- Email: [email protected]
Pre-clinical diagnosis of transmissible spongiform encephalopathies using rapid tests
Transfusion Clinique et Biologique 10 (2003) 19–22 www.elsevier.com/locate/tracli
EUROSAT 2002
Pre-clinical diagnosis of transmissible spongiform encephalopathies using rapid tests Le diagnostic préclinique des encéphalopathies spongiformes subaiguës transmissibles à l’aide de tests rapides Jacques Grassi * CEA, Service de pharmacologie et d’immunologie, bâtiment 136, CEA/Saclay, 981191 Gif-sur-Yvette cedex, France
Abstract During the past few years, important progress has been made in the post-mortem diagnosis of transmissible spongiform encephalopathies (TSEs) (scrapie and BSE) due to the development of the so-called “rapid test” based on the immunological detection of the abnormal form of the prion protein (PrPres) in the central nervous system. These methods now allow routine and high throughput testing, opening the door to large-scale epidemiological studies and systematic testing at slaughterhouses, thus preventing the entry of contaminated carcasses into the human food chain. It has been shown that some of these rapid tests allow pre-clinical diagnosis, anticipating by few months the appearance of clinical signs. In sheep and goat, PrPres can also be detected in peripheral lymphoid tissues a long time before the onset of clinical symptoms. As a consequence, the same rapid tests are suitable for pre-clinical diagnosis of scrapie in these species. It is very likely that the same kind of early diagnosis could be obtained for vCJD. The real challenge in the field of TSE diagnosis is the establishment of a vCJD test, conducted either on blood or urine, since these are the only biological fluids easily accessible from infected people. This is a very important issue to avoid iatrogenic transmission of vCJD within the human population. This is also very difficult because the quantities of infectious agents in the blood are certainly 100–1000 times lower than those present in the brain. © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Résumé Au cours de ces dernières années, des progrès importants ont été obtenus pour le diagnostic post-mortem des ESSTs (tremblante, ESB) grâce aux développement des tests dits “rapides” fondés sur la détection de la forme anormale de la protéine du prion (PrPres) dans le système nerveux central. Ces méthodes permettent maintenant d’analyser en routine un grand nombre d’échantillons ouvrant la porte à des études épidémiologiques à grande échelle et au test systématique à l’abattoir permettant ainsi d’éliminer du circuit de la consommation humaine les carcasses infectées. Il a été montré que certains de ces tests autorisaient une détection préclinique anticipant de quelques mois l’apparition des signes cliniques. Une détection préclinique est aussi possible chez les moutons et les chèvres atteints de tremblante en analysant les organes lymphoïdes périphériques. Dans ce cas la détection peut être très précoce. Le même genre de résultat peut probablement être observé chez l’homme atteint du variant de la maladie de Creutzfeldt-Jakob (vMCJ). Le véritable défi dans le domaine du diagnostic des ESSTs, c’est la mise au point d’un test pour le vMCJ réalisé sur le sang ou l’urine puisque ce sont les deux fluides biologiques facilement accessibles du vivant des personnes infectées. C’est très important pour éviter la transmission iatrogène du vMCJ dans la population humaine. C’est aussi très difficile dans la mesure où les quantités de PrPres dans le sang sont certainement 100 à 1000 fois plus faibles que celles observées dans le cerveau. © 2003 Éditions scientifiques et médicales Elsevier SAS. Tous droits réservés. Keywords: Prion protein; Diagnosis of prion diseases; Rapid tests; Peripheral lymphoid tissues Mots clés : Protéine du prion ; Diagnostic des maladies à prion ; Tests rapides ; Tissus lymphoïdes périphériques
* Corresponding author. E-mail address: [email protected] (J. Grassi). © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. DOI: 1 0 . 1 0 1 6 / S 1 2 4 6 - 7 8 2 0 ( 0 2 ) 0 0 2 7 9 - 3
20
