Sulfated Dextrans Enhance In Vitro Amplification of Bovine Spongiform Encephalopathy PrPSc and Enable Ultrasensitive Detection of Bovine PrPSc

Background Prions, infectious agents associated with prion diseases such as Creutzfeldt-Jakob disease in humans, bovine spongiform encephalopathy (BSE) in cattle, and scrapie in sheep and goats, are primarily comprised of PrPSc, a protease-resistant misfolded isoform of the cellular prion protein PrPC. Protein misfolding cyclic amplification (PMCA) is a highly sensitive technique used to detect minute amounts of scrapie PrPSc. However, the current PMCA technique has been unsuccessful in achieving good amplification in cattle. The detailed distribution of PrPSc in BSE-affected cattle therefore remains unknown. Methodology/Principal Findings We report here that PrPSc derived from BSE-affected cattle can be amplified ultra-efficiently by PMCA in the presence of sulfated dextran compounds. This method is capable of amplifying very small amounts of PrPSc from the saliva, palatine tonsils, lymph nodes, ileocecal region, and muscular tissues of BSE-affected cattle. Individual differences in the distribution of PrPSc in spleen and cerebrospinal fluid samples were observed in terminal-stage animals. However, the presence of PrPSc in blood was not substantiated in the BSE-affected cattle examined. Conclusions/Significance The distribution of PrPSc is not restricted to the nervous system and can spread to peripheral tissues in the terminal disease stage. The finding that PrPSc could be amplified in the saliva of an asymptomatic animal suggests a potential usefulness of this technique for BSE diagnosis. This highly sensitive method also has other practical applications, including safety evaluation or safety assurance of products and byproducts manufactured from bovine source materials.


Introduction
Prions, the infectious agents associated with transmissible spongiform encephalopathies such as scrapie in sheep, chronic wasting disease (CWD) in deer and elk, bovine spongiform encephalopathy (BSE), and Creutzfeldt-Jakob disease (CJD) in humans, are primarily comprised of PrP Sc , a protease-resistant misfolded isoform of the cellular prion protein PrP C [1]. Prion diseases are fatal neurodegenerative disorders, and are characterized by the accumulation of PrP Sc in the nervous tissues of infected subjects [2].
BSE is an emerging disease that first appeared in the United Kingdom in 1986 [3]. The cause of the BSE outbreak in the UK is believed to have been the result of feeding cattle meat and bone meal (MBM) contaminated with PrP Sc acquired from rendering carcasses of BSE-or scrapie-infected ruminants [4,5]. Since variant CJD (vCJD) is suspected to be attributable to infectious agents associated with BSE [6][7][8], prophylactic hygiene dictates that infected cattle be identified and eradicated.
Immunological methods such as enzyme-linked immunosorbent assays and western blotting (WB) have been widely used for the detection of PrP Sc . It is now possible to perform in vitro amplification of hamster scrapie PrP Sc using the protein misfolding cyclic amplification (PMCA) technique [9]. Extremely small amounts of PrP Sc can be detected by combining PMCA with WB [10,11]. PMCA has been used to amplify PrP Sc from mice [12], deer [13], sheep [14], and humans [15]. PMCA has also been applied to the detection of bovine PrP Sc in cattle [16], and serial PMCA has been shown to improve detection sensitivity [17]. Although hyperefficient amplification of a mouse-adapted BSE strain has been demonstrated [18], there are no reports that cite ultrasensitive and direct detection of bovine PrP Sc in cattle using the current PMCA method.
Since the concentration of PrP Sc in tissues or body fluids of BSE-infected animals is expected to be extremely low, the development of a sensitive method for detecting PrP Sc in infected cattle is important. In the present study, we developed an extremely efficient method that is suitable for bovine PrP Sc amplification. The method, which involves amplifying BSE PrP Sc in the presence of sulfated dextran compounds, enables sensitive detection of PrP Sc at levels equivalent to those obtained for detection of hamster PrP Sc [19]. This method is capable of amplifying very small amounts of PrP Sc from the saliva, palatine tonsils, lymph nodes, ileocecal region, and muscular tissues of BSE-affected cattle. The technology will result in a marked improvement in BSE safety.

