Occurence of pathogenic genospecies of Borrelia burgdorferi sensu lato in Ixodes ricinus ticks collected from north-western Poland

In order to learn the heterogeneity of the DNA of B. burgdorferi s.l. and the prevalence of co-infections of B. burgdorferi s.l. genospecies in the populations of I. ricinus, collected in north-western Poland, the nested PCR method was applied, a fragment of the fla gene being used as a marker. Basing on the prevalence data of B. burgdorferi s.l. DNA in I. ricinus ticks in 8 sampling sites during 1998-2001, it may be stated that a risk of contracting Lyme disease exists in forested areas of north-western Poland, the highest in relation to B. burgdorferi s.s. (76.3% infected ticks), lower by B. garinii (2% infected ticks), and minimal threat being posed by B. afzelii (0.3%). I. ricinus ticks collected in north-western Poland pose a risk of contracting double infection by B. burgdorferi s.l. genospecies, i.e. B. burgdorferi s.s. with B. garinii, and B. burgdorferi s.s. with B. afzelii. The north-western part of Poland represents an endemic area for B. burgdorferi s.l.     

Genetic diversity of Borrelia burgdorferi sensu lato in Ixodes ricinus ticks collected in north-west Poland

Borrelia burgdorferi sensu lato (s. l.), the etiological agent of Lyme diesease, is transmitted by the bite of Ixodes ricinus. During May and September 1999, field surveys on Lyme disease spirochetes were conducted in three locations of a region of north-west Poland, known as recreational districts visited by many people. The ticks Ixodes ricinus were collected in natural habitats by dragging a flanel cloth over the vegetation. Sex and developmental stage of each tick were determined. Based on a polymerase chain reaction test with primers that recognize a chromosomal gene of all strains, out of the total 1414 specimens collected, 126 (8.9%) were found to be infected. The species B. burgdorferi s. l. comprises at least three pathogenic genomospecies, B. burgdorferi sensu stricto (s. s.), Borrelia garinii, and Borerelia afzelii, witch could be distinguished in nested-PCR tests with species-specific primers. B. burgdorferi s. s. was most prevalent (96% of infected ticks), followed by B. garinii (1.3%), and B. afzelii. was not found. Of the infected ticks, over the 99% were infected with a single species, one specimens was infected with two species. For 4 ticks, the infecting species could not be identied. The difference in rates of prevalence was observed among the tree locations (17%--5.3%--3.2%).     

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.     

Isolation of Borrelia burgdorferi sensu lato in ticks Ixodes ricinus by polymerase chain reaction (PCR)

Attempts were made to identify the causative orgamsm of Lyme disease in Szczecin from tick Ixodes ricinus as a vector. Ticks were collected in 1997 year in forest areas of Szczecin, from localites associated with numerous attendance of people. The method used in this study was the polymerase chain reaction (PCR) on the flagellin structural gene fla of Borrelia burgdorferi sensu stricto. The flagellin PCR primer set reaction was conservative for B. burgdorferi sensu stricto, B. afzelii and B. garinii. The overall prevalence of B. burgdorferi sensu lato, in tick population studied was 8.8%. The female, nymphs and larves of Ixodes ricinus were infected almost just the some--about 10%, when the male 2.5% only.     

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-D?me. 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-D?me 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-D?me and from 0.8% to 1.4% in Meuse, suggesting a low risk for humans.     

Prevalence and genetic heterogeneity of Borrelia burgdorferi sensu lato in Ixodes ticks in Belgium

Borrelia burgdorferi sensu lato is a genetically diverse group of spirochetes that includes the agent of Lyme borreliosis in which genospecies tend to be associated with specific clinical features. The aim of the study was to determine the prevalence and genetic diversity of Borrelia burgdorferi sensu lato in 524 ticks collected in woods of a western province of Belgium. Presence of spirochetes in ticks was determined by phase contrast microscopy. The mean infection rate of ticks was 12.0%. Variability was observed in the prevalence of infection among the five sites examined, ranging from 2.8 to 21.6%. Identification to genospecies was determined by PCR and sequencing. The most common genomospecies were Borrelia afzelii (55%) and Borrelia garinii (21%). For the first time in Belgium, we detected Borrelia valaisiana and Borrelia spielmanii, representing 14% and 2%, respectively. Borrelia burgdorferisensu stricto counted only for 2%. Co-infections were present in 8% of ticks. We emphasize the need for clinical studies to assess the prevalence of specific genospecies-related clinical manifestations of Lyme borreliosis in Belgium.     

