Progress 05/05/05 to 09/30/07
Outputs
OUTPUTS: Haemonchosis, the disease in ruminants caused by the helminth Haemonchus contortus, may be chronic with minor clinical signs, or acute or hyperacute resulting in death. Treatment and control of this gastrointestinal helminth have relied on anthelmintic drugs. However, this parasite has developed resistance to the three anthelmintic classes - benzimidazoles, imidazothiazoles and macrocyclic lactones - that are currently used for treatment. One of the mechanisms proposed to account for resistance is drug efflux by the transporter P-glycoprotein (Pgp). The goals of this study were to: 1) Determine the resistance status of H. contortus isolates to the different anthelmintic classes, and 2) Compare the transcription of Pgp among known anthelmintic susceptible and resistant H. contortus exposed or not exposed to the anthelmintics. Seven H. contortus isolates were evaluated using the DrenchRite larval development assay. Of the isolates analyzed, only one, Hc-ES (from a sheep ranch
in El Dorado, TX), was susceptible to all of the anthelmintics tested, while the others showed variable levels of resistance to one or more drugs (Table 1). The isolate from a giraffe at a zoo in Florida (Hc-GRF) was the most resistant, with extremely high levels of resistance to benzimidazoles and levamisole. Transcription of Pgp in L3 larvae of the seven different isolates exposed or unexposed to anthelmintics was quantitated using polymerase chain reaction (PCR), with the 18s ribosomal RNA gene serving as the housekeeping gene control. For each test, the same amount of cDNA was used under identical amplification conditions. No Pgp transcription was found by quantitative PCR in any of the isolates under the conditions of this study, whether exposed to anthelmintic or unexposed. The 18s rRNA gene was consistently amplified from the same cDNA samples. Thus, increased Pgp production does not appear to be a primary mechanism of drug resistance in this stage of the worm. It is possible,
however, that Pgp other than the one tested in this study may be involved, or that the mechanism of protection is not increased production but rather increased affinity for the drug(s). This remains to be determined. There are very few reports of helminths in giraffes. To confirm the identity of the helminth obtained from the giraffe in this study, the 18s rRNA gene and the rRNA internal transcribed spacer region (ITS) were amplified, cloned, sequenced, and analyzed. The sequences obtained were consistent with previous reports for H. contortus, confirming that the giraffe was infected with this parasite. This is, to our knowledge, the first report of haemonchosis in a giraffe due to H. contortus resistant to all three classes of anthelmintics.
PARTICIPANTS: Pamela Garretson, graduate student. Graduated August 2007 MS in Veterinary Parasitology. This study comprised her thesis research project. She is currently an instructor in parasitology at Louisiana State University. Elizabeth E. Hammond DVM, veterinarian at the Lion Country Safari, 2003 Lion Country Safari Road, Loxahatchee, Florida 33470, provided an isolate of Haemonchus contortus from an infected giraffe for this study and is collaborating on a manuscript under preparation resulting from this work.
PROJECT MODIFICATIONS: The original protocol outlined an in vivo approach, where animals would be infected with resistant or susceptible Haemonchus contortus isolates, then divided into anthelmintic treated or untreated groups. After treatment, the treated and control animals were to be sacrificed to obtain the adult worms for Pgp testing by quantitative PCR. We elected to use an in vitro approach for evaluating the role of Pgp in resistance for two main reasons: 1) If our approach of assaying L3 larvae were successful, this would provide a rapid method of testing for anthelmintic resistance from by collecting fecal samples (in the lab L3 larvae will develop from eggs taken from feces). If we used adult worms in our assay, these are only obtained from the stomach of the host, hence the animal(s) would have to be sacrificed. We were hopeful that an outcome of our study would be an easy way to assess for drug resistance in worms without harming the animal. 2) We were concerned that due
to the widespread occurrence of Haemonchus in small ruminants in Texas that the animals we obtained would be previously exposed and therefore have developed immunity to the parasite. This would be difficult to control for, would likely affect the worms, and would therefore affect the results of our study.
Impacts
This study identified Haemonchus contortus in a zoo-housed giraffe, and showed that this isolate is resistant to commonly-used anthelmintics. Moreover, this study found that anthelmintic resistance is so common in small ruminants in Texas that it was difficult to find an anthelmintic susceptible population to serve as a control. These findings will contribute to increased awareness of the problem of anthelmintic resistance in various livestock situations, not least of which is the small ruminant industry where haemonchosis contributes greatly to production losses annually. This study comprised the thesis research project for a master of science student who graduated from TAMU in August 2007 and is now an instructor at another university. Isolates evaluated for anthelmintic resistance in this study were provided for another study (M de Lourdes Mottier and R K Prichard, Genetic analysis of a relationship between macrocyclic lactone and benzimidazole anthelmintic
selection on Haemonchus contortus, Pharmacogenetics an dGenomics 18:129-140, 2008).
