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.

Treatment of chickens with lactobacilli prior to challenge with Clostridium perfringens modifies innate responses and gut morphology.

Necrotic enteritis caused by Clostridium perfringens (CP), is a common enteric disease of poultry that has been previously controlled by in-feed antibiotics. However, due to the rapid emergence of antimicrobial resistance, alternatives to antibiotics such as probiotics have received considerable attention because of their immunomodulatory and intestinal health benefits. The present study investigated the effects of probiotic lactobacilli on gut histomorphology and intestinal innate responses in chickens. Day-old male broiler chickens were treated with 1 × 107 or 1 × 108 colony-forming units (CFU) of a lactobacilli cocktail on days 1, 7, 14, and 20 post-hatch, while control groups were not treated with lactobacilli. On day 21, birds in all groups (except the negative control) were challenged with 3 × 108 CFU of CP for 3 days. Intestinal tissue samples were collected before and after the CP challenge to assess gene expression and for histomorphological analysis. Lactobacilli treatment at a dose of 1 × 108 CFU conferred partial protection against NE by lowering lesion scores, increasing villus height in the ileum and reducing crypt depth in the jejunum. In addition, 1 × 108 CFU of lactobacilli enhanced the expression of Toll-like receptor (TLR) 2, interferon-gamma (IFN-γ), interleukin (IL)-10, IL-12, and IL-13 in both the jejunum and ileum at different timepoints and subsequently decreased the expression of transforming growth factor beta (TGF-ß) and IL-1ß post-CP challenge. In conclusion, the results indicate that treatment with lactobacilli mitigated NE in a dose-dependent manner via improvement of intestinal morphology and modulation of innate immune response in chickens.

Efficacy of an inactivated influenza vaccine adjuvanted with Toll-like receptor ligands against transmission of H9N2 avian influenza virus in chickens.

Avian influenza viruses (AIV), including the H9N2 subtype, pose a major threat to the poultry industry as well as to human health. Although vaccination provides a protective control measure, its effect on transmission remains uncertain in chickens. The objective of the present study was to investigate the efficacy of beta-propiolactone (BPL) whole inactivated H9N2 virus (WIV) vaccine either alone or in combination with CpG ODN 2007 (CpG), poly(I:C) or AddaVax™ (ADD) to prevent H9N2 AIV transmission in chickens. The seeder chickens (trial 1) and recipient chickens (trial 2) were vaccinated twice with different vaccine formulations. Ten days after secondary vaccination, seeder chickens were infected with H9N2 AIV (trial 1) and co-housed with healthy recipient chickens. In trial 2, the recipient chickens were vaccinated and then exposed to H9N2 AIV-infected seeder chickens. Our results demonstrated that BPL+ CpG and BPL+ poly(I:C) treated chickens exhibited reduced oral and cloacal shedding in both trials post-exposure (PE). The number of H9N2 AIV+ recipient chickens in the BPL+ CpG group (trial 1) was lower than in other vaccinated groups, and the reduction was higher in BPL+ CpG recipient chickens in trial 2. BPL+ CpG vaccinated chickens demonstrated enhanced systemic antibody responses with high IgM and IgY titers with higher rates of seroprotection by day 21 post-primary vaccination (ppv). Additionally, the induction of IFN-γ expression and production was higher in the BPL+ CpG treated chickens. Interleukin (IL)- 2 expression was upregulated in both BPL+ CpG and BPL+ poly(I:C) groups at 12 and 24 hr post-stimulation.

In ovo feeding of probiotic lactobacilli differentially alters expression of genes involved in the development and immunological maturation of bursa of Fabricius in pre-hatched chicks.

