Characterization of the humoral immune response in dogs after vaccination against the Lyme borreliosis agent A study with five commercial vaccines using two different vaccination schedules

Using five commercially available vaccines, groups of dogs were vaccinated against Lyme borreliosis and followed for 13 months. A modified vaccination schedule was included in the study in an attempt to induce higher and therefore longer-lasting protective antibody levels during the first year of immunization. Following vaccination antibodies were monitored using ELISA and Western blotting. Serum samples were examined either with antigen preparations derived from either Borrelia burgdorferi s. s., B. garinii or B. afzelii lysates or separately with recombinant OspA derived from the three species. Regardless of the vaccine used the third vaccination induced significantly higher antibody levels. Further analyses of the sera with homologue and heterologue detection systems showed in vitro only a minor cross-reactivity of vaccinal antibodies towards antigens derived from heterologous Borrelia species in vitro and consequently only minimal cross-protection can be expected.     

Detection of four species of Borrelia burgdorferi sensu lato in Ixodes ricinus ticks collected from roe deer (Capreolus capreolus) in The Netherlands.

Roe deer (Capreolus capreolus) were investigated for their value as sentinel animals for Lyme borreliosis in the Netherlands. Serum was obtained from 114 roe deer, and 513 Ixodes ricinus, predominantly females (72%), were obtained from 47 animals (41%). The polymerase chain reaction was used to detect DNA of Borrelia burgdorferi sensu lato in a total of 190 ticks, comprising 106 engorged ticks and 84 non-engorged ticks. Borrelia DNA was detected in 24 engorged ticks (23%) and 26 non-engorged ticks (31%). This difference was not significant (P = 0.25). Four species of B. burgdorferi sensu lato were identified in the ticks. B. burgdorferi sensu stricto, Borrelia garinii, Borrelia afzelii and group VS116. B. afzelii was most commonly found and present in 13 mixed infections, and in 28 single infections. Fifteen sera (13%) contained antibodies to Borrelia spp. Ticks are more appropriate sentinel animals for Lyme borreliosis than roe deer, an important host for I. ricinus. Although the viability of borrelia spirochaetes in engorged ticks collected from roe deer was not assessed, a bloodmeal taken from roe deer did not eliminate borrelia spirochaetes from the tick. The relevance of this finding for transovarial transmission of borrelia spirochaetes in ticks is discussed.     

Cloning and expression of recombinant flagellar protein flaB from Borrelia burgdorferi

OBJECTIVE: Lyme borreliosis is an arthropod transmitted infection caused by some species of the Borrelia genus. Current diagnosis employs serological testing and detection of Borrelia-specific antibodies. Using recombinant Borrelia burgdorferi antigens may improve assay specificity and sensitivity. One of the immunodominant Borrelia antigens that elicit a strong and early immune response is FlaB, making it appropriate for recombinant protein based serological diagnostic tests. MATERIAL AND METHODS: Borrelia burgdorferi genomic DNA was isolated and used as a template for the amplification of the flaB gene. The gene was cloned in the expression vector pGEX-2T. RESULTS: The amplified flaB gene was cloned in the expression vector yielding a GST-FlaB fusion gene. The gene ligated in-frame was expressed as the recombinant GST-FlaB protein. After visualization by polyacrylamide gel electrophoresis the successful expression of the FlaB protein was confirmed by immunoblotting. CONCLUSION: The expression and purification of the recombinant FlaB protein is a prerequisite for obtaining large amounts of the product through a simple and labour-free procedure, which will facilitate the diagnosis of Lyme disease.     