J. Grassi / Transfusion Clinique et Biologique 10 (2003) 19–22
During the clinical phase of the disease, transmissible spongiform encephalopathies (TSEs) are characterised by the accumulation, in the central nervous system, of significant amounts of the abnormal form of the prion protein (PrPrsc or PrPres). PrPres derives from the normal form (PrPsens or PrPc) through post-translational modifications, which induce a conformational change and confer on the PrPres a partial resistance to degradation by proteases as well as a marked insolubility in the presence of detergents. During the past few years, important progress has been made in the post-mortem diagnosis of TSEs (scrapie and BSE) due to the development of the so-called “rapid test” based on the immunological detection of PrPres [1,2]. All these tests take advantage of the biochemical properties of PrPres and the availability of antibodies, recognising the denaturated form of PrPres. These methods now allow routine and high through-
put testing, opening the door to large-scale epidemiological studies and systematic testing at slaughterhouses and thus preventing the entry of contaminated carcasses into the human food chain [3]. For instance, in 2001, close to 8.5 million tests were performed in Europe [4]. On risk populations, 771,000 tests were performed and led to the identification of 744 BSE cases, while testing at slaughterhouses identified 279 cases, which were removed from human consumption. It has been shown that some of these rapid tests allow pre-clinical diagnosis, anticipating by few months the appearance of clinical signs [5]. This was demonstrated by analysing samples obtained from the pathogenesis studies conducted in Great Britain between 1991 and 1995 [6–8]. The objective of this study was to determine the spatial and temporal development of infectivity and pathological changes in cattle following an oral exposure to a single dose
Table 1 Design of the pathogenesis study and samples tested with rapid tests (Bio-Rad). Forty Friesian/Holstein calves, born in 1991, were assembled from farms with no history of BSE. At 4 months of age, 30 were challenged orally with 100 g of pooled brain stems from 75 cases of BSE. Ten calves received no treatment and served as controls. Starting at 6 months of age, 2 months after challenge, and then at 4 month intervals until 22 months after inoculation, three of the challenged calves and one of the control calves were killed. Thereafter, challenged and control cattle were killed at discretionary intervals, until the termination of the experiment at 40 months after inoculation. Samples of the brain and spinal cord were taken for examination with a rapid test (Bio-Rad test originally developed at CEA, see [1,2]). Key to abbreviations: BS = brain stem, SCC = spinal cord cervical, SCT = spinal cord thoracic, and SCL = spinal cord lumbar Months post-inoculation
2
6
10
14
18
22
26 28 32 35 36
38
40 Total
Animal number 187 219 198 265 298 199 272 235 276 264 301 222 230 303 229 233 270 271 297 254 232 299 194 231 261 192 300 277 296 74
BS X X X
X X
Challenged animals SCC SCT X X X X X X X X X X X X X
X X X X X X
X
X X X X X
X X X X X X X X
X X X X X X X X X X X X X X X X X X
X X X X X
X
SCL
BS X
X
X
X
315
X
X
X
X
237
X
X
186
X
X
X
X
302
X
X
X
11
SCL
213
X
316
X
X
X
X X X X X X
X X X X X
214
X
X
X
X
217
X
X
X
X
X
X
X
9
8
8
228 X 27
Control animals SCC SCT
Animal number 227
12
24
32
7
J. Grassi / Transfusion Clinique et Biologique 10 (2003) 19–22
21
these species. This is illustrated in Table 2, where results obtained on peripheral lymphoid tissues taken from sheep (sensitive genotype VRQ/VRQ) naturally infected at birth are presented. In this case, detection as early as 21 d after birth was observed. It is very likely that pre-clinical detection is also possible for vCJD in humans, since both PrPres and infectivity were consistently found in peripheral lymphoid tissues (spleen, tonsils, appendix) [9,10]. This is confirmed by the fact that very recently, one vCJD case was identified during a retrospective study conducted in UK on the tonsil and appendix [11].
