Today I made my way across London to London Zoo once again. I was very excited because I knew that today we were going to be completing the analysis on the samples we began using on Monday. In order to do this, we were going to use Quantitative Real Time Polymerase Chain Reaction (qPCR). This has the purpose of amplifying and quantifying a targeted DNA molecule, hence why we had to extract the DNA initially. In this case, we were going to compare the quantity of Batrachochytrium dendrobatidis (the pathogen which causes chytrid) DNA in the samples to standards which have been determines according to the quantities of this DNA present in healthy amphibians. The steps were carried out as follows:
- Defrost samples of DNA which have previously been extracted from swabs.
- Dilute samples with distilled water
- I really enjoyed this step because I got the chance to try a digital pipette. Previously we had used manual micropipettes, where you press the button on top to the point of first resistance to load the set volume, ensuring no air bubbles are drawn up, then dispel the substance by pressing the button all the way down. It was also important to change the tip between every sample to avoid contamination. However, the digital pipette allowed a larger volume to be loaded initially and with a press of a button, the correct amount was delivered into the tubes. This meant that we could proceed quickly without the need to load between each sample. Furthermore as only distilled water was being drawn into the tip, the same one could be used for every sample.
- when diluting the sample it is important to ensure that it if fully mixed, therefore the pipette can be used to suck up then expel the solution several times after the water has been added.
- Create Master Mix
- the master mix is a mixture of reagents needed to prevent inhibition during PCR. It consists of 5 chemicals – water, Taqman, Forward primer, Probe and Reverse primer. Although these names (apart from water) meant nothing to me, it did not prevent me appreciating the importance of this step, especially after seeing the precise volumes of each chemical which are added to the tube before being mixed on a vibrator.
- Place Master Mix, samples, negative and standards in a 96-well PCR plate
- 96-well plate sealed with plastic sheet, ensuring no air bubbles and mixed in centrifuge.
- Place well-plate in PCR machine, set up the computer and leave to run for 2 hours
- during this process, the chytrid DNA will be highlighted with fluorescein so can be detected, creating a graph on which the results can be illustrated.
Whilst the PCR machine was running, we Emma decided to take me pet shop swabbing, for this was the only part of the process which I had not seen. There were only a few pet shops left to swab around London, but nevertheless we boarded a tube and headed to Upper Norwood to find Crystal Palace Reptiles. After entering the shop and talking to the owners, explaining what we were doing and why we proceeded to be shown around all the amphibians in the shop. It was a fantastic shop with over 80 samples to be taken. We made our way around the tanks, wearing gloves which had to be changed between every vivarium to prevent contamination. For each amphibian we brushed off any dirt before wiping the swab over the stomach and legs. The number of wipes taken did not have to be exact as we were testing for presence of chytrid rather than amount. For every sample we had to record the number of the sample, the number of the vivarium, the number of animals in the vivarium, the name of the species, whether they had been wild caught or captive bred and their country of origin. This was all important information in investigating the correlations between positive cases of chytrid. Some of the most interesting species I saw included bufos (toads), a Hong Kong warty newt and a fire salamander. We also saw hairy frogs, which weren’t particularly hairy, but are also very rarely seen in the pet trade as they are very vicious and the shop owners had to handle them very cautiously to avoid being bitten. Furthermore, the pet shop had recently acquired five caecilians from Cameroon as these worm-like amphibians are becoming more popular within the pet trade. They were in a tank full of moist soil and we had to dig around to try and find them although only succeeded in finding two of the five. One was near the surface and moving very slowly. It also had a lesion on its back. These were all clinical signs indicating chytrid as the fungus infects skin cells, causing them to break down leaving lesions in the surface. These can very easily become infected, leading to more problems. In addition to this, the fact that the other caecilians couldn’t be found suggests that they could be dead as when caecilians die, they quickly decompose in the rich, moist soil. Chytrid has recently be found to kill caecilians and previously Emma had found that many samples from individuals imported from Cameroon had tested positive. This was very exciting and indicated that we might have found some very interesting results.
Whilst doing this, it opened up some good opportunities to discuss the pet trade. There are many positives and negatives to the pet trade, many of which are highly controversial. I had not previously considered this and I hope to look into it further.
I later found out that many of the animals, including the caecilians, from Crystal Palace Reptiles tested positive for chytrid, and many were also found to have tuberculosis which is untreatable in amphibians. As a result the shop owners donated all infected amphibians to the ZSL to aid further research. Unfortunately, the decision was made to euthanize all animals to culture chytrid, however for many of them this was best as they were suffering with little chance of full recovery considering the number of them and the possibilities for research involved.