Animal Feed Science and Technology 250 (2019) 41–50

Contents lists available at ScienceDirect

Animal Feed Science and Technology

journal homepage: www.elsevier.com/locate/anifeedsci

Immunity, immunomodulation, and antibiotic alternatives to

T
maximize the genetic potential of poultry for growth and disease
response
⁎
Woo H. Kim, Hyun S. Lillehoj
Animal Biosciences and Biotechnology Laboratory, Beltsville Agricultural Research Center, Agricultural Research Service, USDA, Beltsville, MD 20705,
USA

A R T IC LE I N F O ABS TRA CT

Keywords: Multiple challenges confront the increasing demand for wholesome poultry food products, in-
Immunity cluding governmental restrictions on the use of antibiotic growth promoters (AGPs), nutritional
Immunomodulation requirements to obtain maximum growth potential, understanding crosstalks among the im-
Alternatives to antibiotics munity–microbiota–neuroendocrine system in the gut to maximize intestinal efficiency, high-
Poultry
density production conditions, waste management, and the emergence of infectious pathogens,
particularly those that emerge in the antibiotic-free animal production environment. Although
in-feed antibiotics have dramatically increased the efficiency of commercial poultry production
over the last 50 years, we are now faced with an increasing global crisis concerning the heigh-
tened use of antibiotics in animal agriculture and the emergence of multidrug-resistant superbugs
that threaten disease management in animals and humans. Therefore, much interest has focused
on the development of alternative, antibiotic-free methods of commercial poultry production.
Initially, alternatives to antibiotics included any strategies that replace AGPs, but now include
any feed additives or treatment that will allow antibiotic-free animal production to prevent and/
or treat diseases. These newer disease control strategies can be classified broadly into those that
are directly cytotoxic against infectious agents or remove pathogenic toxins, including vaccines,
hyperimmune antibodies, antimicrobial peptides, and bacteriophages, and those that augment
non-specific host immunity and gut health, including phytochemicals, adjuvants, prebiotics, and
probiotics. Furthermore, because the gut microbiota influences various physiological aspects of
the immune response, brain function, and gut health, most antibiotic alternatives are expected to
promote beneficial microbes that will benefit host physiological responses. However, there is a
timely need to better understand the role of the microbiota in gut health if we want to use
microbes to modulate the host response to enhance growth performance. This review will
highlight current knowledge of host immunity in poultry, and various strategies to modulate host
immunity, growth performance, and disease responses to guide the development of alternatives
to reduce the use of antibiotics, using a few selected alternatives and a description of their ef-
ficacy and modes of action.

⁎
Corresponding author.
E-mail address: hyun.lillehoj@ars.usda.gov (H.S. Lillehoj).

https://doi.org/10.1016/j.anifeedsci.2018.09.016
Received 9 February 2018; Received in revised form 31 July 2018; Accepted 23 September 2018
0377-8401/ © 2018 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/BY-NC-ND/4.0/).
W.H. Kim, H.S. Lillehoj Animal Feed Science and Technology 250 (2019) 41–50

