Prions and Agents of TSEs: Bovine Spongiform Encephalopathy in Cattle

PRIONS AND AGENTS OF TSES

Contents Bovine Spongiform Encephalopathy in Cattle Creutzfeldt–Jakob Disease

Bovine Spongiform Encephalopathy in Cattle D Matthews, United Kingdom r 2014 Elsevier Inc. All rights reserved.

Glossary Oral ID50 The infectious dose, administered by mouth, that is predicted to kill 50% of animals inoculated. Note that the figure quoted for bovine spongiform encephalopathy (BSE) is not an absolute, as it is dependent on the concentration of infectivity in the starting material. Nevertheless, it is sufficient to demonstrate that the amount would be undetectable in contaminated raw ingredients or finished feed and difficult to prevent unless extreme measures are adopted. Peyer’s patches Peyer’s patches are nodules or collections of lymphoid tissue found within the wall of the intestine, especially in the young. They can become continuous and prominent, particularly within the lower small intestine (ileum). In ruminants, they are prominent before maturity but eventually regress in adulthood. Phenotype The biological concept of phenotype is defined as the detectable characteristics displayed by an organism resulting from its interaction with the infected host and its environment or the sum of the observable characteristics embracing the entire physical, biochemical, and physiological make up of an organism. Disease phenotype can be defined as the characteristic clinical signs and pathological changes of a particular form of a disease entity or group, enabling it to be characterized and distinguished by such criteria from other forms of the disease. The frequent detection of infected, but clinically healthy, individuals through surveillance frequently prevents the inclusion of criteria such as clinical signs and pathology in consideration of phenotype. In such circumstances, the term is used more loosely in describing other characteristics, such as patterns of banding obtained by biochemical tests, or the secondary characteristics produced when laboratory rodents are inoculated. Prion The term prion was originally coined to refer to an infectious protein, and at that time conflicted with those that believed that diseases previously known as transmissible spongiform encephalopathies (TSEs) were caused by viruses, or were at least associated with viruses.

Encyclopedia of Food Safety, Volume 2

Although abnormal prion protein is still the only marker consistently associated with disease, it is still not possible to exclude the possibility that it does not act alone. The term is now accepted generically as a substitute for the infectious agent(s) that causes TSEs, acknowledges the role of prion protein in the transmissibility and pathogenesis of the diseases in which protease-resistant protein PrPSc is detected, but accepts that it is not yet possible to exclude a role for other molecules in conferring infectivity on prion protein. PrPSc PrP stands for protease-resistant protein, the protein that is associated with prion diseases, and the product of the PrP gene (PRNP). It can be found in many tissues of healthy individuals although its function remains unclear. In a healthy individual, it is normally designated at PrPC, where the C stands for cellular. PrPSc denotes the disease-specific isoform of PrP, that is folded differently, with a greater proportion of b sheet, and as a result is more resistant to digestion by proteases both within the body and in immunological tests. The Sc arises from its first identification in scrapie in sheep and rodent models, but it is often used generically to refer to the abnormal isoform of PrPC in all affected species. At times, it is referred to as PrPres to denote the protease-resistant property of the pathological protein, PrPd for disease-specific PrP (particularly when detected by methods, such as immunohistochemistry, that do not rely on the use of proteases), or specifically in the case of BSE as PrPBSE. In some instances, such as the World Health Organization (WHO) categorization of infectious tissues, the term PrPTSE is used. Strain Because it is not possible to isolate individual prions and to extract DNA or other agent/strain-specific molecules other than PrP (which is host-derived), science cannot categorize isolates into strains or species/subspecies by means of methods that are appropriate for bacteria or viruses. The characterization of isolates as different from each other relies, therefore, on a combination of approaches. At one time there was reliance on the inoculation of laboratory rodents, followed by a description

doi:10.1016/B978-0-12-378612-8.00001-9

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Prions and Agents of TSEs: Bovine Spongiform Encephalopathy in Cattle

of the ensuing disease, or pathology, such as attack rate, incubation period, and lesion profile of vacuolation produced within the brain. More recently, molecular tools that measure and characterize prion protein extracted from host tissues are also used, but remain relatively crude. Most depend on the characterization of the products of enzyme digestion and their comparison with defined strains from either laboratory rodents or from naturally infected animals or humans. As a consequence, the process of confirming that two isolates are different, identical, or similar is slow and now involves both biochemical and bioassay approaches. Genetically modified rodents may also be used as they can speed up the process and sometimes facilitate infection that would otherwise fail in unmodified animals.

Introduction Bovine spongiform encephalopathy (BSE) is a disease of domesticated cattle, first recorded in November 1986 in the United Kingdom (UK), but subsequently recognized in another 26 countries. It is a disease of the nervous system and is spread between cattle by the consumption of contaminated feed. The epidemic also spread to other countries via exported contaminated feed constituents and infected live cattle. Retrospective investigations suggest that clinical cases of BSE were first seen on British farms early in 1985, but modeling studies indicate a likely origin at least a decade earlier. Although it was the subject of some international concern soon after its first recognition, a larger worldwide crisis of confidence in food safety was triggered almost 10 years later. This followed the recognition in 1996 that BSE had transmitted to humans, giving rise to a disease that was referred to as variant Creutzfeldt–Jakob disease (vCJD). Where this article focuses on events in the UK, this is solely because the size of the epidemic in that country enabled the consequences of exposure of both cattle and humans to be studied more comprehensively than elsewhere. The principles described remain sound in all at-risk countries, although the degree of risk faced by cattle and humans will vary according to the extent of exposure of cattle and the nature of controls introduced.

Nature of the Agent The first diagnosis of BSE was made after the detection of brain lesions similar to those described for a disease of sheep and goats called scrapie. The lesions include vacuolation of neurons identified by the histological examination of fixed tissue sections stained with hematoxylin and eosin. Subsequent studies demonstrated that BSE could be transmitted experimentally to laboratory rodents by inoculation of brain tissue from affected cattle. Consequently, BSE was classified alongside scrapie as a transmissible spongiform encephalopathy (TSE). Chronic wasting disease (CWD) of cervids in North America is another member of this group of disorders.

