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The main goal of this study was to obtain new isolates of Leptospira spp. from sheep. A total of ten kidney samples and 44 blood samples were collected from sheep slaughtered in Pelotas, Southern Brazil. One isolate was obtained which was identified by 16S rRNA gene sequencing and serogrouping to be Leptospira noguchii serogroup Autumnalis. Microscopic agglutination test (MAT) evaluation revealed that 4.5% of the sheep sera reacted against the Autumnalis serogroup. This is the first report of isolation of L. noguchii from sheep. Together these findings indicate that L. noguchii infections may be a potentially important veterinary problem in this domestic animal species.

New vaccine strategies are needed for the prevention of leptospirosis, a widespread human and animal disease caused by pathogenic leptospires. Our previous work determined that a protein leptospiral extract conferred cross-protection in a gerbil model of leptospirosis. The 31- to 34-kDa protein fraction of Leptospira interrogans serovar autumnalis was shown sufficient for this purpose. In the present study, N-terminal sequencing of a 32-kDa fraction and Southern blotting of genomic DNA with corresponding degenerated oligonucleotide probes identified two of its constituents: hemolysis-associated protein 1 (Hap1) and the outer membrane Leptospira protein 1 (OmpL1). Adenovirus-mediated Hap1 vaccination induces significant protection against a virulent heterologous Leptospira challenge in gerbils, whereas a similar OmpL1 construct failed to protect the animals. These data indicate that Hap1 could be a good candidate for developing a new generation of vaccines able to induce broad protection against leptospirosis disease.

The biodiversity of potential leptospiral reservoir hosts is lower in urban than in rural environments. Previous data indicate the potential for bats to act as carriers of Leptospira in regions such as the Amazon of South America and in Australia. Yet, little is known about the contribution of bats to leptospirosis in urban environments in South America. This study aimed to test the hypothesis that bats infected with Leptospira are sources of leptospirosis transmission to humans in São Paulo City, Brazil. Six of 343 bats caught in different districts within the city of Sao Paulo (182 insectivorous, 161 frugivorous or nectarivorous) were polymerase chain reaction (PCR) positive for pathogenic Leptospira; no seropositive bats were found. That few renal carriers of Leptospira were found in the city of Sao Paulo suggests that bats are not important in the transmission of leptospirosis to humans in this, and possibly other urban settings.

Background

The available Leptospira multilocus sequence typing (MLST) scheme supported by a MLST website is limited to L. interrogans and L. kirschneri. Our aim was to broaden the utility of this scheme to incorporate a total of seven pathogenic species.

Methodology and Findings

We modified the existing scheme by replacing one of the seven MLST loci (fadD was changed to caiB), as the former gene did not appear to be present in some pathogenic species. Comparison of the original and modified schemes using data for L. interrogans and L. kirschneri demonstrated that the discriminatory power of the two schemes was not significantly different. The modified scheme was used to further characterize 325 isolates (L. alexanderi [n = 5], L. borgpetersenii [n = 34], L. interrogans [n = 222], L. kirschneri [n = 29], L. noguchii [n = 9], L. santarosai [n = 10], and L. weilii [n = 16]). Phylogenetic analysis using concatenated sequences of the 7 loci demonstrated that each species corresponded to a discrete clade, and that no strains were misclassified at the species level. Comparison between genotype and serovar was possible for 254 isolates. Of the 31 sequence types (STs) represented by at least two isolates, 18 STs included isolates assigned to two or three different serovars. Conversely, 14 serovars were identified that contained between 2 to 10 different STs. New observations were made on the global phylogeography of Leptospira spp., and the utility of MLST in making associations between human disease and specific maintenance hosts was demonstrated.

Conclusion

The new MLST scheme, supported by an updated MLST website, allows the characterization and species assignment of isolates of the seven major pathogenic species associated with leptospirosis.

Author Summary

Leptospirosis is a common zoonotic disease worldwide. Genotyping of the causative organisms provides important insights into disease transmission and informs preventive strategies and vaccine development. Multilocus sequence typing (MLST) is the most widespread genotyping methodology for bacterial pathogens, but the Leptospira scheme supported by a public MLST database is currently only applicable to L. interrogans and L. kirschneri. The purpose of this study was to extend the scheme to a total of seven pathogenic Leptospira species. This was achieved through the development of a modified scheme in which one of the seven MLST loci was replaced, together with newly designed primers for the remaining 6 loci. Comparison of the original and modified scheme demonstrated that they were very similar, hence sequence type (ST) assignments were largely carried over to the modified scheme. Phylogenetic trees reconstructed from concatenated sequences of the seven loci of the modified scheme demonstrated perfect classification of isolates into seven pathogenic species, which resided in clearly distinct phylogenetic clusters. Congruence was low between STs and serovars. The MLST scheme was used to gain new insights into the population genetic structure of Leptospira species associated with clinical disease and maintenance hosts in Asia.

