NR AKOI
AU Schreuder,B.E.C.; van Keulen,L.J.M.; Vromans,M.E.W.; Langeveld,J.P.M.; Smits,M.A.
TI Preclinical test for prion diseases
QU Nature 1996 Jun 13; 381(6583): 563
PT letter
VT
SCIENTIFIC CORRESPONDENCE
Preclinical test for prion diseases
SIR - The recent recognition that there may be a link between bovine spongiforrn encephalopathy (BSE) in cattle and Creutzfeldt-Jakob disease (CJD) in humans[1] highlights the urgent need to find a method for the preclinical diagnosis of transmissible spongiform encephalopathies, or prion diseases. Until then, given the long incubation period of the disease agent, control measures will be hampered. Here we suggest a possible approach to an early preclinical diagnostic method for transmissible spongiform encephalopathies using scrapie in sheep as a model.
Infected animals and humans have neither a disease-speciflc immune response nor consistent biochemical, haematological and gross pathological abnormalities. The early diagnosis of transmissible spongifonn encephalopathies therefore depends on the recognition of clinical signs, electroencephalography or magnetic resonance imaging techniques, or the more invasive method of taking brain biopsies.
Confirmatory diagnosis, however, depends on histological examination of the brain of suspected cases. An altered protein, PrPsc, can be detected in the brain of diseased individuals, by immunohistochemistry or other protein-detection methods. Numerous studies have confirmed PrPsc as a sensitive and specific marker of this group of diseases [2].
Immunohistochemistry does not demonstrate infectivity, but it does have many advantages over the mouse bioassay, which is considered a more sensitive detection method but far too cumbersome and time-consuming to become a practical diagnostic method. Various tissues -blood, urine, tissue fibroblasts and, particularly in animals, lymphoid tissue - have been used in attempts to develop an early-detection technique for these encephalopathies. Most attempts were either negative or inconclusive [3], but we have built on the classic work of Hadlow, who showed that in sheep naturally infected with scrapie, infectivity was detectable in lymphoid tissue as early as 10-14 months of age, well before it occurred in the central nervous system [4,5].
Having demonstrated the consistent presence of the scrapie-associated PrPsc in tonsils of a group (n=55) of naturally infected, clinically positive scrapie sheep by immunohistochemistry[6], using antibodies directed to selected, synthetic PrP-based peptides[7], we have now detected PrPsc in tonsils of sheep in the preclinical stage of the disease, long before the onset of clinical signs.
We selected a group of ten purposely bred lambs, six of them homozygous for the PrPVQ allele (with the residue valine (V) at position 136 of the amino-acid sequence and glutamine (Q) at position 171). The remaining four lambs were heterozygous, possessing one PrPVQ allele and one PrPAR allele (with alanine at position 136 and arginine at position 171). In several breeds, this PrPVQ allele is significantly associated with an increased susceptibility to scrapie, whereas the PrPAR allele is significantly associated with increased resistance of sheep to scrapie[8].
The ten lambs were born and raised on a farrn where scrapie has been occurring for several years. In this flock, we had observed that sheep with the genotype PrPVQ/VQ died from scrapie when about 25 months old, but that most of the sheep with the genotype PrPVQ/AR were still healthy at 70 months. Born and raised in this environment, the PrPVQ/VQ sheep would thus be expected to develop disease symptoms when about 25 months old, whereas the PrPVQ/AR sheep would stay healthy. We therefore regarded these two groups of sheep as a suitable model for studying changes at known stages of the incubation period.
We took tonsillar biopsies from these animals at approximately 10 (range 9.5-10) months after birth, when none of the sheep showed clinical signs of scrapie. We found clear, extensive PrPsc immuno-staining in all six susceptible PrPVQ/VQ sheep (a in the figure) at 10 months of age, but detected no PrPsc immuno-staining in any of the resistant PrPVQ/AR sheep (b in the figure).
