PCR方法特异性鉴定狮源性制品

目的建立可对狮DNA进行特异性检测的PCR方法,应用在检验检疫过程中获得的狮疑似样品和保护执法工作中难以检查辨认的狮产品进行物种鉴定。方法针对狮属动物线粒体细胞色素b(Cytb)基因设计特异性引物,用该引物分别对从亚洲狮、非洲狮和虎、豹、熊等14种非狮源性哺乳动物的肌肉、血液、毛发和肉骨粉中提取的DNA进行PCR(聚合酶链反应)扩增。结果试验表明所设计的引物对狮DNA具有特异性,从而达到了对该物种进行特异性检测鉴定的目的。检测灵敏度可达到在骨粉中检出0.5%含量的狮源性成分。结论该PCR方法具有方便、经济、灵敏度高和重复好等特性,可用于狮源性制品的检测鉴定。     

鸡减蛋综合征病毒Hexon基因SYBR GreenⅠ实时荧光定量PCR检测方法的建立

用PCR方法扩增出鸡减蛋综合征病毒（EDSV）Hexon基因保守片段,经琼脂糖凝胶电泳及序列测定分析扩增产物的特异性。以构建的阳性重组质粒作为标准品,建立SYBR GreenⅠ实时荧光定量PCR反应的扩增曲线和溶解曲线,并绘制标准曲线。结果表明,建立的EDSV荧光定量PCR标准曲线Ct值与1×10^1～1×10^6拷贝/μL的基因拷贝数呈现良好线性关系,灵敏度可达10拷贝,且特异性及重复性良好;说明本试验建立的SYBR GreenⅠ实时荧光定量PCR检测方法可用于EDSV的诊断及病原的定量分析。     

应用SYBR Green Ⅰ实时荧光PCR溶解曲线检测鹌鹁源性成分

根据GenBank中的鹌鹑线粒体DNA(mtDNA)12S RNA基因保守序列,用Primer5.0软件设计了1对针对鹌鹑的特异性扩增引物,建立了一种检测鹌鹑动物源性成分的SYBR Green Ⅰ实时荧光PCR方法.通过SYBRGreen Ⅰ实时荧光PCR反应,同时进行溶解曲线和Tm值分析.结果表明:只有鹌鹑样品在Tm值为82℃下有特异的溶解曲线峰,而参考物种鸽子、鸡、鸭、鹅、鸵鸟、鹧鸪、牛、羊、猪、马、驴、鱼和空白对照,则无特异的溶解曲线峰,说明该引物只对鹌鹑有特异性.测序结果表明,鹌鹑组织SYBR Green Ⅰ实时荧光PCR产物的核苷酸序列与GenBank中检索到的相应序列相吻合;该方法的检测灵敏度为0.001%.     

PCV4 SYBR Green I实时荧光定量PCR方法的建立及应用

为建立一种灵敏、可快速检测猪圆环病毒4型（PCV4）的SYBR Green I实时荧光定量PCR方法,根据PCV4 Rep基因的保守区域设计引物,建立针对PCV4的SYBR Green I实时荧光定量PCR检测方法,并对该方法的特异性、灵敏性和重复性进行评价。结果显示,该检测方法的Ct值与标准品在5.64×102~5.64×109 copies/μL范围内呈良好的线性关系,R2为0.999,斜率为-3.638,检测下限为5.64×102 copies/μL,且无非特异性扩增。利用该方法对56份临床样品进行检测,发现PCV4核酸阳性率为3.57%,比普通PCR更灵敏可靠。结果表明,本试验建立的SYBR Green I实时荧光定量PCR方法可用于PCV4的快速诊断。     

应用SYBR GreenⅠ实时荧光PCR溶解曲线检测鹌鹑源性成分

根据GenBank中的鹌鹑线粒体DNA(mtDNA)12S RNA基因保守序列,用Primer5.0软件设计了1对针对鹌鹑的特异性扩增引物,建立了一种检测鹌鹑动物源性成分的SYBR GreenⅠ实时荧光PCR方法。通过SYBRGreenⅠ实时荧光PCR反应,同时进行溶解曲线和Tm值分析。结果表明:只有鹌鹑样品在Tm值为82℃下有特异的溶解曲线峰,而参考物种鸽子、鸡、鸭、鹅、鸵鸟、鹧鸪、牛、羊、猪、马、驴、鱼和空白对照,则无特异的溶解曲线峰,说明该引物只对鹌鹑有特异性。测序结果表明,鹌鹑组织SYBR GreenⅠ实时荧光PCR产物的核苷酸序列与GenBank中检索到的相应序列相吻合;该方法的检测灵敏度为0.001%。     

