Revisiting anthelmintic resistance in sheep flocks from São Paulo State, Brazil.

Haemonchus contortus and Trichostrongylus colubriformis are the most important gastrointestinal nematodes causing serious losses in sheep production of tropical and subtropical regions. Prophylaxis of gastrointestinal nematode infections is based on anthelmintics use, but their frequent administration selects multiple-resistant parasites. To evaluate how the situation has changed over the last decades, the anthelmintic resistance status of gastrointestinal nematodes in sheep flocks was assessed in the current study and compared to previous surveys. In each one of the 15 flocks evaluated, animals (n ≥ 7) were allocated into at least five groups and treated as follows: 1) untreated control; 2) albendazole; 3) levamisole; 4) ivermectin; and 5) monepantel. If more animals were available, two additional groups were included: 6) closantel, and 7) moxidectin. The faecal egg count reduction test (FECRT) was carried out to evaluate the pre- and post-treatment using the SHINY tool. Haemonchus spp. was the most prevalent nematode from faecal cultures. The mean efficacy of albendazole was 40%. Only in two farms, levamisole presented a relatively high percentage of reduction in the FECRT about 90%, while ivermectin and moxidectin presented the worst mean efficacy of 34% and 21% among all farms, respectively. Like other anthelmintics, closantel demonstrated low efficacy (63%) across all farms evaluated. Monepantel presented an overall mean efficacy of 79%, but it was the only anthelmintic that presented efficacy ≥95%, in five farms. The results revealed that gastrointestinal nematodes with multiple anthelmintic resistance were prevalent in all 15 sheep herds. The research suggests that nematodes are becoming more and more resistant to various anthelmintic compounds, which has made the problem worse. This circumstance highlights the necessity to put into practice sustainable and long-lasting methods to prevent gastrointestinal nematode infections in sheep husbandry.

Concurrent use of two dual-combination drenches containing monepantel/abamectin and oxfendazole/levamisole in sheep: effect on marker residues 21 and 28 days after administration.

AIMS: To determine the concentration, in comparison with the maximum residue limit (MRL), of anthelmintic marker residues in the target tissues (liver and fat) of sheep treated concurrently with two oral drenches, one containing monepantel and abamectin and the other oxfendazole and levamisole. METHODS: On day 0 of the study, 12 sheep (six male and six female; 8-9-months old) were dosed according to individual body weight determined the day prior. Zolvix Plus (dual-active oral drench containing 25 g/L monepantel and 2 g/L abamectin) was administered to all animals prior to administration of Scanda (dual-active oral drench containing 80 g/L levamisole hydrochloride and 45.3 g/L oxfendazole). Six sheep (three male and three female) were slaughtered 21 and 28 days after treatment and renal fat and liver samples were collected.Using validated methods, analyses for monepantel sulfone, abamectin, levamisole and oxfendazole (expressed as total fenbendazole sulfone following conversion of the combined concentrations of oxfendazole, fenbendazole and fenbendazole sulfone) were performed on liver samples while renal fat specimens were analysed for monepantel sulfone and abamectin residues only. Detected concentrations were compared to the established MRL in sheep for each analyte determined by the Ministry for Primary Industries. RESULTS: All residues detected in samples of liver and fat collected 21 and 28 days after treatment were below the MRL for each analyte. All liver samples collected on day 21 had detectable monepantel sulfone (mean 232 (min 110, max 388) µg/kg) and oxfendazole (mean 98.7 (min 51.3, max 165) µg/kg) residues below the MRL (5,000 and 500 µg/kg, respectively). Monepantel sulfone (mean 644 (min 242, max 1,119) µg/kg; MRL 7,000 µg/kg) residues were detected in 6/6 renal fat samples. Levamisole residues were detected in 3/6 livers (mean 40.0 (min 14.3, max 78.3) µg/kg; MRL 100 µg/kg), and abamectin residues in 1/6 livers (0.795 µg/kg; MRL 25 µg/kg) and 2/6 fat samples, (mean 0.987 (min 0.514, max 1.46) µg/kg; MRL 50 µg/kg) 21 days after treatment. CONCLUSION AND CLINICAL RELEVANCE: These results suggest that concurrent administration of Zolvix Plus and Scanda to sheep is unlikely to result in an extended residue profile for any of the active ingredients, with all analytes measured being under the approved New Zealand MRL 21 days after treatment. This work was not completed in line with guidance for establishing official residue profiles, nor is it sufficient to propose a new withholding period.

The proof is in the poo-ding: Benefits of the longitudinal molecular surveillance of drug resistance demonstrated in a New South Wales cattle herd.

