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.

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 microg/ml thiabendazole (TBZ). Final LD50 values (anthelmintic concentration inhibiting development of 50% of eggs into L3 infective larvae) above 0.029 microg/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.

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.

The sexually linked Mpi locus is presumably involved in imidothiazole resistance in Oesophagostomum dentatum parasites.

Information about genetic changes during the selection process could indicate mechanisms underlying the spread of resistance to anthelmintic drugs. For clarification of the role of the Mpi locus encoding mannose-phosphate isomerase enzyme in determining resistance, genotyping of Oesophagostomum dentatum strains was performed using an isoelectrofocusing technique. In levamisole- and pyrantel-selected strains the allele associated with resistance has probably been found. Significant values for genetic differentiation between treated and untreated strains of common origin were recorded by F(st) indices (theta = 0.078; P = 0.0008). The specific genomic makeup of a flubendazole-resistant strain, which did not correlate with that of the remaining isolates, might be ascribed to a different action of the anthelmintic or different environmental conditions under which resistance against this drug arose. The absence of heterozygotes in male populations indicated an XX/X0 system of sex determination for the Mpi locus, thus providing a greater potential for the development of resistance. A possible involvement of alleles linked with mannose-phosphate isomerase in alterations of membrane receptors that can be associated with resistance against imidothiazole-based drugs is discussed.

Lung nematodes of chamois, Rupicapra rupicapra tatrica, from the Tatra National Park, Slovakia.

A larvoscopic examination of faeces collected from localities inhibited by chamois in the Tatra National Park (TANAP) in 1997 demonstrated the presence of the lung nematodes Muellerius spp. (likely to be M. tenuispiculatus and M. capillaris) and Neostrongylus linearis. The overall prevalence of lung nematodes in chamois herds in TANAP was 48.4% with prevalences of 45.6% and 11.9% for Muellerius spp. and N. linearis, respectively. No significant differences in lung nematode prevalences were observed in the biotopes of TANAP with prevalence values of 44.9% being recorded in the High Tatras and 58.5% in the Belianske Tatras. Individual species were in equal proportion in both biotopes, although N. linearis was significantly less prevalent (11.2-13.8%). The prevalence of lung nematodes in the High Tatras varied from 25.0 to 84.2% within individual localities, while in the Belianske Tatras it was more proportionate (50.0-85.7%). In the High Tatras, the prevalence of lung nematodes in the chamois herds peaked during August, declining to its lowest in October. A similar prevalence was also recorded for Muellerius species, while the minimum prevalence of N. linearis was found in July. In the Belianske Tatras, the prevalence of lung nematodes including both species of Muellerius peaked in July and gradually decreased until October. On the other hand, N. linearis was most prevalent in October. The mean L1 count per gram faeces was low (7.6 +/- 13.2 larvae g-1).

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.

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.

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

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 micrograms/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.

Genetic diversity in porcine Oesophagostomum dentatum and O. quadrispinulatum and their delineation by isoenzyme analysis.

The genetic diversity in eight strains of Oesophagostomum dentatum and O. quadrispinulatum was investigated by the electrophoresis study of ten enzyme systems. The loci Idh-2, Fbp, Sdh, and Pgm were found to be diagnostic between the species examined. Both the proportion of fixed allelic differences (26.3%) and the genetic distance coefficient (D = 0.54) are well above the range for differentiation of valid species. Isoenzyme patterns of susceptible and resistant lines of O. dentatum showed at polymorphic loci a reduced genetic heterogeneity in the latter group. No qualitative difference in terms of the presence/absence of alleles was observed among susceptible and resistant isolates with the enzymes studied. The detection of one possible hybrid indicates that introgression in O. dentatum and O. quadrispinuatum may occur.

[Bionomy, occurrence and importance of the giant liver fluke (Fascioloides magna), an important parasite of free-living ruminants]. / Bionómia, výskyt a význam cicavice obrovskej (Fascioloides magna), závazného parazita vol'ne zijúcich prezúvavcov.

The paper summarizes the most important knowledge of the giant liver fluke of ruminants, Fascioloides magna, with respect to its occurrence throughout the Northern Hemisphere, its fenotypic and genetic discriminative characteristics, its pathogenicity in various obligatory and facultative hosts and possible prophylaxis and medical control. The paper emphasizes the fact that new natural foci with heavy F. magna infections of deer regularly occur in Europe.

[Resistance to anthelmintics in nematodes in sheep and goats]. / Rezistencia voci antihelmintikám u nematódov oviec a kôz.

The article offers a brief view on the most important theoretical knowledge of resistance of gastrointestinal nematodes to anthelmintic drugs in sheep and goats. Besides the definition and basic terms, factors of development and occurrence of resistance on farm are analyzed. Furthermore, methods for detection of resistant nematodes as well as complex of recommended preventive measures are given.

[Effectiveness of moxidectin (Cydectin inj.) in mixed Psoroptes ovis and Sarcoptes ovis infestations in sheep]. / Ucinnost moxidectinu (Cydectin inj.) na zmiesanú infekciu svrabovcami Psoroptes ovis a Sarcoptes ovis u oviec.

Field efficacy of moxidectin injectable was evaluated in sheep naturally infected with both Psoroptes ovis and Sarcoptes ovis mange. Three groups of fifteen 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 body weight on Day 0; Group 3 animals received 0.2 mg/kg moxidectin twice on Day 0 and Day 7. Efficacy was assessed by taking skin scrapings from each animal on Days -4, 0, 7, 14, 28 and 35 post treatment (P.T.) and counting viable mite stages and species. In both treated groups the signs of itching disappeared within seven days P.T. 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 P.T. several live mites of both species were present. The second injection of moxidectin removed all living mites and skin scrapings from group 3 animals that were negative for all mites on days 14, 28 and 35. The weight gain on average in group 2 was 2.0 kg and in group 3 2.7 kg, during the 35 days of trial. Untreated control animals have lost on average 3.1 kg from the weight at start of trial. All untreated animals remained positive and suffered from intensive scratching, anorexia and moist active skin lesions. Therefore they were given an emergency moxidectin treatment on days 35 and 37 each animal receiving 0.2 mg/kg b.w., subcutaneously. Moxidectin in both tested doses was well tolerated and no local reaction on injection sites was observed.

Efficacy of moxidectin against multiple resistant Ostertagia spp. in lambs.

Moxidectin was demonstrated to have a high efficacy in lambs against Ostertagia spp. which were resistant to albendazole, levamisole and ivermectin in goats. Moxidectin reduced the number of eggs in faeces by 99.6% and the number of worms found at post-mortem dissection of the lambs by 99.9%. Of the adult worms found in abomasa, 91% were identified as Ostertagia circumcincta and 9% as Ostertagia trifurcata.

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)

[Levamisole- and tetramisole-resistant gastrointestinal nematodes in sheep]. / Nález gastrointestinálnych nematódov u oviec rezistentných k levamizolu a tetramizolu.

Two experiments were carried out with sheep naturally infected with gastrointestinal nematodes to evaluate efficacy of anthelmintics using in vivo faecal egg count reduction (FECR) test. In experiment 1 with 28 ewes, the following efficacy of anthelmintics given at recommended dose rates was observed: albendazole 99.4%, ivermectin 99.3% and levamisole 81.8%. In experiment 2 with 18 ewes, tetramisole exhibited 71.3% efficacy. Suspected resistance to imidothiazole anthelmintics was confirmed by in vitro larval development test (LDT)--minimal inhibition concentration (MIC) values were estimated at 2.0 micrograms/ml. Infective larvae L3 cultivated from eggs produced by the population of resistant helminths were identified as Ostertagia and Trichostrongylus spp.

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.

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 bolus administration of albendazole into the rumen on gastrointestinal nematodes and the Dicrocoelium dendriticum trematode in sheep]. / Ucinnost intraruminálnych albendazolových bólusov na gastrointestinálne nematódy a trematódy Dicrocoelium dendriticum u oviec.

The efficacy of intraruminal albendazole (ABZ) capsules (Proftril-Captec) and the effect of treatment on productivity parameters were studied in two experiments totally on 466 ewes naturally infected with gastrointestinal nematodes and trematodes D. dendriticum. Ovoscopical tests revealed that treated animals remained negative during 10-12 weeks after the administration of capsules and that pasture contamination with helminths was significantly reduced. Necropsy revealed 96.9-99.2% efficacy against nematodes Nematodirus spp., Oesophagostomum spp., Cooperia spp., Trichostrongylus spp. and Trichuris ovis. Priority finding is the efficacy of ABZ capsules against trematodes D. dendriticum which was in the first experiment 88.5% and in the second experiment 91.8%. During the 6-month pasture season treated ewes produced on average 2.56 kg cheese and 0.6 kg wool per ewe more than untreated controls.