We isolated human mesenchymal stem cells (MSC) from bone marrow of young (16–18 years) and old (67–75 years) donors and analyzed their capacity to differentiate and promote neurite outgrowth from dorsal root ganglia (DRG) neurons. Treatment of MSC with growth factors  induced protein expression of the glial cell marker S100 in cultures from young but not old donors. MSC expressed various neurotrophic factor mRNA transcripts. Growth factor treatment enhanced the levels of BDNF and VEGF transcripts with corresponding increases in protein release in both donor cell groups. MSC in co-culture with DRG neurons significantly enhanced total neurite length which, in the case of young but not old donors, was further potentiated by treatment of the MSC with the growth factors. Stem cells from young donors maintained their proliferation rate over a time course of 9 weeks whereas those from the old donors showed increased population doubling times. MSC from young donors, differentiated with growth factors after long-term culture, maintained their ability to enhance neurite outgrowth of DRG. Therefore, MSC isolated from young donors are likely to be a favourable cell source for nerve repair.Clinical efficacy of stem cells for nerve repair is likely to be influenced by issues including donor age and  Peripheral nerve injuries often result in lifelong reduced motor functions and minimal sensory recovery. Typically a gap nerve injury is treated with an autologous nerve graft but these are far from ideal since they involve sacrifice of another nerve with subsequent loss of sensation at the donor site in vitroSchwann cells are the key cells in the peripheral nervous system providing the nervous tissue with cell adhesion molecules and various growth factors following injury in vitro.From a clinical point of view it is of interest to identify the best source of cells in order to effectively provide enough cells to treat nerve injuries. Patient donor age influences the ability to obtain sufficient quantities of cells 2 tissue culture flasks  and incubated at 37°C, 5% (v/v) CO2. After 12–24 h in culture, the supernatant containing non-adherent cells was removed and discarded and fresh medium added. The cells attached to the culture flask were cultured  for 2–3 weeks with medium changes every 48 h. When the cultures had reached 80% confluence, the cells were enzymatically detached from the flask using 0.25% trypsin/EDTA solution (Invitrogen Life Technologies) and re-seeded in new culture flasks at a density of 5×103 cells per cm2.Samples of human bone marrow were obtained from the iliac crests of six healthy donors during reconstructive surgery. The purpose, nature, and potential risks of the study were explained to the patients and informed written consent was obtained from the adult patients or from next of kin in the case of the minors. The donors were designated as follows: young donors  and old donors . Procedures were approved by the Local Ethical Committee for Clinical Research in Umeå University (no 03–425). Modification of a previously described protocol 3 cells per cm2 and cultured for one week (passage 3). The cells were counted and reseeded at the same density and cultured for another week. This was repeated until passage 11. From the cell counts, population doubling (PD) times were calculated.At passage 2 the undifferentiated cells were seeded at a density of 5×10/ml all-trans-retinoic acid (Sigma-Aldrich) followed by growth medium supplemented with 5.7 µg/ml forskolin , 10 ng/ml basic fibroblast growth factor , 5 ng/ml platelet derived growth factor–AA  and 126 ng/ml glial growth factor-2 . The cells were maintained in this supplemented medium for two weeks with medium changes every 72 h to establish differentiated cultures The differentiation process was initiated in sub-confluent MSC at the second and eleventh passage by treatment with medium supplemented with 1 mM β-mercaptoethanol  for 24 h. Cells were then incubated for 72 h with growth medium containing 35 ng2). In another series of experiments, the SH-SY5Y human neuronal cell line  was seeded in α-MEM medium at a density of 5000 cells onto coverslips and allowed to settle for 24 h. Cultures of undifferentiated (uMSC) and differentiated (dMSC) stem cells from the three donors in each group were seeded onto 1.0 µm pore size polyethylene terephthalate membrane cell culture inserts  at a density of 75,000 cells/ml and incubated for 24 h . The inserts were then checked for cell adherence, then placed into wells containing DRG neurons or SH-SY5Y cells to establish the co-culture; these were allowed to incubate together for additional 24 h . Additional controls with the cell-free inserts containing stem cell medium only were also analyzed. Other inserts contained either 10 µg/ml anti-BDNF antibody (Millipore) or 1 µg/ml anti-VEGF antibody . In another series of experiments the conditioned medium was removed from inserts containing the stem cells (plated as above) and transferred to the DRG neuron cultures. After 24 h in co-culture with stem cells in inserts or in the presence of conditioned medium, the neurons on the cover slips were fixed in 4% (w/v) paraformaldehyde  and immunostained for anti-βIII tubulin  with secondary antibody Alexa Fluor 488 goat anti-mouse IgG . The cover slips were mounted with Prolong™ for Fluorescence (Invitrogen) and examined using a fluorescence microscope. The images were captured for quantification at ×10 magnification using an Evolution QEi monochrome digital camera; image analysis was performed using Neurolucida . All neurites were traced to generate total neurite outgrowth data (in µ m). Three independent co-culture experiments were carried out for neurite outgrowth assessment.Dorsal root ganglia (DRG) were harvested from the spinal cords of adult female Sprague-Dawley rats using a previously described protocol The RNeasy™ mini kit  was used according to the manufacturer’s protocol for the isolation of total RNA from the cell pellets of the undifferentiated MSC (uMSC) and differentiated MSC (dMSC)  and then 1 ng RNA was incorporated into the One-Step RT-PCR kit (Qiagen) per reaction mix. Primers were manufactured by Sigma, UK . A thermuMSC and dMSC were seeded at an identical density to those used for neurite analysis; 15000 cells per 200 µl media and maintained for 48 h. The supernatant from the cells was then collected and analysed by ELISA using the ChemiKine™ Brain Derived Neurotrophic Factor (BDNF) sandwich ELISA kits  or Vascular Endothelial Growth Factor (VEGF) sandwich ELISA kit  according to the manufacturer’s protocol. All samples were analysed in triplicate and the absorbance was measured at 450 nm .Statistical analysis was conducted using GraphPad Prism software . The data for neurite outgrowth quantification are expressed as mean ± SEM and analysed by t-test or one-way ANOVA with Newman Kreuls post hoc test where appropriate. Statistical significance was determined as *P<0.05, **P<0.01, ***P<0.001.Stem cells from young or old donors were treated with a mixture of glia growth factors in order to differentiate them towards a glial cell phenotype as previously described by us Next we established co-cultures of early passage human MSC, either undifferentiated (uMSC) or differentiated (dMSC), with rat dorsal rat ganglia (DRG) neurons. The neurite outgrowth evoked by the stem cells was detected by immunocytochemical staining for βIII tubulin . CompareTo investigate the possible growth factors responsible for the enhanced neurite outgrowth of DRG neurons we measured the trophic factor gene transcripts in MSC using semi-qualitative RT-PCR with GAPDH as a house-keeping control gene . UndiffeSince MSC have been shown to exert immune enhancing functions and the DRG neuron co-culture model was xenogeic, we repeated the studies with a human neuronal cell line to ensure our results were not due to a xenogenic effect. Co-culture of differentiated MSC with SH-SY5Y neurons resulted in enhanced neurite outgrowth from the human cells , replicaOur undifferentiated cell cultures showed a classical fibroblast-like morphology at early passage (T2) from both young and old donors . After tCo-cultures between late passage MSC and DRG neurons were also established . The treWe have previously shown that human MSC can be differentiated to express a number of glial cell markers such as S100 and GFAP and that these cells promote neurite outgrowth MSC from young and old donors at early passage (T3) significantly enhanced neurite outgrowth of DRG neurons, most likely as a result of secretion of soluble neurotrophic factors. We found mRNA transcripts for NGF, GDNF, NT3, BDNF and VEGF expressed in undifferentiated MSC. In a previous study, a screen of a human MSC cDNA library also revealed transcripts for BDNF and NGF but not NT-3 The MSC effects were not specific to rat DRG neurons because when co-cultured with human SH-SY5Y neurons, old donor differentiated MSC evoked significantly less neurite outgrowth than from young donor differentiated MSC. We conclude that MSC from both young and old donors express significant levels of neurotrophic factors but that some yet to be identified molecules are not produced (or at significantly lower levels) during the differentiation process in old donor MSC. Alternatively some growth inhibitory molecules produced by old donor cells block the effects of the neurotrophic factors. One way to further determine the differences between young and old donors could be to perform a mixed competitive assay, using different ratios of young:old donor cells  in co-culture with the neurons. In this way, the relative ability of each donor cell population to enhance neurite outgrowth would be determined, enabling a better understanding of the individual neurotrophic phenotypes of the cells. With recent advances in proteomic analysis, in our future studies it might also be possible to characterize new human proteins which modulate neurite outgrowth. This would enable greater understanding of the molecular pathways mediating interactions with neurons and identify new therapeutic targets for treatment of neurological diseases. Recent studies have characterized the cell surface proteome of human MSC which will allow better characterisation of sub-populations of stem cells derived from different tissues MSC from old donors show reduced levels of the proliferation marker ki67 suggestive of a reduced growth rate In summary our results indicate that MSC from young and old donors express significant levels of neurotrophic factors. Treatment of MSC with glial growth factors can boost the levels of BDNF and VEGF but enhanced DRG neurite outgrowth is only achieved with young donor MSC suggesting that other neurotrophic factors play a role. Time in culture does not appear to influence this process indicating that MSC from young donors could be successfully expanded and differentiated in suitable numbers for transplantation and treatment of nerve injuries.Table S1Primer sequences for RT-PCR with annealing temperatures (°C) and cycle number used.(DOC)Click here for additional data file.


