CEA, Service de Neurovirologie, DSV/DRM, CRSSA, BP 6, 6068 avenue du Général Leclerc, 92 265 Fontenay-aux-Roses cedex, France1
Author for correspondence: Jean-Philippe Deslys.Fax + 33 1 46 54 77 26. e-mail jpdeslys{at}cea.fr
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Abstract |
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Introduction |
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With regard to the pathogenesis of TSEs, the role of the LRS in the early steps of infection has been demonstrated in a number of studies. Indeed after intraperitoneal (i.p.) infection, the spleen is infectious and exhibits PrPres accumulation long before the agent has spread to the CNS (Kimberlin & Walker, 1979 , 1989 b
; Doi et al., 1988
). After oral contamination, pathogenesis seems to be less or not dependent on the spleen. Indeed Kimberlin et al. (1989a)
demonstrated that splenectomy had no effect on the incubation period of 139A scrapie administered intragastrically. Moreover, studies performed in the hamster model infected with the 263K scrapie strain pointed out a poor involvement of the spleen in the early pathogenesis of oral infection (Beekes et al., 1996
). Furthermore, these studies identified an alternative neuroinvasion pathway, via the vagal nerve to the medulla (Baldauf et al., 1997
; Beekes et al. , 1998
), in addition to that described in i.p. infection models targeting the thoracic cord with spread of the agent to the brain (Kimberlin & Walker, 1979
).
In this study, we aimed to determine more precisely the propagation pathway of TSE agents after oral contamination. Differences between BSE and scrapie pathogenesis have already been pointed out, particularly concerning the tissue distribution of infectivity. Additionally, the fact that BSE, as opposed to scrapie, has infected carnivores and probably humans via the oral route suggests that the BSE agent could exhibit specific properties with regards to this mode of contamination. Thus, we compared the pathogenesis of oral infection in mice exposed to murine strains of BSE and scrapie. This allowed us to distinguish between the strain-specific characteristics and the common features of the kinetics and the way the TSE agent moves from the digestive tract to the brain.
Practically, we searched for the presence of PrPres, as a marker of infectivity, in 22 organs sequentially throughout the infection from the day of oral dosing until the death of the animal.
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Methods |
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Oral contamination with TSE agents.
We developed an experimental system of oral dosing akin to the natural conditions of food intake. The first day, the mice were accustomed to drink the inoculum. To this purpose, the animals were placed in individual cages equipped with a liquid delivery system consisting of an Eppendorf tube pierced with a 3 mm hole. The tubes were filled with Endolipid, a lipidic injectable emulsion for parenteral nutrition (composition: 20% soya oil, 1·2% egg lecithin, 2·5% glycerol in water) amenable to the preparation of an emulsion with brain homogenate that remains stable during the time of experimentation. Moreover, its appetizing properties encourage rapid consumption by the animals. The next day, the tubes were filled with the infectious Endolipid+brain preparation. Practically, 100 µl of 20% (w/v) scrapie or BSE brain homogenate was mixed with 100 µl Endolipid. Consumption of the infectious preparations was carefully monitored; in all experiments described here, the mice drank the whole inoculum within 15 min.
Fifty-six mice took the inoculum containing either of the two agent strains under monitoring.
Sampling of material.
Mice were sacrificed in duplicate sequentially at 20 days post- inoculation (p.i.), 30 days p.i., 45 days p.i., 60 days p.i. and thereafter at 30-day-intervals until the terminal stage of the disease. Sacrifices were performed by cervical dislocation. Twenty-two organs were harvested per mouse in liquid nitrogen and kept at -80 °C until molecular analysis in the order of the less to the most infectious organs as far as is known from the literature. Samples were as follows: brain, cervical spinal cord, dorsal spinal cord, lumbar spinal cord, digestive tract (stomach, duodenum, jejunum, ileum, caecum, colon, liver, omentum, pancreas and salivary glands) and LRS (Peyer's patches, mesenteric, axillary and sub-maxillary lymph nodes, palatine formations, femoral bone marrow, spleen, thymus).
Intraperitoneal contamination with TSE agents.
Fifty mice were injected by the i.p. route with 100 µl of 2% brain homogenate of a mouse at the terminal stage of scrapie. The mice were sacrificed sequentially in triplicate at 2, 7, 14, 21, 28, 35, 49 and 65 days p.i. The spleen, Peyer's patches, mesenteric and axillary lymph nodes were harvested using the same protocol as in the oral route experiment.
PrPres purification and detection.
The tissues were homogenized at 20% (w/v) in a 5% sterile glucose solution for the voluminous samples and 10% or 5% for the smaller ones, such as lymph nodes. We verified that the efficiency of PrPres purification was the same for the three dilutions of homogenate.
