Some factors controlling the incidence of scrapie in Cheviot sheep injected with a Cheviot-passaged scrapie agent

J.

COMP.

1968.

PATH.

SOME

VOL.

78.

FACTORS

SCRAPIE

IN

313

CONTROLLING CHEVIOT

THE

SHEEP

CHEVIOT-PASSAGED

INCIDENCE

INJECTED

SCRAPIE

WITH

OF A

AGENT

BY

A.R.C.

Animal

A. G.

DICKINSON

Breeding

Research Organisation,

Edinburgh

and

J. T.

C. C.

STAMP, Moredun

RENWICK

Research Institute,

and J. C. Gilmerton,

RENNIE

Edinburgh

INTRODUCTION

The incubation period of scrapie after injection of scrapie agent can vary for a number of reasons, such as the genotype of the recipient animal, whether or not the recipient is of the same species as that from which the agent comes, the dose of agent used, the route of inoculation and the particular strain of scrapie agent which is involved. In general, the incubation period is lengthened when the recipient and donor species are different, when a smaller dose of agent is given or when a peripheral route of injection is employed instead of an intracerebral route. Scrapie agents have now been transmitted to a number of animal species.However only in sheep, which are the natural hostsof the disease,have individuals been reported which appear to be resistant. Some, for example, have resisted the most extensively used scrapie agent, SSBP/ 1, in that clinical diseasehas not developed during the several years they have been kept after challenge and regardless of their age when inoculated (Gordon, 1966). It has also been found that the incidence of scrapie in those sheep which survive a first challenge for long periods and are then rechallenged with the same scrapie agent is no greater than that of sheep not challenged for a second time. It is concluded that this resistance has a positive basis which may be of a genetical or induced irnmunoIogical nature rather than a random failure of the effectiveness of the challenge. This paper is concerned with the influence of the genotype of the sheep and the route of injection on the incidence of scrapie. The experiment is continuing. MATERIAL

AND

METHODS

Sheep. The inoculated sheepwere of the South Country Cheviot breed. Two-thirds were the foundation stock for a selection experiment, still in progress, designed to change the incidence of induced scrapie using a particular scrapie agent; the remainder were first-cross animals born to some of these sheep. The foundation flock was assembledso as to include a wide range of the genotypes present in the breed, the females coming from four farms and representing several different age groups from each location while the foundation rams came from fifteen different flocks. The unselected first-cross lambs were therefore, on average, genetically similar to the

314

FACT~RSC~NTROLLINO~~~~~EIN~HEVIOT~HEEP

foundation population. Breeding ewes were not challenged with scrapie until their lambs were weaned nor breeding rams until the mating period was complete. This procedure which makes the selection process slow and causes a considerable time lag in determining which animals develop scrapie, was unavoidable since the scrapie agent is known to be maternally transmitted while, on occasion, sire transmission appears possible. Breeding and experimental locations. All the matings took place on a farm away from the two Institutes and natural scrapie has not appeared in the Cheviot flock there. Before injection with scrapie the animals were transferred, at ages ranging from 6 months to 6 years, to Moredun Institute where they were subsequently maintained in pens in a large open-sided shed. This shed has been used for other scrapie transmission experiments for some time, but there was no mixing of the Cheviot sheep with other experimental animals and there was strict control of minor surgical procedures such as ear-tagging and therapeutic injections. Scrapie agent, dose and route of injection. The original intention was to have a single pool of agent (SSBP/1/23) f or use throughout the selection experiment. This pool consisted of brains collected from advanced clinical cases of scrapie in Cheviot sheep induced by intracerebral injection with SSBP/ l/22 scrapie agent; inclusion in this pool was restricted to the earlier cases of scrapie. The size of the pool was determined on the assumption that challenge would be by intracerebral injection of all females and those males not used for breeding, but allowing for a relatively large dose to be given subcutaneously to the rams used as sires. After two groups of the foundation stock had been injected intracerebrally it was found that the pool (SSBP/1/23) had a bacterial contaminant which caused the death of 40 per cent. of these groups shortly after injection; subsequent groups were therefore inoculated only subcutaneously. Later in the experiment it became clear that the original pool would be insufficient, because of the larger subcutaneous doses needed, to challenge all the sheep in subsequent selection generations which the interim results warranted : the pool was therefore supplemented with similar, but 24th passage, material (SSBP/1/24) to the extent of 87 per cent. of the new pool. The dose and route of injection for the various groups are shown in Table 1. When the experiment was initiated we were uncertain of the exact relationship between dose, route and incubation period and it was tactically important to aim at sufficiently short average incubation periods which would permit genetical selection to proceed at the rate of one generation per year. It was with this in view that one group was given both intracerebral and subcutaneous injections. Scoring of clinical scrapie. After mating the sheep were checked at intervals of two weeks and scored by one of US (A.G.D.) for evidence of pruritus and incoordination of gait according to the following scheme. Incoordination of gait : -, no + , slight incoordination; + + , obvious incoordination; + + + , incoordination; extreme incoordination with stumbling or obvious limb weakness; + + + +, recumbent. Pruritus : -, no pruritus; +, slight rubbing but no patches of fleece removed; + +, rubbing with patches of fleece missing; + + +, rubbing with patches of fleece missing and some raw skin; + + + +, very extensive pruritus with raw skin. When an individual received a total of four pluses in the combined pruritus and incoordination score this was taken as the end point for calculation of incubation period (injection date being the origin) and the animal was killed at a convenient time during the following two or three weeks for neuropathological examination. In addition to those individuals which were killed in the advanced stage of scrapi,=, a number died or had to be killed for unrelated causes or because of senility; the brains of these animals were also examined histologically for the presence of characteristic lesions of scrapie. Such lesions were found in a few instances in which case the animal was regarded as being a susceptible animal in the early stages of the disease.

