A recent study by our group reported the isolation and partial serological and molecular characterization of four Leptospira borgpetersenii serogroup Ballum strains. Here, we reproduced experimental leptospirosis in golden Syrian hamsters (Mesocricetus auratus) and carried out standardization of lethal dose 50% (LD50) of one of these strains (4E). Clinical disease features and histopathologic analyses of tissue lesions were also observed. As results, strain 4E induced lethality in the hamster model with inocula lower than 10 leptospires, and histopathological examination of animals showed typical lesions found in severe leptospirosis. Gross pathological findings were peculiar; animals that died early had more chance of presenting severe jaundice and less chance of presenting pulmonary hemorrhages (P < 0.01). L. borgpetersenii serogroup Ballum has had a considerable growth in human leptospirosis cases in recent years. This strain has now been thoroughly characterized and can be used in more studies, especially evaluations of vaccine candidates.
Fingerprints for 72 reference serovar strains of pathogenic Leptospira spp. were obtained by pulsed-field gel electrophoresis (PFGE) following NotI restriction digests of the chromosome. These strains included the serovar reference strains of serogroups Australis, Ballum, Bataviae, Grippotyphosa, Panama, Pomona, and Pyrogenes. Sixty-four serovars could be identified by a unique NotI restriction profile. The remaining serovars were differentiated by chromosomal digestion with SgrAI. These included four serovars from serogroup Australis, two serovars from serogroup Ballum, and two serovars from serogroup Bataviae. Thirteen of 18 recent clinical isolates identified by microagglutination test and cross-adsorption procedure were correctly typed by PFGE. The results indicate that PFGE, which is considerably more rapid than serology, should be useful for identification and epidemiological studies.
Rats are known to be the most important reservoirs and transmission sources of leptospirosis. However, the status of leptospirosis in the Philippines regarding reservoirs and transmission remains unknown. A survey was conducted in Metro Manila and Laguna that analyzed samples obtained from 106 rats. Using the microscopic agglutination test, we found that 92% of rat serum samples were positive for anti-Leptospira antibodies; the most common infecting serovars were Manilae, Hebdomadis, and Losbanos. On the basis of pulsed-field gel electrophoresis and gyrase B gene sequence analyses, four groups of rat kidney isolates were found: L. interrogans serovar Manilae, serovar Losbanos, and serogroup Grippotyphosa, and L. borgpetersenii serogroup Javanica. Most isolates were lethal after experimental infection of golden Syrian hamsters. Results showed that these four Leptospira serovars and serogroups are circulating among rats, and that these animals may be one of the possible transmission sources of leptospirosis in the Philippines.
Epidemiological aspects of infection with leptospires of the Ballum serogroup in black rats (Rattus rattus) and brown rats (Rattus norvegicus) are described. Rats inhabiting a variety of habitats were investigated and isolates identifed as belonging to the Ballum serogroup were obtained from 21 of 61 black rats (34%) and 63 of 243 brown rats (26%). The high level of endemic ballum serogroup infection in these species reported here has not been described in other countries. A statistical relationship was shown between the prevalence of infection in brown rat populations and population density but this was not evident for black rats. Epidemiological data indicates that the black rat is a maintenance host for leptospires of the Ballum serogroup in New Zealand. The brown rat does not appear to be an efficient maintenance host for these leptospires, however endemic infection can be maintained in high-density populations inhabiting synanthropic foci. An hypothesis of 'competitive exclusion' (preferential maintenance of a particular serovar by a host species) is introduced with regard to leptospiral infection in brown rats. It is concluded that the establishment and maintenance of an endemic focus of leptospirosis is dependant on: introduction of a particular serovar; a suitable host; and a suitable host habitat. Within a maintenance population direct transmission appears to be more important than indirect transmission via the environment.
