Tight junctions play a crucial role in maintaining the intestinal epithelial cell barrier, protecting the host intestine from pathogens and preventing the transmission of macromolecules (Schneeberger and Lynch, 2004). Tight junction-related proteins include occludin, zonula occludens, and claudins. Eimeria vermiformis-infection inhibits the epithelial cell mRNA expression of zonula occludens-1 in mice (Farid et al., 2008), and zonula occludens-1 downregulation or reduced activity affects the formation of intercellular tight junctions. With higher concentrations of coccidia, the expression of tight junction proteins was dose-dependently upregulated, with a simultaneous increase in gastrointestinal permeability, indicating more severe intestinal damage (Teng et al., 2020). Combined with the disruption of the mucus layer, this damage profoundly alters the interactions between the host and its microflora, allowing for greater microbial contact with the epithelial barrier and even penetration across the interface. After treatment with probiotics, the expression levels of claudin-1 and zonula occludens-1 were increased in the E. tenella-infected chicken (Memon et al., 2020). Probiotics maintain tight junction integrity of intestinal epithelial cells, mainly through the bioactive substances produced by their metabolism, to protect against pathogenic bacteria-induced damage of intestinal epithelial cells. A mixture of Bacillus subtilis and Saccharomyces cerevisiae increased the expression of tight junction-associated proteins, such as occludin, claudin-2, and claudin-3, in broiler chickens (Rajput et al., 2013).

Due to the invasion and replication of coccidia, the host cells are under pressure, which may cause apoptosis. To grow and survive in host cells, coccidia inhibit apoptosis by regulating anti-apoptotic factors. In Eimeria intestinalis-infected rabbits, the percentage of apoptotic cells in the ileum was significantly higher compared with the control group (Abdel-Haleem et al., 2017). Before the development of second-generation schizonts is completed, E. tenella may directly activate the NF-κB pathway in host cells to further inhibit host cell apoptosis. After developmental completion, E. tenella prevent the expression of NF-κB response genes and further reduce the expression of the anti-apoptotic proteins Bcl-2 and Bcl-XL, thereby accelerating host cell apoptosis and promoting the release of merozoites (Del et al., 2004). During their early development, E. tenella inhibit pro-apoptotic proteins by inducing anti-apoptotic factors to protect their cells and ability to proliferate (Del et al., 2004). Using probiotics, including B. subtilis, Clostridium butyricum, and Lactobacillus, we observed upregulated Bax expression and downregulated Bcl-2 levels in the E. tenella-infected chicken (Memon et al., 2020). Zhang et al. (2015) demonstrated that E. tenella promoted the apoptosis of cecal epithelial cells in vitro, especially during the middle to late stages. The use of specific inhibitors significantly decreased DNA injury, apoptosis, and caspase-9 and caspase-3 activity in chick embryo cecal epithelial cells after E. tenella infection (Li et al., 2017). Most probiotics inhibit the NF-κB pathway by impairing epithelial cell protease function and preventing the degradation of NF-κB (IκB) negative regulators (Jiang et al., 2012). The induction of apoptosis may become a new direction in the treatment of coccidiosis. The use of probiotics during the early stage of coccidial infection promotes the apoptosis of intestinal epithelial cells and reduces coccidial colonization and development.

The chemical barrier of the intestine consists of mucin (MUC),antimicrobial peptides (AMPs), regenerating islet-derived protein 3, lysozymes, and other factors (Okumura and Takeda, 2017). Eimeria infection significantly downregulates the gene expression of MUC2 and MUC5ac (Jiang et al., 2013), resulting in a decrease in the content of MUC in the mucus layer. This prevents mucus layer replenishment and further disrupts the integrity of the intestinal mucosal chemical barrier. Mice infected with sporulated Eimeria papillata exhibit marked goblet cell hypoplasia and depleted mucus secretion (Dkhil et al., 2013). The number of colonic cup cells gradually decreases with the development of Eimeria pragensis endogenous life cycle stages (Yunus et al., 2005). Microorganisms, such as Actinobacteria, Bacteroidetes, Firmicutes, and Verrucomicrobia (Tailford et al., 2015), use mucus carbohydrates as a carbon source. Therefore, they may not gain a competitive advantage after a reduction in mucus production. It has been proposed that coccidia stimulate mucus production in vivo, leading to an increase in the relative abundance of MUC-utilizing bacteria, such as Clostridiales (Collier et al., 2008), whose growth in vitro was enhanced by the addition of MUC (Ramanan et al., 2016). The type and glycosylation of mucoproteins in the mucus layer covering the intestinal epithelium are different due to the various colonization sites of coccidia species in the intestine (Moncada et al., 2003), and both the MUC’s composition and glycosylation are known to affect the taxa that use the mucus (Sommer et al., 2014). Therefore, parasite-driven changes in mucus may alter the microbiota.

