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Objective:To study the phenotype and genotype distribution of Yersinia pestis ( Y. pestis) in different natural foci of plague in China, so as to provide scientific basis for plague prevention and control. Methods:A total of 2 184 strains of Y. pestis isolated from different time periods, regions, hosts and vectors in 11 plague natural foci of China since 1943 were selected for biochemical type identification, glycolysis test, virulence factor test [capsule antigen (F1), pesticin Ⅰ (Pst Ⅰ), virulence antigen factor (VWa), pigmentation factor (Pgm)], different region (DFR) typing and clustered regularly interspaced short palindromic repeats (CRISPR) typing. Results:There were 16 biochemical types of Y. pestis in the natural foci of plague in China, and each biochemical type showed obvious regional distribution in each foci. Most strains were positive for ass hide glue glycolysis (89.79%, 1 961/2 184), maltose (80.13%, 1 750/2 184), glycerol (94.23%, 2 058/2 184), and denitrification (82.78%, 1 808/2 184), and negative for rhamnose (88.78%, 1 939/2 184) and melibiose (85.62%, 1 870/2 184). Virulence factor test results showed that 99.95% (2 183/2 184) of Y. pestis were F1 positive; 99.73% (2 178/2 184) of Y. pestis can produce Pst Ⅰ; 73.31% (1 601/2 184) of Y. pestis were VWa positive and 26.69% (583/2 184) were VWa negative; Pgm positive strains accounted for 72.62% (1 586/2 184), Pgm negative strains accounted for 21.52% (470/2 184), and Pgm mixed type strains accounted for 5.86% (128/2 184). According to DFR typing results, there were 52 genotypes in 2 184 strains of Y. pestis, of which 19 were major genotypes and 33 were minor genotypes. CRISPR typing revealed 16 major genotypes, of which 7 were newly discovered. Conclusion:The phenotypes and genotypes of Y. pestis in various natural foci of plague in China are diverse and have geographical distribution characteristics.
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Objective:To learn about the clustered regularly interspaced short palindromic repeats (CRISPR) genotyping of Yersinia pestis in Yushu Tibetan Autonomous Prefecture (Yushu for short), Qinghai Province, and to explore its genetic characteristics. Methods:In this study, 44 representative strains isolated from local natural plague focus in Yushu from 1963 to 2007 were selected as experimental objects to extract DNA. Primers targeting the three CRISPR loci (YPa, YPb, and YPc) were designed for PCR amplification. The amplified products were sequenced and analyzed to identify the CRISPR spacer, and to determine the CRISPR genotypes and clusters.Results:Twenty-three spacers including 14 of YPa, 6 of YPb and 3 of YPc were observed among 44 strains, of which 2 spacers (a106 and a107) were firstly identified. According to the spacer arrays, the strains were divided into 15 CRISPR genotypes and classified into 6 CRISPR clusters which were Cb4, Cc3', Ca7, Ca7', CaΔ5' and Ca35', respectively. Among them, Ca7 was the most epidemic dominant cluster (34 strains) in Yushu.Conclusion:The CRISPR loci of Yersinia pestis in Yushu have multiple genotypes, high genetic polymorphism, and complex population structure.
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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.
