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.

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.

Calcium enhances Acanthamoeba polyphaga binding to extracellular matrix proteins.

PURPOSE: To characterize better the ameba-host interactions that may be involved with the pathogenesis of Acanthamoeba keratitis, the role of calcium (Ca2+) on the binding of Acanthamoeba polyphaga to extracellular matrix proteins was examined in vitro. METHODS: The binding of a metabolically labeled A. polyphaga (CDC:0187:1) isolate from a case of human keratitis to collagen type IV, laminin, and fibronectin was assessed through a range of calcium concentrations in the external fluid. Binding to collagen IV was studied in detail, with and without other divalent cations and calcium channel modulators. RESULTS: Calcium increased binding in a dose-dependent manner, with significant effects at 0.1 to 1.0 microM and near-maximal effects at 1 to 100 microM, depending upon the matrix protein. Magnesium alone had no effect on ameba binding to collagen IV but suppressed the action of calcium. Strontium enhanced ameba binding, with maximal effect at 100 microM. The calcium channel antagonists nifedipine and diltiazem-HCl and a calcium channel activator, Bay-K8644, had no effect on the action of calcium. However, the inorganic calcium antagonists, lanthanum and cobalt, suppressed the effect of calcium. CONCLUSION: Low concentrations of calcium enhance the adhesion of A. polyphaga to extracellular matrix proteins. It remains uncertain whether calcium acts intracellularly or at the cell surface.

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.

Characterization of the clinical and anatomical pathological changes associated with Hepatozoon mocassini infections in unnatural reptilian hosts.

Laboratory-reared Aedes aegypti mosquitoes were employed in the successful transmission of Hepatozoon mocassini from a cotton-mouth moccasin (Agkistrodon piscivorus leucostoma) to 3 lizard species (Sceloporus undulatus, Eumeces obsoletus and Sceloporus poinsetti). Marked to severe lethargy and anorexia developed in the S. undulatus, E. obsoletus and S. poinsetti at 15, 38, and 96 days postinfection (PI), respectively. All 3 lizards developed a leukocytosis and had increased plasma aspartate aminotransferase activity (AST) by 14 days PI. Multifocal random hepatocellular necrosis and intrahepatic aggregates of heterophils centered on mature H. mocassini meronts were demonstrated in all 3 lizards. The pulmonary interstitium was multifocally thickened by aggregates of heterophils centered on meronts. No comparable clinical or anatomical pathological changes were demonstrated in naturally infected snakes. The results of this study suggest that H. mocassini is capable of inducing necrotizing inflammatory by lesions in unnatural reptilian hosts.

Repeated immunogenic amino acid sequences of Plasmodium species share sequence homologies with proteins from humans and human viruses.

The use of recombinant peptides based upon the repeated amino acid sequences of Plasmodium has been proposed for malaria vaccines. By reducing homologies of such peptide vaccines to host proteins, the possibility of autoimmune complications may be reduced, and the effective immune response may be enhanced. The Wilbur and Lipman Wordsearch algorithm was used to identify homologous amino acid sequences between tandemly repeated Plasmodium amino acid sequences and the human and human viral sequences compiled in the National Biomedical Research Foundation database. Six published repetitive immunogenic amino acid sequences from the circumsporozoite (CS) antigen, ring-infected erythrocyte surface antigen (RESA), soluble (S) antigen, and falciparum interspersed repetitive antigen (FIRA) of P. falciparum, and the CS protein of P. vivax, were analyzed by computer. Matches of at least 4 amino acids were found for all sequences. In the database, 29 matches were found for human proteins and 26 matches were found for human viruses with the 6 antigen sequences. Most of the matched proteins, and many of the matched human viruses, are found in blood. The biological significance of these matches remains to be clarified.

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.

Detection of Plasmodium falciparum using a synthetic DNA probe.

A labeled synthetic polynucleotide representing a repetitive sequence from Plasmodium falciparum was hybridized with genomic DNA spotted on nitrocellulose. After an overnight exposure, 0.1 ng of P. falciparum DNA was specifically detected and 0.01 ng was detected after an exposure of 1 week. The synthetic probe showed no cross-hybridization with host DNA or with DNA isolated from other species in the phylum Apicomplexa, P. vivax and Babesia species. Since synthetic DNA is easily prepared, the observed sensitivity and specificity suggests that synthetic DNA probes would be generally useful in diagnosis.

Evaluation of a nonisotopic DNA assay kit for diagnosing Plasmodium falciparum malaria in Madagascar.

