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Objective: To analyze the epidemiological characteristics of anthrax in China from 2017 to 2019 and molecular typing of Bacillus anthracis isolated from some provinces (autonomous regions). Methods: Surveillance data of anthrax cases reported from 2017 to 2019 in the Infectious Disease Surveillance information System of China Disease Prevention and Control and the Public Health Emergency Reporting and Management Information System were collected, and descriptive epidemiological methods were used to analyze the epidemic characteristics, including the temporal, geographic and demographic distribution of this disease. A total of 47 strains of Bacillus anthracis isolated from 2017 to 2019 were analyzed by canSNP and MLVA15. Results: A total of 951 cases of anthrax were reported from 2017 to 2019, of which 938 were cutaneous anthrax, representing 98.63% of the total number reported. It was mainly distributed in the west and northeast of China, and the three provinces with the highest number of cases were Gansu (215), Sichuan (202) and Qinghai (191). Cases had been reported throughout the year, more cases occurred in the summer and autumn, and August was the month with the most cases,66.35% (211/318), 72.32% (243/336) and 68.01% (202/297) of cases were reported during June to September. The age distribution was mainly between 20 and 59 years old, accounting for more than 80% of all cases. The number of male cases was significantly higher than that of female cases, the ratio of male to female was about 3∶1. The occupations were mainly herdsmen and farmers, accounting for 49.70% to 58.18% and 31.45% to 36.70%, respectively. Public health events occurred every year, and 29 events had been reported from 2017 to 2019. canSNP analysis showed that 37 of the 47 strains belonged to the A.Br.001/002 subgroup and 10 belonged to the A.Br.Ames subgroup. MLVA15 analysis showed that there were 17 genotypes, of which 10 genotypes contained only one strain. Conclusion: Cutaneous anthrax was the predominant clinical type in China from 2017 to 2019.The seasonal, geographic and demographic distribution characteristics were evident.Molecular typing methods such as canSNP and MLVA15 can be used to trace the source of infectious diseases and provide technical support for anthrax prevention and control.
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Adult , Female , Humans , Male , Middle Aged , Young Adult , Anthrax/prevention & control , Bacillus anthracis/genetics , China/epidemiology , Molecular Typing , Polymorphism, Single Nucleotide , Skin Diseases, BacterialABSTRACT
On December 14, 2017, a faculty member of a university in Hunan Province reported that an anthrax vaccine strain might have recovered virulence during an undergraduate experiment and potential exposure could not be ruled out for the students involved. Upon receiving the case report, the CDC, health bureaus, and local governments at the county, prefectural, and provincial levels promptly organized experts in different fields (including epidemiologists, biosafety experts, and laboratory testing experts) for case investigation, evaluation, and response. As the investigation results showed, no virulence recovery was identified in the involved anthrax vaccine strain; and no contamination of Bacillus anthracis was detected at the involved areas. Thus, the university returned to normal functioning.
Subject(s)
Humans , Anthrax Vaccines , Bacillus anthracis , Virulence , China , Containment of Biohazards , Laboratories , VirulenceABSTRACT
In order to develop a rapid and reliable method for B. cereus genotyping, factors influencing PFGE results, including preparation of bacterial cells embedded in agarose, lysis of embedded cells, enzymatic digestion of intact genomic DNA, and electrophoresis parameters allowing for reproducible and meaningful DNA fragment separation, were controlled. Optimal cellular growth (Luria-Bertani agar plates for 12-18 h) and lysis conditions (4 h incubation with 500 µg/mL lysozyme) produced sharp bands on the gel. Restriction enzyme NotI was chosen as the most suitable. Twenty-two isolates were analyzed by NotI digestion, using three electrophoretic parameters (EPs). The EP-a was optimal for distinguishing between isolates. The optimized protocol could be completed within 40 h which is a significant improvement over the previous methods.
