Glucose oxidase reportedly exhibits growth-promoting properties in animals, including ducks, broilers and pigs (Liu et al., 2020; Tang et al., 2016; Wu et al., 2019b), while also exerting non-toxic and low-residue antibacterial and antifungal effects (Wang et al., 2018; Wong et al., 2008). In the present study, although GOD supplementation did not markedly increase mouse growth performance, it effectively reduced the weight loss, mortality rate, pathogen colonization and translocation associated with ST infection. Thus, GOD pretreatment could inhibit ST infection and suppress the weight loss and mortality in ST-infected mice.
After infecting the host, Salmonella propagates rapidly and spreads through the bloodstream to invade the liver, spleen, heart, and other organs, causing pathological changes in animal internal organs, which then affect host physiological activities, and ultimately leads to animal death (Herrero-Fresno and Olsen, 2018; Troxell et al., 2015; Wu et al., 2018). The present study illustrated that the relative weight of both liver and spleen did not increase in the GOD group in normal mice, while these parameters increased significantly after ST infection. This symptom may be caused by ST pathological damage to the mice, and is consistent with the results of Wang et al., who reported that the relative weight of the spleen and liver in mice was markedly increased in ST-challenged groups (Wang et al., 2021b). The findings of the current study also indicated that orally administered GOD abrogated the higher liver and spleen index caused by ST infection, indicating that GOD pretreatment, similar to the GM pretreatment, exerted protective effects against tissue injury induced by Salmonella infection.
ST-induced intestinal injury is one of the most common characteristics of its infection. Its major pathological damage to the cell is architectural disorganization, resulting in reduced villi height (Zhang et al., 2020). The findings of the present study show that the villi of ileum were thickened with markedly reduced villi height following ST infection, indicating that the ST-induced intestinal injury model was successfully established. Thus, the height of the villi determines the surface area available for intestinal absorption and affects the growth and development of animals (Wilson et al., 2018). After ST infection, the height of villi and the villi height to crypt depth ratio decreased, indicating that the intestinal structure and its absorption function might be impaired (Li et al., 2021). GOD or GM pretreatment effectively alleviated these effects. These results confirm the findings of previous studies that GOD supplementation increase villi height in the jejunum of ducks (Liu et al., 2020). Hence, GOD pretreatment could alleviate the intestinal damage induced by ST-infected.
ST challenge can lead to impaired intestinal mucosal barrier function (Yu et al., 2018). The epithelial barrier is one of the most important components of the intestinal mucosal barrier to prevent the transmission of macromolecules (Martini et al., 2017). Epithelial cells form a continuous and complete physical barrier, with tight junctions (TJ) between each cell. The destruction of TJs results in increased permeability to luminal antigens and bacteria, and decreased mucosal barrier function. Several bacterial pathogens can alter TJ by altering the TJ-associated proteins (Zhang et al., 2019). Zhang et al., indicated that ST may destroy the structure and function of TJs, thereby destroying the integrity of the epithelial barrier (Zhang et al., 2012). Herein, the qRT-PCR results demonstrated that GOD or GM pretreatment enhanced intestinal TJs by up-regulating the transcription level of tight junction proteins ZO-1, occludin, and claudin-1.
The liver, which is constantly exposed to foreign material, owing to gut translocation and first-pass metabolism from the bloodstream, plays a key role in various physiological activities, such as nutrient metabolism, blood detoxification, and the immune response (Nguyen et al., 2014). Previous studies have shown that Salmonellae infections are accompanied by damage to the liver, spleen and other organs (Fabrega and Vila, 2013; Song et al., 2020), as evidenced by inflammatory cell infiltration, severe congestion, and hepatocyte apoptosis due to the complex interaction between the intestine and liver through the gut-liver axis (Abatemarco et al., 2018; Wei et al., 2019). As expected, ST infection led to the liver cells becoming shrunken and severely congested. We also found that ST infection markedly increased apoptosis and the expression of the pro-inflammatory protein caspase-1, which were effectively inhibited by GOD pretreatment.
