First detection of Borrelia burgdorferi sensu lato DNA in king penguins (Aptenodytes patagonicus halli).

The hard tick Ixodes uriae parasitises a wide range of seabird species in the circumpolar areas of both Northern and Southern hemispheres and has been shown to be infected with Borrelia burgdorferi sensu lato, the bacterial agents of Lyme borreliosis. Although it is assumed that seabirds represent viable reservoir hosts, direct demonstrations of infection are limited to a single study from the Northern hemisphere. Here, the blood of 50 tick-infested adult king penguins (Aptenodytes patagonicus halli) breeding in the Crozet Archipelago (Southern Indian Ocean) was examined for B. burgdorferi sl exposure by serology and for spirochetemia by in vitro DNA amplification. Four birds were found positive by serology, whereas B. burgdorferi sl DNA was detected in two other birds. Our data therefore provide the first direct proof of Borrelia burgdorferi sl spirochetes in seabirds of the Southern hemisphere and indicate a possible reservoir role for king penguins in the natural maintenance of this bacterium. Although the bacterial genetic diversity present in these hosts and the infectious period for tick vectors remain to be elucidated, our results add to a growing body of knowledge on the contribution of seabirds to the complex epizootiology of Lyme disease and the global dissemination of B. burgdorferi sl spirochetes.

Borrelia crocidurae infection of Ornithodoros erraticus (Lucas, 1849) ticks in Tunisia.

Tick-borne relapsing fever (TBRF) is caused by Borrelia species transmitted to humans by infected Ornithodoros sp. ticks. The disease has been rarely described in North Africa, and in Tunisia the local transmission of TBRF seems to have disappeared or is undiagnosed. A longitudinal study was conducted in 14 sites located in four different bioclimatic zones of Tunisia to assess both the distribution of Ornithodoros sp. and their infection rate with the relapsing fever Borrelia sp. Three polymerase chain reaction methods targeting the 16S rRNA, the intergenic spacer, and the fla (flagellin) genes were used and phylogenetic analyses were carried out. Three hundred and fifty-eight specimens of Ornithodoros were collected: O. erraticus (previously termed "small variety") (n = 190) and O. normandi (n = 168). Borrelia crocidurae DNA was detected in 15.1% of O. erraticus (small variety) (24 out of the 159 randomly selected for testing) collected in rodent burrows situated in the arid and Saharan areas in southern Tunisia. Molecular analysis targeting the 16S rRNA gene and the noncoding intergenic spacer domain showed good resolution for this Borrelia sp., although no molecular polymorphism was evidenced according to location. In contrast, none of the 133 O. normandi, also randomly selected for testing, was infected by Borrelia sp. and these ticks were restricted to the subhumid and semiarid zones in northern Tunisia. Both O. erraticus (small variety) and O. normandi were found in Tunisia and the high B. crocidurae infection rate found in O. erraticus highlights the risk of TBRF transmission in the southern part of the country.

Borrelia hispanica relapsing fever, Morocco.

We found that 20.5% of patients with an unexplained fever in northwestern Morocco had tick-borne relapsing fever. Molecular detection specific for the 16S rRNA gene identified Borrelia hispanica. The noncoding intergenic spacer sequence domain showed high sensitivity and good resolution for this species.

Molecular analysis of Leptospira spp. isolated from humans by restriction fragment length polymorphism, real-time PCR and pulsed-field gel electrophoresis.

A total of 17 Leptospira clinical strains isolated from humans in Croatia were serologically and genetically analysed. For serovar identification, the microscopic agglutination test (MAT) and pulsed-field gel electrophoresis (PFGE) were used. To identify isolates on genomic species level, PCR-based restriction fragment length polymorphism (RFLP) and real-time PCR were performed. MAT revealed the following serogroup affinities: Grippotyphosa (seven isolates), Icterohaemorrhagiae (eight isolates) and Javanica (two isolates). RFLP of PCR products from a 331-bp-long fragment of rrs (16S rRNA gene) digested with endonucleases MnlI and DdeI and real-time PCR revealed three Leptospira genomic species. Grippotyphosa isolates belonged to Leptospira kirschneri, Icterohaemorrhagiae isolates to Leptospira interrogans and Javanica isolates to Leptospira borgpetersenii. Genomic DNA from 17 leptospiral isolates was digested with NotI and SgrAI restriction enzymes and analysed by PFGE. Results showed that seven isolates have the same binding pattern to serovar Grippotyphosa, eight isolates to serovar Icterohaemorrhagiae and two isolates to serovar Poi. Results demonstrate the diversity of leptospires circulating in Croatia. We point out the usefulness of a combination of PFGE, RFLP and real-time PCR as appropriate molecular methods in molecular analysis of leptospires.

Multilocus sequence analysis of atypical Borrelia burgdorferi sensu lato isolates--description of Borrelia californiensis sp. nov., and genomospecies 1 and 2.

