Infectious disease.

Abstract

Serapie is a naturally occurring fatal neurodegencrative disease of sheep and goats. Susceptibility to the disease is partly dependent upon the genetic makeup of the host. In a recent study. it was shown that sheep intracercbrally inoculated with a US scrapie agent (No. 13-7) developed scrapie and survived for an average of 19 months post inoculation. In the present study, when this serapie inoculum was further passaged for 3 successive generations, the survival time was reduced by approximately 8 months in scrapie-susceptible (QQ on prion protein gene [PRNP] at codon 171) Suffolk sheep. It is concluded that inoculum No. 13-7 appears to have been stabilized in susceptible (171 QQ) Suffolk sheep and may be considered a specific isolate of sheep serapic agent in the USA and therefore that it can he used to evaluate other isolates of sheep scrapie in this country. Key n'ords: I rnmunohistochemistry PrP genetic susceptibility, sheep scrapic strains transmissible spongilorm encephalopathies (TSEs): Western blot. Scrapie is a naturally occurring fatal neurodegenerative disease of sheep and goats. The susceptibility of' sheep to scrapie is dependent on genetic variation of the host prion protein (PRNP) gene) 410 PRNP genotypes are defined by variations in the amino acids located at codons 136, 154, and 171. At least 5 variant alleles have been found, and they are depicted as ARQ, ARR, VRQ, AHQ, and ARH. The codes represent amino acids at codons 136, 154. and 171, that is, A-136, R-154. and Q-171 (ARQ). where A = alanine, R = arginine. Q = glutamine. H = histidine. and V valine. The level of risk to contract scrapie varies depending on breed and the genotypes found within the fioek ARR are the most resistant, and VRQ are the most Susceptible. 1-4. A study of Suffolk sheep in the United States found 61 of orally inoculated animals developed scrapie. ' All were homozygous for glutanline (QQ) at allele 171 on PRNP gene* In a recent study. it was shown that sheep intracerebrally inoculated with US scrapie agent (inoculum No. 13-7) developed disease within an average of 19 months post inoculation (MPI)) This inoculurn was made with brain tissue of 13 sheep from 7 different source flocks)' This study documents incubation times, pathologic findings, and distribution of abnormal prion Present address: Department of Diagnostic Medicine and Pathobiology. Kansas State University, Manhattan. KS. protein (p1.pSe) by immunohistochemical (IHC) and Western blot (WB) techniques in tissues of genetically susceptible Suffolk sheep (QQ or QH at codon 171 of PRNP gene) that were intracerebrally inoculated with 3 consecutive passages of inoculum No. 13-7 (Table I). The objectives of the study were to reduce the incubation (post inoculation) time of the scrapie inoculum and to stabilize the inoculum so that it can be used to evaluate other isolates of sheep scrapie in this country. Materials and Methods A total of 18 Suffolk lambs (females and castrated males) from National Animal Disease Center's (NADC) scrapie-free flock were obtained for this study and were inoculated with scrapie when they were approximately 4 months of age. All except I sheep (No. 9102, Table I) were AAIRR/QQ at codons 136, 154, and 171. respectively (Table 1). The scrapie inoculum (No. 13-7) was passaged in 4 generations of lambs (from 1999 to 2004) by intraeerebral inoculations (Table 1). Originally this inoculuni was prepared from it pool of 13 scrapie-affected sheep brains (all were positive by IHC technique) from 7 source flocks.' The inoculLim was ground in a mechanical grinder. gentamicin was added at IOU pg/mI, and the final concentration of I 0 (wt/vol) was made with phosphate-buf6red saline. For subsequent passages, the scrapie-infected brain tissue was obtained from the animal with the shortest incubation to terminal disease