Institute for Animal Health, Neuropathogenesis Unit, West Mains Road, Edinburgh EH9 3JF, UK
Correspondence
N. Hunter
nora.hunter{at}bbsrc.ac.uk
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ABSTRACT |
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MAIN TEXT |
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Intracerebral (i.c.) and oral experimental infection of sheep with BSE have shown that infectivity (detected by mouse bioassay) is present in both brain and spleen (Foster et al., 1996). Deposition of the disease-associated isoform of PrP protein, PrPSc, is widespread throughout the central nervous and lymphoid systems, including the gut (Foster et al., 2001a
). This contrasts with clinical cases of BSE in cattle (Wells & Wilesmith, 1995
; Wells et al., 1998
; Terry et al., 2003
), where peripheral PrPSc deposition is very low or non-existent. In sheep BSE, no evidence has been found of PrPSc in reproductive or placental tissues, or fetal brain and spleen (Foster et al., 2001a
), although this contrasts with placental analysis from scrapie studies (Race et al., 1998
; Andreoletti et al., 2002
).
The present study uses sheep from the NPU Cheviot flock, which differ in susceptibility, controlled by PrP genotype, to BSE following experimental oral infection (Goldmann et al., 1994; Foster et al., 2001b
). Genotype here is described using the single letter amino acid code for each of codons 136 and 171 for each allele in turn. [In our original studies of BSE in these sheep, no effect of codon 154 genotype was observed (Foster et al., 2001b
)]. AQ/AQ (Ala136Gln171/Ala136Gln171) sheep develop disease with the shortest but variable incubation period and with 4050 % of challenged sheep remaining clinically unaffected in our hands (see also Foster et al., 2001a
). Another experimental study of BSE in Romney sheep produced 100 % disease incidence in animals left to the end-point with incubation periods of 2028 months (Jeffrey et al., 2001
). The basis for this breed difference in percentage of animals affected, although now well documented, is not understood. Our source of BSE has 103·5 infectious units g1 measured in mice (Foster et al., 2001a
), similar to that used in the Romney sheep study (S. J. Bellworthy, personal communication). The oral doses delivered to the Cheviots were completely ingested by the animals. However, the Cheviot breed has much more complex PrP genetics than is found in Romneys and it is a strong possibility that the variation in response of susceptible Cheviots is because of an additional genetic component within the AQ allele group that remains at present uncertain despite extensive investigation of numerous polymorphic PrP animo acid codons by gene sequencing (W. Goldmann, unpublished). As Cheviot PrP genetics is similar to that found in a wide range of UK sheep, the results, though complex, remain relevant. AQ/AR sheep have only succumbed after i.c., but not oral, inoculation and with very much longer incubation periods than AQ/AQ. The AR/AR genotype is considered the most resistant. However, a number of experimental cases have occurred in this genotype after i.c. BSE-challenge (Houston et al., 2003
).
In our experiments, sheep (n=20) were orally dosed with 5 g of BSE cattle brain. Four uninfected AQ/AQ sheep were mixed with infected sheep as controls for inadvertent contact disease transmission (see Table 3). Ewes were naturally mated with an AQ/AQ or AQ/AR ram each year producing seven lamb crops. Four rams were used, of which two remain alive at >106 months of age and two which died at >106 and >72 months of age with no clinical or histological signs of TSE. Rams were mated to infected and control ewes and there was no evidence for BSE transmission between ewes, or ewe-to-ram by this route.
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A total of 105 lambs are surviving at the time of writing. Twenty progeny are at ages <30 months. Tables 13 show only those 85 animals now over the age of 30 months. This cut-off point was chosen because the earliest we could reasonably expect clinical signs of BSE to appear in offspring is (by comparison with natural scrapie) from about 24 months of age, the shortest oral BSE incubation period in our challenge experiments is 18 months and the mean incubation for all BSE oral challenges in NPU Cheviots is 26 months (J. D. Foster, unpublished). The analysis has also included five offspring of challenged sheep that died of intercurrent illness (not TSE) at 30, 31, 37, 49 and 64 months of age, and one animal culled at 36 months and born to a control, uninfected ewe, see below. None of the intercurrent deaths had clinical signs of TSE or PrPSc; however, transmissions from brain and spleen were set up in mice from two of the culled sheep, the 30 month (AQ/AR) and the 31 month (AR/AR). Both lambs were born to BSE-challenged AR/AR dams, which are still alive. If BSE was present in the culled animals at levels consistent with terminal BSE infection (around 103·5 infectious units g1 in mice), expected mouse incubation periods (Foster et al., 1996
) would be approximately 300350 days in RIII mice and 400450 days in C57 mice. At over 700 days after challenge, the results in both mouse strains for the culled sheep samples are negative.
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The progeny of BSE-challenged ewes can be divided into two groups, those born to ewes which developed clinical signs of BSE, and those born to ewes which remained healthy. Surviving challenged ewes could retain subclinical infection and act as carriers, still having the potential for the transmission of disease.