Subject(s)
Bacillus cereus , Genetics , Bacteriological Techniques , DNA, Bacterial , Chemistry , Genetics , Electrophoresis, Gel, Pulsed-Field , MethodsABSTRACT
Objective To study the characteristic of single nucleotide polymorphism (SNP)in capsule plasmid gene of Bacillus anthracis isolated from China.Methods 95 Bacillus anthracis isolates from different sources were selected.23 SNP sites were amplified by PCR method,sequenced and analyzed by clustering analysis.Results 95 Bacillus anthracis isolates were divided into 5 groups by cluster analysis.The identified isolates had the same sequence features in 17 sites and different nucleotide sequence in the other 6 sites of the 23 SNP sites.17.89％ (17/95) of the isolates had homologous locus sequences compared with the reference strain Pnstuer.38.95％ (37/95) of the isolates had the homologous locus sequences compared with the reference strain Ames Ancestor.The remaining strains were different from those completed sequenced strains.3 strains missed length of about 80 bp sequence in the PS-34 loci amplified gene fragment in which the tested SNP loci were included.9 strains were amplified negative at all SNP loci and Bacillus anthracis capsule plasmid genes were missing which was confirmed by capsule plasmid gene-speeific primers.Conclusion Results through analysis showed that single nucleotide genetic stability and specificity for capsule plasmid gene of Bacillus anthracis did exist in the Chinese isolates.The 6 discriminating SNP sites could be used as indicators in genotyping the Bacillus anthracis.
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Objective Measurement and analysis of the complete genome sequences of Yersinia Pestis from a new plague natural foci and adjacent foci in China, to know the genetic relationship among the epidemic strain isolated in Yulong (D 106004) and Jianchuan strains (D 182038) and the Tibetan strain ( Z 176003 ). Methods Three complete genome sequences were sequenced using the whole-genome shotgun and Solexa method and comparative genomics analysis was done among the three sequences. Genome comparative analysis among the coding sequences was done by BLAST software, SNPs finding was done by the program, genome rearrangements were analyzed using MAUVE software. Results All of the genomes of Yersinia pestis strains D182038, D106004 and Z176003 consist of a single circular chromosome and three virulence plasmids, pMT1, pCD1 and pPCP1. They had similar characteristics in chromosome and plasmid features, and there were no significant difference in coming sequence (CDS) of the cluster of orthologous groups of proteins (COG) functional classification and the number of insertion sequence in the three strains (x2 =3.03, 0.257, all P ＞ 0.05). The comparative genomics results showed that the three bacteria had 2882 genes with 100％ homology, of 3636 genes predicted in D106004, 2994 were identical with D182038's and 3113 with Z176003's, and of which 240 had 90％ homology with D182038's and 200 with Z176003 's. Synonymous single nucleotide polymorphisms(sSNPs) were 59 and 68, and non-synonymous SNPs(nsSNPs) were 104 and 203 between strains D106004 and Z176003/D182038. There were 11 segments rearrangements between D106004 and Z176003, which was less than 16 segments rearrangements between D106004 and D182038. ConclusionsThe three strains are highly homologous, the Yulong strain has more similarity with Tibet strain than with Jianchuan strain, the strain from Yulong foci may be evolved from Tibet foci.
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Objective To study the identification characteristics of rRNA genes on Yersinia (Y.)pestis.Methods By means of comparative genomics,we compared the rRNA genome sequences of nine completely sequenced strains of Y. pestis isolated from China and other countries by Clustal W software.we also compared the 2000 bp sequence adjacent to the rRNA genes,rRNA genes and 16S-23S rRNA spacer region respectively to determine the identification features of rRNA genes for Y. pestis.Results There were 6 rRNA gene clusters in the strains of D182038,D106004,Z176003 and CO92 respectively(6 copies strain).There were 7 rRNA gene clusters in the strains of 91001,KIM,Nepa1516,Antiqua and Pestoides F(7 copies strain).According to the 2000 bp sequence,13 types of rRNA gene clusters could classify the strains between the 6 copies and 7 copies.There were 4 types of tRNA gene among the 16S-23S rRNA spacer region that could classify the strains among the 6 copies and 7 copies strains respectively.The number of point mutation among the 23S rRNA gene was statistically different in some copies under ANOVA analysis(F=0.548,P=0.815>0.05 among the strains and F=5.228,P<0.01 among the copies).Conclusion The 2000 bp sequence adjacent to the rRNA genes,tRNA gene and 23S rRNA gene sequence could serve as the identification sign of rRNA genes for classifing the strains of Y. pestis.
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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.
