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RNA viruses rapidly mutate, which can result in increased virulence, increased escape from vaccine protection, and false-negative detection results. Targeted detection methods have a limited ability to detect unknown viruses and often provide insufficient data to detect coinfections or identify antigenic variants. Random, deep sequencing is a method that can more fully detect and characterize RNA viruses and is often coupled with molecular techniques or culture methods for viral enrichment. We tested viral culture coupled with third-generation sequencing for the ability to detect and characterize RNA viruses. Cultures of bovine viral diarrhea virus, canine distemper virus (CDV), epizootic hemorrhagic disease virus, infectious bronchitis virus, 2 influenza A viruses, and porcine respiratory and reproductive syndrome virus were sequenced on the MinION platform using a random, reverse primer in a strand-switching reaction, coupled with PCR-based barcoding. Reads were taxonomically classified and used for reference-based sequence building using a stock personal computer. This method accurately detected and identified complete coding sequence genomes with a minimum of 20× coverage depth for all 7 viruses, including a sample containing 2 viruses. Each lineage-typing region had at least 26× coverage depth for all viruses. Furthermore, analyzing the CDV sample through a pipeline devoid of CDV reference sequences modeled the ability of this protocol to detect unknown viruses. Our results show the ability of this technique to detect and characterize dsRNA, negative- and positive-sense ssRNA, and nonsegmented and segmented RNA viruses.  相似文献   
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人工驯养野生动物是获取野生动物产品的重要手段,但是非法盗猎的野生动物产品以人工饲养的名义进入市场,就会对野生动物的保护形成挑战。因此,准确鉴别市场上的动物产品是来自养殖场还是野外,是保护野外种群,维持养殖业正常秩序的关键。本研究以北美水貂(Neovison vison)为例,基于颧骨内侧空间(ZAS)、颧宽(ZAA和ZBA)、颅基长(BSL)、下颌长(MDL)、眶后收缩(POC)和最大面部宽度(GFB)等量度,建立了9个头骨形态计量学指标:POC/GFB、POC/BSL、POC/MDL、ZAS SQRT/BSL、ZA SSQRT/MDL、ZBA×BSL/2ZAS、ZBA×MDL/2ZAS、ZBB×BSL/2ZAS和ZBB×MDL/2ZAS。野生组(n=32,9雄23雌)和饲养组(n=45,35雄10雌)的比较表明,除POC/GFB外,所有指标对于在两组样品之间均有显著差异,整体判别正确率为73.2%—85.5%。在性别已知的情况下,雄性的整体正确率为82.4%—93.8%,雌性为41.7%—85.7%。为此,应针对不同性别、不同来源的动物建立相应的参考数据,利用似然比进行鉴别。本研究的结果不仅可用于北美水貂的鉴别,也为其他小型食肉动物的鉴别提供经验。  相似文献   
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OBJECTIVE: To compare sensitivity of a complement fixation (CF) test and competitive ELISA (cELISA) for detection of Anaplasma marginale in experimentally infected steers. ANIMALS: 40 crossbred (Angus-Simmental) steers. PROCEDURES: Steers were inoculated with 2.6 x 10(9) A marginale-infected erythrocytes (day 0). Blood samples were collected on days 9, 13, 20, 28, 34, 41, 61, 96, 126, and 156 days after inoculation. The percentage of parasitized erythrocytes (PPE) was determined by microscopic examination of stained blood films, and sera were evaluated with the CF test and cELISA by use of USDA-approved methods. Sensitivity and agreement (kappa statistic) between the 2 methods were determined. Persistent infections were confirmed by inoculation of blood obtained from infected steers into susceptible, splenectomized calves. RESULTS: 9 days after inoculation, sensitivity of the cELISA was 47.5%, whereas the CF test failed to identify seropositive steers. After day 13, sensitivity of the cELISA and CF test was 100% and 20%, respectively. During peak parasitemia (day 20), sensitivity of the cELISA and CF test was 100%. Thereafter, sensitivity of the CF test fluctuated between 7.5% and 37.5%, whereas sensitivity of the cELISA remained at 100%. Overall sensitivity of the cELISA and CF test was 94.8% and 26.5%, respectively (kappa statistic, 0.039). CONCLUSIONS AND CLINICAL RELEVANCE: The cELISA had superior sensitivity for serologic detection of A marginale.The CF test and cELISA each had a high percentage of false-negative results during the prepatent period. These findings are relevant for export certification and anaplasmosis prevention or eradication programs.  相似文献   
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OBJECTIVE: To assess expression of cyclooxygenase (COX)-1 and -2 in naturally occurring squamous cell carcinomas (SCCs) and the analogous normal tissues in horses. SAMPLE POPULATION: Tissue samples collected from 3 conjunctival, 2 vulvar, 4 preputial, and 5 penile SCCs during surgical excision in 14 horses and from corresponding body regions (conjunctiva [n = 5 horses], vulva [2], prepuce [3], and penis [3]) in 5 horses euthanized for reasons unrelated to neoplasia. PROCEDURES: Tissue samples were snap frozen in liquid nitrogen and stored at -80 degrees C until analysis. Protein was extracted from the frozen tissues, and western blot analyses were performed. Nonneoplastic and abnormal tissues from each body region were run on the same blot, and blots were run in triplicate. Molecular-weight markers and COX-1 and 2 ovine standards (positive control samples) were run concurrently on the gels; negative control samples were not used. RESULTS: All tissues, including the nonneoplastic and SCC tissues, expressed both COX-1 and -2 proteins. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that the expression of COX proteins in both nonneoplastic and SCC-affected tissues in horses is markedly different from that in other species. The reason for the potential benefit of COX-2 inhibitors in horses and other species is unknown. Further research needs to be performed to evaluate the efficacy of COX-2 inhibitors as cancer treatments in horses. Investigation of the mechanisms of tumor development in horses should be performed to increase understanding of this disease and ascertain how the mechanisms differ from those in other animals.  相似文献   
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