Prevalence of Borrelia burgdorferi sensu lato in Ixodes ricinus population in forests of north-western Poland

The ticks Ixodes ricinus were collected during three growing seasons in forested areas of north-western Poland. The ticks were collected by flagging at 30 sites. The specimens collected (nymphs and adults) were subjected, after an appropiate treatment, to indirect immunofluorescence analysis (IFA). In addition, effects of temperature and atmospheric precipitation on the tick infection with the spirochaete Borrelia burgdorferi were investigated based on data supplied by the Institute of Meteorology. A total of 8519 individuals of I. ricinus were collected at the sampling sites within May-October of three consecutive years. The pool of individuals obtained consisted of 7356 nymphs, 559 females, and 604 males. The B. burgdorferri prevalence varied rather widely, from 10 to 19%. Compared to nymphs, adult individuals showed a higher infection rate. A relationship between infection rate and I. ricinus activity in different years and months was studied in detail. The boreliosis risk was found to increase with increasing tick activity. On the other hand, no correlation was found between the meteorological parameters (temperature, precipitation), tick abundance, and the B. burgdorferi infection rate.     

我国部分地区蜱中莱姆病螺旋体的检测与基因分型研究

目的 对我国部分地区的多种蜱类进行莱姆病螺旋体的检测和基因分型。方法 选择我国黑龙江、吉林和浙江省部分林区为调查点，采集当地蜱类，用巢式PCR法进行检测，阳性产物进行限制性片段长度多态性（RFLP）分析和单链构象多态性（SSCP）分析，确定莱姆病螺旋体的基因型。结果 共检测蜱512只，阳性126只，阳性率24．61％。其中吉林全沟硬蜱带菌率为37．00％，黑龙江全沟硬蜱带菌率为20．87％，浙江长角血蜱带菌率为28．07％。RFLP分析表明，蜱中莱姆病螺旋体包括B．garinii和B．afzelii两种基因型。SSCP分析显示为7种亚型，其中B．garinii分为5个亚型，B．afzelii分为2个亚型。发现有3只蜱同时感染不同基因（亚）型莱姆病螺旋体。结论 证实B．garinii和B．afzelii基因型为我国莱姆病螺旋体的优势基因型，并在我国蜱中发现莱姆病螺旋体不同基因（亚）型的混合感染。     

A comparison of two DNA extraction approaches in the detection of Borrelia burgdorferi sensu lato from live Ixodes ricinus ticks by PCR and reverse line blotting

We tested two approaches to extract Borrelia DNA from live Ixodes ricinus ticks before polymerase chain reaction (PCR) and reverse line blotting (RLB): DNA extraction of one half of the tick after incubation in BSK medium and DNA extraction of the other half of the tick directly, using ammonium hydroxide. Among 2079 ticks, 31.2% (n=649) were found to be Borrelia-infected by PCR-RLB test using at least one of the DNA extraction methods. Five hundred four ticks (24.2%) were found infected after incubation in BSK and 481 (23.1%) after direct DNA extraction from the tick. The difference was not significant. However, these prevalences were significantly lower when only one method was applied (23.1% and 24.2%) compared to the prevalence obtained by the use of both methods (31.2%). In 313 infected ticks discordant results were obtained, i.e., one half of the tick was found to be infected whereas the other half was uninfected. Among these ticks, B. garinii and B. burgdorferi sensu stricto (ss) were significantly more frequently identified in the half tick incubated in BSK. No significant differences were observed for B. burgdorferi ss, B. valaisiana, and for undetermined Borrelia species.     

