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Demonstration of lateral transmission of scrapie between sheep kept under natural conditions using lymphoid tissue biopsy
Research in Veterinary Science 76 (2004) 211–217 www.elsevier.com/locate/rvsc
Demonstration of lateral transmission of scrapie between sheep kept under natural conditions using lymphoid tissue biopsy q S. Ryder b
a,*
, G. Dexter a, S. Bellworthy a, S. Tongue
b
a Department of Pathology, Veterinary Laboratories Agency, Woodham Lane, New Haw, Addlestone, Surrey KT15 3NB, UK Centre for Epidemiology and Risk Analysis, Veterinary Laboratories Agency, Woodham Lane, New Haw, Addlestone, Surrey KT15 3NB, UK
Accepted 25 November 2003
Abstract Scrapie free adult sheep were introduced to a sheep flock specifically maintained to maximise scrapie infection. Native born sheep of the highly susceptible VRQ/VRQ genotype in this flock show highly efficient transmission, evidenced by 100% infection, with an age at death of less than 2 years. Infection in introduced sheep was identified by biopsy of tonsilar and nictitating membrane lymphoid tissue. Progeny of these sheep were monitored and clinical disease confirmed by examination of the brain using routine diagnostic methods. Na€ive sheep of New Zealand origin introduced to the flock in adulthood became infected, demonstrating that lateral transmission had occurred. Lambs born to introduced ewes became infected and died at the same age as lambs born to native ewes, consistent with lateral transmission of scrapie to lambs. Although maternal transmission cannot be totally excluded for the lambs in this study, the data are consistent with lateral transmission being the most important means of spread leading to the high incidence of scrapie observed in this flock. Crown Copyright Ó 2004 Published by Elsevier Ltd. All rights reserved.
1. Introduction Scrapie, an invariably fatal nervous disease of sheep, has been known to be infectious since early transmission experiments induced disease in sheep following inoculation of brain material from an affected animal. Although transmission is known to occur under natural conditions (the disease is endemic in the UK and many other northern hemisphere countries) the mode of natural transmission is unknown. For many years scrapie was assumed to be transmitted maternally, due to the relatively high risk of lambs becoming infected if they were born to affected ewes, supported by finding infectivity in placenta but not in any secretions or excretions from affected sheep, reviewed by Hoinville (1996). A number of authors have questioned this conclusion, and it is now generally accepted that it is inheritance of susceptibility from the dam, not maternal transmission, that accounts for this high incidence. Susceptibility is q *
This work was funded by DEFRA. Corresponding author. Tel.: +1932-352345; fax: +1932-357805. E-mail address: [email protected] (S. Ryder).
controlled by the sheep prion protein (PrP) gene, in which polymorphisms at codons 136, 154 and 171 are most important. Sheep carrying the VRQ (valine at 136, arginine at 154 and glutamine at 171) and ARQ (alanine at 136) alleles, inter alia, are susceptible to scrapie, although perhaps to varying extents, and sheep homozygous for the ARR allele are believed to be highly resistant (Dawson et al., 1998; Hunter, 1998). This gene also influences the age at death from the disease under natural conditions, such that in flocks of sheep with a high prevalence of scrapie infection, the risk and timing of death from scrapie is highly predictable (Schreuder et al., 1998). That lateral transmission can occur has been indicated by several studies, however many early experiments suffered from inadequate knowledge of the susceptibility and prior infection status of the animals acquiring infection. The work of Hourrigan and Klingsporn (1996), which showed that incidence of disease was higher in lambs the longer they remained in a contaminated environment, provided the best evidence that lateral transmission to sheep after birth, either via the environment or by close contact, did occur.
0034-5288/$ - see front matter. Crown Copyright Ó 2004 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2003.11.007
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Therefore maternal transmission, if it occurred, was not the only means of spread of the scrapie agent. Foster et al. (1996) showed that transmission from ewe to embryo did not occur within the first 6 days of embryonic life, and that infection occurred in lambs in the absence of infection in the dam, however in this study stringent precautions had been taken to prevent lateral transmission after birth, therefore the source of scrapie infection in lambs was not identified. Nevertheless there have been several reports of infection in placental tissue. Early studies by Pattison et al. (1972, 1974) demonstrated transmission of infection to sheep from the placentae of six scrapie affected ewes. Infectivity was demonstrated by production of disease in recipient sheep and goats after either intracerebral or oral inoculation. This study predated current knowledge of the PrP gene and the status of recipient sheep was not known for certain. Recently disease specific prion protein detection in placenta of infected ewes (Onodera et al., 1993; Race et al., 1998) has raised the profile of maternal transmission of the agent, particularly as no route of excretion of the agent other than placenta is known. It has now been shown that the accumulation of disease specific PrP in the placenta is determined by the genotype of the conceptus; only if the conceptus was of a susceptible genotype was PrP detected in the placenta, however no evidence of transmission to the conceptus was found (Tuo et al., 2001; Andreoletti et al., 2002). Techniques are now available for the diagnosis of scrapie infection in living, non-clinically affected sheep. These methods exploit the widespread accumulation of disease specific PrP in lymphoid tissues of scrapie infected sheep from early in the incubation period (Andreoletti et al., 2000; van Keulen et al., 1996). Biopsy of easily accessible lymphoid tissues of the tonsil (Schreuder et al., 1996) and nictitating membrane (OÕRourke et al., 2000) has enabled this PrP accumulation to be detected in live sheep years before the onset of clinical signs. Studies in this laboratory of the prevalence of detectable PrP in both tonsil and nictitating membrane tissue of clinical scrapie cases of a range of genotypes have shown this to be a reliable phenomenon, but the extent of accumulation varies in sheep of different PrP genotypes. Sheep of the VRQ/VRQ and VRQ/ARQ genotypes, and less so the ARQ/ARQ genotype, have a high prevalence of detectable PrP accumulation in tonsil and nictitating membrane. It was found that PrP detection in tonsil was more reliable than nictitating membrane, with no cases found in which the nictitating membrane was found to be positive and the tonsil negative (unpublished observations). In the present study we report the results of detection of infection in living sheep exposed to scrapie in a high incidence, but naturally infected, sheep flock. We show that lambs of a susceptible genotype born into this flock are
invariably infected, indicating that transmission occurs very efficiently in this environment. Adult sheep introduced to the flock also became infected, demonstrating that lateral transmission had occurred. Lambs of these introduced ewes became infected, in two cases in the absence of detectable infection in the ewe two years after birth. Moreover the age to death of these infected lambs was the same as in lambs born to ‘‘native’’ ewes, indicating that maternal transmission, i.e., transmission from the dam directly and exclusively to itÕs own offspring in utero or during parturition, was unnecessary to produce the pattern of scrapie infection observed in the native born lambs.
