This thesis examines seasonal, diel and vertical distribution patterns of activity of host-seeking Ixodes ricinus (L.) ticks at three localities in south-central Sweden. In addition, by examining the prevalence of infection in ticks with Lyme borreliosis (LB) spirochetes, Borrelia burgdorferi s.l, information for estimating relative LB risk in humans and the effect of control measures directed against this tick vector is provided. The seasonal activity pattern of I. ricinus was, in general, bimodal with peaks of activity in May-June and August-September. Tick densities were generally high at Torö and low at Kungshamn-Morga. The greatest variation in tick density occurred at the sample site level, which indicated a patchy distribution of ticks. The diel activity of adult I. ricinus sampled at Bogesund showed a distinct nocturnal activity peak while nymphal ticks exhibited no particular diel variation. At the meadow site, there was a strong negative association between activity of each tick stage and ambient air temperature, and larval ticks also showed a nocturnal activity peak. I. ricinus of all stages were present in the vegetation up to at least 140 cm above ground level. At Torö, host-seeking larvae were found at significantly lower levels (below 20 cm) in the vegetation compared to nymphs and adults (50-59 and 60-79 cm, respectively). Vegetation structure is likely to be the main factor governing tick vertical distribution at this locality. The northern limit of the geographical distribution of I. ricinus in Sweden corresponds with the southern boundary of the taiga zone, as well as with several other climatic or vegetational isoclines primarily associated with the vegetation period. The prevalence rates of Borrelia spirochetes, recorded by phase-contrast microscopy in host-seeking I. ricinus, were 0% in larvae, 5.8-13.1% in nymphs and 14.5-28.6% in adult ticks. The human LB risk, estimated by the number of Borrelia-infected nymphs per hectare, was greater at Torö than at Kungshamn-Morga and greater in woodland than in open areas. The risk also possessed a bimodal seasonal pattern similar to that of subadult host-seeking activity. Controlling the number of infected nymphs through de-ticking of reservoir hosts seems not to be an effective control measure in Sweden due to the ubiquitous availability of alternative reservoir hosts.

In Europe, Lyme disease is caused by the species Borrelia (B.) burgdorferi sensu stricto, B. garinii and B. afzelii. The disease is the most common vector-borne infection in Europe and the United States, and the resulting manifestation can involve the skin, nervous system, heart and joints. The symptoms that arise are associated with the Borrelia species causing the infection. The species most associated with neuroborreliosis is B. garinii whilst B. burgdorferi sensu stricto is associated with arthritis and B. afzelii is associated with dermatological symptoms. Lyme disease normally has three phases in untreated patients. The first phase is characterised by erythema migrans, a reddening of the skin around the area of the tick bite. If the disease develops to the second phase the patient will suffer from neuroborreliosis which is characterised by neurological symptoms such as headache and peripheral facial paralysis. Cerebrospinal fluid (CSF) analysis is used to diagnose neuroborreliosis. The diagnosis is complicated by variations between the different Borrelia species and that many healthy individuals have antibodies directed against Borrelia. Antibodies in CSF can be found in different diseases. The antibodies can be produced in the central nervous system or come across the blood-brain barrier and thus derive originally from the blood. By measuring the concentration of total albumin in serum and in CSF it can be determined if the antibodies present in the CSF have been produced in the central nervous system or if they originate from the blood. The typical manifestation in the last phase of Lyme disease is severe arthritis. The aim of this examination project was to compare two ELISAs for detection of antibodies directed to Borrelia. Indirect ELISAs from DAKO and Euroimmun were compared for the diagnosis of neuroborreliosis in 100 individuals. Borrelia specific antibodies of class IgM or IgG were found in 16 of 100 patients by DAKO’s ELISA and in 20 of the same 100 patients by Euroimmun’s ELISA. The reason that Euroimmun’s method detected more cases of neuroborreliosis is probably that this method detects antibodies directed to all three pathological species of Borrelia while DAKO’s method only detects antibodies directed to B. burgdorferi. In conclusion, this study indicates that Euroimmun’s method to detect antibodies of class IgM and IgG directed to Borrelia is superior to DAKO’s method. The obtained results were confirmed by Western blot analysis which gave results in accordance with those of Euroimmun’s ELISA.

