Lyme borreliosis incidence in two French departments: correlation with infection of Ixodes ricinus ticks by Borrelia burgdorferi sensu lato.

We conducted a prospective study to estimate the Lyme borreliosis incidence in two rural French departments, Meuse and Puy-de-Dôme. Concurrently, we investigated the prevalence of ticks infected with Borrelia burgdorferi sensu lato (sl) and Anaplasma phagocytophilum. The incidence of Lyme borreliosis decreased from 156 to 109/100,000 inhabitants in Meuse and from 117 to 76/100,000 inhabitants in Puy-de-Dôme in 2004 and 2005, respectively, corresponding to a decrease in the density of Ixodes ricinus nymphs infected with B. burgdorferi sl. During the same period, the density of adult ticks increased. Interestingly, B. valaisiana, a nonpathogenic species, infected adult ticks more often than nymphs. These results confirmed the correlation between the Lyme borreliosis incidence and the density of infected nymphs, a stage preferentially infected with B. afzelii. In contrast, we found a low rate of infection by A. phagocytophilum, ranging from 0% to 0.4% in Puy-de-Dôme and from 0.8% to 1.4% in Meuse, suggesting a low risk for humans.

[Laboratory based human leptospirosis surveillance in New Caledonia (2001-2005)]. / Bilan de cinq années de surveillance biologique de la leptospirose humaine en Nouvelle-Calédonie (2001-2005).

The Pasteur Institute in New Caledonia performs for this territory the biological diagnosis of human leptospirosis; therefore its activity locally gives a rather exhaustive description of this pathology. The results presented here cover the 2001-2005 period and describe the principal epidemiological and biological features of human leptospirosis in New Caledonia. The investigated patients were recruited by the main medical structures: territorial and provincial hospitals, public dispensaries, clinics and general practitioners. The laboratory used the microagglutination test for serological investigations and PCR methods for the early detection of Leptospira genome in clinical samples. 239 cases of leptospirosis were biologically confirmed among 6690 tested patients, giving an average incidence of 21 cases per 100000/year, and a lethality rate of 5.4%. The sex-ratio was 1.8 male/female, patients were predominantly belonging to the 20-50 year age group and were inhabitants from the Northern Province. The circulating serogroups were mainly Icterohaemorrhagiae (69%), Australis (8%) and Pyrogenes (6%). The annual incidence peak occurred in April at the end of the warm season, and the importance of annual outbreaks could be linked with El Ninõ, the main regional climatic phenomenon.

Identification of variable-number tandem-repeat loci in Leptospira interrogans sensu stricto.

Leptospira interrogans sensu stricto is responsible for the most frequent and severe cases of human leptospirosis. The epidemiology and clinical features of leptospirosis are usually associated with the serovars and serogroups of Leptospira. Because of the difficulties associated with serological identification of Leptospira strains, we evaluated a novel PCR-based method for typing L. interrogans serovars. Based upon the genome sequence of L. interrogans serovar Lai type strain 5660, 44 loci were analyzed by PCR for their variability in size due to the presence of variable-number tandem repeats (VNTR). Seven VNTR loci were found to be powerful markers for serovar identification, epidemiology, and phylogenetic studies of L. interrogans. This rapid and easy method should greatly contribute to a better knowledge of the epidemiology of Leptospira.

Guidelines for the diagnosis of tick-borne bacterial diseases in Europe.

Ticks are obligate haematophagous acarines that parasitise every class of vertebrate (including man) and have a worldwide distribution. An increasing awareness of tick-borne diseases among clinicians and scientific researchers has led to the recent description of a number of emerging tick-borne bacterial diseases. Since the identification of Borrelia burgdorferi as the agent of Lyme disease in 1982, 11 tick-borne human bacterial pathogens have been described in Europe. Aetiological diagnosis of tick-transmitted diseases is often difficult and relies on specialised laboratories using very specific tools. Interpretation of laboratory data is very important in order to establish the diagnosis. These guidelines aim to help clinicians and microbiologists in diagnosing infection transmitted by tick bites and to provide the scientific and medical community with a better understanding of these infectious diseases.

Molecular characterization of Leptospira spp. strains isolated from small rodents in Croatia.

We report the isolation and characterization of 16 Leptospira spp. strains isolated from small rodents captured in 11 different regions of inland Croatia. Large NotI and SgrAI restriction fragment allowed us to assign 10 isolates to the serovar istrica, 5 isolates to the serovar tsaratsovo and 1 isolate to the serovar lora. The phylogenetic analysis conducted from the sequences of the first 330 bp from the 16S rDNA gene revealed that the strains belonged to three different species, L. borgpetersenii, L. kirschneri and L. interrogans. Carrier rates in eight rodent species varied from 0 to 71.4%. Mus musculus showed the highest infection level and confirmed its role as a major reservoir of the serogroup Sejroë. For the first time we reported the occurrence of serovars tsaratsovo and lora in Croatia.

