A Three-Dimensional Lung Cell Model to Leptospira Virulence Investigations.

Leptospirosis is a worldwide zoonosis and a serious public health threat in tropical and subtropical areas. The etiologic agents of leptospirosis are pathogenic spirochetes from the genus Leptospira. In severe cases, patients develop a pulmonary hemorrhage that is associated with high fatality rates. Several animal models were established for leptospirosis studies, such as rodents, dogs, and monkeys. Although useful to study the relationship among Leptospira and its hosts, the animal models still exhibit economic and ethical limitation reasons and do not fully represent the human infection. As an attempt to bridge the gap between animal studies and clinical information from patients, we established a three-dimensional (3-D) human lung cell culture for Leptospira infection. We show that Leptospira is able to efficiently infect the cell lung spheroids and also to infiltrate in deeper areas of the cell aggregates. The ability to infect the 3-D lung cell aggregates was time-dependent. The 3-D spheroids infection occurred up to 120 h in studies with two serovars, Canicola and Copenhageni. We standardized the number of bacteria in the initial inoculum for infection of the spheroids and we also propose two alternative culture media conditions. This new approach was validated by assessing the expression of three genes of Leptospira related to virulence and motility. The transcripts of these genes increased in both culture conditions, however, in higher rates and earlier times in the 3-D culture. We also assessed the production of chemokines by the 3-D spheroids before and after Leptospira infection, confirming induction of two of them, mainly in the 3-D spheroids. Chemokine CCL2 was expressed only in the 3-D cell culture. Increasing of this chemokine was observed previously in infected animal models. This new approach provides an opportunity to study the interaction of Leptospira with the human lung epithelium in vitro.

Serological survey and DNA screening of Leptospira spp. in free-living adult tufted capuchin monkeys (Cebus apella nigritus) in a forest reserve Southeast São Paulo State, Brazil.

BACKGROUND: Leptospirosis is an important anthropozoonosis. The study investigated the presence of anti-Leptospira antibodies and detection of Leptospira spp DNA in the urine as well as the biochemical profile in Neotropical wild primates living in a forest reserve from Southeast São Paulo State, Brazil. METHODS: Blood samples were obtained from 50 adult tufted capuchin monkeys (Cebus apella nigritus). Urine samples were obtained only from male primates. The screening for antibodies against Leptospira spp was evaluated by microscopic agglutination test (MAT). Leptospira DNA in the urine was evaluated by polymerase chain reaction (PCR) considering the target gene LipL32. Biochemical profile was evaluated by using a spectrophotometer. RESULTS: The MAT results included 39 (78%) serum reactive animals with the proportions of 28/39 males and 11/39 females. The most frequent reactive serogroups were Icterohemorrhagiae, Canicola, and Autumnalis. All urine samples were negative for leptospiral DNA. There were no significant differences between sexes for aspartate aminotransferase (AST) and alkaline phosphatase values, but alanine aminotransferase (ALT), creatinine, glucose, and urea were significantly higher in males. CONCLUSIONS: Tufted capuchin monkeys were sera reactive against leptospirosis. Prevalence was similar for the 2 sexes. Leptospiral DNA was not detected in the urine of sera reactive primates tested by the MAT method. ALT, creatinine, glucose, and urea values were higher in male animals.

Characterization of a novel protein of Leptospira interrogans exhibiting plasminogen, vitronectin and complement binding properties.

