DOI: 10.15482/usda.adc/30223669
title: Data from: Molecular detection and viability discrimination of zoonotic protozoan pathogens in oysters and seawater

description: <b>Dataset Overview</b>This dataset contains raw output from two laboratory spiking experiments designed to evaluate protozoan detection (Experiment I) and viability discrimination (Experiment II) in oysters and seawater.<b>Experiment I: Protozoan detection in oyster and seawater samples</b>Raw data from Experiment I assessed the performance of a multiplex PCR assay for simultaneous detection of <i>Cryptosporidium</i>, <i>Giardia</i>, <i>Toxoplasma</i>, and <i>Cyclospora</i> DNA in oyster tissues and 10-liter seawater samples. PCR results were compared with microscopy-based detection methods: immunomagnetic separation-direct fluorescent antibody staining (IMS-DFA) for <i>Cryptosporidium</i> and <i>Giardia</i>, and membrane filtration (MF) for <i>Toxoplasma</i>. Each sample was spiked with known quantities of (oo)cysts. Final pellets were split into two subsamples for PCR and microscopy. As a result, the number of (oo)cysts per aliquot represent half the spiked amount. Whole tissue samples were excluded from microscopy analysis, thus the number of (oo)cysts per aliquot equal per sample.Experiment I PCR: From each 50 microliter DNA extract, 5 microliters were used per PCR reaction, making the (oo)cysts per reaction one-tenth of those per aliquot. Detection outcomes were recorded as binary values: 1 for detection, 0 for non-detection.Experiment I Microscopy: Entire aliquots were used for analysis, so (oo)cysts per reaction equal those per aliquot. Due to supply constraints, only three of five oyster replicates were analyzed by IMS-DFA and MF. TNTC indicates "too numerous to count".<b>Experiment II: Detection and discrimination of viable protozoa in oyster and seawater samples</b>Raw data from Experiment II validated molecular assays for detecting and distinguishing viable <i>Cryptosporidi</i><i>u</i><i>m</i>, <i>Giardia</i>, and <i>Toxoplasma</i>. Oyster digestive gland and hemolymph, along with concentrated seawater samples, were spiked with either viable protozoan parasites (Experiment II-A) or defined mixtures of viable and non-viable parasites (Experiment II-B). Final pellets were divided into four aliquots for parallel analysis using reverse transcription qPCR (RT-qPCR), direct fluorescent antibody with propidium iodide staining (DFA-PI), and PCR with or without propidium monoazide (PMA).Experiment II-A RT-qPCR: Protozoa mRNA was quantified and reported as gene copies per oyster tissue or per liter of seawater. Samples with no amplification were labeled ND (non-detect), and those with amplification below the quantification range were labeled DNQ (detected but not quantifiable). For ND and DNQ samples, one-half of the detection limit from the standard curves was substituted and shown in parentheses.Experiment II-B RT-qPCR: Protozoan mRNA was quantified in oyster and seawater samples and reported as gene copies per oyster tissue or milliliter of concentrated seawater. ND and DNQ definitions match those in II-A.Raw data from PMA and DFA-PI assays are not included due to poor performance. These data are available on request.<br>