NR ANCG
AU Fraser,H.; Foster,J.D.
TI Transmission to mice, sheep and goats and bioassay of bovine tissues
QU Transmissible Spongiform Encephalopathies. A Consultation on BSE with the Scientific Veterinary Committee of the Commission of the European Communities held in Brussels, September 14-15, 1993. Edited by R. Bradley and B. Marchant. European Commission, Agriculture, Document VI/4131/94-EN 1994, pp. 145-159
AB Transmissions from cases of bovine spongiform encephalopathy (BSE) using parenteral injection resulted in neurological disease in all recipient mice from about 260 to over 700 days. Major differences in incubation periods between the recipient mouse strains were consistent from all the cases of BSE that have so far been transmitted. RIII mice had the shortest incubation periods. On the basis of these transmissions it appears that the same strain of agent caused the BSE cases. Transmission to sheep and goats was successful by both intracerebral injection and oral dosing. The use of RIII mice to bioassay tissues from BSE affected cattle has failed to identify infection in any non-neural tissue.
VT
INTRODUCTION
The transmissible and infectious nature of the scrapie-like diseases has been established from field observations of spontaneous outbreaks in farmed animals and experimental infection of laboratory animals; the neuropathology and incubation periods of, and susceptibility to, these infections are genetically controlled (Hunter, Foster, Dickinson, & Hope, 1989; Goldmann, Hunter, Foster, Salbaum, Beyreuther & Hope, 1990; Carp, Moretz, Natelli, & Dickinson, 1987; Fraser, 1993; Dickinson, Outram, Taylor, & Foster, 1989; Mohri & Tateishi, 1989; Bruce & Dickinson, 1985; Kingsbury, Kasper, Stites, Watson, Hogan & Prusiner, 1983; Hunter, 1991). Transmissions to and between inbred mice, and between mice and hamsters has shown that many strains of the causal agents of the scrapie-like diseases occur, arising by mutation and selection within different hosts and mouse genotypes (Bruce & Dickinson, 1979, 1987; Kimberlin, Cole & Walker, 1987; Kimberlin, Walker & Fraser, 1989; Bruce & Fraser, 1991; Bruce, McConnell, Fraser, & Dickinson, 1991). A barrier to transmission between species may sometimes be present; this may be an absolute barrier, or be overcome by mutation to a new strain which can replicate in a new host and sometimes not in the originating host (Bruce, 1993; Marsh 1992; Kimberlin, Cole & Walker, 1987; Kimberlin, Walker & Fraser, 1989). Transmission to mice and subsequent serial sub-pasage in mice provides the basis for identifying heritable strain diffences in the causal agent (Bruce, McConnell, Fraser & Dickinson, 1991; Bruce & Fraser 1991), and for analysing the stability properties of different strains with passage in a common host genotype (Bruce, Fraser, McBride, Scott & Dickinson, 1992). BSE appears to be a new strain of a scrapie-like agent, probably from sheep, having undergone mutation in cattle following their exposure initially to the agent in sheep carcasses used in the manufacture of protein feed supplements, before the ban on feeding ruminant protein to ruminants, introduced in July 1988, became effective. This suggests that this cattle-selected strain may have been maintained in the epidemic after the initial exposure to sheep scrapie (Wilesmith, Ryan & Atkinson, 1991), which may or may not have culminated in a recognisable neurological disease of cattle. Primary passage to mice of BSE has shown that transmission is achieved at high efficiency and more rapidly than comparable transmissions of sheep scrapie, that a single major strain of the causal agent predominated in the early part of the epidemic, and that RIII mice have the shortest incubation period (Fraser, Bruce, Chree, McConnell, & Wells, 1992). The cattle-selected mutant strain appears to have infected several other ruminant and non-ruminant species without change in phenotype (Bruce, 1993). BSE could also have arisen from a hitherto unrecognised scrapielike agent of cattle. These alternatives are not mutually exclusive and different strains of agent could co-exist at different frequencies in the present BSE epidemic. If a rare form of cattle scrapie occurs this could be transmitted horizontally, in diet or vertically, whereas a cattle selected mutant originating from sheep scrapie might be a "dead-end" infection. Experimental scrapie in rodents and transmissible mink encephalopathy (TME) are examples of such "dead-end" infections which are not transmitted maternally or vertically (Hartsough & Burger, 1965; Fraser & McBride, 1985).
