The long view: a selective review of 40 years of coccidiosis research

Abstract

This selective review of 40 years of coccidiosis research is one of a number on important diseases of poultry to celebrate the 40th anniversary of the birth of Avian Pathology, the journal of the World Veterinary Poultry Association, and is written for the non-specialist. The intention is to provide a flavour of the field problems and intellectual challenges, with emphasis in the areas of immunology and vaccinology that drove research in the 1970s, and to reflect on research progress since.

Introduction

The 1970s sit in the middle of the modern history of 80 years of research into avian coccidia. The decade now looks backwards to the pioneering work on coccidia and coccidiosis in the late 1920s and early 1930s by researchers who laid the foundations of much fundamental knowledge, including speciation, lifecycles, pathogenicity, host specificity, induction of protective immunity and control by vaccination and chemotherapy; and looks forwards to the modern era in which molecular approaches are being applied to many studies, including Eimeria genomes and genetics, immune mechanisms and protective antigens and host susceptibility with the real prospect that a new generation of innovative vaccines will emerge.

During the 1970s control of coccidiosis took a huge step forward, both with the paradigm-changing introduction of the prophylactic ionophorous antibiotics in the field and, separately, through groundbreaking research in the USA that laid the direct foundations for the introduction of safe vaccines almost two decades later. Many other significant findings were also made in the commercial and academic laboratories worldwide, including: analyses of genetic variation to definitively identify species and some strains; the introduction of a raft of laboratory methods to evaluate the efficacy of new and current drugs and the emergence of drug resistance; and a greater understanding of the mechanisms of protective immunity.

In this short and very highly selective review, the reader will be referred to many of the substantive texts and references to primary data for more detailed information. This review is also mostly written from our personal perspectives so that we can best give insights into how some of the scientific questions of the day were framed and addressed. We therefore apologise to very many colleagues and other coccidiologists whose names and work are either not described at all or only in passing. In particular, whilst the absolutely critical importance of chemotherapy to the control of coccidiosis is highlighted, less attention is given to this topic in our descriptions of scientific progress.

Setting the scene

The laboratories

The increasing significance of coccidiosis and its deleterious consequences for the expanding developing poultry industry (around 3 billion broilers were produced annually in the USA in the 1970s) continued to be a major driver for laboratories in both the commercial and academic sectors to develop better intervention and control strategies. The worldwide financial investment in avian coccidiosis research was probably greater in the 1970s in relation to the size of the global poultry market than in any other decade, before or since. For example, most animal health companies in the USA and Europe had programmes directed towards the chemotherapy of avian coccidia and some companies undertook more fundamental work on the biology of the parasites. The significant volume of scientific activity in the commercial sector directed towards chemotherapy (see, for example, Reid [1970], who set the scene for the proceedings of a symposium on anticoccidial drugs in 1969) was complemented by a large body of research in the academic sector in which the focus was more on the biology of the parasites—a flavour of which can be found in the publication edited by Hammond & Long (1973).

In broad terms, however, many of the laboratories that contributed to the very early work on the avian coccidia in the 1920s/1930s had relinquished their interest in this field by the 1970s and the mantle for research had passed to new institutions (government, university and private). These new laboratories had, for example, the floor-pen facilities for practical studies on control of coccidiosis and/or the resources and specialized buildings and rooms necessary for handling coccidial parasites free from extraneous contaminants in order to undertake critical studies on the basic biology of the organisms. Examples of government-funded laboratories included the Houghton Poultry Research Station (HPRS) and the Central Veterinary Laboratory at Weybridge, both in the UK; INRA, Tours-Nouzilly and Institute d'Elevage de Pathologie et d'Hygiene Alimentaire, Ploufragan (France), the Animal Parasite Diseases Laboratory at Beltsville and the Department of Poultry Science at the University of Georgia, both in the USA. Laboratories of commercial companies included those at Eli Lilly and Company, Merck Sharpe and Dohme, American Cyanamid, Dow Chemicals (all in the USA) and May and Baker (UK).

Since the 1970s, the evolution of research laboratories working on coccidiosis has continued. For example, academic research on coccidia has left the HPRS (later the Institute for Animal Health) and moved to the Royal Veterinary College, London and coccidiosis research at the Central Veterinary Laboratory is minimal. However, new laboratories with programmes on coccidia have appeared, such as those in the Universities of Kebangsaan (Malaysia), Sao Paolo (Brazil) and University of Technology Sydney (Australia). Similarly, most of the commercial companies active in coccidiosis research in the 1970s have given up their interests in the past 40 years through strategic changes and/or takeovers, so in 2012 very few of the big players of the 1970s now have a commitment to coccidiosis.

The scientists

In the 1970s, a new generation of eminent Eimeria researchers had taken over from some of the greats of the late 1920s and early 1930s, and the list of luminaries included Peter Long, Elaine Rose, John Ryley and Len Joyner in the UK; Thomas Jeffers, David Doran, Michael Ruff, Chin Chung Wang, Larry McDougald and W. Malcolm Reid in the USA; Aggie Fernando in Canada; Peter Bedrnik in the Czech Republic; Tamara Beyer and Theresa Shibalova in the USSR; and Peter Yvore in France. These mainstream avian coccidiologists also interacted with other leaders in coccidiosis research, including Datus Hammond, Clarence Speer, J.P. Dubey, Ron Fayer and Erich Scholtyseck (to name only a few).

A new generation of coccidiologists, some of whom benefitted from the mentoring of those above, and who were active for the next 30 years or more, was also entering the scene in the 1970s and included Pat Allen, Patricia Augustine, Harry Danforth, Dennis Schmatz (all in the USA), Ray Williams, David Chapman and Martin Shirley (in the UK).

