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Detection of classical BSE prions in asymptomatic cows after inoculation with atypical/Nor98 scrapie | Veterinary Research

Transmissible spongiform encephalopathies, or prion diseases are fatal neurodegenerative disorders produced by the accumulation of a neurotoxic misfolded isoform (PrPSc) of the cellular prion protein (PrPC). This conformational change provides PrPSc with partial resistance to proteases and a tendency to form aggregates, characteristics that have been extensively used for the diagnosis of these disorders. The presence of the pathogenic protein in a host can be due to three possible origins: sporadic or idiopathic, familial (caused by the mutation of the PRNP gene, encoding PrPC) or acquired through exposure to prions. Prion diseases in animals include scrapie in small ruminants, chronic wasting disease (CWD) in cervids and bovine spongiform encephalopathy (BSE) in cattle. The classical form of BSE (C-BSE) caused one of the most important food safety crises in history, due to its link to the variant form of Creutzfeldt-Jakob disease (vCJD) in humans [1].

Among animal prion diseases, atypical scrapie in sheep and goats has the widest geographical distribution [2, 3]. First described in 1998 in Norway [4], this atypical form presents significant neuropathological, clinical, biochemical and epidemiological differences compared to classical scrapie. The origin of atypical scrapie has been widely discussed. Due to its epidemiological characteristics, it has been suggested that atypical scrapie is a spontaneous prion disease [5]. Results obtained in murine models of spontaneous scrapie seem to indicate that this is indeed the case [6]. In 2019, the emergence of C-BSE from atypical scrapie was demonstrated. The transmission of atypical scrapie isolates to bovine PrP transgenic mice led to the propagation of C-BSE prions. The presence of low levels of the C-BSE agent was also confirmed in the original ovine isolates by protein misfolding cyclic amplification (PMCA) [7], an ultrasensitive technique for the detection of prions. The emergence of C-BSE after interspecies transmission of atypical scrapie was also observed by us in a different host: pigs inoculated with atypical scrapie. Although intracerebral inoculation of the agent did not cause disease in the pigs, C-BSE prions were detected in several brain areas of these animals using PMCA, suggesting that pigs could act as a reservoir for BSE [8].

Due to this link between atypical scrapie and the emergence of BSE, the objective of this study was to determine whether the inoculation of an atypical scrapie isolate could induce BSE-like disease in cattle. In this report, we describe for the first time the emergence of C-BSE-like prions during the in vitro propagation of brain samples from calves inoculated with an atypical scrapie isolate.

Four 10-month-old Pyrenean breed calves were used. Animals were intracerebrally challenged with 1 mL (10% brain homogenate w/v in sterile saline solution) of the PS152 isolate. This isolate was made from the brain of an AFRQ/AFRQ (amino acid at codons 136, 141, 154 and 171 of the PRNP gene) clinical sheep naturally affected by atypical scrapie, and provided by UMR INRAE ENVT 1225, Interactions Hôtes Agents Pathogènes, École Nationale Vétérinaire de Toulouse. Calves were placed under general anesthesia and the trephine was done using a dental drill positioned paramedially in the mid-frontal region of the head, at an angle of 90° with respect to the rostro-caudal slope of the front of the skull. The inoculum was injected into the frontal cortex via a 9-cm-long needle (gauge, 22).

Cows were housed together in the BSL3 facility from Centro de Encefalopatías y Enfermedades Transmisibles Emergentes of the University of Zaragoza, and daily monitored by animal husbandry staff. Veterinary clinical assessments were performed monthly in order to detect any clinical sign compatible with a prion disease. Animals were euthanized by intravenous pentobarbital injection followed by exsanguination without showing clinical signs of prion disease. Post-inoculation periods and reasons for euthanasia are shown in Table 1. Bovine 1 was euthanized due to the compromise of its welfare after the development of concomitant pathologies. The other animals were selectively euthanized to test the evolution of the disease.

