Risk factors for Brucella spp. infection In dairy cattle farms in Asmara, State of Eritrea

A cross-sectional study was conducted to identify risk factors for herd infection by Brucella spp. in dairy cattle in the suburbs of Asmara, Eritrea. Data were collected from 64 herds, randomly selected from a total of 99 herds with a minimum herd size of 9 cows. A questionnaire was used to gather data on management, hygiene and herd structure. Serum samples collected from all pregnant heifers, cows and bulls, were screened for Brucella infection by the Rose Bengal test (RBT), and all RBT-positive sera re-tested with the complement-fixation test (CFT) for confirmation. A seropositive herd was defined as one in which at least one animal tested positive in the CFT. There were 23 (36%) positive herds among the 64 studied. Both multiple logistic and multiple betabinomial regression modeling were used to analyze the data. Mixed-breed herds, compared to single (exotic)-breed herds, were found to be independently associated with increased herd seroprevalence (OR=5.2, 95% confidence interval 1.4–18.7) in the multiple logistic model with the herd infection status as the dependent variable. The importance of this variable was supported by the multiple betabinomial regression model (OR=3.3, 1.4–7.6) with animal-level prevalence within herd as the outcome variable. Both models also revealed the presence of a negative association between seropositivity and cattle stocking density.     

Use of Brucella abortus vaccine strain RB51 in pregnant cows after calfhood vaccination with strain 19 in Argentina

One hundred and seven pregnant cows, which had been calfhood vaccinated with Brucella abortus strain 19 (S-19) were revaccinated with either S-19 or strain RB51 (S-RB51). All S-19-revaccinated animals seroconverted, while none of the RB51-revaccinated animals seroconverted. Two out of 25 (8%) S-19-revaccinated animals aborted, while none of the 57 RB51-revaccinated group aborted. Four of the S-19-revaccinated animals shed S-19 in the milk for at least 7 days, while only 1 cow shed S-RB51 for at least 3 days (but <7 days) post-parturition. Revaccination of strain 19 calfhood-vaccinated, pregnant cattle with S-RB51 appears to be a safe procedure with no diagnostically negative consequences.     

布鲁氏菌pcDNA3．1-omp17．3基因疫苗的研制

采用PCR方法扩增了布鲁氏菌17．3ku外膜蛋白编码基因，并将该基因克隆至真核表达载体pcDNA3．1（＋）中，成功构建了真核表达质粒pcDNA3．1-ompl7．3。pcDNA3．1-omp17．3转染coS-7细胞后，通过Western—blotting检测到了17．3ku蛋白的瞬时表达。将pcDNA3．1-ompl7．3免疫小鼠，三免后经ELISA、流式细胞仪以及ELISPOT技术检测到pcDNA3．1-omp17．3在小鼠体内诱导产生了以Th1型为主的细胞免疫应答。结果表明，构建的基因疫苗可作为潜在的布鲁氏菌新型疫苗，有进一步研究的意义。     

Brucella suis in three dogs: presentation,diagnosis and clinical management

Brucella suis is an emerging, zoonotic disease predominantly affecting dogs and humans that engage in feral pig hunting in Australia and other countries. Although B. suis infection in dogs shares some clinical similarities to the host-adapted species (B. canis), B. suis remains an incompletely understood pathogen in dogs with limited published data on its pathogenesis and clinical features. This case series describes the presentations, diagnosis, and clinical management of B. suis infection in three dogs: (1) a bitch with dystocia, abortion and mastitis; (2) an entire male dog with septic arthritis and presumptive osteomyelitis; and (3) a castrated male dog with lymphadenitis. Unique features of these cases are reported including the first documented detection of B. suis from milk and isolation from lymph nodes of canine patients, as well as the follow-up of pups born to a B. suis-infected bitch. Consistent with previous reports, all three dogs showed a favourable clinical response to combination antibiotic therapy with rifampicin and doxycycline. Individually tailored drug regimens were required based on the clinical presentation and other factors, including owner expectations and compliance with therapy as well as a zoonotic risk assessment (generally considered low, except around time of whelping). The authors include their recommendations for the clinical management of dogs that are at-risk or seropositive for B. suis with or without clinical signs or laboratory-confirmed infection.     