PrPres was purified by centrifugation in the presence of detergents, after adapted proteinase K digestion according to a previously reported scrapie-associated fibril (SAF) protocol (Lasmézas et al., 1997 ). Samples were loaded on a 12% SDS polyacrylamide gel and then transferred onto a nitrocellulose membrane (Schleicher & Schuell). Immunoblotting was performed with a rabbit polyclonal anti-mouse PrP antibody (JB007, produced in the laboratory) at a 1/5000 dilution (Demaimay et al. , 1997
). Immunoreactivity was detected with an enhanced chemiluminescence kit (ECL, Amersham) and visualized on autoradiographic films. For each experiment, a dilution scale of the positive control, the brain of a C57BL/6 mouse at the terminal stage of the disease (C506M3 scrapie strain), was submitted to the same protocol for PrPres detection.
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Results |
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Several segments of the CNS were dissected for analysis of the neuroinvasion. In fact the thoracic, cervical and lumbar spinal cord as well as the brain became positive for PrPres at about the same time, i.e. 270 days p.i. in the scrapie model and 300 days p.i. in the BSE model (Fig. 2 B). While in some cases the signal intensities were similar in the different parts of the CNS (as depicted in the left part of Fig. 2 B
), in other instances the thoracic spinal cord exhibited a stronger PrPres level than the other segments of the spinal cord and the brain.
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Discussion |
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PrPres has been used as a marker for infectivity as it has never been detected in the absence of TSE-associated infectivity. Using stabilized strains of TSE agents, PrPres is closely associated with infectivity. Our threshold level of PrPres detection corresponds to the amount of PrPres present in 2 µg of the brain of an infected mouse at the terminal stage of disease (corresponding to a 1/10000 dilution of our positive control, Fig. 2 A). Thus, the absence of detectable PrPres is interpreted, with the two stabilized strains used in this study, as a level of infectivity at least 10000- fold less than that in the CNS.
Fig. 1 shows the sequence of PrPres detection in the organs that were found positive at least once during the whole incubation period (out of 22 organs or tissues tested).
A different PrPres electrophoretic pattern between the BSE and scrapie strains was constantly observed, as illustrated in the brain positive controls (Fig. 3 B, lanes 1 and 2) and were consistent with the glycotyping studies of new variant CreutzfeldtJakob disease and BSE in mice (Hill et al., 1997
). However, variations in this pattern appeared for the same strain between the different organs and may be linked to differences of the enzymatic capacities between diverse cell populations leading, for example, to the addition of sugar residues of different lengths at the glycosylation sites. Such differences have also been observed by others (Hill et al., 1999
). Even if specific PrPres patterns can be observed after extraction with an SAF-like protocol and can be used for strain characterization, care should be taken, at this stage of knowledge, to compare similar organs.
Analysis of Fig. 1 indicates that the pathway of infection is the same for the two strains of agent: the LRS organs associated with the digestive tract, i.e. Peyer's patches and the mesenteric lymph nodes, appear to be the first sites of replication, followed by the organs of the LRS that are not related to the digestive tract, such as the spleen and the axillary lymph nodes.
This sequence of agent propagation strongly suggests that the agent is carried from the site of entry (gut) to the mesenteric lymph nodes via the lymphatic route; then, it has to travel through the bloodstream to reach the secondary LRS targets (spleen and lymph nodes associated with other organ systems). It is to be noted that some organs such as the pancreas, the salivary glands and the thymus were inconsistently found to be positive.
To be able to evaluate the relevance of this propagation pathway with regard to the oral route of contamination, we compared the first steps of infection observed in the model described hereupon with those occurring after i.p. inoculation with the scrapie strain (Fig. 4). Two major differences were observed. First, in our experimental conditions, the onset of PrPres accumulation at a detectable level occurred much earlier with the i.p. route, namely 5 days after inoculation versus 45 days via the oral route, despite the fact that the dose inoculated i.p. was 10 times lower than that administered orally. The apparent zero phase observed with the oral route could be related to the fact that only a small proportion of the infectious particles present in the inoculum did effectively cross the intestinal barrier. The cell population involved in the transport of the agent remains speculative in this stage, but one hypothesis would implicate M cells, which have been shown to be responsible for the uptake of bacteria (for a review see Trier, 1991
). The conjunction of a limited number of cells able to achieve this transport together with the clearance of the inoculum linked to the intestinal transit would account for the partial uptake of the inoculum.
Secondly, the propagation pathways differed between the two models as, by the i.p. route, the spleen constituted the primary replication site; later, the Peyer's patches and mesenteric lymph nodes were affected, together with lymph nodes unrelated to the digestive tract, such as the axillary lymph nodes. This confirms that after peripheral inoculation of TSE agents the primary replication sites within the LRS depend on the anatomy of the lymphatic/vascular circuitry in the vicinity of the entry site. Whereas the Peyer's patches are in direct contact with the gut epithelium and are connected with lymphatic vessels draining the mesenteric lymph nodes, the lymphoid cells present in the peritoneal cavity enter the bloodstream via the blood capillaries, which brings them first to the spleen. The fact that the spleen is not involved to the same extend, depending on the route of inoculation, has been suggested by Kimberlin & Walker (1989a , b
) who observed that splenectomy does not prolong the incubation period after intragastric infection as opposed to the prolongation observed after infection via the i.p. route.
