Distribution of prion protein genotypes in breeds of sheep in New Zealand

AIM: To use an established high through-put genotyping procedure to gain an estimate of the frequency of alleles of the prion protein (PrP) gene in some common sheep breeds in New Zealand.

METHODS: Using a genotyping procedure based on matrix-assisted laser desorption ionisation-time of flight (MALDI-TOF), DNA samples from 3,024 sheep from New Zealand, including breeds such as Romney, Texel, Coopworth, Merino and mixedbreed, were isolated, genotyped and the results analysed.

RESULTS: The 15 scrapie genotypes commonly reported, and derived from the five commonly reported allelic variants (ARR, ARQ, AHQ, ARH and VRQ), were all observed in the samples analysed. The estimates were indicative of the frequencies in the population of alleles present in breeds of sheep in New Zealand. There was a significant difference between the frequencies of alleles between breeds, but the ARQ, followed by the ARR allele, were, except in Carwell sheep, the most common alleles present.

CONCLUSION: This study gave an indication of the percentages of PrP gene alleles in sheep in New Zealand, including data previously unreported from breeds in this country. It is of interest because of the relatively large size of the sheep population in New Zealand compared with many countries, and it provides some useful information on the genetic susceptibility or resistance of the sheep population in New Zealand to scrapie. The frequencies of the alleles can be different for an individual breed compared between countries.     

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.     

Characterization of the prion protein gene (PRNP) region in Swiss sheep breeds

Polymorphisms in the PRNP gene have both an influence on the progress of scrapie and also of bovine spongiform encephalopathy (BSE) in sheep. In the light that sheep are potentially susceptible to BSE the goal of this project was to characterize 50 animals of each of the four major Swiss sheep breeds, namely Swiss Oxford Down, Swiss Black‐Brown Mountain, Valais Blacknose and Swiss White Alpine with respect to polymorphisms in the PRNP region. A 628‐bp fragment of exon 3 of PRNP comprising codons 112–211 was amplified by PCR and analysed by direct sequencing. Heterozygous genotypes were further verified by restriction fragment length polymorphism analyses. Polymorphisms could be observed at codons 112, 136, 137, 141, 143, 154 and 171 but not at codons 138, 151 and 211. One of the scrapie cases in Switzerland, a sheep of Swiss White Alpine, was also genotyped. Based on the polymorphisms observed at codons 136, 154 and 171 all four sheep breeds should be considered potentially susceptible to scrapie and BSE.     

Influence of prion protein gene polymorphisms on performance traits in German meat sheep breeds

PrP polymorphisms influence the scrapie susceptiblility of sheep. The objective of this study was to analyse the association between performance traits and the PrP genotype in the sheep breeds German black-headed and German white-headed mutton, Bleu du Maine, German mutton merino, Leine, Texel and Suffolk from Lower Saxony and Westphalia. We analysed performance traits such as scores for muscle mass, type and wool quality and the calculated daily weight gain using linear animal models. In all seven breeds no statistically significant associations were found between performance traits and the occurrence of ARR alleles, and the ARR/ARR genotypes, respectively. All genotyped sheep of all breeds investigated showed significantly superior performance traits in comparison to the non-genotyped animals.     

The incidence of genotypes at codon 171 of the prion protein gene (PRNP) in five breeds of sheep and production traits of ewes associated with those genotypes

Scrapie is one of several transmissible spongiform encephalopathies of livestock. Disease susceptibility is linked to polymorphisms in the normal prion protein gene that encodes the mammalian prion precursor. Codon 171 of this gene is a major determinant of scrapie susceptibility. Selection for arginine (R) at codon 171 is encouraged by the USDA to decrease the incidence of scrapie. Objectives of this study were to determine the frequency of R allele variants at codon 171 in a sample of sheep from five breeds (Columbia, Hampshire, Rambouillet, Suffolk, and Targhee) and western white-faced commercial ewes and to determine whether the R allele is associated with ewe and lamb production traits. Genotyping was performed on 532 ewes and 901 lambs from the University of Wyoming flock, in addition to 820 rams from 52 sheep producers from Wyoming and surrounding areas, using a DNA mismatch assay that discriminated the R allele from others at codon 171. Genotyping was performed by DNA sequencing on 127 rams representing all breeds, except Hampshire from the USDA Sheep Experiment Station at Dubois, ID. The 171R allele was found in all five breeds and in the commercial western white-faced ewes. Genotype frequencies varied (P < 0.001) by breed in ewe and ram populations. Influence of R-allele frequency on ewe lambing records and individual lamb records was analyzed for Columbia (62, 161, 121), Hampshire (89, 193, 162), Rambouillet (87, 179, 133), Suffolk (67, 178, 161), and commercial sheep (227, 463, 324) for numbers of ewes, total number of ewe production records, and individual lamb records, respectively. Suffolk ewes without the R allele (non-R/non-R) gave birth to more (P or= 0.08) by ewe genotype. Lamb birth and weaning weights were not influenced (P >or= 0.12) by lamb genotype in any of the breeds or in the commercial flock. In this population, ultimate lamb production was only influenced by genotype at codon 171 in the Suffolk flock.     

