Lyme disease in Italy: Isolation of Borrelia burgdorferi from a patient

A strain of Borrelia burgdorferi was isolated from a 48-year-old female patient suffering from a chronic form of polyarthritis. Significant titers of specific anti-Borrelia antibodies were not found. This is the first report of isolation of Borrelia burgdorferi from a patient in Italy.     

A host-restricted viral vector for antigen-specific immunization against Lyme disease pathogen

Newcastle disease virus (NDV) is an avian virus that is attenuated in primates and is a potential vaccine vector for human use. We evaluated NDV as a vector for expressing selected antigens of the Lyme disease pathogen Borrelia burgdorferi. A series of recombinant NDVs were generated that expressed intracellular or extracellular forms of two B. burgdorferi antigens: namely, the basic membrane protein A (BmpA) and the outer surface protein C (OspC). Expression of the intracellular and extracellular forms of these antigens was confirmed in cultured chicken cells. C3H or Balb/C mice that were immunized intranasally with the NDV vectors mounted vigorous serum antibody responses against the NDV vector, but failed to mount a robust response against either the intracellular or extracellular forms of BmpA or OspC. By contrast, a single immunization of hamsters with the NDV vectors via the intranasal, intramuscular, or intraperitoneal route resulted in rapid and rigorous antibody responses against the intracellular or extracellular forms of BmpA and OspC. When groups of hamsters were separately inoculated with various NDV vectors and challenged with B. burgdorferi (10⁸ cells/animal), immunization with vector expressing either intracellular or extracellular BmpA was associated with a significant reduction of the pathogen load in the joints. Taken together, our studies highlighted the importance of NDV as vaccine vector that can be used for simple yet effective immunization of hosts against bacterial infections including Lyme disease.     

Development of a baited oral vaccine for use in reservoir-targeted strategies against Lyme disease

Lyme disease is a major human health problem which continues to increase in incidence and geographic distribution. As a vector-borne zoonotic disease, Lyme disease may be amenable to reservoir targeted strategies for control. We have previously reported that a vaccinia virus (VV) based vaccine expressing outer surface protein A (OspA) of Borrelia burgdorferi, the causative agent of Lyme disease, protects inbred strains of laboratory mice against infection by feeding ticks and clears the ticks of infection when administered by gavage. Here we extend these studies to develop an effective bait formulation for delivery of the VV based vaccine and test its characteristics under simulated environmental conditions. We show that this vaccine is efficacious in decreasing acquisition of B. burgdorferi by uninfected larval ticks as well as in decreasing transmission from infected ticks to its natural reservoir, Peromyscus leucopus, when fed to mice in oral baits. Using live, in vivo imaging techniques, we describe the distribution of vaccinia virus infection after ingestion of the baited vaccines and establish the use of in vivo imaging technology for optimization of bait delivery. In summary, a VV based OspA vaccine is stable in an oral bait preparation and provides protection against infection for both the natural reservoir and the tick vector of Lyme disease.     

Oral vaccination with vaccinia virus expressing the tick antigen subolesin inhibits tick feeding and transmission of Borrelia burgdorferi

Immunization with the Ixodes scapularis protein, subolesin, has previously been shown to protect hosts against tick infestation and to decrease acquisition of Anaplsma marginale and Babesia bigemina. Here we report the efficacy of subolesin, a conserved tick protein that can act as a regulator of gene expression, expressed from vaccinia virus for use as an orally delivered reservoir - targeted vaccine for prevention of tick infestation and acquisition/transmission of Borrelia burgdorferi to its tick and mouse hosts. We cloned subolesin into vaccinia virus and showed that it is expressed from mammalian cells infected with the recombinant virus in vitro. We then vaccinated mice by oral gavage. A single dose of the vaccine was sufficient for mice to generate antibody response to subolesin. Vaccination with the subolesin expressing vaccinia virus inhibited tick infestation by 52% compared to control vaccination with vaccinia virus and reduced uptake of B. burgdorferi among the surviving ticks that fed to repletion by 34%. There was a reduction in transmission of B. burgdorferi to uninfected vaccinated mice of 40% compared to controls. These results suggest that subolesin has potential as a component of a reservoir targeted vaccine to decrease B. burgdorferi, Babesia and Anaplasma species infections in their natural hosts.     

