Two new subspecies of Bacillus thuringiensis isolated in Japan: Bacillus thuringiensis subsp. kumamotoensis (serotype 18) and Bacillus thuringiensis subsp. tochigiensis (serotype 19)

Bacteriological and serological characteristics of three Bacillus thuringiensis isolates obtained in Japan were investigated. They formed typical rhomboidal parasporal inclusions but flagellar (H) antigens of these isolates were different from those of the known 17 H serotypes of B. thuringiensis. The three isolates were divided into two new serotypes (serotypes 18 and 19). The serotype 18 isolate (3–71) produced thermostable exotoxin and the inclusions of this isolate were toxic to larvae of the silkworm, Bombyx mori, but nontoxic to larvae of the mosquito, Aedes aegypti. The other isolate (119-72) belonging to serotype 18 produced inclusions nontoxic to larvae of B. mori and A. aegypti and did not produce thermostable exotoxin. However, other bacteriological properties of the isolate 119-72 were similar to those of the isolate 3–71. The serotype 19 isolate (117-72) produced inclusions nontoxic to larvae of B. mori and A. aegypti and did not produce thermostable exotoxin. Acid production from saccharose and the production of brownish purple pigment were observed in the two serotype 18 isolates, while neither of them was observed in the serotype 19 isolate. In other 29 biochemical properties tested, there was no difference among the three isolates. Based on these characteristics, the following two subspecies names are proposed: Bacillus thuringiensis subsp. kumamotoensis (serotype 18) for the type strain 3–71 and Bacillus thuringiensis subsp. tochigiensis (serotype 19) for the type strain 117-72.     

Proteome analysis of Streptococcus suis serotype 2

Outbreaks in humans, caused by Streptococcus suis serotype 2 (SS2), were reported in 1998 and 2005 in China. However, the mechanism of SS2-associated infection remains unclear. For the first time, a 2-D gel approach combined with MS was used to establish a comprehensive 2-D reference map for aiding our understanding of the pathogenicity of SS2. The identification of 694 out of 834 processed spots revealed 373 proteins. Most of the identified proteins were located in the cytoplasm and were involved in energy metabolism, protein synthesis, and cellular processes. Proteins that were abundant in the 2-DE gels could be linked mainly to housekeeping functions in carbohydrate metabolism, protein quality control and translation. 2-DE of secretory proteins was performed using IPG strips of pH 4-7. Among the 102 protein spots processed, 87 spots representing 77 proteins were successfully identified. Some virulence-associated proteins of SS2 were found, including arginine deiminase, ornithine carbamoyl-transferase, carbamate kinase, muramidase-released protein precursor, extracellular factor, and suilysin. Enolase and endopeptidase have been proposed as putative virulence-associated factors in this study. The 2-D reference map might provide a powerful tool for analyzing the virulence factor and the regulatory network involved in the pathogenicity of this microorganism.     

New serotype of mutans streptococci isolated from pig oral cavity

Gram-positive streptococcal mutans-like strains, but with clearly different colony formation than S. orisuis on Mitis Salivarius agar, were isolated from the pig oral cavity and identified by 16S rRNA sequencing, G+C content, DNA-DNA homology and extensive biochemical and serological testing. The phenotypic data showed that the strains were similar to S. orisuis except for susceptibility to bacitracin. DNA-DNA homology between the isolates and S. orisuis was 72∼81%. However, serological data showed that they have a different sero-specific antigen from S. orisuis and other mutans streptococci. A new serotype, designated p, strains are classified in a serovar of S. orisuis, one of mutans streptococci.     

Immunoproteomic assay of surface proteins of Streptococcus suis serotype 9

Streptococcus suis is an important swine pathogen responsible for a diverse group of diseases. Studies on vaccines have focused on S. suis serotype 2 strains, which are the most invasive isolates worldwide. However, in China S. suis serotype 9 (SS9) is also a prevalent serotype, which is frequently isolated from diseased pigs. Little is known about immunogenic proteins for SS9. Therefore, an immunoproteomic-based approach was developed to identify immunogenic proteins of SS9. Cell wall proteins extracted from SS9 strain GZ0565 isolated from a diseased pig with meningitis were screened by two-dimensional Western blotting using anti-SS9 sera pooled from specific pathogen-free mice. Protein spots were excised from preparative gels and identified by matrix-assisted laser desorption ionization time-of-flight MS (MALDI-TOF-MS) or MALDI-TOF-TOF-MS, which led to the identification of eight immunogenic proteins (arginine deiminase, extracellular solute-binding protein, translation elongation factor Ts, neprilysin, peptide ATP-binding cassette transporter peptide-binding protein, pyruvate kinase, phosphate acetyltransferase, and fructose-bisphosphate aldolase). These immunogenic proteins, which are encoded by genes that are reasonably conserved among SS9 strains, could be developed as vaccine candidates.     

