Infection, Genetics and Evolution 10 (2010) 955962

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Infection, Genetics and Evolution

journal homepage: www.elsevier.com/locate/meegid

A simple and rapid molecular method for Leptospira species identication

Ahmed Ahmed a, Richard M. Anthony b, Rudy A. Hartskeerl a,*
a

WHO/FAO/OIE and National Collaborating Centre for Reference and Research on Leptospirosis, Department of Biomedical Research, Royal Tropical Institute (KIT), Meibergdreef 39,
1105 AZ Amsterdam, The Netherlands
b
Tuberculosis Research Unit, Department of Biomedical Research, Royal Tropical Institute (KIT), Meibergdreef 39, 1105 AZ Amsterdam, The Netherlands

A R T I C L E I N F O

A B S T R A C T

Article history:
Received 13 April 2010
Received in revised form 28 May 2010
Accepted 2 June 2010
Available online 12 June 2010

Serological and DNA-based classication systems only have little correlation. Currently serological and
molecular methods for characterizing Leptospira are complex and costly restricting their world-wide
distribution and use. Ligation mediated amplication combined with microarray analysis avoids many of
these drawbacks. We demonstrated that this approach used in the Check-Points (CP) assay can
successfully applied for the generic detection of Leptospira and can discriminate between saprophytic,
intermediate and pathogenic species. In addition, the CP assay could unambiguously detect strains of
seven pathogenic species and revealed discrepancies in previous speciation and culture collections. The
method provides a valuable tool adding to the molecular study of leptospires and their local and global
distribution.
2010 Elsevier B.V. All rights reserved.

Keywords:
Leptospira
Molecular
Typing
Micro-array
Ligation
Speciation

1. Introduction
Speciation of Leptospira isolates obtained from clinical material
is important for determining the clinical signicance and probable
source of infection as well as distinguishing sporadic cases from
possible outbreaks. Within the genus Leptospira species are
classied into saprophytic, intermediate and pathogenic groups.
A recent classication of the Leptospira based on genetic data
proposed 20 species, which includes the four most recently
accepted species L. licerasiae, L. wolbachii, L. wolfi and L. kmetyi
(Yasuda et al., 1987; Ramadass et al., 1992; Perolat et al., 1998;
Brenner et al., 1999; Levett et al., 2005, 2006; International
Committee on Systematics of Prokaryotes, 2008; Slack et al., 2008,
2009a; Cerqueira and Picardeau, 2009). Strains belonging to seven
of these 20 species were recognized as the main causative agents of
leptospirosis (Yasuda et al., 1987; Ramadass et al., 1992). The
pathogenic status of intermediate species is unclear or disputable
(Perolat et al., 1998; Matthias et al., 2008), although some
intermediate leptospires seem to be capable of colonizing the
kidneys of host animals while other species contain both
pathogenic and saprophytic leptospires (Brenner et al., 1999;
Victoria et al., 2008).

* Corresponding author.
E-mail addresses: ahmed_aboagla@hotmail.com, r.hartskeerl@kit.nl
(R.A. Hartskeerl).
1567-1348/$ see front matter 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.meegid.2010.06.002

Serotyping of Leptospira isolates is a specialized and complex

procedure that can only be performed at reference centres on
cultures and does not always provide unambiguous identications.
For this reason a number of molecular techniques have been
introduced as alternatives to or to complement serotyping
methods. These include whole genome DNADNA hybridization
analysis (Yasuda et al., 1987; Ramadass et al., 1992; Perolat et al.,
1998; Brenner et al., 1999), randomly amplied polymorphic DNA
(RAPD) ngerprinting (Roy et al., 2004), arbitrarily primed PCR
(AP-PCR) (Ralph et al., 1993), pulsed eld gel electrophoresis
(PFGE) (Herrmann et al., 1992), restriction fragment length
polymorphism (RFLP) analysis (Zuerner et al., 1993), insertion
sequences bacterial typing methods (IS) (Zuerner, 1994; Zuerner
et al., 1995), detection of variable number of tandem repeats
(VNTR) (Majed et al., 2005), rrs sequencing (Morey et al., 2006;
Cerqueira et al., 2010), sequencing of specic genes or gene
fragments including rpoB (La et al., 2006), gyrB (Slack et al., 2006),
secY (Victoria et al., 2008; Ahmed et al., 2009), ligB (Cerqueira et al.,
2009) and recently by multiple locus sequence typing (MLST)
(Ahmed et al., 2006; Thaipadungpanit et al., 2007; Leon et al.,
2010). Most of these techniques clearly differentiate leptospires at
the species level. Furthermore these methods may provide an
appropriate tool for the sub-classication of difcult to distinguish
serovars from the same serogroup but belonging to different
species. Molecular methods are particularly useful when serological methods for discrimination might fail such as in case of
serogroup Grippotyphosa (Hartskeerl et al., 2004) and when the

