牛流行热研究进展

牛流行热,又称为三日热,是由虫媒弹状病毒牛流行热病毒(BEFV)引起的牛和水牛的一种传染病.该病会导致牛发热、四肢僵硬、跛行,通常又会完全恢复.其会引起感染动物的死亡,母牛流产,致使奶牛产奶量下降、公牛体重和生殖能力下降.作者结合国内外对牛流行热病毒的研究,从发病历史、流行病学、病原学、临床症状、致病机理、诊断、防控等方面阐述了牛流行热的研究进展,旨在为该病的深入研究提供参考.     

Prokaryotic Expression and Antigenic Characterization Identification of G Gene for Bovine Ephemeral Fever Virus

In order to explore specific candidate gene of antigens and diagnostics development for the bovine ephemeral fever virus (BEFV), the target gene sequences were derived from G gene by PCR amplification using two specific primers.The PCR products were digested by Xho Ⅰ and Nde Ⅰ and cloned into pET-30 vector, and the recombinant plasmids (480-pET-30, 1107-pET-30) were transformed into E.coil BL21 (DE3) cells, the D_{600 nm} of positive strains were 0.6 to 1.0.The recombinant strains were induced by IPTG (1.0 mmol/L) at 37 ℃.The characteristics of the target proteins were analyzed using SDS-PAGE and the immunogenicity was analyzed through Western blotting.At the same time, the target proteins were used as coating antigens to do ELISA and all rabbits were inoculated with recombinant proteins.The results showed that the expression feature of 1 107 bp gene fragment of G gene for the first time was segmented in vitro as well as has nicer biological activity and specificity, and it more be suitable as a candidate gene for molecular vaccine and diagnostics development.This study provided the theoretical foundation of the establishment of diagnostics technique and vaccine for BEFV.     

牛流行热病毒糖蛋白基因的原核表达与抗原性鉴定

为探索可用来开发牛流行热病毒(bovine ephemeral fever virus,BEFV)疫苗和诊断试剂的候选基因,本研究针对BEFV糖蛋白(G)基因设计了2对特异性引物,用PCR方法扩增基因片段,PCR产物经Xho Ⅰ和Nde Ⅰ双酶切后亚克隆到表达载体pET-30上,将鉴定正确的重组质粒(480-pET-30、1107-pET-30)转化大肠杆菌BL21(DE3)感受态细胞,培养阳性菌株D_{600 nm}值为0.6~1.0,37 ℃下用1.0 mmol/L IPTG诱导表达目的蛋白,将其纯化之后进行SDS-PAGE和Western blotting免疫原性分析。同时,应用间接ELISA、动物免疫试验及交叉反应试验对目的蛋白进行分析。结果表明,首次发现的1 107 bp基因片段是分段表达的,具有很好的生物活性和特异性,更适合作为开发疫苗和诊断试剂的候选基因,为今后建立BEFV的血清学诊断方法及疫苗研发提供了理论基础。     

An epizootic of bovine ephemeral fever in New South Wales in 2008 associated with long-distance dispersal of vectors

Objective To report the rapid transmission of bovine ephemeral fever ( BEF) virus from north-western New South Wales south to the Victorian border in January 2008 and to present data that suggests an uncommon meteorological event caused this rapid southward dispersal of vectors. Procedure The locations of reported clinical cases, data from sentinel herds and results from a survey of cattle in the southern affected area were examined to delineate the distribution of virus transmission. Synoptic weather charts for January 2008 were examined for meteorological conditions that may have favoured movement of vectors in a southerly direction. Results Cases of BEF and exposure to BEF virus in NSW were confirmed west of the Great Dividing Range, extending from the Queensland border to Finley, on the far North Coast and around the Hunter Valley. A low-pressure system moved south across the state on 18–19 January 2008, preceding the first cases of BEF in the south of NSW by 1–2 days. Conclusion Heavy rainfall in December 2007 provided a suitable environment for vector breeding, resulting in the initiation of and support for continuing BEF virus transmission in north-western NSW. The movement of a low-pressure system south across central western NSW in mid-January 2008 after the commencement of BEF virus transmission in the north-west of the state provided a vehicle for rapid southward movement of infected vectors.     