Table 1
Atypical scrapie-challenged cows

Necropsies were conducted systematically, and tissue samples were collected from the central nervous system (CNS) using sterile prion-free material and equipment. All samples were collected in duplicate, one sample was fixed in 10% formalin for the histological analysis and the other sample was stored at −80 °C for further biochemical and in vitro assays.

Formalin-fixed tissues were processed according to standard histopathological procedures. Sections were stained with hematoxylin and eosin for the histopathological study. PrPSc detection in brain sections was performed by immunohistochemistry using the monoclonal primary antibodies L42 and 6H4 (Biopharm, Darmstadt, Germany), as described previously [9, 10].

Samples from frontal cortex (Fc), thalamus (Th), cerebellum (Cbl) and obex from the four inoculated cows and two negative age-paired controls (11 and 12 years old) were subjected to western blotting for PrPSc detection. 20 µL of sample was mixed with 180 µL of 10% negative brain sheep homogenate, as previously described [11]. Then, the TeSeE Western blot kit (Bio-Rad, France) was used following the manufacturer’s recommendations to perform the PrPSc extraction and samples were subjected to a 12% SDS/PAGE and transferred to PVDF membranes. After blocking for 30 min in PBST containing 2% BSA, PrPSc immunodetection was performed with the monoclonal antibodies 12B2 (mAb 1:10 000, Creative Biolabs’) and Sha31 (mAb, 1:8000, SPI-Bio), a secondary antibody HRP-conjugated anti-mouse (1:5000; Bio-rad) and ECL substrate (Pierce) to reveal peroxidase activity.

Homogenates (10% in saline solution) were prepared using tissue from the previously mentioned brain areas and animals. Each homogenate was later diluted 1/50 to prepare the PMCA seeds. These seeds were then subjected to three rounds (24 h each) of PMCA, as previously described [11]. 5 µL of seed and 45 µL of substrate were mixed per well in a 96-well PCR microplate (Axygen Scientific, USA). One Teflon bead (2.381 mm diameter) was added to each well. Amplification was performed in a Qsonica Q700 sonicator, using a water recirculation system. Microplates were then submitted to 96 cycles of 10 s sonication (75% power) followed by a 14 min and 50 s incubation period at 39.5 °C temperature. After the PMCA round (24 h), 5 µL of the reaction product was added to a new microplate containing 45 µL of fresh substrate and a new round with 96 cycles of sonication and incubation was performed. Brains from TgBov mice, overexpressing bovine PrP [12, 13] supplemented with 0.25% final concentration of low molecular weight dextran (Sigma-aldrich, D4911-10G mol wt 6500/10 000) were used as substrate. TgBov mice were selected as the used substrate for PMCA since bovines are the natural hosts of BSE, and therefore this model could correctly mimic BSE prion transmission in natural conditions. Unseeded substrate was used as negative control, and the positive control was performed using cattle C-BSE isolates diluted from a 10−1 to a 10−8 dilution. PrPSc detection after PMCA was performed by western blot. Prior to western blot, a BCA (bicinchoninic acid) protein assay (23227, Thermo Scientific™) was done to determine the total protein concentration of samples and equivalent amount of proteins were loaded in each well. PrPSc band ratio of the resulting PMCA products and 6 C-BSE controls were determined using the Image J software.

Atypical scrapie-inoculated cows were monitored periodically. None of the animals included in the study showed TSE-related clinical signs. Four brain areas (frontal cortex, thalamus, cerebellum and obex) from the inoculated animals and two uninfected age-matched control cows were tested for the detection of PrPSc. None of the tested areas showed PrPSc accumulation by immunohistochemistry (not shown) or western blotting (Figure 1). When evaluating brain sections from the same areas stained with hematoxylin–eosin, no spongiform changes were found.

