Lyme borreliosis (LB) is a common and increasing tick-borne disease in Europe. The risk of acquiring a Borrelia infection after a tick bite is not fully known. Therefore, we investigated the incidence of Borrelia infection after a tick bite and if the Borrelia load and/or the duration of tick-feeding influenced the risk of infection. During 2008-2009, ticks and blood samples were collected from 1546 tick-bitten persons from Sweden and the Åland Islands, Finland. Follow-up blood samples were taken three months after the tick bite. The duration of tick feeding was microscopically estimated and Borrelia was detected and quantified in ticks by real-time PCR. Anti-Borrelia antibodies were detected in sera using ELISA assays and immunoblot.

Even though 28 % of the participants were bitten by a Borrelia-positive tick, only 7.5% (32/428) of them developed a Borrelia infection, half of them LB. All who seroconverted removed “their” ticks significantly later than those who did not. The Borrelia load in the ticks did not explain the risk of seroconversion. Regional as well as gender differences in the Borrelia seroprevalence were found. The risk of developing a Borrelia infection after a bite by a Borrelia-infected tick is small but increases with the duration of tick feeding.

The prevalence of Candidatus Neoehrlichia mikurensis infection was determined in 102 persons bitten by ticks in Sweden. Two infected women had erythematous rashes; 1 was co-infected with a Borrelia sp., and the other showed seroconversion for Anaplasma phagocytophilum. Both patients had increased levels of Neoehrlichia DNA and serum cytokines for several months.

Lyme borreliosis is a tick-borne disease caused by the bacterium Borrelia burgdorferi. The most frequent clinical manifestation is a rash called erythema migrans. Changes in antibody reactivity to B. burgdorferi 3 months after a tick bite are measured using enzyme-linked immunosorbent assays (ELISAs). One assay is based on native purified flagellum antigen (IgG), and the other assay is based on a recombinant antigen called C6 (IgG or IgM). Paired samples were taken at the time of a tick bite and 3 months later from 1,886 persons in Sweden and the Åland Islands, Finland. The seroconversion or relative change is defined by dividing the measurement units from the second sample by those from the first sample. The threshold for the minimum level of significant change was defined at the 2.5% level to represent the random error level. The thresholds were a 2.7-fold rise for the flagellar IgG assay and a 1.8-fold rise for the C6 assay. Of 1,886 persons, 102/101 (5.4%) had a significant rise in antibody reactivity in the flagellar assay or the C6 assay. Among 40 cases with a diagnosis of Lyme borreliosis, the sensitivities corresponding to a rise in antibodies were 33% and 50% for the flagellar antigen and the C6 antigen, respectively. Graphical methods to display the antibody response and to choose thresholds for a rise in relative antibody reactivity are shown and discussed. In conclusion, 5.4% of people with tick bites showed a rise in Borrelia-specific antibodies above the 2.5% threshold in either ELISA but only 40 (2.1%) developed clinical Lyme borreliosis.

We performed a cross-sectional study including 533 persons (median age 61) from the highly TBE endemic Åland Islands in the archipelago between Sweden and Finland. Blood samples, questionnaires and vaccination records were obtained from all study participants. The aim was to investigate if there was any association between TBEV antibody titer and 14 healthrelated factors: [age, gender, number of vaccine doses (0-5), time since last vaccine dose, previous TBE disease, vaccination against other flaviviruses, ≥2 tick-bites during the previous 3 months, pet-ownership, asthma, smoking, allergy, diabetes, medication, and previous tumor]. Measurement of TBEV IgG antibodies was performed using two commercial ELISA assays (Enzygnost and Immunozym), and a third in-house rapid fluorescent focus inhibition test was used to measure TBEV neutralizing antibodies. The age of the person and the number of vaccine doses were the two most important factors determining successful vaccination. The response to each vaccine dose declined linearly with increased age. A 35 year age difference corresponds to a vaccine dose increment from 3 to 4 to achieve the same response. Participants receiving medication and participants previously vaccinated against other flaviviruses had lower TBEV antibody titers on average, while those with self-reported asthma had higher titers. By comparing the 3 serological assays we show that the Enzygnost and Immunozym assay differ due to choice of cutoffs, but not in overall accuracy.

