Detection of four species of Borrelia burgdorferi sensu lato in Ixodes ricinus ticks collected from roe deer (Capreolus capreolus) in The Netherlands

Epiglottic disruption is an uncommon injury usually associated with significant supraglottic trauma. This injury may be overlooked because of the difficulty in examining the larynx or other associated severe injuries. We present two cases of clinically unsuspected epiglottic disruption that were first seen on MR images of the neck.     

Prevalence rates of Borrelia burgdorferi sensu lato in host-seeking Ixodes ricinus ticks in Europe

Ethical attitudes toward in vitro fertilization (IVF) have varied historically. This paper discusses the changes in views about the morality of human IVF by exploring the response to a pair of critical moments in the history of this reproductive technology: the announcement of the first successful IVF experiment by John Rock and Miriam Menkin in 1944 and the first successful IVF live birth of Louise Brown in 1978. By exploring public and private reactions to these two events in the context of wider social and political developments, this paper demonstrates the degree to which our notions of the morality of medical experimentation and of reproductive technologies are socially constructed and historically contingent.     

Characterization of Borrelia burgdorferi isolated from different organs of Ixodes ricinus ticks collected in nature

Borrelia burgdorferi was isolated from 22 out of 133 adult Ixodes ricinus ticks collected from vegetation at two sites in Switzerland. From 17 ticks, spirochetes could be isolated from more than one organ. When the different isolates obtained from one tick were compared by SDS-PAGE analysis, differences in the protein profiles were observed in 8 cases. The isolates were further compared by immunological methods using mono- and polyclonal antibodies. Differences were observed in the proteins of 31-35 kDa and 18-25 kDa. Genetic divergence among isolates was evaluated by use of a B. burgdorferi specific gene probe for ospA. Correlation could be observed between immunological differences in OspA defined by monoclonal antibody LA31 and genetic variation of ospA as judged by restriction fragment length polymorphism (RFLP). Our findings indicate that systemic infection in unfed I. ricinus adults, as reflected by isolation of B. burgdorferi from multiple organs of one tick, is more frequent (8/22, 36%) than previously described (5%). Moreover, the presence of different B. burgdorferi phenotypes/genotypes in one tick is described for the first time. The findings may have bearings (i) on the time of tick attachment required for spirochete transmission since borreliae are already present in the salivary glands of systemically infected ticks at the beginning of the blood meal and (ii) perhaps also on the diversity of B. burgdorferi phenotypes inoculated by these ticks.     

Infection rate of Ixodes ricinus ticks with Borrelia afzelii, Borrelia garinii, and Borrelia burgdorferi sensu stricto in Slovenia

In spring 1993, Ixodes ricinus ticks were collected from six regions of Slovenia to determine their overall rate of infection with Borrelia burgdorferi sensu lato and to assess the frequency of individual species in these tick populations. Ticks were dissected and midgut tissue inoculated into modified Barbour-Stoenner-Kelly (BSK II) medium. Borrelia isolates were differentiated into separate species using species-specific polymerase chain reaction (PCR) primers and by large restriction fragment pattern (LRFP) analysis. Infected ticks were found in all six regions surveyed. Spirochaetes were isolated from 69 of 363 ticks (19%): the isolation rate from adult female ticks was 35% (23/66 ticks cultured), from adult male ticks 22% (20/91), and from nymphal ticks 13% (26/206). Determination of the species of 60 isolates revealed that 32 were Borrelia afzelii (53%), 20 were Borrelia garinii (33%), and 8 were Borrelia burgdorferi sensu stricto (13%). In the Ljubljana region Borrelia afzelii and Borrelia garinii predominated (43% and 40%, respectively), whereas Borrelia burgdorferi sensu stricto constituted only 17% of isolates. In three other regions of the country Borrelia afzelii was isolated exclusively, although the number of isolates investigated was small. This study demonstrates the presence of all three European species of Borrelia burgdorferi sensu lato within the Slovenian tick population and also within a geographic area of less than 100 m2.     