Relapsing fever (RF) is a disease caused by tick- or louse-transmitted bacteria of the genus Borrelia. It occurs worldwide but is most common in Africa where it is one of the most prevalent bacterial diseases. The main manifestation is a recurring fever which coincides with massive numbers of bacteria in the blood. Severity ranges from asymptomatic to fatal. RF is usually considered a transient disease. In contrast, B. duttonii causes a persistent, residual brain infection in C57BL/6 mice which remains long time after the bacteria are cleared from the blood. The host gene expression pattern is indistinguishable from that of uninfected animals, indicating that persistent bacteria are not recognized by the immune system nor do they cause noticeable tissue damage. This is probably due to the quite low number of bacteria residing in the brain. The silent infection can be reactivated by immunosuppression allowing bacteria to re-enter the blood. To investigate if the residual infection is in a quiescent state or if the bacteria are actively dividing, mice with residual brain infection were treated with the cell-wall disrupting antibiotic ceftriaxone, which is only active against dividing bacteria. Since all mice were cured by ceftriaxone we conclude that the bacteria are actively growing in the brain rather than being in a latent, dormant state. The brain is used as an immunoprivileged site to escape host immune defence and probably as a reservoir for bacteria. RF is a common cause of pregnancy complications, miscarriage and neonatal death in sub-Saharan Africa. We established a murine model of gestational relapsing fever to study the pathological development of these complications. B. duttonii infection during pregnancy results in intrauterine growth retardation as well as placental damage and inflammation. Spirochetes cross the maternal-foetal barrier, resulting in congenital infection. Further, pregnancy has a protective effect, resulting in milder disease during pregnancy. A clinic-based study to investigate the presence of RF in Togo was performed. Blood from patients with fever were examined for RF by microscopy, GlpQ ELISA and PCR. About 10% of the patients were positive by PCR and 13% had antibodies to GlpQ. Many RF patients originally had a misdiagnosis of malaria, which resulted in ineffective treatment. The inability of microscopic analysis to detect spirochetes demonstrates the need for tests with greater sensitivity. To provide simple, fast, cheap and sensitive diagnostics using equipment available in small health centres, a method based on enrichment of bacteria by centrifugation and detection by Giemsa staining was developed which detects <10 spirochetes/ml. To study the phylogeny of RF, IGS and glpQ were sequenced and neighbor joining trees were constructed. B. persica and B. hispanica were distant from the other species iswhereas B. crocidurae appeared to be a heterogeneous species. B. duttonii is polyphyletic in relation to B. recurrentis suggesting that the two species may in fact be the same or have a polyphyletic origin.

Relapsing fever (RF) is caused by different species of Borrelia transmitted by soft ticks or by the human body louse. Illness is characterized by reappearing peaks of high concentrations of spirochetes in blood, concordant with fever peaks separated by asymptomatic periods. Neuroborreliosis is one of the most severe manifestations of RF borreliosis. To understand the immune response during early RF, we analyzed immune cells in brain and kidney of mice infected with B. crocidurae during the acute infection. Our results indicate that brain defense is comprised primarily of innate immune cells. Despite the infiltration of innate immune cells, Borrelia was not completely eradicated. A failure of the host brain to clear the bacteria may give the pathogen a niche where it can persist. Using our mouse model, we revealed that Borrelia duttonii could persist in the mouse brain for up to 270 days, without being present in the circulation. The infection was silent with no change in host gene expression, and the spirochetes could re-enter the circulation after immunosuppression. We propose that the brain is used by the pathogen to evade host immunity and serves as a possible natural reservoir for B. duttonii, a spirochete that has rarely been found in any mammalian host other than man. Borrelia-induced complications during pregnancy have been reported, and are especially common in RF. In our established mouse model of gestational RF, we could show that the fetuses suffered from severe pathology and growth retardation, probably as a consequence of placental destruction. We could also show trans-placental transmission of the bacteria leading to neonatal RF. Surprisingly, pregnant dams had a lower bacterial load and less severe disease, showing that pregnancy has a protective effect during RF. We have used the gestational RF model to investigate host factors favoring disease resolution. Because the spleen is the primary organ responsible for trapping and removing blood-borne pathogens, we have compared temporal changes in spleen immune cell populations and cytokine/chemokine induction during the infection. Spleens of pregnant mice had earlier neutrophil infiltration, as well as faster and higher production of pro-inflammatory mediators. This rapid, robust response suggests a more effective host defense. Thus, an enhanced pro-inflammatory response during pregnancy imparts a distinct advantage in controlling the severity of relapsing fever infection.

