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Milbemycins, a group of 16-membered macrocylic lactones with excellent acaricidal, insecticidal and anthelmintic activities, can be produced by several Streptomyces species. For the reason that they have low toxicity in mammals, milbemycins and their derivatives are widely used in agricultural, medical and veterinary industries. Streptomyces bingchenggensis, one of milbemycin-producing strains, has been sequenced and intensively investigated in the past decades. In this mini-review, we comprehensively revisit the progress that has been made in research efforts to elucidate the biosynthetic pathways and regulatory networks for the cellular production of milbemycins. The advances in the development of production strains for milbemycin and its derivatives are discussed along the strain-generation technical approaches of random mutagenesis, metabolic engineering and combinatorial biosynthesis. The research progress made so far indicates that strain improvement and generation of novel milbemycin derivatives will greatly benefit from future development of enabling technologies and deeper understanding of the fundamentals of biosynthesis of milbemycin and the regulation of its production in S. bingchenggensis. This mini-review also proposes that the overproduction of milbemycins could be greatly enhanced by genome minimization, systematical metabolic engineering and synthetic biology approaches in the future.

Milbemycin R, a novel spiro-heterocycle milbemycin, was obtained from the metabolites produced by the mutant strain S. bingchenggensis BCJ60B11. Its structure was determined by spectroscopic and sp...

Exporter engineering is a promising strategy to construct high-yield Streptomyces for natural product pharmaceuticals in industrial biotechnology. However, available exporters are scarce, due to the limited knowledge of bacterial transporters. Here, we built a workflow for exporter mining and devised a tunable plug-and-play exporter (TuPPE) module to improve the production of macrolide biopesticides in Streptomyces. Combining genome analyses and experimental confirmations, we found three ATP-binding cassette transporters that contribute to milbemycin production in Streptomyces bingchenggensis. We then optimized the expression level of target exporters for milbemycin titer optimization by designing a TuPPE module with replaceable promoters and ribosome binding sites. Finally, broader applications of the TuPPE module were implemented in industrial S. bingchenggensis BC04, Streptomyces avermitilis NEAU12 and Streptomyces cyaneogriseus NMWT1, which led to optimal titer improvement of milbemycin A3/A4, avermectin B1a and nemadectin α by 24.2%, 53.0% and 41.0%, respectively. Our work provides useful exporters and a convenient TuPPE module for titer improvement of macrolide biopesticides in Streptomyces. More importantly, the feasible exporter mining workflow developed here might shed light on widespread applications of exporter engineering in Streptomyces to boost the production of other secondary metabolites.

Dirofilaria immitis and Dirofilaria repens are mosquito-borne pathogens responsible for dirofilariasis in humans and animals. Their transmission and spread depend on the activity of vectors and the frequency of hosts in a given area. Here, we investigated the efficacy of a monthly multimodal prophylactic (MMP) strategy against canine dirofilariasis on Corsica Island (France). The study was conducted as evidence of an efficacy trial in which eighty dogs were divided into two groups: (i) one test group consisted of 25 dogs under the MMP (per-os administration of 1.5 tablets of milbemycin-oxime-praziquantel (Milbactor®) and a topical line-on application of a 3.6 mL solution of dinotefuran-permethrin-pyriproxyfen (Vectra® 3D)) and (ii) a control group under different real-life prophylactic treatments (RLP) based on the use of ectoparasiticides (different formulations: deltamethrin, fluralaner, fipronil) and/or macrocyclic lactone-based products (milbemycin-oxime/praziquantel, milbemycin-oxime, moxidectin) during the period from June to October 2017. All animals were followed up for one year, with blood collected at day 0, with follow-up at 6 and 12 months. Samples were tested for Dirofilaria spp. by species-specific qPCR. At the end of the study, no new case of Dirofilaria spp. infection was detected in the test group. However, the cumulative incidence of Dirofilaria spp. infection was 16.4% (n = 9, p = 0.027) in the control group. The data illustrate that, in contrast to RLP treatment, which failed to protect at least 16.4% of dogs, the MMP based on the simultaneous administration of milbemycin oxime-praziquantel and dinotefuran-permethrin-pyriproxyfen efficiently protects dogs in a high-risk area from Dirofilaria spp. infection.

Milbemycin, a group of 16-membered macrolide antibiotics produced by Streptomyces milbemycinicus, has been widely used as an insecticide and an anthelmintic. To enhance the production of milbemycin, ribosomal engineering with heterologous expression of a regulatory gene was used to screen for a high milbemycin-producing mutant. The mutant S. milbemycinicus R2-6-5 isolated after the treatment of S. milbemycinicus A2079 with 6 μg/mL streptomycin produced 172.5 and 163.1% of milbemycins A3 and A4, respectively, compared with original strain. Analysis of the gene rsmG revealed a frameshift mutation, one cytidine unit being inserted into the 21 position (21C → 21CC). The heterologous regulatory gene aveR, which belongs to the LAL-family was integrated into the genome of S. milbemycinicus R2-6-5 denoted S. milbemycinicus J37 to enhance production of milbemycin. The production of milbemycins A3 and A4 in S. milbemycinicus J37 reached 758.9 and 279.0 μg/g respectively, representing 142 and 61% higher yields over S. milbemycinicus R2-6-5. The combination of ribosomal engineering and heterologous regulatory gene expression in S. milbemycinicus J37 resulted in an increase by 12.4-fold for milbemycin A3 and 11.7-fold for milbemycin A4, respectively, when compared to the original strain. Overall, these results demonstrate that combining available technologies for strain modification such as ribosome engineering technology and heterologous regulatory gene expression is an effective approach for development of high milbemycin-producing strains.

Two undescribed milbemycin derivatives, milbemycin P (1) and milbemycin Q (2), were isolated from a culture of the genetically engineered strain Streptomyces bingchenggensis BCJ60. Their structures were elucidated through the interpretation of NMR and HR-ESI-MS spectroscopic data, as well as comparison with previous reports. Their acaricidal and nematocidal activities against Tetranychus cinnabarinus and Bursaphelenchus xylophilus were tested. The results showed that compounds 1–2 possessed potent acaricidal and nematocidal activities.

Milbemycins are used commercially as insect repellents and acaricides; however, their high cost remains a significant challenge to commercial production. Hence, improving the titer of milbemycins for commercial application is an urgent priority. The present study aimed to effectively increase the titer of milbemycins using a combination of genome re-sequencing and metabolic engineering. First, 133 mutation sites were identified by genome re-sequencing in the mutagenized high-yielding strain BC04. Among them, three modifiable candidate genes (sbi_04868 encoding citrate synthase, sbi_06921 and sbi_06922 encoding alpha and beta subunits of acetyl-CoA carboxylase, and sbi_04683 encoding carbon uptake system gluconate transporter) related to primary metabolism were screened and identified. Next, the DNase-deactivated Cpf1-based integrative CRISPRi system was used in S. bingchenggensis to downregulate the transcription level of gene sbi_04868. Then, overexpression of the potential targets sbi_06921-06922 and sbi_04683 further facilitated milbemycin biosynthesis. Finally, those candidate genes were engineered to produce strains with combinatorial downregulation and overexpression, which resulted in the titer of milbemycin A3/A4 increased by 27.6% to 3164.5 mg/L. Our research not only identified three genes in S. bingchenggensis that are closely related to the production of milbemycins, but also offered an efficient engineering strategy to improve the titer of milbemycins using genome re-sequencing. KEY POINTS: • We compared the genomes of two strains with different titers of milbemycins. • We found three genes belonging to primary metabolism influence milbemycin production. • We improved titer of milbemycins by a combinatorial engineering of three targets.