Fig. 1. Results of the rapid tests performed on animals 32 months p.i. or more and scored as positive. White bar: brain stem, black bar: cervical spinal cord, hatched bar: thoracic spinal cord, and cross-hatched bar: lumbar spinal chord.
of BSE-affected brain homogenate (see legend of Table 1). Samples from the central nervous system collected during this study were analysed using a rapid test (Bio-Rad, [5]) and it was shown that PrPres could be detected 32 months postinoculation in animals, at least 3 months before the onset of clinical symptoms (Fig. 1). Depending on the nature of the host and the TSE strain, PrPres can also be detected in peripheral lymphoid tissues in sheep and goats infected by scrapie (including gut Peyer’s patches, lymph nodes, spleen and tonsils) a long time before the onset of clinical symptoms. As a consequence, the same rapid test is suitable for pre-clinical diagnosis of scrapie in
The set of measures taken to control the BSE outbreak and limit the risks of transmission to humans can now be considered to afford effective protection. Apart from the problem posed by the possible presence of BSE in the sheep population, it is now important to avoid iatrogenic transmission within the human population (through medical procedures such as organ grafts, blood transfusion). The goal now is to develop a blood test allowing ante-mortem and pre-clinical diagnosis of vCJD. The stakes are high in terms of public health, but the scientific and technical challenge is daunting because the quantities of infectious agents (and hence of PrPres?) in the blood are certainly 100–1000 times lower than those present in the brain. The development of such a test will therefore require new tools for the extraction and concentration of PrPres as well as the development of much more sensitive detection methods. Recent work has opened up new approaches either by identifying ligands specific to PrPres [12] or by showing that it is possible to amplify this PrPres “in vitro” [13]. Lastly, the demonstration of the pres-
Table 2 Pre-clinical detection of PrPres in peripheral lymphoid tissues of naturally infected sheep. Animals of sensitive genotype (VRQ/VRQ), naturally infected in a flock with endemic scrapie, were killed at sequential time. The PrPres content of peripheral lymphoid tissues was analysed using either Immunohistochemistry (IHC) or the Bio-Rad test (ELISA). Three to six animals of the same age were analysed. The results are expressed in terms of the number of positive samples on the number of samples analysed. Data in bold correspond to those samples for which positive results were recorded. PP = Peyer’s patches, LN = lymph node, MLN = mediastinal lymph node. Data kindly provided by Olivier Andréoletti (INRA/ENVT Toulouse, France, unpublished results) Organ/age Thymus Hepatic LN Spleen Mediastinal LN Prescpular LN Palatin tonsils PP-duodenum PP-jejunum 25% MLN-jejunum 50% PP-jejunum 50% MLN-jejunum 50% PP-jejunum 75% MLN-jejunum 75% PP-ileum MLN-ileum PP-caecum Ileo-coecal LN
IHC 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3
10 d ELISA 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3
IHC 0/3 0/3 0/3 0/3 0/3 0/3 1/6 (d) 2/6 (d-e) 0/3 2/6 (d-e) 0/3 2/6 (d-e) 0/3 4/6 (a-d-e-f) 0/3 0/3 0/3
21 d ELISA 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 1/3 (d) 0/3 0/3 0/3
IHC 0/3 0/3 0/3 0/3 0/3 3/6 (a-d-e) 4/6 (a-d-e-f) 4/6 (a-d-e-f) 4/6 (a-d-e-f) 4/6 (a-d-e-f) 3/6 (a-d-e) 6/6 4/6 (a-d-e-f) 6/6 6/6 3/6 (d-e-f) 3/6 (d-e-f)
64 d ELISA 0/3 0/3 0/3 0/3 0/3 4/6 (a-d-e-f) 2/6 (a-d) 4/6 (a-d-e-f) 1/6 (d) 5/6 (a-b-d-e-f) 1/6 (d) 4/6 (b-c-d-f) 1/6 (d) 6/6 5/6 (a-c-d-e-f) 2/6 (e-f) 2/6 (d-e)
IHC 0/3 4/6 (a-b-d-e) 3/6 (a-b-d) 5/6 (a-b-c-d-e) 4/6 (a-d-c-d) 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6
104 d ELISA 0/3 3/6 (b-c-d) 5/6 (a-b-d-e-f) 5/6 (a-b-c-d-e) 3/6 (a-c-d) 6/6 5/6 (a-b-d-e-f) 6/6 5/6 (a-b-c-d-f) 5/6 (a-b-c-d-f) 6/6 6/6 6/6 6/6 6/6 4/6 (b-d-e-f) 5/6 (a-b-c-d-e)
22
J. Grassi / Transfusion Clinique et Biologique 10 (2003) 19–22
ence of PrPres in the urine of animals or patients with a TSSE raises the prospect of an ante-mortem test usable on a large scale in humans [14]. In view of the number of research teams now focusing on this problem, it is not unreasonable to hope for spectacular progress in the years ahead.