Leptospirosis is a worldwide-distributed zoonosis, endemic in tropical areas. Epidemiologic investigations of leptospirosis still rely on tedious serological identification tests. Recently, molecular typing systems based on variable-number tandem-repeat (VNTR) analysis have been described and have been used to identify Leptospira interrogans strains. Although L. interrogans is the most common Leptospira species encountered in human infections around the world, other pathogenic species, such as Leptospira kirschneri and Leptospira borgpetersenii, are also frequently associated with human leptospirosis. In this study, we aimed to extend multilocus VNTR analysis (MLVA) identification of strains to species other than L. interrogans. We designed primers for VNTR loci found in L. interrogans, L. kirschneri, and L. borgpetersenii. The discriminatory power of the redefined primers was evaluated on collection strains and then on clinical strains. We also carried out a retrospective study on 156 strains isolated from patients and animals from New Caledonia, an area of high endemicity in the South Pacific. Our results show that this simple PCR-based MLVA typing technique is a powerful methodology for the epidemiology of leptospirosis.
Pathogenic Leptospira spp. are the etiological agents of leptospirosis, an important disease of both humans and animals. In urban settings, L. interrogans serovars are the predominant cause of disease in humans. The purpose of this study was to characterize a novel Leptospira isolate recovered from an abandoned swimming pool. Molecular characterization through sequencing of the rpoB gene revealed 100% identity with L. interrogans and variable-number tandem-repeat (VNTR) analysis resulted in a banding pattern identical to L. interrogans serogroup Icterohaemorrhagiae, serovar Copenhageni or Icterohaemorrhagiae. The virulence of the strain was determined in a hamster model of lethal leptospirosis. The lethal dose 50% (LD50) was calculated to be two leptospires in female hamsters and a histopathological examination of infected animals found typical lesions associated with severe leptospirosis, including renal epithelium degeneration, hepatic karyomegaly, liver-plate disarray and lymphocyte infiltration. This highly virulent strain is now available for use in further studies, especially evaluation of vaccine candidates.
Leptospira; Leptospirosis; Virulent; VNTR; rpoB
Leptospirosis is one of the most widespread zoonoses in the world. However, there is a lack of information on circulating Leptospira strains in remote parts of the world. We describe the serological and molecular features of leptospires isolated from 94 leptospirosis patients in Mayotte, a French department located in the Comoros archipelago, between 2007 and 2010. Multilocus sequence typing identified these isolates as Leptospira interrogans, L. kirschneri, L. borgpetersenii, and members of a previously undefined phylogenetic group. This group, consisting of 15 strains, could represent a novel species. Serological typing revealed that 70% of the isolates belonged to the serogroup complex Mini/Sejroe/Hebdomadis, followed by the serogroups Pyrogenes, Grippotyphosa, and Pomona. However, unambiguous typing at the serovar level was not possible for most of the strains because the isolate could belong to more than one serovar or because serovar and species did not match the original classification. Our results indicate that the serovar and genotype distribution in Mayotte differs from what is observed in other regions, thus suggesting a high degree of diversity of circulating isolates worldwide. These results are essential for the improvement of current diagnostic tools and provide a starting point for a better understanding of the epidemiology of leptospirosis in this area of endemicity.
Our objective was to identify local animal reservoirs of leptospirosis to explain the unusual features of Leptospira strains recently described among patients on the island of Mayotte. By means of a microscopic agglutination test using local clinical isolates, we found that 11.2% of black rats were seropositive to Leptospira, whereas 10.2% of flying foxes, 2% of lemurs, 93.1% of domestic dogs, and 87.5% of stray dogs were seropositive. As observed in humans, Mini was the main serogroup circulating in animals, whereas serogroup Icterohaemorrhagiae was absent. Using quantitative polymerase chain reaction, we also showed that 29.8% of rats carried leptospires in their kidneys. The sequencing of 16S rRNA gene sequences of Leptospira found in black rat kidneys identified four genomospecies (Leptospira borgpetersenii, Leptospira interrogans, Leptospira kirschneri, and L. borgpetersenii group B), which established black rats as the major source of leptospirosis transmission to humans. The origins of such a genetic diversity in Leptospira strains are discussed.
