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DNA relatedness was determined among 303 strains of Leptospira and Leptonema. Included in the analysis were reference strains from 228 well-characterized and recognized serovars. The study included 268 serovars from 29 named and one or more unnamed serogroups. The strains clustered into 17 DNA hybridization groups, representing 12 previously described species (292 strains) and five new genomospecies (11 strains). The largest groups included Leptospira interrogans (91 strains from 82 serovars), Leptospira santarosai (65 strains from 59 serovars), Leptospira borgpetersenii (49 strains from 43 serovars), Leptospira kirschneri (29 strains from 26 serovars) and Leptospira noguchii (20 strains from 20 serovars). The new genomospecies include Leptospira genomospecies 1 (two strains, serovars pinagchang and sichuan), Leptospira genomospecies 2 (six strains, serovars lushui, manhao 3, manzhuang, nanding, mengla and yunnan), Leptospira genomospecies 3 (one strain, serovar holland), Leptospira genomospecies 4 (one strain, serovar hualin) and Leptospira genomospecies 5 (one strain, serovar saopaulo). With the exception of Ballum, all serogroups with greater than one serovar studied were genetically heterogeneous. Phenotypic tests, including optimal growth temperature, lipase activity and growth inhibition by copper sulfate or 2,6-diaminopurine, were of little use in differentiating DNA relatedness groups. The name Leptospira alexanderi sp. nov. is proposed for Leptospira genomospecies 2 (type strain L 60T = ATCC 700520T, serovar manhao 3).
A multiplex polymerase chain reaction (PCR) method using primer sets of G1/G2 and B64-I/B64-II was validated to detect pathogenic leptospira serovars from canine urine samples. The PCR method was found to be specific and sensitive with a detection limit of 100 cells of Leptospira icterohaemorrhagiae per milliliter of urine. The primer set previously designated and erroneously transcribed B64-I/B64-II amplified a DNA fragment of 352 base pairs from Leptospira grippotyphosa and Leptospira sejroe but not from Leptospira autumnalis, Leptospira bratislava, Leptospira canicola, Leptospira hardjo, Leptospira icterohaemorrhagiae, and Leptospira pomona. From 100 diagnostic canine urine samples, 5 were found positive for Leptospira grippotyphosalsejroe with a PCR product of 352 base pairs and 6 were positive for other pathogenic leptospira serovars with a PCR product of 285 base pairs. One 285-base pair product was sequenced and found to be 99.3% homologous to the G1/G2 PCR fragment sequence reported previously. All 352-base pair PCR products of clinical samples and pure cultures of L. grippotyphosa and L. sejroe were sequenced. The 352-base pair fragment sequences of L. grippotyphosa and L sejroe were identical. Only 2 base pairs were found different between the sequences from pure cultures and those from clinical samples. Serum samples from 3 positive cases that generated a PCR product of 352 base pairs were tested by the microscopic agglutination test, and 2 were found to be positive for L. grippotyphosa (1:10,240 and 1:5,120), 1 was positive for L. grippotyphosa (1:320) or L. icterohaemorrhagiae (1:320). The results of this study suggest that the multiplex PCR with the primer set G1/G2 and the erroneously transcribed B64-I/B64-II may be able to differentiate L. grippotyphosa or L. sejroe from other pathogenic leptospira serovars commonly tested for in Canadian diagnostic laboratories.
