First human isolate of Leptospira interrogans as serovar bratislava in Italy

Abstract A strain of Leptospira interrogans was isolated from a patient suffering from leptospirosis and was typed by the Gross Agglutination Absorption test using monoclonal antibodies prepared against different serovars of the Australis serogroup. This newly isolated strain belonged to serovar bratislava . It is the first reported isolation from man, in Italy, of Leptospira bratislava , thus supporting the emerging role of this serovar in human leptospirosis.     

Serological and molecular characterization of leptospira serovar Kenya from captive African giant pouched rats (Cricetomys gambianus) from Morogoro Tanzania

Two identical leptospiral isolates coded Sh9 and Sh25 obtained from the urine of captive African giant pouched rats (Cricetomys gambianus), destined for use as biodetector of antipersonnel landmines were typed as serovar Kenya using cross-agglutination absorption test and DNA fingerprinting with the insertion element sequences IS1533 and IS1500 derived primers. The two isolates were previously characterized using cultural and serological-microagglutination test as pathogenic leptospires of the serogroup Ballum, closely related to serovars Kenya and Peru. To our knowledge, this is the first reported in-depth characterization of leptospira isolates from Tanzania.     

表达T~+Pm保护性抗原的重组猪霍乱沙门氏菌C500株的构建及其生物学特性

【目的】本研究利用Asd+平衡致死系统构建表达巴氏杆菌毒素(Pasteurella multocida toxin,PMT)的重组猪霍乱沙门氏菌株,并对重组菌株的生物学特性进行比较研究。【方法和结果】通过基因克隆的方法构建表达PMT的重组质粒pYA-PmtC,再将其电转化减毒猪霍乱沙门氏菌C500的asd基因缺失株C501,构建口服活疫苗菌株C501(pYA-PmtC)。研究结果表明重组菌株C501(pYA-PmtC)的生化特性、血清型和生长速度与亲本菌株C500一致;在没有选择压力的条件下,C501(pYA-PmtC)能够稳定遗传重组质粒及其外源基因片段,并能稳定、高效、分泌性表达30.5kDa的外源保护性抗原rPmtC。C501(pYA-PmtC)腹腔感染BALB/c小鼠的LD50为8.5×106CFU,毒力稍低于C500(LD50为4.4×106CFU);口服接种C501(pYA-PmtC)和C500的所有仔猪未见任何发病症状,两者没有显著差别。【结论】本研究利用Asd+平衡致死系统的原理构建表达T+Pm保护性抗原重组猪霍乱沙门氏菌弱毒菌株C501(pYA-PmtC),为进一步开发猪萎缩性鼻炎-副伤寒的双价基因工程疫苗奠定基础。     

Simple and rapid detection of Salmonella serovar Enteritidis under field conditions by loop‐mediated isothermal amplification

Aim: The objective of this study is to develop a serovar‐specific loop‐mediated isothermal amplification (LAMP) method for sensitive, rapid, and inexpensive detection of Salmonella serovar Enteritidis under field conditions. Methods: A set of six specific primers was designed with Salmonella Enteritidis DNA as the target. LAMP conditions were optimized by incubating the target DNA with the Bst DNA polymerase large fragment in a simple water bath. The sensitivity and specificity of LAMP was then compared with those of fluorescent quantitative real‐time polymerase chain reaction (FQ‐PCR). Results: The results were as follows. (1) Serovar‐specific Salmonella Enteritidis DNA was amplified at 65°C in as early as 20 min in a water bath. (2) A colour change visible to the naked eye indicated a positive amplification reaction. (3) The detection limit of the LAMP assay was 4 copies μl^?1; thus, the sensitivity and specificity of this assay is similar to those of the FQ‐PCR. Conclusions: LAMP is a high‐throughput detection technique with high sensitivity, specificity, and simplicity; these factors make it suitable for specifically detecting Salmonella Enteritidis under field conditions and in laboratory settings. Thus, LAMP eliminates the need for complicated equipment and technical training in the detection of this specific serovar. Significance and impact of the study: This is the first study involving the use of LAMP to detect Salmonella serovar‐specific DNA sequences. It is also the first to report an ideal method of distinguishing between Salmonella Enteritidis and other Salmonella under field conditions.     

北京地区首次发现赖型钩端螺旋体

采用分群和分型因子血清经显微镜凝集试验（ＭＡＴ）对由北京市顺义县某农场稻田周围黑线姬鼠肾组织分离获得的４株钩端螺旋体（钩体）进行了血清学分类检定。均属黄疸出血群赖型钩体。这在北京地区尚属首次分离发现。     

An indirect fluoresent antibody staining procedure for detection of Vibrio cholerae serovar 01 cells in aquatic environmental samples

An immunofluorescence method for detection of Vibrio cholerae serovar 01 in aquatic environmental samples and enrichment broths is described. Antiserum specific for the 01 somatic antigen was produced in rabbits and used in an indirect fluorescent antibody method incorporating fluoresceinisothiocyanate conjugated anti-rabbit globulin goat serum, and rhodamine isothiocynate conjugated bovine serum albumin as background stain. Comparisons of the immunofluorescent procedure and conventional culture methods for isolation of V. cholerae 01 showed that detection occurred significantly more frequently with the fluorescent antibody system.     

