双抗体夹心阻断ELISA检测血清牛病毒性腹泻—粘膜病病毒抗体的研究

本研究建立了检测血清中牛病毒性腹病（ＢＤＶ－ＭＤ）病毒抗体的双抗体夹心阻断ＥＬＩＳＡ，并用其检测了长春地区２９３头奶和２６２头黄牛的血清。结果，奶牛的阳性率为５４．９％，黄牛的阳性率４３．５％。本文法是一种敏感、特异、快速的检测抗体方法。可在基层推广应用。     

口蹄疫病毒A型竞争ELISA抗体检测试剂盒在流行病学调查中的应用

为评价口蹄疫病毒A型竞争ELISA(cELISA)抗体检测试剂盒在流行病学调查中的应用前景,对2017年从福建省三明市采集的336份黄牛、奶牛、羊和猪血清样品,用A型cELISA抗体检测试剂盒进行抗体检测。结果显示,92份黄牛血清、92份羊血清、92份猪血清、60份奶牛血清的A型抗体阳性率分别为13.04%、11.96%、20.65%、86.67%。从上述4种血清中,各挑选10份血清(阴性、阳性各5份)共40份,采用口蹄疫病毒液相阻断ELISA(LPB-ELISA)抗体检测试剂盒进行验证。结果显示:cELISA检测为阳性的20份血清中,用LPB-ELISA检出阳性19份;cELISA检测为阴性的20份血清中,用LPB-ELISA检出阴性17份;两种方法的κ值为0.8,总符合率为90.00%。结果表明,A型cELISA试剂盒与LPB-ELISA试剂盒的符合率和一致性均较高,可用于口蹄疫流行病学调查和血清学监测。     

一种鉴别猪伪狂犬母源抗体与野毒感染的检测方法的建立

为探索一种鉴别16周龄以内仔猪伪狂犬野毒感染与母源抗体的ELISA检测方法,本研究通过对江苏省农科院动物疫病诊断检测中心临床门诊2016年5月份接诊的11头16周龄以内仔猪同时进行PRV-gpl抗体检测,PCR检测和PRV-gb抗体的检测,结果该11头猪的PRV-gpl抗体阳性率为100％,PCR检测阳性率为54.5％ (6/11),同时对上述11份血清进行1∶40稀释后检测PRV-gb抗体,结果PRV-gb抗体阳性率为54.5％(6/11),且PRV-gb抗体阳性猪只与PCR检测阳性猪只的符合率为100％.本检测方法的最大发现在于相同PRV-gb抗体水平的PRV感染仔猪与来自于母源抗体仔猪的血清在经相同倍数的稀释后,PRV-gb抗体阳性率不同.随着血清稀释倍数的不断增大,PRV野毒感染猪只血清PRV-gb抗体下降的速度明显较来自于母源抗体猪只的下降速度慢.并最终确定将血清1∶40稀释为鉴别诊断的最佳稀释倍数.虽然本次检测的样本量只有1 1份,但是100％的符合率仍具有较高的参考价值,为江苏省乃至全国伪狂犬病的诊断与防控提供了有效数据参考,具有推广价值.     

奶牛α-酪蛋白抗体纯化方法的比较及其优化

本研究比较了盐析法和亲和层析法以及两种方法相结合纯化兔抗奶牛α-酪蛋白抗体的效果.试验以兔抗奶牛α-酪蛋白血清为材料,先分别用饱和硫酸铵盐析、蛋白A树脂亲和层析及两种方法相结合进行纯化,然后通过SDS-PAGE检测纯化后的抗体纯度.结果表明,蛋白A树脂纯化抗体的效果优于饱和硫酸铵法,两种方法相结合的纯化效果最佳且延长了蛋白A树脂的使用寿命,Western Blot检测发现纯化后的抗体能够特异性结合奶牛α-酪蛋白     

