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Short Communication |
1 Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy
2 Robert Koch-Institut (P24 – Transmissible Spongiform Encephalopathies), Nordufer 20, 13353 Berlin, Germany
3 Prion and Dementia Research Unit, Department of Neuropathology, University Medical Center, Georg-August University Goettingen, Robert-Koch-Str. 40, 37075 Goettingen, Germany
4 7815 Exeter Road, Bethesda, MD 20814, USA
Correspondence
Franco Cardone
franco.cardone{at}iss.it
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These authors contributed equally to this work. 
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; Manson et al., 2006). Other species, however, are also susceptible to TSEs, as shown by accidental (e.g. feline spongiform encephalopathy) or experimental (e.g. rodents, non-human primates) transmissions. In the central nervous system (CNS), massive replication of infectivity is accompanied by spongiosis, gliosis, neuronal loss and PrPTSE accumulation. Peripheral tissues, like those of the lymphoreticular system (e.g. spleen), viscera (e.g. liver), body fluids (e.g. blood) and muscle, contain lower levels of PrPTSE and infectivity (WHO, 2006).
In recent years, the search for TSE infectivity in muscles has become intensive, due to the importance of this tissue in the human diet and as a potential source of cross-contamination during invasive medical and surgical procedures, with the attendant risk of iatrogenic transmission of disease. The presence and distribution of infectivity or PrPTSE accumulation in muscles of ‘natural’ TSE infections differ in different host–strain combinations (Andréoletti et al., 2004; Angers et al., 2006; Beekes & McBride, 2007; Bosque et al., 2002; Casalone et al., 2005; Glatzel et al., 2003; Herzog et al., 2005; Peden et al., 2006; Thomzig et al., 2004, 2006). This heterogeneity has also been observed in experimental models of TSEs. In hamsters fed with 263K scrapie, PrPTSE and infectivity accumulate in muscles much later than in the CNS, suggesting a centrifugal neural spread of infection from the CNS into muscles (Thomzig et al., 2004). This hypothesis is further supported by the close proximity of PrPTSE deposits to neuromuscular junctions and muscle spindles in scrapie- or TME-infected hamsters (Mulcahy et al., 2004; Thomzig et al., 2004) and in scrapie-infected sheep (Andréoletti et al., 2004). In mice experimentally infected intracerebrally with BSE, the presence of PrPTSE in muscles is more restricted than in hamsters (Thomzig et al., 2003, 2006), but it is similar to that found in CJD patients (Glatzel et al., 2003; Peden et al., 2006) and in sheep with scrapie (Andréoletti et al., 2004). Because the oral route of infection can be important in the occurrence of some ‘natural’ TSEs, as well as variant CJD (Safar et al., 2008; Sigurdson & Aguzzi, 2007; Ward et al., 2006), we studied the chronological deposition of PrPTSE in muscles of mice fed with a mouse-adapted BSE strain.
To prepare the infectious BSE inoculum, brains from terminally ill C57BL/6 mice infected with the BSE isolate 6PB1 (Maignien et al., 1999; kindly provided by Dr Jean-Philippe Deslys, CEA, Fontenay-aux-Roses, France) were homogenized in 9 vols PBS. Clarified inoculum (100 µl) was then absorbed by food pellets and fed immediately to individually caged adult female C57BL/6 mice (n=26) previously subjected to 2 days starvation. After complete ingestion of BSE-infected pellets, the animals were housed (eight per cage) and observed daily for clinical signs of BSE. Groups of five animals were sedated and sacrificed by CO2 asphyxia during the preclinical stage of the disease at 100, 200 and 300 days post-infection (p.i.). One mouse died of intercurrent disease after 290 days. The remaining animals were sacrificed at clinical onset. Control animals (n=9) were fed pellets soaked with normal mouse-brain homogenate and were sacrificed following the schedule described for BSE-infected mice. All animals were autopsied and brain, spleen and a set of nine different muscles were sampled and either fixed in formalin for histological examination or frozen at –70 °C for PrP27–30 (the protease-resistant form of PrPTSE) purification, biochemical and paraffin-embedded tissue (PET)-blot analyses (Thomzig et al., 2003, 2004, 2006).
