J. gen. Virol. (1983), 64, 713-716.
Printed.in Great Britain
713
Key words: scrapie/pathogenesis/centrifugal infection/nervous system
Pathogenesis of Mouse Scrapie: Evidence for Spread of Infection from
Central to Peripheral Nervous System
(Accepted 13 October 1982)
SUMMARY
The onset of replication has been studied in spinal cord, dorsal root ganglia and
spinal nerves of C W m i c e infected intraperitoneally with the 139A strain of scrapie.
The patterns obtained suggest a centrifugal spread of infection from central to
peripheral nervous systems. Infectivity titres in the peripheral nervous system reached
a plateau long before the end of the incubation period, and the maximum titres were
much lower than in the central nervous system. This suggests that there is a restriction
of replication in the peripheral nervous system similar to that already known in
extraneural tissues.
In previous studies, we used a short incubation model of scrapie [strain 139A in Compton
White (CW) mice: Sine~] to investigate how the agent enters the central nervous system (CNS)
after replication in spleen and other visceral lymphoid organs. In the absence of direct methods
of study, we compared the times of onset of replication in different tissues, and in various parts
of the CNS, on the assumption that the sequences obtained reflect the gradual spread of
infectious agent. These studies used different routes of infection [e.g. intravenous, intraperitoneal (i.p.), subcutaneous] and relatively low doses of infectivity so that the detection of the onset
of replication would not be obscured by any uptake and persistence of the original inoculum.
Consistent patterns were obtained showing, successively, replication in spleen and thoracic
spinal cord and then, simultaneously, in brain and lumbar cord (Kimberlin & Walker, 1979,
1980). In more detailed studies with i.p. infected mice, the patterns suggested entry of agent into
spinal cord between thoracic vertebrae 4 and 9, followed by gradual spread to lower spinal cord
and simultaneous forward spread to upper cord, medulla, various mid-brain regions and finally,
anterior brain (Kimberlin & Walker, 1982). The initial onset of agent replication in midthoracic cord is strong circumstantial evidence for centripetal invasion of the CNS by scrapie
via sympathetic nerves serving spleen and visceral lymph nodes.
The present paper is concerned with the possibility that invasion of the CNS by scrapie agent
may be followed by centrifugal spread of infection to other parts of the peripheral nervous
system (PNS). It seemed likely that this would occur since many neurotropic viruses spread both
centripetally and centrifugally (see below). We therefore tested the prediction that spread of
scrapie agent posteriorly to lumbar cord would lead to a later occurrence of replication in lumbar
dorsal root ganglia (DRG). We also examined DRG and spinal nerves of mid-thoracic segments
to test two possibilities: either a similar pattern of centrifugal spread to D R G in both lumbar
and thoracic segments, or a pattern of centrifugal spread in lumbar segments contrasting with
centripetal spread in thoracic segments, which might suggest that the initial invasion of the
CNS occurred via autonomic afferent pathways in DRG and spinal nerves.
Two experiments were carried out using CW mice (Sinc~') infected i.p. with 0.1 ml of brain
homogenates containing 100 to 1000 LDs0 i.p. units of the 139A strain of scrapie agent. Groups
of donor mice were infected each week to provide animals at different stages of incubation on a
given day (Kimberlin & Walker, 1982). Three mice from each group were taken and tissues
removed in the following sequence: brain, thoracic cord (between vertebrae T5 and Tll),
lumbar cord (between vertebrae L1 and L5) followed by the corresponding thoracic spinal
nerves (1 experiment), thoracic DRG and lumbar DRG. With the last three tissues, pairs of
spinal nerves or ganglia were taken from four segments to provide 3 to 11 mg samples from three
mice. The samples of spinal nerve were distal to the DRG. The ganglia samples contained up to
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0022-1317/83/0000-5389 $02.000 1983 SGM
714
Short communications
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Fig. 1. Agent (139A) replication in CNS (solid lines) and PNS (broken lines) of donor mice infected
i.p. ; brain (A), thoracic cord (O), lumbar cord (11), thoracic ganglia (O), thoracic spinal nerves (Z~x)
and lumbar ganglia (C]). The ordinate shows the incubation period in recipient groups of mice
inoculated intracerebrally (i.c.) and indicates relative amounts of infectious agent in tissue
homogenates. The curves are formed from recipient groups where all mice tested developed scrapie.
Points on the abscissa denote recipient groups in which less than 100~ of mice developed scrapie,
indicating very low levels of agent in tissues. The abscissa shows sample times as a percentage of the
incubation period of donor mice; this was 160 + 1 (days + s.E.) for experiment 1 (a to c) and 169 + 1 for
experiment 2 (d to f).
half their weight of attached spinal nerve roots. Different sets o f dissection instruments were
used for different tissues to minimize cross-contamination of scrapie agent between samples
(Kimberlin & Walker, 1979). The pooled tissues from three mice were homogenized in saline at
a concentration of 0.5 % wet weight (experiment 1) or 0.25 % (experiment 2). The homogenates
were injected i.c. into recipient groups of eight female C W mice, which were observed for the
development of clinical scrapie for up to a m a x i m u m of 300 days after injection. The average
incubation period was calculated for all groups with 100% clinical cases. Because o f the inverse
relationship between concentration of agent and length of incubation period ( K i m b e r l i n &
Walker, 1978), replication in donor tissues can be readily detected by a r a p i d reduction in the
average incubation period of consecutive groups of recipient mice. (It is difficult to prove that a
progressive increase in infectivity within a tissue is due to replication and not to accumulation
from elsewhere, but it is a reasonable assumption.)