布鲁菌外膜蛋白Omp22基因的原核表达与生物信息学分析

克隆布鲁菌Omp22基因,原核表达后进行生物学信息分析。根据GenBank中羊种布鲁菌M5-90株基因组PCR扩增出639bp的目的基因片段,构建克隆重组质粒pMD-20T-Omp22,转化入E.coli DH5α。测序正确后构建表达重组质粒pET-28a-Omp22,转化入E.coli BL21(DE3)。IPTG诱导表达,Western blot鉴定诱导融合蛋白。结果表明,成功克隆Omp22基因,构建pET-28a-Omp22原核表达载体,在E.coli BL21(DE3)中表达Omp22基因。DNA Man和BIOEDIT软件生物性息学分析Omp22融合蛋白二级结构中α-螺旋占22.17%;伸展链占19.81%;β-折叠占2.83%;无规卷曲占55.19%。说明该蛋白具有较高亲水性。     

Diagnosis of Canine Brucellosis: Comparison between Serological and Microbiological Tests and a PCR Based on Primers to 16S-23S rDNA Interspacer

A pair of primers directed to 16S-23S rDNA interspacer (ITS) was designed directed to Brucella genetic sequences in order to develop a polymerase chain reaction (PCR) putatively capable of amplifying DNA from any Brucella species. Nucleic acid extracts from whole-blood from naive dogs were spiked with decreasing amounts of Brucella canis RM6/66 DNA and the resulting solutions were tested by PCR. In addition, the ability of PCR to amplify Brucella spp. genetic sequences from naturally infected dogs was evaluated using 210 whole-blood samples of dogs from 19 kennels. The whole-blood samples collected were subjected to blood culture and PCR. Serodiagnosis was performed using the rapid slide agglutination test with and without 2-mercaptoethanol. The DNA from whole blood was extracted using proteinase-K, sodium dodecyl sulphate and cetyl trimethyl ammonium bromide followed by phenol–chloroform purification. The PCR was capable of detecting as little as 3.8 fg of Brucella DNA mixed with 450 ng of host DNA. Theoretically, 3.8 fg of Brucella DNA represents the total genomic mass of fewer than two bacterial cells. The PCR diagnostic sensitivity and specificity were 100%. From the results observed in the present study, we conclude that PCR could be used as confirmatory test for diagnosis of B. canis infection.     

布鲁氏菌BPE159基因缺失株的构建及BPE159蛋白对细胞自噬因子表达的影响

【目的】构建布鲁氏菌BPE159基因缺失株,研究缺失株体外生长变化特征及其在宿主细胞中的存活能力,探究布鲁氏菌感染期间分泌蛋白BPE159对自噬因子表达的影响。【方法】同源重组方法构建布鲁氏菌BPE159基因重组质粒,电转化布鲁氏菌S2308感受态细胞构建BPE159基因缺失株S2308ΔBPE159。PCR扩增BPE159基因,连接转化构建pBBR1MCS-4-BPE159载体,提取质粒进行电转化,构建BPE159基因回补株S2308ΔBPE159-C。琼脂糖凝胶电泳检测缺失株和回补株遗传稳定性。构建布鲁氏菌感染小鼠巨噬细胞RAW264.7模型,实时荧光定量PCR检测布鲁氏菌侵染后自噬细胞因子ATG5、Beclin1、LC3a和LC3b基因表达水平。以S2308、S2308ΔBPE159和S2308ΔBPE159-C株侵染小鼠巨噬细胞,收集细胞总RNA,实时荧光定量PCR检测BPE159基因缺失对布鲁氏菌侵染后自噬细胞因子表达水平的影响。在相同起始浓度下培养S2308、S2308ΔBPE159及S2308ΔBPE159-C株,观察细菌生长变化趋势;评价S2308ΔBPE159株在不同...     

