Chromosomal DNA deletions explain phenotypic characteristics of two antigenic variants, phase II and RSA 514 (crazy), of the Coxiella burnetii nine mile strain.

After repeated passages through embyronated eggs, the Nine Mile strain of Coxiella burnetii exhibits antigenic variation, a loss of virulence characteristics, and transition to a truncated lipopolysaccharide (LPS) structure. In two independently derived strains, Nine Mile phase II and RSA 514, these phenotypic changes were accompanied by a large chromosomal deletion (M. H. Vodkin and J. C. Williams, J. Gen. Microbiol. 132:2587-2594, 1986). In the work reported here, additional screening of a cosmid bank prepared from the wild-type strain was used to map the deletion termini of both mutant strains and to accumulate all the segments of DNA that comprise the two deletions. The corresponding DNAs were then sequenced and annotated. The Nine Mile phase II deletion was completely nested within the deletion of the RSA 514 strain. Basic alignment and homology studies indicated that a large group of LPS biosynthetic genes, arranged in an apparent O-antigen cluster, was deleted in both variants. Database homologies identified, in particular, mannose pathway genes and genes encoding sugar methylases and nucleotide sugar epimerase-dehydratase proteins. Candidate genes for addition of sugar units to the core oligosaccharide for synthesis of the rare sugar 6-deoxy-3-C-methylgulose (virenose) were identified in the deleted region. Repeats, redundancies, paralogous genes, and two regions with reduced G+C contents were found within the deletions.

Differentiation of Aedes triseriatus (Say) from Aedes hendersoni Cockerell (Diptera: Culicidae) by restriction fragment length polymorphisms of amplified ribosomal DNA.

Aedes triseriatus is the primary vector of LaCrosse (LAC) virus, which can cause encephalitis, especially in young children. Aedes hendersoni, a sibling species of Ae. triseriatus, has a salivary gland barrier to LAC virus and, therefore, is not considered a vector of this virus. Adults of Ae. triseriatus are morphologically indistinguishable from those of Ae. hendersoni, and the two species are sympatric in the eastern United States. A definitive method of identifying field specimens is an important part of any disease surveillance program, particularly in the case of LAC virus. This study identifies restriction enzymes that produce species-specific restriction fragment length polymorphisms (RFLPs) from amplified ribosomal (r) DNA. In addition, sequences of the internal transcribed spacers 1 and 2 and the 5.8S regions of the rDNA were used to confirm the RFLP patterns. This study is the first to compare nucleotide sequences from Ae. triseriatus and Ae. hendersoni.

Identification of two genetic markers that distinguish pathogenic and nonpathogenic strains of Acanthamoeba spp.

Species-level identification of Acanthamoeba isolates is difficult and gives little or no indication of the isolate's pathogenicity. We identified two amplification-based genetic markers that were highly correlated with pathogenicity in Acanthamoeba spp. One marker, designed to amplify a 485-bp fragment of the small-subunit ribosomal RNA gene (ssrDNA), was preferentially amplified from the nonpathogenic strains; amplifications from the pathogenic strains yielded anomalous fragments of 650 and 900 bp. A second marker was developed on the basis of the anomalous 650-bp fragment. Primers to this sequence preferentially amplified a noncoding locus (called Ac6) only from the pathogenic strains. These two genetic markers may be useful for identification of pathogenic Acanthamoeba spp. strains.

Restriction analysis of the ribosomal DNA internal transcribed spacer region of Culex restuans and mosquitoes in the Culex pipiens complex.