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Objective To evaluate the method of detecting antibodies to Bacillus anthracis by enzymelinked immunosorbent assay(ELISA)using crude antigen.Methods The anti-Bacillus anthracis antibody levels in sera of 42 healthy people and 42 patients were detected by indirect ELISA.Standard curve was plotted using the data from positive controls,based on which the relative content of each serum was calculated and compared with the result of rLF.Results The median of antibody's relative content in patient group and healthy people group are 1.19 and 0.24,the differences being statistically significant(uc=7.643,P＜0.05).The result of crude antigen is in concordance with rLF(but not parallel absolutely).Conclusions Crude antigen can distinguish most of patients with healthy population effectively.The results suggested that crude antigen is applicable in anti-Bacillus anthracis antibody surveillance.
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Objective To clone and express specific genes (YP01089,psi.ymt) from Yersinia pestis in Escherichia Coli(E.coli)and to analyze the antigenicity of these recombinant proteins.Methods The target genes were amplified by polymerose chmn reaction(PCR).The amplified products were ligated with pET-30a(+) vector after purification and cut by two different restriction enzymes,then these recombinant plasmids were transfefred into the host cells of E.coli BL21(DE3) strain.The target genes were successfully expressed following induction with Isopropyithio-β-D-galactoside(IPTG),and the target proteins were purified by the method of affinity chromatography.Sodium dodecyl sulfate-polyaerylamide gel eleetrophoresis(SDS-PAGE)and Westem blot were used to detect the expressed recombinant protein.Results Three recombinant plasmids were finally constructed. rYP01089,rPst and rYmt were expressed stably and effectively in E.coli thmngh optimizing the induction condition. The Western blot analysis indicated that rPst was capable of binding with positive sernm of phgue.The purity of rest was up to 95%in this stuay.Conclusions This work indicates that the genes of Yersinia pestis are able to be efficiently expressed in the prokaryotie protein expression system.The immune characteristic of rPst is sensitive and specific,80 this study has settled a foundation for developing a new type diagnostic reagent of plague.
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Objective To develop a rapid test for the detection of F1 antigen of Yersinia Pestis based on gold-immunochromatography.Methods F1 antibodies were coupled with colloidal gold to prepare collidal gold reagent,which was used to detect F1 antibodies based on double antigen sandwich.The collidal gold reagent was estimated for its sensitivity specificity and stablity in labs and 1798 samples were detected in 17 surveillance spots.Results The reagent was sensitive to 0.0010 g/L F1 antigens.The reagens kept stable when it had been placed at 4℃ or room-temperature for 12 months and did not react to Yersinia pseudotuberculosis and Yersinia enterolitica.In 17 surveillance labs the reagent was used to test 1798 viscera samples from animal.resulting an accordance rate of 97.11%(1746/1798)to bacterial culture and 96.83%(1741/1798)accordance to reverse indirect hemagglutination assay(RIHA),showing a higher detection rate[9.23%(166/1798)]compared with RIHA[6.79%(122/1798)]and bacterial culture[6.28%(113/1798)].Conclusions The collidal gold reagent,sensitive and specific in diagnosing Yersinia pestis infection of both human and animals,is a rapid method in surveillance spot.
