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PubMed1983

Effects of intrauterine challenge with Leptospira interrogans serovar hardjo on fertility in cattle.

The purpose of this study was to determine the effects of Leptospira interrogans serovar hardio on fertility in cattle. Twenty seronegative mature dairy cows were assigned to two groups. Group I (challenged cows) was bred by a seronegative bull followed by intrauterine infusion (within 30 minutes) of Leptospira interrogans serovar hardjo. Group II was bred by the same bull followed by intrauterine infusion of 5 ml of sterile culture medium. Blood samples were collected at two-day intervals to monitor serum antibody titers. Daily blood cultures for 10 days and weekly urine cultures for five weeks were performed to monitor the animals for leptospiremia and leptospiuria. Cows were slaughtered 35 days post-breeding, and their reproductive tracts were examined. All animals remained clinically normal following intrauterine challenge. There was no difference in pregnancy rates (Group I, 7/10; Group II, 6/10). All embryos, reproductive tracts, and kidneys appeared normal. A microscopic agglutination test (MA) showed that 4 of 10 challenged cows developed serum antibody titers between 8 and 20 days after challenge. However, on the basis of the hamster passive protection test, all challenged cows had serum antibodies present. All blood and urine cultures were negative through the experimental period, as were the final kidney and uterine cultures. In a second experiment, six seronegative cows were infused with killed microorganisms immediately after insemination. Results of a microscopic agglutination test and a hamster passive protection test indicated that these cows did not develop humoral antibodies against serovar hardjo. These results indicated that intrauterine inoculation of Leptospira interrogans serovar hardjo (hamster-adapted strain) following breeding did not affect pregnancy rates despite an intrauterine challenge which caused the development of humoral antibodies.

Journal Article↗
PubMed2000

Rodents and Leptospira transmission risk in Terceira island (Azores).

The role of rodents as Leptospira renal carriers in Terceira island was evaluated (1993-1995) through kidney culture and serology [microscopic aglutination test (MAT)] of 94 mice and rats. Fifty-nine animals were positive (n = 41 by serology + culturing; n = 11 serology; n = 7 culturing), presenting a wide distribution in man-made and natural areas. House mice had the highest bacteriological (82.9%) and serological (90.9%) rates, being strictly related to serovar arborea. Black rats were involved in the dispersion of all isolated L. interrogans sensu lato serovars (arborea, copenhageni and icterohaemorrhagiae). Logistic regression analysis and non-metric multi-dimensional scaling, relating Leptospira infection with biological and environmental variables, expressed that adult males Mus domesticus, sexually active and living in humid biotopes, mainly above 500 m, are the most likely reservoirs. This study emphasizes the role of house-mice in the epidemiology of leptospirosis in Terceira and the need of reducing the risk of Leptospira transmission through integrated control programmes, primarily focusing on adult house-mice in peri-domestic environments, before the breeding season.

Animals↗
PubMed1970

Enhancement of glutamic-pyruvic transaminase activity in leptospirae by addition of rabbit serum.

Glutamic-pyruvic transaminase (GPT) in leptospirae was shown to have an optimal temperature of 30 C and pH 8.0. It was activated by cofactor pyridoxal phosphate, similar to GPT from other sources. However, GPT in all leptospirae, such as Leptospira canicola, L. icterohaemorrhagiae, L. autumnalis, and L. biflexa, evidently had less activity even when pyridoxal phosphate was present, in comparison with the cultivable spirochete Reiter treponeme and Streptococcus faecalis. The low level of activity of leptospiral GPT was enhanced more than 2.5-fold by the addition of about 10% rabbit serum, whereas GPT of S. faecalis did not show such enhancement. Enhancement of leptospiral GPT was greater with rabbit serum than with bovine or horse serum.

Journal Article↗
PubMed1990

Sizing of the Leptospira genome by pulsed-field agarose gel electrophoresis.

