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Emergence of serogroup B meningococci of clonal complex sequence type (ST) 41/44 can cause high levels of disease, as exemplified by a recent epidemic in New Zealand. Multiplication of annual incidence rates (3.1 cases/100,000 population) of meningococcal disease in a defined German region, the city of Aachen and 3 neighboring countries (Greater Aachen) prompted us to investigate and determine the source and nature of this outbreak. Using molecular typing and geographic mapping, we analyzed 1,143 strains belonging to ST41/44 complex, isolated from persons with invasive meningococcal disease over 6 years (2001-2006) from 2 German federal states (total population 26 million) and the Netherlands. A spatially slowly moving clone with multiple-locus variable-number tandem repeat analysis type 19, ST42, and antigenic profile B:P1.7-2,4:F1-5 was responsible for the outbreak. Bactericidal activity in serum samples from the New Zealand MeNZB vaccination campaign confirmed vaccine preventability. Because this globally distributed epidemic strain spreads slowly, vaccination efforts could possibly eliminate meningococcal disease in this area. [ABSTRACT FROM AUTHOR]

Objectives: One of the interventions to reduce risk of central line associated bloodstream infection (CLABSI) is routine replacement of the intravenous administration sets. Guidelines advises a time interval that ranges between four and seven days. However many hospitals replace intravenous administration sets every four days to prevent CLABSI., Research Methodology: In this single centre retrospective study we analysed whether the extension of the time interval from four to seven days for routine replacement of intravenous administration sets had impact on the incidence of CLABSI and colonization of the central venous catheter. Secondary outcomes were the effects on nursing workload, material use and costs., Results: In total, 1,409 patients with 1,679 central lines were included. During the pre-intervention period 2.8 CLABSI cases per 1,000 catheter days were found in comparison with 1.3 CLABSI cases per 1,000 catheter days during the post-intervention period. The rate difference between the groups was 1.52 CLABSI cases per 1,000 catheter days (95% CI: -0.50 to +4.13, p = 0.138). The intervention resulted in a saving of 345 intravenous single use plastic administration sets and 260 hours nursing time, and reduced cost with an estimate of at least 17.250 Euros., Conclusion: Extension of the time interval from four to seven days for routine replacement of intravenous administration sets did not negatively affect the incidence of CLABSI., Implications for Clinical Practice: Additional benefits of the prolonged time interval were saving of nursing time by avoiding unnecessary routine procedures, the reducing of waste because of reducing the use of disposable materials and healthcare costs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Background: Extra Corporeal Life Support (ECLS) may be a life-saving treatment for patients with reversible cardiac and/or respiratory failure. ECLS is associated with a high risk of complications and mortality. Because only a small number of studies have been conducted into the long-term effects of ECLS, we investigated the difference in quality of life, anxiety and depressive complaints and PTSD 3 months after ICU discharge., Method: It is a retrospective case-control study covering the period January 2012 to December 2017. The ECLS patient group was compared to a matched similar patient group in the Intensive Care (IC) that did not have ECLS therapy. Quality of life was measured with the Short-Form-36 (SF-36) questionnaire, anxiety and depression was measured with the Hospital Anxiety and Depression Scale (HADS) questionnaire and for PTSD the Impact of Events Scale (IES) questionnaire was used, comparing sum scores and cut-off points of scores from both groups., Results: Included were 19 patients in the ECLS group and 38 in the control group. The mean sum scores on the sub scales of the SF36 questionnaire were the same for both groups. Only the mean score of 66.2 (scale 0-100) on the domain 'general health experience' was statistically significantly different in the ECLS group than in the control group (56.8, p = .02). There was no significant difference between the sum scores of both groups on anxiety and depressive complaints. In the ECLS group 32% of the patients may have a depressive disorder versus 18% from the control group ( p = .32). And 26% of the patients from the ECLS group may have an anxiety disorder versus 7% from the control group ( p = .51). The incidence of PTSD was 42% in the ECLS group and 24% in the control group ( p = .22)., Conclusion: We found no statistically significant difference in quality of life, anxiety and depressive symptoms and PTSD symptoms between ECLS patients and the matched control group - 3 months after the ICU discharge. The incidence of anxiety and depressive symptoms and PTSD in the ECLS group is higher than in the control group, however, this difference is not significant.

