Researchers have found they can detect viruses in mountain gorilla and golden monkey populations using saliva from remnants of bark, leaves and fruit the animals chewed.
This is a simple, noninvasive alternative to existing sampling options that could help monitor and prevent outbreaks in zoonosis-vulnerable threatened primates.
Chewed material could also be used to identify pathogens in other wildlife populations for whose survival health monitoring is critical.
Preoccupied with snipping a saliva-soaked plant part with a scalpel and placing it in a little tube of viral transport media, veterinary epidemiologist Tierra Smiley Evans thought she was alone, backed up against a big bush. But someone was watching close by with beady eyes. Suddenly, a shaggy black arm darted under her armpit and snatched her sample bag.
The thief? A young mountain gorilla determined to check out that enticing goody bag containing spit-moistened bark, leaves and fruit rejected by one of her group members – material Evans would genetically analyze to help prevent viral outbreaks that could decimate fragile gorilla populations.
Evans, a graduate student at the University of California Davis School of Veterinary Medicine, published a paper detailing this novel disease detection method in the American Journal of Primatology last month. According to its authors, the simple technique can help scientists conduct viral research, monitor threatened wildlife’s health and stop viruses from becoming pandemics without disrupting animals’ natural behaviors.
“I’m hoping it will provide an easier method to noninvasively monitor viruses in primate populations because with species such as mountain gorillas, it’s easier to collect chewed plants than fecal samples,” said Evans.
Finding human diseases in gorilla food scraps
About 880 critically endangered mountain gorillas inhabit the Virunga range across Rwanda, Uganda and the Democratic Republic of Congo (DRC), and the Bwindi Impenetrable Forest in Uganda. Over sixty percent have been habituated to humans for ecotourism, which exposes them to human pathogens. Many gorillas have died, for instance, from human metapneumovirus, which causes the common cold.
The apes spend most of their day eating a variety of plants, spitting out a huge jumble of plant parts they don’t like; abundant food gives them the freedom to be picky. Because park officials and conservationists aim to minimize behavioral disturbance to the gorilla family groups, Evans and her colleagues investigated noninvasive ways to monitor viruses based on the creatures’ table scraps to supplement standard noninvasive fecal testing.
Evans and her colleagues gathered plant material chewed and left behind by 383 wild mountain gorillas and analyzed the samples for viruses shed by mouth. They successfully detected both RNA and DNA viruses; the former are more likely zoonoses. Applied alongside fecal and urine sampling, the new technique could help understand the most common pathways of viral spreading in wild primates.
The researchers tracked 26 gorilla groups, which each took from 30 minutes to six hours to reach depending on its location. Evans watched the animals eat, noting the name of each gorilla she selected a sample from, as individuals are distinguishable by nose prints and other facial features.
“I would observe when they were spending a long period of time chewing on a particular plant and spitting it out. I would watch where they dropped it and collect that piece because those were the best samples,” she said. “When there was a big pile, I would look at all the pieces that were chewed and the ones that looked visibly wet or the ones that looked way more chewed than other pieces, I would prioritize collecting.”
Wearing gloves, the lead author cut the most chewed parts of the plants with a sterile scalpel blade and deposited them into small tubes of viral transport media, a substance that protects viruses. She froze most of the samples in liquid nitrogen, which the scientists had carried into the forest in small tanks, within five to ten minutes of collection. When rough terrain made hiking with heavy packs difficult, she put the samples onto icepacks and transferred them into liquid nitrogen at the forest edge to freeze them quickly.
In the lab, the scientists thawed and centrifuged the samples to separate plant matter from the saliva containing the viruses targeted for investigation. They then processed the resulting liquid with standard nucleic acid extraction kits. Applying conventional PCR (Polymerase Chain Reaction) at Makerere University in Uganda’s capital, Kampala, and at UC Davis, the researchers looked for herpes viruses and simian foamy viruses. If they found a suspect positive sample, they sequenced the PCR products to ensure it was a gorilla virus and identify it further.
