SHORT COMMUNICATION Bolea et al., Journal of General Virology 2017;98:2628–2634 DOI 10.1099/jgv.0.000906 Experimental transmission to a calf of an isolate of Spanish classical scrapie Rosa Bolea,1,*† Carlos Hedman,1† Óscar López-Perez,1 Belen Marín,1 Enríc Vidal,2 Martí Pumarola,2 Fabien Corbiere,3 Antonio Romero,4 Bernardino Moreno,1 Inmaculada Martín-Burriel,1 Olivier Andreoletti3 and Juan Jose Badiola1 Abstract Multiple theories exist regarding the origin of bovine spongiform encephalopathy (BSE). An early and prominent theory proposed that BSE was the result of the adaptation of sheep scrapie to cattle. The reports to date indicate that the distribution of the pathological prion protein (PrPSc) in experimental bovine scrapie is largely restricted to the central nervous system (CNS). Here, we describe pathological findings in a calf intracerebrally inoculated with a Spanish classical scrapie isolate. While clinical disease was observed 30 months after inoculation and PrPSc was detected in the CNS, the corresponding phenotype differed from that of BSE. Immunohistochemistry and PMCA also revealed the presence of PrPSc in the peripheral nerves, lymphoid tissues, skeletal muscle and gastrointestinal tract, suggesting centrifugal spread of the scrapie agent from the brain. To the best of our knowledge, this is the first report describing the detection of PrPSc in tissues other than the CNS after experimental transmission of scrapie to cattle. From a public health perspective, bovine spongiform Depending on the affected species, the strain of the TSE encephalopathy (BSE) is the most important transmissible agent, the prion protein genotype of the recipient and other spongiform encephalopathy (TSE). BSE and variant Creutz- factors, pathological prion protein (PrPSc) can be distributed feldt–Jakob disease (vCJD) in humans share the same causa- in organs outside of the central nervous system (CNS). tive agent [1, 2]. Several theories have been proposed PrPSc has been detected in the lymphoreticular system regarding the origin of BSE. For example, epidemiological (LRS), enteric nervous system (ENS), skeletal muscle, adre- data suggest that BSE originated following the exposure of nal gland, pancreas and other organs in sheep naturally cattle to a scrapie-like agent in ruminant-derived feed [3]. infected with classical scrapie [12–15]. In naturally and Studies of the adaptation of scrapie to cattle have shown experimentally BSE infected cattle, PrPSc is primarily dis- that oral inoculation of cattle with scrapie does not result in tributed in the brain, spinal cord, retina and distal ileum disease transmission [4, 5]. While cattle are susceptible to [16–20]. Moreover, ultrasensitive techniques such as protein different classical scrapie isolates when inoculated intracere- misfolding cyclic amplification (PMCA) [21] have enabled brally [6, 7], in all cases the corresponding clinical presenta- the detection of minuscule amounts of PrPSc in mesenteric tion and neuropathological features [5, 6, 8, 9] can be lymph nodes and other organs from cattle experimentally distinguished from those of BSE, even after a second passage infected with the BSE agent [22]. Although PrPSc has been [8, 10]. Moreover, neither the molecular signature nor strain detected in the CNS of cattle intracerebrally inoculated with typing by mouse bioassay indicate any evidence of classical the classical scrapie agent, a single study in which the [9] or atypical [11] BSE. mesenteric lymph node and spleen were examined by Received 21 July 2017; Accepted 28 July 2017 Author affiliations: 1Centro de Investigación en Encefalopatías y Enfermedades Transmisibles Emergentes (CIEETE), Veterinary Faculty, Universidad de Zaragoza, 50013 Zaragoza, Spain; 2Department of Animal Medicine and Surgery, Veterinary Faculty, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain; 3UMR INRA ENVT 1225, Interactions H^otes Agents Pathog enes, Ecole Nationale V et erinaire de Toulouse, 31076 Toulouse, France; 4Veterinary Hospital, Universidad de Zaragoza, 50013 Zaragoza, Spain. *Correspondence: Rosa Bolea, rbolea@unizar.es Keywords: ovine; scrapie; bovine; BSE; peripheral tissues; prion. Abbreviations: BG, basal ganglia; BSE, bovine spongiform encephalopathy; CNS, central nervous system; Cbl, cerebellum; CSC, cervical spinal cord; ENS, enteric nervous system; FC, frontal cortex; GIT, gastrointestinal tract; Ht, hypothalamus; LSC, lumbar spinal cord; LRS, lymphoreticular system; MO, medulla oblongata; p.i., post-inoculation; OC, occipital