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Roger K Prichard - One of the best experts on this subject based on the ideXlab platform.
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clinical validation of molecular markers of Macrocyclic Lactone resistance in dirofilaria immitis
International Journal for Parasitology-Drugs and Drug Resistance, 2018Co-Authors: Cristina Ballesteros, Catherine Bourguinat, Kathy Keller, Roger K Prichard, Cassan N Pulaski, Timothy G GearyAbstract:Abstract Prophylaxis with Macrocyclic Lactone (ML) endectocides is the primary strategy for heartworm control. Recent evidence has confirmed that ML-resistant Dirofilaria immitis isolates have evolved. Comparison of genomes of ML-resistant isolates show they are genetically distinct from wild-type populations. Previously, we identified single nucleotide polymorphisms (SNPs) that are correlated with phenotypic ML resistance. Since reliable in vitro assays are not available to detect ML resistance in L3 or microfilarial stages, the failure to reduce microfilaraemia in infected dogs treated with an ML has been proposed as a surrogate clinical assay for this purpose. The goal of our study was to validate the genotype-phenotype correlation between SNPs associated with ML resistance and failure to reduce microfilaraemia following ML treatment and to identify a minimal number of SNPs that could be used to confirm ML resistance. In this study, 29 participating veterinary clinics received a total of 148 kits containing supplies for blood collection, dosing and prepaid shipping. Patients recruited after a diagnosis of heartworm infection were treated with a single standard dose of Advantage Multi® and a blood sample taken pre- and approximately 2–4 weeks post-treatment. Each sample was processed by performing a modified Knott's Test followed by isolation of microfilariae, genomic DNA extraction and MiSeq sequencing of regions encompassing 10 SNP sites highly correlated with ML resistance. We observed significant correlation of SNP loci frequencies with the ML microfilaricidal response phenotype. Although all predictive SNP combination models performed well, a 2-SNP model was superior to other models tested. The predictive ability of these markers for ML-resistant heartworms should be further evaluated in clinical and epidemiological contexts.
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genetic profiles of ten dirofilaria immitis isolates susceptible or resistant to Macrocyclic Lactone heartworm preventives
Parasites & Vectors, 2017Co-Authors: Catherine Bourguinat, Kathy Keller, Tom L Mctier, Debra J Woods, Pierre Lepage, Roger K PrichardAbstract:Background For dogs and cats, chemoprophylaxis with Macrocyclic Lactone (ML) preventives for heartworm disease is widely used in the United States and other countries. Since 2005, cases of loss of efficacy (LOE) of heartworm preventives have been reported in the U.S. More recently, ML-resistant D. immitis isolates were confirmed. Previous work identified 42 genetic markers that could predict ML response in individual samples. For field surveillance, it would be more appropriate to work on microfilarial pools from individual dogs with a smaller subset of genetic markers.
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Interaction of Macrocyclic Lactones with a Dirofilaria immitis P-glycoprotein.
International Journal for Parasitology, 2016Co-Authors: Thangadurai Mani, Catherine Bourguinat, Kathy Keller, Byron L Blagburn, Shoaib Ashraf, Roger K PrichardAbstract:Abstract Dirofilaria immitis , a filarial nematode, causes dirofilariasis or heartworm disease in dogs, cats and wild canids. Effective prevention of the disease is mainly by the use of the Macrocyclic Lactone class of drugs as heartworm preventives, and no other class of drugs is effective for preventing infection. Macrocyclic Lactones have been used for prevention of heartworm infection for more than 26 years. However, prevention has been compromised by the development of resistance in recent years. The mechanism of Macrocyclic Lactone resistance in D. immitis has yet to be established. In other parasitic nematodes, P-glycoproteins (PGPs) have been implicated in Macrocyclic Lactone resistance. The presence of two polymorphic loci on D. immitis P-glycoprotein-11 ( Dim - pgp-11 ) correlated with loss of efficacy of Macrocyclic Lactone anthelmintics, suggesting that PGPs may be involved in Macrocyclic Lactone resistance in D. immitis . We have identified the full length of Dim-Pgp-11 cDNA , expressed it in mammalian cells, and studied the functional activity of the expressed protein. We have characterised its interaction with the four Macrocyclic Lactone preventives, ivermectin, selamectin, moxidectin and milbemycin oxime, using the transport of different fluorescent substrates. The inhibitory effect of these Macrocyclic Lactones on the transport of two fluorophore probes, Rhodamine 123 and Hoechst 33342, by Dim- PGP-11 has been studied. The avermectins, ivermectin and selamectin, markedly inhibited Rhodamine 123 transport in a concentration-dependent and saturable manner, whereas the milbemycins, moxidectin and milbemycin oxime, were found to have different inhibition profiles with Rhodamine 123 transport. However, both avermectins and milbemycin preventives inhibited the transport of Hoechst 33342 by Dim- PGP-11 in a concentration-dependent and apparently saturable manner, although differences existed in terms of efficiency and potency of inhibition between the two sub-classes of Macrocyclic Lactones. We postulate that Dim -PGP-11 may have two to three drug binding sites, as with mammalian Pgp, including the ‘R’ site for Rhodamine 123 and the ‘H’ site for Hoechst 33342. The avermectins appear to bind the ‘R’ binding site unlike the milbemycins, whereas both sub-classes of Macrocyclic Lactones might interact with the ‘H’ site of D. immitis PGP-11.