The relatively short incubation periods and high efficiency of primary transmission of BSE to mice, using central nervous system (CNS) tissue, suggested that mouse bioassay provides a sensitive means of identifying infection in cattle tissues. Titration of BSE-infected brain in mice reveals approximately a hundred thousand Karber units of infection per gram (Fraser, Bruce, Chree, McConnell, & Wells, 1992; Taylor, Fraser, McConnell, Brown, Brown, Lamza & Smith, in preparation). Analagous titrations of scrapie-affected sheep brain have revealed somewhat lower levels, but have shown that many non-neural tissues are infected in both affected and clinically normal sheep (Hadlow, Race, Kennedy & Eklund, 1979). RIII mice have therefore been used to bioassay the tissues of cows affected with BSE. BSE has been transmitted experimentally to several species and a closely related infection appears to have arisen in other ruminants and two carnivorous species; in each case the source of infection is assumed to be dietary (Wells & McGill, 1992; Wyatt, Pearson, Smerdon, Gruffydd-Jones, & Wells, 1991; Jeffrey & Wells, 1988; Kirkwood, Wells, Cunningham, Jackson, Scott, Dawson & Wilesmith, 1992). The epidemiology of scrapie in goats is not understood (Wood, Done, Pritchard & Wooldridge, 1992). Although there is some evidence that a spongiform encephalopathy might be transmissible between greater kudu (Cunningham, Wells, Scott, Kirkwood & Barnett, 1993) this is better established for a similar "chronic wasting disease" in mule deer and other Cervidae (Williams & Young, 1992), as it is in scrapie in sheep. In view of these many uncertainties, experiments have been undertaken to infect sheep and goats orally and parenterally, initially only with BSE, to add to knowledge of the biology of this group of infections in these species (Foster, Hope, McConnell, Bruce & Fraser, in press; Foster, Hope, & Fraser, in press).
MATERIALS AND METHODS
The method used in these studies are or are being published in detail elsewhere (Fraser, Bruce, Chree, McConnell, 1992; Foster, Hope, McConnell, Bruce & Fraser, in press; Foster, Hope & Fraser, in press; Taylor, Fraser, McConnell, Brown, Brown, Lamza & Smith, in preparation). Briefly inbred Sinc s7 and Sinc p7 mice and Sinc heterozygote Fl crosses were injected intracerebrally (i.c., 0.02 ml) and intraperitoneally (i.p., 0.l ml) with 10^-1 tissue homogenates of BSE brains obtained by 1989.
End-point tenfold dilution titrations have been carried out with BSE brain using i.c., i.p. and combined i.c.+i.p. injection routes, using these volumes.
South Country Cheviot sheep, selected for different allelisms of the Sip gene (Dickinson & Outram, 1988) and Anglo Nubian goats (Foster & Dickinson, 1988) were injected i.c with 0.5 ml or dosed orally with 50 ml of BSE brain homogenate, as 10^-1 and 10^-2 dilutions respectively.
Frozen, unfixed tissues and fluids from cattle affected with BSE (most by 1989), from different parts of England, are being assessed for BSE agent using RIII mice for bioassay; these tissues are: brain, spinal cord, retina, cerebrospinal fluid, peripheral and optic nerve, cauda equina, spleen, lymph node, tonsil, bone marrow, skin, prostate, skeletal muscle, liver, stomach lining, intestine, buffy coat, clotted blood, serum, fat, pancreas, kidney, udder, lung, heart, ovary, testis, epididymis, semen, uterine caruncle from a gravid cow, placental cotyledon and fluids and foetal blood. Formol-fixed BSE brain has been included.
One scrapie-affected Greyface sheep brain, obtained in Scotland in 1987, was used to compare with these BSE cases.
Incubation periods are obtained or animals allowed to survive until they have to be sacrificed for reasons of old age or intercurrent illness. Diagnosis is confirmed using standard, coded haematoxylin and eosin stained brain sections, and in the case of mouse brains, lesion profiles were constructed, based on the intensity of degenerative spongy pathology in nine grey matter areas, after decoding (Fraser, Bruce, Chree, McConnell & Wells, 1992). All these assessments are carried out using strict coding; if reassessments are repeated on uncoded sections this is stated. The diagnosis of BSE-transmission to sheep and goats also includes the identification of the abnormal, diagnostic-specific PrPsc (Hope, Reekie, Hunter, Multhaup, Beyreuther, White, Scott, Stack, Dawson, & Wells, 1988).
