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1.  Identification of Borrelia burgdorferi ospC genotypes in canine tissue following tick infestation: Implications for Lyme disease vaccine and diagnostic assay design 
Veterinary journal (London, England : 1997)  2013;198(2):10.1016/j.tvjl.2013.07.019.
In endemic regions, Lyme disease is a potential health threat to dogs. Canine Lyme disease manifests with arthritis-induced lameness, anorexia, fever, lethargy, lymphadenopathy and, in some cases, fatal glomerulonephritis. A recent study revealed that the regional mean for the percentage of seropositive dogs in the Northeast of the USA is 11.6%. The outer surface protein C (OspC) of Lyme disease spirochetes is an important virulence factor required for the establishment of infection in mammals. It is a leading candidate in human and canine Lyme disease vaccine development efforts. Over 30 distinct ospC phyletic types have been defined. It has been hypothesized that ospC genotype may influence mammalian host range. In this study, Ixodes scapularis ticks collected from the field in Rhode Island were assessed for infection with B. burgdorferi. Ticks were fed on purpose bred beagles to repletion and infection of the dogs was assessed through serology and PCR. Tissue biopsies (n = 2) were collected from each dog 49 days post-tick infestation (dpi) and the ospC genotype of the infecting strains determined by direct PCR of DNA extracted from tissue or by PCR after cultivation of spirochetes from biopsy samples. The dominant ospC types associated with B. burgdorferi canine infections differed from those associated with human infection, indicating a relationship between ospC sequence and preferred host range. Knowledge of the most common ospC genotypes associated specifically with infection of dogs will facilitate the rational design of OspC-based canine Lyme disease vaccines and diagnostic assays.
PMCID: PMC3872846  PMID: 23962611
Lyme disease; Borrelia burgdorferi; Ticks; Canine; OspC; Vaccine
2.  Microbes Bind Complement Inhibitor Factor H via a Common Site 
PLoS Pathogens  2013;9(4):e1003308.
To cause infections microbes need to evade host defense systems, one of these being the evolutionarily old and important arm of innate immunity, the alternative pathway of complement. It can attack all kinds of targets and is tightly controlled in plasma and on host cells by plasma complement regulator factor H (FH). FH binds simultaneously to host cell surface structures such as heparin or glycosaminoglycans via domain 20 and to the main complement opsonin C3b via domain 19. Many pathogenic microbes protect themselves from complement by recruiting host FH. We analyzed how and why different microbes bind FH via domains 19–20 (FH19-20). We used a selection of FH19-20 point mutants to reveal the binding sites of several microbial proteins and whole microbes (Haemophilus influenzae, Bordetella pertussis, Pseudomonas aeruginosa, Streptococcus pneumonia, Candida albicans, Borrelia burgdorferi, and Borrelia hermsii). We show that all studied microbes use the same binding region located on one side of domain 20. Binding of FH to the microbial proteins was inhibited with heparin showing that the common microbial binding site overlaps with the heparin site needed for efficient binding of FH to host cells. Surprisingly, the microbial proteins enhanced binding of FH19-20 to C3b and down-regulation of complement activation. We show that this is caused by formation of a tripartite complex between the microbial protein, FH, and C3b. In this study we reveal that seven microbes representing different phyla utilize a common binding site on the domain 20 of FH for complement evasion. Binding via this site not only mimics the glycosaminoglycans of the host cells, but also enhances function of FH on the microbial surfaces via the novel mechanism of tripartite complex formation. This is a unique example of convergent evolution resulting in enhanced immune evasion of important pathogens via utilization of a “superevasion site.”
Author Summary
Complement is an important arm of innate immunity. Activation of this plasma protein cascade leads to opsonization of targets for phagocytosis, direct lysis of Gram-negative bacteria, and enhancement of the inflammatory and acquired immune responses. No specific signal is needed for activation of the alternative pathway of complement, leading to its activation on all unprotected surfaces. Pathogenic microbes need to evade this pathway, and several species are known to recruit host complement inhibitor factor H (FH) to prevent the activation. FH is important for protection of host cells, too, as defects in FH lead to a severe autoreactive disease, atypical hemolytic uremic syndrome. We have now identified at the molecular level a common mechanism by which seven different microbes, Haemophilus influenzae, Bordetella pertussis, Pseudomonas aeruginosa, Streptococcus pneumoniae, Candida albicans, Borrelia burgdorferi and B. hermsii, recruit FH. All microbes bind FH via a common site on domain 20, which facilitates formation of a tripartite complex between the microbial protein, the main complement opsonin C3b, and FH. We show that, by utilizing the common microbial binding site on FH20, microbes can inhibit complement more efficiently. This detailed knowledge on mechanism of complement evasion can be used in developing novel antimicrobial chemotherapy.
