Leptospira weilii serovar Topaz, a new member of the Tarassovi serogroup isolated from a bovine source in Queensland, Australia.

This paper reports on a Leptospira isolate of bovine origin and its identification as belonging to a previously unknown serovar, for which the name Topaz is proposed. The isolate (94-79970/3) was cultured from bovine urine from a north Queensland dairy farm in Australia. Strain 94-79970/3 grew at 30 degrees C in Ellinghausen McCullough Johnson Harris (EMJH) medium but failed to grow at 13 degrees C in EMJH medium or in the presence of 8-azaguanine. Serologically, strain 94-79970/3 produced titres against the Leptospira borgpetersenii serovar Tarassovi, the reference strain for the Tarassovi serogroup; however, no significant titres to any other serovars within the serogroup were obtained. Using 16S rRNA and DNA gyrase subunit B gene analysis, strain 94-79970/3 was identified as a member of the species Leptospira weilii. We propose that the serovar be named Topaz, after the location where the original isolate was obtained. The reference strain for this serovar is 94-79970/3 (=KIT 94-79970/3=LT722).

The epidemiology of leptospirosis and the emergence of Leptospira borgpetersenii serovar Arborea in Queensland, Australia, 1998-2004.

Leptospirosis is one of the most commonly encountered zoonoses in both Australia and the rest of the world. The incidence of leptospirosis in Queensland over the 7-year study period (1998-2004) was 3.1/100000 population. Enhanced surveillance questionnaires were used to collect patient data and facilitate an epidemiological investigation of leptospirosis in Queensland. Farming occupations comprised the majority of occupational exposure cases, however, recreational exposure accounted for 18% of the 883 cases. Rainfall and the presence of animal hosts had the most influence on the incidence of leptospirosis. Several trends in serovar numbers over this period are noted, in particular the emergence of L. borgpetersenii serovar Arborea, which accounted for 22% of all leptospirosis cases in Australia and 68% of South-East Queensland cases in 2004. Assessment of epidemiological trends in leptospirosis is important to obtain directed public health intervention and outcomes in the reduction of leptospirosis cases.

Leptospiral antibodies in flying foxes in Australia.

The sera of 271 pteropid bats (or flying foxes) collected from Queensland, New South Wales, Western Australia, and the Northern Territory were screened against a reference panel of 21 Leptospira spp. using the microscopic agglutination test (MAT). Sera were collected from December 1997 through August 1999. The MAT panel represented those serovars previously isolated in Australia, as well as exotic serovars found in neighboring countries. Leptospiral antibodies were detected in 75 (28%) of the sera and represented seven serovars, one of which, L. interrogans serovar cynopteri has been regarded as exotic to Australia. Sixty sera were reactive to one serovar, 12 sera were reactive to two serovars, and three sera were reactive to three serovars. The L. kirschneri serovar australis was most frequently identified (60.2%). The findings suggest a previously unrecognized role of pteropid bats in the natural history of leptospirosis. The potential exists for establishment of infection in new host species, the transmission of new serovars to known host species, and for changes in virulence of leptospires as a result of passage through these species.

Prevalence of antibodies of Leptospira serovars in beef cattle in central Queensland.

OBJECTIVE: To obtain up-to-date data on the prevalence of antibodies to Leptospira serovars in central Queensland beef herds preliminary to assessing their role in bovine subfertility and the role of cattle as a zoonotic reservoir. DESIGN: Sera from 2857 female cattle in 68 central Queensland beef herds were tested for antibodies to 14 Leptospira serovars using the microscopic agglutination test. Vaccination use and age of cattle were collected to enable the calculation of crude and age-stratified seroprevalences. RESULTS: The most commonly detected antibodies were to serovars hardjo (15.8% crude seroprevalence), tarassovi (13.9%), pomona (4.0%) and szwajizak (2.2%). Vaccinates were omitted from the hardjo and pomona seroprevalence data. The seroprevalence for hardjo and pomona tended to increase with age of the animals. CONCLUSION: These results are broadly similar to those of previous serological surveys. The data suggest that serovars other than hardjo, pomona and tarassovi, are unlikely to have a significant role in bovine subfertility and that cattle are unlikely to be a source of human infection with them in central Queensland.