PrP genotypes of rare breeds of sheep in Great Britain

In total 31,669 blood samples were collected from 1187 flocks of 27 rare breeds of sheep in the UK, and their genotype profiles at the prion protein locus were determined. The frequencies of the five alleles varied widely among the breeds and some had only two of the alleles and others had all five; the average was three. The average allele frequencies across all 27 breeds were 49.7 per cent for ARR, 4.4 per cent for AHQ, 2.7 per cent for ARH, 37.4 per cent for ARQ and 5.8 per cent for VRQ. The highest frequencies for each allele were 90.7 per cent for ARR in the Leicester Longwool, 24.7 per cent for AHQ in the Hebridean, 68.7 per cent for ARH in the Manx Loghtan, 98.7 per cent for ARQ in the North Ronaldsay and 28.4 per cent for VRQ in the Boreray. All 27 breeds had the ARR allele, 21 had AHQ, 11 had ARH, 26 had ARQ and 20 had VRQ.     

Variation in the prion protein sequence in Dutch goat breeds

Scrapie is a neurodegenerative disease occurring in goats and sheep. Several haplotypes of the prion protein increase resistance to scrapie infection and may be used in selective breeding to help eradicate scrapie. In this study, frequencies of the allelic variants of the PrP gene are determined for six goat breeds in the Netherlands. Overall frequencies in Dutch goats were determined from 768 brain tissue samples in 2005, 766 in 2008 and 300 in 2012, derived from random sampling for the national scrapie surveillance without knowledge of the breed. Breed specific frequencies were determined in the winter 2013/2014 by sampling 300 breeding animals from the main breeders of the different breeds. Detailed analysis of the scrapie‐resistant K₂₂₂ haplotype was carried out in 2014 for 220 Dutch Toggenburger goats and in 2015 for 942 goats from the Saanen derived White Goat breed. Nine haplotypes were identified in the Dutch breeds. Frequencies for non‐wild type haplotypes were generally low. Exception was the K₂₂₂ haplotype in the Dutch Toggenburger (29%) and the S₁₄₆ haplotype in the Nubian and Boer breeds (respectively 7 and 31%). The frequency of the K₂₂₂ haplotype in the Toggenburger was higher than for any other breed reported in literature, while for the White Goat breed it was with 3.1% similar to frequencies of other Saanen or Saanen derived breeds. Further evidence was found for the existence of two M₁₄₂ haplotypes, M₁₄₂/S₂₄₀ and M₁₄₂/P₂₄₀. Breeds vary in haplotype frequencies but frequencies of resistant genotypes are generally low and consequently selective breeding for scrapie resistance can only be slow but will benefit from animals identified in this study. The unexpectedly high frequency of the K222 haplotype in the Dutch Toggenburger underlines the need for conservation of rare breeds in order to conserve genetic diversity rare or absent in other breeds.     

Enzootic pneumonia of sheep in New Zealand

Extract

Enzootic pneumonia of sheep is a specific condition confined to southern Otago and the Southland district and is commonly referred to as “Southland Pneumonia.” It has been recognized as a distinct clinical entity for many years in that area, heavy losses being reported in the Gore district of Southland in 1924.     