Relationship between Borrelia burgdorferi sensu lato species, red squirrels (Sciurus vulgaris) and Ixodes ricinus in enzootic areas in Switzerland

Self-injurious behavior (SIB) occurs in about 10% of individually housed monkeys. Monkeys with SIB bite their own bodies frequently, occasionally inflicting wounds as a result. At present, there is no standard treatment for this phenomenon. We examined the effectiveness of puzzle feeders in alleviating SIB in monkeys with a veterinary record of self-inflicted wounding. Two groups of monkeys (SIB and controls) were exposed to puzzle feeders for a 6 week period. Three levels of maze difficulty were examined. All monkeys used the feeders, but manipulation was confined to a brief period immediately after the feeders were loaded each day (1000 h) and was infrequent during the later sampling periods (1100 and 1400 h). The most difficult maze yielded a slight increase in usage at 1100 h. During the puzzle feeder phase, whole body stereotypies, including pacing and rocking, were reduced substantially in all monkeys at 1000 h when feeder manipulation was at its highest. However, self-biting in the SIB group was unchanged. Some monkeys actually bit themselves while manipulating the feeder. Long-term effects on abnormal behavior were assessed by comparing behavior during the feeder phase to baseline periods and to a phase in which the monkeys were provisioned with treats placed directly into their food box. Whole body stereotypies, including pacing, were reduced during both treatment phases; however, the reduction was associated only with the 1000 h observation. Puzzle feeders were more effective than treats alone in alleviating whole body stereotypies. Self-biting was unchanged through all phases. Puzzle feeders are beneficial from the perspective of eliciting manipulation. They also yield transient reductions in whole body stereotypy, an effect that does not extend beyond the direct manipulation of the feeder. Puzzle feeders are ineffective in alleviating self-injurious behavior.     

Organization of ribosomal RNA genes in Borrelia burgdorferi sensu lato isolated from Ixodes ovatus in Japan

Borrelia burgdorferi sensu lato was obtained from adult ixodid ticks, Ixodes ovatus, collected in Nagano, Japan, and was named NT112. The genomic DNA was digested with enzymes, electrophoresed, blotted and hybridized with rRNA gene probes obtained from B. burgdorferi sensu stricto B31. The results showed that the borrelial chromosome contains a single rrs (16S rRNA gene) sequence and two copies of rrl/rrf (23S/5S rRNA genes) sequences. The rrl/rrf genes were tandemly repeated at intervals of 3.2 kb and were located separately from the rrs gene on the genome. Our findings indicate that the organization of rRNA genes in Borrelia from I. ovatus ticks is identical to that of B. burgdorferi sensu stricto.     

Simultaneous detection and genotyping of three genomic groups of Borrelia burgdorferi sensu lato in Dutch Ixodes ricinus ticks by characterization of the amplified intergenic spacer region between 5S and 23S rRNA genes

We developed a rapid and reliable method for the identification Borrelia burgdorferi sensu lato species in ticks. We used the DNA sequence polymorphism of the spacer region between 5S and 23S rRNA genes, which has been shown to be able to discriminate between eight genomic groups of B. burgdorferi sensu lato (D. Postic, M. Assous, P. A. D. Grimont, and G. Baranton, Int. J. Syst. Bacteriol. 44:743-752, 1994). Spacer DNA was amplified by PCR and was then hybridized to five membrane-bound oligonucleotides. The oligonucleotides were specific for B. burgdorferi sensu stricto, Borrelia garinii, Borrelia afzelii, and group VS116. A probe which reacted with all genomic groups of B. burgdorferi sensu lato was also used. Ninety-six ticks collected in the field were destructed by bead beating, and the supernatant was used directly in a PCR. B. burgdorferi sensu lato DNA was detected in 6 of 57 adult ticks (11%) and 9 of 39 nymphs (23%). B. garinii was found in three nymphs and four adults, three nymphs carried B. afzelii, and one adult and one nymph carried group VS116. Double infections with B. afzelii and group VS116 were found in two nymphs and one adult. Thus, our method can simultaneously identify three genomic groups of B. burgdorferi sensu lato in ticks collected in the field. This technique provides new ways to study the association of genomic groups present in ticks and the risk of Lyme borreliosis.     

Prevalence of Borrelia burgdorferi infection in Ixodes ricinus in central Italy

A scalp tumor from a 24-year-old male presenting unusual histological finding is described. The tumor was mostly composed of corpuscles resembling Meissner tactile bodies and contained almost no other components. Immunohistochemical studies revealed the tumor cells to be positive for S-100 protein and neuron-specific enolase, so we considered the tumor to be an unusual nevocellular nevus mostly comprised of nevic corpuscles.     

