Testing Pathway
@Roba Olana
- Animal Capture and Sample Collection:
Procedure: (amphibiaweb.org, 2017)
- Preferably, capture amphibians by hand. Wear gloves when swabbing animals and change gloves between animals. If you are using a dip net, be aware that B.dendrobatidis zoospores could be caught on the net and transferred between individuals, therefore, use different nets whenever possible, or disinfect the net as often as you can (there is no perfect solution to this problem).
- Swab the underside or ventrum of adult/metamorphs 30 times. Remember you are in effect scraping small amounts of tissue from the skin. Some pressure must be applied, but this does not mean that you must squash the animal. Areas to target are the drink patch, thighs and webbing between the toes.
- Air dry the swab for approximately 5 minutes, avoid direct sunlight if possible (if conditions are too humid to air dry then store in 95% EtOH).
- Break swab ~3cm from tip and drop into empty screw cap tube. The swab stick should not touch or bump against the top of the vial. Screw the cap on the vial and store in the shade.
- Samples can be kept a room temperature for a week or maybe longer, but it is best to keep the samples cool and placed as soon as possible in a 4° C freezer (the kind you have at home is fine). Avoid extreme high temperature and direct sunlight. Samples may be stored in a freezer for many months without problems.
Videos:
DOD Amphibian Swabbing VideoThis video was created by Zoo Atlanta for the purpose of training Department of Defense biologists how to swab amphibians for the fungus Bd.https://youtu.be/a5CtPrGOK8c
How to collect a chytrid swab from a frog or toadA short training video on how to swab a frog or toad for chytrid. Presented by Dr. Vance Vredenburg, San Francisco State University. Filmed and edited by Hei...
How to collect a chytrid swab from a salamanderA short training video on how to obtain a chytrid swab from a live salamander. Presented by Dr. Vance Vredenburg, San Francisco State University. Filmed and ...
How to swab a preserved amphibianA short training video on how to obtain a chytrid swab on a preserved amphibian. Presented by Dr. Vance Vredenburg, San Francisco State University. Filmed an...
- Comparison of methods for detection of chytrid fungus: (KADEKARU, UNE , 2017)
- "...we compared Bd detection rates in swab-scraped and resected mouthpart samples, using nested polymerase chain reaction (PCR). The resulting detection rates for swab-scraped and resected specimens were 67 and 65%, respectively, with no significant difference. Furthermore, we performed a histopathological examination for Bd distribution in the mouthparts; we found that Bd infection occurred in the tip and basement of the jaw sheaths and tooth rows. We recommend using swab-scraped samples for Bd detection. Moreover, careful attention should be paid to scraping the tip and basement of the jaw sheaths and the entire oral cavity to reduce the rates of false-negative results on nested PCR of the mouthparts of bullfrog tadpoles."
- Swabs are originally placed in a sterile 2.0 microcentrifuge tube and stored on ice until they could be stored at 4°C in the laboratory.
- The temporal gap between collecting the samples and analyzing could range from hours to years depending on the need for testing
- For example, in (D. IWANOWICZ et al, 2017) samples were extracted within four months of collection and analyzed for Bd by qPCR in 2012. All extracted DNA from these 2012 archived samples have been stored for three years at -20°C prior to Bsal analyses for this study in 2015.
- Postmortem testing: directly after death of an animal, it was transferred to a passively aerated plastic container with moist soil and leaf litter to mimic conditions under which dead Bsal-infected salamanders were found in the field. The containers were placed in a climate-controlled room set at 15°C. Directly after death, and after 1, 2, 3, 4, 5, 7, and 10 days, samples (swabs and tissue samples) for the detection of Bsal were taken. Each sampling occasion consisted of collecting swabs of the ventral and dorsal skin by placing a swab on the skin and rotating it in place for 20 full rotations, collecting full-thickness single skin samples of the ventral and dorsal skin with the use of a 3-mm biopsy punch (Stiefel, Germany) and collecting a tail clip of the animal (tissue samples were weighed to allow correction for variability in tissue size). All samples were taken from new, unsampled areas of skin to avoid interference in the detection of Bsal. Swab samples and tail tissues were stored at 20°C and analyzed for the presence of Bsal with use of a Bsal-specific simplex real-time PCR (Blooi et al., 2013). Skin biopsy samples were stored in formaldehyde and processed for histological examination (haematoxylin and eosin [HE] stain) for the detection of Bsal. (Thomas et al, 2017)
- Costs associated with equipment (also represents equipment personnel take with them to the field):
- Nitrile gloves
- Vials: $214.00 - $305.00 for 500 tubes
- Sterile cotton swabs: $392.50 for 1250 swabs
- Plastic bags
- Costs and time associated with labor in field (not quantified):
- Identification
- Capture
- Sampling
💡Important to note that current procedures (including labor) are based on diagnostic methods with low turnover rate, time, and expert sample collection in the field. Value Proposition
- Wet lab work:
- Accuracy: (Thomas et al, 2017) | Standard (OIE, 2019)
- Steps and associated cost and time:
- Dip in buffer solution
- What the buffer solution is
- How much is costs
- For how long
- Potentially helpful: (Strofer et al, 2009)
- Dip in buffer solution
- Result based action