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1.
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.  相似文献   

2.
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.  相似文献   

3.
The objective of this study was to compare the different methods of detecting Toxoplasma gondii in sheep tissue, tested serologically positive by the indirect immunofluorescent antibody test (IFAT). Brain, diaphragm, and blood samples were collected from 522 sheep slaughtered at the S?o Manuel abattoir, S?o Paulo State, Brazil. Brain and diaphragm samples from IFAT seropositive animals were digested by both trypsin and pepsin and then injected into mice. Part of the digested samples was used to prepare slides for Giemsa staining and in the polymerase chain reaction (PCR). Tissue fragments were fixed in formalin and examined using hematoxilin-eosin (HE). Forty of the sheep (7.7%) were IFAT positive. T. gondii was isolated in 23 (59.0%) of the 39 mice with pepsin-digested brain samples and in 27 (69.0%) of the 39 with trypsin-digested brain samples. Injection of diaphragm samples led to T. gondii isolation in 26 (66.7%) of the 39 pepsin-digested samples and 21 (53.8%) of the 39 trypsin-digested samples. Cytological and hystopathological examination of both brains and diaphragms was negative in all examined sheep. PCR was positive in 7 (17.9%) of the trypsin and 2 (5.1%) of the pepsin-digested samples, while 9 (23.1%) of the trypsin and 3 (7.7%) of the pepsin-digested samples showed T. gondii DNA. T. gondii isolation rate in mice (n = 34; 85.0%) was significantly higher than detection by PCR (n = 15; 37.5%).  相似文献   

4.
This study presents preliminary data on the polymorphism in the prion protein gene of Swiniarka sheep using temperature gradient gel electrophoresis (TGGE). Available data indicate that sensitivity to scrapie is associated with polymorphisms in three codons of prion protein gene: 136,154, and 171. The TGGE method was used to detect point mutations in these codons responsible for sensitivity or resistance to scrapie. This study revealed presence of an allele encoding valine (V) in codon 136, which is associated with high sensitivity to scrapie and occurred in the form of heterozygous allele together with alanine (AV). The highest variability was observed in codon 171, with presence of arginine (R) and glutamine (Q) in the homozygous (RR or QQ) as well as the heterozygous form (RQ). The results of examination of fifty sheep DNA samples with mutations in codons 136, 154, and 171 demonstrated that TGGE can be used as a simple and rapid method to detect mutations in the PrP gene of sheep. Several samples can be run at the same time, making TGGE ideal for the screening of large numbers of samples.  相似文献   

5.
为了解新疆南疆部分地方品种羊的无浆体感染情况和分子特征,用PCR法检测新疆南疆5种地方品种绵羊共100份血液DNA样本,发现无浆体总感染率为67.0％(67/100).以多浪羊感染率最高,为100％(20/20),和田羊感染率最低,为44.0％(11/25);散养和圈养羊的无浆体感染率分别为74.0％(37/50)和6...  相似文献   

6.
The misfolded form of cellular prion protein (PrP(C)) is the main component of the infectious agent of transmissible spongiform encephalopathies and the validated biomarker for these diseases. The expression of PrP(C) is highest in the central nervous system and has been found in peripheral tissues. Soluble PrP(C) has been detected in cerebrospinal fluid, urine, serum, milk, and seminal plasma. In this study, attempts were made to characterize prion protein in urine samples from normal and scrapie-infected sheep. Urine samples from scrapie-infected sheep and age-matched healthy sheep were collected and analyzed by Western blot following concentration. A protease K-sensitive protein band with a molecular weight of approximately 27-30 kDa was visualized after immunoblotting with anti-PrP monoclonal antibodies to a C-terminal part of PrP(C), but not after immunoblotting with monoclonal antibodies to an N-terminal epitope of PrP(C) or with secondary antibodies only. The amount of PrP(C) in the urine of 49 animals (control group: n = 16; naturally scrapie-infected group: n = 33) was estimated by comparison with known amounts of ovine recombinant PrP in the immunoblot. Background concentration of PrP(C) in urine was found to be 0-0.16 ng/ml (adjusted to the initial nonconcentrated volume of the urine samples). Seven out of 33 naturally scrapie-infected animals had an elevated level (0.3-4.7 ng/ml) of PrP(C) in urine. The origin of PrP(C) in urine and the reason for the increased level of PrP(C) in scrapie-infected sheep urine has yet to be explored.  相似文献   

