Norwegian moose CWD induces clinical disease and neuroinvasion in gene-targeted mice expressing cervid S138N prion protein.

Chronic wasting disease (CWD) is a prion disease affecting deer, elk and moose in North America and reindeer, moose and red deer in Northern Europe. Pathogenesis is driven by the accumulation of PrPSc, a pathological form of the host's cellular prion protein (PrPC), in the brain. CWD is contagious among North American cervids and Norwegian reindeer, with prions commonly found in lymphatic tissue. In Nordic moose and red deer CWD appears exclusively in older animals, and prions are confined to the CNS and undetectable in lymphatic tissues, indicating a sporadic origin. We aimed to determine transmissibility, neuroinvasion and lymphotropism of Nordic CWD isolates using gene-targeted mice expressing either wild-type (138SS/226QQ) or S138N (138NN/226QQ) deer PrP. When challenged with North American CWD strains, mice expressing S138N PrP did not develop clinical disease but harbored prion seeding activity in brain and spleen. Here, we infected these models intracerebrally or intraperitoneally with Norwegian moose, red deer and reindeer CWD isolates. The moose isolate was the first CWD type to cause full-blown disease in the 138NN/226QQ model in the first passage, with 100% attack rate and shortened survival times upon second passage. Furthermore, we detected prion seeding activity or PrPSc in brains and spinal cords, but not spleens, of 138NN/226QQ mice inoculated intraperitoneally with the moose isolate, providing evidence of prion neuroinvasion. We also demonstrate, for the first time, that transmissibility of the red deer CWD isolate was restricted to transgenic mice overexpressing elk PrPC (138SS/226EE), identical to the PrP primary structure of the inoculum. Our findings highlight that susceptibility to clinical disease is determined by the conformational compatibility between prion inoculum and host PrP primary structure. Our study indicates that neuroinvasion of Norwegian moose prions can occur without, or only very limited, replication in the spleen, an unprecedented finding for CWD.

Extraneural infection route restricts prion conformational variability and attenuates the impact of quaternary structure on infectivity.

Prions can exist as different strains that consist of conformational variants of the misfolded, pathogenic prion protein isoform PrPSc. Defined by stably transmissible biological and biochemical properties, strains have been identified in a spectrum of prion diseases, including chronic wasting disease (CWD) of wild and farmed cervids. CWD is highly contagious and spreads via direct and indirect transmission involving extraneural sites of infection, peripheral replication and neuroinvasion of prions. Here, we investigated the impact of infection route on CWD prion conformational selection and propagation. We used gene-targeted mouse models expressing deer PrP for intracerebral or intraperitoneal inoculation with fractionated or unfractionated brain homogenates from white-tailed deer, harboring CWD strains Wisc-1 or 116AG. Upon intracerebral inoculation, Wisc-1 and 116AG-inoculated mice differed in conformational stability of PrPSc. In brains of mice infected intraperitoneally with either inoculum, PrPSc propagated with identical conformational stability and fewer PrPSc deposits in most brain regions than intracerebrally inoculated animals. For either inoculum, PrPSc conformational stability in brain and spinal cord was similar upon intracerebral infection but significantly higher in spinal cords of intraperitoneally infected animals. Inoculation with fractionated brain homogenates resulted in lower variance of survival times upon intraperitoneal compared to intracerebral infection. In summary, we demonstrate that extraneural infection mitigates the impact of PrPSc quaternary structure on infection and reduces conformational variability of PrPSc propagated in the brain. These findings provide new insights into the evolution of stable CWD strains in natural, extraneural transmissions.

Propagation of PrPSc in mice reveals impact of aggregate composition on prion disease pathogenesis.

Infectious prions consist of PrPSc, a misfolded, aggregation-prone isoform of the host's prion protein. PrPSc assemblies encode distinct biochemical and biological properties. They harbor a specific profile of PrPSc species, from small oligomers to fibrils in different ratios, where the highest infectivity aligns with oligomeric particles. To investigate the impact of PrPSc aggregate complexity on prion propagation, biochemical properties, and disease pathogenesis, we fractionated elk prions by sedimentation velocity centrifugation, followed by sub-passages of individual fractions in cervidized mice. Upon first passage, different fractions generated PrPSc with distinct biochemical, biophysical, and neuropathological profiles. Notably, low or high molecular weight PrPSc aggregates caused different clinical signs of hyperexcitability or lethargy, respectively, which were retained over passage, whereas other properties converged. Our findings suggest that PrPSc quaternary structure determines an initial selection of a specific replication environment, resulting in transmissible features that are independent of PrPSc biochemical and biophysical properties.

Transmission of cervid prions to humanized mice demonstrates the zoonotic potential of CWD.

