Volume 2: Science
2.
The Spongiform Encephalopathies - knowledge existing in 1986
Pathogenesis
Role of the lymphoreticular system (LRS) in the spread of infection
Invasion of the central nervous system
TSEs other than scrapie
Post-1986 research on pathogenesis
Summary
2.158 Most of the knowledge existing in 1986 on pathogenesis (ie, how a TSE spreads in the body) was based on scrapie. Only limited work had been carried out with other TSEs. Most of the studies had been performed with experimentally induced scrapie in mice, sheep or goats, rather than with natural disease in sheep and goats.
2.159 The investigation of pathogenesis was carried out using the so-called 'bioassay', which, to the present day, remains the only means to identify and quantify TSE infective agent in animal tissue. As described in Chapter 1 (paragraph 1.43), tissues are ground up, usually in saline, to produce a suspension which is sequentially diluted. A specified volume of each dilution is then injected into groups of experimental animals which are observed for the development of disease. In this way the titre, or concentration, of infectivity per gram of particular tissue can then be calculated from the last dilution that was sufficient to cause disease in 50 per cent of a group of animals.
2.160 Using this method, it was known by 1960 that tissues other than those of the central nervous system (CNS) could be a source of infection. Experimental production of scrapie in goats and sheep had shown that disease could be produced by inoculating pituitary and adrenal gland, spleen, pancreas and liver from scrapie-infected goats. 1
2.161 In 1962 Pattison had examined the temporal spread of the scrapie agent around the body of goats, following intracerebral inoculation with scrapie goat brain suspension. 2 Donor animals were killed at increasing intervals after injection of scrapie and their tissues sub-inoculated into recipient animals. The infectious agent reached the cerebrospinal fluid (CSF) and pituitary gland after 24 hours, but was not detected in the sciatic nerve, adrenal gland, salivary gland or muscle of donors until four months post-inoculation. Neither spleen nor thymus was tested.
Role of the lymphoreticular system (LRS) in the spread of infection
2.162 The lymphoreticular system (LRS) 3 was first found to have a role in the spread of scrapie in 1967, when infectivity was detected in the spleen and peripheral lymph nodes of subcutaneously infected mice, 4 weeks after inoculation. 4 By 8 weeks after inoculation, the maximum concentration of infectivity had been reached in both of these organs, and was about 50 or 60 times greater than that of the original inoculation. Infectivity was detected in the third important LRS tissue, the thymus, after 8 weeks. Clinical signs appeared in the mice at 23 weeks, by which time infectivity could also be seen in the submaxillary salivary gland, lung, intestine, spinal cord and brain. Further evidence of the importance of the spleen was obtained later, in 1970, when splenectomy was found to lengthen the incubation period of disease in intraperitoneally inoculated mice. 5 Recent evidence obtained in 2000 suggests that destruction of follicular dendritic cells (FDCs) in the spleen with lymphotoxin-beta receptor, impaired prion replication in scrapie-infected mice, and that FDCs are the principal sites of prion replication in the spleen. 6
2.163 In 1982 Hadlow reported observations on the location of infectivity in naturally infected sheep of different ages. 7 He noted the presence of scrapie agent in the tonsil, retropharyngeal lymph node and intestine early in infection, suggesting that infection is initiated by way of the alimentary tract. Furthermore, the site of primary replication was thought to be either the oropharynx, intestine or both. The infective agent was then observed to spread to the lymph nodes and spleen, where it continued to replicate to high titres before spreading to the CNS. The levels of infectivity in the LRS were similar in subclinically affected sheep at 10-25 months of age to levels in the LRS in clinically affected animals aged 34-57 months. Hadlow noted infectivity in the CNS first in a subclinically affected sheep at 25 months. In clinically ill sheep, infectivity was found to be widely dispersed throughout the CNS with titres much higher than in non-neural tissues. The quantitative distribution of infectivity in tissues from sheep of different ages and at different stages of disease is shown in Table 2.1 below. We comment further on this at paragraphs 3.198-3.201 below.