7.
Ovine pulmonary adenocarcinoma (OPA) is a contagious lung tumour of sheep caused by Jaagsiekte sheep retrovirus (JSRV). The disease is a particular problem in flocks in many parts of the world. The aim of the study was to assess screening methods for individual animals as a prelude to future eradication trials. Results of histological examination were used as the standard to evaluate the relative sensitivity and specificity of an established heminested polymerase chain reaction (PCR) test for JSRV proviral DNA from blood and bronchoalveolar lavage (BAL) samples. PCR results from tissue samples are included as control data. PCR testing of blood samples was found to have an estimated sensitivity of only 10% (95% confidence interval (CI) 3-20) while the sensitivity of the PCR test on BAL samples was 89% (CI 79-96) in comparison to the results of histological examination. We conclude that PCR testing of BAL samples is an effective confirmatory test for sheep with suspected clinical OPA. It is also a useful tool for the pre-clinical identification of individual infected sheep within an infected flock and therefore may prove beneficial in future control or eradication programmes.  相似文献   

8.
In recent publications, it was shown that disease-associated prion protein (PrP(d)) accumulates in the lymphoid tissue of the rectal mucosa of a high proportion of scrapie-infected sheep at clinical and preclinical stages, regardless of several host factors; PrP(d) can also be detected in biopsy specimens of rectal mucosa, with an increased probability proportional to age or incubation period and with an efficiency almost identical to that of tonsil biopsies. Rectal biopsies have the advantages of providing higher numbers of lymphoid follicles and of being simpler to perform, which makes them suitable for scrapie screening in the field. In biopsy samples, PrP(d) could be demonstrated by immunohistochemical (IHC) and Western immunoblotting methods, and the purpose of the present study was to optimize and evaluate a "rapid test" for the diagnosis of scrapie in rectal biopsy samples. The HerdChek CWD (chronic wasting disease) antigen EIA (enzyme immunoassay) test was chosen and, once optimized, provided specificity and sensitivity figures of 99.2% and 93.5%, respectively, compared with IHC results in the same samples obtained at a postmortem. The sensitivity of the assay increased from 82.1%, when a single rectal mucosa sample was tested to 99.4% for those sheep in which 3 or more samples were analyzed. Similarly, sensitivity values of the HerdChek CWD antigen EIA test on biopsy samples increased from 95% to 100% for sheep subjected to 1 or 2 sequential biopsies 4 months apart, respectively. Thus, a preclinical diagnosis of scrapie in live sheep can be achieved by a combination of a simple sampling procedure, which can be repeated several times with no detrimental effect for the animals, and a rapid and efficient laboratory method.  相似文献   

9.
There have been no reports of natural scrapie in Irish Blackface Mountain (BM) sheep which account for approximately 16% of the Irish national sheep flock. The aim of this study was to determine if Irish BM sheep had unusual clinical and/or pathological features of scrapie which would account for failure to diagnose the disease in this breed. BM (n=7), Texel (n=3) and Suffolk sheep (n=1) of scrapie-susceptible PrP genotypes (ARQ/ARQ and VRQ/ARQ) were orally challenged with scrapie-infected brain inoculum. The incubation period, clinical signs, pathology and distribution of disease specific prion protein (PrP(d)) in scrapie-affected BM sheep were similar to scrapie in the Texel and Suffolk sheep. It was concluded that there was no evidence to suggest that scrapie in BM sheep differs clinicopathologically from scrapie in other breeds of sheep.  相似文献   

10.
AIMS: To estimate the number of cases of scrapie that would occur in sheep of different prion protein (PrP) genotypes if scrapie was to become established in New Zealand, and to compare the performance of two commercially available, rapid ELISA kits using ovine retro-pharyngeal lymph nodes (RLN) from non-infected and infected sheep of different PrP genotypes.

