Abstract: Objective To understand the phenotypic and genetic characteristics of Yersinia pestis strains isolated from Himalayan marmot natural focus area and domestic rat plague focus area in southern China, and provide reference for mastering the pathogenic characteristics of Yersinia pestis of two plague foci. Methods A total of 412 of Yersinia pestis strains isolated from Himalayan marmot plague focus and domestic rat plague focus of southern China were subjected to to sorbitol fermentation assays, virulence factor, different region (DFR) typing, and clustered regularly interspaced palindromic repeats (CRISPR) typing. Results The biochemical types of Y. pestis from the two plague foci showed distinct regional distribution features. Five biochemical phenotypes were identified in Yersinia pestis isolated from Himalayan marmot natural focus area, while only one biochemical phenotype was identified in strains isolated from the domestic rat plague focus of Southern China. Most of the Yersinia pestis isolated from the two plague foci were capable of producing the virulence factors of Fl and PstI. Among the strains from Himalayan marmot focus, 70.53% (201/285) were VW-positive, 75.09% (214/285) were Pgm-positive, 20.00% (57/285) of the strains were Pgm-negative, and 5.26% (15/285) were Pgm mixed-type strains. Among strains from domestic rat plague focus of southern China, 37.80% (48/127) were VW-positive, 29.13% (37/127) were Pgm-positive, 58.27% (74/127) were Pgm-negative, and 12.60% (16/127) were Pgm mixed-type strains. DFR typing revealed 22 genotypes of Y. pestis from the Himalayan marmot plague focus, with the main genotypes being type 5, 7, 8, 10, 19, 32 and 49. All strains from domestic rat plague focus area in southern China belonged to type 9. CRISPR typing revealed that all strains from the Himalayan marmot natural focus were classified into 7 CRISPR gene clusters and 14 CRISPR genotypes, with the main genotypes being G7, G22, G26-a1'and G22-A1'. All strains from domestic rat plague focus area in southern China belonged to CRISPR genotype G30, with the gene cluster being Ca8. Conclusions The phenotypes and genotypes of the Yersinia pestis of Himalayan marmot plague focus are diverse, with an obvious characteristics of geographical distribution. The phenotype and genotype of the Yersinia pestis of domestic rat plague focus of Southern China are single. DFR and CRISPR genotyping methods with phenotypic characteristics can effectively identify the Yersinia pestis isolated from the two plague foci, thereby meeting the needs of identification and traceability research.

Objective To investigate the clinical manifestations, angiographic features and clinical significance of kinking of extracranial internal carotid artery (ICA). Methods The clinical and radiological data of 21 patients with kinking of extracranial ICA were retrospectively reviewed in our hospital from April 2003 to July 2007. Fisher exact test was performed. Results Of the 21 patients, 7 hod no clinical symptoms, the other 14 showed manifestations of cerebral iachemia with varying degree. One of the characteristic clinical manifestations that neck rotation or specific positions of head and neck might induce the occurrence of clinical symptoms was found in 5 cases. The whole-brain coverage DSA accurately showed the location of kinking of extracranial ICA and the degree of vascular stenosis. In patients with α < 66%, 80% > α≥ 66% and α≥ 80%, clinical symptoms were found in 3 out of 5,7 out of 10 and 4 out of 6 patients, respectively. Fisher exact test revealed that the positive rates of clinical symptoms in three groups had no significant difference (P > 0. 05). Conclusion Kinking of extracranial ICA is a frequent vascular morphologic variation, and it is also a kind of potential disease. The whole-brain coverage DSA is a relatively reliable method to detect this variation.

Objective:To understand whether there are drug resistant and disinfectant resistant Yersinia pestis strains in China, and to provide accurate information for clinical treatment of plague. Methods:A total of 2 753 Yersinia pestis strains isolated from 10 natural plague foci in China from 1943 to 2016 were collected. According to National Center for Biotechnology Information (NCBI) released sequences of aminoglycoside streptomycin resistant genes strA, strB, β-lactam antibiotics resistant genes TEM, SHV and CTX-M, sulfamilamide resistant genes sul1, sul2 and sul3, and disinfectant resistant gene qacE△1-sul1, a pair of primers of each gene was designed for above-mentioned genes. Genomic DNA of 2 753 strains of Yersinia pestis was extracted, and the 9 target genes of all DNA samples were amplified by PCR. Results:Negative and positive controls of PCR detection were established. No corresponding target bands of aminoglycoside streptomycin resistant genes strA, strB, β-lactam antibiotics resistant genes TEM, SHV and CTX-M, sulfamilamide resistant genes sul1, sul2 and sul3, and disinfectant resistant gene qacE△1-sul1 were found in the DNA samples of 2 753 strains of Yersinia pestis.Conclusion:The above-mentioned genes of drug resistance and disinfectant resistance have not been detected in Yersinia pestis of China, but the monitoring of drug resistance of Yersinia pestis still needs to be carried out continuously.