The identification in the UK of 4 v-CJD infected patients thought to be due to the use of transfused Red Blood Cell units prepared from blood of donors incubating v-CJD raised major concerns in transfusion medicine. The demonstration of leucocyte associated infectivity using various animal models of TSE infection led to the implementation of systematic leuco-depletion (LD) of Red Blood cells concentrates (RBCs) in a number of countries. In the same models, plasma also demonstrated a significant level of infectivity which raised questions on the impact of LD on the v-CJD transmission risk. The recent development of filters combining LD and the capture of non-leucocyte associated prion infectivity meant a comparison of the benefits of LD alone versus LD/prion-reduction filters (LD/PR) on blood-borne TSE transmission could be made. Due to the similarity of blood/plasma volumes to human transfusion medicine an experimental TSE sheep model was used to characterize the abilities of whole blood, RBCs, plasma and buffy-coat to transmit the disease through the transfusion route. The impact of a standard RBCs LD filter and of two different RBCs LD/PR prototype filters on the disease transmission was then measured. Homologous recipients transfused with whole-blood, buffy-coat and RBCs developed the disease with 100% efficiency. Conversely, plasma, when intravenously administered resulted in an inconstant infection of the recipients and no disease transmission was observed in sheep that received cryo-precipitated fraction or supernatant obtained from infectious plasma. Despite their high efficacy, LD and LD/PR filtration of the Red Blood Cells concentrate did not provide absolute protection from infection. These results support the view that leuco-depletion strongly mitigates the v-CJD blood borne transmission risk and provide information about the relative benefits of prion reduction filters. Transmissible spongiform encephalopathies (TSE), or prion diseases, are fatal neurodegenerative disorders naturally occurring in sheep (scrapie), cattle , and humans (Creutzfeldt-Jakob disease - CJD).In humans, CJD is a rare disease (about 1 case per million and per year) that usually occurs as either a sporadic (s-CJD), familial or genetic form. Despite their relative rareness, there have been hundreds of iatrogenic CJD cases, following corneal and dura-mater grafts, the use of extractive pituitary hormones or the contamination of deep intracranial electrodes that have been reported over the last 50 years In 1996, a new form of CJD, named variant CJD (v-CJD) was identified in humans. Variant CJD was demonstrated to be due to the same agent that causes BSE in cattle and its emergence in humans was established to be the consequence of a dietary exposure to BSE contaminated products The presence of the v-CJD agent in lymphoid tissues combined with the detection of leucocyte associated infectivity in TSE rodent models Since then, four probable v-CJD transmissions through transfusion were reported in the UK, all the patients having received Red Blood Cell units prepared from donors who developed symptoms of v-CJD 17 months to 3,5 years after donation Before any v-CJD blood borne transmission had been recognized, some countries implemented measures intended to prevent its potential transmission by blood products. Amongst them, France (1998), Ireland, UK, Portugal (1999) and Canada (2000) introduced systematic leuco-depletion (LD) for labile blood products and in 2001 also for plasma in France.10 reduction of infectivity in RBCs spiked with infectious brain homogenate and a reduction to the limit of detection (>1 log10) of the blood endogenous infectivity in rodent TSE models i.e. the administration of large numbers of living cells and volume intravenously to the recipient However, the possible benefits of leuco-depleted red blood cell concentrates (LD-RBCs) use with regards to the v-CJD transmission risk remains unclear. Data obtained in rodent models indicated that only 40% of the infectivity seemed to be associated with the white blood cells (WBC), while plasma would contain 60% Variant CJD’s pathogenesis displays features very similar to natural classical scapie in sheep. In both cases, infection occurs following oral exposure and an early preclinical accumulation of the infectious agent in lymphoid tissues is described In this study, using an experimental scrapie in sheep infection model All animal experiments have been performed in compliance with our institutional and French national guidelines, in accordance with the European Community Council Directive 86/609/EEC. Experimental protocol was approved by the INRA Toulouse/ENVT ethics committee.The VRQ/VRQ sheep were produced in a biosecure unit from a flock that was originally sourced from New Zealand by AHVLA UK. This DEFRA funded TSE free flock’ is a unique source of VRQ/VRQ animals that can be considered safe for classical scrapie The animals included in our experiments were imported in France and housed in a dedicated scrapie free farm situated at 30 km from the experimental facilities before their use in experiments. Dedicated staff, having no contact with the infectious animals, bred them.6.8 ID50/g) Sc detection.The classical scrapie isolate was derived from experimentally VRQ/VRQ affected sheep (PG127 isolate). 6–10 months old TSE-free sheep (AHVLA- Weybridge- UK) were orally challenged with 2 g equivalent of brain material (1% brain homogenate in glucose). This isolate has been previously end-point titrated by intracerebral route in tg338 mice  and Pall Medical Leukotrap Affinity Plus . Both devices complied with the standard requirements for RBCs processing in transfusion medicine. The exact composition/structure of these devices is patented and then protected by commercial confidence. Both devices were demonstrated by manufacturers to achieve a greater than 3 logThe blood collection from the 15 infected sheep was performed in the UMR INRA/ENVT 1225 experimental facilities using Fenwal  disposables R7542 top and bottom (TAB) collection bags. Whole blood units were shipped overnight to the EFS Nord de France for the preparation of the blood components according to the French Transfusion Service processes.Blood from 3 sheep were pooled to obtain a volume higher than 1800 mL : the 3 collected units were mixed in a transfer bag  and then split into 5 Fenwall R7542 TAB collection bags : 1 unit of 200 mL and 4 units of 400 mL .The 200 mL whole blood unit represents Inoculum 1.The 400 ml units (x4) were centrifuged at 3640 rpm for 12 minutes (acceleration and still) at 20°C in a Jouan LR5-22 centrifuge in order to separate the plasma, the buffy-coat and the red cells concentrate (RBCs).The first unit of 400 ml was centrifuged and then separated in plasma, buffy-coat and RBCs (Inoculums 2–3–4).The second unit of 400 ml was centrifuged, to obtain plasma and RBC. RBCs and plasma were then leuco-depleted using the Fenwal R7542 disposal : Sepacell® Pure RC filter from ASAHI for RBCs and Fenwal plasma filter for plasma (Inoculums 5–6).The third and fourth units of 400 ml were also centrifuged and separated in plasma, buffy-coat and RBCs but only the RBCs were leuco-depleted, using LD/PR filter from either manufacturer 1 or manufacturer 2 (Inoculums 7–8).Cryo-precipitation is the first step of the standard procedure applied to extract coagulation factors from plasma. Non leuco-reduced plasma corresponding to the third and fourth units (Inoculum 7 and 8 preparation lines in Recipient sheep were anesthetized by IV administration of Ketamine/Diazepam (respectively 2 mg and 0.1 mg per Kg) and a venal catheter (16 G) was installed (jugular vein). Whole blood (200 mL), plasma (150–200 mL) and RBCs (about 200 mL) were transfused over a 30–40 min period. Buffy-coat samples were gently re-suspended in a 200 mL sterile pouch of isotonic (5%) glucose before administration over a 20–30 min period. Transfusion of the whole blood was performed within the 3 hours following blood collection. In the experiment aiming at the assessment of LD and LD/PR filter efficacy, transfusion of the recipients with the different components was performed within the 5 days following the blood collection.In the plasma titration experiment (by transfusion route), adequate whole plasma volumes were transferred into a new pouch or gently re-suspended in a sterile 5% glucose solution (QSP 200 mL), before IV administration in recipients. Similarly, cryoprecipitate and cryprecipitation supernatants were re-suspended in 5% glucose solution (200 mL) and immediately administered intravenously by gravity over a 30 min period.Sheep were housed in a category 2 animal facility. Donor sheep and recipients were kept separated. Contact controls (n = 5) were housed in the same pen as transfusion recipients sheep. These controls were housed throughout the experiment and none displayed any TSE symptoms. Animals were daily monitored. When displaying symptoms that could impair their capacity to feed, the animals were euthanized by an intravenous injection of T61 (Intervet) followed by exsanguination. Transfused animals that did not display clinical signs after 400–450 days were also euthanized. At the end of the complete experiment, contact controls were also euthanized.Sc Immunohistochemistry investigation and the other was snap frozen at −80°C for Western Blot abnormal PrP detection.Each animal was systematically necropsied and a panel of tissues was collected . The samples were divided in two parts, the first one was formalin fixed for PrPSc extraction. The obtained pellet was denaturated in Laemmli’s buffer (15 µL) before being loaded neat or diluted on a 12% acrylamide gel, before electrophoresis and blotting. Immunodetection was performed using Sha31 monoclonal antibody which recognizes the 145–152 sequence of PrP (YEDRYYRE), conjugated to horseradish peroxidase . Peroxidase activity was revealed using ECL substrate (Pierce) A Western blot kit  was used following the manufacturer’s recommendations. For each sample 250 µL of 10% brain homogenate were submitted to PrPSc IHC detection was performed using 8G8 antibody raised against human recombinant PrP protein and specifically recognising the 95–108 amino acid sequence (SQWNKP) of the PrP protein.This method was performed as previously described Post mortem analysis confirmed the accumulation of abnormal PrP in both their lymphoid tissues and central nervous system (CNS) . At this time point, the animals showed no clinical signs but subsequently developed them and were killed after a mean incubation period of 231+/−15 dpi. em (CNS) . PreviouAfter collection, whole blood from three individuals was pooled in order to obtain pools of 1800 mL . These pThe three others whole blood units (each containing 400 mL) were prepared in plasma and RBCs. One plasma and RBCs units were leuco-depleted using LD filters routinely used to prepare blood components (inoculum 5 and 6). The two last RBCs were processed using LD/PR filters