Biomedical subjects

Thomas R Unnasch

Publications and source records attributed to Thomas R Unnasch.

10 recordsLinked to original sources

PubMed2003

Analysis of the Brugia malayi HSP70 promoter using a homologous transient transfection system.

Biolistic transient transfection of Brugia malayi embryos with constructs driving the expression of a luciferase reporter gene was used to identify regions of the upstream sequence of the heat shock protein 70 (HSP70) gene of B. malayi necessary for transgene expression. Analysis of 1160 nucleotides upstream of the start codon of the HSP70 gene identified several potentially important elements, including putative CAAT and TATA boxes, a core promoter domain, a polypurine stretch, and a spliced leader addition site. Nested deletion analysis of the HSP70 upstream domain mapped the promoter of the HSP70 gene to the region 396 to 31 nucleotides upstream of the start codon. This encompassed the putative CAAT and TATA boxes, and putative core promoter. Deletion of the putative CAAT box did not result in any diminution of reporter activity, while constructs in which the TATA box or core promoter were deleted retained roughly half of the activity of the undeleted construct. Unlike the native gene, transcripts derived from constructs containing the HSP70 upstream sequences were not trans-spliced. However, incorporation of the 495 nucleotides downstream of the start codon (encompassing exon 1, intron 1 and part of exon 2) resulted in the production of transcripts that were correctly cis- and trans-spliced. Similarly, a construct containing the 495 downstream nucleotides in which most of exon 1 was deleted, was correctly cis- and trans-spliced. This finding suggests that downstream intron sequences in addition to the splice leader addition site are necessary for trans-splicing in B. malayi.

Alternative Splicing↗

PubMed2003

Angiogenic activity of an Onchocerca volvulus Ancylostoma secreted protein homologue.

Angiogenesis is an important step in the development of ocular onchocercaisis. In previous studies, it has been demonstrated that Onchocerca volvulus homologues of the Ancylostoma secreted protein family have pronounced angiogenic activity. The overall goal of the current study was to determine if this angiogenic effect is exerted through a direct or indirect mechanism. These studies focused on one member of this family, OvASP-2, as this protein is expressed in microfilaria, the stage of the parasite that causes ocular onchocercaisis. Clones encoding truncated and full length open reading frames were expressed as fusion proteins with Escherichia coli maltose binding protein (MBP), and angiogenic activity was compared in vitro and in vivo with MBP alone. Truncated constructs expressing only the first 105 amino acids of OvASP-2 were as active as the full length protein in inducing new blood vessel formation. The full length fusion protein did not stimulate proliferation or production of vascular endothelial growth factor in vascular endothelial cells in vitro, indicating that OvASP-2 does not directly stimulate angiogenesis. Sequence analysis demonstrated that the gene encoding OvASP-2 contained five introns. Sequence comparisons of the genomic loci from West African blinding and non-blinding strains of O. volvulus revealed that some polymorphism existed among the various isolates tested. However, none of these polymorphisms could be used to differentiate the parasite strains, suggesting that qualitative variation in OvASP-2 could not explain the difference in ocular pathogenic potential of the two parasite strains.

Ancylostoma↗

PubMed2003

Entomological evaluation by pool screen polymerase chain reaction of Onchocerca volvulus transmission in Ecuador following mass Mectizan distribution.

The prevalence of infected and infective black flies was estimated by pool screen polymerase chain reaction (PCR) amplification in the three river basins in Ecuador endemic for onchocerciasis. Mass distribution of ivermectin (Mectizan) resulted in dramatic declines in the prevalence of infected and infective flies. In the Rio Santiago river basin, no infections were detected, suggesting that transmission had ceased. The ratio of infected to infective flies in Simulium exiguum was 10-fold lower than the corresponding ratio for Simulium quadrivittatum, suggesting that S. exiguum is a more-competent vector for Onchocerca volvulus than S. quadrivittatum. However, the prevalence of infective flies in the two species was not different, suggesting that S. quadrivittatum may play an important vectorial role where it is the dominant human-biting species. The data demonstrate that pool screen PCR is an efficient way to monitor transmission in areas subject to control, and to certify an area as free of O. volvulus transmission.

