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PubMed2005

Comparison of immunoreactive proteins of commonly circulating serogroups of Leptospira in Andaman Islands, India.

BACKGROUND & OBJECTIVE: Early diagnosis is the key to the treatment of leptospirosis. For development of rapid diagnostic kits, a thorough knowledge about the nature of the proteins expressed by the pathogen during infection is necessary. The present study was undertaken to understand the nature of immunoreactive proteins from commonly circulating serogroups of Leptospira in the endemic Andaman and Nicobar Islands, India. METHODS: Proteins were extracted from six strains of Leptospira representing five different serogroups following four different preparation methods, viz., whole cell lysis by sonication, detergent solubilization, outer and inner membrane isolations, and were subsequently characterized on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Immunoblots were made from the sonicated proteins using hyperimmune rabbit antisera, homologous and heterologous patient sera separately. RESULTS: The 67, 65, 45, 43, 35, 32 and 18 kDa major proteins in the whole cell lysate were common among all the five serogroups of Leptospira. The 67, 41, 35, 32, 28 and 22 kDa were the major outer membrane proteins, while 94, 32, 25 and 18 kDa protein were in inner membrane. Immunoblots with hyperimmune rabbit antisera detected 67, 65, 60, 45, 43, 41 and 32 kDa common proteins from the whole cell lysates of all strains while homologous and heterologous patient sera detected 32 kDa as the major immunoreactive protein in all pathogenic serogroups. This protein reacted against specific LipL32 antisera indicating that this protein was LipL32. INTERPRETATION & CONCLUSION: The circulating serogroups of Leptospira have common nature of expression of proteins during human infection. Among several immunoreactive proteins, three (67, 45 and 32 kDa) were recognized as major antigens by both rabbit hyperimmune sera and patients sera while the 32 kDa protein was recognized as the major immunoreactive protein by homologous and heterologous patient sera. These conserved immunoreactive proteins could be utilized in developing indigenous diagnostic tests for leptospirosis.

Animals↗

PubMed2006

[Seroprevalence of Leptospira infection in habitants of peripheral neighborhoods in Cali, Colombia].

INTRODUCTION: Leptospirosis is a zoonotic disease with varying modes of transmission and clinical expression that affects humans inhabiting urban and rural areas worldwide. In Colombia, little is known regarding leptospirosis in urban settings. OBJECTIVES: To establish the seroprevalence of leptospiral infection in humans, to assess the serological reactivity and to identify factors associated with seropositivity. MATERIALS AND METHODS: We conducted an infection prevalence study in 259 inhabitants of peripheral neighborhoods in the city of Cali, Colombia, stratified by sex and age. Sociodemographic information, time residing in the same neighborhood, occupation, exposure to potentially infectious sources like water or animals, and serum used in a Microagglutination test against 19 Leptospira serovars were obtained. RESULTS: The population was composed mainly of students (37.1%), housewives (32%) and workers in non-formal activities (24.6%). The prevalence of anti-Leptospira antibodies was 23.3% (C195% 18.3-28.3), with high frequency in people older than 57 years of age and significantly higher in men than women (p=0.045). There was an association between the presence of antibodies and contact with animals (p=0.038). Reactivity was observed against 16 of the 19 serovars evaluated, but the antibody titers were low. CONCLUSIONS: The high infection seroprevalence found in this study suggests frequent contact with Leptospira in putatively high risk neighborhoods of Cali. Contact with animals was the most significant factor associated with the presence of anti-Leptospira antibodies.

Adolescent↗

PubMed2006

Development of a duplex-polymerase chain reaction for rapid detection of pathogenic Leptospira.

