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Excerpts from the article
BSE in Salmon?
Healthy prions in fish brain.
Attack on Hogg over 6,120 land-filled cows
"No country is BSE free"
Germany says it still has no locally-born cases of BSE
Anti-inflammatory drugs may reduce Alzheimer's risk
March 1997 gossip from S. Dealler site

Normal isoform of amyloid protein (PrP) in brains of spawning salmon

Molecular Psychiatry March 1997 Volume 2, Number 2 page 146-147
CJ Gibbs Jr and CL Bolis

... We now report for the first time the detection of this novel protein in the brains of spawning salmon fish collected in Alert Bay, British Columbia, Canada, at the Marine Laboratory. During the course of studying the migratory habits of salmon, brains were collected from spawning and post-spawning, fish. Brains were collected from freshly killed salmon and immediately frozen and shipped on dry ice to the Laboratory of Central Nervous System Studies at the National Institutes of Health, Bethesda, Maryland.

Upon arrival individual brains were tested for the presence of prion protein by Safar modification of the Hilmert and Diringer procedures. As shown in Figure 1, the normal isoform of amyloid precursor protein (PrP was detected in the brains of spawning, and post-spawning migratory salmon. The abnormal isooform of the protein which is proteinase-K resistant was not detected. The failure to detect PK-resistant protein in the few brains tested does not lead to a conclusion that the abnormal.isoform of the amyloid protein does not exist in fish brain. The detection of cellular isoform of PrP suggests that hypothetically as with sporadic cases of CJD the prPc could undergo a rape post-translational modification resulting in the de novo conversion in the abnormal isoform of the protein. Further work is underway in order to determine if captured migratory salmon do, in fact, develop the infectious abnormal isoform of the protein (Pry) on rare occasions.

Figure 1.
western immunoblot defection of normal proton protein  in the brains of salmon fish.

Gel bands:
Scraple Hornstrer Positive Control
Hubert Switbers Positive f''r CdD
Test SPAVINS 25/llt92 PrP 'ic
Test Spawn 25/11/92 PrP:
Fish 3 5/13 PrP Sc
Fish 3 Stl3 PrP C
Female Post Spawn 4/29 PrP C
Female Post Spawn 4/29 FrP Sc  
individual brains were thawed at 4°C for approximately 30 min. Sections of each brain (0.25g) were then separately homogenized in 4.75 ml of 10% Sarcosyl x 10 stroked in a Dounce glass homogenizer and held at room temperature for 30 min. Homogenates were centrifuged at 15 000 rpm in a Beckman Ti SW55 rotor for 30 min at 10°C. The pellet obtained was discarded and the supernatant fluid was collected and recentrifuged in a Beckman Ti SW55 rotor at 47 000 rpm for 2 h at 10°C following which the supernatant fluids were discarded and the pellet resuspended in 4.75 ml of TBS/10% NaCI/1% Sarcosyl and incubated at 37°C for 1 h with microstirring and then centrifuged at 47 000 rpm for 30 min at 10°C. The pellets were collected and each individually resuspended in 0.5 ml of TBS110% NaCl/1% Sarcosyl, treated with proteinase K to a final concentration of 10,ug ml and incubated for 11 h at 37°C with continous stirring after which 2.5 ,~1 PMSF stock solution was vortexed and then centrifuged at 14 000 rprn for 30 min , at 4°C. The pellet was collected and resuspended in , 25''1 of SDS sample (Laemli) buffer, backed for 5 min , at 10°C and tested by SDS page gel and Western immunoblotting employing anti-PrP 3F4 mouse monoclonal antibodies. All specimens were tested before and after a' treatment with proteinase-K.

References

I Gaidusek DC. In Virology Lipincott-Raven 1996 pp2851-2900.
2 Prusiner SB. Novel proteinaceous infectious particles , cause scrapie. Science 1982; 216: 136-144
3 Safar J. Gibbs CI, Gaidusek DC. Biochemical studies of the precursor of scrapie amyloid. Brain Res Rev 1991: 16 103-105.

The following abstracts are intriguing bukt weren't considered --webmaster

Nervous mortality syndrome in farmed Atlantic salmon.

 Vet Rec 137 (24): 616-617 (1995) 
Rodger HD, Turnbull T, Scullion FT, Sparrow D, Richards RH
Institute of Aquaculture, University of Stirling, Scotland.

Linkage relationships reflecting ancestral etraploidy in salmonid fish.

Genetics 116: 579-91 (1987) Johnson KR; Wright JE Jr; May B Fifteen classical linkage groups were identified in two salmonid species ... These linkage relationships support the contention that all extant salmonids arose from a common tetraploid progenitor and that this progenitor may have been a segmental allotetraploid.

