Prions
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Resistant animals could carry BSE
Table with mouse-to-hamster persistence data
TSE persistence: reservoir or replication? Article and commentary
Warwick meeting news 16 April 1998
Three new CJD articles in 25 Apr 98 Lancet

Resistant animals could carry BSE

23 April 98 Nature
April 23 1998 	Times by MICHAEL HORNSBY 
ANIMALS thought to be resistant to "mad cow" disease may be secretly harbouring the infective agent, according to new research. Experiments with mice and hamsters in America have shown that the agent which causes BSE, bovine spongiform encephalopathy, could persist undetected for long periods in resistant species. The findings raise the worrying possibility that poultry and pigs which had eaten BSE-contaminated feed could be infected with the disease while not falling ill themselves.

The research, by Richard Race and Bruce Chesebro, of the National Institute of Allergy and Infectious Diseases in Hamilton, Montana, involved injecting the infectious agent for hamster scrapie - a disease similar to BSE - into the brains of mice. Mice are highly resistant to the hamster disease, and as expected did not develop any symptoms. But the scientists found that a year after the mice received the injections their brain and spleen tissue could cause the disease when put back into hamsters.

Writing in the journal Nature, the scientists acknowledge that, so far, there was no evidence for the secondary transmission of BSE from resistant species to more susceptible ones. But they add: "The results presented here would strongly favour a decision to stop feeding ruminant-derived products to all animal species."

Scrapie infectivity found in resistant species

(Scientific Correspondence) 
R Race & B Chesebro 	770 

The prion's perplexing persistence

Nature 392, 763-764 (1998) 
Adriano Aguzzi and Charles Weissmann

Exposure to infectious prions does not always lead to clinical disease, but a new report indicates that prions can nonetheless persist in the brain. Hamster prions were injected into mice and, although the mice did not develop symptoms, the prions were present for almost the entire life of the mice. What's more, when brain extracts from these mice were injected back into hamsters, the hamsters showed clinical symptoms. These findings could have public-health implications - for example, pigs and poultry, which do not develop disease after eating bovine prions, may be acting as reservoirs for the infectious agent.

Commentary: It seems that even when a 'species barrier' exists, it does and it doesn't. Hamster-to-mouse was supposed to be an example of an absolute species barrier, but now it seems that is just a little slow to develop symptoms relative to livespan and injectable dose, ie, these mice are asymptomatic carriers. Presumbably, knockout controls aren't able to re-infect hamsters, ie, we are not talking about hamster prions merely persisting in mouse for the year.

The evidence supports their policy recommendation to "stop feeding ruminant-derived products to all animal species" which would include pigs, chicken, and fish. Chickens are especially troubling because the White Leghorn has an ominous number of extra repeats already and because of the cow-to-chicken-to-cow amplification cycle. Pigs are a no-brainer being fellow artiodactyls; fish are at the outer limits of reasonability.

Dealler has, "BBC 22.3.98: Realisation that BSE may have gone to chickens and then to humans. This follows an article in Nature by Race and Chesebro indicating that although pigs and chickens did not show any symptoms of BSE it is possible that they (as had taken place in the mice they tried) taken in the bovine prion PrP and simply stored it in brain and spleen. The argument was that it may well have been fed back to cattle from chickens after the feed ban and fed to humans from chickens in the chicken offal. "

Scrapie infectivity found in resistant species

Nature 392:770 23 Apr 98  R Race, Bruce Cheesbro  [B. Caughey not a co-author]
Webmaster commentary of 24 Apr 98
This is a short note, 8 paragraphs and a table. It concerns the persistence of infectivity in clinically resistant (carrier) animals. It is an experiment that must have begun 3-4 years ago.

Donor mouse Recipient hamster Recipient mouse
mouse genotype days post inoc TSE+/total: brain incubation time TSE+/total: spleen TSE+/total: brain
1 +/+ 204 4/4 313 ± 112 ND ND
2 +/+ 310 3/3 178 ± 14 ND ND
3 +/+ 574 7/7 140 ± 4 6/6 0/8
4 +/+ 574 8/8 162 ± 38 6/6 0/8
5 +/+ 693 8/8 156 ± 20 6/6 0/8
6 +/+ 693 8/8 168 ± 42 6/6 0/8
7 +/+ 782 8/8 128 ± 13 ND 0/8
8 0/0 112 0/12 - ND ND
9 0/0 112 2/12 294-334 ND ND
10 0/0 217 0/12 - 1/8 ND
11 0/0 217 4/12 296-352 0/8 ND
12 0/0 330 0/7 - 0/8 ND
13 0/0 330 0/8 - 0/8 ND

It is worth taking a close look at Table 1 -- it contains all the experimental data:

1. The null mouse brain acted up fairly badly, causing 6 hamsters out of 63 (so about 10%, not what I would call 'very rarely') to develop scrapie when they weren't supposed to. Worse, the incubation times (about 320 days) were the same as the 4 hamsters (313 days) inoculated from mouse #1. (Null mouse spleen caused 1 hamster of 32 to succumb.)

