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PubMed2006

In silico and microarray-based genomic approaches to identifying potential vaccine candidates against Leptospira interrogans.

BACKGROUND: Currently available vaccines against leptospirosis are of low efficacy, have an unacceptable side-effect profile, do not induce long-term protection, and provide no cross-protection against the different serovars of pathogenic leptospira. The current major focus in leptospirosis research is to discover conserved protective antigens that may elicit longer-term protection against a broad range of Leptospira. There is a need to screen vaccine candidate genes in the genome of Leptospira interrogans. RESULTS: Bioinformatics, comparative genomic hybridization (CGH) analysis and transcriptional analysis were used to identify vaccine candidates in the genome of L. interrogans serovar Lai strain #56601. Of a total of 4727 open reading frames (ORFs), 616 genes were predicted to encode surface-exposed proteins by P-CLASSIFIER combined with signal peptide prediction, alpha-helix transmembrane topology prediction, integral beta-barrel outer membrane protein and lipoprotein prediction, as well as by retaining the genes shared by the two sequenced L. interrogans genomes and by subtracting genes with human homologues. A DNA microarray of L. interrogans strain #56601 was constructed for CGH analysis and transcriptome analysis in vitro. Three hundred and seven differential genes were identified in ten pathogenic serovars by CGH; 1427 genes had high transcriptional levels (Cy3 signal > or = 342 and Cy5 signal > or = 363.5, respectively). There were 565 genes in the intersection between the set encoding surface-exposed proteins and the set of 307 differential genes. The number of genes in the intersection between this set of 565 and the set of 1427 highly transcriptionally active genes was 226. These 226 genes were thus identified as putative vaccine candidates. The proteins encoded by these genes are not only potentially surface-exposed in the bacterium, but also conserved in two sequenced L. interrogans. Moreover, these genes are conserved among ten epidemic serovars in China and have high transcriptional levels in vitro. CONCLUSION: Of the 4727 ORFs in the genome of L. interrogans, 226 genes were identified as vaccine candidates by bioinformatics, CGH and transcriptional analysis on the basis of the theory of reverse vaccinology. The proteins encoded by these genes might be useful as vaccine candidates as well as for diagnosis of leptospirosis.

Bacterial Outer Membrane Proteins↗
PubMed1996

Protective activity of rabbit polyclonal anti-idiotype antibody against Leptospira interrogans infection in hamsters.

We prepared an anti-idiotype (Id) antibody against leptospirosis. Serum from rabbit immunized with monoclonal antibody (MAb) LW2, which reacted to the main protective antigen prepared from Leptospira interrogans serovar lai, inhibited agglutination of the organism by MAb LW2. The immune rabbit serum was applied to a column coupled with normal mouse IgG as a ligand (first column), and the unbound fraction eluted was applied to a column coupled with MAb LW2 as a ligand (second column). The bound fraction (anti-Id antibody) eluted from the second column inhibited the binding of MAb LW2 to sonicated leptospiral cells in ELISA. Mice produced antibodies against Leptospira by intraperitoneal immunization with the anti-Id antibody at doses of 2 mu g/mouse or more. Hamsters were protected by immunization with the anti-Id antibody at doses of 2 and 20 mu g/hamster from the lethal infection of Leptospira. This is the first report concerning the use of an anti-Id antibody against leptospirosis.

Agglutination↗
PubMedDate not supplied

[Prevalence of dengue and leptospira antibodies in the state of Veracruz, Mexico].

OBJECTIVE: To discover the prevalence of antibodies against denguevirus and leptospira. MATERIAL AND METHODS: A cross-sectional study was carried out in 500 inhabitants of Veracruz, Mexico through household sampling, administration of a questionnaire and collection of blood samples.The presence of IgG antibodies was determined and factors associated with seroprevalence were evaluated using a logistic regression model. RESULTS: Total denguevirus seroprevalence was 79.6% (IC 95% 76-81). The main factors associated with infection were age and residence in neighborhoods with higher vectorial indices (RMP = 2.94, IC 95% 1.46-6.1). Overall seroprevalence against leptospira was 4% (IC 95% 2-6). Prevalence was higher in the 25 to 44 years age group (35%, IC 95% 15-59). Seroprevalence in individuals who referred to living with dogs, pigs, cows and rats was 25% (IC 95% 0.63-80). Eighty-five percent of individuals who were leptospira positive were also dengue positive. CONCLUSIONS: The prevalence of denguevirus antibodies suggests the risk of serious dengue hemorrhagic outbreaks. Although leptospirosis prevalence was low, the coexistence of both agents was confirmed.

