Creutzfeldt-Jakob disease and the risk from blood or blood products.

The occurrence of iatrogenic cases of Creutzfeldt-Jakob disease (CJD) and the isolation of infectivity in some laboratory transmission studies in transmissible spongiform encephalopathies raises the possibility that CJD might be accidentally transmissible through blood or blood products. Epidemiological evidence, although not conclusive, does not suggest that classical CJD is transmitted through this route. However, new variant CJD (nvCJD) might pose greater risks of accidental transmission of infection and mechanisms to reduce the theoretical risk are under consideration. The theoretical risks from CJD and nvCJD must be balanced against the established therapeutic benefits of blood and blood products.

Bovine spongiform encephalopathy. Epidemiology, low dose exposure and risks.

BSE has occurred in the U.K. as an extended common source epidemic since 1985/86. The vehicle of infection was concentrated feeds containing meat and bone meal produced by the rendering of ovine, bovine and other animal wastes. The epidemic was probably initiated in 1981/82 when a sudden decline in the use of solvents in rendering allowed a low incidence of scrapie-like infection to occur in cattle. However, the presence in feed of bovine material that, from 1984/85 (or earlier), was increasingly infected with a cattle-adapted strain of agent amplified the epidemic greatly. Nevertheless, the incidence of BSE cases nationally has been low because of the generally low effective exposure of cattle to infection in feed. This, and a combination of risk factors that were probably unique to the U.K. can explain why relatively few cases of BSE have occurred in other countries. The feeding of ruminant-derived protein to all species of ruminants was banned in Great Britain in 1988, and in Northern Ireland in 1989. A more selective approach was subsequently adopted to minimize the risks of BSE infection of other species, including man. This was based on excluding from food a small number of bovine offals whose use and predicted infectivity titers would constitute the greatest potential source of infection. Recent studies of BSE support the basis of the specified bovine offals ban and suggest that more tissues were restricted than may have been necessary.

Bovine spongiform encephalopathy: an appraisal of the current epidemic in the United Kingdom.

Bovine spongiform encephalopathy (BSE) is a food-borne infection of cattle caused by the use of contaminated meat and bone meal in concentrated feeds. The UK epidemic was initiated by a sudden exposure to infection in 1981-1982, which was associated with a dramatic reduction in the use of organic solvents in the manufacture of meat and bone meal. This change almost certainly removed two partial disinfection steps and allowed enough contamination with a scrapie-like agent to infect cattle. Although it is assumed that the epidemic originated with scrapie infection crossing the species barrier, cattle-to-cattle recycling of infection, via feed, amplified the epidemic very considerably. There would have been a strong tendency for recycling to select a single cattle-adapted strain of agent, and this strain of BSE could well be different from scrapie. There is evidence to support both predictions. Because the median incubation period of BSE is 4-6 years, clinical cases did not appear until 1985-1986, by which time the recycling of infection in cattle was probably well established. However, the average food-borne exposure to infection has remained low resulting in a mainly sporadic occurrence of BSE. Signs of an imminent decline in the epidemic were unmistakable early in 1993, which is over 4 years after the feeding of ruminant-derived protein was banned to prevent new infections of cattle.

Bovine spongiform encephalopathy.

A detailed account is given of the occurrence of bovine spongiform encephalopathy (BSE), current research into the aetiology of this new disease of cattle, and the relationship between BSE, scrapie and other similar diseases. Epidemiology, clinical signs, pathology, diagnosis, prevention and control are described.

Scrapie-infected spleens: analysis of infectivity, scrapie-associated fibrils, and protease-resistant proteins.

Scrapie-associated fibrils (SAF) and protease-resistant proteins (PrP) were isolated from spleens and brains of clinical animals (mice and hamsters) from three scrapie agent-host strain combinations, and their concentrations were compared with infectivity levels. The spleens of infected animals contained lower levels of infectivity, PrP, and SAF than did brains. Regardless of the route of infection, both SAF and infectivity were detected in spleen before brain. Infectivity increased in brains and spleens of 139A-infected mice before the detection and increase in SAF, suggesting that the synthesis of SAF and PrP may not be the limiting factor in agent replication. In contrast to those in ME7- and 263K-infected animals, the Western blot profiles for PrP from brain and spleen of 139A-infected mice exhibited distinct differences. Results indicate that SAF and PrP found in the spleens are both organ- and scrapie strain-specific.

An overview of bovine spongiform encephalopathy.

