On the steppes of Askania Nova

The circles are the parts of the field that are irrigated, while the drier ‘corners’ are planted with different crops.

Southern Ukraine. From Zaporizhza to Askania Nova we pass field after field on the straight (but bumpy) road. The fields are huge, bigger than any fields I’ve seen. This is farming on the industrial scale. Once upon a time this was the river bed of one of Europe’s largest river, which deposited a thick layer of soil perfect to till. But it is dry, and without pumping water from Dnepr most of the fields would be steppe.

Johannes Rydström and I have traveled here to meet with Denys Muzika and his team of ornithologists and virologist. Over the course of a week, we try and catch ducks to equip them with GPS loggers that allow us to study migratory connectivity and influenza A virus dispersal. Just a few weeks earlier I was doing similar work in Bangladesh, and the contrasts in temperature, landscape and number of people couldn’t be larger.

Our base is Askania Nova, a pristine steppe reserve in the southern part of the country. It is a popular tourist destination and the site has a very ambitious zoo with large ungulates and birds, and a huge park with a collection of diverse trees. It is a gem and as a birdwatcher the steppe birds are amazing to see, with a constant background of singing Calandra larks.

Our team scouted different wetlands in the area and we tried to capture birds most nights using mist nets and duck calls. Depite our efforts and some amazing wetlands, we were not as successful as we had hoped. But at the end of the trip we can note 9 mallards equipped with loggers, of which one directly migrated to Russia. A big part of the trip was to connect and build for the future, because this is a site of strategic importance for influenza and duck research, on the gateway to Europe on the Caspian/Black Sea flyway.

We will be back.

A male mallard is about to be equipped with a logger. This bird is currently in southern Ukraine. Photo Johannes Rydström.

Several nights we worked in a beautiful steppe lake, putting up mist nets to catch ducks. This is Denys in action. Photo Johannes Rydström.

Sweden-Ukraine Duck Team (Denys, Sasha, Raysa, Jonas and Kolja) Photo Johannes Rydström.

This is me!

Tanguar Haor – a legendary wetland

IMG_0414There’s no business, like duck business

This spring I have been going places. First Bangladesh, and then Ukraine. Both trips connected by ducks, and the hopes of using telemetry to infer migratory connectivity of waterfowl populations and the transmission risk of avian influenza viruses.

Together with our colleagues at IUCN Bangladesh we spent some magnificent weeks in the wetlands of northeastern Bangladesh catching wintering ducks. I am writing up a longer piece of this trip for Birdlife Sweden’s magazine Vår fågelvärld which I hope to share with you in a couple of months. In the meantime, I’d like to refer you to an excellent article by Abida Rahman Chowdhury, a journalist from The Daily Star who visited us in the field in Tanguar Haor – the gem of wetlands in the north. Please read it on this link.

How to infect your duck, with science

How to infect a duck?

A critical parameter for the spread of a pathogen is the mode of transmission. Some pathogens have evolved to use mosquitoes or ticks as transmission vectors; others rely on direct contact, such as via body fluids during sex, and a score of pathogens travel by air, water or soil to reach the next host. Which route that is optimal depends on the interplay between the pathogen, the host(s) and the environment they occupy.

If we think about ducks, it makes sense to consider water as an effective medium for pathogen transmission. This indeed the case for several duck pathogens, and perhaps most notoriously for low-pathogenic avian influenza viruses. In ducks these viruses are common, causing mild gastrointestinal infections, and infected virus particles are shed in high numbers in feces. The conventional wisdom has been that the fecal-oral infection route is the most important, strengthen by the feeding habits of dabbling ducks where they skim the surface for food items, thereby exposing themselves for newly excreted viruses from their ducky friends.

But if you look yonder, at the ducks bobbing around in the pond, you will notice that they do other things as well. Of course, they dabble their bills in the surface waters, but they occasionally stretch the head and neck down to nibble at food stuff further down in the water. To keep the plumage nice and clean – and their bodies dry – they spend a significant proportion of their time carefully preening their feathers.

Such observations have resulted in alternative infection mode hypotheses, but until now we haven’t been able to disentangle them. In a seminal publication, Wille and co-workers at Uppsala University tested to what extent low-pathogenic avian influenza viruses can infect mallard ducks via the process of cleaning their feathers, or via the rear end, in a process called ‘cloacal drinking’. The drinking part refers to that when pressures are posed when ducks poo, it may create a vacuum through which a little volume of water enters the cloaca, which if containing influenza virions may cause an infection in the lower intestinal tract, bypassing the more traditional mechanism of swallowing viruses.