It should be noted that the work in both the commercial and academic laboratories was carried out in a great spirit of cooperation and that the 1970s were further characterized by three international conferences and symposia on most aspects of coccidiosis control in the field and basic research on the parasites.

Some research in the 1970s

Research into avian coccidiosis in the 1970s may be viewed as a golden period, especially in relation to the practical control of coccidiosis and the improved methodologies to evaluate its impact, changing prevalence and emergence of drug-resistant populations in response to the increasing importance of chemotherapy. An active commercial sector was nicely complemented by substantive academic programmes and, in combination, a broad range of topics was being studied. Within the academic sector in the 1970s it was typical for researchers to continue to address big questions around the host–parasite relationship that were considered in earlier years. Most of these questions still provide a backdrop to the current work of many molecular and cellular biologists, who now have the tools to dissect the biology of Eimeria spp. and gain the detailed insights and nuances of parasite behaviour that were never possible 40 years ago with a limited range of technical and analytical methodologies.

A few typical examples of the sort of questions being posed in the 1970s are as follows:

•	What laboratory studies are best for evaluating the efficacy of new drug candidates?
•	What laboratory approaches will be useful in determining prospects for drug-resistance in the field?
•	How can the efficacy of anticoccidial drugs be best determined in the field?
•	How do parasites become drug resistant?
•	Can novel drug-resistance phenotypes be produced by genetic recombination?
•	How do the parasites establish the infection process in the host?
•	How do the parasites become intracellular?
•	Why are the parasites host and site specific?
•	How do the organelles function and what do the organelles look like?
•	What are the mechanisms of protective immunity?
•	What are the differences between the innate and adaptive immune responses?
•	What are the roles of Paneth, goblet and crypts cells in infection?
•	Is protective immunity absolutely species specific?
•	What are the roles of host genetic factors, age and immune status with regard to outcome of infection?
•	What is the biochemical make-up of Eimeria spp.?
•	How can the parasites be best identified?
•	Can lines of Eimeria tenella be maintained by serial passage through the chorioallantoic membranes (CAMs) of embryonated eggs?
•	Is the prepatent time a genetically stable trait?

Chemotherapy

Chemotherapy against coccidiosis was under very active study in the 1970s by many of the leading coccidiologists in the USA, including W. Malcolm Reid (University of Georgia), Allen Edgar (Auburn University), Don McLoughlin (USDA, Beltsville), Emanuel Waletzky (American Cyanamid Company, and a lead scientist for the introduction of robenidine), and Ray Schumard and Maury Callender (Eli Lilly and Company, and leads for the introduction of monensin). Peter Long (HPRS), Len Joyner (Weybridge) and John Ryley (ICI) led some of the UK research. These scientists collectively contributed to many aspects of chemotherapy, including the introduction of new anticoccidial drugs; analyses of drug resistance; the genetics and transfer of drug resistance; and methodologies around lesion scoring and oocysts counting procedures to evaluate control in the field and the development of immunity in medicated birds. This was truly an exceptional period, and the technical advances pioneered 40 years ago remain in use today and have not been altered significantly. Much of the typical progress around this time may be gleaned from the 1970 proceedings of an international gathering that met in May 1969 at the University of Georgia, USA to consider the “Methodology for the development, selection, and testing of anticoccidial drugs for use in controlling coccidiosis” (introduced by Reid, 1970). Academia-based research in the 1970s was also defined by the early careers of David Chapman (HPRS, UK and University of Arkansas, USA) and Larry McDougald (University of Georgia), who between them were to make significant contributions for the next 40 years to the practical understanding of chemotherapy, including the sensitivity of contemporaneous field isolates to various drugs, importance and role of immunity in drug-medicated birds and the emergence of drug resistance under field and laboratory conditions.

July 1971 was highly notable for the paradigm-changing introduction of the polyether ionophorous antibiotic, monensin, by Eli Lilly and Company. This product abruptly transformed the control of coccidiosis in the worldwide intensive poultry industry and, 40 years later, the ionophores still constitute the main method of chemotherapy against the avian coccidia. The success of monensin, comprehensively reviewed by Chapman et al. (2010), was almost instantaneous and prompted the discovery of other ionophores within Eli Lilly and Company and elsewhere, and, perversely, probably contributed to the longer-term demise of coccidiosis research within the commercial sector because a “blockbuster” anticoccidial drug became the “norm” and nothing less than another de novo blockbuster was likely to be considered a viable proposition. Three further new anticoccidial drugs were introduced in the 1970s—namely, Robenidine, Statyl and Lasalocid (the latter being an ionophore)—and the reader is referred to the timeline in the review by Reid (1990) for the history of the control of coccidiosis. The last new anticoccidial drug (of any class) was introduced in 1989 and, where research impinges on the identification of new chemotherapeutic leads, a critical early question is still “How might the performance of any new compound compare with that of the ionophores?”A short history of anticoccidials has been written recently by Jeffers (2011).

Basic biology

When reflecting on the science being undertaken in the 1970s, it is appropriate to acknowledge the pioneering studies carried out in the 1920s and 1930s by Walter Johnson at the Oregon State University Experiment Station and Ernest Tyzzer at the Harvard Medical School in the 1920s (Johnson, 1923; Johnson, 1923/1924; Tyzzer, 1929; Tyzzer et al., 1932) upon which the 1970s research on the species and strains of Eimeria was still building. Anyone with an interest in the biology of the avian coccidia is encouraged to read the two seminal texts by Tyzzer (1929, 1932) because they give wonderful descriptions of the parasites, contain exquisite drawings and provide data that remain relevant today. In addition, see also the review by Chapman (2003).

In order to undertake critical studies on pure parasites, both Johnson and Tyzzer appreciated the importance of isolating species from single oocysts. This approach remained unchallenged technically until the 1970s when, in studies to determine the basis of clonality in the Eimeria, lines were propagated from infections with single sporozoites (for example, Shirley & Millard, 1976). These lines constituted the first true clonal populations of Eimeria and this cloning approach was to prove useful in later decades, both as part of a mapping strategy to determine the molecular basis of specific genetic traits (for example, Shirley & Harvey, 2000) and for the derivation of pure lines of the Houghton strain of E. tenella prior to whole genome sequencing in the 2000s (for example, Ling et al., 2007) and reviewed by Chapman & Shirley (2003); see also below.

The 1970s also saw some of the first major attempts, both in the academic and commercial sectors, to understand more about the biochemical workings of the coccidia. Leaders of the field, typically based in the commercial animal health sector, were Wang and colleagues (Merck Institute for Therapeutic Research), Smith and colleagues (at Eli Lilly and Company) and Ryley and colleagues (ICI, UK). These researchers contributed significantly, both to fundamental knowledge of Eimeria biochemistry and physiology (often in the context of understanding interactions of the parasite and anticoccidial drug) (reviews by Ryley, 1973; Wang, 1982) and to the introduction of numerous methodologies for the separation and purification of different lifecycle stages—most of which have stood the test of time. One strand of biochemical research was to gain an understanding of the mode of action of different anticoccidial drugs, the greatest success of which was with the ionophores (see review by Chapman et al., 2010). In brief, the ionophores interfered with the ionic balance across the parasite surface membrane and accumulated in the extracellular stages (for example, Smith & Strout, 1979), always allowing a few parasites to complete their lifecycles. This unique interaction was later shown to underpin the slow emergence of (incomplete) resistance and the contribution of immunity to control in the presence of ionophore-based chemotherapy.

Work on biochemistry in the 1970s was also interlinked with further attempts to improve and refine the in vitro growth of parasites, typically E. tenella, from which access to highly defined host–parasite systems gave “cleaner” biochemical data. In practice, the analyses were generally restricted to the early portfolio of events following host cell penetration because growth of parasites in vitro worsened with increasing time of development. Much credit should be accorded to several scientists, such as David Doran, who continued to challenge, probe and enhance the experimental systems for the culture of different species and strains of Eimeria in cell culture throughout the 1970s.

The ultrastructure of Eimeria was another important area of fundamental study during the 1970s; again tapping in to some of the advances in cell culture to derive images of the parasites that would have been far more difficult to obtain in vivo. Teams led by John F. Dubremetz (France), Bill Chobotar (USA) and Erich Scholytseck (Germany) were especially active and their work provided an excellent complement to that of the biochemists, so that by the end of the 1970s the structures and functions of many of the parasite organelles were generally known and more specific impacts of some anticoccidial drugs on the growing parasites were better understood.

Vaccinology: growing parasites in embryonating eggs

Control of avian coccidiosis worldwide in the 1970s was almost exclusively reliant upon the introduction of a series of prophylactic anticoccidial drugs. However, in some countries the vaccine “Coccivac” was also being used (albeit on a small scale); the history of this product has been reviewed comprehensively by Williams (2002). The natural virulence of the wild-type strains that comprised the live vaccine was a relative weakness and some of the work in the 1970s was directed towards the derivation of a set of live attenuated strains representative of, at the very least, the major economic species of Eimeria.

A substantial body of work led by Peter Long in the UK in the 1970s was directed towards addressing the interlinked questions “Can lines of E. tenella be maintained by serial passage through the CAMs of embryonated eggs?” and, if so, “Are they different to wild types?” and, if so, “Might they have utility as live vaccines?”

This body of work built on research started by Peter in the previous decade to consider the broader aspects of host and site specificity and an early consideration of factors that might influence the parasite phenotype. Most practical progress was made with E. tenella and steady improvements in the yields of oocysts with increasing passage were coupled with a steady decrease in the pathogenicity of the emerging lines for the host embryo, defined by: the emergence of a line characterized by excellent growth and production of very large numbers of oocysts in the CAM; and attenuation of virulence through the emergence of small second-generation schizonts that developed only in the most superficial layers of the CAM to replace the large and deeply located schizonts that defined the wild-type parent strain. In a complementary manner, growth of the embryo-adapted line in the chicken decreased with increasing passage in the CAM, and the phenotype of a line that had undergone around 70 passages in the CAM was identified as a laboratory candidate for the first live attenuated vaccine against coccidiosis. Indeed, small-scale laboratory trials with the TA line of E. tenella gave highly encouraging results but, whilst no practical outcomes were to define the UK work, the approach of serial passage of E. tenella in embryonated eggs later found utility in the hands of Peter Bedrnik in the Czech Republic, who in the late 1980s introduced the live attenuated vaccine “Livacox” that contained an egg-adapted line of E. tenella (for example, see Shirley & Bedrnik, 1997).

Whilst the work to derive egg-adapted lines was later expanded and was successful with Eimeria necatrix, Eimeria mitis and Eimeria brunetti (although more limited), the consistent failure to serially passage Eimeria acervulina, Eimeria maxima and Eimeria praecox meant that the approach was ultimately flawed and would not provide a way forward in the selection of all lines of Eimeria necessary for a new vaccine. However, the significant vision of Peter Long in the 1970s who, along with some others, believed that vaccination with safe, attenuated, lines of Eimeria spp. was the long-term way forward for the control of coccidiosis, both kick-started and maintained the necessary momentum in this new area of research that would later lead to the selection of a full set of precocious lines that now dominate the portfolio of attenuated vaccines used worldwide.

