Dietary Intervention to Reduce E. coli Infectious Diarrhea in Young Pigs

Postweaning piglets are immediately imposed to remarkable environmental and psychosocial stressors, which adversely affect their intestinal development and health and predispose them to diarrhea. The ratio of postweaning mortality is 6–10% and may rise up to 20% with poor management strategies. Diarrhea per se accounts for 20–30% of cases of mortality in weanling pigs. E. coli postweaning diarrhea is one of the most important causes of postweaning diarrhea in pigs. This diarrhea is responsible for huge economic losses due to high mortality and morbidity, weight loss, and cost of medication. Burgeoning evidence suggested feed-based intervention are one of the promising measures to prevent postweaning diarrhea and to enhance overall health of weaned pigs. Although the exact protective mechanisms may vary and are still not completely understood, a number of feed ingredients or feed additives are marketed to assist in boosting intestinal immunity and regulating gut microbiota. The promising results have been demonstrated in several nutrients (i.e., functional amino acids, organic acids, micro minerals, nondigestible carbohydrates, and antimicrobial peptides), non-nutrients (i.e., phytochemicals and probiotics), and many other feed additives. The efficiencies of each candidate may differ based on their exact modes of action, the basal diet formulation, and the health status of pigs.


Introduction
Escherichia coli (E. coli), a Gram-negative rod-shaped bacterium, was first discovered in 1885 by Theodor Escherich, who noted that E. coli are highly prevalent in the intestinal microflora of healthy individuals and have potential to cause disease when directly inoculated into extraintestinal sites. Diarrheagenic E. coli can be further divided into six groups: enterotoxigenic E. coli (ETEC), enteropathogenic E. coli, enterohemorrhagic E. coli, enteroinvasive E. coli, diffusely adhering E. coli, and enteroaggregative E. coli [1]. Different groups of diarrheagenic E. coli express different virulence genes, exhibit different adhesion characteristics, and therefore have different mechanisms of pathogenicity. This book chapter only covers the infection caused by ETEC.
ETEC is the major etiological agent causing acute watery diarrhea in postweaning piglets. The duration of diarrheal symptom may be shortened by After adhering to the small intestinal surface, ETEC induce enteric infectious disease and diarrhea through release of enterotoxins, which stimulate copious secretion by the small intestinal mucosa. The enterotoxins include heat-labile toxin, heat-stable toxin, LPS, and Shiga toxins.
Heat-labile toxins. Heat-labile toxin mainly accumulates in the periplasmic space, with limited amount appears on the surface of the bacteria. Heat-labile toxin consists of a single A subunit and five B subunits. The binding of B subunits to the monosialotetrahexosylganglioside (GM1) ganglioside on the cell surface facilitates the translocation of a fragment of A domain into the cell, which then activates the adenylate cyclase system and increases the expression of cyclic adenosine monophosphate (cAMP) [12]. Several physiological changes are mediated by the increased cAMP. First, cAMP stimulates the phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR), inducing chloride secretion from the apical region of enterocytes [31]. Second, cAMP stimulates the activation of an apical chloride channel and a basolateral Na/K/2Cl cotransporter, which results in the release of prostaglandin E2 and vasoactive intestinal peptide and loosening of tight junctions [32,33]. These activities all contribute to increased chloride secretion, reduced sodium absorption, and a concomitant massive loss of water into the intestinal lumen. The effect of heat-labile toxin is irreversible [12].

