Methods Development and Application: Pathogenic Leptospira in Surface Waters

1. Methods Development and Application: Pathogenic Leptospira in Surface Waters Mark Walker University of Nevada Reno, Nevada

2. Overview • Leptospira: difficulties with assessing environmental occurrence • Antibodies as a means of specific detection • Sampling natural waters for microbial pathogens • Analysis: examples of application of immunochemistry • Experimental plan and preliminary results

3. Background • Leptospira: pathogenic and non-pathogenic spirochetes • Pathogenic forms have distinctive hook-like shape (like a question mark) • Very difficult to detect microscopically • Different serogroups, with one or more serovars, which differ from one another partly in seriousness of infection

4. Exposures • Direct contact with infected animals • Indirect contact with animal urine, contaminated water, soil • Occupational • Farmers, veterinarians, meat processors • Sewer workers, soldiers, taro and banana farmers • Recreational - water Clark, T., CDC

5. Animal hosts • Maintenance • Maintain infection in nature(non lethal – a carrier) • Chronic infection of kidneys • Excretion in urine • Accidental • Infected by maintenance hosts Clark, T., CDC

6. Maintenance hosts and associated serogroups and serovars Clark, T., CDC

7. Leptospirosis in the Pacific Clark, T., CDC

8. Katz, et al, 2002

9. Maintenance hosts and associated serogroups and serovars 1 4 7 Australis Bataviae Sejroe Unknown 2 4 6 3 Clark, T., CDC Katz, et al, 2002

10. Katz, et al, 2002

11. Difficulties with assessing environmental occurrence • Current sampling techniques rely on small volumes and culturing to determine presence of spirochetes • Culturing requires lengthy, fastidious conditions (6-8 wks) • Serovar determination follows successful culturing

12. Using antibodies for specific detection • Antibody: an infection-fighting protein molecule in blood or secretory fluids that tags, neutralizes, and helps destroy pathogenic microorganisms (eg, bacteria, viruses) or toxins. • Antibodies are made and secreted in response to stimulation by antigens. • Each antibody bindsto the specific antigen that stimulated its production.

13. Sampling natural waters for microbial pathogens • Principles: for pathogens that occur in small numbers, need large sample volumes • Bacteria are as small as clay particles • Large sample volumes pass through filters to retain particles (some of which may be target pathogens) • Filters clog if too many particles are present in water • Particles must be removed from the filter • After removal, particles must be examined to determine if pathogens are present

14. Analysis: application of antibodies • Antibodies are applied to concentrated sediments and particles • Antibodies seek specific sites to bind with • Secondary antibodies are designed to bind against proteins produced by host animal that produced primary antibodies • Commercially available, with a fluorescent label

15. Example: Cryptosporidium in water • Single stage antibody reaction (fluorescent labels on antibodies to Cryptosporidium) • Top: light microscopy • Bottom: same image, with fluorescent light source and appropriate filters H.D.A. Lindquist, U.S. EPA

16. Quantitative detection: sensitivity and specificity (pomona) Natural water sampling: feasibility and recovery Combination and application • Natural water sampling: feasibility and recovery Evaluate recovery efficiency in bench-scale studies with filtration Beginning with pomona serovar-specific antibody Evaluate recovery efficiency in bench scale studies with pomona seeded in natural waters Quantitative detection: sensitivity and specificity Develop protocol to apply antibody with secondary anti-rabbit conjugate to solution seeded with pomona Beginning with pomona serovar-specific antibody Expand scope to include other pathogenic serovars Evaluate specificity with application to solutions seeded with mixed serovars Test applications with natural water samples (seeded and unseeded) Evaluate use of flow cytometry for detection Sequence of Experimental Activities Optional Step Critical Point Evaluate sensitivity and specificity of two stage antibody detection of pathogenic leptospires in natural waters Experimental plan and preliminary results

17. Results to date: • Working with pomona antibody provided by R. Lefebvre, UC-Davis, • Successful application • Identified optimal working dilution of primary and secondary antibodies • Spirochetes appear as apple-green under fluorescent light under laboratory conditions

18. 10 m

19. Next Steps: • Secure funding from EPA through RARE program • Establish lab and working relationships at University of Hawaii • Identify potential sources of primary antibodies for other serovars • Conduct laboratory trials with detection and isolation • Limited field application, if successful

20. Many thanks to… • Mr. Jimmy Torio, Anahola Homesteaders’ Council • USEPA Region IX RARE program • Dr. Rance Lefebvre, UC-Davis • USDA CSREES Regional Water Quality Initiative