While the transmission of BSE from cattle to humans via oral exposure has been proposed as the origin of vCJD, the risk posed to humans from BSE infection in other species currently is unknown. Sheep can be experimentally infected with BSE, and it has long been a concern that sheep have become infected during the BSE epidemic. Although no evidence of such infection has been identified in the field, cases of BSE in goats have been reported (14
). Here, we have shown that the inoculation of experimental sheep BSE into gene-targeted HuMM transgenic mice resulted in the identification of TSE-related pathology (PrP deposition, vacuolation, and gliosis) in ~70% of the animals overall and 100% of HuMM mice surviving more than 600 days. PrPSc
was detected in brain tissue by immunoblotting and in spleen tissue of several mice using the IDEXX HerdChek assay. Some of the oldest HuMM mice (623 to 750 days postinoculation) that showed PrP deposition in the brain did not have detectable PrPSc
in spleen. This variability is not unusual, as extremely old mice that show PrPSc
in the brain often have no corresponding deposition in the spleen. This likely is due to the loss of germinal centers in the spleen caused by aging (7
). No evidence of disease transmission was observed in HuMV or HuVV mice, mirroring the prevalence of vCJD disease observed to date in the United Kingdom population. Although the presence of disease pathology indicates agent replication and early-phase disease, we cannot predict whether these mice would have developed clinical disease if their life spans had been extended, or if this represents a persistent subclinical state. The subpassage of brain material from HuMM mice will be performed to confirm agent replication and assess the adaptation and host range of the agent.
Our results cast new light on the existing data concerning BSE transmission to humans. Since the identification of the link between BSE and vCJD, many studies have been performed to demonstrate or model the transmission of BSE to humans using in vitro
conversion techniques or by the inoculation of transgenic mice expressing human PrP. In these transmission studies, cattle BSE has shown limited transmissibility to human PrP transgenic mice (~0 to 30%), and considerable variation in susceptibility exists between different transgenic lines with various constructs and protein expression levels (1
). The highest levels of susceptibility to BSE in mice expressing human PrP with codon 129-methionine (~30%) were reported by Asante et al. (1
) in the two-times overexpression Tg35 model (Hu-129 M), which included the identification of both limited clinical disease and subclinical disease. However, lower attack rates of approximately 20% have been reported in Tg650 mice, which have a higher expression level of 129-Met human PrP of around 5- to 8-fold (3
). The gene-targeted transgenic mice utilized in our studies, which express wild-type levels of human PrP from the endogenous mouse Prnp
locus, previously showed no incidence of disease following cattle BSE inoculation (4
). These observations have led to the assumption that the overexpression of PrP is essential to model human disease susceptibility in mice, and that rodent models with wild-type physiological levels of PrP expression do not live long enough to display signs of disease, as would be seen in the longer-life-span human species. It therefore is significant that the inoculation of experimental sheep BSE described here has resulted in the identification of TSE-related pathology in the gene-targeted human PrP transgenic mice. Additionally, previously published data have shown that short incubation times can be achieved in HuMM and HuVV gene-targeted mice (5
). Our data show clearly that gene-targeted transgenic lines are useful in the study of cross-species susceptibility, and that such susceptibility depends on the agent/host combination rather than the life span of a mouse. The inclusion of data obtained from both overexpressing and gene-targeted transgenic mice therefore may inform more accurately on the assessment of the true zoonotic potential of a particular TSE isolate.
The reasons for the increased susceptibility of HuMM mice to experimental sheep BSE in respect to cattle BSE currently are unknown and are the subject of further investigation in our laboratory. One possible explanation is that our BSE-infected sheep brain contained a significantly higher titer of BSE than that found in the cattle BSE brainstem pool, resulting in the shortened incubation times in control 129/Ola and Bov6 mice with experimental sheep BSE inoculum 2 compared to that of cattle BSE, and the pathological features observed in HuMM mice. Incubation times for control mice also were shorter for experimental sheep BSE inoculum 2 than for inoculum 1, although the ratio between 129/Ola and Bov6 mice was similar for each inoculum. Such variation in incubation time on the primary passage of experimental sheep BSE is, however, common, and it has been observed in previous experiments (see Table S1 in the supplemental material). The observed difference in incubation times between inoculum 1 and inoculum 2 therefore is not unexpected. Previous studies by Gonzalez et al. (22
) have shown relatively high infectivity titers of sheep-passaged BSE in RIII mice, which were equivalent to those obtained in Romney sheep. The infectivity titer of the cattle BSE brainstem pool used in our transmissions was 103.3
U/g in RIII mice (R. Lockey and M. Simmons, personal communication). Those reported by Gonzalez et al. (22
) for sheep-passaged BSE were 105
U/g in RIII mice, suggesting that higher titers may indeed be attained in sheep brain. However, reported infectivity titers for BSE in cattle have been variable (6
). We therefore are performing the titration analyses of experimental sheep BSE brainstem in Bov6 mice to provide a direct comparison to titration data already available for the cattle BSE brainstem pool.