956

A. Ahmed et al. / Infection, Genetics and Evolution 10 (2010) 955962

agglutination pattern is indistinguishable in the instance of

serogroup Sejroe strains Hardjoprajitno and Lely 607 belonging
to L. interrogans and L. borgpetersenii, respectively (van Eys et al.,
1991). However, the drawbacks of these methods excluding VNTR
and sequence based typing are their lack of directly generating
electronic portable data and the difcultly in establishing a central
database, along with their low reproducibility, the need of viable
cultures or their labour intensive and time consuming and costly
nature. Recently, oligonucleotide-based DNA microarray technology has been combined with probe ligation technology resulting in
a simple, fast, specic, portable and powerful multiplex technique
for bacteria typing (Wattiau et al., 2008). The Check-Points (CP)
system based on detection of single nucleotide polymorphisms
(SNPs) allows single-tube processing of samples and requires only
general molecular biological skills. Moreover, no expensive
facilities are needed. In this study we developed species-specic
probes based on previously dened and newly identied SNPs at
distinct loci, which successfully discriminate between different
Leptospira species using the CP technique.
2. Materials and methods
2.1. Leptospira strains
65 Leptospira strains from pathogenic, non-pathogenic and
intermediate Leptospira spp. (Table 1) were used in this study.
Leptospira strains were obtained from the collection of the WHO/
FAO/OIE and National Leptospirosis Reference Centre in Amsterdam, The Netherlands.
2.2. DNA extractions
Leptospira strains were propagated at 30 8C in EMJH liquid
media according to Ellinghausen and McCullough (1965) as
modied by Johnson and Harris (1967). The number of bacteria
per ml was determined by counting in a Helber bacteria chamber
(Weber Scientic International, West Sussex BN15 8TN England)
according to the standard protocol. All genomic DNA from
leptospires in culture medium were extracted, puried and eluted
using the QIAamp DNA extraction kit according to the manufacturers instructions (Qiagen, GmbH, D-40724 Hilden, Germany).
The quantity of Leptospira genomic DNA was estimated by
measuring the absorbance of DNA using the Spectrophotometer
ND-1000 Nanodrop (3411 Silverside Rd, Bancroft Building,
Wilmington, DE 19810, USA).
2.3. The technique and the analysis
The analysis was performed according to the instructions of the
manufacturer (Check-Points, Wageningen, The Netherlands). The
principle, equipment and test procedure of the CP method has been
detailed previously (Wattiau et al., 2008). Briey the system uses a
multiplex ligation detection reaction (LDR) technique that
produces circular DNA molecules after a ligation step. The circular
molecules are then subjected to amplication using a universal
single pair biotin labelled primer that composes of sequences
complement to both 50 and 30 end of the probes. The LDR probe
includes a ZIP code, which is complementary to a unique
oligonucleotide (cZIP) immobilized on the microarray on the
bottom of an array tube. The PCR products are hybridized to the
immobilized probes in the array tube, followed by several wash
steps to remove unbound products. Subsequently, the hybridization signals are detected and analysed by the Check-Points tube
(CPT) reader camera connected to a standard computer using
custom made software (Check-Made software, Check-Points,
Wageningen, The Netherlands).

