Survey of anthelmintic resistance in a Romanian horse stud using three different methods.

Resistance of cyathostomins to benzimidazole (BZ) anthelmintics is widespread in horses in many parts of the world. This study compared three methods for the determination of benzimidazole resistance of Cyathostominae in 18 horses from a stud farm in Romania. The horses were treated with Fenbendazole. The resistance test was performed by FECRT, ERP and PCR. On Day 0, larvae of species belonging to the Cyathostominae subfamily, types A, B, C, D and Gyalocephalus, as well as Strongylus vulgaris species of the Strongylinae subfamily, were identified. At 42 days post treatment with fenbendazole only larvae of Cyathostominae, types A and D were identified. Resistance to Fenbendazole was found in one horse, using the FECRT and ERP tests. Both genetic resistance and susceptibility to BZ anthelmintics was observed in 13 samples (72.22%) using the PCR test. However, three samples (16.67%) showed only the BZ-susceptibility gene. In 2 samples, (11.11%) only the resistance gene to BZ anthelmintics was identified. Several inconsistencies in the evidence of resistance to benzimidazole were observed between the PCR test and the other two methods, which indicates that several methods for determining and controlling the resistance should be used in practice.

Multiple resistance in equine cyathostomins: a case study from military establishments in Rio Grande do Sul, Brazil.

Semi-intensive equine breeding system favors gastrointestinal nematode infections. The treatment of these infections is based on the use of anthelmintics. However, the inappropriate use of these drugs has led to parasitic resistance to the available active principles. The objective of this study was to evaluate the efficacy of the main classes of antiparasitic (ATP) used in control in adult and young animals, including: benzimidazoles (fenbendazole), pyrimidines (pyrantel pamoate), macrocyclic lactones (ivermectin and moxidectin), as well as the combination of active ingredients (ivermectin + pyrantel pamoate). The study was carried out in two military establishments, located in Rio Grande do Sul (RS), from January to December, 2018. The intervals between the treatments of the animals were performed from 30 to 90 days. Coproparasitological evaluations were determined by the egg count reduction in the faeces. Cyatostomine larvae were identified in pre and post-treatment cultures. The results demonstrated the multiple parasitic resistance of cyathostomins to fenbendazole, moxidectin in young animals, and to fenbendazole, pyrantel pamoate in adult animals. Thus, it is necessary to define or diagnose parasitic resistance to assist in the creation of prophylactic parasitic control, using suppressive treatment with ATP associated with integrated alternatives. The progress of parasitic resistance can be slowed.

Cytokine and goblet cell gene expression in equine cyathostomin infection and larvicidal anthelmintic therapy.

AIMS: The role of the immune response to cyathostomin infections in horses remains unknown. Intestinal goblet cell hyperplasia has previously been noted as a component in cyathostomin infection; however, the function is unclear. The goal of this study was to evaluate the local and systemic gene expression to cyathostomin infections following larvicidal treatment and explore their relation to goblet cells. METHODS AND RESULTS: Thirty-six ponies with naturally acquired cyathostomin infections were randomly allocated into three groups: fenbendazole-treated (10 mg/kg PO 5 days), moxidectin-treated (0.4 mg/kg PO once) and untreated control. Whole blood from all horses was collected weekly, and tissue samples from the large intestine collected during necropsy at 2 and 5 weeks post-treatment (WPT). Gene expression of interleukin (IL)-4, IL-5, IL-6, IL-10, IL-13, IL-17A, IL-22, IFN-γ, resistin-like molecule beta (RELM-ß), Mucin 2 (MUC2) and tumour necrosis factor (TNF)-α was measured using qRT-PCR. There were statistically significant linear correlations between luminal worm burdens and MUC2 (r = -.2358) and RELM-ß (r = -.2261). CONCLUSION: This suggests an active role of immune system post-treatment in parasite expulsion, specifically in goblet cells, and that the organs respond differently to treatment and the larvae themselves. This may have implications in the disease process and treatment.

