Construction of sRNA-deficient and-overexpressing strains of Yersinia pestis / 军事医学

Objective To construct small RNA deletion and overexpression strains with a length of less than 100 nt in Yersinia pestis.Methods Deletion mutants of the target sRNAs were constructed by increasing the length of homologous regions.Meanwhile, the high copy plasmid pBAD/HisA was modified into an inducible transcriptional vector as an sRNA-overexpression plasmid by using QuikChange lightning site-directed mutagenesis kit .The presence , size, and transcription-al initiation sites of the indicated sRNA were predicted by transcriptome sequencing , primer extension , and previous stud-ies.The full-length DNA fragments of target sRNAs were transformed into the transcriptional vector .The overexpressing strains of sRNAs were identified by Northern Blot .Results and Conclusion Four sRNAs deletion mutants of sR01, sR02, sR03 and HmsA and three sRNAs overexpression mutants MicF , HmsA and CpxQ were successfully constructed .A method of construction of sRNA deficient and overexpressing strains of Y.pestis has been quickly and efficiently established by λ-Red homologous recombination technology and QuikChange ? lightning site-directed mutagenesis kit.

Construction of biofilm formation related mutants in Vibrio parahaemolyticus / 中华预防医学杂志

<p><b>OBJECTIVE</b>To construct the mutants of biofilm related genes in Vibrio parahaemolyticus and confirm the mutants.</p><p><b>METHODS</b>The homologous upstream and downstream flanking fragments of target gene were amplified by using PCR, and the fusion homologous fragment was amplified by using the two flanking fragments as template. Then the fusion homologous fragment was digested by restriction enzyme and cloned into suicide plasmid pDS132. The recombinant plasmid was transferred into Vibrio parahaemolyticus RIMD 2210633 through conjugation. The mutants were screened and identified by PCR and the phenotype of one mutant was analyzed in order to verify that the mutants were constructed successfully.</p><p><b>RESULTS</b>Six recombinant plasmids carrying the fusion homologous fragments of genes vbfR, crp, hns, swrZ, swrT and cpsR respectively were constructed and identified by PCR. The amplification products of 1190, 1128, 1136, 953, 1242 and 1112 bp were obtained respectively. The six mutants (ΔvbfR, Δcrp, Δhns, ΔswrZ, ΔswrT and ΔcpsR) were constructed using recombinant plasmids. Verified by PCR, the size of amplification products of mutants (1190, 1128, 1136, 953, 1242 and 1112 bp respectively) was less (610, 739, 421, 542, 427 and 1367 bp respectively) than the corresponding positive control. Meanwhile, none of the products was amplified using the primers locating on the target gene. One mutant Δhns was selected to test the ability of biofilm formation. The result showed that the ability of biofilm formation of mutant Δhns was increased compared with the wild type.</p><p><b>CONCLUSION</b>Six mutants of biofilm related genes in Vibrio parahaemolyticus were constructed and tested by molecular and phenotype experiment to confirm that the mutants were constructed successfully.</p>

Ecological-geographic landscapes of natural plague foci in China VIII. Typing of natural plague foci / 中华流行病学杂志

Since plague is an important natural focus zoonosis, the typing of natural plague foci becomes one of the elements in understanding the nature and developing related prevention program of the disease. Natural foci of plague are composed by four fundamental parts which include Eco-geographical landscape (natural plague foci), hosts, vectors and pathogens (Yersinia pestis) that comprehensively interact through the large temporal scale of evolution. Human activities have had great impact on the foci of natural plague. Based on the published serial research papers, we tried to integrate the knowledge of each factor in natural plague foci and focusing on theoretical aspects, so as to strengthen the prevention and surveillance programs of plague to be extrapolated to other zoonosis.

