蓝舌病毒RT-PCR检测方法的建立及对云南省库蠓蓝舌病流行病学检测

建立蓝舌病毒(bluetongue virus,BTV)RT-PCR检测方法,用于2018年云南省库蠓的蓝舌病(BT)流行病学检测。设计2对保守引物,优化RT-PCR方法的退火温度、循环次数、退火时间和延伸时间,并进行灵敏性和特异性评价。采集了昭通市3个县及普洱市澜沧县的BT传播媒介库蠓共25万余只,进行研磨后,用建立的RT-PCR方法检测样本。筛选出1对引物BTVS10-F和BTVS10-R作为蓝舌病毒RT-PCR检测引物,敏感度达到10~(2.5) TCID_(50)/0.1 mL,通过对BTV16型的阳性病毒检测,证实该方法的可行性。     

竞争酶联免疫吸附试验检测蓝舌病抗体的研究

用已研制的BTV－11型VP7单克隆抗体建立了竞争酶联免疫吸附试验（C－ELISA）检测蓝舌病抗体的方法，并与琼脂免疫扩散试验（AGID）进行了检测比较，C－ELISA特异性强，不与相关环状病毒发生交叉反应，敏感性比AGID高。用研究制备的C－ELISA诊断试剂盒和美国、澳大利亚制备的诊断试剂盒对1377份临床样品的检测，以及对实验动物人工感染后抗体动脉检测。三种诊断试剂盒检测结果一致，且重复性好。本研究建立的蓝舌病C－ELISA是一种特异性强、敏感性高的蓝舌病抗体检测方法。     

用VP7—ELISA检测牛,羊血清蓝舌病抗体的研究Ⅰ：VP7—ELISA检测蓝舌病抗体方

将表达有蓝舌病毒ＶＰ７蛋白的Ｓｆ２１细胞超声波处理制备ＶＰ７抗原,建立了ＶＰ７－ＥＬＩＳＡ检测蓝舌病抗体的方法,确定阴性阳性判定界值及最佳反应条件：待检牛血清阳性下限为３．０,羊血清阳性下限为２．４,ＶＰ７抗原包被浓度为１：８００,用含５％健康鸡血清的磷酸盐缓冲液作封闭液,待检血清１：１００稀释,豚鼠抗牛羊ＩｇＧ－ＨＲＰ结合物１：５００稀释,３７℃避免显色４ｍｉｎ～６ｍｉｎ。试验结果表明：ＶＰ７可与２３个不同血清型ＢＴＶ抗体反应,具有较高的群特异性和敏感性。     

The epidemiology of bluetongue

The perception that bluetongue virus (BTV), once introduced to a country, would decimate its sheep industry, grew from the acceptance in the late 1950s that it was an emerging virus with Africa as its source. Epidemiological studies in the 1960s and early 1970s confirmed that the geographic distribution of BTV infections included regions of the world, outside the traditionally defined areas where BT was observed. This was interpreted at the time as representing serious and rapid spread of the virus.

This review provides evidence to rebut this earlier view. What has emerged through the 1980s is: (a) the recognition that BTV is a common infection of ruminant livestock throughout the tropics and sub-tropics apparently within several separate ecosystems; (b) in most areas of the world, infection is sub-clinical; (c) incursions of virus (with accompanying disease) into temperate climates do occur at certain key locations, but “die out” usually within the same year; (d) recognition of the vector competence of Culicoides spp in the different ecosystems of the world is critical for understanding the epidemiology of disease; (e) persistent infection in ruminants is no longer considered important in the long term perpetuation of the virus.     

蓝舌病病毒重组VP7蛋白单克隆抗体制备及竞争ELISA检测方法的建立

为了建立蓝舌病(BT)的血清学诊断方法,本研究利用原核表达的蓝舌病病毒(BTV)血清型12型VP7纯化蛋白免疫BALB/c小鼠,制备2株单克隆抗体(MAb),分别命名为BTV-2D10和BTV-4H7。IFA试验表明,2株MAb均能与BTV 24个血清型发生特异性反应,而与茨城病病毒(IBAV)、中山病病毒(CV)、赤羽病病毒(AKAV)、牛病毒性腹泻病毒(BVDV)、牛传染性鼻气管炎病毒(IBRV)、牛轮状病毒(BRV)、牛肠道病毒(BEV)、牛呼肠孤病毒(RV)及口蹄疫病毒(FMDV)无交叉反应,表明2株MAb均为BTV群特异性抗体。采用重组表达的VP7蛋白作为包被抗原建立的竞争ELISA方法证明,BTV-4H7 MAb对不同血清型BTV阳性血清具有良好的阻断效果,而对AKAV、IBAV、BRV和FMDV阳性血清无阻断作用。本研究建立的竞争ELISA方法与IDEXX公司的试剂盒检测包括65份已知背景血清和322份采自广西省的山羊血清样品,检测结果符合率分别达100%和98%。该竞争ELISA方法的建立为BTV抗体的监测提供了安全、快速、准确的技术手段。     