We performed a cross-sectional study including 533 individuals (median age 61) from the highly TBE endemic A land Islands in the archipelago between Sweden and Finland. Blood samples, questionnaires and vaccination records were obtained from all study participants. The aim was to investigate if there was any association between TBEV antibody titer and 12 health-related factors. Measurement of TBEV IgG antibodies was performed using two commercial ELISA assays (Enzygnost and Immunozym), and a third in-house rapid fluorescent focus inhibition test was used to measure TBEV neutralizing antibodies. The age of the individual and the number of vaccine doses were the two most important factors determining the immunological response to vaccination. The response to each vaccine dose declined linearly with increased age. A 35 year age difference corresponds to a vaccine dose increment from 3 to 4 to achieve the same immunological response. Participants previously vaccinated against other flaviviruses had lower odds of being seropositive for neutralizing TBEV antibodies on average, while participants with self-reported asthma had higher odds of being seropositive. By comparing the 3 serological assays we show that the Enzygnost and Immunozym assay differ due to choice of cutoffs, but not in overall accuracy.

The risk of tick-borne encephalitis virus (TBEV) infection after a tick bite remains largely unknown. To address this, we investigated the presence of TBEV in ticks detached from humans in an attempt to relate viral copy number, TBEV subtype, and tick feeding time with the serological and clinical response of the tick-bitten participants. Ticks, blood samples, and questionnaires were collected from tick-bitten humans at 34 primary health care centers in Sweden and in the Aland Islands (Finland). A total of 2167 ticks was received from 1886 persons in 2008-2009. Using a multiplex quantitative real-time PCR, 5 TBEV-infected ticks were found (overall prevalence 0.23%, copy range <4 X 10(2)-7.7 X 10(6) per tick). One unvaccinated person bitten by a tick containing 7.7 x 10(6) TBEV copies experienced symptoms. Another unvaccinated person bitten by a tick containing 1.8 x 10(3) TBEV copies developed neither symptoms nor TBEV antibodies. The remaining 3 persons were protected by vaccination. In contrast, despite lack of TBEV in the detached ticks, 2 persons developed antibodies against TBEV, one of whom reported symptoms. Overall, a low risk of TBEV infection was observed, and too few persons got bitten by TBEV-infected ticks to draw certain conclusions regarding the clinical outcome in relation to the duration of the blood meal and virus copy number. However, this study indicates that an antibody response may develop without clinical symptoms, that a bite by an infected tick not always leads to an antibody response or clinical symptoms, and a possible correlation between virus load and tick feeding time. (C) 2013 Elsevier GmbH. All rights reserved.

Lyme borreliosis (LB) is the most common tick-borne disease in the northern hemisphere. The infection is caused by spirochaetes from the Borrelia (B.) burgdorferi sensu lato (s.l.) group. The clinical outcome after B. burgdorferi s.l. infection differs between individuals from asymptomatic infection without history of LB to individuals who experience persistent symptoms post-treatment for more than six months after treatment. The difference in clinical outcome is not thought to be associated with persistent infection, but could instead be affected by the host’s ability to mount an optimal immune response to the spirochaete.

The hypothesis of this thesis was that a strong inflammatory Th1-like immune response is required in the early stage of infection in order to achieve both an optimal eradication of the B. burgdorferi s.l. bacteria and a good clinical outcome. The inflammatory response must be down-regulated by an anti-inflammatory response in order to avoid excessive immune responses that will end in tissue injury. The proper down-regulation will also protect against development of a chronic Th1-like inflammatory response, with activated cytotoxic cells, which may lead to LB with persistent symptoms post-treatment.

