Does in Vitro and in Vivo Exposure To Medicinal Herbs Cause Structural Cuticular Changes in Haemonchus Contortus?

The health and production of small ruminants in constantly menaced by parasitic infections, especially those caused by the blood-sucking gastrointestinal nematode Haemonchus contortus. The aim of this study was to assess the structural cuticular changes in adult H. contortus induced by the use of extracts from local medicinal plants and to examine their ovicidal activity. Previous studies have confirmed the beneficial effect of herbal mixtures in preventing haemonchosis in lambs by lowering fecal egg production and improving immunocompetence. We exposed adult H. contortus to Herbmix (a mixture of medicinal plants) under in vivo and in vitro conditions for observation by scanning electron microscopy (SEM). For the in vivo observations, adult worms were isolated from the abomasa of experimentally infected lambs from a Herbmix group and a control group. Surface structure did not differ significantly between the exposed and control groups. The ovicidal activity of an aqueous Herbmix extract was assessed in vitro, establishing the inhibition of hatching with an ED50 of 6.52 mg/mL. Adult worms for in vitro examination were isolated from experimentally infected lambs and incubated in Herbmix aqueous extracts for 24 h. SEM observations indicated that none of the worms had prominent ultrastructural changes on their cuticles. This study suggests that previously demonstrated antiparasitic effects of medicinal plants did not negatively affect adult parasites by damaging their external structures.

Multiple Anthelmintic Resistance at a Goat Farm in Slovakia.

Cases of parasite resistance to the main classes of anthelmintics are increasingly reported from small ruminants at farms in Europe. We visited a goat farm in Slovakia in November 2019 with suspected parasite problems that reduced productivity and performed an in vivo faecal egg count reduction test (FECRT) and an in vitro larval development test (LDT) for all three main classes of anthelmintics. The lowest efficacy (60 %) detected by FECRT was for ivermectin (IVM). Benzimidazole (BZ) efficacy ranged between 80.3 and 86.5 %, and levamisole (LEV) efficacy was 94 %. The results from the in vivo FECRT test were confirmed by the in vitro LDT. Minimum inhibitory concentration (MIC) 173.6 ng/ml for IVM several times exceeded the recommended threshold of 21.6 ng/ml. Mean LD50 for BZ was equal to the threshold concentration, but the other threshold criteria indicated a low level of resistance in the population. The LDT did not indicate the presence of resistance only for LEV. The MIC 1.0 µg/ml for LEV was evaluated as susceptible with respect to species composition. Teladorsagia was the dominant genus after treatment with BZ, IVM, and LEV. Haemonchus contortus was identifi ed after treatment with BZ and LEV.

Changes in Haematological Parameters in Wild Ruminants Experimentally Infected with Haemonchus Contortus.

Our study describes changes in haematological parameters in wild ruminants with parasitic infection. Six European mouflons (Ovis musimon), six fallow deer (Dama dama) and six roe deer (Capreolus capreolus) were experimentally infected with the resistant strain of the model parasite 8000 L3 Haemonchus contortus. The blood samples were collected on Day 0, 16, 37, 58, 77, and 99 of the experiment. Mild anaemia was observed in mouflons and roe deer while red blood cells increased in red blood counts (total erythrocytes, haematocrit and haemoglobin). As for the white blood cells count, leucopenia with neutrophilia and lymphopenia was recorded in mouflons, in the fallow deer and roe deer leucocytosis with neutropenia and lymphocytosis were observed. Changes in the dynamics of haematological parameters were statistically insignificant.

Effectiveness of benzimidazole treatments against Haemonchus contortus in sheep and goats - Do they produce similar responses?

