Parasitic infections are commonly associated with inadequate management conditions in the commercial poultry production sector (Salem and Attia, 2021; Salem et al., 2022 a; Grace et al., 2024). Both external and internal parasites pose a serious hazard to the poultry industry, resulting in huge economic losses (Salem et al., 2022 b, c; Mathis et al., 2024). Avian coccidiosis is one of the most important enteric protozoan parasites that cause high mortalities and reduce birds’ productivity (Chapman and Rathinam, 2022; Durairaj et al., 2023). Although control of coccidiosis using several anticoccidial medications, such as coccidiostats and ionophores, has been a regular practice in modern chicken production for a long time, alternative control strategies to antibiotics are needed (Lillehoj et al., 2005 b). Because of this, much effort has been done into developing alternative therapies, including vaccinations and dietary plans such as phytochemicals, bacteriophages, prebiotics, probiotics, and hyperimmune antibodies (Salem et al., 2023; El-Ratel et al., 2024; Alagawany et al., 2023, 2025). Since the 1950s, coccidiosis has been effectively managed in commercial chicken production with attenuated or live vaccines (Soutter et al., 2020).
Although a host is essential in live vaccines to multiply the parasites and develop active immunity, it is considered the main disadvantage because it is time- and money-consuming and significantly increases the chance of causing subclinical coccidiosis (Lee et al., 2022; Ahmad et al., 2024). Furthermore, the prevalence of bacterial enteritis increases due to live vaccines in cases lacking growth promotors (Williams, 2002). These problems have increased the demand for safe and effective anticoccidial vaccines at reasonable costs. Immunological solutions provide an alternative to avoid the problems of traditional ways of coccidiosis prevention (Attia et al., 2023). Immunological solutions such as vaccination with cytokines or recombinant vaccines and antiparasitic substitutes boost the host’s innate immunity with antiparasitic host peptides like NK-lysin (Wickramasuriya et al., 2021).
To effectively use new immunotherapeutics, including vaccinations, in clinical practice, it is essential to thoroughly investigate the life cycle of Eimeria, the immune response of the intestine, and the complicated interplay between the gut microbiome and the parasites. This study analyzes the host’s immune reactions against coccidial infection in poultry, explores the effectiveness of several Eimeria vaccine-candidate antigens that are used to protect against coccidiosis in hens, and delves deep into defenses such as probiotics, prebiotics, anticoccidial peptides, and hyperimmune antibodies.
Eimeria species harms the intestinal tract, the principal organ performing digestion and nutrient absorption functions (Blake et al., 2015). The seven pathogenic Eimeria species that initiate coccidiosis to develop intracellularly in a site-specific manner are E. acervulina, E. maxima, E. praecox, E. mitis, E. necatrix, E. tenella and E. brunetti (El-Shall et al., 2022). Each Eimeria spp. targets a particular intestinal site, for example, the cecum targeted by E. tenella and duodenum infected by E. acervulina (Williams, 1998). Coccidiosis causes poor feed consumption, delayed growth, higher mortality rates, and acute drop in egg production resulting in severe economic consequences (Salem et al., 2022 d). Additionally, infection with E. maxima is considered one of the predisposing factors of necrotic enteritis because it weakens the integrity of the gut and encourages the propagation of toxigenic Clostridium perfringens (Jang et al., 2013 b; Park et al., 2008).
As seen in Figure 1, chickens get coccidiosis by consumption of the sporulated oocysts which are ground physically when they pass through the gastrointestinal tract, then their walls are ruptured through digestive enzymes in the stomach producing four sporocysts with two sporozoites (El-Shall et al., 2022). Eimeria invasive form, known as a sporozoite, invades certain regions of the gut epithelium by an unidentified mechanism to initially develop intracellularly and produces merozoites during the merogony cycle through asexual reproduction (El-Shall et al., 2022). Around 1,000 merozoites are produced from one sporozoite, and this cycle can occur up to four times. After the asexual lifetime, the gametogenic phase starts in the sexual cycle, forming female and male gametes, then male and female gametes are fertilized to produce zygotes with thick outer walls that grow to form oocysts and shed with the feces in the litter (El-Shall et al., 2022). Eimeria parasites are highly host- and tissue-specific; however, the underlying mechanism is unknown (El-Shall et al., 2022).
Eimeria life cycle. Birds get infected by fecal matter, and protozoan reproduction occurs in the intestinal cells, damaging the intestinal wall
Coccidiosis is influenced by many factors, including sex, genetics, immunity, biochemistry, and nutrition, and the interaction of these factors determines the outcome (Lillehoj and Okamura, 2003). Oocysts increase exponentially in the environment with each consecutive cycle; thus, rapid, massive exposure to infectious sporulated oocysts substantially affects native chickens (El-Shall et al., 2022). Anticoccidials can help to alleviate this case (Saeed and Alkheraije, 2023). Depending on established research and the facts about the Eimeria life cycle, Eimeria species have a variety of antigenic variations, which is critical for the advance of vaccines (El-Shall et al., 2022).
The poultry sector has successfully managed the most infectious diseases using various techniques (Chaudhari et al., 2020; Jang et al., 2011; Lee et al., 2018). However, coccidiosis continues to be the most challenging disease to control in poultry farms due to the ability of Eimeria parasites to maintain their power to infect poultry for a prolonged duration and to overcome weather changes (Lee et al., 2018). Vaccination is the most efficient and secure strategy to stop coccidiosis in chickens, and many vaccines containing attenuated or live whole parasites were produced (Liu et al., 2023).
The digestion of food by grinding and the absorption of nutrients into the bloodstream is the principal role of the gut; as a result, the intestinal epithelium is always exposed to both local microorganisms and food as well as an enormous number of potentially hazardous pathogens (Saleh et al., 2023). The intestine mucosa includes mucosal-associated lymphoid tissues (MALTs) (Mayer, 1997). The largest immune system compartment is gut-associated lymphoid tissue (GALTs); most GALTs comprise MALT. The mucosal surfaces protect the body from a large spectrum of harmful pathogens. The primary role of GALT is to stop the proliferation of systemic infection by identifying and getting rid of infectious microorganisms as soon as possible.
Hens possess highly advanced MALTs, serving as the initial defense against various pathogenic antigens (Matsumoto and Hashimoto, 2000). The GALTs are the first line of protection against coccidiosis; since Eimeria parasites infect the intestine, GALT plays a crucial role in defending against coccidial infestation by regulating antigen processing and presenting immunogenic epitopes, stimulating intestinal antibody release, and activating cell-mediated immunity (Shivaramaiah et al., 2014). GALT is a tissue with multiple layers: the basement membrane, a row of lymphocytes, and an epithelial cell on the outer layer (Broom and Kogut, 2018; Mosa et al., 2023). The lamina propria, just below the foundation membrane, contains lymphocytes and submucosa (Broom and Kogut, 2018). Chickens also have developed cecal tonsils, the bursa of Fabricius and Peyer’s patches, to protect the host against penetrating infections (Lillehoj, 1998; Lillehoj and Trout, 1996).
The host immunological response in GALTs is regulated by complex processes such as cytokine generation, lymphocyte activation, and the stimulation of local immune cells (Dalloul and Lillehoj, 2006). At the site of GALTs, antigen detection and immune activation predominate; they involve Peyer’s patches in the intestinal lamina propria (Shivaramaiah et al., 2014). B and T lymphocytes found in GALTs are crucial for avian coccidiosis-acquired immunity (Lillehoj and Lillehoj, 2000). In addition, the mucous layer is a physical barrier to prevent the penetration of pathogens (Cornick et al., 2015). Goblet cells release a glycoprotein mucus gel that covers the majority of the gastrointestinal epithelium; the intestinal tract has also been found to have several non-specific barriers, including endogenous cationic peptides, lysozymes, gastric secretion, microbial flora, and bile salts (Lillehoj and Lillehoj, 2000).
Defensin is a key host protective peptide that offers immediate defense from bacterial penetration (Broom and Kogut, 2018). Gallinacin is an epithelial defensin that protects against invading microbes and is mostly found in the trachea, bursa of Fabricius, and the chicken tongue (Zhao et al., 2001). However, the main function of local defense to prevent infections with coccidia has not been fully analyzed (Broom and Kogut, 2018; Abd El-Hack et al., 2022 a). Regularly, GALTs come into contact with various self-antigens and pathogenic and nonpathogenic microbes, however, a deeper investigation of GALT in poultry is necessary to develop potential anti-inflammatory medicines, antibiotic-alternative feed additives, or oral vaccines to keep homeostasis in the gastrointestinal tract throughout the infections (Sunkara et al., 2011).
The adaptive immune system comprises a humoral immune response involving soluble antibodies and a cell-mediated immune (CMI) reaction by T cells (Broom and Kogut, 2018). The two responses are essential in preventing intracellular and extracellular antigens, while the CMI response is the predominating effector against intracellular pathogens (Attia et al., 2023). As presented in Figure 2, the CMI machinery typically targets endogenous antigens developed inside the cell, such as residues left over after the cell’s neoplastic transformation and viral proteins or exogenous antigens introduced inside the cells through the endocytic route (Erf, 2004).
