**Molecular Genetics and Genome Biology of Goats**

**Molecular Genetics and Genome Biology of Goats**

DOI: 10.5772/intechopen.72414

Kingsley Ekwemalor, Sarah Adjei-Fremah, Emmanuel Asiamah and Mulumebet Worku Emmanuel Asiamah and Mulumebet Worku Additional information is available at the end of the chapter

Kingsley Ekwemalor, Sarah Adjei-Fremah,

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.72414

#### **Abstract**

Information on goat genome has led to a better understanding of the genetics of goats, its response to infection and the underlying immune response mechanism. Natural productbased therapeutic can therefore be utilized to target genes important for goat immunity. In this chapter, we have summarized the effect of diet and dietary supplements as immune modulators in goats. These modulators affect the expression of genes and secreted proteins associated with innate and adaptive immune response and homeostasis. Probiotics, mushroom extracts, plant polyphenol extracts, Sericea lespedeza (SL) and cowpea diet affect key molecular pathways including Toll-like receptor (TLR) pathway, Wnt signaling pathway and cytokine-mediated signaling pathway. Results from various studies reviewed in this chapter suggest that utilization of dietary immunomodulators has beneficial effects on goat health and production.

**Keywords:** blood, gene expression, transcription, translation, modulation, innate immunity, homeostasis

#### **1. Introduction**

The domestic goat (*Capra hircus*) is an important farm and companion animal species. They are descendants of the bezoar (*Capra aegagrus*) goat. The world goat population has been on the increase during the last three decades and is estimated to be 1 billion with the global genetic diversity characterized by more than 590 breeds [1]. Breeds have various advantageous characteristics such as adaptation to harsh environmental conditions, capacity to convert poor quality fibrous feedstuff into animal proteins and resistance to diseases. Known as 'the poor man's cow,' goats are primarily reared for meat production, but different breeds of goat are often used as a source of milk and wool. Goats are also used for carrying small loads and for land management and kept as pets.

Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons

In the United States, production of small ruminants is a growing industry as a result of high demand for grass-fed or organically produced livestock [2]. Compared to all other livestock enterprise, goat production requires minimal capital input and low cost of breeding stock [3]. Globally, healthy goats are crucial for the long-term success of the goat industry. Production is negatively challenged by factors such as feed toxins, respiratory diseases (pneumonia) and other infectious diseases. Goats are also susceptible to viral diseases (foot and mouth diseases) and bacterial diseases (mastitis). Gastrointestinal (GI) nematode infection is considered the most important limiting factor in goat production systems around the world and results in huge economic losses to producers.

various mechanisms to eliminate or withstand the impact of external agents. The animal's immune system is composed of two related functional elements: the innate immune system and the adaptive immune system [11]. Both function in coordination to protect against invading microorganisms [12]. Innate immunity is the first line of defense against organisms; it acts in a nonspecific way through anatomical barriers (skin, mucus membrane), secretions, cells and other elements. The adaptive immune system is the second line of defense which responds slower than the innate immunity system. Innate immune defense plays a key role in affording protection [13]. Unlike the innate immune system, the adaptive immune system has the ability to 'memorize' infectious agents allowing the adaptive immune system to serve as a rapid response system if pathological agents are encountered again [12]. The innate immune system consists of natural killer cells, T-cell and B-cell, basophils, eosinophils, monocytes, macrophages and polymorphonuclear neutrophils. These cells are called white blood cells or leukocytes and are also divided into two groups based on their morphology: granulocytes and agranulocytes. Granulocytes include eosinophils, neutrophils and basophils, and agranulocytes are lymphocytes (T and B cells) and macrophages [14]. A differential white blood cell count is an important tool used to provide clinical diagnosis and for monitoring of disease and blood disorders [15]. This system quantifies and differentiates white blood cells at one particular time giving an insight into infection and checking whether treatments are working [15].