METHODS: Using published data on the distribution of PrP genotypes within the New Zealand sheep flock and the prevalence of cases of scrapie in these genotypes in the United Kingdom, the annual expected number of cases of scrapie per genotype was estimated, should scrapie become established in New Zealand, assuming a total population of 28 million sheep. A non-infected panel of RLN was collected from 737 sheep from New Zealand that had been culled, found in extremis or died. Brain stem samples were also collected from 131 of these sheep. A second panel of infected samples comprised 218 and 117 RLN from confirmed scrapie cases that had originated in Europe and the United States of America, respectively. All samples were screened using two commercial, rapid, transmissible spongiform encephalopathy ELISA kits: Bio-Rad TeSeE ELISA (ELISA-BR), and IDEXX HerdChek BSE-Scrapie AG Test (ELISA-ID).

RESULTS: If scrapie became established in New Zealand, an estimated 596 cases would occur per year; of these 234 (39%) and 271 (46%) would be in sheep carrying ARQ/ARQ and ARQ/VRQ PrP genotypes, respectively. For the non-infected samples from New Zealand the diagnostic specificity of both ELISA kits was 100%. When considering all infected samples, the diagnostic sensitivity was 70.4 (95% CI=65.3–75.3)% for ELISA-BR and 91.6 (95% CI=88.2–94.4)% for ELISA-ID. For the ARQ/ARQ genotype (n=195), sensitivity was 66.2% for ELISA-BR and 90.8% for ELISA-ID, and for the ARQ/VRQ genotype (n=107), sensitivity was 81.3% for ELISA-BR and 98.1% for ELISA-ID.

CONCLUSIONS: In this study, the ELISA-ID kit demonstrated a higher diagnostic sensitivity for detecting scrapie in samples of RLN from sheep carrying scrapie-susceptible PrP genotypes than the ELISA-BR kit at comparable diagnostic specificity.

CLINICAL RELEVANCE: The diagnostic performance of the ELISA-ID kit using ovine RLN merits the consideration of including this assay in the national scrapie surveillance programme in New Zealand.  相似文献   

11.
The aim of the study reported here was to estimate the sensitivity and specificity of the gamma interferon (IFN-gamma) ELISA for paratuberculosis in sheep using receiver-operating characteristic analysis. Bacteriologic culture of tissues was used to define the reference positive population (n = 33). Two reference negative populations were used: culture-negative sheep from infected flocks (n = 77), and sheep from noninfected flocks (n = 358). We also evaluated the accuracy of 2 Mycobacterium avium subspecies paratuberculosis (MAP) antigen preparations, a whole-cell sonicate (MpS) and a johnin purified-protein derivative (PPDj). The source of the reference negative sheep used in the analysis affected overall accuracy of the IFN-gamma ELISA. The area under the curve was 0.683 (95% confidence interval 0.574-0.787), using culture-negative sheep from infected flocks, was 0.831 (0.764-0.889), using sheep from noninfected flocks for the MpS, and was 0.809 (0.726-0.881) and 0.897 (0.862-0.925) for the PPDj, respectively. Using the MpS, the cut point that classified the most sheep correctly was an optical density reading of 0.20, for sensitivity of 40.7% (19.4-57.6) and specificity of 88.7% (77.0-95.7) or 97.6% (93.04-99.5), depending on the reference negative population used. Using the PPDj, the cut point that classified the most sheep correctly was 0.25 for sensitivity of 66.7% (47.2-82.7) and specificity of 93.5% (85.5-97.9) or 98.3% (96.4-99.4), respectively. The PPDj was more accurate at identifying MAP-infected sheep than was the MpS (P = 0.034).  相似文献   

12.
This study aimed to establish the seroprevalence and risk factors associated with neosporosis in sheep and dogs from rural properties. 1497 blood samples were collected from sheep and 42 from dogs that cohabited with sheep from 16 farms located in the central region of S?o Paulo State, Brazil. For the detection of N. caninum antibodies it was performed the indirect fluorescent antibody test (IFAT ≥ 25). For the epidemiological study it was applied a questionnaire for the owners or responsible from the sheep and dogs regarding informations related to neosporosis. The seroprevalence obtained out of the 1497 sheep sera tested was 8.0% (CI95%=6.7-9.2%) and out of the 42 dogs 4.8% (CI95%=0-7.2%). Variables statistically related to seropositivity for N. caninum in sheep were: dams well as water supply (P=0.0004; OR=2.15), presence of other domestic canids (P=0.0013; OR=2.38) and presence of reproductive problems (P=0.0031; OR=1.75).  相似文献   

