16S rRNA gene clone library analysis of bacterial communities of the tick with infection of 4 species of pathogens / 中国地方病学杂志

Objective To develop the method of 16S rRNA gene clone library for tick bacterial flora analysis, and to analyze the detection effective of pathogens in tick and capacity of bacterial flora diversity. Methods Primers were designed according to the specific gene of Borrelia burgdorferi, Bartonella henselae, Anaplasma phagocytophilum, Ehrlichia chaffeensis and templates were choosen by positive PCR result to amplify the DNA extracted from the ticks. One set of primers targeting 16S rRNA gene conserved region were chosen to amplify certain fragments, DNA extraction, PCR reaction, cloning and sequencing. Nucleotide sequences were compared with GenBank database. Calculated Coverage values of clone library and Shannon-Wiener diversity index. Results Sixteen defined genus-or species-bacteria were detected in 103 valid sequences. Eight species were edge type (Clone No. > 5). Three kinds of pathogens were identified (Borrelia burgdorferi, Bartonella henselae and Rickettsia sp). Three kinds of pathogens were not edge type(Clone No. < 5). Coverage value was 96.11%, and Shannon-Wiener index was 2.40. Analysis results of cloning sequence showed that tick-parasitic bacteria mainly were α and γ deformation mycetes which accounted for 56.25% (9/16). Conclusions The 16S rRNA gene sequences technology could make relative quantitative of bacterial flora, and detect many kinds of pathogens in tick. It's a good method for detection of pathogens and bacterial flora analysis.

Locus-specific analysis of IS100 insertion within Yersinia pestis genome / 中国地方病学杂志

Objective To study the genotyping distribution of the Yersinia pestis(Y.pestis)strains by characterizing the diversity of the insertion sequence IS100 within the Y.pestis genome.Methods Derived fromthe known sequence of oriental strain CO92,5 pairs of locus-specific primers originating from both sides of the adjacent region of IS100 copies were designed,and two other complementary primers inside the IS100 sequence were designed to correspond with the outer primers.Then,91 Y.pestis strains and l pseudotubebculosis strain were tested by the specific PCR method using the primers described above and the PCR products were conformed by the sequence analysis,then further analysis WaS performed after the IS100 status was marked on the map of the plague focus type of china.Results The 91 Y.pestis strains had different IS100 status in their genome on tested loci.some possessed IS100 insertion,some didn't,and others changed their genome constitution.The IS100 possession on the 5 loci also suggested a distribution of regionality.Conclusion The analysis of some IS100 insertion element loci reveals that the IS100 genotyping distribution is consistent with the plague focus of type of China.And IS100genotyping pattern of the Y.pestis stains well reflects its genome constitution and the high flowability in its natural evolution.

Epidemiologie investigation on murine typhus in Hongta areas of Yuxi city, Yunnan province of China / 中华流行病学杂志

<p><b>OBJECTIVE</b>To identify epidemic status of murine typhus in Hongta areas of Yuxi city and to provide evidence for control and prevention of the disease.</p><p><b>METHODS</b>Serologic survey was conducted among residents and rodents. Isolation of Rickettsia moseri was performed.</p><p><b>RESULTS</b>The overall infection rate among general population was 28.92% (96/332) with geometric meantiter (GMT) as 10.83 and there was no difference between males and females (26.71%, 43/161 vs. 30.99%, 53/171, P > 0.05). Significant differences were found between age groups (P < 0.05) with positive rates of 29.63% (8/27), 18.06% (13/72), 39.62% (42/106), 27.50% (22/80) and 23.40% (11/47) among age groups 0-6, 7-18, 19-39, 40-59 and over 60, respectively. The overall rate of infection in mouse was 44.95% (89/198) with GMT as 30.30. Five isolates of R. moseri from mouse specimen, three from fleas plus one case of murine typhus were diagnosed. Rattus norvegicus and Rattus flavipectus were the predominant species of rodent animals (99.49%, 197/198) and Xenopsylla cheopis was the major species of vector (74.26%, 303/408). Flea index and mouse density were 2.06 and 11.13% respectively.</p><p><b>CONCLUSION</b>High infection rates on R. moseri were demonstrated in rodents and residents as well as high risk of murine typhus outbreak might occur in these areas.</p>

Surveillance on Rickettsia in epidemic areas of scrub typhus in Xinyang areas of Henan / 中华流行病学杂志

<p><b>OBJECTIVE</b>To understand the epidemic status of Rickettsia in Xinyang areas of Henan province.</p><p><b>METHODS</b>Samples including liver, spleen, kidney from mouse and chigger mites from Xinyang areas and serum samples were detected by nested-polymerase chain reaction (PCR) and indirect immunofluorescence assay (IFA).</p><p><b>RESULTS</b>In 62 viscus samples from mice organs, the positive rates were 16.13%, 8.06% and 6.45% for Orientia tsutsugamushi, R. typhii and Spotted fever group rickettsiae respectively. In blood clots samples from mice, the positive rates were 8.06%, 6.45% and 1.61 % for O. tsutsugamushi, R. typhii and Spotted fever group rickettsiae respectively. Three out of 26 mouse serum samples were positive for the predicted fluorexcent intensity O. tsutsugamushi.</p><p><b>CONCLUSION</b>Using nested-PCR and IFA methods, O. tsutsugamushi, R. typhii and Spotted fever group rickettsiae were detected in the captured mice living in Xinyang areas of Henan province. Results showed that there were intensive natural reserviors of Rickettsia in Henan province, suggesting that the risk of outbreak of Rickettsia in these areas was high.</p>

