1. INTRODUCTION

Intestinal coccidiosis is caused by the intracellular growth and replication of coccidian (Shirley & Schnitzler, 1999; Belli et al., 2006; Lim et al., 2012). Chicken has become the host to seven species Eimeria which are E. tenella, E. maxima, E. acervulina, E. brunette, E. necatrix, E. praecox, E. mitis and each species is responsible for a different form of coccidiosis. The three most pathogenic Eimeria species which cause the most economically significant are E. tenella, E. acervulina and E. maxima. Each parasite is responsible for a different form of coccidiosis (Shirley et al., 2004). Eimeria tenellacause coccidiosis in chickens which is a serious intestinal disease leading to impaired nutrient absorption, weight loss, diarrhea and severe cases of death (Belli et al., 2004). Ceacum is the part of intestinal tract location that will infected by E. tenella (Barta, 1997). On the other hand, three different phases of the life cycle of Eimeria are sporogony (the unsporulated oocysts shed in the faeces of the host undergo sporulation in the environment to become infective), schizogony (an expansive form of asexual reproduction) and gametogony, a sexual phase (which leads to the formation of female and male gametes, and end with the formation of oocysts) (Shirley & Schnitzler, 1999). The control of coccidiosis depends on prophylactic chemotherapy and also vaccination (Shirley et al., 2004). Whereas Mcpherson-K. J. L (2008) state that the current strategies to control coccidiosis in commercial poultry include anticoccidial medication, vaccination and also the use of probiotics. For a long-term control of coccidiosis, the identification of new targets within Eimerian parasite is required and this imperative underpins the need for the genome sequencing (Shirley et al., 2004).

1.1 PROBLEM STATEMENT

Coccidiosis is one of the most important and common diseases that affect poultry, it results in a great economic loss all over the world (S. Al-Quraishy, A.S. Abdel-Baki, M.A. Dkhil, 2009). It is caused by the genus Eimeria of an apicomplixa protozoan parasite (Shirley, 1995). This parasitic infection occurs in the epithelial cells of the intestine, despite the advances in nutrition, chemotherapy, management and genetics (Jamal Gharekhani, Zivar Sadeghi-Dehkordi, and Mohammadali Bahrami,2014). Most Eimeria species affect birds between 3 and 18 weeks of age and can cause high mortality in young chicks(McDougald and Reid, 1997). Eimeria typically infect defined regions of the gastrointestinal tract leading to impaired nutrient absorption, weight loss, diarrhea and in severe cases mortality (Belli et al., 2004). The poultry industry incurs major economic losses since chemoprophylaxis, the preferred method of preventing and controlling the disease, is ineffective because the resilient parasites do not respond to therapy.

Infections of chickens begin after the uptake of oocysts when sporozoites penetrate the epithelium of the villi. They enter crypt epithelial cells after passing through the lamina propria, where they will undergo several rounds of asexual and sexual proliferation, resulting in formation of merozoites and later, gametocytes (Jeurissen SH, Janse EM, Vermeulen AN,Vervelde L, 1996). When macrogametes are fertilized by microgametes, forming zygote that will develop into oocysts and eventually shed in the faeces. In contrast to the malaria parasites, Eimeria spp. has not been proved pathogenic for man. The organism has never been found in intestinal tissue and no specific lesion has been demonstrated. Many therapeutic agents have been used, including bismuth, iodide, quinine, gentian violet, emetine, anthelmintics and others without conclusive evidence of results (R. M. Kiskaddon, M.D.; R. J. F. Renshaw, M.D.,1945).

Nowadays, coccidiosis is prevented by anticoccidial drugs that are added to food, but continuously usage of these drugs will leads to unavoidably emergence of resistant Eimeria strains (Jeurissen SH, Janse EM, Vermeulen AN, Vervelde L, 1996). This prolonged use of drugs have many side effects such as decrease fertility (Joyner, 1964) and encourage to the development of drug-resistant strains (McLoughlin and Gardiner, 1963). It will also interfere with immunity (Davies and Kendall, 1955; Reid, 1960), Moreover, the present drugs that available do not offer effective protection against all Eimeria parasitic species in chickens and most of the current coccidiostats are not suitable to use for prolonged periods intended for human consumption. In spite of the high efficacy of modern coccidiostats, upsurge of coccidiosis may occur due to high levels of contamination in the environment, the development of drug-resistance strains as well as reduced the usage of the drug and a high degree of susceptibility (Joyner, 1970).

1.2 OBJECTIVES

1. To amplify and sequence the surface antigen from apicomplexan parasite, Eimeria Tenella.
2. To clone the surface antigen of apicomplexan parasite, Eimeria Tenella.