The thesis aimed to investigate the immunological mechanisms behind the optimal resolution of human B. burgdorferi s.l. infection and to define the aberrant mechanisms leading to development of persisting symptoms.

prospective study on newly tick-bitten individuals showed that although 25% of the collected ticks were infected with B. burgdorferi s.l. very few individuals bitten by infected ticks developed LB (3.7%). In addition, 4.9% of the individuals bitten by infected ticks developed asymptomatic infection, i.e. B. burgdorferi s.l.-specific antibody seroconversion without LB. Approximately one third of all tick-bitten study subjects reported self-experienced symptoms possibly associated with LB. Individuals bitten by infected ticks were more likely to report experience of symptoms than those bitten by uninfected ticks. Thus, only 8.6% of the individuals bitten by B. burgdorferi s.l.-infected ticks were infected, verified by seroconversion, and out of them 57% were asymptomatic.

A prospective study on EM patients showed that a good clinical outcome was associated with a strong early Th1 immune response since EM patients with persistent symptoms six months after treatment had reduced expression of Th1 cytokines in their EM lesions compared with EM patients without symptoms.

The investigation of blood samples from newly tick-bitten individuals, for detection of possible early immune biomarkers indicating good clinical outcome of LB, showed that none of the investigated markers clearly discriminated between the individuals who developed LB, asymptomatic individuals, or non-infected individuals. However, tick-bitten individuals who developed asymptomatic infection showed an increase of early Th1-associated biomarkers in blood compared to individuals who developed clinical LB.

In an experimental study, Th2-immune-deviated mice had more pronounced clinical signs of infection and could not eradicate the spirochaete as efficiently as non-deviated B. burgdorferi sensu stricto (s.s.)-infected mice. Non-deviated B. burgdorferi s.s.-infected mice showed a decrease of mRNA expression associated with Th2, anti-inflammatory and Treg/Th1 responses during the course of infection, which suggested a termination of the inflammatory response – something that was not seen in the immune-deviated mice. Trends for increased expression of pro-inflammatory GM-CSF and Treg marker Foxp3 in immune-deviated mice suggested on-going inflammation. Non-deviated B. burgdorferi s.s.-infected mice showed increased systemic expression of the Th1-associated CXCL9 and CXCL10 during the course of infection, while immune-deviated mice showed an initial decrease in both chemokines at day 15 p.i. compared with day 0 p.i.

In conclusion, the risk of developing LB after a tick bite is low, and no infection or asymptomatic infection are the most common outcomes after a tick bite. The early immune response in humans and the immune response towards B. burgdorferi s.s. infection in mice support the hypothesis that a strong pro-inflammatory Th1 response is needed for an optimal clinical outcome and eradication of bacteria.

BACKGROUND:

The common tick Ixodes ricinus is the main vector in Europe of the tick-borne encephalitis virus and of several species of the Borrelia burgdorferi sensu lato complex, which are the etiological agents of Lyme borreliosis. The risk to contract bites of I. ricinus is dependent on many factors including the behaviour of both ticks and people. The tick's site of attachment on the human body and the duration of tick attachment may be of clinical importance. Data on I. ricinus ticks, which were found attached to the skin of people, were analysed regarding potentially stage-specific differences in location of attachment sites, duration of tick attachment (= feeding duration), seasonal and geographical distribution of tick infestation in relation to age and gender of the tick-infested hosts.

METHODS:

During 2008-2009, 1770 tick-bitten persons from Sweden and the Åland Islands removed 2110 I. ricinus ticks. Participants provided information about the date of tick detection and location on their body of each attached tick. Ticks were identified to species and developmental stage. The feeding duration of each nymph and adult female tick was microscopically estimated based on the scutal and the coxal index.

RESULTS:

In 2008, participants were tick-bitten from mid-May to mid-October and in 2009 from early April to early November. The infestation pattern of the nymphs was bimodal whereas that of the adult female ticks was unimodal with a peak in late summer. Tick attachment site on the human body was associated with stage of the tick and gender of the human host. Site of attachment seemed to influence the duration of tick feeding. Overall, 63% of nymphs and adult female ticks were detected and removed more than 24 hours after attachment. Older persons, compared to younger ones, and men, compared to women, removed "their" ticks after a longer period of tick attachment.