Given the current state of knowledge and the logistical difficulties of processing many isolates, the categorization of isolates into strains remains relatively crude. Transmissible spongiform encephalopathy (TSE) Before the adoption of prion for the categorization of diseases such as scrapie, BSE, chronic wasting disease, and Creutzfeldt–Jakob disease, they were grouped under the term TSEs. The term describes the pathology of the diseases in the brain of affected individuals, but acknowledges that each can be experimentally transmitted to laboratory rodents. The association of prion protein with these diseases has resulted in the more common reference to prion diseases, rather than TSEs, although the term remains both accurate and appropriate.

TSEs are now more commonly referred to as prion diseases. The ‘prion hypothesis’ is the favored hypothesis for the nature of the infectious agent and mechanisms of transmission. It has been proved difficult to demonstrate the involvement of alternative putative infectious agents, such as viruses or nonviral nucleic acid. Common to the various diseases is the presence in the central nervous system (CNS), and occasionally in other tissues, of an abnormal isoform (prion protein, PrPSc) of a host-encoded protein (PrPC). When PrP, which is encoded by the PrP gene (PRNP), is viewed three-dimensionally, more than 40% of the molecule is organized into a-helix. It contains no b-sheets, but conversion to PrPSc involves refolding into an isoform that comprises both b-sheets and a-helices, with the former being predominant. PrPSc is prone to aggregation and accumulation in affected tissues. Prion diseases are part of a wider group of disorders in which abnormalities of protein folding occur, but natural transmissibility has so far only been demonstrated for the prion diseases. The prion hypothesis ascribes infectivity to protein alone, with transmissibility between individuals being influenced by the degree of homology between their PrP gene sequences. Significant differences can prevent transmissibility, whereas small differences result in greater difficulty of transmission and longer incubation periods. This variability in susceptibility to transmission between species is normally referred to as the species barrier. Although PrPSc is the only protein that appears to be associated with infectivity, leading to its use as a marker for infection, there are instances where it is undetectable despite the presence of significant amounts of infectivity. This may simply reflect the lack of sensitivity of the tools used to detect PrPSc, but such instances fuel debate about the potential involvement of another factor in conferring the infectious state. In the absence of a clearly defined pathogen, there are significant challenges in characterizing isolates of the infectious agent from animals or humans. Biological characteristics of disease produced by inoculation of experimental animals, mostly rodents, have been used to define different ‘strains’ of isolates. Molecular tools are overtaking these traditional bioassay approaches, which are too expensive and slow for routine use. Cell cultures remain of limited use at present, as no model can support replication of most natural

Prions and Agents of TSEs: Bovine Spongiform Encephalopathy in Cattle

isolates. Available molecular diagnostics, involving western immunoblotting and enzyme-linked immunosorbent assay (ELISA) in particular, remain rather unsophisticated. Their dependence on the visualization or quantification of PrP after enzymatic degradation makes it difficult to standardize and control methods to facilitate comparison of results obtained in different laboratories. These tools cannot, in isolation, definitively identify a strain or subtype, or confirm an association between BSE in cattle and prion disease in other species. Nevertheless, with the introduction of immunoblotting into the diagnostic armory for surveillance, two additional phenotypes of BSE have been recognized in cattle. Commonly referred to as atypical BSE, they have been classified as H-type and L-type on the basis of their banding pattern on western blot. ‘H’ and ‘L’ denote high and low positions for the unglycosylated band relative to classical BSE, which is referred to as C-BSE for the sake of consistency. Such variants are however rare. Irrespective of the components of the BSE pathogen, it is, along with other prions, relatively resistant to normal approaches to decontamination, disinfection, or sterilization. In the context of food safety, it is clearly not possible to rely on cooking to eliminate infectivity. Regulatory approaches rightly aim to exclude infectious tissues from the food chain.

Epidemiology The visible face of BSE in the UK was a large epidemic of clinically affected animals. Almost 200 000 such animals were identified by the end of 2009. By 2003, however, it was estimated that more than 4 million cattle had actually been infected. The majority died or were slaughtered for human consumption, without being recognized as infected. Epidemics in all other countries have been significantly smaller. In some, the risk of a large long-term epidemic was small for a variety of reasons: exposure levels may have been low; factors required for the escalation of infectivity levels in feed were absent; or, based on British experience, it was possible to

introduce control measures sooner than was possible in the UK. Early epidemiological investigations identified one factor in common for all cases detected. They had all consumed commercial feed that contained meat-and-bone meal (MBM) derived from ruminants (cattle or sheep). This was a commonly used ingredient in feed for dairy cattle in the UK before 1988. In common with many countries, animals that died on farm were usually rendered (cooked), and the residual solid material, MBM, was considered to be a valuable source of protein. Waste material from animals slaughtered for human consumption also entered the rendering system. Before the recognition of BSE, the safety of MBM as an ingredient of animal feed had focused largely on potential contamination with salmonella species. With the benefit of hindsight, it is clear that rendering systems were at that time unable to inactivate BSE infectivity, and the absence of any species barrier between cattle meant that transmission was relatively easy. Although the exact origin of the first case of BSE still remains in doubt, it is clear that the majority of cattle that became infected did so when fed MBM derived from cattle. Although sheep scrapie is known to transmit naturally from mother to lamb (maternal transmission) and from sheep to sheep (horizontal transmission), there remains no evidence that BSE transmits horizontally, and evidence of maternal transmission is slender and improbable. Also, there is no suggestion that the disease is transmitted via germ plasm (semen, ova, and embryos). The cycle of infection, therefore, relied on infectious tissues from cattle being rendered and converted into MBM, followed by subsequent consumption by more cattle, especially in their first year of life (Figure 1). This cycle has not been demonstrated for atypical BSE, primarily because cases have been detected in insufficient numbers to enable detailed epidemiological investigations. It is postulated that they may represent a natural scenario of low prevalence spontaneous disease, which may indeed have become the origin of the C-BSE epidemic. If truly spontaneous, it is possible that the total removal of regulatory controls may enable BSE to return, or an escalation of atypical BSE, especially in countries where

Infected cattle on farm

Fallen stock

Abattoir − healthy and casualty

Carcasses or offal rendered

3

Feed

MBM

Figure 1 The epidemiology of BSE. Crosses denote points at which the cycle of re-infection needs to be broken. It is normally insufficient to rely on breaking the cycle at a single point, but this decision may be influenced by estimates of risk. It is essential to ensure that accidental cross-contamination does not perpetuate risk despite prohibition on the inclusion of raw material or by-product in products at the next stage of the cycle.

