Atlantic Salmon and Maine’s renewable wood pulp industry may sound like an unlikely duo at first, but Deborah Bouchard and Sarah Turner from the Aquaculture Research Institute (ARI) have recognized the potential this pairing could have for the future of environmental, economic, and social sustainability for the US aquaculture industry. Debbie and Sarah Bouchard are to researching a new generation of safer, more sustainable, and cheaper vaccines for finfish. Here is where Atlantic Salmon meets its unlikely match, wood pulp. This abundant polymer is biocompatible, biodegradable, quite versatile, and easily modified – making it a phenomenal candidate for drug delivery. Tune in to learn more and hear about their results so far!

and Vibrio ordalii. This adjuvant, a component of vaccines to improve and prolong immune

response, showed no evidence of cellular damage and no effects on growth for the fish while

inducing a strong immune and antibody boosting response. With less side effects and cheaper to

manufacture than existing vaccines these preliminary results are exciting, highlighting the efficacy

of nanocellulose adjuvants.

ISAv and V. ordalii alone can cause farmers to lose 90% of their stocks, amounting to over $1

billion annually. Development of this new, safe, and affordable vaccine using nanocellulose is not

only huge for Atlantic salmon farmers but other food production systems as well. This new

generation of low cost, tunable vaccine formulation is paving a way toward more sustainable

aquaculture, with potential applicability for other fish species and even terrestrial animals as well.

Leveraging direct industry support from Cooke Aquaculture, Kennebec River Biosciences,

Benchmark Animal Health LTD, this interdisciplinary team from UMaine’s Aquaculture Research

Institute and the Chemical and Biomedical Engineering Department has immense experience and

resources relating to fish health, immunobiology, and vaccine formulation. Results will be shared

directly through ARI’s web site, Cooperative Extension and Maine Sea Grant extension

professionals, national conferences, and peer-reviewed publications.

Corinne

Welcome back to the Salty Talks podcast. I'm your host, Corinne Noufi, the aquaculture communications specialist with the Aquaculture Research Institute. And today I wanted to talk about the importance of animal health and fish vaccines. So I've brought on Debbie Bouchard, the director of the Aquaculture Research Institute, and Sarah Turner, a current PhD candidate, to help me do this. So will the both of you briefly introduce yourself and talk more about your background?

Debbie

So I'll go first. I am an aquatic animal health specialist. Is that going to wreck it? I'm an aquatic animal health specialist, and I've worked with both private sector and the industry, 1s with Cooperative Extension Now and the University of Maine.

Sarah

I'm Sarah Turner, and I've worked as an aquatic animal health research specialist for about 15 years with the University of Maine.

Corinne

All right. Like humans and terrestrial animals, viruses, bacteria, and parasites can cause adverse health effects and spread through populations, especially when organisms are within close proximity to each other. I wanted to start off talking about why it's important that we put time and effort into vaccine research in general for farmed animals.

Debbie

Well, vaccines are actually very efficient at controlling or mitigating disease, and all animals in an agricultural setting or even in wildlife are susceptible to disease based on the environment that they're in. Vaccine research has really improved the efficiency and productivity of aquaculture worldwide. Right now, if you look at the number one impediment for aquaculture production, it actually is disease. It accounts for 40% loss of production, and that's estimated at 102,000,000,000 in losses annually. I don't think this is any different than any other agricultural area where you're producing animals, but vaccines have been very efficient in helping mitigate that and why we continue working on improving them.

Corinne

And these diseases that you're talking about spreading, is this only in aquaculture or do these diseases also exist in wildfish populations?

Debbie

So one of the things I always say is diseases don't spontaneously generate and they have to originate from somewhere. And that said, 1s in an agriculture aquaculture environment where you have your tend to be raising things in high density they become sort of sentinel to what's out there with disease and it's easier to spread it where animals, in this case fish, congregate and then the environmental conditions and impact will impact the way that disease spreads in that population making them sort of more susceptible to spreading disease.

Corinne

So are there any other outside factors that contribute to disease spread? I guess in particular, I'm thinking about warming temperatures or other external factors.

Debbie

I think climate change will play a role in 2s having disease maybe become more prevalent in two ways. One is going to be a stressor to the animal. And when animals are stressed, their immune response may be lowered and they're more susceptible to disease. But also the environment may change the actual that's causing the disease, the pathogen, to be more virulent in a certain environmental condition. So as temperatures are warming, like even in Maine, we're seeing organisms come in, microbial organisms that we haven't seen before.

