Using environmental DNA for detection of Bsal
@Zerina Rahic
Current process
Active surveillance for Bsal is performed by collecting skin swabs from the amphibian host (Hyatt et al., 2007), and then analyzing these swabs for the presence of Bsal DNA using quantitative PCR (qPCR) techniques (Blooi et al., 2015a; 2015b). The reliable detection of Bsal at low prevalence requires sampling a large number of individuals (DiGiacomo & Koepsell, 1986) and is therefore expensive. Passive surveillance for infection in newts during their aquatic phase is unreliable, as hosts can carry infections asymptomatically (Stegen et al., 2017), and dead newts quickly decompose in water.
Alternative method
eDNA approach has been proven to have a higher detection capability and cost‐effectiveness compared with traditional methods, for example, (Biggs et al., 2015). eDNA techniques have been demonstrated to be a reliable tool for the detection of Batrachochytrium dendrobatidis (Bd), and ranavirus in the environment even at early stage before any amphibian mortality has occurred (e.g., Kamoroff & Goldberg, 2017; Miaud, Arnal, Poulain, Valentini, & Dejean, 2019).
Method 1: Primer and probe validation
Species‐specific primers and a probe were designed in this study for the amplification of a short Bsal DNA fragment of 54 bp by PCR. A 788 bp genomic sequence containing internal transcribed spacer 1 (partial), 5.8S ribosomal RNA (complete), and internal transcribed spacer 2 (partial) was used for the primer design (GenBank accession number KC762295). The PrimerQuest program (IDT, Coralville, USA; retrieved December 12, 2012 from http://www.idtdna.com/Scitools) was used to design the primers and probes that amplified a short fragment of 54 bp (primers excluded). The primers amplified the same internal region which is used for the detection of Bsal in skin samples by PCR (Martel et al., 2013). To test primer specificity, they were first validated in silico using the ecoPCR program (Bellemain et al., 2010; Ficetola et al., 2010) on the EMBL‐Bank release 123, allowing three mismatches per primer. Then, an in vitro validation was undertaken using real‐time PCR on one DNA sample from Bsal (183 GE) and B. dendrobatidis (Bd, 100 GE). The real‐time qPCR was carried out using a final volume of 25 µl, containing 3 μl of template DNA, 12.5 μl of TaqMan Environmental Master Mix 2.0 (Life Technologies), 6.5 μl of ddH2O, 1 μl of forward primer (Bsal_F: CACATTGCACTCTACTTT, 10 μM), 1 μL of reverse primer (Bsal_R: AAGACAAGGAAATGAATTAAA, 10 μM), and 1 μl of probe (Bsal_Pr: 6‐FAM‐ TGATTCTCAAACAGGCATACTCTAC ‐BHQ‐1, 2.5 μM) using thermal cycling at 50°C for 5 min and 95°C for 10 min, followed by 50 cycles at 95°C for 30 s and 53.3°C for 1 min. To test the sensitivity of primer and probes, the limit of detection (LOD, i.e., the minimum amount of target DNA sequence that can be detected in the sample) and the limit of quantification (LOQ, i.e., the lowest amount of target DNA that yields an acceptable level of precision and accuracy) were calculated by running a dilution series of a known amount of total Bsal DNA, ranging from 100 ng/µl (1.83 x 104 GE/µl) to 10–10 ng/µl (1.83 x 10–8 GE/µl) with 12 qPCR replicates per concentration below 10–3 ng/µl (1.83 x 10–1 GE/µl). Total Bsal DNA was extracted from a 5‐day‐old culture containing both sporangia and zoospores using Prepman Ultra reagent (Applied Biosystems), and the final DNA concentration was measured using a NanoDrop spectrophotometer. LOD and LOQ were calculated using the method proposed in Klymus et al. (2019).
The final validation was performed by spiking two liters of distilled water with a known amount of Bsal zoospores. Bsal zoospores in distilled water were collected from the Bsal type strain AMFP1, grown in TGhL broth as described before (Martel et al., 2013). The water samples were spiked with one, 10, 100, 1,000, or 10,000 Bsal spores, and three samples replicates were made per spore concentration.
Study site
A garden pond has be sampled (water surface 58 m2). In the pond, reproduction of smooth newts (Lissotriton vulgaris), alpine newts (Ichthyosaura alpestris), and common frogs (Rana temporaria) occur annually. Other present, but not reproducing species are common toad (Bufo bufo) and water frogs (Pelophylax sp.). These anuran species are considered to be resistant to a Bsal infection (Martel et al., 2014).
