Validation of a microwave-assisted micella rextraction method for the oxibendazole determination in soil samples

Oxibendazole is a veterinary drug used as an antihelmintic for companion and farm animals. This drug may enterin to the environment directly, via excretion of faeces and urine by grazing animals, or indirectly, when manure of treated farm animals are used as soil organic amendment. In this work, a simple, fast, reliable and sensitive method based on microwave-assisted micellar extraction (MAME) procedure, using Genapol X-080 (0.5%) as surfactant, has been developed for the quantification of oxibendazole in soil samples. Two classical methods, based on mechanical shaking and ultrasonic assisted extraction followed by solid phase extraction, were compared with the MAME procedure. Oxibendazole was determined by high-performance liquid chromatography (HPLC) with fluorescence detector. In the optimization of the MAME procedure were evaluated four parameters: surfactant volume, stirring, irradiation power and extraction time. Optimum conditionswere 20 ml of Genapol X-080 (0.5%), irradiation power of1000 W, extraction time of 2 minutes and stirring. At these MAME conditions, good recoveries (around 90%) were obtained, significantly higher than those found by classical method (below 55%). Two different soils from Madrid(central Spain) were included in the study. Results confirmed the extraction effectiveness is strongly influenced by physico-chemical soil properties

Chemometric investigation of the effects of chemical properties and concentrations on the extractability of benzimidazoles with supported liquid membrane

Principle component analysis (PCA) and a two-way analysis of variance (ANOVA) were employed in the study of factors affecting extractability of benzimidazole anthelmintics using supported liquid membrane (SLM) in liver, kidney, milk and urine at four concentration levels. The SLM extraction process was monitored by liquid chromatography - mass spectrometer (LC-MS). The results showed that the extractability of benzimidazoles is dependent on both the concentration levels and the chemical properties of compounds. Based on chemical properties, extraction of the compounds from the liver matrix showed no significant difference (p = 0.05) for the following pairs; albendazole and oxibendazole, thiabendazole and mebendazole, oxibendazole and fenbendazole, and oxibendazole and mebendazole. At some of the concentration levels, mainly between 1000 and 100, 100 and 10, and 10 and 1 μg/Kg, there was no significant difference. It was also found that, there was significant difference (at p = 0.05) in the extractability in milk between oxibendazole and albendazole, and also oxibendazole and fenbendazole. For milk also, the concentration range from 10 to 100 μg/L, showed no significant difference (p = 0.05). Urine matrix on the other hand, showed significant difference in the recoveries at all concentration levels

Voltammetric Determination of a Benzimidazole Anthelmintic Mixture at a Poly(3-methylthiophene)-modified Glassy Carbon Electrode

The voltammetric determination of benzimidazole anthelmintics at a glassy carbon rotating-disk electrode modified with poly(3-methylthiophene) is presented. The purpose of surface modification was to improve the sensitivity and limits of detection for determination of the compounds in a standard mixture. Thus, five compounds, namely thiabendazole, mebendazole, albendazole, fenbendazole and oxibendazole have been studied using square wave voltammetry. It has been possible to resolve four of the compounds, mebendazole, fenbendazole, oxibendazole and thiabendazole, in a mixture. Investigations of a number of parameters, including the mode of potential application, cathodic reduction versus anodic oxidation, the type of electrode, effect of pH and speed of electrode rotation, among others, are reported. South African Journal of Chemistry Vol.56 2003: 5-

Detection of benzimidazole carbamates and amino metabolites in liver by surface plasmon resonance-biosensor

This research was funded by the Irish Department of Agriculture, Fisheries and Food under the Food Institutional Research Measure as part of the National Development Plan (Project 05/R&D/TN/355)peer-reviewedTwo surface plasmon resonance (SPR) biosensor screening assays were developed and validated to detect 11 benzimidazole carbamate (BZT) and four amino-benzimidazole veterinary drug residues in liver tissue. The assays used polyclonal antibodies, raised in sheep, to detect BZTs and amino-benzimidazoles. A modified Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) extraction method was developed to isolate benzimidazole carbamate residues. Liver samples were extracted using an acetonitrile extraction method. BZTs were purified by dispersive solid phase extraction (d-SPE) using C18 sorbent. Residues of amino-benzimidazoles were effectively cleaned-up using a simple cyclohexane defatting step. The assays were validated in accordance with the performance criteria described in 2002/657/EC. The BZT assay limit of detection was calculated to be 32 μg kg−1, the detection capability (CCβ) was determined to be 50 μg kg−1 and the mean recovery of analytes was in the range 77–132%. The amino-benzimidazole assay limit of detection was determined to be 41 μg kg−1, the CCβ was determined to be 75 μg kg−1 and analyte recovery was in the range 103–116%. Biosensor assay performance was tested by analysing liver tissue from animals treated with benzimidazole drugs and comparing the results with an ultra high performance liquid chromatography tandem mass spectrometry (UHPLC–MS/MS) confirmatory method. All non-compliant samples were identified using the biosensor assays.Department of Agriculture, Food and the Marin