that were developed by the two different manufacturers (inoculum 7 and 8). All steps were carried out using standard equipment and procedures employed by transfusion service and the two LD/PR filters were used under the supervision of the manufacturers’ staff.6 leukocytes per RBCs unit, which complies with the current requirements in transfusion medicine.At different points in the processes, samples were analyzed in order to characterize the efficacy of LD . The preSigns of clinical TSE infection were observed in all VRQ/VRQ sheep that received whole blood, Buffy-coat or RBCs . No clinOnly 3 out the 5 fresh plasma  recipient sheep developed disease , and oneIn parallel, an end-point titration experiment was carried out in TSE free sheep using four out of these five non leuco-depleted plasma: 200 mL, 20 mL, 2 mL of plasma  used in the titration experiment were submitted to cryo-precipitation. The supernatant and precipitate obtained from each of the plasma were re-suspended in 5% glucose solution (200 mL) and intravenously administrated to VRQ/VRQ recipient sheep . Althougpost mortem none of the 5 VRQ/VRQ contact controls, who were housed with transfusion recipients, displayed abnormal PrP deposition in their lymphoid tissues or CNS, demonstrating that lateral or environmental cross contamination is unlikely as an explanation for disease in any of the recipients.On On the basis of the relative infectious titre in plasma, as assessed in rodent models, LD has been considered as a potentially beneficial at reducing the risk of v-CJD transmission associated with RBCs transfusion but this risk redution was insufficient measure The results obtained in this experiment using the PG127 sheep infection model indicated that the transfusion of standard RBCs, prepared using 400 mL of blood, transmitted the disease with 100% efficiency compared to only one out of the five recipients of the LD-RBCs. This efficacy of disease transmission by the transfusion of LD-RBCs (that corresponded to 400 mL of whole blood) was lower than the one reported in the same model using 200 µL of whole blood To date, 4 v-CJD cases have been identified in patients who received non leuco-depleted RBCs In the UK (n = 24/176) and France (n = 3/25) patients that had developed v-CJD had been blood donors. Plasma from these patients entered in the composition of several hundreds of batches of plasma products and in France the number of patients that were intravenously treated with those products exceeded 50 000 Our results indicate that, in the PG127 scrapie model, plasma displayed limited capacity to transmit the disease by transfusion. This observation concurs with the results recently reported in both BSE infected sheep and Chronic Wasting Diseases infected cervids The lack of disease transmission observed in sheep that received the plasma cryoprecipitate or the cryo-precipitation supernatant is surprising as the results obtained in rodents TSE models demonstrated that during cryo-precipitation process plasma infectivity split between cryoprecipitate and supernatant without significant reduction of the global infectivity amount 10Based on the assumption that leuco-depletion would be insufficient to prevent v-CJD transmission by the transfusion of RBCs, several manufacturers developed blood prion-reduction filters. These filters were designed to capture TSE infectivity that would not be associated with leucocytes. The low infectious titre in whole blood  was a major limitation to assessing the efficacy of these filters In this experiment, no clinical cases were observed in recipients transfused with LD/PR filtered RBCs but one apparently healthy recipient was found infected after culling, suggesting its exposure to low titre infectious material. Therefore, the comparison of the transmission rates obtained with the LD filter alone and the LR/LP filters, does not appear to support the view that prion-reduction filters had a significant additional beneficial effect on the disease transmission. However considering the limits of our experiment (only 5 recipients were transfused for each filtration device) and the fact that no infection was observed in animals that were transfused with RBC filtered with one of the two tested of LD/PR filter, additional experiments would be necessary to statistically assess the potential benefits of this device. If such experiment should be carried out, it would be critically important to determine whether the additional beneficial effect of the LD/PR filter could be due to its ability to achieve higher level of leuco-reduction  than the one obtained by standard LD filter.Currently, the distribution and level of infectivity in blood components of v-CJD affected patients remains undocumented. In that context, even if appropriate caution should be taken when interpreting the data, TSE animal models represent the main source of information for assessing v-CJD blood borne transmission risk. Under the hypothesis that the blood transfusion model in sheep represents a pertinent model for v-CJD blood borne TSE transmission, the results we obtained indicate that plasma is significantly less efficient than RBCs and buffy-coat to transmit the disease by the transfusion route. Our results also strongly support the view that RBCs and plasma leuco-reduction has a major impact on the mitigation of the v-CJD transmission risk in transfusion medicine and provide information to help with the assessment of the relative benefits of prion reduction filters.


A new variant of Creutzfeldt Jacob Disease (vCJD) was identified in humans and linked to the consumption of Bovine Spongiform Encephalopathy (BSE)-infected meat products. Recycling of ruminant tissue in meat and bone meal (MBM) has been proposed as origin of the BSE epidemic. During this epidemic, sheep and goats have been exposed to BSE-contaminated MBM. It is well known that sheep can be experimentally infected with BSE and two field BSE-like cases have been reported in goats. In this work we evaluated the human susceptibility to small ruminants-passaged BSE prions by inoculating two different transgenic mouse lines expressing the methionine (Met) allele of human PrP at codon 129 (tg650 and tg340) with several sheep and goat BSE isolates and compared their transmission characteristics with those of cattle BSE. While the molecular and neuropathological transmission features were undistinguishable and similar to those obtained after transmission of vCJD in both transgenic mouse lines, sheep and goat BSE isolates showed higher transmission efficiency on serial passaging compared to cattle BSE. We found that this higher transmission efficiency was strongly influenced by the ovine PrP sequence, rather than by other host species-specific factors. Although extrapolation of results from prion transmission studies by using transgenic mice has to be done very carefully, especially when human susceptibility to prions is analyzed, our results clearly indicate that Met129 homozygous individuals might be susceptible to a sheep or goat BSE agent at a higher degree than to cattle BSE, and that these agents might transmit with molecular and neuropathological properties indistinguishable from those of vCJD. Our results suggest that the possibility of a small ruminant BSE prion as vCJD causal agent could not be ruled out, and that the risk for humans of a potential goat and/or sheep BSE agent should not be underestimated. Prion diseases, also referred as transmissible spongiform encephalopathies, are fatal neurodegenerative diseases caused by proteinaceous infectious particles denominated “prions.” Prion diseases acquired their first real public relevance with the outbreak of bovine spongiform encephalopathy (BSE) (“mad cow disease”) in the United Kingdom in the 80s and its link with the appearance of a new, variant form of Creutzfeldt-Jakob disease in humans. Recycling of ruminant tissues in meat and bone meal has been proposed as origin of the BSE epidemic. During this episode, sheep and goats have also been exposed to BSE-contaminated meal, so transmission to this species may have occurred. We analyzed the human susceptibility to sheep and goat passaged-BSE prions by using transgenic mice expressing human prion protein (PrP). When different sheep and goat BSE isolates were inoculated in these transgenic mice, higher susceptibility than that observed for cattle BSE was detected and the disease manifestation was similar to that observed in mice inoculated with the new variant of Creutzfeldt-Jakob disease. Our findings suggest that humans are at least equally, and might be even more, susceptible to a sheep or goat BSE agent compared to a cattle BSE one. Sc) of the cellular prion protein (PrPC) Sc is widely distributed in lymphoid tissues of experimentally BSE-infected sheep Transmissible Spongiform Encephalopathies (TSEs) are fatal neurodegenerative diseases which include Scrapie in sheep and goats, Bovine Spongiform Encephalopathy (BSE) and Creutzfeldt-Jakob disease (CJD) in humans. Prions, the causal agents of these diseases are thought to be infectious protein particles essentially composed of a misfolded isoform  without clear clinical signs. The remaining inoculated mice failed to develop a clinical disease or to accumulate detectable levels of PrPres in the brain up to ∼700 days after inoculation. On second passage performed with brain homogenate from a PrPres-negative mouse (succumbed at 576 dpi) from the first passage (BSE2 isolate), 3 out of 4 inoculated tg340 mice tested positive for brain PrPres by western blot with a survival time of 572±37 d.p.i. It is important to note that all the cattle BSE isolates tested in this study were transmitted as efficiently as vCJD isolates or other BSE-related sources to bovine PrP transgenic mice , respectively. These features were stable upon subpassaging, suggesting an absence of transmission barrier for this agent  and 3. Inic mice  and 3, tres detection in the brain) with almost all the sheep and goat BSE isolates used  contained higher PrPres levels in their brains than the sheep and goat BSE isolates  and prominent diglycosylated species was consistently observed in the challenged, PrPres-positive mice. This signature clearly differed from that observed after inoculation of mice with sporadic CJD (89WGQGG93 according to the human PrP sequence) is known to be poorly protected from proteinase K digestion 2) in tg340 mice, one of the three positive mice presented a brain PrPres profile clearly distinct from PrPvCJD, that was comparable to that of typeI sCJD-inoculated tg340 mice with predominantly monoglycosylated and higher size fragments (∼21 kDa for the aglycosyl band) and preserved detection by 12B2 antibody  and on 3rd passage in tg650. As illustrated in The regional distribution of PrPAt microscopic level, abundant amyloid-like plaques were present , as sugg2) was passaged into bovine (tg110) and ovine PrP transgenic mice  2/TgBov and Ca-BSE2/TgOv, 2/TgBov isolate did not induce a