Lactobacillus casei ATCC 393 was kindly provided by the Netherlands NIZO Institute and grown anaerobically in de Man, Rogosa, and Sharpe (MRS) broth at 37°C without shaking. The Lactobacillus constitutive expression plasmid pPG-T7g10-PPT (Song et al., 2014; encoded resistance to chloramphenicol and contained an HCE promoter, PgsA anchor, T7g10 enhancer, and rrnBT1T2 terminator), Escherichia coli pMD19-T-6Ds/TG1, pMD19-T-COE/TG1, and pMD19-T-VP4/TG1 were constructed and preserved in our laboratory; the PEDV GT/2017 strain, belonging to the G2b subtype, was isolated from clinical samples and identified in 2017 in our laboratory; the TGEV TH-98 and PoRV JL94 strains were isolated in our laboratory.

Twenty-four antibody-seronegative, healthy newborn piglets were purchased from the Acheng Experimental Practice Base of Northeast Agricultural University (Harbin, China). The piglets were breastfed until day 7 after birth and then fed with animal milk powder every 6 h. Animal studies were performed according to the regulations of the Animal Experiment Ethics Committee of the Northeast Agricultural University, China (review number: NEAUEC20210337).

A schematic diagram of the recombinant plasmid construction is shown in Figure 1. The details of primers used in this paper are shown in Table 1. Briefly, primers F1 and R1 were used to amplify the 6Ds gene using the plasmid pMD19-T-6Ds as a template. Primers F2 and R2 were used to amplify the VP4 gene using pMD19-T-VP4 as a template. Using plasmid pMD19-T-COE as a template, the primers F3/R3 and F4/R4 were used to amplify the COE gene, linker sequence, and SacI, KpnI, BamHI, SphI, and ApaI sites. After digesting the vector at restriction enzyme sites SacI and ApaI, the genes 6Ds, VP4, and COE were inserted into the expression plasmid pPG-T7g10-PPT, giving rise to recombinant plasmids pPG-T7g10-6Ds, pPG-T7g10-VP4, and pPG-T7g10-COE, respectively. The 6Ds fragment was inserted into the KpnI/BamHI sites of 19 T-simple-COE to generate the plasmid pMD19-T-6Ds-COE. The VP4 fragment was inserted into the SphI/ApaI sites of the recombinant plasmid pMD19-T-6Ds-COE to generate the plasmid pMD19-T-6Ds-COE-VP4. The 6Ds-COE-VP4 fragment was inserted into the SacI and ApaI sites of the expression plasmid pPG-T7g10-PPT to generate the plasmid pPG-T7g10-6Ds-COE-VP4. All four recombinant expression plasmids were verified using polymerase chain reaction (PCR) and sequencing. Finally, the recombinant plasmids were transformed into L. casei 393 via electroporation to generate the recombinant strains pPG-T7g10-6Ds/LC393, pPG-T7g10-COE/LC393, pPG-T7g10-VP4/LC393, and pPG-T7g10-6Ds-COE-VP4/LC393.

To analyze the expression of the proteins of interest in recombinant strains pPG-T7g10-6Ds/LC393, pPG-T7g10-COE/LC393, pPG-T7g10-VP4/LC393, and pPG-T7g10-6Ds-COE-VP4/LC393, the identified strains were cultured in MRS medium containing chloramphenicol (10 mg/ml) at 37°C for 16 h and then harvested by centrifugation (Heraeus Pico17 centrifuge, Thermo Fisher Scientific, Waltham, MA, United States) at 10000 × g for 2 min. After cell lysis with lysozyme (10 mg/ml) and washing with sterile deionized water, the bacterial precipitate was resuspended in phosphate-buffered saline (PBS) and mixed with 5× sodium dodecyl sulfate buffer to obtain the protein samples. These samples were subjected to western blotting using mouse anti-6Ds serum, rabbit anti-COE serum and rabbit anti-VP4 serum (1:200 dilution) prepared in our laboratory. Anti-FLAG tag mouse monoclonal antibody (1:1000 dilution) was used as the primary antibody, and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and HRP-conjugated goat anti-rabbit IgG (1:5000 dilution) were used as secondary antibodies. Finally, the target protein was detected and visualized using western enhanced chemiluminescence substrate and visualized.

To further evaluate the expression of the target protein, recombinant strains were cultured in MRS medium at 37°C for 16 h, and 500 μl of the culture was collected and centrifuged at 3500 × g for 5 min. The bacterial precipitate was washed three times with PBS, and the bacterial sediment was resuspended in mouse anti-6Ds serum, rabbit anti-COE serum, rabbit anti-VP4 serum, and anti-FLAG tag mouse monoclonal antibody (1:100 dilution) at 37°C for 1 h. Subsequently, the cells were incubated with fluorescein isothiocyanate-conjugated goat anti-mouse IgG and fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (1:200 dilution) at 37°C for 45 min in the dark. The cells were visualized using fluorescence microscopy.

