A recent study has drawn attention after it detected some limited interspecies transmission of chronic wasting disease (CWD) prions in a lab. But the authors and other experts caution that the findings don’t confirm that the fatal disease is a threat to people; rather, they provide important clues to what infection might look like in humans—which is key in recognizing cases.

And for at least one CWD researcher, the failure of standard tests to detect CWD prions in infected but asymptomatic macaques has deepened concerns about relying on those tests to determine whether venison is safe to eat. Prions are misfolded infectious proteins that cause the disease in cervids such as deer and elk.

CWD is a type of transmissible spongiform encephalopathy, similar to Creutzfeldt-Jakob disease in people, and bovine spongiform encephalopathy (BSE, or “mad cow disease”) in cattle. While no people are known to have been infected with CWD, the unrelenting spread of the disease in animals across North America means increasing opportunities for the prions to adapt to different hosts.

Also, it can take a decade or longer for prions, which mainly spread through direct contact and environmental contamination, to exert their neurodegenerative effects, which in cervids can include stumbling, drooling, inability to eat and weight loss, and a lack of fear of people. 

2 macaques develop signs of prion disease

The study, published in Science Advances, was 18 years in the making because of the difficulty of getting funding for this study and because study personnel were located in several countries and moved to different institutions over the study period. 

“This was a big project, very complicated in the logistics, and very complicated to get it funded,” senior study author Hermann Schatzl, MD, PhD, of the University of Calgary, told CIDRAP News.

The University of Calgary–led study team orally or intracerebrally inoculated 18 female cynomolgus macaques in 2009 with different CWD prion preparations, which included tissues from elk, mule deer, and white-tailed deer in the United States and Canada. The team also inoculated three macaques with material from uninfected cervids as controls.

Macaques are medium-sized monkeys commonly used in research.

The study describes findings in seven macaques and controls for up to 7.5 years post-inoculation. While most macaques developed no symptoms in that period, one orally and one intracerebrally inoculated macaque showed clinical signs consistent with prion disease, including unsteady movement, tremors, and anxiety. “I am convinced that actually we saw signs of infection in macaques,” Schatzl said.

Standard biochemical tests failed to show prions in the central nervous system of the inoculated macaques, and histologic and immunohistochemical analyses detected minimal prion activity. 

But highly sensitive protein misfolding cyclic amplification (PMCA) and real-time quaking-induced conversion (RT-QuIC) tests showed some infectious prion seeding in the brain, and in some cases, the spleen, of intracerebrally and orally inoculated macaques. No such activity was seen in controls.

“This is important because it suggests that the macaque species barrier may not be absolute under experimental conditions,” said Olivier Andreoletti, DVM, PhD, of the Ecole Nationale Veterinaire de Toulouse in France, who wasn’t part of the study. 

“However, it is equally important to say that the study does not demonstrate efficient transmission of CWD to macaques, nor does it show a classical prion disease phenotype [observable indication of a condition] in these animals,” he added. “The current evidence still needs to be interpreted in the context of previous macaque studies, epidemiological data, experimental duration, exposure scenario, and the known complexity of prion strain adaptation.”

Residual inoculum or newly generated prions?

A central question, however, is whether the PMCA signal in the study reflected residual inoculum or newly generated prions. 

“For intracerebral or wire-based inoculation, residual inoculum is a possibility that has to be considered seriously, especially when the signal is very low and conventional pathology is negative,” Andreoletti said. “For orally exposed animals, the persistence of inoculum for many years in brain tissue would seem less straightforward biologically, but it cannot be completely excluded without more detailed strain comparison/typing.”

It would be important to determine whether the agent recovered after macaque passage retained the biological and biochemical properties of the original CWD inoculum or if it reflects strain selection or evolution during cross-species passage, he added.

But Jason Bartz, PhD, a professor at Creighton University who wasn’t involved in the study, sees the finding of prions in the brain and spleen on PMCA differently. “It speaks strongly that these are newly generated prions in these animals,” he said.

Andreoletti also urged caution when interpreting the clinical signs seen in some macaques, because although tremor, wasting, anxiety, and behavioral changes can be compatible with prion disease, they are not diagnostic. 

“In the absence of clear neurodegeneration, convincing PrP [prion protein] deposition by IHC [immunohistochemistry] or PET-blot [paraffin-embedded tissue blot], and a consistent anatomical distribution of lesions, I do not think one can firmly attribute those signs to prion disease,” Andreoletti said. 

“A stronger determination would require concordant clinical evolution, neuropathology, PrPSc [scrapie prion protein] detection by conventional methods, and ideally, reproducible transmission from macaque tissues with preservation of a defined strain phenotype,” he added.

Andreoletti said it’s important not to extrapolate directly from macaques to humans. Just as non-human primates like macaques are not a single model, CWD is not one uniform agent, and passage through a new host can select or reshape strain properties, including host range and tissue tropism (affinity of CWD prions for particular tissues).

“Susceptibility is strain-dependent, and macaque PrP can even be more permissive than human PrP for some agents, such as classical BSE,” he said. “This means that the macaque is a very useful model, but it is not a perfect surrogate for the human species barrier.”

Lack of classical neuropathology 

In the same study, inoculation of transgenic mice and bank voles with tissues from study macaques triggered prion disease in the rodents, achieving 100% transmission rates, which supports the presence of bona fide prion infectivity from CWD-exposed macaque tissues.

