An isolate of Haemonchus contortus, UGA/2004, highly resistant to benzimidazoles, levamisole, and... more An isolate of Haemonchus contortus, UGA/2004, highly resistant to benzimidazoles, levamisole, and ivermectin was isolated from sheep at the University of Georgia, and passaged through experimentally infected goats. We measured the expression of twenty-nine mRNAs encoding drug targets and P-glycoproteins (P-gps), comparing the results to a fully susceptible laboratory passaged isolate. Expression levels of some nicotinic acetylcholine receptor mRNAs were markedly different in UGA/2004. Levels of the Hco-acr-8b mRNA, encoding a truncated subunit, were very high in resistant L3, but undetectable in susceptible larvae, with expression of the full-length Hco-acr-8a mRNA also significant increased. Expression of Hco-unc-63 and Hco-unc-29.3 mRNAs was significantly reduced in the resistant larvae. Expression of the Hco-glc-3 and Hco-glc-5 mRNAs, encoding glutamate-gated chloride channel subunits, were slightly reduced in resistant larvae. We observed significant increases in the expression of the Hco-pgp-2 and Hcopgp-9 mRNAs in the UGA/2004 larvae, consistent with previous reports; we also saw a decrease in the levels of Hco-pgp-1 mRNA. Treatment of the larvae with ivermectin and moxidectin in vitro produced variable and inconsistent changes in P-gp mRNA levels. The sequences of the -tubulin isotype 1 mRNAs showed that the resistant larvae had a resistance-associated allele frequency of >95% at codon 200 and ∼40% and codon 167. No changes at codon 198 were present. The presence of the truncated acr-8b mRNA may be a reliable indicator of levamisole resistance, but complex changes in gene expression associated with macrocyclic lactone resistance make the identification of a single genetic marker for this resistance difficult.
International Journal for Parasitology: Drugs and Drug Resistance, 2013
For more than 20 years, anthelmintics of the macrocyclic lactone (ML) drug class have been widely... more 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 MLsusceptible laboratory strain of D. immitis. A nonlinear regression model was fit to the dose-response data, from which IC 50 values were calculated; the mean IC 50 and 95% confidence interval for IVM was 4.56 lM (1.26-16.4 lM), greater than that for EPR at 2.02 lM (1.68-2.42 lM), and this difference was significant (p = 0.0428). The R 2 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.
Gastrointestinal nematodes of the family Trichostrongylidae are the most important parasites of s... more Gastrointestinal nematodes of the family Trichostrongylidae are the most important parasites of sheep, goats and other ruminants worldwide. Of this group, Haemonchus contortus is usually the most damaging species, particularly in warmer climates or during the summer. It is therefore useful to be able to rapidly differentiate infections with this nematode from other, less pathogenic, species. However, aside from Nematodirus spp., there are only subtle differences between the egg morphology within the trichostrongyles, making it very difficult to identify eggs to the species level. It has been shown previously that peanut agglutinin specifically binds to Haemonchus eggs and not those of other trichostrongyle species. By using this lectin conjugated to fluorescein isothiocyanate, binding to Haemonchus eggs can be visualized under ultraviolet illumination. We adapted this test using eggs purified by routine sugar centrifugation methods and evaluated 26 diagnostic samples from ruminants ...
International journal for parasitology. Drugs and drug resistance, 2014
A major hindrance to evaluating nematode populations for anthelmintic resistance, as well as for ... more A major hindrance to evaluating nematode populations for anthelmintic resistance, as well as for screening existing drugs, new compounds, or bioactive plant extracts for anthelmintic properties, is the lack of an efficient, objective, and reproducible in vitro assay that is adaptable to multiple life stages and parasite genera. To address this need we have developed the "Worminator" system, which objectively and quantitatively measures the motility of microscopic stages of parasitic nematodes. The system is built around the computer application "WormAssay", developed at the Center for Discovery and Innovation in Parasitic Diseases at the University of California, San Francisco. WormAssay was designed to assess motility of macroscopic parasites for the purpose of high throughput screening of potential anthelmintic compounds, utilizing high definition video as an input to assess motion of adult stage (macroscopic) parasites (e.g. Brugia malayi). We adapted this ass...
