Classical and atypical TSE in small ruminants

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1 Published December 22, 2014 Classical and atypical TSE in small ruminants V. Beringue* and O. Andreoletti * UR892 Virologie et Immunologie Moléculaires Centre de Recherche de Jouy-en-Josas F Jouy-en-Josas, France UMR INRA ENVT 1225, Interactions Hôtes Agents Pathogènes, Ecole Nationale Vétérinaire de Toulouse, 23 Chemin des Capelles Toulouse, France Key words: control, scrapie, small runimants, transmissible spongiform encephalopathy, zoonosis Transmissible Spongiform Encephalopathy in Small Ruminants: An Emerging Issue? Classical Scrapie Scrapie in small ruminants is a disease that has been described for several centuries. It was reported for the first time in sheep in the United Kingdom in 1732 and few years later in 1759 in Germany. In the following centuries, scrapie endemically affected flocks in several countries (Detwiler, 1992). The disease is thought to have spread across the world through the export of asymptomatic infected animals (for review, see Detwiler and Baylis, 2003). Scrapie is now commonly referred to as classical scrapie following the recent discovery of atypical scrapie cases (cf. infra). Beringue and O. Andreoletti doi: /af Implications Scrapie is present in domestic sheep and goat herds for several centuries. During the first part of the 20th century, most of the countries that had been endemically affected by this disease simply made the choice to live with it. Following, the bovine spongiform encephalopathy (BSE) crisis and its sanitary/economic consequences on the ruminant industry, the situation dramatically changed. The control and eradication of transmissible spongiform encephalopathy (TSE) in the population of small ruminant turned out to be a priority in many western countries. However, the classical sanitary policy (deflocking and restocking) that was applied to control and eradicate transmissible disease in animals appeared to have a limited efficacy. This led to the development of a large-scale selection program for genetically resistant animals. While, in the long term, this strategy seems to be efficient, recent investigations revealed the existence of a new atypical and worldwide spread form of TSE in sheep and goat. This discovery raised new issues about the dietary exposure to small-ruminant TSE agents and also prompts us to re-examine the still pending question of the zoonotic potential of small-ruminant TSE agents. Under natural exposure, contamination with classical scrapie mainly occurs around birth (materno-lateral transmission). The disease is then characterized by long asymptomatic incubation periods that usually range between 2 and 7 years and during which infected animals are a source of contamination. The clinical phase of the disease can last several months. In its earlier stages, the disease is usually characterized by a progressive loss of body weight (with preserved appetite) and slight behavioral changes. At later stages, a progressive pruritus with hyperesthesia and incoordination of gait usually develop (van Keulen et al., 1996; Andreoletti et al., 2002b; Baylis et al., 2004). Over the decades, the identification of classical scrapie has relied on clinical suspicion (passive surveillance). Since 2001 and 2002, in the European Union (EU), an active surveillance of TSE in small ruminants has been implemented and in the later years in a number of countries like the United States (US) and Canada. Active surveillance relies on the systematic testing of a proportion of the slaughtered or found-dead animals for the detection of an abnormal isoform of the prion protein (PrP), called PrP Scrapie (PrP Sc ), in the posterior brainstem. The data collected through this surveillance system have clearly demonstrated that earlier evaluations on the prevalence of TSE in small ruminants and its geographical spreading (on the basis of passive surveillance) were largely underestimated (Fediaevsky et al., 2008). The implementation of active surveillance programs in the early 2000s has provided a better picture of the apparent disease prevalence in certain countries (like EU members states, the US, or Canada). Between 2002 and 2008 in the EU, about 3.5 million sheep were tested and more than 4,700 cases were identified in most of countries (21 out of 27) with remarkable geographical differences and the occurrence of a major outbreak in Cyprus. During the 2002 to 2008 period the EU-wide apparent crude prevalence of scrapie, when the Cypriot outbreaks are excluded, was equal to about 11 cases per 10,000 tested animals (7 and 16 among healthy slaughtered animals and fallen stock, respectively). The withinflock prevalence was, on average, 20 times greater than the apparent prevalence in the general population identified by active surveillance (Fediaevsky et al., 2008). These values are likely underestimated. Indeed a recent study performed using the measured performance of tests used in active surveillance, have shown that whatever would be the proportion of the tested animals in the healthy slaughtered or found-dead animals, a large proportion of infected flocks/herds would be missed (Corbiere et al., 2013a). Atypical Scrapie In 1998, the spectrum of transmissible spongiform encephalopathy (TSE) in sheep was extended by the discovery in Norway of an experimentally Jan. 2014, Vol. 4, No. 1 33

2 transmissible, PrP-related, neurological disease of sheep that was clearly distinguishable from classical scrapie cases that had been reported so far and was therefore considered to be an atypical form of scrapie, also named Nor98 scrapie (Benestad et al., 2008). Following its recognition in sheep, Atypical/ Nor98 was detected in goats (Arsac et al., 2007). In Atypical scrapie/nor98 cases, the abnormal PrP Sc that accumulates in the brain of positive animals is only partially proteinase K (PK) resistant and displayed a multi-band pattern as showed by Western Blot that contrasted with those normally observed in small-ruminant TSE cases (Benestad et al., 2003). The clinical features observed in sheep affected by atypical scrapie also significantly differ from those observed in sheep affected by classical scrapie (Konold et al., 2007), and identified atypical scrapie cases are unusually older than classical scrapie cases (Benestad et al., 2008). After 2001 and the implementation of active TSE surveillance plans, a number of similar cases were identified in most EU members states as well in many other countries, like the US and Canada (Benestad et al., 2008). More recently, atypical scrapie was also detected in Australia and New Zealand (Kittelberger et al., 2010), two countries that had been considered so far by the World