Milestones in avian coccidiosis research: A review

INVITED REVIEW Milestones in avian coccidiosis research: A review H. D. Chapman1 Department of Poultry Science, University of Arkansas, Fayetteville 72701 search that continues today. The topics covered and the references provided are selective and include life cycles and biology, pathology, ultrastructure, biochemistry, immunity, genetics, host cell invasion, species identification, taxonomy, chemotherapy, vaccination, and literature concerned with avian coccidiosis. This review is primarily concerned with the avian species of Eimeria that infect poultry, but some important advances, principally in immunology, have been made using species that infect rodents and rabbits. These are included where appropriate.

Key words: Eimeria coccidiosis, avian, poultry 2014 Poultry Science 93:501–511 http://dx.doi.org/10.3382/ps.2013-03634

INTRODUCTION

the genetics of Eimeria and mechanisms of parasite invasion made possible by advances in molecular biology and cell biology. These aspects are described in more detail in Chapman et al. (2013). A summary of some important milestones in coccidiosis research is provided in Table 1. Although our understanding of the basic biology of Eimeria has lagged behind that of related apicomplexans such as Toxoplasma and Plasmodium, the imminent completion of the genome sequence of all species of Eimeria that infect the fowl promises great progress in the future. In this review, some of the important advances in avian coccidiosis research are described.

Ninety years ago, 2 classic papers concerned with coccidiosis were published in Poultry Science (Johnson, 1923, 1923/1924). The latter publication was prepared especially for the journal following requests to the editor from the readership for more information on poultry diseases. Coccidiosis is a parasitic disease caused by apicomplexan protozoa of the genus Eimeria, and today it may be difficult to appreciate the devastating effects that these parasites once had on poultry flocks. Indeed, without adequate means of control, both by chemotherapy and vaccination, it is possible that the modern poultry industry could not have developed to its present extent. Early research was concerned with establishing details of the life cycle of these organisms, their morphological characteristics, pathogenicity, host specificity, and species identification. This was followed by more detailed investigations of their ultrastructure, pathology, biochemistry, and immunogenicity. During this period, much progress was made in the control of the disease, principally by the discovery of many effective anticoccidial drugs and the introduction of coccidiosis vaccines. Recent research has focused mainly on

LIFE CYCLES AND BIOLOGY At the turn of the 20th century, much confusion existed as to the organism that caused the disease coccidiosis in poultry and other agricultural livestock. Railliet and Lucet (1891a,b) observed oocysts, the transmission stage of the parasite, in ceca from the chicken and conducted the first experimental infections. They named the parasite Coccidium tenellum but subsequently changed this to Eimeria tenella (Railliet, 1913). A review of this early literature is provided by Tyzzer (1929). Fantham was the first to provide a detailed description of the life cycle of an eimerian parasite in an avian host. He was employed to investigate mortality in Red Grouse (Lagopus lagopus) on the grouse moors of

©2014 Poultry Science Association Inc. Received September 19, 2013. Accepted December 3, 2013. 1 Corresponding author: [email protected]

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ABSTRACT This article describes some of the milestones in research concerned with protozoan parasites of the genus Eimeria that infect birds and cause the disease coccidiosis. The time period covered is from 1891, when oocysts were first found in the ceca of diseased chickens, to the present. Progress in our understanding has lagged behind that of other protozoan parasites such as Toxoplasma and Plasmodium despite the enormous importance of Eimeria to animal livestock production. Nevertheless, applied research by universities, government agencies, and private industry has resulted in the successful development of methods of control, re-

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Table 1. Some milestones in avian coccidiosis research Milestone1

Railliet and Lucet, 1891a,b Fantham, 1910 Johnson, 1923, 1923/1924, 1927 Tyzzer, 1929; Tyzzer et al., 1932 Scholtyseck and Mehlhorn, 1970 Ferguson et al., 1976 Dubremetz and Torpier, 1978 Stotish and Wang, 1977; Belli et al., 2003 Wang, 1978 Ryley et al., 1969; Schmatz et al., 1989 Cai et al., 2003 Rose and Long, 1962; Rose and Hesketh, 1979 Rose et al., 1979, 1991, 1992 Hong et al., 2006; Gadde et al., 2013 Joyner, 1969; Blake et al., 2011a Canning and Anwar, 1968; Shirley, 1994 Shirley and Millard, 1976; Chapman and Rose, 1986 Jeffers, 1974; Shirley and Harvey, 2000 Shirley, 2000; Blake et al., 2011b, 2012 Kelleher and Tomley, 1998; Clark et al., 2008 Tomley et al., 1991; Cowper et al., 2012 Schnitzler et al., 1998; Fernandez et al., 2003 Barta et al., 1997; Chapman et al., 2013 Levine, 1939; Cuckler et al., 1955 Grumbles et al., 1948 Cuckler and Malanga, 1955 Shumard and Callender, 1967 Wang, 1975; Smith and Galloway, 1983 Unpublished3 Jeffers, 1975; Shirley, 1989 Chapman, 1994b, 2000 Wallach et al., 1992 Becker, 1934; Davies et al., 1963; Pellérdy, 1974 Brackett, 1949; Reid, 1970; Long et al., 1978