In this study, 191 culture isolates were recovered from suspected samples of animals and humans in Ellinghausen McCullough Johnson and Harris (EMJH) medium and assessed for its morphological features by dark field microscopy. Extracted DNA from individual culture was subjected to different PCR assays for identification and characterization of leptospira. Out of 99 positive leptospira cultures, 52 pathogenic leptospira isolates were characterized at species level by using partial RNA polymerase β-subunit (rpoB) gene sequences. Phylogenetic analysis of the nucleotide sequences revealed that 30, 8, and 14 isolates belong to L. borgpetersenii / L. interrogans, L. kirschneri, and Leptospira intermediate species, respectively. Based on analysis of 99 leptospira isolates, the prevalent Leptospira species were L. borgpetersenii or L. interrogans (30.30%), L. kirschneri (8%) and Leptospira intermediate species (14.14%) in animals and humans. To the best of authors knowledge, this is the first study to use rpoB gene nucleotide sequence based phylogenetic analysis to identify/detect Leptospira intermediate species (L. wolffii) in animals and humans in India. Hence, the prevalence of this species will surely emphasize the importance of consideration of Leptospira intermediate species and formulate a way for further studies especially in understanding the newly emerging Leptospira in animals and humans and to combat the problem associated with the disease conditions.
Leptospira; Animals; Human; Characterization; Prevalence; Intermediate species
We describe the geographic distribution and variation in host-pathogen specificity for Leptospira-infected small mammals collected concurrently from three Hawaiian Islands over a period of 14 years: 1990–2003. Four serogroups (Icterohaemorrhagiae, Ballum, Sejroe, and Australis) were identified from the 15,171 animals tested. Serogroup prevalence differed across host species and islands (P < 0.0001 for each), but not across years. The host associations and biogeographic patterns of Leptospira in Hawaii indicate a pathogen community shaped by ecological factors.
Leptospirosis has been implicated as a severe and fatal form of disease in Mayotte, a French-administrated territory located in the Comoros archipelago (southwestern Indian Ocean). To date, Leptospira isolates have never been isolated in this endemic region.
Methods and Findings
Leptospires were isolated from blood samples from 22 patients with febrile illness during a 17-month period after a PCR-based screening test was positive. Strains were typed using hyper-immune antisera raised against the major Leptospira serogroups: 20 of 22 clinical isolates were assigned to serogroup Mini; the other two strains belonged to serogroups Grippotyphosa and Pyrogenes, respectively. These isolates were further characterized using partial sequencing of 16S rRNA and ligB gene, Multi Locus VNTR Analysis (MLVA), and pulsed field gel electrophoresis (PFGE). Of the 22 isolates, 14 were L. borgpetersenii strains, 7 L. kirschneri strains, and 1, belonging to serogoup Pyrogenes, was L. interrogans. Results of the genotyping methods were consistent. MLVA defined five genotypes, whereas PFGE allowed the recognition of additional subgroups within the genotypes. PFGE fingerprint patterns of clinical strains did not match any of the patterns in the reference strains belonging to the same serogroup, suggesting that the strains were novel serovars.
Preliminary PCR screening of blood specimen allowed a high isolation frequency of leptospires among patients with febrile illness. Typing of leptospiral isolates showed that causative agents of leptospirosis in Mayotte have unique molecular features.
Leptospirosis has been recognized as an increasing public health problem affecting poor people from developing countries and tropical regions. However, the epidemiology of leptospirosis remains poorly understood in remote parts of the world. In this study of patients from the island of Mayotte, we isolated 22 strains from the blood of patients during the acute phase of illness. The pathogenic Leptospira strains were characterized by serology and various molecular typing methods. Based on serological data, serogroup Mini appears to be the dominant cause of leptospirosis in Mayotte. Further molecular characterization of these isolates allowed the identification of 10 pathogenic Leptospira genotypes that could correspond to previously unknown serovars. Further progress in our understanding of the epidemiology of Leptospira circulating genotypes in highly endemic regions should contribute to the development of novel strategies for the diagnosis and prevention of this neglected emerging disease.
An investigation was undertaken to assess the present importance of leptospiral infections in Northern Ireland, and in particular to look for evidence of infection by leptospiral serotypes other than L. icterohaemorrhagiae and L. canicola.