The paper is essentially an ‘how to infect your duck’ guide, complete with some clever appliances and boxes, and rounds of disinfections, to clearly separate the different modes of infection. And, yes, there are indeed many ways to infect a duck, as both preening and cloacal drinking also resulted in infections. Overall It is time for broadening our view of possible infection routes for flu, and other pathogens, especially those that are transmitted through water.

Link to the paper:

Wille, M., Bröjer, C., Lundkvist, Å. & Järhult, J. 2018. Alternate routes of influenza A virus infection in Mallard (Anas platyrhynchos). Veterinary Research 49:110

 

Duck (and virus) movements from afar

A wigeon track on the undulating tribituary of the Pechora river

Before I was a researcher, I was a birder. I spent my free time either birding, or thinking about birds. And my favorite place was Ottenby Bird Observatory. This is where my formative years took place and where I made friends for life. A focus point in my existence to this day. I spent countless mornings ringing birds at the observatory. Sleep deprived, sustained by coffee, sandwiches and tobacco we young ringers often talked about what would happen with the birds we released. Where would they go, what would they do? We marveled about the epic journeys they would undertake, connecting distant parts of the globe.

Sometimes we got answers, for one benefit of ringing is that the rings transform birds into individuals, and hence make possible to follow if they are trapped again, resighted or found dead. The downside is that these are all rare events, especially for smaller birds. For instance, the chance of getting a ring recovery of a willow warbler on wintering grounds in East Africa is very low, somewhere around 1 out of 100,000 ringed birds. For other birds like the mallard, the chance of a recovery is closer to 10% – a considerable difference. In any case, the information you get is limited and usually shown as a dot on a map.

But times have changed. I am older, greyer and possible wiser, a professor working with bird borne infections (but not birding as much as I would like to). I am still very interested in the question of where birds go, and what they do. Fortunately, tracking technology has taken giant leaps and we can now do studies that were unheard of when I was a young ringer. In recent years, my laboratory has been involved in studies investigating movement behavior of mallards. Together with Martin Wikelski’s team in Constance, we have looked at home range sizes and habitat selection of mallards during migratory stopovers, tested the hypothesis that influenza A virus infection impairs movements of mallards, and even made translocation experiments between Sweden and Germany to repeat Perdeck’s classic starling study. We have used Argos loggers, radio-frequency loggers and GSM-loggers, and for each study the loggers have become better and lighter and data ever more detailed.

Right now, we are a part of DELTA-flu, a Horizon2020 EU-project with several European partners. Our role is to investigate the migratory connectivity of waterfowl in Eurasia in light of HPAI virus transmission. Can we use loggers to answer the question about possible routes of virus transmission across continent?

An urban mallard in Roskilde, Denmark, presently hanging out on the Roskilde Festival camping site

The loggers we use come from the company Ornitela in Lithuania, and weigh 10, 15 or 25g depending on which duck species we target. The general rule of thumb is that a logger shouldn’t weigh more than 3% of the bird’s mass, as not to impair it unnecessarily. These loggers are little marvels; they transfer data via the mobile phone network and can be programmed remotely. So far we have deployed loggers in Sweden, Lithuania, Netherlands and Georgia, and are planning to work in Ukraine, South Korea and Bangladesh. We are also waiting for the next leap in telemetry: the ICARUS project onboard the International Space Station. With this technology, loggers may reach 2.5g and hence be put on a larger range of species. What all these loggers do is to provide a real-time window into birds’ movements: Where they are and what they are doing, sometimes even what they avoid or what caused their deaths. We can follow the lives of ducks in great detail.

There is a veritable flood of data, with more than one million GPS points collected already. It is easy to get lost in time just watching the latest whereabouts of the tagged ducks, from the tundra regions east of the Ural mountains to a gravel pit outside Bremen. I hope to write here more frequently, because there is a lot of exciting stuff happening in the lab at the moment – until then, have fun!

Flu transmission – a review

Hi there everyone, it is time for ducks and flu again! Just the other week we published a review on how host and virus traits affect transmission of low-pathogenic avian influenza viruses in wild birds. You should check it out – it is freely available in Current Opinion in Virology.