Leptospirosis is a serious infection disease caused by pathogenic strains of the Leptospira spirochetes, which affects not only humans but also animals. It has long been expected to find an effective vaccine to prevent leptospirosis through immunization of high risk humans or animals. Although some leptospirosis vaccines have been obtained, the vaccination is relatively unsuccessful in clinical application despite decades of research and millions of dollars spent. In this review, the recent advancements of recombinant outer membrane protein (OMP) vaccines, lipopolysaccharide (LPS) vaccines, inactivated vaccines, attenuated vaccines and DNA vaccines against leptospirosis are reviewed. A comparison of these vaccines may lead to development of new potential methods to combat leptospirosis and facilitate the leptospirosis vaccine research. Moreover, a vaccine ontology database was built for the scientists working on the leptospirosis vaccines as a starting tool.

Leptospira interrogans is a bacterium that is capable of infecting animals and humans, and its infection causes leptospirosis with a range of symptoms from flu-like to severe illness and death. Despite being a bacteria, Leptospira interrogans contains a plastidic class ferredoxin-NADP(H) reductase (FNR) with high catalytic efficiency, at difference from the bacterial class FNRs. These flavoenzymes catalyze the electron transfer between NADP(H) and ferredoxins or flavodoxins. The inclusion of a plastidic FNR in Leptospira metabolism and in its parasitic life cycle is not currently understood. Bioinformatic analyses of the available genomic and proteins sequences showed that the presence of this enzyme in nonphotosynthetic bacteria is restricted to the Leptospira genus and that a [4Fe-4S] ferredoxin (LB107) encoded by the Leptospira genome may be the natural substrate of the enzyme. Leptospira FNR (LepFNR) displayed high diaphorase activity using artificial acceptors and functioned as a ferric reductase. LepFNR displayed cytochrome c reductase activity with the Leptospira LB107 ferredoxin with an optimum at pH 6.5. Structural stability analysis demonstrates that LepFNR is one of the most stable FNRs analyzed to date. The persistence of a native folded LepFNR structure was detected in up to 6 M urea, a condition in which the enzyme retains 38% activity. In silico analysis indicates that the high LepFNR stability might be due to robust interactions between the FAD and the NADP+ domains of the protein. The limited bacterial distribution of plastidic class FNRs and the biochemical and structural properties of LepFNR emphasize the uniqueness of this enzyme in the Leptospira metabolism. Our studies show that in L. interrogans a plastidic-type FNR exchanges electrons with a bacterial-type ferredoxin, process which has not been previously observed in nature.

Background

Leptospirosis has become an urban health problem as slum settlements have expanded worldwide. Efforts to identify interventions for urban leptospirosis have been hampered by the lack of population-based information on Leptospira transmission determinants. The aim of the study was to estimate the prevalence of Leptospira infection and identify risk factors for infection in the urban slum setting.

Methods and Findings

We performed a community-based survey of 3,171 slum residents from Salvador, Brazil. Leptospira agglutinating antibodies were measured as a marker for prior infection. Poisson regression models evaluated the association between the presence of Leptospira antibodies and environmental attributes obtained from Geographical Information System surveys and indicators of socioeconomic status and exposures for individuals. Overall prevalence of Leptospira antibodies was 15.4% (95% confidence interval [CI], 14.0–16.8). Households of subjects with Leptospira antibodies clustered in squatter areas at the bottom of valleys. The risk of acquiring Leptospira antibodies was associated with household environmental factors such as residence in flood-risk regions with open sewers (prevalence ratio [PR] 1.42, 95% CI 1.14–1.75) and proximity to accumulated refuse (1.43, 1.04–1.88), sighting rats (1.32, 1.10–1.58), and the presence of chickens (1.26, 1.05–1.51). Furthermore, low income and black race (1.25, 1.03–1.50) were independent risk factors. An increase of US$1 per day in per capita household income was associated with an 11% (95% CI 5%–18%) decrease in infection risk.

Conclusions

Deficiencies in the sanitation infrastructure where slum inhabitants reside were found to be environmental sources of Leptospira transmission. Even after controlling for environmental factors, differences in socioeconomic status contributed to the risk of Leptospira infection, indicating that effective prevention of leptospirosis may need to address the social factors that produce unequal health outcomes among slum residents, in addition to improving sanitation.

Author Summary

Leptospirosis, a life-threatening zoonotic disease, has become an important urban slum health problem. Epidemics of leptospirosis now occur in cities throughout the developing world, as the growth of slum settlements has produced conditions for rat-borne transmission of this disease. In this prevalence survey of more than 3,000 residents from a favela slum community in Brazil, Geographical Information System (GIS) and modeling approaches identified specific deficiencies in the sanitation infrastructure of slum environments—open sewers, refuse, and inadequate floodwater drainage—that serve as sources for Leptospira transmission. In addition to the environmental attributes of the slum environment, low socioeconomic status was found to independently contribute to the risk of infection. These findings indicate that effective prevention of leptospirosis will need to address the social factors that produce unequal health outcomes among slum residents, in addition to improving sanitation.

