Establishment of Double-antigen Sandwich Time-resolved Fluorescence Immunoassay for Detection of Pest des Petits Ruminants Virus

[Objectives]This study was conducted to explore rapid and large-scale screening and detection of peste des petits ruminants(PPR),so as to provide important technical means for prevention,control and purification of PPR.[Methods]Soluble N protein and NH fusion protein were successfully obtained in an Escherichia coli expression system by optimizing E.coli codon and expression conditions.Furthermore,based on purified soluble N protein and NH fusion protein,a double-antigen sandwich time-resolved fluorescence immunoassay method for detection of peste des petits ruminants virus(PPRV)was established.[Results]The method has high sensitivity and specificity and can specifically detect the antibody against PPRV in sheep serum,and it has no cross reaction with other related diseases.The method was used to detect 292 clinical samples,and compared with French IDVET competition ELISA kit.The coincidence rates of positive samples and negative samples from the two kinds of test kits were 92.47%and 97.26%,respectively,and the overall coincidence rate was 94.86%.The intra-group and inter-group coefficients of variation in the repeatability test were less than 10%.[Conclusions]Compared with the traditional ELISA method,the double-antigen sandwich time-resolved fluorescence immunoassay for detection of PPRV has equivalent sensitivity and specificity,and simple and rapid operation,and thus high application and popularization value.

Monitoring and Tracking on Immune Antibody of Sheep Peste des Petits Ruminants

Peste ties petits ruminants is a kind of acute eontagious disease infecting goats anti sheep. In this study, antibtly monitoring and tracking of healthy goat and sheep immunized by peste des petits ruminants vaccine in Changping District of Beijing City were conducted, aiming at providing a reference for the devel- opment of effective immunization procedures.

Seroprevalence and Associated Risk Factors of Peste des Petits Ruminants among Sheep and Goats in Kassala State, Sudan

Peste des petits ruminant (PPR) is a contagious disease of small ruminants caused by a virus that belongs to the genus Morbillivirus of the family Paramyxoviridae. This study aimed to determine the seroprevalence of PPR disease in sheep and goats and its associated risk factors in Kassala State, Eastern Sudan. Across sectional study was conducted during the period from 30th August to 25th November 2015. The study was carried out using a structured questionnaire survey and a total of 918 blood samples were collected from apparently healthy unvaccinated sheep and goats in different localities in State of Kassala. A total of 546 sheep and 372 goats were tested for specific antibodies to nucleoprotein (NP) by competitive enzyme linked immunosorbent assay (cELISA). The apparent overall prevalence of PPR antibodies in Kassala was 58.2% while the true prevalence was calculated to be 61.3%. The apparent prevalence in sheep and goats was 68.1% and 43.5% respectively. Univariate analysis showed that the risk factors had significant associations with a cELISA positive status: locality, species, age, breed, husbandry system, housing mode, animals movement (p = 0.000) and animals sharing pasture and water (p = 0.003), while sex and newly introduced animals were not significant risk factors (p = 0.771) (p = 0.050) respectively. Factors found that significantly associated (p < 0.05) with increased odds of being cELISA positive in multivariate analysis were localities, species, age and newly introduced animals. The prevalence differed between localities and was the highest in the River Atbara (84.0%) locality, whereas it was lowest in Delta North (29.0%). No significant difference was observed among the sexes. However, the prevalence differed in different age groups and was 52.25% in animals of less than six months old;49.3% were between seven months and two years old and 65.5% were above two years old. In different husbandry systems, the prevalence was 47.9%, 73.0% and 49.2% in intensive, open grazing and pastoral systems respectively. Housing type effects were also observed;the highest prevalence was in animals housed in metal fence (83.3%). The movement pattern showed significant effect, where the prevalence was the highest (81.3%) in animals that move inter-states/inter-localities. It is concluded that the disease is endemic in Kassala State, high prevalent in sheep and goats, posing a threat to animal exportation, and may have a serious economic influence. Owners and herders should compulsorily vaccinate their animals yearly and animals should be investigated periodically for implementation of crucial eradication program.

