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1.  Serovar Diversity of Pathogenic Leptospira Circulating in the French West Indies 
Background
Leptospirosis is one of the most important neglected tropical bacterial diseases in Latin America and the Caribbean. However, very little is known about the circulating etiological agents of leptospirosis in this region. In this study, we describe the serological and molecular features of leptospires isolated from 104 leptospirosis patients in Guadeloupe (n = 85) and Martinique (n = 19) and six rats captured in Guadeloupe, between 2004 and 2012.
Methods and Findings
Strains were studied by serogrouping, PFGE, MLVA, and sequencing 16SrRNA and secY. DNA extracts from blood samples collected from 36 patients in Martinique were also used for molecular typing of leptospires via PCR. Phylogenetic analyses revealed thirteen different genotypes clustered into five main clades that corresponded to the species: L. interrogans, L. kirschneri, L. borgpetersenii, L. noguchi, and L. santarosai. We also identified L. kmetyi in at least two patients with acute leptospirosis. This is the first time, to our knowledge, that this species has been identified in humans. The most prevalent genotypes were associated with L. interrogans serovars Icterohaemorrhagiae and Copenhageni, L. kirschneri serovar Bogvere, and L. borgpetersenii serovar Arborea. We were unable to identify nine strains at the serovar level and comparison of genotyping results to the MLST database revealed new secY alleles.
Conclusions
The overall serovar distribution in the French West Indies was unique compared to the neighboring islands. Typing of leptospiral isolates also suggested the existence of previously undescribed serovars.
Author Summary
Leptospirosis is an emerging zoonotic disease caused by infection with pathogenic strains of Leptospira. Isolation of Leptospira strains is rare, making it difficult to assess their distribution worldwide. In this study, we characterized cultures of Leptospira obtained from more than one hundred leptospirosis patients from the French West Indies by serology and various molecular typing methods to identify the strains circulating in this endemic region. Typing of leptospiral isolates showed that causative agents of leptospirosis in the French West Indies are mainly from the serogroups Icterohaemorrhagiae and Ballum, but we also identified new genotypes. We also found that the distribution of the predominant pathogenic leptospiral serovars differed between the Caribbean islands. A better understanding of the epidemiology of leptospirosis will improve our knowledge in the distribution of this emerging neglected tropical disease worldwide. The identification of the circulating etiological agents of leptospirosis in the French West Indies will also help establish appropriate control and prevention measures in this area where the disease is endemic.
doi:10.1371/journal.pntd.0002114
PMCID: PMC3597474  PMID: 23516654
2.  Determining Risk for Severe Leptospirosis by Molecular Analysis of Environmental Surface Waters for Pathogenic Leptospira 
PLoS Medicine  2006;3(8):e308.
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)
doi:10.1371/journal.pmed.0030308
PMCID: PMC1551915  PMID: 16933963
3.  CHARACTERIZATION OF VIRULENCE OF Leptospira ISOLATES IN A HAMSTER MODEL 
Vaccine  2008;26(31):3892-3896.
Effort has been made to identify protective antigens in order to develop a recombinant vaccine against leptospirosis. Several attempts failed to conclusively demonstrate efficacy of vaccine candidates due to the lack of an appropriate model of lethal leptospirosis. The purposes of our study were: (i) to test the virulence of leptospiral isolates from Brazil, which are representative of important serogroups that cause disease in humans and animals; and (ii) to standardize the lethal dose 50% (LD50) for each of the virulent strains using a hamster (Mesocricetus auratus) model. Five of seven Brazilian isolates induced lethality in a hamster model, with inocula lower than 200 leptospires. Histopathological examination of infected animals showed typical lesions found in both natural and experimental leptospirosis. Results described here demonstrated the potential use of Brazilian isolates as highly virulent strains in challenge experiments using hamster as an appropriate animal model for leptospirosis. Furthermore these strains may be useful in heterologous challenge studies which aim to evaluate cross-protective responses induced by subunit vaccine candidates.
doi:10.1016/j.vaccine.2008.04.085
PMCID: PMC2519131  PMID: 18547690
Leptospira; leptospirosis; lethal dose; isolation; animal model; virulence
4.  Serologic and Molecular Studies of Leptospira and Leptospirosis among Rats in the Philippines 
Rats are known to be the most important reservoirs and transmission sources of leptospirosis. However, the status of leptospirosis in the Philippines regarding reservoirs and transmission remains unknown. A survey was conducted in Metro Manila and Laguna that analyzed samples obtained from 106 rats. Using the microscopic agglutination test, we found that 92% of rat serum samples were positive for anti-Leptospira antibodies; the most common infecting serovars were Manilae, Hebdomadis, and Losbanos. On the basis of pulsed-field gel electrophoresis and gyrase B gene sequence analyses, four groups of rat kidney isolates were found: L. interrogans serovar Manilae, serovar Losbanos, and serogroup Grippotyphosa, and L. borgpetersenii serogroup Javanica. Most isolates were lethal after experimental infection of golden Syrian hamsters. Results showed that these four Leptospira serovars and serogroups are circulating among rats, and that these animals may be one of the possible transmission sources of leptospirosis in the Philippines.