Leptospirosis is a severe zoonosis caused by pathogenic species of the genus Leptospira. This work focuses on a hypothetical protein of unknown function, encoded by the gene LIC13259, and predicted to be a surface protein, widely distributed among pathogenic leptospiral strain. The gene was amplified from L. interrogans serovar Copenhageni, strain Fiocruz L1-130, cloned and the protein expressed using Escherichia coli as a host system. Immunofluorescence assay showed that the protein is surface-exposed. The recombinant protein LIC13259 (rLIC13259) has the ability to interact with the extracellular matrix (ECM) laminin, in a dose-dependent manner but saturation was not reach. The rLIC13259 protein is a plasminogen (PLG)-binding protein, generating plasmin, in the presence of urokinase PLG-activator uPA. The recombinant protein is able to mediate the binding to human purified terminal complement route vitronectin, C7, C8 and C9, and to recruit and interact with these components from normal human serum (NHS). These interactions are dose-dependent on NHS increased concentration. The binding of rLIC13259 to C8 and vitronectin was slight and pronounced inhibited in the presence of increasing heparin concentration, respectively, suggesting that the interaction with vitronectin occurs via heparin domain. Most interesting, the interaction of rLIC13259 with C9 protein was capable of preventing C9 polymerization, suggesting that the membrane attack complex (MAC) formation was inhibited. Thus, we tentatively assign the coding sequence (CDS) LIC13259, previously annotated as unknown function, as a novel protein that may play an important role in the host's invasion and immune evasion processes, contributing to the establishment of the leptospiral infection.

Binding of human plasminogen by the lipoprotein LipL46 of Leptospira interrogans.

Leptospirosis is a widespread zoonosis caused by pathogenic Leptospira. Bacteria disseminate via the bloodstream and colonize the renal tubules of reservoir hosts. Leptospiral surface-exposed proteins are important targets, because due to their location they can elicit immune response and mediate adhesion and invasion processes. LipL46 has been previously reported to be located at the leptospiral outer membrane and recognized by antibodies present in serum of infected hamsters. In this study, we have confirmed the cellular location of this protein by immunofluorescence and FACS. We have cloned and expressed the recombinant protein LipL46 in its soluble form. LipL46 was recognized by confirmed leptospirosis human serum, suggesting its expression during infection. Binding screening of LipL46 with extracellular matrix (ECM) and plasma components showed that this protein interacts with plasminogen. The binding is dose-dependent on protein concentration, but saturation was not reached with the range of protein concentration used. Kringle domains of plasminogen and lysine residues of the recombinant protein are involved in the binding because the lysine analog, amino caproic acid (ACA) almost totally inhibited the reaction. The interaction of LipL46 with plasminogen generates plasmin in the presence of plasminogen activator uPA. Because plasmin generated at the leptospiral surface can degrade ECM molecules and decrease opsonophagocytosis, we tentatively infer that Lip46 has a role in helping the invasion process of pathogenic Leptospira.

Genomic characterisation of Leptospira inadai serogroup Lyme isolated from captured rat in Brazil and comparative analysis with human reference strain.

Leptospira inadai is classified as a species of the Leptospira intermediate group that has been poorly studied due to its apparent insignificance to human and animal health. Nevertheless, over the last two decades the species has been described in human cases in India and in carrier animals in Ecuador. Here, we present the first identification and genomic characterisation of L. inadai serogroup Lyme isolated from captured rodent in Brazil. Even though the M34/99 strain was not pathogenic for hamsters, it was able to establish renal colonisation. The M34/99 strain presented high similarity with L. inadai serogroup Lyme human reference indicating that animal strain could also infect humans, although it does not represent high risk of severe disease. An extrachromosomal sequence was also identified in M34/99 strain and presented high identity with previously described L. inadai phage LinZ_10, suggesting that phage-like extrachromosomal sequence may be another feature of this understudied species.

Comparative genomics of pathogenic Leptospira interrogans serovar Canicola isolated from swine and human in Brazil.

Leptospira interrogans serovar Canicola is one of the most important pathogenic serovars for the maintenance of urban leptospirosis. Even though it is considered highly adapted to dogs, serovar Canicola infection has already been described in other animals and even a few human cases. Here, we present the genomic characterisation of two Brazilian L. interrogans serovar Canicola strains isolated from slaughtered sows (L0-3 and L0-4) and their comparison with human strain Fiocruz LV133. It was observed that the porcine serovar Canicola strains present the genetic machinery to cause human infection and, therefore, represent a higher risk to public health. Both human and porcine serovar Canicola isolates also presented sequences with high identity to the Chinese serovar Canicola published plasmids pGui1 and pGui2. The plasmids identification in the Brazilian and Chinese serovar Canicola strains suggest that extra-chromosomal elements are one more feature of this serovar that was previously unnoticed.