Organisation for Animal Health (OIE) as TSE free. A retrospective study performed in tissue banks allowed the identification of atypical scrapie cases in sheep samples collected in the UK in 1987 (Webb et al., 2009). The analysis of data collected through the epidemiosurveillance system in the UK between 2002 and 2006 suggested that Atypical/Nor98 prevalence in this population could have remained stable over this period (McIntyre et al., 2008). Together these elements suggest that atypical scrapie might have been present but undetected in the small ruminant population for several decades. Atypical scrapie now represents a substantial part of the TSE cases identified in the EU small-ruminant population. Several studies described the apparent prevalence of atypical scrapie in sheep slaughtered for human consumption (healthy slaughtered animals) or collected as fallen stock. In a study that included 11 European countries that reported atypical scrapie between 2002 and 2007, the mean prevalence of this disease was estimated to reach 5.5 (cases per 10,000 in abattoir surveillance, and 8.1 cases per 10,000 in fallen stock; Fediaevsky et al., 2010). The apparent prevalence of atypical scrapie in sheep did not present important variations between countries (Fediaevsky et al., 2008) or over time (Fediaevsky et al., 2008; McIntyre et al., 2008). However, these values are likely to be underestimates of the real situation. Indeed a recent study provided evidence that samples from the central nervous system (CNS) containing high infectious titer (as assessed by bioassay) could remain PrP Sc negative in tests used for TSE field detection in active surveillance programs (Andreoletti et al., 2011). This suggests that a significant number of atypical scrapie incubating animals remain undetected even when tested. Diversity of Small-ruminant TSE Agents Despite the relative uniformity of the clinical signs in the natural host, classical scrapie can be caused by a variety of distinct TSE agents or strains that harbor different biological features. Historically, the appreciation of the potential scrapie diversity has relied on serial passaging of natural isolates to a panel of inbred mouse lines. The strains that preferentially replicated in the mice were isolated and the biological phenotype was compared. The considerable strain variations initially described (up to 20 strains; Dickinson, 1976) using this system might finally be restricted to a varying combination of three distinct strains (Bruce et al., 2002). However, the relationship between these mouse-adapted TSE agents and those initially present in the TSE isolates is a matter of debate. The propagation of natural TSE isolates into inbred mice lines implies the passage through a species or transmission barrier (see below) that can result in a different outcome, including a radical and irreversible evolution of the biological properties of the agent, a phenomenon called mutation. Moreover, wild-type mice have been described to be refractory to most scrapie isolates (Foster and Dickinson, 1988; Bruce et al., 2002) including atypical scrapie (Le Dur et al., 2005; Bruce et al., 2007). In that context, it is difficult to consider that the conventional mice models provided a comprehensive and reliable picture of the diversity of the TSE agents in small ruminants. Transgenic mice that overexpress the ovine, caprine, bovine, or even the murine protease-resistant protein (PRNP) genes have been demonstrated to display a reduced transmission barrier to scrapie agents than conventional mouse models (Scott et al., 1989; Groschup and Buschmann, 2008). As a striking example, atypical scrapie cases propagate without apparent barrier in transgenic mice overexpressing the sheep VRQ allele (Le Dur et al., 2005). To date, all available information seems to indicate that atypical scrapie is likely to be caused by a single TSE agent (Le Dur et al., 2005; Arsac et al., 2007; Griffiths et al., 2010). The diversity of classical scrapie agents is currently explored in this model. Serial transmission of about 80 isolates from Europe in the same transgenic mouse line has so far permitted to identify at least four phenotypically distinct classes (Beringue et al., 2007; Thackray et al., 2007; Beringue et al., 2008b; Tixador et al., 2010; Thackray et al., 2012). The transgenic mice are valuable models that will probably enable a more comprehensive view of strain diversity in small ruminants in the near future. Meanwhile, it remains difficult to appreciate the real diversity of TSE agents circulating in small ruminants or the potential relationships that might exist between agents causing scrapie in small ruminants and prion disease in other species. Together, these elements show that our knowledge concerning the diversity, spread, and true prevalence of TSE in populations of small ruminants is still limited. In that respect, the fact that atypical scrapie remained unrecognized for decades and has now been identified everywhere is particularly striking. istockphoto/rekemp 34 Animal Frontiers

3 Towards Control and Eradication of TSE in Small Ruminants In 1754 in England, the sheep breeders petitioned the House of Commons requesting new regulations for the sale and circulation of sheep. This was apparently the consequence of a scrapie epidemic that caused major damages to wool production (pruritus in affected animals), which at that time was a strategic industry for the UK. In the following centuries, scrapie has been reported to cause significant losses to sheep farmers in specific areas. However, the global economic impact of scrapie on the international sheep industry remained negligible, and most affected countries made the choice of living with the disease. Approximately 250 years later, the Bovine spongiform encephalopathy (BSE) crisis completely changed this picture. The hypothesis that the BSE epidemic in the United Kingdom may have originated from scrapieinfected sheep and the anticipated existence of BSE in small ruminants due to their exposure to the BSE agent via contaminated food products lead to the implementation of drastic restrictions in the sheep and sheep product exchanges in several countries. The presence of scrapie became a sufficient reason to limit or stop the imports of live sheep, embryos, semen, and ovine products. According to countries and specific economic interests, there is considerable variation on the type and severity of restrictions. Nevertheless, TSE in small ruminants turned out to be an important issue in countries having a significant sheep industry, and over the last three decades, many of them have spent considerable resources to control and eradicate the disease. Sanitary Measures to Control