United States
Environmental Protection
Agency
Method Development and
Preliminary Applications
for Leptospira Spirochetes
in Water Samples
0°,
Office of Research and Development
National Homeland Security Research Cente
-------
-------
EPA/600/R-08/017 | February 2008 www.epa.gov/ord
Method Development and
Preliminary Applications for
Leptospira Spirochetes
in Water Samples
Prepared for:
National Homeland Security Research Center
U.S. Environmental Protection Agency
Prepared by:
Mark Walker, Ph.D.
University of Nevada
Department of Natural Resources and Environmental Sciences
MS 370, FA 132
1664 N. Virginia Street
Reno, NV 89557
mwalker@cabnr.unr.edu
775-784-1938
Office of Research and Development
National Homeland Security Research Center, Decontamination and Consequence Management
-------
Disclaimer
Any opinions expressed in this report are those of the authors and do not necessarily reflect
the official positions and policies of EPA. Any mention of products or trade names does not
constitute recommendation for use by EPA.
-------
Table of Contents
Abstract vi
Foreword vii
List of Figures ix
List of Tables x
Acronyms and Abbreviations xi
Acknowledgements xii
Chapter 1 Isolation of Leptospira spirochetes using filtration techniques 1
Hypotheses 1
Methods and Materials 1
Outcomes 3
Chapter 2 Application of FITC-labeled antibodies for Leptospira spirochete detection 7
Hypotheses 7
Methods and Materials 7
Outcomes 7
Chapter 3 Preliminary Field Application - Use of PCR for detection of Leptospira spirochetes
in natural water samples 9
Hypotheses 9
Methods and Materials 9
Outcomes 10
Chapter 4 Recommendations for Further Work 13
Isolation using nitration 13
Application of PCR 13
Field applications 13
References cited in the text 15
-------
Abstract
Leptospirosis is an increasingly important zoonosis
that infects humans and animals through contact with
contaminated water. The primary challenges for carrying out
water sampling for Leptospira spirochetes include isolation,
concentration, and quantitative detection of small numbers of
target organisms in water.
The objectives of this work were to 1) develop a protocol to
apply fluorescently labeled antibodies to water samples to
detect virulent serovars of Leptospira, 2) develop a protocol
to isolate Leptospira spirochetes from water samples, and 3)
use these techniques to assess the occurrence of Leptospira in
recreational waters in watersheds on Oahu and Kauai, in the
Hawaiian Islands.
Fluorescently labeled antibodies caused agglutination in
samples, which obscured individual spirochetes, so this
approach was not pursued further. When nitrocellulose filters
(0.45 (jum pore diameter) were used to isolate spirochetes
from stock suspensions, they retained approximately 90% of
the spirochetes. Because these filters are commonly used for
evaluating the presence and number of indicator organisms
in water samples, it seemed appropriate to use them to
concentrate spirochetes from natural waters. However, it may
be equally useful to concentrate small-volume samples (50-
100 ml) by centrifugation and to work directly with pelleted
debris and polymerase chain reaction (PCR), thus avoiding
inefficiencies associated with filtration. Finally, PCR was
found to be potentially more useful than microscopy for
environmental sampling, although this technique was
qualitative rather than quantitative.
This report was submitted in fulfillment of grant
#CR83271701 by Dr. Mark Walker under the partial
sponsorship of the United States Environmental Protection
Agency. This report covers a period from October 2005
to December 2006, and the work was completed as of
May 2007.
-------
Foreword
Leptospirosis
Leptospirosis is considered a reemerging disease
(Levett, 1999) that infects people who have contact with
contaminated water, soil, or urine from infected animal hosts
(Levett, 2001). The disease is commonly associated with
flooding and is prevalent in flood-prone areas (Morshed,
Konishi et al., 1994).
Leptospirosis is characterized by some researchers as the
most common waterborne illness in the world (Bharti, Nally
et al., 2003). Formerly, the number of cases in the United
States was compiled by the Centers for Disease Control and
Prevention (CDC), because leptospirosis was a reportable
disease. However, leptospirosis was removed from the
reportable disease list late in the last century, although it
still is maintained as a reportable disease in many states,
especially for veterinary cases.
Leptospirosis is caused by serovars of at least eight species
of spirochetes (Slack, Symonds et al., 2006), which are
the environmentally transmitted form of the pathogen.
Leptospira spp. are further subdivided into serogroups and
serovars, which are differentiated by commonly observed
immunological reactions to different phenotypes within
these categories. The classification arises in part from
diagnostic techniques that use a library of spirochete stocks
of different serogroups and serovars that react with blood
serum from patients with suspected infection. Common
pathogenic serogroups within L. interrogates include
Canicola (associated with infected dogs) and Copenhagen!
strain M20 (also classified as Icterohaemorrhagiae and
associated with infected rodents). The current taxonomic
approach, developed primarily using immunology, is
being supplemented by genotypic information. One of the
difficulties in identifying spirochetes using the phenotypical
approach is that antibodies in serum may not react
exclusively with a single serovar. In fact, a single diagnostic
test may indicate infection with more than one serovar.
Leptospira biflexa is also found in the environment but is
nonpathogenic. Because L. interrogates and L. biflexa species
are morphologically similar, they cannot be differentiated
with microscopy. Leptospira interogans species spirochetes
are helical and motile with dimensions of approximately
0.2-0.3 um in diameter by 6-30 um in length. Pathogenic
leptospires belong to any of more than 200 known serovars,
which are organized into at least 23 serogroups. Each serovar
may be adapted to infect a particular reservoir host that
sheds spirochetes primarily in urine (Levett, 2001). Common
serogroups identified in patients with leptospirosis in Hawaii
between 1974 and 1998 include (in descending order of
prevalence) Icterohaemorrhagiae, Australis, Ballum, Bataiae,
Sejroe, and Pomona (Katz, Ansdell et al., 2002).
The spirochetes survive well in fresh water, soil, and mud
in tropical and temperate climates (CDC, 1998). One of
the primary challenges for sampling water for spirochetes
is isolation and concentration of these pathogens from
large volumes of water. Research has focused on efficient
techniques for isolating and detecting Leptospira spirochetes
from bodily fluids and tissue samples (LeFebvre, Foley et al.,
1985; LeFebvre, 1987; Faber, Crawford et al., 2000; Levett,
2001; Bunnell, Bushon et al., 2003). Although antibodies
for serovars of Leptospira interrogans have been developed
as clinical diagnostic and research tools, they have not
been applied for water sample analysis. Culturing methods
for Leptospira are also available, but the recommended
incubation periods are exceedingly long (16-26 weeks)
(Wilson and Fujioka, 1995).
Infection may result from contact with contaminated water
or urine from infected animals, especially through skin
abrasions and mucus membranes. Symptoms of illness
range from mild febrile reactions to sometimes fatal disease.