Adolescent↗
PubMed2000

Reservoir hosts of Leptospira inadai in India.

Isolation of Leptospira from the kidneys of Rattus rattus wroughtoni hinton, Rattus rattus rufescens, Bandicota bengalensis and Bandicota indica was attempted in Bangalore in southern India. In total, 296 spirochaetes were isolated from 1,348 kidney cultures (an isolation rate of 22%). A batch of fifty-six isolates from India was identified, based on serological and polymerase chain reaction analysis, of which twenty-three isolates were identified as L. inadai by the World Health Organization/Food and Agriculture Organization Collaborating Centre for Reference and Research on Leptospirosis, in Brisbane. This is the first record of isolation of L. inadai from rodents. The preponderance of L. inadai in four different species of rodents suggests that these animals could be the natural reservoir hosts of L. inadai, and raises a critical question as to the likely impact of this species of Leptospira on the renal carrier status of other Leptospira pathogenic to humans and animals in this part of India. Virulence studies conducted at the University of Trieste in Italy, revealed that isolates of L. inadai from India were moderately or totally serum resistant when subjected to a serum killing test. To establish the possible seroprevalence of this species in the population, the inclusion of L. inadai in the battery of leptospiral antigens used for sero-epidemiological studies is recommended.

Animals↗
PubMed2005

Evaluation of a monoclonal antibody-based dot-blot ELISA for detection of Leptospira spp in bovine urine samples.

OBJECTIVE: To evaluate the efficacy of a novel monoclonal antibody (MAb)-based dot-blot ELISA for detection of Leptospira antigens in urine samples of cattle. SAMPLE POPULATION: Blood and urine samples of 45 test cattle from 5 farms in Chonburi province and 20 control cattle from 2 farms in Khon Kaen province in Thailand. PROCEDURE: Blood and urine samples were assayed (microscopic agglutination test and urine antigen test) for Leptospira infection by use of an MAb-based dot-blot ELISA, and results for the ELISA were compared with those for dark-field microscopy (DFM), microbial culture, and a polymerase chain reaction (PCR) assay. RESULTS: All urine samples with positive results for DFM, microbial culture, PCR assay, or > 1 of these tests also had positive results when tested by use of the MAb-based dot-blot ELISA, except for 1 sample that had positive results only for the PCR assay. Detection limits of the dot-blot ELISA were 10(3) leptospires/mL of urine and 9.3 ng of Leptospira homogenate. Comparison revealed that the diagnostic sensitivity, specificity, efficacy (accuracy), positive predictive value, and negative predictive value for the ELISA were in agreement with results for DFM (100%, 72.72%, 80%, 57.14%, and 100%, respectively), microbial culture (100%, 61.54%, 66.62%, 28.57%, and 100%, respectively), and PCR assay (95.45%, 100%, 91.77%, 100%, and 95.83%, respectively). CONCLUSIONS AND CLINICAL RELEVANCE: The MAb-based dot-blot ELISA is suitable as a tool for detecting leptospires in urine samples of cattle.

Animals↗
PubMed2006

Production of reactive oxygen species and expression of inducible nitric oxide synthase in rat isolated Kupffer cells stimulated by Leptospira interrogans and Borrelia burgdorferi.

AIM: To evaluate the production of reactive oxygen species (ROS) and the expression of inducible nitric oxide synthase (iNOS) in rat isolated Kupffer cells (KCs) stimulated by Leptospira interrogans and Borrelia burgdorferi. METHODS: Rat Kupffer cells were separated by perfusion of the liver with 0.05% collagenase, and purified by Percoll gradients. Purified Kupffer cells were tested in vitro with alive L. interogans and B. burgdorferi preparations. The production of ROS was determined by chemiluminescence, whereas iNOS protein expression was evaluated by Western blot assay using anti-iNOS antibodies. RESULTS: B. burgdorferi and to a less extent L. interrogans induced ROS production with a peak 35 min after infection. The chemiluminescence signal progressively diminished and was undetectable by 180 min of incubation. Leptospirae and borreliae induced an increased iNOS expression in Kupffer cells that peaked at 6 hours and was still evident 22 h after infection. CONCLUSION: Both genera of spirochetes induced ROS and iNOS production in rat Kupffer cells. Since the cause of liver damage both in leptospiral as well as in borrelial infections are still unknown, we suggest that leptospira and borrelia damage of the liver can be initially mediated by oxygen radicals, and is then maintained at least in part by nitric oxide.