Leptospira autumnalis isolated from a human case from Avadi, India, and the serovar's predominance in local rat and bandicoot populations.

Leptospira were successfully isolated from the urine of an Indian patient who had been clinically diagnosed as having leptospirosis. In an attempt to determine the source of this infection, 28 rats (Rattus rattus) and 58 bandicoots (Bandicota bengalensis) living in the vicinity of the patient's home in Avadi, a suburban area of the city of Chennai (Madras), India, were then investigated. Each animal was checked for infection by microscopical examination of fresh and stained urine, serological analysis of serum, and the culture of urine and kidney samples. Direct, dark-field, observation of fresh urine samples and examination of urine samples after Fontana's silver staining were found to be the least sensitive of the tests used. The results of the serological microscopic agglutination test (MAT) indicated that four (14.3%) of the rats and nine (16.1%) of the bandicoots had significant agglutinins, predominantly for the serogroups icterohaemorrhagiae and autumnalis. Leptospira were isolated from at least one culture of samples from one rat and each of four bandicoots. Each of these rodent isolates and the human isolate were typed as Leptospira interrogans serovar autumnalis.

Reservoir hosts of Leptospira inadai in India.

Isolation of Leptospira from the kidneys of Rattus rattus wroughtoni hinton, Rattus rattus rufescens, Bandicota bengalensis and Bandicota indica was attempted in Bangalore in southern India. In total, 296 spirochaetes were isolated from 1,348 kidney cultures (an isolation rate of 22%). A batch of fifty-six isolates from India was identified, based on serological and polymerase chain reaction analysis, of which twenty-three isolates were identified as L. inadai by the World Health Organization/Food and Agriculture Organization Collaborating Centre for Reference and Research on Leptospirosis, in Brisbane. This is the first record of isolation of L. inadai from rodents. The preponderance of L. inadai in four different species of rodents suggests that these animals could be the natural reservoir hosts of L. inadai, and raises a critical question as to the likely impact of this species of Leptospira on the renal carrier status of other Leptospira pathogenic to humans and animals in this part of India. Virulence studies conducted at the University of Trieste in Italy, revealed that isolates of L. inadai from India were moderately or totally serum resistant when subjected to a serum killing test. To establish the possible seroprevalence of this species in the population, the inclusion of L. inadai in the battery of leptospiral antigens used for sero-epidemiological studies is recommended.

Identification of Leptospira biflexa by real-time homogeneous detection of rapid cycle PCR product.

Sequence analysis of 16S rRNA genes extracted from nucleic acids databases enabled the identification of a Leptospira biflexa (L. biflexa) signature sequence, against which a reverse primer designated L613, was designed. This primer, when used in conjunction with a universal bacterial specific forward primer designated Fd1, enabled the development of a LightCycler-based PCR protocol in which fluorescence emission due to binding of SYBR Green I dye to amplified products could be detected and monitored. A melting temperature (Tm), determined from the melting curve of the amplified product immediately following the termination of thermal cycling, confirmed that the product was that of L. biflexa. Agarose gel electrophoresis therefore was not necessary for identification of PCR products. The PCR protocol was very rapid, and consisted of 30 cycles with a duration of 20 s for each cycle with the monitoring of the melting curve requiring an additional 3 min. The whole protocol was completed in less than 20 min. The PCR protocol was also specific and enabled the identification of 18 strains of L. biflexa, whilst excluding 14 strains of L. interrogans and Leptonema illini. Two examples of its utility in improving work flow of a Leptospira reference laboratory are presented in this article. The use of a simple boiling method for extraction of DNA from all the members of the Leptospiraceae family DNA further simplifies the procedure and makes its use conducive to diagnostic laboratories.

Leptospira interrogans antibodies in feral pigs from New South Wales.

