山羊地方性鼻内肿瘤病毒TaqMan荧光定量RT-PCR检测方法的建立

为建立山羊地方性鼻内肿瘤病毒(Enzootic nasal tumor virus of goats,ENTV-2)快速检测方法，根据ENTV-2 env基因保守区域设计引物和TaqMan探针，通过优化反应条件，建立TaqMan荧光定量RT-PCR检测方法，并对其特异性、灵敏性、重复性与临床检测效果进行验证。结果显示，该方法建立的标准曲线在重组质粒浓度为1.019×10⁹—1.019×10²copies/μL时，相关系数R²为0.998,Ct值和重组质粒浓度有良好的线性关系；与相关的其他5种羊常见病原无交叉反应，最低检测限度为10.19 copies/μL，组间和组内的变异系数均在1.5%内。利用本研究建立的方法与常规RT-PCR对20份临床样品进行对比检测，两种方法的ENTV-2阳性检出率分别为60%(12/20)和30%(6/20)，两种方法的总符合率为70%(14/20)。结果表明，本研究建立的方法特异性强、灵敏度高、重复性好，为ENTV-2的诊断和流行病学调查提供技术手段。     

山羊地方性鼻内肿瘤病毒MAb的制备及间接ELISA方法的建立

为建立检测山羊地方性鼻内肿瘤病毒(ENTV-2)的间接ELISA方法，本研究以纯化的ENTV-2免疫BALB/c小鼠，采用间接ELISA方法筛选阳性细胞克隆，经3次克隆纯化后获得1株能够稳定分泌ENTV-2特异性单克隆抗体(MAb)的细胞株，命名为6E4，并采用鼠MAb亚类鉴定试剂盒鉴定6E4 MAb的亚类，结果显示，MAb 6E4的亚类为κ链、Ig G1亚型。进一步以6E4为检测抗体，经各反应条件的优化建立了检测鼻液样品中ENTV-2的间接ELISA方法，该方法的临界值为0.323。利用该方法 检测羊口疮病毒(ORFV)、绵羊肺炎支原体(Movi)、丝状支原体山羊亚种(Mmc)以及纯化的ENTV-2和ENTV-2患病羊鼻液样品，结果显示，除纯化的ENTV-2及ENTV-2患病羊鼻液样品的检测结果呈阳性外，ORFV、Movi、Mmc的检测结果均为阴性，该方法特异性较强。将纯化的ENTV-2 (以蛋白浓度换算) 2倍倍比稀释(20μg/mL～0.08μg/mL)后，利用建立的间接ELISA方法检测，结果显示，纯化的ENTV-2稀释至0.3125μg/mL时检测结果仍可判定为阳性，该方法敏...     

蓝舌病病毒8型实时荧光定量RT-PCR检测方法的建立与应用

根据蓝舌病病毒8型(BTV-8) VP2基因序列(Seg-2)设计特异性引物和探针,建立了BTV-8实时荧光定量RT-PCR(RT-qPCR)检测方法。结果表明该法特异性强,能准确检测BTV-8核酸,与蓝舌病病毒其他血清型病毒、鹿流行性出血热病毒(EHDV)、牛病毒性腹泻病毒(BVDV)、口蹄疫病毒(FMDV)等病原核酸无交叉反应;最低可检测核酸浓度为2.8×10~3 copies/mL;Ct值组内和组间变异系数(CV)均小于2%。用所建立方法对115份临床样品进行检测,结果均为阴性,与文献报道的方法检测结果一致。建立的RT-qPCR方法可用于BTV-8的临床检测和流行病学调查。     

牛轮状病毒SYBR GreenⅠ实时荧光定量PCR检测方法的建立及应用

旨在建立一种SYBR GreenⅠ实时荧光定量PCR(RT-qPCR)检测牛轮状病毒(BRV)的方法,并采用针对BRV vp6基因序列保守区设计的引物,使用RT-qPCR方法进行目的基因扩增,优化反应条件,制备阳性标准品,建立标准曲线,确定重复性、敏感性和特异性,与常规PCR方法比较。结果显示,建立的RT-qPCR方法最佳引物浓度为5μmol/L,退火温度为58℃,40个循环,熔解温度为77℃±0.5℃,最小检出量8.13copies/μL。两种方法检出率分别为30.4%和19.6%,RT-qPCR具有较高敏感性。建立的RT-qPCR可为BRV感染早期诊断、分子流行病学调查及定量检测分析等提供技术保障。     

