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Roger K Prichard

Publications and source records attributed to Roger K Prichard.

15 recordsLinked to original sources
PubMed2005

Characterization of a half-size ATP-binding cassette transporter gene which may be a useful marker for ivermectin selection in Onchocerca volvulus.

ATP-binding cassette (ABC) transporters comprise a large paralogous protein family and several confer drug resistance. Ivermectin (IVM) is the only drug approved for treatment of onchocerciasis and is a substrate for some ABC transporters. Furthermore, there is accumulating evidence that IVM selects on some ABC transporter genes in Onchocerca volvulus and other parasitic nematodes. The onchocerciasis control programs rely on community based treatment with IVM each year to reduce morbidity and decrease parasite transmission. This appears to be imposing selection pressure on O. volvulus. A half-size ABC transporter cDNA has previously been reported for O. volvulus, however, the full length gene has not been previously characterized and investigated for possible selection by IVM. Genes under selection may be identified by patterns of linkage disequilibrium (LD) and a loss of genetic polymorphism at physically linked loci. Twelve genetic markers spanning the full gene were examined in O. volvulus from non-treated and IVM treated populations in Ghana. Analysis of the genomic organization of the half-size ABC transporter (OvPLP) indicates that it spans approximately 3.8 kb comprising nine exons. Worms from treated people showed a reduction in gene diversity, a loss of genetic polymorphism at several markers, a selection for specific alleles, and a reduction in the number of regions in LD; these effects were more pronounced as treatment increased. These changes suggest that IVM is imposing selection pressure on this gene. The region between transmembrane domains four and five may be a useful marker for IVM selection in O. volvulus.

ATP-Binding Cassette Transporters↗
PubMed2005

Genomic organization and effects of ivermectin selection on Onchocerca volvulus P-glycoprotein.

Ivermectin (IVM) was first developed for use with livestock. It is now the only drug used for mass treatment of onchocerciasis. It is difficult to prove whether reports of sub-optimal responses to IVM in some Onchocerca volvulus infected patients are a result of drug resistance, as procedures typically used to examine IVM efficacy in livestock can not be performed on humans. To determine the effects of IVM on O. volvulus, one approach is to examine allele frequencies before and after treatment. Allele(s) linked to resistance may increase in frequency after repeated treatment. Mass treatment of large human populations to reduce transmission of O. volvulus will impose selection pressure for resistance. P-glycoprotein has been implicated as a candidate IVM resistance gene in nematodes. In this study, the intron-exon structure of O. volvulus P-glycoprotein (OvPGP) has been defined. The gene spans 10.6 kb, is AT-rich, contains 24 exons and a high proportion of class 0 introns. The genetic diversity of 28 loci spanning the entire OvPGP gene was examined in four O. volvulus populations from the Volta Region of Ghana. Worms collected in 1999 and 2002 from IVM treated patients showed reduced genetic polymorphism and an increase in the number of loci not in Hardy-Weinberg equilibrium. Changes in allelic patterns and a reduction in diversity at many loci in P-glycoprotein in the parasites from IVM treated patients in 1999 and 2002 suggest that IVM is imposing selection on this gene, consistent with a possible development of IVM resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2005

Detection of benzimidazole resistance-associated mutations in the filarial nematode Wuchereria bancrofti and evidence for selection by albendazole and ivermectin combination treatment.

The Global Program to Eliminate Lymphatic Filariasis has been implemented to reduce human microfilaremia to levels low enough to break the transmission of the disease by using single annual doses of albendazole in combination with diethylcarbamazine or ivermectin. Many veterinary helminth parasites have developed resistance against both albendazole and ivermectin. Resistance to albendazole in veterinary nematodes is known to be caused by either of two single amino acid substitutions from phenylalanine to tyrosine in parasite beta-tubulin at position 167 or 200. We have developed assays capable of detecting these single nucleotide polymorphisms (SNPs) in Wuchereria bancrofti, and have applied them to microfilaria obtained from patients in Ghana and Burkina Faso. One of the SNPs was found in worms from untreated populations in both locations. Worms from treated patients had significantly higher frequencies of these mutations. These findings indicate that a beta-tubulin allele associated with benzimidazole resistance is being selected in these populations.

