Risk Of CWD - Research
On Transgenic Mice

From Patricia Doyle, PhD

From: TSS Subject: Re: Transmission of Elk and Deer Prions to Transgenic Mice Date: November 28, 2006
In Reply to: Transmission of Elk and Deer Prions to Transgenic Mice posted by TSS on September 1, 2006 at 1:07 pm:
FURTHER INTO THIS STUDY, some might find interest ---
Prions are transmissible pathogens that accumulate in the central nervous system (CNS) and cause fatal neurodegeneration (34). Prions are composed of an alternatively folded isoform of the prion protein (PrP), denoted PrPSc. The precursor of PrPSc is a cellular protein designated PrPC that is encoded by a chromosomal gene. Prion diseases afflict humans as well as livestock, such as cattle, goats, and sheep; additionally, prions cause CNS disease in captive and wild populations of deer and elk (51, 53). In contrast to scrapie of sheep and goats, bovine spongiform encephalopathy (BSE) in cattle has been transmitted to humans and has killed more than 170 teenagers and young adults as variant Creutzfeldt-Jakob disease (vCJD) (44, 49, 51, 52). BSE prions have been experimentally transmitted to sheep and appear to be transmitted naturally among sheep (6). Recently, BSE prions were found in goats (14).
The transmission of BSE prions to humans has elevated concern about the possibility of the zoonotic transmission of chronic wasting disease (CWD) from deer and elk to humans (5, 56). Hunters and other consumers of venison are potentially at risk to acquire prion disease from infected deer and elk. CWD was first observed in 1967 in cervids and was recognized as a prion disease a decade later (54). CWD has been reported in 14 U.S. states and 2 Canadian provinces.
The epidemiology of CWD is unclear. In contrast to BSE and scrapie, CWD is highly transmissible among cervids. In some captive mule deer (Odocoileus hemionus) herds, 90% of the animals have been reported to be infected with CWD prions (28). The prevalence of CWD cases in free-ranging deer populations can be up to 30% (53). The number of cases of CWD that arises spontaneously and then spreads horizontally, however, is unknown. Commercial farming and trade with cervids may foster horizontal transmission of CWD. It seems likely that, as surveillance improves, the known geographic distribution of CWD will increase.
Cases of CWD have been reported in South Korea where elk had been imported from Canada (21). Furthermore, free-ranging elk and deer occasionally share the same pastures with cattle and sheep. It is therefore of concern whether CWD prions can be transmitted to livestock and on to humans. The passage of prions into a new host species can alter the host range: hamsters are resistant to CWD prions from deer and elk but are susceptible to CWD prions previously passaged in ferrets (4).
While our work was in progress, two reports appeared describing the transmission of CWD prions to transgenic (Tg) mice expressing cervid PrP (9, 23). The first study showed transmission of CWD prions to Tg(CerPrP) mice expressing the S2 PrP allele (GenBank accession no. AF009180) of mule deer, and the second study reported transmission of CWD prions to Tg12 mice expressing the eGMSE PrP allele (Gen- Bank accession no. AF156183) of Rocky Mountain elk (Cervus elaphus nelsoni). The respective alleles are expressed exclusively in deer or elk and give rise to PrP molecules that differ only at residue 226, which is glutamine in deer PrP and glutamate in elk PrP. Transmission of a CWD brain sample from Rocky Mountain elk to Tg(CerPrP) mice resulted in incubation times of 240 days. Transmission times of several CWD brain samples from mule deer were between 230 and 260 days. On second passage, the incubation time was 160 days in mice
homozygous for the transgene (9). The second study showed transmission of CWD prions from elk to Tg12 mice within 120 to 140 days; no change in incubation time was observed on second passage (23).
In the findings reported here, we describe studies with Tg mice expressing either elk PrP [Tg(ElkPrP)] or deer PrP [Tg(DePrP)]. The Tg(ElkPrP) mice used in this study express the same PrP as Tg12 mice (23), and the Tg(DePrP) mice express the same PrP as Tg(CerPrP) mice (9). We generated four lines of Tg(ElkPrP) mice, one of which was bred to homozygosity, and two lines of Tg(DePrP) mice. We inoculated all lines with CWD prions from elk (n 1). Additionally, we inoculated Tg(ElkPrP) and Tg(DePrP) lines with CWD prions from mule deer (n 2) and white-tailed deer (Odocoileus virginianus; n 2). Tg(ElkPrP) mice succumbed to prion disease within 180 to 200 days and Tg(DePrP) mice, with slightly lower DePrP transgene expression levels, within 300 to 400 days. Neuropathologic analysis showed spongiform degeneration, florid PrP amyloid plaques, and astrocytic gliosis in ill mice.
