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.

Retrospective review of leptospirosis in Guadeloupe, French West Indies 1994-2001.

Demographic, clinical, biological and personal data were obtained from patients hospitalized with symptoms of leptospirosis in the Hospital of Pointe a Pitre, Guadeloupe, French West Indies from 1994 to 2001. Of the 897 screened patients, 212 were acute cases, 607 were non-infected and 78 were undetermined cases. There was no predominant age group. Leptospirosis transmission followed the rainfall cycle and was greater in rural areas. Jaundice and conjunctival suffusion were significantly more frequent in cases than non-cases. Males, professions considered to be at risk and contact with swine or bovine were associated with infection. Serogroups Icterohaemorrhagiae, Cynopteri, Australis, Sejroe, Pomona and Ballum were serovars presumed responsible for acute cases.

Diversity of ribosomal DNA fingerprints of Leptospira serovars provides a database for subtyping and species assignation.

Ribosomal DNA fingerprints from 103 pathogenic Leptospira strains were examined using EcoRI restriction and fragment length polymorphisms of rRNA genes. Sixty-nine new leptospiral ribotypes were described, in addition to 49 previously observed. Except for 5 strains, a good correlation between DNA homology data and ribotyping was observed. The genospecies of 31 reference strains could be presumed, since they shared 13 ribotypes with strain(s) previously studied by DNA homology. Furthermore, the definition of common rRNA hybridization fragments in each recognized DNA hybridization group provides information about the status of Leptospira reference strains yet to be classified. With 118 ribotypes now defined among the validated serovar reference strains, rRNA fingerprints constitute a database for subtyping Leptospira species.

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.

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.

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.

A new genomic species in Borrelia burgdorferi sensu lato isolated from Japanese ticks.

Five Borrelia strains (Ika2, HO14, Cow611C, 0612 and F63B) isolated from Ixodes ovatus ticks in Japan were analysed by DNA-DNA hybridization experiments, ribotyping, pulsed-field gel electrophoresis and protein electrophoresis. DNA relatedness set these strains in a new genomic species within the Borrelia burgdorferi complex; this species appears to be restricted to Japan and could be non-pathogenic for humans. The ribotype and pulsotype of strain Ika2 were atypical of the new genomic species.

Borrelia burgdorferi sensu lato in Russia and neighbouring countries: high incidence of mixed isolates.

A total of 365 isolates of Borrelia burgdorferi sensu lato from 12 major administrative territories of Russia (from St. Petersburg in the west to South Sakhalin in the east) and from the Czech Republic, Estonia, Lithuania, Byelorussia, Moldavia, Ukraine and Kirghizia were identified by analysis of restriction polymorphism of ribosomal rrf-rrl spacer amplicons. The isolates were obtained mainly from ixodes persulcatus and I. ricinus ticks. Other sources included small mammals, human patients and I. trianguliceps ticks. The results showed that B. garinii (two variants) together with B. afzelii circulated throughout the territories studied. The distribution of the variant NT29 of the species B. garinii, the most frequently isolated, was associated with that of I. persulcatus ticks. B. burgdorferi sensu stricto, and the species B. valaisiana and B. lusitaniae (formerly the genomospecies VS116 and PotiB2, respectively) were isolated only from I. ricinus ticks in the western part of the studied territories. None of these three species were found in 327 isolates from Russia where I. persulcatus is the most frequently distributed vector. This work also provides evidence for a high incidence of mixed Borrelia infections within vectors and hosts (9.3% of isolates were mixtures of Borrelia species). A detailed analysis of Borrelia species distribution over the territories studied is presented.

Two genomic species in Borrelia burgdorferi.

A total of 13 Borrelia burgdorferi strains (responsible for Lyme borreliosis) and representatives of 3 other Borrelia species (B. hermsii, B. parkeri, B. turicatae) associated with relapsing fever were studied by DNA/DNA hybridization and rRNA gene-restriction patterns. Two genomic DNA hybridization groups were observed which could be differentiated by rRNA gene-restriction patterns. Moreover, the number and size of restriction fragments suggest the existence of a single set of 16 and 23 S rRNA genes in Borrelia.

Species of Borrelia distinguished by restriction site polymorphisms in 16S rRNA genes.

Three phyletic groups of Borrelia associated with Lyme disease, B. burgdorferi, B. garinii and group VS461 can be distinguished from each other and other species of Borrelia by BfaI restriction site polymorphisms in PCR amplified 16S rRNA genes. One strain isolated from an Ixodes pacificus tick in California that was previously unclassifiable was distinguishable from B. burgdorferi by an Mn/I restriction site polymorphism.

Individualisation of two new genomic groups among American Borrelia burgdorferi sensu lato strains.

We developed a quick typing method for Borrelia burgdorferi sensu lato strains using a fla gene-based PCR assay, followed by dot blot hybridization with non-radioactive species-specific probes. Thirty-six out of 46 strains belonged to one of the four described species (B. burgdorferi sensu stricto n = 11, B. garinii n = 11, B. afzelii n = 9 and B. japonica n = 5) and hybridized with its own species-specific probe. Among the 10 remaining American strains, two new additional genomic groups were identified. This finding was confirmed by direct sequencing of the fla gene-derived amplicons and whole DNA hybridization.

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.

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.

Infection of Ixodes ricinus (Acari:Ixodidae) by Borrelia burgdorferi in Ile de France.