1Selected

milestones in coccidiosis research and references for the subject areas listed. More are included in the text. = internal transcribed spacer 1; SCAR = sequence-characterized amplified region. 3Reviewed by Williams (2002). 2ITS1

Scotland and from these he isolated a parasite that he named Eimeria avium (Fantham, 1910). He was aware of the pioneering work undertaken in Italy, France, and Germany in which 3 essential phases that characterize the life cycle of Eimeria (multiplication and sexual reproduction in the intestine, sporulation in the external environment) were described. Fantham recorded key aspects of the biology of these organisms including the liberation of sporozoites from the sporocysts, cell penetration by sporozoites, schizogony and merozoite formation (asexual multiplication), formation of macro- and microgametocytes (sexual reproduction), oocyst wall formation, and sporogony (spore formation). He provided the first detailed drawing of the life cycle illustrating these phases of development (Chapman, 2003). The intestinal phases of the life cycle have been confirmed in many other studies, and an extraintestinal

phase, involving sporozoite migration to the spleen and liver, has also been observed (Fernando et al., 1987). Significant progress in our understanding of coccidiosis was made by Johnson (Johnson, 1923; Johnson, 1923/1924), the latter publication being lavishly illustrated with photomicrographs of many of the life cycle stages of Eimeria that can be found in mucosal scrapings taken from chickens. Plates I and III, which show oocysts of at least 3 species, and the large schizonts of E. tenella, are reproduced here (Figure 1). Contrary to opinion, at the time Johnson considered that coccidia were not responsible for blackhead disease (subsequently shown by Tyzzer to be caused by the flagellate Histomonas meleagridis), and various other pathological conditions that affected the fowl. Johnson pointed out that infection is self-limiting, that the severity of coccidiosis is proportional to the numbers of sporulated

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Life cycles and biology   Oocysts identified in chicken ceca   Life cycle of Eimeria in an avian host   Self-limitation, specificity, immunity   Species described, pathology, and so on Ultrastructure   Subcellular organelles   Intracellular stages   Pellicle structure Biochemistry   Oocyst wall   Ribosomes, ribosomal RNA   Amylopectin, mannitol cycle  Apicoplast Immunity   Immune response   CD4+, CD8+ T lymphocytes   Cytokines, chemokines   Immunological variation Genetics   Meiosis, chromosomes   Cloning with single sporozoites   Recombination, linkage map   Genomics, proteomics   Transient, stable transfection Host cell invasion   Rhoptry and microneme proteins Species differentiation   ITS1 PCR assays, SCAR markers2 Taxonomy   Consensus tree Chemotherapy   Synthetic drugs  Prophylaxis   Drug resistance  Ionophores   Mode of action Vaccination   First live vaccine introduced (1952)   Life cycle attenuation   Restoration of drug sensitivity   First subunit vaccine Literature  Textbooks   Conference proceedings

Reference

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oocysts ingested, and that transmission within the egg was improbable. Johnson found that a species of Eimeria that infects chickens was unable to infect other avian hosts, thus demonstrating the phenomenon of host specificity, and he also noticed distinct variations in the size and shape of oocysts suggesting that more than one species might occur in the chicken. Johnson was the first to investigate the acquisition of immunity and showed that resistance to a challenge dose of oocysts was not due to age but dependent on prior exposure to infection. He anticipated the possibility of vaccinating chickens by infecting them with live oocysts and instigated experiments to immunize birds by incorporating repeated small doses of oocysts in the feed (Johnson, 1927). He also was the first to observe Eimeria praecox and Eimeria necatrix, which were independently discovered and described in detail by Tyzzer et al. (1932). Johnson’s many contributions to our basic understanding of coccidiosis have been reviewed (Chapman, 2004). The papers by Tyzzer, published in the American Journal of Hygiene in 1929 and 1932, are widely recognized as the most significant milestone in coccidiosis research (Tyzzer, 1929; Tyzzer et al., 1932). Tyzzer presented exquisitely detailed drawings of the life cycle stages of E. tenella and 3 new species that he had isolated from chickens (Eimeria acervulina, Eimeria maxima, and Eimeria mitis). He also described 3 species