Gut-associated T cells, macrophages, and the schematic process of immune response of chickens to herbal anticoccidial compounds
The most significant contribution in responding to severe or primary coccidiosis is played by T cells (Kim et al., 2019). The bulk of the CMI and humoral immunity is controlled by a variety of T-cell subpopulations that exhibit unique T-cell receptor (TCR) repertoires that can recognize a variety of antigens (Kim et al., 2019). The principal T cell types in chickens, same as in mammals, are a cluster of differentiation (CD4+), CD8+ cytotoxic T cells (TC), and helper T cells (TH) (Arstila et al., 1994). Adaptive immunity in mammals and chickens heavily depends on CD4+ TH cells (Arstila et al., 1994). MHC antigens determine the stimulation of T cells; foreign antigens correlated with MHC class I molecules and antigens associated with MHC class II molecules are recognized by cytotoxic T lymphocytes and T helper cells, respectively (Wieczorek et al., 2017). Co-stimulatory signals are required to activate T cells completely in this mechanism. T cells were found to support hens’ anticoccidial immunity (Min et al., 2013). Numerous TCs exhibited CD8 as cell surface antigens in chickens with initial Eimeria infection (Lillehoj and Bacon, 1991; Breed et al., 1996). Furthermore, CD4+ and CD8+ T lymphocytes had different roles in the initial and subsequent Eimeria infections. The immune-regulatory cytokine interferon (IFN), which is secreted by the increasing number of T cells after coccidiosis (Shah et al., 2010), activates the pro-inflammatory route and suppresses the increase of intracellular Eimeria (Lillehoj and Choi, 1998). This is due to the recruitment of the circulating effector memory T cells in the epithelium of the intestinal tract, where Eimeria parasites develop intracellularly (Beura et al., 2018).
More research is necessary to comprehend the distinct lymphocyte effector populations present in the intestine and their responsibility in the emergence of secondary coccidiosis resistance (Wang and Suo, 2020). As TCR transgenic animals were significantly vulnerable to secondary infection with Eimeria vermiformis and still very sensitive to future infections, T cells have a crucial action in the immune memory response of mice infected with E. vermiformis against murine coccidiosis (Smith and Hayday, 2000). T cells may quickly generate and develop IFN-γ and, like T cells, take part in memory responses (Sheridan et al., 2013). CMI responses mediate macrophages’ natural killer (NK) cells, as well as antigen-specific and non-specific T lymphocyte stimulation (Chai and Lillehoj, 1988).
Removing endogenous and external antigens is the responsibility of macrophages and cytotoxic T lymphocytes (Erf, 2004). There is increasing interest in human and chicken immunology, focusing on the lymphoid population in the gut mucosa, specifically the intestinal intraepithelial natural killer cells (IEL NK cells). Although the exact process is unclear, NK mononuclear cells have cytotoxic activity and contribute to protecting against Eimeria infection through IFN release (Erf, 2004). It was noticed that NK cell subpopulations cause spontaneous cytotoxicity in chicken intestine IEL, suggesting their roles in intestinal immunity (Chai and Lillehoj, 1988). NK cells were found in chicken’s thymus (Chai and Lillehoj, 1988), spleen (Lam and Linna, 1979), bursa of Fabricius (Boo et al., 2020), peripheral blood (Leibold et al., 1980), and gut (Göbel et al., 2001).
The jejunum and ileum had the highest intestinal NK cell activity levels compared to other gut parts (Attia et al., 2023). When NK cell activity was studied in chickens with Eimeria infection, it was found that Eimeria severely decreased the effects of NK cells in intestinal IEL and lymphocytes in the spleen throughout the first stages of coccidiosis (Lillehoj, 1989; Attia et al., 2023). Even so, the activities of NK cells were restored to standard levels about a week after the initial infection; during the early phase of secondary infection, there was a significant elevation in the activity of IEL and NK cells in the intestine and spleen (Attia et al., 2023). NK cells are essential in coccidial infection, although the resident host response varies according to the Eimeria species (Cornelissen et al., 2009).
Dendritic cells (DCs) possess a remarkable role in innate immunity, as they are involved in adaptive immunity by acting as antigen-presenting cells (APCs) (Shoaib et al., 2017). DCs aid in the proliferation of immune cells and the formation of cytokine (Zmrhal and Slama, 2020). These features make DCs a crucial constituent of any immunization strategy and are sometimes known as “nature’s adjuvants” (Steinman and Hemmi, 2006). Therefore, after coccidial infection, DCs are essential in the first stages of antigen presentation and serve as a crucial connection between adaptive and innate immunity (Zmrhal and Slama, 2020).
Lysosomal proteases in APCs break down the antigen’s protein before the presentation (Liu, 2016). Naive T lymphocytes are stimulated throughout this stage by exosomes produced by APCs (Delcayre and Le Pecq, 2006). Based on this procedure, exosomes produced by DCs have been suggested as a new immunization approach to prevent coccidia infection (del Cacho et al., 2012).
Vaccination of chicks with intestinal dendritic cells’ exosomes of hens that were stimulated with sporozoites of E. tenella, E. acervulina, and E. maxima showed a stronger infection resistance than unvaccinated chicks (del Cacho et al., 2012). Interestingly, there was an increase in Th1 immune response in the spleen, Peyer’s patches, and cecal tonsils in either immunized or Eimeria-infected hens (del Cacho et al., 2012; Attia et al., 2023). Also, compared to non-inoculated hens, chickens inoculated with exosomes acquired more body weight, released fewer oocysts, and had a lower mortality rate (del Cacho et al., 2012).
The coccidiosis infection process is mediated through a series of cytokines and chemokines such as interleukins (IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-15, IL17, and IL-18), as well as IFN-γ, TGF-1-4, lipopolysaccha-ride-induced TNF-factor (LITAF) and TNF-superfamily 15 (TNFSF15) (Hong et al., 2006 b, c; Kim et al., 2019). These immunological molecules participate in the immunoregulation of the host through primary or secondary pathogens (Hong et al., 2006 c; Zhang et al., 2013). According to RNA-sequencing in two inbred lines of White Leghorn hens (line C.B12 and line 15I), they possess resistance and susceptibility, respectively (Bremner et al., 2021). The cornerstone of coccidiosis resistance may be the early stimulation of IL-10, IFN- γ, and IL-21 (Bremner et al., 2021).
The resistant line (C.B12) displayed continuously rising IFN-γ and IL-10 formation at the second and fourth days post-infection (dpi) (Bremner et al., 2021). Still, the susceptible line’s production of these cytokines was very limited at the second and fourth dpi but significantly increased at the sixth and eighth dpi (line 15I) (Smith and Hayday, 1998). Therefore, the impact of coccidiosis infection is linked to the host’s genetic characteristics and the timing of the primary innate immune response (Smith and Hayday, 1998). Due to its direct preventive activity on the intracellular proliferation of coccidia, IFN-γ, a typical immunomodulator involved in coccidiosis, has attracted much interest (Rose et al., 1991). IFN-γ has been noticed to have a vital role throughout infection with the intestinal parasite in mice (Rose et al., 1991; Smith and Hayday, 1998).
Following exposure to E. acervulina, the inbred chicken (B2B2) showed an increase in the cecal tonsil and splenic IFN expression but a decrease in the duodenum IFN expression (Choi et al., 1999). Similarly, in chickens with E. tenella infection, IFN-γ was expressed in IELs, cecal tonsils, and spleens following primary and secondary infections with E. tenella (Maes, 2011). Peripheral blood lymphocytes in coccidiosis-infected chickens produce IFN-γ when stimulated by Eimeria (Breed et al., 1997 a). It was later shown that specific T lymphocytes stimulated by either mitogens or antigens in the blood of hens with Eimeria infection release IFN-γ (Breed et al., 1997 b). In light of these results, various researchers (Leibold et al., 1980; Lowenthal et al., 1997) have tried to look into the IFN-γ potential defensive action to prevent coccidiosis. Recombinant IFN-treated birds were compared to control chickens after E. acervulina infection. The findings revealed that recombinant IFN-inoculated chicks acquired significantly more weight than the control group (Leibold et al., 1980). Additionally, in vitro, the IFN-γ hindered the entry of sporozoites from E. tenella into chicken cells, and a study found the same result in vivo (Dimier et al., 1998; Lee et al., 2022). Recombinant IFN-γ immunized hens showed significantly lower shedding in oocyst and increased body weight after infection with E. acervulina than noninoculated chickens (Lowenthal et al., 1997). The physically identical proteins IL-1 and IL-18 are two principal cytokines contributing to the onset of the immune reaction (Yamada et al., 2001). Inflammatory cells were found to be recruited through chemokines synthesized through the action of IL-1 (Yamada et al., 2001), but the role of IL-8 was linked to IFN-γ production (Cornelissen et al., 2009). After primary infection, the cecum, duodenum, and jejunum of hens with E. maxima and E. tenella infections showed noticeably elevated IL-1 expression (Hong et al., 2006 c; Laurent et al., 2001).
