NR AHXB
AU Marsh,R.F.; Hanson,R.P.
TI Transmissible mink encephalopathy: infectivity of corneal epithelium.
QU Science 1975 Feb 21; 187(4177): 656
PT journal article
AB Corneal epithelium from hamsters dying of transmissible mink encephalopathy contained a virus titer of 10-4.8 times the 50 percent lethal dose (10-4.8 LD50) per 0.05 milliliter when assayed as a cell suspension derived directly from the infected animal. After one passage in tissue culture, an equivalent concentration of cells contained only 10-0.8 LD50 per 0.05 milliliter.. It is concluded that corneal tissues are infectious; the infectivity may be mainly associated with free nerve endings. However, the most important immediate inference is that corneas from human beings affected with Creuzfeldt-Jakob disease are likely to be lethal if transplanted to healthy recipients.
VT
Transmissible mink encephalopathy (TME) is a natural slow virus disease of commercially reared mink which has been experimentally transmitted to seven other species. Scrapie, kuru, Creutzfeldt-Jakob disease described the form a single nosologic entity known as the subacute spongiform encephalopathies (1). A report (2) on the possible person-to-person transmission of Creutzfeldt-Jakob disease described the development of a fatal spongiform encephalopathy in an individual 18 months after receiving a corneal transplant from a donor later diagnosed as having died of Creutzfeldt-Jakob disease. Since the highest estimate for the incidence of Creutzfeldt-Jakob disease in the United States is only 200 cases per year (3), it is unlikely that both donor and recipient developed the same disease by chance. To investigate the infectivity of corneal tissue, we examined an animal model, hamster TME, a disease with etiologic and clinicopathologic features similar to Creutzfeldt-Jakob disease.
Five outbred Syrian hamsters (Lake-view Hamster Colony, Newfield, New Jersey) showing advanced clinical signs of TME were killed, and the aqueous humor was collected by introducing a 26-gauge needle into the anterior chamber at the corneoscleral conjunction. The corneas were then removed and placed in saline. The epithelial layer was separated from the cornea propria with the aid of a dissecting microscope. The epithelium dissected easily, most remaining in large tissue sheets. These epithelial fragments were centrifuged at 500g and the pellet was washed twice in medium 199 containing 10 percent calf serum and antibiotics (100 units of penicillin and 100 µg of streptomycin per milliliter). The epithelium was then gently aspirated through a 22-gauge needle until the large tissue fragments were reduced to free cells or small aggregates. This suspension was adjusted to a concentration of 2 x 10^6 cells per milliliter and divided into two portions. One was subjected to four freeze-thaw cycles, then frozen at - 70° for later animal inoculation. The second portion was incubated in plastic tissue-culture petri dishes at 37° in room air containing 5 percent C02.
After 38 hours, small foci of cells were seen to adhere to the plastic. At this time, the original tissue culture fluid was carefully decanted and fresh medium 199 was added. The cells from the individual foci grew rapidly, becoming almost confluent by 15 days. Most cells retained an epithelioid appearance and were of varying sizes, some approaching 30 µm in diameter. No cytopathic effect was observed. The cells were scraped from the petri dishes after 3 weeks in culture, adjusted to a concentration of 2 x 10^6 cells per milliliter, subjected to four freeze-thaw cycles, and frozen at -70°.
Cell suspensions from both intact and cultured epithelium, aqueous humor, and brain tissue from the same animals were simultaneously assayed by intracerebral inoculation of weanling hamsters. The results (Table 1) show a moderate amount of infectivity in uncultured epithelium which was not sustained after tissue culture. Brain contained a high concentration of the TME agent, as observed earlier (4). No infectivity was detected in aqueous humor.
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Table 1. Infectivity of corneal epithelium and brain tissue frosen hamsters with transmissible mink encephalopathy. The data are presented as number of hamster, developing disease within 6 months of inoculation per number of animals inocalated. The 50 percent lethal dose (LD50) was calculated by the Spearman-Kirber method (11); NT, not tested.
Epithelium
(2 x 10^6 cells
Dilution per milliliter) Brain
Uncultured Cultured
10^-1 4/4 1/4 NT
10^-2 4/4 0/4 NT
10-3 4/4 0/4 NT
10^-4 3/4 0/4 NT
10^-5 2/4 0/4 4/4
10^-6 0/4 0/4 4/4
10-7 NT NT 4/4
10^-8 NT NT 3/4
10-9 NT NT 2/4
10^-10 NT NT 0/4
LD50 per 0.05ml 10^4,8 10^0.8 10^8.8
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The discrepancy between the infectivity of uncultured and cultured epithelium may be explained by the failure of infected epithelial cells to replicate in vitro. Corneal epithelial cells of animals have a high rate of mitosis in vivo, with a life-span of only 7 to 10 days (5). Since the replication of the scrapie agent in vitro coincides with cell division (6), it is possible that corneal epithelial cells may be capable of supporting a moderate level of infection. A second, and perhaps more probable, explanation is that infectivity is associated with the numerous nerve fibers in corneal tissue (7).
Corneal epithelium is richly innervated with free nerve endings present as naked axis cylinders with no myelin, no sheaths of Schwann (8). It is logical to suspect that these unorthodox neuropathic agents are capable of replicating in nerve fibers, as has been speculated in previous studies on TME (9) and scrapie (10).