[7]
Wells GAH, Dawson M, Hawkins SAC, Austin AR, Green RB, Dexter I, et al. Preliminary observations on the pathogenesis of experimental bovine spongiform encephalopathy. In: Gibbs Jr CJ, editor. Bovine spongiform encephalopathy: the BSE dilemma. New York: Springer-Verlag; 1996. p. 28–44.
[8]
Wells GAH, Hawkins SAC, Austin AR, Green RB, Dexter I, Spencer MJ, et al. Preliminary observations on the pathogenesis of experimental bovine spongiform encephalopathy (BSE): an update. Veterinary Record 1998;142:103–6.
[9]
Wadsworth JDF, Joiner S, Hill AF, Campbell TA, Luthert PJ, Collinge J. Tissue distribution of protease resistant prion protein in variant Creutzfeldt-Jakob disease using a highly sensitive immunoblotting. Lancet 2001;358:171–80.
References [1]
[2]
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[4]
[5]
[6]
Moynagh J, Schimmel H. Tests for BSE evaluated. A more detailed version is available at: http: //europa.eu.int/comm/dg24/health. Nature 1999;400:105. Grassi J, Créminon C, Frobert Y, Frétier P, Turbica I, Rezaei H, et al. Specific determination of the proteinase K-resistant form of the prion protein using two-site immunometric assays. Application to the postmortem diagnosis of BSE. Archives of Virology 2000;16((Suppl): 197):205. Deslys JP, Comoy E, Hawkins SAC, Simon S, Schimmel H, Wells GAH, et al. BSE screening of cattle carcasses. Nature 2001;409: 476–8. Report on the monitoring and testing of bovine animals for the presence of bovine spongiform encephalopathy (BSE) in 2001. Report from DG SANCO of the European Commission. http: //europa.eu.int/comm/food/fs/bse/bse45_en.pdf. Grassi J, Comoy E, Simon S, Créminon C, Frobert Y, Trapmann S, et al. Preclinical postmortem diagnosis of BSE in central nervous system tissue using a rapid test. Veterinary Record 2001;149: 577–82. Wells GAH, Dawson M, Hawkins SAC, Austin AR, Green RB, Dexter I, et al. Infectivity in the ileum of cattle challenged orally with bovine spongiform encephalopathy. Veterinary Record 1994;135: 40–1.
[10] Bruce ME, McConnell I, Will RG, Ironside JW. Detection of variant Creutzfeldt-Jakob disease infectivity in extraneural tissues. Lancet 2001;358:208–9. [11] Hilton DA, Ghani AC, Conyers L, Edwards P, McCardle L, Penney M, et al. Accumulation of prion protein in tonsil and appendix: review of tissue samples. British Medical Journal 2002;325:633–4. [12] Fischer MB, Roeckl C, Parizek P, Schwarz HP, Aguzzi A. Binding of disease associated prion protein to plasminogen. Nature 2000;408: 479–83. [13] Saborio GP, Permanne B, Soto C. Sensitive detection of pathological prion protein by cyclic amplification of protein misfolding. Nature 2001;411:810–3. [14] Shaked GM, Shaked Y, Kaviv Z, Halimi M, Avraham I, Gabizon R. A protease resistant PrP isoform is present in urine of animals and humans affected with prion diseases. Journal of Biological Chemistry 2001;276:31479–82.
