Colibacillosis caused by Escherichia coli is one of the most common and important diseases in poultry industry as it leads to reduced weight gain, increased mortality, and reduced flock uniformity (Nolan et al., 2020), causing serious economic losses more than 5.7 billion pounds for producers in 2017 (National Agricutural Statistics Service [NASS] And USDA, 2017; Wibisono et al., 2018). Although E. coli as a normal inhabitant can be found in the intestinal tract of birds, certain strains can cause respiratory and systemic diseases that known as avian pathogenic Escherichia coli (APEC) (Guabiraba and Schouler, 2015). Respiratory tract and oral cavity are the initial sites where APEC enters the whole body. The body recognizes the pathogen-associated molecular patterns (PAMPs) on APEC through Toll-like receptors (TLRs), thus inducing immune response (Roy and Mocarski, 2007). Antibiotics usually used to prevent and control colibacillosis, but the long-term use of antibiotics leads to antibiotic residues in poultry products and the emergence of multidrug-resistant bacteria, making the prevention of colibacillosis more difficult (Yuvraj, 2019). Therefore, how to prevent and control the negative impact of E. coli on the meat duck industry has become a problem that needs to be studied.

Zinc is an essential micronutrient with multiple effects on enzymatic reactions, DNA synthesis and many cellular functions. Its biological functions include promoting animal growth and development, enhancing antioxidant and immune capacity, and improving the quality of poultry products and intestinal health (Chien et al., 2006; Liu et al., 2011). Emerging research shows that zinc has an important function in protecting from infection of pathogens. Zhang et al. (2012) showed that zinc alleviates intestinal injury by increasing the ileal villi height and V/C ratio while upregulating the expression of Occludin and Claudin-1 mRNA in Salmonella-infected broilers. Besides, zinc supplementation increased the populations of Lactobacillus and decreased epithelium cell apoptosis index in broilers challenged with Salmonella (Shao et al., 2014). Inorganic zinc, such as ZnO and ZnSO₄, is mainly supplemented in the diet to meet the zinc requirements of animals due to its low price. In comparison, organic zinc exhibited more stable biochemical properties, higher bioavailability and better resistance to infection (Esfahani et al., 2021). Excessive supply of Zn sulfate (ZnSO₄, 120–240 mg/kg diet) more than the Zn requirement (60 mg/kg diet) recommended by the National Research Council (National Research Council, 1994) in diets designed for ducks is used to promote growth and intestinal health, possibly by modulating the integrity of intestinal barrier function (Wen et al., 2018). Zinc methionine (Zn–Met), as an organic zinc source, is better than inorganic zinc in improving immunity and anti-stress (Kidd et al., 1994; Saleh et al., 2018). Saleh et al. (2018) found that dietary Zn–Met enhances humoral immune response and antioxidant enzyme activity of heat stressed broilers. Wei (2019) reported that supplemented with Zn–Met posed to anti-diarrhea and growth promotion by enhancing average daily gain (ADG) and reducing diarrhea rate and serum D-lactate content, meanwhile upregulating jejunal tight junction protein mRNA expression of Occludin and Claudin-1, ZO-1 mRNA in newborn calves challenged with E. coli K88. In addition, zinc can reduce the expression of E. coli virulence factors, inhibit bacterial attachment and internalization, destroy cell membrane and lead to cell content leakage, and finally kill E. coli (Liu et al., 2009). Zinc can effectively eliminate E. coli by inducing the expression of antibacterial substances such as antibacterial peptide (Talukder et al., 2011). However, up to now, there has been no study examining the use of dietary Zn–Met supplementation and its effects on infected meat ducks. Therefore, the objective of the current experiment was to investigate the effects of supplementation of Zn–Met on growth performance, immune function, and intestinal health in meat ducks challenged with APEC.

