Protective effects of α-terpineol and Bacillus coagulans on intestinal function in weaned piglets infected with a recombinant Escherichia coli expressing heat-stable enterotoxin STa

This study was to investigate the impact of α-terpineol (α-TPN) and Bacillus coagulans (B. coagulans) on weaned piglets infected with Enterotoxigenic Escherichia coli (ETEC). Thirty-two weaned piglets were assigned into four treatments: Control group (basal diet), STa group (basal diet + 1 × 1010 CFU ETEC), TPN+STa group (basal diet + 0.01% α-TPN + ETEC) and BC+STa group (basal diet + 2 × 106 CFU B. coagulans + ETEC). Result showed that both α-TPN and B. coagulans could alleviate diarrhea (decreased diarrhea rate), intestinal injury (improved intestinal morphology, decreased blood I-FABP concentration, increased protein expression level of Occludin), oxidative stress (increased GSH-Px activity and decreased MDA content) and inflammation (altered concentration of TNF-α, IL-1β in blood) induced by ETEC infection. Mechanism investigation further demonstrated that the beneficial effects of α-TPN and B. coagulans supplementation upon ETEC infection may be achieved by decreasing the protein expression levels of caspase-3, AQP4 and p-NF-κB and decreasing the gene expression levels of INSR and PCK1. Besides, α-TPN supplementation could specifically decreased expression level of gene b0,+AT, and B. coagulans supplementation could specifically decreased expression level of gene AQP10 and protein HSP70 in ETEC-infected weaned piglets. These results suggested that α-TPN and B. coagulans can be used as antibiotic alternatives against ETEC infection in weaned piglets.


. Introduction
Enterotoxigenic Escherichia coli (ETEC) strains are the most common bacterial pathogens that cause diarrhea, which is one of the most important diseases in young farm animals, also the second leading cause of death in children under 5 years old (1,2). ETEC-induced postweaning diarrhea in piglets mainly occurs within the 1st week after weaning (3). ETEC infection could result in great economic losses to the swine industry due to reduced growth performance as well as increased morbidity and mortality of piglets. Over two thirds of diarrhea in piglets caused by ETEC are resulted from STa (heat-stable toxin A)-producing ETEC strains, thereby the most heated studies of ETEC frequently focus on STa (4). In order to study the pathology and .
/fvets. . toxicology of STa, a recombinant E. coli expressing STa, named LMG194-STa, was constructed by using E. coli strain LMG194 and the plasmid pBAD202/Dtopo in our previous work. E. coli LMG194-STa has been demonstrated to induce diarrhea and intestinal injury in piglets (5,6).
Since the use of antibiotic in livestock has been reducing in China, many studies on piglet diarrhea have devoted to exploring the possible alternatives to antibiotics against ETEC infection (7). For which, essential oils and probiotics have been considered as ideal alternatives to antibiotics, due to their natural activities against pathogenic bacteria. Plant essential oils contain a wide variety of secondary metabolites that can inhibit or slow the growth of bacteria, yeasts, and molds (8). α-Terpineol (α-TPN) is a volatile monoterpene alcohol, a major component of the essential oils from various plant species (9). Research has demonstrated that it has insecticidal, antimicrobial, antispasmodic, anticonvulsant, antinociceptive and immunostimulant properties (10). It is present in the essential oil of several medicinal plants, such as Punica granatum L. (Pomegranate), Rosmarinus officinalis L. (Rosemary), Psidium guajava L. (Guava), which is popularly used for treating diarrhea. The antidiarrheal activity of α-TPN has been evaluated in mice (11). This evidence suggested that α-TPN could be used to reduce diarrhea and resist ETEC infection and STa toxin.
Bacillus coagulans (B. coagulans) is a lactic acid producing bacterial species, which is catalase positive, spore forming, motile, and a facultative anaerobe (12). Because of the strong resistance, resurrection, and stability, spores of B. coagulans could be activated in the acidic environment of the stomach, then germinate and proliferate in small intestines (13). Such spores could adapt to the gastrointestinal environment and reach the gut smoothly, and thereby play the beneficial impacts in the intestines (13). Due to these characteristics, B. coagulans has been frequently used in animal nutrition, particularly as a probiotic in poultry, cattle and shrimp (14). Our previous works have also demonstrated that supplementation of B. coagulans modulated metabolism and microbiome, alleviate intestinal integrity, oxidative stress and diarrhea (15), which may be also a good option for the treatment of ETEC infection.
Based on our previous studies on the recombinant E. coli LMG194-STa, the present study was conducted to investigate the protective effects of α-TPN and B. coagulans on intestinal function in weaned piglets infected with LMG194-STa, thereby further exploring the possible mechanisms. Findings of this study can contribute to the development of alternatives to antibiotics, and provide a novel strategy against ETEC infection. . Material and methods

