A pioneering method for monitoring wild animal populations, developed in a nature reserve in southwest China may become an important tool in the prevention of future virus outbreaks – and the solution may lie in the gut of another parasite.

Using the latest biotechnology, a team led by Professor Douglas Yu of Britain’s University of East Anglia extracted DNA from digested blood in leeches’ stomachs, determined what animals they had fed on, and then produced a model of the distribution of wild animals in the Ailao Shan Nature Reserve in Yunnan province.

The same DNA analysis method could feasibly be used to examine drain water for evidence of illegal wildlife consumed or traded in markets, Yu says.

It took the team almost five years and more than 30,000 leeches, and now they hope to see the methodology applied to combat illegal trafficking of animals captured in the wild.

Wild animals are a reservoir of viruses that, due to their ability to rapidly change genetic make-up, regularly “jump” to other species, including humans.

Severe acute respiratory syndrome (Sars) is believed to have originated in bats, before the virus jumped to civet cats and then humans.

Human immunodeficiency virus (HIV) is suspected to have made the leap from chimpanzees to humans in the early to mid-1900s. The origin of the 2019 novel coronavirus is still unclear, but pangolins, the world’s most trafficked animal, are thought to be part of the transmission chain. Bats may have passed the virus on to the scaly animals before they infected humans, scientists say.

Hunting, trapping and eating wild animals brings people into contact with these microscopic parasites.

“Wildlife is dangerous. It is dangerous to catch, handle and consume,” says Yu, an American-born Chinese who is also a principal investigator at the Kunming Institute of Zoology in Yunnan.

Yu’s field is ecology, a branch of biology that studies interactions between living organisms and their environment. He is a pioneer in using environmental DNA (eDNA) in ecological research. eDNA is the “molecular soup” of small fragments of DNA that different animals have shed into the environment.

Until recently, biotechnology couldn’t separate these individual bits of DNA in the “soup” to identify which animals they came from. But the latest technology can process multiple DNA molecules at the same time, and it has become a powerful forensic tool.

Without seeing or touching the animals, their presence can be detected from a sample of soil, water – or the remnants of digested blood in a leech’s stomach.

A major problem is knowing exactly what lives in a forest. Camera traps can be used, but they do not generally pick up small animals. Walking across the terrain, trying to spot wildlife, is labour-intensive and ineffective. “Most animals don’t want to be seen,” Yu says.

Land leeches, driven by a voracious appetite for blood, are far better at finding animals in the thick jungle than humans are.
They are not picky eaters – a frog’s blood is as good as a bear’s. They are plentiful, too. Summer visitors to the forests of Yunnan frantically empty their shoes of leeches every few minutes.

In 2015, Yu’s team set out to survey Yunnan’s forests for wildlife through the eyes – or stomachs – of leeches, with the goal of developing a reliable method of wildlife detection from their DNA in their stomachs.

Located 170km south of Kunming, Ailao Shan Nature Reserve is roughly the size of Singapore – a 6km wide and 125km long strip of old-growth forest clinging to “a chain of top halves of mountaintops”. Hemmed in on all sides by agricultural land, the reserve “has some of the best wildlife still in China, but it has not been properly surveyed since 1981”, Yu says. Illegal hunting of wild animals occurs there.

The scientists paid park rangers to collect leeches during their regular patrols and between them covered the entire territory.

Yu’s team extracted DNA from the leeches’ stomachs, then applied sophisticated statistical software that could compare the different DNA sequences against animal DNA sequences in existing databases, similar to how facial recognition software matches an image of an individual face from a set of millions.

The leeches proved to be effective in revealing the animals hiding out in Ailao Shan. The DNA of Asian black bear, sambar deer, macaque, leopard cat, serow antelope, as well as of numerous birds, frogs, and toads was detected in their stomachs. The list included both endangered and commonly hunted animals.

From the data, Yu’s team made a model of the animals’ distribution in Ailao Shan.

The results closely matched the biology of the animals. “The right species were found in the right places,” Yu says. It also gelled with previous records of animal sightings in Ailao Shan. The leeches could be trusted.

At the beginning, however, there had been scepticism. Small pilot studies in 2012 showed promise, but a large-scale project had never been attempted.

“We did everything for the first time – from designing an effective way to pay the rangers for their work collecting leeches, to molecular analysis and statistical models. We had to string together a lot of different methods,” Yu says.

The team also had to improvise. “Normally you would need a specialist, ultra-clean lab [to avoid cross-contamination with DNA from other organisms], but we did not have enough such facilities. So another approach was using new apartments that never been lived in and so are unlikely to have DNA from other sources. We rented a few for a month at a time in Kunming and set up UV lights and DNA extraction equipment.”

The results were timely. “We certainly did not anticipate a coronavirus epidemic happening,” Yu says. “The government now says that it may ban wildlife markets permanently [in China] and also crack down on the wildlife trade.”

Intensive monitoring of wildlife would be a vital part of the ban’s enforcement. First, the baseline of animal abundance must be established, followed by repeated surveys to measure the effectiveness of protection measures. Yu says that his team is “ready for that … we have provided the methodology in anticipation. The same methods could also be applied to drain water from markets to detect wildlife.”

He adds that the method is affordable. “The cost of each survey would be a few hundred thousand yuan. Relative to money spent on many other things in China, it is a small amount.”

Yu points out that the risks from the wildlife trade and consumption do not just come from China.

“There is subsistence hunting everywhere in the world. Only a small proportion of Chinese eat wildlife, but this being China, a small proportion becomes a very large number, and these consumers are financing a supply chain of hunters, transporters and sellers that are exposing themselves to infection by novel viruses, one of which appears to have set off this year’s coronavirus epidemic.

“There is a massive amount of hunting in Southeast Asia, with much of it to supply the Chinese market. I am a little surprised this [novel coronavirus outbreak] had not yet happened where there is even more hunting, like in Vietnam for example,” he adds.

“There is a traditional Chinese medicine idea that wild animals have this authenticity, a ‘wildness’ to them, that it is healthy to eat them and that it makes you stronger. As the current epidemic shows, it is completely the opposite,” he says.

“The safest thing to do is leave wild animals be and then nothing happens. Don’t buy wildlife products, because that pays lots of people to hunt and handle them, and another person will get infected and start yet another epidemic in the future. People have to understand that buying wildlife medicines, meat, bones, horns, scales and skins puts all our lives and whole economies at risk.”