Members of the Culex pipiens Linn. complex in the eastern, southern, and central United States are the primary vectors of St. Louis encephalitis virus. Although species and subspecies in the complex can be identified as 4th-instar larvae and by characters on the male genitalia, adult females cannot be identified accurately. In this study a ribosomal DNA (rDNA) segment that includes the internal transcribed spacer region (ITS) was amplified from Culex pipiens pipiens Linn., Culex quinquefasciatus Say, and Culex restuans Theobald. The DNA was amplified from single abdomens or single legs. The amplified rDNA segment from Cx. restuans is 90 base pairs smaller than those from members of the Cx. pipiens complex. Ribosomal DNA was amplified separately from 3 individuals for each population of Cx. pipiens and analyzed by restriction digestion. Intrapopulation variation is seen, because for each population, bands are present that are common to all 3 individuals within the population, but are also unique to that population. These results indicate that this method may provide a means for distinguishing among the mosquitoes in the Cx. pipiens complex.

Phylogenetic analysis of beta-tubulin sequences from amitochondrial protozoa.

It has been proposed that certain extant anaerobic protozoa are descended from organisms that diverged early in eukaryotic evolution prior to the acquisition of mitochondria. Among these are the extracellular parasites Giardia lamblia, Trichomonas vaginalis, and Entamoeba histolytica, and the obligately intracellular microsporidia. Phylogenetic analysis of rRNA sequences from these amitochondrial organisms suggests that G. lamblia, T. vaginalis, and microsporidia are near the base of the eukaryotic tree, while E. histolytica clusters with mitochondria-containing species. However, since eukaryotes likely evolved by symbiotic associations, it is important to analyze other sequences which may have independent origins. Unlike ribosomes, microtubules appear to be unique to eukaryotes. Complete gene sequences for the beta-tubulin subunit of microtubules from T. vaginalis, E. histolytica, and the microsporidian Encephalitozoon hellem have recently been determined. Phylogenetic relationships among these, G. lamblia, and 20 additional beta-tubulins were analyzed by distance matrix and parsimony methods, using alpha- and gamma-tubulin outgroups. All analyses placed the E. histolytica sequence at the base of the beta-tubulin evolutionary tree. Similar results were obtained for E. histolytica alpha-tubulin using a less representative set of sequences. In contrast, the E. hellem sequence branched considerably higher, within the lineage containing animal and fungal beta-tubulins. Possible explanations are considered for these unexpected differences between the beta-tubulin and rRNA trees.

Evaluation of a reverse transcriptase-polymerase chain reaction assay for detecting St. Louis encephalitis virus using field-collected mosquitoes (Diptera: Culicidae).

A reverse transcriptase-polymerase chain reaction assay (RT-PCR) was compared with a tissue culture assay (TC) and an enzyme immunoassay (EIA) to detect St. Louis encephalitis virus (SLEV) in mosquito pools. Overall, 1,725 mosquito pools with a low viral prevalence (3.3%-5.0%) were tested. The comparison of the EIA to TC showed that the EIA was 0.947 sensitive, 0.988 specific, and 0.987 accurate. Comparison of RT-PCR to TC showed that the RT-PCR was 0.947 sensitive, 0.980 specific, and 0.979 accurate. Comparison of the RT-PCR to EIA showed that the RT-PCR was 0.878 sensitive, 0.987 specific, and 0.982 accurate. Because of speed, accuracy, and cost, either the RT-PCR or the EIA is recommended as the primary screen. RT-PCR has an advantage over EIA, because the amplified product contains sequence information which can confirm its identity. TC, EIA, or both can be applied as a 2nd assay for the confirmation of samples suspected as positives by the RT-PCR.

Mosquito productivity and surveillance for St. Louis encephalitis virus in Chicago during 1993.

The City of Chicago's Department of Health monitors weekly deposition of egg rafts of Culex species, prevalence of St. Louis encephalitis (SLE) virus-specific antibodies in feral birds, and prevalence of the virus in mosquito pools. The total number of Culex egg rafts collected in 1993 (4,623) was 2-fold greater than for the 1992 mosquito season. Virtually all of the early summer egg rafts were identified as Culex restuans. After the week of July 18, Culex pipiens accounted for 20-70% of the total rafts collected weekly. The prevalences for SLE viral antibodies (avian) and RNA (mosquitoes) were 0.2% and 0.02%, respectively. Both values were about 25-fold less than normally occur in epidemic years. It is important for practical considerations to continue this surveillance in order to recommend time- and site-specific mosquito abatement.