Corinne

So you touched on the economic problem of disease spread and production in crop loss. So it sounds like vaccines have an added economic benefit to Aquaculture as well. So besides the economic benefit and increased food production what are some other benefits of having vaccines inAquaculture?

Debbie

The use of vaccines in Aquaculture has greatly reduced dependency on antibiotics. But remember when we say antibiotics, that's for bacterial pathogens. And so there's always a concern with antibiotics that we're developing resistant organisms out there that would then transfer over to humans. So just for that reason, developing vaccines is pretty important.

Sarah

I would maybe just also add that it's more it's better for animal welfare and for economics to prevent a disease outbreak before it happens than to try and treat one.

Corinne

Yeah, that makes sense. You're trying to start proactively instead of coming at it from the defense.

Sarah

Yeah.

Corinne

And are vaccines in general lower cost to manufacture than antibiotics, or does it sort of depend on the

Debbie

I don't think so. And one of the things I want to make sure that we state is that in Aquaculture, they have relatively few antibiotics that are actually approved. So the relative usefulness of antibiotics, even when employed in Aquaculture, is limited. So vaccines can be very expensive. And that's part of why we're doing our research, which is we're going to talk about a little bit later on our novel adjuvants. So, no, the vaccines can be very expensive, and for the most part, 1s fish are actually vaccinated one fish at a time. And so not only is the cost of the vaccine there, but also the labor in vaccinating. Those animals

Corinne

Sarah

Yes.

Corinne

Are they used in a bunch of different fish species, or does it depend on the fish species if they can be vaccinated or not?

Sarah

There's currently approved vaccines for a number of different aquaculture fish species.

Debbie

No, honestly, no. Vaccines are something in aquaculture that are constantly being developed, and most fish species that we culture have some type of vaccine that's produced for them, whether it be immersion injection, and the newer developing ones are in feed. But for vaccinating fish, you're always going to because of the expense of vaccinating, you're going to target the higher value fish species. So we tend to have a lot more vaccines developed for salmon. For the salmons because they're your higher value fish than we might do for tilapia or catfish.

Corinne

Yeah, that makes sense. You mentioned immersion and injection vaccines. What's the difference between the two? And are there any other types of vaccines?

Sarah

Yeah. So immersion is where you dip the fish or immerse it in the vaccine? Yeah. Injection, obviously, is just as it sounds. You're injecting the fish with the vaccine just like you would with terrestrial animals or humans. And then there's also oral feed vaccines where you can dose the fish in the feed.

Debbie

All of those are also subject to the way that the pathogen would actually infect the fish. So well, would be great to have efficacious effective oral vaccines because that's the simplest way. You just feed it. You're really targeting only certain type of pathogens that you can go with an in feed vaccine right now. Predominantly the most effective vaccines for finfish are injected vaccines

Corinne

Ok down to the actual vaccine part of it. You mentioned an adjuvant in vaccine. So vaccines to my knowledge, are composed of an antigen and an adjuvant that exposes the immune system to a pathogen, which creates a memory for the immune system on to fight off a disease. What is the purpose of the antigen and the adjuvant? And do you need to have both of them and are they working together?

Sarah

So the antigen is either the whole pathogen or a part of it that the immune system recognizes, and on its own it creates a weak immune response. So it needs the adjuvant, which is like a delivery agent, to deliver the antigen to the immune cells to create an effective immune response and memory to that pathogen. So both parts are necessary for an effective vaccine.

Corinne

How long does it take for an immune response to develop after the vaccine is administered, or is that dependent on the species?

Sarah

yeah, it's dependent on the dose and the pathogen and the vaccine. I think also the fish species as well. Pretty much the fish species.

Debbie

If it's a fish species, the warmer the temperature that the fish are raised in, the quicker their response is going to be, the colder, the slow lower. When we're doing vaccination, we call it degree days and the average sort of. What we know in our what we what's in the literature out there and what we've always worked in is that you want 600 degree days. So if you have a fish that's being raised at twelve degrees, that's about 60 days before you know that okay, I've probably reached our peak immune response. And if you have a fish obviously growing at 20 degrees, it's going to be a lot shorter time than that. These are all things that have been worked out through research over years. And I think it's probably important to add that just to remember that we're trying to protect the fish over the course of its lifetime. And for Atlantic salmon, that would be about two years. So it's not quite the same as terrestrial animals or humans.

Corinne

this isn't when we go get our annual flu shot every year. This is like a one and done injection,

Sarah

hopefully. Sometimes there's boosters required, depending on the pathogen.

Corinne

And are the vaccines Live or inactive.