Sampling protocol eDNA
Bsal produces motile zoospores and environmentally resistant, encysted spores that float on the water surface (Stegen et al., 2017). Therefore, water samples were collected only from the top 1 cm of the water in 2018, but prior to the Stegen et al. (2017) publication, water samples were also collected from 5–10 cm depth. The eDNA sampling was conducted before the swab sampling of the individual animals (Table 1) on the same days
The field sampling for eDNA was performed following the protocol described in (Miaud et al., 2019). The sampling kits were composed of a sterile water sampling ladle, a self‐supporting sterile Whirl‐Pak® bag, a sterile syringe, gloves to minimize contamination, a VigiDNA® 0.45‐µM cross‐flow filtration capsule (SPYGEN), and a bottle containing 80 ml of CL1 Conservation buffer (SPYGEN). Using the ladle, subsamples of 100 ml water were collected around the pond margin to create a pooled water sample of approximately 2 L in the sterile self‐supporting plastic bag. Samples were collected while the surveyor stood only on the pond bank or on muddy pond edges without entering the water to avoid possible contamination from the surveyors’ boots, or by stirring up sediment. The water sample was homogenized by gently shaking the bag to ensure that eDNA was evenly mixed through the sample, and then the 2 L of sampled water was filtered through the VigiDNA 0.45 μm filter using a sterile 100 ml syringe, directly in the field. Subsequently, 80 ml of CL1 conservative buffer was added to the filter and the filters were stored at room temperature for a maximum period of six weeks before the DNA extraction. The DNA extraction and amplification were performed as described above in the primer and probe validation section.
Sampling individual animals using swabs
Smooth newts and alpine newts were captured using two to six amphibian fykes (type: Laar M2, rectangular 30 x 30 x 50 cm, mesh size 4 mm, distribution: Laar Technology & Consulting Ltd.), that were placed at the edges of the pond for max 12 – 16hr and were left overnight. The owners did not want dip nets to be used and search intensity varied per field visit. During each sampling event, the garden (60 by 20 m) was searched for newts in their terrestrial phase as well. Ventral skin swabs were taken from postmetamorphic newts, using aluminium sterile cotton‐tipped dry swabs (rayon‐dacron, COPAN, UNSPSC CODE 41,104,116) following the procedure and biosecurity measures described in Hyatt et al. (2007) and Van Rooij et al. (2011). All samples were kept frozen at − 20°C until further analysis for the presence of Bsal DNA through real‐time PCR, as described by Blooi et al. (2013). Skin histopathology as described in Martel et al. (2013) was performed to detect Bsal infection on dead newts. The aim during each site visit was to collect and sample at least 30 and maximally 60 specimens/visit.
Results
The primer pair and the probe showed 100% specificity both in silico and in vitro. The LOQ in this study was 4 GE/µl, and the LOD was 2.93e‐06 GE/µl. The threshold cycles observed in the spiked water sample were below the LOQ, and thus it was not possible to correlate the number of spores with the quantity of DNA retrieved. For this reason, the number of replicates amplified in a sample has been used for relative quantification, rather than reporting the amount of eDNA detected quantitatively, as suggested in Biggs et al. (2015). The number of replicates was positively correlated with the number of spores spiked in the water sample (ANOVA p‐value = 0.00245, Figure 1).
Discussion
The aim of this study was to test the potential of eDNA sampling for Bsal range delineation, and scientists have shown that this technique can be applied to detect Bsal in stagnant water.
A noticeable outcome is the detection of Bsal via eDNA but not via the swabs (Table 1), which could be explained by a low prevalence of the fungus. To detect a pathogen that occurs at low prevalence, sampling of a large number of individuals is required (DiGiacomo & Koepsell, 1986). At the time of the sampling (April 21, 2018) only 20 swabs could be collected (no more individuals could be caught). To detect Bsal with this limited sample size, the Bsal prevalence should range, depending on the level of confidence, between 15% and 20% (DiGiacomo & Koepsell, 1986). This prevalence is rather high for endemic situations and has so far only been observed during Bsal outbreaks in fire salamander populations and in Asian urodelan populations (Dalbeck et al., 2018; Laking, Ngo, Pasmans, Martel, & Nguyen, 2017; Spitzen‐van der Sluijs et al., 2016; Stegen et al., 2017; Yuan et al., 2018). The objective to minimally sample 30 individuals/visit was only reached twice during the entire study, and this unpredictability is inherent to fieldwork.
The fire salamander is a mainly terrestrial species, and as such the here described eDNA technique is less suitable to set the range of Bsal in terrestrial environments and in fire salamanders or other terrestrial species, yet the technique will be useful to demarcate the distribution of Bsal by Bsal‐vectoring and susceptible newt species in their aquatic phase. These results highlight the high detection capability and advances of the eDNA technique. It also shows it is a useful detection method complementary to the collection of skin swab samples. Collecting swabs remains necessary in active pathogen surveillance for detecting prevalence and infection intensity. Equally, passive surveillance for dead or moribund animals remains indispensable.
It has been shown that eDNA sampling can be used to detect Bsal in water and as such the technique may be further validated to play a role in Bsal range delineation and surveillance in both natural situations and in collections. eDNA‐based methods allow for rapid, reliable, and cost‐effective screening for Bsal and can therefore be applied to monitor pathogen presence or absence in high‐risk invasion areas or in collections. Also, eDNA‐based methods will help to delineate the outbreak and allow for the evaluation of the effectiveness of management measures.
References