Identification of semiochemicals attractive to Simulium vittatum (IS-7).

Many blackfly species (Diptera: Simuliidae) are economically important insect pests, both as nuisance biters and as vectors of pathogens of medical and veterinary relevance. Among the important blackfly pest species in North America is Simulium vittatum Zetterstedt sensu lato. The objective of this study was to identify compounds excreted by mammalian hosts that are attractive to host-seeking S. vittatum females. The attractiveness of putative compounds to colonized S. vittatum was tested through electrophysiological (electroantennography; n = 58 compounds) and behavioural (Y-tube assays; n = 7 compounds in three concentrations) bioassays. Five compounds were significantly attractive to host-seeking S. vittatum females: 1-octen-3-ol; 2-heptanone; acetophenone; 1-octanol, and naphthalene. These candidate compounds might be useful as attractants in traps that could be developed for use in alternative or complementary management tactics in programmes to suppress nuisance blackfly populations, or for the collection of samples in which to study the transmission ecology of pathogens transmitted by blackflies of the S. vittatum complex.

Isolation and characterization of expression cDNA clones encoding antigens of Onchocerca volvulus infective larvae.

The isolation of recombinant cDNA clones expressing antigens found in Onchocerca volvulus infective larvae is described. To isolate such clones, an expression cDNA library constructed from adult O. volvulus RNA was screened with antiserum raised against infective larvae. One clone, designated lambda RAL-1 was characterized further. The recombinant antigen produced by lambda RAL-1 stimulates proliferation of peripheral blood mononuclear cells from O. volvulus infected humans. Lambda RAL-1 is derived from a 1450 bases message that encodes a protein with an apparent molecular weight of 42,000 in adult O. volvulus. The inserted DNA of lambda RAL-1 contains an open reading frame of 1008 bp. The amino acid sequence predicted by this open reading frame contains three repeats of the sequence KKPEDWD. The identification of clones such as lambda RAL-1 will provide quantities of purified antigens sufficient to begin to study the immune response to and explore the development of immunity against the infectious form of the parasite.

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.

Interruption of the transmission of Onchocerca volvulus in the Kashoya-Kitomi focus, western Uganda by long-term ivermectin treatment and elimination of the vector Simulium neavei by larviciding.

Uganda is the only country in sub-Saharan Africa whose onchocerciasis elimination programme extensively uses vector control and biannual treatment with ivermectin. The purpose of this study was to assess the impact of combined strategies on interrupting onchocerciasis transmission in the Kashoya-Kitomi focus. Mass Drug Administration annually (13 years) followed by biannual treatments (6 years) and ground larviciding (36 cycles in 3 years) with temephos (Abate®, EC500) against Simulium neavei were conducted. Routine fly catches were conducted for over seven years in six catching sites and freshwater crabs Potamonautes aloysiisabaudiae were examined for immature stages of Simulium neavei. Epidemiological assessments by skin snip were performed in 2004 and 2013. Collection of dry blood spots (DBS) from children <10 years for IgG4 antibodies analysis were done in 2010 and 2013. Treatment coverage with ivermectin improved with introduction of biannual treatment strategy. Microfilaria prevalence reduced from 85% in 1991 to 62% in 2004; and to only 0.5% in 2013. Crab infestation reduced from 59% in 2007 to 0% in 2013 following ground larviciding. Comparison of total fly catches before and after ground larviciding revealed a drop from 5334 flies in 2007 to 0 flies in 2009. Serological assays conducted among 1,362 children in 2010 revealed 11 positive cases (0.8%; 95% CI: 0.4%-1.2%). However, assessment conducted on 3246 children in 2013 revealed five positives, giving point prevalence of 0.15%; 95% CI: 0.02%-0.28%. Four of the five children subjected to O-150 PCR proved negative. The data show that transmission of onchocerciasis has been interrupted based on national and WHO Guidelines of 2012 and 2016, respectively.

Ivermectin selection on beta-tubulin: evidence in Onchocerca volvulus and Haemonchus contortus.

