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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.

The present study was undertaken to profile and compare the cecal microbial communities in conventionally (CONV) grown and raised without antibiotics (RWA) broiler chickens. Three hundred chickens were collected from five CONV and five RWA chicken farms on days 10, 24, and 35 of age. Microbial genomic DNA was extracted from cecal contents, and the V4-V5 hypervariable regions of the 16S rRNA gene were amplified and sequenced. Analysis of 16S rRNA sequence data indicated significant differences in the cecal microbial diversity and composition between CONV and RWA chickens on days 10, 24, and 35 days of age. On days 10 and 24, CONV chickens had higher richness and diversity of the cecal microbiome relative to RWA chickens. However, on day 35, this pattern reversed such that RWA chickens had higher richness and diversity of the cecal microbiome than the CONV groups. On days 10 and 24, the microbiomes of both CONV and RWA chickens were dominated by members of the phylum Firmicutes. On day 35, while Firmicutes remained dominant in the RWA chickens, the microbiome of CONV chickens exhibited am abundance of Bacteroidetes. The cecal microbiome of CONV chickens was enriched with the genus Faecalibacterium , Pseudoflavonifractor , unclassified Clostridium_IV , Bacteroides , Alistipes , and Butyricimonas , whereas the cecal microbiome of RWA chickens was enriched with genus Anaerofilum , Butyricicoccu , Clostridium_XlVb and unclassified Lachnospiraceae . Overall, the cecal microbiome richness, diversity, and composition were greatly influenced by the management program applied in these farms. These findings provide a foundation for further research on tailoring feed formulation or developing a consortium to modify the gut microbiome composition of RWA chickens.

•Inactivating influenza viruses can alter their structure and function.•Structural changes of inactivated influenza viruses affect vaccine immune responses.•Beta-propiolactone, formaldehyde, and gamma radiation can be used for inactivation.•Beta-propiolactone inactivated H9N2 virus enhanced immune responses in chickens. Several types of avian influenza virus (AIV) vaccines exist, including live-attenuated, vectored, and whole inactivated virus (WIV) vaccines. Inactivated vaccines offer some advantages compared to other types of vaccines, including ease of production and lack of ability to revert to a virulent state. However, WIV are poorly immunogenic, especially when these vaccines are delivered to mucosal surfaces. There are several factors that contribute to the immunogenicity of vaccines, one of which is the method used to inactivate viruses. Several methods exist for producing influenza WIVs, including formaldehyde, a chemical that affects protein structures leading to virus inactivation. Other methods include treatment with beta-propiolactone (BPL) and the application of gamma radiation, both of which have less effects on protein structures compared to formaldehyde, and instead alter nucleic acids in the virion. Here, we sought to determine the effect of the above inactivation methods on immunogenicity of AIV vaccines. To this end, chickens were vaccinated with three different H9N2 WIVs using formaldehyde, BPL, and gamma radiation for inactivation. In addition to administering these three WIVs alone as vaccines, we also included CpG ODN 2007, a synthetic ligand recognized by Toll-like receptor (TLR)21 in chickens, as an adjuvant for each WIV. Subsequently, antibody- and cell-mediated immune responses were measured following vaccination. Antibody-mediated immune responses were increased in chickens that received the BPL and Gamma WIVs compared to the formaldehyde WIV. CpG ODN 2007 was found to significantly increase antibody responses for each WIV compared to WIV alone. Furthermore, we observed the presence of cell-mediated immune responses in chickens that received the BPL WIV combined with CpG ODN 2007. Based on these results, the BPL WIV + CpG ODN 2007 combination was the most effective vaccine at inducing adaptive immune responses against H9N2 AIV. Future studies should characterize mucosal adaptive immune responses to these vaccines.

Future demands for food will place agricultural systems under pressure to increase production. Poultry is accepted as a good source of protein and the poultry industry will be forced to intensify production in many countries, leading to greater numbers of farms that house birds at elevated densities. Increasing farmed poultry can facilitate enhanced transmission of infectious pathogens among birds, such as avian influenza virus among others, which have the potential to induce widespread mortality in poultry and cause considerable economic losses. Additionally, the capability of some emerging poultry pathogens to cause zoonotic human infection will be increased as greater numbers of poultry operations could increase human contact with poultry pathogens. In order to combat the increased risk of spread of infectious disease in poultry due to intensified systems of production, rapid detection and diagnosis is paramount. In this review, multiple technologies that can facilitate accurate and rapid detection and diagnosis of poultry diseases are highlighted from the literature, with a focus on technologies developed specifically for avian influenza virus diagnosis. Rapid detection and diagnostic technologies allow for responses to be made sooner when disease is detected, decreasing further bird transmission and associated costs. Additionally, systems of rapid disease detection produce data that can be utilized in decision support systems that can predict when and where disease is likely to emerge in poultry. Other sources of data can be included in predictive models, and in this review two highly relevant sources, internet based-data and environmental data, are discussed. Additionally, big data and big data analytics, which will be required in order to integrate voluminous and variable data into predictive models that function in near real-time are also highlighted. Implementing new technologies in the commercial setting will be faced with many challenges, as will designing and operating predictive models for poultry disease emergence. The associated challenges are summarized in this review. Intensified systems of poultry production will require new technologies for detection and diagnosis of infectious disease. This review sets out to summarize them, while providing advantages and limitations of different types of technologies being researched.

