Return to home page

3. Sardinia : scrapie - brief summary of my findings from Autumn 2000 trip.


Plastics factory and industrial area at Ottana, central Sardinia, looking East towards the Barbagian mountains and the scrapie cluster zone of mountain villages 10 -20KM beyond : Gavoi, Olzai, Ollolai, Sarule and Mamoiada (with Mt Ortobene and Orune to the NE)

First scrapie on the Island : 1982.near Iglesias and near Ardara, with subsequent outbreaks near Ozieri, 1985,88,96,99,2000;
Siamanna(near Oristano) 1998; Budduso 2000; Orune 1999,2000; Mte.Ortobene 1989; Mamoiada 1990, 2000; Gavoi 1999; Ollolai 1998,99,2000; Olzai 1997,1998; Sarule 1999; Silanus 2000; Assemini/ Decimomannu.
Some vets think meat and bonemeal feed may be responsible for the outbreaks; other theories involve vaccinations containing mutant prion, but not all sheep had had these.The clustering and the continued occurence in some locations make this very unlikely - why should dodgy feed or vaccines always end up in Ozieri or the western Barbagian for example.

Locations of Sardinian scrapie. see Fig 1.

Siamanna
Siamanna is on the Western slopes of mountainous terrain, 17KM downwind of an industrial centre at Oristano and Stagno di Santa Giusta on the Central Western coast. Visited but no samples taken here.

Iglesias
Early Scrapie outbreak 1982, situated downwind approx 15KM of the industrial town of Iglesias (SW coast) Not visited.

Assemini and Decimomannu
Scrapie a few kilometres to the NW of Airport and the industrial centre of Cagliari.(South) Not visited.

Ozieri and Ardara (Budduso)
The Ottana industry may be of less relevance to these locations but its N orientation is plausible, albeit with a greater distance of 38KM over mountains.
The scrapie locations lie down wind of a large cement factory in SE Sassari and an electrical plant in Codringianos, in the valley bottom between 12- 36KM. There’s also a redundant volcano at Ardara.
The farm at Occhetta (nr Ozieri) although partly low lying does occupy foothills on which the sheep were grazing.
There are some interesting factors on the farm: sheep were being treated for the bacterial disease, ‘foot rot’ with copper sulphate (bluestone) foot bath. The other possible copper connection was the presence of a bank in the farm yard near to the CuSO4 bath which was of a similar blue green colour. The rocky soil was through coloured over a wide area and subsequent investigation revealed other extensive banks at the roadside bounding the farm where the consistency was harder and on the next door farm.

	I also found similar banks in North Ozieri, near San Nicola where the other Scrapie outbreaks have occurred. Sardinia was well known for its copper mines. Notwithstanding this soil turned out to be high in mn and not cu!
Bank outside Occhetta farm.

There were 2 main industrial plants that are likely to be relevant here. A large cement factory (now redundant) is situated SE of the city of Sassari at the head of a valley that leads Eastwards firstly down to N.Ardara 17KM. the location for first Sardinian Scrapie in 1982. To the SW is the extinct volcano Meilogu at Ardara which has left its legacy of basalt in the area. Secondly Ozieri is 35KM east of the factory. Cement manufacture is known to be a major source of polluting chemicals that generate acid rain and metals. A large electricity generating plant at Codrongianos is also situated in this valley 26KM upwind of Ozieri and 10KM to the SW of the Ardara scrapie.

The Barbagian cluster.(Gavoi, Olzai, Ollolai, Sarule and Mamoiada with Mt Ortobene and Orune to the NE)
I propose that the many cases in the Barbagian mountains are related to the industrial centre in Ottana with the large plastics factory being particularly relevant. Firstly the temporal association is strong. The first cases started about 5 years after the factory was built which is consistent with a short period for polluting factors to be established and then the incubation of the disease.
The orientation also fits with this view. The earliest and most intense cluster point is east of the factory, downwind with less intense distribution only beginning after 10 years (ie in Silanus, NW of the factories ).The scrapie is largely occuring on land inclined towards the factory (Western slopes) and also at high altitudes ( approx 400 –800 metres, Mt Ortobene 1000M) where precipitation would be more intense. UV and ozone is also more intense at higher altitudes.