Active and Passive Surveillance and Phylogenetic Analysis of Borrelia burgdorferi Elucidate the Process of Lyme Disease Risk Emergence in Canada

Background

Northward expansion of the tick Ixodes scapularis is driving Lyme disease (LD) emergence in Canada. Information on mechanisms involved is needed to enhance surveillance and identify where LD risk is emerging.

Objectives

We used passive and active surveillance and phylogeographic analysis of Borrelia burgdorferi to investigate LD risk emergence in Quebec.

Methods

In active surveillance, we collected ticks from the environment and from captured rodents. B. burgdorferi transmission was detected by serological analysis of rodents and by polymerase chain reaction assays of ticks. Spatiotemporal trends in passive surveillance data assisted interpretation of active surveillance. Multilocus sequence typing (MLST) of B. burgdorferi in ticks identified likely source locations of B. burgdorferi.

Results

In active surveillance, we found I. scapularis at 55% of sites, and we were more likely to find them at sites with a warmer climate. B. burgdorferi was identified at 13 I. scapularis–positive sites, but infection prevalence in ticks and animal hosts was low. Low infection prevalence in ticks submitted in passive surveillance after 2004—from the tick-positive regions identified in active surveillance—coincided with an exponential increase in tick submissions during this time. MLST analysis suggested recent introduction of B. burgdorferi from the northeastern United States.

Conclusions

These data are consistent with I. scapularis ticks dispersed from the United States by migratory birds, founding populations where the climate is warmest, and then establishment of B. burgdorferi from the United States several years after I. scapularis have established. These observations provide vital information for public health to minimize the impact of LD in Canada.     

Seroepidemiological survey forBorrelia burgdorferi (Lyme disease) in dogs from northwestern of Spain

A random epidemiological study was undertaken to estimate the prevalence of canine borreliosis (Lyme disease) in Castilla y León, the largest region in Spain. The presence of antibodies was determined by indirect immunofluorescence assay (IFA), using theBorrelia burgdorferi B31 strain as antigen. Sera from 308 dogs from 7 provinces in the region were tested. Of all the animals sampled, 37 (21%) were seropositive (titres of 1/64 or above). Almost all the provinces had seroprevalence of 20% or below, except for an important focus of borreliosis in the Zamora province (42.8%). Potential risk factors (age, sex, use, habitat, season, and presence of ticks on the animals) relating to the presence of antibodies were evaluated. Those dogs which had at some time had ticks were more often seropositive, at 24.2%, than those which had never had them, at only 6.2% (p<10^–n). No significant differences were discovered for the remaining factors studied. This work indicates that dogs in Castilla y Leon, even if in most cases they do not develop the disease, are exposed to the Lyme disease agent.     

The outer surface protein A (OspA) vaccine against Lyme disease: efficacy in the rhesus monkey

The efficacy of an outer surface protein A (OspA) vaccine in three different formulations was investigated in the rhesus monkey. The challenge infection was administered using Ixodes scapularis ticks that were infected with the B31 strain of Borrelia burgdorferi. Protection was assessed against both infection and disease, by a variety of procedures. Some of the animals were radically immune suppressed, as an attempt to reveal any putative low level infection in the vaccinated animals. The significant difference found between the spirochaetal infection rates of ticks that had fed on vaccinated vs. control monkeys, lack of seroconversion in the vaccinated animals, and the absence of spirochaetal DNA in the skin of vaccinated animals in the weeks following the challenge, indicate that vaccinated monkeys were protected against tick challenge. The post-mortem immunohistochemical and polymerase chain reaction analyses, however, suggest that these monkeys may have undergone a low-level infection that was transient.     

