Leptospirosis Serodiagnosis by the UNIT 12E.

5
Microscopic Agglutination Test
Marga G.A. Goris1 and Rudy A. Hartskeerl1
1
Royal Tropical Institute (KIT), KIT Biomedical Research, WHO/FAO/OIE and National Col-
laborating Centre for Reference and Research on Leptospirosis, Amsterdam, The Netherlands

ABSTRACT
The microscopic agglutination test (MAT) is the gold standard for sero-diagnosis of
leptospirosis because of its unsurpassed diagnostic specificity. It uses panels of live
leptospires, ideally recent isolates, representing the circulating serovars from the area
where the patient became infected. A dilution series of the patient’s serum is mixed with
a suspension of live leptospires in microtiter plates. After incubating for about 2 hr at
30°C, results are read under the dark-field microscope. The titer is the last dilution in
which 50% of the leptospires have remained agglutinated. Seroconversion or 4-fold
titer rise in paired sera is consistent with current leptospirosis. The significance of a titer
in a single sample depends on the frequency of residual titers due to past infections and
cross-reacting other diseases in the population. Full standardization of the MAT is not
possible, but quality assurance can be achieved by participation in the international MAT
proficiency testing scheme. Curr. Protoc. Microbiol. 32:12E.5.1-12E.5.18.  C 2014 by

John Wiley & Sons, Inc.

Keywords: MAT r agglutination r leptospirosis r antibodies r serology

INTRODUCTION
Leptospirosis is one of the most widespread zoonotic diseases in the world with an
anticipated high veterinary public health impact. The disease is strongly neglected, which
is not justified considering the global incidence. Recent estimates vary but suggest the
occurrence of at least one million severe, mostly hospitalized, human cases per year with
a case fatality rate of nearly 10% (World Health Organization, 2011; ILRI, 2012). The
impact of veterinary leptospirosis is largely unknown but likely surpasses that of human
leptospirosis.

Leptospirosis is a protean disease that manifests from mild to a severe, potentially

fatal form. It presents with a wide variety of mostly uncharacteristic clinical signs
and symptoms and is often confused with similar diseases that are endemic and epi-
demic in the same environmental and climatologic conditions. Typical examples are
dengue, brucellosis, yellow fever, salmonellosis, and rickettsiosis (World Health Organi-
zation, 2003). For further information on the disease and its causative agent, we refer to
UNIT 12E.1 “Laboratory Maintenance of Pathogenic Leptospira.”

Clinical diagnosis of leptospirosis is challenging and laboratory support for confirmation

is indispensable. To date, a variety of serological tests, mostly rapid diagnostic tests,
have been described. These include ELISA, indirect fluorescent antibody test (IFAT),
macroscopic slide agglutination test (MSAT), latex agglutination tests such as the DriDot,
and various lateral flow assays.

The microscopic agglutination test (MAT) is the reference test for the serodiagnosis of
leptospirosis both in humans and in animals (Faine et al., 1999; World Health Organiza-
tion, 2003; OIE, 2008).
Spirochetes

Current Protocols in Microbiology 12E.5.1-12E.5.18, February 2014 12E.5.1

Published online February 2014 in Wiley Online Library (wileyonlinelibrary.com).
DOI: 10.1002/9780471729259.mc12e05s32 Supplement 32
Copyright C 2014 John Wiley & Sons, Inc.
 Table 12E.5.1 List of Globally Representative Leptospira Reference Strains Recommended by
the World Health Organization for Use as Antigens

Serogroup Serovar Strain

Andaman Andaman CH 11
Australis Australis Ballico
Bratislava Jez Bratislava
Autumnalis Autumnalis Akiyami A
Rachmati Rachmat
Ballum Ballum Mus 127
Bataviae Bataviae Swart
Canicola Canicola Hond Utrecht IV
Celledoni Celledoni Celledoni
Cynopteri Cynopteri 3522 C
Grippotyphosa Grippotyphosa Moskva V
Hebdomadis Hebdomadis Hebdomadis
Icterohaemorrhagiae Copenhageni M 20
Icterohaemorrhagiae RGA
Javanica Javanica Veldrat Batavia 46
Poi Poi
Panama Panama CZ 214
Pomona Pomona Pomona
Pyrogenes Pyrogenes Salinem
Sejroe Hardjo Hardjoprajitno
Saxkoebing Mus 24
Sejroe M 84
Semaranga Patoc Patoc I
Shermani Shermani LT 821
Tarassovi Tarassovi Perepelitsin

The MAT is defined as “Serial dilutions of serum kept in contact with an equal volume
of a well grown suspension of leptospires at a certain temperature for a certain period of
time and read microscopically by estimating 50% agglutination as the end point titer of
the reaction mixture.”

Serum dilutions are made in microtiter plates and live leptospires are added. Currently,
over 250 different leptospiral serovars are known worldwide (Adler and de la Pena,
2010). In view of the serovar specificity of the MAT, a leptospiral panel should be
used, ideally consisting of recent isolates and representing the circulating serovars from
the area where the patient contracted the infection. Alternatively, globally represen-
tative Leptospira reference strains recommended by the World Health Organization
(Table 12E.5.1) may be used.

In general, laboratories use 5- to 7-day-old leptospiral subcultures. For standardization,

one may consider adjusting cultures to a concentration of 2 × 108 leptospires per ml,
Leptospirosis
Serodiagnosis by prior to testing. The density of Leptospira cultures can be determined by: (1) count-
the Microscopic ing in a suitable counting chamber (Helber or Petroff-Hausser, cell depth 0.02 mm),
Agglutination Test
(2) measuring optical density (OD) of the culture by spectrophotometry at 420 nm,
12E.5.2
Supplement 32 Current Protocols in Microbiology
(3) using the McFarland scale, and (4) estimating the number of leptospires per field by
dark-field microscopy.

