Species identification of <Emphasis Type="Italic">Ixodes granulatus</Emphasis> (Acari: Ixodidae) based on internal transcribed spacer 2 (ITS2) sequences

To investigate the genetic specificity of Ixodes granulatus ticks collected from Taiwan, the genetic identities and phylogenetic relationships were analyzed by comparing the sequences of the internal transcribed spacer 2 (ITS2) region obtained from 27 strains of ticks representing twelve species of Ixodes. Five major clades can be easily distinguished by neighbour-joining analysis and were congruent by maximum-parsimony method. All these I. granulatus ticks collected from Taiwan and Japan were genetically affiliated to a monophyletic group with highly homogeneous sequences (95.8–99.5% similarity), and can be discriminated from other species and subgenera of Ixodes ticks with a sequence divergence ranging from 13.6% to 62.9%. Moreover, interspecific analysis revealed that four distinct lineages are evident between Ixodes ticks, and all these I. granulatus ticks collected from Taiwan and Japan belong to the same lineage. Our results provide the first investigation on the genetic specificity of I. granulatus ticks, and demonstrate that all these I. granulatus ticks represent a unique lineage distinct from other species and subgenera of Ixodes ticks. The feasibility of ITS2-based genetic analysis for species-specific identification of I. granulatus ticks around East Asia was highly anticipated.     

Molecular detection of <Emphasis Type="Italic">Borrelia valaisiana</Emphasis>-related spirochetes from <Emphasis Type="Italic">Ixodes granulatus</Emphasis> ticks in Taiwan

Borrelia valaisiana-related spirochetes were detected for the first time in Ixodes granulatus ticks collected in Taiwan. The genetic identities of these detected spirochetes were determined by analyzing the gene sequences amplified by a genospecies-specific polymerase chain reaction assay based on the outer surface protein A (OspA) gene of B. burgdorferi sensu lato. Phylogenetic relationships were analyzed by comparing the sequences of OspA gene obtained from 35 strains of Borrelia spirochetes representing six genospecies of Borrelia. Eight major clades can be easily distinguished by neighbour-joining analysis and were congruent by maximum-parsimony method. Except one strain (KH-74), all these Borrelia spirochetes of Taiwan were genetically affiliated to the same clade with highly homogeneous sequences (97.8–100% similarity), and can be discriminated from other groups of B. valaisiana and other genospecies of Borrelia spirochetes with a sequence divergence ranging from 3 to 19.6%. Moreover, intraspecific analysis also revealed that three distinct groups are evident between the same species of B. valaisiana spirochetes detected in Taiwan. Our results provide the first evidence of B. valaisiana spirochetes detected in I. granulatus ticks collected in Taiwan and demonstrate that all these B. valaisiana spirochetes of Taiwan represent three major groups distinct from the European group of B. valaisiana spirochetes.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

Phylogenetic Studies of <Emphasis Type="Italic">Gordonia</Emphasis> Species Based on <Emphasis Type="Italic">gyrB</Emphasis> and <Emphasis Type="Italic">secA1</Emphasis> Gene Analyses

Phylogenetic relations within the genus Gordonia were analyzed using partial gyrB and secA1 gene sequences of 23 type species in comparison with those of 16S rRNA gene. The gyrB and secA1 phylogenies showed agreement with that constructed using 16S rRNA gene sequences. The degrees of divergence of the gyrB and secA1 genes were approximately 3.4 and 1.7 times greater, respectively, than that of 16S rRNA gene. The gyrB gene showed more discriminatory power than either the secA1 or 16S rRNA gene, facilitating clear differentiation of any two Gordonia species using gyrB gene analysis. Our data indicate that gyrB and secA1 gene sequences are useful as markers for phylogenetic study and identification at the species level of the genus Gordonia.     