EUROSAT 2002
Pre-clinical diagnosis of transmissible spongiform encephalopathies using rapid tests Le diagnostic préclinique des encéphalopathies spongiformes subaiguës transmissibles à l’aide de tests rapides Jacques Grassi * CEA, Service de pharmacologie et d’immunologie, bâtiment 136, CEA/Saclay, 981191 Gif-sur-Yvette cedex, France
Abstract During the past few years, important progress has been made in the post-mortem diagnosis of transmissible spongiform encephalopathies (TSEs) (scrapie and BSE) due to the development of the so-called “rapid test” based on the immunological detection of the abnormal form of the prion protein (PrPres) in the central nervous system. These methods now allow routine and high throughput testing, opening the door to large-scale epidemiological studies and systematic testing at slaughterhouses, thus preventing the entry of contaminated carcasses into the human food chain. It has been shown that some of these rapid tests allow pre-clinical diagnosis, anticipating by few months the appearance of clinical signs. In sheep and goat, PrPres can also be detected in peripheral lymphoid tissues a long time before the onset of clinical symptoms. As a consequence, the same rapid tests are suitable for pre-clinical diagnosis of scrapie in these species. It is very likely that the same kind of early diagnosis could be obtained for vCJD. The real challenge in the field of TSE diagnosis is the establishment of a vCJD test, conducted either on blood or urine, since these are the only biological fluids easily accessible from infected people. This is a very important issue to avoid iatrogenic transmission of vCJD within the human population. This is also very difficult because the quantities of infectious agents in the blood are certainly 100–1000 times lower than those present in the brain. © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Résumé Au cours de ces dernières années, des progrès importants ont été obtenus pour le diagnostic post-mortem des ESSTs (tremblante, ESB) grâce aux développement des tests dits “rapides” fondés sur la détection de la forme anormale de la protéine du prion (PrPres) dans le système nerveux central. Ces méthodes permettent maintenant d’analyser en routine un grand nombre d’échantillons ouvrant la porte à des études épidémiologiques à grande échelle et au test systématique à l’abattoir permettant ainsi d’éliminer du circuit de la consommation humaine les carcasses infectées. Il a été montré que certains de ces tests autorisaient une détection préclinique anticipant de quelques mois l’apparition des signes cliniques. Une détection préclinique est aussi possible chez les moutons et les chèvres atteints de tremblante en analysant les organes lymphoïdes périphériques. Dans ce cas la détection peut être très précoce. Le même genre de résultat peut probablement être observé chez l’homme atteint du variant de la maladie de Creutzfeldt-Jakob (vMCJ). Le véritable défi dans le domaine du diagnostic des ESSTs, c’est la mise au point d’un test pour le vMCJ réalisé sur le sang ou l’urine puisque ce sont les deux fluides biologiques facilement accessibles du vivant des personnes infectées. C’est très important pour éviter la transmission iatrogène du vMCJ dans la population humaine. C’est aussi très difficile dans la mesure où les quantités de PrPres dans le sang sont certainement 100 à 1000 fois plus faibles que celles observées dans le cerveau. © 2003 Éditions scientifiques et médicales Elsevier SAS. Tous droits réservés. Keywords: Prion protein; Diagnosis of prion diseases; Rapid tests; Peripheral lymphoid tissues Mots clés : Protéine du prion ; Diagnostic des maladies à prion ; Tests rapides ; Tissus lymphoïdes périphériques
* Corresponding author. E-mail address: [email protected] (J. Grassi). © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. DOI: 1 0 . 1 0 1 6 / S 1 2 4 6 - 7 8 2 0 ( 0 2 ) 0 0 2 7 9 - 3
20
J. Grassi / Transfusion Clinique et Biologique 10 (2003) 19–22