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Prions and Agents of TSEs: Bovine Spongiform Encephalopathy in Cattle

rendering standards have not been modified to inactivate the agent. It should however be stressed that at the time of writing, the oral transmission of atypical BSE to cattle has not been demonstrated. Research to investigate this possibility remains in progress. The initial assumptions on which the control of BSE was based were, therefore, shown to be correct. The disease can be eradicated by breaking the cycle of infection via feed. With hindsight, however, it is clear that breaking the cycle is also difficult and requires the rigorous application of regulations and auditing of compliance. Failure to prevent cross-contamination of feed with traces of bovine-derived infectivity led to the infection of many cattle after the use of ruminant MBM was banned. The initial UK feed ban was still very successful, and it is clear that even the application of incomplete measures in many countries did significantly reduce the likelihood of infection. Although it has been demonstrated that calves can become infected by consuming as little as a milligram of BSE-infected brain tissue, the oral ID50 is now estimated to be 0.16 g. The significance of cross-contamination of feed with such a low infectious dose during the production, storage, and transportation of feed ingredients was not anticipated, and led to incomplete compliance with feed controls. Draconian riskmanagement measures are justified by the need to eliminate such feed-borne exposure sooner rather than later. Exported MBM also proved to be the conduit by which BSE spread to many other countries, some before BSE was even recognized in the UK. Infected cattle were also exported. When infected imported or indigenous cattle died, they initiated epidemics in the importing countries. The timescales from the initiation of recycling of infectivity to the implementation of control measures were usually shorter than in the UK, thus preventing the weight of infectivity in cattle feed from reaching the levels experienced in the UK. Additionally, unlike the UK, in many countries MBM was only an infrequent component of cattle feed. Early in the course of the epidemic in the UK, the recognition of TSEs in animal species in which such diseases had not previously been recorded strengthened concerns that BSE was capable of spreading to nonruminant species. In domestic cats, feline spongiform encephalopathy (FSE) was recognized in 1990, and also occurred in exotic Felidae, including cheetah, lion, tiger, ocelot, puma, Asian golden cat, and leopard cat. Additional ruminant species were also affected and included nyala, greater kudu, and three species of oryx, eland, ankole cattle, and bison. Most of the cases in ruminants and felids, other than domestic cats, were in zoological collections in the UK. Although the exotic felids would most probably have consumed raw infected tissues from cattle, domestic felids and all ruminants are more likely to have consumed MBM incorporated into their feed.

Pathogenesis Because the infectious agent cannot be visualized, even by microscopic examination, the pathogenesis or course of the disease from infection to death has been proved difficult to study. Naturally infected animals were examined early in the British BSE epidemic, but the examination of tissues from

animals killed at clinical end stage cannot determine the behavior and distribution of the agent in the body during the incubation period of several years. Quantification of risk associated with the consumption of tissues, therefore, relied on the examination of materials derived from experimentally infected calves, sequentially slaughtered during the incubation period. The gold standard for determination of the presence of infectivity was the inoculation of inbred laboratory rodents. Bioassay in cattle, and in genetically modified mice, have more recently offered increased assay sensitivity. The timescales involved in awaiting incubation in cattle, followed by incubation to negative end points in the assay model, have been long (up to 14 years). The high cost of such studies has also limited the number of assays such that negative results must be interpreted with caution. At times, the categorization of a tissue as noninfectious may arise from the inoculation of a single sample from a single animal. This does not devalue the result, particularly if interpreted alongside the additional negative results from other tissues. Together, they build up a body of evidence that enables confident classification into risk categories, recognizing that negative transmissions cannot be guaranteed to represent the total absence of infectivity. All assay models have limits to their power to detect infectivity. Once BSE infectivity is ingested, replication of the agent is first detectable, both by bioassay and by immunochemical detection of PrP, in the Peyer’s patches (PPs) of the lower small intestine. Most cattle appear from epidemiological studies to become infected in calf-hood, which partly reflects early exposure through feed, but may also be dependent on the presence of prominent and numerous PPs in the ileum of the young animals. These regress naturally when mature. The next organ in which significant infectivity can be detected is the CNS, appearing almost simultaneously in the brain stem and spinal cord. Thereafter, as infectivity levels rise exponentially and disperse within the CNS (including the eye), there also appears to be gradual retrograde spread into the peripheral nervous system (PNS), including the dorsal root ganglia (DRG), trigeminal ganglion, and peripheral nerve trunks (facial, optic, sciatic, phrenic). DRG lie within the vertebral column, close to the spinal cord, and form the interface between the CNS and motor and sensory peripheral nerve trunks. The route by which infectivity migrates from the intestine to CNS remains uncertain, but most probably involves transport via the autonomic nerves (vagus and splanchnic) and their associated ganglia. These nerves are, therefore, infected early in the pathogenesis, during preclinical stages of disease. The adrenal gland with its rich sympathetic innervations contains infectivity at or close to clinical onset. Although infectivity has not been detected in the thymus or spleen, two of the original tissues designated as potential risks to consumers, or indeed in lymph nodes, traces have been detected in palatine tonsils of experimentally infected cattle. Additionally, single equivocal results from sternal bone marrow collected close to the onset of clinical disease in experimentally infected cattle and from pooled nictitating membranes from naturally infected cattle have not been replicated. Consequently, although infectivity has been demonstrated to circulate in blood in scrapie-infected sheep, it is not