Sero-Prevalence and Risk Factors of Diffusion of Peste des Petits Ruminants in Cameroon

The present study was carried out between April 2015 and January 2016 to estimate the sero-prevalence and identify the risk factors of the peste des petits ruminants (PPR) in Cameroon. A total of 269 herds randomly sampled across the country have been studied and 1622 samples of serum have been levied on the sheep and goat. The c-ELISA has been studied in order to detect the presence of antibodies in small ruminants like an indicator of exposition to PPRV. The results revealed the circulation of PPRV in the country with a total sero-prevalence of 39% [95%CI;37 - 41] and a sero-prevalence of 63.2% [95%CI;57.2 - 69.2] at the herd level. Sero-prevalence was variable in the ten regions ranging from 7% [95% CI;6.2 - 8.4] to 73% [95% CI;62 - 84] with the northern zone (Adamawa, North and Far-North) having 52.3% [95% CI;37 - 60] and southern zone (including the remaining seven regions) recording 29% [95% CI;11 - 57]. Similarly, it was higher in animals found in urban/peri-urban areas than in rural areas with prevalence ratio of 2.9 [95% CI 2.54 - 3.4;p < 0.001] i.e. 3 times more, 1.6 [95% CI 1.36 - 1.90;p < 0.001] i.e. 1.6 times more, and 5.02 [95% CI 3.91 - 6.85;p < 0.001] i.e. 5 times more at national level, in the northern zone and in the southern area, respectively. Five risk factors have been identified: the breeding environment, introduction of new animals into the herds, gathering of animals for pasture and watering, wandering and transhumance. The breeding area appeared to be the most important risk factor associated with disease exposure. The control measures for the eradication of this disease must take into account the epidemiological situation, the breeding environment, animal transhumance and breeding system.

Establishment of High-sensitivity Rapid Fluorescence Quantitative Detection Method for Antibody against Peste des Petits Ruminants Virus

[Objectives]This study was conducted to establish a rapid quantitative method for detecting antibody against Peste des Petits Ruminants Virus(PPR V)in sheep serum.[Methods]Soluble N protein and NH fusion protein were obtained in Escherichia coli prokaryotic expression system by optimizing codons and expression conditions of E.coli.Furthermore,based on the purified soluble N protein and NH fusion protein,a high-sensitivity fluorescence immunoassay kit for detecting the antibody against PPR V was established.[Results]The method could quickly and quantitatively detect PPR V antibody in sheep serum,with high sensitivity and specificity,without any cross reaction to other related sheep pathogens.The intra-batch and inter-batch coefficients of variation were less than 10%and 15%,respectively,and the method had good repeatability.Through detection on 292 clinical serum samples,it was compared with the French IDVET competitive ELISA kit,and the coincidence rate of the two methods reached 93.84%.Compared with the serum neutralization test,the detected titer value of the high-sensitivity rapid fluorescence quantitative detection method was basically consistent with the tilter value obtained by the neutralization test on the standard positive serum(provided by the WOAH Brucellosis Reference Laboratory of France).[Conclusions]This method can realize rapid quantitative detection of PPR V antibody on site,and has high practical value and popularization value.

Reverse Genetics for Peste des Petits Ruminants Virus: Current Status and Lessons to Learn from Other Non-segmented Negative-Sense RNA Viruses

Peste des petits ruminants(PPR) is a highly contagious transboundary animal disease with a severe socio-economic impact on the livestock industry, particularly in poor countries where it is endemic. Full understanding of PPR virus(PPRV)pathobiology and molecular biology is critical for effective control and eradication of the disease. To achieve these goals,establishment of stable reverse genetics systems for PPRV would play a key role. Unfortunately, this powerful technology remains less accessible and poorly documented for PPRV. In this review, we discussed the current status of PPRV reverse genetics as well as the recent innovations and advances in the reverse genetics of other non-segmented negative-sense RNA viruses that could be applicable to PPRV. These strategies may contribute to the improvement of existing techniques and/or the development of new reverse genetics systems for PPRV.