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Objective:To observe the efficacy of levofloxacin and moxifloxacin in the treatment of experimental plague in guinea pigs.Methods:A total of 70 SPF guinea pigs, female, weighing 250 to 300 g, were selected and randomly divided into 7 groups according to body weight by random number table. There were 10 guinea pigs in each group: levofloxacin 24, 48, 72 h groups, moxifloxacin 24, 48, 72 h groups (drug treatment was carried out after being infected with Yersinia pestis for 24, 48 and 72 h, respectively) and control group (without treatment). Experimentally infected plague model was established through guinea pigs subcutaneous injection of 141 strains of Yersinia pestis [1 × 10 7 colony forming unit (CFU)]; referring to the adult dose in the "National Pharmacopoeia of the People's Republic of China", the daily dose of guinea pigs was converted by Shi Xinyou's animal conversion coefficient method for treatment, the doses of levofloxacin and moxifloxacin in each guinea pig were 12.0 and 9.6 mg/d, respectively for 9 days. The guinea pigs were killed 9 days after drug withdrawal for bacteriological examination and pathological observation, and the cure rate was calculated. Results:The cure rates of levofloxacin 24, 48 and 72 h groups were 100.0% (10/10), 70.0% (7/10) and 6/6, respectively. The cure rates of moxifloxacin 24, 48 and 72 h groups were 100.0% (10/10), 100.0% (10/10) and 5/7, respectively, and the cure rate of animals in the control group was 0 (0/10). Compared with the control group, there were significant differences in the efficacy of moxifloxacin 24, 48 and 72 h groups and levofloxacin 24, 48 and 72 h groups ( P < 0.05). There was no significant difference between the two drugs at the same starting time ( P > 0.05). Conclusion:The effects of levofloxacin and moxifloxacin on animal plague infection are ideal and the two drugs can be used as a substitute for streptomycin in plague treatment under special circumstances.
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Objective:To analyze the pathogenic characteristics of Yersinia pestis in a plague natural foci in Qinghai-Tibet Plateau. Methods:In this study, 1 378 strains of Yersinia pestis isolated from different regions, hosts and vectors in Qinghai-Tibet Plateau from 1954 to 2016 were taken as the research objects. Phenotypic characteristics, plasmid spectrum and genotype of the strains were studied by using conventional techniques and molecular biological techniques. The etiology and geographical distribution of the plague were studied. Results:There were 6 biochemical types of Yersinia pestis in Qinghai-Tibet Plateau, namely Qinghai-Tibet Plateau, Qilian Mountain, Gangdis Mountain, Kunlun Mountain A, Kunlun Mountain B and Chuanqing Plateau. This study found that the Qinghai-Tibet Plateau type strain was not only distributed in north Tibet Plateau, but also distributed in south Tibet, and the distribution of Gangdis Mountain type strain extended to south Tibet. Four virulence factors (capsule antigen, yersinin, virulence antigen and pigmentation factor) were found in 79.97% (1 102/1 378) Yersinia pestis. The results also showed that there were 12 kinds of plasmids carried by Yersinia pestis strains in Qinghai-Tibet Plateau, which constituted 17 kinds of plasmid spectrum. There were 3 kinds of the largest plasmids with taxonomic properties, forming their respective relatively independent distribution areas. The study of different regions (DFR) type showed that 5, 8, 14, 19, 32 and 44 of 1 378 strains were the main genotypes, and the main genome types had obvious geographical distribution. Conclusions:All the tested strains have the characteristics of plague pathogen in Qinghai-Tibet Plateau. The polymorphism of the main hosts, vectors and the ecological landscape of plague geography in the plague foci in Qinghai-Tibet Plateau may lead to the diversity of biochemical characters, plasmid spectrum and geno types of Yersinia pestis.
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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.
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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.
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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.
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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.
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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.
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Objective In order to acquaint with the prevalence of Tibetan sheep plague in this area, we conducted a serum epidemiological investigation of Tibetan sheep plague in Qinghai Province. Methods Indirect hemagglutination assay (IHA) and colloidal gold immunochromatography (GICA) were applied to test serum samples of Tibetan sheep and whole blood samples from jugular vein of Tibetan sheep were collected in 8 Prefectures of Qinghai Province from 2013 to 2016. Results A total of 86 positive Tibetan sheep serum samples with plague F1 antibody were detected by both methods, and the positive rate was 0.68％ (86/12710), the samples collected in Xinghai County Hainan Prefecture had the highest positive rate, which was 5.20％ (27/519). The Haixi Prefecture and Yushu Prefecture were historical epidemic areas, the positive rates were 0.65％(15/2313) and 0.26％(6/2293), respectively. Hainan Prefecture, Guoluo Prefacture and Huangnan Prefecture were newly confirmed epidemic areas, the positive rates were 1.61％ (28/1741), 1.01％ (15/1481), and 1.44％(19/1316), respectively. The antibody titers were 1:20 to 1:5120, the samples collected in Maqin County Guoluo Prefecture had the highest titer, namely 1 :5120. Conclusions In Qinghai Province, Tibetan sheep plague is endemic, and there are outbreaks in some regions. So we have to enhance the Tibetan sheep plague monitoring especially in Marmot plague epidemic area.
