DOI: 10.5061/dryad.hdr7sqvp5
title: Environmental DNA-based detection of Batrachochytrium salamandrivorans in captive settings

description: 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.
description: Water or housing substrates were spiked with
 <em>Batrachochytrium salamandrivorans</em>
 (<em>Bsal</em>) and then recovered with filtering water or
 pelletizing materials by centrifugation. In other studies rough-skinned
 newts were experimentally infected with <em>Bsal</em> 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. 
description: The analysis files are generated from Quarto documents run in R.
 They include all of the code needed to reporduce the results in
 R. 
description: # \# 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.