This report illustrates the value of studying animal models of human disease. In this instance, the results are not only applicable to explaining a clinical observation, but they may also be of significance in further understanding the pathogenesis of these diseases.
Richard F. MARSH
Robert P. HANSON
Department o/ Veterinary Science, University o/ Wisconsin, Madison
References and Notes
1.C.J. Gibbs Jr., and D.C. Gajdusek, Science 165, 1023 (1969).
2. P.Duffy, J.Wolf, G.Collins, A.G.DeVoe, B.Streeten, D.Cowen, N.Engl.J.Med. 290, 692 (1974).
3. D.C.Gajdusek, Am.J.Clin.Pathol. 56, 352 (1971).
4.R.F.Marsh and R.H.Kimberlin, J.Infect.Dis., in press.
5.G.K.Smelser and V.Ozanics, in Symposium on the Cornea (Mosby, St. Louis, 1972), p 3.
6.M.C.Clarke and D.A.Haig, Res.Vet.Sci. 11, 500 (1970).
7. F.C.Rodger, Br.J.Ophtholmol. 34, 107 (1950).
8. C.I. Thomas, The Cornea (Thomas, Springfield, III., 1955), p. 39; M.Whitear, J.Anat. 94, 387 (1960).
9. R.F.Marsh, J.M.Miller, R.P.Hanaon, Infect.Immun. 7. 352 (1973).
10. G.W.Outram, A.G.Dickinson, H.Fraser, Nature (Lond.) 249, 855 (1974).
11. R.M.Dougherty, in Techniques in Experimental Virology, R.J.C.Harris, Ed. (Academic Press, New York, 1964), p.183.
12. Supported in part by PHS grant AI 11250.
27 September 1974; revised 19 Novembcr 1974
IN
Die Autoren entnahmen TME-infizierten (TME = übertragbare, schwammförmige Nerzhirndegeneration) Hamstern Wasser der vorderen Augenkammer und Augenhornhaut und trennten von letzterer in Salzlösung das Epithel ab. Epithelfragmente wurden bei 500-facher Erdbeschleunigung zentrifugiert, zweimal in Medium 199 mit 10% Kälberserum und Antibiotika (100 Einheiten Penicillin und 100 µg Streptomycin pro Milliliter) gewaschen. Durch vorsichtiges Aufsaugen und Ausstoßen durch eine Injektionskanüle wurden die Zellverbände in Einzelzellen aufgelöst. In einer Konzentration von 2 Millionen Zellen pro Milliliter wurde eine Hälfte bei -70° eingefroren, während die andere Hälfte in Plastikpetrischalen bei 37° in Medium 199 unter Raumluft mit 5% CO2 kultiviert wurde. Nach 38 Stunden hatten sich die Zellen in kleinen Gruppen angeheftet und das Medium konnte erneuert werden. Innerhalb von 15 Tagen hatten sich dichte epithelartige Zellrasen gebildet. Nach 3 Wochen wurden die Zellen abgeschabt, wie zuvor auf eine Konzentration von 2 Millionen Zellen pro Milliliter gebracht, viermal eingefroren und wieder aufgetaut und schließlich bei -70° eingefroren. Suspensionen der direkt und der nach Kultivierung eingefrorenen Epithelzellen, Augenkammerwasser und Hirngewebe der infizierten Hamster wurden jungen Hamstern intrazerebral inokuliert. Man fand hohe Infektiosität im Hirngewebe, eine moderate Infektiosität im nur gewaschenen und vereinzelten Hornhautepithelzellen, aber fast keine Infektiosität in den kultivierten Hornhautepithelzellen und keine im Kammerwasser. Vielleicht konnten sich die infizierten Epithelzellen in der Kultur nicht vermehren, vielleicht wurde die Infektiosität aber auch einfach mit den Mediumwechseln herausverdünnt. Die Infektiosität des Hornhautepithels könnte aber auch allein von den vielen in die Augenhornhaut ragenden Nervenenden stammen, welche bei der Kultivierung überwachsen werden. Andererseits bestätigt ein tödlich infizierter Hamster nach all den Waschungen und einer dreiwöchigen Kultivierung, dass das Augenhornhautepithel wirklich infektiös war und die Infektiosität nicht etwa die Folge einer Kontamination war.
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2 Millionen
Epithelzellen
Verdünnung pro Milliliter Gehirn
frisch kultiviert
10^-1 4/4 1/4 -
10^-2 4/4 0/4 -
10-3 4/4 0/4 -
10^-4 3/4 0/4 -
10^-5 2/4 0/4 4/4
10^-6 0/4 0/4 4/4
10-7 - - 4/4
10^-8 - - 3/4
10-9 - - 2/4
10^-10 - - 0/4
LD50 per 0.05ml 10^4,8 10^0.8 10^8.8
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MH Animal; Cornea/*microbiology; Corneal Transplantation; Creutzfeldt-Jakob Syndrome/*transmission; *Disease Models, Animal; Hamsters; Human; *Mink; Slow Virus Diseases/*transmission; Support, U.S. Gov't, Non-P.H.S.; Transplantation, Homologous; Virus Replication; Viruses, Unclassified/growth & development
SP englisch
PO USA
OR Prion-Krankheiten M
ZF kritische Zusammenfassung von Roland Heynkes