China is the world’s largest producer and consumer of waterfowl, including the meat duck, egg duck, and meat goose industries. The total value of waterfowl production has exceeded $100 billion, with duck farming accounting for 74.3% of world production and goose farming for 93.3%. They can provide large quantities of high-quality meat and down. In recent years, diseases caused by immunosuppression have become more and more prevalent in large intensive farms, and the direct or indirect losses and hazards caused by them are quite huge. Immunosuppression can lead to retarded weight gain, decreased egg production in laying hens, and decreased litter size in breeding pigs by affecting animal intake and reducing feed conversion ratios. Meanwhile, the animals are vulnerable to infection, erosion by pathogenic microorganisms, and secondary diseases, which can be fatal in serious cases. However, there is still a gap in studies related to immunosuppression in waterfowl compared to reports in chickens, pigs, and rats. The factors leading to immunosuppression are mainly divided into disease factors, human factors, and feeding environment factors. Most of the factors causing immunosuppression in ducks are viral diseases, such as duck circovirus (DCV) (Hong et al., 2018), duck euthero virus (Wang et al., 2020), duck influenza virus, duck herpesvirus type 2, duck distemper virus (DPV) (Dhama et al., 2017), etc. These diseases are characterized by damage to the immune organs and hinder the process of the humoral immune response.

Dexamethasone can cause an immunosuppressive state in animals, and it was selected as an immunosuppressive drug in this test. In experiments studying animal models of dexamethasone-induced immunosuppression pathology, more attention has been paid to changes in leukocytes and immune cells, and a lack of focus on clinical signs such as body weight (Lo et al., 2005; Harada et al., 2011; Hundakova et al., 2022). Immunosuppression led to atrophy of the thymus and the bursin, organ function was affected, and organ indexes showed a significant decrease after modeling, whereas the spleen showed no difference. It was found that dexamethasone-induced immunosuppression significantly reduced splenic lymphoid follicles in the spleen of house sparrows. But did not affect their CD3 immune effect and had a minimal effect on splenic lymphocytes in mice (Jeklova et al., 2008; Crouch et al., 2022).

Research on natural herbal medicines is critical to reducing the risk of drug resistance on farms. Echinacea, as a natural herb, possesses a wide range of medicinal effects, and it contains a great potential medical value that is worth exploring. Echinacea was already used to treat traumatic injuries, septicemia, and toothache by Indians in the 18th century. Nowadays, it is more commonly used to treat skin diseases and to combat respiratory diseases such as influenza and asthma in Western countries (Aarland et al., 2017). A large number of studies have also reported that EE can exert immunomodulatory effects by affecting immune system mechanisms in different ways (Block and Mead, 2003; Randolph et al., 2003; Sharifi-Rad et al., 2018), such as activating immune cells and promoting the secretion of interferon-α (Zhai et al., 2007). However, the effects of its interaction with the intestinal flora on the immune system are still inconclusive.

It has been found that the immune regulation of the body is inseparably related to gut microbiota (Hansen et al., 2010). The gut microbiota is a system composed of a large variety of bacteria, including beneficial, harmful, and neutral bacteria. These microbiotas play a key role in digestion and absorption, growth and development, immune regulation, and physiological and structural changes in the intestine (Liu et al., 2009; Quinteiro-Filho et al., 2012). The immune function of the host is closely linked to the dynamic balance of the gut microbiota (Yamashiro, 2017; Liu et al., 2021). Normal flora has an important role in promoting the maturation of immune cells and tissues, while the presence of imbalances in the flora, is associated with the development of infectious and inflammatory diseases such as bacterial vaginosis, inflammatory bowel disease, and rheumatoid arthritis (Srinivasan et al., 2012; Ferreira et al., 2014; Trompette et al., 2014; Wagenaar et al., 2021). The gut microbiota can affect the host’s immune system in direct or indirect ways. The flora directly eradicates pathogenic competitors by competing for nutrients and ecological niches, acting as a biological barrier together with the intestinal mucosa; or indirectly influencing the host’s immune system through flora metabolites, enhancing its defense mechanisms (Kamada et al., 2013). For example, SCFAs are common metabolites of the flora, mainly produced by Firmicutes and Bacteroidota. They provide energy to intestinal epithelial cells, maintain the integrity of the intestinal mucosa, balance the pH of the intestinal microenvironment, have a positive regulatory effect on intestinal immune cells, and exert an inhibitory effect on intestinal inflammation (Correa-Oliveira et al., 2016; Parada et al., 2019; Blaak et al., 2020).

Abnormalities in the species, ratio, and the number of gut microbiota could occur due to medical origin, drug abuse, and other problems. The immune regulation and metabolic function of gut microbiota will be affected as the homeostasis of the microbial population are out of order. As a result, changes in the intestinal flora may lead to disruption of the normal immune response process and even immunosuppression. It may also lead to changes in the microenvironment in the intestinal tract and abnormalities in the digestive and absorption functions of the animal. This effect can affect the increase in body weight, decrease in meat yield, increase in feed weight ratio, etc., causing economic losses to the farm (Choi et al., 2014).