Compelling evidence indicates that immunological maturation of the gut-associated lymphoid tissues, including the bursa of Fabricius, is dependent upon antigenic stimulation post-hatch. In view of these data, the present study investigated the impact of exposing the immune system of chick embryos to antigenic stimuli, via in ovo delivery of poultry-specific lactobacilli, on the expression of genes associated with early bursal development and maturation. Broiler line embryonated eggs were inoculated with 106 and 107 colony-forming units (CFUs) of an individual or a mixture of Lactobacillus species, including L. crispatus (C25), L. animalis (P38), L. acidophilus (P42), and L. reuteri (P43), at embryonic day 18 (ED18). The bursa of Fabricius was collected from pre-hatched chicks (ED20) to measure the expression levels of various immune system genes. The results revealed that L. acidophilus and the mixture of Lactobacillus species at the dose of 106 CFU consistently elicited higher expression of genes responsible for B cell development, differentiation, and survival (B cell activating factor (BAFF), BAFF-receptor (BAFF-R)), and antibody production (interleukin (IL)-10) and diversification (TGF-ß). Similar expression patterns were also noted in T helper (Th) cell-associated cytokine genes, including Th1-type cytokines (interferon (IFN)-γ and IL-12p40), Th2-type cytokines (IL-4 and IL-13) and Th17 cytokine (IL-17). Overall, these results suggest that the supplementation of poultry-specific lactobacilli to chick embryos might be beneficial for accelerating the development and immunological maturation of the bursa of Fabricius. However, further studies are required to determine if the changes in gene expression are associated with the developmental trajectory and phenotypes of bursal cells.

Efficacy and tolerability of an mRNA vaccine expressing gB and pp38 antigens of Marek's disease virus in chickens.

Marek's disease is a contagious proliferative disease of chickens caused by an alphaherpesvirus called Marek's disease virus. A bivalent mRNA vaccine encoding MDV's glycoprotein-B and phosphoprotein-38 antigens was synthesized and encapsulated in lipid nanoparticles. Tumor incidence, lesion score, organ weight indices, MDV genome load and cytokine expression were used to evaluate protection and immunostimulatory effects of the tested mRNA vaccine after two challenge trials. Results from the first trial showed decreased tumor incidence and a reduction in average lesion scores in chickens that received the booster dose. The second trial demonstrated that vaccination with the higher dose of the vaccine (10 µg) significantly decreased tumor incidence, average lesion scores, bursal atrophy, and MDV load in feather tips when compared to the controls. Changes in expression of type I and II interferons suggested a possible role for these cytokines in initiation and maintenance of the vaccine-originated immune responses.

In ovo administration of retinoic acid enhances cell-mediated immune responses against an inactivated H9N2 avian influenza virus vaccine.

The H9N2 subtype avian influenza virus (AIV) is a low pathogenic AIV that infects avian species and lead to huge economical losses in the poultry industry. The unique immunomodulatory properties of Retinoic acid (RA), an active component of vitamin A, highlights its potential to enhance chicken's resistance to infectious diseases and perhaps vaccine-induced immunity. Therefore, the present study evaluated the effects of in ovo supplementation of RA on the immunogenicity and protective efficacy of an inactivated avian influenza virus vaccine. On embryonic day 18, eggs were inoculated with either 90 µmol RA/200 µL/egg or diluent into the amniotic sac. On days 7 and 21 post-hatch, birds were vaccinated with 15 µg of ß-propiolactone (BPL) inactivated H9N2 virus via the intramuscular route. One group received BPL in combination with an adjuvant, while the other group received saline solution and served as a non-vaccinated control group. Serum samples were collected on days 7, 14, 21, 28, 35, and 42 post-primary vaccination (ppv) for antibody analysis. On day 24 ppv, spleens were collected, and splenocytes were isolated to analyze cytokine expression, interferon gamma (IFN-γ) production, and cell population. On day 28 ppv, birds in all groups were infected with H9N2 virus and oral and cloacal swabs were collected for TCID50 (50 % Tissue Culture Infectious Dose) assay up to day 7 post-infection. The results demonstrated that in ovo administration of RA did not significantly enhance the AIV vaccine-induced antibody response against H9N2 virus compared to the group that received the vaccine alone. However, RA supplementation enhanced the frequency of macrophages (KUL01+), expression of inflammatory cytokines and production of IFN-γ by splenocytes. In addition, RA administration reduced oral shedding of AIV on day 5 post-infection. In conclusion, these findings suggest that RA can be supplemented in ovo to enhance AIV vaccine efficacy against LPAIV.