We have thus detected scrapie-associated PrPsc in tonsils of sheep at less than half the expected incubation period, approximately one year before the expected onset of clinical disease. We did not detect this PrPsc protein in sheep expected to develop scrapie at a much later stage or to remain healthy throughout their lives. Thus, screening tonsillar tissue for PrPsc by immunohistochemistry offers a possibility of preclinical diagnosis in sheep scrapie.
In view of the negative transmission experiments using different peripheral tissues of cattle affected by BSE, our technique may not work in cattle. However, because immunohistochemistry has not been tried in depth as a diagnostic test, and because the mouse bioassay may not be sensitive enough in the case of BSE, it is surely worth investigating our suggestion further.
An early diagnosis of BSE could alleviate the draconian measures proposed in the United Kingdom for culling potentially BSE-infected cattle. The technique of taking tonsillar biopsies in live cattle is feasible and would be even easier than in sheep. In human spongiform encephalopathies, this approach has even brighter prospects, as infectivity has been detected in various lymphoid tissues of CJD patients[10], and tonsil biopsy is a relatively noninvasive, easy technique.
B. E. C. Schreuder, L. J. M. van Keulen M.E.W. Vromans, J. P.M. Langeveld
M. A. Smits
DLO-Institute for Animal Science and Health (ID-DLO), P0 Box 65, 8200 AB Lelystad, The Netherlands
1. Will, R. G. et al. Lancet 374, 921-925 (1996).
2. Prusiner, S., Collinge, J. & Anderton, B. in Prion
Diseases in Humans and Animals (Ellis Horwood,
London, 1992).
3. Schreuder, B. E. C. Vet. Q. 16, 174-181 (1998).
4, Hadlow, W. J., Kennedy, R. C. & Race, R.E. J. infect.
Dis. 146, 657-664 (1982).
5. Hadlow, W. J.et al. Vet. Path. 17, 187-199 (1980).
6. van Keulen, L i M. et al. J. clin. Microbiol. 34,
1228-1231 (1996).
7. van Keulen, L.J.M. et al. Vet. Path. 32, 299-308(1995).
8. Belt, P. B. G.M. et al. J. gen. Viral. 76, 508-517(1995).
9. Fraser, H. & Foster, J. D. in Proc. Consult. BSE sci. vet. Committee CEc 145-160 (Brussels, September 1993).
10. Brown, P. et al. Ann. Neural. 35, 513-519 (1994).
Figure 1
a, Positive PrPsc immuno-staining in lymphoid follicles in tonsillar biopsy from a scrapie-susceptible sheep (PrPVQ/VQ) at age 10 months. Peroxidase-labelled streptavidin-biotin staining.
b, Negatively staining tonsillar biopsy from a PrPVQ/AR sheep at the same age. Scale bars, 100 µm.
IN Die Autoren beobachteten in einer seit Jahren mit Scrapie infizierten Herde, dass homozygote Schafe mit Valin an Position 136 und Glutamin an Position 171 nach rund 25 Monaten erkrankten, während die meisten heterozygoten Schafe mit Valin und Alanin an Position 136, sowie Glutamin und Arginin an Position 171, selbst nach 70 Monaten gesund waren. Mit Antikörpern gegen Prionpeptide fanden die Autoren in den lymphoiden Follikeln der Tonsillen (Gaumenmandeln) bei 6 erst 10 Monate alten, wahrscheinlich scrapieinfizierten und homozygoten Schafen scrapieassoziiertes Prionprotein lange vor dem Ausbruch der Krankheit. Bei 4 ebenfalls wahrscheinlich scrapieinfizierten, jedoch heterozygoten Schafen fanden die Autoren keine Markierung. Der Tonsillen-Test erkannte also Scrapie bereits nach weniger als der Hälfte der normalen Inkubationszeit rund 15 Monate vor dem mutmaßlichen Ausbruch der Krankheit.
ZR 10
MH Alleles; Animal; Human; PrPsc Proteins/*analysis/genetics; Prion Diseases/*diagnosis; Scrapie/diagnosis; Sheep; Time Factors; Tonsil/*metabolism
SP englisch
PO England
OR Prion-Krankheiten 7