However, when using large panels of serovars, the first three approaches are laborious
and needlessly time-consuming. More importantly, one uses the method in a consistent
way according to a standard operation procedure, which has been well-validated (Goris
et al., 2011).

The MAT is read using a dark-field microscope to check for agglutination. This can be
done indirectly, by placing a small drop of the reaction mixture on a microscope slide.
The direct way is to view the microtiter plate directly under a dark-field microscope.

There are several bottlenecks that have prevented widespread use of the MAT. (1) It
is difficult to prepare culture medium. (2) It is difficult to maintain live strains. (3)
Cultures are easily mixed up. Therefore, the identity of diagnostic strains should be
checked using specific antibodies at regular intervals for their identity. (4) Cultures are
easily contaminated by saprophytic (noninfectious, environmental) leptospires and other
bacteria.

This unit describes the methods utilized for performing MAT, either quantitative (Basic
Protocol 1) or for screening (Basic Protocol 2), both by indirect reading. The Alternate
Protocol describes direct reading.

Procedures for sub-culturing of leptospires (Support Protocol 1) and determining the

density of a leptospiral culture by counting with a Helber counting chamber (Support
Protocol 2), measuring of the OD by a spectrophotometer (Support Protocol 3), estimating
with the McFarland scale (Support Protocol 4), and estimating by eye (Support Protocol
5) are also provided.

Besides the use for serodiagnosis, the MAT can be applied for serological typing of
leptospires. The Cross Agglutinin Absorption Test (CAAT), consisting of the cross-
agglutination and the cross-absorption assays, analysis with factor sera, and typing with
monoclonal antibodies, all make use of the MAT technique.

CAUTION: According to the Centers for Disease Control manual Biosafety in Mi-
crobiological and Biomedical Laboratories (http://www.cdc.gov/biosafety/publications/
bmbl5/bmbl5_sect_VIII.pdf), Leptospira interrogans sensu latu is classified as a
Biosafety Level 2 (BSL-2) pathogen that can cause acute, fatal infections in humans
and animals. Follow all appropriate guidelines and regulations for the use and handling
of pathogenic microorganisms.

For handling serum, follow all appropriate guidelines and regulations for the use
and handling of human-derived materials. See UNIT 1A.1 and other pertinent resources
(APPENDIX 1B) for more information.

MICROSCOPIC AGGLUTINATION TEST (MAT), QUANTITATIVE, BASIC

INDIRECT READING PROTOCOL 1
MAT is a serologic technique that detects agglutinating antibodies against serovar-
associated epitopes of the microorganism Leptospira spp. Equal volumes of serial diluted
sera are mixed with live leptospires. The test can be read after 2 to 4 hr incubation at
30°C with a dark-field microscope. Results are expressed as dilution titers of serum, in-
cluding the added antigen. The dilutions may range from 1:20 to 1:20,480, occasionally
more.

Spirochetes

12E.5.3
Current Protocols in Microbiology Supplement 32
 No 1:10 1:20 1:40 1:80 1:160 1:320 1:640 1:1280 1:2560 1:5120 1:10,240
sample Sample
dilution
Control 1:20 1:40 1:80 1:160 1:320 1:640 1:1280 1:2560 1:5120 1:10,240 1:20,480
Final
dilution
1 2 3 4 5 6 7 8 9 10 11 12
Strain 1 A

Strain 2 B

Strain 3 C

Strain 4 D

Strain 5 E

Strain 6 F

Strain 7 G

Strain 8 H

Figure 12E.5.1 Example of microtiter plate lay-out for quantitative MAT.

Materials
Phosphate-buffered saline (PBS; see recipe), pH 7.2
Serum or plasma (it is recommended to heat-inactivate serum for 30 min in a water
bath at 56°C to reduce infection risks; plasma samples cannot be
heat-inactivated)
Live Leptospira cultures at 2 × 108 leptospires/ml of each of the required serovars
96% ethanol
96-well microtiter plate, V-bottom, flat-bottom or U-bottom
Calibrated pipets: 2 to 20 μl and 20 to 200 μl (optional: 50-μl multichannel pipet
and 50-μl multistepper pipet)
Microtiter plate cover
Microshaker, optional
30°C incubator
Microscope slides
Wire loop
Dark-field microscope
1. Fill all 96 wells of a microtiter plate with 50 μl PBS, pH 7.2 (Fig. 12E.5.1).
The pH of the PBS is not stringent; in various protocols pH ranges from 7.2 to 7.6.

2. Add another 40 μl PBS to the wells of column 2.

3. Add 10 μl of serum to the wells of column 2.
The serum is now 1:10 diluted.

4. Make serial doubling dilutions as follows:

a. First mix the contents of the wells in column 2 by pipetting 50 μl up and down
several times.
b. Next, transfer 50 μl to the wells of the next column and so on up to column 12.
Leptospirosis c. Discard the final 50 μl.
Serodiagnosis by
the Microscopic A multichannel pipet can be used.
Agglutination Test

12E.5.4
Supplement 32 Current Protocols in Microbiology
 5. Add 50 μl Leptospira culture to all wells.
The final dilution (including the added Leptospira culture) of the serum in column 2 is
now 1:20, column 3 is 1:40, etc. The wells in column 1 serve as a comparative control
without agglutination and serve to check for auto-agglutination of the leptospires and
sterility of the PBS.

6. Cover the plate with a cover or another microtiter plate.

7. Mix thoroughly on a microshaker or mix by carefully ticking the plate by hand.
8. Incubate 2 hr at 30°C.
The temperature may range from 28° to 32°C. The incubation time is not critical. Incu-
bation times from 30 min up to 4 hr have been documented. For standardization though,
it is important to consistently use one fixed incubation time. As an alternative, incubation
overnight at room temperature might be considered.

9. Thoroughly clean a microscope slide with 96% ethanol.

10. Transfer a droplet of the mixture with a wire loop to the microscope slide.
Do not use a coverslip. A pipet can be used instead of a wire loop.

