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Journal of Clinical Microbiology, January 2000, p. 450-452, Vol. 38, No. 1
National Reference Center for Leptospirosis,
Instituto Oswaldo Cruz, Rio de Janeiro,1 and
Center for Zoonosis Control2 and
University of São Paulo,3
São Paulo, Brazil, and Unité de
Bactériologie Moléculaire et Médicale, Institut
Pasteur, 75724 Paris, France4
Received 28 July 1999/Accepted 22 September 1999
Leptospira is a highly diverse genus comprising many
species and serogroups in Brazil as well as all over the world.
However, a study by arbitrarily primed PCR of 44 leptospiral strains
isolated from humans during three different outbreaks in Brazilian
urban centers reveals that 43 of 44 isolates exhibit very similar
fingerprints. Analysis of these isolates indicates that they belong to
a clonal subpopulation of Leptospira interrogans sensu stricto.
The large diversity within Leptospira interrogans sensu
lato is demonstrated by the 223 serovars presently recognized according to serological criteria (6). The microscopic agglutination and cross-agglutinin absorption tests have been used for identification of leptospires at the serovar level. Serologically related serovars are
functionally clustered within 25 serogroups. Serogrouping is a simple
and reliable test which requires only 25 serum samples. In contrast,
serotyping at the serovar level requires complete collections of both
reference strains (up to 223) and corresponding sera. Cross-adsorption,
a complex and partially subjective procedure, is then performed.
Since the mid-1980s, 10 Leptospira species isolated from
humans or animals have been distinguished on the basis of DNA-DNA hybridization studies, namely, L. interrogans sensu stricto
(hereafter called L. interrogans), L. kirshneri,
L. weilii, L. noguchii, L. borgpetersenii, L. santarosai L. meyeri,
L. inadai, L. fainei, and L. alexanderi (1, 8, 10, 12). It should be emphasized that
serogroups are unequally distributed into species. For example, serogroup Pomona comprises serovars kunning, mozdok, and tsaratsovo, which belong to the species L. kirshneri, while the seven
remaining serovars from this serogroup belong to three other species.
Arbitrarily primed PCR (AP-PCR) allows the direct generation of highly
discriminant fingerprints and is probably the simplest DNA-based
subtyping method described to date. It has successfully been applied to the characterization of Leptospira, showing species
assignments that were in agreement with species assignments based on
DNA-DNA homology (9).
Our objective was to evaluate an AP-PCR method for the rapid comparison
of a large number of leptospiral isolates from different sources,
sites, and years in Brazil and for the establishment of a possible
clonal connection between isolates from a common-source outbreak.
The 44 Leptospira Brazilian isolates from human sources
(Table 1) were provided by the Instituto
Oswaldo Cruz (Rio de Janeiro, Brazil), the Center for Zoonosis Control
(São Paulo, Brazil), and the University of São Paulo
(São Paulo, Brazil). The isolates originated from three outbreaks
of leptospirosis which had taken place in Brazil, in the states of
São Paulo in 1994 and 1995 and Rio de Janeiro in 1996. Strains
(Table 2) from the Collection of the
World Health Organization Collaborating Center for Leptospirosis, Institut Pasteur, Paris, France, were used as reference strains.
0095-1137/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
A Clonal Subpopulation of Leptospira
interrogans Sensu Stricto Is the Major Cause of Leptospirosis
Outbreaks in Brazil
ABSTRACT
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TABLE 1.
Identification of the Leptospira serogroups
and species from isolates involved in human epidemic outbreaks
among humans in Brazil
TABLE 2.
Representative Leptospira strains from the
Pasteur Institute collection
The identification at the serogroup level (Table 1) was carried out, in Brazil, by previously described procedures (4). All SP (São Paulo) isolates belonged to the Icterohaemorrhagiae serogroup but with some uncertainty regarding SP-18, SP-20, SP-53, and SP-167. Isolates 1010 and 1015 also belonged to the Icterohaemorrhagiae serogroup. Isolate SP-58 was from the Pomona serogroup.