Autochthonous Lyme Borreliosis in Humans and Ticks in Korea

ObjectiveThis study aimed at finding epidemiological and clinical features of autochthonous Lyme borreliosis in humans through epidemiological investigations and identifying its vectors and pathogens through analysis of ticks.MethodEpidemiological investigations, including review of the retrospective medical records and patient interviews, were conducted in two cases that occurred in 2012. To identify the vectors and pathogens, ticks were collected between September 23 and October 6, 2012 from the area where the tick bite in the first patient occurred. The ticks were classified, and polymerase chain reaction (PCR) tests and cultures were performed.ResultsThe first patient, a 46-year-old female, visited a forest in Gangwon province, which was 900 m above sea level, where the tick bite occurred. Two weeks after the tick bite, erythema migrans (12 × 6 cm² in size) appeared on the site of tick bite, along with fever, chill, fatigue, myalgia, and arthralgia on shoulders, knees, and hips. The second patient, a 44-year-old male, visited a mountain in Gangwon province, which was 1200 m above sea level, where a tick bite occurred. One month after the tick bite, erythema migrans appeared at the site of the tick bite, along with fatigue, myalgia, and arthralgia on the right shoulder and temporomandibular joint. Indirect fluorescent antibody testing and Western blotting were carried out in these two cases for diagnosis, and positive findings were obtained. As a result, Lyme borreliosis could be confirmed. To estimate the pathogens and vectors, the ticks were collected. A total of 122 ticks were collected and only two species, Haemaphysalis japonica and Haemaphysalis flava, were identified. PCR and culture were performed on ticks. However, Borrelia burgdorferi sensu lato was not isolated from any collected ticks.ConclusionsThis study is significant to confirm Lyme borreliosis officially at first by the national surveillance system, although identification of the mites and pathogens failed.     

Identification and characterization of Borrelia antigens as potential vaccine candidates against Lyme borreliosis

The three Borrelia species, Borrelia afzelii, Borrelia burgdorferi and Borrelia garinii are the main species causing the most common tick-borne zoonosis, Lyme borreliosis. By applying a genomic approach relying on human antibodies we have identified 122 antigenic Borrelia proteins associated with Lyme borreliosis, including already known and published protective antigens. The heterogeneity of the Borrelia species causing Lyme borreliosis makes the search for conserved antigens providing broad protection challenging. Using several in vitro assays we narrowed down the selection to 15 vaccine candidates. These antigens were further analyzed for antigenicity and cross-reactivity using sera from mice infected with the three pathogenic Borrelia species. All antigens analyzed showed a high degree of cross-reactivity between the three Borrelia species, essential for providing cross-protection. We also investigated whether mice infected with B. afzelii through tick exposure are primed to mount cytokine responses. For a selection of these antigens, we observed preferentially a pro-inflammatory response in C3H/HeN mice, while in contrast also a type 2 T cell response was seen in the Borrelia-resistant mouse strain BALB/c. Thus, antigens mounting a type 2 or mixed type 2/type 1 T cell response might be preferred vaccine candidates for evaluation in animal models of Lyme borreliosis.     

Feedback on difficulties raised by the interpretation of serological tests for the diagnosis of Lyme disease

Objectives

We had for objectives: i) to evaluate the accuracy of serologic testing for Lyme borreliosis performed in a private medical laboratory (PML); ii) to evaluate the impact of these tests on the practices of infectious diseases specialists (IDS).

Patients and method

This study was performed in two steps: i) retrospective study of patients followed in a university hospital infectious diseases outpatient clinic for suspected Lyme borreliosis, tested (ELISA and Western blot) by both the PML and the National Reference Center (NRC); ii) national survey on IDS practices concerning patients consulting for suspected Lyme borreliosis.

Results

Between July 2008 and July 2011, 128 patients consulting for suspected Lyme borreliosis were tested by both laboratories. Serological tests came back positive in 91% of cases from the PML versus 8% of cases from the NRC. Lyme borreliosis was the IDS's final diagnosis for 3.6% of patients. The survey on practices revealed that: i) the modal duration of consultation for suspected Lyme borreliosis was 30–60 minutes; ii) for 33% of patients, serologic test results performed at the PML were the only reason to suspect Lyme borreliosis; iii) 60% of patients had no indication for antibiotics.

Conclusion

The serological test performed in the PML were positive most of the time, but were not confirmed by tests performed at the NRC. This discrepancy lead to multiple and prolonged consultations in infectious diseases clinics, and discordance in the indications for antibiotics.     