Objective:To investigate the drug resistance of Yersinia pestis to 11 kinds of antibiotics in the natural foci of plague in Inner Mongolia Autonomous Region, and to provide a theoretical basis for scientifically and effectively selecting antibiotics for treatment of the plague. Methods:A total of 137 strains of Yersinia pestis isolated from the natural foci of plague in Inner Mongolia Autonomous Region at different times, regions, hosts and vectors were collected. According to Clinical and Laboratory Standard Institute (CLSI), the agar plate dilution method was used to determine the minimum inhibitory concentration (MIC) of the 11 kinds of antibiotics against 137 strains of Yersinia pestis, including ofloxacin, ciprofloxacin, kanamycin, streptomycin, ceftriaxone, ampicillin, chloramphenicol, spectinomycin, cefuroxime, tetracycline and sulfamethoxazole-trimethoprim. The MIC 50 and MIC 90 (the minimum concentration of drug which could inhibit 50% and 90% of bacterial growth) were calculated, and their sensitivity was determined according to CLSI standards. Results:Among 137 strains of Yersinia pestis tested, no strains of Yersinia pestis had single or multiple resistance to ofloxacin, ciprofloxacin, kanamycin, streptomycin, ceftriaxone, ampicillin, chloramphenicol, spectinomycin, cefuroxime, tetracycline and sulfamethoxazole-trimethoprim. According to CLSI standards, 137 strains of Yersinia pestis were all sensitive to the 11 kinds of antibiotics; among them, ofloxacin, ciprofloxacin, ceftriaxone and sulfamethoxazole-trimethoprim had higher antibacterial activity, with MIC 90 < 0.250 μg/ ml; the antibacterial activity of spectinomycin was the lowest, with MIC 90 of 16.000 μg/ml. Conclusions:The Yersinia pestis isolated from the natural foci of plague in Inner Mongolia Autonomous Region is not found to have single or multiple resistance to the 11 kinds of antibiotics. Continuous drug resistance monitoring of Yersinia pestis should be carried out to provide a basis for clinical medication.

Objective To investigate the etiology and the epidemiologic features of drug resistance and disinfectant resistance of Yersinia pestis in Hainan,Qinghai Province,in order to provide a scientific basis for prevention and control of the plague in this area.Methods Totally 75 strains were isolated from vary kinds of host in Hainan from 1960 to 2009,and biochemical test,virulence factors evaluation [Fra1 (F1),pesticin Ⅰ (Pst Ⅰ),virulence antigen (VW),pigmentation (Pgm)],plasmid analysis,different region (DFR) genotyping,drug resistance and disinfectant resistance gene test were carried out.Forty-five strains of Yersinia pestis were selected to determine their toxicity in mice,median lethal dose (LD50) was calculated,and LD50 ＜ 1 000 was defined as strongly toxic.Results Sixty of the 75 strains were Qing-Tibet Plateau ecotype,7 strains were Qilian Mountain ecotype,and the remaining 8 were different ecotypes from the plague foci in Qinghai Plateau.Eighty percent (60/75) contained all the four virulence factors;and 97.78％ (44/45) of the strains were velogenic strains;96.00％ (72/75) of the strains contained 3 kinds of plasmids (Mr:6 × 106,45 × 106 and 52 × 106);the DFR strains had 3 genomovars,which were genomovar 8 (65 strains),genomovar 5 (8 strains) and genomovar 21 (2 strains).No strains related to streptomycin,sulfonamides,β-lactam antibiotics and disinfectants had been found in the 75 strains of Yersinia pestis.Conclusions The strains isolated in Hainan have the characteristics of Qinghai-Tibet Plateau plague's pathogen,and they have strong toxicity.In view of high mortality of plague,drug resistance and disinfectant resistance gene test should be put into routine monitoring of the plague.

