The widespread use of antibiotics as therapeutic agents and growth promoters in animal breeding has increased the antibiotic resistance of bacteria, imbalances of normal microflora, and drug residues in food products on a global scale. In Europe and South Korea, growth-promoting antibiotics have been banned since 2006 and 2012, respectively, and similar bans are expected in other nations as well (Lillehoj and Lee, 2012). One alternative method that has been recommended due to its successful application is the use of probiotics (Reuter, 2001). A previous study has suggested that Bacillus subtilis (B. subtilis) var. natto could be promoted as a biological product intended for humans and animals (Samanya and Yamauchi, 2002).

Also known as probiotics, direct-fed microbials (DFMs) are live microorganisms which have beneficial effects on host health (Reid et al., 2003). Probiotics have been demonstrated to not only modulate symbiotic intestinal bacteria that maintain intestinal homeostasis (Sonnenburg et al., 2006; Martin et al., 2008), but also enhance the barrier functions of intestinal epithelial cells and gut health, as indicated by signs of intestinal morphology such as villus height, crypt depth, and villus height to crypt depth ratio (VH:CD). High villus height and VH: CD ratio correlate directly with increased nutrient absorption and epithelial turnover (Fan et al., 1997). Probiotics can be elicited by antagonizing pathogenic bacteria via the reduction of luminal pH, inhibition of bacterial adherence, and production of antimicrobial molecules (Ng et al., 2009). Studies with pig and poultry fed diets supplemented with probiotic products showed reductions in Clostridium and coliform populations in the guts (Choi et al., 2011; Sen et al., 2012), and as expected, probiotics modulated host protective immunity against Salmonella infection (Higgins et al., 2011; Levkut et al., 2012).

In poultry, the advantages of probiotics range from improved metabolism, immuno-stimulation, and anti-inflammation to the elimination of pathogens. Healthy carcasses also enhances nutrient absorption and lowers the risk to consumers (Edens, 2003). Innate immunity is a first-line defense against pathogen colonization, and recent findings confirmed that dietary B. subtilis can modulate immune response in broiler chickens (Lee et al., 2010). Another recent study showed that Bacillus-based DFM significantly induced inflammatory and anti-inflammatory cytokines in the jejunum and ileum of broiler chickens (Rajput et al., 2013).

In poultry, avian colibacillosis induces different syndromes, the most common symptom of which is respiratory disease, as in 3 to 12 weeks old broiler chickens and ducks, usually followed by systemic infection with characteristic fibrinous lesions and fatal septicemia (Schouler et al., 2012). Novel duck reovirus (NDRV) belongs to the genus orthoreovirus in the family Reoviridae, which includes many viruses, not least the rotavirus, which is a major human pathogen. As observed in China's Fujian, Guangdong, and Zhejiang Provinces, NDRV disease, which caused loss of appetite, unstable gait, diarrhea, and death in various species of ducks (Liu et al., 2011). This disease has been as prevalent as other traditional epidemic diseases, such as avian influenza, duck plague, and ducks Tembusu virus. At the height of their outbreaks, avian colibacillosis and NDRV disease in particular resulted in serious threats to China's duck industry.

According to previous research, we hypothesized that B. subtilis can promote growth performance and intestinal morphology in ducks, as well as prevent bacterial and viral infections by stimulating innate immunity. Accordingly, we analyzed the effects of supplementation with DFMs on growth performance, intestinal morphology, innate immunity, and resistance against Escherichia coli (E. coli) and NDRV in Cherry Valley ducks.

The present results indicated that when used in the diet of ducks as a potential growth promoter at hatching, dietary B. subtilis can benefit ducks' performance, immune organ index, intestinal morphology, and resistance against E. coli and NDRV. Moreover, the expression of primary PRRs in spleen were consistent with results in the thymus. Major pro-inflammatory cytokines and antiviral proteins tended to be up-regulated in ducks fed B. subtilis. As concerns the growth promoting effects of B. subtilis, the addition of B. subtilis spores increased the body weight of the ducks (Xing et al., 2015), and the benefits of B. subtilis were also consistent with those detected in piglet and broiler chichens performance studies (Mountzouris et al., 2010; Zhou et al., 2010).