猪星状病毒SYBR Green Ⅰ实时荧光定量PCR检测方法的建立与初步应用

本研究旨在建立一种能够快速、简便、灵敏地检测猪星状病毒的基于SYBR GreenⅠ实时荧光定量PCR方法。根据已报道的猪星状病毒ORF2基因序列,设计并合成1对引物,通过PCR扩增ORF2基因片段,将测序正确的ORF2基因片段克隆入pMD18-T载体,转化大肠杆菌DH5α,经测序鉴定后得到阳性重组质粒,作为标准品模板建立SYBR GreenⅠ荧光定量PCR标准曲线和熔解曲线,并对其灵敏性、特异性和重复性进行验证。结果表明,猪星状病毒荧光定量PCR的标准曲线Ct值与模板浓度呈良好的线性关系,熔解曲线特异,灵敏度可达1×101拷贝,是普通PCR检测方法的100倍,特异性和重复性较好。本次建立的猪星状病毒荧光定量PCR检测方法具有快速、简便、敏感等优点,有重要的实用价值。     

羊口疮病毒SYBR Green Ⅰ实时荧光定量PCR法的建立

为建立快速、灵敏、特异的检测羊口疮病毒(ORFV)的方法,基于羊口疮病毒保守序列B2L基因合成一对用于建立SYBR Green Ⅰ实时荧光定量PCR的特异性引物,将ORFV B2L全长基因和pMD19-T质粒载体连接,构建重组阳性质粒,以构建的重组质粒DNA作为模板,建立ORFV SYBR Green Ⅰ实时荧光定量PCR检测方法。结果显示:所建立的ORFV SYBR Green Ⅰ实时荧光定量PCR方法线性关系良好,重复性好,扩增效率高,无引物二聚体和非特异性扩增,最低检测限为2.13×101copies/μL,比普通PCR高出1 000倍,该方法对绵羊羊痘病毒和山羊痘病毒的核酸cDNA不检出,特异性好;对180份山羊全血样本进行ORFV检测,结果得出常规PCR阳性检出率为2.8%(5/180),荧光定量PCR阳性检出率为4.4%(8/180)。试验表明,本次建立的ORFV SYBR Green Ⅰ实时荧光定量PCR方法特异性强、敏感度高、重复性好,适用于羊口疮病毒临床样本的检测。     

环形泰勒虫SYBR Green I荧光定量PCR方法的建立及应用

为建立一种快速、敏感检测牛环形泰勒虫的方法,本研究根据GenBank中登录的牛环形泰勒虫Tams1基因序列,设计合成1对特异性引物,通过优化反应条件,建立了检测牛环形泰勒虫SYBR Green I荧光定量PCR检测方法。研究结果显示,该方法可以特异地检测牛环形泰勒虫,而对牛巴贝斯虫、反刍动物艾立希体和弓形虫检测均为阴性;该方法的灵敏度可达到180拷贝/μL,比常规PCR敏感10倍;组内和组间变异系数均小于1.0%。对15份临床血液样品和20只璃眼蜱进行检测,SYBR Green I荧光定量PCR和常规PCR的阳性检出率分别为42.86%和28.57%。本研究建立的SYBR Green I荧光定量PCR检测方法具有特异性强、敏感性高的特点,可准确、高效检测牛环形泰勒虫,为牛环形泰勒虫病防控提供技术支持。     

猪源干扰素刺激基因PPBP荧光定量PCR检测方法的建立及初步应用

根据猪源干扰素刺激基因PPBP(pro-platelet basic protein)基因全长序列,使用引物设计软件Primer Premier5.0设计合成特异性引物,经过PCR扩增,将基因扩增的片段连接到相应的载体上,成功构建重组质粒p3XFLAG-CMV-7.1-PPBP。重组质粒经过筛选、鉴定以及纯化后,经10倍系列稀释作为质控样品,用于实时荧光定量PCR中PPBP标准曲线的构建,并检测重复性、灵敏性和特异性。结果显示标准曲线线性关系R20.99;特异性试验中也能检测到PPBP扩增曲线;组间和组内变异系数均小于5%。使用建立的SYBR GreenⅠ荧光定量RT-PCR检测PRRSV感染组织和未感染组织中PPBP的表达情况,能够检测出组织中PPBP的含量存在差异。本研究初步建立了检测猪PPBP基因的SYBR GreenⅠ荧光定量RT-PCR的方法,为后续猪传染性疾病和猪PPBP之间相互关系的研究提供一个特异灵敏的检测和手段。     