from the turkey. In addition to comprehensive descriptions of these parasites, Tyzzer described the lesions in the intestines associated with acute infection and provided much information on morphological characteristics, pathogenicity, host specificity, immunogenicity, and pathology. His work is the platform upon which our modern understanding of coccidiosis is based.

PATHOLOGY In the chicken, Tyzzer and colleagues showed that species of Eimeria develop in different regions in the gut where, depending on the magnitude of infection, they can cause mild to severe lesions and significant pathology. Thus E. acervulina and E. praecox develop in the duodenum extending in heavy infections to the mid-intestine, E. mitis, E. maxima, and E. necatrix develop in the mid-intestine extending to the posterior intestine, and E. tenella develops in the ceca (Joyner, 1978). The discovery of E. brunetti by Levine (1942) added a seventh species that develops in the lower intestine and rectum. Depending on the species, magnitude, and site of infection, coccidiosis can result in a limited enteritis resulting in fluid loss and malabsorption of nutrients (E. acervulina and E. mitis), inflammation of the intestinal wall with pinpoint hemorrhages and sloughing of epithelia (Eimeria brunetti and

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Figure 1. Gut scrapings are still used to diagnose coccidiosis. Photomicrographs of oocysts and schizonts of Eimeria from the intestine. Plates I and III published in Poultry Science, Vol. 3, No. 2; Johnson, 1923/1924. With permission from Poultry Science.

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ULTRASTRUCTURE Studies with the electron microscope carried out in the 1970s by Scholtyseck and others, using Eimeria stiedae from the rabbit, revealed the complexity of subcellular organelles in motile stages of the life cycle, namely the apical complex that consists of polar rings, a conoid, rhoptries, and micronemes (e.g., McLaren and Paget, 1968; Ryley, 1969; Scholtyseck and Mehlhorn, 1970). These investigations indicated similarities with other coccidian genera such as Sarcocystis and Toxoplasma and place Eimeria in the phylum Apicomplexa. The most prominent structures evident in some sporozoites and merozoites are the refractile bodies, electron dense, homogeneous organelles with no apparent substructure (Colley, 1967, 1968). Based on the presence of 2 proteins (Eimepsin and SO7) in sporozoites of E. tenella, plus an additional 30 putative proteins, de Venevelles et al. (2006) proposed that refractile bodies serve as a reservoir for proteins necessary for invasion of host cells. Freeze-fracture studies by Dubremetz and Torpier (1978) using Eimeria nieschulzi from the rat revealed

the highly complex organization of the pellicle of the sporozoite. Studies by Ferguson and colleagues with E. brunetti have also provided insight into the intracellular phases of the life cycle, schizogony and gametogony (e.g., Ferguson et al., 1976, 1977). Three structures were identified in developing macrogamonts including veilforming bodies, and wall-forming bodies of type 1 and 2 that are involved in oocyst wall formation (Ferguson et al., 2003). Recently, structures referred to as volutin granules have been identified in E. tenella and E. acervulina that are thought to have a similar function to acidocalcisomes in Toxoplasma and Plasmodium, involving cation homeostasis, polyphosphate metabolism, and osmoregulation (Soares Medeiros et al., 2011).

