Using Terminal Transferase-mediated dUTP Nick End-labelling (TUNEL) and Caspase 3/7 Assays to Measure Epidermal Cell Death in Frogs with Chytridiomycosis

James Cook University approved animal ethics in applications A1875 for P. corroboree and A1897 and A2171 for L. v. alpina.

1. Animal Husbandry and Monitoring

1. House animals individually, in an environment appropriate for the species, with an appropriate water, feeding and cleaning schedule. Check animals daily.
   1. Use adult individuals of the critically endangered Pseudophryne corroboree (for the TUNEL Assay, Section 4) and the threatened Litoria verreauxii alpina (for the Caspase 3/7 assay, Section 5), donated from captive breeding facilities. Maintain the animals at 15 – 18 °C, on a moss and gravel substrate, mist them daily with reverse osmosis water, and feed 3 times weekly with gut loaded crickets.
2. Collect data on each individual once weekly. Swab the individuals for Bd as described in Step 2.1. Weigh each animal to the neared 0.1 g using a scale, and measure their snout to vent (SVL) length to the nearest 0.01 mm using dial callipers.
3. After inoculation (as described in Section 3), check animals daily for clinical signs of infection (irregular skin slough, inappetence, leg redness, lethargy, or delayed righting reflex) in compliance with animal ethics guidelines. Euthanize animals if clinical signs and morbidity are present.
   1. Euthanize with an overdose of tricaine methanesulfonate (MS222) (0.1% w/v MS222 to pH 6 - 7 with sodium bicarbonate in aged tap water). Keep animals in MS222 for 10 min after all motion ceases and they display no response to stimuli.

2. Testing for Bd Infection

1. Swabbing for Bd
   1. Swab each animal with a new sterile rayon swab (one swab per animal). Use the following swabbing method: five times on the venter, five times on each thigh, side, and limb. This is a total of 45 strokes.
   2. Gently rotate the swab during and between strokes to ensure effective capture of DNA. Break the swab tip off into 1.5 mL microcentrifuge tubes and store at -20 °C until extraction.
2. DNA Extraction and qPCR assay
   1. Extract genomic DNA from the swabs by adding 50 µL of a commercially available DNA extraction reagent with 30 – 40 mg of 0.5 mm silica beads. Bead beat the samples in a bead beater cell disrupter at maximum speed for 2 min. Following the bead beater step, centrifuge the samples at 2,000 x g for 1 min.
   2. Incubate the samples at 100 °C for 10 min, and allow to cool. Centrifuge the samples at 5,000 x g for 3 min, and then transfer the supernatant to individual 1.5 mL micro centrifuge tubes and store at -20 °C until qPCR.
   3. Use quantitative PCR (qPCR) following Boyle et al. 2004¹⁸ to analyze the infection load. Use the following modifications:
      1. Dilute DNA extraction samples 6:100 with double deionized water. Add 0.7 µL of bovine serum albumin (BSA) to each well to help prevent PCR inhibition. Run each sample in singlicate. On each plate use a negative (no template) control and a positive control with a series of dilution standards to estimate zoospore (infection) load.

3. Inoculation

1. Culture Bd
   1. Prepare culture broth by adding 16 g of tryptone, 2 g of gelatin hydrolysate, and 4 g of lactose (TGHL) to 1 L of deionized water. Autoclave (121 °C for 40 min) and allow broth to cool.
   2. Inoculate Bd isolate (in this case, isolated from New South Wales isolate, AbercrombieR-L.booroologensis-2009-LB1, Passage number 11) in a TGHL broth and grow for 7 d at 23 °C, then transfer the broth culture to TGHL agar plates.
   3. To make plates, add 16 g of tryptone, 2 g of gelatin hydrolysate, 4 g of lactose (TGHL), and 10 g of bacteriological agar to 1 L of deionized water and autoclave (121 °C for 40 min). Once the mixture is cool enough to touch, but before it solidifies, pour the TGHL agar into 92 mm diameter culture plates so they are 1/4 to 1/3 full, in a Class II biological safety cabinet.
   4. When the agar has solidified and cooled completely, inoculate each plate with 0.5 mL of Bd from the liquid broth, and spread evenly. Allow Bd broth mixture to dry on plate for approximately 1 h and then seal plates with plastic paraffin film. Incubate plates agar side down at 23 °C for 5 – 7 d.
2. Flood plates for zoospore inoculation solution
   1. Check zoospore motility under an inverted light microscope to ensure viability daily before inoculation. When zoospore release is high, with many zoospores swimming outside the sporangia, the culture is ready for inoculation.
   2. Flood each plate with 3 mL of aged tap water or artificial pond water, by pouring the water into the plate and incubating at room temperature for 10 min to allow the zoospores to release into the water. After the incubation period, decant the zoospore suspension into a new sterile container.
   3. Estimate zoospore concentration following manufacturer's instructions using a haemocytometer. Once the concentration of the zoospore suspension is known, dilute the suspension to a concentration of 1 x 10⁶ zoospores per 3 mL with aged tap water.
3. Inoculate animals
   1. Inoculate each animal by pouring 3 mL of inoculum mixture over its venter¹⁹ in individual 50 mL containers. Allow excess inoculum to collect into the base of the inoculation container. Leave each animal in individual inoculation container for 24 h to ensure infection, and after the 24 h inoculation, return each individual to a disinfected terrarium.
      1. Disinfect terraria using a 13% volume/volume (v/v) commercial bleach solution²⁰, rinse at least twice with water, then allow to dry for no less than 24 h.
   2. For Bd-negative controls, mock inoculate the animals using the same methods as in 3.2 – 3.3, but flood Bd-free agar plates with 5 mL of aged tap water instead of Bd inoculated agar plates.

