Changed epidemiology of anthrax and molecular characteristics of Bacillus anthracis in Inner Mongolia Autonomous Region, China.

Anthrax is a natural foci disease in Inner Mongolia, which poses a severe threat to public health. In this study, the incidence number, rate and constituent ratio were used to describe the epidemiological characteristics of anthrax in the region from 1956-2018. The molecular correlation and genetic characteristics of the strains were investigated using canonical single nucleotide polymorphisms (CanSNP), multiple-locus variable-number tandem repeat analysis (MLVA-15) and whole genome sequencing (WGS). The epidemiological characteristics of anthrax in Inner Mongolia have altered significantly. The incidence of anthrax has decreased annually without vaccination, and the regional distribution of anthrax gradually transferred from central and western regions to the eastern. Moreover, the occupation distribution evolved from multiple early occupations to predominated by farmers and herdsmen. This change is closely related to policy factors and to changes in the means of production and the living habits of the local population. This indicates that reformulating the control and prevention strategies is essential. Both A. Br. Ames and A. Br. 001/002 subgroups were the predominant CanSNP genotypes of Bacillus anthracis in Inner Mongolia. A total of 36 strains constituted six shared MLVA-15 genotypes, suggesting an epidemiological link between the strains of each shared genotype. The six shared genotypes ([GT1, 9, 11 and 15] and [GT8 and 12]) consisting of 2-7 strains confirmed the occurrence of multiple point outbreaks and cross-regional transmission caused by multiple common sources of infection. Phylogenetic analysis based on the WGS core genome showed that strains from this study formed an independent clade (C.V.), and they were positioned close to each other, suggesting a common origin. Further comparison analysis should be performed to ascertain the geographic origin of these strains.

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

[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.

[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.

[The geographical distribution of ribotypes of Yersinia pestis in China].

OBJECTIVE: To type and group the Yersinia pestis strains isolated in China to clarify the geographical distribution of ribotypes of Yersinia pestis. METHODS: Genomic DNA of Yersinia pestis were digested with EcoR I, then hybridized with 16s-23s-5s rRNA gene probe. RESULTS: These tested strains were divided into 3 ribotypes, the profiles obtained were relatively homogeneous, with most of them differed only by the presence or the absence of 1 - 2 restriction fragments. Ribotype A and B were the most common types, which distributed in a large area in China while ribotype C was the least, only limited to a small area. There was certain correlation between the ribotypes and the plague foci, usually only one ribotype was found in one plague foci. CONCLUSION: The ribotypes were stable in the plague foci. Correlation between the ribotypes of Yersinia pestis strains and their geographical origins was noticed. All 3 ribotypes had different origins, however ribotype A and ribotype C seemed to be closer related.

[The 102 kb pigmentation (pgm) locus of Yersinia pestis isolated from Microtus brandti].

OBJECTIVE: To find out the differences between 102 kb pgm locus of Yersinia pestis isolated from Microtus brandti with of other types, and the characters of Yersinia pestis isolated from Microtus brandti caused by their makeup of the 102 kb pgm locus. METHODS: 102 kb pgm locus of Yersinia pestis isolated from Microtus brandti and Yersinia pestis isolated from Marmota himalayana were amplified by polymerase chain reation (PCR) with 25 pair of nested primers. The PCR products of one pair of primer were obviously different, and then cloned and sequenced. Sequences were searched against current protein and nucleotide databases, using BLAST. RESULTS: The 102 kb pgm locus of Yersinia pestis isolated from Microtus brandti was devoid one IS100. In addition, it had more copies than other types in the similar variable-number tandem repeat sequences. CONCLUSION: The 102 kb pgm locus of Yersinia pestis was different from that of other types. It had only one IS100 flanked it, which corresponded to the character that its pgm(+) phenotype was stable. Further study was needed to confirm the relationship between the diminution virulence of Yersinia pestis isolated from Microtus brandti and the loss of IS100 and other changes.