PMCID: PMC3630169  PMID: 23637600
3.  Molecular Signaling Mechanisms of the Periopathogen, Treponema denticola 
Journal of Dental Research  2011;90(10):1155-1163.
In the healthy subgingiva, oral treponemes account for a small percentage of the total bacteria. However, in diseased periodontal pockets, treponemes thrive and become a dominant component of the bacterial population. Oral treponemes are uniquely adept at capitalizing on the environmental conditions that develop with periodontal disease. The molecular basis of adaptive responses of oral treponemes is just beginning to be investigated and defined. The completion of several treponeme genome sequences and the characterization of global regulatory systems provide an important starting point in the analysis of signaling and adaptive responses. In this review, we discuss existing literature focused on the genetic regulatory mechanisms of Treponema denticola and present an overview of the possible roles of regulatory proteins identified through genome analyses. This information provides insight into the possible molecular mechanisms utilized by oral spirochetes to survive in the periodontal pocket and transition from a minor to a dominant organism.
PMCID: PMC3173007  PMID: 21447698
treponemes; two-component regulatory systems; AtcRS; Hpk2; Rrp2; c-di-GMP
4.  Evidence that the BBA68 Protein (BbCRASP-1) of the Lyme Disease Spirochetes Does Not Contribute to Factor H-Mediated Immune Evasion in Humans and Other Animals  
Infection and Immunity  2006;74(5):3030-3034.
BBA68 (BbCRASP-1) of the Lyme disease spirochetes binds human factor H (FH) and FH-like protein 1 (FHL-1). Here we assess transcription of the BBA68 gene and production of BBA68 in infected mice and humans using real-time reverse transcriptase PCR and immunoblotting. The species specificity of FH binding to BBA68 was also tested. The data suggest that BBA68 does not play an important role in immune evasion in animals.
PMCID: PMC1459725  PMID: 16622245
5.  The bdr gene families of the Lyme disease and relapsing fever spirochetes: potential influence on biology, pathogenesis, and evolution. 
Emerging Infectious Diseases  2000;6(2):110-122.
Species of the genus Borrelia cause human and animal infections, including Lyme disease, relapsing fever, and epizootic bovine abortion. The borrelial genome is unique among bacterial genomes in that it is composed of a linear chromosome and a series of linear and circular plasmids. The plasmids exhibit significant genetic redundancy and carry 175 paralogous gene families, most of unknown function. Homologous alleles on different plasmids could influence the organization and evolution of the Borrelia genome by serving as foci for interplasmid homologous recombination. The plasmid-carried Borrelia direct repeat (bdr) gene family encodes polymorphic, acidic proteins with putative phosphorylation sites and transmembrane domains. These proteins may play regulatory roles in Borrelia. We describe recent progress in the characterization of the Borrelia bdr genes and discuss the possible influence of this gene family on the biology, pathogenesis, and evolution of the Borrelia genome.
PMCID: PMC2640845  PMID: 10756144
6.  Genetic Analysis of Borrelia garinii OspA Serotype 4 Strains Associated with Neuroborreliosis: Evidence for Extensive Genetic Homogeneity 
Journal of Clinical Microbiology  1999;37(12):3965-3970.
Infection with Borrelia garinii outer surface protein (Osp) A serotype 4 strains has been correlated with the development of neuroborreliosis in Lyme borreliosis patients in Europe. OspA serotype 4 isolates have been recovered primarily from human cerebrospinal fluid, suggesting a tropism for this environment. Previous studies with monoclonal antibodies directed against OspA and OspC demonstrated that OspA serotype 4 strains are antigenically closely related. In view of the pronounced antigenic and genetic variability that has been noted in the Osps of other Borrelia isolates, we sought to determine if OspA serotype 4 strains represent a recently emerged clonal lineage of B. garinii. Toward this goal, a representative group of OspA serotype 4 strains was analyzed for traits that typically exhibit hypervariability among isolates that cause Lyme borreliosis. The following criteria were assessed: (i) ospC sequences, (ii) plasmid composition, (iii) genomic restriction fragment length polymorphism (RFLP) patterns, and (iv) the RFLP patterns of the upstream homology box (UHB) element which flanks members of the UHB gene family at their 5′ end. Collectively, these analyses demonstrate genetic homogeneity, suggesting that OspA serotype 4 strains are a recently emerged clonal lineage with an apparent tropism for the central nervous system.
