Detection of Brazilian spotted fever infection by polymerase chain reaction in a patient from the state of São Paulo

Brazilian spotted fever (BSF) cases have been increasing in the state of S?o Paulo but no genomic information about local rickettsia isolated from humans has been well documented. We recovered spotted-fever group rickettsiae from a sample of patient blood cultured in Vero cells using the shell vial technique. Rickettsial DNA fragments (gltA, ompA, and, ompB genes) were detected, and analysis of the ompB gene base sequences showed identity with the Rickettsia rickettsii ompB sequence available in the GenBank.     

Detection of Rickettsia rickettsii in the tick Amblyomma cajennense in a new Brazilian spotted fever-endemic area in the state of Minas Gerais

The present study evaluated rickettsial infection in Amblyomma spp. ticks collected in a farm in Coronel Pacheco, a Brazilian spotted fever (BSF) endemic area. A total of 78 A. cajennense and 78 A. dubitatum free-living adult ticks were collected and tested by polymerase chain reaction (PCR) targeting a fragment of the rickettsial gene gltA. Only one pool of three A. cajennense ticks showed the expected product by PCR. This pool was further tested by PCR using sets of primers targeting the rickettsial genes gltA, ompA, and ompB. All reactions yielded the expected bands that by sequencing, showed 100% identity to the corresponding sequences of the Rickettsia rickettsii gene fragments gltA (1063-bp), ompA (457-bp), and ompB (720-bp). The minimal infection rate of R. rickettii in the A. cajennense population was 1.28% (at least one infected tick within 78 ticks).The present study showed molecular evidence for the presence of R. rickettsii in A. cajennense from a BSF-endemic area in Coronel Pacheco, state of Minas Gerais. Although R. rickettsii has been previously reported infecting A. cajennense ticks in Brazil and other Latin American countries, the present study performed the first molecular characterization of R. rickettsii from the tick A. cajennense.     

Isolation of Rickettsia felis in the mosquito cell line C6/36

We report the isolation and establishment of Rickettsia felis in the C6/36 cell line. Rickettsial growth was intense, always with 90 to 100% of cells being infected after few weeks. The rickettsial isolate was confirmed by testing infected cells by PCR and sequencing fragments of three major Rickettsia genes (gltA, ompB, and the 17-kDa protein gene).     

Identification of tlc and gltA mRNAs and determination of in situ RNA half-life in Rickettsia prowazekii.

RNAs of Rickettsia prowazekii, an obligate intracytoplasmic bacterium, have been identified and analyzed by an RNase protection assay. Total RNA, a mixture of host cell RNA and rickettsial RNA, was isolated from rickettsia-infected mouse L929 cells by the hot-phenol method. After hybridization with specific antisense RNA probes and digestion with RNase, the protected products were analyzed by electrophoresis and autoradiography. The results show that there is only one mRNA species for the ATP/ADP translocase gene (tlc) but two mRNA species for the citrate synthase gene (gltA). RNA half-lives were determined by measuring the RNA remaining after addition of rifampin. The half-lives of tlc mRNA, gltA mRNA I, and gltA mRNA II in R. prowazekii are 8.4 +/- 0.6, 12.3 +/- 1.3, and 20.5 +/- 1.8 min, respectively. However, the half-lives of tlc mRNA and gltA mRNA I in recombinant Escherichia coli strains are 2.9 +/- 0.1 and 1.4 +/- 0.1 min, respectively. The 16S rRNA in R. prowazekii was also examined and shown to be stable.     

Sequence analysis of the gene encoding a spotted fever group-specific intracytoplasmic protein PS120 of Rickettsia japonica

The 3,438-nucleotide (nt) sequence containing a 3,054-nt open reading frame of the gene (rps120) encoding an antigenic, intracytoplasmic, spotted fever group-specific and heat-stable 120-kilodalton protein (PS120) of Rickettsia japonica was determined. The nt and deduced 1,018 amino-acid (aa) sequences were compared to those of R. conorii since only those of this species had been determined among SFG rickettsiae. The homologies of these sequences between R. japonica and R. conorii were considerably high at 97 and 95%, respectively. These high homologies were comparable to those of beta-peptides encoded by the ompB genes among SFG rickettsiae. It was also found that the genome of R. prowazekii contained a nt sequence with 68% homology to that of the rps120 gene of R. japonica.     

Genotype characterization of the bacterium expressing the male-killing trait in the ladybird beetle Adalia bipunctata with specific rickettsial molecular tools.

