Brucellosis, a zoonotic disease caused by Brucella, requires rapid, accurate, and sensitive diagnostic methods for effective prevention and control. This study presents the development of a fluorescence microsphere immunochromatographic assay (QDMs-ICA) for detecting anti-Brucella antibodies in bovine serum and milk. Lipopolysaccharide (LPS) from the Brucella abortus strain A19 was immobilized on the nitrocellulose membrane (NC membrane) as the test line (T-line), while rabbit anti-SPG polyclonal antibody was applied as the control line (C-line). Recombinant streptococcal protein G conjugated with quantum dot microspheres (QDMs-SPG) served as the detection conjugate. After optimizing the preparation parameters of QDMs-ICA, the method demonstrated sensitivities of approximately 0.98 IU/mL for bovine serum and 1.56 IU/mL for milk. No cross-reactions were observed with antibody-positive sera from Coxiella burnetii, Mycobacterium avium paratuberculosis, Mycobacterium tuberculosis, Chlamydia abortus, Bacillus anthracis, Escherichia coli O157:H7, Vibrio cholerae or Salmonella, indicating excellent specificity. In intra- and inter-batch repeatability tests, the coefficient of variation (CV) remained below 15%, confirming good reproducibility. The detection limit remained stable after storage at 37 °C for 7 days. Parallel testing of 150 bovine serum samples and 80 milk samples showed a high degree of concordance with the ID-VET commercial kit, with coincidence rates of 97.3% and 96.3%, respectively. These results demonstrate that QDMs-ICA offers high specificity, sensitivity, repeatability, and reliability, making it an effective tool for the rapid detection and epidemiological monitoring of brucellosis.

Brucella melitensis is a significant zoonotic pathogen transmissible via contact, digestive, and respiratory routes, posing severe threats to livestock economies and human health. While its virulence genes critically regulate intracellular survival and replication, the molecular mechanisms underlying pathogenesis remain elusive. This study employed Transposon Directed Insertion-site Sequencing (TraDIS) technology to identify key genes essential for the survival of Brucella melitensis strain 16 M within RAW 264.7 macrophages, resulting in the discovery of 374 anti-phagocytic associated essential genes. Through GO and KEGG analyses, these genes were categorized into eight functional classes, representative genes from each category were selected to generate respective gene-deletion mutants, with bacterial virulence and phenotypes validated via intracellular survival assays, in vitro stress tests, carbon source utilization experiments, and RT-qPCR. All ten constructed mutants exhibited reduced macrophage survival capabilities. Deletion of membrane-related genes cydDC and BME_RS07715 attenuated stress resistance, membrane stability, and immune evasion, knockout of metabolic gene ptsP impaired carbon source utilization and intracellular proliferation, and disruption of regulatory gene BME_RS00125 diminished TCA cycle activity and fatty acid metabolism, compromising energy supply and structural integrity. In this study, some key genes of Brucella surviving in macrophages were verified, and preliminarily elucidated the function of these genes in regulating bacterial virulence, providing a theoretical basis for exploring their mechanism.

Brucellosis remains a major global public health concern, with B. melitensis and B. abortus being the primary causative agents in China. This study describes the development of a multiplex droplet digital PCR (ddPCR) assay for the simultaneous detection and differentiation of B. melitensis and B. abortus. The assay employs TaqMan probes targeting the bcsp31 gene (genus-specific), a transposase gene (B. melitensis-specific) and an autotransporter-associated beta strand repeat-containing protein gene (B. abortus-specific), each labeled with distinct fluorophores (FAM, HEX, ROX). The optimized assay exhibited no cross-reactivity with other pathogens and exhibited significantly higher sensitivity than both qPCR and conventional PCR, with detection limits as low as 2.54-3.11 copies/reaction. Repeatability was excellent, with intra- and inter-assay coefficients of variation below 9%. When validated on a panel of clinical nucleic acid samples, the ddPCR assay showed strong agreement with qPCR (kappa = 0.85), with a sensitivity of 100% (79.42%~100%, 95%CI) and specificity of 95.96% (92.08%~99.84%, 95%CI). These findings establish the multiplex ddPCR as a rapid, sensitive, and highly specific diagnostic platform that improves brucellosis detection accuracy and supports targeted control strategies.