Heat-stable toxins
Heat-stable toxins in porcine isolates are further classified as STa and STb [32]. STa is a small, non-immunogenic protein with a molecular weight of approximately 2 kDa [34]. The major receptor for STa is a particular transmembrane form of guanylate cyclase (GC-C) [35]. Therefore, STa can stimulate the GC-C system, leading to excessive levels of cyclic guanosine monophosphate (cGMP) in enterocytes. Signals resulting from cGMP accumulation induce the activation of CFTR, elevated secretion of Cl-and water in crypt cells, but reduced Na + and Cl − absorption from cells at the tips of villi [11]. Heat-stable toxin a mainly induces small intestinal fluid secretion in newborn but not in weaned pigs [4].
Heat-stable toxin b is a 48-amino acid protein with a molecular weight of approximately 5.1 kDa [36]. STb is antigenically and genetically unrelated to STa and is poorly immunogenic [37]. Production of STb is restricted to porcine ETEC [13]. The mechanisms of action and molecular characteristics of STb are still less known than heat-labile toxins and STa. STb does not stimulate an increase in intracellular levels of either cAMP or cGMP, either Na + or Cl − [38], but stimulates the secretion of HCO 3 − from intestinal epithelial cells [10,39]. Heat-stable toxin b causes mild histological damage in the intestinal epithelium, including loss of villous epithelial cells and villous atrophy. This damage may be responsible for impaired absorption of fluids [40]. Another proposed mechanism of action is that STb could increase the level of prostaglandins by opening a G-protein-linked receptor-operated calcium channel in the plasma membrane and elevating intracellular Ca ++ [41,42]. STb can induce small intestinal fluid secretion in newborn and weaned pigs [4].

LPS
Lipopolysaccharide is the major surface component of the outer membrane of most Gram-negative bacteria, including ETEC [43]. LPS consists of three distinct regions, lipid A, core oligosaccharides, and the O-antigen polysaccharide with the structural variability from low to high. Lipid A is the primary immunostimulatory component of LPS and is highly recognized by numerous cellular signaling pathways in the innate immunity. The receptors that respond to LPS are mainly located on the cells in the innate immune system, such as macrophages and endothelial cells [44]. Therefore, LPS not only contributes to the physiological membrane functions but also plays an important role in the pathogenesis of Gram-negative bacterial infection [44]. The pathogenic impacts of LPS are mainly through stimulating immune cells of the host, resulting in the release of large amounts of cytokines. The CD14, expressed on monocytes or macrophages, are highly involved in this process. Briefly, the soluble CD14 (sCD14) and LPS-binding protein facilitate the interaction of LPS and the membrane CD14 (mCD14). After binding to mCD14 in the cell

Shiga toxin
Some ETEC strains that cause postweaning diarrhea possess additional genes that encode Shiga toxin, allowing them to cause edema disease as well [4]. Similar to heat-labile toxin, Shiga toxin is also a protein toxin, consisting of one A subunit and five B subunits. However, Shiga toxin has completely different mechanisms infecting cells, in comparison with heat-labile toxins. Briefly, Shiga toxin first binds to the cells that possess the glycolipid receptors, globotriaosylceramide (Gb)3 or Gb4 [46,47]. After binding, Shiga toxin is transported to the Golgi apparatus through endocytosis. The Golgi apparatus further transports Shiga toxin to the endoplasmic reticulum, where the subunit A is cleaved by trypsin and is separated into A1 and A2 subunits. The A1 subunit is released into the cytosol and subsequently impacts ribosomes [14]. Shiga toxin can inhibit protein synthesis and induce synthesis of cytokines, including IL-1, IL-6, IL-8, and TNF-α [47,48]. In addition, Shiga toxin also induces DNA degradation and release of the cellular contents that facilitate proteolytic attack on neighboring cells, contribute to cell apoptosis, and have a toxic effect in the whole organism [14].

Dietary intervention on E. coli infectious diarrhea
Nutrients are compounds in feed ingredients that are essential to animal maintenance and production, by providing animals with energy, the building components for repair and growth, and the substances to regulate biological processes. Nutrients are generally grouped into six major classes: water, carbohydrates, proteins, lipids, minerals, and vitamins. With the exception of carbohydrates, all five classes of nutrients are indispensable and have to be provided through animal feed. In addition, a group of specific nutrients, such as functional amino acids, nondigestible carbohydrates, short-chain fatty acids (SCFA), and several micro minerals, has beneficial effects on animal health and performance beyond their nutritional contributions. Recently, a novel concept, non-nutrients, is illuminated to describe a group of dietary compounds, which has no nutrient contribution to animals, but have physiological activities beyond the nutritional pyramid, formulation practices, and feeding methods that similarly alter physiological condition. Emerging evidence suggested that several non-nutrient feed additives (i.e., plant extracts, probiotics, enzymes, etc.) improved animal health through modulating microbial ecology in the digestive tract and/or enhancing immune responses of animals to enteric infections.