11. Inspect for agglutination, preferably at the border of the droplet with a dark-field
microscope using 100-200× total magnifying power.
Do this quickly, before significant evaporation occurs. Be aware of a prozone effect that
might occur, i.e., agglutination occurs at higher dilution ranges, but is not visible at lower
dilutions. In addition, always check higher dilutions in cases where low dilutions do not
show apparent agglutination (see Fig. 12E.5.3, row C). The titer is the highest dilution
in which 50% of the leptospires are still agglutinated—i.e., <50% of the leptospires
are freely moving—compared to the control suspension in the wells of the first column
(which contains only Leptospira culture diluted 1:2 in PBS without serum). The titer is
expressed as the final dilution including the volume of the added Leptospira culture. See
Figure 12E.5.2 for a schematic presentation and Figure 12E.5.3 for pictures.
IMPORTANT NOTE: The size or pattern of clumping should not be used to determine
the endpoint; rather the clarity of the background and percentage of leptospires that are
free (non-agglutinated) should be estimated. Especially “dirty” sera show particles that
resemble the clumps of agglutinated leptospires.

MICROSCOPIC AGGLUTINATION TEST, SCREENING, INDIRECT BASIC

READING PROTOCOL 2
Screening by MAT is done by using one dilution instead of a dilution series as described
in Basic Protocol 1. Please be aware that reactive serum samples can be missed due to
the prozone effect that might occur, notably when high concentrations of anti-Leptospira
antibodies are present in the test sample (Shimabukuro et al., 2013).
Materials
Serum or plasma (it is recommended to heat-inactivate serum for 30 min in a water
bath at 56°C to reduce infection risks; plasma samples cannot be
heat-inactivated)
Phosphate-buffered saline (PBS; see recipe), pH 7.2
Live Leptospira cultures at approximately 2 × 108 cells/ml of each of the required
serovars
96% ethanol
Small tubes
96-well microtiter plate, V-bottom, flat-bottom or U-bottom
Calibrated pipets: 20 to 200 μl (optional: 50-μl multistepper pipet) Spirochetes

12E.5.5
Current Protocols in Microbiology Supplement 32
 No 1:10 1:20 1:40 1:80 1:160 1:320 1:640 1:1280 1:2560 1:5120 1:10,240
sample Sample
dilution
Control 1:20 1:40 1:80 1:160 1:320 1:640 1:1280 1:2560 1:5120 1:10,240 1:20,480
Final
dilution
1 2 3 4 5 6 7 8 9 10 11 12
Strain 1 A

Strain 2 B

Strain 3 C

Strain 4 D

Strain 5 E

Figure 12E.5.2 Schematic representation of agglutination, quantitative method. (A) Negative sample, titer <1:20;
(B) reactive sample, no prozonal effect, titer 1:320; (C) reactive sample, prozonal effect, titer 1:5120; (D) reactive
sample, prozonal effect, titer 1:320; (E) agglutinations ranging from 100% to 0% (dilution 1:20 shows 100% ag-
glutination, 1:40 is 90%, 1:80 is 80%, 1:160 is 70%, 1:320 is 60%, 1:640 is 50%, 1:1280 is 40%, 1:2560 is 30%,
1:5120 is 20%, 1:10240 and 1:20480 show 0% agglutination). Gray area shows 50% or more agglutination, white
area shows more than 50% free leptospires

Microtiter plate cover

Microshaker, optional
30°C incubator
Microscope slides
Wire loop
Dark-field microscope
1. Predilute the sera 1:25 in PBS in small tubes.
For example, 50 μl serum and 1.2 ml PBS, pH 7.2

2. Add 50 μl of the diluted serum to the number of required wells of the microtiter plate.
The number of wells depends on the number of serovars to be used for each serum.

3. Add 50 μl Leptospira culture to all respective wells.

The final dilution (including the added Leptospira culture) is now 1:50. The wells in
column 1 serve as comparative control.

4. Cover the plate with a cover or another microtiter plate.

Leptospirosis
Serodiagnosis by 5. Mix thoroughly on a microshaker or mix by carefully tapping the plate by hand.
the Microscopic
Agglutination Test

12E.5.6
Supplement 32 Current Protocols in Microbiology
 negative control 1:20 1:40 1:80 1:160

Figure 12E.5.3 Dark-field microscopy (total magnifying power 200×) pictures of agglutinations
with various sera. (A) negative serum, titer <1:20; (B) reactive serum, titer 1:80; (C) serum showing
prozone effect, titer 1:160.

6. Incubate for a fixed time interval between 2 to 4 hr at 30°C.

As an alternative, incubation overnight at room temperature might be considered.

7. Thoroughly clean a microscope slide with 96% ethanol.

8. Transfer a loopful of the mixture with a wire loop to the microscope slide.
Do not use a coverslip.

9. Inspect for agglutination, preferably at the side of the droplet, with a dark-field
microscope using 100× to 200× total magnifying power.
Do this quickly, before significant evaporation occurs. Agglutination is present when 50%
of the leptospires have agglutinated compared to the control suspension.
IMPORTANT NOTE: The size or pattern of clumping should not be used to determine the
agglutination. See Figure 12E.5.2 for a schematic representation of 50% agglutination.