Research concerning Yersinia pestis and its significance in military medicine / 解放军医学杂志

As the military medicine progresses, the scope of protective medicine against biological threats should be extended to any facets that can cause biological threats, including biowarfare, bioterrorisms, invasion of alien organisms, loss of biological resources, genetically modified organisms, and emerging infectious diseases. Yersinia pestis is the pathogen for a typical zoonotic disease, plague, and it is also one of important biowarfare or bioterrorism agents. In history, this pathogen once caused three pandemics, and it was employed several times in war causing infection of military personnels many times. Currently, plague is distributed in Asia, former Soviet Union region, Africa and America. In China, there are 12 kinds of natural plague foci at present, distributing in 19 provinces (regions) and covering about 15% of our land area. Plague surveillance demonstrated that animal plague is active in some natural foci, area of plague foci is increasing gradually and extending to the border of cities, indicating that we have faced a great challenge for plague prevention and control. After 9/11 terrorist attack in U. S. A., studies on Y. pestis grew very rapidly and the progress has laid a solid foundation for researches on other bioterrorism-associated pathogens. Source-tracing database for microbial forensics analysis of Y. pestis and the rapid no-site detection method for this pathogen are also excellent experience for establishing other bioterrorism agents.

Use of rich BHI medium instead of synthetic TMH medium for gene regulation study in Yersinia pestis / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>This study is to verify the use of rich BHI medium to substitute synthetic media for gene regulation studies in Yersinia pestis.</p><p><b>METHODS</b>The transcriptional regulation of rovA by PhoP or via temperature upshift, and that of pla by CRP were investigated when Y. pestis was cultured in BHI. After cultivation under 26 °C, and with temperature shifting from 26 to 37 °C, the wild-type (WT) strain or its phoP or crp null mutant (ΔphoP or Δcrp, respectively) was subject to RNA isolation, and then the promoter activity of rovA or pla in the above strains was detected by the primer extension assay. The rovA promoter-proximal region was cloned into the pRW50 containing a promoterless lacZ gene. The recombinant LacZ reporter plasmid was transformed into WT and ΔphoP to measure the promoter activity of rovA in these two strains with the β-Galactosidase enzyme assay system.</p><p><b>RESULTS</b>When Y. pestis was cultured in BHI, the transcription of rovA was inhibited by PhoP and upon temperature upshift while that of pla was stimulated by CRP.</p><p><b>CONCLUSION</b>The rich BHI medium without the need for modification to be introduced into the relevant stimulating conditions (which are essential to triggering relevant gene regulatory cascades), can be used in lieu of synthetic TMH media to cultivate Y. pestis for gene regulation studies.</p>

Ecological-geographic landscapes of natural plague foci in China Ⅶ.Typing of natural plague foci / 中华流行病学杂志

Objective To group and characterize natural plague foci in China.Methods A novel two-class typing method as well as a three-indication nomenclature method were established to group and characterize the natural plague foci,on the basis of eco-geographical landscapes of plague foci,genetics of Yersinia pestis,zoology of rodent reservoirs and the entomology of flea vectors.Results A total of 12 distinct natural plague foci (including 19 subtypes) as well as their biological features were characterized.Conclusion Natural plague foci in China were grouped and characterized in this study.

Different strategies for preparation of non-tagged rV270 protein and its efficacy against Yersinia pestis challenge / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>LcrV is an important component for the development of a subunit vaccine against plague. To reduce immunosuppressive activity of LcrV, a recombinant LcrV variant lacking amino acids 271 to 326 (rV270) was prepared by different methods in this study.</p><p><b>METHODS</b>A new strategy that produced non-tagged or authentic rV270 protein was designed by insertion of rV270-thrombin-hexahistidine fusion gene into the vector pET24a, or by insertion of hexahistidine-enterokinase-rV270 or hexahistitine-factor Xa-rV270 fusion gene into the vector pET32a. After Co(2+) affinity chromatography, a purification strategy was developed by cleavage of His tag on column, following Sephacryl S-200HR column filtration chromatography.</p><p><b>RESULTS</b>Removal of His tag by thrombin, enterokinase and factor Xa displayed a yield of 99.5%, 32.4% and 15.3%, respectively. Following Sephacryl S-200HR column filtration chromatography, above 97% purity of rV270 protein was obtained. Purified rV270 that was adsorbed to 25% (v/v) Al(OH)₃ adjuvant in phosphate-buffered saline (PBS) induced very high titers of antibody to rV270 in BALB/c mice and protected them (100% survival) against subcutaneous challenge with 10⁶ CFU of Y. pestis virulent strain 141.</p><p><b>CONCLUSION</b>The completely authentic rV270 protein can be prepared by using enterokinase or factor Xa, but they exhibited extremely low cleavage activity to the corresponding recognition site. Thrombin cleavage is an efficient strategy to prepare non-tagged rV270 protein and can be easily operated in a large scale due to its relatively low cost and high cleavage efficacy. The recombinant rV270 can be used as a key component to develop a subunit vaccine of plague.</p>