Functional amino acids
Amino acids are defined as organic substances that contain both amino and carboxyl groups. Amino acids are classified according to their molecular weights, chemical structures, the composition of nitrogen and sulfur, and physiological functions. The 20 common proteinogenic amino acids shared by all animal species are further categorized into indispensable, semi-dispensable (conditional essential), and dispensable amino acids, dependent on their dietary essentiality. Functional amino acids are defined with a group of amino acids that are traditionally classified as dispensable amino acids, but with extra biological functions [49]. The well-investigated functional amino acids are the arginine family, which includes arginine, glutamine, glutamate, aspartate, proline, etc. The basic functions of these amino acids have been well summarized by Wu et al. [49,50], which include but not limited to (1) providing substrates for the synthesis of tissue protein; (2) impacting hormone synthesis and secretion; (3) regulating endothelial function, vasodilation, and blood flow; (4) affecting nutrient metabolism; and (5) maintaining acid-base balance and whole-body homeostasis.
Large amounts of literature have reported the impacts of dietary supplementation of functional amino acids on health and performance of newly weaned pigs. For example, supplementation of 0.2 to 1% L-arginine could enhance growth performance and alleviate the negative effects of different insults or challenges in young pigs [51-54]. Administration of proline was shown to improve mucosal proliferation, intestinal morphology, as well as intestinal tight junction of weaned pigs [55]. Dietary supplementation of glutamine or dipeptides that are composed of glutamine has shown positive impacts on intestinal integrity, enzyme activities, and growth performance of weaned pigs [56][57][58]. Several mechanisms are highly involved in the benefits of the arginine family on intestinal health of weaned pigs, which could prevent the intestinal dysfunction caused by E. coli infectious diarrhea in weaned pigs. First, these amino acids could provide major fuel for small intestinal epithelial cell proliferation and provide energy required for intestinal ATP-dependent metabolic processes [59]. Second, catabolism of these amino acids provides precursors or substrates for the synthesis of nitric oxide, polyamines, and creatine, which are important regulators in blood flow, intestinal integrity and secretion, and epithelial cell repair and migration [60-62]. Third, glutamine is also a major substrate for glutathione synthesis, which is an important endogenous antioxidant in cells regulating the homeostasis of free radicals [63]. Fourth, these amino acids could enhance intestinal secretory IgA production via regulating the intestinal microbiota and immunity [53, 64].