Multiple resistance in equine cyathostomins: a case study from military establishments in Rio Grande do Sul, Brazil / Múltipla resistência de ciatostomíneos de equinos: um estudo de caso em estabelecimentos militares no Rio Grande do Sul, Brasil

Abstract Semi-intensive equine breeding system favors gastrointestinal nematode infections. The treatment of these infections is based on the use of anthelmintics. However, the inappropriate use of these drugs has led to parasitic resistance to the available active principles. The objective of this study was to evaluate the efficacy of the main classes of antiparasitic (ATP) used in control in adult and young animals, including: benzimidazoles (fenbendazole), pyrimidines (pyrantel pamoate), macrocyclic lactones (ivermectin and moxidectin), as well as the combination of active ingredients (ivermectin + pyrantel pamoate). The study was carried out in two military establishments, located in Rio Grande do Sul (RS), from January to December, 2018. The intervals between the treatments of the animals were performed from 30 to 90 days. Coproparasitological evaluations were determined by the egg count reduction in the faeces. Cyatostomine larvae were identified in pre and post-treatment cultures. The results demonstrated the multiple parasitic resistance of cyathostomins to fenbendazole, moxidectin in young animals, and to fenbendazole, pyrantel pamoate in adult animals. Thus, it is necessary to define or diagnose parasitic resistance to assist in the creation of prophylactic parasitic control, using suppressive treatment with ATP associated with integrated alternatives. The progress of parasitic resistance can be slowed.

Resumo O sistema semi-intensivo de criação de equinos favorece infecções por nematoides gastrointestinais. O tratamento dessas infecções é baseado no uso de anti-helmínticos. No entanto, o uso inadequado desses medicamentos levou à resistência parasitária aos princípios ativos disponíveis. O objetivo deste estudo foi avaliar a eficácia das principais classes de antiparasitários (ATP), utilizados no controle em animais adultos e jovens, incluindo: benzimidazois (fenbendazol), pirimidinas (pamoato de pirantel), lactonas macrocíclicas (ivermectina e moxidectina), bem como a combinação de ingredientes ativos (ivermectina + pamoato de pirantel). O estudo foi realizado em dois estabelecimentos militares, localizados no Rio Grande do Sul (RS), de janeiro a dezembro de 2018. Os intervalos entre os tratamentos foram realizados de 30 a 90 dias. As avaliações coproparasitológicas foram determinadas pela redução da contagem de ovos nas fezes. Foram identificadas larvas de ciatostomíneos nas coproculturas pré e pós tratamentos. Os resultados demonstraram a resistência parasitária múltipla dos ciatostomíneos ao fenbendazol, moxidectina em animais jovens, febendazole e pamoato de pirantel em animais adultos. Estabelecer o diagnóstico da resistência parasitária auxiliará na elaboração de um controle parasitário profilático, reduzindo o tratamento supressivo com ATP juntamente com alternativas de controle integrado. Dessa forma, o avanço da resistência parasitária poderá ser retardado.

Managing anthelmintic resistance in cyathostomin parasites: Investigating the benefits of refugia-based strategies.

Selective anthelmintic therapy has been recommended as a sustainable strategy for cyathostomin control in horse populations for several decades. The traditional approach has been to determine strongyle fecal egg counts (FEC) for all horses, with treatment only recommended for those exceeding a predetermined threshold. The aims are to achieve a reduction of overall egg shedding, while leaving a proportion of the herd untreated, which lowers anthelmintic treatment intensity and reduces selection pressure for development of anthelmintic resistance. This study made use of the cyathostomin model to evaluate the influence of treatment strategies with between 1 and 8 yearly treatment occasions, where either 1) all horses were treated, 2) a predetermined proportion of the herd remained untreated, or 3) horses were treated if their FEC exceeded thresholds between 100 and 600 strongyle eggs per gram. Weather data representing four different climatic zones was used and three different herd age structures were compared; 1) all yearlings, 2) all mature horses 10-20 years old, and 3) a mixed age structure of 1-20 years of age. Results indicated a consistent effect of age structure, with anthelmintic resistance developing quickest in the yearling group and slowest among the mature horses. Development of anthelmintic resistance was affected by treatment intensity and selective therapy generally delayed resistance. Importantly, the results suggest that the effects of selective therapy on resistance development are likely to vary between climatic zones and herd age structures. Overall, a substantial delaying of resistance development requires that the average number of treatments administered annually across a herd of horses needs to be about two or less. However, results also indicate that an age-structured prioritisation of treatment to younger horses should still be effective. It appears that a 'one-size-fits-all' approach to the management of anthelmintic resistance in cyathostomins is unlikely to be optimal.

