This study comprehensively examined three Brucella phages (A1, NMY-1, and NMY-2) isolated from Inner Mongolia Autonomous Region. Electron microscopy classified them as short-tailed phages. A1 and NMY-1 lysed smooth strains of Brucella abortus, Brucella melitensis, and Brucella suis, while NMY-2 lysed rough strains of Brucella melitensis and Brucella canis. The optimal multiplicity of infection for A1, NMY-1, and NMY-2 was lower than that of TbC. A1 and NMY-2 had short growth cycles, and NMY-1 had a long one. All three phages showed high stability against temperature, pH, and ultraviolet exposure. Their genomes were double-stranded DNA, about 38 kb long with a 48% GC content. For each phage, 53 genes were predicted, with no drug-resistance, virulence, or lysogenic genes identified. SNP and InDel analysis revealed significant differences in genes encoding hypothesized tail-collar proteins. Based on SNP data, the phylogenetic tree indicated that phage BkW (GenBank: KC556893) was the closest relative of A1, NMY-1, and NMY-2. These findings significantly enhance our understanding of Brucella phage diversity, which is crucial for developing phage-based biocontrol strategies. The host-lysis spectra can guide the selection of effective phages for treating Brucella infections. The absence of harmful genes makes these phages potential safe candidates for phage therapy. Moreover, the genetic and phylogenetic insights support further research on phage evolution and classification.

Ochrobactrum anthropi (O. anthropi) is found in water, soil, plants and animals. Even though it has low virulence, it has increasingly been found to cause a number of infectious diseases in people with low immunity. The identification of O. anthropi mainly uses biochemical methods, such as the API 20NE or Vitek-2. The typing studies of O. anthropi have mainly utilized PFGE, rep-PCR, AFLP, 16s rDNA sequencing, RecA-PCR RFLP, and MALDI-TOF MS. This study aims to evaluate the polymorphisms of variable-number tandem-repeats (VNTRs) within genomic DNA of O. anthropi strains. The tandem repeats (TRs) in genomic DNA are discovered using Tandem Repeat Finder software (version 4.09). Twelve different VNTRs are designated and assigned to the nomenclature. The primers for PCR of 12 loci are designed. The PCR product size is converted to the number of tandem repeats in every locus. The relatedness of 65 O. anthropi strains from geographically different countries are analyzed by means of 12-variable-number tandem-repeat analysis(MLVA-12). A total of 51 different genotypes are found in 65 O. anthropi strains. These strains, which were collected from the same environmental samples, hospitals, and countries, are clustered within the same or closely genotypes. The MLVA-12 assay has a good discriminatory power for species determination, typing of O. anthropi, and inferring the origin of bacteria.

BackgroundBacteria are the most diverse and widely sourced microorganisms in the indoor air of subway stations, where pathogenic bacteria can spread through the air, leading to increased health risks. ObjectiveTo understand the status and distribution characteristics of indoor air bacterial pollution in subway stations and compartments in a city of Central South China, and to provide a scientific basis for formulating intervention measures to address indoor air bacteria pollution in subways. MethodsThree subway stations and the compartments of trains parking there in a city in Central South China were selected according to passenger flow for synchronous air sampling and monitoring. Temperature, humidity, wind speed, carbon dioxide (CO2), fine particulate matter (PM2.5), and inhalable particulate matter (PM10) were measured by direct reading method. In accordance with the requirements of Examination methods for public places-Part 3: Airborne microorganisms (GB/T 18204.3-2013), air samples were collected at a flow rate of 28.3 L·min−1, and total bacterial count was estimated. Bacterial microbial species were identified with a mass spectrometer and pathogenic bacteria were distinguished from non-pathogenic bacteria according to the Catalogue of pathogenic microorganisms transmitted to human beings issued by National Health Commission. Kruskal-Wallis H test was used to compare the subway hygiene indicators in different regions and time periods, and Bonferroni test was used for pairwise comparison. Spearman correlation test was used to evaluate the correlation between CO2 concentration and total bacterial count. ResultsThe pass rates were 100.0% for airborne total bacteria count, PM2.5, and PM10 in the subway stations and train compartments, 94.4% for temperature and wind speed, 98.6% for CO2, but 0% for humidity. The overall median (P25, P75) total bacteria count was 177 (138,262) CFU·m−3. Specifically, the total bacteria count was higher in station halls than in platforms, and higher during morning peak hours than during evening peak hours (P<0.05). A total of 874 strains and 82 species were identified by automatic microbial mass spectrometry. The results of identification were all over 9 points, and the predominant bacteria in the air were Micrococcus luteus (52.2%) and Staphylococcus hominis (9.8%). Three pathogens, Acinetobacter baumannii (0.3%), Corynebacterium striatum (0.1%), and Staphylococcus epidermidis bacilli (2.2%) were detected in 23 samples (2.6%), and the associated locations were mainly distributed in train compartments during evening rush hours. ConclusionThe total bacteria count in indoor air varies by monitoring sites of subway stations and time periods, and there is a risk of opportunistic bacterial infection. Attention should be paid to cleaning and disinfection during peak passenger flow hours in all areas.