IntroductionBrucellosis, an infectious zoonotic disease caused by members of the genus Brucella, results in chronic multi-organ injury. Improving the specificity and sensitivity of serological methods for diagnosing brucellosis necessitates the development of novel diagnostic antigens. The twin-arginine translocation (Tat) pathway is responsible for transporting folded proteins across the cytoplasmic membrane and has been implicated in the virulence of Brucella. Three Tat substrate proteins—L,D-transpeptidase ErfK (A0577), linear amide C-N hydrolase YxeI (A1479), and thioesterase domain-containing protein EntF (B0249)—contribute significantly to Brucella virulence. However, the roles of these Tat substrate proteins in diagnosing brucellosis remain unclear.MethodsIn this study, ErfK, YxeI, and EntF were expressed in prokaryotic cells and utilized as diagnostic antigens. The clinical sera from bovines and sheep diagnosed with brucellosis were analyzed using indirect ELISA with these proteins.ResultsFor bovine serum, the combined protein group (ErfK + YxeI + EntF) and YxeI demonstrated the highest diagnostic accuracy of 94.23% and 93.58%, respectively. Meanwhile, the combined protein group showed the strongest ability to detect Brucella in sheep serum, achieving an accuracy of 88.10%. Both the combined protein group and YxeI displayed no cross-reactivity with rabbit serum immunized against Yersinia enterocolitica O9, Escherichia coli O157:H7, Mycobacterium tuberculosis, Vibrio cholerae, Legionella, and Salmonella, indicating relatively good specificity.ConclusionThe findings of this study suggest that Tat substrate proteins serve as promising candidate antigens with significant potential value for the clinical diagnosis of brucellosis.

Brucellosis, a zoonotic infection caused by the intracellular pathogen Brucella, leads to chronic multi-organ damage. Currently, rapid, accurate, and sensitive diagnostic technologies are crucial for the prevention and control of brucellosis. This study describes the development of a chemiluminescent immunoassay (Bru-CLIA) for sheep and bovine brucellosis antibody detection, utilizing Brucella abortus strain A19 lipopolysaccharide-coated magnetic particles (LPS-MPs) as the serum antigen and acridinium ester-labeled recombinant streptococcal protein G (AE-SPG) for signal generation. After optimizing the assay’s parameters, the Bru-CLIA demonstrated a sensitivity of approximately 1 IU/mL and 2 IU/mL for detecting sheep and bovine brucellosis, respectively. No cross-reactivity was observed with sera from animals immunized with Escherichia coli O157:H7, Mycobacterium tuberculosis, Vibrio cholerae, Legionella, Salmonella, Foot and Mouth Disease virus types O and A, Bovine viral diarrhea virus, Sheep contagious pleuropneumonia, Goat pox virus, or Peste des Petits Ruminants virus, indicating strong specificity. The testing of 81 sheep serum samples and 96 bovine serum samples revealed that Bru-CLIA showed 87.65% and 93.75% concordance with the ID-VET commercial kits for sheep and bovine brucellosis detection, respectively. These results demonstrate that Bru-CLIA offers high specificity, sensitivity, repeatability, and reliability, making it a viable rapid diagnostic tool for the epidemiological surveillance of brucellosis.

Bovine viral diarrhea virus (BVDV) is one of the major viral pathogens responsible for respiratory disease complexes in cattle and other ruminants; it has spread worldwide and poses a significant threat to the cattle industry. To understand the prevalence and genetic diversity of BVDV in northern China, this study conducted an epidemiological survey of BVDV in dairy cows across 13 provinces in northern China from June 2022 to June 2024. A total of 2,199 nasal swab samples were analyzed by RT-PCR. The results revealed an overall positive rate of 6.05

