Brucellosis is a disease caused by Gram-negative, facultative, intracellular bacteria belonging to the genus Brucella. It is an emerging zoonosis, and an economically important infection of humans and livestock with a worldwide distribution. Human infection is known to occur through consumption of infected raw milk, milk products and undercooked or raw meat. Serodiagnosis of brucellosis is carried out by detection of antibodies generated against LPS or whole-cell bacterial extracts by ELISA or agglutination tests using colorimetry. The present study was designed to develop a highly sensitive and specific indirect ELISA in both a microtitre plate and dot-blot format employing the recombinant outer-membrane protein 28 (rOmp28). Cloning and expression of Brucella melitensis Omp28 protein, which is a group 3 antigen, was accomplished by PCR amplification and cloning of the gene in a pET-28a expression system, followed by Ni-NTA affinity chromatography purification of the His-tagged recombinant protein. An indirect ELISA in both a microtitre plate and dot-blot format was optimized with sera collected from three groups: cultureconfirmed cases, clinically suspected cases and healthy individuals. The rOmp28 protein reacted only with the culture-confirmed positive samples and no reaction was observed with culturenegative samples, confirming the immunoreactivity of the recombinant protein. The test in both formats had a correlation of approximately 90 % with the Rose Bengal plate agglutination test (RBPT) and a standard tube agglutination test, assays that are routinely performed for the serodiagnosis of brucellosis. The sensitivity and specificity of the assay in the plate format were 97.50 and 85.59 %, and in the dot-blot format were 82.05 and 92.43%, respectively, in comparison with RBPT. The specificity of this assay was further confirmed by testing samples that were positive for malaria and typhoid, which gave negative results. This ELISA system in microtitre plates and a dot-blot format will be useful for the rapid screening of large numbers of samples for the diagnosis of human brucellosis in endemic areas.

Melioidosis is an emerging infectious disease caused by a free living soil dwelling Gram-negative bacterium Burkholderia pseudomallei. The disease is endemic to most parts of Southeast Asia and northern Australia and the organism has been isolated from moist soil and water. In India clinical cases are recently reported from the states of Tamilnadu, Kerala, Karnataka, Maharashtra, Orissa, Assam, West Bengal, Pondicherry and Tripura. This study is aimed to confirm the prevalence of this important bacterial species in soil samples collected from coastal areas of Tamilnadu. Forty five soil samples from five different sites were collected from Parangipettai, Tamilnadu and screened for the presence of B. pseudomallei. The study confirmed 4 isolates as B. pseudomallei with the help of conventional bacteriological methods and molecular methods that include; 16S rDNA sequencing, B. pseudomallei specific PCR, fliC gene RFLP and MALDI-TOF mass spectrometry based bacterial identification. This study reveals the prevalence and distribution of B. pseudomallei in the soil environment in coastal areas of southern India and further necessitates studies from other parts of the country. It will also be helpful to understand the distribution of B. pseudomallei and to access its epidemiological importance.

bBrucellosis is an important zoonotic infectious disease of humans and livestock with worldwide distribution and is caused by bacteria of the genus Brucella. The diagnosis of brucellosis always requires laboratory confirmation by either isolation of pathogens or detection of specific antibodies. The conventional serological tests available for the diagnosis of brucellosis are less specific and show cross-reactivity with other closely related organisms. These tests also necessitate the handling of Brucella species for antigen preparation. Therefore, there is a need to develop reliable, rapid, and user-friendly systems for disease diagnosis and alternatives to vaccine approaches. Keeping in mind the importance of brucellosis as an emerging infection and the prevalence in India, we carried out the present study to compare the recombinant antigens with the native antigens (cell envelope and sonicated antigen) of Brucella for diagnosis of human brucellosis by an indirect plate enzyme-linked immunosorbent assay (ELISA). Recombinant outer membrane protein 28 (rOmp28) and rOmp31 antigens were cloned, expressed, and purified in the bacterial expression system, and the purified proteins were used as antigens. Indirect plate ELISAs were then performed and standardized for comparison of the reactivities of recombinant and native antigens against the 433 clinical samples submitted for brucellosis testing, 15 culture-positive samples, and 20 healthy donor samples. The samples were separated into four groups based on their positivity to rose bengal plate agglutination tests (RBPTs), standard tube agglutination tests (STATs), and 2-mercaptoethanol (2ME) tests. The sensitivities and specificities of all the antigens were calculated, and the rOmp28 antigen was found to be more suitable for the clinical diagnosis of brucellosis than the rOmp31 antigen and native antigens. The rOmp28-based ELISA showed a very high degree of agreement with the conventional agglutination tests and promising results for further use in clinical screening and serodiagnosis of human brucellosis. Brucellosis is one of the world's major zoonoses, caused by bacteria of the genus Brucella. It is a serious public health problem in India and in other developing countries. The transmission of Brucella infection and its prevalence in a region depend upon several factors, like food habits, methods of processing milk and milk products, social customs, husbandry practices, climatic conditions, socioeconomic status, and environment hygiene (1). The disease has been recognized as one of the most common laboratory-acquired infections; it has been reported to occur in clinical research and production laboratories. Human brucellosis is a multisystem disease that may present with a broad spectrum of clinical manifestations, and its diagnosis requires microbiological confirmations by means of isolation from blood cultures or demonstration of the presence of specific antibodies by serological tests (2). Blood cultures provide definite proof of brucellosis but may not provi...

Background & objectives:Brucellosis is endemic in the southern part of India. A combination of biochemical, serological and molecular methods is required for identification and biotyping of Brucella. The present study describes the isolation and biochemical, molecular characterization of Brucella melitensis from patients suspected for human brucellosis.Methods:The blood samples were collected from febrile patients suspected to have brucellosis. A total of 18 isolates were obtained from 102 blood samples subjected to culture. The characterization of these 18 isolates was done by growth on Brucella specific medium, biochemical reactions, CO2 requirement, H2S production, agglutination with A and M mono-specific antiserum, dye sensitivity to basic fuchsin and thionin. Further, molecular characterization of the isolates was done by amplification of B. melitensis species specific IS711 repetitive DNA fragment and 16S (rRNA) sequence analysis. PCR-restriction fragment length polymorphism (RFLP) analysis of omp2 locus and IS711 gene was also done for molecular characterization.Results:All 102 suspected samples were subjected to bacteria isolation and of these, 18 isolates could be recovered on blood culture. The biochemical, PCR and PCR-RFLP and 16s rRNA sequencing revealed that all isolates were of B. melitensis and matched exactly with reference strain B. melitensis 16M.Interpretation & conclusions:The present study showed an overall isolation rate of 17.64 per cent for B. melitensis. There is a need to establish facilities for isolation and characterization of Brucella species for effective clinical management of the disease among patients as well as surveillance and control of infection in domestic animals. Further studies are needed from different geographical areas of the country with different level of endemicity to plan and execute control strategies against human brucellosis.

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