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The Journal of General Virology logoLink to The Journal of General Virology
. 2016 Mar 1;97(Pt 3):803–812. doi: 10.1099/jgv.0.000367

Sensitive and specific detection of classical scrapie prions in the brains of goats by real-time quaking-induced conversion

Rohana P Dassanayake 1,✉, Christina D Orrú 2, Andrew G Hughson 2, Byron Caughey 2, Telmo Graça 1,3, Dongyue Zhuang 4, Sally A Madsen-Bouterse 1, Donald P Knowles 1,4, David A Schneider 1,4
PMCID: PMC5972304  PMID: 26653410

Abstract

Real-time quaking-induced conversion (RT-QuIC) is a rapid, specific and highly sensitive prion seeding activity detection assay that uses recombinant prion protein (rPrPSen) to detect subinfectious levels of the abnormal isoforms of the prion protein (PrPSc). Although RT-QuIC has been successfully used to detect PrPSc in various tissues from humans and animals, including sheep, tissues from goats infected with classical scrapie have not yet been tested. Therefore, the aims of the present study were to (1) evaluate whether prion seeding activity could be detected in the brain tissues of goats with scrapie using RT-QuIC, (2) optimize reaction conditions to improve scrapie detection in goats, and (3) compare the performance of RT-QuIC for the detection of PrPSc with the more commonly used ELISA and Western blot assays. We further optimized RT-QuIC conditions for sensitive and specific detection of goat scrapie seeding activity in brain tissue from clinical animals. When used with 200 mM sodium chloride, both full-length sheep rPrPSen substrates (PrP genotypes A136R154Q171 and V136R154Q171) provided good discrimination between scrapie-infected and normal goat brain samples at 10− 3 dilution within 15 h. Our findings indicate that RT-QuIC was at least 10 000-fold more sensitive than ELISA and Western blot assays for the detection of scrapie seeding activity in goat brain samples. In addition to PRNP WT samples, positive RT-QuIC reactions were also observed with three PRNP polymorphic goat brain samples (G/S127, I/M142 and H/R143) tested. Taken together, these findings demonstrate that RT-QuIC sensitively detects prion seeding activity in classical scrapie-infected goat brain samples.

Introduction

Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative disorders that affect a wide range of animal species, such as goats and sheep (scrapie), cattle (bovine spongiform encephalopathy), and deer, elk and moose (chronic wasting disease), as well as humans (Creutzfeldt–Jakob disease). The infectious agent, or prion, is largely proteinaceous and consists primarily of an abnormal conformational isoform (PrPSc) of the host-encoded normal cellular prion protein PrPC (Bolton et al., 1982; Prusiner, 1982). A characteristic feature of all TSEs is the accumulation of PrPSc in the central nervous system. In most cases of classical scrapie infection in sheep and goats PrPSc accumulates in the lymphoid tissues (Valdez et al., 2003; van Keulen et al., 1996) such that the preclinical stage of classical scrapie infection can be detected by biopsy of the lymphoid follicles in the rectoanal mucosa-associated lymphoid tissues (Espenes et al., 2006; González et al., 2005) or in the nictitating membrane (O'Rourke et al., 1998b, 2000).

The relative susceptibility of sheep to classical scrapie infection depends in part on polymorphisms affecting the amino acid composition of PrPC, especially those occurring at residues 136 [valine (V) or alanine (A)], 154 [arginine (R) or histidine (H)] and 171 [glutamine (Q), R, H or lysine (K)]. Thus, sheep homozygous for the V136R154Q171 (or VRQ) allele show the most susceptibility compared with the A136R154Q171 (or ARQ) allele, whereas sheep homozygous for the A136R154R171 (or ARR) allele are the least susceptible (Clouscard et al., 1995; Goldmann et al., 1994; Hunter et al., 1997). The prion gene (PRNP) in goats is also highly polymorphic (Vaccari et al., 2009; White et al., 2008). Although goats homozygous for PRNP haplotypes 1 and 2 (WT) are susceptible to scrapie infection, scrapie inoculation studies in goats revealed that PRNP polymorphisms at codons 127, 146, 154, 211 and 222 may provide a prolonged incubation period or possibly resistance to scrapie infection (Acutis et al., 2012; Dassanayake et al., 2015; Lacroux et al., 2014; White et al., 2012).