表达牛流行热病毒G基因的牛传染性鼻气管炎病毒转移载体的构建

本试验将克隆到 p BLG中的牛流行热病毒 (BEFV)糖蛋白 G基因亚克隆到含有 CMV启动子和 Poly(A)信号尾的 p CR3- Uni载体上 ,获得 p CR3- G重组体 ,酶切后将含有 CMV、G基因和 Poly(A)的目的片段 (约 3.6 kb)插入通用转移载体 pd TK- CMB中 ,获得 pd TK- CMB- G重组体 ;再将含 SV4 0启动子的 L ac Z报告基因也亚克隆到该载体中获得表达 BEFV G基因的pd TK- L ac Z- G转移载体 ,为开发 BEFV/IBRV二联基因工程疫苗奠定了基础     

牛流行热病毒JB76H株G蛋白基因核苷酸序列分析

通过反转录－聚合酶链反应（ＲＴ－ＰＣＲ）,从中国北京分离株牛流行热病毒ＪＢ７６Ｈ基因组ＲＢＡ中扩增出主要保护性抗原糖蛋白Ｇ的cＤＮＡ。采用Ｓanger’s双脱氧末端终 止法测定cＤＮＡ片段的核苷酸序列,并推导出氨基酸序列。Ｇ基因全长为１８７２个碱基,单一的开放阅读框架阅读框架编码６２３个氨基酸的多肽。将测得的序列与澳大利亚六个分离株进行比较,发现我国分离株与渊大利亚分离株间同源性为９１％,低于澳大利亚各分     

某牛场奶牛A型多杀性巴氏杆菌的分离鉴定

湖北某奶牛场牛群爆发体温升高、呼吸困难、流涎及颈胸皮下气肿等症状的疾病,部分发病牛衰竭死亡,为确诊牛场牛群发病原因并提出防控方案,本试验采集死亡牛的心脏、肝脏、肺脏及气管组织进行病原菌的分离纯化及PCR鉴定,并对鉴定的病原菌进行生化特性鉴定、致病性试验和药物敏感性分析;同时提取患牛血清RNA,开展牛流行热病毒的RT-PCR鉴定。结果显示,病料在类胸膜肺炎固体培养基上不生长,生化特性鉴定显示能形成靛基质、不发酵肝糖和肌醇;牛流行热病毒RT-PCR扩增阴性;所分离的病原菌经16S rRNA及牛A型多杀性巴氏杆菌特异性引物PCR扩增阳性;致病性试验显示该病原可致死小鼠,且能从死亡小鼠体内分离到感染菌;药敏试验结果显示该病菌对头孢哌酮、左氧氟沙星敏感,其他20种临床常见药物表现耐药。综上所述,该牛场患病牛确诊为牛A型多杀性巴氏杆菌感染,建议根据药敏试验结果选择敏感药物,对患病牛进行隔离治疗,疑似患病牛隔离观察,同时加强通风,及时清理污物并消毒,改善饲养管理,避免拥挤、寒冷及长途运输等应激因素。     

Diagnosis and Treatment of Serotype A Pasteurella multocida Infection in a Dairy Farm