2.4. SNPs selection and probe design

Probes for the CP system were designed on basis of single
nucleotide polymorphisms (SNPs) selected to be specic for
distinct species. To identify suitable SNPs, sequences of various
loci were retrieved from the GenBank sequence database (http://
www.ncbi.nlm.nih.gov) or other sources (Ahmed et al., 2006) as
listed in Table 2. SNPs were identied and selected following
multi-alignment analysis. The SNPs and anking sequences are
shown in Table 2. The universal primer set and ZIP sequences are a
design of Check-Points.
2.5. Sequencing, phylogenetic analysis and serotyping
Once the strains from our collection had been analysed using
the (CP) assay all discrepant results were reanalyzed by sequencing
the G1/G2 fragment in secY gene according to Victoria et al. (2008).
Serovars were determined by the microscopic agglutination test
(MAT) using a panel of 43 rabbit anti-Leptospira reference antisera,
representative for 26 pathogenic and three saprophytic serogroups
(Kmety and Dikken, 1993; World Health Organization, 2003).
Retyping of the Leptospira strains with discrepant results at the
serovar level was performed by MAT using panels of monoclonal
antibodies (mAbs), that characteristically agglutinate serovars
from the serogroups Australis (F81C1, F81C3, F81C4, F81C5, F81C6,
F81C8, F90C4, F90C5, F90C6, F90C8, F90C9, F90C12, F132C2 and
F132C7) (Cinco et al., 1989) and (Gravekamp et al., 1991),
Cynopteri (F64C2, F64C6, F64C7, F64C8, F64C10, F65C3, F69C2,
F69C8, F69C9, F69C10, F69C11, F69C12, F69C14 and F69C15) (Alex
et al., 1993) and Javanica (F12C3, F20C3, F20C4, F70C20, F98C4,
F98C5, F98C8, F98C12, F98C17, F98C19 and F98C20) (Alex et al.,
1993). In case of serogroup Manhao, serovars were identied using
specic rabbit antiserum according to Kmety and Dikken (1993).
3. Results
Initially 25 SNPs were selected as a basis for probe design. Five
SNPs sequence were excluded before synthesis because of their
inadequate technical properties including the nature of the SNP,
adjacent sequences or probe annealing properties. The 20
remaining CP probes were designed to hybridize and produce
positive signal with particular species or species groups as follows:
Probe 1 was designed to detect all Leptospira species and probe 3 to
detect only saprophytic species.
Probe 2 was designed to detect all pathogenic species with the
exception of L. borgpetersenii, L. alexanderi and L. weilii. Probes 5, 14,
16 and 17 were selected to specically hybridize to L. interrogans.
Probes 7, 20, 21 were selected to be specic for L. borgpetersenii and
probes 11, 12, 25 specic for L. weilii; probes 6 and 22 for L.
kirschneri; probes 8 and 15 for L. santarosai; and probes 9, 10 and 13
for L. noguchii, L. alexanderi and L. meyeri, respectively (Table 2).
Strains VAR 010, BUT 6 and 10 of intermediate species L. licerasiae,
L. fainei and L. inadai, respectively were expected to produce a
signal only with probe 1 as no appropriate sequences were
available to design specic probes targeting such species.
The predicted specicity of the SNPs sequences was based on an
in silico check with the blast system (National Center for
Biotechnology Information, 2010, http://blast.ncbi.nlm.nih.gov/
blast/). The sensitivity of the assay was estimated at less than 50
genome copies of cultured leptospires per reaction. The CP system
containing all 20 probes was tested for specicity and functionality
against a panel of 65 reference strains belonging to 13 species
including pathogenic, intermediate and saprophytic Leptospira
species (Table 1). Example results of the hybridization patterns
obtained with L. interrogans and L. kirschneri DNA are shown in
Fig. 1. The results of all species in the test panel are summarized in

A. Ahmed et al. / Infection, Genetics and Evolution 10 (2010) 955962

957

Table 1
Leptospira strains used in this study.
No.

Strain

Serovar

Serogroup

Species

Pathogenic status

Reference

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65

Akiyami A
H6
Hardjoprajitno
Hond HC
Hond Utrecht IV
Jez Bratislava
Kantorowic
Lai
LT 398
M 20
Pomona
Rachmat
RGA
RM 2
Swart
Szwajizak
1051
5621
Dyster
Moskva V
Musa
Ndambari
RM1
Wumalasena
507
9160
1161 U
CZ 214 K
M7
Jules
Lely 607
M 84
Mus 127
Nijenga
Perepelicin
Poi
Sari
Sorex Jalna
Veldrat Batavia 46
1019
1342 K
1413 U
Aa 3
HS-616
M 13
M4
Rr 5
TRVL 3214
TRVL 34056
Celledoni
Cox
L105
M 6906
Sarmin
A 23
A 85
ICF
Soa 874
BUT 6
10
VAR 010
Veldrat Semarang 173
WaZ Holland
CH 11
Patoc I

Autumnalis
Malaya
Hardjo
Medanensis
Canicola
Bratislava
Icterohaemorrhagiae
Lai
Manilae
Copenhageni
Pomona
Rachmati
Icterohaemorrhagiae
Muelleri
Bataviae
Szwajizak
Bim
Mozdok
Valbuzzi
Grippotyphosa
Ramisi
Ndambari
Sokoine
Ratnapura
Rushan
Carimagua
Proechimys
Panama
Huallaga
Jules
Hardjo-bovis
Sejroe
Ballum
Kenya
Tarassovi
Poi
Mini
Sorexjalna
Javanica
Varela
Shermani
Gorgas
Fuminense
Alexi
Tingomaria
Huanuco
Rio
Tabaquite
Trinidad
Celledoni
Coxi
Qingshui (Manhao 2)
Mengdeng
Sarmin
Manzhuang
Mengla
Ranarum
Soa
Hurstbridge
Lyme
Varillal
Semaranga
Holland
Andamana
Patoc

