Prion detection in the muscles of CWD-affected Norwegian cervids

Aims: Chronic wasting disease (CWD) is a fatal neurodegenerative prion disease in cervids, caused by the misfolding of the normal prion protein (PrPC) into its pathological form (PrPSc). Since its discovery in the United States 50 years ago, CWD has been reported in Canada, South Korea, and, more recently, Norway, Finland, and Sweden. Infected animals shed prions into the environment via saliva, urine, and feces, facilitating transmission among cervids and raising concerns about potential spillover to non-cervid ruminants. Given these risks, coupled with the unknown zoonotic potential of CWD, European community has established specific surveillance measures aimed at minimizing disease spread and transmission. Here we employed the protein misfolding cyclic amplification (PMCA) to investigate whether also the muscles of CWD-affected Norwegian cervids contain infectious prions. Materials Muscle samples (masseter, triceps brachii, psoas, longissimus dorsi, semitendinosus, ocular, lingual and cardiac), lymphoreticular system (retropharyngeal, parotid, prescapular and popliteal lymph nodes) and peripheral nervous tissues (brachial plexus, ischiatic- and optic nerve) were collected from Norwegian reindeer (n=2), moose (n=3) and red deer (n=2) with CWD. Samples from three healthy cervids (reindeer, moose and red deer) served as negative controls. Methods PMCA was performed using the brain homogenates of Bank vole a reaction substrate. The amplified products were analyzed by Western blot following proteinase K digestion. Each sample was analyzed independently in two laboratories (Norway and Italy). Results The optimized PMCA protocol successfully amplified PrPSc from lymphoreticular tissues (8/19), peripheral nervous tissues (14/15), and muscle tissues (34/48) of Norwegian reindeer, moose, and red deer affected by CWD. No PrPSc was detected in control animals, achieving 100% specificity. The inter-laboratory agreement for these results was 92%. Discussion We show for the first time the presence of prions in the muscles, lymphoreticular and peripheral nervous system tissue collected from Norwegian CWD affected animals. Conclusions These findings highlight the importance of using ultrasensitive tests to detect cases of Norwegian CWD that are likely to be missed by conventional methods. Additionally, these results will aid in controlling disease spread, particularly concerning its potential zoonotic transmission, by enabling the detection of prions in peripheral tissues.