Fatty acids
Dietary fat and lipids are extremely important for animal health and production. They have three major fundamental roles in swine nutrition by providing energy, compound lipids, and steroids to animals. Triglycerides and free fatty acids are the primary forms of metabolic energy storage and transport in the animal body. Shortchain fatty acids and medium-chain fatty acids (MCFA) have recently attracted increased research attention as potential candidates to reduce enteric infectious disease in animal production due to their potential antimicrobial activities [65-67].
SCFA are fatty acids with a chain of less than six carbon atoms, which are primarily produced by hindgut fermentation of dietary fiber. The most abundant SCFA in the gastrointestinal tract are acetic (C2), propionic (C3), and butyric acid (C4). They are the major fuel source for colonocytes and are essential for maintaining the normal metabolism of colon mucosa, including colonocyte growth and proliferation [68,69]. Butyric acid has received particular attention and has been widely investigated to enhance disease resistance of weaned pigs. Addition of this acid directly to a swine diet may be limited because of its pungent odor and unpalatable flavor. Thus, the salt form (sodium or calcium) or glyceryl form (monobutyrin or tributyrin) of butyric acid has been adopted in animal feed industry. One major advantage of glyceryl forms in comparison with salt forms is that they stay intact in the stomach and are slowly released as butyrate and/or monobutyrin in the small intestine where pancreatic lipase appears [ weaned pigs, as they reduced diarrhea, enhanced gut integrity, and improved overall immunity of newly weaned pigs [71][72][73][74]. The mechanisms resulting in improved disease resistance of weaned pigs are highly associated with the antimicrobial activities of SCFA; however, other mechanisms may be also involved. Butyric acid could penetrate into epithelial cells either by simple diffusion or monocarboxylate transporter [75]. Butyric acid could also bind to G-protein-coupled receptor expressed in epithelial cells or immune cells. The binding will mediate a cascade of immune regulation and regulate large amount of gene expression [76][77][78].
MCFA are saturated fatty acids containing 6 to 12 carbon atoms, including caproic (C6), caprylic (C8), capric (C10), and lauric acids (C12). MCFA are naturally occurred lipids that are enriched in animal milk fat and in the oil fraction of various plants, such as coconuts, palm kernels, and Cuphea seeds. Similar to SCFA, MCFA have unpleasant smell; therefore, they are commonly used in their glyceryl forms in animal feed. MCFA may have particular nutritional and metabolic effects on young animals due to their rapid digestion, passive absorption, and obligatory oxidation [79,80]. Although the evidence for the favorable energetic attributes of MCFA is strong, the results of in vivo studies using weaned pigs have been inconsistent [81]. However, the beneficial effects of MCFA on gut health of weaned pigs have been suggested, as they could influence intestinal morphology and physiology, gut microbiome, and intestinal immunity [80]. More research is necessary in the future to explore the influences of MCFA on disease resistance of weaned pigs.

Micro minerals
Minerals required in smaller quantities are called micro minerals or trace minerals, which include Zn, Cu, Mn, Fe, Se, and others. Micro minerals have confirmed physiological roles and are needed for normal bodily functions of pigs. However, unlike most other minerals, Cu and Zn have antimicrobial properties, and they are, therefore, often added to animal feed in quantities greater than the amount of nutritional requirements.
Zinc serves as a component or an activator of several metalloenzymes and is involved in many intracellular and intercellular signaling pathways. Zinc also plays important roles in skin and wound healing and in regulating immune system [82]. Zinc deficiency in weaned pigs leads to growth retardation, loss of appetite, skeletal abnormalities, and parakeratosis if Zn concentration in feed is much lower than the requirement of 80-100 mg/kg for nursery pigs [83][84][85]. However, pharmacological levels (2000-3000 mg/kg) of inorganic Zn in the form of ZnO have been widely adopted to control postweaning diarrhea and enhance feed intake and overall growth performance [86][87][88][89]. The benefits of pharmacological Zn on disease resistance of weaned pigs are likely related to several mechanisms: enhancement of intestinal integrity [90], regeneration of injured intestinal mucosa, stability of intestinal microbiota diversity [91], reduction of intestinal permeability [92], and modulation of intestinal immunity [93]. However, more recent research indicate that feeding pharmacological ZnO could reduce the digestibility of Ca and P, reduce the effectiveness of microbial phytase in pig diet, and increase the abundance of multiresistant bacteria in weaned pigs [94][95][96]. Inclusion of pharmacological levels of ZnO has recently been banned in the European Union due to increased Zn pollution from pigs fed with high Zn diets. Meantime, animal feed industry and nutritionists are actively working together to search alternatives that could replace pharmacological ZnO. For instance, low dose of organic Zn sources (i.e., 125 mg/kg of Zn-methionine) has been confirmed to have beneficial effects that are equivalent to addition of pharmacological ZnO due to their greater bioavailability [97,98].
Copper is also an essential component of several metalloenzymes including cytochrome oxidase and lysyl oxidase. Copper is highly involved in oxidationreduction reactions, transport of oxygen and electrons, antioxidant system, and many other metabolic functions, including cellular respiration, tissue pigmentation, hemoglobin formation, and connective tissue development [82]. In general, neonatal pigs only require 5-6 mg/kg of Cu for normal metabolism [85,99], and Cu requirement decreases as animal gets older. Cu deficiency may lead to critical dysfunctions and hypocuprosis in pigs [100]. Pigs may also suffer from microcytic anemia and bone abnormalities [101,102]. Addition of pharmacological levels of Cu (125-500 mg/kg) in pig diets has been a common practice to reduce postweaning diarrhea and improve growth performance [103][104][105][106]. The beneficial effects of pharmacological Cu have been attributed to its bacteriostatic and bactericidal properties [107,108]. Similar to zinc, many Cu forms could be used in animal feed, including copper sulfate (CuSO 4 ), copper chloride (CuCl 2 ), tribasic copper chloride (Cu 2 (OH) 3 Cl), and copper citrate. Copper sulfate and copper chloride are the most common supplementing forms. Tribasic copper chloride has been suggested to have similar bioavailability but less negative impacts on phosphorus digestibility and intestinal microbiota than copper sulfate [109][110][111]. Chelated Cu, such as Cu citrate, has greater availability than inorganic Cu sources, which may be used in animal feed as low dose, resulting in reduced Cu excretion [112].

