The in vitro assay profile of macrocyclic lactone resistance in three species of sheep trichostrongyloids

Anthelmintic resistance has emerged as an important problem in animal industries. Understanding resistance mechanisms, especially against macrocyclic lactones (MLs), is the first step in developing better diagnostic tools. Effects of several MLs including ivermectins and milbemycins were tested using two well established in vitro assays: the larval development assay (LDA) and the larval migration inhibition assay (LMIA). These were performed on free-living stages of susceptible and ML-resistant isolates of three trichostrongyloid nematode species of sheep. In general, dose response curves shifted to the right in the resistant isolates. Data showed that resistance was present to ivermectin and its two components suggesting that both components contribute to action and resistance. There were no consistent patterns of potency and resistance of the tested substances for the different isolates in the LDA except that moxidectin (MOX) tended to have lower resistance ratios than ivermectin (IVM). MOX was the most potent inhibitor in the LMIA in susceptible Haemonchus contortus while being less potent in Trichostrongylus colubriformis and particularly in Ostertagia circumcincta. MOX showed high resistance ratios in the LMIA in all three species. Based on these results, resistance to MOX has unique characteristics and the LMIA may perform better in detecting resistance to MOX in these parasite species

A quantitative evaluation of the extent of fluralaner uptake by ticks (Ixodes ricinus, Ixodes scapularis) in fluralaner (Bravecto TM ) treated vs. untreated dogs using the parameters tick weight and coxal index

Background Fluralaner is a new antiparasitic drug that was recently introduced as Bravecto TM chewable tablets for the treatment of tick and flea infestations in dogs. Most marketed tick products exert their effect via topical application and contact exposure to the parasite. In contrast, Bravecto TM delivers its acaricidal activity through systemic exposure. Tick exposure to fluralaner occurs after attachment to orally treated dogs, which induces a tick-killing effect within 12 h. The fast onset of killing lasts over the entire treatment interval (12 weeks) and suggests that only marginal uptake by ticks is required to induce efficacy. Three laboratory studies were conducted to quantify the extent of uptake by comparison of ticks’ weight and coxal index obtained from Bravecto TM -treated and negative-control dogs. Methods Three studies were conducted using experimental tick infestation with either Ixodes ricinus or Ixodes scapularis after oral administration of fluralaner to dogs. All studies included a treated (Bravecto TM chewable tablets, MSD Animal Health) and a negative control group. Each study had a similar design for assessing vitality and weighing of ticks collected from dogs of both groups. Additionally, in one study the coxal index (I. ricinus) was calculated as a ratio of tick’s ventral coxal gap and dorsal width of scutum. Tick weight data and coxal indices from Bravecto TM -treated and negative-control groups were compared via statistical analysis. Results Ticks collected from Bravecto TM -treated dogs weighed significantly less (p ≤ 0.0108) than ticks collected from negative-control dogs, and their coxal index was also significantly lower (p < 0.0001). The difference in tick weights was demonstrated irrespective of the tick species investigated (I. ricinus, I. scapularis). At some assessments the mean tick weights of Bravecto TM -treated dogs were significantly lower than those of unfed pre-infestation (baseline) ticks. The demonstrated tick-killing efficacy was in the range of 94.6 – 100 %. Conclusions Tick weights and coxal indices confirm that a minimal uptake results in a sufficient exposure of ticks to fluralaner (Bravecto TM ) and consequently in a potent acaricidal effect

Untersuchung zum Vorkommen von Anthelminthikaresistenzen in norddeutschen Rinderbeständen

Anthelmintic resistance has become a serious problem worldwide especially for small ruminants but it is also rising for cattle. The sustainability to maintain the efficacy of anthelmintics is an important objective. Furthermore, reduction of anthelmintic use is desired to assure safe and high quality food. Therefore targeted selective treatment (TST) systems should be developed. Through TST the use of anthelmintics can be reduced and selection pressure on sensible endoparasite isolates decreases. The current project aims at 1st the investigation of the current efficacy of macrocyclic lac-tone anthelmintics in first season grazing cattle in Northern Germany and 2nd to com-paratively investigate different approaches to TST such as body weight, body condition-ing scores (BCS) and egg output (EPG)

Targeted Selective Treatment (TST) bei Rindern zur Reduzierung der Resistenzbildung bei Endoparasiten