Background

Leptospirosis, a spirochaetal zoonotic disease, is the cause of epidemics associated with high mortality in urban slum communities. Infection with pathogenic Leptospira occurs during environmental exposures and is traditionally associated with occupational risk activities. However, slum inhabitants reside in close proximity to environmental sources of contamination, suggesting that transmission during urban epidemics occurs in the household environment.

Methods and Findings

A survey was performed to determine whether Leptospira infection clustered within households located in slum communities in the city of Salvador, Brazil. Hospital-based surveillance identified 89 confirmed cases of leptospirosis during an outbreak. Serum samples were obtained from members of 22 households with index cases of leptospirosis and 52 control households located in the same slum communities. The presence of anti-Leptospira agglutinating antibodies was used as a marker for previous infection. In households with index cases, 22 (30%) of 74 members had anti-Leptospira antibodies, whereas 16 (8%) of 195 members from control households had anti-Leptospira antibodies. Highest titres were directed against L. interrogans serovars of the Icterohaemorrhagiae serogroup in 95% and 100% of the subjects with agglutinating antibodies from case and control households, respectively. Residence in a household with an index case of leptospirosis was associated with increased risk (OR 5.29, 95% CI 2.13–13.12) of having had a Leptospira infection. Increased infection risk was found for all age groups who resided in a household with an index case, including children <15 years of age (P = 0.008).

Conclusions

This study identified significant household clustering of Leptospira infection in slum communities where recurrent epidemics of leptospirosis occur. The findings support the hypothesis that the household environment is an important transmission determinant in the urban slum setting. Prevention therefore needs to target sources of contamination and risk activities which occur in the places where slum inhabitants reside.

Author Summary

Leptospirosis has emerged to become an urban slum health problem. Epidemics of severe leptospirosis, characterized by jaundice, acute renal failure and haemorrhage, are now reported in cities throughout the developing world due to rapid expansion of slum settlements, which in turn has produced the ecological conditions for rodent-borne transmission of the spirochete pathogen. A survey was performed in the city of Salvador, Brazil, to determine whether the risk of Leptospira infection clustered in households within slum communities in which a member had developed severe leptospirosis. We found that members of households with an index case of leptospirosis had more than five times the risk of having serologic evidence for a prior infection than members of neighbourhood households in the same communities. Increased risk of infection was found among all age groups who resided in these households. The finding that Leptospira infection clusters in specific slum households indicates that the factors associated with this environment are important determinants for transmission. Further research is needed to identify the sources of contamination and risk exposures which occur in the places where slum inhabitants reside such that effective community-based prevention of urban leptospirosis can be implemented.

Leptospirosis is a widespread zoonotic infection that primarily affects residents of tropical regions, but causes infections in animals and humans in temperate regions as well. The agents of leptospirosis comprise several members of the genus Leptospira, which also includes non-pathogenic, saprophytic species. Leptospirosis can vary in severity from a mild, non-specific illness to severe disease that includes multi-organ failure and widespread endothelial damage and hemorrhage. To begin to investigate how pathogenic leptospires affect endothelial cells, we compared the responses of two endothelial cell lines to infection by pathogenic versus non-pathogenic leptospires. Microarray analyses suggested that pathogenic L. interrogans and non-pathogenic L. biflexa triggered changes in expression of genes whose products are involved in cellular architecture and interactions with the matrix, but that the changes were in opposite directions, with infection by L. biflexa primarily predicted to increase or maintain cell layer integrity, while L. interrogans lead primarily to changes predicted to disrupt cell layer integrity. Neither bacterial strain caused necrosis or apoptosis of the cells even after prolonged incubation. The pathogenic L. interrogans, however, did result in significant disruption of endothelial cell layers as assessed by microscopy and the ability of the bacteria to cross the cell layers. This disruption of endothelial layer integrity was abrogated by addition of the endothelial protective drug lisinopril at physiologically relevant concentrations. These results suggest that, through adhesion of L. interrogans to endothelial cells, the bacteria may disrupt endothelial barrier function, promoting dissemination of the bacteria and contributing to severe disease manifestations. In addition, supplementing antibiotic therapy with lisinopril or derivatives with endothelial protective activities may decrease the severity of leptospirosis.

Author Summary

Leptospirosis is a widespread zoonotic infection that primarily affects residents of tropical regions, but is seen occasionally in temperate regions as well. Leptospirosis can vary in severity from a mild, non-specific illness to severe disease that includes multi-organ failure and widespread endothelial damage and hemorrhage. To investigate how pathogenic leptospires affect endothelial cells, we compared the responses of two endothelial cell lines to infection by pathogenic versus non-pathogenic leptospires. Our analyses suggested that pathogenic L. interrogans and non-pathogenic L. biflexa caused changes in expression of genes whose products are involved in cellular architecture and interactions with the matrix, but that the changes were in opposite directions, with infection by L. biflexa primarily maintaining cell layer integrity, while L. interrogans disrupted cell layers. In fact, L. interrogans caused significant disruption of endothelial cell layers, but this damage could be abrogated by the endothelial protective drug lisinopril. Our results suggest that L. interrogans binds to endothelial cells and disrupts endothelial barrier function, which may promote dissemination of the bacteria and contribute to severe disease manifestations. This disruption may be slowed by endothelial-protective drugs to decrease damage in leptospirosis.

