Designing of Western Blot Technique for Glanders Diagnosing in Iran.

Burkholderia mallei is the etiologic agent of glanders. It is difficult to diagnose this zoonotic disease in its early stages. Some methods such as the complement fixation test (CFT) cause some problems for veterinary authorities and financial losses to animal owners due to false-positive results. The mallein test requires appropriate laboratory equipment and skilled personnel. To quickly and accurately diagnose the disease, especially in areas where animals cannot be kept, new methods (such as the Western blot test [WBT]) should be used to identify the disease. This study designed and optimized the Western blot (immunoblot) test using sera from 84 glanderous equids, and the sensitivity and specificity of ELISA and CFT were compared with the WBT. ELISA tests are based on B. mallei antigens whereas a purified lipopolysaccharide-containing B. mallei antigen is used in the WBT. The sensitivity and specificity of the tests were estimated using the cut-off values recommended by the test developers. The WBT and ELISA were significantly more specific than the CFT. The ELISA based on B. mallei antigens was significantly less sensitive than the CFT. Given their comparable sensitivities and specificities, the CFT (95.7%, 98.5%), the WBT (95%, 100%) and the ELISA (85%, 100%) should be further developed. The CFT is still the prescribed technique for serological investigation of equids for trade purposes to certify individual animals without glanders. Therefore, more efforts should be made to further improve and optimize the WBT and ELISA tests.

Evaluate the efficiency of Antigen 60 (A60) protein from BCG strain of Mycobacterium bovis as a diagnostic antigen.

OBJECTIVE/BACKGROUND: Tuberculosis (TB) is one of the most common infectious diseases in Iran and around the world. Diagnosis of this disease in many cases is difficult and often requires the use of paraclinical methods. Current diagnostic methods are either too slow or lack enough sensitivity or specificity. Several mycobacterial antigens are involved in the complex interaction with the immune system of the host. They can be helpful for mycobacteria diagnosis. Antigen 60 (A60) is a thermostable antigen found in the cytosol of Mycobacterium bovis and Mycobacterium tuberculosis. This antigen is used in ELISA systems design for diagnosis of tuberculosis. The aim of this study is purification of A60 from bacterial cytoplasm and to evaluate the efficiency of this antigen and compare it with the production human tuberculin and standard human tuberculin. METHODS: Using gel filtration chromatography with a sepharose 4B column, A60 was purified from other bacterial components. A60 was recognized by agar gel immunodiffusion with anti-BCG and anti-A60 antiserum, where it formed an immunoprecipitation line with anti-BCG antiserum and anti-A60 antiserum. Molecular weight components of the A60 were obtained using electrophoresis. RESULTS: Seven fractions were obtained by chromatography. In analyzing with dot blotting, both the cytoplasm and cell wall of BCG had A60. This test showed that the first fraction creates maximum color intensity and as a result, the highest amount of A60 was in fraction one. In agar gel immunodiffusion, the cytoplasm sample and all fractions obtained from chromatography showed a positive reaction with anti-A60 antiserum, and fraction one had the most among sediment of the other fractions. Molecular weight components of the A60 were identified to be approximately 35kDa, 38kDa, 40kDa, and 65kDa. CONCLUSION: Results of reactions of the injected A60 and standard human tuberculin shows the effectiveness of this antigen in comparison with standard human tuberculin. Detection of antibody in the serum of patients is a rapid and repeatable method. A60 with 89% sensitivity and 94% specificity could be an appropriate matter for the diagnosis of tuberculosis. Because this method can be performed without radioactive materials or advanced and expensive equipment, it will provide results quickly.