Evaluation of immunization protection efficacy of plague subunit vaccine / 中华预防医学杂志

<p><b>OBJECTIVE</b>To evaluate the protective efficacy of plague subunit vaccine, BALB/c mice, guinea pigs and rabbits were used in this study.</p><p><b>METHODS</b>Groups of mice (10 per group), guinea pigs (14 per group) and rabbits (6 per group) were immunized with F1 + rV270 vaccine, EV76 vaccine and alum adjuvant by intramuscular route, respectively. Serum antibody titres of mice, guinea pigs and rabbits were determined by ELISA and the immunized animals were challenged with 10(6) CFU of Y. pestis strain 141 at the 8th week after the primary immunization.</p><p><b>RESULTS</b>The immunized mice, guinea pigs or rabbits with subunit vaccine developed anti-F1 IgG titre of 41 587.3 +/- 2.1, 11 543.7 +/- 2.1 or 522.4 +/- 22.4 and elicited statistical anti-F1 IgG titre difference among them (F = 17.58, P < 0.01). The immunized mice, guinea pigs or rabbits with subunit vaccine had anti-rV270 IgG titre of 15 748.7 +/- 1.6, 12.6 +/- 1.4 or 1648.0 +/- 5.0 and induced statistical anti-rV270 IgG titre difference among them (F value was 16.34, P < 0.01). There was significant anti-F1 IgG titre difference among mice, guinea pigs and rabbits immunized with EV76 vaccine that developed anti-F1 IgG titre of 913.4 +/- 4.5, 937.0 +/- 2.0 or 342.0 +/- 12.0 (F = 23.67, P < 0.01), whereas the immunized mice, guinea pigs and rabbits with EV76 vaccine developed anti-rV270 IgG titre of 12.0 +/- 1.0, 447.0 +/- 10.0, 40.0 +/- 11.0 and there was no anti-rV270 IgG titre difference between them (F = 2.20, P = 0.1314). The immunized mice with subunit vaccine developed significantly higher anti-F1 IgG titres than immunized guinea pigs and rabbits (q value was 30.57 and 19.04, respectively, P < 0.01), and there were no anti-F1 IgG titre differences between the immunized guinea pigs and rabbits (q = 0.04, P = 0.8485). The immunized mice with subunit vaccine developed significantly higher anti-rV270 IgG titres than immunized guinea pigs and rabbits (q value was 27.10 and 19.49, respectively, P < 0.01), and there were no anti-rV270 IgG titre differences between the immunized guinea pigs and rabbits with the subunit vaccine (q = 0.25, P = 0.6187). The immunized mice with EV76 elicited higher anti-F1 IgG titres than immunized guinea pigs and rabbits (q value was 40.67 and 29.10, respectively, P < 0.01), whereas there was no difference of F1 IgG titer between immunized guinea pigs and rabbits (q = 0.06, P = 0.8098). The immunized mice, guinea pigs and rabbits with subunit vaccine provided 100% (10/10), 86% (12/14) and 100% (5/5) protection against 10(6) CFU Y. pestis of challenge, respectively. The immunized mice, guinea pigs and rabbits with EV76 vaccine gave 100% (6/6), 93% (13/14) and 100% (6/6) protection against 10(6) CFU Y. pestis of challenge respectively.</p><p><b>CONCLUSION</b>BALB/c mice is the best small animal model for valuation of protective efficacy of plague subunit vaccine. The guinea pigs showed a high individual variation for this purpose. The rabbits can be used as an alternative model for evaluating plague subunit vaccine.</p>

