Does sheep scrapie infect the embryo?
Neuro-immune connection in spread of prions in the body?
Other 'repeat; diseases: counting DNA triplet repeats
TSE in Chickens: what to expect?
14 Mar 1997 Reed Holyoak A paper by Foster et al., Vet. Rec. 138:559-562, 1996 seems to suggest this. But a little history on the subject is needed.
Does sheep scrapie infect the embryo before its reaches the uterus?
Back almost 20 years ago two separate studies were initiated to look at the role of embryo transfer in scrapie transmission. The first study started in the United States, was designed to wash the embryos in an attempt to block transmission, and the second study started several years later in Scotland, was designed as a worst case scenario to detect any possibility of transmission via embryo transfer. At the end of these studies when the results were reported there was disagreement as to whether scrapie in sheep could be transmitted through embryo transfer.
Looking closer at these studies, in 1980, Warren Foote and colleagues studied reciprocal embryo transfers between scrapie-inoculated Cheviot and Suffolk ewes, and scrapie-free western white-face (Targhee) range ewes. They transferred embryos from experimentally infected donors, inoculated with strain A scrapie (SSBP-1), to scrapie-free recipients, and transferred embryos from scrapie-free donors to scrapie-inoculated recipients, with the lambs taken by Cesarean section.
Offspring from scrapie-free parents (negative) and scrapie-inoculated parents (positive) served as controls. The embryos were surgically recovered, washed three times and surgically transferred. All animals were observed for clinical signs of scrapie for 60 months or until death. None of the offspring 24 months of age or older from any of the test groups or from the negative control group developed scrapie. Of the positive control group, 9.5% developed scrapie. Thus, scrapie did not appear to be disseminated via embryo transfer. However, the infection of only two positive control animals raised questions about the infectivity and/or of the inoculum.
Because of the question of infectivity and because we (reseachers at Utah State University) didnít, and still donít, know the effect of experimental infection on the natural pathogenesis of scrapie, a second study was started. In this study we used a naturally infected flock of Suffolk sheep as embryo donors. With repeated embryo collections and transfers into scrapie-free recipients of Targhee breeding. All embryos were washed 3 times in dilution volumes of medium greater than 100, as before. All viable embryos, including those with damaged zona pellucidae, and those hatching, were transferred. Donor animals were kept at the USDA,APHIS,VS facilities at Mission, Texas, and embryo transfer offspring and their recipients (surrogate mothers) were/are kept in facilities at Utah State University, Logan, Utah.
To date none of the offspring who have reached 60 months of age have been scrapie positive on any tests. None of the remaining live offspring are showing clinical signs of scrapie. Also to date, none of the recipient ewes have developed clinical scrapie and none of those that have died have been positive on histopathologic analysis. The live animal portion of this second study is slated to end August 31, 1998. Neither the first (Foote et al. 1993) nor the second (**please remebr these results are preliminary**) of the US studies detected transmission of scrapie associated with embryo transfer.
Foster et al. (1992) starting in 1988, experimentally infected embryo donor Cheviot ewes carrying the short incubation allele (Sip sAsA or sApA) with scrapie by subcutaneous injections of a supernatant from homogenized scrapie-infected brain, also strain A scrapie (SSBP-1). Long-incubation period (Sip pApA) Cheviot ewes were the recipients. Embryos were surgically recovered and transferred, without washing by laparoscopic techniques into the recipient ewes (as worst case). All six donor ewes inoculated with strain A scrapie, developed clinical scrapie. Also 6 of 20 of their embryo transfer offspring, all of which were homozygous for short-incubation (Sip sAsA) developed scrapie.
Scrapie apparently passed from the dams to their offspring via the preimplantation embryo, although it was not determined whether the cumulus cell mass was infected or whether infection occurred during passage through the oviduct and uterine horn. Route of infection of the donors, unwashed embryos, possible subclinically infected recipients, and contaminated facilities may also have been factors. There were doubts expressed about whether maternal transmission of infection had actually occurred. Others expressed feelings that the disease had arisen de novo in the highly susceptible genotypes. Still others suggested that scrapie transmission may have occurred through environmental contamination to the offspring while neonates, or through subclinically infected recipients to the offspring in utero. These questions were acknowledged by the authors.
