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PubMed1992

Polymerase chain reaction for detection of Leptospira spp. in clinical samples.

A sensitive assay for Leptospira spp., the causative agent of leptospirosis, was developed on the basis of the polymerase chain reaction (PCR). A 331-bp sequence from the Leptospira interrogans serovar canicola rrs (16S) gene was amplified, and the PCR products were analyzed by DNA-DNA hybridization by using a 289-bp fragment internal to the amplified DNA. Specific PCR products also were obtained with DNA from the closely related nonpathogenic Leptospira biflexa but not with DNA from other spirochetes, such as Borrelia burgdorferi, Borrelia hermsii, Treponema denticola, Treponema pallidum, Spirochaeta aurantia, or more distant organisms such as Escherichia coli, Staphylococcus aureus, Mycobacterium tuberculosis, and Proteus mirabilis. The assay was able to detect as few as 10 bacteria. Leptospira DNA was detected in urine from experimentally infected mice. In addition, the test was found to be suitable for diagnosing leptospirosis in humans. Cerebrospinal fluid and urine from patients with leptospirosis were positive, whereas samples from control uninfected patients were negative.

Adolescent↗
PubMed1965

CANICOLA FEVER.

Explore the source record for details and available documents.

Adolescent↗
PubMed1997

Chronic hepatitis associated with leptospiral infection in vaccinated beagles.

Sixteen juvenile Beagle dogs originating from a single breeding colony and regularly vaccinated against Leptospira interrogans (serogroups Canicola and Icterohaemorrhagiae) developed a clinical syndrome characterized by retarded growth, weight loss and often ascites. Over a 10-month period, post-mortem examinations were performed on all affected dogs. Gross lesions were confined to the liver which was often firm, tan-coloured and mottled. Microscopically, hepatic lesions ranged from those of severe chronic hepatitis to mild diffuse hepatocellular vacuolation, with bile stasis, occasional scattered lymphocytic aggregates and haemosiderin granulomas. Special stains and electron microscopy revealed spirochaetes within bile canaliculi. The genus Leptospira was recognized by immunohistochemical methods in nine dogs. Leptospires were isolated from six dogs, but serological tests failed to detect significant titres of antibody to L. interrogans in these animals. A serological survey of 37 kennelmates demonstrated that 20 dogs had high titres of serogroup Australis leptospiral antibody, which could not have resulted from vaccination. These findings strongly suggest a connection between the presence of leptospires and the hepatic lesions.

Animals↗
PubMed2004

Cell aggregation: a mechanism of pathogenic Leptospira to survive in fresh water.

Transmission of leptospirosis is facilitated by the survival of pathogenic leptospires in moist environments outside their mammalian host. In the present study, the survival mechanisms of Leptospira interrogans serovar Canicola in aqueous conditions and lack of nutrients were investigated. In distilled water, leptospires were able to remain motile for 110 days (pH 7.2). However, when incubated in a semi-solid medium composed of distilled water and 0.5% purified agarose (pH 7.2), they survived 347 days. In this viscous environment, aggregates of live spirochetes were observed. Neither antibiotics (e.g. tetracycline and ampicillin) nor nutrients inhibited leptospiral aggregation. Immunoblot analysis suggested that cells incubated in water down-regulate the expression of LipL31, an inner-membrane protein, but retain expression of other membrane proteins. These studies provide insights into the mechanisms by which pathogenic Leptospira survives for prolonged periods of time in natural aqueous environments, a key stage in the leptospiral lifecycle.

Animals↗
PubMed1992

Prevalence of antibody titers to Leptospira spp. in Minnesota white-tailed deer.

Serum samples (n = 204) from 124 white-tailed deer (Odocoileus virginianus) in northeastern Minnesota (USA) were collected from 1984 through 1989 and tested for antibodies to six serovars of Leptospira interrogans (bratislava, canicola, grippotyphosa, hardjo, icterohemorrhagiae, and pomona) using a microtiter agglutination test. Eighty-eight (43%) sera were positive at greater than or equal to 1:100 for antibodies against serovars pomona and/or bratislava; none was positive for any of the other four serovars. None of the 31 sera collected in 1984-85 was positive, whereas all 54 sera collected from 1986 through 1988 had titers of greater than or equal to 1:100. During 1989, only 34 (29%) of 119 sera had titers of greater than or equal to 1:100. Based on these results, we believe there to be wide variability in exposure of Minnesota deer to Leptospira interrogans.

