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Institute for Novel and Emerging Infectious Diseases, Friedrich-Loeffler-Institut, Boddenblick 5a, 17493 Greifswald-Insel Riems, Germany
Correspondence
Martin H. Groschup
martin.groschup{at}fli.bund.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Transmission of BSE prions to man has eventually caused a variant form of Creutzfeldt–Jakob disease in more than 170 humans primarily in the UK, but also in France, Italy, Japan and elsewhere. As a preventive measure in the European Union, the risk of human BSE exposure is minimized by rapid BSE testing of all cattle over 30 months of age and by the removal of specified risk materials from slaughtered cattle that may possibly contain BSE infectivity in incubating animals. These materials currently include the head and backbone, including the central nervous system (CNS) and spinal cord, of cattle over 12 months of age, and the intestine (independent of age). However, it is presently under discussion in the EU Commission to raise the age limit for head and backbone removal from 12 to 24 months.
During the BSE epidemic, cattle were probably infected by oral uptake of infectious foodstuffs (Wilesmith et al., 1988; Paisley & Hostrup-Pedersen, 2004). However, although several approaches addressing the pathogenesis of BSE have been undertaken, the route and time course of the infectious agent from the gastrointestinal tract (GIT) to the CNS is still unknown. To date, very few data for BSE-incubating preclinical cattle are available.
A feature in the early pathogenesis of orally induced prion diseases (including BSE) is the appearance of PrPSc in the ileal Peyer’s patches, the gut-associated lymphoid tissue (Maignien et al., 1999; Beekes & McBride, 2000; Heggebo et al., 2003; Terry et al., 2003). The subsequent spread of the BSE agent from the gut to the CNS is still an enigma. In experimentally orally challenged cattle, infectivity has been demonstrated convincingly solely in the trigeminal ganglia (Wells et al., 1998) of terminally incubating animals. Moreover, infectivity was detected by using a cattle bioassay in the palatine tonsil of animals killed 10 months post-infection, but not in animals assayed later in the incubation period (Wells et al., 2005).
In contrast to this, PrPSc and/or infectivity has been detected within the entire lymphoreticular system (LRS) of naturally or experimentally BSE- or scrapie-infected sheep, mice and hamsters (van Keulen et al., 1996; Andreoletti et al., 2000; Aguzzi, 2003; Glatzel et al., 2004; Press et al., 2004), whereas the paucity of BSE prions in these tissues (with the exception of the above-mentioned result) in cattle is striking. Studies in sheep and hamsters infected orally with scrapie indicate an important role of the autonomic nervous system in the spread of the transmissible spongiform encephalopathy (TSE) agent. Two possible neuroanatomical pathways, which use the intramural ganglia of the gut and the coeliac and mesenteric ganglion complex (CMGC) as intervening relay points, are proposed: along the splanchnic nerve to the midthoracic spinal cord and/or along the vagus nerve to the brainstem (van Keulen et al., 2000; McBride et al., 2001). However, to date in cattle, BSE infectivity has only been detected in different parts of the CNS and peripheral nervous system (PNS) in clinical animals (Buschmann & Groschup, 2005; Wells et al., 1998, 2005).
Hence, the aim of the study was to elucidate the infection route and, in particular, the time course of BSE infection in cattle. New insights into the pathogenesis of BSE are important not only for the design of new diagnostic strategies, but also to minimize the risk of human BSE exposure, especially with regard to setting the age limit for the removal of specified risk material of cattle in the EU.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Immunohistochemistry (IHC).
With some modifications, tissue samples were processed as described previously (Hardt et al., 2000). All tissues samples were fixed in 4 % buffered formalin, treated for 1 h with 98 % formic acid, rinsed for 40 min in tap water, embedded in paraffin, sectioned at 3–4 µm and stained with haematoxylin and eosin.