2013年内蒙古地区蓝舌病流行情况调查

为了解内蒙古地区蓝舌病(BT)流行情况,本研究于2013年对内蒙古存在疑似BT病例地区进行流行病学调查,收集了内蒙古6个盟市不同种类动物的抗凝血及其对应血清样品212份,采用竞争ELISA及套式RT-PCR方法对样品进行检测.竞争ELISA检测结果显示212份血清样品中有63份为BT阳性样品,13份为BT弱阳性,总阳性率为35.85％(76/212).套式RT-PCR检测结果显示212份血液样品中有61份为BT阳性样品,总阳性率为28.77％(61/212),其中绵羊感染率为21.25％(17/80)、绒山羊感染率为35.35％(41/116)、牛感染率为18.75％(3/16).不同的饲养方式对动物感染BT也有影响,其中圈养感染率为16.67％(1/6),散养感染率为29.13％(60/206).本研究结果表明,内蒙古地区BT流行范围比较广泛,因此有必要加强该病的检测及预防.     

A cross-sectional study of bluetongue virus and Mycoplasma bovis infections in dairy cattle: I. the association between a positive antibody response and production efficiency

In a cross-sectional study of bluetongue virus (BTV) and Mycoplasma bovis (M. bovis) infections, a sample of 572 California dairy cows was tested for the presence of antibodies to answer the question: Is it possible to identify and to assess quantitatively the associations between positive antibody test and production? Serum samples collected from these cows during December 1986 were tested for the presence of antibodies to BTV and M. bovis using the enzyme-linked immunosorbent assay (ELISA). Data on milk production were extracted from individual cow sheets of the California Dairy Herd Improvement Association (DHIA) record-keeping system and interfaced with percentage ELISA results for analysis. Univariate and multivariate statistical analyses, using the X ² test for categorical variables or Student's t-test for continuous variables and multiple logistic regression respectively, were carried out to evaluate for possible associations between positive antibody tests to each agent and each production variable of interest. Complete data on all variables studied were obtained for 289 (50.5%) cows for M. bovis and 423 (74%) cows for BTV. For cows with complete data on all variables, estimates of the point prevalence of antibodies to BTV and M. bovis were 70.5% and 66.1%, respectively. Results of this study indicated that Guernsey cows were more likely to have a positive BTV test than Holstein cows and that cows in higher lactations were more likely to test positive to BTV ELISA than those in lower lactations (p<0.05). Because all cows except those on one farm were Holstein, our confidence in the effect of breed is limited. The association between lactation number and BTV seropositive test may be an age factor identified earlier in the study. For M. bovis, the results of the analysis indicated that seropositive cows were more likely to produce less milk, on a mature equivalent basis (OR_adj=0.96, p=0.034), and that they had less extended 305 day milk production potential (OR_adj=0.90, p<0.0001) than seronegative cows.     

A cross-sectional study of bluetongue virus and Mycoplasma bovis infections in dairy cattle: II. The association between a positive antibody response and reproduction performance

In a cross-sectional study to determine the possible relationship between a positive antibody test to bluetongue virus (BTV) or Mycoplasma bovis infections and reproductive performance of dairy cows, data were collected on 572 California dairy cows during December 1986 for analysis. Serum samples were tested using an enzyme-linked immunosorbent assay (ELISA). Data on reproduction variables were extracted from the individual cow sheets of the California Dairy Herd Improvement Association records and interfaced with the serological results for analysis.Similar data analyses for both BTV and M. bovis were performed to identify and quantitatively assess the association of the reproduction variables and each agent. These associations were evaluated unconditionally using the x ² for categorical variables and Student's t-test for continuous variables. Logistic regression analysis was used to determine if reproduction variables with significant unconditional associations remained significant when adjusted for the effects of possible confounding factors.Both the BTV and M. bovis ELISA antibody titres indicated exposure to the agents. The results of the multiple logistic regression analyses indicated that cows seropositive for BTV were significantly older at first calving (p<0.03). For M. bovis, seropositive cows were more likely to have longer intervals from calving to last service and longer intervals from calving to pregnancy diagnosis than seronegative cows (p<0.05). The other reproduction variables examined were not significantly associated with ELISA seropositivity.     

