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.].

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.].

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.

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.

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.

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.

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.

Ex Vivo Differential Responsiveness to Clostridium perfringens and Lactococcus lactis by Avian Small Intestine Macrophages and T Cells.

Tissue resident immune system cells in the chicken intestine play a significant role in the protection against pathogens. However, very little is known about these cells. The current study was conducted to further characterize chicken intestinal immune system cells. Furthermore, this study aimed to assess the immune modulatory action of a highly virulent Clostridium perfringens, a commonly found chicken intestinal microbe, in comparison with a non-commensal, Lactococcus lactis, on intestine-derived immune system cells. The results demonstrated varying distribution of innate and adaptive immune cells along the avian gut-associated lymphoid tissue (GALT) in the duodenum, jejunum, ileum, and cecal tonsils. In addition, steady-state and tissue-specific presence of CD25+ cells among αß and γδ T-cell subsets was assessed along the intestine. Ex vivo stimulation with C. perfringens or L. lactis resulted in a significant increase in the frequency of CD25+ T cells (γδ and αß T cells). In addition, significantly more cell death was observed in ex vivo stimulation with C. perfringens, which was indirectly correlated with a decrease in macrophage activation based on nitric oxide (NO) production with no effect on lymphoid cell responsiveness as per intracellular interferon (IFN)-gamma (γ) staining. Ex vivo stimulation with L. lactis activated γδ T cells and αß T cells, based on intracellular IFN-γ staining, while it had limited effect on macrophages. However, the ability of γδ and αß T cells to produce IFN-γ and the ability of macrophages production of NO was rescued in the presence of L. lactis. These results demonstrate the potential application of L. lactis, as a probiotic, against virulent C. perfringens infection in chicken.

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.