The primary aim of this study was to compare the in vivo responses to orally administered doses of albendazole (5 mg/kg body weight) between experimentally infected sheep and goats. Fifty-four Improved Valachian lambs and 54 Saanen goat kids were split into six groups of nine animals. The sheep and goats were infected with larvae of the gastrointestinal nematode parasite Haemonchus contortus containing 10, 20, 30, 40, 60, and 80 % of the isotype-1 ß-tubulin gene codon 200 alleles previously shown to be associated with benzimidazole (BZ)-resistance. All groups of goats generally had higher mean eggs per gram (EPG) before treatment, which was significant (p<0.05) only for the group with 80 % resistance alleles. An in vivo faecal egg reduction test (FECRT) was used to determine the efficacy of albendazole (ABZ) eight days after treatment. Anthelmintic treatment significantly reduced the EPGs in the groups with 10, 20, and 80 % resistance alleles in sheep and with 10, 20, 30, and 40 % resistance alleles in goats. Differences in efficacy between the sheep and goats after the application of doses of ABZ recommended for sheep mostly ranged from 2 % to 10 %. The largest variation was in the group infected with worms containing 60 % resistance alleles, where the efficacy was 13 % higher in goats. Our secondary aims were to evaluate the data obtained from an in vitro egg hatch test (EHT) in sheep and goats and to compare these data with the results from the isotype-1 ß-tubulin gene codon 200 pyrosequencing and the FECRT. The percentages of the BZ-resistance alleles were comparable with the mean hatching obtained in the EHT and were also supported by the FECRT data for all groups. The results of the in vivo tests should be verified in the future using in vivo surveys conducted in mixed breeds and infections in multiple species.

Development of resistance to eprinomectin in gastrointestinal nematodes in a goat herd with pre-existing resistance to benzimidazoles.

We described a first case of resistance to eprinomectin in goat herd in Poland in which resistance to benzimidazoles had been previously reported. The herd was established in 2011 by purchasing several goats from a single herd in south-eastern Poland. Resistance to benzimidazoles in the herd was first reported in 2017. Shortly after the owner started to signal low effectiveness of the treatment with eprinomectin. In June 2018 the larval development test from pooled faecal sample was performed and the results indicated the presence of resistance to macrocyclic lactones and levamisole. In July 2018 a faecal egg count (FEC) reduction test was performed in 39 animals with levamisole, eprinomectin and one untreated control group. Drugs were used in doses recommended for goats. Three methods of calculation of FEC reduction were compared. After eprinomectin treatment, FEC reduction ranged from 0 to 20%, depending on the method of calculation. FEC reduction following levamisole treatment was 100%. Main species present in the faecal samples after treatment and in larvicidal concentrations in larval development test was Haemonchus contortus. This is the first report of anthelminthic resistance to macrocylic lactones (eprinomectin) in goats in Poland.

Biotransformation of flubendazole and selected model xenobiotics in Haemonchus contortus.

Haemonchus contortus is one of the most pathogenic parasites of small ruminants (e.g., sheep and goat). The treatment of haemonchosis is complicated because of frequent resistance of H. contortus to common anthelmintics. The development of resistance can be facilitated by the action of drug metabolizing enzymes of parasites that can deactivate anthelmintics and thus protect parasites against the toxic effect of the drug. The aim of this project was to investigate the Phase I biotransformation of benzimidazole anthelmintic flubendazole in H. contortus and to determine the biotransformation of other model xenobiotics. For this purpose, in vitro (subcellular fractions of H. contortus homogenate) as well as ex vivo (live nematodes cultivated in flasks with medium) experiments were used. The results showed that cytosolic NADPH-dependent enzymes of H. contortus metabolize flubendazole via reduction of its carbonyl group. The apparent kinetic parameters of this reaction were determined (V'max=39.8+/-2.1 nM min(-1), K'm=1.5+/-0.3 microM). The reduction of flubendazole in H. contortus is stereospecific, the ratio of (-):(+) enantiomers of reduced flubendazole formed was 90:10. Reduced flubendazole was the only Phase I metabolite found. Effective reduction of other xenobiotics with carbonyl group (metyrapon, daunorubicin, and oracin) was also found. Significant activity of carbonyl-reducing enzymes may be important for H. contortus to survive the attacks of anthelmintics or other xenobiotics with carbonyl group.

Anthelmintic resistance in goat herds-In vivo versus in vitro detection methods.