沙眼衣原体15个血清型omp1基因的VS1和VS2区序列分析

目的比较分析沙眼衣原体15个血清型omp1基因VS1和VS2序列的同源性和变异性。方法巢式PCR扩增VS1-VS2基因,自动测序仪测定核苷酸序列,DNAstar生物软件进行比对分析。结果15个血清型沙眼衣原体扩增出大小453bp的VS1-VS2基因。VS1区域序列比对显示血清群B和中间群的VS1核苷酸序列相对保守,而血清群C各型VS1区域表现出较大的核苷酸变异,型间显示1～9个核苷酸替换,且发生在中心区域。血清型VS2序列较VS1存在更多的变异,血清群B中各血清型间均存在2～19个核苷酸的改变,血清群C表现为4～8个核苷酸差异,中间群的F和G型之间存在6个核苷酸差异。结论阐明VS1和VS2区核苷酸的多态性,为下一步进行该蛋白表达和构建寡核苷酸型特异性探针奠定了基础。     

Analysis of Chlamydia trachomatis serovars in endocervical specimens derived from pregnant Japanese women

The polymerase chain reaction (PCR) method has been employed to amplify a chlamydial genome encoding four variable segments of the major outer membrane protein and genotyping of different Chlamydia trachomatis serovars was successfully achieved by means of restriction fragment length polymorphism (RFLP) analysis and sequencing of amplified DNA. These methods were applied to identify the serotypes of C. trachomatis in endocervical specimens obtained from asymptomatic pregnant Japanese women at 28-30 weeks of gestation. Among the 218 specimens, 207 were serotyped 43 (19.3%) as serovar D, 53 (24.3%) as E, 24 (11.0%) as F, 39 (17.9%) as G, 15 (6. 9%) as H, 15 (6.9%) as I, five (2.3%) as J, nine (4.1%) as K and four (1.8%) as mixed. Among the 11 unclassified strains by RFLP, six (2.8%) were identified as serovar B variants and five (2.3%) were identified as D/IC-Cal-8. It was suggested that variants of endemic trachoma serovars also have affinity for the urogenital tract of Japanese pregnant women.     

Assessment of the efficacy of Japanese Bacillus thuringiensis isolates against the cigarette beetle,Lasioderma serricorne (Coleoptera: Anobiidae)

A total of 2,652 Japanese isolates of Bacillus thuringiensis, belonging to at least 54 H serogroups, were examined for assessment of the toxicity against the cigarette beetle, Lasioderma serricorne (Coleoptera: Anobiidae). When tested with spore/parasporal inclusion mixtures, strong larvicidal activities were associated with 28 isolates (1.1%). Serologically, these toxic isolates fell into 4 known H serovars: thuringiensis (9 isolates), kurstaki (2), kenyae (2), and darmstadiensis (15). Purified parasporal inclusions of the 10 selected isolates exhibited no larvicidal activity, while the supernatants of liquid cultures showed larvicidal and/or growth inhibitory effects. The activities were fully retained after heat treatment at 100 degrees C for 10 min. Overall results suggest that beta-exotoxin (or thuringiensin)-related substances are responsible for the toxicity of the present B. thuringiensis isolates against the cigarette beetle.     

Rapid identification of Salmonella enterica serovars, Typhimurium, Choleraesuis, Infantis, Hadar, Enteritidis, Dublin and Gallinarum, by multiplex PCR

Salmonella enterica subsp. enterica poses a threat to both human and animal health, with more than 2500 serovars having been reported to date. Salmonella serovars are identified by slide and tube agglutination tests using O and H antigen-specific anti-sera, although this procedure is both labor intensive and time consuming. Establishment of a method for rapid screening of the major Salmonella serovars is therefore required. We have established multiplex polymerase chain reaction (m-PCR) assays for identification of seven serovars of Salmonella, i.e., Typhimurium, Choleraesuis, Infantis, Hadar, Enteritidis, Dublin and Gallinarum. Three serovar-specific genomic regions (SSGRs) of each serovar were selected using an approach in comparative genomics. The Salmonella-specific invA gene was used to confirm the genetic background of the organisms. The isolates tested were identified as a target serovar when the three selected SSGRs and invA were all positive for amplification. The specificity of each m-PCR assay was investigated using 118 serovars of Salmonella and 12 species of non-Salmonella strains. Although a small number of false-positive results were observed in the m-PCR assays used to identify Typhimurium, Choleraesuis, Enteritidis and Dublin for closely related serovars, false-negative results were not observed in any assays. These assays had sufficient specificity to identify the seven Salmonella serovars, and therefore, have the potential for use as rapid screening methods.