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haemonchus contortus p glycoprotein 2 in situ localisation and characterisation of Macrocyclic Lactone transport
International Journal for Parasitology, 2015Co-Authors: Pablo Godoy, Robin N Beech, Jing Lian, Roger K PrichardAbstract:Abstract Haemonchus contortus is a veterinary nematode that infects small ruminants, causing serious decreases in animal production worldwide. Effective control through anthelmintic treatment has been compromised by the development of resistance to these drugs, including the Macrocyclic Lactones. The mechanisms of resistance in H. contortus have yet to be established but may involve efflux of the Macrocyclic Lactones by nematode ATP-binding-cassette transporters such as P-glycoproteins. Here we report the expression and functional activity of H. contortus P-glycoprotein 2 expressed in mammalian cells and characterise its interaction with the Macrocyclic Lactones, ivermectin, abamectin and moxidectin. The ability of H. contortus P-glycoprotein 2 to transport different fluorophore substrates was markedly inhibited by ivermectin and abamectin in a dose-dependent and saturable way. The profile of transport inhibition by moxidectin was markedly different. H. contortus P-glycoprotein 2 was expressed in the pharynx, the first portion of the worm’s intestine and perhaps in adjacent nervous tissue, suggesting a role for this gene in regulating the uptake of avermectins and in protecting nematode tissues from the effects of Macrocyclic Lactone anthelmintic drugs. H. contortus P-glycoprotein 2 may thus contribute to resistance to these drugs in H. contortus .
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a dyf 7 haplotype causes sensory neuron defects and is associated with Macrocyclic Lactone resistance worldwide in the nematode parasite haemonchus contortus
International Journal for Parasitology, 2014Co-Authors: Ludmel Urdanetamarquez, Robin N Beech, Patrick Janukavicius, Joseph A Dent, Roger K PrichardAbstract:Heavy reliance on Macrocyclic Lactones to treat parasitic nematodes has resulted in the evolution of widespread drug resistance that threatens human and animal health. Management strategies have been proposed that would slow the rise of resistance, however testing these strategies has been hampered by the lack of identified strong-effect resistance markers in parasites. We show that the Caenorhabditis elegans gene Cel_dyf-7, necessary for amphid sensory neuron development, also confers Macrocyclic Lactone sensitivity. In the sheep parasite Haemonchus contortus: (i) strains selected for Macrocyclic Lactone resistance were enriched in a Hco_dyf-7 haplotype that was rare in the drug-naive population, (ii) the resistant haplotype correlated with the sensory neuron defects, and (iii) the resistant haplotype was associated with decreased Hco_dyf-7 expression. Resistant field isolates of H. contortus from five continents were enriched for the resistant haplotype, demonstrating the relevance of the Hco_dyf-7 haplotype to practise and indicating that it is a locus of strong effect. Hemizygosity resulting from sex linkage of dyf-7 likely contributes to the rise of resistance in treated populations.
Angel Rios - One of the best experts on this subject based on the ideXlab platform.