Where chlorinated emissions are present such as in plastics manufacture, these are likely to rise, eventually to the stratosphere, creating additional Cl atoms by photodissociation that are known to break down the stratospheric ozone layer.This allows greater penetration of UV radiation to the earth. There are also likely to be periods of high ground level ozone production derived from industry and traffic that drift and concentrate away from the source.

A similar scenario was found in Slovakia’s High Tatra mountains, western slopes, with CJD and scrapie occurring downwind of steel factories that had failed to remove high mn from their emissions. This added more mn and to an area that already had naturally high levels + acid rain etc.

The site specific Fenton/Haber-Weiss Hydroxyl radical (OH’)/Histidine interaction: the critical initiation event of all TSEs?
This hypothesis suggests that this type of reaction is a likely candidate because of its specificity. Many neurological diseases including TSEs share many non specific markers of OS such as lipofuscin, lipid peroxidation and high intracellular calcium etc which would be expected from the peripheral generalised cellular OS, although not critical in the initiatiation of TSEs. I propose that the event occurs on the cell membrane phase of the prion cycle when there is added risk of interaction with reactive substrates and metals available for catalysis.

The risks for this type of toxic reaction would include :
1) Abundance, lack or dysregulation of metals cu, mn, zn, that are incorrectly bound to PrPc or freed from their protein sinks.
2) A substitution for cu at the octapeptide domain of PrPc, most likely by mn. Mn can scavenge both superoxide(O2-) and hydroxyl(OH') radicals but as PrPc/mn it cannot function as an anti oxidant for long.
3 ) Raised extracellular oxidants, particularly O2- or Hydrogen peroxide(H2O2) that beget the OH’ radical of Fenton or Haber- Weiss.
4) Increase in acidity would exacerbate no.1 ie reduction in binding of many metals is known to occur in mild acidic conditions : Zn to Beta amyloid and cu to caeruloplasmin, Fe to transferrins.( 1p36). Copper binding however increases under mild acidosis resulting in dramatic aggregation of beta amyloid. PrP is very similar to beta amyloid.

Fenton reactions involve the reaction of H2O2 + a metal catalyst such as cu in which the catalytic metal is oxidised to form a 2+ or possibly a 3+ form with the production of a hydroxyl radical :
ie H2O2 + Cu+ = OH' +OH- + Cu2+ (orCu3+) .
Mn is less certain as a catalyst but in the presence of bicarbonate/CO2 Mn(II) is able to catalyse Fenton reactions.
The Haber Weiss reaction is one in which O2- reacts with H2O2 in the presence of a reactive metal such as Cu, Fe
producing OH’ radicals.

The OH’ radical produced is known to cause a contained site specific reaction confined to the residue to which the catalytic metal is bound. This has structural implications potentially causing the misfolding to PrPsc through its alteration of the hydrophobic/philic balance of the protein.
It is proposed that this OH’ radical oxidation of the octapeptide histidine sites involving the bound metal, whether cu or mn, is the critical event that initiates sporadic TSEs.

Environmental factors and the histidine residues to which cu or mn is bound.
It's proposed that increases in free radical and acid generating molecules, such as Ozone(O3) and UV, NO2,SO2 are factors that have lead to scrapie on the Island by creating cellular and soil conditions that favour the formation of pathogenic PrPsc/cu3+ or mn3+.

OZONE
O3 is a toxic gas formed by photodissociation of O2 by sunlight. In the stratosphere the ozone layer protects the earth from excessive penetration of UV.
O3 can accumulate at ground level from the action of sunlight or electrical discharge on gases such as NO2, SO2. The journey and impact of such 'photochemical' smog is related to topography, industrial /traffic pollution and wind direction.

