Resistance of neonatal mice to scrapie is associated with inefficient infection of the immature spleen

Abstract

Previous studies demonstrated that neonatal mice up to about a week old are less susceptible than adult mice to infection by intraperitoneal inoculation with mouse-passaged scrapie. In peripherally inoculated adult mice, scrapie replicates in lymphoid tissues such as the spleen before invading the central nervous system. Here, we investigated scrapie susceptibility in neonatal mice in more detail, concentrating on spleen involvement. First, we demonstrated that neonatal mice are about 10 times less susceptible than adults to intraperitoneal scrapie inoculation. Then we injected mice intraperitoneally with a scrapie dose that produced disease in all mice inoculated at 10 days or older but in only about a third of neonatally inoculated mice. In this experiment, spleens collected 70 days after scrapie injection of mice 10 days old or older almost all contained pathological prion protein, PrPSc, and those that were bioassayed all contained high infectivity levels. In contrast, at this early stage, only two of six spleens from neonatally inoculated mice had detectable, low infectivity levels; no PrPSc was detected, even in the two spleens. Therefore, neonatal mice have an impaired ability to replicate scrapie in their spleens, suggesting that replication sites are absent or sparse at birth but mature within 10 days. The increase in susceptibility with age correlated with the first immunocytochemical detection of the normal cellular form of prion protein, PrPc, on maturing follicular dendritic cell networks. As lymphoid tissues are more mature at birth in sheep, cattle, and humans than in mice, our results suggest that in utero infection with scrapie-like agents is theoretically possible in these species.

FIG. 1.

Incubation period of disease or survival time following i.p. inoculation of adult mice and neonatal mice with 10-fold dilutions of ME7 scrapie-infected brain homogenate. Shaded symbols represent individual terminally affected mice with a histolopathologically confirmed scrapie diagnosis. Unshaded symbols represent mice with a negative clinical and histopathological diagnosis that survived beyond the last clinical case in their challenge age group.

FIG. 2.

Incubation period of disease following i.p. inoculation of 0- to 1-, 10-, 14-, and 29- to 46-day-old mice with a 10⁻² dilution of ME7 scrapie-infected brain homogenate. Shaded symbols represent individual terminally affected mice with a histopathologically confirmed scrapie diagnosis. The numbers of mice surviving to beyond 550 days with a negative histopathological scrapie diagnosis are indicated.

FIG. 3.

Immunoblot analysis of spleen tissue taken from mice inoculated i.p. with ME7 scrapie at 0 to 1, 10, or 14 days old (d.o.). Samples were treated in the presence (+) or the absence (−) of proteinase K (PK) prior to electrophoresis. (a) At 70 d.p.i, no PK resistant accumulations of PrP^Sc were detected in the spleens of mice inoculated neonatally, but, at this stage, PrP^Sc could be detected in the spleens of mice injected at 10 and 14 days old (also at 29 days old [not shown]). (b) PrP^Sc was detected in the spleens of clinically affected mice inoculated as neonates (left hand panel). PrP^Sc was undetectable in the spleens of neonatally inoculated, clinically negative mice, except in one which had readily detectable PK-resistant accumulations at 592 d.p.i. (right hand panel). All gels included spleen extracts from a clinically affected mouse inoculated as an adult as a positive control (+con). Lane M contains molecular size markers.

FIG. 4.

PrP^c detection by immunofluorescent labeling in the spleens of uninfected mice at 8, 10, 14, and 30 days old (do). Labeling was first seen in a proportion of mice at 10 days, associated with some developing follicles (arrowheads), the spleen capsule (C), and trabeculae (T).

FIG. 5.

Double immunofluorescent labeling using PrP- (green) and FDC-M1-, CR1-, or FDC-M2 (red)-specific antibodies in the spleens of 14- and 30-day-old mice. Colocalization of PrP and FDC-M1, CR1, or FDC-M2 (yellow) is seen on FDC networks in developing follicles (arrowheads).