Brucellosis, a widespread zoonosis, requires reliable diagnostic tools capable of differentiating infected from vaccinated animals (DIVA), particularly with the emergence of DIVA-compatible vaccines such as Brucella abortus A19-ΔVirB12. VirB12, a key immunogenic component of the type IV secretion system, represents a promising serological target for this purpose. In this study, a VirB12-based indirect ELISA (iELISA) was developed and systematically evaluated for the detection of specific antibodies Recombinant VirB12 protein was successfully expressed in Escherichia coli, purified, and used to immunize mice, generating high-titer polyclonal antiserum (1:204,800). The established iELISA demonstrated excellent repeatability (intra- and inter-assay CVs <10%), high analytical sensitivity (detection up to a 1:3200 serum dilution), and high specificity with no cross-reactivity against other common bacterial pathogens. Clinical validation was performed using 244 bovine serum samples, including 37 Brucella-positive, 20 A19-ΔVirB12-vaccinated, and 187 negative samples.The iELISA exhibited superior overall concordance (98.36%) compared with the Rose Bengal Test (RBT, 90.98%) and showed a higher overall accuracy than a commercial cELISA (91.80%). Notably, although the commercial cELISA demonstrated high sensitivity for detecting positive sera, it failed to distinguish vaccinated from infected animals (0% identification rate), whereas the iELISA correctly identified 90.00% of vaccinated sera, highlighting its clear advantage in DIVA-based diagnostics. Collectively, this assay represents a practical, reliable, and DIVA-compatible diagnostic tool with strong potential for large-scale brucellosis surveillance and control programs.

(1) Background: Bovine viral diarrhea virus (BVDV) is an economically important pathogen affecting cattle worldwide. The genetic diversity of BVDV-2 in buffalo remains poorly understood. This study aimed to elucidate the genomic and antigenic features of GX24, the first BVDV-2b isolate identified from dairy buffalo in China. (2) Methods: The virus was isolated from a rectal swab through three blind passages in MDBK cells and identified through immunofluorescence and RT-PCR. The near-complete genome was sequenced using the Illumina platform and subjected to phylogenetic and recombination analysis. B-cell epitopes and glycosylation sites were predicted using BepiPred-3.0, Epitope1D, DiscoTope-3.0, NetNGlyc-1.0, and NetO-Glyc-4.0. (3) Results: The 12,267-nt GX24 genome (GenBank: PX682047) was classified as BVDV-2b. Recombination analysis detected a putative recombination signal within the NS5A gene involving Chinese BVDV-2b and Italian BVDV-2a strains. Integrative analyses predicted conserved linear and conformational epitope clusters in the N- and C-terminal regions of the E2 protein. Spatial analysis indicated that several epitope residues may be masked by glycan shielding. (4) Conclusions: This study provides the first genomic evidence of a BVDV-2b isolate from dairy buffalo in China, suggesting that BVDV-2b may be present in this population. The putative recombination signal and antigenic characteristics offer valuable insights for the development of diagnostics and vaccine design in the future.

Brucella abortus is a primary etiological agent of bovine brucellosis, a zoonosis posing significant threats to livestock industries and public health. The recently developed A19-∆VirB12 vaccine strain, which carries a deletion of the VirB12 gene, complicates serological differentiation from wild-type infections. This study aimed to establish a rapid, accurate, and economical detection strategy targeting the VirB12 gene to distinguish the A19-∆VirB12 vaccine strain from wild-type B. abortus. We developed a recombinase-aided amplification (RAA) coupled with CRISPR/Cas12a-based detection method. Following optimization, primer pair C and crRNA1 were selected as optimal components, with 150 nM each of Cas12a protein and crRNA identified as the ideal concentrations in a 50 μL reaction. The assay demonstrated high analytical specificity, showing no cross-reactivity with six non-target bacterial pathogens. Sensitivity analysis established a limit of detection of 102 copies per reaction. When evaluated on 52 clinical samples, the RAA-CRISPR assay detected two positive samples, outperforming conventional PCR which detected only one. Crucially, the method yielded no positive signal when challenged with the A19-∆VirB12 gene-deficient vaccine strain, while successfully detecting wild-type strains A19 and S2, confirming its discriminatory capability. The entire workflow, comprising RAA amplification (30 min) and CRISPR-mediated cleavage (20 min), can be completed within one hour, with results visualized via fluorescence or lateral flow strips. This study successfully establishes a rapid, sensitive, and specific diagnostic method for distinguishing the A19-∆VirB12 vaccine strain from wild-type B. abortus, offering a practical tool for field surveillance and eradication programs.

