M. W. Shirley1 and H. D. Chapman2
1 Institute for Animal Health, Compton Laboratory, Compton, Newbury, Berks. RG 20 7NN, UK, shirley@bbsrc.ac.uk
2 Department of Poultry Science, University of Arkansas, Fayetteville, AR, 72701, USA.

Overview
For around 80 years, the amount of knowledge gathered on the avian coccidial parasites and the disease of coccidiosis has been impressive and research remains at the cutting-edge of what is being undertaken in Microbiology more generally. Research in to avian coccidiosis has been exceptionally well served and the starting point for knowledge gathering of the disease and the parasites of poultry stems from pioneering studies conducted in the 1920’s and 1930’s by investigators who, with limited experimental facilities, were able to establish many basic principles, such as life cycles, epidemiology and control of the Eimeria parasite, that are still relevant today.
In the years that followed, research was conducted at institutions in the academic, government, and private sectors with the specialized facilities, especially access to buildings that prevented chances of extraneous infections of the hosts. Much of the work in the early period focused on the obvious practical control of Eimeria spp. and was supplemented by the acquisition of much basic information on the biology of the parasites, including host-parasite relationships, genetics, immunology, and pathology and some of this information was to lay the foundation for the introduction of a series of strategies to control the parasites by vaccination of the hosts.
Building on the early discoveries, the control of coccidiosis by chemotherapy was introduced and, by eliminating mass outbreaks of disease, it became a critical lynchpin in the expansion of the international poultry industry. Today, chemotherapy remains the dominant method of control but the use of live vaccines has a similar long history through the early work of Allen Edgar and others. The dramatic attenuation of the life cycle of E. tenella, as first described by Thomas Jeffers in the mid 1970’s, was to lead to the selection of “precocious” lines of all species and these parasites, with their abbreviated life cycles and marked attenuation of virulence, proved to be a significant development as they led to a new generation of safe coccidiosis vaccines. Towards the end of the 20th century much interest was being directed towards the greater use of vaccination for the control of coccidiosis and, in contrast to an opposite trend in anticoccidial drugs – especially within Europe - many new products were introduced.
Some of the research conducted in recent years has helped to maintain work on Eimeria at the cutting edge of microbiological science and is providing a wealth of knowledge on the biology of the genome(s) of the parasite. A highlight has been a project to determine a whole genome sequence for Eimeria tenella and this work is currently nearing completion and being supplemented by many complementary studies focusing on gene expression. With access to a genome sequence, future researchers are now offered tremendous opportunities to investigate the biology of Eimerian parasites and, for example, seek those elusive protective antigens that will enable new vaccines and new drugs to be developed.
This short account of coccidiosis research during the past 80 years is intended to provide only a superficial skim and the names of most of the scientists who contributed to a large body of knowledge on the parasites and the diseases they cause will not be given. We will also omit the names of very many other coccidiologists who, whilst primarily studying other coccidia such as Eimeria species from other hosts such as Toxoplasma, etc., nonetheless have contributed much to the body of relevant science on the avian coccidia. Apologies to them all.

Solid beginnings
Present knowledge of the disease coccidiosis of poultry is founded firmly on pioneering studies carried out by Walter Johnson at the Oregon State University Experiment Station and Ernest Tyzzer at the Harvard Medical School in the 1920’s (Johnson, 1923; 1923/1924; Tyzzer, 1929; Tyzzer et al., 1932). Prior to that time coccidiosis was thought to be caused by just one species, Eimeria avium, that could infect many hosts (avian and mammal); clinical effects were confused with those caused by other organisms such as Histomonas and intestinal bacteria, and a range of effects were thought possible, ranging from leg problems to “white diarrhea” (see Chapman, 2003; 2004). Between them, Johnson and Tyzzer described most of the now recognized species, viz. E. acervulina, E. maxima, E. mitis, E. necatrix, E. praecox and E. tenella and correctly identified their specific lesions. They also considered the phenomena of site and host specificity; the self-limiting nature of the Eimerian life cycle; the relationship between magnitude of dose and severity of infection; the phenomenon of acquired immunity, and the epizootiology of coccidiosis infections. Although both Johnson and Tyzzer emphasized the importance of sanitation and hygiene in reducing environmental contamination with the infective stage of the parasites, they realized that eradication was an unrealistic goal for the control of coccidiosis and advocated exposure of the host to low numbers of oocysts to allow them to acquire protective immunity. Indeed Johnson took this further and at the end of his life was working on the possibility of immunizing birds by intentionally introducing controlled numbers of oocysts in the feed. In order to undertake critical studies on pure parasites, both Johnson and Tyzzer appreciated the importance of isolating species from single oocysts and of using sterilized cages sterility for the propagation of Eimeria infections (Tyzzer, 1932) and some of their methodologies form the foundation of modern procedures for working with these parasites (Chapman and Shirley, 2003).
Anyone with an interest in the biology of the avian coccidia is most definitely encouraged to read the two seminal texts by Tyzzer [Tyzzer (1929) Coccidiosis in gallinaceous birds. American Journal of Hygiene 10: 269-383, and Tyzzer et al. (1932) Coccidiosis in gallinaceous birds II. A comparative study of species of Eimeria of the chicken. American Journal of Hygiene 15: 319-393] because they contain wonderful descriptions of the parasites, exquisite drawings and data that are relevant today.