ANTIBODIES AGAINST BRUCELLA ABORTUS AND LEPTOSPIRA SPP. IN CAPTIVE MAMMALS IN THE STATES OF PARá AND RIO GRANDE DO NORTE, BRAZIL.

Brazil has a large variety of wild animal species, but limited data are available on the occurrence of Brucella abortus and Leptospira spp. antibodies in these animals. Sera from 141 captive mammals belonging to 11 different species from the Northern and Northeastern regions of Brazil were screened. Antibodies against B. abortus and Leptospira spp. (24 live serovars) were investigated using the Rose Bengal plate and microscopic agglutination tests, respectively. Associations between the age, gender, and place of captivity were analyzed using the Pearson chi-square or the Fisher exact test. None of the animals were antibody positive for B. abortus. Among the animals tested, 11 (7.8%) were seropositive for Leptospira spp. These included one red brocket deer ( Mazama americana), two tufted capuchin ( Sapajus apella), seven agoutis ( Dasyprocta aguti), and one lowland paca ( Cuniculus paca). No association was observed between sex, age, and the occurrence of Leptospira spp. antibodies ( P > 0.05). However, an association was observed according to the place of captivity ( P = 0.046). From these 11 positive animals, six (54.5%) reacted to the serovars from the Icterohaemorraghiae serogroup, which is mainly responsible for the clinical cases of human leptospirosis in Brazil. To our knowledge, this is the first report of Leptospira spp. antibodies in M. americana and C. paca.

Characterization of two new putative adhesins of Leptospira interrogans.

We here report the characterization of two novel proteins encoded by the genes LIC11122 and LIC12287, identified in the genome sequences of Leptospira interrogans, annotated, respectively, as a putative sigma factor and a hypothetical protein. The CDSs LIC11122 and LIC12287 have signal peptide SPII and SPI and are predicted to be located mainly at the cytoplasmic membrane of the bacteria. The genes were cloned and the proteins expressed using Escherichia coli. Proteinase K digestion showed that both proteins are surface exposed. Evaluation of interaction of recombinant proteins with extracellular matrix components revealed that they are laminin binding and they were called Lsa19 (LIC11122) and Lsa14 (LIC12287), for Leptospiral-surface adhesin of 19 and 14 kDa, respectively. The bindings were dose-dependent on protein concentration, reaching saturation, fulfilling the ligand-binding criteria. Reactivity of the recombinant proteins with leptospirosis human sera has shown that Lsa19 and, to a lesser extent, Lsa14, are recognized by antibodies, suggesting that, most probably, Lsa19 is expressed during infection. The proteins interact with plasminogen and generate plasmin in the presence of urokinase-type plasminogen activator. Plasmin generation in Leptospira has been associated with tissue penetration and immune evasion strategies. The presence of a sigma factor on the cell surface playing a secondary role, probably mediating host -pathogen interaction, suggests that LIC11122 is a moonlighting protein candidate. Although the biological significance of these putative adhesins will require the generation of mutants, our data suggest that Lsa19 is a potential candidate for future evaluation of its role in adhesion/colonization activities during L. interrogans infection.

Multifunctional and Redundant Roles of Leptospira interrogans Proteins in Bacterial-Adhesion and fibrin clotting inhibition.

