羊种布鲁氏菌M5-90疫苗株铁应答调控因子irr和rirA基因缺失株的构建与鉴定

为研究铁调控因子irr和rirA在羊种布鲁氏菌（Brucella melitensis）感染过程中的作用,本研究通过卡那替换的方法构建两个缺失株M5-90Δirr和M5-90ΔrirA,分别将亲本株和缺失株在相同营养条件下培养36 h,观察其振荡培养时的生长变化趋势。分别将1×10⁸ CFU M5-90Δirr、M5-90ΔrirA和M5-90接种到含1.5 mol/L NaCl、pH 2.5、pH 11.5、10 mmol/L H₂O₂的1 mL布鲁氏菌液体培养基,比较缺失株和亲本株在不同条件下的生长特性;分别以1×10⁶ CFU M5-90Δirr、M5-90ΔrirA和M5-90感染小鼠巨噬细胞RAW264.7检测缺失株的黏附、侵袭和胞内生存能力。结果显示,在相同体外培养条件下缺失株M5-90Δirr和M5-90ΔrirA生长速度明显低于亲本株;M5-90Δirr、M5-90ΔrirA在高盐、强酸和强碱环境下生存率均显著或极显著低于亲本株（P<0.05;P<0.01）,在H₂O₂条件下这两个缺失株的生存率却显著高于亲本株（P<0.05）。与亲本株相比,缺失株在小鼠巨噬细胞RAW264.7内的侵袭和黏附能力均减弱,在感染后45 min缺失株M5-90Δirr和M5-90ΔrirA的黏附和侵袭能力均极显著低于亲本株（P<0.01）,但在感染后24 h,这两个缺失株在细胞内繁殖能力与亲本株相比有所增强。本研究报道了irr和rirA基因不仅调控了羊种布鲁氏菌的生长,同时对细菌黏附和侵袭能力也产生一定的影响,M5-90Δirr和M5-90ΔrirA是两株具有潜力的布鲁氏菌候选疫苗株。     

布鲁氏菌分泌蛋白BspJ多克隆抗体的制备

为获得一定纯度及浓度的布鲁氏菌分泌蛋白BspJ并制备多克隆抗体,本研究通过常规PCR方法扩增BspJ基因,连接至pMD19-T克隆载体并筛选阳性克隆;使用限制性内切酶切下目的片段后连接至pET28a(+)载体,双酶切、菌液PCR验证阳性克隆菌并送测序;表达载体pET28a-BspJ转入大肠杆菌DE3后经IPTG诱导表达,收集表达菌进行SDS-PAGE分析;使用His标签蛋白纯化柱纯化目的蛋白rBspJ;将rBspJ蛋白混入弗氏佐剂分4次免疫实验兔,收集兔血清,Western blot鉴定多克隆抗体,饱和硫酸铵法纯化多克隆抗体.结果显示:PCR方法扩增出大小534 bp的目的片段,测序结果正确,表明成功构建出pMD19-T-BspJ克隆载体及pET28a-BspJ表达载体;表达菌表达出大小约24 kDa的rBspJ,纯化后的rBspJ条带明显,基本无杂带,表明成功纯化出rBspJ;真核表达的rBspJ与制备的多克隆抗体反应,表明成功制备多克隆抗体.本研究结果为BspJ蛋白的亚细胞定位分析、功能研究及布鲁氏菌致病机制的研究积累实验数据和奠定基础.     

Brucellosis associated with stillbirth in a bottlenose dolphin in Australia

A captive adult female bottlenose dolphin presented with stillbirth. The placenta appeared oedematous. No other gross lesions were evident in the placenta or the stillborn calf. Histopathology revealed mild multifocal placentitis and foetal encephalitis. Brucella sp. was isolated from lung, liver, spleen and kidney. Sequence and phylogenetic analysis demonstrated this organism to be most similar to Brucella ceti sequence type (ST) 27. Brucella sp. DNA was detected in formalin-fixed paraffin-embedded placenta and brain by real-time PCR using primers targeting the IS711 gene. Immunohistochemical staining revealed Brucella sp. antigen in placental inflammation. This is the first report of isolation of Brucella sp. from a marine mammal in the Southern Hemisphere and the first report of marine Brucella-associated disease in Australia.     

Molecular detection of important abortion-causing microorganisms in preputial swab of breeding bucks using PCR-based assays

Infectious diseases and aetiological agents related to female reproductive systems were extensively covered compared to its male counterpart. There needs a proper study to bridge this gap, where microflora and infectious agents of both male and female reproductive are mutually intelligible. With this study, we aimed to evaluate the microbial contamination of the preputial cavity and also screened for abortion-causing agents which are zoonotic as well. In goats, such types of abortions are caused by Brucella melitensis, Chlamydophila, Campylobacter and Coxiella etc. One of the major sources of contamination of semen is the preputial cavity, which is exposed to the external environment leading to spread of infection into the female via semen straws or by natural service. In the current study, good quality bucks (n = 32, Barbari = 12, Jamunapari = 10, Jakhrana = 10) which were routinely used for semen collection were screened for their preputial swabs, for the presence of the above pathogens. For detection of Brucella melitensis, OMP31 based TaqMan® probe real-time PCR assay was used, and for Chlamydia, 16srRNA gene based SYBR® green real-time PCR assay was employed for detection of Chlamydophila abortus. While for Campylobacter spp. and Coxiella burnetii, 16srRNA gene based conventional PCR and Trans-PCR were used, respectively. In the current study, of the screened preputial swabs, none of them showed positive for Brucella and Coxiella, but of the screened 32 samples 17 showed positive for Chlamydia (53.13%) and two (6.25%) showed positive for Campylobacter spp. The current study emphasizes on the farms and laboratories which were regularly involved in screening of brucellosis also often overlook the other potential non-brucella pathogens, causing abortions eventually incurring severe economic losses to the goat keepers.     

