Methods Development and Application: Pathogenic Leptospira in Surface Waters

Mark Walker

University of Nevada

Reno, Nevada

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

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

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

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

Maintenance hosts and associated serogroups and serovars

Mammal species Serogroup, serovar

Rats Icterohaemorrhagiae, Ballum

Mice Ballum

Dairy cattle hardjo, pomona, grippotyphosa

Pigs pomona, tarrassovi, bratislava

Sheep hardjo, pomona

Dogs canicola

Clark, T., CDC

Leptospirosis in the Pacific

LocationIncidence rate per

100,000 persons/yr

Hawaii 128

New Caledonia 90

Tahiti 30

New Zealand 4.4

Clark, T., CDC

11

2221

27

0

5

10

15

20

25

30

Four year observation period

Num

ber

of c

ases

(all

sero

vars

)

Katz, et al, 2002

Maintenance hosts and associated serogroups and serovars

Mammal species Serogroup, serovar

Rats Icterohaemorrhagiae, Ballum

Mice Ballum

Dairy cattle hardjo, pomona, grippotyphosa

Pigs pomona, tarrassovi, bratislava

Sheep hardjo, pomona

Dogs canicola

Clark, T., CDC

Australis

Bataviae

Sejroe

Unknown

1

2

3

4

4

6

7

Katz, et al, 2002

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

Lanai 1

Molokai 1

Niihau 1

(Unknown) 2

Total 353 Katz, et al, 2002

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

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.

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

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

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

Experimental plan and preliminary

results

Optional StepCritical 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

serovarsEvaluate 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

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

10 m

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

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