Streptomycetes are characterized by their ability to produce structurally diverse compounds as secondary metabolites and by their complex developmental life cycle, which includes aerial mycelium formation and sporulation. The production of secondary metabolites is growth-stage dependent, and generally coincides with morphological development on a solid culture. Streptomyces sp. BB47 produces several types of bioactive compounds and displays a bald phenotype that is devoid of an aerial mycelium and spores. Here, we demonstrated by genome analysis and gene complementation experiments that the bald phenotype arises from the bldA gene, which is predicted to encode the Leu-tRNAUUA molecule. Unlike the wild-type strain producing jomthonic acid A (1) and antarlide A (2), the strain complemented with a functional bldA gene newly produced milbemycin (3). The chemical structure of compound 3 was elucidated on the basis of various spectroscopic analyses, and was identified as milbemycin A4, which is an insecticidal/acaricidal antibiotic. These results indicate that genetic manipulation of genes involved in morphological development in streptomycetes is a valuable way to activate cryptic biosynthetic pathways.

Dramatic decrease of sugar uptake is a general phenomenon in Streptomyces at stationary phase, when antibiotics are extensively produced. Milbemycins produced by Streptomyces bingchenggensis are a group of valuable macrolide biopesticides, while the low yield and titer impede their broad applications in agricultural field. Considering that inadequate sugar uptake generally hinders titer improvement of desired products, we mined the underlying sugar uptake systems and fine-tuned their expression in this work. First, we screened the candidates at both genomic and transcriptomic level in S. bingchenggensis. Then, two ATP-binding cassette transporters named TP2 and TP5 were characterized to improve milbemycin titer and yield significantly. Next, the appropriate native temporal promoters were selected and used to tune the expression of TP2 and TP5, resulting in a maximal milbemycin A3/A4 titer increase by 36.9% to 3321 mg/L. Finally, TP2 and TP5 were broadly fine-tuned in another two macrolide biopesticide producers Streptomyces avermitilis and Streptomyces cyaneogriseus, leading to a maximal titer improvement of 34.1% and 52.6% for avermectin B1a and nemadectin, respectively. This work provides useful transporter tools and corresponding engineering strategy for Streptomyces.

Background The Australian paralysis tick, Ixodes holocyclus, causes tick paralysis in dogs and cats in the eastern coastal regions of Australia. Prevention is the best option to protect dogs against this potentially fatal disease and sarolaner provides rapid and sustained efficacy against I. holocyclus. In this laboratory study, the efficacy of two combination endectocides containing sarolaner + moxidectin + pyrantel (Simparica Trio™) and afoxolaner + milbemycin (NexGard Spectra®) was evaluated against an artificial infestation of I. holocyclus. Methods Twenty-four (n =24) foxhounds were randomly allocated to three treatment groups and artificially infested with 30 adult female viable ticks on Days − 1, 7, 14, 21, 28 and 35. On Day 0, dogs in each treatment group were treated with either Drontal® (control group), Simparica Trio™ at the label dose to provide minimum doses of sarolaner (1.2 mg/kg), moxidectin (24 µg/kg) and pyrantel (5 mg/kg) or NexGard Spectra® to provide minimum doses of afoxolaner (2.5 mg/kg) and milbemycin (0.5 mg/kg). Live tick counts were performed at 48 and 72 hours after treatment and after each re-infestation on Days 7, 14, 21, 28 and 35. Efficacy was determined at each time point relative to counts for control dogs based on geometric means. Results Against an existing infestation, efficacy of both Simparica Trio™ and NexGard Spectra® was 99.6% and 100% at 48 and 72 h time points, respectively (P = 1.000). Against subsequent weekly infestations, treatment with Simparica Trio™ and NexGard Spectra® resulted in efficacy of ≥ 97.7% and ≥ 95.5% (P ≥ 0.0911), respectively at the 48 h time point and at the 72 h time point, Simparica Trio™ and NexGard Spectra® resulted in efficacy of ≥ 99.0% and ≥ 98.4% (P ≥ 0.0511), respectively. There were no treatment-related adverse events in the study. Conclusions Single doses of Simparica Trio™ and NexGard Spectra® were highly efficacious and provided comparable efficacy against the Australian paralysis tick, I. holocyclus for up to 35 days.

Two new milbemycin derivatives, milbemycin M (1) and milbemycin N (2), were isolated from the culture of a genetically engineered strain Streptomyces bingchenggensis BCJ60. Their structures were elucidated through the interpretation of NMR and HR-ESI-MS spectroscopic data, as well as comparison with previous reports. The acaricidal and nematicidal activities of them against Tetranychus cinnabarinus and Bursaphelenchus xylophilus were tested. The results showed that compounds 1-2 possessed potent acaricidal and nematocidal activities.

The study aimed to evaluate the effects of heat on macrocyclic lactone residues in cows' milk. Ivermectin, abamectin, doramectin, eprinomectin and moxidectin were added to raw milk in three concentrations. The milk was then pasteurised (40 seconds at 74°C or 1 minute at 80°C) and boiled (10 minutes at 100°C). The analyses were performed with a validated method: LC-MS/MS. Thermal treatment resulted in a statistically significant decrease in the abamectin, eprinomectin, and moxidectin concentrations in the milk; however, the residues did not completely degrade. Boiling resulted in a greater decrease in the moxidectin concentrations than was observed with pasteurisation. The high pasteurisation and boiling processes had a greater effect on the eprinomectin residues than did the low pasteurisation process. The pasteurisation and boiling processes did not have an effect on the doramectin and ivermectin. The study concluded that the macrocyclic lactones are generally resistant to such processes.

Milbemycins and their semisynthetic derivatives are recognized as effective and eco-friendly pesticides, whereas the high price limits their widespread applications in agriculture. One of the pivotal questions is the accumulation of milbemycin-like by-products, which not only reduces the yield of the target products milbemycin A3/A4, but also brings difficulty to the purification. With other analogous by-products abolished, α9/α10 and β-family milbemycins remain to be eliminated. Herein, we solved these issues by engineering of post-modification steps. First, Cyp41, a CYP268 family cytochrome P450, was identified to participate in α9/α10 biosynthesis. By deleting cyp41, milbemycin α9/α10 was eliminated with an increase of milbemycin A3/A4 titer from 2382.5 ± 55.7 mg/L to 2625.6 ± 64.5 mg/L. Then, MilE, a CYP171 family cytochrome P450, was determined to be responsible for the generation of the furan ring between C6 and C8a of milbemycins. By further overexpression of milE, the production of β-family milbemycins was reduced by 77.2%. Finally, the titer of milbemycin A3/A4 was increased by 53.1% to 3646.9 ± 69.9 mg/L. Interestingly, overexpression of milE resulted in increased transcriptional levels of milbemycin biosynthetic genes and production of total milbemycins, which implied that the insufficient function of MilE was a limiting factor to milbemycin biosynthesis. Our research not only provides an efficient engineering strategy to improve the production of a commercially important product milbemycins, but also offers the clues for future study about transcriptional regulation of milbemycin biosynthesis.

Overexpression of ATP-binding cassette (ABC) transporters is a major cause of drug resistance in fungal pathogens. Milbemycins, enniatin B, beauvericin and FK506 are promising leads for broad-spectrum fungal multidrug efflux pump inhibitors. The characterization of naturally generated inhibitor resistant mutants is a powerful tool to elucidate structure-activity relationships in ABC transporters. We isolated twenty Saccharomyces cerevisiae mutants overexpressing Candida albicans ABC pump Cdr1 variants resistant to fluconazole efflux inhibition by milbemycin α25 (eight mutants), enniatin B (eight) or beauvericin (four). The twenty mutations were in just nine residues at the centres of transmembrane segment 1 (TMS1) (six mutations), TMS4 (four), TMS5 (four), TMS8 (one) and TMS11 (two) and in A713P (three), a previously reported FK506-resistant 'hotspot 1' mutation in extracellular loop 3. Six Cdr1-G521S/C/V/R (TMS1) variants were resistant to all four inhibitors, four Cdr1-M639I (TMS4) isolates were resistant to milbemycin α25 and enniatin B, and two Cdr1-V668I/D (TMS5) variants were resistant to enniatin B and beauvericin. The eight milbemycin α25 resistant mutants were altered in four amino acids: G521R, M639I, A713P and T1355N. These four Cdr1 variants responded differently to various types of inhibitors, and each exhibited altered substrate specificity and kinetic properties. The data infer an entry gate function for Cdr1-G521 and a role for Cdr1-A713 in the constitutively high Cdr1 ATPase activity. Cdr1-M639I and -T1355N (TMS11) possibly cause inhibitor-resistance by altering TMS-contacts near the substrate/inhibitor-binding pocket. Models for the interactions of substrates and different types of inhibitors with Cdr1 at various stages of the transport cycle are presented.