Taxonomy of Borrelia burgdorferi sensu lato (s.l.) was recently improved by the use of multilocus sequence analysis (MLSA), a new approach to replace the cumbersome DNA-DNA hybridization method [Richter et al., 2006. Int. J. Syst. Evol. Microbiol. 156, 873-881]. In this study, we used this methodology to classify B. burgdorferi s.l. strains isolated both in Europe and the United States, the exact taxonomic status of which remained unclear. We conclude that MLSA can surpass the discrimination power of whole DNA-DNA hybridization, and we delineate three new North American B. burgdorferi s.l. species. In contrast, European atypical strains constituted a subgroup of B. burgdorferi sensu stricto (s.s.).

Reservoir role of lizard Psammodromus algirus in transmission cycle of Borrelia burgdorferi sensu lato (Spirochaetaceae) in Tunisia.

To investigate the reservoir role of the lizard Psammodromus algirus for the Lyme disease spirochete, 199 lizards were trapped from April to October 2003 in El Jouza, northwestern Tunisia. In this site, the infection rate of free-living Ixodes ricinus (L.) by Borrelia was evaluated by immunofluorescence as 34.6% for adult ticks and 12.5% for nymphs. Eighty percent of P. algirus (117/146) captured during this study were infested by I. ricinus, the predominant tick species collected from lizards. The intensity of tick infestation of this host by larvae and nymphs ranged from 0.14 to 7.07 and from 1.5 to 6.58, respectively. These immature stages of I. ricinus were found on lizards in spring and the beginning of summer, with a peak of intensity during June (10.16 immature ticks by lizard). Tissue cultures from lizards and xenodiagnosis with larval I. ricinus were used to assess the infection and the ability, respectively, of infected lizards to transmit Borrelia to naive ticks. Seventeen percent of xenodiagnostic ticks (40/229) acquired B. lusitaniae while feeding on P. algirus. Therefore, we demonstrated the ability of the lizards to sustain Borrelia infection and to infect attached ticks, and we proved that P. algirus is a reservoir host competent to transmit B. lusitaniae.

A single-run, real-time PCR for detection and identification of Borrelia burgdorferi sensu lato species, based on the hbb gene sequence.

Lyme borreliosis is the most important vector-borne disease caused by spirochetes within the Borrelia burgdorferi sensu lato (B. burgdorferi sl) complex. There is strong evidence that different species of this group of genetically diverse spirochetes are involved in distinct clinical manifestations of the disease. In order to differentiate species within this bacterial complex, we developed a real-time-PCR protocol, which targets the hbb gene. We designed a fluorescein-labeled probe specific of a region of this gene harboring a polymorphism linked to species. An internally Red640 labeled primer allowed a fluorescence resonance energy transfer to occur. The sensitivity of this method was in the range of 10 bacteria per assay. After amplification, a melting curve was generated for genotyping. Analysis of these melting curves clearly allowed the distinction between the main European species of B. burgdorferi sl. One hundred seventy tick extracts were analysed by this hbb-based method and in parallel by amplification of the 5S-23S intergenic spacer and RFLP analyses. There was a good correlation between these two methods. We conclude that this hbb-based real-time-PCR is suitable for epidemiological studies on field-collected ticks, although rare mutations in the genomic sequence spanned by the probe could lead to misidentification.

Delineation of Borrelia burgdorferi sensu lato species by multilocus sequence analysis and confirmation of the delineation of Borrelia spielmanii sp. nov.

To evaluate multilocus sequence analysis (MLSA) for taxonomic purposes in the delineation of species within Borrelia burgdorferi sensu lato, seven relevant loci of various strains for which extensive DNA-DNA reassociation data were available were sequenced. MLSA delineation proved to be fully concordant with conventional methods. Our analysis confirmed the delineation of a novel species, Borrelia spielmanii sp. nov., previously known as 'Borrelia spielmani' Richter et al. 2004, with strain PC-Eq17N5T (=DSM 16813T = CIP 108855T) as the type strain.

Prevalence of Borrelia burgdorferi sensu lato and Anaplasmataceae members in Ixodes ricinus ticks in Alsace, a focus of Lyme borreliosis endemicity in France.

Due to the high Lyme borreliosis incidence in Alsace, in northeastern France, we investigated in 2003-2004 three cantons in this region in order to determine the density of Ixodes ricinus ticks infected by Borrelia burgdorferi sensu lato and Anaplasmataceae. The peak density of nymphs infected by B. burgdorferi sensu lato at Munster and Guebwiller, where the disease incidence was high, was among the highest reported in Europe (105 and 114 per 100 m(2), respectively). In contrast, the peak density of infected nymphs was low in the canton of Dannemarie (5/100 m(2)), where the disease incidence was low. The two main species detected in ticks were Borrelia afzelii, more frequent in nymphs, and Borrelia garinii, more frequent in adult ticks. The rates of tick infection by Anaplasma phagocytophilum were 0.4% and 1.2% in nymphs and adults, respectively.