Most of O3's affects are mediated through its ability to generate H2O2 and nitric oxide (NO) .
This can have benefits. In some controlled circumstances it can mitigate against ROS ie of hepatic injuries. It can be used in combination with UV and H2O2 to remove some pesticides (atrazine) from surface waters.
Studies on rainbow trout showed that O3 created ROS damage to red cells (hemolysis, methemoglobin, lipid peroxidation) by generating H2O2 in the cell that reacted with Hb independantly of Fe.

• Singlet Oxygen
• Cu(II) has been found to be a very effective scavenger of singlet O - the effectivity was reduced when a cu binding peptide was present. Other metals tried had no quenching effect.The prion may well be involved here as a cu transporter to sites of singlet O generation, releasing it as appropriate.
• Histidine has the highest 2^nd order rate constant for reaction with singlet O (1 p70)
• Singlet oxygen can be generated from phophatidylcholine hydroperoxide (PCOOH) (oxidised biomembrane product) in the presence of Cu(II). Might occur in vivo following the peroxidation of lipids that are found in TSEs.

Fig 1. Map showing distribution of scrapie and some important industrial plants in North Sardinia.

Results of soil and herbage analysis

Sardinia Herbage : Collected 31st October- 5th November 2000
Analysis at Reading and Derby Universities. Results in mg/Kg dry basis or %m/m = mass for mass where marked.

Sardinia Soil (conc in solid) ug/g

These samples were taken at the end of a long spell of dry weather.There was some rain around whilst I was collecting..

Gavoi, farm 1 had scrapie in 1999. It was approx 700 M above sea level, a second farm 1.5 KM to the east had also had scrapie. Farm 3 was 2KM beyond farm 2 to the East and over the moutain on the East side. It had not had scrapie but the sheep had had Bluetongue virus and the whole flock had recently been slaughtered.
The Mn was fairly high in herbage and very high in the soil on all farms except the control where it was within mean.. In Ozieri the herbage mn,cu,fe,zn,cd and pb were raised. Interestingly copper was low in the soil here but very high in the herbage.

Discussion.
These results differ in some respects from other TSE environments studied. The main difference is the v.high Cu in the herbage at Ozieri, an area that has many outbreaks over 16 years. Most other metals are also fairly high in the herbage in that location. Also Ozieri is low lying in contrast to the mountainous regions where most scrapie is found.
In common with other environments there is a high soil Mn level where scrapie occurs although in the herbage mn is not as high as most of the other TSE locations we have tested - Iceland/Slovakia/Colorado.The map illustrates how scrapie is downwind from some industrial factors and is situated in topograhpy where photochemical oxidative factors O3 and UV, would accumulate-cement factory at Sassari and plastics at Ottana being the most important. The area immediately above the polluting source is protected from UV by the scattering and absorbtion properties of many polluting gases such as NO2 and SO2.

Osilo (the control) has slightly raised levels for herbage Cu, but in the case of Mn this is the lowest result.
Gavoi, Farm 3 is not a true control as scrapie may reach some other local farms in the area over the next few years.The metal levels are consistent with other scrapie environments. The main factor that might have protected this farm is its position on Eastern slopes where precipitation affects from industry are likely to be less. I would argue that acid rainfall, altered UV penetration and wavelength, and any pollutants from the industrial area at Ottana, with the large plastics factory built in the late eighties are the key initiating factors in the scrapie that started in the nineties.
Acidity is known to make metals such as mn and aluminium more available for uptake by plants. Slightly acid conditions in cells are also known to alter binding of key metals to proteins like the prion and predispose to formation of PrPsc. Acidity generated by industrial emmissions can be present in fogs, snow as well as rainfall. Dry deposits from emmissions would be highest on the Western foothills close to industrial sources.
Mild increase in acidity in the sheep via lactic acidosis due to increased altitude may also be a factor in the many mountain locations. The increased altitude would result in higher UV levels also make the blood brain barrier more permeable to metal uptake into the brain.