Agglutination Tests↗
PubMed2004

An indirect enzyme linked immunosorbent assay for the detection of bovine antibodies to multiple Leptospira serovars.

An indirect enzyme linked immunosorbent assay was developed for the detection of bovine antibodies to multiple pathogenic Leptospira serovars, including canicola, copenhageni (represents icterohaemorrhagiae), grippotyphosa, hardjobovis, pomona, and sejroe. The antigen utilized in this assay was a sonicated mixture of equal parts of killed whole cells of each of the 6 serovars named above. A mouse monoclonal antibody against bovine immunoglobulin (Ig)G1 that was conjugated with horseradish peroxidase was used for detection of bound antibodies. This assay was evaluated with sera (n = 3107) that were microscopic agglutination test (MAT)-negative (at a 1:100 dilution) for each of the 6 serovars listed above and sera (n = 601) that were MAT-positive (at a 1:100 dilution) for 1, or any combination of the 6 listed serovars. In addition, sera from serial weekly bleedings of cows, which were individually experimentally infected with serovars hardjobovis, copenhageni, grippotyphosa, or canicola, were also tested in this assay. At an optimal cut-off point determined by receiver operating characteristic (ROC) curve analysis, the relative sensitivity and specificity of the assay were 93.5% (95% confidence interval = 91.2% to 95.3%) and 94.7% (95% confidence interval = 93.9% to 95.5%), respectively. This assay was able to detect antibody in the sera of animals experimentally infected with serovar hardjobovis as early as 1 week postinoculation.

Animals↗
PubMedDate not supplied

Detection of antibodies to leptospirosis in experimentally infected dogs using the microcapsule agglutination test.

Six puppies were infected with a virulent strain of Leptospira interrogans serovar icterohaemorrhagiae and another five animals with a virulent strain of Leptospira interrogans serovar canicola, respectively. Antibodies were examined at 3, 5, 7, 11 and 14 days after infection, using the microcapsule agglutination test (MCAT) and the conventional microscopic agglutination test (MAT). Compared with the MAT, the MCAT detected early specific IgM antibody with high sensitivity. The MCAT titres reached a peak at the 7th day after infection and declined gradually after the 11th day, while the MAT titres increased up to the 14th day.

Agglutination Tests↗
PubMed1992

Serodiagnosis of leptospirosis in pigs using an axial filament enzyme-linked immunosorbent assay.

The axial filament (AF) from Leptospira interrogans serovar canicola was isolated by cesium chloride density gradient centrifugation of 2% sarcosyl treated whole cells. Isolation of AF was confirmed by electron microscopic examination, by protein-A immunogold labelling, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and immunoblotting. Analysis by SDS-PAGE of the purified preparation showed relatively weak bands of molecular size 41 kDa and 21 kDa, and strong bands of 35 kDa and 34.5 kDa. Immunoblot analysis using antiserum to the AF against sonicated leptospires of a variety of serovars showed prominent reaction against the 41, 35, and 34.5 kDa protein bands, as well as against minor bands of molecular weight 43, 39, and 37 kDa. Antisera prepared against leptospiral serovars also identified minor bands at 33 and 32 kDa. Immunoblots with antiserum to whole cells of serovar bratislava detected the 35 and 34.5 kDa AF bands of Borrelia burgdorferi moderately and of Treponema hyodysenteriae only slightly in comparison to leptospiral AF. Antibody to B. burgdorferi did not detect the leptospiral AF antigen. Immunoblots with antiserum to T. hyodysenteriae showed a marked reaction with a 41 kDa band of B. burgdorferi but only a very minor reaction with leptospiral AF. The AF was tested in an AF-ELISA against sera from 260 pigs, many of which reacted in the microscopic agglutination test (MAT) against one or more leptospiral serovars. A sensitivity of 97.1% and a specificity of 93.1% was determined in comparison to the MAT. Only moderate correlation was observed between titres detected in the AF-ELISA and the MAT (r = 0.4). When sonicated whole cells (WC) of serovar canicola were used in an ELISA (WC-ELISA), high correlation was observed between AF-ELISA and WC-ELISA (r = 0.97). These findings show that the AF-ELISA can be used effectively as a species-specific antigen for the serological diagnosis of leptospirosis in swine and that sonicated whole cells can substitute excellently for purified AF as the antigen source. These findings may be extrapolated to the use of AF in immunodiagnosis of leptospirosis in other species.

