To understand epidemiology of Bacillus anthracis in Iran, the morphological, biochemical, and virulence specifications of 32 B. anthracis isolates, collected from human, sheep, cattle, goat, and environmental specimens obtained from throughout Iran were examined by conventional and molecular approaches. B. anthracis isolates were characterized in multiple ways: (1) capsule formation both on bicarbonate agar and in defibrinated horse blood, (2) motility of vegetative forms, (3) hemolysis on 5% sheep blood agar, (4) penicillin G susceptibility, (5) lecithinase production on egg yolk agar, (6) gelatin hydrolysis, (7) ability to develop "string of pearls" on tryptose agar, and (8) capability to develop mucoid colonies in presence of CO(2) were assessed. In addition, biochemical properties such as indole, methyl red, catalase, citrate utilization, and finally nitrate reduction tests were used. All the tested isolates produced identical morphological and biochemical patterns with those of the vaccine strain B. anthracis 34F2 Sterne. In order to assess potential virulence of isolates at genomic level, PCR protocols assaying for the pXO1 and pXO2 loci were employed. The intriguing high level of phenotypic similarity between Iranian isolates of B. anthracis and the 34F2 Sterne strain deserves further studies at genomic level.
Immune assays were taken into consideration to diagnose and quantify metabolites such as antigen and antibody. Enzyme-Linked Immunosorbent Assays (ELISAs), which are used to detect antigens and antibodies, generated several periods of infectious and vaccination conditions. There is an extensive range of commercial infectious disease ELISA kits useful for the detection of human and animal IgG, IgA, IgM antibodies and microorganism antigens. Anthrax is one of the serious infectious diseases caused by rod-shaped, gram-positive bacteria known as Bacillus anthracis. Subunit or attenuated vaccines applied against anthrax disease increase the antibody against the Protective Antigen (PA) which has a critical role as a toxin of B. anthracis. Herein, the ELISA was developed using PA domain 4 and anthrax Lethal Factor to detect IgG antibody in serum. Besides, the level of anti-LF antibodies were determined as a complementary test to measure variance in antibody titers associated with vaccination or infection that leads to detection of anthrax in livestock. The results show that we developed high-quality ELISA kit that can be used to test immunogenicity of vaccines and infections in mice. We tried to develop the Anti- PA4 ELISA kit and conduct the validation studies to evaluate the fluctuation level of the antibody in the anthrax vaccine and distinction between disease and vaccination in mice.
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