A duplex-polymerase chain reaction (PCR) for the rapid detection of pathogenic leptospires was developed by using two sets of newly designed primers which amplified in the same reaction two different DNA fragments simultaneously: 279-bp of LipL32 and 430-bp of 16S rRNA. For DNA extraction from bacterial cultures, the silica-based spin column method was found to be more suitable and was selected for the extraction of DNAs from all 92 bacterial strains including 56 strains of pathogenic Leptospira, 15 strains of non-pathogenic Leptospira and 21 other strains of bacteria. The PCR products were analyzed by agarose gel-electrophoresis with confirmation by Southern and dot hybridization using synthetic DNA probe prepared from LipL32 gene of a pathogenic reference strain, L. interrogans serovar pyrogenes. The duplex-PCR allowed detection of two products of 279 bp and 430 bp in all pathogenic Leptospira. Non-pathogenic Leptospira generated a single product of 430 bp. Other bacterial strains failed to reveal any amplification products. As little as 1 pg of pure DNA corresponding to 100 cells could be detected by agarose gel-electrophoresis, and 1-10 fg of pure DNA by hybridization.

DNA Probes↗

PubMed1989

[Use of the coagglutination reaction for typing Leptospira cultures].

The coagglutination test has been employed for the serologic typing of Leptospira cultures. A set of diagnostic agents with a maximum kit of anti-Leptospira sera of various serogroups has been prepared on the basis of a 10% suspension of formalin-treated Cowan 1 staphylococcus strain. To prepare the diagnostic agent, 0.1 portion of the serum and 9 portions of phosphate buffer solution, pH 7.2 should be added to 1 portion of a 10% staphylococcal suspension. 1% staphylococcal suspension has been prepared for control. The diagnosticum and the culture to type (0.01 ml each) have been mixed on the slides; the control test has been carried out with 1% non-sensitized staphylococcal suspension. The test has been read by microscopy in the dark visual field after the slides' 15 min exposure in a humid chamber. Typing of 17 Leptospira cultures of known serologic groups has yielded positive responses with the homologous diagnosticums only; no cross reactions have been recorded. The coagglutination test may be used for the rapid differentiation of mixed Leptospira cultures belonging to different serologic groups.

Agglutination Tests↗

PubMed1985

[Differences in the susceptibility of field mice from the Altai Territory and Moscow Province to Leptospira of serovar mozdok and serogroup Pomona].

Adult striped field mice (Apodemus agrarius) caught in the Altai region proved to be insusceptible to experimental infection when inoculated with Leptospira of serovar mozdok, serogroup Pomona. In pregnant females, though infected with this organism, no leptospiruria was observed. At the same time nonpubescent animals became Leptospira carriers, females becoming carriers 4.5 times more frequently than males. The formation of antibodies to Leptospira in the test rodents was poorly pronounced and did not depend on their sex, age, physiological state and the presence of renal leptospirosis. But all adult striped field mice belonging to the population of the Moscow region became Leptospira carriers in such experiments.

Aging↗

PubMed1984

Immunohistochemical investigations of leptospira-infected golden hamsters in potency tests.

Golden hamsters taken from routine potency testing of leptospirosis vaccines were investigated for leptospirae after a challenge infection. Kidneys, livers, and spleens were examined immunohistochemically by the unlabelled antibody enzyme (PAP) method, and samples of these organs were additionally cultured for the evidence of living leptospirae. In non-vaccinated animals the recultivation method indicated leptospirae during the bacteraemic phase, beginning immediately after infection. Both methods resulted in positive reactions after organ manifestations. Intravascular evidence of leptospirae in the kidneys could be seen first on day 3 after infection. On day 7, there was intracellular and intratubular evidence as well, combined with tubulo-interstitial alterations. In vaccinated animals the bacteraemia after infection was reduced. Organ manifestations were seen rarely; there were less severe or no pathohistological alterations.

Animals↗

PubMed1984

[Territorial dissemination of antibodies against leptospirae at individual farms throughout a district, detected on the basis of serological testing of slaughter cattle and pigs].

The serological reaction of microagglutination-lysis (RMAL) was performed to examine 4995 head of slaughter cattle and 8211 slaughter pigs for the presence of antibodies to leptospirae. The animals came from 33 farms of the Strakonice district. Antibodies to leptospirae were found in slaughter cattle from 25 (75.75%) farms and in slaughter pigs from 24 (72.72%) farms. As suggested by the examination, the antibodies to leptospirae occur in animals throughout the territory of the district, irrespective of the nature of the landscape. The occurrence of the above-mentioned antibodies on farms occurred within the range from 0.52% to 8.33% in slaughter cattle and from 0.32% to 8.10% in slaughter pigs; only rarely did it exceed 9.0%. The most frequent reactions were those with the leptospirae of the L. grippotyphosa serovar.

