Properties and partial genetic characterization of Nepean phage and other lytic phages of Brucella species.

Nepean (Np), a new brucellaphage, was associated with atypical Brucella abortus strains from Ontario cattle. Carriage of Np was associated with loss of smooth lipopolysaccharide, changes in some protein bands in acrylamide gel electrophoresis profiles, increased susceptibility to colistin, and increased resistance to ultraviolet killing. Nepean (Np) was compared with brucellaphages Tb, Fi, Wb, Iz and R/C. All were morphologically identical, with icosahedral capsids (50-65 nm diameter) and short tails (15-25 nm long), but Np had a more restricted host range, replicating only in smooth strains of B. abortus. All six brucellaphages were generally similar in resistance to chemical and physical agents. Brucellaphage DNA was double stranded and unmethylated; its molecular size was 38 kilobase pairs. The DNAs of Tb, Fi, Wb, Iz and R/C could not be differentiated by restriction endonuclease digest profiles produced by BgII, EcoRI, HindIII or PvuII. Nepean (Np) DNA was very similar to that of the other brucellaphages, but with every enzyme used its profile differed in the number and/or position of at least one fragment. However, there was complete cross-hybridization of Tb and Np DNAs. Hybridization techniques failed to detect Brucella DNA in Dp or Tb phages, or phage DNA in Brucella cells. Extrachromosomal plasmid DNA was not detected.     

Use of phage for the identification of Brucella canis and Brucella ovis cultures

The brucella phage strains R, R/O and R/C standardised at routine test dilution on their propagating strains were effective in identifying cultures of Brucella ovis and B canis and in differentiating these from other non-smooth brucella isolates.     

Restriction endonuclease analysis of Brucella ovis and other Brucella species

Brucella ovis DNA was analysed by using 11 different restriction endonucleases. The most clearly resolved DNA fragment patterns were obtained after digestion with the enzyme Hind III. When DNA preparations from 35 strains of B. ovis were digested with this enzyme, the fragment patterns appeared to be identical. The patterns obtained after Hind III digestion of DNA from one strain each of B. abortus, B. canis and B. melitensis were more similar to each other than to the B. ovis pattern.     

Urease activity of Brucella species

Examination of the urease activity of 604 brucella strains showed a limited correlation with species. Most strains of B canis, B neotomae and B suis gave a positive urease reaction within 15 minutes, although some exceptions were noted. A substantial proportion of strains of B abortus and B melitensis also hydrolysed urea as rapidly as most B suis strains. Although most B ovis strains were negative to the urease test, 28.9 per cent of those examined gave positive reactions.     

Expression and lytic efficacy assessment of the Staphylococcus aureus phage SA4 lysin gene

Treatment of bovine mastitis caused by Staphylococcus (S.) aureus is becoming very difficult due to the emergence of multidrug-resistant strains. Hence, the search for novel therapeutic alternatives has become of great importance. Consequently, bacteriophages and their endolysins have been identified as potential therapeutic alternatives to antibiotic therapy against S. aureus. In the present study, the gene encoding lysin (LysSA4) in S. aureus phage SA4 was cloned and the nucleotide sequence was determined. Sequence analysis of the recombinant clone revealed a single 802-bp open reading frame encoding a partial protein with a calculated mass of 30 kDa. Results of this analysis also indicated that the LysSA4 sequence shared a high homology with endolysin of the GH15 phage and other reported phages. The LysSA4 gene of the SA4 phage was subsequently expressed in Escherichia coli. Recombinant LysSA4 induced the lysis of host bacteria in a spot inoculation test, indicating that the protein was expressed and functionally active. Furthermore, recombinant lysin was found to have lytic activity, albeit a low level, against mastitogenic Staphylococcus isolates of bovine origin. Data from the current study can be used to develop therapeutic tools for treating diseases caused by drug-resistant S. aureus strains.     

光滑型布鲁氏菌LPS单克隆抗体的制备及鉴定

为制备抗布鲁氏菌单克隆抗体(MAb),本研究用灭活的布鲁氏菌疫苗M5-90株免疫BALB/c小鼠,利用细胞融合技术获得杂交瘤细胞.以布氏杆菌疫苗株M5-90、S-19及小肠结肠炎耶尔森菌0:9的脂多糖(LPS)分别作为抗原包被酶标板,对杂交瘤细胞进行筛选,获得两株稳定分泌抗光滑型布氏杆菌LPS(S-LPS)MAb的细胞4G6和16C5.特异性分析表明,MAb 4G6除与耶尔森0:9全菌体有轻微的交叉反应外与其它革兰氏阴性菌无交叉,而16C5完全排除了与其他各菌的交叉反应.Ig亚型分析表明,所制备MAb的轻链亚类均为κ,4G6重链为IgM.16C5重链为IgG3.用Protein G亲和层析纯化MAb 16C5腹水并初步用于竞争ELISA方法,检测布氏杆菌抗体水平,结果表明了该实验方法的有效性.本研究制备的MAb 16C5具有高度的特异性和敏感性,排除了与其他革兰氏阴性菌的交叉反应,为建立一种诊断布鲁氏病的快速、有效、敏感的方法奠定了有力的基础.     