This study isolated and identified two novel Chinese bovine enterovirus (BEV) strains, designated as BEV-GX1901 and BEV-GX1902, from newly transported cattle with the diarrheal feces symptom. We also determined their complete genome sequences (7408 and 7405 nucleotides, respectively) and found both strains have a genome organization analogous to that of picornaviruses. To better understand these two novel strains, a detailed analysis was applied to both strains, including the time of the cytopathic effect (CPE) production, TCID50 measurement, trypsin sensitivity test, ether sensitivity test, chioroform sensitivity test, acid and alkali resistance test, and heat resistance test. Our results showed that these two strains are different in physical and chemical properties. Our study also characterized that BEV-GX1901 and BEV-GX1902, both belonging to the BEV-E4 subtype, were closely related to the Australian strains K2577 and SL305, and the Japanese strain IS1 based on their genome sequences and VP1 region characterizations. It is speculated that this may be related to cattle trade and transportation. Additionally, the gene-by-gene or amino acid-by-amino acid comparison of the two strains found they have differences between their 5'UTR, 3'UTR, VP2, VP1, 2A, 3C, and 3D regions. Our results provide an important update of the virus's presence in China and contribute to a better understanding of the distribution and characterization of BEVs in cattle.

Bovine enterovirus (BEV) is a significant pathogen affecting bovine intestinal health, posing challenges to cattle production and public health. Existing detection methods, such as RT-PCR, RT-qPCR, and LAMP, are time-consuming and dependent on specialized laboratory facilities, highlighting the need for rapid and field-deployable diagnostic tools. In this study, we developed a real-time reverse transcription recombinase-aided amplification (RT-RAA) assay designed for BEV detection, capable of delivering results within 20 min at 39℃. The real-time RT-RAA assay showed no cross-reactivity to other eight pathogens, including BCoV, BVDV, BKV, BRV, BAstV, STEC, FMDV, and PEV. The sensitivity assay revealed that the real-time RT-RAA method could reliably detect a minimum plasmid concentration of 5.50 × 10 ¹ copies/reaction. Clinical validation conducted with 780 field samples revealed performance comparable to those of conventional RT-PCR and RT-qPCR methods, achieving a positivity rate of 23.59 %. The RT-RAA method operates at low temperatures, requires minimal instrumentation, and reduces technical demands, making it ideal for resource-limited settings. Its portability, cost-effectiveness, and robustness position it as a transformative tool for BEV surveillance, enhancing outbreak control, livestock health management, and food safety.

Bovine coronavirus (BCoV) is a notable pathogen affecting newly born calves and adult cattle, increasing mortality rates among calves and reducing productivity in meat and dairy industries, thereby causing substantial economic losses. Current primary laboratory methods for detecting BCoV include RT-PCR assay, real-time RT-PCR assay, and ELISA. However, these methods are time-consuming, require specialized technicians, and necessitate a laboratory environment. Consequently, there is an urgent need for a rapid, sensitive, and easy to use diagnostic method to detect BCoV. This study introduces two innovative protocols: the real-time fluorescent reverse transcription recombinase-aided amplification (RT-RAA) and the test strip RT-RAA (RT-RAA-LFD). Our results indicate that real-time RT-RAA can complete the reaction in 20 min at 39°C, while RT-RAA-LFD can achieve detection in just 17.5 min at 35°C. These new approaches offer higher specificity, with no cross-reactivity to other viruses, and significantly enhanced sensitivity compared to existing methods (1.46 × 101 and 1.46 × 102 copies/μL, respectively). We evaluated the performance of our methods using 242 clinical samples, and compared with RT-PCR and RT-qPCR. Both real-time RT-RAA and RT-qPCR yielded similar detection rates, the detection rate of RT-RAA-LFD was better than RT-PCR. The RT-RAA methods developed in this study effectively overcome the limitations associated with both RT-PCR and RT-qPCR by offering advantages including a single, low reaction temperature that allows for room temperature operation. Both methods boast shorter reaction times, simpler and more portable instrumentation, as well as reduced technical and environmental demands. Generally, both RT-RAA methods established in this study offer new avenues for the rapid detection of BCoV, contributing significantly to the monitoring, prevention, and control of the disease in global bovine industry.