doi:10.4269/ajtmh.2010.09-0711
PMCID: PMC2861393  PMID: 20439972
5.  Similarities in Leptospira Serogroup and Species Distribution in Animals and Humans in the Indian Ocean Island of Mayotte 
Our objective was to identify local animal reservoirs of leptospirosis to explain the unusual features of Leptospira strains recently described among patients on the island of Mayotte. By means of a microscopic agglutination test using local clinical isolates, we found that 11.2% of black rats were seropositive to Leptospira, whereas 10.2% of flying foxes, 2% of lemurs, 93.1% of domestic dogs, and 87.5% of stray dogs were seropositive. As observed in humans, Mini was the main serogroup circulating in animals, whereas serogroup Icterohaemorrhagiae was absent. Using quantitative polymerase chain reaction, we also showed that 29.8% of rats carried leptospires in their kidneys. The sequencing of 16S rRNA gene sequences of Leptospira found in black rat kidneys identified four genomospecies (Leptospira borgpetersenii, Leptospira interrogans, Leptospira kirschneri, and L. borgpetersenii group B), which established black rats as the major source of leptospirosis transmission to humans. The origins of such a genetic diversity in Leptospira strains are discussed.
doi:10.4269/ajtmh.2012.12-0012
PMCID: PMC3391038  PMID: 22764304
6.  Epidemiology of Leptospira Transmitted by Rodents in Southeast Asia 
Background
Leptospirosis is the most common bacterial zoonoses and has been identified as an important emerging global public health problem in Southeast Asia. Rodents are important reservoirs for human leptospirosis, but epidemiological data is lacking.
Methodology/Principal Findings
We sampled rodents living in different habitats from seven localities distributed across Southeast Asia (Thailand, Lao PDR and Cambodia), between 2009 to 2010. Human isolates were also obtained from localities close to where rodents were sampled. The prevalence of Leptospira infection was assessed by real-time PCR using DNA extracted from rodent kidneys, targeting the lipL32 gene. Sequencing rrs and secY genes, and Multi Locus Variable-number Tandem Repeat (VNTR) analyses were performed on DNA extracted from rat kidneys for Leptospira isolates molecular typing. Four species were detected in rodents, L. borgpetersenii (56% of positive samples), L. interrogans (36%), L. kirschneri (3%) and L. weilli (2%), which were identical to human isolates. Mean prevalence in rodents was approximately 7%, and largely varied across localities and habitats, but not between rodent species. The two most abundant Leptospira species displayed different habitat requirements: L. interrogans was linked to humid habitats (rice fields and forests) while L. borgpetersenii was abundant in both humid and dry habitats (non-floodable lands).
Conclusion/Significance
L. interrogans and L. borgpetersenii species are widely distributed amongst rodent populations, and strain typing confirmed rodents as reservoirs for human leptospirosis. Differences in habitat requirements for L. interrogans and L. borgpetersenii supported differential transmission modes. In Southeast Asia, human infection risk is not only restricted to activities taking place in wetlands and rice fields as is commonly accepted, but should also include tasks such as forestry work, as well as the hunting and preparation of rodents for consumption, which deserve more attention in future epidemiological studies.
Author Summary
Leptospirosis is the most prevalent bacterial zoonosis worldwide. Rodents are believed to be the main reservoirs of Leptospira, yet little epidemiological research has been conducted on rodents from Southeast Asia. Previous studies suggest that activities which place humans in microenvironments shared by rodents increase the probability of contracting leptospirosis. We therefore investigated the circulation of leptospiral species and strains in rodent communities and human populations in seven localities scattered throughout Southeast Asia; in Thailand, Lao PDR and Cambodia. Molecular typing assays were used to characterize leptospiral species and strains in both rodents and humans, which demonstrated common strains between humans and rodents. Additionally, we observed that the two most abundant leptospiral species; L. borgpetersenii and L. interrogans, have different habitat requirements, which supposes different modes of transmission. Lastly, in Southeast Asia, the risk of leptospiral transmission to humans is not solely limited to wetlands and rice paddy fields, but is also linked to forested areas, and activities such as the hunting and/or preparation of rodents for consumption.
doi:10.1371/journal.pntd.0002902
PMCID: PMC4046967  PMID: 24901706
7.  Isolation and Characterization of New Leptospira Genotypes from Patients in Mayotte (Indian Ocean) 
Background
Leptospirosis has been implicated as a severe and fatal form of disease in Mayotte, a French-administrated territory located in the Comoros archipelago (southwestern Indian Ocean). To date, Leptospira isolates have never been isolated in this endemic region.
Methods and Findings
Leptospires were isolated from blood samples from 22 patients with febrile illness during a 17-month period after a PCR-based screening test was positive. Strains were typed using hyper-immune antisera raised against the major Leptospira serogroups: 20 of 22 clinical isolates were assigned to serogroup Mini; the other two strains belonged to serogroups Grippotyphosa and Pyrogenes, respectively. These isolates were further characterized using partial sequencing of 16S rRNA and ligB gene, Multi Locus VNTR Analysis (MLVA), and pulsed field gel electrophoresis (PFGE). Of the 22 isolates, 14 were L. borgpetersenii strains, 7 L. kirschneri strains, and 1, belonging to serogoup Pyrogenes, was L. interrogans. Results of the genotyping methods were consistent. MLVA defined five genotypes, whereas PFGE allowed the recognition of additional subgroups within the genotypes. PFGE fingerprint patterns of clinical strains did not match any of the patterns in the reference strains belonging to the same serogroup, suggesting that the strains were novel serovars.
Conclusions
Preliminary PCR screening of blood specimen allowed a high isolation frequency of leptospires among patients with febrile illness. Typing of leptospiral isolates showed that causative agents of leptospirosis in Mayotte have unique molecular features.