猪链球菌2型次黄嘌呤核苷酸脱氢酶基因的克隆与鉴定

根据GenBank猪链球菌2型(SS2)报道序列,对江苏分离株SS2-H部分测序,发现位于已知毒力基因orf2与mrp之间存在两个新的开放阅读框序列。选取可能含有抗原决定簇肽段对应的核酸序列,该阅读框(2738~3694)编码319个氨基酸残基,分子量为33.5kDa,与已知任何基因无同源性。通过InterPro、PHD、DNAstar分析阅读框,并定向克隆至pET-32α( )载体中,转化至大肠杆菌BL21,表达出分子量为48kDa的融合蛋白,蛋白免疫转印可被SS2的抗血清识别,具有免疫原性;并且含有IMP dehydrogenase结构域,催化IMP生成XMP;流式细胞仪检测该蛋白可明显影响HEp-2细胞周期。     

Expanding distribution of human serotype G6 rotaviruses in Australia

Serotype G6 rotaviruses are common pathogens of cattle but are rarely found in humans. In Australia, human G6 isolates have previously been detected in two major southern population centres. A new isolate, ASG6.02, was detected in central Australia (Alice Springs) in 1997. Comparison of the deduced amino acid sequence of the major neutralizing antigen, VP7, indicated that ASG6.02 was related to human G6 viruses isolated from children in Italy and Australia. Phylogenetic analysis supported the close relationship between ASG6.02 and other Australian isolates and indicated that G6 VP7 sequences generally clustered according to the species of origin (human, bovine or porcine). The VP4 type of ASG6.02 was determined as P-type [14], in common with other isolates from Australia and Italy. The detection of ASG6.02 indicates that the distribution of this serotype is increasing in this country and may have implications for successful vaccine development.     

鸭肝炎病毒新血清型基因组序列分析

[目的]为了揭示鸭肝炎病毒新血清型(DHV-N)G株的演化及其与DHV-1的基因序列差异.[方法]运用RT-PCR结合5'-/3'-RACE策略对DHV-N G株基因组进行扩增,克隆测序后进行序列分析.[结果]DHV-NG株全基因组序列除12 nt poly(A)尾外全长共7774 nt,含有一个大的开放阅读框,编码2251 aa的蛋白前体,其基因组结构:5'UTR-L-VP0-VP3-VP1-2A1-2A2-2B-2C-3A-3B-3C-3D-3'UTR;3'UTR长为366 nt,比1型鸭肝炎病毒C80株多52 nt;VP1蛋白与DHV-1相比发生较大的变异,特别在其第140～221位;其基因组与DHV-1、韩国DHV-N和台湾DHV-N的同源率分别为72.8%～73.4%、96.3%～96.5%和78.3%.[结论]DHV-N G株基因组结构与DHV-1存在明显差异,在DHV-N成员DHV-N G株与韩国DHV-N关系更为密切.     

金葡菌荚膜多糖血清型的研究进展

金葡菌是引起奶牛乳腺炎的一种主要的致病菌,致病性金葡菌多数含有荚膜成分,金葡菌荚膜多糖有11种血清型。从不同血清型荚膜多糖的结构,基因构成,抗吞噬作用,致病性等方面作了介绍,并简要介绍了金葡菌荚膜多糖血清型的国内外研究现状。     

Epidermolytic toxin serotype B of Staphylococcus aureus is plasmid-encoded

Abstract The gene coding for epidermolytic toxin serotype B ( etb ) was cloned in a plasmid expression vector in Escherichia coli . Its expression was dependent on the vector plasmid's trc promoter. A polypeptide immunochemically indistinguishable from the purified staphylococcal toxin and with the same molecular weight was predominantly localized in the periplasm of E. coli . The etb gene resides on a 1.7 kb Hin dIII fragment of the 42 kb plasmid pTC142 present in the parental Staphylococcus aureus strain.     

Antigenic relationship among the eight prototype and new serotype strains of Orientia tsutsugamushi revealed by monoclonal antibodies.

