Construction of Recombinant Lactiplantibacillus plantarum NC8 Expressing ETEC FaeC and LngC and Preliminary Evaluation of Immune Responses in an ETEC-Challenged Piglet Model.
5 affiliations
- 1College of Veterinary Medicine, Jilin Agricultural University, Changchun 130118, China.
- 2Jilin Provincial Engineering Research Center of Animal Probiotics, Jilin Provincial Key Laboratory of Animal Microecology and Healthy Breeding, Jilin Agricultural University, Changchun 130118, China.
- 3Key Laboratory of Animal Production and Product Quality Safety of the Ministry of Education, Jilin Agricultural University, Changchun 130118, China.
- 4Agriculture and Animal Husbandry Development Center of Tongliao City, Tongliao 028000, China.
- 5College of Animal Science and Technology, Jilin Provincial Engineering Research Center of Animal Probiotics, Jilin Agricultural University, 2888 Xincheng Street, Changchun 130118, China.
Abstract
Enterotoxigenic Escherichia coli (ETEC) is a major pathogen responsible for diarrhea and mortality in piglets, causing substantial economic losses to the swine industry. Its pathogenicity relies on the synergistic action of colonization factors and enterotoxins, which mediate intestinal colonization and induce secretory diarrhea. Commercial vaccines against ETEC remain limited, and their protective efficacy in piglets is often unsatisfactory. Consequently, probiotics have attracted increasing attention as mucosal immunomodulatory agents for the prevention and control of ETEC infection. In this study, ETEC, the minor subunit located at the tip of the fimbriae, FaeC, and the fimbrial assembly accessory protein, LngC, were individually inserted into Lactiplantibacillus plantarum NC8 using the anchoring expression vector pPG612.1, successfully generating the recombinant strains NC8/pPG612.1-FaeC and NC8/pPG612.1-LngC. Western blot analysis confirmed that both recombinant strains correctly expressed the target proteins upon lactose induction, and their growth curves exhibited no significant differences from those of the wild-type strain, indicating that genetic modification did not compromise bacterial proliferation. To evaluate the immune efficacy of the recombinant strains, thirty-six piglets were randomly assigned to six groups: PBS control, ETEC challenge, empty vector, LngC immunization, FaeC immunization, and combined immunization (MIX). After seven oral gavage immunizations, piglets were challenged with ETEC and euthanized on day 7 post-challenge for sample collection. The results showed that immunized piglets resumed body weight gain by day 4 after challenge, whereas the ETEC group exhibited sustained weight loss. Histopathological examination revealed relatively intact jejunal villus architecture and markedly less severe mucosal damage in immunized groups compared with the ETEC-challenged group. RT-qPCR analysis of intestinal tissues and ELISA analysis of serum showed that immunization with the recombinant strains significantly decreased the mRNA expression of pro-inflammatory cytokines (TNF-α, IL-6, and IL-8) in intestinal tissues and their protein levels in serum, while increasing both the mRNA expression and protein levels of the anti-inflammatory cytokine IL-10. Flow cytometric analysis revealed that immunization with the recombinant strains significantly increased the frequencies of CD4⁺IFN-γ⁺ and CD4⁺IL-4⁺T cells and CD21⁺ B cells in MLNs, LPLs, and spleen compared with the control groups. The increased CD4⁺IFN-γ⁺ T-cell population indicated enhanced Th1-mediated cellular immunity, whereas the increased CD4⁺IL-4⁺ T-cell population suggested activation of Th2-associated humoral immunity. The expansion of CD21⁺B cells further indicated enhanced B-cell activation. Collectively, these findings suggest that the recombinant strains induced coordinated mucosal and systemic immune responses. Additionally, the secretory immunoglobulin A (sIgA) concentration in the jejunal contents was markedly increased, suggesting that the intestinal mucosal immune system was effectively activated. Collectively, these findings indicate that NC8/pPG612.1-FaeC and NC8/pPG612.1-LngC can elicit robust mucosal immune responses and alleviate diarrhea induced by enterotoxigenic Escherichia coli (ETEC) in piglets, providing a scientific basis for the development of oral probiotic-based vaccines against ETEC infection in swine.
