Exploiting digital droplet PCR and Next Generation Sequencing technologies to determine the relative abundance of individual Eimeria species in a DNA sample.

DNA-based diagnostic assays for detecting infections with Eimeria species have been limited to providing identification and presence/absence data for samples containing oocysts. Modern technologies that generate quantitative data, such as droplet digital PCR (ddPCR) and Next Generation Sequencing (NGS), utilize a relatively short amplicon size containing sufficient species-specific variation for reliable species level identification. Targeting the cytochrome c oxidase subunit III gene in the mitochondrial genome, we established protocols using these technologies to determine the relative abundance of the number of copies/µL of Eimeria species in a sample. Samples from chickens of known and unknown Eimeria species composition were analyzed to determine the suitability of these technologies as diagnostic assays. All technologies demonstrated robust capability of identifying and quantifying the Eimeria species in samples. The new quantitative assays described herein will produce invaluable detail of Eimeria species infections for an array of situations in commercial chicken production systems, enabling further characterization of the disease profile and allowing for the development or enhancement of new intervention strategies.

Restoration of anticoccidial sensitivity to a commercial broiler chicken facility in Canada.

Increasing resistance of Eimeria species to anticoccidial medications is an issue in the broiler chicken industry. Using drug-sensitive strains in live-coccidiosis vaccines has been shown to improve anticoccidial effectiveness in US-based broiler production. In Canada, litter is removed between flocks, which differ from the US industry practice. Thus, we investigated the use of drug-sensitive vaccine strains in a Canadian broiler production facility with suspected anticoccidial resistance. Weekly fecal samples were collected from flocks before, during, and after vaccine seeding to determine oocyst shedding patterns; following the vaccine seeding, OPG counts from similar aged birds were lower than flocks before live-coccidiosis vaccine use. Eimeria species isolates, collected before and after vaccine seeding, were used in 2 anticoccidial sensitivity tests to evaluate their susceptibility to commercially available anticoccidial medications; a low-dose challenge to define parasite replication, and a high-dose challenge to monitor broiler performance. In both experiments, isolates collected after seeding were more susceptible to almost every anticoccidial medication evaluated compared with the isolates collected before seeding. These results demonstrate an improvement in sensitivity to many anticoccidials after the use of live-coccidiosis vaccines at this facility. However, the regulated removal of litter at the end of each flock required under Canadian broiler chicken production management rules could limit the establishment of vaccine-strain Eimeria species in broiler facilities and could shorten the longevity of improved drug sensitivity observed in this study.

Cystoisospora felis infection in a captive jaguar cub (Panthera onca) in Michoacán, México.

Cystoisospora felis (Wenyon 1923) was identified in a 3-month-old, captive jaguar (Panthera onca) presenting with signs of gastrointestinal distress. The cub was fed beef, chicken, and commercial diet. Examination of fresh feces detected large (47.8 µm × 35 µm) unsporulated oocysts. Sporulated oocysts contained 2 sporocysts, each with 4 sporozoites. Oocyst morphometrics agreed with published features of C. felis described from domestic felines. Partial mitochondrial cytochrome c oxidase subunit I (mtCOI) gene was PCR-amplified and sequenced; the resulting sequence showed 100% identity to a C. felis isolate from a domestic cat. This is the first molecularly confirmed report of C. felis infecting and producing clinically evident, enteric disease in a jaguar cub.

Full Mitochondrial Genome and Nuclear 18S rDNA Sequences Refine the Taxonomic Placement of Choleoeimeria taggarti n. comb. from the Prostate of Antechinus flavipes (Yellow-Footed Antechinus).

An unusual coccidian parasite was described previously from the prostate of a male Antechinus flavipes (family: Dasyuridae; common name: yellow-footed antechinus). Morphometrics and a partial nuclear 18S small subunit rDNA (18S rDNA) sequence were used to assign this parasite to the genus Eimeria; it was named Eimeria taggarti. We generated full nuclear 18S rDNA and mitochondrial genome sequences from this parasite and used the newly completed 18S rDNA and mitochondrial cytochrome c oxidase subunit I (COI) sequences to perform a more in-depth phylogenetic analysis. The parasite clustered closely with Choleoeimeria spp. and Acroeimeria spp. infecting herptiles in a well-supported clade that was the sister lineage to the Eimeriidae sensu stricto. The mitochondrial genome of this parasite contained 2 inverted segments compared to mitochondrial genomes from parasites in the Eimeriidae sensu stricto (i.e., Stieda body-possessing coccidia with 4 dizoic sporocysts); this mitochondrial genome arrangement was shared with the only Choleoeimeria species for which sequence data were available publicly. Examination of histological preparations and TEM images uncovered bivalvate sporocysts and otherwise confirmed previously described morphological features of the parasite. Based on our phylogenetic analyses and histological observations, we propose the generic reclassification of E. taggarti to Choleoeimeria taggarti n. comb.