2. Methods A flock of sheep with a high level of scrapie infection has been built up at the Veterinary Laboratories Agency (VLA) by purchase of susceptible, though clinically normal, sheep from scrapie affected sheep flocks. The flock currently numbers more than 350 breeding ewes of a mixture of breeds and genotypes, predominantly containing VRQ, ARQ, AHQ and ARR alleles. The incidence of clinical scrapie for 2001 was 8.5%. The flock has been housed at the current location since 1998 and lambing of sheep has taken place annually in the spring. Breeding policy aims to maintain a wide range of susceptible genotypes of sheep in the flock. The sheep are maintained under normal farm conditions, at pasture for most of the year. All, including the introduced ewes, were mated to a group of rams also potentially scrapie infected, carrying at least one ARQ or VRQ allele. Lambing follows common practice in British lowland sheep flocks. Briefly, ewes only are housed on deep straw for one to two months for lambing. Placentae are not routinely removed from the lambing sheds. All sheep are lambed in large groups (subdivided by the shepherds into several large pens for purely management reasons) until they lamb, when they are penned with their own lambs for between one and several days to ensure good mothering. Pens are adjacent to the large groups of sheep therefore there is no separation of sheep from the main flock at any time during the lambing period. Ewes with lambs at foot go out to pasture usually within one week of birth. Scrapie free sheep of the VRQ/ARQ genotype of the Cheviot breed and ARQ/ARQ genotype of the Suffolk breed were originally imported from New Zealand and have been housed and bred in a dedicated facility isolated from all other livestock since 1998. Regular monitoring of sheep from this flock has not found any evidence of scrapie infection. These New Zealand derived sheep were introduced to the infected flock on several occasions, at ages 6, 26, 30 and 35 months. Evidence of infection with scrapie was determined in live sheep by immunohistochemical detection of disease
S. Ryder et al. / Research in Veterinary Science 76 (2004) 211–217
specific PrP in biopsies of lymphoid tissues. Biopsy of tonsil tissue was as described by Schreuder et al. (1996) and lymphoid tissue of the nictitating membrane was as described by OÕRourke et al. (1998). Sheep were sedated using 1.5 ml medetomidine (Domitor, Pfizer Animal Health) and 1ml butorphanol (Torbugesic, Fort Dodge Animal Health Ltd.) combined, by intravenous injection. Tonsils were biopsied using 14 in. rectal biopsy forceps inserted into one tonsilar sinus. Local anaesthetic proxymetacaine hydrochloride (Minims, Chauvin Pharmaceuticals Ltd.) was applied to the eye two minutes prior to sampling of the lymphoid tissue on the bulbar surface of the nictitating membrane. Sheep were revived using 1.5 ml atipamezole hydrochloride (Antisedan, Pfizer Animal Health) given intramuscularly. Biopsies were fixed in 10% neutral buffered formalin for up to 6 h then processed to wax blocks on an automated overnight processing schedule. Five micron sections were cut and immunostained using antiserum R486, a rabbit antiserum raised against a peptide, sequence YQRESQAYYQRGA, corresponding to peptides 216–228 of the bovine PrP gene (5 octapeptide repeat allele) using a method as previously described (Ryder et al., 2001). A positive result for biopsy (Fig. 1) was recorded if either lymphoid tissue were found to contain disease specific staining, as defined by van Keulen et al. (1996) in a single lymphoid follicle. A negative result was recorded if either the tonsil biopsy or both the tonsil and nictitating membrane biopsy gave negative results, and at least four follicles were present in each biopsy (Fig. 2). Failure to acquire an adequate tissue sample of both tissues, or failure to acquire an adequate sample of tonsil combined with a negative result on nictitating membrane biopsy was recorded as ‘‘not done’’, because no interpretation could be made.
Fig. 1. Disease specific PrP deposits in lymphoid follicles in a biopsy of tonsil. Twenty month old VRQ/ARQ Swaledale sheep born within flock to native ewe. Immunohistochemistry for PrP, antiserum Rb486. 80.
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Fig. 2. Biopsy specimen of nictitating membrane showing more than four follicles but no staining for PrP. Negative result. Twenty-four month old ARQ/ARQ cheviot sheep born to New Zealand derived ewe. Immunohistochemistry for PrP, antiserum Rb486. 80.
The flock is monitored by animal care staff and any animals showing clinical signs of scrapie euthanased and necropsied. A diagnosis of scrapie was made if sheep showed typical lesions of scrapie and disease specific PrP accumulation in the brain stem.
3. Results 3.1. Native born lambs Details of examination of biopsies of lymphoid tissue from ‘‘native born lambs’’, that is lambs born to ewes either born in the flock or long term resident in the flock and derived from scrapie infected farms, are shown in Table 1. All lambs of the VRQ/VRQ genotype from two birth cohorts, 25 lambs in total, were examined; those born in 1999 and 2000. In the first year, 8 lambs were tested at 12 months of age and all were found to be infected. These lambs all subsequently died of scrapie at 24–25 months of age. In the second year, 17 lambs were tested at 4, 8 or 12 months. All except three lambs were positive at 4 months, and all those tested were positive at 8 and 12 months of age. All subsequently died of scrapie at 21–28 months of age, with the exception of one animal killed for unrelated reasons at 12 months, which did not show evidence of clinical disease. This animal had a mild non-suppurative encephalitis, probably of viral origin. This sheep had given a negative biopsy at 4 months of age and was not tested at 8 months, but at the time of death immunohistochemistry for PrP gave a positive result on palatine tonsils but a negative result on the brain stem. Lambs of the VRQ/ARQ genotype from the 1999 cohort sampled at 10 months of age gave positive
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Table 1 Biopsy results by genotype and age for sheep born in the flock to ‘‘native’’ ewes Genotype
VRQ/VRQ
VRQ/ARQ
Birth year
Age at biopsy (months) number positive/total number tested
1999 2000
8 17
1999
10
2000
32
Number born
4
8
8/11
9/9
10
12
20
8/8 2/2 6/10
0/20
0/12
28/28
results in 6 of 10 lambs. All subsequently died of scrapie at 28–39 months of age. From the 2000 cohort all gave negative results at 4 and 8 months of age, but all were positive when tested at 20 months of age. From this cohort only two sheep have so far succumbed to scrapie; one died at 21 months of age, but this animal did not show vacuolar pathology in the brain, suggesting it was not at end stage clinical scrapie. The second died showing signs of clinical scrapie, confirmed by histopathology of the brain stem at 24 months of age. The rest of this cohort remain alive and clinically normal. 3.2. Introduced adult sheep Details of 6 month old and adult scrapie free sheep introduced to the infected flock are shown in Table 2. Seven VRQ/ARQ Cheviot sheep were introduced to the flock at 6 months of age. Of those tested 24 months later, 3 of 5 gave positive results, and a further 2 tested at 30 months post introduction gave positive results. Sheep introduced at 26 months of age gave positive results in 5 of 6 animals tested 24 months after introduction. Five sheep introduced when 35 months of age gave negative results when biopsied 15 months later. All of these introduced sheep remain alive and clinically normal. Five Suffolk sheep were introduced to the flock at 30 months of age. One of these gave positive biopsy results 20 months later. All remain alive and clinically normal.