In the USA, gold standard testing for scrapie is based on the detection of PrPSc in ante-mortem or post-mortem tissues using mAb-based immunohistochemistry (IHC) and Western blot assay (O'Rourke et al., 1998a; Spraker et al., 2002). Two extremely sensitive in vitro cell-free conversion assays have been developed to assess prions in various biological tissues. Protein misfolding cyclic amplification (PMCA) (Saá et al., 2006) and real-time quaking-induced conversion (RT-QuIC) (Atarashi et al., 2007, 2011; Orrú et al., 2009; Wilham et al., 2010) assays demonstrate a sensitivity for the detection of brain-derived prion seeding activity from several different prion strains comparable to animal bioassays. In PMCA, brain homogenates prepared from uninfected animals or transgenic mice expressing prion protein of the species of interest are used as the misfolding substrate (Madsen-Bouterse et al., 2012; Saá et al., 2006; Thorne & Terry, 2008), whereas in RT-QuIC, bacterially expressed (unglycosylated) recombinant prion protein (rPrPSen) is used as the substrate (Atarashi et al., 2011; Wilham et al., 2010). Previously, PMCA conditions were described for the detection of scrapie prions in sheep (Thorne & Terry, 2008) and goats (Madsen-Bouterse et al., 2012). These studies further confirmed that PMCA is more sensitive than TSE ELISA (O'Rourke et al., 2011) and Western blot (Madsen-Bouterse et al., 2012) assays.

The RT-QuIC assay has been shown to be sensitive and easy to perform (Wilham et al., 2010). Whilst PMCA relies on end-point detection of proteinase K-resistant prions by Western blotting, detection of prion seeding activity in RT-QuIC is monitored in ‘real-time’ by measuring the increase in amyloid formation via binding of the fluorescent dye Thioflavin T (ThT) to the growing amyloid fibrils. Furthermore, compared with IHC or PMCA, RT-QuIC is fast and results can be obtained within 1–2 days. Although RT-QuIC has been widely used to detect prions from a variety of species, including mice (Vascellari et al., 2012), humans (Atarashi et al., 2011), hamsters, deer and sheep (Wilham et al., 2010), no detection of scrapie-misfolded PrP in goats has been described. To investigate the possible use of RT-QuIC for scrapie surveillance in goats, we therefore set forth to evaluate whether prion seeds in the brain of goats with classical scrapie could be detected using RT-QuIC, to optimize RT-QuIC reaction conditions for this purpose, and to compare the sensitivity of RT-QuIC with the sensitivity of the currently used TSE ELISA and Western blot assays.

Results

Immunohistochemical detection of PrPSc in goat brains

To help characterize the brain homogenates used for RT-QuIC analysis, an adjacent section of formalin-fixed obex hindbrain was examined from each donor by PrPSc IHC. Widespread PrPSc immunolabelling was detected in the dorsal motor nucleus of the vagus nerve as well as other regions of the hindbrain obtained from all the scrapie-infected goats, whereas PrPSc immunolabelling was not detected in the hindbrain sections obtained from normal goats (Fig. 1).

Fig. 1.

Fig. 1.

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Immunohistochemical detection of PrPSc in the obex hindbrain of scrapie-infected donor goats. Immunoreactivity for PrPSc (red chromogen) was dense and widespread in the dorsal motor nucleus of the vagus nerve in the three goat subjects with clinical scrapie disease (animal ID: 4427, 4432 and 4514; upper row), but was not detected in the three normal goat subjects (animal ID: 4016, 4187 and 4719; lower row) when labelling tissues with anti-prion mAbs. Haematoxylin counterstain. Bar, 200 μm.

Quantification of PrPSc in brain homogenates by ELISA and Western blot assays

To determine the relative levels of PrPSc in the brain homogenates from scrapie-infected goats, twofold serial dilutions were prepared (0.05–25 μg total protein for ELISA and 0.016–8 μg total protein for Western blots), and ELISA and Western blot assays were performed. Similar high ELISA absorbance values (A450 = 2.75–3.5 for 6.35–25 μg total protein) were observed with the more concentrated brain homogenates tested from all three scrapie-infected goats samples (PRNP WT; Fig. 2a). The last positive PrPSc ELISA signal (above the cut-off value) was observed at ∼0.39 μg total protein. In contrast, the ELISA absorbance values of 12.5 and 25 μg (total protein) of scrapie-uninfected brain homogenates were well below the assay's cut-off value (Fig. 2a).

Fig. 2.

Fig. 2.

