Use of Deep Amplicon Sequencing Based on the Cytochrome Oxidase I Gene to Quantify the Relative Percentages of Eimeria spp. Oocysts in Poultry Litter.

The purpose of this study was to evaluate a deep amplicon sequencing approach for estimating the relative abundances of different Eimeria spp. oocysts in litter from commercial broiler farms that may or may not be experiencing necrotic enteritis (NE) infections. Oligonucleotide primers directed to the mitochondrial cytochrome oxidase I (COI) gene, a sequence that is conserved among all chicken Eimeria spp., were first used to PCR amplify Eimeria acervulina, Eimeria maxima, and Eimeria tenella oocyst DNA. COI amplification was applied to samples containing either a single Eimeria species or an equal mixture of E. acervulina, E. maxima, and E. tenella oocysts. Amplicon sequencing and mapping to the relevant COI sequences in the GenBank database confirmed the expected ∼100% mapping to the appropriate Eimeria sp. and in approximately equal percentages (∼33%) for mixtures of equal numbers of Eimeria spp. oocysts. This approach was then applied to DNA derived from Eimeria oocysts obtained at 0, 2, and 4 wk after chick placement (growout) from a total of 20 individual houses on six different commercial broiler farms. Of the seven Eimeria spp. known to infect chickens, only five were consistently found in litter at each collection time point: E. acervulina, E. maxima, Eimeria mitis, Eimeria praecox, and E. tenella. The relative numbers of E. maxima and non-E. maxima (Eamipt) oocysts in all litter samples as estimated by COI deep amplicon sequencing showed a modest correlation with the respective E. maxima or Eamipt oocyst counts (R ∼ 0.30). The results revealed an interesting phenomenon that supports the role of E. mitis in predisposing chickens to NE. In this study, the percentage of E. mitis as estimated by deep amplicon sequencing at 0, 2, and 4 wk growout showed a strong positive correlation with NE incidence (0 wk, R = 0.57; 2 wk, R = 0.52; 4 wk, R = 0.61). This study provides evidence for the usefulness of a deep amplicon sequencing approach to estimating the relative abundances of different Eimeria oocysts infecting chickens because it allows reactions to take place in a single tube, thus avoiding the time-consuming, labor-intensive, species-specific internal transcribed spacer 1 (ITS1) PCR analyses. More importantly, it allows one to explore relationships between NE incidence and the abundance of minor Eimeria species, which would have been missed by oocyst counting or ITS1 PCR because most Eimeria species are not distinguishable by microscopy, and ITS1 PCR is not quantitative.