To understand the situation of the main pathogenic bacteria of infectious pneumonia in goats in Guangxi and their resistance to antibiotics,573 samples of disease materials from 2017 to 2021 were detected by pathogen isolation,identification and PCR detection methods,respectively,then the drug sensitivity test was carried out for the main pathogenic bacteria.The results showed that 422 strains of 13 species were isolated and identified,with the infection rates of 75.92％(435/573).The Mycoplasma ovipneumoniae,Escherichia coli and Klebsiella pneumoniae were the main pathogen,with the detection rates of 60.7％(348/573),31.94％(183/573),and 15.2％(87/573),respectively.The infection rates of single infection,double infection,triple infection,quadruple in-fection and quintuple infection among the 435 samples were 52.41％(228/435),26.21％(114/435),11.03％(48/435),6.21％(27/435)and 4.14％(18/435),respectively.Mycoplasma ovipneu-moniae was detected in all the mixed infection samples except the double infection of Escherichia coli and Klebsiella pneumoniae.The results of drug sensitivity test showed that Mycoplasma ovi-pneumoniae was resistant to penicillin G,amoxicillin,cefoxitin,streptomycin,azithromycin and doxycycline,and the resistance rate was 60％to 100％.Escherichia coli was resistant to lincomy-cin,penicillin G and amoxicillin,and the resistance rate was 60％to 100％.Klebsiella pneumoniae was resistant to penicillin G,amoxicillin,doxycycline,flufenicol and lincomycin,and the resistance rate was 70％to 100％.This study showed that there were various pathogens causing goat pneu-monia in Guangxi,and Mycoplasma ovipneumoniae,Escherichia coli and Klebsiella pneumoniae were the main pathogens,and the isolates showed multiple drug resistance.

为研究单原子添加剂对大肠杆菌、金黄色葡萄球菌的体外抑菌效果.本研究采用体外共培养的方法,分析单原子添加剂对这2 种常见致病菌的最小抑菌浓度和最小杀菌浓度,同时进行单原子添加剂抑菌活性的酸碱稳定性、热稳定性研究.结果表明:单原子添加剂对大肠杆菌和金黄色葡萄球菌最小抑菌浓度为3.125 mg/mL,最小杀菌浓度为50 mg/mL;除 100℃外,其余温度处理并未对单原子添加剂抑菌效果有显著影响;pH值为4～8 时,单原子添加剂对大肠杆菌和金黄色葡萄球菌的抑菌效果与阴性对照组无明显差异.说明单原子添加剂对金黄色葡萄球菌和大肠杆菌均有很好的抑制作用,且具有良好的热稳定性和酸碱稳定性.

为了了解广西地区奶牛、肉牛的牛库布病毒(bovine Kobuvirus,BKV)感染情况及分子特征,试验采用RT-PCR的方法对从广西地区采集的奶牛粪便177份、肉牛粪便27份(共204份粪便,其中腹泻牛粪便82份,健康牛粪便122份)进行BKV和其他肠道病毒的检测,分析BKV与其他肠道病毒的共感染情况,以及不同地点和不同季节BKV的感染情况,同时对广西地区BKV的3D基因与国内外30株库布病毒的3D基因进行核苷酸序列同源性分析和遗传进化分析.结果表明:广西地区BKV的感染率为2.94％(6/204),均从腹泻牛粪便中检出,其中奶牛和肉牛的感染率分别为2.26％(4/177)、7.41％(2/27),将6株毒株分别命名为BKV-GXGG01(贵港株)、BKV-GXHC01(河池株)、BKV-GXHC02(河池株)、BKV-GXNN01(南宁株)、BKV-GXNN02(南宁株)和 BKV-GXNN03(南宁株);BKV与牛病毒性腹泻病毒、牛肠道病毒和牛星状病毒存在混合感染,感染率为66.67％(4/6),而单一感染率为33.33％(2/6);6株广西地区BKV毒株的3D基因间的核苷酸序列同源性为93.5％～100％,与国内外Aichivirus B类型参考毒株间的核苷酸序列同源性为89.9％～95.1％,与其他库布病毒参考毒株的核苷酸同源性为35.9％～75.2％;6株广西地区毒株在遗传进化分析中均属于Aichivirus B类型,毒株BKV-GXNN01与日本株K-35、河南株B4、内蒙古株H6和H18、山东株C2、黑龙江株SC7-5、辽宁株H9同属一分支,其余5株毒株BKV-GXGG01、BKV-GXHC01、BKV-GXHC02、BKV-GXNN02、BKV-GXNN03与北京株B1213、泰国株CMB02、日本株U-1同属一分支.说明在广西地区奶牛、肉牛的腹泻病例中均存在BKV感染,推测BKV可能与牛腹泻有关.