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<p><b>OBJECTIVE</b>To apply and evaluate new methods regarding specific gene and antigen detection in plague surveillance program.</p><p><b>METHODS</b>1798 samples from natural foci of plague were tested, using internal quality control multiple-polymerase chain reaction, F1 antigen marked by immuno chromatographic assay and enzyme linked immunosorbent assay. Culture of Yersinia pestis and reverse indirect hemagglutination assay were used as reference diagnostic methods.</p><p><b>RESULTS</b>The overall positive rate of culture on Yersinia pestis together with gene and antigen detection was 7.34%, showing an 16.81% increase when comparing to 6.28% using Yersinia pestis culture method alone. The rate of coincidence was 97.13%.</p><p><b>CONCLUSION</b>The new standard being used for specific gene and antigen detection could increase the positive rate of diagnosis on plague.</p>
Subject(s)
Animals , Mice , Bacterial Proteins , Genetics , Allergy and Immunology , Enzyme-Linked Immunosorbent Assay , Plague , Microbiology , Polymerase Chain Reaction , Yersinia pestis , Genetics , Allergy and Immunology , VirulenceABSTRACT
<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>
Subject(s)
Anthrax , Epidemiology , Genetics , Bacillus anthracis , Genetics , China , Epidemiology , Genetic Variation , Genotype , Geography , Polymerase Chain Reaction , Sequence Analysis, DNA , Tandem Repeat SequencesABSTRACT
<p><b>OBJECTIVE</b>For the detection of Yersinia pestis by polymerase chain reaction (PCR), internal control (IC) is required in order to prevent false negative results that might be caused by PCR inhibitors.</p><p><b>METHODS</b>F1 antigen was amplified by PCR with primer F1 and the PCR product of primer F1 were cloned with TOPO TA cloning Kit. The plasmid of positive clone was then digested with HpaI. The digested plasmid and the PCR products of 16S rRNA were ligated with T(4) DNA ligase before the ligated products were transformed. Isolate plasmid DNA on positive clone and its concentration were measured. Plasmid DNA on different concentration by PCR amplification with primer F1 was analyzed and the standard concentration of IC was determined.</p><p><b>RESULTS</b>Constructing an IC by inserting a 16S rRNA amplicon to the original target DNA between the two primer F1 sites, the size was longer than the target DNA. The standard concentration of IC was determined.</p><p><b>CONCLUSION</b>An optimal IC concentration to increase the reliability of the PCR assays might be used to prevent false negative results and appeared to be useful for detection of Yersinia pestis.</p>
Subject(s)
Bacterial Proteins , DNA, Bacterial , False Negative Reactions , Polymerase Chain Reaction , Methods , RNA, Ribosomal, 16S , Sensitivity and Specificity , Yersinia pestisABSTRACT
<p><b>OBJECTIVE</b>To study the prevalence of severe acute respiratory syndrome coronavirus (SARS-CoV) like virus in animals at a live animal market of Guanzhou in 2004 before and after culling of wild animal action taken by the local authority, in order to predict the re-emerging of SARS from animal originals in this region.</p><p><b>METHODS</b>Animals at live animal market were sampled for rectal and throat swabs in triplicate. A single step realtime reverse transcription-polymerase chain reaction (RT-PCR) diagnostic kit was performed for screening SARS-CoV like virus, the manual nested RT- PCR and DNA sequencing were performed for confirmation. Only specimens which tested positive for both of the N and P genes by nested RT-PCR were scored as positive.</p><p><b>RESULTS</b>In 31 animals sampled in January 5 2004 before culling of wild animals at Guangdong Province, including 20 cats (Felis catus), 5 red fox (Vulpes vulpes) and 6 Lesser rice field rats (Rattus losea), 8 (25.8%) animals were tested positive for SARS-CoV like virus by RT-PCR methods, of which 4 cats, 3 red fox and one Lesser rice field rats were included. However, two weeks after culling of animals and disinfection of the market were implemented, in 119 animals sampled in January 20 2004, including 6 rabbits (Oryctolagus cuniculus), 13 cats, 46 red jungle fowl (Gallus gallus), 13 spotbill duck (Anas platyrhynchos), 10 greylag goose (Anser anser), 31 Chinese francolin (Franclinus pintadeanus), only rectal swab from one greylag goose was tested positive for SARS-CoV like virus. Furthermore, in 102 animals that including 14 greylag gooses, 3 cats, 5 rabbits, 9 spotbill duck (Anaspoecilorhyncha), 2 Chinese francolin (Franclinus pintadeanus), 8 common pheasant (Phasianus colchicus), 6 pigeons, 9 Chinese muntjac (Muntiacus reevesi), 19 wild boar (Sus scrofa), 16 Lesser rice field rats, 5 dogs, 1 mink (Mustela vison), 3 goats, 2 green peafowl (Pavo muticus) sampled in April, May, June, July, August and November, only rectal swab from one pig was tested positive. However, of 12 and 10 palm civets sampled in November and December including five of which had been at the live animals market for 2 days, none of them was tested positive.