clinical disease nor PrPres accumulation in tg340 mice while an intermediate passage on the ovine PrPARQ sequence (Ca-BSE2/TgOv) restored the disease susceptibility, with survival times, biochemical and neuropathological features similar to those obtained with experimental sheep BSE isolates  produced the shortest disease in bovine PrP transgenic mice (0/TgOv isolate (which maintains its PrP ovine sequence) showed a full transmission rate in human transgenic mice with similar survival times as those of the original Sh-BSE0 isolate  in bovine PrP transgenic mice  in two different laboratories. In general, the transmission results obtained in both human-PrP transgenic mouse lines were very comparable. Some shortening in survival times was observed in tg650 mice (compared to the tg340 mice line), which was probably due to higher PrP expression levels in this line. Worryingly, our results support the view that an intermediate passage of BSE agent in small ruminants accelerates the appearance of a vCJD-like disease in human PrP mice or markedly increases its transmission efficiency. Because the apparent phenotype of cattle and sheep/goat BSE prions is conserved, these data also unravel an important role of PrPThe transmission efficiency of cattle BSE isolates in both human-PrP transgenic mouse models was apparently low. With all BSE isolates, whose high infectivity has been demonstrated in bovine-PrP transgenic mice  and 3, vSc type of cattle and sheep/goat BSE agents appeared indistinguishable from the vCJD agent propagated in these mice, as previously demonstrated in bovine transgenic mice res appeared similar in human-PrP transgenic mice, other assays are currently performed to further compare the biochemical or biophysical properties of the respective proteins are ongoing.Remarkably, a different picture emerged when the sheep and goat BSE isolates were inoculated to human PrP transgenic mouse models. Attack rates approaching 100% were observed from the primary passage onwards and mean incubation times were more consistent with those measured after transmission of vCJD. On further passaging, the neuropathological phenotype and PrPres content of these isolates. In addition, the data from inoculation to BoPrP-Tg reporter mice suggest that cattle BSE and sheep and goat-BSE isolates could have similar transmission efficiency , suggesting no infection, in accordance with the lack of epidemiological evidence linking scrapie with human TSE. Moreover, the low transmission efficiency observed for the cattle BSE agent is not exclusively linked to the bovine PrP sequence since other uncommon BSE strains (BSE-L) are efficiently transmitted to human-PrP mice C prefers a BSE PrPSc with conformational characteristics templated by the ovine sequence, to a bovine BSE PrPSc. Because a similar increased transmission efficiency of sheep/goat BSE has been reported in wild type mice Sc sequence may not be limited to human PrPC. One explanation might be an alteration in the quaternary structure (after passage into sheep/goat) generating PrPSc polymers less degraded or more rapidly/easily amplified favouring or enhancing the initial conversion. This question is currently being addressed by sedimentation velocity The PrP primary sequence influence seems to depend strongly on the strain involved, since no PrPAlthough extrapolation of results from prion transmission studies by using transgenic mice has to be done very carefully, especially when human susceptibility to prions is analyzed, our results clearly indicate that Met129 homozygous individuals might be susceptible to a sheep or goat BSE agent at a higher degree than to cattle BSE, and that these agents might transmit with molecular and neuropathological properties indistinguishable from those of vCJD. Although no vCJD cases have been described in Val129 homozygous individuals so far it is relevant to analyze if similar results will be observed in this genotype. This issue is currently being addressed in transmission experiments using transgenic mice expressing Val129 human PrP.res detected in brains of our sheep and goat BSE-inoculated mice seem to be indistinguishable from that observed in vCJD. Considering the similarity in clinical manifestation of BSE- and scrapie-affected sheep Sc have been detected in many peripheral organs, small ruminant-passaged BSE prions might be a more widespread source of BSE infectivity compared to cattle Taken all together, our results suggest that the possibility of a small ruminant BSE prion as vCJD causal agent could not be ruled out, which has important implications on public and animal health policies. On one hand, although the exact magnitude and characteristic of the vCJD epidemic is still unclear, its link with cattle BSE is supported by strong epidemiological ground and several experimental data. On the other hand, the molecular typing performed in our studies, indicates that the biochemical characteristics of the PrPAnimal experiments were carried out in strict accordance with the recommendations in the guidelines of the Code for Methods and Welfare Considerations in Behavioural Research with Animals (Directive 86/609EC) and all efforts were made to minimize suffering. Experiments were approved by the Committee on the Ethics of Animal Experiments of the author's institutions (INRA and INIA); Permit Number: RTA06-091 and CT05-036353.The isolates used in this study are described in The tg650 transgenic mouse line over expresses human PrP M129 at a 6-fold level on a mouse PrP null background res) by immunohistochemistry (IHQ) or histoblotting and the other was frozen at −20°C to determine presence of PrPres by Western blot (WB). In all cases, survival time and attack rate were calculated for each isolate. Survival time was expressed as the mean of the survival days post inoculation (d.p.i.) of all the mice scored positive for PrPres, with its correspondent standard error. Attack rate was determined as the proportion of mice scored positive for PrPres from all the mice inoculated. When all mice were scored negative for PrPres, the survival time range was shown. Brain homogenates from PrPres positive mice, when available, were used for further passaging. When all mice were scored negative for PrPres on primary passage, PrPres-negative brain homogenates were used for second passage.All inocula were prepared from brain tissues as 10% (w/v) homogenates. Individually identified 6–10 week-old mice were anesthetized and inoculated with 2 mg of brain homogenate in the right parietal lobe using a 25-gauge disposable hypodermic needle. Mice were observed daily and the neurological status was assessed weekly. When progression of a TSE disease was evident or at the end of lifespan, animals were euthanized because of ethical reasons. Once euthanized, necropsy was performed and brain was taken. A part of the brain was fixed by immersion in 10% formol to quantify spongiform degeneration by histopathology and PK resistant PrP accumulation . 10–50 µl of a 10% (w/v) brain homogenate were diluted in a 10% (w/v) negative sheep brain homogenate, to obtain a 200 µl final volume. Homogenates were incubated for 10 min at 37°C with 200 µl of a 2% proteinase K solution (in buffer A). PrPres was recovered as a pellet after addition of 200 µl of buffer B and a centrifugation at 15,000× g for 7 min at 20°C. Supernatants were discarded and pellets were dried inverted over absorbent paper for 5 min. Pellets were solubilised in Laemmli buffer and samples were incubated for 5 min at room temperature, solubilised, and heated at 100°C for 5 min. Samples were centrifuged at 20,000× g for 15 min at 20°C and supernatants were recovered and loaded on a 12% Bis-Tris Gel . Proteins were electrophoretically transferred onto PVDF or nitrocellulose membranes (Millipore). Membranes were blocked O/N with 2% BSA blocking buffer. For immunoblotting, membranes were incubated with either Sha 31 175±20 mg of frozen brain tissue were homogenized in 5% glucose in distilled water in grinding tubes (Bio-Rad) adjusted to 10% (w/v) using a TeSeE™ Precess 48™ homogenizer (Bio-Rad) following manufacturer instructions. Presence of PrPAll procedures involving mice brains were performed as previously described nd and 3rd passage, using the 3F4 anti-PrP antibody as previously described Brains were rapidly removed from euthanised mice and frozen on dry ice. Thick 8–10 µm cryostat sections were cut, transferred onto Superfrost slides and kept at −20°C until use. Histoblot analyses were performed on 3 brains per infection at 2XhoI restriction enzyme site adjacent to the translation start and stop sites of the human PrP ORF . The PCR fragments obtained were sub cloned into a pGEM-T Easy Vector System (Promega) following manufacturer instructions, and inserts were sequenced to confirm no differences in the inferred amino acid sequence with respect to previously sequenced human PrP genes (GenBank accession number NM_183079) and to confirm the presence of the consequent codon 129 nucleotide variant (MetATG). The human PrP ORF was excised from the cloning vector using the restriction enzyme XhoI and inserted into the expression vector MoPrP.Xho XhoI restriction sites but could be distinguished from the wild type murine PrP gene because of the absence of intron 2. The vector was also digested with XhoI to excise the murine PrP ORF and the correspondent human PrP ORF were inserted by ligation, obtaining the plasmid pMo-huPrP129M.Xho.Tg340 mouse line expressing about 4-fold level of human PrP M129 on a mouse PrP null background has been generated following a similar procedure previously describe for the generation of other transgenic mouse line expressing different species PrP Not I leading to DNA fragments of approximately 12 Kb. Finally, the DNAs were purified and dissolved in TE at a final concentration of 2 to 6 µg/ml and microinjected into pronuclear stage ova collected from super-ovulated B6CBAf1 females mated with 129/Ola males carrying a null mutation in endogenous PrP The human transgene was excised from the plasmid vector using the restriction endonuclease 5′- TAGATGTCAAGGACCTTCAGCC- 3′ and 5′- GTTCCACTGATTATGGGTACC -3′.DNA from founders' tails biopsies was extracted using a Extract-N-Amp Tissue PCR kit (Sigma-Aldrich) following manufacturer instructions. The presence of the human transgene in these founders was identified by PCR amplification using specific primers for the mouse PrP exon 2 and human PrP open reading frame. The absence of the murine PrP ORF in the transgenic mice generated was confirmed by PCR amplification using the primers: C (huPrP) and murine PrPC (muPrP) heterozygous transgenic mice (PrP mu+/− hu+/−) were obtained. The expression of human PrPC in brain of these mouse lines was analyzed and compared with PrPC content in human brain homogenate by western blot using mAb 3F4 which recognizes the 109MKHM112 epitope (numbered according to the human PrP sequence). Human PrPC was detected in 100% of the tested lines (data not shown). From the initial 8 different mouse lines heterozygous for both murine and human PrP genes (PrP mu+/− hu+/−), the mouse line named as tg340 was selected for further experiments on the basis of the level of PrPC expression.Eight different lines (founders) of human PrP−/− (Prnp−/−) to obtain a null murine PrP background (PrP mu−/− hu+/−). Interbreeding within these animals was performed to obtain homozygosis for the human PrP transgen within a murine PrP background (PrP mu−/− hu+/+). The absence of murine PrP gene was determined by PCR using specific primers. Human PrPC expression levels, determined more accurately in brain from homozygous tg340 animals was about 4-fold higher than PrPC levels in human brain homogenates as determined by dilution experiments in western blot (Homozygous Tg340 mouse line was established backcrossing these animals with homozygous null animals MuPrPern blot .Prnp gene used in this paper is NM_183079.The GenBank accession number for the human