BVDV、BRV和BCoV TaqMan三重RT-qPCR检测方法的建立

为建立牛病毒性腹泻病毒(BVDV)、牛轮状病毒(BRV)和牛冠状病毒(BCoV)的快速检测方法,根据GenBank中登录的BVDV 5'-UTR、BRV NSP5和BCoV N基因序列设计特异性引物和探针,通过优化反应体系和条件,建立了同时检测上述3种病毒的TaqMan三重RT-qPCR方法。该方法仅对BVDV 5'-UTR、BRV NSP5和BCoV N基因扩增呈阳性,而对牛传染性鼻气管炎病毒、牛副流感病毒、魏氏梭菌(A型、B型和D型)、多杀性巴氏杆菌(A型和B型)等犊牛腹泻相关病原扩增均呈阴性;最低检出限为10拷贝/μL;组内和组间变异系数均小于2%。利用本研究建立的TaqMan三重RT-qPCR方法对29份临床样品进行检测,获得BVDV、BRV、BCoV的4种混合感染型,其中BVDV与BCoV的混合感染率最高(27.6%);与已有单重RT-qPCR方法比较,发现两种方法检测BVDV、BRV和BCoV的符合率分别为100%、96.5%、100%。结果表明,本研究建立的TaqMan三重RT-qPCR方法具有特异性强、敏感性高、重复性好、可行性高等优点,可为今后BVDV、BRV和BCoV共感染引起的牛腹泻性疾病的鉴别诊断和流行病学调查提供新技术手段。     

鸭坦布苏病毒一步法RT-PCR与荧光定量RT-PCR检测方法的建立

为建立鸭坦布苏病毒(DTMUV)的检测方法,本研究根据GenBank中登录的DTMUV E基因保守区域设计引物和探针,建立了检测DTMUV的一步法RT-PCR和荧光定量RT-PCR方法,并比较了两种方法的敏感性和特异性。结果显示,荧光定量RT-PCR标准曲线的相关系数(R~2)为0.999,DTMUV RNA在10~2拷贝/μL~10~7拷贝/μL范围内与Ct值呈现良好的线性关系;两种方法均仅对DTMUV RNA具有特异性扩增,而对其他常见鸭病原核酸的扩增均为阴性,具有较强的特异性;一步法RT-PCR对鸭胚纯培养病毒RNA的检测下限为10~3拷贝/μL,而荧光定量RT-PCR的检测下限为10~2拷贝/μL;重复性试验的变异系数均小于1.5%。对20份模拟临床样品的检测结果表明,一步法RT-PCR的阳性率为75%(15/20),而荧光定量RT-PCR的阳性率为90%(18/20)。本研究表明所建立的DTMUV荧光定量RT-PCR特异性强、重复性好、敏感性更高,更适用于DTMUV的临床诊断和流行病学调查。     

多重PCR结合基因芯片技术检测5种猪繁殖障碍性病毒病方法的研究

为建立运用多重PCR和基因芯片技术同时检测5种猪繁殖障碍性病毒病的方法。本研究根据GenBank中登录的猪瘟病毒(CSFV)、猪细小病毒(PPV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪日本乙型脑炎病毒(JEV)及猪圆环病毒2型(PCV2)的基因序列设计特异性引物与探针,制备相应的寡核苷酸芯片,检测了5种猪繁殖障碍性疾病病毒的标准毒株,并对16份临床样品进行检测。通过多重PCR扩增出带有荧光标记的5种病毒的特异性基因片段,并与固定有特异性探针的基因芯片杂交。结果显示,本研究建立的多重PCR结合基因芯片检测方法特异性强、稳定性好,灵敏度可达10~2拷贝/μL。16份临床样品检测结果显示阳性率达87.5%(14/16)。以上结果表明该方法特异性好、灵敏度高,可高效检测以上5种病毒,为其临床诊断及流行病学调查提供了有效的检测方法。     

