Genomic analysis of the causative agents of coccidiosis in domestic chickens.

Global production of chickens has trebled in the past two decades and they are now the most important source of dietary animal protein worldwide. Chickens are subject to many infectious diseases that reduce their performance and productivity. Coccidiosis, caused by apicomplexan protozoa of the genus Eimeria, is one of the most important poultry diseases. Understanding the biology of Eimeria parasites underpins development of new drugs and vaccines needed to improve global food security. We have produced annotated genome sequences of all seven species of Eimeria that infect domestic chickens, which reveal the full extent of previously described repeat-rich and repeat-poor regions and show that these parasites possess the most repeat-rich proteomes ever described. Furthermore, while no other apicomplexan has been found to possess retrotransposons, Eimeria is home to a family of chromoviruses. Analysis of Eimeria genes involved in basic biology and host-parasite interaction highlights adaptations to a relatively simple developmental life cycle and a complex array of co-expressed surface proteins involved in host cell binding.

Genetic mapping identifies novel highly protective antigens for an apicomplexan parasite.

Apicomplexan parasites are responsible for a myriad of diseases in humans and livestock; yet despite intensive effort, development of effective sub-unit vaccines remains a long-term goal. Antigenic complexity and our inability to identify protective antigens from the pool that induce response are serious challenges in the development of new vaccines. Using a combination of parasite genetics and selective barriers with population-based genetic fingerprinting, we have identified that immunity against the most important apicomplexan parasite of livestock (Eimeria spp.) was targeted against a few discrete regions of the genome. Herein we report the identification of six genomic regions and, within two of those loci, the identification of true protective antigens that confer immunity as sub-unit vaccines. The first of these is an Eimeria maxima homologue of apical membrane antigen-1 (AMA-1) and the second is a previously uncharacterised gene that we have termed 'immune mapped protein-1' (IMP-1). Significantly, homologues of the AMA-1 antigen are protective with a range of apicomplexan parasites including Plasmodium spp., which suggest that there may be some characteristic(s) of protective antigens shared across this diverse group of parasites. Interestingly, homologues of the IMP-1 antigen, which is protective against E. maxima infection, can be identified in Toxoplasma gondii and Neospora caninum. Overall, this study documents the discovery of novel protective antigens using a population-based genetic mapping approach allied with a protection-based screen of candidate genes. The identification of AMA-1 and IMP-1 represents a substantial step towards development of an effective anti-eimerian sub-unit vaccine and raises the possibility of identification of novel antigens for other apicomplexan parasites. Moreover, validation of the parasite genetics approach to identify effective antigens supports its adoption in other parasite systems where legitimate protective antigen identification is difficult.

Livestock infectious diseases and zoonoses.

Infectious diseases of livestock are a major threat to global animal health and welfare and their effective control is crucial for agronomic health, for safeguarding and securing national and international food supplies and for alleviating rural poverty in developing countries. Some devastating livestock diseases are endemic in many parts of the world and threats from old and new pathogens continue to emerge, with changes to global climate, agricultural practices and demography presenting conditions that are especially favourable for the spread of arthropod-borne diseases into new geographical areas. Zoonotic infections that are transmissible either directly or indirectly between animals and humans are on the increase and pose significant additional threats to human health and the current pandemic status of new influenza A (H1N1) is a topical example of the challenge presented by zoonotic viruses. In this article, we provide a brief overview of some of the issues relating to infectious diseases of livestock, which will be discussed in more detail in the papers that follow.

Conservation of proteins involved in oocyst wall formation in Eimeria maxima, Eimeria tenella and Eimeria acervulina.