Consolidation
The efforts of Johnson and Tyzzer had put an understanding of coccidiosis on a solid footing, and in the years that followed their findings were widely communicated to the developing poultry industry. At this time the industry was progressing from small backyard flocks of a few dozen chickens to poultry farms with houses holding several hundred birds. The introduction of such large scale husbandry practices began to tip the balance in favour of the highly efficient transmission of coccidia between hosts and a rising incidence of disease was the spur for numerous, initially unsuccessful, studies to investigate possible remedies. Nutritional experiments were carried out at the Bureau of Animal Industry in Washington and elsewhere and pathological/physiological investigations were done at the University of Wisconsin. P. P. Levine at Cornell University made several important contributions, including the first description of E. brunetti, but it was his report of the activity of the compound sulphanilamide that was to determine a primary direction of coccidiosis research for the next few decades (Levine, 1939). Interestingly, Levine believed that use of drugs should be an adjunct to management and that medication should not take the place of proper husbandry (Levine, 1945), a viewpoint that is still relevant today. Levine’s work was the first of many studies in several countries in the 1940’s to define optimal conditions for the use of sulphonamides. The most significant study, however, that had the greatest impact on future control methods, was the demonstration by Delaplane and colleagues at the Rhode Island Agricultural Experiment Station that sulphaquinoxaline, administered at low concentrations in the feed, gave an effective control of coccidiosis (Delaplane et al., 1947).
This era of early research culminated in 1949 in the first conference entirely devoted to coccidiosis. It was organized by the New York Academy of Sciences (Anon, 1949) and leading researchers from government, university and industry in the USA attended, including one international delegate from the UK, Clifford Horton-Smith. The significance of the new era of chemotherapy was evident since half (11 of 22) presentations on poultry coccidiosis were concerned with drugs.