Pulsed-field gel electrophoresis allowed the determination of the size of the genome of Leptospira, a bacterium of the spirochete family. The three restriction enzymes, NotI (5'GC/GGCCGC), NheI (5'G/CTAGC), ApaI (5'-GGGCC/C) generated DNA fragments of suitable size. The results are compatible with a size of 5000 kb for the chromosome of both the pathogenic and the saprophytic species of Leptospira.

Chromosomes, Bacterial↗
PubMed1992

Immunoblotting study of the antigenic relationships among eight serogroups of Leptospira.

Seven strains of Leptospira interrogans belonging to seven different serogroups, and one strain of Leptospira biflexa were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with gradient gels and immunoblotting with hyperimmune rabbit sera raised against each strain. The molecular masses of the proteins were calculated with a polynomial regression model. The SDS-PAGE patterns of the L. interrogans strains were similar and characterized by 24 common bands. This profile was not found for L. biflexa. The immunoblots obtained either with the seven anti-L. interrogans sera or the anti-L. biflexa serum allowed a clear distinction between the two species. Taken as a whole, the L. interrogans strain patterns revealed by the seven anti-L. interrogans sera were similar, sharing eight common major bands. A serovar- or serogroup-specific antigenic zone, ranging from 21 to 26 kDa, was also identified.

Animals↗
PubMed1990

rRNA gene restriction patterns of Leptospira: a molecular typing system.

A total of 67 serovar reference strains and 7 isolates belonging to the genus Leptospira were characterized by ribosomal ribonucleic acid (rRNA) gene restriction patterns. Fifty patterns were observed. Strains belonging to different genomic species always gave different patterns. However, genomic species were subdivided into several patterns. Forty-three serovars gave a specific pattern. Some serovars could not be separated by rRNA gene restriction patterns: strains of serovars icterohaemorrhagiae, copenhageni, lai, pyrogenes and jalna gave pattern 1; serovars birkini, mankarso and wolffi gave pattern 4; serovars canicola, gem, hebdomadis, pomona and hardjo (strain hardjoprajitno) gave pattern 12; serovars valbuzzi and zanoni gave pattern 14; serovars jonsis, malaya and sumneri gave pattern 16; serovars arborea, ballum, castellonis and kenya gave pattern 35; and serovars borincana and shermani gave pattern 43. These data provide the bases for a molecular typing system for the genus Leptospira.

Bacterial Typing Techniques↗
PubMed2003

Genetic evidence for the existence of two pathways for the biosynthesis of methionine in the Leptospira spp.

There are two major pathways for methionine biosynthesis: the enterobacterial type transsulfuration pathway and the sulfhydrylation pathway as previously identified in the spirochete Leptospira meyeri. Sequence analysis of the L. meyeri metYX locus allows the identification of a third gene, called metW, which encodes a protein exhibiting similarities with homologs in many organisms belonging to the alpha-, beta-, and gamma-subdivisions of proteobacteria. The metW, metX and metY genes of L. meyeri were disrupted by a resistance cassette by homologous recombination. While the L. meyeri metX mutant shows methionine auxotrophy, the metY mutant (as well as the metW and metYmetW mutants) conserves methionine prototrophy, suggesting the presence of additional route(s) which may bypass the direct sulfhydrylation pathway. In addition, a L. interrogans gene, called metZ, was found to complement an Escherichia coli metB mutant, further suggesting that the transsulfuration pathway is also present in Leptospira spp.

Acetyltransferases↗
PubMed2000

Detection and differentiation of Leptospira spp. serovars in bovine semen by polymerase chain reaction and restriction fragment length polymorphism.

In view of the importance of venereal transmission of bovine leptospirosis, the objective of the present study was to apply the polymerase chain reaction (PCR) to 26 serovars of Leptospira interrogans, L. borgpetersenii, L. santarosai, L. noguchii and L. biflexa, to determine the detection threshold in semen samples and to evaluate the possibility of differentiation among serovars using 19 restriction endonucleases. The results showed that all serovars were amplified and the detection threshold in semen samples of a bull was 100 bacteria/ml. Using endonucleases we could classify the 26 serovars into eight groups. The present results show that PCR is a method of great potential for the detection of Leptospira spp. at bovine artificial insemination centers.

