Monitoring Public and Recreational Waters for Parasites

Q&A with Dr. Patrick Hanington from the University of Alberta

Parasites may get a bad rap overall, but they play a vital role in healthy ecosystems. In this episode, we focus on the role parasites play in freshwater ecosystems. Specifically, we’re talking about avian schistosomes, a very interesting parasite that infects waterfowl and also uses snails as a host in its larval stage. Larvae also infect humans to cause what’s known as swimmer’s itch.

To guide this conversation, we have Dr. Patrick Hanington, associate professor in the School of Public Health at the University of Alberta. As a self-described parasitologist and immunologist, he and his team focus on developing multiplexed PCR-based tests to detect freshwater parasites, including avian schistosomes. Their work benefits locals in his area by monitoring pubic and recreational waters for swimmer’s itch outbreaks, but schistosomiasis is the second-most prevalent human disease worldwide behind malaria and their work also serves as a model for informing human schistosome research.

In our conversation with Patrick, we learn about how they design their assays and why they’re increasingly using dPCR instead of qPCR. Beyond the technical work, we get into how Patrick’s career path developed, how what he loves most about his job has changed and evolved over time, his lessons learned in the lab, and how his research and hobbies have blended over time. And because it’s Absolute Gene-ius, you know we keep it fun with some unexpected movie references and a bit of discussion about how science is represented in television and film.

To learn more, listen to the Absolute Gene-ius dPCR Podcast Episode at www.thermofisher.com/absolutegeneius

Transcript from Absolute Gene-ius episode three “Avoiding the itch with digital PCR”. This transcript has been edited for brevity and clarity.

Parasite detection in freshwater ecosystems

Question: When it comes to detecting parasites in both public and recreational waters, what does that look like? What exactly do you do in terms of looking for parasites and things in water samples?

Dr. Patrick Hanington: We work on a group of parasites that are related to human schistosomes, which are parasites that can cause disease in tropical regions of the world. The ones that we work on, though, in North America, cause swimmer’s itch. It looks like a bunch of mosquito bites. Swimmer’s itch can last for two weeks and is very unpleasant, especially if it happens to young kids.

These parasites are transmitted by freshwater snails. You have to have a snail to continue the lifecycle of the parasite. And for the ones that cause swimmer’s itch, the next host in the parasite lifecycle is usually waterfowl. It’s looking for a bird that’s in the ecosystem and it’ll try to penetrate the skin of that bird. If a swimmer is in the water, they’re a casualty of the process.

Historically, the way that people would look for those parasites was to look at the snails. They would collect a whole bunch of snails and look to see, you know, which snails are releasing what parasites. It works, but it’s a complicated process and there’s a lot of other parasites that go through those snails.

So, it’s really difficult.

Snails and parasite production

Question: When you’re looking at the snails, do you look at the actual snail and kind of project how much potential parasite there might be? Are you just looking to detect the parasite? What does that look like?

Dr. Patrick Hanington: It’s actually a pretty cool process. So, the snail eventually is just hijacked by these parasites. It becomes a little factory that produces the parasite. It can be a pretty insane process. Up to 25% of the snail’s biomass can be taken up by the parasite.

These larval stages look like microscopic tadpoles and they just come out of the snail through the tissues, just right out of the snail. And there’ll be thousands of them.

Explaining parasites with Alien and other movies

You know, when I teach parasitology to students, one of the things that I usually use is an analogy of the movie Alien, where the alien’s bursting out of Ripley’s chest. And I’ve realized that lots of students nowadays have not watched Alien.

Like that movie is not on the must-watch list of students now, so that that analogy doesn’t go anywhere. But it’s a perfect analogy for what’s happening to these snails because the parasites are just like tearing through them. I always feel like I need to assign a required set of moves for the students to watch before coming to class so that all my stories make sense to them.

Question: Do you make them watch Contagion as well? Is that on the list? When I was in virology in college Contagion was the movie that that we were required to watch. I remember that pretty vividly.

Dr. Patrick Hanington: I should put that on there. Yeah, you know, the one that came became before Contagion – Outbreak – and it had Cuba Gooding Jr. and Dustin Hoffman in it.

That one was an awesome movie to use as a sort of a reference point for how technically specific a movie could get about scientific procedures because there’s this part where they find out that a monkey brought this virus into the United States and Cuba Gooding Jr., he’s playing a scientist in the show, and he’s like, “We’ve got to get this back to the lab and do an ELISA.” That’s such a specific scientific test that he’s talking about.

That’s the only movie I could think of where they actually ever said “I’m gonna go back and do an ELISA.”