Despite the success in the 1970s of establishing a complete lifecycle of E. tenella and some other species within the CAM of embryonated eggs, related attempts to maintain the parasites in cell culture generally floundered and, by and large, the questions of “Why are Eimeria spp. host and site specific?” and “What factors influence this phenotype?” still remain to be answered beyond some of the broader principles.

Vaccinology: pioneering research and precocious lines

The entry into the marketplace in the late 1980s of the first live attenuated vaccines derived from work by the author (M.W.S. with Vincent McDonald) and by Peter Bedrnik (Czech Republic) and built on a truly significant piece of work around the middle of the 1970s by Thomas Jeffers (Jeffers, 1974, 1975; McDougald & Jeffers, 1976). Tom Jeffers, working with E. tenella, investigated the “plasticity” of the trait of prepatent time (interval of time between administration of infective oocysts to a susceptible chicken and the first appearance of the new generation of progeny oocysts), the genetic stability of the trait and, subsequently, the utility of the selected parasites as vaccine candidates. The efforts of Tom Jeffers to serially passage the first oocysts to be produced during serial infections were rewarded with the derivation of the first ever “precocious lines” characterized by traits that included an accelerated lifecycle, a marked attenuation of virulence (as a consequence of smaller second-generation schizonts) and a retained immunogenicity. These innovative studies laid the basis for the subsequent extensive work on all seven species of avian Eimeria done elsewhere in the 1980s and for the introduction of the first live attenuated vaccines that are used so successfully today. The pioneering contributions of Tom Jeffers to studies on Eimeria biology and control are described by Chapman in an accompanying review (Chapman, 2012).

Vaccinology: immunology

The need to better understand host immune responses to Eimeria was part of the vaccination story and much emphasis was placed on understanding the host–parasite immunobiology—a daunting task because of the complex antigenicity of the different intracellular lifecycle stages and their antigenic diversity. Once inside cells, coccidian parasites undergo an initial asexual lifecycle and present to the host immune system a continuously changing portfolio of antigenic challenges. Therefore, discerning the relevance of individual components of this intricate immune response to the outcome of infection was very challenging in the 1970s, especially in the absence of defined genetic knock-out chicken models for avian coccidia. Moreover, any rational development of vaccines against coccidiosis was limited by lack of immunoassays to identify vaccine correlates, a relative absence of information on Eimeria genetics, and the unavailability of suitable model systems to dissect the various immune components of poultry. Furthermore, a lack of methodologies to assess innate and adaptive immunities in poultry restricted the progress of basic immunology research.

During the 1970s the most active group studying host immunity in avian coccidiosis was led by Elaine Rose and colleagues at HPRS (UK). Many important questions on immunity to avian coccidia were formulated by Rose, who tackled them with meticulous experimental designs and incorporated murine gene knock-out animal models where necessary. For example, the results of her work shed important new light on the protective immune mechanisms of coccidiosis (for example, Rose, 1972; Rose et al., 1979) and, indeed, much of the work by Rose and colleagues laid the foundation for future research on host–parasite immunobiology that has been undertaken by many international groups.

One of the features of host immunity to avian coccidia is production of parasite-specific serum antibodies, as described in the 1960s, but with some confusion at the time about their role in protective immunity. By the 1970s, the early immune response to coccidiosis was known to be characterized by a serum antibody response that includes at least three different isotypes, IgM, IgA, and IgY, the latter being considered the orthologue of mammalian IgG. Clear evidence that antibodies were not necessary for protective immunity against coccidiosis came from studies in B-cell immunosuppressed chickens (for example, Rose & Hesketh, 1979). With the development of enzyme-linked immunoassays, an anti-Eimeria antibody response was identified using samples from serum, bile and intestinal washings, leading to characterization of the kinetics and isotypes of the antibodies produced against different species of Eimeria. Furthermore, with evidence for the presence of a secretory-type antibody in the intestine of Eimeria-infected chickens, the role of IgA in coccidiosis was investigated.

Another hallmark of host immunity to coccidia came with the study of local inflammation in the gut—a complex innate immune response that results from a multifaceted interaction of many types of immune cells (macrophages, dendritic cells, mast cells, natural killer cells, basophils and eosinophils), non-lymphoid cells (goblet cells, Paneth cells, enterocytes, enteroendocrine cells, and epithelial cells) and their secreted soluble effector molecules (cytokines, chemokines, defensins, leukotrienes, prostaglandins, etc.). Although the role of many different types of lymphoid as well as non-lymphoid cells in coccidiosis was not well understood in 1970, it was clear that local inflammatory responses in the gut contributed to the alteration of gut integrity and that the resulting structural damage was detrimental to the host. Many reports demonstrated that the level of inflammation depended on the strain and species of Eimeria, the infecting dose, as well as host age, immune status (normal vs. immunosuppressed) and the genetic background.

A better understanding of host immunity to coccidia complemented strategies to derive live attenuated vaccines. Comparison of immune responses with “single infections” versus “trickle” infections (giving low doses of parasites, such as 50 oocysts per day, over the first 1 to 2 weeks of life) showed that a trickle immunization method induced slightly higher IgA responses. Unfortunately, other than a morphological description of local gut changes due to infection, there was very limited information on intestinal cell-mediated immunity, which was difficult to measure in the absence of established methodologies and immunological reagents. Delayed-type hypersensitivity, a crude measurement of cell-mediated immunity, was measured by the wattle swelling response, and in the late 1970s Rose showed that there was a reciprocal relationship between serum antibody levels and delayed-type hypersensitivity. Much of the indirect evidence for local cell-mediated immunity in the Eimeria-infected gut was subsequently obtained from pathological and structural changes observed by Aggie Fernando (Canada) in the early 1980s, which included intestinal crypt hyperplasia, villous atrophy, and mast cell and goblet cell proliferation. The role of T cells in local gut inflammatory responses was confirmed from studies that demonstrated a lack of protective immunity in athymic rats (Rose & Hesketh, 1982). Much of the work done by Rose and her colleagues showed clear evidence that a wide spectrum of host immune responses at the local and systemic levels was elicited during coccidiosis. The questions concerning the importance of different arms of immunity in protection against coccidiosis (i.e. humoral vs. cellular), as well as the function of various T-cell subpopulations, was beginning to be addressed using these poultry model systems (Rose, 1972).

