Identification of pathogenic Leptospira species by conventional or real-time PCR and sequencing of the DNA gyrase subunit B encoding gene

BACKGROUND: Leptospira is the causative genus of the disease, leptospirosis. Species identification of pathogenic Leptospira in the past was generally performed by either DNA-DNA hybridisation or 16s rRNA gene sequencing. Both methods have inherent disadvantages such as the need for radio-labelled isotopes or significant homology between species. A conventional and real-time PCR amplification and sequencing method was developed for an alternate gene target: DNA gyrase subunit B (gyrB). Phylogenetic comparisons were undertaken between pathogenic Leptospira 16srRNA and gyrB genes using clustering and minimum evolution analysis. In addition 50 unidentified Leptospira isolates were characterised by gyrB sequencing and compared with conventional 16s rRNA sequencing. RESULTS: A conventional and real-time PCR methodology was developed and optimised for the amplification of the gyrB from pathogenic Leptospira species. Non pathogenic and opportunistic Leptospira species such as L. fainei and L. broomi were not amplified. The gyrB gene shows greater nucleotide divergence (3.5% to 16.1%) than the 16s rRNA gene (0.1% to 1.4%). Minimum evolution analysis reveals that the gyrB has a different evolution topology for L. kirschneri and L. interrogans. When the two genes were compared for the identification of the 50 unknown isolates there was 100% agreement in the results. CONCLUSION: This research has successfully developed a methodology for the identification of pathogenic Leptospira using an alternate gene to 16s rRNA. The gyrB encoding gene shows higher nucleotide/evolutionary divergence allowing for superior identification and also the potential for the development of DNA probe based identification

Development of a Multiple-Locus Variable number of tandem repeat Analysis (MLVA) for Leptospira interrogans and its application to Leptospira interrogans serovar Australis isolates from Far North Queensland, Australia

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 [1]. 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

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

An outbreak of leptospirosis in North Queensland, Australia January to May, 1999

The World Health Organization/Food and Agriculture Organization Collaborating Center for Reference and Research on Leptospirosis, Western Pacific Region, is part of Queensland Health Scientific Services, Queensland Health. In 1999 there was a marked increase in human leptospirosis notifications in North Queensland. Australia, over the period of January to May. There were 216 notifications for the state of Queensland in 1999, compared with 108 in 1998 and 64 in 1997. By April 1999, the Reference Laboratory and Queensland Health's Tropical Public Health Unit (TPHU), Cairns, had reported an outbreak of leptospirosis in North Queensland. Notifications reported for the North Queensland Health districts of Cairns, Tablelands and Innisfail were the highest, with the majority involving workers in the banana industry. The outbreak resulted from infections with a number of serovars, mostly attributed to serovars Australis and Zanoni. Sixty leptospiral isolates were obtained from human cases in North Queensland during the outbreak. The hospitalization rate was in excess of 50%. The Queensland Health's TPHU has implemented a program to educate target industries and the community on the risks and prevention strategies associated with leptospirosis

Rapid distinction between Leptospira interrogans and Leptospira biflexa by PCR amplification of 23S ribosomal DNA

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

Comparison of two PCR methods for rapid identification of Leptospira genospecies interrogans

This article is free to read on the publishers website Based on (i) an analysis of Leptospira 16S rDNA sequences determined by us and of those from databases and (ii) a previously published finding that restriction fragment length polymorphisms (RFLPs) within the Leptospira 16S and 23S rDNA were detected by nine restriction enzymes and these RFLPs allowed categorisation of Leptospira into eight genospecies, we predicted that one particular DdeI restriction site polymorphism within 16S rDNA could be independently used for identifications of Leptospira strains belonging to the genospecies interrogans. Two PCR-based methods, namely allele-specific amplification (ASA) and PCR-RFLP, were tested for the rapid detection of the DdeI restriction site polymorphism. One or two representative strains from each of nine genospecies were tested by ASA, whereas 73 strains from nine genospecies and two field isolates were tested by PCR-RFLP. Our experiments showed that the ASA method was not as specific as intended, but the PCR-RFLP method was useful for rapid identifications of the genospecies interrogans. We have not only confirmed a previous finding and extended the number of samples particularly from the genospecies biflexa, weilii, and inadai, but also simplified a previous PCR-RFLP protocol

Identification of pathogenic Leptospira by TaqMan probe in a LightCycler

The genus Leptospira is divided into two species of which Leptospira interrogans is an animal pathogen which causes infections in humans when in contact with body fluids of infected animals, whereas Leptospira biflexa is a nonpathogenic saprophyte. Leptospires are identified on the basis of serology and DNA–DNA hybridization techniques but alternative molecular typing methods based mainly on PCR have also been developed with some degree of success (1–16). Recently, non-gel electrophoresis-requiring, fluorophore probe-based rapid techniques for detecting and discriminating specific amplicons during PCR have been introduced (17–19). In the TaqMan probe, both donor and acceptor fluorophores are present on the same oligonucleotide that binds to a target site internal to the amplification primers..

Leptospirosis serology in the Common Brushtail Possum (Trichosurus vulpecula) from urban Sydney, Australia

The common brushtail possum (Trichosurus vulpecula) is indeed a common marsupial in major cities of Australia. This species is known to be susceptible to leptospirosis and often lives in close contact with humans, raising concerns about the potential for transmission of this disease in urban areas. A total of 192 brushtail possum blood samples were collected from 136 individuals in suburban areas of metropolitan Sydney from November 2002 to November 2004. Sera were screened against a reference panel of 21 Leptospira spp. using the microscopic agglutination test. Leptospiral antibodies were detected in 9.6% (13/136) of tested brushtail possums and represented two serovars; antibodies to Leptospira interrogans serovar Hardjo were most frequently identified (11/136). A representative of the exotic serogroup Ballum, most likely serovar Arborea, was found in two of 136 brushtail possums. Exposure to leptospirosis seemed to be associated with age, as older animals had a higher incidence, but there was no distinction in relation to gender. Antibody prevalence varied between the different sampling sites and seropositive animals were clustered and restricted to a few sites. These data support the possible role of brushtail possums as a maintenance host for Leptospira spp. in urban environments and also identified them as a previously unknown and potential source of serovar Arborea

Rapid distinction between Leptonema and Leptospira by PCR amplification of 16S-23S ribosomal DNA spacer

This article is free to read on the publishers website The PCR amplification of the genomic DNA of Leptonema illini strain 3055 using primers directed against conserved regions of the rRNA operon provided evidence that the 16S and 23S rRNA genes were linked via an intergenic spacer region. The sequencing of the intergenic spacer region indicated that it was 435 nucleotides in length and sequence similarity searches revealed that it bore no homology to any known sequences including tRNA available in databases. Further investigations using Southern blot hybridization revealed that there were two copies of these linked genes in the genome. However, similar PCR studies on a representative strain from each of the 23 serogroups of Leptospira interrogans, which are pathogenic, and eight strains from the 6 serogroups of Leptospira biflexa, which are non-pathogenic, revealed that the 16S and 23S rRNA genes were not linked