18S ribosomal DNA typing and tracking of Acanthamoeba species isolates from corneal scrape specimens, contact lenses, lens cases, and home water supplies of Acanthamoeba keratitis patients in Hong Kong.

We examined partial 18S ribosomal DNA (Rns) sequences of Acanthamoeba isolates cultured in a study of microbial keratitis in Hong Kong. Sequence differences were sufficient to distinguish closely related strains and were used to examine links between strains obtained from corneal scrape specimens, contact lenses, lens cases, lens case solutions, and home water-supply faucets of patients with Acanthamoeba. We also looked for evidence of mixed infections. Identification of Acanthamoeba Rns genotypes was based on sequences of approximately 113 bp within the genus-specific amplicon ASA.S1. This permitted genotype identification by using nonaxenic cultures. Of 13 specimens obtained from corneal scrapes, contact lenses, lens cases, or lens case solutions, 12 were Rns genotype T4 and the remaining one was Rns genotype T3. The sequences of corneal scrape specimens of two patients also were the same as those obtained from their contact lenses or lens case specimens. A possible triple-strain infection was indicated by three different T4 sequences in cultures from one patient's lenses. Although faucet water used by patients to clean their lenses is a possible source of infections, specimens isolated from the faucets at two Acanthamoeba keratitis patients' homes differed from their corneal scrape or lens specimens. The overall results demonstrate the potential of this Rns region for tracking Acanthamoeba keratitis strains in infections and for distinguishing single-strain and closely related multiple-strain infections even when other microorganisms might be present with the cultured specimens. They also confirm the predominance of Rns genotype T4 strains in Acanthamoeba keratitis infections.

Molecular phylogeny of the snubnose darters, subgenus Ulocentra (genus Etheostoma, family Percidae).

Snubnose darters comprise one of the largest subgenera of the percid genus Etheostoma. Many species are described based on differences in male breeding coloration. Few morphological synapomorphies have been proposed for the subgenus and their relatives, making it difficult to delineate monophyletic clades. The phylogenetic relationships of the 20 snubnose darter species of the subgenus Ulocentra and 11 members of its proposed sister subgenus Etheostoma were investigated with partial mitochondrial DNA sequences including 1033 bp encompassing the entire mitochondrial control region, the tRNA-Phe gene, and part of the 12S rRNA gene. Two hypotheses on the relationship and monophyly of the two subgenera were evaluated. Both maximum-parsimony and neighbor-joining analyses supported monophyly of the subgenus Ulocentra and resolved some species-level relationships. The banded darter, E. zonale, and its sister taxon, E. lynceum, were not closely related to the snubnose darters and appear to be diverged from the other members of the subgenus Etheostoma, fitting their former distinction as the recognized subgenus Nanostoma. The sister group to Ulocentra appears to be a restricted species assemblage within the subgenus Etheostoma containing E. blennioides, E. rupestre, E. blennius, and the E. thalassinum species group. The placement of the harlequin darter, E. histrio, is problematic, and it may represent a basal member of Ulocentra or of the restricted subgenus Etheostoma. Despite recent estimates of divergence times between nominal Ulocentra taxa, each species exhibits its own unique set of mtDNA haplotypes, providing no direct evidence for current genetic exchange between species. The nominal taxa of snubnose darters thus appear to be evolving independently from each other and therefore constitute valid species under the Phylogenetic Species Concept.

Use of subgenic 18S ribosomal DNA PCR and sequencing for genus and genotype identification of acanthamoebae from humans with keratitis and from sewage sludge.