Autumnalis
Canicola
Sejroe
Sejroe
Canicola
Australis
Icterohaemorrhagiae
Icterohaemorrhagiae
Pyrogenes
Icterohaemorrhagiae
Pomona
Autumnalis
Icterohaemorrhagiae
Grippotyphosa
Bataviae
Mini
Autumnalis
Pomona
Grippotyphosa
Grippotyphosa
Australis
Icterohaemorrhagiae
Icterohaemorrhagiae
Grippotyphosa
Australis
Shermani
Pomona
Panama
Djasiman
Hebdomadis
Sejroe
Sejroe
Ballum
Ballum
Tarassovi
Javanica
Mini
Javanica Sorex Jalna
Javanica
Pyrogenes
Shermani
Sejroe
Javanica
Pyrogenes
Cynopteri
Grippotyphosa
Sarmin
Mini
Sejroe
Celledoni
Javanica
Manhao
Celledoni
Sarmin
Hebdomadis
Javanica
Ranarum
Javanica
Hurstbridge
Lyme
Hurstbridge
Semaranga
Holland
Andamana
Semaranga

L. interrogans
L. interrogans
L. interrogans
L. interrogans
L. interrogans
L. interrogans
L. interrogans
L. interrogans
L. interrogans
L. interrogans
L. interrogans
L. interrogans
L. interrogans
L. interrogans
L. interrogans
L. interrogans
L. kirschneri
L. kirschneri
L. kirschneri
L. kirschneri
L. kirschneri
L. kirschneri
L. kirschneri
L. kirschneri
L. noguchi
L. noguchii
L. noguchii
L. noguchii
L. noguchii
L. borgpetersenii
L. borgpetersenii
L. borgpetersenii
L. borgpetersenii
L. borgpetersenii
L. borgpetersenii
L. borgpetersenii
L. borgpetersenii
L. borgpetersenii
L. borgpetersenii
L. santarosai
L. santarosai
L. santarosai
L. santarosai
L. santarosai
L. santarosai
L. santarosai
L. santarosai
L. santarosai
L. santarosai
L. weilii
L. weilii
L. weilii
L. weilii
L. weilii
L. alexanderi
L. alexanderi
L. meyeri
L. meyeri
L. fainei
L. inadai
L. licerasiae
L. meyeri
genomospecies 3
L. biexa
L. biexa

Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Intermediate
Intermediate
Intermediate
Non-pathogenic
Non-pathogenic
Non-pathogenic
Non-pathogenic

Brenner et al. (1999)

Victoria et al. (2008)
Brenner et al. (1999)
Brenner et al. (1999)
Brenner et al. (1999)
Brenner et al. (1999)
Gravekamp et al. (1993)
Brenner et al. (1999)
Brenner et al. (1999)
Brenner et al. (1999)
Brenner et al. (1999)
Brenner et al. (1999)
Brenner et al. (1999)
Hartskeerl et al. (2004)
Dutch clinical isolate
Brenner et al. (1999)
Brenner et al. (1999)
Brenner et al. (1999)
Brenner et al. (1999)
Brenner et al. (1999)
Brenner et al. (1999)
Brenner et al. (1999)
Mgode et al. (2006)
Brenner et al. (1999)
Brenner et al. (1999)
Brenner et al. (1999)
Brenner et al. (1999)
Brenner et al. (1999)
Brenner et al. (1999)
Brenner et al. (1999)
Victoria et al. (2008)
Brenner et al. (1999)
Brenner et al. (1999)
Brenner et al. (1999)
Brenner et al. (1999)
Brenner et al. (1999)
Brenner et al. (1999)
Brenner et al. (1999)
Brenner et al. (1999)
Brenner et al. (1999)
Brenner et al. (1999)
Brenner et al. (1999)
Brenner et al. (1999)
Brenner et al. (1999)
Brenner et al. (1999)
Mgode et al. (2006)
Brenner et al. (1999)
Brenner et al. (1999)
Brenner et al. (1999)
Brenner et al. (1999)
Brenner et al. (1999)
Brenner et al. (1999)
Brenner et al. (1999)
Brenner et al. (1999)
Brenner et al. (1999)
Brenner et al. (1999)
Brenner et al. (1999)
Brenner et al. (1999)
Perolat et al. (1998)
Brenner et al. (1999)
Matthias et al. (2008)
Victoria et al. (2008)
Brenner et al. (1999)
Brenner et al. (1999)
Brenner et al. (1999)

