Prevalence of Bovine Viral Diarrhoea Virus antibodies among the industrial dairy cattle herds in suburb of Mashhad-Iran

Mashhad is a major dairy production in Iran. The subject of this study was to survey the seroprevalence of Bovine Viral Diarrhea Virus (BVDV) infection using an indirect Enzyme-linked immunosorbent assay (ELISA) test in industrial dairy cattle herds in suburb of Mashhad-Iran. Totally, 141 serum samples were tested. None of the herds had been vaccinated against BVDV. Commercial indirect ELISA kit was used. The herds divided to 3 sizes as cow population. They were included: small, medium and large herds. Data were analyzed using Chi-square test. Ninety-seven (68.79%) cows were ELISA seropositive. However, the true BVDV seroprevalence was 72.25%. All of the herds were antibody positive against BVDV. The prevalence ranged from 66 to 100% within the herds. There were no significant differences between the presence of antibodies to BVDV and the herd size (P > 0.05). The prevalence in animals lower than 2 years old differed significantly with cows higher than 2 years old (P < 0.05). According to the results, it is concluded that it is likely the presence of persistently infection (PI) animal(s) within the herds in suburb of Mashhad-Iran, which is responsible for the presence antibody.     

A Universal `One-tube' RT-PCR Protocol for Amplifying Isolates of Bovine Viral Diarrhoea Virus

The increase in the knowledge of the genetic variability of BVDV and the identification of some of the genetic determinants of its pathogenicity require robust and practical tools for rapid molecular characterization of the various genotypes of this virus. This study was undertaken to develop a standard protocol for RT-PCR that allows the amplification of various parts of the genome of BVDV without the need for optimizing each individual reaction. The reaction set-up is very flexible because it consists of two pre-mixes. These are a master mix, with all the required reagents except the desired primers, which are the components of the second pre-mix and are therefore easily interchangeable between the different reactions. After adding any primer-containing pre-mix to the fixed master mix, a non-interrupted cycling protocol led to the generation of amplicons of up to 4 kbp in size in amounts sufficient for subsequent sequencing reactions. The method was applied to five different regions of the BVDV genome: (i) the well-known 5-UTR to differentiate genotypes I and II; (ii) the entire E2 gene, or an approximately 550 bp region within the E2 gene, in order to find the molecular equivalent of antigenic varieties; (iii) the entire structural protein coding region covering the Npro, capsid, E ^RNS, E1 and E2 genes; (iv) a 2.1 kbp region embracing the NS2/3 junction which is known to be cleaved in cytopathic biotypes of BVDV; and (v) the region covering the entire NS4B and NS5A/B genes. All six RT-PCRs were successfully applied using (i) primers with lengths of between 20 and 52 nucleotides, (ii) an aliquot of RNA extracted from either 10⁶ infected bovine embryonal lung cells or the same number of leukocytes from viraemic cattle, and (iii) all the genotype I and II strains of BVDV tested. The technique described was used to generate various Sindbis virus/BVDV recombinants. The correct processing of the amplicon-derived E2 glycoprotein of BVDV strain PT810 was demonstrated by its reaction with a monoclonal antibody in an immunofluorescence assay. Given the variety of RT-PCRs tested, we conclude that this universal protocol may be useful with other RNA viruses.     

北京地区牛病毒性腹泻病毒流行传播的原因分析

在对北京地区20个奶牛群进行抗体水平检测和全群抗原筛查的基础上,将牛群中牛病毒性腹泻病毒流行状况同牛群在3年内是否引入过动物进行相关性比较。结果显示,20个牛群中,引入过动物的8个牛群中后备牛群血清抗体平均水平为80.0%,抗原筛查的总体阳性率为0.61%;未引入过动物的12个牛群中后备牛血清抗体平均水平为65.5%,抗原筛查的总体阳性率为0.15%,两组数据之间差异极显著,表明牛群在3年内是否引进过动物对牛群持续感染牛的分布影响极显著,动物引进可能是造成BVDV在北京地区流行传播的主要原因之一。     

Validation of a bulk tank milk antibody ELISA to detect dairy herds likely infected with bovine viral diarrhoea virus in New Zealand

AIMS: To assess the sensitivity and specificity of a bulk tank milk (BTM) antibody enzyme-linked immunosorbent assay (ELISA) to detect likely infection of a dairy herd with bovine vi- ral diarrhoea virus (BVDV). The ELISA was subsequently used to estimate the prevalence of likely infected herds in parts of the North Island of New Zealand.