Infektionen mit Endorparasiten, insbesondere mit Magen-Darm-Strogyliden (MDS), sind oft ein limitierender Faktor in der Entwicklung und Produktivität von erststömmrigen Rindern. Diese Infektionen haben somit große wirtschaftliche Bedeutung. Die bisher weitverbreitet durchgeführte strategische Entwurmung (planmäßiger Einsatz von Medikamenten) ganzer Herden sollte aus verschiedenen Gründen angepasst werden

Entwicklung eines Serum- und Milch-ELISAs zum Nachweis der Infektion mit Magen-Darm-Strongyliden bei Ziegen

Grazing goats are naturally infected with gastrointestinal nematodes which are an important cause of diseases and economic losses. Targeted selective treatment (TST) is a possibility to avoid spreading of anthelmintic resistances and to reduce the use of anthelmintics. The aim of this study is the development of an goat-milk ELISA for the detection of infection with T. circumcincta in order to provide a monitoring tool for herd infection levels. Therefore a group of kids were artificially infected, blood and faecal samples were taken over a period of 10 weeks. Additional a herd of milk goats,kept on pasture at the experimental farm were used to obtain milk, blood and faecal samples regularly

Field efficacy of four anthelmintics and confirmation of drug-resistant nematodes by controlled efficacy test and pyrosequencing on a sheep and goat farm in Denmark

AbstractWe describe a case of anthelmintic resistance on one of the largest organic small ruminant farms in Denmark. The flock was established in 2007 by purchase of animals from other Danish farms and had history of clinical parasitism, high mortality of young stock and anthelmintic treatment failure. In October 2011, 40 lambs and 40 kids were selected for a faecal egg count reduction test (FECRT) with fenbendazole (FBZ), ivermectin (IVM), moxidectin (MOX) and levamisole (LEV). Lambs were treated with the recommended sheep dose of each product while kids received the sheep dose of IVM, 1.5× sheep dose of MOX and 2× sheep dose of FBZ and LEV. Untreated lambs and kids were also included and three methods for calculating faecal egg count (FEC) reduction were compared. In a subsequent investigation, a controlled efficacy test (CET) with FBZ and IVM was performed in lambs infected with Haemonchus contortus and Trichostrongylus colubriformis isolated from adult goats on the farm. Recovered specimens of H. contortus were subjected to pyrosequencing for detection of single nucleotide polymorphisms (SNPs) related to benzimidazole (BZ) resistance. During the FECRT, FECs in untreated lambs dropped significantly by 47%. No FEC reduction was detected in untreated kids. After FBZ treatments, FEC reductions in lambs and kids ranged from 15 to 54% and 49–56%, respectively, according to the different calculation methods. Post IVM treatments, FEC reductions in lambs and kids varied between 71–90% and 81–83%, correspondingly. LEV and MOX reduced FECs by 98–100% in both species. In the CET, FBZ reduced H. contortus worm counts by 52–56% and no reduction in T. colubriformis counts were detected after treatment. IVM eliminated 100% of H. contortus and reduced T. colubriformis counts by 84–92%, according to different calculation methods. Pyrosequencing of isolated H. contortus revealed increased frequencies of the BZ resistance-related SNP in codon 200 of the β-tubulin isotype 1 gene. Frequency of BZ resistance-related SNPs in codons 167 and 198 were very low and did not exceed levels as obtained in the susceptible reference isolate. Anthelmintic resistance was confirmed in this recently established organic farm and low field efficacy of FBZ was verified by CET and pyrosequencing. BZ-resistant populations of H. contortus and T. colubriformis were isolated for the first time in Denmark. Problems with correct dosing of goats, the observed FEC reduction in untreated lambs and the relevance of including a control group in the FECRT are discussed

Efficacy of imidacloprid 10%/moxidectin 1% spot-on formulation (Advocate®) in the prevention and treatment of feline aelurostrongylosis