What is already known about this topic?:Brucellosis and severe fever with thrombocytopenia syndrome (SFTS) are neglected zoonoses, attributable respectively to Brucella and the SFTS virus (SFTSV). While the incidence of these diseases has been rising, instances of co-infection remain uncommon. What is added by this report?:This represents the first documented case of a rare coinfection involving Brucella and SFTSV. We carried out an epidemiological analysis of patients diagnosed with brucellosis and those with SFTS at Yidu Central Hospital of Weifang. Our findings demonstrate a temporal and spatial overlap among the affected individuals. What are the implications for public health practice?:Our findings suggest that co-infections arising from the spatiotemporal overlap of Brucella and SFTSV are plausible, necessitating heightened awareness and enhanced diagnostic measures.

Background With the development of domestic animal husbandry, the spread of brucellosis has accelerated, and the scope of the epidemic has expanded. The timely and accurate diagnosis of human brucellosis continues to challenge clinicians in endemic areas. Droplet digital PCR (ddPCR) technology can quickly and accurately determine DNA load in samples, providing laboratory evidence for diagnosis, prognosis and management of brucellosis patients. In this study, a ddPCR method was established to accurately quantify Brucella DNA load in whole blood samples, and its diagnostic, prognostic, and therapeutic value for human brucellosis was evaluated. Methods Annealing temperature, primers, and probe targeting the Brucella bcsp31 gene were optimised, and the sensitivity, specificity and repeatability of the ddPCR assay were assessed using 94 whole blood samples from 61 confirmed and 33 suspected cases. Results were compared with those of quantitative PCR (qPCR). Nine follow-up brucellosis patients were also analysed by the two methods after 2 and 6 months of treatment. Results Optimal primer and probe concentrations were 800 nmol/L and 400 nmol/L, respectively, and the optimal annealing temperature was 55.3 °C. The ddPCR results showed that the limit of detection was 1.87 copies per reaction, with high repeatability. The positive rates for ddPCR and qPCR were 88.5% and 75.4% among 61 serum agglutination test (SAT) positive patients. In addition, 57.6% (19/33) of suspected sero-negative samples were positive by ddPCR, but only 36.3% (12/33) were positive by qPCR. Analysis of nine post-therapy follow-up brucellosis patients revealed that the Brucella DNA load in the whole blood samples decreased after 2 and 6 months of treatment, and was slightly increased following relapse and continuous exposure. Conclusion The ddPCR assay showed good accuracy for whole blood samples, and could be a potential diagnostic and prognostic tool for detecting Brucella.

