Macrocyclic Lactone Drug Research Articles

Efficacy of antiparasitic macrocyclic lactone drops against passalurosis and psoroptic mange in rabbits

The purpose of the research is to study the efficacy of a macrocyclic lactone drug using the spot-on method against passalurosis and psoroptic mange in rabbits.Materials and methods. The study was conducted at the Department of Parasitology and Veterinary-Sanitary Expertise of the MVA named after K. I. Skryabin in 2022–2023 to determine the efficacy of antiparasitic drops (active ingredients: phenylpyrazole derivative, pyrazinoisoquinoline derivative, macrocyclic lactone, insect growth regulator, manufactured by AVZ S-P, LLC) against passalurosis and psoroptic mange. The antiparasitic drops were applied by the spot-on method to rabbits of different age and sex groups in doses of 0.5 mL per animal for body weight of up to 4 kg and 1 mL per animal for body weight of 4 to 8 kg against passalurosis and psoroptic mange. Rabbit fecal samples were examined using the Fülleborn method. Skin scrapings from the inner auricle surface were studied for Psoroptes cuniculi mites as per D. A. Priselkova.Results and discussion. The antiparasitic drug was found to have 100 % efficacy against ticks Psoroptes cuniculi and nematodes Passalurus ambigus.

Genome structure and population genomics of the canine heartworm Dirofilaria immitis

The heartworm, Dirofilaria immitis, is a filarial parasitic nematode responsible for significant morbidity and mortality in wild and domesticated canids. Resistance to macrocyclic lactone drug prevention represents a significant threat to parasite control and has prompted investigations to understand the genetic determinants of resistance. This study aimed to improve the genomic resources of D. immitis to enable a more precise understanding of how genetic variation is distributed within and between parasite populations worldwide, which will inform the likelihood and rate by which parasites, and in turn, resistant alleles, might spread. We have guided the scaffolding of a recently published genome assembly for D. immitis (ICBAS_JMDir_1.0) using the chromosomal-scale reference genomes of Brugia malayi and Onchocerca volvulus, resulting in an 89.5 Mb assembly composed of four autosomal- and one sex-linked chromosomal-scale scaffolds representing 99.7% of the genome. Publicly available and new whole-genome sequencing data from 32 D. immitis samples from Australia, Italy and the USA were assessed using principal component analysis, nucleotide diversity (Pi) and absolute genetic divergence (Dxy) to characterise the global genetic structure and measure within- and between-population diversity. These population genetic analyses revealed broad-scale genetic structure among globally diverse samples and differences in genetic diversity between populations; however, fine-scale subpopulation analysis was limited and biased by differences between sample types. Finally, we mapped single nucleotide polymorphisms previously associated with macrocyclic lactone resistance in the new genome assembly, revealing the physical linkage of high-priority variants on chromosome 3, and determined their frequency in the studied populations. This new chromosomal assembly for D. immitis now allows for a more precise investigation of selection on genome-wide genetic variation and will enhance our understanding of parasite transmission and the spread of genetic variants responsible for resistance to treatment.

The first report of macrocyclic lactone resistant cyathostomins in the UK

In recent years, resistance to the benzimidazole (BZ) and tetrahydropyrimidine (PYR) anthelmintics in global cyathostomin populations, has led to reliance on the macrocyclic lactone drugs (ML-of which ivermectin and moxidectin are licensed in horses) to control these parasites. Recently, the first confirmed case of resistance to both ivermectin (IVM) and moxidectin (MOX) was reported in the USA in yearlings imported from Ireland. This suggests that ML resistance in cyathostomins has emerged, and raises the possibility that regular movement of horses may result in rapid spread of ML resistant cyathostomins. Resistance may go undetected due to a lack of surveillance for ML efficacy. Here, we report anthelmintic efficacies in cyathostomins infecting UK Thoroughbreds on four studs. Faecal egg count reduction tests (FECRT) were performed to define resistance (resistance = FECR <95% lower credible interval (LCI) < 90%). Stud A yearlings had FECRs of 36.4–78.6% (CI:15.7–86.3) after three IVM treatments, 72.6% (CI: 50.8–85.2) after MOX, and 80.8% (CI: 61.9–90.0) after PYR. Mares on stud A had a FECR of 97.8% (CI: 93.3–99.9) and 98% (95.1–99.4) after IVM and MOX treatment, respectively. Resistance to MLs was not found in yearlings or mares on studs B, C or D with FECR after MOX OR IVM treatment ranging from 99.8 to 99.9% (95.4–100); although yearlings on studs B, C and D all had an egg reappearance period (ERP) of six weeks for MOX and stud C had a four-week ERP for IVM. This study describes the first confirmed case of resistance to both licensed ML drugs on a UK Thoroughbred stud and highlights the urgent need for a) increased awareness of the threat of ML resistant parasites infecting horses, and b) extensive surveillance of ML efficacy against cyathostomin populations in the UK, to gauge the extent of the problem.

