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PubMed2005

A test strip IgM dot-ELISA assay using leptospiral antigen of endemic strains for serodiagnosis of acute leptospirosis.

A test strip IgM dot-ELISA assay for the detection of leptospire-specific IgM antibodies in human sera was developed. Antigen dotted on a nitrocellulose paper strip was the pool sonicated antigen prepared from three predominant reactive Leptospira serovars currently in endemic area, i.e., Bratislava, Sejroe and Pyrogenes. The ability of the test to diagnose acute leptospiral infection was assessed by testing 343 single serum samples from 96 laboratory-confirmed leptospirosis case patients with positive result in the standard microscopic agglutination test (MAT), 55 serum samples from patients with various diseases other than leptospirosis, and 192 serum samples from healthy individuals. Using the results of the MAT as a gold standard, the sensitivity and specificity of the test strip IgM dot-ELISA assay were 98.96 and 93.93 per cent, respectively. The assay offered relatively high negative predictive values (99.57%) thus making the assay ideally suited for rapid screening. The stability of the test strip was assessed with a panel of five positive and five negative control sera after storage at 4 degrees C and -20 degrees C at different times. The results showed a good performance of the test strip at both storage temperatures for up to one year. In conclusion, the test strip IgM dot-ELISA assay was sufficiently sensitive for use as a screening test for serodiagnosis of acute leptospirosis. The assay was simple, inexpensive, and easy to perform for both a single test format and a large number of specimens. However, further studies are still needed to improve the stability of the test strip and assay reagents at ambient temperature, and to make the assay more rapidly and more user friendly.

Antigens, Bacterial↗
PubMed1962

IN VIVO AND IN VITRO OBSERVATIONS OF LEPTOSPIRA POMONA BY ELECTRON MICROSCOPY.

Miller, Norman G. (University of Nebraska College of Medicine, Omaha) and Richard B. Wilson. In vivo and in vitro observations of Leptospira pomona by electron microscopy. J. Bacteriol. 84:569-576. 1962.-Leptospira pomona 3341 was observed by electron microscopy, after the preparation of thin sections from culture material and from infected hamster tissue. The external membrane of low electron density envelops the entire leptospire and appears to be quite flexible, as suggested by its many folds. The spiral protoplasmic body is tubular in structure with a relatively dense wall and a central area of low electron density. Occasionally, very dark circumscribed bodies were seen imbedded in the protoplasmic wall. Detailed morphology is presented of a knoblike structure located at the end of the axial filament. Bifurcation of the axial filament could be demonstrated in leptospires from cultures. Leptospires were observed free or enclosed in vesicles within the cytoplasm of liver parenchymal and renal tubule cells. Erythrocytes located in kidney tissue also contained leptospires within the cytoplasm. The appearance of intracellular leptospires is much the same as those seen extracellularly or from culture.

Journal Article↗
PubMed1980

Comparative study between microscopic agglutination and complement fixation tests in experimental canine leptospirosis.

In order to evaluate the antibody response to pathologic leptospira from both serovars icterohaemorrhagiae and canicola, dogs were experimentally inoculated with these leptospiras. Each of the serovar were inoculated in 15 dogs and the antibody response in the acute phase of infected was followed by microscopic agglutination and complement fixation tests. Complement fixing antibodies were detected in both groups at the 4th day post infection and agglutinins appeared in circulation at the 7th day post infection when homologous antigens were used. On the 63rd day post infection with serovar canicola and on 56th day post infection with serovar icterohaemorrhagiae, complement fixing antibodies could no longer be detected but, in both experimental groups, specific agglutinins were presented in significant titers.

Agglutination Tests↗
PubMed2004

Alveolar septal deposition of immunoglobulin and complement parallels pulmonary hemorrhage in a guinea pig model of severe pulmonary leptospirosis.

