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 x 10 7 or 1 x 10 8 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 x 10 8 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 x 10 8 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 x 10 8 CFU of lactobacilli enhanced the expression of Toll -like receptor (TLR) 2, interferon-gamma (IFN- gamma), 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-I3) and IL -1I3 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.

Marek’s disease (MD), caused by the Marek’s disease virus, is a lymphoproliferative disease in chickens that can be controlled by vaccination. However, the current vaccines can limit tumor growth and death but not virus replication and transmission. The present study aimed to evaluate host responses following intramuscular injection of an mRNA vaccine encoding gB and pp38 proteins of the MDV within the first 36 h. The vaccine was injected in low and high doses using prime and prime-boost strategies. The expression of type I and II interferons (IFNs), a panel of interferon-stimulated genes, and two key antiviral cytokines, IL-1β and IL-2, were measured in spleen and lungs after vaccination. The transcriptional analysis of the above genes showed significant increases in the expression of MDA5, Myd88, IFN-α, IFN-β, IFN-γ, IRF7, OAS, Mx1, and IL-2 in both the spleen and lungs within the first 36 h of immunization. Secondary immunization increased expression of all the above genes in the lungs. In contrast, only IFN-γ, MDA5, MyD88, Mx1, and OAS showed significant upregulation in the spleen after the secondary immunization. This study shows that two doses of the MDV mRNA vaccine encoding gB and pp38 antigens activate innate and adaptive responses and induce an antiviral state in chickens.

Necrotic enteritis (NE), caused by Clostridium perfringens, remains a major challenge in broiler chicken production. Probiotics represent a promising alternative strategy to mitigate NE by modulating intestinal immunity and microbiota composition. This study evaluated the effects of oral administration of the commensal bacterium Bacteroides thetaiotaomicron on immune responses, microbial profiles, and lesion development in broiler chickens challenged with C. perfringens. One-day-old male broilers (n = 120) were assigned to four groups and received either a low (1 × 10⁷ CFU) or high (5 × 10⁷ CFU) dose of B. thetaiotaomicron on days 1, 7, 14, and 21 post-hatch. Birds were subsequently challenged to induce NE. Ileal and cecal tonsil tissues were collected for gene expression analysis, and cecal contents were analyzed by 16S rRNA gene sequencing. High-dose B. thetaiotaomicron significantly reduced intestinal lesion scores compared with the positive control, whereas the low dose was ineffective. High-dose supplementation enhanced intestinal immune signaling, with increased expression of TLR2 and TLR4, primarily in the ileum. This was associated with increased expression of pro-inflammatory cytokines (IFN-γ and IL-1β) during the early post-challenge phase and sustained upregulation of regulatory cytokines (IL-10 and IL-2). The epithelial defense-associated cytokine IL-22 was upregulated in both the ileum and cecal tonsils, while TGF-β showed transient induction in the ileum. Markers of epithelial barrier integrity were improved, as evidenced by increased occludin expression, whereas ZO-1 expression remained unchanged. Microbiome profiling revealed that high-dose supplementation enriched beneficial taxa such as Lactobacillus while reducing the abundance of the pathogenic species Enterococcus cecorum. These findings highlight the potential of B. thetaiotaomicron as a probiotic strategy to enhance intestinal immunity and resilience against necrotic enteritis in broiler chickens.

Necrotic enteritis (NE), caused by Clostridium perfringens (C. perfringens), presents a challenge to the global broiler industry. Evidence suggests that Toll-like receptor (TLR) ligands can enhance the immune responses in chickens and protect them against infectious diseases. This study investigated the protective effects of TLR21 ligand class B CpG oligonucleotides (ODN) against NE in broiler chickens. On day 21 of age, chickens were injected with 50 or 100 μg CpG intramuscularly, and one group was injected with 50 μg CpG followed by a booster dose on day 22. Subsequently, birds were orally challenged with C. perfringens twice daily for three days, starting on day 22. On day 22, intestinal samples were collected for gene expression analysis. On day 25, all birds were euthanized, intestinal lesions were scored, and tissue samples were collected from the intestine for gene expression analysis, lymphocyte subset determination, and histomorphological analysis. Cecal contents were also collected for microbiome analysis. The results demonstrated that CpG pre-treatment, either at a single dose of 100 μg or two doses of 50 μg per bird, reduced lesion scores compared to the positive control. C. perfringens infection increased crypt depth in both the jejunum and ileum in the positive control group compared to both the CpG-treated group. At 22 days of age, CpG administration at doses of 100 μg per bird enhanced expression of TLR21, interleukin (IL)-2, CXCL8, IL-10, and interferon (IFN)-γ mRNA transcripts in both the jejunum and ileum. Additionally, at 25 days of age, the group pretreated with two doses of 50 μg of CpG per bird showed increased expression of all cytokines in both the jejunum and ileum compared to the control groups. The percentage of intestinal lymphocytes was not affected by CpG pre-treatment. However, CpG pretreatment at doses of 100 μg resulted in a higher abundance of the members of families Lactobacillaceae and Bacteroidaceae, which are crucial for maintaining gut health. In conclusion, our findings suggest that pretreatment of chickens with intramuscular administration of CpG may be effective in maintaining gut health during C. perfringens infection.