目的 对3株分离自内蒙古自治区的布鲁氏菌噬菌体A1、NMY-1和NMY-2生物学特性展开分析,获取生物学信息.方法 采用双层平板法纯化、增殖噬菌体,观察噬斑特征及测定效价;使用透射电镜观察噬菌体形态;用双层平板法和点滴法测定噬菌体宿主谱、最佳感染复数(MOI)、一步生长曲线和理化稳定性.结果 经电镜观察,3株噬菌体均为二十面体结构,头部直径61.0 nm～65.6 nm,具有一个长为16.5 nm～19.2 nm短尾;在常规检测浓度(RTD)和104×RTD下,噬菌体A1和NMY-1可以裂解光滑型牛种、羊种、猪种和沙林鼠种布鲁氏菌,NMY-2能够裂解粗糙型犬种、牛种和羊种布鲁氏菌,且在高浓度(104×RTD)时可裂解的菌株数量增加;A1、NMY-1和NMY-2的MOI分别为0.1、0.001、0.01;一步生长曲线结果显示A1和NMY-2潜伏期约为30～60 min,暴发期20～40 min,暴发量分别为2 210蚀斑形成单位(PFU)/cell和6 000 PFU/cell,NMY-1的潜伏期和暴发期较长,分别为600 min和420 min,暴发量为1.90×105 PFU/cell;3株噬菌体在40～60℃,pH为2～13,紫外线照射30 min时保持良好活性.结论 本研究明确了噬菌体A1、NMY-1和NMY-2的生物学特征和属性,丰富了布鲁氏菌的噬菌体分型系统,为噬菌体疗法治疗耐药布鲁氏菌提供了更多选择.

Objective:To establish a real-time quantitative PCR assay to detect the copy number of IS711 transposase gene (orfA) in Brucella genome, and the assay is applied to identify the species and biovars of Brucella. Methods:To establish an orfA gene copy number detection system based on Taqman real-time quantitative PCR technique. Primers and probes of bcsp31 and orfA genes were designed, the contents of bcsp31 gene and orfA gene in the same strain with the same DNA concentration were simultaneously detected by real-time quantitative PCR assay, and cycle number (CT value) of the two genes were obtained. According to the differences of CT values of bcsp31 gene and orfA gene, the copy number of orfA gene in Brucella genome was calculated. At the same time, the DNA of Brucella 16M strain was double decreasing dilution to verify the stability of the detection system. Results:A real-time quantitative PCR assay was used to detect bcsp31 gene and orfA gene simultaneously, when the DNA concentration difference of 16M strain was 2 times, the mean difference of CT values measured was 1.00, 95% confidence interval was 0.95-1.05, standard deviation was 0.17, and coefficient of variation was 0.17. The orfA gene copy number of 30 Brucella strains was detected by this detection system. It was found that there were 6, 9, and 7 copy numbers in the biovars 1-3 of Brucella melitensis, respectively. The strain of Brucella suis biovar 2 had 10 copy numbers, which were different from those of the other 4 strains of biovars 1, 3-5. There were 37 copy numbers in Brucella ovis strain. The copy numbers were stable at 5-6 copies in 8 biovars (1-7, 9) of Brucella abortus strains. Conclusions:A real-time quantitative PCR assay for detection of orfA gene copy number in Brucella DNA has been established. This method could identify some Brucella species and biovars strains.

Objective:A nested-PCR assay is developed to detect and identify the genomic DNA of Brucella vaccine A19 strain. Methods:The whole genomic sequences of Brucella vaccine A19 strain and other Brucella spp. strains were compared and analyzed. The primers were designed by nucleotide difference sites. The nested-PCR assay was established to detect and identify Brucella vaccine A19 strain. The genomic DNA of Brucella vaccine A19 strain was extracted and diluted. The diluted template DNA was tested for sensitivity of using nested-PCR assay. And the specificity of nested-PCR assay was tested for the genomic DNA of other Brucella spp. strains and non- Brucella spp. strains. Results:The minimum detection limit of the nested-PCR assay was 3.43 fg. The nested-PCR assay established for amplification of Brucella vaccine A19 strain showed 246 bp electrophoresis bands, while other Brucella spp. strains showed 314 bp electrophoresis bands, and non- Brucella spp. strains did not produce electrophoresis bands. Conclusions:The nested-PCR assay established has the characteristics of high sensitivity and specificity. It can be detected when there is one copy of Brucella vaccine A19 strain genomic DNA in the reaction system. This method is particularly suitable for the detection and identification of trace genomic DNA of Brucella vaccine A19 strain in sample.