Table 3. As expected, probe 1, detected all pathogenic, intermediate and saprophytic strains in the panel. The pathogen-specic
probe 2 detected all pathogenic strains except L. borgpetersenii, L.
alexanderi and L. weilii as predicted. None of the intermediate or
saprophytic tested strains gave a positive signal with probe 2.
Probe 3 produced a signal with all saprophytic strains but not with
pathogenic or intermediate strains. Therefore, as anticipated none

of the intermediate strains produced a signal with other probes

than probe 1.
Out of four probes 5, 14, 16 and 17 designed to target L.
interrogans, only probes 16 and 17 consistently generated a signal
with all 16 strains of this species tested; probe 14 detected only six
strains and probe 5 reacted with none of the strains. Of the L.
borgpetersenii-specic (probes 7, 20, and 21), probe 21 gave a signal

A. Ahmed et al. / Infection, Genetics and Evolution 10 (2010) 955962

958
Table 2
SNPs and anking sequences.
Probe ID

Array position

Gene/locus

Specic SNP

SNPs in upper case and anking sequences in lower case

Accession/Reference

Probe
Probe
Probe
Probe
Probe
Probe
Probe
Probe
Probe
Probe
Probe
Probe
Probe
Probe
Probe
Probe
Probe
Probe
Probe
Probe
Probe
Probe
Probe
Probe
Probe

1
2
3
Not
5
6
9
10
11
12
17
18
19
20
21
22
23
Not
Not
24
25
26
Not
Not
27

rrs
rrs
rrs
secY
IcdaA
secY
secY
lipL41
adk
secY
secY
secY
secY
adk
lipL32
secY
AE010300
AE010300
rplC
CP000350
secY
lipL48
rplC
rplD
rplD

All species
Most pathogenic spp.
Saprophytic spp.
L. interrogans
L. interrogans
L. kirschneri
L. borgpetersenii
L. santarosai
L. noguchii
L. alexanderi
L. weilii
L. weilii
L. meyeri
L. interrogans
L. santarosai
L. interrogans
L. interrogans
L. interrogans
L. alexanderi
L. borgpetersenii
L. borgpetersenii
L. kirschneri
L. santarosai
L. santarosai
L. weilii

agagtgaaactcaaaggaattgacgggggTccgcacaagcggtggagca
taaagatttattgctcggagatgagcccgcgTccgattagctagttggtgaggt
gcagggattggttaaagcagcaatgcgctttTagatgggtccatggctgattag
gtatcatgcgttattttatttcgtaatttataccGctttaattgtattctttgcttacttttat
gactttgaaggagccggtgtgatcatgggGcaacataacttggataagtcgat
ggcgccaatgtgatgccgatcatttttgcttcCtctttgattttgtttccgcaaacga
gaacggtgcaaacgtaatgccgatcatcttCgcttcttccttgatcctgtttccac
ataaggaatcttattacaaacttaccgacctTagcaaacgcgccgatattctta
tgataaagcgatcaatcttcaggttccggatAcagaactcttaaaaagactact
aagccagtccattccttttaaagtgaatggGgcaaacgtaatgccgatcatctt
ttgtcttccagtagcgaacagtgggctggCtgggcgatcatcatggactttttca
gtggttgtcttccagcagcgaacagtgggcCggttgggcgatcattatggactt
atagcagtcaggaatgggcgggttgggcCgtgattatggacttcttcaatccat
gctttagatacacttttaaaaaatgaaggCaagtctattgataaagcgattaat
cataaagccaggacaagcgccggatggCttagtcgacggaaacaaaaaa
aaatggtcggaagaaaaatggttcaggcGaagagtcaatctattcctttcaaa
gaatctgggagaatgactgaatctcctttgCcaacacctaacgcacgtaaagc
gagaaacatataaccggtagaatctgttCttccgtccttaattatcctttatttcctt
gAaaagaatcttgtgttcgtgagcgggtcCgttcccggcacggcaaacaca
tatattgtgatcctccctataaggattcacaGcctatactgtacggggctcaatc
tggtaccatgcattgttctactatataatctatActtctttgattatctttttcgcatacttt
aagtggtactactttagatcacgttcaagtCcacagacatttggacgacggtat
gacacggttctcgttttcacaggcatccCggatctatgggagcgaactcaactc
cacagaagtattctaaagaaggaaaGctgatctcagaaatcgaacttccttc
gggtggacacggatctcgttttcacagacatccGggttccatgggagcgaact