Notes on the biology of Orthopodomyia in Illinois.

The principal habitats of immature Orthopodomyia alba and Orthopodomyia signifera are tree holes, though they are occasionally encountered in artificial containers. We collected 929 Or. alba and 17 Or. signifera from 2 plastic trash cans, 2 scrap tires, and a tree hole. All collections were made in an urban residential area in central Illinois.

PCR-based detection of arboviral RNA from mosquitoes homogenized in detergent.

An improved method for the extraction of viral RNAs was developed to facilitate the reverse transcription (RT)-PCR detection of mosquitoes infected with Western equine encephalitis virus or La Crosse virus. The solubilization method, which uses only EDTA and sodium dodecyl sulfate (SDS) followed by dilution of sample, allows accurate viral detection through the use of random hexamers for the RT followed by specific primers for the PCR. Identities of the reaction products were confirmed either by sequencing or restriction endonuclease digestion. Previous methods for the extraction of RNA for the coupled RT-PCR depended on combinations of guanidinium isothiocyanate, acid phenol, detergents and multiple centrifugations. Ideally, routine detection of viral RNAs for diagnostic purposes should bypass many of the above steps, while still providing a sensitive assay. Our level of detection is 1 infected mosquito in a group of 100.

A rapid diagnostic assay for eastern equine encephalomyelitis viral RNA.

Eastern equine encephalomyelitis virus (EEEV) has been a low-frequency, but serious human and veterinary health problem. Increased frequency of this mosquito-borne virus is anticipated as wetlands are maintained and re-established. Control of EEEV has depended on mosquito abatement in response to increasing frequency of EEEV in the environment. A coupled reverse transcription/polymerase chain reaction assay was designed to rapidly, sensitively, and specifically detect EEEV RNA. The assay successfully detected the viral RNA in a single-blind study of a set of field samples composed of either pooled mosquitoes or bird tissue. These results suggest that it would be practical to use this assay for deciding when and where to implement mosquito abatement.

A ribosomal protein in Acanthamoeba polyphaga is conserved in eukaryotic nuclei, organelles and bacteria.

An EcoRI fragment from the mitochondrial DNA of Acanthamoeba polyphaga was cloned and partly sequenced, and the conceptual translation product of the open reading frame (partial sequence) was found to have similarities with rp114, a ribosomal protein. Phylogenetic analysis based on the amino acid (aa) sequences of this conserved protein resolved four branches that consisted of: (1) eubacteria and the chloroplasts of algae and higher plants, (2) ciliate mitochondria, (3) Acanthamoeba, and (4) archaebacteria and the nuclei of eukaryotes. The groupings based on the rp114 aa sequences were consistent with the phylogenies derived by rRNA analysis of these organisms.

Acanthamoeba binds to extracellular matrix proteins in vitro.

PURPOSE: To identify host-tissue amoeba interactions that may be important in the pathogenesis of Acanthamoeba keratitis, the ability of the opportunistic pathogen Acanthamoeba polyphaga to bind various components of the extracellular matrix (collagen type IV, laminin, or fibronectin) was examined in vitro. METHODS: A polyphaga, isolated from a case of human amoebic keratitis, was used in the studies. In the experiments, 96-well plates were coated with 0-, 5-, 10-, 20-, or 50-micrograms/ml solutions of the basal lamina proteins laminin or collagen type IV, the extracellular matrix protein fibronectin, or casein (control). Amoeba were metabolically labeled with 35[S]-methionine, and 1x 10(4) labeled amoeba in phosphate buffered saline (PBS) were seeded per well and allowed to bind for 20 min. After washing with PBS, bound amoeba were solubilized with 1% sodium dodecyl sulphate (SDS) and scintillation counting was used to determine the number of bound amoeba. RESULTS: Counts from casein and protein-free controls were not significantly different from each other (P > 0.05). There was a significant increase in the binding of 35[S]-labeled A. polyphaga to collagen IV, laminin, and fibronectin over controls (P < 0.0001) and the binding was concentration-dependent. The rank order of binding was collagen > or = laminin >> fibronectin. Alpha-methyl-mannopyranoside, but not fucose, inhibited binding of labeled A. polyphaga to collagen IV, laminin, and fibronectin in a concentration-dependent manner. CONCLUSION: In summary, the binding assays show that Acanthamoeba bind preferentially to collagen, laminin, and fibronectin, in that order, and that the adherence process is inhibited by mannose.