Animals↗

PubMed1984

[Incidence of antibodies against leptospira in the blood of breeding sows].

Within a year, 4438 blood sera of sows coming from 25 farms of the Strakonice district were subjected to serological examination by the reaction of microagglutination lysis (RMAL) with 12 strains of Leptospira. The reaction was positive in 3.22% of all the blood sera. Separate significant titres were demonstrated in the reaction with the Leptospira organisms of four serological groups, including Grippotyphosa, Icterohaemorrhagiae, Hebdomadis and Australis. The reactions with the Leptospira of the Canicola serological group were observed only individually as coagglutination with a simultaneous occurrence of antibodies to Leptospira of the Grippotyphosa serological group. The highest number of reactions 119 (75.8%) was demonstrated in titre 400.

Animals↗

PubMed1983

Survey of rats (Rattus norvegicus) in Kuwait for the presence of Leptospira.

A survey of brown rats (Rattus norvegicus) was made in Kuwait in 1979 for the presence of Leptospira organisms. Kidney tissue from 49 rats, trapped mostly from various Kuwait City districts, were cultured on E.M.J.H. and Stuart media. Eight leptospira strains were isolated; all strains were identical and belonged to Leptospira interrogans Canicola serogroup; they were later identified as a new serovar kuwait. The frequency of L. interrogans group Canicola carriers among the local rats in Kuwait was 16.3%, which is higher than reported so far. A summary of published data on the isolation of Leptospira other than L. interrogans serogroup Icterohaemorrhagiae from rats, particularly the brown rat, is presented with emphasis on the Middle East.

Animals↗

PubMed1994

[Results of serological studies of Leptospira antibodies in foxes].

1253 serum samples of foxes were checked for the presence of Leptospira antibodies. 24 serum samples (1.92%) were positive. Leptospira grippotyphosa (10 samples) was the most common serotype, which ist also widespread in other wild and domestic animals. The possibility of distribution of Leptospira in fox populations are discussed. Leptospira antibodies were found in only 0.20% of the examined serum samples of domestic animals of the same territory (South-Thuringia).

Animals↗

PubMedDate not supplied

[The distribution of leptospirae in the icterohaemorrhagiae serogroup].

The capacity of leptospiras to get acclimated in the organism of unusual hosts has been studied. Cultures of Icterohaemorrhagiae from musquashes, house mice and field voles have been isolated of leptospirosis sources under intensive epizootics among rats. A possibility to reproduce Leptospira carrying by the cultures of leptospirae of serogroup icterohaemorrhagiae in musquashes and water voles has been shown. One can conclude that under icterohemorrhagic leptospirosis musquashes and water voles can take the part of additional source of infection but they cannot independently preserve leptospirae in nature as a biological species grey rats being their main hosts.

Animals↗

PubMed1997

Development of a monoclonal antibody-based competitive enzyme-linked immunosorbent assay for the detection of Leptospira borgpetersenii serovar hardjo type hardjobovis antibodies in bovine sera.