Background Mycoplasma ovipneumoniae is a critical pathogen that causes respiratory diseases that threaten Caprini health and cause economic damage. A genome-wide study of M. ovipneumoniae will help understand the pathogenic characteristics of this microorganism. Results Toxicological pathology and whole-genome sequencing of nine M. ovipneumoniae strains isolated from goats were performed using an epidemiological survey. These strains exhibited anterior ventral lung consolidation, typical of bronchopneumonia in goats. Average nucleotide identity and phylogenetic analysis based on whole-genome sequences showed that all M. ovipneumoniae strains clustered into two clades, largely in accordance with their geographical origins. The pan-genome of the 23 M. ovipneumoniae strains contained 5,596 genes, including 385 core, 210 soft core, and 5,001 accessory genes. Among these genes, two protein-coding genes were annotated as cilium adhesion and eight as paralog surface adhesins when annotated to VFDB, and no antibiotic resistance-related genes were predicted. Additionally, 23 strains carried glucosidase-related genes ( ycjT and group_1595 ) and glucosidase-related genes ( atpD_2 ), indicating that M. ovipneumoniae possesses a wide range of glycoside hydrolase activities. Conclusions The population structure and genomic features identified in this study will facilitate further investigations into the pathogenesis of M. ovipneumoniae and lay the foundation for the development of preventive and therapeutic methods.

为了研究不同来源的地衣芽孢杆菌对常见致病菌的抑菌效果等生物学特性,试验采用细菌形态学、生理生化、16S rRNA基因测序、系统发育树分析、牛津杯抑菌试验等检测技术,对5株不同来源(鸡粪、羊粪、牛瘤胃内容物、猪肠内容物及土壤)的地衣芽孢杆菌进行了分离鉴定及体外抑菌活性检测.结果表明:5株分离株B17(鸡粪)、B30(羊粪)、B32(牛瘤胃内容物)、B35(猪肠内容物)和B38(土壤)均可在LB固体培养基上生长,其中有3株不同来源的分离株B30、B35和B38菌落形态相同;镜检可见分离株均为革兰氏阳性杆菌、均有近中生的孢子,符合芽孢杆菌的菌体特征;生理生化特性与芽孢杆菌相符;PCR扩增均得到约为1 500 bp的目的片段;所有分离株与GenBank中的地衣芽孢杆菌在同一簇,均鉴定为地衣芽孢杆菌;在5株地衣芽孢杆菌中,除猪源地衣芽孢杆菌B35外,其余4株均有抑菌作用,羊源及牛源地衣芽孢杆菌B30及B32的抑菌效果较好,抑菌范围更广泛.说明从羊粪及牛瘤胃内容物中分离出的地衣芽孢杆菌可作为益生芽孢杆菌控制病原菌,不同来源的地衣芽孢杆菌的抑菌效果不同.