This study identified subgenic PCR amplimers from 18S rDNA that were (i) highly specific for the genus Acanthamoeba, (ii) obtainable from all known genotypes, and (iii) useful for identification of individual genotypes. A 423- to 551-bp Acanthamoeba-specific amplimer ASA.S1 obtained with primers JDP1 and JDP2 was the most reliable for purposes i and ii. A variable region within this amplimer also identified genotype clusters, but purpose iii was best achieved with sequencing of the genotype-specific amplimer GTSA.B1. Because this amplimer could be obtained from any eukaryote, axenic Acanthamoeba cultures were required for its study. GTSA.B1, produced with primers CRN5 and 1137, extended between reference bp 1 and 1475. Genotypic identification relied on three segments: bp 178 to 355, 705 to 926, and 1175 to 1379. ASA.S1 was obtained from single amoeba, from cultures of all known 18S rDNA genotypes, and from corneal scrapings of Scottish patients with suspected Acanthamoeba keratitis (AK). The AK PCR findings were consistent with culture results for 11 of 15 culture-positive specimens and detected Acanthamoeba in one of nine culture-negative specimens. ASA.S1 sequences were examined for 6 of the 11 culture-positive isolates and were most closely associated with genotypic cluster T3-T4-T11. A similar distance analysis using GTSA.B1 sequences identified nine South African AK-associated isolates as genotype T4 and three isolates from sewage sludge as genotype T5. Our results demonstrate the usefulness of 18S ribosomal DNA PCR amplimers ASA.S1 and GTSA.B1 for Acanthamoeba-specific detection and reliable genotyping, respectively, and provide further evidence that T4 is the predominant genotype in AK.

Isolation of a sex-linked DNA sequence in cranes.

A female-specific DNA fragment (CSL-W; crane sex-linked DNA on W chromosome) was cloned from female whooping cranes (Grus americana). From the nucleotide sequence of CSL-W, a set of polymerase chain reaction (PCR) primers was identified which amplify a 227-230 bp female-specific fragment from all existing crane species and some other noncrane species. A duplicated versions of the DNA segment, which is found to have a larger size (231-235 bp) than CSL-W in both sexes, was also identified, and was designated CSL-NW (crane sex-linked DNA on non-W chromosome). The nucleotide similarity between the sequences of CSL-W and CSL-NW from whooping cranes was 86.3%. The CSL primers do not amplify any sequence from mammalian DNA, limiting the potential for contamination from human sources. Using the CSL primers in combination with a quick DNA extraction method allows the noninvasive identification of crane gender in less than 10 h. A test of the methodology was carried out on fully developed body feathers from 18 captive cranes and resulted in 100% successful identification.

Phylogenetic relationships of the North American sturgeons (order Acipenseriformes) based on mitochondrial DNA sequences.

The evolutionary relationships of the extant species within the order Acipenseriformes are not well understood. Nucleotide sequences of four mitochondrial genes (12S rRNA, COII, tRNA(Phe), and tRNA(Asp) genes) in North American sturgeon and paddlefish were examined to reconstruct a phylogeny. Analysis of the combined gene sequences suggests a basal placement of the paddlefish with regard to the sturgeons. Nucleotide sequences of all four genes for the three Scaphirhynchus species were identical. The position of Scaphirhynchus based on our data was uncertain. Within the genus Acipenser, the two Acipenser oxyrinchus subspecies were very similar in sequence and found to be basal to the remaining Acipenser species examined. Based on our data, Acipenser transmontanus and Acipenser medirostris were sister taxa, as were Acipenser fulvescens and Acipenser brevirostrum. Comparison of our results with hypotheses of sturgeon relationships proposed by previous authors is presented. The sequence data presented here are phylogenetically useful and provide a solid foundation of genetic information for the North American Acipenseriformes that can be expanded to include Eurasian species to provide a global picture of sturgeon evolution.

Evolution of the ribosomal RNA internal transcribed spacer one (ITS-1) in cichlid fishes of the Lake Victoria region.

The nucleotide sequences of the first internal transcribed spacer (ITS-1) of the ribosomal RNA gene cluster have been determined for 11 species of closely related endemic cichlid fishes of the Lake Victoria region (LVR) and 6 related East African cichlids. The ITS-1 sequences confirmed independently derived basal phylogenies, but provide limited insight within this species flock. The line leading to Pseudocrenilabrus multicolor arose early, close to the divergence event that separated the tilapiine and haplochromine tribes of the "African Group" of the family Cichlidae. In this phylogeny, Astatoreochromis alluaudi and the riverine Astatotilapia burtoni are sister taxa, which together are a sister group to a monophyletic assemblage including both Lake Victoria and Lake Edward taxa. The ITS-1 data support the monophyly of haplochromine genera across lakes. Since Lake Victoria is believed to have been dry between 14, 500 and 12,400 BPE, the modern assemblage must have been derived from reinvasion by the products of earlier cladogenesis events. Thus, although the regional superflock is monophyletic, the haplochromines of Lake Victoria itself did not evolve in situ from a single ancestor.