Host defense peptides exhibit direct antibacterial activity after coccidial infection and induce the expression of MUC and tight junctional proteins to enhance mucosal barrier function (Robinson et al., 2015). After infection with Eimeria praecox, several genes were downregulated, including those that encode antimicrobial peptide 2 and the cationic, anionic, and L-type amino acid transporters (Yin et al., 2015). Similar findings were reported for E. maxima (Casterlow et al., 2011) and E. acervulina (Su et al., 2014). E. acervulina and E. maxima challenge resulted in the downregulation of avian beta-defensin, which had antibacterial effects against Actinobacillus, Candida albicans, Escherichia coli, Listeria monocytogenes, and Salmonella typhimurium species (Elahi et al., 2005). The addition of moderate concentrations of quercetin to feed exerts a regulatory effect on the ileal avian beta-defensin and toll-like receptor (TLR) signaling pathways by reducing the abundance of Clostridium and increasing the levels of Bifidobacterium, thereby maintaining the ileal microecological balance and reducing mortality. In other words, increased host antimicrobial peptide production can improve the intestinal microbiota and subsequently ameliorate the symptoms of coccidia. Lactobacillus and some gram-positive bacteria enhance intestinal barrier function by inducing the NF-κB pathway and activating activator protein-1 and mitogen-activated protein kinase to upregulate β-defensin 2 (Schlee et al., 2008). Probiotics stimulate the host to produce active molecules, such as MUC and antimicrobial peptides, which may be one of their action mechanisms to enhance the body’s resistance to coccidial infection.

Host anti-infectious strategies (including immune responses) are elicited following infection with parasites (Zhou et al., 2013). However, the immune system regulates the gut microbiota and their relative abundance to ensure a mutually beneficial host-microbe symbiosis. Eimeria species inhibit host immune responses to promote their invasion and colonization in hosts through negatively regulating the production of inflammatory cytokines (Zhao et al., 2018), thereby altering the gut microbiota.

The specific immune response to coccidiosis involves both cellular and humoral components. In infected animals, the humoral immune response indicates high titers of various antibody classes, beginning with the increase in IgM, followed by IgG, IgA, and others (Hughes et al., 1985). In an ovine model, increases in the IgG level and oocyst shedding occurred simultaneously during the primary infection and then decreased to baseline levels (Dalloul et al., 2005). Matos et al. (2018) demonstrated that Eimeria ninakohlyakimovae infected goats and revealed the increased levels of specific IgG, IgM, and IgA during the host immune response. By measuring the content of immunoglobulins and gut microbiota in inflammatory bowel disease patients, it was observed that IgG, IgM, and IgA had a positive correlation with Enterobacteriaceae and Enterococcus; while a negative correlation with Lactobacillus and Bifidobacterium. This indicates that IgM, IgG, and IgA are closely related to the imbalance in the gut microbiota, which may be caused by changes in the proportion and quantity of gut microbiota, leading to disruption of the intestinal mucosal microecological balance and abnormal immune responses. However, humoral immune reactions cannot eliminate primary coccidial infections (Daugschies and Najdrowski, 2005). Specific antibodies are reportedly produced in response to ruminant Eimeria infections, however, they are not protective. Although the specific mechanism of action by which intestinal IgA provides protection against coccidial infection remains unknown, it is hypothesized that IgA reduces the development of sporozoites or merozoites and prevents host cell invasion (Yun et al., 2000).