International Journal For Parasitology, Aug 31, 2008
Cyathostomins are recognised as the primary parasitic pathogens of horses. Despite the use of ben... more Cyathostomins are recognised as the primary parasitic pathogens of horses. Despite the use of benzimidazole (BZ) anthelmintics in horses for more than 40 years and widespread drug resistance in the field, the mechanisms of resistance to this drug class in cyathostomins are not fully understood. The results presented here constitute a detailed comparison of b tubulin gene mutations and mRNA transcript levels in populations of BZ-susceptible (BZ-S) and -resistant (BZ-R) cyathostomins. Full-length cDNA sequences were generated from individual parasites of four (n = 24) and two (n = 19) cyathostomin species for isotypes 1 and 2, respectively. Levels of intra-and inter-specific nucleotide sequence variation were comparable with previous findings and single amino acid substitutions were observed at several locations. On comparison of BZ-S and BZ-R parasites, differences were consistently observed at only two sites, codons 167 and 200 of the b tubulin isotype 1 gene. Four populations of parasites were genotyped at these two loci by pyrosequencing; one that was fenbendazole (FBZ)-sensitive (FBZ-rS), two that were FBZ-resistant (FBZ-R1 and -R2) and one that was oxibendazole-resistant (OBZ-R), as previously assessed by faecal egg count reduction tests. This analysis revealed statistically significant differences between FBZ-rS and FBZ-R populations at both loci and this was highly significant for codon 167. For the OBZ-R population, the only significant difference compared with the FBZ-rS population was observed at codon 200. These observations suggest that mutations at codons 167 and 200 are important in BZ resistance and raise the possibility that selection at different loci may occur in FBZ-and OBZ-resistant parasites. Multiple parasites (n = 158) were genotyped for both codons 167 and 200, the majority of which showed homozygous 'resistant' mutations at one locus only and none showed homozygous 'resistant' genotypes at both loci. No significant differences in mRNA levels of b tubulin isotypes 1 and 2 were observed between the FBZ-rS and FBZ-R1 populations. Ó
SUMMARY Resistance to anthelmintics in gastrointestinal nematodes of livestock is a serious probl... more SUMMARY Resistance to anthelmintics in gastrointestinal nematodes of livestock is a serious problem and appropriate methods are required to identify and quantify resistance. However, quantification and assessment of resistance depend on an accurate measure of treatment efficacy, and current methodologies fail to properly address the issue. The fecal egg count reduction test (FECRT) is the practical gold standard for measuring
Four species of artiodactylids (scimitar-horned oryx [Oryx dama]), roan antelope [Hippotragus equ... more Four species of artiodactylids (scimitar-horned oryx [Oryx dama]), roan antelope [Hippotragus equinus], blackbuck [Antilope cervicapra]), and blesbok [Damaliscus pygargus phillipsi]) totaling 13 animals were treated with a one-time 12.5-g dose of copper oxide wire particles (COWPs) in a bolus form. Pretreatment, individual trichostrongyle fecal egg counts (FECs) were performed using the McMaster technique. Individual posttreatment FECs were performed every 7 days for 35 days beginning 7 days after bolus administration, and FEC reduction ratios (FECRRs) expressed as percentage reductions from pretreatment values were calculated every 7 days. Mean FECRRs for the 13 animals were 93% +/- 16%, 98% +/- 7%, 91% +/- 28%, 94% +/- 16%, and 90% +/- 13% at 7, 14, 21, 28, and 35 days posttreatment, respectively. These data demonstrate that COWPs in a bolus form were an effective method for reducing FEC in exotic artiodactylids. Based on this limited data, COWPs show promise as an anthelmintic alternative for exotic artiodactylids in zoologic collections.
Gastrointestinal nematode parasites are the single most important health problem of sheep and goa... more Gastrointestinal nematode parasites are the single most important health problem of sheep and goats. Traditionally, parasites have been controlled by frequent administration of anthelmintic drugs. However, the emergence of multiple-drug-resistant parasites now threatens this paradigm of control and new approaches are required. Anthelmintics can no longer be thought of as a convenient and inexpensive management tool for maximizing animal productivity. Instead, anthelmintics must be thought of as extremely valuable and limited resources that should be used prudently. In response to this changing paradigm of anthelmintic use, new recommendations for parasite control now exist. A key tenet of this approach, referred to as "Smart Drenching", is to use the best available knowledge to develop strategies that maximize the effectiveness of treatments while also decreasing the development of drug resistance. Additionally, new innovative schemes using novel and sustainable approaches must be implemented. An overall strategy that integrates these approaches is referred to as "sustainable integrated parasite management" (sIPM). However, by its very nature sIPM is much more complicated and difficult to implement than casual administration of anthelmintics. Consequently, successful implementation of sIPM will only be possible with the help and active involvement of veterinarians and other animal health professionals. Because multiple-drug resistance in parasites is now the status quo, and development of further drug resistance is almost certain to outpace the development of new anthelmintics, it is critical that this new philosophy and approach to parasite control be embraced and implemented immediately.