Moreover, Dalloul et al. (2007) showed considerably elevated IL-1 and IL-18 expression levels in chickens infected with Eimeria. A crucial cytokine contributing to the response of CMI is the growth factor IL-2, accounting for boosting T lymphocyte propagation in chickens and activating NK cells (Lillehoj et al., 2001; Sundick and Gill-Dixon, 1997). IL-2 contributes to chicken coccidiosis in hens via either primary or secondary infections of E. acervulina accompanied by an increased IL-2 expression level in duodenum (Maes, 2011). Hens infected with E. tenella expressed less IL-2, unlike those infected with E. acervulina (Hong et al., 2006 c). It is unclear if this disparity results from different cytokine responses due to the difference in gastrointestinal tract regions between the two species of Eimeria or if additional unidentified factors are involved with the responses of cytokines (Hong et al., 2006 c). When chickens received the 3-1E recombinant protein immunization, a recombinant vaccination experiment showed improved resistance against challenge Eimeria infection when IL-2 was given with the recombinant protein; this proved the significance of IL-2 in host immune response to infection with E. acervulina (Hong et al., 2006 c). Crucial cytokine IL-6 which is mainly produced by macrophages, endothelial cells, and T cells is necessary for the B cells that make antibodies to reach their full maturity. Chickens with E. tenella infections produced IL-6 in serum samples, suggesting that IL-6 may contribute to acquired immunity (Hong et al., 2006 c). The IEL of birds with E. tenella, E. maxima, and E. acervulina infections has also shown elevated IL-6 gene expression (Hong et al., 2006 c).
The approach to tackle the production of IL-1, IL-6, and TNF-γ, the anti-inflammatory cytokine IL-10, suppresses the inflammatory Th1 reaction (Morita et al., 2001). Thus, IL-10 is capable of suppressing the inflammation during toxoplasmosis infection to minimize the immunopathology of the host. IL-10 was found to reduce harmful inflammatory responses in Eimeria infections (Rothwell et al., 2004). Following E. maxima infection, more levels of IL-10 expression were found in the stomach and spleen of susceptible chickens (line 15I) (Roth-well et al., 2004). Consistently, E. tenella infection promoted IL-10 levels (Hong et al., 2006 b). Also, infection with E. acervulina or E. tenella triggers 20-fold higher expression of IL-10 than uninfected chickens. Eimeria spp. may have evolved the capability of triggering IL-10 production by activating Treg cells to promote parasite entry in chicken epithelial cells, which could account for the contribution of IL-10 during coccidiosis infection (Rothwell et al., 2004). Furthermore, the IFN-related IL-10 blocks the Th1 reaction essential for protecting against infection with the parasite (Rothwell et al., 2004).
Treatment with vitamin D increased the levels of IL-10, which correlated to the elevated activity of Treg cells and decreased productivity losses in hens with Eimeria infection (Morris et al., 2015). These data noticed the crucial role of IL-10 and Treg cells in managing protective immunity in coccidiosis (Mousa et al., 2023). Activated macrophages are the main source of TNF production, but antigen-stimulated T cells, NK cells, and mast cells are also involved (Abbas et al., 2007).
The role of TNF in Eimeria infection has been studied in poultry, and the critical role of drawing neutrophils to the infection site has been pointed out (Mousa et al., 2023). Chicken macrophage cells stimulated with sporozoites and merozoites of E. tenella following the primary infection produce a dose-dependent TNF (Zhang et al., 1995 a). However, TNF was not produced due to secondary infections (Zhang et al., 1995 a). Additionally, the function of TNF-like activity in Eimeria infection development was investigated in vivo in inbred scaleless (SC) chickens (Zhang et al., 1995 b). TNF is involved in coccidiosis pathophysiology, as shown by the fact that SC hens infected with E. tenella treated with TNF antibodies have reduced weight loss (Zhang et al., 1995 b). Chemokines are crucial mediators that encourage leukocyte recruitment to sites of inflammation, which activate host defense responses (Ebnet and Vestweber, 1999).
Different cell types typically form these proteins in response to exogenous and endogenous mediators, like TNF, IFN-γ, IL-1, and platelet-derived growth factors (Oppenheim et al., 1991). T lymphocytes, basophils, eosinophils, and monocytes are regulated by C and CC subfamilies of chemokines (Siveke and Hamann, 1998), while neutrophil migration is regulated by CXC subfamily of chemokines (Breed et al., 1997 a). Chemokine secretion was seen in macrophages induced in vitro by sporozoites of Eimeria (Dalloul et al., 2007). Furthermore, there was an elevation in mRNAs coding K203 and MIP-1b genes expression within both ceca and jejunum of hens infected by E. tenella and E. maxima (Laurent et al., 2001).
As presented in Figure 3, the complex ecosystem’s gut microbiome governs the host’s physiology, encompassing immune system development, function, metabolism, and the exclusion of pathogens (Zhao et al., 2013). It also eliminates pathogenic microorganisms and hinders colonization (Diaz Carrasco et al., 2019). According to several studies, gut bacteria’s ability to harvest energy changes has been linked to changes in energy balance, growth efficiency, and chicken feed efficiency (Stanley et al., 2012; Yan et al., 2017). Recently, the utilization of next-generation sequencing led to a deeper insight into the microorganisms linked with the gastrointestinal tract and their potential impacts on animal and human health (Benson et al., 2009; Lazar et al., 2018).
Impact of coccidiosis on bacterial species in the gastrointestinal tract of chickens
The E. tenella 16S rRNA gene sequencing detected significant abnormalities in the cecal microbiome of E. tenella-infested chickens (Huang et al., 2018). The same changes in fecal microbiome have been shown in many chickens spp. (including White Leghorn chicks, Cobb500, and Arbor Acres broilers) (Huang et al., 2018). Additionally, Huang et al. (2018) demonstrated that E. tenella infection directly triggered dysbiosis in the gut accompanied by decreased numbers of Lactobacillus and Faecalibacterium, which are not harmful bacteria. In addition, it increased Clostridium, Lysinibacillus, and Escherichia with their underlying pathogenicity (Huang et al., 2018). The dysbiosis caused by coccidiosis makes the host more susceptible to other diseases.
Ruminococcaceae and other microflora congregating in the cecum or large intestine are considered the origin of nutrients and energy by breaking down non-starch polysaccharides into monosaccharides (Borda-Molina et al., 2018). Meanwhile, Faecalibacterium helps ferment these sugars to produce butyric acid and essential amino acids (Chen et al., 2020). They significantly decrease damage from E. tenella infection and treat chronic inflammation (Chen et al., 2020).
With the coinfection of Clostridium perfringens and E. maxima in the case of necrotic enteritis (NE) disease, the jejunum increased Escherichia, Shigella, Clostridium sensu stricto 1, and Weissella populations while Lactobacillus levels were declining (Bortoluzzi et al., 2019). Decreasing Lactobacillus spp. causes microbiological variety and disturbs metabolic activities (Yan et al., 2017). The loss of Lactobacillus spp. affects metabolic processes and microbial diversity (Yan et al., 2017). Moreover, losing host defense and developing other bacterial species are possible effects of decreased Lactobacillus reuteri, which produces the antibiotic chemical (Greppi et al., 2020). Lactobacillus species showed an inhibitory impact on the invasion of sporozoites of E. tenella (Tierney et al., 2004). Also, E. tenella infection showed significant attenuation with potentially beneficial bacteria in the cecal microbiota (Cui et al., 2017).
By producing substances with broad-spectrum antibiotic effects, these bacteria suppress the multiplication of potentially harmful bacteria and lessen the production of endotoxins (Biggs and Parsons, 2008). Also, E. tenella invasion increased detrimental bacteria such as Enterococcus, Escherichia-Shigella, Staphylococcus, and Bacillus in the cecum of chicks (Cui et al., 2017; Chen et al., 2020).
Eimeria parasites cause crucial damage to the intestinal barrier, making it less effective and result in a subsequent decrease in nutrient absorption (Hessenberger et al., 2016). Furthermore, an imbalance of bacteria in the gut can affect the metabolism of bacteria (Hessenberger et al., 2016). Because of the negative influence of severe coccidiosis on the poultry industry, more research is required to fully understand the sophisticated interactions between Eimeria parasites and the gut microbiome. Also, deep investigation is required to address the consequences of coccidiosis on host physiology, including nutrient uptake and immunity (Cui et al., 2017).
Field coccidiosis is a prevalent gastrointestinal disorder that damages the global chicken industry’s economy (Mousa et al., 2023). Vaccination enhanced weight, shedding fecal oocyst, and reduced gastrointestinal lesions (da Silva Giacomini et al., 2023). So, advances in biochemistry, molecular biology, and genetic engineering technologies have transformed the creation of animal vaccines (da Silva Giacomini et al., 2023). Focuses on vault development, an effective vaccine with minimal side effects (Gaghan et al., 2022). Traditionally, coccidiosis anticoccidial medicines such as toltrazuril, diclazuril and attenuated or live Eimeria strains have been used (Chapman, 1997, 1999). Field coccidiosis can be effectively controlled with live and attenuated parasite vaccines; however, due to the high cost of manufacturing vaccines and limited availability, the increasing prevalence of drug-resistant strains of Eimeria is leading to growing attention in recombinant vaccines as a viable alternative for preventing coccidiosis (Pastor-Fernández et al., 2020).