RESULTS
Transmission of BSE to mice using brain and spinal cord produces scrapie-like disease in 100% of the recipients (Fraser, Bruce, Chree, McConnell, & Wells, 1992). With brain, both the incubation periods and the neuropathology indicated that the BSE cases were very similar, but differed from one contemporaneous and many previous sheep scrapie transmissions, and from one previous goat source (Fraser, 1983; Fraser, Bruce, McBride & Scott, 1989; Fraser, Bruce & McConnell, 1991). The four mouse strains, two representing one and two the other Sinc genotypes, showed widely different average incubation periods: Sinc s7 RIII mice = 316-327 days, C57BL = 407-438 days, Sinc p7 VM = 471-5l8 days, IM = 537-565 days; in each case the standard errors of the means were 1-2% of the mean. Formol-fixed brains produced incubation periods 40-80 days longer in RIII and C57BL recipients and the l00 day difference between the strains was maintained. C57BLxVM crosses (Sinc heterozyotes) had very much longer incubation periods (743+/-14, mean +/- standard error) than the longest parent strain or the IMs. However, although this Fl incubation period was very different from the contemporary scrapie transmission in which it was intermediate between the parents, in previous studies the F1 incubation periods have exceeded that of the 'longer' parent (Fraser, 1983; Fraser, Bruce, McBride & Scott,1989).
There was a large difference in the lesion profiles in both RIII and CS7BL mice between the BSE and Greyface scrapie sources, but a difference was not detectable in the VM or IM mice. Amyloid plaques and focal asymmetrical degenerative neuropathology were conspicuous in the Sinc p7 and Sinc s7p7 mice but not in Sinc s7 mice, both in BSE and the scrapie-affected mice. In general asymmetrical degenerative lesions occur in the prosencephalon and sometimes cerebellum, and co-exist with symmetrical vacuolar pathology in the hindbrain. (Fraser, Bruce & Dickinson, 1974; Bruce & Fraser, 1982). Asymmetrical lesions are frequently associated with the emergence of mutant strains of scrapie and it is even suggested that these are sites of mutant strain generation (Fraser, 1979; Bruce & Dickinson, 1979; Bruce, Dickinson & Fraser, 1989). Transmission to RIII, CS7BL and VM mice of BSE passaged through sheep and goats (see TABLE 3, Foster, Hope, McConnell, Bruce & Fraser, in press) resulted in the same incubation period characteristics as with BSE isolated directly from cattle.
Using RIII mice to bioassay tissues from BSE-affected cattle, no evidence of the BSE agent has been found in non-nervous tissue, although spinal cord produced positive transmission in 100% of recipients after an incubation period of 356+/-8days. These results suggest a Karber titre in the CNS of about 10^4-10^5 i.c. + i.p. ID50's per gram. In 1992, when results from brain titration end-point dilution assays were becoming available, it was recognised that the termination of the tissue bioassays should not be extended to lifespan. The longest incubation periods in RIII mice receiving limiting dilutions of BSE-infected nervous tissue is 550days. The tissues which have been bioassayed are listed in TABLE 1. Samples which are being tested in bioassays which have not lasted 500 days at the time of writing (26.08.93) are retina (2 samples ), optic nerve (2), ovary (2), epididymis (2), seminal vesicles (2), prostate (2), testis (1), udder (1), skin (1), embryos and uterine "flushings" (13) obtained during removal and washing of embryos for embryo transfer experiments (provided by Dr A E Wrathall of the Central Veterinary Laboratory, Weybridge, Surrey). Some embryo samples consisted of 300 embryos. Bioassay of tissues in which the histopathological results are available are given in TABLE 2. In addition to the bioassays using combined i.c. and i.p. injections, spleen (0.3g, 1.0g, 1.1g and 1.26g per mouse, from three BSE cases) and placental cotyledon (0.75g per mouse, from cows a and b) has been fed to RIII mice and no clinical or neuropathological evidence of transmission has been obtained in experiments lasting over the lifespans of these "fed" mice. No "blind" sub-passages of any bioassays have been undertaken. RIII mice are susceptible to intercurrent infections partly due to Staphylococcus aureus which other strains of mice resist (D M Taylor, personal communication).
