Illumina Next Generation Sequencing for the Analysis of Eimeria Populations in Commercial Broilers and Indigenous Chickens

Abstract

Eimeria species parasites can cause the enteric disease coccidiosis, most notably in chickens where the economic and welfare implications are significant. Seven Eimeria species are recognized to infect chickens, although understanding of their regional occurrence, abundance, and population structure remains limited. Reports of Eimeria circulating in chickens across much of the southern hemisphere with cryptic genotypes and the capacity to escape current anticoccidial vaccines have revealed unexpected levels of complexity. Consequently, it is important to supplement validated species-specific molecular diagnostics with new genus-level tools. Here, we report the application of Illumina MiSeq deep sequencing to partial 18S rDNA amplicons generated using Eimeria genus-specific primers from chicken caecal contents collected in India. Commercial Cobb400 broiler and indigenous Kadaknath type chickens were sampled under field conditions after co-rearing (mixed type farms, n = 150 chickens for each) or separate rearing (single type farms, n = 150 each). Comparison of MiSeq results with established Internal Transcribed Spacer (ITS) and Sequence Characterised Amplified Region (SCAR) quantitative PCR assays suggest greater sensitivity for the MiSeq approach. The caecal-dwelling Eimeria tenella and E. necatrix dominated each sample set, although all seven species which infect chickens were detected. Two of the three cryptic Eimeria genotypes were detected including OTU-X and OTU-Y, the most northern report for the latter to date. Low levels of DNA representing other Eimeria species were detected, possibly representing farm-level contamination with non-replicating oocysts or Eimeria DNA, or false positives, indicating a requirement for additional validation. Next generation deep amplicon sequencing offers a valuable resource for future Eimeria studies.

Poultry Coccidiosis: Design and Interpretation of Vaccine Studies

“…Some studies have utilized non-sporulated oocysts for DNA extraction to minimize the impact of sporulation rate on quantification (91). qPCR has also been applied to validate and standardize next generation sequencing analyses, such as deep sequencing or Eimeria species 18S rDNA PCR amplicons (92).…”

Pre-Columbian zoonotic enteric parasites: An insight into Puerto Rican indigenous culture diets and life styles

“…Eimeria spp. and Perkinsus marinus are regarded as epizootic diseases that are easily transmitted to hosts that live in close proximity, particularly livestock [34][35]. Neither are zoonotic infections, for instance, birds are the definitive hosts of E. necatrix whereas bivalves (Crassostrea and Mya) are the definitive host for P. marinus, and there is no evidence of Eimeria spp.…”

Exploring Eimeria Genomes to Understand Population Biology: Recent Progress and Future Opportunities

“…Amplicon sequencing of 18S rDNA was first applied to communities of Eimeria from the Australian brush-tailed rock-wallaby [ 80 ]. A subsequent study involving Eimeria from chickens also focused on the 18S rDNA, using Illumina MiSeq NGS to sequence amplicons produced from commercial and indigenous chickens sampled in India [ 81 ]. Sequence analysis provided a sensitive assessment of Eimeria species occurrence, validated by standard and quantitative species-specific PCR, and successfully detected the cryptic Eimeria genotypes OTUs x and y as well as a range of other related protozoan pathogens.…”

“…However, detection of low levels of DNA representing other Eimeria species not classically associated with chickens did appear to indicate some background noise. Annotation of sequences assigned previously to Eimeria infecting rock partridges ( Alectoris graeca ) and wild turkeys ( Meleagris gallopavo ) may have represented farm-level contamination with non-replicating Eimeria oocysts or DNA, or false positives [ 81 ]. These latter results indicate a requirement for additional validation and/or more specific primers.…”