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PubMed1978

Broadly reacting precipitating and agglutinating antigen of leptospirae.

A saprophytic Leptospira biflexa strain of equine origin was found which cross-reacts with immune rabbit antisera to 14 pathogenic Leptospira interrogans serotypes. Sera from goats experimentally inoculated with the saprophyte showed multiple low-level cross-agglutination reactions against a battery of live L. interrogans serotypes. Sonically treated and saline-extracted suspensions of the L. biflexa strain and serotypes canicola, icterohaemorrhagiae, and pomona yielded a common precipitating protein antigen that was detected by immunodiffusion and immunoelectrophoresis with all of the antileptospiral sera examined. In cross-absorption and gel diffusion tests, the precipitinogen from each of the strains was shown to be identical. Formaldehyde treatments and heating at 100 degree C suggest that the cellular location of the common antigen is either somatic or subsurface, and Sephadex G-200 gel filtration enabled the isolation of the active fraction of the L. biflexa antigen. Monoprecipitin sera against the common antigen of L. biflexa were produced by immunizing rabbits with specific precipitates in agar. In gel diffusion and immunoelectrophoresis tests the antisera with each of the soluble antigens developed a single precipitin formation, and the antisera agglutinated formolized and heated whole-cell suspensions of serotypes canicola, icterohaemorrhagiae, and pomona at low dilutions. The soluble L. biflexa antigen was evaluated as an immunogen and in passive immunity tests for protection against death and kidney infection in hamsters. No cross-protection occurred when the hamsters were challenged with virulent leptospires. In contrast, the animals vaccinated or administered hamster immune serum before challenge died earlier than the control animals.

Animals↗

PubMed1992

Isolation and biological activities of endotoxin from Leptospira interrogans.

Endotoxins extracted with ethylenediaminetetraacetate (EDTA) from Leptospira interrogans serovars icterohaemorrhagiae and canicola and Leptospira biflexa serovar patoc were tested for various biological activities characteristic of endotoxins. The presence of lipopolysaccharide biological activity was demonstrated by the Limulus amoebocyte lysate test, pyrogenicity in rabbits, complement interaction inhibiting the erythrocyte lysis, and chicken-embryo lethality. The lipopolysaccharides did not induce the local Shwartzman reaction. The lipopolysaccharides of serovars icterohaemorrhagiae and canicola were immunogenic in rabbits and were cytotoxic to chicken-embryo fibroblasts.

Animals↗

PubMed1976

Treatment of human leptospira infections with semicillin (ampicillin) or with amoxil (amoxycillin).

Ampicillin (Semicillin) and Amoxycillin (Amoxil) have a strong leptospirocidal effect in vitro and also in vivo. We have tested these drugs on 28 patients suffering from Leptospira infection. The patients generally received per os 3 g Ampicillin (Semicillin) or 2 g Amoxycillin (Amoxil) per day over a period of 6 days. In the groups treated with Ampicillin and Amoxycillin the average durations of fever during the treatment were 1.6 and 1.2 days, respectively. In the first group (Ampicillin) a 'second wave of fever' occurred in one case (5%) and in the second group (Amoxycillin), in 0% (as compared to 22% after treatment with benzylpenicillin). On the basis of our experiences, Ampicillin and Amoxycillin are strongly recommended for the treatment of human Leptospira infections.

Adolescent↗

PubMed2005

Molecular detection of Leptospira kirschneri in tissues of a prematurely born foal.

Leptospirosis was identified to be the possible cause of premature birth in a foal on a farm with a history of repeated abortions. Using an appropriate polymerase chain reaction (PCR) assay, the presence of Leptospira kirschneri was detected in the tissues of the prematurely born foal. Further confirmation of L. kirschneri was obtained by nucleotide sequence analysis of the PCR-amplified DNA fragment and the partial 16S ribosomal RNA gene sequence. This report further supports mounting evidence that a PCR assay capable of detecting L. kirschneri should be included in routine diagnostic investigations in which Leptospira spp. infection is suspected.