Prebiotics and probiotics
Prebiotics are a category of nutritional compounds that may not share similar structures but have the ability to improve the growth of beneficial microorganism in the gastrointestinal tract. Gibson et al. [113] offered a definition of prebiotics, which contains three key aspects: resistance to digestion, fermentation by the large intestinal microbiota, and a selective effect on the microbiota associated with health-promoting effects. Most well-studied prebiotics are nondigestible oligosaccharides or polysaccharides [114]. For instance, inulin-type prebiotics are a group of nondigestible carbohydrates that mainly comprise fructose, including inulin, oligofructose, and fructo-oligosaccharides. They are commonly used in the pig industry and human foods [115]. Galactooligosaccharides that exist in human milk have been reported to have prebiotic effects by enhancing colonic health of breast-fed infants [116]. Many other naturally occurring prebiotics have been reported as well, including polydextrose, trans-galactooligosaccharides, xylo-oligosaccharides, lactulose, pyrodextrins, and isomalto-oligosaccharides. However, a few other nondigestible carbohydrates are not categorized as prebiotics (e.g., mannan-oligosaccharides, β-glucan etc.), but manifest health-promoting functions [117]. For example, a growing evidence demonstrates that β-glucans, either produced by bacteria or extracted from different sources (i.e., cereal, algae, and fungi), could boost host immunity, therefore enhancing disease resistance of human and animals [118][119][120].
Probiotics, also known as direct-fed microbials, are live microorganisms and, when administered in adequate amounts, confer a health benefit on the host [121]. Probiotics are categorized into three main groups, including Bacillus, lactic acidproducing bacteria, and yeast [122]. Based on the Food and Drug Administration instruction, the term probiotics is used for human microbial products, whereas the term direct-fed microbials is used for the US feed industry. However, "probiotics" are interchangeably used with human and animal feed worldwide. Bacillus-based probiotics are spore-forming, which makes them thermostable and able to survive at low pH. Bacillus-based probiotics have been identified as potent producers of extracellular fiber-degrading enzymes, which may aid nutrient digestion and utilization [123]. Lactic acid-producing bacteria are not spore-forming; therefore, their survival during feed processing is of concern [124]. Lactic acid-producing bacteria dominate the gastrointestinal tract of the nursing pig [125], which helps reduce the pH in the gut by producing lactic acid through fermentation, inhibiting enteric pathogens [126], and improving host immunity [124,127]. However, after weaning of pigs, the concentration of lactic acid-producing bacteria diminishes; therefore, supplementation of weaned pig diets with lactic acid-producing probiotics may be beneficial [122]. Yeast include a broad range of products that may be available in pig feed, including whole live yeast cells, heat-treated yeast cells, ground yeast cells, purified yeast cell cultures, and yeast extracts. The efficacy of yeast-based products varies depending on their forms. Yeast or yeast-based product supplementation may boost feed intake and overall growth performance, augment mucosal immunity, promote intestinal development, adsorb mycotoxins, reduce postweaning diarrhea, and modulate gut microbiota in weaned pigs [128][129][130][131].
The most notable effect of prebiotics and probiotics is their modification of intestinal microbiota. They may control or prevent pathogenic bacterial infection by specifically stimulating the growth of beneficial microorganisms in the intestine. The beneficial microorganisms may include but not limited to Bifidobacteria and Lactobacilli, which have confirmed benefit to suppress the growth of pathogenic microorganisms, such as E. coli, through the potential mechanisms described below. For example, the desired bacteria produce SCFA and lactic acid, which may indirectly and specifically kill or inhibit the growth of pathogens [132]. The production of acids may reduce the pH of the intestinal environment, which is unsupportive of the growth of several pathogens [133]. The desired bacteria may produce antimicrobial compounds such as bacteriocins or antibiotics [134]. The desired bacteria compete the available nutrients against pathogens [135].
Many research articles have been published on the impacts of prebiotics and probiotics on infectious diseases in young pigs. For instance, supplementation of 8% inulin reduce the incidence and severity of postweaning diarrhea, probably by increasing SCFA production in the cecum and proximal colon [136]. The addition of fructo-oligosaccharide prevented the mortality and morbidity of weaned pigs infected with K88 ETEC [137]. Supplementation of β-glucan originated from different sources (yeast or algae) could enhance the resistance of pigs against K88 or F18 ETEC infection [120,138]. The α-ᴅ-mannans from yeast could bind to mannosespecific receptors that are present on many bacteria such as E. coli and Salmonella spp., which prevents adhesion of these pathogens to the mannose-rich glycoproteins lining the intestinal lumen [128]. Indeed, pigs supplemented with live yeast or a yeast fermentation product had reduced disease-related stress, diarrhea scores, duration of diarrhea, and shedding of E. coli and enhanced intestinal integrity in pigs challenged with ETEC [139][140][141]. Supplementation of Bacillus subtilis also enhanced disease resistance and growth performance and reduced diarrhea of weaned pigs infected with F18 ETEC [142].

