A total of 12 mouse lemurs of both sexes (Center for Breeding and Experimental Conditioning of Animal Models, University Montpellier 2, Montpellier, France) were maintained in animal Biosafety Level 3 facilities, according to requirements of the French ethics committee (authorization CE-LR-0810). Young and adult lemurs were fed (8 animals) or IC inoculated (4 animals) with 5 or 50 mg of L-BSE–infected brain tissue (10% homogenate in 5% glucose) (Table). The isolate for the L-BSE agent (02–2528) was derived from cattle in France (11). When progression of prion disease was evident, the lemurs were euthanized and their brains were isolated. Brains were processed for Western blot analysis with SHa31 monoclonal antibody against PrP for PrP^res detection, as described in mice (11); for histologic examination by using hematoxylin and eosin staining; and for disease-associated prion protein (PrPd) immunochemical detection by using the paraffin-embedded tissue blot method or immunohistochemical analysis with monoclonal antibody 3F4 against PrP.

Beginning ≈3 months before the terminal stage of the disease (19–22 months after inoculation), neurologic symptoms developed in the 4 mouse lemurs that received IC inoculations (Table). In all 4 animals, initial clinical signs and symptoms were blindness, thigmotaxic behavior, and poor appearance of the fur. Appetite and general fitness were maintained; anxiety and aggressiveness were not observed. Next, locomotion became slower, followed by incoordination and loss of balance in the last month of life. Ipsilateral circling behavior was reported, indicating unilateral degeneration of the striatum. This behavior stopped 15 days after onset, suggesting damage to the contralateral striatum. Disequilibrium, with frequent falls, became more noticeable. At the terminal stage of the disease, the animals were prostrate.

One orally inoculated lemur, which was fed 5 mg of infected brain and euthanized 27 months later, had signs and symptoms of disease similar to those in IC-inoculated animals, except for the ipsilateral circling behavior. In 2 lemurs fed 50 mg and 2 others fed 5 mg of L-BSE–infected brain, clinical signs and symptoms of prion disease developed just a few weeks before the animals were euthanized (18 and 32 months and 33 and 34 months after inoculation, respectively). Disease was characterized by progressive prostration, loss of appetite, and poor appearance of the fur, without incoordination or disequilibrium. The 3 remaining lemurs were orally inoculated at 2 years of age and were still alive and healthy 28 months after inoculation (Table).

Figure 1

Figure 1. Western blot analysis of protease-resistant prion protein in the brain (thalamus/hypothalamus) and spleen of mouse lemurs inoculated with a cattle-derived L-type bovine spongiform encephalopathy (BSE) isolate by oral and intracerebral routes...

PrP^res was readily detected by Western blot analysis in brain extracts (thalamus/hypothalamus region) from 8 of the 9 animals examined (Table), although at lower levels in the lemur that was euthanized earlier (i.e., 18 months after inoculation). Western blot analyses showed uniform PrP^res molecular profiles, irrespective of the route or dose of inoculation, with a low apparent molecular mass (≈19 kDa, similar to the PrP^res in the original cattle brain) (Figure 1). However, the PrP^res profile in mouse lemurs was characterized by a higher proportion of di- and monoglycosylated species (>95% of the total signal) than in the inoculum of the agent of bovine L-BSE (≈80%). In addition, PrP^res was detected by Western blot in the spleens of 3 (1 IC inoculated and 2 fed with 5 mg of cattle brain) of the 9 animals examined (Figure 1).

Figure 2

Figure 2. Histopathologic and disease-associated prion protein (PrPd) immunodetection in the brain of 2 mouse lemurs after intracerebral (5 mg) or oral (50 mg) inoculation with a cattle-derived L-type bovine spongiform encephalopathy isolate....

Histopathologic analysis showed severe spongiform changes in the brains of the 4 IC-inoculated mouse lemurs (Figure 2, panel A). The brains displayed a pattern of vacuolation characterized by intense spongiosis with many confluent vacuoles in the basal telencephalon (septum, striatum, caudate putamen nuclei), midbrain (thalamus, hypothalamus), mesencephalon (colliculi), and in some parts of the brainstem (tegmental ventral area, raphe nuclei). Lesions in the cortex and hippocampus were less severe than in the subcortical areas. Cerebellum showed occasional small-size vacuoles. Among the 5 orally inoculated animals, 2 (1 fed 5 mg, the other fed 50 mg) showed histopathologic features similar to those observed in IC-inoculated animals. In the other 3 orally inoculated animals, spongiosis was characterized by fewer vacuoles and was restricted to the striatum (Figure 2, panel B), thalamus, colliculi, and brainstem.

Distribution of PrPd in the brain was assessed by paraffin-embedded tissue blot (Figure 2, panels C and D) or immunohistochemical analysis with 3F4 antibody (Figure 2, panels E and F). Results for IC-inoculated animals showed that PrPd strongly accumulated in a dense synaptic pattern associated with nonamyloid plaques in the striatum, several thalamic nuclei (Figure 2, panel E), the external cortex of the colliculi, and the tegmental area. Other areas that were slightly less affected (e.g., neocortex and hippocampus) showed few coarse granules and synaptic deposits. The cortical molecular layer and the corpus callosum were devoid of PrPd (Figure 2, panel C). In orally inoculated animals, PrPd was strongly accumulated in the striatum and thalamus (Figure 2, panel D) but weakly accumulated in the cortex. Immunohistochemical analysis showed synaptic deposits (Figure 2, panel F), and some focal deposits were evident in animals that survived longer. No plaques were detected in orally inoculated animals.

We demonstrated that the agent of L-BSE can be transmitted by the oral route from cattle to mouse lemurs. As expected, orally inoculated animals survived longer than IC-inoculated animals. Orally inoculated lemurs had less severe clinical signs and symptoms, with no evidence of motor dysfunction. It was previously suggested that the agent of L-BSE might be involved in the foodborne transmission of a prion disease in mink (11,12), a species in which several outbreaks of transmissible mink encephalopathy had been identified, notably in the United States (13).

Our study clearly confirms, experimentally, the potential risk for interspecies oral transmission of the agent of L-BSE. In our model, this risk appears higher than that for the agent of classical BSE, which could only be transmitted to mouse lemurs after a first passage in macaques (14). We report oral transmission of the L-BSE agent in young and adult primates. Transmission by the IC route has also been reported in young macaques (6,7). A previous study of L-BSE in transgenic mice expressing human PrP suggested an absence of any transmission barrier between cattle and humans for this particular strain of the agent of BSE, in contrast to findings for the agent of classical BSE (9). Thus, it is imperative to maintain measures that prevent the entry of tissues from cattle possibly infected with the agent of L-BSE into the food chain.