April 14, 2022 — Dr. Kelly Diehl speaks with Dr. Michael Povelones from the University of Pennsylvania about mosquitos and heartworm disease in animals. They talk about the role of the mosquito in disease transmission and new research avenues focused on this pathway as a new preventive measure. Not only could this research impact heartworm disease in animals but it could have translational value for other deadly mosquito-borne disease such as malaria and dengue!
Resources:
American Heartworm Society
Morris Animal Foundation grant – Using a heritable immunostimulatory bacterium to block heartworm transmission by mosquitoes
0:00:05.7 Dr. Kelly Diehl: Welcome to Fresh Scoop, Episode 43, Unraveling the Complexities of Heartworm Disease. I'm your host, Dr. Kelly Diehl, Morris Animal Foundation, Senior Director of Science and Communication. And today we'll talk to Dr. Michael Povelones. Dr. Povelones is an Assistant Professor in the Department of Pathobiology at the University of Pennsylvania School of Veterinary Medicine. Welcome, Michael.
0:00:33.3 Dr. Michael Povelones: Thank you. It's a pleasure to be here.
0:00:35.9 DD: Before we get started, can you tell us a bit about yourself and what led you ultimately to the heartworm study that we're going to discuss?
0:00:45.8 DP: Yeah, that's a cool topic, because I didn't come straight into it. I've always been super interested in basic research, and so during my graduate studies, I was focusing on how cells communicate with each other. And when I was doing this in graduate school, it was in using a really basic model that's used a lot. I worked on fruit flies and some really exciting studies were being done in mosquitoes at the time, and so when I was thinking about how I would continue in research, I was drawn into the mosquito field, the genome was sequenced, and there were ways to manipulate mosquitoes in the laboratory that were really interesting. So, I got interested in mosquitoes and the topic that I focused on in my post-doctoral research, which I did in London at a top university there, with some people who are real innovators in mosquito research. So, I was in the laboratory of Dr. Fotis Kafatos and George Christophides at Imperial College in London.
0:02:05.9 DP: And we focused our attention on the mosquito immune system, because it has a pivotal role in protecting the mosquito against infections by pathogens that mosquitoes transmit. And so, our interest, because of the toll it takes worldwide was with malaria parasites. So, for a number of years, I focused my attention on the mosquito's immune system, and we did a very detailed basic research on how the mosquito's immune system can recognize and kill malaria parasites. And the overall concept there is that we want to be able to make mosquitoes that are resistant to malaria parasites and that might aid in preventing malaria transmission. So, as a post-doctoral researcher, I was very interested in other pathogens that mosquitoes encounter, but I never really had the opportunity to look at anything outside of malaria. As I joined the faculty here at the University of Pennsylvania here at the vet school, I then had the opportunity to look at a really interesting disease that's transmitted by mosquitoes, and so that's heartworm.
0:03:21.2 DP: And I was very interested in this, because it's a totally different kind of parasite than what I was previously studying. Malaria parasites are a single-cell protozoan parasite, whereas heartworms are multicellular animals essentially living inside of a mosquito. And so, I was really excited to open this new chapter of research, and I'm super enthusiastic to talk to you about it today.
0:03:45.7 DD: Oh, it's great, and I appreciate you reminding everyone listening, most of us are veterinary or animal health interested in that, but of course, mosquitoes transmit lots of different diseases. I think we are a little lucky in the United States, though, all of us know that those diseases are moving in and we're seeing more of them. To loop back to heartworm disease, and I'm going to again, date myself a little bit. I think all of us who grew up in a specific era, [chuckle] maybe even now, can remember seeing those gross pictures, like your vet would say, "Here's why heartworm preventive is important," and they'd have a picture, or my vet even had like a jar with a heart and heartworms, a great visual aid, which is either gross or fascinating, depending on whether you're going to be a vet or not. But even though I think a lot of us know about heartworm and maybe the basics, it's probably worth doing a quick review, if I can put you on the spot of the life cycle of the heartworm and how pets get infected, because it has a bearing on your work?