Leptospirosis is thought to be substantially under-reported,
because symptoms are easily confused with those associated
with common influenza, dengue fever, and other viral
infections. Incidence of disease among humans has a marked
association with seasonal weather trends. For example, the
number of new cases in regions with endemic leptospirosis
may increase during wet months (Kuriakose, Eapen et al.,
1997; Sarkar, Nascimento et al., 2002). Researchers also
have noted that incidence of leptospirosis in host animal
populations coincides directly with seasonal fluctuations in
rainfall (Shimizu, 1984; Miller, Wilson et al., 1991; Ward,
2002).
Brief History of This Regional Applied Research Effort
The U.S. Environmental Protection Agency's Regional
Applied Research Effort (RARE) sponsored this project,
which was funded in October 2005. The original project
contract period was from September 2005 until August 2006.
In November 2006, permission to continue the project until
December 31, 2006, without additional funding was received.
The Quality Assurance Project Plan (QAPP) was submitted
and revised in October 2005 and January 2006, respectively.
The QAPP was approved by the U.S. Environmental
Protection Agency's National Homeland Security Research
Center in Cincinnati, OH, in January 2006.
The work took place at the University of Hawaii, Manoa,
using laboratory space that the principal investigator
prepared. Approval to operate at the University of Hawaii
laboratory and equivalent space at the University of Nevada
- Reno as Biosafety Level 2 (BSL-2) facilities was received
in January 2006. Laboratory preparation at the University of
Hawaii required extensive cleaning and removal and disposal
of obsolete equipment, which took approximately two
months (from the release of funds until January 2006). This
was followed by inspection and installation of required safety
equipment.
-------
Although work commenced in October 2006, the early stages
were hindered and delayed by lack of BSL-2 approval.
Accordingly, the scope of work was revised in January
2006 to reflect this delay. In January 2006, we obtained a
stock of a single serovar of Leptospira (interrogans serovar
Copenhagen! Icterohaemorrhagiae M-20) from the National
Veterinary Services Laboratory, in Ames, Iowa. Work began
with darkfield microscopy, polymerase chain reaction (PCR),
and filtration trials to test strategies for isolating spirochetes
from natural waters.
In early August 2006, the work initiated with RARE funds
in Hawaii continued at the University of Nevada - Reno and
University of Hawaii, Manoa, and was supplemented with
funding provided by the U.S. Department of Agriculture's
Cooperative State Research, Extension and Education
Service (CSREES). The supplemental funding from CSREES
has allowed expansion of the original RARE project, partly
as an alternative for objective 1 (see below). This additional
work is being carried out at the University of Hawaii by
a graduate student (Ms. Ilima Hawkins) in the Natural
Resources and Environmental Management Department in
the College of Tropical Agriculture and Human Resources,
under the direction of Dr. Carl Evensen.
The goal and objectives of experimental work, as amended
in January 2006, are listed below. This final report discusses
activities and outcomes associated with each objective.
Project Goal, Objectives, and Anticipated Outcomes
Project Goal: The project will develop a method to sample
environmental waters and detect specific pathogenic serovars
of Leptospira.
Project Objectives: The objectives that guide experimental
approaches include the following:
(1) Develop a protocol to apply fluorescently labeled
antibodies to water sample concentrates to detect
virulent serovars ofLeptospira.
(2) Develop a protocol to concentrate Leptospira
spirochetes from water samples.
(3) Using these techniques, assess the occurrence of
Leptospira in recreational waters in watersheds on
Oahu and Kauai, in the Hawaiian Islands.
Anticipated Outcomes:
• an optimized protocol using an indirect antibody
technique to identify serovars ofLeptospira, beginning
with Pomona serovar and progressing to homologous
antigen and antibody provided by the National Veterinary
Services Laboratory (NVSL), with a focus on the
Copenhageni Icterohaemorrhagiae M-20 serovar;
• an expected analytic detection limit for isolating
spirochetes; and
• an optimized and field-tested protocol for isolating
Leptospira spirochetes from natural waters.
-------
List of Figures
Figure 1. Petroff-Hausser bacterial counting chamber at 200 x, with Leptospira spirochetes appearing
as bright curvilinear objects 1
Figure 2. Average percent of starting numbers of spirochetes in stock suspensions that were found in filtrate,
with 95% confidence intervals displayed 3
Figure 3. Leptospira spirochetes on the surface of a 0.22-jjum pore diameter filter
(Isopore polycarbonate membrane) 5
Figure 4. Surface of a 0.45-jjum pore diameter filter (Fisher Cat # 09-719-555, 47 mmdia) 5
Figure 5. Surface of a 0.45-jjim pore diameter filter (Fisher Cat # 09-719-555, 47 mmdia)
in greater detail than Figure 4, demonstrating variation in pore diameters and potential interferences
due to filter matrix materials 6
Figure 6. Sampling locations on Manoa Stream, Honolulu, HI. Labeled locations correspond with results
presented in Figure 7, for PCR followed by amplicon sequencing 10
Figure 7. PCR amplification of leptospires from stream samples in Hawaii using primer pair G1/G2 11
Figure 8. Results of PCR of replicate suspensions of spirochetes, serovar
Copenhageni Icterohaemorrhagiae M-20 11
-------
List of Tables
Table 1. Filter types tested to determine recovery efficiencies from suspensions of pure culture of
Leptospira interrogans Copenhagen! Icterohaemorrhagiae M-20
-------
Acronyms and Abbreviations
BSA Bovine serum albumen
BSL-2 Biosafety Level 2
C Celsius
Cat Catalog
CDC Centers for Disease Control and Prevention
cm centimeter
CSREES U.S. Department of Agriculture's Cooperative State Research, Education and Extension Service
DNA Deoxyribonucleic acid
EMJH Ellinghausen and McCullough culturing medium as modified by Johnson and Harris
F Fahrenheit
FE-SEM Field emission scanning electron microscope
FITC Fluorescein isothiocyanate
g gram
Hg mercury
hr hour
kV kilovolt
LEP-FAC Anti-Leptospira antibody labeled with FITC
LEP-020 Anti-Leptospira antibody, unlabeled
M molar
mA milliamp
mBa millibar r
MgCl2 magnesium chloride
ml milliliter
ul microliter
um micrometer
mm millimeter
mM millimolar
NVSL National Veterinary Services Laboratory
PBS phosphate buffered saline
PCR polymerase chain reaction
QAPP Quality Assurance Project Plan
RARE Regional Applied Research Effort
sec second
USEPA United States Environmental Protection Agency
V volt
wt weight
-------
Acknowledgements
The Groundwater Management Office of the U.S.
Environmental Protection Agency's Region IX Office in
San Francisco helped to design the initial proposal for the
Regional Applied Research Effort (RARE) competition. Ms.