猪流行性腹泻病毒RAA-CRISPR/Cas13a检测方法的建立与初步应用

将重组酶辅助扩增技术(recombinase aided amplification, RAA)与规律间隔性成簇短回文重复序列相关Cas13a蛋白(CRISPR-Cas13a)技术相结合，即RAA-Cas13a,建议建立高效、灵敏、特异的猪流行性腹泻病毒(porcine epidemic diarrhea virus, PEDV)检测方法。针对PEDV N基因保守区设计RAA特异性引物和CRISPR RNA(crRNA),利用RAA技术扩增样本核酸，并进行CRISPR-Cas13a荧光检测，以RT-qPCR为对照方法，评价该方法的灵敏度、特异性及与RT-qPCR法的一致性。结果表明，该方法最低可检测至10¹ copies·μL^-1,且与猪圆环病毒1型、猪圆环病毒2型、猪圆环病毒3型、猪繁殖与呼吸综合征病毒、猪瘟病毒及猪伪狂犬病病毒等常见猪源病原核酸检测无交叉反应。采用RAA-Cas13a检测40份临床样本与RT-qPCR方法阳性符合率为100%,阴性符合率为84.6%,总符合率为95%,Kappa值为0.881。本研究建立的RAA-Cas13...     

多重PCR结合基因芯片技术检测5种猪繁殖障碍性病毒病方法的研究

为建立运用多重PCR和基因芯片技术同时检测5种猪繁殖障碍性病毒病的方法。本研究根据GenBank中登录的猪瘟病毒(CSFV)、猪细小病毒(PPV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪日本乙型脑炎病毒(JEV)及猪圆环病毒2型(PCV2)的基因序列设计特异性引物与探针,制备相应的寡核苷酸芯片,检测了5种猪繁殖障碍性疾病病毒的标准毒株,并对16份临床样品进行检测。通过多重PCR扩增出带有荧光标记的5种病毒的特异性基因片段,并与固定有特异性探针的基因芯片杂交。结果显示,本研究建立的多重PCR结合基因芯片检测方法特异性强、稳定性好,灵敏度可达10~2拷贝/μL。16份临床样品检测结果显示阳性率达87.5%(14/16)。以上结果表明该方法特异性好、灵敏度高,可高效检测以上5种病毒,为其临床诊断及流行病学调查提供了有效的检测方法。     

猪瘟病毒实时荧光定量PCR检测方法的建立及初步应用

为建立一种特异、灵敏、快速及量化的猪瘟病毒(CSFV)检测方法,设计扩增CSFV E2基因的特异性引物,建立Eva Green染料的RT-qPCR检测方法,对其特异性、灵敏性、重复性测试,并检测疑似猪瘟病毒感染的临床样品。结果显示,所建立的RT-qPCR检测方法标准曲线的线性相关系数R~2=1.000,具有良好的线性关系;检测猪流行性腹泻腹泻病毒(PEDV)、猪圆环病毒2型(PCV2)、猪伪狂犬病病毒(PRV)、猪繁殖与呼吸综合征病毒(PRRSV)无交叉反应,特异性强;相比常规RT-PCR检测方法,灵敏度高出100倍;重复试验的变异系数系数小于1.5%,重复性良好。对31份临床样品进行检测,结果比普通PCR多检出4份阳性样品。建立的RT-qPCR检测方法在特异性、灵敏度、重复性方面均表现良好,可用于临床猪瘟病毒的检测。     

Development of a SYBR Green-based real-time quantitative polymerase chain reaction assay to detect enzootic nasal tumor virus in goats

