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PubMed2004

Orangutan herpesvirus.

A male orangutan suffered from ulcers at the buccal mucosa. We obtained swab fluid from the base of both vesicles and ulcers and collected blood for further separation into serum, plasma and peripheral blood mononuclear cells (PBMC) for detection of antibody to herpesvirus by serology and herpesvirus DNA by polymerase chain reaction (PCR) using consensus degenerate primers. Serology was positive for human EBV IgG but negative for Epstein-Barr virus (EBV) immunoglobulin (IgM), as well as for both human cytomegalovirus and herpes simplex virus IgG and IgM. Upon PCR, we obtained a 232-bp product of virus DNA from PBMC, but not from lesions, serum or plasma. We confirmed the positive result by direct sequencing and compared the nucleotide sequence with other nucleotide sequences applying the BLAST program from GenBank. The sequence was similar to lymphocryptovirus of macaque (93%), marmoset (93%), gorilla (90%) and human EBV (90%). We aligned this sequence with other sequences in GenBank and performed phylogenetic analysis, showing that it probably belongs to the gammaherpesvirus group.

Animals↗
PubMed2006

Marmoset glutathione peroxidases: cDNA sequences, molecular evolution, and gene expression.

BACKGROUND: Dysfunction of the cellular antioxidant system and accumulation of reactive oxygen species are involved in the pathophysiology of diseases such as cardiovascular disease, neurodegenerative disorders, tumors, male infertility and aging. Two gluthathione peroxidases play key roles in the cellular protection against oxidative damage. Glutathione peroxidase (GPx-1) removes cytosolic hydroperoxides while phospholipid-hydroperoxide glutathione peroxidase (GPx-4) is a unique enzyme that reduces phospholipid peroxides in membranes. METHODS: We cloned and sequenced the full-length cDNA for GPx-1 (GenBank: AY966403) and GPx-4 (GenBank: AY966404) from the common marmoset (Callithrix jacchus) in order to create a suitable model for studying human diseases related with oxidative stress. RESULTS: The cDNAs encode a 202 amino acid protein for GPx-1 and a 197 amino acid protein for GPx-4. Both proteins include selenocysteine (Sec, in Gpx-1 at position 48; in GPx-4 at position 73) and showed high homology (>90%) with other mammalian GPxs. The relative levels of mRNA expression for GPx-1 and GPx-4 were determined in different marmoset tissues by quantitative real-time reverse transcriptase-polymerase chain reaction using transcription elongation factor-2 as a reference gene. GPx-1 showed increased levels of expression in the liver, heart and kidney while the highest mRNA levels for GPx-4 were detected in the testis, followed by the liver, lung, kidney and spinal cord. CONCLUSIONS: These findings will be of value for studies designed to assess the role of glutathione peroxidases in non-human primate models for a variety of diseases in which increased oxidative stress has been implicated.

Amino Acid Sequence↗
PubMed1996

A molecular perspective of the genetic relationships of G-protein coupled melatonin receptor subtypes.

Successful cloning of melatonin receptors from various target tissues in the past few years has increased our understanding of the molecular signal transduction mechanisms of G-protein coupled melatonin receptors, of which three subtypes (MEL-1A, MEL-1B, and MEL-1C) have been reported in different vertebrates. Based upon melatonin receptor sequences available in the Genbank database, we have performed phylogenetic analyses of the nucleotide and encoded amino acid sequences of G-protein-coupled melatonin receptors, and determined the range of amino acid identities between melatonin receptors of the same and different subtypes. Besides the three well-known subtypes, a potential novel subtype of MEL-1D, as exemplified by unique separation of Xenopus X2.0 sequence (Genbank accession No. U31826) from the others in the protein phylogenetic tree, possibly exists. In addition, one of the chicken brain melatonin receptor sequences has been identified as belonging to the MEL-1B subtype. Our analyses showed that melatonin receptors of the same subtype and different subtypes are likely to share > or = 75% and < 65% amino acid identities, respectively. Phylogenetic analysis based on amino acid comparisons will be needed to determine the subtype status of any pair of melatonin receptor sequences that exhibit > or = 65% to < 75% amino acid identity. Despite the usefulness of genetic relatedness in the subtype classification of G-protein-coupled melatonin receptors, functional correlation of molecular structure may ultimately prove the most comprehensive approach in melatonin receptor classification.

Amino Acid Sequence↗
PubMed2005

Molecular cloning of a novel mouse testis-specific spermatogenic cell apoptosis inhibitor gene mTSARG7 as a candidate oncogene.