牛皮癣病抗体和细胞免疫检测方法的研究

将双向免疫扩散试验和淋巴细胞转化试验用于牛皮癣病的抗体检测和细胞免疫检测，３５头无牛皮癣症状的新生物中血清抗体全部阴，淋巴细胞转化率均在１０％以下，１０头具有牛皮癣症状的病牛，５０％为血清抗体阳性，淋巴细胞转化率为２２％－４６％。３０头注射抗牛皮癣疫苗的牛血清抗体全部由阴转阳，淋巴细胞转化率升到１５％以上的有１６头。     

北疆规模化猪场CSFV、PRRSV、PRV血清抗体水平监测及分析

为了解北疆地区规模化猪场猪瘟,蓝耳,伪狂犬gE抗体水平,本研究选取3个规模化种猪场,共采集296份血清,采用ELISA抗体检测方法,对其进行猪瘟,蓝耳,伪狂犬gE血清抗体检测工作.结果显示,育肥猪猪瘟抗体阳性率为64.40％ ～100.00％,蓝耳病抗体阳性率为72.00％ ～98.33％,伪狂犬gE抗体阳性率为23....     

重庆市部分地区牛传染性鼻气管炎和流行性牛白血病的血清学调查

为了解传染性鼻气管炎（IBR）和流行性牛白血病（BL）的流行情况，从重庆市辖区内11个区县（自治县）采集奶牛、黄牛、水牛血清共；369头份，用酶联免疫吸附试验（HLISA）检测IBR和BL抗体。结果从部分区县（自治县）检测出阳性样品，IBE阳性率介于2．70％～20．00％之间，总阳性率为4．61％；奶牛、黄牛、水牛总阳性率分别为5．34％、4．92％、0％。BL阳性率介于2．70％～52．50％之间，总阳性率为12．20％，奶牛、黄牛、水牛总阳性率分别为20．87％、1．64％、0％，提示该病在我市奶牛和黄牛牛群中存在，污染较广，应引起重视。     

上海地区奶牛犬新孢子虫病血清学抗体检测

为了掌握上海地区奶牛新孢子虫病的流行情况，应用酶联免疫吸附试验对上海地区奶牛血清样品进行新孢子虫病抗体检测。结果表明，被检的41个奶牛场1239份奶牛血清中有63份抗体阳性，阳性率为5．08％。各场阳性率变幅较大（0-44．23％）。     

奶牛α_s-酪蛋白多克隆抗体的制备、纯化及鉴定

本试验旨在制备高纯度和特异性的奶牛αs-酪蛋白多克隆抗体,为鉴定奶牛乳腺合成的αs-酪蛋白提供试验材料。选用4只健康新西兰大白兔,每2周免疫1次奶牛αs-酪蛋白,待血清达到抗体效价后,对兔进行颈动脉放血并分离血清,利用饱和硫酸铵法和蛋白A树脂纯化抗体,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和免疫印迹(Western-blot)法分别用于鉴定纯化后抗体的纯度和特异性。结果表明,经过饱和硫酸铵法和蛋白A树脂2步纯化后得到了纯度较高的抗体,同时可以特异性结合奶牛αs-酪蛋白。本试验制得的抗体可以用于鉴定奶牛乳腺合成的αs-酪蛋白,为进一步研究奶牛αs-酪蛋白合成的调控提供了有效的科研材料。     

ELISA检测卵黄和血清中禽呼肠孤病毒抗体相关性试验

本试验通过选取氯仿抽提卵黄抗体方法,经ELISA检测鸡卵黄和血清中禽呼肠弧病毒(REOV)抗体的相关性试验,确定卵黄抗体和血清抗体的对应关系,并依此建立ELISA试剂盒检测卵黄抗体的检测方法及判定标准.试验结果显示,采用REOV-ELISA检测试剂盒检测鸡血清及对应卵黄中REOV抗体二者具有良好的相关性,相对于血清ELISA检测法,所选样本206份中,检测结果相符的样本有200份,卵黄ELISA检测法的符合率为97.08％,表明可以采用检测血清的REOV-ELISA试剂盒检测卵黄中的REOV抗体水平.     