Borrelia is a Gram-negative, corkscrew-shaped bacterium transmitted by infected ticks or lice. Borreliae are subdivided into pathogens of two diseases: Lyme disease, caused mainly by B. burgdorferi, B. afzelii and B. garinii; and relapsing fever caused primarily by B. duttonii, B. hermsii, B. recurrentis or B. crocidurae. Both diseases differ in their manifestations, duration times and dissemination patterns. Antibiotics are the major therapeutics, although unfortunately antibiotic treatment is not always beneficial. To date, drug resistance mechanisms in B. burgdorferi are unknown. Transporters of the resistance-nodulation-division (RND) family appear to be involved in drug resistance, especially in Gram-negative bacteria. They consist of three components: a cytoplasmic membrane export system, a membrane fusion protein (MFP), and an outer membrane factor (OMP). The major antibiotic efflux activity of this type in Escherichia coli is mediated by the tripartite multidrug resistance pump AcrAB-TolC. Based on the sequence homology we conclude that the besA (bb0140), besB (bb0141) and besC (bb0142) genes code for a similar efflux system in B. burgdorferi. We created a deletion mutant of besC. The minimal inhibitory concentration (MIC) values of B. burgdorferi carrying an inactive besC gene were 4- to 8-fold lower than in the wild type strain. Animal experiments showed that the besC mutant was unable to infect mice. Black lipid bilayer experiments were carried out to determine the biophysical properties of purified BesC. This study showed the importance of BesC protein for B. burgdorferi pathogenicity and resistance to antibiotics, although its importance in clinical isolates is not known. Due to its small genome, Borrelia is metabolically and biosynthetically deficient, thereby making it highly dependent on nutrients provided by their hosts. The uptake of nutrients by Borrelia is not yet completely understood. We describe the purification and characterization of a 36-kDa protein that functions as a putative dicarboxylate-specific porin in the outer membrane of Borrelia. The protein was designated as DipA, for dicarboxylate-specific porin A. DipA was biophysically characterized using the black lipid bilayer assay. The permeation of KCl through the channel could be partly blocked by titrating the DipA-mediated membrane conductance with increasing concentrations of different organic dicarboxylic anions. The obtained results imply that DipA does not form a general diffusion pore, but a porin with a binding site specific for dicarboxylates which play important key roles in the deficient metabolic and biosynthetic pathways of Borrelia species. The presence of porin P66 has been shown in both Lyme disease and relapsing fever spirochetes. In our study, purified P66 homologues from Lyme disease species B. burgdorferi, B. afzelii and B. garinii and relapsing fever species B. duttonii, B. recurrentis and B. hermsii were compared and their biophysical properties were further characterized in black lipid bilayer assay. Subsequently, the channel diameter of B. burgdorferi P66 was investigated in more detail. For this study, different nonelectrolytes with known hydrodynamic radii were used. This allowed us to determine the effective diameter of the P66 channel lumen. Furthermore, the blockage of the channel after addition of nonelectrolytes revealed seven subconducting states and indicated a heptameric structure of the P66 channel. These results may give more insight into the functional properties of this important porin.