Seroprevalence of Borrelia burgdorferi in Romania

In order to assess the seroprevalence of Borrelia burgdorferi in Romania and to define associated risk factors, a cross-sectional, observational study was performed in 13 districts during 1999. Sera from healthy blood donors (1598) and from forestry workers (1048) were tested. A two-step testing strategy was used in which sera were tested for anti-B. burgdorferi antibodies by a commercially available passive hemagglutination assays (PHA). All PHA positive sera were then evaluated by Western blot IgG. Demographic data regarding age, sex, profession, work place/residence, duration of employment (forestry workers), animals in the environment, and tick bites history were collected using a questionnaire. Data obtained from serological study were matched with that obtained from the questionnaire. The seroprevalence of B. burgdorferi in blood donors was of 4.3% (range 1.4–8.7%) and 9.3% (range 2.8–31.7%) in forestry workers. Seroprevalence was higher in forestry workers with a tick bite history (10.7 vs. 4.3%, p < 0.05). The highest seroprevalence in blood donors (8.7%) was noted in Maramures, a northern district of the country, whereas in forestry workers the highest seroprevalence (31.7%) was observed in a western district (Arad), where a previous study in entomology has demonstrated the highest density of Ixodes ricinus ticks in Romania.     

Enhanced protective efficacy of Borrelia burgdorferi BB0172 derived-peptide based vaccine to control Lyme disease

Lyme disease (LD) accounts for over 70% of tick-borne disease reported in the United States. The disease in humans is characterized by skin rash, arthritis, cardiac and neurological signs. Vaccination is the most efficient preventive measure that could be taken to reduce the incidence of the LD worldwide; however, at present no vaccine is available. In this study, evaluation of the Borrelia burgdorferi BB0172-derived peptide (PepB) in conjugated formulations was investigated as a vaccine candidate in murine model of LD. In brief, PepB was conjugated to the Cross-Reacting Material 197 (CRM197) and to Tetanus Toxoid heavy chain (TTHc) molecules, and subsequently used to immunize C3H/HeN mice. Following the challenge with 10⁵ spirochetes/mouse via subcutaneous inoculation, TTHc:PepB construct showed protection in 66% of the immunized animals. Hence, to further evaluate the efficacy of TTHc:PepB, immunized mice were challenged with B. burgdorferi using the tick model of infection. The outcome of this experiment revealed that serum from TTHc:PepB immunized mice was borrelicidal. After tick infection, bacterial burden was significantly reduced (over 70%) in vaccinated animals when compared with the control groups regardless of whether the mice were infested 8 or 12-weeks post-priming. Therefore, we conclude that PepB conjugated antigens can serve as an alternative to prevent LD; nevertheless, further studies will be needed to dissect the mechanisms by which anti-PepB IgG antibodies are able to kill B. burgdorferi in vitro and in vivo to further advance in the development of formulations and delivery alternative to generate a safe anti-LD vaccine.     

The Ixodes ricinus tick as a vector of Borrelia burgdorferi in Slovenia

In many parts of Slovenia, the vegetation, climate and animals form an ideal natural environment for the persistence of different tick species. In this study, Ixodes ricinus ticks, the prevalent tick species in our country, are identified as the vectors of Borrelia burgdorferi in Slovenia. Ticks were collected by flagging between May and October 1990. All the ticks collected from different parts of Slovenia were I. ricinus and each one was examined individually. The ticks were tested to determine the presence of B. burgdorferi by indirect immunofluorescent assay. Among the 496 ticks examined for borreliae there were 85 adults and 411 nymphs. Twenty (23.5%) adults and 18 (4.4%) nymphs were found positive for Borreliae. This study discusses the importance of I. ricinus ticks in the role of Lyme borreliosis in Slovenia.     

105例拟诊莱姆病患者抗伯氏疏螺旋体抗体检查结果分析

目的 分析拟诊莱姆病患者血清学检查结果,明确莱姆病临床病症及区域分布情况,为有效诊治莱姆病提供科学依据.方法 应用间接免疫荧光试验(IFA)和ELISA方法,对来自全国各地的临床拟诊莱姆病患者进行血清抗伯氏疏螺旋体IgM和IgG抗体检查,并结合临床疾患进行综合分析.结果 2007-2008年共检查临床拟诊莱姆病患者105例,血清呈阳性反应者16例,阳性率15.24%.其中神经系统患者44例,阳性4例;心血管系统患者5例,阳性1例;皮肤病变25例,阳性6例;发热患者10例,阳性1例;关节痛14例,阳性2例;精神障碍疾患7例,阳性2例.16例患者应用抗生素治疗后有效率达87.50%.16例莱姆病患者分布在9个省(直辖市、自治区),确诊病例以黑龙江、吉林、内蒙古地区较多.结论 我国人群确实存在莱姆病螺旋体散发感染,通过血清学检查能及早发现病例,提高诊疗效率.     