Journal of the American Veterinary Medical Association, Aug 15, 2003
To determine prevalence of resistance to all anthelmintics that are commonly used to treat gastro... more To determine prevalence of resistance to all anthelmintics that are commonly used to treat gastrointestinal nematodes (GINs) in goats. Prospective study. 777 goats. On each farm, goats were assigned to 1 of 5 treatment groups: untreated controls, albendazole (20 mg/kg [9.0 mg/lb], p.o., once), ivermectin (0.4 mg/kg [0.18 mg/lb], p.o., once), levamisole (12 mg/kg [5.4 mg/lb], p.o., once), or moxidectin (0.4 mg/kg, p.o., once), except on 3 farms where albendazole was omitted. Fecal samples were collected 2 weeks after treatment for determination of fecal egg counts (FECs), and percentage reductions were calculated by comparing data from anthelmintic-treated and control groups. Nematode populations were categorized as susceptible, suspected resistant, or resistant by use of guidelines published by the World Association for the Advancement of Veterinary Parasitology. Resistance to albendazole was found on 14 of 15 farms, and resistance to ivermectin, levamisole, and moxidectin was found on 17, 6, and 1 of 18 farms, respectively. Suspected resistance to levamisole and moxidectin was found on 4 and 3 farms, respectively. Resistance to multiple anthelmintics (albendazole and ivermectin) was found on 14 of 15 farms and to albendazole, ivermectin, and levamisole on 5 of 15 farms. Mean overall FEC reduction percentages for albendazole, ivermectin, levamisole, and moxidectin were 67, 54, 94, and 99%, respectively. Anthelmintic resistance in GINs of goats is highly prevalent in the southern United States. The high prevalence of resistance to multiple anthelmintics emphasizes the need for reexamination of nematode control practices.
International Journal For Parasitology, Mar 31, 2011
Transient transfection of isolated Brugia malayi embryos by biolistics has proven to be useful in... more Transient transfection of isolated Brugia malayi embryos by biolistics has proven to be useful in defining promoter structure and function in this parasite. However, isolated transfected embryos are developmentally incompetent. A method of producing developmentally competent transfected parasites is therefore needed. We report that L3 parasites can be chemically transfected in situ in the peritoneal cavity of a gerbil with a construct consisting of a secreted luciferase reporter gene containing a promoter, the 3' untranslated region and first intron derived from the B. malayi 70kDa heat shock protein gene. The in situ chemically transfected parasites are developmentally competent, producing adult parasites with an efficiency similar to that obtained from implanted untreated L3s. Cultured adult parasites and progeny microfilariae (mf) derived from L3s transfected with this construct secreted luciferase into the culture medium. When the transfected mf were fed to mosquitoes and the resulting L3s collected, the L3s also secreted luciferase into the culture medium. Progeny mf from transgenic adult parasites contained transgenic DNA, and the transgenic mRNA produced in these parasites was found to be correctly cis-and trans-spliced. In situ chemical transformation thus results in developmentally competent transfected B. malayi in which the transgenic sequences remain transcriptionally active in all life cycle stages and are present in the subsequent generation.
Frequent and broad application of anthelmintic drugs for treatment of intestinal parasite infecti... more Frequent and broad application of anthelmintic drugs for treatment of intestinal parasite infection has led to drug resistance that often renders whole populations of livestock unresponsive to treatment. Therefore, it is important to detect mutations associated with drug resistance before it becomes clinically manifest. To monitor developing drug resistance against benzimidazoles (BZ), we developed real-time PCR assays and applied them to analyse the beta-tubulin isotype-1 gene of the hookworm Ancylostoma caninum, an important parasite of dogs. Previously, we developed PCR assays to monitor codon positions 167 and 200. Here, we describe an assay which is able to detect resistance alleles in codon 198. These realtime PCR assays were subsequently applied to screen hookworm specimens recovered from dogs in Georgia. No elevated levels of polymorphisms at the investigated loci were found, suggesting that selection for resistance in the tested samples did not occur.