Recombinant antigens are safer and possess more immunogenic robustness than live and attenuated parasite vaccines (Gaghan et al., 2022). These favorable characteristics were attributed to selection criteria based on their capacity to promote host immunity properties (Gaghan et al., 2022). However, recombinant Eimeria protein has limitations, including a lower range of protective immunogenic response (Pastor-Fernández et al., 2020). Also, variability in Eimeria strains and the high expense of creating various Eimeria parasite strains will limit issues. For successful recombinant vaccine techniques in the field to give good vaccination against coccidiosis (Ding et al., 2004), a new approach must be created to enhance the efficiency of recombinant vaccination (Gaghan et al., 2022).
Although commercially available recombinant vaccines are unknown, many Eimeria antigens are also recognized as effective anticoccidial vaccine candidates (Blake et al., 2017). Five immunodominant Eimeria antigens, including 14-3-3 protein, elongation factor 2 (EF-2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and ubiquitin-conjugating enzymatic domain-containing protein (UCE), were found by Liu et al. (2017). Most anticoccidial Eimeria proteins target the surface and interior antigens of the parasite. These are suitable targets for producing a host’s protective immune response because of the Eimeria invasion and proliferation.
Eimeria species’ amino acid sequences are compared in many studies; E. maxima and E. tenella-infected hens were used by Liu et al. (2017) to test the favorable protective effect of the EF-1 gene extracted from E. tenella sporozoites. In a correlation to non-inoculated hens, chicks immunized with recombinant EF-1 demonstrated better body weight, elevated serum antibody titer versus EF-1, and lower fecal shedding of oocyst following Eimeria challenge with E. tenella or E. maxima. Also, infection with E. tenella, E. acervulina, and E. maxima, or a combination of these pathogens in chicken. According to Tian et al. (2017), E. acervulina and E. maxima have protective effects and good immunogenicity. Compared to uninfected hens, recombinant GAPDH vaccine-vaccinated chicks had larger percentages of CD8+ T cells and CD4+, IgG antibody synthesis, and very high cytokine release (IL-2, IL-4, and IFN-γ) (Ellington et al., 2021).
A body protein with an epitope shared by all Eimeria species is becoming more immunogenic (Liu et al., 2018). Compared to unvaccinated control hens, SO7-immunized chickens excrete fewer parasites (Klotz et al., 2007). In a separate in vivo study, hens administered a recombinant SO7 protein vaccine demonstrated a substantial decrease in oocyst production and accelerated weight gain related to unvaccinated chickens (Rafiqi et al., 2018). Additionally, SO7-immunized hens exhibited elevated serum IgY, IFN-γ, and lymphocyte proliferation levels. Two gametocyte antigens (GAM82 and GAM56) from the Eimeria parasite’s sexual stages are located in the oocyst wall and represent possible transmission-blocking candidates for a vaccine against coccidiosis (Wallach, 1997; Huang et al., 2018). These two antigens can attenuate the shedding of oocysts in infected chickens with E. maxima and increase sera antibody and lymphocyte multiplication following vaccination (Xu et al., 2013 b; Huang et al., 2018). Likewise, the expression of Game 82 and Game 56 antigens and by transgenic plants (tobacco leaves) generated an immunological defense in hens that have received vaccinations, however, not in unvaccinated hens (Kota et al., 2017).
In addition, Liu (2016) explained that the E. tenella Etgam22 antigen is considered an E. necatrix gametocytes ortholog, inferring a possible goal for future vaccinations against coccidiosis with recombinant subunits. The efficiency of immunoprotective measures of recombinant gametocyte antigen 22 originated from E. tenella (EtGam22) was investigated in hens (Rafiqi et al., 2019). Vaccination produced robust cytokine production, such as EtGam22, IL-4, IL-2, IFN-γ, and TGF-increased proliferation of lymphocytes in peripheral blood (Rafiqi et al., 2019). Additionally, there was a considerable decrease in oocyst discharge and attenuated weight loss (Rafiqi et al., 2019). Zhao et al. (2020) employed the E. tenella surface antigens 4 to produce recombinant protein and DNA vaccines (EtSAG4). They demonstrated that in E. tenella-diseased Cobb broilers, the EtSAG4 recombinant protein significantly increased IFN-γ, IgY formation, gained weight, and decreased oocyst production (Zhao et al., 2020). However, compared to EtSAG4 recombinant antigen vaccination, the DNA vaccine composed of plasmids of pEGFP-N1-EtSAG4 offered noticeably greater protection against E. tenella (Lee et al., 2022).
Profilin vaccine, often known as 3-1E (Min et al., 2001; Zhao et al., 2013; Blake et al., 2017), is an antigen expressed superficially on all stages of E. tenella, E. acervulina, and E. maxima merozoites and sporozoites (Venkatas and Adeleke, 2019). The 3-1E protein induces cell-mediated immune response (Tang et al., 2018 a) and has a conserved actin-regulatory protein profilin domain (Ding et al., 2004). In various studies, hens given intramuscular injections of 3-1E antigen gained more weight and excreted less fecal oocysts after exposure to Eimeria (Zhao et al., 2013; Lee et al., 2022). Moreover, compared to non-immunized chickens, serum IgG antibody levels and cytokine production were higher (Jang et al., 2013 a; Zhao et al., 2013; Tang et al., 2018 a).
Using plasmids containing genes producing interleukins 1, 2, 6, 8, 15, 16, or IFN-γ using in ovo delivery, the efficacy of the 3-1E recombinant protein from E. acervulina opposed to coccidiosis was also investigated by Ding et al. (2004). According to the study’s findings, the ovo 3-1E vaccination produced immunity that guards against infection with E. acervulina, which was determined by a decrease in fecal oocyst production and a reduction in body weight compared to unvaccinated chickens. In addition, injecting a plasmid and the 3-1E vaccination encoding chicken cytokine as IL-15, IL-2, IL-18, IFN-γ, or IL-17 improved vaccination effectiveness (Ding et al., 2004). Recombinant 3-1E vaccine and NE, B-like toxin protein significantly protected against NE challenge, according to Lillehoj et al. (2017).
The tubulin protein from E. acervulina was discovered to have protective qualities by Ding et al. (2008). Protein tubulin recombinant immunized chickens had shown a 36% drop in fecal shedding of oocyst, a decrease in GI lesion ratings, and higher weight than non-inoculated chickens. Specific antigens, like the apical membrane antigen 1 (AMA1) and immune-mapped protein-1 (IMP1) from E. maxima, have been demonstrated to be effective vaccine candidates (Li et al., 2013; Jenkins et al., 2018; Pastor-Fernández et al., 2020). Robust cellular and humoral responses against infection of E. maxima were seen in chickens immunized with the AMA1 antigen (Li et al., 2013). On the other hand, immunization with transgenic E. tenella oocysts that express the IMP1 and AMA1 antigens of E. maxima offered some defense versus a high-level E. maxima challenge (Pastor-Fernández et al., 2020).
DNA vaccinations are based on molecular vaccines against coccidiosis since DNA should penetrate the cell to allow the antigen it contains to be expressed (Lillehoj et al., 2000). Subcutaneous immunization with cDNA that codes 3-1E E. acervulina significantly improved resistance to E. acervulina infection and greatly diminished fecal oocyst shedding (Lillehoj et al., 2000). Furthermore, improved vaccine efficacy was obtained by combining 3.1E with IFN-γ or IL-2-expressing plasmid (Lillehoj et al., 2000). Growth performance and parasite fecundity measurements showed that embryo vaccination with 3-1E and plasmids encoding cytokine genes (IL-1, IL-2, IL-15, or IFN- γ genes) versus E. acervulina infection led to increased levels of protection and more robust serum antibody response (Lillehoj et al., 2005 a). Mathew et al. (2016) examined the prospective vaccination potency of the mitogenic signal transduction, cell cycle regulation, and apoptosis associated with E. maxima protein 14-3-3. A combination of 2 DNA vaccines expressing E. tenella TA4 and Et1A sporozoites diminished oocyst release and elevated growth efficiency after E. tenella infection developed protective immunity against coccidiosis (Wu et al., 2004).
Coccidiosis DNA immunization optimization approaches were investigated by Zhang et al. (2019). The recombinant pVAX1-pEtK2-IL-2 showed a protective impact against E. tenella infection in chickens; this vaccine was formed through the cloning process of pEtK2 present in E. tenella as well as the IL-2 coding gene in Pvax1 (Zhang et al., 2019). The vaccine’s favorable results were correlated to an increase in both survival rate and weight, and it also exhibited a low gut lesion score (Zhang et al., 2019). Two injections comprising 80 g of each DNA successfully protected against coccidiosis challenge infection, and intramuscular delivery was the most successful strategy to elicit an immune defense (Zhang et al., 2019; Zhao et al., 2020). Compared to the unvaccinated control, vaccinated chickens performed better clinically regarding body growth, oocyst generation, and scores for gut diseases. They also showed greater IgY secretory antibody levels and enhanced IL-17 and IFN-cytokine activation (Zhao et al., 2020).