BSE was readily transmitted to both the "positive" and "negative" lines of South Country Cheviot sheep by i.c and oral routes without (at the time of writing) significant or clear differences in efficiency or incubation period between routes or lines. Transmissions were confirmed using both histopathology and PrPsc diagnosis (Foster, Hope, & Fraser, in press; Foster, Hope McConnell, Bruce & Fraser, in press). Oral infection of goats produced much longer incubation periods and appeared less efficient than that following i.c. injection (TABLE 3)
DISCUSSION
Based on the primary transmission incubation period and neuropathological characteristics in four strains of inbred mice and Fl crosses, there is no evidence of variation in the scrapielike agent associated with BSE. These characteristics remain unaltered following single passage through sheep or goats. Experimental oral infection of BSE to sheep and goats is surprisingly efficient, and the transmission to sheep selected for differences in susceptibility to scrapie itself failed to identify any similar pattern of resistance to BSE; so called "positive" line (susceptible) and "negative" line (resistant) lines of sheep (Foster & Dickinson, 1988) developed BSE at about the same incidence and after similar incubation periods.
RIII mice, which have the shortest incubation period following infection with BSE nervous tissues, were used for the bioassay of many tissues from BSE-affected cattle. So far no BSE infection has been detected in the non-nervous tissues of these affected cattle. More surprising is the failure to detect the BSE agent in CSF, peripheral nervous tissue, spleen or lymphoid tissue. The histological confirmation of these conclusions is not complete. It is important to recognise the difficulties in the light-microscopic diagnosis of scrapie-like degeneration in older mice (Fraser & McBride, 1985). What is recognised as the pathognomic lesion of scrapie, and that most likely to signify neuronal dysfunction, consists of a vacuolar degeneration of grey and sometimes white matter (Fraser, 1979). This degenerative lesion is sometimes described with the convenient term "spongiosis" or "spongiform change". The distribution and regional intensity of spongiosis are controlled by a number of factors, of which the strain of scrapie agent and the genotype of the host are the two most important. However, this lesion can bear a superficial similarity to a similar vacuolation accompanying old age, and considerable experience is needed to distinguish the two (see Tables 2 & 3, and Figures 9, 10, 11, 12, 13 & 14 in Fraser & McBride, 1985). To overcome the difficulties in histological diagnosis associated with old age, it is now expedient to terminate experiments as early as possible after the last cases infected with 1 ID50 are likely to occur. Therefore for scientific reasons, as well as for reasons of animal welfare, the termination of bioassay experiments is undertaken 650-700 days post-injection (depending on the number of survivors per cage), as this provides a margin of over 100-150 days after the last cases in RIII mice receiving one ID50 unit of infection (Fraser, Bruce, Chree, McConnell & Wells, 1992; Taylor, Fraser, McConnell, Brown, Brown, Lamza, & Smith, in preparation).
Acknowledgements.
The authors would like to acknowledge the assistance of Aileen Chree for carrying out the histological assessments of the mouse brains and of Dawn Drummond and Kate Fergusson who assisted with the bioassay work.
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TABLE 1 Tissues which have failed to transmit disease to RIII mice (24 mice per sample) from BSE-affected cattle injected i.c. (0.02 ml) and i.p. (0.1 ml) as tenfold homogenates into RIII mice. Histopathological status confirming negative status of some these tissues is not complete (see TABLE 2).
