Intestinal microbial flora plays an important role in human and animal health, it has attracted more and more attention in recent years. Gut contains a tremendous amount of microorganisms, which makes a very big contribution to individual health. A healthy human intestine contains more than 10¹⁴ bacteria, which is 10 times the number of all cells in the body (Koboziev et al., 2014). As it has a close relationship with absorption of nutrients, colonization resistance, development of the immune system and other functions in host, intestinal microbial flora is known as an essential organ (Moya and Ferrer, 2016). Although gut microbiota resides in the intestine, it can cause systemic effects (Ho et al., 2015). Not only the intestine got affected by the change of gut microbiota diversity and balance, but the whole body system could get changed (Ho et al., 2015). Diseases caused by imbalance of intestinal microbiota can be treated by treatments involving microbiota, such as probiotics, prebiotics, synbiotics, and FMT (fecal microbiota transplantation) (Ho et al., 2015). FMT refers to the transplantation of fecal suspension from healthy individuals into the gastrointestinal tract of the patient, in order to achieve the treatment of gastrointestinal diseases. The first record about FMT was a Chinese physician named Ge Hong who used FMT to treat food poisoning and severe diarrhea in Jin Dynasty, so far, the medical profession has never stopped exploration and application of FMT technology. The first randomized controlled trial of FMT was reported in 2012, in contrast to vancomycin, infusion of donor fecal bacteria was significantly more effective in treating refractory recurrent Clostridium difficile infection (van Nood et al., 2013). In addition, it also became a tool for functional “knock-in” studies of the microbiota in animal models (Bojanova and Bordenstein, 2016). However, the connections between FMT, the structural changes of gut microbiota and improvements of intestinal health in recipients as well as the mechanism underlying these effects are not fully illustrated.

Intestinal microbiota of a newborn is in an unstable state, its diversity increase gradually and is vulnerable to external environment and other factors (Clemente et al., 2012; Pabst et al., 2016). Therefore, newborn piglets are usually used as a model, which can be a good animal model for studying human nutrition and physiology (Puiman and Stoll, 2008; Merrifield et al., 2011). However, pig diarrhea can cause sucking piglets to die, which would bring significant economic losses to the pig farm. Jinhua pig, one of the most well-known local breeds in China, has the advantages of good meat quality, high rate of reproduction and a better ability to resist the challenge of enterotoxigenic Escherichia coli (ETEC) K88 and maintain the intestinal physiology homeostasis (Gao et al., 2013). At the present study, newborn piglets were selected as the model, to explore the effect of exogenous fecal microbiota on animal health from the perspective of gut microbiota, intestinal physiological function and growth performance. We hypothesized that exogenous fecal microbiota transplantation would modulate the structure of the gut microbiota, improve intestinal barrier and immune function in newborn piglets. Therefore, this study might provide novel insights in understanding this efficient therapy strategy for various gastrointestinal diseases.

The structure of intestinal microbial flora is not only affected by genetic factors, but is also susceptible to antibiotics, diseases, diets, environment as the starting early in life (Schroeder and Bäckhed, 2016). Infancy is a key period of colonization of gut microbial flora, during which microbiota structure is unstable and susceptible to the surrounding environment (Clemente et al., 2012). In this study, DLY newborn piglets were selected to receive fecal microbiota transplantation from healthy adult Jinhua pigs. The results showed that the relative abundance of Firmicutes and Proteobacteria in the colon of recipient piglets was increased, moreover, Firmicutes and Bacteroidetes were the two most abundant phyla of these piglets, and this result was similar to the previous study that Firmicutes and Bacteroidetes had an absolute advantage in mouse gut (Kulecka et al., 2016). In this study, the ratio of Firmicutes to Bacteroidetes was increased in recipient piglets, and the relative abundance of Firmicutes, Oscillospira, Prevotella was also enhanced. Firmicutes can maintain intestinal health by producing short-chain fatty acids, inhibit inflammation and provide energy for intestinal epithelial cells (Gophna et al., 2017). Firmicutes is also closely related to animal energy metabolism, and it is conducive to animal energy absorption of food under the condition that the proportion of Firmicutes is more than Bacteroides (Turnbaugh et al., 2006). Oscillospira can use host glycans as a source of energy, Oscillospira is also probably associated with the production of the short-chain fatty acid butyrate (Konikoff and Gophna, 2016). Prevotella is associated with digestion of carbohydrates (Wu et al., 2011). Veillonellaceae is associated with dietary intervention and pro-inflammatory (an increased abundance of Veillonellaceae was found in IBD, IBS, and cirrhosis patients) (Gevers et al., 2014; Haberman et al., 2014; Shukla et al., 2015; Bonder et al., 2016).

