Synthesis, bioevaluation and docking studies of some 2-phenyl-1H-benzimidazole derivatives as anthelminthic agents against the nematode Teladorsagia circumcincta.

Gastrointestinal nematode infections are the main diseases in herds of small ruminants. Resistance to the main established drugs has become a worldwide problem. The purpose of this study is to obtain and evaluate the in vitro ovicidal and larvicidal activity of some 2-phenylbenzimidazole derivatives on susceptible and resistant strains of Teladorsagia circumcincta. Compounds were prepared by known procedures from substituted o-phenylenediamines and arylaldehydes or intermediate sodium 1-hydroxyphenylmethanesulfonate derivatives. Egg Hatch Test (EHT), Larval Mortality Test (LMT) and Larval Migration Inhibition Test (LMIT) were used in the initial screening of compounds at 50 µM concentration, and EC50 values were determined for the most potent compounds. Cytotoxicity evaluation of compounds was conducted on human Caco-2 and HepG2 cell lines to calculate their Selectivity Indexes (SI). At 50 µM concentration, nine out of twenty-four compounds displayed more than 98% ovicidal activity on a susceptible strain, and four of them showed more than 86% on one resistant strain. The most potent ovicidal benzimidazole (BZ) 3 showed EC50 = 6.30 µM, for the susceptible strain, while BZ 2 showed the lowest EC50 value of 14.5 µM for the resistant strain. Docking studies of most potent compounds in a modelled Teladorsagia tubulin indicated an inverted orientation for BZ 1 in the colchicine binding site, probably due to its fair interaction with glutamic acid at codon 198, which could justify its inactivity against the resistant strain of T. circumcincta.

Teladorsagia circumcincta beta tubulin: the presence of the E198L polymorphism on its own is associated with benzimidazole resistance.

BACKGROUND: Benzimidazole resistance is associated with isotype-1 ß-tubulin gene F200Y, E198A and F167Y SNPs. In this study, the recently described polymorphism E198L was reported and analysed in Teladorsagia circumcincta. METHODS: The benzimidazole phenotypic resistance was measured by the faecal egg count reduction test (FECRT) and the egg hatch test (EHT) using a discriminating dose (DD) in 39 sheep flocks. Around 1000 larvae collected before and after treatment were used for DNA extraction. The resistant species identified in all flocks was T. circumcincta. The resistance alleles frequencies were measured for F200Y and E198A. A 371-bp fragment of the isotype-1 ß-tubulin gene was analysed, including the three codons of interest, and a new pyrosequencing assay was designed for testing E198L. RESULTS: The percentage of resistant flocks was 35% by FECRT or 26% by EHT; however, F200Y and E198A SNPs were absent in T. circumcincta. The amplification of a 371-bp fragment confirmed the absence of F167Y and F200Y in 6 resistant flocks. Regarding codon 198, all samples after treatment carried a leucine (CTA). A pyrosequencing assay analysed the allele frequencies for the first two bases at codon 198 independently, G/C and A/T. The correlation between C and T frequencies was almost 1 (r = 0.929, P < 0.0001) and the mean value of both was calculated to measure the leucine frequency; this value ranged between 10.4-80.7% before treatment, and 82.3-92.8% after treatment. High and similar correlations were reported between the genotypic variables (C frequency, T frequency or mean of both frequencies) and phenotypic resistance (r > 0.720, P < 0.0001), although negatively associated with the FECRT and positively with the EHT. According to multivariate linear regression analysis, the T frequency was the most significant variable influencing the phenotypic resistance (FECRT or EHT; P < 0.0001). In the EHT, 67.1% of the phenotypic variability is associated with the T frequency but in the FECRT only 33.4%; therefore, the EHT using a DD seems to detect the genotypic resistance more accurately than the FECRT. CONCLUSIONS: The E198L polymorphism can confer BZ resistance on its own in T. circumcincta.

Motility based assays using cultured fourth stage larvae fail to provide consistent discrimination between known avermectin-resistant and -susceptible isolates of Cooperia spp.

The fecal egg count reduction test (FECRT) is the only method commonly used for diagnosing anthelmintic resistance in gastrointestinal nematodes of cattle, but this method has several drawbacks that have limited its widescale implementation. Consequently, there exists a need to develop better methods for diagnosing resistance. Assays based on larval motility are used commonly for screening potential drug candidates, and for detecting drug resistance, but previous work in our lab demonstrated that the L3 stage failed to discriminate between avermectin-resistant and susceptible isolates of Cooperia spp. We hypothesized that the L4 may be a better stage for this purpose because it is a parasitic and actively feeding life stage without a double cuticle. L3 larvae of Cooperia spp. were exsheathed and cultured to L4 by maintaining them in media at 37 °C and 20 % CO2, with media changes and observation every 48 h for nine days. Three avermectin-resistant and two avermectin-susceptible GIN isolates (diagnosed by FECRT) containing >88 % Cooperia spp., were used. Three biological replicates were performed for each parasite isolate using both eprinomectin and ivermectin. Eleven drug concentrations from 0.01um to 40um and negative controls were evaluated. Motility readings were taken using the Worminator system before addition of the drug and at 24- and 48 -hs post drug exposure. Resistance ratios for ivermectin and eprinomectin ranged from 0.35 to 2.75 and 0.54-1.03, respectively. Though significant differences (p < 0.05) in percent inhibition were found at some drug concentrations in some assays, there were no consistent significant differences in the dose-response between susceptible and resistant isolates. Inhibition was greater in about half of the assays for the susceptible isolates, and in half the assays for the resistant isolates. The lack of consistency in these data indicate that motility of L4 is not a reliable diagnostic phenotype for measuring resistance to avermectin drugs in Cooperia spp.

Efficacy and production benefits following use of eprinomectin extended-release injection in pastured dairy heifers.

A study was conducted in grazing dairy heifers to assess anthelmintic efficacy and production responses in dairy heifers treated with a single injection of eprinomectin in an extended-release formulation over a 123 day-period. The study was conducted on a pasture-based dairy in the Southeastern United States (North Carolina) over the summer months. Sixty crossbred dairy heifers were weighed and randomly allocated into 2 groups. One group (n = 30) was given 5% eprinomectin subcutaneously in the cervical region while the other group (n = 30) was given an equivalent volume of saline. Calves were weighed every 30 days throughout the trial for calculation of average daily gain and differences in overall weight gain. In addition, fecal samples were collected at days 0, 30, 60, 90 and 123 for worm egg count and coproculture. Both groups of cattle had similar worm egg concentrations at the start of the study. However, the control group had increasing concentrations of fecal worm eggs throughout the summer months while the heifers that received eprinomectin had minimal fecal worm eggs. The primary parasite species identified in this study were Haemonchus placei, Cooperia species and Ostertagia. The heifers that received eprinomectin gained 105 + 2.8 kg during the 123-day study period, representing an average daily gain of 0.85 kg/day compared to 78.3 + 4.1 kg (0.64 kg/day) for the control group. This represented a 33 % increase in average daily gain associated with deworming. The results of this study indicate that a single dose of extended-release eprinomectin was sufficient to control parasites through a 123-day summer grazing season and that administration of the anthelmintic had a significant impact on weight gain.

High frequency of benzimidazole resistance alleles in trichostrongyloids from Austrian sheep flocks in an alpine transhumance management system.

BACKGROUND: Infections of small ruminants with trichostrongyloid nematodes often result in reduced productivity and may be detrimental to the host. Anthelmintic resistance (AR) against most anthelmintic drug classes is now widespread amongst the trichostrongyloids. Baseline establishment, followed by regular monitoring of the level of AR, is necessary for farmers and veterinarians to make informed decisions about parasite management. The detection of single nucleotide polymorphisms (SNPs) is a sensitive method to detect AR against benzimidazoles (BZs), one of the most widely used anthelmintic classes. Alpine transhumance constitutes a special type of pasturing of sheep from many different farms, the aim of this study was to investigate the prevalence of benzimidazole resistance alleles in this particular management system. RESULTS: Sixteen sheep flocks in Styria and Salzburg in Austria were examined by pyrosequencing for SNPs at codons 167, 198 and 200 of the isotype-1 ß-tubulin gene. The frequency of the resistance-associated exchange F200Y was 87-100% for H. contortus, 77-100% for T. colubriformis and <  5-66% for T. circumcincta. Additionally, the F167Y polymorphism was detected in T. colubriformis from two farms at a frequency of 19 and 23% respectively. CONCLUSIONS: The high resistance allele frequency in H. contortus and T. colubriformis in the examined sheep population urgently calls for the development of new treatment strategies to sustainably control trichostrongyloid infections for this kind of pasturing, since the frequent mixing of flocks during the alpine summer grazing must be considered an important risk factor for the spread of resistant nematodes to a large number of farms.