Importation of Macrocyclic Lactone Resistant Cyathostomins on a US Thoroughbred Farm

Anthelmintic resistance in equine cyathostomins is both widespread and highly prevalent in the benzimidazole and tetrahydropyrimidine classes; however, reports of resistance to macrocyclic lactone (ML) drugs are sparse and sporadic. This study reports a case of clear ML resistance in a group of Thoroughbred yearlings imported from Ireland to the US in 2019. Fecal egg count reduction (FECR) following ivermectin administered in February 2020 demonstrated 100% reduction in the US bred yearlings, but 93.5%, 70.5%, and 74.5% reduction in three groups of the imported yearlings. The two former groups were then retreated with ivermectin, yielding FECRs of 33.8% and 23.5%, respectively. Horses from these two groups were then assigned randomly to two possible treatments; moxidectin or a triple combination of moxidectin, oxibendazole, and pyrantel pamoate. The groups treated with moxidectin had FECRs of 90.2%, 57.3%, and 50.0%, while the triple combination had a 100% FECR in all treated groups. Subsequently, the efficacy of ivermectin was reassessed in June 2020 yielding FECRs of 99.8%, 87.7%, and 62.0% in the three imported groups. The FECRs of the US bred yearlings all remained in the 99-100% range. This is the first study to clearly demonstrate ML resistance in cyathostomins and to confirm the suspicion through reassessment. These data demonstrate that ML-resistant cyathostomins were imported from Ireland and serve to illustrate that the global movement of horses has the potential to quickly spread ML-resistant parasite isolates around the world. The equine industry is strongly encouraged to routinely monitor anthelmintic efficacy, so occurrence of ML resistant cyathostomins can be detected and appropriate interventions implemented as early as possible

ANTHELMINTIC RESISTANCE IN EQUINE PARASITES: MECHANISMS AND TREATMENT APPROACHES

Anthelmintic resistance of parasites infecting livestock animals is a global problem resulting in decreased animal welfare and production losses. Horses are not exempt from this issue as wide-spread anthelmintic resistance exists among the equine cyathostomins and Parascaris spp. Of the three drug classes available for treating equine intestinal helminths anthelmintic resistance, defined as less than 90-95% drug efficacy, exist to all three. New pharmaceutical control regimens and the elucidation of parasite drug response mechanisms are needed. Two studies were carried out evaluating combination deworming regimens. A population of cyathostomins with known resistance to the benzimidazole (BZ) and pyrimidine drug classes maintained in a herd of Shetland ponies was used. Fecal egg counts were performed every two weeks and used to evaluate drug efficacy. The first study evaluated the combination of a BZ and pyrimidine drug for four consecutive treatments, and compared the individual drug efficacies before and after combination use. The first combination treatment exhibited an additive effect at 76.6%, but the subsequent three combination treatments decreased to approximately 40%. There was no significant difference between the initial and final efficacies of individual drugs (BZ, p=0.4421; pyrimidine, p=0.8361). It appears the combination treatment selected for double-drug resistant adult parasites. The timeframe of this study (1 year) and the one year lifespan of adult cyathostomins prevented observations of combination treatment on subsequent generations, however given the sustainability of resistance in this cyathostomin population, it seems unlikely efficacy would improve over time. The second study examined the combination of a BZ drug with a macrocyclic lactone (ML) drug. This parasite population was 100% naïve to the ML drug class. This study was carried out in a similar manner to the first, except only two combination treatments were given. ML exhibited 100% efficacy when it was used alone, or in combination. The initial and final BZ efficacy did not significantly differ (p=0.9890). In summary, the results described herein do not support the use of combination treatments where resistance is prevalent, but more long term studies are needed to fully understand the long-term effects on subsequent generations. The in vitro maintenance of Parascaris spp. provides opportunity for various molecular analyses. An objective motility scoring assessment allowed for continuous monitoring of worm viability. In this study, several saline solutions, nutrient supplements, environmental conditions, and Roswell-Park Memorial Institute medium 1640 (RPMI-1640) were evaluated for the longevity and viability of adult Parascaris spp. Overall, RPMI-1640 resulted in better longevity (168 hours) and significantly better viability (pParascaris spp. to in vitro drug exposure. Oxibendazole at 10 µg/mL for 24 hours and ivermectin at 1 µg/mL for three hours were employed, and worms were used for transcriptomic analyses to identify drug response mechanisms. The top four genes which were significantly different between drug treated and control groups were: cyp4504C1, sup-9, frmd4a, and klhdc10. It is hypothesized that cyp4504C1 and klhdc10 are drug detox mechanisms, while sup-9 and frmd4a may be indirect response related to the drug effects. Their expression was further evaluated using quantitative RT-PCR, however there was no significant difference in any gene expression between groups. It should be noted that there are several limitations associated with the qPCR method, and the lack of significance should not rule out the possible involvement of these genes and more research on drug response mechanisms is needed. In summary, there is very little research regarding combination deworming in horses, and their current use is largely due to some success for ruminant parasites, but the current work summarized herein does not support their use. Finally, until now the lack of in vitro methods for equine helminths has significantly delayed the elucidation of drug response mechanisms. This was the first whole-transcriptome approach for any ascarid parasite and uncovered proteins with possible involvement in drug metabolism or compensate for the toxic effects Overall, the research surrounding anthelmintic resistance in livestock helminths, particularly in horses, is lacking and the resistance crisis demands further investigation