The number of South American camelid (SAC; llama and alpaca) farms is growing in the southeastern... more The number of South American camelid (SAC; llama and alpaca) farms is growing in the southeastern United States, and infection with gastrointestinal nematodes (GIN) is a major health concern in this region. There is widespread resistance to anthelmintic remedies in small ruminants (sheep and goats), but a paucity of information on llamas and alpacas. Anthelmintic resistance was evaluated on three SAC farms (two llama; one alpaca) in Georgia in the southern United States using fecal egg count reduction (FECR) tests. For each farm, animals were randomly assigned to 1 of 5 treatment groups based on initial fecal egg count (FEC) and number of animals available (2-5 groups, n=9-11 per treatment). Ivermectin (IVM, subcutaneous injection; 0.3mg/kg body weight (BW)) and a control group were tested on an alpaca farm, and fenbendazole (FBZ, oral; 10mg/kg BW; two farms), moxidectin (MOX oral; 0.2mg/kg BW; two farms), and levamisole (LEV, oral; 8 mg/kg BW; one farm) were added for the llama farms. Anthelmintic efficacy was determined by comparing FEC of treatment and control animals 14 days post-treatment, with resistance evaluated using the World Association for the Advancement of Veterinary Parasitology (WAAVP) guidelines. Based upon these guidelines, there was GIN resistance to IVM in both llamas and alpacas in Georgia and to FBZ on both llama farms where this drug was tested. There was MOX resistance on one llama farm using the FECR test, while there was no resistance to LEV detected in this study. These data demonstrate a serious emerging problem in the United States of llama and alpaca GIN resistant to drugs from two of the three major anthelmintic classes.
Gastrointestinal nematode (GIN) parasites are the single most important health problem of sheep a... more Gastrointestinal nematode (GIN) parasites are the single most important health problem of sheep and goats. Traditionally, parasites have been controlled by frequent administration of anthelmintic drugs. However, the emergence of multiple-drug resistant parasites now threatens this paradigm of control and new approaches are required. Anthelmintics can no longer be thought of as an inexpensive management tool to be used as
Cyathostomins are the most prevalent parasitic pathogens of equids worldwide. These nematodes hav... more Cyathostomins are the most prevalent parasitic pathogens of equids worldwide. These nematodes have been controlled using broad-spectrum anthelmintics; however, cyathostomin resistance to each anthelmintic class has been reported and populations insensitive to more than one class are relatively commonplace. The faecal egg count reduction test (FECRT) is considered the most suitable method for screening anthelmintic sensitivity in horses, but is subject to variation and is relatively time-consuming to perform. Here, we describe a larval migration inhibition test (LMIT) to assess ivermectin (IVM) sensitivity in cyathostomin populations. This test measures the paralysing effect of IVM on the ability of third stage larvae (L3) to migrate through a pore mesh. When L3 from a single faecal sample were examined on multiple occasions, variation in migration was observed: this was associated with the length of time that the L3 had been stored before testing but the association was not significant. Half maximal effective concentration (EC50) values were then obtained for cyathostomin L3 from six populations of horses or donkeys that showed varying sensitivity to IVM in previous FECRTs. Larvae from populations indicated as IVM resistant by FECRT displayed significantly higher EC50 values in the LMIT than L3 from populations classified as IVM sensitive or L3 from populations that had not been previously exposed to IVM or had limited prior exposure. The analysis also showed that EC50 values obtained using L3 from animals in which IVM faecal egg count reduction (FECR) levels had been recorded as <95% were significantly higher than EC50 values obtained using L3 from animals for which FECR was measured as >95%. For one of the populations, time that had elapsed since IVM administration had an effect on the EC50 value obtained, with a longer time since treatment associated with lower EC50 values. These results indicate that the LMIT has value in discriminating IVM sensitivity amongst cyathostomin populations, but several factors were identified that need to be taken into account when executing the test and interpreting the derived data.
An isolate of Haemonchus contortus, UGA/2004, highly resistant to benzimidazoles, levamisole, and... more An isolate of Haemonchus contortus, UGA/2004, highly resistant to benzimidazoles, levamisole, and ivermectin was isolated from sheep at the University of Georgia, and passaged through experimentally infected goats. We measured the expression of twenty-nine mRNAs encoding drug targets and P-glycoproteins (P-gps), comparing the results to a fully susceptible laboratory passaged isolate. Expression levels of some nicotinic acetylcholine receptor mRNAs were markedly different in UGA/2004. Levels of the Hco-acr-8b mRNA, encoding a truncated subunit, were very high in resistant L3, but undetectable in susceptible larvae, with expression of the full-length Hco-acr-8a mRNA also significant increased. Expression of Hco-unc-63 and Hco-unc-29.3 mRNAs was significantly reduced in the resistant larvae. Expression of the Hco-glc-3 and Hco-glc-5 mRNAs, encoding glutamate-gated chloride channel subunits, were slightly reduced in resistant larvae. We observed significant increases in the expression of the Hco-pgp-2 and Hcopgp-9 mRNAs in the UGA/2004 larvae, consistent with previous reports; we also saw a decrease in the levels of Hco-pgp-1 mRNA. Treatment of the larvae with ivermectin and moxidectin in vitro produced variable and inconsistent changes in P-gp mRNA levels. The sequences of the -tubulin isotype 1 mRNAs showed that the resistant larvae had a resistance-associated allele frequency of >95% at codon 200 and ∼40% and codon 167. No changes at codon 198 were present. The presence of the truncated acr-8b mRNA may be a reliable indicator of levamisole resistance, but complex changes in gene expression associated with macrocyclic lactone resistance make the identification of a single genetic marker for this resistance difficult.