A Coxiella burnetti repeated DNA element resembling a bacterial insertion sequence.

A DNA fragment located on the 3' side of the Coxiella burnetii htpAB operon was determined by Southern blotting to exist in approximately 19 copies in the Nine Mile I genome. The DNA sequences of this htpAB-associated repetitive element and two other independent copies were analyzed to determine the size and nature of the element. The three copies of the element were 1,450, 1,452, and 1,458 bp long, with less than 2% divergence among the three sequences. Several features characteristic of bacterial insertion sequences were discovered. These included a single significant open reading frame that would encode a 367-amino-acid polypeptide which was predicted to be highly basic, to have a DNA-binding helix-turn-helix motif, to have a leucine zipper motif, and to have homology to polypeptides found in several other bacterial insertion sequences. Identical 7-bp inverted repeats were found at the ends of all three copies of the element. However, duplications generated by many bacterial mobile elements in the recipient DNA during insertion events did not flank the inverted repeats of any of the three C. burnetii elements examined. A second pair of inverted repeats that flanked the open reading frame was also found in all three copies of the element. Most of the divergence among the three copies of the element occurred in the region between the two inverted repeat sequences in the 3' end of the element. Despite the sequence changes, all three copies of the element have retained significant dyad symmetry in this region.

Detection of St. Louis encephalitis virus in mosquitoes by use of the polymerase chain reaction.

We recently developed an assay using the polymerase chain reaction (PCR) for the specific detection of St. Louis encephalitis (SLE) virus RNA. This assay was tested in a blind study on 7 samples of pooled mosquitoes (50 mosquitoes/pool) which were also characterized for SLE virus by plaque assay in Vero cell culture. One sample was positive for the SLE virus as determined by both the PCR assay and a combination of the plaque assay and the indirect fluorescent antibody assay. The remaining 6 samples were negative for the presence of SLE virus as determined by both methods. These data indicate that this PCR assay can be used to monitor for the presence of SLE virus in pools of homogenized mosquitoes. This approach could provide early data on which to base disease control decisions.

Differential polymerase chain reaction for detection of wild-type and a vaccine strain of Aujeszky's disease (pseudorabies) virus.

A polymerase chain reaction (PCR) strategy for differentiating between a vaccine mutant strain and wild-type (WT) strains of Aujesky's disease (pseudorabies) virus (ADV) was evaluated. With this approach, a single virus or a concurrent WT and vaccine virus infection could be distinguished by targeting the genomic alteration within the vaccine strain. PCR primers were designed for a recombinant vaccine virus that has almost all of the WT gX gene replaced by the lacZ gene. One primer, corresponding to a conserved sequence upstream of the altered region, was selected for common use. The differentiating primers were chosen from the unique WT gX and vaccine lacZ gene sequences. The sensitivity of the differential PCR was analyzed using extracted viral DNA and in vitro infected cell lysates. Approximately 10 and between 10 to 100 molecules of WT and vaccine viral DNAs, respectively, could be detected, regardless of the presence or absence of uninfected cell lysates. Detection of viral DNA from in vitro infected cell cultures approximated this level of sensitivity. The specificity of the amplifications was verified by restriction endonuclease analysis and Southern hybridization. Although the vaccine primer pair target was amplified to a lesser degree as compared to the WT primer pair product, utility of the differential PCR was demonstrated using trigeminal nerve ganglia from swine infected with vaccine virus and WT virus. Both viral targets were detected only by their specific primer pair, in either the single or dual infection.

Identification of Acanthamoeba at the generic and specific levels using the polymerase chain reaction.

We have adapted the polymerase chain reaction to identify strains of Acanthamoeba. Using computer-assisted analysis, primers were designed from an anonymous repetitive sequence and from published sequences of 18S and 5S ribosomal RNA genes of A. castellanii. Amplification of a short ribosomal DNA target (272 base pairs) at restrictive annealing conditions (greater than 50 degrees C) resulted in a single band that was unique for the genus and distinguished Acanthamoeba from Naegleria. This assay functioned with fresh and formalin-fixed cells as starting material. Amplification of longer targets (400-700 base pairs) at less restrictive annealing conditions (less than 47 degrees C) led to more than one band. This multiple banding pattern could reproducibly classify Acanthamoeba at the strain level and was, in certain cases, diagnostic for known pathogenic strains. However, these assays need to be further refined to make them relevant for clinical purposes.

Use of the polymerase chain reaction for the sensitive detection of St. Louis encephalitis viral RNA.

Polymerase chain reaction (PCR) assays were developed for the detection of RNA from the St. Louis encephalitis (SLE) virus. Using computer-assisted analysis of the MSI-7 strain SLE virus genome, two primer pairs were selected from the capsid-coding and the membrane associated protein-coding genes, and one from the envelope-coding gene. Reverse transcription was primed with either specific oligomers or with random hexamers; these methods were compared for cDNA synthesis and subsequent PCR amplification with the oligomeric pairs. Random hexamers provided more sensitive detection of viral RNA. Each primer pair specifically amplified the expected sized fragment from the Parton SLE strain grown in Aedes albopictus cells, but did not amplify Aedes albopictus cell RNA controls. The technique also detected SLE virus RNA in 1 pg of total cellular RNA added to a background of 1 microgram boiled brain tissue, and in 0.5 pg of total RNA added to homogenized mosquito abdomen. PCR-based assays may be adaptable to detect SLE virus RNA in naturally infected mosquitoes, birds, and human cerebrospinal fluid and brain.

Molecular approaches to malaria and babesiosis diagnosis.

The development of additional methods for detecting and identifying Babesia and Plasmodium infections may be useful in disease monitoring, management and control efforts. The preliminary evaluate synthetic peptide-based serodiagnosis, a hydrophilic sequence (DDESEFDKEK) was selected from the published BabR gene of B. bovis. Immunization of rabbits and cattle with the hemocyanin-conjugated peptide elicited antibody responses that specifically detected both P. falciparum and B. bovis antigens by immunofluorescence and Western blots. Using a dot-ELISA with this peptide, antisera from immunized and naturally-infected cattle, and immunized rodents, were specifically detected. Reactivity was weak and correlated with peptide immunization or infection. DNA-based detection using repetitive DNA was species-specific in dot-blot formats for B. bovis DNA, and in both dot-blot and in situ formats for P. falciparum; a streamlined enzyme-linked synthetic DNA assay for P. falciparum detected 30 parasites/mm3 from patient blood using either colorimetric (2-15 h color development) or chemiluminescent detection (0.5-6-min exposures). Serodiagnostic and DNA hybridization methods may be complementary in the respective detection of both chronic and acute infections. However, recent improvements in the polymerase chain reaction (PCR) make feasible a more sensitive and uniform approach to the diagnosis of these and other infectious disease complexes, with appropriate primers and processing methods. An analysis of ribosomal DNA genes of Plasmodium and Toxoplasma identified Apicomplexa-conserved sequence regions. Specific and distinctive PCR profiles were obtained for primers spanning the internal transcribed spacer locus for each of several Plasmodium and Babesia species.