The 1950s-1970s
Increasing recognition of the significance of coccidiosis and its deleterious consequences for the developing poultry industry required a more detailed and better basic understanding of the nature of the parasites and the disease. In retrospect, it is remarkable how much was accomplished prior to the 1950s, since animal facilities at that time were limited and, although the chicken remains inexpensive and relatively easy to maintain, the ubiquity of coccidial oocysts and the consequent difficulty of raising birds in the absence infection introduced a major constraint upon research.
Many of the laboratories that contributed to the very early work on the avian coccidia had by now relinquished their interest in this field and the mantle for research was passed to those institutions (government, university, and private) with the resources and specialized facilities necessary for this type of research. Such laboratories included the Houghton Poultry Research Station (HPRS) and the Central Veterinary Laboratory at Weybridge in the UK, the Parasite Research Laboratory at Beltsville and the Department of Poultry Science at the University of Georgia in the USA
Some of the leading established scientists of the day were Peter Long, Elaine Rose, John Ryley and Len Joyner in the UK; 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; other luminaries they interacted with included Datus Hammond, Clarence Speer, JP Dubey, Ron Fayer and Erich Scholtyseck (to name only a few). A new generation of coccidiologists was also entering the scene in the 1970’s and included Pat Allen, Patricia Augustine, Harry Danforth, Thomas Jeffers, Dennis Schmatz and Ray Williams.
A broad range of scientific activities was undertaken at all the academic institutions and they included a fuller understanding of the life cycles (including descriptions of prepatent and patent times; endogenous stages), growth of parasites in vitro and in embryonating eggs, genetic recombination between strains, the nature of host immune responses and the immunizing abilities of the different parasite life cycle stages, pathology, chemotherapy, epidemiology and diagnosis of Eimeria infections. For many of the more fundamental studies, E. tenella became the most popular species for a number of reasons including its importance in the field, ease of propagation, high virulence and distinctive lesions, ability to grow cell culture and in the chorioallantoic membranes of embryonating eggs, robustness of the sporozoites for biochemical analyses, etc. A number of reference strains were introduced in to common usage from the 1950’s and it was the well characterized Houghton strain of E. tenella, derived from oocysts recovered from a chicken that was sent to the HPRS for post mortem investigation in 1949, that was chosen for the ongoing internationally-coordinated genome sequencing project (Shirley et al., 2004).
A defining characteristic of research on Eimeria most especially during the 1950’s to 1970’s was the duality of programmes in the government and university settings with those in the commercial sector. An obvious broad distinction between the two research environments was that academic scientists had a stronger focus on the biology of the parasites, with some long-term studies on immunity and the potential for developing new vaccines and those in the commercial sector were working towards the development of better anticoccidials. The introduction of modern broad spectrum anticoccidial drugs from research done within companies such as Merck and Eli Lilly was to have a profound effect on the control of coccidiosis and the recognition by Eli Lilly of the anticoccidial efficacy of the ionophores was to dramatically change prospects for the control of coccidiosis – an impact that remains to day. This period also saw some of the first attempts to introduce comprehensive biochemical studies of the coccidia, both in the academic and commercial sectors from which an understanding of the mode of action of the ionophores was to emerge. Sadly, the basis of how most other drugs exerted their lethal effects was not addressed and/or resolved to the same level of detail. Moreover, although relatively little biochemistry of the avian coccidia was to be done after the 1970’s, the availability of an annotated genome sequence for E. tenella now provides an enhanced opportunity for studies on metabolism, etc.
The 1960’s heralded the first work that examined the ability of Eimeria species to develop in cell culture and in the chorioallantoic membranes (CAM) of the embryonating egg. The findings that the life cycles of E. tenella and a few other species of avian Eimeria could be completed within the CAM following injection of sporozoites in to the allantoic cavity led to an evaluation of the effects of serial passage and, from that work, the emergence of the first attenuated lines that had potential as the basis of live attenuated vaccines.
Despite the success of a complete life cycle of E. tenella and some other species within the CAM, attempts to maintain the parasites in cell culture foundered and that specific challenge still remains today.
Whilst control of avian coccidiosis was being achieved through the introduction of a series of prophylactic anticoccidial drugs, the work of Allen Edgar had led to the introduction of the first commercial vaccine, viz. “Coccivac” in the 1950’s, a history reviewed comprehensively by Williams (2002). In comparison to the use of anticoccidials, Coccivac® was (and still remains) a relatively minor product for control of disease in broilers, but along with a similar product, Immucox®, it represented a victory for an immunological approach and the prior acceptance of Coccivac® by the poultry industry was significant many years later when a new generation of attenuated vaccines was introduced. Their entry in to the marketplace stemmed from a truly significant piece of work around the middle of the 1970’s by Thomas Jeffers (Jeffers 1975) who investigated the genetic stability of the prepatent time of E. tenella. His efforts to serially select the first oocysts to be produced during an infection were rewarded with the first ever “precocious line” and he thus laid the basis for the subsequent, and very comprehensive, work done elsewhere that led, around the end of the 1980’s, to the introduction of the first live attenuated vaccines against the avian coccidia.

