Effects of in ovo Inoculation of Multi-Strain Lactobacilli on Cytokine Gene Expression and Antibody-Mediated Immune Responses in Chickens

Abstract

This study was conducted to investigate the effects of various doses of a multistrain lactobacilli mixture (Lactobacillus salivarius, Lactobacillus reuteri, Lactobacillus crispatus, and Lactobacillus johnsonii) on the innate and adaptive immune responses in broiler chickens. At embryonic day eighteen, 200 eggs were injected with PBS, or three different doses of a multi-strain lactobacilli mixture (1 × 10 5 , 1 × 10 6 , and 1 × 10 7 CFU/egg, P1, P2, and P3 respectively) along with a group of negative control. On days 5 and 10 post-hatch, cecal tonsil, bursa of fabricius, and spleen were collected for gene expression and cellular analysis. On days 14 and 21 posthatch, birds were immunized intramuscularly with both sheep red blood cells (SRBC) and keyhole limpet hemocyanin (KLH). Serum samples were collected on days 0, 7, 14, and 21 after primary immunization. The results demonstrated that lactobacilli inoculation increased the splenic expression of cytokines, including interferon (IFN)α, IFN-β, IFN-γ, interleukin (IL)-8, and IL-12 on day 5 post-hatch compared to the control group (PBS). However, in cecal tonsils, lactobacilli treatment downregulated the expression of IL-6 on day 5 post-hatch and IL-2 and IL-8 on day 10 posthatch. No significant differences were observed in the expression of cytokine genes in the bursa except for IL-13 which was upregulated in lactobacilli-treated groups P2 and P3 on days 5 and 10 post-hatch. Flow cytometry analysis showed that the percentage of KUL01, CD4 + and CD8 + splenocytes was not affected by treatments. In addition, no significant differences were observed for antibody titers against SRBC. However, lactobacilli treatment (P1, P2, and P3) was found to increase IgM titers on day 21 post-primary immunization compared to controls. Furthermore, in ovo injection of the highest dose of probiotics (1 × 10 7 , P3) increased serum IgG titers Alizadeh et al.In ovo Application of Lactobacilli against KLH on day 7 post-primary immunization. In conclusion, this study demonstrated that that in ovo administration of lactobacilli can improve antibody-mediated immune responses and differentially modulate cytokine expression in mucosal and systemic lymphoid tissues of chickens.

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

“…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-in ammatory cytokines was downregulated in the cecal tonsils of lactobacilli-treated birds 20 . It should be noted that in the current study, cytokine and chemokine expression was measured only on days 5 and 10 post-hatch.…”

“…Considering the weakened state of the immune system of neonatal chickens, early establishment of gut microbiota with bene cial microbes may reduce the risk of post-hatch infection 21,24 . There is some evidence that early intestinal colonization of bene cial bacteria through in ovo inoculation or oral administration immediately post-hatch modulates innate responses as well as cell-and antibodymediated immune responses to different antigens 20,25,26 . 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.…”

“…Detection of KLH-speci c IgG and IgM titers in sera was performed by indirect enzyme-linked immunosorbent assay (ELISA) as previously described 16,20 Mandel Scienti c, Guelph, ON, CAN) was added to each well. Plates were incubated for 30 min at room temperature in the dark, and absorbance was measured at 405 nm using the microplate reader (Epoch, BioTek Instruments Inc., Winooski, VT).…”

“…In ovo delivery of selected probiotics into the amniotic sac of embryonated eggs, is a candidate route that may facilitate early colonization of bene cial bacteria in the intestine and alleviate the adverse effects associated with environmental stressors and delayed access to feed and water 18 . Some studies have shown that that early bacterial colonization of the intestine with bene cial bacteria can modulate the immune system of chickens and modify gut microbiota compositions by reducing the colonization of pathogens in the intestine 19,20,21,22 . It has also been demonstrated that the continuous administration of probiotics is required to maintain colonization in the intestine and exert health bene ts 23 .…”

Avian leukosis virus subgroup J infection influencing composition of gut microbiota within chicken

“…Probiotics ferment undigested carbohydrate residues to produce high levels of short-chain fatty acids, which create an acidic environment that is not conducive for the growth of pH-sensitive pathogenic bacteria. In vivo administration of lactobacilli can improve antibody-mediated immune responses in chicken [14]. Strains of Lactobacillus acidophilus interfere with a wide range of pathogens [15][16][17].…”