Resistance to classical scrapie in experimentally challenged goats carrying mutation K222 of the prion protein gene

ABSTRACT: Susceptibility of sheep to scrapie, a transmissible spongiform encephalopathy of small ruminants, is strongly influenced by polymorphisms of the prion protein gene (PRNP). Breeding programs have been implemented to increase scrapie resistance in sheep populations; though desirable, a similar approach has not yet been applied in goats. European studies have now suggested that several polymorphisms can modulate scrapie susceptibility in goats: in particular, PRNP variant K222 has been associated with resistance in case-control studies in Italy, France and Greece. In this study we investigated the resistance conferred by this variant using a natural Italian goat scrapie isolate to intracerebrally challenge five goats carrying genotype Q/Q 222 (wild type) and five goats carrying genotype Q/K 222. By the end of the study, all five Q/Q 222 goats had died of scrapie after a mean incubation period of 19 months; one of the five Q/K 222 goats died after 24 months, while the other four were alive and apparently healthy up to the end of the study at 4.5 years post-challenge. All five of these animals were found to be scrapie negative. Statistical analysis showed that the probability of survival of the Q/K 222 goats versus the Q/Q 222 goats was significantly higher (p = 0.002). Our study shows that PRNP gene mutation K222 is strongly associated with resistance to classical scrapie also in experimental conditions, making it a potentially positive target for selection in the frame of breeding programs for resistance to classical scrapie in goats.     

Immunohistochemical detection and distribution of prion protein in a goat with natural scrapie.

Formalin-fixed, paraffin-embedded tissue sections from a 3-year-old female Angora goat suffering from clinical scrapie were immunostained after hydrated autoclaving using a monoclonal antibody (mAb, F99/97.6.1; IgG1) specific for a conserved epitope on the prion protein. Widespread and prominent deposition of the scrapie isoform of the prion protein (PrPSc) was observed in the brain, brainstem, spinal cord, retina, postganglionic neurons associated with parasympathetic ganglia of myenteric and submucosal plexuses, Peyer's patches, peripheral lymph nodes, and pharyngeal and palatine tonsils. The goat was homozygous for PrP alleles encoding 5 octapeptide repeat sequences in the N-terminal region of the prion protein and isoleucine at codon 142, a genotype associated with high susceptibility and short incubation times in goats. The results of this study indicate that mAb F99/97.6.1 is useful for detection of PrPSc deposition, and this is a specific and reliable immunohistochemical adjunct to histopathology for diagnosis of natural caprine scrapie, although precise determination of the diagnostic sensitivity and specificity of the assay as a diagnostic test for scrapie in goats will require examination of a sufficiently large sample size. As with ovine scrapie, prion protein is widely distributed in the central and peripheral nervous systems, gastrointestinal tract, and lymphoid tissues in natural caprine scrapie.     

Development of an assay to determine single nucleotide polymorphisms in the prion gene for the genetic diagnosis of relative susceptibility to classical scrapie in sheep.

The objective of this study was to develop a reliable Taqman 5' Nuclease Assay for genotyping sheep for scrapie susceptibility. The sheep prion gene contains 2 single nucleotide polymorphisms (SNPs) that may mediate resistance to classical scrapie, one at codon 136, alanine (A) or valine (V), and another at codon 171, arginine (R) or glutamine (Q). The R allele appears to confer resistance to classical scrapie, with the AA(136) RR(171) genotype the most resistant to scrapie and QR(171) only rarely infected in the US sheep population. The Assays by Design protocol was used for development of probes and primers for codon 136 and Primer Express for codon 171. Commercially available kits were used to isolate genomic DNA from blood or muscle. For validation, 70 SNP determinations for each codon were compared to commercial testing with an error rate of less than 1%. Then, 935 samples from blood (n = 818) and muscle (n = 117) were tested for both codons with 928 successful determinations and only 7 samples (<1% of total samples) that needed repeating. Genotypes were AA QQ (n = 102; 11.0%), AV QQ (n = 28; 3.0%), AA QR (n = 396; 42.7%), AV QR (n = 54; 5.8%), and AA RR (n = 348; 37.5%). Thus, 86% of the sheep tested (n = 798) contained R at codon 171 and were expected to be scrapie-resistant. This new Taqman 5' Nuclease SNP genotyping assay is accurate, easy to perform, and useful in the study of classical scrapie in sheep and its prevention through selective breeding programs to eliminate highly susceptible animals.     