Genetic variability among Leptospira interrogans (sensu stricto) serovars was assessed by Southern blot hybridization and PCR analyses. The experiments used probes directed to sequences in a recently described insertion element, IS1500. Hybridization analysis showed that IS1500 was present on polymorphic fragments and that differences in these patterns could be used to identify serovars. Hybridization analysis was also useful in discriminating between serovar pomona type kennewicki isolates, making possible the identification of 15 previously unrecognized genetic groups. A PCR assay was developed in which the primers are positioned near the terminal inverted repeats of the element and directed outward. This assay yielded characteristic amplification patterns from isolates, allowing them to be identified. We applied these assays to several new animal isolates of L. interrogans from Nicaragua, which recently had an outbreak of human leptospirosis. Three groups of isolates were identified: one strain of serovar pomona type kennewicki and two genetically distinct groups of isolates which may be genetic intermediates between serovars canicola and portlandvere. The IS-based typing assays described should be useful for epidemiological analysis of leptospirosis.

Human studies support the use of β-lactams and tetracyclines in the treatment of leptospirosis. Additional agents from these and other classes of antimicrobials also have in vitro activity against Leptospira species, though corroborating in vivo data are limited or lacking. We evaluated the therapeutic efficacy of azithromycin, clarithromycin, and telithromycin in a lethal hamster model of leptospirosis using Leptospira interrogans serogroup Canicola serovar Portlandvere. A range of dosages for each antimicrobial was given to the infected animals on days 2 through 7 (5 days) of the 21-day survival model. All untreated control animals survived less than 10 days from infection. Ninety to 100% of doxycycline controls, treated for 5 days with 5 mg/kg of body weight of drug, survived to 21 days. Treatment with azithromycin (daily dose: 6.25, 12.5, 25, 50, 100, or 200 mg/kg) resulted in 100% survival at all evaluated doses. Animals receiving 20 mg/kg or more of clarithromycin (daily dose: 1, 5, 10, 15, 20, 40, 60, or 100 mg/kg) had improved survival. Ninety-eight percent of animals treated with telithromycin (daily dose: 1, 5, 10, 15, 20, or 40 mg/kg) survived. We conclude that all agents tested have demonstrated in vivo efficacy in treating acute leptospirosis. These results provide support for further evaluation of macrolide and ketolide antimicrobial agents in human trials.

Leptospirosis is an emerging zoonosis caused by pathogenic spirochetes belonging to the genus Leptospira. An understanding of leptospiral protein expression regulation is needed to develop new immunoprotective and serodiagnostic strategies. We used the humoral immune response during human leptospirosis as a reporter of protein antigens expressed during infection. Qualitative and quantitative immunoblot analysis was performed using sera from 105 patients from Brazil and Barbados. Sera from patients with other diseases and healthy individuals were evaluated as controls. Seven proteins, p76, p62, p48, p45, p41, p37, and p32, were identified as targets of the humoral response during natural infection. In both acute and convalescent phases of illness, antibodies to lipopolysaccharide were predominantly immunoglobulin M (IgM) while antibodies to proteins were exclusively IgG. Anti-p32 reactivity had the greatest sensitivity and specificity: positive reactions were observed in 37 and 84% of acute- and convalescent-phase sera, respectively, while only 5% of community control individuals demonstrated positive reactions. Six immunodominant antigens were expressed by all pathogenic leptospiral strains tested; only p37 was inconsistently expressed. Two-dimensional immunoblots identified four of the seven infection-associated antigens as being previously characterized proteins: LipL32 (the major outer membrane lipoprotein), LipL41 (a surface-exposed outer membrane lipoprotein), and heat shock proteins GroEL and DnaK. Fractionation studies demonstrated LipL32 and LipL41 reactivity in the outer membrane fraction and GroEL and DnaK in the cytoplasmic fraction, while p37 appeared to be a soluble periplasmic protein. Most of the other immunodominant proteins, including p48 and p45, were localized to the inner membrane. These findings indicate that leptospiral proteins recognized during natural infection are potentially useful for serodiagnosis and may serve as targets for vaccine design.

Leptospirosis is a potentially deadly zoonotic disease that afflicts humans and animals. Leptospira interrogans, the predominant agent of leptospirosis, encounters diverse conditions as it proceeds through its life cycle, which includes stages inside and outside the host. Unfortunately, the number of genetic tools available for examining the regulation of gene expression in L. interrogans is limited. Consequently, little is known about the genetic circuits that control gene expression in Leptospira. To better understand the regulation of leptospiral gene expression, the L. interrogans kdp locus, encoding homologs of the P-type ATPase KdpABC potassium transporter with their KdpD sensors and KdpE response regulators, was selected for analysis. We showed that a kdpE mutation in L. interrogans prevented the increase in kdpABC mRNA levels observed in the wild-type L. interrogans strain when external potassium levels were low. To confirm that KdpE was a positive regulator of kdpABC transcription, we developed a novel approach for constructing chromosomal genetic fusions to the endogenous bgaL (β-galactosidase) gene of the nonpathogen Leptospira biflexa. We demonstrated positive regulation of a kdpA′-bgaL fusion in L. biflexa by the L. interrogans KdpE response regulator. A control lipL32′-bgaL fusion was not regulated by KdpE. These results demonstrate the utility of genetic fusions to the bgaL gene of L. biflexa for examining leptospiral gene regulation.