Assessing anthelmintic resistance risk in the post-genomic era: a proof-of-concept study assessing the potential for widespread benzimidazole-resistant gastrointestinal nematodes in North American cattle and bison.

As genomic research continues to improve our understanding of the genetics of anthelmintic drug resistance, the revolution in DNA sequencing technologies will provide increasing opportunities for large-scale surveillance for the emergence of drug resistance. In most countries, parasite control in cattle and bison has mainly depended on pour-on macrocyclic lactone formulations resulting in widespread ivermectin resistance. Consequently, there is an increased interest in using benzimidazole drugs which have been used comparatively little in cattle and bison in recent years. This situation, together with our understanding of benzimidazole resistance genetics, provides a practical opportunity to use deep-amplicon sequencing to assess the risk of drug resistance emergence. In this paper, we use deep-amplicon sequencing to scan for those mutations in the isotype-1 ß-tubulin gene previously associated with benzimidazole resistance in many trichostrongylid nematode species. We found that several of these mutations occur at low frequency in many cattle and bison parasite populations in North America, suggesting increased use of benzimidazole drugs in cattle has the potential to result in widespread emergence of resistance in multiple parasite species. This work illustrates a post-genomic approach to large-scale surveillance of early emergence of anthelmintic resistance in the field.

Genotypic characterisation of monepantel resistance in historical and newly derived field strains of Teladorsagia circumcincta.

Recent reports of monepantel (MPTL) resistance in UK field isolates of Teladorsagia circumcincta has highlighted the need for a better understanding of the mechanism of MPTL-resistance in order to preserve its anthelmintic efficacy in this economically important species. Nine discrete populations of T. circumcincta were genotypically characterised; three MPTL-susceptible isolates, three experimentally selected MPTL-resistant strains and three field derived populations. Full-length Tci-mptl-1 gene sequences were generated and comparisons between the MPTL-susceptible isolates, MPTL-resistant strains and one field isolate, showed that different putative MPTL-resistance conferring mutations were present in different resistant isolates. Truncated forms of the Tci-mptl-1 gene were also observed. The genetic variability of individual larvae, within and between populations, was examined using microsatellite analyses at 10 'neutral' loci (presumed to be unaffected by MPTL). Results confirmed that there was little background genetic variation between the populations, global FST <0.038. Polymorphisms present in exons 7 and 8 of Tci-mptl-1 enabled genotyping of individual larvae. A reduction in the number of genotypes was observed in all MPTL-resistant strains compared to the MPTL-susceptible strains that they were derived from, suggesting there was purifying selection at Tci-mptl-1 as a result of MPTL-treatment. The potential link between benzimidazole (BZ)-resistance and MPTL-resistance was examined by screening individual larvae for the presence of three SNPs associated with BZ-resistance in the ß-tubulin isotype-1 gene. The majority of larvae were BZ-susceptible homozygotes at positions 167 and 198. Increased heterozygosity at position 200 was observed in the MPTL-resistant strains compared to their respective MPTL-susceptible population. There was no decrease in the occurrence of BZ-resistant genotypes in larvae from each population. These differences, in light of the purifying selection at this locus in all MPTL-resistant isolates, suggests that Tci-mptl-1 confers MPTL-resistance in T. circumcincta, as in Haemonchus contortus, but that different mutations in Tci-mptl-1 can confer resistance in different populations.

Proteolytic and nematicidal potential of the compost colonized by Hypsizygus marmoreus.