cortex; OD, optical density; PrPSc, pathological prion protein; PNS, peripheral nervous sys- tem; PrPres, protease-resistant prion protein; PMCA, protein misfolding cyclic amplification; TPC, temporal/parietal cortex; T, thalamus; TSC, thoracic spinal cord; TBMs, tingible body macrophages; TSE, transmissible spongiform encephalopathy; vCJD, variant of Creutzfeldt–Jakob disease; WB, West- ern blot; GBC, basal ganglia cortex; O, obex. †These authors contributed equally to this work. 000906 ã 2017 The Authors 2628 Bolea et al., Journal of General Virology 2017;98:2628–2634 immunohistochemistry (IHC) found no evidence of periph- Darmstadt, Germany), in order to discriminate between eral distribution of PrPSc [6]. scrapie and BSE prion protein [14]. Here, we describe the clinical signs, neuropathological fea- Homogenates (10 % in saline solution) were prepared using tures and molecular signature of a heifer experimentally tissue from the brain, sciatic nerve, brachial nerve, mesenteric infected with a classical scrapie isolate derived from a field- lymph node, extraorbital muscle, ileum and spleen. These infected sheep of Spanish origin. The objective of this study inocula were then subjected to three rounds (48 h each) of was to determine whether a single isolate from a natural PMCA, as previously described [24]. Tg338 mouse brain scrapie-infected sheep (ARQ/ARQ) could induce BSE-like expressing the high susceptibility allele (VRQ) of the ovine disease in cattle. Our findings describe for the first time the PrP gene [25] was used as the substrate. The reaction was per- distribution of PrPSc in the peripheral nerves, lymphoid tis- formed on a microplate (Axygen, Union City, CA, USA) into sues, skeletal muscle and gastrointestinal tract of a cow which a single ceramic bead was placed [26]. The Dawson infected with classical scrapie. scrapie strain [27] and healthy bovine brain homogenate were used as positive and negative controls, respectively. PMCA A 10-month-old Pyrenean breed calf was used. The inocu- products were analysed by Western blot (WB) using the mAb lum consisted of 1 ml of a 10 % solution of homogenized Sha31 (Bio-Rad Laboratories). obex from a sheep naturally infected with scrapie (ARQ/ ARQ) and diagnosed by the Reference Laboratory for TSE At 30 months post-inoculation (p.i.), the heifer stood as part of the active TSE surveillance programme of Aragón, motionless, with its head lowered or resting against nearby Spain. objects when undisturbed, and did not overreact to external stimuli. Ataxia and severe lethargy were observed. Two The calf was placed under general anaesthesia. The trephine weeks later, the animal showed abdominal distension and was positioned paramedially in the mid-frontal region of discomfort, which resulted in prostration. The animal was the head, at an angle of 90 with respect to the rostro-caudal then euthanized. No macroscopic abnormalities were slope of the front of the skull. The inoculum was injected observed during necropsy, except for distension and the into the frontal cortex via a 9-cm-long needle (gauge, 22). presence of abundant gravel in the small intestine. The animal was monitored daily by animal husbandry staff. IDEXX analysis revealed PrPSc positivity in the CNS. The Clinical assessments were carried out once per week until optical density (OD) values for BSE conjugate varied among the development of clinical signs, and once daily thereafter. CNS samples: the highest OD values were obtained for the The animal was euthanized by exsanguination after intrave- frontal cortex (3.575), followed by the obex (3.507); inter- nous injection of sodium pentobarbital. A systematic post- mediate values were obtained for the LSC (2.506) and cere- mortem examination was conducted and a range of samples bellum (2.480); and the lowest values were obtained for the were collected from the CNS, peripheral nervous system thalamus (2.227). Despite these findings, IDEXX failed to (PNS), LRS, gastrointestinal tract (GIT), skeletal muscle detect PrPSc in PNS, LRS, GIT, or skeletal muscle samples. (extraorbital) and other tissues. The samples were collected Histopathological examination of CNS samples revealed no in duplicate: one was fixed in a solution of 10 % formalde- morphological alterations. Immunohistochemistry revealed hyde in saline and the other one was frozen at 80  C for the distribution of PrPSc throughout multiple neuroanatom- subsequent molecular analyses. ical areas (Fig. 1a). Three distinct PrPSc deposition patterns