Animals↗

PubMed2003

Transmission of eastern equine encephalomyelitis virus in central Alabama.

A site near Tuskegee, Alabama was examined for vector activity of eastern equine encephalomyelitis (EEE) virus in 2001. More than 23,000 mosquitoes representing 8 genera and 34 species were collected during a 21-week period, and five species, Culiseta melanura, Aedes vexans, Coquillettidia perturbans, Culex erraticus, and Uranotaenia sapphirina, were examined for the presence of virus using a nested reverse transcriptase-polymerase chain reaction for EEE virus. Each species was infected at various times of the mosquito season (May-September) with different minimum infection rates (MIRs). Culiseta melanura had the highest MIR (20.2) and positive pools were detected from late May to mid-September. Aedes vexans had an MIR of 2.2 and was infected early in the season (June), while Cq. perturbans exhibited a much higher field infection rate (9.9) with all positive pools collected in August. Culiseta melanura is a likely endemic vector in central Alabama, while Ae. vexans and Cq. perturbans probably function as bridge vectors. Culex erraticus, the most common mosquito in the habitat (54% of total collections), had an MIR of 3.2, and was persistently infected from mid-June to mid-September. This is the first report of high rates of EEE virus infection in this species, a member of the tropical subgenus Melanoconion. Uranotaenia sapphirina, considered to feed on amphibians and possibly reptiles, had an MIR of 5.6, with positive pools spanning a four-month period. This suggests that species other than birds may serve as a reservoir for EEE in hardwood swamps in the Southeastern United States and elsewhere. The lengthy period of mosquito infection with EEE virus, coupled with the diverse habits of the vectors and their proximity to a population center, indicate the importance of monitoring EEE virus activity in the Mid-South.

Alabama↗

PubMed2002

River blindness.

Explore the source record for details and available documents.

Africa↗

PubMed2002

Brugia malayi: transient transfection by microinjection and particle bombardment.

To develop a method for the introduction of DNA into filarial parasites, several methods that have proven successful in other organisms were evaluated for their ability to transform Brugia malayi. Luciferase activity was detectable in embryos bombarded with gold particles coated with a construct consisting of a luciferase reporter gene under the control of the 5S rRNA intergenic spacer (SL promoter). Similar results were seen in adult parasites and infective larvae bombarded with this construct, or in adult female parasites microinjected with the plasmid. In similar experiments employing the SL promoter driving a green fluorescent protein (GFP) reporter, expression of the reporter was detectable in the intrauterine embryos of the microinjected adult parasites, and in the sub-cuticular tissues of biolistically transfected adult female parasites. A similar pattern of GFP expression to that seen in the SL promoter construct transfected parasites was noted in parasites transfected with constructs consisting of the upstream domain derived from an aspartyl aminoacyl tRNA synthetase gene of B. malayi. The ability to transfect B. malayi embryos may provide a foundation for studies of the regulation of gene expression and function in these organisms.

Animals↗

PubMed2002

A comparison of two surface sample collection devices for use in polymerase chain reaction based detection of Pneumocystis carinii in house dust.

A polymerase chain reaction assay was optimized to detect P. carinii cysts in composite dust samples. The optimal assay was capable of detecting as few as 10(3) P. carinii cysts in 50 mg of dust. Two dust collection devices were evaluated for efficiency and precision of collection of bulk dust and compatibility with the optimized PCR protocol for P. carinii DNA detection. A handheld vacuum cleaner equipped with a high-retention bag was found to be superior to a 37-mm filter cassette attached to an electrically powered vacuum pump in terms of dust collection efficiency (87% [n = 37] versus 81% [n = 35]), although the precision of the two devices as assessed by the standard deviation was similar (6.2% versus 6.3%). However, the vacuum cleaner method was not as compatible with the PCR-based detection assay as the filter cassette method. The filter cassette appears to be a better device for use in conjunction with PCR-based detection of P. carinii DNA in bulk dust samples from both smooth and carpeted surfaces.

