Acarologic risk of exposure to Borrelia burgdorferi spirochaetes: long-term evaluations in north-western California, with implications for Lyme borreliosis risk-assessment models

Over a 5-year period (1997-2001) the population densities of Ixodes pacificus Cooley & Kohls (Acari: Ixodidae) nymphs infected with spirochaetes of Borrelia burgdorferi sensu lato (s.l.) were evaluated in areas of 2000 ha at two localities (CHR, nine sites; HREC, seven sites) 25 km apart in Mendocino County, north-western California. The 5-year median density of infected nymphs was significantly higher at CHR than at HREC (0.51 vs. 0.09 per 100 m(2) and site-specific yearly densities exceeding one infected nymph per 100 m2 were 10-fold more likely to occur at CHR than at HREC. The importance of long-term data in acarologic risk assessment was demonstrated by significantly higher median yearly densities of infected nymphs at CHR from 1997 to 1999, whereas both areas had similar densities during 2000-2001. Overall, the causative agent of Lyme borreliosis in North America, B. burgdorferi Johnson et al. sensu stricto (s.s.) accounted for 76% of 46 genetically characterized B. burgdorferi s.l. infections from I. pacificus nymphs. Tremendous variability in acarologic risk was recorded within both areas: yearly densities of infected nymphs varied 11-97-fold between sites at CHR and 8-30-fold at HREC. Part of this variation could be explained by environmental traits, most notably deer usage. However, correlations between environmental factors and density of infected nymphs (for CHR and HREC combined) did not necessarily apply when these areas were considered separately. Thus, a Lyme borreliosis ecology model developed in one of these areas needs testing in the other area.     

Production of tumor necrosis factor alpha by Treponema pallidum, Borrelia burgdorferi s.l., and Leptospira interrogans in isolated rat Kupffer cells

Stimulation of isolated rat Kupffer cells by viable Leptospira interrogans, Treponema pallidum and Borrelia garinii elicited cellular responses resulting in the release of different tumor necrosis factor alpha (TNF-alpha) levels, depending on the spirochetes. L. interrogans induced TNF-alpha levels higher than those achieved with B. garinii and T. pallidum (in this order), but lower than the levels achieved with lipopolysaccharide (LPS). In contrast to L. interrogans, pretreatment of borreliae and treponemes with polymyxin B did not substantially diminish the ability of B. garinii and T. pallidum to stimulate Kupffer cells. Purified T. pallidum lipoproteins TpN47, TmpA, TpN15-TpN17, and B. garinii OspA induced TNF-alpha responses comparable to that achieved by LPS. This response was almost insensitive to the action of polymyxin B.     

Impaired host defense to infection and Toll-like receptor 2-independent killing of Borrelia burgdorferi clinical isolates in TLR2-deficient C3H/HeJ mice

To investigate the role of Toll-like receptor 2 (TLR2)-mediated signaling in host innate defense and development of Lyme disease, the pathogenicity of Borrelia burgdorferi sensu stricto clinical isolates representing two distinct genotypes (RST1 and RST3A) was assessed in TLR2(-/-) C3H/HeJ mice. All TLR2(-/-) mice infected with a B. burgdorferi RST1 isolate developed severe arthritis. The numbers of spirochetes in heart, joint and ear biopsy specimens were significantly higher in TLR2(-/-) mice than in wild-type mice similarly infected as determined by real-time quantitative polymerase chain reaction. Interestingly, despite the higher spirochete levels in heart tissues, milder carditis was observed in TLR2(-/-) than in wild-type mice infected with this RST1 isolate (P=0.02). By contrast, no positive cultures were obtained from any of the blood and tissue specimens from TLR2(-/-) mice inoculated with two RST3A clinical isolates. The data suggest that there is impaired host innate defense against infection and TLR2-independent killing of B. burgdorferi clinical isolates in TLR2-deficient C3H/HeJ mice.     

Longitudinal surveillance of the tick Ixodes ricinus for borreliae

Host-seeking Ixodes ricinus (L.) (Acari: Ixodidae) were monitored for borreliae (Borrelia burgdorferi s.l.) using dark-field microscopy in South Moravia (Czech Republic) each May from 1991 to 2001 (150 nymphs, 100 females and 100 males each year). This survey revealed a mean annual percentage of infected ticks of 16.8% (range, 11.7-24.2) in nymphs, 24.9% (range, 16.5-33.6) in females and 26.1% (range, 17.1-37.3) in males. Annual incidence of Lyme borreliosis in humans of the area in the same period (range, 8.7-41.7 per 100,000) correlated significantly with the frequency (number of ticks per flag per hour) of nymphs infected with >50 borreliae or all nymphal ticks, but not with the frequency of females, infected females or the infection rate (% of ticks infected) of either nymphal or female ticks. A prediction of the annual incidence of Lyme borreliosis, based on the frequency of heavily infected or all nymphal I. ricinus ticks, is feasible. The infection rate in I. ricinus correlated significantly with the North Atlantic Oscillation winter index of the last year (in nymphs) or of the year before last (in adults).     