Blood samples from 335 patients, sent to the laboratory for a variety of tests, were examined. After initial screening with five groups of pooled antigens, tests for leptospiral agglutinins were completed with formolized antigens prepared from 13 different serotypes. In seven patients a diagnosis of acute leptospirosis was made while nine others showed serological evidence of previous leptospiral infection. Attempts to isolate leptospirae by culture from 29 blood samples were unsuccessful.
The serological results indicate that two additional leptospiral serotypes, namely L. ballum and L. bratislava, are causing human infection in Northern Ireland, and presumably also in other parts of the British Isles. Some clinical and epidemiological features associated with different types of leptospiral infection are described. It is stressed that leptospirosis is essentially a febrile illness, that meningeal symptoms are common, and that (contrary to popular belief) jaundice is by no means a constant occurrence.
The implications of these findings are discussed, with special reference to the diagnosis of leptospiral infections. Laboratory diagnostic procedures are briefly reviewed, and the possible deficiencies of the agglutination test commonly used in Britain are pointed out. Some suggestions are made concerning both clinical and laboratory aspects of diagnosis, and the need for a reliable screening test for all forms of leptospiral infection is emphasized.
Leptospira is the causative agent of leptospirosis. The O-antigen is the distal part of the lipopolysaccharide, which is a key component of outer membrane of Gram-negative bacteria and confers serological specificity. The epidemiology and clinical characteristics of leptospirosis are relative to the serology based taxonomic unit. Identification of Leptospira strains by serotyping is laborious and has several drawbacks.
In this study, the O-antigen gene clusters of four epidemic Leptospira serogroups (serogroup Canicola, Autumnalis, Grippotyphosa and Hebdomadis) in China were sequenced and all genes were predicted in silico. Adding published sequences of two serogroups, Icterohaemorrhagiae (strain Lai and Fiocruz L1-130) and Sejroe (strain JB197 and L550), we identified six O-antigen-specific genes for six epidemic serogroups in China. PCR assays using these genes were developed and tested on 75 reference strains and 40 clinical isolates.
The results show that the PCR-based assays can be reliable and alternative means for rapid typing of these six serogroups of Leptospira.
Pathogenic mechanisms of Leptospira interrogans, the causal agent of leptospirosis, remain largely unknown. This is mainly due to the lack of tools for genetic manipulations of pathogenic species. In this study, we characterized a mutant obtained by insertion of the transposon Himar1 into a gene encoding a putative lipoprotein, Loa22, which has a predicted OmpA domain based on sequence identity. The resulting mutant did not express Loa22 and was attenuated in virulence in the guinea pig and hamster models of leptospirosis, whereas the genetically complemented strain was restored in Loa22 expression and virulence. Our results show that Loa22 was expressed during host infection and exposed on the cell surface. Loa22 is therefore necessary for virulence of L. interrogans in the animal model and represents, to our knowledge, the first genetically defined virulence factor in Leptospira species.
The spirochetes, which include medically important pathogens such as the causative agents of Lyme disease, syphilis, and leptospirosis, constitute an evolutionarily unique group of bacteria. Leptospirosis is a zoonotic disease that causes a high rate of mortality and morbidity in humans and animals throughout the world each year. The year 2007 marks the centenary of the discovery of the causative agent of leptospirosis, Leptospira interrogans. Until now, the genetic obstacles posed by leptospires (principally, the difficulties in generating targeted mutants) have hampered the identification of virulence genes. In this study, we describe an avirulent mutant in a pathogenic Leptospira that was obtained via disruption of loa22, a gene that encodes an outer membrane protein containing an OmpA domain. This mutation resulted in an avirulent mutant in the guinea pig model, and reintroduction of loa22 into the mutant restored Leptospira's ability to kill guinea pigs. Our results therefore indicate that loa22 is a virulence determinant that is, to our knowledge, the first identified for this pathogen.
