Atypical Case of Bovine Spongiform Encephalopathy in = an East-Flemish Cow in Belgium

Atypical = Case of=20 Bovine Spongiform Encephalopathy in an East-Flemish Cow in=20 Belgium

H. De Bosschere, DVM, PhD

S. Roels, DVM, PhD

E. Vanopdenbosch, DVM, Lic

Veterinary and Agrochemical Research Centre=20 (CODA/CERVA)

National Reference Laboratorium for Veterinary = TSEs

Groeselenberg 99, B-1180

Ukkel (Brussels), Belgium

KEY = WORDS: Bovine=20 spongiform encephalopathy, BSE, Western blot, atypical = BSE.

ABSTRACT

For many years, researchers believed that only = one bovine=20 spongiform encephalopathy (BSE) strain existed, in contrast to the = many=20 different scrapie strains found. However, only very recently = reports=20 emerged about unconventional BSE strains seen in Italy, France, = and Japan.=20 The present case describes an atypical strain of BSE in Belgium in = a=20 64-month-old East-Flemish cow with an electrophoretic profile and = other=20 features similar to those described in Japan.

INTRODUCTION

Transmissible spongiform encephalopathies = (TSEs), or prion=20 diseases, are a group of fatal neurodegenerative diseases = including sheep=20 and goat scrapie, bovine spongiform encephalopathy (BSE), and=20 Creutzfeldt-Jakob disease (CJD) in humans. They are characterized = by the=20 accumulation of an abnormal protein, called PrPsc, which is formed = post-translationally from the normal isoform (PrPc).1,2 At = present, the=20 agent causing TSEs is still incompletely characterized, although = PrPsc is=20 believed to be its major if not unique constituent.3

Research in mice showed the existence of = different scrapie=20 strains.4,5 Scrapie strain discrimination is currently based on = biologic=20 typing in a panel of inbred mice, using incubation time and brain=20 pathology scoring as criteria.6 However, no large-scale studies of = the=20 molecular features of PrPsc have been reported for bovine BSE to = date.=20 Till now, the BSE strain seemed to maintain constant biologic and=20 molecular properties even after experimental or accidental = passages into=20 different species, such as mice, humans, primates, and = sheep.7=9610 However,=20 very recently, variant forms of BSE have been reported in Japan, = Italy,=20 and France.11-13 These forms were characterized by atypical=20 histopathologic, immunohistochemical, or biochemical phenotypes. = The=20 present case is the description of the first atypical BSE case in=20 Belgium.

MATERIALS AND=20 METHODS

Since January 2001, all cattle older than 30 = months are=20 tested for TSE via a rapid test (TeSeE-kit, Bio-Rad, Nazareth, = Belgium)=20 after EC regulation 999/2001.14,15 Samples positive according to = the=20 enzyme-linked immunosorbent assay (ELISA) screening are further = subjected=20 to scrapie-associated fibrils (SAF), histopathology, = immunohistochemistry,=20 and Western blot (WB) testing16,17 at the National Reference = Laboratory=20 (NRL).

RESULTS

A positive ELISA sample from a 64-month-old = East-Flemish=20 cow or Belgian white and red (Figure 1) was presented at the NRL = for=20 confirmation. The animal was reported healthy before slaughter. = The=20 optical density (OD) titers at the local laboratory were 2.324 and = 2.116.16 The OD titers at the NRL were 0.953 and 0.708 (sample = taken at=20 the contralateral side of the first sampling side of the obex = region). The=20 histopathology of the obex, pons, and midbrain showed no = spongiform=20 changes; immunohistochemistry of the brainstem revealed no signal = of PrPsc=20 accumulation typical for BSE; and SAF was negative. However, WB = analysis=20 (Bovine WB, Bio-Rad, France; antibodies 12F10 and SAF60) of the = same=20 homogenate that was prepared from the obex region for ELISA = revealed a=20 small amount of PrPsc with an electrophoretic profile different = from that=20 of typical BSE-associated PrPsc.18,19 The band on the gel of the=20 non-glycosylated form of PrPsc of the present case clearly showed = a lower=20 migration pattern compared with that of a typical BSE case (Figure = 2).

DISCUSSION

For many years, researchers assumed that only = one BSE=20 strain existed.7=9610 Only in the past months, reports of atypical = BSE cases=20 were announced.11=9613 The Japanese case11 describes a very young = bull (23=20 months) characterized by the absence of spongiform changes and = PrPsc=20 deposits immunohistochemically. The WB analysis revealed an=20 electrophoretic profile different from that of typical BSE, = characterized=20 by low content of the di-glycosylated molecular form of PrPsc and = a faster=20 migration of the nonglycosylated form of PrPsc. In Italy,12 two = BSE=20 affected cattle with a previously unrecognized neuropathologic = profile and=20 PrPsc type were seen. These cases were determined using a = different=20 staining pattern on immunohistochemistry, a difference in size and = glycoform ratio of PrPsc on immunoblot and a difference in = regional=20 distribution of lesions. The two cases in France13 showed variant=20 molecular features with a different PrPsc electrophoretic profile = from=20 other BSE cases, mainly characterized by a higher molecular mass = of the=20 nonglycosylated PrPsc. The present case shows the most = similarities (ie,=20 identical electrophoretic profile, only ELISA and WB positive and=20 histopathology and immunohistochemistry negative) with the = Japanese=20 case,11 although the cow in the Japanese case was only 23 months = old, and=20 the cow in this case was 64 months old.

The fact = that these=20 strains were detected worldwide and in several breeds suggest that = there=20 is no local or breed-dependent feature involved. It could be that = the WB=20 techniques have become more specific within the past year in the = detection=20 of minor differences in di-, mono-, and nonglycosylated molecular = forms of=20 PrPsc. Infection of cattle by scrapie could also be considered = since=20 scrapie can be transmitted by direct contact between animals or = through=20 environmental contamination.13

In = conclusion,=20 this Belgian case should be added to the list of atypical BSE = strains only=20 very recently detected worldwide and may contribute to further = research=20 studies about epidemiologic significance. Current continued = research on=20 BSE would appear to reveal different BSE strains in analogy with = the=20 different scrapie strains.

ACKNOWLEDGMENTS

The authors wish to thank Rita Geeroms, Patrick = Van=20 Muylem, Stephanie Durand, Rapha=EBl Foubert and Amina Chama for = their=20 technical assistance. Mario Vanpoucke is acknowledged for = providing=20 references.

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Figure 1. Photograph of the = East-Flemish cattle=20 breed or the Belgian white and red.

Figure 2. Bovine Western=20 blot (Bio-Rad, France) using antibodies 12F10 and SAF60. MM, Magic = mark;=20 Atyp. BSE, Atypical BSE case (present case); Ref1, Reference 1 of = a=20 classical BSE case; Ref2, Reference 2 of a classical BSE case. The = third=20 band of the non-glycosylated PrPsc of the Atyp. BSE case (left = rectangle)=20 shows a markedly faster migration compared to the Ref1 and Ref2 = cases=20 (right rectangle).