All mice allowed to survive became symptomatic, with a mean±SD incubation period of 368.3±13.7 days (n=10), confirming the efficiency of the oral route of infection in mouse BSE. As shown in Table 1 and Fig. 1, the earliest PrPTSE-positive immunoblots (one Musculus triceps brachii caput laterale and one M. trapezius) occurred in two different mice sacrificed at 100 days p.i. The proportion of PrPTSE-positive mice increased as the incubation period progressed and, at 300 days p.i., multiple muscle involvement began to occur. Heart and tongue samples, as well as muscles from mock-infected animals, were consistently PrPTSE-negative.
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, lanes M2, M5–M9; Fig. 1e, lanes M1–M3), sometimes appearing as a doublet, also visible after omission of the primary anti-PrP antibody, that were clearly distinguishable from PrP27–30 triple bands and were therefore considered unrelated to TSE infection. Analysis of brains from infected mice showed PrPTSE in two of five asymptomatic mice sacrificed at 300 days p.i. (during the preclinical period) and in all symptomatic animals. The electrophoretic pattern of PrP27–30 from muscles and brain was indistinguishable from the typical BSE signature observed in intracerebrally infected mice.
Immunohistochemical studies in preclinical animals showed granular PrPTSE deposition in the brainstem and pontine reticular nuclei (Fig. 2a). PET-blots of M. triceps brachii and M. psoas major were performed in some mice (Table 1) and revealed PrPTSE positivity only in the lymphatic tissue associated with muscle of preclinical animals. In brains from clinically affected animals, granular deposition was associated with PrPTSE plaques in the thalamus, mesencephalon, cerebellar cortex, pons and brainstem, together with spongiosis in the brainstem and cerebellum (Fig. 2b–d). In these animals, PET-blots of M. triceps brachii and M. psoas major revealed PrPTSE in the muscle, single fibres of small nerves and neuromuscular junctions.
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). All but one animal with PrPTSE-positive muscles had moderate to high PrPTSE load in the spleen (Fig. 1h).
The novelty of these data is that, in our rodent model for BSE in cattle, i.e. mice infected orally with a mouse-adapted BSE strain, there is an unexpected early preclinical deposition of PrPTSE in muscles, probably associated with the lymphatic tissues, which occurs as early as during the first one-third of the incubation period. This result is similar to what has been found in sheep infected orally with scrapie, where muscle PrPTSE was detected during the first one-quarter of the incubation period (Andréoletti et al., 2004). Interestingly, this phenomenon does not occur in all TSE models; for example, in hamsters fed with 263K scrapie, muscles only became PrPTSE-positive near clinical onset (Thomzig et al., 2004).
The early involvement of spleen and other lymphatic tissues found in this study is consistent with the observations reported by Maignien et al. (1999) in mice infected with the same BSE strain and by the same route. Interestingly, they also observed that in the later stages of the incubation period, PrPTSE appeared simultaneously in the thoracic spinal cord and brain, suggesting that after oral infection, the BSE agent spreads by blood or lymph to lymphatic tissues associated with muscles, and only at a later stage during the incubation period enters the CNS and is then projected into muscles via nerve fibres.
The late centrifugal spread of PrPTSE from the CNS to muscle in orally BSE-infected mice is also consistent with observations in other experimental models (Thomzig et al., 2004; Herzog et al., 2005) or in scrapie-infected sheep (Andréoletti et al., 2004), where the CNS plays a primary role in the dissemination of infectivity to muscles (Beekes & McBride, 2007).
In the light of these new data, there would seem to be a very low probability that BSE-infected cows harbour infectivity in muscle destined for human consumption, in view of the minimal involvement of the lymphoreticular system in cattle and the continued absence of evidence for infectivity in bovine muscles (Buschmann & Groschup, 2005; Espinosa et al., 2007; WHO, 2006). It is nevertheless possible that limited muscle sampling and methodological insensitivity could fail to detect the irregular distribution of low levels of PrPTSE in muscle. Moreover, the absence of PrPTSE does not necessarily imply absence of infectivity (Barron et al., 2007; Berardi et al., 2006; Lasmézas et al., 1997). A more thorough examination of entire muscle groups using the ultrasensitive protein-misfolding cyclic-amplification technique (Soto et al., 2005) might yield a positive result, but would need to be verified by infectivity bioassay before inferring a risk of disease transmission.
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ACKNOWLEDGEMENTS |
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Received 4 February 2009;
accepted 17 June 2009.
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