Orientia tsutsugamushi, the etiologic agent of tsutsugamushi disease, exhibits great antigenic variation. Three classical strains (Karp, Gilliam, and Kato) and new antigenic types from Thailand (TA686, TA678, TA716, TA763, and TH1817) have been used as prototype strains of O. tsutsugamushi in many studies. In this study, monoclonal antibodies to the five Thailand strains were produced, and their reactivity against prototype strains and newly identified isolates from Korea and Japan was tested. With a panel of these monoclonal antibodies, we could analyze the antigenic relationship among various strains of O. tsutsugamushi from Thailand, Japan, and Korea. Twelve strains of the O. tsutsugamushi tested showed various reactivities to monoclonal antibodies, and no distinct pattern of reactivity was found according to their location of isolation. Although the Boryong and Kuroki strains were similar in reactivities to most monoclonal antibodies, several monoclonal antibodies could differentiate the two strains. These results indicate that the immunofluorescence antibody test using monoclonal antibodies used in this study is valuable for analyzing the antigenic relationship and classification of O. tsutsugamushi.     

猪链球菌2型不同mrp基因型与毒力的关系

【目的】猪链球菌2型(Streptococcus suis serotype 2,SS2)是重要的人畜共患病病原,且有强弱毒株之分,但至今仍缺少合适的毒力标志基因来鉴定致病性SS2。本文旨在研究mrp基因型与SS2毒力的关系。【方法】通过PCR方法鉴定不同SS2菌株的mrp基因型。再通过"内标"化的斑马鱼感染模型和实时荧光定量PCR,分别测定不同mrp基因型菌株的毒力水平和mrp转录水平。【结果】根据PCR结果可将53株SS2分为mrp-A型(27株)和mrp-B型(26株)两种基因型;mrp-A型菌株比mrp-B菌株毒力偏强,且A型菌株中mrp转录水平更高。【结论】发现mrp基因在SS2中分布广泛,但不同菌株中mrp基因型不同,mrp-A型菌株的致病力更强。而且,以mrp非保守区域作为诊断靶点能有效鉴定SS2强毒株。     

2型猪链球菌新毒力因子PlcR的发现

<正>2型猪链球菌(SS2)是一种重要的人畜共患传染病病原体。SS2感染不仅可致猪急性败血症、脑膜炎、关节炎、心内膜炎及急性死亡,并且可通过伤口和呼吸道等传播途径,导致人的感染发病和死亡。1998年和2005年在我国江苏"苏中"地区和四川资阳等市县人群中曾先后两次暴发大规模SS2感染人的事件。人感染病例中出现了从未报道过的链球菌中毒性休克综合征(Streptococcal toxic shock syndrome,STSS),病死率高达80%以上,已成为重要的新发传染病病原体[1-4]。     

高致病性2型猪链球菌plcR基因敲除株的构建及其相关生物学特性的研究

【目的】构建高致病性2型猪链球菌05ZYH33菌株plcR基因敲除株,通过比较突变株与野生株生物学特性的差异,研究plcR基因在2型猪链球菌致病过程中的作用。【方法】利用同源重组技术敲除plcR基因,多重交叉PCR及RT-PCR鉴定并测序验证。比较野生株与突变株基本生物学特性的差异,小鼠攻毒实验分析plcR基因缺失对细菌毒力的影响。【结果】经RT-PCR证实05SSU0241与05SSU0242共转录,通过多重交叉PCR及RT-PCR证实成功构建plcR基因缺失突变株,基本生物学特性显示突变株的生长速率、菌落形态、溶血活性均无显著改变,小鼠致病性试验结果显示,野生株攻毒的小鼠死亡率为70%,突变株攻毒的小鼠死亡率为40%,毒力较野生株显著降低。【结论】plcR基因作为2型猪链球菌有毒株基因组中特有的外源基因,在细菌致病过程中具有重要作用。     

Recombinant Bivalent Vaccine against Foot-and-Mouth Disease VirusSerotype O／A Infection in Guinea Pig

Foot-and-mouth disease (FMD) is a highly contagiousdisease of cloven-hoofed animals such as cattle and pig.The disease causes explosive epidemics and heavyeconomic losses in the agriculture worldwide [1]. FMDvirus (FMDV) shows a high genetic and antigenicvariability, and has seven serotypes: O, A, C, AsiaI, SAT1,SAT2 and SAT3 [2]. The FMDV control is mainly imple-mented using chemically inactivated virus vaccines, whichmay contain residual living virus and pose a risk of virusreleas…     

