Enhanced immunological memory responses to Listeria monocytogenes in rodents, as measured by delayed-type hypersensitivity (DTH), adoptive transfer of DTH, and protective immunity, following Lactobacillus casei Shirota ingestion

Abstract

We have investigated the effect of orally administered Lactobacillus casei Shirota (L. casei) on immunological memory, as measured by delayed-type hypersensitivity (DTH) and acquired cellular resistance (ACR). The studies were performed in animal models in which the animals were rendered immune by a primary Listeria monocytogenes infection. It was shown that orally administered viable L. casei, and not heat-killed L. casei, enhanced significantly the antigen-specific DTH at 24 and 48 h in Wistar rats, Brown Norway rats, and BALB/c mice in a time- and dose-dependent fashion. L. casei had to be administered at least 3 days prior to the DTH assay at a daily dose of 10(9) CFU in order to induce significant effects. Long-term administration of 10(9) CFU of viable L. casei resulted in enhanced ACR, as demonstrated by reduced L. monocytogenes counts in the spleen and liver and diminished serum alanine aminotransferase activity after reinfection. Enhancement of cell-mediated immunological immune responses by L. casei was further established in an adoptive transfer study. Naïve recipient BALB/c mice, which were infused with nonadherent, immunized spleen cells from L. casei-fed donor BALB/c mice, showed significantly enhanced DTH responses at 24 and 48 h compared to recipient mice which received spleen cells from control donor mice. In conclusion, orally administered L. casei enhanced cell-mediated immunological memory responses. The effects relied on lactobacillus dose and viability as well as timing of supplementation and, further, appeared to be independent of host species or genetic background.

FIG. 1.

Effect of L. casei Shirota on L. monocytogenes-specific DTH response in Wistar rats, Brown Norway (BN) rats, and BALB/c mice. Daily oral administration of L. casei started 10 days prior to the L. monocytogenes infection. Following 10 days after the subcutaneous L. monocytogenes infection, DTH responses were measured as the difference in ear thickness (in no. of millimeters × 10⁻²) before and 24 to 48 h after intradermal challenge. Bars represent the means ± standard deviations for six animals per group. Values are significantly different from those for control animals: ∗, P < 0.05; ∗∗, P < 0.01, as calculated with Student's t test.

FIG. 2.

Effect of L. casei Shirota on L. monocytogenes-specific DTH response at 24 and 48 h after challenge in Wistar rats. Daily oral administration of L. casei started 10 days before [L. casei (t = −10)] and 7 days after [L. casei (t = 7)] infection with L. monocytogenes. Following 10 days after (t = 10) the subcutaneous L. monocytogenes infection, DTH responses were measured. Bars represent the means ± standard deviations for six animals per group. Values are significantly different from those for control animals: ∗∗, P < 0.01, as calculated with Student's t test.

FIG. 3.

Effect of differentially dosed, timed, viable, and dead L. casei Shirota on L. monocytogenes-specific DTH responses at 24 and 48 h after challenge in Wistar rats. Rats received a daily oral dose of saline (i), 10⁹ CFU of heat-killed L. casei (ii), or 10⁷ (iii), 10⁸ (iii), or 10⁹ CFU of viable L. casei (ii, iii, and iv) starting 7 days after the L. monocytogenes infection (t = 7) or 10⁹ CFU of viable L. casei starting 8 (t = 8; iv) or 9 days (t = 9; iv) after the L. monocytogenes infection. Following 10 days (t = 10) after the subcutaneous L. monocytogenes infection, DTH responses were measured. Bars represent the means ± standard deviations for four animals per group. Values are significantly different from those for control animals: ∗, P < 0.05; ∗∗, P < 0.01, as calculated with Student's t test. #, identical data bars.

FIG. 4.

Transfer of L. monocytogenes-specific DTH. BALB/c donor mice were subcutaneously infected with viable L. monocytogenes. Starting 10 days prior to infection, the mice received a daily dose of L. casei or saline. Nonadherent spleen cells from L. casei and control-fed BALB/c mice were infused into syngeneic naive recipients 7 days after infection. Twenty-four hours after the transfer, the L. monocytogenes-specific DTH was set, and it was determined 24 and 48 h later for the recipient mice. Bars represent the means ± standard deviations for five animals per group. Asterisks indicate significant differences (∗, P < 0.05; ∗∗, P < 0.01) from the control group as calculated with Student's t test.