Secretory microRNA Profiles of Third- and Fourth-Stage Dirofilaria immitis Larvae with Different Macrocyclic Lactone Susceptibility: In Search of Biomarkers for Early Detection of Infection.

The canine heartworm, Dirofilaria immitis, is among the most important parasites of dogs in the United States and worldwide, and may cause severe and potentially fatal disease. Current diagnostic recommendations rely on serological detection of an adult female antigen, and visualization of microfilariae in the blood. Therefore, a reliable diagnosis can be only performed approximately six months post-infection. There is a growing need to characterize novel diagnostic markers that are capable of detecting the early stages of heartworm infection, in special markers associated with third-stage larvae (L3) and fourth-stage larvae (L4). The early detection of infection would guide medical interventions that could impede the development of patent infections and further parasite transmission. We cultured D. immitis L3 and L4 of two laboratorial strains with different susceptibility statuses to macrocyclic lactone drugs in vitro. Excretory/secretory microRNAs were sequenced and analyzed. We identified two miRNA novel candidates secreted abundantly by both L3 and L4 of both strains. These candidates were previously detected in the secretions of other D. immitis stages and one of them was found in the blood of D. immitis-infected dogs. These miRNAs have not been found in the secretions of other nematodes and could be D. immitis-specific diagnostic biomarkers, which could allow for the early detection of infection.

Ivermectin inhibits extracellular vesicle secretion from parasitic nematodes.

Lymphatic filariasis (LF) is a disease caused by parasitic filarial nematodes that is endemic in 49 countries of the world and affects or threatens over 890 million people. Strategies to control LF rely heavily on mass administration of anthelmintic drugs including ivermectin (IVM), a macrocyclic lactone drug considered an Essential Medicine by the WHO. However, despite its widespread use the therapeutic mode of action of IVM against filarial nematodes is not clear. We have previously reported that filarial nematodes secrete extracellular vesicles (EVs) and that their cargo has immunomodulatory properties. Here we investigate the effects of IVM and other anti‐filarial drugs on parasitic nematode EV secretion, motility, and protein secretion. We show that inhibition of EV secretion was a specific property of IVM, which had consistent and significant inhibitory effects across nematode life stages and species, with the exception of male parasites. IVM inhibited EV secretion, but not parasite motility, at therapeutically relevant concentrations. Protein secretion was inhibited by IVM in the microfilariae stage, but not in any other stage tested. Our data provides evidence that inhibiting the secretion of immunomodulatory EVs by parasitic nematodes could explain, at least in part, IVM mode of action and provides a phenotype for novel drug discovery.

IMPROVING SOME PARAMETERS OF ANTIBODY RESPONSE IN HORSES BY PROBIOTIC AGENTS AGAINST BACKGROUND USE OF MACROCYCLIC LACTONE ANTHELMINTIC

Multiple researches demonstrate that both invasive diseases and use of anthelmintcs adversely affect horse immunity harming the gut flora. Macrocyclic lactone anthelmintics are proved to be highly effective agents but there is insufficient knowledge of their effect on the horse antibody immunity. The issue of immune system correction by the probiotic agents is also underreported. The paper demonstrates the results of the effect made by parascaridosis and strongylidosis invasion and deworming using the macrocyclic lactone drug on some parameters of antibody response in horses. Data on effect of probiotic Bioxymin horse on blood serum bacteriocidal and lysozyme activity were reported for the first time. The research was performed from December 2018 to January 2019. The results demonstrated that helminthic invasion and anthelmintic drug Univerm make depressive effect on some parameters of antibody response in horses. It also has an impact on the extensive efficacy of the anthelmintic agent. Probiotic Bioxymin horse makes a stimulating effect on the antibody response in horses.