Till now, scientists have predominantly used blood samples as well as other direct swabs to identify viral sicknesses in primates, but these samples generally require anesthesia to obtain. Veterinarians anesthetize mountain gorillas only after agreeing that an animal’s illness or injury is severe enough to warrant intervention. Researchers draw as many samples as possible–blood samples, oral swabs, nasal swabs, fecal swabs–from gorillas anesthetized for treatment; these contribute to a biobank for future studies.
“I hope chewed plants start to be used in conjunction with fecal samples when people are trying to do noninvasive studies of great apes and other primates to broaden the scope of viruses we detect,” said Evans of the technique. “I would like to see it used with mountain gorillas in the context of respiratory outbreaks.”
According to Evans, wild celery stems and various species of branches off which the apes ate leaves provided especially valuable samples for gorilla herpes DNA. The celery was effective because the gorillas strip its inner pith and drag it along their gums as they eat it and discard its outer shell. The branches were useful because the animals run stems through their mouth to tear off the leaves, allowing saliva and oromucosal cells to catch in the leaves as they would in a swab. The scientists, however, haven’t determined how long plant samples are valid because they gather them fresh within ten to fifteen minutes of disposal.
The scientists have also used chewed plant samples to identify the prevalences of specific herpes viruses within the gorilla population; results from these studies will be published soon.
Evans thinks the method could also work with unhabituated gorillas if sampling scientists maintained a safe distance from the often unpredictable groups.
Applications and limitations
Additionally, the study followed a troop of golden monkeys, which also inhabit the Rwandan Virunga Mountains, to see if chewed material could be used for virus detection in other species. Although effective, the samples were harder to acquire because the arboreal creatures often dropped chewed items in trees, which required the scientists to climb. Evans believes that although the technique hasn’t been tried with any other species, it could work for other primates.
“It’s just a matter of the researcher knowing how they eat, drop food and whether it’s logistically possible to follow them, observe them and collect a fresh chewed piece of food,” she said.
Evans recalled some challenges to using this new kind of sample. First, the process of obtaining multiple chewed samples for a population-wide study was labor-intensive and time-consuming.
“You’re hiking eight hours on a long day to find the group, spend time with them and get out of the forest,” she said. “It’s tiring, but that’s what researchers like myself love the most, the most fun part of science.”
Second, rain washes away the saliva on the samples, limiting the number of viable ones to those undercover. What’s more, the scientists had to be stealthy while collecting samples to avoid disrupting or being disrupted by the gorillas.
“If you tried to collect a sample before they moved on to a new spot, they would come back and want that pile again. You had to be sneaky about it–make sure they were chewing their food, moving on to another spot and had their back to you while you processed samples,” Evans said. “Particularly with young ones, you didn’t want them to see you with a sample bag or different fun items because they would try and steal them. They’re curious, just like we are. You have something interesting, they want to explore it.”
The researcher completed the study over a year as a Fulbright Scholar. While researching and teaching at Makerere University, she hopped from city to forest and back on two-week sampling trips to Bwindi in southwestern Uganda and the dormant volcanoes of northern Rwanda. As a veterinary student, Evans had spent time with the gorillas and noticed the potential the mass of chewed material they left behind had as samples. She’d known this type of sample had been applied to bats to detect viruses and chimpanzees to detect pathogenic bacteria such as Streptococcusus and Staphylococcus.
The new research was headed by the One Health Institute’s Karen C. Drayer Wildlife Health Center and Gorilla Doctors program at the UC Davis School of Veterinary Medicine. Collaborators included the UC Davis California National Primate Research Center, Makerere University, Uganda Wildlife Authority and Rwanda Development Board. The One Health Institute leads the USAID Emerging Pandemic Threats PREDICT project, which conducts global surveillance to detect and prevent spillover of pathogens of pandemic potential between wildlife and people.
Evans and colleagues’ study reflects the “one health” approach to public health and wildlife conservation to which Gorilla Doctors and PREDICT ascribe. This holistic paradigm emphasizes the interdependence of human, domestic animal, wildlife and environmental health and the need to take all of these factors into account to produce better medical solutions.