Based on the data reported here and those of others cited above, CWD prions from elk, mule deer, or white-tailed deer seem to be equally transmissible among these three cervid species. In addition, CWD prions do not readily transmit disease to Tg mice expressing human, bovine, or ovine PrP. Whether CWD prions cause disease in humans or livestock remains uncertain.
The overexpression of cervid PrPs in mice did not have any deleterious effect on the Tg lines described here. Uninoculated mice from one such Tg line was observed for 650 days (Table 1). The absence of spontaneous disease in these Tg mice allowed us to use them to bioassay prions in the brains of elk and deer that died of CWD.
Brainstem samples from elk, mule deer, and white-tailed deer with CWD were inoculated into five Tg lines expressing ElkPrP and two lines expressing DePrP. Bioassay of the Elk1 inoculum in the seven Tg cervid PrP lines showed that the length of the incubation time is inversely proportional to the level of cervid PrP expression in the brain (Fig. 5; Table 2).
When the level of cervid PrPC expression was similar to that of MoPrPC in WT mice, it was designated 1. In Tg(DePrP) mice expressing DePrP at 1, the incubation time was 300 days, whether the CWD inoculum was from mule deer (MD1) or elk (Elk1). Doubling the level of cervid PrPC to 2 resulted in a reduction of the incubation time to 200 days for the Elk1 and MD1 inocula while tripling the expression of cervid PrPC reduced the incubation time to 100 days for the Elk1 inoculum.
A similar relationship was described earlier for Tg mice expressing SHaPrPC (35); however, both MoPrP and SHaPrP were coexpressed in those Tg lines. In the studies reported here (Fig. 5), the MoPrP gene was disrupted (10) so that the only PrP being expressed was cervid PrPC. In a recent study, Tg mice expressing DePrP at 5 and 3 the level of PrP expressed in WT FVB mice developed neurologic deficits at 235 days after intracerebral inoculation with CWD prions from elk and at 225 to 264 days with CWD prions from mule deer (9). In another study, two lines of Tg mice expressing ElkPrP at 2 developed CNS disease 118 or 142 days after inoculation with CWD prions from elk (23).
The CDI studies of the CWD inocula indicated that the Elk1 and MD1 inocula contained similar levels of PrPSc (Fig. 1A), which is consistent with the indistinguishable incubation times for these inocula in Tg(ElkPrP)12577, Tg(DePrP)10945, and Tg(DePrP)10969 mice (Table 2). Interestingly, the levels of PrPSc varied over a 100-fold range for the first nine cervid brain specimens examined (Fig. 1A). Assessing the level of PrPSc in brain samples in advance of our transmission studies proved to have been quite useful in retrospect (Table 2).
Serial passage of CWD prions in the Tg(ElkPrP) mice resulted in modest reductions in the incubation times, i.e., up to 70 days (Fig. 3A to D; Table 3). This shortening was seen with prions from elk, mule deer, and white-tailed deer. These
results contrast with those for the serial passaging of BSE prions in Tg(BoPrP) mice and the serial passaging of CWD prions in Tg12 mice, for which no changes in the incubation times were observed (23, 42, 44). There are several possible explanations for the shortening of the incubation times upon serial passage in Tg(ElkPrP)12577 mice. First, the level of prions in the brains of cervids may be lower than in Tg(Elk PrP)12577 mice. If that were the case, then the first Tg mouseto- Tg mouse passages would be expected to exhibit shorter incubation times than those found with passages from the cervids to Tg mice. A corollary to this scenario is that the incubation times upon subsequent passage in a given Tg line should remain constant. Second, the cervid brain inocula may be composed of a mixture of strains, and one strain may emerge as the predominant strain over the length of the incubation time.
In this case, the predominant strain in the Tg mouse brain exhibits a shorter incubation time during the next passage, because it exists at a higher titer in the mouse brain than in the cervid brain sample. Third, within a mixture of prion strains, some slow strains may be inhibitory for faster ones as previously reported (13, 22). If this were the case and transmission to Tg mice resulted in the elimination of the slower strain, then on subsequent passage in Tg mice, the incubation time would shorten. Fourth, a posttranslational modification in cervid PrP, such as the N-linked oligosaccharides or the glycosylphosphatidylinositol anchor, might slow replication of cervid prions in the Tg mice. If this were the case, then on subsequent passage, CWD prions formed in a mouse would exhibit shorter incubation times.
Except for the first possibility, for which endpoint titrations can be used to establish the titers of CWD prions in cervid and Tg mouse brains, distinguishing among the possibilities listed above may be difficult. Interpreting such a titration study will be facilitated if endpoint titrations in cervids give results similar to those obtained with the Tg mice. It is notable that endpoint titrations performed with cattle resulted in a titer of 106 50% infective dose units/g of brain tissue from the obexes of BSE-infected cattle, whereas endpoint titrations performed with Tg(BoPrP) mice resulted in a titer of 106.9 50% infective dose units/g of brain tissue (39, 42, 50).
