Moderating the Effect of Coccidia and Necrotic Enteritis Challenge Using Non-Pharmaceutical Means

Components of Gut Health

The intestine is a complex organ that comprises regions with distinct structural and physiological functions specialized in digestion and nutrient absorption. Simultaneously, the gut represents the primary contact site with foreign antigens and pathogens that can enter, reside and disseminate to the internal organs. For this reason, the gut harbors the majority of immune cells, referred to as gut-associated lymphoid tissues, when compared to other tissues. Oral tolerance to feed particles and commensal bacteria and efficient recognition and elimination of pathogens are two gut-specific functions that affect immunity (Smith et al., 2022). Recently, gut health has been gaining great attention for its role in the general health of animals. By definition, gut health is the number of physiological, microbiological, physical, and immunological functions working together to maintain intestinal homeostasis (Kogut, 2019). This paragraph will briefly discuss gut health moving from the intestinal lumen that contains commensal bacteria, mucosal layer, and various antimicrobial peptides to the epithelial monolayer and finally discusses the gut-associated lymphoid organs in the lamina propria.

Commensal bacteria

Commensal bacteria play a major role in gut health by competing with pathogens for attachment sites and nutrients. One of the clear roles of commensal bacteria has been demonstrated by Nurmi and Rantala (1973) that showed that gut microbiota from healthy adult chickens could protect newly born chicks against Salmonella invasion. Commensal bacteria produce different antimicrobial peptides, known as bacteriocins, that have a wide range of antimicrobial activity against various pathogens (Vieco-Saiz et al., 2019). In addition, some commensal bacteria ferment indigestible complex carbohydrates and produce short-chain fatty acids SCFA, primarily acetate, propionate, and butyrate. Short-chain fatty acids have a bactericidal activity mainly against gram-negative bacteria (Ricke, 2003). Butyrate is known to be a source of energy for enterocytes and to modulate gut health in poultry (Bedford and Gong, 2018).

Gut lumen

The first physical barrier against pathogen invasion is mucus, a glycoprotein produced by goblet cells. Mucus traps pathogens that can be expelled from the intestine by the luminal flow. Intestinal IgA is the main antibody class in mucosal secretions and plays a major role in neutralizing gut pathogens and controlling commensal bacteria (Broom, 2018). IgA is in the form of dimer in mucosal secretions and forms a complex with the secretory component from the surface of the epithelial cells that protect the antibody from endogenous proteases (Rautenschlein, 2019). Another major component of the intestinal lumen is host defense peptides (HDP) which are antimicrobial peptides produced by the host and are known to have a wide range of antimicrobial activities against different bacteria, viruses, protozoa, and fungi. Defensins and cathelicidins are the most characterized HDP in chickens (Cuperus et al., 2013).

Epithelial layer

Another physical barrier is the epithelial monolayer after the mucosal lining (lumen). Epithelial cells are held together by complex protein structures known as tight junctions. Villus structures protrude the gut, followed by indentations known as crypts, increasing the gut's surface area. Intestinal epithelial cells play a major role in digestion and their function as a physical barrier. In addition, intestinal epithelial cells have a significant role in innate immunity by expressing pathogen recognition receptors (PRR). PRR monitor and respond to pathogens by recognizing invariant molecular motifs of pathogens known as Pathogen-associated molecular patterns (PAMPs).

Gut-associated Lymphoid Tissue (GALT)

The gut-associated lymphoid tissue is comprised of lymphoid cells distributed in the lamina propria. Chickens lack lymph nodes and rely heavily on lymphoid aggregates within the lamina propria, including Meckel's diverticulum, payer's patches, and cecal tonsils. The GALT is a major immune tissue that harbors most immune cells when compared to other immune tissues (Smith et al., 2022). The epithelial monolayer consists of microfold cells (M cells) that are specialized epithelial cells that sample and deliver contents from the gut lumen to antigen-presenting cells like dendritic cells and macrophages. Antigen-presenting cells delivered antigens to lymphoid follicles rich in B and T lymphocytes. The chicken hindgut is known to harbor more organized lymphoid tissue than the foregut (Smith et al., 2022).

