2011 Annual Report
1a.Objectives (from AD-416)
The project has 4 objectives: (1) Identify conformational and biological correlates of strain variation in the transmissible spongiform encephalopathies, (2) Identify genetic factors associated with horizontal transmission efficiency and susceptibility to the transmissible spongiform encephalopathies (3) Characterize the influence of genetics, strain, and multiple births on placental transmission of small ruminant TSEs; and (4) Devise a model system for assessing methods to reduce persistent environmental contaminations by prions.
1b.Approach (from AD-416)
The current proposal addresses methods for characterizing and controlling classical and novel transmissible spongiform encephalopathies (TSEs)of domestic sheep and of farmed and free ranging deer and elk. The project includes discovery of unique identifiers for the North American TSE strain of small and wild ruminants and development of standardized methods suitable for use by the federal diagnostic reference laboratory and federally approved diagnostic laboratories. The genetic basis for relative transmission efficiency between and within the affected species, a critical element in design of control programs, will be is addressed through identification of haplotypes associated with naturally occurring disease. Allelic frequencies and disease associations are determined from tissue samples of naturally infected sheep, goats, deer, and elk. Genomic DNA is analyzed for the sequence of genomic regions including Prnp, Prnd, and Prnp' (when applicable). Samples of brain from infected animals are evaluated for relatively large changes in the apparent molecular weight of the proteinase K resistant core and for changes in the relative abundance of the variously glycosylated isoforms. The distribution and processing of disease associated PrP will be examined with a panel of monoclonal antibodies using single and double label immunohistochemistry assay. Samples with novel genotypes or prion protein isoforms will be evaluated in vivo when applicable. If novel strains are identified by these methods, standardized reagents and protocols for rapid strain typing of field samples will be developed and transferred to the national reference laboratory and the federally approved veterinary diagnostic laboratories. The role of the shed placenta and other environmental factors in TSE transmission and prion persistence will be examined.
The project (1) addresses critical gaps in our understanding of the distribution of abnormal prion proteins in the tissues of sheep, goats, deer, elk, and small carnivores; (2) determines whether the current diagnostic methods are suitable for animals in each genotype; (3) identifies the limits of genetic resistance to the prion diseases; and (4) identifies environmental reservoirs of the infectious agent. This project provided reagents and methods for the first generation live animal test in sheep and participated in two international collaborations to extend those methods to diagnosis of bovine spongiform encephalopathy by assay of brain tissue. The methods were used to characterize atypical (Nor98) scrapie in Canada and the US. In association with Colorado State University, we described the distribution of the abnormal prion protein in the tissues of Rocky Mountain elk; this work contributed to our understanding of disease transmission and early diagnosis of prion disease in captive elk. In collaboration with the Canadian Food Inspection Agency, we reported the experimental transmission of prion disease from elk to red deer. The project provided information on the role of prion genotype in disease transmission in sheep and information on the effects of chimerism on genotype testing. In collaboration with the Canadian Food Inspection Agency, we described the prion genotypes of scrapie infected Canadian sheep and reported that the susceptibility patterns are similar to those observed in the US sheep. We provided baseline information on prion gene variation in US goats. In collaboration with the Veterinary Genetics Laboratory, we developed methods for determining relatedness in captive herds of white tailed deer with prion disease. In addition, we described the association between tissue mineral levels and prion disease in Rocky Mountain elk. Taken together, studies provided information being used by the regulatory agencies of the US and Canada in developing scrapie eradication programs. New project 5348-32000-030-00D to start 10-1-11.
Prion genotypes of scrapie-infected Canadian sheep. Selection for genetic resistance to scrapie is a critical element in the North American scrapie control programs although data supporting this approach are derived largely from European studies. The Canadian Food Inspection Agency and ARS collaborated on a retrospective study of the prion genotypes of a sample of 249 sheep with confirmed classical scrapie infection representing 98 flocks from 6 provinces and a further case-control analysis of 3 of these flocks comparing the genotypes between infected sheep (n = 72) and those of their healthy flockmates (n = 1990). The incidence of classical scrapie in the Canadian sheep population was highly associated with a single haplotype (91.8%), findings similar to those reported by USDA APHIS for US scrapie-infected sheep. This finding supports the current control program that specifies removal or permanent quarantine of all scrapie-exposed sheep homozygous for that genotype.