1. Immunity and immunomodulation

The major function of the gut is to process food through digestion and to absorb nutrients from the lumen into the bloodstream.
Thus, the intestinal epithelium, which constitutes the interface between the lumen and host tissues, is in contact continuously with
large quantities and a wide variety of antigens from exogenous sources, including food, resident microorganisms, the commensal
flora, and potential pathogenic microbes that could be harmful (Garrett et al., 2010). Accordingly, the intestinal immune system must
trigger a protective immune response against pathogenic microbes while maintaining tolerance to antigens from food and com-
mensals. Gut-associated lymphoid tissues (GALTs) represent the largest compartment of the immune system, and they are affiliated
with the nervous and endocrine systems. Like all other immune systems, a variety of both innate and adaptive immune responses
against pathogenic microbes takes place in the intestine. The first layer of defense against pathogenic microbes in the intestinal
mucosa is the intestinal epithelium that separates the luminal material from the underlying lamina propria (LP) and deeper intestinal
layers. The intestinal epithelium comprises four cell lineages that originate from a common stem cell progenitor: absorptive en-
terocytes, mucus-producing goblet cells, hormone-producing enteroendocrine cells, and antimicrobial peptide-producing Paneth cells
(Yen and Wright, 2006; Abreu, 2010). The luminal side of the intestine is covered by mucus that limits direct contact between
antigens and the intestinal cell lining, and it contains mucins that are secreted by goblet cells. Goblet cells residing throughout the
intestine and the mucus layer protect the mucosa from dehydration and mechanical damage, and they play a dynamic role by
increasing mucous production in response to infection. Initiation of the innate immune response is triggered by the recognition of
pathogen-associated molecular patterns (PAMPs) by pathogen recognition receptors (PRRs) such as Toll-like receptors (TLRs), nu-
cleotide-binding oligomerization domain (NOD) proteins, and nucleotide-binding domain leucin-rich repeat-containing receptors
(Fukata et al., 2009). Once PAMPs from pathogenic microbes are sensed by PRRs, epithelial cells secrete antimicrobial peptides and
cytokines such as interleukin (IL-10) and transforming growth factor-beta (TGF-β), then lymphocytes, including macrophages and
dendritic cells (DCs), become activated to combat the infection in the LP, where immunocompetent cells are found underneath the
epithelium. Another consequence of PRR signaling in the intestine is the production of secretory IgA (sIgA) by B cells. Activation of
TLR4 promotes B cell recruitment to the LP and class switching of B cells to sIgA, resulting in an increase of sIgA, which is useful for
neutralizing pathogenic microbes. In addition, the intestinal epithelium has other strategies for inhibiting microbial invasion, in-
cluding the production of antimicrobial peptides (AMPs) and lectins, such as defensins, which permeabilize the cell walls of microbes.
Paneth cells located at the base of small intestine crypts are the main source of AMPs and lectins.
Although innate immunity is sufficient for protecting the gut in most instances, adaptive immunity confers more specific and
efficient protection against re-encountered pathogens. As the intestine is the largest reservoir of T and B lymphocytes, which are
major effector cells of the memory response, the adaptive immune response in the gut plays a central role in protection against
infection, as well as maintenance of immunological tolerance to harmless food antigens. The immune cells participating in the
adaptive immune response are found mostly in the epithelium, which is home to intraepithelial lymphocytes (IELs), and the LP. The
majority of IELs and LP lymphocytes are T cells that include a variety of effector cells. Cytotoxic CD8 + T cells predominate among
the IELs, but CD4 + T cells are distributed in the LP and lymphoid follicles of the intestine. Among the CD4 + T cell subsets, excessive
responses of T helper 1 (Th1) and Th17 cells are related to gut inflammation. To tightly regulate the activity of inflammatory T cells,
regulatory T (Treg) cells are induced to suppress the inflammatory response through the production of anti-inflammatory cytokines,
including IL-10 and TGF-β, in addition to other mechanisms that inhibit the function of antigen-presenting cells and maintain
intestinal homeostasis.
The chicken (Gallus gallus) is the best-studied non-mammalian species, and it is an invaluable model for investigating basic
immunological mechanisms. Compared with mammals, chickens have different repertories of TLRs, defensins, major histocompat-
ibility complexes (MHCs), cytokines, chemokines, antibodies, and other immune molecules. Chickens are predicted to have two TLR2
isoforms and TLR1/6/10 orthologs, and single isoforms of TLR3, TLR4, TLR5, and TLR7. Interestingly, chickens have two TLRs that
are absent in mammals, namely chTLR15 and chTLR21 (Keestra et al., 2013). Chickens do not have α-defensin, and their β-defensins,
which are the only known defensin family in chickens, contain arginine as the predominant cationic amino acid, compared with
mammalian defensins that contain similar amounts of arginine and lysine (Ganz, 2003; Derache et al., 2009). There are two classes of
MHC genes in chickens: polymorphic MHC class I and class II genes that are localized into two regions (MHC-B and MHC-Y) on the
same chromosome (Miller and Taylor, 2016). Although many of the cytokines and chemokines identified in mammals are also present
in chickens, chickens have a limited repertory of these molecules, especially regarding multigene families of cytokines and che-
mokines (Kaiser et al., 2005). Chicken immunoglobulins consist of three classes, IgM, IgA, and IgY, and there is no evidence they have
IgE and IgD. IgY is a counterpart of mammalian IgG (Warr et al., 1995). Chickens lack highly structured lymph nodes like those found
in mammals, as well as functional eosinophils, and the avian functional equivalent of the mammalian neutrophil is the heterophil, but
they have many distinct lymphoid aggregates lining the gut (Kogut et al., 2005). They have an avian-specific primary lymphoid
organ, the bursa of Fabricius, which is the site of development of their B-cell receptor repertoire (Glick et al., 1956; Cooper et al.,
1965).
Chicken GALT comprises lymphoid cells residing in the epithelial lining, and they are distributed in the underlying LP and in
specialized lymphoid structures, including lymphoid aggregates located within the LP, Meckel’s diverticulum, Peyer’s patches (PPs),
cecal tonsils, and the bursa of Fabricius. Following oral administration of foreign antigens, activation of helper T cells and IgA
precursor B cells in the GALT, especially in PPs, occurs, and these cells migrate to mucosal effector sites, such as the LP, to mediate
antigen-specific sIgA antibody responses. This activation of B and T cells in the GALT is followed by their migration to effector sites
where the mucosal immune response develops. These mucosal effector tissues consist mainly of T cells, predominantly CD4+
memory/effector T cells, but they also contain many B cells and plasma cells (Lillehoj and Trout, 1996). Given that intestinal

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inflammation is associated with the T cell response, it was believed that intestinal inflammation is caused by a disruption in the
balance between Th1 and Th2 cytokine responses until the discovery of Th17 cells as a new lineage of helper T cells that contribute to
gut inflammation (Guglani and Khader, 2010). Th17-related molecules in chickens were discovered and characterized, and their
contribution to intestinal immunity has been investigated (Min and Lillehoj, 2002; Kim et al., 2012, 2014).
In coccidiosis, as an intestinal inflammation model in chickens, Th17 cytokines modulate inflammation, regulate parasite ma-
turation and migration, and contribute to intestinal pathology (Min et al., 2013; Zhang et al., 2013; Del Cacho et al., 2014; Kim et al.,
2014). During the steady-state immune response, the number and activity of pathogenic effector T cells are regulated tightly by an
organized regulatory system consisting of multiple cell types that maintain intestinal homeostasis, because excessive Th1 and Th17
cells can result in inflammation-induced gut pathology. Treg cells are the most important cell types that suppress the immune system.
Chicken CD4+CD25+ T cells, as the only Treg cells identified thus far, are distributed broadly on mucosal surfaces where mucosal
tolerance is maintained (Shanmugasundaram and Selvaraj, 2011). Because meat production is highly dependent on nutrient ab-
sorption in the intestine, maintaining a balanced and healthy gut is a key factor determining the success of the poultry industry.