The avidin–biotin complex (ABC) method was used for PrP-IHC. The paraffin-wax tissue sections were mounted on Superfrost Plus slides (Menzel–Gläser) and rehydrated. The subsequent pretreatment included incubation of the slides in 98 % formic acid for 15 min, a 5 min rinse in tap water, inhibition of the endogenous peroxidase activity with 3 % H2O2 (Merck) in methanol for 30 min, followed by 15 min digestion with proteinase K (4 µg ml–1; Boehringer Mannheim) at 37 °C. The primary monoclonal antibodies (mAbs) were applied at a dilution of 1 : 1800 for mAb 12F10 (Cayman Chemical) and 1 : 250 for mAb L42 (Harmeyer et al., 1998) in goat serum and incubated at 4 °C overnight. Negative-control sections were treated with a mAb against GP5 of Porcine respiratory and reproductive syndrome virus (Weiland et al., 1999). As a secondary (link) antibody, biotinylated goat anti-mouse antiserum (Vector Laboratories) was incubated on the sections in a 1 : 200 dilution for 30 min at room temperature. Immunodetection was amplified by using Vector ABC-elite avidin–horseradish peroxidase–biotin complex (Vector Laboratories). The slides were finally developed in diaminobenzidine tetrahydrochloride (Fluka Feinchemikalien) and counterstained with Mayer’s haematoxylin. All sections were examined by light microscopy.
Western blot (WB) analysis.
Selected tissue samples were investigated for the accumulation of PrPSc by Western blotting using phosphotungstic acid precipitations (PTA-WB), which were carried out according to a protocol established previously (Wadsworth et al., 2001; Glatzel et al., 2004) with some modifications described elsewhere (Gretzschel et al., 2005).
Rapid tests.
To confirm the IHC results at the level of the obex, adjacent brainstem material from the cranial and caudal medulla was examined by using the IDEXX HerdChek rapid test following the manufacturer’s instructions.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Kärber, 1931; Buschmann & Groschup, 2005) (data not shown). The cattle were housed in a special TSE infection facility and were assessed clinically every 2 months. Every 4 months, four or five randomly selected animals were euthanized and necropsied under TSE sterile conditions and more than 150 tissue and bodily fluid samples were collected from each animal.
To determine the location and earliest time point of a PrPSc deposition in the CNS or PNS, as well as in the LRS and viscera, the most relevant tissue samples (Table 1), i.e. brainstem at the level of the obex and distal ileum, were analysed by IHC. Here, results from two clinically normal animals that were killed after 24 months (cow A) and 28 months (cow B) and in which a PrPSc deposition at the obex was detectable at the earliest time point post-oral challenge are described. A full report covering all other animals will be published separately.
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). Both animals showed, like many other cows in the herd, moderate to severe eosinophilic enteritis, predominantly in the small intestine. In cow A, this was accompanied by moderate eosinophilic cholangitis and mild bile ductule proliferation. These alterations were probably due to mild coccidiosis as shown by light microscopy and in the parasitological examination. No other histopathological alterations were observed in the CNS or elsewhere.
Cow A
In the intestine, IHC immunolabelling was confined to the follicles of the Peyer’s patches of the distal ileum. A predominantly globular reaction pattern was observed in the cytoplasm of large mononuclear cells in the light central zone of 12 of 52 follicles examined (Fig. 1a). Additionally, very fine granular immunolabelling was found within single follicles. PrPSc was also found in the ileocaecal plica by PTA-WB, but nowhere else in the GIT (Table 1). BSE-specific PrPSc was not detected in the enteric nervous system (ENS) (myenteric or submucosal plexi) of the distal ileum or elsewhere.
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). Accumulated PrPSc was also seen in satellite cells. Moreover, a similar reaction pattern was observed in the Ganglion mesentericum caudale (GMC). Representing the next step in the autonomic nervous system, almost all segments of the spinal cord, with the exception of the caudal lumbal region and the Cauda equina, were shown to contain BSE-PrPSc by IHC and/or PTA-WB (Table 1). Again, only single neuronal cells in the Substantia intermedia centralis and lateralis displayed a mild intraneuronal PrPSc accumulation and, in some sections, even a weak linear staining pattern. No specific PrPSc staining was detected in ependymal cells or in the dorsal root ganglia of the thoracic spinal cord.