MICROSCOPIC AGGLUTINATION TEST (MAT), DIRECT READING ALTERNATE

PROTOCOL
Direct reading can be done with a suitable dark-field microscope (100-W light source
and long distance objective). This will speed up the reading process. In order to get
a proper view, a clear flat-bottom microtiter plate is required, as well as reducing the
volumes in the wells mentioned in Basic Protocols 1 and 2 by half. Here, we only detail
the alternative of Basic Protocol 1. For the alternative of Basic Protocol 2, please use
the flat-bottom microtiter plate specified below, and use half of the volumes in the wells
described in Basic Protocol 2.
Materials
Spirochetes
Phosphate-buffered saline (PBS; see recipe), pH 7.2
12E.5.7
Current Protocols in Microbiology Supplement 32
 Serum and plasma (it is recommended to heat-inactivate serum for 30 min in a
water bath at 56°C to reduce infection risks; plasma samples cannot be
heat-inactivated)
Live Leptospira cultures at 2 × 108 leptospires/ml of each of the required serovars
96-well microtiter plate, flat-bottom (Greiner Crystal-Clear, cat. no. 655101)
Calibrated pipets: 2 to 20 μl and 20 to 200 μl (optional: 50-μl multichannel pipet
and 50-μl multistepper pipet)
Microtiter plate cover
Microshaker, optional
30°C incubator
Microscope slides
Wire loop
Dark-field microscope suitable for direct reading
1. Fill all 96 wells of a microtiter plate with 25 μl PBS, pH 7.2 (Fig. 12E.5.1).
In various protocols the pH of the PBS ranges from 7.2 to 7.6.

2. Add another 20 μl PBS to the wells of column 2.

3. Add 5 μl of serum to the wells of column 2.
The serum is now 1:10 diluted.

4. Make serial doubling dilutions as follows:

a. First mix the contents of the wells in column 2 by pipetting 25 μl up and down
several times.
b. Next, transfer 25 μl to the wells of the next column, up to column 12.
c. Discard the final 25 μl.
A multichannel pipet can be used.

5. Add 25 μl Leptospira culture to all wells.

The final dilution (including the added Leptospira culture) of the serum in column 2 is
now 1:20, column 3 is 1:40, etc. The wells in column 1 serve as comparative control.

6. Cover the plate with a cover or another microtiter plate.

7. Mix thoroughly on a microshaker or mix by carefully tapping the plate by hand.
8. Incubate for a fixed time within the range 2 to 4 hr at 30°C.
Incubation overnight at room temperature is not an option due to possible evaporation.

9. Remove the cover and read the plate directly under the dark-field microscope.
10. Inspect for agglutination, with a dark-field microscope using 80× to 120× total
magnifying power.
Check all dilutions. The titer is the highest dilution in which 50% of the leptospires are
still agglutinated, i.e., <50% of the leptospires are freely moving, compared to control
suspension in the wells of the first column (which contains only Leptospira culture diluted
1:2 in PBS without serum). The titer is expressed as the final dilution including the volume
of the added Leptospira culture. See Figure 12E.5.2 for a schematic representation and
Figure 12E.5.3 for pictures.
IMPORTANT NOTE: The size or pattern of clumping should not be used to determine
Leptospirosis the endpoint; rather the clarity of the background and percentage of leptospires that are
Serodiagnosis by free (non-agglutinated) should be estimated. Especially “dirty” sera show particles that
the Microscopic resemble the clumps of agglutinated leptospires.
Agglutination Test

12E.5.8
Supplement 32 Current Protocols in Microbiology
GROWTH OF LEPTOSPIRA STRAINS (ANTIGENS) FOR THE SUPPORT
MICROSCOPIC AGGLUTINATION TEST PROTOCOL 1
For MAT, Leptospira serovars are usually grown and maintained in EMJH liquid medium,
although other, less expensive liquid culture media, such as Korthof-Babudieri medium
(World Health Organization, 2003), may also be adequate. The stock culture collection
of leptospires is best maintained at room temperature in closed screw-cap test tubes half-
filled with liquid medium (usually 5 to 6 ml). It is very important that the stock culture
tubes be clearly and correctly labeled and that every effort be made to avoid mislabeling
and contamination during transfers. If many sera are to be examined, additional tubes
of the medium may be inoculated for use as antigens. Periodically, the cultures used as
antigens should be checked by MAT against reference homologous rabbit antisera or
monoclonal antibodies to check the identity of the strains. The selection of leptospiral
serovars for use in the MAT should represent the serogroups of all known serovars
present in the country or area. It is preferable to use local isolates/strains. For additional
information, please also consult UNIT 12E.1.
Materials
Leptospira strains in liquid medium
Liquid medium in tubes (5 to 10 ml; e.g., EMJH; see UNIT 12E.1)
Laminar flow cabinet
Microscope slide
Wire loops, sterile
Pipets, sterile
Dark-field microscope
29°C incubator
1. Work in a laminar flow cabinet to reduce the probability of contaminating the cultures.
2. Place a droplet of the culture with a wire loop onto a microscope slide.
A pipettor may also be used. Make sure not to contaminate the culture at this step.

3. Examine by dark-field microscopy to confirm the presence of viable leptospires and

the absence of contamination.
This can be done outside the laminar flow cabinet.

4. Inoculate 1 volume of the last transfer of each strain to 10 volumes of fresh medium
in duplicate.
For example, 0.5 ml leptospiral strain to 5 ml of fresh medium.

5. Incubate the subcultures at 29°C (±2°C) and check the density of the leptospires and
absence of contamination prior to use (see Support Protocol 5).
Usually, 5 to 10 days of incubation are sufficient. If larger quantities are quickly needed,
use 50- to 500-ml Erlenmeyer flasks in a shaking incubator to speed up growth.
After incubation, store the subcultures in a dark place at 15° to 20°C.
Subcultures can be used for 1 week.