Sulfated dextran compounds enhanced BSE PrP Sc amplification
In general, the efficiency of BSE PrP Sc amplification using PMCA was low compared to amplification of hamster and mouse scrapie PrP Sc ( Figure 1A). We attempted to improve BSE PrP Sc amplification efficiency using high temperature conditions (39uC and higher). Various reagents were screened for their ability to prevent thermal denaturation of the brain homogenate, and we found that sulfated dextran compounds were suitable for this purpose. Unexpectedly, the BSE PrP Sc amplification efficiency was greatly increased when amplification was performed in the presence of sulfated dextran compounds at 37uC.
The signal intensities of amplified PrP Sc upon WB were significantly higher in samples containing sodium dextran sulfate with a high molecular weight of 900-2000 kDa (DSS-H) at a final concentration of 0.25-1% ( Figure 1A, right panel). This enhancement was dependent upon the polarity and molecular size of the dextran compound. Smaller anionic sodium dextran sulfate (DSS-L) and potassium dextran sulfate (DSP) were more effective than the high molecular weight compounds, but positively charged DEAE-dextran and dextrans covering a range of molecular weights had little or no effect on amplification ( Figure 1B). Glycosaminoglycans that are distributed throughout animal tissues and a sulfated polysaccharide were not effective at the concentrations examined ( Figure 1C).
In contrast, in vitro amplification of sheep scrapie PrP Sc was completely inhibited by the addition of DSP to the reaction mixture ( Figure 2A). A similarly inconsistent experimental result Figure 1. The effect of polysaccharides on the amplification of PrP Sc derived from BSE-affected cattle. A. The effect of sodium dextran sulfate with a high molecular weight (MW) ranging from 900 to 2000 kDa (sodium dextran sulfate, DSS-H) on BSE-PrP Sc amplification. The PrP Sc seed (10% brain homogenate) was diluted to 10 22 to 10 23 in PrP C substrate, and the diluted samples were amplified in the presence of DSS-H at 0-1%. The samples before (left panel) and after (right panel) amplification were analyzed by WB after digestion with PK. ''N'' designates controls in which the PrP C substrate alone was treated in the same manner. B. The effect of dextran compounds on BSE-PrP Sc amplification. The PrP Sc seed was diluted to 10 24 , and amplification was performed in the presence or absence (''No additive'') of dextran compounds at 0.5%. ''No seed'' designates the control in which the PrP C substrate alone was amplified without dextran compounds. DSS-L: sodium dextran sulfate with a low MW, ranging from 5 to 6 kDa; DSP: potassium dextran sulfate (1.5 to 1. was obtained with pentosan polysulfate (PPS), which may be effective in treating CJD [20,21]. This reagent acted as an inhibitor of sheep scrapie PrP Sc amplification ( Figure 2A), but induced a low-level, dose-dependent amplification of BSE PrP Sc ( Figure 2B).

Detection limit of BSE PrP Sc using DSP-PMCA techniques
The optimal concentration of DSP was estimated to be in the range of 0.05-0.75% ( Figure 3); therefore, we used 0.5% DSP for subsequent experiments. We determined the detection limit of the DSP-PMCA technique and confirmed that PrP Sc present in a 10 26 dilution of infected brain homogenate could be detected after one round of amplification, and both 10 28 and 10 29 dilutions tested positive for PrP Sc after two rounds of amplification ( Figure 4A). A PrP Sc signal was detected in one of the 10 210 dilution samples, but no signal was detected in the more extreme dilution range, even after four rounds of amplification. Thus, the PrP Sc detection sensitivity was improved 10 8 times compared to no amplification. The 50% lethal dose (LD50) per gram of brain homogenate used as seed was determined in a previous study [22] by a bioassay using Tg(BoPrP)4092HOZ/Prnp 0/0 (TgBoPrP) transgenic mice [23]. The infectious titer was 10 6.7 LD50/g, and infectivity was confirmed in mice intracerebrally inoculated with up to a 10 24 dilution of the 10% brain homogenate. Therefore, our improved method was 10 5 times more sensitive than the bioassay. The generation of spontaneous PrP Sc , as has been reported for amplification in the presence of polyanions [24,25], was not observed with four rounds of amplification ( Figure 4B).

Infectivity of the PMCA product
The PMCA product obtained after six rounds of amplification was diluted 10-fold and inoculated intracerebrally into TgBoPrP transgenic mice that overexpress bovine PrP C . Mice inoculated with the PMCA sample died after a mean of 243 days (Table 1). Control mice administered DSP (0.05%) or PrP Sc (3.2610 212 dilution) at concentrations corresponding to the BSE seed dilution in the PMCA sample survived more than 500 days. The results of immunohistochemical analysis of the habenular nuclei and the midbrains from control and treated mice are shown in Figure 5. Vacuolation and PrP Sc accumulation, which was occasionally observed as plaque-like PrP Sc aggregates, were found in mice inoculated with the PMCA sample or the BSE-affected cattle brain  homogenate, indicating that the amplified PrP Sc was infectious and caused lesions typical of prion diseases.