Shannon Fitzgerald and Mr. Carl Goldstein were instrumental
in supporting this effort. The University of Hawaii's College
of Tropical Agriculture, Horticulture and Human Resources
at Manoa provided laboratory space, office resources, and
material support during the portion of the project that took
place in Hawaii. In particular, Dr. Carl Evensen, chair of
the Department of Natural Resources and Environmental
Management, provided essential support, without which
research could not have been conducted. Dr. Bruce Wilcox,
director of the John A. Burns School of Medicine's Emerging
and Infectious Disease program, provided support as well.
Two graduate students at the University of Hawaii (Ms.
Ilima Hawkins and Ms. Mayee Wong) worked diligently,
patiently, and with great dedication on projects associated
with this research effort. Dr. Frank Schaefer III, of the U.S.
Environmental Protection Agency's National Homeland
Security Research Center, in Cincinnati, OH, served as
project officer for this project and provided guidance and
very constructive suggestions throughout.
-------
Chapter 1
Isolation of Leptospira Spirochetes Using
Filtration Techniques
Hypothesis
The hypothesis that guided this part of the project was that
we could effectively isolate spirochetes from natural waters
using filtration, either with a nested approach (involving
removal of debris and sediment with a coarse filter followed
by a finer filter) or with a simple membrane filtration
approach similar to that used to isolate indicator organisms
from natural waters.
Methods and Materials
Suspensions of pure culture were prepared using liquid
and semi-solid Ellinghausen and McCullough medium as
modified by Johnson and Harris (EMJH) with Leptospira
(Leptospira interrogates Copenhagen! Icterohaemorrhagiae
M-20) obtained from the National Veterinary Services
Laboratory (NVSL) in Ames, Iowa. The semi-solid EMJH
medium (DIFCO EMJH) was prepared with 0.2% noble
agar (wt/wt) using triple-filtered (1.0/0.45/0.22 |jim filters)
distilled, deionized water and was supplemented with 200-
ul/ml 5-fluorouracil (Acros Organics, Cat # 228440050) to
suppress the growth of bacterial contaminants. Liquid EMJH
was prepared as above but without noble agar. Inoculated
EMJH was stored in the dark at 21 °C (70 °F) for 3-5 weeks
(Levett, 2001). When cultures in the semi-solid medium
began to present the characteristic cloudy, compressed layer
of spirochetes approximately 1.5 cm below the surface (the
Dinger's ring), an aliquot of the spirochetes was withdrawn
from the ring and from the well-mixed liquid medium to be
sure that spirochetes were present in both types of culture.
Figure 1. Petroff-Hausser bacterial counting chamber at
200 x, with Leptospira spirochetes appearing as
bright curvilinear objects (representative examples
appear in white circles).
The spirochete suspension density was determined using a
Petroff-Hausser counting chamber, observed at magnification
of 200 x on a Nikon Labophot microscope equipped for
darkfield microscopy (Figure 1). Spirochetes were bright
curvilinear objects, approximately 20 um long, often flexing
or spinning along their long axes in suspension. Ten replicate
counts were averaged to determine each suspension density.
Fifty ml experimental suspensions were prepared containing
approximately 1.5* 106 spirochetes per ml in autoclaved,
filtered 0.01 M phosphate buffered saline (PBS) solution.
Filtration trials used 30 ml of the suspension, with several
filters (see Table 1) mounted in a 47-mm filter holder
(Osmonics, model #PFC0004703), with a vacuum of 5 inches
of Hg to draw the sample into a 50-ml tube. The number of
spirochetes retained on the filter was estimated by comparing
the average often replicate 10-ul aliquots of filtrate with the
numbers of spirochetes present in the stock suspensions.
The filter materials evaluated (Table 1) included
nitrocellulose (0.22- and 0.45-um pore diameters),
polyvinylidene fluoride (Durapore 0.22 jjum and 0.40 jjum
pore diameters), glass fiber (1.0 um), and nylon mesh
(37 |jim). Fisher (Cat # 09-719-555) nitrocellulose (0.45 |jim)
and Millipore Durapore polyvinylidene fluoride filters
(0.22 (jum) were examined by scanning electron microscopy
to verify that spirochetes were present following filtration.
Specimens were prepared by passing 0.200-ml aliquots
from undiluted liquid EMJH cultures through filters at low
vacuum (~ 5 inches Hg), followed by 200 jjul of fixative
(Karnovsky's Fixative, Electron Microscopy Services Cat
# 15720, prepared as 16% paraformaldehyde, 50% electron
microscopy grade glutaradehyde, 0.2 M sodium phosphate
buffer, with distilled water, per manufacturer's instructions)
to preserve organism structure. The fixative was added with
vacuum off. After 20 minutes, excess fixative was drawn
through the filter to waste with vacuum. The specimens were
vacuum freeze-dried (- 0.133 mBar, - 40 °C, with a Labconco
Freeze Dry System - Freezone 18) mounted on a 1.6-cm
diameter carbon stage on a bed of dessicant (anhydrous
calcium sulfate - Drierite®) for 24 hrs. A multimolecular
platinum layer was applied to the filters by sputter-coating
using an EMITECH model K575x Turbo Sputter Coater with
30-mm platinum target, sputter cycle of 20 seconds, under
ultrahigh purity argon gas at 85 mA, in a vacuum of at least
10"5 mBar. The samples were examined using a Hitachi Field
Emission Scanning Electron Microscope (FE-SEM) model
S-4700 type II, operated at a voltage of 10 kV.
-------
VI > "iJil'iiHiv'-i'i;;!!! 11 1"'
> is • ' i s ,' >
GE* Nitrocellulose-Mixed Esters of
Cellulose Membrane (E02WP04700)
Millipore Durapore Membrane filter
(HTTP04700)
Fisher (09-71 9-555)
Millipore (AP1 504700)
Small Parts Inc (CMN-0040)
Millipore Durapore (GVWP)
Millipore Isopore (HTTP)
-Mill,
Hydrophobic membrane for water
sampling
Hydrophobic
Hydrophobic membrane for water
sampling
Hydrophilic prefilter for coarse debris
removal
Hydrophobic nylon mesh sheet
Hydrophobic membrane for liquid
purification
Hydrophilic membrane for filtration of
biological liquids
l',H<| i-.',M'
Nitrocellulose
Polyvinylidene fluoride
Nitrocellulose
Glass fiber
Nylon mesh
Polyvinylidene fluoride
Polycarbonate
II 1 . J I'KIHIIl^l 1-
0.22 |jim
0.40 |jim
0.45 (jum
1.0 (jum
37 (jim
0.22 (jim
0.40 |jim
* http://www.osmolabstore.com/OsmoLabPage.dll?BuildPage&1&1&327
Table 1. Filter types tested to determine recovery efficiencies from suspensions of pure culture ofLeptospira interrogates
Copenhagen! Icterohaemorrhagiae M-20
S "I1
g o
rt MH
£ s
'o
3?