2. Gut microbiota and immunity

The gut represents a continuously evolving ecosystem where there are crosstalks among the largest lymphoid tissue in the body,
the neuroendocrine system, and trillions of commensal bacteria. Increasing evidence indicates that the gut microbiota plays crucial
roles in the development and function of the host immune system. Particularly, the intestinal microflora influences diverse aspects of
host metabolic and immunological functions, and this crosstalk with the various components of mucosal immunity, comprising
cellular and soluble elements, is critical for maintaining the gut homeostasis that promotes animal growth.
As an essential organ of the host mucosal immune system, the gut has evolved to carry out two apparently confounding tasks:
nutrient absorption and pathogen defense. It is well established that a beneficial microbial community has an important role in
maintaining normal physiological homeostasis, modulating the host immune system, and influencing organ development and host
metabolism (Sommer and Bäckhed, 2013). The intestine harbors the highest bacterial densities and diversities within the microbiota
(O’Hara and Shanahan, 2006). To ensure a beneficial and diverse microbiota composition to keep pathobionts in check, the intestinal
immune system developed an innate defense system that includes a robust mucosal layer comprising tightly interconnected intestinal
epithelial cells, soluble sIgA, and AMPs. Although the underlying molecular mechanisms of host–microbe interplay in maintaining
normal physiological homeostasis remain largely unknown, there are numerous examples of animal and human disorders (obesity,
inflammatory enteropathy, and autoimmune diseases) that are linked to an altered microbiota status (Caesar et al., 2010; Wlodarska
and Finlay, 2010). For example, in poultry, an altered microbiota caused by in-feed antimicrobials is associated closely with dysbiosis
(Li et al., 2010a) and enhanced host susceptibility to Clostridium pathogens (e.g., gangrenous dermatitis) (Li et al., 2010b). The
diversity of poultry gut microbiota has been shown to affect the complexity of T cell receptor β repertoire as well as mucin profile and
number of goblet cells in the gut (Forder et al., 2007; Mwangi et al., 2010). Therefore, further research is needed to fully understand
the relationship between the microbiota and immunity, because commonly used antimicrobials in animal production will have a
definite impact on the intestinal microbiota, the gut immune system, and disease susceptibility.

2.1. Microbiota and innate immunity

Non-self-recognition is a distinguishing feature of multicellular organisms that allows them to protect themselves from en-
vironmental pathogens, and it has undoubtedly driven the evolution of the immune system across a wide array of animal phyla
(Garcia-Garcia et al., 2013). Microbiota-driven modulation of host immunity seems to be a conserved feature of the immune system in
both higher and lower vertebrates (Gómez and Balcázar, 2008). In non-vertebrate innate immune systems, defensive cellular re-
sponses are mediated by phagocytosis, encapsulation, and the production of reactive oxygen species, nitric oxide (NO), and AMPs,
whereas molecular defense mechanisms include the production of humoral components such as phenoloxidases, clotting factors,
complement factors, lectins, protease inhibitors, lysozyme, and soluble TLRs (Garcia-Garcia et al., 2013). These mechanisms have
been conserved across vertebrates, except the phenoloxidase-mediated melanization and encapsulation of infectious agents (Garcia-
Garcia et al., 2013), which supports the notion that there is an important interplay between non-vertebrate hosts, their gut micro-
biota, and their immune systems, which shapes the development and functions of the immune system across a wide array of animal
phyla.
In non-vertebrates and vertebrates, non-self-recognition relies on their ability to distinguish between potentially pathogenic
microbial pathogens and harmless antigens via unique microbial signatures called PAMPs. PAMPs are detected by germ line-encoded,
highly conserved host innate immune receptors called PRRs (Akira and Hemmi, 2003). PAMPs, such as lipopolysaccharides (LPSs),
peptidoglycans, flagellin, formylated peptides, and bacterial DNA and RNA that are present on commensal bacteria, can regulate the
intestinal innate immune system by modulating host PRRs, e.g., TLRs, NOD proteins, retinoid-inducible gene 1, and C-lectin binding
receptors. PRRs are expressed on key players of the innate immune response, such as macrophages, neutrophils, DCs, intestinal
epithelial cells, and other cells. The initial recognition of a microbial PAMP by a host PRR triggers a series of complex and so-
phisticated intra- and intercellular signaling pathways that lead to the final activation of nuclear factor-kappa B, a signaling pathway
that leads to cytokine production and the upregulation of co-stimulatory molecules on antigen presenting cells, leading to the
activation of T cells (Akira and Hemmi, 2003). The activation of TLR signaling leads to the maturation of DCs that contribute to the
generation of adaptive immunity and the subsequent differentiation of naïve T helper cells into mature effector cell types with diverse
functions, such as Th1, Th2, Th17, and Treg cells that secrete different types of cytokines including interferon-gamma (IFN-γ), tumor

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necrosis factor-alpha (TNF-α), IL-10, TGF-β, IL-4, and IL-5 (Purchiaroni et al., 2013). The activation of innate immune cells and
complex downstream signaling pathways also leads to the development of an antigen-specific, long-lasting memory response that
represents a secondary line of host defense called adaptive immunity. Recent evidence supports the idea of close crosstalk between
the innate and adaptive components of the host immune system through activated receptors and secreted soluble effector molecules,
and, thus, the type of initial immune response elicited by various gut microbiota antigens has a profound influence on the quality of
the secondary line of the host defense (Purchiaroni et al., 2013).