A novel mouse gene, mTSARG7 (GenBank accession No. AY489184), with a full cDNA length of 2279 bp and containing 12 exons and 11 introns, was cloned from a mouse expressed sequence tag (GenBank accession No. BE644543) that was significantly up-regulated in cryptorchidism. The gene was located in mouse chromosome 8A1.3 and encoded a protein containing 403 amino acid residues that was a new member of the acyltransferase family because the sequence contained the highly conserved phosphate acyltransferase (PlsC) domain existing in all acyltransferase-like proteins. The mTSARG7 protein and AU041707 protein shared 83.9% identity in 402 amino acid residues. Expression of the mTSARG7 gene was restricted to the mouse testis. The results of the in situ hybridization analysis revealed that the mTSARG7 mRNA was expressed in mouse spermatogonia and spermatocytes. Subcellular localization studies showed that the EGFP-tagged mTSARG7 protein was localized in the cytoplasm of GC-1 spg cells. The mTSARG7 mRNA expression was initiated in the mouse testis in the second week after birth, and the expression level increased steadily with spermatogenesis and sexual maturation of the mouse. The results of the heat stress experiment showed that the mTSARG7 mRNA expression gradually decreased as the heating duration increased. The pcDNA3.1 Hygro(-)/mTSARG7 plasmid was constructed and introduced into GC-1 spg cells by liposome transfection. The mTSARG7 can accelerate GC-1 spg cells, causing them to traverse the S-phase and enter the G2-phase, compared with the control group where this did not occur as there was no transfection of mTSARG7. In conclusion, our results suggest that this gene may play an important role in spermatogenesis and the development of cryptorchid testes, and is a testis-specific apoptosis candidate oncogene.

Amino Acid Sequence↗
PubMed2006

Total variation in the penA gene of Neisseria meningitidis: correlation between susceptibility to beta-lactam antibiotics and penA gene heterogeneity.

In recent decades, the prevalence of Neisseria meningitidis isolates with reduced susceptibility to penicillins has increased. The intermediate resistance to penicillin (Pen(i)) for most strains is due mainly to mosaic structures in the penA gene, encoding penicillin-binding protein 2. In this study, susceptibility to beta-lactam antibiotics was determined for 60 Swedish clinical N. meningitidis isolates and 19 reference strains. The penA gene was sequenced and compared to 237 penA sequences from GenBank in order to explore the total identified variation of penA. The divergent mosaic alleles differed by 3% to 24% compared to those of the designated wild-type penA gene. By studying the final 1,143 to 1,149 bp of penA in a sequence alignment, 130 sequence variants were identified. In a 402-bp alignment of the most variable regions, 84 variants were recognized. Good correlation between elevated MICs and the presence of penA mosaic structures was found especially for penicillin G and ampicillin. The Pen(i) isolates comprised an MIC of >0.094 microg/ml for penicillin G and an MIC of >0.064 microg/ml for ampicillin. Ampicillin was the best antibiotic for precise categorization as Pen(s) or Pen(i). In comparison with the wild-type penA sequence, two specific Pen(i) sites were altered in all except two mosaic penA sequences, which were published in GenBank and no MICs of the corresponding isolates were described. In conclusion, monitoring the relationship between penA sequences and MICs to penicillins is crucial for developing fast and objective methods for susceptibility determination. By studying the penA gene, genotypical determination of susceptibility in culture-negative cases can also be accomplished.

Amino Acid Sequence↗
PubMed1999

Cloning, sequencing, and characterization of genomic subtracted sequences from Listeria monocytogenes.

Individual sequences of a genomic subtracted, PCR-amplified, mixed-sequence probe (GS probe) were cloned and sequenced. The GS probe differentiated restriction fragment length polymorphism patterns for Listeria monocytogenes but did not hybridize with members of other bacterial genera. Sequence analysis identified several L. monocytogenes sequences already present in the GenBank database; the putative identities of other sequences were inferred from homology data, and still other sequences did not exhibit significant levels of homology with any GenBank sequences.

Amino Acid Sequence↗
PubMed2000

Complete nucleotide sequence of ubiquitous plasmid pEA29 from Erwinia amylovora strain Ea88: gene organization and intraspecies variation.