Author Summary
Leptospirosis has been recognized as an increasing public health problem affecting poor people from developing countries and tropical regions. However, the epidemiology of leptospirosis remains poorly understood in remote parts of the world. In this study of patients from the island of Mayotte, we isolated 22 strains from the blood of patients during the acute phase of illness. The pathogenic Leptospira strains were characterized by serology and various molecular typing methods. Based on serological data, serogroup Mini appears to be the dominant cause of leptospirosis in Mayotte. Further molecular characterization of these isolates allowed the identification of 10 pathogenic Leptospira genotypes that could correspond to previously unknown serovars. Further progress in our understanding of the epidemiology of Leptospira circulating genotypes in highly endemic regions should contribute to the development of novel strategies for the diagnosis and prevention of this neglected emerging disease.
doi:10.1371/journal.pntd.0000724
PMCID: PMC2889827  PMID: 20582311
8.  Characterization of a virulent Leptospira interrogans strain isolated from an abandoned swimming pool 
Brazilian Journal of Microbiology  2013;44(1):165-170.
Pathogenic Leptospira spp. are the etiological agents of leptospirosis, an important disease of both humans and animals. In urban settings, L. interrogans serovars are the predominant cause of disease in humans. The purpose of this study was to characterize a novel Leptospira isolate recovered from an abandoned swimming pool. Molecular characterization through sequencing of the rpoB gene revealed 100% identity with L. interrogans and variable-number tandem-repeat (VNTR) analysis resulted in a banding pattern identical to L. interrogans serogroup Icterohaemorrhagiae, serovar Copenhageni or Icterohaemorrhagiae. The virulence of the strain was determined in a hamster model of lethal leptospirosis. The lethal dose 50% (LD50) was calculated to be two leptospires in female hamsters and a histopathological examination of infected animals found typical lesions associated with severe leptospirosis, including renal epithelium degeneration, hepatic karyomegaly, liver-plate disarray and lymphocyte infiltration. This highly virulent strain is now available for use in further studies, especially evaluation of vaccine candidates.
doi:10.1590/S1517-83822013005000029
PMCID: PMC3804194  PMID: 24159300
Leptospira; Leptospirosis; Virulent; VNTR; rpoB
9.  A LigA Three-Domain Region Protects Hamsters from Lethal Infection by Leptospira interrogans 
The leptospiral LigA protein consists of 13 bacterial immunoglobulin-like (Big) domains and is the only purified recombinant subunit vaccine that has been demonstrated to protect against lethal challenge by a clinical isolate of Leptospira interrogans in the hamster model of leptospirosis. We determined the minimum number and location of LigA domains required for immunoprotection. Immunization with domains 11 and 12 was found to be required but insufficient for protection. Inclusion of a third domain, either 10 or 13, was required for 100% survival after intraperitoneal challenge with Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130. As in previous studies, survivors had renal colonization; here, we quantitated the leptospiral burden by qPCR to be 1.2×103 to 8×105 copies of leptospiral DNA per microgram of kidney DNA. Although renal histopathology in survivors revealed tubulointerstitial changes indicating an inflammatory response to the infection, blood chemistry analysis indicated that renal function was normal. These studies define the Big domains of LigA that account for its vaccine efficacy and highlight the need for additional strategies to achieve sterilizing immunity to protect the mammalian host from leptospiral infection and its consequences.
Author Summary
Leptospirosis is the most widespread bacterial infection transmitted to humans from host animals that harbor the bacteria in their kidneys. Human infections caused by the bacterium, Leptospira interrogans, frequently result in a life-threatening illness characterized by jaundice and kidney failure. Vaccines are urgently needed to prevent leptospirosis in populations at risk. The leptospiral protein, LigA, is a promising vaccine candidate because it is the first purified protein to be shown to protect animals from fatal leptospirosis. The goal of this study was to determine which of LigA's 13 domains are required for the protective effect. Immunization with domains 11 and 12 was found to be required, but was insufficient, for protection. A third domain, either 10 or 13, was required for 100% survival. As in previous studies, residual bacteria were cultured from the kidneys of survivors. However, in contrast to previous studies, we determined the amount of bacterial DNA in the kidneys as a measure of vaccine efficacy. We also examined the kidneys microscopically for signs of damage and measured blood chemistries to assess kidney function. These are important steps towards developing vaccines that provide protection from kidney damage and infection.
doi:10.1371/journal.pntd.0001422
PMCID: PMC3236721  PMID: 22180800
10.  Preliminary Characterization of Mus musculus–Derived Pathogenic Strains of Leptospira borgpetersenii Serogroup Ballum in a Hamster Model 
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.
doi:10.4269/ajtmh.2010.10-0120
PMCID: PMC2911180  PMID: 20682877
11.  Kinetics of Leptospira interrogans Infection in Hamsters after Intradermal and Subcutaneous Challenge 
Background
Leptospirosis is a zoonosis caused by highly motile, helically shaped bacteria that penetrate the skin and mucous membranes through lesions or abrasions, and rapidly disseminate throughout the body. Although the intraperitoneal route of infection is widely used to experimentally inoculate hamsters, this challenge route does not represent a natural route of infection.
Methodology/Principal Findings
Here we describe the kinetics of disease and infection in hamster model of leptospirosis after subcutaneous and intradermal inoculation of Leptospira interrogans serovar Copenhageni, strain Fiocruz L1-130. Histopathologic changes in and around the kidney, including glomerular and tubular damage and interstitial inflammatory changes, began on day 5, and preceded deterioration in renal function as measured by serum creatinine. Weight loss, hemoconcentration, increased absolute neutrophil counts (ANC) in the blood and hepatic dysfunction were first noted on day 6. Vascular endothelial growth factor, a serum marker of sepsis severity, became elevated during the later stages of infection. The burden of infection, as measured by quantitative PCR, was highest in the kidney and peaked on day 5 after intradermal challenge and on day 6 after subcutaneous challenge. Compared to subcutaneous challenge, intradermal challenge resulted in a lower burden of infection in both the kidney and liver on day 6, lower ANC and less weight loss on day 7.