I propose that this acidity may affect cells at certain peripheral points of the sheep before normal acid /base homeostasis has had time to redress the balance, increasing susceptibility. For example the eye could have long exposure to atmospheric acidity via smogs, rain etc as well as UV light; the feet, when lesioned in foot rot particularly. PrPc is expressed in the skin and at vulnerable interfaces and also in immune cells such as neutrophils. As the sheep at Occhetta had foot rot their immune systems would be expected to mount a phagocytic attack in the foot area. Neutrophils are involved in the lysis of bacteria with H2O2, OH', peroxide radicals via respiratory burst -some chemicals are released into the phagocytic vacuole and some into the extra cellular space. Its feasible that under adverse conditions these products may not be metabolised properly oxidising PrPc on the neutrophil membrane at the burst.

It is intriguing to think that scrapie could be initiated in lesioned feet.- One characteristic of scrapie is the presence of pruritis around the legs/feet. In the case of bacterial foot rot there is the added factor of respiratory phagocytic bursts of Fenton substrates coupled with a) chronic exposure of the blood/lymph and nerves in the lesioned feet directly into the high soil cations and acidity(when waterlogged diffusion would be greater).Sheep often consume soil particles pulled up with their forage.b) treatment with toxic CuSo4 foot baths introduced additional cu presenting a severe challenge to distribution proteins and would no doubt become available for pro oxidant catalytic reactions c) In increasingly acid conditions due to pollution, metal binding would be affected before acid base buffers had kicked in. Once the 'radical fuse' is lit in lesioned areas it could spread by anterograde transport along peripheral nerves and/or blood transmission - known transmission routes of TSEs to the CNS.
Most evidence seems to suggest that sporadic TSEs are diseases initiated by a change in bound cations at the octa repeat region. Mn for Cu being the common one. Does the Ozieri farm suggest another scenario where abnormal octapeptide copper, zinc binding could create scrapie. Much in vitro work suggests that this could indeed occur.

If this is the case then it represents a different risk from traditional cu toxicity in which the cu, although high, is rapidly bound to protective proteins. It's also subject to compartmentalisation protection by the blood brain barrier.Finally in most toxicity cases oxidative stress is less intense than in a TSE environment and this prevents critical site specific Fenton type reactions occurring.
In this case I think the octapeptide bound copper has come into contact with an OH' generating substrate like H2O2 and caused site specific damage to the histidines resulting in conversion to PrPsc. This would produce a different strain/variant from the mn type of scrapie that was occurring in Iceland/Slovakia. Unfortunately no analysis of scrapie PrPsc in relation to bound metals and strains and or different locations has been carried out. This would be worthwhile. The increased acidity of 'our times' bearing on high risk soils in my view is the main factor that has precipitated scrapie in Sardinia..

An alternative explaination is the high herbage cu is a red herring if as suggested in the paragraphs above direct exposure to the soil is the most important factor then this would be consistent with the the high mn TSEs we've found elsewhere.

Another factor of interest is the higher levels of UV that would be present in these mountain villages. Higher levels generally would be expected due to thinning of the stratospheric ozone layer. The eye, brain, feet and spine would be especially at risk. Ground level Ozone, which is increasing in Europe by approx. 1% per year, is also highly likely to be raised in this environment by the action of UV on pollutants. It usually concentrates away from the source and drifts downwind and is more likely to be at the highest concentrations in the mountains.
Mild hypoxia from the altitude may make the blood brain barrier more permeable allowing more metals through to the brain and would also tend to mildly increase acidic conditions (lactic acidosis) that favour deleterious metallic binding changes to the prion protein and increase the risk of PrPsc formation.

Temporal and orientation factors support the role of industrial emmissions initiating scrapie on Sardinia.

The Barbagian cluster is downwind, to the East, SE, NE, 10-20Km of the industrial centre of Ottana. The construction of the main plastics factory predates the scrapie by about 5 years.
Ozieri and Ardara are downwind of large industrial sites- Cement factory SE of Sassari and electricity generation plant.12- 32Km
Silanus is NW of Ottana and scrapie first appeared here in 2000. The lag is probably due to the orientation producing less intense industrial emmissions.
Siamanna is 17Km downind of the industrial areas of Oristano/ San Giusta
Other scrapie locations not visited are also similarly placed in relation to industry.
Sardinia is also volcanic(extinct) and is known for copper mining.