Tissue Source Number Duration of
of bioassay at
samples 7/9/93 (days)
splanchnic nerve cow c 1 >650
sciatic nerve bull a 2 >650
cauda equina bull a 1 >650
CSF(a) cow a 1 >650
spleen cow a 1 >650
lymph nodes(b) bull a 3 >650
tonsil cow b 1 630
semen bull c 1 >650
semen bull b 1 >650
semen bull a 1 >650
testis bull a 1 >650
buffy coat cow b 1 >650
blood clot cow b 1 >650
serum cow e 1 >650
skeletal muscle(c) bull a 1 >650
skeletal muscle(d) cow b 2 >650
skeletal muscle(e) bull a 1 >650
skeletal muscle(f) cow d 1 >650
heart cow b 1 >650
bone marrow cow a 1 >650
bone marrow cow b 1 523
skin cow b 1 627
liver cow a 1 >650
pancreas cow a 1 >650
alimentary tract(g) bull a 8 >650
alimentary tract(h) bull a 3 623 & 633
alimentary tract(i) bull d 1 >650
perirenal fat bull a 1 >650
kidney cow a 1 >650
lung cow b 1 >650
udder cow a 1 >650
uterine caruncle cow e 1 607
placental cotyledon cow b 1 >650
placental cotyledon cow e 2 546 & 607
allantoic fluid cow b 1 633
amniotic fluid cow b 1 >650
foetal heart blood cow a 1 >650
uterine flushings (j) 18 >650
embryos (j) 2 598 & 601
a: cerebrospinal fluid; b: mesenteric, prefemoral, retropharyngeal. c, d, e, f: longissimus, semitendinosus, diaphragm, masseter. g: epithelium of oesophagus, rumen & rumen "pillar", proximal colon, distal ileum, reticulum, omasum, abomasum. h: epithelium of rectum, proximal intestine, distal colon. i: distal small intestine. j: samples taken from BSE-affected cows for embryo transfer experiments by the Ministry of Agriculture by Dr A E Wrathall.
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TABLE 2. Histological confirmation (August 1993) of failure to transmit BSE to RIII mice from samples of tissues (TABLE 1) from affected cows. Twenty four mice used per sample. Intercurrent illness necessitating the killing of mice before 200 dpi excluded.
Tissue Sample Number/ of Survival, days Number
number histological of cases with of mice
examinations "negative" surviving
/number histology >650 or
pending & (mean +/- s.d.) 700 days,
still alive or alive
at 300793
lymph node 1 16/0 222-650(a) 0
(404+/-138)
2 14/0 277-650 0
(472+/-128)
3 7/14 230-498 0
semen 1 21/0 447-861 0
(606+/-132)
2 7/14 216-497 5
testis 1 5/13 209-463 5
placental 1 17/0 378-768) 0
cotyledon (587+/-145)
buffy coat 1 16/0 204-916 0
(641+/-211)
bone marrow 1 17/0 300-793) 0
(554+/-138)
skeletal 1 23/0 268-870 0
muscle (511+/-137)
2 14/0 334-888 0
(562+/-162)
3 5/20 221,356,402, -(b)
405,414(a)
fat 1 4/19 382,407, 6
412,543
kidney 1 3/19 343,393,457 10
oesophagus 1 6/14 245,370,393, 4
421,430,434
distal 1 2/1 335,448 0
ileum(c)
udder 1 8/17 263-531 2
sciatic nerve 1 8/16 248-418 10
2 6/19 211,294,392, 2
426,461,613
cauda equina 1 4/20 268,313, 6
422,532
a: range (& mean +/- standard deviation), individual values when <6;
b: details incomplete;
c: many early losses.
The table analyses experimental data from experiments started by August 1991, but excludes bioassay of pancreas, CSF, liver, rumen epithelium, foetal heart blood, proximal colon epithelium and spleen, which were all "negative".
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Dickinson, A.G., Outram, G.W., Taylor, D.M. & Foster, J.D. (1989). Further evidence that scrapie agent has an independent genome. In (Editors, L. Court, D. Dormont, P. Brown & D. Kingsbury) : "Unconventional Virus Diseases of the Central Nervous System". pp.446-460. La Melleray-deBretagne, Abbay de Melleray.
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IN
Material und Methoden
Homozygoten Sinc s7 und Sinc p7 Mäusen, sowie heterozygoten F1-Mischlingen wurden 1/10 verdünnte Hirnhomogenate von 1989 getöteten BSE-Rindern intrazerebral (20 µl) und intraperitoneal (l00 µl) injiziert.
Endpunkttitrationen in 10er-Verdünnungsschritten wurden mit Hirnhomogenaten von 1989 getöteten BSE-Rindern intrazerebral (20 µl) und/oder intraperitoneal (l00 µl) durchgeführt.
