NR ANBJ
AU Pattison,I.H.; Millson,G.C.
TI Distribution of the scrapie agent in the tissues of experimentally inoculated goats
QU Journal of Comparative Pathology and Therapeutics 1962; 72: 233-44
PT journal article
VT
INTRODUCTION
Scrapie can be produced in sheep or goats by inoculation of brain suspension from scrapie-affected animals (Palmer, 1959; Stamp, Brotherston, Zlotnik, Mackay and Smith, 1959; Pattison, Gordon and Milison, 1959). Experimental production of the disease in sheep by inoculation of scrapie sheep spleen was reported by Gordon (1957), and by inoculation of scrapie sheep lymphatic node or cerebrospinal fluid (C.S.F.) by Stamp et al. (1959). Pattison and Millson (1960) produced the disease in goats by inoculating suspensions of pituitary gland, adrenal gland, spleen, pancreas, or liver from a scrapie affected goat.
It would appear, therefore, that the scrapie agent is widely distributed in the tissues of clinically affected animals. This paper provides some information on the spread of the scrapie agent within the tissues of goats inoculated intracerebrally with scrapie goat brain suspension. The object of the work was to trace the spread of the agent into certain selected body fluids and tissues following its inoculation directly into the substance of the brain. This was done by killing donor animals at varying intervals after injection and subinoculating their tissues into recipient animals.
MATERIALS AND METHODS
Goats.
Some of the animals used were from the Compton flock, others were brought to Compton from a variety of sources when about one week old. The donor animals were approximately three months old when inoculated, and recipient animals varied in age from about three to nine months at the time of inoculation. In scrapie experiments carried out over the past seven years, involving some hundreds of goats, we have found no evidence that age (up to one year), breed, or sex of the animals used has had, any influence on development of the disease.
Scrapie material used for injection of donor goats.
The "strain" of scrapie used for the inoculation of the donor goats was that described by Pattison et al. (1959), passaged three times through goats. It produced disease of the "scratching" type (Pattison and Milison, 1961a). Donor animals received 1.0 ml intracerebrally of the supernatant obtained by centrifugation at 1,500 r.p.m. for 15 minutes of a 10 per cent. w/v suspension of scrapie goat brain.
Preparation of material for inoculation of recipient animals.
Nine body fluids or tissues obtained from each donor goat were used for intracerebral inoculation of the recipient animals. The volume injected was 1.0 ml in every case, of either whole fluid (blood, C.S.F., or urine), or of the supernatant of a 10 per cent. w/v suspension in physiological saline of tissue ground in a mortar with a little sterile sand and centrifuged at 1,500 r.p.m. for 15 minutes. The fluids and tissues were obtained as follows:
Blood: a 20 ml. sample taken from the jugular vein just before slaughter and mixed with 3.0 ml. of 3 per cent. sodium citrate.
Cerebro-spinal fluid: obtained by cisternal puncture immediately after the animal had been electrocuted and the carotid arteries severed.
Urine: by puncture with a hypodermic needle through the bladder wall immediately on opening the abdomen after slaughter.
Adrenal glands: a pooled sample of approximately half each gland.
Sciatic nerve: a piece about 4 cm. in length obtained by cutting down through the overlying muscle.
Muscle: a piece from the inner surface of the biceps femoris.
Salivary glands: a pooled sample consisting of approximately equal amounts of the parotid, submaxillary and sublingual glands.
Brain: a piece from the thalamic-hippocampal region.
Pituitary gland: the whole gland taken from the skull after removal of the brain.
When obtaining these materials, contact between one tissue and another was avoided by using aseptic precautions and different sets of instruments.
All the donor animals were slaughtered at about 9.30 a.m.; tissue suspensions were then prepared and were kept at room temperature until the time of inoculation of the recipient animals, which was, in all cases, at about 2.30 p.m. on the same day.
Recipient animals were kept under observation for approximately 30 months. The nine recipient animals inoculated from each donor were housed together in a loose-box, and a tenth non-inoculated goat was added to each of these groups to check for possible spread of the disease by contact.
Plan of experiment.