Phytochemicals
Phytochemicals are secondary plant metabolites that are either naturally obtained from plant materials or directly synthetized. Phytochemicals are used in solid powder form, as crude extracts, or as concentrated extracts. The extracts are further classified as essential oils or oleoresins based on the extraction methods. Essential oils are volatile lipophilic substances obtained by cold extraction or distillation, whereas oleoresins are derived by nonaqueous solvents [143]. A few examples of well-known phytochemicals are curcumin, flavonoids, phenolic acids, isoflavones, carotenoids, etc. The major bioactive compounds in phytochemicals are polyphenols, terpenoids, alkaloids, or sulfur-containing compounds.
However, the composition and concentration of bioactive compounds in different phytochemicals may vary a lot, completely depending on the types of plant, the parts of plant, geographical origins, growing conditions, harvesting seasons, processing techniques, and storage conditions [144]. The in vitro biological properties of many phytochemicals have been well investigated, including antimicrobial, antioxidant, anti-inflammatory, and antiviral effects [145][146][147][148]. Therefore, phytochemicals have been largely applied in food processing, cosmetics, and other areas related to human nutrition and health.
Various phytochemicals have been reported to exhibit a broad spectrum of antimicrobial activities against Gram-negative and Gram-positive bacteria [149][150][151]. The potential mechanisms of action of antimicrobial activities of phytochemicals are described below. Many phytochemicals are lipophilic, which could damage bacterial membrane, eventually causing the leakage of intracellular materials and cell death [152][153][154][155][156]. In addition, the phenolic compounds possess strong antibacterial properties by inhibiting virulence factors, such as enzymes and toxins [157][158][159]. Lastly, certain bioactive components may also prevent the development of virulent structure (i.e., flagella) in bacteria, therefore inhibiting ETEC adhesion and toxin binding [160,161].
Our previously published research reported that dietary supplementation of 10 mg/kg of capsicum oleoresin, garlic botanical, or turmeric oleoresin reduced the frequency of diarrhea and enhanced disease resistance of pigs infected with F18 ETEC [162]. The active components in these phytochemicals are capsaicin, propyl thiosulfonates, and curcuminoides, respectively. The results of gene expression profiles in ileal mucosa indicated that supplementation of these phytochemicals modified the expression of genes related to mucin production, cell membrane integrity, and antigen processing and presentations in ETEC-infected pigs [163]. In addition to the enhanced intestinal mucosal health, pigs fed with those phytochemicals had less recruitment of macrophages and neutrophils in the ileum [162]. These observations also suggest that the weaned pigs supplemented with those phytochemicals actually had less gut inflammation than infected control. The gene expression profile analysis by microarray also confirmed the reduced gut inflammation by feeding those phytochemicals to weaned pigs [163]. The phytochemicals discussed above can be naturally obtained from seasonings that are commonly used in kitchen. Many other phytochemicals have been thoroughly investigated to against ETEC infection as well. For example, the anti-diarrheal activity of back or green tea extract has been revealed, because the reduced net fluid and electrolyte losses were observed when F4 ETEC-infected jejunal segments were perfused with black or green tea extract [164]. The administration of cranberry extract (1 g/L) in drinking water also reduced the diarrhea of F18 ETEC-infected piglets [165].

