NR ABVM
AU Brown,P.; Gajdusek,D.C.
TI Survival of scrapie virus after 3 years' interment
QU Lancet 1991 Feb 2; 337(8736): 269-70
PT journal article
AB Supernatant fluid from a scrapie-infected hamster brain homogenate was mixed with soil, packed into perforated petri dishes that were then embedded within soil-containing pots, and buried in a garden for 3 years. Between 2 and 3 log units of the input infectivity of nearly 5 log units survived this exposure, with little leaching of virus into deeper soil layers. These results have implications for environmental contamination by scrapie and by similar agents, including those of bovine spongiform encephalopathy and Creutzfeldt-Jakob disease.
VT
Supernatant fluid from a scrapie-infected hamster brain homogenate was mixed with soil, packed into perforated petri dishes that were then embedded within soil-containing pots, and buried in a garden for 3 years. Between 2 and 3 log units of the input infectivity of nearly 5 log units survived this exposure with little leaching of virus into deeper soil layers. These results have implications for environmental contamination by scrapie and by similar agents, including those of bovine spongiform encephalopathy and Creutzfeldt-Jakob disease
Laboratory procedures to inactivate scrapie virus have established its extraordinary resistance, and field experience suggests that the virus might also withstand environmental exposure for several years [1]. We report a study in which the residual infectivity of a mixture of scrapie hamster brain and soil was measured after a 3-year-interment.
263k strain hamster-adapted scrapie virus was used at its fourth passage level. Brains from fourteen terminally ill hamsters were removed (total weight 13g), sonicated to homogeneity as a 10% suspension in phosphate-buffered saline (PBS) pH 7.4, and clarified by centrifugation at 150 g for 5 min. The 100 ml of supernatant fluid was added to 150 g dry screened topsoil. Samples of the supernatant fluid and supernatant-saturated soil were frozen and stored at -70 °. Two plastic petri dishes with holes drilled in their covers (and in the bottom of one dish) were used. Disks of nylon mesh ('Nitex' HC 3-160, 160 (m mesh opening, 52% open area; Tetco, Elmaford, NY) were placed in the bottom of the dishes and in the covers. The dishes were then packed with 90 ml of scrapie-soil mush, covered, and sealed at the sides. Two 15 cm diameter plastic flowerpots were lined with aluminium foil and plastic sheeting and filled with soil. The dishes were placed just below the upper level of each pot, covered with a small amount of additional soil, and the pots were sunk at ground level in a garden and loosely covered with mulch (fig. 1). The ensemble was enclosed in a wire cage and left undisturbed from September, 1986 to August, 1989.
The Washington, DC area has a temperate climate with average annual high and low temperatures between 25 ° and 3°, short-term highs being up to 40 ° and down to minus 20 °; the average annual rainfall is 100 cm, and several ground level freeze-thaw cycles occur each winter [2].
In the laboratory the contents of the petri dishes surrounding soil within each pot were restored to their original consistency with distilled water and mixed with equal volumes of distilled water after the addition of gentamicin sulphate (0.2 mg/ml final concentration). The mixtures were agitated vigorously for 24 h, and samples were ground in a previously unused mortar and centrifuged at 1000 g for 15 min. The supernatant fluids were passed through 450 nm filters before inoculation into animals. End-point dilution titrations were done in 4-week weanling female LGV golden Syrian hamsters (Harlan Sprague Dawley, from the Frederick facility, Maryland); 33 (l serial 10-fold dilutions were inoculated intracerebrally into four to eight hamsters. Animals were then observed for 10 months (as were three cages of unincolulated sentinel hamsters), and the brains from all sick animals were examined histologically for spongiform change. Virus infectivity titres (method of Reed and Meunch) were expressed as logarithms of the median lethal dose (log10LD50) per 33 (l inoculum volume.