100% -
80% -
60% -
40% -
20% -
100.32%
0%
49.35%
i
32.23%
0.57%
0.06%
10.06%
i
£
62.97%
<
Filter Type
Figure 2. Average percent of starting numbers of spirochetes in stock suspensions that were found in
filtrate, with 95% confidence intervals displayed.
Outcomes
Filtration Trials: The filtration results are displayed in
Figure 2. These results suggest several important aspects
of using filters to isolate spirochetes from environmental
samples. First, in order to isolate nearly 100% of spirochetes
from sampled volumes, the optimal pore diameter should
be less than 0.45 jjum [a standard pore size used to detect
indicator organisms in 100 ml of water (Clesceri, Greenberg
et al., 1998)]. Second, the results suggest that the filter
material itself may affect recovery rates. For example,
with pore diameters of 0.4 (hydrophilic polyvinylidene
fluoride Durapore filters) and 0.45 jjum (Fisher nitrocellulose
filters), flow through recovery rates varied from <32% to
<10%, respectively. This effect is also seen in the results
from glass fiber filters and nylon mesh filters. The results
from trials with glass fiber filters may be biased in part,
because glass fiber filters are very similar in appearance to
spirochetes, which likely led to false positive results. Given
the difficulties of counting spirochetes microscopically,
this suggests that glass fiber filters would be unsuitable as
-------
prefilters for natural water samples, unless the analytical
endpoint focuses on detection of DNA rather than the
physical form of the parasite. This is because, using
microscopy, glass fibers could be mistakenly identified as
spirochetes. However, the similar morphology would not
interfere with techniques that rely on detection of specific
DNA sequences.
For environmental samples, in order to sample volumes of
water that are at least comparable to those used for detecting
indicator organisms, it may be best to prefilter the sample
using either a glass fiber filter or a nylon mesh filter to
remove large pieces of debris prior to working with the
0.22-um pore diameter filters. It may be possible to use
0.45-umpore diameter filters (especially the nitrocellulose
filters) to isolate spirochetes from environmental samples,
given that approximately 90% of spirochetes appear to
be isolated on or within the filters. This suggests that a
polymerase chain reaction (PCR) method could be adapted
for detecting the spirochetes directly on the filter itself, rather
than concentrating them from a large-volume filtrate after
coarse filtration.
Scanning Electron Microscopy Results: Scanning electron
microscopy results (Figures 3, 4, 5) support, in part, the
results presented in Figure 2. In Figure 3, spirochetes are
visible on the 0.22-um pore diameter filter (Durapore®
0.22 polyvinylidene fluoride filters). The image of the filter
suggests that a small number of pores are spaced closely
enough to slightly overlap, such that the resulting pore
diameter could be approximately equal to the diameter of a
spirochete. As a consequence, a pressure gradient across the
membrane could force spirochetes through these large pores,
leading to passage through filters that should retain them. In
fact, a small proportion of spirochetes passed through filters
that were expected to completely retain spirochetes under
the experimental conditions [the 0.22-jjum pore diameter
nitrocellulose and 0.22-um Durapore® filters (Figure 2)].
The results of FE-SEM trials with 0.45-jjum pore diameter
nitrocellulose filters are more difficult to interpret than those
with the 0.22-|jjn pore diameter nitrocellulose and Durapore
filters. After four trials, no spirochetes were detected on any
part of the filter. The electron micrograph of the filter surface
has four important discernible features. First, the pores are
irregularly shaped and pore diameter varies widely within
the matrix. Second, the resulting pores have a clear third
dimension, or depth, that cannot be captured well by electron
microscopy because of the narrow field of focus. Third, pore
geometry and orientation vary considerably with depth of the
filter, such that pores are irregular in radius and orientation,
and inconsistent and often tortuous through the fiber matrix.
Fourth, the filter matrix is a composite of homogeneous
fragments of nitrocellulose, each of which is larger in
diameter than spirochetes.
The first characteristic of these filters is important with
respect to expected retention of spirochetes. The pore
diameter of this type of filter is determined by retention
ofSerratia marcescens, a rod-shaped organism that has
a size range of 0.5-0.8 jjum in diameter by 0.9-2.0 jjum in
length. Product certification for pore size is based on overall
retention of the S. marcescens, rather than direct examination
of the filter surface.
Filter performance could be determined by more than one
process, including hydrophobic bonding and mechanical
retention. The tests performed on the filter by the
manufacturer do not differentiate between the mechanisms
of retention. Accordingly, it is possible that even though pore
size appears to be highly variable, in some cases larger than
the 0.45 um specified for these filters, an additional factor
related to sorption, such as hydrophobic binding, could retain
spirochetes on or within the filter. The second factor, depth of
the filter, suggests that spirochetes that could not be found on
the surface of the filter were retained out of the field of focus
within the filter itself. Third, the variation of pore geometry
and orientation may also enhance retention, because
spirochetes may be forced into contact with the filter material
due to the tortuosity of flow paths. This could enhance the
likelihood of contact with the filter matrix, which would
increase the opportunities for sorption. Finally, the actual
surface area of the filters is much larger than the 958-mm2
surface presented in the filter holder. This would enhance the
likelihood of sorption, if hydrophobic binding occurs.
Overall, although filtration can be used to isolate spirochetes
from water samples, it is unclear whether this is a useful
intermediate step with respect to detection. Although a
large proportion of spirochetes can be retained by filters
with a pore diameter commonly used to isolate indicator
organisms from water samples (0.45-jjim pore diameter),
the filters must be processed to recover spirochetes. Given
that sample volumes are likely to be small (100 ml or less if
waters have significant suspended sediment content) and that
under ideal conditions a maximum of 90% of spirochetes
in liquid filtered by a 0.45-jjim pore diameter filter will
be retained, additional inefficiencies will be introduced
during filter processing. This suggests that rather than
relying on processing techniques to obtain spirochetes from
filters, it may be useful to apply a detection technique to
the filter itself. In order to increase efficiency of isolating
spirochetes from liquid, it may be appropriate to work
with filters that have a smaller pore diameter (e.g., the
polyvinylidene fluoride filters with 0.22-jjum pore diameter).
Although microscopy could be useful for this, PCR may
be more appropriate because of difficulties associated with
agglutination (discussed below) and the presence of debris
that may obscure spirochetes.
-------
Sample-2 10.OkV 12.1 mm x13.0k SE(M) 4/26/07
I I I I I I I
4.00um
Figure 3. Leptospira spirochetes on the surface of a 0.22-jjum pore diameter filter (isopore
polycarbonate membrane). Arrows indicate overlapping pores that could be large
enough to allow passage of spirochetes under vacuum.
Figure 4. Surface of a 0.45-jjum pore diameter filter (Fisher Cat #09-719-555, 47 mm diameter).
-------
Figure 5. Surface of a 0.45-jjimpore diameter filter (Fisher Cat # 09-719-555, 47 mm diameter)
in greater detail than in Figure 4, demonstrating variation in pore diameters and
potential interferences due to filter matrix materials.