This study also showed that Escherichia of recipient piglets was decreased. Escherichia coli are a typically pathogenic species that infects intestine, and causes diarrhea and other serious gastrointestinal symptoms, and includes types such as enterotoxigenic and enteropathic E. coli (Camerlink et al., 2010). The above results suggested that intervarietal fecal microbiota transplantation increased the proportion of bacteria that benefit the host, and decreased harmful bacteria in the intestine of recipient piglets. Moreover, homology between the intestinal microbial flora of recipient piglets and donor pigs were closer after FMT, though a portion of bacteria in recipient piglets did not change with the corresponding bacteria in the donor. Similar to the structure of microbiota composition in donor pigs, the relative abundance of Firmicutes, Prevotellaceae, Paraprevotellaceae, Prevotella of recipient was increased. For these bacteria, receptor piglets showed a donor-like succession, which is consistent with the previous reports (Duca et al., 2014). The results also showed that Bacteroidaceae, Veillonellaceae, and Bacteroides of the recipient piglets were lower than the control, although it had a higher proportion in Jinhua pigs. Such shift phenomenon has also been reported in other studies (Pang et al., 2007). It might be due to the complex interaction between microbial flora and colonization. Up to now, there were few studies about intervarietal fecal microbiota transplantation induced changes in the structure of intestinal microbial flora in pigs.

As the gut of a newborn pig is not fully developed, intestinal microbial floras are in an unstable state. At the same time, the development of its immune system is not mature enough, thus piglets are susceptible to a variety of pathogens (Xu et al., 1992) Piglets are weak in resistance, diarrhea would happen under this condition. Diarrhea would lead to the decrease of survival rate and growth retardation of piglets. The proportion of Bifidobacterium would decrease, Enterobacterium, Bacteroides and Clostridium would increase when diarrhea happens (Vondruskova et al., 2010). Clinical practice trials showed that the patients with toxic megacolon caused by C. difficile infection were treated by FMT successfully, and the frequency of diarrhea was decreased rapidly (Gweon et al., 2015). The diarrhea incidence of recipient piglets was decreased significantly in this study. As mentioned above, Escherichia are connect with many diseases including diarrhea (Camerlink et al., 2010). It indicated that the decrease of diarrhea incidence might ascribe to the reduction of colonic Escherichia. Intestinal ecosystem of piglets is closely related to its growth characteristics (Ramayo-Caldas et al., 2016). The microbial structures of the heavier body weight piglets are different from the lighter ones (Han et al., 2016). The increase of bacteria mentioned above, which associate with nutrient absorption and metabolism might also lead to the weight gain of the recipient. Consistently, the piglets inoculated with fecal microbiota suspension performed better than the control piglets in this study, and the differences in the intestinal microbial composition between recipient and donor led to the differences in their weight gain. Moreover, the decrease of diarrhea incidence might also contribute to weight gains of recipient piglets.

The development of intestine in newborn piglets is rapid, which include tissue growth, morphology changes and functions maturity. The villi capillaries of conventional mice were better developed compared to germfree mice, which suggested that the gut microbiota contribution to the development of the intestinal villi (Hooper, 2004). The growth rate of crypts affects growth and atrophy of the intestinal villi, the reduction of crypt depth indicate an increase of epithelial cell maturation rate and absorptive capacity. So the decrease of crypt depth in recipient piglets in this study indicated the enhanced ability of absorption and digestion in the small intestinal epithelium. As shown under microscope, jejunal villi of recipient piglets were smooth with luminal integrity, while the control group showed broken luminal integrity. The scanning electron microscopes result indicated that that intestinal villus morphology was improved by fecal microbiota suspension.