Animals↗

PubMed1989

Evaluation of multivalent Leptospira fluorescent antibody conjugates for general diagnostic use.

Four lots of conjugate were evaluated for optimal dilution and degree of fluorescence produced with reference cultures and bovine and porcine leptospira isolates. One lot that uniformly produced better fluorescence was evaluated for sensitivity and specificity with reference cultures, isolates, culture-positive tissues, and 13 other bacterial species. Further evaluation of the conjugates was done with bovine, porcine, and ovine specimens submitted to a diagnostic laboratory. Leptospires were detected with the fluorescent antibody test (FAT) in 9 of 21 culture-positive bovine kidneys and were detected in diluted cultures when present at concentrations of 10(2)-10(3) organisms/ml. With the exception of Treponema hyodysenteriae, FAT's of other bacterial cultures produced minimal fluorescence or were negative. Positives were characterized by moderate to brilliant fluorescence of typical cell forms, and most nonspecific fluorescence was eliminated with a flazo-orange counterstain. The results indicated that the FAT utilizing multivalent conjugates could be used successfully as an additional method for diagnosis of leptospira infections.

Animals↗

PubMed1995

Prevalence and serovars of leptospira involved in equine abortions in central Kentucky during the 1991-1993 foaling seasons.

In this study, the prevalence of leptospira-induced abortions/stillbirths for the past 3 foaling seasons (1991-1993) was determined, and fetal tissues and/or the mare's urine from positive cases were cultured in an attempt to isolate and identify the leptospira serovars responsible for the abortions. The sensitivity and specificity of the primary diagnostic tests, the fluorescent antibody test (FAT) and the microscopic agglutination test (MAT), used for the diagnosis of leptospirosis were also determined. For the 3 years, 74 (3.3%) of 2,264 abortion/stillborn submissions were diagnosed as leptospirosis. Twelve cases occurred in the 1991, 19 in the 1992, and 43 in the 1993 foaling seasons. Leptospires were isolated from 45 (60.8%) of the 74 cases, and they were identified as serovar kennewicki (43 cases), serovar grippotyphosa (1 case), and a serovar similar to pomona (1 case). Of the 29 culture-negative cases, serologic results indicated that leptospires in the Pomona serogroup (kennewicki is a member of this serogroup) were responsible for 25 abortions, leptospires in the Grippotyphosa serogroup for 1 abortion, and leptospires in the Sejroe serogroup for 1 abortion. The specificities of the FAT on fetal tissues and mare's placenta and of the MAT on fetal fluid were 100%. The sensitivity of the FAT was 98.7%, and that of the MAT was 81.3%.

Abortion, Veterinary↗

PubMed2002

A quantitative PCR (TaqMan) assay for pathogenic Leptospira spp.

BACKGROUND: Leptospirosis is an emerging infectious disease. The differential diagnosis of leptospirosis is difficult due to the varied and often "flu like" symptoms which may result in a missed or delayed diagnosis. There are over 230 known serovars in the genus Leptospira. Confirmatory serological diagnosis of leptospirosis is usually made using the microscopic agglutination test (MAT) which relies on the use of live cultures as the source of antigen, often performed using a panel of antigens representative of local serovars. Other techniques, such as the enzyme linked immunosorbent assay (ELISA) and slide agglutination test (SAT), can detect different classes of antibody but may be subject to false positive reactions and require confirmation of these results by the MAT. METHODS: The polymerase chain reaction (PCR) has been used to detect a large number of microorganisms, including those of clinical significance. The sensitivity of PCR often precludes the need for isolation and culture, thus making it ideal for the rapid detection of organisms involved in acute infections. We employed real-time (quantitative) PCR using TaqMan chemistry to detect leptospires in clinical and environmental samples. RESULTS AND CONCLUSIONS: The PCR assay can be applied to either blood or urine samples and does not rely on the isolation and culture of the organism. Capability exists for automation and high throughput testing in a clinical laboratory. It is specific for Leptospira and may discriminate pathogenic and non-pathogenic species. The limit of detection is as low as two cells.