and Eradicate Scrapie Sanitary approaches to eradicate contagious diseases in farm animals rely on the capacity: To detect infected animals/flocks that could transmit the disease. To efficiently control the movement of animals between premises (individual identification and movement records) To prevent potential occurrence of infection due to the environment (persistence of infectivity/disinfection) During the second half of the 20th century, several countries implemented sanitary control/eradication plans against scrapie. Among them Iceland is certainly the most illustrative. Scrapie was apparently introduced into Iceland in 1878 through the import of sheep originating from the United Kingdom. For the first 70 years of the epidemic, the disease was limited to the northern region of the country. In 1933, Jaagsiekte, Maedi-Visna, and Johne s disease were also introduced to Iceland through the importation of sheep from Germany. Because of the rapid spread of these diseases and the economic loss they caused, it was decided to control and eradicate them. For that purpose, the country was divided into 36 quarantine areas. The eradication strategy relied on the depopulation of affected areas before re-stocking them with animals from non-affected areas. Scrapie was present in three of the quarantine areas. All the sheep in the scrapie-affected areas were destroyed during the years 1946 to Most of the farms which were depopulated were restocked within the same year, but some were left without sheep for up to 3 years. Scrapie recurred within 2 to 4 years following repopulation in certain farms that had displayed high incidence of the disease. By 1953, disease could be observed not only in the original endemic area, but also in other quarantine areas. At the peak of the epidemic (early seventies) the disease had spread to 23 of the 36 quarantine areas. Considering the possibility that scrapie might spread throughout Iceland, leaving no scrapie free areas, it was decided, in 1978, to implement a specific control and eradication policy for scrapie. This plan has been continuously applied since then. In 1993, further enhancements of the program were made, mainly with regard to the practical aspects of handling scrapie cases, and since 2012, different measures have been applied to flocks depending on whether they are infected with classical scrapie or have atypical/nor98 cases. Susan Schoenian, 2004 Jan. 2014, Vol. 4, No. 1 35

4 The approach is based on the total depopulation of affected flocks in the quarantine zone, with restocking not permitted for at least 2 years. During the year before restocking, the housing facilities, equipment, and machinery were cleaned by high-pressure washing and then treated with a sodium hypochlorite solution (one of the few prion-inactivating agents). They were then sprayed with an iodophor disinfectant or burned off with a gas flame. All surfaces in sheep houses and barns up to a height of 1.5 meters were sealed with creosote or oil-based paint. All woodwork and even complete sheep houses that could not be disinfected were burned or buried. Surface soil by barns and other heavily exposed areas were removed and replaced with gravel (4 cm layer) or asphalt. In addition, the first harvest of hay from potentially infected fields was not permitted to be used as forage for the new stock. Government inspection was required to ensure that procedures were completed to the standards set by the scheme. These measures were accompanied with substantial financial support for the disinfection procedures and compensation for impacted farmers (Sigurdsson, 1954; Thorgeirsdottir et al., 1999, 2002; Georgsson et al., 2006). All over this period, extensive surveillance has being maintained on farms and at slaughter to identify newly infected flocks. The surveillance methodologies evolved with the knowledge and techniques in the TSE field. Initially surveillance relied on clinical examination. In 1978, the surveillance was reinforced by random sampling of brain stem (10,000 to 15,000 animals per year) for histological examination (vacuolation) and later PrP Sc immunohistochemistry. In 2004, a retrospective epidemiological study was performed. It indicated that despite the eradication measures, recurrences of the disease had occurred in 33 out of the 397 farms where restocking had been completed. Recurrences were observed most frequently 4 to 7 years after restocking of the farms, but cases were also observed as late as 12 to 19 years following restocking (Georgsson et al., 2006). This scrapie eradication policy in Iceland has resulted in a substantial decrease in disease prevalence. However, despite these 35 years of continuous effort, and the drastic sanitary measures that had been applied, disease has not yet been eradicated. Why Sanitary Eradication Policies are Unlikely to Succeed The limited sensitivity of passive surveillance for detecting TSE in small ruminants is probably one important parameter explaining the limited success of the scrapie control and eradication plans described above for Iceland and the US. The postmortem PrP detection screening tests currently performed on posterior brainstem (obex) clearly improved the diagnostic sensitivity and capacity for monitoring of prion disease in small ruminants. However, their performances in terms of detection of individual infected animals are also limited due to PrP Sc accumulation in the CNS not occurring until late in the incubation phase (Andreoletti et al., 2000; Bellworthy et al., 2005). Nevertheless, active surveillance is now perceived as a possible means for controlling and eradicating TSE in sheep and goat populations. For instance, the animal health authorities of the US have developed the National Scrapie Eradication Program. This program aims, through the postmortem testing of small ruminants, to eradicate classical scrapie from the US small ruminant population within the next 10 years. In a recent study (Corbière et al., 2013b), the performance of active PrP Sc detection in goats was assessed in eight scrapie-infected goat herds. A total of 183 out of 1,961 goats were found to be PrP Sc positive in at least one tissue (posterior brainstem, tonsil, mesenteric lymph node, or ileum). The sensitivity of detection using the posterior brainstem appeared to be strongly dependent on the age of tested individuals, which is consistent with the known pathogenesis of TSE. The test failed to identify any of the infected individuals younger than 2 years old that were incubating scrapie, and it detected only one-third of the asymptomatic positive cases older than 2 years old. However, it identified accurately the infected individuals belonging to the clinical suspect subpopulation. Similar