NR ADZM
AU Field,E.J.; Shenton,B.K.
TI Rapid diagnosis of scrapie in the mouse
QU Nature 1972 Nov 10; 240(5376): 104-6
PT journal article
VT
Rapid Diagnosis of Scrapie in the Mouse
RESEARCH into scrapie, an enigma of veterinary medicine which may embody principles of considerable interest for human disase, commonly involves demonstration of agent activity or its titration. A great step forward was made by Chandler[1] when he showed the disease could be produoed in mice, and biological titration in these animals is currendy widely employed. This takes 6-8 months. From a recent study of multiple sclerosis, with which scrapie has been linked[2], we were led into an immunological study of scrapie itself. We found that brain or spleen from a scrapie mouse when injected into a guinea-pig (adult Hartley) led to the appearance of blood lymphocytes which were more highly sensitized to scrapie brain or spleen than to normal brain or spleen.
In early experiments, guinea-pigs were inoculated intracutaneously with 0.1 ml of 10^-1 suspension of scrapie brain or scrapie spleen in sterile saline. Control animals were similarly injected with brain or spleen from normal mice which had been injected some weeks previously with normal brain suspension. At intervals after 5 days, 2-3 ml of blood was removed by cardiac puncture and lymphocyte sensitization to the scrapie and normal suspensions estimated by the sensitive and highly specific macrophage electrophoretic migration (MEM) method[3]. In principle the method depends on the release by sensitized lymphocyte of a protein which slows migration of normal guinea-pig macrophages in an electric field (macrophage slowing factor (MSF)-which may be identical with macrophage inhibitory factor (MIF)). Our measurements have been carried out in a Zeiss cytopherometer and full experimental details have been given [4].
Guinea-pig blood lymphocytes were prepared by the method of Coulson and Chalmers[5], as modified by Hughes and Caspary[6], and normal guinea-pig macrophages by washing out the peritoneal cavity with heparinized Hanks solution 6-8 days after injection of sterile liquid paraffin. In order to obviate a two-way mixed lymphocyte reaction (at least for the duration of the test) the normal exudate was subjected to 100 rad gamma-irradiation[3].
10^-1 scrapie brain, scrapie spleen, normal brain and normal spleen suspensions were used as test antigen and for later testing lymphocyte sensitization. 0.5 x 10^6 guinea-pig blood lymphocytes were incubated for 90 min at 20°C with 0.1 ml of a 10^-1 suspension of scrapie mouse brain or spleen (cleared by spinning at 1,800g for 10 min) in the presence of 10^7 irradiated normal macrophages. Control tubes comprised lymphocytes and macrophages without antigen. The migration time of macrophages in each specimen was measured by timing ten cells (readily identified by their size and paraffin droplet content) in each direction of the potential difference so that a mean (with SD) from twenty readings could be calculated. A full protocol from one specimen is given by Caspary and Field[4]. All measurements were made "blind" and results unscrambled later. If tc = migration time of control macrophage in the absence of antigen and te = migration time when antigen is present; then in general te > tc and te- tc/tc x 100 is a measure of lymphocyte sensitization to the antigen.
The scrapie brain and spleen with which the guinea-pigs were inoculated were titred out by inoculation into groups of 6 mice at dilutions of 10^-1 through 10^-7. The animals were observed for eight months and all clinical scrapie diagnoses were checked histologically.
Guinea-pigs were immunized with either scrapie or normal mouse brain and spleen, 0.1 ml of a 10^-1 suspension being inoculated intracutaneously in the dorsum of the right foot (Table la, b). The guinea-pig lymphocyte sensitization to normal brain and normal spleen is always greater when the animal has been injected with scrapie material than with normal (as pointed out by Gardiner[7] with respect to circulating antibody in rabbit experiments). Moreover, the scrapie-normal difference (when scrapie brain or spleen is used as test antigen for the lymphocytes) is greater in the guinea-pig immunized with scrapie brain than with normal brain. The difference appears to be greatest between five and thirteen days. Table lb shows that the same is true when guinea-pigs immunized with scrapie or normal spleen are tested for cellular sensitization to scrapie and normal brain and spleen. The results with spleen are particularly interesting since this organ whilst rich in agent (LD50 = l0^-4.7) shows no morphological change[8] so that the test antigen being used might well be the scrapie agent itself (or scrapie-altered but morphologically normal membrane).