The complete sequence of plasmid pEA29 from Erwinia amylovora strain Ea88 consists of 28,185 bp with a 50.2% G+C content. As deletions and insertions were detected in other derivatives of pEA29, its size actually varied from 27.6 to 34.9 kb. Thirteen open reading frames that encoded predicted proteins with similarities to known proteins from other bacteria were identified along with two open reading frames related to hypothetical proteins found in GenBank and six open reading frames with no similarities to existing GenBank entries. Predicted products of open reading frames with similarity to the thiamine biosynthetic genes thiO, thiG, and thiF; a betT gene coding for choline transport; an msrA gene for the enzyme methionine sulfoxide reductase; a putative methyl-accepting chemotaxis gene; an aldehyde dehydrogenase gene; an hns DNA binding gene; a LysR-type transcriptional regulator; and parA and parB partitioning genes were identified. A putative iteron-containing theta-type origin of replication with an AT-rich region and a gene for a RepA protein was identified. PstI and KpnI restriction patterns for pEA29 isolated from tree fruit strains of E. amylovora were homogenous and different from those for pEA29 isolated from Rubus (raspberry) strains. All Rubus derivatives of pEA29 contained a point mutation that eliminated a PstI site and a 1,264-bp region that replaced 1, 890 bp of sequence found in pEA29 from strain Ea88. This change eliminated a second PstI site and increased the length of a KpnI fragment. An insertion sequence, ISEam1, was detected in one Rubus strain, and transposon Tn5393 was detected in three apple strains in two separate locations on the plasmid. Plasmid-cured strains exhibited reduced virulence and modified colony morphology on minimal medium without thiamine, indicating that some of the genes in pEA29 play a role in the physiology or metabolism of E. amylovora.

Base Sequence↗
PubMed2002

16S rRNA-based analysis of microbiota from the cecum of broiler chickens.

The microbiota of the intestinal tract of chickens plays an important role in inhibiting the establishment of intestinal pathogens. Earlier culturing and microscopic examinations indicated that only a fraction of the bacteria in the cecum of chickens could be grown in the laboratory. Therefore, a survey of cecal bacteria was done by retrieval of 16S rRNA gene sequences from DNA isolated from the cecal content and the cecal mucosa. The ribosomal gene sequences were amplified with universal primers and cloned or subjected to temporal temperature gradient gel electrophoresis (TTGE). Partial 16S rRNA gene sequences were determined from the clones and from the major bands in TTGE gels. A total of 1,656 partial 16S rRNA gene sequences were obtained and compared to sequences in the GenBank. The comparison indicated that 243 different sequences were present in the samples. Overall, sequences representing 50 phylogenetic groups or subgroups of bacteria were found, but approximately 89% of the sequences represented just four phylogenetic groups (Clostridium leptum, Sporomusa sp., Clostridium coccoides, and enterics). Sequences of members of the Bacteroides group, the Bifidobacterium infantis subgroup, and of Pseudomonas sp. each accounted for less than 2% of the total. Sequences related to those from the Escherichia sp. subgroup and from Lactobacillus, Pseudomonas, and Bifidobacterium spp. were generally between 98 and 100% identical to sequences already deposited in the GenBank. Sequences most closely related to those of the other bacteria were generally 97% or less identical to those in the databases and therefore might be from currently unknown species. TTGE and random cloning indicated that certain phylogenetic subgroups were common to all birds analyzed, but sequence data from random cloning also provided evidence for qualitative and quantitative differences among the cecal microbiota of individual birds reared under very similar conditions.

Animals↗
PubMed2002

Use of 16S ribosomal DNA for delineation of marine bacterioplankton species.

All of the marine bacterioplankton-derived 16S ribosomal DNA sequences previously deposited in GenBank were reanalyzed to determine the number of bacterial species in the oceanic surface waters. These sequences have been entered into the database since 1990. The rate of new additions reached a peak in 1999 and subsequently leveled off, suggesting that much of the marine microbial species richness has been sampled. When the GenBank sequences were dereplicated by using 97% similarity as a cutoff, 1,117 unique ribotypes were found. Of the unique sequences, 609 came from uncultured environmental clones and 508 came from cultured bacteria. We conclude that the apparent bacterioplankton species richness is relatively low.

Bacteria↗
PubMed2005

Complete genome sequence of phiHSIC, a pseudotemperate marine phage of Listonella pelagia.