. . Animals and design
The animal use protocol for the present study was approved by the Animal Care and Use Committee at Wuhan Polytechnic University (0328-2017-0117). Thirty-two crossbred healthy piglets (Duroc × Landrace × Yorkshire) were weaned at 21 days of age. Each piglet was individually housed in a 1.20 × 1.10 m 2 steel metabolic cage with eight replicates per treatment. After weaning, piglets had a 3-day adaptation. During this period, piglets had free access to the basal diet for adapting to solid food. Then piglets were assigned randomly on the basis of body weight (5.62 ± 0.66 kg) and litter origin to 4 groups: 1) control group-piglets were fed the basal diet; 2) STa group-piglets were fed the basal diet and challenged with 1 × 10 10 E. coli LMG194-STa; 3) TPN+STa group-piglets were fed the basal diet supplemented with 0.01% α-TPN, and challenged with 1 × 10 10 CFU E. coli LMG194-STa and; 4) BC+STa grouppiglets were fed the basal diet supplemented with 2 × 10 6 CFU B. coagulans, and challenged with 1 × 10 10 CFU E. coli LMG194-STa. The composition and nutrient levels of the basal diet are shown in Table 1. From day 1 to day 10 of the trial, piglets were fed the basal diet with or without α-TPN or B. coagulans. All diets were isocaloric. On day 11 and day 12, each piglet was orally inoculated with 1 × 10 10 CFU of E. coli LMG194-STa at both 8:00 and 20:00. The dose of ETEC and B. coagulans were determined based on our previous studies (15,16). The dose of α-TPN was chosen according to the result of a preliminary experiment. The recombinant E. coli LMG194-STa were constructed by our previous work, and cultured in Lysogeny broth liquid medium (5,6). B. coagulans were produced by fermenting; the conditions were 200 r/min of stirring speed and 350 L/h of throughput. The products of fermentation were filtered by organic ceramic membrane filtration equipment, then mixed with mineral adsorbent by 1:1. Final products were a dry powder that were counted by the plate counting method to determine the dosage and then were mixed in the diet. On the 15th day of the trial, blood samples were collected from the anterior vena cava and transferred into a tube with heparin sodium for anticoagulation. Plasma and serum were separated by centrifugation at 3,500 rpm for 15 min and stored at −20 • C. All piglets were then slaughtered under anesthesia. Intestine, and intestinal content were collected and stored at −80 • C until assay.

. . Plasma parameters analysis
The activity of superoxidase dismutase (SOD) and glutathione peroxidase (GSH-Px), and the content of malondialdehyde (MDA) in plasma were determined by spectrophotometry using commercially available kits (Jiancheng Bioengineering Institute, Nanjing, China). The concentration of tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1β), and intestinal fatty-acid binding protein (I-FABP) was determined by ELISA using Quantikine

. . Intestinal morphology
Intestinal tissue samples used for the morphometric study were dehydrated and embedded in paraffin, sectioned at a thickness of 4 mm, and stained with haematoxylin and eosin. Morphological measurements were carried out using a light microscope (Leica microsystems, Wetzlar, Germany) equipped with the Leica Application Suite image analysis software (Leica microsystems, Wetzlar, Germany). Intestinal villus height and width, as well as crypt depth, were measured to calculate both the villus crypt ratio and villous surface area.