Status July 2002
Scrapie confirmed at necropsy
All dead All dead
8/8 (24–25 months) 16/17 (21–28 months) 1 negative, killed at 12 months old
All dead
10/10 (28–39 months) 1 scrapie pos (IHC pos, histopath neg at 20 months) 1 scrapie pos at 24 months
2 dead
3.3. Lambs born to scrapie free sheep after introduction to the infected flock Fifteen New Zealand derived ewes lambed during 2000, at varying times after introduction, as detailed in Table 3. VRQ/ARQ Cheviot ewes produced a mixture of VRQ/VRQ, VRQ/ARQ and ARQ/ARQ lambs. All three of the VRQ/VRQ lambs born to 3 different VRQ/ ARQ ewes succumbed to scrapie at 22 months of age. These lambs were born 5 months after introduction of the ewes to the flock, in their first lambing season. Two of the ewes showed evidence of infection by positive biopsies taken in November 2001, 24 months after introduction to the flock and 17 months following lambing, however one ewe gave negative biopsy results at this time point. All VRQ/ARQ lambs born to VRQ/ARQ ewes gave positive biopsy results by December 2001, at 20 months of age. None of the ARQ/ARQ lambs gave positive results in May 2002, at 25 months of age, regardless of the status of the ewe. All lambs of both genotypes remain clinically normal. Suffolk ewes of the ARQ/ARQ genotype all produced ARQ/ARQ lambs. One of 7 lambs gave positive biopsy results at 25 months of age, but all ewes, including the dam of the infected lamb, gave negative results when tested in November 2001 or May 2002, 19 and 25 months following birth of the lambs (adequate biopsies could not be obtained from 2 of the ewes).
Table 2 Biopsy results of sheep introduced to the flock Genotype
Breed
Age at introduction
Number
Date introduced
Date of biopsy
Result no. pos/no. sampled
Time in flock
Status
VRQ/ARQ VRQ/ARQ
Cheviot Cheviot
6 months 6 months
5 2
Nov 99 Nov 99
Nov 01 May 2002
3/5 2/2
24 months 30 months
Alive Alive
VRQ/ARQ VRQ/ARQ
Cheviot Cheviot
26 months 35 months
6 5
Nov 99 Aug 00
Nov 01 Nov 01
5/6 0/5
24 months 15 months
Alive Alive
ARQ/ARQ
Suffolk
30 months
5
Mar 2000
Nov 01
1/5
20 months
Alive
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Table 3 Progeny of New Zealand derived ewes exposed to scrapie infected sheep Dam genotype
Date and age (months) when introduced
Date of lambing
Cheviot VRQ/ARQ
Nov 99
26
VRQ/ARQ
Nov 99
VRQ/ARQ
Progeny
Dam status June 2002 (biopsy)
Genotype
Biopsy results
Status June 2002
Apr 00
VRQ/VRQ
Pos Nov 01
26
Apr 00
VRQ/VRQ
Pos Nov 01
Nov 99
26
Apr 00
VRQ/VRQ
VRQ/ARQ
Nov 99
26
Apr 00
VRQ/ARQ VRQ/ARQ VRQ/ARQ VRQ/ARQ
Nov Nov Nov Nov
99 99 99 99
26 26 26 26
Apr Apr Apr Apr
Died scrapie Feb 02 Died scrapie Feb 02 Died scrapie Feb 02 Alive Alive Alive Alive Alive Alive Alive
ND Pos Nov 01 Pos Nov 01 Pos Nov 01
Suffolk ARQ/ARQ ARQ/ARQ ARQ/ARQ ARQ/ARQ ARQ/ARQ ARQ/ARQ ARQ/ARQ
Mar Mar Mar Mar Mar Mar Mar
00 00 00 00 00 00 00
30 30 30 30 30 30 30
Alive Alive Alive Alive Alive Alive Alive
Neg ND ND Neg Neg Neg Neg
00 00 00 00
ARQ/ARQ ARQ/ARQ VRQ/ARQ VRQ/ARQ VRQ/ARQ ARQ/ARQ ARQ/ARQ
Neg May 02 Neg May 02 Pos Dec 01 Neg Aug 00 Neg Aug 00 Neg May 02 Neg May 02
Apr 00 Apr 00 Apr 00 Apr 00 Mar 00 Apr 00 Apr 00
ARQ/ARQ ARQ/ARQ ARQ/ARQ ARQ/ARQ ARQ/ARQ ARQ/ARQ ARQ/ARQ
Neg May 02 Neg May 02 Neg May 02 Pos May 02 Neg May 02 Neg May 02 ND
Pos Dec 01 Pos Dec 01
Pos Nov 01 Pos Nov 01 Neg Nov 01
ND
May 02
Nov 01 Nov 01 May 02 Nov 01
ND: not done, or biopsy unsuitable.