Molecular phylogeny of the blind cavefish <Emphasis Type="Italic">Phreatichthys andruzzii</Emphasis> and <Emphasis Type="Italic">Garra barreimiae</Emphasis> within the family Cyprinidae

The phylogenetic relationships of two cavefish, Phreatichthys andruzzii and Garra barreimiae, belonging to the family Cyprinidae, were investigated by sequencing the mitochondrial cytochrome b gene. These cavefish species are native to Somalia (eastern Africa) and Oman (southeastern Arabian peninsula), respectively, and so far no molecular support to their taxonomy and phylogenetic position was ever provided. The analysis of cytochrome b sequences showed that the species are monophyletic taxa, closely related to each other and to other species of the genus Garra. Molecular clock calculations allowed to date the origin of these hypogaean species back to the Plio-Pleistocene and support the hypothesis that African cyprinids originated from Miocenic immigrations of Asian ancestors.     

Detection of a novel spotted fever group rickettsia in <Emphasis Type="Italic">Amblyomma parvum</Emphasis> ticks (Acari: Ixodidae) from Argentina

The present study evaluated the rickettsial infection in Amblyomma parvum ticks collected in Northwestern Córdoba Province, Argentina. Each tick was subjected to DNA extraction and tested by polymerase chain reaction (PCR) targeting fragments of the rickettsial genes gltA and ompB. Nine (69.2%) out of 13 adult ticks yielded expected PCR products for the two rickettsial genes. Products from the ompB PCR were sequenced, generating DNA sequences 100% identical for the nine PCR-positive ticks. Three of these ticks were tested in another battery of PCR targeting fragments of the rickettsial genes gltA, htrA, and ompA. Products from the gltA, htrA, and ompA PCRs were sequenced generating DNA sequences 100% identical for the three PCR-positive ticks. The rickettsia detected in the A. parvum ticks was designated as Rickettsia sp. strain Argentina. Phylogenetic analyses performed with partial sequences of the rickettsial genes gltA, htrA, ompB, and ompA showed that Rickettsia sp. strain Argentina belonged to the spotted fever group, being distinct from all known Rickettsia species and genotypes available in GenBank, representing possibly a new Rickettsia species. This was the first evidence of rickettsial infection in the tick A. parvum, and the third report of rickettsial infection among the Argentinean tick fauna. The role of Rickettsia sp. strain Argentina as a human pathogen is unknown. Further studies are needed to obtain tissue-cultured isolates of Rickettsia sp. strain Argentina, in order to better characterize it and to determine its taxonomic status as a new species.     

IS<Emphasis Type="Italic">Rm31</Emphasis>, a new insertion sequence of the IS<Emphasis Type="Italic">66</Emphasis> family in<Emphasis Type="Italic"> Sinorhizobium meliloti</Emphasis>

Sinorhizobium meliloti natural populations show a high level of genetic polymorphism possibly due to the presence of mobile genetic elements such as insertion sequences (IS), transposons, and bacterial mobile introns. The analysis of the DNA sequence polymorphism of the nod region of S. meliloti pSymA megaplasmid in an Italian isolate led to the discovery of a new insertion sequence, ISRm31. ISRm31 is 2,803 bp long and has 22-bp-long terminal inverted repeat sequences, 8-bp direct repeat sequences generated by transposition, and three ORFs (A, B, C) coding for proteins of 124, 115, and 541 amino acids, respectively. ORF A and ORF C are significantly similar to members of the transposase family. Amino acid and nucleotide sequences indicate that ISRm31 is a member of the IS66 family. ISRm31 sequences were found in 30.5% of the Italian strains analyzed, and were also present in several collection strains of the Rhizobiaceae family, including S. meliloti strain 1021. Alignment of targets sites in the genome of strains carrying ISRm31 suggested that ISRm31 inserts randomly into S. meliloti genomes. Moreover, analysis of ISRm31 insertion sites revealed DNA sequences not present in the recently sequenced S. meliloti strain 1021 genome. In fact, ISRm31 was in some cases linked to DNA fragments homologous to sequences found in other rhizobia species.     