Animals↗
PubMed2006

In vitro susceptibilities of Leptospira spp. and Borrelia burgdorferi isolates to amoxicillin, tilmicosin, and enrofloxacin.

Antimicrobial susceptibility testing was conducted with 6 different spirochetal strains (4 strains of Leptospira spp. and 2 strains of Borrelia burgdorferi) against 3 antimicrobial agents, commonly used in equine and bovine practice. The ranges of MIC and MBC of amoxicillin against Leptospira spp. were 0.05 - 6.25 microgram/ml and 6.25 - 25.0 microgram/ml, respectively. And the ranges of minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of amoxicillin against B. burgdorferi were 0.05 - 0.39 microgram/ml and 0.20 - 0.78 microgram/ml, respectively. The ranges of MIC and MBC of enrofloxacin against Leptospira spp. were 0.05 - 0.39 microgram/ml and 0.05 - 0.39 microgram/ml, respectively. Two strains of B. burgdorferi were resistant to enrofloxacin at the highest concentration tested for MBC (>or=100 microgram/ml). Therefore, the potential role of tilmicosin in the treatment of leptospirosis and borreliosis should be further evaluated in animal models to understand whether the in vivo studies will confirm in vitro results. All spirochetal isolates were inhibited (MIC) and were killed (MBC) by tilmicosin at concentrations below the limit of testing (<or=0.01 microgram/ml).

Amoxicillin↗
PubMed1999

Antibodies against Leptospira interrogans in California sea lion pups from Gulf of California.

One hundred and twenty-five serum samples from California sea lion (Zalophus californianus californianus) pups, and one from an adult female from eight reproductive rookeries located in seven islands in the Gulf of California (Mexico), were collected during the 1994-96 reproductive seasons. These were tested for antibodies to 19 serovars of Leptospira interrogans using a Microscopic Agglutination Test (MAT). Forty-one samples (32%) had antibody levels from 1:20 to 1:320 to one or more serovars. The most frequently detected serotypes were Leptospira interrogans hardjo (n = 13), cynopteri (8), ballum (6), and szwajizak (5). Serovars with the highest prevalence were Leptospira interrogans hardjo and serjoe (1:320), ballum (1:160), and cynopteri, girppotyphosa, and tarassovi (1:80). Based on these results, exposure of sea lions to L. interrogans serovar hardjo seems to be relatively common among colonies located in the islands of the Gulf of California in contrast with those located on the Pacific coast, where the most frequently detected serovar is L. interrogans serovar pomona.

Agglutination Tests↗
PubMed2005

Flying foxes as carriers of pathogenic Leptospira species.

Recent serologic studies have identified flying foxes (Pteropus spp.) as carriers of leptospirosis; however, little is known about the role of flying foxes as carriers of pathogenic Leptospira spp. To determine if Australian Pteropus spp. are carriers of pathogenic Leptospira spp., TaqMan real-time polymerase chain reaction (PCR) was used to detect leptospiral DNA in kidney and urine specimens from four species of flying fox, including the spectacled flying fox (Pteropus conspicillatus), black flying fox (Pteropus alecto), grey-headed flying fox (Pteropus poliocephalus), and little red flying fox (Pteropus scapulatus). Of the 173 kidney samples tested, 19 (11%) were positive for leptospiral DNA. Positive individuals were detected in all four species; significant differences in prevalence were not detected between species, between species within the same geographic area, or between geographically separated samples from the same species. Of the 46 urine samples tested, 18 (39%) tested positive by PCR, confirming that flying foxes shed leptospires into the environment. The detection of leptospiral DNA in the kidneys and urine of flying foxes suggests that flying foxes are carriers of pathogenic Leptospira spp. No evidence collected in the present study, however, suggests that flying foxes pose a significant risk of leptospirosis to the wider community or that humans who are in regular, close contact with flying foxes are at risk for leptospirosis.

Animals↗
PubMed1999

Identification of a serogroup bataviae Leptospira strain isolated from an ox in Zimbabwe.

A strain belonging to the genus Leptospira serogroup Bataviae, isolated from an ox at slaughter in Zimbabwe, was identified by using cross-agglutinin absorption, the monoclonal antibody, restriction fragment length polymorphism, and polymerase chain reaction analyses. Results of both serological tests used showed that the isolate (strain SBF 37) was antigenically similar to reference strain Paidjan and therefore belongs to serovar paidjan. Strain SBF 37 was, however, genetically different from strain Paidjan since their chromosomal DNA had different restriction fragment length polymorphism patterns. The Zimbabwe paidjan strain was identified as belonging to the species Leptospira kirschneri while strain Paidjan reacted as a member of one of the non-kirschneri species in the polymerase chain reaction. The Zimbabwe isolate therefore belongs to Leptospira kirschneri serovar paidjan.