. . Real-time PCR
The gene expression levels were quantitated by the method of real-time PCR. Firstly, RNA from the frozen samples were isolated using the Trizol Reagent protocol (Invitrogen, Carlsbad, CA). Then cDNA was synthesized using a PrimeScript R RT reagent kit (Takara, Dalian, China) with gDNA Eraser. Finally, the real-time PCR was carried out with the SYBR R Premix Ex Taq TM (Takara, Dalian, China) on 7,500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Ribosomal protein L4 (RPL4) was used as the reference gene. Data was analyzed by using the 2 − Ct method as described previously (17). The primer sequences are shown in Table 2.

. . Statistical analysis
All data, expressed as means ± SD, were analyzed by one-way analysis of variance (ANOVA). The normality and constant variance for experimental data were tested by the Levene's test. Differences . /fvets. . . Results

. . Growth performance
During the experimental period, the average daily gain (ADG), average daily feed intake (ADFI), and diarrhea rate (DR) was recorded and calculated (Table 3). There was no significant difference in ADG, ADFI and F/G among these treatments throughout the experiment. However, ETEC infection significantly increased diarrhea rate during day 11 to 15 of the trial, whereas the supplementation of both α-TPN and B. coagulans significantly alleviated diarrhea (P < 0.05).

. . Plasma immunity, intestinal injury, and antioxidant ability parameters
Inflammatory, intestinal injury and redox indexes in plasma are present in Table 4. Compared with control group, ETEC infection significantly increased TNF-α and MDA concentration, decreased GSH-Px activity in plasma (P < 0.05). IL-1β and I-FABP concentration had a tend to be increased by ETEC infection. Compared with STa group, TPN+STa group significantly decreased the concentration of TNF-α, IL-1β, and MDA, and increased GSH-Px activity, I-FABP concentration had a tend to be decreased by α-TPN supplementation; BC+STa group significantly decreased the concentration of IL-1β, I-FABP and MDA in plasma (P < 0.05), and tend to increase GSH-Px activity.

. . Intestinal morphology
The microscopic pictures of intestinal mucosal are shown in Figure 1, and the results of morphology is summarized in Table 5. The statistical analysis showed that compared to control group, ETEC infection significantly decreased villus height, width, surface area and the ratio of villus height to crypt depth in the duodenum, jejunum and ileum, increased crypt depth in the ileum (P < 0.05). Compared to STa group, α-TPN supplementation significantly increased villus surface area in the duodenum, increased villus height, surface area and villus height/crypt depth ratio in the ileum; B. coagulans supplementation significantly increased villus height in the duodenum, villus height, villus width and surface area in the jejunum, villus height and width and villus height/crypt depth ratio in the ileum, decreased crypt depth in the ileum (P < 0.05).

