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.     

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.     

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%).     

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.     

甘肃、湖南和广东三省蜱体内嗜吞噬细胞无形体与伯氏疏螺旋体共感染研究

目的 嗜吞噬细胞无形体和伯氏疏螺旋体是由媒介蜱传播的引起人粒细胞无形体病和莱姆病的病原。目前,对两种病原在蜱体内共感染流行情况的研究相对较少。方法 以甘肃、湖南和广东三个省采集到3个蜱种、共543份样品为研究对象,检测嗜吞噬细胞无形体和伯氏疏螺旋体在蜱体内的感染情况。结果 嗜吞噬细胞无形体和伯氏疏螺旋体在不同地区采集到的蜱体内的感染率不同,分别为3.2%~20.0%和2.3%~19.3%。在检测的样品中,共发现有7份样品中同时感染嗜吞噬细胞无形体和伯氏疏螺旋体。青海血蜱、血红扇头蜱和微小牛蜱中均检测到这两种病原。结论 青海血蜱、血红扇头蜱和微小牛蜱均能够携带嗜吞噬细胞无形体和伯氏疏螺旋体,可能为这两种病原在自然界的持续存在和循环提供了条件。此研究结果丰富了人粒细胞无形体病和莱姆病的流行病学信息,有利于提高这两种病的防控策略。     

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.     

The importance of Ixodes arboricola in transmission of Rickettsia spp., Anaplasma phagocytophilum, and Borrelia burgdorferi sensu lato in the Czech Republic, Central Europe

Wild birds are known to be a reservoir of infectious disease agents and disseminatory hosts of ticks. The purpose of this work was to obtain information about the occurrence of rickettsial, anaplasmal, and borrelial infections in some ticks that parasitize wild birds in the Czech Republic. A total of 549 subadult ticks of three species Ixodes arboricola (75.0%), Ixodes ricinus (23.1%), and Haemaphysalis concinna (1.8%) were collected from 20 species of birds (Passeriformes). Rickettsiae were detected in 44.0% larvae and 24.5% nymphs of I. arboricola collected from Parus major, Poecile palustris, and Sitta europaea. Rickettsiae-positive I. ricinus larvae (13.7%) were collected from P. major, Cyanistes caeruleus, and S. europaea, and 2.6% of nymphs from Erithacus rubecula and Prunella modularis. Comparison of sequences of a gltA gene fragment with data available in GenBank identified Rickettsia helvetica, a spotted fever rickettsia associated with human infections, and other Rickettsia spp. Anaplasma phagocytophilum was found only in two I. ricinus nymphs collected from E. rubecula and P. major. Infections with Borrelia burgdorferi sensu lato were recorded in 1.3% larvae of I. arboricola acquired from P. palustris and P. major and in 11.8% larvae and 25.0% nymphs of I. ricinus collected from P. major, P. palustris, C. caeruleus, Acrocephalus schoenobaenus, Turdus merula, Carpodacus erythrinus, Sylvia atricapilla, P. modularis, and Phylloscopus collybita. Reverse-line blot hybridization showed infections with Borrelia garinii and Borrelia valaisiana and mixed infections with these two genospecies. This is the first record of a high rate of rickettsial infection in I. arboricola subadult ticks acquired from birds in the Czech Republic and in central Europe. Our study suggests that I. arboricola, P. major, and P. palustris play important roles in circulating rickettsiae.     

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.     

Seroprevalence and geographic distribution of Dirofilaria immitis and tick-borne infections (Anaplasma phagocytophilum, Borrelia burgdorferi sensu lato, and Ehrlichia canis) in dogs from Romania

Tick-borne diseases are of great concern worldwide. Despite this, in Romania there is only limited information regarding the prevalence of vector-borne pathogens in dogs. In all, 1146 serum samples were tested by SNAP(?) 4Dx(?) (IDEXX Laboratories, Inc., Westbrook, ME) for Anaplasma phagocytophilum, Borrelia burgdorferi, and Ehrlichia canis antibodies, and for Dirofilaria immitis antigen. The correlation between positive cases and their geographic distribution, as well as potential risk factors (age, sex, breed, type of dog, habitat, and prophylactic treatments) were evaluated. Overall, 129 dogs (11.3%) were serologically-positive to one or more of the tested pathogens. The seroprevalence for the four infectious agents were: A. phagocytophilum 5.5% (63/1146), D. immitis 3.3% (38/1146), E. canis 2.1% (24/1146), and B. burgdorferi 0.5% (6/1146). Co-infection with E. canis and A. phagocytophilum was registered in 2 dogs (0.2%). The geographical distribution of the seropositive cases suggests clustered foci in southern regions and in the western part of the country for D. immitis, and in the southeastern region (Constan?a County) for E. canis. A. phagocytophilum and B. burgdorferi showed a homogenous distribution, with a tendency for Lyme-positive samples to concentrate in central Romania. For D. immitis, A. phagocytophilum, and E. canis, administering prophylactic treatments was a risk factor associated with infection. Another associated risk factor was the type of dog (stray dogs were at risk being positive for D. immitis, shelter dogs for E. canis, and hunting dogs for B. burgdorferi). The prevalence of D. immitis was significantly higher in males and in dogs older than 2 years. This survey represents the first data detailing A. phagocytophilum and E. canis seroprevalence in Romanian dogs, and the most comprehensive epidemiological study on vector-borne infections in dogs from this country.     