International Journal for Parasitology: Drugs and Drug Resistance, 2013
For more than 20 years, anthelmintics of the macrocyclic lactone (ML) drug class have been widely... more 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 MLsusceptible laboratory strain of D. immitis. A nonlinear regression model was fit to the dose-response data, from which IC 50 values were calculated; the mean IC 50 and 95% confidence interval for IVM was 4.56 lM (1.26-16.4 lM), greater than that for EPR at 2.02 lM (1.68-2.42 lM), and this difference was significant (p = 0.0428). The R 2 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.
Gastrointestinal nematodes of the family Trichostrongylidae are the most important parasites of s... more Gastrointestinal nematodes of the family Trichostrongylidae are the most important parasites of sheep, goats and other ruminants worldwide. Of this group, Haemonchus contortus is usually the most damaging species, particularly in warmer climates or during the summer. It is therefore useful to be able to rapidly differentiate infections with this nematode from other, less pathogenic, species. However, aside from Nematodirus spp., there are only subtle differences between the egg morphology within the trichostrongyles, making it very difficult to identify eggs to the species level. It has been shown previously that peanut agglutinin specifically binds to Haemonchus eggs and not those of other trichostrongyle species. By using this lectin conjugated to fluorescein isothiocyanate, binding to Haemonchus eggs can be visualized under ultraviolet illumination. We adapted this test using eggs purified by routine sugar centrifugation methods and evaluated 26 diagnostic samples from ruminants ...
International journal for parasitology. Drugs and drug resistance, 2014
A major hindrance to evaluating nematode populations for anthelmintic resistance, as well as for ... more A major hindrance to evaluating nematode populations for anthelmintic resistance, as well as for screening existing drugs, new compounds, or bioactive plant extracts for anthelmintic properties, is the lack of an efficient, objective, and reproducible in vitro assay that is adaptable to multiple life stages and parasite genera. To address this need we have developed the "Worminator" system, which objectively and quantitatively measures the motility of microscopic stages of parasitic nematodes. The system is built around the computer application "WormAssay", developed at the Center for Discovery and Innovation in Parasitic Diseases at the University of California, San Francisco. WormAssay was designed to assess motility of macroscopic parasites for the purpose of high throughput screening of potential anthelmintic compounds, utilizing high definition video as an input to assess motion of adult stage (macroscopic) parasites (e.g. Brugia malayi). We adapted this ass...