Albendazole↗
PubMed2004

Agonist enhacement of macrocyclic lactone activity at a glutamate-gated chloride channel subunit from Haemonchus contortus.

The mode of action of ivermectin (IVM) in nematodes appears to be the opening of inhibitory ion channels, including the glutamate-gated chloride channel (GluCl). Recently, it has been shown that IVM binds with high affinity to a Haemonchus contortus GluCl subunit (HcGluCla) expressed in COS-7 cells, and this binding is potentiated in the presence of glutamate. To gain further insight into the potentiation of macrocyclic lactone anthlemintics we have screened various glutamatergic and nonglutamatergic ligands for their ability to enhance [ 3H ] IVM binding to HcGluCla. Of the ligands tested, only glutamate and ibotenate potentiated [ 3H ] IVM binding. Interestingly, these ligands have also been shown to open the HcGluCla channel expressed in Xenopus oocytes. We examined the effect of ibotenate on macrocyclic lactone binding in more detail and found that it caused a 7-fold enhancement in [ 3H ] IVM binding affinity and a 4-fold increase in [ 3H ] MOX binding affinity. In in vivo efficacy studies, ibotenate (up to 2mg/kg) had no anthelmintic activity against H. contortus in gerbils. When 1mg/kg ibotenate was used in combination with IVM, IVM efficacy increased by 15% ( P=0.048 ). These results demonstrate that a GluCl agonist enhances IVM activity and provides further information on the mode of action of ivermectin in parasitic nematodes.

Animals↗
PubMed2004

Genomic organization of an avermectin receptor subunit from Haemonchus contortus and expression of its putative promoter region in Caenorhabditis elegans.

Avermectins and milbemycins are believed to exert their anthelmintic effects by binding to glutamate-gated chloride channels (GluCls). Two GluCl subunits have been localized in the pharynx in Caenorhabditis elegans, and the pharynx has been implicated as a major target for avermectins in C. elegans. However, in parasitic nematodes, the pharyngeal localization of the GluCl subunits needs to be determined. The HcGluCla gene encoding an alpha-type GluCl subunit has been cloned from Haemonchus contortus previously. To investigate the expression site of the HcGluCla gene we have isolated a 1439bp 5'-flanking region and determined the genomic organization of this gene. The HcGluCla gene is composed of 12 exons separated by 11 introns and spans approximately 7.3kb of genomic DNA. Analysis of the 1439bp 5'-flanking region of the HcGluCla gene revealed that it contained TATA, CCAAT boxes, and several other consensus transcriptional factor recognition sequences. The 1439bp 5'-flanking region and the first exon and intron and part of the second exon of the HcGluCla gene were fused to green fluorescence protein (GFP) reporter gene and microinjected into the gonads of C. elegans. After microinjection of the construct into C. elegans, four stable transformed lines were established and assayed for GFP expression. The transformed animals exhibited fluorescence in the two pairs of MC and M2 pharyngeal neurons, but no expression was detected in the muscle cells. Expression of HcGluCla in pharyngeal neurons suggests a mechanism for the effects of avermectins and milbemycins on pharyngeal function in parasitic nematodes.

3' Untranslated Regions↗
PubMed2004

Mutations in the extracellular domains of glutamate-gated chloride channel alpha3 and beta subunits from ivermectin-resistant Cooperia oncophora affect agonist sensitivity.