4. Discussion This report provides evidence for the lateral transmission of scrapie from naturally infected British sheep of a variety of breeds to both adult and sub-adult scrapie free Cheviot and Suffolk sheep of New Zealand origin. In addition it provides evidence highly suggestive of lateral transmission to lambs born in a scrapie infected flock. Data on sheep of two susceptible genotypes, VRQ/ VRQ and VRQ/ARQ, presented here demonstrate transmission to 100% of sheep in the groups examined kept under the normal farm conditions practiced in the infected flock. This is supported by further work on necropsy of lambs of these genotypes and monitoring of all scrapie suspects, which has also shown 100% infection in these genotypes (data not shown). The data presented here do not show any evidence for a difference in susceptibility between VRQ homozygous sheep and VRQ/ARQ heterozygotes; for both 100% of lambs became infected, but age at death is greater for heterozygotes. In experimental models in rodents it has been shown that incubation period is dependent on the dose of scrapie agent. It could be argued that an increased age at death in sheep is a consequence of reduced effective exposure, i.e., reduced susceptibility compared to homozygotes given the same level of exposure. However, it is also possible that susceptibility is identical, and the
genotype of the sheep has a direct influence on incubation period. It is often assumed that under most conditions sheep born into a flock in which scrapie is endemic will become infected at an early age, and it is possible that young sheep are more susceptible to scrapie than adults. The transmission to adult and sub-adult sheep introduced to the infected flock in this study shows that sheep are susceptible to infection at various ages, including in adulthood. As none of these sheep have yet been exposed for significantly longer than the age at death of native born lambs, it is not possible to say whether the exposure at a later age results in either lower susceptibility or a prolonged incubation period, but these results provide conclusive evidence of lateral transmission of scrapie between sheep. It is curious that after 24 months exposure sub-adults first introduced at 6 months of age had a lower prevalence of infection than those introduced at 24 months of age. The sheep introduced at 6 months of age in November were too young to lamb in their first Spring, and so were not housed with the rest of the flock for lambing until the following year, 18 months later. Those introduced at 24 months lambed with the flock 6 months later. It is possible therefore that the extent of exposure to infection for those introduced at 6 months of age was low until approximately 18 months following introduction, when they will have been housed for lambing with
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the infected flock. Although there is no statistical evidence to support a true difference between the two groups (p ¼ 0:36, Fishers exact test), the small group sizes may lack the power to detect a true difference. The results of examination of lambs born to New Zealand derived ewes are also highly suggestive of lateral transmission to lambs. In all cases it is unlikely that the ewes were shedding infection at the time of birth of the lambs, given the short period of exposure and the absence of detectable disease specific PrP in sheep of these gentoypes at less than 10 months of age (Table 1). In two cases biopsy of tonsil and nictitating membrane were negative almost two years following lambing. One of these ewes was of the VRQ/ARQ genotype, in which disease specific PrP has been detected in it 20/21, i.e., 95.2% (95% C.I. 76.2–99.9%) of scrapie affected sheep of this genotype tested post-mortem (unpublished observations). Given this high level of sensitivity for PrP detection in this genotype, it is unlikely that this ewe was in an advanced stage of the incubation period. Therefore although a false negative result cannot be ruled out in this case it is unlikely and a negative biopsy result is consistent with either the ewe not being infected at the time of biopsy, or being at an early stage of infection; in either scenario this means it would have been unlikely to be infected at the time of birth of itÕs lambs. The second ewe which gave a negative biopsy result was of the ARQ/ARQ genotype; PrP detection in this genotype is less reliable than in ARQ/VRQ, and the possibility of a false-negative result is greater in this case. Therefore, whilst maternal transmission cannot be excluded as a source of infection in these lambs, it is unlikely, at least for one ARQ/VRQ ewe, almost two years after the birth of the lambs. Yet VRQ homozygous lambs born to these VRQ/ARQ New Zealand derived ewes died from scrapie at the same age as native born lambs; just under two years old despite the probable absence of maternal transmission, indicating a similar level of exposure regardless of the infection status of the ewes. These data are therefore consistent with lateral transmission being the most important route of infection in both lambs and adult sheep. A similar observation was made by Andreoletti et al. (2002) based on identical incidence of infection in lambs, as identified by PrPSc accumulation in lymphoid tissues, regardless of the PrPSc content of the placenta. The mechanism of natural infection with scrapie is unknown. The only known source of contamination of the environment by infectious tissues is via the placenta and fetal fluids. There is no evidence for infectivity in milk, saliva, faeces or urine of scrapie infected sheep (reviewed by Hoinville, 1996). However this is based almost exclusively on failure to detect infectivity by bioassay in these excretions/secretions, and given the likely low level of infectious agent present and the difficulty of bioassay of such contaminated material by
intra-cranial inoculation of mice, these negative findings must be interpreted with caution. Contamination of the environment outside of the lambing period remains a possibility. Abnormal PrP has recently been detected in urine of experimentally infected hamsters and bovine spongiform encephalopathy infected cattle (Shaked et al., 1999). An additional possible route of transmission, which has received little attention, is via contaminated needles. The common use of multiple dose injectors for vaccine delivery may represent a means of transmission between sheep on the same farm. Infectivity has been detected in blood of BSE infected sheep by transfusion of large volumes of blood between sheep (Hunter et al., 2002) but multiple dose injectors are an unlikely vehicle for transmission of contaminated blood. Needles penetrate the skin and subcutaneous tissues and contamination with blood is minimal. Inadvertent puncture of lymphoid tissue and transfer of infected lymphoid tissue would be theoretically possible, but would be a very rare event. Furthermore scrapie has been known for centuries, long before the use of injectable drugs. Therefore the likely potential mechanisms for lateral transmission of scrapie under normal farm conditions are either exchange of body fluids via direct contact (which would include saliva, urine and faeces) or via the environment, in which the agent can persist for a considerable length of time following contamination with placenta and itÕs fluids, urine, faeces, regurgitated rumenal contents or even saliva. At the present time all of these sources of infection must remain possible, but there is no direct evidence for any except placenta. Studies are ongoing in this laboratory to dissect the process of natural transmission of scrapie in this flock by exposure of sheep to defined and restricted parts of the annual husbandry cycle. It is hoped that results of this work will indicate possible routes of excretion of the agent, and help explain when and how during the pathogenesis of this disease the agent is shed from the host. Acknowledgements We are grateful to Hugh Simmons for provision of New Zealand derived sheep, to Tony Duarte for management of the infected flock, to Peter Bellerby for immunohistochemistry, to Roy Jackman for supply of antibody R486 and to Charlotte Cook, Alies Hoek and Linda Hoinville for their contributions to management of the infected flock. References Andreoletti, O., Berthon, P., Marc, D., Sarradin, P., Grosclaude, J., van Keulen, L., Scheler, F., Elsen, J.-M., Lantier, F., 2000. Early
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Demonstration of lateral transmission of scrapie between sheep kept under natural conditions using lymphoid tissue biopsy q S. Ryder b
a,*
, G. Dexter a, S. Bellworthy a, S. Tongue
b
a Department of Pathology, Veterinary Laboratories Agency, Woodham Lane, New Haw, Addlestone, Surrey KT15 3NB, UK Centre for Epidemiology and Risk Analysis, Veterinary Laboratories Agency, Woodham Lane, New Haw, Addlestone, Surrey KT15 3NB, UK
Accepted 25 November 2003
Abstract Scrapie free adult sheep were introduced to a sheep flock specifically maintained to maximise scrapie infection. Native born sheep of the highly susceptible VRQ/VRQ genotype in this flock show highly efficient transmission, evidenced by 100% infection, with an age at death of less than 2 years. Infection in introduced sheep was identified by biopsy of tonsilar and nictitating membrane lymphoid tissue. Progeny of these sheep were monitored and clinical disease confirmed by examination of the brain using routine diagnostic methods. Na€ive sheep of New Zealand origin introduced to the flock in adulthood became infected, demonstrating that lateral transmission had occurred. Lambs born to introduced ewes became infected and died at the same age as lambs born to native ewes, consistent with lateral transmission of scrapie to lambs. Although maternal transmission cannot be totally excluded for the lambs in this study, the data are consistent with lateral transmission being the most important means of spread leading to the high incidence of scrapie observed in this flock. Crown Copyright Ó 2004 Published by Elsevier Ltd. All rights reserved.