Our understanding of anthelmintic resistance in the gastrointestinal nematodes of Australian cattle relies exclusively on small-scale phenotypic reports utilising traditional faecal egg count reduction tests. This approach is not readily scalable to establish the national prevalence of resistance, nor is it conducive of routine longitudinal surveillance for the emergence of resistance in its early stages. This study introduces the benefits of applying mixed amplicon metabarcoding longitudinally for timely and cost-efficient molecular surveillance of multiple anthelmintic resistance mutations, as they emerge on farms. Using opportunistically collected faecal samples from a cattle herd in central west New South Wales (2019-2023), we detected the early emergence of Haemonchus spp. levamisole-resistant S168T shortly after levamisole introduction, while benzimidazole-resistant allele frequencies remained constant. Additionally, we observed the possible spill-over of resistant Haemonchus contortus from sheep, along with variations in faecal burdens and species diversity influenced by climate stochasticity and host immunity. This study emphasises the power of molecular diagnostics for farm-level anthelmintic resistance management, providing essential evidence to support its integration into routine surveillance programmes.

Simultaneous resistance to multiple anthelmintic classes in nematode parasites of cattle in New Zealand.

Resistance to the benzimidazole and macrocyclic lactone anthelmintics is widespread in Cooperia spp. on cattle farms in New Zealand. Since this was first documented in 2006 little has changed in cattle farming systems except for the widespread use of levamisole to control Cooperia spp. in young cattle (i.e., parasite control has maintained an almost total reliance on use of anthelmintics). Here we report the emergence of simultaneous resistance to the benzimidazole, macrocyclic lactone and levamisole anthelmintics in Cooperia spp. and in Ostertagia spp. Anthelmintic efficacy against nematode parasites of cattle was investigated on four commercial farms following reports of poor animal growth rates and welfare, and positive faecal egg counts, despite routine treatment with combination anthelmintics, which included levamisole. Faecal egg count reduction tests involved 15 animals per treatment group, individual egg counts (paired samples) conducted pre- and post-treatment, with eggs counted to ≤ 15 eggs per g faeces and larval cultures for morphological identification. Actives tested varied between farms but always included levamisole alone and several combination products containing levamisole. Of the 20 tests conducted (i.e., 5 products on each of 4 farms) only 3 exceeded 90% efficacy against Cooperia spp. even though 8 of the products tested were combinations containing levamisole and at least one other broad-spectrum anthelmintic. Levamisole used alone achieved efficacies between 44% and 71% against Cooperia spp. across the four trials. The only product to exceed 95% efficacy against Cooperia spp. was a combination of monepantel + abamectin which was 100% effective against all parasites. Resistance to oxfendazole in Ostertagia spp. was indicated on 3 farms, while on one farm efficacy of all the tested products was ≤75% against this parasite. All the farms involved in this study were farming intensive cattle operations with an almost total reliance on anthelmintics to control parasitism. The results clearly demonstrate the emergence of simultaneous resistance to oxfendazole, levamisole and the macrocyclic lactone anthelmintics. Despite years of advice and recommendations to change farming practices away from intensive monocultures, many farmers have continued with the practice, and some are now faced with the very real possibility of being unable to control cattle parasites on their farms.

A mixed amplicon metabarcoding and sequencing approach for surveillance of drug resistance to levamisole and benzimidazole in Haemonchus spp.

Anthelmintic-resistant parasitic nematodes present a significant threat to sustainable livestock production worldwide. The ability to detect the emergence of anthelmintic resistance at an early stage, and therefore determine which drugs remain most effective, is crucial for minimising production losses. Despite many years of research into the molecular basis of anthelmintic resistance, no molecular-based tools are commercially available for the diagnosis of resistance as it emerges in field settings. We describe a mixed deep amplicon sequencing approach to determine the frequency of the levamisole (LEV)-resistant single nucleotide polymorphism (SNP) within arc-8 exon 4 (S168T) in Haemonchus spp., coupled with benzimidazole (BZ)-resistant SNPs within ß-tubulin isotype-1 and the internal transcribed spacer-2 (ITS-2) nemabiome. This constitutes the first known multi-drug and multi-species molecular diagnostic developed for helminths of veterinary importance. Of the ovine, bovine, caprine and camelid Australian field isolates we tested, S168T was detected in the majority of Haemonchus spp. populations from sheep and goats, but rarely at a frequency greater than 16%; an arbitrary threshold we set based on whole genome sequencing (WGS) of LEV-resistant Haemonchus contortus GWBII. Overall, BZ resistance was far more prevalent in Haemonchus spp. than LEV resistance, confirming that LEV is still an effective anthelmintic class for small ruminants in New South Wales, Australia. The mixed amplicon metabarcoding approach described herein paves the way towards the use of large scale sequencing as a surveillance technology in the field, the results of which can be translated into evidence-based recommendations for the livestock sector.

Efficacy of levamisole, ivermectin and moxidectin against Capillaria spp. in European hedgehogs (Erinaceus europaeus).

