Here we report isolation of a new species of Leptospira
with novel biological characteristics that caused in humans a non-specific syndrome of undifferentiated fever. We showed definitively through serological and molecular analysis using 16S rRNA gene sequencing and pulsed field gel electrophoresis that this new leptospire, provisionally named “Leptospira licerasiae
” serovar Varillal strain VAR 010T
, is antigenically unique, is a significant cause of acute leptospirosis in the Peruvian Amazon region of Iquitos, and has a Rattus
reservoir. Recognition of “Leptospira licerasiae”
serovar Varillal strain VAR 010T
as a new serovar is supported by the lack of agglutination of this strain by any serogroup reference serum and the lack of reactivity of anti- VAR 010T
serum raised in rabbits against the serovars of Leptospira
strains representing the nearly comprehensive and standard panel of leptospiral serogroups. A similar situation was found with L. fainei
serovar Hurstbridge, where the following evidence was adduced in support of this novel serovar: lack of significant cross-agglutination was observed with reference antisera representing the 24 pathogenic serogroups and the main saprophytic ones; lack of agglutination by antiserum raised against one of the strains against any serogroup 
. The serological characterization of the new serovar, Varillal, was conducted in two laboratories, one of which was the WHO/FAO/OIE Collaborating Centre for Reference and Research on Leptospirosis, Australia and Western Pacific Region, fulfilling the requirements for recognition of new serovars by the International Committee on Systematics of Prokaryotes, Subcommittee on the Taxonomy of Leptospiraceae 
The genus Leptospira
presently consists of 13 named species and 4 unnamed genomospecies 
. Phylogenetic analysis reveals three clades, representing species that contain pathogenic serovars, non-pathogenic serovars and an intermediate group 
. The latter clade comprises three species, Leptospira broomii
, Leptospira inadai
and Leptospira fainei 
. Based on phylogenetic analysis, L. licerasiae
is classified as an intermediate leptospiral species. Nonetheless, “L. licerasiae”
serovar Varillal strain VAR 010T
shares properties with pathogenic Leptospira
such as sensitivity to 8-azaguanine, has a LipL32-related protein as revealed by Western and Southern blots, but does not appear to contain a LigA
-related gene as determined by Southern blot. In contrast to L. interrogans, “L. licerasiae”
serovar Varillal strain VAR 010T
grew rapidly (similar to L. biflexa
), did not grow significantly in vivo
, and did not cause observable disease in experimentally infected animal. These observations suggest important biological and virulence differences between pathogenic and intermediate Leptospira.
Leptospirosis is typically thought of as an occupational disease originating from contact with water, soil or vegetation contaminated with the infected urine of carrier animals. The literature in recent years has shown that in under-developed areas of the world it is associated with environmental exposure during activities of daily living.1,22 Neither patient had an occupation that would be considered a risk factor for leptospirosis. Patient A in this study was a food vendor and had contact with obvious risk factors having frequented a market area (Belen) with poor sanitation and a high density of rats, and bathed in a natural pool, as there is no running water in her village. Patient B was a female student/domestic worker who lived in the city, did not frequent Belen (the urban slum area of Iquitos) and did not engage in other behavior that would place her at particular risk for contracting leptospirosis. However, both patients recalled seeing rats in and around their homes. Patient B did raise dogs and chickens, and the dogs urinated within the house. There were no established social or professional links between the patients, and their infections occurred in different places and times. Both cases presented with a mild, self-resolving febrile illness without secondary complications and show the ubiquity of exposure as part of the activities of daily living in this region.
Both patients initially had negative MAT and IgM results in their acute serum sample. While MAT of convalescent serum from Patient B was initially positive to a variety of serogroups, both acute and convalescent sera from Patient A were negative. However, when the test was repeated with the patient's own isolate, Patient A was found to have circulating leptospiral antibodies. This pattern of leptospiral seroreactivity, known to be a common problem in the diagnosis of leptospirosis,2 underscores the importance of including region-specific leptospiral isolates in the panel of strains used in MAT for diagnosing leptospirosis and determining its true burden.
A curious finding in this study is that isolation of “L. licerasiae” serovar Varillal from humans was rare, only being obtained from 2 of 881 febrile patients, despite the far higher seroprevalence rate of antibodies to this serovar. Some might raise the concern that this rare isolation rate could reflect a laboratory contamination with this leptospiral species. We believe this possibility is unlikely for two reasons. First, the patients from whom these isolates were obtained seroconverted to this novel serovar: patient 1 seroconverted to L. licerasiae serovar VAR 010T but to no other leptospiral antigen by MAT, while patient 2 seroconverted with the highest titer against her own isolate of L. licerasiae at a titer higher than other leptospires. Second, we never obtained an isolate of L. licerasiae in any other culture of human or animal specimens other than those reported here, making the possibility of contaminated culture medium negligible. While the biological basis for the rare isolation of L. licerasiae remains speculative, we propose two hypotheses. First, it is possible that the two patients from whom this leptospire was isolated had an undefined, undetermined genetic predisposition that led to higher leptospiremia or failure to clear this relatively less virulent leptospire after exposure. Second, it is possible that varying degrees of heterologous, cross-reacting, anti-leptospiral immunity exist in the study population. This latter hypothesis is supported by the very high prevalence of anti-leptospiral antibodies in the Iquitos region, likely due to ubiquity of leptospiral exposure. It may be that these two patients, for some reason, never had been exposed to Leptospira, and thus were immunologically naïve and thus predisposed to a higher level of leptospiral bacteremia. Further prospective, population-based studies are needed to address these important questions.
