Rekha Khushiramani

Evaluation of RAPD-PCR and protein profile analysis to differentiate Vibrio harveyi strains prevalent along the southwest coast of India

Sixty five isolates of Vibrio harveyi were subjected to random amplified polymorphic DNA (RAPD)-PCR analysis and protein profiling to investigate the genetic variability among V. harveyi prevalent along the coast and also assess the discriminating ability of these two molecular methods. A total of 10 RAPD primers were assayed for their specificity in detecting V. harveyi, of which only two primers: PM3 and CRA25 were highly reproducible and found suitable for use in RAPD-PCR. The genetic diversity among V. harveyi isolates assessed by RAPD-PCR using PM3 primer yielded 35 different RAPD patterns which clustered the isolates into 15 groups at 72% similarity level. Similarly, RAPD-PCR with CRA25 clustered the 38 patterns into 10 groups at 74% similarity. The discriminatory index (D) value calculated for RAPD fingerprints generated with PM3 and CRA25 were 0.90 and 0.85, respectively. On the other hand, molecular typing of V. harveyi using whole cell proteins generated profiles that showed no major difference indicating the technique to be not useful in typing strains of this bacterium. However, a few of the isolates showed the presence of unique band of 28 kDa that needs to be further investigated to understand the role of the protein in disease process if any.

Recombinant ferritin protein protects Penaeus monodon infected by pathogenic Vibrio harveyi.

Hemocytes of shrimp perform an essential role in defense against microbial pathogens, involving both cellular and humoral factors. The gene coding for ferritin in black tiger shrimp Penaeus monodon was cloned, sequenced and expressed using pQE-30-UA vector and SG13009 Escherichia coli host cells. The deduced amino acid sequence of P. monodon ferritin showed 32 to 95% similarity with ferritin proteins of other organisms. The recombinant protein was purified by nickel-nitrilotriacetic acid affinity chromatography. A single thick band of recombinant protein of approximately 21 kDa was observed in 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis. Following mild acid treatment, 2 bands of ca. 14 and 7 kDa were produced; aspartine and proline acid cleavage sites were found at amino acid residues 123-124. The purified recombinant ferritin helped in reducing the mortality in shrimp infected with Vibrio harveyi . However, no direct antimicrobial activity against pathogenic V. harveyi was observed.

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.