Group-I introns with unusual sequences occur at three sites in nuclear 18S rRNA genes of Acanthamoeba lenticulata.

Seven of eleven isolates of Acanthamoeba lenticulata were found to have group-I introns located at one of three positions within the 18S rRNA gene. The introns are 636-721-bp long and are absent from mature rRNA. They lack open reading frames that could encode any known endonucleases. Sequences of introns from the same site in different isolates are 86.0-98.9% identical, while from different sites they are 24.2-29.8% identical. The most closely related introns from other organisms are in the 18S rRNA genes of several green algae where the 17.0-23.6% identity is mostly limited to a highly conserved core of base pairs including P, Q, R and S. Because the A. lenticulata introns only occur in one Acanthamoeba lineage, they were probably acquired after the divergence of this species.

The evolutionary history of the genus Acanthamoeba and the identification of eight new 18S rRNA gene sequence types.

The 18S rRNA gene (Rns) phylogeny of Acanthamoeba is being investigated as a basis for improvements in the nomenclature and taxonomy of the genus. We previously analyzed Rns sequences from 18 isolates from morphological groups 2 and 3 and found that they fell into four distinct evolutionary lineages we called sequence types T1-T4. Here, we analyzed sequences from 53 isolates representing 16 species and including 35 new strains. Eight additional lineages (sequence types T5-T12) were identified. Four of the 12 sequence types included strains from more than one nominal species. Thus, sequence types could be equated with species in some cases or with complexes of closely related species in others. The largest complex, sequence type T4, which contained six closely related nominal species, included 24 of 25 keratitis isolates. Rns sequence variation was insufficient for full phylogenetic resolution of branching orders within this complex, but the mixing of species observed at terminal nodes confirmed that traditional classification of isolates has been inconsistent. One solution to this problem would be to equate sequence types and single species. Alternatively, additional molecular information will be required to reliably differentiate species within the complexes. Three sequence types of morphological group 1 species represented the earliest divergence in the history of the genus and, based on their genetic distinctiveness, are candidates for reclassification as one or more novel genera.

Concordance of Porphyromonas gingivalis colonization in families.

Periodontitis is a widespread disease that appears to be due to a specific bacterial infection. Several species of bacteria have been investigated as potential pathogens, and particularly strong evidence links the presence of Porphyromonas gingivalis with indicators of periodontitis. Information concerning the transmission of P. gingivalis between human contacts may be important in determining risk factors for disease and developing preventive strategies. A few small studies have provided some evidence of transmission between related individuals, but no large-scale study of families that would reflect the typical transmission of this pathogen in the population has been reported. The purpose of this study was to investigate the transmission of P. gingivalis within randomly selected, extended families. The colonization status of 564 members of multigeneration families was determined, and the degree of concordance observed among members of these families was then compared to that expected to occur based on the prevalence of colonization in the population studied. A PCR assay was used for detection of P. gingivalis. Concordance in colonization was more frequently observed within entire families (P = 0.0000) and for spouses (P < 0.001), children and their mothers (P < 0.001), children and their fathers (P < 0.01), adults and their mothers (P < 0.005), and siblings (P < 0.05) than would be expected if P. gingivalis were randomly distributed in the population studied. Results showed that contact with an infected family member substantially increased the relative risk of colonization in these intrafamilial pairs. This indicates that P. gingivalis is commonly transmitted by contact with an infected family member.

Subgenus systematics of Acanthamoeba: four nuclear 18S rDNA sequence types.