The initial infectivity of 4.8 log units fell to between 2.2 and 3.0 log units of residual infectivity at the end of the 3 years, and an additional 1.3 log units had leached into the soil lying immediately under the petri dish that was perforated top and bottom. No infectivity was detectable in the lower layer of soil 4-8 cm beneath the bottom of the dish or in the soil surrounding the unperforated dish. None of the sentinel animals caged with the inoculated animals became ill. Before nitrocellusole filtration (to eliminate bacteria) the control virus-soil mixture had an infectivity of 7.5 log units, compared with 4.8 after filtration. If filtration losses were the same for all specimens, the buried samples may have contained nearly 1000-fold more infectivity than is shown below:
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Material Infectivity Specimen volume Total infectious*)
titres* (ml) units
Control viurs/ 4.8 90 170.358.000
soil mixture+)
Top-drilled dish (1)** 2.2 90 428.000
Soil under dish (2) 0§ 1200 0
Top/bottom-drilled 3.0 90 2.700.000
dish (3)
Upper soil layer 1.3 600 359.000
under dish (4)
Lower soil layer 0§ 600 0
under dish (5)
*) Infectivity titre (per 33 (l inoculum) and total infectious units in log10LD50.
+) Specimen stored at -70 ° during 3 year experiment
**Numbers in parentheses correspond to those in the figure
§ No virus detected in undiluted inoculum
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This experiment establishes the durability of scrapie virus exposed to natural environmental conditions for 3 years, and also shows that most residual infectivity remains in the originally contaminated soil, with little leaching. Our data help to explain how, in Iceland, healthy flocks of sheep contracted scrapie after being brought to vacant farmland that had 3 years earlier been grazed by scrapie-affected flocks [3]. Other unconventional viruses, such as Creutzfeldt Jakob agent, all of which are highly resistant to ordinary methods of disinfection, may also survive for a long time in contaminated environments. We do not know whether such persistence has any role in the epidemiology of human disease [4,5] - for example, as a means of species-crossing transmission of scrapie or bovine spongiform encephalopathy (BSE) or as a mechanism for the spead of Creutzfeldt-Jakob disease - but our findings leave these possibilities open to consideration. We do suggest that the practice of ploughing-under carcasses of animals dying of scrapie or BSE, even with the addition of quicklime, be abandoned, and that such animals be excluded as a source of bone meal in fertilisers, unless it is first autoclaved.
REFERENCES
1. Palsson PA. Rida (scrapie) in Iceland and its epidemiology. In: Prusiner SB, Hadlow WJ, eds. Slow transmissible diseases of the nervous system, vol I. New York: Academic Press, 1979: 357-366
2. Anon. Local climatological data: annual summary with comparative data (Washingtron, DC National Airport), 1986-1989. Ashville, North Carolina: National Climatic Data Center
3. Tateishi J, Hikita K, Kitamoto T, Nagara H. Experimental Creutzfeldt-Jakob disease: induction of amyloid plaques in rodents. In: Prusiner SB, McKinley MP, eds. Prions: novel infectious pathogens causing scrapie and Creutzfeldt-Jakob disease, New York: Academic Press, 1987: 424
4. Brown P. An epidemiologic critique of Creutzfeldt-Jakob disease. Epidemiol Rev 1980; 2: 113-35
Brown P, Cathala F, Roubertus RF, Gajdusek DC, Castaigne P. The epidemiology of Creutzfeldt-Jakob disease: conclusion of a 15-year-investigation in France and review of the world literature. Neurology 1987; 37: 895-904
IN Scrapie-infiziertes Hamsterhirn wurde homogenisiert (gemixt) und man wartete, bis sich die Gewebestückchen abgesetzt hatten. Den wässrigen Überstand mischte man mit Erde und packte diese in perforierte Petrischalen. Wiederum eingebettet in mit Erde gefüllte Töpfe verblieben die Plastikgefäße 3 Jahre vergraben in einem Garten. Von ursprünglich 5 logarithmischen infektiösen Einheiten sollen nach diesen 3 Jahren noch 2-3 logarithmische infektiöse Einheiten erhalten gewesen sein. Nur ein kleiner Teil der Infektiosität wurde anschließend in tieferen Erdschichten gefunden. Demnach hätte nur 1/1000 bis 1/100 dieser 3 Jahre in allerdings von der Bodenfauna isoliertem Erdreich befindlichen Infektiosität "überlebt".
MH Animal; Brain Chemistry; Environmental Exposure; Evaluation Studies; Female; Hamsters; Prions/*growth & development/pathogenicity; *Soil Microbiology; Time Factors; Virology/methods; Virus Activation
AD Paul Brown (pwb@codon.nih.gov), Laboratory of Central Nervous System Studies, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA
SP englisch
PO England