Paul A. Fuerst

WHAT MOLECULES CAN TELL US ABOUT POPULATIONS: CHOOSING ANDUSING A MOLECULAR MARKER

The rapid development of molecular techniques offers a palette of technical approaches for population biologists interested in a wide range of questions. For example, these tools can be used to determine individual reproductive success or to measure rates of genetic divergence among populations. Which technique is most appropriate for a par- ticular question depends upon (1) the extent of genetic polymorphism required to best answer the question, (2) the analytical or statistical approaches available for the techniques application, and (3) the pragmatics of time and costs of materials. Here we evaluate the application of several major techniques (protein electrophoresis, nuclear and mitochondrial RFLPs (restriction fragment length polymorphisms), minisatellite and microsatellite VNTRs (variable number tandem repeats), RAPDs (random amplified polymorphic DNA), and DNA sequencing) to an array of questions regarding individual identification, exclusion and assignment of parentage, and various levels of population structure. In our evaluation, we briefly explain the technical components of each molecular approach and assess whether the typical outcomes expected from each approach will provide useful information as applied to each level of inquiry. For studies of population genetic structure, protein electrophoresis remains a powerful tool for most taxa, although techniques based on nucleic acids (par- ticularly DNA sequencing and mitochondrial DNA RFLPs) are useful here as well. Recently developed nucleic acid techniques (e.g., VNTRs) can often identify enough genetic vari- ability to address questions of self-identification or parentage. Some of the newest tech- niques (RAPDs and microsatellites) are potentially useful across a number of levels of inquiry, although procedures for adopting them are still developing.

Use of Subgenic 18S Ribosomal DNA PCR and Sequencing for Genus and Genotype Identification of Acanthamoebae from Humans with Keratitis and from Sewage Sludge

ABSTRACT 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-bpAcanthamoeba-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.

The Evolutionary History of the Genus Acanthamoeba and the Identification of Eight New 18S rRNA Gene Sequence Types

ABSTRACT 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.

Scrub Typhus: The Geographic Distribution of Phenotypic and Genotypic Variants of Orientia tsutsugamushi

Orientia tsutsugamushi is the etiological agent of scrub typhus, an acute, mite-borne, febrile illness that occurs in the Asia-Pacific region. Historically, strain characterization used serological analysis and revealed dramatic antigenic diversity. Eyeing a recommendation of potential vaccine candidates for broad protection, we review geographic diversity and serological and DNA prevalences. DNA analysis together with immunological analysis suggest that the prototype Karp strain and closely related strains are the most common throughout the region of endemicity. According to serological analysis, approximately 50% of isolates are seroreactive to Karp antisera, and approximately one-quarter of isolates are seroreactive to antisera against the prototype Gilliam strain. Molecular methods reveal greater diversity. By molecular methods, strains phylogenetically similar to Karp make up approximately 40% of all genotyped isolates, followed by the JG genotype group (Japan strains serotypically similar to the Gilliam strain but genetically non-Gilliam; 18% of all genotyped isolates). Three other genotype groups (Kato-related, Kawasaki-like, and TA763-like) each represent approximately 10% of genotyped isolates. Strains genetically similar to the Gilliam strain make up only 5% of isolates. Strains from these groups should be included in any potential vaccine.

Identification and Distribution of Acanthamoeba Species Genotypes Associated with Nonkeratitis Infections

ABSTRACT Acanthamoeba is a free-living protozoan genus found in a wide variety of natural habitats, including water, soil, and air. Pathogenic isolates of Acanthamoeba are medically relevant as the causative agent of sight- threatening Acanthamoeba keratitis (AK), serious infections of other organs, and fatal granulomatous amebic encephalitis. Previous work employing DNA sequences of nuclear and mitochondrial small-subunit rRNA genes (SSU rRNA genes) determined the genotypic diversity of Acanthamoeba and found that many named species of Acanthamoeba are associated with particular genotypes. These studies also concluded that nearly all AK infections result from a single molecular genotype: T4. Here, we asked whether Acanthamoeba clinical isolates from non-AK infections are also associated with particular genotypes. DNA sequence determination of nuclear SSU rRNA genes was employed for genotypic identification of 29 isolates of Acanthamoeba from non-AK infections. Sequence analysis demonstrates that T4 is the predominant genotype in non-AK infections, including those in brain, cerebrospinal fluid, nasal passages, skin, and lung. Rare genotypes (T1, T10, and T12) have been isolated from brain infections. We conclude that genotype T4 is the primary genotype in non-AK Acanthamoeba infections, as was the case in AK infections. However, the genotypes that were isolated from brains have not been observed in environmental isolates of Acanthamoeba, and their natural ecological niche is unknown.

