Selection of distinct strain phenotypes in mice infected by ovine natural scrapie isolates similar to CH1641 experimental scrapie

A few cases of transmissible spongiform encephalopathies in sheep have been described in France in which the protease-resistant prion protein (PrP(res)) exhibited some features in Western blot of experimental bovine spongiform encephalopathy in sheep. Their molecular characteristics were indistinguishable from those produced in the CH1641 experimental scrapie isolate. Four of these CH1641-like isolates were inoculated intracerebrally into wild-type C57Bl/6 mice. In striking contrast to previous results in ovine transgenic mice, CH1641 transmission in wild-type mice was efficient. Several components of the strain signature, that is, PrP(res) profile, brain distribution, and morphology of the deposits of the disease-associated prion protein, had some similarities with "classical" scrapie and clearly differed from both bovine spongiform encephalopathy in sheep and CH1641 transmission in ovine transgenic mice. These results on CH1641-like isolates in wild-type mice may be consistent with the presence in these isolates of mixed conformers with different abilities to propagate and mediate specific disease phenotypes in different species.     

Transmission of chronic spongiform encephalopathy with kuru plaques from humans to small rodents.

An unusual case of spongiform encephalopathy was transmitted directly from a human to rats and mice. After serial passages, incubation periods were shortened to about six months in rats and four months in mice. Clinical symptoms were similar in rats and mice, including ruffled fur, arched back, bradykinesia, and hind limb paralysis. Pathologically, a spongy state, proliferation of astrocytes, and neuronal changes were observed. Electron microscopic observation of the parietal cortex of rats and mice disclosed many membrane-bound vacuoles in the neuropil, predominantly in dendrites. In the pons of mice, intramyelinic vacuoles and accumulation of extracellular fluid were prominent. The clinicopathological symptoms of the affected animals resembled those in other experimental spongiform encephalopathies, especially scrapie.     

Genetic control of amyloid plaque production and incubation period in scrapie-infected mice

The extent of amyloid plaque production was investigated in three inbred mouse strains carrying the p7 allele of the scrapie incubation (Sinc) gene (VM, IM and MB). With either ME7 or 87V scrapie, many more plaques were seen in the MB strain than in VM or IM mice. A backcrossing experiment using 87V suggested the involvement of more than one gene. Within this backcrossing experiment there was a positive correlation between mean plaque count and mean incubation period for the various strains and crosses. Also male mice tended to have higher plaque counts and longer incubation periods than female mice of the same genotype. These results suggest that some of the genes controlling minor variation in the incubation period also influence plaque production. This is consistent with previous evidence that the number of amyloid plaques depends, to some extent, on the duration of agent replication within the brain. This study has also identified a high plaque model (MB mice infected with 87V) for future investigation of the nature of the amyloid protein.     

Transmission of prion strains in a transgenic mouse model overexpressing human A53T mutated α-synuclein

There is a growing interest in the potential roles of misfolded protein interactions in neurodegeneration. To investigate this issue, we inoculated 3 prion strains intracerebrally into transgenic (TgM83) mice that overexpress human A53T α-synuclein. In comparison to nontransgenic controls, there was a striking decrease in the incubation periods of scrapie, classic and H-type bovine spongiform encephalopathies(C-BSE and H-BSE), with conservation of the histopathologic and biochemical features characterizing these 3 prion strains. TgM83 mice died of scrapie or C-BSE prion diseases before accumulating the insoluble and phosphorylated forms of α-synuclein specific to late stages of synucleinopathy. In contrast, the median incubation time for TgM83 mice inoculated with H-BSE was comparable to that observed when these mice were uninfected, thereby allowing the development of molecular alterations of α-synuclein. The last 4 mice of this cohort exhibited early accumulations of H-BSE prion protein along with α-synuclein pathology. The results indicate that a prion disease was triggered concomitantly with an overt synucleinopathy in some transgenic mice overexpressing human A53T α-synuclein after intracerebral inoculation with an H-BSE prion strain.     