The male-killing ladybird beetle (LB) bacterium (AB bacterium) was analyzed with specific rickettsial molecular biology tools in the LB Adalia bipunctata strains. Eight phenotype-positive LB strains showing mortality of male embryos were amplified with rickettsial genus-specific primers from the gene for citrate synthase (CS) and the gene for a 17-kDa protein and spotted fever group-specific primers from the gene for the 120-kDa outer membrane protein (ompB). The specificity of amplification was confirmed by Southern hybridization and the absence of the above-listed gene products in three phenotype-negative LB strains. Restriction polymorphism patterns of three examined amplicons from the CS gene, 17-kDa-protein gene, and ompB gene were identical among the eight phenotype-positive LB strains and were unique among all known rickettsiae of the spotted fever and typhus groups. Amplified fragments of the CS genes of the AB bacterium, Rickettsia prowazekii Breinl, Rickettsia typhi Wilmington, Rickettsia canada 2678, and Rickettsia conorii 7 (Malish) were sequenced. The greatest differences among the above-listed rickettsial and AB bacterium CS gene sequences were between bp 1078 and 1110. Numerical analysis based on CS gene fragment sequences shows the close relationships of the AB bacterium to the genus Rickettsia. Expanding of knowledge about rickettsial arthropod vectors and participation of rickettsiae in the cytoplasmic maternal inheritance of arthropods is discussed.     

多位点序列分型分析空肠弯曲菌华东动物源分离株

【目的】研究空肠弯曲菌菌株间的分子特征,对不同宿主来源的空肠弯曲菌进行分子分型研究。【方法】选择空肠弯曲菌的7个看家基因gltA、aspA、glnA、glyA、pgm、tkt和uncA作为目的基因,对2006-2008年间华东地区分离的42株空肠弯曲菌样本进行PCR扩增后测序。将测序结果软件分析并上传到数据库进行比对,将结果制作多位点序列分型(multilocus sequence typing,MLST)遗传进化树并进行分析。【结果】与数据库已有类型比对,发现了24个新的ST型,通过进化树得到其遗传关系。【结论】MLST方法对于研究空肠弯曲菌的菌株群体基因差异与进化趋势具有重要意义。     

嗜肺巴氏杆菌蛋白及抗原图谱初步分析

对不同来源的 1 1株嗜肺巴氏杆菌进行了全菌可溶性蛋白及抗原图谱分析。使用SDS -聚丙烯酰胺凝胶电泳 (SDS -PAGE) ,梯度凝胶电泳结果显示 ,嗜肺巴氏杆菌蛋白主要分布于相对分子质量 ( 1 4 4～ 97 4)× 1 0 3,且带型基本一致。 6株菌与参考菌株有完全相同的蛋白带 ,另外 5株则缺乏 40× 1 0 3带。分别用嗜肺巴氏杆菌免疫血清和自然感染嗜肺巴氏杆菌小鼠血清对 1 1株菌进行了免疫印迹 (Westernblots)试验。与免疫小鼠血清的反应显示 ,1 1株受试菌主要有相对分子质量大约 ( 1 7、31 )× 1 0 3两条反应带 ;在与自然感染血清的反应中主要的反应带在 1 7× 1 0 3处 ,缺乏 40× 1 0 3蛋白的 5株菌有 31× 1 0 3反应带 ,其余 7株均有大约 ( 2 0、2 8)× 1 0 3的反应带 ,故可以认为该菌主要抗原大约为 ( 1 7、31 )× 1 0 3;1 0个流行株根据 40× 1 0 3蛋白和 ( 2 0、2 8)× 1 0 3抗原的有无可被分为两型。本研究为精制血清学方法的诊断抗原 ,以及将SDS -PAGE和Westernblot法用于嗜肺巴氏杆菌检测奠定了基础。     

A 29.5 kDa heat-modifiable major outer membrane protein of Rickettsia prowazekii, putative virulence factor, is a peptidyl-prolyl cis/trans isomerase

Allelic genes from three Rickettsia prowazekii strains encoding parvulin-like protein (Plp), a heat-modifiable 29.5 kDa major outer membrane protein, were earlier cloned into expression vector pQE 30. In this work, recombinant proteins were overproduced in E. coli, purified, and found to exhibit an expected peptidyl-prolyl cis/trans isomerase activity of a parvulin type in vitro with oligopeptide substrates. Native polypeptide of prototype virulent Breinl strain is known to differ by SDS-PAGE mobility from those of both vaccine Madrid E and virulent EVir isolates. Being different in electrophoretic behavior, heat-unmodified forms of the three strains were shown to migrate apart from lipopolysaccharides. A EVir Plp gene was sequenced, and deduced protein sequence was found to be identical to previously published Breinl and Madrid E. Present data indicate that unknown post-translational modification(s) in rickettsiae are responsible for both interstrain difference and heat-modifiability of Plp.     

Genetic characterization of a Chlamydophila pneumoniae isolate from an African frog and comparison to currently accepted biovars

The amphibian isolate DE177 identified as Chlamydophila (C.) pneumoniae was sequenced in five genomic regions: 16S ribosomal RNA gene, 16-23S intergenic spacer, ompA, ompB, and groESL genes. Comparison with corresponding sequences of the currently accepted equine, human and koala biovars of C. pneumoniae revealed that koala strains represented the most closely related taxon, although sequence dissimilarities in the ompA (VD4) and ompB gene regions were noted. In this respect, the present isolate is distinct from a previously described frog isolate (Berger et al., 1999) whose sequence analysis yielded identity to the koala biovar. As three of the nucleotide substitutions in ompA (VD4) of DE177 will be translated into two altered amino acids the possible existence of another biovar is discussed.     

