Commentary - Oral infection by the bovine spongiform encephalopathy prion, PNAS, April 27, 1999

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tent/full/96/9/4738

Proceedings of the National Academy of Science

Vol. 96, Issue 9, 4738-4739, April 27, 1999

Commentary

Oral infection by the bovine spongiform encephalopathy prion

R. G. Will and J. W. Ironside

National Creutzfeldt-Jakob Disease Surveillance Unit, Western General

Hospital, Edinburgh, EH4 2XU, United Kingdom

The route by which prion infection spreads from peripheral tissues to the

brain has been the subject of interest and research for decades. The seminal

pathogenesis studies by Hadlow and colleagues (1, 2) demonstrated that in

natural scrapie in Suffolk sheep infectivity initially was detected at 10-14

months of age in tonsil, lymph nodes, spleen, and intestine, including ileum

and upper colon. The tissue distribution of infectivity was consistent with

uptake from the alimentary tract and, by implication, oral exposure as the

likely portal of entry of infection. By the time clinical disease developed,

peripheral tissues continued to exhibit a similar distribution and titer of

infectivity, but there was also evidence of infectivity in the central

nervous system, with higher titers of infectivity, initially in the medulla

and diencephalon. Laboratory studies of oral scrapie infection in rodents

have confirmed these findings and suggest that infection spreads from the

lymphoreticular system to the spinal cord, presumptively via the autonomic

nervous system, and thence rostrally to the brain (3). The importance of

peripheral pathogenesis is underlined by the marked increase in incubation

time in mice after splenectomy (4).

There is, however, variation in pathogenesis that is determined by factors

including the interaction between host genome and agent strain. Some breeds

of sheep affected by natural scrapie, for example, Montadales, have no

detectable infectivity in peripheral tissues, and the distribution of

infectivity in the brain may vary according to the breed of sheep (5). In

bovine spongiform encephalopathy (BSE) infectivity has not been detected in

peripheral tissues in natural disease, except for dorsal root ganglia and

possibly bone marrow, although infectivity has been found in terminal ileum

after experimental oral challenge with BSE brain (6). An important

implication of this data is that, accepting the limits of the sensitivity of

bioassay systems, there may be variation in pathogenesis in different

host/agent combinations and extrapolation from date on scrapie, either

natural or experimental, to other agent strains in other species, such as

primates, may be misleading.

In primates only a small and unpredictable proportion of animals develop

disease after oral exposure to tissues containing a high titer of

infectivity such as brain (7). There is little information on pathogenesis

in nonhuman primates after oral exposure, not least because the relative

inefficiency of the oral route makes such experiments difficult to carry out

in practice. In kuru, which is presumed to be caused by peripheral, and

perhaps oral, exposure to infection through ritual cannibalism, infection

has been found by bioassay in lymph nodes, kidney, and spleen (8). Similar

experiments in Creutzfeldt-Jakob disease (CJD) have demonstrated infectivity

in liver, kidney, lung, and lymph nodes. Examination of peripheral tissues

by immumocytochemical techniques, either histologically or by Western blot,

in human prion disease has been fairly limited and has not documented

evidence of peripheral pathogenesis, although these techniques have a

limited sensitivity (9).

More widespread immumocytochemical staining of components of the

lymphoreticular system has been found in new variant CJD (nvCJD) (9),

raising the possibility of a different pathogenesis from sporadic CJD. The

study by Bons et al. (10) in a previous issue of the Proceedings provides

new data on the pathogenesis of prion disease in one primate species after

experimental oral exposure to BSE agent. This paper significantly extends

the available information on the tissue distribution of infectivity in the

preclinical phase of the incubation period in primates and confirms that

primates can be susceptible to oral exposure to the BSE agent. The findings

will be of interest and concern to the scientific community and others in

view of the hypothesis that nvCJD is causally linked to BSE, presumptively

through oral exposure to the BSE agent (11, 12).

Consideration of methodological issues is an important prerequisite to the

interpretation of the results. Immunocytochemical methods for the detection

of disease associated prion protein (PrPsc) depend on the, presumed,

denaturation of normal prion protein (PrPc), because none of the available

antibodies can readily distinguish between the isomers. This differentiation

usually involves partial protein digestion using proteinase K, but this

methodology was not used in this study (10) because only fixed tissues were

examined. Bons et al. have addressed this issue by carefully considering a

range of criteria for the interpretation of PrP antibody immunocytochemical

staining. Although it is highly likely that the positive PrP staining in

this report indicates the presence of PrPsc, the findings would be

strengthened if they were backed up by other techniques such as Western

blotting.

