Pcr-hybridization Based Detection of Pathogenic Leptospira in Environmental Water

Leptospirosis, a major health problem worldwide, is known to be endemic in the northeastern part of Thailand with the risk of infection by exposure to pathogenic Leptospira in contaminated aquatic environment. A method based on PCR-hybridization detection of pathogenic Leptospira in water was established. The method included filtration of water sample through membrane filters of two pore sizes, DNA extraction from filters using a guanidine thiocyanate extraction method, a duplex-PCR assay with two primer pairs, and hybridization with a synthetic LipL32 DNA probe. The duplex-PCR allowed detection of two products of 279 bp for LipL32 gene and 430 bp for 16S rRNA gene. In water samples artificially seeded with serovar bratislava, at least 103 cells could be analyzed by PCR-agarose gel electrophoresis and 1-10 cells by PCR-Southern blot hybridization. The protocol was applied to the detection of pathogenic Leptospira in environmental waters collected from endemic areas in the northeast region of Thailand. Of 100 water samples analyzed, 23 samples were positive for pathogenic Leptospira with PCR performed with Southern blot hybridization only, but none was detected by PCR-agarose gel-electrophoresis. However, PCR performed with the chemiluminescent LipL32 probe using the Fluorescein ULS® labeling facilitated the detection of low numbers of pathogenic Leptospira in water. This method should prove useful for monitoring of pathogenic Leptospira pollution in environmental waters, and has the potential to become a valuable tool to the surveillance of leptospirosis in endemic areas, thus leading to enhanced public health protection. SOUTHEAST ASIAN J TROP MED PUBLIC HEALTH 730 Vol 37 No. 4 July 2006 ming, fishing, canoeing and rafting (Wilkins et al, 1988). Occupation is a significant risk factor for humans (Waitkins, 1986) and accounts for most infections in rice field workers, fish farmers, soldiers, veterinarians and sewer workers, etc (Levett, 2001). Water and soil contaminated with the urine of infected animals are the sources of pathogenic Leptospira, and the role of water as an important vehicle of transmission for pathogenic leptospires is well known (Henry and Johnson, 1978). In Thailand, leptospirosis is found to be sporadic in many regions of the country. In 1996, the outbreak of a re-emerging leptospirosis occurred and expanded to many provinces in the northeast region and the outbreak corresponded with the rainy season and most infections occurred in agricultural workers, primarily rice producers (Tangkanakul et al, 2005). Nowadays, the outbreak of disease has spread extensively, with higher incidence in northeastern region than in the other regions of Thailand. Accordingly, leptospirosis is now concerned to be the significant health problem in Thailand. Therefore, new methods for detection of Leptospira in environmental samples are essential for epidemiological studies that can provide the surveillance program for protection against this disease. Conventional laboratory methods for detection of pathogenic leptospires in aquatic environments include culture isolation of the organism (Henry and Johnson, 1978) and animal inoculation (Baker and Baker, 1970). However, analysis by culture method or direct inoculation into animal to recover pathogenic Leptospira from environmental samples is laborious and time-consuming. Furthermore, culture of pathogenic leptospires may be hampered by the predominance in a water environment of saprophytic leptospires which are morphologically similar and grow faster than pathogenic ones (Murgia et al, 1997), and the disadvantages of animal inoculation include limited susceptibility of the test animal to the wide range of Leptospira strains and requirement for confirmation of the results by culture and serological characterization of the isolated strains (Alexander et al, 1975). Recently, a number of PCR-based methods for leptospirosis detection have been published (Gravekamp et al, 1993) and successfully used in the clinics (Bal et al, 1994; Merien et al, 1992, 1995). Few reports, however, deal with molecular methods for specific detection of pathogenic leptospires in analyses of natural water. Recently we have developed a duplex PCR-based method using two sets of newly design primers based on 16S rRNA and LipL32 genes, which amplifies in the same reaction two different DNA fragments, the 279bp LipL32 and the 430-bp 16S rRNA (Tansuphasiri et al, 2006). This PCR method and amplicon detection by Southern blot hybridization (SBH) was utilized successfully for differentiation of pathogenic from non-pathogenic Leptospira. In this study, we have applied this PCRbased method to the detection of pathogenic Leptospira in environmental water samples collected from endemic areas in the northeast region of Thailand. The purposes of the study were (i) to compare two methods for DNA isolation, (ii) to determine the sensitivity for detection of pathogenic Leptospira in seeded water samples, and (iii) to determine the occurrence of pathogenic Leptospira in natural water samples collected from two endemic areas of leptospirosis. MATERIALS AND METHODS Water samples and collection Water samples used for seeding experiment were obtained from 3 sites of the Chao Phya River and Samsen Canal in Bangkok area. One liter of sample from each site was collected in sterile glass bottle and transported in an icebox. For field study, a total of 100 samples from different water sources (canals, PCR-HYBRIDIZATION BASED DETECTION OF PATHOGENIC LEPTOSPIRA IN ENVIRONMENTAL WATER Vol 37 No. 4 July 2006 731 creeks, rivers, fen, marsh, ponds, rice-fields and sewages) were obtained from two endemic areas, Khon Kaen and Nakhon Ratchasima Province during November 2005 to January 2006. Samples were collected by dipping a 300-ml sterile plastic bottle directly into the water. Other parameters measured included temperature and pH using a handheld probe (IQ Scientific Instruments, USA). The samples were transported in an icebox to the laboratory and processed within 24 hours.