2.2. Microbiota and acquired immunity

In the LP of the GALT, many macrophages, DCs, T cells, and IgA-secreting B cells are important mediators of the acquired immune
response (Campbell and Butcher, 2002). Regulatory cells are also present in the gut immune organs to maintain tolerance to foods
and self-antigens. In healthy individuals, T-cell unresponsiveness is maintained by resident DCs that induce the differentiation of
naïve T cells into various effectors cells (Th1, Th2, and Th17) or Treg cells (Tr1 and Th3) and maintain tolerance toward commensal
and food antigens (Kamada et al., 2013) through a complex network of cellular and molecular responses. Although the precise role of
commensal bacteria-specific acquired immunity against invasive pathogens remains poorly understood, there is evidence indicating
that the gut microbiota plays a critical role in limiting the systemic dissemination of commensal bacteria (Slack et al., 2009).
Increasing evidence indicates the important role of the resident microbiota in the gut mucosa in regulating a sophisticated im-
munoregulatory network of acquired immunity to prevent collateral damage that is often associated with pathogen infection and
epithelial barrier disruption, and it may contribute to the elimination of pathogens through opsonization or other immune me-
chanisms.

3. Alternative strategies for maximizing the efficiency of growth performance and immunity

With increasing regulatory restrictions on the use of antibiotics in animal agriculture, there is an urgent need for the development
of alternative strategies to combat many infectious diseases of poultry, especially avian coccidiosis and necrotic enteritis (NE).
Antibiotic alternatives that are used commonly in animal industry include prebiotics, probiotics, phytonutrients (herbs and essential
oils), hyperimmune antibodies, bacteriophages, AMPs, and minerals, but some have challenged their unknown mode of action,
inconsistency, and variability (Gadde et al., 2017a). In recent years, it has been shown that a combination of additives (e.g., pre- and
probiotics) may better synergize to obtain the maximum intended effects to compensate for production losses in the absence of
antibiotic growth promoters (AGPs) while still providing good economic returns (Lillehoj and Lee, 2012). Since our recent review
detailed different classes of antibiotic alternatives and the current understanding of their modes of action (Gadde et al., 2017b), here
we will highlight a few selected examples of promising alternatives that our laboratory has been studying. Considering the speculated
modes of action of AGPs (microbiome and immune-modulating activities), practical alternatives for AGPs should possess both of
these properties, in addition to having a positive impact on feed conversion and/or growth.

3.1. Phytochemicals

Phytochemicals are plant-derived chemicals with many proven beneficial effects. An increasing number of studies has docu-
mented that many of the health-promoting activities of phytochemicals are mediated through their ability to enhance host defenses
against microbial infections and tumors (Lillehoj et al., 2011). In recent years, Phytogenic feed additives (PFAs) have been used as
natural growth promoters in the pig and poultry industries. These PFAs include a wide variety of herbs and spices, as well as various
essential oils (thymol, carvacrol, cinnamaldehyde, essential oils (EO) from clove, coriander, star anise, ginger, garlic, rosemary,
turmeric, basil, caraway, lemon, and sage), which have been used either individually or as blends to improve animal health and
performance. Variable results have been reported with the use of EO in poultry diets. A blend of thymol and cinnamaldehyde in feed
was shown to improve bodyweight gain in broilers (Lillehoj et al., 2011). Nevertheless, one commercial blend of phytonutrients
(containing carvacrol, cinnamaldehyde, and capsicum oleoresin) was approved in the European Union as the first botanical feed
additive for improving performance in broilers. Several research trials performed with this commercial blend demonstrated a con-
sistent improvement in growth and feed efficiency (Bravo et al., 2014). A meta-analysis of 13 broiler studies involving the use of this
commercial blend showed that its inclusion in diets increased bodyweight gain and decreased the feed conversion ratio (FCR) and
mortality (Bravo and Ionescu, 2008).
The immunological basis for many of the known beneficial effects of phytochemicals has been evaluated in vitro using avian cell
lines and lymphocytes. Organic phase extracts from milk thistle (Silybum marianum), turmeric (Curcuma longa), reishi mushrooms
(Ganoderma lucidum), and shiitake mushrooms (Lentinus edodes) have been tested for their effects on chicken innate immunity and
tumor cell cytotoxicity (Lee et al., 2010a). Cinnamaldehyde ((2E)-3-phenylprop-2-enal) is a constituent of cinnamon (Cinnamomum
cassia), a widely used flavoring compound, and it was reported to possess antioxidant, antimicrobial, and anticancer activities. In
vitro stimulation of chicken spleen lymphocytes with 25 mg/ml of cinnamaldehyde induced greater cell proliferation, compared with
medium controls (Lee et al., 2011a). At 1.2 mg/ml, cinnamaldehyde activated cultured macrophages to produce higher NO levels,
and at 0.6 mg/ml, it inhibited the growth of chicken tumor cells, compared with untreated controls. Cinnamaldehyde also reduced
the in vitro viability of Eimeria tenella sporozoites at 10 mg/ml, compared with medium controls. The beneficial effects of two
organosulfur secondary metabolites of garlic (Allium sativum), propyl thiosulfinate (PTS) and PT oxide (PTSO), on chicken leukocytes
have been reported (Kim et al., 2013a). Garlicon40 (Pancosma S.A., Geneva, Switzerland) is a commercial product, 40% of which