Brucella BP26 proves to be a highly immunogenic antigen with excellent specificity in brucellosis detection. In China, the authorized use of the Bp26-deleted vaccine M5ΔBP26 for preventing small ruminant brucellosis highlights the importance of developing accurate detection methods targeting BP26, particularly for the diagnosis of differentiation between infected and vaccinated animals (DIVA). Using the traditional mouse hybridoma technique, we successfully obtained 12 monoclonal antibodies (mAbs) targeting BP26. The efficacy of these mAbs in detecting various animal brucellosis cases using the competitive ELISA method was evaluated. Among them, only the E10 mAb exhibited significant efficiency, being inhibited by 100, 97.62, and 100% of brucellosis-positive sera from cattle, small ruminants, and canines, respectively. The E10-based competitive enzyme-linked immunosorbent assay (cELISA) outperformed the BP26-based indirect enzyme-linked immunosorbent assay (iELISA) in accuracy, particularly for cattle and small ruminant brucellosis, with cELISA sensitivity reaching 97.62% compared to 64.29% for iELISA for small ruminants. Although cELISA showed slightly lower specificity than iELISA, it still maintained high accuracy in canine brucellosis detection. The epitope of mAb E10 was identified in the amino acid sequence QPIYVYPDDKNNLKEPTITGY, suggesting its potential as a diagnostic antigen for brucellosis. In conclusion, the E10-based cELISA presents an effective means of detecting animal brucellosis, particularly significant for DIVA diagnosis in China, where the BP26-mutant vaccine is widely used.

IntroductionTuberculosis, caused by Mycobacterium tuberculosis complex (MTBC), remains a global health concern in both human and animals. However, the absence of rapid, accurate, and highly sensitive detection methods to differentiate the major pathogens of MTBC, including M. tuberculosis, M. bovis, and BCG, poses a potential challenge.MethodsIn this study, we have established a triplex droplet digital polymerase chain reaction (ddPCR) method employing three types of probe fluorophores, with targets M. tuberculosis (targeting CFP-10-ESAT-6 gene of RD1 and Rv0222 genes of RD4), M. bovis (targeting CFP-10-ESATs-6 gene of RD1), and BCG (targeting Rv3871 and Rv3879c genes of ΔRD1), respectively.ResultsBased on optimization of annealing temperature, sensitivity and repeatability, this method demonstrates a lower limit of detection (LOD) as 3.08 copies/reaction for M. tuberculosis, 4.47 copies/reaction for M. bovis and 3.59 copies/reaction for BCG, without cross-reaction to Mannheimia haemolytica, Mycoplasma bovis, Haemophilus parasuis, Escherichia coli, Pasteurella multocida, Ochrobactrum anthropi, Salmonella choleraesuis, Brucella melitensis, and Staphylococcus aureus, and showed repeatability with coefficients of variation (CV) lower than 10%. The method exhibits strong milk sample tolerance, the LOD of detecting in spike milk was 5 × 103 CFU/mL, which sensitivity is ten times higher than the triplex qPCR. 60 clinical DNA samples, including 20 milk, 20 tissue and 20 swab samples, were kept in China Animal Health and Epidemiology Center were tested by the triplex ddPCR and triplex qPCR. The triplex ddPCR presented a higher sensitivity (11.67%, 7/60) than that of the triplex qPCR method (8.33%, 5/60). The positive rates of M. tuberculosis, M. bovis, and BCG were 1.67, 10, and 0% by triplex ddPCR, and 1.67, 6.67, and 0% by triplex qPCR, with coincidence rates of 100, 96.7, and 100%, respectively.DiscussionOur data demonstrate that the established triplex ddPCR method is a sensitive, specific and rapid method for differentiation and identification of M. tuberculosis, M. bovis, and BCG.

炭疽病是由炭疽杆菌引起的一种急性、接触性人兽共患病,是一种自然疫源性疫病,各种家畜和野生动物对其均易感.炭疽杆菌在体外可以形成芽孢,对外界具有很强的抵抗力.该病在世界各国均有流行,人主要通过接触患病动物、污染的动物制品或环境而感染.该病严重危害人畜安全,是阻碍畜牧业发展的主要疫病之一.近几年国内多个省份报道了炭疽病的发生,引起社会广泛关注.本文整理、归纳、分析了动物炭疽病的病原学特征、流行病学特点、危害、诊断方法和防控措施等,以期为该病防控提供参考.

