Introduction
The poultry industry has experienced unprecedented growth in the past 70 years due to improved genetics, nutrition, housing, water quality and veterinary care driven by post-WWII consumer demands (Havenstein, et al., 2003). Modern-day poultry efficiently convert feed to meat and eggs, but genetic selection for improved performance may have inadvertently selected for a negative correlative response to other traits such as immune function (Cheema et al., 2003). The reader may consider what occurs when they are ill to understand the phenomenon. When an individual is sick, they do not eat, or drink and activity is reduced. Through a process called cachexia, the body uses amino acids and energy from muscles and fats, which is incompatible with growth and reproduction. Therefore, it is reasonable why selecting for a feed efficient bird may not always result in improved immune function.
Poultry are exposed to numerous bacteria, fungi, viruses, protozoa, and parasites throughout their production cycle (Kogut et al. 2020). These microbes can replicate and mutate very quickly, presenting a challenge for the immune system. They have evolved over millions of years and have developed many ways to evade detection and destruction. Further reading on pathogen evasion mechanisms can be reviewed in Finlay and McFadden’s manuscript (2006). While many would consider the integument to have the greatest exposure to microbes, human mucosal surfaces are estimated to have a total surface area of approximately four hundred square meters and harbor over 13 trillion bacteria in the hindgut (Murphy et al., 2022). Mucosal surfaces have the greatest exposure to the outside world; therefore, most of the immune system can be found nearby in mucosal or gut associated lymphoid tissues (MALT/GALT). The purpose of this proceedings is to give a brief immunology primer and then discuss the proposed mechanisms of activity of antibiotic alternatives and their potential effects on the microbiota and gut health. Please be reminded that the simple answer is not always the most correct answer, but we will cover the topic at 30,000 feet to give the reader a basic understanding.
Innate Immunity
For ease of understanding, the immune system can be divided into an innate and adaptive immune system. Innate immunity begins with physical barriers to the outside world such as skin, mucus, stomach acid, digestive enzymes, nutrient binding (ovotransferrin), tears (lysozyme), changes in pH, oxygen tension (foregut versus hindgut), competitive exclusion by microflora, antimicrobial peptides (e.g., defensins produced by Paneth cells) and temperature differences. The innate immune system is characterized as being fast acting, non-specific and “what you are born with.” It is found in more primitive organisms such as fruit flies and was marginalized until the recent discovery of pattern recognition receptors and the understanding that the innate leads the acquired immune system (Janeway and Medzhitov, 2002). Cells can communicate by producing a multitude of soluble factors, called cytokines, or membrane bound receptors(Kaiser et al., 2005). A subgroup of these cytokines are called chemokines which allow for movement of immune cells to tissues that are damaged or infected. An example of this, would be a gut epithelial cell infected with a bacterium causing it to secrete CXCL-8 (formerly interleukin-8 or IL-8) to promote chemotaxis of immune cells to the site of infection. Microbial associated molecular peptides (MAMPs) are cues immune cells may use to identify a potential pathogen via germ line encoded pattern recognition receptor (PRRs). These MAMPs are typically microbial components that cannot be changed, such as peptidoglycan which gives structure to a bacterial cell membrane. These MAMPs (formerly pathogen associated molecular patterns, PAMPs) allow for a phagocyte to discover, consume, and kill a microbe. In addition to MAMPs, danger associated molecular patterns (DAMPs) are needed for an effective immune response to be mounted. These DAMPs are necessary redundancies. Most microbes, that an individual may encounter, are not harmful but they still exhibit these MAMPs.
Heterophils, macrophages and dendritic cells are white blood cells that eat (or phagocytize) and kill pathogens. Heterophils, the avian equivalent to a mammalian neutrophil, respond to an infection very quickly and kill microbes through oxidative burst, nitric oxide, degranulation and netosis. These phagocytes cause inflammation by releasing cytokines and other immunogenic factors in addition to what is released when the pathogen is killed, such as nucleic acids. Macrophages and dendritic cells arrive shortly after the heterophils. They can kill microbes, like the heterophil, but they also have the advantage of serving as an antigen presenting cell. What this means, is that they can present a small piece or epitope of the pathogen via MHC II (major histocompatibility complex) to other immune cells to coordinate or direct the adaptive immune response. Dendritic cells can cover a large surface area and interact with more cells due to its dendrites, which are analogous to roots of a tree. The natural killer cell is comes from a lymphoid progenitor cell that kills abnormal, or virus infected cells through programmed cell death. These cells are identified by unique PRRs and the absence of MHC I, which is present on all normal nucleated cells. MHC I present endogenous antigens such as a virus fragments as well as self-antigens. This is useful, as a phagocytic cell would not be able to determine that a cell was infected using its PRRs.