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Objective To establishment a method for detection of multiple drug resistance gene of Yersina pestis using polymerase chain reaction(PCR), to provide a guidance for treatment of plague. Methods According to National Center for Biotechnology Information (NCBI) released sequences of aminoglycoside resistant genes of streptomycin resistant,strB,strA,beta lactam antibiotics resistant genes tem,shv,and ctx-m,sulfamilamide resistant genes sul1, sul2, and sul3, a pair of primers of each gene was designed. DNAs of 282 strains isolated from plague natural foci in Qinghai Province were amplified by PCR using every pair of primers. The products were separated using gel electrophoresis, and the results were visualized through a gel imaging system. The susceptibility of 282 Yersina pestis to streptomycin, sulfamethoxazole and ceftriaxone was tested by drug sensitivity test. Results The PCR amplification results of all samples were negative,and strains with streptomycin,sulfamilamide and beta lactam antimicrobial drug resistance genes were not found. Drug sensitivity test showed that 282 strains were highly sensitive to streptomycin,sulfamethoxazole and ceftriaxone sodium.The diameter of bacteriostasis ring>19,17,21 mm, respectively. Conclusions It is a feasible method to use PCR technology to detect the multiple drug resistance genes of Yersinia pestis. Using this method to systematically monitor the resistance gene of Yersinia pestis is an efficient, economical and practical experimental method, which can provide guidance for the treatment of plague disease.
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Objective To study the biological characteristics and epidemiological significance of Yersinia pestis in Chengduo County of Qinghai Province,in order to provide scientific basis for plague prevention and control in this area.Methods Thirty one strains of Yersinia pestis isolated from Chengduo County of Qinghai Province from 1980 to 2011 were selected as study subjects.Biochemical test,virulence factors evaluation [Fra1 (F1),pesticin Ⅱ (Pst Ⅱ),virulence antigen (VW),pigmentation (Pgm)] and different region (DFR) genotyping were carried out.Nineteen of the 31 strains Yersinia pestis were selected according to different time,different areas and different hosts to determine their toxicity in mice,MLD ≤ 10 000 was strong toxic strain,10 000 ＜ MLD ＜ 100 000 was moderate toxic strain.Results Among thirty one strains of Yersinia pestis,23 strains were isolated from human,the Himalaya marmot and its fleas and lice,and their biological type was classical,biochemical type was Qinghai-Tibet plateau;21 strains genotype was type 5,1 was type 16,1 was type 32,and they contained all four kinds of virulence factors (F1,Pgm,Pst Ⅱ,VW),and toxicity test showed all strains (14) were strong toxic strains.The rest 8 strains of Yersinia pestis isolated from the Microtus fuscus and its fleas,and their biological type was Microtus,biochemical type was Chuanqing plateau;they could produce F1 and Pgm,of which 87.5％ (7/8) strains could produce Pst Ⅱ,but could not produce VW antigen factor,the genotype was 14,and the toxicity results showed that they were strong (3)and moderate (2) toxic strains.Conclusion The strains separated in Chengduo County of Qinghai Province from 1980 to 2011 have the pathogen characteristics of Qinghai-Tibet plateau plague,they are mainly strong toxic strains;the work on prevention and control of plague should not be neglected.
