Mouse bioassays are commonly used to characterize prion strains. We inoculated transgenic mice that overexpress bovine PrPC with bovine adapted TME (bTME), ovine passaged bTME, L-BSE, and C-BSE and compared the incubation period, attack rate, vacuolation lesion profile, amount of PrPSc, western blot profile, and PrPSc fibril stability for each inoculum. These data were used to evaluate strain differences and transmission efficiencies of selected TSEs. We were interested in determining the effects of multiple interspecies transmission events on strain phenotype. We found that both the TSE agent-type and the sheep donor genotype influenced the transmission efficiency. We also observed that o-bTMEVV, bTME, and L-BSE shared phenotype characteristics, and o-bTMEAV was different from o-bTMEVV, bTME, and L-BSE.
To compare the effectiveness of prion strains to replicate and cause disease in mice, several outcomes are easily measurable including the attack rate (AR) and incubation period (IP). These parameters are considered partially representative of the strain phenotype. Between L-BSE, bTME, and o-bTMEVV, there was little disparity between ARs and IPs. For example, the AR was slightly higher in o-bTMEVV compared to L-BSE, but the IP was longer compared to bTME. Recently, a computative ratio called the Transmission Efficiency (TE) combined these parameters into a single number [25]. L-BSE had the highest TE in TgBovXV mice followed by bTME. The TE of o-bTMEVV was similar to C-BSE; however, o-bTMEAV had the lowest TE. The less efficient transmission of o-bTME compared to bTME is most likely due to the species barrier effect (sheep PrP to bovine PrP); whereas, the other isolates represent intraspecies passages of bovine prion protein to bovinized mouse prion protein. Nonetheless, the high attack rates and high EIA results for o-bTME isolates indicate the absence of a robust species barrier.
The phenomenon of altered transmissibility after passage through an intermediate species has been previously documented. For example, the passage of BSE through sheep results in a decrease in incubation period in BoPrP-Tg110 mice [19]. Furthermore, transmission of TSEs to intermediate species can expand the host range to include species that were not susceptible to the original TSE [18, 27]. Additional studies would be necessary to determine the complete host range of TME after modification by interspecies transmission.
The sheep donor genotype also influences the efficacy of interspecies prion transmission. Ovine bTME isolates from VRQ/VRQ and VRQ/ARQ genotype sheep have notably different TEs in bovinized mice. The bovine adapted TME agent has a high attack rate in bovinized mice inoculated with brain homogenate from VRQ/VRQ genotype sheep; however, the transmission efficiency is reduced after passage through sheep with the VRQ/ARQ genotype. This pattern of host genotype versus disease susceptibility is similar to that observed in classical scrapie-affected sheep [20, 28–32]. The present study demonstrates that sheep donor genotype also influences the transmission efficiency of a non-scrapie TSEs to other species.
Divergent neuropathology and western blot profiles arose after passage of bovine adapted TME through different ovine host genotypes: VRQ/VRQ and VRQ/ARQ. Since other authors have reported similarities between L-BSE and bovine passaged TME from the Stetsonville, WI outbreak [11, 12], we compared the ovine passaged isolates to L-BSE in TgBovXV mice. The VRQ/VRQ donor material (o-bTMEVV) had a similar pattern and vacuolar lesion scores to L-BSE and bTME. Both bTME and o-bTMEVV had similar western blot migration patterns to L-BSE. The lesion profile of o-bTMEAV was notably different compared to o-bTMEVV, bTME, and L-BSE. Another difference with o-bTMEAV isolates was a larger fraction of diglycosylated PrPSc. Even though o-bTMEAV and C-BSE had similar glycosylation fractions, 77% and 85%, respectively, the molecular weight of the diglycosylated band in C-BSE was greater than o-bTMEAV. Brains from C-BSE inoculated mice also contained florid plaques that were absent from o-bTMEAV and other isolates.
To evaluate the transmission efficiency between species, a first-passage transmission study is compulsory. In this study, ovine passaged bTME is a first-passage interspecies transmission event. However, the effect of host adaptation on disease phenotype cannot be fully accounted for in the lesion profiles derived from first passage mice. Subsequent repeated intraspecies passages is required to sift out strain variants and select for a new variant [15, 17, 33–38]. This strain selection results in a decreased IP and stabilized neuropathology [39]. Subsequent intraspecies passages would result in increased severity of vacuolation with retention of regional distribution [17]. Therefore, a shortcoming of the present study is the use of first passage interspecies transmission mice to construct lesion profiles. The lesion profiles of C-BSE and o-bTMEAV had similar regional distribution, but o-bTMEAV generally had more severe vacuolation than C-BSE. Consequently, upon subsequent passages, it could be expected that the magnitude of vacuolation would increase further for o-bTMEAV leading to a greater discrepancy with C-BSE. To the contrary for o-bTMEVV and L-BSE, where the magnitude of vacuolation is usually lower for o-bTMEVV. Repeated passages in TgBovXV mice would be expected to increase the degree of vacuolation possibly making lesion profiles for o-bTMEVV and L-BSE more similar. To evaluate these hypotheses, second passage transmissions of o-bTME in TgBovXV mice are planned.
We sought to compare the PrPSc fibril stabilities of bovine spongiform encephalopathies with host adapted bTME and non-host adapted ovine bTME. The stabilities of the non-host adapted o-bTME isolates were different than C-BSE and L-BSE; whereas, bovine adapted TME fibrils displayed intermediate stability. Previous work has demonstrated differences in the fibril stabilities between prion strains [40, 41]. In the present experiment, the midpoint of the GdnHCl fibril unfolding curve of bovine adapted TME was not significantly different from C-BSE and L-BSE. The o-bTME isolates were indistinguishable from each other. Therefore, fibril stability analysis wasn’t an all-inclusive means for strain differentiation in this experiment.
Our findings differed from previous work that demonstrated a positive correlation between incubation period and conformational stability in mice inoculated with various TSE isolates [42, 43]. Namely, shorter incubation periods in mice were associated with lower stability. Lower conformational stability is postulated to allow increased exposure of PrPSc to bind PrPC resulting in more rapid propagation of PrPSc that shortens the incubation period [44]. This correlation has been similarly identified in naturally occurring TSEs [45, 46]; however, in other laboratory animal models of prion disease, different observations have been made. Short incubation hamster-adapted prion strains have higher fibril stability while strains with longer incubation periods exhibit lower fibril stability [47]. In the present work, three isolates most recently passaged in cattle exhibited higher fibril stability and shorter incubation periods compared to ovine passaged isolates. The two ovine passaged bTME isolates exhibited no significant difference in fibril stability compared to each other despite having different incubation periods. The o-bTMEVV isolate had an incubation period similar to L-BSE and C-BSE; however, its fibril stability was lower. Our observations could be due to species barrier effects. It is possible that subsequent passage and host adaptation could result in changes to either fibril stability or incubation time.