-------
-------
Chapter 2
Application of FITC-Labeled Antibodies for
Leptospira Spirochete Detection
Hypothesis
The hypothesis that guided this portion of the experimental
work focused on application of antibodies to specific serovars
of Leptospira. The antibodies were labeled either directly
with fluoroscein isothiocyanate (FITC) or used with a
secondary fluorescently labeled antibody that reacted with the
primary avti-Leptospira antibody.
Methods and Materials
The purpose of this experimental work was to enhance the
visibility of spirochetes with a labeled antibody that would
be species and serovar specific and could be applied using a
simple protocol. The approach was to use the specificity of
the antibody binding to exclude nonpathogenic, saprophytic
spirochetes that could be observed using darkfield
microscopy, but could not otherwise be distinguished from
pathogenic spirochetes. It was thought that epifluorescent
microscopy with high-resolution optics would make it
possible to overcome the interferences of background debris
that obscure spirochetes using darkfield microscopy. A
key element of this approach involved use of 40 x and 60 x
objectives, which would increase magnification available
using darkfield microscopy (a maximum of 200x, using a
20x objective and 10x eyepiece).
The NVSL Leptospira Copenhagen! Icterohaemorrhagiae
M-20 cultured organisms were tested with two homologous
antibodies (either FITC-labeled or unlabeled, with a
complementary goat-anti-mouse antibody provided by
Invitrogen (Spectra-Alexa Fluor 488 goat-dervied anti-
mouse IgG antibody). An antibody application and rinsing
protocol was adapted from the procedure developed for
immunofluorescent staining of Cryptosporidium oocysts in
water samples (USEPA, 2001). The specifics of the antibody
staining protocol are detailed below:
• Dilutions of LEP-FAC antibody (LEP-FAC is a FITC-
labeled mouse-derived anti-Leptospira Copenhagen!
Icterohaemorrhagiae M-20 antibody available from
NVSL) were prepared using filtered, sterilized (2
fiberglass Millipore Cat # APFD 04700 prefilters),
followed by a 0.45-um nitrocellulose filter (Fisher Cat #
09-719-555), followed by a 0.22 polyvinylidene fluoride
filter (Fisher Cat # 09-719-2B) 0.01-M PBS solution
containing 1% bovine serum albumen (BSA) (wt/wt)
to avoid nonspecific binding. In addition, the PBS also
contained 0.2% (wt/wt) 5-fluorouracil added to suppress
microbial contamination.
• 0.010-ml aliquots of diluted LEP-FAC were added to
0.100 ml of prepared stock suspensions in 1.5-ml snap-
cap microcentrifuge tubes and incubated at 21 °C (70 °F)
for 30 minutes in the dark.
• For trials with the unlabeled primary antibody (LEP-
020) and complementary Spectra-Alexa Fluor 488
antibody, the same diluent and incubation protocol [21°C
(70 °F) for 30 minutes in the dark] were used, with an
intermediate rinse and centrifugation step to remove
excess primary antibody. The rinse took place by adding
1.4 ml of 0.01 M PBS to the 0.1 ml of suspension used
for application of the unlabeled primary antibody,
followed by light vortexing (10 sec), and centrifugation
at 6,000xg for 3 minutes to precipitate spirochetes.
Aspiration reduced the volume to 0.1 ml. Trials were
carried out with dilutions of secondary antibody
including 1:100 and 1:200, volume Spectra-Alexa Fluor
488 antibody :volume 0.01 M PBS with 1% wt/wt BSA
as diluent. A total volume of 0.01 ml of diluted secondary
antibody was added to stock suspensions, which was
incubated at 21 °C (70 °F) for 30 minutes in the dark.
• 1,000 ml of PBS with 2% wt/wt DABCO (1,4-diazabicyc
lo[2.2.2]octane, Sigma Chemical Co. Cat # D-2522) was
added to reduce fluorescence quenching and mixed by
light vortexing (10 sec.).
• Spirochetes were precipitated by centrifugation (6,000 xg)
for 3 minutes.
• The supernatant was removed by aspiration to 0.1 ml.
• The pellet was examined with fluorescent and darkfield
microscopy at 200 x magnification for the presence of
spirochetes.
Outcomes
Immunofluorescent staining experiments with Leptospira
serovar Pomona began in October 2005, using a multivalent
mouse-derived antibody provided by Dr. Ranee LeFebvre
(University of California - Davis, School of Veterinary
Medicine). Working with Leptospira Pomona and an anti-
mouse labeled antibody coupled with the secondary antibody
(Spectra-Alexa Fluor 488), initial promising results were
obtained; however, clumps of spirochetes were clearly visible
at 600 x magnification.
During these limited trials, the National Veterinary Services
Laboratory (NVSL: Ames, Iowa) was contacted to discuss
obtaining a serovar that could be produced in large quantities,
as a standard, along with complementary antibodies.
The NVSL maintains serovar Leptospira Copenhagen!
-------
Icterohaemorrhagiae M-20 and two mouse-derived
antibodies, one with a FITC-label and one without. The
antibodies are for experimental and diagnostic applications,
primarily for use with blood from infected animals from
which clotting factors have been removed. Clinical diagnostic
work is based on an immunoreaction [the microscopic
agglutination test (Cumberland, Everard et al., 1999)] that
relies on using a standard suite ofLeptospira serovars and
antibodies isolated from serum obtained from infected
hosts. The NVSL's stocks are primarily applied as positive
controls for diagnosis. The diagnostic technique relies
on agglutination of spirochetes with surface proteins and
carbohydrates specific to individual serovars. The presence of
spirochetes is inferred from visible agglutination on darkfield
microscopic examination of individual reaction wells.
Single-stage antibodies - LEP-FAC: Initial results with a
1:20 dilution led to agglutination of spirochetes. In addition,
the sample, which consisted only of spirochetes that had
been washed and resuspended in distilled water, contained
small fluorescing particles that were filamentous, though too
wide and too long to be mistaken for spirochetes. However,
these filamentous particles fluoresced so brightly in some
microscopic fields that the spirochetes were obscured.
Attempts were made to dilute the antibody to reduce
agglutination(1:10, 1:20, 1:30, 1:40, 1:60). Ata 1:60
dilution, the spirochetes were no longer agglutinated, but
they also were not labeled sufficiently to be visible by
epifluorescence microscopy.
Two-stage antibodies - LEP-020 and Spectra-Alexa Fluor
488: The two-stage antibody approach yielded results similar
to those obtained from the single-stage antibody approach.
Spirochetes were agglutinated and not easily distinguished
from other types of autofluorescing debris present in the
sample. As with application of single-stage antibodies alone,
we could not identify a dilution that avoided agglutination
but also clearly labeled spirochetes.
Application of antibodies to filters: Another approach was
to use a nitrocellulose filter (Fisher Cat # 09-719-555, 47
mm diameter) to isolate spirochetes and then to apply the
antibody and fluorescence preservative solutions to the
filter directly rather than rely on centrifugation, mixing by
vortexing, and application of antibodies to the concentrate.
