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.

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.

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.

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.