1. Introduction Scrapie, an invariably fatal nervous disease of sheep, has been known to be infectious since early transmission experiments induced disease in sheep following inoculation of brain material from an affected animal. Although transmission is known to occur under natural conditions (the disease is endemic in the UK and many other northern hemisphere countries) the mode of natural transmission is unknown. For many years scrapie was assumed to be transmitted maternally, due to the relatively high risk of lambs becoming infected if they were born to affected ewes, supported by finding infectivity in placenta but not in any secretions or excretions from affected sheep, reviewed by Hoinville (1996). A number of authors have questioned this conclusion, and it is now generally accepted that it is inheritance of susceptibility from the dam, not maternal transmission, that accounts for this high incidence. Susceptibility is q *
This work was funded by DEFRA. Corresponding author. Tel.: +1932-352345; fax: +1932-357805. E-mail address: [email protected] (S. Ryder).
controlled by the sheep prion protein (PrP) gene, in which polymorphisms at codons 136, 154 and 171 are most important. Sheep carrying the VRQ (valine at 136, arginine at 154 and glutamine at 171) and ARQ (alanine at 136) alleles, inter alia, are susceptible to scrapie, although perhaps to varying extents, and sheep homozygous for the ARR allele are believed to be highly resistant (Dawson et al., 1998; Hunter, 1998). This gene also influences the age at death from the disease under natural conditions, such that in flocks of sheep with a high prevalence of scrapie infection, the risk and timing of death from scrapie is highly predictable (Schreuder et al., 1998). That lateral transmission can occur has been indicated by several studies, however many early experiments suffered from inadequate knowledge of the susceptibility and prior infection status of the animals acquiring infection. The work of Hourrigan and Klingsporn (1996), which showed that incidence of disease was higher in lambs the longer they remained in a contaminated environment, provided the best evidence that lateral transmission to sheep after birth, either via the environment or by close contact, did occur.
0034-5288/$ - see front matter. Crown Copyright Ó 2004 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2003.11.007
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Therefore maternal transmission, if it occurred, was not the only means of spread of the scrapie agent. Foster et al. (1996) showed that transmission from ewe to embryo did not occur within the first 6 days of embryonic life, and that infection occurred in lambs in the absence of infection in the dam, however in this study stringent precautions had been taken to prevent lateral transmission after birth, therefore the source of scrapie infection in lambs was not identified. Nevertheless there have been several reports of infection in placental tissue. Early studies by Pattison et al. (1972, 1974) demonstrated transmission of infection to sheep from the placentae of six scrapie affected ewes. Infectivity was demonstrated by production of disease in recipient sheep and goats after either intracerebral or oral inoculation. This study predated current knowledge of the PrP gene and the status of recipient sheep was not known for certain. Recently disease specific prion protein detection in placenta of infected ewes (Onodera et al., 1993; Race et al., 1998) has raised the profile of maternal transmission of the agent, particularly as no route of excretion of the agent other than placenta is known. It has now been shown that the accumulation of disease specific PrP in the placenta is determined by the genotype of the conceptus; only if the conceptus was of a susceptible genotype was PrP detected in the placenta, however no evidence of transmission to the conceptus was found (Tuo et al., 2001; Andreoletti et al., 2002). Techniques are now available for the diagnosis of scrapie infection in living, non-clinically affected sheep. These methods exploit the widespread accumulation of disease specific PrP in lymphoid tissues of scrapie infected sheep from early in the incubation period (Andreoletti et al., 2000; van Keulen et al., 1996). Biopsy of easily accessible lymphoid tissues of the tonsil (Schreuder et al., 1996) and nictitating membrane (OÕRourke et al., 2000) has enabled this PrP accumulation to be detected in live sheep years before the onset of clinical signs. Studies in this laboratory of the prevalence of detectable PrP in both tonsil and nictitating membrane tissue of clinical scrapie cases of a range of genotypes have shown this to be a reliable phenomenon, but the extent of accumulation varies in sheep of different PrP genotypes. Sheep of the VRQ/VRQ and VRQ/ARQ genotypes, and less so the ARQ/ARQ genotype, have a high prevalence of detectable PrP accumulation in tonsil and nictitating membrane. It was found that PrP detection in tonsil was more reliable than nictitating membrane, with no cases found in which the nictitating membrane was found to be positive and the tonsil negative (unpublished observations). In the present study we report the results of detection of infection in living sheep exposed to scrapie in a high incidence, but naturally infected, sheep flock. We show that lambs of a susceptible genotype born into this flock are
invariably infected, indicating that transmission occurs very efficiently in this environment. Adult sheep introduced to the flock also became infected, demonstrating that lateral transmission had occurred. Lambs of these introduced ewes became infected, in two cases in the absence of detectable infection in the ewe two years after birth. Moreover the age to death of these infected lambs was the same as in lambs born to ‘‘native’’ ewes, indicating that maternal transmission, i.e., transmission from the dam directly and exclusively to itÕs own offspring in utero or during parturition, was unnecessary to produce the pattern of scrapie infection observed in the native born lambs.