Identification of group VS116 strains among Borrelia burgdorferi sensu lato grown from the hard tick, Ixodes ricinus (Linnaeus, 1758) by off-coupled restriction fragment length polymorphism analysis

The degree of chemical shift similarity for homologous proteins has been determined from a chemical shift database of over 50 proteins representing a variety of families and folds, and spanning a wide range of sequence homologies. After sequence alignment, the similarity of the secondary chemical shifts of C alpha protons was examined as a function of amino acid sequence identity for 37 pairs of structurally homologous proteins. A correlation between sequence identity and secondary chemical shift rmsd was observed. Important insights are provided by examining the sequence identity of homologous proteins versus percentage of secondary chemical shifts that fall within 0.1 and 0.3 ppm thresholds. These results begin to establish practical guidelines for the extent of chemical shift similarity to expect among structurally homologous proteins.     

Novel Borrelia burgdorferi isolates from Ixodes scapularis and Ixodes dentatus ticks feeding on humans

Seven cultures of Borrelia burgdorferi differing from strains B31 and ZS7 were identified from among 99 isolates from Ixodes scapularis ticks and from white-footed mice (Peromyscus leucopus) and 1 isolate from an Ixodes dentatus tick. Five of the six novel isolates from I. scapularis and the isolate from I. dentatus were from ticks feeding on humans. The six isolates from I. scapularis lacked OspA and OspB, four possessed an OspD band, and two reacted with an anti-OspC monoclonal antibody. Restriction fragment length polymorphisms of HindIII-digested DNAs from six OspA-negative isolates did not hybridize with radiolabeled ospA or LA88 DNA, and only isolate 46047 hybridized with the pG gene. Fragments similar to those recorded for the standard B. burgdorferi sensu stricto strains B31 and ZS7 were obtained with the fla and the HSP70 genes. Pulsed-field gel electrophoresis patterns of DNA digested with MluI included the specific B. burgdorferi sensu stricto band at 135 kbp for the five OspA-negative isolates from I. scapularis ticks. The six novel isolates apparently lack the 55-kbp plasmid encoding OspA. The pG-containing plasmid may be missing from all but isolate 46047. The isolate from the I. dentatus tick was similar to previous isolates from I. dentatus ticks feeding on rabbits. None of the isolates could be recovered from inoculated C3H/HeNCrlBR or white-footed mice. All isolates reacted with sera from humans with early or late Lyme disease. Our studies demonstrate that these borreliae occur in ticks feeding on humans, and therefore, at least some humans in the northeastern United States are likely being exposed to borreliae other than the classic B31-type strains that have thus far been isolated from humans.     

Arguments against the involvement of Borrelia burgdorferi sensu lato in Alzheimer''s disease

The posterior prostatomembranous urethral stricture or distraction defect has historically been the most formidable challenge of stricture surgery. This uncommon lesion occurs most often as the sequelae of pelvic fracture injuries, or straddle trauma, and is associated with serious urethral disruption and separation--an injury that is often complicated by inappropriate initial management using substitution skin flap techniques with the development of recurrent stenosis, irreversible impotence, and occasional incontinence. Management by endoscopic techniques may be possible in patients with short strictures or in those after prostatectomy, but they rarely play a role in resolving the complex obliterated urethra with a significant defect [1]. Resolution of post-traumatic posterior urethral distraction defects and other posterior urethral pathologic conditions has dramatically improved over the past two decades despite an inaccessible subpublic location involving exposed sphincter-active and erectile neurovascular anatomy. The contemporary, perineal, one-stage bulboprostatic anastomotic operation as popularized by Turner-Warwick [20] with selective scar excision is a versatile procedure with a high patent lumen success. Patients undergoing anastomotic urethroplasty have a substained patent urethral lumen success rate approaching 100% versus those who have undergone urethral skin flap or patch repair, where the restricture rate in 5 and 10 years increases twofold to threefold [1, 20]. A patent urethra after an anastomotic urethroplasty at 6 months is free from further recurrent stricture and gives credence to Mr. Turner-Warwick's admonition that "urethra is the best substitute for urethra".     

