Intestinal microbiome profiles in broiler chickens raised without antibiotics exhibit altered microbiome dynamics relative to conventionally raised chickens.

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.

Corrigendum: Effects of in ovo inoculation of multi-strain lactobacilli on cytokine gene expression and antibody-mediated immune responses in chickens.

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

Corrigendum: In ovo and oral administration of probiotic lactobacilli modulate cell- and antibody-mediated immune responses in newly hatched chicks.

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

Effect of treatment with Lactococcus lactis NZ9000 on intestinal microbiota and mucosal immune responses against Clostridium perfringens in broiler chickens.

Alterations in intestinal microbiota can modulate the developing avian intestinal immune system and, subsequently, may impact on resistance to enteric pathogens. The aim was to demonstrate that early life exposure to Lactococcus lactis, could affect either susceptibility or resistance of broilers to necrotic enteritis (NE). L. lactis NZ9000 (rL. lactis) pre-treatment at 1, 7, 14 and 21 days of age (DOA) led to a significant decrease in NE lesion scores in Clostridium perfringens infected chickens. C. perfringens Infection was associated with spatial and temporal decreases in mononuclear phagocytes and CD4+ αß T cells. However, rL. Lactis pre-treatment and subsequent C. perfringens infection led to a significant increase in mononuclear phagocytes, CD8α + γδ T, αß T cells (CD4+ and CD8α+) and B cells (IgM+, IgA+ and IgY+), as well as IL-12p40, IFN-γ and CD40. Differential expression of interleukin (IL)-6, IL-8, IL-10, IL-13, IL-18, IL-22, and transforming growth factor (TGF)-ß were observed in L. lactis treated chickens when compared to C. perfringens infected chickens. Microbiota analysis in C. perfringens infected chickens demonstrated an increase in abundance of Bacillota, Bacteroidota, Pseudomonadota and Actinomycetota. These findings suggests that modulation of the chicken intestinal immune system by L. lactis confers partial protection 30 against NE.

Transmission of H9N2 Low Pathogenicity Avian Influenza Virus (LPAIV) in a Challenge-Transmission Model.

Migratory birds are major reservoirs for avian influenza viruses (AIV), which can be transmitted to poultry and mammals. The H9N2 subtype of AIV has become prevalent in poultry over the last two decades. Despite that, there is a scarcity of detailed information on how this virus can be transmitted. The current study aimed to establish a direct contact model using seeder chickens infected with H9N2 AIV as a source of the virus for transmission to recipient chickens. Seeder chickens were inoculated with two different inoculation routes either directly or via the aerosol route. The results indicate that inoculation via the aerosol route was more effective at establishing infection compared to the direct inoculation route. Shedding was observed to be higher in aerosol-inoculated seeder chickens, with a greater percentage of chickens being infected at each time point. In terms of transmission, the recipient chickens exposed to the aerosol-inoculated seeder chickens had higher oral and cloacal virus shedding compared to the recipient chickens of the directly inoculated group. Furthermore, the aerosol route of infection resulted in enhanced antibody responses in both seeder and recipient chickens compared to the directly inoculated group. Overall, the results confirmed that the aerosol route is a preferred inoculation route for infecting seeder chickens in a direct contact transmission model.

Effects of therapeutic levels of dietary antibiotics on the cecal microbiome composition of broiler chickens.

Dietary antibiotics, including antibiotic growth promoters (AGPs), have been commonly used to improve health and growth of poultry. The present study investigated the effects of therapeutic doses of dietary antibiotics, including bacitracin methylene disalicylate (BMD), penicillin G potassium (PP) and an ionophore (salinomycin, SA), on the cecal microbiome of chickens. BMD and SA treatments were given as dietary supplements from d 1 to 35 of age. The SAPP (salinomycin+ penicillin G potassium) group was given SA as a dietary supplement from d 1 to 35 of age and PP was added to drinking water from d 19 to 24 of age to simulate common practices for control of necrotic enteritis in broilers. The cecal contents were collected from all treatment groups on d 10, 24, and 35 of age and DNA was extracted for metagenomic analysis of the cecal microbiome. The results revealed that dietary or water supplementation of therapeutic levels of antibiotics and ionophores to chickens significantly altered the cecal microbial homeostasis during different stages of the chicken life. The alpha diversity analysis showed that BMD, SA, and SAPP treatments decreased diversity and evenness of the cecal microbiome of treated chickens on d 10 of age. Species richness was also reduced on d 35 following treatment with BMD. Beta diversity analyses revealed that SAPP and BMD induced significant changes in the relative abundance of Gram-positive and -negative bacteria on d 10, while no significant differences were observed on d 24. On d 35, the non-treated control group had higher relative abundance of unclassified Gram-positive and -negative bacteria compared to SA, SAPP, and BMD treatment groups. Overall, despite their beneficial role in controlling necrotic enteritis outbreaks, the findings of this study highlight the potential negative effects of dietary supplementation of therapeutic levels of antibiotics on the gut microbiome and suggest that adjusting gut bacteria may be required to restore microbial richness and diversity of the gut microbiome following treatment with these antibiotics.