Enzootic origins for clinical manifestations of Lyme borreliosis

Both early localized and late disseminated forms of Lyme borreliosis are caused by Borrelia burgdorferi senso lato. Differentiating between the spirochetes that only cause localized skin infection from those that cause disseminated infection, and tracing the group of medically-important spirochetes to a specific vertebrate host species, are two critical issues in disease risk assessment and management. Borrelia burgdorferi senso lato isolates from Lyme borreliosis cases with distinct clinical manifestations (erythema migrans, neuroborreliosis, acrodermatitis chronica atrophicans, and Lyme arthritis) and isolates from Ixodes ricinus ticks feeding on rodents, birds and hedgehogs were typed to the genospecies level by sequencing part of the intergenic spacer region. In-depth molecular typing was performed by sequencing eight additional loci with different characteristics (plasmid-bound, regulatory, and housekeeping genes). The most abundant genospecies and genotypes in the clinical isolates were identified by using odds ratio as a measure of dominance. Borrelia afzelii was the most common genospecies in acrodermatitis patients and engorged ticks from rodents. Borrelia burgdorferi senso stricto was widespread in erythema migrans patients. Borrelia bavariensis was widespread in neuroborreliosis patients and in ticks from hedgehogs, but rare in erythema migrans patients. Borrelia garinii was the dominant genospecies in ticks feeding on birds. Spirochetes in ticks feeding on hedgehogs were overrepresented in genotypes of the plasmid gene ospC from spirochetes in erythema migrans patients. Spirochetes in ticks feeding on hedgehogs were overrepresented in genotypes of ospA from spirochetes in acrodermatitis patients. Spirochetes from ticks feeding on birds were overrepresented in genotypes of the plasmid and regulatory genes dbpA, rpoN and rpoS from spirochetes in neuroborreliosis patients. Overall, the analyses of our datasets support the existence of at least three transmission pathways from an enzootic cycle to a clinical manifestation of Lyme borreliosis. Based on the observations with these nine loci, it seems to be justified to consider the population structure of B. burgdorferi senso lato as being predominantly clonal.     

Co-feeding transmission and its contribution to the perpetuation of the lyme disease spirochete Borrelia afzelii

To determine whether direct passage of spirochetes between co-feeding vector ticks contributes to the likelihood that the Lyme disease spirochete Borrelia afzelii will perpetuate in nature, we compared the effects of time and space on transmission efficiency between simultaneously feeding ticks. The likelihood of co-feeding transmission increases with duration of attachment of the infecting tick. Co-feeding transmission becomes less efficient as distance from the infecting tick increases. Approximately 6 times as many ticks acquire infection when feeding on infected mice than when co-feeding with infected ticks. Both subadult stages of the wood tick Ixodes ricinus infrequently co-infest mice and voles in nature; on approximately 1 in 20 small rodents, larvae co-feed with spirochete-infected nymphs. Because only 1 in 100 larvae in nature appear to acquire spirochetal infection when co-feeding with infected nymphs, perpetuation of B. afzelii depends largely on horizontal transmission of such pathogens from previously infected mice to noninfected larvae.     

Guideline 'Lyme borreliosis'

Borrelia burgdorferi is the causative bacterial agent of Lyme borreliosis, a tick-transmitted infectious disease. The Dutch Institute for Health Care Improvement (CBO) has now issued a guideline on 'Lyme borreliosis'. Lyme borreliosis is classified as 'early', 'early disseminated', 'late' or as 'post-infectious complaints and symptoms'. Erythema migrans is the most common manifestation of early Lyme borreliosis. Frequent neurological manifestations of 'early disseminated Lyme borreliosis' include meningoradiculitis, meningitis and peripheral facial palsy, but Lyme carditis and arthritis also occur. Late Lyme borreliosis is characterised by skin abnormalities (acrodermatitis chronica atrophicans), chronic neuroborreliosis or chronic arthritis. Confirmation serology with respect to Borrelia is the most commonly used laboratory technique, but in early Lyme borreliosis the immune response may be absent. In addition, the mere presence of antibodies in the serum is no proof of an active infection with Borrelia and serology may yield false-positive reactions. Doxycycline and ceftriaxone are the most commonly used antibiotics in the various stages of Lyme borreliosis. Lyme borreliosis may be prevented by avoiding high-risk areas, keeping the skin covered as much as possible, and inspection of the skin after possible exposure to remove ticks within 24 hours. Laboratory tests after a tick bite are not recommended, nor is prophylactic treatment with antibiotics.     