International Journal For Parasitology, Aug 31, 2008
Cyathostomins are recognised as the primary parasitic pathogens of horses. Despite the use of ben... more Cyathostomins are recognised as the primary parasitic pathogens of horses. Despite the use of benzimidazole (BZ) anthelmintics in horses for more than 40 years and widespread drug resistance in the field, the mechanisms of resistance to this drug class in cyathostomins are not fully understood. The results presented here constitute a detailed comparison of b tubulin gene mutations and mRNA transcript levels in populations of BZ-susceptible (BZ-S) and -resistant (BZ-R) cyathostomins. Full-length cDNA sequences were generated from individual parasites of four (n = 24) and two (n = 19) cyathostomin species for isotypes 1 and 2, respectively. Levels of intra-and inter-specific nucleotide sequence variation were comparable with previous findings and single amino acid substitutions were observed at several locations. On comparison of BZ-S and BZ-R parasites, differences were consistently observed at only two sites, codons 167 and 200 of the b tubulin isotype 1 gene. Four populations of parasites were genotyped at these two loci by pyrosequencing; one that was fenbendazole (FBZ)-sensitive (FBZ-rS), two that were FBZ-resistant (FBZ-R1 and -R2) and one that was oxibendazole-resistant (OBZ-R), as previously assessed by faecal egg count reduction tests. This analysis revealed statistically significant differences between FBZ-rS and FBZ-R populations at both loci and this was highly significant for codon 167. For the OBZ-R population, the only significant difference compared with the FBZ-rS population was observed at codon 200. These observations suggest that mutations at codons 167 and 200 are important in BZ resistance and raise the possibility that selection at different loci may occur in FBZ-and OBZ-resistant parasites. Multiple parasites (n = 158) were genotyped for both codons 167 and 200, the majority of which showed homozygous 'resistant' mutations at one locus only and none showed homozygous 'resistant' genotypes at both loci. No significant differences in mRNA levels of b tubulin isotypes 1 and 2 were observed between the FBZ-rS and FBZ-R1 populations. Ó
SUMMARY Resistance to anthelmintics in gastrointestinal nematodes of livestock is a serious probl... more SUMMARY Resistance to anthelmintics in gastrointestinal nematodes of livestock is a serious problem and appropriate methods are required to identify and quantify resistance. However, quantification and assessment of resistance depend on an accurate measure of treatment efficacy, and current methodologies fail to properly address the issue. The fecal egg count reduction test (FECRT) is the practical gold standard for measuring
Four species of artiodactylids (scimitar-horned oryx [Oryx dama]), roan antelope [Hippotragus equ... more Four species of artiodactylids (scimitar-horned oryx [Oryx dama]), roan antelope [Hippotragus equinus], blackbuck [Antilope cervicapra]), and blesbok [Damaliscus pygargus phillipsi]) totaling 13 animals were treated with a one-time 12.5-g dose of copper oxide wire particles (COWPs) in a bolus form. Pretreatment, individual trichostrongyle fecal egg counts (FECs) were performed using the McMaster technique. Individual posttreatment FECs were performed every 7 days for 35 days beginning 7 days after bolus administration, and FEC reduction ratios (FECRRs) expressed as percentage reductions from pretreatment values were calculated every 7 days. Mean FECRRs for the 13 animals were 93% +/- 16%, 98% +/- 7%, 91% +/- 28%, 94% +/- 16%, and 90% +/- 13% at 7, 14, 21, 28, and 35 days posttreatment, respectively. These data demonstrate that COWPs in a bolus form were an effective method for reducing FEC in exotic artiodactylids. Based on this limited data, COWPs show promise as an anthelmintic alternative for exotic artiodactylids in zoologic collections.
Gastrointestinal nematode parasites are the single most important health problem of sheep and goa... more Gastrointestinal nematode parasites are the single most important health problem of sheep and goats. Traditionally, parasites have been controlled by frequent administration of anthelmintic drugs. However, the emergence of multiple-drug-resistant parasites now threatens this paradigm of control and new approaches are required. Anthelmintics can no longer be thought of as a convenient and inexpensive management tool for maximizing animal productivity. Instead, anthelmintics must be thought of as extremely valuable and limited resources that should be used prudently. In response to this changing paradigm of anthelmintic use, new recommendations for parasite control now exist. A key tenet of this approach, referred to as "Smart Drenching", is to use the best available knowledge to develop strategies that maximize the effectiveness of treatments while also decreasing the development of drug resistance. Additionally, new innovative schemes using novel and sustainable approaches must be implemented. An overall strategy that integrates these approaches is referred to as "sustainable integrated parasite management" (sIPM). However, by its very nature sIPM is much more complicated and difficult to implement than casual administration of anthelmintics. Consequently, successful implementation of sIPM will only be possible with the help and active involvement of veterinarians and other animal health professionals. Because multiple-drug resistance in parasites is now the status quo, and development of further drug resistance is almost certain to outpace the development of new anthelmintics, it is critical that this new philosophy and approach to parasite control be embraced and implemented immediately.