A. 0. DICKINSON

315

6’f al.

RESULTS

Incubation Periods The distribution of incubation periods in the various groups of sheep is shown in Fig. 1 and is summarised in Table 2. In sheep receiving an intracerebral injection the distribution of incubation periods is clearly discontinuous. Because of the small number of animals involved it is uncertain whether the cases occurring after 500 days are part of a common, very variable, distribution or whether this distribution is multimodal, being made up of several distinct incubation period classes. However, the short incubation period (SIP) sheep are quite distinct from the rest and for purposes of description this term is used for cases with incubation periods up to 450 days following intracerebral injection or up to 750 days following subcutaneous injection. The mean incubation period (SIP) in the sheep challenged intracerebrally is 197 days and in those challenged subcutaneously is 3 13 days. In the injection groups there are no mean incubation periods (SIP) which differ significantly one from the other when the inoculation route is the same (P > 0.05) so there can be no objection on thii ground to the pooling of results from the seven groups which received only a subcutaneous challenge and, similarly, the results from the two groups of sheep given intracerebral injections can be pooled. However, since the incubation periods in some groups of subcutaneously inoculated sheep are more variable than in others, pooling of results may not be entirely justified ; especially is this so in the case of Group 6 where the variance is four times the average in the others. There is no obvious biological explanation for this variation, but some is probably an artifact of the scale used since the groups with the higher means also tend to have the higher variances. This scaling bias is also shown in the symmetry of some of the distributions. Of the ones which can be tested two are virtually symmetrical, two are slightly skewed with a long tail to the right and Group 6 is very skewed in the same direction, while Group 4 is skewed to the left.

TABLE

Group

:

SSBP/1/23 SSBP/1/23

3

SSBP/1/23

4 5 6

SSBP/1/23 SSBP/1/23 SSBP/1/23 SSBP/ l/23 SSBP/I 123 SSBP/l/23-24

i 9

* Excluding

animals

1

Dostf

Routt

ml. 10 1 5

SIC

{

: % 2 2 4

7 10-l Saline twqmaiqn. dying due to contamination

i/c SIC i/c SIC S/C SIC sic SIC s/c

of the inoculum.

.hhba

injtckd*

:z 53 51 21 :: 89 9

316

FACTORS

CONTROLLING

SCRAPIE TABLE

INCIDENCE

OF

SCRAPIE

AND

Sheep challenged by different short (SIP) and longer (LIP)

Route

of injection

Intracerebral or intracerebral plus subcutaneous Subcutaneous

Number injecfed

Incidence SIP-type

IN CHEVIOT

SHEEP

2 INCUBATION

PERIODS

routes and classified into incubation period types Mean incubation period (days) LIP-type(s) SIP-type

of cases LIP-type(s)

73

39oj,

20q;