To our knowledge, avian colibacillosis is triggered by APEC, induces a lot of deleterious effect, such as high mortality and decreased productivity, which imparts a severe economic loss on poultry production. The side effect of E. coli challenged on growth performance was reported in chicken (Zhang et al., 2014), piglet (Rodrigues et al., 2020), calf (Bi et al., 2017) and was confirmed in this study. In the present study, BW and BWG were reduced and mortality was sharply increased after APEC challenge, whereas dietary Zn–Met tended to alleviate the reduced BW and BWG. In addition, C120 significantly decreased mortality compared to C0. Although zinc is beneficial to animals in normal physiological states, there were only a few reports about Zn–Met on E. coli infection so far. Wei (2019) reported that dietary Zn–Met significantly improved growth performance by increasing ADG and decreasing the incidence of diarrhea of newborn dairy calves challenged with E. coli. Chen et al. (2019) also found weaned piglets fed with Zn–Met exhibited higher ADG and lower diarrhea rate after E. coli infection compares to control group. Furthermore, organic zinc exerts a protective role in other pathogens challenged model, such as coccidia (Bortoluzzi et al., 2019) and Salmonella (2014). The promotion effect on growth performance may be attributed to the fact that Zn–Met, as a source of organic zinc can be absorbed more efficiently than inorganic zinc, especially in case of impaired intestine (Yu et al., 2017; Wu et al., 2019). However, further investigations are needed to validate the results of this study due to the lack of literature about the effect of Zn–Met on E. coli challenged model in poultry.

The immune organ index is one of the important indicators to evaluate the development status of immune organs and immune function of poultry, birds with heavier immune organ usually show stronger immunity (Jin et al., 2019). The thymus is the site of T-cell differentiation and maturation, and plays an important role in the establishment of the immune function (Miller, 2002). The spleen, as an important peripheral lymphoid organ, has a filtering effect on all types of tumor cells and harmful microorganisms (Lewis et al., 2019). The bursa of fabricius is a unique immune organ of birds and plays a vital role in the development of the specific B-lymphocyte lineage which produced antibody in birds (Nera et al., 2015). Consistent with the previous studies (Zhang et al., 2020), a reduction of both thymus and bursa of fabricius index induced by APEC challenge in the current study indicated an obstruction of development of immune organs, whereas the increase in spleen index may be due to congestion and enlargement of spleen result from colibacillosis (Wani et al., 2017). Moreover, in our study, Zn–Met had a promotional effect on thymus organogenesis to combat the adverse impact on thymus ensued from APEC infection. A similar study showed that the weight of the spleen and bursa of fabricius is higher in Eimeria tenella infected broilers supplemented with ZnSO₄ (Sajadifar, 2013). It may be explained by Zn is a pro-mitogen that can promote cell mitosis and affect the differentiation and growth of lymphocytes, while Zn deficiency restrains the development of lymphoid organs and the maturity and population of peripheral blood T-lymphocytes (Cui et al., 2004). These results suggest Zn–Met has a positive effect on immune organ development under challenge.

Owing to the important role in the immune system, immunoglobulins, and complement components are commonly used to evaluate the immune status of animals, and three main types, IgA, IgG, and IgM, are present in birds (Zhang et al., 2014). Immunoglobulin A plays a key role in defending mucosal surfaces against attack by infectious microorganisms (de Sousa-Pereira and Woof, 2019). Immunoglobulin G is the only immunoglobulin that can be transmitted to the offspring and function in recognition of antigens on the surfaces of invading viruses and bacteria and recruitment of effector molecules (Yanaka et al., 2020). Immunoglobulin M, as the first antibody in response to an invading pathogen, plays an important role in combating microbial infection and regulating the immune response (Gong and Ruprecht, 2020). The complement system consists of a tightly regulated network of proteins that play a key role in host homeostasis, inflammation, and in the defense against pathogens (Sarma and Ward, 2011). In this study, APEC challenge significantly increased serum IgA, IgM, and IgG, which suggests that the immune system responds to pathogen invasion even when immune organ is impaired. Similar finding was reported in previous studies by Zhang et al. (2014) in broilers. Zinc is necessary for maintenance of phagocytosis of macrophages and neutrophils (Driessen et al., 1994), and Kidd et al. (1994) found Zn–Met boosted clearance of E. coli from blood as a result of enhanced leading to a reduction of immune load. We also found that supplemental Zn–Met resulted in increased IgA, IgM, IgG, and C3 in serum. Our results are consistent with a report by Hamidi and Pourreza (2009) who observed increased serum IgA and antibody response to sheep red blood cell (SRBC) in mixed coccidial infected broilers fed with 60-mg/kg Zn–Met. In another study, Akhavan-Salamat and Ghasemi (2019) also found improvement of total and IgG antibody titers against SRBC for primary responses in Zn–Met supplemented broilers under high ambient temperature condition compared to birds fed ZnO, but no differences were observed in secondary responses. These results suggest that Zn–Met could exert a protective role in controlling APEC infection by improving immunity.