不同种型布鲁菌标准株的比较基因组学研究

探究不同种型布鲁菌标准株的基因组构成差异。通过布鲁菌全基因组DNA芯片技术对19株不同种型布鲁菌标准株进行比较基因组学研究。结果显示：19株不同种型布鲁菌标准株之间存在大量缺失基因,同时发现一些基因以多拷贝形式存在。缺失基因的功能大致分为4类：信息储存和传递;胞内活动处理;营养代谢、功能未知或仅了解部分功能,共鉴定到这类基因211个。深入认识了19株不同种型布鲁茵标准株基因组组成上的差异,为我们进一步认识不同种以及亚型在毒力以及宿主特殊性提供了依据。大量缺失基因在19株布鲁菌标准株出现,构成了布鲁菌标准株不同种以及亚型之间的遗传学基础。     

Brucella species lacking the major outer membrane protein Omp25 are attenuated in mice and protect against Brucella melitensis and Brucella ovis

To aid in the development of novel efficacious vaccines against brucellosis, Omp25 was examined as a potential candidate. To determine the role of Omp25 in virulence, mutants were created with Brucella abortus (BA25), Brucella melitensis (BM25), and Brucella ovis (BO25) which contain disruptions in the omp25 gene (Deltaomp25 mutants). Western immunoblot analysis and PCR verified that the Omp25 protein was not expressed and that the omp25 gene was disrupted in each strain. BALB/c mice infected with B. abortus BA25 or B. melitensis BM25 showed a significant decrease in mean CFU/spleen at 18 and 4 weeks post-infection, respectively, when compared to the virulent parental strain (P<0.05, n=5). Mice infected with B. ovis BO25 had significantly lower mean CFU/spleen counts from 1 to 8 weeks post-infection, at which point the mutant was cleared from the spleens (P<0.01, n=5). Murine vaccination with either BM25 or the current caprine vaccine B. melitensis strain Rev. 1 resulted in more than a 2log(10) reduction in bacterial load following challenge with virulent B. melitensis (P<0.01, n=5). Vaccination of mice with the B. ovis mutant resulted in clearance of the challenge strain and provided 2.5log(10) greater protection against virulent B. ovis than vaccine strain Rev. 1. Based on these data, the B. melitensis and B. ovis Deltaomp25 mutants are interesting vaccine candidates that are currently under study in our laboratory for their safety and efficacy in small ruminants.     

Molecular typing of Brucella species isolates from livestock and human

Although host specificity has been observed in different species of Brucella, crossing the animal host boundary is likely to occur at any time. In this study, Bruce ladder PCR and abortus–melitensis–ovis–suis (AMOS) PCR assays were used to characterize 47 Brucella isolates from Indian origin in order to know exact species for understanding epidemiology of brucellosis. Out of them, 28, 14, and 5 isolates were found to be Brucella abortus, Brucella melitensis, and Brucella suis, respectively. Further analysis by AMOS PCR has identified that all the B. abortus isolates belong to any one of the biovar 1, 2, or 4; of the five B. suis isolates, three belong to biovar 1 and two belong to any one of the biovar 2, 3, 4, or 5. Although this multiplex Bruce ladder PCR is useful in differentiating all Brucella species, elaborate study is required to further characterize the isolates at exact biovar level.     

Comparison of protein components of different species and strains of Brucella

The proteins constituents of Brucella abortus, Brucella melitensis and Brucella ovis were analyzed SDS-PAGE. From the comparison appears that the three species of Brucella studied shows a different electrophoretic pattern specially at the level of small peptides. On the contrary when two strains of B. abortus are analyzed no differences can be noticed.     

Differential reactivity of bovine lymphocytes to species of Brucella

The reactivity of bovine lymphocytes to 4 species of Brucella was tested in thymidine-uptake assays, using long-term cultured lymphocytes and freshly obtained blood mononuclear cells. Lymphocytes were taken from cows that had been challenge exposed with a virulent strain of B abortus at midgestation. The cows were classified retrospectively as being naturally resistant or susceptible to brucellosis. Lymphocytes taken from these cows had 3 patterns of reactivity with species of Brucella: pattern 1 was defined by reactivity with 4 species (B abortus, B canis, B suis, and B melitensis); pattern 2 was defined by reactivity with all these species, except B melitensis; pattern 3 was defined by reactivity with B abortus and B canis, but not with B suis or B melitensis. There was a statistically significant correlation between susceptibility to brucellosis and expression of lymphocyte cross-reactivity with B suis (P less than 0.01) and with B melitensis (P less than 0.001).     

IS711和omp2作为布氏杆菌种属及种株间分子鉴别诊断标记的研究

布氏杆菌为世界性具重要公共卫生意义的人兽共患疫病原，分6个种。建立种间及种株间安全敏感、经济有效的快速鉴别诊断方法对布病防制及分子流行病学研究具有重要意义。布氏杆菌IS711和omp2基因具有种属特异性，可用于布氏杆菌的PCR分子诊断。其中IS711为转座因子，在不同种布菌种存在插入位置的多态性，外膜蛋白OMP2编码基因则存在反向重复序列及种株间的多态性。为此，分别采用复式-PCR、PCR和限制性酶切片段长多态性（RFLP）分析，对分属于B．mclitcnsis、B．suis和B．abortus的不同种布氏杆菌的不同种株，M5、M16、S2、S6和S19进行分子鉴别诊断。结果显示，根据IS711基因特定PCR扩增片段长多态性，可进行布氏杆菌种间的快速鉴别；而omp2编码基因PCR扩增片段PsrⅠ、KpnⅠ、NcoⅠ和Eco47 Ⅲ等4种限制酶片段长多态性，则可成为布氏杆菌菌株间特异的分子鉴别诊断标记，甚至疫苗株M5和野毒株M16之间的分子诊断标记。