Objective:To establish a quantitative real-time PCR detection system for Brucella S2 vaccine strain. Methods:Based on the differences in the entire genome sequence between Brucella S2 vaccine strain and other reference strains of Brucella, primers and probes were designed to establish a quantitative real-time PCR detection system for Brucella S2 vaccine strain. The DNA of 22 reference strains of Brucella and 8 non- Brucella control strains were obtained from the National Institute for Infectious Disease Control and Prevention of the Chinese Center for Disease Control and Prevention. At the same time, environmental samples were obtained from the brucellosis vaccine manufacturers, and bacterial DNA from environmental samples was extracted using a blood/tissue genomic DNA extraction kit. The obtained DNA was pre-amplified by conventional PCR, and then subjected to quantitative real-time PCR secondary amplification (nested fluorescence quantitative PCR) using the amplified PCR product as a template. The specific fluorescence curve and corresponding number of cycles (Ct value) were observed, and the sensitivity was tested. Results:The quantitative real-time PCR detection system established did not detect specific fluorescence curves (without Ct values) for 21 reference strains of Brucella and 8 non- Brucella control strains, except for S2 vaccine strains. The established detection system had a minimum detection limit of 4.34 fg (genomic DNA) for detecting the DNA of Brucella S2 vaccine strain; DNA of Brucella S2 vaccine strain was detected in 3 of the 14 environmental samples collected. Conclusion:The quantitative real-time PCR detection system established can detect Brucella S2 vaccine strain in samples, with good sensitivity and specificity.

目的 应用巢式聚合酶链式反应(PCR)方法检测布鲁氏菌病(布病)患者血液中布鲁氏菌DNA,分析其与临床症状和抗体水平之间的关系.方法 对门诊就诊患者进行个案调查,包括临床症状、既往史、接触史、用药史等.布病可疑患者,采集血液,分离血清,进行血清试管凝集试验(SAT),检测布鲁氏菌抗体,按照《布鲁氏菌病诊断》(WS 269-2019)进行布病临床诊断.同时对患者抗凝全血进行细菌DNA提取,应用巢式PCR方法检测血液中布鲁氏菌DNA.对不同症状、抗体水平血液中布鲁氏菌细菌数之间的关系进行统计学分析.结果 118例布病患者中,男性78例,女性40例.患者的临床症状主要有发热、乏力、头痛、胸背酸痛、腰痛、四肢酸痛、关节肌肉疼痛、出汗、失眠和睾丸痛等.118例布病患者血液标本中,布鲁氏菌DNA阳性率为63.56％(75/118),布鲁氏菌细菌数中位数(MED)为7拷贝/mL血液,四分位数(IQR)为2-17拷贝/mL血液.118例布病患者中,SAT检测阳性率为38.14％(45/118).118例布病患者中,新发病例和复诊病例分别占55.08％(65/118)和44.92％(53/118),新发病例和复诊病例血液中布鲁氏菌DNA阳性率分别为72.31％(47/65)和52.83％(28/53).47例新发病例血液标本中布鲁氏菌细菌数MED为3拷贝/mL血液,IQR为2～26拷贝/mL血液;28例复诊病例血液标本中布鲁氏菌细菌数MED为4拷贝/mL血液,IQR为2～12拷贝/mL血液.结论 巢式PCR方法检测布病疑似患者血液中布鲁氏菌DNA,有助于布鲁氏菌感染或血清抗体阴性患者的诊断,血液中布鲁氏菌DNA的检测和血清学检查相结合可以有效地诊断布病.

