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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
956
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
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
958
Table 2
SNPs and anking sequences.
Probe ID
Array position
Gene/locus
Specic SNP
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)
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.
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
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
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
961
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