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

Animals.Male specified-pathogen-free Wistar (U:Wu) rats, Brown Norway (Rij:Hsd) rats, and BALB/c mice were obtained from the animal facility of the Utrecht University (Utrecht, The Netherlands), Harlan Netherlands B.V. (Horst, The Netherlands), and the National Institute of Public Health and the Environment (Bilthoven, The Netherlands), respectively. The rats and mice, 6 to 7 weeks of age, were randomly allocated and kept in filter-topped cages (two animals per cage). The animals were provided with autoclaved food pellets and drinking water ad libitum. The animals were housed in the barrier under standard conditions (50 to 60% humidity, 12-h dark-12-h light cycle). All animal studies were approved by an independent ethical committee of the Utrecht Faculty of Veterinary Medicine, as required by Dutch law.

Bacteria. Lactobacillus casei Shirota strain YIT9029 (L. casei) was obtained from the Yakult Central Institute for Microbiological Research (Tokyo, Japan). L. casei was grown overnight at 37°C in MRS broth (CM359; Oxoid, Haarlem, The Netherlands) and resuspended in sterile saline (0.9% NaCl) to a final concentration of 2 × 10⁹, 2 × 10⁸, or 2 × 10⁷ CFU/ml, respectively. The number and viability of the lactobacilli were determined by aerobic culturing on MRS plates (Oxoid CM361). Heat-killed L. casei was obtained by heating a viable L. casei suspension of 2 × 10⁹ CFU for 15 min at 65°C. The animals received 0.5 ml of saline (control) or bacterial suspensions in saline (experimental groups) per day by a feeding tube placed in the esophagus.

Listeria monocytogenes strain L242/73 type 4b was obtained from the National Institute of Public Health and the Environment (Bilthoven, The Netherlands). To prepare activated L. monocytogenes for oral infections, an egg passage was performed as described by Ruitenberg et al. (32). The stimulated bacteria were maintained in brain heart infusion BHI broth (Oxoid CM225) at −80°C. In each experiment, an aliquot was thawed and clear-cultured on BHI (Oxoid CM375) agar plates. Inocula of L. monocytogenes were grown for 18 h in BHI broth and resuspended in sterile saline to the appropriate concentrations. The L. monocytogenes concentration was confirmed by aerobic culturing on both BHI and modified Oxford agar (MOA) plates. The MOA medium was modified from a selective medium for L. monocytogenes as described elsewhere (5).

Experimental design.In various independent experiments, the effect of orally administered Lactobacillus casei Shirota strain YIT9029 on L. monocytogenes-specific DTH responses and ACR in mice and rats was studied. The studies were designed to study whether the immune effects induced by oral L. casei intake are dependent on lactobacillus dose, viability, and administration timing and host genetics. In all experiments, the moment of L. monocytogenes infection was defined as time zero (t = 0).

Host genetics.In three separate experiments, the effect of L. casei on the DTH response in Wistar rats, Brown Norway rats, and BALB/c mice was studied. Starting 10 days (t = −10) before the subcutaneous L. monocytogenes infection, the animals (n = 6 per group) were daily administered in 0.5-ml suspensions, which contained 10⁹ CFU of viable L. casei. Control animals received saline instead of lactobacilli. At day 10 postinfection (t = 10), the L. monocytogenes-specific DTH was measured.

Administration timing.Starting 10 days before (t = −10) or 7 days after (t = 7) the subcutaneous L. monocytogenes infection, rats were daily administered 0.5-ml suspensions with 10⁹ CFU of viable L. casei. Control animals received saline instead of lactobacilli (n = 6 rats per group). At t = 7, the host has completely cleared the bacterial pathogen (data not shown), which means that the control and experimental groups have been exposed to the same amounts of Listeria antigen, since the animals were not L. casei treated before day 7 postinfection. Differences found in DTH responses at day 10 would be due only to ingestion of L. casei from day 7 till day 10 postinfection. At day 10 post infection (t = 10), L. monocytogenes-specific cell-mediated immunity was measured using the DTH assay.

Lactobacillus dose, viability, and administration timing.Starting 7 (t = 7), 8 (t = 8), or 9 (t = 9) days after the subcutaneous L. monocytogenes infection, rats were daily administered 0.5-ml suspensions containing 10⁹ CFU of viable L. casei. Additionally, the effects of orally administered 10⁹ heat-killed, 10⁸, and 10⁷ CFU of viable L. casei on the DTH response were analyzed. Control animals received saline instead of lactic acid bacteria (n = 4 rats per group). At day 10 postinfection (t = 10), L. monocytogenes-specific cell-mediated immunity was measured using the DTH assay. Subsequently, rats received daily the corresponding L. casei suspensions for an additional 8 weeks. At day 63 (t = 63) the animals were reinfected with L. monocytogenes, and 36 h later the rats were killed and analyzed for bacterial burden in the liver and the spleen as well as serum alanine aminotransferase (ALT) activity. ACR was determined by the capacity of the rats to eliminate L. monocytogenes cells from the spleen and liver after the challenge.