Classification of Acanthamoeba at the subgenus level has been problematic, but increasing reports of Acanthamoeba as an opportunistic human pathogen have generated an interest in finding a more consistent basis for classification. Thus, we are developing a classification scheme based on RNA gene sequences. This first report is based on analysis of complete sequences of nuclear small ribosomal subunit RNA genes (Rns) from 18 strains. Sequence variation was localized in 12 highly variable regions. Four distinct sequence types were identified based on parsimony and distance analyses. Three were obtained from single strains: Type T1 from Acanthamoeba castellanii V006, T2 from Acanthamoeba palestinensis Reich, and T3 from Acanthamoeba griffini S-7. T4, the fourth sequence type, included 15 isolates classified as A. castellanii, Acanthamoeba polyphaga, Acanthamoeba rhysodes or Acanthamoeba sp., and included all 10 Acanthamoeba keratitis isolates. Interstrain sequence differences within T4 were 0%-4.3%, whereas differences among sequence types were 6%-12%. Branching orders obtained by parsimony and distance analyses were inconsistent with the current classification of T4 strains and provided further evidence of a need to reevaluate criteria for classification in this genus. Based on this report and others in preparation, we propose that Rns sequence types provide the consistent quantititive basis for classification that is needed.

Characterization of the SF agent, an Ehrlichia sp. isolated from the fluke Stellantchasmus falcatus, by 16S rRNA base sequence, serological, and morphological analyses.

The organism designated the SF agent was originally isolated in Japan in 1962 from Stellantchasmus falcatus metacercaria parasitic on gray mullet fish. The SF agent resembles members of the genus Ehrlichia morphologically and exhibits weak antigenic cross-reactivity with Ehrlichia sennetsu. This organism causes mild clinical signs in dogs, but severe splenomegaly and lymphadenopathy in mice. This suggests that the SF agent may be similar to either Neorickettsia helminthoeca, an intracellular parasite of a fluke and the cause of salmon poisoning disease in dogs, or E. sennetsu, the causative agent of human sennetsu ehrlichiosis in Japan and Malaysia. In order to determine the phylogenetic relationship between the SF agent and other ehrlichial species, the 16S rRNA gene was amplified by the PCR and sequenced. The SF agent sequence was most closely related to the sequences of Ehrlichia risticii (level of sequence similarity, 99.1%), the causative agent of Potomac horse fever, and E. sennetsu (level of sequence similarity, 98.7%). The next most similar sequence was that of N. helminthoeca, but the level of sequence similarity was only 93.7%. E. sennetsu, E. risticii, the SF agent, and N. helminthoeca formed a distinct cluster that was separated from all other ehrlichial species. As determined by immunofluorescence labeling, antiserum against the SF agent cross-reacted strongly with E. sennetsu, E. risticii, and N. helminthoeca. When three genetically distinct ehrlichial isolates obtained from horses with Potomac horse fever were compared with the SF agent, we found that the SF agent was most closely related to Ohio isolate 081, followed by IllinoisT (T = type strain) and a Kentucky isolate. We observed strong antigenic cross-reactivities and similarities in Western blot (immunoblot) reaction profiles when we compared the SF agent, E. risticii, and E. sennetsu; however, weaker antigenic cross-reactivity was observed when the SF agent and N. helminthoeca were compared. Our results indicate that the SF agent is antigenically more closely related to E. risticii and E. sennetsu than to N. helminthoeca. The biological and antigenic characteristics and the 16S rRNA sequence data suggest that the SF agent is a new species that belongs to the genus Ehrlichia.

16S rRNA gene sequence of Neorickettsia helminthoeca and its phylogenetic alignment with members of the genus Ehrlichia.

Neorickettsia helminthoeca (tribe Ehrlichieae, family Rickettsiaceae) is the agent of salmon poisoning disease, which affects members of the family Canidae. This bacterium is unusual in that it is the only known obligately intracellular bacterium that is transmitted via a helminth vector. The nucleotide sequence of the N. helminthoeca 16S rRNA gene was determined and compared with the sequences of intracellular bacteria belonging to the alpha subgroup of the Proteobacteria. The N. helminthoeca sequence was most similar to the sequences of two Ehrlichia species, Ehrlichia risticii and Ehrlichia sennetsu (levels of sequence similarity, > 95%). All other members of the tribe Ehrlichieae, including members of the other Ehrlichia species, and the related species Cowdria ruminantium and Anaplasma marginale, were only distantly related phylogenetically (levels of sequence similarity, 84 to 86%). Our results corroborate the results of previous ultrastructural and Western blot (immunoblot) comparisons of N. helminthoeca with other ehrlichial species. The genus Ehrlichia is phylogenetically incoherent and can be separated into three identifiable clusters of species. Each cluster is closely associated with a species classified in another non-Ehrlichia bacterial genus. The close relationships among N. helminthoeca, E. risticii, and E. sennetsu and the striking differences between these organisms and other members of the tribe Ehrlichieae suggest that in the future, these organisms should be treated as members of a new bacterial genus separate from the genus Ehrlichia.