Molecular and cellular characterization of immunity conferred by lactobacilli against necrotic enteritis in chickens.

Necrotic enteritis is an important enteric disease of poultry that can be controlled with in-feed antibiotics. However, with the concerns over antimicrobial resistance, there is an increased interest in the use of alternatives. Probiotics are one of the alternatives that have gained considerable attention due to their antimicrobial and immunomodulatory activities. Therefore, in the present study, we evaluated the effects of two different Lactobacillus species alone or as a cocktail on prevention of necrotic enteritis. Day-old male broiler chickens were divided into five groups and on days 1, 8, 15, and 22, birds in groups 2 and 3 received 1×108 colony forming units (CFU) of L. johnsonii and L. reuteri, respectively. Group 4 received probiotic cocktails containing both bacteria (108 CFU/bird) and the negative and positive control groups did not receive any lactobacilli. Starting on day 23 post-hatch, birds in all groups (except the negative control group) were orally challenged twice per day with 3×108 CFU of a pathogenic C. perfringens strain for 3 days. Tissue and cecal samples were collected before and after challenge to assess gene expression, lymphocyte subsets determination, and microbiome analysis. On day 26 of age, lesion scoring was performed. The results demonstrated that the group that received the lactobacilli cocktail had significantly reduced lesion scores compared to the positive control group. In addition, the expression of interleukin (IL)-12 in the jejunum and CXC motif chemokine ligand 8 (CXCL8), IL-13, and IL-17 in the ileum were downregulated in the group that received the lactobacilli cocktail when compared to the positive control. Treating chickens with the lactobacilli cocktail prior to challenge enhanced the percentage of CD3-CD8+ cells and Bu-1+IgY+ B cells in the ileum and increased the frequency of monocyte/macrophages, CD3-CD8+ cells, Bu-1+IgM+, and Bu-1+IgY+ B cells in the jejunum. Treatment with the lactobacilli cocktail reduced the relative expression of Gamma-Protobacteria and Firmicutes compared to the positive control group. In conclusion, the results presented here suggest that treatment with the lactobacilli cocktail containing L. johnsonii and L. reuteri reduced necrotic enteritis lesions in the small intestine of chickens, possibly through the modulation of immune responses.

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.].

The tumor microenvironment generated by Marek's disease virus suppresses interferon-gamma-producing gamma delta T cells.

The tumor microenvironment (TME) is generated by the cross-talk among tumor cells, immune system cells, and stromal cells. The TME generated by Marek's disease virus (MDV) is suggested to display an immunosuppressive milieu due to immune inhibitory molecules and cytokines which are possibly induced by MDV-transformed cells and regulatory T cells. Both anti-tumor and pro-tumor gamma delta (γδ) T cells are reported in human cancer. Although anti-tumor like and pro-tumor like γδ T cells are found in MDV-infected chickens at the later phase of infection, how the TME affects circulating and tissue-resident γδ T cells has not been investigated. Here, we demonstrated that the supernatant of the cultured splenocytes derived from MDV-challenegd chickens inhibited interferon (IFN)-γ production and CD25 expression by T cell receptor (TCR)γδ-stimulated tissue-resident γδ T cells, but the supernatant of the cultured MDV-transformed cell line did not affect γδ T cell activation. TCRγδ-stimulated circulating γδ T cells were influenced neither by the supernatant of the cultured splenocytes derived from MDV-challenegd chickens nor by the supernatant of the cultured MDV-transformed cell line. Taken together, activation and IFN-γ production by tissue-resident γδ T cells can be inhibited in the TME generated by MDV while tumor attracted circulating γδ T cells may not be influenced in activation and IFN-γ production by the TME generated by MDV.