BSE‐associated polymorphisms in the prion protein gene: an investigation

The aim of this study was to determine the frequency of the 12‐bp and 23‐bp indel polymorphisms in the prion protein gene (PRNP) in cattle and to investigate the association between these frequencies and the occurrence of bovine spongiform encephalopathy (BSE). There was no significant difference in the 12‐bp indel frequency between the BSE animals and control group. For the 23‐bp indel, the BSE animals had a significantly lower + + (insins) genotype frequency and + allele frequency compared with the control animals. The ? ? / ? ? genotype frequency in the BSE animals was not significantly higher when compared with the control animals. One ? allele increased the risk of BSE by a factor of 1.55 (i.e. by 55%) for the 12‐bp indel and by a factor of 2.10 for the 23‐bp indel. When both indels are considered, one ? allele increased the risk of BSE by a factor of 1.54.     

Novel polymorphisms at codons 146 and 151 in the prion protein gene of Cyprus goats, and their association with natural scrapie

To discern whether an association exists between specific combinations of polymorphisms of the prion protein (PrP) and natural scrapie in Cyprus goats, 250 goats were examined, including 164 histologically positive cases. Previously reported amino acid polymorphisms were detected at codons 154 (R-->H), 168(P-->Q), 220(Q-->H) and 240 (S-->P) and nucleotide alterations at codons 42 (a-->g) and 138 (c-->t). Additionally, novel amino acid polymorphisms were detected at codons 146 (N-->S or D) and 151 (R-->H) and new "silent" mutations were found at codons 179 (V,g-->t), 181 (D,c-->t) and 219 (T,c-->t). The two novel polymorphisms at codon 146 were found only in the healthy control and scrapie-negative goats. By comparison, none of the scrapie-affected goats encoded these polymorphisms.     

High incidence of single nucleotide polymorphisms in the prion protein gene of native Brazilian Caracu cattle

Different alleles of the human and ovine prion protein gene correlate with a varying susceptibility to transmissible spongiform encephalopathies. However, the pathogenic implications of specific polymorphisms in the bovine prion protein gene (PRNP) are only poorly understood. Previous studies on the bovine PRNP gene investigated common European and North American cattle breeds. As a consequence of decades of intensive breeding for specific traits, these modern breeds represent only a small fraction of the bovine gene pool. In this study, we analysed PRNP polymorphisms in the native Brazilian Caracu breed, which developed in geographical isolation since the 16th century. A total of 10 single nucleotide polymorphisms (SNPs) were discovered in the coding region of the Caracu PRNP gene. Eight of the SNPs occurred at high frequencies in Caracu cattle (variant allele frequencies = 0.10–0.76), but were absent or only rarely observed in European and North American breeds. One of the Caracu SNPs was associated with an amino acid exchange from serine to asparagine (f = 0.17). This SNP was not detected in Holstein–Friesian, Simmental and German Gelbvieh and was only rarely detected in beef cattle (f = 0.01). We found 17 haplotypes for PRNP in the Caracu breed.     

Prion protein (PrP) gene polymorphisms associated with natural scrapie cases and their flock-mates in Ireland

The PrP genotypes associated with natural scrapie in Ireland were determined and a comparison was made between genotypes found in scrapie-infected sheep and those found in healthy animals from scrapie-infected flocks. Seven PrP genotypes were identified in scrapie-infected animals: VV(136)RR(154)QQ(171),VA(136)RR(154)QQ(171),VA(136)RR(154)QR(171),VA(136)RR(154)QH(171),AA(136)RR(154)QQ(171),AA(136)RR(154)QH(171) and AA(136)RR(154)HH(171). Of 11 scrapie-infected flocks, 15 genotypes were identified in the healthy flock-mates. The genotypes identified in scrapie-affected animals were also all identified in healthy flock-mates. In 9 of the 11 flocks studied, the genotype frequencies among scrapie-infected animals were significantly different from those among healthy flock-mates. The results show that there is a significant risk of developing the clinical signs of scrapie associated with particular PrP genotypes in the Irish sheep population. The association between the V(136)R(154)Q(171) allele and scrapie was evident, as was the association between A(136)R(154)R(171) and resistance to developing the clinical signs of scrapie. The presence of the A(136)H(154)Q(171) allele in the flocks examined resulted in a decreased risk of developing scrapie compared to the presence of the A(136)R(154)Q(171).     