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OBJECTIVE: To determine the existence of genus-specific antigens in outer membrane proteins (OMPs) of leptospira with different virulence. METHODS: Microscope agglutination test (MAT) was applied to detect the agglutination between commercial rabbit antiserum against leptospiral genus-specific TR/Patoc I antigen and 17 strains of Leptospira interrongans belonging to 15 serogroups and 2 strains of Leptospira biflexa belonging to 2 serogroups. The outer envelopes (OEs) of L.interrogans serogroup Icterohaemorrhagiae serovar lai strain lai (56601) with strong virulence and serogroup Pomona serovar pomona strain Luo (56608) with low virulence, and L.biflexa serogroup Semaranga serovar patoc strain Patoc I without virulence were prepared by using the method reported in Auran et al.(1972). OMPs in the OEs were obtained by treatment with sodium deoxycholate. SDS-PAGE and western blot were used for analyzing the features of the OMPs on electrophoretic pattern and the immunoreactivity to the antiserum against TR/Patoc I antigen, respectively. RESULTS: All the tested strains belonging to different leptospiral serogroups agglutinated to the antiserum against leptospiral genus-specific TR/Patoc I antigen with agglutination titers ranging from 1:256-1:512. A similar SDS-PAGE pattern of the OMPs from the three strains of leptospira with different virulence was shown and the molecular weight of a major protein fragment in the OMPs was found to be approximately 60 KDa. A positive protein fragment with approximately 32 KDa confirmed by Western blot, was able to react with the antiserum against leptospiral genus-specific TR/Patoc I antigen, and was found in each the OMPs of the three stains of leptospira. CONCLUSION: There are genus-specific antigens on the surface of L.interrogans and L.biflexa. The OMP with molecular weight of 32 KDa may be one of the genus-specific protein antigens of leptospira.
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Arbitrarily primed PCR (AP-PCR) assays can be used to discriminate between species of Leptospira. Comparative analysis of the fingerprints obtained from representative sets of serovar reference strains of Leptospira interrogans sensu stricto, L. borgpetersenii, and L. kirschneri and the reference strains of the other Leptospira spp. revealed species-specific DNA fragments. These species-specific sequences were reamplified in order to produce digoxigenin-11-dUTP-labeled genomic DNA probes that could be used to identify Leptospira species. Three probes (specific for L. interrogans sensu stricto, L. borgpetersenii, and L. kirschneri) were selected and tested with 72 representative serovar reference strains, all of which had previously been studied by DNA-DNA hybridization. The two techniques were in general agreement, and hybridization with AP-PCR-derived probes was shown to be a useful approach for rapid species determination of leptospires, without the prior need for DNA sequence information. These nonradioactive probes can be used to identify Leptospira species in nonspecialized laboratories, and this should contribute to a better knowledge of the molecular epidemiology of leptospirosis.
Murine monoclonal antibodies were produced by immunizing BALB/c mice with a killed whole-cell antigen prepared from Leptospira borgpetersenii serovar hardjo type hardjobovis. Six of these antibodies recognized epitopes on the homologous antigen and on whole-cell antigen prepared from Leptospira interrogans serovar hardjo type hardjoprajitno. These antibodies did not cross-react with whole-cell antigens prepared from L. borgpetersenii serovar sejroe, 10 other pathogenic Leptospira serovars, or the saprophytic Leptospira biflexa serovar patoc. Three other monoclonal antibodies reacted with antigens prepared from the 2 hardjo serovars and serovar sejroe but not with antigens from the 10 other pathogenic serovars, or serovar patoc. The epitopes recognized by all of the hardjo-specific antibodies and 2 of the 3 hardjo/sejroe-specific antibodies were susceptible to sodium meta-periodate oxidation. All of the antibodies were characterized by Western blots with the hardjobovis whole-cell antigen. Each of the 9 monoclonal antibodies was inhibited from binding to the hardjobovis antigen by bovine sera which were obtained from cattle experimentally infected with hardjobovis and from field cattle, with anti-serovar hardjo microscopic agglutination test antibody titres ranging from 100 to 12800. Some of these antibodies may be suitable for incorporation into competitive enzyme immunoassays for the specific detection of antibodies to either of the hardjo serovars.