Seasonality of Ixodes ricinus ticks on vegetation and on rodents and Borrelia burgdorferi sensu lato genospecies diversity in two lyme borreliosis-endemic areas in Switzerland

We compared Ixodes ricinus questing density, the infestation of rodents by immature stages, and the diversity of Borrelia burgdorferi sensu lato (sl) in questing ticks and ticks collected from rodents in two Lyme borreliosis (LB)-endemic areas in Switzerland (Portes-Rouges [PR] and Staatswald [SW]) from 2003 to 2005. There were variations in the seasonal pattern of questing tick densities among years. Questing nymphs were globally more abundant at PR than at SW, but the proportion of rodents infested by immature ticks was similar (59.4% and 61%, respectively). Questing tick activity lasted from February to November with a strong decline in June. The seasonal pattern of ticks infesting rodents was different. Ticks infested rodents without decline in summer, suggesting that the risk of being bitten by ticks remains high during the summer. Rodents from SW showed the highest infestation levels (10±21.6 for larvae and 0.54±1.65 for nymphs). The proportion of rodents infested simultaneously by larvae and nymphs (co-feeding ticks) was higher at SW (28%) than at PR (11%). Apodemus flavicollis was the species the most frequently infested by co-feeding ticks, and Myodes glareolus was the most infective rodent species as measured by xenodiagnosis. At PR, the prevalence of B. burgdorferi sl in questing ticks was higher (17.8% for nymphs and 32.4% for adults) than at SW (10.4% for nymphs and 24.8% for adults), with B. afzelii as the dominant species, but B. garinii, B. burgdorferi sensu stricto, and B. valaisiana were also detected. Rodents transmitted only B. afzelii (at PR and at SW) and B. bavariensis (at SW) to ticks, and no mixed infection by additional genospecies was observed in co-feeding ticks. This implies that co-feeding transmission does not contribute to genospecies diversity. However, persistent infections in rodents and co-feeding transmission contribute to the perpetuation of B. afzelii in nature.     

Indication of Borrelia burgdorferi sensu lato in the taiga tick larvae obtained from spontaneously infected females

Hungry larvae from 17 clutches by spontaneously infected Ixodes persulcatus females were examined by inoculation of the BSK II medium, by microscopy of fixed smears, and by polymerase chain reaction (PCR) with the primers specific toward the conserved sequences of Borrelia burgdorferi 16S ribosomal RNA gene. A study of 781 larvae individually or as pools could not reveal Borrelia DNA by PCR. Inoculations of the BSK II medium with 600 larvae yielded no positive results either; immobile spiral forms of Bacillus spp. were detected in 16 (26.7 +/- 5.7%) cultures of the larvae obtained from 7 females. Microscopy of 1416 fixed smears showed typical Borrelia in 7 (0.5 +/- 0.4%) specimens of larvae resulting from the same hatch; spirochaete-like cells were present in 13 (0.9 +/- 0.5%) cases. The single female from those of the 17 hatches transmitted Borrelia through eggs to filial-generation larvae. The infection rate of the larvae emerged from the eggs of this hatch was 7% and their individual infection rates were 0.4-0.8 Borrelia per 100 fields of vision. The findings suggest that there may a transovarian transmission of Borrelia in I. persulcatus ticks, but its likelihood is very little.     

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.     

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.     

Prevalence of Anaplasma phagocytophila and Borrelia burgdorferi in Ixodes persulcatus ticks from northeastern China

A total of 1,345 Ixodes persulcatus ticks collected from northeastern China were investigated for the presence of Anaplasma phagocytophila and Borrelia burgdorferi by a nested polymerase chain reaction (PCR). Sixty-two (4.6%) ticks were positive for A. phagocytophila and 454 (33.8%) were positive for B. burgdorferi. Seven (0.5%) were coinfected with both agents. Sequence analysis of 919-basepair PCR amplicons revealed three types of A. phagocytophila. Type 1 was identical to the published sequences of A. phagocytophilas responsible for human granulocytic ehrlichiosis (HGE). The other two variants differed from the HGE agent sequence at one and four positions, respectively. These findings imply that infection with A. phagocytophila poses a potential health threat to both humans and animals in northeastern China, and that ehrlichiosis should be considered in the differential diagnosis of febrile patients with a history of tick bite, particularly when clinical manifestations are atypical for Lyme disease.     

Prevalence of antibodies to Anaplasma phagocytophilum and Borrelia burgdorferi in forestry workers from the Lublin region

Ticks are recently regarded as ones of the most important vectors of emerging diseases. One of these diseases is human granulocytic ehrlichiosis caused by obligatory intracellular microorganism recently named as Anaplasma phagocytophilum. 334 sera from forestry workers and 56 sera from blood donors (as a control group) were collected and examined for antibodies to A. phagocytophilum and B. burgdorferi. The antibodies were found statistically more frequently in sera from foresters compared to sera of the control group, both to A. phagocytophilum (19.8% vs. 5.4%, p < 0.001) and B. burgdorferi (32.0% vs. 7.1%, p < 0.01). Only 15 sera (4.5%) were found positive for both examined bacteria suggesting inhibition of coexistence although it was not statistically significant. No correlation was found between percentages of antibodies to A. phagocytophilum and to B. burgdorferi from different locations.     

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.     

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.