Fig. 1 (a, d) confirms the results of previous experiments with i.p. infected mice ( K i m b e r l i n &
Walker, 1980, 1982) that replication in thoracic cord precedes that in brain and l u m b a r cord.
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Short communications
715
Table 1. Infectivity titres in various tissues during the incubation period of scrapie
Percent of incubation period
,A
Tissue
Brain
Thoracic cord
ganglia
nerves
Lumbar cord
ganglia
57*
3"8t
5.6
~<2.9
ND~:
~<3.2
~<2.6
6l*
4.5
5-8
3.6
ND
4-1
3.4
66*
5.3
5.9
3-6
ND
4.8
3.5
70*
4.8
5-5
3-9
ND
4-5
3.6
87
6.3
6.1
4-8
4.7
6.1
4.3
95
6.2
6.3
4-6
4"6
5.8
4.4
* Data taken from experiment 1; the other times from experiment 2.
t Infectivity titres expressed as -log~o LD50 i.e. units per 0.03 g of tissue.
:~ND, Not done.
There were three new findings. First, the onset of replication in both lumbar and thoracic D R G
was 4 to 13% of the incubation period (1 to 3 weeks) later than the onset of replication in the
corresponding segments of cord (Fig. 1 b, c, e,f). This suggests that there is a centrifugal spread
of infection from CNS to PNS, and the observation in experiment 2 that replication in thoracic
spinal nerves occurred later than in thoracic ganglia (Fig. I e) supports this view. Hence the
initial centripetal invasion of the CNS in mid-thoracic cord (Kimberlin & Walker, 1980, 1982)
probably occurs via pathways not associated with D R G and spinal nerves; the most likely
pathway from spleen is via postganglionic fibres to the coeliac ganglion and then along
preganglionic fibres to the sympathetic trunk and ventral spinal roots.
The second finding was that the curves for brain, thoracic cord and lumbar cord tended
towards plateaus of similar height, representing similar concentrations of infectivity in these
tissues at the end of incubation (Fig. 1 d). Thirdly, the curves for thoracic spinal nerves, thoracic
ganglia and lumbar ganglia reached definite plateaus quite early in the incubation period but at
levels much lower than in the CNS.
The third finding was unexpected and led us to carry out complete titrations of infectivity in
selected samples that had been stored as 0-5 or 0.25% homogenates at - 2 0 °C for 7 to 10
months. The results showed that after 87 and 95 % of the incubation period, the infectivity titres
in brain, thoracic cord and lumbar cord were similar (5-8 to 6-3 loglo units/0-03 g; Table 1). The
titres of agent in ganglia and spinal nerves were also similar to one another (4.3 to 4.8 logt0
units/0.03 g) but were on average 1-5 log~o units lower than in the CNS.
The present and previous studies suggest that both centrifugal and centripetal spread of
infection occurs between CNS and PNS, but along different pathways. In this respect, scrapie
may resemble neurotropic viruses such as rabies (Murphy, 1977), herpes simplex (Wildy et al.,
1982) and pseudorabies (Field & Hill, 1974). There is one study (with a different combination of
scrapie strain and mouse genotype) showing intraneuronal transport of agent from eye to
contralateral superior colliculus (Fraser, 1982), but otherwise nothing is known of which cells
transport scrapie in nerves or in the CNS. However, the apparent rate of spread of scrapie seems
generally to be much slower than with the neurotropic viruses. For example, retrograde axonal
transport of herpes simplex, rabies, pseudorabies and polio occurs at rates of 50 to 250 mm/day
(Kristensson, 1978; Wildy et al., 1982) whereas the estimated rate of spread of scrapie agent
within the CNS is only 0.5 to 1.0 mm/day (Kimberlin & Walker, 1982). A similar slow rate
probably applies to the centrifugal spread of scrapie agent, otherwise the patterns shown in Fig.
1 would not have been detectable using 1 week intervals between samples. Assuming that there
is intraneuronal spread, the estimates for scrapie are approximately equal to the slow
axoplasmic transport of cytoskeletal proteins (Black & Lasek, 1980).