Molecular Genetics and Genome Biology of Goats http://dx.doi.org/10.5772/intechopen.72414 5

Animals live in a wide variety of microbe-rich environments, and hence, it is crucial to have a sensitive innate defense mechanism which relies in part by recognizing conserved molecules that are unique to some classes of potential pathogens [16]. It is very important to understand the innate immunity against microbial components and its critical role in host defense against infection. Toll-like receptors (TLRs) have been shown to participate in the recognition

Toll-like receptors (TLRs) are a highly conserved group of proteins that have been identified in mammals [17]. The TLR family consists of 10 receptors: TLR-1-10, which are very important in the identification of microbes [18]. The coding regions within the goats, TLR-1-10 genes, have been sequenced and found to be conserved and highly similar in nucleotide composition [10]. With the discovery of Toll-like receptors (TLRs), studies have shown that pathogen recognition by the innate immune system is broadly specific, which relies on germline-encoded pattern-recognition receptors (PRRs) to detect relatively conserved components of pathogens referred to as pathogen-associated molecular patterns (PAMPs) [19]. The PAMPs recognized by TLRs include lipids, lipoproteins, proteins and nucleic acids derived from a wide range of microbes such as bacteria, viruses, parasites and fungi [20] which initiates a complex signaling cascade to activate a wide variety of transcription factors and inflammatory cytokines [18].

Cytokines are small proteins that transmit information from one cell to another. The analysis of cytokines secreted by immune cells in response to infectious agents is crucial to understand pathogenesis and immunity. Most cells in the body produce cytokines during inflammatory

**2.2. Toll-like receptors**

**2.3. Cytokines**

of pathogens by the innate immune system.

However, resistance to current drugs and lack of interest in developing new drugs by companies pose a challenge for sustainable ruminant production. The widely used approach for the treatment of infection by parasite is drug treatment. Measures used to reduce parasite infection include the reduction of stock density and the maximization of pasture to reduce parasite numbers. Plant-based anthelmintic is also being explored for use in the elimination of gastrointestinal parasites including extracts such as: garlic, neem, wormwood, tobacco, cowpea [4–6] and Sericea lespedeza [7]. Several other alternatives that have been proposed include the use of nonchemical additives such as probiotics [8] and prebiotics, the improved production practices and the use of genetics-based breeding schemes. Improvement in animal nutrition can also impact the immune response including gene response, protein synthesis, modification and degradation, metabolism, signal transduction and cellular proliferation [9]. The understanding of protective mechanisms regarding the initial steps of the host's response to pest or parasite-derived molecules that can correlate with resistance or susceptibility to pathogens needs to be explored. This understanding will aid the design of immunomodulatory strategies to induce a change in the magnitude of immune or nonimmune responses.

#### **2. The goat genome**

In the last decade, molecular genetics has led to the discovery of individual genes or candidate genes with substantial effects on our understanding of homeostasis and immunity. The goat genome has been sequenced, and raw sequences have been deposited in NCBI (GenBank, CapAeg\_1.0 (GCA\_000978405.1) under the accession no. SRA184825. Utilizing information on the goat genome enables a better understanding of the genetics of the goat and how it responses to infection and disease and fights it naturally. The sequencing of the goat genome has led to increased understanding of the genetics underlying immune response mechanism [10]. Genes significant for goat immunity can be targeted by using natural products and plantbased therapeutics for improving goat health. This eliminates the need for chemical treatment, buildup of antibiotic resistance and food insecurity concerns among goat consumers.

#### **2.1. Innate immune system**

The main function of the immune system is to distinguish between own cells and tissues from external cells and tissues in order to protect against infestation. The immune system has various mechanisms to eliminate or withstand the impact of external agents. The animal's immune system is composed of two related functional elements: the innate immune system and the adaptive immune system [11]. Both function in coordination to protect against invading microorganisms [12]. Innate immunity is the first line of defense against organisms; it acts in a nonspecific way through anatomical barriers (skin, mucus membrane), secretions, cells and other elements. The adaptive immune system is the second line of defense which responds slower than the innate immunity system. Innate immune defense plays a key role in affording protection [13]. Unlike the innate immune system, the adaptive immune system has the ability to 'memorize' infectious agents allowing the adaptive immune system to serve as a rapid response system if pathological agents are encountered again [12]. The innate immune system consists of natural killer cells, T-cell and B-cell, basophils, eosinophils, monocytes, macrophages and polymorphonuclear neutrophils. These cells are called white blood cells or leukocytes and are also divided into two groups based on their morphology: granulocytes and agranulocytes. Granulocytes include eosinophils, neutrophils and basophils, and agranulocytes are lymphocytes (T and B cells) and macrophages [14]. A differential white blood cell count is an important tool used to provide clinical diagnosis and for monitoring of disease and blood disorders [15]. This system quantifies and differentiates white blood cells at one particular time giving an insight into infection and checking whether treatments are working [15].

