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1.  Highly Virulent Leptospira borgpetersenii Strain Characterized in the Hamster Model 
A recent study by our group reported the isolation and partial serological and molecular characterization of four Leptospira borgpetersenii serogroup Ballum strains. Here, we reproduced experimental leptospirosis in golden Syrian hamsters (Mesocricetus auratus) and carried out standardization of lethal dose 50% (LD50) of one of these strains (4E). Clinical disease features and histopathologic analyses of tissue lesions were also observed. As results, strain 4E induced lethality in the hamster model with inocula lower than 10 leptospires, and histopathological examination of animals showed typical lesions found in severe leptospirosis. Gross pathological findings were peculiar; animals that died early had more chance of presenting severe jaundice and less chance of presenting pulmonary hemorrhages (P < 0.01). L. borgpetersenii serogroup Ballum has had a considerable growth in human leptospirosis cases in recent years. This strain has now been thoroughly characterized and can be used in more studies, especially evaluations of vaccine candidates.
doi:10.4269/ajtmh.2011.11-0013
PMCID: PMC3144824  PMID: 21813846
2.  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
3.  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
4.  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
5.  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
6.  Live Imaging of Bioluminescent Leptospira interrogans in Mice Reveals Renal Colonization as a Stealth Escape from the Blood Defenses and Antibiotics 
Leptospira (L.) interrogans are bacteria responsible for a worldwide reemerging zoonosis. Some animals asymptomatically carry L. interrogans in their kidneys and excrete bacteria in their urine, which contaminates the environment. Humans are infected through skin contact with leptospires and develop mild to severe leptospirosis. Previous attempts to construct fluorescent or bioluminescent leptospires, which would permit in vivo visualization and investigation of host defense mechanisms during infection, have been unsuccessful. Using a firefly luciferase cassette and random transposition tools, we constructed bioluminescent chromosomal transformants in saprophytic and pathogenic leptospires. The kinetics of leptospiral dissemination in mice, after intraperitoneal inoculation with a pathogenic transformant, was tracked by bioluminescence using live imaging. For infective doses of 106 to 107 bacteria, we observed dissemination and exponential growth of leptospires in the blood, followed by apparent clearance of bacteria. However, with 2×108 bacteria, the septicemia led to the death of mice within 3 days post-infection. In surviving mice, one week after infection, pathogenic leptospires reemerged only in the kidneys, where they multiplied and reached a steady state, leading to a sustained chronic renal infection. These experiments reveal that a fraction of the leptospiral population escapes the potent blood defense, and colonizes a defined number of niches in the kidneys, proportional to the infective dose. Antibiotic treatments failed to eradicate leptospires that colonized the kidneys, although they were effective against L. interrogans if administered before or early after infection. To conclude, mice infected with bioluminescent L. interrogans proved to be a novel model to study both acute and chronic leptospirosis, and revealed that, in the kidneys, leptospires are protected from antibiotics. These bioluminescent leptospires represent a powerful new tool to challenge mice treated with drugs or vaccines, and test the survival, dissemination, and transmission of leptospires between environment and hosts.
Author Summary
Leptospirosis is a worldwide neglected disease caused by the pathogenic bacterium named Leptospira interrogans. Some rodents, such as rats, do not get sick from leptospirosis and constitute a reservoir. They carry leptospires in their kidneys and excrete the bacteria in the environment. L. interrogans are mobile and penetrate their hosts through abraded skin or mucosa. Infected humans may develop mild to severe leptospirosis, potentially leading to death. Leptospires are difficult to cultivate and to genetically manipulate, impairing the study of leptospirosis. Here, we constructed bioluminescent leptospires, and monitored infection in live mice by tracking bioluminescence. In the first days after infection, a rapid dissemination and growth of bacteria was observed in the blood circulation, followed around one week after the infection by their apparent disappearance. However, the leptospires reemerged and multiplied in the kidneys, to reach sustained levels three weeks after infection. The use of antibiotics showed that antibiotic-susceptible L. interrogans are very difficult to eradicate once they are settled in the kidneys. Mice infected with bioluminescent leptospires represent a pertinent model to study leptospirosis. These bioluminescent leptospires are novel tools that will be useful to test the efficacy of treatments or vaccines against leptospirosis.