Density of questing Ixodes ricinus nymphs and adults infected by Borrelia burgdorferi sensu lato in Switzerland: spatio-temporal pattern at a regional scale

Lyme borreliosis, the most important vector-borne disease in the Northern hemisphere, causes health problem for populations in endemic areas. In the present study, the density of questing Ixodes ricinus ticks and their infection with Borrelia burgdorferi sensu lato (sl) was examined in 11 areas located on the Swiss Plateau and in an alpine valley. From 1999 to 2001, free-living I. ricinus ticks were collected on a monthly basis by flagging vegetation in these areas. Each tick was examined for the presence of B. burgdorferi sl using direct fluorescent antibody assay, and for isolation of the bacteria. Borreliae were characterized by PCR followed by RFLP. Density of questing ticks varied greatly between studied areas. Borreliae were observed in ticks collected in all investigated sites. However, the prevalence of infection differed significantly among areas. Infection prevalence varied from 9% to 40% in nymphs and from 22% to 47% in adults. Adult ticks were significantly more infected (129/366, 35%) than nymphs (109/552, 20%). There was no correlation between nymphal density and infection prevalence as well as between adult density and infection prevalence, but there was a correlation between density of ticks and density of infected ticks. During the spring peak of questing tick density, a range of 2-30.3 infected ticks per 100 m(2) was observed. B. burgdorferi sl isolates (n = 129) were obtained from ticks collected in 10/11 areas. Five Borrelia species were identified: B. garinii, B. burgdorferi sensu stricto, B. afzelii, B. valaisiana, B. lusitaniae, and six mixed infections were also obtained. Borrelia species were heterogeneously distributed in the different areas.     

Presence of Borrelia burgdorferi sensu lato in mites parasitizing small rodents

Ticks (especially those in the family Ixodidae) are the primary vectors of the infectious agent Borrelia burgdorferi sensu lato, which causes Lyme disease. To determine the potential role of mites as vectors of B. burgdorferi sensu lato, mites were collected from wild rodents in the Bazantula region of North Moravia (Czech Republic). These samples were examined for the presence of Borreliae by using DFM and PCR methods. Mites positive for the presence of DNA of B. burgdorferi sensu lato were determined as members of the families Haemogamasidae and Parasitidae. One sample from a mite of genus Haemogamasus was successfully isolated, and the specimen was confirmed as B. afzelii by using PCR-RFLP and by gradient SDS-PAGE. This suggests the possible participation of gamasid mites in borrelial circulation in nature and also points to the utility of further such studies to identify potential vectors (other than ticks) of the spirochete.     

Investigations on the mode and dynamics of transmission and infectivity of Borrelia burgdorferi sensu stricto and Borrelia afzelii in Ixodes ricinus ticks

Borrelia burgdorferi sensu lato (sl), the agent of Lyme disease, is transmitted to the host during the blood meal of Ixodes ticks. In most unfed ticks, spirochetes are present in the midgut and migrate during blood feeding to the salivary glands, from which they are transmitted to the host via saliva. In the present study, the efficiency of Ixodes ricinus ticks to transmit B. afzelii and B. burgdorferi sensu stricto (ss) and their infectivity for mice were examined in relation to the duration of the blood meal. In addition, we investigated whether these two Borrelia species can penetrate intact skin. Three modes of infection of mice were studied: tick-bite infection, inoculation of tick homogenates, and transcutaneous infection by topical application of tick homogenates on mouse skin. Transmission of B. burgdorferi sl from I. ricinus nymphs to mouse increased with duration of tick attachment. B. afzelii-infected ticks start to transmit infection earlier (< or = 48 h) than B. burgdorferi ss-infected ticks. As previously shown for B. burgdorferi ss in Ixodes scapularis, B. burgdorferi ss and B. afzelii in unfed I. ricinus were noninfectious for mice when tick homogenates were inoculated. However, the inoculation of homogenates of ticks fed for 24 h readily produced infection in mice. Therefore, B. burgdorferi ss and B. afzelii spirochetes are potentially infectious in the tick before natural transmission can occur. None of the mice (n = 33) became infected by transcutaneous transmission when tick homogenates were applied on mouse skin, showing that B. burgdorferi ss and B. afzelii are unable to penetrate intact skin, in contrast to relapsing fever spirochetes. This study also shows that B. afzelii is transmitted by I. ricinus to the host earlier than B. burgdorferi ss and that I. ricinus seems to be a more efficient vector of B. afzelii than B. burgdorferi ss.     

