Environmental DNA-based detection of Batrachochytrium salamandrivorans in captive settings
Data files
Sep 11, 2023 version files 264.49 KB
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AllExposed.csv
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Experiment1.csv
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Experiment2.csv
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Experiment3.csv
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Experiment4.csv
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Experiment5.csv
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README.md
Abstract
Detecting pathogens in the live animal trade is critical for tracking and preventing their movement, introduction, and spillover into susceptible fauna. However, the scale of the live animal trade makes individually testing animals infeasible for all but the most economically important taxa. For instance, while the fungal pathogen, Batrachochytrium salamandrivorans (Bsal), threatens amphibian, particularly caudate diversity, in Europe and the Americas, screening even a fraction of the millions of live amphibians imported into the United States, alone, is impractically laborious and expensive. A promising alternative to individual-level sampling (e.g., swabbing the skin of salamanders) is to instead collect DNA from the animals’ environment (e.g., housing container or water) which allows us to screen a whole group of animals at a time.
We used a series of experiments with Bsal-spiked water and substrates and experimentally infected rough-skinned newts (Taricha granulosa) to determine how best to collect Bsal environmental DNA (eDNA) samples, that is, which methods yield the greatest recovery of Bsal eDNA, and evaluate the capacity of these methods to detect Bsal-infected animals in conditions that might be found in captive settings and trade.
We found that filtering water housing infected animals for even an hour can consistently recover detectable levels of Bsal eDNA, that there is little evidence of Bsal eDNA being clumped in housing containers or being swamped or inhibited under realistically dirty conditions, and that eDNA-based methods achieves an equivalent or higher chance of detecting Bsal infections in a group of co-housed newts with fewer samples than traditional methods of individually swabbing.
By sampling the genetic materials shed or produced by a whole group of animals, eDNA-based methods are a powerful means of detecting pathogens, such as Bsal, in shipment and captive population. These methods bring routine pathogen surveillance into reach in many more contexts and can thus be an important tool in conservation and disease control.
README: # Environmental DNA-based detection of Batrachochytrium salamandrivorans in captive settings
These data come from a series of experiments designed to understand how best to collect use environmental DNA (eDNA) to detect the amphibian pathogen, Batrachochytrium salamandrivorans (Bsal), in captive settings (e.g., trade or collections). Water or housing substrates were spiked with Batrachochytrium salamandrivorans (Bsal) and then recovered with filtering water or pelletizing materials by centrifugation. In other studies rough-skinned newts (Taricha granulosa) were experimentally infected with Bsal and samples were collected from their water, housing substrates, or from skin swabs. In each case DNA was extacted and then screened for Bsal genes with a quantitative realtime PCR (qPCR) assay.
Description of the data and file structure
Data are estimated copy number from the qPCR assay, as well as columns specifying whether we had detected inhibition in an exogenous internal positive control assay added to the third well of each triplicate of wells used per sample and then whether the sample was cleaned up with a Zymo PCR Inhibitor Removal kit or diluted 1-to-10 (in which case the quantity was adjusted). See the main text for details.
Each file corresponds to the data from one study:
Experiment1.csv includes data from experiments focused on determining the limits of detection for two methods of collecting eDNA: 1) filtering water and 2) centrifuging water to pelletize the DNA-bearing materials. The columns are:
- Sample.ID / sampleID: a unique identifier for the individual sample
- Trial: This experiment was conducted three times using different volumes and concentrations of zoospores (see paper for details). A, B, and C represent the trial.
- Zoospores.Processed: The expected number of zoospores processed by the sample collection. Is equal to the volume processed times the concentration of zoospores.
- Sample.Method: Whether eDNA in the sample was collected from the water by filtering or pelletization.
- Pos: Binary variable indicating whether the sample was scored as positive (i.e., Bsal DNA was detected in the quantitative realtime PCR reaction; = 1) or not (= 0). See the paper for details.
- Quantity: The number of Bsal gene copies detected in the, averaged across the three wells of the qPCR reaction.
- ExptMethod: An indicator variable for the combination of the trial (A, B, or C) and the sampling method (filtering or pelletization)
Experiment2.csv includes data from a study in which each of five volumes of Bsal-spiked water were filtered and the number of Bsal gene copies in the eDNA samples determined. The columns unique to this experiment are:
- Experiment: An identifier that these samples came from experiment "X2"
- Treatment: All samples were in the "Bsal" treatment (as opposed to mock-spiked controls in other experiments)
- Volume: The volume of water filtered.