Detection of <Emphasis Type="Italic">Candida</Emphasis><Emphasis Type="Italic">dubliniensis</Emphasis> in Venezuela

Over the past decades there has been a significant increase in fungal infections caused by Candida species, and continues to be common in immunocompromised individuals infected with the human immunodeficiency virus (HIV). Although Candida albicans remains the fungal species most frequently isolated as an opportunistic oral pathogen, other non-albicans are often identified in this cohort of patients, including C. dubliniensis. This yeast is closely related to and shares many phenotypic characteristics with C. albicans. Colonies of these two species appear morphologically identical when not grown on special media. The shared phenotypic characteristics of C. dubliniensis and C. albicans suggest that many C. dubliniensis isolates may have been misidentified as C. albicans in the past. The present studies aim is to recover and identify C. dubliniensis, and presumptive clinical C. albicans, from the oral cavities of HIV-seropositive individuals, comparing conventional media to obtain a simple, low-cost and reliable identification system for C. dubliniensis. A total of 16 isolates (3,98%) had been obtained from 402 HIV infected individuals with recurrent oropharyngitis and were identified as C. dubliniensis. Out of these C. dubliniensis isolates 19% were resistant, with MICs above 64 μg/ml to fluconazole. This constitutes, to the authors knowledge the first recovery of this organism in Venezuela.     

Identification of <Emphasis Type="Italic">S</Emphasis>-haplotype-specific F-box gene in Japanese plum (<Emphasis Type="Italic">Prunus salicina</Emphasis> Lindl.)

Self-incompatibility has been studied extensively at the molecular level in Solanaceae, Rosaceae and Scrophulariaceae, all of which exhibit gametophytic self-incompatibility controlled by a single polymorphic locus containing at least two linked genes, i.e., the S-RNase gene and the pollen-expressed SFB/SLF (S-haplotype-specific F-box/S-locus F-box) gene. However, the SFB gene in Japanese plum (Prunus salicina Lindl.) has not yet been identified. We determined eight novel sequences homologous to the SFB genes of other Prunus species and named these sequences PsSFB. The gene structure of the SFB genes and the characteristic domains in deduced amino acid sequences were conserved. Three sequences from 410 to 2,800 bp of the intergenic region between the PsSFB sequences and the S-RNase alleles were obtained. The eight identified PsSFB sequences showed S-haplotype-specific polymorphism, with 74–83% amino acid identity. These alleles were exclusively expressed in the pollen. These results suggest that the PsSFB alleles are the pollen S-determinants of GSI in Japanese plum. Nucleotide sequence data reported are available in the NCBI database under the accession numbers DQ849084–DQ849090 and DQ849118.     

Evidence of <Emphasis Type="Italic">Babesia microti</Emphasis> infection in multi-infected <Emphasis Type="Italic">Ixodes persulcatus</Emphasis> ticks in Russia

To detect Babesia-infected Ixodes persulcatus Shulze in a suburb of St. Petersburg, Russia, 738 adult ticks were studied using Babesia specific primers and PCR techniques. The entire sample (more than 1,200 individuals) was screened for the presence of Borrelia spp., Ehrlichia spp. and tick-borne encephalitis virus (TBEV). All 7 ticks infected with Babesia microti, were also infected with other pathogens (all 7 among 417 infected ticks, zero amongst the remaining 321 naive ones (χ² = 5.25, p < 0.05). Babesia microti occurred twice with Borrelia afzelii, 3 times with Borrelia garinii, once with both, and once with both B. garinii and TBEV. The prevalence of infection with Borrelia spp. was 34.0%, with Ehrlichia spp. 6.2%, with TBEV 1.5%, and with Ba microti 0.9%. Babesia microti infection was not found in combination with Ehrlichia sp. or Borrelia burgdorferi sensu stricto. The latter pathogen (prevalence 2.6%), just like Ba. microti, was not encountered as a monoinfection. The data suggest that Ba. microti infection can only survive in I. persulcatus in combination with Borrelia spp. (7 of 7 infections). The disease in humans is more severe and longer-lasting when more than one pathogen is involved. Our observations show that the well known St. Petersburg focus of tick-borne encephalitis and Lyme disease is also a focus of ehrlichiosis and babesiosis. This revised version was published online in July 2006 with corrections to the Cover Date.     