Parasite lifecycles and the effect on snail hosts

Question: So, I’m relatively ignorant when it comes to parasites. You say that these things sort of burst out of the snails but, what’s that really like? Are they harmful to the snails? Are the snails exploding or is it not harmful to them?

Dr. Patrick Hanington: There is certainly some negative effects on the snail. But when we bring a snail that’s infected into our lab, and we have like a whole room that’s just for maintaining snails, we can keep them alive for months. They’ll live like that and produce parasites almost every morning.

The parasites come out of the snail when the sun comes up and they’re positively phototactic. They’ll rise to the surface of the water and they’ll just sit there and if they encounter a bird, or a swimmer for that matter, they’ll penetrate through your skin. And within a minute they’ll be in your skin, so it doesn’t take a long time for them to get in.

If you’re working with human parasites, then the parasite will enter the bloodstream and move around and develop into an adult worm, but for the ones that cause swimmers itch, they just get killed in your skin and there’s a local inflammatory response. Our goal was really to try to find a better way to evaluate the risk of swimmer’s itch at a recreational water site. That’s what got us into this track of applying a recreational water quality monitoring lens that is often used for monitoring for things like enteric bacteria.

Using PCR, qPCR, we were able to piggyback off of an existing publication that developed a test that would detect all of the species of swimmers-itch-causing parasite from a water sample. Then, we further validated that test to allow us to quantify those parasites and standardize sample collection.

So, when we go out and sample now we use a 20 micron zooplankton collecting net. This is a three-foot-long mesh net with a cup at the bottom. We pour a set volume of water through that, and then it all collects into this bottom cup that we can then pass through a smaller filter that concentrates everything that was in that water sample onto a little filter disk that we can extract DNA from. Then we just run qPCR to analyze that sample and quantify the number of parasites. W

hat we’ve tried to do is evolve from the test that just allows us to quantify all the swimmer’s-itch-causing parasites to get down to the level of species identification using PCR.

Question: When you’re using PCR for things like this, do you have to have multiple different targets that you’re that you’re looking at to identify these parasites? Or is it one that’s a species-conserved region that you’re looking at?

Dr. Patrick Hanington: Yeah, it’s, it is a single gene target that we look for. But we’re often looking at slightly different regions of that gene for each of the different species of the parasite. So far, that’s worked out okay. And luckily, these parasites are relatively distinct within an aquatic ecosystem compared to all the other things that are in a water sample.

And that’s one of the biggest challenges for us doing this sort of level of environmental microbiology or parasitology: we have to be really careful that we’re not cross reacting with all the other possible organisms that are leaving behind DNA or present in that water sample. There’s a lot of test validation that has to be done and we need to have known quantities.

We have to have pure specimens of each of the different species that we’re trying to detect and all the ones that might cross react. It’s taken a lot of time for us to develop and accumulate all that sample material that allows us to go in and develop these tests now. We now have almost 100 different species of flatworm parasite DNA samples.

That’s really helpful because we can then use those to confirm that we’re not going to get false positives or false negatives or things like that on our tests.

Goals and implications of parasite research

Question: Now, ultimately, what are your goals when you’re looking at this? Is it just to understand and research more of the parasite? What exactly is the ultimate goal for your research?

Dr. Patrick Hanington: One of them is just to understand more about the specific swimmer’s-itch-causing parasites, these avian schistosomes. And the reason for that is because I think they’re interesting parasites but they also, working on those parasites, allows us to make a lot of inferences about the human parasites which we also work on in my lab. From an ecosystem perspective, we can test a lot of hypotheses about environmental factors and different drivers of the abundance of those parasites in an aquatic ecosystem.

We can test that using swimmer’s-itch-causing parasites, which are much lower-consequence and a lot easier to design experiments on than human-disease-causing parasites from the tropics. It’s a nice system to test hypotheses about how these parasites behave in an aquatic ecosystem. And then I think, more broadly, at a sort of a bigger picture level, it’s really emerging that we’re able to use these parasites more broadly, not just the schistosomes, but all the flatworm parasites that are in a sample as an indication of aquatic ecosystem biodiversity.

So, we can predict the birds that are present, and we can predict the mammals that are present. Often, some of those parasites are going through aquatic invertebrates. And so, as we piece together all of the species that we have in our aquatic environments, then we can use something like eDNA meta-barcoding to create a parasite profile for that aquatic ecosystem, and then predict what the aquatic ecosystem biodiversity is.

It’s a way of unifying around a single sort of sampling strategy. But you get this huge, comprehensive profile.

Environmental impact and invasive species

Question: Something like, say you had some sort of invasive species come in, would that greatly shift the meta-barcoding? I’m assuming these things would change over time, depending on how the ecosystem itself will change. Yeah?