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validation of a screening method for rapid control of Macrocyclic Lactone mycotoxins in maize flour samples
Analytical and Bioanalytical Chemistry, 2008Co-Authors: Mohammed Zougagh, Helena Tellez, Alberto Sanchez, Manuel Chicharro, Angel RiosAbstract:A procedure for the analytical validation of a rapid supercritical fluid extraction amperometric screening method for controlling Macrocyclic Lactone mycotoxins in maize flour samples has been developed. The limit established by European legislation (0.2 mg kg−1), in reference to zearalenone (ZON) mycotoxin, was taken as the reference threshold to validate the proposed method. Natural ZON metabolites were also included in this study to characterize the final screening method. The objective was the reliable classification of samples as positive or negative samples. The cut-off level was fixed at a global concentration of mycotoxins of 0.17 mg kg−1. An expanded unreliability zone between 0.16 and 0.23 mg kg−1 characterized the screening method for classifying the samples. A set of 30 samples was used for the final demonstration of the reliability and usefulness of the method.
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supercritical fluid extraction of Macrocyclic Lactone mycotoxins in maize flour samples for rapid amperometric screening and alternative liquid chromatographic method for confirmation
Journal of Chromatography A, 2008Co-Authors: Mohammed Zougagh, Angel RiosAbstract:Abstract A rapid and simple method for the direct screening of Macrocyclic Lactone mycotoxins (zearalenone, ZON; α-zearalenol, α-ZOL; and β-zearalenol, β-ZOL) in maize flour samples is proposed. The sample screening method comprises supercritical fluid extraction (SFE) and clean-up on Florisil adsorption cartridge of the selected toxic compounds, followed by continuous flow electrochemical detection. Those samples for which the total concentration is close to or above the threshold limit established by legislation (0.200 mg kg−1) are subjected to preconcentration on C18 chromatographic material and liquid chromatographic separation for confirmation purposes. This confirmation method allows the determination of ZON, α-ZOL and β-ZOL in the range between 30 and 300 μg kg−1, with a average relative standard deviation lower than 5.2 in all cases.
Ray M Kaplan - One of the best experts on this subject based on the ideXlab platform.
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using population genetics to examine relationships of dirofilaria immitis based on both Macrocyclic Lactone resistance status and geography
Veterinary Parasitology, 2020Co-Authors: Julie Sanchez, Cassan N Pulaski, Adrian J Wolstenholme, Guha Dharmarajan, Melissa M George, John S Gilleard, Ray M KaplanAbstract:Abstract Prevention of infection with canine heartworm (Dirofilaria immitis) is based on the compliant administration of Macrocyclic Lactone (ML) drugs. Resistance to ML drugs is well documented in D. immitis; however, there remains a paucity of information on the spatial distribution and prevalence of resistant isolates. This project aims to improve understanding of ML-resistance by using a population genetic approach. We developed a large panel of microsatellite loci and identified 12 novel highly polymorphic markers. These 12, and five previously published markers were used to screen pools of microfilariae from 16 confirmed drug-susceptible, 25 confirmed drug-resistant, and from 10 suspected drug-resistant field isolates. In isolates where microfilarial suppression testing indicated resistance, Spatial Principal Component Analysis (sPCoA), Neighbor Joining Trees and Bayesian clustering all revealed high genetic similarity between pre- and post-treatment samples. Somewhat surprisingly, the Neighbor Joining tree and sPCoA generated using pairwise Nei’s distances did not reveal clustering for resistant isolates, nor did it reveal state-level geographic clustering from samples collected in Georgia, Louisiana or Mississippi. In contrast, Discriminant Analysis of Principle Components was able to discriminate between susceptible, suspected-resistant and resistant samples. However, no resistance-associated markers were detected, and this clustering was driven by the combined effects of multiple alleles across multiple loci. Additionally, we measured unexpectedly large genetic distances between different passages of laboratory strains that originated from the same source infection. This finding strongly suggests that the genetic makeup of laboratory isolates can change substantially with each passage, likely due to genetic bottlenecking. Taken together, these data suggest greater than expected genetic variability in the resistant isolates, and in D. immitis overall. Our results also suggest that microsatellite genotyping lacks the sensitivity to detect a specific genetic signature for resistance. Future investigations using genomic analyses will be required to elucidate the genetic relationships of ML-resistant isolates.