Experimental transmission of human subacute spongiform encephalopathy to small rodents

Summary Experimental transmission of subacute spongiform encephalopathy from three human cases to small rodents is reported. The first case with atypical CJD with spongiform change, kuru plaques, and leukomalacia was transmitted directly to mice, rats, and guinea pigs and indirectly to hamsters and Mongolian gerbils through rats. From two other typical SSE cases the disease was also successfully transmitted; from the second case to mice and rats, and from the third case to guinea pigs. Brain showed the highest infectivity; the spleen, liver, blood, and cerebrospinal fluid of diseased animals were also infective. Intracerebral inoculation was the route for the fastest transmission, followed by intrathecal, intraperitoneal, submucosal, and subcutaneous routes. The incubation periods and clinical features were characteristic in each inoculated species and did not vary within several passages, except for the shortening of incubation period from the first to the second passage. Histologically, a marked spongy state and proliferation of astrocytes were observed in all diseased animals, though the distribution of the lesion was peculiar to each species. The severe lesion in the white matter in mice was similar to that seen in mice inoculated with scrapie and also to that seen in the first case.     

Vacuolization, incubation period and survival time analyses in three mouse genotypes injected stereotactically in three brain regions with the 22L scrapie strain

In previous studies we showed that C57BL mice injected stereotactically in the cerebellum with the 22L scrapie strain had a significantly shorter incubation period than those injected with the same agent in other brain regions. In mice injected in the cerebellum, vacuolization was limited to the cerebellum, medulla and mesencephalon, whereas injection into forebrain regions resulted in vacuolization in all brain regions. The studies suggested that the cerebellum had a selective vulnerability for 22L. In this study we examined the interaction between host genotype and selective vulnerability of specific brain regions. The mouse gene that has the most profound effect on pathogenesis, particularly incubation period, is termed Sinc (scrapie incubation). Groups of mice with three genotypes of Sinc (s7s7, p7p7 and their F1 cross, s7p7) were injected with 22L into the cerebral cortex, thalamus or cerebellum. Analysis of incubation periods showed that, regardless of the host genotype, the cerebellum injection group had a significantly shorter incubation period than groups injected in other regions. After cerebellum injection vacuolization was limited to the cerebellum, medulla and mesencephalon in all three host genotypes. The location of vacuoles within the cerebellum differed depending upon the host strain. Vacuolization developed almost exclusively in grey matter in s7s7 mice, mainly in white matter in p7p7 mice, and in both grey and white matter in F1 mice. These results demonstrate that the selective vulnerability of the cerebellum to induction of clinical disease by 22L does not depend on host genotype, but host genotype does affect lesion distribution within the cerebellum.     

Experimental scrapie in mice: ultrastructural observations

Scrapie, kuru, and Creutzfeldt-Jakob disease are characterized by a similar spongiform pathology, prolonged incubation periods, and an agent with unique physical, chemical, and biological properties. Swiss mice were inoculated with the scrapie agent and sacrificed three to five months later for light and electron microscopy. At three months, small vacuoles were seen within the neuropil of the cerebral cortex and basal ganglia. By the fifth month these vacuoles had increased in number and size and were accompanied by moderate astrocytic proliferation. The brainstem, cerebellum, and spinal cord showed variable changes of much less intensity. Many dilated postsynaptic processes contained osmiophilic particles in random or crystalline arrays. The particles, measuring approximately 23 nm in diameter, appeared consistently in postsynaptic processes of brain from scrapie-infected mice, were lacking in controls, and were a size consistent with sedimentation and filtration data for the scrapie agent. Whether these particles represent the scrapie agent must await further studies.     

Comparison of spongiform lesions in experimental scrapie and rabies in skunks

Summary Striped skunks were inoculated intracerebrally with the scrapie agent (suspension of brain from a naturally infected Suffolk sheep) or intramuscularly with street rabies virus (suspension of salivary glands from naturally infected skunks). Those given the scrapie agent developed clinical signs of weakness, posterior ataxia, and emaciation after incubation periods of 8 to 23 months. Those inoculated with rabies virus developed clinical signs of rabies (aggressive behavior, hyperexcitability, ataxia and paralysis) after incubation periods of 20 to 62 days. The gross lesions in the brains of the skunks given the scrapie agent consisted of marked atrophy of the thalamus and moderate atrophy of the cerebrum. No gross lesions occurred in the rabid skunks. Histologically, the type of spongiform lesion in rabies was the same as that in scrapie. However, spongiform change of rabies infected brains was less extensive (only rarely affected the basal ganglia, hippocampus or hypothalamus) than that of brains infected with the scrapie agent and was characterized by fewer numbers of small vacuoles (as a proportion of total number of vacuoles) than occurred in scrapie spongiform change.     