958 lung tissue samples of swine influenza were collected in Guangxi from 2013 to 2014 to further study the pathogenic mechanism and prevention and control technology of influenza virus, and influenza virus isolation and biological characteristics analysis of Eurasian avian influenza (ER-H1N1) were performed. The positive rate of the 14 strains of ER-H1N1 swine influenza virus isolated was 1.46%, with 8 gene pieces all from avian sublineages, followed by recombinant Pdm/09 H1N1 fragments. Although their HA cleavage site is PSIQSR↓GLF or GIF, which has typical low-pathogenicity characteristics, the isolated virus strain grows well on the cell, and after the BALB/c mouse challenge experiment, the evidence of the isolated strain multiplying in the respiratory tract of mice is obvious, and the mice lose weight quickly, all die within a week and are accompanied by severe systemic infection. Based on changes in the amino acid residues of the A/swine/Guangxi/6/2013 (No.6) strain, it is thought that the NA protein E119G mutation is the main site where highly dangerous changes happen. D N mutations in the PB2 strains G2, g14, g21, g30, S2, 32, 7, and 8 show that these strains are gradually adapting to human sources. According to the traits of strains 30, G2, G14, and G21, NP develops 375 , which boosts pathogenicity. In summary, the ER-H1N1 subtype influenza virus is widespread in the Guangxi swine herd, and it is pathogenic to mice, with the trend of genomes derived from Pdm/09 H1N1 fragments increasing.

数字PCR(dPCR)是一种新兴的核酸检测技术,继承了PCR和荧光定量PCR(qPCR)的优点,并开创了新的发展方向.dPCR最独特之处在于,它可以在无需标准曲线的情况下,对核酸样本实现绝对定量分析.而多重数字PCR(mdPCR)更是dPCR的主要发展方向.在众多研究领域中,特别是疾病检测方面,mdPCR已展现出卓越的优势和广阔的应用前景.本文介绍了dPCR和mdPCR的原理,探讨了mdPCR在细菌检测、病毒检测、癌症诊断等方面的研究现状,并深入分析了mdPCR的优势及不足,以期为未来mdPCR的应用提供重要的理论指导.

为解决牛病毒性腹泻病毒(BVDV)现场快速诊断难点,提升应急响应速度,根据BVDV 1型基因组5'-UTR保守区序列设计特异性引物和探针,建立了BVDV 1型重组酶聚合酶等温核酸扩增方法(reverse transcription recombinase aid amplification,RT-RAA),并对该方法的特异性、敏感性和重复性进行评估.结果显示:该方法在39℃等温条件下检测时间为20 min,对BVDV 1型核酸检测下限为1.2×102 copies/μL;3个浓度梯度的核酸组内重复性试验的变异系数(CV)分别为3.24％、9.48％和8.79％,均在10.00％以内,重复性好;建立的方法与猪瘟病毒、牛冠状病毒、牛传染性鼻气管炎病毒等无特异性反应.对66份临床样品进行检测,发现所建立的方法与荧光定量PCR检测结果符合率为98.48％,Kappa值为0.881(P<0.001).结果表明,本研究建立的RT-RAA快速诊断方法特异性强、灵敏度高、操作便捷,可用于BVDV 1型的现场快速检测.

针对我国外来动物疫病(简称"外疫")传入风险高、病种多的现实,为提升外疫早期预警、快速诊断、应急处置能力,中国动物卫生与流行病学中心联合农业、卫生、海关、教育、科技等部门共10家单位共同承担了国家重点研发计划"重大动物源性病原体传入风险评估和预警技术研究"项目(项目编号:2017YFC1200500).