Prions cause infectious and fatal neurodegenerative diseases in mammals. Chronic wasting disease (CWD), a prion disease of cervids, spreads efficiently among wild and farmed animals. Potential transmission to humans of CWD is a growing concern due to its increasing prevalence. Here, we provide evidence for a zoonotic potential of CWD prions, and its probable signature using mice expressing human prion protein (PrP) as an infection model. Inoculation of these mice with deer CWD isolates resulted in atypical clinical manifestation with prion seeding activity and efficient transmissible infectivity in the brain and, remarkably, in feces, but without classical neuropathological or Western blot appearances of prion diseases. Intriguingly, the protease-resistant PrP in the brain resembled that found in a familial human prion disease and was transmissible upon second passage. Our results suggest that CWD might infect humans, although the transmission barrier is likely higher compared to zoonotic transmission of cattle prions. Notably, our data suggest a different clinical presentation, prion signature, and tissue tropism, which causes challenges for detection by current diagnostic assays. Furthermore, the presence of infectious prions in feces is concerning because if this occurs in humans, it is a source for human-to-human transmission. These findings have strong implications for public health and CWD management.

New and distinct chronic wasting disease strains associated with cervid polymorphism at codon 116 of the Prnp gene.

Chronic wasting disease (CWD) is a prion disease affecting cervids. Polymorphisms in the prion protein gene can result in extended survival of CWD-infected animals. However, the impact of polymorphisms on cellular prion protein (PrPC) and prion properties is less understood. Previously, we characterized the effects of a polymorphism at codon 116 (A>G) of the white-tailed deer (WTD) prion protein and determined that it destabilizes PrPC structure. Comparing CWD isolates from WTD expressing homozygous wild-type (116AA) or heterozygous (116AG) PrP, we found that 116AG-prions were conformationally less stable, more sensitive to proteases, with lower seeding activity in cell-free conversion and reduced infectivity. Here, we aimed to understand CWD strain emergence and adaptation. We show that the WTD-116AG isolate contains two different prion strains, distinguished by their host range, biochemical properties, and pathogenesis from WTD-116AA prions (Wisc-1). Serial passages of WTD-116AG prions in tg(CerPrP)1536+/+ mice overexpressing wild-type deer-PrPC revealed two populations of mice with short and long incubation periods, respectively, and remarkably prolonged clinical phase upon inoculation with WTD-116AG prions. Inoculation of serially diluted brain homogenates confirmed the presence of two strains in the 116AG isolate with distinct pathology in the brain. Interestingly, deglycosylation revealed proteinase K-resistant fragments with different electrophoretic mobility in both tg(CerPrP)1536+/+ mice and Syrian golden hamsters infected with WTD-116AG. Infection of tg60 mice expressing deer S96-PrP with 116AG, but not Wisc-1 prions induced clinical disease. On the contrary, bank voles resisted 116AG prions, but not Wisc-1 infection. Our data indicate that two strains co-existed in the WTD-116AG isolate, expanding the variety of CWD prion strains. We argue that the 116AG isolate does not contain Wisc-1 prions, indicating that the presence of 116G-PrPC diverted 116A-PrPC from adopting a Wisc-1 structure. This can have important implications for their possible distinct capacities to cross species barriers into both cervids and non-cervids.

Cervid Prion Protein Polymorphisms: Role in Chronic Wasting Disease Pathogenesis.

Chronic wasting disease (CWD) is a prion disease found in both free-ranging and farmed cervids. Susceptibility of these animals to CWD is governed by various exogenous and endogenous factors. Past studies have demonstrated that polymorphisms within the prion protein (PrP) sequence itself affect an animal's susceptibility to CWD. PrP polymorphisms can modulate CWD pathogenesis in two ways: the ability of the endogenous prion protein (PrPC) to convert into infectious prions (PrPSc) or it can give rise to novel prion strains. In vivo studies in susceptible cervids, complemented by studies in transgenic mice expressing the corresponding cervid PrP sequence, show that each polymorphism has distinct effects on both PrPC and PrPSc. It is not entirely clear how these polymorphisms are responsible for these effects, but in vitro studies suggest they play a role in modifying PrP epitopes crucial for PrPC to PrPSc conversion and determining PrPC stability. PrP polymorphisms are unique to one or two cervid species and most confer a certain degree of reduced susceptibility to CWD. However, to date, there are no reports of polymorphic cervid PrP alleles providing absolute resistance to CWD. Studies on polymorphisms have focused on those found in CWD-endemic areas, with the hope that understanding the role of an animal's genetics in CWD can help to predict, contain, or prevent transmission of CWD.

Large-scale prion protein genotyping in Canadian caribou populations and potential impact on chronic wasting disease susceptibility.