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Table 1 Demonstration of Scrapie Activity in Mouse Brain and Spleen
a, Mouse scrapie brain injected guinea-pig l0^-1
5 days 13 days 22 days 34 days
EF 12.0 15.3 11.1 4.7
Normal brain 11.3 12.5 10.3 3.4
Scrapie brain 16.5 17.9 14.3 5.4
Brain difference 5.2 5.4 4.0 2.0
Normal spleen 5.2 6.1 6.1 1.7
Scrapie spleen 9.6 11.1 10.4 3.5
Spleen difference 4.4 5.0 4.3 1.8
Mouse normal brain injected guinea-pig
5 days 13 days 22 days 34 days
EF 12.0 13.5 9.6 3.0
Normal brain 9.9 13.7 9.4 2.5
Scrapie brain 12.1 15.5 10.3 3.2
Brain difference 2.2 1.8 0.9 1.3
Normal spleen 4.0 5.9 6.2 1.2
Scrapie spleen 6.2 7.7 7.6 2.2
Spleen difference 2.2 1.8 1.4 1.0
b, Mouse scrapie spleen injected guinea-pig 10^-1
7 days 11 days 25 days 35 days
EF 6.6 9.2 11.3 2.2
Normal brain 3.4 8.9 7.6 0.8
Scrapie brain 10.1 14.2 12.3 2.6
Brain difference 6.7 5.3 4.7 1.8
Normal spleen 11.5 12.2 10.8 1.8
Scrapie spleen 17.4 17.9 15.3 3.9
Spleen difference 5.9 5.7 4.7 2.1
Mouse normal spleen injected guinea-pig
7 days 11 days 25 days 35 days
EF 6.2 8.7 8.2 2.4
Normal brain 4.0 6.2 6.9 1.5
Scrapie brain 5.7 8.2 8.4 2.4
Brain difference 1.7 2.0 1.5 0.9
Normal spleen 13.9 13.9 11.4 2.7
Scrapie spleen 14.6 15.7 13.3 3.5
Spleen difference 0.7 1.8 1.9 0.8
Adult Hartley guinea-pigs inoculated intracutaneously in the dorsum of the right foot with 0.1 ml. 10^-1 scrapie brain or spleen (titre l0^-6; 10^-4.7 respectively) and lymphocyte sensitization to scrapie and normal brain measured at intervals by the macrophage electrophoresis method of Field and Caspary[3]. Results expressed as percentage slowing (loc. cit.).
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In the case of inoculation with scrapie brain, however, the difference may perhaps be attributed to morphological changes (especially astrocyte increase) in the inoculated material. Having established the quantitative difference in response to scrapie as opposed to normal tissue we used this method to titre out scrapie activity.
Guinea-pigs were injected with scrapie brain at dilutions of l0^-1 to 10^-6 and the lymphocytes examined for sensitization to scrapie brain or spleen and normal brain or spleen at six or seven days and at seventeen or eighteen days. For comparison a similar study was made in guinea-pigs injected with normal brain (Tables 2 and 3). In the scrapie brain sensitized guinea-pig the difference in lymphocyte sensitization to scrapie as compared with normal brain is 2.5% (P<0.01) even at l0^-6 original inoculum level when the guinea-pig is tested at six days but falls to 2.0% (P= 0.1 -0.05) by sixteen days. When sensitization to scrapie spleen and normal spleen in these animals is compared, the difference is significant only in guinea-pigs which have received 10-4 scrapie brain.
Animals injected with normal brain showed no significant difference when their lymphocytes were tested with scrapie as opposed to normal brain or spleen (though in general the values with the former were higher).
Guinea-pigs were sensitized by injecting scrapie spleen at 10^-1 to l0^-6 and their lymphocytes tested for sensitization to scrapie brain and spleen (Table 3). A significant difference between sensitization to scrapie and normal spleen still exists at 10^-5 (P< 0.01) with spleen as antigen but barely with brain (2.1 % difference; P= 0.05), showing apparent scrapie antigenicity in the 10^-5 dilution.