Sc distribution patterns in a well characterized transgenic mouse line (Tg338) expressing the ovine VRQ prion allele, following inoculation with brain from scrapie infected goats.The United States control program for classical ovine scrapie is based in part on the finding that infection is typically spread through exposure to shed placentas from infected ewes. Transmission from goats to sheep is less well described. A suitable rodent model for examining the effect of caprine scrapie isolates in the ovine host will be useful in the ovine scrapie eradication effort. In this study, we describe the incubation time, brain lesion profile, glycoform pattern and PrPFirst passage incubation times of caprine tissue in Tg338 ovinized mice varied widely but second passage intervals were shorter and consistent. Vacuolation profiles, glycoform patterns and paraffin-embedded tissue blots from terminally ill second passage mice derived from sheep or goat inocula were similar. Proteinase K digestion products of murine tissue were slightly smaller than the original ruminant inocula, a finding consistent with passage of several ovine strains in previous reports.These findings demonstrate that Tg338 mice propagate prions of caprine origin and provide a suitable baseline for examination of samples identified in the expanded US caprine scrapie surveillance program. Ovine and caprine scrapie are transmissible spongiform encephalopathies (TSEs) of small ruminant livestock. TSEs are also reported in domestic cattle and exotic zoo-raised bovids  and in farm-raised and free-ranging cervids . Human TSEs include a varied group of familial, sporadic, iatrogenic and infectious disorders. Human variant Creutzfeldt Jakob disease (vCJD) is a novel infectious TSE apparently originating from exposure to infectious bovine tissues,2 and trSc) of the normal cellular prion protein (PrPc), encoded by the PRNP gene[Sc. In all TSEs, prions and PrPSc accumulate in the central nervous system but significant accumulation also occurs in the lymphoid tissue and placenta[c[TSEs are characterized by accumulation of a relatively protease resistant isoform  and one reference domestic sheep (case number 3178) with naturally-acquired classical scrapie. All four animals had been identified as scrapie suspect animals based on antemortem detection of PrPPRNP genotyping was performed on the 4 donor samples. The reference sheep 3178 was homozygous for the wild type ovine allele, encoding alanine at codon 136, arginine at codon 154, and glutamine at codon 171 (ARQ/ARQ). This genotype is identified in more than 90% of the scrapie cases diagnosed in the US and CanaSc levels using serial endpoint dilutions in western blot and ELISA . Thus, these two data subsets were combined for calculation of the median survival time reported for this sample in TableShown in FigureSc. Representative data are shown in FigureWestern blot analysis of brain homogenates from donor goats n = 3) and sheep (n = 1) and bioassay recipient Tg338 mice  showed the characteristic banding pattern of proteinase K resistant PrP and sheeSc from the brain tissue of P1  of each P2 mouse brain homogenate were loaded, the relative amount of PrPSc in each sample slightly varied among mice . Western blots were scanned and the percent of the total density represented in each of the 3 PrPd Figure.Distribution and severity of vacuolization was assessed in P2 Tg338 mice from each of the 4 inoculum groups. Twelve regions  of each Mean vacuolization scores and variation for P2 Tg338 mice are shown in FigureParaffin embedded tissue (PET) blot analysis was performed on brain sections from one mouse in each P1 inoculum group and 4 to 9 mice in each of the P2 groups. Representative data are shown in Figurec in the central nervous system and lymphoreticular system[Sc accumulation in sheep and goats. This transgenic mouse line has been useful in characterizing atypical (Nor98)[Bioassay of tissues from scrapie infected sheep and goats demonstrated the transmissible nature of the disorder and the novel characteristics of the infectious agent well before the introduction of the prion hypothesis,17. Oralr system, sites o (Nor98),33 and c (Nor98),35. Seve (Nor98),37. In tIncubation times for all 4 inoculum groups were prolonged and variable upon primary passage to Tg338 mice; mean incubation times were not related to ELISA/Western blot titer. The sample from goat 3538 showed the widest variation in survival time on primary passage, with insidious onset at as early as 199 days and clinical signs of ataxia, weight loss and kyphosis by 206 days; survival times in some mice in this group exceeded 300 days. Mice were euthanized at 199–228 days (n = 3 short incubation), at 253–263 days (n = 4 mid-range incubation), and at greater than 331 days (n = 3 long incubation). Subpassage of samples from mice in the short (228 days) and long (331 days) incubation groups resulted in shorter incubation periods and mean survival times with no significant difference between the short and long incubation sample recipients (137 +/−2 and 135 +/−5 respectively); likewise, there was no significant difference among the groups of P2 mice from the other inocula.Several phenotypes have been described following passage of tissue from scrapie infected sheep to Tg338 mice. Notably, differences in incubation time on primary and secondary passage associated with differences in electrophoretic profiles have been described,38, partPET blots, glycoform analysis, and lesion profiles of the Tg338 samples derived from caprine brain inoculation were remarkably consistent. The marked uniformity in survival time following secondary passage and the consistent patterns in PET blot, glycoform analysis and electrophoretic profile suggest that the caprine isolates, derived from separate herds, represent a single strain with characteristics of a predominant US ovine scrapie strain. This finding is not surprising; scrapie appears to have been introduced into the US in sheep and is observed almost exclusively in ARQ homozygous sheep. Subsequent spread to goats is reported only rarely and there may be a limited number of caprine scrapie strains in the population. The US will expand caprine scrapie surveillance over the next 3 years as ovine scrapie nears eradication; as additional samples from goats are identified, monitoring of caprine scrapie strains in the US using biochemical methods and bioassay in Tg338 mice should be useful in identifying any additional strains.The Tg338 mouse strain expressing the VRQ allele of the ovine PRNP gene is useful in detection and analysis of ovine prions. In this study, this murine strain was found to propagate prions following intracerebral inoculation with brain homogenates from goats with naturally occurring scrapie. The electrophoretic profile and incubation time on secondary passage were similar to those observed by passage of ARQ homozygous sheep. The high attack rate and uniform findings on PET blot, glycoform analysis and lesion profile provide a suitable baseline for examination of caprine samples identified in the expanded US caprine scrapie surveillance program.The Washington State University and University of Washington Institutional Animal Care and Use Committees granted approval for the study before it was conducted, under agreements 3811, 3815 and 1640. Breeding pairs of transgenic Tg338 mice were kindly provided by Hubert Laude and held in a breeding colony at the University of Washington. The presence of the transgene was confirmed by DNA analysis of tail snips.Sc deposits detected by monoclonal antibody immunohistochemistry[Scrapie infected goats and sheep were diagnosed by antemortem analysis of rectal mucosal associated lymphoid tissue or post hemistry. Donor tDiploid PRNP genotypes were determined by polymerase chain reaction amplification of the open reading frame followed by DNA sequence analysis of both strands using standard methods,42.Brain tissue from the three goats with naturally acquired scrapie and the reference sheep with classical ovine scrapie were prepared as 10% (w/v) homogenates in normal saline. Relative titer of each homogenate was estimated from serial dilution and endpoint titration using a commercial enzyme linked immunosorbent assay and western blot analysis as described below. Total protein levels varied slightly among the inocula: 2 mg starting wet weight tissue yielded 76 (goat 3538), 71 (goat 3558), 85 (goat 30–75) and 99 (sheep 3178) μg total protein as determined by BCA protein assay. Passage 1 (P1) Tg338 mice were inoculated with 20 μl of 10% (w/v) sheep or goat brain homogenate (2 mg starting wet weight tissue) by the intracerebral route in the right parietal lobe. Serial passage of murine brain  was performed using 20 μl of a 10% (w/v) homogenate of brain from clinically affected P1 mice administered by the intracerebral route as above. An additional group of mice was inoculated intraperitoneally with brain homogenate from sheep 3178 using 2 mg starting wet weight tissue in a final volume of 200 μl. Inoculated animals and age matched uninoculated controls were monitored daily for appearance of clinical signs suggestive of scrapie  and culled at terminal disease. Brain and spleen were collected from mice at necropsy. Samples were fixed in 10% buffered formalin or frozen at–20C for western blot analysis.Survival time following intracerebral challenge with brain homogenates from goats or sheep (P1) or mouse (P2) was recorded as days post-inoculation (dpi). Recipient animals terminated for reasons other than clinical scrapie disease were considered censored data. The survival function of each scrapie isolate was determined using the Kaplan-Meier product-limit estimation method . Median survival times with 95% confidence limits were determined by the log(−log) method.Sc in brain homogenates from three goats and one reference sheep with scrapie were determined by serial endpoint dilution followed by enzyme linked immunosorbent assay and Western blot analysis