The observations in this paper give some indication as to how prion

infection might spread after oral exposure. Of particular interest is the

staining of the epithelium of the gut and tonsil, which raises the

possibility of a number of mechanisms of onward transmission of infection

and subsequent disease. One possibility is that the infectious agent

penetrates the epithelial cells and replicates, another that the gut

epithelial cells express PrPc that acts as a receptor down which the agent

passes, without necessitating entry to cell cytoplasm. It is not known

whether gut epithelial cells express PrPc, but PrPsc can be detected in

gut-associated lymphoid tissue, particularly follicular dendritic cells

(13). An important question is how the infectious agent is transmitted from

the gut lumen to these cells. Immunocytochemical staining was found in

specialized M cells and lymphocytes within the gut epithelium and

lymphoreticular system. These interesting findings may stimulate further

experiments to address this important issue. It is of note that a recent

study has implicated B lymphocytes as necessary for neuroinvasion in one

model of experimental scrapie (14).

A further important observation in the study is the immumocytochemical

detection of PrPsc accumulation in the ventral and dorsal root ganglia

throughout the spinal cord and in the cerebral cortex in the preclinical

phase of the incubation period. This finding is consistent with models of

scrapie pathogenesis. However, the exact mechanism of transmission from

lymphoreticular system to central nervous system has not been precisely

defined. Possibilities include transmission via the neuro-immune connection

through the autonomic nerves in organized lymphoid tissue, such as spleen,

or via the autonomic nervous system in the gut. Further studies are needed

to investigate these interesting possibilities.

From an epidemiological perspective the study by Bons et al. (10) raises a

number of important issues. First, spongiform change and/or PrP

immunostaining were found in the brains of 20 lemurs from three different

primate centers in France and a simian in Montpellier zoo. The prevalence of

prion infection in the Montpellier primates overall was five of 61 animals,

although it is of note that autopsy examination was not carried out in nine

of the 26 animals that had died. Although only two of these animals had

signs of a neurological disease, 18 lemurs from three other primate

facilities in France were apparently healthy and exhibited positive PrP

immunocytochemical staining. The proportion of primates that had died with

evidence of preclinical or clinical prion disease after presumed oral

exposure to BSE was 19%, which may be an underestimate.

On the other hand, requests for information from other zoos in France on

neurological disease in primates was largely negative, although the response

rate was low. Furthermore, in Montpellier only 8% of primates, including

those still living were PrP-positive. Even this figure is a matter for great

concern if humans had a similar sensitivity to oral BSE exposure. However,

this hypothesis would assume that humans are identical to lemurs in their

susceptibility to BSE, an assumption that may not be justifiable.

Transmission of BSE to marmosets by intracerebral inoculation was reported

in 1993 (15), and this experiment was interpreted by the authors as follows:

" Our experiments suggested that there was no specific reason to suppose that

the BSE agent was more transmissible to primates than was the scrapie agent "

(16). Scrapie is not thought to be a human pathogen.

No primates are reported to have developed a spongiform encephalopathy in

British zoos, despite the heightened awareness of these diseases after the

identification of a BSE-like illness in a range of potentially exposed zoo

species (17) (although only in a minority of such species). It is of

interest that BSE is experimentally transmissible to mice but not hamsters,

species that are genetically closely related (18).

Second, although two lemurs were PrP-positive after experimental oral

exposure to the BSE agent, it is relevant to question the source of

infection in the primates with apparently " natural " transmission. Three of

the lemurs, which died of spongiform encephalopathy in Montpellier, were

almost certainly exposed to oral infection at the zoo, but the source of

exposure in the other positive animals in Montpellier and the other three

zoos is necessarily uncertain. The animals all were exposed to a food

supplement containing cracklings, the " fourth quarter of beef, " which might

have originated in the United Kingdom. It would be of great interest to

determine the constituents of this material and in particular whether it

contained central nervous system tissue. Marmosets, which are susceptible to

BSE by intracerebral inoculation, were fed in the United Kingdom throughout

life with a supplement containing meat and bonemeal potentially contaminated

with BSE between 1980 and 1990, but none of more than 100 uninoculated

animals developed a spongiform encephalopathy (19). However, Bons et al.

(10) provide data indicating that the neuropathological profile in the

lemurs orally exposed to 0.5-1 g of bovine brain was similar to that in

lemurs with a " natural " spongiform encephalopathy, suggesting that the

disease in this group also may have been caused by BSE.

The fact that primates are susceptible to a prion disease through oral

exposure to the BSE agent is a finding of great interest. Extrapolating from

this finding to human disease is problematic as the sensitivity of one

species to a particular prion strain does not necessarily indicate the

sensitivity of another species, even if this species is closely related.

Because of this constraint, the study by Bons et al. (10) may discourage

further experiments in other primates if these are aimed at quantifying the

risk posed to public health by oral exposure to the BSE agent, as the

results of such experiments can never provide hard information on what may

happen to the human population after oral exposure to BSE. However, the

paper should stimulate further research into pathogenesis of prion diseases

in view of the intriguing findings in peripheral tissues, and there is

clearly a need for more information on the peripheral pathogenesis of human

prion diseases, not least because of possible public health implications.

FOOTNOTES

The companion to this Commentary is published on page 4046 in issue 7 of

volume 96.

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