Spent mushroom compost (SMC) is a residue generated in edible mushrooms production, such as Hypsizygus marmoreus. Its genome was recently sequenced, demonstrating cuticle-degrading protease genes. The present work aims to investigate the proteases from H. marmoreus spent mushroom compost (SMC) by verifying its action on nematode larvae. The extraction of the crude extract directly with water from H. marmoreus SMC proved to be efficient for proteases obtainment, with proteolytic activity of 195.36 ±â€¯18.38 U g-1 of compound. Moreover, the zymogram and SDS-PAGE indicated the presence of two proteases with estimated molecular weights of 30.2 and 33.7 kDa. Due to the protease activity present in H. marmoreus SMC extract, there was a significant reduction in the number of Panagrellus redivivus and L3 in treated group compared to control group (p < 0.01), with 52% and 26% of reduction, respectively. A0A151VWY3 mature protein is composed of 296 amino acid residues, exhibiting molecular weight and pI of 29.5 kDa and 6.72. A0A151WD28 mature protein is composed of 343 amino acid residues, exhibiting molecular weight and pI of 34.4 kDa and 8.04. In the present work it was demonstrated that SMC from H. marmoreus has easily extracted protease content, presenting two proteases, possibly with cuticle-degrading activity, which had significant nematicidal effect on P. redivivus and bovine infective larvae.

Hidden in plain sight - Multiple resistant species within a strongyle community.

Ovine parasitic gastroenteritis is a complex disease routinely treated using anthelmintics. Although many different strongyle species may contribute to parasitic gastroenteritis, not all are equally pathogenic: in temperate regions, the primary pathogen is Teladorsagia circumcincta. In this study we investigated benzimidazole and ivermectin resistance on a commercial sheep farm in southeast Scotland. We assessed the impact of species diversity on the diagnosis of resistance using the faecal egg count reduction test and in vitro bioassays, and correlated the results with the frequency of benzimidazole resistance-associated genotypes measured in the T. circumcincta population by pyrosequencing of the ß-tubulin isotype-1 gene. Faecal egg count reduction test results showed efficacies of 65% for albendazole and 77% for ivermectin, indicating moderate resistance levels on the farm. However, PCR speciation of the same populations pre- and post-treatment revealed that removal of susceptible species had masked the presence of a highly resistant population of T. circumcincta. Less than 25% of individuals in the pre-treatment populations were T. circumcincta, the remainder consisting of Cooperia curticei, Chabertia ovina, Oesophagostomum venulosum and Trichostrongylus spp. In contrast, post-treatment with albendazole or ivermectin, the majority (88% and 100% respectively) of the populations consisted of T. circumcincta. The egg hatch test for benzimidazole resistance and the larval development test for ivermectin resistance were carried out using eggs obtained from the same populations and the results were broadly consistent with the faecal egg count reduction test. Thirty individual T. circumcincta from each sampling time point were assessed for benzimidazole resistance by pyrosequencing, revealing a high frequency and diversity of resistance-associated mutations, including within the population sampled post-ivermectin treatment. These results highlight the potential diversity of parasite species present on UK farms, and their importance in the diagnosis of anthelmintic resistance. On this particular farm, we demonstrate the presence of a highly dual-resistant population of T. circumcincta, which was strongly selected by treatment with either benzimidazoles or ivermectin, while other potentially less pathogenic species were removed.

Effect of Gliricidia sepium leaves intake on larval establishment of Cooperia punctata in calves and bio-guided fractionation of bioactive molecules.

