Jpn. J. Infect. Dis., 52, 242‑244, 1999

Short Communication

Exacerbated Spong血‑ Lesions in the Cerebral Cortex in Japanese Sheep,in an Outbreak of Scrapie during 1984‑1987
ShigeruMizoi, Tomoo Yoshinol , Eiichi Momotani2, Atsutaka Kubosaki3,
Yuko Nakamura3and Takashi Onodera3*
Hokkaido Nemwo Livestock Hygene Service Center,
Betukai‑cho, Hokkaido 086‑0214,
1Rakuno Gakuen University, Ebetsu, Hokkaido 069‑8501 ,2National Institute ofAnin∽l Health, 7Tsukuba, Ibaraki 305‑0856 and3FacultyofAgricullure, Universityof Tokyo,
Yayoi, Bunkyo‑ku, To砂0 113‑0032, Japan(Received November I , 1999. Accepted December 3, 1999)

StJMMARY: The present study dealt withthe pathology of natural scraple in Japanese Suffolk sheep ln a Certain
selected area･ Vacuolations in the cytoplasm of neurons were conspICuOuS･ They were particularly evident inmany areas of the medulla and pons, extending Into andthroughpedunculus cerebriandthalamus tothe septal
areaand olfactory tubercle. Proliferation of astrocytes wasalso easily observed with glial fibrillary acidic protein

staining･ Neural vacuolations in the cerebralcortex were observed in 73% of the cases･ Abnormal pnon protein

deposits were seen inall cases observed by hydrolytic autoclaving,and subsequentperoxidaseandanti‑peroxidase
immunostaining･ Abnomal pnon protein staining was the most consplCuOuS in the polymo叩hic layers of thehipp∝ampus.

Worldwide attention has been focused on scrapie because

sacrificed. A flock of about 100 sheep was confined to a 20‑of experimental results revealing the oral transmission of

hectar field where lambing has been conducted for the pastbovine spongifbm encephalopathy (BSE) to sheep (1) and

20 years. Scrapie‑affected cases were removed from theplausible transmission to beef‑consuming humans (2). If

restricted field andpenned separately when symptoms txcame

contaminated meatand bone mealS托d to sheep affect humanS,obvious. The sheep were sacrificed at an advanced stage ofthen it is possible that an ovine fbm of BSE, clinically
identical to scraple, may exist. The Japanese Ministry ofAgriculture, Forestryand Fisheries has declared scraple athe disease,and scraple Was then confirmed by pathologlCal

notifiable disease on April 26, 1996, and decided to organize

The histopathologiCaltissues studied included the splnal cord,

examination of the brain and spinal sections (3). Sheep thatwere killed or died of other causes were similarly examined.

medulla oblongatae (at least two), cerebellum, pons, mesen‑a national suⅣeillance scheme against this disease.

It has long been establishedthat there is much variation in

cephalon, diencephalon, septalarea, basalganglia,and frontal

the pathology of natural scrapie. The pattem and distribution

cortex.

of lesions vary with the breed of sheep; the nature of lesions

Table l shows that the dominant sign in the fully developed

also differs within breeds (3). The present study considers

disease was pruritus. Ataxia was a common finding, which

the pathology of natural scraple in Japanese Su恥lk sheep lna certain selected area. This paper describes the results of apresented as gall incoordination, hypermetria, trotting, orbunny hopplng. Some sheep were hypersensitive to sound,study designed to detemine whether any temporal changes

movement, Or touching (stimulation). Not all sheep demon‑in the pathology of naturally occurrlng SCraple in sheep

strated all signs, which are progressive and nomally last

occurred concurrently with the epidemic of BSE in Englandseveral weeks or months.

since 1986.

In all cases, there was moderate to severe neuropil vacuola‑

Suffolk or Corriedale sheep were bred in a restricted areation in many areas of the medulla and pons, extending into

in Hokkaido, Japan. Sheep were fed with grasses and silage

and through pedunculus cerebriand thalamustothe septal

without the addition of processed food (4). In England, thearea and olfactory tubercle.Lesions were visible in theBSE epidemic originated by the use of processed food (4).