2.0 LITERATURE REVIEW

2.1 APICOMPLEXAN PARASITES

The Apicomplexa are a phylum from the group of diverse obligate intracellular parasites containing Toxoplasma gondii and Cryptosporidium parvum which are the opportunistic pathogens of immunocompromised individuals, Plasmodium spp., the parasites that cause malaria and also Eimeria spp. and the Theileria spp., the parasites that consider as agricultural importance(Naomi S. Morrissette and L. David Sibley, 2002). Parasitic protozoans of the apicomplexa are the most frequentt and successful pathogens known to the world. Infection by this parasites causes incalculable morbidity and mortality to humans and agricultural animals(Aikawa, M, 1988). Presently, more than 50 billion livestock for food production especially for the poultry, suffer from debilitating intestinal diseases that caused by many species of apicomplexan parasites such as Eimeria, Theileria, and Babesia (Tomley and Shirley, 2009). Besides, half of the world’s population is at risk of getting malaria that caused by Plasmodium species (Guerra et al., 2006). Eimeria is the cause of of coccidiosis in chickens while Theileria, the cattle parasite is characterized by anemia and high death rate especially in pregnant cows. Plasmodium infects red blood cells in bird species and cause malaria as well as in several other vertebrate including human. In Africa, almost one million human died because of malaria each year, which mean that a child dies every 30 seconds of this disease (Coombs and Muller, 2002; Shirley et al., 2005).

All of those apicomplexan parasites share distinguishing morphological features, cytoskeleton organization and the way of motility, invasion and also replication(Naomi S. Morrissette and L. David Sibley, 2002). These parasites have an elongated shape and a clearly visible specialization of the apical region (Aikawa,M., 1998). Many of the distinct characteristics compose of a collection of unique organelles termed the apical complex(Naomi S. Morrissette and L. David Sibley, 2002). Theapical complexis the flag trait required for classification asApicomplexa (Lee et al., 2000, Levine, 1973). It is a components found at the anterior end of certain stages, most notably at the infective stages, replacing the nucleus and mitochondria towards the posterior end (Aikawa et al., 1978). Upon contact with a suitable host cell, apicomplexans can invade within seconds, with minimal apparent disturbance of the infected cell (Boris Striepenmail, Carly N Jordan, Sarah Reiff, Giel G van Dooren, 2007).

Figure 2.1 : The morphology of apicomplexan parasites

Copyright© 2002, American Society for Microbiology

2.2 THE ROLE OF GLIDING MOTILITY OF THE APICOMPLEXA IN CELL INVASION

The members of Apicomplexa invade host cell by substrate-dependent forward locomotion known as gliding motility(Pinder et al., 2000; Opitz & Soldati, 2002). Apicomplexa does not possess cilic, flagella, type IV pili or other locomotory organelles (Russell & Sinden,1981), nor do they crawl like amoeba (Mitchison & Cramer, 1996) or deform their membrane. The gliding movement is actin–myosin motor dependent (Hakansson et al.,1998; Pinder et al., 2000)which coupled with the substratum, presumably by transmembrane proteins such as circumsporozoite-and-TRAP related protein (CTRP) and thrombospondin-related adhesive protein (TRAP) which have cytoplasmic sequences (Menard, 2000). Motility can be visualized in real time with video microscopy (Russell &Sinden, 1981; Morisaki et al., 1995) or by the formation of surface membrane traits that can be labelled with immunofluorescence assays (Arrowood et al., Stewart & Vanderberg, 1988; Hakansson et al., 1999). Circular gliding occurs when a parasite lies on its right side(where the apex is defined as the top of the parasite and dorsal is defined as the convex surface of the parasite) and moves around in a counter clockwise circle. Upright twirling takes place when a parasitic stands on its posterior and spins in a clockwise circle. Furthermore, helical gliding occurs when the parasite begins on its left side and initiates a clockwise revolution around its long axis while moving forward one body length. The parasite then flips onto its left side while undergoing little forward motility. Helical gliding allows a curved parasite to propel itself straight across substrate (Hakansson et al., 1999).

Host-cell invasion by apicomplexan parasites involves the successive exocytosis of three different secretory organelles which are micronemes, rhoptries and dense granules. Rhoptries, dense granule and micronemes are unique secretory organelles containing the products that need for motility, adhesion to host cells, invasion of host cells, and formation of the parasitophorous vacuole (N. S. Morrissette, A. Mitra, D. Sept and L. D. Sibley, 2004). Micronemes are used for host-cell recognition, binding, and possibly motility. Rhoptries are used for parasitophorous vacuole formation whilst dense granules used for remodeling the vacuole into a metabolically active compartment (Dubremetz JF et al., 1998).

Attachment to the host cell is started via interaction of the surface protein of the parasites with the plasma membrane of the host cell(Grimwood and Smith, 1996). The apical region of the infecting parasite which is called zoite connects to the host cell, creating a depression in the cell plasma membrane and taking the shape of the zoite while forming a condensed, electron-dense area at the point of attachment (Aikawa et al., 1978). Rhoptry ducts extend from the apical complex and through the junction formed between the two cells (Aikawa et al., 1978). This step is proceed by the microneme and rhoptry that release vast array proteins which have the capacity to encourage formation of the protective parasitophorous vacuole that surrounds the parasite once inside the host cell (Bannister and Mitchell, 1989). As conclusion, the actual invasion of the parasite is intervene by the formation of a moving junction around the infecting parasite which is so named because it moves along the length of the parasite resulting in the engulfment of the parasite within the host cell (Besteiro et al., 2009).