Figure 1
figure 1

No PrPSc accumulation by western Blot is detected in brains from cows intracerebrally challenged with atypical scrapie. Frontal cortex (Fc), thalamus (Th), cerebellum (Cbl) and obex homogenates from the four atypical scrapie-challenged cows and a negative control cow were subjected to PrPSc detection by western blot using the Sha31 antibody. No positivity was detected in any of the experimentally inoculated cows. Brain homogenates from a classical scrapie-infected sheep (Scrapie + C) and a BSE-infected cow (BSE + C) were used as positive controls.

The previously mentioned brain samples from cows were subjected to PMCA in order to determine whether C-BSE prions could emerge in the atypical scrapie-challenged cows. Brain samples from the negative aged-matched cows were included as a control for the spontaneous generation of prions in vitro. Each brain area was tested in duplicate. After in vitro propagation, positive PMCA amplification was detected in reactions seeded with brain material from 3 out of the 4 atypical scrapie-inoculated cows, in the areas of frontal cortex, thalamus and/or cerebellum. None of the obex samples showed seeding activity (results not shown). No positivity was detected in reactions seeded with the uninfected cows’ brain samples or unseeded PMCA reactions. The glycosylation pattern of the positive PMCA reactions observed in western blot using the Sha31 antibody was characterized by a predominance of the diglycosylated band and a non-glycosylated band at ~19 kDa. No PrPres was detected when using the 12B2 antibody (Figure 2). The observed features are indistinguishable from C-BSE prions and PMCA products from reactions seeded with C-BSE prions. Duplicates of the tested samples revealed identical results when tested by western blot (results not shown).

Figure 2
figure 2

C‑BSE prions emerge during the in vitro propagation by PMCA of brain isolates from cows inoculated with atypical scrapie. A PMCA reaction products from the frontal cortex (Fc), thalamus (Th) and cerebellum (Cbl) from the four atypical scrapie-challenged cows and a negative control cow were subjected to PrPSc detection by western blot. Positivity was detected in Th and Cbl samples from Bovine 1; Fc and Cbl samples from Bovine 2; and Fc and Th samples from Bovine 3. No positive reactions were detected in PMCA reaction products from the negative control. PMCA reaction products from a second negative control cow were also subjected to PrPSc detection by western blot, showing no positive results (results not shown). A classical scrapie isolate (Scrapie + C) and a C-BSE isolate were used as positive controls for western blot. B Relative amounts of diglycosylated (black bar), monoglycosylated (grey bar) and unglycosylated (clear bar) glycoforms of PMCA products seeded with brain samples from bovines challenged with atypical scrapie and six C-BSE controls were determined by Image J software. Results are presented as mean ± standard deviation. No significant differences in the relative amount of PrPSc glycoforms were observed between samples from the study and C-BSE controls.

The atypical scrapie isolate PS152 used to inoculate the cows intracerebrally was subjected to PMCA using the previously described conditions (TgBov substrate supplemented with 0.25% final concentration of low molecular weight dextran). The inoculum was diluted at 1/50 and then tested by PMCA in duplicate. After three rounds of PMCA, products were tested by western blot using the Sha31 antibody and amplification was observed. PMCA samples seeded with the original inoculum displayed a C-BSE-like pattern, showing a non-glycosylated band at ~19 kDa and a predominant diglycosylated band (Figure 3).

Figure 3
figure 3

The atypical scrapie inoculum PS152 shows a C‑BSE signature after propagation in PMCA using TgBov substrate. PMCA reaction products from the PS152 inoculum (PMCA-PS152) were subjected to PrPSc detection by western blot. Positive PMCA reactions from cattle C-BSE (PMCA-BSE + C) and scrapie (PMCA-Scrapie C+) propagated in tgBov substrate were used as controls. A classical scrapie isolate (Scrapie + C) and a C-BSE isolate (BSE + C) were used as positive controls for western blot. Using the Sha31 antibody, PS152 PMCA products displayed an indistinguishable glycoform pattern to that of cattle C-BSE and cattle C-BSE propagated by PMCA.

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