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comprises a mixture of 33% (w/w) PTS and 67% (w/w) PTSO. In in vitro assays, PTS and PTSO dose-dependently reduced the
viability of invasive Eimeria acervulina sporozoites and stimulated higher chicken spleen cell proliferation, compared with untreated
controls. The effects of plant extracts on poultry innate immunity that have been demonstrated by in vitro studies also have been
shown to protect against Eimeria infections in vivo. Chickens fed a diet supplemented with cinnamaldehyde at 14.4 mg/kg had up to
47-fold greater levels of gene transcripts encoding IL-1β, IL-6, IL-15, and IFN-γ in intestinal lymphocytes, compared with chickens
given an unsupplemented diet (Lee et al., 2011a). Cinnamaldehyde-fed chickens had 17% and 42% increased bodyweight gains
following E. acervulina or Eimeria maxima experimental infections, respectively, 40% reduced E. acervulina oocyst shedding, and 2.2-
fold higher E. tenella-stimulated parasite antibody responses, compared with unsupplemented controls. Chickens fed from hatch with
a PTSO/PTS mixture at 10 ppm and orally challenged with live E. acervulina oocysts had increased bodyweight gain, decreased fecal
oocyst excretion, and greater E. acervulina profilin serum antibody responses, compared with chickens fed an unsupplemented diet
(Kim et al., 2010; Lillehoj et al., 2011). In uninfected chickens, in vivo dietary supplementation with a PTS/PTSO mixture increased
the levels of transcripts encoding IFN-γ, IL-4, and the antioxidant enzyme paraoxonase 2, compared with chickens given an un-
supplemented diet (Kim et al., 2013a). By contrast, transcripts for peroxiredoxin-6 were decreased in the PTS/PTSO-treated group,
compared with controls. In E. acervulina-infected chickens given a PTS/PTSO-supplemented diet, transcripts for TNF superfamily
member 15 (TNFSF15), catalase, and paraoxonase 2 were increased, while those for IL-10 were reduced, compared with un-
supplemented controls.
Synergistic effects of multiple phytochemicals against experimental avian coccidiosis have been demonstrated. Dietary supple-
mentation of chickens with a mixture of C. longa, Capsicum annuum (pepper), and L. edodes resulted in improved bodyweight gains,
reduced fecal oocyst shedding, and higher serum antibody titers against profilin following challenge infection with E. acervulina,
compared with birds given an unsupplemented diet or a diet containing Capsicum plus Lentinus alone (Lee et al., 2011b). The levels of
transcripts for IL-1β, IL-6, IL-15, and IFN-γ in gut lymphocytes also were greater in the Curcuma/Capsicum/Lentinus-fed group,
compared with the standard diet, Curcuma only, or Capsicum/Lentinus-only groups. In a follow-up report, feeding chickens a com-
bination of carvacrol (5-isopropyl-2-methylphenol), an active component of oregano (Origanum vulgare), thyme (Thymus vulgaris),
cinnamaldehyde, and Capsicum oleoresin (a mixture of essential oils and resins), or Capsicum oleoresin plus Curcuma oleoresin,
increased protective immunity against experimental E. tenella infection following immunization with profilin, compared with un-
treated and immunized controls (Lee et al., 2011b). Birds fed either supplemented diet had increased bodyweight gain, greater
profilin antibody levels, and/or greater lymphocyte proliferation, compared with unsupplemented controls. Immunized chickens fed
the carvacrol/cinnamaldehyde/Capsicum-supplemented diet had increased numbers of macrophages in their intestine, while those
given the Capsicum/Curcuma oleoresin-supplemented diet had increased numbers of intestinal T cells, compared with untreated
controls.
While numerous studies have shown the disease prevention or immune-enhancing effects of phytochemicals, few reports have
examined the underlying mechanisms that are involved. Some phytochemicals inhibit innate immune responses by targeting PPRs or
their downstream signaling molecules. In chickens, the effects of carvacrol, cinnamaldehyde, and Capsicum oleoresin on the reg-
ulation of the expression of genes associated with immunology, physiology, and metabolism have been investigated using a high-
throughput microarray analysis (Kim et al., 2010). This study revealed that Capsicum oleoresin stimulated the greatest number of
gene changes, compared with unsupplemented controls, and many of the altered genes were associated with metabolism and im-
munity. The most reliable genetic network induced by dietary cinnamaldehyde treatment was related to the functions of antigen
presentation, humoral immunity, and inflammatory disease. Further, dietary supplementation with these three phytochemicals was
associated with increased protective immunity following live E. acervulina challenge infection, based on increased bodyweight gain
and reduced parasite fecal shedding, compared with unsupplemented controls. Further studies to delineate the intestinal immune
pathways affected by phytochemical feeding were conducted via an mRNA microarray hybridization (Kim et al., 2010). Compared
with chickens fed an unsupplemented diet, carvacrol-fed chickens showed altered levels of 74 gene transcripts in their gut lym-
phocytes (26 increased, 48 decreased), and cinnamaldehyde supplementation was associated with altered levels of 62 mRNAs (31
increased, 31 decreased), while Capsicum oleoresin-fed chickens had altered levels of 254 mRNAs (98 increased, 156 decreased),
compared with unsupplemented controls. Among the transcripts that showed greater than two-fold altered expression levels, most
were encoded by genes associated with metabolic pathways. In the case of Capsicum oleoresin, these included pathways for lipid
metabolism, small molecule biochemistry, and cancer. In another investigation, a global gene expression analysis via a microarray
hybridization identified 1,810 transcripts (677 increased, 1,133 decreased) whose levels were altered significantly in the intestinal
lymphocytes of anethole-fed birds, compared with unsupplemented controls (Kim et al., 2013a). Of these, 576 corresponding genes
were identified that were related to the inflammatory response.
The effect of dietary phytochemicals on the gut microbiota was studied in three major commercial broiler chickens fed Capsicum
and C. longa oleoresins (Kim et al., 2015). Among the three chicken breeds, Cobb, Hubbard, and Ross, oleoresin supplementation was
associated with altered intestinal microbiota. The results suggest that dietary feeding of Capsicum and C. longa oleoresins reduces the
negative consequences of NE, in part through the alteration of the gut microbiome. Although these are preliminary characterizations
of the effects of dietary phytochemicals on gut microbiota, they document the role of dietary Capsicum and C. longa oleoresins in
regulating susceptibility to NE and altering the intestinal microbiota of commercial broiler chickens. Future studies on the role of the
avian gut microbiome in immune regulation and host–pathogen interactions are expected to shed new light on the host response to
NE, which will be beneficial for practical poultry husbandry. In conclusion, we have observed that dietary Capsicum and C. longa
oleoresins regulate susceptibility to experimental avian NE and alter the intestinal microbiota of commercial broiler chickens. Dietary
phytonutrients exert beneficial effects on gut health to reduce the negative consequences of NE, and a nutratherapeutic mechanism
may involve altering gut microbial communities. These new findings increase our understanding of: (1) the positive effects of dietary