2021—2023年云南临沧市小反刍兽疫病原学和免疫抗体检测分析

[目的]为全面掌握临沧市小反刍兽疫免疫效果,科学评估其感染状况。[方法]随机采集2021—2023年来自规模场和散养户的羊血清4 524份、羊眼鼻棉拭子1 000份,采用阻断ELISA方法进行小反刍兽疫抗体检测,病毒核酸荧光RT-PCR进行病原学检测。[结果]小反刍兽疫免疫抗体合格率,2022年比2021年提高了7.44百分点,2023年比2022年提高了3.11百分点,病原学未检测出小反刍兽疫病毒核酸阳性样品。[结论]临沧市以“人病兽防、关口前移”各项防控措施为抓手,小反刍兽疫的免疫效果总体较好。

小反刍兽疫疫苗免疫效果监测

为监测小反刍兽疫-山羊痘二联活疫苗对小反刍兽疫的免疫效果,结合桐城市“先打后补”项目的实施,分别抽取3个规模养殖场免疫注射后7 d、35 d羊血清样品180份,用竞争ELISA法进行检测。结果表明,小反刍兽疫-山羊痘二联活疫苗在免疫注射后7 d产生免疫抗体;35 d抗体滴度高,抗体阳性率高达92.2%,符合国家规定的群体免疫抗体合格率在70%以上要求,免疫效果确实,可有效阻遏小反刍兽疫的发生与流行。

2021—2023年贵州省凯里市牛羊重要疫病免疫效果调查分析

[目的]为做好牛羊疫病防控专项工作,科学评估牛羊口蹄疫和小反刍兽疫疫苗免疫效果。[方法]用ELISA方法对2021—2023年采集的凯里市牛羊血清进行免疫抗体检测。[结果]2021—2023年,规模场牛群O型口蹄疫免疫平均抗体合格率87.69%~89.31%,散养户牛群平均合格率65.91%~67.20%。规模场牛群A型口蹄疫免疫平均抗体合格率71.15%~72.07%,散养户牛群平均合格率55.91%~57.60%。规模场羊群O型口蹄疫免疫平均抗体合格率82.94%~85.56%,散养户平均合格率70.53%~72.27%。羊A型口蹄疫免疫平均抗体合格率65.88%~67.22%,散养户平均合格率53.68%~55.91%。规模场羊群小反刍兽疫免疫抗体合格率80.00%~81.11%,散养羊群平均抗体合格率71.05%~73.18%。[结论]贵州省凯里市牛羊O型口蹄疫免疫效果较好,牛羊A型口蹄疫免疫效果相对较差,羊小反刍兽疫免疫效果很好,但牛羊O/A型口蹄疫免疫效果散养户比规模场差,规模场羊群小反刍兽疫免疫抗体合格率比散养户高。需加强规模场、散养户牛羊A型口蹄疫免疫和检测,同时加强散养户羊群小反刍兽疫免疫与效果评估,确保不发生重大动物疫情。

小反刍兽疫病毒的研究进展

小反刍兽疫(peste des petits ruminants,PPR)是由一种副黏病毒科麻疹病毒属病毒引起的主要感染山羊、绵羊的急性高治病性传染病,但牛感染后临床症状不明显。世界动物卫生组织(OIE)将其列为A类疫病,感染率和死亡率高达90%。由于绵羊和山羊是非洲和西亚贫困人民的重要生产资料,PPR对食品安全和摆脱贫困构成巨大威胁。小反刍兽疫病毒(PPRV)和牛瘟病毒(RPV)与麻疹病毒(MV)联系紧密。通过大规模接种疫苗牛瘟已被彻底消除。虽然可以利用弱毒疫苗免疫机体预防PPRV感染,但由于其在亚热带气候中的热不稳定性、使用时所需剂量的不确定性及接种范围的不足导致该病仍未得到有效控制。另外已有证据表明,在疫苗与不同毒株之间存在很少的交叉中和,使得目前流通的PPRV疫苗的保护功效被质疑。文章简要介绍了目前全世界对PPRV的高关注度及PPRV的生物学特性、发病机理等。

