Veterinary Research Communications, 31 (2007) 509511

DOI: 10.1007/s11259-007-3535-1


C Springer 2007

Short Communication
Phylogenetic Analysis of LipL32 Gene Sequence of Different
Pathogenic Serovars of Leptospira
Sohini Dey1,2, , C. Madhan Mohan1,3 , P. Ramadass1 and K. Nachimuthu1
1
Department of Animal Biotechnology, Madras Veterinary College, Chennai; 2 Division of
Biological Products, 3 Division of Veterinary Biotechnology, Indian Veterinary Research
Institute, Izatnagar, Bareilly, India

Correspondence: E-mail: sohinimadhan@yahoo.com

Keywords: Leptospira, LipL32, sequence, phylogenetic tree

Abbreviations: MEGA, Molecular Evolutionary Genetic Analysis; NJ, neighbour joining; PCR, polymerase
chain reaction

Leptospirosis is a zoonosis of worldwide significance caused by infection with pathogenic

leptospiral species (Levett, 2001). The leptospiral outer membrane proteins may be important in the diagnosis of leptospirosis. The major outer membrane protein, LipL32 is a
prominent immunogen in human (Haake et al., 2000) and canine (Dey et al., 2004) leptospirosis. The serovar pomona strain Pomona and serovar icterohaemorrhagiae strain RGA
of Leptospira interrogans were chosen for the cloning and sequencing of the LipL32 outer
membrane protein gene. In this study, the forward and reverse primers for amplifying 665
bp of the LipL32 gene were based on the LipL32 gene sequence of L. kirschneri species
(Haake et al., 2000). The restriction enzyme sites for XbaI and KpnI were incorporated at
the 5 ends of the primers for directional cloning in the pBluescript KS(+) cloning vector
(Stratagene, La Jolla, CA, USA). The primers were able to amplify only the pathogenic
serovars and not the non-pathogenic serovars of Leptospira. The PCR-amplified LipL32
genes of Pomona and RGA strains were cloned into pBluescript KS(+) cloning vector.
The presence of insert was checked by colony PCR using vector specific primer T3 as the
forward primer and the reverse gene-specific primer. The restriction enzyme analysis with
XbaI and KpnI also confirmed the presence of insert in the recombinant plasmid pKSP4.
The portion of the structural gene sequences of LipL32 of the strains Pomona and RGA
were submitted to GenBank under accession numbers AY223718 and AY423075, respectively. The nucleotide sequences of LipL32 of these two serovars were stored along with
the sequence of the other available pathogenic serovars in FASTA format and used as the
input file for the program CLUSTAL X 1.8. The MegAlign package was used to align all the
sequences in the worktable using the CLUSTAL V method developed by Higgins and Sharp
(1989). MEGA software V1.02 was used to identify the amino acid variations between the
serovars. After multiple alignments, a neighbour-joining method (Saitou and Nei, 1987)
was employed to reconstruct phylogeny for the putative alignment.
The neighbour-joining (NJ) tree file produced three groups (Figure 1). Group I consisted
of L. interrogans and L. kirschneri species; group II was represented by L. borgpetersenii

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Figure 1. Phylogenetic tree based on the nucleotide sequence alignment of the LipL32 gene of different
serovars of Leptospira

serovar hardjo strain 203 (AF 181554); and group III included L. santarosai serovar tropica
strain CA 299 (AF 181555). Group I, consisting of serovar grippotyphosa strain RM 52 (AF
121192) of L. kirschneri species showed relatedness with L. interrogans species, namely
serovars pomona (AF 181553), australis (AB 094437), canicola (AB 094434), icterohaemorrhagiae (AY 423075) and autumnalis (AB 094435). The Pomona and RZ II strains of
serovar pomona showed no difference. The Hond Utrecht IV strain of serovar canicola and
Akiyami C strain of serovar australis again showed no difference; the Akiyami A strain
of serovar autumnalis and RGA strain of serovar icterohaemorrhagiae showed similarity.
Comparison of the deduced nucleotide and amino acid sequences of the LipL32 variants
revealed an average DNA sequence identity of 97.42% (Table I) and amino acid sequence
identity of 98.06% (range, 96.7199.06%). Most of the sequence polymorphisms detected
(39 of 56) were silent.
TABLE I
Nucleotide similaritya and divergenceb of the sequences of serovars of Leptospira
L. autum. L. borg. L. canicola L. ictero. L. kirsch. RM52 L. sant. L. australis
L. autum.
L. borg.
L. canicola
L. ictero.
L. kirsch. RM52
L. sant.
L. australis
L. pomona RZ11
L. pomona

95.5
4.5

0.2
4.4
0.6
4.9
1.3
4.2
6.9
5.9
0.2
4.4
0.5
4.4
1.1
5
L. autum. L. borg.

99.8
95.6

0.8
1.1
6.8
0
0.3
0.9
L. canicola

99.1
95
98.9

1.6
7.3
0.8
0.8
1.4
L. ictero.

98.7
95.8
98.9
98.3

7.3
1.1
1.1
1.7
L. kirsch. RM52

93.1
99.8
94.2
95.6
93.3
100
92.6
98.9
92.8
98.9

93.3
6.8
6.8
0.3
7.5
0.9
L. sant. L. australis

a Percentage similarity in upper (right) triangle

b Percentage divergence in lower (left) triangle

L. autum; L. autumnalis; L. borg; L. borgpetersenii; L. ictero; L. icterohaemorrhagiae; L. kirsch; L. kirschneri;

L. Sant; L. santarosai

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The principles involved in the analysis of phylogenetic relationship between isolates are
similar whether the data consist of nucleotide sequences or amino acid sequences. The first
approach is based on a distance matrix generated from all pairwise comparisons of DNA
sequences. The phylogeny can then be inferred by applying the NJ method because it is
extremely fast and highly efficient at finding the correct tree. Hence the NJ method was
used in arriving at the phylogenetic tree.

ACKNOWLEDGEMENTS
The authors thank the Dean, Basic Sciences, Madras Veterinary College, Chennai for providing necessary facilities to carry out the research work.

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Molecular Biology and Evolution, 4, 406425
(Accepted: 31 January 2006; Published online: 11 January 2007)