Agglutination Tests↗
PubMed1997

Demonstration of leptospiral antigens on tissues using monoclonal antibodies and avidin-biotin peroxidase staining.

Glycolipoprotein (GLP) cytotoxin was extracted from Leptospira interrogans serovar canicola. The silver staining profile of GLP subjected to SDS-PAGE under denaturing conditions showed a number of bands in the mol. weight range of 14-66 kDa. Mouse Monoclonal Antibodies (MAbs) IgG3 recognizing a band near to 24 kDa of leptospiral GLP were produced (clone number MGLP-01). The agglutinating property of MAbs was established by microscopic agglutination test (MAT) using 25 different serovars as antigens. Only the homologous serovar was agglutinated by MAbs suggesting that the recognized epitope is a specific surface-exposed antigen. The MAbs were applied to demonstration of leptospiral antigens in tissue damage by avidin-biotin immunoperoxidase staining. Golden hamsters were experimentally infected with a virulent strain of L. interrogans serovar canicola. Histologically kidneys stained by routine hematoxylin and eosin showed changes characterized by injury of tubular epithelial cells leading to acute tubular necrosis (ATN). Typical, well-defined morphologic leptospires or finely granular deposits were found by immunoperoxidase staining near to blood vessels, within inflammatory infiltrates and intraluminal in proximal and distal parts of the nephron. Binding of leptospiral antigens to capillary endothelial cells, tubular epithelial cells and macrophages were also demonstrated. This entails a basis for further studies either in research or in diagnostic histopathology.

Animals↗
PubMed1989

Unique organization of Leptospira interrogans rRNA genes.

We cloned Sau3AI fragments containing the rRNA genes for Leptospira interrogans serovar canicola strain Moulton in the BamHI site of lambda EMBL3 bacteriophage DNA. Physical maps of the fragments were constructed, and the locations of the rRNA genes were determined by Southern blot hybridization and S1 protection. Each fragment of the 23S or the 16S rRNA gene contained at least one copy of the 23S or the 16S sequence. Genomic hybridization showed that there were two genes for the 23S rRNA and the 16S rRNA but only one gene for the 5S rRNA on the chromosome of L. interrogans. The results revealed the important fact that each rRNA gene is located far from the other rRNA genes. Our findings, accordingly, also suggest that these rRNA genes are expressed independently in this organism.

Blotting, Southern↗
PubMed1990

Isolation and characterization of the 5S rRNA gene of Leptospira interrogans.

The gene encoding the 5S rRNA for Leptospira interrogans serovar canicola strain Moulton was isolated and sequenced. The 5S rRNA gene occurs as a single copy within the genome and encodes a 117-nucleotide-long RNA molecule. The 5S rRNA gene is flanked at both the 5' and 3' ends by regions of A + T-rich sequences, and the 5'-flanking region contains a promoter sequence. L. interrogans has a unique and remarkable organization of the 5S rRNA gene. The 5S rRNA molecule exhibits a strong similarity to typical eubacterial 5S rRNA in terms of overall secondary structure, while the primary sequence is conserved to a lesser degree. Restriction analysis of the 5S rRNA gene indicated that the DNA sequence including the 5S rRNA gene is highly conserved in the genomes of parasitic leptospires.

Base Sequence↗
PubMed1991

Pulsed-field gel electrophoretic analysis of leptospiral DNA.

The genomic structures of spirochete species are not well characterized, and genetic studies on these organisms have been hampered by lack of a genetic exchange mechanism in these bacteria. In view of these observations, pulsed-field gel electrophoresis was used to examine the genomes of Leptospira species. Live cells, prepared in agarose plugs, were lysed in situ, and the DNA was analyzed under different electrophoretic conditions. Pulsed-field gel electrophoresis of DNA digested with infrequently cutting restriction enzymes showed that the genome of Leptospira interrogans serovar canicola is approximately 3.1 Mb, while that of the saprophytic L. biflexa serovar patoc I is 3.5 Mb. DNA forms of approximately 2,000 and 350 kb which were present in samples from L. interrogans serovars were not readily detected in nonpathogenic serovars. Three distinct populations, designated type alpha, beta, and gamma, of L. interrogans DNA molecules were further analyzed with two-dimensional gel electrophoresis. Evidence suggested that two of these DNA forms, type alpha and gamma, were linear structures. Pulsed-field gel electrophoresis has proven to be a valuable tool with which to size bacterial genomes and to take the first steps toward characterization of a form of leptospiral DNA which behaves as a linear molecule and which may be related to the virulence of L. interrogans.