Diagnosis and control of anthelmintic-resistant Parascaris equorum

Since 2002, macrocyclic lactone resistance has been reported in populations of Parascaris equorum from several countries. It is apparent that macrocyclic lactone resistance developed in response to exclusive and/or excessively frequent use of ivermectin or moxidectin in foals during the first year of life. The development of anthelmintic resistance was virtually inevitable, given certain biological features of Parascaris and unique pharmacologic characteristics of the macrocyclic lactones. Practitioners can utilize the Fecal Egg Count Reduction Test to detect anthelmintic resistance in Parascaris, and the same technique can be applied regularly to confirm the continued efficacy of those drugs currently in use. In the face of macrocyclic lactone resistance, piperazine or anthelmintics of the benzimidazole or pyrimidine classes can be used to control ascarid infections, but Parascaris populations that are concurrently resistant to macrocyclic lactones and pyrimidine drugs have been reported recently from Texas and Kentucky. Compared to traditional practices, future recommendations for ascarid control should feature: 1) use of only those anthelmintics known to be effective against indigenous populations, 2) initiation of anthelmintic treatment no earlier than 60 days of age, and 3) repetition of treatments at the longest intervals which prevent serious environmental contamination with Parascaris eggs. In the interest of decreasing selection pressure for anthelmintic resistance, horse owners and veterinarians must become more tolerant of the passage of modest numbers of ascarid eggs by some foals. Anthelmintic resistance is only one of several potential responses to genetic selection. Although still only theoretical, changes in the immunogenicity of ascarid isolates or reduction of their prepatent or egg reappearance periods could pose far greater challenges to effective control than resistance to a single class of anthelmintics

Efficacy of oxibendazole in the treatment and prophylaxis of worm infections in swine.