为探究感染大片形吸虫后不同时间水牛脊髓全基因组DNA甲基化的类型、占比及其感染后差异甲基化区域(DMR)涉及的功能及信号通路,本研究将大片形吸虫囊蚴经口灌胃感染8～10月龄水牛,分别于感染后3d(J01)、10d(J02)、28 d(J03)、42 d(J04)、70 d(J05)和98 d(J06)采集水牛脊髓,利用全基因组重亚硫酸盐测序技术(WGBS)对基因组DNA的甲基化测序,测序数据经过滤筛选后,采用Bismark软件分析各组水牛脊髓基因组DNA甲基化的类型及其占比(某种类型甲基化序列在该组全部可用测序序列中的占比以及该组某种C类型甲基化的数量在全部C类型甲基化中的占比),并采用weight methylation level对各组水牛脊髓基因组DNA 7个功能区域的甲基化进行聚类分析.WGBS测序结果经过滤后显示,平均每组测序长度为100.29 Gp,Q20％(质量值≥20的碱基占总碱基数的百分比)和Q30％分别达到95％和85％以上,6组样品亚硫酸盐(BS)转化率均大于99％,表明WGBS测序结果准确可靠;各组样品DNA甲基化类型和占比的分析及统计结果显示,J01～J06组基因组分别包含CG、CHG、CHH 3种类型的甲基化(mCG、mCH及mCHH),且以mCG类型占比最多(63.1％～71.7％,80.11％～85.73％),mCHH类型(0.9％～1.2％,11.27％～15.18％)及mCHG类型均较少(1.0％～1.2％,3.00％～4.84％);聚类分析结果显示,上述3种类型的甲基化主要分布在水牛脊髓基因组第1内含子和内部内含子.上述结果表明,感染大片形吸虫后水牛脊髓基因组DNA甲基化类型以mCG为主,且第1内含子和内部内含子的甲基化可能影响该两个区域相关基因的正常表达.利用R软件包分析各组水牛脊髓基因组之间是否存在DMR,并采用基于模型的亚硫酸氢盐测序数据分析(MOABS)筛选并统计各组之间的DMR及DMR的数量;采用DAVID软件分析各组DMR在其相应基因组中的分布;采用R语言对各组的DMR进行GO功能注释和KEEG富集分析.DMR的筛选及统计结果显示,各基因组间均存在DMR,且各组间均以CG类型DMR数量的差异最大.其中,J06与J01(7 134个)、J06与J02(7 174个)、J06与J03(6 743个)、J04与J03组(11 611个)之间DMR数量的差异最大,且96.42％～99.04％的DMR分布在基因组基因间区,0.96％～3.59％的DMR分布在基因组启动子区.DMR的GO功能分析显示,各组间CG类型DMR的GO功能注释结果基本相似,主要富集在细胞进程、生物调节、代谢过程、结合及催化活性等生物学过程;KEGG分析结果显示,各组间尤其是在感染后期(J06组和J04组)DMR主要富集在癌症及PI3K-Akt等信号通路中.上述结果表明,在大片吸虫慢性感染的过程中,尤其在感染中后期对水牛脊髓基因组基因间区相关基因的表达有影响,且甲基化的DNA可能主要通过以上两个信号通路影响相关基因的表达,进而影响水牛脊髓的各种生物学功能,最终引起水牛中枢神经系统疾病.这在一定程度阐释了寄生在肝脏的片形吸虫影响宿主中枢神经系统的机制.本研究为深入探究大片形吸虫感染对水牛神经系统的影响机制奠定了实验基础.

Astroviruses (AstVs) are widespread and can infect multiple species. Porcine astroviruses produce mainly gastroenteritis and neurological diseases in pigs.

为研究饲料中添加不同剂量单原子添加剂对三黄鸡的生长性能和免疫功能的影响,选用150只1日龄的健康三黄鸡,随机分成5组,每组30只.试验分为空白组、对照组、低剂量组(0.5 g/kg单原子添加剂)、中剂量组(1.5 g/kg单原子添加剂)和高剂量组(3.0 g/kg单原子添加剂).试验期42 d,分为试验前期(1～21日龄)和试验后期(22～42日龄)两个阶段.结果表明:试验全程,高剂量组和中剂量组平均日增重(ADG)均显著高于对照组和空白组(P<0.05);在试验前期,中剂量组脾脏指数显著高于对照组(P<0.05);高剂量组的血清免疫球蛋白A(IgA)、免疫球蛋白M(IgM)和免疫球蛋白G(IgG)的含量均显著高于空白组(P<0.05);低剂量组血清禽流感(AI)抗体效价显著高于空白组(P<0.05),对照组血清新城疫(ND)抗体效价显著高于空白组(P<0.05);中剂量组空肠和十二指肠绒毛高度(VH)显著高于空白组(P<0.05),高剂量和低剂量组空肠和十二指肠隐窝深度(CD)显著低于空白组(P<0.05).在试验后期,高剂量组的血清IgM含量显著高于其他试验组(P<0.05),高剂量组的空肠和十二指肠VH显著高于空白组(P<0.05),高剂量和中剂量组空肠CD显著低于其他各组(P<0.05).结果表明,日粮中添加单原子添加剂可通过完善肠道结构、调节免疫相关因子和特异性抗体的分泌水平,提高三黄鸡的生长性能和免疫功能,且日粮中最佳的添加剂量为3 g/kg.

