Classical Bovine Spongiform Encephalopathy (cBSE), the first prion disease identified in cattle, was initially reported in 1986 in the UK. Food-borne transmission of cBSE to humans was observed ten years later as a variant form of Creutzfeldt-Jakob Disease (vCJD) [1], leading to a major public health crisis.

This strain of cBSE is now rapidly disappearing as a result of appropriate containment measures. However, atypical forms of BSE have recently been identified in Europe and North America as a consequence of cBSE testing performed in these countries [2]–[4]. Because these cases are only found sporadically in older animals (≥8 years) coming to slaughter with few or no signs of disease, it would be plausible to suppose that atypical forms of BSE may have a lower virulence than cBSE and be innocuous to humans. However, recent studies suggest that one of the two main forms of atypical BSE, initially discovered in Italy and referred to as the bovine amyloidotic spongiform encephalopathy (BASE), might be at the origin of the cBSE epidemic: inoculation of the BASE strain into transgenic and inbred mice showed an apparent natural evolution towards the typical BSE strain [5], [6]. Moreover, a possible link has been suggested between BASE and one subtype (MV2) of human sporadic CJD (sCJD) on the basis of biochemical similarities [2], [7]. In contrast to vCJD, sCJD is believed to occur de novo without food-borne transmission. However, specific contaminating events by ingestion are difficult to rule out because human prion diseases can have silent incubation periods exceeding 50 years, as demonstrated for kuru [8].

One strategy to evaluate the risk of BASE for humans consists in assessing the susceptibility to disease transmission and the degree of pathogenicity in a non-human primate model that has already been shown to have characteristic clinical signs, histopathological lesions and PrPres profiles following infections with either BSE or vCJD [9], [10]. We therefore inoculated cynomolgus macaque monkeys (Macacca fascicularis) intracerebrally with BASE, cBSE and vCJD prion strains. The BASE strain, prepared from brain extract of a 15-year-old asymptomatic cow induced a distinctive and more rapidly fatal disease than cBSE, and showed a biochemical signature similar to that of the MM2 cortical subtype of human sCJD.

We have shown that BASE, the first identified atypical strain of BSE [2], originating from asymptomatic cattle, is transmissible by i.c. inoculation to a species of non-human primate. Although this observation concerned only one animal, its survival was substantially shorter than for all the macaques inoculated with classical BSE as well as the majority of those inoculated with human vCJD. Moreover, in earlier experiments by others on a total of 6 macaques inoculated i.c. with 50 mg of cBSE brain, none had an incubation period of less than 30 months [17], and humanized transgenic mice have been found to be highly susceptible to infection with BASE, and completely resistant to infection with cBSE [18]. If BASE is more pathogenic than classical BSE for primates, it could indicate a more readily transmissible infection from cattle to humans than previously suspected. A preliminary trial of oral transmission is currently ongoing for alimentary risk assessment: 49 months after oral dosing there is no indication of transmission; however, the incubation period following similar oral challenge with cBSE in an already completed experiment was 60 months.

The disease induced by BASE was different in all respects from that induced by classical BSE. The clinical presentation was characterized by mild tremors and myoclonus, progressing to a marked cognitive disorder, including spatial disorientation but without anxiety, aggressiveness or loss of appetite. In contrast, cBSE presented signs of anxiety and aggressiveness together with progressive difficulties in locomotion as well as cerebellar signs (major ataxia), and severe decrease of appetite with concurrent weight loss. The widespread spongiform lesions and loss of pyramidal cells in the third and fifth layers of the frontal cortex together with the severe parietal lesions could explain the prominent cognitive signs and the spatial disorientation seen in the BASE-infected monkey, contrasting with the severity of lesions in the obex and cerebellum consistent with the incoordination seen in animals inoculated with cBSE. Amyloid plaques, the hallmark of BASE in cattle, are not produced in the Macaque monkey, and conversely, cBSE does not produce plaques in cattle, but does so in the Macaque [9], a clear indication that plaque deposition depends as much on the host as the prion strain.