OspA antibodies inhibit the acquisition of Borrelia burgdorferi by Ixodes ticks

Ixodes ticks are infected by Borrelia burgdorferi when larvae feed on spirochete-infected mice. We studied the acquisition of B. burgdorferi by larval ticks, characterized the production of outer surface protein A (OspA) by spirochetes entering larvae, and examined the effects of OspA antibodies on the establishment of B. burgdorferi infections in ticks. Most larvae were infected by spirochetes 24 to 48 h after placement on mice. OspA antibodies stained the first spirochetes observed in larvae, suggesting that OspA is synthesized early during the colonization of the vector. When OspA antibodies were administered to B. burgdorferi-infected mice and larvae were then placed on the animals, the severity of larval infection and the number of infected ticks (7 of 16) were decreased compared with that of controls (15 of 16). The inhibitory effects of OspA antibodies were observed with passive antibody transfer as well as active host-generated immunity. The lower larval infection rate observed in the presence of OspA antibodies was exacerbated after the larval molt since only 1 of 12 nymphs was infected, and none of the mice that were fed upon by these nymphs became infected with B. burgdorferi. Therefore, an OspA antibody response in mice altered the reservoir competence of the vertebrate host by inhibiting the movement of B. burgdorferi from the host to the vector.     

Distribution of Borrelia burgdorferi sensu lato genomic groups in Europe, a review

The survey is based on a total of 1263 records (738 isolations and 525 molecular DNA detections) of five Borrelia burgdorferi s.l. genomic groups available from 26 European countries: B. burgdorferi sensu stricto, B. afzelii, B. garinii, B. valaisiana (= VS116) and B. lusitaniae (= PoTiB2). It shows the geographic distribution, the source (ixodid ticks 802 records, fleas 2 records, mosquitoes 2 records, wild mammals 66 records, human patients 391 records) and the association of the genomic groups with particular clinical manifestations of Lyme borreliosis in humans (B. afzelii significantly prevails in skin lesions whereas B. garinii is more often associated with neuroborreliosis). The most frequent genomic groups in Europe are B. garinii (501 records) and B. afzelii (469 records). They occur across the continent and islands, whereas the third frequent genomic group, B. burgdorferi s.s. (201 records), has only rarely been isolated in eastern Europe. The remaining genomic groups, i.e. B. valaisiana (85 records) and B. lusitaniae (7 records) have only been isolated from, or detected in, Ixodes ricinus ticks in a few European countries.     

Borrelia burgdorferi sensu lato in Russia and neighbouring countries: high incidence of mixed isolates

A total of 365 isolates of Borrelia burgdorferi sensu lato from 12 major administrative territories of Russia (from St. Petersburg in the west to South Sakhalin in the east) and from the Czech Republic, Estonia, Lithuania, Byelorussia, Moldavia, Ukraine and Kirghizia were identified by analysis of restriction polymorphism of ribosomal rrf-rrl spacer amplicons. The isolates were obtained mainly from ixodes persulcatus and I. ricinus ticks. Other sources included small mammals, human patients and I. trianguliceps ticks. The results showed that B. garinii (two variants) together with B. afzelii circulated throughout the territories studied. The distribution of the variant NT29 of the species B. garinii, the most frequently isolated, was associated with that of I. persulcatus ticks. B. burgdorferi sensu stricto, and the species B. valaisiana and B. lusitaniae (formerly the genomospecies VS116 and PotiB2, respectively) were isolated only from I. ricinus ticks in the western part of the studied territories. None of these three species were found in 327 isolates from Russia where I. persulcatus is the most frequently distributed vector. This work also provides evidence for a high incidence of mixed Borrelia infections within vectors and hosts (9.3% of isolates were mixtures of Borrelia species). A detailed analysis of Borrelia species distribution over the territories studied is presented.     