In this experiment, we analyzed the effect of EE on the treatment of the dexamethasone immunosuppressed duck model by the 16 s-RNA intestinal flora sequencing method and explored the relationship between the immunomodulatory effect of EE and intestinal flora.

Dexamethasone-induced immunosuppression significantly inhibited the growth performance of ducks. It included a significant slowing of body weight gain, and a marked reduction in the thymus and bursal index (p < 0.001). In the trial, immunosuppression damaged the normal structure of the bursa of Fasciola and reduced the number of lymphocytes. And this damage was significantly relieved by the administration of Echinacea extract and restored the number of lymphocytes to a healthy level. The above results indicated that Echinacea extract could effectively repair the damage of the bursa of Fasciola, promote lymphocyte proliferation and improve the immune organ index. It was reported in several studies that the immune-enhancing effects of the polysaccharide components of herbal medicine were mainly achieved by significantly increasing the levels of TNF-α, IFN-γ, and IL-2 in serum (Fan et al., 2013; Zhou et al., 2018; Liu et al., 2022; Nam et al., 2022), and Echinacea extract also increased the levels of these three cytokines in the serum in this trial. It is worth considering that there is a link between changes in serum levels of immune-related cytokines and changes in the intestinal flora. Several studies have reported that the flora is involved in host immunity mainly through their metabolites as signaling factors, acting on immune cells and regulating the expression as well as the release of anti-inflammatory or pro-inflammatory factors. For example, butyric acid in SCFAs can inhibit the proliferation of Th1 cells (Guilloteau et al., 2010), the main cytokines secreted by Th1 cells are TNF-α, IFN-γ, and IL-2, so butyric acid can inhibit the secretion of pro-inflammatory factors and play an immunomodulatory role; or lipopolysaccharide in the flora can promote the secretion of pro-inflammatory factors and induce chronic systemic inflammation (Nicholson et al., 2012).

The intestinal flora, as another organ of the animal body, is not only involved in the digestion and absorption process but also influences the immune process of the animal body by metabolizing and synthesizing essential nutrients needed by the body. The intestinal flora has a crucial role in the development and maturation of the immune system. Lack of colonization of the intestinal flora reduces metabolites associated with the development of the body’s immune organs and tissues, thereby inhibiting the development of the body’s immune function, which may be defective, as is common in germ-free and neonatal animals (Ennamorati et al., 2020; Yang and Cong, 2021). During colonization, infection training enhances the resistance of the microbiota to infection, while stimulating the host immune system to respond, which can promote the development and maturation of the immune system (Butel et al., 2018; Stacy et al., 2021). The absence of specific intestinal flora may affect the maturation and differentiation of immune cells, such as CD4+ T cells in the spleen (Ostman et al., 2006) and Th17 cells in intestinal lymphoid tissue (Ivanov et al., 2009). Conversely, deletion of immune organs can likewise affect the stability of the gut microbiota, as splenectomy can result in abnormal intestinal flora composition in mice (Wei et al., 2021). Immunosuppression can lead to changes in the intestinal flora, which in turn can cause many problems. In this study, Echinacea purpurea was found to regulate the changes in flora caused by immunosuppression.

The sequencing results revealed a recovery trend in the EE group. Although no significant differences were seen in the alpha-diversity indices (p > 0.05), the EE group showed an opposite trend in each index compared to the M group, gradually returning to healthy levels. There were similar reports in the intestine of immunosuppressed mice (Fang et al., 2019; Li et al., 2021). Remarkably, immunosuppression significantly increased the abundance of Megamonas and Prevotellaceae_UCG_001, while Akkermansia, Alistipes, and Butyricicoccus were significantly reduced. In contrast, Echinacea extract is effective in alleviating these changes in flora and may even increase the abundance of beneficial bacteria to improve the immune deficiency of the body. The relative abundance of Prevotella、Prevotellaceae_UCG_001 was significantly higher in the immunosuppressed group. Both could be potential biomarkers in dexamethasone-induced immunosuppression observed in the Lefse analysis. Prevotella is strongly associated with systemic and chronic inflammation. Prevotella copri may increase the probability of developing colitis by affecting the structure of the flora when colonized in the mouse intestine (Scher et al., 2013). Prevotella intestinalis colonization may affect the metabolic processes of the intestinal flora, exacerbating intestinal inflammation and potentially systemic autoimmunity (Iljazovic et al., 2021). Some studies have reported a positive association between Prevotella and HIV-induced intestinal inflammation (Dillon et al., 2016). However, further research is needed to uncover the relationship between Prevotella and immunosuppression.