EU159693
EF536982
AY631897
S81410
Ahmed et al.
EU358044
EU358070
AY461965
Ahmed et al.
EU365964
EU358065
EU358009
EU365965
Ahmed et al.
Ahmed et al.
EU358053
AE010300
AE010300
EU365946
CP000350
EU358070
AF394742
EU365938
EU365938
EU365942

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25

included

included
included

included
included

with all 10 strains tested, while probe 7 was only positive for 2
strains and probe 20 reacted with none of the strains. Four of the
ve strains of L. weilii reacted with at least one of the probes 11, 12,
25 (probe 11 one strain, probe 12 three strains and probe 25 two
strains). Surprisingly L. weilii strain L105 did not react with any of
these probes but instead gave a signal with the L. alexanderispecic probe 10. Probe 6 selected to target L. kirschneri, generated
a signal with all eight strains in the test panel while probe 22 only
detected 4 strains. The L. santarosai probe 8 detected 4 of the 10
strains whereas probe 15 detected 9 strains only failing to detect M
13, which reacted with probe 9. Probe 9 selected to target L. noguchi
[(Fig._1)TD$IG]only failed to detect strain 507, which gave a positive signal with

(2006)

(2006)

(2006)
(2006)

the L. meyeri probe 13. Probe 10 produced a signal with both L.

alexanderi strains in the test panel. Probe 13 for L. meyeri detected
its pathogenic strain ICF but as expected, not saprophytic strain
Veldrat Semarang 173 and did not detect strain Soa 874 that
reacted with L. borgpetersenii probe 21.
All unexpected results, i.e. L105 (supposed to belong to L. weilii),
M 13 (supposedly L. santarosai), 507 (supposedly L. noguchi), and
Soa 874 (supposedly L. meyeri) were checked both by resequencing the secY region of extracted DNA and by sequencing
newly extracted DNA from freshly cultures strains from the
reference collection in addition to serotyping as mentioned earlier
in the materials and methods section. On the basis of resequencing the CP-based identity as L. alexanderi, L. noguchii, L.
meyeri and L. borgpetersenii, for strains L105, M 13, 507 and Soa
874, respectively was conrmed. Subsequent serotyping categorized these strains as serovar Manhao 2, Huallaga, Rushan and Soa
respectively. Thus the discrepant results do not appear to be due to
errors in the assay but obviously reected historical inaccuracies in
the characterisation of the tested panel.
4. Discussion

Fig. 1. Images of the Check-Points tube (CPT) hybridization detected by the CPT
reader camera and probes position in the system. (a) Probes position in the CPT, G:
alignment control; H: hybridization control. (b and c) Hybridization patterns
obtained with L. interrogans and L. kirschneri DNA respectively.

Current characterization methods for Leptospira have many

drawbacks. Serological classication with the serovar as a basic
taxon is tedious, laborious and restricted to three international
reference centres. Thus, the shipment of viable pathogens which
must comply with increasingly stringent international shipment
regulations at high cost is required resulting in considerable delay
and additional costs connected to serotyping. There are several
molecular techniques for DNA-based classication, including,
PFGE, AP-PCR, RFLP, VNTR, and MLST (Herrmann et al., 1992;
Ralph et al., 1993; Zuerner et al., 1993; Majed et al., 2005; Ahmed
et al., 2006), which either have limited applicability (VNTR), low
reproducibility (AP-PCR), require large amounts of cultured
leptospires (PFGE, RFLP), or require expensive investment in
equipment and expertise in the application of the methods (PFGE,
MLST). Additionally, only MLST produces unequivocal digital data
that can be easily compared between laboratories. Costs,
complexity and shipment requirements also hamper the wide
distribution of such techniques. The CP assay addresses many of

Table 3
Hybridization results of different Leptospira strains tested with the Check-Points system.
No.
1

Probe
01

A
23
A 85
Patoc I
CH 11
Sari
Jules
Mus 127
Veldrat Batavia 46
Poi
Sorex Jalna
Perepelicin
Nijenga
Lely 607
M 84
BUT 6
WaZ Holland
10
Szwajizak
Jez Bratislava
Swart
H6
M 20
RGA
Pomona
RM 2
LT 398
Hardjoprajitno
Lai
Hond HC
Hond Utrecht IV
Kantorowic
Rachmat
Akiyami A
Wumalasena
5621
RM1
Dyster
1051
Moskva V
Musa
Ndambari
Soa 874
Veldrat
Semarang 173
ICF
M7
9160
507
CZ 214 K
1161 U
TRVL 3214
TRVL 34056
M 13
M4