Despite wide variation among natural languages, there are linguistic properties thought to be universal to all or nearly all languages. Here, we consider universals at the semantic level, in the domain of quantifiers, which are given by the properties of monotonicity, quantity, and conservativity, and we investigate whether these universals might be explained by differences in complexity. First, we use a minimal pair methodology and compare the complexities of individual quantifiers using approximate Kolmogorov complexity. Second, we use a simple yet expressive grammar to generate a large collection of quantifiers and we investigate their complexities at an aggregate level in terms of both their minimal description lengths and their approximate Kolmogorov complexities. For minimal description length we find that quantifiers satisfying semantic universals are simpler: they have a shorter minimal description length. For approximate Kolmogorov complexity we find that monotone quantifiers have a lower Kolmogorov complexity than non-monotone quantifiers and for quantity and conservativity we find that approximate Kolmogorov complexity does not scale robustly. These results suggest that the simplicity of quantifier meanings, in terms of their minimal description length, partially explains the presence of semantic universals in the domain of quantifiers., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Although life-saving in selected patients, ECMO treatment still has high mortality which for a large part is due to treatment-related complications. A feared complication is ischemic stroke for which heparin is routinely administered for which the dosage is usually guided by activated partial thromboplastin time (aPTT). However, there is no relation between aPTT and the rare occurrence of ischemic stroke (1.2%), but there is a relation with the much more frequent occurrence of bleeding complications (55%) and blood transfusion. Both are strongly related to outcome., Methods: We will conduct a three-arm non-inferiority randomized controlled trial, in adult patients treated with ECMO. Participants will be randomized between heparin administration with a target of 2-2.5 times baseline aPTT, 1.5-2 times baseline aPTT, or low molecular weight heparin guided by weight and renal function. Apart from anticoagulation targets, treatment will be according to standard care. The primary outcome parameter is a combined endpoint consisting of major bleeding including hemorrhagic stroke, severe thromboembolic complications including ischemic stroke, and mortality at 6 months., Discussion: We hypothesize that with lower anticoagulation targets or anticoagulation with LMWH during ECMO therapy, patients will have fewer hemorrhagic complications without an increase in thromboembolic complication or a negative effect on their outcome. If our hypothesis is confirmed, this study could lead to a change in anticoagulation protocols and a better outcome for patients treated with ECMO., Trial Registration: ClinicalTrials.gov NCT04536272 . Registered on 2 September 2020. Netherlands Trial Register NL7969., (© 2022. The Author(s).)

Theory of mind refers to the human capacity for reasoning about others' mental states based on observations of their actions and unfolding events. This type of reasoning is notorious in the cognitive science literature for its presumed computational intractability. A possible reason could be that it may involve higher-order thinking (e.g., 'you believe that I believe that you believe'). To investigate this we formalize theory of mind reasoning as updating of beliefs about beliefs using dynamic epistemic logic, as this formalism allows to parameterize 'order of thinking.' We prove that theory of mind reasoning, so formalized, indeed is intractable (specifically, PSPACE-complete). Using parameterized complexity we prove, however, that the 'order parameter' is not a source of intractability. We furthermore consider a set of alternative parameters and investigate which of them are sources of intractability. We discuss the implications of these results for the understanding of theory of mind.

Introduction: Delirium in critically-ill patients is a common multifactorial disorder that is associated with various negative outcomes. It is assumed that sleep disturbances can result in an increased risk of delirium. This study hypothesized that implementing a protocol that reduces overall nocturnal sound levels improves quality of sleep and reduces the incidence of delirium in Intensive Care Unit (ICU) patients., Methods: This interrupted time series study was performed in an adult mixed medical and surgical 24-bed ICU. A pre-intervention group of 211 patients was compared with a post-intervention group of 210 patients after implementation of a nocturnal sound-reduction protocol. Primary outcome measures were incidence of delirium, measured by the Intensive Care Delirium Screening Checklist (ICDSC) and quality of sleep, measured by the Richards-Campbell Sleep Questionnaire (RCSQ). Secondary outcome measures were use of sleep-inducing medication, delirium treatment medication, and patient-perceived nocturnal noise., Results: A significant difference in slope in the percentage of delirium was observed between the pre- and post-intervention periods (-3.7% per time period, p=0.02). Quality of sleep was unaffected (0.3 per time period, p=0.85). The post-intervention group used significantly less sleep-inducing medication (p<0.001). Nocturnal noise rating improved after intervention (median: 65, IQR: 50-80 versus 70, IQR: 60-80, p=0.02)., Conclusions: The incidence of delirium in ICU patients was significantly reduced after implementation of a nocturnal sound-reduction protocol. However, reported sleep quality did not improve., (Copyright © 2017. Published by Elsevier Ltd.)