COUNTING LEPTOSPIRES IN A HELBER COUNTING CHAMBER SUPPORT

PROTOCOL 2
The Helber bacteria counting chamber consists of a microscope slide with grids and
a special cover glass. The grids form 20 by 20 squares with standardized dimensions
(Fig. 12E.5.4). A Petroff-Hausser counting chamber can also be used; see APPENDIX 4A.
Spirochetes

12E.5.9
Current Protocols in Microbiology Supplement 32
 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20

Figure 12E.5.4 Schematic presentation of a Helber counting chamber. The lines represent the
horizontal and vertical grids indicating separate counting areas. The small squares represent the
fields that should be counted (e.g., no. 2 vertical and no. 2 horizontal is one square)

Materials
Phosphate-buffered saline (PBS; see recipe), pH 7.2
Formalin (37% formaldehyde)
Suspension of leptospires to be counted
70% (v/v) ethanol
Distilled water
2- to 20-μl and 20- to 200-μl micropipets
Small tubes
Vortex mixer
Helber bacteria counting chamber with special slide cover
Dark-field microscope
Tally counter, optional
1. Pipet the following in a small tube:

180 μl PBS
10 μl formalin (final concentration of formalin is 5%; this equals 1.8%
formaldehyde)
10 μl of the suspension of leptospires.
This results in a 20-fold dilution of leptospires in 1.8% formaldehyde.

2. Mix briefly on a vortex.

Leptospires are killed.

3. Make sure the Helber counting chamber is clean and dry, see step 9.
4. Pipet 6 μl from the tube into the middle of the counting chamber.
Leptospirosis
Serodiagnosis by 5. Cover the counting chamber carefully with the special slide cover.
the Microscopic
Agglutination Test
6. Leave for 5 min at room temperature to allow the leptospires to settle down.
12E.5.10
Supplement 32 Current Protocols in Microbiology
 7. Place the counting chamber under a dark-field microscope (total magnifying power
200×, with a 20× ocular).
8. Randomly count 20 squares in the counting chamber.
A hand tally counter saves memorizing numbers during the counting.
Include leptospires that touch the left and upper grids. Ignore those that touch the right
and lower grids.
A sample giving one to five leptospires per square is convenient to count. Adapt the
dilution of the leptospires suspension if needed.
9. Clean the counting chamber with 70% ethanol and subsequently with distilled water
and dry carefully with a tissue. Avoid scratching the surface.
10. Calculate the leptospiral density of the suspension using the following criteria.
Dimensions of the counting chamber:
Length: 1 mm, 20 squares
Width: 1 mm, 20 squares
Depth: 0.02 mm
N = number of leptospires counted in 20 squares
Suspension has been diluted 20-fold (10 μl + 190 μl) in PBS containing 1.8%
formaldehyde.
Number of leptospires/ml suspension: 106 × N × 20
Adapt the calculation accordingly when the leptospires suspension is diluted differently
from 1:20.

USING OPTICAL DENSITY TO DETERMINE NUMBER OF LEPTOSPIRES SUPPORT

PROTOCOL 3
The OD is measured on a spectrophotometer with visible light at 420 nm. Culture medium
without leptospires is used as the blank.

Figure 12E.5.5 shows a generalized relationship between the density of the suspension of
leptospires in culture medium and the OD at 420 nm. Although this diagram can be used
directly, it is recommended to make well-defined dilutions of a suspension of leptospires
with known density, established by counting the master culture (see Support Protocol 2),
prior to OD determination.
Materials
Distilled water
Culture medium
Leptospires
Cuvettes
Spectrophotometer with visible light at 420 nm
1. Fill a cuvette with distilled water.
2. Fill a cuvette with culture medium.
This is the medium used to grow the leptospires.

3. Fill a cuvette with leptospiral culture.

4. Measure the OD at 420 nm:

a. Measure the OD of the cuvette with distilled water.

b. Measure the medium compared to the distilled water.
Usually, OD of the medium is <0.1.
Spirochetes

12E.5.11
Current Protocols in Microbiology Supplement 32
 0.4

0.35

0.3

0.25
OD 420 nm

0.2

0.15

0.1

0.05

0
0 2 4 6 8 10 12 14
Number of leptospires ⫻ 108/ml

Figure 12E.5.5 Graph showing relation between density of Leptospira culture and OD at 420 nm.

c. Lastly, measure the Leptospira culture against medium as a blank.

5. Compare the OD with ODs and densities found on the graph.

SUPPORT THE McFARLAND NEPHELOMETRIC METHOD FOR DETERMINING THE

PROTOCOL 4 DENSITY OF CULTURES
In this method, suspensions of barium sulfate are made with different densities; these
have been calculated to be comparable to the densities of suspensions of bacteria with
known numbers of bacteria. In order to estimate the numbers of bacteria in suspensions
from cultures, the density of the experimental suspension (absorbance or transmission) is
compared to that of the set of reference tubes in the series. Different proportions of barium
chloride and sulfuric acid are used to prepare a range of barium sulfate concentrations
for the McFarland nephelometry standards.
Materials
Barium chloride (BaCl2 ; anhydrous)
Distilled water
Sulfuric acid (H2 SO4 ; 95% to 97%)
Leptospiral culture
Fume hood
Tubes, appropriate size for nephelometer
Nephelometer
1. Prepare 1% aqueous barium chloride by dissolving 1 g barium chloride in 100 ml
Leptospirosis distilled water.
Serodiagnosis by
the Microscopic 2. Prepare 1% aqueous sulfuric acid by adding 1 ml sulfuric acid to 99 ml distilled water.
Agglutination Test
Add the sulfuric acid to water; perform this step in a fume hood.
12E.5.12
Supplement 32 Current Protocols in Microbiology
Table 12E.5.2 Composition of McFarland Suspensions and Corresponding Density of Bacteria

Tube ml barium ml sulfuric Corresponding density of

number chloride (1% w/v) acid (1% v/v) bacteria suspension (106 /ml)
1 0.1 9.9 300
2 0.2 9.8 600
3 0.3 9.7 900
4 0.4 9.6 1200
5 0.5 9.5 1500
6 0.6 9.4 1800
7 0.7 9.3 2100
8 0.8 9.2 2400
9 0.9 9.1 2700
10 1.0 9.0 3000

3. Prepare the reference tubes by adding 1% aqueous barium chloride and 1% aqueous
sulfuric acid to tubes of a size appropriate for the nephelometer.
The amounts of each stock and the corresponding densities of bacteria are given in
Table 12E.5.2.