Villi height, CD and villi height/CD are the most widely used as a marker to evaluate the digestive and absorptive capacity of the gut and the maturation rate of intestinal epithelial cells (Lamb-Rosteski et al., 2008). Increasing the villi height increases and decreasing the CD together improve the nutrient absorption capability (Chwen et al., 2013). Villous atrophy and intestinal morphology disruption in broiler chickens challenged with E. coli (Mohebodini et al., 2019). Our study showed that APEC challenge decreased the jejunal VH and V/C, indicated a disturbance of intestinal villi-crypt structure. Furthermore, dietary Zn–Met supplementation increased the jejunal VH and V/C at 2 DPI and 8 DPI whether with or without APEC challenge. Similar studies have shown that the VH and V/C enhanced when feeding 120-mg/kg Zn in broilers challenged with Salmonella (Shao et al., 2014). Previous studies in vitro found that Zinc may promote intestinal epithelial wound healing by enhancement of epithelial cell restitution through a TGFβ-independent mechanism (Cario et al., 2000). In addition, zinc can effectively inhibit apoptosis by suppressing oxidative stress and death receptor-mediated pathways (Zhou et al., 2008). These results indicate that zinc can repair the intestinal injury resulted from APEC infection in meat ducks.

Inflammation is a pervasive phenomenon coordinated by multiple cytokines and immune signaling molecules that can arise in response to infection, injury and other adverse factors (Ashley et al., 2012). Although inflammation is a normal process for repair of tissue after an injury and cytokines are the bridge of cell–cell communications, uncontrolled inflammation represented by cytokine storm causes unwarranted energy wastage, irreversible organ damage, and unbearable pain (Gupta et al., 2019). Infections caused by pathogens are usually accompanied by a surge in pro-inflammatory cytokines, such as IL-1β and IL-6 (Oh et al., 2019). In this study, the mRNA expression of IL-1β, IL-6, IFN-γ, and TNF-α was significantly upregulated by APEC challenge compared with unchallenged group, which is consistent with the observations in broiler chickens (Ateya et al., 2019) and piglets (Wan et al., 2019). Meanwhile, dietary Zn–Met supplementation tended to downregulate IL-1β (p = 0.061) at 2 DPI. Similarly, Bortoluzzi et al. (2019) reported that organic zinc supplementation downregulated IL-8 mRNA expression and upregulated IL-8 in jejunum of broiler chickens challenged with coccidia plus Clostridium perfringens. Moreover, organic zinc was found to be more effective in reducing serum IL-1β concentration of hens after administered with lipopolysaccharide (LPS) compared to ZnSO₄ (Cheng and Guo, 2004).

Pattern recognition receptors (PRRs), which are specific molecules on host cells that recognize PAMPs, the specific target molecules of pathogens, and TLRs are crucial members of the PRRs (Kawai and Akira, 2010). Generally, TLR4 primarily recognizes LPS in the cell wall of host extracellular pathogens, and E. coli can produce a variety of toxins, including LPS. When TLR4 is activated, the cytosolic TIR domain of TLR4 binds to myeloid differentiation factor 88 (MyD88) and then activates NF-κB through cascade reaction, which eventually induces the expression of pro-inflammatory cytokines (such as IL-1β, IL-6, TNFα), chemokines, and immune co-stimulatory molecules (Liu et al., 2017). We found that, the mRNA expression of TLR4, Myd88 and NF-κB increased in APEC challenge ducks compared to non-infected ducks, which is similar to Huang et al. (2019) who reported that the mRNA and protein abundance of TLR4, Myd88, NF-κB in jejunum and ileum in E. coli-challenged piglets. Zn–Met supplementation tended to downregulated the mRNA expression of TLR4 and NF-κB in jejunum. The results are similar to previous studies (Hu et al., 2017), it was found that CS-Zn supplementation downregulated the protein expression of TLR4 and its downstream signals NF-κB, IKKβ, and IκBα, along with a reduction of IL-2, TNF-α, and IFN-γ. According to previous studies, zinc acts as a crucial immune mediator against infection and inflammation, zinc deficiency enhances the generation of IL-1β and TNF-α in myeloid cells via epigenetic effects (Wessels et al., 2013). Zinc supplementation inhibited the activation of NF-κB by upregulating the gene expression of A20 and the binding of its transactivating factor to DNA, leading to decreased production of inflammatory cytokines (Prasad et al., 2011). Combined with a reduced expression of pro-inflammatory cytokines, it can be concluded that Zn–Met alleviates the inflammatory response by reducing the expression of inflammatory cytokines possibly through suppressing the TLR4/MyD88/NF-κB pathway, which may partially explain the reduction of mortality.