Figure 2.2 : Current model of the motor protein complex driving gliding motility.

(Adapted from Soldati et al (2004) Current Opinion in Cell Biology 16, 32-40.)

2.3 EIMERIA TENELLA

Eimeria Tenellais one of seven species that cause coccidiosis in chickens (Shirley MW, Smith AL, Tomley FM, 2005). It is one of the most pathogenicEimeriaspp. that inflicts economic losses on the poultry industry all over the world (Dalloul RA, Lillehoj HS, 2006). Eimeria tenella can be found in the feces of the infected chicken (Michael G. Wallach mail, Udi Ashash, Amnon Michael, Nicholas C. Smith, 2008) and they have complex developmental life cycles with an exogenous phase in the environment during which oocysts excreted from the chicken undergo sporulation and become infective while the endogenous phase in the intestine during which there are two or more rounds of discrete depending on the species, expansive asexual reproduction (schizogony) followed by sexual differentiation, fertilization and shedding of unsporulated oocysts (Kalpana Lal, Elizabeth Bromley, Richard Oakes, Judith Helena Prieto, Sanya J Sanderson, Dominic Kurian, Lawrence Hunt, John R Yates, III, Jonathan M Wastling, Robert E Sinden, Fiona M Tomley, 2009).

The unsporulated oocyst will develops by the deposition of proteinsfrom two visible wall forming bodies becoming a multi-layered oocyst cell wall (Ferguson DJ, Belli SI, Smith NC, Wallach MG, 2003). After shedding, the unsporulated oocysts will make contact with moisture and air then rapidly undergo meiosis and mitosis to produce 8 haploid sporozoites (Ryan R, Shirley M, Tomley F, 2000). In the case of Emeria tenella, sporozoites will migrate to the caecum where they invade villus enterocytes and undergo their entire endogenous development within enterocytes of the crypts (Rose ME, Lawn AM, Millard BJ, 1984). Eimeria tenellaundergoes two distinct and massive waves of schizogony in the crypts, which produce large numbers of first and second generation merozoites. A third round of schizogony, begin by invasion of second generation merozoites and characterized by much smaller schizonts, is known to occur and may be mandatory although it is possible that invasion of second generation merozoites can also initiate gametogony (McDonald V, Rose ME, 1987). Sporozoites and merozoites ofEimeria tenellahave many features related to their invasive natures including micronemes that release protein, which are very important for host binding and invasion (Periz J, Gill AC, Hunt L,Brown P,Tomley FM, 2007) , the use of actin based ‘glideosome’ to power up the host invasion(Bumstead J, Tomley F,2000) and the secretion of rhoptry proteins to form the parasitophorous vacuole within which the parasite resides during the invasion (Greif G, Entzeroth R,1996).

As a conclusion, the life cycle can be divided into three distinct phases which include sporogony (the unsporulated oocysts shed in the faeces of the host undergo sporulation in the environment to become infective), schizogony (an expansive form of asexual reproduction) and a sexual phase, gametogony (which leads to the formation of female and male gametes and terminates with the formation of oocysts) (Shirley & Schnitzler, 1999).

Figure 2.3 : The life cycle of Eimeria

(United States Department of Agriculture (USDA)

2.4 SURFACE ANTIGEN

Surface antigens are those expressed on the surface of infected cells that can induce a cytotoxic phenomenon leading to a destruction of host cells or to activation of the killing mechanism in the host cell itself. However, little is known about their role in parasite development (Tabarés et al., 2004). Some of these surface antigens have been associated with a variety of functions in host cell invasion, pathogenicity as well as the immune avoidance and also known to draw out strong immune responses (Jung C, Lee CYF, Grigg M, 2004). Many research has been carried out to study the role of surface antigens in the growth, development, and also the survival of the parasites. Glycosylphosphatidylinositol (GPI)-anchored surface antigens (SAGs) of Eimeria Tenellaare among the major surface molecules of the parasite and many of the SAGs are expressed during the development of second generation merozoitesmaking them good targets for host innate and adaptive immune responses. Other apicomplexan parasites such as Plasmodium falciparum, Sarcocystis neuronaand Toxoplasma gondii also have the GPI-linked antigens expressed on their surfaces (Gilson PR, Nebl T, Vukcevic D, Moritz RL, Sargean T, 2006). Besides, SAG proteins may be used by Eimeria tenellato confuse the host immune system and improve the survival of the parasites. The chicken immune response might be misdirect towards the antibody production because of the simultaneous expression of multiple SAG proteins rather than the cellular mediated immune responses required to eliminateEimeria Tenella, therefore, allowing the parasites to avoid the first line defense mechanisms of the host and multiply more easily (Yock-Ping Chow, Kiew-Lian Wan, Damer P. Blake, Fiona Tomley, Sheila Nathan, 2011).