用聚合酶链方法对北京林区莱姆病疫源地及莱姆病病原体基因型的探索性研究

目的：进一步明确北京林区是否存在莱姆病的自然疫源地及其分布，方法基于莱姆病螺旋体外膜蛋白A基因建立半巢式聚合酶链式反应(polymerase chain reactio,PCR)方法，对从北京6个林区采集的蜱和鼠进行检测和基因分型，选择阳性标准本进行克隆和序列测定，与已知序进行同源性比较，间接免疫荧光法检测抗莱姆病螺旋体IgG抗体，从长角血蜱中分离莱姆病螺旋体。结果：从门头沟区东灵山采集的标本中检测到莱姆病螺旋体DNA片段，3只游离全沟硬蜱1只检测阳性，57只寄全沟硬蜱若蜱中1只检测阳性；119只野鼠中9只检测阳性，其中8只，B.garinii阳性，1只B.afzelii阳性。50份野鼠血清有5份莱姆病螺旋体IgG抗体阳性，采集采的160只长角血蜱（20只／组）。未分离到莱姆病螺旋体菌株。结论：北京门头沟区东灵山可能存在莱姆病的自然疫源地，包括两个基因型，全沟硬蜱可能是莱姆病的传播媒体，野鼠可能是贮存宿主。     

陕西省莱姆病自然疫源地调查研究

目的调查了解陕西省莱姆病自然疫源地。方法人群莱姆病螺旋体血清抗体检查,采用间接免疫荧光法(IFA);莱姆病患者,采用个案调查;蜱样,采用拖旗法采集;莱姆病螺旋体检测,采用巢式PCR法;莱姆病螺旋体基因分型,采用RFLP法。结果通过血清流行病学调查,陕西省富县张家湾镇、南郑县碑坝、陇县八渡镇3个农林地区人群莱姆病螺旋体自然感染率分别为8.33%(6/113)、8.88%(3/36)和7.51%(4/45)(P0.05),发现莱姆病患者14例,蜱类以嗜群血蜱和达吉克斯坦革蜱为优势种,检测蜱样1 116只。检测出阳性138只,总阳性率为12.35%。物种的阳性率分别为嗜群血蜱30.06%(107/356),达吉克斯坦革蜱3.88%(29/747),日本血蜱15.38%(2/13);检测出阳性蜱样138只,其中嗜群血蜱107只,84只为伽氏疏螺旋体基因型,23只为阿弗西尼疏螺旋体基因型;达吉克斯坦革蜱29只,24只为伽氏疏螺旋体基因型,5只为阿弗西尼疏螺旋体基因型;日本血蜱2只,均为伽氏疏螺旋体基因型。结论研究提示陕西省富县张家湾镇、南郑县碑坝、陇县八渡镇3个农林地区存在莱姆病自然疫源地。     

Migratory passerine birds as reservoirs of Lyme borreliosis in Europe

To define the role of birds as reservoirs and disseminators of Borrelia spirochetes, we characterized tick infestation and reservoir competence of migratory passerine birds in Sweden. A total of 1,120 immature Ixodes ricinus ticks were removed from 13,260 birds and assayed by quantitative polymerase chain reaction (PCR) for Borrelia, followed by DNA sequencing for species and genotype identification. Distributions of ticks on birds were aggregated, presumably because of varying encounters with ticks along migratory routes. Lyme borreliosis spirochetes were detected in 160 (1.4%) ticks. Borrelia garinii was the most common species in PCR-positive samples and included genotypes associated with human infections. Infestation prevalence with infected ticks was 5 times greater among ground-foraging birds than other bird species, but the 2 groups were equally competent in transmitting Borrelia. Migratory passerine birds host epidemiologically important vector ticks and Borrelia species and vary in effectiveness as reservoirs on the basis of their feeding behavior.     