This randomised study aimed to assess and compare the efficacy of treatment protocols containing levamisole, ivermectin, or moxidectin against Capillaria spp. in naturally infected European hedgehogs (Erinaceus europaeus) presented to a British wildlife rehabilitation centre. Faecal analysis, consisting of wet mount and flotation, was performed for 229 hedgehogs weighing ≥200g. Animals testing positive for Capillaria spp. (81%), excluding pregnant females, were randomly allocated a treatment protocol. Initially, hedgehogs (n = 50) received one of six 'pilot' protocols, whereas the remaining animals (n = 97) received one of three 'main' protocols. Faecal analysis was repeated on day 8 and day 12 after treatment initiation. Efficacy of each treatment was assessed based on Capillaria reduction rate (CRR), weight gain, presence of respiratory clinical signs, and outcome. Pilot protocols containing only moxidectin had a significantly lower CRR (≥28.1%) compared to those with levamisole or ivermectin (≥86.6%), whereas the main protocols containing levamisole had a significantly higher CRR (≥93.0%) compared to those containing only ivermectin (≥69.3%). Clinical parameters did not differ significantly between treatments, but animals with respiratory clinical signs at the end of the trial were significantly more likely to have lower CRR and test positive for Crenosoma striatum. C. striatum often appeared refractory to treatment, and managing these infections requires additional anthelmintic therapy. Based on the formulations and dosages trialled, moxidectin is not recommended for treating capillariosis in European hedgehogs, whereas levamisole given orally for two consecutive days at 25-35 mg/kg is suggested as the treatment of choice.

Dose confirmation of a novel fixed dose combination injectable (0.2 mg/kg doramectin + 6.0 mg/kg levamisole hydrochloride) against naturally acquired gastrointestinal nematodes in US cattle.

Macrocyclic lactone (ML) resistance in cattle gastrointestinal nematodes (GINs) is an increasing problem. Concurrent combination anthelmintic therapy incorporating an existing ML with a second drug class has been proposed to control cattle GINs while slowing the development of ML resistance. Two dose confirmation studies were conducted to investigate the efficacy of a new fixed-dose combination injectable (FDCI) anthelmintic against common cattle GINs known to negatively impact production. The FDCI is formulated with 5 mg/ml doramectin and 150 mg/ml levamisole hydrochloride (HCl). Cattle enrolled in the two studies were sourced from either the Southern (Study 1, n = 30) or Midwest (Study 2, n = 36) United States. Animals with GIN infections confirmed by fecal egg count (FEC) were randomly allocated to one of three treatment groups. On Day 0, cattle with positive FECs on Day -5( ± 2) were weighed and administered a single subcutaneous injection of either saline (0.9% sodium chloride) at 0.04 ml/kg, 10 mg/ml doramectin at 0.02 ml/kg (to provide 0.2 mg/kg doramectin) or the FDCI at 0.04 ml/kg (to provide 0.2 mg/kg doramectin and 6.0 mg/kg levamisole HCl). On Day 14, fecal samples were collected, animals were euthanized, and worms were collected from the intestinal tract of each animal. Treatment efficacy was calculated using worm burdens and the fecal egg count reduction test (FECRT). Pre-treatment (Day -5, Study 1; Day -3, Study 2) mean FECs were 999.4-1136.2 eggs per gram (EPG) in Study 1 and 137.1-226.6 EPG in Study 2. The FDCI was active against cattle GIN populations in both studies, with FECRT ≥ 99.98% in both studies. Compared to saline-treated cattle, FDCI-treated cattle had significantly fewer adult and immature worms of all identified species on Day 14. In Study 1, Day 14 efficacy of the FDCI was 96.9% for Cooperia spp. (C. oncophora (99.7%) and C. punctata (95.9%)), 99.1% for Nematodirus helvetianus, and 99.8% for Ostertagia spp. In Study 2, the FDCI provided 100% efficacy against all adult GIN species identified, including all GINs identified in Study 1 and Trichostrongylus axei. The FDCI also provided 95.5% efficacy against immature Ostertagia spp. and 100% efficacy against immature Cooperia spp. (Study 2). Doramectin was effective against all adult cattle GINs (except N. helvetianus) in Study 2 but was only effective against adult Ostertagia spp. in Study 1. Additionally, doramectin was only effective against immature Cooperia spp. (and not immature Ostertagia spp.) in Study 2. A single administration of the doramectin + levamisole HCl FDCI provides a new and effective approach to the treatment and control of common cattle GINs, including those exhibiting decreased susceptibility to doramectin alone.

Comparative growth performance of backgrounded beef heifers treated with an injectable fixed-dose combination (0.2 mg/kg doramectin + 6.0 mg/kg levamisole hydrochloride) or single-active (0.2 mg/kg ivermectin) endectocide.