Vaccination with proteins from gametocytes of Eimeria maxima protects chickens, via transfer of maternal antibodies, against infection with several species of Eimeria. Antibodies to E. maxima gametocyte proteins recognise proteins in the wall forming bodies of macrogametocytes and oocyst walls of E. maxima, Eimeria tenella and Eimeria acervulina. Homologous genes for two major gametocyte proteins - GAM56 and GAM82 - were found in E. maxima, E. tenella and E. acervulina. Alignment of the predicted protein sequences of these genes reveals that, as well as sharing regions of tyrosine richness, strong homology exists in their amino-terminal regions, where protective antibodies bind. This study confirms the conservation of the roles of GAM56 and GAM82 in oocyst wall formation and shows that antibodies to gametocyte antigens of E. maxima cross-react with homologous proteins in other species, helping to explain cross-species maternal immunity.

Challenges in the successful control of the avian coccidia.

Eimeria species infect livestock in a host-specific manner and are the cause of the disease, coccidiosis. Control of Eimeria species is essential and is currently dominated by chemotherapy; with vaccination using formulations of live wild-type or attenuated parasites an increasing option. A new generation of subunit, live-vector or DNA vaccination strategies is being sought and determining the identity of suitable antigens remains difficult. Some past and present methods of controlling avian coccidia are discussed briefly and we describe progress with a novel approach to identify immunoprotective antigens as vaccine candidates.

Eimeria maxima: the influence of host genotype on parasite reproduction as revealed by quantitative real-time PCR.

The influence of host genotype on susceptibility to infection with Eimeria species has long been recognised, but beyond monitoring pathological severity or magnitude of oocyst excretion attempts to quantify fluctuations in parasite reproduction within the host have previously relied upon labour-intensive microscopic analysis. The development and application of a quantitative real-time PCR assay has opened this biological 'black box', permitting the sensitive and reproducible enumeration of parasite genomes throughout the course of infection. Generic and species-specific quantitative PCR methods are described, based upon the conserved 5S ribosomal RNA coding sequence of nine avian and murine Eimeria species and the Eimeria maxima MIC1 gene, respectively. These complementary assays have been applied to study the influence of host genotype on resistance to infection with E. maxima, revealing significant differences in parasite load between 'resistant' Line C and 'susceptible' Line 15I inbred chickens 5 days after infection. Parasite DNA remained detectable up to 20 days post-infection; 11 days after the last oocysts had been detected leaving the host.

The biology of avian Eimeria with an emphasis on their control by vaccination.

Studies on the biology of the avian species of Eimeria are currently benefiting from the availability of a comprehensive sequence for the nuclear genome of Eimeria tenella. Allied to some recent advances in transgenic technologies and genetic approaches to identify protective antigens, some elements are now being assembled that should be helpful for the development of a new generation of vaccines. In the meantime, control of avian coccidiosis by vaccination represents a major success in the fight against infections caused by parasitic protozoa. Live vaccines that comprise defined populations of oocysts are used routinely and this form of vaccination is based upon the long-established fact that chickens infected with coccidial parasites rapidly develop protective immunity against challenge infections with the same species. Populations of wild-type Eimeria parasites were the basis of the first live vaccines introduced around 50 years ago and the more recent introduction of safer, live-attenuated, vaccines has had a significant impact on coccidiosis control in many areas of the world. In Europe the introduction of vaccination has coincided with declining drug efficacy (on account of drug resistance) and increasing concerns by consumers about the inclusion of in-feed medication and prospects for drug residues in meat. The use of attenuated vaccines throughout the world has also stimulated a greater interest in the vaccines that comprise wild-type parasites and, during the past 3 years worldwide, around 3x10(9) doses of each type of vaccine have been used. The need for only small numbers of live parasites to induce effective protective immunity and the recognition that Eimeria spp. are generally very potent immunogens has stimulated efforts to develop other types of vaccines. None has succeeded except for the licensing, within several countries in 2002, of a vaccine (CoxAbic vaccine; Abic, Israel) that protects via the maternal transfer of immunoglobulin to the young chick. Building on the success of viral vaccines that are delivered via the embryonating egg, an in ovo coccidiosis vaccine (Inovocox, Embrex Inc.) is currently in development. Following successful field trials in 2001, the product will be ready for Food and Drug Administration approval in 2005 and a manufacturing plant will begin production for sale in late 2005. Limited progress has been achieved towards the development of subunit or recombinant vaccines. No products are available and studies to identify potential antigens remain compromised by an absence of effective in vitro assays that correlate with the induction of protective immunity in the host. To date, only a relatively small portfolio of molecules has been evaluated for an ability to induce protection in vivo. Although Eimeria are effective immunogens, it is probable that to date none of the antigens that induce potent protective immune responses during the course of natural infection has been isolated.

metaSHARK: software for automated metabolic network prediction from DNA sequence and its application to the genomes of Plasmodium falciparum and Eimeria tenella.

