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D F Wirth

Publications and source records attributed to D F Wirth.

At least 73 records · Page 4Linked to original sources
PubMed1989

Plasmodium falciparum: DNA probe diagnosis of malaria in Kenya.

We previously reported isolation of DNA probe which specifically recognizes Plasmodium falciparum and developed a simple method for its use. The sensitivity and specificity of this DNA probe method have now been extensively field tested in comparison with those of conventional microscopic examination of blood films in two separate studies in Malindi, Kenya, involving a total of 1179 patients. In the second study, which used improved techniques, sensitivity of the DNA probe was 89% when compared to microscopy. We conclude that the DNA probe method compares favorably with conventional microscopy in detecting parasite densities as low as 25 parasites per microliter of blood. A significant advantage of the DNA probe method is that it utilizes a standardized procedure which can simultaneously and reproducibly analyze a large number of samples without opportunity for significant reader bias.

Adult↗
PubMed1989

Cloning and characterization of an Onchocerca volvulus specific DNA sequence.

A cloned sequence, pOvs134, was isolated from a genomic library prepared from Onchocerca volvulus of savanna origin in the plasmid pUC9. pOvs134 hybridizes to all the geographic isolates of O. volvulus tested from both the New and the Old World, but not to the species Onchocerca gibsoni, Onchocerca gutturosa, Onchocerca ochengi, Onchocerca cervicalis, the filarial parasites Brugia malayi, or Dirofilaria immitis, nor to human or simuliid DNA. As little as 250 pg of DNA can be detected on a dot blot hybridization, suggesting that pOvs134 is sensitive enough to detect a single third stage larva. DNA sequence analysis of the inserted DNA of pOvs134 revealed that it consisted of twelve examples of a 149-bp repeat. The sequence of this repeat is strikingly similar to that of two O. volvulus genomic clones previously described, one of which has been reported to be specific for forest form O. volvulus, and one of which hybridizes to genomic DNA of several species of Onchocerca. These results suggest that the 149-bp repeat sequence is highly repeated in the genome of O. volvulus, and that variants of this repeat with different specificities exist.

Animals↗
PubMed1989

Characterization of a RNA virus from the parasite Leishmania.

We were interested in screening a series of isolates of the protozoan Leishmania for the presence of viruses. The experimental procedure we used was based on an enzymatic assay originally developed for viral RNA-dependent RNA polymerases. Simultaneously, total promastigote nucleic acid preparations were analyzed for the presence of viral genome and/or transcripts. Two isolates, both classified as L. braziliensis guyanensis, were found to be positive for RNA polymerase activity and to carry a large (6 kilobases) RNA species. The polymerase reaction products hybridized to the 6-kilobase RNA, believed to be the viral genome. In conjunction with electron microscopical observations these results indicate the presence of an RNA virus in these Leishmania isolates.

Animals↗
PubMed1989

Transfection of Leishmania enriettii and expression of chloramphenicol acetyltransferase gene.

We report a transient expression transfection system in Leishmania enriettii. A hybrid gene containing an intergenic region of the alpha-tubulin cluster and the bacterial chloramphenicol acetyltransferase (CAT; EC 2.3.1.28) gene is expressed after transfection of L. enriettii with the hybrid plasmid. The expression of the CAT gene is dependent on the presence of sequences from the alpha-tubulin gene. The hybrid gene is also active in Leishmania braziliensis and Leishmania major.

Animals↗
PubMed1989

Sequence identification of cytochrome b in Plasmodium gallinaceum.

We have identified a gene that encodes the polypeptide cytochrome b in the avian malarial parasite Plasmodium gallinaceum. The gene containing the open reading frame was found to be located on a 6.2-kilobase multimeric extrachromosomal element. The amino acid translation from this gene demonstrated significant similarities to cytochrome b sequences from yeast, mammal, and fungus genomes. We present evidence that the P. gallinaceum cytochrome b transcript is part of a larger primary transcript from the element that is subsequently processed. The message for P. gallinaceum cytochrome b was found to be 1.2 kilobases in size. This is the first report identifying a mitochondrial nucleic acid sequence in malaria-causing organisms and suggests that a functional cytochrome system may exist in these parasites.

Amino Acid Sequence↗
PubMed1989

Characterization of a conserved extrachromosomal element isolated from the avian malarial parasite Plasmodium gallinaceum.

We have identified a conserved, repeated, and highly transcribed DNA element from the avian malarial parasite Plasmodium gallinaceum. The element produced multiple transcripts in both zygotes and asexual blood stages of this parasite. It was found to be highly conserved in all of five malarial species tested and hybridized at reduced stringency to other members of the phylum Apicomplexa, including the genera Babesia, Eimeria, Toxoplasma, and Theileria. The copy number of the element was about 15, and it had a circularly permuted restriction map with a repeat unit length of about 6.2 kilobases. It could be separated from the main genomic DNA by using sucrose gradients and agarose gels, and it migrated separately from the recognized Plasmodium chromosomes on pulse-field gels. In the accompanying paper (S. M. Aldritt, J. T. Joseph, and D. F. Wirth, Mol. Cell. Biol. 9:3614-3620, 1989), evidence is presented that element contains the mitochondrial genes for the protein cytochrome b and a fragment of the large rRNA. We postulate that this element is an episome in the mitochondria of the obligate parasites belonging to the phylum Apicomplexa.