Animals↗
PubMed1987

Serological studies on British isolates of the Sejroe serogroup of leptospira. II. An evaluation of the factor analysis method of identifying leptospires using strains belonging to the Sejroe serogroup.

Twelve British isolates of leptospira belonging to the Sejroe serogroup were examined using a series of six factor sera prepared by a number of different absorption methods. Ten of the isolates were identified as Leptospira interrogans serovar hardjo and two as L. interrogans serovar saxkoebing. These isolates had previously been identified using the cross agglutination absorption method.

Agglutination Tests↗
PubMed2006

Genome reduction in Leptospira borgpetersenii reflects limited transmission potential.

Leptospirosis is one of the most common zoonotic diseases in the world, resulting in high morbidity and mortality in humans and affecting global livestock production. Most infections are caused by either Leptospira borgpetersenii or Leptospira interrogans, bacteria that vary in their distribution in nature and rely on different modes of transmission. We report the complete genomic sequences of two strains of L. borgpetersenii serovar Hardjo that have distinct phenotypes and virulence. These two strains have nearly identical genetic content, with subtle frameshift and point mutations being a common form of genetic variation. Starkly limited regions of synteny are shared between the large chromosomes of L. borgpetersenii and L. interrogans, probably the result of frequent recombination events between insertion sequences. The L. borgpetersenii genome is approximately 700 kb smaller and has a lower coding density than L. interrogans, indicating it is decaying through a process of insertion sequence-mediated genome reduction. Loss of gene function is not random but is centered on impairment of environmental sensing and metabolite transport and utilization. These features distinguish L. borgpetersenii from L. interrogans, a species with minimal genetic decay and that survives extended passage in aquatic environments encountering a mammalian host. We conclude that L. borgpetersenii is evolving toward dependence on a strict host-to-host transmission cycle.

Animals↗
PubMed2004

Identification and analysis of genes present in Leptospira interrogans serovar lai but absent in L. biflexa serovar monvalerio.

Genes present in virulent bacterial strains but absent in avirulent close relatives can be of great biologic and clinical interest. This project aimed to identify strain specific DNA sequences of Leptospira interrogans serovar lai, which is absent in the saprophytic L. biflexa serovar monvalerio, via suppression subtractive hybridization with the former as the tester while the latter as the driver. The mixture of PCR amplified DNA fragments from two subtractive hybridization experiments were cloned into pMD18-T vector and the positive clones were identified by dot blotting against the chromosome DNA of the two strains individually. After DNA sequencing and analysis, the distribution of these genomic fragment sequences in a panel of pathogenic and nonpathogenic leptospires was investigated employing dot blot analysis. Among the 188 positive clones randomly chosen, 24 contained the tester strain specific genomic regions, of which, 5 were non-coding fragments while the others contained 23 distinct protein coding sequences. Besides 9 genes encoding functional proteins, 12 genes encode unknown proteins and the rest two genes encode proteins with recognizable domain structures, one for a putative leucine-rich repeats (LRR) family protein while the other as an outer-membrane protein. Our experiment results indicated that suppression subtractive hybridization is effective for screening specific DNA sequences between two leptospiral strains, and some of these sequences might be responsible for virulence determination. Further analysis of these DNA sequences will provide important information on the pathogenesis of Leptospira.

DNA, Bacterial↗
PubMed1963

NUTRITION OF LEPTOSPIRA POMONA. II. FATTY ACID REQUIREMENTS.