绵羊感染牛病毒性腹泻-粘膜病病毒的调查研究

应用双抗体夹心ELISA对长春、内蒙等地区的244只绵羊进行了牛病毒性腹泻-粘膜病病毒(BVD—MDV)感染调查.结果,BVD—MDV的阳性感染率为14.6％～83.3％.应用电镜对部分ELISA阳性和阴性样品进行观察,结果7份阳性样品中均见有BVD—MDV粒子,5份阴性样品均未观察到BVD—MDV粒子.定期采取11只自然感染BVD—MD病毒的绵羊粪便进行检查,结果自然感染BVD—MDV的绵羊,排毒持续时间至少为3个月.本试验表明,羊感染BVD—MDV可长时间排毒,是潜在的危险传染源.     

Anti-3AB antibodies in the Chinese yellow cattle infected by the O/Taiwan/99 foot-and-mouth disease virus.

The O/Taiwan/99 foot-and-mouth disease virus (FMDV), a South Asian topotype of serotype O, was introduced into Taiwan in 1999. The Chinese yellow cattle infected by the virus did not develop clinical lesions under experimental and field conditions. A blocking enzyme-linked immunosorbent assay (ELISA) kit with the 3AB antigen, a polypeptide of FMDV non-structural (NS) proteins, was used to evaluate the development and duration of anti-3AB antibodies, proving active viral replication, in the Chinese yellow cattle. The specificity of the assay was 99%, as was established with negative sera from regularly vaccinated and from na?ve cattle. The sensitivity tested with sera from naturally infected animals was approximately 64% and it was lower than that obtained by serum neutralization (SN) test. Under experimental infection, the Chinese yellow cattle developed lower anti-3AB antibodies than that developed in other species. Duration of anti-3AB antibodies was traced in two herds of naturally infected animals, indicating that anti-3AB antibodies persisted for approximately 6 months after outbreaks. On the basis of this study, we propose that the Chinese yellow cattle may have natural resistance, which limits viral replication and reduces the development of anti-3AB antibodies.     

Relationship between the anti-FMD virus antibody reaction as measured by different assays, and protection in vivo against challenge infection.

The antibody response of cattle after vaccination against foot-and-mouth disease (FMD) virus was monitored using the serum neutralization test (SNT), the sandwich ELISA, liquid-phase ELISA, sandwich competition ELISA, liquid-phase competition ELISA, and the liquid-phase sandwich competition (blocking) ELISA. The competition ELISAs (in particular the "blocking" ELISA) were the most effective at detecting reactivity in these cattle sera. However, 95% of negative sera also competed in the most sensitive ELISA (the "blocking" ELISA) to titres of 1:32 (4% of the sera competed to a titre of 1:128). Comparisons between the different ELISAs, and between these ELISAs and the SNT, demonstrated that the tests were not measuring exactly the same reaction of antibody with FMD virus. With respect to the capacity of animals to resist FMD virus challenge, neither the SNT nor the competition ELISAs were consistently able to identify such animals. The anti-FMD virus antibody titres obtained could be classified into three zones; the "white zone" wherein antibody titres were high and donor animals likely to be protected; the "black zone" wherein antibody titres were low and donor animals likely to be susceptible to infection; the "grey zone" wherein the antibody titres were intermediary and no interpretation could be made with respect to protection. Assays such as ELISA and SNT cannot and do not measure immunological protection; they are a measure of antibody responses and nothing more, and should be interpreted in terms of the "three zone" phenomenon.     