Strategies for enhancing immunity against avian influenza virus in chickens: a review.

Poultry infection with avian influenza viruses (AIV) is a continuous source of concern for poultry production and human health. Uncontrolled infection and transmission of AIV in poultry increase the potential for viral mutation and reassortment, possibly resulting in the emergence of zoonotic viruses. To this end, implementing strategies to disrupt the transmission of AIV in poultry, including a wide array of traditional and novel methods, is much needed. Vaccination of poultry is a targeted approach to reduce clinical signs and shedding in infected birds. Strategies aimed at enhancing the effectiveness of AIV vaccines are multi-pronged and include methods directed towards eliciting immune responses in poultry. Strategies include producing vaccines of greater immunogenicity via vaccine type and adjuvant application, and increasing bird responsiveness to vaccines by modification of the gastrointestinal tract (GIT) microbiome and dietary interventions. This review provides an in-depth discussion of recent findings surrounding novel AIV vaccines for poultry, including reverse genetics vaccines, vectors, protein vaccines and virus-like particles, highlighting their experimental efficacy among other factors such as safety and potential for use in the field. In addition to the type of vaccine employed, vaccine adjuvants also provide an effective way to enhance AIV vaccine efficacy; therefore, research on different types of vaccine adjuvants and vaccine adjuvant delivery strategies is discussed. Finally, the poultry gastrointestinal microbiome is emerging as an important factor in the effectiveness of prophylactic treatments. In this regard, current findings on the effects of the chicken GIT microbiome on AIV vaccine efficacy are summarized here.

In ovo co-administration of vitamins (A and D) and probiotic lactobacilli modulates immune responses in broiler chickens.

There is evidence that probiotic lactobacilli, in addition to essential vitamins, such as vitamin A and D, have immunomodulatory properties that enhance immune response of neonatal chickens against infections. The present study evaluated the effects of in ovo administration of retinoic acid (RA), 25-Hydroxyvitamin D3 (VitD), and a lactobacilli cocktail on cytokine gene expression, antibody responses and spleen cell subsets in chickens. RA (90 µmol/egg) and VitD (0.6 µg/egg) were administered in ovo, either alone or in combination with lactobacilli (107 CFU/egg), at embryonic d 18. On d 5 and 10 posthatch, gene expression and cellular composition were analyzed in the bursa of Fabricius and spleen. Birds were immunized on d 14 and 21 posthatch with 2 T-dependent antigens, sheep red blood cells (SRBC) and keyhole limpet hemocyanin (KLH), to assess their antibody responses. Sera were collected from the immunized chickens on d 14, 21, 28, and 35 posthatch. The results demonstrated that lactobacilli treatment increased the number of monocyte/macrophages (KUL01+) and CD3+CD4+ T cells in the spleen, and enhanced serum anti-KLH IgM and IgY on d 14 postprimary immunization (P < 0.05). RA significantly increased serum IgY and IgM titers to KLH and enhanced the expression of interferon (IFN)-α, interleukin (IL)-1ß, IL-6, IL-8, IL-12, IL-13, and transforming growth factor-ß (TGF-ß) in the bursa of Fabricius (P < 0.05). The percentage of CD3+CD8+ T cells, and monocyte/macrophages (KUL01+) was elevated in the spleen as well (P < 0.05). These findings reveal that prehatch administration of RA improves immunocompetency of neonatal chickens by increasing the production of cytokines that regulate innate immunity and through enhancing antibody-mediated response against T-dependent antigens.

