(UNESCO / Japan Young Researchers' fellowships programme)

Detection of Entamoeba histolytica and Flavivirus Vectors in Irrigation and Recreational Waters

Summary of research carried out: 
Detection of Entamoeba histolytica and Flavivirus Vectors in Irrigation and Recreational Waters

Contamination of drinking and recreational water by pathogenic microorganisms associated with faecal waste has a significant impact on public health. Rivers are exposed to a wide spectrum of potential contaminants as a result of many recreational uses and close proximity to the agricultural industry.

These increase the risk of accidental release and raise concern about agents of disease entering the rivers, making them an excellent testing ground for pathogens and vectors that transmit viral diseases. We are developing a method to determine contaminants in such recreational and drinking water. To begin this study, we determined the specific biomarkers in a microarray design through a proteomic analysis of water samples collected in Mexico City and the surrounding areas.

To investigate the most frequent contaminants, these samples were analysed in 3200 QTrap proteomic equipment with an HPLC Agilent 1200. The results showed that the water contained Aedes aegypti, Culex pipiens and Entamoeba histolytica peptides. Aedes aegypti and Culex pipiens are very significant in the transmission of dengue and West Nile viruses, respectively. Interestingly, we also detected the following major pathogens – E. coli, Proteus mirabilis, Salmonella, Shigella, Vibrios including Vibrio cholerae, Pseudomonadales and Plasmodium. On the basis of these results, the DNA of six samples of the collected water was obtained and tested using fluorochrome-labelled specific primers. Ae. aegypti, C. pipiens and E. histolytica DNA from laboratory cultures were used as positive controls in our tests. In addition, RT-PCR fragments of NS3 and NS5 c-DNA from DEN 2 were also obtained to test the specificity of two labelled primers to identify dengue. The DNA in DMSO 50% was spotted in amino-silane slides to bind it covalently using the Robot GeneTACTM G3. All DNA spots were detected with SYBR green. All specific primers used to detect the DNA from the different samples (C. pipiens, E. histolytica and the genes for NS3 and NS5 of the dengue virus) were designed previously. The specific primer for E. histolytica and C. pipians were labelled with P1-Texas red and R22-Cy5 respectively. DNA that was covalently bound to the slides was hybridized with the labelled primers. The primers recognized the spots that contained the DNA for E. histolytica or C. pipiens in the microarray. The primer labelled with F-HEX specific for NS5 had high specificity for the NS5 RT-PCR product in the microarray compared to the NS3 primer (DSP2) labelled with 6-FAM. The primer (Ae. aegypti specific) ITS1A labelled with 6 FAM did not recognize the mosquito DNA, although the DNA binding was detected with SYBR green.


18 September 2008