Clustered cases of leptospirosis in Rochefort, France, June 2001.

Five clustered cases of leptospirosis were diagnosed in the area of Rochefort, France, in June 2001, among teenagers who had swum in the Genouillé canal. The symptoms included fever, headache, abdominal pain and vomiting, chills and myalgia. Three cases were confirmed by PCR and serology. The mean cumulative duration of bathing was significantly higher in cases (23.8 hours) compared to controls (14.4 hours). No other particular risk factor was observed. The environmental investigation revealed the presence of rodents excreting of leptospires near the bathing area. For all antigens considered, the occurence of seropositive rodents was 30.8%, L. icterohaemorrhagiae being the predominant serogroup (23,1%).

High prevalence of Borrelia lusitaniae in Ixodes ricinus ticks in Tunisia.

To investigate whether ticks of the genus Ixodes are infected by Borrelia burgdorferi complex, 490 unfed Ixodes ricinus ticks were collected by flagging in three different areas of Tunisia in 1998. DNAs extracted from 81 adults, 60 nymphs and 38 larvae were analysed after genic amplification of the non-coding spacer between the two copies of the rrl-rrf genes of B. burgdorferi sl. The prevalence of B. burgdorferi sl. in adults, nymphs and larvae was found to be 34, 33.3 and 2.6%, respectively. All DNAs (n = 61) but one were identified as belonging to different genotypes of B. lusitaniae by analysis of the restriction fragment length polymorphism of amplification products. In addition, 290 adults, 14 nymphs and 7 larvae were used to inoculate BSK-H medium to isolate spirochetes. Fifteen strains were isolated from adult ticks in the humid areas of Tunisia, whereas only one was obtained from larvae. Isolates were identified as B. lusitaniae (15/16) and B. garinii (1/16). These results provide new evidence for the existence of Lyme borreliosis in North Africa.

The implications of a low rate of horizontal transfer in Borrelia.

The nature and rate of recombination can be studied by comparing the sequences of multiple genes across a set of strains. When this approach is applied to Borrelia burgdorferi, four results emerge: (1) chromosomal genes are clonal; (2) there is little or no plasmid exchange; (3) the major mode of horizontal transfer of genetic material inserts a small fragment of DNA, typically <1 kb, during recombination; and (4) the level of horizontal transfer in Borrelia is so low that there is evidence for horizontal transfer only in genes where there is positive selection for diversity, that is, positive selection for the recombinant. Thus, Borrelia can serve as a model of a low recombination taxon. The implications of these results lead us to postulate that an unknown agent that is part of the Borrelia genome mediates the horizontal transfer of small fragments of DNA; the rare transfer of small fragments of DNA excludes both DNA parasites and virulence factors from the genome.

First evidence for a restriction-modification system in Leptospira sp.

The LE1 leptophage exhibited a host range restricted to the saprophytic Leptospira biflexa [Saint Girons et al., Res. Microbiol. 141 (1990) 1131-1133] and mainly to the Patoc 1 strain (hereafter called PFRA) kept in the Paris, France collection. Results of titration of LE1 lysates indicated the presence of a host-controlled modification and restriction system within PUSA (Patoc 1 strain maintained in the Morgantown, WV, USA collection) that was absent in PFRA. Because genomic DNA of PITAL (Patoc 1 strain maintained in Trieste, Italy) appeared smeared in pulsed field gel electrophoresis (PFGE), this strain is likely to contain nucleases that are activated upon DNA isolation. Moreover, comparative NotI digestions of PUSA and PFRA DNAs, as visualized by PFGE, indicated that PUSA belonged to a different serovar than PFRA. Finally, 16S ribosomal sequence analysis indicated that PUSA belonged to the saprophytic Leptospira meyeri species, while PITAL and PFRA appertained to L. biflexa. The evolutionary significance and the importance of the restriction and modification enzymes or non-specific nucleases within strains for genetic experiments are discussed.

Distinct levels of genetic diversity of Borrelia burgdorferi are associated with different aspects of pathogenicity.