To boost the efficiency of recombinant vaccines, it is imperative to examine alternative adjuvants and antigen delivery mechanisms (Lillehoj and Lillehoj, 2000). Some adjuvants are being added, such as montanide IMS or an ISA; these substances originated from a mixture of specified, supplemented light mineral oil proved significant efficacy in promoting recombinant vaccine beneficial impact (Jang et al., 2013 a; Lillehoj et al., 2017; Rafiqi et al., 2018). Oppositely, after embryo vaccination by an Eimeria profilin antigen, Lee et al. (2010 b) addressed a better immunoprotecting impact accompanied by improved weight gain and bowel cytokine activity.
Ginsenosides produced from ginseng plant material can be utilized as an adjuvant to target E. tenella infection (Zhang et al., 2012). Profilin and ginsenoside extract showed a synergetic impact on antibody synthesis, diminished gastrointestinal pathologies, and decreased oocyst production compared to treatment with profilin alone. Additionally, Min et al. (2001) examined several adjuvants, such as IL-1, IL-2, IL-8, IL-15, IFN, TGF-4, and lymphotactin to boost 3-1E efficacy and addressed significant stimulation in CD3+ upon using IL-8 or IL-15 together with 3-1E.
The antigen presentation process can modulate the effect level of recombinant vaccines; thus, the antigen delivery method can trigger a more robust immune defense against infection (Trovato and De Berardinis, 2015). Numerous delivery vectors were investigated in association with immunomodulatory Eimeria antigens (Salmonella strains and pVAX1) serving as eukaryotic vectors (Venkatas and Adeleke, 2019). In addition, Saccharomyces cerevisiae is an example of yeast vectors, and pMV361 and pET32a from the bacterial origin (Liu et al., 2017; Sun et al., 2014; Wang et al., 2014). In hens, Saccharomyces cerevisiae vector enhanced EtMic2 microneme protein vaccination and was accompanied by improved body weight, lower cecal pathology, and decreased fecal oocyst shedding (Sun et al., 2014). Moreover, pVAX1 and pET32a (+) delivery vectors significantly boosted the impact of E. maxima 14-3-3 antigen. This approach showed minimal jejunum lesions, reduced oocyst output, slower body weight loss, and improved immunological responses as observed by greater portions of CD4+ cells and increased serum antibody titers (Liu et al., 2017). The favorable immune response of mucosa that protects against pathogens has been achieved by introducing foreign antigens to weakened strains of Salmonella enterica serovar Typhi (S. Typhi) or S. Typhimurium (Galen et al., 2009).
The ability of pMV361 to enhance vaccination efficacy was indicated in both rBCG pMV361-rho and pMV361-IL2 (Wang et al., 2014). Together they showed a decline in oocyst output and cecal lesions (Wang et al., 2014; Venkatas and Adeleke, 2019). Eimeria parasites have been investigated as vectors for delivering more antigens (Liu et al., 2017; Tang et al., 2018 a). The Eimeria parasite delivery system provides several advantages due to its strong host specificity, safety, and enormous genomic size (Shirley et al., 2004; Vrba and Pakandl, 2015). E. tenella oocysts can be employed as a vaccine vector during expression of E. maxima antigens, including EmAMA1 and EmIMP1, according to research by Tang et al. (2018 b) and Pastor-Fernández et al. (2020). Thus, oral immunization with the E. tenella vector expressing a Campylobacter jejuni antigen (CjaA) revealed 90% protection versus Campylobacter jejuni infection (Clark et al., 2012).
A serum antibody response was produced by transgenic E. tenella oocysts that expressed two antigens originating from the infectious laryngotracheitis virus (ILTV) and infectious bursal disease virus (IBDV) (Marugan-Hernandez et al., 2016). Moreover, experiments using plant-derived vectors have been made public (Kota et al., 2017). Plant-based vaccines have a large capacity for replication, are affordable, and are not tainted by illnesses from animals (Shanmugaraj and Ramalingam, 2014).
As seen in Figure 4, antimicrobial alternatives are widely described as any material that can substitute therapeutic medications, which are becoming increasingly inefficient against harmful bacteria, viruses, or parasites (Lillehoj et al., 2018). Several antiparasitic-alternative techniques are available, in addition to developing vaccines, such as phytochemicals, probiotics, prebiotics, hyperimmune egg yolk antibodies, and host immune peptides were effectively created to combat coccidiosis (Gadde et al., 2017). In addition, using different organic acids and feed enzymes has garnered significant interest. Several investigations were adopted to evaluate the effectiveness of numerous feed additives, such as phytobiotics, as antibiotic-free remedies for chicken coccidiosis (Table 1). Most of these feed additives have significant potential since they lessen gut injury, fight pathogens, and increase local immunity in the chicken bowels (Gadde et al., 2017).
Anticoccidial drug alternatives to control coccidiosis in chickens
Phytobiotics against Eimeria infections
| Phytobiotic | Active parts | Active compound | Anticoccidial action | References |
|---|---|---|---|---|
| 1 | 2 | 3 | 4 | 5 |
| Allium sativum | Aerial parts | Diallyl disulphide, alliin, and allicin, propyl thiosulfinate, propyl thiosulfinate oxide. S-allyl cysteine sulfoxide, phenols, flavonoids, diallyl sulphide (DAS), diallyl disulphide (DADS), or diallyl trisulphide (DATS) | Allicin and diallyl disulfide in garlic powder have antioxidant and anti-inflammatory activities, inhibiting Eimeria oocyst sporulation. The S-allyl cysteine sulfoxide, phenols, and flavonoids in the aqueous garlic extract inhibit Eimeria tenella sporulation efficiently in vitro. Furthermore, they enhance the intestinal microbiota and reduce the number of oocysts in the feces of Eimeria tenella in vivo. Essential oil of garlic contains diallyl sulphide (DAS), diallyl disulfide (DADS), or diallyl trisulphide (DATS) have anti-inflammatory and immunostimulatory characteristics and inhibit Eimeria sporulation. Propyl thiosulfinate and propyl thiosulfinate oxide inside garlicon alters the expression levels of 1,227 transcripts associated with intestinal intraepithelial lymphocytes in chickens. | (Adjei-Mensah and Atuahene, 2022; Ali et al., 2019; Hussein et al., 2021) |
| Artemisia annua; Artemisia sieberi | Aerial parts | Artemisinin, Artemisia ketone; artemisinic acid; scopoletin, α-caryophyllene; germacrene D; Acenaphthene; 2-cyclohexen-1-one, 3-methyl-6-(1-methylethyl) | Artemisinin inhibits the growth of Eimeria sp and its sporulation. It reduces the formation of lesion score by degrading the bacterial iron-peroxide complex via the production of free radical oxygen. Coroian et al. (2022) declared that the number of oocysts per gram of feces in chickens and their lesion score decreased after treatment by Artemisia annua and Artemisia sieberi. Furthermore, it affects the gametocyte sexual cycle of Eimeria tenella via termination of the sarcoplasmic-endoplasmic reticulum calcium ATPase enzyme expression. Other components in Artemisia annua and Artemisia sieberi improve the immune response, food absorption, and digestion by increasing the beneficial intestinal microflora. | (Coroian et al., 2022; Jiao et al., 2018) |
| Curcuma longa (turmeric) | Rhizomes | Curcumin diferuloylmethane, demethoxycurcumin, and bis-demethoxycurcumin | Curcumin inhibits sporozoites by interfering with the life cycle of Eimeria. It keeps the integrity in the gut and promotes humoral immunity in the host. Besides, it alters the gut microbial population in chickens by enhancing the Lactobacilli population while lowering the Selenihalanaerobacter population. | (Lee et al., 2020; Yadav and Jha, 2019; Yadav et al., 2020) |
| Bidens pilosa | Leaves, flowers, seeds, stems, roots | Polyacetylenes, flavonoids, triterpenes, some essential oils | These active components inside Bidens pilosa have an anticoccidial effect against Eimeria tenella. They also have an immune impact by enhancing T-cell production and anti-parasitic effects against Eimeria species. Chen et al. (2020) observed that these active components treat chickens infected with E. tenella and reduce the percentage of mortalities, oocyst count, and intestinal lesions. Furthermore, they increase the body weight and gut microflora of treated chicken. | (Chen et al., 2020; Yang et al., 2019) |
| Origanum vulgare (oregano) | Leaves | Carvacrol, p-cymene, c-terpinene, limonene, terpinene, ocimene, caryophyllene, β-bisabolene, linalool, and 4-terpineol thymol, eugenol | Oregano oil has anticoccidial activity against Eimeria sp by interfering with its life cycle with the destruction of the membrane of sporozoite, resulting in a decreased number of oocysts. Furthermore, oregano oil alters the gut physiology of broiler chickens, improving growth performance and intestinal barrier function. Finally, it is observed that these compounds in the oregano oil improve the carcass traits of infected broiler chickens with coccidiosis. | (Pop et al., 2019; Tauer et al., 2018) |