Ivermectin resistance is common in trichostrongylid nematodes of livestock, such as Haemonchus contortus. This anthelmintic is the only drug approved for mass administration to control onchocerciasis caused by the nematode parasite, Onchocerca volvulus. In parts of West Africa up to 18 rounds of ivermectin treatment have been administered to communities and there are reports of poor parasitological responses to treatment. Understanding ivermectin resistance and ivermectin selection is an important step to reduce selection pressure for resistance, and to develop molecular markers which can be used to monitor the development of resistance and its spread. Here we report evidence that ivermectin selection changes the frequency of beta-tubulin alleles in both the sheep parasite, H. contortus, and the human parasite, O. volvulus. In O. volvulus we have been able to look at the frequency of beta-tubulin alleles in O. volvulus obtained before any ivermectin was used in humans in Africa, and following its widespread use. In H. contortus, we have been able to look at the frequency of beta-tubulin alleles in a strain which has not seen any anthelmintic selection and in an ivermectin selected strain derived from the unselected strain. We have found ivermectin selects on beta-tubulin in both of these nematode species. In the case of O. volvulus, we had previously reported that ivermectin selects for specific single nucleotide polymorphisms in the O. volvulus beta-tubulin gene. This polymorphism results in three amino acid changes in the H3 helix of beta-tubulin, as well as deletions in an associated intron. We report a simple PCR assay to detect the amplicon length polymorphism, resulting from these intronic deletions, which can be used to monitor the frequency of the beta-tubulin allele selected for by ivermectin in O. volvulus.

Genetic evidence for assortative mating between 13-year cicadas and sympatric "17-year cicadas with 13-year life cycles" provides support for allochronic speciation.

Thirteen-year cicadas of brood XIX from northern Arkansas, Missouri, and southern Illinois (lineage A) are known to be genetically different at two marker loci (mitochondrial DNA and abdominal color) from 13-year cicadas to the south (lineage B) that emerge in the same year. Because 17-year cicadas from all broods (year classes) are indistinguishable from lineage A at these two marker loci, previous workers suggested that the lineage A cicadas of 13-year brood XIX were derived from 17-year cicadas by life-cycle switching (allochrony). Data presented here show that, over the same northern geographic range, lineage A is also present in 13-year cicadas belonging to brood XXIII (which always emerges four years later than brood XIX). Detailed sampling along the putative life-cycle-switching boundary in 13-year brood XXIII revealed a previously unsuspected broad zone of overlap where populations contained individuals of both lineages A and B. Despite this sympatry, and previous reports of a lack of behavioral barriers to interbreeding, a strong correlation between mitochondrial haplotype and abdominal color suggests that assortative mating has taken place. Lineage A 13-year cicadas from both broods XIX and XXIII are only found within a gap in the spatial distribution of 17-year cicadas. This, in combination with the lack of differentiation between lineage A 13- and 17-year cicadas at the marker loci and new behavioral data for 13-year brood XIX, suggests a recent derivation of all northern 13-year cicadas from the 17-year cicadas via life-cycle switching. We discuss the implications of these allochronic shifts for speciation.

Analysis of apicoplast targeting and transit peptide processing in Toxoplasma gondii by deletional and insertional mutagenesis.

Deletion and insertion mutagenesis was used to analyze the targeting sequence of the nuclear encoded apicoplast protein, the ribosomal protein small subunit 9 of Toxoplasma gondii. Previous studies have shown that nuclear encoded apicoplast proteins possess bipartite leaders having characteristic signal sequences followed by serine/threonine rich transit sequences. Deletion analysis demonstrated that the first 55 amino acids of the rps9 leader were sufficient for apicoplast targeting. Insertional mutagenesis tagging the leader sequence with a hemagglutinin (HA) tag was used to study the events involved in the targeting pathway. Transfectants with insertions near the N-terminus of the transit displayed HA tagged precursors outside of the apicoplast, in the perinuclear region. In contrast, transfectants with the HA tag inserted near the carboxyl end of the transit-like region had apicoplast labeling. Western blot analysis of HA tagged stable isolates suggested that processing of the HA tagged leaders was a multi-step process, with processing occurring both outside of and at or within the apicoplast.

The mitochondrial genome of Onchocerca volvulus: sequence, structure and phylogenetic analysis.