BIOCHEMISTRY The work of Wang and colleagues in the 1970s indicated various unique aspects of the biochemistry of avian coccidia and the mode of action of several anticoccidial drugs widely used at the time by the poultry industry (Wang, 1978). He noted the difficulty of isolating viable schizonts or gametocytes and obtaining pure preparations free of host and bacterial contamination, which hindered progress in understanding the biochemistry of intracellular life cycle stages. However, oocysts could be purified in large numbers and it was possible to investigate their structure and biochemical events that occur during sporogony. The oocyst wall was shown to have 2 layers, the inner layer a glycoprotein (Stotish and Wang, 1977) and the outer layer n-hexacosanol, a C-26 primary fatty alcohol (Weppelman et al., 1976). Wang predicted that the inner layer comprised a network of small peptides interlinked by disulfide bonds (Wang, 1978). Recently, Belli and colleagues have shown that during development of the oocyst wall 2 tyrosine-rich proteins derived from the wall-forming bodies of macrogamonts are processed into smaller glycoproteins. They are then cross-linked to form dityrosine matrices that are believed to contribute to the resilience of the oocyst and its survival in the environment (Belli et al., 2003). Recently, a sugar polymer β-1,3-glucan has been found to form a porous trabecular scaffold in the inner wall of oocysts of Eimeria (Bushkin et al., 2012). It is not found in sporocysts. The outer wall of the oocyst was shown to contain a complex set of triglycerides rich in long fatty acyl chains that form a waxy coat of acid-fast lipids that confer environmental resistance (Bushkin et al., 2013). Ribosomes have been isolated from E. tenella and shown to contain ribosomal RNA composed of 5S, 16S, and 23S units similar to those of bacteria (Wang, 1976a). Ribosomal proteins also showed markedly different profiles from those of the chicken. Other prokaryotic characteristics displayed by Eimeria include a mitochondrial enzyme, superoxide dismutase, which is activated by manganese, as in Escherichia coli. Amylopectin is a plant storage polysaccharide that has been identified as a carbohydrate energy reserve

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E. maxima), or complete villar destruction resulting in extensive hemorrhage and death (E. necatrix and E. tenella). Eimeria praecox is generally considered to produce little pathology and to have little pathogenicity. These characteristics and others have been utilized in the widely accepted visual system for scoring the severity of lesions in different regions of the gut (Johnson and Reid, 1970). Many studies have investigated the effects of different dietary components, such as l-arginine, threonine, various vitamins, and so-called plant-based natural products on coccidiosis (e.g., Allen et al., 1998; Allen and Fetterer, 2002; Wils-Plotz et al., 2013). The effect of coccidiosis on the absorption of glucose, oleic acid, vitamin A, carotenoids, calcium, and trace minerals such as zinc in the duodenum of birds infected with E. acervulina has been investigated and in most cases impaired absorption demonstrated (e.g., Turk and Stephens, 1967; Kouwenhoven and van der Horst, 1969; Southern and Baker, 1983). Few studies have been carried out with other species of Eimeria (Turk, 1978). The absorption of amino acids and glucose is complicated because impaired absorption in the duodenum may be compensated by increased uptake in the ileum (Ruff, 1974). Infection may cause changes in the permeability of the mucosa resulting in leakage of plasma proteins into the intestinal lumen, increased pH and decreased gut motility (Schildt and Herrick, 1955; Stephens, 1965; Preston-Mafham and Sykes, 1967). Infection with E. maxima results in a marked hypoproteinemia, a decline in plasma concentrations of the hormones thyroxine and triiodothyronine, and an increase in plasma corticosterone and prolactin (Chapman et al., 1982; Davison et al., 1985). No changes in growth hormone were observed, indicating that this hormone is not involved in the decreased growth observed during infection.

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IMMUNITY Work by Rose and colleagues established the basic principles of immunity resulting from infection with Eimeria such as specificity, in which one species of Eimeria elicits little protection in birds against challenge with others (Rose and Long, 1962). Cell-mediated immune responses were shown to be most important, humoral immunity playing little role in protection (Rose and Hesketh, 1979). Many types of cells of the immune system, including macrophages, dendritic cells, mast cells, natural killer cells, basophils, and eosinophils, were shown to be involved in the innate immune response (Rose, 1996). The cytokine IFN-γ was found to be important in immunoregulation (Rose et al., 1991), and subsequently, numerous cytokines and chemokines have been shown to be involved in primary and secondary responses to infection (Hong et al., 2006; Gadde et al., 2013). Recently evidence has been obtained that Toll-like receptors (TLR4 and TLR15) play a role in the innate immune response in eimerian infections (Zhou et al., 2013). A murine model indicated that acquired immunity is dependent on T cell lymphocytes and that B cells are not involved (Rose et al., 1979). Subsequently, the differential role of CD4+ and CD8+ T lymphocytes was described (Rose et al., 1992; Rothwell et al., 1995), and the importance of the CD4+ αβ T cell subset demonstrated (Roberts et al., 1996). Much work has been concerned with how dietary ingredients and plant extracts may modulate the immune response (e.g., Allen, 2003; Lee et al., 2011).