Formalin-fixed tissues were trimmed and processed accord- were identified: a granular cytoplasmic pattern in neuronal ing to standard histopathological procedures. A 4-µm-thick perikarya; an intra-glial pattern (Fig. 1b, c); and a less fre- tissue section from each sample was stained with haematox- quently observed particulate neuropil-associated pattern ylin and eosin. Adjacent sections were immunolabelled (Fig. 1d). PrPSc was not observed in control tissues. using the monoclonal antibody (mAb) L42 for detection of No histopathological alterations were observed in tissues PrPSc deposition by IHC, as described previously [23]. outside the CNS. However, small deposits of PrPSc were dis- Corresponding tissues from a healthy heifer were used as tributed throughout several peripheral tissues. In the PNS, control samples for IHC. periaxonal immunolabelling was observed in transverse sec- Samples of frontal cortex (FC), thalamus (T), cerebellum tions of the sciatic nerve (Fig. 2a). In the LRS, PrPSc was (Cbl), obex (MO) and lumbar spinal cord (LSC) were ana- detected in tingible body macrophages (TBMs) present in lysed by enzyme immunoassay [IDEXX HerdChek BSE- the mesenteric lymph nodes (Fig. 2b). This same pattern was observed outside the lymphoid follicles in the tonsils scrapie antigen test kit (IDEXX)]. A variety of peripheral and the submandibular and prescapular lymph nodes. No tissues were also tested using conjugates for the detection of clear PrPSc staining was detected in the spleen. In the GIT, both BSE and scrapie. The average mean result obtained for immunoreactivity was observed in intracytoplasmic gran- the negative control (+0.180) was used as the cut-off value. ules within the neurons of the ENS in the duodenum, jeju- CNS samples were analysed using the Bio-Rad TESeE num, ileum (Fig. 2c) and caecum. PrPSc immunoreactivity Western blot method (Bio-Rad Laboratories, Marnes-La- was also detected in the muscle spindles of the extraorbital Coquette, France) with the mAbs 6H4 and P4 (Biopharm, muscle (Fig. 2e). 2629 Bolea et al., Journal of General Virology 2017;98:2628–2634 Fig. 1. PrPScdistribution and deposition patterns in different areas of the CNS in a scrapie-infected calf. (a) FC, frontal cortex; BG, basal ganglia; GBC, basal ganglia cortex; T, thalamus; Ht, hypothalamus; TPC, temporal/parietal cortex; OC, occipital cortex; Cbl, cerebellum; P, pons; O, obex; CSC, cervical spinal cord; TSC, thoracic spinal cord; and LSC, lumbar spinal cord. (b) Thalamus. Immuno- labelling is predominantly intracellular (intraneuronal and intraglial). (c) Cervical spinal cord. Immunolabelling is predominantly intra- neuronal. (d) Basal ganglia caudate nucleus. Immunolabelling is predominantly detected in the neuropil. While protease-resistant prion protein (PrPres) was detected scrapie glycosylation pattern, revealing an unglycosylated in the CNS by WB, the molecular profile differed from that band of a molecular mass higher than that detected for of the original scrapie inoculum, and that of classical BSE. bovine scrapie. No PrPSc was amplified in brachial nerve or The mAb 6H4 revealed an unglycosylated band of a lower negative control homogenates (Fig. 3d). molecular mass than that of natural scrapie. This profile This study presents a thorough neuropathological charac- was similar to that observed for the BSE sample, but con- terization, achieved using IHC, molecular tests and in vitro tained monoglycosylated and diglycosylated bands of lower PrPres amplification assays, of a calf intracerebrally inocu- masses than those observed for BSE. The mAb P4 failed to lated with a single classical scrapie isolate. The animal detect PrPres in either the bovine scrapie samples or the con- trol BSE samples, but did detect the classical scrapie strain developed clinical signs suggestive of a TSE at 30 months (Fig. 3a). WB using the mAb Sha 31 failed to detect the p.i., in accordance with previous scrapie infection studies in presence of PrPres in peripheral tissues (Fig. 3b). cattle describing incubation periods of between 14 [6] and 48 months [7]. Furthermore, the clinical presentation was A third round of PMCA at a dilution of 10 2 succeeded in characterized by general listlessness (weakness and leth- amplifying PrPres in the brain, sciatic nerve, mesenteric argy), in line with the ‘dull form’ previously described in lymph node, extraorbital muscle and ileum samples, all of sheep scrapie-infected cattle [9]. While previous studies which were positive by IHC. A signal was detected