The infectivity titres in ganglia and spinal nerves were different from those in the CNS in two
respects; they reached a plateau long before the end of the incubation period and the plateau
titres were about 30-fold less than the maximum CNS titres (Fig. 1, Table 1). Other studies, of
the same scrapie model, have shown that these two features also occur in extraneural tissues
where the agent replicates, namely spleen (Clarke & Haig, 1971 ; Kimberlin & Walker, 1979),
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Short communications
716
cervical lymph nodes (R. H. K i m b e r l i n & C. A. Walker, unpublished observations) and
submaxillary salivary gland (MiUson et al., 1979). The differences in infectivity titres between
spleen and brain have been seen in other models of scrapie and interpreted as evidence for a
more limited n u m b e r of replication sites in extraneural tissues compared to the C N S (Dickinson
& Outram, 1979). It is therefore of great interest that a similar restriction seems to apply to
replication in the PNS. What this means, however, in terms of different populations of cells in
CNS, PNS and extraneural tissues is completely unknown.
Previous studies indicated that scrapie agent eventually spreads to most, if not all, parts of the
CNS (Kimberlin & Walker, 1982). Spread of infection from brain to optic nerve and retina has
been suggested recently for a hamster model of scrapie (Buyukmihci et al., 1980; Hogan et al.,
1981). The present studies indicate that scrapie infection may spread to much of the P N S as well.
Hence, the pruritus which frequently occurs in natural sheep scrapie could be a consequence of
agent in the PNS, or even in skin, rather than in the CNS, as is often assumed. If our
observations with serapie apply to the related diseases, then spread of Creutzfeldt-Jakob agent
to the PNS could account for the recent findings of autonomic dysfunction in two cases of
Creutzfeldt-Jakob disease in m a n ( K h u r a n a & Garcia, 1981).
IARC & MRC Neuropathogenesis Unit, Edinburgh EH9 3JQ
2Department of Pathology, University of Cambridge
Tennis Court Road, Cambridge CB2 1QP
3ARC Institute for Research on Animal Diseases
Compton, Newbury RG16 ONN, U.K.
R. H. KIMBERLIN1.
H. J. FIELD2
CAROL A. WALKER3
REFERENCES
BLACK, M. M. & LASEK, R. J. (1980). Slow c o m p o n e n t s o f a x o n a l t r a n s p o r t : two c y t o s k e l e t a l n e t w o r k s . Journalof Cell
Biology 86, 616-623.
BUYUKMIHCI,N., RORVIK,M. & MARSH,R. F. (1980). Replication of the scrapie agent in ocular neural tissues.
Proceedings of the National Academy of Sciences of the UnitedStates of America 77, 1169-1171.
CLARKE,M. C. &HAIG,D. A. (1971). Multiplication of scrapie agent in mouse spleen. Researchin VeterinaryScience
12, 195-197.
DICKINSON,A. G. & oLrrRAM, G. W. (1979). The scrapie replication-site hypothesis and its implications for
pathogenesis. In Slow TransmissibleDiseasesof the NervousSystem, vol. 2, pp. 13-31. Edited by S. B. Prusiner
& W. J. Hadlow. New York: Academic Press.
FIELD,H. J. &HILL,T. J. (1974). The pathogenesis of pseudorabies in mice followingperipheral inoculation. Journal
of General Virology 23, 145-157.
FRASER,H. (1982). Neuronal spread of scrapie agent and targeting of lesions within the retino-tectal pathway.
Nature, London 295, 149-150.
HOGAN, R. N., BARINGER, J. R. & FRUSINER, S. B. (1981). P r o g r e s s i v e retinal d e g e n e r a t i o n in s c r a p i e - i n f e c t e d
hamsters. Laboratory Investigation 44, 34-42.
KHURANA,R. K. &GARCIA,J. H. (1981). Autonomic dysfunction in subacute spongiform encephalopathy. Archivesof
Neurology 38, 114-117.
KIMBERLIN,R. H. & WALKER,C. A. (1978). Pathogenesis of mouse scrapie: effect of route of inoculation on
infectivity titres and dose-response curves. Journalof Comparative Pathology 88, 39-47.
KIMBERLIN, R. H. &WALKER,C. A. (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, 551-562.
KIMBERLIN,R. H. &WALKER,C. A. (1980). Pathogenesis of mouse scrapie: evidence for neural spread of infection to
the CNS. Journal of General Virology 51, 183-187.
KIMBERLIN,R. H. &WALKER,C. A. (1982). Pathogenesis of mouse scrapie: patterns of agent replication in different
parts of the CNS following intraperitoneal infection. Journalof the Royal Society of Medicine 75, 618-624.
KRISTENSSON,K. (1978). Retrograde transport of macromolecules in axons. Annual Reviewof Pharmacology and
Toxicology 18, 97-110.
MILLSON, G. C., KIMBERLIN, R. H., MANNING, E. J. & COLLIS, S. C. (1979). Early distribution of radioactive liposomes
and scrapie infectivity in mouse tissues followingadministration by different routes. VeterinaryMicrobiology
4, 89-99
MURPHY,F. A. (1977). Rabies pathogenesis. Archives of Virology 54, 279-297.
WlLDY,P., FIELD,H. J. &NASH,A. A. (1982). Classical herpes latency revisited. In Virus Persistence,pp. 133-167.
Edited by B. W. J. Mahy, A. C. Minson & G. K. Darby. Cambridge: Cambridge University Press.
(Received 12 August 1982)
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