Urban woodland habitat is important for tick presence and density in a city in England

Urban green spaces provide an opportunity for contact between members of the public and ticks infected with pathogens. Understanding tick distribution within these areas and the drivers for increased tick density or Borrelia infection are important from a risk management perspective. This study aimed to generate data on tick presence, nymph density and Borrelia infection across a range of urban green space habitats, in order to identify those that may potentially present a higher risk of Lyme borreliosis to members of the public. Several sites were visited across the English city of Bath during 2015 and 2016. Tick presence was confirmed in all habitats surveyed, with increased likelihood in woodland and woodland edge. Highest nymph densities were also reported in these habitats, along with grassland during one of the sampling years. Adult ticks were more likely to be infected compared to nymphs, and the highest densities of infected nymphs were associated with woodland edge habitat. In addition to Lyme borreliosis causing Borrelia genospecies, Borrelia miyamotoi was also detected at several sites. This study adds to the growing evidence that urban green space habitats present a public health risk from tick bites, and this has implications for many policy areas including health and wellbeing, climate adaptation and urban green space planning.     

Seroepidemiology of Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum in wild mice captured in northern Turkey

An expedition across the Asian part of the Black Sea coast and national parks of Northern Turkey was organized in the summer of 2001 to investigate the presence of Borrelia burgdorferi sensu lato (s.l.), Lyme borreliosis agent, and Anaplasma phagocytophilum, human granulocytic ehrlichiosis, agent, in wild mice. A total of 65 Apodemus flavicollis, Apodemus sylvaticus, Microtus epiroticus, Crocidura suaveolens and Mus macedonicus, were captured. Two out of 22 Apodemus sylvaticus specimens were seropositive for B. afzelii by enzyme-linked immunosorbent assay as confirmed by Western blotting, however cultures of skin and bladder samples from all small mammals in Barbour-Stoenner-Kelly's medium-II remained negative for B. burgdorferi s.l. All sera tested were negative for Anaplasma phagocytophilum by indirect immunofluorescent assay. The prevalence of B. burgdorferi s.l. and Anaplasma phagocytophilum is low in wild mice of the Asian part of Northern Turkey.     

Borrelia burgdorferi hijacks cellular metabolism of immune cells: Consequences for host defense

Changes in cellular metabolism have proven to be important factors in driving cell behavior. It has been shown that cellular metabolism of immune cells changes when exposed to or infected by several pathogens: while this is often an adaptation of the host cells to the infection, sometimes it represents a mechanism through which the pathogens evade immune activation. Borrelia burgdorferi sensu lato, the causative agent of Lyme borreliosis, is a pathogen that highly depends on the host to survive, as the bacterium lacks many central metabolic pathways to generate its own nutrients. It is therefore quite likely that the bacterium interacts with host cells to obtain these metabolites and thereby affects metabolism in the host. Previously, several studies have assessed metabolic pathways in B. burgdorferi s.l. and how it adapts to its different host species. However, few studies have looked into how the interaction with the bacterium might affect the host cell metabolism. In this review we present the major metabolic pathways activated during Lyme borreliosis, viewed from both bacterium and host metabolism, and we discuss how these pathways interact with each other, and how they influence pathogenesis of Lyme borreliosis.     

Tick zoonoses in the southern part of Switzerland (Canton Ticino): occurrence of Borrelia burgdorferi sensu lato and Rickettsia sp.

The diversity and the distribution of tick species and their infection rates by the pathogenic micro-organism Borrelia burgdorferi sensu lato, the etiologic agent of Lyme borreliosis, and Rickettsia sp., were studied in Canton Ticino (the southern part of Switzerland). Ticks specimens collected from animals and humans were classified and analysed for the presence of both pathogens. In particular, PCR analysis was performed for the detection of Borrelia spirochetes in Ixodes ricinus and Ixodes hexagonus, and the hemolymph test was done on Rhipicephalus sanguineus for the detection of Rickettsia sp. PCR assays, performed on 424 of the 989 collected ticks, revealed a low rate of infection (around 2%) of both vectors I. ricinus and I. hexagonus by B. burgdorferi sensu lato. These results are in agreement with the modest number of Lyme borreliosis cases yearly recorded in Ticino. Further, through analysis of DNA sequences, the strains carried by the infected ticks were identified as belonging to the genomic group VS116. The widespread finding of the Mediterranean species Rhipicephalus sanguineus in different locations from July 1994 to October 1995 demonstrates its establishment in Ticino. Of the 210 specimens collected, 70 were analysed and one was infected by Rickettsia sp.     