Energy is the common currency of life. To guarantee a homeostatic supply of energy, multiple neuro-endocrine systems have evolved in vertebrates; systems that regulate food intake, metabolism, and distribution of energy. Even subtle (lasting) dysregulation of the delicate balance of energy intake and expenditure may result in severe pathologies. Feeding-related pathologies have fueled research on mammals, including of course the human species. The mechanisms regulating food intake and body mass are well-characterized in these vertebrates. The majority of animal life is ectothermic, only birds and mammals are endotherms. What can we learn from a (comparative) study on energy homeostasis in teleostean fishes, ectotherms, with a very different energy budget and expenditure? We present several adaptation strategies in fish. In recent years, the components that regulate food intake in fishes have been identified. Although there is homology of the major genetic machinery with mammals (i.e., there is a vertebrate blueprint), in many cases this does not imply analogy. Although both mammals and fish must gain their energy from food, the expenditure of the energy obtained is different. Mammals need to spend vast amounts of energy to maintain body temperature; fishes seem to utilize a broader metabolic range to their advantage. In this review, we briefly discuss ecto- and endothermy and their consequences for energy balance. Next, we argue that the evolution of endothermy and its (dis-)advantages may explain very different strategies in endocrine regulation of energy homeostasis among vertebrates. We follow a comparative and evolutionary line of thought: we discuss similarities and differences between fish and mammals. Moreover, given the extraordinary radiation of teleostean fishes (with an estimated number of 33,400 contemporary species, or over 50% of vertebrate life forms), we also compare strategies in energy homeostasis between teleostean species. We present recent developments in the field of (neuro)endocrine regulation of energy balance in teleosts, with a focus on leptin.

Background: Treatment of community acquired pneumonia (CAP) patients with antibiotics before laboratory-confirmed diagnosis leads to loss of knowledge on the causative bacterial pathogen. Therefore, an increasing number of pneumococcal infections is identified using non-culture based techniques. However, methods for serotyping directly on the clinical specimen remain scarce. Here we present three approaches for detection and serotyping of pneumococci using samples from patients with CAP., Methods: The first approach is quantitative PCR (qPCR) analysis on blood samples (n = 211) followed by capsular sequence typing (CST) to identify the serotype. The second approach, a urinary antigen assay (n = 223), designated as inhibition multiplex immunoassay (IMIA), is based on Luminex technology targeting 14 serotypes. The third approach is a multiplex immunoassay (MIA) (n = 171) also based on Luminex technology which detects serologic antibody responses against 14 serotypes. The three alternative assays were performed on samples obtained from 309 adult hospitalized CAP patients in 2007-2010 and the results were compared with those obtained from conventional laboratory methods to detect pneumococcal CAP, i.e. blood cultures, sputum cultures and BinaxNOW urinary antigen tests., Results: Using qPCR, MIA and IMIA, we were able to detect the pneumococcus in samples of 56% more patients compared to conventional methods. Furthermore, we were able to assign a serotype to the infecting pneumococcus from samples of 25% of all CAP patients, using any of the three serotyping methods (CST, IMIA and MIA)., Conclusion: This study indicates the usefulness of additional molecular methods to conventional laboratory methods for the detection of pneumococcal pneumonia. Direct detection and subsequent serotyping on clinical samples will improve the accuracy of pneumococcal surveillance to monitor vaccine effectiveness.