Immunological variation between strains of the same species was first reported by Joyner for E. acervulina (Joyner, 1969) and subsequently shown to occur in E. maxima (Norton and Hein, 1976). These were the first of several studies showing that inherent intraspecific variation could occur in biological characteristics of Eimeria (e.g., Jeffers, 1978). Other examples include oocyst morphology, pathogenicity, and drug sensitivity. Several genotypic tools, including random amplification of polymorphic DNA-PCR, RFLP, amplified fragment length polymorphism, and gross chromosomal size polymorphism as revealed by pulsed-field gel electrophoresis, have recently been used to define interand intraspecific variation (Chapman et al., 2013). Immunological variation between strains has been used by Blake and colleagues to map loci that encode strain-specific immunoprotective antigens (Blake et al., 2011a). From a practical perspective, the importance of immunological variation is that it could confound the use of vaccines because they might not confer protection against all strains of a species in the field. Recent transcriptome, proteome, and genome investigations by Tomley and colleagues have identified many antigens on the surface of sporozoites, some of which may suppress cell-mediated immunity and also contribute to proinflammatory responses and the associated pathology seen during infection (Lal et al., 2009).

GENETICS The occurrence of meiosis in the zygote during the sexual phase of the life cycle of Eimeria was first proposed by Canning and colleagues, who noted the presence of 2 rows of 5 condensed chromosomes (Canning and Anwar, 1968; Canning and Morgan, 1975); subsequently, 14 chromosomes were recognized (Shirley, 1994). Nuclear reduction division occurs during sporulation, and therefore a genetically homogenous population requires the establishment of an infection from a single sporozoite or sporocyst (Shirley and Millard, 1976; Chapman and Rose, 1986). Classical genetic studies became possible when Jeffers demonstrated that recombinant phenotypes could be developed utilizing strains resistant to different anticoccidial drugs (Jeffers, 1974). In other studies, recombinant phenotypes could not be obtained between lines where close physical linkage of loci for resistance was thought to occur (Joyner and Norton, 1978). Eventually similar work led to the development of a genetic linkage map of E. tenella (Shirley and Harvey, 2000) and the identification of hot- and cold-spots of genetic recombination (Blake et al., 2011b). The last 20 yr have seen major advances in our knowledge of the molecular organization of the Eimeria genome, in the case of E. tenella comprising 50 to 60 Mb DNA (Shirley, 2000) with a predicted 8,786 proteins and a gene density of 160 per Mb−1 (Chapman et al., 2013). Other genomes in eimerian parasites include those in the mitochondria and the apicoplast

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that is used during sporulation in E. tenella (Ryley et al., 1969). Another energy reserve is mannitol, a sugar alcohol that has been found in large quantities in unsporulated oocysts of this species (Schmatz et al., 1989). Enzyme components of a unique biochemical pathway, the mannitol cycle, previously found exclusively in fungi, were described. A functional tricarboxylic acid cycle has been demonstrated in sporulating oocysts (Wang, 1978), but failure to isolate coupled mitochondria has hampered studies of oxidative phosphorylation. Based on studies with quinolone drugs that inhibit electron transport, Wang has proposed that coccidia have a branched electron transport chain (Wang, 1975). Further work by Fry and Williams (1984) suggested the presence of 2 functional terminal oxidases in the mitochondria of E. tenella. Recent high-throughput RNA sequencing provides transcriptional evidence of aerobic respiratory enzymes in sporulating oocysts and the existence of glycolytic, tricarboxylic acid, and pentosephosphate pathways (Matsubayashi et al., 2013). Like other apicomplexa, E. tenella has a nonphotosynthetic plastid-like organelle referred to as an apicoplast (Cai et al., 2003). An enzyme, enoyl reductase, was localized to this organelle and was considered to be involved in type II fatty acid synthesis during schizogony and gametogony (Ferguson et al., 2007).