Subgenus systematics of Acanthamoeba : Four nuclear 18S rDNA sequence types

ABSTRACT 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.

Evolutionary analysis of the spotted fever and thyphus groups of Rickettsia using 16S rRNA gene sequences

Summary The bacterial genus Rickettsia is traditionally divided into three biotypes, the spotted fever group (SFG), the typhus group (TG), and the scrub typhus group (STG) based on vector host and antigenic cross-reactivity. DNA sequence data were gathered from the 16S ribosomal RNA gene of several SFG and TG species. Comparative sequence analysis shows that: i) all species of Rickettsia are closely related, exhibiting 0.3–2.6% sequence divergence; ii) although there are identifiable clusters corresponding to the SFG and TG, species of Rickettsia fall into more than two distinct phylogenetic groups; iii) the tick-borne species Rickettsia bellii and Rickettsia canada diverged prior to the schism between the spotted fever and typhus groups; iv) the newly described AB bacterium is clearly a member of Rickettsia , but its phylogenetic placement within the genus is problematic; v) the mite-borne Rickettsia akari , the tick-borne Rickettsia australis and the recently described flea-borne ELB agent form a loose cluster that cannot be definitively associated with either the TG or the traditional SFG cluster. This latter Glade may represent a unique group(s) distinct from the main cluster of spotted fever and typhus group species. The divergence of Rickettsia was an ancient event within the α-subclass of the proteobacteria. The sequence divergence between Rickettsia and Ehrlichia , the closest known genus to Rickettsia , is nearly equal to the sequence divergence between Rickettsia and all other α-subclass proteobacterial taxa included in the analysis. When Rickettsia was compared to a representative group of the α-subclass, twenty-eight nucleotide sites were identified which uniquely characterize the 16S rRNA sequences of all species of Rickettsia . The approximate time of divergence between the various species of Rickettsia , estimated from the bacterial 16S rRNA molecular clock, coincides with the approximate divergence time of the hard body ticks which are the arthropod hosts of many Rickettsia . Thus, the possibility of coevolution between these intracellular bacteria and their tick hosts exists.

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)

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.

The relative value of confocal microscopy and superficial corneal scrapings in the diagnosis of Acanthamoeba keratitis.

Purpose: To compare the relative diagnostic value of confocal microscopy and superficial corneal cultures in the diagnosis of Acanthamoeba keratitis by using clinical and microbiologic definitions of disease. Methods: Results of confocal microscopy, superficial corneal smear, and superficial corneal culture were analyzed for validity against 2 different microbiologic and a clinical composite standard for Acanthamoeba keratitis. Results: In patients with both clinical characteristics and objective evidence of Acanthamoeba keratitis, confocal microscopy exhibited a sensitivity of 90.6% (95% confidence interval [CI]: 79.3%-96.9%) and a specificity of 100% (95% CI: 95.0%-100%). In patients with either positive culture or smear evidence of Acanthamoeba keratitis, confocal microscopy showed a sensitivity of 90.9% (95% CI: 78.3%-97.5%) and specificity of 90.1% (95% CI: 81.5%-95.6%). In strictly culture-positive patients, confocal microscopy showed a sensitivity of 92.9% (95% CI: 76.5%-99.1%) and a specificity of 77.3% (95% CI: 67.7%-85.2%). Of the 53 patients with Acanthamoeba keratitis, confocal microscopy was positive in 48 patients, whereas corneal smears and cultures were positive in 30 of 41 and 23 of 42 patients, respectively. Sensitivity of Acanthamoeba culture was 52.8% (95% CI: 38.6%-66.7%) in patients with a clinical diagnosis of Acanthamoeba keratitis. Simultaneous testing of smear and superficial corneal scraping resulted in a sensitivity of 83.0% (95% CI: 70.2%-91.9%), independent of the results of confocal microscopy. Conclusions: As confocal microscopy comes into wider clinical use, it remains in need of clinical and pathologic correlation. When performed and interpreted by an experienced operator, confocal microscopy is both sensitive and specific in the diagnosis of Acanthamoeba keratitis. Contemporaneous corneal scrapings are independently sensitive in the detection of Acanthamoeba keratitis, and a combination of both diagnostic modalities offers the highest likelihood of rapidly and accurately diagnosing Acanthamoeba keratitis in patients with atypical keratitis.