Pathogenic Leptopira is the etiological agent of leptospirosis, the most widespread zoonotic infection in the world. The disease represents a major public health problem, especially in tropical countries. The present work focused on two hypothetical proteins of unknown function, encoded by the genes LIC13059 and LIC10879, and predicted to be surface-exposed proteins. The genes were cloned and the proteins expressed using E. coli as a host system. We report that the recombinant proteins interacted with extracellular matrix (ECM) laminin, in a dose-dependent fashion and are novel potential adhesins. The recombinant proteins were called Lsa25.6 (rLIC13059) and Lsa16 (rLIC10879), for Leptospiral surface adhesins, followed by the respective molecular masses. The proteins attached to plasminogen (PLG), generating plasmin, in the presence of PLG-activator uPA. Both proteins bind to fibrinogen (Fg), but only Lsa25.6 inhibited fibrin clotting by thrombin-catalyzed reaction. Moreover, Lsa16 interacts with the mammalian cell receptor E-cadherin, and could contribute to bacterial attachment to epithelial cells. The proteins were recognized by confirmed leptospirosis serum samples, suggesting that they are expressed during infection. The corresponding leptospiral proteins are surface exposed based on proteinase K accessibility assay, being LIC10879 most probably exposed in its dimer form. The data of this study extend the spectrum of surface-exposed proteins of L. interrogans and indicate a possible role of the originally annotated hypothetical proteins in infection processes.

The role of Lsa23 to mediate the interaction of Leptospira interrogans with the terminal complement components pathway.

Leptospirosis is a severe worldwide zoonotic disease caused by pathogenic Leptospira spp. It has been demonstrated that pathogenic leptospires are resistant to the bactericidal activity of normal human serum while saprophytic strains are susceptible. Pathogenic strains have the ability to bind soluble complement regulators and these activities are thought to contribute to bacterial immune evasion. One strategy used by some pathogens to evade the complement cascade, which is not well explored, is to block the terminal pathway. We have, thus, examined whether leptospires are able to interact with components of the terminal complement pathway. ELISA screening using anti-leptospires serum has shown that the pathogenic, virulent strain L. interrogans L1-130 can bind to immobilized human C8 (1 µg). However, virulent and saprophyte L. biflexa strains showed the ability to interact with C8 and C9, when these components were employed at physiological concentration (50 µg/mL), but the virulent strain seemed more competent. Lsa23, a putative leptospiral adhesin only present in pathogenic strains, interacts with C8 and C9 in a dose-dependent mode, suggesting that this protein could mediate the binding of virulent Leptospira with these components. To our knowledge, this is the first work reporting the binding of Leptospira to C8 and C9 terminal complement components, suggesting that the inhibition of this pathway is part of the strategy used by leptospires to evade the innate immunity.

Leptospira interrogans Lsa23 protein recruits plasminogen, factor H and C4BP from normal human serum and mediates C3b and C4b degradation.

It has been reported that pathogenic Leptospira are resistant to normal human serum (NHS) due to their ability to evade the complement immune system by interacting with factor H (FH) and C4b-binding protein (C4BP) regulators. Moreover, plasmin generation on the leptospiral surface diminishes C3b and IgG deposition, decreasing opsonophagocytosis by immune competent cells. We have previously reported that Lsa23 (LIC11360) is a multipurpose protein capable of binding purified extracellular matrix molecules, FH, C4BP and plasminogen (PLG)/plasmin in the presence of PLG activators. In this work, we provide further evidence that Lsa23 is located at the bacterial surface by using immunofluorescence microscopy. We show that Lsa23 has the ability to acquire FH, C4BP and PLG from NHS, and use these interactions to evade innate immunity. The binding with the complement regulators FH and C4BP preserves factor I (FI) activity, leading to C3b and C4b degradation products, respectively. C3b and C4b alpha-chain cleavage was also observed when Lsa23 bound to PLG generating plasmin, an effect blocked by the protease inhibitor aprotinin. Lsa23 also inhibited lytic activity by NHS mediated by both classical and alternative complement pathways. Thus, Lsa23 has the ability to block both pathways of the complement system, and may help pathogenic Leptospira to escape complement-mediated clearance in human hosts. Indeed, NHS treated with Lsa23 confers a partial serum resistance phenotype to Leptospira biflexa, whereas blocking this protein with anti-Lsa23 renders pathogenic L. interrogans more susceptible to complement-mediated killing. Thus, Lsa23 is a multifunctional protein involved in many pathways, featuring C4b cleavage by plasmin, knowledge that may help in the development of preventive approaches to intervene with human complement escape by this versatile pathogen.