At the terminal stage of disease after oral infection, the distribution of PrPres for both scrapie and BSE mouse-adapted strains involved the LRS organs, the CNS and occasionally the pancreas and the salivary glands but not, for example, the liver or the omentum (Fig. 3). This distribution corresponds to that classically described in natural and experimental sheep scrapie (Hadlow et al. , 1982
, 1984
) but is different from that observed in field cases of BSE, where only the CNS was found positive, and in experimental cattle infection via the oral route, where the ileum and peripheral nervous system were the only positive organs in addition to the CNS (Wells et al., 1998
). The tissue distribution of PrPres in our model is closer to that found in sheep experimentally infected with BSE via the oral route (Foster et al., 1996
). These findings show once more that the tissue distribution of the BSE agent can vary considerably, even for the same route of inoculation, depending on the genetic background of the host.
In the scrapie model only, the digestive tract was found to be positive from the stomach to the colon, including the ileum after removal of the Peyer's patches (Fig. 3). The fact that the ileum was negative in the case of BSE, although the Peyer's patches exhibited the same amount of PrPres as in the case of scrapie, indicates that the vast majority of PrPres found in the ileum of scrapie-infected mice was located outside these lymphoid formations. This means that PrPres is distributed all along the digestive tract, most probably either in the diffuse gut-associated lymphoid tissue (GALT) and/or in the plexuses of the enteric nervous system as described in immunohistochemical studies conducted in natural sheep scrapie (L. van Keulen, personal communication). The presence of PrPres within these plexuses that are connected to parasympathetic neurons of the vagus nerve would be consistent with the findings, obtained in the hamster model, that PrPres appears in the dorsal motor nucleary of the vagus nerve (DMNV) and solitary tract nucleus (SN) contemporary to the thoracic spinal cord and the suggestion of the important role of the vagal circuit in the spread of infectivity from visceral organs to the CNS (Baldauf et al., 1997
; Beekes et al., 1998
), in addition to the neuroinvasion through the thoracic spinal cord (Kimberlin & Walker, 1979
, 1982
, 1986
, 1989a
). In fact, both parasympathetic fibres of the vagal nerve (targeting the DMNV and SN) and sympathetic fibres of the celiac nerve complex (targeting the thoracic spinal segment) innervate the digestive system, including the pancreas and the mesenteric lymph nodes. Thus, it is not possible at this stage to know whether these pathways correspond to a direct neuroinvasion from the plexuses of the visceral organs as previously suggested (Kimberlin & Walker, 1989a
) or to a propagation to the aforementioned nerve fibres after prior replication in the GALT and lymph nodes. Both routes are probably possible and the preferential use of one of them may depend on the neuroinvasive properties of the TSE strain considered.
The propagation of the agent to the CNS occurs at 300 days p.i. with the BSE strain versus 270 days p.i. with the scrapie strain. PrPres was detected at the same time-point in the three segments of the spinal cord and the brain, suggesting a multisite entry in the CNS corresponding to sympathetic fibres projecting to the thoracic and lumbar cord and the vagus nerve targeting the medulla (Fig. 3). However, in some cases, the thoracic spinal cord exhibited a stronger PrPres level than the other segments of the spinal cord and the brain. This may be relevant to the pathogenesis of the neuroinvasion, although the precise observation of this process was hindered by the 30 day delay between the sacrifices.
The terminal stage of the disease was, similarly to the neuroinvasion, delayed by about 1 month in the BSE model when compared to the scrapie model (Fig. 1)
In conclusion this study shows that the propagation pathway is similar in C57BL/6 mice infected via the oral route with the BSE agent and one scrapie strain derived from sheep, and is clearly different from that of infection by the i.p. route.
Moreover, in the case of scrapie only, the digestive tract was found to be strongly positive for PrPres. This also means that the faeces might be infectious in these diseases, as they are in direct contact with the intestinal wall during the whole transit time and that cell exfoliation is very important in the intestinal lumen. Providing experimental evidence for this, however, entails substantial methodological difficulties due to the insolubility and the volume of the material to test (dilution of potential infectivity in a large amount of faeces produced per day). In our model, the identification of the cell type(s) responsible for the accumulation of PrPres in the digestive tract will provide further possibilities to appreciate the risk linked to faeces. It is to be noted that the intestine had been reported to harbour infectivity in one study conducted in sheep (Hadlow et al., 1982 ) and that lateral contamination can occur under normal field conditions (Dickinson et al., 1974
; Dickinson, 1976
). Our findings add weight to the suspicion that contamination via faeces could play an important role in the maintenance of endemic natural scrapie.
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Acknowledgments |
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We are grateful to Dr Karim Tarik Adjou for his critical reading of the manuscript. We gratefully acknowledge the expert care of the animals provided by René Rioux, Jean- Claude Mascaro and Denis Farrand.
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References |
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Received 15 February 1999;
accepted 20 July 1999.