The sera of 195 hunter-killed feral pigs (Sus scrofa), collected in New South Wales (Australia) from April to November 1995, were screened against a reference panel of 14 Leptospira interrogans serovars using a microscopic agglutination test (MAT). The panel represented those serovars previously isolated from wild and domestic mammals in mainland Australia. Antileptospiral agglutinins were detected in 20% of the sera tested and included nine L. interrogans serovars. The majority of serological reactors (63%) were to L. interrogans serovar pomona. Sera from 26% of immunoreactors cross reacted with antigens from one or more serovars. No differences were noted in the prevalence of L. interrogans antibodies between the sexes, or between pigs from areas of low and high rainfall. The implications of leptospirosis in feral pigs on the transmission of leptospires to wildlife, livestock, and humans are discussed.

Identification of Leptospira inadai by continuous monitoring of fluorescence during rapid cycle PCR.

Seven new Leptospira isolates from rats, a buffalo, and contaminated media showed either reactive serology against more than 1 serogroup or no reactive serology against a reference panel of 22 serovars in the microscopic agglutination test (MAT). Because of these inconclusive results, the 16S rDNA sequences of these isolates were determined and found to resemble that of the type strain of Leptospira inadai (L. inadai), serovar lyme strain 10, which is considered to be nonpathogenic for humans. Comparative analyses of other Leptospira 16S rDNA sequences from databases revealed a L. inadai-specific signature sequence, against which an amplification primer was designed. This primer when used in conjunction with an universal primer enabled the trial of a rapid PCR protocol in which fluorescence emissions due to binding of SYBR Green I dye to PCR products were continuously monitored during rapid thermal cycling. A melting curve acquired immediately after PCR was used to distinguish the intended product. The thermal cycling and continuous monitoring of fluorescence emission were accomplished by the LightCycler; the whole procedure of 30 PCR cycles and melting curve acquisition required only 20 minutes. The primer achieved the required specificity, as the intended PCR product resulted only from 6 confirmed L. inadai reference strains and 7 field isolates that had been verified as L. inadai by the 16S rDNA sequencing, but not from 16 reference strains of Leptospira belonging to 7 other genospecies. Furthermore, these experiments showed that the PCR protocol was robust because target DNA of different conditions, which were extracted by either 1 of the 4 methods used, could be detected.

Real-time homogeneous assay of rapid cycle polymerase chain reaction product for identification of Leptonema illini.

Partial 16S rDNA sequences of eight Leptospira-like field isolates that reacted weakly or not at all to microscope agglutination test were found to be similar to the 16S rDNA sequence of the nonpathogen Leptonema illini-type strain 3055. Comparison of these sequences with those of Leptospira 16S rDNA sequences revealed a Leptonema species signature sequence for which a forward amplification primer was designed. This primer was used in conjunction with a bacterial-specific 16S rDNA universal reverse primer for developing a LightCycler-based rapid PCR protocol in which fluorescence emission due to the binding of SYBR green I dye to the amplified products was continuously monitored. A melting temperature (T(m)) determined from the melting curve of the amplified product immediately after PCR confirmed that the product was of Leptonema. The protocol for 24 samples consisting of 30 PCR cycles and melting curve acquisitions required 30 min to complete and agarose gel electrophoresis of the PCR products was not necessary. The method was specific as PCR products were detected from the seven Leptonema reference strains and the eight field isolates that had been previously verified as Leptonema by 16S rDNA sequencing, but not from the two representative strains from each of the eight Leptospira genospecies tested.

Identification of pathogenic Leptospira genospecies by continuous monitoring of fluorogenic hybridization probes during rapid-cycle PCR.

Partial sequences of 23S rRNA gene PCR products from 23 strains of 6 pathogenic Leptospira genospecies and from 8 strains of the saprophytic Leptospira biflexa were determined. Sequence analyses enabled Leptospira genus-specific amplification primers and pathogen-specific fluorogenic adjacent hybridization probes to be designed and synthesized. A PCR protocol was developed in which changes in fluorescence emission resulting from specific annealing of fluorogenic adjacent hybridization probes to the target DNA were continuously monitored. Nine strains of the pathogenic Leptospira genospecies could be differentiated from Leptonema illini, Escherichia coli, and eight strains of Leptospira biflexa. The PCR method was rapid, requiring 18 min for the completion of 45 cycles. It was also simple and flexible, as DNA templates prepared by four different methods, including the simple boiling method, could be used without adverse effects. Two hundred copies of target, equivalent to 100 cells, could be detected.

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

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.

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.

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

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.