Cheviot-Schafe mit unterschiedlichen Allelen des Sip-Genes und Ziegen (Anglo Nubian) wurden intrazerebral mit 500 µl oder oral 50 ml 1/10 oder 1/100 verdünntem BSE-Hirnhomogenat inokuliert.
Per RIII-Mausbioassay versuchte man, BSE-Infektiosität in gefrorenen, aber nicht fixierten Geweben und Flüssigkeiten von BSE-Rindern aus verschiedenen Teilen Englands (die meisten aus dem Jahr 1989) nachzuweisen. Im Einzelnen waren dies Gehirn (auch formalinfixiert), Rückenmark, Retina, Cerebrospinalflüssigkeit, periphere und Sehnerven, Ende des Rückenmarks, Milz, Lymphknoten, Mandeln, Knochenmark, Haut, Prostata, Skelettmuskel, Leber, Magenschleimhaut, Dünndarm, buffy coat, Blutkuchen, Blutserum, Fett, Pankreas, Niere, Eutergewebe, Lunge, Herz, Eierstock, Hoden, Nebenhoden, Samen, Uteruskarunkel einer schwangeren Kuh, Keimblätter und Flüssigkeiten aus Plazenta, sowie fötales Blut.
Zum Vergleich wurde das Gehirn eines 1987 in Schottland an Scrapie erkrankten Greyface-Schafes verwendet.
Die Empfängermäuse wurden erst getötet, wenn sie zu krank oder altersschwach wurden. TSE-Diagnosen erfolgten histopathologisch nach Haematoxylin- und Eosinfärbung von Hirnschnitten. Zusätzlich wurden Schafe und Ziegen immunologisch diagnostiziert und bei Mäusen wurden Läsionsprofile durch die Einstufung von 9 verschiedenen Hirnregionen nach dem Grad der Schädigung erstellt.
Resultate
Hinsichtlich der Inkubationszeiten und der Schädigungsmuster in den Hirnen der Empfängermäuse lieferten die Übertragungsexperimente mit den Gehirnen der verschiedenen BSE-Fälle sehr ähnliche Ergebnisse. Diese unterschieden sich jedoch deutlich von verschiedenen mit Scrapie bei Schaf und Ziege erzielten Daten. Mit den BSE-Hirnhomogenaten betrugen die Inkubationszeiten bei den RIII-Mäusen (Sinc s7) 316-327 Tage, bei den C57BL-Mäusen (Sinc s7) 407-438 Tage, bei den VM-Mäusen (Sinc p7) 471-5l8 Tage und bei den IM-Mäusen (Sinc p7) 537-565 Tage. Bei Sinc-heterozyoten C57BLxVM-Mischlingen waren die Inkubationszeiten mit 743+/-14 Tagen wesentlich länger. Im Gegensatz dazu lagen die Inkubationszeiten der Sinc-heterozygoten Mäuse nach Inokulation mit Schafscrapie zwischen den Inkubationszeiten der Sinc s7 und der Sinc p7 Mäuse.
Die Inokulation von Hirnhomogenaten BSE-infizierter Schafe und Ziegen resultierte in den verschiedenen Mausstämmen in BSE-typischen Inkubationszeitmustern.
Formalin-fixierte Hirnhomogenate produzierten in RIII- und C57BL-Mäusen 40-80 Tage längere Inkubationszeiten.
Bei den Sinc s7 Mäusen, nicht jedoch bei den Sinc p7 Mäusen erzeugten Scrapie-Infektionen andere Schädigungsmuster als die BSE-Infektionen. Amyloid plaques waren nur bei den Sinc p7 Mäusen und den Mischlingen, nicht jedoch bei den Sinc s7 Mäusen deutlich erkennbar.
Alle mit Rückenmark inokulierten RIII-Mäuse erkrankten mit Inkubationsperioden von 356+/-8 Tagen. Die Autoren leiten davon Rückenmarkstiter von 10^4-10^5 i.c. + i.p. ID50-Einheiten pro Gramm ab. Interessant ist, dass auch bei Verdünnungsreihen die Inkubationszeiten bei BSE-infizierten RIII-Mäusen nie länger als 550 Tage waren. Aber mittels RIII-Maus-Bioassay wurde keine Infektiosität in nichtnervösen Geweben der BSE-kranken Rinder gefunden. Selbst in Cerebrospinalflüssigkeit, peripheren Nerven, Milz und Lymphknoten wurde keine Infektiosität nachgewiesen.