Improved growth performance with probiotics occurs primarily via provided nutrient and enzymatic digestion (Sahu et al., 2008). Bacillus sp. can produce certain essential nutrients (e.g., amino acids), vitamins K and B12, and extracellular enzymes (e.g., proteases and lipases), as well as by providing necessary growth factors to promote host growth (Rosovitz et al., 1998; Sanders et al., 2003). Previous research has reported that the relative weights of the liver and bursa of Fabricius were unaffected by diets containing 10⁸ cfu/kg B. subtilis in broiler chickens (Zhang et al., 2012). However, another study showed that the relative weight of the spleen increased by adding B. subtilis in broiler diets (Awad et al., 2009). The present study indicated that the relative weights of the bursa of Fabricius, thymus, and spleen increased significantly due to dietary supplementation with B. subtilis at 14 dph. Such inconsistencies could stem from differences among animal species or among concentrations and species of direct-fed probiotics. Measuring immune organ weight is a common method of evaluating the immune status in chickens (Heckert et al., 2002). Enhanced immune ability might also explain increased body weight gain. In any case, B. subtilis exerted some immune-related benefits in ducks.

The crypt is the site of the proliferation and differentiation enterocytes that migrate and boost villus growth (Uni and Perry, 2006). B. subtilis promotes shallower crypt depth, which results in longer villi, greater villi surface area and more absorptive epithelial cells. Furthermore, shallower crypt depth promotes rapid epithelial turnover in response to inflammation from pathogenic bacteria (Al-Fataftah and Abdelqader, 2014). Changes in small intestinal morphology and in particular, increased villus height and VH: CD ratio in ducks fed diets supplemented with B. subtilis indicate improved gut health and digestive capacity (Caspary, 1992). The results are consistent with the results of broiler chickens fed diets supplemented with B. subtilis LS 1-2 (Sen et al., 2012).

The innate immunity system is highly conservative, triggered by the activation of PRRs, and plays an essential role in defending against pathogenic microorganisms (Barbalat et al., 2011). In this study, the expression of MDA5 and TLR3 was up-regulated in the thymus and spleen at 28 dph. The activation of PRRs induces the expression of cytokines and antiviral proteins. Cytokines are immune regulatory peptides with relatively small molecular weights that participate in innate and adaptive host immune responses (Lee et al., 2015). Among our results, dietary B. subtilis significantly up-regulated pro-inflammatory cytokines IL-1β, IL-6, IL-8, and IFN-γ in the thymus of ducks at 28 dph. IFN-γ is a common marker of cellular immunity (Lillehoj and Choi, 1998), whereas IL-1β, produced by macrophages, monocytes and dendritic cells, is a major pro-inflammatory cytokine that mediates innate immunity. A recent study moreover revealed that a Bacillus-based diet significantly induced inflammatory and anti-inflammatory cytokines in the jejunum and ileum of broiler chickens (Rajput et al., 2013).

Type I IFN production is a typical innate defense against viral infection and the expression of antiviral proteins contributes to viral clearance. The expression of MX and OAS increased in the thymus and spleen of mice infected with West Nile virus (WNV), thereby suggesting that the expression could be involved in protection against WNV (Venter et al., 2005). In this study, IFN-α/β expression were significantly induced in the thymus and spleen at 14 dph. Accordingly, the expression of MX, PKR, and OAS increased significantly in the spleen at 14 dph, which suggests that type I IFNs can activate the expression of many IFN-stimulated genes (ISGs), including MX, PKR, and OAS, which can interfere with virus replication. Although the expression of type I IFNs in the thymus was also up-regulated at 14 dph, no significant difference occurred in the ISGs, not even a slight down-regulation. In response to that, the underlying mechanisms to this observation should be studied.