Ehrlichia muris sp. nov., identified on the basis of 16S rRNA base sequences and serological, morphological, and biological characteristics.

The 16S rRNA gene of a new infectious agent, strain AS145T (T = type strain), which was isolated from a wild mouse in Japan, was amplified by using the PCR. The amplimers were directly sequenced by dideoxynucleotide methods with Taq DNA polymerase. Sequence comparisons with other members of the tribe Ehrlichieae and related species revealed that the infectious agent isolated from the mouse is a new species of the genus Ehrlichia that is most closely related to Ehrlichia chaffeensis (level of sequence similarity, 97.9%), an agent of human ehrlichiosis in the United States. This result was consistent with the results of an immunoblot analysis performed with immune sera against different ehrlichiosis agents. On the basis of these findings and other morphological, biological, and serological characteristics of the organism, we propose that ehrlichiae with these properties belong to a new species, Ehrlichia muris.

Diversity of 16S rRNA genes of new Ehrlichia strains isolated from horses with clinical signs of Potomac horse fever.

Ehrlichia risticii is the causative agent of Potomac horse fever. Variations among the major antigens of different local E. risticii strains have been detected previously. To further assess genetic variability in this species or species complex, the sequences of the 16S rRNA genes of several isolates obtained from sick horses diagnosed as having Potomac horse fever were determined. The sequences of six isolates obtained from Ohio and three isolates obtained from Kentucky were amplified by PCR. Three groups of sequences were identified. The sequences of five of the Ohio isolates were identical to the sequence of the type strain of E. risticii, the Illinois strain. The sequence of one Ohio isolate, isolate 081, was unique; this sequence differed in 10 nucleotides from the sequence of the type strain (level of similarity, 99.3%). The sequences of the three Kentucky isolates were identical to each other, but differed by five bases from the sequence of the type strain (level of similarity, 99.6%). The levels of sequence similarity of isolate 081, the Kentucky isolates, and the type strain to the next most closely related Ehrlichia sp., Ehrlichia sennetsu, were 99.3, 99.2, and 99.2%, respectively. On the basis of the distinct antigenic profiles and the levels of 16S rRNA sequence divergence, isolate 081 is as divergent from the type strain of E. risticii as E. sennetsu is. Therefore, we suggest that strain 081 and the Kentucky isolates may represent two new distinct Ehrlichia species.

Ancestral divergence of Rickettsia bellii from the spotted fever and typhus groups of Rickettsia and antiquity of the genus Rickettsia.

The eubacterial genus Rickettsia belongs to the alpha subgroup of the phylum Proteobacteria. This genus is usually divided into three biotypes on the basis of vector host and antigenic cross-reactivity characteristics. However, the species Rickettsia bellii does not fit into this classification scheme; this organism has characteristics common to both the spotted fever group and the typhus group biotypes and also exhibits some unique features. Sequences of the 16S rRNA and 23S rRNA genes from Rickettsia rickettsii (spotted fever group), Rickettsia prowazekii (typhus group), and R. bellii were studied to determine the position of R. bellii in the rickettsial classification scheme. The 23S rRNA gene sequences described in this paper are the first 23S rRNA sequences reported for any member of the Rickettsiaceae. The 23S rRNA gene contains substantially more phylogenetic information than is contained in the 16S rRNA sequences, and the 23S rRNA gene sequence has diverged about 1.9 times faster in the three Rickettsia species which we studied. Taken together, the molecular data obtained from the two genes indicate that R. bellii is not a member of either the spotted fever group or the typhus group; rather, this organism appears to be the product of a divergence which predates the separation of the genus into the spotted fever group and the typhus group. Consequently, different combinations of the ancestral characteristics retained by R. bellii have been retained in the more derived lineages of the genus.(ABSTRACT TRUNCATED AT 250 WORDS)

Sequence variations in small-subunit ribosomal RNAs of Hartmannella vermiformis and their phylogenetic implications.