During the clinical phase of the disease, transmissible spongiform encephalopathies (TSEs) are characterised by the accumulation, in the central nervous system, of significant amounts of the abnormal form of the prion protein (PrPrsc or PrPres). PrPres derives from the normal form (PrPsens or PrPc) through post-translational modifications, which induce a conformational change and confer on the PrPres a partial resistance to degradation by proteases as well as a marked insolubility in the presence of detergents. During the past few years, important progress has been made in the post-mortem diagnosis of TSEs (scrapie and BSE) due to the development of the so-called “rapid test” based on the immunological detection of PrPres [1,2]. All these tests take advantage of the biochemical properties of PrPres and the availability of antibodies, recognising the denaturated form of PrPres. These methods now allow routine and high through-
put testing, opening the door to large-scale epidemiological studies and systematic testing at slaughterhouses and thus preventing the entry of contaminated carcasses into the human food chain [3]. For instance, in 2001, close to 8.5 million tests were performed in Europe [4]. On risk populations, 771,000 tests were performed and led to the identification of 744 BSE cases, while testing at slaughterhouses identified 279 cases, which were removed from human consumption. It has been shown that some of these rapid tests allow pre-clinical diagnosis, anticipating by few months the appearance of clinical signs [5]. This was demonstrated by analysing samples obtained from the pathogenesis studies conducted in Great Britain between 1991 and 1995 [6–8]. The objective of this study was to determine the spatial and temporal development of infectivity and pathological changes in cattle following an oral exposure to a single dose
Table 1 Design of the pathogenesis study and samples tested with rapid tests (Bio-Rad). Forty Friesian/Holstein calves, born in 1991, were assembled from farms with no history of BSE. At 4 months of age, 30 were challenged orally with 100 g of pooled brain stems from 75 cases of BSE. Ten calves received no treatment and served as controls. Starting at 6 months of age, 2 months after challenge, and then at 4 month intervals until 22 months after inoculation, three of the challenged calves and one of the control calves were killed. Thereafter, challenged and control cattle were killed at discretionary intervals, until the termination of the experiment at 40 months after inoculation. Samples of the brain and spinal cord were taken for examination with a rapid test (Bio-Rad test originally developed at CEA, see [1,2]). Key to abbreviations: BS = brain stem, SCC = spinal cord cervical, SCT = spinal cord thoracic, and SCL = spinal cord lumbar Months post-inoculation
2
6
10
14
18
22
26 28 32 35 36
38
40 Total
Animal number 187 219 198 265 298 199 272 235 276 264 301 222 230 303 229 233 270 271 297 254 232 299 194 231 261 192 300 277 296 74
BS X X X
X X
Challenged animals SCC SCT X X X X X X X X X X X X X
X X X X X X
X
X X X X X
X X X X X X X X
X X X X X X X X X X X X X X X X X X
X X X X X
X
SCL
BS X
X
X
X
315
X
X
X
X
237
X
X
186
X
X
X
X
302
X
X
X
11
SCL
213
X
316
X
X
X
X X X X X X
X X X X X
214
X
X
X
X
217
X
X
X
X
X
X
X
9
8
8
228 X 27
Control animals SCC SCT
Animal number 227
12
24
32
7
J. Grassi / Transfusion Clinique et Biologique 10 (2003) 19–22
21
these species. This is illustrated in Table 2, where results obtained on peripheral lymphoid tissues taken from sheep (sensitive genotype VRQ/VRQ) naturally infected at birth are presented. In this case, detection as early as 21 d after birth was observed. It is very likely that pre-clinical detection is also possible for vCJD in humans, since both PrPres and infectivity were consistently found in peripheral lymphoid tissues (spleen, tonsils, appendix) [9,10]. This is confirmed by the fact that very recently, one vCJD case was identified during a retrospective study conducted in UK on the tonsil and appendix [11].
Fig. 1. Results of the rapid tests performed on animals 32 months p.i. or more and scored as positive. White bar: brain stem, black bar: cervical spinal cord, hatched bar: thoracic spinal cord, and cross-hatched bar: lumbar spinal chord.
of BSE-affected brain homogenate (see legend of Table 1). Samples from the central nervous system collected during this study were analysed using a rapid test (Bio-Rad, [5]) and it was shown that PrPres could be detected 32 months postinoculation in animals, at least 3 months before the onset of clinical symptoms (Fig. 1). Depending on the nature of the host and the TSE strain, PrPres can also be detected in peripheral lymphoid tissues in sheep and goats infected by scrapie (including gut Peyer’s patches, lymph nodes, spleen and tonsils) a long time before the onset of clinical symptoms. As a consequence, the same rapid test is suitable for pre-clinical diagnosis of scrapie in