Although both cellular and humoral immunity are activated in response to coccidial infections (Daugschies and Najdrowski, 2005), several studies have shown that the cellular immune response mediated by T cells plays a key role in the protective immunity against coccidia. T-cell-mediated immune responses reduce the excretion of oocysts in animals infected with Eimeria bovis and mainly involve CD4⁺ and CD8⁺ lymphocytes (Sühwold et al., 2010). Matos et al. (2018) infected 3-, 4-, and 5-week-old goat kids with sporulated oocysts and subjected them to a homologous challenge 3 weeks later. The results demonstrated higher eosinophils and lymphocytes compared with challenged groups infected at 6, 7, and 8 weeks old. The activation of antigen-specific T cells from Eimeria-immune mice, cattle, and chickens has been demonstrated by lympho-proliferation assays (Lillehoj, 1986). In addition, the gut microbiota and its metabolites induce the differentiation of T cells by direct or indirect mechanisms, including T-bet⁺ Th1 cells, RORγt⁺ Th17 cells, Treg cells and GATA3⁺ Th2 cells (Lee and Kim, 2017), and coccidia colonization primarily mediates Th1 cell responses. E. bovis-mediated T cell activation was accompanied by increased levels of certain cytokines (such as IL2, IL4, and IFN-γ) known to participate in the regulation of complex networks, thereby activating the migration of immune cells to the site of infection (Taubert et al., 2008). E. tenella strongly induces an immune response and increases IL-8 and IL-6 expression in the cecum (Yu et al., 2020). Macrophages isolated from chickens infected with E. tenalla or E. maxima produced IL-1 in vitro and showed 80-fold increased mRNA levels of jejunal and cecum IL-1β after 7 days of culture. IL family members have a wide range of immunomodulatory functions and are highly beneficial for the host’s defense against coccidial infection. The administration of B. subtilis to chickens infected with coccidia increased the level of specific antibodies and regulated intestinal immunity by modulating the expression of IL-1β, IFN-γ, and CXCLi2 in the intestine (Lee et al., 2013). Lactobacillus-based feed products increased intestinal IFN-γ and IL-2 expression in chickens, resulting in a 14% reduction in fecal oocysts compared with the control group (Chaudhari et al., 2020).

Several cytokines are produced after coccidial infection, most of which have a coccidial suppressive effect in vivo or in vitro. However, some may have both pathological and immunophysiological effects. Significantly increased TLR2, TLR4, and TLR15 expression is observed after infection by coccidia (Zhou et al., 2013), and the upregulation of TLRs typically induces pro-inflammatory cytokines that regulate the immune response against bacterial infections. TLR2 mediates intestinal repair and barrier function to prevent pathogenic microorganism invasion by recognizing the cell wall components of gram-positive bacteria. In chickens, the expression level of TGF-β4 in intestinal intraepithelial lymphocytes was increased by 5- to 8-fold after coccidial infection (Jakowlew et al., 1997), and the expression of TGF-β4 in the spleen and cecum tonsils was increased by 3-fold (Song et al., 2010). The increased expression of TGF-β4 decreases the expression of IFN-γ, preventing excessive inflammation from causing damage to the organism. This may be a potential mechanism regulating mucosal inflammatory responses against intestinal microbes to maintain intestinal immune homeostasis. The current literature on immune-mediated interactions between coccidia and the microbiota is limited, and most previous studies focused on how microorganisms enhance immunity against coccidia without considering the opposite circumstance. For example, Toxoplasma gondii was found to induce TLR2, TLR4, and TLR9 signaling through the stimulation of gut microbiota and indirectly stimulate dendritic cells to activate innate and adaptive immune responses (Benson et al., 2009). In healthy organisms, the gut microbiota activates B cell receptors or TLRs to promote antigen presentation and antibody production (Buchta and Bishop, 2014). Collectively, these results suggest that coccidia and the microbiota have a complex relationship and interact across the mechanical barrier, chemical barrier and immune system ( Figure 1 ).