Gastrointestinal nematodes (GINs) can negatively impact all production classes of cattle, particularly growing cattle. A global decline in efficacy of broad-spectrum single-active anthelmintics requires alternative GIN control methods without the aid of novel drug classes. Here, we present a new fixed-dose combination injectable (FDCI) endectocide for cattle that combines doramectin (5 mg/ml) and levamisole hydrochloride (150 mg/ml). A 56-day comparative performance confinement backgrounding trial was conducted in stocker beef heifers (n = 1548) with confirmed GIN infections to (1) compare the Day 14 post-treatment effectiveness of the new FDCI endectocide to pen mates treated with the injectable single-active endectocide ivermectin, as evidenced by fecal egg counts (FECs) conducted for a randomly selected subset (10%) of both treatment groups, and (2) determine if the greater GIN control by the FDCI evidenced in the subsample improved growth performance in all FDCI-treated heifers. Heifers were procured in four cohorts, with a 10-week timeframe between enrollment of the first and last cohort. Treatment groups were comingled within dirt-floor pens (n = 31; 7-8 per cohort) and offered a standard backgrounding diet ad libitum for the study duration. Heifers with enrollment FEC ≥ 30 eggs per gram (EPG) were randomly allocated to receive the FDCI (n = 773) or ivermectin (n = 775) on Day 0. Day 0 FECs conducted on 10% of enrolled heifers (FDCI, n = 78; ivermectin, n = 79) were not different between treatment groups (p = 0.491). Day 14 FECs for the same heifers were reduced compared to Day 0 within each treatment group. Heifers given the FDCI had lower Day 14 AM FECs and higher FEC reduction test (FECRT) result (0.07 EPG; 0.999) than ivermectin-treated heifers (21.58 EPG; FECRT = 0.850). Mean body weight (BW) was not different between treatment groups on Day 0 (p = 0.2762) and Day 14 (p = 0.2010) but was significantly greater (p = 0.0007) for FDCI-treated heifers compared to ivermectin-treated heifers on Day 56. Compared to ivermectin-treated heifers, overall average daily gain from all evaluation periods (Day 0-14, Day 14-56, and Day 0-56) was greater (p ≤ 0.0052) in FDCI-treated heifers, and FDCI-treated heifers had 4.223 kg greater total weight gain over the 56-day study. The FDCI (0.2 mg/kg doramectin + 6.0 mg/kg levamisole hydrochloride) was highly effective in reducing GIN infections and thus promoted improved growth performance in beef heifers over a 56-day backgrounding period.

Efficacy of a new fixed-dose combination injectable (0.2 mg/kg doramectin + 6.0 mg/kg levamisole hydrochloride) in Australian cattle against artificial infections of gastrointestinal nematodes.

We describe a new fixed-dose combination injectable (FDCI) formulated with doramectin and levamisole hydrochloride (HCl) to target broad and overlapping spectra of gastrointestinal nematodes (GINs) through two distinct modes of action. Here, we demonstrate the superior efficacy of the FDCI against mixed populations of cattle GINs in two dose confirmation studies conducted in Australia using artificially induced adult (Study 1) and immature (Study 2) GIN infections. Artificial infections consisted of Cooperia spp., Haemonchus placei, Ostertagia ostertagi, and Trichostrongylus axei. In both studies, cattle were inoculated with third-stage larvae and infections were confirmed by fecal egg count (FEC). Treatment groups in both studies were as follows: (1) negative control (saline, 0.9% sodium chloride), (2) positive control injectable endectocide (Study 1-0.2 mg/kg ivermectin; Study 2-0.2 mg/kg doramectin), (3) positive control injectable anthelmintic (7.5 mg/kg levamisole HCl), and (4) FDCI (0.2 mg/kg doramectin + 6.0 mg/kg levamisole HCl). Cattle were treated either 28 days (Study 1) or 6 days (Study 2) post-infection. On Days 14-16 (Study 1) or Days 20-21 (Study 2) post-treatment, cattle were euthanized and necropsied for the recovery, identification, and enumeration of worms. Treatment efficacy was calculated as reduction in worm burdens of treated cattle compared to saline-treated cattle, and treatments were considered effective if the geometric mean worm burden in the treatment group was reduced by ≥ 95% compared to the negative control group. In both studies, saline-treated cattle remained positive for GIN infections for the study duration. Ivermectin was less than 95% effective against Cooperia spp. (80.2%) and H. placei (24.8%) in Study 1, and levamisole HCl was less than 95% effective against Ostertagia spp. (47.1%) in Study 2. In contrast, the novel FDCI was 100% effective in treating adult and immature life stages of all cattle GINs included in the artificial infections, with no worms recovered at necropsy from doramectin + levamisole HCl-treated cattle. These data show a single administration of the FDCI provides broad-spectrum treatment of economically important cattle GINs.

Efficacy of a fixed-dose combination injectable (0.2 mg/kg doramectin + 6.0 mg/kg levamisole hydrochloride) in Australian cattle against naturally acquired gastrointestinal nematode infections.