Prevalence of Borrelia burgdorferi sensu lato genospecies in host-seeking Ixodes ricinus ticks in selected South Bohemian locations (Czech Republic)

In selected localities of Ceské Bud?jovice and Cesky, Krumlov districts, well known by stable high incidence of tick-borne encephalitis (TBE) human cases but with low incidence of Lyme borreliosis, monitoring of Borrelia burgdorferi sensu lato (s.l.) in Ixodes ricinus ticks was performed. Research was also aimed at the spread of I. ricinus to mountain areas of this region (National Park Sumava), as well as at investigating this tick for B. burgdorferi s.l. genospecies and TBE virus infection. Altogether 498 nymphs, 88 females and 11 males of I. ricinus from lower locations and 58 nymphs from mountain locations (760-1080 m above sea level) were tested by polymerase chain reaction. In lower locations total prevalence of Borrelia burgdorferi s.l. in Ixodes ricinus ticks was 35%. Single infection of Borrelia afzelii, B. garinii and B. burgdorferi sensu stricto (s.s.) was found in 59, 50 and 63 ticks, respectively (i.e. in 12.8, 11.2 and 14.1%). Double infection was found in 42 ticks (6.0%) and triple infection in three ticks (0.4%). The high frequency of B. burgdorferi s.s. exceeds the as yet reported occurrence in Central Europe. These circumstances are discussed. In mountain locations B. afzelii was found in five ticks, that including two co-infection with B. garinii, in elevations of 762 m and 1024 m above sea level, respectively. This fact signals a real danger of human infections in a region that was previously deemed to be without risk. Moreover, this region is more and more the target destination of tourist activities. The results also suggest that the penetration of infection can be rapid and formation and establishment of natural focus of Lyme borreliosis might be rather quick.     

Protective efficacy of an oral vaccine to reduce carriage of Borrelia burgdorferi (strain N40) in mouse and tick reservoirs

Lyme disease is caused by the spirochete Borrelia burgdorferi, which is transmitted through the bite of infected Ixodes ticks. Vaccination of mice with outer surface protein A (OspA) of B. burgdorferi has been shown to both protect mice against B. burgdorferi infection and reduce carriage of the organism in feeding ticks. Here we report the development of a murine-targeted OspA vaccine utilizing Vaccinia virus to interrupt transmission of disease in the reservoir hosts, thus reducing incidence of human disease. Oral vaccination of mice with a single dose of Vaccinia expressing OspA resulted in high antibody titers to OspA, 100% protection of vaccinated mice from infection with B. burgdorferi, and significant clearance of B. burgdorferi from infected ticks fed on vaccinated animals. The results indicate the vaccine is effective and may provide a manner to reduce incidence of Lyme disease.     

Epidemic spread of Lyme borreliosis, northeastern United States

We examined the degree of host specialization of different strains of Borrelia burgdorferi, the tickborne pathogen that causes Lyme borreliosis in the northeastern United States. We first assessed the genetic population structures of B. burgdorferi in ticks obtained from different mammalian host species and in questing ticks sampled in a woodland ecosystem in Connecticut. By comparing the patterns found in our study with data from another cross-sectional study, we demonstrate that B. burgdorferi is a generalist microparasite and conclude that efficient cross-species transmission of B. burgdorferi is a key feature that has allowed the rapid spread of Lyme borreliosis across the northeastern United States.     

The Ixodes ricinus salivary gland proteome during feeding and B. Afzelii infection: New avenues for an anti-tick vaccine