Free-living nymphs and adults from Rambouillet and Fontainebleau, 2 major forests of Ile de France, were collected to determine the infection rates of Ixodes ricinus L. by Borrelia burgdorferi (Johnson, Schmid, Hyde, Steigerwalt & Brenner). Field-collected I. ricinus nymphs and adults were screened for the presence of B. burgdorferi using direct fluorescent antibody assay. The infection rates of nymph, male and female were 12.4% (314), 2.8% (35), and 2.9% (34). No difference in infection rates of nymphs among sites were detected. Spirochetes was isolated from both sites. Isolates from Rambouillet and Fontainebleau were identified as B. burgdorferi sensu stricto and B. garinii, respectively (Baranton, Postic, Saint-Girons, Boerlin, Piffaretti, Assous, and Grimont).

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%).

Molecular epidemiology of the aetiological agents of Lyme borreliosis.

Ten species are up to now recognized among Borrelia burgdorferi sensu lato complex. Among those, only three (Borrelia burgdorferi sensu stricto, B. garinii and B. afzelii) are reported to be pathogenic for humans and each responsible for a predominant clinical form of Lyme borreliosis. Each species is characterized by its vectors (Ixodidae), its host spectrum, its organotropism (for the pathogenic ones) and its geographical repartition. Borrelia are strictly parasitic and essentially clonal bacteria. Our goal was to explore the diversity of this bacterial complex. We selected, by several molecular markers, atypical isolates and compared them to already known species representative strains by RFLP or sequencing. The results show an unexpected diversity at a level which could be a species one leading to the conclusion that the structure of the Borrelia burgdorferi sensu lato complex is a high number of small (by their populations) clones among which emerge some large ones usually corresponding to the pathogenic species. Our data also allow to speculate on when, where and how these species evolved and migrated.

First isolation and characterisation of Borrelia garinii, agent of Lyme borreliosis, from Irish ticks.

Nymphal Ixodes ricinus, the tick vector of Lyme borreliosis, were collected from the edges of paths in Muckross Demesne, Killarney National Park, Co. Kerry, Ireland. Examination of some of these nymphs by indirect immunofluorescence showed an infection prevalence of 12% with Borrelia burgdorferi sensu lato, the spirochaete agent of Lyme borreliosis. Gerbils (Meriones unguiculatus) were infected by infesting them with other nymphs from the same batch. Subsequently uninfected laboratory larvae were applied to the gerbils and the contents of the resulting infected engorged ticks were then placed in media and the spirochaetes cultured. The spirochaetes were identified as B. burgdorferi sensu lato by indirect immunofluorescence using monoclonal antibodies and they were further characterised by polymerase chain reaction and pulsed-field gel electrophoresis. Both of these latter techniques showed that spirochaetes in all samples belonged to the genomic species, Borrelia garinii.

[First isolation of Borrelia afzelii in France (Marne) from Ixodes ricinus]. / Premier isolement en France (Marne) de Borrelia afzelii à partir d'Ixodes ricinus.

A strain of Borrelia has been isolated from Ixodes ricinus in France (Marne). The DNA analysis by PCR showed that it is identical to B. afzelii.

[Research of Lyme's disease in facial paralysis. A French multicenter study]. / Recherche de maladie de Lyme dans les paralysies faciales. Une étude multicentrique Française.

OBJECTIVES: Apparent a frigore facial palsy could possibly mask manifestations of unrecognized Lyme's disease. Since commonly used corticosteroid treatment could be deleterious if Borrelia burgdorferi infection was indeed the cause, we conducted a prospective study to search for possible infections in cases of recently diagnosed a frigore facial palsy. METHODS: For 3 years, 1990-1992, 49 French centres diagnosed a facial palsy in 346 patients (310 adults, 36 children under 15 years of age; mean age 38; range 16 months to 83 years). The patients were divided into three groups: a) facial palsy alone, b) zoster origin recognized due to outer ear eruption and c) cases with meningoradiculitis or joint signs or cases with facial diplegia. A questionnaire was used to evaluate exposure to risk of tick bits. A control group was established with 246 serum samples from subjects matched with the patients for age, geographical origin and exposure to risk of tick bits. Laboratory tests (indirect immunofluorescence and Western blot) were performed to search for anti-Borrelia burgdorferi antibodies in serum samples, and cerebral spinal fluid when possible, collected at presentation, on day 30 and on day 90. RESULTS: Sixty percent of the patients were urban dwellers, 15% lived in rural and 25% in semi-rural areas. There were 294 patients with facial palsy alone and their serum results were compared with those of the matched controls. There was no significant difference in the positivity for Borrelia burgdorferi antibodies between these two groups. CONCLUSION: These findings indicate that, unless there are clinical signs suggestive of borreliosis, it would not be necessary to test for Lyme's disease in patients with apparent a frigore facial palsy.

[Three bacterial species associated with Lyme borreliosis. CLinical and diagnostic implications]. / Trois espèces bactériennes associées à la borréliose de Lyme. Conséquences cliniques et diagnostiques.

DNAs from various Borrelia associated with Lyme disease were reciprocally hybridized. This genomic taxonomy method showed that Lyme disease agent comprised three genomic species. Two species could be differentiated by phenotypic characters as major proteins molecular weights and monoclonal antibodies reactivity. Western-blot with sera from patients suffering from different clinical forms of Lyme disease: arthritis, meningoradiculitis and Acrodermatitis Chronicum Atrophicans showed that each of these evolutive forms was preferentially associated with one of the species, respectively: Borrelia burgdorferi sensu stricto, Borrelia garinii and Borrelia group VS 461. Furthermore, geographical repartition of these three species was heterogeneous. B. burgdorferi sensu stricto seems to be the only one present in the United States of America, whereas it coexists in Europe mainly with B. garinii in Western Europe and Borrelia group VS 461 in Northern Europe.