Mast cells, basophils and eosinophils are granulocytes that can be activated by the antibody isotype IgE, PRRs and complement. Allergic individuals are called atopic and may have up to 10 times the normal amount as the rest of the population. While severe allergies are harmful, these atopic individuals may have increased resistance to parasitic infections. Mast cells are the primary immune cell that mediates inflammation, releasing factors such as histamine, leukotrienes, and arachidonic acids. Inflammation is characterized by redness, swelling, heat and pain. The process prepares a tissue for battle by allowing for the migration of fluids, nutrients, and white blood cells to an injured or infected area. Eosinophils and basophils primarily focus on multicellular parasites, such as helminths (reviewed by Stone et al., 2010).
Complement may be described as innate humoral immunity. It’s a complex system of 35 proteins that may be triggered by MAMPs, or antibody bound to its respective antigen. The ultimate effect is to produce holes in the offending organism, via the membrane attack complex (MAC), allowing for leakage of it internal contents which also will be immunogenic. Complement opsonins may also act similarly to an antibody by binding to a pathogen and serving as a ligand for the complement receptor on phagocytes.
Adaptive Immunity
The adaptive immune system can take longer to respond to infection but is extremely specific and diverse. Estimates vary from 1011 to 1018 unique epitopes that can be identified by the adaptive immune system, which is an incredible number! While the innate system is always ready to respond quickly, the adaptive immune system can take weeks to months to mount an effective immune response as it goes through a complicated and stringent negative selection process where 95-98% of the lymphocytes are destroyed. It also has the added advantage of creating memory cells, which allows for vaccination to occur once a viable solution has been found.
The cells of the adaptive immune system are lymphocytes called B and T cells. Glick and colleagues discovered B cells by serendipity when they discovered that bursectomized chickens were not able to produce antibodies (1956). It was later discovered that the bursa of Fabricius allowed for a subset of lymphocytes, which produce antibodies, to develop. Its equivalent has not been found in mammals, where B cell maturation occurs in the bone marrow. Antibodies are extremely specific to what they bind to. They may be found on mucosal surfaces, in circulation, in tissues and bound to cells receptors. In a best-case scenario, they may neutralize a pathogen on a mucosal surface allowing it to simply float away in the lumen with no added response required. They may also help cells of the innate immune system to become much more specific by a process called antibody dependent cellular cytotoxicity, in effect serving as a specialized receptor for an innate immune cell.
T cells were later discovered and found to mature in the thymus (Cooper, 2015). These cells primarily produce cytokines to direct, improve or reduce the immune response. The major subtypes are T helper 1, T helper 2, T helper 17, T regulatory and cytotoxic T cells (Th1, Th2, Th17, Treg, and Tc). T helper 1 lymphocytes facilitate cell mediated immunity. These cells produce cytokines that help phagocytic cells to better respond to an infection. T helper 2 cells drive the humoral immune response helping B cells to produce additional and over time, better antibodies. T helper 17 cells produce a cytokine called IL-17 which improves neutrophil/heterophil function. These three cell types would be categorized as mediating a type I, II, or III immune response, respectively. T regulatory cells produce IL-10 and TGF-beta to return cell mediated immunity to baseline conditions prior to an infection. Interestingly, Th1 and Th2 cells produce factors that inhibit the other, so they may act in a regulatory fashion as well. Cytotoxic T cells are like natural killer cells functionally, but they are much more specific searching for a distinct epitope instead of a MAMP or absence of MHC I expression. These cells are particularly important as viruses can be difficult to identify when hiding inside of the host’s cell. Cytotoxic T cells make use of MHC I which presents endogenous antigens from within a cell. Some of these may be self-antigen, but it may also present material related to production of a virus. Fortunately, all lymphocytes have been intensively selected to only respond to pathogens and not self-antigen. These cells are especially important when cells become malignant. It is thought that incidence of cancer increases as an individual ages to due to decreased immune surveillance.