Research on the procedure for recovery and species identification of Legionella from surface environmental water / 中华预防医学杂志

<p><b>OBJECTIVE</b>To establish a set of procedure for recovery and species identification of Legionella from the surface environmental water.</p><p><b>METHODS</b>Forty-four water samples were collected in eight parks of Guangzhou city from August to November in 2006. The bacteriologic examination was performed by cultivation on BCYEalpha plate, and 108 presumptive Legionella colonies were picked and their homogeneous relationship was analyzed by using an amplified fragment length polymorphism (AFLP) method. Species identification was carried out by latex agglutination test, biochemical characterization, analysis of cellular fatty acids composition, 16 S rRNA gene and mip gene sequencing.</p><p><b>RESULTS</b>Legionella was recovered among 27 (61.36%) samples of all eight parks, and 31 different strains were identified from those 108 presumptive Legionella isolates by AFLP method, including 20 strains of L. pneumophila, five strains of L. feeleii, four strains of L. longbeachae, one strain of L. oakridgensis and one strain of L. sainthelensi, and L. pneumophila could be easily differentiated by phenotypic and biochemical characteristics, latex agglutination test or analysis of the cellular fatty acids composition . However, uncertain factors were existing in those phenotypic identification methods as compared to the sequence analysis.</p><p><b>CONCLUSION</b>The taxonomic analysis of the Legionellae family should be dependent on the 16 S rRNA gene or mip gene.</p>

Antibacteral effect of six Chinese traditional medicines on Yersinia pestis / 中国地方病学杂志

Objective To screen the antibacterial activity of Chinese traditional medicines against Yersinia pestis.Methods Six Chinese traditional medicines(Coptis Chinesis etc)were selected and extracted with pure water to make a concentration of 1 mg/L.Yersinia pestis strain 201 and EV 76 were used to determine the minimal inhibitory concentrations(MIC)of these selected medicines in vitro with liquid dilution method.Results Three herbs had inhibition effects on the strain 201 and EV76 in different extents,among which Rheum palmatum had the strongest effect and MIC was 0.025 00 mg/L.Furthermore,the Chinese traditional medicine had the same MIC on both strain 201 and EV76.Conclusions Chinese traditional medicines commonly used have inhibiting effect on Yersinia pesti.

Study of Rhubarb anti-Yersina pestis based on DNA microarray / 中国地方病学杂志

Objective To establish a method for studying molecular mechanism of Rhubarb inhibiting anti-Yersinia pesti based on DNA microarray.Methods A whole genome DN A microarray containing 4005 annotated genes of Yersiniapesti Was used.The minimal inhibitory concentration(MIC)of Rhubarb to Yersiniapestiwas determined by liquid dilution method.The gene expression profile of Yersinia pesti was performed after the exposure to Rhubarb at a concentration of 10×MIC for 30 minutes.The total RNA extracted and purified from Yersinia pesti Was reversely transfected to cDNA and labeled by Cy3-Cy5 dye.The labeled probes were hybridized to the microarray anti the results were obtained by a laser scanner and the microarray data was confirmed by real-time quantitative RT-PCR.Results The platform of the DNA microarray-based bacteria transcriptional profile was established.A total of 498 genes of Yersinia pesti changed significantly in response to Rhubarb.Among them.358 genes were up-regulated,140 down-reguated.Conclusions The whole genome DNA microarray can be used in the studying of molecular anti-Yersinia pesti mechanism of Rhubarb.