Background Extracellular vesicles (EVs) released by helminths play an important role in parasite-host communication. However, little is known about the characteristics and contents of the EVs of Fasciola gigantica, a parasitic flatworm that causes tropical fascioliasis. A better understanding of EVs released by F. gigantica will help elucidate the mechanism of F. gigantica-host interaction and facilitate the search for new vaccine candidates for the control and treatment of fascioliasis.Methods Two different populations of EVs (15k EVs and 100k EVs) were purified from adult F. gigantica culture media by ultracentrifugation. The morphology and size of the purified EVs were determined by transmission electron microscopy (TEM) and by the Zetasizer Nano ZSP high performance particle characterization system. With the aim of identifying diagnostic markers or potential vaccine candidates, proteins within the isolated 100k EVs were analyzed using mass spectrometry-based proteomics (LC-MS/MS). Mice were then vaccinated with excretory/secretory products (ESPs; depleted of EVs), 15k EVs, 100k EVs and recombinant F. gigantica heat shock protein 70 (rFg-HSP70) combined with alum adjuvant followed by challenge infection with F. gigantica metacercariae. Fluke recovery and antibody levels were used as measures of vaccine protection.Results TEM analysis and nanoparticle tracking analysis indicated the successful isolation of two subpopulations of EVs (15k EVs and 100k EVs) from adult F. gigantica culture supernatants using differential centrifugation. A total of 755 proteins were identified in the 100k EVs. Exosome biogenesis or vesicle trafficking proteins, ESCRT (endosomal sorting complex required for transport) pathway proteins and exosome markers, heat shock proteins and 14-3-3 proteins were identified in the 100k EVs. These results indicate that the isolated 100k EVs were exosome-like vesicles. The functions of the identified proteins may be associated with immune regulation, immune evasion and virulence. Mice immunized with F. gigantica ESPs, 15k EVs, 100k EVs and rFg-HSP70 exhibited a reduction in fluke burden of 67.90%, 60.38%, 37.73% and 56.6%, respectively, compared with the adjuvant control group. The vaccination of mice with F. gigantica 100k EVs, 15k EVs, ESP and rFg-HSP70 induced significant production of specific immunoglobulins in sera, namely IgG, IgG1 and IgG2a.Conclusion The results of this study suggest that proteins within the exosome-like vesicles of F. gigantica have immunomodulatory, immune evasion and virulence functions. This knowledge may lead to new strategies for immunotherapy, vaccination and the diagnosis of fascioliasis.

为探究马脓皮病的病原种类及特性,从病马的皮肤脓汁中分离到一株革兰氏阳性菌,经16SrDNA鉴定为金黄色葡萄球菌.经致病性试验、药敏试验、毒力基因和耐药基因等检测.结果显示,以浓度为9.3×108 CFU/mL菌液攻毒小鼠,致死率为40％;分离菌对氧氟沙星等14种药物高敏,具有fnbA、hly/hla、hysA、clfA和clfB等5个毒力基因和norA和gyrA 2个耐药基因.此结果可作为诊治马脓皮病用药参考.

为建立一种快速、准确并可定量检测溶血性曼氏杆菌的环介导等温扩增(LAMP)方法,本研究针对溶血性曼氏杆菌gcp基因设计5条特异性引物,在链置换DNA聚合酶的作用下,优化反应条件,并进行特异性和敏感性试验.特异性检测结果表明,建立的LAMP扩增方法可以在65 min内完成,并具有良好的特异性,检测化脓隐秘杆菌、肺炎克雷伯氏菌、大肠杆菌、牛支原体、鼠伤寒沙门菌、溶血性巴氏杆菌等其他6种常见牛呼吸道综合征致病菌和空白对照(水)时结果均为阴性;灵敏性检测结果表明,建立的LAMP方法检测溶血性曼氏杆菌质粒标准品的最低检出浓度为0.855×10-4 ng/μL;在对临床样品的检测中,本研究建立的LAMP方法对溶血性曼氏杆菌的检出率为52.63％(10/19)大于PCR方法的检出率47.36％(9/19).以上结果表明,本研究建立的基于gcp基因的溶血性曼氏杆菌LAMP检测方法为溶血性曼氏杆菌的快速检测提供了一种新的技术手段,该方法特异性强、灵敏度高、快速简便,可将该方法推广至基层和流行病调查一线,应用于该病原的快速检测.