To answer these questions and others, this group produced the more recent report of the second embryo transfer project (Foster et al. 1996) that Roland referred to. In this study both their test and control groups of embryo transfer offspring had animals die of scrapie infection. Scrapie occurred in offspring resulting from both washed (test) and unwashed (control) embryos. Also the rams used to sire these offspring died of scrapie. Rather than providing clear answers, the study could not differentiate disease transmission by contaminated premises, from subclinically infected recipient ewes, nor from infected semen. They stated in the article that they could not rule out the possibility of the progeny from the embryo transfers being infected at or around the time of lambing.
It appears probable that the most likely cause of scrapie infection. was due to environmental contamination rather than the SSBP-1 inoculations. I say this because the clinical signs and the histological brain lesions in most of the cases bore close resemblance to those of the natural type of scrapie endemic in the Cheviot flock used in the study and not the distinctive changes normally associated with the SSBP-1 inoculum.
NOW to sum up these studies and to put things in perspective, despite the considerable research done in both the US and Scotland, the question of whether scrapie may be transmitted by embryos REMAINS UNANSWERED.
Perhaps we are dealing with strain and/or gentic differences, or it may be something else. One thing I do know. I know almost all of the people involved in all of these studies. They are good people and solid respected researchers. These are just tough diseases, with very few easy answers!
References:Foote W.C., Clark W., Maciulis A., Call J.W., Hourrigan J., Evans R.C., Marshall M.R. and M. de Camp. 1993. Prevention of scrapie transmission in sheep using embryo transfer. AJVR, 54:1863-1868. Foster J.D., McKelvey W.A.C., Mylne M.J.A., Williams A., Hunter N., Hope J. and H. Fraser. 1992. Studies on maternal transmission of scrapie in sheep by embryo transfer. Vet. Rec. 130:341-343. Foster J.D., Hunter N., Williams A., Milne M.J.A., McKelvey W.A.C., Hope J., Fraser H., and C. Bostock. 1996. Observations on the transmission of scrapie in experiments using embryo transfer. Vet. Rec. 138:559-562.
Other perspectives on sheep embryos and scrapie15 Mar 1997 Roland Heynkes
There was no reason to reference the article of Foote et al. because it was totally inconclusive. A few healthy lambs from scrapie inoculated donor or recipient mothers do not prove anything. Especially not if more than one third of the inoculated ewes and 18 of 20 positive controls did not develope scrapie. Obviously their inoculation method was not very effective and this may be the reason for their lack of success. In view of the fact that only 2 to 20 positive controles developed scrapie, the numbers of embryo transfers were to small and only 19 of 25 lambs from inoculated recipient ewes reached the end of the experiment after 60 months.
If the study of Foote et al. was designed to demonstrate a protective effect of washing the embryos before the transfer, why didn't they transfer some embryos without washing? Could scrapie have arisen de novo in 6 of 20 of the embryo transfer offspring?
Adriano Aguzzi Department of Pathology, University Hospital, Zurich CH-8091, Switzerland
Neuro-immune connection in spread of prions in the body?
Lancet Volume 349, Number 9054 - Saturday 15 March 1997
The enigma surrounding the nature of the infectious agent causing transmissible spongiform encephalopathies (the "prion") captivates the attention of scientists and media alike. Do prions really consist only of PrPSc (an abnormally folded cellular protein1), or do they contain additional, undiscovered components?2 For all its fascination, this controversy threatens to obscure the many other questions about prion diseases that demand urgent answers, especially since the occurrence of "new variant" Creutzfeldt-Jakob disease (nvCJD), which is thought to result from the ingestion of bovine spongiform encephalopathy prions.