Enteric infections in poultry: The case of coccidiosis and necrotic enteritis

The gut is in constant contact with pathogens, and the mucosal layer and the epithelial layer are the first physical barriers to entry. Gut pathogens can be localized, colonize and persist in the intestinal lumen and cause little damage, such as Campylobacter. Another type of pathogens is intracellular pathogens that reside in the intracellular epithelial layer and can cause acute damage, such as Eimeria. Other pathogens utilize the intestine to disseminate into internal tissues, and therefore localized immune response is critical for controlling the systemic infection. Innate immunity is the first line of defense activated by pathogen recognition receptors (PRR) such as TLR that are expressed in epithelial cells. These receptors respond to pathogens by recognizing invariant molecular motifs of pathogens known as Pathogen-Associated molecular patterns (PAMPs). In addition, the GALT, such as payer's patches, is lined with M cells that constantly sample the lumen to detect pathogens. Once the innate immune system initiates pathogen recognition, heterophils infiltrate to kill the pathogen or act as antigen-presenting cells (APC) that help recruit other immune cells. NK cells, macrophages, and TCR γδ+ T cells play a major role in limiting infection during the early stages (Smith et al., 2022). After the breakdown and presentation of the pathogen on MHC of APC, the adaptive immune cells play a major role in the resolution of the infection. Adaptive immunity is mainly divided into humoral immune response mediated by B cells that later differentiate into plasma cells and cell-mediated immunity that is mediated by CD4+ helper T cells that help B cells T cells and macrophages, and CD8+ cytotoxic T cells (Rautenschlein, 2019). Immune response against infectious agents does not always lead to the clearance of the pathogen and can often be damaging to the host's tissues.

Coccidiosis

Coccidiosis is a disease caused by a protozoan parasite of the genus Eimeria. Eimeria species are transmitted via the fecal-oral route and are intracellular parasites that enter the intestinal tract, replicate and cause damages to the epithelial layers (McDougald and Fitz-Coy, 2008). This disruption to the gut barrier, commonly called "leaky gut," results in detrimental effects that include reduction in feed intake, digestibility and nutrient absorption, blood loss, and dehydration. Coccidiosis can be mild or severe depending on the number of ingested oocysts. In poultry, nine Eimeria species are known to infect chickens and cause distinct lesions that affect different parts of the GIT (Long, 1985; Pellerdy, 1974). Gross lesion scores of different sections of the GIT are recorded to determine the severity of infection. The most commonly used lesion scoring system uses a graded scale from 0-4, where 0 is for the absence of lesions and 4 is the maximum lesion (Johnson and Reid, 1970). The eimerian life cycle involves oral ingestion of sporulated oocyst (usually sporulate in wet poultry litter), invasion of enterocytes (zoite stage), development of asexual (schizonts) and sexual (gametocytes) reproduction phases, fertilization, and release of oocysts in feces (Smith et al., 2022). Therefore, efficient immune response to Eimeria has to be an intracellular response, rapid, and dependent on the primary immune response (Smith et al., 2022). The immune response to Eimeria is species-specific and, in some cases, strain-specific such as E. maxima. Eimeria maxima are the most immunogenic Eimeria species. Studies have shown that IFN- -γ has anticoccidial properties and is critical in controlling Eimeria infection and cycling. Rothwell et al. (2004) showed that chicken lines more susceptible to E. maxima infection have a higher expression of IL-10 mRNA both pre and post-infection in the spleen and higher expression of IL-10 mRNA in the intestine post-infection when compared to resistant lines. Infection with Eimeria induces heterophil infiltration, activation of innate NK cells, and antigen-specific B and T cells (Shirley et al., 2005). However, the immune response of chickens to Eimeria depends on the age, host genetics, and species of Eimeria (Smith et al., 2002). Historically, coccidiosis has been controlled by using different chemotherapeutics and live vaccines. Even though live and attenuated vaccines have been used to decrease the disease severity and oocysts shedding, live vaccines can induce a light infection and decrease body weight and feed conversion. Coccidiosis vaccines rely mainly on inducing immunological memory to protect against secondary infections. A study by Rose and Hesketh (1979) found that B cells play a limited role in the primary infection and re-challenge infection in bersectomised chickens. The authors concluded that CD4+ (helper T cells) but bot CD8+ were essential in resisting E. maxima primary infection. Depleting chicken CD4+ helper T cells using anti-CD4 demonstrated that CD4+ cells play a role in E. tenella primary infection but not E. acervulina (Trout and Lillehoj,1996). Eimeria infection, mainly E. maxima and E. acervulina, has been shown to increase the susceptibility of birds to Necrotic Enteritis that involves the Gram-positive anaerobic bacterium Clostridium perfringens (Immerseel et al., 2004).