There is some evidence that lactobacilli can strengthen the immune system of chickens. This study evaluated the effects of in ovo and oral administration of a lactobacilli cocktail on cytokine gene expression, antibody-mediated immune responses, and spleen cellularity in chickens. Lactobacilli were administered either in ovo at embryonic day 18, orally at days 1, 7, 14, 21, and 28 post-hatches, or a combination of both in ovo and post-hatch inoculation. On day 5 and 10 post-hatch, spleen and bursa of Fabricius were collected for gene expression and cell composition analysis. On days 14 and 21 post-hatch, birds were immunized with sheep red blood cells (SRBC) and keyhole limpet hemocyanin (KLH), and sera were collected on days 7, 14, and 21 post-primary immunization. Birds that received lactobacilli (10 7 CFU) via in ovo followed by weekly oral administration showed a greater immune response by enhancing antibody responses, increasing the percentage of CD4 + and CD4 + CD25 + T cells in the spleen and upregulating the expression of interferon (IFN)-α, IFN-β, interleukin (IL)-8, IL-13, and IL-18 in the spleen and expression of IFN-γ, IL-2, IL-6, IL-8, IL-12, and IL-18 in the bursa. These findings suggest that pre-and post-hatch administration of lactobacilli can modulate the immune response in newly hatched chickens.

[This corrects the article DOI: 10.3389/fimmu.2021.664387.].[This corrects the article DOI: 10.3389/fimmu.2021.664387.].

This study was conducted to investigate the effects of various doses of a multi-strain lactobacilli mixture ( Lactobacillus salivarius, Lactobacillus reuteri, Lactobacillus crispatus , and Lactobacillus johnsonii ) on the innate and adaptive immune responses in broiler chickens. At embryonic day eighteen, 200 eggs were injected with PBS, or three different doses of a multi-strain lactobacilli mixture (1 × 10 5 , 1 × 10 6 , and 1 × 10 7 CFU/egg, P1, P2, and P3 respectively) along with a group of negative control. On days 5 and 10 post-hatch, cecal tonsil, bursa of fabricius, and spleen were collected for gene expression and cellular analysis. On days 14 and 21 post-hatch, birds were immunized intramuscularly with both sheep red blood cells (SRBC) and keyhole limpet hemocyanin (KLH). Serum samples were collected on days 0, 7, 14, and 21 after primary immunization. The results demonstrated that lactobacilli inoculation increased the splenic expression of cytokines, including interferon (IFN) - α, IFN - β, IFN - γ, interleukin (IL)-8, and IL-12 on day 5 post-hatch compared to the control group (PBS). However, in cecal tonsils, lactobacilli treatment downregulated the expression of IL-6 on day 5 post-hatch and IL-2 and IL-8 on day 10 post-hatch. No significant differences were observed in the expression of cytokine genes in the bursa except for IL-13 which was upregulated in lactobacilli-treated groups P2 and P3 on days 5 and 10 post-hatch. Flow cytometry analysis showed that the percentage of KUL01, CD4 + and CD8 + splenocytes was not affected by treatments. In addition, no significant differences were observed for antibody titers against SRBC. However, lactobacilli treatment (P1, P2, and P3) was found to increase IgM titers on day 21 post-primary immunization compared to controls. Furthermore, in ovo injection of the highest dose of probiotics (1 × 10 7 , P3) increased serum IgG titers against KLH on day 7 post-primary immunization. In conclusion, this study demonstrated that that in ovo administration of lactobacilli can improve antibody-mediated immune responses and differentially modulate cytokine expression in mucosal and systemic lymphoid tissues of chickens.

Vaccines against Marek’s disease can protect chickens against clinical disease; however, infected chickens continue to propagate the Marek’s disease virus (MDV) in feather follicles and can shed the virus into the environment. Therefore, the present study investigated if MDV could induce an immunoregulatory microenvironment in feathers of chickens and whether vaccines can overcome the immune evasive mechanisms of MDV. The results showed an abundance of CD4+CD25+ and CD4+ transforming growth factor-beta (TGF-β)+ T regulatory cells in the feathers of MDV-infected chickens at 21 days post-infection. In contrast, vaccinated chickens had a lower number of regulatory T cells. Furthermore, the expression of TGF-β and programmed cell death receptor (PD)-1 increased considerably in the feathers of Marek’s disease virus-infected chickens. The results of the present study raise the possibility of an immunoregulatory environment in the feather pulp of MDV-infected chickens, which may in turn favor replication of infectious MDV in this tissue. Exploring the evasive strategies employed by MDV will facilitate the development of control measures to prevent viral replication and transmission.

[This corrects the article DOI: 10.3389/fvets.2020.00105.].[This corrects the article DOI: 10.3389/fvets.2020.00105.].

Objective Infection of chickens with low pathogenic avian influenza virus, such as H9N2 virus, culminates in decreased egg production and increased mortality and morbidity if co-infection with other respiratory pathogens occurs. We have previously observed the induction of antibody- and cell-mediated immune responses after intramuscular administration of an H9N2 beta-propiolactone inactivated virus vaccine to chickens. Given the fact that in ovo vaccination represents a practical option for vaccination against H9N2 AIV in chickens, in the current study, we set out to characterize immune responses in chickens against a beta-propiolactone inactivated H9N2 virus vaccine after primary vaccination in ovo on embryonic day 18, and secondary intramuscular vaccination on day 14 post-hatch. We also included the Toll-like receptor 21 ligand, CpG ODN 2007, and an oil emulsion adjuvant, AddaVax™, as adjuvants for the vaccines. Results Antibody-mediated immune responses were observed after administering the secondary intramuscular vaccine. Cell-mediated immune responses were observed in chickens that received the beta-propiolactone inactivated H9N2 virus combined with AddaVax™. Our results demonstrate that adaptive immune responses can be induced in chickens after a primary in ovo vaccination and secondary intramuscular vaccination.