The metabolic SearcH And Reconstruction Kit (metaSHARK) is a new fully automated software package for the detection of enzyme-encoding genes within unannotated genome data and their visualization in the context of the surrounding metabolic network. The gene detection package (SHARKhunt) runs on a Linux system and requires only a set of raw DNA sequences (genomic, expressed sequence tag and/or genome survey sequence) as input. Its output may be uploaded to our web-based visualization tool (SHARKview) for exploring and comparing data from different organisms. We first demonstrate the utility of the software by comparing its results for the raw Plasmodium falciparum genome with the manual annotations available at the PlasmoDB and PlasmoCyc websites. We then apply SHARKhunt to the unannotated genome sequences of the coccidian parasite Eimeria tenella and observe that, at an E-value cut-off of 10(-20), our software makes 142 additional assertions of enzymatic function compared with a recent annotation package working with translated open reading frame sequences. The ability of the software to cope with low levels of sequence coverage is investigated by analyzing assemblies of the E.tenella genome at estimated coverages from 0.5x to 7.5x. Lastly, as an example of how metaSHARK can be used to evaluate the genomic evidence for specific metabolic pathways, we present a study of coenzyme A biosynthesis in P.falciparum and E.tenella.

The influence of immunizing dose size and schedule on immunity to subsequent challenge with antigenically distinct strains of Eimeria maxima.

Eimeria maxima, the most immunogenic of the Eimeriidae that infect the chicken, is characterized by the presence of antigenic diversity within field isolates. In priming/challenge experiments immunity to homologous infection is essentially complete while immunity against challenge by a heterologous strain is often only partial. The phenotype "escape from immune protection" is known to be influenced by both host and parasite genotypes but the impact of varied immunization dose and schedule remains poorly documented. In this manuscript we report that an immunizing dose between 99.99%) protective immunity against challenge by 100 oocysts of a homologous strain. In contrast, complete immunity against a heterologous strain was never observed, although increasing the immunizing dose size did frequently reduce oocyst production arising from subsequent heterologous challenge. Differences in cross-protective immunizing capacity between two strains of E. maxima were evident as the H strain consistently stimulated a more potent protective immune response than the W strain. Similarly, increasing the number of immunizing doses of the E. maxima W strain (but not the H strain) increased immune protection against subsequent heterologous challenge. When combined with previously published data the results described here suggest that the E. maxima genome encodes a pool of antigens that are capable of stimulating an immune response cross-protective against more than one strain. These antigens supplement a separate restricted pool of antigens that are capable of stimulating stronger, but strain-specific, protective immune responses.

Parasite genetics and the immune host: recombination between antigenic types of Eimeria maxima as an entrée to the identification of protective antigens.

The genomes of protozoan parasites encode thousands of gene products and identification of the subset that stimulates a protective immune response is a daunting task. Most screens for vaccine candidates identify molecules by capacity to induce immune responses rather than protection. This paper describes the core findings of a strategy developed with the coccidial parasite Eimeria maxima to rationally identify loci within its genome that encode immunoprotective antigens. Our strategy uses a novel combination of parasite genetics, DNA fingerprinting, drug-resistance and strain-specific immunity and centres on two strains of E. maxima that each induce a lethal strain-specific protective immune response in the host and show a differential response to anti-Eimeria chemotherapy. Through classical mating studies with these strains we have demonstrated that loci encoding molecules stimulating strain-specific protective immunity or resistance to the anti-coccidial drug robenidine segregate independently. Furthermore, passage of populations of recombinant parasites in the face of killing in the immune host was accompanied by the elimination of some polymorphic DNA markers defining the parent strain used to immunise the host. Consideration of the numbers of parasites recombinant for the two traits implicates very few antigen-encoding loci. Our data provide a potential strategy to identify putative antigen-encoding loci in other parasites.