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HOST CELL INVASION In recent years, progress has been made in our understanding of the mechanisms involved in host cell invasion by sporozoites and merozoites. In the 1990s, subcellular fractionation techniques facilitated the isolation of organelles such as micronemes, rhoptries, and dense granules from sporozoites and the eventual characterization of many proteins that are secreted during parasite invasion of host cells (Tomley et al., 1991). One such protein from micronemes of E. tenella, known

as EtMIC4, was shown to contain tandem arrays of epidermal growth factor-like repeats and thrombospondin type-1 repeats (Tomley et al., 2001). Invasion is an active process that involves the formation of a moving junction at the parasite/host cell membrane, which migrates to the posterior end of the parasite (Russell, 1983). A detailed proteomic comparison of different life cycle stages of E. tenella was provided by Tomley and colleagues and 1,868 proteins were identified including the moving junction proteins known as RON2, RON4, RON5, AMA-1, and AMA-2 that are involved in host cell invasion (Lal et al., 2009). The RON2 was found in sporozoites and merozoites but AMA-1 and RON4 only in merozoites and AMA-2 and RON5 only in sporozoites, suggesting stage-specific moving junction proteins. Microneme and most rhoptry proteins were found only after sporulation. Sequencing of a gene encoding an immunodominant microneme protein from sporozoites of E. tenella indicated that it is involved in cell-cell or cellmatrix interactions. A continuation of this work has led to structural and functional information on the role of microneme proteins in host cell recognition in extraordinary atomic detail (Cowper et al., 2012).

SPECIES IDENTIFICATION AND TAXONOMY Traditional methods of species identification have involved assessment of several phenotypic characteristics such as site of development, cross-immunity, characteristic lesions, and pathogenicity (Joyner, 1978); these are time consuming and expensive. Although still necessary, they are now complemented by molecular methods that involve PCR diagnostic assays based on DNA amplification. MacPherson and Gajadhar (1993) employed arbitrary primers under low stringency and Stucki et al. (1993) developed a specific PCR assay based on E. tenella 5S rDNA. This was followed by assays capable of detecting and differentiating all 7 species that infect chickens using the internal transcribed spacer 1 regions of rDNA. These contain sufficient interspecific variation to enable the selection of primers that can be used in PCR analyses to differentiate species (Schnitzler et al., 1998). An alternative recently developed by Gruber and colleagues involves the conversion of anonymous random amplified polymorphic DNA markers into sequence-characterized amplified region markers (Fernandez et al., 2003). A set of molecular (PCR-based) and computational tools involving morphological characteristics (COCCIMORPH) has been developed for species differentiation (Castañón et al., 2007). Early work on the taxonomy of Eimeria and their relationship to other Apicomplexa was based on morphological and biological characteristics (Levine et al., 1980). Characterization of small subunit (18S) rDNA gene sequences by Barta and colleagues has allowed the phylogenetic relationships of Eimeria species infecting the fowl to be inferred and compared with other Apicomplexa (Barta et al., 1997). A maximum likeli-

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that have recently been sequenced (Cai et al., 2003; Lin et al., 2011), and double-stranded RNA associated with virus-like particles (Lee and Fernando, 2000). The nuclear genome of E. tenella has been sequenced in a collaborative venture with the Institute for Animal Health, UK, the Wellcome Trust Sanger Institute, and other organizations. This was extended recently to include E. maxima (Blake et al., 2012) and has culminated in the imminent completion of nuclear genome sequences of all 7 species that infect the fowl (F. Tomley, Royal Veterinary College, London, UK, personal communication). Chromosome 1 of E. tenella was shown to have an unusual segmented structure with 3 repeat-rich segments containing large numbers of simple sequence repeats, an organization that may be present throughout the genome (Lim et al., 2012). Repetitive DNA sequences in eimerian genes were first demonstrated by Jenkins and shown to comprise numerous trinucleotide GCA repeats (Jenkins, 1988). Genetic manipulation of Eimeria has proved possible using transfection techniques. In 1998, development of a transient transfection system was reported by Kelleher and Tomley (1998), and 10 yr later a stable system was described (Clark et al., 2008). Transient transfection was achieved by electroporation with plasmids; PCR amplified DNA or fragmented genomic templates that encode the exogenous DNA, flanked by Eimeriaspecific regulatory sequences. Stable transfection was achieved by use of the mutated dihydrofolate reductasethymidylate synthase gene (which confers resistance to pyrimethamine, a drug used to potentiate the action of the sulfonamides) as a selection marker, fluorescenceactivated cell sorting of fluorescence reporter proteins, and restriction-enzyme-mediated integration, to boost transfection efficiency. A commercially available “cutand-paste” transposon (PiggyBac) has recently been used successfully to achieve targeted insertional mutagenesis in Eimeria (Su et al., 2012). According to Clark et al. (2008), transfection of Eimeria species is limited by the inability to transfect oocysts and sporocysts, the difficulty of obtaining single-sporocyst-derived recombinant clones, and the obligate requirement of in vivo amplification and selection of stably transfected parasites. Nevertheless, genetic manipulation of Eimeria utilizing high transfection efficiencies may better our understanding of the biology of these organisms and facilitate their use as vaccine vectors.