2. Methods A flock of sheep with a high level of scrapie infection has been built up at the Veterinary Laboratories Agency (VLA) by purchase of susceptible, though clinically normal, sheep from scrapie affected sheep flocks. The flock currently numbers more than 350 breeding ewes of a mixture of breeds and genotypes, predominantly containing VRQ, ARQ, AHQ and ARR alleles. The incidence of clinical scrapie for 2001 was 8.5%. The flock has been housed at the current location since 1998 and lambing of sheep has taken place annually in the spring. Breeding policy aims to maintain a wide range of susceptible genotypes of sheep in the flock. The sheep are maintained under normal farm conditions, at pasture for most of the year. All, including the introduced ewes, were mated to a group of rams also potentially scrapie infected, carrying at least one ARQ or VRQ allele. Lambing follows common practice in British lowland sheep flocks. Briefly, ewes only are housed on deep straw for one to two months for lambing. Placentae are not routinely removed from the lambing sheds. All sheep are lambed in large groups (subdivided by the shepherds into several large pens for purely management reasons) until they lamb, when they are penned with their own lambs for between one and several days to ensure good mothering. Pens are adjacent to the large groups of sheep therefore there is no separation of sheep from the main flock at any time during the lambing period. Ewes with lambs at foot go out to pasture usually within one week of birth. Scrapie free sheep of the VRQ/ARQ genotype of the Cheviot breed and ARQ/ARQ genotype of the Suffolk breed were originally imported from New Zealand and have been housed and bred in a dedicated facility isolated from all other livestock since 1998. Regular monitoring of sheep from this flock has not found any evidence of scrapie infection. These New Zealand derived sheep were introduced to the infected flock on several occasions, at ages 6, 26, 30 and 35 months. Evidence of infection with scrapie was determined in live sheep by immunohistochemical detection of disease
S. Ryder et al. / Research in Veterinary Science 76 (2004) 211–217
specific PrP in biopsies of lymphoid tissues. Biopsy of tonsil tissue was as described by Schreuder et al. (1996) and lymphoid tissue of the nictitating membrane was as described by OÕRourke et al. (1998). Sheep were sedated using 1.5 ml medetomidine (Domitor, Pfizer Animal Health) and 1ml butorphanol (Torbugesic, Fort Dodge Animal Health Ltd.) combined, by intravenous injection. Tonsils were biopsied using 14 in. rectal biopsy forceps inserted into one tonsilar sinus. Local anaesthetic proxymetacaine hydrochloride (Minims, Chauvin Pharmaceuticals Ltd.) was applied to the eye two minutes prior to sampling of the lymphoid tissue on the bulbar surface of the nictitating membrane. Sheep were revived using 1.5 ml atipamezole hydrochloride (Antisedan, Pfizer Animal Health) given intramuscularly. Biopsies were fixed in 10% neutral buffered formalin for up to 6 h then processed to wax blocks on an automated overnight processing schedule. Five micron sections were cut and immunostained using antiserum R486, a rabbit antiserum raised against a peptide, sequence YQRESQAYYQRGA, corresponding to peptides 216–228 of the bovine PrP gene (5 octapeptide repeat allele) using a method as previously described (Ryder et al., 2001). A positive result for biopsy (Fig. 1) was recorded if either lymphoid tissue were found to contain disease specific staining, as defined by van Keulen et al. (1996) in a single lymphoid follicle. A negative result was recorded if either the tonsil biopsy or both the tonsil and nictitating membrane biopsy gave negative results, and at least four follicles were present in each biopsy (Fig. 2). Failure to acquire an adequate tissue sample of both tissues, or failure to acquire an adequate sample of tonsil combined with a negative result on nictitating membrane biopsy was recorded as ‘‘not done’’, because no interpretation could be made.
Fig. 1. Disease specific PrP deposits in lymphoid follicles in a biopsy of tonsil. Twenty month old VRQ/ARQ Swaledale sheep born within flock to native ewe. Immunohistochemistry for PrP, antiserum Rb486. 80.
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Fig. 2. Biopsy specimen of nictitating membrane showing more than four follicles but no staining for PrP. Negative result. Twenty-four month old ARQ/ARQ cheviot sheep born to New Zealand derived ewe. Immunohistochemistry for PrP, antiserum Rb486. 80.
The flock is monitored by animal care staff and any animals showing clinical signs of scrapie euthanased and necropsied. A diagnosis of scrapie was made if sheep showed typical lesions of scrapie and disease specific PrP accumulation in the brain stem.
3. Results 3.1. Native born lambs Details of examination of biopsies of lymphoid tissue from ‘‘native born lambs’’, that is lambs born to ewes either born in the flock or long term resident in the flock and derived from scrapie infected farms, are shown in Table 1. All lambs of the VRQ/VRQ genotype from two birth cohorts, 25 lambs in total, were examined; those born in 1999 and 2000. In the first year, 8 lambs were tested at 12 months of age and all were found to be infected. These lambs all subsequently died of scrapie at 24–25 months of age. In the second year, 17 lambs were tested at 4, 8 or 12 months. All except three lambs were positive at 4 months, and all those tested were positive at 8 and 12 months of age. All subsequently died of scrapie at 21–28 months of age, with the exception of one animal killed for unrelated reasons at 12 months, which did not show evidence of clinical disease. This animal had a mild non-suppurative encephalitis, probably of viral origin. This sheep had given a negative biopsy at 4 months of age and was not tested at 8 months, but at the time of death immunohistochemistry for PrP gave a positive result on palatine tonsils but a negative result on the brain stem. Lambs of the VRQ/ARQ genotype from the 1999 cohort sampled at 10 months of age gave positive
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Table 1 Biopsy results by genotype and age for sheep born in the flock to ‘‘native’’ ewes Genotype
VRQ/VRQ
VRQ/ARQ
Birth year
Age at biopsy (months) number positive/total number tested
1999 2000
8 17
1999
10
2000
32
Number born
4
8
8/11
9/9
10
12
20
8/8 2/2 6/10
0/20
0/12
28/28
results in 6 of 10 lambs. All subsequently died of scrapie at 28–39 months of age. From the 2000 cohort all gave negative results at 4 and 8 months of age, but all were positive when tested at 20 months of age. From this cohort only two sheep have so far succumbed to scrapie; one died at 21 months of age, but this animal did not show vacuolar pathology in the brain, suggesting it was not at end stage clinical scrapie. The second died showing signs of clinical scrapie, confirmed by histopathology of the brain stem at 24 months of age. The rest of this cohort remain alive and clinically normal. 3.2. Introduced adult sheep Details of 6 month old and adult scrapie free sheep introduced to the infected flock are shown in Table 2. Seven VRQ/ARQ Cheviot sheep were introduced to the flock at 6 months of age. Of those tested 24 months later, 3 of 5 gave positive results, and a further 2 tested at 30 months post introduction gave positive results. Sheep introduced at 26 months of age gave positive results in 5 of 6 animals tested 24 months after introduction. Five sheep introduced when 35 months of age gave negative results when biopsied 15 months later. All of these introduced sheep remain alive and clinically normal. Five Suffolk sheep were introduced to the flock at 30 months of age. One of these gave positive biopsy results 20 months later. All remain alive and clinically normal.