Frequencies of PrP genotypes in meat breeds of Japanese sheep and trail of selective breeding in experimental sheep flock

The selection of sheep with scrapie-resistant PrP genotypes is one of the control measures for transmissible spongiform encephalopathies in ruminants. In this study, we investigated the frequencies of PrP genotypes in meat breeds in Japan. The nationwide surveillance revealed that nearly half of the Suffolk sheep, a major meat breed in Japan, carried scrapie-susceptible AQ/AQ and AQ/VQ genotypes. In addition, the VQ haplotype, which confers high susceptibility to scrapie within sheep, was also found in Poll Dorset sheep. A trial of selective breeding using sires with scrapie-resistant PrP genotypes AQ/AR and AR/AR could raise the ratio of scrapie-resistant sheep from less than 50% to 80% within 3 years. However, the use of sires with the AR/AR genotype and the selection of ewes would be required to achieve a higher ratio of scrapie-resistant sheep.     

Scrapie in imported sheep in New Zealand

Extract

The disease known as Scrapie was diagnosed in New Zealand for the first time in June, 1952. Two positive cases and, one suspected case occurred in Canterbury in Suffolk sheep which were imported from England early in 1950. The disease appeared after a typiqally long incubation period and ran a characteristic course in the affected sheep. The outbreak of Scrapie in New Zealand and the drastic measures necessary to control it have placed new emphasis on the difficulty of preventing the introduction of this and other diseases which develop insidiously after a long incubation period, and for which no biological test is available. The same factors have also been responsible for the peculiar difficulties encountered by overseas workers in investigating Scrapie and in establishing clearly the facts of its etiology and transmission.     

绵羊不同器官中PrP分布的免疫组织化学研究

传染性海绵状脑病(TSE)是由朊病毒(Prion)引起的人和多种哺乳动物以神经退行性变化为主要特征的一种慢性消耗性传染病。引起这类疾病的病原因子是一种编码宿主蛋白的PrPC转变为异常的具有致病性的PrPSc,二者具有相同的氨基酸序列,只是其空间结构发生变化后由正常的以α螺旋为主     

Multifocal retinitis in New Zealand sheep dogs

Thirty-nine percent of 1,448 working sheep dogs were affected with varying degrees of multifocal retinal disease on ophthalmoscopic examination. Lesions consisted of localized areas of hyperreflexia in the tapetal fundus, often associated with hyperpigmentation. Severely affected animals had widespread hyperreflexia with retinal vascular attenuation. Only 6% of 125 New Zealand dogs raised in urban environment were similarly affected. Both eyes of 70 dogs from New Zealand were examined histologically. Forty-seven of 70 dogs had ocular inflammatory disease. Ten other dogs had noninflammatory eye disease, and 13 dogs had normal eyes. Histologically, eyes with inflammatory disease were divided into three categories: Dogs 3 years of age or less with active inflammatory disease of the retina, uvea, and vitreous. Four dogs in this group had migrating nematode larvae identified morphologically as genus Toxocara. Diffuse retinitis and retinal atrophy in conjunction with localized retinal necrosis and choroidal fibrosis. Dogs in this category were severely, clinically affected. Chronic, low-grade retinitis with variable retinal atrophy. Most dogs in this category were over 3 years of age, and many were visually functional. The existence of a definable spectrum of morphological changes associated with inflammation, suggests that Toxocara sp. ocular larva migrans may be the cause of a highly prevalent, potentially blinding syndrome of working sheep dogs in New Zealand.     

朊蛋白在奶牛生殖系统表达的定位

用免疫组织化学方法对奶牛生殖系统中朊蛋白进行定位研究,结果表明:在雌奶牛卵巢、子宫阜、子宫呈阳性,输卵管呈弱阳性。雄奶牛睾丸、附睾呈现弱阳性,其他器官为阴性。这说明在这些组织中有正常的PrP存在,为探索Prion致病机理、感染途径提供基础数据,也为朊病毒是否可以垂直传播提供了基础数据。     

藏绵羊朊蛋白基因的原核表达

从藏绵羊全血中提取基因组总DNA，用所设计的引物以聚合酶链式反应扩增出细胞型朊蛋白（PrP^c）基因。测序分析表明，所克隆的羊PrP^c基因片段大小为771bp，包含了羊朊蛋白基因的完整编码区序列，其与国内外报道的序列基本相同。将目的基因与经EcoRⅠ和XhoⅠ酶切的载体pGEX-4T-1连接后转化宿主菌BL21（DE3），挑选重组阳性菌用IPTG诱导表达，收集不同培养时间的菌液进行SDS-PAGE和Western-blotting。结果表明，PrP基因在大肠杆菌中成功表达，并能被抗牛PrP^c单抗4C11识别。凝胶薄层扫描结果显示，表达蛋白约占菌体总蛋白的309／6～45％，目的蛋白以包涵体的形式存在，包涵体经变性裂解、纯化和复性后得到具有一定生物学活性的目的蛋白。     

