Ticks on wings – screening for Candidatus Neoehrlichia mikurensis in birds

neo

Neoehrlichia mikurensis is a recently described zoonotic bacterium with unknown biology. It did NOT get its name from Neo in the Matrix movies, but it would have been cool if it had.

Ticks are fascinating creatures. Slightly alien, with a hard carapace and saw-toothed mouth parts, and all too many crawly legs – little nightmare creatures hiding in the backyard waiting to draw your blood. Sane people tend to detest them, but many scientists (me included) have taken a liking to them. Not only do ticks have an interesting biology – being parasites with distinct development stages, each stage associated with a blood meal from an (unwilling) host – they also are perfect little transport vessels for various pathogens that seek shelter and future transmission opportunities. The ability to use ticks as vectors seems to be a fitness bonanza, given the range of known tick borne pathogens. A specific bond exists between small rodents and tick borne diseases, but also larger mammals and birds can host zoonotic tick borne pathogens. And birds and zoonoses are what we love in our laboratory, so of course we have to study ticks on birds!

What does a tick on a bird tell you about the epidemiology of a tick borne disease? Actually quite a lot, if you consider both the biology of the bird and the tick. Some ticks have a broad range of hosts, such as Ixodes ricinus – the most common tick in Europe – while others have a more narrow host range, sometimes even restricted to a single host species. Although many tick species favors mammal hosts, there are those that are strict bird eaters (ornithophagus) – for instance, there is a tick specialized on Sand Martins. Thus knowing the species, and the development stage of the tick can tell you whether the bird is merely carrying infected ticks, or if it can be infected too – factors that influence the bird’s capacity to affect disease transmission over short or long temporal and spatial scales. Similarly, on the host side, knowing the breeding biology and migration ecology of a species allow you to say something on the current and future geographical range of the ticks and their pathogens.

Generally, as there are more bird lovers than tick lovers, the overwhelming majority of ecological data comes from the bird side, and very little from the tick side. We wanted to properly investigate birds as carriers of tick borne infections and carried out a very large sampling effort a few years ago. From roughly a thousand ticks, collected in a standardized way during a full year at Ottenby Bird Observatory, we extracted RNA and DNA for molecular screening for a smorgasbord of tick borne infections. These samples have been doing rounds in different Swedish labs, and the idea is to have an end-product where several pathogens are analyzed in parallel. We are not there yet, but a few weeks ago we published one article from this material, analyzing the prevalence of a particular bacterial species: Candidatus Neoehrlichia mikurensis. This novel bacterium has recently been linked to disease in immunocompromised patients, and exploratory studies have shown that it is widely distributed in Ixodes ricinus ticks and different rodent and mammal hosts in Europe. But was is the role of migratory birds?

Our first line of tick identification was digital photos. Each tick was photographed on both dorsal and ventral sides, just like flipping hamburgers on the grill, and the photos captured by a little USB-driven microscope with a camera. Although convenient in the field, the device didn’t really produce high quality pictures, making separation of similar looking species difficult. Ticks are hard to identify, and you need clear views of distinguishing characters (such as the shape of the mouth parts) to be certain of species identity. It is like when you renew your passport: there are specific guidelines for how your ears and nose should be portrayed. Our tick photo booth was more of the party pic variety, and we were therefore forced to set up molecular typing methods in the lab to complement the morphology-based identification. This turned out to be surprisingly difficult. A number of typing methods have been proposed, often targeting mitochondrial or ribosomal genes, but in most cases these protocols have not been evaluated on a large range of species, and their performance turned out to be variable. After much tinkering we settled with two methods that together worked well on our assemblage of bird borne ticks.