Comparison of western blot and microimmunofluorescence as tools for lyme disease seroepidemiology

Sera from a population of 212 farmers of the Puy de Dôme (Midlands of France) who are in close contact with Ixodes ricinus, the vector of Lyme disease in Europe, and sera from 100 urban blood donors from the nearly city of Clermont Ferrand were examined by microimmunofluorescence (MIF) for antibodies reacting with Borrelia burgdorferi. The results showed a higher seroprevalence of IgG > 1/100 in farmers (25%) than in blood donors (10%). Using western blot with antibody at a 1/200 dilution, and regarding sera as being positive if they contained IgG reactive with more than 5 bands, 93/212 farmers (44%) and 20% of blood donors were positive. Reactions with specific protein bands (94, 73, 30 and 21 KDa) by western blot were demonstrated in 83/212 sera from farmers (39%) and 16% of blood donors. Both methods showed a higher seroprevalence in the farmer population. Western blot is a sensitive and specific test for seroepidemiology but, in highly endemic areas, it is not diagnostic for active Lyme borrelliosis.     

Geographic Distribution and Expansion of Human Lyme Disease,United States

Lyme disease occurs in specific geographic regions of the United States. We present a method for defining high-risk counties based on observed versus expected number of reported human Lyme disease cases. Applying this method to successive periods shows substantial geographic expansion of counties at high risk for Lyme disease.     

Geographic Differences in Genetic Locus Linkages for Borrelia burgdorferi

Borrelia burdorferi genotype in the northeastern United States is associated with Lyme borreliosis severity. Analysis of DNA sequences of the outer surface protein C gene and rrs-rrlA intergenic spacer from extracts of Ixodes spp. ticks in 3 US regions showed linkage disequilibrium between the 2 loci within a region but not consistently between regions.     

Occurrence of Borrelia burgdorferi sensu stricto, Borrelia garinii and Borrelia afzelii in the Ixodes ricinus ticks from Eastern Slovakia

A total of 2816 unfed adults nymphs of Ixodes ricinus ticks were collected from vegetation in Koice (Eastern Slovakia) from 1994 to 1997. Prevalence of Borrelia burgdorferi s. 1. in I. ricinus ticks, detected by dark field microscopy, varied and depended upon the year and the habitat of the collected ticks. The lowest prevalence was observed in 1994 (4.8%). During 1995 it increased to 17.2% and during the next two years decreased to 15.5% and 14.2%. The rate of infection varied from 2.1 to 23.3% within 10 examined habitats of the Koice area. A different value of relative density of ticks was observed in various habitats. It ranged from 9–212 ticks per collecting hour within one flagged area (600m²) which is 1.5–35.5 ticks per 100m². Eight isolates were obtained from the infected ticks. Electrophoresis and immunoblotting with 6 monoclonal antibodies were used for the identification of Borrelia strains. Three tick isolates were identified as B. burgdorferi s. s. and the other three isolates were found to be B. garinii. One strain reacted as a mixed culture of B. burgdorferi s. s., and B. garinii. The strain originated from the Vihorlat Mountains habitat and was detected by PCR-SSCP as B. burgdorferi s. s. with a small amount of B. afzelii. The obtained results emphasize the epidemiological importance not only of B. garinii and B. afzelii but also of B. burgdorferi s. s. in Central Europe.     

Oligoarthritis Caused by Borrelia bavariensis,Austria, 2014

A case of Lyme oligoarthritis occurred in an 11-year-old boy in Vienna, Austria. DNA of Borrelia bavariensis was detected by PCR in 2 aspirates obtained from different joints. Complete recovery was achieved after a 4-week course with amoxicillin. Lyme arthritis must be considered in patients from Europe who have persisting joint effusions.