doi:10.1371/journal.pntd.0003359
PMCID: PMC4256284  PMID: 25474719
7.  Identification of the Hemolysis-Associated Protein 1 as a Cross-Protective Immunogen of Leptospira interrogans by Adenovirus-Mediated Vaccination 
Infection and Immunity  2001;69(11):6831-6838.
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.
doi:10.1128/IAI.69.11.6831-6838.2001
PMCID: PMC100061  PMID: 11598056
8.  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
9.  Cross-protection between experimental anti-leptospirosis bacterins 
Brazilian Journal of Microbiology  2014;45(3):1083-1088.
We investigated the existence of cross-protection between two anti-leptospirosis monovalent experimental bacterins produced with two strains of Leptospira serogroup Pomona: Fromm strain of serovar Kennewicky, isolated from pigs in the United States, and strain GR6 of serovar Pomona isolated from pigs in Brazil. Both were added of aluminum hydroxide as an adjuvant. Experimental bacterins were tested with the hamster potency test in order to assess protection provided against the disease and against the establishment of kidney infection. Controls were polyvalent commercial vaccine produced with Leptospira strains isolated outside Brazil, which included a representative of Pomona serovar, or Sorensen solution added of aluminum hydroxide adjuvant. The challenge was performed with cross-strains of serogroup Pomona tested in accordance with international standards established for the potency test. After 21 days of the challenge, survivors were killed to evaluate the condition of Leptospira renal carrier. Experimental bacterins protected hamsters against homologous and heterologous strains, demonstrating the existence of cross-protection. The commercial vaccine protected the hamsters challenged with both strains, but there was a high proportion of animals diagnosed as renal carriers when the challenge was performed with strain GR6, isolated from pigs in Brazil.
PMCID: PMC4204950  PMID: 25477946
Pomona; hamsters; bacterin; cross-protection
10.  Cross-protective Immunity Against Leptospirosis Elicited by a Live, Attenuated Lipopolysaccharide Mutant 
The Journal of Infectious Diseases  2011;203(6):870-879.
Background. Leptospira species cause leptospirosis, a zoonotic disease found worldwide. Current vaccines against leptospirosis provide protection only against closely related serovars.
Methods. We evaluated an attenuated transposon mutant of Leptospira interrogans serovar Manilae (M1352, defective in lipopolysaccharide biosynthesis) as a live vaccine against leptospirosis. Hamsters received a single dose of vaccine and were challenged with the homologous serovar (Manilae) and a serologically unrelated heterologous serovar (Pomona). Comparisons were made with killed vaccines. Potential cross-protective antigens against leptospirosis were investigated.
Results. Live M1352 vaccine induced superior protection in hamsters against homologous challenge. The live vaccine also stimulated cross-protection against heterologous challenge, with 100% survival (live M1352) versus 40% survival (killed vaccine). Hamsters receiving either vaccine responded to the dominant membrane proteins LipL32 and LipL41. Hamsters receiving the live vaccine additionally recognized LA3961/OmpL36 (unknown function), Loa22 (OmpA family protein, recognized virulence factor), LA2372 (general secretory protein G), and LA1939 (hypothetical protein). Manilae LigA was recognized by M1352 vaccinates, whereas LipL36 was detected in Pomona.