Journal of the American Veterinary Medical Association, Aug 15, 2003
To determine prevalence of resistance to all anthelmintics that are commonly used to treat gastro... more To determine prevalence of resistance to all anthelmintics that are commonly used to treat gastrointestinal nematodes (GINs) in goats. Prospective study. 777 goats. On each farm, goats were assigned to 1 of 5 treatment groups: untreated controls, albendazole (20 mg/kg [9.0 mg/lb], p.o., once), ivermectin (0.4 mg/kg [0.18 mg/lb], p.o., once), levamisole (12 mg/kg [5.4 mg/lb], p.o., once), or moxidectin (0.4 mg/kg, p.o., once), except on 3 farms where albendazole was omitted. Fecal samples were collected 2 weeks after treatment for determination of fecal egg counts (FECs), and percentage reductions were calculated by comparing data from anthelmintic-treated and control groups. Nematode populations were categorized as susceptible, suspected resistant, or resistant by use of guidelines published by the World Association for the Advancement of Veterinary Parasitology. Resistance to albendazole was found on 14 of 15 farms, and resistance to ivermectin, levamisole, and moxidectin was found on 17, 6, and 1 of 18 farms, respectively. Suspected resistance to levamisole and moxidectin was found on 4 and 3 farms, respectively. Resistance to multiple anthelmintics (albendazole and ivermectin) was found on 14 of 15 farms and to albendazole, ivermectin, and levamisole on 5 of 15 farms. Mean overall FEC reduction percentages for albendazole, ivermectin, levamisole, and moxidectin were 67, 54, 94, and 99%, respectively. Anthelmintic resistance in GINs of goats is highly prevalent in the southern United States. The high prevalence of resistance to multiple anthelmintics emphasizes the need for reexamination of nematode control practices.
International Journal For Parasitology, Mar 31, 2011
Transient transfection of isolated Brugia malayi embryos by biolistics has proven to be useful in... more Transient transfection of isolated Brugia malayi embryos by biolistics has proven to be useful in defining promoter structure and function in this parasite. However, isolated transfected embryos are developmentally incompetent. A method of producing developmentally competent transfected parasites is therefore needed. We report that L3 parasites can be chemically transfected in situ in the peritoneal cavity of a gerbil with a construct consisting of a secreted luciferase reporter gene containing a promoter, the 3' untranslated region and first intron derived from the B. malayi 70kDa heat shock protein gene. The in situ chemically transfected parasites are developmentally competent, producing adult parasites with an efficiency similar to that obtained from implanted untreated L3s. Cultured adult parasites and progeny microfilariae (mf) derived from L3s transfected with this construct secreted luciferase into the culture medium. When the transfected mf were fed to mosquitoes and the resulting L3s collected, the L3s also secreted luciferase into the culture medium. Progeny mf from transgenic adult parasites contained transgenic DNA, and the transgenic mRNA produced in these parasites was found to be correctly cis-and trans-spliced. In situ chemical transformation thus results in developmentally competent transfected B. malayi in which the transgenic sequences remain transcriptionally active in all life cycle stages and are present in the subsequent generation.
Frequent and broad application of anthelmintic drugs for treatment of intestinal parasite infecti... more Frequent and broad application of anthelmintic drugs for treatment of intestinal parasite infection has led to drug resistance that often renders whole populations of livestock unresponsive to treatment. Therefore, it is important to detect mutations associated with drug resistance before it becomes clinically manifest. To monitor developing drug resistance against benzimidazoles (BZ), we developed real-time PCR assays and applied them to analyse the beta-tubulin isotype-1 gene of the hookworm Ancylostoma caninum, an important parasite of dogs. Previously, we developed PCR assays to monitor codon positions 167 and 200. Here, we describe an assay which is able to detect resistance alleles in codon 198. These realtime PCR assays were subsequently applied to screen hookworm specimens recovered from dogs in Georgia. No elevated levels of polymorphisms at the investigated loci were found, suggesting that selection for resistance in the tested samples did not occur.
The number of South American camelid (SAC; llama and alpaca) farms is growing in the southeastern... more The number of South American camelid (SAC; llama and alpaca) farms is growing in the southeastern United States, and infection with gastrointestinal nematodes (GIN) is a major health concern in this region. There is widespread resistance to anthelmintic remedies in small ruminants (sheep and goats), but a paucity of information on llamas and alpacas. Anthelmintic resistance was evaluated on three SAC farms (two llama; one alpaca) in Georgia in the southern United States using fecal egg count reduction (FECR) tests. For each farm, animals were randomly assigned to 1 of 5 treatment groups based on initial fecal egg count (FEC) and number of animals available (2-5 groups, n=9-11 per treatment). Ivermectin (IVM, subcutaneous injection; 0.3mg/kg body weight (BW)) and a control group were tested on an alpaca farm, and fenbendazole (FBZ, oral; 10mg/kg BW; two farms), moxidectin (MOX oral; 0.2mg/kg BW; two farms), and levamisole (LEV, oral; 8 mg/kg BW; one farm) were added for the llama farms. Anthelmintic efficacy was determined by comparing FEC of treatment and control animals 14 days post-treatment, with resistance evaluated using the World Association for the Advancement of Veterinary Parasitology (WAAVP) guidelines. Based upon these guidelines, there was GIN resistance to IVM in both llamas and alpacas in Georgia and to FBZ on both llama farms where this drug was tested. There was MOX resistance on one llama farm using the FECR test, while there was no resistance to LEV detected in this study. These data demonstrate a serious emerging problem in the United States of llama and alpaca GIN resistant to drugs from two of the three major anthelmintic classes.