The Regulatory Microenvironment in Feathers of Chickens Infected with Very Virulent Marek's Disease Virus.

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-ß)+ 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-ß 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.

Effects of Treatment with Lactobacilli on Necrotic Enteritis in Broiler Chickens.

Growth promoter antibiotics have been commonly used for the control of necrotic enteritis (NE) in broilers for decades. However, due to a ban on the use of these antibiotics, alternatives such as probiotics have been tested widely for NE control. The present study tested the efficacy of four different species of lactobacilli (two isolates of Lactobacillus johnsonii and one of Ligilactobacillus (L.) salivarius, Limosilactobacillus (L.) reuteri, and L. crispatus) against NE. Day-old male broiler chickens were divided into six groups and orally inoculated with 1 × 107 or 1 × 108 colony-forming units (CFU) of lactobacilli on 1, 7, 14, and 20 days of age. While negative and positive control groups did not receive lactobacilli, the latter was challenged with Clostridium perfringens (CP). Chickens, at 21 days old, were challenged for 3 days with 3 × 108 CFU of a virulent strain of CP. Tissues were collected for immune system gene expression, immunophenotyping, intestinal histomorphometry, and microbiota analysis. Lactobacilli inoculation conferred partial protection in chickens against NE, marked by lowered lesion scores and improved villus:crypt ratio. Immunomodulatory effects were demonstrated by the significant alteration of interferon (IFN)-γ, interleukin (IL)-1ß, IL-2, IL-12p35, IL-17, and transforming growth factor beta (TGF-ß) gene transcription in the duodenum and jejunum as well as subtle changes in the frequency of CD8 + T cells and B cells in the cecal tonsil of the treated chickens. Microbiota analysis showed increased levels of some bacterial phyla including Actinobacteria, Lactobacillaceae, and Firmicutes. In conclusion, these findings suggest that the use of certain lactobacilli can reduce NE severity and modulate immune responses and intestinal microbiota composition in chickens.

In Ovo and Oral Administration of Probiotic Lactobacilli Modulate Cell- and Antibody-Mediated Immune Responses in Newly Hatched Chicks.

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 (107 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-ß, 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.

Within-host model of respiratory virus shedding and antibody response to H9N2 avian influenza virus vaccination and infection in chickens.

Avian influenza virus (AIV) H9N2 subtype is an infectious pathogen that can affect both the respiratory and gastrointestinal systems in chickens and continues to have an important economic impact on the poultry industry. While the host innate immune response provides control of virus replication in early infection, the adaptive immune response aids to clear infections and prevent future invasion. Modelling virus-innate immune response pathways can improve our understanding of early infection dynamics and help to guide our understanding of virus shedding dynamics that could lead to reduced transmission between hosts. While some countries use vaccines for the prevention of H9N2 AIV in poultry, the virus continues to be endemic in regions of Eurasia and Africa, indicating a need for improved vaccine efficacy or vaccination strategies. Here we explored how three type-I interferon (IFN) pathways affect respiratory virus shedding patterns in infected chickens using a within-host model. Additionally, prime and boost vaccination strategies for a candidate H9N2 AIV vaccine are assessed for the ability to elicit seroprotective antibody titres. The model demonstrates that inclusion of virus sensitivity to intracellular type-I IFN pathways results in a shedding pattern most consistent with virus titres observed in infected chickens, and the inclusion of a cellular latent period does not improve model fit. Furthermore, early administration of a booster dose two weeks after the initial vaccine is administered results in seroprotective titres for the greatest length of time for both broilers and layers. These results demonstrate that type-I IFN intracellular mechanisms are required in a model of respiratory virus shedding in H9N2 AIV infected chickens, and also highlights the need for improved vaccination strategies for laying hens.

Effects of administration of probiotic lactobacilli on immunity conferred by the herpesvirus of turkeys vaccine against challenge with a very virulent Marek's disease virus in chickens.