| Aloe excelsa Aloe vera | Stem and fleshy, deeply channeled leaves | Acemannan chromone, anthraquinone aloe-emodin, aloin, aloesin, emodin | The active compounds inside Aloe excelsa, especially acemannan, induce immunomodulatory responses after binding with mannose receptors on the surface of macrophages. The immunomodulatory response stimulates the production of inflammatory cytokines, including IL-1, IL-6, and TNF-α, and inhibits the infection of Eimeria sp. It is observed that Aloe vera treats the infected chicken and decreases the number of droppings oocyst while improving the chicken’s weight. | (Akhtar et al., 2012; Chen et al., 2020; Darabighane and Nahashon, 2014) |
| Cinnamomum zeylanicum (cinnamon) | Inner stem bark | Cinnamaldehyde, cinnamate, and cinnamic acid | It is observed that the active compounds inside cinnamon have an anticoccidial effect against Eimeria species by promoting and increasing the production of T helper cells and cytokines with increasing body weight. Furthermore, these compounds improve the growth performance and change the caecal microbiota composition. | (Lee et al., 2011; Yang et al., 2020) |
| Beta vulgaris (beet) | Leaves taproot | Betaine, betacyanin, betaxanthin, betacyanin, phenolic acid, flavonoid | Active compounds inside Beta vulgaris improve phagocyte and lymphocyte production in the intestines of chickens infected with Eimeria sp. Also, they enhance the chicken’s weight and improve feeding efficiency by stabilizing and protecting the metabolism of intestinal epithelial cells of chicken where the Eimeria sp are multiplied and increased. They can protect intestinal cells from osmotic stress through cell membrane stabilization by a methyl group donor. Abbas et al. (2017) observed that B. vulgaris is avian coccidiosis because it contains antioxidant compounds that minimize xenobiotic-oxidative stress and stabilize the intestinal cell structures against E. maxima in broilers. | (Abbas et al., 2017; Klasing et al., 2002) |
| Triticum aestivum Saccharum officinarum | Endosperm and embryo of the wheat plant, root of Saccharum officinarum | Arabinoxylans, sugar cane | Arabinoxylan inside Triticum aestivum and Saccharum officinarum have an anticoccidial effect against coccidiosis, improving body weight, oocyst shedding, and lesion score. Arabinoxylan and sugar cane improve the natural adaptive immune response inside infected chicken against Eimeria sp. | (Awais and Akhtar, 2012) |
| Pinus radiata (pine tree) | Cones, seeds, needles, pollen, bark | Tannins | Tannins inside Pinus radiata have anticoccidial activity against Eimeria sp. Tannins penetrate the wall of Eimeria oocyst and inactivate the endogenous enzymes important for sporulating the oocyst. And so, the abnormal shapes of sporocysts inhibit the life cycle of E. tenella, E. maxima, and E. acervulina. | (Molan et al., 2009; Muthamilselvan et al., 2016) |
| Fomitella fraxinea (mushroom) | Fruiting bodies | Lectin, urushiols, polyphenols, gallotannins | It is observed that lectin inside Fomitella fraxinea has an anticoccidial effect against Eimeria sp through the improvement of both cellular and humoral immune responses inside the host. Mushrooms containing macro and micronutrients enhance immune protection and improve poultry growth. | (Dalloul et al., 2006) |
| Olea europaea (olive tree) | Leaves. olive fruits | Maslinic acid, secoiridoids, oleuropein, and oleuropein-aglycone, flavonoids, rutin and luteolin-7-glucoside, simple phenols, hydroxytyrosol, and tyrosol | Maslinic acid has anticoccidial activity and reduces the number of oocysts, lesions, and anticoccidial indices in E. tenella, increasing the treated chickens. Polyphenols, including oleuropein content, have an anti-hypertensive effect and minimize the incidence of ascites. Simple phenols penetrate and interact with cytoplasmic membranes and alter their cation permeability, impairing significant biological processes in coccidia cells and death. | (Almuhayawi et al., 2023; De Pablos et al., 2010; Debbou-Iouknane et al., 2021; Selim et al., 2022) |
| Grape seed | Seeds | Flavan-3-ol proanthocyanidins catechins | Proanthocyanidin has antioxidant activity and reduces the infection of E. tenella. It also improves the chicken’s weight and intestinal cell structure while reducing the number of mortalities. | (Wang et al., 2008) |
| Areca catechu (areca nut) | Seeds | Arecoline, arecaidine, guvacine, guvacoline | These compounds improve immunity through the production interleukins-2 and decrease the scores of the caecal lesion. Wang et al. (2018) declared that areca nut has anticoccidial characteristics in infected broiler chicks with coccidiosis. | (Wang et al., 2018 a) |
Moreover, prebiotic treatment with probiotics demonstrated a potent synergy-mediated effect (Ren et al., 2019; Villagrán-de la Mora et al., 2019). However, caution must be used when picking combinations of anticoccidials (Ren et al., 2019). We must understand their action pathways and verify their effectiveness in diverse field conditions using sufficient studies on target animals.
IgY antibodies have been utilized effectively to prevent and treat different enteric illnesses in pigs and cattle (Wang et al., 2019 b; Vega et al., 2020). Moreover, IgY has been successfully utilized in treating disease and preventing enteric pathogen infections in poultry and people (Khalf et al., 2016). It is an effective counterpart of mammalian IgG (Wang et al., 2019 b). IgY is predominantly present in the yolk sac during early embryogenesis, allowing for rapid extraction and purification (Cook, 2011). There are several advantages when the antibody production process is conducted through comparatively non-invasive methods and can be applied on a big scale (Cook, 2011). The IgY antibodies, owning specificity to pathogens, can be utilized as a passive immunization technique for treating several human and animal disorders and an adequate replacement for antibiotics (Cook, 2011; Hussein et al., 2020).
As a result, it offers an effective substitute for producing antibodies (Gadde et al., 2015). This approach can be taken with vaccines to boost the immunity and resistance of animals against infections (Ren et al., 2019; Villagránde la Mora et al., 2019). Using IgY antibodies in passive vaccination provides numerous benefits, including environmental friendliness, nontoxicity, and a reduction in the number of animals necessary for antibody generation (Lee et al., 2009 a). In poultry and humans, a unique delivery mechanism will boost the field application of IgY for treating disease and preventing enteric pathogen infections (Lee et al., 2009 a).
Supplementing chickens’ feed with hyperimmune IgY egg yolk concentrations of 10% or 20% (Supracox®) defended them from further E. acervulina challenges (Lee et al., 2009 a). Chickens fed 10% or 20% Supracox® supplemented meals gained more weight and produced fewer oocysts than chickens consuming the control diet. While with lower amounts of Supracox® (0.01, 0.02, 0.05, or 0.5%), there was a decrease in fecal oocyst shedding, but it did not impact the rate of weight gain. Comparable results were observed in hens infected with E. maxima and E. tenella, including attenuated rate of weight loss, relief of intestinal disorders, and formation of fecal oocysts compared to chicks given a regular diet (Lee et al., 2009 a).
Data was detected from hens inoculated with 3 Eimeria species (E. acervulina, E. maxima, and E. tenella) that induced hyperimmune IgY (Lee et al., 2009 b), together with the work of Xu et al. (2013 a), who utilized hyperimmune IgY generated from hens inoculated with 5 Eimeria species (E. acervulina, E. necatrix, E. maxima, E. tenella, and E. praecox). They demonstrated its preventive efficacy in chickens with E. tenella infection. Additionally, hyperimmune IgY supplemented in diet indicated increased weight gain, higher survival, improved cecal lesion scoring, and decreased oocyst output in contrast to regular diet feeding (Lee et al., 2009 b).
Probiotics are dietary supplements with live microorganisms that advance the gut microbiome balance of the host animal (El-Saadony et al., 2022 a). On the other hand, prebiotics are non-digestible feed components that influence the host favorably by selectively boosting the growth and activity of one or a restricted count of gut bacteria (Lee et al., 2007 a; Park et al., 2021).
Synbiotics are prebiotic and probiotic mixtures with a synergistic effect (Gadde et al., 2017). According to reports, prebiotics, probiotics and synbiotics, decrease the number of harmful bacteria and could compete with coccidial infection in the chicken gastrointestinal tract while concurrently raising the beneficial microbiota, resulting in an enhanced growth rate (Table 2). The gut microbiome is an essential component of the first line of defense in humans and animals (Abd El-Hack et al., 2022 a). It has been demonstrated that supplementation of the probiotic of the intestinal microflora in chicken positively regulates the intestinal microbiota and inhibits the expansion of pathogen colonies (Gadde et al., 2017).