Suggested treatment. EDTA is a chelator that is known to be effective in removal of iron fron site specific protein Fenton type radical damage. Its probably effective with cu and mn as well.

I'm currently preparing a full paper for journal submission on Sardinia and some of its implications for TSEs

References: See the 4 papers by Mark on this site that cover UV etc.
Most of these abstracts and some full texts are available free online via medline
They provide direct and backgroud refs to aspects of these cases studies.

1. Halliwell B.and Gutteridge JMC. Free Radicals in Biology and Medicine. Oxford: Clarendon Press 1989.
   
2. Wong BS, Chen SG, Colucci M, Xie Z, Pan T, Liu T, Li R, Gambetti P, Sy MS, Brown DR.
   Aberrant metal binding by prion protein in human prion disease. J Neurochem. 2001 Sep;78(6):1400-8.
3. Brown D R.et al. Consequences of manganese replacement of copper for prion protein function and proteinase resistance. The EMBO Journal Vol 19 No 6 pp 1180-1186. 2000. Full text
4. Jackson GS, Murray I, Hosszu LL, Gibbs N, Waltho JP, Clarke AR, Collinge J. Location and properties of metal-binding sites on the human prion protein
5. Bost M, Kopp N, Giraud P. Copper status and screening for other trace elements in CJD patients; preliminary results. J of Alzheimers Disease 2000; 2: poster 27.
6. Fischer TW, Scholz G, Knoll B, Hipler UC, Elsner P. Melatonin reduces UV-induced reactive oxygen species in a dose-dependent manner in IL-3-stimulated leukocytes. J Pineal Res 2001 Aug;31(1):39-45
7. Ryoo YW, Suh SI, Mun KC, Kim BC, Lee KS. The effects of the melatonin on ultraviolet-B irradiated cultured dermal fibroblasts. J Dermatol Sci 2001 Nov; 27(3):162-9
8. Paretzoglou A, Stockenhuber C, Kirk SH, Ahmad SI.Generation of reactive oxygen species from the photolysis of histidine by near-ultraviolet light: effects on T7 as a model biological system.
   J Photochem Photobiol B. 1998 May 15;43(2):101-5.
9. Seifert J, Thanit P. Alteration of mice L Tryptophan metabolism by the organophosphorous acid triester diazinon. Biochem Pharmacol 1992; 44: 2243-50
10. Oya-Ohta Y, Ueda A, Ochi T, Harada M, Yamamoto K. The biological activity of hydrogen peroxide VII. L-Histidine increases incorporation of H(2)O(2) into cells and enhances formation of 8-oxodeoxyguanosine by UV-C plus H(2)O(2) but not by H(2)O(2) alone. Mutat Res 2001 Jul 1;478(1-2):119-27
11. Joshi PC.Copper(II) as an efficient scavenger of singlet molecular oxygen.
    Indian J Biochem Biophys. 1998 Aug;35(4):208-15.
    
12. Requena JR, Groth D, Legname G, Stadtman ER, Prusiner SB, Levine RL.Copper-catalyzed oxidation of the recombinant SHa(29-231) prion protein.
    Proc Natl Acad Sci U S A. 2001 Jun 19;98(13):7170-5
13. Jobling MF, Huang X, Stewart LR, Barnham KJ, Curtain C, Volitakis I, Perugini M, White AR, Cherny RA, Masters CL, Barrow CJ, Collins SJ, Bush AI, Cappai R. Copper and zinc binding modulates the aggregation and neurotoxic properties of the prion peptide PrP106-126.
    Biochemistry. 2001 Jul 10;40(27):8073-84.
    
