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 <i>Eimeria</i> 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 <i>Eimeria</i> spp., were first used to PCR amplify <i>Eimeria acervulina</i>, <i>Eimeria maxima</i>, and <i>Eimeria tenella</i> oocyst DNA. COI amplification was applied to samples containing either a single <i>Eimeria</i> species or an equal mixture of <i>E. acervulina</i>, <i>E. maxima</i>, and <i>E. tenella</i> oocysts. Amplicon sequencing and mapping to the relevant COI sequences in the GenBank database confirmed the expected ∼100% mapping to the appropriate <i>Eimeria</i> sp. and in approximately equal percentages (∼33%) for mixtures of equal numbers of <i>Eimeria</i> spp. oocysts. This approach was then applied to DNA derived from <i>Eimeria</i> 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 <i>Eimeria</i> spp. known to infect chickens, only five were consistently found in litter at each collection time point: <i>E. acervulina</i>, <i>E. maxima</i>, <i>Eimeria mitis</i>, <i>Eimeria praecox</i>, and <i>E. tenella</i>. The relative numbers of <i>E. maxima</i> and non-<i>E. maxima</i> (<i>Eamipt</i>) oocysts in all litter samples as estimated by COI deep amplicon sequencing showed a modest correlation with the respective <i>E. maxima</i> or <i>Eamipt</i> oocyst counts (<i>R</i> ∼ 0.30). The results revealed an interesting phenomenon that supports the role of <i>E. mitis</i> in predisposing chickens to NE. In this study, the percentage of <i>E. mitis</i> as estimated by deep amplicon sequencing at 0, 2, and 4 wk growout showed a strong positive correlation with NE incidence (0 wk, <i>R</i> = 0.57; 2 wk, <i>R</i> = 0.52; 4 wk, <i>R</i> = 0.61). This study provides evidence for the usefulness of a deep amplicon sequencing approach to estimating the relative abundances of different <i>Eimeria</i> 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 <i>Eimeria</i> species, which would have been missed by oocyst counting or ITS1 PCR because most <i>Eimeria</i> species are not distinguishable by microscopy, and ITS1 PCR is not quantitative.