Fortunately, some of these questions can be addressed before certainty has been attained about the physical make-up of prions. For example, the remarkable ability of this agent to invade the nervous system deserves some close attention. What happens, precisely, when prions gain access to the gastrointestinal tract? Which pathways are exploited by prions to spread across the body and reach the central nervous system (CNS)? Are there "reservoirs" in the body where prions multiply silently during the incubation phase of the disease? Answering these questions may help in devising ways to interfere with the march of prions from peripheral sites to CNS.
Hill and colleagues3 have provided striking evidence for invasion of the human immune system by nvCJD prions. "Type 4"-PrPSc, which is the hallmark of the new disease,4 accumulates in the lymphoid tissue of tonsil in such large amounts that it can easily be detected with antibodies on histological sections. Besides providing a useful diagnostic tool, this discovery highlights the fact that nvCJD affects more systems of the body than only the CNS.
That prions can "go immune" is not new. A wealth of early studies points to the importance of prion replication in lymphoid organs.5 In mice, infectivity can be demonstrated in the spleen as early as 4 days after both intraperitoneal and, surprisingly, intracerebral infection. Replication of the infectious agent in the spleen precedes intracerebral replication, even if the infection is introduced intracerebrally. Infectivity can accumulate in all components of the lymphorecticular system, including lymph nodes and intestinal Peyer's patches, where it replicates almost immediately following oral administration of prions.
The nature of the cells supporting prion replication within the lymphoreticular system is uncertain. Splenectomy experiments after intraperitoneal infection have suggested that the critical cells are long lived. Follicular dendritic cells would be a prime candidate, and indeed PrPSc accumulates in such cells of wild-type and nude mice (which have a selective T-cell defect), but intraperitoneal infection does not lead to replication of prions in the spleen or to cerebral scrapie in mice with severe combined immunodeficiency (SCID) (whose follicular dendritic cells are thought to be functionally impaired).6 Transfer of spleen cells to SCID mice restores ability to infect organs within the peritoneal cavity.7 This finding suggests that cellular requirements for prion replication may be different from those for prion transport: the former may need follicular dendritic cells, whereas the latter may be dependent on lymphocytes.
The findings referred to above suggest that prions "misuse" immune cells to travel from the site of infection to the lymphoreticular system. Do immune cells suffice to transport the agent all the way from lymphoreticular system to the CNS (figure, A)? This is unlikely, since lymphocytes do not normally cross the blood-brain barrier (unless they have a specific reason to do so). Moreover, disease and prion replication occur first in the CNS segments to which the sites of peripheral inoculation relate,8 implying that the agent spreads through the peripheral nervous system, the way the rabies virus and herpesviruses do.
How then might prions spread in the body? Perhaps prions injected intraperitoneally are first brought to follicular dendritic cells by mobile immune cells. Then peripheral nerve endings are invaded in a lymphocyte-dependent fashion. Eventually, the CNS is reached, and further spread occurs transsynaptically and along fibre tracts (figure, B).
Is it possible to interfere with this chain of events without resorting to physical ablation of prion-carrying cells (as in the case of SCID mice)? The normal prion protein, PrPC, may offer an intriguing handle. PrPC is crucial for prion spread within the CNS,9 and it is not unlikely to be required also for spread of prions from peripheral sites to CNS. If the latter suspicion is confirmed, a true opportunity may arise for interference with prion spread and, therefore, for secondary prevention of encephalopathy after exposure to prions. Given the availability of transgenic and knockout mice10,11 for PrPC, answers to these questions should not be far away.1 Prusiner SB. Novel proteinaceous infectious particles cause scrapie. Science 1982; 216: 13644. 2 Lasmezas CI, Deslys JP, Robain O, et al. Transmission of the BSE agent to mice in the absence of detectable abnormal prion protein. Science 1997; 275: 40205. 