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

Abstract

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.

Keywords: Eimeria; chickens; coccidiosis; food security; population structure.

Fig. S1.

The geographic origin of chicken fecal samples collected for use during these studies. The number within each node indicates the number of samples collected. The individual states sampled in Northern and Southern India are indicated in the expanded boxes.

Fig. 1.

Median-joining phylogenetic NETWORK showing the relationships between Sequenom MassARRAY haplotypes for E. tenella samples collected in Northern India (yellow), Southern India (green), Egypt and Libya combined (North Africa, red), and Nigeria (blue). Node size indicates the frequency of haplotype occurrence. Nodes surrounded by a heavy black boundary indicate the haplotypes used in the cross-protection studies.

Fig. S2.

Estimate of the ancestral population number (K) for E. tenella produced using STRUCTURE. (A) Plot of the mean log likelihood of the data [Ln P(D)] and associated SDs. Data points colored red were selected for further population structure illustration in C–E. (B) Plot of the mean difference between successive likelihood values of K [L′(K)]. (C–E) Population structure calculated using ancestral population sizes (K) of 7 (C), 8 (D), and 9 (E).

Fig. S3.

Cross-protective ability of European and Asian E. tenella isolates. (A) High-level immunizing dose. Total parasite burden (E. tenella genomes per milligram of cecal tissue, determined using species-specific quantitative PCR normalized against the host genome number 5 d postchallenge) following homologous or heterologous challenge of 6-wk-old birds immunized by infection with 1,000 and then 4,000 sporulated oocysts of the Houghton (H; European) or Northern Indian (NI; Asian) strains at 3 and 4 wk of age, respectively. The challenge dose was 250 sporulated oocysts per bird. Birds given no previous infection were included as a control. nd, no parasite genomes detected (the limit of detection was 17 genomes per gram). Parasite escape was calculated as the average number of parasite genomes detected in the immunized groups as a percentage of the number detected in the challenge strain-matched unimmunized group. (B) Low-level immunizing dose. Total parasite replication (determined as in A) following homologous (NI) or heterologous (H) challenge of 6-wk-old birds immunized by infection with 100 and then 1,000 sporulated oocysts of the NI strain at 3 and 4 wk of age, respectively.

Fig. 2.

The extent and impact of sequence diversity within the E. tenella AMA11 (EtAMA1) coding sequence. (A) Nonsynonymous polymorphism within the EtAMA1 coding sequence. “Major” denotes the more common sequence type identified; “minor” the less common. (B) Sliding window plot of EtAMA1 coding sequence nucleotide diversity calculated using π with the Jukes Cantor correction (multiplied by 100; green line, open circle markers) and plots of neutrality using Tajima’s D (red, open triangles) and Fu and Li’s F* (black, crosses). The window length was 100 bp with a 25-bp step size. No points were statistically significant. The EtAMA1 domain structure indicated by the blue lines was predicted based on cysteine residue locations. (C) Heat map representing the F_ST calculated for EtAMA1. Incremental intensity indicates elevated F_ST. 1, Europe; 2, Nigeria; 3, Asia; 4, the Americas; 5, North Africa.