Primary and challenge infections.Rats and mice were subcutaneously infected at day zero with 5.10⁵ or 2.10⁴, respectively, viable L. monocytogenes cells at five different sites, 0.1 ml at two sites in the left and right thighs, 0.1 ml at two sites in the left and right forelimbs, and 0.1 ml in the neck region. Challenge infections were performed by intraperitoneal infusion of 5.10⁷ viable L. monocytogenes cells in both rats and mice.

Adoptive transfer of immunity.Starting 10 days previously (t = −10), subcutaneously L. monocytogenes-infected donor mice received orally 10⁹ CFU of L. casei or saline per day (n = 10 mice per group). Seven days (t = 7) after the primary L. monocytogenes infection, single-cell suspensions were prepared from the spleens of donor mice. After passage over a column packed with glass wool fiber, 10⁸ wool-nonadherent spleen cells of five mice of each group were infused via the orbital plexus into syngeneic naive recipients. The recipient mice received no lactobacilli. Twenty-four hours after the transfer (t = 8), the DTH was measured in the recipient mice and the remaining five donor mice of each group. Twenty-four and forty-eight hours after the challenge, the increase in ear thickness was measured. Five days after setting the DTH assay (t = 13), all mice were reinfected with live L. monocytogenes. The mice were killed 24 h later and analyzed for L. monocytogenes counts in the liver and spleen. ACR was determined by the capacity to eliminate L. monocytogenes cells after challenge.

DTH assay.Animals in the experimental and control groups were challenged in left and right ear pinnas with 25 μl (10⁷ CFU) (for rats) 10 μl (10⁷ CFU) of heat-killed L. monocytogenes (HKLM) under light ether anesthesia. The application and preparation of HKLM in DTH assays have been described in previous studies (1, 12, 14). The absolute increase in ear thickness was measured with a digital engineer's micrometer (Mitutoyo, Veenendaal, The Netherlands) 24 and 48 h after challenge. The swelling was calculated according to the following equation: net swelling = (T₂₄ − T₀) − (P₂₄ − P₀); T₀ is thickness of ear before HKLM challenge, T₂₄ is thickness of ears 24 h after HKLM antigen challenge, P₀ is thickness of ear before phosphate-buffered saline injection, and P₂₄ is thickness of ear 24 h after phosphate-buffered saline injection. The same type of equation for DTH measurements at 48 h after challenge was performed. It was shown that challenging the ear pinnas in noninfected L. casei-fed rats with heat-killed L. monocytogenes or challenging the ear pinnas of L. monocytogenes-immunized rats with heat-killed L. casei did not result in a significant net swelling response. This excludes possible cross-reactivity of L. monocytogenes antigens with L. casei antigens.

Bacteriological analysis.Spleens and livers were removed aseptically from the animals. The numbers of viable L. monocytogenes were determined by culturing 100 μl of serial 10-fold dilutions of organ homogenates in saline on MOA plates. The numbers of L. monocytogenes bacteria were expressed as CFU per gram of spleen and/or liver. Detection limit was 100 CFU/g of tissue.

ALT.ALT levels in rate serum were analyzed with an automated chemistry analyzer (Beckman Synchron CX7; Beckman-Coulter Ned., Mijdrecht, The Netherlands). Alanine aminotransferase is a liver-specific enzyme in rats (3). Analyses were performed at the Department of Clinical Sciences of Companion Animals of the Utrecht University, The Netherlands.

Statistics.Statistical analysis was performed with SPPS software (SPPS version 7.5.2.). Significant differences were revealed by analysis of variance followed by a Student's t test for comparison between control and experimental groups. A P value of <0.05 is considered significantly different. A low number (100 CFU) was assigned to animals from whom no L. monocytogenes could be recovered in order to calculate a group mean. To analyze correlations between (i) dose of L. casei and DTH responses, (ii) timing of L. casei administration and DTH responses, and (iii) L. monocytogenes counts in liver and ALT levels in serum, the Pierson correlation test was applied. Values are expressed as means ± standard deviations (SD).