#### **2.2. Toll-like receptors**

In the United States, production of small ruminants is a growing industry as a result of high demand for grass-fed or organically produced livestock [2]. Compared to all other livestock enterprise, goat production requires minimal capital input and low cost of breeding stock [3]. Globally, healthy goats are crucial for the long-term success of the goat industry. Production is negatively challenged by factors such as feed toxins, respiratory diseases (pneumonia) and other infectious diseases. Goats are also susceptible to viral diseases (foot and mouth diseases) and bacterial diseases (mastitis). Gastrointestinal (GI) nematode infection is considered the most important limiting factor in goat production systems around the world and results

However, resistance to current drugs and lack of interest in developing new drugs by companies pose a challenge for sustainable ruminant production. The widely used approach for the treatment of infection by parasite is drug treatment. Measures used to reduce parasite infection include the reduction of stock density and the maximization of pasture to reduce parasite numbers. Plant-based anthelmintic is also being explored for use in the elimination of gastrointestinal parasites including extracts such as: garlic, neem, wormwood, tobacco, cowpea [4–6] and Sericea lespedeza [7]. Several other alternatives that have been proposed include the use of nonchemical additives such as probiotics [8] and prebiotics, the improved production practices and the use of genetics-based breeding schemes. Improvement in animal nutrition can also impact the immune response including gene response, protein synthesis, modification and degradation, metabolism, signal transduction and cellular proliferation [9]. The understanding of protective mechanisms regarding the initial steps of the host's response to pest or parasite-derived molecules that can correlate with resistance or susceptibility to pathogens needs to be explored. This understanding will aid the design of immunomodulatory strategies to induce a change in the magnitude of immune or nonimmune responses.

In the last decade, molecular genetics has led to the discovery of individual genes or candidate genes with substantial effects on our understanding of homeostasis and immunity. The goat genome has been sequenced, and raw sequences have been deposited in NCBI (GenBank, CapAeg\_1.0 (GCA\_000978405.1) under the accession no. SRA184825. Utilizing information on the goat genome enables a better understanding of the genetics of the goat and how it responses to infection and disease and fights it naturally. The sequencing of the goat genome has led to increased understanding of the genetics underlying immune response mechanism [10]. Genes significant for goat immunity can be targeted by using natural products and plantbased therapeutics for improving goat health. This eliminates the need for chemical treatment, buildup of antibiotic resistance and food insecurity concerns among goat consumers.

The main function of the immune system is to distinguish between own cells and tissues from external cells and tissues in order to protect against infestation. The immune system has

in huge economic losses to producers.

4 Goat Science

**2. The goat genome**

**2.1. Innate immune system**

Animals live in a wide variety of microbe-rich environments, and hence, it is crucial to have a sensitive innate defense mechanism which relies in part by recognizing conserved molecules that are unique to some classes of potential pathogens [16]. It is very important to understand the innate immunity against microbial components and its critical role in host defense against infection. Toll-like receptors (TLRs) have been shown to participate in the recognition of pathogens by the innate immune system.

Toll-like receptors (TLRs) are a highly conserved group of proteins that have been identified in mammals [17]. The TLR family consists of 10 receptors: TLR-1-10, which are very important in the identification of microbes [18]. The coding regions within the goats, TLR-1-10 genes, have been sequenced and found to be conserved and highly similar in nucleotide composition [10]. With the discovery of Toll-like receptors (TLRs), studies have shown that pathogen recognition by the innate immune system is broadly specific, which relies on germline-encoded pattern-recognition receptors (PRRs) to detect relatively conserved components of pathogens referred to as pathogen-associated molecular patterns (PAMPs) [19]. The PAMPs recognized by TLRs include lipids, lipoproteins, proteins and nucleic acids derived from a wide range of microbes such as bacteria, viruses, parasites and fungi [20] which initiates a complex signaling cascade to activate a wide variety of transcription factors and inflammatory cytokines [18].

#### **2.3. Cytokines**

Cytokines are small proteins that transmit information from one cell to another. The analysis of cytokines secreted by immune cells in response to infectious agents is crucial to understand pathogenesis and immunity. Most cells in the body produce cytokines during inflammatory processes which represent a large series of regulatory proteins of the immunologic system. Many cytokines are referred to as interleukins, a name indicating that they are secreted by some leukocytes and act upon other leukocytes. Two general patterns of cytokine secretion by such cells have been described. In the Th1 response, cytokines initiate cell-mediated reactions defined as the activation of macrophages to combat infectious pathogens by releasing IL-1, IL-2, IL-8, and IL-12 to activate inflammation [21]. In the Th2 response, T-helper cells activate B-cells; interleukins IL-4, IL-5, IL-6, IL-10 and IL-13 are released to counter infectious agents caused by extracellular organisms [21]. Studies have shown that the release of cytokines is essential for host survival from infection and is also required for tissue repair.