Two full-length glutamate-gated chloride channel (GluCl) cDNAs, encoding GluClalpha3 and GluClbeta subunits, were cloned from ivermectin-susceptible (IVS) and -resistant (IVR) Cooperia oncophora adult worms. The IVS and IVR GluClalpha3 subunits differ at three amino acid positions, while the IVS and IVR GluClbeta subunits differ at two amino acid positions. The aim of this study was to determine whether mutations in the IVR subunits affect agonist sensitivity. The subunits were expressed singly and in combination in Xenopus laevis oocytes. Electrophysiological whole-cell voltage-clamp recordings showed that mutations in the IVR GluClalpha3 caused a modest but significant threefold loss of sensitivity to glutamate, the natural ligand for GluCl receptors. As well, a significant decrease in sensitivity to the anthelmintics ivermectin and moxidectin was observed in the IVR GluClalpha3 receptor. Mutations in the IVR GluClbeta subunit abolished glutamate sensitivity. Co-expressing the IVS GluClalpha3 and GluClbeta subunits resulted in heteromeric channels that were more sensitive to glutamate than the respective homomeric channels, demonstrating co-assembly of the subunits. In contrast, the heteromeric IVR channels were less sensitive to glutamate than the homomeric IVR GluClalpha3 channels. The heteromeric IVS channels were significantly more sensitive to glutamate than the heteromeric IVR channels. Of the three amino acids distinguishing the IVS and IVR GluClalpha3 subunits, only one of them, L256F, accounted for the differences in response between the IVS and IVR GluClalpha3 homomeric channels.

Amino Acid Sequence↗
PubMed2003

Haemonchus contortus: HcGluCla expressed in Xenopus oocytes forms a glutamate-gated ion channel that is activated by ibotenate and the antiparasitic drug ivermectin.

Ion channels are targets for many drugs including insecticides and anthelminthic agents such as ivermectin (IVM) and moxidectin (MOX). IVM has been shown to activate glutamate-gated chloride channels (GluCls) from the free-living nematode, Caenorhabditis elegans. Haemonchus contortus is a parasitic nematode that is also extremely sensitive to IVM. The high sensitivity of H. contortus to IVM is probably the result of the fact that, like C. elegans, H. contortus also express GluCls. To investigate the potential physiological response to IVM in H. contortus we have expressed a GluCl from this parasite (H. contortus glutamate-gated chloride channel, HcGluCla) in Xenopus oocytes. HcGluCla expressed in oocytes formed a homomeric channel that responded to glutamate and ibotenate as well as the anthelmintics IVM and MOX. The response to glutamate and ibotenate was fast acting and reversible whereas the response to IVM and MOX was a slower activating channel that was essentially irreversible. These results suggest that IVM toxicity in H. contortus is the result of its irreversible activation of GluCls.

Animals↗
PubMed2003

Selection at a gamma-aminobutyric acid receptor gene in Haemonchus contortus resistant to avermectins/milbemycins.

Gamma-aminobutyric acid (GABA) Type A receptors are inhibitory chloride channels in membranes of vertebrate and invertebrate neuromuscular cells. Gating of the channels by GABA leads to an influx of chloride ions into, and hyperpolarisation of, the cell. GABA receptors are believed to form channels by the association of five protein molecules of varying subunit types, with the second transmembrane (M2) domain of each protein molecule forming a central pore through which chloride ions can pass. We have analysed by single-strand conformation polymorphism the genetic variation of a GABA-receptor gene, HG1, from two sets of unselected and anthelmintic-selected strains of the parasitic nematode Haemonchus contortus. Significant differences in allele frequencies were detected between one unselected strain and its derived ivermectin-selected strain and between the other unselected strain and its derived ivermectin- and moxidectin-selected strains. In each set of strains, one allele increased substantially in frequency in the drug-selected strains relative to their respective unselected strains. The selected allele, however, differed between the two sets of strains. Similar analyses were performed on a phosphoenolpyruvate carboxykinase gene and a nicotinic acetylcholine receptor subunit gene. No significant differences were found in allele frequencies between the unselected and their derived anthelmintic-selected strains. These results indicate the GABA receptor as a possible site of action for avermectins and milbemycins, and suggest its involvement in resistance to these anthelmintics.

Animals↗
PubMed2002

A glutamate-gated chloride channel subunit from Haemonchus contortus: expression in a mammalian cell line, ligand binding, and modulation of anthelmintic binding by glutamate.