Effect of Pre-Treatment with a Recombinant Chicken Interleukin-17A on Vaccine Induced Immunity against a Very Virulent Marek's Disease Virus.

The host response to pathogenic microbes can lead to expression of interleukin (IL)-17, which has antimicrobial and anti-viral activity. However, relatively little is known about the basic biological role of chicken IL-17A against avian viruses, particularly against Marek's disease virus (MDV). We demonstrate that, following MDV infection, upregulation of IL-17A mRNA and an increase in the frequency of IL-17A+ T cells in the spleen occur compared to control chickens. To elaborate on the role of chIL-17A in MD, the full-length chIL-17A coding sequence was cloned into a pCDNA3.1-V5/HIS TOPO plasmid. The effect of treatment with pcDNA:chIL-17A plasmid in combination with a vaccine (HVT) and very virulent(vv)MDV challenge or vvMDV infection was assessed. In combination with HVT vaccination, chickens that were inoculated with the pcDNA:chIL-17A plasmid had reduced tumor incidence compared to chickens that received the empty vector control or that were vaccinated only (66.6% in the HVT + empty vector group and 73.33% in HVT group versus 53.3% in the HVT + pcDNA:chIL-17A). Further analysis demonstrated that the chickens that received the HVT vaccine and/or plasmid expressing IL-17A had lower MDV-Meq transcripts in the spleen. In conclusion, chIL-17A can influence the immunity conferred by HVT vaccination against MDV infection in chickens.

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.].

Cellular Immune Responses in Lymphoid Tissues of Broiler Chickens Experimentally Infected with Necrotic Enteritis-Producing Clostridium perfringens Strains.

Host cellular responses against Clostridium perfringens (CP), the causative agent of necrotic enteritis (NE) in chickens, are poorly understood. In the present study, we first tested the NE-producing ability of seven netB+ CP strains (CP5, CP18, CP26, CP64, CP67, CP68, and NCNE-1), using an experimental infection model of broiler chickens. Evaluation of intestinal gross lesions showed that all the strains, except CP5, were able to produce NE, while CP26 and CP64 strains produced relatively more severe lesions when compared with other groups. Next, cellular responses in the cecal tonsil (CT), bursa of Fabricius, and spleen were evaluated in chickens infected with strains representing variation in the level of virulence, namely, avirulent CP5, virulent CP18, and a relatively more virulent CP26 strain. Immunophenotyping analysis showed that CT or splenic macrophage frequencies were significantly higher in CP18- and CP26-infected chickens compared with uninfected controls, while the frequencies of γδ T-cells and B-cells in the CT of CP26-infected chickens were significantly higher than those in the uninfected, CP5- or CP18-infected groups. The T-cell analysis showed that chickens infected with CP18 and CP26 had a significantly higher number of splenic CD4+ and CD8+ T-cells expressing CD44 and CD28 activation molecules, while CP26-infected chickens also had significantly increased CT frequency of these activated CD4+ and CD8+ T-cells when compared with uninfected or CP5-infected groups. Collectively, our findings suggested that cellular responses, including activation of T-cells, are selectively induced against virulent CP strains and that the NE-producing characteristics of this pathogen may influence the outcome of immunity to NE.