The effects of autolysis on detection of abnormal prion proteins. Diagnosis and characterization of scrapie strains is made by a combination of biochemical tests for the abnormal prion protein PrP-Sc. Tissues submitted for analysis are often of poor quality, with varying levels of autolysis noted but the effect of autolysis on detection and characterization of the prion protein is not known. The Canadian Food Inspection Agency, in collaboration with ARS, completed a study of experimentally autolyzed tissues to address this knowledge gap. This study showed that the amount of PrP-Sc in lymphoid and central nervous system (CNS) tissues from elk and sheep decreased gradually as a result of autolysis, but PrP-Sc was still detectable after 5 and 15 d incubation at 37°C using either of two detection methods. The study demonstrated that the western blot technique can be used for diagnosis of scrapie using autolyzed samples.
Scrapie control program. The scrapie control program is based on the observations that ovine scrapie is transmitted effectively at the time of lambing and that transmission by unbred ewes or males rarely occurs. Similar data on transmission of scrapie in goats is lacking. In this study, ARS scientists in Pullman, WA examined the placentas of goats with naturally acquired scrapie and determined that the levels of abnormal prion protein are significantly lower than those observed in infected sheep, although the progeny of these goats were shown to develop the disease. This finding suggests that control programs specific for goats may be warranted, with attention to alternative modes of transmission.
Resistance of fallow deer to chronic wasting disease under field conditions. Chronic wasting disease in farmed cervids is managed on a species basis by a coordinated series of state and federal regulations. The regulations were written for the most commonly affected species (mule deer, white tailed deer and Rocky Mountain elk). The susceptibility of fallow deer, another commonly held species, to experimental infection by the intracerebral route has been established but the susceptibility of the species to natural challenge is not known. ARS contributed to a study conducted by USDA APHIS and the Colorado Division of Wildlife, in which fallow deer were held in pens with mule deer; all mule deer in the study died with evidence of CWD infection and none of the fallow deer showed either clinical disease or evidence of abnormal prions in any of the examined tissues. This finding suggests that the species is relatively resistant to natural transmission and may represent the only naturally resistant deer species reported to date.
Association analysis of prion gene region sequences with chronic wasting disease in Rocky Mountain elk. Genetic resistance to ovine scrapie is a cornerstone of the current control program and a similar approach to chronic wasting disease (CWD) would be beneficial to the industry. In association with Colorado State University and USDA APHIS, ARS scientists in Pullman, WA, analyzed prion gene sequences in a sample of 559 captive and free ranging Rocky Mountain elk, of which 120 were considered positive for CWD. The previously reported mutation at codon 132 cut the odds of CWD by half but no association with CWD was found for any additional variants in the PRNP region (P > 0.05). This finding supports the current control programs, which consider that all elk exposed to CWD must be considered at risk of CWD.
White, S.N., Spraker, T.R., Reynolds, J.O., Orourke, K.I. 2010. Association analysis of PRNP gene region with chronic wasting disease in Rocky Mountain elk. BMC Research Notes. 3:314.
O'Rourke, K., Zhuang, D., Truscott, T.C., Yan, H., Schneider, D.A. 2011. Sparse PrP-Sc accumulation in the placentas of goats with naturally acquired scrapie. BioMed Central (BMC) Veterinary Research. 7(1):7.
Rhyan, J.C., Spraker, T.R., Mccollum, M., Nol, P., Wolfe, L., Miller, M.W., Davis, T., Creekmore, L., Orourke, K.I. 2009. Resistance of fallow deer (dama dama) to chronic wasting disease under natural exposure in a heavily contaminated environment. Journal of Wildlife Diseases. 47(3):739-744.
Harrington, N., Orourke, K.I., Feng, Y., Rendulich, J., Difruscio, C., Balachandran, A. 2010. Prion genotypes of scrapie-infected Canadian sheep 1998-2008. Canadian Journal of Veterinary Research. 74(3):228-232.
Huang, H., Soutyrine, A., Rendulich, J., Orourke, K.I., Balachandran, A. 2011. Investigation of the effects of experimental autolysis on the detection of abnormal prion protein in lymphoid and central nervous system tissues from elk and sheep using the Western blotting method. Canadian Journal of Veterinary Research. 75(1)69-72.