Molecular typing of Borrelia burgdorferi sensu lato by randomly amplified polymorphic DNA fingerprinting analysis

To study whether pathogenic clusters of Borrelia burgdorferi sensu lato strains occur, we typed 136 isolates, cultured from specimens from patients (n = 49) with various clinical entities and from ticks (n = 83) or dogs (n = 4) from different geographic regions, by randomly amplified polymorphic DNA (RAPD) fingerprinting with four arbitrary primers. The RAPD patterns were reproducible up to the 95% similarity level as shown in duplicate experiments. In these experiments the purified DNAs prepared on different days, from different colonies, and after various passages were used as templates. With an intergroup difference of 55%, the 136 strains could be divided into seven genetic clusters. Six clusters comprised and corresponded to the established species B. burgdorferi sensu stricto (n = 23), Borrelia garinii (n = 39), Borrelia afzelii (n = 59), Borrelia japonica (n = 1), Borrelia valaisiana (n = 12), and genomic group DN127 (n = 1). One strain from a patient with erythema migrans (EM) did not belong to any of the species or genomic groups known up to now. The RAPD types of B. burgdorferi sensu stricto, B. garinii, and B. afzelii isolates, which may give rise to human Lyme borreliosis (LB), were associated with their geographic origins. A high degree of genetic diversity was observed among the 39 B. garinii strains, and six subgroups could be recognized. One of these comprised eight isolates from patients with disseminated LB only and no tick isolates. B. afzelii strains from patients with EM or acrodermatitis chronica atrophicans were not clustered in particular branches. Our study showed that RAPD analysis is a powerful tool for discriminating different Borrelia species as well as Borrelia isolates within species.     

Molecular characterization of Borrelia burgdorferi sensu lato from Slovenia revealing significant differences between tick and human isolates

The aim of the study was to evaluate the morphological and functional status of the liver in acute, oral cholinesterase inhibitors poisoning using static scintigraphy, hepatography and measurements of chosen enzymes activity. Considering the different clinical picture of cholinesterase inhibitors poisonings in people, it was necessary to estimate the poisoning severity and its dependence on the frequency and intensification of the liver lesion. Under examination there were 37 cholinesterase inhibitors orally poisoned patients, treated at the Department of Toxicology in the years 1992-1995. The examined group comprised 7 women (19%) and 30 men (81%). Organophosphate compounds poisoning was noted in 14 patients, and carbamates poisonings in 23 patients. The reference group comprised 30 healthy men aged 24 to 59 years not exposed to hepatotoxic agents. More than 90% of patients were classified as severe poisoned. Any fatal case was not noted. A differently intensified pathological changes of the liver dependent on age and poisoning severity were found in 97.2% of patients and their frequency was significantly higher than in the control group. Hepatographic picture revealed in 96.6% of cases the liver lesion. Hepatographic picture of the liver was also dependent on poisoning severity. The higher activity of AST, ALT, AP and higher bilirubin concentration in blood were noted in poisoned men compared to the control group. Control scintigraphic examination revealed a considerable improvement in the intensification of the liver scintigraphic picture in 40% of the patients and a higher intensification in 13% of the subjects. In 46.6% of the patients the intensification of scintigraphic changes remained at the same level. Considering arbitrary criteria for the degree of the liver lesion, the improvement in the intensification of hepatographic changes was noted in 42.8% of the patients; the intensification of the liver lesion was not noted even in one case. Analyzing the percentage of the liver lesion for each individual patient, improvement was noted in 92.8% of the examined patients, and the changes with the same level of intensification in 7.2%. Deterioration was not noted at all. Conclusion: The liver scintigraphy and hepatography combined with biochemical analysis allows to assess the liver condition in acute cholinesterase inhibitors poisoning.     

Rickettsia helvetica in Ixodes ricinus ticks in Sweden

In the present study further characterization of the amplified sequence of the citrate synthase gene of the spotted fever group Rickettsia isolated from Ixodes ricinus ticks in Sweden showed that it has 100% homology with the deposited sequence of the citrate synthase gene of Rickettsia helvetica. The restriction fragment length polymorphism (RFLP) pattern of an amplified 382-bp product of the citrate synthase sequence, defined by primers RpCS877 and RpCS1258, yielded fragments for our isolate that could be visualized as a double band that migrated at approximately 44 bp, another double band at 85 bp, and a single band at nearly 120 bp after digestion with the restriction enzyme AluI. When calculating a theoretical PCR-RFLP pattern of the sequence of the citrate synthase gene of R. helvetica from the known positions where the AluI enzyme cuts, we arrived at the same pattern that was obtained for our isolate, a pattern distinctly different from the previously published PCR-RFLP pattern for R. helvetica. Investigation of 125 living I. ricinus ticks showed a higher prevalence of rickettsial DNA in these ticks than we had found in an earlier study. Rickettsial DNA was detected by amplification of the 16S rRNA gene, for which a seminested primer system consisting of two oligonucleotide primer pairs was used. Of the 125 ticks, some were pooled, giving a total of 82 tick samples, of which 20 were found to be positive for the rickettsial DNA gene investigated. When considering the fact that some of the positive samples were pooled, the minimum possible prevalence in these ticks was 20 of 125 (16%) and the maximum possible prevalence was 46 of 125 (36.8%). These prevalence estimates conform to those of other studies of spotted fever group rickettsiae in hard ticks in Europe.     