Objective:To establish the minimum inhibitory concentration (MIC) detection method of Yersinia pestis by determining MIC of 11 kinds of antibiotics against Yersinia pestis, to master the inhibition range of common antibiotics on Yersinia pestis, and provide baseline data for clinical treatment of plague. Methods:According to Clinical Labor Standard Institution (CLSI), the agar plate dilution method was used to determine the MIC of 11 kinds of antibiotics against 118 strains of Yersinia pestis, including ofloxacin, ciprofloxacin, trimethoprim-sulfamethoxazole, kanamycin, streptomycin, ceftriaxone, ampicillin, chloramphenicol, spectinomycin, cefuroxime and tetracycline. MIC 50 and MIC 90 (the minimum concentration of drug which could inhibit 50% and 90% of bacterial growth) were calculated. The consistency was observed by comparing the results with those of the disk diffusion method. One hundred and eighteen strains of Yersinia pestis were isolated from natural plague foci of Qinghai Province and preserved by Qinghai Institute for Endemic Disease Prevention and Control. Results:Among 118 strains of Yersinia pestis tested, no single or multiple strains of Yersinia pestis resistant to 11 kinds of antibiotics were found, which was consistent with the results of the disk diffusion method. The MIC 50 and MIC 90 of 11 kinds of antibiotics against 118 strains of Yersinia pestis were obtained. Conclusions:The MIC detection method of Yersinia pestis is successfully established. This method can be used to measure the MIC of antibiotics against Yersinia pestis in high throughput and evaluate the sensitivity of Yersinia pestis to antibiotics. It is an efficient, economical and practical experimental method.

Objective:To investigate the genotypes of clustered regularly interspaced short palindromic repeats (CRISPR) of Yersinia pestis ( Y. pestis) and it's regional distribution in the plague natural foci of Qinghai-Tibet Plateau. Methods:In this study, 1 004 strains of Y. pestis isolated from different hosts and vectors in different regions from 1954 to 2011 were selected as experimental objects, which preserved in Qinghai Institute for Endemic Disease Prevention and Control, and DNA of Y. pestis was extracted by traditional phenol-chloroform method. Three CRISPR loci (YPa, YPb and YPc) were amplified by PCR and sequenced, respectively, and the CRISPR sequences were compared with the CRISPRDictionary database recently reported in the literature to identify CRISPR spacer. For the spacer discovered for the first time at each CRISPR locus, Blast sequence alignment was performed in the National Center for Biotechnology Information (NCBI) database to speculate the source of gene sequence. Genotyping of Y. pestis from Qinghai-Tibet Plateau was performed based on the polymorphism of CRISPR spacer array. Results:A total of 53 spacers were found in 1 004 strains of Y. pestis, of which 6 spacers were newly discovered, namely a105, a106, a107, b51, b52 and c14. The 1 004 strains of Y. pestis were divided into 10 groups with 44 different CRISPR genotypes, and 15 new genotypes were found. The dominant genotypes of Y. pestis were G26-a1', G7, G22, G24-a1', G22-a1', G9 and G26-a1'a60 in Himalayan Marmot plague natural foci, and G37-a6' in Qinghai Microtinae plague natural foci. Conclusion:The CRISPR genotypes of Y. pestis in the plague natural foci of Qinghai-Tibet Plateau are high diverse and have significant regional distribution characteristics.