Both the glycoform abundance patterns and the distribution of neuropathologic lesions in CWD-inoculated Tg(ElkPrP) and Tg(DePrP) mice argue for a single prion strain. The molecular masses of the di-, mono-, and unglycosylated PrPSc fragments from all CWD isolates were similar before (Fig. 2A) and after passaging in Tg(ElkPrP)12577 (Fig. 2B and D) and Tg(DePrP)10945 (Fig. 2C) mice. Moreover, the relative abundance of these glycoforms did not change upon passage in the Tg mice (see Fig. S1 in the supplemental material).
Neuropathologic examination of ill Tg(ElkPrP) and Tg(DePrP) mice demonstrated similar CNS lesions in all of the mice inoculated with CWD prions from elk, mule deer, and whitetailed deer (Fig. 6). The deposition patterns and neuroanatomic distribution of both PrPSc deposition (Fig. 7) and florid PrP amyloid plaques (Fig. 6) were similar for all inocula but differed somewhat in intensity. Upon serial passage in Tg(Elk PrP) mice, the CNS lesions remained unchanged (data not shown). Overall, our neuropathologic findings for CWD-infected Tg mice expressing DePrP or ElkPrP did not differ substantially from those reported by others (9, 23).
In Tg(MoPrP)4053 mice inoculated with CWD prions, both the morphology of the lesions and distribution of PrP amyloid plaques (Fig. 6I) were different from those found in Tg(Elk PrP) and Tg(DePrP) mice. In contrast to RML prions that produce finely granular deposits of PrPSc in the absence of amyloid plaques, the CWD prion strain was amyloidogenic in the brains of Tg(MoPrP)4053 mice.
In contrast to Tg mice expressing cervid PrP, Tg mice expressing human, bovine, or ovine PrP did not succumb to prion disease after inoculation with CWD prions after 500 days (Table 4). Our results agree with those of others who reported that Tg mice expressing human PrP were resistant to CWD prions but susceptible to sCJD prions (23). Despite our con- firmation of an earlier study demonstrating that Tg mice expressing HuPrP(M129) do not develop prion disease when inoculated with CWD prions, any conclusions from such negative data need to be tempered (7), especially in light of a recent study with squirrel monkeys. Intracerebral inoculation of CWD prions into squirrel monkeys (Saimiri sciureus) demonstrated transmission to a nonhuman primate, arguing that humans might be susceptible to CWD prions (27).
While our studies of Tg(BoPrP) mice inoculated with CWD prions also gave negative results, a recent study reported that five of 13 cattle inoculated with CWD prions produced PrPSc, based on limited proteolysis and immunohistochemical staining of brain sections (16). These studies were terminated 6 years after intracerebral inoculation, before any of the cattle developed neurologic dysfunction. In other studies, sheep scrapie prions were inoculated into elk (17). After more than three years, PrPSc was found in the brains of three elk that developed neurologic deficits before dying. Our negative results with CWD prions inoculated into Tg(HuPrP), Tg(Bo PrP), and Tg(OvPrP) mice might reflect low levels of prion replication that are insufficient to produce disease during the 500-day observation period. Several investigators have described situations in which prions from one species replicate too slowly in another species to cause signs of neurologic dysfunction but do produce disease with serial passage (19, 36).
In the studies reported here, we did not choose to passage serially the brains of asymptomatic, CWD-inoculated Tg(Hu PrP), Tg(BoPrP), and Tg(OvPrP) mice (Table 4). An alternative explanation for our negative results may reside in the strain(s) of CWD prions that we used for inoculation. While our CWD prions were unable to initiate the conversion of HuPrPC, BoPrPC, or OvPrPC into PrPSc, some other CWD strains might be able to do so. Precedent for the latter has been seen with human prions: human vCJD prions replicate well in Tg(BoPrP) mice but multiply slowly in Tg(HuPrP) mice and in Tg(MHu2M) mice expressing a chimeric human-mouse PrP (2, 24, 42, 44).
Whether hunters, cervid farmers, and aficionados of venison are at increased risk for prion disease remains to be established. Recently, CWD prions were also reported in the skeletal muscles of infected deer, indicating a possible hazard for the oral transmission of CWD prions (1). Tg(ElkPrP) and Tg(DePrP) mice provide a sensitive and specific bioassay for determining the levels of CWD prion infectivity in cervid tissues and for studying the biology of these particular prions. These Tg mice make it possible to determine the levels of CWD prion titers in brain, muscle, liver, pancreas, kidney, and
other tissues as well as in the blood, urine, feces, and saliva of both elk and deer. Elucidating the mode of CWD prion spread among elk, deer, and moose will be important for understanding why CWD prions are so contagious for domesticated and free-ranging cervids. Such information may prove useful in learning how to restrict the epizootic spread of CWD prions to humans and livestock. ......snip.......end



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