Bioassay of tissues from scrapie infected sheep and goats demonstrated the transmissible nature of the disorder and the novel characteristics of the infectious agent well before the introduction of the prion hypothesis
]. Oral and parenteral challenge demonstrated the susceptibility of goats to ovine scrapie
]. Reciprocal studies examining the susceptibility of sheep to caprine scrapie are less well described. Studies in the natural ruminant host are limited by the prolonged incubation time and the expense of housing large animals in biocontainment. Transgenic mice expressing a PRNP gene from a species of interest are a suitable surrogate host for some studies of prion disease in humans, cattle, sheep and deer
]. Considerable variation is noted among transgenic strains, however, with differences in outcome associated with route of inoculation
], transgene sequence of the regulatory and coding regions of PRNP
], and transgene expression level
]. The murine Tg338 line expresses the ovine PRNP VRQ allele under the ovine PRNP promotor with overexpression of PrPc
in the central nervous system and lymphoreticular system
], sites of PrPSc
accumulation in sheep and goats. This transgenic mouse line has been useful in characterizing atypical (Nor98)
] and classical scrapie in sheep of various genotypes
]. Several ovine isolates have been further characterized by serial passage to identify candidate classical scrapie strains differing in biochemical or clinical profiles in Tg338 mice
]. In this study, we examined the use of Tg338 mice as a surrogate for characterizing caprine scrapie bioassayed in the ovine host. We examined incubation time, neuropathological phenotype, glycoform profile, and proteinase K cleavage pattern following primary and secondary passage of brain homogenates from three goats with naturally acquired scrapie. Brain tissue from a sheep homozygous for the PRNP ARQ allele was used as a reference ovine scrapie case.
Incubation times for all 4 inoculum groups were prolonged and variable upon primary passage to Tg338 mice; mean incubation times were not related to ELISA/Western blot titer. The sample from goat 3538 showed the widest variation in survival time on primary passage, with insidious onset at as early as 199
days and clinical signs of ataxia, weight loss and kyphosis by 206
days; survival times in some mice in this group exceeded 300
days. Mice were euthanized at 199–228
3 short incubation), at 253–263
4 mid-range incubation), and at greater than 331
3 long incubation). Subpassage of samples from mice in the short (228
days) and long (331
days) incubation groups resulted in shorter incubation periods and mean survival times with no significant difference between the short and long incubation sample recipients (137 +/−2 and 135 +/−5 respectively); likewise, there was no significant difference among the groups of P2 mice from the other inocula.
Several phenotypes have been described following passage of tissue from scrapie infected sheep to Tg338 mice. Notably, differences in incubation time on primary and secondary passage associated with differences in electrophoretic profiles have been described
], particularly in sheep with alleles occurring on the ARQ haplotype. These cases have been reported as homozygous for alleles encoding ARQ at codons 136, 154 and 171, and the effect of other polymorphisms (nonsynonymous changes at codon 141 or 241 for instance, on the ARQ backbone in donor sheep) has not been determined. Multiple electrophoretic patterns arising from a single ovine homogenate occur as individual or mixed profiles, even when this variation was not noted in the original ovine sample
]. In our study, the electrophoretic profiles from all murine samples showed only a fast migrating unglycosylated band corresponding to a reduction of ~1.5 to 2
kDa in apparent molecular mass when compared to the ruminant derived samples. The shift in PK cleavage site was confirmed by marked reduction of reactivity by mAb P4, which recognizes an epitope at ovine residues 93–99. Western blot analysis with antibody SAF84 showed no evidence of a 14
kDa band, ruling out a strain CH1641 pattern
]. The pattern of prolonged incubation associated with reduced apparent molecular weight product in Western blot is consistent with ARQ isolates characterized by Thackray et al.
], Beck et al.
], and Tixador et al.
]. Our observation was not based on an inherent characteristic of the Tg338 mouse line or our methods, as samples from mice infected with VRQ ovine tissues migrate at ~21
kDa (data not shown).
PET blots, glycoform analysis, and lesion profiles of the Tg338 samples derived from caprine brain inoculation were remarkably consistent. The marked uniformity in survival time following secondary passage and the consistent patterns in PET blot, glycoform analysis and electrophoretic profile suggest that the caprine isolates, derived from separate herds, represent a single strain with characteristics of a predominant US ovine scrapie strain. This finding is not surprising; scrapie appears to have been introduced into the US in sheep and is observed almost exclusively in ARQ homozygous sheep. Subsequent spread to goats is reported only rarely and there may be a limited number of caprine scrapie strains in the population. The US will expand caprine scrapie surveillance over the next 3
years as ovine scrapie nears eradication; as additional samples from goats are identified, monitoring of caprine scrapie strains in the US using biochemical methods and bioassay in Tg338 mice should be useful in identifying any additional strains.