The experimental plan is given in Table I, from which it can be seen that 11 donor goats were killed during the pre-clinical stage of the disease, four in the first month after inoculation and the remainder at approximately monthly intervals thereafter. Symptoms of scrapie were noted in the remaining donor animals at about the 240th day after inoculation; thus, the 12th donor was killed at an early clinical stage of the disease, the 13th after symptoms had been evident for about a month and the 14th after clinical disease had progressed for about three months.
RESULTS
Development of Scrapie in the Recipient Animals
The overall occurrence of scrapie in the recipient animals is shown in Table 1, from which it will be seen that the Scrapie agent was detected in the brains of all donor animals except the 7th which died of intercurrent disease, and in other tissues with the following frequency: pituitary gland 11/14; C.S.F., 6/13; sciatic nerve, 5/14; adrenal gland, 5/13; salivary gland, 5/14; muscle, 1/14. The agent apparently reached the C.S.F. and the pituitary gland of the donor animals within 24 hours of injection into the brain, but it was not detected in the sciatic nerve, adrenal gland, salivary gland, or muscle of donors until at least four months after injection.
Examination of Table I and Fig. I shows that there was a general, and statistically significant, tendency for the incubation period in recipient animals to become shorter as disease in the donor animals progressed (p = less than 0.0012). A possible exception to this general rule was that the incubation periods in recipient goats inoculated with brain, pituitary gland, C.S.F., or adrenal gland, but not sciatic nerve, from the 14th donor that was killed in a late stage of the clinical disease, were slightly longer than in animals injected with the same tissues from the 13th donor. Table I also shows that of the 46 cases of scrapie that occurred in recipient animals, the 12 shortest incubation periods of less than 300 days were related to injection of brain (six cases), pituitary gland (four cases), C.S.F. (one case), and adrenal gland (one case).
Neither blood nor urine produced disease in any animal, and in the case of all tissues except brain several recipient animals failed to develop disease.
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Table 1
SUBINOCULATIONS OF VARIOUS TISSUES FROM DONOR GOATS
INOCULATED I.C. ON 3.6.58 WITH SCRAPIE GOAT BRAIN SUSPENSION
.......................................................................
Donor Killed/ Symptoms Appearance of scrapie (days after inoculation)
number days of in recipient goat inoculated with:-
after scrapie
inocu- CSF Brain Sciatic Pituitary Adrenal Muscle Salivary
lation nerve gland gland gland
1 1 No 495 621 461
2 8 No 365
3 15 No 376 344
4 22 No 323 614
5 36 No 323 336
6 56 No 342
7 92 No 316
8 121 No 341 260 354 241
9 147 No 232 323 5o8
10 176 No 306 222 264 523 502
11 217 No 334 516 266 334 494
12 243 Yes 281 224 385 322 447 385
13 279 Yes 236 398 251 393
14 344 Yes 573 271 305 369 305 445 334
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Fig. 1
INCUBATION PERIODS FOR THE APPEARANOC OF SCRAPIE IN RECIPIENT GOATS INOCULATED WITH VARIOUS TISSUES
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Failure of scrapie to occur among in-contact animals
Scrapie did not occur in any of 13 goats kept in immediate contact with the various groups of recipient animals (see Table I). It would appear reasonable, therefore, to accept that in all cases scrapie in the recipient animals was the result of intracerebral injection of material from the donor animals. The failure of the disease to spread by contact among housed goats is in accord with our earlier observations (Pattison and Millson, 1961a) on the apparent difficulty of spreading scrapie in this manner. It is appropriate, therefore, to summarise here our experience to date on this aspect of the disease.
Firstly, a summary is given of all the goats we have injected intra-cerebrally since 1956 with scrapie goat brain, i.e., with the inoculum most likely to reproduce the disease (Table 2). The figures in this table show that in 24 different experiments, using brain material from a variety of sources and goats of different breeds and from different populations, scrapie occurred in all of 170 animals inoculated. From this we conclude that a very high proportion of British goats are susceptible to scrapie provided that the causal agent is suitably presented to the host. And from this we would argue that goats of similar kind would be suitable for examining the possibility that scrapie behaves like a contagious disease among housed animals.
