Nature 423, 498 (2003); doi:10.1038/423498a
Prion diseases: BSE in sheep bred for resistance to infection
Selective breeding for disease-resistant genotypes is being pursued as a means of eradicating scrapie (and bovine spongiform encephalopathy (BSE), if it is present) from sheep flocks. Here we show that the genotype associated with the highest resistance can still be infected with BSE by intracerebral inoculation. Although the relevance of this finding to sheep exposed to natural infection remains to be determined, it may have important implications for disease-eradication strategies.
Susceptibility and resistance to transmissible spongiform encephalopathies (TSEs) in sheep is largely controlled by polymorphisms at codons 136, 154 and 171 of the gene that encodes prion protein (PrP). Representing the resulting alleles as the amino acids at these positions
susceptibility to scrapie is strongly associated with valine (V) at position 136 and/or glutamine (Q) at position 171, and susceptibility to experimental BSE is associated predominantly with the alanine-arginine-glutamine (ARQ) allele. The ARR allele is associated with resistance to natural scrapie — the disease is rare in ARR heterozygotes and only one unconfirmed case has ever been reported in a homozygote. The limited data available suggest that ARR/ARR sheep are also resistant to experimental oral challenge with scrapie and BSE. Britain has therefore initiated the National Scrapie Plan, which aims eventually to eradicate scrapie (as well as any BSE concealed in sheep) by means of increasing the frequency of the ARR allele and decreasing that of the VRQ allele.
We investigated whether ARR/ARR sheep are still resistant to transmission of BSE when inoculated intracerebrally with infected cattle-brain homogenate (0.05 g). Groups of New Zealand (scrapie-free) sheep of three different breeds (Suffolk, Cheviot and Poll Dorset) and six different PrP genotypes (VRQ/VRQ, VRQ/ARQ, VRQ/ARR, ARQ/ARQ, ARQ/ARR and ARR/ARR) were tested.
As expected, the incubation period (495–671 days) was shortest in ARQ/ARQ sheep, with 17 out of 19 being affected. So far, four out of ten VRQ/VRQ and two out of ten VRQ/ARQ Cheviot sheep have also succumbed to disease after incubation periods of 881–1,092 days. Unexpectedly, 3 out of 19 BSE-challenged ARR/ARR sheep also showed clinical symptoms after incubation periods of 1,008, 1,124 and 1,127 days, respectively, but none of the ARR heterozygotes has been clinically affected to date.
Western-blot analysis of the abnormal protease-resistant fraction of PrP (PrPSc) (results not shown) and immunohistochemical labelling of disease-associated PrP in brain sections from the ARR/ARR sheep (Fig. 1) were consistent with experimental BSE in ARQ/ARQ sheep.
This contrasts with the results of in vitro cell-free conversion experiments, in which conversion of the sheep ARR PrP protein to PrPSc was extremely inefficient.
Immunohistochemical labelling of abnormal PrP protein in the dorsal motor nucleus of the vagus of a sheep that is homozygous for the ARR allele (see text) and has been infected with bovine spongiform encephalopathy (BSE). The susceptibility of ARR/ARR sheep to intracerebral injection with BSE indicates that these animals cannot be regarded as having absolute genetic resistance to TSE infection. The significance of this result for sheep that are exposed to natural TSE infection remains to be determined. In ongoing experiments, ARR/ARR sheep orally dosed with BSE have remained free from clinical disease or evidence of preclinical infection for up to 2,100 days after BSE challenge (ref. 6 and J.F., unpublished results). However, the number of animals in these studies is probably too small to rule out susceptibility even if the surviving animals remain free of infection.
Our results may reflect those found for pigs, in which no natural case of BSE has been reported. Pigs inoculated with BSE simultaneously by intracerebral and peripheral routes developed TSE within 520–1,130 days, whereas those exposed to repeated oral challenge remained healthy for up to 2,550 days after infection.
Even if ARR/ARR sheep exposed to natural TSE infection do not succumb to disease, they might be able to act as subclinical carriers of infection. However, no abnormal PrP (and, by implication, infectivity) was detected in the lymphoid tissues of any of the ARR/ARR sheep cases of BSE reported here. Likewise, neither clinically normal ARR/ARR sheep in scrapie-endemic flocks, nor those challenged orally with TSEs, show evidence of abnormal PrP deposition in peripheral tissues — although here again the number of animals examined is small. These results indicate that ARR/ARR sheep are unlikely to excrete the infectious agent, although transmission of infection to in-contact animals in the absence of detectable abnormal PrP cannot be ruled out.
We have shown that ARR/ARR sheep can become infected when inoculated intracerebrally with a high dose of BSE, but there is, as yet, no evidence to suggest that they are susceptible when exposed by natural routes of infection. But the fact that the ARR PrP protein can undergo conversion to a pathological isoform in vivo raises questions about the basic mechanisms that underpin genetic resistance to TSEs.
FIONA HOUSTON*, WILFRED GOLDMANN†, ANGELA CHONG†, MARTIN JEFFREY‡, LORENZO GONZÁLEZ‡, JAMES FOSTER†, DAVID PARNHAM† & NORA HUNTER† * Institute for Animal Health, Compton, Newbury, Berkshire RG20 7NN, UK † IAH Neuropathogenesis Unit, Ogston Building, Edinburgh EH9 3JF, UK
‡ Veterinary Laboratories Agency Lasswade, Pentlands Science Park, Penicuik, Midlothian EH26 0PZ, UK
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