Status July 2002
Scrapie confirmed at necropsy
All dead All dead
8/8 (24–25 months) 16/17 (21–28 months) 1 negative, killed at 12 months old
All dead
10/10 (28–39 months) 1 scrapie pos (IHC pos, histopath neg at 20 months) 1 scrapie pos at 24 months
2 dead
3.3. Lambs born to scrapie free sheep after introduction to the infected flock Fifteen New Zealand derived ewes lambed during 2000, at varying times after introduction, as detailed in Table 3. VRQ/ARQ Cheviot ewes produced a mixture of VRQ/VRQ, VRQ/ARQ and ARQ/ARQ lambs. All three of the VRQ/VRQ lambs born to 3 different VRQ/ ARQ ewes succumbed to scrapie at 22 months of age. These lambs were born 5 months after introduction of the ewes to the flock, in their first lambing season. Two of the ewes showed evidence of infection by positive biopsies taken in November 2001, 24 months after introduction to the flock and 17 months following lambing, however one ewe gave negative biopsy results at this time point. All VRQ/ARQ lambs born to VRQ/ARQ ewes gave positive biopsy results by December 2001, at 20 months of age. None of the ARQ/ARQ lambs gave positive results in May 2002, at 25 months of age, regardless of the status of the ewe. All lambs of both genotypes remain clinically normal. Suffolk ewes of the ARQ/ARQ genotype all produced ARQ/ARQ lambs. One of 7 lambs gave positive biopsy results at 25 months of age, but all ewes, including the dam of the infected lamb, gave negative results when tested in November 2001 or May 2002, 19 and 25 months following birth of the lambs (adequate biopsies could not be obtained from 2 of the ewes).
Table 2 Biopsy results of sheep introduced to the flock Genotype
Breed
Age at introduction
Number
Date introduced
Date of biopsy
Result no. pos/no. sampled
Time in flock
Status
VRQ/ARQ VRQ/ARQ
Cheviot Cheviot
6 months 6 months
5 2
Nov 99 Nov 99
Nov 01 May 2002
3/5 2/2
24 months 30 months
Alive Alive
VRQ/ARQ VRQ/ARQ
Cheviot Cheviot
26 months 35 months
6 5
Nov 99 Aug 00
Nov 01 Nov 01
5/6 0/5
24 months 15 months
Alive Alive
ARQ/ARQ
Suffolk
30 months
5
Mar 2000
Nov 01
1/5
20 months
Alive
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Table 3 Progeny of New Zealand derived ewes exposed to scrapie infected sheep Dam genotype
Date and age (months) when introduced
Date of lambing
Cheviot VRQ/ARQ
Nov 99
26
VRQ/ARQ
Nov 99
VRQ/ARQ
Progeny
Dam status June 2002 (biopsy)
Genotype
Biopsy results
Status June 2002
Apr 00
VRQ/VRQ
Pos Nov 01
26
Apr 00
VRQ/VRQ
Pos Nov 01
Nov 99
26
Apr 00
VRQ/VRQ
VRQ/ARQ
Nov 99
26
Apr 00
VRQ/ARQ VRQ/ARQ VRQ/ARQ VRQ/ARQ
Nov Nov Nov Nov
99 99 99 99
26 26 26 26
Apr Apr Apr Apr
Died scrapie Feb 02 Died scrapie Feb 02 Died scrapie Feb 02 Alive Alive Alive Alive Alive Alive Alive
ND Pos Nov 01 Pos Nov 01 Pos Nov 01
Suffolk ARQ/ARQ ARQ/ARQ ARQ/ARQ ARQ/ARQ ARQ/ARQ ARQ/ARQ ARQ/ARQ
Mar Mar Mar Mar Mar Mar Mar
00 00 00 00 00 00 00
30 30 30 30 30 30 30
Alive Alive Alive Alive Alive Alive Alive
Neg ND ND Neg Neg Neg Neg
00 00 00 00
ARQ/ARQ ARQ/ARQ VRQ/ARQ VRQ/ARQ VRQ/ARQ ARQ/ARQ ARQ/ARQ
Neg May 02 Neg May 02 Pos Dec 01 Neg Aug 00 Neg Aug 00 Neg May 02 Neg May 02
Apr 00 Apr 00 Apr 00 Apr 00 Mar 00 Apr 00 Apr 00
ARQ/ARQ ARQ/ARQ ARQ/ARQ ARQ/ARQ ARQ/ARQ ARQ/ARQ ARQ/ARQ
Neg May 02 Neg May 02 Neg May 02 Pos May 02 Neg May 02 Neg May 02 ND
Pos Dec 01 Pos Dec 01
Pos Nov 01 Pos Nov 01 Neg Nov 01
ND
May 02
Nov 01 Nov 01 May 02 Nov 01
ND: not done, or biopsy unsuitable.
