- Dirk, Adeline
Wildlife diseases - insights into lab work
Updated: Dec 14, 2019
Have you been wondering, how scientists can check for tiny little pathogens invisible to the naked eye? In the past, microscopes have been used to magnify what was invisible. But carrying a microscope into the mountains is (by far) not very practical.... Nowadays, things are done a bit differently and the film below shows how.
Of course, first there is the sampling, and that is the fun part of all. Even though it is not always easy to find the target hosts and that in sufficient numbers. We often aimed to at least catch and sample 30 host individuals. That is, because this way we have a 95% chance to detected infections as low in prevalence as 5% in an infinite population (which means when only 5 individuals out of 100 are carrying the disease). There is, however, still the probability that we miss an infection in a population, in that case we talk about false negatives. Anyway, in our other blog entries you can see and read how our fieldwork looks like. Here, we give you an insight in our lab work.
After hours of sampling in the field, it is hours and hours of lab work to analyze our samples. Each sample is a swab, a sterile cotton-bud which was gently rubbed against the amphibian's skin. If an amphibian is infected with the pathogen, the individual has DNA of the pathogen on its skin, so when we swab the skin, we also take the pathogen DNA on the swab, and then check in the lab if there is pathogen DNA or not on that swab. So, what do we do in the lab? To see if an individual is infected or not by a pathogen, there are several steps. We need to organize our samples and put them in reaction tubes. We need to be careful not to mix them up. Then we extract the DNA from each sample and place each of them on a "plate" comprising 96 wells (we call that a 96-well plate). This is a long and repetitive process. Each sample has its own well, and we need to be very careful not to mix the different samples, to avoid what we call "cross contamination". A single piece of DNA in the wrong well, and our sample is cross contaminated.
Then we need to find the right qPCR programm. What is qPCR? The q stands of quantity and PCR for polymerase chain reaction. A PCR is a process in which even minimal amounts of DNA will be amplified thanks to an enzyme (a protein) by multiplying the DNA during a repetitive automated process. To activate and inactivate the process, the enzyme and DNA are submitted to specific temperatures during specific periods of time, that have to be carefully chosen (machine programming). In a qPCR, we also put, in the machine and in specific wells, known quantities of the pathogen DNA, they are called standards. This will allow the machine to calculate the amount of the pathogen DNA in our samples. At the end, the machine delivers the curves you see towards the end of the video (and below). Once we have that information, we know which of our samples has tiny amounts of pathogen DNA in it. This way, when we have been careful, we will know which individual from where and when has been infected. The overall results tell us if a population is at risk of local extinction or if the infection prevalence is stable or if we actually are observing a process of a species extinction.
Climate change and human impact are known factors influencing the occurrence of pathogens, especially in mountain ecosystems. P³ is investigating why, where, and how pathogens spread and what harm it does to mountain freshwater ecosystems.
Here now the video: