ABSTRACT Multilocus sequence analysis (MLSA) was used to clarify the taxonomic status of a virulent Borrelia organism previously isolated from patients with relapsing fever and from ticks in Spain that is designated the Spanish relapsing fever (SRF) Borrelia . This species has been used extensively in experimental infection models because of its continued virulence. Seven genes were amplified to analyze the phylogenetic relationships among several Spanish isolates of SRF Borrelia and other relapsing fever Borrelia species. The genes targeted in this study included rrs and flaB , which have commonly been used in phylogenetic studies; the rrf-rrl intergenic spacer (IGS), which is highly discriminatory; and four additional genes, p66 , groEL , glpQ , and recC , which are located on the chromosome and which have therefore evolved in a clonal way. The species included in this study were Borrelia duttonii , B. recurrentis , B. crocidurae , and B. hispanica as Old World Borrelia species and B. turicatae and B. hermsii as New World Borrelia species. The results obtained by MLSA of the SRF Borrelia on the basis of 1% of the genomic sequence data analyzed confirmed that the SRF Borrelia isolates are B. hispanica . However, the prototype isolates of B. hispanica used in this study have an uncertain history and display unique phenotypic characteristics that are not shared with the SRF Borrelia . Therefore, we propose to use strain SP1, isolated from a relapsing fever patient in 1994 in southern Spain, as the type strain for B. hispanica .

Hypothalamic-pituitary-adrenocortical (HPA) regulation in adults is influenced by early psychosocial adversity, but the role of infectious disease history is poorly understood. We studied the association between cumulative pathogen burden and cortisol profile over the day in a sample of 317 healthy men and women aged 51-72 years. Cumulative pathogen burden was defined as positive serostatus for Chlamydia pneumoniae, cytomegalovirus (CMV) and herpes simplex virus 1 (HSV-1). Salivary cortisol was sampled repeatedly over the day. The cortisol slope was defined as the decrease across the day and evening. Age, gender, grade of employment, body mass index, smoking status, self-rated health, cardiovascular medication, depressed mood and time of waking were included as covariates. The pathogen burden averaged 1.76 (S.D. = 0.92). The cortisol slope was inversely associated with pathogen burden after controlling for covariates. When individual pathogens were studied, only CMV was associated with flatter cortisol rhythms in isolation. We conclude that pathogen burden is independently associated with flatter cortisol slopes over the day, and may contribute to disturbed neuroendocrine regulation.

The distributional area of the tick Ixodes ricinus (L.), the primary European vector to humans of Lyme borreliosis spirochaetes (Borrelia burgdorferi sensu lato) and tick-borne encephalitis virus, appears to be increasing in Sweden. It is therefore important to determine which environmental factors are most useful to assess risk of human exposure to this tick and its associated pathogens. The geographical distribution of I. ricinus in Sweden was analysed with respect to vegetation zones and climate. The northern limit of I. ricinus and B. burgdorferi s.l. in Sweden corresponds roughly to the northern limit of the southern boreal vegetation zone, and is characterized climatically by snow cover for a mean duration of 150 days and a vegetation period averaging 170 days. The zoogeographical distribution of I. ricinus in Sweden can be classified as southerly-central, with the centre of the distribution south of the Limes Norrlandicus. Ixodes ricinus nymphs from 13 localities in different parts of Sweden were examined for the presence of B. burgdorferi s.l. and found to be infected with Borrelia afzelii and Borrelia garinii. Tick sampling localities were characterized on the basis of the density of Borrelia-infected I. ricinus nymphs, presence of specific mammals, dominant vegetation and climate. Densities of I. ricinus nymphs and Borrelia-infected nymphs were significantly correlated, and nymphal density can thus serve as a general indicator of risk for exposure to Lyme borreliosis spirochaetes. Analysis of data from this and other studies suggests that high densities of Borrelia-infected nymphs typically occur in coastal, broadleaf vegetation and in mixed deciduous/spruce vegetation in southern Sweden. Ixodes ricinus populations consistently infected with B. burgdorferi s.l. can occur in: (a) biotopes with shrews, rodents, hares and birds; (b) biotopes with shrews, rodents, hares, deer and birds, and (c) island locations where the varying hare (Lepus timidus) is the only mammalian tick host.

ABSTRACT The presence of heat shock protein 60 (Hsp60) in human plasma has been linked with cardiovascular disease (CVD). In this study, the examination of the relationship between Hsp60 in plasma and seropositivity for three microbial agents, which are thought to be risk factors for CVD, surprisingly revealed a negative association between Hsp60 and seropositivity, suggesting a protective effect of this circulating stress protein.

Objective: Socioeconomic status ( SES) is inversely associated with coronary heart disease ( CHD) risk. Cumulative pathogen burden may also predict future CHD. The hypothesis was tested that lower SES is associated with a greater pathogen burden, and that pathogen burden accounts in part for SES differences in cardiovascular risk factors.Methods: This was a cross- sectional observational study involving the clinical examination of 451 men and women aged 51 - 72 without CHD, recruited from the Whitehall II epidemiological cohort. SES was defined by grade of employment, and pathogen burden by summing positive serostatus for Chlamydia pneumoniae, cytomegalovirus and herpes simplex virus 1. Cardiovascular risk factors were also assessed.Results: Pathogen burden averaged 1.94 ( SD) 0.93 in the lower grade group, compared with 1.64 ( 0.97) and 1.64 ( 0.93) in the intermediate and higher grade groups ( p = 0.011). Pathogen burden was associated with a higher body mass index, waist/ hip ratio, blood pressure and incidence of diabetes. There were SES differences in waist/ hip ratio, high- density lipoprotein- cholesterol, fasting glucose, glycated haemoglobin, lung function, smoking and diabetes. The SES gradient in these cardiovascular risk factors was unchanged when pathogen burden was taken into account statistically.Conclusions: Although serological signs of infection with common pathogens are more frequent in lower SES groups, their distribution across the social gradient does not match the linear increases in CHD risk present across higher, intermediate and lower SES groups. Additionally, pathogen burden does not appear to mediate SES differences in cardiovascular risk profiles.

During a period of 3 years, 1998–2000, 1047 faecal swabs from Black-headed gulls were sampled at one location in Southern Sweden. Salmonella spp. was found in 28 individuals (2·7%) and the dominating serotype found was S . Typhimurium (83%). Twenty-five per cent of the Salmonella -infected gulls were later recaptured and re-sampled. We found that Salmonella infection in Black-headed gulls was of short duration, and that infection in this bird species was predominantly expressed as carriage without disease manifestations. All S . Typhimurium isolates were subjected to antibiotic resistance profiling and molecular characterization by pulsed-field gel electrophoresis and IS 200 polymerase chain reaction. The S . Typhimurium gull isolates were compared to human and domestic animal isolates of the same serotype and phage type. We found genetic relatedness of S . Typhimurium DT195 isolates from gulls, domestic animals and humans, indicating that Black-headed gulls might play a role in the spread of S . Typhimurium in Sweden.

The occlusion of vessels by packed Plasmodium falciparum-infected (iRBC) and uninfected erythrocytes is a characteristic postmortem finding in the microvasculature of patients with severe malaria. Here we have employed immunocompetent Sprague-Dawley rats to establish sequestration in vivo. Human iRBC cultivated in vitro and purified in a single step over a magnet were labeled with 99mtechnetium, injected into the tail vein of the rat, and monitored dynamically for adhesion in the microvasculature using whole-body imaging or imaging of the lungs subsequent to surgical removal. iRBC of different lines and clones sequester avidly in vivo while uninfected erythrocytes did not. Histological examination revealed that a multiadhesive parasite adhered in the larger microvasculature, inducing extensive intravascular changes while CD36- and chondroitin sulfate A-specific parasites predominantly sequester in capillaries, inducing no or minor pathology. Removal of the adhesive ligand Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), preincubation of the iRBC with sera to PfEMP1 or preincubation with soluble PfEMP1-receptors prior to injection significantly reduced the sequestration. The specificity of iRBC binding to the heterologous murine receptors was confirmed in vitro, using primary rat lung endothelial cells and rat lung cryosections. In offering flow dynamics, nonmanipulated endothelial cells, and an intact immune system, we believe this syngeneic animal model to be an important complement to existing in vitro systems for the screening of vaccines and adjunct therapies aiming at the prevention and treatment of severe malaria.

The clinical course and outcome of several infectious diseases are dependent on the type of immune response elicited against the pathogen. In adults with neuroborreliosis (NB), a type 1 response with high production of Borrelia-specific IFN-gamma, but no IL-4, has been reported. Since children have a more benign course of NB than adults, we wanted to investigate type 1 and type 2 responses in children with NB. Cerebrospinal fluid (CSF) and blood were collected from children during the acute stage of 'confirmed NB' (n = 34), 'possible NB' (n = 30) and 'non-NB' (n = 10). The number of Borrelia-specific IL-4- and IFN-gamma-secreting cells was measured by enzyme-linked immunospot assay. Borrelia-specific secretion of both IL-4 and IFN-gamma was increased in CSF in confirmed (P < 0.05) and possible (P < 0.01) NB, when compared with non-NB controls. Furthermore, children with NB had significantly higher Borrelia-specific IL-4 secretion in CSF than an adult reference material with NB (P < 0.05). There were no differences in cytokine secretion in relation to onset or recovery of neurological symptoms. Since IL-4 is known to down-regulate the pro-inflammatory and possibly harmful effects of prolonged IFN-gamma responses, the prominent IL-4 response observed in the central nervous system compartment might contribute to the more benign disease course seen in children with Lyme NB.

Innate immunity is important for early defence against borrelia spirochetes and should play a role in the clinical outcome of the infection. In order to study early cytokine responses, in vitro differentiated dendritic cells (DCs) and whole blood cells from 21 patients with different clinical outcomes of Lyme neuroborreliosis were stimulated with live borrelia spirochetes. The borrelia-induced secretion of interleukin (IL)-4, IL-10, IL-12p70, interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha in DCs and IL-1 beta, IL-6, IL-8, IL-10, IL-12p70, TNF-alpha, regulated upon activation normal T cell expressed and secreted (RANTES), monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1 alpha, MIP-1 beta and eotaxin in whole blood cells was measured by enzyme-linked immunospot (ELISPOT) and multiplex arrays, respectively. We found increased numbers of TNF-alpha-secreting DCs (P = 0.018) in asymptomatic seropositive individuals compared to patients with subacute neuroborreliosis and seronegative controls. Asymptomatic individuals were also found to have elevated levels of IL-12p70 (P = 0.031) in whole blood cell supernatants compared to seronegative controls. These results are in line with previous experiments using cells of the adaptive immune response, indicating that strong T helper type 1 (Th1) proinflammatory responses might be associated with a successful resolution of Lyme disease.

ABSTRACT Small regulatory RNAs (sRNAs) have recently been shown to be the main controllers of several regulatory pathways. The function of sRNAs depends in many cases on the RNA-binding protein Hfq, especially for sRNAs with an antisense function. In this study, the genome of Borrelia burgdorferi was subjected to different searches for sRNAs, including direct homology and comparative genomics searches and ortholog- and annotation-based search strategies. Two new sRNAs were found, one of which showed complementarity to the rpoS region, which it possibly controls by an antisense mechanism. The role of the other sRNA is unknown, although observed complementarities against particular mRNA sequences suggest an antisense mechanism. We suggest that the low level of sRNAs observed in B. burgdorferi is at least partly due to the presumed lack of both functional Hfq protein and RNase E activity.

ABSTRACT A gene encoding a putative carboxyl-terminal protease (CtpA), an unusual type of protease, is present in the Borrelia burgdorferi B31 genome. The B. burgdorferi CtpA amino acid sequence exhibits similarities to the sequences of the CtpA enzymes of the cyanobacterium Synechocystis sp. strain PCC 6803 and higher plants and also exhibits similarities to the sequences of putative CtpA proteins in other bacterial species. Here, we studied the effect of ctpA gene inactivation on the B. burgdorferi protein expression profile. Total B. burgdorferi proteins were separated by two-dimensional gel electrophoresis, and the results revealed that six proteins of the wild type were not detected in the ctpA mutant and that nine proteins observed in the ctpA mutant were undetectable in the wild type. Immunoblot analysis showed that the integral outer membrane protein P13 was larger and had a more acidic pI in the ctpA mutant, which is consistent with the theoretical change in pI for P13 not processed at the carboxyl terminus. Matrix-assisted laser desorption ionization—time of flight data indicated that in addition to P13, the BB0323 protein may serve as a substrate for carboxyl-terminal processing by CtpA. Complementation analysis of the ctpA mutant provided strong evidence that the observed effect on proteins depended on inactivation of the ctpA gene alone. We show that CtpA in B. burgdorferi is involved in the processing of proteins such as P13 and BB0323 and that inactivation of ctpA has a pleiotropic effect on borrelial protein synthesis. To our knowledge, this is the first analysis of both a CtpA protease and different substrate proteins in a pathogenic bacterium.

The Borrelia-specific interferon (IFN)- gamma and interleukin (IL)-4 responses of 113 patients and control subjects were analyzed using the sensitive enzyme-linked immunospot method. Cerebrospinal fluid (CSF) and blood samples were obtained, during the course of disease, from patients with chronic or nonchronic neuroborreliosis (NB) and from control subjects without NB. Blood samples were obtained from patients with Lyme skin manifestations and from healthy blood donors. Early increased secretion of Borrelia-specific IFN- gamma (P<.05) and subsequent up-regulation of IL-4 (P<.05) were detected in the CSF cells of patients with nonchronic NB. In contrast, persistent Borrelia-specific IFN- gamma responses were observed in the CSF cells of patients with chronic NB (P<.05). In patients with erythema migrans, increased IFN- gamma (P<.001) was observed in blood samples obtained early during the course of disease, whereas increased IL-4 (P<.05) was observed after clearance. On the contrary, patients with acrodermatitis chronica atrophicans had Borrelia-specific IFN- gamma (P<.001), but not IL-4, detected in blood samples. The present data suggest that an initial IFN- gamma response, followed by up-regulation of IL-4, is associated with nonchronic manifestations, whereas a persistent IFN- gamma response may lead to chronic Lyme borreliosis.

The aetiological agent of Lyme disease, Borrelia burgdorferi cycles between its tick vector and mammalian hosts, implying that it can sense different environments and consequently change the expression of genes encoding several surface-associated proteins. The genome of the type strain B. burgdorferi B31 has revealed 175 different gene families. The p13 gene, situated on the chromosome, encodes a channel-forming protein that belongs to the gene family 48 consisting of eight additional paralogous genes. The heterogeneity of the P13 protein from different Lyme disease Borrelia strains was investigated. The predicted surface-exposed domains are the most heterogeneous regions and contain probable epitopes of P13. The membrane-spanning architecture of P13 was determined and a model for the location of this protein in the outer membrane is presented. The transcription of the paralogues of gene family 48 during in vitro culturing and in a mouse infection model was also analysed. The bba01 gene is the only p13 paralogue present in all three Lyme-disease-causing genospecies; it is stable during cultivation in vitro and the BBA01 protein was expressed in all Borrelia strains investigated. Conversely, paralogues bbi31, bbq06 and bbh41 were only detected in B. burgdorferi and the corresponding plasmids harbouring bbi31 and bbh41 were lost during in vitro passage. Finally, p13 and bbi31 are the only members of gene family 48 that are transcribed in mice, suggesting their importance during mammalian infection.

Selected bacterial pathogens, such as Helicobacter pylori and Mycobacterium tuberculosis, establish persistent infections in mammalian hosts despite activating inflammatory and antimicrobial responses. The strategies used to overcome host defense responses vary with the anatomical location of the infection but often rely on deliberate manipulations of the host cell responses. Phylogenetically unrelated bacteria can share similar strategies for the establishment of persistence and, in selected examples, one even can define homologous "persistence" genes. Such observations suggest that persistent infection is a specific phase in infection pathogenesis rather than a fortuitous imbalance in the host-pathogen interaction.

The immuno-pathogenetic mechanisms underlying chronic Lyme neuroborreliosis are mainly unknown. Human Borrelia burgdorferi (Bb) infection is associated with Bb-specific secretion of interferon-gamma (IFN-gamma), which may be important for the elimination of Bb, but this may also cause tissue injury. In order to increase the understanding of the pathogenic mechanisms in chronic neuroborreliosis, we investigated which cell types that secrete IFN-gamma. Blood mononuclear cells from 13 patients with neuroborreliosis and/or acrodermatitis chronicum atrophicans were stimulated with Bb antigen and the phenotypes of the induced IFN-gamma-secreting cells were analyzed with three different approaches. Cells expressing CD8 or TCRgammadelta, which both have cytolytic properties, were the main phenotypes of IFN-gamma-secreting cells, indicating that tissue injury in chronic neuroborreliosis may be mediated by cytotoxic cells.

