Genomic Sequencing of Single Ciliate Protozoa Cells from Equine Fecal Material

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University of Delaware
The equine hindgut is home to a myriad of microbes including bacteria, archaea, fungi, and protozoa that allow for digestion and usage of a forage-based diet. This study sought to expand the current 18S database for equine gut protozoans by optimizing a ciliate-specific PCR protocol. Fresh equine fecal samples were collected and combined with 1X PBS to create a slurry solution. This slurry underwent filtration steps which included a series of five filters of decreasing size (200µm, 100µm, 70µm, 40µm, 10µm). Samples were observed under an inverted microscope and protozoans were manually single sorted. Cells were washed from debris and stored at -80°C in a 1-µL droplet in a PCR tube. DNA extraction optimization included two different series of freeze/thaw steps and a Chelex 100 mechanical disruption protocol. PCR amplification was optimized using ThermoFisher Scientific Phusion™ High-Fidelity DNA Polymerase and P-SSU54F or 82F and P-SSU1747R or EkyB primers (Ito et al., 2014, Sylvester et al., 2004). Custom designed inner primers, VegaF0721 and VegaR0821, were used in a nested primer approach for Sanger sequencing. Data from previously unsequenced species Blepharoconus benbrooki and Blepharocorys valvata, were obtained, as well as sequences from already sequenced species Cochliatoxum periachtum, and Tripalmaria dogieli. It was observed that the forward reads for each sequence were of high quality, and thus the forward primer proved to be more successful. It is recognized that the reverse primer produced poor reads and these sequences were not used within the study and the VegaR0821 inner primer needs to be re-evaluated for effectiveness. A small equine dataset (n=45) was obtained from the Equine Microbiome Project (Berg et al., 2017) to determine the prevalence of the five species obtained from single sorting experiments in equine fecal samples. Results showed that the five single-sorted species were highly prevalent within the samples, validating the approach and need for an expansion of the 18S database. Neighbor Joining trees revealed that the putative P46-Blepharoconus benbrooki and P42- Blepharocorys valvata samples did not cluster close to other species in their genus. It is to be noted that the Blepharocorythidae family has the possibility of being polyphyletic (Cedrola et al., 2021) and thus may account for the differences seen within the Blepharocorys valvata samples.
Equine science, Microbiome, Protozoans, Equine gut