Development of amplicon sequencing for the analysis of benzimidazole resistance allele frequencies in field populations of gastrointestinal nematodes.

Anthelmintic resistant gastrointestinal helminths have become a major cause of poor health in sheep and goats. Sensitive and specific molecular markers are needed to monitor the genotypic frequency of resistance in field parasite populations. Gastrointestinal nematode resistance to benzimidazole is caused by a mutation in one of three positions within the isotype 1 ß-tubulin gene. In the absence of markers for resistance to the other broad spectrum anthelmintic classes, these provide a relevant study example. Determination of the prevalence of these single nucleotide polymorphisms in field nematode populations can be impractical using conventional molecular methods to examine individual parasites; which can be laborious and lack sensitivity in determining low levels of resistance in parasite populations. Here, we report the development of a novel method based on an Illumina MiSeq deep amplicon sequencing platform to sequence the isotype 1 ß-tubulin locus of the small ruminant gastrointestinal nematode, Teladorsagia circumcincta, and determine the frequency of the benzimidazole resistance mutations. We validated the method by assessing sequence representation bias, comparing the results of Illumina MiSeq and pyrosequencing, and applying the method to populations containing known proportions of resistant and susceptible larvae. We applied the method to field samples collected from ewes and lambs on over a period of one year on three farms, each highlighting different aspects of sheep management and approaches to parasite control. The results show opportunities to build hypotheses with reference to selection pressures leading to differences in resistance allele frequencies between sampling dates, farms and ewes or lambs, and to consider the impact of their genetic fixation or otherwise. This study provides proof of concept of a practical, accurate, sensitive and scalable method to determine frequency of anthelmintic resistance mutations in gastrointestinal nematodes in field studies and as a management tool for livestock farmers.

Current anthelmintic treatment is not always effective at controlling strongylid infections in German alpaca herds.

BACKGROUND: Endoparasites are considered a major health problem of South American camelids as shown in a recent survey among German and Austrian camelid owners. Although prophylactic and therapeutic measures such as application of anthelmintics are commonly used, treatment efficacy is usually not assessed. Owners have expressed significant concerns regarding the effect of antiparasitic therapy, so this study aimed to evaluate the outcome of anthelmintic treatment in German alpaca herds with different drugs. RESULTS: Overall, 617 samples from 538 clinically healthy alpacas > 1 year-old from 27 farms (n = 11-157 animals/herd) were examined. The most common parasites detected by flotation were Eimeria spp. (75.1%) followed by strongylids (55.0%), Nematodirus spp. (19.3%), cestodes (3.1%) and Trichuris (2.7%). After initial coproscopical examination by flotation and strongylid egg quantification by the McMaster technique, positive animals excreting at least 150 eggs per gram of faeces were included in a faecal egg count reduction test (FECRT) using fenbendazole (n = 71 samples), moxidectin (n = 71) or monepantel (n = 66). Pre-treatment larval cultures (n = 23 positive pooled farm samples) revealed Haemonchus (87% of the farms), Cooperia (43.5%), Trichostrongylus (21.7%), Ostertagia (13.0%), Nematodirus and Oesophagostomum (4.3% each). Fenbendazole treatment reduced egg excretion by 45%, moxidectin by 91% and monepantel by 96%. On the farm level, 13/18 farms that used fenbendazole, 6/6 farms that used moxidectin and 2/5 farms that used monepantel had individual FECR values < 90% (fenbendazole) or < 95% (moxidectin, monepantel). Haemonchus and Cooperia were overrepresented on the farms with reduced treatment efficacy. CONCLUSIONS: Gastrointestinal strongylids are common in German alpacas and fenbendazole in particular was not sufficiently effective to reduce strongylid egg excretion. Although the FECRT could not unambiguously determine anthelmintic resistance in the present study, the finding that small ruminant strongylids, especially Haemonchus, are common in alpacas indicates that determination of effective anthelmintic doses, monitoring of efficacy and adapted (selective) treatment regimens must be implemented as part of sustainable deworming practices in this species in accordance with recommendations for ruminants.