More specifically, the number of strongyle eggs and coccidia oocytes is measured using the modified McMaster method [31]. The fecal eggs counted are multiplied by 50, and resulting

The molecular effects of immunomodulators have been evaluated in goats at the gene transcription and protein levels using different techniques including real-time polymerase chain

Quantitative real-time PCR is used to measure messenger RNA (MRNA) levels [77]. For realtime PCR analysis, total RNA is isolated from whole blood cell pellets using Trizol method or QuickRNA MiniPrep Kit (Zymo Research) as per manufacturer's procedure. The concentration and purity of the RNA are checked on NanoDrop Spectrophotometer (ND-1000; Thermo Fisher). Mostly, a pure RNA typically yields a 260/280 ratio of ~2.0 and this is considered ideal. A 260/280 ratio below 2.0 suggests protein contamination [82]. In addition, the integrity of the RNA (RNA integrity number (RIN)) can be measured with a bioanalyzer, and a RIN <7.0 indicates a good RNA. Since RNA is not stable, it is converted into more stable complimentary DNA (cDNA) using cDNA conversion kits containing oligo (DT) and random primers, reverse transcriptase, and other needed reagents as specified in the manufacturer's manual. Real-time PCR is performed with reaction mixture comprising of cDNA template, primers and SYBR Green [78]. Housing-keeping genes such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), *β*-actin (*ACTB*), ribosomal protein L32 (RPL32), TATA sequence binding protein (TBP) and cyclophilin are used as internal controls for normalization of the RT-PCR data obtained [32]. The RT-PCR data are analyzed by calculating fold change in the expression of the specific genes tested using

Enzyme-linked immunosorbent assay (ELISA) is a molecular assay used for analytical detection and quantification of specific antigens or antibodies in a given sample. It uses the concept of an antigen binding to its specific antibody which enables detection of antigens such as proteins, peptides and antibodies [33]. With ELISA, goat serum or plasma is evaluated for the levels of immune response and inflammation biomarkers such as cytokines, prostaglandin and immunoglobulins. Cytokines measured in goat serum following dietary supplementation include TNFα, IL-1β, IL-8, GCSF, GMCSF, Rantes and IFNγ [7, 8, 34]. The levels of secreted prostaglandin E, an eicosanoid and also an inflammation mediator have also been measured

Goats rely on pasture as their main source of feed. Studies have been done to elucidate the effects of different PAMPs, microbe-associated molecular pattern (MAMP) and plant polyphenol

T or Livak's method).

Molecular Genetics and Genome Biology of Goats http://dx.doi.org/10.5772/intechopen.72414 7

total is expressed as eggs per gram (epg) of fecal sample per animal [14].

reaction (PCR) and enzyme-linked immunosorbent assay (ELISA).

statistical approaches including the comparative CT (also known as 2−ΔΔ*<sup>C</sup>*

*3.3.1. Enzyme-linked immunosorbent assay*

in goat serum and plasma with ELISA [34–36].

**3.4. Effect of pathogen-associated molecular patterns**

**3.2. Molecular techniques**

**3.3. Real-time PCR**

#### **2.4. Wingless pathway**

The Wingless (Wnt) signaling pathway is a conserved pathway in mammals. It involves Wnts, which are secreted glycoproteins that are associated with the Wnt-1 and Wingless gene products of Drosophila [22]. Activation of Wnt signaling happens when Wnt ligands binds to Frizzled receptors together with other receptors lipoprotein receptor-related protein (LRP) 5 and 6 [23–25]. About 19 Wnt ligands and 10 Frizzle receptors have been identified in metazoan mammals. The receptor-ligand interaction leads to downstream signal regulation which is categorized into two: canonical (Wnt/β-catenin) and noncanonical pathways. The former is dependent on β-catenin, but the latter is not. The noncanonical pathway is further subdivided into the planar cell polarity and the Wnt/Ca2+ pathways. The Wnt signaling pathway function in cellular processes includes cell proliferation, cell differentiation, cell migration, cell polarity and cell fate determination and has recently been implicated in stem cell renewal [26]. Wnt signaling has also been associated with various biological processes including adipogenesis, myogenesis, embryogenesis and meat quality. In addition, Wnt signaling has been associated with innate immune and inflammation responses via a cross talk with the TLR and NF-κB pathways [27, 28]. Therefore, a defective or deregulated Wnt signaling has detrimental effect on developing embryo (birth defects) and also affects a number of pathological disease conditions.