PrP Sc distribution in the tissues of BSE-affected cattle
Using our improved method, the distribution of PrP Sc was examined in cattle that were orally administered a brain homogenate prepared from BSE-infected cattle. In the terminal disease stage in BSE-affected cow 5550, PrP Sc was detected by conventional WB in several peripheral nervous tissues and the adrenal glands (Table 2). Moreover, PrP Sc was detected by serial DSP-PMCA in the palatine tonsils, lymph nodes, ileocecal region, and muscular tissues ( Figure 6A), whereas no PrP Sc signal was detected in the corresponding tissue samples from uninfected control cow 2914 (Table 2 and Figure 6B).
We could not detect PrP Sc in the spleen, blood, or cerebrospinal fluid (CSF) from BSE-affected cow 5550 in the end stage of disease, even after four rounds of amplification. Although an additional five tissue pieces were cut from both the central portion of the spleen and the splenic hilum for amplification, splenic PrP Sc was not detected in any of these 10 tissue samples ( Figure 7A). The distribution of PrP Sc in the spleen was examined further in two more terminal disease-stage BSE-affected cattle, numbers 5499   and 5468. As was the case with cow 5550, splenic PrP Sc was not detected in any of the 10 tissue samples from cow 5499 ( Figure 7B). However, a PrP Sc signal was detected in 3 of 10 tissue pieces from the spleen of cow 5468 ( Figure 7C).

PrP Sc distribution in the bodily fluids of BSE-affected cattle
Before amplification, PrP Sc accumulation was confirmed using conventional WB analysis in the peripheral nervous tissues of cows 5499 and 5468 and in the adrenal gland of cow 5468 (Table 2). Individual differences in the distribution of PrP Sc in CSF samples were observed in terminal-stage animals. PrP Sc signals were detected in duplicate samples from cow 5468 after three rounds of amplification ( Figure 7C) but were not detected in samples from cow 5499 ( Figure 7B), as confirmed in samples from cow 5550 ( Figure 6A).
We also examined the distribution of PrP Sc in the salivary glands and saliva obtained from BSE-affected cattle. PrP Sc was detected in the submandibular and parotid glands of cows 5468 and 5499 after two rounds of amplification ( Figure 8A). In cow 5550, PrP Sc was detected in the submandibular and sublingual glands ( Figure 8B). Moreover, a PrP Sc signal was detected in two of the quadruplicate saliva samples from cow 5550 after four rounds of amplification. The presence or absence of salivary PrP Sc was investigated in intracerebrally infected cow 9007. PrP Sc accumulation in the brain stem was found by conventional WB analysis 17 months after inoculation ( Table 2), but the animal remained asymptomatic until sacrifice. PrP Sc was detected in the sublingual and parotid glands after two rounds of amplification. In cow 9007, one of the duplicate saliva samples was found to be positive for PrP Sc after four rounds of amplification ( Figure 8C).