Polymorphisms within the prion protein gene (Prnp) are an intrinsic factor that can modulate chronic wasting disease (CWD) pathogenesis in cervids. Although wild European reindeer (Rangifer tarandus tarandus) were infected with CWD, as yet there have been no reports of the disease in North American caribou (R. tarandus spp.). Previous Prnp genotyping studies on approximately 200 caribou revealed single nucleotide polymorphisms (SNPs) at codons 2 (V/M), 129 (G/S), 138 (S/N), 146 (N/n) and 169 (V/M). The impact of these polymorphisms on CWD transmission is mostly unknown, except for codon 138. Reindeer carrying at least one allele encoding for asparagine (138NN or 138SN) are less susceptible to clinical CWD upon infection by natural routes, with the majority of prions limited to extraneural tissues. We sequenced the Prnp coding region of two caribou subspecies (n = 986) from British Columbia, Saskatchewan, Yukon, Nunavut and the Northwest Territories, to identify SNPs and their frequencies. Genotype frequencies at codon 138 differed significantly between barren-ground (R. t. groenlandicus) and woodland (R. t. caribou) caribou when we excluded the Chinchaga herd (p < .05). We also found new variants at codons 153 (Y/F) and 242 (P/L). Our findings show that the 138N allele is rare among caribou in areas with higher risk of contact with CWD-infected species. As both subspecies are classified as Threatened and play significant roles in North American Indigenous culture, history, food security and the economy, determining frequencies of Prnp genotypes associated with susceptibility to CWD is important for future wildlife management measures.

Cellulose ether treatment in vivo generates chronic wasting disease prions with reduced protease resistance and delayed disease progression.

Chronic wasting disease (CWD) is a prion disease of free-ranging and farmed cervids that is highly contagious because of extensive prion shedding and prion persistence in the environment. Previously, cellulose ether compounds (CEs) have been shown to significantly extend the survival of mice inoculated with mouse-adapted prion strains. In this study, we used CEs, TC-5RW, and 60SH-50, in vitro and in vivo to assess their efficacy to interfere with CWD prion propagation. In vitro, CEs inhibited CWD prion amplification in a dose-dependent manner. Transgenic mice over-expressing elk PrPC (tgElk) were injected subcutaneously with a single dose of either of the CEs, followed by intracerebral inoculation with different CWD isolates from white tailed deer, mule deer, or elk. All treated groups showed a prolonged survival of up to more than 30 % when compared to the control group regardless of the CWD isolate used for infection. The extended survival in the treated groups correlated with reduced proteinase K resistance of prions. Remarkably, passage of brain homogenates from treated or untreated animals in tgElk mice resulted in a prolonged life span of mice inoculated with homogenates from CE-treated mice (of + 17%) even in the absence of further treatment. Besides the delayed disease onset upon passage in TgElk mice, the reduced proteinase K resistance was maintained but less pronounced. Therefore, these compounds can be very useful in limiting the spread of CWD in captive and wild-ranging cervids.

Comparison of molecular and conventional methods for the diagnosis of Fasciola hepatica infection in the field.

The liver fluke, Fasciola hepatica, is one of the major parasite threats to livestock industries world-wide. In sheep and cattle, F. hepatica infection is commonly diagnosed using a range of methods. Aside from conventional coprological and serological diagnostic methods, there are also several molecular methods available based on the detection of liver fluke DNA in faeces. In this study, the outcomes of faecal egg count (FEC), serology and coproantigen ELISA (cELISA) were compared with the performance of polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) in diagnosis of F. hepatica from naturally infected cattle and sheep. A total of 64 individual faecal and serum samples were collected from four sheep and beef cattle herds with previous histories of F. hepatica infection. FEC and coproantigen levels were measured in faecal samples and anti-F.hepatica antibody levels were measured in serum samples. DNA samples isolated from faeces were examined both by PCR and LAMP, targeting the internal transcribed spacer 2 (ITS2) region of the F. hepatica genome. Results showed that F. hepatica eggs were present in 28 animals, while coproantigen and specific anti-F. hepatica antibodies were detected in 36 and 53 animals, respectively. Only 3 and 6 samples were positive by PCR and LAMP, respectively. To calculate method specificity and sensitivity, a combination of FEC and cELISA was selected as the composite reference standard (CRS). When compared to the CRS, PCR had a sensitivity of 10.7% and specificity of 100%, whereas LAMP had a sensitivity and specificity of 17.9% and 97.2%, respectively. PCR and LAMP in this field study were highly specific, but both had poor sensitivity compared with FEC and cELISA. Potential reasons for PCR and LAMP failure were inadequate amounts of amplifiable F. hepatica DNA, possibly due to the choice of DNA extraction procedure, amount of faecal material processed, as well as different faeces consistency and composition between different animal species.