0:04:48.6 DP: Certainly, yeah. A very mosquito-centric point of view [chuckle] on this topic, but I'll start with, so the infection begins when a mosquito lands on a host, on a dog, an infected mosquito, and it's doing this to obtain blood so that it can make eggs and reproduce. So, the infected mosquito lands on the skin of the host and deposits infectious larvae onto the skin of the host. And when the mosquito flies away, those larvae can migrate into the tissue, into the skin, into the subcutaneous tissues. And over a long period of time, maybe six to nine months, those larvae find their way into the vasculature, eventually into the pulmonary artery, and there they find their permanent home.
0:05:39.9 DP: Male and female worms in the pulmonary artery will mate, they'll make babies, those babies are shed into the blood, the circulating blood of the dog. And there they can be picked up by another mosquito. So, when the mosquito take... Another mosquito comes and lands on this animal and takes a blood meal, they ingest these motile... They're essentially glorified eggs. They're motile little larvae that are in the dog's blood called microfilaria. So, this is really interesting, the microfilaria, they enter the blood, they enter the blood meal of the mosquito and they're in the gut of the mosquito, but they don't stay there. So immediately after they come into the mosquito, they find their way into the renal organ of the mosquito called the malpighian tubules. So, this is essentially the mosquito's kidneys.
0:06:37.6 DP: They rightfully have no reason to go into that tissue, blood doesn't go into that tissue, in fact, stuff flows out of that tissue. So, they're actually doing something really interesting there that I don't think is understood at all, but essential to their life cycle. They migrate into these tubules, they migrate into the cells of those tubules and there they make a home for a few weeks. They molt from this microfilaria, they turn into an L1 larvae, they molt again to an L2 larvae, and then eventually they form this infectious L3 larvae. Eventually, they're too big to reside in the cells of these malpighian tubules anymore, they essentially destroy part of that tissue upsetting the renal function of the animal and impose a fitness cost on the mosquito. The mosquito may die from this infection. They will enter the body cavity of the mosquito, and eventually, they'll go to the proboscis of the mosquito, where they lie in wait. This is again, now when the mosquito takes a blood meal, the larvae that are in the proboscis of the mosquito have a chance to infect a new host.
0:07:50.1 DP: And this is really fascinating. So, something I didn't mention at the beginning, so now I've completed the cycle. We now have the microfilaria entering the mosquito and then converting into this third stage infected larvae. What's really cool is, unlike malaria parasites or Zika virus or other things that mosquitoes infect or transmit to a host, these larvae aren't injected into the host, but they actually break out of the proboscis of the mosquito. So, they basically make a hole in the animal, it's very small. This, as far as we understand, doesn't hurt the mosquito, but they end up on the skin, they're not injected into the mosquito or into the dog. And this is an important aspect of our work, because actually that knowledge and the idea that we could actually simulate mosquitoes fighting an animal in the laboratory is the basis of... One of the really important bases of this project. So, we can trick infected mosquitoes and the larvae inside the infected mosquitoes into thinking they're on a dog by taking the mosquitoes and putting them in warm buffer.
0:09:13.9 DP: So, if we put a mosquito that's harboring infectious L3 larvae into warm buffer, essentially the worms think that they're... The larvae think that they're on the skin of the dog, and they come out of the proboscis. And we do this in a laboratory in petri dishes, and we can then count the number of larvae that are in an individual mosquito. And this is a really important aspect of our work, because now when we do a manipulation of the mosquito, we have the ability to see how that manipulation may impact the ability of a mosquito to transmit parasites. So, the take home message here is that the dog is the primary host for the parasites. This is where the heartworms can mate and have offspring. The mosquito is a vector, but it's also a host that really essential parts of the life cycle of Dirofilaria immitis, the canine heartworm, are accomplished in the mosquito. And so, blocking the parasite either from infecting the dog or blocking the parasites in the mosquito, both of these things would disrupt their life cycle, and that's kind of the focus of our lab is that, that latter part, blocking the parasites in the mosquito.
0:10:35.2 DD: Wow. That's really... It's a pretty fascinating story, and I appreciate you reminding us that the mosquito isn't really a... They're transferring the infection, but they're an important part of the life cycle, stuff has to happen, they're not just passively. But I think most of us, and I could have been mistaken all along, used to think the mosquito injected these things. They're like, "The larvae went out and they sucked in a blood meal and then they moved on," versus this concept of them actually breaking out and being on the skin of the dog, that I think, that's really interesting. Another interesting aspect, and when I made this question up, I said I might be dating myself, because when I went to vet school in the '80s, we didn't really know much about this interesting organism called Wolbachia, which is a... I want you to review for everyone what exactly is this organism Wolbachia, and how it interacts with heartworms, because it's another taught area that people are targeting research?