Vaccinology: better identification of species and strains and composition of new live vaccines

Until the mid-1970s, the criteria used to identify the avian coccidia were virtually unchanged from those described in the 1920s. The introduction of a more objective methodology in the 1970s to analyse genetic variation in electrophoretic mobility of enzymes paved the way for more critical analyses of laboratory-held stocks of Eimeria (for an early review, see Shirley & Rollinson, 1979).

From this work, the number of recognized species of Eimeria from the chicken was revised down to seven; this revision is generally accepted today but still a topic of controversy in some laboratories. Greater clarity on the identity of species proved to be of considerable practical benefit in all subsequent decades when new live vaccines could be registered as containing defined lines and/or strains representative of specific economically important species; including E. mitis, whose taxonomic status was re-established from work started in the mid-1970s.

Work in the 1970s also laid the foundation for an ongoing consideration in the formulation of live vaccines—namely, the typical inclusion of two strains of E. maxima in order to protect chickens against strains in the field characterized by a spectrum of “antigenic diversity”. As first demonstrated by Long & Millard (1979), the most effective solution to this potentially very important problem is the inclusion within a vaccine of two populations of E. maxima that represent “extremes” of antigenic diversity. For example, the two strains included within Paracox-5 in the late 1980s were chosen for their ability to complement each other immunologically and to protect chickens exposed to a range of immunological unrelated strain(s)—a scenario that has so far stood the test of time.

Some scientific progress since the 1970s.

For the past 40 years, chemotherapy has remained the absolute heart of coccidiosis control in the field and its importance to the success of the modern poultry industry cannot be over-estimated. Much excellent work on the emergence, transmission and stability of drug resistance, and so forth, has underpinned this highly effective approach to practical control (for example, see reviews by Chapman 2000, 2001), but our greater focus in the following sections will be on laboratory-based research directed towards an understanding of the basic biology of the parasites.

The 1980s

The “molecular” era of coccidiosis research really began to take off in the 1980s. A new generation of coccidiologists was entering the field and introducing a variety of molecular studies to investigate the biology of Eimeria spp. at an entirely new level of detail and, when set against the bigger picture of control, to work towards deriving vaccines based on defined antigens or to identify new targets for chemotherapy.

Monoclonal antibody technologies were a starting point in several laboratories as an entree to identifying molecules of different lifecycle stages critically involved in features such as invasion of the host cell, site specificity, endogenous development and induction of (protective) immune responses (for example, see Danforth, 1986). A second strand of work in these and other laboratories was often the manipulation of Escherichia coli as an expression vector, both to augment studies to identify and express candidate antigens and their subsequent administration and evaluation for vaccine efficacy in chickens.

Most research activities aligned to mainstream immunology in 1980s remained focused on addressing fundamental mechanistic questions around the role of host innate and adaptive immunities and the nature of protective immunity during infection. Many further questions were related to host and site specificity of infection, the role of the host genetic background in parasite transmission, species specificity of protective immunity and the different immunological characteristics of primary and secondary immune responses to coccidia. As an immunologist joining the coccidiosis group at the USDA in 1984, one of the authors (H.S.L.) found it quite daunting to study immunity because of the limited availability of immunological assays and immune reagents for poultry.

In the 1980s, most immunology research was still done by Rose and colleagues at HPRS, with others entering the field, including Pascale Quere at INRA (France) and Arno Vermeulen (the Netherlands). One challenging goal for these researchers was to tease apart the immune mechanisms to determine whether a single response might protect the host against all species of Eimeria. However, most of the evidence indicated that Eimeria elicits species-specific immunity, which is not cross-protective—equivalent to what is seen in the field. Furthermore, E. maxima, known to be the most immunogenic of the poultry species, was shown to be defined by considerable antigenic diversity suggesting that an understanding of host immune responses to each Eimeria species would probably be needed for the design of novel immunological interventions.

Studies on Eimeria-specific T-lymphocyte activation to selectively suppress T-cell immunity in the 1980s led to many findings that demonstrated the abrogation of protection in T-cell-deficient chickens, but not in B-cell-deficient chickens (Lillehoj, 1987). Additional evidence for the protective role of T cells came from adoptive cell transfer studies in which peripheral blood lymphocytes and splenocytes from E. maxima-immune chickens protected syngeneic recipients against a live parasite challenge infection (Rose & Hesketh, 1982). With the development of poultry lymphocyte-specific immune reagents in the late 1980s, the nature of T lymphocytes involved in host–Eimeria interactions became clearer. Flow cytometric analyses of intestinal intraepithelial lymphocytes obtained from the Eimeria-infected chicken gut showed that coccidial infection of naïve and previously-infected chickens elicited innate and adaptive immunities, respectively, and identified the many different types of intestinal lymphocytes responsible for local defence against the parasite (Lillehoj, 1998).