Prions and Agents of TSEs: Bovine Spongiform Encephalopathy in Cattle

possible to conclusively demonstrate a role for hematogenous spread through the body in BSE-infected cattle. Indeed, blood is accepted as a safe commodity as long as there is no risk of contamination with brain tissue at slaughter. The only additional tissue in which there is published evidence of transmission is muscle. This result, from a single naturally infected and clinically affected cow, was based on evidence of transmission to only one of 10 inoculated transgenic mice expressing the bovine PrP gene. This transgenic mouse, estimated to be even more susceptible than a calf to BSE, otherwise produced results that were consistent with all other bioassays of bovine tissues. Consequently, muscle is not considered to be infected, although the presence of peripheral nerves within muscle masses gives rise to potential risk if consumed after the onset of clinical disease. Arising from the analysis of pathogenesis is a general theme that in the early stages of infection, the greatest risk is associated with the intestine and particularly the ileum. In the later stages of incubation, intestinal infectivity levels are Table 1

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exceeded by CNS infectivity, which reach maximal levels after onset of clinical disease. Bioassay results have demonstrated that infectivity can be present marginally before the CNS tests positive with a postmortem test. It is not possible to specify when the CNS will become infected in any individual animal. There is considerable variation in incubation period, with naturally infected and clinically affected cattle in the UK ranging from 20 months to 22 years of age when identified. Incubations will have been marginally shorter than lifespan. In experimentally infected cattle, incubation period is generally dose-related, but even within a single dose group it can be highly variable. The assumption that risk is primarily associated with CNS from cattle aged 30 months or more, as used in defining regulations for the removal of specified risk materials (SRMs), derives from the pragmatic interpretation of epidemiological data, taking into account the decline of risk of infection in young calves by the time the rule was first adopted in the UK in 1996. It is however supported by subsequent evidence from

Tissues in which infectivity is confirmed or presumed to be present

Tissue Higher levels of infectivity Brain and spinal cord Eye (retina) and optic nerve Trigeminal ganglion Spinal ganglia Lower levels of infectivity Ileum (lower small intestine)

Gastrointestinal tract excluding the ileum

Peripheral nerve trunks Facial Sciatic Phrenic Autonomic nerves and ganglia, vagus/splanchnic nerves Tonsil

Bone marrow – equivocal result Third eyelid (nictitating membrane) – equivocal result Muscle (equivocal result)

Not detected but designated as SRMs Thymus Spleen Lymph nodes

When infectious

Designated SRMs

Late incubation – shortly before onset of clinical disease After onset of clinical disease After onset of clinical disease After onset of clinical disease

Yes

Probably throughout incubation, but with infectivity levels greatest in first two years of incubation Probably throughout incubation, but with infectivity levels greatest in first two years of life. Infectivity levels are much lower than detected in the ileum. Not yet classified by WHO as infectious After onset of clinical disease

Probably from early stages of incubation, and up to clinical onset Probably for significant part of incubation period, but only demonstrated in experimentally infected cattle Not clear, but possibly close to onset of clinical disease Not clear – may be a rare event

Yes – may be incorporated into definition of skull Yes – may be incorporated into definition of skull Yes – removed with vertebral column Yes

In EU, still assumed to be infected, and, partly for ease of risk management, entire intestine is defined as SRM. Other countries restrict definition to ileum No – but likely to be partially removed during carcass dressing/deboning and jointing of meat

No – partial removal likely during carcass dressing Yes

No No – but will be removed along with skull and eyes

Not clear – evidence is unconvincing, has not yet been repeated

No

No infectivity detected No infectivity detected No infectivity detected

Yes originally, but now delisted Yes originally, but now delisted No – but visible lymph nodes removed along with major peripheral nerves during deboning process

Note: Infectivity levels and risk levels are not synonymous. Many factors determine the degree of risk to consumers, and will include not only the quantity of tissue to which they are exposed, but also the stage of incubation of the source animal (early or late), and the route of exposure. For example, oral exposure is regarded as relatively inefficient in comparison with direct inoculation into the brain or blood stream.

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Prions and Agents of TSEs: Bovine Spongiform Encephalopathy in Cattle

calves experimentally exposed to low doses of infectivity, to simulate the most likely dose received naturally, and provides a considerable margin of safety to consumers. Tissues in which infectivity or PrPSc have definitely been detected are summarized in Table 1, which is based on data collated and tabulated on behalf of the World Health Organization (WHO).

Controls The principles of risk management for animals and humans were, and indeed still are, similar. They involve a precautionary approach, based on known science, but taking into account the need for feasible regulatory measures. The aim is to eliminate or minimize the risk of exposure by mouth, the only routes by which BSE is considered to be naturally transmitted to cattle and humans. The initial steps were taken before there was any understanding of the pathogenesis of BSE, and were based on what was already known about sheep scrapie. With the benefit of hindsight, this proved to be appropriate. Measures were, however, tempered by the recognition of uncertainty regarding the science of BSE, necessitating a balance between the maintenance of viable food production and allied industries and reduction of putative risk. Excessive regulation, unsupported by scientific evidence, could have generated resistance to the implementation of controls, and put lives at risk.

Protection of Animal Health Because cattle became infected through the consumption of ruminant-derived MBM, the key control was the prevention of the recycling of ruminant protein. Therefore, the first feed ban in the UK prohibited the feeding of ruminant-derived MBM to ruminants. The World Organisation for Animal Health (OIE) continues to retain this position, insisting that the mechanism of implementation and enforcement of such a prohibition is an issue for individual countries. In time it became clear that some cattle were still becoming infected in the UK even if born after the introduction of the ruminant-to-ruminant feed ban. In part, this arose because ruminant-derived MBM still contained some infectivity (not all infected tissue had been identified and excluded at that time), and the use of such material in feed for pigs and poultry was still permitted. Rendering standards had not been changed at this point to maximize the inactivation of infectivity in source tissues. These changes are described below. The presence of contaminated MBM in facilities and equipment that produced or stored feed for ruminants led to a real risk of cross-contamination. Furthermore, it was not possible to test the feed for evidence of random contamination, and uncertainty remained with respect to verification of the species of origin of feed ingredients. No tests introduced to audit feed production systems actually attempt to identify infectivity. They simply aim to detect the presence of tissues derived from prohibited species. As a result, from 1994 the use of any mammalian-derived MBM (MMBM – to include porcine protein) in ruminant feed