0:11:41.9 DP: Yeah, it's really fascinating. And you're not really dating yourself, it's a very active area of research still. My colleague here at the vet school in our parasitology course, when he shows students, when we start off talking about the basic biology of heartworm, he'll show a picture of a heartworm, and then... This is Professor Sparky Lok, James Lok. He'll show a picture of the parasite, and he'll say, what you're looking at is not one organism, it's two. It's two organisms in one. So Wolbachia to heartworm is an essential endosymbiont. It's important for their fecundity and for their long-term survival in the dog. And so, these are intracellular bacteria, they're actually bacteria, they live inside of certain cells in the heartworm, and it's not well understood, but there is sort of a mutualistic interaction with the worm. The worm needs these bacteria to be able to reproduce, to produce offspring. And without them, without those bacteria, the worms can't survive as well or reproduce.
0:13:03.1 DP: And similarly, because they're intracellular bacteria, those Wolbachia, they don't exist outside of the worm, they need the worm as well for their survival. So, it's really fascinating, it's a very active area of research. What exactly do those bacteria provide to the worms that allows them to have their... Be able to reproduce. And exactly, I think targeting the Wolbachia in the parasites, in the heartworms is a really interesting concept for dealing with heartworm infection. And I think it's important to point out here that there are a wide variety of Wolbachia bacteria. So, this is... Wolbachia is a genus of bacteria, there are many different species of Wolbachia, and the ones that are associated with nematodes like heartworm are a specific clade of Wolbachia that are specific to these nematode parasites. Mosquitoes and other insects, they have their own Wolbachia, but in the case of mosquitoes and in other insects, they're not always dependent on those Wolbachia, like the worms are. And so, there are some big differences in the different types of Wolbachia and the different associations with hosts.
0:14:33.6 DD: So yeah, that's good to know, because if I'm not mistaken, Wolbachia is of interest in other mosquito transmitted diseases as well as heartworm. Moving along, because this seems like a good progression, and so again, if you could go briefly over how heartworm is treated and prevented. And I know that's not exactly your area of expertise completely, but you know this better than I do now, because it has changed. We've changed a lot from... I don't even want to... Tell you what, when we used to put nerve-racking IV arsenic compounds into jaws to treat them, and if you got it outside the vein, it was really bad. So, if you could give us a quick overview, that would be great.
0:15:25.5 DP: Well, I think that's a really great segue here is that... I would like to start off this answer by saying that prevention is a lot better than treatment, okay? And things have moved on and you're right about that, but still for treatment, you're talking about treating an animal with something called melarsomine, which is basically at the basis in the fundamental-like way that it works is it's based on arsenic, which people have heard about, which is a poison. And so, this is the way that we can treat adult infections and during treatments, animals are exercise intolerant and have to be closely monitored so that there's no adverse effects to treatment. And it's still somewhat risky, and so, I think a really strong message to get out there, and this would be... I think every veterinarian would tell you, the American Heartworm Society would tell you that prevention is the name of the game here.
0:16:33.4 DP: I think another important point to point out here is that there is no vaccine, there's no vaccine for heartworm. So really you're talking about chemical preventatives, drugs, basically. And a lot of the drugs are all based on a similar chemistry, they're macrocyclic lactones and related compounds. A very famous one of these compounds would be ivermectin, and there's other related compounds, but basically, there's different formulations, different brand names and different ways to dose animals. But all of these compounds work based on killing the immature stages of the parasite that are delivered by the mosquito. So, the L3 we talked about enter the skin, and they take six to nine months to get... Before their adult parasites that are reproductive. There is a window from when they're L3 and L4 larvae where they are susceptible to treatment with these macrocyclic lactone compounds.