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Relative levels of PrPSc in brain homogenates from scrapie-infected goats. Twofold serial dilutions of scrapie-infected and normal (uninfected) goat brain homogenates were analysed by IDEXX TSE ELISA (total protein 0.05–25 μg per well) and Western blot assays as described in Methods. (a) A450 results for each dilution. The cut-off value for TSE ELISA (black line, A450 = 0.257) was determined from the negative control samples provided with the kit. (b–d) Brain homogenates prepared from scrapie-infected [animal ID: 4427 (b), 4432 (c) and 4514 (d)] and normal [lane 11, animal ID: 4016 (b), 4187 (c) and 4719 (d)] goats were incubated with proteinase K (50 μg ml− 1 for 60 min at 37 °C) and twofold serial diluted with sample loading buffer and loaded into the gels (total protein: lane 1, 8.0 μg; lane 2, 4.0 μg; lane 3, 2.0 μg; lane 4, 1.0 μg; lane 5, 0.5 μg; lane 6, 0.25 μg; lane 7, 0.125 μg; lane 8, 0.063 μg; lane 9, 0.031 μg; lane 10, 0.016 μg) and Western blot analysis was performed with anti-prion mAb P4. PrPres bands were not observed with normal goat brain homogenates (lane 11, 8 μg total protein). Molecular mass markers (in kDa) are shown to the left of the panels.

To estimate the level of proteinase K-resistant prion protein in these samples, we performed Western blot analysis of these same brain homogenates from scrapie-infected goats (PRNP WT). The typical three-band pattern of proteinase K-resistant PrPSc was observed in all reactions up to ∼0.250 μg total protein (Fig. 2b–d). The last serial dilution with a detectable proteinase K-resistant PrPSc band (diglycosylated) varied among the samples, but was in the range of 0.063–0.125 μg total protein. As expected, proteinase K-resistant prion protein (PrPres) bands were not detected with normal brain homogenates even at the highest total protein level tested (8 μg; Fig. 2b–d, lane 11). The results indicated that the sensitivity of these Western blots was at best in the low nanogram range for total protein.

RT-QuIC detection of goat scrapie prion seeds in brain samples

A previous study has shown that scrapie prion seeds in the brain samples from classical scrapie-infected sheep can be detected by RT-QuIC (Wilham et al., 2010) using sheep VRQ (aa 25–234) rPrPSen as the substrate. To investigate if RT-QuIC could be used to detect goat scrapie prion seeding activity, we used scrapie-infected (animal ID: 4514) and normal (animal ID: 4016) brain homogenates prepared from two PRNP WT goats (Table 1) as seeds (10− 1 to 10− 8 brain tissue dilutions; ∼15 μg to 1.5 pg total protein) with full-length ovine VRQ rPrPSen (aa 25–234) substrate and the reaction conditions (300 mM NaCl, 0.002 % SDS and 0.1 mg rPrPSen ml− 1 final concentrations) as previously optimized for sheep (Wilham et al., 2010). As previously observed for brain homogenate samples from sheep (Wilham et al., 2010) and possibly due to an inhibitory effect of the brain tissue matrix, the lowest homogenate dilutions (10− 1 and 10− 2; ∼15 and ∼1.5 μg total protein) did not yield a positive ThT signal (Fig. 3a). However, a rapid increase in ThT fluorescence (mean of four replicate wells), indicative of newly formed seeded rPrP amyloid fibrils, was observed in each quadruplicate reaction seeded with 10− 3 (∼150 ng total protein) and 10− 4 (∼15 ng total protein) brain tissue dilutions within 15 h incubation. Reactions seeded with more diluted scrapie samples (10− 5 to 10− 7 brain tissue dilutions; ∼1.5 ng to 15 pg total protein) were positive by 40 h incubation (Fig. 3a). Although positive reactions were also observed ∼65 h with the most dilute scrapie samples (i.e. 10− 8; 1.5 pg total protein), spontaneous amyloid formation (rPrPspon) was also observed in one of the quadruplicate reactions seeded with 10− 8 and 10− 3 normal brain tissue dilutions at ∼55 and 70 h, respectively (Fig. 3b). We therefore chose 50 h as our reaction cut-off as no increase in fluorescence was observed in normal brain homogenates reactions at any of the dilutions tested within a 50 h incubation period.

Table 1. Donor information.

Hindbrain tissues collected from normal or clinically scrapie-infected goats were used to prepare homogenates.

Animal ID PRNPhaplotype* Scrapie status Age at necropsy (months)
4016 1,1 Uninfected 79
4187 1,1 Uninfected 67
4719 1,2 Uninfected 7
4427 1,2 Infected (clinical) 41
4432 1,1 Infected (clinical) 40
4514 1,1 Infected (clinical) 31
30–75 1,3 Infected (clinical) 84
3558 1,4 Infected (clinical) 82
4412 2,6 Infected (clinical) 38
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*

Haplotype: 1 and 2, WT (G127, I142, H143, N146, R142, R211, Q222, P/S240); 3, S127; 4, M142; 6, R143.

