1 AFSSA Lyon, 31 avenue Tony Garnier, 69364 Lyon cedex 07, France
2 CEA, Service de Pharmacologie et d'Immunologie, CEA/Saclay, France
Correspondence
J.-Y. Madec
jy.madec{at}lyon.afssa.fr
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ABSTRACT |
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MAIN TEXT |
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Using Western blotting, we previously studied 26 sheep from one scrapie-infected flock (Madec et al., 2000) and demonstrated that PrPres levels in the brain varied according to their prnp genotype. All sheep sampled were 2 years of age, the only group showing symptoms in this flock at the time of this study. In the present work, we used immunohistochemistry (IHC) and a sandwich ELISA to investigate 23 of these 26 sheep further (Table 1
). Initially, we considered a subset of 11 animals, all with detectable PrPres in the brain, as detected by Western blotting, which all carried at least one VRQ allele.
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PrPres was purified from the brain of all 11 sheep, following the protocol specified in the Bio-Rad BSE purification kit, and analysed by a sandwich ELISA using mAbs SAF-34 as the primary antibody (Demart et al., 1999) and 12F10 as the secondary antibody (Krasemann et al., 1996
, 1999
). The four brain sites previously found to be positive in Western blots in these 11 sheep (cerebellum, brain stem, mesencephalon and frontal cortex) were also positive for ELISA, confirming the suitability of the sandwich ELISA for the detection of PrPres in sheep tissues, as reported elsewhere (Bennion & Daggett, 2002
). The PrPres load was highest in the cerebellum, as reported previously (Ersdal et al., 2003
), and the PrPres distribution in brain appeared to be highly consistent with that previously detected using Western blotting (Madec et al., 2000
), showing a similar gradient in the levels of PrPres in positive animals from the cerebellum to the frontal cortex (Begara-McGorum et al., 2002
; Gonzalez et al., 2002
; Ligios et al., 2002
; Wood et al., 1997
; Zlotnik, 1958
).
Next, we investigated the 12 sheep from the same flock (Table 1) that had previously had negative results for the detection of PrPres in Western blots (Madec et al., 2000
), including sheep with possible early symptoms of scrapie (S18 and S22) and asymptomatic sheep (S1517, S1921 and S2326). Two had the susceptible allele VRQ, but in both cases this was with the resistant ARR allele. Overall, 9 of these 12 sheep carried this ARR allele.
All 12 sheep were diagnosed as negative for the detection of PrPres by IHC. However, four animals were positive for the detection of PrPres in the cerebellum by ELISA, and two were also positive by ELISA for detection of PrPres in the brain stem and/or mesencephalon (giving a specific signal 2·5 times greater than the cut-off level in three separate experiments). All positive results were obtained using pure 20 % homogenates and negative results were obtained at a 1 : 10 dilution, thus indicating low levels of accumulated PrPres. Two of these four sheep, including one VRQ/ARR sheep (S18), had been classified as having possible symptoms (Madec et al., 2000), including some wool loss and emaciation but no trembling, hyperexcitability or paresthesias. However, since marked clinical signs can be associated with a low amount of brain PrPres (as in sheep S9), it was reasonable to assume that a less obvious clinical status might also be associated with a very low level of PrPres accumulation, possibly only detectable by ELISA. By comparison, the two animals in which PrPSc was only found in the DMNV (S9 and S14, see above) by IHC displayed OD values by ELISA at least 2·5-fold higher than those obtained for the two IHC-negative sheep with possible symptoms (S18 and S22). These results suggested that ELISA may have a higher sensitivity than IHC for the detection of PrPres in these sheep and under these experimental conditions, whereas Western blotting and IHC may have a comparable sensitivity, as also suggested by other studies (Jeffrey et al., 2002
).
Interestingly, three of these four asymptomatic (or possibly asymptomatic for scrapie) and ELISA-positive animals carried the ARR allele (Table 1). The ARR allele is associated with a protective effect against, if not resistance to, clinical scrapie. Our results thus raise some concern that PrPSc can be found not only in preclinical stages of the disease in susceptible animals, but also in relatively resistant animals. It is generally considered that ARH/ARR animals are either not expected to develop scrapie during their life span or that they will develop the disease only after 70 months (Andréoletti et al., 2000
; van Keulen et al., 1996
). Therefore, these findings suggest that scrapie-infected sheep with genetic resistance may behave as healthy carriers or as only very slightly affected animals in flocks. The possibility of a carrier state has already been suggested in certain rodent models (Collis & Kimberlin, 1985
; Race & Chesebro, 1998
; Race et al., 2001
) and this could help to explain the persistence of the natural disease in some cases. It may also have significance for breeding and lambing management programmes for the eradication of scrapie.
It is not known whether both PrPs encoded by the ARH and ARR alleles or only the PrP encoded by the most susceptible alleles (ARH or VRQ) are converted into a pathological form in these sheep during the natural process of scrapie infection. It is notable that the sheep PrP encoded by the ARR allele was successfully converted into its pathological counterpart in experimental BSE challenge of sheep (Houston et al., 2003). A recent study also reported that some atypical ELISA-positive sheep remained negative using either Western blotting or, in some cases, IHC (Buschmann et al., 2003
), and this work also included a number of sheep carrying the ARR or AHQ allele associated with a relatively high resistance to clinical scrapie. Similar observations were also made by another group in sheep with so-called Nor98 scrapie carrying the AHQ allele, displaying clinical signs of scrapie (Benestad et al., 2003
). The data presented here on asymptomatic animals with partial genetic resistance to the disease reinforce the possible association between these alleles and an unusual biochemical behaviour or low levels of PrPSc, according to the method used for its identification. Attempts to transmit the disease to ovine-transgenic mice, which have been shown to be more susceptible to scrapie than conventional rodents (Crozet et al., 2001
; Vilotte et al., 2001
), will help to determine the potential infectivity of these samples.
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ACKNOWLEDGEMENTS |
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Received 23 April 2004;
accepted 26 July 2004.