Emergency Response for a Laboratory Biosafety Incident.

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.

Genetic source tracking of an anthrax outbreak in Shaanxi province, China.

BACKGROUND: Anthrax is an acute zoonotic infectious disease caused by the bacterium known as Bacillus anthracis. From 26 July to 8 August 2015, an outbreak with 20 suspected cutaneous anthrax cases was reported in Ganquan County, Shaanxi province in China. The genetic source tracking analysis of the anthrax outbreak was performed by molecular epidemiological methods in this study. METHODS: Three molecular typing methods, namely canonical single nucleotide polymorphisms (canSNP), multiple-locus variable-number tandem repeat analysis (MLVA), and single nucleotide repeat (SNR) analysis, were used to investigate the possible source of transmission and identify the genetic relationship among the strains isolated from human cases and diseased animals during the outbreak. RESULTS: Five strains isolated from diseased mules were clustered together with patients' isolates using canSNP typing and MLVA. The causative B. anthracis lineages in this outbreak belonged to the A.Br.001/002 canSNP subgroup and the MLVA15-31 genotype (the 31 genotype in MLVA15 scheme). Because nine isolates from another four provinces in China were clustered together with outbreak-related strains by the canSNP (A.Br.001/002 subgroup) and MLVA15 method (MLVA15-31 genotype), still another SNR analysis (CL10, CL12, CL33, and CL35) was used to source track the outbreak, and the results suggesting that these patients in the anthrax outbreak were probably infected by the same pathogen clone. CONCLUSIONS: It was deduced that the anthrax outbreak occurred in Shaanxi province, China in 2015 was a local occurrence.

Optimization of Pulsed-field Gel Electrophoresis Procedure for Bacillus cereus.

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.

[Study on the single nucleotide polymorphism in capsule plasmid gene of Bacillus anthracis in the China isolates].

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 Pastuer. 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-specific 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.

[Study on the identification characteristics of rRNA genes on Yersinia pestis].

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, Nepal516, 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.

[Study on the application and evaluation of methods for gene and antigen detection in plague surveillance program].

OBJECTIVE: To apply and evaluate new methods regarding specific gene and antigen detection in plague surveillance program. METHODS: 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. RESULTS: 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%. CONCLUSION: The new standard being used for specific gene and antigen detection could increase the positive rate of diagnosis on plague.

[Use of variable-number tandem repeats to examine genetic diversity of Bacillus anthracis].

OBJECTIVE: To study the genotyping of Bacillus anthracis based on multiple-locus variable-number tandem repeats(VNTR) in the B. anthracis genome. METHODS: 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. RESULTS: (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. CONCLUSION: 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

[Study on the internal control on polymerase chain reaction in Yersinia pestis detection].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

[Surveillance on severe acute respiratory syndrome associated coronavirus in animals at a live animal market of Guangzhou in 2004].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

[Molecular biological characteristics and genetic significance of Yersinia pestis in China].

OBJECTIVE: To understand the molecular biological characteristics in order to analyse the genetic background of Yersinia pestis in China. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

[Study on the relation between the absence of one IS100 in 102 kb pgm locus of Yersinia pestis and the stability of pigmentation phenotype].

OBJECTIVE: 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(+)). METHODS: 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. RESULTS: 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. CONCLUSION: 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.

[Genetic analysis of Yersinia pestis strains isolated in China].

OBJECTIVE: The strains of Yersinia pestis isolated in different period and different natural foci in China were analyzed. METHODS: 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. RESULTS: The strains could be divided into 15 genetic types by those 6 characters with each of them covered an isolated geographical territories. CONCLUSION: 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.