Isolation of Rickettsia rhipicephali and Rickettsia bellii from Haemaphysalis juxtakochi ticks in the state of São Paulo, Brazil

In the present study, attempts to isolate Rickettsia in cell culture were performed individually in seven specimens of Haemaphysalis juxtakochi ticks collected in the state of S?o Paulo (southeastern Brazil). Rickettsia was successfully isolated by the shell vial technique and established in Vero cell culture from six ticks (six isolates). DNA extracted from infected cells of these isolates was tested by PCR and DNA sequencing, using genus-specific Rickettsia primers targeting the genes gltA, htrA, ompA, and ompB. After the generated sequences were compared with available sequences in GenBank, five out of the six isolates were identified as Rickettsia bellii (isolates HJ#1, HJ#2, HJ#3, HJ#4, and HJ#7). The sixth isolate (HJ#5) was closest to Rickettsia sp. strain R300, previously detected in H. juxtakochi in northern Brazil, and to Rickettsia rhipicephali, isolated from ticks in the United States. Following recent gene sequence-based criteria proposed for the identification of Rickettsia isolates, both isolate HJ#5 and strain R300 were identified as South American strains of R. rhipicephali, which was confirmed in this continent for the first time. Isolation of R. bellii from H. juxtakochi ticks, added to eight other tick species that have been reported to be infected with this bacterium in Brazil, indicates that R. bellii is indeed the most frequent Rickettsia species infecting ticks in Brazil. Currently, the role of both R. rhipicephali and R. bellii as human pathogens is regarded as unknown.     

天津地区气单胞菌分离株的鉴定与多位点序列分型

[目的]研究气单胞菌菌株分类情况,并分析其致病性.[方法]采集环境样品和鱼类标本,分离并鉴定气单胞菌菌株,并运用多位点序列分型(Multilocus sequence typing,MLST)方法进行分类研究,利用PCR和测序方法分析毒力基因Aera、Hly、Aha1、GCAT和Nuc的分布.[结果]通过对分离菌株的16S rRNA基因进行分析,确认属于4种不同气单胞菌的7个分离株.发现所有菌株至少有1种毒力基因阳性,其中3株具有4种毒力基因.药物敏感实验显示,6株分离株对3种或3种以上抗菌素具有多重耐药性.最后,对看家基因gyrB、groL、gltA、metG、ppsA和recA进行分析,与MLST数据库中的等位基因序列比对,发现7株分离株均为新的不同的序列型(Sequence type,ST).[结论]气单胞菌具有较高的遗传多样性.     

Molecular cloning of four tricarboxylic acid cyclic genes of Escherichia coli.

A fragment of DNA (3.1 kilobases [kb]) from a ColE1 Escherichia coli DNA hybrid plasmid containing the bacterial citrate synthase gene (gltA) was subcloned in both orientations into phage lambda vectors by in vitro recombination. The resulting phages were able to transduce gltA and, as prophages, complemented the lesion of a gltA mutant, showing that a functional gltA gene is contained in the 3.1-kb fragment. The segment of E. coli DNA cloned in these lambda gltA phages was extended in vivo by prophage integration and aberrant excision in the gltA region. Plaque-forming derivatives, carrying up to three additional tricarboxylic acid cycle genes, succinate dehydrogenase (sdh), 2-oxoglutarate dehydrogenase (sucA), and dihydrolipoamide succinyltransferase (sucB), were isolated and characterized by their transducing and complementing activities with corresponding mutants, and the order of the genes was confirmed as gltA-sdh-sucA-sucB. Physical maps of a variety of the transducing phages showed that the four tricarboxylic acid cycle genes are contained in a 12.8-kb segment of bacterial DNA. The four gene products, plus a possible succinate dehydrogenase small subunit, were identified in postinfection labeling studies, and the polarities of gene expression were defined as counterclockwise for gltA and clockwise for sdh, sucA, and sucB, relative to the E. coli linkage map.     

Isolation and characterization of the Rickettsia prowazekii recA gene.

The recA gene has been isolated from Rickettsia prowazekii, an obligate intracellular bacterium. Comparison of the amino acid sequence of R. prowazekii RecA with that of Escherichia coli RecA revealed that 62% of the residues were identical. The highest identity was found with RecA of Legionella pneumophila, in which 69% of the residues were identical. Amino acid residues of E. coli RecA associated with functional activities are conserved in rickettsial RecA, and the R. prowazekii recA gene complements E. coli recA mutants for UV light and methyl methanesulfonate sensitivities as well as recombinational deficiencies. The characterized region upstream of rickettsial recA did not contain a sequence homologous to an E. coli LexA binding site (SOS box), suggesting differences in the regulation of the R. prowazekii recA gene.