Short report: dual infections with Puumala virus and Leptospira interrogans serovar lora in a bank vole (Clethrionomys glareolus).

Leptospirosis and hemorrhagic fever with renal syndrome are public health problems in Croatia. Diagnosis and epidemiology of these diseases are complicated because these two diseases are sympatric in certain areas. We describe a natural dual infection of Puumala virus and a leptospire in a bank vole (Clethrionomys glareolus).

Borrelia burgdorferi sensu stricto invasiveness is correlated with OspC-plasminogen affinity.

Borrelia burgdorferi sensu lato, the causative agent of Lyme borreliosis, is transmitted through tick bite. Lyme borreliosis evolves in two stages: a primary red skin lesion called erythema migrans; later on, invasive bacteria disseminate to distant sites inducing secondary manifestations (neuropathies, arthritis, carditis, late skin disorders). It has been previously suggested that the ospC gene could be associated with invasiveness in humans depending on its sequence. Here, we confirm the pattern of invasiveness, according to B. burgdorferi sensu stricto (B. b. ss) ospC group, using the mouse as an experimental host of B. b. ss. As it has been shown that the host plasminogen activation system is used by B. burgdorferi to disseminate throughout the host, we studied the interaction of plasminogen with OspC proteins from invasive and non-invasive groups of B. b. ss. Using two methods, ELISA and surface plasmon resonance, we demonstrate that indeed OspC is a plasminogen-binding protein. Moreover, significant differences in binding affinity for plasminogen are correlated with different invasiveness patterns in mice. These results suggest that the correlation between ospC polymorphism and Borrelia invasiveness in humans is linked, at least in part, to differences in OspC affinity for plasminogen.

Trends in leptospirosis epidemiology in France. Sixty-six years of passive serological surveillance from 1920 to 2003.

OBJECTIVES: The incidence of leptospirosis, a worldwide zoonosis, has significantly decreased in Western Europe during the last decades. Our aim was to analyse all reliable data collected at the Institut Pasteur from 1920 to 2003 to evaluate more precisely the evolution of the incidence of leptospirosis in France. METHODS: The passive surveillance system used as early as 1920 allows the evaluation of leptospirosis incidence. Serological results inferred from the microagglutination procedure were used to evaluate variation in the incidence of the disease and the evolution of the Leptospira serogroups involved in the human disease. RESULTS: No significant variation either in the number of leptospirosis cases or in the incidence of the disease was observed. However, the period of the 1970s was characterized by a rather low incidence. The weather plays a major role by modifying fresh water abundance, rodent populations and human behaviour. However the weather's influence is not the sole factor involved in the incidence rate. No cyclic variation was evident. CONCLUSION: Although France has the highest incidence of leptospirosis in Europe, the analysis of serological data collected over 66 years has allowed us to conclude that in France, the incidence is slowly decreasing.

Characterization of Borrelia lusitaniae isolates collected in Tunisia and Morocco.

Borrelia lusitaniae is a species within the complex Borrelia burgdorferi sensu lato and is infrequently isolated in Europe. In contrast, this species is by far the most predominant in North Africa and in Portugal. In this study, we analyzed the genetic diversity, at several loci, of a large population of isolates from free-living Ixodes ricinus ticks collected in Tunisia and Morocco. We found a moderate diversity of the whole genome by using pulsed-field gel electrophoresis as well as in the ospA gene sequences, compared to a high level of strain homogeneity in the small noncoding ribosomal spacer. In contrast, a high diversity of this locus has been previously reported for Portuguese isolates. We hypothesize that B. lusitaniae strains isolated in North Africa constitute a clone of Portuguese origin.

Detection and identification of Ehrlichia spp. in ticks collected in Tunisia and Morocco.

A broad-range 16S rRNA gene PCR assay followed by partial sequencing of the 16S rRNA gene was used for the detection of members of the family Anaplasmataceae in ticks in North Africa. A total of 418 questing Ixodes ricinus ticks collected in Tunisia and Morocco, as well as 188 Rhipicephalus ticks from dogs and 52 Hyalomma ticks from bovines in Tunisia, were included in this study. Of 324 adult I. ricinus ticks, 16.3% were positive for Ehrlichia spp., whereas only 3.4 and 2.8% of nymphs and larvae, respectively, were positive. A large heterogeneity was observed in the nucleotide sequences. Partial sequences identical to that of the agent of human granulocytic ehrlichiosis (HGE) were detected in I. ricinus and Hyalomma detritum, whereas partial sequences identical to that of Anaplasma platys were detected in Rhipicephalus sanguineus. However, variants of Anaplasma, provisionally designated Anaplasma-like, were predominant in the I. ricinus tick population in Maghreb. Otherwise, two variants of the genus Ehrlichia were detected in I. ricinus and H. detritum. Surprisingly, a variant of Wolbachia pipientis was evidenced from I. ricinus in Morocco. These results emphasized the potential risk of tick bites for human and animal populations in North Africa.

