Detection of pathogenic bacteria in shellfish using multiplex PCR followed by CovaLink NH microwell plate sandwich hybridization

Outbreak of diseases associated with consumption of raw shellfish especially oysters is a major concern to the seafood industry and public health agencies. A multiplex PCR amplification of targeted gene segments followed by DNA-DNA sandwich hybridization was optimized to detect the etiologic agents. First, a multiplex PCR amplification of hns, spvB, vvh, ctx and tl was developed enabling simultaneous detection of total Salmonella enterica serotype Typhimurium, Vibrio vulnificus, Vibrio cholerae and Vibrio parahaemolyticus from both pure cultures and seeded oysters. Amplicons were then subjected to a colorimetric CovaLink NH microwell plate sandwich hybridization using phosphorylated and biotinlylated oligonucleotide probes, the nucleotide sequences of which were located internal to the amplified DNA. The results from the hybridization with the multiplexed PCR amplified DNA exhibited a high signal/noise ratio ranging between 14.1 and 43.2 measured at 405 nm wavelength. The sensitivity of detection for each pathogen was 10(2) cells/g of oyster tissue homogenate. The results from this study showed that the combination of the multiplex PCR with a colorimetric microwell plate sandwich hybridization assay permits a specific, sensitive, and reproducible system for the detection of the microbial pathogens in shellfish, thereby improving the microbiological safety of shellfish to consumers.     

PCR detection of a newly emerged pandemic Vibrio parahaemolyticus O3:K6 pathogen in pure cultures and seeded waters from the Gulf of Mexico

This study describes the optimization of PCR parameters and testing of a wide number of microbial species to establish a highly specific and sensitive PCR-based method of detection of a newly emerged pandemic Vibrio parahaemolyticus O3:K6 strain in pure cultures and seeded waters from the Gulf of Mexico (gulf water). The selected open reading frame 8 (ORF8) DNA-specific oligonucleotide primers tested were found to specifically amplify all 35 pathogenic V. parahaemolyticus O3:K6 pandemic isolates, whereas these primers were not found to detectably amplify two strains of V. parahaemolyticus O3:K6 that were isolated prior to the 1996 outbreaks, 122 non-O3:K6 strains of V. parahaemolyticus, 198 non-V. parahaemolyticus spp., or 16 non-Vibrio bacterial spp. The minimum level of detection by the PCR method was 1 pg of purified genomic DNA or 10(2) ORF8-positive V. parahaemolyticus O3:K6 cells in 100 ml of water. The effectiveness of this method for the detection of ORF8-positive isolates in environmental samples was tested in gulf water seeded with 10-fold serial dilutions of this pathogen. A detection level of 10(3) cells per 100 ml of gulf water was achieved. Also, the applicability of this methodology was tested by the detection of this pathogen in gulf water incubated at various temperatures for 28 days. This PCR approach can potentially be used to monitor with high specificity and well within the required range of sensitivity the occurrence and distribution of this newly emerged pathogenic V. parahaemolyticus O3:K6 strain in coastal, marine, and ship ballast waters. Early detection of V. parahaemolyticus O3:K6 will help increase seafood safety and decrease the risk of infectious outbreaks caused by this pathogen.     

Control of malaria and filariasis vectors in South India

Community participation is increasingly seen as a major component of successful control programmes against parasitic diseases. One of the strongest exponents of this has been the Vector Control Research Centre (VCRC) in Pondicherry, South India (Fig. 1), who have successfully motivated and involved village communities in vector control activities with considerable success against malaria and filariasis vectors. Their largest single programme, the Filariasis Control Demonstration Project in Pondicherry Town, has now been handed to the Pondicherry State Government, while the VCRC's work on malaria and filariasis control is now expanding throughout other parts of India. As this article shows, much has been learnt from these projects, not just about control techniques but also about education, administration and decision-making.     

Ventricular tachycardia as the presenting feature in two patients with cardiac lipoma and cardiac fibroma

We hereby present two patients with benign cardiac tumours presenting as ventricular tachycardia (VT). Most such tumours have a favorable prognosis, unless complicated by arrhythmias. Intracavitary tumours are easily diagnosed by echocardiography. Intramural tumours as in our patients may be missed at times by echocardiography. Multimodality imaging helped confirm the diagnosis and etiology, since biopsy was not safe. Surgical removal was not feasible due to extensive infiltration. The patients are so far doing well on medical therapy.     

Diversity of free-living ‘naked’ amoeboid organisms

Amoeboid organisms are phylogenetically diverse, some being more closely related to plants or metazoans than to each other. Amoeboid organisms are ecologically successful, having been isolated on all continents, including Antarctica, as well as being the main predators controlling bacterial populations in soil. The classification of these organisms has historically relied upon morphological characteristics. The application of electron microscopy, comparison of enzymic profiles after electrophoretic separation, and analysis of nucleic acid fractions have provided reliable bases for classifying amoeboid organisms. The extent of diversity of these organisms has been recognized, as methods to detect, culture, characterize and identify them has increased. It is reasonable to anticipate that the current 40 000 species of protists will increase substantially as amoeboid organisms are cultivated from poorly accessible niches and from extreme environs.