4. Add leptospiral culture with an unknown density to a tube.

5. Measure all tubes in the nephelometer, or compare the turbidity by eye.
6. Read the unknown density of the leptospiral strain in Table 12E.5.2.

ESTIMATING CULTURE DENSITY BY EYE SUPPORT

PROTOCOL 5
Only experienced staff should visually judge the density of a Leptospira culture directly
by dark-field microscopy. Do this by placing a small drop on a microscope slide.

A specimen is fully grown when all inspected fields are completely covered with well-
shaped leptospires and no other bacteria, yeast, or fungi are observed. This is indicated
by 4+, and “shows” similar to the density of the negative control wells depicted in Figure
12E.5.3. This is the correct density for use in the MAT.

3+ 75% of the microscope field is covered by leptospires as compared to 4+.

2+ 50% of the microscope field is covered by leptospires as compared to 4+.
1+ 25% of the microscope field is covered by leptospires as compared to 4+.
0 only a few or none leptospires are observed.

REAGENTS AND SOLUTIONS

Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see
APPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX.

Phosphate-buffered saline (PBS), 10× concentrated stock

For 5 liters:
425 g NaCl
42.8 g Na2 HPO4 ·2H2 O
12.7 g KH2 PO4
Bring volume to 5 liter with distilled water
Mix to dissolve the salts
Divide into aliquots in 1-liter bottles Spirochetes

continued 12E.5.13
Current Protocols in Microbiology Supplement 32
 Store up to 1 year room temperature in a dark place
The high concentration of salts prevents the growth of microorganisms.

Phosphate-buffered saline (PBS), pH 7.2

For 1 liter:
100 ml phosphate-buffered saline (PBS), 10× concentrated stock (see recipe)
Add 900 ml distilled water
Mix, and adjust the pH to 7.2 with diluted HCl or NaOH
Divide into aliquot in 100-ml bottles, and use the same day
Alternatively, the PBS can be autoclaved
Autoclaved PBS is stable for 1 year at room temperature in a dark place. The PBS needs
autoclaving to avoid growth of saprophytic leptospires and other microorganisms, which
disturb reading the MAT.