in spleen have described a clinical course of between 6 and 10 weeks, samples by PMCA alone (Fig. 3c). Furthermore, amplifica- in the present case the animal presented severe abdominal tion of bovine scrapie in this substrate restored the original discomfort and was killed 2 weeks after the clinical signs 2630 Bolea et al., Journal of General Virology 2017;98:2628–2634 Fig. 2. Immunohistochemical findings in peripheral tissues in a scrapie-infected calf. The arrows indicate PrPSc immunolabelling (mAb L42) in the (a) sciatic nerve, (b) mesenteric lymph node, (c) myenteric plexus of the ileum and (d) muscular spindle of the extraor- bital muscle. associated with the scrapie infection started. The absence of lower than the corresponding bands detected in BSE sam- spongiform changes in the brain was in agreement with ples, indicating that the glycosylation pattern of bovine scra- previous reports [6], but contrasted with the results pie differed from that of BSE. Subsequent characterization obtained in natural and experimental BSE infections [28]. of the same two scrapie pools in a mouse bioassay revealed The pattern of PrPSc distribution also closely resembled no evidence of classical or atypical BSE [11]. that described in previous studies of intracerebral transmis- In contrast to these studies, our calf was inoculated with a sion of scrapie agent to cattle, consisting of a predominantly single scrapie isolate. This approach ruled out the possibility intraneuronal pattern [6] in the trigeminal nuclei, olive that competition between coexisting prions strains could nucleus and reticular area. No involvement of the dorsal interfere with the development of a BSE-like disease. How- nuclei of the vagus nerve (data not shown) or the solitary ever, the replication of the classical scrapie isolate in a heifer tract was observed, in contrast to natural cases of BSE in used in the present experiment failed to produce BSE. cattle [29]. No PrPSc amyloid plaques were observed in any of the brain regions analysed, in contrast to findings While IDEXX failed to detect the presence of PrPSc in peripheral tissues, it should be noted that rapid tests may be reported in L-type BSE [30]. less sensitive than IHC in cases in which there is minimal Analysis of bovine scrapie samples by discriminatory WB PrPSc accumulation [31]. IHC revealed for the first time the using mAb P4, which binds ovine PrP in the N-terminal of presence of PrPSc in organs outside the CNS, such as PNS, the PrPres, detected no signal. This lack of detection could LRS, extraorbital muscle and GIT from a heifer inoculated have some resemblance to the BSE agent. Similarly, Konold with the classical scrapie agent. In a previous similar study, and coworkers [9] reported two distinct prion disease phe- no PrPSc was detected in either the mesenteric lymph node notypes in cattle inoculated with two different scrapie pools. or the spleen [6]. The accumulation and distribution of One of the phenotypes was positive to mAb P4 and one was PrPSc in peripheral tissues may be influenced by several fac- negative. However, in agreement with our findings, the tors. Gonzalez and coworkers reported that in sheep orally authors found that the mAb 6H4 (which binds both bovine infected with scrapie both the PrP genotype of the recipient and ovine PrP) in the second phenotype, revealed diglycosy- and that of the infecting source may contribute to the lated and monoglycosylated bands of molecular masses resulting neuropathological phenotype [32]. The route of 2631 Bolea et al., Journal of General Virology 2017;98:2628–2634 Fig. 3. Western blot analysis. (a) BSE and bovine scrapie were detected by discriminatory Western blot using the 6H4 but not the P4 antibody. Western blot using Sha31 antibody before (b) and after (c) PMCA assay in scrapie-infected heifer and control (d): brain (1), sciatic nerve (2), brachial nerve (3), mesenteric lymph node (4), extraorbital muscle (5), ileum (6) and spleen (7). 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The Microbiology Society is a not-for-profit organization. 2. We offer fast and rigorous peer review – average time to first decision is 4–6 weeks. 3. Our journals have a global readership with subscriptions held in research institutions around the world. 4. 80% of our authors rate our submission process as ‘excellent’ or ‘very good’. 5. Your article will be published on an interactive journal platform with advanced metrics. Find out more and submit your article at microbiologyresearch.org. 2634