Evidence of associations between free-living amoebas and human disease has been increasing in recent years. Knowledge about phylogenetic relationships that may be important for the understanding of pathogenicity in the genera involved is very limited at present. Consequently, we have begun to study these relationships and report here on the phylogeny of Hartmannella vermiformis, a free-living amoeba that can harbor the etiologic agent of Legionnaires' disease. Our analysis is based on studies of small-subunit ribosomal RNA genes (srDNA). Nucleotide sequences were determined for nuclear srDNA from three strains of H. vermiformis isolated from the United Kingdom, Germany, and the United States. These sequences then were compared with a sequence previously obtained for a North American isolate by J. H. Gunderson and M. L. Sogin. The four genes are 1,840 bp long, with an average GC content of 49.6%. Sequence differences among the strains range are 0.38%-0.76%. Variation occurs at 19 positions and includes 2 single-base indels plus 14 monotypic and 3 ditypic single-base substitutions. Variation is limited to eight helix/loop structures according to a current model for srRNA secondary structure. Parsimony, distance, and bootstrap analyses used to examine phylogenetic relationships between the srDNA sequences of H. vermiformis and other eukaryotes indicated that Hartmannella sequences were most closely related to those of Acanthamoeba and the alga Cryptomonas. All ditypic sites were consistent with a separation between European and North American strains of Hartmannella, but results of other tests of this relationship were statistically inconclusive.

Detection and strain identification of Actinobacillus actinomycetemcomitans by nested PCR.

By using PCR, Actinobacillus actinomycetemcomitans strains were identified directly from plaque samples without the need to isolate or culture bacteria. DNA fragments were generated by a nested, two-step PCR amplification of the ribosomal spacer region between the 16S and 23S rRNA genes. For the first amplification, primers homologous to sequences common to all bacterial species were used. This was followed by a second amplification with primers specific to A. actinomycetemcomitans. The ribosomal DNA spacer region was amplified from as few as 10 bacterial cells within a total population of 10(8) cells (0.00001%), and cross-reactivity between species was not observed. DNA fragments specific for Porphyromonas gingivalis were generated from the same samples by using a P. gingivalis-specific primer, and equivalent sensitivity and specificity were observed. A. actinomycetemcomitans was detected in 60% and P. gingivalis was detected in 79% of 52 subjects tested. Sequence analysis of the spacer region DNA fragment for A. actinomycetemcomitans gave precise strain identification, producing unique sequences for seven reference strains and identification of nine plaque-derived isolates. A phylogenetic tree based on quantitative sequence relationships was constructed. Two-step PCR amplification directly from plaque samples combined with sequence analysis of the ribosomal DNA spacer region provides a sensitive assay for detection and strain identification of multiple species directly from a single plaque sample. This simplified approach provides a practical method for large-scale studies on the transmission and pathogenicity of periodontitis-associated bacteria.

Discovery of group I introns in the nuclear small subunit ribosomal RNA genes of Acanthamoeba.

The discovery of group I introns in small subunit nuclear rDNA (nsrDNA) is becoming more common as the effort to generate phylogenies based upon nsrDNA sequences grows. In this paper we describe the discovery of the first two group I introns in the nsrDNA from the genus Acanthamoeba. The introns are in different locations in the genes, and have no significant primary sequence similarity to each other. They are identified as group I introns by the conserved P, Q, R and S sequences (1), and the ability to fit the sequences to a consensus secondary structure model for the group I introns (1, 2). Both introns are absent from the mature srRNA. A BLAST search (3) of nucleic acid sequences present in GenBank and EMBL revealed that the A. griffini intron was most similar to the nsrDNA group I intron of the green alga Dunaliella parva. A similar search found that the A. lenticulata intron was not similar to any of the other reported group I introns.