Effects of Recombinant Porcine Lactobacillus reuteri Secreting Expression of Bovine Lactoferrin Peptides on Growth Performance of Newborn Piglets and Protective Effect against Diarrhea Virus Infection

WANG Zhaorui; WANG Xueying; XIE Weichun; SONG Liying; WANG Xiaona; CUI Wen; JIANG Yanping; ZHOU Han; WANG Li; QIAO Xinyuan; XU Yigang; LI Yijing; TANG Lijie

doi:10.3969/j.issn.1006-267x.2022.01.017

2022 , Vol. 34 >Issue 1: 159 - 176

DOI: https://doi.org/10.3969/j.issn.1006-267x.2022.01.017

RESEARCH PAPER

Effects of Recombinant Porcine Lactobacillus reuteri Secreting Expression of Bovine Lactoferrin Peptides on Growth Performance of Newborn Piglets and Protective Effect against Diarrhea Virus Infection

WANG Zhaorui ,
WANG Xueying ,
XIE Weichun ,
SONG Liying ,
WANG Xiaona ,
CUI Wen ,
JIANG Yanping ,
ZHOU Han ,
WANG Li ,
QIAO Xinyuan ,
XU Yigang ,
LI Yijing ,
TANG Lijie

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1. College of Veterinary Medicines, Northeast Agricultural University, Harbin 150030, China;
2. Northeast Scientific Inspection Observation, Key Laboratory of Animal Pathogen Biology of Ministry of Agriculture, Harbin 150030, China

Received date: 2021-05-31

Online published: 2022-01-18

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Abstract

This experiment was conducted to investigate the effects of recombinant porcine Lactobacillus reuteri secreting bovine lactoferrin peptide (LFCA) on growth performance of newborn piglets and the protective effect on porcine transmissible gastroenteritis virus (TGEV)infection which caused piglet diarrhea. Experiment 1:thirty-six one-day-old newborn piglets with an average body weight of about 1.5 kg were randomly divided into 3 groups, which were pPG-LFCA/LR-CO21 group, pPG/LR-CO21 group and control group, each group with 12 piglets. Piglets in each group were orally administered recombinant porcine Lactobacillus reuteri expressing LFCA pPG-LFCA/LR-CO21, containing empty vector plasmid PPG/LR-CO21 and equal volume phosphate buffer (PBS); oral administration continued for 3 days, and the observation time after oral administration was 14 d. During the period, piglets were fed freely, and the changes of body weight and diarrhea were recorded. Experiment 2:thirty one-day-old newborn piglets with an average body weight of about 1.5 kg were randomly divided into 5 groups and given TGEV with a half tissue culture infection dose (TCID₅₀) of 10^-7.50/mL by oral administration of 1, 3, 6, 9 and 12 mL, respectively. The observation period of 7 d was set to analyze the conditions of half lethal dose. Experiment 3:another thirty-two newborn piglets with an average body weight of about 1.5 kg were selected as experimental animals and randomly divided into 4 groups, with 8 piglets in each group. The groups were pPG-LFCA/LR-CO21 group, pPG/LR-CO21 group, control group and TGEV infect group. There were 8 replicates in each group and 1 piglet in each replicate. Each head of the experimental group was orally fed ppG-LFCA/LR-CO21, pPG/LR-CO21 and equal volume of PBS at a dose of 2×10¹⁰ CFU per day for 1 consecutive week. At 8 days of age, TGEV was infected by oral administration at half lethal dose, and samples were collected after 7 days of infection. The weight change and diarrhea of each group of piglets were recorded; hematoxylin-eosin staining was used to detect the length of intestinal villi and the depth of crypts; enzyme linked immunosorbent assay (ELISA) was used to determine total serum total immunoglobulin G (IgG) and total secretory immunoglobulin A (sIgA) antibody contents. RT-qPCR was used to detect the mRNA relative expression levels of Claudin-1, Occludin, tight junction protein-1 (ZO-1), inflammatory cytokines interleukin-6 (IL-6), interleukin-8 (IL-8), interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) and Toll-like receptor 2 (TLR2). The flora structure of the contents of the piglet's cecum was analyzed. After oral recombinant porcine Lactobacillus reuteri, compared with the control group, the average daily gain of newborn piglets in the pPG-LFCA/LR-CO21 group was significantly increased (P<0.01), while the diarrhea rate was significantly decreased (P<0.01). Compared with TGEV infection group, the average daily gain of piglets in pPG-LFCA/LR-CO21 group was increased and diarrhea rate was decreased, and the differences were significant (P<0.05). Villus height and the ratio of villus height to crypt depth in jejunum and ileum were significantly increased (P<0.05). The contents of total IgG and intestinal mucosal total sIgA antibody in serum of piglets were significantly increased (P<0.05); the mRNA relative expression levels of tight junction protein-related genes Claudin-1, Occludin and ZO-1 in intestinal mucosal tissue were extremely significantly increased (P<0.01), and the serum TNF-α content was extremely significantly decreased (P<0.01). Serum IFN-γ, IL-6, IL-8 and TLR2 contents were significantly increased (P<0.01), and the survival rate of piglets was improved. The analysis of the bacterial diversity in the contents of the piglets' cecum showed that the proportion of normal intestinal flora of piglets decreased after TGEV infection. Compared with the TGEV infect group, the proportion of pathogenic bacteria Bacteroides in piglet's intestinal flora decreased by oral recombinant bacteria (P<0.05), the diversity of the flora was closer to the control group, and the metabolism of the intestinal flora and the processing of genetic information had been enhanced. In summary, the recombinant porcine Lactobacillus reuteri expressing LFCA is successfully constructed, which can promote the growth of newborn piglets and reduce the diarrhea rate of newborn piglets after oral feeding. Feeding the recombinant bacteria to newborn piglets infected with TGEV by oral route can improve the intestinal environment, reduce the damage of TGEV to piglets' intestine, and provide effective protection against TGEV infection.

Key words： bovine lactoferrin peptides; recombinant porcine Lactobacillus reuteri; mucosal immunization; anti-TGEV infection

Cite this article

WANG Zhaorui , WANG Xueying , XIE Weichun , SONG Liying , WANG Xiaona , CUI Wen , JIANG Yanping , ZHOU Han , WANG Li , QIAO Xinyuan , XU Yigang , LI Yijing , TANG Lijie . Effects of Recombinant Porcine Lactobacillus reuteri Secreting Expression of Bovine Lactoferrin Peptides on Growth Performance of Newborn Piglets and Protective Effect against Diarrhea Virus Infection[J]. Chinese Journal of Animal Nutrition, 2022 , 34(1) : 159 -176 . DOI: 10.3969/j.issn.1006-267x.2022.01.017

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