蜱传脑炎病毒(TBEV)SYBR Green Ⅱ荧光定量PCR检测方法的建立

根据TBEV NS5基因序列设计特异性引物,构建标准品,优化荧光定量PCR的反条件和反应体系,将标准品按10~(8 )～10~1拷贝/μL梯度稀释,建立标准曲线。同时分析其敏感性和特异性,并对155份牛血清样品进行检测验证。结果显示,建立了TBEV基于SYBR GreenⅡ荧光定量PCR的检测方法。标准曲线在8个浓度梯度间呈现良好的线性关系,扩增效率为97%,组间和组内变异系数均低于2%且无非特异杂峰。敏感性检测显示,荧光定量检测下限为10~1拷贝/μL,灵敏度比半巢式PCR高10倍。该方法对LCMV、SFTSV及TBEV毒株进行检测,仅TBEV出现特异性扩增。临床样品检测结果显示,荧光定量PCR检出TBEV阳性率为14.2%,比半巢式PCR检测阳性率提高了5.2%,两者检测一致率为63.6%。结果表明,成功建立TBEV基于SYBR GreenⅡ荧光定量PCR的检测方法,为蜱传病防控奠定基础。     

Presumed primary thiamine deficiency in a young African lion (Panthera leo).

A 1-yr-old intact male African lion (Panthera leo) fed only beef muscle meat was evaluated for episodes of hypermetric ataxia, generalized weakness and tonic-clonic front limb movements. A hemogram, biochemical profile, blood lead, electrocardiogram, survey radiographs, and brain computed tomography were normal. Cerebral spinal fluid analyses suggested mild inflammation. Acetylcholine receptor antibody and serologic tests for all infectious agents tested were negative. Clinical signs resolved completely 9 days after instituting oral thiamine (3 mg/kg/day) and a completely nutritional diet. This lion's pretreatment thiamine blood value (11 nmol/L) was markedly lower than that of a healthy lion (191 nmol/L) and a proposed reference range for adult African lions (160-350 nmol/L). The lion remained clinically normal 2 yr later when his blood thiamine value was 340 nmol/L. African lions can develop clinical primary thiamine deficiency and may respond favorably when thiamine treatment and adequate diet are instituted prior to irreversible neuronal necrosis.     

Prevalence of Bartonella infection in wild African lions (Panthera leo) and cheetahs (Acinonyx jubatus)

Bartonella species are emerging pathogens that have been isolated worldwide from humans and other mammals. Our objective was to estimate the prevalence of Bartonella infection in free-ranging African lions (Panthera leo) and cheetahs (Acinonyx jubatus). Blood and/or serum samples were collected from a convenience sample of 113 lions and 74 cheetahs captured in Africa between 1982 and 2002. Whole blood samples available from 58 of the lions and 17 of the cheetahs were cultured for evidence of Bartonella spp., and whole blood from 54 of the 58 lions and 73 of the 74 cheetahs tested for the presence of Bartonella DNA by TaqMan PCR. Serum samples from the 113 lions and 74 cheetahs were tested for the presence of antibodies against Bartonella henselae using an immunofluorescence assay. Three (5.2%) of the 58 lions and one (5.9%) of the 17 cheetahs were bacteremic. Two lions were infected with B. henselae, based on PCR/RFLP of the citrate synthase gene. The third lion and the cheetah were infected with previously unidentified Bartonella strains. Twenty-three percent of the 73 cheetahs and 3.7% of the 54 lions tested by TaqMan PCR were positive for Bartonella spp. B. henselae antibody prevalence was 17% (19/113) for the lions and 31% (23/74) for the cheetahs. The prevalence of seropositivity, bacteremia, and positive TaqMan PCR was not significantly different between sexes and age categories (juvenile versus adult) for both lions and cheetahs. Domestic cats are thus no longer the only known carriers of Bartonella spp. in Africa. Translocation of B. henselae seronegative and TaqMan PCR negative wild felids might be effective in limiting the spread of Bartonella infection.     

济南动物园孟加拉虎和非洲狮行为节律与游客行为的关系

动物园是迁地保护的重要场所,研究动物行为与游客行为可以为动物和游客管理提供有价值的信息。本文分析了济南动物园孟加拉虎、非洲狮的日行为活动节律,以及其行为与游客行为之间的关系。结果显示:孟加拉虎的日行为中走动时间所占比例最大,占45. 47%;其次是睡眠,占43%,卧息占10. 88%,其他行为仅占0. 65%;非洲狮卧息时间所占比例最大,占62. 76%;其次是睡眠,占27. 82%,走动行为占7. 24%;其他行为仅占2. 18%。二者行为与游客召唤、拍照行为以及游客停留时间存在一定的相关关系。游客停留时间与孟加拉虎的活跃状态存在显著相关性;当其睡眠时,游客的召唤行为增加,游客的停留时间也随之增长。游客拍照行为与非洲狮卧息行为有一定相关性。因此动物园应增加环境丰富度,促进动物自然、多样的行为表达;加强游客教育,为游客创造更好的观赏体验,以提高动物园的综合效益。     

一例非洲狮异氟醚吸入麻醉病例报告

内蒙古呼和浩特市某野生动物园3岁雌性非洲狮产前出现里急后重、外阴流出黏液性分泌物等症状,通过临床、影像学及实验室综合性诊断为胎儿死亡,随即进行外科剖宫产手术。手术过程中对非洲狮采取赛拉嗪镇静、丙泊酚诱导麻醉和异氟醚维持麻醉。手术持续52 min,术中非洲狮体心率、收缩压、血氧饱和度和呼吸等体征控制良好,麻醉保定效果确实,基本满足了手术要求。     

Polymorphic expression in the CD8alpha chain surface receptor of African lions (Panthera leo).