IntroductionBorrelia burgdorferi sensu lato, the causative agents of Lyme borreliosis, are transmitted by Ixodes ticks. Tick saliva proteins are instrumental for survival of both the vector and spirochete and have been investigated as targets for vaccine targeting the vector. In Europe, the main vector for Lyme borreliosis is Ixodes ricinus, which predominantly transmits Borrelia afzelii. We here investigated the differential production of I. ricinus tick saliva proteins in response to feeding and B. afzelii infection.MethodLabel-free Quantitative Proteomics and Progenesis QI software was used to identify, compare, and select tick salivary gland proteins differentially produced during tick feeding and in response to B. afzelii infection. Tick saliva proteins were selected for validation, recombinantly expressed and used in both mouse and guinea pig vaccination and tick-challenge studies.ResultsWe identified 870 I. ricinus proteins from which 68 were overrepresented upon 24-hours of feeding and B. afzelii infection. Selected tick proteins were successfully validated by confirming their expression at the RNA and native protein level in independent tick pools. When used in a recombinant vaccine formulation, these tick proteins significantly reduced the post-engorgement weights of I. ricinus nymphs in two experimental animal models. Despite the reduced ability of ticks to feed on vaccinated animals, we observed efficient transmission of B. afzelii to the murine host.ConclusionUsing quantitative proteomics, we identified differential protein production in I. ricinus salivary glands in response to B. afzelii infection and different feeding conditions. These results provide novel insights into the process of I. ricinus feeding and B. afzelii transmission and revealed novel candidates for an anti-tick vaccine.     

Characterization and optimization of a novel vaccine for protection against Lyme borreliosis

Lyme borreliosis (LB) is the most common vector-borne disease in the northern hemisphere and there is no vaccine available for disease prevention. The majority of LB cases in Europe are caused by four different Borrelia species expressing six different OspA serotypes, whereas in the US only one of these serotypes is present. Immunization with the outer surface protein A (OspA) can prevent infection and the C-terminal part of OspA is sufficient for protection against infection transmitted by Ixodes ticks. Here we show that the order of the stabilized monomeric OspA fragments making up the heterodimers in our LB vaccine does not influence the induced immunogenicity and protection. Using bioinformatics analysis (surface electrostatics), we have designed an improved version of an LB vaccine which has an increased immunogenicity for OspA serotype 3 and an optimized expression and purification profile. The OspA heterodimers were highly purified with low amounts of endotoxin, host cell proteins and host cell DNA. All three proteins were at least 85% triacylated which ensured high immunogenicity. The LB vaccine presented here was designed, produced and characterized to a level which warrants further development as a second generation human LB vaccine.     

Lyme arthritis, Lyme carditis and other presentations potentially associated to Lyme disease

Lyme disease or Lyme borreliosis is the most common tick-transmitted disease in the Northern hemisphere and is caused by Borrelia burgdorferi spirochetes. Lyme disease commonly begins with a characteristic skin lesion, erythema migrans. Weeks or months later, the patients may have neurologic, joint, or cardiac abnormalities. Some patients may still present persistent deep fatigue and various unspecific symptoms after standard courses of antibiotic treatment for Lyme disease. This constellation of symptoms has been variously referred to as "chronic Lyme disease", or "post-Lyme disease syndrome". The first French National Consensus Conference on Lyme Disease was the reason to review all aspects of articular and cardiac manifestations of Lyme disease after a synthesis of recent literature. The involvement of Borrelia species in chronic Lyme disease and other pathologies is discussed.     

Peripheral facial palsy in children: test for lyme borreliosis only in the presence of other clinical signs

Peripheral facial palsy (PFP) may be the only sign of Lyme borreliosis in children. A literature study prompted by three children with PFP showed that a positive test for Borrelia burgdorferi antibodies in the blood or cerebrospinal fluid in combination with pleocytosis in the cerebrospinal fluid makes a diagnosis of Lyme borreliosis extremely probable. At least half of the children with PFP as a result of Lyme borreliosis show other signs or symptoms suggesting this disease. No data are available on the prevalence of Lyme borreliosis among children with PFP in the Netherlands; in adults, however, the prevalence is very low. Therefore, assuming a similar low prevalence in children with PFP, the predictive value of serological testing in children may be limited, in which case serological screening is not useful. Moreover, treatment with antibiotics does not seem to improve the prognosis of PFP significantly. Based on these data, serological testing and lumbar puncture is justified only in children with PFP associated with other signs or symptoms of Lyme borreliosis, such as bilaterality of the PFP, signs of meningeal irritation, a recent history of erythema migrans, arthritis or a recent tick bite.