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Objective To understand the epidemic trend of Tibetan sheep plague in Guoluo Prefecture,Qinghai Province,we detected the plague F1 antibody in Tibetan sheep serum in this area.Methods Indirect hemagglutination test (IHA) and colloidal gold immunochromatography (GICA) were applied to test serum samples of Tibetan sheep which were separated from 5 ml whole blood drew from jugular vein in Maqin County,Maduo County,Gande County,Banma County,Jiuzhi County and Dari County in 2014 and 2015.Results We collected 1 481 serum samples,566 from Maqin County,315 from Maduo County,150 from Gande County,150 from Banma County,150 from Jiuzhi County and 150 from Dari County.Totally 14 serum samples showed F1 antibody positive,the positive rate was 0.95％ (14/1 481),and they were all from Maqin County.Conclusions This area has the prevalence of Tibetan sheep plague.Therefore,the monitoring work of Tibetan sheep plague should be strengthened.
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Objective To investigate the biological characteristics and epidemiological significance of Yersinia pestis strains in Qilian County,Qinghai Province,in order to provide a scientific basis for plague prevention and control.Method Totally 67 strains were separated from kinds of host in Qilian County,Qinghai Province from 1958 to 2011,to do biochemical test,toxicity test,virulence factors evaluation,plasmid analysis and different region (DFR) genotyping.Results According to biochemical typing,48 of the 50 strains tested were Qing-Tibet Plateau ecotype,15 were Qilian Mountain ecotype,and the remaining 4 were different ecotypes from the plague foci in Qinghai plateau.The strains had 8 genomovars,and were given priority to genomovar8 (42 strains),secondly,genomovar44 (15 strains),genomovar5 (4 strains),genomovar7 (2 strains),genomovar19 (1 strain),genomovar30 (1 strain),genomovar32 (1 strain),and genomovar34 (1 strain).A proportion of 95.52％ (64/67) of the strains contained 3 kinds of plasmid-6 × 106,45 × 106,and 52 × 106;85.07％ (57/67) contained all the four virulence factors,and 96.00％ (48/50) were velogenic strains.Conclusion The strains separated in Qilian County,Qinghai Province have the characteristics of Qinghai-Tibet Plateau plague's pathogen and have strong toxicity,so we should enhance the plague monitoring and give more publicity to plague prevention to prevent animal plague spreading to human.
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Objective@#To investigate the CRISPR genotypes (clusters) and regional distribution of Yersinia pestis in Qinghai-plateau.@*Methods@#One hundred and two isolates of Y. pestis isolated from human plague patients, host animal and insect vectors from Qinghai-plateau were selected. The DNAs were extracted using the traditional sodium dodecyl sulfate decomposition and phenol-chloroform method. Three CRISPR loci YPa, YPb and YPc of 102 isolates of Y. pesits were amplified and sequenced, and then the CRISPR sequence analysis was carried out by comparing the latest published CRISPR spacer dictionary and the NCBI database to identify the spacer and spacer array. CRISPR genotyping of isolates of Y. pesits were finally conducted according to the polymorphism of the spacer arrays and the regional distribution pattern of isolates of Y. pesits in Qinghai-plateau was described.@*Results@#Forty spacers including 22 of YPa, 13 of YPb and 5 of YPc were observed among 102 isolates of Y. pestis in Qinghai-plateau, of which 5 spacers (a1', a103, a104, b4'' and b4''') were firstly identified. Meanwhile, 16, 10, and 5 different spacer arrays were obtained in YPa, YPb and YPc respectively, including 11 new spacer arrays detected in this study. One hundred and two isolates were divided into 24 CRISPR genotypes and classified into 9 CRISPR clusters (Cb4, Cb4', Cb2, Ca37, Ca7, Ca7', CaΔ5', Ca35' and Cc3'). Each dominant cluster presented significant aggregation geographically: Ca7 were found in Yushu, Nangqian, Chenduo, Zaduo, Zhiduo and Qumalai countries. Ca7' were found in Xunhua, Tongren, Zeku, Tongde, Maqin and Guinan countries. CaΔ5' were restricted to Qilian, Gangcha, Menyuan and Datong countries. CaΔ35' were found in Huangyuan, Haiyan, Gangcha, Tianjun, Delingha, Wulan, Doulan, Gonghe, Xinghai, Guide and Tongde countries.@*Conclusion@#CRISPR-based genotyping analyses showed complicated population of Y. pestis in Qinghai-plateau. Four clusters, Ca7, Ca7', CaΔ5' and Ca35' were the most epidemic dominant four clusters and presented obvious regional distribution patterns, which instructed us to strengthen the surveillance and prevention and control by CRISPR-genotyping technique.
