A sensitive and specific PCR hybridization assay was developed for the simultaneous detection and identification of Ehrlichiaand 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 EhrlichiaDNA. 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. burgdorferigenospecies. In more than 70% of the ticks 16S rRNA gene sequences forBartonella species or other species closely related toBartonella were found. In five of the ticks bothEhrlichia and B. burgdorferi species were detected.

We developed an efÞcient molecular method for the identiÞcation of the bloodmeal sources in the tick Ixodes ricinus (L.), the European vector of the agents of Lyme borreliosis and tick-borne encephalitis. A 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 ampliÞcation of the 12S rDNA molecular marker by using nondegenerate primers followed by a reverse line blot hybridization assay by using speciÞc 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 blood-meal 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 identiÞcation 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 Þeld-collected I. ricinus ticks. Moreover, the bloodmeal source was identiÞed 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.

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.

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 m 2 were observed, and declined during the summer months to densities as low as 20 ticks/100 m 2 . 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.

PurposeWe investigated the hypothesis that many total hip arthroplasty revisions that are classified as aseptic are in fact low-grade infections missed with routine diagnostics.MethodsIn 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.Results7 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.InterpretationAlthough 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.

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. phagocytophiluminfected lambs from one sheep flock in Norway and were stored at ؊70°C with 10% dimethyl sulfoxide as a cryoprotectant before being inoculated intravenously into susceptible lambs. The infectious blood contained, per ml, approximately 0.5 ؋ 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.

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.Neisseria meningitidis remains a major cause of meningitis and septicemia worldwide (4, 24). On the basis of the structure of its capsule polysaccharide, 13 serogroups are recognized. Polysaccharide vaccines against serogroups A, C, Y, and W135 are available. Due to poor immunogenicity and cross-reactivity with neural tissue, a vaccine based on the serogroup B polysaccharide is not available. A licensed vaccine against serogroup B meningococci based on other components of the pathogen will not become available for some time. While disease due to serogroup A, W135, and C meningococci is prevalent in Africa and Asia, in Europe and the Americas serogroup B meningococci are causing most of the cases of meningococcal disease. Study of the epidemiology of N. meningitidis increases knowledge about the spread of the bacterium and has identified particular clones with apparent increased virulence (15,20). Many different typing techniques have been employed to characterize meningococci. This is particularly true for the molecular techniques, which range from multilocus enzyme electrophoresis to PorA variable region typing and multilocus sequence typing (MLST) (1,3,5,20,27,33). MLST can now be considered the gold standard for genotyping N. meningitidis, and a large database is accessible via the Internet (http://pubmlst.org/neisseria/). MLST of N. meningitidis is a method using sequence data obtained from seven housekeeping genes. The alleles from these housekeeping genes are assigned allele numbers, and the combination of these allele numbers makes up a sequence type. MLST is a portable technique yielding unambiguous results and has been shown to be very suited for global epidemiology of meningococci (9,20,21,31). However, despite these obvious advantages, MLST is a costly and labor-intensive typing technique. To type a single strain, seven PCRs and 14 sequence reactions are required. Recently, multiple-locus variable-number ta...