The integrity of the intestinal barrier structure is the basis for maintaining normal intestinal function. Tight junction is an important part of intestinal mucosa mechanical barrier function, which prevents bacteria and other antigens from spreading over the epithelial cells (Ulluwishewa et al., 2011). Tight junction proteins are often used as an indicator for the function of the barrier and the permeability in the intestine, including: occluding, ZO-1, and cladding. Improvements of intestinal barrier integrity were associated with tight junction proteins expression levels (Ulluwishewa et al., 2011). Microbiota can lead to the increase of tight junction proteins at cell boundaries, and prevent or reverse the harmful effect of pathogens in the intestine in some cases (Ulluwishewa et al., 2011). Studies have shown that probiotics can regulate tight junctions through bacterial metabolites (Hsieh et al., 2015). The expressions of tight junction proteins in the intestine of recipients were increased in this study.

As the first line of defense against intestinal microbial invasion host, goblet cells play a key role in the identification of intestinal microorganism (Shen, 2009). Intestinal microbes can directly regulate the goblet cell function by delivering local biologically active factors. And the functions of goblet cells can also be changed by host-derived bioactive factors which are produced by active epithelial or underlying lamina propria cells after contacting with intestinal bacteria (Deplancke and Gaskins, 2001). Intestinal goblet cells in the sterile-fed mice were fewer in number and smaller in volume as compared with that of the conventionally fed mice, which demonstrated that microbe regulates the number of goblet cells (Deplancke and Gaskins, 2001). In the present study, the number of goblet cells in the colon was higher than in jejunum both on days 12 and 27 which was consistent with the Karam (1999) report. From duodenum to the ileum, and jejunum, the ratio of goblet cells in all epithelial cells occupied 4, 6, and 12%. In the distal colon, the goblet cells proportion increased to 16%. This coincides with a gradual increase in the number of microbes from the proximal intestine to the colon. Goblet cells mainly secrete making that cover intestinal epithelial surface, and play the role of mechanical protective barrier (Linden et al., 2008). Mucins lubricate intestine and protect it from the damage of potential pathogens, mutagens, as well as mechanical and chemical damage (Neutra and Forstner, 1987). The predominant gel-forming mucin in the intestine is MUC2, which could be expressed by goblet cells throughout intestine (Johansson and Hansson, 2016). Without microbiota in the intestinal tract, the thickness of the mucous layer will become thinner, and bacteria can contact with the epithelium directly, translocate and trigger the submucosal immune system to cause an immune response (van Vliet et al., 2010; Johansson and Hansson, 2016). Different bacteria species in the colon of mouse also led to the differences in mucin production capacity (Caballero et al., 2015). So in this study, the different structure of intestinal microbial flora may also cause the different expressions of MUC2 in the intestine. The intestinal microbiota induced mucin production and antibiotic administration resulted in thinning of the mucus barrier, thereby increasing susceptibility to bacterial invasion (Wlodarska et al., 2011). The present study showed that mRNA and protein expressions of MUC2 were up-regulated in recipient piglets. The increase of MUC2 might be attributed to the increase of the number of goblet cells. The results indicated that the exogenous fecal microbiota suspension had a beneficial effect on the development of intestinal mucous barrier in recipient piglets. So in this study, intestinal morphology and intestinal barrier function was improved via intervarietal fecal microbiota transplantation.

There are a wide variety of antimicrobial peptides, which aid congenital barrier resist microbial infection. As the first line of innate immunity in mammals, antibacterial peptides are a class of small molecules that are widely found in animals. The expression of β-defensin 2 gene is inducible and induced by intestinal microbes in the intestine of pigs (Veldhuizen et al., 2006). The immune system of newborn piglets is imperfect, antimicrobial peptides are the key effector molecules of innate immunity at this stage. The expression of antimicrobial peptide gene in the intestine of mice increased gradually with age after birth (Ménard et al., 2008). β-defensin 2 increased gradually with age was also found in this study, the expressions of β-defensin 2 in ileum of recipient piglets on day 27 were higher than that of piglets on day 12. Moreover, the expressions of β-defensin 2 in recipient piglets were enhanced. The expressions of defensin vary in breeds of piglets. It has been reported that the expression level of defensin gene in the intestine of Jinhua pig was higher than that in Landrace pig (An et al., 2011). And they consider that β-defensin play the key role during each stage of piglets growth and development that the expression of β-defensin is different in different breeds might contribute to the various disease resistance of pigs. We found that the expressions of β-defensin 2 in recipient piglets were higher than that of the control in this study. It indicated that intervarietal fecal microbiota promoted expressions of intestinal antimicrobial peptides in DLY recipient piglets. Increased expressions of antimicrobial peptide contributed to the improvement of the resistance of recipients to diseases.