The complete DNA sequence of the mitochondrial genome of Onchocerca volvulus is described. The O. volvulus mitochondrial genome is 13747 bp, slightly smaller than the mitochondrial genomes of the nematodes Ascaris suum and Caenorhabditis elegans, and the smallest metazoan mitochondrial DNA molecule reported to date. The O. volvulus mitochondrial genome contains genes for two ribosomal RNAs, 22 transfer RNAs and 12 proteins. Consistent with the small size of the genome, four gene pairs overlap and eight contain no intergenic regions. Only 17 intergenic regions are found, ranging in size from 1 to 46 bp. As in C. elegans and A. suum, the O. volvulus mitochondrial genome lacks an open reading frame encoding ATPase subunit 8, and all genes are apparently transcribed in the same direction. However, the mitochondrial gene order of O. volvulus differs from that of A. suum, C. elegans and other metazoan mitochondrial genomes. A total of 20 of the 22 transfer RNAs encoded in the O. volvulus mitochondrial genome have the potential to fold into secondary structures lacking the TpsiC arm, as has been reported in other nematodes. The genome exhibits a striking codon bias, with 15/20 amino acids having a single codon preference of > 70%.

The primary structure of the rRNA insertions of Plasmodium lophurae.

The DNA sequences of the novel insertion in the 17s rRNA gene and the large insertion in the 25s rRNA gene in the cloned rDNA unit of the avian malaria parasite Plasmodium lophurae are presented, together with a partial sequence of the flanking regions, which code for the mature rRNA. The homology of the mature rRNA coding regions with the rRNA sequences of other eukaryotic organisms is extensive, indicating that the plasmodium rRNA is structurally similar to other eukaryotes. Sequence data also reveal that the region 3' to the insertion in the 17s rRNA contains a second small inserted DNA sequence, in contrast to other known small rRNA sequences. The region containing the 25s insertion shares sequence homology and some secondary structure characteristics with the terminal direct repeat of the Drosophila melanogaster transposable element copia. This is the first such sequence described in plasmodia. The direction of transcription of the cloned rDNA unit of P. lophurae has also been determined. As in other organisms, the direction of transcription is found to be 5' 17s-25s 3'.

Design of Onchocerca DNA probes based upon analysis of a repeated sequence family.

Repeated DNA sequences have been instrumental in the development of DNA probes for many different parasites. Isolation of such DNA probes has generally been accomplished by differential screening of genomic libraries with total genomic DNA preparations. In the current work, a rational design strategy is presented for the development of oligonucleotide probes based upon repeated sequence families. A repeated sequence family present in the genome of Onchocerca parasites, designated O-150, has been amplified from various samples of genomic DNA using PCR. DNA sequence analysis of the resulting PCR products demonstrated that the sequences may be arranged into clusters within which the individual sequences are identical or nearly identical. Differences among the cluster consensus sequences have been exploited to explain the specificities of previously isolated O-150 based probes and to develop two new oligonucleotide probes. One of these probes hybridizes specifically to Onchocerca volvulus O-150 PCR products, while the second hybridizes specifically to O-150 PCR products from the closely related bovine parasite O. ochengi. These oligonucleotide probes have been used to characterize Onchocerca infective larvae isolated from wild caught infected flies in West Africa. Because repeated sequence families are a common feature of most genomes, including those of parasites, this method should be applicable to the rational design of oligonucleotide probes for other parasitic infections.

Characterization and expression of enzymatically active recombinant filarial prolyl 4-hydroxylase.

The cuticle of parasitic nematodes consists primarily of a network of collagen molecules. The enzyme responsible for collagen maturation is prolyl 4-hydroxylase, making this enzyme a central activity in cuticle biosynthesis and a potentially important chemotherapeutic target. Adult and embryonic Brugia malayi are shown to be susceptible to inhibitors of vertebrate prolyl 4-hydroxylase, with exposed parasites exhibiting pathologies consistent with a disruption in cuticle biosynthesis. A full-length cDNA (Ov-phy-1) encoding a catalytically active alpha-subunit of Onchocerca volvulus prolyl 4-hydroxylase was isolated and characterized. The derived amino acid sequence of Ov-phy-1 encoded a peptide that was most similar to the two Caenorhabditis elegans prolyl 4-hydroxylase homologues and to the isoform II enzymes of vertebrates. Expressed sequence tag (EST) analysis and developmental polymerase chain reaction (PCR) studies demonstrated that Ov-phy-1 was expressed in L3 and adult parasites. The gene encoding the Ov-phy-1 open reading frame contained 11 introns, similar in structure to the gene encoding human prolyl 4-hydroxylase isoform I. Genomic Southern blot, EST and genomic PCR studies demonstrated that the O. volvulus genome contained between three and eight genes closely related to Ov-phy-1. Co-expression of Ov-phy-1 with the O. volvulus homologue of protein disulfide isomerase in a baculovirus system resulted in the production of enzymatically active O. volvulus prolyl 4-hydroxylase. In vitro production of enzymatically active O. volvulus prolyl 4-hydroxylase should facilitate identification of specific inhibitors of the parasite enzyme.