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Table 2 Titration of Scrapie Activity from Mouse Brain
Scrapie brain
Lymphocytes tested at 6 days
Antigen 10^-1 10^-2 l0^-3 10^-4 10^-6
Normal mouse brain 11.3 11.0 8.1 5.7 3.7
Scrapie brain 16.5 14.1 11.7 9.6 5.2
Brain difference 5.2 3.1 3.6 3.9 2.5
Normal mouse spleen 5.2 6.1 4.7 3.9 1.7
Scrapie mouse spleen 9.6 10.8 8.1 7.4 3.2
Spleen difference 4.4 4.7 3.4 3.5 1.5
% macrophage slowing
Lymphocytes tested at 16 days
Antigen l0^-2 10^-4 10^-6
Normal mouse brain 11.1 5.9 3.9
Scrapie brain 14.7 9.9 5.9
Brain difference 3.6 4.0 2.0
Normal mouse spleen 5.9 4.7 2.9
Scrapie mouse spleen 10.1 8.1 4.4
Spleen difference 4.2 3.4 1.5
% macrophage slowing
Normal brain
Lymphocytes tested at 6 days
Antigen 10^-1 10^-2 l0^-3 10^-4 10^-6
Normal mouse brain 8.9 7.1 5.9
Scrapie mouse brain 10.3 7.9 6.7
Brain difference 1.4 0.6 0.8
Normal mouse spleen 5.4 2.5 3.0
Scrapie mouse spleen 6.2 3.8 4.2
Spleen difference 0.8 1.3 1.2
% macrophage slowing
Lymphocytes tested at 16 days
Antigen l0^-2 10^-4 10^-6
Normal mouse brain 9.6 5.5 4.4
Scrapie brain 10.1 6.5 5.2
Brain difference 0.5 1.0 0.8
Normal mouse spleen 6.1 4.4 3.5
Scrapie mouse spleen 7.2 5.7 4.5
Spleen difference 1.1 1.3 1.0
% macrophage slowing
Guinea-pigs inoculated with scrapie mouse brain with a titre of 10^-5.6. 0.1 ml inoculated at different dilutions.
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Table 3 Titration of Scrapie Activity from Mouse Spleen
Scrapie spleen
Lymphocytes tested at 7 days
Antigen l0^-1 10^-2 10^-3 10^-4 10^-5 10^-6
Normal mouse brain 3.4 4.5 2.7 1.0 1.7 0
Scrapie brain 10.1 10.3 7.2 4.4 3.8 1.2
Brain difference 6.7 5.8 4.5 3.4 2.1 1.2
Normal mouse spleen 11.5 12.6 10.4 10.6 8.1 4.5
Scrapie mouse spleen 17.4 17.0 15.3 14.4 10.8 6.3
Spleen difference 5.9 4.4 4.9 3.8 2.7 1.8
% macrophage slowing
Lymphocytes tested at 17 days
Antigen 10^-2 10^-4 l0^-6
Normal mouse brain 4.7 1.5 0.7
Scrapie brain 10.1 4.9 1.5
Brain difference 5.4 3.4 0.8
Normal mouse spleen 11.1 10.0 4.4
Scrapie mouse spleen 15.0 13.8 6.2
Spleen difference 3.9 3.8 1.8
% macrophage slowing
Normal spleen
Lymphocytes tested at 7 days
Antigen l0^-1 10^-2 10^-3 10^-4 10^-5 10^-6
Normal mouse brain 4.5 1.5 1.0
Scrapie mouse brain 5.9 2.9 1.2
Brain difference 1.4 1.4 0.2
Normal mouse spleen 11.1 9.1 4.7
Scrapie mouse spleen 12.4 10.2 6.4
Spleen difference 1.3 1.1 1.7
% macrophage slowing
Lymphocytes tested at 17 days
Antigen 10^-2 10^-4 l0^-6
Normal mouse brain 6.4 3.0 1.8
Scrapie mouse brain 7.6 4.4 2.9
Brain difference 1.2 1.4 1.1
Normal mouse spleen 10.5 7.9 4.7
Scrapie mouse spleen 11.5 8.9 5.6
Spleen difference 0.9 1.0 0.9
% macrophage slowing
Guinea-pigs inoculated with spleen suspension with a titre of 10^6.