data were obtained from studies of a large UK goat herd (Gonzalez et al., 2009). These data confirm that the age of the tested individuals (incubation stage), strongly influences the diagnostic performance of PrP detection methods in scrapie-infected small ruminants. Beyond this, they also provided quantitative parameters to estimate the performance of scrapie active surveillance at the population level through simulation studies of the capacity of TSE surveillance programs to detect classical scrapie-infected herds under different testing scenarios. According to the mathematical model, if in a population of one million adult animals, 20,000 tests would be randomly performed each year in fallen stock (10,000 animals) and healthy slaughtered animals (10,000 animals), only 12% the scrapie-infected herds would be detected. In a scenario where 100% of the animals aged over 2 years that are slaughtered for human consumption or eliminated at a rendering plant would be tested, the estimated proportion of infected herds that would be identified after 1 year of surveillance using postmortem PrP Sc detection tests on the posterior brainstem would be 50%. These estimates are consistent with those reported by Hopp et al. (2003) when assessing the performance of TSE active surveillance in the Norwegian sheep population. Beyond this, the occurrence of environmental contamination is probably also a likely cause of the limited efficacy of sanitary control/eradication measures against TSE in small ruminants. Contamination with classical scrapie was reported in sheep that were introduced into an infected flock after they had reached adulthood (Detwiler and Baylis, 2003). The efficacy of such transmission appeared to be less in older animals than in younger animals. The origin of such contamination remains unclear, and both inter-individual horizontal transmission and/or environmental sources could be at their origin. The role of the environment as a source of contamination has now been unambiguously demonstrated by infection of naïve animals that were introduced into an infected environment without contact with animals (Ryder et al., 2009). The implication of environment in classical scrapie transmission is a likely explanation for the failure of the stringent eradication policy that was applied in Iceland since 1947, with the recording of new contamination after stamping out infected flocks and reflocking with scrapie-free animals (Georgsson et al., 2006). Placentas from scrapie-incubating ewes that are released at lambing appear as a major source for environment contamination (Andreoletti et al., 2002c). Once shed into the environment, TSE agents have been shown to resist degradation over long periods in soil (Genovesi et al., 2007; Wiggins, 2009), and their high capacity to resist chemical and physical decontamination treatments has been documented extensively (Dickinson and Taylor, 1978; Taylor et al., 1994,1999; Taylor, 2000). Genetic Control of TSE in Small Ruminants Classical scrapie is the sole infectious disease of small ruminants for which the susceptibility is so strongly influenced genetically. In sheep, the polymorphism of the PRNP gene that encodes for PrP at codons 136 (A or 36 Animal Frontiers

5 V), 154 (R or H), and 171 (R, Q, or H) have been demonstrated to be of major importance (Clouscard et al., 1995; Hunter et al., 1996). Under natural exposure conditions, VRQ/VRQ, ARQ/VRQ, and ARQ/ARQ genotype animals are considered the most susceptible to classical scrapie, whereas homozygous or heterozygous AHQ and heterozygous ARR animals only show a marginal susceptibility. ARR/ARR sheep are considered to be strongly (but not absolutely) resistant to classical scrapie (Elsen et al., 1999; Groschup et al., 2007). Beside those three major polymorphisms, other polymorphisms like the K176 or the T137 have more recently been recognized to be associated to resistance to scrapie infection (Vaccari et al., 2009). In goats, the knowledge related to PrP polymorphism-associated susceptibility to classical scrapie is far more limited than in sheep; data available in goats have mainly resulted from case control field studies. These usually involved only a limited number of animals, especially positive animals and herds (limited range of TSE agents). In addition, goats carrying PrP polymorphisms are less usually observed than sheep. Case/control studies performed in Italy and France have demonstrated a potentially high protective effect of the K 222 allele (Acutis et al., 2006; Vaccari et al., 2006; Barillet et al., 2009; Corbiere et al., 2013b) against classical scrapie. This view was recently reinforced by the apparent resistance of the K 222 allele to infection following an experimental challenge with a scrapie isolate (Acutis et al., 2012). Similarly, the Q211 allele has been associated with an increase in resistance to classical scrapie in French infected herds (Barillet et al., 2009; Corbiere et al., 2013b). The H 154 allele has also been associated with some resistance to classical scrapie in studies performed in Greece, France, Italy and Cyprus (Barillet et al., 2009; Billinis et al., 2002; Corbiere et al., 2013b). Similarly, studies conducted in Cyprus support the view that S 146 and D 146 polymorphisms of the PRNP gene are likely to be associated with substantial resistance to classical scrapie in goats naturally exposed to classical scrapie. This contention was further supported by the results of an experimental challenge of S 146 allele carrier goats conducted in the US (White et al., 2012). The PrP genetic sensitivity to atypical scrapie is totally different from that observed in classical scrapie. While a clearly increased risk for developing atypical scrapie is associated with AF 141 Q and AHQ alleles, the VRQ allele seems to be at lower risk. Strikingly, ARR allele carriers (both homozygous and heterozygous) can develop the disease (Moum et al., 2005; Arsac et al., 2007; Moreno et al., 2007). In goats, the AHQ allele is clearly associated with a greater susceptibility to atypical scrapie (Colussi et al., 2008). istockphoto/mjhollinshead The differences of susceptibility observed between classical and atypical scrapie illustrate that susceptibility of sheep and goat to TSE strongly depends on the nature of the agent involved. Despite these limitations, the EU along with the US and several other countries have implemented breeding policies in sheep that intend to control and eradicate classical TSE by the use of reproducers bearing the ARR allele. These long-term policies have clearly demonstrated their efficacy in controlling classical scrapie outbreaks in affected flocks (Dawson et al., 2008). Similarly, the development of a PrP genotype