A murine monoclonal antibody (designated M553) that binds to an epitope on whole cell antigens prepared from Leptospira borgpetersenii serovar hardjo type hardjobovis and Leptospira interrogans serovar hardjo type hardjoprajitno, was produced and incorporated into a competitive enzyme-linked immunosorbent assay for the detection of bovine antibodies to serovar hardjo. The epitope recognized by M553 was susceptible to periodate oxidation. The M553 antibody was characterized by western blot with hardjobovis whole cell antigen. This antibody does not cross-react with whole cell antigens prepared from 11 other pathogenic Leptospira serovars, or, Leptospira biflexa serovar patoc. The sensitivity estimate of the competitive ELISA was 100% with field sera (n = 165) with serovar hardjo microscopic agglutination test (MAT) titres of > or = 100. The specificity estimate was 100% with sera (n = 128) obtained from a specific pathogen free herd of cattle that were negative in the MAT at a dilution of 1:100 for serovars hardjo, pomona, sejroe, copenhageni, canicola, and grippotyphosa. The specificity estimate with field sera (n = 301) with serovar hardjo MAT titres of < 100, was 98% (95% confidence interval = +/- 1.58%). There was no cross-reactivity with field sera (n = 306) with serovar pomona titres > or = 100 and serovar hardjo titres < 100. The specificity estimate with the combined populations of sera with serovar hardjo MAT titres of < 100 (n = 735) was 99.18% (95% confidence interval = +/- 0.65%). There was a high level of agreement (kappa = 0.977) between the results of the competitive ELISA and those of the MAT.

Animals↗

PubMed1997

[Relation between characteristics of the small mammal population and occurrence of Leptospira antibodies].

Five rodent species (Apodemus flavicollis, A. agrarius, A. microps, Clethrionomys glareolus and Microtus arvalis) were examined serologically for presence of antibodies to leptospira. Small mammals were caught in several areas of eastern Slovakia, an important part of host material coming from East Slovakian Lowland (45.7%), south part of Kosice basin (26.5%) and Slovak carst (10.1%). Serological examinations were used by Kmety and Bakoss (1978), the sexual condition of rodents were ascertained after Pelikán (1965). Altogether, 2,113 individuals of rodent species were examined during five years (1991-1995), antibodies to leptospires were demonstrated in 5.02% of examined hosts. In examined hosts antibodies to six serovars of leptospira (Leptospira grippothyphosa, L. sejroe, L. bataviae, L. bratislava, L. pomona and L. sorex-jalna) were found. The most frequently observed antibodies were those of the serovar L. grippothyphosa (65.1%) and L. sejroe (25.5%). Slightly higher percentage of positive hosts in mature group rodents (5.4%) comparing with immature rodents (4.6%) were statistically insignificant. Similarly, no differences were found between percentage of positive rodent females (5.3%) and group of males (4.7%). No statistical differences were found in positivities to leptospira antibodies among sexual and age groups of individual rodent species, too. Proportion of serological positivities to L. grippothyphosa and L. sejroe in compared sex and age rodent groups were very similar. In evaluated rodent species trend of increases of serological positive values from spring to autumn was observed, but it was statistically confirmed only in A. agrarius.

Age Factors↗

PubMed1992

[The identification of Leptospira strains of different origins].

Leptospirosis is at present an ever-increasing problem in human and animal health. By means of the Korthof medium, 43 Leptospira strains were isolated from samples of human blood, water and soil. For their identification the microagglutination technique was used. The strains corresponded to the species Leptospira biflexa and Leptospira interrogans.

Cuba↗

PubMed2004

Effects of static magnetic field on growth of leptospire, Leptospira interrogans serovar canicola: immunoreactivity and cell division.

The effects of the exposure of the bacterium, Leptospira interrogans serovar canicola to a constant magnetic field with magnetic flux density from a permanent ferrite magnet=140+/-5 mT were studied. Changes in Leptospira cells after their exposure to the field were determined on the basis of changes in their growth behavior and agglutination immunoreactivity with a homologous antiserum using dark-field microscopy together with visual imaging. The data showed that the exposed Leptospira cells have lower densities and lower agglutination immunoreactivity than the unexposed control group. Interestingly, some of the exposed Leptospira cells showed abnormal morphologies such as large lengths. We discussed some of the possible reasons for these observations.

Journal Article↗

PubMed1993

The prevalence of small terrestrial mammals infected with tick-borne encephalitis virus and leptospirae in the foothills of the southern Bavarian forest, Germany.