DNA, Bacterial↗
PubMed2003

Leptospirosis in dogs: a serologic survey and case series 1996 to 2001.

All leptospirosis microscopic agglutination test titers for the Leptospira serovars icterohaemorrhagiae, canicola, grippotyphosa, bratislava, hardjo, and pomona conducted on 1,260 blood samples from dogs at the University of Illinois Veterinary Diagnostic Laboratory between March 1996 and March 2001 were evaluated. Low titers (1:100 to 1:400) were predominantly L. icterohaemorrhagiae and L. canicola, which represented the predominant serovars (65.4%) among all positive samples with low titers. L. grippotyphosa was the predominant serovar (72.1%) among samples with clinically significant titers (greater than 1:800). The medical records of 87 dogs with a titer greater than 1:800 that were patients at the Veterinary Teaching Hospital of the University of Illinois were reviewed. A clinical diagnosis of leptospirosis was made in 15 cases (17.2%) based on the elevated titer, appropriate clinical signs, lack of recent vaccination, and lack of concurrent disease that could explain the clinical signs present. Renal disease was present in 10 of the cases, concurrent renal and hepatic disease in two, and hepatic disease in three. In 12 cases, the predominant serovar was L. grippotyphosa; titers to L. grippotyphosa and L. bratislava were equal in magnitude in three cases.

Animals↗
PubMed2003

Leptospirosis in dogs: a serologic survey and case series 1996 to 2001.

All leptospirosis microscopic agglutination test titers for the Leptospira serovars icterohaemorrhagiae, canicola, grippotyphosa, bratislava, hardjo, and pomona conducted on 1,260 blood samples from dogs at the University of Illinois Veterinary Diagnostic Laboratory between March 1996 and March 2001 were evaluated. Low titers (1:100 to 1:400) were predominantly L. icterohaemorrhagiae and L. canicola, which represented the predominant serovars (65.4%) among all positive samples with low titers. L. grippotyphosa was the predominant serovar (72.1%) among samples with clinically significant titers (greater than 1:800). The medical records of 87 dogs with a titer greater than 1:800 that were patients at the Veterinary Teaching Hospital of the University of Illinois were reviewed. A clinical diagnosis of leptospirosis was made in 15 cases (17.2%) based on the elevated titer, appropriate clinical signs, lack of recent vaccination, and lack of concurrent disease that could explain the clinical signs present. Renal disease was present in 10 of the cases, concurrent renal and hepatic disease in two, and hepatic disease in three. In 12 cases, the predominant serovar was L. grippotyphosa; titers to L. grippotyphosa and L. bratislava were equal in magnitude in three cases.

Animals↗
PubMed2003

Comparison of the efficacy of three commercial bacterins in preventing canine leptospirosis.

Twenty-four specific pathogen-free beagles were randomly allocated into four groups (three vaccinated groups and one control group) and inoculated at nine and 12 weeks of age with one of three commercial inactivated Leptospira vaccines: A (Vanguard 7; Pfizer Santé Animale), B (Dohyvac 7L; Fort Dodge), and C (Nobivac DHPPi + Lepto; Intervet International); the control group received Nobivac DHPPi (Intervet International). Seven weeks after the second vaccination all the dogs were challenged with Leptospira interrogans serogroup canicola. All the vaccinated dogs developed a mild serological response (microscopic agglutination titres) after the booster vaccination. A significant serological response after the challenge was observed, particularly in the controls. The challenge induced fever and clinical disorders in the control group, whereas in the vaccinated groups the clinical signs were mild. Blood cultures became positive in all control dogs, and in one of six dogs vaccinated with vaccine A and two of four dogs vaccinated with vaccine B; none of the six dogs vaccinated with vaccine C was leptospiraemic at any stage of the experiment. Urine cultures were positive in all the control dogs two weeks after the challenge. One of six dogs vaccinated with vaccine A and two of four dogs vaccinated with vaccine B shed bacteria in their urine after the challenge, but none of the dogs vaccinated with vaccine C shed bacteria in their urine at any time during the experiment.

Animals↗