Two new sixteen-membered macrolides, Δ2,3-tenvermectin A (1) and 13α-hydroxy milbemycin β11 (2), were isolated from the fermentation broth of the genetically engineered strain Streptomyces avermitilis MHJ1011. Their structures were determined based on extensive spectroscopic analysis and comparison with related known compounds. Compounds 1 and 2 exhibited potent acaricidal activity against Tetranychus cinnabarinus.

Polyketides represent an important class of biologically active and structurally diverse compounds found in nature. They are biosynthesized from acyl CoA precursors by polyketide synthases (PKSs). The use of combinatorial biosynthesis to form hybrid PKSs is considered to be an excellent approach for the development of novel polyketides. Here, 10 new 16-membered macrolide compounds were isolated from the broth of the genetically engineered strain Streptomyces avermitilis TM24, in which the PKS gene aveA3 was seamlessly replaced by the milbemycin PKS gene milA3. Their structures were elucidated on the basis of NMR and MS spectroscopic analyses. The acaricidal and nematicidal activities of them against Tetranychus cinnabarinus and Bursaphelenchus xylophilus were tested. The results indicated that compound 1 had potent acaricidal activity against adult mites with an LC50 value of 0.0022 mg L-1, while compounds 5 and 7 possessed potent nematicidal activity with LC50 values of 4.56 and 4.30 mg L-1, respectively.

A new β-class milbemycin, 13α-hydroxy milbemycin β6 (1), was isolated from the fermentation broth of a mutant of genetically engineered strain Streptomyces avermitilis AVE-H39. Its structure and absolute configuration were elucidated by extensive spectroscopic methods and confirmed by single crystal X-ray diffraction.

Introduction: Moxidectin is a milbemycin endectocide recently approved for the treatment of human onchocerciasis. Onchocerciasis, earmarked for elimination of transmission, is a filarial infection endemic in Africa, Yemen, and the Amazonian focus straddling Venezuela and Brazil. Concerns over whether the predominant treatment strategy (yearly mass drug administration (MDA) of ivermectin) is sufficient to achieve elimination in all endemic foci have refocussed attention upon alternative treatments. Moxidectin’s stronger and longer microfilarial suppression compared to ivermectin in both phase II and III clinical trials indicates its potential as a novel powerful drug for onchocerciasis elimination. Areas covered: This work summarizes the chemistry and pharmacology of moxidectin, reviews the phase II and III clinical trials evidence on tolerability, safety, and efficacy of moxidectin versus ivermectin, and discusses the implications of moxidectin’s current regulatory status. Expert opinion: Moxidectin’s superior clinical performance has the potential to substantially reduce times to elimination compared to ivermectin. If donated, moxidectin could mitigate the additional programmatic costs of biannual ivermectin distribution because, unlike other alternatives, it can use the existing community-directed treatment infrastructure. A pediatric indication (for children

This study examines the therapeutic and year-round prophylactic efficacy of different formulations used in dogs in three Spanish areas where canine thelaziosis is endemic. The study was conducted as a Good Clinical Practice, multicentre, randomised field study in privately owned outdoor dogs naturally infected with Thelazia callipaeda. The active pharmaceutical ingredients tested were: an oral formulation of milbemycin oxime 12.5 mg combined with praziquantel 125 mg (A), a subcutaneous sustained-release formulation of moxidectin 10 g (B), a moxidectin 2.5% weight/volume (w/v) spot-on formulation combined with imidacloprid 10% w/v (C), and an eye drop formulation (6 µg) of ivermectin 10 mg/ml diluted 10% in propylene glycol (D). Infected dogs were randomly allocated to treatment Groups A, B, C and D. Dogs testing negative for T. callipaeda inspection in two visits (Day 7/Day 14 and D30) were enrolled in the prophylaxis trial and reallocated to the corresponding study group (A, B, C or D). Treatment efficacy ranged from 70.4% recorded in Group A 1 week after treatment, to 100% recorded in Group C on Day 30 and in Group B on Day 60. Treatment was more efficacious in Group D (85.7% 1 week after treatment) than A, but was never 100% efficacious as in Groups B and C. Year-round prophylactic efficacy was 83.3% in Group A, 100% in Group B, 93.5% in Group C and 87.5% in Group D. In conclusion, products containing moxidectin were highly efficacious both in treating and preventing canine thelaziosis. Milbemycin also emerged as a good option. However, the off-label use of topical or subcutaneous ivermectin should be avoided due to possible adverse reactions such as pruritus, irritation or redness. In endemic areas, monthly prophylaxis to limit the spread of T. callipaeda to new areas across Europe and reduce zoonotic risks is essential.

Two novel milbemycin derivatives, 5,27-epoxy-13α-hydroxy milbemycin β11 (1) and 5,27-epoxy-13α-hydroxy-25-ethyl milbemycin β11 (2), were isolated from the genetically engineered strain Streptomyces avermitilis AVE-H39. Their structures were elucidated through the interpretation of HR-ESIMS and extensive NMR spectroscopic data. Compounds 1 and 2 exhibited moderate acaricidal and nematicidal activities.

Two new milbemycin metabolites, 13α-hydroxymilbemycin β13 (1) and 26-methyl-13α-hydroxymilbemycin β13 (2), were isolated from the fermentation broth of a genetically engineered strain Streptomyces avermitilis AVE-H39. Their structures were determined by the comprehensive spectroscopic data, including 1 D, 2 D NMR, MS spectral analysis and the comparison with data from the literature. Compounds 1 and 2 not only exhibited potent acaricidal activities against Tetranychus cinnabarinus, but also had nematocidal activity against Bursaphelenchus xylophilus.

The very first development of avermectins was done from isolation in Kitasato Institute laboratories, using a novel soil-dwelling bacterium. It was transmittal to the Merck & Co. research laboratory incorporation. These belong to the 16-membered, closely related family, which are macrocyclic lactones constituting of four major and four minor homologous compounds. One of the macrocyclic lactone compounds is milbemycin, which is a group of chemical related to the avermectins and was first isolated from Streptomyces hygroscopicus in 1972. They are a group of macrolides chemically associated to the ivermectins. Milbemycin, a commercially available insecticide, consists of milbemycin A3 and milbemycin A4 (30% and 70%, respectively). Milbemycin and avermectin anthelmintic groups share a common action mechanism, but the moxidectin molecular structure differs from avermectin anthelmintics, which afford much potency and high lipid solubility and therefore perseverance. Abamectin is the only compound that belongs to the family avermectin and has some application in crop protection from parasites. Apart from it, abamectin also causes oral and dermal toxicity. Other members of the macrolide group are used in antiparasitic medicines, in order to inhibit the animal products contaminating parasites. Remains of macrocyclic lactones compound including avermectin and milbemycin are used in veterinary medicines to inhibit parasites found in meat and milk.

