Lack of detectable short-term effects of a single dose of ivermectin on the human immune system.

BACKGROUND: Ivermectin is widely used in human and animal medicine to treat and prevent parasite nematode infections. It has been suggested that its mode of action requires the host immune system, as it is difficult to reproduce its clinical efficacy in vitro. We therefore studied the effects of a single dose of ivermectin (Stromectol®-0.15 mg/kg) on cytokine levels and immune cell gene expression in human volunteers. This dose reduces bloodstream microfilariae rapidly and for several months when given in mass drug administration programmes. METHODS: Healthy volunteers with no travel history to endemic regions were given 3-4 tablets, depending on their weight, of either ivermectin or a placebo. Blood samples were drawn immediately prior to administration, 4 h and 24 h afterwards, and complete blood counts performed. Serum levels of 41 cytokines and chemokines were measured using Luminex® and expression levels of 770 myeloid-cell-related genes determined using the NanoString nCounter®. Cytokine levels at 4 h and 24 h post-treatment were compared to the levels pre-treatment using simple t tests to determine if any individual results required further investigation, taking p = < 0.05 as the level of significance. NanoString data were analysed on the proprietary software, nSolver™. RESULTS: No significant differences were observed in complete blood counts or cytokine levels at either time point between people given ivermectin versus placebo. Only three genes showed a significant change in expression in peripheral blood mononuclear cells 4 h after ivermectin was given; there were no significant changes 24 h after drug administration or in polymorphonuclear cells at either time point. Leukocytes isolated from those participants given ivermectin showed no difference in their ability to kill Brugia malayi microfilariae in vitro. CONCLUSIONS: Overall, our data do not support a direct effect of ivermectin, when given at the dose used in current filarial elimination programmes, on the human immune system. Trial registration ClinicalTrials.gov NCT03459794 Registered 9th March 2018, Retrospectively registered https://clinicaltrials.gov/ct2/show/NCT03459794?term=NCT03459794&draw=2&rank=1 .

Development of a broad-spectrum monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay for the multi-residue detection of avermectins in edible animal tissues and milk.

Avermectins (AVMs) are a group of anti-parasitic agents that have been widely used in food-producing animals. To monitor the residue of the AVMs, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was developed with a simple sample preparation procedure. Conjugates of 4″-HS-IVM/AVM on three different proteins were used to raise a broad-spectrum monoclonal antibody (mAb), 6D4, that had IC50 values for avermectin, ivermectin, eprinomectin and emamectin of 7.2, 10.4, 19.8 and 20.8 µg L-1, respectively. The limit of detection and limit of quantitation of this method for AVMs in various matrix samples ranged from 0.5 to 5.4 µg L-1 and 1.0 to 10.3 µg L-1, respectively. The recoveries of the samples spiked with AVMs were in the range of 78.1-110.5% with coefficients of variation below 14%. The ic-ELISA was applied to monitor ivermectin in the milk samples, and the results showed a good correlation with that of HPLC-MS/MS.

A monoclonal antibody-based ELISA for multiresidue determination of avermectins in milk.

Due to the widespread use and potential toxicity of avermectins (AVMs), multi-residue monitoring of AVMs in edible tissues, especially in milk, has become increasingly important. With the aim of developing a broad-selective immunoassay for AVMs, a broad-specific monoclonal antibody (Mab) was raised. Based on this Mab, a homologous indirect enzyme-linked immunosorbent assay (ELISA) for the rapid detection of AVMs in milk was developed. Under the optimized conditions, the IC50 values in assay buffer were estimated to be 3.05 ng/mL for abamectin, 13.10 ng/mL for ivermectin, 38.96 ng/mL for eprinomectin, 61.00 ng/mL for doramectin, 14.38 ng/mL for emamectin benzoate. Detection capability (CCß) of the ELISA was less than 5 ng/mL and 2 ng/mL in milk samples prepared by simple dilution and solvent extraction, respectively. The optimized ELISA was used to quantify AVMs in milk samples spiked at different amounts. The mean recovery and coefficient of variation (CV) were 95.90% and 15.42%, respectively. The Mab-based ELISA achieved a great improvement in AVMs detection. Results proved this broad-selective ELISA would be useful for the multi-residue determination of AVMs in milk without purification process.

Selection of a single chain variable fragment antibody against ivermectin from a phage displayed library.