Although in Japan, Such tainted feed is not given to sheep,

neocortex of 73% of the cases (Table 2), centered aroundthe
superior frontal gyrus. Vacuolation of the neuronal perikarya,

yearly surveillance is necessary to exclude accidentaluse ofand vacuolation of the surrounding neuropil were observed

contaminated products. For this study, we used 15 sires thatas occumng with variable seventy. On the other hand, thefrequency of lesions in the neocortex was relatively rare (37%)

displayed clinical signs of the disease. The average age ofsheep at the onset of scraple Symptoms is 3.5 years. Mostin the case of British sheep (5). The severity of histologiCal

animals between the ages of 2.5 and 4.5 years were affected.

lesions in the brain exacerbated progressively, resulting ln a

Clinical aspects including age, duration of disease, and symp‑

shorter lifespan of the animals. A major difference between

toms from 15 cases were recorded befわre the animals werethis study and most previous reports was the finding of*Corresponding author: Tel: +8 I‑3‑5841 ‑5 196, Fax :+81 13‑5841 ‑of histopathologlCal lesions in brain cortex exacerbatedvacuolarchange inthe cerebralcortex (1bble 2). The severity
8020, E‑mail: aonoder@mail.eec.u‑tokyo.acJP

progressively in succeeding generations within 4 years･ White242

Table l･ Reladonship between year of incidence, age, and clinicalsign
C‑eS S S S S S S S 1234/b一781 1 1 1I

Ye‑111111111111111

糾 糾 糾 糾86 78 7折節折節q′ 9 q/ 9 9 0′ 9 9 0′ 0′ 9Breeds S CS S S S S(:eg;)

2Sex

4F F F に▲ F F F F FPl)I++十十+++++++++++

D

SlIIlIIl + IIIIJlJ

一一一一一一 l + + + 一一 t 一lIII + + + ll 1 + + lI

4 56 323NR服NRNRN

0 1 2 3 4 5 AV 7i)P: pruritus, I: incoordination, D: depression, S:Stimulation･ 2)S:Suffolk,
C: Corriedale. 3)NR: not recorded. 4)F: female.

Table 2･ Histopathology of the frontal cortex (superior frontalgyruS)S S S S S S S S 1 2 3 4 ′0 7 0 1 1 1 I1Intra‑cytopl as mi cSpongifom orIncreas￠ inWhite Matter Gray Matteras trocytes

v acuol ation
centralperiph･+ + + +一一一F I 什 l + + l lFig. l･ a) Immunodetection of abnormalprion protein in the hippo‑

Campalarea observed by in situ immunohistochemistry using anti‑
SAF (scrapie‑associated fibri1) rabbit amibody (6)･ AbnormalpriOnprotein was seen as dark spots in the stratum multifome･ Bar shows

100 Elm. b) Immunohistochemistry directed against GFAP (glial二･･十小二…叫.}fibrillary acidic protein, a specific marker of astr∝yte cytoplasm)

usinganti‑humanGFAP rabbit antibody (8), inthe hippocampalarea･
A: stratum multiforme, B: stratum reticulare, C:alveus. Bar shows
200〝m.

0 1 2 3 4 5 ′b 7

Tris‑buffered saline (TBS, pH 7.6, I.6% sodium chloride),and then incubated with a mixture of lO% (V/V) normal goat

serum and 3% (W/V) bovine serup albumin in TBS for 15min. After drainlng, Sections were Incubated ovemight at 40Cwith a rabbit antisemm to the prion protein (7) at a dilutionof l :200. This antiserum has been characterized by Western+++

Limited, often confined to a slngle brain nucleus･

blottlng and has been shown to identify ovine pnon protein

Moderate, present in more than one nucleus.

十十十Severe, spongifom in many nuclei and neuropils.

(7). After washing with TBS, all sections were incubated atperiph∴ peripheral

room temperature with goat anti‑rabbit immunoglobulins(Dako Japan, Kyoto, 1 :50), and then with rabbit Iap complex(1:320), each for 30 min, witha TBS wash afterthe incuba‑tions. Diaminobenzidine tetrahydrochloride (DAB) was usedmatter vacuolation is not normally associated with naturalscrapie, as is addressed in a previous report (3). However,
the white matter vacuolation found in the present study,

to visualize immunoreactivity, and the sections were lightly

counterstained with Mayers haematoxylin.

especially the vacuolation found in polymorphic layers (stra‑

Glial fibrillary acidic protein (GFAP) staining revealed

tum multiforme) of the hippocampus, was associated withactive astrocyte proliferation in the hippocampus(Fig･ 1 b)(8)･

abnormalprion protein by immunoperoxidase staiming (Fig. 1 a)

In the same area, positive stainlng Of abnomal prlOn prOteins(6).
For immunostaining of abnormal pnon protein, peroxidase‑

was observed, even in the absence of vacuolated neuronal cells.