We sought an optimal dilution for application of labeled
and unlabeled primary antibodies and found significant
background fluorescence. The background fluorescence
obscured the spirochetes, which could also not be seen with
darkfield microscopy because of the filter matrix.
Assessment of antibody use for identifying pathogenic
spirochetes in samples: Based on the two types of trials
(application of antibodies with and without fluorescent
labels to small-volume suspensions of spirochetes and
application of antibodies to spirochetes isolated on filters), it
was apparent that epifluorescence microscopy will not be a
feasible way to detect spirochetes in environmental samples.
Microscopy is limited in several ways including:
• Agglutination
• Presence of interfering debris
• Lack of resolution with epifluorescent light sources,
leading to uncertainties in identification
Agglutination produces large masses of spirochetes that
cannot be distinguished from the autofluorescing debris in
samples. Although the antibodies bound to spirochetes and
fluoresced brightly, they also caused agglutination, which
was the primary purpose for which these reagents were
developed. Consequently, it is unlikely that application
of antibodies would be useful for environmental samples,
especially because of autofluorescing particles in samples
that closely resemble agglutinated spirochetes.
Autofluorescing particles also interfere with visualizing
labeled spirochetes. Even when the individual spirochetes
were visible under darkfield microscopy, fluorescing
background debris obscured or blocked them. Given that
the sample matrix was extremely simple (pure cultures
with triple-filtered diluent), it is likely that background
interferences would be increased in natural water samples,
which would magnify the difficulty of finding and identifying
labeled spirochetes.
Although epifluorescence microscopy offers the benefit
of excluding nonfluorescing particles from observed
specimens, it does not offer the same level of resolution
obtained by high-contrast darkfield microscopy, even with
high magnification objectives (>20*). Epifluorescence
does not appear to have the contrast and resolution needed
to distinguish spirochetes from other small, fluorescing,
linear objects. Samples observed through the microscope
were unsatisfactory as well, because very few details, such
as the tightly wound coils, were visible with the limited
light source available when epifluorescence was applied. In
addition, the spirochetes were no longer motile, as they often
are when using darkfield microscopy. This adds uncertainty
about specificity, because other linear autofluorescing
particles could be mistakenly identified as spirochetes under
epifluorescence alone.
-------
Chapter 3
Preliminary Field Application - Use of PCR for
Detection of Leptospira Spirochetes in Natural
Water Samples
Hypothesis
PCR could be a very sensitive, qualitative technique to
determine whether spirochetes are present in natural waters.
It could be adapted for use with spirochetes retained on filter
surfaces or used directly with concentrates from natural
waters developed with centrifugation.
Methods and Materials
Preliminary sampling of natural waters to detect pathogenic
spirochetes: Natural water samples (50 ml) were collected
from Manoa Stream in Honolulu on Oahu Island, Hawaii,
(Figure 6) using a modified version of method 9222-D
(Clesceri, Greenberg et al., 1998). These were transported on
ice and centrifuged upon arrival at 1500*g for 15 minutes.
The resulting pellet (~1 ml) was vortexed to resuspend the
solids and transferred to 1.5-ml microcentrifuge tubes. We
carried out DNA extractions (as described below) directly
with the pellet and residual liquid.
Sensitivity: We evaluated the sensitivity of the PCR
using Leptospira Copenhagen! Icterohaemorrhagiae M-
20 in serially diluted suspensions. This step relied on the
same diluent as that applied for experiments described in
Chapter 2. DNA from pellets was then extracted using a
DNeasy® Blood and Tissue Kit (Qiagen, Inc., Valencia, CA.,
Cat # 69506) and amplified by PCR. Controls consisted of
a negative control and a positive control drawn from stocks
provided by the University of Hawaii. Aliquots of samples
also were cultured using semi-solid and liquid preparations
of EMJH medium.
DNA extraction and amplification by PCR: DNA was
extracted from liquid or liquid/sediment mixtures using the
DNeasy® Blood and Tissue Kit. For each 52-ul reaction, the
PCR reaction used the following:
distilled H20
MgCl2
Buffer
Gl primer 1
G2 primer 2
Taq
DNA sample
27.5
5
15
0.5
Samples were processed in a GeneAmp 9700 Thermacycler
(Applied Biosystems, Foster City, CA.) at 94 °C for
5 minutes, followed by 30 cycles at the following
temperatures:
94 °C for 1 minute
51.7 °C 1 minute
72 °C 1.5 minutes
Final extension at 72 °C for 10 minutes
The reactions used Gl and G2 primers (as described by
Gravekamp et al., 1993), Gl Sequence: CTG AAT CGC
TGTATAAAAGT; G2 Sequence: GGAAAACAAATG
GTC GGA AG, and positives were verified by amplification
of a 16S rRNA region by an external laboratory (Vinetz, I,
U.C. San Diego), followed by sequence analysis (Matthias,
Diaz et al., 2005). Amplicons were separated by an electrical
potential of 90 V applied for 1.5 hours to a 1% agarose gel
(developed in a 40-mM tris acetate buffer).
After immersing the gel in an ethidium bromide solution
for 10-20 minutes, the position of fluorescing bands was
evaluated using imaging software.
-------
Worth
Figure 6. Sampling locations on Manoa Stream, Honolulu, HI. Labeled locations correspond
with results presented in Figure 7, for PCR followed by amplicon sequencing. MS 1,
Manoa Sample 1; MS 2, Manoa Sample 2; MS 3, Manoa Sample 3.
Outcomes
Field samples: Amplicon from one sample (MS 2), collected
at the base of Manoa Falls, appeared to have DNAfrom
a pathogenic serovar of Leptospira, when compared with
signals from controls in the electrophoresis gels. After
further amplification, we submitted the sample to the
University of Hawaii's Department of Microbiology Gene
Sequencing Facility. The results indicated that serovar
Icterohaemorrhagiae was likely present in the original
sample. Of eleven samples collected from Manoa Stream,
three samples were found to be positive (Figure 7). To further
verify the results, a laboratory maintained at University
of California San Diego by Dr. Joseph Vmetz analyzed
amplicons from all samples with extracted DNA from a blind
reference strain. Dr. Vinetz's laboratory identified spirochetes
in three samples (MS 1, MS 2, MS 3) that included two
pathogenic (MS 1, MS 2) and one nonpathogenic form
(MS 3). These were identified by sequence analysis as L.
alexanderi (MS 1), L. borgpetersenii (MS 2), and L. biflexa
(MS 3), as was a blind reference strain (L. borgpeterseni).
Sensitivity: Serial 1:10 dilutions of a passaged NVSL stock
were prepared, beginning with approximately 915,000
spirochetes in 1 ml of suspension and progressing to an
estimated 9 spirochetes in 1 ml. Unknown triplicate replicates
of these dilutions along with blanks were prepared. Ms.