We searched 5365 birds of 65 species for ticks, and screened the resulting 1150 ticks for Neoehrlichia. Neo was found in a low prevalence in Ixodes ricinus ticks, roughly 2%, but not in other tick species, although the sample size for the latter was rather low. Furthermore, the bacterium was only detected in nymphs, and not in the numerous larvae, suggesting that the tick primarily got the infection via the first blood meal, and that it is less likely to be spread by vertical transmission (i.e. from mother to egg). Some birds carried more than one infected ticks, but we couldn’t find conclusive evidence whether they acquired the infection via a bacterimic host, by feeding close to each other on a non-bacterimic host, or whether the ticks had independently acquired the infection via earlier blood meals. Unfortunately, we did not have any blood samples from the birds, thus at present it is unknown whether birds can be competent hosts for Neoehrlichia or not. As regards which species that had infected ticks, there wasn’t any revolutionary patterns – the majority of birds were species that are heavily infested with ticks, such as ground-dwelling birds like Robins, Blackbirds and Wrens.

Neoehrlichia is a new kid on the block in tick borne disease research, and a lot of fundamental information is still missing. However, things are happening at the moment, and the knowledge gaps filled with speed. We are still hampered by the lack of culturing tools for this pathogen – hence the prefix Candidatus – as well as the lack of genomic data. The data at hand suggest very little genetic diversity, regardless of from where, and from what host the bug is detected. This may be artefactual, caused by phylogenies based on a gene with low phylogenetic resolution (i.e. “conserved”), or, more likely, suggests it to have a clonal population structure.

You can read the paper here:

Labbé Sandelin L, Tolf C, Larsson S, Wilhelmsson P, Salaneck E, Jaenson TGT, et al. (2015) Candidatus Neoehrlichia mikurensis in Ticks from Migrating Birds in Sweden. PLoS ONE 10(7): e0133250. doi:10.1371/journal.pone.0133250

Call of the wild: gulls as sentinels of antibiotic resistant bacteria

Gulls are our model species for antibiotic resistance dissemination. Picture from Wikimedia Commons.

Gulls are our model species for antibiotic resistance dissemination. Picture from Wikimedia Commons.

By Jonas Waldenström

What scare you the most, little one? Is it the monsters under your bed? Warfare, missiles and guns? Climate change? Spiders? Dogs with fangs? Yes, all of them are scary, and most are nasty. But if I have to choose among all the nastiness of this world I’d say antibiotic resistance scares me the most.

You have certainly heard about it, again and again. A researcher, or physician in a white lab coat saying that the situation is “worrisome” or even “alarming”. But it doesn’t really sink in. We don’t want to listen. We hurry, rush the kids to school and daycare, work dull hours in the office and dream about a better life. Or at least considers the substitutes for happiness:  a nicer car, a holiday in the sun, or a tight bum. But dreams sift like sand and we settle for a bottle of wine and a piece of beef on a Friday night. We are too occupied to ponder the grander questions, and leave the future for another day.

But if? Think about it.

What if antibiotics didn’t work? What kind of world would that be? We take these drugs for granted, but they are fairy stuff; twinkling wondrous inventions that can be gone in wink, rendered useless by evolution. You see, no one can escape evolution, and we have played the game wrong for as long as we have had antibiotics at our disposal. Instead of safeguarding, we have peppered the bacteria in animals, ourselves and the environment with antibiotics thereby increased the reward for resistance mutations to the point that they rapidly increase in frequency. In our hospitals and stables we have created environments were bacteria could mingle in antibiotic-cladded environments, promoting bacteria to share plasmid-borne resistance markers through horizontal transmission. Bad play, really bad play.

We talk misuse on an epic scale: an ever-accelerated prescription of drugs for any types of infections, even viral infections, where, of course, antibiotics do little good. We have used our drugs on food animals to treat infections, but also as growth promoters in chickens and swine; we have poured bucket loads of antibiotics in ponds to grow shrimps in clear-cut mangrove swamps; we have sprayed antibiotics on apple and fruits to keep them fresh until the retail level. But all this came with a cost, an interest rate we were not considering at the time. And soon it is time to settle the bill, but like Greece we have little means to pay.