13.
This report describes the genetics of the prion protein gene (PRNP) at codons 136, 154, and 171 for sheep diagnosed with naturally acquired classical scrapie in Canada between 1998 and 2008. Genotyping analysis was performed on 249 sheep with confirmed classical scrapie infection representing 98 flocks from 6 provinces. A further case-control analysis of 3 of these flocks compared 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 the ARQ haplotype (91.8%) and the ARQ/ARQ genotype (91.6%). In addition, the ARQ haplotype was found at significantly higher frequency in scrapie-infected sheep when compared with their healthy flockmates. Comparison with other published data suggests that the scrapie risk of PRNP genotypes differs between Canada and countries where the VRQ allele is associated with the highest susceptibility to infection.  相似文献   

14.
Scrapie and bovine spongiform encephalopathy (BSE) are major global concerns and the emergence of variant Creutzfeldt-Jakob disease (vCJD) has caused turmoil for blood transfusion services and hospitals worldwide. Recent reports of iatrogenic CJD (iCJD) cases following blood transfusions from Transmissible Spongiform Encephalopathies (TSE)-infected donors have fuelled this concern. Major diagnostic tests for BSE and scrapie are conducted post-mortem from animals in late stages of the disease. Although the lymphoreticular system is involved in the earlier pathogenesis of some forms of sheep scrapie and vCJD, which presents great opportunity for diagnostic development, other TSE diseases (some strains of scrapie, sporadic CJD (sCJD) and bovine BSE) do not present such a diagnostic opportunity. Thus, there is an urgent need for pre-mortem tests that differentiate between healthy and diseased individuals at early stages of illness, in accessible samples such as blood and urine using less invasive procedures. This review reports on the current state of progress in the development and use of prion and non-prion biomarkers in the diagnosis of TSE diseases. Some of these efforts have concentrated on improving the sensitivity of PrPSc detection to allow in vivo diagnosis at low abundances of PrPSc whilst others have sought to identify non-prion protein biomarkers of TSE disease, many of which are still at early stages of development. In this review we comment upon the limitations of prion based tests and review current research on the development of tests for TSE that rely on non-prion disease markers in body fluids that may allow preclinical disease diagnosis.  相似文献   

15.
In 2000 Guarnera et al. proposed using ELISA in canine faeces collected from the ground to detect dogs infected with Echinococcus granulosus, thus determining sheep farms with active transmission. The objective was to evaluate the prevalence of E. granulosus infection in sheep farms of the Patagonia. Sheep farms were randomly selected in the Provinces of Río Negro, Chubut, Neuquén, Santa Cruz and Tierra del Fuego (areas with control programs) and La Pampa (comparison area). From one to three samples of fecal matter were obtained for each sheep farm, which were processed by means of copro-ELISA test with confirmation of positive samples by copro-Western blot. A total of 1042 samples were obtained from 352 sheep farms, 26 (7.3%) proving positive. Of these 5 (6.3%) were from La Pampa, 9 (13.8%) from Neuquén, 4 (4.7%) from Río Negro, 2 (2.9%) from Chubut, 1 (5.9%) from Santa Cruz and 5 (13.9%) from Tierra del Fuego. The identification of parasitized dogs is an essential activity upon which rests the strategy of control and surveillance. Arecoline tests or coproantigen test with fecal matter obtained directly from the dog contribute information on individual prevalence, while the use of coproantigens detected in ground-collected samples transfers the dog unit of observation to units of greater epidemiological value. In the present experience, the technique employed seems promising for its application in systems of epidemiological surveillance of cystic echinococcosis and in drawing a baseline on which to measure the progress of control programs in the Argentine Patagonia in subsequent years.  相似文献   