Conclusions/Significance
The intradermal and subcutaneous challenge routes result in significant differences in the kinetics of dissemination and disease after challenge with L. interrogans serovar Copenhageni strain Fiocruz L1-130 at an experimental dose of 2×106 leptospires. These results provide new information regarding infection kinetics in the hamster model of leptospirosis.
Author Summary
Leptospirosis is the most widespread bacterial infection transmitted from animals to man. Humans are exposed to infection when host animals that harbor the bacteria in their kidneys shed them in their urine. Human infections, caused by the bacterium Leptospira interrogans, frequently result in a life-threatening illness characterized by liver and kidney failure. In the hamster model of leptospirosis, signs of hepatic and renal dysfunction developed on days 6 and 7, respectively, after intradermal and subcutaneous inoculation of L. interrogans. Renal dysfunction was preceded by the development of inflammatory changes and the appearance of large numbers of leptospires in the kidney on day 5. On day 6, animals began to lose weight, became dehydrated, and had elevated numbers of neutrophils circulating in their bloodstream. Importantly, animals inoculated intradermally had lower numbers of bacteria in their liver and kidneys on day 6 than animals inoculated subcutaneously and lower weight loss and circulating neutrophil levels on day 7. These studies show that the hamster model of leptospirosis is similar to human infection and indicate that the route of infection has significant effects on the course of the illness.
doi:10.1371/journal.pntd.0003307
PMCID: PMC4239013  PMID: 25411782
12.  Establishment of a leptospirosis model in guinea pigs using an epicutaneous inoculations route 
Background
Leptospires are presumed to enter their host via small abrasions or breaches of the skin. The intraperitoneal route, although commonly used in guinea pig and hamster models of leptospirosis, does not reflect conditions encountered during natural infection. The aim of this study is to develop a novel leptospirosis guinea pig model through epicutaneous route and to elucidate the pathogenesis of leptospirosis in experimental guinea pigs by comparing the data from other studies using different infection routes.
Methods
The guinea pigs were inoculated with 5 × 108 Leptospira interrogans strain Lai onto either shaved-only or abraded skin. The guinea pigs were sacrificed at 2, 8, 24, 48, 72, 96 and 144 h post-infection (p.i.) followed by harvest of the lungs, liver, kidneys, spleen, and the skin around the inoculated sites for further examinations. Hematoxylin and eosin (HE) staining and electron microscopy were used to detect the pathologic changes. Real time PCR and immunohistochemistry staining were performed to detect dynamic distribution of leptospires in blood and tissues, respectively.
Results
In the guinea pigs with abraded skin inoculations, leptospires were detected in blood as early as 2 h post infection (p.i.) and then disseminated to the liver, lungs and kidneys of almost all animals within 96 h p.i.. Leptospires were also detected engulfed in the swelling vascular endothelial cells and were frequently aggregated around the capillaries in the dermis and subcutaneous tissue under the inoculated site. For the guinea pigs with abraded skin inoculations, hemorrhage at the dermis around the inoculated site was found before the appearance of internal organs hemorrhage, severe lesions such as hemorrhages in the lungs, nephritis, jaundice, haematuria were also observed, and two of seven guinea pigs died at 144 h p.i. while no lesions and leptospires were detected in the shaved-only guinea pigs using the same dose of strain Lai.
Conclusion
Intact keratinocyte layer is a very efficient barrier against leptospires, and intact skin can prevent the infiltration of leptosipres to the host. Leptospires can penetrate abraded skin and quickly establish a systemic infection by crossing tissue barriers. We have successfully established a novel leptospirosis guinea pig model through epicutaneous inoculations route, which replicates a natural course of infection and appears to be an alternative way to investigate the pathogenesis of leptospirosis, especially in terms of early stage of host-pathogen interactions. This novel model may also be advantageous for studies of the mechanisms involved in cutaneous barriers and epidermal interactions with this organism.
doi:10.1186/1471-2334-12-20
PMCID: PMC3329641  PMID: 22273178
13.  Human Leptospirosis Caused by a New, Antigenically Unique Leptospira Associated with a Rattus Species Reservoir in the Peruvian Amazon 
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.
doi:10.1371/journal.pntd.0000213
PMCID: PMC2271056  PMID: 18382606
14.  Oral Immunization with Escherichia coli Expressing a Lipidated Form of LigA Protects Hamsters against Challenge with Leptospira interrogans Serovar Copenhageni 
Infection and Immunity  2014;82(2):893-902.