高致病性2型猪链球菌截短型类枯草杆菌丝氨酸蛋白酶基因的鉴定和功能研究

【目的】克隆表达高致病性2型猪链球菌05ZYH33株的SspA截短型基因,验证其是否具有酶学活性,并构建该基因的缺失突变株细菌,探讨其在2型猪链球菌致病过程中所起的作用【。方法】构建SS2的SspA截短型基因05SSU0811原核表达质粒,表达并纯化05SSU0811蛋白,运用丝氨酸蛋白酶底物Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide(pNa),通过显色反应检测表达产物的酶学活性;运用同源重组技术敲除05SSU0811基因,多重交叉PCR筛选敲除株并测序鉴定,动物试验分析05SSU0811基因缺失对细菌毒力的影响。【结果】成功表达并纯化05SSU0811蛋白,浓度约为3.5 g/L。丝氨酸蛋白酶活性测定试验证实其具有酶学活性;获得05SSU0811基因缺失突变株,小鼠攻毒试验表明,野生株攻毒的20只小鼠全部死亡,基因缺失突变株攻毒组死亡9只,死亡率45%,两组间死亡率有显著性差异。表明05SSU0811基因缺失的菌株毒力较野生株明显下降。【结论】05SSU0811基因编码的截短型丝氨酸蛋白酶仍然具有酶学活性,SS2的截短型基因SspA在高致病性2型猪链球菌的致病性方面具有一定作用。     

Streptococcus suis serotype 2 binding to extracellular matrix proteins

Streptococcus suis serotype 2 is a major swine and human pathogen that causes septicemia and meningitis. The ability of S. suis serotype 2 to bind to different extracellular matrix (ECM) proteins was evaluated by ELISA. All 23 strains tested bound to plasma and cellular fibronectin and collagen types I, III, and V, some to fibrin, vitronectin, and laminin, and none to the other ECM proteins tested. An unencapsulated isogenic mutant bound to ECM proteins better than its parental encapsulated strain, suggesting that the polysaccharide capsule interfered with binding. Cross-inhibition was observed between soluble plasma fibronectin and collagens in the ECM adherence assay, indicating that binding domains for both proteins exist on the same or nearby bacterial surface molecules. On the other hand, pre-incubation with plasma fibronectin increased binding to collagen IV, suggesting that S. suis might use fibronectin as a bridging molecule. The results of heat treatment and proteolytic digestion suggest that adhesins for these ECM proteins are proteinaceous in nature.     

Biological activity and identification of a peptide inhibitor of LuxS from Streptococcus suis serotype 2

The virulence of bacterial communities may be regulated by mechanisms involving the synthesis of the quorum-sensing signal autoinducer 2 (AI-2), which allows both intra- and interspecies communication. AI-2 is produced in bacteria that express the gene luxS . In the present study, expressed and purified LuxS from Streptococcus suis serotype 2 (SS2) was used to catalyze the substrate S -ribosylhomocysteine in a reaction that leads to the production of AI-2. The biological activity of the in vitro synthesized AI-2 was demonstrated in a Vibrio harveyi strain BB170 bioassay; real-time PCR results showed that biosynthesis of AI-2 can increase the virulence of SS2. Phage-encoded peptides that specifically interact with the LuxS enzyme were selected following three rounds of phage display. One such peptide inhibitor (TNRHNPHHLHHV) of LuxS was shown to partially inhibit the activity of the enzyme. Furthermore, 14 peptides containing the consensus sequence HSIR showed high affinity with LuxS. The selected and characterized specific inhibitor as well as the high-affinity ligands may facilitate the identification of new vaccination targets, opening up new approaches to the development of therapeutic drugs.     

Monoclonal antibodies against serotype specific and conserved epitopes in morphotype E Escherichia coli flagellins

Monoclonal antibodies (mAbs) were used to examine the interrelationships between morphologically identical flagellar filaments from Escherichia coli H serotype strains belonging to morphotype E. Serotype specific mAbs recognised epitopes exposed on the surface of flagellar filaments from H1, H7, H23, H49 and H51, but were inaccessible to immunolabelling in H45. Several mAbs which recognised conserved epitopes were also examined. mAb 7-56.1 recognised an epitope present in all morphotype E flagellins but not expressed on the filament surface. Similarly, mAb 1-5.1 recognised an internal epitope shared only by serotypes H1 and H12. Serotype H23 expressed a surface epitope which was present but not surface exposed in H7, H1 and H45 filaments.