Conclusion. This study demonstrated that a live, attenuated vaccine can stimulate cross-protective immunity to L. interrogans and has identified antigens that potentially confer cross-protection against leptospirosis.
doi:10.1093/infdis/jiq127
PMCID: PMC3071135  PMID: 21220775
11.  A Prime-Boost Strategy Using the Novel Vaccine Candidate, LemA, Protects Hamsters against Leptospirosis 
Toward developing an effective vaccine capable of conferring heterologous protection, the putative lipoprotein LemA, which presents an M3 epitope similar to that of Listeria, was evaluated as a vaccine candidate in the hamster model of leptospirosis. LemA is conserved (>70% pairwise identity) among the pathogenic Leptospira spp., indicating its potential in stimulating a cross-protective immune response. Using different vaccination strategies, including prime-boost, DNA vaccine, and a subunit preparation, recombinant LemA conferred different levels of protection in hamsters. Significant protection against mortality was observed for the prime-boost and the DNA vaccine strategies, which showed 87.5% (P < 0.01) and 62.5% (P < 0.05) efficacy, respectively. Although the subunit vaccine preparation protected 50.0% of immunized hamsters, the level of protection was not significant. None of the hamsters in the control groups survived challenge with a virulent strain of Leptospira interrogans serogroup Icterohaemorrhagiae. Characterization of the immune response found that the strongest antibody response was stimulated by the subunit vaccine preparation, followed by the prime-boost strategy. The DNA vaccine failed to elicit an antibody response in immunized hamsters.
doi:10.1128/CVI.00034-13
PMCID: PMC3647757  PMID: 23515012
12.  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
13.  Subunit Approach to Evaluation of the Immune Protective Potential of Leptospiral Antigens ▿ 
Clinical and Vaccine Immunology : CVI  2011;18(12):2026-2030.
Leptospirosis is the most widespread zoonosis in the world. Current vaccines are based on whole-cell preparations that cause severe side effects and do not induce satisfactory immunity. In light of the leptospiral genome sequences recently made available, several studies aimed at identification of protective recombinant immunogens have been performed; however, few such immunogens have been identified. The aim of this study was to evaluate 27 recombinant antigens to determine their potential to induce an immune response protective against leptospirosis in the hamster model. Experiments were conducted with groups of female hamsters immunized with individual antigen preparations. Hamsters were then challenged with a lethal dose of Leptospira interrogans. Thirteen antigens induced protective immune responses; however, only recombinant proteins LIC10325 and LIC13059 induced significant protection against mortality. These results have important implications for the development of an efficacious recombinant subunit vaccine against leptospirosis.
doi:10.1128/CVI.05297-11
PMCID: PMC3232701  PMID: 22030369
14.  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
15.  Characterization of the ompL1 gene of pathogenic Leptospira species in China and cross-immunogenicity of the OmpL1 protein 
BMC Microbiology  2008;8:223.
Background
The usefulness of available vaccine and serological tests for leptospirosis is limited by the low cross-reactivity of antigens from numerous serovars of pathogenic Leptospira spp. Identification of genus-specific protein antigens (GP-Ag) of Leptospira would be important for development of universal vaccines and serodiagnostic methods. OmpL1, a transmembrane porin of pathogenic leptospires, was identified as a possible GP-Ag, but its sequence diversity and immune cross-reactivity among different serovars of pathogenic leptospires remains largely unknown.
Results
PCR analysis demonstrated that the ompL1 gene existed in all 15 official Chinese standard strains as well as 163 clinical strains of pathogenic leptospires isolated in China. In the standard strains, the ompL1 gene could be divided into three groups (ompL1/1, ompL1/2 and ompL1/3) according to their sequence identities. Immune electron microscopy demonstrated that all products of the different gene types of ompL1 are located on the surface of leptospires. The microscopic agglutination test revealed extensive yet distinct cross-immunoagglutination among the antisera against recombinant OmpL1 (rOmpL1) and leptospiral strains belonging to different ompL1 gene types. These cross-immunoreactions were further verified by ELISAs using the OmpL1 proteins as the coated antigens in serum samples from 385 leptospirosis patients. All the antisera against rOmpL1 proteins could inhibit L. interrogans strain Lai from adhering to J774A.1 cells. Furthermore, immunization of guinea pigs with each of the rOmpL1 proteins could cause cross-immunoprotection against lethal challenge with leptospires from different ompL1 gene types.