Recent evidence based on the current epidemiological situation suggests that vaccines against canine leptospirosis in Europe should be directed against infection with Leptospira interrogans (sensu lato) serogroups Canicola, Icterohaemorrhagiae, Grippotyphosa and Australis. In the eight studies presented here, dogs were vaccinated with Nobivac L4 (MSD Animal Health), a new tetravalent inactivated vaccine containing antigen from four strains representing these four serogroups. The dogs were then challenged, together with unvaccinated control dogs, using heterologous strains from the same four serogroups. In four of the studies, pups without agglutinating antibodies against the four serogroups were vaccinated with Nobivac L4 vaccine. In a further four studies, Nobivac L4 vaccine was given 48 hours after administration of antiserum from vaccinated dogs designed to mimic the serological status of pups with maternally derived antibodies against these serogroups. In all eight studies, vaccine efficacy was assessed in terms of antibody response, clinical signs, fever, thrombocyte count, frequency of positive isolation of challenge organisms from blood, urine and kidney and frequency of interstitial nephritis. The results demonstrate that Nobivac L4 vaccine induces sterile immunity against leptospiraemia and renal infection with strains of serogroups Canicola, Icterohaemorrhagiae and Grippotyphosa, and induces sterile immunity against leptospiraemia with a strain of serogroup Australis. Since sterile immunity was achieved in pups pretreated with antiserum as well, it can be concluded that this vaccine is also likely to be efficacious in the face of maternally derived antibodies in pups from the age of six weeks.
Leptospira; Vaccines; Dogs; Zoonoses
Leptospirosis is one of the most important neglected tropical bacterial diseases in Latin America and the Caribbean. However, very little is known about the circulating etiological agents of leptospirosis in this region. In this study, we describe the serological and molecular features of leptospires isolated from 104 leptospirosis patients in Guadeloupe (n = 85) and Martinique (n = 19) and six rats captured in Guadeloupe, between 2004 and 2012.
Methods and Findings
Strains were studied by serogrouping, PFGE, MLVA, and sequencing 16SrRNA and secY. DNA extracts from blood samples collected from 36 patients in Martinique were also used for molecular typing of leptospires via PCR. Phylogenetic analyses revealed thirteen different genotypes clustered into five main clades that corresponded to the species: L. interrogans, L. kirschneri, L. borgpetersenii, L. noguchi, and L. santarosai. We also identified L. kmetyi in at least two patients with acute leptospirosis. This is the first time, to our knowledge, that this species has been identified in humans. The most prevalent genotypes were associated with L. interrogans serovars Icterohaemorrhagiae and Copenhageni, L. kirschneri serovar Bogvere, and L. borgpetersenii serovar Arborea. We were unable to identify nine strains at the serovar level and comparison of genotyping results to the MLST database revealed new secY alleles.
The overall serovar distribution in the French West Indies was unique compared to the neighboring islands. Typing of leptospiral isolates also suggested the existence of previously undescribed serovars.
Leptospirosis is an emerging zoonotic disease caused by infection with pathogenic strains of Leptospira. Isolation of Leptospira strains is rare, making it difficult to assess their distribution worldwide. In this study, we characterized cultures of Leptospira obtained from more than one hundred leptospirosis patients from the French West Indies by serology and various molecular typing methods to identify the strains circulating in this endemic region. Typing of leptospiral isolates showed that causative agents of leptospirosis in the French West Indies are mainly from the serogroups Icterohaemorrhagiae and Ballum, but we also identified new genotypes. We also found that the distribution of the predominant pathogenic leptospiral serovars differed between the Caribbean islands. A better understanding of the epidemiology of leptospirosis will improve our knowledge in the distribution of this emerging neglected tropical disease worldwide. The identification of the circulating etiological agents of leptospirosis in the French West Indies will also help establish appropriate control and prevention measures in this area where the disease is endemic.