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evaluation of the larval migration inhibition assay for detecting Macrocyclic Lactone resistance in dirofilaria immitis
Veterinary Parasitology, 2017Co-Authors: Christopher C Evans, Andrew R Moorhead, Byron L Blagburn, Adrian J Wolstenholme, Bobby E Storey, Ray M KaplanAbstract:Abstract Anthelmintics of the Macrocyclic Lactone (ML) drug class are widely used as preventives against the canine heartworm ( Dirofilaria immitis ). Over the past several years, however, reports of ML lack of efficacy (LOE) have emerged, in which dogs develop mature heartworm infection despite the administration of monthly prophylactics. More recently, isolates from LOE cases have been used to infect laboratory dogs and the resistant phenotype has been confirmed by the establishment of adult worms in the face of ML treatment at normally preventive dosages. Testing for and monitoring resistance in D. immitis requires a validated biological or molecular diagnostic assay. In this study, we assessed a larval migration inhibition assay (LMIA) that we previously optimized for use with D. immitis third-stage larvae (L 3 ). We used this assay to measure the in vitro ML susceptibilities of a known-susceptible laboratory strain of D. immitis and three highly suspected ML-resistant isolates originating from three separate LOE cases; progeny from two of these isolates have been confirmed ML-resistant by treatment of an infected dog in a controlled setting. A nonlinear regression model was fit to the dose-response data, from which IC 50 values were calculated. The D. immitis LMIA yielded consistent and reproducible dose-response data; however, no statistically significant differences in drug susceptibility were observed between control and LOE parasites. Additionally, the drug concentrations needed to paralyze the L 3 were much higher than those third- and fourth-stage larvae would experience in vivo. IC 50 values ranged from 1.57 to 5.56 μM ( p ≥ 0.19). These data could suggest that ML resistance in this parasite is not mediated through a reduced susceptibility of L 3 to the paralytic effects of ML drugs, and therefore motility-based assays are likely not appropriate for measuring the effects of MLs against D. immitis in this target stage.
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a diagnostic algorithm for evaluating cases of potential Macrocyclic Lactone resistant heartworm
Parasites & Vectors, 2017Co-Authors: Andrew R Moorhead, Christopher C Evans, Ray M KaplanAbstract:The emergence of Macrocyclic Lactone resistance in canine heartworm poses a substantial threat to what is currently the only effective, FDA-approved available method of prevention. Further study of the biotypes is necessary to understand the mechanism of resistance and evaluate novel prevention options. Identifying cases of drug-resistant infection remains problematic, however, especially when poor compliance and insufficient testing are concerns. Furthermore, a definitive demonstration of resistance requires experimental infection and treatment, which is prohibitively costly. With the aim of identifying likely cases of Macrocyclic Lactone-resistant heartworm and preventing their continued spread, we describe an algorithm for determining the likelihood of drug resistance and appropriate treatment strategies for each case. This algorithm relies on the microfilarial suppression test (MFST), which has been used previously as an efficient and discrete measure of suspected resistance. By standardizing this method in a format that is readily available to practitioners, it could become possible to preliminarily survey the emergence and spread of resistance. Heartworm isolates identified through this method can be used in research to better understand Macrocyclic Lactone resistance so prevention strategies can be adapted.
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A diagnostic algorithm for evaluating cases of potential Macrocyclic Lactone–resistant heartworm
Parasites & Vectors, 2017Co-Authors: Andrew R Moorhead, Christopher C Evans, Ray M KaplanAbstract:The emergence of Macrocyclic Lactone resistance in canine heartworm poses a substantial threat to what is currently the only effective, FDA-approved available method of prevention. Further study of the biotypes is necessary to understand the mechanism of resistance and evaluate novel prevention options. Identifying cases of drug-resistant infection remains problematic, however, especially when poor compliance and insufficient testing are concerns. Furthermore, a definitive demonstration of resistance requires experimental infection and treatment, which is prohibitively costly. With the aim of identifying likely cases of Macrocyclic Lactone-resistant heartworm and preventing their continued spread, we describe an algorithm for determining the likelihood of drug resistance and appropriate treatment strategies for each case. This algorithm relies on the microfilarial suppression test (MFST), which has been used previously as an efficient and discrete measure of suspected resistance. By standardizing this method in a format that is readily available to practitioners, it could become possible to preliminarily survey the emergence and spread of resistance. Heartworm isolates identified through this method can be used in research to better understand Macrocyclic Lactone resistance so prevention strategies can be adapted.