of paired samples. Aliquots of the 10% homogenate of sheep or goat tissue prepared for inoculation were adjusted with 2 volumes of lysis buffer  using a table top homogenizer . Preliminary assays demonstrated that the use of this lysis buffer did not affect ELISA readings (data not shown) and the initial dilution in lysis buffer provided a homogeneous mixture suitable for aliquoting into paired samples for ELISA and Western blot. Additional serial two fold dilutions were prepared in lysis buffer and assayed in duplicate using a commercially available PrPSc detection kit (ELISA) . Mean absorbance values (A450) were plotted against starting wet weight equivalents of brain. The cutoff value was determined using the internal standard provided in the kit.Relative amounts of PrPSc was detected by Western blot assay as described previously[1 secondary Ab  or a peroxidase labeled F(ab’)2 fraction of goat anti-mouse IgG2 . Membranes were exposed to radiographic films  for exposure times of 1–10 min. Digital images of the radiographic films were obtained with an Alpha Innotec image analyzer  and saved in tiff file format. Density of the diglycosylated band was plotted against starting wet weight of tissue to estimate relative endpoints.PrPeviously-43 with Sc. For each group of mice, mean density of each band was plotted into Tri-plot Excel template. The tri-plot excel template was downloaded fromhttp://www.lboro.ac.uk/research/phys-geog/tri-plot[Brain tissues of 20 inoculated mice (five from each donor group) were selected for glycoform analysis and tested in three independent western blot assays. For each sample, triplicate aliquots of 50 μl brain homogenate were incubated with PK  at 37C for 90 min. Aliquots equivalent to 300 μg wet brain weight equivalents were loaded into individual lanes of 12-well 12% polyacrylamide gels and analyzed by western blot assay as described above, using mAb F99/97.6.1. Digital images of the radiographic films were obtained with an Alpha Innotec image analyzer and saved in tiff file format. These files were analyzed with AlphaEaseFC software Version 4.0 to identify the relative densities of unglycosylated, monoglycosylated and diglycosylated forms of PrP/tri-plot.Formalin fixed brains were processed routinely and sectioned at 5 μm for hematoxylin and eosin staining. Tissues were analyzed for vacuolation profile using standard methods as described. Lesion Sc labeling in P2 Tg338 mouse brains were evaluated in paraffin embedded tissue blots. The brain sections were dissected at the levels of frontal cortex (level 1), hypothalamus (level 2), hippocampus (level 3) and cerebellum-medulla (level 4) and probed with mAb F99/97.6.1 as described[Regional distribution and intensity of PrPescribed-48, withescribed. Eight PKIO developed the study design, contributed to design of the glycoform analysis, participated in histologic scoring, performed analysis of genotypes, and drafted the manuscript. DAS performed the survival curve analysis and the vacuole pattern analysis. TRS performed the histopathological examinations. RPD performed the glycoform analysis and assisted with PET blot analysis. DZ performed the western blots, ELISA and PET blots and provided data analysis for ELISA and Western blots. MAH analyzed the PET blot data. TCT performed the histologic preparation of all small ruminant and murine tissues. All authors participated in writing the manuscript and have read and approved the final manuscript. Brain tissue from 20 Tg338 passage 2 mice (five from each sheep- or goat-inoculated P1 donor) were selected for western blot assay and glycoform analysis. Three hundred μg wet brain weight equivalent volumes of each of the mouse brain were digested with proteinase K and loaded into SDS-PAGE gels followed by western blot assay using mAb F99/97.6.1 and chemiluminescent imaging. Films  were used to obtain relative densities of un-, mono- and di-glycosylated forms of PrPSc bands for glycoform analysis.Click here for file Glycoform patterns of PrPSc  from 3 goats and a reference sheep with naturally occurring scrapie and from Tg338 mice from each inoculum group.Click here for file


Aetobatus narinari) is the most abundant inshore elasmobranch in subtropical Bermuda, but its predatory role remains unexamined despite suspected abundance increases and its hypothesized specialization for mollusks. We utilized a combination of acoustic telemetry, benthic invertebrate sampling, gut content analysis and manipulative experiments to assess the impact of spotted eagle rays on Bermudian shellfish resources. Residency and distribution of adult spotted eagle rays was monitored over two consecutive summers in Harrington Sound (HS), an enclosed inshore lagoon that has historically supported multiple recreational and commercial shellfish species. Telemetered rays exhibited variable fidelity (depending on sex) to HS, though generally selected regions that supported relatively high densities of potential mollusk prey. Gut content analysis from rays collected in HS revealed a diet of mainly bivalves and a few gastropods, with calico clam  representing the most important prey item. Manipulative field and mesocosm experiments with calico clams suggested that rays selected prey patches based on density, though there was no evidence of rays depleting clam patches to extirpation. Overall, spotted eagle rays had modest impacts on local shellfish populations at current population levels, suggesting a reduced role in transmitting cascading effects from apex predator loss. However, due to the strong degree of coupling between rays and multiple protected mollusks in HS, ecosystem-based management that accounts for ray predation should be adopted.Declines of large sharks and subsequent release of elasmobranch mesopredators  may pose problems for marine fisheries management as some mesopredators consume exploitable shellfish species. The spotted eagle ray ( Rhinoptera bonasus) transmit the effects of large shark declines down to bay scallop (Argopecten irradians) populations due to the specialist and density-dependent foraging behavior of the rays Declines in large sharks have been demonstrated in the northwest Atlantic for some time Aetobatus narinari) is a large tropical to warm-temperate ray that consumes benthic invertebrates and is considered highly migratory A. narinari appears to specialize on clams Strombus gigas) may dominate the diet of individuals found in the Bahamas and Caribbean A. narinari appears to be a key predator of giant clams (Tridacna and Hippopus spp.) and is considered a pest to farmers of these bivalves The spotted eagle ray . Though fishery-independent data for sharks is lacking for the Bermuda Islands, landings suggest declining abundance of “dusky” sharks  and increasing exploitation of larger tiger sharks . The potential “mesopredator release” of spotted eagle rays from large shark removals has raised concerns of apex predator declines cascading down to shellfish populations in Bermuda, many of which are protected . Without knowledge of the habitat use and diet of these mesopredatory rays, however, the dynamics of potential cascading effects cannot be understood.In subtropical Bermuda, the spotted eagle ray is reportedly common in seagrass beds and sand flats Despite its prevalence in the study of movements of other marine vertebrates, acoustic telemetry has only gained popularity in the study of batoids (skates and rays) over the last decade Aetobatus narinari) in Harrington Sound (HS), Bermuda. Because of their highly mobile nature, we tracked and monitored rays with acoustic telemetry and analyzed their distribution relative to various benthic habitats in HS. During the monitoring period, benthic communities were sampled using bottom survey techniques to examine potential prey available and were compared to prey items consumed by rays. The spatial distribution and residency data of spotted eagle rays were then used to guide enclosure and field manipulation (exclosure) experiments aimed at quantifying the impact of rays on calico clam Macrocallista maculata, an abundant and protected bivalve species in HS.In this study, we utilized a suite of approaches to examine the foraging ecology and habitat use of spotted eagle rays (This study was conducted in accordance with the laws of the State of Alabama and under IACUC protocols (Permit # 05043-FSH) approved by the University of South Alabama. All efforts were made to minimize animal suffering during tagging procedures.Thalassia testudinum) algal mats (Cladophora prolifera), sand flats, relic Oculina reefs, and rubble, mud and shell bottoms. A wide variety of infaunal and epifaunal benthic invertebrates characterize the heterogeneous benthos of HS, including commercially and recreationally important mollusks such as the calico clam, turkey wing (Arca zebra), Bermuda scallop Pecten ziczac, Atlantic Pearl Oyster (Pinctada imbricada) and conch (Strombus costatus and S. gigas). With the exception of turkey wing, all aforementioned mollusks have been protected in Bermuda waters since 1978 The Bermuda Islands are limestone-capped volcanic pedestals situated within the Sargasso Sea and include several large inshore sounds and harbors. Harrington Sound (HS), the most land-locked of the inshore sounds, has a single navigable connection to the open ocean through Flatts Inlet (FI) (Thomas 2003). Surface temperatures of HS range from 16°C in winter to 30°C in summer. Mean tidal range in HS is 19 cm, lagging approximately 2 hr 53 min behind oceanic tides . HS is host to a wide variety of habitats, including seagrass beds  from a moving skiff. Once located, a nylon seine net (100 m×5 m) was corralled around the animals and the encircled rays were brought aboard. Captured animals were measured , photographed for future identification, and assessed for level of sexual maturity when possible. Rays then received one of four treatments prior to release with an external tag: 1) fitted with an acoustic transmitter (n = 18), 2) pulsed gastric lavage (n = 18), 3) transported to enclosure pen for foraging experiments (n = 7), or 4) given no additional treatment (n = 12).Acoustic telemetry was employed to determine habitat use and residency of spotted eagle rays in Harrington Sound. Acoustic transmitters were wrapped in a neoprene sleeve to reduce abrasion of the ray’s soft skin and attached to monofilament dart tags using cable ties and superglue. Following Silliman and Gruber (1999), tags were secured to the dorsal “saddle” region of spotted eagle rays . In eachAcoustic monitoring array size, deployment length and locations differed between 2007 and 2008. In the 2007 study, 6 total Lotek Wireless Hydrophone Systems (WHS) 3050 (Lotek Inc.) were utilized; 5 throughout Harrington Sound (HS) and one unit at the junction between HS and Flatts Inlet. Hydrophones were straddled on either side of the junction at Flatts Inlet to serve as “gatekeepers”, which monitored the ingress and egress of the rays. The full 