When the magic bullets don’t hit their mark we will face a harsh reality. A post-antibiotic world. Science writer Maryn McKenna recently wrote an essay in Medium on how this brave new world would be. A world where simple infections could kill, a return to an era before the two world wars. No more knee implants and hip replacements, no more bowel surgery or nose jobs. A long kiss goodnight on our future health care.

But it can’t be that bad can it, you may ask yourself. They must exaggerate! Clearly, if things were so dire, the Government would do something about it! Well, truth is, we do far too little, governments included. We are standing with one foot leaning over the Pit of Doom (to use Fantasy jargon) and only a concerted action could take us out of it. Simply put, it is tragedy of the commons, where many small decisions end up in a big bad one.

To turn the tiller and set a new course we all need to chip in. Governments need to stimulate research in new drug developments. Global action needs to be taken for how to use antibiotics; these drugs are too potent to be sold over the counter without prescription. And antibiotics should not be used when they are not needed – and farm size and practices need to be addressed in the light of reducing consumption of antibiotics, not in the light of maximizing profit. And there need to be basic science. Our research group addresses the occurrence of resistance in the environment. Using gulls as model species we have travelled wide and far, from pole to pole, and sampled birds for antibiotic resistant bacteria. A few days ago we published our latest article on resistance dissemination in Europe. And it is a scary read.

Antibiotic resistance can attack you when you least expect it. Picture from Wikmedia Commons

Antibiotic resistance can attack you when you least expect it. Picture from Wikmedia Commons

A problem in most investigations, especially those conducted on wildlife, is that studies have been small – often a bunch of samples collected without proper sampling design or power calculation. Few studies have addressed larger spatial scales, beyond the country level. Graduate student Johan Stedt set out to change this, and his thesis – that he will defend in June – investigates the occurrence and frequency of resistance markers in gull Escherichia coli on a global scale. In the summer of 2009 (time flies fast in science between fieldwork and published articles) we sent out three teams of trained fieldworkers. In each car there was a liquid nitrogen dewar and sacks of sterile cotton wool swabs. Using our network of ornithologists across Europe, put in place by earlier flu virus studies, we were able to sample gull breeding colonies in nine European countries, from Spain and Portugal in the south, to Scandinavia in the north and the Baltic states in the east. All in all 3152 samples were collected during two weeks of fieldwork. This is by far the largest study conducted in wildlife across Europe.

In the lab, Johan spent months and months going through the samples. First, a primary isolation was done to get putative E. coli isolates. Then the identity of the bacterium needed to be validated with phenotypic test, and then the susceptibility of each isolate was tested with disc diffusion against a panel of 10 antibiotic agents. For the untrained ear they have strange, but beautiful names: ampicillin, cefadroxil, chloramphenicol, nalidixic acid, nitrofurantoin, mecillinam, tetracycline, tigecycline, streptomycin and trimethoprim/sulfamethoxazole. They are, however, commonly used in human and veterinary medicine.

But how is resistance quantified? It is actually rather simple. The isolate is inoculated onto an agar plate where little discs containing antibiotics are attached. The plate goes into a 37C heating cabinet for 24 hours and then one measure how close to the antibiotic discs the bacteria grow. A susceptible bacterium cannot grow close to the disc (where the antibiotic is leaked or ‘diffused’ into the agar), leaving a large zone devoid of growth called the inhibition zone. A resistant bug, on the other hand, can cope with the antibiotic compound and will therefore grow closer to the disc. Resistance is not always a black or white thing. Rather, there is a range of phenotypes with varying susceptibility to a compound. In clinical practice, breakpoints have been established for different bacteria and antibiotics by plotting the range of phenotypes for a large number of samples. This usually gives a normal distribution around the mean for susceptible bacteria, and a hump of isolates as outliers representing the resistant fraction.

