Transmissible spongiform encephalopathies (TSEs), also know as prion diseases, represent a group of fatal neurodegenerative disorders in humans and several free-ranging and captive mammalian species. A report of scrapie in mouflon from England and the fact that Germany’s free-ranging mouflon population is the second largest in the world raised the question if mouflon in Germany are affected by TSEs. Therefore, the aim of this survey was (1) to examine the prevalence of prion diseases in mouflon from Germany, and (2) to determine the genetic susceptibility of the populations. Thus a population screening for prion diseases and a sequence analysis of the prion protein gene (PRNP) were carried out. According to the results, a detailed risk assessment was aimed for.
All administrative districts in Germany were divided into risk categories defined by the number of reported scrapie cases in domestic sheep, the number of hunted mouflon per year, and the abundance of sheep. Screening included districts of risk categories III and II where the probability to discover TSEs in mouflon was highest. Mouflon ewes and rams older than 18 months collected from hunting bags during the hunting seasons 2006 / 2007 and 2007 / 2008 were sampled. In order to detect classical and atypical scrapie cases, samples were taken from brain stem, cerebellum and retropharyngeal lymph nodes. The samples were analysed by an accredited laboratory using a highly sensitive rapid test (IDEXX HerdChek BSE-scrapie).
Mouflon of all screening districts were chosen at random for PRNP genotyping. Brain tissue samples were used for DNA extraction and subsequent PCR amplification and sequencing of an 862 base pair long DNA fragment. All sequences received were compared with gene bank submissions of sheep and mouflon and translated into their amino acid sequences. Focus was the 771 base pair long prion protein coding sequence, especially codons 136, 154, and 171, since these are associated to TSE susceptibility.
A total of 2.453 tissue samples of 823 mouflon from 39 administrative districts were analysed. Protease resistant prion protein (PrPSc) was not detected in any of the examined brain and lymph node samples; all mouflon were TSE negative. All samples from districts of the same risk category were pooled for further analysis and estimation of the detection limits with a probability of 95 percent. TSE prevalence in adult mouflon during the sample period was less than 0.56 % in districts of risk category III and 1.2 % in districts of risk category II.
Genotyping of the PRNP sequence of 246 mouflon was performed. The sequence-based analysis showed that all tested mouflon carried the same ARQ/ARQ genotype. Thus the genotype is independent of the geographic origin and appears to reflect naturalisation history. Accordingly, it can be assumed that ARQ/ARQ is the only existing genotype in mouflon in Germany.
This study represents the most comprehensive survey on TSE and prion protein genotypes in free-ranging mouflon. At present, evidence of prion diseases in the German mouflon population has not been found. However, it cannot be entirely ruled out that prion diseases are present in the populations at very low prevalence, since a sample survey was performed. Results of the genotyping indicate that mouflon are genetically highly susceptible to classical scrapie and BSE and susceptible to atypical scrapie. In addition a potential direct and indirect contact to scrapie-infected domestic sheep results in a high exposure risk for mouflon in certain areas. Mouflon should be included in the TSE surveillance to ascertain a sound free-ranging population and to meet the precaution principle. Passive surveillance should be performed on all clinically conspicuous mouflon, including a veterinary examination and a rapid test. Active surveillance, i.e. examination of all hunted and fallen mouflon, is advisable for all areas with a high exposure risk to classical scrapie.
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