Animals↗
PubMed1998

[Cloning, characterization and expression in E. coli of the flagellin gene from Leptospira interrogans Serovar lai].

To obtain a large amount of recombinant endoflagellin protein of leptospsira, we have cloned the fla B gene from Leptospira interrogans serovar lai strain 017, which encodes the core flagellar protein. The entire open reading frame was amplified by polymerase chain reaction, which was digested with endonucleases Sal I and Cla I, then was orientationally cloned into the expression vector, pT7-7. The recombinant plasmid was transformed into Escherichia coli JM109 (DE3) for expression. After induction with IPTG, a 34 kd expression protein was detected by SDS-PAGE. The amount of recombinant protein was estimated as 11.8% of the whole bacterial lysate proteins. Probed with insert fragments, Southern blot analysis indicated that possibly two gene related to fla B were present in the genome of Leptospira interrogans serovar lai stain 017. Western blot analysis showed that the 34 kd expression protein reacting with rabbit polyclonal antiserum raised against leptospira endflagellin.

Cloning, Molecular↗
PubMed1975

[Sensitivity of certain strains of pathogenic Leptospira to streptomycin, the nature of resistant variants and the frequency of their occurrence].

Streptomycin sensitivity of 112 Leptospira strains isolated from domestic and wild animals was studied. Independent of the isolation period and nature the strains proved to be highly sensitive to the antiobiotic (MIC 0.1 to 0.5 gamma/ml). On the example of a clone strain of Leptospira it was shown with a fluctuation test that streptomycin resistance occurred spontaneously. Frequency of the streptomycin resistant mutations was determined for 5 clone strains of L. interrogans. It was 10-9 to 10-7 per a Leptospira.

Animals↗
PubMed2000

Use of locally isolated saprophytic Leptospira strain for serological testing of human leptospirosis.

A saprophytic Leptospira isolate recovered from tap water was utilized for serological testing. One hundred-twenty Serum samples comprising 55 cases from PUO/febrile jaundice and 65 samples from apparently healthy individuals were tested by MAT and HA using this environmental saprophytic strain and the results compared with that of Leptospira biflexa semaranga patoc, the standard saprophytic strain commonly employed for sero-diagnosis of leptospirosis. The MAT data showed 96.4 per cent correlation between the two strains. Similarly, the HA results were matching to the extent of 94.5 per cent. Results, therefore, suggest that local saprophytic Leptospira strain may serve as a substitute to serovar patoc for serodiagnosis of leptospirosis.

Antibodies, Bacterial↗
PubMed2003

Prevalence of antibodies to Leptospira serovars in rodents and shrews trapped in low and high endemic areas in Thailand.

OBJECTIVE: To investigate the prevalence of antibodies to Leptospira serovars in rodents and shrews trapped in urban and rural areas in low and high endemic areas in Thailand. MATERIAL AND METHOD: A total of 1,664 serum samples were collected from rodents and shrews in areas of low and high endemicity for leptospirosis. Four areas classified by case rates (CR) per 100,000 population of leptospirosis were urban Area I Bangkok (CR = 0.07), rural Area II (CR = 0.24), rural Area III (CR = 1.97) and rural Area IV (CR = 48.20). All serum samples were investigated for antibodies to leptospires by microscopic agglutination test (MAT) using antigens from each of the 22 pathogenic serovars of Leptospira interrrogans: australis, autumnalis, ballum, bangkok, bataviae, bratislava, canicola, celledoni, copenhageni, djasiman, grippotyphosa, hardjo, hebdomadis, icterohaemorrhagiae, javanica, pomona, pyrogenes, rachmati, saigon, sejroe, tarassovi and wolffi and one non-pathogenic strain of L. biflexa serovar patoc. RESULTS: Ninety-four (5.6%) serum samples were positive for Leptospira antibodies. The most commonly detected antibodies were to serovars pyrogenes (39.1%), sejroe (19.1%), bataviae (10.0%), pomona (6.4%), autumnalis (5.5%), copenhageni (3.6%) and javanica (3.6%). The positive rates in Area I, II, III and IV were 7.6 per cent, 2.9 per cent, 4.6 per cent and 7.1 per cent, respectively. The seroprevalence in rural areas tended to increase significantly with high endemicity for leptospirosis (Chi-square for trend, p = 0.04). The seropositive rates by animal species were 39/496 (7.9%), 22/322 (6.8%), 23/492 (4.7%), 6/170 (3.5%), 4/175 (2.3%), 0/4 (0%) and 0/5 (0%) in Rattus norvegicus, Rattus exulans, Rattus rattus, Bandicota indica, Bandicota savilei, Mus musculus and Suncus murinus, respectively. There was a statistical trend between seropositive rates in R. exulans and endemicity for leptospirosis (Chi-square for trend, p = 0.04). CONCLUSION: The 5.6 per cent of rodents and shrews trapped in urban and rural areas in Thailand were reservoirs of leptospires. The results of high seroprevalence in rats also indicate the high endemicity for leptospirosis.