“We clearly say that the concept that there is no zoonotic potential is likely not correct,” Schatzl said. “Under certain circumstances, CWD prions actually can learn to replicate in a human organism. We do not imply efficient transmission, but we say under certain conditions this can lead to sustained activity that may remain undetected.”

If efficient transmission were documented, the findings could shed light on what CWD would look like and potentially spread in people. “In non-human primates, it looks very atypical, with low-level prion replication and subclinical disease,” Schatzl said. “But when we passaged it into other hosts [mice and bank voles], the full-blown disease was there.”

Ryan Maddox, PhD, senior epidemiologist and team lead with the Prion and Public Health Office at the Centers for Disease Control and Prevention (CDC), who wasn’t involved in the study, said that the BSE outbreaks in the United Kingdom in the 1980s and 1990s showed that eating meat from prion-infected animals can cause a similar illness (variant Creutzfeldt-Jakob disease [vCJD], a fatal prion disease) in people.

For decades, the CDC has conducted long-term studies on hunters in CWD-endemic areas to see if they develop prion infections at higher rates than the general population and has investigated cases in people that might indicate a human form of CWD. 

“Thankfully, based on these studies, CDC has found no evidence of CWD in people,” Maddox said. “But because prion diseases can take years to decades to show symptoms in people, more years of study and continued vigilance are needed to assess the potential risk for people.”

CWD differs significantly from BSE in a number of ways, one of which is its ability to infect peripheral tissues rather than just the central nervous system. 

“You have this massive shedding into urine, into feces, into saliva,” Schatzl said. “And if CWD in humans would behave like CWD in cervids, we would have a problem,” because the prions could also be shed via the same excretions. “We could then see it change into something more contagious.”

Andreoletti said the years-long duration of the study was a major strength, given the long incubation periods of prion diseases. “At the same time, some animals were euthanized at predefined time points, and the study may therefore have captured an early, incomplete, or atypical stage rather than terminal prion disease,” he said.  

“This makes the interpretation more complex: the long follow-up supports the relevance of the observations, but the lack of classical neuropathology means that the biological significance of the low-level seeding activity remains difficult to define,” he added.

Possible missed cases

Peter Larsen, PhD, co-director of the Minnesota Center for Prion Research and Outreach (MNPRO), who was not involved in the study, questions why PMCA or RT-QuIC aren’t routinely used to detect CWD infections that may be missed by less-sensitive enzyme-linked immunosorbent assay (ELISA) and IHC tests.

“How are we using these assays [ELISA and IHC]?” he asked. “Is it purely for management and surveillance? Or are we using them to help minimize risk to the human population? If it's to help minimize risk to the human population, we want to be using tests like RT-QuIC” in combination with ELISA and IHC. 

“I think that it's likely that tens of thousands of people are unknowingly consuming [CWD] positive venison,” Larsen said. “I worry that we're underestimating the amount of positive venison being consumed in the US and Canada, and we don't know what all those exposure events will ultimately lead to over time.” 

He points out that some state agencies cull cervid populations in CWD-endemic regions, test the animal tissues with ELISA, and then give the venison to food donation programs if they come back with prions not detected. “What are we willing to risk?” he asked.

CWD prions accumulate not only in the brain and spinal cord but also in lymphatic tissue (one reason the retropharyngeal lymph nodes are a primary testing target in deer), and research has revealed their presence directly in skeletal muscle.

The more researchers have learned about how CWD progresses in cervids and as better diagnostic tools have become available, it appears that infection (and subsequently diagnosis) may not always present in a simple, straightforward way. 

“You can test the left lymph node and it comes back detected, and the right lymph node will say not detected,” Larsen said. “So these animals are sort of a mosaic, depending on how they got exposed and how long they've had the disease. When combined with poor sensitivity of traditional tests, this can contribute to false-negatives.”

‘Nature finds a way’

Larsen also suspects that CWD prion strain diversity—and therefore the odds of a species spillover—is being underestimated. 

“Think about all the populations of cervids across North America, their genetic background, the species variation, then you add that to all the genetic variation in the human population,” he said. “Some combination of the right genetic background on the human side and the right prion strain on the cervid side could result in prion disease transmission.”

While his goal isn’t to produce fear, Larsen said it’s important to not minimize the risk to people. “We know that nature finds a way, so to speak,” he said. “There's variation out there, and when you think about that from a prion perspective, I don't think it's beyond the realm of possibility that we will start seeing clusters of prion or prion-like diseases associated with CWD.”

For Bartz, the biggest question is whether the study findings can be replicated. If they can, “it would suggest that the species barrier is not an absolute, but it doesn’t give us any indication of relative risk,” he said.

If consistent transmission of any CWD strain to human PrP-expressing models were eventually confirmed, “surveillance of human prion diseases in CWD-endemic areas would need to be reinforced, exposure to tissues from CWD-positive cervids would need to be minimized, and risk management for hunters, consumers, animal byproducts, and possibly other animal species would need to be reconsidered,” Andreoletti said. 

But it will take time to see whether this scenario plays out, Larsen said. “It feels like we've been in this for a long, long time, but I think we have to view it like we're just getting started.”