Human patients suffering from leptospirosis present with a diverse array of clinical manifestations, including the more severe and often fatal pulmonary form of the disease. The etiology of pulmonary hemorrhage is unclear. Isolates of Leptospira acquired from patients suffering from pulmonary hemorrhage were used to develop a guinea pig model of pulmonary hemorrhage. Gross findings post-infection confirmed extensive hemorrhage in the lungs and on peritoneal surfaces as the likely cause of death. Immunohistochemistry confirmed the presence of large numbers of leptospires in kidney, liver, intestinal tissues, and spleen, but few inflammatory cells were seen. In marked contrast, few leptospires were detected in infected hemorrhagic lung tissue. Blood chemistries and hematology did not reveal the etiology of the hemorrhage observed. There was no chemical or microscopic evidence for disseminated intravascular coagulation. To ascertain an immunopathologic role during disease, immunofluorescence was performed on infected lung tissues and confirmed the presence of IgM, IgG, IgA, and C3 along the alveolar basement membrane. This suggests that an autoimmune process may be the etiology of fatal pulmonary hemorrhage in leptospirosis.

Animals↗
PubMed1996

Molecular cloning and sequence analysis of the gene encoding LipL41, a surface-exposed lipoprotein of pathogenic Leptospira species.

We report the cloning of the gene encoding a surface-exposed leptospiral lipoprotein, designated LipL41. In a previous study, a 41-kDa protein antigen was identified on the surface of Leptospira kirschneri (D. A. Haake, E. M. Walker, D. R. Blanco, C. A. Bolin, J. N. Miller, and M. A. Lovett, Infect. Immun. 59:1131-1140, 1991). We obtained the N-terminal amino acid sequence of a staphylococcal V8 proteolytic-digest fragment in order to design an oligonucleotide probe.A Lambda ZAP II library containing EcoRI fragments of L. kirschneri DNA was screened, and a 2.3-kb DNA fragment which contained the entire structural lipL41 gene was identified. The deduced amino acid sequence of LipL41 would encode a 355-amino-acid polypeptide with a 19-amino-acid signal peptide, followed by an L-X-Y-C lipoprotein signal peptidase cleavage site. A recombinant His6-LipL41 fusion protein was expressed in Escherichia coli in order to generate specific rabbit antiserum. LipL41 is solubilized by Triton X-114 extraction of L. kirschneri; phase separation results in partitioning of LipL41 exclusively into the detergent phase. At least eight proteins, including LipL41 and the other major Triton X-114 detergent phase proteins, are intrinsically labeled during incubation of L. kirschneri in media containing [3H] palmitate. Processing of LipL41 is inhibited by globomycin, a selective inhibitor of lipoprotein signal peptidase. Triton X-100 extracts of L. kirschneri contain immunoprecipitable OmpL1 (porin), LipL41, and another lipoprotein, LipL36. However, in contrast to LipL36, only LipL41 and OmpL1 were exposed on the surface of intact organisms. Immunoblot analysis of a panel of Leptospira species reveals that LipL41 expression is highly conserved among leptospiral pathogens.

Amino Acid Sequence↗
PubMed1997

[Frequency and etiology of calf losses and calf diseases in cow-calf farms. III. seroprevalence of selected diseases and prevalence of endoparasites and weaning age].

In a cross sectional study in 38 cow-calf farms blood and fecal samples were taken from 207 calves at an age of 8-10 months and their darms, in order to determine the seroprevalence of Leptospira hardjo, Coxiella burnetii, Toxoplasma gondii and the presence of the BVD-Antigen as well as the prevalence of endoparasites. The study was conducted in fall 1994 at the end of the grazing period. Eggs or larvaes of gastrointestinal helminths were detected in 74% of calves that were dewormed before the grazing period and in 88% of untreated calves. The difference between the two groups was statistically significant. In 83% of the weaned calves oocysts of Eimeria spp. were diagnosed. No significant difference in weight gain was observed between animals without endoparasites and animals infected with either helminths and/or Eimeria. In cows the prevalence was 61.5% for helminths and 45.7% for Eimeria spp. In calves the seroprevalence was 4% for Leptospira hardjo, 0.5% for Coxiella burnetii and 14.0% for Toxoplasma gondii. The seroprevalence in cows were 18% for Leptospira hardjo, 7.6% for Coxiella burnetii and 14.3% for Toxoplasma gondii. The prevalence of the BVD-Virus antigen was 0.5% for calves and cows.