Avalia-se a eficiência e o efeito ovicida da droga oxibendazole (metil 5n-Propoxi-2 benzimidazole - carbamato), em animais bovinos naturalmente parasitados por Áscaris suum, Trichuris suis, Oesophagostomum sp e Strongyloides ransomi e em suínos desafiados com Oesophagostomum sp, T. suis e A. suum. A droga foi administrada misturada a ração em dose única de 15 mg/kg,em doses fracionadas de 100 ppm por seis dias, a 15 ppm por 50 dias e 60 dias. Os resultados de necrópsia dos animais revelaram que nos tratamentos com oxibendazole a 15 mg/kg, 100 ppm/seis dias e 15 ppm/50- dias, foi 100% eficaz na remoção de A. suum e Oesophagostomum. A eficácia na remoção de T. suis foi de 74,5, 80,4 e 100% respectivamente, nas dosagens de 15 mg/kg, 15 ppm/50 dias e 100 ppm/ seis dias. Com base apenas em exame de fezes dos animais o anti-helmintico foi 100% eficaz na eliminação dos ovos de S. ransomi, nas três dosagens utilizadas. Os resultados dos exames e Culturas de fezes revelaram que o oxibendazole na concentração de 15 ppm na ração, reduziu em 95,5% a eliminação de ovos de A. suum e em 91.0% a capacidade de embrionia dos ovos, após cinco dias de medicação. Os resultados dos exames de fezes e de necropsia revelaram que este anti-helmintico, adicionado à ração na concentração de 15 ppm durante 60 dias, foi 100% eficiente na prevenção de infecções por formas adultas de A. suum e Oesophagostomum sp. Com relação a Trichuris suis apesar da detecção de ovos nas fezes de animais que estavam recebendo ração medicada os helmintos foram posteriormente eliminados tendo atingido 100% de eficiência no final dos 60 dias de tratamento.The efficacy af oxibendazole (methyl 5n-propoxy-2 benzimidazole - carbamate) as an anthelmintic for swine was evaluated in animals harboring natural infections by Ascaris suum, Oesophagostomum sp, Trichuris suis and Strongyloides ransomi. The prophylactic effect was conducted with A. suum, Oesaphagostomum sp, and Trichuris suis. The levels of the anthelmintic tested were 15 mg/kg body weight, 100 ppm (100 g of active per metric ton of feed) during six consecutive days and 15 ppm during 50 and 60 consecutive days, all treatments administered, in the feed. The results at necropsy showed an efficacy of 100% in the treatment of pigs naturally infected by A. suum and Oesophagostomum sp. The efficacy against T. suis was 74.5%, 80.4% and 100% for 15 mg/kg b.w., 15 ppm during 50 days, and 100 ppm during six days, respectively. All treatments eliminated fecal egg counts of S. ransomi. The administration of oxibendazole in the feed at 15 ppm reduced 95.5% of the egg output of Ascaris in the feces after five days of medication and had an ovicidal effect of 91.0% at that time. The low level anthelmintic in feed 15 ppm was 100% effective in preventing infections at different levels of challenge with A. suum and. Oesophagostomum sp. Eggs of T. suis were found in feces of pigs receiving medicated feed at 15 ppm but no worms were present at necropsy on the 60th day after medication

RP-HPLC method development and validation of Albendazole and its impurity

Oxibendazole is a type of benzimidazole that is commonly used as an antiparasitic medication for both humans and animals. However, it is also a significant impurity found in albendazole, and it is crucial to follow the ICH Q3B criteria when analysing oxibendazole impurities. Therefore, it is recommended to use a simple, fast, sensitive, and precise RP-HPLC approach to identify oxibendazole impurities in bulk and pharmaceutical formulations of albendazole.To separate the oxibendazole impurity, acetonitrile and 10 nM potassium phosphate were used as a mobile phase. Orthophosphoric acid was used to accurately adjust the pH of the mobile phase to 2.03, ensuring optimal conditions. A nucleosil C18 column (250 x 4.6 mm, 5 µm) was used for the separation process, and it effectively provided the necessary separation. The gradient elution was set at a wavelength of 235 nm and a flow rate of 1 mL/min. The analytical technique was successfully designed and validated. The AQbD technique was used to optimize the analytical conditions for the suggested methodology, and the Design Expert 13® trial version was used for the central composite design optimization of analytical conditions. The procedure's linearity was verified using a regression coefficient of 0.999 within a working range of 0.5 to 3 μg/ml. Accuracy research showed results of 99.94–100.10% at 50, 100, and 150% levels of the working concentration. The oxibendazole impurity's average retention time was found to be 6.40 minutes, with a relative standard deviation of less than 2%, indicating high accuracy. The limits of detection (LOD) and quantification (LOQ) were found to be 0.073 and 0.091 μg/ml, respectively. Following the ICH Q2(R1) criteria, other validation criteria, such as robustness, were also evaluated. In conclusion, the proposed approach is suitable for analysing albendazole and oxibendazole in bulk and pharmaceutical formulations, making it ideal for detecting oxibendazole impurities

Anthelmintic resistance of gastrointestinal cattle nematodes

Anthelmintic resistance of parasites in small ruminants, cattle and horses is increasing worldwide as a consequence of the over usage of the currently available products. In Belgium, Cooperia oncophora is the most common cattle nematode in which resistance, especially against macrocyclic lactones, occurs. Once resistance has been diagnosed, a change to another drug with a different mode of action is advised. However, effective anthelmintics will be hardly available in the near future. Therefore, it is important that farmers and veterinarians find a balance between achieving good parasite control and the sustainability of their control strategies. In this way, anthelmintic resistance may be delayed, and the effectiveness of anthelmintic drugs may be prolonged. This requires sensitive detection tools. With a sensitive detection technique, anthelmintic resistance can be diagnosed in a very early stage. Hence, the spread of resistance alleles in the parasite population may be prevented. In this review, different diagnostic assays for the detection of anthelmintic resistance are discussed, an overview is given of the current status of anthelmintic resistance in Belgian cattle, and measures are suggested to avoid or delay the development of anthelmintic resistance