Macrocyclic lactones (MLs) are chemical compounds administered to livestock for parasite control. These compounds are poorly metabolized and pass relatively unchanged into the dung of treated animals. When coprophagous insects such as dung beetles (Coleoptera: Scarabaeoidea) are exposed to ML residues while feeding on dung, lethal and sublethal effects are often observed. This can lead to ML residues impairing ecosystem functions that underpin agricultural production. A strategy to reduce these negative effects involves the use of compounds that offer lower risk to non-target invertebrates, such as the ML moxidectin. Considering two dung beetle species with differing sensitivities to agricultural intensification, we asked whether exposure to moxidectin residues influenced survival, reproductive output, and functioning (short- and long-term estimates of dung removal). When exposed to moxidectin, adults of the sensitive species (Geotrupes spiniger Marsham) experienced a 43% reduction in survival. In contrast, survival of the non-sensitive species (Aphodius rufipes L.) was unaffected. We were unable to determine whether exposure affected reproductive output of either species. We found little evidence to suggest moxidectin impaired dung removal. However, high densities of a species with relatively low individual functional importance (A. rufipes) can compensate for the loss of a functionally dominant species (G. spiniger). Over a longer timeframe, earthworms decomposed dung fully, irrespective of moxidectin residues. This functional redundancy reinforces that wider justifications for the conservation of biodiversity should remain integrated into agricultural policy and practice.

Streptomyces Sp FJS31-2 is a strain isolated from special habitat soils in the early stage of our laboratory for producing a new type of halogenated type II polyketide antibiotic with good anti-MRSA activity. In this experiment, a variety of chromatographic and spectroscopic methods was used to isolate and identify a milbemycin compound VM48130 from the ethyl acetate extract of the fermentation products. To investigate its bioactivity, Cell Counting Kit-8 (CCK-8) assay was used to test the cytotoxic activity of the compound against a variety of cancer cells (human liver cancer cell line MHCC97H and SK-Hep1, human nasopharyngeal carcinoma cell line CNE1, mouse melanoma cell line B16, human colon cancer cell line LOVO, human lung adenocarcinoma cell line A549) and normal cells (human bronchial epithelial cell line 16HBE, human normal liver cell line L02, human nasopharyngeal epithelial cell line NP69). The results showed that the compound had significant cytotoxic activity against the above cancer cells, and the IC50 values were 21.96 ± 1.45, 22.18 ± 0.55, 19.42 ± 0.71, 18.61 ± 1.68, 18.62 ± 0.67, 18.52 ± 0.64 μM, respectively. Furthermore, the CCK-8 method was used to evaluate the compound's reversal of cisplatin resistance in multidrug resistant cisplatin-resistant human lung adenocarcinoma (A549/DDP) cells. The results indicated that when the compound concentration was 0.5 μM, the reversal fold (RF) reached 6.25 and showed a dose-dependent effect. At 5 μM, the RF reached 8.35, which was approximately equivalent to the reversal effect of the positive drug verapamil at the same concentration. The expression of MDR1, MRP1, LRP, MAST1 resistance genes and the corresponding proteins were analyzed by quantitative RT-PCR and Western blot assay, and found that the compound could significantly down-regulate the expression of these genes and proteins. These results indicated that VM48130 had the potential of being a lead compound for the treatment or adjuvant treatment of cancer.

Motility is a commonly used in vitro phenotype for assessing anthelmintic activity of candidate compounds, and for detecting anthelmintic resistance in nematodes. Third-stage larvae (L3) of parasitic nematodes are commonly used in motility-based assays because L3 are simple to obtain and can remain viable in storage for extended periods. To improve the measurement of motility of microscopic stages of nematodes, our laboratory developed the Worminator, which quantitatively measures motility of parasites. Using the Worminator, we compared the dose-response characteristics of several avermectin/milbemycin (AM) compounds using L3 from both AM-susceptible and AM-resistant Cooperia spp. (abamectin, doramectin, eprinomectin, ivermectin, moxidectin) and Haemonchus contortus (eprinomectin, ivermectin, moxidectin). Concentrations tested with the Worminator ranged from 0.156 to 40 μM. Differences in EC50 between AM-susceptible and AM-resistant isolates of Cooperia spp. and Haemonchus contortus were small, with resistance ratios ranging from 1.00 to 1.34 for Cooperia spp., 0.99 to 1.65 for Haemonchus contortus. Larval migration inhibition assays were conducted using the same isolates and were equally ineffective for detection of resistance with resistance ratios less than 2.0. These results contrast with those of the Larval Development Assay where we obtained a resistance ratio of 16.48 using the same isolates of Haemonchus contortus. Moreover, even at the highest concentration tested (40 μM), 100% inhibition of motility was never achieved and EC50 for Worminator assays were more than 100× higher than peak plasma levels achieved in vivo following treatment. These data demonstrate that dose-response characteristics for inhibition of motility in L3 of gastrointestinal nematodes of livestock do not significantly differ for AM-susceptible and AM-resistant isolates. These data challenge the suitability of motility as a phenotype for detecting and measuring resistance to AM drugs in gastrointestinal nematodes of livestock., Graphical abstract Image 1, Highlights • Motility of L3 is a poor phenotype for detection of avermectin resistance. • Resistance ratios were less than 2.0 between susceptible and resistant isolates. • Confidence intervals overlapped between susceptible and resistant isolates. • Concentration to inhibit L3 motility is 100× peak plasma concentration in vivo.

Pearsonema (syn. Capillaria) plica is a nematode that resides in the urinary bladder of canids, felids and mustelids (definitive hosts) and is classified in the same class as Trichuris spp. Epidemiological and clinical data on Pearsonema plica infection in domestic animals are limited. The nematode has an indirect lifecycle that involves earthworms as intermediate hosts. A six-year-old crossbred dog from Greece, presented a history of intermittent pollakiuria and hematuria. At urine analysis, P. plica eggs were found in the urine sediment. The dog was successfully treated with a double dose of milbemycin. To the best of our knowledge this is the first case of urinary capillariasis diagnosed in a domestic animal in Greece.

Background The macrocyclic lactones abamectin and milbemectin are frequently used to control phytophagous mites such as Tetranychus urticae. Consequently, resistance has developed and was genetically linked with substitutions in the glutamate-gated chloride channel (GluCl) subunits TuGluCl1 and TuGluCl3. Here, we functionally validated a G326E substitution in TuGluCl3 by functional expression in Xenopus laevis oocytes followed by two-electrode voltage-clamp electrophysiology. Results Homomeric wild-type and mutated GluCl3 were successfully expressed. L-glutamic-acid-induced currents exhibited a rapid onset equal in both channels and EC50 for glutamate was in the micromolar range (384.2 μM and 292.7 μM, respectively). Abamectin and milbemycin A4 elicited sustained currents in wild-type GluCl3 channels, but the G326E substitution completely abolished the agonistic activity of macrocyclic lactones. Conclusion A target-site mutation in GluCl3 contributes to avermectin resistance in T. urticae. However, given the multitude of channel genes and the potential additive or synergistic effects of mutations, to what extent mutations determine the often extremely strong resistance phenotype in the field deserves further study.

Summary Herein, we examined two dogs from central Italy with chronic severe upper respiratory signs associated with Eucoleus boehmi eggs shedding at coproscopy. In the first one, improvement in clinical signs was observed after fenbendazole administration, but complete negativity at coprological analysis was gained only after treatment with milbemycin oxyme by mouth. The second one failed to respond to two monthly administrations of a spot-on formulation with imidacloprid/moxidectin, and improvement in clinical signs and negativity at coproscopy were gained only after administration of milbemycin oxyme by mouth. Results here obtained underline the importance to perform further studies to evaluate epidemiological and clinical behavior of this neglected nematode.