Background

Leptospirosis is a world-widely distributed zoonosis. Humans become infected via exposure to pathogenic Leptospira spp. from contaminated water or soil. The availability of genomic sequences of Leptospira interrogans serovar Lai and serovar Copenhageni opened up opportunities to identify genetic diversity among different pathogenic strains of L. interrogans representing various kinds of serotypes (serogroups and serovars).

Results

Comparative genomic hybridization (CGH) analysis was used to compare the gene content of L. interrogans serovar Lai strain Lai with that of other 10 L. interrogans strains prevailed in China and one identified from Brazil using a microarray spotted with 3,528 protein coding sequences (CDSs) of strain Lai. The cutoff ratio of sample/reference (S/R) hybridization for detecting the absence of genes from one tested strain was set by comparing the ratio of S/R hybridization and the in silico sequence similarities of strain Lai and serovar Copenhageni strain Fiocruz L1-130. Among the 11 strains tested, 275 CDSs were found absent from at least one strain. The common backbone of the L. interrogans genome was estimated to contain about 2,917 CDSs. The genes encoding fundamental cellular functions such as translation, energy production and conversion were conserved. While strain-specific genes include those that encode proteins related to either cell surface structures or carbohydrate transport and metabolism. We also found two genomic islands (GIs) in strain Lai containing genes divergently absent in other strains. Because genes encoding proteins with potential pathogenic functions are located within GIs, these elements might contribute to the variations in disease manifestation. Differences in genes involved in O-antigen biosynthesis were also identified for strains belonging to different serogroups, which offers an opportunity for future development of genomic typing tools for serological classification.

Conclusion

CGH analyses for pathogenic leptospiral strains prevailed in China against the L. interrogans serovar Lai strain Lai CDS-spotted microarrays revealed 2,917 common backbone CDSs and strain specific genes encoding proteins mainly related to cell surface structures and carbohydrated transport/metabolism. Of the 275 CDSs considered absent from at least one of the L. interrogans strains tested, most of them were clustered in the rfb gene cluster and two putative genomic islands (GI A and B) in strain Lai. The strain-specific genes detected via this work will provide a knowledge base for further investigating the pathogenesis of L interrogans and/or for the development of effective vaccines and/or diagnostic tools.

Leptospirosis is an important zoonosis of worldwide distribution. Humans become infected via exposure to pathogenic Leptospira spp. from infected animals or contaminated water or soil. The availability of genome sequences for Leptospira interrogans, serovars Lai and Copenhageni, has opened up opportunities to examine global transcription profiles using microarray technology. Temperature is a key environmental factor known to affect leptospiral protein expression. Leptospira spp. can grow in artificial media at a range of temperatures reflecting conditions found in the environment and the mammalian host. Therefore, transcriptional changes were compared between cultures grown at 20°C, 30°C, 37°C, and 39°C to represent ambient temperatures in the environment, growth under laboratory conditions, and temperatures in healthy and febrile hosts. Data from direct pairwise comparisons of the four temperatures were consolidated to examine transcriptional changes at two generalized biological conditions representing mammalian physiological temperatures (37°C and 39°C) versus environmental temperatures (20°C and 30°C). Additionally, cultures grown at 30°C then shifted overnight to 37°C were compared with those grown long-term at 30°C and 37°C to identify genes potentially expressed in the early stages of infection. Comparison of data sets from physiological versus environmental experiments with upshift experiments provided novel insights into possible transcriptional changes at different stages of infection. Changes included differential expression of chemotaxis and motility genes, signal transduction systems, and genes encoding proteins involved in alteration of the outer membrane. These findings indicate that temperature is an important factor regulating expression of proteins that facilitate invasion and establishment of disease.

We diagnosed leptospirosis in 2 patients exposed to southern flying squirrels imported from the United States to Japan. Patients worked with exotic animals in their company. Leptospira isolates from 1 patient and 5 of 10 squirrels at the company were genetically and serologically identical and were identified as Leptospira kirschneri.

Background

Although previous data indicate that the overall incidence of human leptospirosis in the Peruvian Amazon is similar in urban and rural sites, severe leptospirosis has been observed only in the urban context. As a potential explanation for this epidemiological observation, we tested the hypothesis that concentrations of more virulent Leptospira would be higher in urban than in rural environmental surface waters.