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hood consensus tree was constructed from many Eimeria species and has shown that the clade of eimeriid coccidia is not monophyletic, indicating that the taxonomy of this genus is far from resolved (Chapman et al., 2013). The cecal species of coccidia from chickens and turkeys (i.e., E. necatrix, E. tenella, and Eimeria adenoeides) were found to form a monophyletic group to the exclusion of the other species that infect the fowl (Miska et al., 2010).

CHEMOTHERAPY

VACCINATION Although the concept of vaccination using live oocysts was conceived by Johnson (1927), it was Edgar who first turned this into a commercial reality (Edgar, 1956; see Williams, 2002). His vaccine (Coccivac), which consists of a mixture of oocysts of important species to be administered at a low dose to birds, was introduced in 1952 and is still widely employed today in various forms. Technical information on the efficacy of this vaccine was never reported in the scientific literature. A key to the recent success of vaccination with live oocysts, especially in broilers, is the realization that protective immune responses can be produced following immunization with low doses of oocysts in dayold chicks (Long et al., 1986). Also the development of practical means of delivery, such as the use of spray cabinets in the hatchery, thus avoiding the need to vaccinate birds in the poultry house, has facilitated the adoption of vaccination with live oocysts as a practical means of control. The induction of solid immunity can be achieved by repeated inoculation of birds with low numbers of oocysts (Joyner and Norton, 1973). Davis et al., (1986) proposed that such a “trickle” exposure could be achieved by encapsulating oocysts in gel beads that could be fed to chickens, a concept that has recently been revived (Jenkins et al., 2012). Jeffers (1975) demonstrated that E. tenella could be attenuated by selection for precocious (early) development. The line so developed had reduced patho-

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Several important contributions to our knowledge of coccidiosis were made by Levine, the most significant of which was his study of the drug sulfanilamide (Levine, 1939). Many claims had been made for the efficacy of various substances in controlling coccidiosis, but sulfanilamide, although toxic if used for long periods, was the first compound shown to have true efficacy against Eimeria. Sulfanilamide and other sulfonamides were the first synthetic drugs (also known as “chemicals”) that are produced by chemical synthesis. In subsequent years, many other synthetic compounds were discovered with excellent broad-spectrum efficacy against Eimeria including nicarbazin, introduced in 1955, which is still widely used today (Cuckler et al., 1955). The mode of action of nicarbazin is not known but thought to involve inhibition of oxidative phosphorylation mechanisms in coccidia (Wang, 1978). Today, nicarbazin is principally used in the starter feed provided to broilers (Chapman, 1994a). Several other synthetic drugs have fascinating modes of action such as amprolium, which was shown to competitively inhibit the uptake of thiamine by second generation schizonts of E. tenella (James, 1980), and the quinolone drugs that inhibit respiration by blocking electron transport in the parasite mitochondrion (Wang, 1976b). A landmark contribution to the control of coccidiosis was the demonstration by Grumbles and colleagues that the disease could be controlled by the inclusion of a low concentration of sulfaquinoxaline continuously in the feed (Grumbles et al., 1948). The principle involved (prophylaxis or prevention) has had a major impact on the ability to grow poultry under intensive conditions and it is likely that the poultry industry could not have developed to its present extent without the advent of drugs that could be used in this manner. An insight in this early work was that prophylactic use of a drug did not necessarily prevent the acquisition of immunity, an important finding that helps explain the continued efficacy of some anticoccidials today. A few years later, the first reports of the development of resistance in field strains of Eimeria appeared (Cuckler and Malanga, 1955) to be followed by many subsequent reports of resistance to anticoccidial agents (e.g., Bafundo et al., 2008); indeed the acquisition of resistance in Eimeria species has been demonstrated for every drug that has been introduced (Chapman, 1997).

