Population, genetic, and antigenic diversity of the apicomplexan Eimeria tenella and their relevance to vaccine development.

The phylum Apicomplexa includes serious pathogens of humans and animals. Understanding the distribution and population structure of these protozoan parasites is of fundamental importance to explain disease epidemiology and develop sustainable controls. Predicting the likely efficacy and longevity of subunit vaccines in field populations relies on knowledge of relevant preexisting antigenic diversity, population structure, the likelihood of coinfection by genetically distinct strains, and the efficiency of cross-fertilization. All four of these factors have been investigated for Plasmodium species parasites, revealing both clonal and panmictic population structures with exceptional polymorphism associated with immunoprotective antigens such as apical membrane antigen 1 (AMA1). For the coccidian Toxoplasma gondii only genomic diversity and population structure have been defined in depth so far; for the closely related Eimeria species, all four variables are currently unknown. Using Eimeria tenella, a major cause of the enteric disease coccidiosis, which exerts a profound effect on chicken productivity and welfare, we determined population structure, genotype distribution, and likelihood of cross-fertilization during coinfection and also investigated the extent of naturally occurring antigenic diversity for the E. tenella AMA1 homolog. Using genome-wide Sequenom SNP-based haplotyping, targeted sequencing, and single-cell genotyping, we show that in this coccidian the functionality of EtAMA1 appears to outweigh immune evasion. This result is in direct contrast to the situation in Plasmodium and most likely is underpinned by the biology of the direct and acute coccidian life cycle in the definitive host.

Cryptic Eimeria genotypes are common across the southern but not northern hemisphere.

The phylum Apicomplexa includes parasites of medical, zoonotic and veterinary significance. Understanding the global distribution and genetic diversity of these protozoa is of fundamental importance for efficient, robust and long-lasting methods of control. Eimeria spp. cause intestinal coccidiosis in all major livestock animals and are the most important parasites of domestic chickens in terms of both economic impact and animal welfare. Despite having significant negative impacts on the efficiency of food production, many fundamental questions relating to the global distribution and genetic variation of Eimeria spp. remain largely unanswered. Here, we provide the broadest map yet of Eimeria occurrence for domestic chickens, confirming that all the known species (Eimeria acervulina, Eimeria brunetti, Eimeria maxima, Eimeria mitis, Eimeria necatrix, Eimeria praecox, Eimeria tenella) are present in all six continents where chickens are found (including 21 countries). Analysis of 248 internal transcribed spacer sequences derived from 17 countries provided evidence of possible allopatric diversity for species such as E. tenella (FST values ⩽0.34) but not E. acervulina and E. mitis, and highlighted a trend towards widespread genetic variance. We found that three genetic variants described previously only in Australia and southern Africa (operational taxonomic units x, y and z) have a wide distribution across the southern, but not the northern hemisphere. While the drivers for such a polarised distribution of these operational taxonomic unit genotypes remains unclear, the occurrence of genetically variant Eimeria may pose a risk to food security and animal welfare in Europe and North America should these parasites spread to the northern hemisphere.

An optimised protocol for molecular identification of Eimeria from chickens.

Molecular approaches supporting identification of Eimeria parasites infecting chickens have been available for more than 20 years, although they have largely failed to replace traditional measures such as microscopy and pathology. Limitations of microscopy-led diagnostics, including a requirement for specialist parasitological expertise and low sample throughput, are yet to be outweighed by the difficulties associated with accessing genomic DNA from environmental Eimeria samples. A key step towards the use of Eimeria species-specific PCR as a sensitive and reproducible discriminatory tool for use in the field is the production of a standardised protocol that includes sample collection and DNA template preparation, as well as primer selection from the numerous PCR assays now published. Such a protocol will facilitate development of valuable epidemiological datasets which may be easily compared between studies and laboratories. The outcome of an optimisation process undertaken in laboratories in India and the UK is described here, identifying four steps. First, samples were collected into a 2% (w/v) potassium dichromate solution. Second, oocysts were enriched by flotation in saturated saline. Third, genomic DNA was extracted using a QIAamp DNA Stool mini kit protocol including a mechanical homogenisation step. Finally, nested PCR was carried out using previously published primers targeting the internal transcribed spacer region 1 (ITS-1). Alternative methods tested included sample processing in the presence of faecal material, DNA extraction using a traditional phenol/chloroform protocol, the use of SCAR multiplex PCR (one tube and two tube versions) and speciation using the morphometric tool COCCIMORPH for the first time with field samples.