Different species of pathogenic Borrelia show different symptoms and tick vector specificity. Even within regions where only one species is found, Lyme disease progresses very differently from one patient to another. Since Borrelia shows very little recombination either within or between species, alleles of a gene can be used to mark clones. The ospC gene is highly variable within each species and can be used to define groups of related clones. It has been previously shown that only four out of seventeen ospC groups of Borrelia burgdorferi sensu stricto cause invasive forms of the disease. Other groups cause erythema migrans, a skin rash at the site of the tick bite, but not invasive disease, while still other groups seem to be nonpathogenic to humans. In this study we extend the analysis of the ospC gene to the other pathogenic species, Borrelia garinii and Borrelia afzelii. Only two groups in B. afzelii and four groups in B. garinii cause invasive disease. Thus, only ten out of the 58 defined ospC groups cause invasive and presumably chronic Lyme disease.

Interest of partial 16S rDNA gene sequences to resolve heterogeneities between Leptospira collections: application to L. meyeri.

This paper describes the advantage of using the first 330 bp (positions 46 to 375, Escherichia coli numbering) of the 16S rDNA gene for comparison of Leptospira isolates. Phylogenetic analysis conducted from the whole 16S rDNA sequences available in databanks as well as that conducted from the partial sequences yielded quite similar results, in accordance with data inferred from previous DNA-DNA relatedness studies. This tool was used for the comparison of Leptospira strains from different reference collections. Consistent results were obtained from the analysis of the polymorphism generated by pulsed-field gel electrophoresis. The study focused on different serovars of L. meyeri species, the classification of which has been controversial. The results revealed large collection heterogeneities, and suggest that the classification of the L. meyeri species should be revised.

Analysis of Leptospira isolates from mainland Portugal and the Azores islands.

From 228 recent Leptospira isolates from mainland Portugal and Azorean wild mammals, 149 were characterized at the serovar level by monoclonal antibodies (MAbs), a quick serological method in epidemiological studies. In order to compare this antigenic information with that from new genetic techniques, a sample of isolates was analyzed through pulsed-field agarose gel electrophoresis (PFGE) (n=71), mapped restriction site polymorphisms (MRSPs) in PCR-amplified rRNA genes (n=45, including 13 saprophytes) and arbitrarily primed polymerase chain reaction (AP-PCR) fingerprinting (n=32). MRSP and AP-PCR lead to species identification of the studied 32 pathogenic isolates: Leptospira interrogans (n=3), Leptospira kirschneri (n=8) and Leptospira borgpetersenii (n=21). MAbs and PFGE characterized pathogenic isolates at the serovar level and resulted mainly in agreement (64%) although many discrepancies (35%) were observed.

Identification of a 36-kDa fibronectin-binding protein expressed by a virulent variant of Leptospira interrogans serovar icterohaemorrhagiae.

We investigated the ability of a virulent strain of Leptospira interrogans serovar icterohaemorrhagiae, its isogenic avirulent variant and a saprophytic strain to bind fibronectin using alkaline phosphatase-labelled fibronectin. A single 36-kDa fibronectin-binding protein was expressed only by the virulent strain and was located in the outer sheath according to proteinase K treatment results. The interaction of this protein with fibronectin was specific and the region of fibronectin bound to this potential adhesin overlapped the gelatin-binding domain. The inability of a RGDS synthetic peptide to inhibit the binding of fibronectin indicated that the cell-binding domain was not involved in this interaction. Considering the wide distribution of fibronectin within a host and the diversity of mammals involved in the epidemiology of leptospirosis, its implication in the cell attachment process of virulent leptospires is coherent with the multiplicity of target cells.

Antigenic variation of Borrelia turicatae Vsp surface lipoproteins occurs in vitro and generates novel serotypes.

As a means of avoiding the host immune response, the tick-borne relapsing fever spirochete Borrelia turicatae undergoes antigenic variation in its abundant surface lipoproteins. In this study, B. turicatae strain Oz1, serotype B, was subcultured in vitro and cloned by limited dilutions after 50 passages. Four different serotypes (serotypes A, B, E, and F) differing by their expressed Vsp lipoproteins were isolated. Using pulsed-field gel electrophoresis, we showed that the variability in surface-exposed proteins is correlated with rearrangement between different linear plasmids, defining serotype-specific plasmid profiles. Moreover, we determined the nucleotide sequence of genes encoding the VspE and VspF lipoproteins, corresponding to the two novel serotypes E and F, respectively. Our results showed that antigenic variation in B. turicatae occurs spontaneously in vitro, in the absence of immune selection.

A clonal subpopulation of Leptospira interrogans sensu stricto is the major cause of leptospirosis outbreaks in Brazil.

Leptospira is a highly diverse genus comprising many species and serogroups in Brazil as well as all over the world. However, a study by arbitrarily primed PCR of 44 leptospiral strains isolated from humans during three different outbreaks in Brazilian urban centers reveals that 43 of 44 isolates exhibit very similar fingerprints. Analysis of these isolates indicates that they belong to a clonal subpopulation of Leptospira interrogans sensu stricto.