Mayee Wong of the John A. Burns School of Medicine used
Gl and G2 primers (see PCR protocol above for further
information about primers) and performed the PCR to
evaluate the sensitivity of this assay. The results are depicted
in Figure 8.
The results indicate that blank samples were contaminated
at some stage in the process. In tracing our own procedures
and discussing this with Ms. Wong, the contamination
likely occurred during sample processing for the PCR rather
than in our laboratory. All blanks were positive, which
renders the rest of the results suspect. However, when very
few spirochetes were present (in sample A) results were
only partially positive, which suggests that the threshold
for detection may be less than 9 spirochetes. When this
experiment is repeated, these trials will be carried out with
dilutions beginning with lower numbers per ml (possibly 103)
and progressing in 50% dilutions.
-------
LI 23456
I
Figure 7. PCR amplification of leptospires from stream sampling in Hawaii, using primer pair
Gl / G2 (Gravekamp, et al, 1993). From left to right: lane L) size ladder; 1) positive
control; 2) negative control (water); 3) Manoa Stream sample MS-1; 4) MS-2; 5) MS-
3; 6) reference sample from Hawaii, L. borgpeterseni.
Figure 8. Results of PCR of replicate suspensions of spirochetes, serovar Copenhageni
Icterohemorrhagiae M-20. The labels indicate sample dilutions (A-G) and replicate
number (1-3). Dilutions are ten-fold beginning with F (915,000/ml) through E, D, C,
B and A (9/ml). Sample G is a blank.
-------
-------
Chapter 4
Recommendations for Further Work
Several aspects of the research discussed in Chapters 1-3
could be explored further to develop a sampling method with
well-characterized sensitivity that could be applied for field
application. These include using filters to isolate spirochetes
from natural waters, with the ultimate goal of applying PCR
to qualitatively determine whether pathogenic spirochetes
have been isolated from water samples, working directly
with debris pellets developed by centrifugation to apply PCR
(without filtering), and evaluating the application of either
method to natural waters. Each is discussed below.
Isolation using filtration
The results of trials indicate that a substantial proportion of
spirochetes in pure suspensions can be isolated from water
with a 0.45-um pore diameter nitrocellulose filter. The filter
is readily available and is commonly applied to determine
whether indicator organisms, including E. coli and, more
generally, fecal conform, are present in water samples. One
of the concerns about field application of this method is
whether interfering debris and sediment will limit the volume
sampled to less than 50 ml. In several trials with soil/water
slurries (~1 g soil/100 ml water, representing ~10 mg/1 total
suspended solids, work not discussed in the report), filters
clogged and failed before relatively small volumes (<10 ml)
could be processed. It is unclear whether such a concentration
of total suspended solids will be often equaled or exceeded in
natural waters. However, during high-flow events, especially
in erosion prone watersheds with unstable, steep headwater
areas, it is possible that total suspended solid concentrations
could reach and exceed this level. Given the link between
flooding and outbreaks of leptospirosis, such events may be
important to sample. However, it may be most efficient to
concentrate sediment and spirochetes in samples collected
from such events directly by centrifugation of volumes of
50-250 ml. In either case, whether the sample is concentrated
on a filter or pelleted by centrifugation, the concentrate will
be a compact sample that likely can be transported without
significant loss during shipping, especially if genomic DNA
from pathogenic spirochetes is the analytic target rather than
direct examination of the filter using microscopy.
One promising technique that could be explored further is use
of a water DNA isolation kit (for example, the UltraClean™
water kit, Cat # 14800-10, MoBio Laboratories, Inc.) These
kits are designed for extracting microbial DNA from filters
used to isolate microorganisms from water samples and have
been in use for several years but have not been applied for
use with Leptospira spirochetes. When coupled with PCR
and pathogen-specific primers, as described by Smythe
et al. (2002), the combination of filtration to concentrate
spirochetes and qualitative or quantitative PCR for detection
could yield a process that provides field researchers with
a technique to collect a highly portable set of samples for
transport and analysis in laboratories equipped to perform
either type of analysis.
Application of PCR
The preliminary applications of PCR described in this
report were successful in determining that pathogenic
and nonpathogenic spirochetes were present in water
samples concentrated by centrifugation, though the
determinations were qualitative. It is unlikely that such a
procedure (preliminary amplification of DNA, followed by
reamplification and genetic sequencing of the amplicon)
would be practical for field use because of the expense
and need for specialized equipment and training in two
laboratories.
In terms of sensitivity and the potential for application, PCR
appears more practical than microscopy for determining the
presence or absence of pathogenic spirochetes in concentrates
from natural waters. It can also be applied for quantitative
and very specific real-time sampling (Slack, Symonds et al.,
2006). In fact, Ganoza et al. (2006) recently reported results
from an extensive survey of water quality in Peru using PCR
as a quantitative method of detection. The results indicated
the presence of pathogenic spirochetes in samples from three
different environments, in concentrations ranging from 2
to 17, 147 pathogenic spirochetes/ml in positive samples.
However, it was unclear whether the authors determined a
lower limit of detection for the method that they applied, or
how the lower limit of detection might change in the presence
of chemical and biological interferences with the PCR
reaction. This is an important step that should be completed,
especially if surveys relying on PCR are conducted to
determine risk associated with contact with or consumption
of specific water sources.
Field applications
In addition to establishing a laboratory limit of detection for a
sampling protocol, it will be important to carry out field trials
with natural waters to quantify expected limits of detection in
the presence of naturally occurring chemical and biological
compounds. This includes humic acids and sediments, both
of which may be important in tropical surface waters. This
could be done with seeding and direct use of DNA extracts
from standardized suspensions of spirochetes and serial
dilutions in natural waters.
With a method limit of detection estimated for field
application, it will also be important to publish a complete
protocol that can be included as a standard reference. The
current standard method for collecting and processing
samples for Leptospira spirochetes [method 9260 I (Clesceri,
-------
Greenberg et al, 1998)] specifies culturing and animal application of filters, or centrifugation, to isolate and
inoculation as potential analytic endpoints, with a clear concentrate spirochetes, followed by a filter processing,
statement that successful cultures may contain a mixture DNA extraction protocol, and details of the PCR, would
of saprophytic and pathogenic spirochetes, as well as other be useful, especially if accompanied by guidelines about
microbial contaminants. It does not provide expected expected sensitivity and specificity of the entire field and
limits of detection for either field or laboratory methods. laboratory protocol.
A complete description of a field method that describes
-------
References Cited in the Text
Bharti, A. R., J. E. Nally, J. N. Ricaldi, M. A. Matthias, M. M. Diaz, M. A. Lovett, P. N. Levett, R.
H. Oilman, M. R. Willig, E. Gotuzzo, and J. M. Vinetz (2003). "Leptospirosis: a zoonotic disease of
global importance." Lancet Infectious Diseases 3(12): 757-771.