The implementation of nationwide pneumococcal vaccination may lead to alterations in the pneumococcal population due to selective pressure induced by the vaccine. To monitor such changes, pneumococcal isolates causing invasive pneumococcal disease (IPD) before (2004-2005, n=1154) and after (2008-2009, n=1190) the implementation of the 7-valent pneumococcal vaccine (PCV7) in 2006 in the national immunization program (NIP) of The Netherlands were characterized by molecular typing using multiple-locus variable number tandem repeat analysis (MLVA) and capsular sequence typing (CST). The IPD incidence after the implementation of PCV7 in children <5 years of age declined, mainly due to an impressive reduction of cases caused by vaccine serotypes. In the age group of patients ≥5 years of age, the overall IPD incidence remained constant, but the IPD incidence due to vaccine serotypes declined in this age cohort as well, indicating herd immunity. IPD incidence of non-vaccine serotypes 1 and 22F isolates increased significantly and a shift in genetic background of the isolates belonging to these serotypes was observed. In general the composition of the pneumococcal population remained similar after the introduction of PCV7. Both before and after introduction of the vaccine several possible capsular switch events were noticed. We found 4 isolates from the pre-vaccination period in which the serotype 19F capsular locus had been horizontally transferred to a different genetic background. Remarkably, none of the 5 post-vaccination isolates in which we observed possible capsule switch belonged to the 19F serotype, possibly due to vaccine induced pressure. In the post-vaccine implementation period we found no evidence for capsular switch of a vaccine serotype to a non-vaccine serotype, indicating that capsular switch is not the main driving force for replacement. This study provides insights into the effects of nationwide vaccination on the pneumococcal population causing IPD., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The introduction of nationwide pneumococcal vaccination may lead to serotype replacement and the emergence of new variants that have expanded their genetic repertoire through recombination. To monitor alterations in the pneumococcal population structure, we have developed and utilized Capsular Sequence Typing (CST) in addition to Multiple-Locus Variable number tandem repeat Analysis (MLVA).To assess the serotype of each isolate CST was used. Based on the determination of the partial sequence of the capsular wzh gene, this method assigns a capsular type of an isolate within a single PCR reaction using multiple primersets. The genetic background of pneumococcal isolates was assessed by MLVA. MLVA and CST were used to create a snapshot of the Dutch pneumococcal population causing invasive disease before the introduction of the 7-valent pneumococcal conjugate vaccine in The Netherlands in 2006. A total of 1154 clinical isolates collected and serotyped by the Netherlands Reference Laboratory for Bacterial Meningitis were included in the snapshot. The CST was successful in discriminating most serotypes present in our collection. MLVA demonstrated that isolates belonging to some serotypes had a relatively high genetic diversity whilst other serotypes had a very homogeneous genetic background. MLVA and CST appear to be valuable tools to determine the population structure of pneumococcal isolates and are useful in monitoring the effects of pneumococcal vaccination.

The main virulence factor of Streptococcus pneumoniae is the capsule. The polysaccharides comprising this capsule are encoded by approximately 15 genes and differences in these genes result in different serotypes. The aim of this study was to investigate the sequence diversity of the capsular genes of serotypes 6A, 6B, 6C, 19A and 19F and to explore a possible effect of vaccination on variation and distribution of these serotypes in the Netherlands. The complete capsular gene locus was sequenced for 25 serogroup 6 and for 20 serogroup 19 isolates. If one or more genes varied in 10 or more base pairs from the reference sequence, it was designated as a capsular subtype. Allele-specific PCRs and specific gene sequencing of highly variable capsular genes were performed on 184 serogroup 6 and 195 serogroup 19 isolates to identify capsular subtypes. This revealed the presence of 6, 3 and a single capsular subtype within serotypes 6A, 6B and 6C, respectively. The serotype 19A and 19F isolates comprised 3 and 4 capsular subtypes, respectively. For serogroup 6, the genetic background, as determined by multi locus sequence typing (MLST) and multiple-locus variable number of tandem repeat analysis (MLVA), seemed to be closely related to the capsular subtypes, but this was less pronounced for serogroup 19 isolates. The data also suggest shifts in the occurrence of capsular subtypes within serotype 6A and 19A after introduction of the 7-valent pneumococcal vaccine. The shifts within these non-vaccine serotypes might indicate that these capsular subtypes are filling the niche of the vaccine serotypes. In conclusion, there is considerable DNA sequence variation of the capsular genes within pneumococcal serogroup 6 and 19. Such changes may result in altered polysaccharides or in strains that produce more capsular polysaccharides. Consequently, these altered capsules may be less sensitive for vaccine induced immunity.