Pilot study evaluating the efficacy of a topical formulation containing emodepside and praziquantel in the treatment of natural feline troglostrongylosis.

BACKGROUND: Troglostrongylus brevior, a lungworm usually affecting wild felids, has been recently recorded in a number of cases in domestic cats, mainly in Mediterranean areas. Although feline troglostrongylosis is a severe and life-threatening disease, especially in young cats, treatment options are very limited. The present study evaluated the efficacy and safety of a spot-on formulation containing emodepside 2.1% and praziquantel 8.6% (Profender®, Bayer), which is licensed for treatment of the more common cat lungworm Aelurostrongylus abstrusus, for the treatment of natural troglostrongylosis. METHODS: Sixteen cats enrolled in the study were 1:1 allocated to two groups, i.e. Group T, treated with Profender® spot-on on days 0 and 14 (± 2) at the recommended clinical dose, and Group C which remained untreated. After study completion, the control cats received two rescue treatments with Profender® on days 28 (± 2) and 42 (± 2). The primary efficacy criterion was the absence of T. brevior L1 following treatment. Other efficacy parameters were the quantitative comparison of L1 presence before (baseline) and after treatment in both groups, and the comparison of clinical signs pre- and post-treatment. RESULTS: In terms of stopping larval shedding, Profender® showed an efficacy of 97% and 97.5% (arithmetic and geometric means, respectively) for group T, 97.1% and 98.5% for group C after one administration, and 100% for both groups after two doses. Overall, 12 cats showed clinical signs related to T. brevior. Specifically, 9 were clinically affected before treatment while clinical signs appeared after the first treatment in 3 cats. At the end of the study, all symptomatic cats fully recovered with the exception of 3 cats that showed clinical signs similar to those observed at the pre-treatment examination at the end of the study. CONCLUSIONS: This study shows that Profender® is effective against T. brevior.

The saprophytic fungus Fusarium solani increases the insecticidal efficacy of the entomopathogenic nematode Steinernema diaprepesi.

In two field surveys, high proportions of Galleria mellonella L. (Lepidoptera: Pyralidae) sentinel larval cadavers were infected by Fusarium solani without evidence of concomitant entomopathogenic nematode (EPN) or entomopathogenic fungus (EPF) reproduction. Because F. solani is not considered entomopathogenic, the survey suggested the possibility that F. solani competes with EPNs. We tested the hypotheses that F. solani attracts the EPN, Steinernema diaprepesi, to facilitate infection of Diaprepes root weevils (Diaprepes abbreviatus L.) and thereafter competes with the nematode in the insect cadaver. In two-choice olfactometer assays where one side was treated with F. solani mycelia and conidia, juvenile S. diaprepesi were attracted to the fungus, in either raw soil, or in autoclaved soil in the presence or absence of insects. However, this attraction was attenuated as the habitat became more complex, by using raw soil in combination with insect larvae. Fusarium oxysporum did not recruit the nematode. When soil microcosms were tested with F. solani conidia and S. diaprepesi, the concomitant infection increased the mortality of the insect (P = 0.02) to 83%, compared to 58% and 0% mortality when nematodes or fungi were individually applied, respectively. Concomitant inoculation also increased the number of cadavers that supported nematode reproduction and increased the population density of fungus in soil. The number of IJs entering the host insect was not affected by F. solani. These results support the possibility that F. solani can facilitate the insecticidal efficiency of S. diaprepesi in order to exploit the resources in the cadaver.

Evaluation of the anthelminthic potential of Duranta erecta L. (Verbenaceae) fruits used in Nigerian ethnomedicine as a vermifuge.