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TABLE 2
OCCURRENCE OF SCRAPIE IN GOATS INOCULATED INTRACEREBRALLY WITH SCRAPIE GOAT BRAIN
Experiment Date of Number of Cases of
number experiment animals scrapie
# 2 18. 1.56 16 16
# 4 17. 2.56 10 10
# 8 31. 7.56 10 10
# 14 16.11.56 6 6
# 22 5. 2.57 10 10
# 28 21. 5.57 6 6
# 31 30. 4.57 4 4
# 32 9. 5.57 2 2
# 34 4. 6.57 4 4
# 35 2. 7.57 6 6
# 36 8. 7.57 3 3
# 45 22. 4.58 6 6
# 47 14. 5.58 2 2
# 49 3. 6.58 20 20
# 51 17. 7.58 2 2
# 53 16.12.58 2 2
# 54 1.12.58 8 8
# 62 13. 7.59 2 2
# 63 24. 9.59 6 6
# 64 8.10.59 2 2
# 68 18.11.59 30 30
# 73 27. 4.60 6 6
# 74 15. 6.6o 3 3
# 88 24.11.60 4 4
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# 24 170 170
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Secondly, a summary is given of all the goats and sheep that we have held in contact indoors with scrapie-affected animals, without on any occasion observing lateral spread of the disease (Table 3). In this context, contact, as far as the goats are concerned, means that the animals were housed and exercised in a building that continuously contained clinical cases of goat scrapie, and that they had direct physical contact with affected goats for several months or in some cases for more than a year. The sheep included in the table were kept together in a yard and were in direct and continuous physical contact with clinically affected sheep and/or goats. The 210 animals included in this table fall into two categories (a) not inoculated (b) inoculated intracerebrally with materials such as saline, distilled water, milk, blood, urine etc. that did not produce the disease. The maximum period of observation was five years. From these observations we conclude that if the disease can indeed spread by direct contact indoors, then such cases must be very rare and/or they only occur after contact over very long periods.
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TABLE 3
FAILURE OF SCRAPIE TO SPREAD AMONG HOUSED GOATS AND SHEEP
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Description Total Number of animals in
number contact with scrapie for
of various periods (years)
animals 1-2 2-3 3-4 4-5
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Not inoculated 37 goats 3 17 7 10
18 sheep 0 18 0 0
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Inoculated with 155 goats 43 96 14 2
materials that did not
produce scrapie, e.g.
saline, milk, urine etc.
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Total 210 46 131 21 12
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Incubation Period for the Development of Scrapie in Intracerebrally Inoculated Goats
The longest incubation period for the occurrence of scrapie in a recipient animal was 621 days (Table 1). All animals were kept under observation for 30 months, at which time the experiment was discontinued. It is pertinent, therefore, to consider whether this period of observation was long enough to cover the development of most cases of the disease. Table 4 shows the incubation periods we have observed for the development of scrapie in 248 goats inoculated intra-cerebrally with scrapie goat tissues. From these figures we conclude that the 30-month period of observation used in the experiment described in this paper was long enough to cover the likely development of scrapie in recipient animals.
Our colleague Dr. H. H. Holman has pointed out that the frequencies for the occurrence of scrapie in inoculated goats give the sigmoid curve expected in certain other diseases, in which a few highly susceptible animals have a short incubation period, a large number of moderately susceptible animals have an average incubation period and a small number of relatively resistant individuals have a long incubation period. The very close similarity of the results in Table 4 to a theoretical sigmoid curve can be shown by expressing incubation time (in months) as logarithms, and the scrapie infection rate as probits. This has been done in Fig. 2. The obvious interpretation of this finding is that goats vary considerably in their susceptibility to the scrapie agent, and that a variable incubation period following experimental inoculation reflects a variation in susceptibility of the animals inoculated. It cannot be doubted that there is some variation in susceptibility among goats to an inoculum containing the scrapie agent, for this has been observed in many experiments. However, Pattison, Gordon and Millson (1959) and Pattison and Millson (1960) have drawn attention to an apparent correlation between the scrapie-producing power of an inoculum and the incubation period shown by goats inoculated with it. The validity of this observation has been strengthened in this present work by the statistically significant association of longer incubation periods with the earlier donors and shorter incubation periods with the later donors. It seems likely, therefore, that the sigmoid curve given by the figures in Table 4 is an expression of two complementary factors (a) some degree of individual variation among goats to the same scrapie inoculum and (b) an overall sensitivity of goats to differing amounts of the scrapie agent in different inocula.