Discussion
The present study is the first report describing ultrasensitive detection of BSE PrP Sc in cattle. The determination that sulfated dextran compounds enhance the efficiency of in vitro BSE PrP Sc amplification was unexpected because sulfated dextran has long been known as an antiscrapie agent both in vivo [26] and in vitro [27]. In vitro studies using recombinant PrP [28] and PrP C purified from brain homogenate [29] have suggested that various polyanionic compounds increase the amplification of protease-resistant PrP. Therefore, endogenous polyanionic molecules such as sulfated glycans [28,29] and RNA [29] could be cofactors required to facilitate the propagation of PrP Sc . Small and negatively charged dextran compounds (DSP and DSS-L) were most effective for in vitro amplification of bovine PrP Sc in this study. These dextran compounds may accelerate the rate of PrP Sc formation by acting as accessory molecules that facilitate or stabilize interactions between PrP Sc , PrP C and cofactors contained in brain homogenates. Since species-specific differences in cofactor preference for in vitro amplification of PrP Sc have been reported [30], dextran compounds may interfere with PrP Sc formation by acting as competitive inhibitors of the cofactors required for propagation of scrapie PrP Sc .
The theory that PrP Sc is widely distributed in various peripheral tissues in the terminal disease stage is strongly supported by our  experimental results as well as previous findings, which demonstrated a low level of infectivity in the tonsils [31] and muscles [32] of cattle that were orally administered a brain homogenate prepared from BSE-infected cattle. With regard to the level of PrP Sc detected in muscle tissue, the level estimated from the amplification factor in PMCA was lower than that found in a brain homogenate diluted to 10 26 because no PrP Sc signal was detected in the first round of amplification. In the mesenteric and Rouviere lymph nodes as well as the ileocecal region, only one or two of the quadruplicate samples tested positive, even after four rounds of amplification. Given that PrP Sc tends to aggregate, partial detection of PrP Sc in the reaction may be due to the near absence of PrP Sc ; PrP Sc levels in these tissues would have been equivalent to the levels found in brain homogenate dilutions of 10 210 to 10 211 . Although significant prionemia [33] and PrP Sc distribution in the blood [34] have been demonstrated in scrapie-infected animals, the presence of PrP Sc in the blood was not substantiated in the BSE-affected cattle used in our study, despite a dramatic improvement in the sensitivity of PrP Sc detection. This observation was in agreement with the previous result showing that the disease is not transmitted through the blood of BSE-affected cattle [32,35]. Together with the scattered accumulation of PrP Sc in the spleen and the low-level accumulation of PrP Sc in the lymph nodes, the absence of detectable PrP Sc in the blood of BSEaffected cattle suggests a neuronal rather than lymphoreticular progression of BSE PrP Sc to the brain.
With regard to other bodily fluids, a bioassay of saliva from CWD-affected deer showed significant levels of infectious prions [36]. The presence of PrP Sc in the salivary glands [37] and the amplification of PrP Sc in concentrated buccal swab samples [38] have been reported in scrapie-infected sheep. Ours is the first report describing PrP Sc detection in both the salivary glands and saliva of BSE-infected cattle. The salivary glands are regulated by parasympathetic nerves arising from the salivary nuclei of the medulla oblongata and sympathetic nerves arising from the thoracic portion of the spinal cord. Thus, it is possible that PrP Sc accumulated in the central nervous system and spread to the salivary glands through the autonomic nervous system, and that very small amounts of PrP Sc were secreted into the saliva in BSEinfected cattle.
Our study demonstrated that the distribution of PrP Sc was not restricted to the nervous system, and that PrP Sc was able to spread to most of the peripheral tissues examined in the terminal disease stage. The finding that PrP Sc could be amplified in saliva taken from an asymptomatic animal suggests a potential usefulness of this technique for BSE diagnosis. Detailed examinations of the temporal course of BSE infection and the incidence of PrP Sc appearance in saliva, as well as studies of how infection route affects salivary PrP Sc accumulation, will be necessary to confirm whether salivary PrP Sc can serve as a reliable marker for BSE infection. The highly sensitive method we describe has other practical applications as well, such as evaluating the safety of livestock products and raw feed materials, and safety assurance of pharmaceutical and cosmetic products manufactured from bovine source materials.

Ethics Statement
All animal experiments were approved by the Animal Care and Use Committee (approval IDs: 450 and 08-009) and Animal Ethics Committee (approval IDs: 04-III-7 and 08-IV-32) of the National Institute of Animal Health.

Cattle
The cattle used in this study were imported from Australia. A c-BSE-infected brain homogenate (50 ml of a 10% homogenate) was orally administered to cattle (cows 5499, 5468, and 5550) ranging from 10 to 12 months in age. After 34 to 57 months, the animals were sacrificed and dissected in the terminal disease stage. Cow 9007 was intracerebrally inoculated with c-BSE-infected brain homogenate (1 ml of a 10% homogenate), sacrificed and dissected 17 months following inoculation, and the tissues were used for the analysis of salivary PrP Sc . Normal 3-to 4-month-old cattle (cows 2914 and 5660) were used as controls.

Sample preparation
Tissues, white blood cells (WBCs), plasma, serum, CSF, and saliva were collected upon dissection and stored in small aliquots at 280uC. Oral cavity saliva was collected by aspiration. Samples from each tissue (0.2 g) and WBCs (approximately 2610 8 cells) were homogenized and suspended at 20% (w/v) in phosphatebuffered saline (PBS) containing 26 complete protease inhibitors (Roche Diagnostics).