Glutamate-gated chloride channels (GluCls) are inhibitory ion channels that are sensitive to the antiparasitic drugs ivermectin (IVM) and moxidectin (MOX). We have transiently transfected COS-7 cells with a subunit of a GluCl (HcGluCla) from the parasitic nematode Haemonchus contortus. This subunit bound [3H]-IVM and [3H]-MOX with K(d) values of 0.11+/-0.021 and 0.18+/-0.02nM, respectively. Displacement analysis revealed that IVM and MOX bind to the same site on HcGluCla and that this site is likely distinct from the glutamate binding site. Glutamate was found to be an allosteric modulator of [3H]-MOX and [3H]-IVM binding and increased the affinity of [3H]-MOX for HcGluCla by more than 50% and that of [3H]-IVM by more than 7-fold. These results point to both similarities and differences in the interactions of IVM and MOX with the GluCl. Aspartate, which is structurally similar to glutamate, had little or no effect on [3H]-IVM and [3H]-MOX binding, suggesting that this ligand does not induce the conformational change necessary to potentiate macrocyclic lactone binding. These results also indicate that it may be possible to enhance the efficacy of macrocyclic lactone anthelmintics by administering these compounds with ligands acting allosterically to enhance their binding.

Animals↗
PubMed2002

Immunization with a tubulin-rich preparation from Trypanosoma brucei confers broad protection against African trypanosomosis.

Tubulin from Trypanosoma brucei was purified to near homogeneity using a protocol which involved treatment with urea with subsequent renaturation and was then used to immunize mice. Renatured tubulin further purified by SDS-PAGE (denatured), synthetic tubulin peptides (STP), and rat brain tubulin (RbTub) were also used. Immunized mice were challenged with T. brucei, Trypanosoma congolense or Trypanosoma rhodesiense. Renatured T. brucei tubulin (nTbTub) induced protection in all mice tested, of which 60-80% (n = 81) was complete and the remainder partial. Denatured T. brucei tubulin (dTbTub), STP, or RbTub induced lower antibody levels than nTbTub and did not offer protection. However, in culture, the antibodies against dTbTub or STP killed trypanosomes although at lower dilutions than nTbTub, but those against RbTub did not. In Western blots anti-trypanosome antibodies recognized the tubulin of all the trypanosome species investigated but not vertebrate tubulin, whereas the anti-RbTUB antibodies recognized both trypanosome and vertebrate tubulin. Of the five mice given passive immunity by the transfer of anti-nTbTub serum, four were completely protected and one partially protected. These data suggest that tubulin is the relevant immunogen in the preparation used and could therefore be a promising target for the development of a parasite-specific, broad spectrum vaccine.

Animals↗
PubMed2002

Study of the nematode putative GABA type-A receptor subunits: evidence for modulation by ivermectin.

Two alleles of the HG1 gene, which encodes a putative GABA receptor alpha/gamma subunit, were isolated from Haemonchus contortus. These two alleles were shown previously to be associated with ivermectin susceptibility (HG1A) and resistance (HG1E), respectively. Sequence analysis indicates that they differ in four amino acids. To explore the functional properties of the two alleles, a full-length cDNA encoding the beta subunit, a key functional component of the GABA receptor, was isolated from Caenorhabditis elegans (gab-1, corresponding to the GenBank locus ZC482.1) and coexpressed in Xenopus oocytes with the HG1 alleles. When gab-1 was coexpressed with either the HG1A allele or the HG1E allele in Xenopus oocytes, gamma-aminobutyric acid (GABA)-responsive channels with different sensitivity to the agonist were formed. The effects of ivermectin on the hetero-oligomeric receptors were determined. Application of ivermectin alone had no effect on the receptors. However, when coapplied with 10 micro m GABA, ivermectin potentiated the GABA-evoked current of the GAB-1/HG1A receptor, but attenuated the GABA response of the GAB-1/HG1E receptor. We demonstrated that the coexpressed HG1 and GAB-1 receptors are GABA-responsive, and provide evidence for the possible involvement of GABA receptors in the mechanism of ivermectin resistance.

Alleles↗
PubMed2002

Classical ligands bind tubulin of trypanosomes and inhibit their growth in vitro.