Aging↗
PubMed1976

[Review of the incidence of antibodies to various serological groups of the species Leptospira interrogans in a number of farm animals in the Netherlands (author's transl)].

Report on the results of serological studies on the species Leptospira interrogans in cattle (19,607), swine (6,348), dogs (182) and horses (88) from the Netherlands during the period from 1969 to 1974. Living cultures of the serotypes of pomona, icterohaemorrhagiae, canicola, guidae (Tarassovi serological group), grippotyphosa and sejroe were used as antigen in the micro-agglutination test. The numerical findings showed that antibodies to serotypes of the species Leptospira interrogans were present in 7.67 per cent of the cattle, 22.21 per cent of the pigs, 36.81 per cent of the dogs and 92.05 per cent of the horses studied. Infection with the serotypes of icterohaemorrhagiae and grippotyphosa was most common in cattle and horses, icterohaemorrhagiae and tarassovi were most common in swine and icterohaemorrhagiae and canicola were most common in dogs. The presence of pomona is not a factor in the Netherlands. In view of statistical findings, grippotyphosa infections in cattle may be assumed to result in abortion.

Abortion, Septic↗
PubMed1993

[Evaluation of the counterimmunoelectrophoresis technique for the serologic diagnosis of leptospirosis].

The counterimmunoelectrophoresis (CIE) technic was assessed for leptospirosis serological diagnosis by using an antigenic preparation from Leptospira biflexa Patoc I strain. It was showed that the CIE technic had 82% sensitivity and 100% specificity. The antigen used showed a gender-specific reactivity on detecting antibodies in patients infected by different leptospirosis serogroups by microagglutination test. Antigen stability for CIE technic was 6 months without loss of titre.

Antigens, Bacterial↗
PubMed1995

T cell response to Borrelia garinii, Borrelia afzelii, and Borrelia japonica in various congenic mouse strains.

The infectivity and T cell response to Borrelia garinii SIKA2, Borrelia afzelii BFOX, and Borrelia japonica 0612, the organisms that cause Lyme disease in Japan, were examined in various inbred and congenic strains of mice. Infectivity differed among the species: B. garinii SIKA2 and B. afzelii BFOX were each able to infect 90% to 100% of C3H/He mice; B. japonica 0612 was able to infect only 20% of C3H/He mice. The pattern of infectivity to various inbred and congenic strains of mice may influence the pathogenicity of the organism and the clinical signs of Lyme disease. Cross-reactivity between Borrelia antigens was observed, but there was no cross-reactivity between Borrelia antigens and Leptospira antigens. We evaluated the genetic control of the delayed-type hypersensitivity (DTH) reaction in the form of footpad swelling produced by Borrelia antigens using viable or sonicated bacteria as sensitization. Differences in strains of mice infected by viable antigen were observed. However, all strains of mice showed a strong DTH reaction using sonicated antigens without genetic background. A DTH reaction in the form of footpad swelling did not appear to be associated with genetic background. The footpad reaction was mediated by CD4+8- and Ia- T cells, as revealed by in vitro monoclonal antibody treatment. However, CD8+ T cells did not suppress footpad swelling. These results indicate that many antigenic epitopes of the Borrelia spirochete can stimulate the DTH reaction.

Animals↗
PubMed1980

Detection of leptospiral antibodies in animal sera by means of fractionated antigenic extracts.