2. This favors persistance of original inoculum for a year or more in null mice as an unavoidable background level. Some of the brain inoculum is getting to the spleen and evidently persisting there too. However, inoculum from wild-type mice was significantly more infectious, 46 of 46 hamsters developing TSE with a 150 day incubation period (half the incubation time). This could be taken as replication in addition to persistence, or as better persistence when host prion protein is present.

3. If mice instead of hamster are used as recipients, no mice became ill. This has been taken to favor simple persistence; if agent had replicated in the donors, this mouse prion agent would have no species barrier to mouse except that a hamster strain began the whole business. Unfortunately, no data is given on time the mice were kept under observation. A further passage of these mice might do the trick. Further, no control mice were studied at all (inoculated from null mice).

4. The authors can apparently read off the ID-50 from incubation time from some earlier publication, saying hamster incubation times here reflect about 100 units per gm, down from the 10 million unit inoculum per brain the wild-type mice received. They don't cite any source, making it difficult to evaluate the ID-50 difference between 128 and 313 day incubation periods. The graph of this must be dramatically non-linear. (ID-50: number of infectious doses causing 50% to develop clinical symptoms)

5. The donor mice, in pairs, were sampled for infectivity to mice and hamster every 4 months or so. There is somewhat of a trend over time of incubation in mouse to shorter incubation periods in hamster, favoring replication (or recovery of hidden persistance).

The bottom line: these data don't distinguish between simple persistence, facilitated persistence, persistance through replication, and various mixes. For pig and chicken policy, it doesn't matter: like they say, stop feeding ruminant protein to the other species.

They begin by noting that BSE has transmitted orally or by injection to numerous species, but not to hamsters or chickens (citing letter by Hill AF et al, Nature 1997 Oct 2;389 (6650):448-450 which I do not think contains any data on chickens, I don't think adequate data on chickens exists).

10 million half-maximal doses of hamster scrapie strain 263K were injected intra-cerebrally into the same line of regular and knockout mice. No mice developed symptoms. However both brain and spleen from regular mice at days 204 and 782 could infect hamsters (but not mice) but only with long incubation periods (indicative of only 100 infectious units per gram in the donor brains). All recipient hamsters were positive.

However null mice _were_ able to infect the hamsters too, "very rarely." (One hopes there were good controls for laboratory and facility contamination.) Null mice are not the ideal control because they make anti-prion antibody that could clear circulatory or spleen prions, not so clearly intra-cerebral prions. They read this as normal mouse prion facilitating the persistence of hamster prion, saying there is "no evidence for any replication of the hamster scrapie agent."

But did they look for any, and if so, how? All I could see was a lack of mouse-to-mouse transmission, which I wouldn't expect to work despite like-like amino acid sequences given conformational persistence of what is to mouse a manifestly unfavorable conformation [strain], initial failure of transmission, and hugely lower doses.

I could not read the table well enough to see if day 782 mice had higher (amplification model) or lower (persistence model) incubation times in hamsters; it seems that any increase of infectivity over time is not statistically significant but this is net units, so amounts to rate of loss of hamster prion as compensated by rate of de novo recruitment of mouse prion and its turn-over.

Of course, there are several logs of infectivity lost, though the dilution factor is never stated (ie, 1 microliter injected, 1 gm whole brain extract?) and I don't believe that they measured infectious units recoverable from freshly injected brains as opposed to infectious units from the hamster inoculum itself, which would totally negate any inference from loss of titre per se.

They write, "the prolonged survival of a foreign scrapie agent raises the possibility that BSE infectivity might persist in various 'resistant' species exposed to BSE-contaminated feeds. Of particular concern would be domestic animals such as poultry which are raised for human consumption.... The results presented here would strongly favour a decision to stop feeding ruminant-derived products to all animal species."

Further experiments they want include titration in cattle, ie, take the chicken fed offal and see if the chicken is infectious back in calves (most sensitive indicator species?).

Aguzzi and Weissmann, in the accompaning News and Views piece, cite a couple of earlier papers showing "traces of infectivity that were occasionally found in the brain several months after infection." They point out the uncomfortable anomaly of uniformly fed, genetically homogeneous cattle herds where only a single or few animals go clinical, the rest perhaps a silent reservoir.

They provide cites buttressing the point I made above, noting "after a few days, mouse prions injected into mice become virtually undetectable," which favors replication over reservoir, and note also the possible primacy of conformation over sequence.

This is a catchy phrase but in error: it is actually conformational cooperativity overwhelming monomer conformation, peer pressure as it were. (It is not sequence per se because the same sequence may assume different fibril conformations.) That is, if a longish hamster fibril is already developed as the template, the energetics of its subunit cooperativity overwhelms the propensity of a new unit of mouse amino acid sequence to form the fibril that it would otherwise form. In other words, there is persisting conformational trapping by the nucleating seed of new recruits to a potential well that is not a global minimum for a fibril of pure recruit. This is like crystallizing table salt off a body-centered cubic versus face-centered lattice.