Anthelmintic resistance (AR) is a serious threat to animal health and has a major economic impact worldwide due to production and financial losses. The aim of this study was to determine the occurrence of AR on 30 goat farms in Slovakia during the pasturing seasons and to compare three widely used in vitro and in vivo methods for detecting AR in field conditions. A three-year survey was conducted during the pasturing seasons of 2014-2016. Goats on each farm were split into treated and control groups and were treated by recommended (5 mg/kg body weight) and double doses (10 mg/kg b.w.) of albendazole. Comparisons between percent reduction in a faecal egg count reduction test (FECRT) and an egg hatch test (EHT) and the presence of L3 larvae in a larval development test (LDT) using resistant concentrations of benzimidazole (BZ) were monitored after treatment. The FECRT indicated percent reductions of 69.2-86.2% for the single dose and of 36.3-45.4% for the double dose. The EHT indicated that all farms had BZ-resistant nematodes. Low (<15% hatching) and high (>15% hatching) levels of resistance were detected on 13 and 17 farms, respectively. The LDT failed to detect resistant larvae on seven farms but detected low and high levels of resistance on seven and 14 farms, respectively. The data indicate a moderate correlation between in vitro and in vivo tests for detecting BZ resistance among the 30 goat farms. The hatching detected by the EHT and the presence of L3 larvae by the LDT at resistant BZ concentrations provided reasonable identification of low levels of resistance in the parasite populations, but the use of a double dose for a treatment may underestimate the real occurrence of low levels of resistant parasites on goat farms.

In vitro detection of benzimidazole resistance in Haemonchus contortus: egg hatch test versus larval development test.

The present study was designed to compare the egg hatch test (EHT) and the larval development test (LDT) as in vitro tools for detection of benzimidazole (BZ) resistance in Haemonchus contortus, a nematode parasite of small ruminants. Comparisons were made during a course of infection and changes in both EHT and LDT were monitored to measure the correlation between resistance and susceptibility in different parasite stages (eggs and larvae). In addition, mixed doses of known numbers of susceptible and BZ-resistant H. contortus eggs were used to assess the sensitivity of LDT for the detection of low levels of resistance. The degree of resistance for each test was expressed as resistance factor (RF). The LDT showed a greater ability to distinguish between four susceptible and four resistant isolates of H. contortus with higher resistance factors compared to the EHT. For the EHT the RF by using ED(50) criterion ranged from 3.2 to 13.3 and from 7.4 to 25.2 by using LC(99). For LDT the resistant isolates were 4.3-63.1 times more tolerant than the susceptible isolates using the ED(50) criterion and 91.1-1411.0 times more tolerant using the LC(99) criterion. The LDT was also able to clearly indicate the presence of low level (4%) of resistant larvae amongst a susceptible background population.

A survey on anthelmintic resistance in nematode parasites of sheep in the Slovak Republic.

The prevalence of anthelmintic resistance on 27 sheep farms in Slovakia was investigated in 2003 and 2004 using the faecal egg count reduction test (FECRT) according to the WAAVP guidelines. Resistance to albendazole was detected on one farm (3.7%) and suspected on two farms (7.4%) out of 27 sheep flocks. Resistance to ivermectin was tested on 26 farms. On six (23.1%) farms, results indicated the presence of ivermectin resistance. Resistance to ivermectin was suspected on eight farms (30.8%). However, it is also possible that generic ivermectin anthelmintics used in survey have a lower efficacy against sheep nematodes.

Treatment of multiple resistant field strain of Ostertagia spp. in Cashmere and Angora goats.

A multiple resistant field strain of gastro-intestinal nematodes was detected in Cashmere and Angora goats imported from New Zealand. Different treatments with three types of broad spectrum anthelmintics (albendazole, levamisole/tetramisole and ivermectin) at various dose rates are described. Satisfactory effect in Angora goats was not achieved until all three anthelmintics were administered simultaneously at twice the normal sheep dose. Post mortem worm counts and identification revealed that the resistant population consisted of 89% Ostertagia circumcincta, 6% Ostertagia ostertagi and 5% Ostertagia trifurcata. However, egg hatch assay carried out seven months later detected the presence of resistant nematodes again.

