Progress 08/01/09 to 09/30/10
Outputs
OUTPUTS: The activities we carried out during this period were designed to throw light on changes in gene expression associated with the development of anthelmintic resistance in the small ruminant parasite, Haemonchus contortus. We examined the expression of candidate resistance genes in two isolates, a laboratory-passaged drug sensitive isolate and 3XR, an isolate obtained from the University of Georgia sheep flock that is fully resistant to thiabendazole, levamisole, ivermectin and moxidectin when assayed in the Drenchrite in vitro assay. We isolated RNA from cultured L3 larvae of both isolates and synthesized cDNA. Three different larval cultures were used from each isolate to provide three distinct cDNA samples. In each sample we used quantitative real-time PCR to measure the amount of mRNA coding for about 30 candidate resistance genes. The genes studied included those coding for nicotinic acetylcholine receptor subunits - the target of levamisole - ligand-gated chloride channel subunits and P-glycoproteins. The latter two gene families represent the target and possible detoxification mechanisms of the macrocylic lactones (which include ivermectin and moxidectin). We also examined the sequences of the beta-tubulin isotype 1 cDNA from both isolates, since known changes in this gene are associated with resistance to the benzimidazole anthelmintics (which include thiabendazole). A new graduate student, Shannon Kraft, was mentored and introduced to these techniques during her rotation in the Infectious Diseases PhD program. We attended two events during this project; Dr Williamson attended the International Congress of Parasitology (ICOPA) in Melbourne, Australia, and Dr Wolstenholme attended a symposium on Molecular Helminthology in Hydra, Greece. Presentations on the work were made at both events. Products: As part of the experimentation that we performed we had to develop novel assays for many of the target genes, and for the detection and measurement of the expression of a novel mRNA species, acr-8b, from Haemonchus contortus. PARTICIPANTS: Dr Wolstenholme acted as PI for this project. Though not supported by USDA funds, Dr Williamson performed all the experiments described. Collaborators at the University of Georgia College of Veterinary Medicine were Dr Ray Kaplan and Mr Bob Storey, who provided the worms used in the study. TARGET AUDIENCES: Target audiences for efforts to improve diagnosis of resistance, the long-term objective of the work described here, are livestock veterinarians and farmers. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The changes in knowledge that took place during the project relate to our improved understanding of the changes in gene expression that accompany the development of anthelmintic resistance. We found that expression of the mRNA, acr-8b, that encodes a truncated subunit of the nicotinic acetylcholine receptor, is massively increased (>100-fold) in the resistant worms, and we believe that this may form the basis for a new molecular test for levamisole resistance. We also found that expression of another nicotinic receptor subunit, UNC-29, may be reduced in the resistant worms. Many changes in expression were discovered for genes implicated in resistance to the macrocyclic lactones, ivermectin and moxidectin. We found changes in expression of two genes that encode ivermectin-sensitive subunits of the glutamate-gated chloride channel; expression of both glc-3 and glc-5 was reduced in the resistant worms. We also found multiple changes in the expression of the P-glycoproteins, pumps that have been associated with resistance; we saw small decreases in the mRNA levels for some of these pumps, but increases in the expression of others, especially pgp-2 and pgp-9. These finding imply that either there are multiple changes associated with ivermectin and moxidectin resistance, or that a single change affects the expression of multiple genes. We confirmed the presence of the known benzimidazole-resistance polymorphisms in the beta-tubulin sequence of the 3XR isolate, at positions 167 and 200.
Publications
- Williamson, S.M., Wolstenholme, A.J. (2011) P-glycoproteins of Haemonchus contortus: development of real-time PCR assays for gene expression studies. J. Helminthol. (submitted).
- Wolstenholme, A., Kaplan, R. (2011). Monsters Inside Our Animals. VMES Annual Report.