Johnson, R. C. (Fort Detrick, Frederick, Md.) and N. D. Gary. Nutrition of Leptospira pomona. II. Fatty acid requirements. J. Bacteriol. 85:976-982. 1963.-The albumin fraction of rabbit serum, obtained by (NH(4))(2)SO(4) fractionation, supported good growth of Leptospira pomona Wickard when added to a medium containing phosphate buffer, NH(4)Cl, and thiamine. Extraction of the albumin fraction with ethanol and ether (3:1) resulted in a loss of its growth-supporting activity, which could be restored upon the addition of the extract or various fatty acids. The growth-supporting activity of fatty acids containing 2 to 18 carbon atoms was investigated with the extracted albumin medium. The activity of the fatty acid was found to be related to the number of carbon atoms in the molecule. Maximal growth was obtained with palmitic, heptadecanoic, stearic, and oleic acids. The amount of growth of L. pomona increased at a fixed concentration of albumin as the molecular ratio of extracted albumin to the long-chain fatty acids increased from 1:1 to 1:6. At higher ratios, growth decreased sharply. Direct utilization of fatty acids during growth of L. pomona was shown by the incorporation of palmitic acid-1-C(14) or -2-C(14) into cellular material. A medium composed of 0.02 m Na(2)HPO(4)-KH(2)PO(4) buffer (pH 7.4), 0.8% extracted albumin, 4 x 10(-4)m palmitic acid, 10(-3)m NH(4)Cl, and 5 mug/ml of thiamine supported good growth. L. pomona was transferred ten times, and seven other serotypes were transferred five times, in this medium without any decrease in the amount of growth. Growth of L. pomona was initiated with approximately four organisms per ml.

Ammonium Chloride↗
PubMed1963

NUTRITION OF LEPTOSPIRA POMONA. III. CALCIUM, MAGNESIUM, AND POTASSIUM REQUIREMENTS.

Johnson, R. C. (Fort Detrick, Frederick, Md.) and N. D. Gary. Nutrition of Leptospira pomona. III. Calcium, magnesium, and potassium requirements. J. Bacteriol. 85:983-985. 1963.-The cation requirements for Leptospira pomona Wickard were investigated employing a basal medium prepared with 10% deionized rabbit serum and containing 10(-3)m NH(4)Cl, 5 mug/ml thiamine, and 0.01 m Na(2)HPO(4) buffer (pH 7.4). Rabbit serum was deionized by passage through a column of Amberlite IRC-50 resin. This basal medium did not support growth when either Ca(++) or Mg(++) was added to it. However, when both Ca(++) (3 x 10(-5)m) and Mg(++) (5 x 10(-6)m) were present, growth was obtained. Although an absolute requirement for K(+) could not be demonstrated, an increase in the amount of growth did occur if K(+) (5 x 10(-5)m) was incorporated in addition to Ca(++) and Mg(++). When adequate concentrations of Ca(++) and K(+) were present with a limiting concentration of Mg(++) (less than 5 x 10(-7)m), filamentous cells were observed.

Animals↗
PubMed1965

ELECTRON MICROSCOPY OF LEPTOSPIRES. I. ANATOMICAL FEATURES OF LEPTOSPIRA POMONA.

Ritchie, A. E. (National Animal Disease Laboratory, Ames, Iowa), and Herman C. Ellinghausen. Electron microscopy of leptospires. I. Anatomical features of Leptospira pomona. J. Bacteriol. 89:223-233. 1965.-Anatomical features of Leptospira pomona are described. Most cells in the logarithmic phase of growth, when examined in whole cell "negative-stained" preparations, display a homogeneous finely granular protoplasmic cylinder. Some cells contain discrete or complex lamellar structures. The cell termini are attached to the protoplasmic cylinder by a membrane system. Each organism appears to have two independent axial filaments originating from opposite ends of the cell. The axial filaments are composed of a closely knit bundle of some 12 to 15 individual strands. A simple model suggesting a locomotory function for the axial filaments is presented. A superficial callouslike mass is occasionally observed in the cell's terminal region. The enveloping sheath is similar to the cytoplasmic membrane; both are triple-layered and ca. 50 A thick. The sheath preferentially forms blebs at the cell termini and midway along the protoplasmic cylinder. Septal-wall formation occurs at the mid region of the cell, and it is concluded that simple transverse binary fission is the predominant mode of reproduction.

Animals↗
PubMed1986

In vitro evaluation of combined usage of fosfomycin and 5-fluorouracil for selective isolation of Leptospira species.