275

37ql

0%

197 -c 7 313

f

9

917 i 90 -

Whether the animal in Group 6, in which the incubation period was 748 days, should be considered as part of the main SIP distribution or whether it ought to be regarded as having a longer incubation period (LIP) is debatable. Excluding this observation the distribution of the other incubation periods is still moderately skewed in the same direction and therefore about 3 per cent. of the incubation periods would be expected to be greater than 560 days. It therefore seemsreasonable that the sheep in which the incubation period was 748 days should be a member of the SIP group. It would indeed be incorrect to place this animal in the LIP group since the shortest incubation period that could be expected in such sheep when subcutaneously inoculated is about 800 days for it is known that the extraneural route increasesthe incubation period by at least 50 per cent. Although their incubation periods (SIP) do not differ in mean, data from the two groups of sheep given intracerebral injections may not entirely justifiably be pooled because the variances are significantly different (P < *OOl). This may be due to the sheep of one group having also received a subcutaneous injection. However, for present purposes the difference between the groups is probably of no relevance and pooling is assumedto be legitimate. The average long incubation period is 917 days, but whether this has any biological meaning is doubtful for the animals may be genetically heterogeneous with respect to the control of incubation period. It is nevertheless evident that scrapie following a long incubation period has only occurred in those sheep inoculated intracerebrally and not in the subcutaneously inoculated sheep which have been observed for 1600 days and many of which are senile after this time. It was not practicable to inject the sheep at a given age, but there is no apparent relationship between the age of animal at injection and the length of incubation period, the age at time of injection being randomly distributed between the intracerebral and the subcutaneous groups. Also, sheep of all ages at time of inoculation have developed scrapie following long incubation periods. Incidence

of

Scrapie

in Progeny

of

Diferent

Mating

Types

Since only sheep that develop scrapie after a short incubation perid could be adequately classified in this way not more than two classesof animals, namely, SIP-scrapie-positive and SIP-scrapie-negative (the latter probably b&g a h&r* geneous group) need to be considered.

A. G. DICKINSON

317

et al.

Group

4.

40%

2 (

1

S/C

affected \1

01111 Group

2

I/C

40+15*/.affected I

O-

I

\1

I

Group3 SIC 8 I/C 36+ 21’1. affected

4.

Group 4 SIC 32% affectod -.L

I !I

4. -

% P i

.

Group 5 S/C 61% affected

2: 0.

\L

II

1 4.

Group6 S/C 34% affected

2. o+

II1

1

\1

I

Group7 S/C 41% affected

4. 2. O-

IIUI

\1

I

Group6 S/C 40% affected

Group9 S/C .22X affected

42. 0.

&

I 500

1000 INCUBATION

Fig. 1.

PERIOD

1500 - DAYS

Distribution of incubation periods and incidence of scrapie indicates latest time to which each group has been observed.

in each injection

group.

Arrow

Table 3 gives the incidence of scrapie-positive progeny in the four mating classes. The different incidence in the two reciprocal mating groups and the lower incidence of positive progeny in the three mating classes involving sub cutaneously injected dams are probably due to random deviations (P, O-1 to

318

FACTORS

CONTROLLING

SCRAPIE

IN CHEVIOT

SHEEP

O-05) so that these features do not constitute serious inconsistencies in the data. A summary of the findings is that none of the progeny was scrapie-positive when both parents were negative, about 44 per cent. were scrapie-positive when one parent was of this type and 70 per cent. were scrapie-positive in matings between two such parents. These findings, along with the fact that approximately 38 per cent. of the parental population developed scrapie (SIP), are consistent with the hypothesis that this type of scrapie disease is largely controlled by a dominant gene for susceptibility which had an allele frequency of the order of 20 per cent. in the foundation stock. This would give expected frequencies of 0, 56 and 80 per cent. respectively in the above mating classes and 36 per cent. in the foundation population. This hypothesis can only be proved if homozygous and heterozygous animals can be identified and this has not been feasible up to the present time. One-in-nine of the foundation stock which developed scrapie should be homozygous, but as there were only 6 scrapie-affected rams and since segregation occurred in each of these rams’ progeny groups it can be presumed that no affected ram was homozygous. The progeny of each scrapie-positive ewe are too few in number to allow any assessment to be made and subsequent generations have been ins&iciently tested as yet. Those few cases where it has been possible to classify the sheep which were susceptible, but with a long incubation period (LIP), appear to have had little effect on the expression of the SIP-genotype in the progeny (Table 4). The incidence of SIP scrapie in such progeny is broadly similar to that in the progeny of ewes which remained unaffected for over 1600 days even though they had received an intracerebral challenge. The data are too few to draw any other genetical inferences regarding this LIP type. 3

TABLE INCIDENCE

Various

OF SUSCEPTIBLE

types of matings between parents (but excluding Mating Sire

Dam

+ (SS

----

Mating -

Dam

Sire

+ (LIP)

-

+ (LIP)

+ (SIP)

of progeny

$9 530; 70%

:;

OF SUSCEPTIBLE (SIP) SUSCEPTIBLE

and non-susceptible parents).