In this piece, Jacintha van Dijk, Josanne Verhagen, Michelle Wille and I synthesized the current knowledge of wild bird/flu interactions focusing on exposure and susceptibility. It is always challenging to write a review, especially when there are restrictions on length. But it is also fun (especially with such a talented team). We identified nine key host traits that can affect transmission: migration, non-migratory movements (e.g. dispersal), foraging, molt, reproduction, age, sex, pre-existing immunity and body condition, and provided the most recent findings from the literature regarding these traits. We also looked at five virus traits that can affect LPIAV transmission: virus stability, virus binding, virus replication, and the ability of the virus to evade the host innate and adaptive immune response. Many of these traits are not mutually exclusive, some have inherent spatial and temporal variation and can be affected by other confounding or unidentified factors.

Compared to many other wildlife pathogens, there is actually quite a lot of studies on LPIAV disease ecology to draw from. Yet, there is a clear need for additional and more integrative studies. You could say there are two sides: one more traditional approach with controlled infection experiment, and one more ecological approach with field samples and observations. Both are good, but neither is perfect. In lab studies, there is uncertainty in how well the experiment mimics the natural situation, and in field studies there are often many uncontrolled factors or results are correlative. We argue that these lines of research should be combined more often, either to use field studies to generate hypotheses to test in the lab with higher ecological realism, or to do semi-natural approaches in the field. A particularly challenging part is to study virus in free-living wild birds. Hopefully, the ongoing developments of remote-tracking could also be used to follow individual birds in the field for assessments of movement ecology, contact rates and other parameters of importance for predicting LPIAV maintenance.

Anyway, since the article is open access I strongly recommend you to click on this link and download the full review.

van Dijk, JGB., Verhagen, JH, Wille, M. & Waldenström, J. 2017. Host and virus ecology as determinants of influensa A virus transmission in wild birds. Current Opinion in Virology 28: 26-36.

Littlest workshopTM – small, but productive meetings are the best

A duck and computer, all wrapped up in a nice package - extend this to a workshop and you'll have the LittlestTM Workshop.

A duck and computer, all wrapped up in a nice package – extend this to a workshop and you’ll have the LittlestTM Workshop.

By Jonas Waldenström

Last week we organized a duck immunology workshop here in Kalmar that brought together people with various backgrounds in pathogen research, immunology, and movement ecology.

And it was a great meeting! Over the course of two days we presented data, discussed findings, and crafted possible research roads for the future. We also ate out on restaurants, and went to Ottenby Bird Observatory for some hands-on experience of birds. Some of the folks had met before, but most had not. My co-organizer Robert Kraus (Max Planck Institute for Ornithology) and I wanted to have a small meeting that fostered interactions. And it small it was, actually only 14 people. But we coined it the first International Duck Immunology Workshop (IDIW), partly for fun and partly because we would like to see this series to continue.

Me, Martin Wikelski and a duck - as well as a slightly tilted horizon. Photo Helena Westerdahl.

Me, Martin Wikelski and a duck – as well as a slightly tilted horizon. Photo Helena Westerdahl.

Anyway, what are the benefits of a small meeting?

To start with, everyone gets involved, and you have plenty of time to talk to each other. In my experience, lasting collaborations depend on social interactions – you are more inclined to do good science with someone you know, than with someone you never met. With time, such collaborations turn into friendship, and it is incredible how much you can do with a set of friends. Actually, I think most of the stuff I have done in my career would have been impossible without good friends that chipped in with ideas and analyzes.

Secondly, ideas come more easily in shared brainstorming. By connecting disparate dots, a cohesive picture may appear. The opposite is also true – your wonderful idea perhaps wasn’t properly thought through, and comments from folks with a different background may help you find the weak spots.

Thirdly, if we want to foster a new generation of scientists, we PIs need to provide a space for our students. Newly started PhD students are often intimidated at big scientific conferences, overwhelmed by it all, and old PIs tend to talk with other old PIs, rather than with unknown students. In this meeting we had two fresh PhD students that were given time to present their ideas of what to do in their projects, and to get direct feedback on their plans. Quite brave of them, but also very fruitful.

So, yes, size of a scientific meeting matter. A lot! Larger meetings have the benefit of attracting a bigger crowd, but if carefully crafted a small meeting can give all the output from a large one, but in distilled form. Let’s go for more Littlest WorkshopsTM in the future, shall we?

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Disease is a property of the individual

Ecologists are obsessed with variation, in any form, the more bizarre, the better. We really love it! But why?

The textbook explanation is that variation among individuals, if heritable, work as a template for selection and thus drives evolution. Without variation, little can change. Evolutionary important variation relates to genetic traits that make the organism better adapted to its environment, a better competitor, more disease resistant, or relates to traits that make him/her more attractive to the other sex, thereby increasing the likelihood of siring offspring.