- Zsp.Conc: The concentration of zoospores in the water (number / milliliter)
- Total.Zsp: The expected number of zoospores processed (=Zsp.Conc * Volume). Like "Zoospores.Processed" in Experiment1.csv.
- Replicate: The experimental replicate (=container of water) from which the sample was collected.
- Diluted: Whether the extracted DNA sample was diluted 1:10 due to signs of PCR inhibition in initial qPCR. Y= Yes, N = No.
- Zymogen: Whether the extracted DNA sample was run through a Zymo Research OneStep PCR Inhibitor Removal kit due to signs of PCR inhibition in initial qPCR. Y= Yes, N = No.
- Inhibition: Whether the sample displayed signs of PCR inhibition in initial qPCR reactions. Y= Yes, N = No.
Experiment3.csv includes data from a study in which eDNA was collected from Bsal-spiked animal housing substrates. The columns unique to this experiment are:
- Substrate: The type of housing substrate (Paper Towel or Sphagnum Moss)
- Zsp.Dose: The number of zoospores added to the substrate
- Sample.Method: Whether the sample was directly subsampled (=Direct subsample) or soaked and then filtered (=Soak then filter) prior to filtering to collect eDNA.
Experiment4.csv includes data from a study in which Bsal-infected newts were housed in bags of fresh water for different durations of time (1, 2, 3, 6, and 12 hours; see paper for details) and then the water was filtered to collect eDNA. The columns unique to this experiment are:
- Animal.ID / Animal: A unique identifier for the individual animal
- sampID: a unique identifier for the samples from each animal
- Time: the time in hours from the start of this study when the water sample was collected and the animal moved to new water
- Duration: the duration of time in hours the animal had been in the water prior to the sampling being collected and the animal moved to new water.
- Date.Collected: the date the sample was collected.
Experiment5.csv includes data from a study in which Bsal-infected newts were housed for 24 hours in 10L of either clean water, which was then sampled (control treatment) or homogenized with an immersion blender for 1 min (homogenized treatment), or in water that had previously housed 20 uninfected newts for the prior 72 hours (dirty water treatment). The columns unique to this experiment are:
- Sort.ID: a variable used to enforce an ordering of the animals for plotting purposes
- Replicate: The number of the eDNA sample, out of ten, collected from a given animal in a given treatment.
The AllExposed.csv file includes all of the samples collected from each Bsal-exposed animal over the duration of the experiment. (Note: unexposed controls never tested positive for Bsal). The unique columns in this study are:
- Sample: Whether the sample consisted of Paper towel or Spagnum moss substrate, a skin swab from the animal (see paper for details), or a sample collected from the water (=WaterBath).
- DPE: Days post exposure to Bsal at which time the sample was collected.
In all data file "NA" means the particular value is "not available," if the measurement was lost or otherwise missing, or "not applicable" if this value is not relevant or applicabple for a given study, treatment, etc.
Code/Software
The Bsal_eDNA_analyses_X1.pdf and similar files contain the R code and output of that code using the data files for each experiment. The Bsal_eDNA_analyses_AllAnimals.pdf file contains the analysis comparing eDNA-based detection with traditional sampling with swabs from individual animals. Each piece is standalone except where priors are derived from previous analysis, as noted. See the paper for more detail.
Methods
Water or housing substrates were spiked with Batrachochytrium salamandrivorans (Bsal) and then recovered with filtering water or pelletizing materials by centrifugation. In other studies rough-skinned newts were experimentally infected with Bsal and samples were collected from their water, housing substrates, or from skin swabs. In each case DNA was extacted and then screened for Bsal genes with a quantitative realtime PCR assay. Data are estimated copy number from the qPCR assay, as well as columns specifying whether we had detected inhibition in an exogenous internal positive control assay added to the third well of each triplicate of wells used per sample and then whether the sample was cleaned up with a Zymo PCR Inhibitor Removal kit or diluted 1-to-10 (in which case the quantity was adjusted). See the main text for details.
Usage notes
The analysis files are generated from Quarto documents run in R. They include all of the code needed to reporduce the results in R.