The assay was aimed to clarify the reasons why dairy cattle became ill with the symptoms including fever,breathing difficulty,salivation and subcutaneous emphysema at neck or chest in a dairy cow farm at Hubei province,even part of dairy cows were dead for exhaustion.The disease was diagnosed by laboratory methods,and treatment scheme was proposed.The samples from heart,liver,lung or trachea tissues were collected,then M.bovis and pathogenic bacteria were isolated,respectively.Biochemical characteristics,pathogenicity test and drug sensitivity analysis were performed on the isolated pathogenic bacteria.RNA was extracted from bovine serum,then bovine ephemeral fever virus was identified by RT-PCR.The results showed that nothing grew in PPLO medium,and the isolated bacteria could form indole,but did not ferment glycogen and inositol in the biochemical tests.The isolated pathogenic bacteria was conformed as serotype A P.multocida by PCR while bovine ephemeral fever virus of RT-PCR result was negative.The pathogen could kill mice and the pathogenic bacteria could be isolated from heart blood of the dead mice in pathogenicity test.The isolated bacteria was sensitive to cefoperazone or levofloxacin in drug sensitivity analysis.In conclusion,the disease was diagnosed as bovine serotype A P.multocida infection.Some treatments including choosing sensitive antibiotic drugs based on the drug sensitivity tests,holding sick cattle under quarantine treatment and isolating suspected cattle should be taken.Moreover,some complex measures such as keeping the air flowing,cleaning up in time,improving the management,and preventing stress from congestion,cold and long distance transport were very important to prevent and control serotype A P.multocida infection in dairy cows.     

徐州地区牛流行热病毒的分离与鉴定

1991年夏秋之交徐州市各奶牛场暴发牛流行热。为确诊该病,采集了发病高热期抗凝血,进行了病毒分离和鉴定试验。病料经处理后,直接接种于BHK_21细胞,传至第二代可见典型的CPE。电镜下观察到80—150nm弹状病毒颗粒。分离毒的理化特性、核酸类型及中和试验结果均与标准BEFV相一致,证实该分离病毒为牛流行热病毒。     

Complete genome sequencing and assessment of mutation-associated protein dynamics of the first Indian bovine ephemeral fever virus (BEFV) isolate

BackgroundBovine ephemeral fever (BEF) is a re-emerging disease caused by bovine ephemeral fever virus (BEFV). Although it poses a huge economic threat to the livestock sector, complete viral genome information from any South Asian country, including India, lacks.AimGenome characterization of the first Indian BEFV isolate and to evaluate its genetic diversity by characterizing genomic mutations and their associated protein dynamics.Materials and MethodsOf the nineteen positive blood samples collected from BEF symptomatic animals during the 2018-19 outbreaks in India, one random sample was used to amplify the entire viral genome by RT-PCR. Utilizing Sanger sequencing and NGS technology, a complete genome was determined. Genome characterization, genetic diversity and phylogenetic analyses were explored by comparing the results with available global isolates. Additionally, unique genomic mutations within the Indian isolate were investigated, followed by in-silico assessment of non-synonymous (NS) mutations impacts on corresponding proteins’ secondary structure, solvent accessibility and dynamics.ResultsThe complete genome of Indian BEFV has 14,903 nucleotides with 33% GC with considerable genetic diversity. Its sequence comparison and phylogenetic analysis revealed a close relatedness to the Middle Eastern lineage. Genome-wide scanning elucidated 30 unique mutations, including 10 NS mutations in the P, L and G_NS proteins. The mutational impact evaluation confirmed alterations in protein structure and dynamics, with minimal effect on solvent accessibility. Additionally, alteration in the interatomic interactions was compared against the wild type.ConclusionThese findings extend our understanding of the BEFV epidemiological and pathogenic potential, aiding in developing better therapeutic and preventive interventions.     

Development and application of G1-ELISA for detection of antibodies against bovine ephemeral fever virus

The gene encoding antigenic site G₁ of bovine ephemeral fever virus (BEFV) was highly expressed in the host cell Escherichia coli. An indirect G₁-ELISA with the recombinant protein as the coating antigen was established to detect antibodies against BEFV. The result revealed that the optimal concentration of the coated antigen was 0.5 μg/well and the dilution of serum was 1:20. It was optimal that sera with P/N value  2.2 were considered positive, P/N value  2.0 negative, and between 2.0 and 2.2 ambiguous. The G₁-ELISA method gave a sensitivity of 97.6% and a specificity of 98.6% by testing 590 field serum samples. These results suggest that the G₁-ELISA may be a good alternative tool for seroepidemiological surveys.     