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phytonutrients as alternatives to antibiotics; (2) host genetics in host–pathogen interactions in NE; and (3) the possible role of the gut
microbiota in local immune regulation in broiler chickens. Further studies of the effects of dietary phytonutrients on the gut mi-
crobiota in commercial broiler breeds are needed to develop alternative ways to reduce or replace antibiotics in poultry disease
control. Future studies of the roles of the avian intestinal microbiome in immune regulation and host–pathogen interactions are
expected to shed new light on the host response to NE, which will be beneficial for practical poultry husbandry.
The newly acquired knowledge that has increased our understanding of the importance of the gut environment and barrier
function in health should aid the development of phytochemical-based feed additive products that can deliver the benefits of AGPs
without causing an increase in the emergence of drug resistance. For example, when we consider using phytochemicals as antibiotic
alternatives, we need to consider: (1) the dose versus bacteriostatic/bactericidal effects in target animals; (2) variations in active
compounds in plants and plant-derived products; (3) unexplored concurrent effects of phytochemicals (antiviral and antineoplastic);
(4) target organs/tissues affected by phytochemicals; (5) safety of phytochemical residues in humans; and (6) the long-term effect of
using phytochemicals in animals on developing resistance.

3.2. Hyperimmune egg yolk antibodies

Passive immunization of protective immunity using hyperimmune, pathogen-specific antibodies is an alternative control strategy
that is potentially applicable to intestinal diseases such as avian coccidiosis and NE. As opposed to the pathogen-specific immunity
achieved by active vaccination with live or inactivated microorganisms, or subunits derived from these pathogens, passive im-
munization relies on the transfer of humoral immunity in the form of active antibodies from one individual to another (Gadde et al.,
2015). Polyclonal antibodies from mammals, such as rabbits and goats, have been used commonly for passive immunization, but
rising concerns over animal welfare issues are prompting the pharmaceutical industry to explore less invasive alternatives for pro-
ducing therapeutic antibodies. In this regard, chicken hyperimmune egg yolk IgY antibodies offer a practical alternative to mam-
malian serum antibodies because of their feasibility for large-scale commercial production and the relative noninvasive methods used
for their preparation (Gadde et al., 2015). Maternal IgY is concentrated in the yolk sac of eggs during embryogenesis, allowing it to be
easily collected, purified, and used to passively immunize mammals.
Hyperimmune egg yolk antibodies (IgY), which are produced by the repeated immunization of hens with specific antigens and the
collection of antibodies thereafter from their egg yolks, have been employed commonly in the prevention and treatment of various
enteric diseases in humans and animals (Gadde et al., 2015). Limited research exists on the use of egg yolk antibodies as viable
alternatives to AGPs in improving the growth and feed efficiency in poultry (Cook, 2004). Earlier studies focused on generating egg
antibodies in breeding hens, which could be transferred passively to their progeny to improve their productivity. As IgY technology
evolved, subsequent research trials involved the use of antibodies in feed to improve performance or enhance disease-specific host
immunity (Lee et al., 2009a, 2009b). For example, the protective effect of the IgY fraction of egg yolk from hens hyperimmunized
with Eimeria oocysts was evaluated in young broilers with experimental coccidiosis (Lee et al., 2009a, 2009b). Chickens that had been
fed continuously from hatch with a standard diet supplemented with 10% or 20% (wt/wt) of a freeze-dried egg yolk powder from
hens hyperimmunized with E. acervulina showed significantly increased bodyweight gain and reduced fecal oocyst shedding fol-
lowing experimental E. acervulina infection, compared with control birds given an unsupplemented diet. Still lower doses of egg yolk
supplementation (0.01%–0.05%) also reduced fecal oocyst shedding, but with no differences in bodyweight gain. Nevertheless, it is
encouraging to note the reduced oocyst shedding by chickens fed low-dose egg yolk diets, which suggests that this immunization
strategy may prove beneficial for disrupting the infectious cycle of Eimeria parasites in the field.
These promising preliminary studies provide the impetus for future field investigations to evaluate the effects of hyperimmune
antibodies at low-dose exposures, and for in vivo mechanistic studies to determine how passively administered antibodies bestow
protection against coccidia challenge infection. Finally, elucidating the parasite components that are recognized by antigen-specific
IgY antibodies in egg yolk will facilitate the discovery of novel coccidiosis vaccines. The use of egg yolk antibodies offers several
advantages. Large quantities of antibodies can be produced in laying hens and non-invasively collected. Their use is environmentally
friendly and less toxic, and it does not lead to resistance. Although the existing results seem encouraging, much more research is
needed on using egg antibodies for growth promotion in poultry.