Objective To analyze the biological characteristics of Yersinia pestis strains in Haixi Prefecture,Qinghai Province,in order to provide a scientific basis for plague prevention and control in future.Methods Totally 181 strains were separated from variety kinds of host in Haixi Prefecture,Qinghai Province from 1957 to 2011,and these strains were conducted biochemical test,virulence factor evaluation,plasmid analysis,different region (DFR) genotyping,drug and disinfectant resistant genes detection;79 of the 181 strains were examined by toxicity test and classified according to the criteria (minimum lethal dose:MLD≤ 10 000 was velogenic strain,10 000 ＜ MLD ＜ 100 000 was moderate virulence strain,MLD ≥ 100 000 was hypovirulent strain).Results According to six biochemical typing about gelatin candy,rhamnose,maltose,melibiose,glycerin and denitrification,the 181 strains of Yersinia pestis were antique biovar and Qing-Tibet Plateau ecotype.Aproportion of 81.22％ (147/181) of Yersiniapestis strains contained all the four virulence factors (F1,Pst Ⅰ,VW,Pgm).Totally 63.54％ (115/181) of the strains contained 3 kinds of plasmid-6 × 106,45 × 106,and 52 × 106;31.49％ (57/181) of the strains contained 3 kinds of plasmid-6 × 106,45 × 106,and 65 × 106.The strains had 8 genomovars,and were given priority to genomovar 8 (109 strains),secondly,genomovar 32 (33 strains),genomovar 5 (20 strains),genomovar 1b(i4 strains),genomovar 44 (2 strains),genomovar 7 (1 strain),genomovar 37 (1 strain),and genomovar 49 (1 strain).Among the 181 Yersinia pestis strains,strains with genes related to streptomycin resistance,sulfanilamide resistance,beta lactam resistance and disinfectant resistance were not found;and 75 of 79 strains were velogenic strains by toxicity test (MLD ≤ 10 000),accounted for 94.94％ (75/79).Conclusion The strains separated in Haixi Prefecture,Qinghai Province have the characteristics of Qinghai-Tibet Plateau plague's pathogen and have strong toxicity;all strains don't have the characteristics of drug and disinfectant resistance genes.

Objective To explore the scan technique and image quality of coronary artery imaging with dual-source CT without oral Betaloc preparation.Methods Plain and enhanced dual-source CT coronary artery imaging without oral Betaloc preparation was performed in 215 patients with clinically suspected coronary heart disease or early-stage coronary lesions.Calcium scoring with plain scan images and multi-planar reconstruction(MPR),maximum intensity projection(MIP)and volume rendering technique (VRT)reconstruction with enhanced scan images were made in all cases.The scan technique and post reconstruction experience was summarized.The image quality was classified as three grades,and coronary segments classified according to AHA standards were evaluated.Results The median of calcium score of the 215 cases was 82.2(2.3—1827.9).The average heart rate of the enhanced scan was(80.6?15.3) (57—139)bpm.The post reconstruction methods with which coronary segments could be shown as best as possible consisted of(1)multiphases screening methods,(2)bi-phase or multiple-phase complement method,and(3)premature beat removing or arrhythmia shifting method.Altogether 3026 coronary segments were evaluated,among them 97.5% were evaluated as grade 1 image quality,2.0% were evaluated as grade 2 and 0.5% were evaluated as grade 3.The coronary segments in 91 cases were completely normal, while 112 segments with

Objective:To study the clustered regularly interspaced short palindromic repeats (CRISPR) genotype of Yersinia pestis and its regional distribution characteristics in natural plague focus of Tibet Autonomous Region. Methods:A total of 125 representative Yersinia pestis strains isolated from natural plague focus in Tibet Autonomous Region at different times, regions, hosts and vectors were selected as experimental strains, and the phenol chloroform mixed extraction method was used to extract Yersinia pestis DNA. Three pairs of CRISPR primers (for YPa, YPb, YPc locus) were used to amplify the DNA of the experimental strains, and the CRISPR genotype of Yersinia pestis was determined by sequencing. Results:All 125 strains of Yersinia pestis had three CRISPR locus: YPa, YPb, and YPc. A total of 18 spacer were found, including 8 in YPa loci, 6 in YPb loci, and 4 in YPc loci. Two new types of spacers had been discovered, namely b52 and c14. CRISPR typing revealed 10 genotypes, including G1, G7, G7-b4''', G7-b52, G7-c2 -, G8, G22, G22-a4 -, G22-b4''', and G22-c14, of which 6 were newly discovered genotypes. Among the 125 experimental strains, G7 was the main genotype, accounting for 65.6% (82/125), which was distributed in 6 prefecture level citys and 1 region of Tibet Autonomous Region. Next were G22 and G7-c2 - genetypes, accounting for 14.4% (18/125) and 11.2% (14/125), respectively. G22 gene type was distributed in Nagqu, Changdu, Lhasa citys, and Ngari Prefecture, while G7-c2 - genetype was distributed in Shigatse and Shannan cities. Conclusion:The CRISPR locus of Yersinia pestis in natural plague focus of Tibet Autonomous Region is highly polymorphic, and the Yersinia pestis strains with different genotypes have obvious regional distribution characteristics.