selection program is now considered in certain countries as a potential tool for the control and eradication of classical scrapie in the goat population. Food-Borne Exposure to TSE Agents Pathogenesis of Classical and Atypical Scrapie Most of the available data related to the dissemination of the classical scrapie agent in small ruminants that have been naturally infected were obtained in VRQ/VRQ sheep born and raised in two individual flocks (one in the Netherlands and one in France). According to these studies, infection apparently occurs via the Gut Associated Lymphoid Tissues (GALT) before a rapid spread of the agent to draining mesenteric lymph nodes and later to all lymph nodes, including those that remain on prepared carcasses (Andreoletti et al., 2000; van Keulen et al., 2000). The amount of PrP Sc in lymphoid formations increases with age before reaching a plateau level. The TSE agent disseminates to the CNS (brain and spinal cord), which is considered to accumulate TSE agents until around half of the incubation period apparently via the enteric nervous system and its nerve fibres (Andreoletti et al., 2000; van Keulen et al., 2000). From there, the agent redistributes (centrifugally) to the peripheral nervous system and skeletal muscle (Andreoletti et al., 2004). In blood, the infectious agent can be detected as early as 3 months of age and persists throughout the incubation period (Lacroux et al., 2012). This dissemination scheme is consistent with most of the data reported with regard to natural classical scrapie cases in small ruminants. However VRQ/VRQ sheep are considered to be the most sensitive sheep PrP genotype to the majority of TSE agents responsible for classical scrapie. In sheep bearing other PrP genotypes as well as in goats, the kinetics of the agent distribution in the organism of the affected animals can vary substantially. Moreover, in heterozygote ARR sheep and under natural exposure conditions, the PrP Sc distribution seems to be mostly confined to the CNS (van Keulen et al., 1996; Andreoletti et al., 2002a). Additionally, in several classical scrapie cases in ARQ/VRQ and ARQ/ARQ sheep (Jeffrey et al., 2002; Ligios et al., 2006) and in goats (Gonzalez et al., 2009), PrP Sc accumulation in the CNS was reported in the absence of detectable PrP Sc in the lymphoid tissues. Atypical scrapie pathogenesis remains poorly documented. Abnormal PrP has never been evidenced in peripheral tissues collected from field cases or experimental atypical scrapie cases (Benestad et al., 2008; Andreoletti et al., 2011). It suggests that the infectious agent would be restricted to the CNS, which was interpreted as a support to the contention that atypical scrapie could be a spontaneous disorder of PrP folding and metabolism, occurring in aged animals without external cause (Benestad et al., 2008). In addition, no statistical difference in the detectable atypical scrapie frequencies was observed between the general population and the flocks where a positive case had been identified (Fediaevsky et al., 2010). Jan. 2014, Vol. 4, No. 1 37

6 This findings has been interpreted by some as an evidence that atypical scrapie could be only slightly contagious or not contagious at all. The transmissibility of the atypical scrapie agent by the intracerebral route is clearly established in both rodent models (transgenic animals overexpressing the ovine PrP gene) and in sheep (Le Dur et al., 2005; Simmons et al., 2007; Fediaevsky et al., 2010). However, the contagiousness of atypical scrapie in natural conditions is still debated. Data from an oral challenge study indicate that very early exposure (within 24 h of birth) can lead to transmission of the disease (Simmons et al., 2010). The analysis of the data collected through the active surveillance program support the view that inter-individual transmission of atypical scrapie under field conditions is at most low (Fediaevsky et al., 2010). In most of the atypical scrapie flocks, only a single case is found. However, there are reports of several cases in sheep that originated from the same flock (Benestad et al., 2008). This and the suboptimal sensitivity of TSE tests for detecting pre- or sub-clinically infected animals implies that we cannot at this stage exclude the idea that atypical scrapie is a contagious disease. However, recent information obtained in both natural and experimental atypical scrapie cases demonstrated that reduced infectivity amounts can be present in skeletal muscle, peripheral nerves, and lymphoid tissues of animals incubating or affected with atypical scrapie (Andreoletti et al., 2011). These data also indicated that CNS can contain a massive amount of infectivity but remain negative for abnormal PrP detection using the current most sensitive diagnostic tests. Finally, atypical scrapie can transmit via the oral route. Experimentally challenged animals display a similar clinic-pathological pattern to those observed in natural cases (Simmons et al., 2010). Therefore, it is probably too early to conclude if atypical scrapie is a spontaneous and non-contagious disorder or if it behaves like the other TSE agents circulating in small ruminants. Scrapie Agents Shedding in Milk In 2005, disease-associated prion protein (PrP Sc ) accumulation was reported in mammary glands from three scrapie-affected ewes (Ligios et al., 2005). PrPSc deposits were associated with mammary ectopic lymphoid follicles that develop in response to retroviral infection (Maedi-Visna virus; Cutlip et al., 1985; Ligios et al., 2005). In another study (Lacroux et al., 2008), the presence PrP Sc granules was reported in lacteal ducts and mammary acini lumen in sheep naturally infected by classical scrapie and harboring ectopic lymphoid follicles typical of Maedi-Visna virus infection. Such PrP Sc deposits were observed in ewes bearing different PrP genotypes (ARQ/ARQ, ARQ/VRQ, and VRQ/VRQ), but no PrP Sc deposits were observed in ewes infected with scrapie and displaying a healthy mammary gland. These observations indicated that in sheep incubating natural scrapie, the TSE agent could be shed in milk. The feeding of TSE-free lambs with milk/colostrum from infected ewes is certainly the most relevant experiment for assessing the potential mother-offspring transmission of the TSE agent through mammary secretion. However, such experiment is difficult to organize since it requires not only long observation periods and specific facilities (avoiding cross-contamination risk) but also the use of TSE-free animals and appropriate contact controls to prevent bias in the interpretation of results. Two independent studies based on such experimental design have been performed (Konold et al., 2008; Ligios et al., 2010). The