Sodium dodecyl sulphate extracts of the reference strains Mus 127, Castellón 3 and Arborea of the Ballum agglutinogenic serogroup of Leptospira interrogans (the species of pathogenic leptospira), of strain Patoc 1 of the saprophytic species of L. biflexa, and of strain 3055 of illini serotype, the sole representative of L. illini, were each fractionated by ultracentrifugation in a sucrose density gradient into 10 fractions. The fractions were tested by complement fixation and immunodiffusion against the sera of animals during the process of immunisation and during the course of naturally occurring infections. The fractions could be divided into three main pools of serological activity: pool I (fractions 1, 2 and 3), pool II (fractions 4, 5 and 6), pool III (fractions 7, 8 and 9). Pool I was species/genus specific; fraction 1 tended to be species specific while fraction 2 reacted with antisera to all strains whether pathogenic or saprophytic. Pool II was serogroup specific and reacted only with antisera to members of the same serogroup. Pool III was serotype (serovar) specific and revealed the identity of the infecting strain at an early stage of infection.

Agglutination Tests↗
PubMed1991

Anticardiolipin antibodies in leptospirosis.

The clinical course and serology of 16 cases of leptospirosis in an area with an unusually high endemic infection rate were studied to gain further insight into the pathology of the secondary immune phase that is typical of the disease. IgG anticardiolipin antibody concentrations were measured by immunoassay and found to be increased in eight serologically confirmed cases with severe complicated disease, compared with eight patients with relatively uncomplicated leptospirosis who had IgG anticardiolipin concentrations within the control reference range. This previously unreported association suggests that leptospira may induce vascular endothelial injury in severe cases and expose crypt antigens or induce conformational change of cell surface phospholipids. Leptospirosis may provide a model for an infective origin of some cases of the antiphospholipid syndrome.

Autoantibodies↗
PubMed2001

Immunoperoxidase and histopathological examinations of leptospiral nephritis in cattle.

This study was designed to investigate the role of leptospirae in interstitial nephritis. Sixty-eight white-spotted kidneys and 30 grossly normal kidneys from slaughtered cattle were examined histologically and immunohistochemically for the presence of Leptospira interrogans antigens. The presence of L. interrogans antigens was found in 21 of 68 white-spotted kidneys and in four of 30 normal kidneys. In conclusion, the detected incidence of infection with L. interrogans was not high, but there was a relationship between the presence of interstitial nephritis and leptospiral antigens.

Animals↗
PubMed2005

Diversity of bat-associated Leptospira in the Peruvian Amazon inferred by bayesian phylogenetic analysis of 16S ribosomal DNA sequences.

The role of bats as potential sources of transmission to humans or as maintenance hosts of leptospires is poorly understood. We quantified the prevalence of leptospiral colonization in bats in the Peruvian Amazon in the vicinity of Iquitos, an area of high biologic diversity. Of 589 analyzed bats, culture (3 of 589) and molecular evidence (20 of 589) of leptospiral colonization was found in the kidneys, yielding an overall colonization rate of 3.4%. Infection rates differed with habitat and location, and among different bat species. Bayesian analysis was used to infer phylogenic relationships of leptospiral 16S ribosomal DNA sequences. Tree topologies were consistent with groupings based on DNA-DNA hybridization studies. A diverse group of leptospires was found in peri-Iquitos bat populations including Leptospira interrogans (5 clones), L. kirschneri (1), L. borgpetersenii (4), L. fainei (1), and two previously undescribed leptospiral species (8). Although L. kirschenri and L. interrogans have been previously isolated from bats, this report is the first to describe L. borgpetersenii and L. fainei infection of bats. A wild animal reservoir of L. fainei has not been previously described. The detection in bats of the L. interrogans serovar Icterohemorrhagiae, a leptospire typically maintained by peridomestic rats, suggests a rodent-bat infection cycle. Bats in Iquitos maintain a genetically diverse group of leptospires. These results provide a solid basis for pursuing molecular epidemiologic studies of bat-associated Leptospira, a potentially new epidemiologic reservoir of transmission of leptospirosis to humans.

Animals↗