4. Discussion This report provides evidence for the lateral transmission of scrapie from naturally infected British sheep of a variety of breeds to both adult and sub-adult scrapie free Cheviot and Suffolk sheep of New Zealand origin. In addition it provides evidence highly suggestive of lateral transmission to lambs born in a scrapie infected flock. Data on sheep of two susceptible genotypes, VRQ/ VRQ and VRQ/ARQ, presented here demonstrate transmission to 100% of sheep in the groups examined kept under the normal farm conditions practiced in the infected flock. This is supported by further work on necropsy of lambs of these genotypes and monitoring of all scrapie suspects, which has also shown 100% infection in these genotypes (data not shown). The data presented here do not show any evidence for a difference in susceptibility between VRQ homozygous sheep and VRQ/ARQ heterozygotes; for both 100% of lambs became infected, but age at death is greater for heterozygotes. In experimental models in rodents it has been shown that incubation period is dependent on the dose of scrapie agent. It could be argued that an increased age at death in sheep is a consequence of reduced effective exposure, i.e., reduced susceptibility compared to homozygotes given the same level of exposure. However, it is also possible that susceptibility is identical, and the
genotype of the sheep has a direct influence on incubation period. It is often assumed that under most conditions sheep born into a flock in which scrapie is endemic will become infected at an early age, and it is possible that young sheep are more susceptible to scrapie than adults. The transmission to adult and sub-adult sheep introduced to the infected flock in this study shows that sheep are susceptible to infection at various ages, including in adulthood. As none of these sheep have yet been exposed for significantly longer than the age at death of native born lambs, it is not possible to say whether the exposure at a later age results in either lower susceptibility or a prolonged incubation period, but these results provide conclusive evidence of lateral transmission of scrapie between sheep. It is curious that after 24 months exposure sub-adults first introduced at 6 months of age had a lower prevalence of infection than those introduced at 24 months of age. The sheep introduced at 6 months of age in November were too young to lamb in their first Spring, and so were not housed with the rest of the flock for lambing until the following year, 18 months later. Those introduced at 24 months lambed with the flock 6 months later. It is possible therefore that the extent of exposure to infection for those introduced at 6 months of age was low until approximately 18 months following introduction, when they will have been housed for lambing with
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the infected flock. Although there is no statistical evidence to support a true difference between the two groups (p ¼ 0:36, Fishers exact test), the small group sizes may lack the power to detect a true difference. The results of examination of lambs born to New Zealand derived ewes are also highly suggestive of lateral transmission to lambs. In all cases it is unlikely that the ewes were shedding infection at the time of birth of the lambs, given the short period of exposure and the absence of detectable disease specific PrP in sheep of these gentoypes at less than 10 months of age (Table 1). In two cases biopsy of tonsil and nictitating membrane were negative almost two years following lambing. One of these ewes was of the VRQ/ARQ genotype, in which disease specific PrP has been detected in it 20/21, i.e., 95.2% (95% C.I. 76.2–99.9%) of scrapie affected sheep of this genotype tested post-mortem (unpublished observations). Given this high level of sensitivity for PrP detection in this genotype, it is unlikely that this ewe was in an advanced stage of the incubation period. Therefore although a false negative result cannot be ruled out in this case it is unlikely and a negative biopsy result is consistent with either the ewe not being infected at the time of biopsy, or being at an early stage of infection; in either scenario this means it would have been unlikely to be infected at the time of birth of itÕs lambs. The second ewe which gave a negative biopsy result was of the ARQ/ARQ genotype; PrP detection in this genotype is less reliable than in ARQ/VRQ, and the possibility of a false-negative result is greater in this case. Therefore, whilst maternal transmission cannot be excluded as a source of infection in these lambs, it is unlikely, at least for one ARQ/VRQ ewe, almost two years after the birth of the lambs. Yet VRQ homozygous lambs born to these VRQ/ARQ New Zealand derived ewes died from scrapie at the same age as native born lambs; just under two years old despite the probable absence of maternal transmission, indicating a similar level of exposure regardless of the infection status of the ewes. These data are therefore consistent with lateral transmission being the most important route of infection in both lambs and adult sheep. A similar observation was made by Andreoletti et al. (2002) based on identical incidence of infection in lambs, as identified by PrPSc accumulation in lymphoid tissues, regardless of the PrPSc content of the placenta. The mechanism of natural infection with scrapie is unknown. The only known source of contamination of the environment by infectious tissues is via the placenta and fetal fluids. There is no evidence for infectivity in milk, saliva, faeces or urine of scrapie infected sheep (reviewed by Hoinville, 1996). However this is based almost exclusively on failure to detect infectivity by bioassay in these excretions/secretions, and given the likely low level of infectious agent present and the difficulty of bioassay of such contaminated material by
intra-cranial inoculation of mice, these negative findings must be interpreted with caution. Contamination of the environment outside of the lambing period remains a possibility. Abnormal PrP has recently been detected in urine of experimentally infected hamsters and bovine spongiform encephalopathy infected cattle (Shaked et al., 1999). An additional possible route of transmission, which has received little attention, is via contaminated needles. The common use of multiple dose injectors for vaccine delivery may represent a means of transmission between sheep on the same farm. Infectivity has been detected in blood of BSE infected sheep by transfusion of large volumes of blood between sheep (Hunter et al., 2002) but multiple dose injectors are an unlikely vehicle for transmission of contaminated blood. Needles penetrate the skin and subcutaneous tissues and contamination with blood is minimal. Inadvertent puncture of lymphoid tissue and transfer of infected lymphoid tissue would be theoretically possible, but would be a very rare event. Furthermore scrapie has been known for centuries, long before the use of injectable drugs. Therefore the likely potential mechanisms for lateral transmission of scrapie under normal farm conditions are either exchange of body fluids via direct contact (which would include saliva, urine and faeces) or via the environment, in which the agent can persist for a considerable length of time following contamination with placenta and itÕs fluids, urine, faeces, regurgitated rumenal contents or even saliva. At the present time all of these sources of infection must remain possible, but there is no direct evidence for any except placenta. Studies are ongoing in this laboratory to dissect the process of natural transmission of scrapie in this flock by exposure of sheep to defined and restricted parts of the annual husbandry cycle. It is hoped that results of this work will indicate possible routes of excretion of the agent, and help explain when and how during the pathogenesis of this disease the agent is shed from the host. Acknowledgements We are grateful to Hugh Simmons for provision of New Zealand derived sheep, to Tony Duarte for management of the infected flock, to Peter Bellerby for immunohistochemistry, to Roy Jackman for supply of antibody R486 and to Charlotte Cook, Alies Hoek and Linda Hoinville for their contributions to management of the infected flock. References Andreoletti, O., Berthon, P., Marc, D., Sarradin, P., Grosclaude, J., van Keulen, L., Scheler, F., Elsen, J.-M., Lantier, F., 2000. Early
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