CONCLUSIONS:

The infestation behaviour of the different tick stages concerning where on the host's body the ticks generally will attach and when such ticks generally will be detected and removed in relation to host age and gender, should be of value for the development of prophylactic methods against tick infestation and to provide relevant advice to people on how to avoid or reduce the risk of tick infestation.

The incidence of Lyme borreliosis (LB) in a region may reflect the prevalence of Borrelia in the tick population. Our aim was to investigate if regions with different LB incidences can be distinguished by studying the prevalence and diversity of Borrelia species in their respective tick populations. The Borrelia load in a feeding tick increases with the duration of feeding, which may facilitate a transmission of Borrelia Spirochetes from tick to host. Therefore, we also wanted to investigate how the Borrelia load in ticks that have fed on humans varies with the duration of tick feeding. During 2008 and 2009, ticks that had bitten humans were collected from four regions of Sweden and Finland, regions with expected differences in LB incidence. The duration of tick feeding was estimated and Borrelia were detected and quantified by a quantitative PCR assay followed by species determination. Out of the 2,154 Ixodes ricinus ticks analyzed, 26% were infected with Borrelia and seven species were identified. B. spielmanii was detected for the first time in the regions. The tick populations collected from the four regions exhibited only minor differences in both prevalence and diversity of Borrelia species, indicating that these variables alone cannot explain the regions different LB incidences. The number of Borrelia cells in the infected ticks ranged from fewer than ten to more than a million. We also found a lower number of Borrelia cells in adult female ticks that had fed for more than 36 hours, compared to the number of Borrelia cells found in adult female ticks that had fed for less than 36 hours.

The clinical outcome following infection with Borrelia (B.) burgdorferi sensu lato (s.l.) differs between individuals, ranging from asymptomatic infection to Lyme borreliosis (LB) with persistent symptoms post-treatment. Previous studies in mice and humans have generated the hypothesis that a successful outcome of B. burgdorferi s.l. infection is associated with an early strong pro-inflammatory T helper (Th)1-like immune response. The aim of this study was to assess the early course of events in B. burgdorferi s.l.-associated inflammation by screening for possible early immune biomarkers in peripheral blood from newly tick-bitten persons. The study subjects bitten by B. burgdorferi s.l.-infected ticks were divided into (1) those later developing clinical LB, (2) those who developed anti-B. burgdorferi s.l. antibodies but not clinical LB, (3) those who neither developed antibodies nor clinical LB. A fourth group consisted of bitten study subjects without development of antibodies or clinical LB. Two sets of samples, both comprising all four groups, were collected in order to repeat the analyses and confirm the data. Sera or plasma collected a few days after the tick bite were analysed for 18 biomarkers (IL-1β, IL-6, CXCL8/IL-8, IL-12p70, IL-17A, IL-27, TNF, CCL18, CCL20, CCL22, CXCL1, CXCL9, CXCL10, CXCL11, calprotectin, MMP-3, MMP-8, MMP-9) by multiplex bead assay and ELISA. In the first set of samples, the neutrophil activation marker calprotectin was increased in subjects who developed clinical LB compared with subjects who developed antibodies against B. burgdorferi s.l. but did not develop LB. However, the finding could not be confirmed in the second set of samples, thus the study failed to identify an early prognostic marker for development of clinical LB. Interestingly, both sets of samples showed increases in two different Th1-associated markers, CXCL10 and IL-12p70, respectively, in subjects who following a bite by a B. burgdorferi s.l.-infected tick developed antibodies against B. burgdorferi s.l. but did not develop LB compared with subjects who developed clinical LB, thus supporting the hypothesis of an early strong Th1-response being important for optimal resolution of B. burgdorferi s.l. infection.