Bunnell, J. E., R. N. Bushon, D. M. S. Stoekel, A. M. Gifford, M. Beck, H. E. Lerch, R. Shi,
B. McGee, B. C. Hanson, J. Kolak, and P. D. Warwick (2003). "Preliminary geochemical,
microbiological, and epidemiological investigations into possible linkages between lignite aquifers,
pathogenic microbes, and kidney disease in northwestern Louisiana," U.S. Geological Survey Open-
File Report 2003-374, 59 pages.
CDC (Centers for Disease Control and Prevention) (1998). "Update: Leptospirosis and unexplained
acute febrile illness among athletes participating in triathlons - Illinois and Wisconsin, 1998
(Reprinted from MMWR 47: 673-676, 1998)." Journal of the American Medical Association 280(17):
1474-1475.
Clesceri, L. S., A. Greenberg and A. Eaton, Eds. (1998). Standard Methods for the Examination of
Water and Wastewater. 20th ed. Washington, D.C., American Public Health Association, American
Water Works Association, Water Environment Federation.
Cumberland, P., C. O. R. Everard, and P. N. Levett (1999). "Assessment of the efficacy of an IgM-
elisa and microscopic agglutination test (MAT) in the diagnosis of acute leptospirosis." American
Journal of Tropical Medicine and Hygiene 61(5): 731-734.
Faber, N. A., M. Crawford, R. B. LeFebvre, N. C. Buyukmihci, J. E. Madigan, and N. H. Willits
(2000). "Detection ofLeptospira spp. in the aqueous humor of horses with naturally acquired
recurrent uveitis." Journal of Clinical Microbiology 38(7): 2731-2733.
Ganoza, C. A., M. A. Matthias, D. Collins-Richards, K. C. Brouwer, C. B. Cunningham, E. R. Segura,
R. H. Oilman, E. Gotuzzo, and J. M. Vinetz (2006). "Determining risk for severe leptospirosis by
molecular analysis of environmental surface waters for pathogenic LeptospiraT Plos Medicine 3(8):
1329-1340.
Gravekamp, C., H. Vandekemp, M. Franzen, D. Carrington, G. J. Schoone, G. Vaneys, C. O. R.
Everard, R. A. Hartskeerl, and W. J. Terpstra (1993). "Detection of 7 species of pathogenic leptospires
by PCR using 2 sets of primers." Journal of General Microbiology 139: 1691-1700.
Katz, A. R., V. E. Ansdell, P. V. Effler, C. R. Middleton, and D. M. Sasaki (2002). "Leptospirosis in
Hawaii, 1974-1998: Epidemiologic analysis of 353 laboratory-confirmed cases." American Journal of
Tropical Medicine and Hygiene 66(1): 61-70.
Kuriakose, M., C. K. Eapen, andR. Paul (1997). "Leptospirosis in Kolenchery, Kerala, India:
Epidemiology, prevalent local serogroups and serovars and a new serovar." European Journal of
Epidemiology 13(6): 691-697.
LeFebvre, R. B. (1987). "DNA probe for detection of the Leptospira-Interrogans serovar Hardjo
genotype Hardjo-Bovis." Journal of Clinical Microbiology 25(11): 2236-2238.
LeFebvre, R. B., J. W. Foley, and A. B. Thiermann (1985). "Rapid and simplified protocol for isolation
and characterization of leptospiral chromosomal DNA for taxonomy and diagnosis." Journal of
Clinical Microbiology 22(4): 606-608.
Levett, P. N. (1999). "Leptospirosis: re-emerging or re-discovered disease?" Journal of Medical
Microbiology 48(5): 417-418.
-------
Levett, P. N. (2001). "Leptospirosis." Clinical Microbiology Reviews 14(2): 296-326.
Matthias, M. A., M. M. Diaz, K. J. Campos, M. Calderon, M. R. Willig, V Pacheco, E. Gotuzzo, R.
H. Oilman, and J. M. Vinetz (2005). "Diversity of bat-associated Leptospira in the Peruvian Amazon
inferred by Bayesian phylogenetic analysis of 16S ribosomal DNA sequences." American Journal of
Tropical Medicine and Hygiene 73(5): 964-974.
Miller, D. A., M. A. Wilson, and G. W. Beran (1991). "Relationships between prevalence of
Leptospira Interrogans in cattle, and regional, climatic, and seasonal factors." American Journal of
Veterinary Research 52(11): 1766-1768.
Morshed, M. G., H. Konishi, Y. Terada, Y. Arimitsu, and T. Nakazawa (1994). "Seroprevalence
of leptospirosis in a rural flood prone district of Bangladesh." Epidemiology and Infection 112(3):
527-531.
Sarkar, U., S. F. Nascimento, R. Barbosa, R. Martins, H. Nuevo, I. Kalafanos, I. Grunstein, B.
Flannery, J. Dias, L. W. Riley, M. G. Reis, and A. I. Ko (2002). "Population-based case-control
investigation of risk factors for leptospirosis during an urban epidemic." American Journal of Tropical
Medicine and Hygiene 66(5): 605-610.
Shimizu, M. M. (1984). "Environmental and biological determinants for the prevalence of
Leptospirosis among wild small mammal hosts, island of Hawaii." International Journal ofZoonoses
11(2): 173-188.
Slack, A. T., M. L. Symonds, M. F. Dohnt, and L. D. Smythe (2006). "Identification of pathogenic
Leptospira species by conventional or real-time PCR and sequencing of the DNA gyrase subunit B
encoding gene." BioMedCentralMicrobiology 6: doi: 10.1186/1471-2180-6-95.
Smythe, L. D., I. L. Smith, G. A. Smith, M. F. Dohnt, M. L. Symonds, L. J. Barnett, and D. B.
McKay (2002). "A quantitative PCR (TaqMan) assay for pathogenic Leptospira spp" BioMedCentral
Infectious Diseases 2(13: doi:10.1186/1471-2334-2-13).
U.S. EPA (2001). Method 1622: Cryptosporidium in water by filtration/IMS/FA. Washington, D.C.,
Office of Water.
Ward, M. P. (2002). "Seasonality of canine leptospirosis in the United States and Canada and its
association with rainfall." Preventive Veterinary Medicine 56(3): 203-213.
Wilson, R. and R. Fujioka (1995). "Development of a method to selectively isolate pathogenic
Leptospira from environmental samples." Water Science and Technology 31(5-6): 275-282.
-------
-------
&EPA
United States
Environmental Protection
Agency
PRESORTED STANDARD
POSTAGES FEES PAID
EPA
PERMIT NO. G-35
Office of Research and Development
National Homeland Security Research Center
Cincinnati, OH 45268
Official Business
Penalty for Private Use
$300
Recycled/Recyclable
Printed with vegetable-based ink on
paper that contains a minimum of
50% post-consumer fiber content
processed chlorine free
------- |