Sarah

Yes. So the most common antigen used in Aquaculture is an inactivated antigen or what's also called the bactrian. And that's used primarily because it's the most cost effective and it's the most safe. So the parts of the pathogen used for the antigen are not active pathogen.

Debbie

There's rare cases in Finfish where you have what's called an attenuated antigen or pathogen where they've done something in the laboratory that has changed it so it won't produce the disease anymore. But the fish's response to it is a strong immune response. That one's. Like now we're getting way into the weeds of science.

Corinne

That is good. I like the weeds of science.

Debbie

Okay, great.

Corinne

I think we should move into the nanocellulose research now and Sarah, you are doing your PhD on this, correct?

Sarah

Yeah. So this was a grant that I actually started writing for class credit, and then once it was finished, reached out. Debbie and I reached out to the engineering department here at the University of Maine for some collaboration, and we submitted it. It's a USDA afri Foundational grant. 1s And it was awarded So it's a three year project. We are currently about a year and a half in.

Corinne

And so your project is looking at new things to use as adjuvants in vaccines. And you're using nanosellulose, right?

Sarah

Yeah. So the current adjuvant used in commercial aquaculture vaccines is oil based, and it works really well in terrestrial animals. But fish are a little bit different. And when you put it into fish, it can create a strong inflammatory response, which can result in some negative consequences, such as pigmentation adhesions, some negative growth consequences, and it's very expensive. So Nanocellulose is a biocompatible, biodegradable more economically effective possibility for an adjuvant for fish vaccines.

Corinne

The vaccine that you are looking at is specifically for what type of pathogen or disease.

Sarah So we chose a bacterial pathogen called Vibrio Angularum, and that causes a disease called Vibriosis, which can result in skin lesions and 2s high mortality in aquaculture facilities. We chose that bacteria because it's really well all studied and characterized. So it helps to take out a variable in our experiments because we already know how it's going to behave and the impact it will have.

Debbie

Part of the when you're evaluating new vaccine and new vaccine formulations, it's always good to go with what we know is out there and what is working before you target the hardest pathogen in the world. So, like Sarah was saying, you knock out a variable. So we've taken vibrio, angularum causes vibriosis globally in many, many different kinds of species, finfish species. So a good target organism to try the new adjuvant formulation with, because we can compare it to an already commercially available vaccine that's working efficiently. It's a win win if we even have this vaccine work just as well, because the cost of production is going to be so less. So we're working on two fronts there. We're making it one. We're working not only with a biocompatible, biodegradable substance, which is an oil based adjuvant, and I lost my flow of thought there. What was I going to say after that? But then we're reducing the cost. Again, one of the reasons why we chose that particular antigen or pathogen is that it is a global concern and there are efficacious vaccines and we really need to compare our new formulations against something known.

Corinne

So then would the idea be that you could then use this to treat other diseases in salmonids, like salmon, anemia and other things, if this works out?

Debbie

So what we're testing again, as Sarah was saying, is the effect of our novel nanocellulose as either a depot or an adjuvant. And if it's effective with a known pathogen, then yes, we can definitely apply it to other new and emerging pathogens. That's one thing that's never going to disappear in any type of living system of animals is new and emerging pathogens will always surface.

Sarah

And I think I would just add that we chose a bacterial pathogen to do this research with, but it could very easily translate to using a viral antigen or other potential bacterial antigens.

Corinne

So why did you pick nanocellulose as your adjuvant? Like you've said, it's, um, biodegradable and environmentally friendly. But I'm sure there's other particles that are also biodegradable and environmentally friendly as well.

Sarah

So nanocellulose is produced from cellulose, which actually is the most predominant biopolymer on earth. So we're sourcing it here from trees. We chose it because it's readily available to us here at the university of Maine. And it has a lot of great potential has a lot of great potential to work as an adjuvant and deliver the antigen. Yeah, we actually became more aware of it by Debbie. And I took a tour, the engineering facility, and they were showing it to us, and Debbie was the one that said, hey, this looks like something that could serve as an adjuvant.

Debbie

The other thing about it, too, is it is basically the predominant nanocellulose does come from wood pulp. Of course, Maine is a forestry state, and so it was just serendipitous that we actually saw this. But yet 1s the way that, again, it's renewable. It's a renewable source 1s for producing this product as well. And then a cellulose is used in many applications already, and that's what we learned. I mean, it's used for drug delivery, for cosmetics, and the idea of it, because it's so inert and I guess what you would call tunable as well.

Corinne

Tunable meaning, like, you can modify the characteristics of it to deliver.