The modern era
The ‘politics’ around in-feed medication of livestock have continued to evolve (especially within Europe) and an overall more negative view on the use of prophylactic chemotherapy is proving to be a spur for an increasing interest in the immunological control of avian coccidiosis. Not surprisingly, since Paracox® and Livacox® vaccines were developed in the early 1980’s and introduced commercially towards the end of the 1980’s, a slew of other live attenuated vaccines has been introduced worldwide (e.g. ADVENT®, Eimerivac®, Eimeriavax; Gelcox®, Inovocox® Nobilis® CoxATM, etc.). The new vaccines make use of different combinations of the seven recognized species and different methods of administration and this range of products is now supplemented with the first subunit vaccine (CoxAbic) that was developed from the efforts and insights of Michael Wallach.
In contrast to the enhanced research activities towards the development of vaccines, there is now significantly less research interest in new anticoccidials. A number of factors contribute to this and, in addition to the poorer political climate for the use of in-feed antibiotics and other drugs, new drugs are increasingly expensive to get to market and drug-resistance remains an inevitable threat to the financial investment. Thus whilst the marketplace for anticoccidials is still dominated by the use of drugs, the greatest financial investment (albeit limited) for future products within the commercial sector lay not now with the large multinationals but with smaller companies (and some might be described as “bijoux”) that are interested in live, and other, vaccines.

One noteworthy aspect of the current use of many live vaccines is that research has shown that introduction of drug-sensitive vaccinal strains (most were isolated before the onset of global chemotherapy) in to the field leads to a decrease in the numbers of drug-resistant parasites (e.g. Chapman, 1994). Thus ability of coccidiologists to help restore drug-sensitivity in the field through the tactical deployment of drug-sensitive strains is a considerable benefit arising from the use of some vaccines and may be unique in the field of microbiology.
The study of host immune responses to coccidial infection has also continued to run as a significant strand of international research from the 1970’s to the present day and the highly productive efforts of Elaine Rose and Hyun Lillehoj are especially noteworthy.
The so-called modern era might also be thought of as the “molecular” era and an understanding of the biology of the parasites has been extended to new levels of detail and technical sophistication. Examples of areas where progress has, and is still being, made include a better understanding of parasite motility; the mechanisms of host cell invasion allied to the function of sub-cellular organelles, especially rhoptries and micronemes; the basis of virulence; host specificity and the process of differentiation as the life cycle progresses through the different asexual and sexual stages.
Diagnosis of infections and the definitive identification of the different species was once limited by consideration of the appearance of gross lesions, etc., but has now been transformed (at least in the laboratory setting) through the availability of DNA markers that permit rapid and unequivocal discrimination, not only between different species but also between some strains.
A highly significant outcome of current research is the close integration of many laboratories worldwide to form an “Eimeria Genome Consortium” that is working with a major genome sequencing facility in the UK (The Sanger Institute) to derive a sequence for the 55 million units (bases) of DNA that make up the 14 chromosomes within the nucleus of E. tenella. This global consortium is reflective both of the way in which large research projects are undertaken and funded and of the changing scientific grouping whereby smaller research groups around the world with distinctive and, sometimes, unique, expertise are able to tackle specific scientific problems. In the context of the Eimeria genome Consortium it is worth recording that membership comprises three laboratories in the UK; one in the USA; one in Brazil, one in Malaysia and one in China. The inputs of the group at the Institute for Animal Health in the UK (Martin Shirley and Fiona Tomley), Arthur Gruber at the University of Sao Paolo, Brazil and Wan Kiew-Lian at the Universiti Kebangsaan, Malaysia were at the core of the project to derive and annotate a whole genome sequence through funding of more than £1M (~$1.8M) from the Biotechnology and Biological Sciences Research Council (BBSRC) in the UK where the work was done at the Sanger Institute near Cambridge (http://www.sanger.ac.uk/Projects/E_tenella/), coordinated by Al Ivens and Matt Berriman. Complementing to this whole genome sequencing and annotation work are more detailed efforts on specific chromosomes (Malaysia), studies on expressed sequence tags (Brazil; Kate Miska and colleagues at the USDA, Beltsville and Jianping Cai, Guangdong Academy of Agricultural Sciences, China) and physical (HAPPY) mapping of chromosomes (Paul Dear, laboratory of Molecular Biology, Cambridge, UK). The sum total of this activity is that critical resources are now in place to assist the next generation of scientists address further important questions. Even after 80 years some fundamental questions remain to be answered. For example, “what are the molecules within the parasite that induce protective immune responses within the host?”, “how do the avian species locate their preferred sites of development within the intestine?”, “why does asexual reproduction end and sexual reproduction begin?”, “what are metabolic pathways that the parasites use throughout their life cycles, both inside and outside of the chicken?”. Answers to these questions might be needed to make the leap to the next generation of control measures.
Perhaps one of the real surprises from work on the genomes of Eimeria spp. and related parasites was the finding that they contain three genomes; viz. nuclear, mitochondrial, and a plastid (vestigial plant-like genome). A chance now to look at the roles that the other genomes play in facilitating the life style of parasitism (especially the plastid) offers further hopes for better control.
Finally, the ability to genetically engineer Eimeria parasites is now being achieved with some marked success. This considerable technical development by Fiona Tomley and colleagues (this meeting) potentially paves the way forward for the development of live vaccines in which it might be envisaged that one species of Eimeria could be engineered to deliver protective antigens of a number of species.