This study evaluated a nonisotopic DNA assay kit for diagnosing Plasmodium falciparum malaria in an area of Madagascar where all Plasmodium species of human malaria are present and where malaria is endemic. Blood samples from 440 healthy children and 20 healthy adults were processed and assayed in a single day in a blind protocol. The parasitemia levels of the four Plasmodium species were determined by microscopic examinations and by counts of numbers of malaria parasites per 1,000 white blood cells. Relative to P. falciparum infections, the DNA assay results agreed with those of microscopy for 207 positive and 239 negative samples; two samples were scored as positive by the DNA probe that were not detected by microscopy, and 12 samples were scored as positive by microscopy but were not detected by the assay. Relative to microscopy, the sensitivity of the assay was 95%, the specificity was 99%, and the effective sensitivity threshold of the DNA probe assay was approximately 30 parasites/mm3 of blood. The assay did not detect infections with either P. vivax, P. malariae, or P. ovale alone, but detected mixed infections of P. falciparum with either P. vivax or P. ovale. With this nonisotopic DNA probe assay, we were able to process large numbers of samples efficiently and to detect P. falciparum malaria infections with high sensitivity and specificity in a population that did not display overt disease symptoms.

Comparison of thick films, in vitro culture and DNA hybridization probes for detecting Plasmodium falciparum malaria.

Using blood from volunteers with sporozoite induced malaria, a comparison was made of the sensitivity and specificity of Giemsa stained thick film examination, in vitro culture, and 4 different DNA probes for detecting parasitemia. Between 9 and 13 days after sporozoite inoculation, patent parasitemia (4-550 parasites/microliters) was detected by thick film examination of 0.5 microliters blood in 7 volunteers. Cultures of 1 ml blood obtained 7 days after sporozoite inoculation were positive in all volunteers who eventually developed patent parasitemia. The DNA hybridization probes detected parasites in only 5-28% of smear- or culture-positive samples.

Assessment of a synthetic DNA probe for Plasmodium falciparum in African blood specimens.

Synthetic DNA oligomers homologous to 21-base long repetitive sequences of Plasmodium falciparum DNA were labeled with 32P using T4 kinase, and were hybridized with purified DNA and with processed blood samples from Africa. The sequence PFR1, its antiparallel oligomer PFR1R, and PFR1 covalently attached to biotin hybridized similarly to P. falciparum DNA. One-microliter aliquots of blood from Zaire spotted on prewet nylon filters and hybridized with PFR1 gave detectable autoradiogram signals from samples with parasitemias as low as 1,000 parasites/mm3. Blood lysis and protein digestion followed by alkylation allowed dot-blot processing of larger aliquots of blood. After hybridization with PFR 1 and autoradiography, 26 samples were scored positive visually, compared with 34 scored positive by microscopy. The effective sensitivity for processed 10-microliter samples was about 500 parasites/mm3. Signals from hybridized probes were quantitated by liquid scintillation counting and densitometry, and were proportional to the amounts of purified P. falciparum DNA applied to the filter. Autoradiogram signals also were roughly proportional (correlation coefficient, r = 0.77) to the number of parasites/mm3 of blood from field samples as determined by microscopic examination.

Regulation of the plasma membrane potential in Pneumocystis carinii.

Many protists use a H(+) gradient across the plasma membrane, the proton motive force, to drive nutrient uptake. This force is generated in part by the plasma membrane potential (DeltaPsi). We investigated the regulation of the DeltaPsi in Pneumocystis carinii using the potentiometric fluorescent dye bisoxonol. The steady state DeltaPsi in a buffer containing Na(+) and K(+) (standard buffer) was found to be -78+/-8 mV. In the absence of Na(+) and K(+) (NMG buffer) or Cl(-) (gluconate buffer), DeltaPsi was not significantly changed suggesting that cation and anion conductances do not play a significant role in the regulation of DeltaPsi in P. carinii. The DeltaPsi was also not affected by inhibitors of the Na(+)/K(+)-ATPase, ouabain (1 mM), and the K(+)/H(+)-ATPase, omeprazole (1 mM). In contrast, inhibitors of the plasma membrane H(+)-ATPase, dicyclohexylcarbodiimide (100 microM), N-ethylmaleimide (100 microM) and diethylstilbestrol (25 microM), significantly depolarized the DeltaPsi to -43+/-7, -56+/-5 and -40+/-12 mV, respectively. The data support that the plasma membrane H(+)-ATPase plays a significant role in the regulation of DeltaPsi in P. carinii.

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 differentiation of two new world saurian Plasmodium species in different host species using the polymerase chain reaction.

Several isolates of Plasmodium floridense obtained from naturally infected Anolis carolinensis and Anolis sagrei, and 2 isolates of Plasmodium chiricahuae obtained from Sceloporus jarrovi were characterized at the ribosomal DNA (rDNA) locus using the polymerase chain reaction and agarose gel electrophoresis. Enzymatic amplification of the rDNA locus from both Plasmodium species resulted in the generation of a 590-base pair (bp) DNA fragment. The results obtained with all isolates of P. floridense appeared as a doublet, with the second fragment being approximately 630 bp in size. Isolates of P. floridense obtained from A. carolinensis from ecologically different northern and southeastern Florida, and from A. sagrei a the same southeastern Florida site, were demonstrated to be molecularly similar. Plasmodium floridense and P. chiricahuae were molecularly distinct at the 18s rDNA locus, thus confirming their morphological and morphometrical distinction as taxonomic species. Anolis sagrei is a third natural host species for P. floridense in Florida.