Decrease in antithrombin III and prothrombin serum levels contribute to coagulation disorders during leptospirosis.

Pathogenic bacteria of the genus Leptospira are the causative agent of leptospirosis, an emergent infectious disease that affects humans and animals worldwide. Severe forms of the disease in humans include jaundice, multiple organ failure and intense haemorrhage. Up to now, mechanisms associated with the haemorrhage foci are poorly understood. We report in this work that, despite the low levels of antithrombin III in convalescent human serum samples, virulent, culture-attenuated and saprophyte strains of Leptospira are unable to bind and/or degrade this thrombin inhibitor, suggesting an indirect mechanism of pathogenesis. Lower levels of prothrombin were found in serum samples at the onset and convalescent phase of the disease when compared to normal human sera. The concomitant decreased levels of antithrombin III and prothrombin suggest a process of stimulated coagulation, which is corroborated by the increase of prothrombin fragment F1+2 in the serum samples. Data obtained with hamsters experimentally infected with virulent Leptospira interrogans serovars Kennewicki and Canicola strongly point out that haemorrhage is correlated with decreased levels of thrombin inhibitors and prothrombin. Activated coagulation might lead to an overconsumption of coagulation factors ultimately leading to bleeding and organ failure.

Mammalian cell entry (Mce) protein of Leptospira interrogans binds extracellular matrix components, plasminogen and ß2 integrin.

A severe re-emergingzoonosis, leptospirosis, is caused by pathogenic spirochetes of the genus Leptospira. Several studies have identified leptospiral surface proteins with the ability to bind ECM and plasma components, which could mediate adhesion and invasion through the hosts. It has been shown that Mce of pathogenic Leptospira spp. is an RGD (Arg-Gly-Asp)-motif-dependent virulence factor, responsible for infection of cells and animals. In the present article, we decided to further study the repertoire of the Mce activities in leptospiral biological properties. We report that the recombinant Mce is a broad-spectrum ECM-binding protein, capable of interacting with laminin, cellular and plasma fibronectin and collagen IV. Dose--r-esponse interaction was observed for all the components, fulfilling ligand--receptor requirements. Mce is a PLG binding protein capable to recruit this component from NHS, generating PLA in the presence of PLG activator. Binding of Mce was also observed with the leukocyte cell receptors αLß2 [(CD11a/CD18)-LFA-1] and αMß2 [(CD11b/CD18)-Mac-1], suggesting the involvement of this protein in the host immune response. Indeed, virulent Leptospira L1-130 was capable of binding both integrins, whereas culture-attenuated M-20 strain only bind to αMß2 [(CD11b/CD18)-Mac-1]. To the best of our knowledge, this is the first work to describe that Mce surface protein could mediate the attachment of Leptospira interrogans to human cell receptors αLß2(CD11a/CD18) and αMß2(CD11b/CD18).

Novel Leptospira interrogans protein Lsa32 is expressed during infection and binds laminin and plasminogen.

Pathogenic Leptospira is the aetiological agent of leptospirosis, a life-threatening disease of human and veterinary concern. The quest for novel antigens that could mediate host-pathogen interactions is being pursued. Owing to their location, these antigens have the potential to elicit numerous activities, including immune response and adhesion. This study focuses on a hypothetical protein of Leptospira, encoded by the gene LIC11089, and its three derived fragments: the N-terminal, intermediate and C terminus regions. The gene coding for the full-length protein and fragments was cloned and expressed in Escherichia coli BL21(SI) strain by using the expression vector pAE. The recombinant protein and fragments tagged with hexahistidine at the N terminus were purified by metal affinity chromatography. The leptospiral full-length protein, named Lsa32 (leptospiral surface adhesin, 32 kDa), adheres to laminin, with the C terminus region being responsible for this interaction. Lsa32 binds to plasminogen in a dose-dependent fashion, generating plasmin when an activator is provided. Moreover, antibodies present in leptospirosis serum samples were able to recognize Lsa32. Lsa32 is most likely a new surface protein of Leptospira, as revealed by proteinase K susceptibility. Altogether, our data suggest that this multifaceted protein is expressed during infection and may play a role in host-L. interrogans interactions.