ABSTRACT We have studied the immune response to a variable surface-exposed loop region of the P66 outer membrane protein from Borrelia burgdorferi sensu lato by using an enzyme immunoassay. Lyme borreliosis populations found in North America and Sweden were preferentially more seroreactive to P66 from their respective regional species, namely, B. burgdorferi sensu stricto and B. garinii and B. afzelii, respectively.

ABSTRACT Campylobacteriosis is a zoonotic disease in which birds have been suggested to play an important role as a reservoir. We investigated the prevalence of Campylobacter jejuni subsp. jejuni in black-headed gulls (Larus ridibundus) in southern Sweden with the aim of examining the nature of C. jejuni infection in this bird species. Birds were sampled in four sampling series each year during 1999 (n = 419) and 2000 (n = 365). Longitudinally sampled C. jejuni isolates from individual gulls were subjected to macrorestriction profiling (MRP) by pulsed-field gel electrophoresis to investigate the genotypical stability during the natural course of infection. Furthermore, a subset (n = 76) of black-headed gull isolates was compared to isolates from broiler chickens (n = 38) and humans (n = 56) originating from the same geographic area. We found a pronounced seasonal variation in C. jejuni carriage, with the highest rates found in late autumn. MRP similarities were higher between isolates of human and broiler chicken origin, than between those of wild bird origin and either of the other two hosts. However, identical MRPs were found in two gull isolates and one human isolate after digestion with two restriction enzymes, strongly indicating that they may have been colonized by the same clone of C. jejuni. The MRPs most prevalent in gull isolates did not occur among isolates from humans and broiler chickens, suggesting the existence of a subpopulation of C. jejuni adapted to species-specific colonization or environmental survival.

ABSTRACTP13 is a chromosomally encoded 13-kDa integral outer membrane protein of the Lyme disease agent,Borrelia burgdorferi. The aim of this study was to investigate the function of the P13 protein. Here, we inactivated thep13gene by targeted mutagenesis and investigated the porin activities of outer membrane proteins by using lipid bilayer experiments. Channel-forming activity was lost in thep13mutant compared to wild-typeB. burgdorferi, indicating that P13 may function as a porin. We purified native P13 to homogeneity by fast performance liquid chromatography and demonstrated that pure P13 has channel-forming activity with a single-channel conductance in 1 M KCl of 3.5 nS, the same as the porin activity that was lost in thep13mutant. Further characterization of the channel formed by P13 suggested that it is cation selective and voltage independent. In addition, no major physiological effects of the inactivatedp13gene could be detected under normal growth conditions. The inactivation ofp13is the first reported inactivation of a gene encoding an integral outer membrane protein inB. burgdorferi. Here, we describe both genetic and biophysical experiments indicating that P13 inB. burgdorferiis an outer membrane protein with porin activity.

The Lyme Borrelia genospecies Borrelia afzelii and B. garinii have previously been isolated using a culture method in Swedish patients with Lyme borreliosis (LB). There are reports suggesting that the genospecies distribution in human tissue specimens as determined by molecular methods is different from that obtained by culture. In the present study, we developed a nested PCR for detection of Lyme Borrelia-specific DNA in cerebrospinal fluid from Swedish patients with LB. The genospecies were subsequently identified by sequence analysis in a total of 7 PCR-positive patients. Two sequences were identified as B. burgdorferi sensu stricto (s. s.), 1 as B. afzelii and 4 as B. garinii. These are the first reported cases in which B. burgdorferi s. s. has been shown to be the causative agent of human LB in Sweden. The results of our study confirm that the use of direct molecular analytical methods for Borrelia genospecies identification in clinical specimens can provide epidemiological information additional to that obtained by culture.

The case is reported of a 26-y-old woman with latent Lyme borreliosis that was concurrently activated with a herpes simplex virus type 1 infection. Immune suppression by stress may have caused activation of both infections.