Angiogenic activity of Onchocerca volvulus recombinant proteins similar to vespid venom antigen 5.

Although the mechanisms underlying the host inflammatory response in ocular onchocerciasis have been examined, the role of particular parasite proteins in this process remains largely unexplored. Recently, it was found that one of the most abundant expressed sequence tags in Onchocerca volvulus infective larvae encoded a protein with similarities to a component of vespid venom. This clone was designated O. volvulus Activation associated Secreted Protein -1 (Ov-asp-1). We report the characterization of three members of a family of proteins, designated the Ov-ASP family, of which Ov-ASP-1 is a member. Sequence based and phylogenetic analyses suggest that these proteins form a filarial specific protein family related to both the vespid venom antigen 5 and the vertebrate CRISP/Tpx family of proteins. The three members of the Ov-ASP family exhibit distinct patterns of expression in the life cycle of O. volvulus. Genomic Southern blot analyses indicate that several genes encoding sequences related to the Ov-asp family are present in the genome of O. volvulus. Recombinant proteins expressed from full length cDNAs encoding two members of the Ov-asp family were found to induce an angiogenic response after injection into corneas of naive mice, and vessel formation was associated with only minor inflammatory cell infiltration. These data suggest that Ov-ASP proteins may directly induce an angiogenic response and may therefore contribute to corneal neovascularization in onchocercal keratitis.

Characterization of a ribosomal DNA clone of Brugia malayi.

The structure of ribosomal DNA (rDNA) clone pBmr7 from microfilariae of the human parasite Brugia malayi has been examined in detail by Southern blot analysis and S-1 mapping techniques. The results demonstrate that this clone contains regions homologous to 28S, 18S and 5.8S rDNAs. A noncoding or 'spacer' region lies between the 3' end of 28S rDNA and the 5' end of 18S rDNA. An AccI-Sau3AI fragment of approximately 900 bp from this spacer region cross-hybridizes to genomic DNA fragments of different sizes from Brugia pahangi and Dirofilaria immitis. The differences observed in hybridization suggest that this rDNA fragment can be used to differentiate between various filariid species.

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.

Identification of an epitope of a recombinant Onchocerca volvulus protein that induces corneal pathology.

Ocular onchocerciasis results from immune recognition of parasite proteins released into the eye by degenerating microfilariae. Previous studies have shown that pathology similar to human ocular onchocerciasis can be induced in sensitized mice by intracorneal injection with Onchocerca volvulus antigens. In the current study, we used this murine model to map the segments of O. volvulus protein disulfide isomerase (OvPDI) associated with the development of corneal pathology. Subclones of OvPDI were constructed encompassing one or more predicted T cell epitopes. Keratitis was induced in BALB/c mice after subcutaneous immunizations with OvPDI, followed by intracorneal challenge of OvPDI constructs. Truncated OvPDI proteins containing amino acids 450-481 of OvPDI were found to induce keratitis, whereas constructs that did not include this region did not induce corneal pathology. Consistent with this observation, two peptides derived from the 450-481 region stimulated T cell proliferation to a greater degree than control carrier protein. DNA sequence analysis of cDNAs encoding OvPDI from blinding and non-blinding strains of O. volvulus indicated no differences in the primary amino acid sequence of the 450-481 domain. Immunization of animals with OvPDI induced antibodies recognizing a 55 kDa host protein, identical to the predicted molecular weight of the mouse PDI homologue. Together, these data implicate specific antigenic epitopes of OvPDI in the development of O. volvulus mediated corneal pathology.

Characterization of genes encoding members of the nuclear hormone receptor superfamily from Onchocerca volvulus.