In the brain, PrPSc deposits were confined, bilaterally symmetrically, to the dorsal motor nucleus of the vagus (DMNV) at the level of the obex and were characterized by a diffuse distribution with slightly stronger immunolabelling in the medial parts of the DMNV. The mild reaction patterns were intraneuronal, perineuronal and linear when adjacent to neuronal cells, as well as intraglial and diffuse granular in the neuropil (Fig. 1c). Additionally, a weak intraneuronal staining reaction of single cells was detectable in the Medulla oblongata, confirmed by the results of PTA-immunoblotting (Fig. 2), and in the pons region, but not in the midbrain (Table 1).
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Cow B
In the entire GIT, neither the lymphoid tissues of the gut nor the compartments of the ENS showed detectable amounts of PrPSc (Table 1). Moreover, PrPSc was not detected in the autonomic nervous system or the entire spinal cord of this animal. However, mild perineuronal immunostaining of single cells was observed at the ventrolateral margin of the DMNV at the level of the obex. No immunoreactivity was found in the adjacent parts of the Medulla oblongata.
Interestingly, a negative result in the rapid test was obtained for the cranial and caudal parts of the Medulla oblongata (both adjacent to the level of the obex) of this animal.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Wells et al., 2005). These differences may be due to a considerable biological variation in the timing of the pathogenesis of BSE in cattle in individual animals. Although unlikely, the influence of the breed of cattle examined (Holstein–Friesian cattle used in UK studies versus Simmental cross-breed calves used here) cannot be ruled out. Similarly, it is conceivable that the gut-associated inflammatory processes, observed in both cows, may have had an effect on the progress of disease, a phenomenon that has already been shown in mice (Thackray et al., 2003). The BSE-challenged cows described here were clearly at the threshold of the earliest detection of PrPSc by currently available IHC and PTA-WB methods. The IHC examination was carried out with extreme scrutiny in order to discover even minor traces of PrPSc staining that may not be detected under routine conditions. There was just a mild immunostaining of single neuronal cells in the brainstem of cow A. Moreover, it should be emphasized that, in cow B, the IHC reaction was restricted to the DMNV at the obex. This limited IHC staining (as confirmed by analysing different parts of the brainstem, including the midbrain of cow A) indicates that no spread of BSE prions to other brain areas had taken place before this early time point post-infection.
In accordance with previous studies, an accumulation of PrPSc was observed in cow A in the Peyer’s patches of the distal ileum, mostly in tingible body macrophages and, to a lesser extent, in follicular dendritic cells, which are characterized by a fine reticular staining pattern (Andreoletti et al. 2000). However, PrPSc deposition was not detected in the ENS of the entire GIT. Both results are in contrast to the reported distribution of the scrapie agent in sheep (Heggebo et al., 2003), where PrPSc was consistently found in the ENS when there was also abundant PrPSc deposition in the gut-associated lymphoid tissue. To verify the sensitivity of our IHC technique, intestine samples from scrapie-diseased sheep were also examined; clear and strong PrPSc immunolabelling was observed in the ENS as expected (data not shown). Therefore, two possible routes may be considered for BSE prion spread in cattle in the GIT: (i) a direct transmission of the BSE agent from the gut lumen to the network of nerve fibres in the submucosa (Balemba et al., 1999) without further replication and subsequent accumulation in the neurons of the ENS and in the lymphoid tissue. The PrPSc accumulation in the Peyer’s patches reported here and elsewhere (Terry et al., 2003) could be due to tingible body macrophages involved in PrPSc clearance. (ii) An alternative explanation would be locally restricted uptake of BSE prions in the ileum and subsequent replication in the local follicles. This hypothesis is supported by the small amount of randomly distributed follicles that were positive in cow A and the lack of detectable amounts of PrPSc in cow B (tissue sections as thin as 3 µm may not be representative of the complete ileum when only a few follicles are affected).