Molecular phylogenetic analysis shows that <Emphasis Type="Italic">Amanita ponderosa</Emphasis> and <Emphasis Type="Italic">A. curtipes</Emphasis> are distinct species

Amanita curtipes and A. ponderosa are two Mediterranean taxa sharing a number of morphological features as well as their habitat. Their synonymy or variety status has been proposed by several authors. To clarify this taxonomic issue we have sequenced the D1-D2 domains of the 28S rRNA gene as well as the complete ITS1-5.8S-ITS2 region of several specimens of the two species collected in Spain, and aligned these sequences with those from other Amanita species. Molecular phylogenetic analysis based on the two regions revealed that A. ponderosa and A. curtipes are clearly distinct species. The distribution of Amanita species in the phylogenetic trees was consistent with the division of the genus in subgenera and sections as proposed by previous authors. Sequences of A. ponderosa and A. curtipes were grouped in a monophyletic cluster together with other species of the section Amidella. However, A. ponderosa was closer to other species in the section, such as A. peckiana and A. volvata, than to A. curtipes. We also indicate the macromorphological characters that are most useful to reliably distinguish A. ponderosa and A. curtipes.     

Trans-specific <Emphasis Type="Italic">S</Emphasis>-RNase and SFB alleles in <Emphasis Type="Italic">Prunus</Emphasis> self-incompatibility haplotypes

Self-incompatibility in the genus Prunus is controlled by two genes at the S-locus, S-RNase and SFB. Both genes exhibit the high polymorphism and high sequence diversity characteristic of plant self-incompatibility systems. Deduced polypeptide sequences of three myrobalan and three domestic plum S-RNases showed over 97% identity with S-RNases from other Prunus species, including almond, sweet cherry, Japanese apricot and Japanese plum. The second intron, which is generally highly polymorphic between alleles was also remarkably well conserved within these S-allele pairs. Degenerate consensus primers were developed and used to amplify and sequence the co-adapted polymorphic SFB alleles. Sequence comparisons also indicated high degrees of polypeptide sequence identity between three myrobalan and the three domestic plum SFB alleles and the corresponding Prunus SFB alleles. We discuss these trans-specific allele identities in terms of S-allele function, evolution of new allele specificities and Prunus taxonomy and speciation. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

<Emphasis Type="Italic">Drosophila</Emphasis><Emphasis Type="Italic">P</Emphasis> transposons of the urochordata <Emphasis Type="Italic">Ciona intestinalis</Emphasis>

P transposons belong to the eukaryotic DNA transposons, which are transposed by a cut and paste mechanism using a P-element-coded transposase. They have been detected in Drosophila, and reside as single copies and stable homologous sequences in many vertebrate species. We present the P elements Pcin1, Pcin2 and Pcin3 from Ciona intestinalis, a species of the most primitive chordates, and compare them with those from Ciona savignyi. They showed typical DNA transposon structures, namely terminal inverted repeats and target site duplications. The coding region of Pcin1 consisted of 13 small exons that could be translated into a P-transposon-homologous protein. C. intestinalis and C. savignyi displayed nearly the same phenotype. However, their P elements were highly divergent and the assumed P transposase from C. intestinalis was more closely related to the transposase from Drosophila melanogaster than to the transposase of C. savignyi. The present study showed that P elements with typical features of transposable DNA elements may be found already at the base of the chordate lineage. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Invasion of the Lyme Disease Vector <Emphasis Type="Italic">Ixodes scapularis</Emphasis>: Implications for <Emphasis Type="Italic">Borrelia burgdorferi</Emphasis> Endemicity