Histological Examination of the Brains of Donor Goats.
Hadlow (1961) examined histologically the brains of some of the donor animals described in this paper and found evidence of scrapie in the 10th and 11th donors, but not in the 9th. Thus, histological lesions characteristic of scrapie were not present in an animal killed 92 days before the time of onset of clinical disease in the remaining animals, but were present in two animals killed 65 days or 23 days before that time. Zlotnik (1958), working with sheep, also found histological abnormalities in the brain during the pre-clinical stage of experimental scrapie, and he concluded that vacuolation of nerve cells in the medulla oblongata preceded the appearance of clinical disease.
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TABLE 4
INCUBATION PERIODS FOR THE OCCURRENCE OF SCRAPIE IN GOATS INOCULATED BY THE INTRACEREBRAL ROUTE WITH SCRAPIE GOAT TISSUES
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Kind of Number Incubation period: months
scrapie of 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
inoculum animals
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Untreated 170 6 22 19 29 18 24 23 10 10 3 0 2 0 2 1 1 0
goat brain
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Other goat 78 1 6 8 13 9 11 8 4 8 3 2 1 2 1 0 0 1
tissues
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Average: untreated brain - 12 months other inocula - 12 months
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Fig. 2
REGRESSION OF LOGARITHMS OF SCRAPlE INCUBATION TIME IN MONTHS WITH PROBITS OF PERCENTAGE AFFECTED
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DISCUSSION
The results presented here confirm our earlier observations, and those of others, that the scrapie agent is widely distributed in the tissues of clinically affected animals. They indicate also that this wide distribution probably occurs a considerable time before clinical disease is obvious.
The variation in incubation period noted in recipient animals might well be related, in part at least, to the amount of the scrapie agent present in the material injected, as suggested by Pattison and Millson (1960). If this were so, it would indicate that, in general, smaller amounts of the agent were present in the tissues of donors in the early stages of the disease than in later donors, and that there were greater concentrations of the agent in brain, pituitary gland, and possibly, in C.S.F. and adrenal gland, than in sciatic nerve, salivary gland or muscle.
It may be significant that the incubation periods noted in recipient animals inoculated with tissues from the 14th donor, that had shown symptoms of scrapie for some three months when destroyed, were slightly longer in the case of brain, pituitary gland, C.S.F., and adrenal gland inocula (but not the sciatic nerve) than were those of recipient animals injected with the same tissues from the 13th donor that had shown clinical evidence of disease for only one month when slaughtered. If this slight increase in incubation period-indicative of a lower scrapie-producing potential of the tissues inoculated-was in fact real, it would confirm the observation of Wilson, Anderson and Smith (1950) that reproduction of scrapie in sheep may be most readily accomplished by using inocula obtained from animals in an early clinical stage of the disease.
The observations that neither blood nor urine produced scrapie in any recipient animals and that in the case of all tissues except brain some recipient animals failed to develop the disease, must not be taken to indicate necessarily that the scrapie agent was absent from these inocula; it might not have been present in sufficient quantity, or in a form suitable to produce clinical scrapie. The blood inocula were different from all others in that they contained sodium citrate as an anti-coagulant. It might be argued that this substance blocked the activity of the scrapie agent. We do not consider this to be likely, however, because in other experiments (unpublished) we have produced scrapie in goats by intracerebral inoculation of a saline suspension of scrapie goat brain to which was added an amount of sodium citrate equivalent to that added to the blood in this experiment. To the best of our knowledge, no report exists in the literature to indicate that scrapie can be reproduced in sheep or goats by inoculation of blood from a scrapie-affected animal, and it is clear that this phenomenon requires further investigation.