Whilst there was a significant research effort on completely new approaches to vaccination in the 1980s, work on more traditional live vaccines was making greater progress. Precocious lines of all species were first derived in the 1980s (see above) and the first live attenuated vaccines, namely Paracox^® and Livacox^®, based on these parasites were introduced within Europe and elsewhere. One noteworthy aspect of the use of these live vaccines (and some others that followed) is that they contain drug-sensitive parasites (derived from laboratory strains that were isolated before the onset of global chemotherapy) and their widespread use thus helped restore drug sensitivity in the field—a benefit that may be unique in the field of microbiology.

In a separate study, Wallach and colleagues (Pugatsch et al., 1989) began to demonstrate that antigens recovered from the sexual stages of E. maxima and injected with an adjuvant could protect the young offspring of vaccinated hens against coccidial infections; that is, through the passively transferred maternal IgG-mediated immunization (Wallach et al., 1995). Protection afforded by this strategy was wider than with live vaccines (chickens vaccinated with antigens from E. maxima alone were protected against subsequent challenge with other species) and in the 1990s the first sub-unit vaccine against coccidiosis, Coxabic^®, was introduced.

The 1990s

Ground-breaking molecular and cellular studies over the next 20 years were started in the late 1980s/early 1990s by Fiona Tomley at the Institute for Animal Health (IAH, UK), and the data and techniques that have been derived now have the potential to deliver a new generation of vaccines (see later).

Throughout the 1990s, Fiona Tomley and colleagues interrogated the biology of early lifecycle stages and especially the structure and molecular organization of surface membranes and apical (secretory) organelles of sporozoites and merozoites, including the sequence and structure of genes encoding key proteins.

An impressive portfolio of knowledge, reagents and techniques was introduced from studies on E. tenella and the powerful combination of, for example, subcellular fractionation techniques to isolate microneme and rhoptry organelles (for example, Kawazoe et al., 1992) together with molecular cloning and sequencing led to a far greater understanding of the role(s) of Eimeria proteins during invasion (for example, Tomley, 1994; Tomley et al. 1991, 1996).

Work on molecular karyoptypes for Eimeria was started in the laboratory of one of the authors (M.W.S.) and 14 chromosomes ranging in size between 1 and >6 Mbp DNA were identified in E. tenella (Shirley et al., 1990). This work proved to be a considerable catalyst for the later genome sequencing studies (see below).

Immunology

In the 1990s, host–parasite studies were focused on identifying and characterizing the chicken immune effector molecules involved in coccidial infection. For example, identification of the subpopulations of intestinal lymphocytes that respond to infection in the gut led to the notion that locally produced cytokines and chemokines regulate the quality of host immune response to Eimeria. Most advances in understanding the nature of T-cell immunity in protection against coccidia continued from studies started by Rose and colleagues working with the natural host and genetically-defined lines of mice and, for example, they showed that the activation of local dendritic cells and macrophages elicited a diverse array of chemokines and cytokines that initiated and amplified host immune pathways (for example, Wakelin et al., 1993). It became clear in coccidia-immune hosts that parasites entered the gut early after infection but were prevented from further development—indicating that acquired immunity to coccidiosis most probably involved mechanisms that stopped the natural progression of parasite development. For example, cytotoxic T lymphocytes expressing the CD8 antigen markedly increased in White Leghorn chickens soon after a primary infection with E. tenella (Breed et al., 1996, 1997), which was accompanied by increased interferon-γ production. It was later shown that interferon-γ inhibits intracellular development of Eimeria (Lillehoj & Choi, 1998).

Vaccinology

In the 1990s (and 2000s), further live attenuated vaccines were introduced in many different countries (e.g. ADVENT^®, Eimerivac^®, Eimeriavax^®; Gelcox^®, Inovocox^®, Nobilis^®, CoxATM^®, etc.) and they were characterized by variations of numbers of species (typically three to eight) with one or two lines of E. maxima (the most antigenically diverse species). The identification of species and strains was also progressing, and work in the 1990s switched from electrophoretic variation of isoenzymes to electrophoretic polymorphisms in DNA—finally giving coccidiologists access to techniques that could be used rapidly and unequivocally to identify small numbers of parasites in laboratories with standard molecular apparatus. This work progressed further in the 2000s and a variety of definitive identification methods were described, especially by Gruber and colleagues in Brazil (for example, Fernandez et al., 2003).

Host genetics of avian coccidiosis

As early as the 1950s, there was clear evidence for genetic variability in susceptibility to coccidiosis among genetically-divergent chicken lines. Heritability of coccidiosis resistance and susceptibility was confirmed later by the ability to select for coccidiosis resistance traits (Rosenberg et al., 1954; Johnson & Edgar, 1982). In the 1980s and 1990s, Bumstead and colleagues at IAH (UK), Pinard-Van Der Laan at INRA (France) and one of the authors (H.S.L.) actively investigated the genetics of avian coccidiosis using inbred, outbred and B-congenic chickens, the latter differing only at the major histocompatibility complex. In developing a genetic selection strategy, the USDA group applied DNA marker technology to identify quantitative trait loci and genes underlying disease resistance (for example, Zhu et al., 2003) and were the first to identify a coccidiosis quantitative trait loci, on chromosome 1, associated with disease resistance. With the availability of a complete chicken genome sequence in 2004 and relevant high-throughput gene expression technology shortly thereafter, quantitative trait loci analysis was combined with gene expression analysis to identify candidate genes associated with coccidiosis resistance (Lillehoj & Hong, 2008). Further fine mapping and gene expression analyses led to the discovery of several novel single nucleotide polymorphism candidate markers for coccidiosis resistance for potential application to the development of marker-assisted selection strategy in the future (Kim et al., 2010).

The 2000s

The 2000s are important to the history of coccidiosis research because of significant advances in studies on genomes and genetics and the allied development of technologies for manipulating genes, including their transfer between coccidia.