Publications
- No publications reported this period
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Progress 01/01/06 to 12/31/06
Outputs
The anthelmintic resistance/susceptibility status of populations of Haemonchus contortus was determined using the Drenchrite larval development assay (LDA). This assay determines the level of resistance to three classes of anthelmintics (benzimidazoles, macrocyclic lactones, and imidazothiazoles) at different concentrations based on worm survival and development to the L3 stage. Of the populations tested, three were found to be relatively susceptible and two were found to be resistant to all three anthelmintics. The L3 larvae that survived the assay were collected, graded, and stored in 50 worm aliquots according to anthelmintic resistance/susceptibility status for later use in molecular analysis. Based on the desired parameters, L3 larvae aliquots (both susceptible and resistant) collected from the LDA were selected for molecular analysis. Ribonucleic acid (RNA) was isolated for selected sample, quantitated and bioanalyzed. 3' cDNA was synthesized from the RNA for
quantitative PCR of P-glycoprotein transcripts. Gene specific primers were designed for P-glycoprotein 1 and P-glycoprotein A amplification from cDNA. Housekeeping genes evaluated for use as internal standards included actin, ATP synthase, and small subunit ribosomal RNA (SSU rRNA). The latter was selected and optimized for use as an internal control standard. Currently, P-gp transcription expression has been analyzed by reverse transcriptase polymerase chain reaction (RT-PCR) for one set of larvae from a susceptible strain, encompassing aliquots of worms that had been exposed to each of the three drugs and unexposed worms. No P-gp transcription was found among these samples. The remaining samples are under analysis.
Impacts
Haemonchus contortus resistance to all three major groups of anthelmintics has been recorded across the globe. Lack of genetic reversion, presence of side resistance, and lack of universality contribute to the problem. Diagnosis and surveillance are essential for resistance management and diagnostics based on genotypes would provide sensitive molecular tools for monitoring resistance. The focus of this proposed project is drug efflux mediated by Pgp molecules as a common mechanism of resistance in MDR H. contortus. Pgp in field-selected MDR strains resistant to the 3 major classes of anthelmintics will be evaluated alongside a susceptible strain. We will identify which Pgp are directly associated with the resistance for each drug. The long term goal is to identify specific markers for anthelmintic resistant H. contortus.
Publications
- No publications reported this period
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Progress 01/01/05 to 12/31/05
Outputs
Graduate student Pamela Garrettson is assisting with this project to fulfill the research requirement to matriculate the Master of Science degree in Veterinary Parasitology, Department of Veterinary Pathobiology, Texas A&M University. P-glycoproteins (Pgp) are implicated in Haemonchus contortus resistance to ivermectin as facilitating the efflux of the drug, thus abrogating the toxic effect on the primary drug target, but the role of Pgp in resistance to other antihelmintics has not been explored. In this study, we propose to determine if parasite P-glycoproteins are overexpressed on challenge with benzimidazole, avermectin, and levamisole, to determine if specific H. contortus Pgp are associated with resistance for each anthelmintic, and to acquire full-length gene sequences for Pgp from multiply drug resistant and anthelmintic susceptible H. contortus strains. Samples of H. contortus include two field acquired larval isolates from sheep, designated H916 and H992,
resistant to benzimidazoles and Ivermectin, but susceptible to moxidectin and levamisole, and two field acquired adult worm isolates from goats, G-079 and LTAMU, both of which show similar resistance and susceptibility. DNA has been purified from the samples and primer sets designed from H. contortus Pgp-A and Pgp-1 sequences in the GenBank database. Total RNA was extracted from larval and adult worms not challenged with anthelmintics and cDNA prepared. Pgp-A (GenBank accession AF003908) primers yielded no product from larval worm cDNA, however, an approximate 4 kb band was acquired with cDNA from adult worms. Partial gene sequence analysis confirms that Pgp-A was amplified, with 97% identity to the GenBank sequence. Sequencing of the remainder of the gene is underway. Haemonchus contortus gDNA from both larval and adult worms yielded bands in excess of the expected size. Sequencing of the product from the larval worms revealed the presence of introns, which account for the increased
size observed. Pgp-1 primers were designed from GenBank accessions U94401, AF182012, AF182011 and AF055166, which share the same sequence. Amplification products from genomic DNA from adult and larval worms were of the expected 800 bp size. Sequencing of the larval gene product confirmed that Pgp-1 was amplified. However, no bands were obtained from either adult or larval cDNA with these primers. The results to date indicate that Pgp-A is transcribed at detectable levels in adult worms unchallenged by anthelmintics, but not in unchallenged larval worms. Pgp-1, however, is not transcribed in great enough quantity, if at all, to detect in either adult or larval worm cDNA when not challenged. Both genes are present, however, in all of the H. contortus genomic DNA preparations tested to date. An impediment to this project at the current time is the failure to identify an anthelmintic susceptible population of H. contortus to use as a control in addition to internal controls. To date a
number of isolates have been tested for resistance in an effort to find an adequate external control, but none was fully susceptible to all classes of anthelmintic.
Impacts
Haemonchus contortus resistance to all three major groups of anthelmintics has been recorded across the globe. Lack of genetic reversion, presence of side resistance, and lack of universality contribute to the problem. Diagnosis and surveillance are essential for resistance management and diagnostics based on genotypes would provide sensitive molecular tools for monitoring resistance. The focus of this proposed project is drug efflux mediated by Pgp molecules as a common mechanism of resistance in MDR H. contortus. Pgp in field-selected MDR strains resistant to the 3 major classes of anthelmintics will be evaluated alongside a susceptible strain. We will identify which Pgp are directly associated with the resistance for each drug. The long term goal is to identify specific markers for anthelmintic resistant H. contortus.
Publications
- No publications reported this period
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