On policy and risks, they support the authors, stressing the need to test pigs and chickens for BSE using calves as indicator species. They hint of having immuno-compromised mice already in the experimental pipeline to augment the knock-out controls.

The bottom line: (1) Infectivity surprisingly can persist a long time in asymptomatic animals, (2) reservoir persistence vs recruitment is completely up in the air, (3) ruminant offal should not be fed to any vertebrate species, especially not pigs or poultry.

Warwick meeting 16 April 1998

Second Annual CJD Conference
Organised by the CJD support network of the Alzheimer's Disease Society. 
Thursday 16 April 1998 at the University of Warwick 
For further information: Gill Turner tel 01630 673993

Webmaster opinion:

What I heard is that an unpublished paper by James Hope indicates a match between a particular Cheviot line of scrapie and Collinge's nv-CJD strain-type, which would support (but not prove) a conventional scrapie origin of BSE (remembering that there are many, many strains of scrapie that presumably didn't give this result).

Glenn Telling is reporting differences in Collinge strain-typing between samples taken from different organs. I don't know if this includes different regions of the brain or whether this detracts from using glycoforms as a diagnostic signature for nvCJD or whether nvCJD might be broader or non-existent as a distinctly recognizable.

There will be a summary of the conference in the group's next newsletter which will be published in June or July.

Three new CJD articles in 25 Apr 98 Lancet

Lancet Volume 351, Number 9111  25 April 1998

New-variant Creutzfeldt-Jakob disease and treatment of haemophilia

C A Ludlam on behalf of Executive Committee UK Haemophilia Directors' Organisation
Offline to non-subscribers, no details available.

US guidelines aim to reduce CJD risk from dura mater

by Steven Mitchell 
Despite WHO recommendations not to use dura mater and the report from Japan in November, 1997, of 43 people who contracted Creutzfeldt-Jakob disease (CJD) after cadaveric dura-mater allografts, such transplants are still allowed in the USA. For this reason, the US Food and Drug Administration is rapidly developing guidelines to minimise the risk of transmitting CJD through infected dura mater. The FDA's transmissible spongiform encephalopathies (TSE) advisory committee ratified the proposed safety measures at a meeting held in Bethesda, MD, USA, on April 16.

The committee concluded that the risk of CJD transmission would approach zero if the proposed guidelines were followed, even though the infectious agent seems capable of surviving procedures, including sterilisation, pasteurisation, and ultraviolet irradiation, that kill most other pathogens.

The committee agreed that requiring dura mater to come from risk-free donors--defined as having no evidence of neurological disease and no family history of neurological degenerative disease--would make it almost impossible for an undetected case of CJD to become a donor. However, additional precautions would help to reduce the risk further.

Because of mounting, although not conclusive, evidence that an abnormal conformation of prion protein (PrPres) is the infectious agent in CJD and other TSEs, the committee agreed that immunocytochemistry for PrPres would help eliminate infected tissue. But a full brain autopsy, including gross and histological examination, for tissue that passed the PrPres test was necessary to completely rule out CJD.

Disinfection of dura mater with 1 M sodium hydroxide for 1 hour can substantially reduce the amount of infectious agent, but does not completely remove it, the committee heard. Finally, the committee agreed that archiving a sample of dura mater and a 5-10 g portion of the frontal lobe of the donor's brain at -70° C would help future analysis.

Genotype at codon 129 and susceptibility to Creutzfeldt-Jakob disease

Jean-Philippe Deslys, Alexandre Jaegly, Jerôme Huillard d'Aignaux, Franck Mouthon, Thierry Billette de Villemeur, Dominique Dormont
Volume 351, Number 9111  25 April 1998
This is a very sketchy research note concerning the 54 cases of growth hormone CJD in France, which mimicked the US experience. The only figure shows met/met and val/val quite similar and met/val lagging by 5 years, in terms of onset. Heterozygotes are underrepresented by a factor of 7.5. Left as homework for the reader are val/val and met/met over-representations, at 1.9 and 1.6 resp., that is, val/val are more sensitive than met/met here.

In other words, the situation does not mimic the nvCJD story because there would have been 6-7 val/val nvCJd cases already in their scenario. So I see no basis whatsoever for their conclusion, "Our evidence suggests that [nvCJD] cases may appear in people who are heterozygous over the next few years." Even if the numbers did line up, there still would be no basis as nvCJD is cross-species.

Val/val cases should precede val/met yet the time scale for the former is impossible to predict. The met/met cases are poorly understood within the Uk population and prion promoter genotypes or possible trisomy have not been determined for any of the nvCJD victims.

The other curious question is whether they are out of the woods yet with 5% illness out of 968 exposed or whether there will be trailings or multiple peaks years or decades later on in the epidemic. -- webmaster commentary

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