Ticks on wings – screening for Candidatus Neoehrlichia mikurensis in birds

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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

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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.

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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.

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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