Fig. 3.

Fig. 3.

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Detection of prion seeding activity in the scrapie-infected goat brain samples by RT-QuIC. Tenfold serial dilutions (10− 1 to 10− 8; ∼15 μg to 1.5 pg total protein) of (a) scrapie-infected (animal ID: 4514) and (b) scrapie-uninfected (animal ID: 4016) goat 10 % (w/v) brain homogenates were seeded, and RT-QuIC reactions were performed with 300 mM NaCl, 0.002 % SDS and sheep full-length VRQ rPrPSen (aa 25–234) as the substrate. Data are presented as mean ThT fluorescence [relative fluorescence units (RLU)] of quadruplicate reactions.

Optimization of RT-QuIC for the detection of goat scrapie prion seeding activity

To improve RT-QuIC for the detection of goat scrapie prion seeding activity, we tested the effects of SDS (0 versus 0.002 %), NaCl concentration (130–600 mM) and different rPrPSen substrates (ARQ versus VRQ, aa 25–234) on the sensitivity and specificity of the assay. RT-QuIC reactions seeded with 10− 4 brain tissue dilutions (∼15 ng total protein) using either ARQ or VRQ rPrPSen as the substrate in the presence of 0.002 % SDS show similar amplification kinetics in which the presence of SDS reduced the initial lag phase for prion seeded reactions by ∼10 h (compared with no SDS) for each rPrPSen substrate (Fig. 4a, b). Next, we assessed the effect of NaCl on the ability of RT-QuIC to detect goat scrapie seeding activity. Quadruplicate reactions were seeded with 10− 6 dilutions of brain tissue (∼150 pg total protein) from scrapie-positive or -negative goats. Full-length ARQ or VRQ rPrPSen were used as the substrate in the presence of 0.002 % SDS. The results show that when the conversion buffer contained the highest NaCl concentrations (500–600 mM) and VRQ rPrPSen was used as the substrate, goat scrapie prion seeded and normal goat brain seeded reactions had a similar lag phases with ThT fluorescence increasing at ∼1–30 h (Fig. 5a, b). Conversely, when the same substrate was incubated with a final concentration of 130 or 200 mM NaCl (Fig. 5b) normal brain homogenate seeded reactions showed no increase in fluorescence out to 120 and 90 h, respectively. Furthermore, goat scrapie prion seeding activity was readily detected by ∼60 h with 130 mM NaCl and even faster at ∼30 h with 200 mM NaCl (Fig. 5a). Similar NaCl titration results were also found with ARQ rPrPSen (data not shown). Collectively, our findings indicated that faster detection of scrapie seeding activity in goat brain samples and better specificity could be achieved when using VRQ or ARQ aa 25–234 rPrPSen substrates in the presence of a final concentration of 200 mM NaCl.

Fig. 4.

Fig. 4.

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Comparison of RT-QuIC using full-length (a) ARQ or (b) VRQ rPrPSen as the substrate with or without SDS for assay of scrapie-infected goat brain samples. RT-QuIC reactions were seeded with 10− 4 dilutions (∼15 ng total protein) of scrapie-infected goat (animal ID: 4514), scrapie-infected sheep (animal ID: 4393) and scrapie-uninfected goat (animal ID: 4016) brain homogenates using either full-length ARQ or VRQ rPrPsen (aa 25–234) as substrates with or without the addition of 0.002 % SDS (final concentration). Data are presented as mean ThT fluorescence [relative fluorescence units (RLU)] of quadruplicate reactions.

Fig. 5.

Fig. 5.

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RT-QuIC sodium chloride titration for scrapie-infected and uninfected goat brain samples. RT-QuIC reactions were seeded with 10− 6 dilution (∼150 pg total protein) of (a) scrapie-infected (animal ID: 4514) and (b) uninfected (animal ID: 4016) goat brain homogenates using a range of NaCl concentrations (130–600 mM) with the full-length sheep VRQ rPrPSen (aa 25–234) as substrate. Data are presented as mean ThT fluorescence [relative fluorescence units (RLU)] of quadruplicate reactions.