ETHNOPHARMACOLOGICAL RELEVANCE: Duranta erecta L. fruits have been reported to have in vitro anthelminthic properties. People living in the tropical South-Eastern part of Nigeria use the plant in folk medicine for the treatment of malaria, abscesses and as a vermifuge. Although there are a few reports about its in vitro anthelminthic activity against different worm categories, scientific reports regarding its in vivo anthelminthic activity are limited. AIM OF THE STUDY: This study was designed with the aim of determining the potential of the plant as an anthelminthic and to verify the claims made by its local users. MATERIALS AND METHODS: Acute toxicity of the plant extract was determined using Lorke's method. Anthelminthic activity was investigated using adult male albino mice experimentally infected with Heligmosomoides bakeri infective L3. Graded ascending doses of the plant extract and Albendazole respectively were orally administered to the mice in the infected groups. Corprological and haematological parameters were recorded within the study period. Twenty-eight (28) days post-infection, all infected mice were humanely sacrificed and the Post-Mortem Adult Worm Burden (WB) was estimated and recorded. RESULTS: The results showed that the extract had an LD50 greater than 5000 mg/kg BW and therefore was not acutely toxic for oral use. It also showed that the plant extract was unable to eliminate the faecal egg output or adult worms in the gastrointestinal tract of infected animals even at the high doses used in the study. This was in contrast to Albendazole which significantly (p < .05) reduced faecal egg counts and worm burdens by 71% and 92% respectively in treated mice. Following infection, there was anaemia in all infected groups seen from results of erythrocytic parameters. Treatment with the plant extract, regardless of the dose, was unable to effectively reverse the effect of parasite infection on erythrocytic parameters. However, treatment with Albendazole positively reversed the anaemia, restoring the mice to pre-infection values by the end of the experiment. The results showed significant (p < .05) increase in WBC counts across all groups following infection with the parasite. Treatment with the plant extract and Albendazole respectively, significantly (p < .05) reduced the WBC counts to near pre-infection values in most treatment groups. CONCLUSION: As a result of the poor anthelminthic effects recorded in the study, it is therefore recommended that Duranta erecta L. fruits be explored for its other useful effects rather than as an anthelminthic.

Diagnosis of resistance alleles in codon 167 of the beta-tubulin (Cya-tbb-1) gene from third-stage larvae of horse cyathostomins.

Anthelmintic resistance is a serious problem for the control of equine gastrointestinal nematodes. In the present survey, 173 third stage larvae of cyathostomins were investigated from three different locations for the presence of the resistant genotype at codon 167 of the beta-tubulin gene, as this is the most prevalent mutation. The larvae from the state of Parana (n=67), Sao Paulo (n=54) and Santa Catarina (n=52), showed 61.2; 31.5 and 38.5% of the heterozygous resistant genotype - TTC/TAC, respectively. An unpublished mutation at codon 172 that results in a serine (S) to threonine (T) substitution was found in 17.9% (12/67) of samples from Parana; and 13.0% (7/54) of samples from Sao Paulo. We have compared the molecular diagnostic with the fecal egg count data (R2=-0.79) from the same farms, and consider that the use of routine molecular diagnostic in individual larva may help to determine the population genetic distribution that is associated with drug failure.

Papaya latex supernatant has a potent effect on the free-living stages of equid cyathostomins in vitro.

The control of equid gastrointestinal nematodes in developed countries, in particular the cyathostomins, is threatened by high levels of anthelmintic resistance. In recent years, there has been increasing interest in the evaluation of traditional 'ethnoveterinary' medicines as alternatives to chemical anthelmintics. The cysteine proteinases (CPs), a group of enzymes derived from fruits such as papaya (Carica papaya), pineapple (Ananas comosus) and figs (Ficus spp.), have shown good efficacy against adult stages of a range of parasitic nematodes, in vitro and in vivo. The efficacy of CPs against cyathostomins remains to be explored. In this study, the efficacy of a crude preparation of CPs, papaya latex supernatant (PLS), against the free-living stages of cyathostomins was evaluated using two in vitro tests, the egg hatch test (EHT) and the larval migration inhibition test (LMIT). It was demonstrated that PLS had a potent effect in the EHT, with EC-50 values in the range of 0.12-0.22µM. At concentrations above 6.25µM the eggs did not develop, below this concentration the L1 developed but they lost integrity of the cuticle upon hatching. These effects were inhibited by pre-incubation of PLS with the CP inhibitor L-trans-epoxysuccinyl-l-leucylamido-(4-guanidino butane) (E64), indicating that CPs were responsible for the anti-parasitic activity. A dose-dependent inhibition of migration of third stage larvae (L3) in the LMIT was demonstrated at higher concentrations of PLS, with EC-50 values in the range of 67.35-106.31µM. Incubation of PLS with E64 prior to use in the LMIT did not reverse the anti-migratory effect, suggesting that CPs were not responsible for the reduced migration of cyathostomin L3 and that PLS also contains an additional active compound. This is the first report of PLS and/or CPs showing activity against the free-living stages of a parasitic helminth. In addition, it suggests that cyathostomins are highly sensitive to the effects of CPs and further evaluation of their efficacy against parasitic stages and in vivo are strongly indicated.