results obtained in both studies clearly demonstrated that a limited amount of colostrum/ milk from classical scrapie infected ewes is able to transmit classical scrapie to lambs. Lympho-proliferative mastitis seems to enhance the efficacy of the transmission; nevertheless, colostrum/milk collected in ewes displaying an apparently healthy mammary gland was also efficient in transmitting the disease. To estimate the infectious titre, colostrum and milk samples were collected in naturally infected ewes and tested by intra-cerebral bioassay in tg338 mice, which overexpress the VRQ ovine PrP variant (Lacroux et al., 2008). The bioassay was performed after fractionation of the milk into three different components: cell pellet, casein whey, and cream. The animals were all sampled during their first lactation (lactation starts at months of age) and developed clinical scrapie when between 19 and 36 months old. All three fractions prepared from milk (cell pellet, casein whey, and cream) transmitted disease to tg338 mice. In that study, the infectivity found in one liter of sheep milk was estimated to be comparable to the infectivity contained in about g of brainstem material from a terminally affected sheep. Despite in-depth investigations, using immuno-concentration steps, no PK-resistant PrP could be detected in milk and colostrum fractions that were found positive by bioassay in tg338 mice. Such discrepancies were likely to be due to the sensitivity limit of the used PrP Sc detection methods. Indeed, using protein misfolding cycle assay (PMCA), an in vitro technique that allows the amplification of abnormal PrP molecule and infectivity, PrP Sc was evidenced in milk samples collected in symptomatic and presymptomatic classical scrapie infected sheep (Maddison et al., 2009). There is no available data regarding the possible presence of an atypical scrapie agent in the milk of infected ewes or goats. Dietary Exposure to Scrapie Agents In BSE-affected cattle, the infectious agent has a limited dissemination in tissues other than the central and peripheral nervous system (Wells et al., 2007; Arnold et al., 2009). In countries that were affected by BSE, the systematic retrieval of certain tissues (named SRM for specific risk materials) from cattle above a certain age that are slaughtered for consumption was implemented. The SRM tissue list (mainly the skull excluding the mandible, brain, eyes, spinal cord, tonsils, intestines, and the mesentery) corresponds to tissues described as containing significant levels of BSE infectivity. A SRM retrieval policy is a key measure for preventing the dietary exposure to a BSE agent. Because the potential occurrence of BSE epidemics in small ruminants and the difficulties that could have existed in differencing BSE in sheep from scrapie, a number of countries (in particular, the European Union members states) have implemented SRM retrieval measures in sheep and goat. The skull, CNS, tonsils, ileum, and spleen from sheep and goats above a certain age slaughtered for food are systematically retrieved because those tissues contain high levels of infectivity in TSE-infected small ruminants (Hadlow et al., 1982). In that respect, SRM measures significantly mitigate the potential dietary exposure of the consumers to scrapie agent(s). However, the infectious agent is also present in a large variety of tissues (like the skeletal muscle and the blood, see above; Andreoletti et al., 2004,2011,2012). Therefore, the final/real efficiency and the cost/benefits of SRM measures in small ruminants has always been a matter of debate. Beyond those considerations, these data indicate that despite the protective measures implemented by a number of countries, classical and atypical scrapie agents have continuously entered into the food chain during the past decades. This raises some questions about the capacity of these agents to propagate in other species and in particular about their zoonotic potential. 38 Animal Frontiers

7 Zoonotic Potential of Small-Ruminant TSE Agents Transmission Barriers and its Consequences Whereas the homospecific (within the same species) transmission of TSE agents is usually quite efficient, their heterospecific transmission is generally difficult. In a number of cases, no transmission can be observed on first passage, and when transmission occurs, it displays a long incubation period and incomplete attack rate. Subsequent passages of the agent in its new host species result in its adaptation as shown by a reduction and a stabilization of the incubation period. This phenomenon has been initially named the species barrier and is now usually referred to as the transmission barrier (Pattison and Millson, 1961; Dickinson et al., 1968; Beringue et al., 2008b). Prion transmission barriers have major consequences for animal and public health. They strongly limit the risk for individual dietary exposure to a TSE agent from a different species to develop a disease. Without the existence of transmission barrier, considering the nearly one million BSEinfected and undetected cattle that entered the food chain during epidemics in Europe in the 1980s and 1990s, it is likely that the magnitude of the Creutzfeldt Jakob varient disease (vcjd) epidemics in exposed human consumers would certainly have been much more dramatic. Early studies suggested that the transmission barrier resides essentially in differences between the host and donor species in the primary structure of the PrP. In seminal transgenesis experiments, the recognized resistance of the mouse to hamster scrapie (Kimberlin and Walker, 1978) was abrogated by expressing hamster PrP C in mice (Scott et al., Susan Schoenian, ), demonstrating that the PrP sequence of the recipient host is a key component of the transmission barrier phenomenon. The TSE agent also plays a pivotal role in inter-species transmission events. Whereas many TSE agents are difficult to transmit to other species, the BSE agent transmits fairly in a broad range of species such as exotic ruminants, mice, primates, felines, and humans (Collinge et al., 1996; Bruce et al., 1997a; Scott et al., 1999). The transmission of sporadic and genetic CJD cases to inbred mice is extremely inefficient. However, despite the divergence between human and bank vole PrP sequences, these human isolates propagated in bank voles with very low, if any, transmission barrier (Nonno et al., 2006). Conversely, transmission to mice of vcjd isolates is efficient (Bruce et al., 1997b), whereas its transmission to bank voles is inefficient (Beringue et al., 2008b). The nature of the donor host can also strongly alter the leakiness of the transmission barrier. For instance, whereas cattle BSE propagates with low efficacy on