An especially significant collaboration in the 2000s was the close integration of several coccidia laboratories worldwide, together with the Sanger Institute in Cambridge, UK to form an “Eimeria Genome Consortium” working to derive a sequence for the genome of E. tenella. The collaborators included Martin Shirley and Fiona Tomley (IAH/RVC), Kiew-Lian Wan (University of Kebaansan, Malaysia) and Arthur Gruber (University of Sao Paolo, Brazil). Some of the data from this large body of work have now been published (for example, Ling et al., 2007) and all of the sequence data are placed within the public domain to provide a rich resource for future generations of protozoologists and others. In brief, the E. tenella sequencing project at the Sanger Institute derived and/or confirmed the following top-level data:

•	A genome of approximately 60 MB in size.
•	A GC content of approximately 53%.
•	Two major ribosomal DNA clusters, which account for ~2.5% of the genome.
•	Abundant tracts of repeats of the nucleotides GCA in coding and non-coding regions.

Transcriptome sequencing is now being used to both improve the annotation of the genome and to examine changes in gene expression between different life-stages. Moreover, work is ongoing in the laboratories of the current collaborators to extend the work to other avian species of Eimeria.

Work led by Fiona Tomley on the molecular and cellular biology of invasion and parasite development continued apace. Apical organelle proteins were newly identified and further characterized (for example, Tomley et al., 2001; Bromley et al., 2003; Periz et al., 2007) from a variety of approaches, including proteomics studies performed in combination with data derived from the E. tenella genome sequencing project.

The laboratories of Fiona Tomley and Damer Blake at the Royal Veterinary College (UK), with further collaboration from Adrian Smith (University of Oxford), are looking at the vaccine potential of several proteins from apical organelles and definitively immune-protective molecules of E. maxima rationally identified within the genome of E. maxima (Blake et al., 2004, 2011). The latter strategy uniquely combined parasite genetics, DNA fingerprinting and the use of two selectable biological markers (drug resistance and strain-specific immunity); amongst other findings, the likely involvement of an E. maxima homologue of apical membrane antigen-1 was identified.

The different strands of work to identify protective antigens are now being brought together through the pioneering advances of Eimeria transfection technologies by Fiona Tomley and colleagues (for example, Yan et al., 2009). These powerful methodologies continue to take to a new level the ability of coccidiologists to evaluate the role and utility of critical gene products, including: those that are involved with parasite motility and invasion of the host cell; those contributing to the structure and function of sub-cellular organelles, especially rhoptries and micronemes; those responsible for traits such as virulence and host specificity and lifecycle differentiation through the different asexual and sexual stages; and those involved in induction of protective immune responses.

In the early 2000s, extensive experimental evidence existed to support the notion that immunity mediated by lymphocytes and their secreted products led to antigen-specific protection against challenge infection with Eimeria (see review by Lillehoj, 1998), and the discovery of several chicken cytokines homologous to mammalian counterparts reinforced this connection. The application of high-throughput sequencing of intestinal-expressed sequence tag cDNA libraries from Eimeria-infected chickens (Min et al., 2005) quickly led to the identification of many further cytokines, such as interleukin-16 and interleukin-17, which were elevated in Eimeria-infected tissues indicating their potential role in regulating local immune responses to coccidia. Moreover, recent studies with quantitative real-time polymerase chain reaction techniques have identified more than 30 different cytokines and chemokines involved in coccidia infection to further illustrate the complexity of the host immune response to Eimeria (Hong et al., 2007).

The availability in late 2000 of a complete sequence for the poultry genome, along with various tissue-specific chicken microarrays, has recently led to large-scale functional genomic analyses of chickens. Gene expression analyses using microarrays have become powerful tools to evaluate the complexity of host–pathogen immunobiology and, most specifically, genomics technologies combined with immunology (immunogenomics) have enabled in-depth analyses of complex immunological processes based on large-scale genomic approaches. The laboratory of the author (H.S.L.) developed the first chicken intestinal cDNA array (Min et al., 2005), which was used to reveal differential local gene expression profiles in host innate and adaptive immune responses to different Eimeria species. Most recently, a second-generation avian intestinal intraepithelial lymphocyte cDNA microarray was constructed and revealed that primary infection with Eimeria spp. significantly modulated the levels of mRNAs for genes involved in innate immunity and the metabolism of lipids and carbohydrates genes (Kim et al., 2011), consistent with the gut damage caused by the asexual replicative phase of the parasite lifecycle. Future gene expression analyses will lead to the comprehensive identification of immune-related transcripts and their immune pathways that are modulated during infection, together with other important genes related to cellular metabolisms that are perturbed during coccidiosis.

Molecular vaccines

In 2000, molecular vaccine technology was applied to coccidiosis by several laboratories. Unlike protein-based vaccines, DNA vaccines comprise genes encoding immunogenic proteins of pathogens rather than the proteins. They are administered directly in conjunction with appropriate regulatory elements (i.e. promoters, enhancers), thus permitting the encoded protein to be expressed in its native form and presented in the context of MHC class I and II. They are thereby recognized by the host's immune system in a manner that simulates natural infection. Immune protection manifested by significantly reduced faecal oocyst shedding in chickens vaccinated with a DNA vaccine encoding E. acervulina profilin protein was observed (Lillehoj et al., 2005) and greater protection was obtained when the vaccine was administered with cDNAs encoding chicken cytokines such as interferon-γ or interleukin-2.