Stalheim, O. H. V. (University of Wisconsin, Madison), and J. B. Wilson. Cultivation of leptospirae. I. Nutrition of Leptospira canicola. J. Bacteriol. 88:48-54. 1964.-The nutrition of Leptospira canicola was investigated by use of synthetic media of suitable ionic strength. At an incubation temperature of 30 C, the minimal components were calcium, iron, magnesium, and ammonium ions, thiamine, and a fatty acid source; barium and strontium replaced calcium. Aspartic acid, glutamic acid, or methionine stimulated the rate and amount of growth; the best growth occurred in medium containing additional amino acids. Additions of cyanocobalamin or biotin permitted growth at 37 C. The stimulatory effects of added cyanocobalamin, biotin, pyridoxine, pantothenate, lipoic acid, or nicotinic acid were additive at 37 C, but not at 30 C. Fatty acids containing 14, 16, 17, or 18 carbon atoms supported growth; linoleic and linolenic acids were toxic. Glyceryl monooleate or trioleate, or Tween 40, 60, or 80 supported moderate to good growth; a mixture of monoolein and Tween 60, or Tweens 60 and 80 supported the best growth. Ten strains of L. canicola cultivated in a synthetic medium containing Tweens 60 and 80 attained cellular densities per ml of 10(7) to 4.0 x 10(7) organisms. L. canicola cells, resuspended in medium containing oleic-1-C(14) acid, incorporated label primarily into cellular lipids; a lesser amount was located in the protein fraction, and only trace amounts were found in the nucleic acid fraction. The rate of incorporation was not affected by added sodium acetate. L. canicola was found to have fatty acid decarboxylase activity.
A new repetitive DNA element was identified in an isolate of Leptospira interrogans serovar copenhageni from a patient in Salvador, Brazil. A Sau3A genomic library from this strain was constructed and screened for repetitive DNA elements. An insert of 438 bp (Rep1) from one library clone hybridized to multiple chromosomal DNA fragments resolved electrophoretically after digestion with BamHI, HindIII, and MfeI. A single oligonucleotide primer, designated iRepl, was designed to generate multiple PCR amplicons of various electrophoretic mobilities in a PCR typing method. The method distinguished strains belonging to the eight pathogenic and three saprophytic species of the genus Leptospira. Clinical isolates obtained during urban epidemics between 1996 and 1998 in Salvador, Brazil, were analyzed by this PCR method. Although the iRep1 primer was unable to discriminate strains among L. interrogans serovar copenhageni isolates, it was able to differentiate strains belonging to different species and serogroups of Leptospira identified in Salvador. This PCR-based method may provide a faster and less expensive alternative to serologic tests used in reference laboratories.
BACKGROUND: Leptospirosis is a zoonotic disease caused by the genus, Leptospira. Leptospira interrogans is the most common genomospecies implicated in the disease. Epidemiological investigations are needed to distinguish outbreak situations or to trace reservoirs of the organisms. Current methodologies used for typing Leptospira have significant drawbacks. The development of an easy to perform yet high resolution method is needed for this organism. METHODS: In this study we have searched the available genomic sequence of L. interrogans serovar Copenhageni strain Fiocruz L1-130 for the presence of tandem repeats. These repeats were evaluated against reference strains for diversity. Six loci were selected to create a Multiple Locus Variable Number of Tandem Repeats (VNTR) Analysis (MLVA) to explore the genetic diversity within L. interrogans serovar Australis clinical isolates from Far North Queensland. RESULTS: The 39 reference strains used for the development of the method displayed 39 distinct patterns. Diversity Indexes for the loci varied between 0.80 and 0.93 and the number of repeat units at each locus varied between less than one to 52 repeats. When the MLVA was applied to serovar Australis isolates three large clusters were distinguishable, each comprising various hosts including Rattus species, human and canines. CONCLUSION: The MLVA described in this report, was easy to perform, analyse and was reproducible. The loci selected had high diversity allowing discrimination between serovars and also between strains within a serovar. This method provides a starting point on which improvements to the method and comparisons to other techniques can be made.
One of the genes encoding the 5S ribosomal ribonucleic acid (rRNA) for the Leptospira biflexa strain Patoc I was isolated and sequenced. The physical maps of the 5S rRNA genes in Leptospira were constructed. The strains of Leptospira biflexa had two genes on their chromosome; these two 5S rRNA genes were located several kb apart and sequences flanking these genes were divergent. In contrast to saprophytic leptospires, maps in parasitic leptospires that had only one gene for 5S rRNA on their genome were highly conserved and the physical maps of the genes in almost all strains were similar.