Global gene expression of berberine against Yersiniapestis in vitro / 中国地方病学杂志

Objective To investigate the antibacterial molecular mechanism of Traditional Chinese Medicine Coptis rhizome against Yersinia pestis(Y.pestis).Methods The method based on whole genome DNA micrnarray of Y.pestis was used.The minimal inhibition concentration(MIC)of berberine to Y.pestis was determined with liquid dilution method.Then gene expression profile of Y.pestis was performed after exposed to berberine at the concentration of 10×MIC for 30 minutes.Total RNA extracted and purified from Y.pestis and reverse-transcribed to cDNA,then labeled by Cy-dye.Finally,the labeled probes were hybridized to the microarray and the results were obtained by a laser scanner and analyzed by the SAM software.Results The gene expression profile data revealed that the response of Y.pestis to berberine was a global phenomenon.A total of 360 genes changed significantly.Among them,333 genes were up-regulated,27 down-regulated.These differentially expressed genes were further classified into 24 different functional categories based on the genomie annotation of Y.pestis CO92,in which the number of mainly related genes were 83,75 and 48,including cell envelop,unkown,transport/binding proteins functions.The 40 genes related to the metabolism were upregulated,which was a remarkable change.Conclusion Our results have revealed the general gene expression changes of Y.pestis in response to berberine and demonstrated the antibacterial molecular mechanism of the Coptis rhizome.The major mechanism of Y.pestis in response to berberine is the upregulation of genes related to the metabolism.

A molecular epidemiological study on human plague fulminant epidemic in Qinghai, 2004 / 中华流行病学杂志

<p><b>OBJECTIVE</b>To study the epidemiology of genotyping Yersinia pestis isolated in the fulminant epidemics of human plague in Qinghai province in 2004.</p><p><b>METHODS</b>Primer pairs targeting the twenty-three different identified regions (DFRs) were designed to detect the presence or deletion of each DFR in 13 strains of Yersinia pestis isolated from the fulminant epidemic of human plague in Qinghai province in 2004.</p><p><b>RESULTS</b>There were 4 genomovars, i.e. Genomovar 8, 10, 15 and 16 in the 13 strains of Yersinia pestis identified. The genomovar of all the strains of Yersinia pestis isolated from Nangqian county was Genomovar 10. Among the two strains of Yersinia pestis isolated from Wulan county, the genomovar of one strain was Genomovar 8 and the other was Genomovar 10. The genomovars of all the strains of Yersinia pestis isolated from Qilian, Qumalai and Chengduo county belonged to Genomovar 16.</p><p><b>CONCLUSION</b>It was demonstrated that the genotyping of Yersinia pestis appeared to be a powerful tool for investigating human plague epidemics.</p>

Study on the genotyping and microevolution of Yersinia pestis in the Qinghai-Tibet Plateau / 中华流行病学杂志

<p><b>OBJECTIVE</b>To study the distribution of genomovars and microevolution of Yersinia pestis in the Qinghai-Tibet Plateau.</p><p><b>METHODS</b>Primer pairs targeting the twenty-two different regions(DFRs) were designed for detecting the presence or deletion of each DFR in 297 strains isolated from the Qinghai-Tibet Plateau.</p><p><b>RESULTS</b>9 genomovars, i. e. Genomovar 1, 5, 6, 7, 8, 10, 11, new type and Ype-ancestor were identified in the Marmota himalayana plague focus of the Qinghai-Tibet Plateau. Among these genomovars, genomovar 5,8 and 10 were dominant types. The total rate of the three genomovars was 80.6% (204/253) and the genomovars in different regions were different. All of 44 strains of Y. pestis in the Microtus fuscus plague focus of the Qinghai-Tibet Plateau belonged to genomovar 14.</p><p><b>CONCLUSION</b>The distribution of genomovars of Y. pestis in the Qinghai-Tibet plateau had remarkable characteristics geographically. Based on the distribution of genomovars of Y. pestis, the routes of transmission and microevolution of Y. pestis were proposed.</p>