RT-QuIC sensitivity for detection of goat scrapie prion seeds

To evaluate the sensitivity of RT-QuIC under our newly optimized conditions [i.e. 200 mM NaCl, 0.002 % SDS and rPrPSen (VRQ) substrate at 0.1 mg ml− 1] we performed RT-QuIC end-point dilution analysis of three representative clinical scrapie-infected goat (PRNP WT) brain homogenates. As a specificity control, quadruplicate reactions were seeded with 10− 3 (∼150 ng total protein) tissue dilutions of a normal goat brain. Positive reactions were observed in all quadruplicate reactions seeded with 10− 3 and 10− 4 scrapie-positive brain tissue dilutions (∼150–15 ng total protein) within 15 h (Fig. 6a–c). In two out of the three animals tested, prion seeding activity was detected down to 10− 7 tissue dilutions (∼15 pg total protein) (Fig. 6a, c) within a 50 h incubation time. Similar results were also observed when full-length ARQ rPrPSen was used as the substrate (Fig. 6d). In a few instances, occasional rPrPspon formation occurred in one of the quadruplicate reactions seeded with normal brain homogenate at ∼59 h (Fig. 6b).

Fig. 6.

Fig. 6.

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Relative sensitivity of RT-QuIC for the detection of prion seeds in scrapie-infected goat brain samples. (a–c) Tenfold serial dilutions (10− 3 to 10− 8; ∼150 ng to 1.5 pg total protein) of scrapie-infected [animal ID: 4427 (a), 4432 (b) and 4514 (c)] and scrapie-uninfected [animal ID: 4187 (a), 4719 (b) and 4016 (c)] goat brain homogenates were seeded, and RT-QuIC reactions were performed with 200 mM NaCl, 0.002 % SDS and sheep full-length VRQ rPrPSen (aa 25–234) as the substrate. (d) Similar results were also observed when full-length ARQ rPrPSen (aa 25–234) was used as the substrate. Data are presented as mean ThT fluorescence [relative fluorescence units (RLU)] of quadruplicate reactions.

RT-QuIC detection of goat scrapie prion seeds from PRNP polymorphic samples

As our RT-QuIC was able to detect scrapie prion seeds in PRNP WT scrapie brain samples, it was of interest to assess whether the newly optimized assay conditions supported the detection of prion seeding activity in PRNP polymorphic goat scrapie brain samples. All three PRNP polymorphic goat brain samples (G/S127, I/M142 and H/R143) used for RT-QuIC were previously characterized by IHC and Western blot (Madsen-Bouterse et al., 2015; O'Rourke et al., 2012) for PrPSc, and two of the three samples were also assessed for infectivity using bioassay studies in Tg338 mice (O'Rourke et al., 2012). Although there was a slight delay in the amplification of PrPSc between WT and PRNP polymorphic samples, positive reactions were observed in all quadruplicate samples seeded with 10− 3 dilutions (∼150 ng total protein) of PRNP polymorphic goat scrapie brain samples incubated with both VRQ (Fig. 7a) and ARQ rPrPSen (Fig. 7b) within 20–40 h.

Fig. 7.

Fig. 7.

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Detection of prion seeds in PRNP polymorphic scrapie-infected goat brain samples by RT-QuIC. RT-QuIC reactions were seeded with 10− 3 dilution (∼150 ng total protein) of scrapie-infected PRNP polymorphic (G/S127, animal ID: 30–75; I/M142, animal ID: 3558; H/R143, animal ID: 4412), WT (animal ID: 4514) and uninfected (animal ID: 4719) goat brain homogenates, and RT-QuIC reactions were performed with 200 mM NaCl, 0.002 % SDS and sheep full-length (a) VRQ rPrPSen or (b) ARQ rPrPSen as the substrates. Data are presented as mean ThT fluorescence [relative fluorescence units (RLU)] of quadruplicate reactions.

Discussion

Several highly sensitive in vitro prion seeding activity assays have been described for the detection of scrapie in sheep (Atarashi et al., 2011; Colby et al., 2007; Saá et al., 2006; Wilham et al., 2010), yet classical scrapie prion seeding activity in goats has only been reported using PMCA (Madsen-Bouterse et al., 2012). To develop a faster and more practical test for caprine scrapie, we evaluated the ability of RT-QuIC to detect prion seeds in the brain of classical scrapie-infected goats. We initially used previously described assay conditions for detecting seeding activity associated with classical scrapie in sheep brain (Wilham et al., 2010) and found that RT-QuIC could specifically detect scrapie prion seeds in the brain of scrapie-infected goats. Recently published research indicates that the assay can be further optimized by varying SDS, NaCl as well as rPrPSen substrate concentrations (Orrú et al., 2015b; Vascellari et al., 2012; Wilham et al., 2010). Our findings indicate that using full-length ARQ or VRQ rPrPSen with 0.002 % SDS and 200 mM NaCl allowed rapid and sensitive amplification of classical scrapie prion seeding activity in goat brain samples within a 50 h incubation period.

