Analysis of epidemic characteristics of anthrax in China from 2017 to 2019 and molecular typing of Bacillus anthracis / 中华预防医学杂志

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.

Detection of Bacillus anthracis and Bacillus anthracis-like spores in soil from state of Rio de Janeiro, Brazil

BACKGROUND Bacillus anthracis is the aetiologic agent of anthrax, a re-emerging, septicaemic, haemorrhagic and lethal disease that affects humans, domestic ruminants and wildlife. Plasmids pXO1 and pXO2 are attributes that confer pathogenicity to B. anthracis strains. This bacterium was used as biological weapon in the World Wars and in the biological attack in the United States of America at 2001. B. anthracis is classified as a Tier 1 bioterrorism agent by the Centers for Diseases Control and Prevention. Anthrax is recognised as a re-emerging disease. Several studies concerning the dynamics of B. anthracis cycle in soil revealed that nonpathogenic B. anthracis strains due to lack of pXO2 plasmid are commonly found in some types of soil. OBJECTIVES This study aimed isolation and identification of B. anthracis spores in soil samples of the state of Rio de Janeiro, Brazil. METHODS Phenotypic and genotypic approaches were used to identify isolates including MALDI-TOF/MS, motility test, susceptibility to gamma phage and penicillin, survey for pag and cap genes as surrogates of pXO1 and pXO2 plasmids, respectively, and sequencing of 16SrRNA-encoding gene. Physicochemical analysis of the soil samples were carried out to describe soil characteristics. FINDINGS We observed the presence of one B. anthracis pXO1+ and pXO2- isolated from clay loam soil; one B. anthracis-like strain pXO1+ and pXO2-isolated from loamy sand; and 10 Bacillus spp. strains sensitive to phage-gamma that need better characterisation to define which their species were recovered from loamy sand. MAIN CONCLUSIONS This work showed promising results and it was the first study to report results from an active surveillance for B. anthracis in Brazil.

Genome sequencing of two Bacillus anthracis strains: a virulent strain and a vaccinal strain

ABSTRACT Bacillus anthracis strain SPV842_15 was isolated from bovine fetus, while B. anthracis strain Brazilian vaccinal was recovered from a commercial vaccine. We report here the genome sequences of both strains. The SPV842_15 genome is composed of a single circular chromosome with a length of 5,228,664 base pairs, and comprises 5911 coding sequences. In turn, the Brazilian vaccinal genome remains in 201 contigs with 5733 coding sequences. Both genomes have an overall C + G content of 35.4%, and 11 genes encoding the ribosomal RNAs (rRNAs) 5S, 16S and 23S. Only the plasmid pX01 sequence, which carries genes for toxins synthesis, was detected and completely assembled for both strains. These plasmids have a length of 181,684 base pairs and a C + G content of 32.5%. These genomic data generate insights about vaccinal B. anthracis virulence.

Bacillus anthracis: una mirada molecular a un patógeno célebre / Bacillus anthracis: a molecular look at a famous pathogen

Bacillus anthracis es un bacilo gram positivo del grupo Bacillus cereus, que posee un genoma extremadamente monomórfco y comparte gran similitud fsiológica y de estructura genética con B. cereus y Bacillus thuringiensis. En este artículo se describen nuevos métodos moleculares para la identifcación y tipifcación de B. anthracis, basados en repeticiones en tándem de número variable o en diferencias genéticas detectadas por secuenciación, desarrollados en los últimos años. Los aspectos moleculares de los factores de virulencia tradicionales, cápsula, antígeno protector, factor letal y factor edema se describen en profundidad, junto con factores de virulencia recientemente propuestos, como los sideróforos, petrobactina y bacilibactina, la adhesina de la capa S y la lipoproteína MntA. También se detalla la organización molecular de los megaplásmidos pXO1 y pXO2, incluyendo la isla de patogenicidad de pXO1. El esqueleto genético de estos plásmidos se ha encontrado en otras especies relacionadas, probablemente debido a eventos de transferencia lateral. Finalmente, se presentan los dos receptores celulares del antígeno protector, ANTXR1/TEM8 y ANTXR2/CMG2, esenciales en la interacción del patógeno con el hospedador. Los estudios moleculares realizados en los últimos años han permitido aumentar enormemente el conocimiento de los diferentes aspectos de este microorganismo y su relación con el hospedador, pero a la vez han abierto nuevos interrogantes sobre este notorio patógeno.