Tubulin ligands known to be toxic to certain organisms or cells were tested for their ability to inhibit proliferation of trypanosomes in culture. Tubulin was purified from Trypanosoma brucei brucei or rat brain by poly-L-lysine affinity chromatography and used in binding studies in order to compare the binding of [3H]mebendazole to trypanosome and mammalian tubulin. All the compounds tested in culture inhibited trypanosome proliferation in a concentration-dependent manner. The concentration required to inhibit trypanosome proliferation by 50 or 90% (IC50 or IC90) in 24 hr was determined for each compound. There were no significant differences (P > 0.05) among the benzimidazoles (BZs), but colchicine and vinblastine caused significantly greater inhibitions than the BZs (P < 0.02 and P < 0.005, respectively). Increasing the incubation time to 72 hr caused a 2- to 4-fold lowering of the IC50 and IC90 values for all the drugs. In the binding assays, there was higher total binding of [3H]mebendazole to trypanosome than rat brain tubulin. The results suggest that the inhibition of trypanosome growth was caused by the specific interaction of these ligands with trypanosome tubulin. Trypanosome tubulin is, therefore, a reasonable target against which novel drugs can be developed to control trypanosomiasis.

Animals↗
PubMed2002

Localization of p-glycoprotein mRNA in the tissues of Haemonchus contortus adult worms and its relative abundance in drug-selected and susceptible strains.

Membrane transporter P-glycoproteins (Pgps) are present in a number of nematode species, including Haemonchus contortus. Allelic variation in some Pgp genes has been found to be associated with resistance to the anthelmintic ivermectin (IVM), although functional verification of a role for Pgps in IVM resistance has yet to be demonstrated. By in situ hybridization, the distribution of Pgp mRNA was visualized in transverse cryosections of adult H. contortus, using a digoxigenin-labeled cDNA probe encoding the ATP-binding region of an H. contortus Pgp. The probe sequence targeted a conserved ATP-binding region of Pgp-A (97.9% identity). It also shared 49.7-71.1% identity with 11 other Pgp sequences previously identified in H. contortus and may hybridize these sequences to give an overall measure of the total P-glycoprotein mRNA. Staining was predominately localized along the intestinal tract of the worms, with the most intense staining localized in the pharynx and anterior intestine. In the mid- and posterior intestinal regions, staining was restricted to the luminal side of the intestine. Some staining was also associated with the vas deferens and the lateral hypodermal chords anterior to the nerve ring. Using densitometry, the levels of Pgp mRNA in the pharynges of unselected and IVM- and moxidectin (MOX)-selected strains of male and female H. contortus were compared. No differences were detected between the levels of expression of Pgp in the susceptible strain versus the IVM- or MOX-selected strains. Evidence in the literature suggests that not all Pgp homologues are linked to chemical resistance phenotypes. It is thus possible that expression of I of the H. contortus Pgps is altered in IVM-resistant strains but that this phenomenon was undetectable in our experiments.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMedDate not supplied

Trypanosoma brucei: anti-tubulin antibodies specifically inhibit trypanosome growth in culture.

We previously observed that trypanosome tubulin immunizes mice against infection by this parasite. Here we describe the direct effect of anti-tubulin antibodies on trypanosomes, using rabbit antibodies to renatured (nTbTub) or SDS-PAGE denatured (dTbTub) Trypanosoma brucei tubulin. We also evaluate antibodies to synthetic tubulin peptides (STP) and rat brain tubulin (RbTub). The anti-nTbTub serum strongly inhibited trypanosome proliferation in culture, and immunoagglutinated trypanosomes even after heat inactivation of complement. The anti-dTbTub and the anti-STP sera also inhibited trypanosome growth and immunoagglutinated trypanosomes, but to a lesser extent than the anti-nTbTub, whereas the anti-RbTub serum had no effect. In Western blots these antibodies were species specific. Immunofluorescence showed that the surface of intact trypanosomes was not uniformly stained by any of these antibodies, but cells that had been permeabilised were labeled throughout the cytoplasm. This suggests that the variant surface glycoproteins (VSG) played no part in the generation of these inhibitory antibodies.

Agglutination Tests↗