3.3. Antimicrobial peptides

Host-derived AMPs are promising drug alternatives that are being developed as natural therapeutics for animal treatment. These
are considered natural antimicrobials because of their broad-spectrum antimicrobial activity and their different activation me-
chanisms. AMPs are diverse in size, sequence, and localization, and they consist of four major structural groups, including amphi-
pathic α-helical, β-sheet, β-hairpin or loop, and extended variants (Kim et al., 2016). AMPs have been considered as potential
therapeutic sources of natural antimicrobials, given their broad-spectrum antimicrobial activities and different activation mechan-
isms compared with classical antimicrobials (Kim et al., 2016). AMPs are used widely as defense mechanisms by animals, plants, and
bacteria, and over 2500 AMPs consisting of 10–40 amino acid residues have been discovered and can be found in the Antimicrobial
Peptide Database (http://aps.unmc.edu/AP/main.php). Recently, with the discovery of granulysin and its multi-species homologs, a
new class of AMPs, called NK-lysins, has been recognized (Bruhn et al., 2003). In contrast to classical AMPs, granulysin and NK-lysins
are much larger (74–78 amino acids) and possess a globular three-dimensional structure. They are also members of the saposin-like
protein family, which includes amoebapores found in Entamoeba histolytica (Bruhn and Leippe, 2001). The common functional
features of the saposin-like protein family include their ability to kill a wide range of microbes through interactions with target

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membranes.
The chicken NK-lysin variant is the most extensively studied AMP in avian species and consists of 140 amino acids, including a
saposin B-type domain and six conserved cysteine residues (Hong et al., 2006). Interestingly, the antimicrobial domains of cNK-lysin,
granulysin, and porcine NK-lysin share conserved structural characteristics. Avian NK-lysins have been identified in chickens (Kim
et al., 2016), with genes consisting of four exons and three introns, while the mammalian NK-lysin protein shares 14%–17% amino
acid sequence identity with mammalian NK-lysins and 49%–88% amino acid sequence identity with other avian NK-lysins. Although
the sequence identity is low, antimicrobial cNK-lysin domains, granulysin, and porcine NK-lysin share common structural char-
acteristics enabling antimicrobial activities (Hong et al., 2006, 2008). Our laboratory has been studying cNK-2, a synthetic peptide
similar to the core α-helical region of cNK-lysin, which exhibits antimicrobial activity against apicomplexan parasites, including
Eimeria and Neospora, in vitro and protects against live parasite infection following live parasite challenge (Lee et al., 2013). An
enhanced understanding of the structure-function relationship of AMPs will facilitate their application in animal agriculture as
antibiotic alternatives. In addition to the antimicrobial activity of AMPs, the immunomodulatory activity of AMPs mediated by their
interactions with host cells is increasingly recognized. In a recent report, by evaluating the induction of chemokines, anti-in-
flammatory properties, and the activation of signalling pathways, we showed that cNK-2 modulates the immune response in the
chicken macrophage cell line HD11 and in chicken primary monocytes (Kim et al., 2017). cNK-2 induced the expression of chemokine
(C-C motif) ligand 4 (CCL4), CCL5, and IL-1β in HD11 cells and CCL4 and CCL5 in primary monocytes. Furthermore, cNK-2 sup-
pressed the LPS-induced inflammatory response by abrogating IL-1β expression. The immunomodulatory activity of cNK-2 involves
the mitogen-activated protein kinases-mediated signalling pathway, including p38, extracellular signal-regulated kinase 1/2, and c-
Jun N-terminal kinases, as well as the internalization of cNK-2 into cells. These results indicate that cNK-2 is a potential novel
immunomodulating agent, rather than an antimicrobial agent.

3.4. Probiotics

Direct-fed microbials, often referred to as probiotics, represent a non-antibiotic nutritional approach to modulate gut function and
enhance intestinal health in chickens (Lee et al., 2010b). A variety of bacterial species (including Bacillus, Bifidobacterium, En-
terococcus, Lactobacillus, Streptococcus, and Lactococcus spp.), yeast (Saccharomyces), and in some cases undefined cultures have been
tested as probiotics in poultry (extensively reviewed in Kabir, 2009). Most of the conducted research was aimed specifically at
investigating the effects of probiotics in reducing the numbers of pathogenic microorganisms in the gastrointestinal tract. However, a
considerable amount of research has also examined the effects of probiotics on improving growth and performance in poultry without
apparent disease. They were shown to regulate intestinal epithelial cell function and apoptosis, influence T-lymphocyte populations,
modulate cytokine profiles, and enhance antibody secretions (Lee et al., 2010b; 2011). For example, Bacillus spp. have been tested as
probiotics in commercial poultry applications and have been shown to improve performance, positively modulate intestinal mi-
croflora, and inhibit pathogen colonization (Gadde et al., 2017a and 2017b).
The beneficial effects of Bacillus-based (B. subtilis and B. amyloliquefaciens) probiotic diet supplementation in broiler chickens have
been well documented. In recent studies, we investigated the effects of one mono-strain and two multi-strain B. subtilis-based pro-
biotics on the growth performance of commercial broilers (Gadde et al., 2017a, 2017b). These results showed that probiotic-sup-
plemented chickens had significantly higher bodyweights at 14 days of age, compared with non-supplemented controls, and the
increase in bodyweight was similar to that of antibiotic-fed chickens. The FCR was significantly lower in all the Bacillus-supplemented
groups, compared with controls. In general, differences in the strains used, the administration level, application methods, diet
composition, and hygiene status all affect the results of Bacillus trials. In our recent study (unpublished observation), we evaluated the
effects of dietary supplementation with novel B. subtilis strains isolated from environmental sources on performance, the gut immune
response, and epithelial barrier integrity in broilers. The objective of this study was to investigate the effects of B. subtilis-based
probiotic supplementation of broiler chicken diets on growth performance, feed efficiency, and intestinal cytokine and tight junction
(TJ) protein mRNA expression. Zero-day-old broiler chicks (n = 140) were assigned randomly to one of five dietary treatments: a
basal diet (CON), the basal diet supplemented with either antibiotic bacitracin methylene disalicylate (BMD) or probiotics, namely B.
subtilis strain 1781 (PB1), a combination of B. subtilis strain 1104 + strain 747 (PB2), or B. subtilis strain 1781 + strain 747 (PB3).
Bodyweight and feed intake were measured at 14 days of age, and the FCR was calculated. At 14 days of age, ileal samples were
collected and used for intestinal cytokine, TJ protein, and mucin gene expression analyses using quantitative real-time polymerase
chain reactions. The chickens supplemented with BMD or B. subtilis strain 1781 alone (PB1) had significantly higher bodyweights,
compared with controls of the same age. Dietary supplementation with BMD or probiotics (PB1, PB2, or PB3) significantly improved
the feed efficiency as evidenced by a decreased FCR, compared with controls. No differences were observed in the expression of IL-1β,
IL-17 F, IFN-γ, and the mucin 2 genes among the different treatment groups. However, elevated expression of IL-6 (BMD, PB1, and
PB2), IL-8 (PB2), and TNFSF15 (PB1, PB2, and PB3), compared with controls, was observed in the ileum. IL-2 and IL-10 expression
was upregulated in chicks in the PB2 and PB3 groups, and IL4 was elevated in the PB1 group. IL13 was elevated in all the probiotic-
fed groups (PB1, PB2, and PB3). Probiotic supplementation also significantly increased the expression of TJ proteins junctional
adhesion molecule 2, zonulin 1 (PB2 and PB3), and occludin (PB1 and PB2). Taken together, B. subtilis supplementation altered
intestinal immune activity and influenced gut barrier integrity through increased TJ gene expression.
In a recent study, we investigated the role of probiotics in regulating immune responses during the acute phase response and the
maintenance of intestinal integrity (Gadde et al., 2017b). Especially, we studied how Bacillus supplementation modulates the host
inflammatory response and changes in intestinal TJ gene expression induced by LPS challenge upon the supplementation of diets with
probiotics, compared to non-supplemented controls or those supplemented with antibiotics. Chickens fed probiotics weighed

