1 Laboratório Nacional de Investigação Veterinária, Lisboa, Portugal
2 Instituto Gulbenkian de Ciência, Rua da Quinta Grande 6, 2780-156 Oeiras, Portugal
3 Laboratório de Microbiologia, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal
Correspondence
J. Pedro Simas
jpsimas{at}igc.gulbenkian.pt
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ABSTRACT |
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MAIN TEXT |
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In Portugal, despite the completion of previous scrapie surveillance plans, both at the clinical and histopathological levels, as yet no scrapie cases have been diagnosed. The absence of scrapie in Portugal cannot be attributed to a genetic basis and the possibility has been suggested for subclinical or other forms of scrapie strains to be present (Orge et al., 2003). In this study, as a result of the EU active scrapie surveillance plan, we have identified the first cases of sheep with detectable PrPres deposition in the central nervous system (CNS) in Portugal.
Since its implementation in 2002 (according to EU regulation 999/2001 and subsequent alterations) until February 2004, a total of 30 269 sheep over 18 months of age were screened for the presence of PrPres in the brainstem by the Bio-Rad rapid test done according to manufacturer's instructions. All the cases that tested positive by the rapid test had been selected for human consumption. These cases were analysed further for the presence of vacuolation by histopathology, for the deposition of PrPres by immunohistochemistry and for the analysis of the PrPres electrophoretic profile by Western immunoblotting, and the Prnp (codons 136, 154 and 171) genotypes were also determined. Brainstems were divided sagittally with one half frozen for rapid test analysis, Western immunoblotting and DNA extraction, and the other half fixed in 10 % saline formalin and processed for routine paraffin histology. For immunohistochemistry, three anti-PrP monoclonal antibodies, 2G11 (Institute Pourquier-Farmoquil), SAF84 (SPI-bio) and L42 (RIDA; R-Biopharm), were used in three confirmatory independent assays essentially as previously described (Orge et al., 2000). For 2G11, sections were pretreated with formic acid followed by autoclaving. In the case of SAF84 and L42, sections were, in addition to formic acid treatment, pretreated with proteinase K as described previously (Bencsik et al., 2001
; Hardt et al., 2000
). The specificity of PrPres immunolabelling was controlled using sections from known scrapie-positive [kindly supplied by the Veterinary Laboratory Agency (VLA) archive] and scrapie-negative cases. For Western immunoblotting analysis of PrPres, frozen brainstem tissue was processed according to a new protocol from Bio-Rad developed for ovine tissues. For Prnp genotyping, genomic DNA extracted from frozen brainstem tissue was subjected to PCR with Prnp specific primers (upper and lower primers: 321340 and 572590 nt, respectively, according to GenBank accession no. AF180389) to yield an amplicon that encompassed codons 136, 154 and 171. PCR products were then submitted to automated cycle sequencing in both orientations using an ABI Prism 377 DNA sequencer (Perkin Elmer).
As a result of the rapid test screening, seven sheep slaughtered for human consumption that tested positive for the presence of PrPres (Table 1) were confirmed for the deposition of PrPres in the brainstem at the level of the obex, both by us and the VLA despite the absence of obvious vacuolation. All cases showed the same granular PrPres deposition in the neuropil of the spinal tract nucleus of the trigeminal nerve (STN V) (Fig. 1
b, e, h and k). Remarkably, no PrPres deposition could be detected in the neuropil of the dorsal vagal nucleus (DVN) (Fig. 1a, d, g and j
), which is the neuronal nucleus that is always associated with PrPres deposition in scrapie. In three sheep (Table 1
; cases 4, 6 and 7), focal granular immunostaining in the neuropil of the nucleus of the solitary tract (NST) was also observed (Fig. 1c, f and i
). This distribution is unusual because of the consistent absence of detectable PrPres in the DVN and the deposition in the STN V. In scrapie, it has been extensively reported that the DVN is the first CNS nucleus to show vacuolation and PrPres deposition (Andreoletti et al., 2000
; Ligios et al., 2002
; Ryder et al., 2001
; van Keulen et al., 1995
; Wood et al., 1997
), hence, is the elected nucleus used for routine diagnosis. Thus, the distribution of PrPres deposition obtained with three different anti-PrP specific antibodies was atypical in all seven cases reported here. Recently, scrapie cases have been reported that also diverged phenotypically from typical scrapie (Benestad et al., 2003
; Buschmann et al., 2004
). In particular, in several atypical scrapie cases that have been reported in Norway, PrPres deposition was observed in the cerebellum, midbrain and cerebral cortex, but no PrPres could be detected in the obex region (Benestad et al., 2003
). In these cases, designated Nor98, although inconsistently, PrPres deposition was also detected in the STN V. These findings are consistent with those described here in that no PrPres could be detected in the DVN.
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In order to gain further insight into the epidemiological and public health relevance of this apparent atypical scrapie, it is extremely important to show if it is transmissible and assess if it constitutes a new scrapie strain like Nor98. The occurrence of such a putative atypical scrapie strain, independently of the Prnp genotype, may be of significance for current EU sheep breeding programmes for selection of non-susceptible haplotypes. Furthermore, given the recent BSE epidemic in Portugal (Donnelly et al., 1999), and thus the geographical risk of transmission of BSE into sheep, a possible link to BSE cannot be excluded. Although, we cannot rule out the possibility of adaptation of BSE in sheep following natural transmission, the fact that the PrPres electrophoretic profile described for this putative atypical scrapie strain is distinct from that observed in BSE experimentally infected sheep, makes this link less probable.
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ACKNOWLEDGEMENTS |
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Received 30 April 2004;
accepted 24 July 2004.