Lyme disease risk is increasing in the United States due in part to the spread of blacklegged ticks Ixodes scapularis, the principal vector of the spirochetal pathogen Borrelia burgdorferi. A 5-year study was undertaken to investigate hypothesized coinvasion of I. scapularis and B. burgdorferi in Lower Michigan. We tracked the spatial and temporal dynamics of the tick and spirochete using mammal, bird, and vegetation drag sampling at eight field sites along coastal and inland transects originating in a zone of recent I. scapularis establishment. We document northward invasion of these ticks along Michigan’s west coast during the study period; this pattern was most evident in ticks removed from rodents. B. burgdorferi infection prevalences in I. scapularis sampled from vegetation in the invasion zone were 9.3% and 36.6% in nymphs and adults, respectively, with the majority of infection (95.1%) found at the most endemic site. There was no evidence of I. scapularis invasion along the inland transect; however, low-prevalence B. burgdorferi infection was detected in other tick species and in wildlife at inland sites, and at northern coastal sites in years before the arrival of I. scapularis. These infections suggest that cryptic B. burgdorferi transmission by other vector-competent tick species is occurring in the absence of I. scapularis. Other Borrelia spirochetes, including those that group with B. miyamotoi and B. andersonii, were present at a low prevalence within invading ticks and local wildlife. Reports of Lyme disease have increased significantly in the invasion zone in recent years. This rapid blacklegged tick invasion—measurable within 5 years—in combination with cryptic pathogen maintenance suggests a complex ecology of Lyme disease emergence in which wildlife sentinels can provide an early warning of disease emergence.     

Characterization of the horse (<Emphasis Type="Italic">Equus caballus</Emphasis>)<Emphasis Type="Italic"> IGHA</Emphasis> gene

Nucleotide sequences of the immunoglobulin constant heavy chain genes of the horse have been described for IGHM, IGHG and IGHE genes, but not for IGHA. Here, we provide the nucleotide sequence of the genomic IGHA gene of the horse (Equus caballus), including its secretion region and the transmembrane exon. The equine IGHA gene shows the typical structure of a mammalian IGHA gene, with only three exons, separated by two introns of similar size. The hinge exon is located at the 5 end of the CH2 exon and encodes a hinge region of 11 amino acids, which contains five proline residues. The coding nucleotide sequence of the secreted form of the equine IGHA gene shares around 72% identity with the human IGHA1 and IGHA2 genes, as well as the bovine, ovine, porcine and canine IGHA genes, without distinct preference for any of these species. The same species also cluster together in a phylogenetic tree of the IGHA coding regions of various mammals, whereas rodent, rabbit, marsupial and monotreme IGHA genes each build a separate cluster.The nucleotide sequences reported in this paper have been assigned the EMBL/GenBank accession numbers AY247966 and AY351982     

Molecular Detection of <Emphasis Type="Italic">Streptococcus pyogenes</Emphasis> and <Emphasis Type="Italic">Streptococcus dysgalactiae</Emphasis> subsp. <Emphasis Type="Italic">equisimilis</Emphasis>

We developed molecular diagnostic assays for the detection of Streptococcus pyogenes (GAS) and Streptococcus dysgalactiae subsp. equisimilis (SDSE), two streptococcal pathogens known to cause both pharyngitis and more invasive forms of disease in humans. Two real-time PCR assays coupled with an internal control were designed to be performed in parallel. One assay utilizes a gene target specific to GAS, and the other utilizes a gene target common to the two species. Both assays showed 2–3 orders of magnitude improved analytical sensitivity when compared to a commercially available rapid antigen test. In addition, when compared to standard culture in an analysis of 96 throat swabs, the real-time PCR assays resulted in clinical sensitivity and specificity of 91.7 and 100%, respectively. As capital equipment costs for real-time PCR can be prohibitive in smaller laboratories, the real-time PCR assays were converted to a low-density microarray format designed to function with an inexpensive photopolymerization-based non-enzymatic signal amplification (NESA™) method. S. pyogenes was successfully detected on the low-density microarray in less than 4 h from sample extraction through detection.     