Several vaccines have been used to control Marek's disease (MD) in chickens. However, the emergence of new strains of Marek's disease virus (MDV) imposes a threat to vaccine efficacy. Therefore, the current study was carried out to investigate whether concurrent administration of probiotics with the herpesvirus of turkeys (HVT) vaccine enhances its protective efficacy against MDV infection. In this regard, a cocktail comprised of four Lactobacillus species was administered with HVT to chicken embryos at embryonic day 18 (ED18) and/or from day 1 to day 4 post-hatch. The results revealed that the administration of a probiotic Lactobacillus with HVT at ED18 followed by oral gavage with the same lactobacilli cocktail to newly hatched chicks for the first 4 days post-hatch increased the expression of major histocompatibility complex (MHC) II on macrophages and B cells in spleen and decreased the number of CD4+CD25+ T regulatory cells in the spleen. Subsequently, chicks were infected with MDV. The chickens that received in ovo HVT and lactobacilli or HVT had higher expression of IFN-α at 21dpi in the spleen compared to the chickens that were challenged with MDV. Also, the expression of IFN-ß in cecal tonsils at 10dpi was higher in the groups that received in ovo HVT and lactobacilli and oral lactobacilli compared to the group that received in ovo HVT alone. Moreover, the expression of tumor growth factor (TGF)-ß4 at 4 days post-infection was reduced in the group that received both HVT and probiotics at ED18. Additionally, concurrent probiotics administration reduced tumor incidence by half when compared to HVT vaccine alone indicating enhancing effect of lactobacilli with HVT vaccine on host immune responses. In conclusion, these findings suggest the potential use of probiotic lactobacilli as adjuvants with the HVT vaccine against MDV infection in chickens.

Centennial Review: Effects of vitamins A, D, E, and C on the chicken immune system.

Vitamins are nutritional elements which are necessary for essential activities such as development, growth, and metabolism of cells. In addition to these conventional functions, vitamins A, D, E, and C have vital roles in normal function of the immune system as their deficiency is known to impair innate and adaptive host responses. By altering transcription of multiple immune system genes and contributing to antioxidant activities, these vitamins influence the immune system in different ways including modulation of cell-mediated and antibody-mediated responses, immunoregulation, and antiinflammatory effects. Furthermore, supplementation of these vitamins to poultry may assist the immune system to combat microbial pathogens while reducing detrimental effects associated with stress and enhancing responses to vaccines. In this article, the relationship between the chicken immune system and vitamins A, D, E, and C is reviewed, and evidence from the literature pertaining to how these vitamins exert their antiinflammatory, regulatory, and antimicrobial effects is discussed.

In Ovo Inoculation of Vitamin A Modulates Chicken Embryo Immune Functions.

Vitamin A mediates many important biological functions in humans and animals. Presence of vitamin A receptors on immune system cells emphasizes their role in immune functions. To assess the effects of in ovo inoculation of vitamin A on the immune system of chicken embryos, 18 days old embryonated eggs were inoculated with 3 different concentrations of retinoic acid (the active form of vitamin A) at 30, 90, and 270 µmol/egg via the amniotic sac. After 6, 18, and 24 h, the spleen and bursa of the embryos were collected for RNA extraction and real-time polymerase chain reaction. The results were dose dependant. After 24 h, inoculation with 270 µmol/egg downregulated relative expression of interferon IFN-α, IFN-ß, IFN-γ, interleukin (IL)-1ß, IL-2, CXCLi2, IL-12, and IL-13 compared to control in the spleen, indicating an anti-inflammatory effect at this concentration. In comparison, 90 µmol/egg induced greater expressions of the above genes at the same timepoint compared to the 270 µmol. The results of this study indicate that in ovo inoculation of vitamin A can modulate immune functions of the chicken embryo, which might be beneficial for induction of immune responses by in ovo vaccines.

Necrotic enteritis in chickens: a review of pathogenesis, immune responses and prevention, focusing on probiotics and vaccination.

Necrotic enteritis (NE), caused by Clostridium perfringens (CP), is one of the most common of poultry diseases, causing huge economic losses to the poultry industry. This review provides an overview of the pathogenesis of NE in chickens and of the interaction of CP with the host immune system. The roles of management, nutrition, probiotics, and vaccination in reducing the incidence and severity of NE in poultry flocks are also discussed.

Gut microbiota is associated with protection against Marek's disease virus infection in chickens.