Has eight decades of research on Eimeria in poultry made a difference?
It might be argued with some confidence that research on Eimeria has been one of the success stories associated with livestock production.
· The challenge of bringing under good control seven genetically complex species of Eimeria has been met through chemotherapy and vaccination. Control strategies have been developed both through knowledge gained empirically and that accumulated from studies on the basic biology of the parasites.
· Both the commercial and academic sectors have played a significant part in improving control of coccidiosis
· The two sectors have worked synergistically together through collaborative scientific and technical projects; meetings on coccidiosis arranged jointly; scientific exchanges between laboratories – both information and personal visits
· A steady stream of complementary publications has been delivered from both sectors in to the more popular press to ensure that the poultry industry is well informed
· Government funding in the UK and USA has provided a long term commitment to research on avian coccidiosis
· A constant stream of effective scientists in to the field has ensured that work on Eimeria spp. remains near to the cutting-edge of microbiological sciences.

References
- Anonymous. (1949). Coccidiosis. Annals of the New York Academy of Sciences 52: 429-624.
- Chapman, H. D. (1994). Sensitivity of field isolates of Eimeria to monensin following the use of a coccidiosis vaccine in broiler chickens. Poultry Science 73: 476-478.
- Chapman, H. D. (2003). Origins of coccidiosis research in the fowl - the first fifty years. Avian Diseases 47: 1-20.
- Chapman, H. D. (2004). Walter T. Johnson (1892 to 1937): pioneer of coccidiosis research in the fowl. Avian Pathology 33: 107-116.
- Chapman, H. D. and Shirley, M.W. (2003). The Houghton strain of Eimeria tenella: a review of the type strain selected for genome sequencing. Avian Pathology 32: 115-127.
- Delaplane, J. F., R. M. Batchelder, and T. C. Higgins. (1947). Sulfaquinoxaline in the prevention of Eimeria tenella infections in chickens. North American Veterinarian 28: 19-24.
- Jeffers, T. K. (1975). Attenuation of Eimeria tenella through selection for precociousness. Journal of Parasitology 61: 1083-1090.
- Johnson, W.T. (1923). Avian coccidiosis. Poultry Science 2: 146-163.
- Johnson, W.T. (1923/1924). Eimeria avium and the diagnosis of avian coccidiosis. Poultry Science 3: 41-57.
- Levine, P. P. (1939). The effect of sulfanilamide on the course of experimental avian coccidiosis. Cornell Veterinarian 29: 309-320.
- Levine, P. P. (1945). Specific diagnosis and chemotherapy of avian coccidiosis. Journal of the American Veterinary Medical Association 106: 88-103.
- Shirley M. W., Ivens, A., Gruber, A., Madeira, A. M. B., Wan, K-L., Dear, P. H. and Tomley, F. M. (2004). The Eimeria genome projects: a sequence of events. Parasitology Today 20: 199-201.
- Tyzzer, E.E. (1929). Coccidiosis in gallinaceous birds. American Journal of Hygiene 10: 269-383.
- Tyzzer, E.E. (1932). Criteria and methods in the investigation of avian coccidiosis. Science 75: 324-328.
- Tyzzer, E.E., Theiler, H. & Jones, E.E. (1932). Coccidiosis in gallinaceous birds II. A comparative study of species of Eimeria of the chicken. American Journal of Hygiene 15: 319-393.
- Williams, R.B. (2002). Fifty years of anticoccidial vaccines for poultry (1952-2002). Avian Diseases, 46: 775-802.

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