Oral treatment of chickens with lactobacilli influences elicitation of immune responses

Abstract

Commensal microbes in the intestine are in constant interaction with host cells and play a role in shaping the immune system. Lactobacillus acidophilus, Lactobacillus reuteri, and Lactobacillus salivarius are members of the chicken intestinal microbiota and have been shown to induce different cytokine profiles in mononuclear cells in vitro. The objective of the present study was to examine the effects of these bacteria individually or in combination on the induction of antibody- and cell-mediated immune responses in vivo. The birds received lactobacilli weekly via oral gavage starting on day of hatch and subsequently, at 14 and 21 days, were immunized with sheep red blood cells (SRBC), keyhole limpet hemocyanin (KLH), Newcastle disease virus vaccine, and infectious bursal disease virus vaccine. Antibody responses in serum were measured weekly for 4 weeks beginning on the day of primary immunization. The cell-mediated immune response was evaluated at 21 days postimmunization by measurement of gamma interferon (IFN-γ) production in splenocytes stimulated with inactivated vaccine antigens. L. salivarius-treated birds had significantly more serum antibody to SRBC and KLH than birds that were not treated with probiotics. L. salivarius-treated birds also had decreased cell-mediated immune responses to recall antigen stimulation. L. reuteri treatment did not significantly affect the systemic immune response, while L. acidophilus treatment increased the antibody response to KLH. These results indicate that systemic antibody- and cell-mediated immune responses can be modulated by oral treatment with lactobacilli but that these bacteria may vary in their ability to modulate the immune response.

Fig. 1.

Serum anti-SRBC antibody titers determined by a direct hemagglutination assay. Error bars represent standard errors of the means, and letters that are different within a time point denote significant differences among groups (P < 0.05). Chicks received 1 × 10⁷ CFU/chick of either L. acidophilus, L. reuteri, L. salivarius, or an equal combination (total of 1 × 10⁷ CFU/chick) of all three bacteria (mix) or PBS (control group) by oral gavage weekly starting on the day of hatch and throughout the trial. The groups were as follows: gavaged with PBS and not immunized (PBS), gavaged with a mixture of L. acidophilus, L. reuteri, and L. salivarius and not immunized (Mix), gavaged with PBS and immunized with SRBC (PBS + SRBC), gavaged with L. acidophilus and immunized with SRBC (L. acidophilus + SRBC), gavaged with L. reuteri and immunized with SRBC (L. reuteri + SRBC), gavaged with L. salivarius and immunized with SRBC (L. salivarius + SRBC), and gavaged with a mixture of L. acidophilus, L. reuteri, and L. salivarius and immunized with SRBC (Mix + SRBC).

Fig. 2.

Anti-KLH antibodies in the serum. Sample-to-positive (Sp) ratios are presented, and the error bars represent standard errors of the means. Chicks received 1 × 10⁷ CFU/chick of either L. acidophilus, L. reuteri, L. salivarius, or an equal combination (total of 1 × 10⁷ CFU/chick) of all three bacteria (mix) or PBS (control group) by oral gavage weekly starting on the day of hatch and throughout the trial. The groups were as follows: gavaged with PBS and not immunized (PBS), gavaged with a mixture of L. acidophilus, L. reuteri, and L. salivarius and not immunized (Mix), gavaged with PBS and immunized with KLH (PBS + KLH), gavaged with L. acidophilus and immunized with KLH (L. acidophilus + KLH), gavaged with L. reuteri and immunized with KLH (L. reuteri + KLH), gavaged with L. salivarius and immunized with KLH (L. salivarius + KLH), and gavaged with a mixture of L. acidophilus, L. reuteri, and L. salivarius and immunized with KLH (Mix + KLH). Anti-KLH IgM antibody responses (A) and anti-KLH IgG antibody responses (B) on 0, 7, 14, and 21 days post-primary immunization are shown. Letters that are different within a time point denote significant differences among groups (P < 0.05).

Fig. 3.

Serum anti-NDV antibody titers as determined by ELISA. Error bars represent standard errors of the means, and letters that are different within a time point denote significant differences amoung groups (P < 0.05). Chicks received 1 × 10⁷ CFU/chick of either L. acidophilus, L. reuteri, L. salivarius, or an equal combination (total of 1 × 10⁷ CFU/chick) of all three bacteria (mix) or PBS (control group) by oral gavage weekly starting on the day of hatch and throughout the trial. The groups were as follows: gavaged with PBS and not immunized (PBS), gavaged with a mixture of L. acidophilus, L. reuteri, and L. salivarius and not immunized (Mix), gavaged with PBS and immunized with NDV (PBS + NDV), gavaged with L. acidophilus and immunized with NDV (L. acidophilus + NDV), gavaged with L. reuteri and immunized with NDV (L. reuteri + NDV), gavaged with L. salivarius and immunized with NDV (L. salivarius + NDV), and gavaged with a mixture of L. acidophilus, L. reuteri, and L. salivarius and immunized with NDV (Mix + NDV).

Fig. 4.

IFN-γ concentrations in the supernatants of cultured spleen mononuclear cells as determined by ELISA. Chicks received 1 × 10⁷ CFU/chick of either L. acidophilus, L. reuteri, L. salivarius, or an equal combination (total of 1 × 10⁷ CFU/chick) of all three bacteria (mix) or PBS (control group) by oral gavage weekly starting on the day of hatch and throughout the trial. Spleens were isolated from chickens immunized with live NDV and Vaxxitek vaccines at 2 weeks of age. Spleens were collected at 21 days postimmunization, and mononuclear cells were cultured for 24 and 48 h in the presence of ConA, UV-inactivated NDV, or UV-inactivated Vaxxitek vaccine. Error bars represent standard errors of the means, and letters that are different within a time point denote significant differences among groups (P < 0.05).

Fig. 5.

Relative expression of cytokine transcripts (IFN-γ, IL-12p40, IL-13, and IL-6) by spleen mononuclear cells after culture for 24 h with medium only, UV-inactivated NDV, or UV-inactivated Vaxxitek. Chicks received 1 × 10⁷ CFU/chick of either L. acidophilus, L. reuteri, L. salivarius, or an equal combination (total of 1 × 10⁷ CFU/chick) of all three bacteria (mix) or PBS (control group) by oral gavage weekly starting on the day of hatch and throughout the trial. Spleens were isolated from chickens immunized with live NDV and Vaxxitek vaccines at 2 weeks of age. Data are expressed as the relative expression of cytokine mRNA levels normalized to the expression of β-actin. *, treatment significantly different (P < 0.05) from the corresponding unstimulated medium control. Error bars represent standard errors of the means, and different letters indicate that the treatments are significantly different from the other treatments within the group.