was prohibited in the European Union (EU). The continuing evidence for the role of cross-contamination in premises manufacturing animal feed led to an even wider prohibition in the UK in 1996. From this point, MMBM was excluded from all feed for farmed animals, including fish and horses, and indeed also for pets where there was a risk of contamination of feed intended for ruminants. Feed prohibitions were similarly extended in the EU at the beginning of 2001, although the prohibition referred to processed animal protein rather than MMBM. Minor exclusions are permitted, such as the feeding of milk or blood meal. Three additional measures reinforce the protection of cattle, namely the slaughter and destruction of clinically affected cases, a prohibition on the use of high-risk tissues (SRMs) for animal feed (although now encompassed in rules for their segregation and destruction) and improved rendering standards. Because clinically affected cattle represent the greatest source of infectivity, in particular within the CNS, their exclusion from the rendering system will have reduced the infectious load entering the process. SRMs were originally defined for the protection of human health, and were removed from healthy animals at slaughter. Their removal from animal feed, including animals that die on farm from which SRMs are not removed, protects animal health by also reducing the infectious load entering the rendering and animal feed chains. Although retrospective audits and advancement of the underlying science confirmed that some infectivity continued to enter the rendering plants in the UK in the early 1990s, the absence of cases of FSE in cats born in the UK after the first exclusion of SRMs from animal feed demonstrates the effectiveness of the measure. Research conducted in the EU into the effectiveness of rendering processes in inactivating BSE, and scrapie demonstrated that most could not guarantee the elimination of infectivity. This led to changes in EU regulations, both in terms of processing standards (e.g., higher temperatures) and the segregation of raw materials into risk categories. The end result, a regulation that prescribes processes for the handling of all wastes, demands the most rigorous standards for tissues and carcasses that potentially carry a risk of transmitting TSEs. Animal by-products, other than SRMs, arising from animals that are healthy and fit for human consumption are subject to less rigorous processing standards. In non-EU countries, rendering processing standards have not necessarily been adjusted, however, particularly where risk is perceived as being low. In summary, the removal, segregation, and processing of SRMs, or carcasses containing SRMs, reinforce the wider feed prohibition in order to minimize the risk of continued exposures and infection of cattle through the cross-contamination of feed. Protection of animal health inevitably delivers a reduction in risk for humans, albeit over a longer time frame. With the incubation period averaging 5 years in the UK, and frequently much longer elsewhere, it is usually necessary to wait many years before controls visibly reduce the prevalence of disease. Subtle effects may be seen sooner, but a decline in case numbers is likely to take 5–6 years. This is why it is recommended that preventive measures are implemented before an epidemic is detected in countries classified as being at risk. The consequences of the introduction of controls, in the

Prions and Agents of TSEs: Bovine Spongiform Encephalopathy in Cattle

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FB 2 FB 3

FB 1 40 000

Year of birth Year of detection

35 000 30 000

Cases

25 000 20 000 15 000 10 000 5000

19 8 19 0 81 19 8 19 2 8 19 3 8 19 4 8 19 5 8 19 6 87 19 8 19 8 8 19 9 9 19 0 91 19 9 19 2 93 19 9 19 4 9 19 5 9 19 6 97 19 9 19 8 99 20 0 20 0 01 20 0 20 2 0 20 3 0 20 4 0 20 5 06 20 0 20 7 0 20 8 09

0 Year of birth or detection Figure 2 Graphic of British epidemic, showing by date of birth and date of confirmation – with feed bans superimposed, based upon data supplied by the Veterinary Laboratories Agency, United Kingdom. The two lines represent all cases detected, predominantly as clinical cases, the first plotted by date of birth (infection will have occurred shortly afterwards), the second by date of detection. The second peak includes animals for which no accurate date of birth was available, and therefore represents more animals than in the first peak, but this does not detract from the trends depicted, and their relationship to feed bans. The interval between peaks represents the mean incubation period of 5 years. FB1: first feed ban – ruminant protein to ruminants; FB2: second feed ban – mammalian MBM to ruminants; FB3: third feed ban – MMBM to all livestock.

form of graphic representation of the UK epidemic, on which the different feed bans are superimposed, are provided in Figure 2.

Protection of Human Health The eradication of BSE offers the greatest protection to consumers, but interim measures are necessary to mitigate risk of exposure until this is achieved. The most obvious protective measure is the exclusion of clinically affected cases of BSE from the human food chain. These represent the greatest risk because infectivity levels in the brain and spinal cord are at maximal levels. It is important, however, to appreciate that the clinical signs of BSE can be very subtle in their early stages, and will not always guarantee detection and exclusion of the affected animal from the food chain. By late 1989, it proved possible to define and introduce additional controls in the UK that recognized the fact that infected cattle, destined to be consumed by humans, remained alive but undetectable on British farms. They could not be tested while alive to determine whether they were infected, and this remains unchanged at the time of writing. Based on knowledge of the pathogenesis of sheep scrapie, a short list of tissues was identified that appeared to represent the greatest risk to consumers if eaten. At the time, there were no data about the actual risk associated with these tissues in cattle, nor evidence that humans were susceptible to BSE, but they were

excluded from the human food chain on a precautionary basis. With time, research into BSE has confirmed the potentially infectious status of some of these tissues (brain, spinal cord, intestine), but did not confirm the categorization of others (thymus, spleen). Additional tissues have been added to the list of SRMs on the basis of new research findings, but it is interesting that results arising late in the EU epidemic, when prevalence of infection was low, and consumer concerns reduced, did not trigger the extension of the list of SRMs. For example, peripheral nerves have never been classified as SRMs, but are thought only to become infectious at about the time of clinical onset. With respect to the likely impact of such tissues on human exposure levels, it is necessary to appreciate that the amounts of infectivity estimated to be present in nerves are substantially less than in the brain and spinal cord. There continues to be some international disagreement in the definition of SRMs, with the EU adopting a more precautionary approach than elsewhere. This is primarily because of the scale of the crisis in consumer confidence that occurred in Europe in 1996. Consumer risk was inevitably greater in the EU by virtue of the large number of infected cattle present, but relaxation of regulations is anticipated as risk falls to acceptable levels. It is, however, more difficult to relax regulations than to introduce them, because the relaxation inevitably increases risk to consumers even though the residual risk may still be extremely low. SRM measures in Europe, North America, and Japan as at January 2009 are summarized in Table 2. Some SRMs, such as skull and vertebral column,