全球小反刍兽疫流行趋势

2013年以来,全球共43个国家报告发生小反刍兽疫(PPR)。其中,疫情仍然集中在亚洲和非洲。本文介绍了全球小反刍兽疫的发生情况,尤其是我国周边国家的疫情情况,分析了全球小反刍兽疫的扩散趋势。

小反刍兽疫病毒的分子生物学特性及其在全球的流行

小反刍兽疫由副粘病毒科麻疹病毒属小反刍兽疫病毒引起,经直接或间接接触传染,主要感染山羊、绵羊等小反刍动物,严重影响畜牧业,造成巨大经济损失.1942年首次发现于西非科特迪瓦,逐渐扩散至非洲及亚洲大部,涉及四十余国家.2007年7月首次传入中国西藏阿里地区,并于2008年6月再度爆发,且呈由边境逐渐向内传播的趋势.该地区有多种珍贵濒危野生反刍动物,是小反刍兽疫病毒的潜在宿主,它们随季节迁徙及夏季产仔的习性更增加其受小反刍兽疫感染并传播小反刍兽疫的可能.因此,对小反刍兽疫病毒的生物学特征、流行特点和防控措施进行综述,必将有利于正确评估小反刍兽疫对野生有蹄类动物的威胁并采取合适的措施预防与控制小反刍兽疫疫情.

小反刍兽疫活疫苗的安全性评价试验

2007年小反刍兽疫(PPR)在我国西藏首次暴发,在西藏和新疆部分地区使用PPR Nigeria 75/1疫苗株制造的疫苗进行免疫接种。为明确疫苗的安全性,中国兽医药品监察所国家牛瘟参考实验室对其安全性能进行了系统评价。健康易感山羊、绵羊及怀孕山羊、怀孕绵羊按不同剂量接种疫苗后,均未观察到异常临床反应;怀孕母羊所产羔羊数量与对照组无明显差异。疫苗对小白鼠、豚鼠的非特异性安全试验表明,所有接种动物均健活。结果表明该疫苗安全性良好,可在田间大规模使用。

小反刍兽疫免疫荧光诊断技术研究

为了建立小反刍兽疫(PPR)荧光抗体诊断方法,研究选用小反刍兽疫病毒(PPRV)Nigeria75/1减毒疫苗株制备抗原,免疫试验山羊,制备小反刍兽疫高免血清,用硫酸铵盐析法粗提免疫球蛋白(IgG),再应用提取的IgG制备异硫氰酸荧光素标记抗体。结果表明:所制备的荧光抗体对试验用Vero细胞组织抗原和犬瘟热病毒(CDV)抗原无反应性,对小反刍兽疫病毒感染细胞的检出敏感性为1×106稀释度。

小反刍兽疫诊断技术的研究进展

小反刍兽疫是由小反刍兽疫病毒引起的一种重大烈性传染病,主要发生于山羊和绵羊等小反刍兽。该病无特效治疗方法,主要通过早期诊断和疫苗免疫的方式控制。本文主要就小反刍兽疫诊断技术的研究进展作一综述。

小反刍兽疫临床诊断技术研究进展

小反刍兽疫是一种高传染、高致病、高死亡的疫病,被国家列为一类疫病,给养殖业带来巨大威胁。简单、快捷、廉价的小反刍兽疫诊断方法对控制甚至消灭小反刍兽疫至关重要。本文从临床角度对小反刍兽疫的临床症状、剖检变化、胶体金和分子生物学技术在小反刍兽疫临床诊断的发展情况进行论述,为基层畜牧工作者对小反刍兽疫的诊断提供参考。

小反刍兽疫流行状态及检测方法研究进展

小反刍兽疫(PPR)是由小反刍兽疫病毒(PPRV)引起小反刍动物的一种急性接触性传染病。近年来,我国西藏、辽宁等地陆续暴发该病,给当地养羊业带来了严重经济损失。本文介绍了PPR的流行现状、传播途径、易感动物,阐述了PPRV的实验室检测方法,包括常规诊断、核酸检测方法(c DNA探针杂交、RT-PCR方法、实时荧光定量PCR技术、反转录环介导等温扩增技术)、血清学诊断(竞争ELISA、间接ELISA、夹心ELISA方法)等,以期对该病的防控提供借鉴。