Leptospirosis is a potentially fatal zoonosis transmitted by reservoir host animals that harbor leptospires in their renal tubules and shed the bacteria in their urine. Leptospira interrogans serovar Copenhageni transmitted from Rattus norvegicus to humans is the most prevalent cause of urban leptospirosis. We examined L. interrogans LigA, domains 7 to 13 (LigA7-13), as an oral vaccine delivered by Escherichia coli as a lipidated, membrane-associated protein. The efficacy of the vaccine was evaluated in a susceptible hamster model in terms of the humoral immune response and survival from leptospiral challenge. Four weeks of oral administration of live E. coli expressing LigA7-13 improved survival from intraperitoneal (i.p.) and intradermal (i.d.) challenge by L. interrogans serovar Copenhageni strain Fiocruz L1-130 in Golden Syrian hamsters. Immunization with E. coli expressing LigA7-13 resulted in a systemic antibody response, and a significant LigA7-13 IgG level after the first 2 weeks of immunization was completely predictive of survival 28 days after challenge. As in previous LigA vaccine studies, all immunized hamsters that survived infection had renal leptospiral colonization and histopathological changes. In summary, an oral LigA-based vaccine improved survival from leptospiral challenge by either the i.p. or i.d. route.
doi:10.1128/IAI.01533-13
PMCID: PMC3911400  PMID: 24478102
15.  Application of Multilocus Variable-Number Tandem-Repeat Analysis for Molecular Typing of the Agent of Leptospirosis 
Journal of Clinical Microbiology  2006;44(11):3954-3962.
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.
doi:10.1128/JCM.00336-06
PMCID: PMC1698352  PMID: 17088367
16.  The OmpL37 Surface-Exposed Protein Is Expressed by Pathogenic Leptospira during Infection and Binds Skin and Vascular Elastin 
Pathogenic Leptospira spp. shed in the urine of reservoir hosts into freshwater can be transmitted to a susceptible host through skin abrasions or mucous membranes causing leptospirosis. The infection process involves the ability of leptospires to adhere to cell surface and extracellular matrix components, a crucial step for dissemination and colonization of host tissues. Therefore, the elucidation of novel mediators of host-pathogen interaction is important in the discovery of virulence factors involved in the pathogenesis of leptospirosis. In this study, we assess the functional roles of transmembrane outer membrane proteins OmpL36 (LIC13166), OmpL37 (LIC12263), and OmpL47 (LIC13050), which we recently identified on the leptospiral surface. We determine the capacity of these proteins to bind to host tissue components by enzyme-linked immunosorbent assay. OmpL37 binds elastin preferentially, exhibiting dose-dependent, saturating binding to human skin (Kd, 104±19 nM) and aortic elastin (Kd, 152±27 nM). It also binds fibrinogen (Kd, 244±15 nM), fibrinogen fragment D (Kd, 132±30 nM), plasma fibronectin (Kd, 359±68 nM), and murine laminin (Kd, 410±81 nM). The binding to human skin elastin by both recombinant OmpL37 and live Leptospira interrogans is specifically enhanced by rabbit antiserum for OmpL37, suggesting the involvement of OmpL37 in leptospiral binding to elastin and also the possibility that host-generated antibodies may promote rather than inhibit the adherence of leptospires to elastin-rich tissues. Further, we demonstrate that OmpL37 is recognized by acute and convalescent leptospirosis patient sera and also by Leptospira-infected hamster sera. Finally, OmpL37 protein is detected in pathogenic Leptospira serovars and not in saprophytic Leptospira. Thus, OmpL37 is a novel elastin-binding protein of pathogenic Leptospira that may be promoting attachment of Leptospira to host tissues.
Author Summary
Leptospirosis is a potentially fatal disease in humans and livestock caused by Leptospira bacteria. Effective antibiotic treatment depends on timely, accurate diagnosis. However, current diagnostic and vaccine options are limited by their specificity for the lipid-sugar coat of leptospires, which varies among 200 serum-reactive groups. We aim to understand how leptospires infect a host, and in turn, to develop broadly effective diagnostic and immunization products. We recently described OmpL37, a new protein on the surface of leptospires. Here, we show it is made by pathogenic strains, suggesting it can be a target for detecting and protecting against a wide range of Leptospira. Moreover, leptospirosis patients and hamsters infected with leptospires make antibodies against OmpL37. Purified OmpL37 binds host proteins, including human elastin, fibrinogen, fibronectin, and mouse laminin. Although other leptospiral proteins bind multiple host proteins, OmpL37 has novel preferential affinity for skin and aorta elastin, suggesting a role in a common route of transmission through abraded skin and exposed blood vessels. Indeed, OmpL37 binding and leptospiral attachment to elastin are both enhanced by OmpL37 antiserum, further implicating a possible role for OmpL37 during infection. Thus, OmpL37 may mediate host attachment and has potential clinical application with a broad range of Leptospira.
doi:10.1371/journal.pntd.0000815
PMCID: PMC2935396  PMID: 20844573
17.  Human Leptospira Isolates Circulating in Mayotte (Indian Ocean) Have Unique Serological and Molecular Features 
Journal of Clinical Microbiology  2012;50(2):307-311.
Leptospirosis is one of the most widespread zoonoses in the world. However, there is a lack of information on circulating Leptospira strains in remote parts of the world. We describe the serological and molecular features of leptospires isolated from 94 leptospirosis patients in Mayotte, a French department located in the Comoros archipelago, between 2007 and 2010. Multilocus sequence typing identified these isolates as Leptospira interrogans, L. kirschneri, L. borgpetersenii, and members of a previously undefined phylogenetic group. This group, consisting of 15 strains, could represent a novel species. Serological typing revealed that 70% of the isolates belonged to the serogroup complex Mini/Sejroe/Hebdomadis, followed by the serogroups Pyrogenes, Grippotyphosa, and Pomona. However, unambiguous typing at the serovar level was not possible for most of the strains because the isolate could belong to more than one serovar or because serovar and species did not match the original classification. Our results indicate that the serovar and genotype distribution in Mayotte differs from what is observed in other regions, thus suggesting a high degree of diversity of circulating isolates worldwide. These results are essential for the improvement of current diagnostic tools and provide a starting point for a better understanding of the epidemiology of leptospirosis in this area of endemicity.