14. Gaubert S, Bouchaut M, Brumas V, Berthon G.Copper--ligand interactions and the physiological free radical processes. Part 3. Influence of histidine, salicylic acid and anthranilic acid on copper-driven Fenton chemistry in vitro.
    Free Radic Res. 2000 May;32(5):451-61
15. Beltran FJ, Rivas J, Acedo B. Atrazine removal by ozonation processes in surface waters.
    J Environ Sci Health B. 1999 May;34(3):449-68.
16. Peralta C, Leon OS, Xaus C, Prats N, Jalil EC, Planell ES, Puig-Parellada P, Gelpi E, Rosello-Catafau J. Protective effect of ozone treatment on the injury associated with hepatic ischemia-reperfusion: antioxidant-prooxidant balance. Free Radic Res 1999 Sep; 31(3) : 191-6
    
17. Moldau H. Ozone detoxification in the mesophyll cell wall during a simulated oxidative burst.
    Free Radic Res. 1999 Dec;31 Suppl:S19-24.
    
18. Fukunaga K, Nakazono N, Suzuki T, Takama K. Mechanism of oxidative damage to fish red blood cells by ozone.
    IUBMB Life. 1999 Dec;48(6):631-4.
    
19. Valacchi G, Bocci V. Studies on the biological effects of ozone: 11. Release of factors from human endothelial cells.
    Mediators Inflamm. 2000;9(6):271-6.
20. Balcioglu IA, Arslan I. Partial oxidation of reactive dyestuffs and synthetic textile dye-bath by the O3 and O3/H2O2 processes.
    Water Sci Technol. 2001;43(2):221-8.
    
21. Pellinen R, Palva T, Kangasjarvi J. Short communication: subcellular localization of ozone-induced hydrogen peroxide production in birch (Betula pendula) leaf cells. Plant J. 1999 Nov;20(3):349-56.
22. Andreozzi R, Caprio V, Marotta R, Tufano V. Kinetic modeling of pyruvic acid ozonation in aqueous solutions catalyzed by Mn(II) and Mn(IV) ions. Water Res. 2001 Jan;35(1):109-20.
    
23. Korczynski RE.Occupational health concerns in the welding industry.
    Appl Occup Environ Hyg. 2000 Dec;15(12):936-45.
    
24. Weller BL, Witschi H, Pinkerton KE. Quantitation and localization of pulmonary manganese superoxide dismutase and tumor necrosis factor alpha following exposure to ozone and nitrogen dioxide.
    Toxicol Sci. 2000 Apr;54(2):452-61.
    
25. Kim MJ, Nriagu J. Oxidation of arsenite in groundwater using ozone and oxygen.
    Sci Total Environ. 2000 Feb 28;247(1):71-9.
    
26. McDonald RJ, Usachencko J. Neutrophils injure bronchial epithelium after ozone exposure. Inflammation 1999 Feb;23(1):63-73
27. White AR, Collins SJ, Maher F, Jobling MF, Stewart LR, Thyer JM, Beyreuther K, Masters CL, Cappai R. Prion protein-deficient neurons reveal lower glutathione reductase activity and increased susceptibility to hydrogen peroxide toxicity.
    Am J Pathol. 1999 Nov;155(5):1723-30
28. Brown DR, Besinger A.Prion protein expression and superoxide dismutase activity.
    Biochem J. 1998 Sep 1;334 ( Pt 2):423-9.
29. Huang X, Cuajungco MP, Atwood CS, Hartshorn MA, Tyndall JD, Hanson GR, Stokes KC, Leopold M, Multhaup G, Goldstein LE, Scarpa RC, Saunders AJ, Lim J, Moir RD, Glabe C, Bowden EF, Masters CL, Fairlie DP, Tanzi RE, Bush AI. Cu(II) potentiation of alzheimer abeta neurotoxicity. Correlation with cell-free hydrogen peroxide production and metal reduction. J Biol Chem. 1999 Dec 24;274(52):37111-6.
    
30. Atwood CS, Moir RD, Huang X, Scarpa RC, Bacarra NM, Romano DM, Hartshorn MA, Tanzi RE, Bush AI Dramatic aggregation of Alzheimer abeta by Cu(II) is induced by conditions representing physiological acidosis. J Biol Chem. 1998 May 22;273(21):12817-26