Two recombinant DNA probes-PLIpso1 (15kb) and PLIEc34 (4kb), derived from Leptospira serovaricterohaemorrhagiae genomic libraries, were applied for the hybridization and identification of 13 strains of Leptospira in serogroup icterohaemorrhagiae. Difference in hybridization signal in combination with the banding pattern provide a good way for identification of serovars and strains. The recombinant DNA, specific to L. serogroup icterohaemorrhagiae, hybridized with a limited number of DNA fragments which had been digested by several restriction endonucleases. The less complex banding pattern and higher sensitivity facilitate characterization of various serovars and strains in serogroup icterohaemorrhagiae. In general the DNA patterns recognized by both probes have extensive genomic homology in same serovar (but still can distinguish strains in same serovar by some unique bands) and apparent difference in various serovars (especially serovars naam, nanxi and honghe). The results indicated that Southern blotting with recombinant DNA probe might provide tools for identification, characterization and analysis of leptospira.
The affinity constants of anti-canicola and anti-hebdomadis monoclonal antibodies and the numbers of antigenic determinants per organism of each serovar of leptospira were determined, and their influence on the results of microscopic agglutination test (MAT) and ELISA were examined. In the combinations of a monoclonal antibody and some serovar which showed higher affinity constants at levels of 10(7) to 10(8)/M order, both MAT and ELISA were positive except for one combination, while in the combinations which gave lower affinity constants at levels of 10(6)/M order, MAT was positive but ELISA was negative. The number of antigenic determinants seemed to have no significant influence on the results. The disagreement due to the difference in the threshold affinities of MAT and ELISA should be considered in the use of monoclonal antibodies in the taxonomy of leptospira.
The gene organization in the lipopolysaccharide biosynthetic (rfb) locus was analyzed in seven Leptospira interrogans serovars within serogroup Icterohemorrhagiae, seven non-Icterohemorrhagiae serovars and one Leptospira borgpetersenii serovar. Two groups of loci were delineated based on DNA hybridization and sequence analysis. Group 1 contained the two Hardjo subtypes, Hardjoprajitno and Hardjobovis. Group 2 (containing Copenhageni, Pomona, Naam, Mwogolo, Smithi, Lai, Canicola, Autumnalis, Pyrogenes, Australis and Icterohemorrhagiae) differed from Group 1 in its organization upstream of orf11, where five ORFs (32, 33, 34, 35, 37) were identified that were not contained in the Group 1 loci. These ORFs encoded a putative epimerase (orf32), a glycosyltransferase (orf33), two integral membrane proteins (orfs 34 and 35), and a galactosyltransferase (orf37). Serovars Australis, Pomona and Autumnalis did not contain orf37. Serovar Bataviae was excluded from the grouping because of its unique genetic organization upstream of orf13. In the Group 2 loci, comparison of the genetic layout at the 5' end revealed differences which included mutations disrupting reading frames in either or both orf34 and orf35 and apparent allelic differences between orf33 homologs that may be sufficient to account for the genetic basis of serovar identity.
Bacterial specific primers were used to amplify 23S rRNA genes from a representative strain from each of the 23 serogroups of the pathogenic Leptospira interrogans and 8 strains from 6 serogroups of the non-pathogenic Leptospira biflexa. Only regions of extreme variability, which had been identified on the basis of homology-based search of all the 23S rRNA sequences available in GenBank database, were sequenced from the amplified products. PCR primers that had the potential to distinguish L. interrogans from L. biflexa species were designed from the derived sequences and a sensitive PCR protocol developed. The PCR method enabled the differentiation of the 59 strains of the 23 serogroups of L. interrogans from the 8 strains of 6 serogroups of L. biflexa. Further investigation by 16S rDNA sequencing of two strains of L. interrogans, which gave unexpected PCR results, provided evidence that they had been misclassified and hence we propose to reassign them to L. biflexa.