In 1967, a series of publications reported the discovery of a novel drug, monensin, the introduction of which was to have a profound effect on the control of coccidiosis (Shumard and Callender, 1967). Unlike previous anticoccidials that were produced by chemical synthesis, monensin is an antibiotic belonging to the family of polyether ionophores, and is produced by fermentation. This was followed by the discovery of other ionophores, including lasalocid, narasin, and salinomycin, which, like monensin, have a broad spectrum of activity against different species of Eimeria (McDougald, 1982). The mode of action, involving disruption of ion gradients across the cell membrane (Smith and Galloway, 1983), is unique and unlike most other drugs resistance is difficult to induce and slow to develop. Ionophores and a combination of nicarbazin with narasin are widely used for the control of coccidiosis. An important innovation was the introduction of the shuttle program in which 2 or more different drugs, usually with different modes of action, were provided in different feeds during the life of a flock. Another innovation was the rotation program in which different drugs were employed in successive flocks. It is thought that both programs may have helped prolong the effective life of anticoccidials in the face of constant selection for drug resistance. Both programs are widely used by the broiler industry (Chapman, 2001).

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LITERATURE A description of the milestones in avian coccidiosis research would not be complete without mentioning significant literature that has contributed to a better understanding of avian Eimeria, much of which cannot readily be found in online databases. Coccidia and Coccidiosis by Pellérdy, first published in 1965 with a revised version in 1974, is perhaps the most valuable resource for those interested in the classical description of these organisms (Pellérdy, 1974). Significant educational textbooks include those by Becker (1934), Davies et al. (1963), ongoing chapters in Diseases of Poultry (first published in 1944), which has been revised by different authors to the present day (Swayne et al., 2013), The Coccidia (Hammond and Long, 1973), and the Biology of the Coccidia (Long, 1982). The proceedings of several conferences are also a valuable resource because they contain original data and important reviews that cannot readily be found elsewhere. Examples include the first coccidiosis conference (Brackett, 1949), the symposium on methodology for testing anticoccidial

drugs (Reid, 1970), the 13th Poultry Science Symposium (Long et al., 1978), the Georgia Coccidiosis Conference (McDougald et al., 1986), and the Vth International Coccidiosis Conference (Yvore, 1989). Color illustrations of the lesions of coccidia and diagnostic charts have been reproduced in various books, articles, and pamphlets, and have been a valuable resource for veterinarians and others needing to diagnose coccidiosis in the field (e.g., Long et al., 1976; McDougald and Reid, 1991).

CONCLUSIONS In this article, some of the milestones in avian coccidiosis research are described. The time period covered is from 1891 when oocysts were first found in the ceca of diseased chickens to the present. Topics covered have included life cycles, biology, biochemistry, ultrastructure and genetics of the parasite, pathology caused by infection, host cell invasion and acquisition of immunity by the host, species identification and taxonomy, and control of coccidiosis by chemotherapy and vaccination. Significant literature concerned with avian coccidiosis is described. Although progress in our basic understanding of Eimeria has lagged behind that of other apicomplexan parasites, in recent years the modern tools of molecular biology and cell biology have been used to greatly expand our knowledge of these organisms. The resilience of the oocyst and its ubiquitous presence wherever poultry are reared provides a continuing threat to the health of poultry, and therefore there will be a continued need for basic and applied research into all aspects of the biology of these organisms. The recent completion of the genome sequence of all species that infect the fowl holds out great promise for improved control in the future.

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genicity compared with the parent line but retained its immunogenicity. Histological examination revealed that the second generation schizonts developed earlier than those of the control line and were either defective or contained fewer merozoites. Indeed, continued selection for the precocious trait eventually resulted in the complete loss of the second generation of schizonts, sexual development occurring following the first generation (McDougald and Jeffers, 1976). Development of the smaller schizonts in the intestine resulted in only moderate tissue damage, thus accounting for the loss of virulence. This discovery led to the commercial introduction of an attenuated vaccine containing all species that infect the fowl (Shirley, 1989). Jeffers (1986) suggested that alternating cycles of immunization and chemotherapy might provide effective long-term control of coccidiosis, and Chapman (1994b) subsequently demonstrated that sensitivity to monensin could be restored following use of a live vaccine in commercial broiler production. Similar observations were made for the synthetic drug diclazuril (Mathis and Broussard, 2006). Various programs involving alternation of vaccination with the use of drugs have therefore been proposed with the object of achieving sustainable coccidiosis control (Chapman, 2000). The identification of gametocyte antigens from E. maxima by Wallach and colleagues has led to a different approach to vaccination in which hens are injected with 2 proteins (gam56 and gam82) derived from the wallforming bodies of macrogamonts (Wallach et al., 1989, 1992). The resultant IgY antibodies are transferred via egg yolk to chicks and confer protection against E. maxima and other species early in life. A vaccine based on this principle, the first subunit vaccine for an apicomplexan parasite, is now used commercially in some countries.

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