doi:10.1128/JCM.05931-11
PMCID: PMC3264139  PMID: 22162544
18.  Leptospira Interrogans Induces Fibrosis in the Mouse Kidney through Inos-Dependent, TLR- and NLR-Independent Signaling Pathways 
Background
Leptospira (L.) interrogans are bacteria responsible for a worldwide reemerging zoonosis. Rodents carry L. interrogans asymptomatically in their kidneys and excrete bacteria in the urine, contaminating the environment. Humans get infected through skin contact and develop a mild or severe leptospirosis that may lead to renal failure and fibrosis. L. interrogans provoke an interstitial nephritis, but the induction of fibrosis caused by L. interrogans has not been studied in murine models. Innate immune receptors from the TLR and NLR families have recently been shown to play a role in the development and progression of tissue fibrosis in the lung, liver and kidneys under different pathophysiological situations. We recently showed that TLR2, TLR4, and NLRP3 receptors were crucial in the defense against leptospirosis. Moreover, infection of a human cell line with L. interrogans was shown to induce TLR2-dependent production of fibronectin, a component of the extracellular matrix. Therefore, we thought to assess the presence of renal fibrosis in L. interrogans infected mice and to analyze the contribution of some innate immune pathways in this process.
Methodology/principal findings
Here, we characterized by immunohistochemical studies and quantitative real-time PCR, a model of Leptospira-infected C57BL/6J mice, with chronic carriage of L. interrogans inducing mild renal fibrosis. Using various strains of transgenic mice, we determined that the renal infiltrates of T cells and, unexpectedly, TLR and NLR receptors, are not required to generate Leptospira-induced renal fibrosis. We also show that the iNOS enzyme, known to play a role in Leptospira-induced interstitial nephritis, also plays a role in the induction of renal fibrosis.
Conclusion/significance
To our knowledge, this work provides the first experimental murine model of sustained renal fibrosis induced by a chronic bacterial infection that may be peculiar, since it does not rely on TLR or NLR receptors. This model may prove useful to test future therapeutic strategies to combat Leptospira-induced renal lesions.
Author Summary
Leptospirosis is a bacterial disease transmitted by asymptomatic rodents to humans. The symptoms may be mild, or severe with kidney failure. Renal fibrosis, occurring during inflammatory situations, is characterized by the pathological accumulation of extra-cellular matrix components and can compromise the kidney functions of patients with leptospirosis. Recent research revealed that both innate and adaptive immune responses are involved in the establishment of fibrosis, in several organs and in different pathophysiological situations. In the present study, we characterized a mouse model of chronic infection with Leptospira that provokes mild renal fibrosis. We show that fibrogenesis requires the presence of live Leptospira in the kidney and that B and T cells from the adaptive immune response do not participate in the induction of renal fibrosis. Unexpectedly, we also found that innate immune receptors, TLRs and NLRs, are not involved in the Leptospira-induced fibrosis. Finally, we show that the enzyme responsible for NO production, iNOS, known to participate in renal inflammatory lesions induced by Leptospira, is also involved in renal fibrosis. Our work provides a novel mouse model to study fibrosis occurring due to leptospirosis.
doi:10.1371/journal.pntd.0002664
PMCID: PMC3907306  PMID: 24498450
19.  The OmpA-Like Protein Loa22 Is Essential for Leptospiral Virulence 
PLoS Pathogens  2007;3(7):e97.
Pathogenic mechanisms of Leptospira interrogans, the causal agent of leptospirosis, remain largely unknown. This is mainly due to the lack of tools for genetic manipulations of pathogenic species. In this study, we characterized a mutant obtained by insertion of the transposon Himar1 into a gene encoding a putative lipoprotein, Loa22, which has a predicted OmpA domain based on sequence identity. The resulting mutant did not express Loa22 and was attenuated in virulence in the guinea pig and hamster models of leptospirosis, whereas the genetically complemented strain was restored in Loa22 expression and virulence. Our results show that Loa22 was expressed during host infection and exposed on the cell surface. Loa22 is therefore necessary for virulence of L. interrogans in the animal model and represents, to our knowledge, the first genetically defined virulence factor in Leptospira species.
Author Summary
The spirochetes, which include medically important pathogens such as the causative agents of Lyme disease, syphilis, and leptospirosis, constitute an evolutionarily unique group of bacteria. Leptospirosis is a zoonotic disease that causes a high rate of mortality and morbidity in humans and animals throughout the world each year. The year 2007 marks the centenary of the discovery of the causative agent of leptospirosis, Leptospira interrogans. Until now, the genetic obstacles posed by leptospires (principally, the difficulties in generating targeted mutants) have hampered the identification of virulence genes. In this study, we describe an avirulent mutant in a pathogenic Leptospira that was obtained via disruption of loa22, a gene that encodes an outer membrane protein containing an OmpA domain. This mutation resulted in an avirulent mutant in the guinea pig model, and reintroduction of loa22 into the mutant restored Leptospira's ability to kill guinea pigs. Our results therefore indicate that loa22 is a virulence determinant that is, to our knowledge, the first identified for this pathogen.