Australian producers have long used macrocyclic lactones (MLs) to successfully control cattle gastrointestinal nematodes (GINs) and consequently improve production parameters. However, the trajectory of ML resistance development in cattle GINs is following that of small ruminant nematode populations, highlighting a need for novel treatment options to provide efficacy in the current environment and interrupt the long-term establishment of ML-resistant GIN populations in Australian cattle. Here, we describe three field studies conducted in Australia to evaluate the efficacy of a single administration of a novel fixed-dose combination injectable (FDCI) endectocide against naturally acquired infections of cattle GINs. The FDCI is administered subcutaneously to deliver 0.2 mg/kg doramectin and 6 mg/kg levamisole hydrochloride (HCl). Study sites consisted of three farms in New South Wales (n = 2) and Victoria (n = 1). At each site, cattle were randomly allocated into one of three treatment groups: (1) untreated control (saline), (2) FDCI (0.2 mg/kg doramectin, 6 mg/kg levamisole HCl) or (3) positive control (0.2 mg/kg ivermectin). All treatments were administered on Day 0. Fecal samples were collected prior to treatment on Days -1 (Study 3) or 0 (Studies 1 and 2) and again on Day 14 (post-treatment) to evaluate efficacy via fecal egg count (FEC) and for coproculture. Adequacy of infection was confirmed at all three study sites, with Day 14 geometric mean (GM) FECs for saline-treated cattle ranging from 32.5 eggs per gram (EPG) to 623.7 EPG. FECs for FDCI-treated cattle were significantly reduced compared to saline-treated cattle (p ≤ 0.0001) on Day 14, with GM-based efficacy ≥ 99.7% at all three study sites. In contrast, ivermectin was 97.4% effective against cattle GINs in Study 1 but was only 47.2% and 39.8% effective at study site 2 and 3, respectively. Genus-specific efficacies suggest the presence of ivermectin-resistant Cooperia spp. (Study 1), Haemonchus spp. (Study 2) and Ostertagia spp. (Study 3) populations in the naturally infected cattle used in these studies. The post-treatment FEC and genus-specific efficacy estimations indicate the doramectin + levamisole HCl FDCI was highly efficacious against cattle GINs even in the face of ivermectin LOE at study sites 2 and 3. The efficacy of the new FDCI against both ML-susceptible and ML-resistant economically important cattle GINs in Australia affirms it is a valuable treatment option for producers operating in an environment of ML loss of efficacy.

Effectiveness of a fixed-dose combination injectable (0.2 mg/kg doramectin + 6.0 mg/kg levamisole hydrochloride) against Rhipicephalus microplus and sucking lice infesting cattle.

Unmanaged tick and sucking lice infestations negatively impact the health and production potential of cattle. Described herein are two non-interference dose confirmation studies evaluating the efficacy of a single administration of a new fixed-dose combination injectable (FDCI) endectocide consisting of 0.2 mg/kg doramectin + 6.0 mg/kg levamisole hydrochloride, against either laboratory-induced Rhipicephalus microplus infestations in Australia or naturally acquired sucking lice (Linognathus vituli) infestations in the US. This FDCI is available as Dectomax V® in Australia and New Zealand and as Valcor® in the United States. To evaluate therapeutic efficacy against R. microplus, 12 calves were each exposed to 10 infestations of ∼5000 larvae per infestation between Days -24 and -2. Calves were either treated on Day 0 with the FDCI or left untreated (control). Additional R. microplus infestations of ∼5000 larvae were conducted on Day 2 and then three times weekly to also evaluate persistent efficacy of the FDCI. Tick collections were conducted daily from Day -3. Group mean live tick counts, egg production, and egg viability were analyzed for significant differences between the two groups. To determine efficacy of the FDCI against lice, 24 cattle with active sucking lice infestations based on Day -7 counts were allocated to two groups and treated on Day 0 with either saline (control) or the FDCI. Lice counts were conducted weekly from Day 14 through 42 and again on Day 56. Mean group lice counts on each count day were compared between treatment groups. In the R. microplus study presented here, cattle in Queensland, Australia treated with the FDCI (Dectomax V®) showed > 90 % reduction in tick counts based on arithmetic means within 48 h of treatment when compared to untreated cattle, and counts were > 95 % reduced from post-treatment Day 5 through Day 30. In the sucking lice study conducted in the US, the FDCI (Valcor®) displayed 100 % efficacy against sucking lice infestations (L. vituli) from first count day (Day 14 post-treatment) through Day 35 and then 99.9 % efficacy through Day 56 post-treatment. No treatment-related adverse events were reported for cattle in either study. Using R. microplus and sucking lice as representative ectoparasites, these studies demonstrate the ectoparasite activity of doramectin is retained in the new FDCI.

Efficacy in US cattle of a novel fixed-dose combination injectable (0.2 mg/kg doramectin + 6.0 mg/kg levamisole hydrochloride) against naturally acquired gastrointestinal nematode infections.