Lymphocyte Recirculation/Antigen Trapping
Blood vessels can be considered leaky pipes that transport nutrients to various tissues. When the pressure increases, as seen with hypertension or ascites syndrome more leakage will occur, and tissues swell. Fortunately, these tissues are drained by an efficient lymphatic system that returns the extracellular fluid to the heart via the thoracic duct. There is some similarity between the lymphatic system and a water shed where smaller streams combine forming large rivers. Multiple afferent lymphatic vessels are consolidated at lymph nodes which will have only one efferent vessel. This system provides a very efficient means for white blood cells to travel and to communicate with each other. Interestingly, while the chicken has an effective lymphatic system, it does not have defined lymph nodes. It is thought that other lymphatic tissues found in the Harderian gland, Meckel’s diverticulum, Peyer’s patches, cecal tonsils, and bursa of Fabricius make up for the lack of avian lymph nodes (Kim and Lillehoj, 2019). Lymphocytes can make copies of themselves within these specialized environments leading to lymph node enlargement that may be observed during an infection. Lymphocyte recirculation is especially important as there may be only one B cell that has to meet only one T cell that are both specific for a single extremely specific epitope to be effective. Once this match has been made, these cells will make many copies of themselves to expand the immune response. They will refine the receptors and antibodies that are produced to mount an improved immune response over time. Further, some of these cells will become memory cells, which may last a lifetime, and are ready to respond quickly upon a secondary infection. Macrophages and dendritic cells also use this recirculation phenomenon, presenting new epitopes to these lymphocytes. Dendritic cells may migrate from the lamina propria of the gut or the epidermis of the skin (Langerhans’s cells) to a lymph node to share this information with lymphocytes.
Antibiotic Alternatives
Antibiotics were used in animal production for many years. It is thought that these products fed at a sub-therapeutic level enhanced growth production by reducing microbial competition for nutrients. They also prevent infections, such as caused by Clostridium perfringens in necrotic enteritis, which would counter nutrient absorption by the gut (reviewed in Bedford, 2000). Consumer demand and government restrictions have significantly curtailed the use of antibiotic usage in farm animals. It has been contended that these impediments to veterinary care may prevent sick animals from being treated, thereby causing a reduction in animal welfare, and increasing production costs (Singer et al., 2019). Antibiotic alternatives are being looked at closely to fill this void and improve animal health.
Vaccination may be one of the most successful alternatives to antibiotics, but it is not always a perfect solution. Despite significant growth rates and size, broiler chickens are still juveniles. They are harvested at 4-9 weeks of age, while puberty occurs much later at 18-22 weeks. This shortened timeline may not allow for adaptive immunity to develop before the bird is exposed to a pathogen. Maternal antibodies, found in the yolk sac and in circulation, may interfere with vaccination at the hatchery, which is the most convenient time to vaccinate. Microbes have many ways to evade the immune system, changing what it “looks” like making a vaccine ineffective. Poultry appear to be challenged with a constant onslaught of viruses. By the time that the etiology has been decided, the virus has run its course and a new one arises. This obviously makes vaccination difficult, but several of these viruses are also immunosuppressive, which could have a negative effect for vaccinating against another pathogen. Added booster vaccinations are usually needed to achieve maximum protection against a pathogen, but this adds to the cost, labor and handling stress to the animal. Predicting what pathogens will affect a flock can be exceedingly difficult, but a significant amount of time is also needed for the bird’s immune system to be properly prepared to defend itself. Vaccinating during an infection won’t be helpful and could exacerbate the problem. There are many types of vaccines available. They may be classified as living or not living. Both vaccine types will have the necessary PAMPs to be recognized by PRRs but sometimes that isn’t enough. Living vaccines can produce or express DAMPs which are a necessary secondary signal to prevent the immune system from “misfiring”. Unfortunately, low dose or weakened live vaccines can also cause the disease in rare cases. Non-living vaccines typically need something else to be noticed by the immune system. A material known as an adjuvant (Latin adjuvere - to help) is used in combination with the vaccine to help the immune system to recognize these PAMPs. These adjuvants cause inflammation and may cause localized tissue damage in extreme circumstances, which could be a negative if it affects muscles consumed as meat.
While a modern hatchery is an environmentally controlled and clean environment, it isolates the neonate from the mother hen thereby interfering with establishment of a healthy microbiota via coprophagy (Nurmi and Rantala, 1973). Prebiotics and probiotics are a means to improve the gut microflora of an individual. Prebiotics are indigestible nutrients to the host but can be used by and favor the growth of certain beneficial bacteria. Probiotics are beneficial bacteria that may be administered via the feed or water to colonize the gut with beneficial bacteria. Both solutions work by a process known as competitive exclusion. Microbes compete for attachment space and nutrients. They produce substances, such as antimicrobial bacteriocins, to kill and out compete other microbes. A major issue with antibiotic usage is that these beneficial bacteria may be indiscriminately killed allowing the gut to be reseeded with bad microbes causing diarrhea and poor nutrient absorption.
