Location: Animal Parasitic Diseases Laboratory
Project Number: 8042-32000-113-008-R
Project Type: Reimbursable Cooperative Agreement
Start Date: Jan 1, 2023
End Date: Dec 30, 2024
Objective:
The inability to propagate Cyclospora hinders viability assessment. Recent attempts to assess viability, using PCR or qPCR, have reportedly failed to discriminate live from dead parasites. Hence, we propose to determine the utility of PCR-suppressing dyes and droplet-digital PCR to assess and enumerate viable Eimeria and Cyclospora parasites.
Approach:
1) Adapt and validate sensitive biomarkers for risk assessments:
We will target ten genes that are highly-expressed in mature oocysts. Our prior study suggests promising candidates. For each gene, we will design two sets of primers (for short and long amplicons). Detection specificity will be determined by assessing cross-amplification and by BLAST-searching primer sequences against sequences of related taxa deposited to GenBank. Additionally, we will sequence amplified products to verify that the product represents targeted parasites.
Assay sensitivity will be established via limiting dilution of parasites oocysts, purification of genomic DNA, and amplification using ddPCR. This will define the limit of detection (the concentration of oocysts necessary to consistently generates a signal in ddPCR). After validating the specificity and sensitivity of the ddPCRs using all ten genes, we will seek to dev¬elop a multiplex assay for simultaneous detection of 4 genes with the highest specificity and sensitivity. We will then seek to translate these assays to homologues of Cyclospora.
2) Develop quantitative viability assays for Eimeria and Cyclospora, using ddPCR system and PMA:
We will evaluate PMA treatment as a means to discriminate viable from nonviable parasites, and evaluate ddPCR as a basis for quantifying live parasites. We will vary several treatment conditions to optimize the PMA protocol, including:
• PMA concentration (25 to 150 µM)
• Incubation time in the dark (5 to 60 mins)
• Light exposure, which causes PMA to crosslink DNA to which it is bound (15 to 30 mins).
We will also evaluate bead-beating and freeze-thaw as means to facilitate penetration of PMA through the oocysts wall of dead parasites. Additionally, we will test the effect of the addition of SDS or PMA enhancer on dead cell signal suppression by PMA-ddPCR.
After PMA treatment, DNA will be isolated for PMA-ddPCR. To test the discrimination power between live and dead parasites, we will conduct a spiking experiment, generating defined mixtures varying in the proportion of heat-inactivated parasites (0%, 25%, 50%, 75%, and 100%) in mixtures containing 10 oocysts per ml. Assays will be performed in triplicate and repeated in three independent experiments. We will analyze the effect of amplicon size, sample composition, and treatment condition using two-way ANOVA with Tukey’s multiple comparisons test.