Early behavioural changes in mice infected with BSE and scrapie: automated home cage monitoring reveals prion strain differences

Mice inoculated with transmissible spongiform encephalopathies (TSE) show behavioural abnormalities well before the appearance of clinical signs. TSE strains are obtained by serial re-infection of infectious brain homogenates in laboratory rodents. They are characterized by strain-typical brain lesion profiles, which implies that they might be differentiated behaviourally as well. Seventy female C57BL/6 mice were tested, 14 per group. Controls received no or sham inocula, two other groups received scrapie strains adapted to mice (139A, ME7) and one group a mouse-adapted BSE strain (301C). From week 7 until the end of the incubation period, 8 mice per group were subjected once every 2 weeks to open-field and hot-plate tests. Assessment of clinical signs, and measuring of body weight, food and water consumption were carried out weekly on the remaining animals kept in single cages. In addition, locomotor activity was recorded continuously in these mice by means of infrared detectors. Monitoring of circadian activity revealed early significant TSE strain differences, most pronounced during the nocturnal active phase. Behavioural changes in open-field tests also occurred before the appearance of clinical signs, and differences in rearing, wall rearing and sniffing were strain-specific, however, such differences varied according to the period of testing. Hind paw lick latencies increased equally in all groups after week 19, jump latencies also increased in the two scrapie groups but not in the BSE group. It was at this time that clinical signs first appeared consisting of ataxia, lack of balance, motor dyscoordination, and lordosis. These data imply that automated assessment of circadian activity in mice is a powerful and economical tool for early behavioural typing of TSE strains.     

Dendritic and synaptic alterations of hippocampal pyramidal neurones in scrapie-infected mice

Neurone damage and eventual loss may underlie the clinical signs of disease in the transmissible spongiform encephalopathies (TSEs). Although neurone death appears to be through apoptosis, the trigger for this form of cell death in the TSEs is not known. Using two different murine scrapie models, hippocampal pyramidal cells were studied through microinjection of fluorescent dye, and synaptic integrity, using p38-immunoreactivity (p38-IR), both visualized using confocal laser scanning microscopy. Intradendritic distensions and dendritic spine loss were found to co-localize to areas of vacuolar and prion protein pathology in the hippocampus of mice infected with ME7 or 87 V scrapie. A significant reduction in p38-IR was found concomitantly in the hippocampus in ME7 scrapie mice. These results indicate that both pre- and post-synaptic sites are altered by scrapie infection; this would disrupt neuronal circuitry and may initiate apoptotic cell death, giving rise to the neurological disturbances manifested in clinical TSE cases.     

In vivo toxicity of prion protein in murine scrapie: ultrastructural and immunogold studies

Prion protein (PrP) is a cell surface, host coded, sialoglycoprotein which accumulates in excess in scrapie, Creutzfeldt‐Jakob disease, bovine spongiform encephalopathy and other transmissible spongiform encephalopathies. Infection of mice with the 87 V or ME7 scrapie strains results in distinctive and very different light microscopical patterns of vacuolation and disease specific PrP accumulation. In both of these scrapie strains immunogold electron microscopy was used to locate PrP to the plasmalemma of neurons from where it was released into the neuropil. Initial PrP accumulation around neurons and in early plaques lacking amyloid fibrils was generally not associated with morphological changes either of the neuron or dendrite releasing the PrP or in the adjacent neuropil in which excess PrP accumulated. However, accumulation of pre‐amyloid PrP in some brain areas was associated with specific degeneration of dendritic spines and axon terminals. Initial PrP aggregation into fibrils was also associated with tissue damage with both ME7 and 87 V plaques and diffuse accumulations. Tissue damage associated with fibrillogenesis was localized and would not be expected to have clinical significance. We conclude that pre‐amyloid PrP release and accumulation is not invariably toxic, either to the neuron releasing PrP or to the neuropil into which it is released. However, axon terminal degeneration and dendritic spine loss in some neuroanatomical areas may be indicative of specific PrP toxicity and may be the main cause of neurological dysfunction in murine scrapie.     