In vitro characterization of anthelmintic susceptibility of field isolates of the pig nodular worm Oesophagostomum spp., susceptible or resistant to various anthelmintics.

A larval development assay (LDA) and an egg hatch paralysis assay (EHPA) were used to measure the sensitivity to anthelmintics of eggs and larvae of nodular worms (Oesophagostomum spp.) in pigs. The tests were carried out using in vivo defined resistant and susceptible isolates of Oesophagostomum dentatum, O. quadrispinulatum and Oesophagostomum spp. For measurement of pyrantel/morantel and levamisole sensitivity the LDA was found able to distinguish between susceptible or resistant isolates of Oesophagostomum. The EHPA was able to detect levamisole resistance, but the test failed to show differences in response to pyrantel between pyrantel susceptible and resistant lines. The possible routine application of LDA and EHPA in the diagnosis of anthelmintic resistance in Oesophagostomum spp. is discussed.

In vitro characterization of lines of Oesophagostomum dentatum selected or not selected for resistance to pyrantel, levamisole and ivermectin.

Lines of Oesophagostomum dentatum artificially selected or not selected for resistance to pyrantel, levamisole and ivermectin were used in this study. From the 10th generation of selection eggs were collected from each line and subjected to an in vitro larval development assay (LDA) and an egg hatch assay (EHPA). Significant differences were observed between an unselected line of O. dentatum and the lines selected for resistance to levamisole or pyrantel in both assays. The LDA was more sensitive than EHPA in detecting anthelmintic resistance in O. dentatum. The results obtained from the LDA confirmed side-resistance between levamisole and morantel/pyrantel. The in vitro tests failed to show significant differences between ivermectin-sensitive and resistant lines.

The efficacy of ivermectin against nodular worms of pigs: the response to treatment using three different dose levels against Oesophagostomum dentatum and Oesophagostomum quadrispinulatum.

Anthelmintic efficacies of 3 different doses of ivermectin (IVM) were evaluated in 3 isolates of nodular worms in pigs. An isolate of Oesophagostomum quadrispinulatum (OQ) was recently obtained from a commercial farm where poor efficacy of IVM at the recommended dose (300 micrograms.kg-1 body weight) was detected. On this farm, IVM had been used for treatment of sows twice yearly for 6 years. Two other isolates, an O. dentatum (OD) and a mixed Oesophagostomum dentatum and Oesophagostomum quadrispinulatum isolate (ODQ) were obtained from a farm where anthelmintics had never been used. Efficacies of IVM against adult worms of the OQ-isolate at dose rates of 150, 300 and 600 micrograms.kg-1 body weight ranged from 40.5-78.6%. Efficacies against larval stages (L3 and L4) were superior. Efficacies against the OD-isolate were 88.7, 96.1 and 99.6%, respectively. In the ODQ-isolate the efficacies of IVM against adult stages furnished similar results. In conclusion, the efficacy of IVM against O. dentatum was high but against both isolates of O. quadrispinulatum poorer. This suggests that IVM is intrinsically less effective against O. quadrispinulatum and therefore not indicative of acquisition of anthelmintic resistance in the OQ-isolate.

An in vivo dose-response study of fenbendazole against Oesophagostomum dentatum and Oesophagostomum quadrispinulatum in pigs.

