Utveckling av en "loop-mediated isothermal amplification (LAMP)" och en "polymerase chain reaction (PCR)" assay för diagnos av Fasciola hepatica i djuravföring och jämförelser med traditionella diagnostiska metoder

The liver fluke Fasciola hepatica is a parasitic trematode prevalent in mammals, primarily in sheep and cattle. There is a wide range of methods for diagnosis of F. hepatica infections, such as coproscopy, coproantigen ELISA, serum ELISA, PCR and Loop-mediated isothermal amplification (LAMP). As with all diagnostic methods, each presents benefits and disadvantages. Coproscopy requires no sophisticated equipment, but its robustness is limited due to difficulty of species identification and inability to detect early F. hepatica infections. Coproantigen ELISA can detect infections during the pre-patent period, yet its sensitivity in field applications is still debated. Serum ELISA is a good method for large herd screening, although it provides less insight to the infection status. PCR can differentiate between species using primers targeting the ITS2 region of F. hepatica genome. LAMP is a molecular method based on rapid amplification of target DNA under isothermal conditions. Both PCR and LAMP have only recently been attempted for F. hepatica identification in faeces. The aim of the study was to develop and set up LAMP and PCR methods for diagnosis of F. hepatica in ruminant faeces and to compare these molecular techniques with coproscopy, coproantigen detection and serology. A total of 64 faecal and blood samples were collected from 64 sheep and cattle from four farms in Sweden. Faecal samples were examined by faecal egg counts (FEC) with a sedimentation method and coproantigen ELISA using the Bio-X Bovine Fasciola hepatica Antigen ELISA Kit (Bio-X Diagnostics, Belgium). Serologic testing with an in-house ELISA was conducted on all serum samples. PCR and LAMP were performed with DNA extracted directly using PowerFecal® DNA isolation kit (MO BIO, USA) from faecal samples. F. hepatica eggs were present in 28 animals, while coproantigen and antibodies were detected in 36 and 53 animals respectively. PCR and LAMP managed to amplify only 3 and 6 samples respectively. Based on a composite reference standard, results showed that LAMP and PCR had a sensitivity of 14% and 8% respectively, which was much lower compared to the 78% sensitivity of FEC and 100% sensitivity of both coproantigen and serum ELISA. FEC, coproantigen ELISA and PCR all had 100% specificity, while LAMP and serum ELISA had 96% and 39% specificity respectively. In conclusion, FEC and coproantigen ELISA were good diagnostic tools for detection of patent F. hepatica infections. PCR and LAMP results could possibly improve with further development of faecal DNA extraction techniques

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 Columb

A Prion Protein Gene Polymorphism at Codon 138 Modulates Chronic Wasting Disease Pathogenesis

Prion diseases are fatal and infectious neurodegenerative diseases caused by prions. Chronic wasting disease (CWD) is a prion disease of cervids found in North America (NA), Scandinavia and South Korea. Although there are no reports of CWD in caribou (Rangifer tarandus spp.) in NA so far, previous findings show that reindeer (R. t. tarandus) are susceptible to CWD. Single amino acid substitutions (SAAS) within the cervid prion protein (PrP) sequence have been shown to prolong survival times and produce incomplete attack rates upon CWD infection. Prion protein SAAS have been found in caribou populations in NA, including a serine to asparagine substitution at codon 138 (S138N). Previous studies reported that animals harboring the N variant at this codon were either resistant or less susceptible to natural CWD prion exposure. Based on these reports, we hypothesized that the S138N PrP amino acid substitution modulates CWD pathogenesis. We report that the 138N allele frequency is rare among caribou in areas with high risk of contact with CWD-infected species, particularly in woodland caribou (R. t. caribou) herds in Saskatchewan and Alberta. We also report that the barren-ground caribou (R. t. groenlandicus) herds have higher frequencies of the 138N allele. We found that the S138N SAAS did not alter endogenous PrP properties, but rather impairs the prion conversion process. Transgenic knock-in (KI) mice expressing the 138NN PrP genotype did not develop clinical disease up to 700 days post-inoculation (dpi), whilst their wild-type deer (138SS) counter parts succumbed to CWD between ~450-580 dpi. The 138NN KI mice did, however, harbor prions capable of inducing conversion in an in vitro prion conversion assay. Remarkably, even upon intracerebral prion inoculation, seeding activity was first detected in the spleens of these KI mice. Our findings provide new insights into the role of PrP genotype in tissue tropism of prion replication. Caribou in NA are a Threatened species and an essential resource for Indigenous people. Thus, determining the mechanisms by which the 138N allele modulates CWD pathogenesis is important for future CWD management strategies, especially in areas where caribou are at a high risk of contracting the disease

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

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

International audiencePrions 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