Although various levels of protective immune response have been achieved using recombinant vaccine technologies, better definition of protective epitopes is still needed to identify proteins that are specific to different stages of the parasite lifecycle, including those involved in invasion, survival, virulence, pathogenesis, and transmission. Recent studies of recombinant proteins expressed by sporozoites, merozoites or gametocytes and DNA preparations have shown promise as vaccine candidates, but have yet to be proven in commercial applications. A highly conserved sporozoite and merozoite Eimeria profilin protein that is a ligand for Toll-like receptor is a promising vaccine candidate since it stimulated strong cell-mediated immunity and induced protection against a live coccidial challenge when injected in ovo (Lillehoj et al., 2005).

Novel adjuvants

In 2010, a significant effect of the oil-based ISA 71 VG adjuvant was demonstrated in combination with the recombinant Eimeria profilin subunit antigen in broiler chickens when T-cell immune responses were measured (Jang et al., 2010). Furthermore, aqueous nanoparticle-based Montanide IMS 1313 N VG (IMS 1313) in combination with profilin augmented protective immunity against multiple Eimeria infections (Jang et al., 2011), and a new adjuvant complex comprising saponin Quil A, cholesterol, dimethyldioctadecylammonium bromide and the acrylic acid polymer Carbopol showed similar promise (Lee et al., 2010) and induced significant intestinal gene expression with the majority of altered transcripts related to the immune response (Kim et al., 2012). These latest studies have opened other doors for the development of recombinant vaccines against coccidiosis and illustrate the importance of elucidating the underlying molecular mechanism of vaccination.

The future

Critical control of coccidiosis will be required by the poultry industry in the future. The human population is set to rise to circa 9 billion by 2050 and chickens provide a major and increasing supply of the world's animal protein. It is hard to imagine disease control in the field without the use of anticoccidial drugs. Indeed, based on the huge global success of the “potentiated ionophore” strategy in which birds are medicated with a combination of an ionophore with a chemical anticoccidial drug (e.g. Maxiban), the incomplete resistance associated with ionophores, the benefit of protective immunity associated with use of ionophores, an ionophore-mediated control of bacteria (including Clostridia) and the very low unit cost of chemotherapy, it is probable that the current methods of control will continue unabated. Certainly, no new anticoccidial drugs are expected because the costs of R&D to introduce new veterinary drugs continue to increase and are typically prohibitive unless the new compound is guaranteed a large marketplace. Moreover, any new anticoccidial has to compete with the reality and reputation of the current drug strategies, and an additional problem is the likely emergence of drug resistance that could seriously threaten the considerable financial investment.

There are, however, increasingly negative political views towards in-feed medication of livestock (especially within Europe) and an overall more negative view on the use of prophylactic chemotherapy provides a significant spur for work on the immunological control of avian coccidiosis. In the USA, the market-leading vaccine in terms of sales is Coccivac B—a cheap product comprising wild-type strains of Eimeria that has been available for more than 50 years. The price of any new vaccines will continue to be a major consideration to the global poultry industry and any significantly higher price for the control of coccidiosis associated with vaccines will only be accepted if, for example, the use of drugs were ever to become more restricted.

Application of the recently described innovative and ground-breaking Eimeria transfection technologies by Tomley and colleagues (see above) may offer one chance of success on a grand(er) scale. These technologies are being allied to the results of other studies in the UK directed towards identification of the naturally protective antigens of Eimeria so that, in combination, there is the real prospect that transgenic lines of Eimeria can be derived which express the protective antigens of, at least, the most important species of avian coccidia.

New vaccines based on this approach might be considered as natural successors to those based on precocious lines because: the vaccine would be a live vaccine; a precocious line could serve as the “parent” to receive protective antigens; only a single line might be needed—making propagation easier and possibly reducing the overall cost; it might be possible to produce the vaccine on a much larger scale than hitherto; and the delivery method is optimal for the induction of protective innate and adaptive immune responses at the tissue sites where parasites invade and undergo intracellular development.

Evidence for conserved coccidia proteins that induce cross-protective immunity to multiple species of Eimeria in combination with other advanced vaccinology technologies, including novel adjuvants to overcome the weak immunogenicity of peptide antigens and that can target specific host immune cells (Lee et al., 2010; Jang et al., 2011), also offer further opportunities for new recombinant vaccines. New research leads will continue to open up as the genome data are further interrogated and their annotation improves and becomes more extensive. One focus is likely to be on the discovery of new conserved pattern-recognition motifs that can activate innate immunity against multiple strains of Eimeria spp.

In the future, the control of coccidiosis may become more sustainable and the feasibility of using natural products, such as antimicrobial peptides, hyperimmune IgY, probiotics and plant-derived phytochemicals, will need review. Furthermore, advances in bioinformatics should provide additional state-of-the-art tools for allowing rapid gene discovery for coccidiosis resistance for developing marker-assisted selection strategies. Genetic selection of disease-resistant chickens offers a powerful method of disease control, especially when this technology is combined with other strategies utilizing new molecular genetic and functional genomics tools. With rapidly developing technologies in functional genomics and computational biology, it is anticipated that new paradigms for coccidiosis control will be formulated. It is possible that the use of genetic tools could become one way of combating parasites, in synergy with other strategies of coccidiosis control such as vaccination, nutrition, and management.

Finally, in linking back to research in the 1970s at the time that Avian Pathology was launched, it is possible that some of the big questions being asked then—such as “How do the avian species locate their preferred sites of development within the intestine?”, “Why does asexual reproduction end and sexual reproduction begin?” or “What metabolic pathways do the parasites use through their lifecycles, both inside and outside of the chicken?”—might be answered in the coming years through both the application of new technologies and new reagents derived from analyses of the genome sequence. In another 40 years, as the world becomes ever more dependent upon poultry, coccidia will still require control and researchers will need to provide the farmers with a larger armoury of control strategies.