Reports of macrocyclic lactone (ML) loss of efficacy suggest ML resistance in gastrointestinal nematodes (GINs) is a growing problem in the US cattle industry. Empirical and modeling data support combining an ML and second anthelmintic from a different drug class to help ML resistance development while effectively treating existing resistant parasite populations. Here, we present a novel fixed-dose combination injectable (FDCI) solution for cattle that delivers 0.2 mg of doramectin and 6.0 mg of levamisole hydrochloride (HCl) per kg of body weight. Field studies were conducted at six sites across the Midwest United States to investigate the efficacy of a single administration of the FDCI in treating common cattle GINs. Cattle (n = 425) with GIN infections confirmed by fecal egg count (FEC) on Day -10( ± 2) were randomly allocated to the control (saline) or treatment (FDCI) group. On Day 0, pre-treatment fecal samples were collected, and cattle were administered a single subcutaneous injection of saline (n = 106) or FDCI (n = 319). Post-treatment fecal samples were collected on Day 14. Fecal egg count reduction tests (FECRTs) were conducted using Day 0 and Day 14 FECs. Efficacy was evaluated using Day 14 FECs (FDCI-treated versus saline-treated). Within treatment, samples collected on Days 0 and 14 with ≥ 20 eggs per gram (EPG) were cultured for nematode larvae recovery and identification. Day -10 FECs for enrolled animals were similar between treatment groups. Coprocultures from cattle with FEC ≥ 20 EPG (n = 68 saline; n = 211, FDCI) on Day 0 showed the presence of Cooperia punctata, Ostertagia spp., Haemonchus spp., C. oncophora, Oesophagostomum spp. and Trichostrongylus spp. Day 14 FECs for FDCI-treated cattle were significantly reduced (0-3 EPG) compared to saline-treated cattle (0-1252 EPG) (p ≤ 0.0042). At all study sites, the efficacy of the new FDCI was ≥ 99.4% and the FECR was 0.99 or 1.00. Day 14 coprocultures from control cattle showed infections of common GIN genera, confirming the efficacy of the FDCI against GINs in the field. A single administration of the doramectin + levamisole HCl combination injectable effectively treats common and economically important cattle GINs.

Efficacy of a fixed-dose combination injectable (0.2 mg/kg doramectin + 6.0 mg/kg levamisole hydrochloride) in New Zealand cattle against naturally acquired gastrointestinal nematode populations with demonstrated resistance to doramectin.

Intensive farming practices and heavy reliance on anthelmintics have contributed significantly to the problem of macrocyclic lactone (ML) resistance in New Zealand. Farmers now have few options for effectively controlling cattle gastrointestinal nematodes (GINs) and regularly experience sub-optimal efficacy against economically important species. We present a novel fixed-dose combination injectable (FDCI) that simultaneously delivers 0.2 mg/kg doramectin and 6 mg/kg levamisole hydrochloride (HCl) to target a broad spectrum of cattle GINs in a single dose, providing an additional solution to endoparasite control in an environment of anthelmintic resistance. A dose confirmation study was conducted using naturally acquired infections of GINs in beef cattle in New Zealand. Cattle with GIN infections confirmed by fecal egg count (FEC) were randomly allocated (n = 12 per group) to the control (saline-treated), FDCI-treated or doramectin-treated group. On Day 0, cattle were weighed and administered a single subcutaneous injection of saline or endectocide. Rectal fecal samples were collected from each animal on Day 7 for individual duplicate fecal egg count (FEC) analysis, and coprocultures were conducted on pooled fecal samples within each treatment group. All animals were euthanized and necropsied for worm recovery on Days 14 through 16. Treatment efficacy was calculated based on reduction in FECs and worm burdens. All enrolled cattle were positive for GINs based on Day -5 FECs, with geometric mean (GM) FECs ranging from 337 to 521 eggs per gram (EPG). All saline-treated cattle remained positive for GIN infections for the study duration (Day 7 GM FEC = 427 EPG). Necropsy and worm recoveries revealed the presence of doramectin-resistant Cooperia oncophora, C. surnabada and Trichostrongylus longispicularis, as evidenced by ≤ 72.3 % efficacy of doramectin against these species. The new FDCI was ≥ 99.8 % effective against all GIN species, including ML-resistant C. oncophora, C. surnabada and T. longispicularis, providing broad-spectrum efficacy and eliminating economically important cattle GINs, including ML-resistant populations.

Levamisole derivatives as immune modulators for the treatment of amyotrophic lateral sclerosis (ALS).

Aim: To discover derivatives of the anthelmintic drug levamisole, which has been reported to possess immune-modulatory properties, as treatments for amyotrophic lateral sclerosis (ALS), which has been suggested to be in part an autoimmune disease. Results: We have synthesized ten analogs of the racemic version of levamisole, tetramisole, as well as eleven analogs on a related system. All of the analogs have been tested for their ability to affect the response of five ALS-relevant cytokines. Conclusion: We have discovered a number of interesting derivatives that have encouraging cytokine response data and good metabolic stability, with the potential to have a positive impact on ALS either as single agents, or in combination.

Aim: To discover derivatives of the antiparasitic worm drug levamisole, which has been reported to be able to modulate the immune response, as treatments for amyotrophic lateral sclerosis (ALS), which has been suggested to be in part an autoimmune disease. Results: We have synthesized ten analogs of a variation of levamisole, tetramisole, as well as 11 analogs on a related system. All of the analogs have been tested for their ability to affect the response of five ALS-relevant immune-modulatory substances (cytokines). Conclusion: We have discovered a number of interesting derivatives that have encouraging cytokine response data and good metabolic stability, with the potential to have a positive impact on ALS either as single agents, or in combination.

Levamisole hydrochloride as an anthelmintic against Neoechinorhynchus buttnerae in juvenile Colossoma macropomum.