L. alexanderi

L. alexanderi
L. biexa
L. biexa
L. borgpetersenii
L. borgpetersenii
L. borgpetersenii
L. borgpetersenii
L. borgpetersenii
L. borgpetersenii
L. borgpetersenii
L. borgpetersenii
L. borgpetersenii
L. borgpetersenii
L. fainei
genomospecies 3
L. inadai
L. interrogans
L. interrogans
L. interrogans
L. interrogans
L. interrogans
L. interrogans
L. interrogans
L. interrogans
L. interrogans
L. interrogans
L. interrogans
L. interrogans
L. interrogans
L. interrogans
L. interrogans
L. interrogans
L. kirschneri
L. kirschneri
L. kirschneri
L. kirschneri
L. kirschneri
L. kirschneri
L. kirschneri
L. kirschneri
L. meyeri
L. meyeri

X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X

L.
L.
L.
L.
L.
L.
L.
L.
L.
L.

X
X
X
X
X
X
X
X
X
X

meyeri
noguchii
noguchii
noguchi
noguchii
noguchii
santarosai
santarosai
santarosai
santarosai

Probe
02

Probe
03

Probe
05

Probe
06

Probe
07

Probe
08

Probe
09

Probe
10

Probe
11

Probe
12

Probe
13

Probe
14

Probe
15

Probe
16

Probe
17

Probe
20

Probe
21

Probe
22

Probe
25

X
X
X
X
X
X
X
X
X
X
X
X
X
X

X
X

X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X

X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X

X
X
X
X
X
X

X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X

X
X
X
X
X
X
X
X

X
X
X
X
X

X
X
X
X
X
X
X
X
X
X
X

X
X
X
X
X
X
X
X
X
X

959

44
45
46
47
48
49
50
51
52
53

Species

A. Ahmed et al. / Infection, Genetics and Evolution 10 (2010) 955962

2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43

Strain

A. Ahmed et al. / Infection, Genetics and Evolution 10 (2010) 955962

X
X
X
X
X
X

X
X

X
X

X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
santarosai
santarosai
santarosai
santarosai
santarosai
santarosai
weilii
weilii
weilii
weilii
weilii
licerasiae
1019
Aa 3
1413 U
Rr 5
1342 K
HS-616
Sarmin
Celledoni
L105
Cox
M 6906
VAR 010
54
55
56
57
58
59
60
61
62
63
64
65

L.
L.
L.
L.
L.
L.
L.
L.
L.
L.
L.
L.

Strain
No.

Table 3 (Continued )