Blood Specimen Collection↗

PubMed2006

Multilocus sequence typing method for identification and genotypic classification of pathogenic Leptospira species.

BACKGROUND: Leptospira are the parasitic bacterial organisms associated with a broad range of mammalian hosts and are responsible for severe cases of human Leptospirosis. The epidemiology of leptospirosis is complex and dynamic. Multiple serovars have been identified, each adapted to one or more animal hosts. Adaptation is a dynamic process that changes the spatial and temporal distribution of serovars and clinical manifestations in different hosts. Serotyping based on repertoire of surface antigens is an ambiguous and artificial system of classification of leptospiral agents. Molecular typing methods for the identification of pathogenic leptospires up to individual genome species level have been highly sought after since the decipherment of whole genome sequences. Only a few resources exist for microbial genotypic data based on individual techniques such as Multiple Locus Sequence Typing (MLST), but unfortunately no such databases are existent for leptospires. RESULTS: We for the first time report development of a robust MLST method for genotyping of Leptospira. Genotyping based on DNA sequence identity of 4 housekeeping genes and 2 candidate genes was analyzed in a set of 120 strains including 41 reference strains representing different geographical areas and from different sources. Of the six selected genes, adk, icdA and secY were significantly more variable whereas the LipL32 and LipL41 coding genes and the rrs2 gene were moderately variable. The phylogenetic tree clustered the isolates according to the genome-based species. CONCLUSION: The main advantages of MLST over other typing methods for leptospires include reproducibility, robustness, consistency and portability. The genetic relatedness of the leptospires can be better studied by the MLST approach and can be used for molecular epidemiological and evolutionary studies and population genetics.

Animals↗

PubMed1989

Monoclonal antibody against Leptospira interrogans serovar canicola.

Nine cell lines producing monoclonal antibodies (MAbs) against Leptospira interrogans serovar canicola strain Moulton were established by the cell fusion technique. The immunological reactivity of these MAbs with various kinds of serogroups, serovars and strains were examined by microscopic agglutination test (MAT) and enzyme-linked immunosorbent assay (ELISA). MAbs W1-W3 derived from mice, which were immunized with whole cells of the strain Moulton, reacted with the serogroups Canicola, Icterohaemorrhagiae and Pyrogenes. On the other hand, MAbs A1-A6 derived from mice immunized with the outer envelope (OE) fraction, which showed a potent protective activity and was extracted with ammonium hydroxide from the strain Moulton, reacted specifically with the serogroup Canicola alone in MAT. A difference in antigenic structure between subserogroup A (canicola subgroup) and subserogroups B (schüeffneri subgroup) of the serogroup Canicola was demonstrated by MAT using the MAbs. All the MAbs clearly agglutinated serovars of subserogroup A except for serovars kamituga, jonsis and bindjei, but did not react with any serovars of subserogroup B. These findings suggest that MAb highly specific to each serovar is readily available by OE immunization and is useful for the classification of Leptospira.

Animals↗

PubMedDate not supplied

A comparison of three DNA extractive procedures with Leptospira for polymerase chain reaction analysis.

Three DNA extraction methods were evaluated in this study: proteinase K followed by phenol-chloroform; a plant proteinase (E6870) followed by phenol-chloroform; and boiling of leptospires in 0.1 mM Tris, pH 7.0 for 10 min at 100 degrees C, with no phenol treatment. Every strain treated with proteinase K or E6870 afforded positive polymerase chain reaction (PCR) reaction. On the other hand, from five strains extracted by the boiling method, three did not feature the 849 bp band characteristic in Leptospira. We also evaluated by RAPD-PCR, DNAs from serovars isolated with proteinase K and proteinase 6870 with primers B11/B12. Each of the DNA samples provided PCR profiles in agreement with previous data. Moreover, the results with E6870 showed less background non-specific amplification, suggesting that removal of nucleases was more efficient with E6870. The limit for detection by PCR using Lep13/Lep14 was determined to be 10(2) leptospira, using the silver stain procedure.