The aims of this study were: 1) to assess the anthelmintic effect of Gliricidia sepium on the establishment of C. punctata third-stage larvae (L3) in calves, and 2) to isolate and to elucidate an anti-exsheathment phytochemical from the plant offered during the trial. Twelve ¾ Holstein × Zebu calves were divided in two experimental groups: control (T1) and treatment (T2) (n = 6). After adaptation, each calf was infected with an oral dose of 400 C. punctata L3/Kg LW. Basal diet consisted of Digitaria decumbens hay (6.27% CP) and commercial concentrate (12% CP). In addition, during the experimental period T2 received fresh G. sepium leaves (26.88% CP) ad libitum. On day 9 post-infection, three calves per treatment were randomly selected for slaughter, and worm counts were performed. Larval establishment rates obtained were 13.44 ±â€¯0.13% and 3.1 ±â€¯1.42% for T1 and T2, respectively (P < .05). The reduction of larval establishment was 76.9%. The total length of worms recovered from the animals was also affected by the intake of G. sepium (P < .05). Phytochemicals present in G. sepium leaves offered to calves were isolated through silica gel columns and elucidated through Magnetic Nuclear Resonance (1H and 13C). Bio-guided isolation procedures lead to the elucidation of Oxytroside (Kaempferol 3-O-rhamnopyranosyl-(1 → 6)-ß-D-glucopyranoside-7-O-rhamnopyranoside), which fully inhibited the C. punctata exsheathment process (2400 µg mL-1). Gliricidia sepium represents an alternative to prevent severe C. punctata infections by reducing larval establishment in cattle.

Combination deworming for the control of double-resistant cyathostomin parasites - short and long term consequences.

Equine cyathostomin are pervasive gastrointestinal parasites with wide-spread resistance to the benzimidazole and tetrahydropyrimidine drug classes worldwide. Combination deworming has been proposed as a more sustainable parasite control strategy. Simulation studies have found combination deworming to be effective in controlling drug resistant ovine trichostrongylid parasites. One equine study demonstrated an additive effect of a combination of oxibendazole and pyrantel pamoate against cyathostomins. However, this is the only equine study evaluating combination therapy, and the effects of repeated combination treatments administered over time remain unknown. The purpose of this study was to observe the efficacy of repeated oxibendazole/pyrantel pamoate combination therapy administered over one year against a cyathostomin population with resistance to benzimidazole and pyrantel products. Fecal egg counts were determined for the entire herd (N = 21) at the day of anthelmintic treatment and at two-week intervals for eight weeks post treatment. Starting efficacies of oxibendazole (OBZ, 10 mg/kg) and pyrantel pamoate (PYR, 6.6 mg base/kg) were 66.7% and 63.3%, respectively. Hereafter, the herd was treated four times with an oxibendazole/pyrantel pamoate combination, eight weeks apart, followed by repeating the single active treatments before concluding the study. While the first combination treatment exhibited an additive effect of the two active ingredients, this efficacy was not sustained over the course of the study. Mean fecal egg count reduction (FECR) was significantly greater for the first combination treatment (76.6%) than the second (42.6%, p = 0.0454), third (41.6%, p = 0.0318), and fourth (40.7%, p = 0.0372) combination treatments. The final single active mean FECRs were 42.3% for oxibendazole, and 42.7% for pyrantel pamoate. These efficacies were not significantly different from the initial single active efficacies (OBZ, p = 0.4421; PYR, p = 0.8361). These results suggest that combination therapy against double resistant equine cyathostomin populations is not sustainable, when using actives with markedly decreased starting efficacies.

The effect on liveweight gain of using anthelmintics with incomplete efficacy against resistant Cooperia oncophora in cattle.

A replicated field trial was conducted to measure the effect on liveweight gain of failing to adequately control anthelmintic resistant populations of Cooperia oncophora and to determine whether populations, and hence production losses, increased with time. Eight mobs of 10 Friesian-Hereford calves were run on independent farmlets from January to December, over each of two years. All mobs were routinely treated with a pour-on formulation of eprinomectin every six weeks, which controlled parasites other than Cooperia. Four mobs also received six weekly treatments with an oral levamisole plus albendazole combination anthelmintic to control Cooperia. Liveweights, condition scores, faecal egg counts and larval numbers on pasture were measured throughout. In the first year animals treated with eprinomectin alone were 12.9 kg lighter in November than those treated with eprinomectin plus albendazole and levamisole, however, in the second year there was no difference between the treatment groups. The data, therefore, support the view that while C. oncophora is less pathogenic than other cattle parasite species it can still cause production losses when present in sufficient numbers. In the first year of the study, parasite load, as measured by faecal nematode egg count and larval numbers on herbage, tended to be higher and calf growth rates lower than in the second year. In both years, counts of infective larvae on herbage declined over winter-spring to be at low levels before mid-summer. This suggests that the carry-over of infection from one crop of calves to the next was relatively small and hence that the level of challenge to the young calves at the start of each year was largely due to the effectiveness of the quarantine treatments administered when the animals arrived on the trial site. Low survival of larvae on pasture between grazing seasons, resulting in small larval populations on pasture when drenching programmes start each summer, might help to explain the widespread development of anthelmintic resistance in this parasite under New Zealand grazing systems.