Epitope Tagging of the rpoS gene of Y. pestis by Recombineering Technique / 中国生物工程杂志

Objective: To facilitate the functional analysis of chromosomal genes and their products, the recombineering technique to epitope tagging of chromosomal genes of Y. pestis was adapted. Methods: The epitope tag was generated by primer annealing and then fused with resistance gene by fusion PCR. The epitope-resistance cassette was inserted into pBluecript, resulted in the template plasmid, pBS-MH. The tagging cassette for rpoS was obtained by PCR amplification from pBS-MH with primers containing homology specific to the target gene. PCR products were transformed into recombination competent cells and recombinants were selected. PCR and DNA sequencing were used to confirm the correct tagging event. The expression of the tagged protein was detected with Western blot by using monoclonal antibody to the epitope. Results: The template plasmid containing fusion of epitope and resistance gene was successfully constructed. The sigma factor gene, rpoS, was tagged with a myc-his tag at the COOH terminus. Expression of the tagged rpoS was successfully detected indirectly by the antibody against His tag. Conclusion: The chromosomal gene tagging by recombineering technique represents a powerful tool in the functional study of bacterial genes and their products.

Development of a new sampling medium for bioaerosols / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To develop a new sampling medium for detecting of bioaerosols.</p><p><b>METHODS</b>The sampling media were tested by using Escherichia coli, Staphylococcus aureus and Serratia marcescens under static and active conditions, preliminary applications were performed using AGI-10 and high volume sampler.</p><p><b>RESULTS</b>The average recovery rates were raised to 24.7%, 58.2%, 40.5%, 44.1%, 20.5%, and 15.4%, respectively in six consecutive experiments under static condition for 60 min at room temperature. Four kinds of sampling media were singled out after static experiments, which were referred to as "samplutions" PD1, PX2, TD1, and TX2, respectively. Under the active condition, the protective efficacy of PD1, PX2, TD1, and TX2 was 226% (153/47), 553% (111/17), 150% (120/48), and 268% (419/114), respectively.</p><p><b>CONCLUSION</b>The samplutions have some effects on the subsequent nucleic acid detection, which could be avoided by employing standard nucleic acid extraction procedure. The newly developed samplution can be applied to the detection of bioaerosols.</p>

Relationship between core promoter mutation, clinical features and virus replication in HBV carriers / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To elucidate the relationship between HBV core promoter mutation and clinical features as well as its effects on serum e system and viral replication.</p><p><b>METHODS</b>Semi-nested mutation specific PCR (msPCR) was employed for detecting core promoter mutation at nt 1 762-1 764 in 97 patients with HBV infection.</p><p><b>RESULTS</b>The msPCR method was demonstrated to be specific and reliable for the mutation detection by sequencing the PCR products. The detection ratio of the mutation in patients with acute hepatitis, mild, moderate and severe chronic hepatitis and liver cirrhosis was 2/5, 7/43, 10/31, 1/3 and 7/15, respectively. The detection rate of the mutation in liver cirrhosis was significantly higher than that in light chronic hepatitis (P < 0.025). In 92 patients with chronic HBV infection, HBeAg positive rate in wild (25/92), mutant (42/92) and mixed (25/92) strain infection was 80.0%, 56.0% and 64.3%, HBV DNA level was (4.4 +/- 8.5) x 10(8), (1.1 +/- 1.6) x 10(9) and (1.4 +/- 1.8) x 10(9) copies/ml, the rate of abnormal ALT was 44.0%, 52.0% and 42.6%; ALT level was (58.6 +/- 79.0), (57.1 +/- 75.2) and (62.6 +/- 90.3) IU/L, respectively (P > 0.05).</p><p><b>CONCLUSIONS</b>The msPCR method for detecting core promoter mutation at nt 1 762-1 764 is specific and reliable. Core promoter mutation is associated with the severity of liver disease, but neither related to the status of e system in serum nor to the virus replication.</p>