Respuestas inmunes celulares en tejidos linfoides de pollos de engorde infectados experimentalmente con cepas de Clostridium perfringens productoras de enteritis necrótica. Las respuestas celulares del huésped contra Clostridium perfringens (CP), el agente causante de la enteritis necrótica (NE) en pollos, son poco conocidas. En el presente estudio, primero se analizó la capacidad de producción de enteritis necrótica de siete cepas de C. perfringens netB+ (CP5, CP18, CP26, CP64, CP67, CP68 y NCNE-1), utilizando un modelo de infección experimental de pollos de engorde. La evaluación de las lesiones macroscópicas intestinales mostró que todas las cepas, excepto CP5, podían producir enteritis necrótica, mientras que las cepas CP26 y CP64 produjeron lesiones relativamente más severas en comparación con los otros grupos. Posteriormente, se evaluaron las respuestas celulares en las tonsilas cecales (CT), la bolsa de Fabricio y en el bazo de pollos infectados con cepas que representan variaciones en el nivel de virulencia, por ejemplo las cepas CP5 avirulenta, CP18 virulenta y la cepa CP26 relativamente más virulenta. El análisis de inmunofenotipado mostró que las frecuencias de los macrófagos esplénicos o de las tonsilas cecales fueron significativamente más altas en los pollos infectados con las cepas CP18 y CP26 en comparación con los controles no infectados, mientras que las frecuencias de células T γd y células B en tonsilas cecales de los pollos infectados con la cepa CP26 fueron significativamente más altas que las de los pollos de los grupos no infectados, o infectados con las cepas CP5 o CP18. El análisis de células T mostró que los pollos infectados con las cepas CP18 y CP26 tenían un número significativamente mayor de células esplénicas T CD4+ y CD8+ que expresaban moléculas de activación CD44 y CD28, mientras que los pollos infectados con la cepa CP26 también tenían una frecuencia significativamente mayor en las tonsilas cecales de estas células T CD4+ y CD8+ activadas en comparación con grupos no infectados o infectados con la cepa CP5. En conjunto, estos hallazgos sugirieron que las respuestas celulares, incluida la activación de las células T, se inducen selectivamente contra las cepas virulentas de C. perfringens y que las características productoras de enteritis necrótica de este patógeno pueden influir en el resultado de la inmunidad contra la enteritis necrótica.

Credibility of vaccine-related content on Twitter during COVID-19 pandemic.

During national COVID-19 vaccine campaigns, people continuously engaged on Twitter to receive updates on the latest public health information, and to discuss and share their experiences. During this time, the spread of misinformation was widespread, which threatened the uptake of vaccines. It is therefore critical to understand the reasons behind vaccine misinformation and strategies to mitigate it. The current research aimed to understand the content of misleading tweets and the characteristics of their corresponding accounts. We performed a machine learning approach to identify misinformation in vaccine-related tweets, and calculated the demographic, engagement metrics and bot-like activities of corresponding accounts. We found critical periods where high amounts of misinformation coincided with important vaccine announcements, such as emergency approvals of vaccines. Moreover, we found Asian countries had a lower percentage of misinformation shared compared to Europe and North America. Our results showed accounts spreading misinformation had an overall 10% greater likelihood of bot activity and 15% more astroturf bot activity than accounts spreading accurate information. Furthermore, we found that accounts spreading misinformation had five times fewer followers and three times fewer verified badges than fact-sharing accounts. The findings of this study may help authorities to develop strategies to fight COVID-19 vaccine misinformation and improve vaccine uptake.

Predictive analysis for pathogenicity classification of H5Nx avian influenza strains using machine learning techniques.