Yeast fermentation products were initially produced as a by-product of alcohol production. These products can be fractioned into yeast cell wall fragments, mono-oligosaccharide (MOS), and beta-glucans (reviewed by Sanchez et al., 2021). Yeast beta-glucans have been shown to be immune-stimulatory in chickens (Lowry et al., 2005). Mono-oligosaccharides bind pathogenic bacteria in the gut, thereby neutralizing the pathogen preventing attachment to the epithelium (Peisker, 2017).
Fungi have been shown to be immunogenic and recognized by numerous PRRs as reviewed by Patin and colleagues (2019), but they may also supply nutrients for beneficial bacteria and the host.
Phytogenic feed additives are derived from plants that may have beneficial effects. Essential oils, tannins, herbs and spices are examples of these products. They are reported to have immune-modulatory and antimicrobial properties. El-Hack and colleagues recently published an extensive review concerning this category of antibiotic alternatives (2022)
Bacteriophages are viruses that infect a specific host bacterium. They have shown to be effective when fed to live birds or applied to carcasses but must also be paired with the correct microbe which can be problematic. There are estimated to be more than 10 times as many bacteriophages than bacteria (reviewed by Zbikowska et al., 2020). Therefore, it’s possible that a bacteriophage may exist for every or at least many pathogens.
Meat birds typically undergo 8-10 hours of feed withdrawal prior to processing to reduce contamination by ingesta and fecal matter. Unfortunately, the birds are still hungry and begin to eat litter and darkling beetles which may carry foodborne pathogens. They will also consume more water to reach satiety. Products have been administered via the drinker system such as pre/probiotics and acids to counteract this phenomenon. Byrd and colleagues evaluated the administration of acetic, formic and lactic acids in drinking water and found the most improvement with lactic acid reducing Campylobacter and Salmonella contamination in broilers. Pineda and colleagues had some success reducing Salmonella Heidelberg biofilm and cecal colonization using sodium bisulfate in water (2021). While acidification of the foregut is possible, it is quickly countered by the dilution by ingesta and the bicarbonate buffering system. Foodborne bacteria can survive a wide range of pH, which makes them difficult to kill without injuring the host or significantly reducing water consumption.
The importance of nutrition should not be discounted when discussing immunity. Energy and amino acids are needed for an effective immune response, including clonal expansion, production of antibodies, complement and cytokines. While least cost feed formulation allows for meeting all the bird’s nutrient needs at the lowest cost, additional factors may need to be considered concerning immunity, microbiota and overall gut health. Cod liver oil may be one of the earliest examples of using a nutraceutical to treat tuberculosis in the 1800’s (Grad, 2004). Cod liver oil is rich in omega-3 fatty acids and vitamins A and D which are both touted to affect immunity. Calder reviews the importance of zinc, copper, iron and vitamins A, B6, B9, B12, C, D and E to immune function in his recent paper (2021). While malnutrition will have negative health effects, fasting or reduced consumption of calories can be beneficial. In fact, overeating can cause chronic inflammation which can lead to other maladies such as cancer and heart disease (Collins and Belkaid, 2022).
Enzymes can make nutrients more available to the host and potentially the microflora within the host. Fernandez and colleagues reported that the addition of xylanase to a wheat-based broiler diet reduced mucin viscosity and colonization by Campylobacter jejuni. Campylobacter use mucin as a nutrient source and the xylanase increased production of mucin by goblet cells within the gut reducing viscosity and creating a less hospitable environment for the pathogen (2000).
Conclusion
It is amazing to think that trillions of microorganisms are living on and within our bodies trying to eke out an existence. They have had millions of years to adapt and improve through random mutations and shared genes. Most microbes do no harm and are in fact beneficial for our development and nutrient absorption. These organisms can even influence behavior as seen with infections such as rabies and Toxoplasma gondii. The immune system is a formidable weapon that keeps these microflorae in check and even encourages growth of the advantageous ones. Production agriculture will continue to use and improve antibiotic alternatives to modulate and improve immune function thereby improving animal welfare, gut health and nutrient absorption, but also decreasing waste which is beneficial for the environment.
Presented at the 2022 Animal Nutrition Conference of Canada. For information on the next edition, click here. 