COMMENTARY
Background Information
In 1918, Martin and Pettit described the
phenomenon of agglutination and “lysis” with
sera (Martin and Pettit, 1918). Since then,
the method has been improved by others
(Schuffner and Mochtar, 1926; Borg-Petersen
and Fagraeus, 1949; Wolff, 1954; Carbrey,
1960; Cole et al., 1973). These investigators
have optimized factors like incubation time
and temperatures, reading of the endpoint titer,
and density and age of the culture. There are
several obstacles in the wide-scale applica-
tion of the MAT, which also impede its in-
troduction in nonspecialized laboratories. Cul-
ture medium is expensive and demanding to
prepare. Maintenance of diagnostic strains is
laborious and time consuming. A pitfall of
culturing is that during preparation of culture
medium and sub-culturing, contamination oc-
curs easily, either by other bacteria or yeasts
(which can be observed by microscopy), or
worse by saprophytic leptospires (which are
morphologically similar to pathoghenic lep-
tospires) (see UNIT 12E.1). Additionally, serovar
labels might become mixed up. One may de-
mand that laboratories retain a high standard
of accurate and aseptic working, but it must be
conceded that no laboratory is perfect and that
unwittingly and unwillingly accidental con-
tamination or switching of tubes/serovars oc-
curs. Reference laboratories are no exception
in this respect, as was shown in past years with
disputes on the identity of reference strains and
the loss of strains (Anonymous, 2011). Profi-
ciency testing has shown that test results with
the MAT may differ from one laboratory to an-
other (Chappel et al., 2004). Discordant results
are partly attributable to this lack of standard-
Leptospirosis
Serodiagnosis by ization, but in addition, subjectivity in reading
the Microscopic agglutination and determination of the end-
Agglutination Test
point of titration plays a role.
12E.5.14
Supplement 32
Critical Parameters and
Troubleshooting
Antigen
The titers of agglutination tests may be
affected by the quality of the antigen sus-
pensions (i.e., cultures) and the strains used.
Borg-Petersen and Fagraeus (1949) assessed
the optimal density of the Leptospira antigen
using comparisons with the McFarland scale.
Culture densities can also be estimated by vi-
sual inspection of the suspension by dark-field
microscopy or determined by nephelometry
and counting of leptospires using a Helber or
Petroff Hauser counting chamber (cell-depth
0.02 mm). The density of the leptospires sus-
pension has a marked effect on the titer in the
MAT. A decrease of the antigen density corre-
lates with an increase of the serum titer (Car-
brey, 1960). On the contrary, increasing age
of an antigen culture causes some decrease in
its sensitivity. The agglutinability of certain
Leptospira strains, especially those which are
already maintained and sub-cultured in refer-
ence collections for decades, might also affect
the MAT results. Local strains and fresh iso-
lates for a certain serovar tend to react bet-
ter than reference strains for the same serovar.
Using local isolates as MAT antigens may be
an advantage especially when a serum sam-
ple is taken very early in the disease, when
antibody levels are still very low. Cultures
with clumps of leptospires, often referred to
as “nests” or precipitates, may be filtered, or
centrifuged lightly, as long as the final density
is not below the preferred density of 2 × 108
leptospires/ml.
If the causative serovar is not present in the
diagnostic panel, the diagnosis may be missed,
as agglutination may not be observed (unless
low titer cross-reactions with other serovars
Current Protocols in Microbiology
occur). If the local strains are not or only partly
known, it is advised to add representative
strains of other groups to a panel. Addition-
ally, the saprophytic Leptospira biflexa serovar
Patoc might be added to the panel. Patoc usu-
ally cross-agglutinates at high titers, notably
in acute serum samples, and might alert the
investigator of infections with a serovar that
is not included in the panel. Please note that
agglutinations of Patoc as such, have no diag-
nostic value.
Culturing of patient material (e.g., blood,
urine) is advised for routine diagnosis, as it
may reveal the presence of new, thus far un-
known strains/serovars. Antibiotic treatment
in the early phase of the disease may suppress
antibody production.
Some patients tend to produce antibodies
reacting with broadly reactive antigen rather
than agglutinating antibodies or vice versa.
Therefore, it is advised to combine the MAT
with an (IgM) test using broadly reactive anti-
gen (e.g., the ELISA).
Formalinized antigen
Antigens can be preserved by adding for-
malin (final concentration 0.5% v/v) and thus
be “standardized.” Formalin-preserved anti-
gens are stable for several weeks when stored
at 2° to 8°C. Formalin-preserved leptospires
tend to give more cross-reactions and thus lose
serovar-specificity, which frequently is not de-
sirable in veterinary testing. On the other hand,
formalin-preserved antigen is reactive slightly
earlier in the course of the disease than live
leptospires. Notice that with formalin-killed
antigens, the end-point titer is set at the high-
est dilution still giving agglutination. Thus, the
50% agglutination rule is not applied in this
case. An additional advantage of using killed
leptospires is a reduced risk of a laboratory
infection.
Serum specimens
Turbid sera, containing debris or contami-
nating bacteria may interfere with reading of
the MAT. Live bacteria are killed by inactiva-
tion at 56°C in a water bath for 30 min. Bacte-
ria and debris can be pelleted by centrifugation
(e.g., for 5 min at 10,000 × g, room tempera-
ture, in a microcentrifuge) and the clear super-
natant can be used.
Lipemic sera (milky appearance) also dis-
turb reading of the MAT. These sera should be
centrifuged for 15 min at 15,000 × g, 4°C, in a
microcentrifuge. In this case, the serum layer
below the lipid top layer should be used.
Current Protocols in Microbiology
Incubation: Temperature and time
Some investigators claim that agglutination
occurs within several minutes at room temper-
ature (Ryu, 1970), but most laboratories allow
an incubation period of at least 2 hr at 30°C
before reading the test. Obviously, at lower
temperatures, a longer incubation time will be
needed. For the sake of standardization, it is
advised to incubate at a fixed time and temper-
ature, preferably 2 hr at 30°C. Only when un-
avoidable, incubation can be done for a longer
period (e.g., overnight) at room temperature;
this will not risk significant changes in titer
values.
Endpoint titer
The degree of agglutination ranges from
100% when no free leptospires can be dis-
covered under the microscope to zero with
all leptospires free. The endpoint titer of the
MAT with live leptospires is the final dilu-
tion of serum at which 50% of the leptospires
are agglutinated (i.e., 50% of leptospires re-
main free as compared to the control suspen-
sion). The estimation of 50% agglutination to
50% free leptospires is not always very clear.
The transition from clumps of leptospires to
free non-agglutinated leptospires may not be
abrupt but may occur gradually over a few dilu-
tions, which make it difficult to determine the
endpoint titer. Furthermore, there is a person-
to-person, as well as a laboratory-to-laboratory
variability due to the application of differ-
ent criteria. Use of control sera promotes re-
producibility. Participation in the international
MAT proficiency testing enables quality assur-
ance of the method.
Microscope
For reading the MAT, it is highly recom-
mended to use a dark-field microscope with
a strong contrast between the leptospires and
the dark background.
An optimal dark-field microscope meets the
following criteria.
(1) A 12-V, 100-W light source
(2) A high numerical aperture dry dark
field
(3) High numerical aperture (0.4 NA) 10×
objective (Olympus, Nikon and Wild) or, al-
ternatively, the 13× or 16× (0.4 NA) objective
of Leitz
(4) A tube factor of c1.5
(5) 15× eyepieces—preferably wide field
for rapid reading
For indirect reading of agglutination, a 30-
W light source and 80× to 200× total magni-
Spirochetes
fying power will suffice.
12E.5.15
Supplement 32
 For direct reading, a dark field microscope
with a 100-W light source and a long work-
ing distance objective is mandatory. The total
magnifying power for direct reading of agglu-
tination should be 80× to 120×; otherwise de-