Methods and Findings

A quantitative real-time PCR assay was used to compare levels of Leptospira in urban and rural environmental surface waters in sites in the Peruvian Amazon region of Iquitos. Molecular taxonomic analysis of a 1,200-bp segment of the leptospiral 16S ribosomal RNA gene was used to identify Leptospira to the species level. Pathogenic Leptospira species were found only in urban slum water sources (Fisher's exact test; p = 0.013). The concentration of pathogen-related Leptospira was higher in urban than rural water sources (~103 leptospires/ml versus 0.5 × 102 leptospires/ml; F = 8.406, p < 0.05). Identical 16S rRNA gene sequences from Leptospira interrogans serovar Icterohaemorrhagiae were found in urban slum market area gutter water and in human isolates, suggesting a specific mode of transmission from rats to humans. In a prospective, population-based study of patients presenting with acute febrile illness, isolation of L. interrogans-related leptospires from humans was significantly associated with urban acquisition (75% of urban isolates); human isolates of other leptospiral species were associated with rural acquisition (78% of rural isolates) (chi-square analysis; p < 0.01). This distribution of human leptospiral isolates mirrored the distribution of leptospiral 16S ribosomal gene sequences in urban and rural water sources.

Conclusions

Our findings data support the hypothesis that urban severe leptospirosis in the Peruvian Amazon is associated with higher concentrations of more pathogenic leptospires at sites of exposure and transmission. This combined quantitative and molecular taxonomical risk assessment of environmental surface waters is globally applicable for assessing risk for leptospiral infection and severe disease in leptospirosis-endemic regions.

Vinetz and colleagues used a quantitative real time PCR assay combined with molecular taxonomic analysis to quantify Leptospira in environmental surface waters in the Peruvian Amazon region of Iquitos.

Editors' Summary

Background.

Humans catch many diseases from animals—so-called zoonotic infections. Often, these occur in limited regions of the world. However, one—leptospirosis—occurs in temperate and tropical climates, and in urban and rural settings, making it the most widespread zoonotic disease. Leptospirosis is caused by Leptospira, a large group of closely related spiral-shaped bacteria that live in both domestic animals (for example, cattle) and wild animals (particularly rats). Millions of humans become infected each year with leptospires through close contact with water, food, or soil contaminated with the urine of infected animals—swimming or wading in contaminated water is particularly hazardous. Some infected people have no symptoms; others develop a flu-like disease that clears up within a few days. However, in 5%–10% of infected people, the disease progresses to a second, sometimes fatal phase. This is usually characterized by jaundice, kidney problems, and an enlarged spleen (it's then called Weil disease) but can also involve the lungs (pulmonary leptospirosis). Leptospirosis can be successfully treated with antibiotics if treatment is started soon after infection.

Why Was This Study Done?

In a recent study in the Peruvian Amazon, half of the people visiting urban hospitals and rural health posts with acute fever had antibodies in their blood to Leptospira, suggesting that they had acute leptospirosis. However, only patients living in urban areas developed pulmonary leptospirosis. In this study, the researchers tested the hypothesis that this pattern arose because more virulent types of Leptospira were present at higher levels in urban environmental surface water than in rural water sources.

What Did the Researchers Do and Find?

Between June 2003 and March 2004, the researchers isolated strains of Leptospira from patients with acute fever who visited a hospital in the town of Iquitos or clinics in nearby villages. Early in 2004, they also collected a large number of different water samples from an urban slum in Iquitos and from a nearby rural community. They measured the concentrations of Leptospira in these samples by using a molecular technique called real-time PCR (polymerase chain reaction) to detect and quantify a type of RNA found only in disease-causing Leptospira. They also identified which specific Leptospira were present in the water samples and the patient samples by sequencing this RNA. The researchers found that leptospires were present in both urban and rural water samples (particularly in samples from gutters and puddles in the urban slum's market area) but that their concentration in the positive water samples from the urban sites was 20 times that in the positive samples from the rural sites. Furthermore, the distribution of different Leptospira types isolated from the patients mirrored that of the bacteria in the local environment. So, one particular type of Leptospira interrogans known as icterohaemorrhagiae—the leptospire most commonly associated with severe leptospirosis in the patients—was found more often in the urban water samples than in the rural ones. Finally, the researchers discovered a new group of Leptospira in the rural environment. This group may contain one or several new species of Leptospira but whether any of them causes human disease is unknown.

What Do These Findings Mean?

These results support the researchers' hypothesis that pulmonary leptospirosis in urban areas of the Peruvian Amazon is associated with high environmental levels of specific disease-causing leptospires. The researchers were able to discover this link only by using molecular techniques—this sort of study is impossible with traditional bacteriological techniques because Leptospira are hard to grow in the laboratory and cannot be isolated efficiently from environmental water sources. Different types can't be identified using a microscope. The researchers' findings need to be validated in other settings, but they suggest that environmental interventions such as reducing sources of standing water and clearing away garbage in urban areas might reduce the number of cases of severe leptospirosis. The distribution of different Leptospira types also suggests that whereas rats may be the main disease reservoir in towns, cattle, pigs, and bats may be more important in rural settings in Peru and presumably elsewhere. Overall, this new information, together with the availability of molecular methods for rapid clinical diagnosis and environmental risk assessment, should aid attempts to control leptospirosis around the world.