Lyme borreliosis was first described some 30 years ago in the USA. Today, it is the most common vector borne disease in Europe and the USA. The disease can have multiple stages and symptoms can manifest from various parts of the body; joints, skin heart and nervous system. In Europe, neuroborreliosis is the most frequent late stage diagnosis. Although Lyme borreliosis is treatable with antibiotics and the causative spirochete has not been shown to be resistant to drugs, some patients do not recover completely. They have persistent symptoms and are diagnosed with chronic or persistent Lyme borreliosis. The mechanism behind the lingering symptoms is unclear but might be due to tissue damage caused by the immune system. The aim of this thesis was to study the immunological differences between patients with different outcome of Lyme borreliosis, i.e. chronic, subacute and asymptomatic, and various factors that might influence the course of the disease. The Borrelia-specific IFN-γ and IL-4 secretion was detected in blood and cerebrospinal fluid from patients with chronic and subacute neuroborreliosis during the course of the disease. Blood samples were also obtained from patients with erythema migrans (EM) and acrodermatitis chronicum atrophicans. An early increase of IFN-γ with a later switch to an IL-4 response was observed in patients with a subacute disease course whereas the IFN-γ secretion continued to be elevated in chronic patients. The Borrelia-specific Th1-response was further investigated in chronic, subacute and asymptomatic individuals by studying the expression of the Th1-marker IL-12Rβ2, on a protein and mRNA level. The cytokine secretion and Foxp3, a marker for regulatory T-cells, were also analyzed. Chronic patients had a lower IL-12Rβ2 expression on CD8+ T-cells and a lower number of Borrelia-specific IFN-γ secreting cells compared to asymptomatic individuals. Chronic patients also displayed a higher expression of Borrelia-specific Foxp3 than healthy controls. The conclusions for these tow studies were that a strong Th1-response early in the infection with a later switch to a Th2-response is beneficiary whereas a slow or weak Th1-response corresponds to a prolonged disease course. The influence of a previous infection with another pathogen, seen to suppress the immune response in animals, and the possible gender difference in immune response was also investigated. Patients with EM were screened for antibodies to Anaplasma phagocytophilum (Ap) as a sign of a previous exposure to these tick-borne bacteria. Blood lymphocytes from Ap seronegative, Ap seropositive and healthy controls were stimulated with Borrelia antigen and the secretion of IL-4, IL-5, IL-12, IL-13 and IFN-γ was detected by ELISPOT. Ap seropositive patients had a lower number of cells responding with IL-12 secretion compared to the other groups which might indicate an inhibited Th1-response. Reinfections with Lyme borreliosis was in a previous study, done by Bennet et al, found to be more frequent in postmenopausal women than in men. To investigate if there was an immunological explanation to the gender discrepancy, blood lymphocytes from individuals reinfected with Lyme borreliosis and individuals infected only once were stimulated with various antigens. The cytokine secretion was detected by ELISPOT, ELISA and Immulite. There were no differences between reinfected and single infected individuals. However, women, regardless of times infected, displayed a Th2-derived and anti-inflammatory spontaneous immune response compared to men. A previous infection with the bacteria Ap might possibly have a long term effect on the immune system and might be of disadvantage when mounting a Th1-response to a Borrelia infection. Also, the Th2-derived response displayed by postmenopausal women could indicate why more women than men get reinfected with Borrelia burgdorferi. / On the day of the public defence date of the doctoral thesis the status of article III was Accepted; the status of article IV was Submitted and the title was "Importance of induction and secretion of interferon-gamma for optimal resolution of human Lyme borreliosis: differencesbetween different outcomes of the infection".

Medicinsk entomologi

The present study aimed detection of a human pathogen B. bugdorferi sensu lato species in suspected Lyme borreliosis (LB) patients in Turkey by PCR analysis and supportive serologic tests. The 152 clinical samples (140 serum and blood, 10 cerebrospinal fluid (CSF), 1 synovial fluid, 1 skin biopsy specimens) from 140 patients sent from 22 different cities of Turkey to The Spirochetal Diseases Diagnosis Laboratory of Central Veterinary Control and Research Institute were analysed. Serum samples were subjected to ELISA with a commercial kit and all of the blood, CSF, synovial fluid and skin biopsy samples were examined by PCR. In PCR analysis two primer sets targeting the ospA gene located on the plasmid and ribosomal 23S rRNA gene of B. burgdorferi sensu lato were used. The results indicated that 32,1% (45 of 140) seropositivity was detectable by ELISA. Our results support that there is a risk of acquiring LB in different regions of Turkey. Although considerable positive detections were recorded using serologic tests,none of the specimens were positive in PCR analysis. Further studies on PCR based methods for detection of B. burgdorferi sensu lato in patients with a high clinical probability of LB apparently may require that the specimen should be taken in the early phases and before the administration of any therapeutic agent.

The objectives of this study were to determine the distribution of Borrelia burgdorferi (Bb), the bacterium that causes Lyme disease, and its tick vector (Ixodes scapularis) in the Thousand Islands in Ontario, and to assess the effects of various factors, including host populations, temperature, and vegetation, on the distribution of Bb and ticks in this new endemic region. Data were collected via drag sampling and small mammal trapping at 12 sites and multimodel inference was used to evaluate variables. There was inter-island variation in the abundance of ticks and prevalence of Bb in ticks. Important predictors of tick numbers and infection prevalence included deer abundance, distance to the United States, temperature, species richness, and relative mouse abundance. These results will contribute to management strategies to reduce Lyme disease risk in the Thousand Islands and to our understanding of the effects of biodiversity on disease risk. / Canadian Cooperative Wildlife Health Centre; Ontario Ministry of Natural Resources; Parks Canada; Public Health Agency of Canada; Ontario Graduate Scholarship

No description available.