In goats, PRNP WT haplotype 2 is identical to the sheep PRNP ARQ allele and goat haplotype 1 is identical except for a serine-to-proline substitution occurring at codon 240 (White et al., 2008). The full-length rPrPSen used in RT-QuIC contains aa 25–234, and both ARQ and VRQ rPrPSen lack a serine or proline residue at codon 240. Therefore, no attempts were made to clone WT PRNP from goats, but rPrPSen produced from the sheep ARQ allele construct was considered as goat WT PrPC. Furthermore, we also used the sheep PRNP VRQ allele rPrPSen as the substrate for RT-QuIC. Although PMCA detection of prion seeding activity in goat brains using Tg338 mice (expressing the VRQ allele) as the substrate has been described, our improved RT-QuIC protocol which uses ARQ or VRQ rPrPSen has the potential to significantly reduce the time and cost required for testing (Madsen-Bouterse et al., 2012). Furthermore, using a recombinantly expressed PrP produced in Escherichia coli removes the need for animal donors for substrate preparation.

Our previous bioassay studies in Tg338 mice inoculated with the brain homogenates prepared from PRNP WT and polymorphic (G/S127 and I/M142) scrapie clinical goats produced clinical scrapie with relatively longer survival times in first-passage mice (O'Rourke et al., 2012). Although the survival times of second-passage Tg338 mice became shorter and more consistent for both PRNP WT and polymorphic genotypes, clinical signs of scrapie were observed at ∼4 months post-inoculation (O'Rourke et al., 2012). Although PMCA is faster compared with bioassay studies in Tg338 mice, it still requires several weeks and multiple rounds of amplification followed by Western blot analysis (Madsen-Bouterse et al., 2012). However, we as well as others have shown that RT-QuIC is much faster than PMCA and is overall less technically challenging (compared with PMCA) due to the lack of maintenance of mouse colonies, addition of substrate during the assay, proteinase K digestion and Western blotting. In order to minimize the assay-to-assay variation, all the experiments (ELISA, Western blot and RT-QuIC assays) were performed with the same batch of brain homogenate preparations. It would have been ideal if a HerdChek Scrapie-BSE Ag Test kit had been used instead of a HerdChek CWD Ag Test kit to quantify PrPSc in scrapie brain samples. As we used similar conditions described for sheep scrapie samples with a HerdChek CWD Ag Test kit (González et al., 2008), we expect minimal changes in ELISA sensitivities between the two kits. The lowest total protein levels from which caprine PrPSc could be reliably detected by TSE ELISA and Western blot assays (all three bands) for PRNP WT goats were ∼390 and 250 ng, respectively. Using the RT-QuIC method, the lowest total protein levels of PRNP WT caprine brain with reliably detectable seeding activity were in the range of 14.2–15.2 pg (10− 7 dilutions). Therefore, our comparison of the sensitivity and specificity of RT-QuIC with the routinely used TSE ELISA and Western blot assays indicates that RT-QuIC is at least 10 000-fold more sensitive than either ELISA or Western blot tests for PRNP WT caprine scrapie brain tissues. Taken together, these findings indicate that, as shown for many other prion strains, assay by RT-QuIC is a more rapid, sensitive and specific test for quantitative evaluation of the levels of seeding activity in caprine brain samples.

In the USA, scrapie surveillance and eradication programs are currently based on histopathological examination of brain tissues for characteristic microscopic lesions of scrapie, protease-resistant protein analysis methods including IHC and Western blotting on brain and peripheral tissues from live or dead animals, bioassay, scrapie-associated fibril detection by electron microscopy, or any other approved test methods in accordance with the ‘Scrapie Eradication Uniform Methods and Rules’ (9 CFR 54.10). Most of the above approved scrapie diagnostic tests can take days to weeks to identify scrapie prions. Furthermore, identification of scrapie status of goats can be complicated due to the existence of several polymorphisms in the PRNP coding sequence which can delay the scrapie incubation period and PrPSc accumulation (Dassanayake et al., 2015; Goldmann et al., 2011; Lacroux et al., 2014). A recent study by us revealed that the polymorphisms in caprine PRNP can also affect the sensitivity of PrPSc detection in brain samples by anti-prion mAb-based immunoassays such as IHC and Western blot analysis (Madsen-Bouterse et al., 2015). It therefore becomes important to have a rapid, sensitive, PRNP polymorphism-independent as well as antibody-independent detection test to detect scrapie. Furthermore, IHC assays depend on time-consuming preparation steps (e.g. fixation and paraffin embedding) and thus may take up to 1 week to complete. Once rPrPSen is prepared from bacterial cultures, the RT-QuIC assay can be set up within a few hours after which results can be available within 1–2 days. Although Western blot analysis is arguably faster than IHC, we have shown in this study that the RT-QuIC assay is more sensitive. Furthermore, our study also shows that the RT-QuIC assay was able to detect PrPSc from both PRNP WT and polymorphic goat brain samples. However, further studies are necessary to assess the ability of RT-QuIC to detect prion seeding activity prior to the onset clinical signs in biological samples of diagnostic interest. Therefore, use of RT-QuIC could be considered for small ruminant TSE surveillance, and might become a useful technique for more rapid and efficient scrapie eradication programs in the USA.