Both hypotheses are supported by the data reported by Terry et al. (2003), which show sparse immunolabelling only in neurons of the ENS in two animals (38 and 40 months after exposure). Apart from the local Peyer’s patch invasion, the lymphoreticular tract seems not to be affected in cattle, as the adjacent Lymphonodi ileales (similar to the Lnn. jejunales, lienales and colici) were free of any detectable PrPSc.
The detection of PrPSc reported here in the DMNV, CMGC and GMG, as well as in the Substantia intermedia centralis and lateralis of the spinal cord of BSE-infected cattle, suggests two routes that BSE prions may take to the brain (Fig. 3). Both CMGC and GMC ganglia contain sympathetic and parasympathetic nerve fibres. Therefore, the most likely route by far follows the efferent sympathetic fibres of the Nervi splanchnici majores and minores, which contain nerve fibres crossing over in the CMGC, to the thoracic and/or lumbal spinal cord (T6–L2). It must be emphasized that both parts of the spinal cord can innervate the same part of the intestine (Fig. 3a). The importance of this pathway is also supported by immunolabelling of the sympathetic (in parts, splanchnici-associated) pre-ganglionic neuronal cells in the Substantia intermedia centralis and lateralis of the spinal cord. However, other than in the hamster scrapie model, in which an initial PrPSc accumulation in the mid-thoracic region and delayed deposition in other areas of the spinal cord (Beekes et al., 1996; Baldauf et al., 1997) were observed, all thoracic spinal cord segments, with the exception of the most caudal part (Th12), were affected evenly in the BSE-infected cow A, indicating an almost-simultaneous prion invasion through the Nn. splanchnici. However, this pattern could also have arisen from a focal invasion followed by a subsequent retro- and anterograde spread in the spinal cord. Moreover, the lack of involvement of the thoracic dorsal root ganglia, which contain the afferent neurons, also indicates the spread of PrPSc along the efferent nerve fibres via the Radix ventralis directly to the pre-ganglionic neuronal cells (Fig. 3b).
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The second possibility for the spread of BSE prions from the CMGC to the brain follows the parasympathetic nerve fibres of the vagus nerve, although it was not possible to demonstrate PrPSc accumulation in this nerve itself. This result supports the hypothesis of McBride et al. (2001) that PrPSc is in transit in nerve fibres rather than actively replicated. However, both cows showed a clear DMNV-associated PrPSc immunostaining of singleton neuronal cells at the level of the obex and cow B displayed minute immunostaining at this site without any other neuronal nuclei involved. This pattern is indicative of anterograde spread along the parasympathetic nerve fibres, which has already been described in hamsters infected orally with scrapie (McBride & Beekes, 1999; McBride et al., 2001). Moreover, in cow B, there was no PrPSc immunoreactivity detectable by IHC or PTA-immunoblot in the spinal cord. This result also indicates early BSE prion transmission along the vagus nerve.
In conclusion, results obtained here clearly suggest a neuronal rather than a lymphoreticular progression of BSE prions to the brain. Moreover, a simultaneous spread in the early pathogenesis of BSE is postulated: along the parasympathetic nerve fibres of the vagus nerve to the brain and via the sympathetic splanchnic nerves to the spinal cord and subsequently to the brain. The relatively short period of 24 months for the appearance of PrPSc deposition in the brain is of particular interest. This finding, although produced after challenge with a massive dose of BSE infectivity, which has been obtained 8 months earlier than described in similar experiments, should be taken into consideration during the discussion on the age limit for removal of brain and spinal cord as specified risk material of cattle in the EU.
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ACKNOWLEDGEMENTS |
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Received 8 May 2006;
accepted 18 November 2006.
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