Evaluation of occurrence of spirochetes Borrelia burgdorferi sensu lato in Ixodes ricinus ticks in selected areas of the Lublin region by polymerase chain reaction method (PCR)

During the period 2001-2002, 1098 Ixodes ricinus ticks were collected at forest sampling sites and the degree of their infection with Borrelia burgdorferi spirochetes was determined by means of polimerase chain reaction (PCR). The presence of Borrelia burgdorferi genetic material was noted in 69 cases (6.3%). It was confirmed that the frequency of infection of adult forms of ticks (males and females) was nearly twice as high as nymphs. The highest degree of infection was observed in females--9.5%. The degree of infection among males and nymphs was smaller--5.9% and 4.4% respectively in individual provinces. The percentage of infected females ranged from 7.9% in the Zamo?? Province to 13.6% in the W?odawa Province. In males, the percentage of infected ticks remained within the range from 3.1% in the Lublin Province to 13.3% in the Lubartów Province.     

Genetic diversity of Borrelia burgdorferi sensu lato isolates from Northeastern China

Thirty-two strains of Borrelia burgdorferi sensu lato were isolated from Ixodes persulcatus ticks collected from northeastern China from May to June in 2004 and 2005. Restriction fragment length polymorphism (RFLP) analysis and sequence analysis of 5S-23S rRNA intergenic spacer revealed that 29 (90.6%) belonged to Borrelia garinii, demonstrating B, C, and a unique pattern. The remaining three isolates (9.4%) were Borrelia afzelii with pattern D. The phylogenetic analysis based on 5S-23S rRNA intergenic spacer showed that B. garinii and B. afzelii genospecies clustered into two separate lineages. B. garinii strains were classified into three different branches: All the strains with RFLP pattern C were in the same branch, strain VH10 with a unique RFLP pattern clustered with strains VH9 and MDH2 with pattern B, and the rest of the strains with pattern B constitute another branch. These findings demonstrate the genetic diversity of B. burgdorferi sensu lato isolates from northeastern China.     

Molecular and serological evidence of Borrelia burgdorferi sensu lato in wild rodents in the Czech Republic

The aim of the present study was to determine the frequency and spatial distribution of the Borrelia species in wild rodents in the Czech Republic. In total, 293 muscle tissue samples and 106 sera from 293 wild rodents captured in North Bohemia and North-East and South Moravia were examined for the presence of Borrelia spp. and antibodies. Muscle samples were investigated with real-time polymerase chain reaction (PCR) with a recA primer set, with DNA quantification and melting curve analysis, and with restriction fragment length polymorphism (RFLP) analysis of the 5S-23S rDNA intergenic spacer. Infection with Borrelia burgdorferi sensu lato was found in 16.4% of the muscle samples. The most abundant genospecies was Borrelia afzelii (11.3%), followed by Borrelia burgdorferi sensu stricto (4.8%) and Borrelia garinii (0.7%). Borrelia infection was more frequently observed in Clethrionomys glareolus than in Apodemus spp. Sera were analyzed with an enzyme-linked immunosorbent assay (ELISA) test, yielding the total seropositivity rates of 24.5% for anti-Borrelia IgM antibodies and 25.5% for IgG antibodies. Total seroprevalence was higher in Apodemus spp. than in C. glareolus. In conclusion, our data indicate that in the Czech Republic small wild rodents can serve as hosts for B. burgdorferi s. s. as well as for B. afzelii.     