The genome for the marine pseudotemperate member of the Siphoviridae phiHSIC has been sequenced using a combination of linker amplification library construction, restriction digest library construction, and primer walking. phiHSIC enters into a pseudolysogenic relationship with its host, Listonella pelagia, characterized by sigmoidal growth curves producing >10(9) cells/ml and >10(11) phage/ml. The genome (37,966 bp; G+C content, 44%) contained 47 putative open reading frames (ORFs), 17 of which had significant BLASTP hits in GenBank, including a beta subunit of DNA polymerase III, a helicase, a helicase-like subunit of a resolvasome complex, a terminase, a tail tape measure protein, several phage-like structural proteins, and 1 ORF that may assist in host pathogenicity (an ADP ribosyltransferase). The genome was circularly permuted, with no physical ends detected by sequencing or restriction enzyme digestion analysis, and lacked a cos site. This evidence is consistent with a headful packaging mechanism similar to that of Salmonella phage P22 and Shigella phage Sf6. Because none of the phage-like ORFs were closely related to any existing phage sequences in GenBank (i.e., none more than 62% identical and most <25% identical at the amino acid level), phiHSIC is unique among phages that have been sequenced to date. These results further emphasize the need to sequence phages from the marine environment, perhaps the largest reservoir of untapped genetic information.

Electrophoresis, Gel, Pulsed-Field↗
PubMed2006

Oligonucleotide array for identification and detection of pythium species.

A DNA array containing 172 oligonucleotides complementary to specific diagnostic regions of internal transcribed spacers (ITS) of more than 100 species was developed for identification and detection of Pythium species. All of the species studied, with the exception of Pythium ostracodes, exhibited a positive hybridization reaction with at least one corresponding species-specific oligonucleotide. Hybridization patterns were distinct for each species. The array hybridization patterns included cluster-specific oligonucleotides that facilitated the recognition of species, including new ones, belonging to groups such as those producing filamentous or globose sporangia. BLAST analyses against 500 publicly available Pythium sequences in GenBank confirmed that species-specific oligonucleotides were unique to all of the available strains of each species, of which there were numerous economically important ones. GenBank entries of newly described species that are not putative synonyms showed no homology to sequences of the spotted species-specific oligonucleotides, but most new species did match some of the cluster-specific oligonucleotides. Further verification of the specificity of the DNA array was done with 50 additional Pythium isolates obtained by soil dilution plating. The hybridization patterns obtained were consistent with the identification of these isolates based on morphology and ITS sequence analyses. In another blind test, total DNA of the same soil samples was amplified and hybridized on the array, and the results were compared to those of 130 Pythium isolates obtained by soil dilution plating and root baiting. The 13 species detected by the DNA array corresponded to the isolates obtained by a combination of soil dilution plating and baiting, except for one new species that was not represented on the array. We conclude that the reported DNA array is a reliable tool for identification and detection of the majority of Pythium species in environmental samples. Simultaneous detection and identification of multiple species of soilborne pathogens such as Pythium species could be a major step forward for epidemiological and ecological studies.

Oligonucleotide Array Sequence Analysis↗
PubMed2003

Differences in clinical manifestations and hematological and serological responses after experimental infection with genetic variants of Anaplasma phagocytophilum in sheep.

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

Anaplasma phagocytophilum↗
PubMed2000

rpoS mutants in archival cultures of Salmonella enterica serovar typhimurium.

Long-term survival under limited growth conditions presents bacterial populations with unique environmental challenges. The existence of Salmonella enterica serovar Typhimurium cultures undisturbed in sealed nutrient agar stab vials for 34 to 45 years offered a unique opportunity to examine genetic variability under natural conditions. We have initiated a study of genetic changes in these archival cultures. We chose to start with examination of the rpoS gene since, among gram-negative bacteria, many genes needed for survival are regulated by RpoS, the stationary-phase sigma factor. In each of 27 vials examined, cells had the rpoS start codon UUG instead of the expected AUG of Salmonella and Escherichia coli strains recorded in GenBank. Ten of the 27 had additional mutations in the rpoS gene compared with the X77752 wild-type strain currently recorded in GenBank. The rpoS mutations in the 10 strains included two deletions as well as point mutations that altered amino acid sequences substantially. Since these stored strains were derived from ancestral cells inoculated decades ago and remained undisturbed, it is assumed that the 10 rpoS mutations occurred during storage. Since the remaining 17 sequences were wild type (other than in the start codon), it is obvious that rpoS remained relatively stable during decades of sealed storage.

Amino Acid Substitution↗
PubMed1999

Distribution of a Nocardia brasiliensis catalase gene fragment in members of the genera Nocardia, Gordona, and Rhodococcus.