Bacillus anthracis, a gram-positive rod belonging to the Bacillus cereus group, has an extremely monomorphic genome, and presents high structural and physiological similarity with B. cereus and Bacillus thuringiensis. In this work, the new molecular methods for the identifcation and typing of B. anthracis developed in the last years, based on variable number tandem repeats or on genetic differences detected through sequencing, are described. The molecular aspects of traditional virulence factors: capsule, protective antigen, lethal factor and edema factor are described in depth, together with virulence factors recently proposed, such as the siderophores petrobactin and bacillibactin, the S-layer adhesin and the MntA lipoprotein. It is detailed the molecular organization of megaplasmids pXO1 and pXO2, including the pathogenicity island of pXO1. The genetic skeleton of these plasmids has been observed in related species, and this could be attributed to lateral gene transfer. Finally, the two anthrax toxin protective antigen receptors, ANTXR1/TEM8 and ANTXR2/CMG2, essential for the interaction of the pathogen with the host, are presented. The molecular studies performed in recent years have greatly increased knowledge in different aspects of this microorganism and its relationship with the host, but at the same time they have raised new questions about this noted pathogen.

Construction of a eukaryotic plasmid encoding bacillus anthracis protective antigen, a candidate for DNA vaccine

DNA immunization with plasmid DNA encoding bacterial, viral, parasitic and tumor antigens has been reported to trigger protective immunity. To evaluate the use of a DNA immunization strategy for protection against anthrax, a plasmid was constructed. The partial sequence of protective antigen of Bacillus anthracis, amino acids 175-764, as a potent immunogenic target was selected. The DNA encoding this segment was utilized in the construction of pcDNA3.1+PA plasmid. After intramuscular injection of rats with pcDNA3.1+PA plasmid, the expression of PA was assessed by RT-PCR and immunohistochemistry at RNA and protein levels, respectively. We also evaluated the presence of anti-PA antibodies in sera of immunized mice with pcDNA3.1+PA construct using immunoblotting. The integrity of pcDNA3.1+PA construct was confirmed with restriction analysis and sequencing. The expression of PA was detected at RNA and protein levels. The presence of anti-PA antibodies in immunized mice with pcDNA3.1+PA construct was also confirmed. Our results indicate that pcDNA3.1+PA eukaryotic expressing vector could express PA antigen, induce antibody response and may be used as a candidate for DNA vaccine against anthrax

Cloning of protective antigen gene from Bacillus anthracis into Bacillus subtilis

Protective antigen [PA] of Bacillus anthracis is used as anthrax vaccine. Cloning and expression of the PA gene in various strain such as E.coli and Bacillus subtilis was reported that most expression was in B. subtilis up to 160 microg/ml. The objectives of this study were: to clone the gene of PA in an expression vector [pWB980] and then transformation into the B. subtilis WB600 strain. The pXOl plasmid was separated from the strain stern of B. anthracis with alcalin method and the PA gene with 2.4kb sequence amplified by PCR. Then the amplified fragment was directly cloned into pTZ57R plasmid as T-vector and transferred into E.coli DH5 alpha using CaC12 method. After that the gene was separated from the T-vector by enzymatic digestion [Sa1I and KpnI]. Ligation between the purified gene fragment of the PA and the vector was carried out. Then it was transferred into B. subtilis WB600 by electroporation method in 1000 V. In this study we isolated PA gene from B. anthracis strain stern with PCR and was cloned into pTZ57R plasmid. The Presence of the gene was confirmed by restriction analysis, PCR and sequencing. Then the PA gene was cloned into pWB980 and B. subtilis and the presence of the gene in two kanamycin resistant colonies [AMN1 and AMN3] was confirmed by restriction analysis and PCR. We may conclude that by making modification in the methods used and using pWB980 expression vector, we were able to clone the PA gene into B. subtilis. This is the first research project in Iran that the PA gene is isolated and cloned and B. subtilis is used as host