Use of deep amplicon sequencing based on the cytochrome oxidase I gene to quantify the relative percentage of Eimeria spp. oocysts in poultry litter

Authors: Jenkins, Mark; Parker, Carolyn; Campos, Philip; Grimm, Kenneth; Davies, Cary; Tucker, Matthew; HEEDER, CARL - Mountaire Farms, Inc; QUIST, MICHAEL - Mountaire Farms, Inc; Thompson, Peter

Submitted to: Avian Diseases
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/28/2025
Publication Date: 5/9/2025
Citation: Jenkins, M.C., Parker, C.C., Campos, P.M., Grimm, K.G., Davies, C.L., Tucker, M.S., Heeder, C., Quist, M., Thompson, P.C. 2025. Use of deep amplicon sequencing based on the cytochrome oxidase I gene to quantify the relative percentage of Eimeria spp. oocysts in poultry litter. Avian Diseases. 69:177-182. https://doi.org/10.1637/aviandiseases-D-25-00006.
DOI: https://doi.org/10.1637/aviandiseases-D-25-00006

Interpretive Summary: Protozoans in the genus Eimeria cause avian coccidiosis, inflicting over $ 1billion in losses to the U.S. poultry industry by injuring the intestine, reducing weight gain, and decreasing feed conversion efficiency. Further, diseased chicks succumb disproportionately to infections with Clostridium bacteria that induce necrotic enteritis (NE), increasing chick mortality and compromising poultry safety. Here, ARS scientists in Beltsville, Maryland devised a means to rapidly characterize which species of Eimeria predominate in the litter of any given farm. Using this technique, which uses deep amplicon sequencing targeting mitochondrial DNA, they discovered Eimeria mitis as unexpectedly frequent on farms experiencing outbreaks of NE. This discovery reinforces anecdotal reports of veterinarians associating NE with E. mitis, a parasite that has not previously garnered priority for control. These data highlight a need to control E. mitis via drugs or vaccination. Success would help broiler growers and poultry companies combat avian coccidiosis and NE. These data will be of interest to veterinarians, poultry producers, food safety authorities, pathologists, and diagnosticians.

Technical Abstract: The purpose of this study was to evaluate a deep amplicon sequencing approach for estimating the relative abundance of different Eimeria spp. oocysts in litter from commercial broiler farms that may or may not be experiencing necrotic enteritis (NE). Oligonucleotide primers directed to the mitochondrial cytochrome oxidase I (COI) gene whose sequence is conserved among all chicken Eimeria were firstly used to PCR amplify E. acervulina, E. maxima, and E. 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 GenBank database confirmed the expected ~ 100% mapping to the appropriate Eimeria sp. and in about 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 weeks growout from a total of 20 individual houses on 6 different commercial broiler farms. Of the 7 Eimeria sp. known to infect chickens, only 5 were consistently found in litter at each collection timepoint- E. acervulina, E. maxima, E. mitis, E. praecox, and E. tenella. The relative number of E. maxima and non-E. maxima (Eamipt) 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). Being able to estimate the relative numbers of Eimeria spp. 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 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 abundance 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 ITS1 PCR analyses. More importantly, it allows one to discover 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.