The use of discriminant analysis in predicting the distribution of bluetongue virus in Queensland,Australia

The climatic variables that were most useful in classifying the infection status of Queensland cattle herds with bluetongue virus were assessed using stepwise linear discriminant analysis. A discriminant function that included average annual rainfall and average daily maximum temperature was found to correctly classify 82.6% of uninfected herds and 72.4% of infected herds. Overall, the infection status of 74.1% of herds was correctly classified. The spatial distribution of infected herds was found to parallel that of the suspected vector,Culicoides brevitarsis. This evidence supports the role of this arthropod species as a vector of bluetongue viruses in Queensland.The effect of potential changes in temperature and rainfall (the so-called global warming scenario) on the distribution of bluetongue virus infection of cattle herds in Queensland was then investigated. With an increase in both rainfall and temperature, the area of endemic bluetongue virus infection was predicted to extend a further 150 km inland in southern Queensland. The implications of this for sheep-raising in Queensland are discussed.Abbreviations AGID agar gel immunodiffusion     

A seroepidemiological study on bluetongue virus in dairy cattle in the central valley of California

A seroepidemiological study on bluetongue virus (BTV) infection in California dairy cattle was conducted to estimate the prevalence and distribution by age and season of BTV group-reactive antibodies and to look for possible associations between the presence of antibodies and cattle age or breed and farm. Between December 1985 and March 1987, a sample of cattle was tested at approximately two-month intervals for BTV group-reactive antibodies using an enzyme-linked immunosorbent assay (ELISA). Data taken during the month of December 1986 were used to evaluate possible associations between a positive antibody test and certain intrinsic (age, breed) and extrinsic (farm) factors.Univariate and multivariate statistical analyses using the -square test for associations and multiple logistic regression, respectively, were carried out for possible associations between positive antibody tests to BTV and each factor of interest. The strengths of the associations were determined using estimates of the odds ratio.Of the 3774 serum samples tested, 238 (6.3%) were from calves, 1045 (27.6%) were from heifers and 2492 (66.0%) were from cows. Seroprevalence varied from nil in calves on two occasions to over 90% on several occasions in cows. Cows consistently had higher prevalence rates than heifers or calves across all test dates (p<0.05). The seroprevalence of BTV group-reactive antibodies also showed a seasonal fluctuation, with the highest rates occurring during the warmer months of the year. These highest prevalence rates coincided with heavy activity of the known vector of BTV, Culicoides spp. Breed and farm effects were not statistically significant (p>0.05). With the exception of one farm, all cattle were of the Holstein breed, which reduced confidence in assessing any breed effect in this study. Relative estimates of the sensitivity and specificity of BTV ELISA were 87% and 100% respectively, compared to the standard agar gel immunodiffusion (AGID) test.The observations support previous findings of seasonal distribution of BTV antibodies and suggest an age relationship, whereby older cattle are more likely to be positive to BTV group-reactive antibodies than younger cattle.     

抗蓝舌病病毒VP6和VP7蛋白单克隆抗体的制备及鉴定

为制备针对蓝舌病病毒(BTV)的单克隆抗体(MAb),本研究利用血清型1型BTV(BTV1)免疫BALB/c鼠,将其脾淋巴细胞与SP2/0进行融合,并用BTV1包被ELISA板,通过间接ELISA方法筛选出3株稳定分泌抗BTV1的MAb的杂交瘤细胞株(2B10、3D4和4H8)。利用表达BTV1主要蛋白的真核表达重组质粒转染BHK-21后,对所制备的杂交瘤细胞株上清进行间接免疫荧光(IFA)以及western blot鉴定,结果显示:2B10和4H8与VP7蛋白反应,而3D4与VP6蛋白反应。同时,IFA鉴定结果进一步表明,3株MAb与24个血清型的BTV均可以发生反应。本研究制备的MAb为建立BTV免疫学检测方法和相关病毒蛋白的功能研究奠定了基础。