Sarah

Yeah. So there's a lot of different ways that you can modify it. The first being in production, depending on how you produce it, you can get a lot of different versions that have different structural properties, different chemical properties. So you can see nanocellulose being used as a film or a solid or a gel. And then on top of different ways to produce it, you can also modify it chemically by adding different molecules to it. It's very easily manipulated, I guess is how to put it.

Corinne

So what does this vaccine look like when it's going into the fish? Like, we go to the doctor and we get our shots, and I think we can all picture a needle full of some sort of fluid or something. Is that essentially what it's like for the fish?

Sarah

Yeah. So we've got a couple of different variations that we're looking at. One is more like. Fluid or injectable. Goes in with a syringe. Yeah. Goes in with a needle and syringe. We just recently completed a study looking at a hydrogel formulation. And a hydrogel is a biopolymer that absorbs a lot of water but maintains its shape and structure really well. So what our thoughts were with the hydrogel was that if we could implant this into the fish at a young age, early on, it would last the entire duration of the fish's life span. So it would slowly kind of

Corinne

who's texting you?

Debbie

I didn't want I turned it off. Sorry.

Sarah

The hydrogel, so it's more like a pellet that over time would release this antigen with the adjuvant. So it's like a slow release of immune response. Right. So the hydrogel, what it would allow us to do is to control the release rate of the antigen to the immune cells over a long period of time to provide building amore response. Right. Theoretically, you'd only have to do this one time. Right? Yeah. Got you.

Corinne

So I think the last time I read about this project, there was not results yet. I think it was very new here. You have some results.

Sarah

We do have some results. So we performed a safety study where we implanted the hydrogel into the fish and then monitored the fish over 600 degree days, like Debbie said. And what we found there were some pros and some cons to it. The pros were that the fish had no adverse reactions acutely. So they went back to eating and swimming and behaving as normally growing. Well, the cons were that the hydrogel actually. By the 600 degree day mark, it had actually triggered a strong inflammatory response called the foreign body reaction, which it's this highly conserved immune response across many different species, where when the body is exposed to a foreign material, it actually walls off into a granuloma or like a cyst to sort of protect the body from it. So that's not ideal for our purposes.

Debbie

It almost wants to inject. It was like, what is that? It was like pimple, pushing it back out.

Sarah

If you imagine if you've ever gotten a splinter implanted into your skin and then just gets inflamed and works the splinter back out, that's kind of what we were seeing. So really, we know a little bit about the foreign body reaction, and it's typically triggered from specific surface properties of the material. So we know that we can modify the surface of the material to avoid that.

Corinne

Um, do you mean, like I guess I don't know how, like, pelody this was, but do you mean change sort of like the viscosity of it? Like, if it was

Sarah

yeah, it was a rough, rough surface, very porous, and some of the chemical some of the chemical components of the hydrogel likely 1s interacted with the immune cells in a way that we didn't want.

Debbie

Always soliciting an immune response is a good thing. Sometimes it just goes overboard. This one a little overboard. And now, one, it was great, hey, we got an immune response. Two, it was like, oh, we got an immune response. And so with that knowledge, though, that's what research is about. We will reformulate. What's the next step that we're doing for reformulating?

Sarah

So the next formulation we're looking at is called Shear Thinning. And so what that is, is it's a liquid in the syringe. It's injectable so a lot less invasive than our implanting process, which the implanting process might have played a role in that foreign body reaction. So this will be injectable because of the pressure of pushing the nanocellulose through the needle and then also a slight PH change within the fish. That liquid will then form a sort of viscous gel inside the fish. So we hope to gain some of the advantages of the hydrogel without triggering that foreign body reaction to the stiff, rough material.

Debbie

One of the unique things is it's sort of multidisciplinary. You've got biologists working with engineers, which sometimes has its

Sarah

we're speaking two different languages sometimes

Debbie

but we definitely have a strong industry support for this type of work. And we have commercial companies that we engage with 2s that are waiting for the results for this. Moving forward, this might be with success and easily licensable technology.

Corinne

You better get going on it, then. This is a question I probably should have asked, like, 20 minutes ago, but how do you inject a vaccine into a fish without it flopping around all over the place? Does it? It sounds kind of hard.

Sarah

So what we do is we actually do a light anesthetic. So the fish is very calm. Even in industry and commercial aquaculture settings, it really is individually injecting every single fish. So it is, unfortunately, a laborious process. But again, because each fish is getting a targeted dose, it's a very effective process.

Debbie

This was like no, we did quite a bit of detail with the formulations and stuff, but you can edit.

Corinne

Thanks, both of you. I think this was really great and super informative about fish vaccines, something that probably not a lot of people know much about.