Antimicrobial peptides
Antimicrobial peptides, also called host defense peptides, are polypeptides that are naturally occurring molecules in various organisms from prokaryotes to mammals. Antimicrobial peptides can be synthesized as recombinant molecules, such as recombinant lactoferrin, or can be isolated from bacteria, insects, vertebrates, or plants, such as bovine lactoferrin and plant defensins [166,167]. Most of antimicrobial peptides are cationic (positively charged) and amphiphilic (hydrophobic and hydrophilic). Antimicrobial peptides were firstly discovered in the 1980s. They can be classified into different groups based on the different amino acid components, structures, and biological function. The antimicrobial peptides derived from mammals are mainly classified into two families, defensins or cathelicidins. Defensins are further subgrouped into α-, β-, and θ-defensins according to the spacing patterns of their cysteine residues [168]. Defensins are more abundant in epithelial cells and phagocytic cells, whereas cathelicidins are highly expressed in mammalian neutrophils [169].
Antimicrobial peptides possess a strong and wide-spectrum activity against Gram-negative and Gram-positive bacteria, as well as against parasites, fungi, and viruses [170]. One potential advantage of antimicrobial peptides is that they may kill pathogenic bacteria that are resistant to specific medically important antibiotics [168,171]. Most antimicrobial peptides are small, positively charged, and amphipathic molecules that allow them to actively interact with bacterial membranes through different models, such as barrel-stave model, carpet model, or toroidal pore model [172,173]. These properties will also allow them to disrupt cell membrane structure, penetrate into cells, regulate intracellular signaling pathways, and ultimately cause bacterial cell death. Many research findings have demonstrated that antimicrobial peptide treatment could inhibit protein and nucleic acid synthesis, suppress bacterial cell wall synthesis, as well as inhibit enzyme activities in bacteria [174]. In addition to their antibacterial properties, antimicrobial peptides may also act as epithelial "preservatives" or immunomodulators to protect host against enteric infectious agents [166,175,176].
The protective effects of antimicrobial peptides on infectious diarrhea and intestinal integrity have been reported in weaned pigs [177,178]. For example, feeding 0.4% of a mixture of bovine lactoferrin, plant defensins, and active yeast increased intestinal integrity and reduced gut permeability of weaned pigs [178]. Addition of cecropin AD reduced incidence of diarrhea and enhanced intestinal Lactobacilli counts in E. coli-challenged piglets [177]. The regulation of gut microbiota may also attribute to the potential benefits of antimicrobial peptides. Supplementation of recombinant lactoferrin or lactoferramoin-lactoferricin reduced the total viable counts of E. coli and Salmonella but enriched the abundance of Lactobacillus and Bifidobacterium in the colon of weaned pigs [179,180].