Chloroform↗

PubMed2006

Molecular characterization of Leptospira sp. strains isolated from human subjects in São Paulo, Brazil using a polymerase chain reaction-based assay: a public health tool.

A polymerase chain reaction (PCR)-based assay which amplifies repetitive DNA elements present within bacterial genomes was used to characterize and differentiate Leptospira sp. Thirty-five strains from a reference culture collection and 18 clinical isolates which had been previously analyzed by cross agglutinin absorption test (CAAT) were evaluated by this technique. PCR results from analysis of the reference culture collection showed no bands corresponding to serogroups Australis, Autumnalis, Bataviae, Celledoni, Cynopteri, Djasiman, Panama, Pomona, Pyrogenes, and Tarassovi. However, the PCR method was able to clearly discriminate the serogroups Andamana, Ballum, Canicola, Grippotyphosa, Hebdomadis, Icterohaemorrhagiae, Javanica, Sejroe, Semaranga, and Shermani. Clinical isolates previously characterized by CAAT as serovar Copenhageni, serovar Castellonis, and as serovar Canicola were in agreement with PCR results. The clinical isolate previously characterized as serovar Pomona was not differentiated by PCR. Forty additional clinical isolates from patients with leptospirosis obtained in São Paulo, Brazil were also evaluated by this PCR method. Thirty-nine of these were determined to belong to serogroup Icterohaemorrhagiae (97.5%) and one to serogroup Sejroe (2.5%). These results demonstrate that the PCR method described in this study has utility for rapid typing of Leptospira sp. at the serogroup level and can be used in epidemiological survey.

Agglutination Tests↗

PubMed2006

[Occurrence of Leptospira spp. soropositive stray dogs in Itapema, Santa Catarina, Brazil].

This study aimed to verify Leptospira spp. serovar infections in stray dogs in Itapema, Santa Catarina, Brazil. Serum samples were collected from 590 stray dogs and tested against 25 Leptospira spp. serovars using the microscopic agglutination test. Prevalence of anti-leptospiral antibodies against one or more serovars was 10.5%. The most frequent serovar was pyrogenes, positive in 26 (18.0%) samples, followed by canicola with 20 (13.8%) and icterohaemorragiae and copenhageni with 18 (12.5%, with antibody titers from 1:100 to 1:3,200). Significant prevalence (10.4 to 11.1%) was also detected against serovars castellonis, butembo, and grippothyphosa.

Agglutination Tests↗

PubMed2000

Leptospira outer membrane protein activates NF-kappaB and downstream genes expressed in medullary thick ascending limb cells.

Tubulointerstitial nephritis is the main manifestation of acute renal damage caused by leptospirosis, but the mechanism remains unexplored. Patients infected with LEPTOSPIRA: shermani in Taiwan disclosed tubular dysfunction particularly in the medullary thick ascending limb of loop of Henle (mTAL), and the related renal damage seems to be underestimated. To elucidate the mechanism of tubular damage, outer membrane protein extract from LEPTOSPIRA: was administered to a model of cultured mTAL cells derived from normal mice. The addition of outer membrane protein extract from L. shermani to cultured mTAL cells induced a significant nuclear DNA binding of the NF-kappa B transcription factor by electrophoresis mobility shift assay. Forty-eight h after adding the outer membrane protein extract (0.2 microg/ml) to the cultured cells, the expression of inducible nitric oxide mRNA increased by 4.2-fold, monocyte chemoattractant protein-1 by 3-fold, and tumor necrosis factor-alpha by 2.4-fold when compared with untreated cells examined by reverse transcription competitive-PCR. Supernatant nitrite, monocyte chemoattractant protein-1, and tumor necrosis factor-alpha protein levels also increased by 1.8-, 7.1-, and 5-fold, respectively. An antiserum raised against L. shermani largely prevented these effects. Outer membrane protein extract from L. bratislava induced fewer effects than L. shermani, and the avirulent nonpathogenic L. biflexa serovar patoc did not induce significant effects in the mTAL cells. In conclusion, L. shermani infection may cause mTAL cell damage and inflammation through the NF-kappa B-associated pathway. Findings of this study may be important in understanding the pathogenesis of tubulointerstitial nephritis caused by these organisms.