A dose-response study using fenbendazole (FBZ) was carried out in pigs infected with O. dentatum and O. quadrispinulatum to determine the minimum effective dose rate of the drug. Thirty pigs were randomly divided into 6 groups of 5 pigs and infected with 5000 infective larvae each. The animals were re-infected 5 days before treatment (Day 30 after the first infection) with the same number of larvae. On Day 35 the pigs in groups 1-5 were treated with FBZ at the following dose rates: 2.5 mg kg-1 (i.e. 50% of the registered dose level), 1.0 mg kg-1 (20%), 0.25 mg kg-1 (5%), 0.1 mg kg-1 (2%) and 0.05 mg kg-1 (1%), respectively. Pigs in group 6 served as non-treated controls. Seven days after treatment (Day 42 after infection) the pigs were slaughtered, worms recovered from the large intestine and counted. The species and sex of adult worms was determined. A high faecal egg count reduction (FECR) after treatment was observed in groups 1, 2 and 3 (98%, 88% and 91%, respectively), while in groups 4 and 5 the egg counts were not affected by treatment. The mean worm count reduction was high in groups 1, 2 and 3 (100%, 99.9% and 98.6%, respectively), but declined in groups 4 and 5 (77% and 40%, respectively). FBZ showed a high efficacy against immature worms in groups 1 and 2, while in groups 3, 4 and 5 counts were not reduced. Species differentiation revealed a higher effect of FBZ against O. dentatum than against O. quadrispinulatum. Sex differentiation indicated a slightly higher (not significant) efficacy against females than males in both species. This study demonstrated a high efficacy of FBZ against the nodular worms in pigs, even at 5% of the currently registered dose level.

Comparison of six in vitro tests in determining benzimidazole and levamisole resistance in Haemonchus contortus and Ostertagia circumcincta of sheep.

Six in vitro methods for the detection of anthelmintic resistance were compared using benzimidazole/levamisole-resistant Haemonchus contortus and benzimidazole/levamisole/ivermectin-resistant Ostertagia circumcincta as well as susceptible strains of both parasite species. The degree of resistance to thiabendazole and levamisole was compared by (1) an egg hatch assay, (2) an egg hatch paralysis assay, (3) a larval development assay, (4) a larval paralysis assay (5) a larval paralysis assay with physostigmine and (6) larval micromotility assay. The degree of resistance for each assay was expressed as resistance factor--RF. For the detection of thiabendazole and levamisole resistance, the larval development test was observed as the most sensitive to measure quantitatively a degree of resistance between susceptible and resistant strains. For this test the RF for thiabendazole and levamisole was 14.3 and >32.5, respectively in H. contortus strains and 21.1 and 3.5 in strains of O. circumcincta. Egg hatch assay was also found to be sensitive and accurate in determining of resistance to benzimidazole. For measurement of levamisole resistance the egg hatch paralysis assay and larval paralysis assay were found to be able to distinguish between strains, but some disadvantages of these techniques make it unsuitable for field detection of levamisole resistance. Other in vitro assays as larval paralysis assay with physostigmine and larval micromotility assay were also investigated. Significant differences in paralysis of the larvae were observed using larval paralysis assay.

Benzimidazole resistance in equine cyathostomes in Slovakia.

The present study included 19 stud farms, including 243 horses, that were investigated for the occurrence of anthelmintic resistant cyathostomes. The number of horses on the farms varied from nine to more than 100, and horses of all ages were included. A minimum of seven horses were used for faecal egg count reduction (FECR) tests. The anthelmintics included were: fenbendazole (paste formulation), ivermectin (paste formulation) and pyrantel (powder). Resistance to benzimidazoles was detected on 14 farms, with FECR values ranging from 65.1 to 86.3%. Larval cultures after fenbendazole treatment revealed exclusively cyathostome larvae. Ivermectin was tested on eight farms and proved to be effective on all. Pyrantel was tested on two farms and FECR test indicated high efficacy (92-97%). Egg hatch assay (EHA) results showed that mean concentrations of thiabendazole that inhibited hatching in 50% of the eggs (ED(50)) in resistant populations were over 0.1 microg ml(-1). The results of our study suggest widespread resistance to fenbendazole in equine cyathostomes in Slovakia, and possible strategies to delay anthelmintic resistance are discussed briefly.

Efficacy of injectable moxidectin against mixed (Psoroptes ovis and Sarcoptes scabiei var. ovis) mange infestation in sheep.