Enzootic nasal adenocarcinoma is a contagious respiratory disease in goats that is caused by the enzootic nasal tumor virus 2 (ENTV-2). In order to increase the number of available detection methods for ENTV-2, we developed a SYBR Green real-time polymerase chain reaction (SGrPCR) assay that targets the gag gene of ENTV-2. The low limit of detection of the assay was 3.68 × 10¹ copies/μL, a hundredfold more sensitive than conventional PCR. The melt curve showed a single sharp melt peak at 83°C, which indicated that there was no non-specific amplification or primer dimer formation. The intra-assay and inter-assay coefficients of variation were 1.58% and 1.82%, respectively. There was no cross-reactivity with closely related goat viruses (i.e., orf virus, peste des petits ruminants virus, goatpox virus, foot-and-mouth disease virus) and endogenous retroviruses. In conclusion, the SGrPCR assay is specific for the gag gene of ENTV-2 and provides a rapid and sensitive approach for detecting ENTV-2 in clinical samples.     

山羊地方性鼻内肿瘤病毒全基因组序列分析及其gag基因促肿瘤发生的机制研究

本研究旨在获得山羊地方性鼻内肿瘤病毒（enzootic nasal tumor virus 2,ENTV-2）全基因组序列并进行分析。从福建某羊场的山羊鼻内肿瘤组织取样,针对ENTV-2设计引物,通过RT-PCR方法从肿瘤组织中分段扩增出6个特异性产物,测序后利用生物学软件DNAStar进行拼接,获得长度为7 443 bp全基因组序列,将其命名为ENTV-2-FJ。ENTV-2-FJ基因组结构与其他逆转录病毒类似,具有5'-U5-gag-pro-pol-env-U3-3'典型结构,包含4个开放阅读框和侧翼非编码区以及末端重复序列。为制备兔抗ENTV-2 gag蛋白多克隆抗体,将特异性扩增的gag基因克隆至pET-21a （+）载体,构建重组质粒pET-21a-gag,鉴定后转化至BL21（DE3）细胞并经IPTG诱导成功表达分子量约为70 ku的融合蛋白。Western blot分析表明,制备的兔抗gag蛋白抗体能在患病山羊肿瘤组织和表达gag基因的细胞中特异性地检测出gag蛋白。为了深入研究ENTV-2-FJ致瘤机制,作者构建了过表达gag基因的K562细胞系,并进行裸鼠致瘤试验。结果显示, gag蛋白通过激活JAK2-STAT5信号通路促进肿瘤的生长。综上,本研究获得了ENTV-2-FJ全基因组序列并对其进行分析,制备并验证了gag蛋白的多克隆抗体,揭示了gag蛋白的作用机制,这些结果为阐明ENTV-2的致病机制提供了科学依据。     

Detection of enzootic nasal tumor virus (ENTV) in a sheep flock in southern Brazil

Enzootic nasal tumor (ENT) is a contagious neoplasm associated with enzootic nasal tumor virus (ENTV), which may induce disease in sheep (ENTV-1) and goats (ENTV-2). This study aimed to describe the occurrence of ENT in two Texel sheep (Ovis aries) from a 75-sheep flock, located in the city of Gravataí, southern Brazil. Animals used to be purchased from different origins, and no specific tests for disease monitoring or quarantine procedure were performed. Affected animals presented respiratory distress, anorexia with severe weight loss, and mucopurulent unilateral nasal discharge. Necropsy was performed in both animals and nasal cavity masses were observed. Histopathological analysis demonstrated an epithelial neoplasm compatible with nasal adenocarcinoma. PCR using a protocol that amplifies a 591 bp sequence of 5’LTR-gag region of ENTV1 was performed followed by DNA sequencing. Both samples were positive, and the sequences obtained presented highest identity (97%) with ENTV strain TN28 (GenBank accession number MH899613) detected in a Texel sheep from Scotland. This is the first report of ENTV-1 leading to enzootic nasal tumor in sheep in Latin America, which confirms the presence of the retrovirus in sheep flocks in the Brazilian territory.