BSE in Salmon?

"BSE-Verdacht bei Lachsen" (Salmons suspected of having BSE).
FOCUS Magazine ... 10 Mar 97

According to a Focus announcement, scientists detected prions in the brain of salmons. These prions are suspected of being involved in the origin of the mad cow disease and the Creutzfeld-Jakob-Disease with human beings. The discovery of prions in salmons does not prove that the fish can catch BSE. Martin Groschup from the "Bundesforschungsanstalt fuer Viruskrankheiten der Tiere" in Tuebingen warns that a transfer to the fish might not be excluded any more.

According to Hans-Jürgen Schlotfeld, Staatlicher Fischgesundheitsdienst Niedersachsen, Salmons can get in contact with BSE infected feed. However, the use of meat meal in rearing was not the usual practice. What actually got into the fish feed was really a mystery.

Commentary:

Your webmaster finds this a little fishy but who knows. My question is "detecting prion in salmon." Who did this, how can they be reached, where will it be published, is it just immunological or did they sequence cDNA? It would be amazing if the species barrier can be crossed because teleost fish separated off from cow lineage 420 million years ago and chicken prions [at 300 mya] are already very different C-terminally. The only explanation could be that the prion protein residues104-120 comprising the toxic core is still conserved and sufficient to convert normal prion conformer to rogue.

Amazingly, dog and cat are not even sequenced even though they are important to the species barrier understanding, dogs not seeming to get BSE from their dogfood. Wilfred Goldmann said in the July 4 1996 Nature they had cat sequenced, but it is not on any public database [as of last night] and I don't have an email for him. They had oryx sequence too, which got BSE from zoo food. Says positions146 and 158 differ from cattle. Only carnivores known are mink and ferret. Good species barrier question. Ostrich and macaw reports of TSE also intriguing if doubtful, but we do really really need more bird/herptile/amphibian sequences to understand structre/function of domains.

Listserve 10.03.97
As to salmon,. the UK banned the inclusion of MBM in fish feed last April on just suspicions. As a side issue, since PrP seems to be a universal for vertebrates, and given the auto-carnivorous nature of the sea-fish community, may there not be a number of TSEs native to sea fish anyway?

Chicken update:
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. I read this as negative so far, with the caution that 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 sub-passaging of the daffyest chickens in mice.


"Healthy prions" in the fish brain.

FRANKFURTER RUNDSCHAU, 11 Mar 1997

Scientists at the National Health Institutes in Bethesda (Maryland/USA) and the Pharmacological Institute of the University of Milan have discovered the molecular basis for a transfer of BSE to salmons. The study was published in "Molecular Psychiatry".

Clarence Gibbs (NIH) and Liana C. Bolis (Milan) succeeded in demonstrating prion proteins in salmon brains while examinating free living salmons in Canada. For the first time in the history of the prion research they found the normal prion protein PrPc in fish brains. However, they did not detect the abnormal form PrPsc which is regarded as trigger of the diseases BSE with cattle, scrapie with sheep and CJD with human beings. They do not yet exclude that fish could be able to have the pathogenic form of the prions too. Prion proteines have only been demonstrated in mammals and Drosophila [?] up to now.

According to Dirk Willem Kleingeld, researcher at the "Staatlicher Fischseuchenbekämpfungsdienst und Fischgesundheitsdienst Niedersachsen" in Hannover, the fish are fed with animal meal at many salmon farms -- if the feed was contaminated, they could also catch the agent.

Hans-Juergen Schlotfeld, head of this authority, thinks that feeding of salmon with animal meal is not worthwhile for economical reasons. However, it was still kept in the dark what kind of meat actually went into the feed and where it came from. He said that as long as meat production was a subsidised business, it invited fraud and took up Mafia associatiations. The producer of fish feed "Biomar" stated they use neither MBM nor products of ruminants.

It remains uncertain whether fish eaters are also endangered. Martin Groschup of the "Bundesforschungsanstalt für Viruskrankheiten der Tiere" in Tübingen is of the opinion that it is not proved by the study that salmons can get the infection. But even without findings of prions, you could not exclude a transmission of the epidemic from warm-blooded animals into fish.

Webmaster Commentary: I haven't seen the article so I am just commenting as best I can from the title:

Normal isoform of amyloid protein (PrP) in brains of  spawning salmon
CJ Gibbs, Jr and CL Bolis  Molecular Psychiatry     
March 1997, Volume 2, Number 2 page 146
I would read into 'normal isoform' that they treated extracted salmon prion with Protease K and lost their band on the gel. Rogue _mammalian_ prion isoform is resistant to this particular proteasse associated with the alpha helix to beta shift. Whether this should hold in salmon [and chicken] too seems problematic a priori. Protease K is a non-specific fungal enzyme; resistance is a fairly crude and over-rated diagnostic tool. A protein could undergo a major conformational change and still be sensitive. 'Amyloid' is an old-fashioned name this lab gives to prion protein and does not imply they found amyloid deposits in salmon brains.