doi:10.1371/journal.ppat.0030097
PMCID: PMC1914066  PMID: 17630832
20.  Pathogenomic Inference of Virulence-Associated Genes in Leptospira interrogans 
Leptospirosis is a globally important, neglected zoonotic infection caused by spirochetes of the genus Leptospira. Since genetic transformation remains technically limited for pathogenic Leptospira, a systems biology pathogenomic approach was used to infer leptospiral virulence genes by whole genome comparison of culture-attenuated Leptospira interrogans serovar Lai with its virulent, isogenic parent. Among the 11 pathogen-specific protein-coding genes in which non-synonymous mutations were found, a putative soluble adenylate cyclase with host cell cAMP-elevating activity, and two members of a previously unstudied ∼15 member paralogous gene family of unknown function were identified. This gene family was also uniquely found in the alpha-proteobacteria Bartonella bacilliformis and Bartonella australis that are geographically restricted to the Andes and Australia, respectively. How the pathogenic Leptospira and these two Bartonella species came to share this expanded gene family remains an evolutionary mystery. In vivo expression analyses demonstrated up-regulation of 10/11 Leptospira genes identified in the attenuation screen, and profound in vivo, tissue-specific up-regulation by members of the paralogous gene family, suggesting a direct role in virulence and host-pathogen interactions. The pathogenomic experimental design here is generalizable as a functional systems biology approach to studying bacterial pathogenesis and virulence and should encourage similar experimental studies of other pathogens.
Author Summary
Leptospirosis is one of the most common diseases transmitted by animals worldwide. It is important because it causes an often lethal febrile illnesses in tropical and subtropical areas associated with poor sanitation and agriculture. Leptospirosis may be epidemic, associated with natural disasters and flooding, or endemic in tropical regions. It is unknown how Leptospira cause disease and why different strains cause different severity of illness. In this study we attenuated (weakened) a highly virulent strain of L. interrogans by culturing it in vitro over several months. Comparison of the whole genome sequence before and after the attenuation process revealed a small set of genes that were mutated, and therefore associated with virulence. We discovered a putative soluble adenylate cyclase with host cell cAMP elevating activity, with implications for immune evasion and a new gene family that is upregulated in vivo during acute hamster infection. Interestingly, both Bartonella bacilliformis and Bartonella australis also have this unique gene family we describe in pathogenic Leptospira. This information aids in our understanding of Leptospira evolution and pathogenesis.
doi:10.1371/journal.pntd.0002468
PMCID: PMC3789758  PMID: 24098822
21.  Leptospiral Outer Membrane Proteins OmpL1 and LipL41 Exhibit Synergistic Immunoprotection 
Infection and Immunity  1999;67(12):6572-6582.
New vaccine strategies are needed for prevention of leptospirosis, a widespread human and veterinary disease caused by invasive spirochetes belonging to the genus Leptospira. We have examined the immunoprotective capacity of the leptospiral porin OmpL1 and the leptospiral outer membrane lipoprotein LipL41 in the Golden Syrian hamster model of leptospirosis. Specialized expression plasmids were developed to facilitate expression of leptospiral proteins in Escherichia coli as the membrane-associated proteins OmpL1-M and LipL41-M. Although OmpL1-M expression is highly toxic in E. coli, this was accomplished by using plasmid pMMB66-OmpL1, which has undetectable background expression without induction. LipL41-M expression and processing were enhanced by altering its lipoprotein signal peptidase cleavage site to mimic that of the murein lipoprotein. Active immunization of hamsters with E. coli membrane fractions containing a combination of OmpL1-M and LipL41-M was found to provide significant protection against homologous challenge with Leptospira kirschneri serovar grippotyphosa. At 28 days after intraperitoneal inoculation, survival in animals vaccinated with both proteins was 71% (95% confidence interval [CI], 53 to 89%), compared with only 25% (95% CI, 8 to 42%) in the control group (P < 0.001). On the basis of serological, histological, and microbiological assays, no evidence of infection was found in the vaccinated survivors. The protective effects of immunization with OmpL1-M and LipL41-M were synergistic, since significant levels of protection were not observed in animals immunized with either OmpL1-M or LipL41-M alone. In contrast to immunization with the membrane-associated forms of leptospiral proteins, hamsters immunized with His6-OmpL1 and His6-LipL41 fusion proteins, either alone or in combination, were not protected. These data indicate that the manner in which OmpL1 and LipL41 associates with membranes is an important determinant of immunoprotection.
PMCID: PMC97069  PMID: 10569777
22.  Variation Between Strains of Hamsters in the Lethality of Pichinde Virus Infections 
Infection and Immunity  1977;16(2):413-421.
Infection by Pichinde virus, a member of the arenavirus group, was studied in Golden Syrian hamsters (Mesocricetus auratus) with regard to possible mechanisms of resistance to virus infection in adult hamsters. Two hamster strains were found to differ in their susceptibility to lethal Pichinde virus infection. LVG/Lak randomly bred hamsters were found to be 100% susceptible to low doses of Pichinde virus during the first 6 days of life, but after 8 days of life, mortality was uncommon. Peak virus titers in the serum of animals infected at 3 days of life were 4 logs greater than in animals infected at 12 days. MHA/Lak inbred hamsters, in contrast, were found to be susceptible to lethal virus infection both as newborns and as adults. Peak virus titers of greater than 108 plaque-forming units/ml were observed in serum 8 days after infection of adult MHA hamsters as compared with less than 103 plaque-forming units/ml in the serum of adult LVG hamsters. Cultured primary kidney cells and peritoneal macrophages from either hamster strain supported Pichinde virus replication equally well in vitro. Antibodies to the complement-fixing antigens and to antigens at the surface of virus-infected cells were produced by both strains of hamsters. Cyclophosphamide immunosuppression rendered adult LVG animals susceptible to lethal infections, and virus grew to high titers in the treated animals. These findings suggest that immunological factors that appear early in life in LVG hamsters and are deficient in MHA hamsters limit Pichinde virus infection. Unlike previously reported arenavirus diseases, the observations suggest that death is produced by a direct viral effect and not through immunopathological mechanisms.