0.1 ml inoculated at different dilutions.
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The titre of the scrapie brain inoculated into the test guinea-pigs was calculated to be LD50= 10^-5.6 We noted that 1/6 mice developed scrapie at 10^-6 and 1/6 at 10^-7 and the immunological test suggests that some activity is still present at 10^-6. The titre of spleen used for immunizing the guinea-pigs was LD 50=10^-5. The results of in vivo titration therefore agree with the in vitro immunological assay, and the significance of these results is two-fold. (1) Attempts to show the existence of circulating antibody and/or specific antigen in scrapie disease have not been successful[7,8]. Gardiner did show by inoculation of rabbits with scrapie and non-scrapie spleen material that the latter had increased antigenicity in vivo. The present work extends these very important findings in that it suggests that this is true also of lymphocyte sensitization and that the reactivity is greater against scrapie material (both brain and spleen) than against normal material. This suggests that some specific antigen-perhaps the agent itself, or specifically agent-altered material since the spleen is so potent as a testing tissue-may be active. The macrophage electrophoretic migration (MEM) method is exquisitely responsive to minor changes in antigenic determinant structure and appears to be able to distinguish between normal and scrapie tissue. It is, of course, not possible to decide whether the difference resides in the presence of a specific scrapie agent or whether we are dealing with a structural (antigenic) change induced by tbe agent. However, if scrapie infected brain is split into a neuronal and glial compartment by the method of Giorgi (unpublished) then, contrary to expectations based on the precocious glial hypertrophy, higher scrapie titre is associated with the neuronal rather than the glial fraction and it is in the former that particles with the size and structural characters postulated for scrapie agent have been found[9]. (2) Lymphocyte sensitization test enables the presence of scrapie to be established and titration carried out within 8 days, an important saving in time during the study of this fascinating condition. Further experiments are in progress involving the use of Freund's adjuvant to boost the responses. A full account of these experiments will be published elsewhere.
E. J. FIELD
B. K. SHENTON
Medical Research Council, Demyelinating Diseases Unit, Newcastle General Hospital, Westgate Road, Newcastle upon Tyne NE4 6BE
Received July 25, 1972.
1 Chandler, R. L., Lancet, ii, 1378 (1961).
2 Field, E. J., Int. Rev. Exp. Path., 8, 129 (1969).
3 Field, E. J., and Caspary, E. A., Lancet, ii, 1337 (1970).
4 Caspary, E. A., and Field, E. J., Brit. Med. J., 2, 613 (1971).
5 Coulson, A. S., and Chalmers, D. G., Immunology, 12, 417 (1967).
6 Hughes, D., and Caspary, E. A., Int. Arch. Allergy, 37, 506(1970).
7 Gardiner, A. C., Res. Vet. Sci, 7, 190 (1966).
8 Chandler, R. L., Vet. Rec., 71, 58 (1959).
9 Narang, H. K., Shenton, B., Giorgi, P. P., and Field, E. J., Nature, 240, 106 (1972).
IN Field und Shenton vom Newcastle General Hospital injizierten verdünntes Hirn- oder Milzgewebe von gesunden oder scrapieinfizierten Mäusen in die Haut von Meerschweinchen. Wenige Tage danach wurde den Meerschweinchen Blut entnommen und einem sogenannten Makrophagenmigrationstest unterzogen. Nach Zugabe des zur Sensibilisierung verwendeten Antigens produzieren sensibilisierte Lymphozyten einen Botenstoff, der die Fortbewegung von Makrophagen in einem elektrischen Feld reduziert. Es zeigte sich, dass die Injektion von Hirn- und Milzgewebe scrapieinfizierter Mäuse auch noch bei Verdünnungen von mehreren Größenordungen in Meerschweinchen Lymphozyten sensibilisiert. Der Makrophagenmigrationstest konnte dies binnen rund einer Woche nachweisen.
MH Animal; Brain/immunology; *Cell Migration Inhibition; Guinea Pigs/immunology; Immunization; Lymphocyte Activation; Macrophages/*immunology; Mice; Scrapie/*diagnosis/immunology; Sheep; Spleen/immunology
SP englisch
PO England
OR Prion-Krankheiten 3
ZF kritische Zusammenfassung von Roland Heynkes