Although the scrapie agent has been shown to be widely distributed within the body of the inoculated animal its method of spread remains obscure, particularly because it could not be demonstrated in circulating blood. The infectivity of sciatic nerve is of much interest and raises the possibility of spread by way of the nervous system. But if this were so, how could the agent reach adrenal gland, salivary gland and muscle in quantities sufficiently large to be so readily detectable? Would the concentration in fine nerve branches and nerve endings in these organs be sufficient to be detectable by animal inoculation? It might be postulated that these tissues contained small amounts of a living agent capable of multiplication when inoculated into a suitable host ; but it is difficult to believe that similarly small amounts would not be constantly present in say, C.S.F. or pituitary gland so that these tissues would always produce the disease. Indeed, the results suggest to us that a certain minimum amount of the agent is necessary to produce clinical scrapie; and it may well be that other factors, as yet undetermined, may prevent the reproduction of clinical disease even though the agent is present in an inoculum.
Examination of the distribution of cases of scrapie in recipient goats, as shown in Table 1, suggested an early spread of the agent through the brain and to the C.S.F. and pituitary gland, and later spread to the other organs examined. The wide distribution of the agent in tissues of donor animals killed in the late pre-clinical and the clinical stages of the disease might reasonably be taken as an indication of increase in quantity of the agent following inoculation, for it would seem to be likely that dilution throughout the tissues of the actual material injected would be so great that an insufficient concentration to produce disease would be present in tissues remote from the brain. This conclusion is similar to that reached by Stamp et al. (1959) who demonstrated the continued presence of the scrapie agent in sheep brain after five blind passages at two-monthly intervals.
We have discussed on a previous occasion (Pattison and Millson, 1960) the length of time that animals inoculated with material suspected to contain the scrapie agent should be held under observation. This point is of considerable practical importance because of the difficulties associated with maintaining large numbers of sheep or goats for long periods. We suggested "that experiments in which the scrapie-potential of the inocula may have been reduced . . . must not be discontinued until long after the time at which clinical evidence of the disease might, under normal circumstances, have been expected." We believe that this general statement still holds good, but in Table 4 we have presented more complete evidence to suggest that a reasonable time to hold under observation goats inoculated intracerebrally with scrapie goat tissue is not less than two years. In other work, however (Pattison and Millson, 1961a), we have shown that when sheep scrapie tissue is inoculated into goats, or when goat scrapie tissue is inoculated into goats by a route other than the intracerebral, the incubation period may be greatly extended and that experiments should not be accepted as negative for at least four years.
The further evidence presented here of the great difficulty of spreading scrapie among sheep or goats housed indoors is not to be confused with our earlier assessment (Pattison and Millson, 1961b) of possible contact spread among animals at pasture. We have no further evidence that would contribute towards reconciling the observations that have been made regarding the possibility of contact spread among animals at pasture, with the apparent difficulty of achieving this among housed animals.
CONCLUSIONS
Donor goats inoculated intracerebrally with scrapie goat brain were destroyed from one to 344 days after injection and selected tissues and body fluids were inoculated intracerebrally into recipient goats. The 11 donor animals destroyed between the first and 217th day after inoculation were not showing signs of scrapie, but those killed on the 243rd, 279th and 344th days were clinically affected with the disease.
Over an observation period of 30 months, scrapie occurred in recipient animals inoculated with the various donor tissues as follows: brain, 13/13 ; pituitary gland, 11/14 ; cerebro-spinal fluid, 6/13; sciatic nerve, 5/14; adrenal gland, 5/13; salivary gland, 5/14; muscle 1/14.
Scrapie did not occur in recipient goats inoculated with blood or urine, nor in non-inoculated animals kept in contact with recipient animals.
There was a general tendency for the incubation period in recipient animals to be longer when material from donors in the early pre-clinical stages of the disease was used. This might reflect the presence of less of the scrapie agent in the tissues of the early donors.
The scrapie agent was widely distributed in the tissues of recipient goats injected from the later donors, and it appeared likely that the agent had increased in quantity following injection.
Further evidence was obtained that it is apparently very difficult to spread scrapie by contact among housed animals, but attention is drawn to evidence from other sources which indicates that the disease may, very rarely, spread by contact among animals at pasture.
Incubation periods for the development of scrapie in goats inoculated intracerebrally with goat tissues in this experiment were related to figures obtained from earlier work, and it was concluded that animals thus treated showed an average incubation period of 12 months, but that the appearance of the disease might be delayed up to about two years.
Histological evidence of scrapie was not found in the 9th donor goat killed 92 days before the expected time of onset of clinical disease, but was found in the 10th and 11th donors killed 65 or 23 days respectively, before that time.