Early unsuspected neuron and axon terminal loss in scra pie-infected mice revealed by morphometry and immunocytochemistry

Neuronal loss is often quoted as an element of the pathology of the transmissible spongiform encephalo-pathies, but few data are published. To determine whether neuronal loss is a salient feature of murine scrapie, and whether there is a relationship with the other hallmark lesions of scrapie we compared the numbers of neurons, severity of vacuolation, axonal bouton density and distribution of prion protein (PrP) in the dorsal lateral geniculate nucleus (dLGN) following intraocular infection of C57BL/FaBtDk mice with ME 7 scrapie. This route of infection limits the initial spread of infection to the retinal efferents, thus directing infectivity and subsequent pathological changes to the dLGN which is a major projection of the optic nerve. Morphometric assessment of neuron number in the dLGN was made on semi-serial sections from five infected and five normal brain injected controls at four 50-day intervals during the incubation period, and on terminally affected mice. The number of neurons decreased from around 20 000 at 50 days to under 1000 in the terminal group. Significant loss was identified in individual mice at 150 days post-infection, coincident with the onset of vacuolation: neuron number was found to have an inverse relationship to the severity of vacuolation. Axonal boutons in the dLGN (demonstrated by synaptophysin immunolabelling) were reduced at 200 days, and virtually absent in terminal mice. The intensity of PrP immunostaining progressively increased from 150 days, and in a separate experiment PrP was detected from 175 days by polyacrylamide gel electrophoresis of brain extracts. These results show that early neuronal loss is a significant feature of experimental scrapie infection, and the possible mechanisms of this degeneration are discussed.     

Differentiation of prion protein glycoforms from naturally occurring sheep scrapie,sheep-passaged scrapie strains (CH1641 and SSBP1), bovine spongiform encephalopathy (BSE) cases and Romney and Cheviot breed sheep experimentally inoculated with BSE using two monoclonal antibodies

A panel of ruminant brain tissues were subjected to a Western immunoblotting technique using two monoclonal antibodies (mAbs). The resultant prion protein (PrP) glycoforms showed that three distinctions can be made between natural ovine scrapie cases and sheep experimentally inoculated with bovine spongiform encephalopathy (BSE). Differentiation between BSE-infected cattle and natural cases of sheep scrapie was also possible using these two antibodies. There were subtle differences in the molecular weight positions of the di-glycosylated, mono-glycosylated and unglycosylated forms of the abnormal PrP (PrP(Sc)) associated with these ruminant transmissible spongiform encephalopathies. In particular, a distinct difference for the unglycosylated protein band was observed. For ovine scrapie samples, this band was noticeably of a higher molecular weight than that found for brain samples from the Romney and Cheviot breed sheep infected with BSE and, to a lesser degree, higher than that observed for bovine BSE samples. Using the comparison of glycoform ratios, the technique provided a distinction between the sheep experimentally infected with BSE and natural cases of sheep scrapie but did not provide a distinction between natural cases of bovine BSE and ovine scrapie. The sheep-passaged CH1641 scrapie strain gave molecular weights similar to, but not identical to BSE, and a glycoform ratio similar to ovine scrapie cases. The SSBP1 experimental scrapie strain gave molecular weights that were akin to natural scrapie cases but the glycoform ratio was different to that found for all the other samples. When mAb P4 was substituted for mAb 6H4 in the technique, only the natural scrapie samples and SSBP1 gave strong signals. BSE in sheep and the CH1641 strain gave weak reactions and PrP(Sc) from BSE-infected cattle could not be detected at all. The results suggest that this combination of molecular weight and glycoform ratio analyses, and differentiation with two specific antibodies could be used to provide a possible screening test for BSE in the UK sheep flock, if confirmed as accurate by bioassay and lesion profile analysis in mice inoculated with brain tissue from suspect field cases.     