The mechanisms of terbinafine resistance in a set of clinical isolates of Trichophyton rubrum have been studied recently. Of these isolates, TIMM20092 also showed reduced sensitivity to azoles. The azole resistance of TIMM20092 could be inhibited by milbemycin oxime, prompting us to examine the potential of T. rubrum to develop resistance through multidrug efflux transporters., The mechanisms of terbinafine resistance in a set of clinical isolates of Trichophyton rubrum have been studied recently. Of these isolates, TIMM20092 also showed reduced sensitivity to azoles. The azole resistance of TIMM20092 could be inhibited by milbemycin oxime, prompting us to examine the potential of T. rubrum to develop resistance through multidrug efflux transporters. The introduction of a T. rubrum cDNA library into Saccharomyces cerevisiae allowed the isolation of one transporter of the major facilitator superfamily (MFS) conferring resistance to azoles (TruMFS1). To identify more azole efflux pumps among 39 ABC and 170 MFS transporters present within the T. rubrum genome, we performed a BLASTp analysis of Aspergillus fumigatus, Candida albicans, and Candida glabrata on transporters that were previously shown to confer azole resistance. The identified candidates were further tested by heterologous gene expression in S. cerevisiae. Four ABC transporters (TruMDR1, TruMDR2, TruMDR3, and TruMDR5) and a second MFS transporter (TruMFS2) proved to be able to operate as azole efflux pumps. Milbemycin oxime inhibited only TruMDR3. Expression analysis showed that both TruMDR3 and TruMDR2 were significantly upregulated in TIMM20092. TruMDR3 transports voriconazole (VRC) and itraconazole (ITC), while TruMDR2 transports only ITC. Disruption of TruMDR3 in TIMM20092 abolished its resistance to VRC and reduced its resistance to ITC. Our study highlights TruMDR3, a newly identified transporter of the ABC family in T. rubrum, which can confer azole resistance if overexpressed. Finally, inhibition of TruMDR3 by milbemycin suggests that milbemycin analogs could be interesting compounds to treat dermatophyte infections in cases of azole resistance.

Rabbits (Oryctolagus cuniculi) are very popular as pets. However, problems of otitits caused by Psoroptes cuniculi are one of the main reasons to visit the veterinarian. Isoxazolines are an alternative treatment to treat this mite, and therefore, an evaluation of the effectiveness of oral afoxalaner with milbemycin oxime in rabbits infected with P. cuniculi was carried out. Nineteen rabbits, of New Zealand breed, with otitis due to an infection with P. cuniculi, were treated, whereas six rabbits were left untreated and formed the control group. The ear canals of each individual were examined, through the collection of otic exudate samples with cotton swabs. These were visualized under the microscope to identify the ectoparasite. Each animal was treated with a single oral dose of 2.50 mg / kg of afoxolaner, and 0.50 mg / kg of milbemycin oxime. Clinical signs and lesions associated with the infection, such as the presence of detritus, cerumen and / or scabs, and erythema, were evaluated. After receiving the treatment, all the lesions were classified as: mild, moderate and intense, with a visual analog scale. A week after providing medication, there was a decrease in the lesions of the group treated with Nexgard Spectra®, without further topical or systemic treatment. The decrease was gradual in the treated group and no recurrence was detected of P. cuniculi infection in both ears. Thus, the administration of a single oral dose of afoxolaner with milbemycin oxime was effective for the treatment of P. cuniculi infection in rabbits.

Agradecimentos: The authors gratefully acknowledge financial support from the São Paulo State Research Foundation (FAPESP, grants #2013/09543-7 and #2018/03571-2) and the Brazilian National Council for Scientific and Technological Development (CNPq, grant #301737/2017-7). FAPESP provided a scholarship to Fabrício de Oliveira Ferreira (grant #2013/25670-9) Abstract: Avermectins and moxidectin are antiparasitics widely used as active pharmaceutical ingredients in veterinary medicine, as well as in pesticide formulations for pest control in agriculture. Although the use of these compounds provides benefits to agribusiness, they can impact the environment, since a large part of these substances may reach the soil and water from the excreta of treated animals and following direct applications to crops. The present work had the objective of evaluating the dissipation behaviors of abamectin, doramectin, eprinomectin, ivermectin, and moxidectin in four native Brazilian soils of different textural classes (clay, sandy-clay, sandy, and sandy-clay-loam), following OECD Guideline 307. The studies were conducted in a climate chamber at 22 °C, 71% relative humidity, and protected from light. The dissipation studies were carried out with all drugs together, since no difference was verified when studies were done with each drug separately. The concentrations of the drugs in the soils were determined using an ultra-high performance liquid chromatograph coupled to a fluorescence detector or a tandem mass spectrometer. The dissipation half-life (DT50) values ranged from 9 to 16 days and the calculated GUS index values were in the range from -1.10 to 0.08, indicating low mobility of the drugs in the soils evaluated and low tendency for leaching. In addition, a field study was carried out to evaluate the dissipation of abamectin after application of a foliar pesticide in an orange crop. A DT50 of 9 days was determined, which was similar to that obtained under controlled conditions in the climate chamber (12 days), indicating that biotransformation was the primary process influencing the overall dissipation CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQ FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP Fechado

Two new milbemycin derivatives, 27-methoxylmilbemycin α31 (1) and 27-oxomilbemycin α31 (2), were isolated from the genetically engineered strain Streptomyces bingchenggensis BCJ60. Their structures were determined by 1D NMR, 2D NMR and HR-ESI-MS spectral analysis, and comparison with previous reports. The acaricidal and nematocidal capacities of compounds 1 and 2 were evaluated against Tetranychus cinnabarinus and Bursaphelenchus xylophilus, respectively. The results showed that the two new macrocyclic lactones 1 and 2 possessed potent acaricidal and nematocidal activities.

Dirofilaria immitis, a filarial nematode, causes dirofilariasis or heartworm disease in dogs, cats and wild canids. Effective prevention of the disease is mainly by the use of the macrocyclic lactone class of drugs as heartworm preventives, and no other class of drugs is effective for preventing infection. Macrocyclic lactones have been used for prevention of heartworm infection for more than 26years. However, prevention has been compromised by the development of resistance in recent years. The mechanism of macrocyclic lactone resistance in D. immitis has yet to be established. In other parasitic nematodes, P-glycoproteins (PGPs) have been implicated in macrocyclic lactone resistance. The presence of two polymorphic loci on D. immitis P-glycoprotein-11 (Dim-pgp-11) correlated with loss of efficacy of macrocyclic lactone anthelmintics, suggesting that PGPs may be involved in macrocyclic lactone resistance in D. immitis. We have identified the full length of Dim-Pgp-11 cDNA, expressed it in mammalian cells, and studied the functional activity of the expressed protein. We have characterised its interaction with the four macrocyclic lactone preventives, ivermectin, selamectin, moxidectin and milbemycin oxime, using the transport of different fluorescent substrates. The inhibitory effect of these macrocyclic lactones on the transport of two fluorophore probes, Rhodamine 123 and Hoechst 33342, by Dim-PGP-11 has been studied. The avermectins, ivermectin and selamectin, markedly inhibited Rhodamine 123 transport in a concentration-dependent and saturable manner, whereas the milbemycins, moxidectin and milbemycin oxime, were found to have different inhibition profiles with Rhodamine 123 transport. However, both avermectins and milbemycin preventives inhibited the transport of Hoechst 33342 by Dim-PGP-11 in a concentration-dependent and apparently saturable manner, although differences existed in terms of efficiency and potency of inhibition between the two sub-classes of macrocyclic lactones. We postulate that Dim-PGP-11 may have two to three drug binding sites, as with mammalian Pgp, including the 'R' site for Rhodamine 123 and the 'H' site for Hoechst 33342. The avermectins appear to bind the 'R' binding site unlike the milbemycins, whereas both sub-classes of macrocyclic lactones might interact with the 'H' site of D. immitis PGP-11.

Three new 16-membered macrolide compounds, 13α-O-α-l-oleandrosyl milbemycin β3 (1), 13α-O-α-l-oleandrosyl-25-ethyl milbemycin β3 (2), 13α-O-α-l-oleandrosyl-25-isopropyl milbemycin β3 (3), were isolated from the genetically engineered strains Streptomyces avermitilis MHJ1011. Their structures were determined on the basis of spectroscopic analysis, including 1D and 2D NMR techniques as well as ESI-MS and comparison with data from the literature. Both compounds 1–3 displayed impressive acaricidal activity against larval mites with the IC50 values of 0.0327, 0.0276 and 0.0235 mg/L, respectively, which are higher than those of 13α-hydroxy milbemycin β3 and 13α-hydroxy-25-ethyl milbemycin β3.