4. TUNEL Assay

1. Euthanize animals that are showing clinical signs of chytridiomycosis, as described in step 1.3.1, and an equal number of Bd-negative control animals.
2. Dissect skin (dorsal, ventral, and thigh) samples from each animal. Fix the skin samples for 2 h in 4% v/v phosphate buffered formaldehyde. The short and consistent fixing time allows the tissues to be fully fixed, but also allows for effective and accurate immunohistochemistry staining. Then transfer to 80% ethanol until embedding for sectioning.
3. Embed skin in paraffin wax for histological preparation following standard methods²¹. Briefly, the protocol is as follows:
   1. Dehydrate tissues in a graded series of ethanol, and clear the ethanol with xylene. Embed the tissue in paraffin wax, and place all three skin samples for each individual in one paraffin block.
4. Section skin using a microtome following standard histological preparation²¹. Section the tissue serially at 5 µm and then affix to hydrophilic glass slides. Place four serial histosections per skin type per slide. Make three slides per individual with serial skin sections, remember that each slide has four sections of each tissue affixed.
5. Stain the slides in the following order:
   1. Stain the first slide with hematoxylin followed by eosin counterstaining (H&E). This slide is to visualize the location of Bd sporangia within the skin.
   2. Stain the second slide following a commercially available TUNEL assay for histological preparation and follow manufacturer's instructions, which are described below.
      1. First, deparaffinize the tissue sections in a coplin jar by washing the slides with three changes of xylene for 5 min per wash, and follow with two changes of 100% ethanol for 5 min each wash. Follow with one wash of 95% ethanol for 3 min and then 70% ethanol for 3 min. Finish with one wash of PBS for 5 min.
      2. Pretreat the tissue with freshly diluted protein digesting enzyme (proteinase K at a concentration of 20 μg/mL diluted in PBS), and add directly to the slide. Allow to incubate for 15 min. Wash with two changes of PBS in a coplin jar for 2 min each.
      3. Tap off the excess liquid and quench in 3.0% hydrogen peroxide in PBS in a coplin jar for 2 min at room temp. Rinse twice with PBS, for five minutes each wash.
      4. Tap off the excess liquid, then apply 75 µL/5 cm² of the equilibrium buffer directly onto the tissue on the slide. Incubate for 10 s. Tap off the excess liquid around the section and apply 55 µL/5 cm² of working terminal deoxynucleotidyl tranferase (TdT) enzyme. Incubate in a humidified chamber for 1 h at 37 °C.
      5. After the TdT incubation, put the slide in a coplin jar of working strength stop/wash buffer, agitate for 15 s and incubate for 10 min at room temperature. Wash the slide with three changes of PBS for 1 min per wash. Tap off the excess liquid.
      6. Apply anti-digoxigenin conjugate (rhodamine) that has been warmed to room temperature to the tissue, 65 µL/5 cm². Incubate in a humidified chamber for 30 min at room temp and avoid exposure to light. Wash with four changes of PBS for 2 min per wash. Tap off the excess liquid.
      7. Finish by adding 15 µL of 0.5 – 1 µg/mL DAPI (4',6-diamidino-2-phenylindole) in slide mounting medium to the slide, which acts as a counter stain. Then cover with a cover slip and seal with nail polish or rubber cement. Allow slides to dry in the dark as the assay is light sensitive.
   3. Stain the third slide is stained using the TUNEL assay and DAPI counterstain, but use as a positive and negative control for each sample.
      NOTE: Of the 4 histosections on the control slides, the first 2 are positive control replicates and the last 2 are negative control replicates.
      1. For the positive controls, instead of step 4.5.2.2, pre-treat the tissue with DN buffer (30mM trizma base, pH 7.2, 4mM MgCl₂, 0.1mM DDT) and let incubate at room temperature for 5 minutes. Then dissolve Dnase I in DN buffer for a final concentration of 0.1ug/mL, and apply it directly to the slide. Incubate for 15 minutes at room temperature.  Then, wash the slide with 5 wash of dH₂O for 3 minutes each wash. Blot off the excess liquid. Then resume TUNEL assay as directed in section 4.5.2.3.
      2. For the negative controls, do not add the terminal deoxynucleotidyl tranferase (TdT) enzyme to those samples (as described in section 4.5.2.4).
6. Observe TUNEL slides immediately upon completion of the assay.
   1. Take photos at random intervals along each skin section at 200X using a fluorescent microscope, using filters for both the rhodamine stain, which reveals the apoptotic cells and appears red, and DAPI which reveals all nuclei and appears blue, so that an overlay of cells can be generated. Ensure at least 100 cells are photographed per skin section per sample. Store the slides in a dark microscope box at -20 °C.
   2. Count cells (blue DAPI stained) per image. Ensure at least 100 cells are counted per skin section. To reach 100 cells, count all cells within the image. If less then 100 cells are present in one image, count another image until at least 100 cells are reached per skin section per animal.
   3. Next, count the number of TUNEL positive cells in the same images (red rhodamine stained). TUNEL positive cells, which indicate apoptosis and not necrosis or background, are single cells with clear cell edges (membranes are intact with no lysis). Sometimes the cells shrink to form apoptotic bodies.
   4. Locate the areas counted in the TUNEL assay and analyze the corresponding regions on the H&E stained histosections. Confirm that the H&E stained sections correspond to sites of Bd infection, which ensures Bd-infected sites are used when counting apoptotic cells in the TUNEL assay.