Field efficacy of injectable moxidectin was evaluated in sheep naturally infested with both Psoroptes ovis and Sarcoptes scabiei var. ovis mange. Three groups of 15 ewes were selected from the flock based on parasitological and clinical status. Group 1 remained as untreated controls; Group 2 animals received a subcutaneous injection of moxidectin at 0.2 mg kg-1 body weight on Day 1; Group 3 animals received 0.2 mg kg-1 moxidectin twice on Day 1 and Day 7. Efficacy was assessed by taking skin scrapings from each animal on Days -4, 0, 7, 14, 28 and 35 post-treatment and counting viable mite stages and species. In both treated groups the signs of itching disappeared within 7 days post-treatment. Rapid clinical improvement was associated with reduction in numbers of mites compared with initial score which was over 90%. However, in skin scrapings on Day 7 post-treatment several live mites of both species were present. The second injection of moxidectin removed all living mites, and skin scrapings from Group 3 animals were negative for all mites on Days 14, 28 and 35. The weight gain on average was 2.0 kg in Group 2 and 2.7 kg in Group 3 during the 35 days of trial. Untreated control animals lost on average 3.1 kg from their weight at the start of the trial. All untreated animals remained positive and suffered from intensive scratching, anorexia and moist, active skin lesions. They were given an emergency moxidectin treatment on Days 35 and 42, each animal receiving 0.2 mg kg-1 body weight, subcutaneously.(ABSTRACT TRUNCATED AT 250 WORDS)

A survey of anthelmintic resistance in Slovakia.

A survey of prevalence of anthelmintic resistant nematode strains was carried out in 77 sheep farms in the Slovak Republic. An egg hatch assay was used for the detection of resistance to the benzimidazole group of anthelmintics. Results could not be obtained from five farms because of low egg counts in faecal samples. On 64 farms, the effective dose (ED50) values were under 0.1 microgram ml-1 while on six farms, trichostrongylid populations with resistance to benzimidazoles were found. Their ED50 values ranged from 0.10 to 0.12 microgram ml-1.

Efficacies of different doses of ivermectin against male, female and L4 Oesophagostomum dentatum in pigs.

Efficacies of ivermectin against larval stages and adult males and females of Oesophagostomum dentatum were investigated in two slaughter assays. In Experiment A, 20 pigs were each infected with 6000 third-stage larvae on Day 0 and Day 24. Pigs were ivermectin treated on Day 28 at dose rates of 0, 75, 150, and 300 micrograms kg-1 bodyweight (bw) and slaughtered 6 days after treatment. In Experiment B, 20 pigs each received 6000 third-stage larvae and were treated 35 days after infection at dose rates of 0, 150, 300 and 600 micrograms kg-1 bw. Pigs were slaughtered 14 days after treatment. In Experiment A, the adult worm burden was reduced by 69.1% at a dose rate of 300 micrograms kg-1 bw and the larval burden was reduced by 68.7 and 90.9% at 150 and 300 micrograms kg-1 bw, respectively. In Experiment B, the adult worm burden was reduced by 88.8, 96.2 and 99.6% at dose rates of 150, 300 and 600 micrograms kg-1 bw, respectively. In the control group of Experiment A, the mean proportion of females among adults worms was 57.6%, but this decreased to 19.7% after ivermectin treatment at 300 micrograms kg-1 bw. In Experiment B, at 300 micrograms kg-1 bw, this proportion was reduced from 46.0% to 0.8%. The fecundity of female worms was reduced at dose rates of 150 and 300 micrograms kg-1 bw in Experiment A, but not in Experiment B. It is concluded that in O. dentatum, ivermectin not only reduces the egg output of female worms, but also is more effective against female than male worms.

The in vitro motility response to various anthelmintics of third-stage larvae of Oesophagostomum spp. from pigs.

The in vitro activities of thiabendazole, levamisole, pyrantel, morantel and ivermectin against Oesophagostomum spp., the nodular worm of pigs, were determined and compared. The study was carried out using isolates of O. dentatum and O. quadrispinulatum, which had been defined in vivo. Infective larvae were exposed to the anthelmintics for 24 h and then placed in a micromotility meter. All the treatments significantly reduced the motility of the ensheathed L3 larvae, but the micromotility meter was not able to differentiate between anthelmintic resistant and anthelmintic susceptible isolates.