The intestinal barrier ensures the entrance of essential nutrients from the intestinal lumen and also effectively prevents harmful substances in the intestinal lumen (such as bacteria and various toxins) from entering the blood circulation and internal organs through the intestinal mucosa (Vancamelbeke and Vermeire, 2017). The barrier is consisted of physical, chemical, immunological, and microbiological components. The impaired intestinal barrier allows pathogens and toxins to enter the blood circulation more easily, thus causing the translocation of bacteria and toxins, which can easily lead to enterogenic infections and even to systemic inflammatory responses or multi-organ failure (Wageha et al., 2017). The physical barrier is composed of intestinal epithelium and junctional complexes, which mainly include desmosomes, adherens junctions (also known as zonula adherens), and tight junctions (such as occludin and claudins) (Groschwitz and Hogan, 2009). In this study, the mRNA expression of ZO-1, ZO-2, ZO-3, OCLN were downregulated in jejunum after APEC challenge, indicating the destruction of intestinal physical barrier, which is consistent with report of Gao et al. (2013) and Yang et al. (2014). Additionally, we found that dietary supplementation of Zn–Met to meat ducks challenged by APEC remarkably reversed the physical barrier injury to a certain extent by the improved level of ZO-2, ZO-3 and OCLN. This was consistent with the report of Zhang et al. (2012) that Zn improved relative expression of Claudin-1 and OCLN in ileum of broiler chickens challenged with Salmonella.

Secretory IgA (sIgA) secreted by epithelial cell is the main effector of the immunological barrier, its role is not only in the protection of the intestinal epithelium from enteric pathogens and toxins, but is also important for communication with gut-associated lymphoid tissue and restoration of homeostasis in pathology (Corthésy, 2013). Avian β-defensins (AvBD) are important components of host defense peptides (HDPs) that targeting a broad spectrum of pathogens through direct binding and lysis of microbial membranes, and neutralizing bacterial endotoxins (Dijk et al., 2008). Several HDPs possess different degrees of antibacterial activity for E. coli, S. Enteritidis, S. Typhimurium, C. jejuni, C. perfringens in vitro and protection from early chick mortality caused by APEC (Nguyen et al., 2021). The APEC challenge stimulated the mRNA expression of sIgA and AvBD2, which are similar to Cao et al. (2013) and Li et al. (2016). Zn–Met supplementation had no effects on the mRNA expression of sIgA and AvBD2, but Bun et al. (2011) found that organic zinc addition increased the cecal concentration of sIgA in broilers challenged with E. tenella compared to the control. The discrepancy may result from the difference of zinc sources and pathogens.