Host genetics and the effects of oral L. casei administration on DTH.Figure 1 shows that L. casei, following oral delivery, significantly enhanced the L. monocytogenes-specific DTH response in Wistar rats at 24 h (P < 0.05) and 48 h (P < 0.05), compared to results for control animals. In a parallel study it was shown that orally administered L. casei enhanced significantly the expression of DTH at 24 h (P < 0.05) and 48 h (P < 0.05) in the Brown Norway rats (Fig. 1). Further, in two experiments with L. monocytogenes-immunized BALB/c mice, it was demonstrated that L. casei ingestion enhanced the antigen-specific DTH in rats. In the first experiment the DTH was significantly enhanced at 24 h (P < 0.01) and 48 h (P < 0.01) after challenging the ear pinna (Fig. 1). In the second experiment DTH values in lactobacillus-treated mice were significantly higher at 24 h (P < 0.001) and 48 h (P < 0.05) (Table 1). In these experiments a dose of 10⁹ CFU of viable L. casei was orally administrated on a daily basis, starting 10 days prior to the subcutaneous L. monocytogenes infection.

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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.

The effect of L. casei administration timing on DTH.Starting daily, oral L. casei administration at a dose of 10⁹ CFU, 10 days prior to as well as 7 days after the primary Listeria monocytogenes infection in Wistar rats, resulted in significantly enhanced antigen-specific DTH expression, as shown in Fig. 2. The magnitude of the DTH responses at 24 and 48 h was comparable for the two groups of rats which received L. casei 20 or 3 consecutive days prior to the DTH assay.

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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.

The effect of oral administration of 10⁹ CFU of viable L. casei 3 days prior to the DTH assay in Wistar rats was reproduced, as shown in Fig. 3. A significantly enhanced Listeria-specific DTH reaction was measured at 24 h (P < 0.05) and 48 h (P < 0.01) in animals which received L. casei starting 3 days before the DTH assay. DTH values at 24 and 48 h after ingestion of 10⁹ CFU of lactobacilli at 2 days or 1 day prior to the DTH assay did not significantly differ from those for control rats.

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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.

Decreasing the number of lactobacillus applications prior to the DTH assay resulted in a strong, but not significant, tendency to correlate with decreasing DTH values at 24 h. This correlation was significant at 48 h (r = 0.55; P < 0.05).

The effect of L. casei viability on DTH.Ingestion of heat-killed L. casei did not affect DTH expression. As shown in Fig. 3, DTH values at 24 and 48 h were not significantly different from control values after oral administration of 10⁹ CFU of heat-killed L. casei 3 days prior to the DTH assay.

Effect of L. casei dose on DTH.DTH values at 24 and 48 h were not significantly different from control values after oral administration of 10⁸ and 10⁷ viable L. casei bacteria 3 days prior to the DTH assay, as denoted in Fig. 3. A significant correlation was observed between lactobacillus dose, administered 3 days prior to the DTH assay, and DTH values, measured at 24 and 48 h after challenge (r = 0.61 and P < 0.05 [24 h]; and r = 0.71 and P < 0.01 [48 h]).

Effects of long-term oral administration of viable L. casei on ACR.Long-term (i.e., approximately 8 weeks) oral supplementation of viable L. casei at a dose of 10⁹ CFU, but not 10⁷ or 10⁸ CFU, enhanced ACR as measured by reduced counts of viable L. monocytogenes in the spleen and liver at 36 h after reinfection compared to results for control animals (Table 2). Immunologically primed animals responded to reinfection in a more vigorous and effective manner, as shown in Table 2. Unprimed control animals (control-1) had approximately 10 times higher Listeria counts in the visceral organs, spleen (P < 0.01), and liver (P < 0.01), as well as significantly higher serum ALT activity (P < 0.01) than primed control animals (control-2).

Further, L. monocytogenes growth was not affected by long-term oral administration of heat-killed L. casei. Serum ALT activity was significantly lower in two (out of three) groups of animals which were fed viable L. casei Shirota at a dose of 10⁹ CFU per day for 63 consecutive days, compared to the control group (control-2), as shown in Table 2. Other experimental groups did not affect significantly the ALT activity. In spite of that, ALT values were significantly correlated with bacterial load in the liver (r = 0.75; P < 0.01).

Adoptive transfer of immunity.It was shown that expression of antigen-specific DTH was significantly enhanced in recipient BALB/c mice which received nonadherent spleen cells from L. casei-fed donor BALB/c mice. In Fig. 4, it is shown that DTH was enhanced at 24 h (P < 0.01) and 48 h (P < 0.05). The magnitude of the DTH values of recipient mice, as depicted in Fig. 4, was comparable to DTH values of the remaining, corresponding groups of mice (Table 1) that were assayed parallel to the spleen cell-infused recipient mice, following 7 days after the primary infection with L. monocytogenes. Ten days after the DTH assay, all groups of mice were reinfected with viable L. monocytogenes. Numbers of Listeria bacteria in the spleen appeared reduced, but not significantly, in normal L. casei-fed mice as well as in reconstituted recipients which received spleen cells from L. casei-supplemented donor animals, compared to the respective controls.

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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.