Single chain variable fragment antibodies (scFvs) have considerable potential in immunological detection of pesticides, veterinary and other residues. In this study, a large human synthetic phage displayed library was used to select scFvs against ivermectin (IVM) by subtractive panning. After four rounds of panning, seven monoclonal phage particles capable of binding with the IVM were obtained. One of the identified novel anti-IVM scFv, hsIVM8, was expressed in Escherichia coli HB2151 and purified with Ni metal ion affinity chromatography. Sodium dodecyl sulfate polyacrylamine gel electrophoresis (SDS-PAGE) indicated that the relative molecular mass of hsIVM8 was estimated at 28 kDa. The purified hsIVM8 was used to develop a competitive indirect enzyme-linked immunosorbent assay (ci-ELISA) for IVM. The linear range of detection for standards in this ci-ELISA was approximately 0.1-5 microg/mL and 50% inhibition of control (IC(50)) was 4.11 microg/mL for IVM. These results showed promising applications of hsIVM8 for detecting IVM in agricultural and environmental matrices.

Preparation and characterization of monoclonal antibodies against avermectin.

The coupled antigen, avermectin (AVM) and bovine serum albumin (BSA), was artificially synthesized. Female 8-week-old BALB/c mice were immunized with AVM-BSA emulsified in Freund's adjuvant. Two monoclonal antibodies (MAbs) against AVM, named 2F2 and 2H10, were prepared by lymphocyte hybridoma technique. Ascitic fluid titers of the MAbs 2F2 and 2H10 were, respectively, 1:6400 and 1:25,600, and their subtypes were all IgMkappa. In competitive indirect ELISA (Ci-ELISA), 50% inhibiting concentration (IC(50)) of the MAb 2H10 for avermectin was 101 ng/mL. In specific assay, the MAb 2H10 could not react with ivermectin, doramectin, erythromycin, and albomycin. These data demonstrated that the MAb 2H10 will have a potential use for developing diagnostic reagents of avermectin residues.

[Immunogenic properties of the colloidal gold]. / Immunogennye svoistva kolloidnogo zolota.

We studied the capacity of colloidal gold for enhancing specific and nonspecific immune response in laboratory animals (rabbits, rats, and mice) immunized with antigens of various nature. The antibody titers obtained with colloidal gold as a carrier were higher as compared to the standard immunization techniques (free antigen or Freund's adjuvant). Application of colloidal gold increased nonspecific immune responses as well: lysozyme concentration in the blood, activity of the complement system proteins, as well as phagocytic and bactericidal activities. The obtained antibodies were tested by immunodot assay using gold markers. Immunization of the animals with colloidal gold conjugates with haptens as well as complete antigens was shown to induce formation of highly active antibodies without using other antigens such as complete Freund's adjuvant. In addition, antigen quantities for animal immunization with colloidal gold was by one order of magnitude lower as compared to the complete Freund's adjuvant immunization. This fact can point to direct adjuvant activity of colloidal gold.

Efficacy of selamectin in the treatment and control of clinical signs of flea allergy dermatitis in dogs and cats experimentally infested with fleas.

OBJECTIVE: To determine whether treatment with selamectin would reduce clinical signs of flea allergy dermatitis (FAD) in dogs and cats housed in flea-infested environments. DESIGN: Randomized controlled trial. ANIMALS: 22 dogs and 17 cats confirmed to have FAD. PROCEDURE: Animals were housed in carpeted pens capable of supporting the flea life cycle and infested with 100 fleas (Ctenocephalides felis) on days -13 and -2 and on alternate weeks with 10 to 20 fleas. On day 0, 11 dogs and 8 cats were treated with selamectin (6 mg/kg [2.7 mg/lb]). Dogs were retreated on day 30; cats were retreated on days 30 and 60. All animals were examined periodically for clinical signs of FAD. Flea counts were conducted at weekly intervals. RESULTS: Throughout the study, geometric mean flea counts exceeded 100 for control animals and were < or = 11 for selamectin-treated animals. Selamectin-treated cats had significant improvements in the severity of miliary lesions and scaling or crusting on days 42 and 84, compared with conditions on day -8, and in severity of excoriation on day 42. In contrast, control cats did not have any significant improvements in any of the clinical signs of FAD. Selamectin-treated dogs had significant improvements in all clinical signs on days 28 and 61, but in control dogs, severity of clinical signs of FAD was not significantly different from baseline severity at any time. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that topical administration of selamectin, even without the use of supplementary environmental control measures and with minimal therapeutic intervention, can reduce the severity of clinical signs of FAD in dogs and cats.

Detection of unwanted residues of ivermectin in bovine milk by dissociation-enhanced lanthanide fluoroimmunoassay.