目的 比较不同提取方法对血培养瓶培养的抗凝血中布鲁氏菌检出率的影响.方法 将定量检测的灭活菌液/基因组DNA梯度添加至含抗凝血的血培养瓶中,选择不同的方法提取核酸,利用荧光定量检测其含量.结果 磁珠法和裂解法最低能检测添加10 2 CFU/mL的灭活菌液以及10-3 ng/μL的基因组DNA;离心柱法无法检测未经红细胞裂解液处理的添加106 CFU/mL的灭活菌液以及10 ng/μL的基因组DNA,经红细胞裂解液处理后也只能检测到添加104 CFU/mL的灭活菌液以及1 ng/μL的基因组DNA的模拟样品.结论 红细胞裂解液处理模拟样品能够增加核酸的提取量,裂解法提取效果最好,其次是磁珠法,再次是离心柱法.本研究为疑似布鲁氏菌病样品血培养物的核酸检测提供了方法参考,为提高布鲁氏菌病的诊断率提供了新的思路.

Brucella spp. are facultative intracellular pathogens that can persistently colonize animal host cells and cause zoonotic brucellosis. Brucellosis affects public health and safety and even affects economic development. Our lab found that a Brucella strain isolated from Marmota himalayana exhibited amikacin resistance. To annotate and analyze the potential resistance genes in this strain, we utilized sequencing platforms in this study and cloned potential resistance genes. The findings showed that the isolated strain belonged to B. abortus biovar 1 and was similar to B. abortus 2308. The isolate had amikacin resistance genes encoding aminoglycoside 3'-phosphotransferase. Based on the results of genome analysis, the isolated strain may have obtained amikacin resistance genes from Salmonella spp. through Tn3 family transposons. Notably, this study establishes a foundation for further research on the resistance mechanism of Brucella spp. and provides data that may be useful for the prevention and control of drug-resistant Brucella strains.

目的 探讨应用多重荧光定量PCR方法检测待检者血液、血清以及环境样本中布鲁氏菌的价值.方法 根据149名待测者的就诊情况进行分类,其中疑似组75人、治疗组74人.对收集的环境和待检者的样本应用多重荧光定量PCR方法检测,结果进行统计分析.将3对引物、探针的目的基因克隆到PUC57载体上制作阳性标准品,进行灵敏度检测和标准曲线的绘制.结果 通过对149名待检者的样本进行荧光定量PCR检测,单重、双重和三重方法的阳性率分别为79.2％、34.2％和27.5％.单重与双重,单重与三重荧光定量方法的阳性率差异均有统计学意义,X2值分别为55.42和73.42,P＜0.05.布鲁氏菌属的单重荧光定量PCR结果生成的标准曲线为y=-3.773 2x+43.188,R2=0.9953,线性关系良好,最低检测范围为101 copies/μL.经过多重荧光定量方法检测的环境样本结果全部为阳性.结论 单重荧光定量PCR方法灵敏度较好,建议与血清学方法联合使用对高危人群进行定期筛查,三重荧光定量方法可以在一次实验中既能完成属水平的鉴定又能区分牛种和羊种布鲁氏菌,对于环境样品具有良好的扩增效果,2种方法对于布鲁氏菌病的预防控制具有重要作用.

目的 对陕西省1952-2019年以来保存的布鲁氏菌分离株进行MLVA分型研究,了解陕西省布鲁氏菌遗传变异情况,为制定预防控制策略提供依据.方法 基于MLVA技术对收集的121株布鲁氏菌分离株基因型进行研究,利用Bio Numerics 7.6软件进行聚类分析,计算辛普森指数.结果 基于MLVA-8分型,分为7种,即羊种菌5种(42、43、63、83及115型),猪种菌1种(6型)及牛种菌1种(38型),其中羊种42、43型占总数的91.74％;42型在全省10个市区均有分布;榆林、延安均有4种基因型,多样性较其他市区更丰富;基于MLVA-11,分为11种,基于MLVA-16对121株菌种进行聚类分析结果显示,所有菌株分为3个群,96个基因型,呈现基因型别的多样性,辛普森指数在0.000 ～0.800之间,Panel 1和Panel 2A多样性均明显低于Panel 2B.结论 MLVA技术可用于种属亲缘关系的确立、菌株遗传多样性分析以及分子流行病学溯源调查,陕西省布鲁氏菌MLVA分型显示高度基因多态性,提示陕西省布病流行呈零星和散发特征,因此,降低传染源在区域之间的频繁流动,是控制布病疫情发生的有效手段.

