H. Bjørn

Resistance of Oesophagostomum spp. in pigs to pyrantel citrate

This study was undertaken to determine whether anthelmintic-resistant Oesophagostomum spp. populations occur in Danish swine herds. A controlled field trial on selected sow herds suggested resistance to pyrantel citrate in a herd where faecal egg count depression in response to treatment was only 28.3%. This was confirmed by subsequent experimental infection of pigs where the suspected resistant Oesophagostomum isolate was compared with a susceptible worm isolate. After treatment with the recommended dose of the drug, worm burdens of the suspected isolate were only reduced by 42.6% (p greater than 0.05) in contrast to the susceptible isolate, which was reduced by 94.6% (p less than 0.01). Differential counts of the resistant isolate suggested that O. dentatum might be more resistant than O. quadrispinulatum. The resistant isolate originated from a herd, where the anthelmintic had been administered four times a year over a period of 7 years. The potential for development of anthelmintic resistance in Oesophagostomum spp. under management practices in our country is discussed in the light of the current views on predisposing factors.

Recovery of Oesophagotomum dentatum from pigs by isolation of parasites migrating from large intestinal contents embedded in agar-gel

Four groups with three pigs in each group were inoculated with Oesophagostomum dentatum larvae (L3 larvae). Groups 1 and 3 were inoculated with 20,000 larvae, and Groups 2 and 4 with 200,000 larvae. On Days 11 and 34, respectively, Groups 1 and 2 and Groups 3 and 4 were slaughtered, and the contents from the large intestines collected. Subsamples of intestinal contents were mixed with agar to a final concentration of 1% agar and allowed to set. The worms were allowed to migrate from the agar-gel into 38 degrees C 0.9% saline overnight. Then the worms were collected on a sieve (38 microns mesh) and counted. The worms retained in the agar-gel were counted after pouring the melted agar through a sieve (38 microns mesh). The results showed that more than 95% of the worms migrated out of the agar-gel, and subsequently were available for counting in an almost clean suspension. Additionally the method yielded a high worm recovery; all stages were recovered. The recovery percentage was not significantly affected by either the dose of parasites or the time interval from slaughtering to start of incubation (37-128 min).

A comparison of in vitro tests and a faecal egg count reduction test in detecting anthelmintic resistance in horse strongyles

This study reports a comparison between faecal egg count reduction test (FECRT), egg hatch assay (EHA) and larval development assay (LDA) for detecting anthelmintic resistance in equine strongyles. Resistance to benzimidazoles was demonstrated in 33 of 42 (79%) farms tested by FECRT and in 32 (62%) of the 52 farms tested by EHA. As the reference strain used was not fully susceptible to benzimidazoles it was not possible to determine the level of resistance by LDA. Pyrantel resistance was indicated on three of 15 farms by faecal egg count reduction. Resistance was also indicated by LDA for one of these farms. In addition resistance was indicated by LDA on two more farms that were not tested by FECRT. Further testing is needed to confirm if these findings are truly indicative of resistance. Generally, correlations between the tests were poor and it was not possible to use the outcome of one test to predict the outcome of another.

Resistance to levamisole and cross-resistance between pyrantel and levamisole in Oesophagostomum quadrispinulatum and Oesophagostomum dentatum of pigs