Available serological diagnostics do not allow the confirmation of clinically suspected leptospirosis at the early acute phase of illness. Several conventional and real-time PCRs for the early diagnosis of leptospirosis have been described but these have been incompletely evaluated. We developed a SYBR Green-based real-time PCR targeting secY and validated it according to international guidelines. To determine the analytical specificity, DNA from 56 Leptospira strains belonging to pathogenic, non-pathogenic and intermediate Leptospira spp. as well as 46 other micro-organisms was included in this study. All the pathogenic Leptospira gave a positive reaction. We found no cross-reaction with saprophytic Leptospira and other micro-organisms, implying a high analytical specificity. The analytical sensitivity of the PCR was one copy per reaction from cultured homologous strain M 20 and 1.2 and 1.5 copy for heterologous strains 1342 K and Sarmin, respectively. In spiked serum & blood and kidney tissue the sensitivity was 10 and 20 copies for M 20, 15 and 30 copies for 1342 K and 30 and 50 copies for Sarmin. To determine the diagnostic sensitivity (DSe) and specificity (DSp), clinical blood samples from 26 laboratory-confirmed and 107 negative patients suspected of leptospirosis were enrolled as a prospective consecutive cohort. Based on culture as the gold standard, we found a DSe and DSp of 100% and 93%, respectively. All eight PCR positive samples that had a negative culture seroconverted later on, implying a higher actual DSp. When using culture and serology as the gold standard, the DSe was lower (89%) while the DSp was higher (100%). DSe was 100% in samples collected within the first – for treatment important - 4 days after onset of the illness. Reproducibility and repeatability of the assay, determined by blind testing kidney samples from 20 confirmed positive and 20 negative rodents both appeared 100%. In conclusion we have described for the first time the development of a robust SYBR Green real-time PCR for the detection of pathogenic Leptospira combined with a detailed assessment of its clinical accuracy, thus providing a method for the early diagnosis of leptospirosis with a well-defined satisfactory performance.
The main goal of this study was to evaluate the prevalence of leptospirosis among field rodents of Tiruchirappalli district, Tamil Nadu, India. In total 35 field rats were trapped and tested for seroprevalence by the microscopic agglutination test (MAT). Isolation of leptospires was performed from blood and kidney tissues and characterized to serovar level. Genomospecies identification was carried out using 16S rRNA and lipL32 gene sequencing. The molecular phylogeny was constructed to find out species segregation. Seroprevalence was about 51.4 %, and the predominant serovars were Autumnalis, Javanica, Icterohaemorrhagiae and Pomona. Two isolates from the kidneys were identified as serovar Javanica of Serogroup Javanica, and sequence based molecular phylogeny indicated these two isolates were Leptospira borgpetersenii.
Leptospirosis; Leptospira borgpetersenii; lipL32; 16S rRNA
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.
Leptospirosis is a worldwide zoonosis caused by pathogenic Leptospira. The whole-genome sequence of L. interrogans serovar Copenhageni together with bioinformatics tools represent a great opportunity to search for novel antigen candidates that could be used as subunit vaccine against leptospirosis. We focused on six genes encoding for conserved hypothetical proteins predicted to be exported to the outer membrane. The genes were amplified by PCR from Leptospira interrogans genomic DNA and were cloned and expressed in Escherichia coli. The recombinant proteins tagged with N-terminal hexahistidine were purified by metal-charged chromatography. The immunization of hamsters followed by challenge with lethal dose of virulent strain of Leptospira showed that the recombinant proteins Lsa21, Lsa66 and rLIC11030 elicited partial protection to animals. These proteins could be used combined or in a mixture with novel adjuvants in order to improve their effectiveness.
Leptospira interrogans; leptospirosis; recombinant protein; vaccine.
The clinical and epidemiological features associated with the first reported outbreak in the British Isles of benign anicteric leptospirosis, due to strains belonging to the hebdomadis subgroup, are described. Four cowherdsmen working in two dairy farms in Surrey developed a febrile illness which was not associated with jaundice, but aseptic meningitis was a feature. Microscopic agglutination tests with formolized antigen suspensions revealed a significant rise in the agglutinin titres against various serotypes belonging to the hebdomadis serogroup of Leptospira interrogans. The probable source was eventually traced to the cattle, which showed serological evidence of infection with the same serogroup.
The agents of leptospirosis, a zoonosis with worldwide distribution, are pathogenic
spirochetes belonging to the genus Leptospira. The leptospiral life cycle
involves transmission via fresh water and colonization of the renal tubules of their
reservoir hosts. Infection of accidental hosts, including humans, may result in
life-threatening sequelae. Bacterial outer membrane proteins (OMPs), particularly those
with surface-exposed regions, play crucial roles in pathogen virulence mechanisms and
adaptation to environmental conditions, including those found in the mammalian host.