     

一起山羊地方性鼻内肿瘤的诊断

陕西省某山羊养殖场发生一起山羊鼻内肿瘤病,通过流行病学和临床症状调查、病理剖检、病理切片检查进行初步诊断。其次根据GenBank收录的山羊地方性鼻内肿瘤病毒基因组序列设计1对特异性引物,通过PCR方法从肿瘤组织中扩增获得目的片段并测序。序列比对分析表明,该目的基因和山羊地方性鼻内肿瘤病毒高度相似,最终确定该病为山羊地方性鼻内肿瘤病毒引起的山羊地方性鼻内肿瘤(ENT)。     

CHRONIC INTERSTITIAL PNEUMONIA IN ASSOCIATION WITH A GRANULOMATOUS ENCEPHALITIS IN A GOAT

SUMMARY A 30-month-old British Alpine goat had clinical and pathological findings consistent with the diagnosis of a chronic interstitial pneumonia. The goat also had lesions in the brain similar to those described for granulomatous encephalomyelitis (GE) of goats. Lesions similar to GE were reproduced experimentally in feral goats following intracerebral inoculation of a lung homogenate from the naturally occurring case. The differential diagnosis of chronic interstitial pneumonia in goats, the differentiation of GE from viral leucoencephalomyelitis, and the possible aetiological role of retroviruses in these diseases is discussed.     

The possible role of Ostertagia circumcincta, coccidiosis and dietary protein level in the development of swelling disease in Angora goat kids

A trial was carried out to investigate the possible role of Ostertagia circumcincta, coccidiosis and level of protein in the diet in the development of swelling disease in Angora goat kids. Eighty Angora goat kids were bought from 5 producers whose flocks had a history of swelling disease. These kids were kept in enclosures at Grootfontein Agricultural Development Institute near Middelburg (Eastern Cape) for the duration of the experiment. The animals received a combination of the following treatments: (a) high protein vs normal protein diet; (b) Ostertagia circumcincta infection or no O. circumcincta infection; (c) coccidial infection or no coccidial infection. Data recorded included weekly body weight, weekly total plasma protein levels (TPP), weekly faecal egg counts, weekly coccidial oocyst counts and haematology at Weeks 1, 5, 9, 13 and 16. The goats were also monitored daily for any clinical symptoms. There was no specific trend in any of the parameters measured among the different treatment groups at any stage during the experimental period. The goats were shorn during Week 10 of the experiment. On Monday 6 September 2004 (Week 12 of the study), 19 of the goats developed some subcutaneous oedema. The Saturday (4 September 2004) was rather hot (30 degrees C), followed by very cold rainy conditions (11 degrees C) on Sunday (5 September 2004). Twelve of the goats developed what can be described as little oedema, while 7 developed moderate oedema. The number of goats that developed oedema was fairly evenly distributed among the various treatment groups. As far as the specific treatments are concerned, more goats on the normal protein diet developed moderate oedema than the goats on the high protein diet. Body weights of goats that developed moderate oedema were lower throughout the experimental period than body weights of goats that developed little or no oedema, while TPP of goats that developed moderate oedema were lower from Week 5 of the study onwards. There were also no significant differences at any stage throughout the experimental period in faecal egg counts, faecal coccidial oocyst counts or any of the blood parameters between goats that developed moderate oedema, little oedema and those that did not develop any oedema. No goats developed full-blown swelling disease during the course of the experiment. It is possible that the treatments applied in this study are not inductive of the disease, or the effects of the treatments were not severe enough to induce swelling disease.     

Isolation of caprine arthritis encephalitis virus from goats in Mexico.

A lentivirus was isolated from 2 goats in Mexico that were seropositive to caprine arthritis encephalitis virus (CAEV) by the agar gel immunodiffusion (AGID) test. The lentivirus was identified as CAEV by the observation of giant multinucleated cells (syncytia) in goat synovial membrane (GSM) monolayers co-cultivated with blood mononuclear (BMN) cells from the seropositive goats, and by amplifying a DNA segment of the CAEV gag gene using the polymerase chain reaction (PCR) technique. Subsequently, cell supernatants from the GSM cells co-cultivated with BMN cells were used to infect 2 CAEV-seronegative goats. These goats seroconverted to CAEV as determined by the AGID test, and CAEV was re-isolated from these goats. One of the goats developed polyarthritis 8 mo after inoculation. Previous serological surveys indicate that infection with CAEV is prevalent among goats in Mexico. To our knowledge this is the first report of CAEV isolation in Mexico. Because of globalization of markets and increased trading among nations, the rapid identification and reporting of diseases such as CAEV are important to prevent the dissemination of these diseases.     