Proportion suxeptible (SIP)

zi”8

TABLE INCIDENCE

PROGENY

Number of Prwnv

+ (SIP) 1 -+ (SIP).

+ G)

(SIP)

susceptible (SIP) parents known susceptible (LIP)

4

PROGENY IN MATINGS (LIP) DAMS

Number of

PrDyenr

-

INVOLVING

Proportion of strcccgible (SZP) progeny

10

0%

5

80%

A. 0. DICKINSON

f?t d.

319

DISCUSSION

There is evidence that scrapie can spread by contact from mouse to mouse (Dickinson, Mackay and Zlotnik, 1964 ; Pattison, 1964 ; Morris, Gajdusek and Gibbs, 1965) from sheep to goats (Stamp, 1962; Brotherston, Renwick, Stamp, Zlotnik and Pattison, 1968) and from sheep to sheep (Dickinson, Young, Stamp and Renwick, 1965 ; Brotherston et al., 1968), although the incidence following such contact is probably low, except perhaps where there is continual intimate exposure on a lifetime basis. It is important to exclude from the data any cases of scrapie which could have arisen as a result of contagion if some of the deductions made are to be justified. There is no reason to suspect that contagious transmission involving the same agent occurred while the sheep were on the breeding farm since no transmission experiments are carried out there and no natural cases have occurred among the Cheviot breeding flock. Contagion involving the agent used for challenge is also not likely to be relevant at some stage after the sheep were inoculated for two reasons. Exposure to contagion was similar for all groups irrespective of route of injection whereas the late developing cases-which could conceivably be the result of contagion-are confined to intracerebrally challenged groups. Also, the size of dose given, based on previous evidence, must have been an effective challenge to every individual. That this was so is indicated by the equal incidence of scrapie following a short incubation period in both the subcutaneously and intracerebrally inoculated groups. A possibility to be considered is that the scrapie cases, which occurred after a long incubation period, arose as a result of contagion involving some other strain of scrapie agent than the one being used experimentally and to which some of the animals were susceptible even though they resisted challenge with the SSBP/1/23-24 agent. There is no reason to think that this is the explanation for the appearance of scrapie years after the original challenge injection since all the animals were equally liable to have contact with other agents regardless of which route of inoculation was used and yet only those sheep inoculated intracerebrally developed scrapie after the very prolonged intervals in question. Since 20 per cent. of these latter animals were affected the difference in incidence is not due to chance (P < O*OOl). The possibility that the inoculum used for the intracerebral injections was accidentally contaminated with another type of scrapie agent is an unlikely one which cannot be checked. Scrapie is known to occur naturally in the Cheviot breed and even though it has not occurred among our breeding flock it is possible that it could have arisen only among the injected sheep, as they were, on average, a few years older than those being bred on the farm. That this is dismissed as an explanation for the late occurrence of scrapie is again due to the fact that such cases only appeared in the intracerebralinjection groups and these represent only one-fifth of the sheep challenged. The only other feasible explanations involving natural scrapie are the possibilities that either the intracerebral injection stimulated a latent scrapie agent hating a very 10% incubation period which was not stimulated when the injection was by the subcutaneous route or that subcutaneous injection suppressed the development of late natural scrapie. Attempts to stimulate such hypothetical latent scrapie agents by intracerebral inoculation of normal brain and other materials