Lysozyme
Lysozyme, also known as muramidase or N-acetylmuramide glycanhydrolase, is an antimicrobial enzyme that is naturally present in body fluids of all mammalian species [181][182][183]. Lysozyme could catalyze the hydrolysis of its natural substrate peptidoglycan that is the major component of bacterial cell wall. The hydrolysis of peptidoglycan eventually results in cell lysis. Gram-positive bacteria have cell walls composed of thick layers of peptidoglycan; they are, therefore, more sensitive to the enzymatic degradation of lysozyme [184]. However, a growing evidence supports that lysozyme also displays bactericidal activity against a variety of Gram-negative strains through non-enzymatic mechanisms [184][185][186]. For instance, lysozyme has been found to act synergistically with antimicrobial peptides, lactoferrin, in killing Gram-negative bacteria, such as E. coli [181,187]. In addition to its antimicrobial activity, lysozyme also exhibited anti-inflammatory property that was mediated through inhibiting neutrophil migration and shown the ability to modulate intestinal microbiota [188].
Consumption of lysozyme-rich milk significantly enhanced the relative abundance of Bifidobacteriaceae and Lactobacillaceae in feces of weaned pigs [189]. Those bacterial families are known for their health-promoting functions in lower gastrointestinal tract of human and pigs. Interestingly, consumption of lysozyme-rich milk reduced the relative abundance of bacteria (Mycobacteriaceae, Streptococcaceae, Campylobacterales) that are associated with diseases in pig feces [189]. In another trial from the same research group, feeding of lysozyme milk reduced the incidence of diarrhea and reduced total bacteria translocation into the mesenteric lymph nodes by 83% in ETEC (O149:F4 strain)-infected pigs [190]. Feeding lysozyme-rich milk also tended to reduce fecal Enterobacteriaceae family, in which many prevalent enteric pathogens such as E. coli and Salmonella belong to [190]. Similar results were also reported in one animal trial focusing on human lysozyme-rich milk [191]. In this experiment, neonatal pigs were used and infected with F4 ETEC. Consumption of human lysozyme-rich milk (approximately 1300 mg/L lysozyme) increased survival rate, reduced diarrhea, and facilitated the recovery of infected pigs [191]. Lysozyme treatment also increased the relative abundance of Lactobacillus in feces and enhanced intestinal integrity and mucosa immunity of these neonatal pigs [191].

Conclusions
Accumulating evidence has confirmed the importance of nutritional interventions, including modified feeding strategies and nutrient supplements, in the control of diarrheal diseases, and preventing enteric infection as the use of antibiotics will be progressively restricted in many countries. Interest is particularly growing  [120] 1 Modified from Liu and Ji [192]. in the use of probiotics and/or prebiotics to increase the populations of target microbes in the digestive tract, thereby improving gut health and performance of animals. Phytochemicals can be an additional tool that producers use to keep pigs healthy and reduce the negative impacts of disease. Dietary supplementation of certain phytochemicals may enhance disease resistance of pigs by improving gut mucosal integrity and optimizing immune response. There are much more candidates of feed additives/nutritional interventions, which may be effective in regulating intestinal environments and immunity and alleviating postweaning enteric infection (Table 1) [192]. It is very important to keep in mind that the efficiencies of each candidate may differ on the basis of their modes of action, the basal diet formulation, and the health status of pigs.

Conflict of interest
The authors declare no conflict of interest.
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