大额牛与黄牛本交的效果观察

[目的]通过大额牛与黄牛杂交的试验来为探索种间杂交的研究提供依据。[方法]用2头大额牛公牛与20头黄牛母牛圈舍混养分别进行本交试验。[结果]表明,大额牛能与黄牛进行本交使母牛受孕,受孕率达50%。[结论]不仅证实了大额牛与黄牛的种间杂交可行性,也为牛种间杂交改良提供强有力的科学依据。     

Culling decisions of dairy farmers during a 3-year Salmonella control study

Salmonella enterica subsp. enterica-serotypes lead to periodically increased morbidity and mortality in cattle herds. The bacteria can also lead to serious infections in humans. Consequently, Denmark has started a surveillance and control programme in 2002. The programme focuses on Salmonella Dublin which is the most prevalent and most persistent serotype in the Danish cattle population. A field study in 10 dairy herds with persistent Salmonella infections was carried out over three years to gain experience with control procedures including risk assessment, targeted control actions and test-and-cull procedures. From autumn 2003 until end of 2006 quarterly milk quality control samples from all lactating cows and biannual blood samples from all young stock above the age of three months were tested using an indirect antibody ELISA. The most recent and previous test results were used to categorise all animals into risk groups. These risk groups and all individual ELISA-results were communicated to the farmers as colour-coded lists four to six times per year. Farmers were advised to manage the risk of Salmonella transmission from cattle with repeatedly high ELISA results (flagged as "red") or cows with at least one recent moderately high ELISA result (flagged as "yellow") on the lists. Risk management included, e.g. culling or separation of the cows at calving. We analysed culling decisions using two models. For heifers a hierarchical multivariable logistic model with herd as random effect evaluated if animals with red and yellow flags had higher probability of being slaughtered or sold before first calving than animals without any risk flags. For adult cows a semi-parametric proportional hazard survival model was used to test the effect of number of red and yellow flags on hazards of culling at different time points and interactions with prevalence in the herd while accounting for parity, stage of lactation, milk yield, somatic cell count and the hierarchical structure of the data with animals clustered at herd level. This study illustrates how investigation of culling decisions made by herd managers when they have access to test-status of individual animals and overall apparent prevalence during control of an infection can lead to useful new knowledge. Overall herd managers were more likely to cull cattle with increasing number of yellow and red flags than animals with no flags. However, cattle were more likely to be culled with yellow and red flags during times with low or medium high within-herd seroprevalence than at times with high seroprevalence. These results are valuable knowledge for modelling and planning of control strategies and for making recommendations to farmers about control options.     

Challenges for bovine viral diarrhoea virus antibody detection in bulk milk by antibody enzyme-linked immunosorbent assays due to changes in milk production levels

Background

Bovine viral diarrhoea (BVD) is considered eradicated from Denmark. Currently, very few (if any) Danish cattle herds could be infected with BVD virus (BVDV). The Danish antibody blocking enzyme-linked immunosorbent assay (ELISA) has been successfully used during the Danish BVD eradication program, initiated in 1994. During the last decade, the cattle herd size has increased while the prevalence of BVDV has decreased. In this study, we investigated how these changes could affect the performance of the Danish blocking ELISA and of the SVANOVIR^®BVDV-Ab indirect ELISA. The latter has successfully been used to eradicate BVD in Sweden.Data (2003–2010) on changes in median herd size and milk production levels, occurrence of viremic animals and bulk milk surveillance were analysed. Additionally, the Danish blocking ELISA and the SVANOVIR ELISA were compared analyzing milk and serum samples. The prevalence of antibody positive milking cows that could be detected by each test was estimated, by diluting positive individual milk samples and making artificial milk pools.

Results

During the study period, the median herd size increased from 74 (2003) to 127 cows (2010), while the prevalence of BVDV infected herds decreased from 0.51 to 0.02 %. The daily milk yield contribution of a single seropositive cow to the entire daily bulk milk was reduced from 1.61 % in 2003 to 0.95 % in 2010 due to the increased herd size. It was observed that antibody levels in bulk milk decreased at national level. Moreover, we found that when testing bulk milk, the SVANOVIR^®BVDV-Ab can detect a lower prevalence of seropositive lactating cows, compared to the Danish blocking ELISA (0.78 % vs. 50 %). Values in the SVANOVIR^®BVDV-Ab better relate to low concentrations of antibody positive milk (R² = 94-98 %), than values in the blocking ELISA (R² = 23–75 %). For sera, the two ELISAs performed equally well.