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.

Granulomatous hepatitis associated with Hepatozoon sp. meronts in a southern water snake (Nerodia fasciata pictiventris).

A wild-caught adult female southern water snake (Nerodia fasciata pictiventris) did poorly in captivity. A peripheral blood-film examination demonstrated numerous hemogregarines characterized as fusiform nondividing intraerythrocytic gametocytes. Xenodiagnostic typing in laboratory-reared mosquitoes demonstrated the parasite to be of the genus Hepatozoon. Gross and histopathologic examination of the liver demonstrated numerous granulomas centered on groups of one to six Hepatozoon sp. meronts, an unusual finding in naturally infected wild-caught snakes.

Thermal ecology of Naegleria fowleri from a power plant cooling reservoir.

The pathogenic, free-living amoeba Naegleria fowleri is the causative agent of human primary amebic meningoencephalitis. N. fowleri has been isolated from thermally elevated aquatic environments worldwide, but temperature factors associated with occurrence of the amoeba remain undefined. In this study, a newly created cooling reservoir (Clinton Lake, Illinois) was surveyed for Naegleria spp. before and after thermal additions from a nuclear power plant. Water and sediment samples were collected from heated and unheated arms of the reservoir and analyzed for the presence of thermophilic Naegleria spp. and pathogenic N. fowleri. Amoebae were identified by morphology, in vitro cultivation, temperature tolerance, mouse pathogenicity assay, and DNA restriction fragment length analysis. N. fowleri was isolated from the thermally elevated arm but not from the ambient-temperature arm of the reservoir. The probability of isolating thermophilic Naegleria and pathogenic N. fowleri increased significantly with temperature. Repetitive DNA restriction fragment profiles of the N. fowleri Clinton Lake isolates and a known N. fowleri strain of human origin were homogeneous.

Restriction fragment length polymorphisms of the DNA of selected Naegleria and Acanthamoeba amebae.

Fourteen strains of Naegleria fowleri, two strains of N. gruberi, and one strain each of N. australiensis, N. jadini, N. lovaniensis, Acanthamoeba sp., A. castellanii, A. polyphaga, and A. comandoni isolated from patients, soil, or water were characterized by restriction fragment length polymorphisms. Total cellular DNA (1 microgram) was digested with either HindIII, BglII, or EcoRI; separated on agarose gels; and stained with ethidium bromide. From 2 to 15 unusually prominent repetitive restriction fragment bands, totaling 15 to 50 kilobases in length and constituting probably more than 30% of the total DNA, were detected for all ameba strains. Each species displayed a characteristic pattern of repetitive restriction fragments. Digests of the four Acanthamoeba spp. displayed fewer, less intensely staining repetitive fragments than those of the Naegleria spp. All N. fowleri strains, whether isolated from the cerebrospinal fluid of patients from different parts of the world or from hot springs, had repetitive restriction fragment bands of similar total lengths (ca. 45 kilobases), and most repetitive bands displayed identical mobilities. However, polymorphic bands were useful in identifying particular isolates. Restriction fragment length polymorphism analysis generally was consistent with taxonomy based on studies of infectivity, morphology, isoenzyme patterns, and antibody reactivity and suggests that this technique may help classify amebae isolated from clinical specimens or from the environment.

Comparison of genomic, plasmid, synthetic, and combined DNA probes for detecting Plasmodium falciparum DNA.

Total genomic Plasmodium falciparum DNA, the plasmid clone pRepHind, and a 21-base-long synthetic DNA probe (PFR1), the sequence of which was derived from pRepHind, were hybridized with DNA from various species of the phylum Apicomplexa. The genomic probe hybridized with P. reichenowi and P. falciparum DNA and significantly cross-hybridized with DNA of all the other Plasmodium species tested. The synthetic and plasmid probes hybridized to P. falciparum DNA and at reduced levels to P. reichenowi but did not hybridize to P. vivax, P. malariae, P. ovale, P. fragile, P. inui, P. knowlesi, Babesia bovis, B. microti, B. bigemina, Anopheles sp., Pan sp., Aotus sp., or human DNA. Southern blot analysis indicated that approximately 60 distinct restriction enzyme fragments from P. falciparum DNA were similarly detected by PFR1 and pRepHind. A method was developed by using a second brief hybridization with synthetic DNA to amplify signals from samples that were previously hybridized with plasmid-borne repetitive DNA. This amplification procedure was shown to allow the detection of 0.005% P. falciparum parasitemias from 10-microliter samples of blood from patients in Kenya.