Various studies have demonstrated the complex interactions of coccidia with bacteria, fungi, viruses or other intestinal parasites (Motha and Egerton, 1984; Fukata et al., 1984; Ruff and Rosenberger, 1985), which may lead to more severe clinical manifestations and economic losses. Changes in the gut microbiota caused by coccidial infection provide an environment that is conducive for the reproduction of pathogenic bacteria. Coccidial infections not only enhance the colonization of Campylobacter jejuni (Macdonald et al., 2019), Clostridium perfringens (Ficko-Blean et al., 2012), Salmonella (Kogut et al., 1994) and other bacteria but also increase their pathogenicity (Dykstra and Reid, 1978). This increases livestock and poultry diseases, thereby reducing animal performance, reproductive capacity and egg production and potentially leading to death. And coccidial infection causes a marked inflammatory response in the intestine, and the presence of inflammation favors the colonization of aerobic bacteria, especially Enterobacteriaceae (Lupp et al., 2007), which have been shown to exacerbate the increase in pathogenic bacteria. Enterobacteriaceae and Lactobacillus are antagonistic, and an increase in the number of Enterobacteriaceae may inhibit the intestinal colonization by Lactobacillus (Tortuero, 1973). A reduction in anaerobic bacteria in the intestine after coccidial infection in chickens was suggested to potentially decrease the concentration of volatile fatty acids in the cecum and induce changes in pH and oxidation-reduction potential in the intestine, which may directly lead to enhanced pathogenic infection (Qin et al., 1995). For example, a reduction in Lactobacillus after coccidial infection prevents the production of large amounts of lactic and acetic acid to effectively inhibit the invasion of Salmonella enteritidis (Bjerrum et al., 2006). The damage induced by coccidia appears to promote the spread and colonization of C. perfringens deep in the mucosa, and in some cases, this extends to the crypts and causes focal necrosis (Ficko-Blean et al., 2012), leading to secondary necrotic enteritis (Hofacre et al., 1998). The severity of necrotic enteritis has been reported to be associated with an increase in Proteobacteria and a decrease in Firmicutes (Xu et al., 2018), and these changes occurred during coccidial infection. Firmicutes were important for suppressing or eliminating C. perfringens and restoring intestinal homeostasis (Fasina et al., 2016). In addition, coccidial infections significantly increased Bacteroidetes, including Bacteroidaceae and Rikenellaceae. Bacteroidetes can damage intestinal epithelial cells and increase the invasion of other pathogens, thereby inducing or exacerbating enteritis. We speculate that the increase in Bacteroidetes and decrease in Firmicutes may be related to secondary infections with bacterial diseases.

Coccidial infection, which reduces the abundance of the microbes, is associated with low immunity. Virus-coccidial co-infection reportedly increased viral replication and delayed the clearance of viruses, such as avian leukosis virus (Cui et al., 2017), Marek’s disease virus (Biggs et al., 1968), infectious bursal disease virus (Giambrone et al., 1977), reticuloendotheliosis virus (Motha and Egerton, 1984) and reoviruses (Ruff and Rosenberger, 1985). Many conditionally pathogenic bacteria were significantly enriched in the intestine of coccidia-infected chickens, including Firmicutes and Proteobacteria. The significant enrichment of these conditionally pathogenic bacteria may be a key factor in the increased occurrence of secondary infections of avian leukosis virus (Dong et al., 2015). On the other hand, coccidia parasitize the intestinal epithelium and cause changes in the intestinal environment, like changes in metabolites such as SCFAs, which will influence the antiviral immune response (Chapman et al., 2013; Budden et al., 2017). Subdoligranulum, which decreases dramatically after coccidial infection, belongs to the subgroup of Clostridiales and is capable of butyrate production (Bjerrum et al., 2006). Butyrate reduces chronic inflammation by modulating the immune system, and its reduction may lead to increased chronic inflammation and immune disorders (Lund et al., 2010). Meanwhile, coccidial specific antigens can affect the activity of lymphocytes and suppress the immune response (Rose and Hesketh, 1984). When damaged the gut microbiota of chickens, we can observed higher cloacal and oropharyngeal shedding of avian influenza H9N2 in chickens, with the compromised type I IFNs and IL-22 expression (Yitbarek et al., 2018). So we may conclude that the coccidial infection may contribute the replication of virus. And dual infection of coccidia and virus will extend the replication time of the virus (Gao et al., 2015), which exacerbates clinical symptoms and leads the increased mortality (Giambrone et al., 1977).