Risk factors for leptospirosis in metropolitan France: results of a national case-control study, 1999-2000.

Risk factors for leptospirosis in France were investigated to improve the vaccination program for this disease. Data from 90 hospitalized case patients and 169 matched control subjects were analyzed in a case-control study. Skin lesions, canoeing, contact with wild rodents, and country residence were independently associated with leptospirosis, emphasizing that leisure activity is a risk factor for this illness.

Genetic diversity among Borrelia strains determined by single-strand conformation polymorphism analysis of the ospC gene and its association with invasiveness.

Lyme borreliosis (LB) is a tick-borne spirochetal infection caused by three Borrelia species: Borrelia afzelii, B. garinii, and B. burgdorferi sensu stricto. LB evolves in two stages: a skin lesion called erythema migrans and later, different disseminated forms (articular, neurological, cardiac.). Previous research based on analysis of ospC sequences allowed the definition of 58 groups (divergence of <2% within a group and >8% between groups). Only 10 of these groups include all of the strains isolated from disseminated forms that are considered invasive. The aim of this study was to determine whether or not invasive strains belong to restricted ospC groups by testing human clinical strains isolated from disseminated forms. To screen for ospC genetic diversity, we used single-strand conformation polymorphism (SSCP) analysis. Previously known ospC sequences from 44 different strains were first tested, revealing that each ospC group had a characteristic SSCP pattern. Therefore, we studied 80 disseminated-form isolates whose ospC sequences were unknown. Of these, 28 (35%) belonged to previously known invasive groups. Moreover, new invasive groups were identified: six of B. afzelii, seven of B. garinii, and one of B. burgdorferi sensu stricto. This study confirmed that invasive strains are not distributed among all 69 ospC groups but belong to only 24 groups. This suggests that OspC may be involved in the invasiveness of B. burgdorferi.

First isolation of Borrelia burgdorferi sensu lato from Ixodes ricinus ticks in Morocco.

To determine the infection rate of Ixodes ricinus (I. ricinus) ticks with Borrelia burgdorferi sensu lato (B. burgdorferi sl) and to assess the frequency of the individual Borrelia species in this tick species, a total of 295 I. ricinus were collected in Taza region (Northeast of Morocco), from January to June 2002. The presence of B. burgdorferi sl was determined by direct fluorescence antibody assay (DFA) and by PCR after culture. B. burgdorferi sl isolates were identified at the species level by restriction fragment length polymorphism analysis of amplified products. The mean rate of I. ricinus infection with B. burgdorferi sl was 47.8%. Isolation attempts in BSK II medium resulted in 26 pure isolates. However, PCR performed on culture medium allowed to identify 82 Borrelia DNAs. B. lusitaniae has been identified from 76 out of 82 infected I. ricinus ticks (92.7%). Three ticks were infected by B. burgdorferi ss, and three other ticks were infected by B. garinii. This is the first report of the presence of B. burgdorferi sl in Morocco and more specifically of B. burgdorferi ss in North Africa.

Molecular diversity of the ospC gene in Borrelia. Impact on phylogeny, epidemiology and pathology.

Lyme borreliosis is a 2 steps disease: i) Localized erythema migrans ii) occasionally a disseminated disease. Three out of the 10 up to now described Borrelia species are pathogenic for man and each of them exhibits its own organotropism: joints for Borrelia burgdorferi sensu stricto (B.b. ss), nervous system for B. garinii, skin for B. afzelii, ospc gene is subject to lateral transfer leading to a huge diversity among corresponding encoded proteins. This allows the spirochete to develop a repertoire of epitopes to escape the host immune response. We noticed that the European endemic ospc repertoire is only a subset of the American one. This bottleneck situation transduces a "founder's event" suggesting B.b. ss has been imported from North America to Europe at historical times. Another valuable observation is the fact that isolates from disseminated forms (called "invasive") of the disease, all are distributed in only ten out of the 70 ospc genotypes. The conclusion is that in human, some OspC conformations are associated with the invasive potential of a given Borrelia isolate.

Human infection caused by Leptospira fainei.

We report a human case of leptospirosis in which the spirochete was detected by dark-field microscopy examination of cerebrospinal fluid (CSF) and isolated from both CSF and blood. Leptospira fainei was identified by sequencing the 16S rDNA gene, which had been amplified by polymerase chain reaction. This case confirms the role of L. fainei as a human pathogen and extends its distribution to southern Europe.