Over the past decades, avian influenza (AI) outbreaks have been reported across different parts of the globe, resulting in large-scale economic and livestock loss and, in some cases raising concerns about their zoonotic potential. The virulence and pathogenicity of H5Nx (e.g., H5N1, H5N2) AI strains for poultry could be inferred through various approaches, and it has been frequently performed by detecting certain pathogenicity markers in their haemagglutinin (HA) gene. The utilization of predictive modeling methods represents a possible approach to exploring this genotypic-phenotypic relationship for assisting experts in determining the pathogenicity of circulating AI viruses. Therefore, the main objective of this study was to evaluate the predictive performance of different machine learning (ML) techniques for in-silico prediction of pathogenicity of H5Nx viruses in poultry, using complete genetic sequences of the HA gene. We annotated 2137 H5Nx HA gene sequences based on the presence of the polybasic HA cleavage site (HACS) with 46.33% and 53.67% of sequences previously identified as highly pathogenic (HP) and low pathogenic (LP), respectively. We compared the performance of different ML classifiers (e.g., logistic regression (LR) with the lasso and ridge regularization, random forest (RF), K-nearest neighbor (KNN), Naïve Bayes (NB), support vector machine (SVM), and convolutional neural network (CNN)) for pathogenicity classification of raw H5Nx nucleotide and protein sequences using a 10-fold cross-validation technique. We found that different ML techniques can be successfully used for the pathogenicity classification of H5 sequences with ∼99% classification accuracy. Our results indicate that for pathogenicity classification of (1) aligned deoxyribonucleic acid (DNA) and protein sequences, with NB classifier had the lowest accuracies of 98.41% (+/-0.89) and 98.31% (+/-1.06), respectively; (2) aligned DNA and protein sequences, with LR (L1/L2), KNN, SVM (radial basis function (RBF)) and CNN classifiers had the highest accuracies of 99.20% (+/-0.54) and 99.20% (+/-0.38), respectively; (3) unaligned DNA and protein sequences, with CNN's achieved accuracies of 98.54% (+/-0.68) and 99.20% (+/-0.50), respectively. ML methods show potential for regular classification of H5Nx virus pathogenicity for poultry species, particularly when sequences containing regular markers were frequently present in the training dataset.

Treatment with Toll-like Receptor (TLR) Ligands 3 and 21 Prevents Fecal Contact Transmission of Low Pathogenic H9N2 Avian Influenza Virus (AIV) in Chickens.

Transmission of H9N2 avian influenza virus (AIV) can occur in poultry by direct or indirect contact with infected individuals, aerosols, large droplets and fomites. The current study investigated the potential of H9N2 AIV transmission in chickens via a fecal route. Transmission was monitored by exposing naïve chickens to fecal material from H9N2 AIV-infected chickens (model A) and experimentally spiked feces (model B). The control chickens received H9N2 AIV. Results revealed that H9N2 AIV could persist in feces for up to 60-84 h post-exposure (PE). The H9N2 AIV titers in feces were higher at a basic to neutral pH. A higher virus shedding was observed in the exposed chickens of model B compared to model A. We further addressed the efficacy of Toll-like receptor (TLR) ligands to limit transmission in the fecal model. Administration of CpG ODN 2007 or poly(I:C) alone or in combination led to an overall decrease in the virus shedding, with enhanced expression of type I and II interferons (IFNs) and interferon-stimulating genes (ISGs) in different segments of the small intestine. Overall, the study highlighted that the H9N2 AIV can survive in feces and transmit to healthy naïve chickens. Moreover, TLR ligands could be applied to transmission studies to enhance antiviral immunity and reduce H9N2 AIV shedding.

Intracloacal Inoculation of Broiler Chickens with Clostridium perfringens Strains: Evaluation of Necrotic Enteritis Disease Development and Lymphoid Immune Responses.

Necrotic enteritis (NE) is an economically important disease of chickens. We have recently shown that inflammatory responses in chickens inoculated orally with virulent Clostridium perfringens were spatially regulated. Here, we used previously virulence-characterized netB+C. perfringens strains, avirulent CP5 and virulent CP18 and CP26, to assess the severity of NE and immune responses in broiler chickens when inoculated intracloacally. The results showed that CP18- and CP26-infected birds had a reduced weight gain and developed milder/less severe NE lesions, as determined by the gross lesions scores, suggesting a subclinical-grade infection. Gene expression analysis in infected birds revealed three statistically significant observations compared to uninfected-control: (1) Increased expression of anti-inflammatory/immunoregulatory interleukin (IL)-10/transforming growth factor (TGF)ß in cecal tonsil (CT) and bursa of Fabricius in the CP18/CP26-infected groups. (2) Increased CT transcription of pro-inflammatory IL-1ß, IL-6 and interferon (IFN)γ and decreased Harderian gland (HG) expression of IFNγ in the CP18/CP26-infected birds. (3) Increased HG or bursal expression of IL-4 and IL-13 in CP5-infected birds. Collectively, intracloacal C. perfringens inoculation seems to induce a highly regulated inflammatory response in the CT and other mucosal lymphoid organs and an intracloacal infection model may be useful in evaluating immune responses in chickens with subclinical NE.