Probiotics and prebiotics against Eimeria infections
| Probiotic bacteria | Anticoccidial action | References |
|---|---|---|
| Bacillus cereus | Bacillus cereus competes with the Eimeria by improving the capability of beneficial intestinal bacteria to occupy the epithelium cell receptors and adhere to the gut epithelium instead of the parasite, resulting in parasite growth inhibition. Bacillus cereus facilitates the digestion of indigestible fibers into fatty acids and butyrate, which are important for extra nutrients and energy. Bacillus cereus stimulates intestinal epithelium proliferation. Furthermore, treating Bacillus cereus protects against Eimeria sp., by promoting the host immune response and regulating T-helper cells. | (Gu et al., 2020; Leung et al., 2019) |
| Bacillus subtilis | Bacillus subtilis modifies the composition of the microbiota in the gut and results in the enhancement of growth performance. Bacillus subtilis increases the number of intestinal Bacteroidetes and promotes nutrient digestion in broilers. Also, it alters microbial communities and increases predominant species, resulting in a decrease in bacterial diversity in the caecum of the infected chicken. | (Erdoğmuş et al., 2019; Wang et al., 2019 a) |
| Enterococcus faecium | Enterococcus faecium alters the composition and number of beneficial microflora in the intestine. Besides, it induces the production of anti-inflammatory cytokines with cell-mediated and humoral immunity responses in infected chickens with Eimeria sp., and decreases the severity effect of intestinal lesions. | (Wu et al., 2019) |
| Pediococcus | Pediococcus induces an immune response against Eimeria acervulina or Eimeria tenella. It protects against growth reduction and oocyst shedding. It is also observed that combining Pediococcus with saccharomyces improves the immune response against Eimeria acervulina or Eimeria tenella. | (Lee et al., 2007) |
| Bacillus DFMs | Combining Bacillus direct-fed microbials (DFMs) alone or with xylanase has anticoccidial activity against Clostridium perfringens and Eimeria sp. Furthermore, this combination improves body weight growth and decreases the mortality rate in infected broilers. Moreover, Bacillus DFMs alone or with xylanase reduces the severity and gross lesion scores in the small intestine and improves the growth performance of broilers. | (Nusairat et al. 2018) |
| A mixture of lactic acid bacteria (Lactobacillus species) |
| (Alagawany et al., 2018; Behnamifar et al. 2019; Royan, 2019) |
| Prebiotic | ||
| Arabinoxylooligosaccharides (AXOS) | Arabinoxylan is partially hydrolyzed to give Arabinoxylooligosaccharides, which have an anti-bacterial effect against Salmonella sp., by decreasing the bacterial shedding in the feces. Also, it passes from the small intestine to the lower gut. It becomes available for probiotic bacteria and enhances feed efficiency in broilers by improving the quality of short-chain fatty acids (SCFAs). AXOS enhances the selective stimulation of the beneficial bacteria and inhibits pathogenic bacteria, resulting in improved colon function and enhanced immune response. It also decreases the severity degree of lesions in the intestine and oocyst excretion in broilers. | (Al-Sheraji et al., 2013; Eeckhaut et al., 2008) |
| Fructooligosaccharides (FOS) | Fructooligosaccharides are structurally oligosaccharides extracted from garlic, onion, and chicory. It has an anticoccidial effect against Eimeria sp. It has a significant role in inducing the activity of commensal bacteria, excluding pathogenic bacteria in the gut. FOS induces the accumulation of probiotics in the gut, inhibits pathogens, and enhances the immune response in broilers. | (Nopvichai et al., 2019; Ocejo et al., 2019; Yadav and Jha, 2019) |
| Isomaltooligosaccharides (IMOS) | Isomaltooligosaccharides are extracted from the main components of starch in a multistage process. Isomaltooligosaccharide enhances the broiler’s growth performance and controls the microbiota in the intestine by improving the numbers of Lactobacillus in the ceca with broilers infected with Escherichia coli O78. | (Tarabees et al., 2019) |
| Mannan-oligosaccharides (MOS) | Mannan-oligosaccharides comprise ten mannose units linked via α-(1,3) and α-(1,6) bonds extracted from the yeast’s cell wall and Saccharomyces. MOS decreases the degree of the severity of E. acervulina lesions and oocyst fecal shedding in infected broilers with Eimeria sp. mixtures. MOS improves broilers’ growth and feed and decreases the degree of severity of lesions in broilers infected with Eimeria spp. | (Angwech et al., 2019; Bozkurt et al., 2014) |
| Soy oligosaccharides (SBM) Soybean meal-based diets | Soybean meal-based diets (SBM) enhance feed intake, weight gain, and production of short-chain fatty acids. It induces cytokines production in the duodenal in the broilers infected with E. acervulina, which is related to boiler feed improvement. | (Faber et al., 2012) |
| Xylooligosaccharides (XOS) | Lin et al. (2022) declared that xylooligosaccharides enhance nutrient utilization and growth performance in infected chicken with Eimeria sp by increasing the production of branched-chain fatty acids isobutyrate and isovalerate. | (Lin et al., 2022) |
| Quercetin | Quercetin is used as a flavonoid prebiotic, which can modify the cecal microflora of broilers by decreasing the numbers of P. aeruginosa, S. enterica, S. aureus, and E. coli and increasing the numbers of Lactobacillus and Bifidobacterium. Quercetin inhibits microbial growth of E. coli and S. aureus in vitro by cell wall and cell membrane destruction. | (Wang et al., 2018 b) |
Therefore, using and developing probiotics cultivates and maintains favorable intestinal microflora, thereby enhancing the host’s resistance to intestinal pathogens (Lee et al., 2007 b; Talebi et al., 2008). Although multiple reports addressed that oral administration of probiotics prevents disease and boosts the immune system, few studies have studied their positive effects versus coccidiosis (Lee et al., 2007; Talebi et al., 2008). Several investigations by Dalloul et al. (2003, 2005) demonstrated that local immunity can be boosted by probiotics from Lactobacillus, allowing more protection against coccidiosis. Lee et al. (2010 a) investigated dietary Bacillus-based direct-fed microorganisms (3AP4, LSSAO1, Bs2084, 15AP4, Bs18, Bs27, Bs278, and 22CP1) that were infected with E. maxima, chickens showed reduced clinical symptoms and improved immunity. Saccharomyces and Pediococcus-based probiotics (MitoMax®) lowered oocyst shedding in chickens infested by E. tenella or E. acervulina (Lee et al., 2007 a).
Additionally, a meal supplemented with 0.1% Mito-Max® enhanced the amount of serum antibodies specific to Eimeria (Lee et al., 2007 a). Recent research adopted by Park et al. (2020) examined the impact of food supplementation with Bacillus subtilis 1781 or 747 on chickens with E. maxima infection. They found that dietary treatment with B. subtilis 747 following E. maxima infection increased gut defenses and the health of the epithelial barrier (Park et al., 2020). Recently, beneficial gut bacteria were found to be involved in the production of a novel category of feed additives known as “postbiotics,” which was formed and identified as having a favorable impact on host health through utilizing cutting-edge “omics” technologies (Park et al., 2020). The Bacillus subtilis 1781 strain caused a change in chicken gut metabolites connected to the benefits of B. subtilis probiotics for stimulating growth and the immune system (Park et al., 2021). Maltol, which is a naturally occurring organic compound that is used primarily as a flavor enhancer significantly impacts gut metabolites with the capacity to exhibit several physiological functions, especially in both antioxidant and anti-inflammation processes (Park et al., 2020). Collectively, we suggest that more research is required to investigate the minute gut metabolites linked to the favorable impacts of probiotics (Park et al., 2020). This will aid in discovering new postbiotic candidates that may enhance chicken growth and immune response without anticoccidials.
Prebiotics are non-digestible oligosaccharides that can modulate the intestinal microbiome, subsequently increasing probiotics’ growth and activity in the gut (Slawinska et al., 2019). Prebiotics typically utilized in poultry include fructooligosaccharides (FOS), arabinoxylooligosaccharides (AOS), mannan-oligosaccharides (MOS), isomaltooligosaccharides (IMOS), pyrodextrins, inulin, soy oligosaccharides (SOS), and xylooligosaccharides (XOS) (Al-Sheraji et al., 2013; Muthamilselvan et al., 2016). Most of these prebiotics come from tomatoes, artichokes, onions, garlic, chicory, and leeks (Al-Sheraji et al., 2013). Feeding chickens with prebiotics enhances their defense against pathogens and attenuates mortality (Ganguly, 2013; Sugiharto, 2016). Prebiotics significantly selectively boost good bacteria growth and subsequently increase the helpful microbiome in the digestive tract (Sayed et al., 2023).
Due to the preponderance of beneficial bacteria in the intestinal system of chickens (Muthamilselvan et al., 2016), dangerous pathogens are excluded. In several investigations adopted by Angwech et al. (2019) and Elmusharaf et al. (2007), prebiotics have been shown to prevent Eimeria infection in hens. Treatment with dietary MOS (10 g/kg feed) significantly tackled oral infection of chicken with a mixture of E. maxima, E. acervulina, and E. tenella. Interestingly, MOS attenuated oocyst fecal shedding and lessened the severity of E. acervulina lesions using subclinical dosages (900 mg, 570 mg, and 170 mg, respectively) (Angwech et al., 2019). Bozkurt et al. (2014) observed that a daily supply of MOS with a concentration of 1 g/kg in diet effectively improved feed conversion efficiency and growth rate, decreasing lesions’ severity in mixed Eimeria spp. infection. Angwech et al. (2019) evaluated the efficacy of Bi2tos (trans-galactooligosaccharides), a commercial prebiotic administered in ovo and found that the prebiotics attenuated the burden of intestinal lesions and oocyst discharge generated by three distinct Eimeria species when administered in ovo. Prebiotics and probiotics share several antimicrobial mechanisms (Abd El-Hack et al., 2022 b). Consequently, prebiotics are emerging as a novel method of coccidiosis control (El-Ashram et al., 2019).