primary passage in transgenic mice expressing the porcine PrP, BSE passaged in sheep propagates in the same host without a noticeable barrier (Castilla et al., 2004; Espinosa et al., 2009). Finally, there is clear evidence that the transmission barrier can be broken insidiously in the absence of clinical symptoms and of detectable PrP Sc in the brains of the infected animals. Prions introduced from other species can persist or even replicate at low levels in brains during most of the life span of the inoculated host. Those silent prions can efficiently elicit a disease when retrotransmitted to the original host or to a third host species. Foreign prions can also multiply efficiently in the lymphoid tissue without necessarily invading the brain, even at the end of life, revealing a tissue-specific transmission barrier (Beringue et al., 2012). These examples illustrate the extreme complexity of the interactions between host and TSE agents. The mechanisms that control the permeability of the transmission barrier remain therefore largely unknown. In that context, it is still currently impossible to predict, in the absence of experimental data, the capacity of a TSE agent from a donor host to transmit to a specific host belonging to another species. Assessing the Zoonotic Potential of TSE Agents Classically, the recognition of the zoonotic features of an infectious agent relies on the combination of epidemiological and laboratory data. The epidemiology allows the identification of spatiotemporal relations between the occurrence of human cases and the exposure to animal products or infected animals. The laboratory data allow the confirmation that the same infectious agent can be found in diseased humans and the suspected animal source. The identification of the zoonotic capacity of the cattle BSE agent has relied on this approach. The occurrence of massive epidemics of TSE in the UK cattle population at the end of the 1980s followed a few years later by the emergence of an apparently new type of spongiform encephalopathy in young humans in the same country lead naturally to the suspicion that there was a link between both events (Collinge, 1996). Strain typing in wild-type mice definitively confirmed the suspicion of the implication of the same etiologic agent in human vcjd and cattle BSE (Bruce et al., 1997b; Brandner et al., 1999). However this success story should not overestimate our power to identify the zoonotic capacity of one or several agent causing scrapie. Indeed, TSE has circulated in small-ruminant populations for centuries, and they are unlikely to provoke now the emergence of a new disease that modern epidemiological monitoring systems would instantly spot. Jan. 2014, Vol. 4, No. 1 39

8 Epidemiological Aspects In humans, the identification of TSE cases (mainly CJD) rely on passive surveillance (clinical suspicion followed by confirmation) only. The sensitivity of such a monitoring system remains very difficult to evaluate. However, the active surveillance system of TSE in animal populations highlighted the limits of passive surveillance systems. Most countries only report anecdotally TSE in humans, and assessing the epidemiological situation in these geographical areas is impossible. In countries with specific TSE monitoring and reporting systems, the incidence of sporadic CJD (the most common form of human TSE) ranges between 1 and 1.5 cases per million and per year (Ladogana et al., 2005). The rarity of identified cases renders the epidemiological analysis of the data collected over short periods (decades) delicate. The most common argument used to rule out the hypothesis of a link between the occurrence of certain TSE cases in human and scrapie in small ruminants is the lack of correlation between the frequency of TSE cases in humans and the recognized presence/apparent absence of TSE cases in small ruminants. The recent discovery of atypical scrapie in New Zealand and Australia, two countries that were considered free of animal TSE, clearly illustrates the limits of this argument (Kittelberger et al., 2010 and Australia ref). Similarly, the increase in international trade (including food) also implies that dietary exposure to TSE agents in a country or a geographical area cannot be solely inferred to the domestic prevalence of the disease. In conclusion, the recognition by epidemiological approaches of the zoonotic power of a scrapie agent would require a systematic and reliable TSE epidemiological surveillance in both small ruminants and humans. Considering the limits of the past and current TSE surveillance systems in animals and humans, this requirement cannot be considered to be fulfilled. Phenotyping of TSE Agents in Humans and Animals Classically, the comparison between two TSE isolates from hosts belonging to different species is made by transmission of these agents to a third species host. Bioassays in rodent models formed the basis of the TSE strain-typing methodology developed in Scotland in the 1960s and 1970s. It was extremely useful for demonstrating that the same TSE agent was at the origin of BSE in cattle and vcjd in humans (Bruce et al., 1997b). It also confirmed the existence a certain number of differentiable strains in sheep and goat scrapie isolates (Bruce et al., 1991; Bruce and Fraser, 1991; Baron et al., 1999,2000). However, as already stated, because of the existence of a transmission barrier, a significant proportion of the scrapie field isolates cannot be propagated in conventional mice models (Bruce et al., 1997b; Beck et al., 2012). Moreover, the effect of the passage over this transmission barrier can impact the TSE strain properties. Together, these elements represent major limitations for describing the classical scrapie biodiversity using conventional mice models. In addition, most of the scjd isolates failed to propagate in these mice models (Bruce et al., 1997b; Hill et al., 1997), which limits the interest of conventional mice bioassay for comparing classical scrapie and human TSE isolates. Nevertheless, in C57Bl6 mice, a natural scrapie isolate was reported to show, after first passage, a phenotype similar to sporadic and iatrogenic CJD (Lasmezas et al., 2001). However, considering the potential effect of transmission barrier passage on the strain properties, this result remains difficult to interpret. More recently, bioassay in bank voles (Myodes glareolus) have been proposed as a model to compare human and animal TSE isolates. Indeed, both scjd isolates and animal TSE isolates seem to propagate efficiently in this model (Nonno et al., 2006). Available results did not indicate similarities between any of the investigated CJD cases and the ruminant TSE isolates. However, the limited diversity