Two strains of Oesophagostomum spp., consisting of both O. quadrispinulatum and O. dentatum, were subjected to a controlled in vivo assay for resistance to levamisole and pyrantel by comparison with susceptible isolates. One strain (LEM) was recently isolated from a commercial herd, where sows showed high numbers of strongyle eggs in faeces within 2 weeks of farrowing and following treatment with levamisole at the manufacturers recommended dose rate 1 week before farrowing. Levamisole had been used as the sole anthelmintic for treatment for at least 7 years on this farm. Treatment with pyrantel in this herd also indicated cross-resistance to this drug. A mixed population of O. quadrispinulatum and O. dentatum of this strain was subjected to controlled in vivo assays. Faecal egg count reduction (FECR) was found to be -573.3% (P greater than 0.05) and worm count reductions (WCR) of O. quadrispinulatum and O. dentatum were estimated as 44.5% (P greater than 0.05) and 96.4% (P less than 0.001), respectively. Treatment with pyrantel showed that cross-resistance existed to this drug, with FECR of 10.4% (P greater than 0.05) and WCR of 64.5% (P greater than 0.05) and 90.7% (P less than 0.05) for O. quadrispinulatum and O. dentatum, respectively. Another strain (VJ) was isolated from another commercial pig herd, which was dosed with pyrantel citrate four times a year for at least 8 years. This strain showed resistance to pyrantel, with FECR of 43.8% (P greater than 0.05) and WCR of 65.9% (P greater than 0.05) and 49.4% (P greater than 0.05) for O. quadrispinulatum and O. dentatum, respectively. However, both species were susceptible to levamisole. Our results suggested that selection with levamisole gave rise to levamisole resistance and automatically conferred resistance to pyrantel, whereas selection with pyrantel only resulted in resistance to this drug alone. These findings are discussed in relation to the location of the two species of Oesophagostomum in the large intestine of pigs and the mode of action of this class of anthelmintics.

Experimental Oesophagostomum dentatum infections in the pig: Worm populations resulting from trickle infections with three dose levels of larvae

This paper describes the worm populations in pigs experimentally infected by trickle infections with different dose levels of the nodular worm, Oesophagostomum dentatum. Four groups each of 20 helminth naïve pigs, 10-12 weeks old, were inoculated with 0 (group 1), 100 (group 2), 1000 (group 3), or 10,000 (group 4) infective larvae twice weekly, and the pigs were killed after 10-13 weeks. No overt clinical signs were observed, and only group 4 had slightly lower food conversion rate (P < 0.05) than the controls. Faecal egg counts revealed that the nodular worms in pigs of groups 2 and 3 had a short prepatent period (3-4 1/2 weeks) and a fairly stable egg output, while the worms in the pigs of group 4 had prepatent periods of 3-10 weeks and low, unstable egg excretion. The mean worm burdens increased with the dose rate (group 2: 929 worms; group 3: 7467 worms; group 4: 19,847 worms), but detailed analyses of the worm populations from 10 pigs from each of the infected groups revealed a clear dose-dependency in worm recovery, percentage adult worms, worm lengths and female fecundity, as all these measures declined significantly with increasing dose level. The adult worms seemed to be shorter and less fertile when they were located posteriorly to their predilection site, and especially in group 4 many stunted infertile adults measuring only 2-5 mm were found in the posterior half of the colon, but there were no indications of worm expulsion. Superimposed on the main experiment was a cohort study in which 4 pigs of group 3 were given a single dose of 1000 pyrantel resistant larvae at day 56 (all other larvae were pyrantel sensitive), treated with 28 mg pyrantel per kg body weight at day 85 and killed at day 90. Appropriate control groups were included. The mean establishment of the cohort was similar to previously uninfected controls, but between-animal variation was much higher in the trickle infected group.

Use of two in vitro methods for the detection of benzimidazole resistance in equine small strongyles (Cyathostoma spp.)

Ten stables were included in a study to evaluate two in vitro methods for the detection of anthelmintic resistance in cyathostomes by comparing a faecal egg count reduction test (FECRT) to a larval development assay (LDA) and an egg hatch assay (EHA). The LDA was used in seven stables and EHA in the last three. On the basis of FECR values, resistance to benzimidazoles was detected in eight of the ten small strongyle populations. Resistance to pyrantel pamoate and ivermectin was not detected. The mean concentrations that inhibited hatching in 50% of the eggs (EC50), using thiabendazole (TBZ) in an EHA, were 1.02 microM in resistant populations and 0.37 microM in susceptible or suspected resistant ones. In the LDA, TBZ concentrations preventing 50% development by first/second stage larvae to the third larval stage (LC50) were 3.8 times lower than EC50 values in resistant worm populations. Mean LC50 for morantel, levamisol, ivermectin monosaccharide and avermectin-B2 in small strongyle populations susceptible to pyrantel and ivermectin was 8.0 microM, 0.99 microM, 15.6 nM and 2.93 nM, respectively. Data on pyrantel and ivermectin resistant populations could not be obtained as no resistant populations were detected. This study concludes that in vitro tests may be useful as a supplement to FECRT for the detection of benzimidazole resistance in cyathostomes, even if reference populations to be used as controls in the assays were not available. It is suggested that EC50 values for TBZ > 0.6 microM in LDA and > 0.5 microM in EHA strongly indicate benzimidazole resistance in equine small strongyles.