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Objective By the method of multiple polymerase chain reaction (PCR),we intend to amplify different regions (DFR) of Yersinia pestis DNA,and to establish a multiple DFR genotyping technique for detection of Yersinia pestis.Methods According to the product size of 23 DFRs and pMT plasmid,24 primers were optimized and combined,then multiple primers in one PCR reaction system were added,and positive template DNA was amplified.Meanwhile,200 wild strain DNAs were amplified by multiple PCR and normal PCR,to verify the coincidence rate of the two methods.Results Totally 24 target segments were amplified through the positive DNA template.Through different permutation and combination,24 primers were optimized and combined into 9 groups.Totally 200 wild strain DNAs were used for verification,the coincidence rate of multiple PCR and normal PCR was 100％.Conclusions Multiple PCR is applicable and feasible for DFR genotyping of Yersinia pestis.It is an efficient,economic and high accuracy experimental method for large quantities of Yersinia pestis DFR genotyping.
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<p><b>OBJECTIVE</b>To identify the epidemiology and etiology characteristics of Tibetan sheep plague in Qinghai plateau.</p><p><b>METHODS</b>The background materials of Qinghai Tibetan sheep plague found during 1975 to 2009 were summarized, the regional, time and interpersonal distribution, infection routes, ecological factors for the spread were used to analyze; followed by choosing 14 Yersinia pestis strains isolated from such sheep for biochemical test, toxicity test, virulence factors identification, plasmid analysis, and DFR genotype.</p><p><b>RESULTS</b>From 1975 to 2009, 14 Yersinia pestis strains were isolated from Tibetan sheep in Qinghai province. Tibetan sheep, as the infection source, had caused 10 cases of human plague, 25 plague patients, and 13 cases of death. All of the initial cases were infected due to eating Tibetan sheep died of plague; followed by cases due to contact of plague patients, while all the initial cases were bubonic plague. Cases of bubonic plague developed into secondary pneumonic plague and septicemia plague were most popular and with high mortality. Most of the Tibetan sheep plague and human plague occurred in Gannan ecological zone in southern Gansu province, which was closely related to its unique ecological and geographical landscape. Tibetan sheep plague coincided with human plague caused by Tibetan sheep, especially noteworthy was that November (a time for marmots to start their dormancy) witnesses the number of Yersinia pestis strains isolated from Tibetan sheep and human plague cases caused by Tibetan sheep. This constituted the underlying cause that the epidemic time of Tibetan sheep plague lags obviously behind that of the Marmot plague. It was confirmed in the study that all the 14 strains were of Qinghai-Tibet Plateau ecotype, with virulence factors evaluation and toxicity test demonstrating strains as velogenic. As found in the (Different Region) DFR genotyping, the strains isolated from Yushu county and Zhiduo county were genomovar 5, the two strain isolated from Nangqian county were genomovar 5 and genomovar 7, while those isolated Delingha region were genomovar 8.</p><p><b>CONCLUSION</b>Tibetan sheep were vulnerable to plague infection, hence causing human plague as the infectious source. The Yersinia pestis strains isolated from Tibetan sheep plague carried pathogen characteristics of Qinghai-Tibet plateau plague, developing many new characteristics of such plague.</p>