At the molecular level, under conditions of high proteinase pre-treatment and detection using two antibodies reacting with either an epitope in the N terminal octapeptide repeat region or the core of PrP, BASE and cBSE were clearly distinguishable in primate. BASE was detectable only by the core antibody, whereas cBSE was detectable by both antibodies. We estimated that the proportion of octapeptide-resistant PrPres molecules in the BASE brain homogenate was only a small fraction (≤1/10) of that of the cBSE brain homogenate. The difference in octapeptide sensitivity to PK between cBSE and vCJD in macaques on the one hand, and Type 1 sporadic CJD in humans on the other hand, is similar to what was observed between cBSE and classical scrapie in sheep. This method can now be used to test both ruminant and human samples to identify similarities and differences in their molecular protein signatures, and to implement the classification of ruminant and possibly human strains.

Although classical epidemiological studies have not found any link between scrapie in sheep and goats and human CJD, newer molecular biological studies now indicate that about half of all cases of scrapie are due to previously undetected atypical strains [19] that are experimentally transmissible to sheep and mice [20]. Their risk for humans is unknown and is the subject of current studies in experimental models, including primates. cBSE has been shown to be responsible for human cases of vCJD, but the comparative risk for humans of BASE and other atypical strains of BSE is still unknown, and its clarification will require many years of epidemiological surveillance and molecular biological testing of both bovine and human populations.

The first cases of BASE in cattle had PrPres electrophoretic profiles similar to the MV2 subtype of sporadic CJD patients [2] that, together with the presence of amyloid plaques in both the cattle and the patients, suggested a possible link between BASE and this subtype of sCJD. However, our PrPres typing technique has shown that, in the primate, PrPres of other MV2 sCJD patients exhibited a resistance to proteolysis different from the BASE-infected primate, whereas PrPres from vCJD-infected patients and primates behave similarly. This observation, together with the absence of amyloid plaques in the BASE-infected primate, weakens the likelihood of a direct link between BASE and MV2 subtype sCJD patients.

In contrast, the specific signature of PrPres in the BASE-infected primate was similar to that seen in three of four patients with the MM2 cortical subtype of sporadic CJD [7]. It is interesting that an important feature of the clinical-pathological syndrome in this BASE-infected macaque –the absence of cerebellar involvement – is also a common element in patients with the MM2 subtype of human sporadic CJD (Supplementary Figure S2). However, as illustrated by the clinical details of our four tested MM2 cases, there is considerable patient-to-patient variation, just as there can be variation among individual animals experimentally inoculated with a given strain of TSE. [21], [22].

It is not known whether atypical strains of BSE have been circulating for years, or represent new forms of disease, and continuing research is clearly needed to answer both this and the equally important question about a possible relationship to at least certain forms of what are presently regarded as sporadic cases of human disease (sCJD) [4], [23]. Moreover, the BASE strain has been described to evolve naturally towards BSE after successive transmissions in inbred mice [6]. The stability and pathogenicity of this strain in humans remains to be determined, and it is worth recalling that the stability of the cBSE/vCJD strain, which retains its specific molecular signature in different infected hosts, is the exception rather than the rule. As has been previously observed [24]–[26], one patient (Case No. 4, cf. figure 5 sample MM2#4) exhibited both types of PrP, i.e. type 2 typical of the MM2 subtype and type 1 observed in the MM1 subtype. On the one hand, this demonstrates the interest of such a simple biochemical test to refine PrP analysis, and on the other hand it raises a question about the existence of different PrPres signatures in the same patient, i.e., different prion strains linked to multiple infections or to variants selected by the host.

In summary, we have transmitted one atypical form of BSE (BASE) to a cynomolgus macaque monkey that had a shorter incubation period than monkeys infected with classical BSE, with distinctive clinical, neuropathological, and biochemical features; and have shown that the molecular biological signature resembled that seen in a comparatively uncommon subtype of sporadic CJD. We cannot yet say whether BASE is more pathogenic for primates (including humans) than cBSE, nor can we predict whether its molecular biological features represent a clue to one cause of apparently sporadic human CJD. However, the evidence presented here and by others justifies concern about a potential human health hazard from undetected atypical forms of BSE, and despite the waning epizoonosis of classical BSE, it would be premature to abandon the precautionary measures that have been so successful in reversing the impact of cBSE. We would instead urge a gradual, staged reduction that takes into account the evolving knowledge about atypical ruminant diseases, and both a permanent ban on the use of bovine central nervous system tissue for either animal or human use, and its destruction so as to eliminate any risk of environmental contamination.