As predominant immunoglobulin isotype on most mucosal surfaces (Otten and van Egmond, 2004; Snoeck et al., 2006), secretory immunoglobulin A (sIgA) can protect intestinal tract from dietary and microbial antigens (Bakker-Zierikzee et al., 2006). Except for the inhibition of adherence and invasion of potentially harmful antigens into mucosa, sIgA also can neutralize toxins and virulence factors from microbial pathogens (Brandtzaeg, 2003). The innate inflammatory response of neonates is not mature enough to prevent pathogen invasion (Walker, 2010). The relationship between microbes and sIgA is mutual adjustment. SIgA in the intestinal mucosa of mice that the intestinal microbial flora imbalanced caused by antibiotic were increased after receiving fecal microbiota suspension (Li et al., 2015). The balance of original intestinal microbiota in recipient piglets would be disrupted after fecal suspension was treated and the optical density of sIgA⁺ cells in the colon was also increased in recipient piglets consistently. More and more bacterial colonization facilitates the maturation of mucosal immune system and is associated with the production of the large amount of sIgA, which stabilizes the microbiota-host interaction (Pabst et al., 2016). Exogenous fecal microbiota suspension stimulated proliferation of sIgA⁺ cells and the development of the intestinal immune system of piglets.

The composition of intestinal microbiota and function of the immune system is closely related (Round and Mazmanian, 2009). As a sensor for microbial infection, TLRs are critical for initiation of inflammation and immune defense responses (Rakoff-Nahoum et al., 2004). TLR recognized bacterial ligands that are not only unique to pathogens, but shared by all bacteria, and also produced by symbiotic microorganisms (Chen et al., 2010). Previous studies have shown that intestinal microbiota can activate TLR2/TLR4 on the luminal surface of epithelial cells and may subsequently improve intestinal barrier function through promoting the assembly of intestinal tight junction-associated molecules as well as regulating the proliferation and apoptosis of epithelial cells (Rakoff-Nahoum et al., 2004; Fukata et al., 2006; Cario et al., 2007). Stimulated by exogenous fecal bacteria, TLR2 and TLR4 of recipient piglets on day 12 were increased. As mentioned above, exogenous microbial flora might break the original balance of microbiota, therefore, TLRs in the intestine of piglets were significantly increased by the stimulation of exogenous fecal microbiota. However, there were no significant differences in the expressions of TLRs on day 27, which might indicate that intestinal microbiota of recipient piglets was reestablished and the new homeostasis stablished. The activation of TLRs by commensal bacteria is essential for maintaining homeostasis in the colon (Ben-Neriah and Schmidt-Supprian, 2007). Sensing of commensal bacteria by TLRs in epithelial does not elicit an inflammatory cascade but is required for the maintenance of the epithelial barrier and contributes to TLR-dependent intestinal homeostasis observed in vivo (Rakoff-Nahoum et al., 2004; Cario and Podolsky, 2005). Moreover, the induced TLR receptors in intestinal epithelium also can stimulate an innate immune response and activate the PP to secrete antimicrobial peptides (Wang et al., 2016), and lead to the induction of MUC2 (Ikeda et al., 2007). The study indicated that the expression of TLRs were up-regulated under the condition that intestinal microbial flora was imbalanced and didn’t change when the structure of intestinal microbiota restored to steady state.

In summary, some characteristics of donor pigs could be transferred to recipient neonatal piglets by fecal microbiota suspension in this study. Exogenous fecal microbiota suspension changed the structure of intestinal microbial flora as well as contributed to the improvement of intestinal morphology, the development of the intestinal mucosal barrier and innate immunity in recipient piglets. Therefore, the recipients’ resistance to disease was enhanced, diarrhea was reduced and weight gain was raised. Fecal microbiota transplantation might be an efficient way to improve young animals’ resistance to disease by changing the structure of intestinal microbial flora.

XH conceived and designed the experiments. LH and SG performed the experiments, analyzed the data and drafted the manuscript. YL, SC, XF, and XY performed the experiments and analyzed the data.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.