Additional Information.

Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030308.

US Centers for Disease Control and Prevention, information for patients and professionals on leptospirosis

The Leptospirosis Information Center, information and advice on human leptospirosis for the public and medical professionals

MedlinePlus encyclopedia entry on leptospirosis

NHS Direct Online, patient information on leptospirosis from the UK National Health Service online encyclopedia

Wikipedia pages on leptospirosis (note: Wikipedia is a free online encyclopedia that anyone can edit)

Leptospirosis is a worldwide-distributed zoonosis, endemic in tropical areas. Epidemiologic investigations of leptospirosis still rely on tedious serological identification tests. Recently, molecular typing systems based on variable-number tandem-repeat (VNTR) analysis have been described and have been used to identify Leptospira interrogans strains. Although L. interrogans is the most common Leptospira species encountered in human infections around the world, other pathogenic species, such as Leptospira kirschneri and Leptospira borgpetersenii, are also frequently associated with human leptospirosis. In this study, we aimed to extend multilocus VNTR analysis (MLVA) identification of strains to species other than L. interrogans. We designed primers for VNTR loci found in L. interrogans, L. kirschneri, and L. borgpetersenii. The discriminatory power of the redefined primers was evaluated on collection strains and then on clinical strains. We also carried out a retrospective study on 156 strains isolated from patients and animals from New Caledonia, an area of high endemicity in the South Pacific. Our results show that this simple PCR-based MLVA typing technique is a powerful methodology for the epidemiology of leptospirosis.

One study described the frequency of pre-breeding vaccination for leptospirosis in 205 cow-calf herds from across western Canada and the prevalence of positive Leptospira antibody titers in unvaccinated, weaned calves from 61 of these herds. The percentages of herds vaccinated for leptospirosis were 13.7% in 2001 and 8.4% in 2002. Of 1539 calves examined, 13 (0.8%) had a positive antibody titer for a Leptospira serovar; the most common serovar detected was hardjo. A second study examined the prevalence of positive Leptospira antibody titers during the summer grazing season in 313 vaccinated and 478 unvaccinated cows from 40 cow-calf herds in southern Saskatchewan. Antibody titers for 7 Leptospira serovars were measured during the grazing season. Of the non-vaccinated cows, 9.6% were positive in the spring for serovar pomona, 6.7% for serovar grippotyphosa, and 6.1% for serovar icterohaemorrhagiae; the corresponding percentages for the fall were 5.5%, 3.0%, and 1.3%, respectively. Of 781 vaccinated and unvaccinated cows that were sampled twice, 11.3% of vaccinated cows and 2.3% of unvaccinated cows had increases in Leptospira antibody titers during the grazing season.

As part of a prospective study of leptospirosis and biodiversity of Leptospira in the Peruvian Amazon, a new Leptospira species was isolated from humans with acute febrile illness. Field trapping identified this leptospire in peridomestic rats (Rattus norvegicus, six isolates; R. rattus, two isolates) obtained in urban, peri-urban, and rural areas of the Iquitos region. Novelty of this species was proven by serological typing, 16S ribosomal RNA gene sequencing, pulsed-field gel electrophoresis, and DNA-DNA hybridization analysis. We have named this species “Leptospira licerasiae” serovar Varillal, and have determined that it is phylogenetically related to, but genetically distinct from, other intermediate Leptospira such as L. fainei and L. inadai. The type strain is serovar Varillal strain VAR 010T, which has been deposited into internationally accessible culture collections. By microscopic agglutination test, “Leptospira licerasiae” serovar Varillal was antigenically distinct from all known serogroups of Leptospira except for low level cross-reaction with rabbit anti–L. fainei serovar Hurstbridge at a titer of 1∶100. LipL32, although not detectable by PCR, was detectable in “Leptospira licerasiae” serovar Varillal by both Southern blot hybridization and Western immunoblot, although on immunoblot, the predicted protein was significantly smaller (27 kDa) than that of L. interrogans and L. kirschneri (32 kDa). Isolation was rare from humans (2/45 Leptospira isolates from 881 febrile patients sampled), but high titers of MAT antibodies against “Leptospira licerasiae” serovar Varillal were common (30%) among patients fulfilling serological criteria for acute leptospirosis in the Iquitos region, and uncommon (7%) elsewhere in Peru. This new leptospiral species reflects Amazonian biodiversity and has evolved to become an important cause of leptospirosis in the Peruvian Amazon.

Author Summary

Leptospirosis has emerged as a globally important infectious disease. Its impact on public health is often difficult to determine, sometimes because of low clinical suspicion, or, as is more common, difficulty in laboratory diagnosis. Gold-standard serology-based diagnosis has a number of important limitations, including the need to use live leptospires that have a sufficient diversity of antigens to be able to detect specific anti-leptospiral antibodies; such antigens vary greatly from region to region. In this paper, we report the discovery of a new species of Leptospira in the highly biodiverse region of the Peruvian Amazon, and demonstrate that the animal source of infection for humans is the domestic rat. Detailed biological characterization of this new species shows that it is antigenically unique and represents a new serogroup and serovar, proposed as Leptospira licerasiae serogroup Iquitos serovar Varillal. Incorporation of this new isolate into serological testing of patients presenting with acute febrile illness in Iquitos, Peru, showed a far higher incidence of leptospirosis than previously suspected, showing the important of using region-specific Leptospira in diagnosis. The field-to-laboratory approach presented here has general application to the discovery of other emerging pathogens and their impact on human health.