Recombinant Lactobacillus strains were cultured in MRS medium for 16 h without agitation, washed with sterile PBS, and resuspended in PBS to a final concentration of 10¹⁰ colony-forming units (CFU)/ml. Twenty-four newborn piglets were divided into three groups. Six piglets were orally immunized with a mixture of 2 × 10¹⁰ CFU/ml pPG-T7g10-6Ds/LC393, pPG-T7g10-COE/LC393, and pPG-T7g10-VP4/LC393 (mixed group); six piglets were orally immunized with 2 × 10¹⁰ CFU/ml pPG-T7g10-6Ds-COE-VP4/LC393 (fused group), and the remaining 12 piglets were orally administered 2 ml of sterile PBS (PBS group). The immunization program was conducted daily for seven consecutive days after birth.

After immunization, anal and nasal swabs of piglets were collected daily and soaked in 1 ml of cold sterile PBS at 4°C overnight. After centrifugation at 10000 × g for 10 min at 4°C, the supernatant was stored at −40°C. Specific sIgA levels in the samples were detected using enzyme-linked immunosorbent assay (ELISA) (Zhao et al., 2017). Briefly, polystyrene microtiter plates were coated with purified TGEV/PEDV/PoRV for 12 h at 4°C, washed three times with PBS containing 0.05% Tween-20 (PBST), blocked at 37°C for 2 h with 5% skim milk in PBST, washed with PBST, and incubated for 2 h at 37°C. After washing, commercial HRP-conjugated goat anti-porcine sIgA diluted to 1:5000 was added to the plate, incubated at 37°C for 1 h, and washed with PBST, followed by color development using 3,3′,5,5′ tetramethylbenzidine chromogenic solution at 37°C for 10 min. The reaction was stopped by adding 2 N H₂SO₄, and the optical density at 450 nm was determined using an ELISA reader (SpectraMax® ABS, Molecular Devices, Sunnyvale, CA, United States). Each sample was tested in triplicate.

Blood flow from the anterior vena cava was collected from piglets in each group on days between 0 and 7 post-immunization. Serum was prepared, and serum-specific IgG responses were measured using ELISA. The method was the same as that for sIgA, but the secondary antibody was HRP-conjugated goat anti-porcine IgG (1:5000).

After 7 days of continuous immunization, piglets were randomly selected from the mixed, fused, and PBS groups (three piglets per group) for oral administration of 4 ml PEDV (10⁷ of the median tissue culture infectious dose), and the piglets were grouped as shown in Figure 2.

The piglets were weighed prior to challenge and at 7 days post-challenge to calculate body weight gain, and the clinical symptoms of piglets were recorded daily and scored according to the guidelines in Table 2.

Anal swabs were collected from the piglets daily after the challenge, and virus in the anal swabs was detected using reverse transcription PCR (RT-PCR). Briefly, one anal swab from each pig was suspended in 1 ml PBS at 4°C overnight, and the supernatant was harvested by centrifugation at 5000 × g for 3 min. RNA was extracted and then reverse transcribed into cDNA, and PCR was performed using primers ORF3-F and ORF3-R.

All experimental piglets were euthanized on day 8 after challenge and dissected to observe lesions in the intestinal tract. In addition, jejunum samples were embedded in paraffin wax, and pathological sections were prepared and stained with hematoxylin and eosin.

The data were analyzed using GraphPad Prism 6 software (GraphPad, Inc., La Jolla, CA, United States), and all data are expressed as the mean ± standard deviation. Data were analyzed using two-way analysis of variance, with at least three independent experiments. The results were considered as significant at p < 0.05 and highly significant at p < 0.01.

To improve the expression of antigens and ensure their localization on the surface of LAB, the previously constructed expression vector pPG-T7g10-PPT was used. This vector contains the enhancer T7g10 fragment, HCE constitutive promoter, and PgsA anchor sequence (Liu et al., 2011). The protective antigen (6Ds, COE, and VP4) genes of TGEV, PEDV, and PoRV were inserted into pPG-T7g10-PPT separately or combined into the same vector (Figure 1). Four types of recombinant lactobacilli expression vectors were identified using PCR and sequencing. The sequencing results showed that the above vectors were successfully constructed.

Recombinant Lactobacillus was obtained by electroporation of the recombinant plasmid into Lactobacillus cells. Cell lysates of the recombinant strains were analyzed using western blotting after 16 h of culture. Correctly sized proteins were detected in all inserts of pPG-T7g10-6Ds/LC393, pPG-T7g10-COE/LC393, pPG-T7g10-VP4/LC393, and pPG-T7g10-6Ds-COE-VP4/LC393. The negative control, pPG-T7g10-PPT/LC393, did not display a corresponding immunoreactive band (Figure 3A). Immunofluorescence assays were performed to analyze the expression of the proteins of interest. As shown in Figure 3B, green fluorescence was observed for recombinant Lactobacillus but not for pPG-T7g10-PPT/LC393. These results indicate that the proteins of interest were successfully expressed in recombinant Lactobacillus.

