Abstract

   Porcine epidemic diarrhea virus (PEDV) has become a challenging problem
   in pig industry worldwide, causing significant profit losses.
   Lactobacillus rhamnosus GG (LGG) has been regarded as a safe probiotic
   strain and has been shown to exert protective effects on the intestinal
   dysfunction caused by PEDV. This study evaluated the effect of LGG on
   the gut health of lactating piglets challenged with PEDV. Fifteen
   piglets at 7 days of age were equally assigned into 3 groups (5 piglets
   per group): 1) control group (basal diet); 2) PEDV group: (basal diet +
   PEDV challenged); 3) LGG + PEDV group (basal diet + 3×10^9 CFU/pig/day
   LGG + PEDV). The trial lasted 11 days including 3 days of adaptation.
   The treatment with LGG was from D4 to D10. PEDV challenge was carried
   out on D8. PEDV infection disrupted the cell structure, undermined the
   integrity of the intestinal tract, and induced oxidative stress, and
   intestinal damage of piglets. Supplementation of LGG improved
   intestinal morphology, enhanced intestinal antioxidant capacity, and
   alleviated jejunal mucosal inflammation and lipid metabolism disorders
   in PEDV-infected piglets, which may be regulated by LGG by altering the
   expression of TNF signaling pathway, PPAR signaling pathway, and fat
   digestion and absorption pathway.

   Keywords: Lactobacillus rhamnosus GG, PEDV, gut health, gut functions,
   piglets

1. Introduction

   Porcine epidemic diarrhea (PED) is a highly contagious intestinal
   disease caused by the porcine epidemic diarrhea virus (PEDV) and is
   considered one of the most significant threats to the pig farming
   industry. In recent years, the global spreading of PED has caused
   enormous economic losses to the pig farming industry. PEDV, as well as
   transmissible gastroenteritis virus and porcine enteric A coronavirus,
   belongs to the genus α-coronavirus in the family Coronaviridae
   ([45]Gong et al., 2017). The main PEDV transmission route is
   fecal-oral, but airborne transmission via the fecal-nasal route may
   play a role in pig-to-pig and farm-to-farm spread ([46]Gerber et al.,
   2014; [47]Yuan et al., 2022). Piglets up to 7 days of age are
   particularly susceptible to the virus and thus cause acute diarrhea,
   vomiting, dehydration and high mortality in newborn piglets ([48]Sun
   et al., 2016). In recent years, various mutations and recombinations
   have occurred between different strains and even between PEDV and other
   coronaviruses. The lack of updated effective vaccination in controlling
   PEDV infections has caused continuous problems in the pig industry
   ([49]Du et al., 2019).

   Lactobacillus rhamnosus GG (LGG), a Gram-positive parthenogenetic
   anaerobic bacterium first extracted from the gastrointestinal tract of
   healthy human beings in 1983 ([50]Shi et al., 2019), is one of the most
   widely studied probiotic organisms for atopic diseases. The main
   characteristics of LGG are the strong ability to survive and reproduce
   under low pH conditions of gastric acid and bile, easy to colonize and
   function in the intestinal tract, with a high level of immunological
   activity ([51]Hou et al., 2023). Some studies have shown that
   colonization of LGG in neonatal mice enhances intestinal functional
   maturation and IgA production and confers lifelong health consequences
   on protection from intestinal injury and inflammation ([52]Yan et al.,
   2016). Oral administration of Lactobacillus rhamnosus powder to mice
   alleviated antibiotic-induced intestinal injury ([53]Cresci et al.,
   2013). Moreover, LGG may be effective in ameliorating E. coli
   K88-induced diarrhea in weaned piglets by modulating the intestinal
   flora, enhancing the intestinal antibody defenses, and modulating the
   production of systemic inflammatory factors ([54]Zhang et al., 2010).
   In addition, dietary Lactobacillus rhamnosus GG supplementation
   improves the mucosal barrier function in the intestine of weaned
   piglets challenged by Porcine Rotavirus ([55]Mao et al., 2016).
   Moreover, studies have shown that treatment with L. rhamnosus can
   significantly increase the villus height of ileum, promote the
   proliferation of T Cell proliferation, and therefore improve the
   absorption function and immune capacity of intestines in piglets
   ([56]Shonyela et al., 2020).

   Lactobacillus rhamnosus has been applied in medical research and animal
   husbandry production, but there are few reports on the resistance of
   Lactobacillus rhamnosus powder to PEDV infection. Our research group
   has previously established the intestinal injury model of PEDV-infected
   piglets. Therefore, the present study aimed to explore the protective
   effect of Lactobacillus rhamnosus GG powder on the intestinal tract of
   PEDV-infected piglets.

2. Materials and methods

2.1. Animal care and diets

   All animal procedures used in the present study were approved by the
   Institutional Animal Care and Use Committee of Wuhan Polytechnic
   University (Index number: 202205003). A total of 15 healthy 7-day-old
   piglets (Duroc × Landrace × Yorkshire) were used for the experiment.
   Piglets were housed separately in two environment-controlled nursery
   rooms (28-30°C) to avoid cross-infection and given ad libitum access to
   water throughout the study. The freeze-dried Lactobacillus rhamnosus GG
   powder containing 3 ×10^9 colony forming units (CFU)/g was purchased
   from Hubei Haohua biotechnology Co., Ltd (Wuhan, China), and stored in
   the sealed packet at 4°C until used. A commercial milk replacer from
   Shanghai Nouriz Dairy Co., Ltd (Shanghai, China) was served as a basal
   diet for piglets, formulated to satisfy the requirements of suckling
   piglets. Before feeding, the milk replacer was dissolved in warm water
   (45–55°C) to form a liquid feed (dry matter content of 20%). Pigs were
   fed with the liquid feed every 3 h between 8:00 am and 8:00 pm.