Host defense peptides (HDPs), essential innate immune system effector molecules found in various species, constitute the initial line of defense for the host (Cuperus et al., 2013). Because they can inhibit bacterial growth in vitro, they are also known as antimicrobial peptides (AMPs) (Cuperus et al., 2013). Four significant structural groups have been noted by Kim et al. (2016) as amphipathic α-helical, ß-sheet, ß-hairpin or loop, and extended variations. Antibiotic activity of HDP peptides is against mycobacteria, fungi, gram-positive and gram-negative bacteria, and enveloped viruses (Yount et al., 2006). A few investigations by Hong et al. (2006 a) and Su et al. (2017) have revealed the protective effect of HDPs against coccidiosis in hens. Antimicrobial peptides can be used as an alternative for antibiotics due to their broad-spectrum effectiveness and selective action against bacteria (Sumners et al., 2011). They are proficient against several bacterial strains, consisting of those resistant to a wide range of antimicrobial medications due to their cationic nature. AMPs disrupt the bacterial membrane and exert their effect (Kim et al., 2017).
The complex immunogenic function of cationic AMPs consists of immunology regulation, such as cytokines and chemokines induction, inflammatory modulator, direct chemotaxis, wound healing, apoptotic activity, angiogenesis, and their role as an adjuvant (Sumners et al., 2011). Our preliminary research noted that chicken NK-lysin (cNK-lysin), a cationic amphiphilic AMP and human granulysin homolog, owns cytolytic properties versus Eimeria infection (Sumners et al., 2011). Chicken NK-lysin-2 (cNK-2) is a naturally occurring lytic peptide that disrupts the sporozoite membrane to exert cytotoxic activities versus apicomplexan parasites such as Eimeria (Kim et al., 2017). Eimeria species are eliminated in vitro and in vivo by cNK-2, produced by chicken cytotoxic lymphocytes during coccidiosis, and generated from the cNK-lysin protein’s cationic core region (Kim et al., 2016).
Infection with E. maxima, E. acervulina, and E. tenella causes a significant increase in NK-lysin in CD4+ and CD8+ intestinal IELs (Hong et al., 2006 a). NK-lysin originates from NK cells and T lymphocytes (Hong et al., 2006 a; Lee et al., 2013). Synthetic peptide (cNK-2) contains an estimated membrane-permeating, amphipathic alpha-helix of the full-length chicken NK-lysin. The anti-parasitic efficacy of synthetic peptide (cNK-2) was assessed (Lee et al., 2013). Cytotoxicity of E. tenella and E. acervulina sporozoites was affected by the cNK-2 peptide in a dose- and time-dependent manner as obtained in in vitro model (Lee et al., 2013). It triggered the destruction of the outer plasma membrane of E. tenella sporozoites and the loss of intracellular contents identified through transmission electron microscopy (Lee et al., 2013). The delivery of cNK-2 peptide in vivo exhibited a unique antiparasitic action, as indicated by promoted weight gain and attenuated fecal oocyst shedding (Lee et al., 2013).
AMPs administered using sustained delivery showed an increase in the host’s local immunity, prevented the formation of parasites, and promoted a balanced population of gut bacteria (Sumners et al., 2011). Also, efficient industry-friendly distribution methods for antibiotic substitutes will save labor costs (Sumners et al., 2011). Additionally, they positively impact animal production without antimicrobials such as the oral delivery of Bacillus spores to the colon, where Eimeria parasites engage the host’s gut epithelial cells (Wickramasuriya et al., 2021).
The cNK-2 peptide-carrying B. subtilis strain was administered orally to young broiler hens infected with live E. acervulina oocysts (Lee et al., 2023). The outcomes indicated that B. subtilis-cNK-2 could be a potential and efficient substitute for anticoccidial for coccidiosis through attenuation of parasite survival, ameliorating reduction in body weight, and reducing gut damage (Lee et al., 2023). This favorable impact was attributed to elevated protein expression, integrity, and intestinal health. Su et al. (2017) analyzed the expression of 2 innate immune system proteins, avian beta-defensin (AvBD) and hepatic-expressed antibacterial peptide 2 (LEAP2), in response to infection with Eimeria spp. Nevertheless, LEAP2 expression was consistently attenuated in the duodenum, jejunum, ileum, and ceca of hens infected with E. acervulina, E. maxima, or E. tenella (Su et al., 2017). The overall AvBD response to an Eimeria challenge was uneven, and this finding suggests that LEAP2 directly helps manage Eimeria infections (Su et al., 2017).
Dietary acetic acid supplementation enhanced broiler chickens’ pathological indicators, including decreased mortality, improved lesion scores, and oocyst shedding (Abbas et al., 2011; El-Saadony et al., 2023). Also, it improved the weight gain and feed consumption ratio (Abbas et al., 2011). On the other hand, butyric acid glycerides (lactobutyrin) and clopidol showed anticoccidial action when tested in chickens infested with E. maxima (Ali et al., 2014).
Organic acids improve health by lowering the pH of the digestive tract and altering the microbiota; chickens can become more resistant to disease (Dittoe et al., 2018). Elevated levels of organic acids might hurt plant growth (Versteegh and Jongbloed, 1999). Recently, there have been no commercially available organic acids for coccidian treatment, and the bulk of experimental findings have been verified in broiler chickens grown intensively (Kiarie et al., 2019). Infection with coccidia suggestively elevated the host’s maintenance energy requirements (Kiarie et al., 2019). Moreover, it has been noted that the transcript levels of nutrition transporters and digestive enzymes are decreased in E. acervulina and E. maxima infections (Leung et al., 2019; Su et al., 2014). The effectiveness of feed enzymes as an alternate strategy to supply enzyme deficiencies brought on by coccidiosis has not received as much research attention (Bortoluzzi et al., 2019; Scapini et al., 2019).
Chickens supplemented with mannanase after being given a 20x dose of a commercial live-attenuated coccidiosis vaccine experienced an increase in Lactobacillus, Ruminococcaceae, and Akkermansia in their intestinal flora and a decrease in Bacteroides, which is the cause of the poor feed efficacy in chickens (Bortoluzzi et al., 2019). Also, mannanase enhanced intestine health, as shown by better villi, crypt proportion, and the number of goblet cells in the gut mucosa (Scapini et al., 2019), with no effect on their productive performance. Dersjant-Li et al. (2016) reported that combining Bacillus spp. with enzymes could lessen the injury and ameliorate performance losses caused by coccidiosis.
Three strains of Bacillus spp. and three enzymes (xylanase, amylase, and protease) lowered the inflammatory response in hens exposed to a six-fold dose of coccidial vaccine (Dersjant-Li et al., 2016). They continued to perform at a level comparable to that of unopposed birds to that of unchallenged birds (Dersjant-Li et al., 2016). Novel techniques are utilized to find new feed additives, such as quorum sensing, bacteriophages, and small molecular chemical compounds, that increase animals’ physiological defenses against enteric infections and the feed additives already described above (El-Saadony et al., 2022 b). Alternatives to antibiotics are a significant unmet need in the livestock industry; however, multiple elements predict its success or failure (Dersjant-Li et al., 2016; El-Saadony et al., 2022, 2023).
Kurt et al. (2019) provided a methodology for assessing alternative possibilities, allowing federal agencies, nonprofits, and other significant stakeholders to prioritize financing for antimicrobial alternatives methodically and transparently. Since economic viability is the cornerstone of long-term commercial success, this process starts by analyzing the costs and advantages of the new alternative (Kurt et al., 2019). This data might have been easily accessible before or during the early stages of research and development. Passing alternatives to anticoccidial (ATA) to market ultimately entails evaluating the product’s acceptability, efficacy, and practicability (Abd El-Hack et al., 2022 c). As a result, assessing the potential effectiveness of a new option from various angles may be helpful. This strategy was promoted to design our initial survey and workshop (Kurt et al., 2019). For instance, earlier funding decisions for research may include input from farmers, veterinarians, and agricultural advisors (Abd El-Hack et al., 2022 d). Although creating novel antibiotic alternatives is challenging, doing so has enormous potential for improving animal health and combating antibiotic resistance (Abd El-Hack et al., 2022 e). Funders can use this method to assess alternatives during the earliest research and development phases and devote limited resources to the most promising ATAs (Salem et al., 2022 e; Sayed et al., 2023).
Chicken is among the most widely accessible animal protein resources and their requests are increasing worldwide. The lack of new anticoccidials and the decline in antimicrobial growth promoters (AGPs) drive the hunt for antimicrobial alternatives. After genetic engineering technology was developed, the pharmaceutical sector became interested in several logical antibiotic-independent solutions, including the recombinant vaccine technique. Determining the characteristics of immunogenic parasite antigens that trigger immune responses from the host similar to those caused by living parasites has proven to be complicated. Owing to Eimeria parasites’ high degrees of mutability, the large production of these recombinant proteins is prohibitively expensive. Besides, there are no acceptable delivery methods for recombinant proteins for the sustainable field vaccination of large flocks. Because of the intricacy of host-parasite immunobiology, emerging solutions for efficiently avoiding coccidiosis require a thorough, in-depth examination of molecular and cellular connections between hosts and parasites. More studies on the function of the gut microbiome in the role and function of GALT, the relationship between the gut microbiota and immunity, and recent thoughts on gut-brain relation are also necessary. As we strive to reduce the number of antibiotics used in animal and poultry farming, enhancing performance and integrating the optimum mixes of numerous alternatives will increase with the appropriate management and agricultural practices.