16.
ABSTRACT: Interspecies transmission studies afford the opportunity to better understand the potential host range and origins of prion diseases. The purpose of this experiment was to determine susceptibility of white-tailed deer to the agent of scrapie after intracerebral inoculation and to compare clinical signs and lesions to those reported for chronic wasting disease (CWD). Deer (n = 5) were inoculated with 1 mL of a 10% (wt/vol) brain homogenate derived from a sheep clinically affected with scrapie. A non-inoculated deer was maintained as a negative control. Deer were observed daily for clinical signs of disease and euthanized and necropsied when unequivocal signs of scrapie were noted. One animal died 7 months post inoculation (pi) due to intercurrent disease. Examinations of brain tissue for the presence of the disease-associated abnormal prion protein (PrPSc) by western blot (WB) and immunohistochemistry (IHC) were negative whereas IHC of lymphoid tissues was positive. Deer necropsied at 15-22 months pi were positive for scrapie by IHC and WB. Deer necropsied after 20 months pi had clinical signs of depression and progressive weight loss. Tissues with PrPSc immunoreactivity included brain (at levels of cerebrum, hippocampus, colliculus, cerebellum, and brainstem), trigeminal ganglion, neurohypophysis, retina, spinal cord, and various lymphoid tissues including tonsil, retropharyngeal and mesenteric lymph nodes, Peyer's patches, and spleen. This work demonstrates for the first time that white-tailed deer are susceptible to sheep scrapie by intracerebral inoculation. To further test the susceptibility of white-tailed deer to scrapie these experiments will be repeated with a more natural route of inoculation.  相似文献   

17.
A cross-sectional serological survey was conducted in order to evaluate, irrespective of abortion, the Toxoplasma gondii infection in pastured sheep from the Campania region of southern Italy. A geographical information system was used in order to uniformly sample the ovine farms (n=117) throughout the entire region. Blood and milk samples were collected from 10 adult sheep (>18 months) on each farm (total number=1170 sheep). Serum samples were tested for the presence of IgG antibodies to T. gondii using a commercial indirect fluorescent antibody test. For each farm, the 10 milk samples collected were pooled in order to obtain a single milk sample per farm (total number=117 milk samples). The 77.8% (91/117) of the farms and the 28.5% (333/11,170) of the sheep resulted positive by serology. In addition, the presence of T. gondii DNA was detected by PCR in 4 milk samples out of the 117 examined (3.4%).  相似文献   

18.
The purpose of this study was to enhance the sensitivity of the Western blot (WB) test for use as an alternative and confirmatory method for the diagnosis of scrapie and chronic wasting disease (CWD) in Canada by comparing 2 sample preparation procedures: an abnormal prion protein (PrPSc) concentration procedure using sodium phosphotungstic acid (PTA) precipitation and a procedure using crude sample without precipitation. A total of 100 cerebrum samples (52 sheep and 48 elk), including 66 negative (31 sheep, 35 elk) and 34 positive (21 scrapie and 13 CWD positive) samples diagnosed by using immunohistochemistry (IHC) on retropharyngeal lymph node (RPLN) and medulla oblongata at obex, were tested by using WB with the 2 sample preparation procedures. The WB using non-PTA enriched sample (crude extract) detected, on average, only 71.7% (9 of 15, 60.0% for scrapie, 5 of 6, 83.3% for CWD) of the samples that tested positive by using WB with PTA enriched samples. No case was positive by WB using crude extract but negative by WB using PTA enriched sample. No false positive was found. Serial dilution of PTA precipitated samples demonstrated that the technique increases the detection limit approximately 100 fold. Additionally, the comparison of the WB and IHC on cerebrum from all the positive cases demonstrated that WB following PTA precipitation and IHC had 100% agreement by detecting 6 positive for CWD on cerebrum; while IHC detected scrapie in only 14 out of 15 positive cerebrum samples by using WB following PTA precipitation. Phosphotungstic acid precipitation is therefore a useful adjunct to WB analysis of scrapie and CWD and tissues.  相似文献   

19.
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20.
The first results of the Hungarian sheep prion protein (PrP) genotyping programme are discussed in this paper. To obtain initial genotype frequency data 10 commercial (Hungarian Merino, German Mutton Merino, Merino Landschaf, German Blackheaded, Suffolk, Texel, Ile de France, Charollais, Lacaune, British Milksheep) and 4 indigenous (Gyimes Racka, Hortobágy Racka, Tsigaja, Cikta) breeds were sampled in 2003 and 2004, and the PrP genotypes were determined by microsequencing analysis with capillary electrophoresis. In all commercial breeds, a higher number of sheep were genotyped in 2005 (3648) and in 2006 (3834) within the breeding programme to increase scrapie resistance, and the estimated frequency data were compared to the initial figures to evaluate the efficiency of selection. The new developments arising from the identification of the so-called 'atypical' scrapie cases are also discussed.  相似文献   

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