Purpose: We investigated the hypothesis that many total hip arthroplasty revisions that are classified as aseptic are in fact low-grade infections missed with routine diagnostics., Methods: In 7 Dutch hospitals, 176 consecutive patients with the preoperative diagnosis of aseptic loosening of their total hip arthroplasty were enrolled. During surgery, between 14 and 20 tissue samples were obtained for culture, pathology, and broad-range 16S rRNA PCR with reverse line blot hybridization. Patients were classified as either not being infected, suspected of having infection, or infected according to strict, predefined criteria. Each patient had a follow-up visit after 1 year., Results: 7 patients were classified as infected, 4 of whom were not identified by routine culture. 15 additional patients were suspected of having infection. 20 of these 22 patients received a cemented prosthesis, fixated with antibiotic-loaded bone cement. All 22 patients received prophylactic systemic antibiotics. 7 of them reported complaints one year after surgery, but only one showed signs of early loosening. However, additional surgery was not performed in any of the patients., Interpretation: Although the proportions were not as high as previously reported in the literature, between 4% and 13% of patients with the preoperative diagnosis of aseptic loosening were infected. However, as thorough debridement was performed during surgery and prophylactic antibiotics were used, the diagnosis of infection did not have any obvious clinical consequences, as most patients performed well at the 1-year follow-up. Whether this observation has implications for long-term implant survival remains to be seen.

We developed an efficient molecular method for the identification of the bloodmeal sources in the tick Ixodes ricinus (L.), the European vector of the agents of Lyme borreliosis and tick-borne encephalitis. An approximately 145-bp orthologous fragment of the vertebrate mitochondrial 12S rDNA was used as a molecular marker to discriminate host vertebrate species. The method consists of a single run polymerase chain reaction amplification of the 12S rDNA molecular marker by using nondegenerate primers followed by a reverse line blot hybridization assay by using specific oligonucleotide probes. The palette of probes allowed us to distinguish major groups of host vertebrates (e.g., mammals, small rodents, artiodactyls, birds, lizards) and to identify the bloodmeal sources at the genus or species level. External primers were designed and used to sequence the 12S rDNA molecular marker of a broad range of known or potential host vertebrate species (n = 60), including mammal (n = 28), bird (n = 31), and reptile (n = 1) species. The use of this technique coupled with known methods for identification of tick-borne pathogens (e.g., Borrelia burgdorferi sensu lato) allowed us to determine the source of infective bloodmeal and to identify reservoir species. The present method was successfully used to identify the source of bloodmeals in all feeding I. ricinus ticks and in half of questing field-collected I. ricinus ticks. Moreover, the bloodmeal source was identified in 65% of ticks infected with B. burgdorferi sensu lato. Further development of this technique may be envisaged for the detection of other vector-borne pathogens and their reservoir hosts.

We identified many variable-number tandem repeat (VNTR) loci in the genomes of Neisseria meningitidis serogroups A, B, and C and utilized a number of these loci to develop a multiple-locus variable-number tandem repeat analysis (MLVA). Eighty-five N. meningitidis serogroup B and C isolates obtained from Dutch patients with invasive meningococcal disease and seven reference strains were analyzed using MLVA and multilocus sequence typing (MLST). MLVA, based on eight VNTR loci with limited variability in the number of repeats, yielded clustering of the strains similar to that obtained by MLST, with congruence between both methods amounting to 69%. The ability to recognize clonal complexes makes MLVA a valuable high-throughput method to serve as a tool complementary to MLST. Four highly variable VNTR loci were used in a second assay to analyze N. meningitidis serogroup C strains collected during an outbreak of meningococcal disease in The Netherlands. Typing based on the latter VNTR loci enabled differentiation of isolates with identical MLST sequence types and grouped epidemiologically related strains.

Recently, there has been an increase in The Netherlands in the number of cases of invasive disease caused by Haemophilus influenzae serotype b (Hib). To study a possible change in the Hib population that could explain the rise in incidence, a multiple-locus variable number tandem repeats analysis (MLVA) was developed to genotype H. influenzae isolates. The MLVA enabled the differentiation of H. influenzae serotype b strains with higher discriminatory power than multilocus sequence typing (MLST). MLVA profiles of noncapsulated H. influenzae and H. influenzae serotype f strains were more heterogeneous than serotype b strains and were distinct from Hib, although some overlap occurred. The MLVA was used to genotype a collection of 520 H. influenzae serotype b strains isolated from patients in The Netherlands with invasive disease. The strains were collected from 1983 from 2002, covering a time period of 10 years before and 9 years after the introduction of the Hib vaccine in the Dutch national vaccination program. MLVA revealed a sharp increase in genetic diversity of Hib strains isolated from neonates to 4-year-old patients after 1993, when the Hib vaccine was introduced. Hib strains isolated from patients older than 4 years in age were genetically diverse, and no significant change in diversity was seen after the introduction of the vaccine. These observations suggest that after the introduction of the Hib vaccine young children no longer constitute the reservoir for Hib and that they are infected by adults carrying genetically diverse Hib strains.