Milbemycins, a group of 16-membered macrolide antibiotics, are used widely as insecticides and anthelmintics. Previously, a limited understanding of the transcriptional regulation of milbemycin biosynthesis has hampered efforts to enhance antibiotic production by engineering of regulatory genes. Here, a novel ArpA/AfsA-type system, SbbR/SbbA (SBI_08928/SBI_08929), has been identified to be involved in regulating milbemycin biosynthesis in the industrial strain S. bingchenggensis BC04. Inactivation of sbbR in BC04 resulted in markedly decreased production of milbemycin, while deletion of sbbA enhanced milbemycin production. Electrophoresis mobility shift assays (EMSAs) and DNase I footprinting studies showed that SbbR has a specific DNA-binding activity for the promoters of milR (the cluster-situated activator gene for milbemycin production) and the bidirectionally organized genes sbbR and sbbA. Transcriptional analysis suggested that SbbR directly activates the transcription of milR, while represses its own transcription and that of sbbA. Moreover, 11 novel targets of SbbR were additionally found, including seven regulatory genes located in secondary metabolite biosynthetic gene clusters (e.g., sbi_08420, sbi_08432, sbi_09158, sbi_00827, sbi_01376, sbi_09325, and sig24sbh ) and four well-known global regulatory genes (e.g., glnRsbh , wblAsbh , atrAsbh , and mtrA/Bsbh ). These data suggest that SbbR is not only a direct activator of milbemycin production, but also a pleiotropic regulator that controls the expression of other cluster-situated regulatory genes and global regulatory genes. Overall, this study reveals the upper-layer regulatory system that controls milbemycin biosynthesis, which will not only expand our understanding of the complex regulation in milbemycin biosynthesis, but also provide a basis for an approach to improve milbemycin production via genetic manipulation of SbbR/SbbA system.

none 5 no The use of a low-dose infusion of intravenous lipid emulsion (ILE) in five young cats presented with acute neurotoxicity is reported. Suspected toxic compounds were permethrin (three, dermal), fipronil/eprinomectin/praziquantel (one, oral) and milbemycin (one, oral). Cats presented with acute onset of generalised tremors of variable severity. Initial treatment included toxin decontamination whenever possible, and control of neurological signs by administration of benzodiazepine and eventually a continuous rate infusion of propofol. Due to persisting clinical signs, a low-dose ILE therapy was additionally given. Discontinuation of treatment, once a resolution of neurological signs was achieved, ranged between two and nine hours from hospital admission. All the cats fully recovered and were discharged within 18–92 hours. No side effects were noticed and follow-up was normal. The authors report the use of a low-dose infusion of ILE as an adjunctive and safe treatment for the management of lipophilic intoxications in cats. mixed Marco Pelizzola, Clara Mattavelli, Roberta Troìa, Elsa Murgia, Massimo Giunti Marco Pelizzola, Clara Mattavelli, Roberta Troìa, Elsa Murgia, Massimo Giunti

Macrocyclic lactones (MLs) comprised of a number of compounds (doramectin, eprinomectin, ivermectin, milbemycin, moxidectin and selamectin) that are commonly used as insecticides, acaricides and nematicides in various animal species. Any species can be poisoned with a large acute dose of these compounds, but most often dogs are intoxicated following accidental exposure to the products meant for cattle, sheep or horses. Individuals of over 11 different dog breeds have been found to possess defects in the ABCB1 gene which result in a defective p-glycoprotein in the blood–brain barrier, and makes these individuals particularly sensitive to these compounds. MLs act as agonists of the neurotransmitters glutamate and gamma-aminobutyric acid (GABA), which are major inhibitory neurotransmitters. The clinical signs are of CNS toxicity, which can occur at lower doses in individuals with defective or immature blood–brain barriers than in normal individuals. Poisoned animals may also exhibit the signs of ataxia, diarrhea, dyspnea, miosis, etc. which may be associated with GABA-mediated cholinergic effects. There is no specific antidote available, although the use of intravenous lipid infusions has recently been shown to reduce the duration and severity of signs in some cases of ML toxicosis. In refractory cases, treatment rests with symptomatic and supportive therapies.

Moxidectin is a macrocyclic lactone belonging to milbemycin family closely related to ivermectin and is currently progressing towards Phase III clinical trial against human infection with the filaria Onchocerca volvulus (Leuckart, 1894). There is a single report on the microfilaricidal and embryostatic activity of moxidectin in case of the human lymphatic filarial parasite Brugia malayi (Brug, 1927) in Mastomys coucha (Smith) but without any adulticidal action. In the present study, the in vitro and in vivo antifilarial efficacy of moxidectin was evaluated on, B. malayi. In vitro moxidectin showed 100% reduction in adult female worm motility at 0.6 µM concentration within 7 days with 68% inhibition in the reduction of MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide dye) (which is used to detect viability of worms). A 50% inhibitory concentration (IC50) of moxidectin for adult female parasite was 0.242 µM, for male worm 0.186 µM and for microfilaria IC50 was 0.813 µM. In adult B. malayi-transplanted primary screening model (Meriones unguiculatus Milne-Edwards), moxidectin at a single optimal dose of 20 mg/kg by oral and subcutaneous route was found effective on both adult parasites and microfilariae. In secondary screening (M. coucha, subcutaneously inoculated with infective larvae), moxidectin at the same dose by subcutaneous route brought about death of 49% of adult worms besides causing sterilisation in 54% of the recovered live female worms. The treated animals exhibited a continuous and sustained reduction in peripheral blood microfilaraemia throughout the observation period of 90 days. The mechanism of action of moxidectin is suggested to be similar to avermectins. The in silico studies were also designed to explore the interaction of moxidectin with glutamate-gated chloride channels of B. malayi. The docking results revealed a close interaction of moxidectin with various GluCl ligand sites of B. malayi.

Background Monthly topical and sustained-release injectable formulations of moxidectin are currently marketed; however, an oral formulation, while approved at a dose of 3 μg/kg, is not currently marketed in the United States. Although resistance of heartworms to all macrocyclic lactone (ML) heartworm preventives (ivermectin, milbemycin, selamectin and moxidectin) has been demonstrated, to date no data have been reported on the effectiveness of oral moxidectin against recent isolates of Dirofilaria immitis. Methods A total of nine studies were conducted to determine the efficacy of moxidectin against a range of older and recently sourced heartworm isolates. Dogs (groups of three to eight) were inoculated with 50 D. immitis infective larvae (L3) from nine different isolates (MP3, Michigan, JYD-34, ZoeMO-2012, ZoeKy-2013, ZoeLA-2013, GCFL-2014, AMAL-2014 and ZoeAL-2015) and treated 28–30 days later with single oral doses of 3 μg/kg of moxidectin. Additionally, one group of dogs that was inoculated with JYD-34 was treated monthly for 3 consecutive months beginning 30 days post inoculation. Dogs were held for approximately 4 months after the initial (or only) treatment and then necropsied for recovery of adult heartworms. Results A single dose of 3 μg/kg of moxidectin was 100% effective in preventing the development of five of nine heartworm isolates (MP3, Michigan, ZoeKy, GCFL and ZoeAL isolates), confirming their susceptibility to oral moxidectin at this dose. MP3 and Michigan are isolates sourced from the field more than 9 years ago, while ZoeKy, ZoeAL and GCFL were isolated from the field within the past 2 to 3 years. Against JYD-34, ZoeMO, ZoeLA and AMAL isolates, a single dose of 3 μg/kg of moxidectin was not completely effective, with efficacies of 19%, 82%, 54% and 62%, respectively, demonstrating resistance of these heartworm isolates to oral moxidectin at this dosage. Three consecutive monthly doses of 3 μg/kg of moxidectin were also incompletely effective against the JYD-34 isolate, with an efficacy of 44%. JYD-34 was originally isolated in 2010, while ZoeMO, ZoeLA and AMAL were isolated within the past 2 to 3 years. Conclusions A single oral dose (3 μg/mg) of moxidectin was 100% effective in preventing the development of ML-susceptible heartworm isolates while being incompletely effective against ML-resistant isolates.