Practical and low cost whole-organism motility assay: A step-by-step protocol.

Here, we provide a step-by-step protocol for a practical and low cost whole-organism assay for the screening of chemical compounds for activity against parasitic worms. This assay has considerable advantages over conventional methods, mainly in relation to ease of use, throughput, time and cost. It is readily suited to the screening of hundreds to thousands of compounds for subsequent hit-to-lead optimisation, and should be applicable to many different parasites and other organisms commensurate with the size of wells in the microtiter plates used for phenotypic screening.

Efficacy of selected anthelmintic drugs against cyathostomins in horses in the federal state of Brandenburg, Germany.

Cyathostomins are currently the most common internal parasites of horses. With the intensive use of anthelmintic drugs over the past decades, resistance of cyathostomins to anthelmintics is becoming a growing problem in many countries. The aim of this study was to assess the current situation on horse farms in the German federal state of Brandenburg. A pre-selected population of horses from 24 premises that had shown a prevalence of cyathostomins higher than the average in a previous study was examined for anthelmintic efficacy. Faecal egg count reduction tests (FECRTs) were performed for ivermectin (IVM) and pyrantel (PYR). For IVM, the egg reappearance period (ERP) was also examined, as a shortened ERP can be indicative of developing resistance. The efficacy of IVM on cyathostomins was high: 99.1 % of 224 horses had a zero egg count 14 days after treatment. No shortening of the ERP was detected. For the data of the FECRT for PYR, three different methods of calculation were employed: (a) the method as recommended by the World Association for the Advancement of Veterinary Parasitology (WAAVP), (b) a bootstrapping method and (c) a Markov chain Monte Carlo method. Two methods of interpretation for these data were used: Resistance was declared (a) when FECR was <90 % and the lower 95 % confidence interval (LCL) <80 % and (b) when additionally the upper 95 % confidence level (UCL) was <95 %. When applying the first interpretation, resistance against PYR was found on four yards, while, when considering the UCL, all three methods for calculation only detected resistance on one single yard. Twelve species of cyathostomins were detected in larval cultures derived from strongyle egg positive faecal samples collected 14 days after treatment with PYR by reverse line blot hybridization (RLB). In order to generate comparable data, it is suggested to establish international standards for the calculation of FECRT data.

A highly conserved, inhibitable astacin metalloprotease from Teladorsagia circumcincta is required for cuticle formation and nematode development.

Parasitic nematodes cause chronic, debilitating infections in both livestock and humans worldwide, and many have developed multiple resistance to the currently available anthelmintics. The protective collagenous cuticle of these parasites is required for nematode survival and its synthesis has been studied extensively in the free-living nematode, Caenorhabditis elegans. The collagen synthesis pathway is a complex, multi-step process involving numerous key enzymes, including the astacin metalloproteases. Nematode astacinsare crucial for C. elegans development, having specific roles in hatching, moulting and cuticle synthesis. NAS-35 (also called DPY-31) is a homologue of a vertebrate procollagen C-proteinase and performs a central role in cuticle formation of C. elegans as its mutation causes temperature-sensitive lethality and cuticle defects. The characterisation of DPY-31 from the ovine gastrointestinal nematode Teladorsagia circumcincta and its ability to rescue the C. elegans mutant is described. Compounds with a hydroxamate functional group have previously been shown to be potent inhibitors of procollagen C-proteinases and were therefore examined for inhibitory activity against the T. circumcincta enzyme. Phenotypic screening against T. circumcincta, Haemonchus contortus and C. elegans larval stages identified compounds that caused body morphology phenotypes consistent with the inhibition of proteases involved in cuticle collagen synthesis. These compounds correspondingly inhibited the activity of recombinant T. circumcincta DPY-31, supporting the hypothesis that this enzyme may represent a potentially novel anthelmintic drug target.