Dr. Patrick Hanington: Yeah. And we just got a grant from one of our provincial funding organizations to look at exactly that question about how invasive species influence this sort of aquatic ecosystem broadly. It relies on eDNA meta-barcoding and we compare it to digital PCR assessments of the presence or absence of those aquatic invasive species.

We are comparing the very high sensitivity, high specificity of digital PCR assessment to what is probably slightly lower sensitivity and specificity of meta-barcoding, just because you have some organisms are going to be very abundant in an aquatic ecosystem eDNA sample and you might lose that signal from what is a rare invasive species.

For an early detection type of an approach, digital PCR could be a really powerful tool. If you have the right information, and you can continue to survey for those organisms using digital PCR, I think it’s a really exciting tool.

Question: Patrick, I know you know we are big digital PCR fans here at Absolute Gene-ius. Can you elaborate a little more how you utilize digital PCR in your workflow?

Dr. Patrick Hanington: Like I mentioned earlier, we have a lot of experience using qPCR, quantitative polymerase chain reaction. It was just a few years ago now that we realized some of the limitations of using qPCR in an environmental context. A lot of them have been addressed by digital PCR.

The big one that we often struggle with is PCR inhibitors in a sample because we’re often working with water samples, and you guys probably haven’t been to Alberta before, but the lakes here are pretty gunky.

Question: Why even for clear water, I can imagine you get you get mud, you get sediment, you get plants, you get lots of stuff in there?

Dr. Patrick Hanington: Well, we have cyanobacterial blooms in every one of our central Alberta lakes. We’re always wrestling with PCR inhibitors from cyanobacteria. And you know, we can do cleanup procedures of the DNA when we do the DNA extraction, we can try to clean that sample up a little bit.

And often those work pretty well to get rid of inhibitors, but it doesn’t take much to impact the qPCR reaction. One of the big advantages of digital PCR, from our perspective, is that it does a much better job of minimizing the impact of inhibitors on a reaction. That is the number one big advantage for us in the environmental space, I think.

And then the other is that we’ve actually found that it’s a little bit easier to multiplex than the qPCR reactions are. So, that’s become really advantageous for this invasive species work that we’re doing. This idea that we can create these customized digital PCR panels for particular invasive species groups and just run a single digital PCR plate to assess for all four of them is really exciting.

Advantages of digital PCR in Multiplexing

Question: What benefits does digital PCR allow in that in that multiplexing area?

Dr. Patrick Hanington: Related to that question, one of the really big advantages that we find with the Absolute Q system is that we can use a qPCR assay on the Absolute Q system. So, we can do a lot of the sort of traditional validation steps for the assay performance and all that kind of stuff. And then we move it on to the Absolute Q system, and it works fine.

In terms of multiplexing, we always struggle with the presence or absence of inhibitors, but an additional part of our challenge sometimes is that we’re working with assays that that can cause problems during multiplexing depending on the fluorophore or what we’re amplifying. We’ve often defaulted to just always running a single fluorophore assay with the qPCR. It’s not that we can’t multiplex on the qPCR platforms, but there’s a lot more that we have to do in terms of compensation and figuring out exactly how the two different assays perform with each other.

The thing that we found with the Absolute Q was that that’s essentially set up so that the three, three of the fluorophores for sure are very distinct and then the fourth one is usually also pretty good. We find that it’s a lot easier to just run those four and we can confirm that there’s no bleed over a lot easier with that software. So, it’s just a very simple multiplexing platform.

And because of the simplicity of the way that the system works, generally, it’s just really easy for us to just load it up and run the samples with four different fluorophores in there and then get the data we need from all four. With the qPCR, we often found that we had to work on optimizing enzyme amount versus DNA that we loaded into the reaction, versus the presence of like all the different components of the reaction, and Absolute Q system just works a lot easier to do that multiplexing.

We’ve played around with how much DNA we load versus water versus enzyme and all that stuff in our dPCR system, and we basically found that like, the amount that it says to use is just the right amount to use. So, it’s a lot easier that way. That’s helpful just because, often, within our lab we’re you know, it’s MSc students or PhD students or sometimes even undergrad students that are working on this stuff.

And it’s helpful just to know that you can multiplex with these four things and often that you’re going to be okay, and that it’s really easy for them to go in and know whether or not there’s a problem rather than having to have like all that technical background of understanding how it works, the qPCR system, part of it is that it’s a lot easier.

End of Transcript.

To get more information and hear Dr. Hanington’s career advice, listen to the Absolute Gene-ius dPCR Podcast Episode at www.thermofisher.com/absolutegeneius

Learn more about digital PCR at www.thermofisher.com/absoluteq