termination of endpoint readings may become
inaccurate.
Microtiter plate
For direct reading, the optical clarity of the
microtiter plate is important to facilitate read-
ing of agglutination. Do not use round-bottom
plates. For indirect reading, the type and clar-
ity of the microtiter plate is not critical.
Anticipated Results
The MAT is the cornerstone of a correct
sero-diagnosis (Wolff, 1954). There is prob-
ably no test presently available that surpasses
the MAT in diagnostic specificity. On the other
hand, the MAT is disappointingly insensi-
tive, reaching only 81.7% in human diagnostic
testing (Goris et al., 2011; Limmathurotsakul
et al., 2012). Sensitivity might even be lower in
veterinary testing (OIE, 2008). One can detect
antibodies to leptospires 5 to 10 days after the
onset of the disease. When receiving a blood
sample during the first week of the disease, it
is important to examine a follow up sample
after another 1 to 2 weeks to check for sero-
conversion or a rise in titer. Seroconversion or
a 4-fold rise in titer is an indication for cur-
rent leptospirosis. When only a single serum
sample is available, the diagnostic significance
depends on the magnitude of the titer within
the background of cross-reacting or persistent
agglutinating antibodies in the population. The
diagnostic significance of a given titer should
be determined by evaluation of sera from cul-
ture confirmed cases and from laboratory con-
firmed controls with diseases other than lep-
tospirosis and symptoms and signs similar to
leptospirosis (Goris et al., 2011). A negative
MAT reaction even in serial samples does not
rule out the possibility of infection, since the
patient may be infected with a serovar not
included in the battery of antigens. Further-
more, early antibiotic treatment may suppress
the development of anti-Leptospira antibodies.
In the MAT, representative serogroup antigens
should be used. The number and choice of
strains should be adapted to the aim of the in-
vestigations and preferably consist of locally
circulating strains. When a serum reacts with
Leptospirosis strains of different serovars, it is generally as-
Serodiagnosis by sumed that the infection was caused by lep-
the Microscopic tospires of the same serogroup or serovar as the
Agglutination Test
strain which gives the highest titer. However, it
12E.5.16
Supplement 32
should be noted that deduction of the infecting
serovar from diagnostic MAT results is noto-
riously unreliable (Levett, 2003). At the best,
the highest titer can be used for a presumptive
serogroup indication. Deduction of the pre-
sumptive infecting Leptospira serovar will be-
come more reliable when investigating a popu-
lation with well-established local serovars and
provided that the serum sample is taken suffi-
ciently late in the disease when antibodies have
a higher serovar specificity. Levels of cross-
reacting antibodies to heterologous serovars
in past infections will decrease more rapidly
than titers with the homologous agent. Borg-
Petersen (1949) described that it is common
that heterologous reactions are stronger than
the homologous reactions in the first weeks of
illness. The homologous reaction may even be
absent. This phenomenon is called “paradoxi-
cal reaction” (Kmety, 1958).
Serodiagnosis of leptospirosis by MAT in
itself is not particularly difficult, but the value
of the test depends on the availability of a suf-
ficiently large number of representative strains
of leptospires and on special experience. Apart
from variables inherent in the serological tests,
the antibody response of the host to leptospiral
infections is also variable. The results must be
considered for each individual case and eval-
uated in relation to all other, especially clini-
cally and epidemiological data. The presence
of agglutinating antibodies is evidence of in-
fection, present or past. If a second serum sam-
ple is tested after an interval of at least one
week, the increase of the titer probably sig-
nifies an acute infection. Observation of sim-
ilar titers with several serovars in both sam-
ples or even a reduction of some titers in
the second sample agglutination is support-
ive of another physical disorder, either caused
by an infectious agent or not, causing cross-
agglutinations. A highly strain-specific agglu-
tination, usually at low titer, supports a past
infection. Antibiotic usage may interfere with
the development of the antibody response.
In this unit, the serial dilution of patient
samples in the MAT begin at 1:20, but other
starting dilutions and dilution schemes are be-
ing used. Often, low agglutination titers are
ignored; sera are screened at a 1:50 dilution
and only further diluted in case of a positive
result. This saves time and labor but holds
the risk of a false negative outcome because
of an ignored prozone effect. The use of the
1:20 dilution-based protocol offers a number
of advantages. Firstly, although low titers may
arise from cross-reactions with other illnesses,
it also might signify the start of antibody
Current Protocols in Microbiology
production in an early acute infection, or a low
level of antibodies persisting long after infec-
tion. Prompt consultation with the clinician on
these aspects, when finding a low titer, might
help starting a timely adequate antibiotic treat-
ment and hence will be beneficial for the indi-
vidual patient. Secondly, MAT is particularly
insensitive in case of host-adapted leptospiral
infections and may be as low as 67% even
when using a minimum titer of 1:10. There-
fore, in veterinary testing, 1:20 is strongly rec-
ommended (OIE, 2008).
For epidemiological purposes, panels of
different serovars are needed, which repre-
sent the locally circulating strains causing dis-
ease. There is no guarantee that the panel is
complete or the unknown serovars are ade-
quately represented by another antigenically
similar (reference) strain if one is investigat-
ing serum samples from a previously unsur-
veyed area. New serovars, which may occur,
will of course not be represented in the panel
and as a consequence antibodies to this serovar
may be missed. The inclusion of the cross-
reacting saprophytic Leptospira serovar Patoc
in the panel is recommended to alert in case of
an infection with an unknown or unexpected
serovar. Agglutinations of Patoc as such have
no diagnostic value.
Time Considerations
Leptospires are grown in EMJH medium,
incubated for 5 to 7 days at 30°C (after a
1:10 inoculation from a full-grown culture),
and checked by dark-field microscopy for ad-
equate density and absence of contaminating
bacteria. When the strains have to be cultured
from semi-solid media or frozen stocks it will
take longer until the strain has grown to an
acceptable density (see also UNIT 12E.1).
To make a full titration of a patient serum
and adding of 28 antigens will take 15 to 30
min. The incubation is 2 to 4 hr or overnight,
and indirect reading of these plates will take 15
min. Testing more samples takes relatively less
time, as does the application of direct reading.
Direct reading however has to be done within
2 to 4 hr of incubation.
Leptospiral cultures can be used for one
week. When a panel of 28 strains has to be
maintained, sub-culturing and checking the
strains will take 2 hr once a week. It is rec-
ommended to check every two months the
strains for contamination and switches. This
will take 3 hr. Determination of the density
of the strains by counting with a Helber count-
ing chamber will take 15 min for each strain.
Measuring of OD will take 15 min for 5 strains.
Current Protocols in Microbiology
When a nephelometer is used, it will take 2 hr
for the complete procedure. Estimating by eye
can be done in one min.
Acknowledgements
We thank Dr. W.A. Ellis for his recommen-
dations for the dark-field microscope and Dr.
L.D. Smythe for providing his protocol on di-
rect reading of the MAT.
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12E.5.18
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Key References
Faine et al., 1999. See above.
This book provides comprehensive coverage of all
aspects of leptospirosis.
World Health Organization. 2003. See above.
This book provides basic information on human lep-
tospirosis and details diagnostic methods and pub-
lic health related issues.
OIE manual. See above.
This book provides comprehensive information on
veterinary leptospirosis, diagnosis and vaccine
requirements.
Current Protocols in Microbiology