牛病毒性腹泻病毒在猪瘟疫苗中的污染情况及其检测方法的研究进展

牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV)和猪瘟病毒(classical swine fever virus,CSFV)同属黄病毒科瘟病毒属,猪瘟疫苗中污染BVDV可引起免疫失败。但由于两者在病毒粒子结构、基因组结构和抗原特性等方面均很接近,在血清学上存在交叉反应,因此难以检测猪瘟疫苗中污染的BVDV。文章对BVDV在猪瘟疫苗中的污染情况和检测方法进行了论述,旨在为猪瘟疫苗污染BVDV的检测提供理论基础。     

牛流行热病毒灭活疫苗免疫后牛外周血淋巴细胞亚类分布的研究

对牛流行热病毒灭活疫苗免疫并攻每后，牛外周血淋巴细胞亚类的变化进行了研究。结果表明，灭活苗免疫后，牛的CD4^ 显著升高，可能与参与辅助B淋巴细胞合成抗体有关，攻毒后，γδT细胞均显著上升，免疫组在攻毒后3周仍保持在相当的高水平，IL－2Rα阳性细胞在攻毒后高热期也有显著升高，而CD8^ 细胞没有明显变化。     

奶牛流行热的诊断及中西医结合疗法

奶牛流行热是奶牛的一种急性热性传染病,奶牛感染此病后,发病率高,给养殖户带来的损失严重。对一例奶牛流行热的诊疗新技术的研究供广大临床工作者及研究人员参考。     

Pestivirus infections in Norway. Serological investigations in cattle, sheep and pigs.

Serum samples from 1,133 dairy cows (187 herds), 3,712 ewes (103 flocks) and 1,317 adult pigs (877 herds), were tested for neutralizing antibodies against the NADL strain of bovine virus diarrhoea virus. The prevalence rate of seropositive animals was 18.5% in cattle, 4.5% in sheep and 2.2% in pigs, such seroreactors being found in 28% of the cattle herds and 18% of the sheep flocks. In all three species the rate showed considerable herd and geographical variation. In cattle the seroreactor rate was similar in herds with normal reproduction and in 62 herds with problems of repeat breeding. Of 31 pig sera containing antibodies against the NADL strain, 27 were also positive in a neutralization test for antibodies against swine fever virus (Baker strain). However, all sera showed a higher titre of antibodies against the bovine strain than against the swine fever virus. It was concluded that the immune response of the pigs had been induced by ruminant pestivirus, and not by swine fever virus.     

应用套式RT—PCR检测猪瘟制品中牛病毒性腹泻病毒污染的研究

为建立猪瘟制品中牛病毒性腹泻病毒（BVDV）污染的诊断方法,本研究根据GenBank公布的BVDV全基因组序列,选择BVDV保守性比较高的5′非编码区,利用Olig06．0软件设计了2对特异性引物,建立了检测BVDV的套式RT-PCR方法。该方法极大提高了常规RT-PCR检测方法的特异性和敏感性,重复性好、特异性强、敏感性高,可以准确快速检测出极低含量的BVDV,为动物与疫苗生物制品原辅料中BVDV的快速低含量检测提供了一种简单、特异、敏感、快速的检测方法。     

牛流行热病毒感染白纹伊蚊细胞免疫调控相关基因的差异表达分析

为了阐明牛流行热病毒(bovine ephemeral fever virus,BEFV)在媒介蚊形成持续感染过程中免疫相关基因的调控作用,利用白纹伊蚊幼蚊细胞C6/36从牛体传代抗凝血分离BEFV JT02L株,用geNorm和NormFinder软件分析感染细胞看家基因的mRNA表达水平稳定性,筛选获得最稳定表达的...