Five-month-old lambs were experimentally infected with two 16S rRNA genetic variants of Anaplasma phagocytophilum, variants 1 (GenBank accession no. M73220) and 2 (GenBank accession no. AF336220). Additional sequencing of the groESL heat shock operon gene indicated that these variants differ in three nucleotides at positions 782, 824, and 890. The variants were obtained by blood sampling of A. phagocytophilum-infected lambs from one sheep flock in Norway and were stored at -70 degrees C with 10% dimethyl sulfoxide as a cryoprotectant before being inoculated intravenously into susceptible lambs. The infectious blood contained, per ml, approximately 0.5 x 10(6) neutrophils infected with either of the variants. Six weeks after the primary inoculation, the lambs were challenged with the same infectious dose of the heterologous variant. The results of the study indicate a marked difference in clinical manifestation, neutropenia, antibody response, and cross-protection after experimental infection with the two variants of A. phagocytophilum.

A total of 41 blood samples were collected from 40 Anaplasma phagocytophila-infected sheep in 11 sheep flocks from four different counties of southern Norway. The presence and nature of the Anaplasma species were identified by microscopic detection of morulae, PCR, reverse line blot hybridization, and 16S rRNA gene sequencing. A. phagocytophila was identified in all of the samples, and sequencing of the 16S rRNA gene revealed the presence of four variants of A. phagocytophila. Two of these variants have been described before, but two were newly identified 16S rRNA variants of this species. A. phagocytophila variant 1 was found in nine flocks, A. phagocytophila variant 2 was found in four flocks, the A. phagocytophila prototype was found in two flocks, and A. phagocytophila variant 5 was found in one flock. In two flocks, some sheep were infected with A. phagocytophila variant 1, whereas others were infected with A. phagocytophila variant 2, and in three animals a double infection with two variants was registered. Analyses of the blood samples revealed that blood from sheep infected with A. phagocytophila variant 2 contained nearly twice as many neutrophils and eight times as many Anaplasma-infected neutrophils as blood from sheep infected with the A. phagocytophila variant 1. Furthermore, only 43% of the A. phagocytophila variant 2-infected sheep displayed antibody responses in an immune fluorescence assay, whereas 93% of the sheep with the A. phagocytophila variant 1-infected sheep were seropositive.

Bulgarian Ixodes ricinus ticks were examined for Ehrlichia and Borrelia coinfection: 34 and 32% of adult ticks and at least 2 and 10% of nymphs were positive for these infections, respectively. Coinfections and dual or triple Borrelia infections were frequent, although Ehrlichia phagocytophila heterogeneity was minimal. Multiple tick-borne bacteria coexist in I. ricinus ticks in southeastern Europe.

We report the results of a study of the prevalence of Ehrlichia and Borrelia species in 341 questing Ixodes ricinus ticks from two locations in southern Norway. The prevalences of Borrelia burgdorferi sensu lato and Ehrlichia spp. were, respectively, 16 and 11.5% at site 1 and 17 and 6% at site 2. Prevalence and species composition of Borrelia and Ehrlichia varied with location and date of collection. The dominant Borrelia species at both sites was Borrelia afzelii, followed by Borrelia burgdorferi sensu stricto. Borrelia garinii was found in only a single tick. The dominant member of the Ehrlichia group was a recently described Ehrlichia-like organism related to the monocytic ehrlichiae. Variants of Ehrlichia phagocytophila and the agent of human granulocytic ehrlichiosis were also found. The highest prevalences for B. afzelii, B. burgdorferi sensu stricto, and the Ehrlichia-like organism were observed in May. B. afzelii was most prevalent in females, less prevalent in nymphs, and least prevalent in males, while the prevalence of Ehrlichia was highest in nymphs, lower in females, and least in males. Double infections with B. afzelii and B. burgdorferi sensu stricto and with B. afzelii and the Ehrlichia-like organism were significantly overrepresented. Tick densities were highest in May, when densities of more than 200 ticks/100 m2 were observed, and declined during the summer months to densities as low as 20 ticks/100 m2. We conclude that estimates of the prevalence of tick-borne bacteria are sensitive to the choice of date and site for collection of ticks. This is the first study of tick-borne Borrelia and Ehrlichia in Norway and the lowest reported B. garinii prevalence in Northern Europe. The prevalence of the Ehrlichia-like organism is described for the first time in questing ticks.