目的 比较5种布鲁氏菌核酸实时荧光PCR检测试剂盒的一致性和检出能力,为临床实验室选择检测方法和布鲁氏菌的诊断提供参考依据.方法 选用经病原学检测确定为布鲁氏菌阳性的血液样本38份,健康人的血液样本24份,潘氏变形杆菌、溶藻弧菌、河弧菌、铜绿假单胞菌、肺炎克雷伯菌DNA各1份,使用5种试剂盒(编号A-E)分别进行核酸检测,比较5种试剂盒临床样本检测的一致性;选择1份阳性样本核酸用无RNA酶水梯度稀释得到5个浓度(浓度1:4 453.13 fg/μL,浓度2:1 113.28 fg/μL,浓度3:278.32 fg/μL,浓度4:69.58 fg/μL,浓度5:17.40 fg/μL),每个浓度使用5种试剂盒(编号A-E)分别进行3次检测,比较5种试剂盒的阳性检出率及批内重复性.结果 5种试剂盒检测67份DNA样品的符合率稍有不同,试剂盒ABDE的符合率均为100％,试剂盒C的符合率为98.51％.批内重复性显示5种试剂盒在浓度1、浓度2、浓度3水平重复检测DNA的Ct值变异系数均＜5％;在浓度1与浓度4梯度区间,试剂盒的阳性检出能力比较显示试剂盒A、B、D较高,为11/12,试剂盒C和E较低,为8/12.结论 5种试剂盒的真实性和可靠性较好,灵敏度和符合率稍有差别,特异度均为100％;重复性较好,检测性能良好.部分试剂盒对弱阳性样本的检出能力不强,该类样本可使用多种试剂盒复核,以保障结果的准确性.

目的 使用多位点可变数目串联重复序列分析(MLVA)对四川省2015-2019年人间布鲁氏菌病(布病)分离菌株进行分型研究,为当地人间布病防控工作提供参考.方法 选用MLVA-16方法对四川省布鲁氏菌株进行分型实验,通过在线数据库对比MLVA-8型别,利用Bio Numerics对MLVA-16位点重复数进行聚类分析.结果 四川省布鲁氏菌株被分为29个MLVA-16基因型,遗传相似度在74.6％～100.0％,具有42、43、83共3个MLVA-8型,三者均起源于东地中海群,42型为主要MLVA-8基因型.MLVA-8方法分辨率为0.356,MLVA-16分辨力为0.991,其中Bruce04、Bruce16及Bruce30位点分辨力较高,可采用MLVA-16方法作为本地暴发调查和散发疫情监测手段.结论 四川省主要流行株为42型(MLVA-8型别)羊种布鲁氏菌,与全国流行株一致,提示该省布病疫情与北方疫情存在关联.本研究首次将四川省布鲁氏菌进行MLVA分型,利用其构建的数据库将对四川省未来的布病监测及分子溯源工作提供基础数据支持.

目的 建立可对布鲁氏菌准确定量的微滴式数字PCR(Droplet digital PCR,ddPCR)方法.方法 使用以布鲁氏菌bscp 31基因为靶标的引物和探针,摸索最佳的引物和探针工作浓度,确定反应体系和扩增条件,评价所建立方法的灵敏性、特异性及对疫情相关血液标本的检测效果.结果 ddPCR反应体系中最佳引物和探针浓度分别为0.8 μmol/L和0.4μmol/L;最佳扩增条件:95℃10 min;95℃30 s,60℃1 min,循环40次;98℃酶失活10 min.布鲁氏菌核酸DNA为0.96 ng/反应～3.68 fg/反应时,ddPCR检出靶标基因拷贝数为2.00×105拷贝/反应～1拷贝/反应,ddPCR测得值的对数值与DNA浓度的对数值有线性关系.在疫情相关血液标本DNA的检测中,ddPCR阳性率远高于培养法和试管凝集法;16份人血液标本中靶标基因浓度为5～65拷贝/mL血液(平均29拷贝/mL血液),5份羊血液标本中靶标基因浓度为25～245拷贝/mL血液(平均98拷贝/mL血液).结论 ddPCR灵敏性高,可检测微量核酸DNA,适合临床血液标本的检测以及布病病原体的分子流行病学调查.