Adaptation of a 96-well plate larval migration inhibition test for measuring the sensitivity of cyathostomins to macrocyclic lactone anthelmintics

The use of macrocyclic lactone drugs for control of equine cyathostomins is threatened by increasing levels of resistance. Detection of changes in drug sensitivity is important for effective and sustainable management of cyathostomins, however, at present such detection relies on the use of the faecal egg count reduction test, which is known to be an insensitive method. The present study therefore aimed to examine the use of a 96-well plate larval migration inhibition test for detection of resistance to macrocyclic lactone drugs in cyathostomins. We optimised conditions for migration of larvae, and examined the effects of larval storage time on drug dose responses. The modified test was able to define the sensitivity of cyathostomin isolates to ivermectin and eprinomectin in terms of dose response curves, and IC50 and IC95 values. The IC95 showed much greater consistency than the IC50 with larvae that had been stored for different periods prior to the test. Comparisons between two isolates, which had both been defined previously as susceptible using faecal egg count reduction tests, showed more variation at the IC50 compared to the IC95. Limitations of the test included the degree of variation in control-well migration despite optimisation of migration incubation conditions, and the need to incorporate a method to determine the species composition of the larval populations to account for possible species differences in drug sensitivity among cyathostomins. Validation of the technique on reference susceptible and resistant isolates of known species composition is still required.

Gastrointestinal Parasite Community in a New Population of the Przewalski’s Horse (Equus Ferus Przewalskii) in the Orenburg State Reserve, Russia

AbstractThe Przewalski’s horse (Equus ferus przewalskii, Poljakov, 1881) is an endangered subspecies of wild horses (Equus ferus) native to steppes of Central Asia. In 2015, the Program of Establishing of a Semi-Free Population of the Przewalski’s Horse in the Orenburg Reserve was launched by FGFI “Orenburg Reserves”. The first group of 6 Przewalski’s horses (2 males and 4 females) born in the semi-reserve Le Villaret, France, was transported to the Orenburg Reserve. Th e aim of this work was to investigate the species composition of the intestinal parasite community and to monitor the dynamics of the parasite infection of the newly established Przewalski’s horse population. The level of infection by gastrointestinal parasites within the horses was examined by the McMaster method. Gastrointestinal parasites were collected in vivo after deworming of the horses with macrocyclic lactone drug “Univerm” (0.2 % aversectin C, PharmBioMed, Russia). Totally, 20 species of parasites were found: 19 species of nematodes (species of the family Strongylidae and Habronema muscae) and one species of botflies from the genus Gasterophilus. Th e widest species diversity (18 species from 8 genera) was observed in strongylids: 2 species from the subfamily Strongylinae and 16 species from Cyathostominae. Distribution of strongylid species between ten prevalence classes revealed a bimodal structure (“core-satellite” mode) of the strongylid community. Th e results obtained in this study are to be considered as the initial data for the further parasitological monitoring of Przewalski’s horses at the Orenburg State Reserve.

Effects of third generation P-glycoprotein inhibitors on the sensitivity of drug-resistant and -susceptible isolates of Haemonchus contortus to anthelmintics in vitro

P-glycoproteins (P-gps) play an important role in the sensitivity of nematodes to anthelmintic drugs. They have been implicated in a number of anthelmintic resistances, particularly for macrocyclic lactone drugs. Hence, inhibition of nematode P-gps has been suggested as a means of reversing some types of anthelmintic resistance. The present study aimed to investigate the ability of the most-recently developed group of P-gp inhibitors (the so-called ‘third generation’ of inhibitors) including tariquidar, zosuquidar and elacridar, to increase the sensitivity of Haemonchus contortus larvae to various anthelmintics (ivermectin, levamisole and thiabendazole) in vitro. We compared these compounds to some older P-gp inhibitors (e.g. verapamil and valspodar). Larval migration and development assays were used to measure the sensitivity of larvae to anthelmintics alone, or in combination with P-gp inhibitors. Significant increases in sensitivity to ivermectin were observed with zosuquidar and tariquidar in larval migration assays (synergism ratios up to 6-fold). Several of the inhibitors increased the sensitivity of both the drug-resistant and -susceptible isolates (e.g. tariquidar with ivermectin in migration assays, zosuquidar with ivermectin in larval development assays), while others had significant effects on the resistant isolate only (e.g. zosuquidar with ivermectin in migration assays, verapamil with ivermectin in development assays). This suggests that some of the inhibitors interact with P-gps representing intrinsic pathways present across nematode populations with quite different drug sensitivities, while other inhibitors interact with P-gps of significance only to resistant nematodes, and hence most likely representing an acquired resistance mechanism. The study highlights the potential of the third generation of P-gp inhibitors for increasing the sensitivity of nematodes to anthelmintics.