PMCID: PMC85856  PMID: 10565915
8.  Molecular and evolutionary analyses of a variable series of genes in Borrelia burgdorferi that are related to ospE and ospF, constitute a gene family, and share a common upstream homology box. 
Journal of Bacteriology  1996;178(19):5615-5626.
In this study we report on the molecular characterization of a series of genes that constitute a gene family related to ospE and ospF. Some members of this family appear to represent recombined or variant forms of ospE and ospF. Variant ospE and ospF genes were found in several Borrelia burgdorferi isolates, demonstrating that their occurrence is not a phenomenon relevant to only a single isolate. Hybridization analyses revealed that the upstream sequence originally identified 5' of the full-length ospEF operon exists in multiple copies ranging in number from two to six depending on the isolate. This repeated sequence, which we refer to as the upstream homology box (UHB), carries a putative promoter element. In some isolates, UHB elements were found to flank copies of ospE and ospF that exist independently of each other. We refer to this group of UHB-flanked genes collectively as the UHB gene family. The evolutionary relationships among UHB gene family members were assessed through DNA sequence analysis and gene tree construction. These analyses suggest that some UHB-flanked genes might actually represent divergent forms of other previously described genes. Analysis of the restriction fragment length polymorphism patterns of the UHB-flanked genes among B. burgdorferi isolates demonstrated that these patterns are highly variable among isolates, suggesting that these genes are not phylogenetically conserved. The variable restriction fragment length polymorphism patterns could indicate recombinational activity in these sequences. The presence of numerous copies of the UHB elements and the high degree of homology among UHB-flanked genes could provide the necessary elements to allow for homologous recombination, leading to the generation of recombination variants of UHB gene family members.
PMCID: PMC178399  PMID: 8824605
9.  Analysis of linear plasmid dimers in Borrelia burgdorferi sensu lato isolates: implications concerning the potential mechanism of linear plasmid replication. 
Journal of Bacteriology  1996;178(11):3357-3361.
The Borrelia genome is composed of a linear chromosome and a number of variable circular and linear plasmids. Atypically large linear plasmids of 92 to 105 kb have been identified in several Borrelia burgdorferi sensu lato isolates and characterized. These plasmids carry the p27 and ospAB genes, which in other isolates reside on a 50-kb plasmid. Here we demonstrate that these plasmids are dimers of the 50-kb ospAB plasmid (pAB50). The 94-kb plasmid from isolate VS116, pVS94, was an exception and did not hybridize with any plasmid gene probes. When this plasmid was used as a probe, homologous sequences in other isolates were not detected, suggesting that it is unique to isolate VS116. These analyses provide insight into the mechanism of linear plasmid replication and the mechanisms by which plasmid variability can arise.
PMCID: PMC178094  PMID: 8655522
10.  Assessing users' satisfaction through perception of usefulness and ease of use in the daily interaction with a hospital information system. 
The present study deals with the assessment of the subjective perception of the clinical core of the hospital information system (HIS) we are building. This HIS is not in use on a voluntary basis, but physicians and nurses use it for all the aspects of their inpatient care that have been informatized. Two questionnaires, aimed at the assessment of users perceived usefulness and ease of use of information technology, were utilized to: obtain feedback of the actual users' satisfaction as a predictive factor for the future life of the system, assess the real influence of the often-mentioned problems of age and unfamiliarity with computers of potential users, learn about the aspects which would enhance users' acceptance. The analysis of answers to the questionnaires has indicated a substantially positive perception of the system in terms of both usefulness and ease of use. This constitutes a good reason to keep on investing in the project. Even though this study has the intrinsic limit of the small dimension of the inquired population (53 users, equivalent to 98% of the personnel of the assessed clinical units), our data confirm the inconsistency of the relationship between perception of usefulness and age, and show "unfamiliarity with computers" as commonplace. On the other hand, it seems that the keystone for usefulness perception is the knowledge the users have of the system. An effort by the technical personnel in establishing a broader collaboration with the users, and in providing more exhaustive training and support may well be worthwhile.