0:17:44.2 DP: And so, as long as you're keeping up with the regimen that's specified by whatever preventative that you're using, you can basically... It's interesting, you're basically reaching back and killing the parasites that entered previously. So, if your dog got bit by a mosquito and got some infectious L3 larvae in it, and then you, two weeks later on schedule gave it its treatment, you would reach back and kill those parasites, which were wandering around in the skin of the animal. And then, the mosquito, it might get some more infectious bites and then next month you treat again and then boom, you kill all the parasites. So, I think that's an interesting way. It's not protecting forward, it's reaching back and killing parasites that have entered.
0:18:38.0 DD: Right. And an adjacent question that many of our listeners may have heard that heartworms are becoming resistant and in the veterinary literature, it can be a little bit conflicting report, but what have you heard? And why is it happening?
0:19:01.0 DP: Well, so I think that it's easier just to start with... I think that there's no question that there are resistant parasites. Whether... So, I think the... I think some of the considerations are that not what you do with that knowledge. So, does that mean that you shouldn't give an animal preventatives? No, absolutely not. You should still be maintaining your animal on preventatives despite that knowledge. And so, yeah. And I think that's one of the issues that's so important to point out, and then whether... Then the question becomes, why do we see resistance? And I don't think that actually has an answer at this point. In other settings with other infections, the heavy use of preventatives can drive resistance. I don't know if that's the case with heartworm at this point, and I wouldn't really want to speculate about that.
0:20:10.7 DP: It could be that there are resistant parasites circulating, and that basically when we do our treatments, we see them. And so, whether prevention is driving it or they're just there and they're being revealed, but there is no question that there are existing parasites. We've actually made use of them through a collaborator in some of our work because we're interested in the topic as well.
0:20:40.2 DD: Right. And this is the segue [chuckle] to your research, and we wanted to talk about your foundation funded project, because it's pretty interesting and unique. Can you start by giving us a high-level overview and then we'll dive down?
0:20:58.9 DP: Yeah. Definitely. So, we talked about resistance, and there's no vaccine for heartworm, so that's part of the backdrop of being interested in this topic is that, well, we need alternative approaches, we need options, if resistance starts to become a wider spread issue, how are we going to deal with heartworm infections? Another thing we haven't touched on yet is, it's not just our companion animals that become infected with heartworm, there are wild animal reservoirs, there are lots of untreated animals, and then like we talked about these resistant parasites. So, we've been very interested in the mosquito's role in heartworm transmission to maybe tackle some of those things. Because again, like I said earlier, when we were talking about the life cycle, if you block the parasites in the mosquito, you will block the transmission not only to our companion animals, but also to any other wild canids. And so, maybe we can even make inroads... If we were targeting parasites in the mosquito, maybe we could make inroads into the wild reservoirs of these parasites.
0:22:10.8 DP: But that's jumping pretty far ahead of where we're at right now. And so, what we're doing now is... This project is really exciting because the work leading up to it in my lab has shown that if we enhance the mosquitoes' immune system genetically, we can block parasites from being... We can block the transmission stages of the parasites. So, what do I mean by that? I mean that if we use a genetic tool where we can silence a gene in the mosquito, and we're going to... I'm going to invoke this double negative here. So, what we do is we target something that negatively regulates or slows down the mosquito's immune response. So, if we remove the brakes, we overstimulate the mosquito's immune system, and then we challenge it with heartworms. And remember this assay we were talking about before, so we can take the infective mosquitoes. We have some that are treated and some that are sort of mock treated, our control group, and then we take them 14 days after infection, we do this trick to them, we put them in the warm buffer, and we see how many parasites can come out of the mosquito.
0:23:28.8 DP: And in that study, we found that there was a dramatic decrease, almost completely wiping out the parasite's capability of emerging from the mosquito when we over-stimulated the mosquito's immune system. Which was super exciting, because not only that, but we could figure out... We figured out that the majority of the mosquitoes that weren't able to successfully complete their lifecycle in mosquito were stuck in that kidney tissue. So, we kind of... We knew where they were, we could find them, we saw where they were arrested, and we saw that we could... The most important point was that we could shut down their ability to leave the mosquito. And that would basically block the infection cycle. So why this project? Why is this interesting? Well, you beautifully segued this for me as well, which is that the approach that we're using in this project in the foundation is that instead of using a genetic tool to overstimulate the mosquito's immune system, we are going to use a Wolbachia infection of the insect, of the mosquito.