为比较几种常用布鲁氏菌病(简称布病)检测方法特性,在某疫苗免疫的奶牛场随机选取40头奶牛,采集血清和奶样各40份,然后对采集的血清进行虎红平板凝集、试管凝集、胶体金、酶联免疫吸附试验(iELISA和cELISA)和琼脂扩散方法检测,对采集的奶样进行病原学检测(qPCR)和抗体检测(胶体金).结果显示:4种国标方法(虎红平板凝集、试管凝集、iELISA和cELISA)检出的阳性样品数量有较大差距,分别为16、6、23和33份,其中经试管凝集试验检出的阳性血清,经其他3种国标方法也检测为阳性;通过胶体金法检出阳性血清数(9份)略高于试管凝集试验,且血清及牛奶样品经胶体金检测结果基本一致;琼扩法检出12份阳性血清,且显示抽检奶牛中存在野毒感染;qPCR和胶体金试纸条对采集奶样的检测结果有较大差距,经qPCR检测仅1份样品呈阳性.结果表明:国标方法中,试管凝集试验特异性最高,iELISA和cELISA敏感性较高;病原学检测临床样品检出率较低,琼脂扩散试验敏感性有限,胶体金法敏感性适中,操作简便,适合布病免疫奶牛场自检.本研究为牛场布病不同检测目的方法选择提供了参考.

Background:Brucellosis is one of the most important zoonotic diseases in the world. Canine brucellosis, caused mainly by Brucella canis, is seriously neglected, and there is a lack of accurate diagnostic tools.Methods:In this study, to compare BP26, Omp25, Omp31 and a multiepitope-based fusion protein in the serological detection of canine brucellosis, using 34 brucellosis-positive dog sera and 62 negative control sera, the Brucella outer membrane proteins Omp31, BP26, Omp25 and a multiepitope-based fusion protein were evaluated by iELISA for their potential use as antigens in the serological diagnosis of canine brucellosis.Results:The results showed that the multiepitope-based fusion protein performed best in distinguishing brucellosis-positive and brucellosis-negative dog sera, with a positive predictive value (PPV) of 100% and a negative predictive value (NPV) of 98.41%. BP26 and Omp31 showed excellent sensitivity in detecting brucellosis-positive dog sera, but their cross reaction to sera infected with Vibrio parahaemolyticus and Listeria monocytogenes may hinder their application as diagnostic reagents. Omp25 lacked sufficient sensitivity and showed limited ability in distinguishing positive and negative dog sera.Conclusion:The multiepitope-based fusion protein can be used as an ideal antigen for serologically diagnosing canine brucellosis currently prevalent worldwide.

本试验旨在评价下排气式灭菌器对锐器盒的灭菌效果,确定适合的灭菌程序,并找出锐器盒的冷点.在锐器盒的上、中、下层和灭菌器内放置包内化学指示卡和生物指示物,关闭锐器盒盖子后放入灭菌器.运行程序1:121℃、20 min,程序2:121℃、30 min,程序3:121 C、60 min,程序4:132℃、10 min,程序5:132℃、20 min,程序6:132℃、30 min.运行程序6时,在锐器盒的上、中、下层和灭菌器内分别放置1个温度探头.结果显示,唯有程序6可以使各层的化学和生物指示物都指示合格.锐器盒上、中、下层的平均温度依次降低.结果表明程序6适于锐器盒灭菌.下层是锐器盒的冷点,可以仅通过观察置于底部的指示物,判断整个锐器盒是否灭菌合格.

戊型肝炎(hepatitis E,HE)是由戊型肝炎病毒(hepatitis E virus,HEV)引起的一种病毒性人兽共患传染病.自1997年在美国首次从家猪体内发现HEV以来,已确定该病毒能够感染人类以及猪、鹿、兔、骆驼、大鼠等多种动物.HEV通常会导致肝衰竭、慢性肝炎以及肝外神经和肾脏疾病.该病主要通过粪口途径传播,也可以通过血液传播.据估计,全球每年约有2000万人感染HEV,年轻人病死率为0.5％～3.0％,而孕妇感染后死亡率可高达30％,因此HEV对公共卫生安全构成了巨大的威胁.本文主要对HEV的病原学、流行病学、诊断、防控等方面的研究情况进行综述,旨在进一步提高相关从业人员等对本病的认识,以便做好研究和防控应对工作.