PMCID: PMC2233074  PMID: 8947766
11.  Identification of novel insertion elements, restriction fragment length polymorphism patterns, and discontinuous 23S rRNA in Lyme disease spirochetes: phylogenetic analyses of rRNA genes and their intergenic spacers in Borrelia japonica sp. nov. and genomic group 21038 (Borrelia andersonii sp. nov.) isolates. 
Journal of Clinical Microbiology  1995;33(9):2427-2434.
Borrelia spp. associated with Lyme disease possess an rRNA gene organization consisting of a single 16S rRNA gene followed by a spacer of several kilobases and a tandem repeat of a 23S (rrl)-5S (rrf) rRNA gene cluster. The restriction fragment length polymorphism (RFLP) patterns for these genes have been widely used to classify Lyme disease spirochete isolates. We analyzed the rRNA gene organization and sequences for two Ixodes ovatus isolates from Japan (IKA2 and HO14) and two group 21038 isolates associated with Ixodes dentatus ticks or rabbits from North America (isolates 21038 and 19857). This analysis revealed unique polymorphisms not previously described in other Lyme disease spirochete isolates. The molecular basis of these polymorphisms was determined by Southern blotting and PCR analyses. Only one continuous copy of the rrl-rrf gene cluster was identified in isolates IKA2, 19857, and 21038. The second rrl-rrf gene cluster is entirely absent from the IKA2 genome. In isolates 19857 and 21038, an intervening sequence is present, resulting in a fragment rrlB gene. The insertion site of this intervening sequence element differed in each isolate. While isolates 19857 and 21038 were found to carry a fragmented rrlB gene, they lacked rrfB. To determine if these rRNA polymorphisms were indicative of an underlying phylogenetic divergence, sequence analysis of the 16S rRNA (rrs) genes was conducted. The phylogenies inferred from rrs sequence analysis suggest that the polymorphisms resulted from recent mutational events.(ABSTRACT TRUNCATED AT 250 WORDS)
PMCID: PMC228430  PMID: 7494041
12.  Interaction and dialogue between the users and the patient record core of hospital information system: looking for a solution. 
The lack of good user interface, in terms of both modality of dialogue and system behaviour is the major impediment to the acceptance and routine use of the computer based patient record (CPR) core of a hospital information system. We describe here the adopted approach to face the daily users' needs, overcoming the pitfalls of the paper based patient record (PPR), and giving the physicians an exhaustive modality for CPR inspection.
PMCID: PMC2579138  PMID: 8563328
13.  Analysis of the distribution and molecular heterogeneity of the ospD gene among the Lyme disease spirochetes: evidence for lateral gene exchange. 
Journal of Bacteriology  1994;176(15):4572-4582.
Analysis of the ospD gene has revealed that this gene is not universal among Lyme disease spirochete isolates. The gene was found to be carried by 90, 50, and 24% of the Borrelia garinii, B. afzelii, and B. burgdorferi isolates tested. Size variability in the ospD-encoding plasmid was also observed. Sequence analysis has demonstrated the presence of various numbers of a 17-bp repeated sequence in the upstream control (promoter) region of the gene. In addition, a region within the coding sequence where various insertions, deletions, and direct repeats occur was identified. ospD gene sequences from 31 different isolates were determined and utilized in pairwise sequence comparisons and construction of a gene tree. These analyses suggest that the ospD gene was the target of several recombinational events and that the gene was recently acquired by Lyme disease spirochetes and laterally transferred between species.
PMCID: PMC196277  PMID: 7913928
14.  gyrB mutations in coumermycin A1-resistant Borrelia burgdorferi. 
Journal of Bacteriology  1994;176(10):3072-3075.
We have isolated and characterized mutants of Borrelia burgdorferi that are resistant to the antibiotic coumermycin A1, which targets the B subunit of DNA gyrase. Mutants had either 100- or 300-fold higher resistance to coumermycin A1 than wild-type B. burgdorferi. In each case, a single point mutation in the gyrB gene converted Arg-133 to Gly or Ile. Mutations in the homologous Arg residue of Escherichia coli DNA gyrase are also associated with resistance to coumarin antimicrobial agents.