0:24:39.2 DP: And this has been shown previously to have... It has several effects on the mosquito, but one of them is that it can enhance the mosquito's immune response. Because at the end of the day, it is a bacteria and bacteria can provoke the mosquitoes' immune system like I have other pathogens do. And so, what's really interesting about this approach and why it's attractive is that, because Wolbachia are naturally found in insects and in mosquitoes in certain populations of mosquitoes, and they're not otherwise modified, they're naturally occurring Wolbachia, it's not a transgenic approach. We're not making genetically modified mosquitoes in this approach. We're just associating them with the natural insect bacteria. And the real champion of this approach was... It's used in a project that used to be called Eliminate Dengue, and I think has another name now, but Wolbachia association with this mosquito, Aedes aegypti, is being used around the world to basically prevent infections by dengue virus.
0:25:57.5 DP: And what's... The final thing that's interesting about this approach is that Wolbachia do one little trick to the mosquito that sort of ensures their survival. They do this thing, it's called cytoplasmic incompatibility, but at the end of... Which is kind of a complicated term, but what it really means is that the infected mosquitoes bias mating with uninfected mosquitoes, and we don't need to talk about the details, but what it means at the end of the day, the upshot is that the infection of Wolbachia can sort of spread to the population. So, imagine you want to do an intervention, you want to get some mosquitoes out there that harbor this beneficial Wolbachia bacteria to prevent these infections. Well, you could either release lots and lots and lots and lots of mosquitoes and keep doing that over and over again, or you can have a way where a smaller seed of the population could spread throughout the population, and Wolbachia has that in addition to having the immuno-stimulatory feature as well, and it's been successful with this dengue, and we hope that it might be successful one day for something like heartworm.
0:27:21.5 DD: So, talk a little bit about how this vaccine might work then, knowing what you know now.
0:27:32.0 DP: Yeah so, what we're trying to do now is some really basic look at what's happening inside of the mosquito, so our preliminary data that was the basis for this proposal was that we associated the mosquitoes with Wolbachia, we infected them by basically pricking the mosquitoes with needles, so with Wolbachia, then we gave them a heartworm infection, we fed them the Microfilaria and then we performed this assay where we warmed them up. And we found that the infected mosquitoes that were harboring the Wolbachia produced less, fewer of these infectious third stage larvae. But well, we don't know anything more about it at this point, and so what we're doing in the proposal is trying to figure out what's happened to the mosquito, what's happened to the parasites, and what are some of the molecules that are involved that are really the mechanistic details of how is the mosquito shutting down this infection when we associate it with Wolbachia. And so those are the core ideas and the core goals of the proposal.
0:28:57.2 DP: A long-term goal would be basically similar to what's happening with the dengue work, is that if we could associate, we're working with laboratory mosquitoes at this point, because it's difficult to work with, like in the way that we're trying to do it, it's difficult to work with field-caught mosquito, that's field isolated mosquitoes. But in the future, it would be interesting to see if we could take a natural vector and associate it with Wolbachia and see if they could still, in our laboratory setting, shut down heartworm infection. And there then there'll be a lot of possibility for maybe releasing mosquitoes that have Wolbachia to reduce transmission, and I think it would probably be best targeted in areas where there are a few dominant vectors, something we haven't talked about, but an underlying complication for this type of approach is that there are several mosquito vectors for heartworm, so an area where there is a predominant vector would be a good area to test out something like this, where you just have to associate one particular vector with the Wolbachia, and especially in areas where there's very high transmission, like in the Mississippi River basin or something like that, where there's a lot of transmission and where we have the strongest evidence for these drug-resistant parasites.
0:30:36.7 DD: So, in its most simplistic, if I can boil what you're hoping to look for is to, first of all, amp up the immune system of the mosquito, and I think a lot of us who are veterinarians know that bacteria can hide out. If they hide out in cells, they're often protected from the immune system, but as you pointed out, Wolbachia is still a bacteria. So, if we can somehow manipulate that relationship such that ultimately fewer larvae are being transmitted or none are being transmitted, then we've attacked heartworm from a completely different angle than just treating the dog once it gets infected or trying to prevent, which is still, I agree with you. I think that we're never going to maybe not give them preventive, but we may be able to attack this from another standpoint, that we don't... I grew up in New Jersey as you did too, those trucks that would just spray stuff to wipe out mosquitoes, this is a much more elegant way of targeting this. And you talked about the outcome of your study. What do you think you would do next with this, let's say it all works really well? And do you think this could be a model? You talked about dengue and that, is this going to have maybe applicability from a broader sense as far as mosquito transmitted diseases?