Genospecies identification and characterization of Lyme disease spirochetes of genospecies Borrelia burgdorferi sensu lato isolated from rodents in Taiwan

Lyme disease spirochetes of the genospecies Borrelia burgdorferi sensu lato were identified and characterized for the first time in Taiwan. Seven isolates, designated TWKM1 to TWKM7, were purified from the ear tissues of three species of rodents captured from seven localities of Taiwan. The immunological characteristics of these Taiwan isolates were compared with those of other genospecies of Lyme disease spirochetes by analyzing the protein profiles and reactivities with B. burgdorferi-specific monoclonal antibodies (MAbs). The genospecies of these Taiwan isolates were also identified by the similarities in their plasmid profiles and differential reactivities with genospecies-specific PCR primers. Although two distinct protein profiles were observed among the seven Taiwan isolates, the MAb reactivities against the outer surface proteins of B. burgdorferi of all of these isolates were consistent with those of B. burgdorferi sensu lato. The similarities of the plasmid profiles also confirmed the identities of these Taiwan isolates. PCR analysis indicated that all of these Taiwan isolates were genetically related to the genospecies B. burgdorferi sensu stricto. These results demonstrate the first identification of Lyme disease spirochetes in Taiwan and also highlight the increasing demand for defining the reservoirs and vector ticks of B. burgdorferi. A serosurvey for Lyme disease infection in the human population of Taiwan may also be required.     

Seroprevalence of Borrelia burgdorferi sensu lato infection in blood donors and park rangers in relation to local habitat

Serum samples were obtained from blood donors in eleven selected locations in Ireland and tested for antibodies to Borrelia burgdorferi s.l. by enzyme immunoassay (EIA) and immunoblot (IB). The highest seroprevalence (8.7%) was found in Portumna, an area rated as high risk because of the presence of public access woodland harbouring both ticks and spirochaete reservoir hosts. The lowest seroprevalence (0.0%) was found in Ballina, an area where there is no suitable woodland though ticks are common on pastures grazed by cattle and sheep. These results support the findings of an earlier less extensive study and suggest that infection with B. burgdorferi s.l. in Ireland is mainly associated with mixed woodland containing deer and a variety of spirochaete reservoir hosts. The lack of association of tick-infested farmland with seropositive samples suggests that exposure to ticks alone is not a reliable indicator of risk of Lyme borreliosis. This is supported by the fact that none of 38 park rangers, all of whom are regularly bitten by ticks, were seropositive for B. burgdorferi s.l. The low overall seroprevalence of 3.4% in Ireland correlates with the rarity of clinical cases compared with continental European countries, and is due in part to the scarcity of high risk Lyme borreliosis habitat.     

Concomitant infection with tick-borne encephalitis virus and Borrelia burgdorferi sensu lato in patients with acute meningitis or meningoencephalitis

Chronic lymphocytic leukemia involves the skin in a small percentage of patients and may portend an ominous prognosis in some patients. We report a series of eight biopsies from seven different patients in which the cutaneous leukemic infiltrate was confined exclusively to the region immediately surrounding primary epithelial neoplasms including squamous cell carcinoma (6), basal cell carcinoma (1) and actinic keratosis (1). The malignant lymphocytes appear to constitute a host response to these neoplasms and do not appear to suggest a rapid downhill course for these patients. These observations serve to 1) suggest a new pattern of cutaneous involvement by leukemic cells and 2) offer some insights into potential cellular trafficking patterns of these neoplastic cells.