Animals↗

PubMed2001

Use of a monovalent leptospiral vaccine to prevent renal colonization and urinary shedding in cattle exposed to Leptospira borgpetersenii serovar hardjo.

OBJECTIVE: To determine whether a monovalent Leptospira borgpetersenii serovar hardjo (type hardjobovis) vaccine commercially available in Australia, New Zealand, Ireland, and the United Kingdom would protect cattle from renal colonization and urinary shedding when exposed to a US strain of Leptospira borgpetersenii serovar hardjo. ANIMALS: 24 Hereford heifers that lacked detectable antibodies against serovar hardjo. PROCEDURE: Heifers received 2 doses, 4 weeks apart, of the commercial hardjo vaccine (n = 8) or a monovalent US reference hardjo vaccine (8) or were not vaccinated (controls; 8). Heifers were challenged 16 weeks later by intraperitoneal inoculation or conjunctival instillation. Serum antibody titers were measured weekly, and urine samples were examined for leptospires. Heifers were euthanatized 11 to 14 weeks after challenge, and kidney tissue was examined for evidence of colonization. RESULTS: All 8 heifers vaccinated with the reference vaccine were found to be shedding leptospires in their urine and had evidence of renal colonization. All 4 control heifers challenged by conjunctival instillation and 2 of 4 control heifers challenged by intraperitoneal inoculation shed leptospires in their urine, and all 8 had evidence of renal colonization. In contrast, leptospires were not detected in the urine or tissues of any of the 8 heifers that received the commercial hardjo vaccine. Heifers that received the commercial hardjo vaccine had significantly higher antibody titers than did heifers that received the reference vaccine. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that cattle that received 2 doses of the commercial hardjo vaccine were protected against renal colonization and urinary shedding when challenged with L borgpetersenii serovar hardjo strain 203 four months after vaccination.

Agglutination Tests↗

PubMed2001

Serum and vitreous humor antibody titers in and isolation of Leptospira interrogans from horses with recurrent uveitis.

OBJECTIVE: To measure antibody titers against Leptospira interrogans in serum and vitreous humor and determine the prevalence of L interrogans in vitreous humor of horses with recurrent uveitis. DESIGN: Cross-sectional study. ANIMALS: 242 horses (270 eyes) with recurrent uveitis undergoing vitrectomy and 39 control horses (54 eyes) without any history or clinical signs of recurrent uveitis undergoing euthanasia or enucleation for unrelated reasons. PROCEDURE: Serum and vitreous humor were tested for antibodies against 13 serovars of L interrogans. Vitreous humor was submitted for leptospiral culture; isolates were typed to the serogroup level. RESULTS: Leptospira interrogans was isolated from vitreous humor from 120/229 (52%) horses (126/252 [50%] eyes) with recurrent uveitis but was not isolated from vitreous humor from 36 eyes of 21 control horses. Duration of recurrent uveitis was > or = 1 year for 45 of the 120 (38%) horses from which the organism was isolated. Geometric mean antibody titers against L interrogans in the vitreous humor and serum of horses with recurrent uveitis were 1:1,332 and 1:186, respectively. Only 91 of 120 (76%) horses from which the organism was isolated had a 4-fold or greater difference between serum and vitreous humor antibody titers. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that persistent ocular infection with L interrogans is common in horses with recurrent uveitis. A 4-fold increase in vitreous humor versus serum antibody titers may not be a sensitive test for the diagnosis of L interrogans-induced recurrent uveitis. We hypothesize that the immune component of recurrent uveitis can be directly induced and maintained by persistent infection of the eye with L interrogans.

Animals↗

PubMed2003

Markedly prolonged jaundice from simultaneous infection with hepatitis E virus and leptospira.