Inhibition zones encircle the antibiotic discs in susceptible isolates. Picture from Wikimedia Commons

Inhibition zones encircle the antibiotic discs in susceptible isolates. Picture from Wikimedia Commons

Let’s return to the gulls. It pretty soon became apparent that resistant bacteria were common and widespread in European gulls. In fact, roughly a third of the isolates retrieved were resistant to at least one antibiotic compound, and a fair proportion was resistant to several. Looking at specific resistance profiles, the most frequently recovered phenotypes were resistant to tetracycline or ampicillin. These results are in concordance with other studies on gulls and may reflect the fact that these antibiotics have been commonly used in both veterinary and human medicine for decades. The occurrence of other resistance phenotypes, such as mecillinam and nalidixic acid resistance, was more surprising. Tigecycline was the only tested antibiotic that we did not find any resistance to; perhaps due to it being a relatively new antibiotic, used for skin-structure infections and complicated intra-abdominal infections.

A striking finding was the geographical variation in resistance levels. Have a look at the map above. Samples from the Iberian Peninsula were on average more often resistant than samples from gulls in more northern countries. This was true for all tested antibiotics (and also for ESBLs, but that is something that will be covered in another publication) and is also mirrored in similar data from humans and food animals in the EU. This south-to-north gradient can have many explanations, but likely reflects true differences in usage of antibiotic compounds across Europe.

Why gulls? What our research has indicated in this and other studies is that gulls are very convenient model species for dissemination of resistant bacteria in the environment. They are everywhere, especially where there is concentration of people, animals, and waste products. The twist in this study was to sample the birds during breeding times, when birds are most sedentary and where results therefore are more likely to depict the local situation.

Finally, what does this tell us? Should we worry about some resistant bugs in gull? The answer is yes, we should. You, me, we all should care – and we should act! Clearly there are no strict boundaries between the everyday lives of humans and the organisms that occur in the environment. The bugs we select for in our hospitals and in agriculture do not stay there – they leak, finding their way out into nature. The gull story shows that similar patterns occur across Europe, the situation in gulls is mirroring the situation in anthropogenic sources. They are our canaries for the antibiotic mines, our whistleblowers. And it is a vivid illustration of the magnitude of the resistance problem we are facing in the future.

Link to the paper:

Stedt, J., Bonnedahl, J., Hernandez, J., McMahon, B.J., Hasan, B., Olsen, B., Drobni, M. & Waldenström, J. 2014. Antibiotic resistance patterns in Escherichia coli from gulls in nine European countries. Infection Ecology and Epidemiology  4: 21565

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Smell the roses –how does the fecal aroma differ between infected and uninfected Mallards?

By Jonas Waldenström

Dogs do it, mice do it, and probably a bunch of other mammals do it too! Do what? Use their noses to sniff the disease status of conspecifics. Chances are, you do it too – certain diseases give an odor taint, a recognizable miasma, suggesting us to keep our distance. For a dog, the world is made up of fragrances, and a sniff in the but is a social call and a way of catching up on the latest developments (e.g. what you ate, which reproductive state you currently are at, and your disease status). However (unfortunately?), humans very rarely smell each other’s bottoms – it is simply not socially acceptable, and we are not very bendy animals. Even though our noses are better than we give them credit for.

A social call of fragrances that tells the receiver all the things she/he needs to know

A social call of fragrances that tells the receiver all the things she/he needs to know

But what about ducks? How and what do they smell? In the rear end? After infection? These questions are about to finally get an answer through the very recent publication of Dr Kimball and his USDA and Chicago State University colleagues in the journal PLOS One! Read it, it has already gone viral on the internet – some studies are just so unexpected that they flutter into the limelight for a while. But similar to IgNoble prizewinning studies, it does tell you some things worth remembering.

Ever wondered how a duck's rear end smells?

Ever wondered how a duck’s rear end smells?