Therefore, elucidation and characterization of the surface-exposed OMPs of
Leptospira spp. is of great interest in the leptospirosis field. A
thorough, multi-pronged approach for assessing surface exposure of leptospiral OMPs is
essential. Herein, we present evidence for a sub-surface location for most or all of the
major leptospiral lipoprotein, LipL32, based on surface immunofluorescence utilizing three
different types of antibodies and four different permeabilization methods, as well as
surface proteolysis of intact and lysed leptospires. We reevaluate prior evidence
presented in support of LipL32 surface-exposure and present a novel perspective on a
protein whose location has been misleading researchers, due in large part to its
extraordinary abundance in leptospiral cells.
Most of the current knowledge of leptospirosis epidemiology originates from serological results obtained with the reference Microscopic Agglutination Test (MAT). However, inconsistencies and weaknesses of this diagnostic technique are evident. A growing use of PCR has improved the early diagnosis of leptospirosis but a drawback is that it cannot provide information on the infecting Leptospira strain which provides important epidemiologic data. Our work is aimed at evaluating if the sequence polymorphism of diagnostic PCR products could be used to identify the infecting Leptospira strains in the New Caledonian environment.
Both the lfb1 and secY diagnostic PCR products displayed a sequence polymorphism that could prove useful in presumptively identifying the infecting leptospire. Using both this polymorphism and MLST results with New Caledonian isolates and clinical samples, we confirmed the epidemiological relevance of the sequence-based identification of Leptospira strains. Additionally, we identified one cluster of L. interrogans that contained no reference strain and one cluster of L. borgpetersenii found only in the introduced Rusa deer Cervus timorensis russa that is its probable reservoir.
The sequence polymorphism of diagnostic PCR products proved useful in presumptively identifying the infecting Leptospira strains. This could contribute to a better understanding of leptospirosis epidemiology by providing epidemiological information that cannot be directly attained from the use of PCR as an early diagnostic test for leptospirosis.
Serological surveys of leptospiral antibodies in cattle were carried out in Macon and the surrounding counties of East Central Alabama. A total of 286 bovine serum samples were screened for the presence of antibodies against live antigens from twelve pathogenic leptospiral serotypes using a microscopic agglutination test. The most frequently encountered serotypes were Leptospira hardjo (47%), Leptospira wolffi (34%), Leptospira canicola (12%), Leptospira pomona (10%) and Leptospira ballum (10%). Leptospira autumnalis, Leptospira grippotyphosa, Leptospira icterohemorrhagiae, Leptospira pyrogenes and Leptospira tarassovi were observed in less than 5% of the samples.
Leptospirosis is a widespread zoonosis characterized by multiple organ failure and variable host susceptibility toward pathogenic Leptospira strains. In this study, we put the role of inflammatory mediators in parallel with bacterial burdens and organ lesions by comparing a susceptible animal model, the hamster, and a resistant one, the Oncins France 1 (OF1) mouse, both infected with virulent Leptospira interrogans serovar Icterohaemorrhagiae strain Verdun. Histological observations evidenced edema, congestion, hemorrhage, and inflammatory infiltration in the organs of hamsters, in contrast to limited changes in mice. Using reverse transcription-quantitative PCR techniques, we showed that the relative Leptospira burden progressively increased in hamster tissues, while a rapid clearance was observed in mouse tissues. The early regulation of the proinflammatory mediators interleukin-1β (IL-1β), IL-6, tumor necrosis factor alpha, and cyclo-oxygenase-2 and the chemokines gamma interferon-inducible protein 10 kDa/CXCL10 and macrophage inflammatory protein-1α/CCL3 in mouse tissues contrasted with their delayed and massive overexpression in hamster tissues. Conversely, the induction of the anti-inflammatory cytokine IL-10 was faster in the resistant than in the susceptible animal model. The role of these cytokines in the pathophysiology of leptospirosis and the implications of their differential regulation in the development of this disease are discussed.