Table 2.1: A - Distribution of scrapie infectivity in non-neural tissues of subclinically infected sheep
B - Distribution of scrapie infectivity in nervous tissue of clinically affected sheep
C - Distribution of scrapie infectivity in non-neural tissues of clinically affected sheep
Invasion of the central nervous system
2.164 Work during the early 1980s had indicated that the thoracic spinal cord was the first site in the CNS to be invaded by scrapie following peripheral infection; the agent moved to the CNS from the LRS via sympathetic nerve fibres. The infection then spread along the spinal cord in both directions, at a rate of approximately 1mm/day. 8 The invasion of the CNS had been shown to be a relatively controlled event, with replication in the brain detectable after 40 to 50 per cent of the incubation period, whichever peripheral route of inoculation was used. 9
2.165 By 1986 knowledge of the efficiency of different routes of experimental transmission of scrapie had also provided clues to the understanding of the pathogenesis of the disease. Intraspinal inoculation was known to be the most efficient route, followed by intracerebral. 10 Among the non-neural routes, intravenous injection was more efficient than subcutaneous. 11 In addition, scrapie infectivity after injection by intracerebral, intravenous, intraperitoneal and subcutaneous routes was shown to be widely distributed in blood and many other tissues after 30 minutes. 12 However, the amount of infectivity in the bloodstream quickly dropped to undetectable levels within seven hours, in accordance with previous reports on the infectivity of blood in the first few hours after infection. 13 The shorter incubation times with the direct routes (intraspinal and intracerebral) into the CNS were explained by the fact that there were areas in the CNS where replication of the agent occurred in nerve cells, and resulted in their degeneration. With inoculation via peripheral routes, replication in the lymphoreticular system was thought to be necessary before invasion of the CNS, which explained the delay in disease development. 14
2.166 Little work had been carried out on the pathogenesis of other TSEs. In 1969 analysis of the distribution of infectivity in terminally affected mink inoculated intramuscularly or intracerebrally with TME had shown low concentrations of the agent in non-neural tissues (liver, spleen, kidneys, bladder, muscles and faeces). 15 However, the pathogenesis of this disease was not studied in detail until 1987.
Post-1986 research on pathogenesis
2.167 In order to set the position in 1986 in context, we briefly describe subsequent work. Since 1986 it has been confirmed that following non-neural peripheral infection of mice with scrapie agent, the earliest sites of detectable levels of agent replication are tissues of the lymphoreticular system. Much has been done to find out which specific cells are involved. Depending on the strain of scrapie used, studies have implicated either follicular dendritic cells 16 or B lymphocytes 17 in disease pathogenesis. However, in the latter case, results suggested that the role of B lymphocytes was transporting prions from the site of replication to the CNS rather than prion replication itself. Nevertheless, the fact that both of these cell types were shown to be vital for scrapie replication suggests that different strains may utilise different cell populations in the LRS. Recent work on the propagation of BSE via the oral route in mice showed infectivity in Peyer's patches at 45 days post-inoculation and in the LRS one to three months later. Scrapie infection (oral) was shown to differ from BSE in that it was detected in the stomach and colon. This work indicated that the agent is propagated in Peyer's patches, moves via lymphatics to mesenteric lymph nodes, and then by blood to secondary replication sites in the LRS. 18
2.168 After replication in the mouse lymphoreticular system, scrapie infection spreads via nerve fibres of the autonomic nervous system 19 to the clusters of nerve cells alongside each vertebral body known as pre-vertebral (or dorsal root) ganglia, and thence into the spinal cord. Other minor points of entry have been discovered into the spinal cord in the lower neck and in the lower back after intraperitoneal inoculation. 20 Once the central nervous system has been affected, replication becomes independent of the lymphoreticular system. Finally, after certain target areas of the brain have been reached and sufficient agent replication and functional damage has occurred, clinical disease develops. 21
2.169 An alternative route of access to the brain, bypassing the spinal cord, has also been implicated following peripheral inoculation of mice. 22 Evidence suggests that the fibres of the autonomic vagus nerve, 23 which runs directly to the brain, are the most likely direct pathway to the brain, although a blood or spinal fluid access has not been completely ruled out.
2.170 The earlier results concerning the pathogenesis of natural scrapie in sheep have also been confirmed, although the portal of entry to the central nervous system is still unclear. PrPSc has been detected in the enteric (intestinal) nervous system of sheep with natural scrapie. 24 This suggests that oral infection via the gastrointestinal tract could spread from the neurons of the enteric nervous system, via the autonomic vagus nerve, to the brain, or through other autonomic nerve fibres to the spinal cord.
2.171 Studies with scrapie provided most of the information in 1986 on the spread of TSE infectivity within animals, from the point of infection until the animals succumbed to the disease. These studies suggested that the route of natural infection was generally through the gastrointestinal system and then onwards via the lymphatic tissues of the small intestine (Peyer's patches) to the mesenteric lymph glands and then to the spleen and thymus. During this progression through the LRS the scrapie agent replicated principally within the follicular dendritic cells of the spleen and lymph glands. The agent was thought to find its way to the CNS mainly via the network of nerves of the autonomic nervous system of the gut, entering the spinal cord by way of the dorsal route ganglia and passing into the brain. Transmission to the brain via the bloodstream could also occur, with B lymphocytes transporting the agent. This process could take between 18 months and 3 years in sheep, indicating that the tissues of the animal contained the infective agent long before the development of clinical disease.