Motility in the L3 stage is a poor phenotype for detecting and measuring resistance to avermectin/milbemycin drugs in gastrointestinal nematodes of livestock.

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

Gastrointestinal parasites of captive European bison Bison bonasus (L.) with a sign of reduced efficacy of Haemonchus contortus to fenbendazole.

The history of European bison Bison bonasus Linnaeus, 1758 has been stormy since its extinction in the wild after the First World War. Due to the fact that the species was restored from just 12 founders, further expansion has suffered from low genetic variability, rendering the bison vulnerable to various pathogens due to inbreeding depression. Parasites are recognised as a key biological threat to bison population. Thus, parasitological examination including monitoring of the level of anthelmintic resistance in a herd should be a routine procedure involved in management and protection of European bison. This study was conducted in a group of 27 bison kept in a European bison breeding centre in Sweden. In April 2015, a faecal egg count reduction test (FECRT) was performed in animals with ≥ 100 gastrointestinal nematode (GIN) eggs per gram faeces, to determine effectiveness of fenbendazole (FBZ) treatment. Additionally, the third stage larvae were cultured for molecular examination by a conventional PCR as well as by real-time quantitative PCR (q-PCR) for detection of the blood-sucking nematode Haemonchus contortus. Faecal sampling was conducted 1 day before and 8 days after deworming each animal. Anthelmintic treatment turned to be entirely efficient toward intestinal nematodes of genera Nematodirus and Trichuris, whereas shedding of strongylid eggs from the subfamily Ostertagiinae was reduced from 81 to 30%. Polymerase chain reaction (PCR) on cultured third-stage larvae (L3) before treatment was positive for H. contortus, Ostertagia ostertagi and Cooperia oncophora, whereas post-treatment examination revealed exclusively the DNA of H. contortus. Thus, only H. contortus was involved in post-treatment faecal egg count (FEC). FECRT showed that the reduction in strongylid FEC to FBZ in the examined bison herd was 87% (95%-confidence intervals [95% CI] = 76-93), suggesting reduced efficacy of FBZ to strongylid GIN including mainly H. contortus.

Effects of selected Palestinian plants on the in vitro exsheathment of the third stage larvae of gastrointestinal nematodes.

BACKGROUND: Gastrointestinal parasites are one of the main restrictions to small ruminant production. Their pathological importance is primarily related to the major production losses, in quantity or quality, induced by the direct action of worms. Control of these parasites is based exclusively on the frequent use of anthelmintic drugs. However, the resistance to anthelmintics in worm populations after commercialisation of chemical drugs is now widespread. Therefore, there is a need to find new natural resources to ensure sustainable and effective treatment and control of these parasites. The aim of this study was to evaluate the anthelmintic activity, as minimum inhibitory concentration (IC 50 mg/mL), of different plant extracts using larval exsheathment inhibition assay using a two-species but steady population of parasitic nematodes (ca. 20% Teladorsagia circumcinta and 80% Trichostrongylus colubriformis). RESULTS: The study showed that the ethanolic extracts of 22 out of the 48 plant extracts, obtained from 46 plant species, have an inhibitory effect >50% (at concentrations of 100 mg/mL) on the third stage larvae (L3) of the nematodes exhibited the strongest inhibition activity (94%) with IC 50 of 0.02 mg/mL, where other members of the Rhamnaceae family have shown to possess strong anthelmintic activity (70-89%). CONCLUSIONS: Plant extracts are potential rich resources of anthelmintics to combat helminthic diseases. Our results suggest that extracts from Rhamnus elaternus, Epilobium hirsutum, Leucaena leucocephala and Rhamnus palaestinus have promising anthelmintic activity, with potential applications in animal therapeutics and feed.

An attempt to replace an ivermectin-resistant Cooperia spp. population by a susceptible one on grazing pastures based on epidemiological principles and refugia management.