Immune evasion by pathogenic Leptospira strains: the secretion of proteases that directly cleave complement proteins.

Leptospirosis is an infectious disease of public health importance. To successfully colonize the host, pathogens have evolved multiple strategies to escape the complement system. Here we demonstrate that the culture supernatant of pathogenic but not saprophytic Leptospira inhibit the three complement pathways. We showed that the proteolytic activity in the supernatants of pathogenic strains targets the central complement molecule C3 and specific proteins from each pathway, such as factor B, C2, and C4b. The proteases cleaved α and ß chains of C3 and work in synergy with host regulators to inactivate C3b. Proteolytic activity was inhibited by 1,10-phenanthroline, suggesting the participation of metalloproteases. A recombinant leptospiral metalloprotease from the thermolysin family cleaved C3 in serum and could be one of the proteases responsible for the supernatant activity. We conclude that pathogenic leptospiral proteases can deactivate immune effector molecules and represent potential targets to the development of new therapies in leptospirosis.

Functional and immunological evaluation of two novel proteins of Leptospira spp.

This work shows the production and characterization of two novel putative lipoproteins encoded by the genes LIC10645 and LIC10731 identified in the genome sequences of Leptospira interrogans. In silico conservation analysis indicated that the proteins are well conserved among pathogenic leptospiral serovars and species. Recombinant proteins were obtained in Escherichia coli BL21(DE3) Star pLysS strain, purified by metal-affinity chromatography, and used for characterization and immunological evaluations. Recombinant proteins were capable of eliciting a combination of humoral and cellular immune responses in animal models, and could be recognized by antibodies present in human serum samples. The recombinant proteins Lsa44 and Lsa45 were able to bind laminin, and were named Lsa44 and Lsa45 for leptospiral surface adhesins of 44 and 45 kDa, respectively. The attachment to laminin was dose-responsive with KD values of 108.21 and 250.38 nM for Lsa44 and Lsa45, respectively. Moreover, these proteins interact with plasminogen (PLG) with KD values of 53.56 and 36.80 nM, respectively. PLG bound to the recombinant proteins could be converted to plasmin (PLA) in the presence of an activator. Cellular localization assays suggested that the Lsa44 and Lsa45 were surface-exposed. These are versatile proteins capable of interacting with laminin and PLG/PLA, and hence could mediate bacterial adhesion and contribute to tissue penetration.

Sero-epidemiological survey for brucellosis, leptospirosis, and toxoplasmosis in free-ranging Alouatta caraya and Callithrix penicillata from São Paulo State, Brazil.

BACKGROUND AND METHODS: Sera were tested for Brucella spp., Leptospira spp. and Toxoplasma gondii antibodies in 68 free-ranging New World monkeys from a forest fragment of the Brazilian Cerrado. RESULTS AND CONCLUSION: All animals were negative for Brucella spp. and Leptospira spp. However, 75% of Alouatta caraya and 16.6% of Callithrix penicillata were positive for T. gondii. The implications for conservation and health management are discussed.

Interaction of Leptospira interrogans with human proteolytic systems enhances dissemination through endothelial cells and protease levels.