Each isolate was grown to the exponential phase of growth in EMJH
medium (3, 5) at 30°C with shaking. Approximately 5 × 108 cells of each strain were used for DNA extraction,
which was performed with the Dynabeads kit (Dynal) according to the
instructions of the supplier. DNA was eluted from the Dynabeads complex
in 40 µl of TE (10 mM Tris-HCl, 1 mM EDTA [pH 8]) and was kept at 
20°C. Five microliters was used for each AP-PCR experiment.
Fingerprints were generated as described previously (9) by
using the KG primer (5'-CAC ACG CAC ACG GAA GAA-3';
purchased from Eurogentec). We compared the AP-PCR patterns of
the isolates tested in this study with those of strains of the
Icterohaemorrhagiae and Pomona serogroups from the collection, as
identified above, which belonged to diverse pathogenic species. The
fingerprints of 38 isolates were similar to those of a copenhageni
serovar strain from the collection (Fig.
1A, lanes 2 to 8, 11 to 16, 18 to 20, 22 to 30, 32 to 33 compared to lane 1, and Fig. 1B, lanes 2 to 6 and 8 to 13 compared to lanes 1 and 18). The fingerprints of five isolates (SP-28, SP-53, SP-13, SP-167, and SP-68, Fig. 1A, lanes 9, 17, 21, and
31, and Fig. 1B, lane 7, respectively) showed DNA profiles closest to
that of the canicola serovar reference strain (Fig. 1B, lanes 20 and
21).
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While the fingerprints of the reference strains of serovars copenhageni and canicola have a great deal of similarity, there are recognizable differences in major bands (in Fig. 1B, the two upper arrows indicate bands present in the profile for serovar canicola and absent from the profile for serovar copenhageni, while the lower arrow indicates a band present in the profile for serovar copenhageni and absent from the profile for serovar canicola). The 38 isolates serotyped to the Icterohaemorrhagiae serogroup and the 5 strains serotyped to the Canicola serogroup were found to be similar to serovar copenhageni strains and serovar canicola strains from collections, respectively. However, small differences in two or three minor bands could be found. Since serovars canicola and copenhageni belong to the L. interrogans species, it can be concluded that with one exception (strain SP-58 [Fig. 1A, lane 10]) all of the 44 human isolates are L. interrogans. Isolate SP-58 showed a profile similar to that of serovar grippotyphosa and was identified as L. kirshneri (Fig. 1C, lane 1 compared to lane 2).
The 43 strains represent a cluster of isolates that are quite similar, even though they were isolated from human infections that occurred in different places and at different times during so-called epidemic periods. It should be noted that a very large set of serovars belonging to several serogroups and species does exist in wildlife in Brazil (2, 7, 11). In addition, the 10 Brazilian serovars described in the Leptospira collection (1) correspond to eight serogroups and four species. The present data confirm the predominance of a clonal subpopulation of L. interrogans as the cause of human leptospirosis in the large urban centers of Brazil. Similar strains have been isolated (data not shown) from dogs and rodents, which are the main reservoirs and shedders of leptospires in urban areas.
AP-PCR fingerprinting analysis is a rapid method for use in epidemiological studies. It is possible to compare the profiles of a large number of strains and reach meaningful and epidemiologically relevant conclusions concerning the geographic distributions of Leptospira populations.
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ACKNOWLEDGMENTS |
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We thank Antonio José Alves and Leticia Bastos for technical assistance in the cultivation of leptospires. We thank Danielle Margarita, Elisabeth Bellenger, Edith Fournié-Amazouz,and Natacha Sertour for help during the course of this study.
This work was supported by grants from the National Council for Research and Technological Development (fellowship to M. M. Pereira) and the National Foundation for Health/Brazilian Ministry of Health.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Bacteriology, Oswaldo Cruz Institute/FIOCRUZ, Avenida Brasil, 4365, 21045-900. Rio de Janeiro, Brazil. Phone: 55-21-270-3545. Fax: 55-21-590-3545. E-mail: mpereira{at}gene.dbbm.fiocruz.br.
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