The maintenance of anthelmintic-susceptible parasite refugia to delay the onset of anthelmintic resistance is an almost impossible effort in many grazing livestock production countries given that current refugia consist of already resistant parasites. Rather, efforts could be focused on replacing the resistant parasite refugia by susceptible parasite ones and implementing sustainable parasite control measures from then on. To this purpose, a trial was conducted to attempt to establish a new population of ivermectin-susceptible Cooperia sp. on a beef cattle farm with proven problems of ivermectin-resistant Cooperia. During two consecutive years, 82 (Year 1) and 100 (Year 2) recently weaned and parasite-free heifers were inoculated with 40,000 or 30,000 susceptible Cooperia L3, respectively, at a time when levels of resistant parasite refugia were normally low. The animals were subsequently allowed to graze on the problem pastures during autumn until the end of spring. Levels of parasitism in the animals and on pasture were monitored monthly and animals were treated with levamisole when needed. The combination of parasitological monitoring and local epidemiological knowledge was essential to determine when treatments were to be administered. No clinical signs of gastrointestinal parasitosis in the herd were observed throughout the study and unnecessary treatments were avoided. Faecal egg counts reduction tests (FECRT) and controlled efficacy tests (CET) employing worm counts were carried out at different times throughout the study to determine the clinical efficacy (FECRT) and the absolute efficacy (CET) of ivermectin, respectively. The clinical efficacy of ivermectin increased from an initial 73% to 99.4%, while the absolute efficacy increased from 54.1% to 87.5% after just two animal production cycles. The switch from a resistant parasite population to a susceptible one requires knowledge of parasitological epidemiology, especially in relation to seasonal variations of parasite populations in both the host and in refugia.

In Vitro, in Vivo, and Interaction Studies of Nematophagous Fungus Arthrobotrys thaumasia ( Monacrosporium thaumasium) with the Larvae of Trichostrongylides of Sheep.

It is important to isolate potential candidates from the local isolates of nematophagous fungi and to investigate interaction between the fungal strains and gastrointestinal nematodes for the biological control of parasitic nematodes in livestock. In the present study, we assessed the in vitro predatory activity and the viability of isolates of Arthrobotrys thaumasia ( Monacrosporium thaumasium) after passage through the gastrointestinal tract of sheep. The predatory process of a representative isolate selected against the larvae of trichostrongylids was prepared with a scanning electron microscope (SEM). In vitro experiments tested the ability of 9 native isolates of A. thaumasia to prey on larvae of feces of sheep infected with natural mixed nematodes ( Haemonchus contortus, Trichostongylus colubriformis, Marshallagia mongolica). These isolates of A. thaumasia decreased infectivity of third stage infective larvae (L3) by 75.54-99.97%; 7 isolates decreased infectivity by more than 90%. In vivo experiments also demonstrated significant reductions of L3 numbers in the feces treated with the 9 isolates after passing through the gastrointestinal tract of sheep, and these decreases ranged from 51.68 to 88.16%. The isolates tested were re-isolated in 5-g sub-samples of feces from sheep in each treatment group, indicating that these isolates had the capacity to prey upon larvae of trichostrongylids after the passage through gastrointestinal tract. SEM shows that at 6 hr after the larvae were added, including the second stage larvae (L2) and L3 of trichostrongylids, the isolate NBS 005 caught them; at 8 hr after being caught L2 was penetrated by the fungus while penetration of L3 occurred at 12 hr; at 78 hr post-capture L2 was completely destroyed by the fungus while complete digestion of L3 occurred at 84 hr.

Genomic introgression mapping of field-derived multiple-anthelmintic resistance in Teladorsagia circumcincta.