In total, nine of the most BSE-susceptible AQ/AQ lambs are amongst the 13 progeny that survive at >30 months, and born to ewes which developed clinical disease (Table 1). One of the AQ/AQ lambs born in 1999 (to 52x11) was culled at 49 months and was negative for TSE by histology and ICC; the remaining 8 AQ/AQ lambs are still living at >30 months of age (Table 1
). The upper 95 % confidence limit for the probability of transmission has been calculated by assuming that the number of lambs, of a particular genotype, contracting the disease have a binomial distribution. In this study, the probability of transmission is defined as the probability that a lamb aged at least 30 months shows clinical signs of the disease. Using this analysis, and if maternal transmission of BSE exists in the most susceptible genotypes and with proven clinical illness developing in the mother, the maximum rate of transmission would be less than 0·28. It should also be noted that six of the progeny were born over half-way through the BSE incubation period of their mothers, thereby increasing any putative maternal challenge. There are also four AQ/AR lambs surviving at 69 months of age having been born to clinically affected ewes. This genotype is susceptible to BSE experimentally inoculated by the i.c. route with a mean incubation period of 67 months (Foster et al., 2001b
).
Seventeen ewes were challenged with BSE but remained apparently healthy, apart from four intercurrent (non-TSE) deaths: one 4 months after challenge that produced no lambs, three at 14, 51 and 75 months after challenge. All four intercurrent deaths were negative for PrPSc in brain and lymphoid tissue. Of the 16 ewes that produced lambs, four were AQ/AQ genotype, six were AQ/AR and 6 were AR/AR. All these genotypes are susceptible to BSE-challenge, although some only with the i.c. route (AQ/AR and AR/AR) and so all have the potential to act as asymptomatic carriers of infection. They have produced 81 lambs to date, 55 surviving at ages >30 months, plus four lambs that were culled following intercurrent illness at 3064 months old. Because of the small numbers, progeny from these ewes have been considered as a single group (n=59) but the full dataset from the >30 month old progeny is presented in Table 2. Twenty are of AQ/AQ genotype, 31 are AQ/AR, 7 are AR/AR genotype and one of incompletely determined genotype (AX/AR). Considering the most susceptible AQ/AQ offspring alone, this suggests that BSE transmission from subclinically infected carrier mothers could only occur at a rate of less than 0·14. If all AQ/AQ progeny (29) from challenged dams are considered the likelihood of transmission is less than 0·1 (95 % confidence limit). Transmissions to mice from two of this group of progeny have proved negative (see above in discussion of intercurrent deaths in older offspring).
Four AQ/AQ uninfected-control ewes produced 16 lambs, 14 at >30 months of age (Table 3), all mixed with infected sheep for the lifetime of the experiment. Eleven control AQ/AQ lambs are alive at >30 months of age, the oldest are >81 months of age. One additional AQ/AQ lamb died at 36 months (not TSE) and was included in the analysis, which gives a rate for contact transmission for the lambs of less than 0·22. However, one of four uninfected ewes developed an apparent neurological condition (shaking, not rubbing and loss of condition over 4 months) and was culled 48 months after joining the infected sheep groups. ICC detection of PrPSc in brain was inconclusive, with some sparse deposits in the cerebellum, and completely negative in lymphoid viscera, which is not indicative of terminal BSE, or indeed of natural scrapie, in sheep (Foster et al., 2001a
). Mouse transmissions have also been set up using brain and spleen tissues from this unchallenged ewe and are negative (at the time of writing) after 570 days, around 270 days later than the earliest expected for BSE in RIII mice, and will continue to be observed. It is unlikely, however, from the ICC results, that this sheep developed a TSE infection. One other unchallenged ewe became ill (not TSE) at >72 months of age and was negative by immunostaining for PrPSc.
Our results suggest that maternal transmission of BSE, even in the most favourable circumstances, could only occur in NPU Cheviots at a maximum rate of less than one in four. It is difficult to prove a negative result; however, from other studies experimental BSE in goats resulted in no clinical cases in progeny with a maximum possible transmission rate of <5 % (Foster et al., 1999) and natural maternal transmission of BSE from cow-to-calf was estimated at a maximum rate of 10 % (Wilesmith et al., 1997
). Our mixing of unchallenged ewes and their offspring with the experimentally challenged groups suggests that contact transmission of BSE is also inefficient.
All progeny will continue to be monitored. The possibility of hidden infection, or carrier-state, could have serious consequences for the UK National Scrapie Plan, which seeks to eradicate all TSEs in sheep by increasing the frequency of the PrP AR allele. However, our results should give some degree of confidence that if sheep-to-sheep transmission of BSE were to occur, it would probably only delay, and not prevent, BSE eradication.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 8 March 2004;
accepted 21 June 2004.