Factors associated with mortality of geriatric horses in the United Kingdom

Geriatric horses (aged≥15 years) now represent a substantial proportion of the equine population, yet mortality rates for aged horses in the UK have not previously been described. Although post-mortem studies have provided some data regarding specific causes of death, "old age" is a common owner-reported reason for euthanasia of adult horses, indicating further elucidation of reasons for death or euthanasia is required for the geriatric equine population. The objective of this cohort study was to describe mortality rates, causes of death or euthanasia and factors associated with mortality in geriatric horses and ponies. Veterinary registered horses randomly selected for a cross-sectional questionnaire survey on geriatric health were enrolled in the cohort study (n=908). Follow-up information was obtained over an 18-month period via telephone questionnaires and mortality questionnaires, providing data regarding reasons for death or euthanasia, were completed for 118 cases of mortality. Overall and stratified mortality rates were calculated and Cox proportional hazards models were used to investigate risk factors associated with mortality. The majority (94%) of mortalities were euthanised, most frequently due to lameness (24%) and colic (21%). Veterinary advice was important in owner decision making regarding euthanasia of cases of colic or other acute illnesses, while poor quality of life was considered an important factor where euthanasia was due to chronic diseases or lameness. Overall mortality rate was 11.1 (95% C.I. 9.2-13.2) per 100 horse-years at risk, with the mortality rate of animals >30 years of age over five times the rate in horses aged 15-19 years. On multivariable analysis, increasing age was associated with increasing mortality and Cob/Cob crossbreeds and Thoroughbred/Thoroughbred crossbreeds had an increased risk of mortality. Horses considered to be underweight had a greater risk of mortality than those in good condition. Increasing number of owner-reported clinical signs and the degree to which pain was reported to limit normal daily activities were associated with increased risk of mortality. The results of this study provide useful information about rates and factors associated with mortality in geriatric horses in the UK, which should be relevant to veterinary surgeons involved in the treatment of aged horses.     

Conformational change in hamster scrapie prion protein (PrP27-30) associated with proteinase K resistance and prion infectivity

The scrapie prion protein (PrP27-30) is a crucial component of the prion and is responsible for its transmissibility. Structural information on this protein is limited because it is insoluble and shows aggregated properties. In this study, PrP27-30 was effectively dispersed using sonication under the weak alkaline condition. Subsequently, the small PrP27-30 aggregates were subjected to different pH, heat, and denaturing conditions. The loss of proteinase K (PK) resistance of PrP27-30 and prion infectivity were monitored along with spectroscopic changes. Prion inactivation could not be achieved by the loss of PK resistance alone; a significant loss of the PrP27-30 amyloid structure, which was represented by a decrease in thioflavin T fluorescence, was required for the loss of transmissibility.     

Lameness prevalence and risk factors in organic and non-organic dairy herds in the United Kingdom

This study aimed to compare the prevalence of lameness on organic and non-organic dairy farms in the United Kingdom (UK) and to assess which cow and farm factors influenced lameness levels. Forty organic and 40 non-organic dairy farms across the UK were repeatedly visited over a 2.5 year period. On each visit all milking cows were locomotion scored, and information about farm housing, management and husbandry practices was recorded on-farm. Over the whole study, the mean herd lameness prevalence was 16.2%, 16.3% and 19.3% in the autumn, winter and spring observation periods, respectively. Lameness prevalence was lower (P=0.012) on organic farms compared to non-organic farms. Numerous specific factors were found to significantly influence the prevalence of lameness. This study provided evidence that organic management reduced herd lameness. It supported previous research which suggested that lameness is a serious problem on many farms in the UK and further emphasised the multi-factorial aetiology of lameness problems.     

Degradation of scrapie associated prion protein (PrPSc) by the gastrointestinal microbiota of cattle

A food-borne origin of the transmission of bovine spongiform encephalopathy (BSE) to cattle is commonly assumed. However, the fate of infectious prion protein during polygastric digestion remains unclear. It is unknown at present, whether infectious prion proteins, considered to be very stable, are degraded or inactivated by microbial processes in the gastrointestinal tract of cattle. In this study, rumen and colon contents from healthy cattle, taken immediately after slaughter, were used to assess the ability of these microbial consortia to degrade PrP(Sc). Therefore, the consortia were incubated with brain homogenates of scrapie (strain 263K) infected hamsters under physiological anaerobic conditions at 37 degrees C. Within 20 h, PrP(Sc) was digested both with ruminal and colonic microbiota up to immunochemically undetectable levels. Especially polymyxin resistant (mainly gram-positive) bacteria expressed PrP(Sc) degrading activity. These data demonstrate the ability of bovine gastrointestinal microbiota to degrade PrP(Sc) during digestion.     