Several lines of evidence suggest that molting in parasitic nematodes is controlled through the action of steroid molting hormones, or ecdysones. In other organisms, the central mediator of steroid hormone action is the hormone receptor. These receptor molecules are members of a superfamily of proteins called the nuclear hormone receptor family. Using an oligonucleotide derived from the amino-acid sequence of the Drosophila melanogaster ecdysone receptor, genes encoding homologues of the nuclear hormone receptor family were identified in the genome of the human filarial parasite Onchocerca volvulus. The O. volvulus genome contains at least three genes that encode putative members of the nuclear hormone receptor superfamily. A complete cDNA for one of these genes, designated OvNHR-1, has been isolated and characterized. The OvNHR-1 cDNA was 2378 bp in length, and contained a single open reading frame of 1104 bp. The open reading frame encoded a peptide with all of the features characteristic of a member of the nuclear hormone receptor superfamily of proteins. OVNHR-1 appeared to be encoded by a single-copy gene. Expression of the mRNA corresponding to OvNHR-1 was developmentally regulated, with maximal expression occurring during early embryogenesis. The polypeptide encoded by the OvNHR-1 open reading frame is antigenic in a minority of individuals exposed to O. volvulus.

Characterization of a putative nuclear receptor from Onchocerca volvulus.

Steroids and retinoids are important regulators of development in invertebrates and vertebrates. The central mediators of action of these compounds are their cognate receptors, which together form a family of proteins known as the nuclear receptor family. Previous studies have demonstrated that the genome of Onchocerca volvulus encodes at least three members of the nuclear receptor family. Here, the characterization of one member of this family from O. volvulus, designated OvNR-2, is described. OvNR-2 was found to be most similar to a number of vertebrate retinoic acid receptors and to the Drosophila melanogaster EiP78c protein. Modeling studies suggest that OvNR-2 forms a boot shaped ligand-binding cavity of a shape and size that can bind steroids. Expression of the mRNA corresponding to OvNR-2 is tightly regulated in adult parasites, appearing only in the extended intrauterine microfilariae. The protein derived from expression of the OvNR-2 cDNA in a bacterial system is recognized by serum antibodies in a majority of individuals infected with O. volvulus.

The Onchocerca volvulus homologue of the multifunctional polypeptide protein disulfide isomerase.

Protein disulfide isomerase (PDI) functions to catalyze the formation of correct disulfide bonds in nascent proteins, and also acts as one of the subunits of prolyl-4 hydroxylase, the enzyme responsible for the oxidative maturation of procollagen. Since the cuticle of parasitic nematodes consists primarily of a network of collagen molecules which are connected through intermolecular disulfide bonds, PDI might be expected to be involved in the process of cuticle biosynthesis. The isolation and characterization of a cDNA encoding the PDI homologue of Onchocerca volvulus is described. This cDNA contains a single, long open reading frame that encodes sequence motifs identical to the two known active sites of PDI for isomerase activity. The O. volvulus PDI appears to be encoded by a single copy gene. Both in situ hybridization and immunolocalization data suggest that PDI is both spatially and temporally regulated in O. volvulus. The pattern of spatial and temporal regulation is consistent with the involvement of PDI in the biosynthesis of the parasite cuticle. The parasite protein appears to be an antigen recognized by a minority of individuals exposed to O. volvulus.

Transformation of DNA sequence data into geometric symbols.

Comparative analysis of related DNA sequences has been simplified by the transformation of data in the standard A, G, C, T format into a set of geometric symbols that promote pattern recognition. Previously, comparing more than 2 or 3 sequences simultaneously has been difficult because of the monotonous patterns established by letters. Here 33 sequences are simultaneously compared to demonstrate the ease with which nucleotide substitutions are accurately identified. This has been accomplished by writing a Word-Perfect macro program to facilitate this transformation. Since this word processing program is widely used, performing this kind of analysis is readily achievable in most laboratories involved in DNA sequence analysis.

Technical report. Optimizing probe selection in directed heteroduplex analysis using HDprobe 1.1.

Directed heteroduplex analysis (DHDA) has proven to be a powerful technique for rapid geotyping in human populations. This strategy should also have widespread utility in differentiating closely related organisms of medical and public health importance through identification of DNA sequence polymorphisms. Identifying an optimal probe sequence for use in DHDA has required empirical testing of both the positive and negative strands of a number of potential probes. To identify optimal probes more efficiently, a computer program has been developed that predicts the number of potential stable and unstable mismatches between a probe and its target sequences in DHDA. This information can then be used to predict--from among a group of potential probes--which one will be the most successful in differentiating closely related homologues of a targeted gene sequence. This approach was tested on a number of probe and target sequences derived from the mitochondrial NADH dehydrogenase subunit 4 gene of the West African black fly, Simulium damnosum sensu lato. The number of unstable mismatches predicted to occur in a given heteroduplex by the computer program was found to be important in differentiating closely related species. Therefore, this strategy is useful in identifying informative probes in the development of new DHDA-based assays.