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And additional explanation, and sometimes equally important, is that it is fun with variation: an animal may be short or long, have a peculiar nostril shape, vary in the curvature of antlers, or have striking plumage colors. Simply, humans like variation, and the diversity in itself therefore drives curiosity-driven researchers.

This said, when it comes to disease in animals most researchers tend to neglect variation. Disease is commonly treated as a constant; the animal is either infected with parasite X or is not. However, in reality what the researcher denotes as parasite X may actually be a plethora of different pathogen genotypes, all seemingly dressed in the same costume (the phenotype), or sometimes even consist of cryptic species. This is dangerous, as things that look the same in the microscope (or in a conserved gene used for molecular screening) may have fundamental differences in traits that are relevant for infection processes, such as pathogenicity, transmission and virulence. Simply, we may run the risk of not seeing patterns that are there, or jump to the wrong conclusion based on simplified assumptions.

Further, surprisingly often wildlife diseases are treated at the level of the population (especially abundant in veterinary medicine), and not at the level of the individual animal. For instance, prevalence, the proportion of individuals carrying a particular disease at a given time, is much more frequently used than estimates of incidence, which relates to the risk of acquiring infection. In the former you can adhere to a ‘hit and run’ sampling approach, in the latter you need to monitor individuals across time and take repeated samples.

For a long time, actually since 2002, we have studied influenza A virus in a migratory population of Mallards in SE Sweden. We also started at the level of population, describing temporal variation in influenza A virus prevalence in the duck population, and describing differences in prevalence among ages and sexes. And yes, we treated the virus as pathogen X, not at the level of subtype (which there are many of in flu). But with time we have moved to assessing what is happening at the individual level, and how differences among individuals in susceptibility drive disease dynamics, and how disease histories and immunity patterns in turn drive evolution in the virus.

These efforts are starting to pay, and in a paper published this week (http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0061201) we address the issue of individual variation among Mallards in influenza A virus infection risk. The question we asked is how individuals with the same background, in a shared environment with similar exposure to influenza, differ in disease histories and immune responses.

In our monitoring program we use a large duck trap to catch wild ducks. By providing grain we give the birds an incentive to visit the trap, and as additional attraction we have a compartment with lure ducks, that are supposed to get the wild ducks to enter. In this study, we used the lure ducks as a natural infection experiment. Ten immunologically naïve, juvenile Mallards from a farm were placed in the trap and were then followed throughout an autumn season, and then for the next spring, summer and autumn. Fecal samples were collected daily and blood samples approximately every second week. A lot of samples, and collected with a precision that allowed us to give very detailed infection histories for each individual.

cropped-oimg_3582.jpg

In turned out that our study ducks varied tremendously in disease patterns, despite being of the same age, raised in the same farm, sharing the same little experimental enclosure and being exposed to the same environmental variation. All ducks became infected with flu within the first five days of being placed in the trap, but the number of infection days varied tremendously. And so did the number of retrieved virus subtypes, thus different individuals were infected with varying number of virus variants, in this case equal to different infection events.

Furthermore, we got really nice long-term patterns. After the initial primary infections early on in the first autumn, and a number of secondary infections later the same autumn, we recorded only a single infection day the next spring and summer. It wasn’t until the second autumn, when migration of wild ducks started in earnest again, that new infections were seen in the lure ducks. And in this case, no infection was of a subtype the individual had experienced the year before, suggesting very strong and long-lasting homosubtypic immunity.

Individuals also varied profoundly in their immune responses. We measured the humoral immune response, manifested as anti-influenza-antibodies (raised against the conserved nucleoprotein of the virus), across time. Have a look at the figure below; it really shows variation both on a temporal scale, but also at the individual scale, both in patterns and in height of response.

journal.pone.0061201.g004

So what does it tell us? To start with, there is a large difference between individuals in resistance/susceptibility to influenza A virus infection in Mallards. This difference is not only manifested in different infection histories, but also as very variable immune responses. Second, these differences are very likely determined by genetic differences, meaning that there are heritable differences, and thus traits that could be selected for by natural selection. Not all ducks are equal – and this important for our ability to model disease dynamics in this system. Is it really the mean that is important for assessing the transmission probabilities along migration? Perhaps it is the outliers that are driving the processes?

This study is a first step to adress individual variation, and there are already a couple of follow-up publications in the peer-review tube, so we will have opportunities to get back to this topic.

That’s all for now. Live long and prosper – and don’t treat disease simply as a property of the population.

Jonas Waldenström