Detection of ivermectin resistance by a larval development test—Back to the past or a step forward?

The aim of this study was to evaluate the potential of the larval development test for the detection of ivermectin (IVM) resistance in Haemonchus contortus of sheep. Single infections with 5000 third-stage larvae of five resistant and two susceptible isolates of H. contortus were given to sheep. Fecal samples were collected four times during patency, and the micro-agar version of the larval development test (MALDT) was performed. Three macrocyclic lactone drugs (IVM, eprinomectin and IVM aglycone) were tested. The results of the tests are presented as LC50 and LC99 values. The MALDT was well able to distinguish between susceptible and resistant isolates. Resistance factors (RF) for the LC99 values were generally higher than those obtained by comparing LC50 values. The highly resistant isolates were readily distinguishable from the susceptible isolates, particularly when using IVM aglycone and eprinomectin, with RFs above 20.

Equine rotavirus detection: sensibility and specificity of two commercially available diagnostic kits

different schemes of parasite control from “zero-treatment” to regular deworming 4-5 times per year. Strongylid nematodes were collected from all equids in vivo after their deworming with the macrocyclic lactone drug “Univerm” (0.2% aversectin C, Russia). Totally, 62,722 specimens were collected and identified. Thirty-six strongylid species were found – 9 species of large strongyles (subfamily Strongylinae) and 27 species of small strongyles (Cyathostominae). In domestic horses, 27 strongylid species from 12 genera were found. The highest biodiversity of the strongylid community (27 species) was observed in horses from farms with rare anthelmintic treatments (1 time per year or less); from 8 to 20 species (average 12.1 3.4) parasitized per host. The lowest biodiversity (19 species) was observed in horses from farms with deworming 4-5 times per year; from 3 to 16 (6.7 2.6) per horse. Eleven cyathostome species (Cylicocyclus nassatus, C. ashworthi, C. leptostomus, Cyathostomum catinatum, C. pateratum, Cylicostephanus calicatus, C. longibursatus, C. goldi, C. minutus, Coronocyclus coronatus, and C. labiatus) were found to be resistant to benzimidazole anthelmintics. In donkeys, 26 strongylid species were found. In donkeys from the “Askania-Nova” reserve with rare anthelmintic treatment, 25 strongylid species were found; from 11 to 16 species (13.2 1.7) per host. In donkeys regularly treated with BZ and PYR, 14 species were found; from 3 to 10 (5.6 1.9) per host. In Przewalski’s horses from the “Askania-Nova” reserve, 31 strongylid species were found; there were 11 to 18 species (15.2 1.9) per host. Przewalski horses from zoos harbored 21 strongylid species; from 7 to 13 (10.4 2.1) per host. The structure of the strongylid community was bimodal (with dominant and rare species) in all regularly dewormed equids. Multimodal structure (with dominant, subdominant, background and rare species) was observed in never treated animals or with rare dewormings. Future studies are necessary to clear up whether such a decrease in biodiversity and change in strongylid community structure caused by deworming programs can lead to increasing the pathogenic influence of these parasites in horses or provoke development and spreading of anthelmintic resistance in strongylids.