PMCID: PMC205466  PMID: 8188609
15.  Identification of a protein in several Borrelia species which is related to OspC of the Lyme disease spirochetes. 
Journal of Clinical Microbiology  1993;31(10):2577-2583.
Using oligonucleotide probes which have previously been shown to be specific for the ospC gene found in the Lyme disease spirochete species Borrelia burgdorferi, B. garinii, and group VS461, we detected an ospC homolog in other Borrelia species including B. coriaceae, B. hermsii, B. anserina, B. turicatae, and B. parkeri. In contrast to the Lyme disease spirochetes, which carry the ospC gene on a 26-kb circular plasmid, we mapped the gene in other Borrelia species to linear plasmids which varied in size among the isolates tested. Some isolates carry multiple copies of the gene residing on linear plasmids of different sizes. The analyses conducted here also demonstrate that these Borrelia species contain a linear chromosome. Northern (RNA) blot analyses demonstrated that the gene is transcriptionally expressed in all species examined. High levels of transcriptional expression were observed in some B. hermsii isolates. Transcriptional start site analyses revealed that the length of the untranslated leader sequence was identical to that observed in the Lyme disease spirochete species. By Western blotting (immunoblotting) with antiserum (polyclonal) raised against the OspC protein of B. burgdorferi, we detected an immunoreactive protein of the same molecular weight as the OspC found in Lyme disease spirochete species. The results presented here demonstrate the presence of a protein that is genetically and antigenically related to OspC which is expressed in all species of the genus Borrelia tested.
PMCID: PMC265939  PMID: 8253952
16.  Variability of osp genes and gene products among species of Lyme disease spirochetes. 
Infection and Immunity  1993;61(6):2611-2617.
A comparison of the osp operon in 24 Lyme disease isolates, including representatives from each of the three established species, Borrelia burgdorferi, Borrelia garinii, and group VS461, was conducted. Several properties were assessed to determine whether the variability observed in this operon was reflective of the species of the isolate. At the transcriptional level, start site and Northern (RNA) blot analyses were conducted. B. garinii and VS461 group isolates were found to possess an untranslated leader sequence 6 nucleotides longer than that observed in B. burgdorferi isolates. By Northern blot analyses all Lyme disease isolates, except the B. garinii isolate VS102, were found to produce a polycistronic full-length ospAB message. Isolate VS102 produced a truncated message lacking the ospB portion of the transcript. Southern blot analyses suggest that the deletion occurred at the DNA level and was not due to a posttranscriptional event. Analysis of the outer surface proteins by two-dimensional gel electrophoresis demonstrated that the OspB isoelectric points were variable, with the OspB of B. garinii isolates exhibiting a pronounced acidic shift. The reactivity of different isolates to OspA and -B monoclonal antibodies and to a hyperimmune anti-ospAB serum was also variable. The results presented here demonstrate genotypic and phenotypic heterogeneity in the osp operon at both the inter- and intraspecies levels. The results have implications concerning the use of the osp genes or their gene products in the development of a Lyme disease vaccine, as diagnostic markers of Lyme disease, and in subtyping of Lyme disease isolates.
PMCID: PMC280891  PMID: 8500899
17.  Transcriptional analyses and mapping of the ospC gene in Lyme disease spirochetes. 
Journal of Bacteriology  1993;175(4):926-932.
In Lyme disease spirochetes, the ospC gene encodes a 22.7-kDa protein referred to as either the pC or the OspC protein. Using a variety of electrophoretic approaches followed by Southern blotting and probing with oligonucleotide probes, we mapped the ospC gene to a circular 26-kb plasmid. The ospC gene represents the first gene to be mapped to a circular plasmid in Lyme disease spirochetes. The occurrence of this gene in isolates belonging to each of the three Lyme disease-associated species, Borrelia burgdorferi, Borrelia garinii, and the VS461 group, was evaluated. The ospC gene was found to occur in all 21 isolates tested from each of the three species. Differential hybridization with a series of ospC probes in both Northern (RNA) and Southern blot analyses demonstrated that there is sequence variability in the ospC gene among isolates. While the gene was found to be present in all isolates, not all actively transcribed the gene. Transcriptional start site analyses suggest that the gene may be under the control of multiple promoters that are highly similar in nucleotide sequence.