Avermectins are frequently used to control parasitic infestations in many animal species. Previous studies have shown the long-term persistence of unwanted residues of these drugs in animal tissues and fluids. An immunoassay screening test for the detection and quantification of ivermectin residues in bovine milk has been developed. After an extensive extraction procedure, milk samples were applied to a competitive dissociation-enhanced lanthanide fluoroimmunoassay using a monoclonal antibody against an ivermectin-transferrin conjugate. The monoclonal antibody, raised in Balb C mice, showed cross-reactivity with eprinomectin (92%), abamectin (82%) and doramectin (16%). The limit of detection of the assay (mean + 3 SD), calculated from the analysis of 17 known negative samples, was calculated as 4.6 ng/mL. Intra- and inter-assay RSDs were determined as 11.6% and 15.8%, respectively, using a negative bovine milk sample fortified with 25 ng/mL ivermectin. Six Friesian milking cows were treated with ivermectin, three with a pour-on formulation of the drug and three with an injectable solution at the manufacturer's recommended dose rate. An initial mean peak in ivermectin residue concentration was detected at day 4 (mean level = 47.5 ng/mL) and day 5 post-treatment (mean level = 26.4 ng/mL) with the injectable form and pour-on treatment, respectively. A second peak in residue concentration was observed using the DELFIA procedure 28 days post-treatment in both treatment groups (23.1 ng/mL injectable and 51.9 ng/mL pour-on). These second peaks were not confirmed by HPLC and must at this time be considered to be false-positive results. By day 35 after treatment the mean ivermectin residue concentration of both groups fell below the limit of detection of the assay.

Induction of protective immunity to Dictyocaulus viviparus in calves while under treatment with endectocides.

Three groups of five parasite-naive calves were used. The treatments were: (a) Group 1 calves were weighed on Day 0 and injected with doramectin at 200 microg/kg. From Day 1 to 19 they were dosed orally with 2000 infective larvae of Dictyocaulus viviparus. On Day 28 they were again injected with doramectin, and infected with D. viviparus larvae from Days 33 to 41. They were then left untreated until Day 81 when they were infected with 20 infective larvae of D. viviparus per kg body weight. They were killed on Day 110 and lungworms were counted; (b) Group 2 calves were immunised with oral lungworm vaccine on Days 0 and 28, and infected and slaughtered as Group 1 on Days 81 and 110, respectively; (c) Group 3 calves acted as infection controls. Blood samples were taken at Days 0, 21, 49, 77 and 110 for antibody tests to D. viviparus. At autopsy there were no significant differences between the number of lungworms from Groups 1 and 2 (Means 17.4 and 31.3, respectively); Group 1 had significantly less value than Group 3 (Mean 228) (p < 0.05). Increased antibody titres to the larval sheath of the infective larvae were observed from Groups 1 and 2, showing that the larvae in Group 1 had penetrated the intestine before being killed by the circulating anthelmintic. This experiment shows that if calves are exposed to infective larvae while under systemic endectocide cover, an immune reaction is stimulated.

Immunoaffinity column cleanup procedure for analysis of ivermectin in swine liver.

A simple and sensitive method for the analysis of ivermectin (22,23-dihydroavermectin B1) in swine liver based on immunoaffinity column cleanup is described. The immunosorbent was prepared by coupling polyclonal anti-ivermectin antibodies to carbonyl diimidazole-activated Sepharose CL-4B. After extraction with methanol, ivermectin was cleaned up on an immunoaffinity column, and determined by reversed-phase liquid chromatography with UV absorbance detection at 245 nm. Recoveries of ivermectin from fortified samples of 5-10 micrograms kg-1 levels ranged 85-102%, with coefficients of variation of 6-12%. The limit of detection was 2 micrograms kg-1 in a 5-g samples.

Simple and sensitive enzyme-linked immunosorbent assay for ivermectin.

A sensitive and reproducible enzyme-linked immunosorbent assay (ELISA) for the determination of the concentration of ivermectin (IVM) in biological fluids was developed. A conjugate of IVM on bovine serum albumin and poly-L-lysine was used to produce antibodies in rabbits and served as a solid-phase marker for titration of antibodies, respectively. The competitive ELISA was conducted by simultaneously incubating IVM and IVM-biotin conjugate with anti-IVM antiserum over goat anti-rabbit IgG (Fc) and then determining the amount of bound IVM-biotin with avidin-peroxidase conjugate as a tracer. The coefficient of variation for the assay was less than 10% in the range of 0.3-10 ng/ml. The limit of detection was 0.1 ng/ml. The cross-reactivities of anti-IVM antiserum with some anthelmintic drugs were negligible. Using this ELISA, serum levels of IVM were easily determined in Mongolian jirds (Meriones unguiculatus) up to 72 hr following a single oral dose of 500 microgram/kg of body weight.