An immunodominant protein from Nocardia brasiliensis, P61, was subjected to amino-terminal and internal sequence analysis. Three sequences of 22, 17, and 38 residues, respectively, were obtained and compared with the protein database from GenBank by using the BLAST system. The sequences showed homology to some eukaryotic catalases and to a bromoperoxidase-catalase from Streptomyces violaceus. Its identity as a catalase was confirmed by analysis of its enzymatic activity on H2O2 and by a double-staining method on a nondenaturing polyacrylamide gel with 3,3'-diaminobenzidine and ferricyanide; the result showed only catalase activity, but no peroxidase. By using one of the internal amino acid sequences and a consensus catalase motif (VGNNTP), we were able to design a PCR assay that generated a 500-bp PCR product. The amplicon was analyzed, and the nucleotide sequence was compared to the GenBank database with the observation of high homology to other bacterial and eukaryotic catalases. A PCR assay based on this target sequence was performed with primers NB10 and NB11 to confirm the presence of the NB10-NB11 gene fragment in several N. brasiliensis strains isolated from mycetoma. The same assay was used to determine whether there were homologous sequences in several type strains from the genera Nocardia, Rhodococcus, Gordona, and Streptomyces. All of the N. brasiliensis strains presented a positive result but only some of the actinomycetes species tested were positive in the PCR assay. In order to confirm these findings, genomic DNA was subjected to Southern blot analysis. A 1.7-kbp band was observed in the N. brasiliensis strains, and bands of different molecular weight were observed in cross-reacting actinomycetes. Sequence analysis of the amplicons of selected actinomycetes showed high homology in this catalase fragment, thus demonstrating that this protein is highly conserved in this group of bacteria.

Actinomycetales↗
PubMed2000

16S ribosomal DNA sequence analysis of a large collection of environmental and clinical unidentifiable bacterial isolates.

Some bacteria are difficult to identify with phenotypic identification schemes commonly used outside reference laboratories. 16S ribosomal DNA (rDNA)-based identification of bacteria potentially offers a useful alternative when phenotypic characterization methods fail. However, as yet, the usefulness of 16S rDNA sequence analysis in the identification of conventionally unidentifiable isolates has not been evaluated with a large collection of isolates. In this study, we evaluated the utility of 16S rDNA sequencing as a means to identify a collection of 177 such isolates obtained from environmental, veterinary, and clinical sources. For 159 isolates (89.8%) there was at least one sequence in GenBank that yielded a similarity score of > or =97%, and for 139 isolates (78.5%) there was at least one sequence in GenBank that yielded a similarity score of > or =99%. These similarity score values were used to defined identification at the genus and species levels, respectively. For isolates identified to the species level, conventional identification failed to produce accurate results because of inappropriate biochemical profile determination in 76 isolates (58.7%), Gram staining in 16 isolates (11.6%), oxidase and catalase activity determination in 5 isolates (3.6%) and growth requirement determination in 2 isolates (1.5%). Eighteen isolates (10.2%) remained unidentifiable by 16S rDNA sequence analysis but were probably prototype isolates of new species. These isolates originated mainly from environmental sources (P = 0.07). The 16S rDNA approach failed to identify Enterobacter and Pantoea isolates to the species level (P = 0.04; odds ratio = 0.32 [95% confidence interval, 0.10 to 1.14]). Elsewhere, the usefulness of 16S rDNA sequencing was compromised by the presence of 16S rDNA sequences with >1% undetermined positions in the databases. Unlike phenotypic identification, which can be modified by the variability of expression of characters, 16S rDNA sequencing provides unambiguous data even for rare isolates, which are reproducible in and between laboratories. The increase in accurate new 16S rDNA sequences and the development of alternative genes for molecular identification of certain taxa should further improve the usefulness of molecular identification of bacteria.

Animals↗
PubMed2000

Identification by 16S rRNA gene analyses of a potential novel mycobacterial species as an etiological agent of canine leproid granuloma syndrome.