The bottom line is that there was enough prion sequence and structure conserved for salmon prion to cross-react with antibody to mammalian prion. And that could mean that transmission to penned salmon from ruminant pellets needs to be seriously considered.

The cost of a single issue to this journal is 40 pounds sterling. Fax: +44 1256 810526 Attention: Matt Smith.


Commission to discuss ways to cut mad cow risk

Reuter Information Service Mar 10, 1997

BRUSSELS - The European Commission plans to discuss with EU member states ways to reduce the risk of mad cow disease and to improve detection methods report, a commission spokesman said Monday. The spokesman was commenting after release of a report showing widespread shortcomings in dealing with the fatal brain wasting disease known as bovine spongiform encephalopathy (BSE).

"No country can say 'We are 100 percent free of BSE," Commission agriculture spokesman Gerard Kiely said. "Individual country reports will be discussed with member states in the next few days or weeks," he added.

The initial report, which did not identify culprits, urged strict EU-wide enforcement of a ban on infected meat and bone meal, believed to be the main cause of BSE, in cattle and sheep feed. A scientific conference on this issue is planned in June. It also recommended closer monitoring of animal waste disposal and meat heat processing rules.

An EU-wide BSE surveillance network should be created, data on suspected cases distributed to all member states and standardized training to diagnose cases stepped up, it added. The report was compiled after visits by EU inspectors to 13 member states between October and December 1996. Britain and Portugal were inspected separately as they already had BSE control and eradication programs. The report's findings reinforce British claims that BSE has been under-reported on the European continent and support demands by EU Farm Commissioner Franz Fischler for wider measures to eradicate BSE.

Fischler was angered last December when EU farm ministers rejected a proposal to ban from the human food chain offal from sheep, cattle and goats that can carry scrapie or BSE. Scrapie, like BSE, is a brain wasting disease and has affected sheep for centuries.

Shortcomings identified in EU member states included:

- Reliability of official scrapie figures.
- Large variation in scrapie measures ranging from killing a herd to doing nothing.
- Unequal application of EU trade rules to prevent the spread of scrapie.
- Unequal implementation of animal waste disposal measures and heat processing of meat and
bone meal to kill infection.
- Lack of officials trained in BSE detection, controls and cattle processing rules.
- Illegal presence of up to five percent meat and bone meal in animal feed.
- Cross-contamination of more than 10 percent between pig, poultry and cattle rations in feed
mills.

Germany says it still has no locally-born cases of BSE

Reuter Information Service Mar 10, 1997

BONN ( 11:49 a.m. EST) - Germany said Monday a calf originally thought to have been the first locally-born animal to die of mad cow disease was actually imported from Britain, and that Germany's own livestock was free of the disease. "After this report Germany can once again say it is BSE free," agriculture state secretary Franz-Josef Feiter told a news conference to present the results of genetic tests into the animal's origin.

The calf died of bovine spongiform encephalopathy (BSE) last December, sparking fears that it could be the first German-born cow to fall victim to the disease, believed to be a threat to humans. But Feiter said it had been established with 95 percent certainty that the calf was an import from Britain called Scottish Queen, and that it had almost certainly been infected there by contaminated feed.

After the calf's death, German authorities ordered the slaughter of more than 5,000 cows imported from Britain and Switzerland, the only countries where BSE is widespread. Feiter said he expected the German states to vote on Friday to make this emergency measure legally binding.

Until December there had been 165,000 BSE cases in Britain, but just four in Germany, all in cows brought from Britain. Germany had always maintained it was free of BSE because there is no evidence of any German livestock farmer using contaminated protein feed -- the standard source of infection. The only other infection route which scientists consider potentially viable is from mother to calf. But the ministry said the fact that the cow was directly imported from Britain meant it was probably not a case of maternal infection.

The result means German authorities are unlikely to order the slaughter of 14,000 cows directly descended from British and Swiss imports, which they had been considering. The genetic tests on the cow were ordered after confusion over its ear-tag cast doubt on its origin and prompted worries that controls to prevent the spread of the disease to Germany were not strict enough.


Strang in attack on Hogg over BSE error

March 11 1997 ... POLLY NEWTON ... The Times

DOUGLAS HOGG was criticised last night after he admitted giving inaccurate information to his Labour Shadow about the disposal of cattle infected with BSE. The mistake prompted Labour calls for a Commons statement from the Agriculture Minister on the Government's handling of the BSE crisis.