3 Hill AF, Zeidler M, Ironside J, Collinge J. Diagnosis of new variant Creutzfeldt-Jakob disease by tonsil biopsy. Lancet 1997; 349: 99. 4 Collinge J, Sidle KCL, Meads J, Ironside J, Hill AF. Molecular analysis of prion strain variation and the aetiology of "new variant" CJD. Nature 1996; 383: 68588. 5 Eklund CM, Kennedy RC, Hadlow WJ. Pathogenesis of scrapie virus infection in the mouse. J Infect Dis 1967; 117: 1522. 6 Muramoto T, Kitamoto T, Hoque MZ, Tateishi J, Goto I. Species barrier prevents an abnormal isoform of prion protein from accumulating in follicular dendritic cells of mice with Creutzfeldt-Jakob disease. J Virol 1993; 67: 680810. 7 Lasmezas CI, Cesbron JY, Deslys JP, et al. Immune system-dependent and independent replication of the scrapie agent. J Virol 1996; 70: 129295. 8 Kimberlin RH, Walker CA. Pathogenesis of mouse scrapie: evidence for neural spread of infection to the CNS. J Gen Virol 1980; 51: 18387. 9 Brandner S, Raeber A, Sailer A, et al. Normal host prion protein (PrPC) required for scrapie spread within the central nervous systme. Proc Natl Acad Sci USA 1996; 93: 1314851. 10 Bueler H, Aguzzi A, Sailer A, et al. Mice devoid of PrP are resistant to scrapie. Cell 1003; 73: 1339-47. 11 Fischer M, Rulicke T, Raever A, et al. Prion protein (PrP) with amino-proximal deletions restoring susceptibility of PrP knockout mice to scrapie. Embo J 1996: 15: 1255-64.
Dorothy Bonn ,,, The Lancet Volume 349, Number 9054 - Saturday 15 March 1997
Anticipating the future by counting DNA triplet repeats
Friedreich's ataxia (FA) is an inherited, progressive, fatal disease characterised by spinocerebellar degeneration. The first sign of the disease -- almost invariably ataxia -- appears in childhood or adolescence. "But early clinical diagnosis is difficult, because the full clinical presentation is seen only several years after onset", says Alexis Brice, a neurogeneticist at the Salpêtrière Hospital, Paris, France. "Unfortunately", he adds, "delayed diagnosis hinders genetic counselling", so people with FA may start families without knowing their genetic legacy.
But now the results of molecular studies by Brice and his colleagues in Paris and at the Institut de Génétique et de Biologie Moléculaire et Cellulaire (Strasbourg, France) raise the possibility of prenatal diagnosis. The new genetic information also suggests how the disease might eventually be treated.
The genetic error in FA has been pinned down to an unusual mutation, an "expanded triplet repeat", in the gene for frataxin, a protein of unknown function. Expanded triplet repeats are multiple, tandem copies of a trinucleotide sequence. Repetition of short DNA sequences is common throughout the genome, and blocks of up to 30 or so repeats cause no problems. However, if the number of repeats expands beyond the normal range, gene function can be deleteriously altered. This is what happens in the handful of known "triplet repeat diseases", most of which are neurodegenerative disorders.
In FA, the commonest of the hereditary ataxias, unstable expansion of GAA repeats in a non-coding region of the frataxin gene causes loss of function. "This is important", says Brice, "because it has implications for treatment. In disorders caused by gain-of-function expansions [where the function of the encoded protein is altered], for example Huntington's disease, a way will have to be found to suppress the pathological consequences of the expansion. Treatment of FA is theoretically simpler, because all that is needed is to replace the missing protein".
At least seven adult-onset neurodegenerative diseases--Huntington's disease, spinocerebellar ataxia types 1, 2, 3 , and 6, Kennedy disease, and dentatorubropallidoluysian atrophy (DRPLA)--have a variably expanded CAG triplet within the coding region of the affected gene. The CAG tract encodes a polyglutamine domain in the expressed protein, and abnormal expansion of the repeat causes a deleterious gain of function. James Burke and colleagues at Duke University Medical Center (Durham, NC, USA) reasoned that the underlying defect might be the same in all these disorders. "The size range of the repeats is similar", says Burke. "Unaffected people have fewer than 30 CAG repeats, whereas those affected generally have more than 40." Maybe, thought Burke and his colleagues, Jeffery Vance and Warren Strittmatter, there are specific brain proteins that selectively interact with the expanded polyglutamine domains to cause the clinical manifestations of all these disorders.