So lets tease this study apart and look at the rationales and the results. First of all, influenza A virus infection in Mallards is a gastrointestinal infection, with viruses primarily replicating in the cells of the smaller intestine. Virus progeny is released in huge amounts with feces out into the environment. These well-known facts make the rear end a good starting point for studies on the possible olfactory difference between infected and uninfected birds. Mallards are fairly gregarious, although not extremely social birds, and the viruses released from the behind of one individual need to find its way to the front of the next individual. Mallards spend a large part of their lives dabbling – a behavior where surface water is taken into the mouth, the beak closes and the tongue presses upward, forcing the water through thin lamellae on the side of the beak. The yummy stuff, and perhaps viruses too, are stuck on the lamellae and is swallowed down. This behavior is thought to be one reason why Mallards (and other dabbling ducks) are especially frequent influenza A virus hosts.

Given this, it would actually be beneficial for a duck to be able to tell if the duck ‘over there’ is excreting viruses or not. Kimball et al. took this to heart and infected six domestic Mallards with an H5N2 low-pathogenic avian influenza virus. The researchers collected Mallard feces (the fancy word for bird shit) before experimental infection, and up till 10 days post infection. These fecal samples were then used to train mice – yes, you read it right: MICE – to differentiate between duck feces from infected and uninfected individuals. In the training sessions, mice were rewarded if they went to the right fecal sample (i.e. the infected one), and this was later tested in a double-blind test where both the mice and the operator had no clue where the different samples went in.

The tests showed that mice were actually quite good at learning to differentiate which birds that had been positive. The remainder of the paper examines which volatile compounds that were present in the feces and that may have given the results. For those of you that are keen chemists, I suggest you read the original article. For the rest of us, let’s just settle with that there were differences in the chemical spectra of the two groups. Particularly, acetoin (3-hydroxy-2-butanone) was more prevalent in the fecal samples from the infected birds.

Let us pause here and summarize. Mice can be trained to distinguish the smell of fecal samples from an influenza-infected duck. End of story. But a rather fun story. And not too far-fetched. The volatiles associated with infections are coming more and more in medicine, all the way from dogs trained at sniffing out cancers, to breath test to diagnose Helicobacter pylori infections. Smell is the future. However, the results of the current publication are not strong enough to say much on the use of olfactory cues among Mallards. Unfortunately, the paper is very thin in the material and methods section and the scant information on the infection protocol, the methods used for detecting influenza A virus in the samples, and for how long individual birds where shedding virus aren’t very helpful. If we are to believe the results we also need to be able to read all relevant information. For instance, the sex of each bird, whether control birds (if they indeed where controls) were housed together with the others, or separately, whether the diet and husbandry was the same for all birds etc. Many small questions, but where answers are important for interpretation.

And the bigger question, of course, is whether ducks themselves can distinguish the fragrance of infection. And whether that translates into a modified behavior. My personal feeling is that a duck with its bill spooning up water with muck and filth like a boss probably doesn’t take the time to smell the roses.

Don't forget to stop and smell the roses!

Don’t forget to stop and smell the roses!

Citation: Kimball BA, Yamazaki K, Kohler D, Bowen RA, Muth JP, et al. (2013) Avian Influenza Infection Alters Fecal Odor in Mallards. PLoS ONE 8(10): e75411. doi:10.1371/journal.pone.0075411

How do you do, the things that you do, Mr. Flu?

The influenza A virus is a simple little thing. Just eight strands of RNA packed inside an envelope consisting of a host cell membrane. No metabolism, no fuzz, all very simple.

But, but, but – it is not very simple after all. In the flu business we still have large problems understanding many basic things this virus does. For instance, how does it survive in the environment? What governs host shifts? What makes a virus airborne, and what determines virulence properties? It is, in truth, a virus full of surprises.

This week, we published an article in Virology (http://www.sciencedirect.com/science/article/pii/S0042682213002638) on a particular aspect of flu – the way it changes genotype and phenotype through reassortment. Does this sound dull? NO, this is extremely interesting – follow me and see. It is evolution in the fast lane!

