J. Čorba

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.

Comparison of in vitro methods and faecal egg count reduction test for the detection of benzimidazole resistance in small strongyles of horses

The objective of the study was to compare the in vitro egg hatch test (EHT), larval development test (LDT) and in vivo faecal egg count reduction test (FECR test) for the detection of benzimidazole resistance in equine strongyles. The presence of resistant or susceptible strongyle populations was determined in 25 stud farms using the in vivo FECR test and in vitro EHT. On the basis of the FECR values, resistance to fenbendazole was detected on 15 of the 25 farms (60%). The ED50 value (anthelmintic concentration producing 50% inhibition of hatching) for suspected resistant populations varied from 0.110 to 0.222 μg/ml thiabendazole (TBZ). Final LD50 values (anthelmintic concentration inhibiting development of 50% of eggs into L3 infective larvae) above 0.029 μg/ml TBZ in the in vitro larval development test on samples from 11 stud farms revealed the presence of populations of small strongyles suspected of being benzimidazole-resistant.

Comparison of two versions of larval development test to detect anthelmintic resistance in Haemonchus contortus

Larval development (LDT) and micro-agar larval development tests (MALDT) were used to compare the reliability and sensitivity of two methods for detecting anthelmintic resistance in Haemonchus contortus. The tests were conducted using three resistant and four susceptible isolates of H. contortus. Both versions of the tests provided comparable results with regard to the characterization of benzimidazole and levamisole susceptibility but neither test was sufficiently sensitive to discrimination between an ivermectin (IVM) susceptible and an IVM resistant isolate. Each test has its own merits with the LDT having the advantage of being less time-consuming.

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.

Effects of free and liposomized praziquantel on the surface morphology and motility of Mesocestoides vogae tetrathyridia (syn. M. corti; Cestoda: Cyclophyllidea) in vitro

Abstract The effects of in vitro exposure to praziquantel (PZQ), liposomized PZQ (lip.PZQ), and empty liposomes on the surface morphology and motility of Mesocestoides vogae tetrathyridia were investigated using scanning electron microscopy (SEM) and a motility apparatus. Examination of treated larvae showed an effect that was concentration- and time-dependent, involving morphological damage that was similar in character for all of the treated groups. The most marked effects were a flattening and elongation of the larval body accompanied by irregularities in the surface architecture involving the development of tegumental protuberances and depressions. Erosion of the surface microvillous layer occurred only after overnight incubation, being most pronounced after treatment with lip.PZQ. The motility index of treated tetrathyridia corresponded well to the SEM observations. The frequency of contractions was maximal in worms treated with free PZQ at 25 μg/ml in both regimens. However, after incubation with lip.PZQ the increase in motility was concentration-dependent and of a greater extent. Empty liposomes and lipid mixtures of the same concentration and composition resulted in increased motility in treated larvae as compared with controls. More extensive tegumental damage and higher motility of larvae occurred after lip.PZQ treatment, perhaps resulting from a synergistic action of the drug and its associated lipid.

Worm control practices on sheep farms in the Slovak Republic

A questionnaire to obtain information on worm control practices and sheep management was performed on 49 sheep farms in 2003 and 2004. The majority of Slovak farms kept native breeds Tsigai (22 farms) and Improved Valachian (14 farms). Farms were divided according to their altitude to lowland, upland and lower highland farms. Sizes of pastures and stocking rates for lowland, upland and lower highland farms were 81.5, 269.2, and 316.7 ha and 6.3, 2.6, and 2.9 sheep/ha, respectively. One third of farmers (33.3%) used permanent pastures and two thirds of breeders (66.7%) rotated sheep between pastures. Mean drenching rate for lambs and yearlings/adults was 1.76 and 1.70, respectively. The most frequently used drugs during period from 1999 to 2004 were albendazole and ivermectin. On 13 farms benzimidazole drugs were applied in spring before turn out and macrocyclic lactones in autumn before turn in. Benzimidazoles and macrocyclic lactones were used almost exclusively on 7 and 9 farms, respectively. Visual appraisal was the most common practice to determine weight of animals (87.8% of farmers). Weights of the heaviest animals were used for determination of anthelmintic doses only on 16.7% of farms. Coprological examinations were performed on 47.9% of farms, usually in frequency once per year (75%).

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.

Effect of a controlled-release albendazole capsule on parasitism and productivity of sheep

The efficacy of intraruminal albendazole (ABZ) capsules (Profitril-Captec) and the effect of treatment on productivity were studied in 300 ewes infected with gastrointestinal nematodes and the trematode Dicrocoelium dendriticum. Coprological tests revealed that treated animals remained negative for 10 weeks after the administration of capsules. Contamination of pasture with nematode larvae was significantly reduced during the whole experiment. Necropsy of 14 animals (seven treated and seven untreated) showed 96.9-99.2% efficacy against the nematodes Nematodirus spp., Oesophagostomum spp., Cooperia spp., Trichostrongylus spp. and Trichuris ovis, while efficacy was 88.5% against D. dendriticum. During the 6 month pasture season (May-October 1989), treated ewes produced on average 2.56 kg cheese and 0.6 kg wool per ewe more than untreated controls. Our study confirms the reliability of the ABZ slow-release capsules over 90 days and the positive effect of treatment on nematode contamination of pasture and ewe productivity.