Conclusion
Three types of the ompL1 gene are present in pathogenic leptospires in China. OmpL1 is an immunoprotective GP-Ag which should be considered in the design of new universal vaccines and serodiagnostic methods against leptospirosis.
doi:10.1186/1471-2180-8-223
PMCID: PMC2632671  PMID: 19087358
16.  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
17.  LipL21 Is a Novel Surface-Exposed Lipoprotein of Pathogenic Leptospira Species  
Infection and Immunity  2003;71(5):2414-2421.
Leptospira is the etiologic agent of leptospirosis, a bacterial zoonosis distributed worldwide. Leptospiral lipopolysaccharide is a protective immunogen, but the extensive serological diversity of leptospires has inspired a search for conserved outer membrane proteins (OMPs) that may stimulate heterologous immunity. Previously, a global analysis of leptospiral OMPs (P. A. Cullen, S. J. Cordwell, D. M. Bulach, D. A. Haake, and B. Adler, Infect. Immun. 70:2311-2318, 2002) identified pL21, a novel 21-kDa protein that is the second most abundant constituent of the Leptospira interrogans serovar Lai outer membrane proteome. In this study, we identified the gene encoding pL21 and found it to encode a putative lipoprotein; accordingly, the protein was renamed LipL21. Southern hybridization analysis revealed the presence of lipL21 in all of the pathogenic species but in none of the saprophytic species examined. Alignment of the LipL21 sequence from six strains of Leptospira revealed 96 to 100% identity. When specific polyclonal antisera to recombinant LipL21 were used, LipL21 was isolated together with other known leptospiral OMPs by both Triton X-114 extraction and sucrose density gradient membrane fractionation. All nine strains of pathogenic leptospires investigated by Western blotting, whether culture attenuated or virulent, were found to express LipL21. In contrast, the expression of LipL21 or an antigenically related protein could not be detected in nonpathogenic L. biflexa. Infected hamster sera and two of eight human leptospirosis sera tested were found to react with recombinant LipL21. Native LipL21 was found to incorporate tritiated palmitic acid, consistent with the prediction of a lipoprotein signal peptidase cleavage site. Biotinylation of the leptospiral surface resulted in selective labeling of LipL21 and the previously known OMPs LipL32 and LipL41. These findings show that LipL21 is a surface-exposed, abundant outer membrane lipoprotein that is expressed during infection and conserved among pathogenic Leptospira species.
doi:10.1128/IAI.71.5.2414-2421.2003
PMCID: PMC153295  PMID: 12704111
18.  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
19.  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
20.  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
21.  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
22.  Detection and Quantification of Leptospira interrogans in Hamster and Rat Kidney Samples: Immunofluorescent Imprints versus Real-time PCR 
PLoS ONE  2012;7(2):e32712.
A major limitation in the clinical management and experimental research of leptospirosis is the poor performance of the available methods for the direct detection of leptospires. In this study, we compared real-time PCR (qPCR), targeting the lipL32 gene, with the immunofluorescent imprint method (IM) for the detection and quantification of leptospires in kidney samples from the rat and hamster experimental models of leptospirosis. Using a virulent strain of Leptospira interrogans serovar Copenhageni, a chronic infection was established in the rat model, which were euthanized 28 days post-infection, while the hamster model simulated an acute infection and the hamsters were euthanized eight days after inoculation. Leptospires in the kidney samples were detected using culture isolation, qPCR and the IM, and quantified using qPCR and the IM. In both the acute and chronic infection models, the correlation between quantification by qPCR and the IM was found to be positive and statistically significant (P<0.05). Therefore, this study demonstrates that the IM is a viable alternative for not only the detection but also the quantification of leptospires, particularly when the use of qPCR is not feasible.