Marek's Disease Virus (MDV) infects chickens via respiratory route and causes lymphomas in internal organs including gastrointestinal tract. MDV infection causes a shift in the gut microbiota composition. However, interactions between the gut microbiota and immune responses against MDV infection are not well understood. Therefore, the current study was performed to understand the effect of the gut microbiota on Marek's disease (MD) pathogenesis. The findings showed that depletion of gut microbiota increased the severity of MD in infected chickens. In addition, an increase in the transcription of interferon (IFN)-α, IFN-ß and IFN-γ in the bursa of Fabricius at 4 days post-infection (dpi) was observed in the gut microbiota depleted chickens. The observations in this study shed more light on the association between the gut microbiota and MDV infection in chickens. More research is needed to explore the mechanisms of involvement of the gut microbiota in immunity against MD in chickens.

Probiotic Lactobacilli Enhance Immunogenicity of an Inactivated H9N2 Influenza Virus Vaccine in Chickens.

Avian influenza viruses (AIVs) infect a wide range of hosts, including humans and many avian species. Efforts have been made to control this pathogen in chickens using vaccination programs, but that has been met with varying degrees of success. Therefore, identification of more efficacious vaccination strategies is warranted. This study was undertaken to investigate the potential effects of probiotics on the immunogenicity of a beta-propiolactone-whole inactivated virus (WIV) vaccine of H9N2 subtype adjuvanted with the Toll-like receptor-21 ligand, CpG oligodeoxynucleotides 2007 (CpG). Eighty-four 1-day-old White Leghorn layers were allocated into six groups. Two out of six groups received a mixture of probiotic Lactobacillus spp. (PROB) biweekly from days 1 35 of age. Chickens were intramuscularly vaccinated with WIV either alone or adjuvanted with AddaVax™ (WIV+Add) or CpG (WIV+CpG), and one group received saline (phosphate-buffered saline). Primary and secondary vaccinations occurred at days 14 and 28 of age, respectively. The results revealed that the group that received probiotics and was vaccinated with CpG-adjuvanted WIV H9N2 vaccine had higher hemagglutination inhibition titers than the other treatment groups at days 14 and 21 postprimary vaccination. Probiotics did not induce higher IgM or IgY titers in chickens receiving the WIV vaccine only. Concerning their effect on cell-mediated immune responses, probiotics enhanced interferon-gamma (IFN-γ) gene expression and significantly increased secretion of IFN-γ protein by splenocytes in chickens vaccinated with CpG-adjuvanted WIV H9N2. Together, these findings suggest the use of probiotics to enhance the immunogenicity of CpG-adjuvanted WIV H9N2 vaccines. Additional studies are required to better understand the specific interactions between probiotics and the gut microbiota and different types of cells of the gastrointestinal tract to decipher the underlying mechanisms of how probiotics modulate immune responses to vaccines.

Campylobacter-derived ligands induce cytokine and chemokine expression in chicken macrophages and cecal tonsil mononuclear cells.

Campylobacter jejuni colonizes the chicken gut at a high density without causing disease. However, consumption of poultry products contaminated with this bacterium causes gastroenteritis in humans. Therefore, it is critically important to reduce the Campylobacter burden in poultry products to prevent transmission to humans. Evidence indicates that enhancing intestinal mucosal immune responses is of paramount importance for preventing or reducing Campylobacter colonization in chickens. In view of this, the present study was undertaken to evaluate host responses to different C. jejuni-derived ligands, including lipooligosaccharide (LOS), outer membrane proteins (OMPs), and genomic DNA, with the ultimate goal of identifying a ligand with potent immunostimulatory capacity to serve as a mucosal vaccine adjuvant against enteric infections in chickens. The results revealed that C. jejuni pathogen-associated molecular patterns (PAMPs) varied in their ability to induce the expression of cytokines and chemokines in chicken macrophages and cecal tonsil mononuclear cells and nitric oxide production in macrophages. In addition, C. jejuni OMPs demonstrated superior activity over LOS and DNA ligands in eliciting cytokine expression associated with T helper (Th)1 and Th2 responses (interferon [IFN]-γ and interleukin [IL]-13, respectively), in addition to expression of pro-inflammatory cytokines (IL-1ß), chemokine (CXCLi2), and regulatory cytokines (IL-10 and TGFß1/4) in cecal tonsil cells. Importantly, in addition to their ability to induce innate responses, OMPs could also function as antigens to elicit C. jejuni-specific antibody responses and thereby confer dual protection against C. jejuni infection. Further studies are required to assess the protective efficacy of C. jejuni OMPs against C. jejuni infection in chickens.