Vijai Pal

Use of a recombinant burkholderia intracellular motility a protein for immunodiagnosis of glanders.

ABSTRACT Glanders, caused by the Gram-negative, nonmotile bacterium Burkholderia mallei, is a contagious and highly fatal disease of equines. During the last decade, the number of glanders outbreaks has increased steadily. The disease also has high zoonotic significance and B. mallei is listed biological warfare agent. The complement fixation test (CFT) is a routinely used and internationally recognized test to screen equine sera for the glanders. However, discrepant results have been observed using the CFT. The low sensitivity and specificity of the CFT and enzyme-linked immunosorbent assay (ELISA) have been linked to the use of crude test antigens. We expressed a novel recombinant Burkholderia intracellular motility A (rBimA) protein in Escherichia coli for the diagnosis of equine glanders. Purified rBimA was used in an indirect ELISA format. All of the 21 true-positive serum samples used in the study tested positive, whereas only 17 of the 1,524 potentially negative sera tested positive by indirect ELISA, thus exhibiting 100% sensitivity and 98.88% specificity. Also, rBimA protein did not react with melioidosis patient and normal healthy human serum samples, showing its high specificity. The developed assay can be used as a simple and rapid tool for diagnosis of glanders in equine serum samples. An Indian patent (1328/DEL/2010) has been filed for the reagent.

Evaluation of Recombinant Proteins of Burkholderia mallei for Serodiagnosis of Glanders

ABSTRACT Glanders is a contagious disease caused by the Gram-negative bacillus Burkholderia mallei. The number of equine glanders outbreaks has increased steadily during the last decade. The disease must be reported to the Office International des Epizooties, Paris, France. Glanders serodiagnosis is hampered by the considerable number of false positives and negatives of the internationally prescribed tests. The major problem leading to the low sensitivity and specificity of the complement fixation test (CFT) and enzyme-linked immunosorbent assay (ELISA) has been linked to the test antigens currently used, i.e., crude preparations of whole cells. False-positive results obtained from other diagnostic tests utilizing crude antigens lead to financial losses to animal owners, and false-negative results can turn a risk into a possible threat. In this study, we report on the identification of diagnostic targets using bioinformatics tools for serodiagnosis of glanders. The identified gene sequences were cloned and expressed as recombinant proteins. The purified recombinant proteins of B. mallei were used in an indirect ELISA format for serodiagnosis of glanders. Two recombinant proteins, 0375H and 0375TH, exhibited 100% sensitivity and specificity for glanders diagnosis. The proteins also did not cross-react with sera from patients with the closely related disease melioidosis. The results of this investigation highlight the potential of recombinant 0375H and 0375TH proteins in specific and sensitive diagnosis of glanders.

Evaluation of the Recombinant 10-Kilodalton Immunodominant Region of the BP26 Protein of Brucella abortus for Specific Diagnosis of Bovine Brucellosis

ABSTRACT Brucellosis is a disease with worldwide distribution affecting animals and human beings. Brucella abortus is the causative agent of bovine brucellosis. The cross-reactions of currently available diagnostic procedures for B. abortus infection result in false-positive reactions, which make the procedures unreliable. These tests are also unable to differentiate Brucella-infected and -vaccinated animals. The present work is focused on the use of a nonlipopolysaccharide (LPS) diagnostic antigen, a recombinant 10-kDa (r10-kDa) protein of B. abortus, for specific diagnosis of brucellosis. The purified recombinant protein was used as a diagnostic antigen in plate enzyme-linked immunosorbent assay (p-ELISA) format to screen 408 bovine serum samples (70 presumptively negative, 308 random, and 30 vaccinated), and the results were compared with those of the Rose Bengal plate agglutination test (RBPT) and the standard tube agglutination test (STAT). Statistical analysis in presumptive negative samples revealed 100 and 98.41% specificity of p-ELISA with RBPT and STAT, and an agreement of 91.43% with the tests using Cohens kappa statistics. In random samples, the agreement of p-ELISA was 77.92% and 80.52% with RBPT and STAT, respectively. p-ELISA investigation of vaccinated samples reported no false-positive results, whereas RBPT and STAT reported 30% and 96.6% false-positive results, respectively. The data suggest that p-ELISA with r10-kDa protein may be a useful method for diagnosis of bovine brucellosis. Furthermore, p-ELISA may also be used as a tool for differentiating Brucella-vaccinated and naturally infected animals.

Improvement of recombinant-truncated Burkholderia motility protein A (BimA)-based indirect ELISA for equine glanders

ABSTRACT Glanders is a contagious and highly fatal disease of equines with zoonotic potential. It is caused by a Gram-negative, nonmotile bacterium Burkholderia mallei. Complement fixation test (CFT) is one of the most commonly used tests for diagnosis of glanders; however, it has some limitations. A recombinant-truncated Burkholderia intracellular motility A (BimA) protein-based indirect enzyme-linked immunosorbent assay (iELISA) was previously reported by us for glanders diagnosis, which has been re-optimized in this study using a panel of glanders positive (n = 75) and glanders negative (n = 227) serum samples. The improved iELISA exhibited 96% sensitivity and 90.75% specificity. The assay had 98.56% negative predictive value. In the improved iELISA, background for negative samples was reduced and a rational assay cut-off based on ROC curves was introduced. Intra laboratory repeatability of the iELISA was tested by 3 different operators with 100% correlation. The BimA-coated ELISA plates could be used without significant decrease in diagnostic efficacy even after their storage at room temperature or 37°C for 90 days. Overall, the improved iELISA is a sensitive, specific, reproducible, and easy-to-use assay that has potential in serodiagnosis of glanders, more suitably to demonstrate freedom from B. mallei infection in a population.

Development of a TaqMan Real-Time Polymerase Chain Reaction Assay for Detection of Burkholderia mallei

Abstract Burkholderia mallei is the causative agent of glanders, a highly contagious and reemerging zoonotic disease. B. mallei is closely related to Burkholderia pseudomallei, the etiologic agent of melioidosis. The genetic proximity between B. mallei and B. pseudomallei had always been a problem in development of specific molecular detection tests for B. mallei. In the present investigation, we describe a rapid, sensitive, and specific TaqMan real‐time polymerase chain reaction (PCR) assay for detection of B. mallei. The TaqMan real‐time PCR assay could detect as little as 1 picogram of B. mallei genomic DNA and 100 copies of the target DNA cloned in pGEM‐T Easy vector. In spiked human blood, the assay could detect as few as 5.5 × 103 CFU/mL of B. mallei. The assay did not cross‐react with other bacterial strains used in the study and was found specific for B. mallei. TaqMan real‐time PCR assay can dramatically decrease the turnaround time for diagnostic results, and hence, appropriate action can be initiated at early stage to control and contain the B. mallei infection. HighlightsTaqMan real‐time polymerase chain reaction assay for detection of Burkholderia mallei is proposed.The assay is rapid and could detect 1 pg genomic DNA of B. mallei.The assay is specific for B. mallei and did not react with other bacterial strains.The assay could detect as few as 5.5 × 103 CFU of B. mallei per mL of spiked blood.