PMCID: PMC420966  PMID: 193786
23.  Gene Expression Profiles of Immune Mediators and Histopathological Findings in Animal Models of Leptospirosis: Comparison between Susceptible Hamsters and Resistant Mice ▿  
Infection and Immunity  2011;79(11):4480-4492.
Leptospirosis is a widespread zoonosis characterized by multiple organ failure and variable host susceptibility toward pathogenic Leptospira strains. In this study, we put the role of inflammatory mediators in parallel with bacterial burdens and organ lesions by comparing a susceptible animal model, the hamster, and a resistant one, the Oncins France 1 (OF1) mouse, both infected with virulent Leptospira interrogans serovar Icterohaemorrhagiae strain Verdun. Histological observations evidenced edema, congestion, hemorrhage, and inflammatory infiltration in the organs of hamsters, in contrast to limited changes in mice. Using reverse transcription-quantitative PCR techniques, we showed that the relative Leptospira burden progressively increased in hamster tissues, while a rapid clearance was observed in mouse tissues. The early regulation of the proinflammatory mediators interleukin-1β (IL-1β), IL-6, tumor necrosis factor alpha, and cyclo-oxygenase-2 and the chemokines gamma interferon-inducible protein 10 kDa/CXCL10 and macrophage inflammatory protein-1α/CCL3 in mouse tissues contrasted with their delayed and massive overexpression in hamster tissues. Conversely, the induction of the anti-inflammatory cytokine IL-10 was faster in the resistant than in the susceptible animal model. The role of these cytokines in the pathophysiology of leptospirosis and the implications of their differential regulation in the development of this disease are discussed.
doi:10.1128/IAI.05727-11
PMCID: PMC3257942  PMID: 21844232
24.  Leptospira species and serovars identified by MALDI-TOF mass spectrometry after database implementation 
BMC Research Notes  2014;7:330.
Background
Leptospirosis, a spirochaetal zoonotic disease of worldwide distribution, endemic in Europe, has been recognized as an important emerging infectious disease, though yet it is mostly a neglected disease which imparts its greatest burden on impoverished populations from developing countries. Leptospirosis is caused by the infection with any of the more than 230 serovars of pathogenic Leptospira sp. In this study we aimed to implement the MALDI-TOF mass spectrometry (MS) database currently available in our laboratory with Leptospira reference pathogenic (L. interrogans, L. borgpetersenii, L. kirschneri, L. noguchii), intermediate (L. fainei) and saprophytic (L. biflexa) strains of our collection in order to evaluate its possible application to the diagnosis of leptospirosis and to the typing of strains. This was done with the goal of understanding whether this methodology could be used as a tool for the identification of Leptospira strains, not only at species level for diagnostic purposes, but also at serovar level for epidemiological purposes, overcoming the limits of serological and molecular conventional methods. Twenty Leptospira reference strains were analysed by MALDI-TOF MS. Statistical analysis of the protein spectra was performed by ClinProTools software.
Results
The spectra obtained by the analysis of the reference strains tested were grouped into 6 main classes corresponding to the species analysed, highlighting species-specific protein profiles. Moreover, the statistical analysis of the spectra identified discriminatory peaks to recognize Leptospira strains also at serovar level extending previously published data.
Conclusions
In conclusion, we confirmed that MALDI-TOF MS could be a powerful tool for research and diagnostic in the field of leptospirosis with broad applications ranging from the detection and identification of pathogenic leptospires for diagnostic purposes to the typing of pathogenic and non-pathogenic leptospires for epidemiological purposes in order to enrich our knowledge about the epidemiology of the infection in different areas and generate control strategies.
doi:10.1186/1756-0500-7-330
PMCID: PMC4048046  PMID: 24890024
Leptospira sp.; MALDI-TOF MS; Identification; Database implementation
25.  The terminal portion of leptospiral immunoglobulin-like protein LigA confers protective immunity against lethal infection in the hamster model of leptospirosis 
Vaccine  2007;25(33):6277-6286.
Subunit vaccines are a potential intervention strategy against leptospirosis, which is a major public health problem in developing countries and a veterinary disease in livestock and companion animals worldwide. Leptospiral immunoglobulin-like (Lig) proteins are a family of surface-exposed determinants that have Ig-like repeat domains found in virulence factors such as intimin and invasin. We expressed fragments of the repeat domain regions of LigA and LigB from Leptospira interrogans serovar Copenhageni. Immunization of Golden Syrian hamsters with Lig fragments in Freund’s adjuvant induced robust antibody responses against recombinant protein and native protein, as detected by ELISA and immunoblot, respectively. A single fragment, LigANI, which corresponds to the six carboxy-terminal Ig-like repeat domains of the LigA molecule, conferred immunoprotection against mortality (67-100%, P <0.05) in hamsters which received a lethal inoculum of L. interrogans serovar Copenhageni. However, immunization with this fragment did not confer sterilizing immunity. These findings indicate that the carboxy-terminal portion of LigA is an immunoprotective domain and may serve as a vaccine candidate for human and veterinary leptospirosis.
doi:10.1016/j.vaccine.2007.05.053
PMCID: PMC1994161  PMID: 17629368
Leptospirosis; subunit vaccine; Leptospiral immunoglobulin-like protein; recombinant protein; immunity; antibodies; hamsters

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