The set of measures taken to control the BSE outbreak and limit the risks of transmission to humans can now be considered to afford effective protection. Apart from the problem posed by the possible presence of BSE in the sheep population, it is now important to avoid iatrogenic transmission within the human population (through medical procedures such as organ grafts, blood transfusion). The goal now is to develop a blood test allowing ante-mortem and pre-clinical diagnosis of vCJD. The stakes are high in terms of public health, but the scientific and technical challenge is daunting because the quantities of infectious agents (and hence of PrPres?) in the blood are certainly 100–1000 times lower than those present in the brain. The development of such a test will therefore require new tools for the extraction and concentration of PrPres as well as the development of much more sensitive detection methods. Recent work has opened up new approaches either by identifying ligands specific to PrPres [12] or by showing that it is possible to amplify this PrPres “in vitro” [13]. Lastly, the demonstration of the pres-
Table 2 Pre-clinical detection of PrPres in peripheral lymphoid tissues of naturally infected sheep. Animals of sensitive genotype (VRQ/VRQ), naturally infected in a flock with endemic scrapie, were killed at sequential time. The PrPres content of peripheral lymphoid tissues was analysed using either Immunohistochemistry (IHC) or the Bio-Rad test (ELISA). Three to six animals of the same age were analysed. The results are expressed in terms of the number of positive samples on the number of samples analysed. Data in bold correspond to those samples for which positive results were recorded. PP = Peyer’s patches, LN = lymph node, MLN = mediastinal lymph node. Data kindly provided by Olivier Andréoletti (INRA/ENVT Toulouse, France, unpublished results) Organ/age Thymus Hepatic LN Spleen Mediastinal LN Prescpular LN Palatin tonsils PP-duodenum PP-jejunum 25% MLN-jejunum 50% PP-jejunum 50% MLN-jejunum 50% PP-jejunum 75% MLN-jejunum 75% PP-ileum MLN-ileum PP-caecum Ileo-coecal LN
IHC 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3
10 d ELISA 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3
IHC 0/3 0/3 0/3 0/3 0/3 0/3 1/6 (d) 2/6 (d-e) 0/3 2/6 (d-e) 0/3 2/6 (d-e) 0/3 4/6 (a-d-e-f) 0/3 0/3 0/3
21 d ELISA 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 1/3 (d) 0/3 0/3 0/3
IHC 0/3 0/3 0/3 0/3 0/3 3/6 (a-d-e) 4/6 (a-d-e-f) 4/6 (a-d-e-f) 4/6 (a-d-e-f) 4/6 (a-d-e-f) 3/6 (a-d-e) 6/6 4/6 (a-d-e-f) 6/6 6/6 3/6 (d-e-f) 3/6 (d-e-f)
64 d ELISA 0/3 0/3 0/3 0/3 0/3 4/6 (a-d-e-f) 2/6 (a-d) 4/6 (a-d-e-f) 1/6 (d) 5/6 (a-b-d-e-f) 1/6 (d) 4/6 (b-c-d-f) 1/6 (d) 6/6 5/6 (a-c-d-e-f) 2/6 (e-f) 2/6 (d-e)
IHC 0/3 4/6 (a-b-d-e) 3/6 (a-b-d) 5/6 (a-b-c-d-e) 4/6 (a-d-c-d) 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6
104 d ELISA 0/3 3/6 (b-c-d) 5/6 (a-b-d-e-f) 5/6 (a-b-c-d-e) 3/6 (a-c-d) 6/6 5/6 (a-b-d-e-f) 6/6 5/6 (a-b-c-d-f) 5/6 (a-b-c-d-f) 6/6 6/6 6/6 6/6 6/6 4/6 (b-d-e-f) 5/6 (a-b-c-d-e)
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J. Grassi / Transfusion Clinique et Biologique 10 (2003) 19–22
ence of PrPres in the urine of animals or patients with a TSSE raises the prospect of an ante-mortem test usable on a large scale in humans [14]. In view of the number of research teams now focusing on this problem, it is not unreasonable to hope for spectacular progress in the years ahead.
[7]
Wells GAH, Dawson M, Hawkins SAC, Austin AR, Green RB, Dexter I, et al. Preliminary observations on the pathogenesis of experimental bovine spongiform encephalopathy. In: Gibbs Jr CJ, editor. Bovine spongiform encephalopathy: the BSE dilemma. New York: Springer-Verlag; 1996. p. 28–44.
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Wells GAH, Hawkins SAC, Austin AR, Green RB, Dexter I, Spencer MJ, et al. Preliminary observations on the pathogenesis of experimental bovine spongiform encephalopathy (BSE): an update. Veterinary Record 1998;142:103–6.
[9]
Wadsworth JDF, Joiner S, Hill AF, Campbell TA, Luthert PJ, Collinge J. Tissue distribution of protease resistant prion protein in variant Creutzfeldt-Jakob disease using a highly sensitive immunoblotting. Lancet 2001;358:171–80.
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