None of the diseases caused by the "unconventional slow viruses" is highly infectious in its natural host. Transmission of infection to other species only occurs if the effective dose is high enough to overcome the species barrier. The current epidemic of bovine spongiform encephalopathy (BSE) in the U.K. is believed to have been initiated by scrapie infection of cattle via contaminated meat and bone meal in concentrated feedstuffs. But the effective exposure was extremely low. Subsequently the epidemic was driven by the re-cycling of infected cattle material which by-passed the species barrier and effectively "passaged" the infection within the cattle population. It is important that re-cycling would not apply to any public health risks arising from BSE. The safety of food can be ensured by excluding all potentially high titre tissues from the food chain. With medicines, risks can most simply be avoided by not using bovine materials from U.K. sources.

Transmissible encephalopathies in animals.

Scrapie in sheep and goats is the best known of the transmissible encephalopathies of animals. The combination of maternal transmission of infection and long incubation periods effectively maintains the infection in flocks. A single sheep gene (Sip) controls both experimental and natural scrapie and the discovery of allelic markers could enable the use of sire selection in the control of the natural disease. Studies of experimental rodent scrapie show that neuroinvasion occurs by spread of infection from visceral lymphoreticular tissues along nerve fibers to mid-thoracic cord. The slowness of scrapie is due to restrictions on replication and cell-to-cell spread of infection affecting neuroinvasion and subsequent neuropathogenesis. Probably both stages in mice are controlled by Sinc gene, the murine equivalent of Sip. The glycoprotein PrP may be the normal product of Sinc gene. Posttranslationally modified PrP forms the disease specific "scrapie associated fibrils" and may also be a constituent of the infectious agent. Scrapie-like diseases have been reported in mink and several species of ruminants including cattle. All of them may be caused by the recycling of scrapie infected sheep material in animal feed. The human health implications are discussed.

Intraperitoneal infection with scrapie is established within minutes of injection and is non-specifically enhanced by a variety of different drugs.

Single intraperitoneal (i.p.) doses of 16 different drugs were given to mice 2 h before injecting scrapie i.p. Scrapie was injected as serial ten-fold dilutions of standard inocula and the effective titres obtained were used as a measure of the relative efficiency of infection in treated compared to saline injected mice. Despite the wide variety of drugs tested, most of them increased, non-specifically, the efficiency of infection by 0.6 to 2.1 log10 i.p. LD50 units (i.e., 4 to 126-fold), but only when both drug and scrapie were given i.p. The effect was greatest with a 2 h or a 6 h interval suggesting an involvement either of resident peritoneal cells or of elicited cells such as polymorphonuclear neutrophils. There was no increase in the efficiency of infection after intervals of 2 or 7 days when induced macrophages would predominant. The reverse sequence of injections (scrapie-0.5 h-drug) had no effect despite the persistence of high scrapie titre in the peritoneum at the time of drug injection. However, the effect was restored by a second injection of scrapie in the sequence, scrapie-drug-scrapie. It is concluded that scrapie infection is established within minutes of injection but much of the inoculum is associated with peritoneal cells which are irrelevant to pathogenesis. Drugs may enhance the infection of relevant peritoneal cells or their targeting to the visceral lymphoreticular tissues where early replication takes place.

Polyclonal increase in certain IgG subclasses in mice persistently infected with the 87V strain of scrapie.

Eight different combinations of seven strains of scrapie agent and the three known Sinc genotypes of mice were screened for changes in the concentration of IgG in serum. A single radial immunodiffusion assay was used to measure IgG throughout the incubation period which in different models ranged from an average of 125 days to longer than the maximum observation period of about 600 days. The only major changes occurred with the 87V strain of scrapie injected intracerebrally (i.c.) or intraperitoneally (i.p.) into mouse strains of the Sinc genotype p7p7. IgG concentration reached 1.5 to 2.0 times the control values in i.c. infected mice, which developed clinical disease after 270 to 320 days and also in i.p. infected mice, which did not develop the disease within the 600-day observation period. At very high IgG concentration, the increase was polyclonal; it involved the IgG 1 subclass more than the others and was accompanied by an increased rate of IgG clearance from serum. It is suggested that some scrapie infections of mice (and sheep) may upset the control of IgG production. The underlying mechanism may involve cell-pathogen interactions which are common to all scrapie infections, but only lead to gross changes in IgG in some combinations of agent strain and host genotype.

The genomic identity of different strains of mouse scrapie is expressed in hamsters and preserved on reisolation in mice.