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A diagnostic algorithm for evaluating cases of potential Macrocyclic Lactone–resistant heartworm
Parasites & Vectors, 2017Co-Authors: Andrew R Moorhead, Christopher C Evans, Ray M KaplanAbstract:Abstract Background The emergence of Macrocyclic Lactone resistance in canine heartworm poses a substantial threat to what is currently the only effective, FDA-approved available method of prevention. Further study of the biotypes is necessary to understand the mechanism of resistance and evaluate novel prevention options. Identifying cases of drug-resistant infection remains problematic, however, especially when poor compliance and insufficient testing are concerns. Furthermore, a definitive demonstration of resistance requires experimental infection and treatment, which is prohibitively costly. Methods With the aim of identifying likely cases of Macrocyclic Lactone-resistant heartworm and preventing their continued spread, we describe an algorithm for determining the likelihood of drug resistance and appropriate treatment strategies for each case. Results This algorithm relies on the microfilarial suppression test (MFST), which has been used previously as an efficient and discrete measure of suspected resistance. By standardizing this method in a format that is readily available to practitioners, it could become possible to preliminarily survey the emergence and spread of resistance. Conclusion Heartworm isolates identified through this method can be used in research to better understand Macrocyclic Lactone resistance so prevention strategies can be adapted
Adrian J Wolstenholme - One of the best experts on this subject based on the ideXlab platform.
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using population genetics to examine relationships of dirofilaria immitis based on both Macrocyclic Lactone resistance status and geography
Veterinary Parasitology, 2020Co-Authors: Julie Sanchez, Cassan N Pulaski, Adrian J Wolstenholme, Guha Dharmarajan, Melissa M George, John S Gilleard, Ray M KaplanAbstract:Abstract Prevention of infection with canine heartworm (Dirofilaria immitis) is based on the compliant administration of Macrocyclic Lactone (ML) drugs. Resistance to ML drugs is well documented in D. immitis; however, there remains a paucity of information on the spatial distribution and prevalence of resistant isolates. This project aims to improve understanding of ML-resistance by using a population genetic approach. We developed a large panel of microsatellite loci and identified 12 novel highly polymorphic markers. These 12, and five previously published markers were used to screen pools of microfilariae from 16 confirmed drug-susceptible, 25 confirmed drug-resistant, and from 10 suspected drug-resistant field isolates. In isolates where microfilarial suppression testing indicated resistance, Spatial Principal Component Analysis (sPCoA), Neighbor Joining Trees and Bayesian clustering all revealed high genetic similarity between pre- and post-treatment samples. Somewhat surprisingly, the Neighbor Joining tree and sPCoA generated using pairwise Nei’s distances did not reveal clustering for resistant isolates, nor did it reveal state-level geographic clustering from samples collected in Georgia, Louisiana or Mississippi. In contrast, Discriminant Analysis of Principle Components was able to discriminate between susceptible, suspected-resistant and resistant samples. However, no resistance-associated markers were detected, and this clustering was driven by the combined effects of multiple alleles across multiple loci. Additionally, we measured unexpectedly large genetic distances between different passages of laboratory strains that originated from the same source infection. This finding strongly suggests that the genetic makeup of laboratory isolates can change substantially with each passage, likely due to genetic bottlenecking. Taken together, these data suggest greater than expected genetic variability in the resistant isolates, and in D. immitis overall. Our results also suggest that microsatellite genotyping lacks the sensitivity to detect a specific genetic signature for resistance. Future investigations using genomic analyses will be required to elucidate the genetic relationships of ML-resistant isolates.