PCR amplifications of the 16S rRNA gene were performed on 46 specimens obtained from 43 dogs with canine leproid granuloma syndrome to help determine its etiology. Sequence capture PCR was applied to 37 paraffin-embedded specimens from 37 dogs, and nested PCR was attempted on DNA from 9 fresh tissue specimens derived from 3 of the 37 aforementioned dogs and from an additional 6 dogs. Molecular analyses of the paraffin-embedded tissues and fresh tissue specimen analyses were performed at separate institutions. PCR products with identical sequences over a 350-bp region encompassing variable regions 2 and 3 of the 16S rRNA gene were obtained from 4 of 37 paraffin-embedded specimens and from all 9 specimens of fresh tissue originating from 12 of the 43 dogs. Identical sequences were determined from amplicons obtained from paraffin-embedded and fresh specimens from one dog. The consensus DNA sequence, amplified from paraffin-embedded tissue and represented by GenBank accession no. AF144747, shared highest nucleotide identity (99.4% over 519 bp) with mycobacterial strain IWGMT 90413 but did not correspond exactly to any EMBL or GenBank database sequence. With a probe derived from the V2 region of the novel canine sequence, reverse cross blot hybridization identified an additional four paraffin-embedded specimens containing the same novel sequence. In total, molecular methodologies identified the proposed novel mycobacterial sequence in 16 of 43 dogs with canine leproid granuloma syndrome, indicating that the species represented by this sequence may be the principal etiological agent of canine leproid granuloma syndrome.

Animals↗
PubMed2000

Identification of Aspergillus species using internal transcribed spacer regions 1 and 2.

Aspergillus species are the most frequent cause of invasive mold infections in immunocompromised patients. Although over 180 species are found within the genus, 3 species, Aspergillus flavus, A. fumigatus, and A. terreus, account for most cases of invasive aspergillosis (IA), with A. nidulans, A. niger, and A. ustus being rare causes of IA. The ability to distinguish between the various clinically relevant Aspergillus species may have diagnostic value, as certain species are associated with higher mortality and increased virulence and vary in their resistance to antifungal therapy. A method to identify Aspergillus at the species level and differentiate it from other true pathogenic and opportunistic molds was developed using the 18S and 28S rRNA genes for primer binding sites. The contiguous internal transcribed spacer (ITS) region, ITS 1-5.8S-ITS 2, from referenced strains and clinical isolates of aspergilli and other fungi were amplified, sequenced, and compared with non-reference strain sequences in GenBank. ITS amplicons from Aspergillus species ranged in size from 565 to 613 bp. Comparison of reference strains and GenBank sequences demonstrated that both ITS 1 and ITS 2 regions were needed for accurate identification of Aspergillus at the species level. Intraspecies variation among clinical isolates and reference strains was minimal. Sixteen other pathogenic molds demonstrated less than 89% similarity with Aspergillus ITS 1 and 2 sequences. A blind study of 11 clinical isolates was performed, and each was correctly identified. Clinical application of this approach may allow for earlier diagnosis and selection of effective antifungal agents for patients with IA.

Aspergillosis↗
PubMed2000

Genetic characteristics of Borrelia coriaceae isolates from the soft tick Ornithodoros coriaceus (Acari: Argasidae).

Two Borrelia isolates (CA434 and CA435) cultured from the soft tick Ornithodoros coriaceus were analyzed by contour-clamped homogeneous electric field gel electrophoresis of unrestricted and ApaI-restricted DNA, standard electrophoresis of BamHI- and HindIII-restricted DNA, Southern hybridization, restriction fragment length polymorphism and sequencing of the 16S rRNA gene, and amplification of the 5S-23S intergenic spacer region. These isolates were compared with Borrelia coriaceae type strain Co53, B. burgdorferi sensu stricto strain CA4, and the relapsing-fever spirochete B. parkeri (undesignated). The 16S rRNA region of CA434 and CA435 differed from that of B. coriaceae type strain Co53 by the presence of 1 base (C) at position 367 (GenBank accession no. U42286). The linear plasmid profile of CA434 was similar to that of Co53, and the ApaI, BamHI, and HindIII restriction fingerprints of the total cellular DNA of CA434 and Co53 were similar. In contrast, CA435 differed somewhat from CA434 and Co53, which demonstrates that B. coriaceae is genetically diverse. Southern hybridization showed that the DNAs of CA434 and CA435 hybridized strongly with the digoxigenin-labeled DNA of Co53. Low homology was found between the DNA of Co53 and that of B. parkeri. The 16S rRNA sequence of B. parkeri was identical to previously published results for B. parkeri strain M3001 (GenBank accession number U42296). CA434 and CA435 represent only the second and third isolates of B. coriaceae obtained from any source since its initial isolation from an O. coriaceus tick in 1985. All three B. coriaceae isolates were derived from adult ticks collected from the same locality in northwestern California. Difficulties encountered in detecting B. coriaceae in, and isolating this spirochete from, the tissues of O. coriaceus are discussed. The lack of concordance between different detection or isolation methods suggests that reliance upon a single technique may grossly underestimate the true prevalence of spirochetal infection in wild-caught O. coriaceus ticks.

Animals↗