METHODS: BTM samples from 724 randomly selected dairy herds in the Waikato, Bay of Plenty and Northland regions of New Zealand were tested for BVDV antibodies. From this group, 20 herds were again randomly selected from each of the quartiles of the ELISA percentage inhibition (%INH) result. From each participant herd, serum from 15 randomly selected calves aged 6–18 months and 15 cows was collected and tested using an indirect blocking ELISA for BVDV antibodies.

RESULTS: Among serum results from calves from 50 herds available for analysis, 34 (68%) herds were classified as likely non-infected (0-3 seropositive among 15 calves) and 16 (32%) as likely infected (5–15 seropositive among 15 calves). Receiver- operator characteristic (ROC) analysis identified an optimal cut-off for BTM of 80%INH associated with 81% sensitivity and 91% specificity for likely herd infection. The prevalence of BVDV antibodies in cows within herds and %INH for BVDV in bulk milk were positively correlated (p<0.01). The association between bulk milk %INH and the prevalence of BVDV antibodies in calves was stronger than the same association in cows. Based on the threshold of 80%INH, the 95% confidence interval (CI) for prevalence of likely infection in the 724 herds in the Waikato, Bay of Plenty and Northland regions of New Zealand was 12–17%. Vaccination against BVDV was not significantly associated with the likely infection status of the herd based on prevalence of BVDV antibodies among calves.

CONCLUSION: An ELISA test result for BVDV antibodies in BTM ≥80%INH can be used as a threshold to indicate the presence of likely infection with BVDV in dairy herds in New Zealand, with 81% sensitivity and 91% specificity.     

Monitoring and identification of residues of anabolic preparations in slaughtered cattle by HPLC with diode array detection

Summary

The new combination of isocratic high performance liquid chromatography (HPLC) with on line UV spectrum detection via a diode array configuration has been applied to the detection and identification of anabolics present in application sites of cattle.

Combination of the characteristic retention time in the HPLC chromatogram and a comparison of the full spectrum between 190–400 nm of the anabolic components with that of a standard resulted in a very reliable identification. By means of this method 117 samples of application sites were investigated for the presence of anabolic residues. Of the xenobiotic anabolics, 19‐nortestosterone (NT) was found most frequently (in 96 cases), whereas diethylstilbestrol (DES) was found in only 11 cases.

In all samples the identification of NT and DES was confirmed by high resolution gas chromatography‐mass spectrometry (GCMS).     

分泌抗BVDV McAb杂交瘤细胞株的建立

用牛病毒性腹泻病毒（ＢＶＤＶ）感染ＭＤＢＫ细胞,将感染细胞培养物冻融后离心取上汪经ＰＥＧ沉淀浓缩抗原免疫ＢＡＬＢ／Ｃ小鼠,取血清抗体效价高遥免疫鼠的脾细胞与ＳＰ２／Ｏ细胞在ＰＥＧ作用下融合,产生杂交瘤细胞,用间接ＥＬＩＳＡ法检测杂交瘤细胞生长孔上清,筛选阳性杂交瘤细胞株。以有限稀法克隆２－３次,得到８ 泌抗ＢＶＤＶ的单克隆抗体（ＭｃＡｂ）杂交瘤细胞株。８株ＭｃＡｂ对ＢＶＤＶ,猪瘟均呈阳性反应。杂交     

新疆某规模化牧场中引发犊牛腹泻的病因调查

多种病毒、细菌、寄生虫均可导致犊牛发生腹泻。为了查明新疆某规模化牧场发生犊牛腹泻的原因,2020年12月采集该牧场48 份犊牛粪便及血液,通过血清学结合实验室方法进行检测。牛病毒性腹泻病毒(BVDV)抗原检测结果阳性率为8.33%(4/48);犊牛腹泻4联抗原快速检测结果显示,大肠杆菌阳性率为43.75%(21/48);BVDV病原逆转录-聚合酶链反应(RT-PCR)检测阳性率为29.17%(14/48)。结果显示,该规模化牧场的犊牛腹泻主要由BVDV和大肠杆菌引起,本试验为该牧场的后续治疗和防控提供了科学依据。     

牛病毒性腹泻病毒E2结构蛋白的原核表达、纯化及鉴定

根据GenBank中已发表的牛病毒性腹泻病毒囊膜糖蛋白E2基因设计1对特异性引物,以Oregon CV24株为模板,PCR扩增目的片断后与pET-32a原核表达载体连接,转化大肠杆菌Transetta（DE3）感受态细胞,用IPTG诱导表达,SDS-PAGE电泳分析表达产物,结果表明重组E2蛋白在体外得到较好表达,表达的融合蛋白分子量为57.7 ku。可溶性分析表明重组E2蛋白以包涵体的形式存在,经镍柱His标签蛋白纯化后进行western-blot检测。结果表明：E2重组蛋白均具有反应原性,能与BVDV的阳性血清进行反应。