This prospective study of acute febrile illness in Peru has shown that “L. licerasiae” is an important cause of fever in the Iquitos area and its surroundings, as evidenced by the number of patient sera that reacted predominantly or solely with serovar Varillal (298/425; 70%). Isolation of “L. licerasiae” from rats suggests that this leptospiral species has at least Rattus spp. as a major reservoir host; we have not found this leptospiral species in other rodent, bat and marsupial species in the Peruvian Amazon (Dr. Monica Diaz and J.M. Vinetz et al, data not shown). Domestic rats are common in Iquitos: R. norvegicus and R. rattus are closely associated with human settlements in the area. R. norvegicus is more often encountered in urbanized areas, while R. rattus is the predominant rural species (data not shown). Six isolates identical to those isolated from both patients were recovered from rats caught in Belen, a city slum where sanitation is poor, rats are common and the risk of transmission to man is high. A further two VAR 010T-related isolates were recovered from rural rats. The isolation of a strain common to rats and found in 2 clinical cases, the ubiquity of the rat in Iquitos and the poor sanitation in most areas make the rat the likely source of leptospires in Iquitos. In other studies, we have succeeded in isolating L. interrogans serovar Icterohaemorrhagiae from 22 to 48% of peri-domestic Rattus species in villages near to and within urban areas of Iquitos (unpublished observations).
Molecular and serological analysis of human- and rat-derived strains revealed that they comprise a single novel leptospiral species and serovar. The fact that the PFGE fingerprint patterns were found to be identical to each other, but did not match any of the patterns in the CDC database (P.N. Levett and R. Galloway, unpublished data) supports our contention that the strains are novel leptospires. The isolates are members of a new serovar and serogroup as none were agglutinated by any of the reference anti-sera in our panel, although they had trace reactions to serogroup Hurstbridge. Given the lack of reactivity to the leptospiral serogroups represented by the rabbit reference sera used in the present study, the reference serological test, the cross-absorption agglutination test, was not necessary to define Varillal as a new serovar or antigenic type, similar to what was found for L. fainei
serovar Hurstbridge 
. Phylogenetic analysis of 16S rRNA gene sequence demonstrated that the strains comprised a homogenous genetic group separate from all other described leptospiral species. These strains, much like the recently described L. broomii 
, L. fainei
serovar Hurstbridge 
and L. inadai
serovar Lyme 
, are intermediate between the two larger saprophytic and pathogenic groups of Leptospira
and, as such, share characteristics similar to both pathogenic and saprophytic leptospires. DNA-DNA hybridization further confirmed that L. licerasiae
is a new Leptospira
species. Our logic was similar to that taken in using DNA-DNA hybridization to further confirm L. broomii
as a new Leptospira
. Because 16S rRNA gene sequencing places L. licerasiae
into the intermediate Leptospira
group close to L. fainei, L. broomii
, etc., the only relevant DNA-DNA hybridization analysis is to differentiate the closest known clade partners identified by 16S rRNA gene sequence, so as to be able to confirm the distinctness of these 16S-rRNA gene-defined intermediate Leptospira
species. The DNA-DNA hybridization analysis reported here does indeed confirm the distinctness of L. licerasiae
from the other known intermediate Leptospira
We report the existence of a new species of leptospire, provisionally named “Leptospira licerasiae”
serovar Varillal, which causes acute leptospirosis in the Peruvian Amazon. We have proposed this name to recognize the contribution of Professor Julia Liceras de Hidalgo who obtained the first leptospiral isolates in Peru 
. We propose a new serogroup, Iquitos, based on the lack of agglutination with a comprehensive panel of reference antisera comprised of all serogroups except for Lyme and Sehgali (which in the case of serovar Lyme had cross-reaction with serovar Celledoni at a titer of 1
, and in the case of Sehgali had a broad level of cross-reactivity 25 serovars and 12 serogroups ranging from titers of 1
80 to 1
Based on serological data that take advantage of its antigenic uniqueness, “Leptospira licerasiae” serovar Varillal appears to be an important cause of leptospirosis in the Peruvian Amazon region, but is uncommon elsewhere in Peru. The peridomestic rat is likely the major reservoir of this new species. Elucidation of virulence differences between pathogenic and intermediate leptospires will provide insight into leptospiral evolution and disease mechanisms, and may contribute to the control and amelioration of leptospirosis in the developing world.
Note on Taxonomy
To fulfill the rules of the International Code of Nomenclature of Bacteria [Lapage SP, Sneath PHA, Lessel EF, Skerman VBD, Seeliger HPR, Clark WA. International code of nomenclature of bacteria (1990 revision). Washington, DC: American Society for Microbiology, 1992.], we provide the following description of the novel species identified in this report.
Description of Leptospira licerasiae sp. nov. Leptospira licerasiae
(li.ce.ra' si.ae. N.L. fem gen. n. licerasiae of Liceras, to honor Dr. Julia Liceras de Hidalgo, who obtained the first leptospiral isolates in Peru). Isolated from the blood of human patients with febrile illness and from kidneys of rats in Peru. Morphology is as described previously for the genus 
. The G+C ratio is 43.9 mol%. The type strain is VAR 010T
WPR VAR 010T
), and has been deposited at the American Type Culture Collection, Manassas, Virginia, the National Veterinary Services Laboratory, U.S. Department of Agriculture, Ames, Iowa, and the WHO/FAO/OIE Collaborating Centre for Reference and Research on Leptospirosis, Australia and Western Pacific Region.
The 16S rRNA sequences for the Leptospira licerasiae isolates reported in this paper have been deposited in GenBank with the following accession numbers (strain, accession number): CEH006, EF612278; CEH011, EF612279; CEH033,EF612280; CEH044, EF612281; CEH162, EF612282; MMD735, EF612283; VAR 010T, EF612284; CEH010, EF612285; CEH174, EF612286; MMD835, EF612287; HAI029, EF612288.