. Discussion
Diarrhea in piglets is one of the most challenging problems in the pig industry, which causes a huge economic loss nowadays (18). A major finding of this study is that diarrhea rate was markedly decreased by both α-TPN and B. coagulans supplementation, indicating that α-TPN and B. coagulans supplementation could effectively alleviate diarrhea in the weaned piglets. Furthermore, diarrhea rate in the TPN+STa group was lower than that in the BC+STa groups, suggesting that supplementation of α-TPN had a greater impact on alleviating diarrhea in the piglets infected with ETEC. The anti-diarrheal activity of α-TPN was also observed in mice, and this activity may due to its ability to block PGE2 and GM1 receptors (11). Further studies are needed to verify its anticholinergic action in piglets.
Intestinal morphology indexes such as villus height, surface area, crypt depth, and the ratio of villus height to crypt depth are the common indicators of intestinal morphologic development and intestinal morphological integrity (16). Usually, the increases in villus height, villus surface area, as well as the ratio of villus height to crypt depth reflect the improvement of intestinal absorption capacity and intestinal health (19). In this study, STa group decreased the height, width and surface area of villus and the ratio of villus height to crypt depth in the duodenum, jejunum and ileum, increased the crypt depth in the ileum, reflecting that ETEC infection markedly damaged intestinal integrity. However, both TPN+STa and BC+STa group increased villus height, width and surface area as well as the ratio of villus height to crypt depth, indicating that supplementation of α-TPN and B. coagulans can repair the intestinal injury caused by ETEC infection and enhance intestinal integrity. This was further supported by the finding that lower plasma I-FABP concentration was detected in BC+STa and TPN+ STa groups compared to STa group. As I-FABP leakages into the circulation from enterocytes only when intestinal mucosal damage occurs (20). Moreover, the improvement of intestinal morphology would partially explain the alleviation of diarrhea by α-TPN and B. coagulans.
Activation of the host immune system in response to pathogen infection results in the production of many cytokines that act as mediators of disease activity (21). In particular, proinflammatory cytokines, such as IL-1β, and TNF-α appear to play important roles in the pathophysiology of inflammatory diseases (22). In this study, STa group increased plasma TNF-α and IL-1β concentration, indicating that ETEC infection had obviously proinflammatory effect on piglets. Additionally, compared to STa group, TPN+STa group decreased the concentration of TNF-α and IL-1β; BC+STa group . /fvets. .   significantly decreased the concentration of IL-1β. These results suggested that both α-TPN and B. coagulans supplementation could relieve inflammation induced by ETEC infection and had antiinflammatory properties. Oxidant stress reflects the unbalance between the systematic phenomenon of reactive oxygen species and the capacity of biosystem to readily detoxicate the reactive intermediaries or to renovate the resulting injury (23). Nevertheless, cells protect themselves from hydroxyl radicals and other oxygenates by antioxidant enzymes, such as SOD and GSH-Px (24). MDA can induce noxious stress in cells and constitute homopolar protein adducts known as advanced lipoxidation end-products (ALEs), which is usually utilized as a marker to evaluate the oxidant stress levels (25). In the present study, STa group significantly increased MDA content and decreased GSH-Px activity in plasma, suggesting that ETEC infection could reduce the anti-oxidative capacity of piglets and induce oxidative stress. However, compared with STa group, both the TPN+STa and BC+STa group decreased the content of MDA and increased GSH-Px activity in plasma. These results indicated that α-TPN and B. coagulans supplementation could alleviant oxidative stress caused by ETEC infection, and supplementation of α-TPN and B. coagulans could enhance anti-oxidative capacity.
The injury and slow growth as well as the barrier function reducing of intestinal mucosa were probably associated with some genes such as villin, I-FABP, Occludin, and MMP3. Villin is one kind of actin binding protein and a marker of villus cell differentiation, which conduce to prop up the microfilaments of the microvilli of the mucosal villus (26). Occludin integrates such diverse processes as gene transcription, tumor suppression, and cell proliferation to modulate the intestinal mucosal structure and function (27). MMP3 as well as their inhibitors play a crucial role in the maintenance of extracorpuscular matrix homeostasis, which is expressed at high levels in the intestine of clinical IBD and celiac diseases (28). In response to stress, HSP70 is expressed at elevated levels to promote refolding and prevent aggregation of partially denatured proteins, thereby, protecting cells from injury (29). Results of this study showed that STa group decreased expression level of gene MMP3 and protein Occludin, increased expression levels of gene villin and I-FABP and protein HSP70 and I-FABP. Whereas, compared with STa group, the TPN+STa and BC+STa group increased expression level of protein Occludin, which is beneficial for the repairment of intestinal barrier. The BC+STa group decreased expression level of protein HSP70, indicating that B. coagulans supplementation could promote the recovery from ETEC infection.