Prevalence of Anaplasma phagocytophilum and coinfection with Borrelia burgdorferi sensu lato in the hard tick Ixodes ricinus in the city of Hanover (Germany)

The castor bean tick Ixodes ricinus has been found to be the main vector for Lyme borreliosis spirochetes and Anaplasma phagocytophilum in Central Europe. 1646 I. ricinus ticks from Hanover, a city located in Northern Germany, were examined for infection with A. phagocytophilum and coinfection with Borrelia burgdorferi sensu lato (sl) to obtain so far missing prevalence data for this region. The total A. phagocytophilum infection rate was 3.2% (52/1646 ticks), divided into 4.1% (32/777) adults and 2.3% (20/869) nymphs. Coinfections with B. burgdorferi sl were found in 0.9% of all tick stages. The detected genospecies were B. afzelii, B. garinii, B. burgdorferi sensu stricto (ss), and B. garinii, which was the most frequent species in coinfected ticks.     

Serological reactivity to Borrelia burgdorferi sensu lato in dogs and horses from distinct areas in Romania

Lyme disease is a perfect model of the complex relationship between host, vector, and the vector-borne bacteria. Both dogs and horses in Romania are exposed to infection. The aim of the present study was to assess the seroreactivity against Borrelia burgdorferi sensu lato in dogs and horses from different regions of Romania. 276 samples from dogs and 260 samples from horses located in different regions of Romania were analyzed by ELISA and IFA, respectively. The effect of several factors potentially affecting seroreactivity (location, age, gender, occupation, and vector exposition risk) was evaluated using Fisher's exact test (R 2.12.0). The overall prevalence of anti-Borrelia antibodies was 6.52% (18/276) in dogs, with a significantly higher positivity (46.15%, 6/13, p = 0.0005) recorded in a midcountry region. Seroreactivity was correlated with occupation, with working dogs being more exposed. The results may indicate that Lyme borreliosis foci are restricted to small areas, but further studies on Borrelia prevalence in tick populations are needed to confirm this hypothesis. In horses, a global seroprevalence of 11.92% (31/260) was observed. No correlations were found between positive results and age, sex, county, or occupation. This is the first serological survey on antibodies to B. burgdorferi sensu lato in Romanian dogs and horses.     

Changes in the protein profile and antigenicity of different Borrelia burgdorferi strains after reintroduction to Ixodes ricinus ticks

Eight Swiss strains of Borrelia burgdorferi, with various protein profiles and the North-American strain B31 were artificially introduced into Ixodes ricinus ticks and reisolated 10 days later. All isolates were subsequently examined by SDS-PAGE analysis. Comparing initial isolates with the reisolates, we observed that 7 out of 9 strains changed their protein pattern with respect to the major proteins OspA, OspB and the 22 kDa protein after passage in the tick. The strains NE2, NE4 and NE83 with the initial phenotype of OspA and 22 kDa proteins changed to the phenotype of OspA and OspB, the strains B2 and NE202 with the initial phenotype of OspA acquired an additional protein of 22 kDa and the strain NE58 with the initial phenotype of OspA also acquired a protein of 22 kDa. Examination of these isolates by Western blot analysis demonstrated that the reaction with the monoclonal antibody H5332 and a monospecific polyclonal antibody PoAb/anti-22 kDa differed between the initial isolates and the reisolates.     

First isolation of Borrelia lusitaniae DNA from Ixodes ricinus ticks in Poland

European genospecies of B. burgdorferi sensu lato were identified by examining unfed I. ricinus ticks collected from 10 locations in northwest Poland. Research was conducted using 3 methods: PCR amplification of the fla gene with the FLA1 and FLA2 primer set conserved in all European species of B. burgdorferi sensu lato, PCR-RFLP, and sequencing. There were 5 restriction patterns obtained in this study: 4 characteristic for genospecies B. burgdorferi sensu stricto, B. garinii, B. valaisiana, and B. afzelii and 1 untypical restriction pattern type. PCR products for all restriction patterns were sequenced. Polish sequences of the fla gene for B. burgdorferi s.s., B. garinii, B. valaisiana, and B. afzelii were identical with sequences from GeneBank at 99.79%, 99.58%, 100% and 100% respectively. The fifth sequence demonstrated 99.79% identity with sequences of a B. lusitaniae PotiB2 isolate from Portugal, and also clusters with this strain. B. lusitaniae DNA in I. ricinus was detected in 3 out of 10 localities and constituted 5.9% of infected individuals with I. ricinus.