Nematode resistance to ivermectin (630 and 700µg/kg) in cattle from the Southeast and South of Brazil.

Two high doses of ivermectin (630µg/kg and 700µg/kg) that are sold commercially in Brazil were evaluated in dose-and-slaughter trials with 144 naturally nematode-infected cattle from eight regions within the states of Minas Gerias, São Paulo and Rio Grande do Sul in Brazil. Treatment groups were based on fecal egg counts 1, 2, and 3days before treatment; all animals studied had a minimum egg count of at least 500 eggs per gram of feces (EPG). Post-mortem analyses were conducted on day 14. The highest levels of resistance to ivermectin were found for Haemonchus placei, Cooperia punctata and Oesophagostomum radiatum; all populations of H. placei were resistant to the 630µg/kg dose, and 67% were resistant to 700µg/kg; 86% of C. punctata were resistant to the 630µg/kg dose, and 33% were resistant to 700µg/kg. A combined analysis revealed that 57% of O. radiatum were resistant to the lower dose of ivermectin. H. placei, C. punctata and O. radiatum, in order, were the nematode populations with the highest indices of resistance, whereas Trichostrongylus axei was the most susceptible to 630 and 700µg/kg dosages of ivermectin. The results of helminthic resistance to ivermectin for different populations of H. placei and C. punctata described in the present study support previous literature data, in which a small decrease in the average parasitic burden of C. punctata and a consequent increase of H. placei were observed in cattle from the Southeast, South and Center-West regions of Brazil.

Evaluation of enhanced virgin coconut oil and senduduk (Melastoma malabathricum) as anthelmintics against caprine strongyle nematodes.

A study was conducted to evaluate the anthelmintic properties of enhanced virgin coconut oil (EVCO) and senduduk (Melastoma malabathricum) plant against strongyle nematodes in goats. Two preparations of 10% EVCO dissolved in 90% virgin coconut oil and 10% EVCO dissolved in 90% palm oil, were given orally to two groups of mixed breeds goats. The efficacy test indicated that EVCO was insufficiently active as an anthelmintic. Four concentrations of senduduk solution (1.25, 2.5, 5.0 and 10 mg ml(-1)) were compared with a control and albendazole in an in vitro test for larvicidal effect. There was no significant larval mortality using senduduk solution. An in vivo test of senduduk was conducted by comparing three groups of goats, namely control, levamisole and treatment groups that were given a daily oral dose of senduduk crude extract with 1g kg(-1) from Day 0 to Day 12 and 2 g kg(-1) from Day 13 to Day 30. This efficacy test with senduduk also gave negative results. The findings obtained indicated that EVCO and senduduk were ineffective as anthelmintics against caprinestrongyle nematodes at the concentrations used.

Control trial and fecal egg count reduction test determinations of nematocidal efficacies of moxidectin and generic ivermectin in recently weaned, naturally infected calves.

An anthelmintic efficacy study was performed with young, naturally infected beef-type calves obtained at local farms. Presumably, the study calves had been recently weaned and had not been treated with a parasiticide prior to study acquisition. After blocking the 24 study calves in accordance with calculated Cooperia spp EPG counts, the calves were randomly allocated within each block to one of four treatment groups, resulting in 6 animals per treatment group (untreated controls, topical ivermectin at the rate of 500 mcg/kg BW [Noromectin Pour-On(®) Norbrook], topical moxidectin at the rate of 500 mcg/kg BW [Cydectin Pour-On(®) Boehringer Ingelheim Vetmedica (BIVM)] and injectable moxidectin at the rate of 200 mcg/kg BW [Cydectin(®) BIVM]. After treatment, the animals were penned by treatment group until necropsy. Fecal, strongyle egg count reduction percentages at 2 days post-treatment were 13, 52, 81 and 93 for control, topical ivermectin, topical moxidectin and injectable moxidectin treatment groups, respectively. In the same order as above, egg count reduction percentages at necropsy (15-18 days post-treatment) were -14, 91, 94 and 97. Based on geometric means for worm burdens quantified at necropsy, anthelmintic efficacies ranged from 96 to 100% for adult Oesophagostomum radiatum, Ostertagia ostertagi, Haemonchus placei and Trichostrongylus axei. Against adult Nematodirus helvetianus, percent efficacies based on geometric means were 56.7, 98.3 and 82.2 for topical ivermectin, topical moxidectin and injectable moxidectin, respectively; an observation that is guarded, as only 5 control animals were infected with adult N. helvetianus. Respective anthelmintic efficacies (%'s) against adult Cooperia oncophora and C. punctata were 93.0 and 73.4 (topical ivermectin), 99.3 and 99.9 (topical moxidectin) and 46.1 and 93.6 (injectable moxidectin). Judging from these data, it appears that treatment of calves soon after weaning with topical moxidectin is effective (>90% efficacy) for all common nematodes in cattle, but injectable moxidectin and topical ivermectin have limited effectiveness against Cooperia spp. With Cooperia spp and H. placei infections, the fecal egg count reduction test and the control trial determinations of anthelmintic effectiveness were in disagreement regarding injectable moxidectin and topical ivermectin.