Methods

Normal and scrapie-positive goat brain tissue

All the experimental protocols used in this study were approved by the Institutional Care and Use Committee at Washington State University. Information regarding tissue donor animals is summarized in Table 1. Scrapie-uninfected donor goats (n = 3) were born and housed in a scrapie-free farm at Washington State University with no direct contact with scrapie-infected animals. Of the scrapie-infected donor goats, those bearing WT prion gene (PRNP) haplotypes (n = 3) were born and raised by scrapie-infected does at the scrapie quarantine farm of the US Department of Agriculture (USDA) Animal Disease Research Unit (Pullman, WA, USA), whereas those heterozygous PRNP with a minor allele (n = 3) had been referred to us by the USDA Animal and Plant Health Inspection Service (Madsen-Bouterse et al., 2015; O'Rourke et al., 2012). The PRNP genotypes of goats were determined by sequencing the ORF of PRNP as described previously (White et al., 2008). All scrapie-infected goats were clinical at the time of tissue collection. All tissues were collected post-mortem and saved; half were fixed in formalin, and half were frozen fresh and stored at − 80 °C. Brain tissues were further processed according to standard IHC conditions to detect PrPSc using an automated immunolabeller and a mixture of mAbs F89/160.1.5 (O'Rourke et al., 1998a) and F99/97.6.1 (O'Rourke et al., 2000), as described previously (Dassanayake et al., 2013).

Preparation of brain homogenates

Brain homogenates (10 %, w/v) from normal goats and goats infected with classical caprine scrapie were prepared as described previously (Saá et al., 2006). Briefly, ∼100 μg each frozen hindbrain (anterior to the obex) was dissected and homogenized in 900 μl lysis buffer [PBS (pH 7.4) containing 150 mM sodium chloride, 0.5 % Triton X-100, 1 mM EDTA and complete mini protease inhibitor cocktail (Roche Life Science)] using a table-top tissue homogenizer. Samples were centrifuged briefly (1000 g for 2 min) and supernatants stored at − 80 °C in aliquots. Total protein concentration of brain homogenates was measured using a commercially available BCA Protein Assay kit (Thermo Fisher Scientific). All the experiments were performed with the same batch of brain homogenate preparations.

Scrapie ELISA of goat brain homogenates

The relative levels of PrPSc in PRNP WT scrapie brain homogenates were determined using a commercially available TSE ELISA. Briefly, twofold serial dilutions of brain homogenates from scrapie-infected goats were prepared and total protein concentrations in the range 0.05–25 μg per well were loaded in duplicate into a PrPSc ELISA plate (Chronic Wasting Disease antigen test kit EIA; IDEXX Laboratories). Total protein levels of 12.5 and 25 μg normal goat brain homogenate per well were also evaluated. ELISA was performed as described by the manufacturer. The cut-off value for ELISA was determined by the negative control samples provided with the kit (0.15 plus negative control sample value). In diluted samples, PrPSc detection was considered positive if the corrected A450 exceeded the assay cut-off value of 0.257.

Western blot analysis of goat brain homogenates for PrPSc

The relative levels of proteinase K-resistant prion protein (PrPres) in PRNP WT scrapie brain homogenate were determined by Western blot analysis. An aliquot of each brain homogenate was incubated with 50 μg ml− 1 (final concentration) proteinase K at 37 °C for 60 min after which the sample was immediately diluted with 2 ×  Bolt LDS sample loading buffer (Invitrogen). Twofold serial dilutions were then prepared in lysis buffer (final total protein ranging from 8 to 0.016 μg) and loaded into each well of 15-well 4–12 % Novex Bistris Plus gels (Invitrogen). After electrophoresis, proteins were transferred onto PVDF membranes, blocked with commercial casein blocker (Thermo Fisher Scientific) and incubated with primary mAb P4 (R-Biopharm; 1 : 10 000 dilution) overnight at 4 °C followed by incubation with a HRP-conjugated anti-mouse secondary antibody (1 : 20 000 dilution; SouthernBiotech) at room temperature for 60 min. Peroxidase activity was detected following incubation with SuperSignal West Pico chemiluminescence substrate (Thermo Fisher Scientific) and exposure to premium radiographic films (Phenix Research Products).

