Considering the weakened state of the immune system of neonatal chickens, early establishment of gut microbiota with beneficial microbes may reduce the risk of post-hatch infection21,24. There is some evidence that early intestinal colonization of beneficial bacteria through in ovo inoculation or oral administration immediately post-hatch modulates innate responses as well as cell- and antibody-mediated immune responses to different antigens20,25,26. The present study was conducted to evaluate and compare the effects of in ovo or oral administration of probiotic bacteria on innate and adaptive immune responses of newly hatched chicks.
Given the important role of cytokines in immune system activation27, the expression of cytokine genes in lymphoid organs of chickens was assessed following in ovo and oral administration of probiotic lactobacilli. In general, combined administration of 107 CFU of lactobacilli induced overall greater cytokine and chemokine expression in spleen and bursa of Fabricius than did either administration method alone. Noticeably, in birds that received 107 CFU of lactobacilli via both routes, significantly higher expression of IFN-γ, IFN-β, IL-12 and IL-13 was observed in the spleen and significantly higher expression of IFN-γ, IL-2, IL-6, IL-8, IL-12, and IL-18 was observed in the bursa of Fabricius on day 10 post-hatch. When administered orally after hatching, 107 CFU of lactobacilli enhanced the expression of IFN-β, IL-1 β, IL-6, and IL-13 in the bursa of Fabricius at day 10 post-hatch. In lieu of this, weekly oral administration of 106 CFUs of lactobacilli enhanced the expression of only IL-2 in the spleen and IFN-γ in the bursa of Fabricius at day 10. These findings indicate that the number of lactobacilli as well as the route and frequency of administration are crucial to generate robust immune responses. In line with these observations, Brisbin and colleague (2011) demonstrated that oral treatment of chickens with 107 CFU of lactobacilli (L. salivarius, L. acidophilus, L. reuteri) increased expression of IFN-γ and IL-12 in splenic mononuclear cells16. Wang and colleagues (2015) also showed that L. plantarum P-8 enhanced the expression of T helper (Th)-type 1 and Th-type 2 cytokines in the small intestine of chickens28. In another study, Taha-Abdelaziz and colleagues (2019) investigated the effects of different Lactobacillus strains (L. salivarius, L. johnsonii, L. reuteri, L. crispatus, and L. gasseri) on cytokine gene expression using a chicken macrophage cell line, with results showing that all Lactobacillus isolates, either alone or in combination, increased the expression of IFN-γ, IL-1β, IL-12p40, and IL-1029.
In the present study, irrespective of the number of lactobacilli, in ovo administration of lactobacilli downregulated the expression of IFN-β, IFN-γ, TGF-β and IL-8 in the spleen but had no effect on cytokine and chemokine expression in the bursa of Fabricius. A previous study by our group demonstrated that in ovo lactobacilli supplementation did not alter the expression of cytokines in the bursa of Fabricius; additionally, it was shown that expression of pro-inflammatory cytokines was downregulated in the cecal tonsils of lactobacilli-treated birds20. It should be noted that in the current study, cytokine and chemokine expression was measured only on days 5 and 10 post-hatch. Considering that cytokines and other immune system genes are inducible and transiently expressed, it is plausible that expression of some genes studied here (especially in in ovo groups) might have been altered at some point prior to the sampling time point. Thus, future studies are required to capture the expression of these genes at earlier time points.
In the chicken immune system, CD4+ T cells play a key role in adaptive immunity by activation of B cells in addition to their role in the induction and recruitment of macrophages to the site of infection30. In the present study, the groups that received 107 CFU of lactobacilli in ovo, and those that received combined administration (107 CFU), had higher numbers of CD4+ cells compared to the control. The notable increase in CD4+ T cells population following in ovo and post-hatch lactobacilli administration indicates that these bacteria could promote the development of lymphoid organs, thus potentially improve resistance of young chickens to microbial pathogens. In agreement with these results, Asgari and colleagues (2016) demonstrated that dietary supplementation with L. acidophilus (109 CFU/kg) significantly increases the percentage of CD4+ T cells in chickens’ peripheral blood31. Bai and colleagues (2013) have also demonstrated that dietary supplementation of L. fermentum significantly increases proportions of CD3+ and CD4+ T lymphocytes in intestinal intraepithelial lymphocytes (IELs) of chickens32. Another study demonstrated that oral treatment with L. reuteri, L. salivarius and L. acidophilus increased the number of CD4+ T lymphocytes in chicken intestine33. Interestingly, despite their ability to increase the number of CD4+ T cells, the number of cytotoxic CD8+ T cells was not affected by any of the concentrations or delivery routes of lactobacilli used in this study. These results are inconsistent with previous studies that demonstrated that lactobacilli supplementation significantly increases CD8+ T lymphocytes in IELs and spleen31–33. This could be explained by differences in strains and concentration of lactobacilli used in these studies.