Molecular epidemic survey on co-prevalence of scrub typhus and marine typhus in Yuxi city, Yunnan province of China / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Human rickettsioses are worldwide zoonoses and it is not easy to differentiate them from other infectious diseases because of their atypical manifestation. In recent years the number of patients with fever of unknown causes from Hongta District CDC, Yuxi city of Yunnan Province has been increasing significantly in the summer. Diagnosis of scrub typhus was made by local clinicians. In order to ascertain the disease, we undertook a laboratory investigation for such patients from August 18 to 26, 2005.</p><p><b>METHODS</b>Active surveillance was conducted by Hongta District CDC Yuxi city of Yunnan Province from 2002 to 2004 and basic data were obtained from cases confirmed according to clinical definitions. Average incidences and town-level incidences were calculated during the study periods. Blood samples were analyzed by PCR and serological test. Based on the groEL gene sequences a paired general outer primers (Gro-1 and Gro-2) targeting typhus, spotted fever as well as scrub typhus and two paired inner primers (SF1, SR2 and TF1, TR2) for typhus together with spotted fever and scrub typhus, respectively, were designed to perform a multiplex-nested PCR. Serological assay was carried out by indirect immunofluorescence assay with 7 different rickettsial antigens, i.e., R.mossori, R.sibirica, R.conorii, O.tsutsugamushi, B.quintana, B.henselae and Coxilella burnetii phase II Ag.</p><p><b>RESULTS</b>Epidemiological surveillance showed that from 2002 to 2004, the average incidences of the scrub typhus or scrub typhus with murine typhus were 222.1/10(5), 204.3/10(5) and 109.6/10(5), respectively. Of 13 blood samples taken during acute stage of illness, 6 showed the amplified products for scrub typhus and the sequenced products showed 100%, 99%, 99%, 99%, 99%, 99% similarity to O.tsutsugamushi Karp but they shared the same deduced amino acid sequences, which indicated 100% identity with the heat shock protein of the O.tsutsugamushi Karp strain. Five yielded PCR products for murine typhus and their corresponding nucleotide sequences exhibited 100%, 100%, 99%, 99% and 99% similarity to R. mossori Wilmington and the analyses of predicted amino acid sequences indicated 100%, 100%, 98%, 98% and 98% identity with the heat shock protein of R. mossori Wilmington strain. Of the 8 PCR positive patients, 3 showed a co-infection of scrub typhus with murine typhus. All the 13 serum samples from febrile patients were positive against O. tsutsugamushi and 8 of them were positive against R. mossori. All of the 8 paired specimens had four-fold elevation of antibody against O. tsutsugamushi, and seroconversion for typhus was demonstrated in 3 paired serum samples. Another finding in the study was that a high seropositive prevalence (76.9%) of Q fever was detected.</p><p><b>CONCLUSION</b>It's confirmed that co-prevalence of scrub typhus with murine typhus are occurring in Yuxi city of Yunnan province, China. Other rickettsial diseases also need to be investigated in these areas.</p>

Use of variable-number tandem repeats to examine genetic diversity of Bacillus anthracis / 中华流行病学杂志

<p><b>OBJECTIVE</b>To study the genotyping of Bacillus anthracis based on multiple-locus variable-number tandem repeats(VNTR) in the B. anthracis genome.</p><p><b>METHODS</b>We selected 13 VNTR loci (which cited from published articles) to study 88 strains of B. anthracis isolated from China. The methods used were: (1) Selecting the primers which were at both ends of the tandem repeat locus; (2) Amplifying the sequence of the locus by PCR; (3)cDetecting the PCR products by agarose gel and polyacrylamide electrophoresis; (4)Analyzing the PCR products and computing the molecular weight by analysis software of gel images;(5) Double-checking with sequencing results; (6)Reckoning the repeat numbers and study the VNTRs loci characters.</p><p><b>RESULTS</b>(1) We used multiple-locus variable-number tandem repeat analysis (MLVA) to characterize 88 B. anthracis isolates from diverse geographic locations which were divided into 45 MLVA genotypes and 3 groups through cluster analysis. The genotypes was relative to restricted geographical region. It seemed clear that the multiple isolates from the same anthrax outbreak frequently having identical genotypes. (2)Results from VNTR analysis showed that A16R vaccine strain isolated from China was having the nature of representativeness in the country.</p><p><b>CONCLUSION</b>Analysis showed that the VNTR patterns was an appropriate study method for B. anthracis genetic diversity from different geographical areas and different time. Isolates from the same anthrax outbreak had identical</p>