UTS2R gene polymorphisms are associated with fatty acid composition in Japanese beef cattle

Fatty acid composition of beef adipose tissue is one of its important traits because a high proportion of monounsaturated fatty acid is related to favorable beef flavor and tenderness. In this study, we searched polymorphisms in full length coding DNA sequence of urotensin 2 recepter and investigated the effects on fatty acid composition (C14:0, C14:1, C16:0, C16:1, C18:0, C18:1, C18:2, monounsaturated fatty acid, saturated fatty acid). Eight single nucleotide polymorphisms (SNP) were identified by sequence comparison among eight animals, including five Japanese Black and three Holstein cattle. One of these SNP (c.866C>T) was predicted to cause amino acid substitutions (P289L) and the other seven synonymous SNP, including c.267C>T, were presumed to be in linkage disequilibrium. Therefore we selected two SNP (c.267C>T and c.866C>T) for further analysis. We investigated associations between these genotypes and fatty acid composition in three Japanese Black populations (n = 560, 245 and 287) and a Holstein population (n = 202). Tukey‐Kramer's honestly significant difference test revealed that CC genotype in c.267C>T indicated lower C14:0 and higher C18:1 than the other genotypes in Japanese Black cattle and CC genotype in c.866C>T showed lower C16:1 than CT genotype in Holstein cattle (P < 0.05). These results suggested that these genotypes would contribute to production of high‐grade meat as selection markers in beef cattle.     

A real-time polymerase chain reaction assay to detect single nucleotide polymorphisms at codon 171 in the prion gene for the genotyping of scrapie susceptibility in sheep.

The objective of this study was to report a reliable real-time polymerase chain reaction assay compatible with the Roche LightCycler 2.0 capable of genotyping sheep for scrapie susceptibility at codon 171. The single nucleotide polymorphisms (SNPs) in the prion protein gene in sheep that may govern resistance to scrapie at codon 171 encode for lysine (K), histidine (H), glutamine (Q), and arginine (R). A modified proteinase K method for leukocytes or whole blood was used to isolate genomic DNA from sheep blood. Fluoresentric developed and optimized primers and probes for the codon 171 SNP assay. The assay was initially validated using 218 determinations from whole blood of known genotypes with 100% correct identity. The assay was further validated through a whole-blood check test provided annually by the National Veterinary Services Laboratory with a correct identification rate of 100%. From January 2005 to December 2006, 3,672 samples from blood were genotyped at codon 171. The genotypes were QR(171) (n = 1,838, 50.05%), RR(171) (n = 1,423, 38.75%), QQ(171) (n = 407, 11.08%), HR(171) (n = 2, 0.05%), and HQ(171) (n = 2, 0.05%). The combination of this simple extraction method and the novel Fluoresentric assay is very accurate, is capable of identifying all 4 SNPs at codon 171 in one reaction, and has proven to be a useful tool for producers in their selective breeding programs.     

Detection of prion protein gene polymorphisms in Baltic breeds of sheep

A total of 167 sheep belonging to the Estonian whiteheaded mutton, Estonian blackheaded mutton, Lithuanian coarsewool native, Lithuanian blackface and Latvian darkheaded mutton breeds, and a population of sheep kept isolated on the Estonian island of Ruhnu, were sequence-analysed for polymorphisms in the prion protein (PrP) gene, to determine their genotype and the allele frequencies of polymorphisms in PrP known to confer resistance to scrapie. A 939 base pair fragment of exon 3 from the PrP gene was amplified by pcr and analysed by direct sequencing. For animals showing polymorphism at two nucleotide positions, both haplotypes of these double-heterozygous genotypes were further verified by pcr cloning and sequence analysis. Known polymorphisms were observed at codons 136, 154 and 171, and six different haplotypes (arr, ahq, arh, ahr, arq and vrq) were determined. On the basis of these polymorphisms, the six populations of sheep possessed the resistant arr haplotype at different frequencies. The high-risk arq haplotype occurred in high frequencies in all six populations, but vrq, the haplotype carrying the highest risk, occurred at low frequencies and in only three of the populations.     

Detection of prions in the faeces of sheep naturally infected with classical scrapie

ABSTRACT: Classical scrapie is a naturally transmitted prion disease of sheep and goats. Contaminated environments may contribute to the spread of disease and evidence from animal models has implicated urine, blood, saliva, placenta and faeces as possible sources of the infection. Here we sought to determine whether sheep naturally infected with classical scrapie shed prions in their faeces. We used serial protein misfolding cyclic amplification (sPMCA) along with two extraction methods to examine faeces from sheep during both the clinical and preclinical phases of the disease and showed amplification of PrPSc in 7 of 15 and 14 of 14 sheep respectively. However PrPSc was not amplified from the faeces of 25 sheep not exposed to scrapie. These data represent the first demonstration of prion shedding in faeces from a naturally infected host and thus a likely source of prion contamination in the environment.