DNA, Fungal↗

PubMed2002

Host-feeding habits of Culex and other mosquitoes (Diptera: Culicidae) in the Borough of Queens in New York City, with characters and techniques for identification of Culex mosquitoes.

The host-feeding patterns of mosquitoes (n = 247) collected in the Borough of Queens in New York City in July and August 2000 were investigated using an indirect ELISA and a polymerase chain reaction (PCR)-heteroduplex assay. Culex pipiens L. and Cx. restuans Theobald fed primarily on birds, and their feeding habits support their implication as enzootic vectors of West Nile virus. Culex salinarius Coquillett and Coquillettidia perturbans (Walker) fed mainly on mammals, with fewer blood meals taken from birds, and these two species are potential bridge vectors of West Nile virus. Culex mosquitoes took blood meals (n = 54) from 11 different avian species. Only the northern cardinal (Cardinalis cardinalis), American robin (Turdus migratorius), and Brown-headed cow bird (MolIothrus ater) were fed upon by all three Culex species. Multiple blood feedings on avian hosts were detected in Cx. pipiens and Cx. restuans. Species identifications of Culex mosquitoes made using morphological characteristics were confirmed with a PCR assay that employed species-specific primers. All Cx. pipiens (n = 20) and Cx. salinarius (n = 10) specimens were correctly identified, but three (20%) of 15 Cx. restuans were misidentified as Cx. pipiens.

Animals↗

PubMed2002

Identification of mosquito avian-derived blood meals by polymerase chain reaction-heteroduplex analysis.

A polymerase chain reaction (PCR) heteroduplex assay (HDA) was developed to identify avian derived mosquito blood meals to the species level. The assay used primers amplifying a fragment of the cytochrome B gene from vertebrate but not invertebrate species. In Culex tarsalis fed on quail, PCR products derived from the quail cytochrome B gene were detected seven days post-engorgement. In an analysis of wild-caught mosquitoes, 85% of blood-fed mosquitoes produced detectable PCR products. Heteroduplex patterns obtained from bird-derived PCR products were found to permit the unambiguous identification of all species examined. No intraspecific variation in HDA patterns was found. The PCR-HDA was used to characterize blood meals in wild caught Cx. tarsalis. Of the 67 blood meals analyzed, 60% were derived from avian sources. Of the avian blood meals, 65% were derived from a single host, the common grackle.

Alabama↗

PubMed2002

Simultaneous detection of three mosquito-borne encephalitis viruses (eastern equine, La Crosse, and St. Louis) with a single-tube multiplex reverse transcriptase polymerase chain reaction assay.

Three mosquito-borne human encephalitis viruses (eastern equine encephalitis virus [EEE], St. Louis encephalitis virus [SLE], and La Crosse encephalitis virus [LAC]) are sympatric in the southeastern United States. However, little is known concerning the temporal and spatial pattern of the distribution of these viruses in this area. As part of surveillance activities to detect the transmission of these 3 viruses in the Tennessee Valley area, we developed a single-tube multiplex reverse transcriptase polymerase chain reaction (RT-PCR) assay capable of detecting these 3 mosquito-borne viruses in a single reaction. Three viruses were differentiated by size of amplified products. Sensitivities of the multiplex RT-PCR assay for SLE, EEE, and LAC were 1-3 log median tissue culture infective doses per pool, roughly comparable to the reported sensitivity of PCR detection assays for the individual viruses, and 1 log more sensitive than antigen-capture assays for SLE and EEE. The sensitivity of the multiplex PCR was not changed significantly when carried out in the presence of extracts prepared from 50 uninfected mosquitoes. The cost of the assay is estimated at $2.98 per test, similar to the cost of other RT-PCR-based assays for viruses. However, adaptation of the RT-PCR to a multiplex format adds less than $0.01 to the per-unit cost of an RT-PCR assay targeting a single virus species. Analysis of these data suggests that the single-tube multiplex RT-PCR assay represents a sensitive, specific, cost-effective, and rapid method for monitoring activities of the 3 endemic mosquito-borne human encephalitis viruses in mosquito populations in the southeastern United States.

Animals↗