Prepatent periods of different Oesophagostomum spp. isolates in experimentally infected pigs

To define prepatent periods of different Oesophagostomum spp. isolates we carried out two separate experiments, one using two monospecific laboratory isolates and another using laboratory isolates as well as isolates obtained from pig herds having different management systems and with different anthelmintic treatment histories. Pigs were inoculated with 1,000–2,000 infective larvae. Fecal samples were collected daily beginning on days 15 and 16 postinoculation (p.i.). Fecal cultures were set up at different times to yield larvae that could be identified by DNA analyses. All pigs started to excrete eggs on days 18–24 p.i. The mean prepatent period was 20.2 ± 1.4 days, with no significant difference being observed between species and isolates. Prepatent periods of 17–19 days were found for the monospecific laboratory isolates of O. dentatum and O. quadrispinulatum. These findings conflict with parasitology textbooks; therefore, suggestions as to the possible reasons for the observed short prepatent periods are given.

Growth rate and trapping efficacy of nematode-trapping fungi under constant and fluctuating temperatures.

Abstract The effect of temperature on radial growth and predatory activity of different isolates of nematode-trapping fungi was assessed. Four isolates of Duddingtonia flagrans and one isolate of Arthrobotrys oligospora were inoculated on petri dishes containing either corn-meal agar (CMA) or faecal agar and then incubated for 14 days under three different constant and fluctuating temperature regimes. The radial growth was similar on the two substrates at each temperature regime. All fungal isolates showed a higher growth rate at a constant 20 °C. At 10° and 15 °C, all D. flagrans isolates showed very similar patterns of radial growth at both constant and fluctuating temperatures. At 20 °C, they grew significantly faster at constant than at fluctuating temperatures. A. oligospora grew significantly faster than all D. flagrans isolates except when incubated at a fluctuating 20 °C. Spores of each fungal isolate were added to faecal cultures containing eggs of Cooperia oncophora at a concentration of 6250 spores/g faeces. The cultures were incubated for 14 days at the same temperature regimes described above. Control faeces (without fungal material) were also cultured. More larvae were recovered from the fungus-treated cultures incubated at a constant 10° or 15 °C than from those incubated at the respective fluctuating temperatures, except for one D. flagrans isolate. Incubation at 20 °C showed the opposite effect. The general reduction observed in the number of nematode larvae due to fungal trapping was 18–25% and 48–80% for a constant and fluctuating 10 °C, 70–96% and 93–95% for a constant and fluctuating 15 °C, and 63–98% and 0–25% for a constant and fluctuating 20 °C, respectively.

A new in vitro assay of benzimidazole activity against adult Oesophagostomum dentatum.

A new in vitro assay of benzimidazole activity against adult Oesophagostomum dentatum is described. The method is based on the ability of O. dentatum to migrate through polyamide nets after exposure to various concentrations of benzimidazole. To determine an appropriate mesh size, control worms and worms exposed to 10 microM oxfendazole for 24 h were allowed to migrate through nets with various mesh sizes (300-500 microns) for up to 1 h. A mesh size of 350 microns and migration periods of 10, 20 and 30 min were selected. Exposure to oxfendazole at 10 microM for 24, 48 and 72 h inhibited the migration in a time-dependent manner. After 72 h of exposure and with a 20-min migration period, the EC50 of oxfendazole for O. dentatum was 0.564 microM. In further studies the activities of albendazole sulphoxide, albendazole, cambendazole, fenbendazole, flubendazole, luxabendazole, mebendazole, oxfendazole, oxibendazole, parbendazole and thiabendazole were compared. The worms were exposed to each drug at two concentrations (0.1 and 3.16 microM) for 72 h. At 3.16 microM there were no significant differences in the activity of the drugs. At 0.1 microM significant differences in activity were found. Albendazole sulphoxide and oxfendazole were poor inhibitors of migration compared with their parent compounds, albendazole and fenbendazole.

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.