2021年6月,海南省白沙黎族自治县一村民被诊断为布鲁氏菌病.为查明畜间疫情情况,及时控制疫情,通过现场走访、实地采样、实验室检测等方式开展了紧急流行病学调查.调查发现,该村共有羊养殖户4户,调查时共计存栏120只.对该村的4户养殖户以及1户流行病学关联养殖场户采集血清进行检测,结果均发现布鲁氏菌病阳性.对村养羊户主及密切接触者采集血清20人份进行布鲁氏菌抗体检测,结果9人确诊为阳性.病原分离鉴定发现,分离菌株为羊种布鲁氏菌3型.经与布鲁氏菌MLVA国际数据库的菌株信息比对发现,该菌株的MLVA8、MLVA11基因型为我国北方地区和中亚地区羊种布鲁氏菌常见基因型,而该菌的MLVA16基因型在数据库中没有匹配的菌株信息,说明该基因型为新发现的基因型.调查表明,此起疫情病原由外地传入的可能性较大,并有在本地定殖趋势,人畜再次感染风险较大.因此,海南省需加强动物检疫监管,采取多项防控措施,开展综合防控,消除人畜间布鲁氏菌病疫情,保障海南自由贸易港的公共卫生安全.

为提升我国跨境动物疫病风险预警、识别和快速响应的能力,在国家重点研发计划"生物安全关键技术研发"重点专项支持下,中国动物卫生与流行病学中心黄保续团队牵头,联合中国疾病预防控制中心病毒病预防控制所、中国农业科学院哈尔滨兽医研究所、上海市检验检疫科学技术研究院、中国科学院地理科学与资源研究所、云南省畜牧兽医科学院、新疆畜牧科学院兽医研究所、武汉大学、中国农业大学、浙江农林大学等多家单位实施了"重大动物源性病原体传入风险评估和预警技术研究"项目.该项目基于全球疫情数据、畜牧业生产贸易数据、气象地理数据,研究建立重大动物源性病原体跨境传播风险评估与监测预警系统,对82种以上重要疫病及主要风险因素进行动态监视,对外疫传入风险进行实时预警,提升防范目标识别力;建成非洲猪瘟、口蹄疫、禽流感和小反刍兽疫4种重大动物病原体传入扩散风险评估模型,提升防控策略针对性;针对牛羊神经系统、禽呼吸系统、猪猝死、犬病毒病四大征候群和多种动物虫媒病等5类31种病原体,建成72小时快速鉴定平台体系,提升应急响应速度,并配套研发9种疫病的3小时现场快速检测方法,提升一线筛检识别力.

为了了解2018年4月-2019年12月山东省部分地区规模化奶牛场牛病毒性腹泻病毒(BVDV)的感染情况,采用商品化酶联免疫吸附(ELISA)试剂盒对山东省13个地市5901份送检样品进行了BVDV抗原检测,879份送检样品(823份血样、56份大缸奶样)进行了BVDV抗体检测.结果 显示,所检BVDV抗原样本总阳性率为0.49％、血样BVDV抗体样本总阳性率为75.94％、大缸奶BVDV抗体样本总阳性率为94.64％.菏泽地区BVDV抗原样本阳性率最高,为1.07％;滨州地区血样BVDV抗体样本阳性率最高,为96.77％;德州、聊城、潍坊、日照、威海、泰安地区均在送检奶样中检测到BVDV抗体,样本阳性率达100％.2018年4-6月送检样品BVDV抗原阳性率最高,为1.94％;2019年1-3月送检血样BVDV抗体阳性率最高,为90.91％.中规模牧场送检样品BVDV抗原阳性率最高,为0.79％;大规模牧场送检血样BVDV抗体阳性率最高,为84.47％.结果 表明山东地区BVDV的感染情况较为严重,存在地域和季节性差异,应引起有关单位高度重视,本调查以期为山东省牛病毒性腹泻病毒的防控与净化提供帮助.