Efficacy of albendazole and moxidectin and resistance to ivermectin against Libyostrongylus douglassii and Libyostrongylus dentatus in ostriches.

Anthelmintic resistance has emerged globally as a problem amongst nematode of livestock and has been particularly well documented in equine and small ruminants. There are no studies regarding the efficacy of anthelmintics against the hematophagous nematodes in ostriches, Libyostrongylus dentatus; and just a few on L. douglassii. Here the efficacy of albendazole, ivermectin and moxidectin were evaluated against these two species in an ostrich farm in Minas Gerais state, Brazil. The feces were collected on the day of treatment and after 13 days of an oral dose of albendazole (6 mg/kg), or an injected dose (0.2mg/kg) of ivermectin or moxidectin. The fecal egg count reduction test and coprocultures were performed to determine possible resistance against the drugs used. An efficacy of 60% was found for ivermectin, while albendazole and moxidectin were 100% effective. Both worm species appeared to have reduced sensitivity to ivermectin.

Management and environmental factors related to benzimidazole resistance in sheep nematodes in Northeast Spain.

A survey to determine the level of parasite resistance to benzimidazoles (BZ) under field conditions was performed on 107 commercial sheep farms located in the Aragon region of northeast Spain. Resistance was measured using the discriminant dose, a simplified form of the in vitro egg hatch assay (EHA). Taking into account the spatial structure of the data, a multivariate approach was applied to management and environmental variables as well as to their relationships with BZ resistance levels compiled from each flock. Results estimated that 11% of flocks had resistant parasite populations, although we suspected the presence of BZ-resistant parasite strains in 98% of the sample. Resistance levels were more similar among the nearest flocks, suggesting a contagious spatial distribution of resistance (i.e., resistance at neighbouring farms was not independent from one another). Management variables such as frequency of deworming, grazing in private pastures and underdosing were positively related to resistance levels, whereas only the use of BZ was negatively related to resistance levels, likely because BZ were replaced by other anthelmintics in flocks where reduced BZ efficacy was suspected. In addition to climatic conditions and seasonality, land use was an environmental variable associated with observed BZ resistance levels. Generally, resistance was highest in cooler and wetter areas but was lower in flocks sampled during January-March compared to flocks sampled in April-June or October-December. Variation partitioning procedures showed that the variation of resistance explained by the effect of environmental variables was higher than management variables. The effects of both variable groups, however, highly overlapped with the spatial structure of resistant levels, which suggests that a considerable amount of the effects attributable to both variable groups may be actually due to the spatial distribution of resistance. The resistance variation explained by the spatial component suggested that other uncontrolled factors acting at short spatial scale (e.g., common management and environmental variables; the importation of resistant strains and their posterior spread across neighbouring flocks; the selection history of the worms carried out by historical management events previous to this survey; and genetic, physiological or both types of parasite population variation) could yield this contagious spatial structure of BZ resistance. Although further research is needed, both seasonal variation and the dependence of resistance levels among neighbouring flocks should be taken into account in the design of future research or observational resistance programmes to minimise spatial and temporal pseudo-replication. Thus, research would avoid biased estimations of resistance prevalence or of its relationship with putative factors.