A reappraisal of the relative sensitivity of nematode pharyngeal and somatic musculature to macrocyclic lactone drugs

Macrocyclic lactone (ML) drugs inhibit pharyngeal pumping, motility and egg laying in parasitic nematodes. Previous work has indicated that in vitro effects on worm feeding occurred at lower ivermectin concentrations than effects on worm motility, suggesting that the pharynx musculature was a more important target site for the ML drugs than somatic musculature. We have reassessed this issue of relative sensitivity by examining the response of drug-susceptible and -resistant adult Haemonchus contortus worms to abamectin in vitro using both feeding and motility assays. The motility assay involved observation of changes in the form and degree of movement of individual worms in response to the drug. A comparison of the data from the two assays indicated that worm motility was affected at drug concentrations below those required to inhibit feeding. Analysis of the motility data using different levels of sensitivity (varying in the degree to which they accounted for subtle vs. more profound changes in worm motility) provided an explanation as to why earlier reports had observed feeding to be the more sensitive target. Motility IC50 values shifted from being less than feeding IC50s to being greater than the feeding IC50s as the motility assay analysis method became less sensitive. The present study indicates that when sensitive worm motility assessment methods are utilised, worm motility is affected at lower abamectin concentrations than worm feeding, and hence highlights somatic musculature as a more important target site for this ML drug, and most likely for ML drugs in general.

Determination of Macrocyclic Lactone Drug Residues in Animal Muscle by Liquid Chromatography/Tandem Mass Spectrometry

A robust, credible, and practical multiresidue method based on liquid chromatography/tandem/mass spectrometry (LC/MS/MS) was developed for the simultaneous determination of 9 macrocyclic lactone drugs (abamectin B1a, doramectin, erythromycin, ivermectin B1a, josamycin, kitasamycin, roxithromycin, tilmicosin, and tylosin A) in bovine, porcine, chicken, and sheep muscles. The drugs were extracted with acetonitrile, and the extracts were defatted with n-hexane and further cleaned up on a C18 solid-phase extraction cartridge. LC/MS/MS data acquisition was achieved by using the multiple-reaction monitoring (MRM) mode, i.e., 2 transitions, to provide a high degree of sensitivity and repeatability. Matrix-matched standard calibration curves were used to achieve the best accuracy of the method by compensating for the matrix effect. The calibration graphs were linear (r > 0.998) from 10 to 1000 ng/mL for erythromycin, josamycin, kitasamycin, roxithromycin, tilmicosin, and tylosin, and from 5 to 250 ng/mL for abamectin, doramectin, and ivermectin. The average recoveries of the 9 drugs were between 64.5 and 105%, calculated by using matrix-matched calibration, with relative standard deviation values ranging from 1.6 to 14%. The limits of detection were 0.1 microg/kg for erythromycin, josamycin, roxithromycin, and tylosin; 0.2 microg/kg for tilmicosin and kitasamycin; and 0.5 microg/kg for abamectin, doramectin, and ivermectin. For confirmation, the MRM ratios for the 9 drug residues in the samples and the solvent were evaluated and found to be within the ratio criteria set by the guidelines of the European Union.

Is anthelmintic resistance a concern for heartworm control?: What can we learn from the human filariasis control programs?

Heartworm prophylaxis is currently largely dependent on the ability of avermectins and milbemycins to arrest the development of third and fourth stages of Dirofilaria immitis for prolonged periods, without producing adulticidal effects. Major control programs, dependent on the activity of ivermectin, are being implemented for human onchocerciasis and lymphatic filariasis. The avermectins and milbemycins act on glutamate-gated and γ-aminobutyrate-gated chloride channel subunit proteins in nematodes. Ivermectin resistance has been widely described in trichostrongylid nematodes of ruminants. There is evidence that when ivermectin resistance occurs in nematodes, there may be selection on some, but not all of the genes that code for ligand-gated chloride channel subunit proteins as well as on some ABC-transporter genes, whose products may be involved in regulating macrocyclic lactone drug concentrations at receptors, and on some structural protein genes of amphidial neurones. Although ivermectin resistance has not been reported in filarial nematodes, there have recently been reports of suboptimal responses to ivermectin in Onchocerca volvulus. Evidence has been found of ivermectin selection on at least ABC-transporter genes and some neuronal structural protein genes in O. volvulus. To date, there is no evidence of avermectin/milbemycin resistance in D. immitis, also a filarial nematode. Chemotherapy against trichostrongylids of animals, human filariae, and D. immitis, relies on avermectins or milbemycins. However, control involves targeting different stages or processes in the nematode life cycles, different control strategies, different proportions of the nematode population in refugia, and different drug dosage rates. Consideration of the proportion of the D. immitis population normally in refugia, the life cycle stage targeted, and the anthelmintic dosages used suggest that it is unlikely that significant avermectin/milbemycin resistance will be selected in D. immitis with current treatment strategies.