Improved amplification tests can help detect chronic wasting disease (CWD) in the saliva, urine, and feces of white-tailed (WTD) before clinical signs appear, which may be useful in disease surveillance, Colorado State University researchers reported yesterday in Emerging Infectious Diseases.
CWD, a neurologic disease caused by misfolded infectious proteins called prions, affects cervids such as deer, elk, moose, and reindeer. Prions are extremely resilient and can persist in the environment for years. CWD poses an ongoing threat to cervids, because it can spread from animal to animal and through environmental contamination.
The study authors tracked CWD prion shedding in the saliva, urine, and feces of 12 WTD for 66 months after they were given small amounts of CWD-positive brain tissue or saliva by mouth. Two uninoculated deer that were housed separately at the research facility served as controls. The team also determined the prion protein PRNP genotype of the deer and collected tonsil and recto-anal lymphoid tissue 3 months after they were inoculated (given infected tissue).
Then they analyzed prion seeding activity through iron-oxide bead capture along with conventional serial protein misfolding amplification (sPMCA) and real-time quaking-induced conversion (RT-QuIC) to concentrate prions and reduce or bypass interfering factors, boosting CWD detection and reducing false-positive results.
"Our goal was to provide a more complete understanding of prion shedding onset and duration that contributes to CWD pathogenesis and transmission and aid in early antemortem detection by using minimally invasive methods," the researchers wrote.
Saliva may be more infectious than urine, feces
As soon as 6 months after infection, CWD prion shedding was detected in all six deer expressing the wild-type 96GG genotype. Seeding was consistently detected (four or more consecutive positive time points) in four of six deer and infrequently detected (three or fewer time points) in two.
Among deer with other polymorphisms (possible variations of a trait on a gene; 96GS or 103NT), one or more positive fecal results were noted in four of six deer at 9 to 15 months. In one deer, seeding activity was identified in three consecutive fecal samples.
We also found more consistent CWD prion shedding in saliva than urine for both cohorts, suggesting that saliva might be a plausible vector for efficient disease transmission.
In total, prion shedding was found in 27 (64%) of 42 fecal samples from the 96GG cohort and 10 (19%) of 52 fecal samples from 96GS/103NT deer. All 21 fecal samples from controls stayed negative.
At 18 months post-inoculation, CWD prion shedding was seen in the urine of one of two 96GG deer. Three of six deer shed prions from 18 months until euthanasia, and two were positive only at the final urine collection. One of six deer never shed prions in urine. CWD prion seeding was detected in urine from only one of six 96GS/103NT deer at 27 months, coinciding with the first positive tonsil test.
Overall, CWD prion shedding was observed in 9 of 16 urine samples from deer in the 96GG group and in 1 of 50 urine samples from 96GS/103NT deer. All 28 urine samples from control deer stayed negative. "We show that CWD-infected deer expressing the 96GG genotype shed prions in their urine during later disease stages and did so more frequently and consistently than deer expressing alternate polymorphisms," the authors wrote.
As soon as 9 months post-inoculation, CWD prion shedding was seen in the saliva of two of six 96GG deer, at the same time as the first positive tonsil test. Intermittent shedding occurred in five of the deer 0 to 6 months after the first positive tonsil biopsy. In the 96GS/103NT cohort, prion shedding was detected in three of the deer at 21 months, 3 to 9 months after the first positive tonsil test result.
Fifteen of 47 saliva samples from 96GG deer and 8 of 70 samples from 96GS/103NT deer showed prion seeding. None of the 28 saliva samples from control deer demonstrated seeding.
"We also found more consistent CWD prion shedding in saliva than urine for both cohorts, suggesting that saliva might be a plausible vector for efficient disease transmission," the investigators wrote. "This finding reinforces previous studies reporting that saliva is more infectious than urine or feces after experimental CWD prion inoculation."
Certain genotypes may be tied to more prion shedding over lifetime
PRNP codon 96 polymorphisms are known to lower CWD susceptibility and slow disease progression in WTD, the authors noted, and the study results bore this out. One of six deer expressing the 96GG genotype had positive lymphoid biopsies at 6 months, as did all six at 18 months.
Our findings suggest that deer expressing alternative PRNP polymorphisms might live longer and, although they shed fewer prions throughout CWD course, might over their extended lifespan increase CWD prions in the environment.
In contrast, 96GS/103NT deer took 9 months longer to become CWD-positive status, and the first positive tonsil tests occurred at 15 to 27 months. All 96GG deer were euthanized by 39 months because of disease progression, while three of six 96GS/103NT deer displayed no clinical signs when euthanized at 48 (96GS) or 66 (103NT) months.
"Our findings suggest that deer expressing alternative PRNP polymorphisms might live longer and, although they shed fewer prions throughout CWD course, might over their extended lifespan increase CWD prions in the environment," the authors wrote.
The results suggest the role of prion shedding in CWD spread, especially because prion load in saliva, urine, and feces is presumably low, the researchers said. "Our findings demonstrate that improved amplification methods can be used to identify early antemortem CWD prion shedding, which might aid in disease surveillance of cervids," they concluded.
CWD found in 35 US states
CWD has been confirmed in 35 US states, five Canadian provinces, and in Norway, Finland, Sweden, and South Korea.
Signs of the disease include weight loss, uncoordinated movement, listlessness, excessive thirst or urination, drooling, drooping ears, and behavioral changes.
While CWD isn't known to infect people, the World Health Organization and the US Centers for Disease Control and Prevention recommend against eating meat from an infected animal and urge taking precautions when field-dressing or butchering cervids.