Anti-TGEV/PEDV/PoRV-specific sIgA was assessed to evaluate the ability of recombinant Lactobacillus strains to induce mucosal immune responses in piglets. Anal and nasal swabs were collected from the piglets at 0–7 days post-immunization, and specific sIgA levels in the samples were detected using ELISA. As shown in Figure 4, anti-TGEV/PEDV/PoRV-specific sIgA in the nasal swabs and anal swabs post-immunization was detected in both the mixed group and fused group and gradually increased over time. No significant differences were observed in the PBS group before and after immunization. Anti-TGEV/PEDV/PoRV-specific sIgA levels in the nasal swabs and anal swabs from piglets in the mixed and fused groups were significantly higher than those in the PBS group from days 3 to 7 post-immunization (p < 0.05). The levels of sIgA in the mixed and fused groups did not exhibit significant differences; however, sIgA was produced earlier in the mixed group than in the fused group.

Anti-TGEV/PEDV/PoRV-specific IgG was assessed to evaluate the ability of recombinant Lactobacillus to induce humoral immune responses in piglets. Twenty-four newborn piglets were divided into three groups for immunization (mixed, fused, and PBS groups). Serum collected from piglets between days 0 and 7 post-immunization was prepared, and serum-specific IgG responses were measured using ELISA. As shown in Figure 5, anti-TGEV/PEDV/PoRV-specific IgG was detected at day 7 post-immunization in both the mixed and fused groups. No significant differences were observed in the PBS group before and after immunization. The levels of serum anti-TGEV/PEDV/PoRV-specific IgG in the mixed and fused groups were significantly higher than those in the PBS group (p < 0.01). However, there was no significant difference between the mixed and fused groups.

The challenge test using PEDV as an example was carried out after seven consecutive days of immunization; the piglets were grouped as shown in Figure 2. The clinical symptoms of the piglets in each group were observed after challenge and scored as shown in Table 1. Briefly, the piglets that were orally administered PBS followed by PEDV challenge (PBS + PEDV³) showed typical PEDV infection symptoms, including acute watery diarrhea, depression, and drowsiness, with the most severe symptoms observed between days 4 and 5 after challenge (Figure 6A). The immunized piglets (mixed+PEDV¹ and fused+PEDV²) had slight diarrhea and a normal appetite and showed a significant difference from the PBS + PEDV³ group after 2 days (Figure 6A). None of the cohabiting piglets (mixed¹ and fused²) developed diarrhea during the experiment and showed significant differences from unimmunized piglets (PBS¹ and PBS²). In addition, before and after challenge, the weight of immunized piglets was significantly higher than that of unimmunized piglets (Figure 6B).

Anal swabs were collected from the piglets daily after grouping, and PEDV RNA in the anal swabs was detected using RT-PCR. The PBS + PEDV³ group began shedding the virus on day 1, which continued for the next few days (Figure 7). However, PEDV RNA was detected at 1–4 days in immunized piglets (mixed+PEDV¹ and fused+PEDV²) and then disappeared (Figure 7). Among the cohabiting piglets, PEDV RNA was detected in unimmunized piglets (PBS¹ and PBS²) 2–4 days after cohabitation; almost no fecal virus shedding was observed in the mixed¹ and fused² groups (Figure 7). Oral immunization with recombinant Lactobacillus inhibited PEDV shedding through the feces of piglets and reduced the infection risk of the cohabiting piglets. The inhibitory effect on fecal PEDV shedding in the mixed group was slightly stronger than that in the fused group.

All piglets were euthanized for macroscopic examination at 7 days after the challenge. Piglets in the PBS + PEDV³ group exhibited moderately thin and transparent intestinal walls in the small intestine, and large amounts of fluid accumulated in the intestinal lumen (Figure 8A). Cohabiting piglets (PBS¹ and PBS²) exhibited similar or even more serious symptoms. In contrast, there were no observable changes in the small intestines of immunized piglets (mixed+PEDV¹, fused+PEDV², mixed¹, and fused²) compared with those of the piglets in the PBS⁴ group (Figure 8A). Histopathological observations indicated that infection by PEDV caused extensive damage to the jejunum of piglets in the PBS + PEDV³ group, characterized by large amounts of fragmentation and shedding of intestinal villi (Figure 8B). Interestingly, the PBS¹ and PBS² groups exhibited more severe pathological damage to the jejunum. In contrast, damage to the jejunum of immunized piglets (mixed+PEDV¹ and fused+PEDV²) was minor compared to that in the PBS + PEDV³ group. No pathological damage was observed in the piglets of the mixed¹ and fused² groups (Figure 8B).