Activated Chicken Gamma Delta T Cells Are Involved in Protective Immunity against Marek's Disease.

Gamma delta (γδ) T cells play a significant role in the prevention of viral infection and tumor surveillance in mammals. Although the involvement of γδ T cells in Marek's disease virus (MDV) infection has been suggested, their detailed contribution to immunity against MDV or the progression of Marek's disease (MD) remains unknown. In the current study, T cell receptor (TCR)γδ-activated peripheral blood mononuclear cells (PBMCs) were infused into recipient chickens and their effects were examined in the context of tumor formation by MDV and immunity against MDV. We demonstrated that the adoptive transfer of TCRγδ-activated PBMCs reduced virus replication in the lungs and tumor incidence in MDV-challenged chickens. Infusion of TCRγδ-activated PBMCs induced IFN-γ-producing γδ T cells at 10 days post-infection (dpi), and degranulation activity in circulating γδ T cell and CD8α+ γδ T cells at 10 and 21 dpi in MDV-challenged chickens. Additionally, the upregulation of IFN-γ and granzyme A gene expression at 10 dpi was significant in the spleen of the TCRγδ-activated PBMCs-infused and MDV-challenged group compared to the control group. Taken together, our results revealed that TCRγδ stimulation promotes the effector function of chicken γδ T cells, and these effector γδ T cells may be involved in protection against MD.

Intervention Strategies to Control Campylobacter at Different Stages of the Food Chain.

Campylobacter is one of the most common bacterial pathogens of food safety concern. Campylobacter jejuni infects chickens by 2-3 weeks of age and colonized chickens carry a high C. jejuni load in their gut without developing clinical disease. Contamination of meat products by gut contents is difficult to prevent because of the high numbers of C. jejuni in the gut, and the large percentage of birds infected. Therefore, effective intervention strategies to limit human infections of C. jejuni should prioritize the control of pathogen transmission along the food supply chain. To this end, there have been ongoing efforts to develop innovative ways to control foodborne pathogens in poultry to meet the growing customers' demand for poultry meat that is free of foodborne pathogens. In this review, we discuss various approaches that are being undertaken to reduce Campylobacter load in live chickens (pre-harvest) and in carcasses (post-harvest). We also provide some insights into optimization of these approaches, which could potentially help improve the pre- and post-harvest practices for better control of Campylobacter.

Determining the Protective Efficacy of Toll-Like Receptor Ligands to Minimize H9N2 Avian Influenza Virus Transmission in Chickens.

Low-pathogenicity avian influenza viruses (AIV) of the H9N2 subtype can infect and cause disease in chickens. Little is known about the efficacy of immune-based strategies for reducing the transmission of these viruses. The present study investigated the efficacy of Toll-like receptor (TLR) ligands (CpG ODN 2007 and poly(I:C)) to reduce H9N2 AIV transmission from TLR-treated seeder (trial 1) or inoculated chickens (trial 2) to naive chickens. The results from trial 1 revealed that a low dose of CpG ODN 2007 led to the highest reduction in oral shedding, and a high dose of poly(I:C) was effective at reducing oral and cloacal shedding. Regarding transmission, the recipient chickens exposed to CpG ODN 2007 low-dose-treated seeder chickens showed a maximum reduction in shedding with the lowest number of AIV+ chickens. The results from trial 2 revealed a maximum reduction in oral and cloacal shedding in the poly(I:C) high-dose-treated chickens (recipients), followed by the low-dose CpG ODN 2007 group. In these two groups, the expression of type I interferons (IFNs), protein kinase R (PKR), interferon-induced transmembrane protein 3 (IFITM3), viperin, and (interleukin) IL-1ß, IL-8, and 1L-18 was upregulated in the spleen, cecal tonsils and lungs. Hence, TLR ligands can reduce AIV transmission in chickens.