An alternative hypothesis is that the passage of BSE through a sheep has altered the strain characteristics of the agent, producing a variant with increased virulence and/or host range. This possibility is supported by recent data describing the enhanced virulence of experimental sheep BSE in bovine PrP transgenic mice (BoPrP-Tg110) and porcine PrP transgenic mice (PoPrP-Tg001) compared to that of cattle BSE (15
). BoPrP-Tg110 mice and PoPrP-Tg001 mice (which overexpress PrP 8× and 4×, respectively) produced significantly shorter incubation times following inoculation with an experimental sheep BSE brainstem pool than with cattle BSE isolates. The differences in incubation time observed in BoPrP-Tg110 mice were maintained on subpassage (15
), indicating that the original variation probably was not due to infectivity titer discrepancies between the two BSE sources. However, the full titration of these tissue homogenates in mice would be required to confirm that this was indeed the case. In PoPrP-Tg001 mice, incubation times shortened significantly on subpassage and were maintained on further subpassage, indicating adaptation to the new host (16
). In the study described here, lesion profiles obtained from control 129/Ola mice and Bov6 transgenic mice were similar for both cattle and experimental sheep BSE. Although we were unable to resolve the size of the PrP-res low-molecular-weight band in both the experimental sheep BSE brain homogenate and the experimental sheep BSE-infected HuMM mouse, both showed reduced staining with MAb 12B2, which is characteristic of BSE infection (27
). Therefore, there were no differences in strain characteristics between experimental sheep BSE and cattle BSE, with the exception of the transmissibility to HuMM mice (which could be due to increased infectivity titer).
The altered agent properties of sheep BSE observed by Espinosa and colleagues (15
) suggest that passage through a sheep causes BSE to transmit in a manner more similar to that of natural scrapie than that of cattle BSE. To investigate this, we inoculated our transgenic panel with two isolates of natural sheep scrapie. No disease pathology was observed in any transgenic mice following inoculation with either isolate of natural scrapie. Hence, in the experiments described here, the susceptibility of the HuMM mice to experimental sheep BSE does not appear to be due to a general susceptibility to ovine prions but is instead linked specifically to the replication of the BSE agent strain in sheep brain. Although agent strain characteristics of BSE are not altered when assayed in Bov6 or 129/Ola mice following passage in sheep, both samples of experimental sheep BSE did show positive TSE pathology in HuMM transgenic mice, which has not been seen previously with cattle BSE inoculations in these mice. Whether this is due simply to agent infectivity titer or a more subtle change in agent characteristics is the subject of further analysis in our laboratory.
Although sheep can be experimentally infected with BSE by oral, intravenous, or intracerebral exposure (18
), no cases of sheep BSE have been reported in the field. The possible increased risk of disease transmission identified in these studies thus is not of major concern to the public at present. Natural BSE infection has, however, been identified in goats (14
), indicating that small ruminants have been exposed to sources of contamination. We cannot rule out the possibility that sheep have been infected with BSE during the height of the BSE epidemic, as these animals undoubtedly were exposed to similar feed sources (although with different levels of exposure compared to those of cattle). Such infection may have been limited and/or localized and resolved very quickly. BSE in small ruminants may, however, represent an increased risk to humans due to the wider distribution of BSE infectivity identified in peripheral sheep tissues (2
) compared to that of BSE in cattle, mainly which is restricted to the CNS (10
). While TSEs remain in the environment and continue to infect animals (even at low prevalence), there remains the potential for cross-species transmission and the emergence of TSE isolates with altered strain properties or host ranges. Our data therefore emphasize the need for continued surveillance to identify, monitor, and characterize any new emerging TSE agents that are identified in ruminants and the assessment of the potential risks posed to other species.