anti‑peroxidase (PAP) method was used. Sections werelesions in the cerebral cortex (Table 2). Wood et al. (5)deparaffinized and dehydrated, washed in runnlng tap Water

recently completed a report on the neuropathology of naturaland rinsed with distilled water before pretreatment withsheep. Scrapie inthe UnitedKingdom (submitted to the Central
Vetennary Laboratory, Weybridge, U･K･, for more than aIn Japanese natural cases, a majority showed spongiform

hydrolytlC autOClavlng. For the hydrolytic autoclavlng

HCl and autoclaved at 121oC. Endogenous peroxidase wasdecade)･ According to their study, in son.e cases, lesions in
the cerebral cortex afforded certain discrlminatlng Patterns

blocked by immerslng Sections in freshly prepared 3%of the disease between different breeds. Japanese scraple Cases

hydrogen peroxide (100 volumes) in methanol for 10 min.

in 1984 matched well with Wbod's type III, and cases in 1987
corresponded well to Wood's type IV･ In cases 6‑8, nopretreatment, sections were completely immersed in 2.5 mM

Sections were washed in mnnlng tap Water, rinsed with243

925.

spongiform lesions were observed in the frontal cortex.

However, GFAP staining showed an increased number of

3. Zlotnik, Ⅰ. and Rennie, ∫. G. (1962): The pathology ofastrocytes in the same area.

the brain of mice inoculated with tissues from scraple

The lesions in cattle with BSE differ from those of Japanesesheep. ∫. Comp. Patho1., 72, 360‑382.

scraple in that the fbmer advance more rostrally and are

4. Wilesmith, J. W., Wells, G. A. H., Cranwell, M. P. andconcom血nt with increaslng Severity of vacuolar change at

Ryan, ∫. (1988): Bovine spongiform encephalopathy:

the level of the obex (3). By Westem blotting, our cases (frozenepidemiologlCal studies. Vet. Rec., 123, 638‑644.

5. Wtxxl, J. L N., McGill, I. S., Done, S. H.and Bradley, R.

brain samples) were shown to co汀eSpOnd well to Collimge'sglycosylation type IV: new variant Creutzfeldt‑Jakob disease( 1997): Neuropathology of scrapie: a study of the distribu‑(CJD), and to the BSEtype (7, 9). We still need to clarify thetion pattems of brain lesions in 222 cases of naturalscraple

in sheep, 1982‑1991. Vet. Rec., 140, 167‑174･relationship between the Japanese scraple agent and theBritish BSE agent; this can be accomplished by companng

6. Haritani, M., Spencer, Y. I. and Wells, G. A. H. (1994):

pathologicallesionsin the brain uslng Several different strains

Hydrated autoclave pretreatment enhancement of priOnof mice, as has been studied previously (10).

protein immunoreactivity in formalin ‑fixed bovine
spongiform encephalopathy‑affected brain･ Acta

Neuropatho1., 87. 86‑90.

AC KNOWL E GMENTS

7. Onodera, T., Ikeda, T., Muramatsu, Y. and Shinagawa,M. (1993): Isolation of scrapie agents from the placentaof sheep affected with naturalscraple in Japan. Microbiol.

This work was supported by a grant‑in‑aid for Science

Research from the Ministry of Education, Science, Sports

Immuno1., 37, 31ト316.

and Culture of Japan and by Special Coordination Funds fわr

8. Dahl, D., Grossi, M. and Bignami, A. (1974): Maskingof epitopes in tissue sections. A study of glial fibrillarypromoting Science and Technology from the Science andTechnology Agency in Japan.
The authors are grateful to Dr. Yasuyuki Nakashima foracidic (GfA) protein with antisera and monoclonal anti‑bodies. Histochemistry, 8 I , 525‑53 I.

his invaluable assistance in the pathologICal studies.

9. Collinge, J., Sidle, K. C. L., Meads, J･, Ironside, J･andHill, A. 氏 (1996): Molecular analysis of strain variationREFERENCES

and aetiology of "new variant''CJD. Nature, 383, 685‑
690.

l･ Foster, ∫. D., Hope, ∫. and Fraser, H. (1993): Transmis‑

10. Bruce, M. E., Wlll, R. G., Ironside, J. W., McConnell, I.,sion of bovine spongiform encephalopathy to sheep andDrummond, D., Suttie,A., McCardle, L., Chree,A., Hope,goats. Vet. Rec., 133, 339‑341.

2. Wlll, R. G., Ironside, J. W., Zeider, M., Cousens, S. N.,

J., Birkett, C., Cousens, S., Fraser, H. and Bostock, C. ∫.

Estibeiro, K., Alperovitch, A., Poser, S., Pocchiari, M.,( 1 997): Transmissions to mice indicate that 'new variant'Hofman, A. and Smith, P. G. (1996): A new variant ofCJD is caused by BSE agent. Nature, 389, 498‑501･Creutzfeldt‑Jakob disease in the U.K. Lancet, 347, 921‑244