Recombinant prion protein purification

E. coli (Rosetta strain) transformed with pET41 vector containing the full-length sheep ARQ or VRQ PRNP alleles (mature PrP with residues from aa 25 to 234; GenBank accession numbers: AJ567984 and AJ567988) were kindly provided by Dr Byron Caughey (Rocky Mountain Laboratory, Hamilton, MT, USA). Expression and purification of sheep recombinant prion protein (rPrPSen) was performed as described previously (Wilham et al., 2010), but using a newer FPLC machine (ÄKTA Avant 25 chromatography system; GE Healthcare). rPrPSen protein concentration was determined using a spectrophotometer by measuring A280 and rPrPSen was stored at − 80 °C in aliquots.

RT-QuIC analysis of brain homogenates

Initial RT-QuIC was performed using the full-length sheep VRQ rPrPSen (aa 25–234) with the reaction conditions described previously for scrapie sheep brain homogenates (Wilham et al., 2010). Brain homogenates from normal and scrapie-infected goats (PRNP WT) were initially serial tenfold diluted in PBS containing 0.1 % SDS and N2 media supplement (Gibco). Aliquots of 98 μl RT-QuIC reaction buffer (10 mM phosphate buffer, pH 7.4, 300 mM NaCl, 0.1 mg VRQ rPrPSen ml− 1, 10 μM ThT and 1 mM EDTA) were loaded into each well of a black 96-well plate with an optical bottom (Nunc; Thermo Fisher Scientific). Then, 2 μl diluted (10− 1 to 10− 8; ∼15 μg to 1.5 pg total protein) scrapie or normal brain homogenates was added into each well in quadruplicates, thus giving 0.002 % SDS final concentration. Similarly prepared aliquots of a brain homogenate prepared from a sheep infected with classical scrapie (10− 4) were used as a positive control for the assay (Fig. 4). Each plate was sealed with a plate sealer film and incubated in a BMG Labtech FLUOstar Omega plate reader at 42 °C with 1 min shake (700 r.p.m., double orbital pattern) and 1 min rest for 120 h. ThT fluorescence measurements (excitation 450 ± 10 nm, emission 480 ± 10 nm, bottom read, 20 flashes per well, manual gain 1850 and 20 μs integration time) were recorded every 45 min.

To optimize RT-QuIC conditions, assays using scrapie-infected goat brain homogenates and normal goat brain homogenates were also performed with (1) ARQ and VRQ rPrPSen, (2) without and with SDS (0.002 % final concentration), and (3) at different NaCl concentrations (130, 200, 300, 400, 500 and 600 mM). Once optimized, RT-QuIC was performed using quadruplicates of serial tenfold dilutions (10− 3 to 10− 8; ∼150 ng to 1.5 pg total protein) of normal and scrapie goat brain homogenates (PRNP WT) and freshly prepared RT-QuIC buffer containing 10 mM phosphate buffer (pH 7.4), 0.1 mg rPrPSen (ARQ or VRQ) ml− 1, 200 mM NaCl, 0.002 % SDS, 10 μM ThT and 1 mM EDTA. All reactions for each dilution and each sample were performed in quadruplicates in three independent RT-QuIC assays.

Data analysis

ThT fluorescence data in graphs are displayed as the mean ThT fluorescence of four technical replicates for each time point. To consider a given dilution positive for seeding activity, the ThT fluorescence of at least two replicate reactions must exceed a pre-defined positive threshold. The positive threshold was calculated as 10 sd above the mean ThT fluorescence (∼40 000 relative fluorescence units) of normal goat brain homogenates (Orrú et al., 2014, 2015a).

Acknowledgements

This study was funded by the US Department of Agriculture Agricultural Research Service (CRIS 2090-32000-030-00D) and also in part by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases. We thank Katherine O'Rourke (Washington State University) for critical reading of the manuscript. The authors would also like to thank Linda Hamburg, Lori Fuller, Laetisha O'Rourke, Deborah Wolheter, Jan Luft, Karel Emma and Desiree Lesiak for technical assistance. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture.

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