5. Caspase 3/7 Assay

1. Collect toe tips from each animal (both Bd– exposed and Bd– negative) once weekly through week 3 post inoculation, and fortnightly through the end of the experiment, up to 8 toes per individual.
   1. Cut the toe tip at the second phalange with flame-sterilized scissors, and place in a 1.5 mL microtube and freeze immediately at -80 °C.
2. Extract proteins from frozen toe sample.
   1. Place samples in 100 µL of sample buffer (25 mM HEPES pH 7, 5 mM MgCl₂) with two stainless steel beads (3.2 mm) in a 1.5 mL screw cap microtube. Lyse samples by 4 cycles of 1 min bead beating at maximum speed (in the same bead beater as used in step 1.2.1) followed by 3 min on ice. After lysis, centrifuge samples at 12,000 x g and 4 °C for 5 min. Collect supernatant to use in the assay.
3. Quantify protein concentration per sample using the Bradford assay.
   1. In a 384 well plate, mix 10 µL of protein extract with 10 µL of Bradford reagent, and incubate for 2 min at room temperature. Perform each sample in duplicate, along with a series of 5 fold BSA dilution standards. Read the absorbance at 595 nm on an absorbance plate reader.
4. Alternatively, if the toe tip samples are too small (the protein concentration is near the lowest standard as determined from the Bradford assay in step 4.3.1, or if the frogs that are sampled are under 3 g total weight), standardize samples by estimating skin surface area using photographs, instead of the Bradford assay.
   1. Before extracting the proteins from the sample for the caspase assay, photograph each toe at 40X magnification using an inverted light microscope.
   2. Analyze the toe sample using a computer imaging software, by estimating area of the toe. Do this by drawing a line around the edge of the toe clip, and have the imaging software estimate area within the shape. Then multiply that area by pi (3.14), which will approximate the surface area of the 3-dimentional skin sample (as length x cross-sectional area (pi x diameter) gives the outer surface area of a tube).
5. Perform the caspase 3/7 assay.
   1. In a 384 well luminescence plate, add 10 µL of commercially available caspase 3/7 reagent and 10 µL of protein extract. Run each sample in triplicate.
   2. Mix the reagents by shaking the plate slowly for 15 s. Incubate the plate away from light for 30 min at room temperature. Measure luminescence using a luminescent plate reader.