小反刍兽疫和山羊痘二联灭活疫苗的制备及其免疫原性

为了研制小反刍兽疫(Peste des petits ruminants,PPR)和山羊痘(Goat pox,GTP)二联油剂灭活疫苗,以Vero细胞增殖小反刍兽疫病毒Nigeria75/1 Clone 9株和山羊痘病毒CVCC AV41株,通过细胞病变观察、间接免疫荧光试验和荧光定量PCR确定接种方式、接毒剂量和收毒时间对病毒增殖的影响。采用细胞病变观察、SDS-PAGE和Western-blot比较甲醛、二乙烯亚胺和β-丙内酯灭活病毒的效果。随后在灭活的病毒悬液中加入等体积的油佐剂进行乳化,制备PPRV-GTPV二联油剂灭活疫苗,并参照《中国兽药典》检测疫苗理化性状。为评价疫苗的免疫原性,将24只2月龄临床健康的雷州山羊随机分为3组(每组8只),分别肌肉注射生理盐水、PPRV-GTPV二联灭活疫苗、商品化PPRV-GTPV弱毒疫苗。免疫后14 d和28 d分别检测试验山羊血清PPRV和GTPV中和抗体。结果表明,PPRV和GTPV以100 TCID50/mL的剂量共感染Vero细胞84 h后的病毒滴度较高。用终浓度为0.025%的β-丙内酯4℃处理24 h,能有效灭活PPRV和GTPV;制备的灭活疫苗符合《中国兽药典》相关要求。PPRV和GTPV二联灭活疫苗免疫14 d后,山羊血清PPRV和GTPV中和抗体水平与商品化弱毒疫苗相仿;免疫28 d后,山羊血清PPRV中和抗体显著高于商品化弱毒疫苗,GTPV抗体呈上升趋势,但显著低于商品化弱毒疫苗。试验山羊均未见明显不良反应。综上说明,PPRV-GTPV二联灭活疫苗安全、有效,有望在小反刍兽疫和山羊痘的防制中发挥重要作用。

规模羊场小反刍兽疫弱毒活疫苗免疫效果的监测评估

为评估规模化羊场小反刍兽疫弱毒活疫苗免疫效果,对开阳县2个绵羊养殖场、1个山羊养殖场的羊群进行小反刍兽疫活疫苗免疫接种,采用C-ELISA方法对3个免疫羊群免疫抗体消减进行监测。结果显示,免疫后羊群无不良反应和异常情况发生,1次免疫后30 d,3个养殖场免疫抗体平均阳性率为95.11%,免疫120 d后抗体阳性率为98.22%,免疫270 d后抗体阳性率为99.56%。结果表明,小反刍兽疫活疫苗免疫安全,且能诱导较强的体液免疫应答,免疫抗体持续时间长;弱毒活疫苗免疫是预防小反刍兽疫的一种可行方法。

辽宁省一起小反刍兽疫疫情的紧急流行病学调查

2020年5月15日,辽宁省抚顺市清原县动物疫病预防控制中心报告,该县某养羊户发生山羊急性发病,且死亡率较高;5月16日,经市级专家组现场临床诊断和省级实验室复核,诊断为疑似小反刍兽疫疫情,经国家小反刍兽疫参考实验室检测,确诊为小反刍兽疫。5月18日,农业农村部公布了该起疫情。为追溯疫情可能来源,分析疫情扩散风险,科学指导疫情处置工作,开展了紧急流行病学调查。现场调查结果显示,该起疫情的袭击率为72.7%(136/187),病死率为55.9%(76/136)。溯源调查表明,疫情由羊贩运人及其运输车辆带毒传入和外购未经检疫羔羊带毒传入的风险较大,新生或外购羔羊未及时免疫小反刍兽疫疫苗是内在风险因素;追踪调查结果显示,疫情局限于该养羊户,向外扩散的风险较低。本起疫情提示,应做好养殖场区内的生物安全综合防控,重点加强相关人员及运输车辆、工具的移动控制,及时做好羔羊的疫苗免疫,降低疫情发生风险。