The presence and distribution of Ehrlichia spp. and Borrelia burgdorferi sensu lato was demonstrated among ixodid ticks collected in the Baltic regions of Russia, where Lyme borreliosis is endemic. A total of 3,426 Ixodes ricinus and 1,267 Ixodes persulcatus specimens were collected, and dark-field microscopy showed that 265 (11.5%) I. ricinus and 333 (26.3%) I. persulcatus ticks were positive. From these samples, 472 dark-field-positive and 159 dark-field-negative ticks were subjected to PCR and subsequent reverse line blot hybridization. Fifty-four ticks (8.6%) carried Ehrlichia species, and 4 (0.6%) carried ehrlichiae belonging to the Ehrlichia phagocytophila complex, which includes the human granulocytic ehrlichiosis agent. The E. phagocytophila complex and an Ehrlichia-like species were detected only in I. ricinus whereas Ehrlichia muris was found exclusively in I. persulcatus, indicating a possible vector-specific infection. Borrelia garinii was found predominantly in I. persulcatus, but Borrelia afzelii was evenly distributed among the two tick species. Only two I. ricinus ticks carried B. burgdorferi sensu stricto, while Borrelia valaisiana and a newly identified B. afzelii-like species were found in 1.7 and 2.5% of all ticks, respectively. Of the dark-field-positive ticks, only 64.8% yielded a Borrelia PCR product, indicating that dark-field microscopy may detect organisms other than B. burgdorferi sensu lato. These observations show that the agent of human granulocytic ehrlichiosis may be present in ticks in the Baltic regions of Russia and that clinicians should be aware of this agent as a cause of febrile disease.

A case of granulocytic ehrlichiosis in a moose calf (Alces alces) in Norway is described. The animal was heavily infested with ticks (Ixodes ricinus), and died from a Klebsiella pneumoniae septicemia. Examination of blood smears from the calf revealed cytoplasmic inclusions (morulae) typical of infection with Ehrlichia phagocytophila in the granulocytes. Ehrlichia sp. was detected by polymerase chain reaction (PCR) in blood from the calf, and in the ticks. Sequence determination identified it as E. phagocytophila. This is the first report of ehrlichiosis in moose.

A sensitive and specific PCR hybridization assay was developed for the simultaneous detection and identification of Ehrlichia and Borrelia burgdorferi sensu lato. In separate assays the 16S rRNA gene of Ehrlichia species and the 23S-5S rRNA spacer region of B. burgdorferi sensu lato were amplified and labeled by PCR. These PCR products were used in a reverse line blot hybridization assay in which oligonucleotide probes are covalently linked to a membrane in parallel lines. Hybridization of the samples with the oligonucleotide probes on this membrane enabled the simultaneous detection and identification of Ehrlichia, B. burgdorferi, and Bartonella species in 40 different samples. The application of the assay to DNA extracts from 121 Ixodes ricinus ticks collected from roe deer demonstrated that 45% of these ticks carried Ehrlichia DNA. More than half of these positive ticks carried species with 16S rRNA gene sequences closely related to those of E. phagocytophila and the human granulocytic ehrlichiosis agent. The majority of the other positive ticks were infected with a newly identified Ehrlichia-like species. In addition, 13% of the ticks were infected with one or more B. burgdorferi genospecies. In more than 70% of the ticks 16S rRNA gene sequences for Bartonella species or other species closely related to Bartonella were found. In five of the ticks both Ehrlichia and B. burgdorferi species were detected.

Tissues from Dutch family dogs symptomatic for borreliosis according to established criteria and from infected but asymptomatic dogs were tested for Borrelia burgdorferi sensu lato DNA using a polymerase chain reaction. Subsequently, B. burgdorferi sensu stricto, B. garinii, B. afzelii, and B. valaisiana were identified by hybridization. Symptomatic dogs showed a higher prevalence of Borrelia in liver samples (9 of 15) than asymptomatic dogs (9 of 43) p = 0.0049. Overall, B. garinii was the most prevalent species and occurred together with up to three other species in on liver sample. B. burgdorferi sensu stricto however, was predominantly detected in samples of synovial membranes, skin, cerebrospinal fluid, bladder, heart, and bone marrow. Nine out of 10 symptomatic dogs with a very high antibody titre were positive for Borrelia DNA by PCR in one or more of these tissues. We conclude that dissemination in naturally infected European dogs occurs and that the two most prevalent species, B. burgdorferi sensu stricto and B. garinii, differ in their tropism.