To our knowledge, this is the first report of the innate immune response to dietary B. subtilis in ducks. B. subtilis was recognized by PRRs, which triggers the innate immune response of the host. Moreover, changes in the gut microbiota due to the B. subtilis addition may have also led to the different activation of PRRs. In this study, the expression of TLRs and RIG-I-like receptors (RLRs) were up-regulated in the thymus and spleen. Previous research showed that TLR3 plays a critical role in the expression of pro-inflammatory cytokines, such as IL-6 and IL-8 (Le et al., 2007). The activation of duRIG-I signaling induces the expression of downstream innate immune genes including Mx, PKR, and IFN-β (Barber et al., 2013). Since MDA5 is a strong inducer of antiviral molecules and pro-inflammatory cytokines such as IL-2, IL-6, IFN-α, and IFN-γ (Wei et al., 2014), the upregulation of cytokines and antiviral proteins expression might have stemmed from the activation of these receptors.

Previous studies have reported that the lipopolysaccharide and bacterial modulins of bacteria induce cytokines (Aldapa-Vega et al., 2016). Bacterial adhesion can induce the release of cytokines. Pathogens are able to not only stimulate host cells to produce cytokines, but also to synthesize proteins or toxins directly or indirectly inhibit the production of cytokines in host cells and cause diseases (Henderson et al., 1996). B. subtilis has many beneficial effects on hosts, it activated the immune response of the spleen, thymus and other organs related, and regulated host innate immunity (Lee et al., 2015). However, innate immune responses differed in the thymus and spleen with dietary B. subtilis supplementation in our research. Considering the complexity of the mucosal immune system, drawing conclusions about results from immunomodulatory studies tend to be rather difficult. For that reason, increased, decreased, or unchanged gene expression of different cytokines cannot be interpreted strictly as a benefit or detriment (Lähteinen et al., 2014). As such, more detailed information about active crosstalk between supplemented probiotics and host mucosal immune system is clearly necessary to better understand immune-related alterations induced in the ducks gut.

Probiotics enhance the resistance of aquatic animals against pathogens by improving the non-specific immune system as previously described (Nayak, 2010). Dietary administration of lactic acid bacteria significantly increased the survival rate in orange grouper fish or Epinephelus coioides challenged with Streptococcus sp. and grouper iridovirus (Son et al., 2009), as well as in white shrimp or Litopenaeus vannamei, challenged with Vibrio alginolyticus (Chiu et al., 2007). The results of the current study indicated that dietary B. subtilis immunologically modulated hosts to increase their defense capability against E. coli and NDRV infection. The researchers detected that the mRNA expression of TLR3 and MDA5 were significantly up-regulated in DFM before the challenge. It is generally known that TLR3 and MDA5 can recognize viral dsRNA and activate the IFN signal pathway, thereby promoting the production of cytokines (Takeda and Akira, 2005). Since the genome of NDRV is dsRNA, TLR3, and MDA5 might have recognized the NDRV and promoted the antiviral response in the thymus and spleen. Probiotics benefit hosts by producing inhibitory compounds, competing for adhesion sites, stimulating immune function, and improving the microbial balance (Fuller, 1989; Mccracken et al., 1999; Verschuere et al., 2000). As a result, the survival rate of ducks fed B. subtilis was higher than that of controls after challenged with E. coli. This conclusion takes support from increased body weights and the enhanced expression of major innate immunity genes involved in initiating and regulating immune response against E. coli and NDRV in B. subtilis-fed ducks compared with those in controls.

In sum, B. subtilis improved growth performance, immune response and disease resistance in Cherry Valley ducks. Such awareness of resistance to pathogenic microorganisms caused by probiotics should encourage the development of probiotics, and these results in general inform the relationship of probiotics and host immune response.

MG and NL designed and conducted the study, performed most of the experiments, and wrote the manuscript. GH and RL performed the calculation with support from TC. LW and BW collected samples. MG and NL performed the biological experiments. TC and LW discussed the results and revise the manuscript.