2.2. Experiment design

   The healthy piglets (half male and half female) at 7 day of age (Duroc
   × Landrace × Yorkshire, BW = 3.51 ± 0.33 kg) were used in this
   experiment. Piglets were housed in clean pens with strict control of
   cross-infection.The trial lasted 11 days (D0-D10), including three days
   of adaptation (D1-D3). The treatment with LGG was from D4 to D10. The
   piglets were orally administered with PEDV at a dose of 10^6 TCID[50]
   (50% tissue culture infectious dose) per pig on D8, We used the dose of
   infection in our previous study and pretest ([57]Zhang et al., 2022;
   [58]Song et al., 2024), while the control group was orally administered
   with an equal volume of PBS solution. Fifteen healthy crossbred piglets
   were chosen and the commercially available milk replacer (Nouriz
   shanghai, China) was used as a basic diet. After 3-day adaptation, the
   piglets were assigned randomly, based on body weight (BW) and litter
   origin, to 3 groups: (5 piglets/group): 1) control group - piglets fed
   the basal diet; 2) PEDV group: piglets fed the basal diet, and were
   orally administered with 3 ml 1×10^6 TCID[50]/pig PEDV; 3) LGG+PEDV
   group-piglets fed the basal diet supplemented with 50 mg/kg BW LGG
   supplementation in diet, and were orally administered with 3 ml 1×10^6
   TCID[50]/pig PEDV.

2.3. Samples collection

   Intestine sample was collected from the jugular vein of piglets on D11
   and then all animals were slaughtered under sodium pentobarbital
   anesthesia (50 mg/kg BW, iv). The abdomen of piglets was opened
   immediately and the small intestine was separated from the mesentery
   onto a chilled stainless-steel tray, then the 5-cm segment samples were
   cut respectively at distal duodenum, mid-jejunum and mid-ileum. The
   samples were gently flushed with ice-cold PBS and placed in chilled
   formalin solution (10%), mucosa was collected by scraping using a
   sterile glass microscope slide at 4°C, rapidly frozen in liquid
   nitrogen, then processed by embedding and staining for the observation
   of intestinal morphology. All the samples were collected within 10 min.
   Intestinal tissues were stored at −80°C until analysis. Piglet
   challenge PEDV, administration of LGG powder and sample collection
   process are shown in [59]Figure 1 .

Figure 1.

   [60]Figure 1
   [61]Open in a new tab

   Workflow for PEDV challenge, administration of LGG powder, and sample
   collection in piglets.

2.4. Intestinal morphology

   The procedure of preparing samples for measuring intestinal morphology
   was performed as described previously ([62]Wu et al., 2018). Briefly,
   the samples were dehydrated and embedded in paraffin, sectioned at a
   thickness of 4 mm, and stained with haematoxylin and eosin.
   Morphological examination was conducted with a light microscope (Leica
   microsystems, Wetzlar, Germany) with the Leica Application Suite image
   analysis software (Leica microsystems, Wetzlar, Germany). Eight areas
   per slide were inspected randomly in a double-blind manner. Intestinal
   villus height and crypt depth were measured to calculate the ratio of
   villus height to crypt depth.

2.5. Antioxidant capacity of intestine mucosa

   Mucosa of duodenum, jejunum, ileum, and colon were used for analysis of
   antioxidative enzymes and oxidation-related products. The activities of
   catalase (CAT), total superoxide dismutase (T-SOD), myeloperoxidase
   (MPO), and the concentration of hydrogen peroxide (H[2]O[2]) and
   malondialdehyde (MDA) were determined by using commercially available
   kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China),
   according to the protocols of manufacturer.

2.6. Quantitative RT-PCR assay

   The gene expression levels were quantitated using the method of qRT-PCR
   assays and the primers used in the present study was shown in
   [63]Table 1 , which were either verified experimentally using the
   conditions in previous publications including the housekeeping gene
   encoding ribosomal protein L19 (RPL19) or were designed using Primer
   Express software version 3.0 (Applied Biosystems). The qRT-PCR was
   performed using the SYBR ^® Premix Ex Taq TM (Takara, Dalian, China) on
   an Applied Biosystems 7500 Fast qRT-PCR System (Foster City, CA). The
   total volume of PCR reaction system was 50 μL, containing 0.2 µM of
   each primer, 25 µL of SYBR ^® Premix Ex Taq TM (2×), and 4 µL of cDNA.
   All PCR assays were performed in triplicate on a 96-well real-time PCR
   plate (Applied Biosystems) under the following conditions (two-step
   amplification): 95°C for 30 sec, followed by 40 cycles of 95°C for 5
   sec and 55-65°C for 31 sec depending on the melting temperature of each
   pair of primers. A subsequent melting curve (95°C for 15 sec, 60°C for
   1 min and 95°C for 15 sec) with continuous fluorescence measurement was
   constructed. Data were analyzed using the 2-ΔΔCt method as described
   ([64]Yi et al., 2016).

Table 1.

   Primer sequences of qRT-PCR.
   Gene Sequences (5′–3′) Product (bp) Accession Numbers References