Background: In three randomized, controlled laboratory efficacy studies, the efficacy in the prevention of patent infections of a topical combination of imidacloprid 10%/moxidectin 1% (Advocate® spot-on formulation for cats, Bayer Animal Health GmbH) against larval stages and immature adults of Aelurostrongylus abstrusus, as well as the treatment efficacy of a single or three monthly treatments against adult A. abstrusus, were evaluated. Methods: Cats were experimentally inoculated with 300–800 third-stage larvae (L3). Each group comprised 8 animals and the treatment dose was 10 mg/kg bodyweight (bw) imidacloprid and 1 mg/kg bw moxidectin in each study. Prevention of the establishment of patent infections was evaluated by two treatments at a monthly interval at three different time points before and after challenge infection. Curative efficacy was tested by one or three treatments after the onset of patency. Worm counts at necropsy were used for efficacy calculations. Results: In Study 1, the control group had a geometric mean (GM) of 28.8 adult nematodes and the single treatment group had a GM of 3.4 (efficacy 88.3%). In Study 2, the control group had a GM of 14.3, the prevention group had a GM of 0 (efficacy 100%), while the treatment group had a GM of 0.1 (efficacy 99.4%). In Study 3, the GM worm burden in the control group was 32.6 compared to 0 in all three prevention groups (efficacy 100% for all of those groups). Conclusions: The monthly administration of Advocate® reliably eliminated early larval stages and thereby prevented lung damage from and patent infections with A. abstrusus in cats. Regarding treatment, a single application of Advocate® reduced the worm burden, but it did not sufficiently clear the infection. In contrast, three monthly treatments were safe and highly efficacious against A. abstrusus

Development of a multiplex fluorescence immunological assay for the simultaneous detection of antibodies against Cooperia oncophora, Dictyocaulus viviparus and Fasciola hepatica in cattle

Background A major constraint for the effective control and management of helminth parasites is the lack of rapid, high-throughput, routine diagnostic tests to assess the health status of individual animals and herds and to identify the parasite species responsible for these helminthoses. The capability of a multiplex platform for the simultaneous detection of three pasture associated parasite species was evaluated and compared to existing ELISAs. Methods The recombinant antigens 14.2 kDa ES protein for Cooperia oncophora, major sperm protein for Dictyocaulus viviparus and Cathepsin L1 for Fasciola hepatica were recombinantly expressed either in Escherichia coli or Pichia pastoris. Antigens were covalently coupled onto magnetic beads. Optimal concentrations for coupling were determined following the examination of serum samples collected from experimentally mono-infected animals, before and after their infection with the target species. Absence of cross-reactivity was further determined with sera from calves mono-infected with Haemonchus contortus, Ostertagia ostertagi and Trichostrongylus colubriformis. Examination of negative serum samples was characterised by low median fluorescence intensity (MFI). Results Establishment of the optimal serum dilution of 1:200 was achieved for all three bead sets. Receiver Operating Characteristic analyses were performed to obtain cut-off MFI values for each parasite separately. Sensitivity and specificity at the chosen cut-off values were close to, or 100 % for all bead sets. Examination of serum samples collected on different days post infection from different animals showed a high reproducibility of the assays. Serum samples were additionally examined with two already established ELISAs, an in-house ELISA using the recombinant MSP as an antigen and a DRG ELISA using Cathepsin L1 for liver fluke. The results between the assays were compared and kappa tests revealed an overall good agreement. Conclusions A versatile bead-based assay using fluorescence detection (xMAP® technology) was developed to simultaneously detect antibodies against C. oncophora, D. viviparus and F. hepatica in cattle serum samples. This platform provides rapid, high-throughput results and is highly sensitive and specific in comparison to existing serological as well as coproscopical diagnostic techniques

Impacts, Trends and Sustainable Solutions

Infections with parasitic helminths (nematodes and trematodes) represent a significant economic and welfare burden to the global ruminant livestock industry. The increasing prevalence of anthelmintic resistance means that current control programmes are costly and unsustainable in the long term. Recent changes in the epidemiology, seasonality and geographic distribution of helminth infections have been attributed to climate change. However, other changes in environment (e.g., land use) and in livestock farming, such as intensification and altered management practices, will also have an impact on helminth infections. Sustainable control of helminth infections in a changing world requires detailed knowledge of these interactions. In particular, there is a need to devise new, sustainable strategies for the effective control of ruminant helminthoses in the face of global change. In this paper, we consider the impact of helminth infections in grazing ruminants, taking a European perspective, and identify scientific and applied priorities to mitigate these impacts. These include the development and deployment of efficient, high- throughput diagnostic tests to support targeted intervention, modelling of geographic and seasonal trends in infection, more thorough economic data and analysis of the impact of helminth infections and greater translation and involvement of end-users in devising and disseminating best practices. Complex changes in helminth epidemiology will require innovative solutions. By developing and using new technologies and models, the use of anthelmintics can be optimised to limit the development and spread of drug resistance and to reduce the overall economic impact of helminth infections. This will be essential to the continued productivity and profitability of livestock farming in Europe and its contribution to regional and global food security. View Full-Tex