Caspase-3 is one of the key components of the apoptotic pathway in the small intestine, this protein is either partially or totally responsible for the proteolytic cleavage of many key proteins (30). The expression of caspase-3 was increased by ETEC infection whereas decreased by α-TPN and B. coagulans supplementation, which might contribute to enterocyte proliferation.
NF-κB is involved in activation of an exceptionally large number of genes in response to infections, inflammation, and other stressful situations requiring rapid reprogramming of gene expression (31). NF-κB is normally sequestered in the cytoplasm of non-stimulated Frontiers in Veterinary Science frontiersin.org . /fvets. . cells and consequently must be translocated into the nucleus to stimulate gene transcription. The subcellular location of NF-κB is controlled by a family of inhibitory proteins, IκBs, which bind NF-κB and mask its nuclear localization signal, thereby preventing nuclear uptake (32). Exposure of cells to a variety of extracellular stimuli leads to the rapid phosphorylation, ubiquitination, and ultimately proteolytic degradation of IκB, which frees NF-κB to translocate to the nucleus where it regulates gene transcription (33). Mx proteins form a family of interferon (IFN)-induced GTPases with potent antiviral activity against various single-stranded RNA viruses in mammals (34). In the present study, STa group decreased expression level of NF-κB, increased expression levels of Mx1 and p-NF-κB, whereas both the TPN+STa and BC+STa group increased expression level of NF-κB, decreased expression level of Mx1 and p-NF-κB. The decline of p-NF-κB expression was in accordance with the decrease of TNF-α and IL-1β concentration in plasma. Together, these results demonstrated that ETEC infection activated the immune response in the piglets, while α-TPN and B. coagulans supplementation could alleviate the immune stress in the piglets during ETEC infection. AQP4 and AQP10 are two of the most important water channel proteins and mainly distributed in intestinal epithelial cells and facilitated the transport of water across epithelial cell (35). KCNJ13 is an ATP-dependent calcium channel that transports calcium out of cells, which plays a very considerable role in calcium homeostasis (36). b 0,+ AT plays a role in the high-affinity and sodium-independent transport of cystine and neutral and dibasic amino acids and appears to function in the reabsorption of cystine in the kidney tubule (37). Increased level of genes b 0,+ AT, AQP10, KCNJ13, and protein AQP4 were observed in the STa group, indicated that ETEC infection could increase intestinal transportation, which could be ascribed to the damage of intestinal barrier and may the mechanism of diarrhea. Additionally, α-TPN and B. coagulans supplementation alleviated the intestinal transport dysfunction in the piglets during ETEC infection, as presented by decreased level of gene b 0,+ AT in the TPN+STa group and gene AQP10 in the BC+STa group, as well as protein AQP4 in both groups.
INSR is a transmembrane receptor which can binds insulin, IGF-I, and IGF-II and belongs to the large class of tyrosine kinase receptors. Binding of insulin or other ligands to this receptor activates the insulin signaling pathway, which regulates glucose uptake and release, as well as the synthesis and storage of carbohydrates, lipids and protein (38). PCK1 is a main control point for the regulation Frontiers in Veterinary Science frontiersin.org . /fvets. . of gluconeogenesis and can be regulated by insulin, glucocorticoids, glucagon, cAMP, and diet (39). LPL is expressed in heart, muscle, and adipose tissue, which functions as both triglyceride hydrolase and ligand/bridging factor of receptor-mediated lipoprotein uptake (40). This study observed that STa group decreased expression level of LPL, increased expression levels of INSR and PCK1; whereas both the TPN+STa and BC+STa group decreased the level of INSR and PCK1 and the TPN+STa group increased expression level of LPL.
The opposite results on metabolic regulation between ETEC and two supplements indicated the beneficial impact of α-TPN and B. coagulans on intestinal metabolism in piglets during ETEC infection.

. Conclusion
The present study investigated the beneficial impact of dietary supplementation of α-TPN and B. coagulans in piglets infected with ETEC. Both α-TPN and B. coagulans supplementation could relieve diarrhea, inflammation, intestinal injury, oxidative and immune stress caused by ETEC infection, by means of modulation of genes and proteins associated with intestinal barrier, immune response, transport and metabolism. Findings of this study demonstrated that both α-TPN and B. coagulans can be used against ETEC infection, and could be considered as ideal alternatives to antibiotics.

Data availability statement
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.