Creutzfeldt-Jakob disease

The historical aspects of spongiform encephalopathies, Creutzfeldt-Jakob disease (CJD) and kuru of man, as well as scrapie and transmissible mink encephalopathy, are outlined. Transmissions of these diseases to animal hosts are presented, with emphasis on CJD transmissions to guinea pigs, hamsters, and mice. The relationship of CJD to scrapie with reference to the pathological findings is discussed. In CJD the incubation period is cut in half in guinea pigs and hamsters in the second passage. The spongiform changes occurring in the neuropil are reviewed. These changes are related to the type of inoculum, e.g., there is more vacuolization after inoculation with brain, and less after inoculation with spleen. Spongiform changes are also dependent upon the route of inoculation; these are more severe in intracerebral inoculation compared to intraperitoneal inoculation. Viremia is present. Maternal transmission and lateral transmission are absent. No virus-like particles are detected, and no other organisms are visible by electron microscopy. Isolations of the causative agent and strains of the agent in spongiform encephalopathies remain elusive. The hypotheses concerning the nature of the agent are critically reviewed. Novel data on the production of tumors derived from CJD brains are presented. Tissue culture cells arising from such brains become permanent lines and are similar to neoplastic lines. When such CJD lines are injected subcutaneously into nude mice, malignant neoplasms are formed. No evidence of an infectious etiology in Alzheimer's disease exists. Reported similarities between this disease and CJD are reviewed. Animal models of CJD are useful for the investigation of dementias.     

The sequential development of spongiform change and gliosis of scrapie in the golden Syrian hamster

The lesion profiles of spongiform change and gliosis in the hamster occurring after intracerebral (IC) inoculation of scrapie virus, are calculated and compared to the lesion profile of spongiform change of scrapie in mice and of scrapie and Creutzfeldt-Jakob disease (CJD) in the squirrel monkey. The profile of scrapie in hamsters differs considerably from that of a closely related strain of scrapie in mice, and both differ from scrapie and CJD in the squirrel monkey. These differences emphasize the effect of the host on the distribution of pathological changes in these unconventional virus infections. The sequential development of the lesions in the hamster shows that the earliest changes are detectable before the onset of clinical disease 49-57 days after inoculation, as assessed by light microscopy. Gliosis is detectable by indirect immunofluorescence 35-39 days after inoculation by use of a monoclonal antibody directed against astrocytes.     

Immunological studies of scrapie infection

Scrapie is a show, degenerative nervous system disease of sheep and goats. No obvious inflammatory or immunological response occurs despite early replication of the srapie agent in the spleen. The extend earlier investigations (Garlin et al. 1978a, b) on the immunorcactivity of lymphocytes during scrapie infection, purified populations of T and B cells were prepared from BALB/c mice. No consistent differences in the response to mitogens of T and B cells from scrapie-infected and control mice were observed. An assessment of T-dependent B cell function was obtained using a hemolytic plaque-forming cell assay, but again no differences between scrapie-infected and control splenocytes could be detected. To study the possible influence of B cell maturation on scrapie infection, incubation periods were measured in CBA/N mice, bearing an X-linked deficiency of mature B cells. Scrapie incubation periods were identical in affected males and unaffected females, indicating that B cell maturation does not influence the course of scrapie. Using highly enriched preparations of the scrapie agent, we attempted to raise antibodies in rabbits using an extensive immunization protocol. Scrapie-specific antibodies could not be detected using either indirect immunofluorescence or enzyme-linked immunosorbent assay (ELISA). Some antisera reacted more strongly with scrapie than with control preparations; however, these differences disappeared when the antisera were     

Marked influence of the route of infection on prion strain apparent phenotype in a scrapie transgenic mouse model

Prion strains yield specific neuropathological features including spongiform degeneration and deposition patterns of pathological prion protein. Their invariant regional distribution, following variations in the infection route, has led to the proposal that prions replicate preferentially in defined neuro-anatomical areas. The molecular mechanisms underlying this apparent strain-specific neuronal tropism are currently unknown. However, a possible explanation may be that prion replication is relatively innocuous, resulting in long-term propagation, thus masking initial regional distribution variations linked to different infection routes. This “low neurotoxicity” may be imputable either to the rodent model used or the prion strain(s) inoculated. To investigate this possibility, we studied prion pathogenesis in a prototypal short-incubation disease model consisting of 127S scrapie strain propagated in tg338 transgenic mice expressing the VRQ allele of ovine PrP. This prion strain derives from a natural sheep scrapie isolate that was serially transmitted to tg338 mice without any obvious transmission barrier and biologically cloned by limiting dilution. We compared the pathology induced by the peripheral or intracerebral inoculation of 127S strain. Surprisingly, we found that the disease greatly differed in clinical signs, abnormal prion protein levels, and neuropathology among the routes of infection. Secondary transmission performed with brain material from mice inoculated either intracranially or intraperitoneally produced similar neuropathological features. These results therefore indicate that the route of infection can strongly influence the apparent phenotype of a scrapie strain.     