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significantly more than controls at 15 days of age, irrespective of immune challenge. LPS challenge significantly reduced weight gain
at 24 h post-injection, and the probiotics did not alleviate the LPS-induced reduction of weight gain. Serum α-1-AGP levels were
significantly higher in LPS-injected chickens, and probiotic supplementation significantly reduced their levels. The percentages of
CD4+ lymphocytes were significantly higher in probiotic groups in the absence of immunological challenge, but were reduced
during LPS challenge, compared with controls. CD8+ lymphocytes were significantly less abundant in probiotic-fed birds. The LPS-
induced increases in the expression of the cytokines IL-8 and TNFSF15 were reduced by probiotic supplementation, and IL-17 F and
inducible NO synthase expression was significantly higher in probiotic-fed birds subjected to LPS challenge. The reduced gene
expression of TJ proteins (junctional adhesion molecule 2, occludin, and zonulin 1) and mucin 2 induced by LPS challenge was
reversed by probiotic supplementation. The results indicate that B. subtilis-based probiotics differentially regulate intestinal immune
and TJ protein mRNA expression during states of LPS-mediated immunological challenge. We hypothesize that dietary probiotics
modulate the inflammatory activities that occur in response to LPS challenge and play a role in the restoration of cytokine balance to
minimize inflammation-induced damage.
Though evidence exists showing that probiotics communicate with intestinal epithelial cells, macrophages, DCs, and lymphocytes
in the gut, the mechanisms underlying these interactions have yet to be defined. Further studies involving the characterization of the
biological signaling pathways involved in the intestinal modulation of immune activities by probiotics should be performed. An ideal
probiotic organism should be able to withstand processing and storage, survive in the gastric acidic environment, adhere to the
epithelium and/or mucus in the intestines, produce antimicrobial compounds, and modulate immune responses (Lee et al., 2010b;
Gadde et al., 2015). However, not all strains exhibit all these properties, and care must be taken to select the strains or combinations
of strains that will achieve a maximum beneficial effect in vivo. Measures to protect organisms during their passage through the
upper alimentary tract, such as a microencapsulation, should be considered to ensure viability and colonization in the intestine (Han
et al., 2013). Overall, probiotics can serve as potential alternatives to antibiotics for increasing poultry performance.

4. Conclusions

With the increase in regulations regarding the use of AGPs and the rise in consumer demand for poultry products from “Raised
Without Antibiotics” or “No Antibiotics Ever” flocks, the quest for alternative products or approaches has intensified in recent years.
A great deal of research has focused on the development of antibiotic alternatives to maintain or improve poultry health and per-
formance. This review describes poultry immunity, immune modulation, and the potential for the various alternatives to modulate
poultry immunity and productivity and enhance poultry performance so that chickens can fulfil their genetic potential under existing
commercial conditions. The classes of alternatives described include probiotics, prebiotics, synbiotics, organic acids, enzymes,
phytogenics, AMPs, hyperimmune egg antibodies, bacteriophages, and clay. There are still challenges in understanding the me-
chanism of action, efficacy, and advantages and disadvantages of their use. Although the beneficial effects of many of these alter-
natives have been well demonstrated, the general consensus is that these products lack consistency and the results vary greatly from
farm to farm. Furthermore, their modes of action need to be better defined. Optimal combinations of various alternatives, coupled
with good management and husbandry practices, will be the key to maximize performance and maintain animal productivity while
we move forward with the ultimate goal of reducing antibiotic use in the animal industry.

Conflict of interest

The authors declare no conflict of interest.

Acknowledgements

This research was supported by Development of Poultry Immune Reagent program (award number: 2017-67015-26793) funded
by USDA/NIFA.

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