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Tabelle 1
Gewebe, von denen 1/10 verdünnte Homogenate pro Probe jeweils 24 RIII-Mäusen intrazerebral (20 µl) und zusätzlich intraperitoneal (100 µl) injiziert wurden, ohne daß auch nur eine Empfängermaus an BSE erkrankte. Bei einigen dieser Gewebe stand allerdings die histopathologische Bestätigung der negativen Resultate noch aus (Siehe Tabelle 2).
Gewebe Quelle Proben- Inkubations-
anzahl periode bis
zum 7.9.93
Eingeweidenerv Kuh c 1 >650
Ischiasnerv Bulle a 2 >650
Cauda equina Bulle a 1 >650
CSF(a) Kuh a 1 >650
Milz Kuh a 1 >650
Lymphknoten(b) Bulle a 3 >650
Gaumenmandeln Kuh b 1 630
Sperma Bulle c 1 >650
Sperma Bulle b 1 >650
Sperma Bulle a 1 >650
Hoden Bulle a 1 >650
buffy coat Kuh b 1 >650
Blutkuchen Kuh b 1 >650
Serum Kuh e 1 >650
Skelettmuskel(c) Bulle a 1 >650
Skelettmuskel(d) Kuh b 2 >650
Skelettmuskel(e) Bulle a 1 >650
Skelettmuskel(f) Kuh d 1 >650
Herz Kuh b 1 >650
Knochenmark Kuh a 1 >650
Knochenmark Kuh b 1 523
Haut Kuh b 1 627
Leber Kuh a 1 >650
Pankreas Kuh a 1 >650
Verdauungstrakt(g) Bulle a 8 >650
Verdauungstrakt(h) Bulle a 3 623 & 633
Verdauungstrakt(i) Bulle d 1 >650
perirenales Fett Bulle a 1 >650
Niere Kuh a 1 >650
Lunge Kuh b 1 >650
Euter Kuh a 1 >650
Uteruskarunkel Kuh e 1 607
Plazentalappen Kuh b 1 >650
Plazentalappen Kuh e 2 546 & 607
Allantoisflüssigkeit Kuh b 1 633
Amnionflüssigkeit Kuh b 1 >650
fötales Herzblut Kuh a 1 >650
Uterusspülungen (j) 18 >650
Embryonen (j) 2 598 & 601
a) CSF: Cerebrospinalflüssigkeit, b) aus dem Mesenterium, prefemoral, retropharyngeal. c) Musculus longissimus, d) Musculus semitendinosus, e) Diaphragma, f) Musculus masseter, g) Epithelium von Ösophagus, Pansen & rumen "pillar", proximalem Kolon, distalem Ileum, Reticulum, Omasum und Abomasum. h: Epithelium aus dem Rektum, proximalem Darm (wahrscheinlich Dünndarm) und distalem Kolon. i) distaler Dünndarm. j) von Dr. A.E. Wrathall des britischen Ministry of Agriculture BSE-kranken Kühen für Embryotransferexperimente genommene Proben.
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Die Autoren berichten aber auch von zum Zeitpunkt ihres Redaktionsschlusses am 26.8.1993 noch laufenden Übertragungsexperimenten (intrazerebral plus intraperitoneal) mit Retina (2 Proben), Sehnerven (2), Eierstock (2), Nebenhoden (2), Vesikula seminalis (Samenbläschen) (2), Prostata (2), Hoden (1), Euter (1), Haut (1), Embryonen und Uterusspülungen (13).
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TABLE 2. Histologische Bestätigung (August 1993) nicht gelungener Übertragungen von BSE auf RIII-Mäuse mit den Proben der Tabelle 1 und jeweils 24 Empfängertieren. Bis zu 200 Tage nach der Inokulation gestorbene Tiere wurden ausgeschlossen.