Megamonas, together with Bifidobacterium, can act as beneficial bacteria to regulate the composition of the gut microbiota to promote the synthesis and secretion of SCFAs (Dillon et al., 2016; Wu et al., 2022). In the immunosuppression model group, its elevated abundance may be more associated with the positive aspects. The decrease in its relative abundance correlates with the activation of abnormal immune responses, such as in the intestine of patients with Crohn’s disease (Maldonado-Contreras et al., 2020), immune thrombocytopenia (Yu et al., 2022), or IgA nephropathy (Dong et al., 2020), where its abundance is significantly reduced. It suggests that the relative abundance of Megamonas is related to the immune status of the organism. Its abundance increases when the immunity declines, while it decreases significantly with abnormal activation in the immune response.

Akkermansia is a genus of beneficial bacteria that has received recent attention in research reports. Its Akkermansia muciniphila could enhance the activity of immune cells by being injected intravenously into mice to reduce the tumor burden in mice (Dong et al., 2020; Luo et al., 2021). Its colonization of the intestine increases the expression of genes involved in the immune response, producing IL-8 to participate in the host’s mucosal immune regulation. It also produces mucins that positively works on intestinal epithelial cells to maintain the integrity of the intestinal epithelial mucosa (Derrien et al., 2011; Reunanen et al., 2015). Immunosuppression significantly reduced the relative abundance of Akkermansia in the gut to only 0.67% in the immunosuppressed model group. Its low abundance may lead to the absence of the functions described above and put the already immune dysregulated hosts at increased risk of disease infection. However, its relative abundance was significantly higher in the EE group supplemented with Echinacea extract, enhancing the protective effect on the intestine and modulating mucosal immune function. It also displays significant anti-inflammatory properties in the intestine, effectively relieving DSS-induced acute colitis (Qu et al., 2021). Echinacea extract may improve intestinal mucosal immune function and restore host immunity by increasing the abundance of Akkermansia in the gut of immunosuppressed ducks. It also enhances the immunity by increasing the abundance of Alistipes. Because Alistipes could bind to TLR4 and activate the expression of TNF to enhance the immune clearance of tumor cells (Iida et al., 2013). However, there is no definitive evidence for the main components of Echinacea extract that act with the flora.

The gut microbiota interacts with the host primarily through metabolites produced during the metabolism of the flora. The prediction of the metabolic function of the flora revealed that immunosuppression had a significant effect on the metabolism of the flora and involved metabolic pathways associated with some human diseases and organism systems. While Echinacea extract antagonized the effect of immunosuppression on the mycota and increased the abundance of mycota associated with metabolic functions of human diseases and organic systems. The classification based on the KO database revealed that KO3671 is associated with the immune system. It has a regulatory role not only in plant immune responses (Mata-Perez and Spoel, 2019) but also in mammals，playing a role in the regulation of immune signal release (Kim et al., 2008; Mougiakakos et al., 2011). It mainly through its protection of cells against oxidation and thus reducing immune cell apoptosis positively affects the immune system (Lu and Holmgren, 2012). Akkermansia, Alistipes, Butyricoccus, and Bacteroides, whose relative abundance increased in the EE group, were found to have genes corresponding to KO3671 in the functional prediction. We speculate that the increased abundance of the genus mentioned above may have increased the Trx content in the intestine, exerting its enhancing and modulating effects on the immune system. It could be one of the pathways of immune function modulation by Echinacea extract, but more evidence is needed to prove it.

To sum up, Echinacea extract can significantly alleviate the immunosuppressive effect of dexamethasone on ducks. It mainly contributes by improving the growth performance of immunosuppressed ducks, restoring the function of immune organs, and regulating the level of immune-related cytokines in the serum. 16 s-rRNA sequencing identified Prevotella as a potential biomarker for dexamethasone-induced immunosuppression. Echinacea extract may modulate intestinal immune function by increasing the abundance of beneficial bacterial genera such as Akkermansia and Alistipes in the intestine. The trial provides a possibility for the application of Echinacea in waterfowl and enriches the research on immunosuppression in waterfowl.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: https://www.ncbi.nlm.nih.gov/, PRJNA895924.

The animal study was reviewed and approved by the Institutional Animal Welfare and Research Ethics Committee of South China Agricultural University, Guangzhou, China.

RL, CZ, YS, JC, DG, and SL: were responsible for study conception and design. DS: revised the manuscript. RL, CZ, YS, JC, DG, SL, and DS: were involved in the drafting of the manuscript. All authors contributed to the article and approved the submitted version.

The study was supported by the General Project of Guangdong Provincial Natural Science Foundation (2021A1515011010), the Key R&D Project of Guangzhou City (202206010189) and the Project of Young Innovative Talents of Guangdong General Universities (2022KQNCX269).

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.

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