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 Mammal speciesSerogroup, serovar RatsIcterohaemorrhagiae, Ballum MiceBallum Dairy cattlehardjo, pomona, grippotyphosa Pigspomona, tarrassovi, bratislava Sheephardjo, pomona Dogscanicola Clark, T., CDC

7. Leptospirosis in the Pacific Location Incidence rate per 100,000 persons/yr Hawaii128 New Caledonia90 Tahiti30 New Zealand4.4 Clark, T., CDC

8. Katz, et al, 2002

9. Maintenance hosts and associated serogroups and serovars Mammal speciesSerogroup, serovar RatsIcterohaemorrhagiae, Ballum MiceBallum Dairy cattlehardjo, pomona, grippotyphosa Pigspomona, tarrassovi, bratislava Sheephardjo, pomona Dogscanicola Clark, T., CDC Australis Bataviae Sejroe Unknown 1 2 3 4 4 6 7 Katz, et al, 2002

10. Number observed Estimated annual incidence rate (per 100,000) Island 1974 – 1998 (percent of total) Hawaii 176 (50.1%)5.85 Kauai 100 (28.5%)7.85 Oahu 67 (19.1%)0.32 Maui 5 (1.4%)0.22 Lanai1 Molokai1 Niihau1 (Unknown)2 Total353 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 binds to 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. Experimental plan and preliminary results Optional Step Critical Point Beginning with pomona serovar-specific antibody Evaluate recovery efficiency in bench-scale studies with filtration Evaluate recovery efficiency in bench scale studies with pomona seeded in natural waters Expand scope to include other pathogenic serovars Natural water sampling: feasibility and recovery Test applications with natural water samples (seeded and unseeded) Sequence of Experimental Activities Beginning with pomona serovar-specific antibody Develop protocol to apply antibody with secondary anti-rabbit conjugate to solution seeded with pomona Evaluate specificity with application to solutions seeded with mixed serovars Evaluate use of flow cytometry for detection Evaluate sensitivity and specificity of two stage antibody detection of pathogenic leptospires in natural waters Quantitative detection: sensitivity and specificity Quantitative detection: sensitivity and specificity (pomona) Natural water sampling: feasibility and recovery Combination and application

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