Development of a diagnostic system for Burkholderia pseudomallei infections.

Monoclonal antibodies were generated against whole cell lysate of Burkholderia pseudomallei. Two out of 6 monoclonal antibodies were found specific and exhibited high affinity against B. pseudomallei, one of which, was utilized to develop sandwich ELISA for detection of specific B. pseudomallei antigen. Immunoassays were found to be specific as no reaction was observed with closely related Burkholderia and Pseudomonas species. Blood samples from experimentally infected mice were found positive for isolation till 4 days post infection (DPI) and ELISA till 10 DPI. One out of 40 sick animal serum samples tested in Thailand was found positive by sandwich ELISA that was earlier confirmed by isolation of B. pseudomallei. The results indicate the potentiality of the assay for its applicability in specific diagnosis of septicaemic melioidosis.

Evaluation of a digoxigenin-labelled probe for detection of Aeromonas spp.

An outer membrane protein-based Digoxigenin (DIG)-labelled DNA probe was developed for the specific detection of Aeromonas sp. from food/environmental/clinical samples. Dot blot reaction answered for all the Aeromonas isolates and was negative for Escherichia coli, Pseudomonas sp., Klebsiella sp., Vibrio parahaemolyticus, V. harveyi, V. alginolyticus, V. vulnificus. Edwardsiella tarda and Staphylococcus sp. As this protein is highly conserved in various Aeromonas species, the probe has the potential for use as a rapid and reliable diagnostic tool.

Molecular typing of Vibrio parahaemolyticus isolated from seafood harvested along the south-west coast of India.

AIMS: Evaluation of protein profiling for typing Vibrio parahaemolyticus using 71 strains isolated from different seafood and comparison with other molecular typing techniques such as random amplified polymorphic DNA analysis (RAPD) and enterobacterial repetitive intergenic consensus sequence (ERIC)-PCR. METHODS AND RESULTS: Three molecular typing methods were used for the typing of 71 V. parahaemolyticus isolates from seafood. RAPD had a discriminatory index (DI) of 0.95, while ERIC-PCR showed a DI of 0.94. Though protein profiling had less discriminatory power, use of this method can be helpful in identifying new proteins which might have a role in establishment in the host or virulence of the organism. CONCLUSIONS: The use of protein profiling in combination with other established typing methods such as RAPD and ERIC-PCR generates useful information in the case of V. parahaemolyticus associated with seafood. SIGNIFICANCE AND IMPACT OF THE STUDY: The study demonstrates the usefulness of nucleic acid and protein-based studies in understanding the relationship between various isolates from seafood.

Characterization of a newly established potato tuber moth (Phthorimaea operculella Zeller) cell line.

BACKGROUND & OBJECTIVE: Potato tuber moth (PTM), Phthorimaea operculella Zeller is a widely distributed, devastating pest of potatoes attacking the foliage and infest the tubers in both field and store causing serious economic damage. As application of PTM granulovirus (PTM-GV) has shown significant reduction in damage, attempts were made to develop a new cell line from this insect to grow PTM-GV for use as a biopesticide. METHODS: Approximately 100 mg of insect eggs were collected, surface sterilized and crushed gently in a boiling tube aseptically. The tissues were washed with physiological saline, suspended in growth medium and incubated stationary at 28 degrees C. Morphology of cells was studied after staining with Giemsa. Besides karyological and growth curve studies, PCR amplification was also done for rapid amplified polymorphic DNA pattern. RESULTS: A new cell line from the embryonic tissue of PTM was maintained in Mitsuhashi Maramorosch medium supplemented with 10 per cent foetal bovine serum. It is in the 78th passage level and designated as NIV-PTM-1095. Random amplified polymorphic DNA profile analysis indicated this as a new cell line from potato tuber moth and differed from the profiles of two other lepidopteran cell lines maintained in the laboratory. Three different cell types were observed at the 40th passage level and comprised of epithelial-like cells (77%), fibroblast-like cells (20%) and giant cells (3%). The chromosome number varied from 54-176. The cell line had a cell doubling time of approximately 42 h during the logarithmic phase of growth. The cell line did not support the multiplication of any of the baculoviruses used in the study. INTERPRETATION & CONCLUSION: Since the new cell line is found to replicate PTM-GV, it may be useful for the propagation of PTM-GV in large scale. Studies to scale up the production of the GV in the cell line and field trials may lead to its widespread use as an eco-friendly biopesticide.