Subject(s)
Animals , Humans , Ecology , Genotype , Geography , Marmota , Plague , Epidemiology , Plasmids , Sheep , Microbiology , Tibet , Epidemiology , Yersinia pestisABSTRACT
<p><b>OBJECTIVE</b>To analyze the plasmid features and geographical distribution characteristics of Yersinia pestis of different plague foci in China.</p><p><b>METHODS</b>A total of 2 213 Yersinia pestis strains were colected from 11 Chinese plague foci separated during 1943 to 2012, and plasmid DNA according to alkali cracking method, and measured the relative molecular mass (Mr) of plasmid DNA based on the standard plasmid contrast method, then analyzed the plasmid profiles by agar gel electrophoresis.</p><p><b>RESULTS</b>A total of 2 213 strains had 16 kinds of plasmids with different Mr, including 4×10(6), 6×10(6), 7×10(6), 13×10(6), 16×10(6), 20×10(6), 22×10(6), 23×10(6), 27×10(6), 30×10(6), 36×10(6), 45×10(6), 52×10(6), 65×10(6), 72×10(6) and 90×10(6). Plasmid were classified into 26 kinds of plasmid profiles. A total of 2 213 Yersinia pestis strains contained 4 large plasmids, 52×10(6), 65×10(6), 72×10(6) and 90×10(6), whose ratio was 22.10% (589/2 213), 75.60% (1 672/2 213), 0.17% (4/2 213), 2.12% (47/2 213), respectively. Among which, strains with plasmid 52×10(6), 65×10(6), 90×10(6) distributed in Qinghai-Tibet plateau Himalayan Marmot natural plague foci, strains with 72×10(6) plasmid only distributed in Inner Mongolia Meriones unguiculatus natural plague foci and Junggar Basin R. opimus natural plague foci, and 65×10(6) plasmid distributed in all the other foci.</p><p><b>CONCLUSION</b>Strains in Chinese 11 plague foci contained 4 kinds of large plasmid, the Mr respectively were 52×10(6), 65×10(6), 72×10(6), 90×10(6), which were classified into 26 kinds of plasmid profiles with other plasmid. These plasmid profiles distributed in relatively independent epidemic focus.</p>
Subject(s)
Animals , China , Genotype , Plague , Plasmids , Yersinia pestisABSTRACT
<p><b>OBJECTIVE</b>To study the biological and genetic characteristics of 119 strains of Yersinia (Y.) pestis isolated from plague patients in Qinghai province, from 1958-2012.</p><p><b>METHODS</b>Both regular methods and different region(DFR)molecular typing techniques were used to study the epidemiological characteristics on 119 strains of Y. pesticin Qinghai during 1958-2012. Sources of Y. pestis from two outbreaks, in Nangqian county in 2004 and in Xinghai county in 2009,Qinghai province were also analyzed.</p><p><b>RESULTS</b>105 strains of Y. pestis were identified as Qinghai-Tibet Plateau Ecotype while the other 6 strains as Qilian Mountains Ecotype. 84.03% (100/119) of the tested strains carried 4 virulence factors F1(+), Pst I(+), VW(+) and Pgm(+)). 97.30% (72/74) of the tested strains showed high virulence. Strains that carrying 52×10(6), 65×10(6), 92×10(6) plasmids were distributed in Hainan, Haibei, Haixi,Yushu,Guoluo, Huangnan and Huangyuan counties. Genomovar 5 and 8 were the main gene types that circling around Qinghai Lake. Genomovar 10 was found in strains of Y. pesticin Nangqian county while Genomovar 8 was found in the strains isolated from human plague patient during the epidemics in Xinghai county in Qinghai.</p><p><b>CONCLUSION</b>Data from biological and genetic analyses on the epidemics of human plague in Nangqian county in 2004 and in Xinghai county in 2009 demonstrated that methods as DFR genotyping and virulence factors profiles, as well as plasmids profiles were powerful tools in confirming the human plague epidemics and sources of infection.</p>