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Prions and Agents of TSEs: Bovine Spongiform Encephalopathy in Cattle

Table 2 A summary of bovine-derived SRMs in Europe, North America, and Japan as at January 2009 European Union and Switzerland Cattle Skull (including brain and eyes) Tonsils Spinal cord Vertebral column (including dorsal root ganglia – DRG – but excluding vertebrae of the tail and the transverse processes of lumbar and thoracic vertebrae) Intestines and mesentery

412 months All ages 412 months 430 months

All ages USA

Cattle Skull (including brain, eyes, and trigeminal ganglia) Tonsils Spinal cord Vertebral column (including dorsal root ganglia – DRG – but excluding vertebrae of the tail and the transverse processes of lumbar and thoracic vertebrae, and wings of sacrum) Distal ileum

430 months All ages 430 months 430 months

All ages Canada

Cattle Skull (including brain, eyes, and trigeminal ganglia) Tonsils Spinal cord Dorsal Root Ganglia (Vertebral column, excluding vertebrae of the tail and the transverse processes of lumbar and thoracic vertebrae, and wings of sacrum, is not defined in law as SRM, but removal from the human food chain is ensured by administrative action through meat hygiene controls.) Distal ileum

430 months 430 months 430 months 430 months

All ages Japan

Cattle Head (including brain, eyes, and tonsils, but excluding tongue and cheek meat) Spinal cord Vertebral column (including dorsal root ganglia) Distal ileum

All ages All ages All ages All ages

are not inherently infected. They are designated as SRMs because they are closely associated with the brain and spinal cord, and it is assumed that they are either contaminated during the slaughter and carcass dressing process or that it is impossible to completely separate the CNS tissues from the bones encasing them. There were two further examples of protective measures that were driven by potential contamination of raw materials during the process of slaughter and meat production, rather than the inherent presence of infectivity. These affected procedures for the stunning of cattle and the production of mechanically separated meat. When cattle were stunned

before slaughter with a mechanism that injected air into the skull, particles of brain tissue were subsequently detected in the blood stream. This meant that any tissues exposed to that blood could potentially be contaminated. Blood itself has not been shown to be infectious. The prohibition of the use of such methods minimized the risk of accidental contamination of muscle and other organs. Mechanically separated meat, however, was a process by which the vertebral column and some other bones were subjected to further treatment after the primary dressing of the carcass. This process mechanically stripped residual meat from the bone. Any residual infectivity attached to the vertebral column after carcass splitting, via retained DRG, incomplete removal of spinal cord, or contamination of the cut surface caused by the splitting saw, presented a risk that the stripped meat could be contaminated. Controls were, therefore, necessary to prohibit the use of bovine bones in the manufacture of such products. It is interesting that no cases of vCJD have been identified in the UK in individuals that were born after the introduction of the first SRM prohibition. Although with hindsight it is now known that this measure did not remove all infectivity from the food chain, it appears to have had a significant effect in reducing human exposures and likelihood of infection. Two further measures were perceived to have possible additional protective effect for humans. First, milk from clinically affected cattle was excluded from the human food chain (UK only). Although infectivity has not been detected in milk from BSE-affected cattle, it has been demonstrated in sheep with scrapie. Second, the identification of birth or feed cohorts of affected cattle, and their exclusion from the food chain, was adopted internationally. Birth or feed cohorts represent animals that were born or reared at about the same time as an infected animal, on the same farm, and were potentially exposed to the same feed source. They clearly represent a greater risk than the population at large, but with the exception of cattle born in the UK before the first feed ban came into force in 1988, the numbers of infected animals actually detected in cohorts has been extremely low. A final protective measure, not specifically introduced to reduce the risk for BSE, now included in international BSErelated guidelines for trade in beef includes the removal of visible lymph nodes and nerves during the process of dressing carcasses following slaughter. This applies particularly to deboned beef, where preparation of the meat for sale, storage, and transit has involved the removal of such tissues for many years. Infectivity has never been detected in bovine regional carcass lymph nodes, or in bones, but such a measure provides the final level of protection should clinically affected cattle be inadvertently slaughtered. The introduction of postmortem testing of healthy cattle of more than 30 months of age in Europe, and later in Japan even in younger cattle, was frequently portrayed and perceived as a risk reduction measure. There is no doubt that it increased consumer confidence, but in reality, SRM removal ensured that the vast majority of infectivity was removed from the food chain anyway. Furthermore, SRM removal protected consumers from instances where infectivity was present in the brain, but the postmortem test result was negative. Following research into the time-course of detection of PrPSc by

Prions and Agents of TSEs: Bovine Spongiform Encephalopathy in Cattle

postmortem tests on experimentally infected cattle, it has been estimated that postmortem testing would only detect approximately 50% of infected animals at 1.7 months before onset of clinical signs. Detection would fall close to zero at 3 months before onset of disease, or 97% of the incubation period. In most countries, where epidemics are likely to be small and infectivity levels in cattle feed low, incubation periods are likely to be very long. Therefore, infected cattle that are slaughtered while still healthy are most unlikely to be detected by postmortem testing. At the time of writing, investigations into cases of vCJD have not identified any route of infection directly from cattle to humans other than through contaminated food produced before the introduction of regulatory controls.