泛素结合酶UBE2T基因在布鲁氏菌感染过程中的作用

【目的】探讨泛素结合酶E2T（UBE2T）在布鲁氏菌感染过程中的功能。【方法】构建UBE2T基因过表达载体pLVX-puro-UBE2T,对其进行PCR和EcoR Ⅰ/BamH Ⅰ双酶切验证,并将其转染小鼠巨噬细胞RAW264.7,利用实时荧光定量PCR（qRT-PCR）检测UBE2T基因过表达效果。设计并合成3条UBE2T基因沉默表达的siRNA（UBE2T-379-siRNA、UBE2T-599-siRNA和UBE2T-661-siRNA）,将其转染小鼠巨噬细胞RAW264.7,利用qRT-PCR检测UBE2T基因沉默表达效果。采用四甲基偶氮唑盐（MTT）试验,检测UBE2T沉默或过表达对RAW264.7细胞活性的影响。利用qRT-PCR,检测M5-90感染对RAW264.7细胞UBE2T基因相对表达量的影响,以及对过表达或沉默表达UBE2T基因的RAW264.7细胞中含pyrin结构域NOD样受体家族3（NLRP3）和半胱氨酸天冬氨酸蛋白水解酶1（Caspase-1）基因相对表达量的影响。用布鲁氏菌M5-90菌株感染过表达和沉默UBE2T基因的RAW264.7细胞,用活菌计数法检测UBE2T沉默或过表达对RAW264.7细胞内布鲁氏菌增殖的影响,利用相应的试剂盒检测细胞乳酸脱氢酶（LDH）水平、半胱氨酸天冬氨酸蛋白水解酶3（Caspase-3）活性及白细胞介素6（IL-6）、IL-18、IL-1β和γ-干扰素（IFN-γ）等细胞因子的水平。【结果】成功构建了UBE2T基因过表达载体pLVX-puro-UBE2T,过表达效果良好。3条siRNA中,UBE2T-661-siRNA的干扰效果最佳,其对UBE2T基因的沉默效率为84%。UBE2T沉默或过表达对RAW264.7细胞活性无影响。M5-90感染可提高RAW264.7细胞UBE2T的表达量;沉默UBE2T可提高M5-90介导的NLRP3和Caspase-1的表达,而过表达UBE2T会抑制M5-90介导的NLRP3表达,提高M5-90介导的Caspase-1表达。沉默表达UBE2T可抑制胞内M5-90的增殖,而过表达UBE2T可促进胞内M5-90的增殖。沉默表达UBE2T可抑制M5-90介导的LDH水平,提高Caspase-3活性及IFN-γ、IL-6和IL-1β水平;而过表达UBE2T可提高M5-90介导的LDH水平,抑制Caspase-3的活性及IFN-γ、IL-1β水平。【结论】泛素结合酶UBE2T对胞内布鲁氏菌增殖具有正调控作用。     

Use of Brucella abortus vaccine strain RB51 in pregnant cows after calfhood vaccination with strain 19 in Argentina

One hundred and seven pregnant cows, which had been calfhood vaccinated with Brucella abortus strain 19 (S-19) were revaccinated with either S-19 or strain RB51 (S-RB51). All S-19-revaccinated animals seroconverted, while none of the RB51-revaccinated animals seroconverted. Two out of 25 (8%) S-19-revaccinated animals aborted, while none of the 57 RB51-revaccinated group aborted. Four of the S-19-revaccinated animals shed S-19 in the milk for at least 7 days, while only 1 cow shed S-RB51 for at least 3 days (but <7 days) post-parturition. Revaccination of strain 19 calfhood-vaccinated, pregnant cattle with S-RB51 appears to be a safe procedure with no diagnostically negative consequences.     

呼和浩特布鲁氏菌分离株bp26基因的原核表达

目的:克隆布鲁氏菌外膜蛋白bp26基因并构建原核表达系统.方法:用聚合酶链式反应技术扩增得到布鲁氏菌bp26基因片段,与PEASY-E1载体链接,转入BL21(DE3)感受态细胞中,经IPTG诱导后,经SDS-PAGE检测重组蛋白表达,用Western blot鉴定目的蛋白的生物活性.结果:DNA测序结果显示,重组质粒bp26基因的插入位点正确,成功构建了重组质粒PEASY-E1-bp26,经IPTG诱导,表达出大小为27kDa的bp26融合蛋白.结论:获得布鲁氏菌外膜蛋白bp26基因片段,并在大肠杆菌中表达出具有生物活性的bp26融合蛋白.