Brucella is gram-negative, facultative,intracellular bacteria implicated in infectious zoonosis diseases, particularly among domestic animals, that are also transmittable to humans. The genus Brucella is classified based on the primary host preferences, pathogenicity, host preference, and phenotypic characteristics of its species [1] .

Background: The prevalence of human brucellosis in Qinghai Province of China has been increasing rapidly, with confirmed cases distributed across 31 counties. However, the epidemiology of brucellosis transmission has not been fully elucidated. To characterize the infecting strains isolated from humans, multiple-locus variable-number tandem repeats analysis (MLVA) and whole-genome single-nucleotide polymorphism (SNP)-based approaches were employed. Methods: Strains were isolated from two males blood cultures that were confirmed Brucella melitensis positive following biotyping and MLVA. Genomic DNA was extracted from these two strains, and whole-genome sequencing was performed. Next, SNP-based phylogenetic analysis was performed to compare the two strains to 94 B. melitensis strains (complete genome and draft genome) retrieved from online databases. Results: The two Brucella isolates were identified as B. melitensis biovar 3 (QH2019001 and QH2019005) following conventional biotyping and were found to have differences in their variable number tandem repeats (VNTRs) using MLVA-16. Phylogenetic examination assigned the 96 strains to five genotype groups, with QH2019001 and QH2019005 assigned to the same group, but different subgroups. Moreover, the QH2019005 strain was assigned to a new subgenotype, IIj, within genotype II. These findings were then combined to determine the geographic origin of the two Brucella strains. Conclusions: Utilizing a whole-genome SNP-based approach enabled differences between the two B. melitensis strains to be more clearly resolved, and facilitated the elucidation of their different evolutionary histories. This approach also revealed that QH2019005 is a member of a new subgenotype (IIj) with an ancient origin in the eastern Mediterranean Sea.

BACKGROUND:Brucellosis is an infectious-allergic zoonotic disease caused by bacteria of the genus Brucella. Early diagnosis is the key to preventing, treating, and controlling brucellosis. Fluorescence polarization immunoassay (FPA) is a new immunoassay for relatively rapid and accurate detection of antibodies or antigens based on antigen-antibody interaction. However, there is no report on FPA-based detection of human brucellosis in China. Therefore, this study is to evaluate the value of FPA for the diagnosis of human brucellosis in China.METHODS:We recruited 320 suspected brucellosis cases who had the clinical symptoms and epidemiological risk factors between January and December, 2019. According to China Guideline for Human Brucellosis Diagnosis, the Rose Bengal test (RBT) was used for the screening test, and the serum agglutination test (SAT) was used as the confirmatory test. Brucellosis was confirmed only if the results of both tests were positive. Additionally, FPA and enzyme linked immune sorbent assay (ELISA) were compared with SAT, and their sensitivity, specificity, coincidence rate and consistency coefficient (Kappa value) as diagnostic tests were analyzed individually and in combination. The optimal cut-off value of FPA was also determined using the receiver operator characteristic (ROC) curve.RESULTS:The optimum cut-off value of FPA was determined to be 88.5 millipolarization (mP) units, with a sensitivity of 94.5% and specificity of 100.0%. Additionally, the coincidence rate with the SAT test was 96.6%, and the Kappa value (0.9) showed excellent consistency. The sensitivity and specificity of FPA and ELISA combined were higher at 98.0% and 100.0% respectively.CONCLUSIONS:When the cut-off value of FPA test is set at 88.5 mP, it has high value for the diagnosis of brucellosis. Additionally, when FPA and ELISA are combined, the sensitivity of diagnosis is significantly improved. Thus, FPA may have potential in the future as a diagnostic method for human brucellosis in China.