Segmental axonopathy of Merino sheep in New Zealand

AIM: To investigate an axonopathy of Merino sheep that caused progressive hindlimb ataxia and slight to moderate paresis, with the purpose of understanding its pathogenesis.

METHODS: Tissues were fixed in buffered paraformaldehyde or paraformaldehyde and glutaraldehyde, processed into wax and epoxy resin, respectively, and examined by light and electron microscopy. Fresh frozen spinal cord and trigeminal nerve roots were subjected to homogenisation, centrifugation and two-dimensional electrophoresis. Selected protein spots were identified using matrix-assisted laser desorption ionisation (MALDI) mass spectrometry.

RESULTS. By light microscopy, there were large pale foamy spheroidal axonal swellings affecting peripheral as well as central axons. By electron microscopy, these were shown to contain many membrane-bound vesicles. The main abnormalities in expressed proteins involved cytoskeletal elements and myosin heavy chain, the latter interpreted as associated with the molecular motor myosin Va.

CONCLUSIONS: The disorder is the same as that described in Merinos in Australia as segmental axonopathy, and believed to have an inherited aetiology. The lesions and protein changes indicate abnormalities of the cytoskeleton, its relationship with the myelin sheath, and myosin Va molecular motor. The consequence appears to be abnormal axonal transport and inability to maintain the integrity of axons and their myelin sheaths.     

The protein retention capacity of piglets of different breeds and genotypes

The genetically caused intensity of growth from piglets (barrows) of four breeds and two crossbreeds was evaluated by estimation of maximum daily capacity per LW 0.67 kg of N-balance plus N-maintenance-requirement (A) on the basis of a total of 130 N balance measurings on 66 piglets in the live weight range between 10 and 20 kg. The estimation was realized by using the N utilization model by Gebhardt (1963) and of its further development. In comparison with Landrace (A = 2575 mg) the breeds Edelschwein (A = 3,242 mg) and Leicoma (A = 3,038 mg) had a higher A in the midst of the piglet period (9th and 10th week of life), but not so the breed Schwerfurter (A = 2,350 mg). The capacity of the following N balances during growing and fattening stages is not derivable from these values. The experimentally evaluated A of crossbreeds is in good agreement with the calculated A on the basis of the used races for breeding. The product A.b = 1.15 is characterizing the quality of the feed protein (a mixture of wheat and casein in the relation 2.2:1), independently of the live weight and also of the breed or the genotype of the pig.     

Segmental axonopathy of Merino sheep in New Zealand

AIM: To investigate an axonopathy of Merino sheep that caused progressive hindlimb ataxia and slight to moderate paresis, with the purpose of understanding its pathogenesis. METHODS: Tissues were fixed in buffered paraformaldehyde or paraformaldehyde and glutaraldehyde, processed into wax and epoxy resin, respectively, and examined by light and electron microscopy. Fresh frozen spinal cord and trigeminal nerve roots were subjected to homogenisation, centrifugation and two-dimensional electrophoresis. Selected protein spots were identified using matrix-assisted laser desorption ionisation (MALDI) mass spectrometry. RESULTS. By light microscopy, there were large pale foamy spheroidal axonal swellings affecting peripheral as well as central axons. By electron microscopy, these were shown to contain many membrane-bound vesicles. The main abnormalities in expressed proteins involved cytoskeletal elements and myosin heavy chain, the latter interpreted as associated with the molecular motor myosin Va. CONCLUSIONS: The disorder is the same as that described in Merinos in Australia as segmental axonopathy, and believed to have an inherited aetiology. The lesions and protein changes indicate abnormalities of the cytoskeleton, its relationship with the myelin sheath, and myosin Va molecular motor. The consequence appears to be abnormal axonal transport and inability to maintain the integrity of axons and their myelin sheaths.