Animals↗
PubMed1989

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.

Animals↗
PubMed1989

Detection of Plasmodium falciparum infection in human patients: a comparison of the DNA probe method to microscopic diagnosis.

We have previously reported the isolation and testing of a DNA probe specific for the detection of Plasmodium falciparum. Field studies to compare the sensitivity and specificity of the DNA probe with that of light microscopy have been performed. In 2 studies in Thailand, 1,397 patients were tested. Microscope slides were prepared in a standard fashion and examined by clinical technicians and expert microscopists. The DNA probe method compares favorably in sensitivity with routine microscopy, detecting parasite densities as low as 40 parasites/microliters blood in the first study and, after modifications, 20-25 parasites/microliters blood in the second. Modifications included the elimination of salt from the lysis buffer, increasing the pH of the lysis buffer, and use of nylon based hybridization membranes instead of nitrocellulose. The DNA probe method offers the advantage of a standardized procedure that can be used in a batchwise fashion on a large number of samples.

Adolescent↗
PubMed1988

Generation of sequence diversity in the kinetoplast DNA minicircles of Leishmania mexicana amazonensis.

In order to understand the mechanisms which generate minicircle sequence diversity, we sequenced three minicircles belonging to the same or closely related sequence classes from the kinetoplast DNA of Leishmania mexicana amazonensis strains, PH8, Raimundo, and Josefa. Closely related minicircles from PH8 and Raimundo were unexpectedly found to differ at 11% of positions within the evolutionarily conserved region, but at only 3.9% of positions in the variable region. It thus appears that accumulation of point mutations will not account for the wide intra-strain and intra-subspecies divergence of the variable region. Comparison of more distantly related minicircles from PH8 and Josefa revealed only two short stretches of 70% homology within the variable region. These stretches of homology are not located in the same positions relative to the conserved regions in their respective minicircles. They may represent vestiges of recombinational events responsible for the rapid divergence of minicircle variable regions.

Animals↗
PubMed1988

trans splicing in Leishmania enriettii and identification of ribonucleoprotein complexes containing the spliced leader and U2 equivalent RNAs.

The 5' ends of Leishmania mRNAs contain an identical 35-nucleotide sequence termed the spliced leader (SL) or 5' mini-exon. The SL sequence is at the 5' end of an 85-nucleotide primary transcript that contains a consensus eucaryotic 5' intron-exon splice junction immediately 3' to the SL. The SL is added to protein-coding genes immediately 3' to a consensus eucaryotic 3' intron-exon splice junction. Our previous work demonstrated possible intermediates in discontinuous mRNA processing that contain the 50 nucleotides of the SL primary transcript 3' to the SL, the SL intron sequence (SLIS). These RNAs have a 5' terminus at the splice junction of the SL and the SLIS. We examined a Leishmania nuclear extract for these RNAs in ribonucleoprotein (RNP) particles. Density centrifugation analysis showed that the SL RNA is predominantly in RNP complexes at 60S, while the SLIS-containing RNAs are in complexes at 40S. We also demonstrated that the SLIS can be released from polyadenylated RNA by incubation with a HeLa cell extract containing debranching enzymatic activity. These data suggested that Leishmania enriettii mRNAs are assembled by bimolecular or trans splicing as has been recently demonstrated for Trypanosoma brucei. Furthermore, we determined the partial sequence of the Leishmania U2 equivalent RNA and demonstrated that it cosediments with the SL RNA at 60S in a nuclear extract. These RNP particles may be analogous to so-called spliceosomes that have been demonstrated in other systems.

Animals↗
PubMed1988

Detection of Leishmania within sand flies by kinetoplast DNA hybridization.

A kinetoplast DNA hybridization probe method was used to detect Leishmania within sand flies and to distinguish it from the non-pathogenic flagellate, Endotrypanum. Eighty-one sand flies (74 Lutzomyia umbratilis, 1 Lu. anduzei, and 6 Lu. shannoni) collected outside Manaus, Brazil were dissected. Forty-four of these were found to be infected with flagellates, and 2 hybridized with a Leishmania braziliensis probe. Thirty-three of sixty-one flies reprobed with an Endotrypanum probe were positive.

Animals↗
PubMed1987

Characterization of the filarial genome.

Filarial parasites are just beginning to be studied at the genetic level. The potential of recombinant DNA technology for identifying parasite genes that are important in the pathogenesis of filarial disease or for the survival of the parasite is enormous. Work in several laboratories has already identified genes which encode ribosomal RNAs, as well as highly repeated DNA sequences that can be used as diagnostic probes. In addition, new methods to separate chromosomes will allow the physical mapping of parasite genes without the requirement for classical genetic analysis, which would be difficult in filariids.