Acanthocephalosis is caused by the endoparasite Neoechynorhyncus buttnerae and affects fish farmed in the Amazon. This study assessed the efficacy of therapeutic levamisole hydrochloride (LVC) baths against N. buttnerae and its effects on juvenile tambaqui blood parameters. In vitro and in vivo tests were carried out, the latter employing two experimental therapeutic LVC bath protocols. Concerning in vitro efficacy, the T75 (75 mg.L-1 LVC) and T100 (mg.L-1 LVC) treatments were 100% effective in 15 min, while the T50 (50 mg.L-1 LVC) and T25 (25 mg.L-1 LVC) treatments required parasite exposure for 45 and 60 min, respectively. During exposure, the parasites displayed reduced motility, proboscis retraction, coiling into a spiral shape, body rigidity and swelling. The LVC LC50-72h for juvenile tambaqui was 115 mg.L-1 . Regarding in vivo efficacy for Protocol I (8-h bath), the T125 resulted in 82% effectiveness, while in Protocol II (two 8-h baths with a 24-h interval), the T115 treatment (115 mg.L-1 LVC) achieved 95.6% effectiveness without clinical intoxication signs, despite behavioural changes. No significant changes were observed in fish blood parameters. LVC was, therefore, highly effective both in vitro and in vivo in controlling the acanthocephalan N. buttnerae without compromising tambaqui juvenile homeostasis.

Novel insights on the therapeutic effect of levamisole on the chronic toxoplasmosis in mice model.

Latent toxoplasmosis mostly reactivates which could result in acute encephalitis. Chronic toxoplasmosis treatments are severely constrained by Toxoplasma cyst resistance. Novel therapeutic approaches are therefore becoming more essential. In this study, the effects of levamisole (LEVA) and spiramycin on the early and late stages of experimental toxoplasmosis are investigated. MATERIALS AND METHODS: Seventy-five Me49 Toxoplasma gondii infected Swiss albino mice were divided into five groups; (GI): noninfected control group; (GII): infected untreated control group; (GIII): infected- LEVA treated group; (GIV): infected and received combination of spiramycin and LEVA and (GV): infected-spiramycin treated group. The impact was assessed through brain cyst count by Quantitative Real-Time Polymerase Chain Reaction (PCR), interferon gamma (IFN-γ) assay, histopathological study, and total blood counts. RESULTS: The progression of chronic toxoplasmosis could only be partially controlled by using either levamisole or spiramycin as a separate drug. The combined spiramycin and levamisole treatment significantly decreased the burden of Toxoplasma brain cyst, increased IFN-γ level, total blood parameters and improved the histopathological features especially at the late stage of infection. IN CONCLUSION: Levamisole effectively modulated Toxoplasma-induced immune responses, resulting in chronic toxoplasmosis remission. Further clinical trials will be needed to study the effect of these combination in HIV/AIDS (human immunodeficiency virus) patients with toxoplasmosis.

Anthelmintic resistance in sheep in the semiarid region of Minas Gerais, Brazil.

Anthelmintic efficacy was evaluated among sheep that had become naturally infected with gastrointestinal nematodes in 17 flocks located in the semiarid region of Minas Gerais, Brazil. Feces were collected individually from 1021 hairy sheep to determine the number of eggs per gram of feces (EPG) and for coprocultures to identify nematode genera the nematodes. Only the animals that presented EPG counts greater than or equal to 200 were included in the study (totaling 381 sheep). The animals were divided into three treatment groups: albendazole, ivermectin and levamisole. Fourteen days after the administration of anthelmintics, fecal samples were taken from all animals. In each flock, the pre-treatment and post-treatment arithmetic mean EPG were used to calculate the efficacy (FECR) for each of the treatment groups and the lower 95% confidence limit. Data were analyzed with the "eggCounts 2.3" package in RStudio, using a Bayesian model for paired design. The anthelmintics were classified as being efficacious (when the FECR was both equal to or above 95% and the lower 95% confidence limit was equal to or above 90%) or as encountering anthelmintic resistance (when the FECR was below 95% and the lower 95% confidence limit was below 90%) or inconclusive (when none of the other criteria were fulfilled). Albendazole and ivermectin were not effective in any of the flocks. Levamisole was effective against gastrointestinal nematodes in 25% of the flocks studied. Haemonchus, Trichostrongylus and Oesophagostomum genera were identified in this study in a semiarid region of Minas Gerais, Brazil. The genus Haemonchus was the most prevalent, followed by Trichostrongylus and Oesophagostomum. After anthelmintic treatment, the most prevalent genus was Haemonchus, followed by Trichostrongylus; the genus Oesophagostomum was not detected. The highest percentage of Haemonchus larvae was observed after treatment with ivermectin, followed by albendazole and levamisole. This study revealed the existence of gastrointestinal nematodes in sheep that present multiple resistance to all three main classes of anthelmintic drugs.

Pharmacokinetics and therapeutic efficacies of fenbendazole in comparison with levamisole in helminth-infected Caspian turtles (Mauremys caspica).