Gewebe Proben- histologisch Überlebenszeit mindestens
nummer untersucht / und (mittlere 650 oder 700
noch lebend Überlebenszeit Tage oder
mit Standard- am 30.7.93
abweichung) immer noch
lebende Mäuse
Lymphknoten 1 16/0 222-650(a) 0
(404+/-138)
2 14/0 277-650 0
(472+/-128)
3 7/14 230-498 0
Samen 1 21/0 447-861 0
(606+/-132)
2 7/14 216-497 5
Hoden 1 5/13 209-463 5
Plazentalappen 1 17/0 378-768) 0
(587+/-145)
buffy coat 1 16/0 204-916 0
(641+/-211)
Knochenmark 1 17/0 300-793) 0
(554+/-138)
Skelettmuskel 1 23/0 268-870 0
(511+/-137)
2 14/0 334-888 0
(562+/-162)
3 5/20 221,356,402, -(b)
405,414(a)
Fett 1 4/19 382,407, 6
412,543
Niere 1 3/19 343,393,457 10
Ösophagus 1 6/14 245,370,393, 4
421,430,434
distales Ileum(c) 1 2/1 335,448 0
Euter 1 8/17 263-531 2
Ischiasnerv 1 8/16 248-418 10
2 6/19 211,294,392, 2
426,461,613
Cauda equina 1 4/20 268,313, 6
422,532
a: minimaler bis maximaler Wert (Mittelwert +/- Standardabweichung), bei weniger als 6 individuelle Werte;
b: Details unvollständig
c: viele frühe Verluste
Die Tabelle summiert die Daten von im August 1991 begonnenen Experimenten, läßt aber die Bioassays mit Pankreas, Cerebrospinalflüssigkeit, Leber, Pansenepithelium, fötales Herzblut, proximales Kolonepithelium und Milz, die aber auch alle "negativ" waren.
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RIII-Mäuse blieben über ihre gesamte normale Lebensspanne gesund, nachdem sie mit 0,3 Gramm, 1,0 Gramm, 1,1 Gramm bzw. 1,26 Gramm Milz von 3 BSE- Fällen, oder mit je 0,75 Gramm Plazentakeimblättern von 2 BSE-Fällen gefüttert worden waren.
BSE wurde mit ähnlichen Übertragungswahrscheinlichkeiten und Inkubationszeiten intrazerebral sowie oral auf scrapieempfängliche und scrapieresistente Cheviot-Schafe übertragen. Im Gegensatz dazu waren BSE-Übertragungen auf Ziegen oral weniger effizient und resultierten in wesentlich längeren Inkubationszeiten als intrazerebrale Inokulationen.
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Tabelle 3 Orale und intrazerebrale Übertragung von BSE auf Schafe (South country) und Ziegen (Anglo Nubian)
Empfänger- Art der Anteile Inkubations-
tiere Inokulation erkrankter zeiten in
Tiere (a) Tagen
"positive" i.c. 2/4 724,880
line
Cheviots oral 2/6(b) 538,994
"negative" i.c. 2/6(c) 440,487
line
Cheviots oral 1/6(d) 734
Anglo i.c. 3/3 506,509,570
Nubian
goats oral 2/3 941,1501
a: Überlebende 1800 Tage nach der Inokulation zum Zeitpunkt der Publikation. Zwischenzeitlich 665(b), 883(c) bzw. 620(d) Tage nach der Inokulation gestorbene Tiere wurden als Überlebende gezählt.
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Diskussion
Die in verschiedenen Mausstämmen produzierten Inkubationszeiten und Schädigungsmuster wiesen bei verschiedenen BSE-Fällen und auch bei Bse-infizierten Schafen und Ziegen keine Unterschiede auf. Dies spricht für nur einen BSE-Stamm mit selbst nach Übertragung auf andere Spezies unverändert bleibt. So ganz kann dies allerdings nicht stimmen, denn immerhin findet man bei BSE-infizierten Schafen die Infektiosität im ganzen Körper.
Die orale Übertragung von BSE auf Schafe funktionierte erstaunlich gut und zwar bei zumindest gegenüber einem bestimmten Scrapiestamm empfänglichen bzw. resistenten Schafen etwa gleich gut.
AD Institute for Animal Health, AFRC and MRC Neuropathogenesis Unit, Edinburgh, U.K.
SP englisch
PO Belgien
OR Prion-Krankheiten 3
ZF kritische Zusammenfassung von Roland Heynkes