of the animal and human TSE isolates tested by this approach remains too limited at this stage to make any definitive conclusions. Models for the Human Transmission Barrier First evidence of the transmissibility of human TSE isolates (Kuru and scjd) were obtained in primate models. The infected primates displayed clinical and pathological similarities with human diseases (Brown et al., 1994). Because of these similarities and the high sequence homology of the human/primate PrP genes (96 to 99% according to primate species), primate models have been considered for decades as the best proxy for assessing TSE-associated risk in humans. Primates demonstrated the transmission capacity of low amounts of BSE-infected tissue following dietary exposure (Lasmezas et al., 2005) and highlighted the risk of vcjd secondary transmission by transfusion (Herzog et al., 2005). A very limited number of classical scrapie isolates have been inoculated into primates. Importantly, the infectious material was rarely originating directly from small ruminants or from field isolates and was rather propagated in one or several intermediate hosts, including laboratory rodents, before inoculation to primates. In one single study, a natural sheep classical scrapie isolate transmitted in two out of two intra-cerebrally challenged marmosets. The incubation period observed with this scrapie isolate was shorter than with a cattle BSE isolate, suggesting that that transmission barrier towards both isolates might not be different (Baker et al., 1993). In another experiment, a sheep scrapie isolate referred to as Compton, which had been serially propagated in goats and then in conventional mice (eight passages), caused a clinical TSE in Cynomologus monkeys after a 5-year incubation period (intracerebral challenge; Gibbs and Gajdusek, 1972). The Compton isolate, after three additional passages in hamsters, transmitted to squirrel monkeys both after an intracerebral challenge (incubation period 14 months) or an oral challenge (incubation period 25 to 32 months). In this model, sporadic CJD (scjd)isolates propagated with comparable incubation periods (11 to 48 months for intracerebral and 23 to 27 months for oral route; Gibbs and Gajdusek, 1973; Gibbs et al., 1980). These latter results remain very difficult to interpret since the serial sub-passages of the original TSE agent into laboratory rodents are likely to have permanently altered its scrapieness. Moreover, if the chimpanzee and the other old-world monkeys are evolutionarily close to humans, the new-world monkeys (marmosets and squirrel monkeys) are phylogenetically more distant. This may limit the pertinence of the model for studying the human transmission barrier. There is now clear evidence that that using transgenic animals expressing a PRNP gene identical to that of the donor (Scott et al., 1989) virtually abrogates the transmission barrier. A number of transgenic mice lines for mammalian PrP, including notably human PrP (hereafter referred to as tghu) (Wadsworth et al., 2007; Groschup and Buschmann, 2008), have been successfully developed. 40 Animal Frontiers

9 A high transmission barrier with low attack rates and long incubation periods has been reproducibly observed in several tghu lines following inoculation of cattle BSE isolates (Asante et al., 2006; Beringue et al., 2008a,2008b). This is in line with the apparently low BSE transmission efficacy observed in the UK human population (despite a massive exposure). These findings support the view that tghu lines could provide highly pertinent data when assessing the relative permeability of the human transmission barrier to animal prions. A number of projects aimed at characterizing the capacity of field classical and atypical scrapie isolates to overcome the transmission barriers are currently ongoing using human transgenic mice models. One of the major difficulties in this approach is to constitute a panel of TSE isolates that representatively cover 1) the full diversity of the TSE agents that circulate in sheep and goat populations and 2) the full PrP polymorphisms of sheep and goats as the PrP primary sequence can influence the issue of the cross-species transmission. For example, the BSE agent passage in sheep or goats transmits in tghu lines with much better efficacy than the cattle BSE agent. To date, results from such experiments remain extremely limited and insufficient to draw any definitive conclusion. Several additional years will certainly be necessary before comprehensive results become available. Conclusions Scrapie is considered as the archetype of prion diseases, and it has been the subject of scientific studies for almost a century. The pathogenesis of the disease and its transmission modalities are now well established. 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Heasman, R. Warner, and S.J. Moore Accumulation and dissemination of prion protein in experimental sheep scrapie in the natural host. BMC Vet. Res. 5:9. Scott, M., D. Foster, C. Mirenda, D. Serban, F. Coufal, M. Wälchli, M. Torchia et al Transgenic mice expressing hamster prion protein produce speciesspecific scrapie infectivity and amyloid plaques. Cell 59: About the Authors Dr. Vincent Beringue is Research Director and Head of Laboratory in the Virology Immunology Department at the National Institute for Agricultural Research (INRA) in Jouy-en-Josas (France). His research interests are focused on mammalian prion diseases. Particular emphasis is placed on studying prion diversity and evolution under selective pressure, identifying the physical structure of prion infectious particles, and characterizing the cell biology and neurotoxicity of prions. Dr. Andreoletti (DVM-PhD) is Senior Scientist at INRA and Head of Prion Team in the UMR INRA ENVT 1225 since His scientific expertise involves the characterization of TSE agent biological and biochemical properties by using various animals and cellular model of TSE agents. Results produced (70 publications) over the last decade have substantially contributed to the understanding of pathogenesis of TSE, TSE risk evaluation, and TSE agent detection methodologies in various species (including human). Correspondence: o.andreoletti@envt.fr Scott, M., R. Will, J. Ironside, H.-O.B. Nguyen, P. Tremblay, S.J. DeArmond, and S.B. Prusiner Compelling transgenetic evidence for transmission of bovine spongiform encephalopathy prions to humans. Proc. Natl. Acad. Sci. USA 96: Sigurdsson, B Rida, a chronic encephalitis of sheep with general remarks on infections which develop slowly and some of their special characteristics. Br. Vet. J. 110: Simmons, M.M., T. Konold, H.A. Simmons, Y.I. Spencer, R. 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