Leptospirosis is the most widespread zoonosis throughout the world and human mortality from severe disease forms is high even when optimal treatment is provided. Leptospirosis is also one of the most common causes of reproductive losses in cattle worldwide and is associated with significant economic costs to the dairy farming industry. Herds are tested for exposure to the causal organism either through serum testing of individual animals or through testing bulk milk samples. Using serum results from a commonly used enzyme-linked immunosorbent assay (ELISA) test for Leptospira interrogans serovar Hardjo (L. hardjo) on samples from 979 animals across 12 Scottish dairy herds and the corresponding bulk milk results, we develop a model that predicts the mean proportion of exposed animals in a herd conditional on the bulk milk test result. The data are analyzed through use of a Bayesian latent variable generalized linear mixed model to provide estimates of the true (but unobserved) level of exposure to the causal organism in each herd in addition to estimates of the accuracy of the serum ELISA. We estimate 95% confidence intervals for the accuracy of the serum ELISA of (0.688, 0.987) and (0.975, 0.998) for test sensitivity and specificity, respectively. Using a percentage positivity cutoff in bulk milk of at most 41% ensures that there is at least a 97.5% probability of less than 5% of the herd being exposed to L. hardjo. Our analyses provide strong statistical evidence in support of the validity of interpreting bulk milk samples as a proxy for individual animal serum testing. The combination of validity and cost-effectiveness of bulk milk testing has the potential to reduce the risk of human exposure to leptospirosis in addition to offering significant economic benefits to the dairy industry.

The purpose of this study was to perform a 16S sequence-based quality control of two Leptospira strain collections. 16S rRNA gene sequencing was used to verify two Leptospira reference collections provided by the World Health Organization and maintained at a reference laboratory for leptospirosis in Brazil. Among the 89 serovars evaluated, four conflicting strains were identified in one of the collections. Although 16S rRNA gene sequencing cannot identify Leptospira beyond the species level, it is suitable for the identification of contamination and quality control of leptospiral reference collections. This study highlights the importance of the availability of high-quality 16S rRNA sequences in public databases. In addition, it emphasizes the need for periodical verifications and quality control of Leptospira reference collections.

Sheep red cells, sensitized with a genus-specific leptospiral substance extracted from a water leptospira, were preserved by freeze drying. The serological reactivity of the preparation was tested against a total of 41 sera from proven cases of human leptospirosis and 27 from proven cases of animal leptospirosis. The sensitized, freeze-dried red cells appeared to be a sensitive reagent for the detection of leptospiral antibodies by haemagglutination. The use of this reagent as a screening test for leptospiral diagnosis is discussed. In the reported haemagglutination test on human and rabbit antisera the findings suggested that only IgM (19S) antibodies took part in the reaction.

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Background

Leptospira are the parasitic bacterial organisms associated with a broad range of mammalian hosts and are responsible for severe cases of human Leptospirosis. The epidemiology of leptospirosis is complex and dynamic. Multiple serovars have been identified, each adapted to one or more animal hosts. Adaptation is a dynamic process that changes the spatial and temporal distribution of serovars and clinical manifestations in different hosts. Serotyping based on repertoire of surface antigens is an ambiguous and artificial system of classification of leptospiral agents. Molecular typing methods for the identification of pathogenic leptospires up to individual genome species level have been highly sought after since the decipherment of whole genome sequences. Only a few resources exist for microbial genotypic data based on individual techniques such as Multiple Locus Sequence Typing (MLST), but unfortunately no such databases are existent for leptospires.

Results

We for the first time report development of a robust MLST method for genotyping of Leptospira. Genotyping based on DNA sequence identity of 4 housekeeping genes and 2 candidate genes was analyzed in a set of 120 strains including 41 reference strains representing different geographical areas and from different sources. Of the six selected genes, adk, icdA and secY were significantly more variable whereas the LipL32 and LipL41 coding genes and the rrs2 gene were moderately variable. The phylogenetic tree clustered the isolates according to the genome-based species.

Conclusion

The main advantages of MLST over other typing methods for leptospires include reproducibility, robustness, consistency and portability. The genetic relatedness of the leptospires can be better studied by the MLST approach and can be used for molecular epidemiological and evolutionary studies and population genetics.

Human and animal leptospirosis caused by Leptospira spp. belonging to serogroup Ballum has increased worldwide in the past decade. We report the isolation and serologic and molecular characterization of four L. borgpetersenii serogroup Ballum isolates obtained from Mus musculus, and preliminary virulence studies. These isolates are useful for diagnosis of leptospirosis and for epidemiologic studies of its virulence and pathogenic mechanisms.