In the district of Grafenau/Freyung (Bavaria, Germany), 266 specimens of small terrestrial mammals of 8 species were captured using live traps. From these mammals, Apodemus flavicollis (42.1%) and Clethrionomys glareolus (39.5%) were prevalent. All animals were tested for neutralizing antibodies to tick-borne encephalitis (TBE) virus and agglutinating antibodies to leptospirae. Seropositivity against TBE virus was 14.0% and against leptospirae 7.9%, respectively. Seropositivity to leptospirae appeared to be primarily to Leptospira grippotyphosa, less to Australis and occasionally to Javanica serovars. Only one A. flavicollis specimen was positive to both pathogens tested. The parasitocoenosis of trapped micromammals with ectoparasites consisted of 69.5% from ticks (mainly Ixodes ricinus, less from I. trianguliceps), 16.1% of mites (primarily Laelaps agilis) and 14.3% of fleas (mostly Ctenophthalmus agyrtes).

Animals↗

PubMed2000

Control of immunologically crossreactive leptospiral infection by administration of lipopolysaccharides from a nonpathogenic strain of Leptospira biflexa.

In our previous paper (Matsuo, K., Isogai, E., and Araki, Y., Carbohydr. Res., 328: 517-524, 2000), antigenic polysaccharides obtained from the lipopolysaccharide (LPS) fraction of a nonpathogenic leptospira, Leptospira biflexa patoc Patoc I, are shown to be broadly crossreactable with most rabbit antisera elicited by immunization with various pathogenic leptospires. The result led us to test a protective effect of the same LPS in a hamster model system by heterologously challenging with a pathogenic leptospira, L. interrogans manilae UP-MMG. Firstly, a similarity in the antigenic epitopes of L. biflexa and L. interrogans was confirmed by the following assays. In the microscopic agglutination test (MAT), a hamster antiserum elicited by immunization with the L. biflexa-LPS preparation was shown to agglutinate cells of L. interrogans. Contrarily, in the enzyme-linked immunosorbent assay (ELISA), the L. biflexa-LPS preparation was shown to crossreact with a hamster antiserum elicited by immunization with whole cells of L. interrogans. These results suggest that the same or closely related antigens may be present on the cell surfaces of both L. biflexa patoc Patoc I and L. interrogans manilae UP-MMG. Furthermore, in a protective assay, the prior administration of a L. biflexa-LPS preparation resulted in raising a protective response in hamsters against challenge by L. interrogans without any side effect. The protective effect was strongly dependent on the dose amounts and/or administration times of L. biflexa-LPS. Thus, L. biflexa-LPS preparations can use as a potent vaccine against leptospirosis caused by various leptospires.

Agglutination Tests↗

PubMed2004

Broth microdilution susceptibility testing for Leptospira spp.

Leptospirosis in humans has traditionally been treated with penicillin or doxycycline. The choice of therapy offered at the time of initial patient presentation is often empirical, as definitive diagnosis can take weeks. Determining the activity of numerous antimicrobial agents against a wide range of Leptospira serovars may broaden empirical therapeutic options. Various antimicrobials have been shown to be active against a limited number of serovars in in vitro studies, chiefly by the use of broth macrodilution techniques. We developed a broth microdilution technique using the commercially available growth indicator alamarBlue. MICs produced by this technique were compared to MICs and minimal bactericidal concentrations produced by the traditional broth macrodilution technique. The internal validity of our methods was assessed with 11 runs over numerous days with a single isolate of Leptospira interrogans serovar Icterohaemorrhagiae. By either method, the MICs for these internal-validity runs fell within 2 dilutions of each other for more than 90% of antimicrobials. A broader application of these two techniques included 12 serovars (including seven species) of Leptospira and six antimicrobials (penicillin G, doxycycline, chloramphenicol, erythromycin, cefotaxime, and ciprofloxacin). Observed reproducibility fell within 2 dilutions for 99% of the duplicate result sets for the MIC microdilution method, compared to 89% for the MIC macrodilution method. The macrodilution method tended to have a higher MIC at which 90% of the isolates were inhibited (MIC(90)) than did the microdilution method, but the MIC(90)s of both methods were within 2 dilutions of each other for all six drugs. The macrodilution and microdilution techniques produced similar results, with microdilution allowing a faster, more streamlined method of producing MIC results.

Anti-Bacterial Agents↗