"We took polystyrene beads coated with either a synthetic 20-glutamine peptide [representing the domain size of the normal protein] or a 60-glutamine peptide [representing the mutant protein domain] and passed brain homogenate over them", recounts Burke. "Only a few brain proteins bound to the synthetic peptides, and even fewer bound preferentially to the 60-glutamine peptide." Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was one of the latter, and the scientists were able to show that GAPDH binds both to the DRPLA protein and to huntingtin, the protein encoded by the Huntington disease gene. "GAPDH fits the bill as a candidate protein for involvement in neurodegeneration", says Burke, "because it has many functions. It is essential in glycolysis, which fits in with the proposed role of impaired energy generation in the pathogenesis of Huntington's disease. It also binds RNA, ATP, actin, and tubulin".
A characteristic feature of the triplet repeat diseases is anticipation--increasing severity and earlier disease onset in successive generations. Although the size of the triplet repeat in the normal population is relatively stable, "Expanded alleles are highly unstable in both the germline and the soma. This goes a long way towards accounting for the unusual genetics of triplet repeat diseases", explains Darren Monckton (University of Glasgow, UK). "In fragile X syndrome, for instance, the CGG repeat in the gene for FMR1 is extremely unstable and is frequently enlarged on transmission from mother to child." The genetic instability of expanded repeats probably also explains the phenotypic variability of triplet diseases, adds Monckton. "There is an association between repeat number and disease severity. In some diseases, for example fragile X, there is a threshold effect--only males with more than 220 CGG repeats in the gene for FMR1 have the syndrome. In others--for example, myotonic dystrophy [panel]--the correlation between disease severity, age at onset, and the size of the expanded array is progressive."
Meanwhile, Brice's studies and those of Michel Koenig in Strasbourg have indicated that FA has a wider clinical spectrum than previously recognised. Of 187 patients with progressive ataxia, 140 were homozygous for a frataxin GAA expansion of between 120 and 1700 repeats. In a quarter of these patients the disease was atypical. "These patients were older [than usual] and/or had intact tendon reflexes." One patient was 51 years old at diagnosis--disease onset is usually before the age 25. Brice suggests that the usual diagnostic criteria are not sensitive enough to include all patients homozygous for the GAA expansion, adding that direct molecular diagnosis of FA by determination of the size of the GAA expansion is "a real advance".
Listserve 13.03.97 discussion
TSE in Chickens: what to expect?
Narang has examined the brains, finding no signs of tumour, no obvious vacuolation, but some neuronal loss and shrinkage. So far, he has not done PrPsc immunostaining. We cannot be certain what BSE would look like in a chicken. Certainly MAFF's attempts to infect chickens did not give rise to the familiar pathology, and they are as yet only 11 months into the subpassaging of the daffyest chickens in mice.
The clinical symptoms and the epidemiological findings speak for a TSE. Any other differential diagnosis is rather improbable:
1. Newcastle Disease: As this is a highly contagious disease, it is absolutely impossible that only one chicken of the population comes down with it. And apart from that, one of chicken had been vaccinated against ND according to a press release. Moreover the disease mainly strikes chicks and young chickens.
2. Infectious avian encephalomyelitis: Mainly in chicks. Lymphocytes are found histologically (inflammation!).
3. Fowl paralysis (Mareksche Krankheit): Mainly in the chicks in a whole population. Thickened nerves (neuritis) and lymphoma are found pathologically-anatomically.
4. Rabies: Not present in GB.
5. Lack of vitamin B1: mainly in chicks
With chicken, homology to mammals peters out C-terminal in the midst of important secondary structural elements. It helps alignment to use glycosylated asparagines 1 and 2 instead of 1 and 3. After considering a bunch of exotic hypotheses, I am back to thinking that helix 2 to helix 3 homology is no worse than, say, the stop transfer region. So what it means is that in the old days this region just functioned for GPI, carbohydrate, disulphide bond, and not much else; later an additional selected function was acquired in mammalian line (but not in chicken), accounting for the subsequent slow evolution in mammal.