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

Abstract

The purpose of this study was to evaluate a deep amplicon sequencing approach for estimating the relative abundances of differentspp. 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 chickenspp., were first used to PCR amplify,, andoocyst DNA. COI amplification was applied to samples containing either a singlespecies or an equal mixture of,, andoocysts. Amplicon sequencing and mapping to the relevant COI sequences in the GenBank database confirmed the expected ∼100% mapping to the appropriatesp. and in approximately equal percentages (∼33%) for mixtures of equal numbers ofspp. oocysts. This approach was then applied to DNA derived fromoocysts 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 sevenspp. known to infect chickens, only five were consistently found in litter at each collection time point:,,,, and. The relative numbers ofand non-() oocysts in all litter samples as estimated by COI deep amplicon sequencing showed a modest correlation with the respectiveoroocyst counts (∼ 0.30). The results revealed an interesting phenomenon that supports the role ofin predisposing chickens to NE. In this study, the percentage ofas estimated by deep amplicon sequencing at 0, 2, and 4 wk growout showed a strong positive correlation with NE incidence (0 wk,= 0.57; 2 wk,= 0.52; 4 wk,= 0.61). This study provides evidence for the usefulness of a deep amplicon sequencing approach to estimating the relative abundances of differentoocysts 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 minorspecies, which would have been missed by oocyst counting or ITS1 PCR because mostspecies are not distinguishable by microscopy, and ITS1 PCR is not quantitative.