Milbemycins are macrolide antibiotics with a broad spectrum of nematocidal, insecticidal, and acaricidal activity. To obtain milbemycin A3/A4 derivatives suitable for chemical conjugation to protein carriers (milbemycin haptens), succinate linker and a novel 17-atom-long linker containing a terminal carboxylic acid group were attached to the milbemycin core in a protecting group-free synthesis. The obtained milbemycin A3/A4 derivatives were coupled to Potato virus Y-like nanoparticles by the activated ester method. The reaction products were characterized and used in mice immunization experiments. It was found that the mice developed weak specific immune responses toward all tested milbemycin haptens.

Background Milbemycins, produced from Streptomyces hygroscopicus subsp. aureolacrimosus and Streptomyces bingchenggensis, are 16-membered macrolides that share structural similarity with avermectin produced from Streptomyces avermitilis. Milbemycins possess strong acaricidal, insecticidal, and anthelmintic activities but low toxicity. Due to the high commercial value of the milbemycins and increasing resistance to the avermectins and their derivatives, it is imperative to develop an efficient combinatorial biosynthesis system exploiting an overproduction host strain to produce the milbemycins and novel analogs in large quantities. Results The respective replacement of AveA1 and AveA3 (or module 7 in AveA3) of the avermectin polyketide synthase (PKS) in the avermectin high-producing strain S. avermitilis SA-01 with MilA1 and MilA3 (or module 7 in MilA3) of the milbemycin PKS resulted in the production of milbemycins A3, A4, and D in small amounts and their respective C5-O-methylated congener milbemycins B2, B3, and G as major products with total titers of approximately 292 mg/l. Subsequent inactivation of the C5-O-methyltransferase AveD led to a production of milbemycins A3/A4 (the main components of the commercial product milbemectin) in approximately 225 and 377 mg/l in the flask and 5 l fermenter culture, respectively, along with trace amounts of milbemycin D. Conclusions We demonstrated that milbemycin biosynthesis can be engineered in the avermectin-producing S. avermitilis by combinatorial biosynthesis with only a slight decrease in its production level. Application of a similar strategy utilizing higher producing industrial strains will provide a more efficient combinatorial biosynthesis system based on S. avermitilis for further enhanced production of the milbemycins and their novel analogs with improved insecticidal potential. Electronic supplementary material The online version of this article (doi:10.1186/s12934-017-0626-8) contains supplementary material, which is available to authorized users.

International audience; Faecal egg count reduction tests (FECRTs) were performed on 21 goat farms in Guadeloupe (FWI). Anthelmintic resistance (AR) to netobimin (benzimidazole) was found in all 15 herds in which it was tested. AR to ivermectin (avermectin) and levamisole (imidazothiazole) were also very largely spread (14 out of 17 farms and 7 out of 9 farms, respectively). AR to the final moxidectin (milbemycin) released was already present in 2 out of 9 farms in which it was tested. Haemonchus was the dominant genus of gastrointestinal nematodes and was more frequently found to be resistant to netobimin, ivermectin and moxidectin than Trichostrongylus, the latter appeared to be more often resistant to levamisole. A first survey 15 years ago revealed only AR to benzimidazoles and one suspected case of AR to ivermectin.

Azole resistance in Candida glabrata, a pathogenic yeast, has prompted studies of compounds that have therapeutic potential by reversing azole resistance. Milbemycin A4 oxime blocked azole efflux and enhanced azole susceptibility four-fold in 28 clinical isolates of C. glabrata. Specificity of the milbemycin A4 oxime effect depended on the drug transporter and the substrate being effluxed. The major effect of milbemycin A4 oxime was inhibition of azole and rhodamine 6G efflux by the ATP-Binding Cassette (ABC) transporters CgCDR1 and PDH1. Milbemycin A4 oxime effect did not extend to oligomycin, transported by the ABC transporter YOR1 or to benomyl, transported by the Major Facilitator Superfamily transporter, CgFLR1. Milbemycin A4 oxime did not suppress transcription of CgCDR1 but increased CgCDR1 expression 126-fold. Selectivity of the effect is compatible with the concept that milbemycin A4 oxime may interact directly with a one or more drug-binding sites of the major azole transporters.

Summary Anthelmintics of the avermectin/milbemycin class continue to demonstrate the highest efficacy against cyathostomin nematodes; however, over-reliance on these drugs is increasing the likelihood that recent emerging resistance to these drugs will worsen. Use of benzimidazole and pyrantel compounds given concurrently may provide sufficiently high efficacy to serve as a viable treatment alternative to the avermectin/milbemycin drugs, thus reducing the selective pressures for resistance development. The study was conducted to determine if oxibendazole (OBZ) and pyrantel pamoate (PYR) would have greater efficacy when used in combination vs. what either drug could achieve individually, and to determine if the combination protocol would consistently achieve faecal egg count reduction (FECR) rates >90%, the general threshold for acceptable efficacy. Horses of various ages and breeds on 11 horse farms were assigned randomly to treatment with OBZ (n = 34), PYR (n = 35), or a combination of both (n = 61). A faecal egg count was performed for each horse prior to treatment and 9–14 days post treatment. Mean FECR percentages were calculated for each treatment group on individual farms, and for each treatment group across all farms. Combination treatment achieved >90% mean FECR on all 11 farms, and >95% on 9 of the 11 farms. Overall arithmetic mean FECR rates were 90.03%, 81.10% and 96.35% for horses treated respectively with OBZ, PYR, and the 2 drugs in combination. Combination treatment with OBZ and PYR demonstrated an additive effect whereby horses given both anthelmintics concurrently had mean FECR percentages that were significantly greater than in horses given either drug alone. Combination treatment with OBZ and PYR can be an efficacious and viable treatment choice for controlling cyathostomins, thereby reducing the over-reliance on avermectin/milbemycin drugs.

The aim of this study was to compare the liveweight gain of lambs, infected by multidrug-resistant nematodes, treated by conventional schemes of helminth control or using a schedule based on fecal egg count reduction test (FECRT). The flock was selected after a FECRT (experiment 1) which revealed a parasite population resistant to benzimidazoles, imidazothiazoles, macrocyclic lactones (ivermectin), salicylanilides, nitrophenols, and organophosphates. Despite the parasite resistance to ivermectin (an avermectin), the moxidectin (a milbemycin) was effective against the gastrointestinal nematodes (PR > 90 %). In experiment 2, 48 suckling lambs were distributed in four randomized blocks (G1, G2, G3, and G4) by previous body weighings. G1 was kept as untreated control; G2 was treated following a FECRT-based schedule with drugs chosen based on fecal analysis (first drench with moxidectin, second drench with a combination of moxidectin and levamisole, and third drench with praziquantel, an anti-cestode drug); G3 and G4 received three drenches with ivermectin or disophenol, respectively. Body weighings and fecal analysis of these lambs were performed every 2 weeks over a 98-day period. An effective control of gastrointestinal nematodes was obtained with two nematicidal drenches following the FECRT-based schedule of treatments. On the other hand, eggs per gram of feces (EPG) counts were no different among untreated control, G3, and G4. Lambs treated using the FECRT-based schedule had the greatest liveweight gain among the groups tested. Additionally, liveweight gain was no different among the groups G3, G4, and G1. The FECRT-based schedule of anthelmintic treatments was beneficial regarding productivity and sustainability of helminth control in lambs infected by multidrug-resistant nematodes.

