggest that vaccines may be losing efficacy or, possibly, that vaccine strains are causing disease. Thus, the goal of the current study was to investigate the diversity of HEV isolates from fattening turkey flocks between 2008 and 2012 by characterizing the open reading frame (ORF)1 gene at its 5' and 3' ends. Analyses of ORF1 sequences of field isolates and comparison with sequences present in databases revealed that in many cases (13 out of 16 samples), vaccine (avirulent) strains were present. In addition, data indicated the circulation of suspected virulent field isolates and these isolates (3 out of 16) cluster with an early isolate from Germany in the 1980s, but show some mutations in the predicted amino acid (aa) sequences of ORF1 compared to the early isolate. These virulent isolates clearly differ from the spleen-derived avirulent Domermuth vaccine strain used in Germany. In this study, a unique isolate was identified and showed unusual nucleotide mutations that resulted in aa exchanges at the 5' end of ORF1 between aa positions 34 and 174. This genetic drift suggests evolution of HEV including virulent and vaccine-derived strains in the field. This may lead to evasion of vaccinal immunity by drifted viruses and/or an increase in the virulence of field strains.

Gene expression profiling of chicken cecal tonsils and ileum following oral exposure to soluble and PLGA-encapsulated CpG ODN, and lysate of Campylobacter jejuni.

Campylobacter jejuni (C. jejuni) is a leading bacterial cause of food-borne illness in humans. Contaminated chicken meat is an important source of infection for humans. Chickens are not clinically affected by colonization, and immune responses following natural infection have limited effects on bacterial load in the gut. Induction of intestinal immune responses may possibly lead to a breakdown of the commensal relationship of chickens with Campylobacter. We have recently shown that soluble and poly D, L-lactic-co-glycolic acid (PLGA)-encapsulated CpG oligodeoxynucleotide (ODN) as well as C. jejuni lysate, are effective in reducing the intestinal burden of C. jejuni in chickens; however, the mechanisms behind this protection have yet to be determined. The present study was undertaken to investigate the mechanisms of host responses conferred by these treatments. Chickens were treated orally with soluble CpG ODN, or PLGA-encapsulated CpG ODN, or C. jejuni lysate, and expression of cytokines and antimicrobial peptides was evaluated in cecal tonsils and ileum using quantitative RT-PCR. Oral administration of soluble CpG ODN upregulated the expression of interferon (IFN)-γ, interleukin (IL)-1ß, CXCLi2, transforming growth factor (TGF)-ß4/1, IL-10 and IL-13, while treatment with PLGA-encapsulated CpG ODN upregulated the expression of IL-1ß, CXCLi2, TGF-ß4/1, IL-13, avian ß-defensin (AvBD) 1, AvBD2 and cathelicidin 3 (CATHL-3). C. jejuni lysate upregulated the expression of IFN-γ, IL-1ß, TGF-ß4/1, IL-13, AvBD1, and CATHL-3. In conclusion, induction of cytokine and antimicrobial peptides expression in intestinal microenvironments may provide a means of reducing C. jejuni colonization in broiler chickens, a key step in reducing the incidence of campylobacteriosis in humans.

Infectious bursal disease virus in poultry: current status and future prospects.

Infectious bursal disease virus (IBDV) affects immature B lymphocytes of the bursa of Fabricius and may cause significant immunosuppression. It continues to be a leading cause of economic losses in the poultry industry. IBDV, having a segmented double-stranded RNA genome, is prone to genetic variation. Therefore, IBDV isolates with different genotypic and phenotypic diversity exist. Understanding these features of the virus and the mechanisms of protective immunity elicited thereof is necessary for developing vaccines with improved efficacy. In this review, we highlighted the pattern of virus evolution and new developments in prophylactic strategies, mainly the development of new generation vaccines, which will continue to be of interest for research as well as field application in the future.

Local Innate Responses to TLR Ligands in the Chicken Trachea.

The chicken upper respiratory tract is the portal of entry for respiratory pathogens, such as avian influenza virus (AIV). The presence of microorganisms is sensed by pathogen recognition receptors (such as Toll-like receptors (TLRs)) of the innate immune defenses. Innate responses are essential for subsequent induction of potent adaptive immune responses, but little information is available about innate antiviral responses of the chicken trachea. We hypothesized that TLR ligands induce innate antiviral responses in the chicken trachea. Tracheal organ cultures (TOC) were used to investigate localized innate responses to TLR ligands. Expression of candidate genes, which play a role in antiviral responses, was quantified. To confirm the antiviral responses of stimulated TOC, chicken macrophages were treated with supernatants from stimulated TOC, prior to infection with AIV. The results demonstrated that TLR ligands induced the expression of pro-inflammatory cytokines, type I interferons and interferon stimulated genes in the chicken trachea. In conclusion, TLR ligands induce functional antiviral responses in the chicken trachea, which may act against some pathogens, such as AIV.

Characterization of host responses induced by Toll-like receptor ligands in chicken cecal tonsil cells.

The innate responses of cecal tonsils against invading microorganisms are mediated by conserved pattern recognition receptors (PRRs) such as the Toll-like receptors (TLRs). TLRs expressed by mammalian and avian immune system cells have the capability to recognize pathogen-associated molecular patterns (PAMPs). Although, the role of TLR ligands in innate and adaptive responses in chickens has been characterized in spleen and bursa of Fabricius, considerably less is known about responses in cecal tonsils. The aim of the current study was to assess responses of mononuclear cells from cecal tonsils to treatment with the TLR2, TLR4 and TLR21 ligands, Pam3CSK4, lipopolysaccharide (LPS), and CpG oligodeoxynucleotide (ODN), respectively. All three ligands induced significant up-regulation of interferon (IFN)-γ, interleukin (IL)-1ß, IL-6 and CxCLi2/IL-8, whereas no significant changes were observed in expression of IL-13 or the antimicrobial peptides, avian ß-defensin (AvBD) 1, AvBD2 and cathelicidin 3 (CATHL-3). In general, CpG ODN elicited the highest cytokine responses by cecal tonsil mononuclear cells, inducing significantly higher expression compared to LPS and Pam3CSK4, for IFNγ, IL-1ß, IL-6 and CxCLi2 at various time points. These findings suggest the potential use of TLR21 ligands as mucosal vaccine adjuvants, especially in the context of pathogens of the intestinal tract.

Expression profiles of antiviral response genes in chicken bursal cells stimulated with Toll-like receptor ligands.

Cells of the adaptive immune system express Toll-like receptors (TLRs) and are able to respond to TLR ligands. With this in mind, the goal of the current study was to determine the expression of antiviral response genes in the cells of the chicken bursa of Fabricius (BF) to stimulation with TLR ligands. We investigated initially the response of bursal B cells to CpG-ODN, lipopolysaccharide (LPS) and poly(I:C) treatment. The expression level of type I interferons (IFNs) and interferon regulatory factor 7 (IRF7) did not differ between CpG-ODN and LPS treated groups compared to the non-stimulated cells. Poly(I:C) was the only TLR ligand, which has induced significant expression of antiviral innate immune response genes from bursal cells. Further in vitro and in vivo studies need to examine the efficacy of these antiviral responses against avian viruses.