Free-ranging African lion (Panthera leo) peripheral blood mononuclear cells (PBMC) were examined using flow cytometry and antibodies developed for use in the domestic cat to determine if phenotypic changes occurred in lion lymphocytes as a result of feline immunodeficiency virus (FIV) infection. The percentage of CD8 cells from lion peripheral blood was considerably lower than in the domestic cat. Lions with elevated levels of CD8+ cells were typically infected with FIV, similar to observations in the domestic cat. Antibodies against the alpha chain of the CD8 receptor (monoclonal antibody (mAb) 3.357) did not react consistently in all lions examined. Flow cytometric analysis determined that approximately 82 and 80% of the animals from Kruger and Hluhluwe-Umfolozi National Parks in South Africa reacted with the monoclonal antibody against the alpha chain of CD8 receptor, while only 17% of the lions in Etosha National Park in Namibia cross-reacted with the CD8alpha chain. There was no apparent correlation between FIV status and CD8alpha chain reactivity. The relative isolation of Etosha from the other two parks could explain the marked difference in CD8alpha chain expression and suggests that lions similar to other mammalian species demonstrate polymorphic expression of the CD8alpha chain (197).     

Veterinary dental health science. 18. Chronic molar fistula in a lion: case discussion

In this case-report immobilization, examination and treatment of a lion (Panthera leo) with a chronic fistulation of a carnassial tooth are described.     

Blastomycosis in nondomestic felids.

Blastomycosis was diagnosed in six nondomestic felids from eastern Tennessee, including two Asian lions (Panthera leo persicus), one African lion (Panthera leo), one Siberian tiger (Panthera tigris), one cheetah (Acinonyx jubatus), and one snow leopard (Panthera uncia). Clinical signs included lethargy, anorexia, weight loss, dyspnea, sneezing. ataxia, and paresis. Variable nonspecific changes included leukocytosis, monocytosis, moderate left shift of neutrophils, moderate hypercalcemia, hyperproteinemia, and hyperglobulinemia. Thoracic radiographs revealed interstitial and alveolar changes, consolidation or collapse of a lung lobe, bullae formation, and a pulmonary mass. Agar gel immunodiffusion (AGID) serology for Blastomyces dermatitidis was performed in five felids and was positive in three. The tiger had cerebral blastomycosis and was positive for AGID serologic tests of both cerebrospinal fluid and serum. One percutaneous lung aspirate in the snow leopard and one bronchial aspirate in an Asian lion demonstrated B. dermatitidis organisms. whereas tracheal wash samples and a nasal discharge were nondiagnostic in others. Treatment with itraconazole was attempted in four cats. The tiger improved before euthanasia, whereas the others did not survive beyond initial treatments. In four felids, B. dermatitidis was found in the lungs and tracheobronchial lymph nodes associated with a florid pyogranulomatous reaction; the tiger had a pyogranulomatous encephalomyelitis, and the cheetah had a single pulmonary granuloma. Thoracic radiography, cytologic examination of lung lesion aspirates, and B. dermatitidis AGID serology should be performed on clinically ill zoo felids in endemic areas to rule out blastomycosis.     

Treatment of malignant lymphoma in an African lion (Panthera leo).

A 14 yr-old male, vasectomized African lion (Panthera leo) exhibited mild weight loss despite adequate appetite. Splenomegaly was diagnosed on physical examination. On the basis of hematology and clinical pathology, malignant lymphoma with chronic lymphocytic leukemia was diagnosed. Abdominal exploratory surgery and splenectomy were performed. Histologic examination and immunohistochemistry confirmed a small cell peripheral T-cell lymphoma. Initial treatments consisted of doxorubicin and prednisone, with later addition of lomustine. The lion remained in clinical remission at 2 mo, 6 mo, and 12 mo postchemotherapy physical examinations. The lion survived 504 days from initial diagnosis. At necropsy, the only lesions consistent with lymphoma were localized epitheliotrophic infiltrates of small neoplastic T lymphocytes within the nasopharyngeal epithelium and the underlying submucosa observed on microscopic examination.