In a written parliamentary answer last Thursday, Mr Hogg told Gavin Strang, the Shadow Agriculture Minister, that three cattle suspected of having "mad cow" disease, had been buried rather than incinerated last year. Yesterday, the Ministry of Agriculture said the three carcasses had in fact been used for research.

European and government guidelines say carcasses infected with BSE should be burned to avoid any possibility of land or water supplies becoming contaminated. The Ministry of Agriculture has told Dr Strang that the remains of 6,120 BSE-infected cattle were buried at landfill sites before the practice was stopped, but it refuses to give details of where and when on the grounds of "disproportionate cost".

Dr Strang yesterday sent a letter to the minister demanding that information. The Labour backbencher Helen Jackson, who has put down several questions about BSE, also raised the issue in the Commons.

Anti-inflammatory drugs may reduce Alzheimer's risk

Reuter Information Service Mar 9, 1997

WASHINGTON - Scientists reported Monday that common anti-inflammatory drugs, including certain over-the-counter painkillers, may reduce the risk of Alzheimer's Disease. Scientists from the National Institute on Aging and Johns Hopkins University found that taking ibuprofin for as little as two years reduced the likelihood of getting Alzheimers. Aspirin and acetaminophen did not reduce the risk, although the researchers suggested that further study was warranted of people taking larger doses of aspirin.

The study participants taking the non-steroidal anti-inflammatory drugs had half the risk of developing the degenerative brain disease than those who were not, according to the research appearing in the journal Neurology. However, long-term use of anti-inflammatories such as ibuprofin may create other health problems, such as ulcers and kidney problems, and the authors of this study said clinical trials were needed to weigh the risks and benefits.

Other studies have found a similar effect from non-steroidal anti-inflammatory drugs. However, this report was a larger, longer-term study. The study relied on data from the Baltimore Longitudinal Study of Aging, which has examined more than 2,300 people since 1958, tracking numerous health factors including brain and mental function. Since 1978, the study also has tracked use of non-steroidal anti-inflammatory drugs.

"Many scientists now believe that inflammation may be an important component of the Alzheimer's disease process," said co-author Dr. Claudia Kawas of Johns Hopkins School of Medicine. The tangles and plaques found in Alzheimer's patients' brains "may be indicative of an inflammatory response," she said. The anti-inflammatory drugs may interfere with certain proteins involved in this response.


March 1997

Independent 10.3.97 Buried BSE cattle pose health risk Following a question asked in the house of commons it showed that 6,120 cases were buried in landfill sites. ( This is probably an underestimate, and can be calculated from the level of incineration plant available in different years minus the number of cases reported - Ed)


Guardian 8.3.97 Food on the face of ministers Does anyone still trust them. An editorial about how the Agriculture ministers again misled the House of Commons and gave the data given to parliament was not a good indication of what actually happened concerned with BSE and E.coli. The fact that the truth could be found out simply by reading the newspapers made the MPs look shabby. The editorial gives a number of examples.
Farmers Guardian 7.3.97 Big drop in confirmned BSE cases. A dramatic delcine inthe number in Gratbritain since the start of 1997. 650 reported but only 70 confirmed. i.e 7 per week (This seems statistically very odd - Ed) Apparently the people at CVL would not comment on the meaning of the findings.

Farmers Guardian 7.3.97 Cover up in reporting is alleged Suggestions that there has been a cover up in the reportng of BSE have been made by Professor Richard Lacey of Leeds. "if BSE was related to the feed then there should have been far more cases in Europe because we have exported have exported large quantities of meat and bone meal. My analysis suggests a signficant degreee of animal to animal transmission" he said.

Farmers Guardian 7.3.97 Minister quizzed on costs of carcase storage Tony Baldry told Alan Williams (Labour Carmarthen)that by mid February there were still 208,000 carcases of animals slaughtered under the over 30 month scheme. (about a million have been slaughtered, and only 2,000 tons of rendered material are permitted to be burnt per annum. At times the figures are difficult to understand - Ed), also 160,000 tons of rendered material in store currently, and costing the Intervention Board about 60,000 pounds per week but only the Fawley plant in Southampton of Rechem had been given the OK (under EC regulations) to burn it. Also there is 101,800 tonnes of tallow in storage. During 1996/7 rendering companies received 100 million pounds in support based on loss of income compared with 1995/6, with adjustments to take broad account of their levels of throughput and costs this year. One of the problem was that there was inadequate compettition and the Government wanted to get this going. This is followed by a list of budget costs until year 2000 for BSE .

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