doi:10.1371/journal.pone.0032712
PMCID: PMC3290571  PMID: 22393440
23.  A Conserved Region of Leptospiral Immunoglobulin-Like A and B Proteins as a DNA Vaccine Elicits a Prophylactic Immune Response against Leptospirosis 
The leptospiral immunoglobulin-like (Lig) proteins LigA and LigB possess immunoglobulin-like domains with 90-amino-acid repeats and are adhesion molecules involved in pathogenicity. They are conserved in pathogenic Leptospira spp. and thus are of interest for use as serodiagnostic antigens and in recombinant vaccine formulations. The N-terminal amino acid sequences of the LigA and LigB proteins are identical, but the C-terminal sequences vary. In this study, we evaluated the protective potential of five truncated forms of LigA and LigB proteins from Leptospira interrogans serovar Canicola as DNA vaccines using the pTARGET mammalian expression vector. Hamsters immunized with the DNA vaccines were subjected to a heterologous challenge with L. interrogans serovar Copenhageni strain Spool via the intraperitoneal route. Immunization with a DNA vaccine encoding LigBrep resulted in the survival of 5/8 (62.5%) hamsters against lethal infection (P < 0.05). None of the control hamsters or animals immunized with the other vaccine preparations survived. The vaccine induced an IgG antibody response and, additionally, conferred sterilizing immunity in 80% of the surviving animals. Our results indicate that the LigBrep DNA vaccine is a promising candidate for inclusion in a protective leptospiral vaccine.
doi:10.1128/CVI.00601-12
PMCID: PMC3647749  PMID: 23486420
24.  Evaluation of Immunoprotective Activity of Six Leptospiral Proteins in the Hamster Model of Leptospirosis 
Leptospirosis is a worldwide zoonosis caused by pathogenic Leptospira. The whole-genome sequence of L. interrogans serovar Copenhageni together with bioinformatics tools represent a great opportunity to search for novel antigen candidates that could be used as subunit vaccine against leptospirosis. We focused on six genes encoding for conserved hypothetical proteins predicted to be exported to the outer membrane. The genes were amplified by PCR from Leptospira interrogans genomic DNA and were cloned and expressed in Escherichia coli. The recombinant proteins tagged with N-terminal hexahistidine were purified by metal-charged chromatography. The immunization of hamsters followed by challenge with lethal dose of virulent strain of Leptospira showed that the recombinant proteins Lsa21, Lsa66 and rLIC11030 elicited partial protection to animals. These proteins could be used combined or in a mixture with novel adjuvants in order to improve their effectiveness.
doi:10.2174/1874285801206010079
PMCID: PMC3502890  PMID: 23173023
Leptospira interrogans; leptospirosis; recombinant protein; vaccine.
25.  Protection against Lethal Leptospirosis after Vaccination with LipL32 Coupled or Coadministered with the B Subunit of Escherichia coli Heat-Labile Enterotoxin 
Leptospirosis, a worldwide zoonosis, lacks an effective, safe, and cross-protective vaccine. LipL32, the most abundant, immunogenic, and conserved surface lipoprotein present in all pathogenic species of Leptospira, is a promising antigen candidate for a recombinant vaccine. However, several studies have reported a lack of protection when this protein is used as a subunit vaccine. In an attempt to enhance the immune response, we used LipL32 coupled to or coadministered with the B subunit of the Escherichia coli heat-labile enterotoxin (LTB) in a hamster model of leptospirosis. After homologous challenge with 5× the 50% lethal dose (LD50) of Leptospira interrogans, animals vaccinated with LipL32 coadministered with LTB and LTB::LipL32 had significantly higher survival rates (P < 0.05) than animals from the control group. This is the first report of a protective immune response afforded by a subunit vaccine using LipL32 and represents an important contribution toward the development of improved leptospirosis vaccines.
doi:10.1128/CVI.05720-11
PMCID: PMC3346321  PMID: 22379066

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