Agglutination Tests↗
PubMed1992

[The immunochemical and biological properties of Leptospira membranes].

The immunochemical and biological properties of purified membrane fractions obtained from Leptospira interrogans, serovar copenhageni, strain Rat 2, and Leptospira biflexa, strain Patoc 1, were studied. The presence of genus-specific and group-specific antigens in leptospiral membranes was established by the methods of immunodiffusion analysis, the microagglutination (MA) and lysis tests. In animal experiments cell membrane preparations produced no toxic and allergic effects. Leptospiral membranes obtained from strain Rat 2 ensured the protection of golden hamsters infected with Leptospira virulent culture and induced antibody production in high titers, detected with the use of the MA test, the lysis test and the enzyme immunoassay, in rabbits immunized in two injections.

Animals↗
PubMed2004

Evidence of rickettsial and leptospira infections in Andean northern Peru.

Between May and October 2002, a cluster of acute febrile illnesses occurred in the subtropical Andean foothills of Peru. Serologic evidence in villages where disease had been documented showed that the prevalence of IgM antibody to Leptospira ranged from 6% to 52%, that of IgM antibody to spotted fever group (SFG) rickettsia ranged from 10% to 19%, and that of IgM antibody to Coxiella burnetii from 1% to 15%. Measurement of IgG antibodies for SFG rickettsiae suggested that this disease was endemic. In contrast, IgG antibodies against C. burnetii were largely absent. In humans, microagglutination tests identified pathogenic variants of Leptospira. The presence of an SFG rickettsial infection was confirmed in four febrile patients following polymerase chain reaction and sequencing of the conserved 17-kD common antigen gene (htrA). Collectively, these analyses indicated that Rickettsia sp., C. burnetii, and Leptospira sp. were circulating in the region during the time of disease outbreak and implicate the involvement of an as yet undetermined SFG rickettsia in northwestern Peru.

Adolescent↗
PubMed2006

Neospora caninum and Leptospira serovar serostatus in dairy cattle in Ontario.

No significant association existed between Neospora caninum titer and serostatus to Leptospira serovar hardjo, icterohaemorrhagiae, or pomona in cattle on 78 dairy herds in Ontario. Leptospira titer increased with parity. Amongst herds not vaccinated against Leptospira, the proportions of herds with > or = 1 animal seropositive to serovar hardjo, icterohaemorrhagiae, or pomona were 45%, 42%, and 58%, respectively.

Abortion, Veterinary↗
PubMedDate not supplied

[Modified EMJH medium for cultivation of Leptospira interrogans serogroup ballum].

Strains within the Ballum serogroup of spirochete Leptospira show fastidious growth with more exigent nutritional requirements than those of other Leptospira pathogenic strains. The influence of 37 nutritional compounds on the growth of Leptospira interrogans serogroup Ballum was investigated employing the synthetic EMJH medium as the base for the study. Microbial growth was estimated spectrophotometrically and direct counts were performed with a Petroff-Hausser counting chamber. Virulence stability was evaluated by calculating the mean lethal dose in hamsters. Antigenicity stability was evaluated by Western blotting using a specific antiserum. Cell yields commonly obtained in EMJH were triplicated without virulence or antigenicity depletions after culturing in a modified EMJH medium with an increased concentration of Tween 80, and the incorporation of sodium acetate and beef extract. Neither the increased concentration of at least 6 components of EMJH nor the incorporation of a variety of new nutrients stimulated cell yields or the growth rate of the microorganism. The results allow us to make use of an enriched culture medium that promotes high cell yields of this fastidious serogroup most prevalent in humans in Cuba.

Culture Media↗