Dot-ELISA检测莱姆病血清抗体的实验研究

经实验研究,建立了检测莱姆病血清IgG的Dot-ELISA方法。阻断试验表明,本法具有特异性。经对10例伯氏疏螺旋体人工感染兔血清和24例正常兔血清标本的检测证实,该法与常规ELISA法检测的符合率为100％。重复性试验表明其重复性良好。     

Seroprevalence study of Tick-borne encephalitis, Borrelia burgdorferi, Dengue and Toscana virus in Turin Province

Tick borne encephalitis virus (TBEV) is present in some European countries and it is transmitted by a tick bite. Ixodes ricinus is the main vector of the infection in Italy, where fortunately clinical neurological manifestations, typical of the more serious phase of the disease, are very rarely observed. This behaviour is different from other endemic Euroasiatic areas where numerous cases of encephalitis are described. However TBE transmission has not been widely investigated in Italy and available epidemiological data have been obtained only by studies performed in Central and Northern Regions of the country. In addition seroepidemiological researches were made prevalently on subjects at high risk of tick bite, such as hunters or forest guards from Trentin and Central Italy. No precise information about TBE virus diffusion was available in the Piedmont before our investigations. We found that hunters and wild boar breeders seem to be particularly exposed to the risk of TBE virus infection in Turin Province and in particular in the Susa valley, although no neurological involvement was observed in our population. In particular a seroprevalence of about 5% was detected by the use of purified antigens ELISA test, amongst the subjects at high risk of tick bite. Moreover low risk individuals showed a seroprevalence of below 2%. In addition a parallel seroepidemiological study was performed in Turin Province for Borrelia burgdorferi, the aetiological agent of Lyme disease, also transmitted by tick bite (e.g. Ixodes ricinus), for Dengue and Toscana (TOS) arboviruses, respectively transmitted by Aedes mosquitoes and phlebotomes. Data reported here demonstrate only a sporadic presence in our population of antibodies against Borrelia and Dengue infection. Moreover using an ELISA test performed with viral purified nucleoprotein, we reported a total percentage of about 3% of subjects positive for TOSV.     

Strain diversity of Borrelia burgdorferi in ticks dispersed in North America by migratory birds

The role of migratory birds in the dispersal of Ixodes scapularis ticks in the northeastern U.S. is well established and is presumed to be a major factor in the expansion of the geographic risk for Lyme disease. Population genetic studies of B. burgdorferi sensu stricto, the agent of Lyme disease in this region, consistently reveal the local presence of as many as 15 distinct strain types as designated by major groups of the ospC surface lipoprotein. Recent evidence suggests such strain diversity is adaptive to the diverse vertebrate hosts that maintain enzootic infection. How this strain diversity is established in emergent areas is unknown. To determine whether similar strain diversity is present in ticks imported by birds, we examined B. burgdorferi strains in I. scapularis ticks removed from migrants at an isolated island site. Tick mid‐guts were cultured and isolates underwent DNA amplification with primers targeting ospC. Amplicons were separated by gel electrophoresis and sequenced. One hundred thirty‐seven nymphal ticks obtained from 68 birds resulted in 24 isolates of B. burgdorferi representing eight ospC major groups. Bird‐derived ticks contain diverse strain types of B. burgdorferi, including strain types associated with invasive Lyme disease. Birds and the ticks that feed on them may introduce a diversity of strains of the agent of Lyme disease to emergent areas.     

An in vitro study of the susceptibility of mobile and cystic forms of Borrelia burgdorferi to hydroxychloroquine

In this work the susceptibility of mobile and cystic forms of Borrelia burgdorferi to hydroxychloroquine (HCQ) was studied. The minimal bactericidal concentration (MBC) of HCQ against the mobile spirochetes was >32 μg/ml at 37 °C, and >128 μg/ml at 30 °C. Incubation with HCQ significantly reduced the conversion of mobile spirochetes to cystic forms. When incubated at 37 °C, the MBC for young biologically active cysts (1-day old) was >8 μg/ml, but it was >32 μg/ml for old cysts (1-week old). Acridine orange staining, dark-field microscopy and transmission electron microscopy revealed that the contents of the cysts were partly degraded when the concentration of HCQ was ≥MBC. At high concentrations of HCQ (256 μg/ml) about 95% of the cysts were ruptured. When the concentration of HCQ was ≥MBC, core structures did not develop inside the cysts, and the amount of RNA in these cysts decreased significantly. Spirochetal structures inside the cysts dissolved in the presence of high concentrations of HCQ. When the concentration of HCQ was ≥MBC, the core structures inside the cysts were eliminated. These observations may be valuable in the treatment of resistant infections caused by B. burgdorferi, and suggest that a combination of HCQ and a macrolide antibiotic could eradicate both cystic and mobile forms of B. burgdorferi. Electronic Publication