1 Pattison, I. and Millson, G. (1960) Further Observations on the Experimental Production of Scrapie in Goats and Sheep, Journal of Comparative Pathology, 70, 182-93
2 Pattison, I. and Millson, G. (1962) Distribution of the Scrapie Agent in the Tissues of Experimentally Inoculated Goats, Journal of Comparative Pathology, 72, 233-44
3 The lymphoreticular system (also termed the lymphatic system) is composed of the tissues and organs that produce and store cells that fight infection and of the network of vessels that carry these cells
4 Eklund, C., Kennedy, R. and Hadlow, W. (1967) Pathogenesis of Scrapie Virus Infection in the Mouse, Journal of Infectious Diseases, 117, 15-22
5 Fraser, H. and Dickinson, A. (1970) Pathogenesis of Scrapie in the Mouse: The Role of the Spleen, Nature, 226, 462-3
6 Montrasio, F., Frigg, R., Glatzel, M., Klein, M.A., Mackay, F., Aguzzi, A. and Weissmann, C. (2000) Impaired prion replication in spleens of mice lacking functional follicular dendritic cells, Science, 288(5469), 1257-9
7 Hadlow, W., Kennedy, R. and Race, R. (1982) Natural Infection of Suffolk Sheep with Scrapie Virus, Journal of Infectious Diseases, 146, 657-64
8 Kimberlin, R. and Walker, C. (1980) Pathogenesis of Mouse Scrapie: Evidence for Neural Spread of Infection to the CNS, Journal of General Virology, 51, 183-7; Kimberlin, R. and Walker, C. (1982) Pathogenesis of Mouse Scrapie: Patterns of Agent Replication in Different Parts of the CNS Following Intraperitoneal Infection, Journal of the Royal Society of Medicine, 75, 618-24; Kimberlin, R. (1986) Scrapie: How much do we Really Understand? Neuropathology and Applied Neurobiology, 12, 131-47
9 Kimberlin, R. and Walker, C. (1979) Pathogenesis of Mouse Scrapie: Dynamics of Agent Replication in Spleen, Spinal Cord and Brain after Infection by Different Routes, Journal of Comparative Pathology, 89, 551-62
10 Kimberlin, R., Hall, S. and Walker, C. (1983) Pathogenesis of Mouse Scrapie Evidence for Direct Neural Spread of Infection to the CNS after Injection of Sciatic Nerve, Journal of the Neurological Sciences, 61, 315-25
11 Kimberlin, R. and Walker, C. (1979) Pathogenesis of Mouse Scrapie: Dynamics of Agent Replication in Spleen, Spinal Cord and Brain after Infection by Different Routes, Journal of Comparative Pathology, 89, 551-62
12 Millson, G., Kimberlin, R., Manning, J. and Collis, S. (1979) Early Distribution of Radioactive Liposomes and Scrapie Infectivity in Mouse Tissues Following Administration by Different Routes, Veterinary Microbiology, 4, 89-99
13 Field, E.J., Caspary, E.A. and Joyce, G. (1968) Scrapie Agent in Blood, Veterinary Record, 83, 109-10
14 Kimberlin, R. (1986) Scrapie: How much do we Really Understand? Neuropathology and Applied Neurobiology, 12, 131-47
15 Marsh, R., Burger, D. and Hanson, R. (1969) Transmissible Mink Encephalopathy: Behaviour of the Disease Agent in Mink, American Journal of Veterinary Research, 30, 1637-42
16 The follicular dendritic cells are immune cells which are involved in the recognition of non-host proteins, found in specific areas of the spleen and lymph nodes; Mabbott, N., Farquar, C., Brown, K. and Bruce, M. (1998) Involvement of the Immune System in TSE Pathogenesis, Immunology Today, 19, 201-3
17 B lymphocytes are a type of white blood cell involved in antibody production and are a component of the LRS; Klein, M., Frigg, R., Flechsig, E., Raeber, A., Kalinke, U., Bluethmann, H., Bootz, F., Suter, M., Zinkernagel, R. and Aguzzi, A. (1997) A Crucial Role for B Cells in Neuroinvasive Scrapie, Nature, 390, 687-90
18 Maignien, T., Lasmezas, C.I., Beringue, V., Dormont, D. and Deslys, J.P. (1999) Pathogenesis of the oral route of infection of mice with scrapie and bovine spongiform encephalopathy agents, Journal of General Virology, 80(11), 3035-42
19 The autonomic nervous system is the name given to the nerves that are not under conscious control. The autonomic nervous system controls the heart, intestines, muscle in blood vessel walls, etc
20 Baldauf, E., Beekes, M. and Diringer, H. (1997) Evidence for an Alternative Direct Route of Access for the Scrapie Agent to the Brain Bypassing the Spinal Cord, Journal of General Virology, 78, 1187-97
21 Recent work has shown that the hydrophobic core sequence in PrP106-126 peptide is neurotoxic and may affect aggregation. See Jobling, M.F., Stewart, L.R., White, A.R., McLean, C., Friedhuber, A., Maher, F., Beyreuther, K., Masters, C.L., Barrow, C.J. and Collins, S.J. (1999) The hydrophobic core sequence modulates the neurotoxic and secondary structure properties of the prion peptide 106-126, Journal of Neurochemistry, 73(4), 1557-65
22 Ibid.
23 The major nerve that provides connections to the ear, pharynx and tongue
24 Van Keulen, L., Schreuder, B., Vromans, M., Langereld, J. and Smits, M. (1999) Scrapie-Associated Prion Protein in the Gastro-Intestinal Tract of Sheep with Natural Scrapie, Journal of Comparative Pathology, 121, 55-63
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