</p><p><b>CONCLUSION</b>This findings revealed that animals being sampled in April, May, June, July, August and November of 2004, only one rectal swab from a pig was tested positive as SARS-CoV like virus, much lower than the results from the previous year, suggesting that the possibility of re-emerging of human infection from animal origins is low for the winter of 2004-2005.</p>
Subject(s)
Animals , Animals, Wild , Virology , China , DNA, Viral , Felidae , Virology , Reverse Transcriptase Polymerase Chain Reaction , Severe acute respiratory syndrome-related coronavirusABSTRACT
<p><b>OBJECTIVE</b>To understand the molecular biological characteristics in order to analyse the genetic background of Yersinia pestis in China.</p><p><b>METHODS</b>Primary datum on ribotyping, pulsed field gene electrophoresis (PFGE), random amplified polymorphic DNA (RAPD) and insertion sequence (IS) of Yersinia pestis were used and under cluster analysis. Genetic interval and various methods of recognized molecular feature between different strains were evaluated.</p><p><b>RESULTS</b>Ribotypes the PFGE types seemed to be corresponding. Stains from Microtus fuscus and area in Tibet Zhongba belonged to 7 copy rRNA gene and the genetic interval were the far more with 6 copy rRNA gene stains, and not definite with RAPD, but with many exceptions. The genetic interval between strains were showed by resemble value.</p><p><b>CONCLUSION</b>Yersinia pestis in China had its own manifold, particular molecular biological characteristics due to natural barriers, geographical complex, circumstances in Tianshan Mountains and Gandise Mountains areas. Yersinia pestis were limited to separateness, evoluted only in certain areas to form a great many gene types.</p>
Subject(s)
Animals , Humans , Mice , China , Cluster Analysis , DNA, Bacterial , Genetics , Electrophoresis, Gel, Pulsed-Field , Genetic Drift , Genotype , Geography , Random Amplified Polymorphic DNA Technique , Ribotyping , Yersinia pestis , Classification , GeneticsABSTRACT
<p><b>OBJECTIVE</b>To study the relation between the absence of one IS100 in the 102 kb pgm locus of Yersinia pestis and the stability of pigmentation phenotype (pgm(+)).</p><p><b>METHODS</b>We amplified the segment including IS100 in 102 kb pgm locus of Yersinia pestis that isolated from all ecotypes in China by polymerase chain reaction (PCR). There were 171 strains isolated from 18 ecotypes in this study. One strain was chosen to be cloned and sequenced.</p><p><b>RESULTS</b>Besides the type of Microtus brandti, the types of East-North Tianshan, A and B of West-North Tianshan, Microtus Qinghai had one band with about 2560 bp. These strains lost one IS100 in 102 kb pgm locus of Yersinia pestis. Their pgm(+) phenotype was stable. Some strains of ecotypes from Qilian Mountain, Qinghai-Tibet Plateau, Gangdisi Mountain, West Yunnan Mountain had no bands in the PCR products. Negative strains would lose the whole 102 kb pgm locus. The others had one band with 4492 bp. These strains had two IS100 which flanked the 102 kb pgm locus but the pgm(+) phenotype was unstable.</p><p><b>CONCLUSION</b>Yersinia pestis which had only one IS100 would flank the 102 kb pgm locus and had stable pgm(+) phenotype while the Yersinia pestis that having two IS100 flanked the 102 kb pgm locus would have unstable pgm(+) phenotype.</p>
Subject(s)
Bacterial Proteins , Genetics , Metabolism , Cloning, Molecular , DNA, Bacterial , Genetics , Genetic Variation , Genomic Instability , Phenotype , Pigmentation , Genetics , Yersinia pestis , GeneticsABSTRACT
<p><b>OBJECTIVE</b>The strains of Yersinia pestis isolated in different period and different natural foci in China were analyzed.</p><p><b>METHODS</b>Traditional and molecular biological methods were used. Rhamnose fermentation, rRNA gene copy number, nitrite reduction, and the glycerol fermentation were important characters for typing, and pulse field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD) profile could reflect the genetic distance between the strains.</p><p><b>RESULTS</b>The strains could be divided into 15 genetic types by those 6 characters with each of them covered an isolated geographical territories.</p><p><b>CONCLUSION</b>The characters of strains were described; the genetic relationship of different types, their evolution, and the forming and shift of plague natural foci were analyzed.</p>