PrP C substrate
To avoid contamination, brain homogenates were prepared in a laboratory that had never contained infected materials. The brains of bovine PrP C -overexpressing TgBoPrP transgenic mice and PrP knockout (PrP 0/0 ) mice were homogenized separately in 20% (w/v) PBS containing 26 complete protease inhibitors. The homogenates were mixed with an equal volume of elution buffer (PBS containing 2% Triton X-100 and 8 mM EDTA) and incubated at 4uC for 1 h with continuous agitation. After centrifugation at 45006g for 5 min, the supernatants were mixed in a 5:1 proportion of PrP 0/0 :TgBoPrP. This mixture was used as the PrP C substrate. Dextrans (Nacalai), dextran compounds (sodium dextran sulfate (DSS), potassium dextran sulfate (DSP), Nacalai; DEAE-dextran, Sigma), pentosan polysulfate (PPS, Elmiron, Janssen-Ortho), sulfated polysaccharide (l-carrageenan (l-Cag), Nacalai), glycosaminoglycans (sodium chondroitin sulfate C (CSS), sodium heparan sulfate (HSS), Nacalai), and heparan sulfate proteoglycan (HSPG, Sigma) were dissolved in PBS or distilled water and added to the PrP C substrate at the concentration indicated in the figures. For the amplification of sheep scrapie PrP Sc , normal ICR mouse brains were homogenized in 10% (w/v) PBS containing complete protease inhibitors, 1% Triton X-100, and 4 mM EDTA. After centrifugation at 45006g for 5 min, the supernatant was used as the PrP C substrate.

PMCA
To examine the bovine PrP Sc detection sensitivity, 100 ml of PrP C substrate containing 0.5% DSP was mixed with 1/100 volume of 10% brain homogenate from cattle infected with c-BSE (infectivity titer = 10 6.7 LD50/g) [20], and serial 10-fold dilutions were prepared of the PrP C substrate containing 0.5% DSP. Homogenates of each tissue and WBCs, plasma, serum, CSF, and saliva were diluted 1:20 with the PrP C substrate containing 0.5% DSP (total volume 100 ml) in an electron beam-irradiated polystyrene tube. Amplification was performed with a fully automatic cross-ultrasonic protein-activating apparatus (Elestein 070-CPR, Elekon Science Corporation) using 40 cycles of sonication in which a 3-s pulse oscillation was repeated five times at 1-s intervals, followed by incubation at 37uC for 1 h with agitation. For the amplification of PrP Sc in various tissues from BSE-inoculated and control cattle (Figure 6), the PrP C substrate containing 0.5% DSP was mixed with 1/20 volume of homogenized samples or bodily fluids (total volume 80 ml) in an electron beam-irradiated 8-strip polystyrene tube specially designed for PrP Sc propagation. PMCA was performed using 40 cycles of sonication (a pulse oscillation for 5 s, repeated five times at 1-s intervals), followed by incubation at 37uC for 1 h with agitation. The 1:5 dilution of the PMCA product and subsequent amplification was repeated three times.

Western blotting
Samples (10 ml) were mixed with 10 ml of proteinase K (PK) solution (100 mg/ml) after each round of amplification and incubated at 37uC for 1 h. The digested materials were mixed with 20 ml of 26 SDS sample buffer and incubated at 100uC for 5 min. The samples were separated by SDS-PAGE and transferred onto a polyvinylidene fluoride membrane (Millipore). After blocking, the membrane was incubated for 1 h with a horseradish peroxidase (HRP)-conjugated T2 monoclonal antibody [39] diluted 1:10 000. After washing, the blotted membrane was developed using the Immobilon Western Chemiluminescent HRP Substrate (Millipore) according to the manufacturer's instructions. Chemiluminescence signals were analyzed with a Light Capture system (Atto).

Bioassay
A 10% brain homogenate from c-BSE-infected cows was diluted to 10 28 with PrP C substrate containing 0.5% DSP and amplified. The PMCA product was diluted 1:5 with the PrP C source containing 0.5% DSP, and a second round of amplification was performed. The 1:5 dilution of the PMCA product and its subsequent amplification were repeated five times. The product from the sixth round was diluted 1:10 with PBS and inoculated intracerebrally into TgBoPrP mice (20 ml per mouse). The PrP C substrate containing 0.05% DSP and the PrP C substrate containing the PrP Sc seed diluted to 3.2610 212 were inoculated as dilution controls for DSP and the PrP Sc seed, respectively.

Immunohistological analysis
The left hemisphere of the brain was fixed in 10% buffered formalin for neuropathological analysis. Coronal brain sections were immersed in 98% formic acid to reduce infectivity and embedded in paraffin wax. Sections of 4-mm thickness were cut and stained with hematoxylin and eosin (HE). Immunoreactive PrP Sc was detected in brain sections using anti-PrP monoclonal antibody F99/97.6.1 (VMRD) or 12F10 (SPI-bio) as the primary antibody. An anti-mouse universal immunoperoxidase polymer (Histofine simple stain MAX-PO (M), Nichirei) was used as the secondary antibody, and 3,39-diaminobenzidine tetrachloride served as the chromogen.