We have recently reported the ability of Leptospira to capture plasminogen (PLG) and generate plasmin (PLA) bound on the microbial surface in the presence of exogenous activators. In this work, we examined the effects of leptospiral PLG binding for active penetration through the endothelial cell barrier and activation. The results indicate that leptospires with PLG association or PLA activation have enhanced migration activity through human umbilical vein endothelial cell (HUVEC) monolayers compared with untreated bacteria. Leptospira cells coated with PLG were capable of stimulating the expression of PLG activators by HUVECs. Moreover, leptospires endowed with PLG or PLA promoted transcriptional upregulation matrix metalloprotease 9 (MMP-9). Serum samples from patients with confirmed leptospirosis showed higher levels of PLG activators and total MMP-9 than serum samples from normal (healthy) subjects. The highest level of PLG activators and total MMP-9 was detected with microscopic agglutination test (MAT)-negative serum samples, suggesting that this proteolytic activity stimulation occurs at the early stage of the disease. Furthermore, a gelatin zymography profile obtained for MMPs with serum samples from patients with leptospirosis appears to be specific to leptospiral infection because serum samples from patients with unrelated infectious diseases produced no similar degradation bands. Altogether, the data suggest that the Leptospira-associated PLG or PLA might represent a mechanism that contributes to bacterial penetration of endothelial cells through an activation cascade of events that enhances the proteolytic capability of the organism. To our knowledge, this is the first proteolytic activity associated with leptospiral pathogenesis described to date.

Adhesin activity of Leptospira interrogans lipoprotein identified by in vivo and in vitro shotgun phage display.

Leptospira interrogans causes leptospirosis, one of the most common zoonotic diseases in the world. This pathogenic spirochete is able to bind to extracellular matrix, to express virulent factors and to cause host death. Until now, there is no effective human vaccine for the disease. Shotgun phage display genomic libraries of L. interrogans were constructed and used for in vivo biopanning in hamsters and screened for ligands able to bind to LLC-PK1 epithelial cells. In both panning procedures, clones coding for the putative lipoprotein LIC12976 were identified and, in order to confirm its adhesin activity, a recombinant protein was produced in Escherichia coli and showed to interact with A31 fibroblasts, LLC-PK1 and Vero epithelial cells in vitro. Moreover, rLIC12976 was shown to bind to laminin, indicating an adhesin function. This protein was also detected in extracts of L. interrogans from different serovars and it was found to be conserved among pathogenic leptospires. Further, the protein was tested as vaccine candidate and immunization of hamsters with LIC12976 did not confer protection against a lethal challenge with the homologous L. interrogans serovar Copenhageni. Nevertheless, LIC12976 seems to act as an adhesin, and may be important for the host-pathogen interaction, so that its study can contribute to the understanding of the virulence mechanisms in pathogenic leptospires.

Serological analysis by enzyme-linked immunosorbent assay using recombinant antigen LipL32 for the diagnosis of swine leptospirosis.

Leptospirosis is an important global zoonotic disease caused by pathogenic Leptospira spp. species. Swine leptospirosis has a major economic impact because pigs are sources of animal protein and by-products. The signs of swine leptospirosis are abortion, stillbirth, birth of weak or ill piglets, appearing 14-60 days after infection. The reference method for diagnosis of leptospirosis is the microscopic agglutination test (MAT), in which serum samples are reacted with live antigen suspensions of leptospiral serovars. However, MAT is laborious and time consuming as a diagnostic procedure when dealing with a large number of samples; therefore, efforts are being made to develop novel, sensitive, and specific diagnostic tests for leptospirosis. In this study, a recombinant LipL32 based on enzyme-linked immunosorbent assay (rLipL32/ELISA) was evaluated as a screening test for the detection of pathogenic leptospiral-specific antibodies. A total of 86 swine serum samples tested by MAT were used to develop rLipL32/ELISA. Compared to positive and negative sera tested by MAT, rLipL32/ELISA showed 100 % sensitivity, 85.1 % specificity, and 91.86 % accuracy. No positive reaction for other bacterial diseases (enzootic pneumonia and brucellosis) was observed. The rLipL32/ELISA reported in this study is a specific, sensitive, and convenient test for the detection of antibodies against swine leptospiral infection and can be used as a rapid screening test in epidemiological surveys.