REFERENCES
Gordon,W.S. The Veterinary Record 1957; 69: 1318
Hadlow,W.J. Research in Veterinary Science 1961; 2: 289
Palmer,A.C. Vet. Rev. & Annot. 1959; 5: 1
Pattison,I.H., Gordon,W.S., and Milison, G. C. Journal of comparative Pathology 1959; 69: 300
Pattison,I.H., and Milison,G.C. Journal of International Biomedical Information and Data 1960; 70: 182
Pattison,I.H., and Milison,G.C. Journal of International Biomedical Information and Data 1961a; 71: 101
Pattison,I.H., and Milison,G.C. Journal of International Biomedical Information and Data 1961b; 71: 171
Stamp,J.T., Brotherston,J.G., Zlotnik,I., MacKay,J.M.K., Smith,W., Journal of International Biomedical Information and Data 1959; 69: 267
Wilson, D.R., Anderson, Ruth D., and Smith, W. Journal of International Biomedical Information and Data 1950; 60: 267
Zlotnik, I. Journal of International Biomedical Information and Data 1958; 68: 428
[Received for publication, December 14th, 1961]
IN
Alle 170 seit 1956 von der Gruppe intrazerebral infizierten Ziegen erkrankten an Scrapie. Bei mit Ziegen-Scrapie intrazerebral infizierten Ziegen ließ sich die Infektiosität durch intrazerebrale Übertragung auf weitere Ziegen vom ersten Tag an - wenn auch nicht mit jedem Spendertier erfolgreich - in Gehirn, Rückenmarksflüssigkeit und Hirnanhangdrüse nachweisen. Im Ischiasnerv wurde die Infektiosität vom 121. Tag (nach 17 Wochen) an nachgewiesen. In den Speicheldrüsen gelang dies ab dem 147. Tag (nach 21 Wochen) und in den Nebennierendrüsen ab dem 176. Tag (nach 25 Wochen). Muskelfleisch erwies sich nur bei einer bereits seit 344 Tagen (49 Wochen) infizierten Ziege als infektiös. Die ersten Symptome wurden nach 243 Tagen (fast 35 Wochen) erkennbar. Mit Blut, Milch und Urin gelang der Nachweis von Infektiosität nicht und als Negativkontrollen mit Wasser injizierte Ziegen erkrankten ebenfalls nicht.
Über einen Beobachtungszeitraum von 30 Monaten erkrankten von den intrazerebral inokulierten Empfängern verschiedener Gewebe: Gehirn 13/13, Hypophyse (Hirnanhangdrüse) 11/14, CSF (Cerebrospinal- oder Rückenmarksflüssigkeit) 6/13, Ischiasnerv 5/14, Nebennierendrüse 5/13, Speicheldrüse 5/14, Muskel 1/14.
Tendenziell wurden die Inkubationszeiten der Empfängertiere kürzer, je länger sie bei den Spendertieren gedauert hatten. Eine deutliche Verkürzung der Inkubationszeiten bei den Empfängertieren aufgrund exponentieller Vermehrung der Infektiosität bei den Spendertieren wurde aber nicht beobachtet.
Von 13 nicht inokulierten, aber zur Kontrolle gegen denkbare horizontale Übertragung mit den infizierten Tieren zusammen gehaltenen Ziegen, erkrankte ebenfalls keine an Scrapie. Insgesamt wurden in dem Institut 210 nicht oder nicht tödlich infizierte Schafe und Ziegen 1-5 Jahre lang gemeinsam mit infizierten und teilweise bereits kranken Schafen und Ziegen gehalten. Dabei wurde nie eine horizontale Übertragung beobachtet.
Bei Schafen wurden 23 und 65 Tage vor dem ersten Einsetzen klinischer Symptome histopathologisch Scrapie-Läsionen beobachtet. Bei einem 92 Tage vor dem ersten Einsetzen klinischer Symptome war hingegen der histopathologische Befund negativ.
AD Agricultural Research Council, Institute for Research on Animal Disease (Field Station), Compton, Newbury, Berkshire
SP englisch
PO England
OR Prion-Krankheiten 6
ZF kritische Zusammenfassung von Roland Heynkes