Graphical abstract, Highlights • Glutamate-gated chloride channels (GluCl) are targets for avermectin/milbemycin (A/M) anthelmintics. • Little is known regarding the mechanism of A/M anthelmintics to reduce microfilaria production and release in filarial worms. • We show that two GluCl subunits gene are expressed in developing embryos, and reproductive tissues of adult worms. • The results may explain the temporary sterilizing effects of A/M drugs on filarial worms., Glutamate-gated chloride channels (GluCl) are targets for avermectin/milbemycin (A/M) anthelmintics such as ivermectin that cause paralysis of somatic and pharyngeal muscles in gastrointestinal nematodes. Ivermectin is useful for onchocerciasis control programs because of its activity against microfilariae that often cause ocular disease and severe dermatitis. However, mechanisms responsible for reduced microfilaria production by adult worms following ivermectin treatment are poorly understood. We synthesized subunit-specific RNA probes for the Brugia malayi GluCl gene avr-14 (BmAVR-14) to localize expression of this gene in adult filarial worms. Both subunits of BmAVR-14 exhibited very similar expression patterns. In female worms, strong expression signals were detected in the ovary, developing embryos and lateral hypodermal chords, with moderate expression in the uterus wall adjacent to stretched microfilariae. These genes were also highly expressed in adult male worms (in spermatogonia, in the wall of the vas deferens, and in the lateral chords, but not in mature spermatozoa). In addition, avr-14 was highly expressed in somatic muscles adjacent to the terminal end of the vas deferens which contains mature sperm. These results show that avr-14 is highly expressed in B. malayi developing embryos and reproductive tissues, and they provide evidence for the involvement of GluCl in gamete production and embryogenesis in filarial worms. This may explain the observed suppression of microfilaria (Mf) production by female worms following treatment with avermectin/milbemycin anthelmintics.

Fish lice, ectoparasites of the genus Argulus, are branchurian crustaceans that can significantly impact fish health by causing mechanical damage to cutaneous barriers and increasing susceptibility to other infections. While many treatments have been reported in teleosts and invertebrates, there are no published treatments for elasmobranchs. This study evaluated the safety and efficacy of a commercial formulation of milbemycin oxime and lufenuron in freshwater stingrays for treatment of Argulus spp. Seven juvenile Magdalena river stingrays (Potamotrygon magdalenae) and 10 juvenile smooth back river stingrays (Potamotrygon orbignyi) had severe infestations of Argulus spp. that were identified visually and microscopically. Animals were treated with milbemycin oxime and lufenuron (at 0.015 mg/L and 0.30 mg/L, respectively) in a 6-hr immersion once weekly for two treatments. They were visually examined for skin lesions as well as behavior and appetite daily by animal care staff. A subset of animals was euthanized and necropsied on days 8, 9, 43, and 78 after treatment initiation. There were no Argulus spp. detected at the time of the second treatment. Complete gross and histologic evaluations were completed for all animals. At all time points, no gross abnormalities were detected with the exception of thin body condition in some animals; no Argulus spp. were noted. Histologic lesions were all attributed to poor nutritional state at the time of acquisition. No histologic evidence of acute or chronic toxicosis was detected. The commercial formulation of milbemycin oxime and lufenuron, applied at the dose and for the exposure time used in this study, effectively eradicated Argulus spp. in a population of juvenile P. magdalenae and P. orbignyi, and did not cause mortality or clinical gross or histologic evidence of acute or chronic toxicity.

ATP-binding cassette transporter G2/breast cancer resistance protein (ABCG2/BCRP) mediates drug–drug interactions that affect the secretion of drugs into milk. The aims of this study were: (1) to determine whether the major plasma metabolites of the flukicide triclabendazole (TCBZ), triclabendazole sulfoxide (TCBZSO) and triclabendazole sulfone (TCBZSO 2 ), inhibit ovine and bovine ABCG2 and its Y581S variant in vitro, and (2) to examine whether coadministration of TCBZ with the ABCG2 substrates danofloxacin (a fluoroquinolone) and moxidectin (a milbemycin) affects the secretion of these drugs into the milk of sheep. TCBZSO and TCBZSO 2 inhibited ruminant ABCG2 in vitro by reversing the reduced mitoxantrone accumulation and reducing basal to apical transport of nitrofurantoin in cells transduced with bovine variants (S581 and Y581) and the ovine variant of ABCG2. Coadministration of TCBZ with moxidectin or danofloxacin to sheep resulted in significantly reduced levels of moxidectin, but not danofloxacin, in the milk of TCBZ-treated sheep compared to sheep administered moxidectin or danofloxacin alone. The milk area under concentration time curve (AUC 0–48 h) was 2.99 ± 1.41 μg h/mL in the group treated with TCBZ and moxidectin, and 7.75 ± 3.58 μg h/mL in the group treated with moxidectin alone. The AUC (0–48 h) milk/plasma ratio was 37% lower in the group treated with TCBZ and moxidectin (7.34 ± 1.51) than in the group treated with moxidectin alone (11.68 ± 3.61). TCBZ metabolites appear to inhibit ruminant ABCG2 and affect the secretion of ABCG2 substrates into milk of sheep.

The objective of this randomized, blinded, placebo controlled laboratory study was to confirm the efficacy of a single oral administration of two marketed formulations of milbemycin oxime (Interceptor(®) Flavor Tabs(®) and Sentinel(®) Flavor Tabs(®)) at a minimum dose of 0.5 mg/kg (0.23 mg/lb) against natural infections of Ancylostoma braziliense in dogs. Thirty-six hookworm infected dogs, a minimum of 10 weeks of age and of various breeds and genders were used. Fecal egg counts were done on three separate days prior to treatment for randomization purposes. Dogs were ranked by descending order of the fecal egg count arithmetic means and randomly assigned to either the two milbemycin treatment groups or the placebo control group in blocks of three dogs each, 12 dogs per group. Dogs were dosed according to the product label with blinding maintained by separation of function. Worm counts were done at necropsy 7 days after treatment. Reduction in A. braziliense worm counts in the milbemycin groups were compared to the placebo control group using analysis of variance of the A. braziliense logarithmic mean worm counts and percent efficacy was based on geometric means. Efficacy was defined as the ability of the test products to significantly (p≤0.05) reduce parasite load by 90% or greater in treated dogs when compared to adequately infected placebo control dogs. The placebo control group had a geometric mean worm count of 19.2. The Interceptor treated group had a geometric mean worm count of 0.38 representing a 98% reduction in parasite load and the Sentinel treated group had a geometric mean worm count of 0.98 representing a 95% reduction in parasite load. Both reductions were highly significant (p0.0001). In this study, milbemycin oxime, when administered as two marketed formulations at a minimum dose of 0.5 mg/kg (0.23 mg/lb), was efficacious for removing adult A. braziliense in naturally infected dogs.

Macrocyclic lactones (MLs), specifically the avermectins and milbemycins, are known for their effectiveness against a broad spectrum of disease-causing nematodes and arthropods in humans and animals. In most nematodes, drugs in this class induce paralysis, resulting in starvation, impaired ability to remain associated with their anatomical environment, and death of all life stages. Initially, this was also thought to be the ML mode of action against filarial nematodes, but researchers have not been able to validate these characteristic effects of immobilization/starvation of MLs in vitro, even at higher doses than are possible in vivo. Relatively recently, ML receptor sites exclusively located proximate to the excretory-secretory (ES) apparatus were identified in Brugia malayi microfilaria and an ML-induced suppression of secretory protein release by B. malayi microfilariae was demonstrated in vitro. It is hypothesized here that suppression of these ES proteins prevents the filarial worm from interfering with the host’s complement cascade, reducing the ability of the parasite to evade the immune system. Live microfilariae and/or larvae, thus exposed, are attacked and presented to the host’s innate immune mechanisms and are ultimately killed by the immune response, not the ML drug. These live, exposed filarial worms stimulate development of innate, cellular and humoral immune responses that when properly stimulated, are capable of clearing all larvae or microfilariae present in the host, regardless of their individual sensitivity to MLs. Additional research in this area can be expected to improve our understanding of the relationships among filarial worms, MLs, and the host immune system, which likely would have implications in filarial disease management in humans and animals.