Similar to the results obtained for CD4+ T cells, in ovo administration of 107 CFU of lactobacilli, either as a single dose or combined with post-hatch oral treatment, increased the number of CD4+CD25+ T cells. In chickens, CD4+ CD25+ T cells have been shown to exhibit immunoregulatory properties by secreting immunosuppressive cytokines such as IL-10 and TGF-β that limit inflammatory response towards the end of the inflammatory processes34. Therefore, the higher number of CD4+CD25+ T cells observed in lactobacilli-treated group supports the role of probiotic lactobacilli in maintaining immune system homeostasis35,36. Results from some human studies suggest a role for lactobacilli in the modulation of monocyte-derived dendritic cells (DCs) and the development of Treg cells37,38. However, the exact mechanism by which these lactobacilli affect T cells is still unclear. It has been suggested that some lactobacilli (such as L. reuteri and L. casei) modulate monocyte-derived DCs by binding to the lectin DC-specific intercellular adhesion molecule 3-grabbing nonintegrin which triggers DCs to induce the development of IL-10-producing Treg cells.
While probiotic lactobacilli demonstrated the ability to increase the numbers of different T cell subsets, they do not seem to demonstrate such effects on Bu-1+ IgM+ B cell populations in the spleen. In agreement with our results, previous studies showed that different Lactobacillus species and isolates had no significant effects on Bu-1 mRNA expression levels32,39. It was suggested that lactobacilli modulate the immune system mainly through interaction with T lymphocytes rather than B cells. With regards to macrophages, our results showed that in ovo administration of 107 CFU of lactobacilli increased the number of KUL01+ cells in the spleen on day 5 post-hatch, while no significant difference was observed in chickens that received lactobacilli through both in ovo and oral routes, indicating the importance of early lactobacilli colonization on macrophage populations. This observation also suggests that higher numbers of lactobacilli or more frequent administration of lactobacilli may not always lead to enhanced immune responses. Macrophages play a key role in host immune defense through phagocytosis of pathogens and activation of lymphocytes by processing and presenting antigens to T lymphocytes40,41. Therefore, higher numbers of KUL01+ cells in the spleens of chickens that received 107 CFU of lactobacilli via in ovo administration suggests the importance of early inoculation of probiotics in chicken immune defense. In agreement with our finding, a previous study demonstrated that Lactobacillus-based probiotic culture significantly increases the number of macrophages in the intestine of chickens42.
In addition to their role in eliciting innate and cell-mediated immune responses, probiotic lactobacilli have shown to enhance antibody-mediated responses to various antigens43–45. Both SRBC and KLH are considered thymus-dependent antigens, which require the cooperation of T helper cell for B cell activation and proliferation46. The results of the current study demonstrated that in ovo administration of 107 CFU of lactobacilli, either as a single dose or combined with post-hatch oral treatment, significantly enhanced antibody responses to SRBC, suggesting the importance of early inoculation of lactobacilli in adaptive immune response activation. With respect to anti-KLH antibody titers, the group that received the combination of in ovo and weekly oral administration of lactobacilli (107 CFU) showed consistently increased IgM and IgG titers on days 7 and 14- and day 21 post-primary immunization, respectively. These results are consistent with our earlier findings that oral and in ovo administration of lactobacilli enhanced IgG and IgM antibody responses against KLH16,20. Collectively, the notable increase in SRBC and KLH antibody responses following treatment with lactobacilli is indicative of adjuvant properties of probiotic lactobacilli. The exact mechanisms underlying this effect remains to be elucidated. It could be attributed to their interactions with pattern recognition receptors (expressed by cells of innate immune system) and subsequent production of cytokines (IL-4, IL-10, and IL-13) involved in B cell development and antibody production47.
In conclusion, the results of the present study demonstrated that pre-hatch administration of a mixture of lactobacilli via in ovo route or early post-hatch administration via oral gavage could trigger innate and adaptive immune response. Administration of lactobacilli could also enhance the development of lymphoid organs, providing early protection to hatchlings. More importantly, overall improved immune responses were attained when these lactobacilli were administered in ovo followed by weekly oral administration to hatched chicks. Further studies are required to evaluate the effects of these lactobacilli on the development of gut-associated lymphoid tissues and other lymphoid organs and assess their protective efficacy against infectious agents.