Specific Food Products To the consumer, the relationship between a food product and a specific source tissue may not always be obvious. This is particularly so where a manufacturing process is involved. Additionally, the pooling of materials from different animals and regions compromises traceability. Regulatory authorities have used the evolving knowledge of the pathogenesis of BSE to ensure that food, whether processed or unprocessed, is safe by excluding certain tissues from the food chain, but their impact on certain commodities may not be obvious. In view of their widespread use, it is appropriate to discuss certain commodities and their safety in a little more detail. It is important to remember that safety is assured by two measures: the exclusion of infectivity by the removal of tissues in which the presence of infectivity is suspected or confirmed and the prevention of contamination with infected tissues during the manufacturing process. At no time have authorities accepted the principle that food can be made safe solely by the application of specific processing standards. Heating, or cooking, has never been accepted as sufficient to remove infectivity, and even in the production of animal feed, it is only expected to reduce infectivity levels rather than guarantee elimination. The most obvious commodity that receives minimal processing before consumption is milk. The initial exclusion of milk from clinically affected cows from the human food chain in the UK was precautionary. It presumed that because sheep scrapie appeared to transmit from ewe to lamb, this may have been via milk. There is now sound scientific support for such transmission in sheep, but none for transmission of BSE via bovine milk. For this reason the OIE, supported by the WHO, lists bovine milk as a commodity that can be traded safely irrespective of the BSE status of the country of origin. Gelatine is an animal by-product that is incorporated into a wide range of food and pharmaceutical products. It is derived from two source tissues, hides or bones, and the extraction of gelatine involves rigorous chemical processes. Infectivity has not been detected in hides. Gelatine derived from skin or hide is, therefore, considered to be free of risk. Although infectivity has not been detected in bone, the close association of the skull and spinal (vertebral) column with the brain and spinal cord means that the bones are inevitably contaminated with CNS tissue during the process of carcass

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dressing. Despite the rigor of the process for extracting gelatine from bones, it has not been possible to conclusively demonstrate that it is free of infectivity when derived experimentally from raw materials that contained BSE infectivity. In part, this is because of the limitations of sensitivity of the assay methods. The outcome of extensive consideration by expert committees is a requirement to exclude bovine skull and vertebral column from the manufacturing process in BSE-affected countries. Tallow is a product that most consumers are unaware of, but is an essential ingredient of much processed food and of pharmaceuticals. It is produced by rendering tissues resulting in three by-products, namely water, fat, and protein. The water is driven off as steam, the fat is separated from the protein, and the latter is further processed into MBM. The separated fat is processed further to produce a range of end products or derivatives. There are several categories of tallow determined in part by the nature of the raw materials and by the type of rendering process used. Tallow intended for use in food products would be derived from animals and raw materials already inspected and passed fit for human consumption. Although there is no published evidence that infectivity is found in tallow after rendering, there is both unpublished data and scientific opinion that suggest that such a risk cannot be totally ignored. As for gelatine, this subject has been debated extensively, concluding that any residual infectivity is likely to be associated with traces of protein that may remain in the tallow. Consequently, trade rules focus on establishing maximal levels for insoluble impurities in tallow if it is allowed to be traded freely. Levels of contamination above this maximum require the application of additional conditions to the production process, which may include the exclusion of certain SRMs. It is worth stressing, however, that the residual risks estimated to be associated with gelatine and tallow are extremely low. Furthermore, these commodities are not inherently risky. If there is no BSE in the cattle from which source tissues are derived, then there will be no scope for infectivity to be present in the end product unless contaminated from another source after production.

Diagnosis At the time of writing, there is no live animal test for BSE. Claims that tests can detect BSE in vivo must be treated with caution unless accompanied by evidence of thorough evaluation by a third party. Clinical signs alone are insufficient to enable detection of the majority of infected animals. In most countries that have experienced BSE, such cases are too rare to reinforce diagnostic skills, and only the most extreme clinical signs are recognized. In accordance with rules established by the OIE, BSE should be legally notifiable. This ensures that suspicion of disease is reported to national authorities, and that tests used to confirm a diagnosis are appropriate. The accepted approaches for postmortem diagnosis are established in the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial animals, and accommodate the likely range of technical facilities that may be present in member countries.

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Prions and Agents of TSEs: Bovine Spongiform Encephalopathy in Cattle

BSE Risk – Categorization of Countries Traditional approaches to international trade in animals and animal products require the declaration of health status of source countries, zones, farms, or animals. This may involve the testing of individual animals or herds. Because of the difficulties encountered in identifying BSE-infected animals while still alive, national and international organizations have adopted alternative approaches to define BSE-related risk. Additionally, it is accepted that the risk mitigation measures that need to be introduced in a country should be driven by the estimated risk within it, as determined by independent scrutiny of national data. In other words, countries that represent a low BSE risk require fewer control measures and smaller surveillance programs than those with significant risk. A proportionate response to risk is considered appropriate. Although with other diseases the incidence of disease is normally an appropriate indicator of risk, the recognition that clinically affected cases of BSE are difficult to detect and that they represent only a small proportion of infected animals challenges this traditional approach to categorization. The European Commission was advised by its former Scientific Steering Committee (SSC) to apply a comprehensive qualitative risk-assessment approach, based initially on likelihood of exposure from the UK through the importation of MBM, and potential for further amplification of infectivity after importation. This approach proved to be very successful, and was reinforced by the later results of active surveillance. Infected cattle were detected in several countries that previously claimed freedom from disease, and which may have exported BSE-infected cattle or contaminated products before indigenous cases were identified. The SSC methodology was eventually updated by the European Food Safety Authority (EFSA), and took into account changes adopted in the OIE rules for categorization and surveillance. It is important to recognize the fact that statements of BSE-related risk are most robust where countries have been categorized as being of negligible risk or controlled risk. Remaining countries, which are of undetermined risk, may not have been exposed to BSE. It would be wrong, therefore, to presume that their inclusion within this broad category is an indicator of risk, but the absence of external evaluation of risk means that statements of safety must be interpreted with caution. Future categorization will take into account the establishment of surveillance programs that target the most appropriate population of cattle (potential clinical cases in particular), but also recognize the infrastructure in individual countries. A one-size-fits-all program is not possible, other than in the establishment of a target for each to achieve in order to facilitate categorization. Such programs are intended to detect trends in prevalence of infection, as a means of monitoring the effectiveness of controls.

