Bacillus anthracis, a Gram positive bacterium causes a highly dreadful disease and its use as a bioweapon has highlighted the urgent need to understand the pathogenesis and to design effective strategies to combat it. Thus, to develop a highly efficient anthrax vaccine it is essential to understand the protective efficacy and the basis of possible attenuation by characterization of the unstudied B. anthracis strains. Hence, in the current study, an attempt was made to evaluate the characteristics of unstudied IVRI strain by conventional means following comparison with Sterne strain. Simultaneously, antibiogram was also studied, which showed almost similar pattern for both the strains except co-trimoxazole resistance by IVRI strain. In addition, growth pattern studies were also executed in which faster growth rate and short generation time was shown by the IVRI strain.

Anthrax is an often fatal, bacterial infection affecting various animal species which also has serious impact on human health. Major virulence factors produced by different strains of Bacillus anthracis include polyglutamic acid capsule and three-component toxin. Nevertheless, involvement of other pathological features cannot be overruled. Therefore, the present study was planned to determine, production of extracellular DNAse by B. anthracis for the first time and was compared with other principal bacterial species that have already been reported to produce the extracellular DNAse. Two distinct DNAse activity assays namely plate and tube assays were used to demonstrate DNAse activity. The B. anthracis strains used in the study showed DNAse production in both the assays, however further intensive research is required to understand its role in the pathogenesis of anthrax and its possible mechanism of action.

Anthrax is a fatal septicaemic disease affecting a wide range of species, including humans. The disadvantages of live spore veterinary vaccine justify the extensive interest in development of improved safer vaccines against anthrax. In present study, ethidium bromide, the DNA-intercalating agent was used as a mutagenic agent to create various undefined mutations. The mutants so developed were evaluated for various virulence indicators like hydrophobicity, which ranged from 10.53 - 30%, adhesibility, which ranged from 13.11 -55.37% and antibiotic sensitivity test where all the mutants showed somewhat similar pattern except strain M2, M3 and M7, which were resistant to co-trimoxazole. Remarkable differences observed among the developed mutants of Bacillus anthracis clearly indicates strain variability, which might have been occurred because of mutational events.

An attempt was made in the present study to create a dominant phenotype of protective antigen (PA), which can block the lethal activities of native anthrax toxin by a rapid, cheap and efficient method named PCR based site-directed mutagenesis. The mutation was carried out in two step PCR reaction at positions 1279-1280 so that phenyl alanine at 427 position was changed to aspartic acid. For this, the first PCR was carried out with mutation primer pairs viz., PA-F & mut PA-R and mut PA-F & PA-R, which amplified the required large fragment and small fragment from pX-O1 plasmid of B. anthracis strain 34F2. Then a second PCR was run in which products of the first PCR were employed as template and PA-F & PA-R as primers, so that the full length of pag containing desired mutation was amplified. Subsequently, the mutated pag was cloned into pJET. The positive clones were confirmed by colony PCR, plasmid PCR and sequencing. Nucleotide sequencing of recombinant plasmid revealed that the nucleotides at 1279-1280 positions were mutated without any other alterations in nucleotide sequence of pag gene. Now, further studies are required to express pag and confirm the therapeutic potential of this mutated PA against anthrax.

A protective antigen (PA) based coagglutination test was optimized in the present study for the specific and sensitive identification of bacteria causing anthrax in a cost effective and less risky manner. The test showed 100% specificity and sensitivity up to 9 × 10(3) formalinized vegetative cells or 11 ng of PA. The optimized test also detected anthrax toxin directly from the serum as well as blood of anthrax infected animals indicating the potential application for direct diagnosis of anthrax under field conditions.

UNLABELLED:A specific latex agglutination test (LAT) based on anti-PA (protective antigen) antibodies having detection limit of 5 × 10(4) formalin treated Bacillus anthracis cells or 110 ng of PA was optimized in this study. The optimized LAT could detect anthrax toxin in whole blood as well as in serum from the animal models of anthrax infection. The protocol is a simple and promising method for the specific detection of bacteria causing anthrax under routine laboratory, as well as in field, conditions without any special equipments or expertise. SIGNIFICANCE AND IMPACT OF THE STUDY:The article presents the first report of a latex agglutination test for the specific identification of the cultures of bacteria causing anthrax. As the test is targeting one of anthrax toxic protein (PA), this can also be used to determine virulence of suspected organisms. At the same time, the same LAT can be used directly on whole blood or sera samples under field conditions for the specific diagnosis of anthrax.