263K is the most widely used strain of agent in scrapie research because it produces very short incubation periods in golden hamsters and exceptionally high infectivity titres in clinically affected brain. 263K is also remarkable in having a very low pathogenicity for mice. Evidence is presented that 263K originated as a mutant that was strongly selected on passage in hamsters. Seven new passage lines have been established in hamsters using well characterized strains of mouse scrapie representing the 'drowsy goat' and SSBP/1 families of scrapie strains, and one natural scrapie source. Considerable differences between scrapie strains were found in hamsters using incubation period criteria alone. There was evidence that the parent strain of 263K might be 79V or a strain like it in the 'drowsy goat' family. Four of the hamster passage lines were established from scrapie strains that had been cloned in mice. Reisolates in mice were compared with original strains. By the criteria used, two of the reisolates were the same as the original strains. Two others were mutants with incubation periods longer than those of their parental strains but the mutants were different from one another. It is concluded that passage between mice and hamsters can select mutants that would otherwise be lost but there is also clear evidence that the genotypic identity of some scrapie strains is preserved on passage between different host species. These findings are important in the search for the putative nucleic acid genome of the scrapie agent.

The role of the spleen in the neuroinvasion of scrapie in mice.

The pathogenesis of 139A scrapie has been studied in CW mice infected intraperitoneally (i.p.), intravenously (i.v.) or subcutaneously (s.c.). In mice splenectomised before i.p. infection, the evidence points to a neuroinvasive pathway from visceral lymph nodes (and other sites of scrapie replication in the peritoneum) to the thoracic spinal cord. However, in non-splenectomised mice, the major neuroinvasive pathway is clearly from spleen to thoracic cord because i.p. incubation periods are shorter and replication in the thoracic cord starts correspondingly earlier than in splenectomised mice. Studies of splenectomy at different times after i.p. infection show that pathogenesis becomes independent of the spleen once infection has initiated scrapie replication in the spinal cord. The simplest interpretation of all the evidence favours the spread of scrapie infection along splenic nerve fibres to the thoracic spinal cord. The same neuroinvasive pathway is suggested by the findings using the s.c. and i.v. routes of infection. In addition it was found that the 100-fold greater efficiency of infection by the i.v. compared to the i.p. route was entirely dependent on the spleen, because splenectomy before i.v. infection reduced its efficiency to the same as that found in i.p. infected (non-splenectomised) mice.

Pathogenesis of scrapie in mice after intragastric infection.

Infection via the gastrointestinal tract is likely to be a natural route of scrapie infection in sheep. This paper describes the pathogenesis of the 139A strain of scrapie introduced intragastrically (i.g.) into CW mice. There was an almost immediate uptake of infectivity and onset of replication in Peyer's patches which preceded replication in spleen. Splenectomy had no effect on incubation period suggesting that, in contrast to the intraperitoneal route, the spleen plays little or no role in the pathogenesis of 139A scrapie administered intragastrically. Replication in the CNS was first detectable in the thoracic spinal cord and later in brain. The evidence is consistent with neural spread of infection from the gastrointestinal tract, via the enteric and sympathetic nervous systems to spinal cord. Neuroinvasion may be initiated either via infection of Peyer's patches or directly by infection of nerve endings in the gut wall. The latter possibility means that pathogenesis may be completely independent of the lymphoreticular system.

Introduction to scrapie and perspectives on current scrapie research.

Scrapie is the prototype of a family of six transmissible diseases which includes Creutzfeldt-Jakob disease, the only known transmissible human dementia. Bovine spongiform encephalopathy is the most recent addition to the family. Alzheimer's disease is not known to be transmissible. However the pathogenesis of both scrapie and Alzheimer's disease is associated with the modification of (different) normal brain proteins to form various types of amyloid. In scrapie, the evidence suggests that a membrane glycoprotein, PrP, is the precursor of the scrapie associated fibrils (SAF) found in brain extracts, and also of the extracellular deposits of cerebral amyloid which resemble the Alzheimer plaque cores. The host gene that makes PrP is closely linked to the gene Sinc which controls the incubation period of the many different scrapie strains in mice. It is possible that these two genes are the same, i.e., that PrP is the product of Sinc gene. In addition, scrapie infectivity copurifies with modified PrP (SAF). Modified PrP may be the protective protein coat of the independent scrapie genome which some think is a very small nucleic acid. Others believe that the amyloidogenic form of PrP is itself the infectious scrapie agent. Whatever the true situation, there appear to be very short pathways between scrapie infection and amyloidogenesis.

Incubation periods in six models of intraperitoneally injected scrapie depend mainly on the dynamics of agent replication within the nervous system and not the lymphoreticular system.