DNA elements modulating the <Emphasis Type="Italic">KARS12</Emphasis> chromosomal replicator in <Emphasis Type="Italic">Kluyveromyces lactis</Emphasis>

Eukaryotic chromosomal DNA replication is initiated by a highly conserved set of proteins that interact with cis-acting elements on chromosomes called replicators. Despite the conservation of replication initiation proteins, replicator sequences show little similarity from species to species in the small number of organisms that have been examined. Examination of replicators in other species is likely to reveal common features of replicators. We have examined a Kluyeromyces lactis replicator, KARS12, that functions as origin of DNA replication on plasmids and in the chromosome. It contains a 50-bp region with similarity to two other K. lactis replicators, KARS101 and the pKD1 replication origin. Replacement of the 50-bp sequence with an EcoRI site completely abrogated the ability of KARS12 to support plasmid and chromosomal DNA replication origin activity, demonstrating this sequence is a common feature of K. lactis replicators and is essential for function, possibly as the initiator protein binding site. Additional sequences up to 1 kb in length are required for efficient KARS12 function. Within these sequences are a binding site for a global regulator, Abf1p, and a region of bent DNA, both of which contribute to the activity of KARS12. These elements may facilitate protein binding, protein/protein interaction and/or nucleosome positioning as has been proposed for other eukaryotic origins of DNA replication.     

Patterns of DNA Variation Among Three Centromere Satellite Families in <Emphasis Type="Italic">Arabidopsis halleri</Emphasis> and <Emphasis Type="Italic">A</Emphasis>. <Emphasis Type="Italic">lyrata</Emphasis>

We describe patterns of DNA variation among the three centromeric satellite families in Arabidopsis halleri and lyrata. The newly studied subspecies (A. halleri ssp. halleri and A. lyrata ssp. lyrata and petraea), like the previously studied A. halleri ssp. gemmifera and A. lyrata ssp. kawasakiana, have three different centromeric satellite families, the older pAa family (also present in A. arenosa) and two families, pAge1 and pAge2, that probably evolved more recently. Sequence variability is high in all three satellite families, and the pAa sequences do not cluster by their species of origin. Diversity in the pAge2 family is complex, and different from variation among copies of the other two families, showing clear evidence for exchange events among family members, especially in A. halleri ssp. halleri. In A. lyrata ssp. lyrata there is some evidence for recent rapid spread of pAge2 variants, suggesting selection favoring these sequences. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Brian Morton]     

mtDNA Diversity and genetic lineages of eighteen cattle breeds from <Emphasis Type="Italic">Bos taurus </Emphasis>and<Emphasis Type="Italic"> Bos indicus</Emphasis> in China

In order to clarify the origin and genetic diversity of indigenous cattle breeds in China, we carried out phylogenetic analysis of representatives of those breeds by employing mitochondrial gene polymorphism. Complete cyt b gene sequences, 1140 bp in length, were determined for a total of 136 individuals from 18 different breeds and these sequences were clustered into two distinct genetic lineages: taurine (Bos taurus) and zebu (Bos indicus). In analysis of the cyt b gene diversity, Chinese cattle showed higher nucleotide (0.00923) and haplotype diversity (0.848) than the reports from other studies, and the animals from the taurine lineage indicated higher nucleotide diversity (0.00330) and haplotype diversity (0.746) than the ones from the zebu lineage (0.00136; 0.661). The zebu mtDNA dominated in the southern breeds (63.3–100%), while the taurine dominated in the northern breeds (81.8–100%). Six cattle breeds from the central area of China exhibited intermediate frequencies of zebu mtDNA (25–71.4%). This polymorphism revealed a declining south-to-north gradient of female zebu introgression and a geographical hybrid zone of Bos taurus and Bos indicus in China.