We treated a patient simultaneously infected with hepatitis E virus and Leptospira interrogans, both acquired in China. Severe hyperbilirubinemia required nearly 200 days to resolve, transminase elevation showed a fluctuating course, and liver biopsy specimens showed fibrosis unusual for hepatitis E. Leptospirosis appeared to have altered the course of hepatitis E virus infection in this patient, even though infection with Leptospira was cleared with antibiotics by 50 days after the onset of the hepatitis symptoms.

Hepatitis E↗

PubMed2005

[Apoptosis and ultrastructural lesions in Vero and J774A.1 cells induced by Leptospira interrogans].

OBJECTIVE: To determine the ability of adhering and invading to host cells and the related pathologic changes of Leptospira spp. with different virulence. METHODS: A special Fontana silver staining method was developed to observe the ability of L.interrogans serogroup Icterohaemorrhagiae serovar lai strain 56601 and serogroup Pomona serovar pomona strain 56608, and L.biflexa serogroup Samaranga serovar patoc strain Patoc I to adhere Vero and J774A.1 cells. Ultrastructural lesions of the infected cells were examined by electron microscopy. By using flow cytometry with fluorescein labeling of FITC-Annexin V/PI,apoptosis and necrosis of the Vero and J774A.1 cells induced by the leptospiral strains before and after inactivation with ultraviolet treatment were detected, respectively. RESULTS: The adhering rates of L.interrogans strains 56601 and 56608 to the Vero cells were 24.2% and 22.9% (P>0.05), while to the J774A.1 cells were 49.0% and 46.9% (P>0.05), respectively. L.biflexa strain Patoc I did not adhere to these two host cells. After the two strains of L.interrogans invaded two different cell lines, the special phagosomes containing the Leptospira were formed and similar cell ultrastructural lesions, such as chromatilysis and chromatin condensation, vacuolar degeneration, mitochondrion swelling and mitochondrial crista disappearance, and endoplasmic reticulum swelling and paramembranous ribosome disappearance, were observed. The apoptosis rates in the Vero cells caused by the L.interrogans strains 56601 and 56608 before and after ultraviolet inactivation were 84.4%, 82.8% and 77.9%, 86.1%, respectively. The L.interrogans strain 56601 mainly induced the terminal apoptosis in Vero cells with the rates of 68.0% and 52.9% before or after inactivation, while the L.interrogans strain 56608 mainly induced the early apoptosis in Vero cells with apoptosis rates of 64.1% and 50.1% before or after inactivation. In the J774A.1 cells, the L.interrogans strain 56608 caused cell necrosis (before and after inactivation) and apoptosis that was dominated by terminal apoptosis. CONCLUSION: The established Fontana silver staining method can be used to observe adhesion of L.interrogans.L.interrogans can invade host cells through endocytosis which causes untrastructural lesions. The necrosis or apoptosis induced by L.interrogans are affected by different host cell lines but not the virulence of L.interrogans strains.

Animals↗

PubMed1999

Three case studies involving Leptospira interrogans serovar pomona infection in mixed farming units.

Three case studies involving Leptospira interrogans serovar pomona outbreaks within mixed farming systems in South Africa are described. On 2 farms, pigs constituted the main enterprise with cattle and sheep of secondary importance. On each of these 2 farms, abortion due to L. pomona in sows was confirmed by culture, and antibody titres to pomona were detected in cattle, sheep, horses and dogs. On the 3rd farm, a piggery was of secondary importance to cattle farming. Abortion and death in cows occurred on this farm and serology showed titres to various serovars, including pomona. L. pomona was also isolated from bovine urine, an aborted bovine foetus and kidneys from slaughtered pigs. This particular case study was regarded as clinically atypical in that adult Jersey cattle died of acute leptospirosis in a semiarid region of South Africa. In all 3 case studies, the poor management of pig effluent and of the drinking water and its sources played a pivotal role in the transmission of the disease. Inadequate vaccination of animals against Leptospira and poor record-keeping within the secondary farming enterprises were also contributing factors to the spread of leptospirosis.

Abortion, Veterinary↗