Interview with the author: Detecting pathogens in koalas – dogs versus qPCR

Detecting pathogens in wild populations can be an enormous challenge. This is particularly the case for one of Australia’s most iconic but threatened animals – the koala. Koala populations face numerous threats, but one threat that has been difficult to quantify is infections from Chlamydia bacteria that can cause significant mortality in this species. Romane Cristescu (a postdoc at the University of the Sunshine Coast) gives us a behind the scenes look at the innovative paper she published recently with colleagues in Molecular Ecology Resources.

Find the full paper here: https://onlinelibrary.wiley.com/doi/full/10.1111/1755-0998.12999

Lead author Dr Romane Cristescu routinely works with detection dogs for ecological surveys, so naturally when the molecular tools proved imperfect, the team thought of training a disease detection dog (credits Marie Colibri)

What led to your interest in this topic / what was the motivation for this study?

Koalas are a very iconic species – in fact they are one of the most recognised animals on Earth. Yet they are also threatened, with most of their populations now in decline. They face many threats – some are easy to see and study, such as habitat loss, but some are harder. This is the case of the disease Chlamydia. To study the impact of Chlamydia on koalas, in the wild, at a broader landscape level, has been difficult. This is because koalas are hard to find (they are both cryptic and often occur at low density), and expensive to catch and sample. This led our team’s interest in Chlamydia detection from non-invasive samples: koala scats (droppings).

What difficulties did you run into along the way?

In this research project, we were interested in ascertaining both the sensitivity (the ability to correctly identify individuals with a pathogen) and the specificity (the ability to correctly identify individuals without a pathogen) of Chlamydia detection from koala scats using a well-proven method: qPCR. We unexpectedly found that qPCR had quite low sensitivity in our study (58% for koalas with urogenital infection, 78% for koalas with urogenital disease). This led us to try another molecular method: next generation sequencing. Again, we were taken aback when sensitivity was low. We then had to think outside the box. We already work with detection dogs, in fact they are the method we use to collect the scats to start with. Logically (for us), we decided to train and test a Chlamydia detection dog.

What is the biggest or most surprising innovation highlighted in this study?

When comparing two molecular (qPCR and next generation sequencing) methods and a detection dog to detect Chlamydia from koala scats, we did not expect that the dog would come out on top! The dog had both 100% sensitivity and specificity. This was a sobering outcome – no matter how fancy and high tech we have become in the laboratory, sometimes we are still no match to our old best friend – a dog. We were absolutely flabbergasted when we also found that the dog could detect ocular infection from the scats – we still do not know how this happens. Just that somehow the smell of the scat is affected by the eye infection. The volatile organic compounds (our likely suspects) involved are still unknown.

Moving forward, what are the next steps in this area of research?

There are two areas our team is keen to move on to. First, next generation sequencing using a specific Chlamydia panel. We expect this will dramatically increase the ability of next generation sequencing to detect Chlamydia. Second, and because not everyone can have a detection dog, we’d be keen to team up (please contact us!) and investigate ‘artificial noses’ to detect the volatile compounds – as these might be more robust against environmental conditions than DNA (non-invasive samples, such as scats, can be degraded by the elements). But beyond the method used to detect the presence of a chlamydial infection, we are still at the infancy of understanding the link between infection and disease (i.e. clinical signs). This is the most important aspect requiring clarification before we can effectively study the impact of chlamydial disease at the landscape level and, in particular, under which conditions this disease can threaten a population’s survival.

What would your message be for students about to start developing or using novel techniques in Molecular Ecology?

Our strongest advice in molecular ecology is to understand, and test, your methodology, especially if it is novel, but also if you are using a well-proven method in a novel way. An essential step is to quantify a novel technique’s characteristics and limitations (in our example, sensitivity and specificity of chlamydial detection tests from non-invasive (scat) samples). A risk here is to be tempted to compare results that have different methods to draw general conclusions. If you compare the results of one population using a particular test to another study of a different population and test, you must remember to also compare both methods using samples with known outcomes: how consistent are the results of different tests compared to the reality? This is costly and time consuming, but nonetheless necessary before novel methods can be widely applied.

What have you learned about methods and resources development over the course of this project?

Resilience is key – the first method you try might not give you the answer you want, nor the second. You haven’t failed yet – you have found many ways that do not work. Keep on keeping on, think outside the box, collaborate with others outside your area of expertise, who will bring skills and ideas you would never think of: this is how scientific advances are made.

Describe the significance of this research for the general scientific community in one sentence.

This research highlights that sometimes the best method can be unexpected – to do successful research takes both creativity and perseverance.

Describe the significance of this research for your scientific community in one sentence.

We need to continue developing non-invasive, affordable, accurate tools if we are to understand how hosts, pathogens and landscapes interact to create a perfect extinction vortex; for koalas and Chlamydia – we are not there yet.

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