PMCID: PMC193003  PMID: 7679385
18.  Development of polymerase chain reaction primer sets for diagnosis of Lyme disease and for species-specific identification of Lyme disease isolates by 16S rRNA signature nucleotide analysis. 
Journal of Clinical Microbiology  1992;30(11):2830-2834.
We have determined and compared partial 16S rRNA sequences from 23 Lyme disease spirochete isolates and aligned these with 8 sequences previously presented. The 16S rRNA signature nucleotide compositions were defined for each isolate and compared with the genomic species signature nucleotide sets previously established. To identify positions truly indicative of species classification which could serve as targets for polymerase chain reaction species-specific identification primers, 16S rRNA-based phylogenetic analyses were conducted. On the basis of the identified signature nucleotides, we designed polymerase chain reaction primer sets which (i) amplify all spirochete species associated with Lyme disease and (ii) differentiate between these species. The primer sets were tested on 38 Borrelia isolates associated with Lyme disease and were found to be sensitive and specific. All Lyme disease isolates tested were amplification positive. These primers allow for the rapid species identification of Lyme disease isolates.
PMCID: PMC270537  PMID: 1280643
20.  Species-specific identification of and distinction between Borrelia burgdorferi genomic groups by using 16S rRNA-directed oligonucleotide probes. 
Journal of Clinical Microbiology  1992;30(3):628-632.
Examination of a number of previously published aligned Borrelia 16S rRNA sequences revealed the presence of regions which could serve as oligonucleotide probe targets for both species-specific identification of Borrelia burgdorferi and distinction between genomic groups. Total cellular RNA isolated from Borrelia cultures was used in slot blot analysis. Radiolabeled oligonucleotides designed to hybridize to specific 16S rRNA targets were used as probes. These probes allowed for both species-specific identification and genomic group typing of B. burgdorferi.
PMCID: PMC265123  PMID: 1372620
21.  Phylogenetic analysis of the genus Borrelia: a comparison of North American and European isolates of Borrelia burgdorferi. 
Journal of Bacteriology  1992;174(1):241-244.
We have sequenced the 16S rRNA molecules from four species of Borrelia and from six isolates of Borrelia burgdorferi via the reverse transcriptase primer extension method. The sequences were aligned and evolutionary relationships were determined, including the calculation of evolutionary distances and the construction of a phylogenetic tree. These analyses demonstrate significant divergence among B. burgdorferi isolates, with the European isolates G1 and G2 residing most distant from the main cluster. Signature nucleotides which distinguish B. burgdorferi from all other members of this genus and which distinguish the European isolates G1 and G2 from the North American isolates B31, Sh-2-82, and 1352 were identified. Finally, Southern blot analyses were performed to compare the restriction patterns of the genes coding for rRNA and to relate our data to the grouping scheme of Postic et al. (D. Postic, C. Edlinger, C. Richaud, F. Grimont, J. Dufresne, P. Perolat, G. Baranton, and P. A. D. Grimont, Res. Microbiol. 141:465-475, 1990).
PMCID: PMC205701  PMID: 1370282
22.  Identification of defined sequences in domain V of E. coli 23S rRNA in the 50S subunit accessible for hybridization with complementary oligodeoxyribonucleotides. 
Nucleic Acids Research  1988;16(4):1603-1615.
The accessibility of specific sequences in domain V of E. coli 23s rRNA in the 50S subunit to complementary oligodeoxyribonucleotides (cDNA) has been investigated. The apparent percentage of subunits engaged in complex formation was determined by incubation of radiolabeled cDNA probe with 50S subunits, followed by nitrocellulose membrane filtration of the reaction mixtures and measurement of the bound radiolabeled cDNA probes by liquid scintillation counting of the filters. The site(s) of hybridization were determined by digestion of the RNA in the RNA/DNA heteroduplex by RNase H. The results of this study indicated that single-stranded sequences, 2058-2062, 2448-2454, 2467-2483, and 2497-2505 were available for hybridization to cDNA probes. Bases 2489-2496, which have been postulated to be base paired with 2455-2461 were also accessible for hybridization.
PMCID: PMC336338  PMID: 3279396

Results 1-22 (22)