Necrotic enteritis

Necrotic enteritis is associated with the Gram-positive anaerobic bacterium Clostridium perfringens. The disease is mainly caused by C. perfringens toxins that induce intestinal necrosis. Clostridium perfringens is a ubiquitous and commensal bacterium that is commonly found in the chicken gut. NE is primarily diagnosed by the presence of macroscopic lesions with the presence of Gram-positive Clostridium perfringens. Necrosis is not limited to intestinal villi but can also reach internal tissues like the liver, heart, kidney, and bursa. Necrotic enteritis usually causes a sudden spike in mortality. In addition, NE has subclinical forms that cause reduced growth and efficiency, leading to significant economic losses to the poultry industry. Clostridium perfringens strains are classified into seven types A – G based on the toxins produced. Poultry pathogenic C. perfringens strains belong to types A, C, and G. The pathogenesis on NE is not completely understood. Researchers focused initially on the CPA toxin, and studies later showed that CPA-deleted mutants could still be infectious in vivo. The focus shifted to the NetB toxin, and studies showed that NetB positive strains could reproduce NE lesions but not NetB-negative strains. Recently studies showed that NetB is associated with virulent CP strains; however, it is not the only virulent factor since some NetB negative strains could still cause NE lesions experimentally (Rood et al., 2016). Historically, NE was controlled by controlling the growth of Clostridium perfringens by the use of antibiotics, namely bacitracin (narrow-spectrum) and virginiamycin (broad-spectrum) activity against Gram-positive bacteria (LaVorgna et al., 2013). In addition, NE was controlled by controlling the exposure to coccidiosis by anticoccidial drugs such as ionophores and chemicals (Opengart and Songer, 2008). Most NE outbreaks occur at around three weeks or later and could be associated with maternal anti-CPA antibodies that usually wane at three weeks of age (Heier et al., 2001). Numerous attempts have been made to develop NE vaccines utilizing different C. perfringens toxins and delivery systems (Opengart and Songer, 2008). The immune response of chickens to necrotic enteritis is mainly linked to the NE challenge model utilized in the studies. Table 1 summarizes the various NE challenge models. Necrotic enteritis models rely on either creating an ecological niche or providing nutrients, mainly proteins and amino acids, for the proliferation of C. perfringens (Antonissen et al., 2016). Necrotic enteritis challenge models induce bacterial dysbiosis in the gut that is observed by a reduced abundance of immune-modulating segmented filamentous bacteria and lactic acid and butyrate-producing bacteria (Antonissen et al., 2016).

Table 1. Published necrotic enteritis challenge models.

Non-Antibiotic Growth Promoters for alleviating coccidiosis and necrotic enteritis

The poultry industry has been under immense pressure to reduce the reliance on antibiotics in feeds. Non-antibiotic growth promotors (non-AGPs), such as feed additives, have been tested for their use as on-farm interventions that can alleviate the detrimental effects of coccidiosis and necrotic enteritis. Research feed additives against coccidiosis and necrotic enteritis include probiotics, prebiotics, phytogenic compounds, enzymes, organic acids, and organic trace minerals. Table 2 summarizes the hypothesized mechanism of action for every feed additive intervention and brief literature results. It is worth noting that feed additive combinations also provide an advantage for providing synergistic biological activity. However, combinations should also be economical (below $2/short ton). Product combinations can also be beneficial for decreasing pathogen resistance by avoiding the repetitive use of a single product. One clear example of the benefit of feed additive combinations could be the bioshuttle coccidiosis control programs in no-antibiotics-ever (NAE) production.

Table 2. Non-pharmaceutical interventions for alleviating necrotic enteritis and coccidiosis in poultry.

Necrotic enteritis and coccidiosis interventions beyond feed additives

Poultry producers today rank restrictions on antibiotics as a key factor impacting the cost of production (Watt Poultry Survey, 2021). A widespread reduction of antibiotic usage increased broiler health and, consequently, also production challenges not seen before implementing these changes. It might be natural to consider only nutrition to help with these issues since antibiotics and ionophore coccidiostats were mostly added via feed, which means that the replacement strategies should also be feed-based. However, the issue is larger than just nutrition. Experience has shown us that nutritional interventions alone cannot fully compensate for performance losses associated with approaches like NEA (no antibiotics ever). Management, in particular, is an important factor since much needs to be done to control coccidiosis and necrotic enteritis. Issues such as bird density (Tsiouris et al.,2015a), the occurrence of wet litter (Hermans and Morgan, 2007), the use of coccidia vaccine (Williams, 2002), and temperature stress (Tsiouris et al., 2015b) can all impact NE. Limiting exposure to infectious agents through biosecurity might be a tool to reduce the incidence of NE and improve gut health (Shawkat et al., 2015). Among the general nutrition topics impacting NE and its severity are mycotoxin contaminations (Antonissen et al., 2014) but also other water and feed quality parameters such as the condition of oils and fats, presence of antinutrients in soybean meal, and retrograded starch and reduction of protein solubility in corn (Oviedo-Rondon, 2019). Grain source (Branton et al., 1987), type and level of protein (Drew et al., 2004), and mineral levels (Paiva et al., 2013) can also impact NE.

In conclusion, necrotic enteritis and coccidiosis are the top two enteric challenges facing as poultry production moves towards no-antibiotics ever production systems (NAE). Research methods are constantly advancing, especially in the field of omics that provide a valuable tool for the comprehensive understanding of challenges in the biological system (Karahalil, 2016). Therefore, research aimed at controlling coccidiosis and necrotic enteritis and understanding the functionality of omics data will elucidate the complex host-microbiome-pathogen interactions. This effort would require cross-functional teamwork between poultry producers, veterinarians, nutritionists, microbiologists, immunologists, and bioinformaticians.

Presented at the 2022 Animal Nutrition Conference of Canada. For information on the next edition, click here.