TRANSMISSIBLE MINK ENCEPHALOPATHY (TME) IN CHINESE HAMSTERS: IDENTIFICATION OF TWO STRAINS OF TME AND COMPARISONS WITH SCRAPIE

TME from a single source was transmitted by intracerebral injection to Chinese hamsters, producing clinical disease in all seven animals after incubation periods of over 600 days. The brain from each of the primary cases was used to establish separate intracerebral passage-lines of TME and this led to the isolation of two different strains of agent, designated 333K and 333W. These strains were easily distinguished by the incubation periods they produced (about 130 and 230 days, respectively) under standard conditions of infection, and by the characteristic profiles of vacuolation seen in different regions of the brain. Comparisons were made with a strain of scrapie passaged in Chinese hamsters, designated 34W, which could be distinguished from both strains of TME. Nevertheless the properties of the scrapie and TME strains overlapped, with one of the TME strains (333K) resembling the 34W strain of scrapie in Chinese hamsters more closely than the other TME strain (333W). These similarities strengthen the view that TME and scrapie are caused by a similar type of infectious agent. The very large 'species barrier effect' on transmitting TME to Chinese hamsters was in marked contrast to the minimal effect seen with scrapie and an explanation for this is suggested. Two interesting pathological features of the study were (a) the severe loss of pyramidal cells produced in the hippocampus by the 34W strain of scrapie, and (b) the focal, symmetrical vacuolation of the thalamus caused by 333K TME.     

CEREBRAL AMYLOIDOSIS IN SCRAPIE IN THE MOUSE: EFFECT OF AGENT STRAIN AND MOUSE GENOTYPE

Cerebral amyloid deposits, predominantly in the form of discrete plaques are common in mice infected with certain scrapie isolates. In this paper the incidences of cerebral amyloidois occurring with different combinations of agent strain and mouse genotype are investigated. Seven different agents from four primary sources were studied in three mouse genotypes (C57BL, VM and their F₁ cross). It is shown that the degree of amyloidosis is a passageable characteristic of the agent depending also on host genotype. However, the amyloid incidence is not influenced by the agent and mouse genotype independently but depends on a specific interaction between the two. For two scrapie agents, 22A and ME7 there is a correlation between incubation period and the incidence of cerebral amyloid in the three mouse genotypes. The degree of cerebral amyloid for ME7 remains unaffected by passage through the two genotypes of mouse, C57BL and VM, and through another species, a Cheviot sheep.     

EVIDENCE THAT TRANSMISSIBLE MINK ENCEPHALOPATHY AGENT IS BIOLOGICALLY INACTIVE IN MICE

Transmissible mink encephalopathy (TME) is probably a form of the sheep disease, scrapie, introduced by accidentally feeding mink with scrapie-infected sheep tissues. Although no successful transmissions of TME to mice have been achieved previous work has involved various limitations. To maximize the possibility of transmission, 176 mice, representing 14 different genotypes mostly not previously tested with TME, were injected with TME-infected mink brain from three sources with different histories. No scrapie-like disease was detected clinically or histologically in these mice or in a further 111 which were subsequently injected with brain or spleen material from 10 of the TME-injected mice killed when senile. Furthermore, a series of experiments involving seven strains of scrapie, demonstrated that prior injection of mice with TME failed to affect the normal progress of scrapie infection indicating that TME agent had not occupied scrapie replication sites or otherwise influenced the pathogenesis of scrapie. The overall conclusion from these experiments is that TME is biologically inactive in mice. Although many strains of natural scrapie can be transmitted to laboratory mice, this has not been possible with all strains and it is concluded that one or more of such strains is likely to be the cause of TME in mink.