The pathogenesis of intraperitoneally injected ME7 scrapie has been studied in two Sinc genotypes of mice which gave predictable but widely different incubation periods. Comparisons were made with three other mouse scrapie models and one model in hamsters (involving different strains of agent and an untyped isolate from sheep). Average incubation periods ranged from 114 days in the fastest model (263K/hamsters) to 482 days in the slowest (ME7/Sincp7 mice). There were only small differences between models in the times of onset of replication in spleen and cervical lymph nodes. We suggest that the lymphoreticular stage of pathogenesis initiates neuroinvasion in the peripheral nervous system within a few days to a few weeks of infection. Thereafter, pathogenesis appears to be dominated by neural events and replication in brain becomes detectable after approximately 54% of the remaining incubation period has elapsed, irrespective of its length. It is concluded that the differences between incubation periods of the six scrapie models depend mainly on the rate of a continuous process of replication and spread of infection in the peripheral and central nervous system, which is predetermined by scrapie strain and host genotype. The unpredictability of some other scrapie models (and the natural disease) could be explained by additional factors which restrict neuroinvasion from the lymphoreticular system.

Molecular pathology of scrapie-associated fibril protein (PrP) in mouse brain affected by the ME7 strain of scrapie.

Scrapie-associated fibrils (SAF) are disease-specific structures found in extracts of the brains of animals affected with scrapie. These structures are pathological aggregates of a normal host protein (PrP). Abnormal post-translational modification of PrP has been suggested to explain its aberrant properties in scrapie-affected brains and although there is a form of PrP in SAF indistinguishable in size from the protein in uninfected brain, lower-molecular-mass variants of PrP are also found in SAF fractions. We report the characterisation of the multiple forms of PrP found in SAF fractions purified from mouse brain affected by the ME7 strain of scrapie. The quantitatively major forms of PrP in SAF prepared without the use of proteinase K have the amino-terminal sequence Lys-Lys-Arg-Pro-Lys-Pro-Gly-Gly-, identical to that predicted for the amino-terminus of normal mouse brain PrP. However N-terminal cleavage of some PrP does occur in vivo within a domain of repetitive sequences at sites similar to but distinct from those cut by proteinase K in vitro. This suggests the conformation of the protein in aggregates in vivo does not differ extensively from that in detergent-treated SAF in vitro. We conclude that the size diversity of PrP in SAF is only partly due to N-terminal proteolysis and is independent of the proteolysis that occurs if proteinase K is used in the purification of SAF. Apart from proteolytic changes in the structure of PrP, we found a novel, as yet unidentified, amino-acid derivative of the arginine residue at position 3 in mouse PrP, which may predispose PrP to form SAF.

Pathogenesis of experimental scrapie.

Most of our understanding of the pathogenesis of the unconventional slow infections comes from studies of experimental scrapie in mice and hamsters. After injection by non-neural peripheral routes, pathogenesis necessarily involves the lymphoreticular system (LRS) before the central nervous system (CNS). Available evidence indicates haematogenous spread from the site of injection to the scrapie replication sites in the LRS; later, infection spreads along visceral autonomic nerves from the LRS to the thoracic spinal cord, and thence to brain. The cells in the LRS which are important to scrapie pathogenesis are long lived. Neuroinvasion and spread of infection within the CNS probably involve neuronal pathways. We suggest that disease develops after infection has reached certain clinical target areas in the CNS but only when scrapie replication there has caused sufficient functional damage. Restriction of the replication process in both LRS and CNS is indicated by the occurrence of plateau concentrations of infectivity, especially in some long incubation scrapie models. A remarkable feature of these is that both neuroinvasion and clinical disease occur long after infectivity plateaux have been reached in the LRS and CNS, respectively. We propose that the slowness of scrapie is related to (1) limitations of cell-to-cell spread of infection from LRS to CNS, and (2) limitations on spread between neurons, coupled with restrictions on replication in brain.

Temporary and permanent modifications to a single strain of mouse scrapie on transmission to rats and hamsters.

The interspecies transmission of scrapie is frequently associated with exceptionally long incubation periods at first passage in the new host compared to later passages (the species barrier effect). The basis of this was investigated using the 139A strain of scrapie which had been cloned by three serial passages in mice at limiting infectious doses. Cloned scrapie was passaged through hamsters (twice) or rats (thrice) and then reisolated in mice. Large species barrier effects were encountered on mouse-to-hamster and hamster-to-mouse passage resulting in the isolation of a mutant strain, 139-H/M, with properties very different from 139A. In contrast, the strain reisolated from rats was indistinguishable from 139A. However, a large species barrier was encountered at the mouse-to-rat passage but not at the rat-to-mouse passage. It is suggested that the transmission of scrapie between species may be associated with no change in properties or a permanent change in the scrapie genome due to the selection of mutants. A third possibility, the donor species effect, is a temporary change occurring only at first passage in the new host species which is largely or entirely caused by the introduction of material from the previous host. We speculate that the donor species effect could be explained if some host protein forms a functional part of the infectious agent.