Inflammation is considered to be the basic mechanism of pathophysiology in intestinal injury (Ma et al., 2021). The relative increase in the abundance of certain inflammatory factors may weaken the expression of ZO-1, cause intestinal barrier dysfunction, and promote inflammation of the intestinal mucosa (Ogawa et al., 2018). NO is a highly soluble, reactive free radical as well as a potent intercellular and intracellular signaling molecule that acts as a pro-inflammatory agent (Beckman et al., 1990; Levy et al., 2005). Moreover, NO can increase the cellular expression of TNF-α and IL-1β, and enhance the ability of the cell to produce H2O2 (Lander et al., 1993; Magrinat et al., 1992). We found that ST infection resulted in a dramatic increase in NO abundance in both the ileum and colon, as well as increased MPO activity, an indicator of an increase in the number of neutrophilic granulocytes. Interestingly, the adverse effects of ST infection were abrogated by GOD or GM pretreatment, indicating a reduction in tissue injury and neutrophil infiltration.
Increasing experimental evidence agrees with the viewpoint that the abnormal regulation of the mucosal immune system in response to infection by enteric bacteria leads to intestinal injury (Guarner 2005; Nozawa et al., 2004). Moreover, inflammation is also a key factor affecting the intestinal mucosal barrier (Huang et al., 2021). Cytokines IL-6, TNF-α, and IFN-γ are primarily derived from mononuclear phagocytes and other antigen-presenting cells, which play an important role in promoting cell infiltration and damage to resident tissues, characteristic of inflammation (Santamaria, 2003). In addition, IL-10 is a multicellular and multifunctional cytokine that is considered to be an inflammatory and immunosuppressive factor, that regulates cell growth and differentiation and participates in inflammation and immune responses (Huang et al., 2018). Therefore, the decreased levels of IL-6, IL-12, TNF-α, and IFN-γ, as well as the increased IL-10 levels induced by GOD pretreatment suggest its anti-inflammation response and protective effects on the mucosal barrier.
Ig is a specific active protein that is transformed into antibodies (IgG, IgA, IgM, IgE, and IgD) following antigen induction (Liu et al., 2020). sIgA, secreted by intestinal epithelial cells, is critical for preventing the damage induced by inflammatory bowel disease by inhibiting the attachment of pathogenic microorganisms to the gastrointestinal tract (Lammers et al., 2010). In the present study, GOD significantly enhanced the production of sIgA in normal mice. It should be noted that this also occurred in the ileum and colon after ST infection, which may be due to the adhesion of a large number of pathogenic bacteria, such as ST, in the intestine. Similar results also reported that GOD promotes serum Ig levels so as to enhance the ability to eliminate pathogenic microorganisms (Wu et al., 2019).
Previous studies have shown that certain adverse factors are associated with the application of antibiotics, particularly the disruption of intestinal bacterial diversity in antibiotic-supplemented groups (Liu et al., 2020). However, GOD supplementation helped overcome these shortcomings, which was in agreement with the findings of our current study on mice. The intestinal microbiota plays a vital role in host defense, as evidenced by its ability to regulate innate and acquired immunity at the local and systemic levels (Colliou et al., 2017). Most importantly, changes in microbial populations may be closely related to the degree of intestinal inflammation, which is also a characteristic of ST infection (Bronner et al., 2018). Throughout history, there have been reports of protection against ST infection via regulation of intestinal microbiota (Baba et al., 1991; Deriu et al., 2013). Herein, 16S rRNA sequencing indicated that GOD treatment or pretreatment had considerable regulatory effects on the intestinal microbiota; PL-SDA results further revealed that oral GOD administration restructured the flora of diseased intestines to be similar to that of the CK group.
Ruminococcaceae are primarily responsible for fermenting dietary fiber and other plant components, such as inulin and cellulose, to produce SCFAs, which can be used as energy by the host (Scheppach and Weiler, 2004) and exhibit anti-inflammatory properties in the intestine (Kles and Chang, 2006). An abundance of Lactobacillaceae is associated with T cell counts in the peripheral blood, while Lactobacillus, specifically, can effectively enhance the innate and adaptive immunity of the host (Ma et al., 2017). Furthermore, Lactobacillus species are also promising probiotic candidates as they exhibit antimicrobial activities, which are attributed to the secretion of bacteriocins or the production of organic acids. In the present study, GOD markedly increased the relative abundance of Ruminococcus and Lactobacillaceae in the intestines of normal mice. Furthermore, the decreased relative abundance of Lactobacillales, Lactobacillaceae and Lactobacillus following ST infection were abrogated by GOD pretreatment. These findings indicate that GOD can regulate the abundance of specific intestinal microbiota to provide protection against ST infection, which may also account for why GOD pretreatment was found to effectively reduce ST colonization and translocation. Nevertheless, the differences induced in the diversity of mouse fecal microbiota following application of the six treatments requires further investigation, particularly with respect to the association between the specific characterization of GOD and the metabolic products of gastrointestinal tract microbiota.