Preventive chemotherapy has long been practiced against nematode parasites of livestock, leading to widespread drug resistance, and is increasingly being adopted for eradication of human parasitic nematodes even though it is similarly likely to lead to drug resistance. Given that the genetic architecture of resistance is poorly understood for any nematode, we have analyzed multidrug resistant Teladorsagia circumcincta, a major parasite of sheep, as a model for analysis of resistance selection. We introgressed a field-derived multiresistant genotype into a partially inbred susceptible genetic background (through repeated backcrossing and drug selection) and performed genome-wide scans in the backcross progeny and drug-selected F2 populations to identify the major genes responsible for the multidrug resistance. We identified variation linking candidate resistance genes to each drug class. Putative mechanisms included target site polymorphism, changes in likely regulatory regions and copy number variation in efflux transporters. This work elucidates the genetic architecture of multiple anthelmintic resistance in a parasitic nematode for the first time and establishes a framework for future studies of anthelmintic resistance in nematode parasites of humans.

Anthelmintic effect of 2H-chromen-2-one isolated from Gliricidia sepium against Cooperia punctata.

Gliricidia sepium is a tropical legume with known anthelmintic-like properties. The aim of this study was to: (1) perform a bio-guided fractionation of an acetonic extract of G. sepium leaves using the egg hatch assay (EHA); (2) elucidate the anthelmintic (AH)-like phytochemical using nuclear magnetic resonance (NMR); and (3) assess the ultrastructural damage of the Cooperia punctata treated eggs. The anthelmintic activity of G. sepium was traced from an acetonic extract using the EHA. Phytochemicals were isolated through silica gel columns and elucidated through spectroscopic measurements (1H and 13C). Final fraction was evaluated with EHA at decreasing concentrations of: 1.100; 0.500, 0.250, 0.125, 0.060, 0.001 and 0.00001 mg mL-1. Egg hatching inhibition was calculated using the formula: 100*(1-HT/HC). The maximal half of effective concentration (EC50) was calculated with GraphPad. Bio-guided isolation procedures lead to the elucidation of 2H-chromen-2-one, which inhibited both hatching and embryo development of C. punctata (EC50 of 0.024 ± 0.082 mg mL-1) (P < 0.05). Scanning and Transmission Electron Microscopy (SEM and TEM) revealed electrodensity alterations and fractures in the eggshell layers. After toxicity evaluations and in vivo assessment, 2H-chromen-2-one can be suggested as a novel AH-phytochemical for reducing larval density in pastures and worm burdens inside the host.

Benzimidazole resistance survey for Haemonchus, Teladorsagia and Trichostrongylus in three European countries using pyrosequencing including the development of new assays for Trichostrongylus.

Resistance to benzimidazoles (BZs) in trichostrongyloid nematodes is a worldwide problem for livestock production, particularly regarding small ruminants. Sensitive and reliable methods are required to assess anthelmintic resistance status. Currently available methods for BZ resistance detection can be divided into three main groups, in vivo (e.g. faecal egg count reduction test), in vitro (e.g. egg hatch assay) and molecular tests. Three single nucleotide polymorphisms (SNPs) in the isotype-1 ß-tubulin gene of various nematode species correlate with BZ resistance. While PCR-based methods have been reported for the three most economically important nematodes of sheep, namely, Trichostrongylus, Haemonchus and Teladorsagia, pyrosequencing assays are so far only available for the latter two. Here, the design and evaluation of pyrosequencing assays for isotype-1 and isotype-2 ß-tubulin genes of Trichostrongylus colubriformis are described. PCR fragments carrying the susceptible and corresponding resistant genotype were combined in defined ratios to evaluate assay sensitivity and linearity. The correlation between the given and the measured allele frequencies of the respective SNPs (codons F167Y, E198A and F200Y) was very high. Pyrosequencing assays for Haemonchus, Teladorsagia and Trichostrongylus were subsequently used for a BZ resistance survey, carried out in the three European countries, namely Ireland, Italy and Switzerland. Larval cultures obtained from field survey samples in 2012 and 2013 were used for pyrosequencing. The test was applied when the target species represented at least 10% of the sample. Trichostrongylus and Teladorsagia were detected in all countries' samples whereas Haemonchus was not detected in samples from Ireland. SNPs in isotype-1 associated with resistance were detected for all three species, with frequencies at codon F200Y far exceeding those at codons F167Y and E198A. Elevated SNP frequencies in isotype-2 of Trichostrongylus were only rarely detected. Farms with BZ resistance-associated SNP frequencies above 10% were most often found in Switzerland followed by Ireland and Italy.