The pharmacokinetics and bioavailability of fenbendazole and levamisole were determined in Caspian turtles after a single intravenous (i.v.) and subcutaneous (s.c.) administration. Thirty turtles diagnosed as naturally infected with Serpinema microcephalus and Falcaustra armenica nematodes received fenbendazole (50 mg/kg) or levamisole (10 mg/kg) by i.v. and s.c. administrations. Blood samples were collected at time 0, 0.125, 0.25, 0.5, 1, 2, 4, 8, 12, 24, and 48 h after drug administration. Plasma drug concentrations were determined by a validated high-performance liquid chromatography method. Data were analyzed by noncompartmental methods. The mean elimination half-life of levamisole was 5.16 h and 12.03 h for i.v. and s.c. routes, respectively, and for fenbendazole, the mean elimination half-life was 25.38 h (i.v.) and 29.77 h (s.c.). The total clearance and volume of distribution at steady-state for levamisole and fenbendazole following i.v. administration were 0.22, 0.44 ml/g/h, and 1.06 and 7.35 ml/g, respectively. For the s.c. route, the peak plasma concentration of levamisole and fenbendazole was 10.53 and 5.24 µg/mL, respectively. The s.c. bioavailability of levamisole and fenbendazole was complete. Considering high anthelmintic efficacy and bioavailability after s.c. administration of levamisole and fenbendazole, and the absence of adverse effects, this route of administration is an easy and efficacious way of treating nematodes in Caspian turtles.

Signs of multiple anthelmintic resistance in sheep gastrointestinal nematodes in Sweden.

Gastrointestinal nematodes in small ruminants are clinically and economically important parasites that often are controlled with anthelmintics. In this study, we compiled information on the anthelmintic efficacy collected on sheep farms according to routines established by Farm & Animal Health in Sweden. The efficacies of benzimidazoles (i.e. albendazole or fenbendazole, n = 30), ivermectin (n = 47), levamisole (n = 2) or moxidectin (n = 2) were examined between 2015 and 2021 in 81 treatment groups on 49 non-randomly selected farms in south-central Sweden. Drug efficacies were estimated with the faecal egg count reduction test. In addition, efficacy data were in most cases supplemented with data on the abundance of the three most common nematode genera in sheep by performing droplet digital (dd) PCR on coprocultures. Efficacies of <95% for benzimidazoles or ivermectin were identified in 37% and 77% of the tested groups, respectively. In addition, on 27 (55%) of the 49 farms where both benzimidazoles and ivermectin were tested, multiple resistance was found on 8 (30%). In contrast, on each of the two farms tested for levamisole and moxidectin both drugs proved to be 100% effective. However, because post-sampling was performed earlier than recommended in several susceptible groups (benzimidazoles = 15, and ivermectin = 10 groups), this could have underestimated the severity of the situation. Mainly larvae from the genus Haemonchus were detected in post-treatment coprocultures, in all groups with declared resistance, suggesting that this parasite was primarily associated with anthelmintic resistance. Unexpectedly, the DNA of larvae, which survived treatment, was also detected on farms declared as susceptible. Taken together, this indicates that the situation regarding the anthelmintic efficacy has deteriorated compared with the latest nationwide study on Swedish sheep farms conducted more than a decade ago. Unlike the previous study, the farm selection here was not strictly randomized but rather opportunistic i.e., only farms with a recognized parasite problem were included. Thus, there is a need for a truly randomized study to get an update on the extent of the situation of anthelmintic resistance at a national level, as well as to identify risk factors involved in the resistance selection. Research is also required to establish the optimal intervals for sampling post-treatment.

Pharmacokinetics and therapeutic efficacy of levamisole in Ascaridia galli experimentally infected ducks.

This study investigated the pharmacokinetics and therapeutic efficacy of levamisole (LVS) after intravenous (i.v.) and oral administrations to healthy and Ascaridia galli-infected ducks by developing an infection model. Twenty-four two-week old ducklings were experimentally infected with A. galli. The ducks were monitored for the development of infection and after 8 weeks they were administered with LVS at a single dose of 30 mg/kg by oral or i.v. administration. Sixteen healthy ducks were subjected to the same treatment and served as control. Serial blood samples were taken for LVS determination with HPLC-UV and pharmacokinetic analysis was carried out based on the non-compartmental approach. The LVS therapeutic efficacy was determined 1 week post drug administration by intestinal worm count at necropsy. In vivo data on development of ascariasis in ducks showed that 8 weeks post inoculation the number of eggs per gram of feces reached at least 100 in each bird. After a single dose of LVS, no parasites were recovered upon necropsy. Results of the pharmacokinetic study showed no statistical differences between infected and non-infected birds for both routes of administration. The mean oral bioavailability was slightly below 50% in both experimental groups. In conclusion, the pharmacokinetics of LVS in ducks was not affected by experimentally-induced ascariasis. A single dose of LVS was found to be efficient against experimental ascariasis in ducks induced by in field isolates of A. galli.