Molecular karyotypes of Eimeria tenella resolved by PFGE: an evaluation of different agaroses.

Molecular karyotypes that revealed size polymorphisms between homologous forms of chromosomes 1, 2, 3 or 4 in three strains of the coccidial parasite Eimeria tenella were derived by pulsed field gel electrophoresis (PFGE) using brands of agaroses marketed either specifically for PFGE or for purposes such as protein electrophoresis or the separation of small molecules of DNA. Chromosomes up to approximately 4 Mbp were well resolved in agaroses marketed for non-PFGE purposes and these products clearly provide useful alternatives when supplies of dedicated PFGE agaroses are limited or withdrawn.

Comparative EST analyses provide insights into gene expression in two asexual developmental stages of Eimeria tenella.

The protozoan parasite Eimeria tenella has a complex life cycle that includes two major asexual developmental stages, the merozoite and the sporozoite. The expressed sequence tag (EST) approach has been previously used to study gene expression of merozoites. We report here the generation and analysis of 556 ESTs from sporozoites. Comparative analyses of the two datasets reveal a number of transcripts that are preferentially expressed in a specific stage, including previously uncharacterised sequences. The data presented indicate the invaluable potential of the comparative EST analysis for providing information on gene expression patterns in the different developmental stages of E. tenella.

Antigenic diversity in Eimeria maxima and the influence of host genetics and immunization schedule on cross-protective immunity.

Eimeria spp. are a group of highly successful intracellular protozoan parasites that develop within enterocytes. Eimeria maxima from the chicken is characterized by high immunogenicity (a small priming infection gives complete immunity to subsequent homologous challenge) and naturally occurring antigenically variant populations that do not completely cross-protect. In this study we examined the expression of antigenic diversity in E. maxima, as manifested by cross-strain protection in a series of inbred chicken lines. The IAH line of Light Sussex chickens and all lines of inbred White Leghorns were susceptible to primary infections with either of two strains (H and W) of E. maxima and were protected completely against challenge with the homologous strain of parasite. The extent of cross-protection against the heterologous parasite strain varied from 0 to almost 100% depending on host genetics. Interestingly, in one inbred line of chickens (line 15I) the cross-protective phenotype was directional and intensely influenced by the infection history of the host. The basis for the observed variation in cross-protection is not known, but our results suggest that the major histocompatibility complex is not a major genetic component of the phenotype. These results are discussed in relation to the number of protective antigens presented by complex pathogens and the development of immunoprotective responses in hosts of different genetic backgrounds.

Immunological aspects of infections with Eimeria maxima: A short review.

Eimeria maxima is one of the seven species of protozoan parasites that cause intestinal coccidiosis in chickens. Discovered and first described exactly 70 years ago, the parasite has some unique features that both distinguish it from the other members of the genus and make it of relevance to the world's poultry industry. Of special interest is the balanced immunological relationship that E. maxima achieves with its host. The marked immunogenicity of the parasite results in swift and complete protection to homologous challenge, but is counterbalanced by the ubiquity of antigenically different strains that provide a significantly heterologous challenge and are only poorly controlled-essentially two sides of the same coin. In this paper, we will describe some of the features of the biology of E. maxima, with an emphasis on the immunogenicity and antigenic diversity of the parasite, and discuss some of the ways in which these features impinge on attempts to control infection. The review is, of necessity, brief and selective by choice; for more information, the reader is referred to the definitive paper on E. maxima by Tyzzer (1929) and to reviews on other aspects of Eimeria spp., including E. maxima, by Rose (1997) and Chapman (1997).