One thing that we are interested in in my research group is how influenza A viruses evolve. Being a RNA-virus with a sloppy RNA polymerase (equivalent to the virus’ genome copy machine) it mutates a lot all the time, much more than eukaryotes (animals like you, me, amoebae, fish, insects and whales). Thus, natural selection has a lot of variation to act upon, and hence the virus’ evolutionary trajectories may spurt out in different directions. Sometimes very fast, other times more slowly, when balanced by evolutionary constraints imposed by the host.

Long-term evolution in hominids

Long-term evolution in hominids

Long-term evolution in equines

Long-term evolution in equines

But the influenza A virus has another tool in its toolbox when it comes to evolutionary change. The reassortment tool. Reassortment starts when two viruses infect the same cell. The cell machinery is hitchhiked and forced into making new copies of the virus genome and making new virus proteins. And we are talking many, many, many copies of virus genomes!

The newly borne RNA segments are embedded with certain viral proteins and migrate to the cell surface. There they are stuffed into small pockets of cell membrane and are released to the outside by the scissor-like protein neuraminidase, that cuts the last anchors binding the virus to the cell surface. Off they go to infect new cells! But, the process of stuffing RNA segments into the virus bag is a rather random process (at least we think it is), which means that RNA segments from one virus can suddenly get in a genome constellation with RNA segments from another virus.

Reassortment of two influenza A viruses in a duck

Reassortment of two influenza A viruses in a duck

This means that the new virus progeny can consist of segments (each coding for one, or two proteins; thus having a function) with completely different descent. Different evolutionary histories! This is also the process that has started several of the flu pandemics we have seen in humans during the last century. In this context, it is often talked about the pig as a mixing vessel where flu viruses with human origin can meet viruses with an avian origin, since the pig has receptors on its cells that allows both virus types to enter the cell. Coinfections in the pig could then give rise to reassortant viruses, sometimes with the mammalian transmission adaptions and novel antigenic properties from the avian side. Wham-bam – no prior immunity in the population of humans and loads of sick people! Thus, understanding reassortment is important also for our own health.

The picture below summarizes how drastically the genotype/phenotype could change with reassortment.

flu reassortment

Reassortment creates genome constellations where segments may have completely different descent.

But how common is reassortment in nature? We know it should be fairly common, but can we enumerate it? I have previously written a post on individual disease histories and immune responses in Mallards. That article, published in PLoS ONE in April (http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0061201), followed 10 semi-domestic lure ducks in our duck trap at Ottenby for 1.5 years. The Virology article is a follow-up study on the viruses collected from these ducks in 2009.

It is a fairly complex story, and the most devoted readers of this blog should have a look at the original article (if you don’t have access contact me and we’ll sort it out). From the ten Mallards, we managed to retrieve 92 viruses of 15 different subtypes. With a detailed sampling scheme, where each bird was sampled daily, we could really determine the sequence of infection in each duck from primary infections, to secondary infections, and to study how different RNA segments were exchanged among viruses in different hosts across time.

In order to quantify reassortment, we used a combination of phylogeny-based and network-based tools (and we even needed to invent some novel ways of analyzing data, to make it happen). The level of reassortment was extremely high in our population of Mallards, and we estimated that >50% of the viruses were reassortants.

In a way this doesn’t make sense. If segments are exchanged so frequently, we would expect the influenza A virus gene pool to be in a panmictic stage, and that there shouldn’t be any linkage between segments. The solution, we believe, is that there is a strong selection afterwards, in the environment, or in transmission, where certain combinations of segments have a higher fitness. The discrepancy between the structured populations, where certain subtypes such as H4N6 or H1N1 are very common, and others combinations such as H4N1 or H1N6 are very rare, and the promiscuous panmictic reassortment levels suggest very strong selection pressures outside the host cells.

This is also the reason why unicorns are rare.

Jonas Waldenström

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.

l_015_04_l

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