Species

Probe
01

Probe
02

Probe
03

Probe
05

Probe
06

Probe
07

Probe
08

Probe
09

Probe
10

Probe
11

Probe
12

Probe
13

Probe
14

Probe
15

Probe
16

Probe
17

Probe
20

Probe
21

Probe
22

X
X

Probe
25

960

these shortcomings. The CP system is a multiplex ligase-depended

probe amplication (MLPA) in which the distinct products are
detected via hybridization to specic probes bound to a solid
support (Bergval et al., 2008; Wattiau et al., 2008). The method is
simple and rapid to execute and does not require sophisticated
equipment keeping labour and investment costs low.
To investigate the potential of the CP system we designed an
array to rapidly distinguishing pathogenic Leptospira species. The
array consisted of 20 probes suitable for the specic detection of
the seven major pathogenic Leptospira species. The system has a
good sensitivity on DNA extracted from leptospires culture and is
applicable on clinical and environmental materials. However,
residual inhibitors present in such materials may reduce the
sensitivity (Ahmed et al., 2009). The CP system was evaluated with
65 reference strains classied into 13 species, which included
species with incomplete or no genetic information in the database
such as L. licerasiae strain VAR 010, Genomospecies 3 strain WaZ
Holland and L. fainei strain BUT 6. Three rrs-derived probes were
designed for control purposes targeting specic groups of species,
one (probe 1) for all strains in the genus Leptospira, one (probe 2)
for most pathogenic species and one (probe 3) for all saprophytic
species. The generic and saprophytic specic probes 1 and 3
consistently produced signals for the strains included in the study
and the pathogenic probe 2 showed a high specicity in detecting
pathogenic species with the exception of three species (Table 3).
However, when combined with the other species-specic probes
these three species are identied and thus the system can clearly
differentiate between pathogenic and saprophytic species.
The phylogenetic analysis of rrs sequences of intermediate
species forms a separate clade between the pathogenic and
saprophytic Leptospira (Morey et al., 2006). Our observation that
the intermediate strains L. inadai strain 10, L. fainei strain BUT 6 and
L. licerasiae strain VAR 010 did not produce a signal with either rrs
probe 2 or probe 3 is consistent with the presence of these species
in a separate clade as previously suggested (Morey et al., 2006).
For most species we designed several probes and tested them
against a reference panel. Highly variable performance was
observed for various probes; from 100% sensitivity for some
probes (e.g. 16, 17 and 21) to 0% for probes 5 and 20. There are
several reasons for this varying performance (i) uniform reaction
conditions for the multiplex system at ligation, amplication and
hybridization might not be optimal for all probes and (ii)
sequences selected for identifying SNPs might have contained
errors. While inadequate reaction conditions might be an
acceptable explanation for probes only generating a signal for
part of the strains in the respective species, the complete absence
of a signal for probe 5 and 20 is more probably due to an absence of
the species-specic SNPs because of sequence errors. Clearly
failing probes have to be excluded from the assay but in this
multiplex assay partly successful probes remain useful and can be
retained and used in combination to identify specic clades. The
latter is particularly true for probes 11, 12 and 25, which, only in
combination could detect all strains of L. weilii. It is conceivable
that these variations may ultimately be in themselves informative
but more strains will need to be screened before the very
implications of this variation can be determined.
In addition to the moderate sensitivity of some probes, others
showed an apparent lack of specicity by reacting to DNA of strains
from different species. To rule out errors in the original
identications of the DNA reference samples, we re-tested the
DNA originally used in the CP assay and DNA newly extracted from
the respective reference cultures in our collection by sequencing.
We did not identify any discrepancy between each pair of DNA
samples and thus conrmed the CP system results were correct.
Serotyping conrmed strains 507, Soa 874, and L105 as serovar
Rushan, Soa and Manhao 2 (alternative nomination is Qingshui),

A. Ahmed et al. / Infection, Genetics and Evolution 10 (2010) 955962

respectively but classied the assumed serovar Tingomaria strain

M 13 (L. santarosai) as serovar Huallaga that belongs to L. noguchii.
Indeed, previous speciation of serovar Manhao 2, strain L105 is
ambiguous. Brenner et al. (1999) denoted this strain both as L.
weilii and as non-classiable. In addition, Kmety and Dikken (1993)
indicated that the strain is serologically indistinguishable from the
serovars Manhao 1 (alternative name is Lushui) strain L 70 and
Manhao 3, strain L 60, which both belong to species L. alexanderi
(Brenner et al., 1999). The latter is in accordance with our nding
and considering the doubt in speciation (Brenner et al., 1999) we
argue that serovar Mianhao strain L105 belongs to the species L.
alexanderi. Incorrect speciation in previous studies has been
reported before (Victoria et al., 2008; Letocart et al., 1997; Slack
et al., 2009b) and our observation of serovars Rushan and Soa in
other species than previously reported might well be another
example of such errors. However, we will need to investigate the
corresponding strains in other Leptospira collections to unambiguously rule out a mislabelling of strains at our centre, as apparently
occurred with strain M 13. The apparent mislabelling of this strain
in our collection as revealed by the CP assays emphasizes the
complexity of performing and interpreting the results of serological techniques and supporting the reliability of the array method.
In conclusion the CP system developed in this study proved to
be more accurate and technically less demanding than the
standard serological typing methods such as monoclonal antibody typing and the cross agglutinin absorption test. This system
does not require expensive or complex equipment and is a
standardized method that could be performed in any molecular
biological laboratory and produces unequivocal results that can
easily be digitized and compared. The use of dened SNPs in the
assay allows re-checking of the results by alternative methods
such as sequencing which is important for quality control. With
the expected availability of many more Leptospira sequences in
the near future, the CP will have the potential to be further
developed into a strain-specic assay. Such an assay would allow
molecular studies to be performed simply and rapidly for the
purpose of local and global epidemiology. We believe this method
has the potential to become a standard typing method and allow
Leptospira typing to be carried out in many more laboratories than
is currently feasible.
Acknowledgments
The authors wish to thank the staff of the Check-Points,
Wageningen, The Netherlands for their contribution in the design
of the CP system and the execution of part of the experiments. This
work was supported by the EC grant ICA4-CT-2001-10086.
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