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BibTeX
@article{niu2026construction,
title = {{Construction of Recombinant Lactiplantibacillus plantarum NC8 Expressing ETEC FaeC and LngC and Preliminary Evaluation of Immune Responses in an ETEC-Challenged Piglet Model.}},
author = {Tianming Niu and Tianyuan Niu and Hongyan Wang and Mingxiao Liu and Kuipeng Gao and Yupeng Wu and Chenxi Jiang and Shi Zhang and Qiong Wu and Shuzhen Zhang and Wentao Yang and Chunwei Shi and Zhipeng Li and Chunfeng Wang},
journal = {Veterinary sciences},
year = {2026},
month = oct,
doi = {10.3390/vetsci13101098},
url = {https://europepmc.org/article/MED/42944192},
note = {CC BY},
}
RIS
TY - JOUR
TI - Construction of Recombinant Lactiplantibacillus plantarum NC8 Expressing ETEC FaeC and LngC and Preliminary Evaluation of Immune Responses in an ETEC-Challenged Piglet Model.
AU - Tianming Niu
AU - Tianyuan Niu
AU - Hongyan Wang
AU - Mingxiao Liu
AU - Kuipeng Gao
AU - Yupeng Wu
AU - Chenxi Jiang
AU - Shi Zhang
AU - Qiong Wu
AU - Shuzhen Zhang
AU - Wentao Yang
AU - Chunwei Shi
AU - Zhipeng Li
AU - Chunfeng Wang
PY - 2026
DA - 2026/10/15
JO - Veterinary sciences
DO - 10.3390/vetsci13101098
UR - https://europepmc.org/article/MED/42944192
AB - Enterotoxigenic Escherichia coli (ETEC) is a major pathogen responsible for diarrhea and mortality in piglets, causing substantial economic losses to the swine industry. Its pathogenicity relies on the synergistic action of colonization factors and enterotoxins, which mediate intestinal colonization and induce secretory diarrhea. Commercial vaccines against ETEC remain limited, and their protective efficacy in piglets is often unsatisfactory. Consequently, probiotics have attracted increasing attention as mucosal immunomodulatory agents for the prevention and control of ETEC infection. In this study, ETEC, the minor subunit located at the tip of the fimbriae, FaeC, and the fimbrial assembly accessory protein, LngC, were individually inserted into Lactiplantibacillus plantarum NC8 using the anchoring expression vector pPG612.1, successfully generating the recombinant strains NC8/pPG612.1-FaeC and NC8/pPG612.1-LngC. Western blot analysis confirmed that both recombinant strains correctly expressed the target proteins upon lactose induction, and their growth curves exhibited no significant differences from those of the wild-type strain, indicating that genetic modification did not compromise bacterial proliferation. To evaluate the immune efficacy of the recombinant strains, thirty-six piglets were randomly assigned to six groups: PBS control, ETEC challenge, empty vector, LngC immunization, FaeC immunization, and combined immunization (MIX). After seven oral gavage immunizations, piglets were challenged with ETEC and euthanized on day 7 post-challenge for sample collection. The results showed that immunized piglets resumed body weight gain by day 4 after challenge, whereas the ETEC group exhibited sustained weight loss. Histopathological examination revealed relatively intact jejunal villus architecture and markedly less severe mucosal damage in immunized groups compared with the ETEC-challenged group. RT-qPCR analysis of intestinal tissues and ELISA analysis of serum showed that immunization with the recombinant strains significantly decreased the mRNA expression of pro-inflammatory cytokines (TNF-α, IL-6, and IL-8) in intestinal tissues and their protein levels in serum, while increasing both the mRNA expression and protein levels of the anti-inflammatory cytokine IL-10. Flow cytometric analysis revealed that immunization with the recombinant strains significantly increased the frequencies of CD4⁺IFN-γ⁺ and CD4⁺IL-4⁺T cells and CD21⁺ B cells in MLNs, LPLs, and spleen compared with the control groups. The increased CD4⁺IFN-γ⁺ T-cell population indicated enhanced Th1-mediated cellular immunity, whereas the increased CD4⁺IL-4⁺ T-cell population suggested activation of Th2-associated humoral immunity. The expansion of CD21⁺B cells further indicated enhanced B-cell activation. Collectively, these findings suggest that the recombinant strains induced coordinated mucosal and systemic immune responses. Additionally, the secretory immunoglobulin A (sIgA) concentration in the jejunal contents was markedly increased, suggesting that the intestinal mucosal immune system was effectively activated. Collectively, these findings indicate that NC8/pPG612.1-FaeC and NC8/pPG612.1-LngC can elicit robust mucosal immune responses and alleviate diarrhea induced by enterotoxigenic Escherichia coli (ETEC) in piglets, providing a scientific basis for the development of oral probiotic-based vaccines against ETEC infection in swine.
SN - 2306-7381
ER -