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Macrocyclic Lactone anthelmintic induced leukocyte binding to dirofilaria immitis microfilariae influence of the drug resistance status of the parasite
International Journal for Parasitology-Drugs and Drug Resistance, 2019Co-Authors: Tessa Berrafato, Ruby Coates, Barbara J Reaves, Daniel Kulke, Adrian J WolstenholmeAbstract:: The Macrocyclic Lactone anthelmintics are the only class of drug currently used to prevent heartworm disease. Their extremely high potency in vivo is not mirrored by their activity against Dirofilaria immitis larvae in vitro, leading to suggestions that they may require host immune functions to kill the parasites. We have previously shown that ivermectin stimulates the binding of canine peripheral blood mononuclear cells (PBMCs) and polymorphonuclear leukocytes (PMNs) to D. immitis microfilariae (Mf). We have now extended these studies to moxidectin and examined the ability of both drugs to stimulate canine PBMC and PMN attachment to Mf from multiple strains of D. immitis, including two that are proven to be resistant to ivermectin in vivo. Both ivermectin and moxidectin significantly increased the percentage of drug-susceptible parasites with cells attached at very low concentrations ( 100 nM) were required to increase the percentage of the two resistant strains, Yazoo-2013 and Metairie-2014, with cells attached. Moxidectin increased the percentage of the two resistant strains with cells attached at lower concentrations (<10 nM) than did ivermectin. The attachment of the PBMCs and PMNs did not result in any parasite killing in vitro. These data support the biological relevance of the drug-stimulated attachment of canine leukocytes to D. immitis Mf and suggest that this phenomenon is related to the drug resistance status of the parasites.
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evaluation of the larval migration inhibition assay for detecting Macrocyclic Lactone resistance in dirofilaria immitis
Veterinary Parasitology, 2017Co-Authors: Christopher C Evans, Andrew R Moorhead, Byron L Blagburn, Adrian J Wolstenholme, Bobby E Storey, Ray M KaplanAbstract:Abstract Anthelmintics of the Macrocyclic Lactone (ML) drug class are widely used as preventives against the canine heartworm ( Dirofilaria immitis ). Over the past several years, however, reports of ML lack of efficacy (LOE) have emerged, in which dogs develop mature heartworm infection despite the administration of monthly prophylactics. More recently, isolates from LOE cases have been used to infect laboratory dogs and the resistant phenotype has been confirmed by the establishment of adult worms in the face of ML treatment at normally preventive dosages. Testing for and monitoring resistance in D. immitis requires a validated biological or molecular diagnostic assay. In this study, we assessed a larval migration inhibition assay (LMIA) that we previously optimized for use with D. immitis third-stage larvae (L 3 ). We used this assay to measure the in vitro ML susceptibilities of a known-susceptible laboratory strain of D. immitis and three highly suspected ML-resistant isolates originating from three separate LOE cases; progeny from two of these isolates have been confirmed ML-resistant by treatment of an infected dog in a controlled setting. A nonlinear regression model was fit to the dose-response data, from which IC 50 values were calculated. The D. immitis LMIA yielded consistent and reproducible dose-response data; however, no statistically significant differences in drug susceptibility were observed between control and LOE parasites. Additionally, the drug concentrations needed to paralyze the L 3 were much higher than those third- and fourth-stage larvae would experience in vivo. IC 50 values ranged from 1.57 to 5.56 μM ( p ≥ 0.19). These data could suggest that ML resistance in this parasite is not mediated through a reduced susceptibility of L 3 to the paralytic effects of ML drugs, and therefore motility-based assays are likely not appropriate for measuring the effects of MLs against D. immitis in this target stage.