Conclusions

The SVANOVIR ELISA is recommended for analysis of bulk milk samples in the current Danish situation, since infected dairy herds e.g. due to import of infected cattle can be detected shortly after BVDV introduction, when only few lactating cows have seroconverted. In sera, the two ELISAs can be used interchangeably.     

西藏日喀则市黄牛改良现状与对策

为提高西藏黄牛生产性能,使其成为当地的优良品种,利用优秀奶牛冻精使用胚胎移植的方法,或引进优质的种公牛对西藏黄牛进行改良.结果表明,改良后的黄牛经济效益显著,相当于两头以上当地母牛的产奶量,每头改良母牛增加收入1 755元,而且提前一年半投产.     

Evaluation of an antigen capture ELISA for the early diagnosis of Hypoderma lineatum in cattle under field conditions

An antigen capture or sandwich ELISA (sELISA) was evaluated for the diagnosis of Hypoderma lineatum in cattle under field conditions in northwestern Spain. The kinetics of circulating hypodermin C (HyC) and specific antibodies during the course of natural infestation were determined in a group of 10 Frisian calves. In addition, oesophagi and blood samples were taken from 105 cows at a slaughterhouse in order to compare three methods for the diagnosis of H. lineatum: sandwich ELISA for the detection of the antigen HyC (sELISA), indirect ELISA for the detection of antibodies anti-HyC (iELISA) and the detection of first instars (L1) in the oesophagus. In naturally infested cattle, HyC was present in circulation at low levels during the early and late phases of the infestation. However, in the middle phase, coinciding with the presence of L1 in the oesophagus, two peaks of increased HyC concentration were observed. Specific antibodies increased progressively until the first appearance of larvae in warbles on the back. There was no correlation between antigen or antibody levels and the number of grubs in the back. Prevalence of first instars in the oesophagi of slaughtered cows was 21.9% (23/105). The percentage of cattle that were positive for circulating antigen was slightly higher (24.8%), suggesting the recent destruction of migrating larvae in some animals. However, there was no correlation between the number of L1 and HyC levels. With the iELISA, 79% of the animals were positive to Hypoderma, which means that a high percentage of those animals have been exposed to the parasite but they had no apparent current infestation. The sELISA is a good tool to follow larval development within the host; however, the episodic elevation of HyC levels limits the usefulness of this test for the early diagnosis of Hypoderma under field conditions.     

Comparative evaluation of indirect and sandwich ELISA for the detection of antibodies to bovine respiratory syncytial virus (BRSV) in dairy cattle

Seroprevalence of bovine respiratory syncytial virus (BRSV) infection in both exotic and crossbred cattle were described. A baculovirus expressed recombinant purified nucleocapsid (N) protein was used in indirect and sandwich ELISA for screening of 499 bovine sera samples from all over the state for the presence of BRSV antibodies. The seroprevalence rate of BRSV was found to be 46.09% through indirect ELISA while it would found to be 65.33% by sandwich ELISA. The result also indicated that exotic breeds were more susceptible to BRSV infection compared to crossbred cattle. A comprehensive analysis on susceptibility to BRSV as regards to various factors like age and sex was also summarized.     

徐闻黄牛与海南黄牛的RAPD分析

本文用ＲＡＰＤ方法以荷斯坦牛作对照分析了徐闻黄牛与海南黄牛的关系．结果说明徐闻黄牛与海南黄牛相似性极大，ＲＡＰＤ方法检出的两个品种的ＤＮＡ多态性十分丰富。本文还讨论了ＲＡＰＤ方法在家畜品种分类与起源研究中应用的前景。