近年来,畜禽规模化养殖蓬勃发展,但我国畜禽养殖业标准化工作相对滞后,而畜禽养殖业的标准化建设是畜禽养殖业发展方式转型升级的必由之路.只有加快我国畜禽养殖业标准化工作,才能促进畜禽养殖业产业化、品牌化发展,助力乡村振兴.本文分析加快我国畜禽养殖业标准化工作的重要意义,并提出相应的解决方法.

为了筛选出一种适合体外测定益生菌抑菌活性的试验方法,同时筛选出具有较好抑菌活性的益生菌株,试验采用牛津杯法、琼脂平板扩散法(打孔法)、竞争排斥法分别测定实验室分离鉴定的9株益生菌(副干酪乳杆菌、植物乳杆菌、屎肠球菌、干酪乳杆菌、瑞士乳杆菌、嗜酸乳杆菌、粪肠球菌、鼠李糖乳杆菌、枯草芽孢杆菌)对大肠杆菌、金黄色葡萄球菌的抑菌活性.结果表明:除干酪乳杆菌对大肠杆菌无抑菌活性外,其余8株益生菌对大肠杆菌均有一定的抑菌活性;除嗜酸乳杆菌对金黄色葡萄球菌无抑菌活性外,其余8株益生菌对金黄色葡萄球菌都有一定的抑菌活性;但牛津杯法和打孔法测定的结果有一定差异,在对大肠杆菌、金黄色葡萄球菌有抑菌活性的益生菌中,牛津杯法测定的抑菌圈直径比打孔法测定的抑菌圈直径大.竞争排斥法测定9株益生菌对致病菌的抑菌活性除干酪乳杆菌和粪肠球菌对金黄色葡萄球菌抑菌活性与牛津杯法测定结果不一致外(竞争排斥法中金黄色葡萄球菌对干酪乳杆菌低度敏感,而牛津杯法中为中度敏感;竞争排斥法中金黄色葡萄球菌对粪肠球菌低度敏感,而牛津杯法中为不敏感),其余菌株与牛津杯法测定结果均一致.说明体外测定益生菌抑菌活性牛津杯法比打孔法好,而竞争排斥法较牛津杯法操作更简便;植物乳杆菌、枯草芽孢杆菌对大肠杆菌、金黄色葡萄球菌的抑菌活性最好.

通过GenBank中登录号(神经型BufAstV-GX-NN-ORF2-14、肠道型BufAstV-GX-NN-ORF2-12)合成神经型ORF2-14-1与肠道型ORF2-12-1基因序列,运用PCR技术扩增神经型ORF2-14与肠道型ORF2-12目的片段的基因并克隆到pET-32a(+)载体,成功构建了重组质粒神经型pET-32a-ORF2-14与肠道型pET-32a-ORF2-12,经鉴定正确后转化BL21(DE3)感受态细胞中成功诱导表达,纯化后的重组蛋白利用SDS-PAGE、Western blot进行鉴定.以纯化的神经型pET-32a-ORF2-14与肠道型pET-32a-ORF2-12重组蛋白为诊断抗原,成功建立牛星状病毒(bovine astrovirus,BoAstV)神经型与肠道型的间接ELISA抗体检测方法.运用建立的间接ELISA对2019-2020年广西省部分地区采集的712份牛血清进行BoAstV抗体的检测,结果显示肠道型共检出170份抗体阳性血清,BoAstV抗体阳性率为23％;神经型共检出124份抗体阳性血清,BoAstV抗体阳性率为17％;其中,2种型共同感染阳性率占总份数10％.结果表明,本试验成功建立了对BoAstV的神经型与肠道型分型检测方法,为临床上对BoAstV分型检测提供良好的检测方法打下基础.