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development of an in vitro bioassay for measuring susceptibility to Macrocyclic Lactone anthelmintics in dirofilaria immitis
International Journal for Parasitology-Drugs and Drug Resistance, 2013Co-Authors: Christopher C Evans, Andrew R Moorhead, Adrian J Wolstenholme, Bobby E Storey, Ray M KaplanAbstract:For more than 20 years, anthelmintics of the Macrocyclic Lactone (ML) drug class have been widely and effectively used as preventives against the canine heartworm, Dirofilaria immitis. However, in recent years an increased number of lack of efficacy (LOE) cases are being reported, in which dogs develop mature heartworm infections despite receiving monthly prophylactic doses of ML drugs. While this situation is raising concerns that heartworms may be developing resistance to MLs, compelling evidence for this is still lacking. Resolution of this dilemma requires validated biological or molecular diagnostic assays, but, unfortunately, no such tests currently exist. To address this need, we developed and optimized a larval migration inhibition assay (LMIA) for use with D. immitis third-stage larvae. The LMIA was used to measure the in vitro dose–response of two ML drugs (ivermectin and eprinomectin) on a known ML-susceptible laboratory strain of D. immitis. A nonlinear regression model was fit to the dose–response data, from which IC50 values were calculated; the mean IC50 and 95% confidence interval for IVM was 4.56 μM (1.26–16.4 μM), greater than that for EPR at 2.02 μM (1.68–2.42 μM), and this difference was significant (p = 0.0428). The R2 value for EPR assays (0.90) was also greater than that for IVM treatment (0.71). The consistency and reproducibility of the dose–response data obtained with this assay suggests that it may be a useful technique for investigating the relative susceptibilities to ML drugs in other D. immitis populations.
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nematode ligand gated chloride channels an appraisal of their involvement in Macrocyclic Lactone resistance and prospects for developing molecular markers
Parasitology, 2007Co-Authors: S Mccavera, Tom Walsh, Adrian J WolstenholmeAbstract:Ligand-gated chloride channels, including the glutamate-(GluCl) and GABA-gated channels, are the targets of the Macrocyclic Lactone (ML) family of anthelmintics. Changes in the sequence and expression of these channels can cause resistance to the ML in laboratory models, such as Caenorhabditis elegans and Drosophila melanogaster. Mutations in multiple GluCl subunit genes are required for high-level ML resistance in C. elegans, and this can be influenced by additional mutations in gap junction and amphid genes. Parasitic nematodes have a different complement of channel subunit genes from C. elegans, but a few genes, including avr-14, are widely present. A polymorphism in an avr-14 orthologue, which makes the subunit less sensitive to ivermectin and glutamate, has been identified in Cooperia oncophora, and polymorphisms in several subunits have been reported from resistant isolates of Haemonchus contortus. This has led to suggestions that ML resistance may be polygenic. Possible reasons for this, and its consequences for the development of molecular tests for resistance, are explored.
Mohammed Zougagh - One of the best experts on this subject based on the ideXlab platform.
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validation of a screening method for rapid control of Macrocyclic Lactone mycotoxins in maize flour samples
Analytical and Bioanalytical Chemistry, 2008Co-Authors: Mohammed Zougagh, Helena Tellez, Alberto Sanchez, Manuel Chicharro, Angel RiosAbstract:A procedure for the analytical validation of a rapid supercritical fluid extraction amperometric screening method for controlling Macrocyclic Lactone mycotoxins in maize flour samples has been developed. The limit established by European legislation (0.2 mg kg−1), in reference to zearalenone (ZON) mycotoxin, was taken as the reference threshold to validate the proposed method. Natural ZON metabolites were also included in this study to characterize the final screening method. The objective was the reliable classification of samples as positive or negative samples. The cut-off level was fixed at a global concentration of mycotoxins of 0.17 mg kg−1. An expanded unreliability zone between 0.16 and 0.23 mg kg−1 characterized the screening method for classifying the samples. A set of 30 samples was used for the final demonstration of the reliability and usefulness of the method.
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supercritical fluid extraction of Macrocyclic Lactone mycotoxins in maize flour samples for rapid amperometric screening and alternative liquid chromatographic method for confirmation
Journal of Chromatography A, 2008Co-Authors: Mohammed Zougagh, Angel RiosAbstract:Abstract A rapid and simple method for the direct screening of Macrocyclic Lactone mycotoxins (zearalenone, ZON; α-zearalenol, α-ZOL; and β-zearalenol, β-ZOL) in maize flour samples is proposed. The sample screening method comprises supercritical fluid extraction (SFE) and clean-up on Florisil adsorption cartridge of the selected toxic compounds, followed by continuous flow electrochemical detection. Those samples for which the total concentration is close to or above the threshold limit established by legislation (0.200 mg kg−1) are subjected to preconcentration on C18 chromatographic material and liquid chromatographic separation for confirmation purposes. This confirmation method allows the determination of ZON, α-ZOL and β-ZOL in the range between 30 and 300 μg kg−1, with a average relative standard deviation lower than 5.2 in all cases.