Mucin 2 (MUC2) is the major gel-forming mucin of the chemical barrier, which serves as a biophysical layer against the luminal hostile environment (Liu et al., 2020). The jejunal MUC2 concentration reduced after APEC challenged, the result is consistent with the report of Lee et al. (2017) that a reduction of ileal MUC2 mRNA expression in LPS-induced broiler chicken. Meanwhile, the jejunal MUC2 concentration increased with the level of Zn–Met supplementation in ducks whether APEC challenge or not. Similar study was reported by Wen et al. (2018) who found that ZnSO₄ supplementation markedly upregulated the MUC2 mRNA expression of jejunum in Pekin ducks. But Bortoluzzi et al. (2019) showed that neither ZnSO₄ nor organic Zn had an effect on the MUC2 mRNA expression in the jejunum of broiler chickens challenged with coccidia plus C. perfringens. The reduction of MUC2 secretion may be due to a decrease in the number goblet cells at early infection, however, MUC secretion is increasing with the restoration of immune function and the positive effect of zinc (Kim et al., 2015; Zhang et al., 2016). A study in vitro found that Zn supplementation enhanced intestinal barrier function by activating of PI3K/AKT/mTOR signaling. In addition, Song et al. (2015) reported that ZnO improved intestinal barrier function of weaned pigs by activating the ERK1/2 signaling pathway and inhibiting the JNK and p38 signaling pathway. So, more investigations are needed to elucidate the underlying mechanism of the effects of zinc on intestinal barrier.

Numerous commensal microbiota inhabited in the gastrointestinal tract can not only participate in the digestion and absorption of nutrients, but also plays a crucial role in the prevention and resolution of pathogenic infections. On the one hand, microbiota can resist pathogens by producing bacteriocins or microcins with antibacterial and bactericidal activity in a direct manner. On the other hand, microbiota defense against intestinal pathogens by regulating the processes of differentiation of T-cells, B-cell development, and antibody production in an indirect manner (Ubeda et al., 2017). Intestinal microbiota homeostasis is disrupted after pathogen infection, which may affect animal growth (Chen et al., 2020). In the current study, the cecal microbial population were measured by q-PCR. It was observed that the number of E. coli was increased, while the number of Bifidobacterium and Lactobacillus was reduced after APEC infection, which is similar to Zhang et al. (2014) reported in broiler chickens, indicating the out-of-balance of intestinal microbiota in meat ducks. Dietary Zn plays an important role in modulating the gut microbiota, massive studies have shown that pharmacological dose of Zn possess a positive effect on concentrations of short chain fatty acids, lactic acid bacterial, and other anaerobic bacterial growth, bacterial richness and diversity (Xia et al., 2017; Sun et al., 2019). The Zn deficiency leads to distinct taxonomic changes and reduction of overall species richness and diversity, establishing a microbial profile resembling that of various other pathological states (Spenser et al., 2015). In this study, we found that Zn–Met supplementation reversed the deleterious effect of APEC challenge by increasing Bifidobacterium and Lactobacillus counts, and decreasing E. coli counts. Shao et al. (2014) demonstrated that Zn supplementation increased the populations of the total bacteria and Lactobacillus group in cecal contents of Salmonella-infected broilers. Through 16S rRNA sequencing analyses, Feng et al. (2020) reported that dietary chitosan-chelated zinc (CS-Zn) reduced the relative abundance of Proteobacteria, as a potential diagnostic signature of dysbiosis and risk of disease, at the phylum level, while decreased the relative abundance of Desulfovibrio, Peptococcus, and increased Lactobacillus, Romboutsia, Anaerotruncus at the genera level in rats challenged with E. coli. Further research found the function of enrichment of carbohydrate metabolism and the metabolism of cofactors and vitamins were dramatically increased in CS-Zn treatment with the change of intestinal flora. However, our results did not evaluate gut microbiota community structure from different taxonomic levels, which should be further investigated.

In conclusion, dietary Zn–Met supplementation alleviated the deleterious effects induced by APEC challenge. Inclusion of 120-mg/kg Zn–Met enhanced immune function by stimulating the development of immune organ, secretion of immunoglobulins, and promoted intestinal health by improving intestinal barrier downregulating of pro-inflammatory cytokines and regulating of the cecal microbiota, ultimately improving the growth performance in APEC-challenged meat ducks.

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

The animal study was reviewed and approved by the Animal Health and Care Committee of Sichuan Agricultural University (No. 20180718).

JM: conceptualization, methodology, and writing original draft preparation. YC and ZZ: software, data curation, and revising the manuscript. TY, FW, and HZ: visualization and investigation. BW, GL, XC, GT, and JC: supervision, software, and validation. GJ: writing–reviewing and editing. All authors contributed to the article and approved the submitted version.

BW was employed by the company Chelota Group. The remaining 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.

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