Animals↗
PubMed1987

Kinetoplast DNA minicircles: regions of extensive sequence divergence.

Previous work has shown that the kinetoplast minicircle DNA of Leishmania species exhibits species-specific sequence divergence and this observation has led to the development of a DNA probe-based diagnostic test for leishmaniasis. In the work reported here, we demonstrate that the minicircle is composed of three types of DNA sequences with differing specificities reflecting different rates of DNA sequence change. A library of cloned fragments of kinetoplast DNA (kDNA) from Leishmania mexicana amazonensis was prepared and the cloned subfragments were found to contain DNA sequences with different taxonomic specificities based on hybridization analysis with various species of Leishmania. Four groups of subfragments were found, those that hybridized with a large number of Leishmania sp. as well as sequences unique to the species, subspecies, or isolate. Analysis of nested deletions of a single, full-length minicircle demonstrates that these different taxonomic specificities are contained within a single minicircle. This implies that different regions of a single minicircle have DNA sequences that diverge at different rates. These sequences represent potentially valuable tools in diagnostic, epidemiologic, and ecological studies of leishmaniasis and provide the basis for a model of kDNA sequence evolution.

Animals↗
PubMed1987

Characterization of an Onchocerca-specific DNA clone from Onchocerca volvulus.

A genomic library of a savanna isolate of Onchocerca volvulus was screened to detect recombinant plasmids containing highly repeated DNA sequences of this parasite. Four recombinant plasmids were identified which hybridized specifically to Onchocerca DNA, but not to DNA from humans, black flies, Brugia malayi, B. pahangi, or Wuchereria bancrofti. The recombinant plasmids had a low level of homology to Dirofilaria immitis. All recombinant plasmids contain related DNA sequences based on Southern hybridization analysis. Sequences related to these recombinant plasmids are present in different geographic isolates of O. volvulus and O. ochengi, an animal parasite. Two of the recombinant plasmids contain sequences also found in O. lienalis. One recombinant plasmid, puOvs3, has been characterized in detail, including DNA sequence determination. Radiolabeled puOvs3 is able to detect 100 pg of genomic DNA isolated from O. volvulus worms from both savanna and forest regions. It can differentiate O. volvulus from O. ochengi by Southern blot analysis.

Animals↗
PubMed1986

Leishmaniasis and malaria: new tools for epidemiologic analysis.

Parasitic diseases are still prevalent in many parts of the world, causing both human suffering and economic loss. Recent developments in biotechnology, such as the use of monoclonal antibodies and recombinant DNA, have the potential for providing both more extensive and detailed information on the parasite in the infected human and in insect vectors. New methods of detection, both in man and insect vectors, have been developed for two parasitic diseases, leishmaniasis and malaria. These new methodologies will be important in epidemiologic studies on the prevalence and transmission of these parasitic diseases.

Antibodies, Monoclonal↗
PubMed1986

Cloning and characterization of a Leishmania gene encoding a RNA spliced leader sequence.

Recent studies on leishmania enriettii tubulin mRNAs revealed a 35 nucleotide addition to their 5' end. The gene that codes for this 35 nucleotide leader sequence has now been cloned and sequenced. In the Leishmania genome, the spliced leader gene exists as a tandem repeat of 438 bases. There are approximately 150 copies of this gene comprising 0.1% of the parasite genome. This gene codes for a 85 nucleotide transcript that contains the spliced leader at its 5' end. The 35 nucleotide sequence and the regions immediately 5' and 3' to it are highly conserved across trypanosomatids. We have detected a RNA molecule that is a putative by-product of the processing reaction in which the 35 nucleotide spliced leader has been transferred to mRNA. We suggest that this molecule is the remnant of the spliced leader transcript after removal of the 35 nucleotide spliced leader.

Animals↗
PubMed1986

Specific DNA probe for the diagnosis of Plasmodium falciparum malaria.

Malaria can be diagnosed either by direct microscopic examination of blood smears, which is time consuming and requires expertise, or by immunological techniques, which are effective but do not distinguish between past and present infections. In this study, a simple procedure was developed for spotting lysed blood from infected patients directly onto nitrocellulose paper and identifying the malaria species on the basis of hybridization of parasite DNA with a species-specific probe. A genomic DNA library of Plasmodium falciparum was screened to detect clones containing DNA sequences that are highly repeated within the parasite genome. Several such clones were further analyzed to identify those that hybridize specifically with P. falciparum DNA but not with DNA from humans, P. vivax, or P. cynomolgi. This technique appears to be sensitive enough to detect 10 picograms of purified P. falciparum DNA (equivalent to 100 parasites) and in field studies is able to detect approximately 40 parasites per microliter of blood.

Cloning, Molecular↗