טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentRaz Nili
SubjectDevelopment of Rapid Methods for Monitoring Vibrio cholerae
in Endemic Areas
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Professor Yechezkel Kashi
Full Thesis textFull thesis text - English Version


Abstract

 Vibrio cholerae is the etiological agent of cholera. Its natural reservoir is the aquatic environment. To date, over two hundred different serogroups are known, of which only O1 and O139 serogroups can cause widespread pandemics. Recent findings indicated chironomids (Diptera: Chironomidae) as a possible natural reservoir of these bacteria in the aquatic environment.

Here we study the role of chironomids as a natural reservoir and carrier of V. cholerae in endemic areas. Since many pathogenic bacteria are present in the natural environment only at low cell densities, it is essential to develop efficient methods for their identification. Thus, a novel protocol for rapid extraction of high quality DNA was developed, as well, low levels of the toxigenic bacteria in drinking water were detected as more than 10 liters of water could be rapidly filtered through a ROAK-packed column (in collaboration with Dr. Mor's Group), both yielded relatively high detection limits of pathogenic                  V. cholerae O1 (10 CFU/ml) using real-time PCR.

During a survey conducted in India (June 2006), 282 V. cholerae non-O1/non-O139 isolates were isolated from chironomid egg masses, larvae and exuviae from 14 out of 23 monitored different ponds. None of the isolates carried the cholera toxin (ctxA), yet, a NAG (virulence gene, found in various environmental strains) positive isolate from exuviae was isolated during cholera outbreak, demonstrating that exuviae are potential and useful tool (which was not used yet) for monitoring V. cholerae in endemic areas. Variation among 102 representative isolates was tested at Simple Sequence Repeats (SSR) loci,          a source of high genomic polymorphism. Typing by SSR-based method revealed 62 different strains (60%) out of the 102, reflecting the high variation of V. cholerae strains distributed in the environment. Several isolates that were isolated during cholera outbreak, were clustered together with non- O1/O139 environmental pathogenic cluster consists of strains that were proved before to be responsible for localized cholera outbreaks. Finding two identical isolates, both from chironomid exuviae in two different 2 km distant ponds during cholera outbreak, demonstrates the existing potential for epidemic spread by chironomids; transferring the bacteria from one water body to another, thus disseminate cholera in the environment. This environmental monitoring showed that chironomids are able to carry wide diversity of V. cholerae strains, including pathogenic strains. Tracing V. cholerae on chironomids in endemic areas during cholera outbreak may improve monitoring and may assist in prediction of cholera epidemics.