The combined usage of fosfomycin (FOM) and 5-fluorouracil (5-FU) as selective agents for the isolation of leptospires from contaminated materials was investigated. Additive or synergistic antibacterial activity was apparent with the combination compared with each agent used separately. Of 54 bacterial strains tested, 52 were inhibited, while all 5 Leptospira strains tested were unaffected by the combined addition of FOM (400 micrograms/ml) and 5-FU (100 micrograms/ml) to Korthof medium. Furthermore, this combination successfully supported the selective growth of Leptospira interrogans serovar copenhageni in experimentally contaminated specimens. This FOM-5-FU combination is surmised to be useful for the selective isolation of leptospires from contaminated clinical, pathological, or environmental materials.

Bacteriological Techniques↗
PubMed2002

Serovar distribution of a DNA sequence involved in the antigenic relationship between Leptospira and equine cornea.

BACKGROUND: Horses infected with Leptospira present several clinical disorders, one of them being recurrent uveitis. A common endpoint of equine recurrent uveitis is blindness. Serovar pomona has often been incriminated, although others have also been reported. An antigenic relationship between this bacterium and equine cornea has been described in previous studies. A leptospiral DNA fragment that encodes cross-reacting epitopes was previously cloned and expressed in Escherichia coli. RESULTS: A region of that DNA fragment was subcloned and sequenced. Samples of leptospiral DNA from several sources were analysed by PCR with two primer pairs designed to amplify that region. Reference strains from serovars canicola, icterohaemorrhagiae, pomona, pyrogenes, wolffi, bataviae, sentot, hebdomadis and hardjo rendered products of the expected sizes with both pairs of primers. The specific DNA region was also amplified from isolates from Argentina belonging to serogroups Canicola and Pomona. Both L. biflexa serovar patoc and L. borgpetersenii serovar tarassovi rendered a negative result. CONCLUSIONS: The DNA sequence related to the antigen mimicry with equine cornea was not exclusively found in serovar pomona as it was also detected in several strains of Leptospira belonging to different serovars. The results obtained with L. biflexa serovar patoc strain Patoc I and L. borgpetersenii serovar tarassovi strain Perepelicin suggest that this sequence is not present in these strains, which belong to different genomospecies than those which gave positive results. This is an interesting finding since L. biflexa comprises nonpathogenic strains and serovar tarassovi has not been associated clinically with equine uveitis.

Animals↗
PubMed2003

Prevalence of Leptospira and Brucella antibodies in wild boars (Sus scrofa) in Tuscany, Italy.

Five hundred sixty-two blood samples were collected from wild boars (Sus scrofa) shot in six districts of Tuscany, central Italy, between 1997 and 2000. Sera were examined for antibodies specific for Leptospira interrogans by microagglutination test and Brucella spp. by the Rose Bengal test and indirect enzyme-linked immunosorbent assay. Thirty-four (6.0%) samples tested positive for anti-Leptospira antibodies, 29 (5.1%) sera were positive for anti-L. interrogans serovar bratislava antibodies (titres ranging from 1:100-1:400), and 5 (0.9%) sera were positive for anti-L. interrogans serovar icterohaemorrhagiae antibodies (titres 1:100). All the examined sera were negative for anti-Brucella antibodies.

Animals↗
PubMedDate not supplied

[Gene diagnostics of acute persistent Leptospira infection].

The results of clinical trials carried out in different foci have demonstrated high diagnostic value of analysis made with the use of the polymerase chain reaction (PCR) at early stages of Leptospira infection caused by infective agents of the serogroups Grippotyphosa, Canicola and Icterohaemorrhagiae. The possibility of leptospiremia lasting considerably longer than heretofore believed to be possible, as well as the persistence of leptospires in the liquor of patients after the acute phase of the disease is over, i.e. during the early and late convalescence periods, has been shown. This is indicative of good prospects of using the PCR analysis not only for early rapid diagnostics of Leptospira infections, but also for controlling the course of the infection, for prognosticating early and late complications of the disease, as well the mechanisms of pathogenesis.

Acute Disease↗