Common ancestry of Borrelia burgdorferi sensu lato strains from North America and Europe.

Ten atypical European Borrelia burgdorferi sensu lato (Borrelia spp. ) strains were genetically characterized, and the diversity was compared to that encountered among related Borrelia spp. from North America. Phylogenetic analyses of a limited region of the genome and of the whole genome extend existing knowledge about borrelial diversity reported earlier in Europe and the United States. Our results accord with the evidence that North American and European strains may have a common ancestry.

Isolation of Lyme disease Borrelia from puffins (Fratercula arctica) and seabird ticks (Ixodes uriae) on the Faeroe Islands.

This is the first report on the isolation of Lyme disease Borrelia from seabirds on the Faeroe Islands and the characteristics of its enzootic cycle. The major components of the Borrelia cycle include the puffin (Fratercula arctica) as the reservoir and Ixodes uriae as the vector. The importance of this cycle and its impact on the spread of human Lyme borreliosis have not yet been established. Borrelia spirochetes isolated from 2 of 102 sampled puffins were compared to the borreliae previously obtained from seabird ticks, I. uriae. The rrf-rrl intergenic spacer and the rrs and the ospC genes were sequenced and a series of phylogenetic trees were constructed. Sequence data and restriction fragment length polymorphism analysis grouped the strains together with Borrelia garinii. In a seroepidemiological survey performed with residents involved in puffin hunting on the Faeroe Islands, 3 of 81 serum samples were found to be positive by two commonly used clinical tests: a flagellin-based enzyme-linked immunosorbent assay (ELISA) and Western blotting. These three positive serum samples also had high optical density values in a whole-cell ELISA. The finding of seropositive Faeroe Islanders who are regularly exposed to I. uriae indicate that there may be a transfer of B. garinii by this tick species to humans.

In vivo apoptosis of hepatocytes in guinea pigs infected with Leptospira interrogans serovar icterohaemorrhagiae.

To investigate the contribution of the previously demonstrated in vitro apoptosis to the pathogenesis of leptospirosis, guinea pigs were infected with Leptospira interrogans serovar icterohaemorrhagiae strain Verdun and sequentially killed to collect target organs involved in the natural history of the disease (liver, kidneys, lungs, spleen and heart). The combination of histopathological procedures and a specific TUNEL assay showed a significant Leptospira-induced programmed cell death of hepatocytes with a peak at 48 h post inoculation. Hepatocyte nuclei showed morphological changes including fragmented and condensed nuclei. This phenomenon occurred early in the course of the disease at a time where infecting leptospires were present at a low density between the liver parenchyma cells.

Expanded diversity among Californian borrelia isolates and description of Borrelia bissettii sp. nov. (formerly Borrelia group DN127).

Up to now, the only species in the complex Borrelia burgdorferi sensu lato known to cause Lyme borreliosis in the United States has been B. burgdorferi sensu stricto. However, some atypical strains closely related to the previously designated genomic group DN127 have been isolated in the United States, mostly in California. To explore the diversity of B. burgdorferi sensu lato group DN127, we analyzed the nucleotide sequences of the rrf-rrl intergenic spacer regions from 19 atypical strains (18 from California and one from New York) and 13 North American B. burgdorferi sensu stricto strains (6 from California). The spacer region sequences from the entire B. burgdorferi sensu lato complex available in data banks were used for comparison. Phylogenetic analysis of sequences shows that the main species of the B. burgdorferi sensu lato complex (B. afzelii, B. garinii, B. andersonii, B. japonica, B. burgdorferi sensu stricto, B. valaisiana, and B. lusitaniae) each form a coherent cluster. A heterogeneous group comprising strains belonging to the previously designated group DN127 clustered separately from B. burgdorferi sensu stricto. Within this cluster, the deep branches expressing the distances between the rrf-rrl sequences reflect a high level of divergence. This unexpected diversity contrasts with the monomorphism exhibited by B. burgdorferi sensu stricto. To clarify the taxonomic status of this highly heterogeneous group, analysis of the rrs sequences of selected strains chosen from deeply separated branches was performed. The results show that these strains significantly diverge at a level that is compatible with several distinct genomic groups. We conclude that the taxonomy and phylogeny of North American B. burgdorferi sensu lato should be reevaluated. For now, we propose that the genomic group DN127 should be referred to as a new species, B. bissettii sp. nov., and that other related but distinct strains, which require further characterization, be referred to as Borrelia spp.