Conclusions There are two reasons for anticipating that BSE will eventually assume a more appropriate position in the spectrum of foodborne zoonoses to which consumers are exposed. First,

however incomplete, the research conducted over the past 20 or more years indicates that irrespective of disease incidence in a source country, the distribution of infectivity within the body of infected cattle lends itself to practical approaches to risk reduction. Second, the natural experiment conducted in the UK before the introduction of control measures, when exposure levels were high, demonstrates that exposure of both animals and humans can be controlled. The steep and continued decline in prevalence of BSE in cattle following the introduction of the feed ban illustrates this statement, albeit with a time lag that is determined by the long incubation period. In addition, the absence of vCJD cases in humans born after the first ban on consumption of SRM in 1989 will, if sustained, confirm the effectiveness of excluding targeted high-risk tissues in protecting consumers. Although the consequences of infection are fatal, the likelihood of infection after the initial SRM regulation was low relative to the quantity of infectivity that entered the food chain beforehand. Evidence for transmission between humans, for example via blood transfusions, does not undermine such a statement. The first announcement of the likely transmission of BSE to humans prompted unprecedented concerns about human safety. Uncertainty prompted prognostications of doomsday scenarios, with millions of human deaths. They also failed to recognize the fact that the human cases arose from historical exposure, before the introduction of protective measures. Global events drove the establishment of international protective measures to unprecedented levels, but the process of relaxation has begun and may conclude with residual, but focused, measures that may remain in place in perpetuity. Countries that have taken the trouble to determine their status vis-a`-vis BSE risk should be congratulated, and others encouraged to transparently eliminate any uncertainty surrounding the status of their national herds.

See also: Analytical methods: Transmissible Spongiform Encephalopathy Diagnosis. Prions and Agents of TSEs: Creutzfeldt–Jakob Disease

Further Reading Arnold ME, Ryan JBM, Konold T, et al. (2007) Estimating the temporal relationship between PrPSc detection and incubation period in experimental Bovine Spongiform Encephalopathy of cattle. Journal of General Virology 88: 3198–3208. EC (2005) TSE Roadmap. Comm. 15 July, 2005. 322 Final. http://ec.europa.eu/ food/food/biosafety/bse/roadmap_en.pdf European Food Safety Authority (EFSA) (2005) Quantitative assessment of the residual BSE risk in bovine-derived products. The EFSA Journal 307: 1135. http://www.efsa.europa.eu/EFSA/efsa_locale-1178620753812_;1211902588886. htm European Food Safety Authority (EFSA) (2007) Opinion of the scientific panel on biological hazards on the revision of the geographical BSE risk assessment (GBR) methodology. The EFSA Journal 463: 1–35. http://www.efsa.europa.eu/ EFSA/efsa_locale-1178620753812_1178620775032.htm European Food Safety Authority (EFSA) (2009) Scientific opinion of the scientific panel on biological hazards on BSE risk in bovine intestines. The EFSA Journal 1317: 1–19. http://www.efsa.europa.eu/en/scdocs/doc/ 1317.pdf Ferguson NM and Donnelly CA (2003) Assessment of the risk posed by bovine spongiform encephalopathy in cattle in Great Britain and the impact of potential

Prions and Agents of TSEs: Bovine Spongiform Encephalopathy in Cattle

changes to current control measures. Proceedings of the Royal Society Biological Science Series B 270: 1579–1584. Hornlimann B, Riesener D, and Kretzshmar H (eds.) (2006) Prions in Humans and Animals. Berlin/New York: De Gruyter. World Organisation for Animal Health (OIE) (2009) Bovine spongiform encephalopathy. Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, Vol. 2. Paris: OIE. Ch. 2.4.6. http://www.oie.int/fileadmin/Home/eng/ Health_standards/tahm/2.04.06_BSE.pdf World Organisation for Animal Health (OIE) (2009) Bovine spongiform encelphalopathy. Terrestrial Animal Health Code, Vol. 2. Paris: OIE. Ch. 11.5. http://www.oie.int/index.phpid=169&L=0&htmfile=chapitre_1.11.5.htm World Organisation for Animal Health (OIE) (2003) Risk analysis of prion diseases in animals. Review scientific et technique Office International des E´pizooties 22(1): 1-345. http://web.oie.int/boutique/index.phppage=ficprod&id_produit=84&fichrech=1& lang=en&PHPSESSID=143851c9c46131fd298bfe153a922c60 Wells GAH, Konold T, Arnold ME, et al. (2007) Bovine spongiform encephalopathy: The effect of oral exposure dose on attack rate and incubation period. Journal of General Virology 88: 1363–1373. World Health Organization (WHO) (2010) WHO Tables on Tissue Infectivity Distribution in Transmissible Spongiform Encephalopathies. http://www.who.int/ bloodproducts/tablestissueinfectivity.pdf

Relevant Websites http://www.defra.gov.uk/animal-diseases/a-z/bse/ Defra (2010) Department for Environment, Food and Rural Affairs – Home page on all TSEs.

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http://ec.europa.eu/food/food/biosafety/tse_bse/index_en.htm European Commission (2009) DG Health and Consumers: Biological Safety of Food – BSE and scrapie homepage, legislative chronology and links to scientific opinions. http://www.efsa.europa.eu/EFSA/ScientificPanels/efsa_locale1178620753812_BIOHAZ.htm European Food Safety Authority (EFSA) (2009) Scientific Panel on Biological Hazards. Opinions and reports on BSE. http://www.tafsforum.org/ International Forum for Transmissible Animal Diseases and Food Safety (2009) Guidance documents (position papers) on BSE and scrapie. http://www.cjd.ed.ac.uk/index.htm National CJD Surveillance Unit, United Kingdom (2009) Home page on CJD and human surveillance data. http://web.archive.org/web/20010203064300/bseinquiry.gov.uk/ The BSE Inquiry (2000) The Inquiry into BSE and variant CJD in the United Kingdom. Homepage for historical data on BSE in the United Kingdom, 1986–1996. http://www.defra.gov.uk/vla/science/sci_tse_rl_web.htm Veterinary Laboratories Agency (2009). TSE Web resource home page of OIE Reference Laboratory, with links to multiple sources of information. http://www.who.int/topics/spongiform_encephalopathies_transmissible/en/ World Health Organization (WHO) (2009) Bovine spongiform encephalopathy homepage. http://www.oie.int/en/animal-health-in-the-world/bse-specific-data/ World Organisation for Animal Health (OIE). Animal disease data by disease.