0:32:09.1 DP: Yeah, well, I guess time will tell there. I think my colleagues in the vector community are having a... There's a lot of interest in this approach for malaria as well, and there's a lot of active research going on with this Wolbachia approach for the problem of malaria. It's already been shown to be successful for reducing dengue transmission it seems, and I do think in general, it has some interesting potential for sure. Time will tell with this one, but you know what, we still need to do a lot of basic research to understand what's going on.
0:33:00.7 DP: Potential and echoing what you said earlier, which is... You gave a perfect description of what we're trying to do. It's not to supplant anything that we're currently doing, anything targeting mosquitoes is certainly complementary to our current approaches, in fact, targeting mosquitoes with insecticides and things like that, have been very successful in the short term for preventing disease transmission by mosquitoes, but as everybody, intuitively knows, if the overall outcome is death. You're using insecticides, you have a very strong selection for resistance, and so insecticide resistance is a big problem. In this case, we're sort of turning the tables a little bit where we're not...
0:34:00.6 DP: Unfortunately, your picnics are still going to be ruined, the plan now isn't necessarily to reduce mosquito populations, so we don't have that selection pressure against the mosquito. The objective is to make the mosquito refractory to an infection, so basically shut down the parasite but not the mosquito. And so, this is a more surgical approach into the problem than just wiping out the mosquitoes in general, which is effective in the short term, but not necessarily in the long term.
0:34:34.2 DD: Right, that's a good point. So, I'm looking at the time and our time is running out, so I think you've said this, but say it again for me, what is your take home message, really, for the listeners.
0:34:46.7 DP: So, my big take-home message would be that heartworm is a terrible problem, and if you look at the transmission maps that are made for the past years, it's widespread in the US, there are certainly hot spots, but it's present in all 50 states, and I don't see a lot of change in these maps where heartworm is reported, so it's a problem that's with us. There are some scary things, we hear about resist... Parasites that might be resistant to our preventatives, and then there are just complications that mean that those maps might not change like these wild animal reservoirs we're talking about. So, I think mosquitoes are definitely something that we should be looking at whether they will have a long-term impact on this issue, will remain to be seen, but I think there's... There are a lot of reasons to think that they may. And so there needs to be some basic research, and I'm really happy that the Morris Foundation is providing some ability to do these really basic experiments to explore this exciting potential, and I think something that we haven't really touched on, but I think it's worth pointing out, right here at the end is that what we're learning also impacts diseases beyond canine heartworm. So, this... What the heartworm is teaching us about the mosquito's immune system is applicable to other parasites that are transmitted by mosquitoes.
0:36:32.2 DP: So, what we learned with the canine heartworm, we applied to a human infectious worm, the one that causes elephantiasis or lymphatic filariasis, this disfiguring disease that people get. We took some of the same strategies that we learn from our heartworm model and applied them to mosquito infections with these human parasites and found that they also... We could also shut down those parasites. So beyond just canine health, what we're learning here, I think, has the potential to impact human health as well, which is really exciting and something that I think it's an exciting opportunity as well, these are neglected diseases and they need research like this to help reduce their disease burden.
0:37:26.9 DD: Oh great. Well, that does it for this episode of Fresh Scoop, and once again, thanks to Dr. Michael Povelones for joining us. We'll be back with another episode next month that we hope you'll find just as informative. And the science of animal health is ever-changing, and all of us need cutting edge research information to help our pets, the wildlife we love, our patients, for those of us who are veterinarians and keep them healthy, and that's why we're here. You can find us on iTunes, Spotify, Google Podcast and Stitcher. And if you like today's episode, we sure appreciate, if you could take a moment to rate us, that will help others find us, of course. And if you want to learn more about the foundation and work like what Dr. Povelones is doing, please go to morrisanimalfoundation.org, and there you'll see just how we merge science and resources to advance the health of animals. You can also follow us on Facebook, Twitter and Instagram. I'm Dr. Kelly Diehl, and we'll talk soon.