6 hydrophone array monitored tagged animal movements across HS from 21 May 2007 to 25 May 2007 with the two gatekeepers remaining until 28 July 2007 between FI and HS. All WHS 3050 units were vertically mounted to PVC poles secured to concrete moorings along the sea floor. In 2008, the acoustic array was expanded to 10 total WHS units, and included 9 WHS units in HS and one at the mouth of FI to serve as a gatekeeper . Monitoriw) for each hydrophone:io  =  proportion of the detections at hydrophone i, and πi = proportion of available resource units (i.e. volume) for that hydrophone. A iw value larger than 1 indicates a positive selection for the resource and a value less than 1 indicates avoidance At the end of the monitoring period, data files (.txt format) were downloaded from the WHS 3050 units, filtered and imported into MATLAB  for analysis and processing. To examine potential habitat preferences tagged rays within Harrington Sound, we performed a likelihood chi-square analysis on the observed number of detections recorded at each hydrophone with FishTel 1.4  telemetry analysis program. This program accounted for habitat availability  for each hydrophone, as well as the individual variability among rays (replicates) and determined a selection ratio , sex and the interaction (year x sex) on Harrington Sound proportional habitat use  and maximum residency interval (maximum time in Harrington Sound) of spotted eagle rays using univariate two-way ANOVA. We also examined the influence of ray size  on proportional residency in Harrington Sound with linear regression. Proportional data were arc-sine transformed prior to analysis, and ANOVA post-hoc analyses were performed using Tukey’s HSD iR is the number of detections a hydrophone i, iP is the power of detections at hydrophone i, and iX and iY are the x and y coordinates of hydrophone ihttp://www.spatialecology.com) was then used to examine overall distribution of COAs relative to various benthic habitats within the convex polygon of hydrophones in HS.Fifteen minute centers of activity (COA) were determined for acoustically tagged rays to examine use of the interior portion of the Harrington Sound monitoring array. Individual COA positions were determined using a weighted activity cell method developed by Yergey et al. (2012), which was adapted from Simpfendorfer et al. (2002):To estimate fine-scale (±10 m) habitat use by spotted eagle rays, we conducted a series of mobile tracking surveys (Protocol S4). Tracking surveys were conducted July 2–4, and in September 19–21, 2008. After data collection, Synthetic Aperture Positioning System  software was used to estimate positions of the transmitting tags. Convergent position estimates were plotted in ArcMap 9.2, and localizations within various habitat types were extracted from digitized maps of Harrington Sound using Hawth’s Tools. To examine benthic habitat use of individuals tagged with depth-sensing transmitters, we used the intersect function in Hawth’s Tools to extract the position estimates within 1 m of the benthos.2) weighted PVC frame four times along a 25 m transect. At each quadrat we excavated the upper 15 cm of the area by hand and collected all epi- and infaunal invertebrates by passing excavated sediment through a 2 mm mesh bag. Excavated fauna were identified to species level and enumerated. For each species, we determined density per square meter by pooling the counts of the four 0.25 m2 quadrats per site. Due to the restricted spatial coverage of the benthic sampling, we interpolated densities of dominant benthic fauna using the inverse distance weighted (IDW) method in the Spatial Analyst extension of ArcMap 9.2. We elected IDW interpolation due to its utility in analyzing relatively sparse or irregularly spaced data sets . Interpolations were used to assess spatial trends in faunal abundance across HS and qualitatively compared with spotted eagle ray distribution.In order to examine the distribution of prey available to spotted eagle rays, we conducted benthic quadrat surveys concurrently with acoustic monitoring at ten sites throughout Harrington Sound and Flatts Inlet . At eachSa is the number of stomachs containing food group a, and S is the total number of stomachs containing food Na).Gut contents of spotted eagle rays were sampled opportunistically using pulsed gastric lavage (PGL) on live animals. During PGL, a plastic tube was placed into the esophagus of the ray and a gentle stream of ambient water was run into the stomach to flush out the contents. This technique was a non-lethal alternative to sacrificing rays for dietary information. We verified the efficacy of this technique by sacrificing three individuals after PGL was conducted, and found that PGL successfully removed 100% of the gut content. All animals sampled survived the PGL procedure. Recovered gut content was identified from photos of local benthic invertebrate tissue. For each prey item, we calculated percent frequency of occurrence: 2 regions of limestone rock. Prior to experimentation, the sandy area within the enclosure was excavated by SCUBA divers to remove any ambient infauna and epifauna to a depth of 15 cm. Calico clams collected from nearby locations in Harrington Sound were distributed among 9 separate 1 m2 vexar screen trays filled with sand. During each trial, trays were randomly assigned a number between 1 and 3, and given a density of 10, 20 or 40 calico clams per square meter . Calico clams were introduced to the surface of the trays 24 h after rays were placed inside the enclosure. Trays were checked daily for evidence of predation and restocked when necessary. Analyses of variance (ANOVA) were used to test the impact of clam density on the number of clams consumed and mortality rate (proportion of clams consumed per day). To better meet the assumptions of ANOVA, all proportional tray mortality rates were arc-sine square root transformed prior to analysis and checked for normality and heterogeneity of variances.To examine small-scale foraging preferences of spotted eagle rays among heterogeneous prey densities, we conducted a series of patch selection experiments within an experimental enclosure. The enclosure measured 46 m×22 m and was 1–2 m in depth. The border of the enclosure was constructed from plastic fencing material with a 2.54 cm diameter styrofoam float line at the top and a lead-line sewn into the bottom. Floats were placed evenly at 5 m intervals across the top of the net to ensure flotation during high tide. Additional anchors were used to secure the enclosure in place. The substrate within the enclosure was primarily characterized by coarse sand with a few 10 m2) square patches with 75 cm tall steel poles spaced 25 cm apart around the perimeter. These dimensions were chosen because all rays sampled were 60–170 cm in disk width, and thus were assumed to be capable of accessing open patches but too large to enter stockades. Yellow flagging tape was stretched across the top of the exclosure to deter animals from entering from above. Excavated “open” patches (4/site) were placed haphazardly nearby (≥3 m) the exclosures. Corners of the open patches were marked with short metal stakes and flagging tape. In July 2009, SCUBA divers excavated these patches of infauna and epifauna to a depth of 15 cm. Ten marked calico clams were placed haphazardly throughout patch and tethered to the substrate at least 50 cm from the edge. Patches were re-excavated in October 2009 using the same method and the number of marked clams and unmarked clams was recorded at each patch. Due to the high variability in clam mortality rates among sites, separate independent samples t-tests were run at each site comparing the number of marked-tethered clams recovered between exclosure and open patches. It was assumed that marked-tethered clams missing from patches were killed by predators. Shells that were emptied , but not crushed, were not considered killed by rays or smaller predators (crabs). Shells broken into large pieces were considered killed by crabs. The various species-specific fracture morphologies of calico clam shells were confirmed through tests with captive predators at the Bermuda Aquarium Museum and Zoo.To estimate the contribution of spotted eagle ray predation to calico clam mortality in Harrington Sound, we conducted a 3-month field study in 2009 where we introduced marked calico clams to unprotected (open) patches and protected (exclosure) patches. Four replicate open and exclosure patches  were constructed at 3 different sites : Major’s Bay, Tuckers Bay and Trunk Island . The thrMt  =  the number of captured individuals at time t; Ft = the number of free marks, or total individuals previously marked in the population at time t; Rt  =  the number of recaptured individuals at time t; and N-hat  =  the estimated population size. We used a modification of this index to incorporate movement rates of spotted eagle rays in a “closed” population model (Protocol S5).We used tag-recapture data to develop an approximate population estimate for spotted eagle rays in Harrington Sound. Population size was assessed with the Schnabel method, which is an extension of the single sample Lincoln-Peterson model of population estimation to include a series of samples 29]. Each sampling trip (2007–2010) was used as a “sample” because we captured, examined for previous marks, marked and released animals on each of these trips and revisited the same sites continuously throughout Harrington Sound and Flatts Inlet. We then used the following formula to estimate the population:Because daily rations of elasmobranchs (including batoid rays) range from 0.3–4.3% of body weight per day Spotted eagle rays were sighted along shallow sand flats <5 m in depth within Harrington Sound and Flatts Inlet . The majp>0.05). However, when maximum residency was utilized as the response variable, a significant sex effect was found . Mean maximum residency (m = 37.9±6.6 d) for females was found to be significantly higher than mean maximum residency (m = 16.7±6.3 d) for males . No significant relationship was found between ray size and proportional residency in Harrington Sound .We monitored movements of four female (121–161 cm DW) and four male (120–137 cm DW) spotted eagle rays in 2007 between Harrington Sound and Flatts Inlet with gatekeepers only. In 2008, four males (120–133 cm DW) and six females (99–161 cm DW) were monitored across the extended Harrington Sound acoustic array. Monitoring periods ranged from 3–67 days; with 94% of tagged individuals detected by the acoustic array . Neither2 = 1251886.01, df = 64; p<0.0001). The highest number of proportional detections (detections per available volume) was recorded in the shallow southwest portion of the Sound. In this region, selection ratios were all greater than 1 , indicating observed detections were greater than expected by the total available habitat (H8w = 1.21) at the northeast corner of HS, which was also situated in