Gastrointestinal nematode (GIN) parasites are the single most important health problem of sheep a... more Gastrointestinal nematode (GIN) parasites are the single most important health problem of sheep and goats. Traditionally, parasites have been controlled by frequent administration of anthelmintic drugs. However, the emergence of multiple-drug resistant parasites now threatens this paradigm of control and new approaches are required. Anthelmintics can no longer be thought of as an inexpensive management tool to be used as
Cyathostomins are the most prevalent parasitic pathogens of equids worldwide. These nematodes hav... more Cyathostomins are the most prevalent parasitic pathogens of equids worldwide. These nematodes have been controlled using broad-spectrum anthelmintics; however, cyathostomin resistance to each anthelmintic class has been reported and populations insensitive to more than one class are relatively commonplace. The faecal egg count reduction test (FECRT) is considered the most suitable method for screening anthelmintic sensitivity in horses, but is subject to variation and is relatively time-consuming to perform. Here, we describe a larval migration inhibition test (LMIT) to assess ivermectin (IVM) sensitivity in cyathostomin populations. This test measures the paralysing effect of IVM on the ability of third stage larvae (L3) to migrate through a pore mesh. When L3 from a single faecal sample were examined on multiple occasions, variation in migration was observed: this was associated with the length of time that the L3 had been stored before testing but the association was not significant. Half maximal effective concentration (EC50) values were then obtained for cyathostomin L3 from six populations of horses or donkeys that showed varying sensitivity to IVM in previous FECRTs. Larvae from populations indicated as IVM resistant by FECRT displayed significantly higher EC50 values in the LMIT than L3 from populations classified as IVM sensitive or L3 from populations that had not been previously exposed to IVM or had limited prior exposure. The analysis also showed that EC50 values obtained using L3 from animals in which IVM faecal egg count reduction (FECR) levels had been recorded as <95% were significantly higher than EC50 values obtained using L3 from animals for which FECR was measured as >95%. For one of the populations, time that had elapsed since IVM administration had an effect on the EC50 value obtained, with a longer time since treatment associated with lower EC50 values. These results indicate that the LMIT has value in discriminating IVM sensitivity amongst cyathostomin populations, but several factors were identified that need to be taken into account when executing the test and interpreting the derived data.
A major hindrance to evaluating nematode populations for anthelmintic resistance, as well as for ... more A major hindrance to evaluating nematode populations for anthelmintic resistance, as well as for screening existing drugs, new compounds, or bioactive plant extracts for anthelmintic properties, is the lack of an efficient, objective, and reproducible in vitro assay that is adaptable to multiple life stages and parasite genera. To address this need we have developed the ''Worminator'' system, which objectively and quantitatively measures the motility of microscopic stages of parasitic nematodes. The system is built around the computer application ''WormAssay'', developed at the Center for Discovery and Innovation in Parasitic Diseases at the University of California, San Francisco. WormAssay was designed to assess motility of macroscopic parasites for the purpose of high throughput screening of potential anthelmintic compounds , utilizing high definition video as an input to assess motion of adult stage (macroscopic) parasites (e.g. Brugia malayi). We adapted this assay for use with microscopic parasites by modifying the software to support a full frame analysis mode that applies the motion algorithm to the entire video frame. Thus, the motility of all parasites in a given well are recorded and measured simultaneously. Assays performed on third-stage larvae (L3) of the bovine intestinal nematode Cooperia spp., as well as microfilariae (mf) of the filarioid nematodes B. malayi and Dirofilaria immitis, yielded reproducible dose responses using the macrocyclic lactones ivermectin, doramectin, and moxidectin, as well as the nicotinic agonists, pyrantel, oxantel, morantel, and tribendimidine. This new computer based-assay is simple to use, requires minimal new investment in equipment, is robust across nematode genera and developmental stage, and does not require subjective scoring of motility by an observer. Thus, the ''Worminator'' provides a relatively low-cost platform for developing genera-and stage-specific assays with high efficiency and reproducibility, low labor input, and yields objective motility data that is not subject to scorer bias.
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Papers by Ray Kaplan