布鲁菌缺失疫苗株M5-ΔznuA和M5-Δbp26-ΔznuA的构建及毒力和免疫原性的评估

【目的】利用分子生物学技术,通过同源重组的方法构建了新型的羊种布鲁菌Brucella melitensis减毒活疫苗株,为布鲁菌的防治提供新的选择.【方法】以羊种布鲁菌减毒活疫苗M5株及缺失bp26基因的M5-Δbp26缺失突变株为亲本株,利用电击转化法,将含有znuA基因上下游同源臂的自杀质粒转入到亲本株中,通过同源重组敲除znuA基因.对构建的新型基因缺失株进行生物学特性及毒力的鉴定与分析.【结果和结论】PCR及核苷酸序列测序结果表明,羊种布鲁菌减毒活疫苗znuA单基因缺失株和bp26、znuA双基因缺失株均构建成功,分别将其命名为M5-ΔznuA和M5-Δbp26-ΔznuA.与亲本株相比,2种缺失突变株的生物学特性没有显著性差异;体外连续传至20代后,菌落PCR和核苷酸测序结果显示M5-ΔznuA和M5-Δbp26-ΔznuA株具有良好的遗传稳定性.小鼠脾脏质量和脾脏菌落数表明,M5-Δbp26-ΔznuA双基因缺失株毒力最弱,单基因缺失株M5-ΔznuA和M5-Δbp26次之.血清中抗体水平的监测显示,znuA基因的缺失对布鲁菌减毒活疫苗诱导机体体液免疫的能力没有影响.获得了免疫原性保持良好而毒力减弱的羊种布鲁菌减毒活疫苗基因突变株M5-ΔznuA和M5-Δbp26-ΔznuA.     

The Fluorescent Characterization and Analysis of Two Brucella Attenuated Vaccine Infecting Mouse Macrophagocyte

The experiment was conducted to discuss the difference of binding time of green fluorescent protein B.melitensis M5 (GFP-M5) and B.abortus S19 (GFP-S19) infecting the mouse macrophagocyte (RAW264.7),lysosome,endoplasmic reticulum and golgi body in the initial stage and compare the binding rate of GFP-M5,GFP-S19 with organelle in different timeline,respectively,by confocal laser scanning microscope (CLSM) and flow cytometry.The result showed that GFP-M5 and GFP-S19 were successfully constructed.The intracellular survival ability of Brucella M5,Brucella S19,GFP-M5 and GFP-S19 were not obvisouly affected after infecting RAW264.7.GFP-M5 and GFP-S19 could enter the macrophagocyte in 30 mins,and in 2 h the Brucella could reach lysosome,endoplasmic reticulum and golgi body.In addition,the binding time for two attenuated vaccine did not show differences in 1,2,3 and 4 h.The content of GFP⁺ cell produced by RAW264.7 infected by GFP-M5 and GFP-S19 did not show significant differences (P>0.05).Therefore,the two strains did not have significant differences in the invasion ability in the initial stage of infecting host cell.     

羊布鲁菌16MOmp25真核表达载体的构建

利用聚合酶链式反应（PCR）,以羊布鲁菌16M基因组为模板克隆Omp25基因,设计含有EcoRI和xhoI酶切位点的引物序列,定向克隆到真核表达载体pCMV-HA上,构建真核表达重组质粒pCMV-HA～Omp25。经测序及双酶切验证正确,证明成功构建了pCMV-HA—Omp25真核表达质粒,为表达纯化外膜蛋白Omp25,研究其功能奠定基础。     

Analysis of a survey of metabolic disorders

Extract

This survey was instigated in an endeavour to determine the overall incidence of metabolic disorders in dairying herds in the area covered by the Huntly District Veterinary Club. Response to treatment and mortality figures are also included.     

A study of periodontal disease in sheep over a twelve month period

Sheep affected by broken mouth periodontal disease (P.D.) were examined over a twelve month period for different clinical parameters. It is suggested that P.D. in sheep is an episodic phenomenon similar to human P.D., and that only a few animals with signs of P.D. may undergo clinically significant destruction over a yearly period. No single parameter could reliably predict future deterioration in other parameters.     

Atypical symptoms in a case of sleepy foal disease (Actinobacillus equuli infection)

Extract

It was reported that a maiden thoroughbred marc was unwilling to feed her foal in spite of persistent efforts by the foal during the first eight hours after birth.