Immune response to pulmonary injection of Pasteurella haemolytica-impregnated agar beads followed by transthoracic challenge exposure in goats

A method of inducing Pasteurella haemolytica serotype 1 (Ph1) lung infection in goats, using low numbers of bacteria and without impairing host immunity, was developed. Two trials were conducted. Results of trial 1, using 10 principals (Ph1 agar beads) and 6 controls (agar beads alone), indicated that Ph1 organisms imbedded in agar beads could survive host lung defenses for 32 days. Results of trial 2 indicated that lung immunity in the inoculated goats (principals) was high and they were more protected than controls against a transthoracic challenge of Ph1 (1.18 x 10(7) colony-forming units) injected into a lung of each goat on posttreatment day 35. When comparing challenge-exposed principals with controls, the controls developed rectal temperatures above normal for a longer time, duration of anorexia was longer, and signs of depression were seen. The controls developed large areas of consolidated lung tissue, more Ph1 isolates were recovered from nasal turbinates and lung tissue, and higher Ph1 concentrations were found in the lungs. The serum Ph1 indirect hemagglutination antibody titers in the principals of both trials increased, compared with titers in controls. Principal goats in trial 2 had higher Ph1 indirect hemagglutination antibody titers after injection of Ph1-impregnated agar beads and less severe lung lesions after challenge exposure than did controls. The small pneumonic consolidated lesions in the principals, compared with extensive lesions in controls after Ph1 challenge exposure, indicated a high degree of immunity after exposure to Ph1 organisms imbedded in agar beads.     

绒山羊皮肤毛囊兴盛前期组织形态、chi-miR-107-3p与RHBDF2基因表达分析

为了探究chi-miR-107-3p和菱形家族蛋白2（rhomboid family member 2,RHBDF2）基因在不同品种（系）和光控增绒绒山羊兴盛前期（5~7月份）皮肤毛囊重建时的表达差异,本试验在7月份采集内蒙古阿拉善型绒山羊、敏盖绒山羊和光控增绒技术处理的阿尔巴斯型绒山羊体侧皮肤毛囊组织,采用组织切片技术对组织形态比较分析,实时荧光定量PCR法检测chi-miR-107-3p和RHBDF2基因的表达量。结果显示,在皮肤毛囊兴盛前期,阿拉善型绒山羊次级毛囊正处于重建阶段,敏盖绒山羊和阿尔巴斯型绒山羊（光控增绒）次级毛囊重建已基本完成;在阿拉善型绒山羊皮肤组织中chi-miR-107-3p表达量极显著高于敏盖绒山羊和阿尔巴斯型绒山羊（光控增绒）（P<0.01）,而RHBDF2基因表达量极显著低于其他两品种（系）（P<0.01）,且RHBDF2基因表达量在敏盖绒山羊皮肤组织中显著低于阿尔巴斯型绒山羊（P<0.05）。不同品种（系）和光控增绒绒山羊皮肤毛囊兴盛前期组织显微结构与chi-miR-107-3p、RHBDF2基因在皮肤组织中的表达差异分析结果相一致,次级毛囊重建初期chi-miR-107-3p表达量较高,而RHBDF2基因表达量极低,随着次级毛囊重建完成,chi-miR-107-3p表达量明显降低,RHBDF2基因表达量逐渐增高,且在不同品种（系）内蒙古绒山羊绒毛生长发育过程中的表达机制基本相同。因此,chi-miR-107-3p和RHBDF2基因是绒山羊皮肤毛囊生长发育的重要调控因子。