shallow waters. Low use and thus avoidance (iw <1) was generally observed for deeper hydrophones of the interior of HS, and the lowest amount of total detections was recorded at the hydrophone stationed at Flatts Inlet (H0).Based on 2008 acoustic monitoring data, spotted eagle rays habitat use was not evenly distributed throughout Harrington Sound . However, 90–95% of the acoustic detections occurred in the upper 10 m . Tag depOculina debris and shell sand, all typically >15 m in depth. Primary prey density was generally high within the 50% KUD for most individuals, although the core use region between H7–H9 was devoid of clams  within sub-arrays H1-H2-H3 and H2-H3-H5, and between H7 and H9 . Notablyof clams .2 = 84.83, df = 45; p<0.001). Similar to the qualitative COA analysis, towed hydrophone telemetry processed with SYNAPS showed rays used overlying waters of mud, relic Oculina debris, shell-sand and sand habitats, while fringing rock and rubble habitats of the immediate coastline were avoided , milk conch (Strombus costatus), purple urchin (Lytechinus variegatus) and two species of sea cucumbers  was determined to be the prey item of highest importance, both by frequency (78.6%) and by number (86.3%) for 14 individuals , eared ark  and purplish tagelus . One individual ray  was found to consume two species of gastropods, the moon snail  and milk conch . Most individuals (80%) possessed a single prey item type in their gut content, with generally no shell fragments.Opportunistic sampling of spotted eagle ray gut content revealed a diet dominated by mollusks . Though ividuals . In factF2,11 = 5.523, p<0.05; p<0.01) treatment, though medium density mortality  was not found to be different from the high or low density clam mortality rates .After complete excavation in July 2009, unmarked calico clams colonized open and exclosure patches at the three manipulation sites by October 2009. Clam immigration (denoted by the number of unmarked clams) was highest at Tuckers Bay  followedt(4) = 2.353; p = 0.019; −2). No significant patch effects were found at the Tuckers Bay (t(4)  =  −0.866; p = 0.386) or Majors Bay (t(4)  =  −0.662; p = 0.508) manipulation sites  at the Trunk Island site  of 1.38 km2 and average density of 16.55± clams · m−2 across all habitats where clams were collected in 2008 and 2009. Using the average weight of 35 kg for all rays collected in HS, rays would consume 1.05 kg (3.0% of BW) of foodstuff per individual per day. For rays feeding on calico clams (mean clam tissue wet weight of 7.765 g from benthic sampling), this translated to a feeding rate of 135 clams per day. Of the 55 individual rays collected in Harrington Sound, four were identified as recaptures (7.3% recapture rate). All recaptures were large mature females  originally tagged within Harrington Sound. Based on these recapture rates, the Schnabel method estimated 307 individuals inhabited Harrington Sound (6 clams (13.7% of the standing stock).The calico clam population in Harrington Sound was estimated at 2.01±0.72×10on Sound . Given aOculina, etc.), they were rarely positioned at or near the benthos in these regions. The lack of substrate use in these habitats suggests they were not used for foraging purposes since eagle rays prey solely on benthic organisms. Along sandy habitats of Tuckers Bay, Trunk Island and Church Bay, on the other hand, tracked rays were observed on or near bottom. While these areas were indeed shallower, rays were never detected in the upper depth layer when positioned in these sandy regions. Conversely, rays were commonly positioned in surface waters overlying deeper regions of HS. Reduced movement of rays in the sand habitat, as indicated by motion sensor tags, may be indicative of foraging behavior as rays must cease forward swimming to excavate prey from the benthos.Our combined acoustic monitoring and tracking work showed that mobile spotted eagle rays did not evenly partition available habitat within a semi-enclosed lagoon, and exhibited a strong affinity to shallow (<10 m) sand and sand-shell bottom habitats. This core use area was situated among the highest densities of their preferred prey  recorded in benthic surveys of Harrington Sound. Although our active tracking transects positioned spotted eagle rays over various other habitat types  was the largest ray by weight (73 kg) captured over the sampling period. The same individual also consumed calico clams and ark clams (Anadara notabilis), the latter of which was only observed in one other similarly large individual. Though the reduced sample size may not encompass the full dietary breadth of spotted eagle rays from Harrington Sound, the discovery of ark clams and conch in two larger individuals suggests that rays exhibit some degree of ontogenetic diet variation. This is supported by the work of Schluessel et al. (2011), who observed that spotted eagle rays from the Indopacific switch from a crustacean  diet to a hard-shelled mollusk diet with ontogeny. A comprehensive diet study encompassing all maturity levels is needed to elucidate these potential ontogenetic patterns in resource use.Our observation of spotted eagle ray predation on bivalves and gastropods in Bermuda is supported by previous studies that report this species as mollusk-specialized Rhinichthys cataractae) on benthic cobble communities are significantly greater on stones with higher biomass of invertebrates, implying selection for these patches when resources are patchily distributed Callinectes sapidus) also exhibit high prey density-dependence when foraging on infaunal Macoma balthica with proportionally more clams consumed at higher density patches in enclosure experiments Cetorhinus maximus, also appear to select habitats based on threshold prey densities when foraging on plankton Foraging patch selection in aquatic benthic vertebrates and invertebrates is commonly based on resource availability. In freshwater streams, foraging rates by longnose dace , suggesting potential preference for these habitats. This apparent patch selection behavior exhibited by the spotted eagle rays conforms to optimality models that predict higher consumption of prey by predators in more “profitable” habitat types Argopecten irradians) to complete extirpation off North Carolina Though spotted eagle rays demonstrated preferences for habitats that supported dense populations of important shellfish, given their combined lack of patch-depleting foraging behavior and relatively low population densities, it is unlikely that rays would completely deplete calico clam from Harrington Sound. The larger size, higher site fidelity and solitary foraging behavior of spotted eagle rays may account for their reduced consumptive effects on manipulated prey patches when compared to schooling cownose rays. Schools of fall-migrating cownose rays have been observed to deplete patches of bay scallop  with field sampling and experimentation can elucidate the habitat use and potential impact of large benthic mesopredators. Given the high mobility of mesopredators and relatively ephemeral nature in benthic systems, methods to derive residency patterns and fine-scale habitat use of these animals must be implemented in order to guide manipulative experiments capable of detecting foraging effects. In our example, we coupled active and passive telemetry with field surveys and enclosure/exclosure experiments to unveil the potential impacts of highly mobile fish on a relatively stationary mollusk species. While our approach is highly applicable to other predator-prey systems that include mobile predators and fixed prey, we caution that researchers should have a strong underpinning of the dynamics of their study system prior to implementing these experimental approaches.Protocol S1Transmission scheme for pressure/motion sensor data from acoustic transmitters.(DOCX)Click here for additional data file.Protocol S2Methods for determining total available habitat at each receiver station.(DOCX)Click here for additional data file.Protocol S3Methods for determining long-term residency in Harrington Sound.(DOCX)Click here for additional data file.Protocol S4Towed hydrophone tracking survey methodology.(DOCX)Click here for additional data file.Protocol S5Technique for meeting assumptions of the Schnabel population estimation method.(DOCX)Click here for additional data file.


Dedicated to Prof. Bernhard Dick on the occasion of his 60th birthdayNowadays it is well established that photochemically induced electron transfer (ET) through DNA in solution is limited to relatively short distances. In most experimental studies, photochemically induced ET was observed over less than 10 base pairs bp; 3.4 nm),.4 nm,3 HHalobacterium salinarum is a hollow spherical dodecameric protein, which binds oxidized flavins with high affinity, whereas flavin reduction induces the dissociation of the holoprotein.Herein we use the riboflavin binding protein dodecin as a redox-sensitive probe to study ET processes through DNA monolayers. The ET signal is translated into a change of surface-bound mass, thus allowing further analytical techniques, such as surface plasmon resonance (SPR) or quartz crystal microbalance (QCM) measurements, to be used to trace ET. Dodecin from n) were synthesized. Dodecin was reconstituted with these ligands and X-ray structures were measured. In Figure n O5 with a hexyl, butyl, propyl, and ethyl group  at the N(10) of the isoalloxazine moiety are shown. For all the flavin–DNA ligands, electron density can be traced for the isoalloxazine moiety (and the first two CH2 groups). For the CofC4_O5 ligand, additional electron density is present, probably caused by weak binding of the linker and the first DNA base. For the CofC2_O5 ligand further electron density indicates binding between apoprotein and linker plus the first two DNA bases. As shown by superposition of the ligands in Figure Kd of 10 μm vs. 400 nm), but finds a deeper position, and previously a deeper position has been correlated with a higher binding affinity.For the adsorption of disulfide-modified ss-DNA onto gold, three 1,2-dithiane rings forming six gold–sulfur bonds are used  isoalloxazine moiety, appeared to be the best compromise between high affinity binding and short ET distance. Dodecin was reconstituted in a flow cell for combined electrochemistry and SPR measurements. The SPR kinetic scans collected during the stepwise reconstitution of dodecin on the flavin terminated ds-DNA monolayer and subsequent electrochemical experiments are shown in Figure For electrochemical measurements a ligand with To demonstrate that the release of tE upon reduction is indeed possible, a chemical reduction step was carried out by addition of sodium dithionite . For this purpose dodecin was reconstituted as before on a template stripped gold (TSG) slide with flat surface.While DNA-mediated charge transfer between DNA repair proteins has been proposed as a long-range signaling mechanism to trace damages in the genome,