320

FACTORS

CONTROLLING

SCRAPIE

IN CHEVIOT

SHEEP

have not succeeded (Stamp, unpublished). It can be argued that occult brain damage in the surviving intracranially inoculated sheep as a result of infection with the contaminating bacterium altered in some way their scrapie resistance, but it seems more likely that this would shorten rather than lengthen the incubation period. In mice, the genotype of the animal exerts extensive contro1 over the incubation period of the ME7 scrapie agent (Dickinson and Mackay, 1964). Further work with the same agent in over 20 strains of mice has shown that the incubation period in any one strain is increased by 70 to 90 per cent. when a relatively high dose of the agent is given intraperitoneally instead of intracerebrally (Dickinson, unpublished). In the present instance the 55 per cent. increase in incubation period (SIP) of scrapie in sheep when the subcutaneous rather than the intracerebral route is used is of a comparable order. Most of the sheep inoculated subcutaneously have been observed for twice as long as the time required for the first intracerebrally-inoculated sheep to develop the late form of scrapie (LIP and although uncertainty about the homogeneity of the sheep developing scrapie after a long incubation period makes a direct comparison with the above results difficult the time would appear to be sufficiently long to accept that similar cases are not likely to occur in the subcutaneously-inoculated animals. It is of course a possibility that scrapie could yet develop, but the stage has now been reached when a number of the animals under observation are having to be killed because of senility. For a longer period of observation it would have been necessary to inject the lambs soon after birth and this was not feasible. Finally, it could be argued that if the size of the subcutaneous inoculum had been larger some cases of scrapie would have developed after a long incubation period (LIP), but this in itself would be a biological situation different from anything previously observed with this disease. The evidence is fairly clear that the genetical control of the SIP phenotype is largely determined by a single gene with a fully dominant allele for susceptibility. In general with diseaseresistance phenomena, with a wide range of hosts, bacteria and viruses, where a single gene has been found, most of these genes have shown full dominance of one allele (Dickinson and Mackay, 1967). The finding of full dominance permits certain deductions about the mechanism involved, to the extent that the SIP resistant sheep are presumably lacking, or almost entirely so, in a chemical component present in the SIP-susceptible individuals and which is necessary for clinical scrapie to develop. The only other gene yet known to affect scrapie is present in mice where it controls the incubation period of the ME7 agent, neither of the alleles being dominant. The main attribute of this gene, which has been found so far, is that the allele giving a prolonged incubation period seems to have little effect on the rate of replication of scrapie extraneurally but delays the onset of multiplication in the brain and in some ways appears to control the effective access of the agent to brain cells even though injection may be intracerebral (Dickinson, Meikle and Fraser, 1968). A possibility which therefore needs to be investigated is whether there is a difference in the rate of replication of the agent in extraneural sites in the three SIP-genotypes which have been defined (homozygous and heterozygous sus-

A. G. DICKINSON

321

et cd.

ceptible, and homozygous resistant). If the main difference were to be found, as in the mice, in terms of access to the central nervous system and multiplication there, then this could help to explain those LIP cases in which otherwise resistant sheep had occasionally suffered a breakdown of barriers to the central nervous system either simply because of the site of inoculation or from resulting trauma. SUMMARY

Evidence is given for the occurrence in Cheviot sheep of a gene controlling susceptibility to clinical scrapie following injection with the SSBP/l allele confers susceptibility strain of scrapie agent. The fully dominant with the clinical signs appearing 197 + 7 days after intracerebral injection with the particular dose used or 313 -t 9 days when injection was subcutaneous. A number of animals apparently homozygous for the recessive resistant allele of this gene developed scrapie after a very long interval, but these cases were confined to those animals receiving an initial intracerebral challenge. Possible reasons for these long-incubation-period cases are discussed. ACKNOWLEDGMENT

We are indebted to Dr. I. Zlotnik and Mr. W. Smith for their extensive cooperation, without which these experiments would not have been undertaken. This work was supported by a grant from the United States Department of Agriculture administered under PL 480. REFERENCES

Brotherston, J. G., Renwick, C. C., Stamp, J. T., Zlotnik, I., and Pattison, I. H. (1968). J. camp. Path., 78, 9. Dickinson, A. G., and Mackay, J. M. K. (1964). Heredity, 19, 279; (1967). Methods in Virology, I, Ch. 2, Ed. Maramorosch and Koprowski. Academic Press, New York. Dickinson, A. G., Mackay, J. M. K., and Zlotnik, I. (1964). 1. camp. Path., 74, 250. Dickinson, A. G., Meikle, V. M. H., and Fraser, H. (1968). Ibid., 78, 293. Dickinson, A. G., Young, G. B., Stamp, J. T., and Renwick, C. C. (1965). Heredity, 20, 485. Gordon, W. S. (1966). U.S.D.A. Report of Scrapie Seminar ARS9153, 19. Morris, J. A., Gajdusek, D. C., and Gibbs, C. J., Jr. (1965). Nut. Inst. Neurological Diseases and Blindness, Monograph No. 2, p. 273, U.S. Public Health Service, Publ. No. 1378; Washington, D.C. Pattison, I. H. (1964). Vet. Rec., 76, 333. Stamp, J. T. (1962). Ibid., 74, 357. [Received

for publication,

October

16th, 19673