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Progress 07/01/09 to 12/31/09
Outputs
OUTPUTS: Our activities during this period have concentrated on conducting and analyzing experiments described in the proposal and designed to identify strong candidate genes associated with resistance to the macrocyclic lactone anthelmintics. The first stage of this work was to reanalyze the fragmented and unannotated genome sequence of Haemonchus contortus to identify the number of potential candidate resistance genes, specifically members of the P-glycoprotein gene family, are present in this parasite. Based on this analysis, oligonucleotide primers were designed to amplify portions of the P-gp genes as templates for quantitative real-time PCR. These partial cDNA clones were sequenced, to confirm their identity, and then used in the optimisation of the PCR reactions. Six P-Gp genes were found to be expressed in L3 larvae at levels high enough to be detected by RT-PCR. We optimized the amplification conditions for these transcripts and determined the efficiency of the amplification - both necessary controls if the quantitative data were to be reliable. In several cases this required multiple sets of primers to be synthesized and tested to ensure that amplification was both specific i.e. only one band, corresponding to the target transcript, was produced, and at least 60% efficient. The expression levels of the six P-gp mRNAs were measured in L3 larvae from the ivermectin-susceptible ISE and -resistant ISE-IVMR isolates of H. contortus. We used the Pfaffl ratiometric method, comparing expression of the target transcripts to those of a standard transcript from a house-keeping gene, in this case beta-actin, to determine the relative changes in expression level found in the resistant nematodes. There have been recent suggestions that ivermectin resistance might also select for polymorphisms in beta-tubulin genes that are known to be associated with resistance to the benzimidazole class of anthelmintics: we therefore measured the allele frequency of both strains at codon 200 of beta-tubulin for both strains of parasite using our previously described PCR methods for populations and individuals. There have been limited events during this period, as this was early in the project, but an abstract has been submitted for oral presentation at the British Society for Parasitology spring meeting to be held in Cardiff, UK. An internal presentation was made to members of the Parasitology group in Infectious Diseases at UGA in November 2009. PARTICIPANTS: The PI, Dr Wolstenholme, directed the research and helped analyse the data. Dr Williamson, post-doctoral associate in the Dept of Infectious Diseases, carried out the experiments and analysed the data. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Changes in Knowledge. Our re-examination of the still unfinished H. contortus genome sequence led to the identification of twelve P-gp related genes. When these were compared to the fourteen functional P-gp genes in C. elegans, we concluded that pgp-5, pgp-6, pgp-7 and pgp-8 are absent in the parasite genome, nor could we find a convincing orthologue for pgp-13. However, pgp-9 has been duplicated, possibly twice, to produce three parasite orthologues (Hco_pgp9.1, 9.2 and 9.3) and either pgp-11 or pgp-12 may also have undergone gene duplication - the genome sequence is not yet well enough assembled to be certain. Of these twelve genes, eight (Hco_pgp-1,-2, -3, -4, -9.2, -10, -11 and -12) were expressed at levels high enough in the L3 stage to permit quantitative real-time PCR to take place, however we were unable to optimize the reactions for pgp-1 or pgp-2 sufficiently well to have confidence in the data produced. Quantitative RT-PCR data were analysed by the Pfaffl method, using actin as the standard. We have not detected any significant changes in the expression of these genes between the ivermectin-susceptible and -resistant larvae, except for a possible fall in the expression of a novel transcript with similarities to both pgp-12 and pgp-14.This transcript was reduced 4-6 fold in two experiments; additional data are required before we can suggest that this represents a candidate resistance-associated transcript. When we measured the allele frequency at codon 200 of beta-tubulin in the same populations, we found that the benzimidazole resistance-associated allele (encoding a tyrosine) was present at a frequency of 0.3% in the ivermectin-susceptible worms but at 15.6% in the resistant population. This supports previous suggestions that selection for ivermectin resistance may also tend to select for benzimidazole resistance. Changes in Actions. We are adopting a non-hypothesis based approach to the identification of candidate resistance genes, and plan to use 454 next generation sequencing to analyse the transcriptomes of both resistant and susceptible nematodes. Unlike older technologies, such as microarrays, this does not rely on a properly annotated genome sequence in order to carry out the studies. It is too early in these studies for any Changes in Conditions to have resulted.
Publications
- No publications reported this period
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