EFFECTIVENESS OF BROAD SPECTRUM ANTHELMINTICS AGAINST SELECTED STRAINS OF TRICHOSTRONGYLUS COLUBRIFORMIS

SUMMARY A field population of Trichostrongylus colubriformis was divided into 4 lines for exposure to selection in the laboratory. The first line was selected with 50 mg/kg thiabendazole, the second with 4 mg/kg morantel tartrate, the third with 50 mg/kg thiabendazole followed by 4 mg/kg morantel tartrate and the fourth line was not selected for drug resistance. Following at least 9 generations of selection there was no difference in LD₅₀ or LD₉₅ between the unselected and single selected strains of worms. The strain selected by⁵⁰both thiabendazole and morantel tartrate had a significantly higher LD₅₀ against thiabendazole, morantel tartrate and levamisole than did the other three strains. The single selected strains had LD₉₅'s of 172, 21.5 and 2.3 mg/kg for thiabendazole, morantel tartrate and levamisole respectively, compared with corresponding values of 111, 17.3 and 2.4 in the unselected strain and 124, 15.5 and 3.0 in the double selected strain. The estimated efficiency of the recommended dose of each anthelmintic against the unselected field strain was:—thiabendazole (44 mg/kg) 50% efficient, morantel tartrate (8.8 mg/kg) 76% efficient and levamisole (7.0 mg/kg) 99.9% efficient.     

Reliability and reproducibility of the larval paralysis test as an in vitro method for the detection of anthelmintic resistance of nematodes against levamisole and morantel tartrate

In order to study the reliability of the larval paralysis test as an in vitro assay for the detection of resistance of nematodes to levamisole and morantel tartrate, the influence of different parameters was evaluated using resistant and susceptible Ostertagia ostertagi strains. The operator, the sample (10% of the larvae present in the suspension), the incubation time (24, 48 or 72 h), the incubation temperature (20 or 25 degrees C) and the observation period of the larvae (5 or 15 s) had no statistically significant influence on the test results. Statistical differences were obtained only when L3 larvae of different ages (1 or 2 months) were used. Reversibility of the paralysis did not occur when concentrations of levamisole of less than or equal to 200 micrograms ml-1 or morantel tartrate of less than 2000 micrograms ml-1 were used. The reproducibility of the test was fairly good, with a mean standard deviation of 21.3% for the LC50 values. Morantel resistance in a strain of O. ostertagi was not confirmed as such by the larval paralysis test. The resistance factor was less than or equal to 1, in spite of an efficacy of morantel of 77.4% as shown in vivo.     

Effectiveness of morantel tartrate and naphthalophos against levamisole resistantOstertagia in sheep

Adult worms from a levamisole resistant population ofOstertagia consisting ofO. circumcincta andO. trifurcata showed no dose response to morantel tartrate. The lowest dose rate of 2.2 mg/kg removed 30.8% of worms while the highest dose rate of 17.6 mg/kg removed 39.2%. A dose response was obtained against naphthalophos and the LD₅₀ and LD₉₅ were determined as 12.54 mg/kg and 23.25 mg/kg respectively. It was concluded that the strain was cross resistant to morantel tartrate but not to naphthalophos.     

Trichostrongylus colubriformis and Ostertagia circumcincta resistant to levamisole, morantel tartrate and thiabendazole: occurrence of field strains

Field strains of Trichostrongylus colubriformis and Ostertagia circumcincta, designated PF4 and PF5 respectively, were recovered from a farm on which the sole use of levamisole over a preceding 12 year period led to the development of anthelmintic resistance. The results of field observations and preliminary critical trials in both Merino and crossbred sheep showed that both species have varying degrees of resistance to three major anthelmintics; levamisole, morantel tartrate and thiabendazole. Mean worm count reductions for adult T colubriformis (PF4) for therapeutic doses of morantel tartrate, thiabendazole and levamisole in crossbreds were 45.7 per cent, 97.3 per cent and zero respectively, and for Merinos 80.7 per cent, 88.3 per cent and 92.0 per cent respectively. Against O circumcincta the corresponding reductions for crossbreds were 51.4 percent, 95.4 per cent and 20.3 per cent and for Merinos, 52.5 per cent, 73.1 per cent and 29.8 per cent. There was no statistically significant difference in the responses of both parasite species to either levamisole or morantel. This result suggests that resistance to the two chemically unrelated drugs may be co-inherited.     

Resistance of Trichostrongylus colubriformis to levamisole and morantel: differences in relation to selection history

Two field strains of Trichostrongylus colubriformis were tested by in vitro and in vivo methods for resistance to morantel, levamisole and thiabendazole and compared with an anthelmintic-naive laboratory-passaged strain (McM). One field strain (TAS) was isolated from a dairy goat herd which had experienced severe helminthiasis despite intensive anthelmintic usage. The other (BCK) was isolated from sheep which had been treated solely with levamisole over a 6-year period. The BCK strain had very high levels of both levamisole and morantel resistance. In contrast the TAS strain was resistant to morantel but highly susceptible to levamisole. This finding is contrary to the expectation that selection with morantel automatically confers resistance to levamisole, the converse of which was shown to apply in the BCK strain. Although the TAS strain was exposed to levamisole prior to isolation, examination of the drug's pharmacokinetics in goats indicated that it exerted little if any anthelmintic effect, and therefore selection pressure, on the parasite population. This study suggests that the mechanism of levamisole resistance covers a wide spectrum, and embraces that for morantel. It also suggests that in order to conserve the effectiveness of the levamisole/morantel group of broad spectrum anthelmintics, morantel should be used to the exclusion of levamisole until resistance is detected, at which time levamisole may be introduced to re-establish high levels of control.     

A new anthelmintic assay using rats infected with Trichostrongylus colubriformis.

A new anthelmintic assay is described which uses immunosuppressed (60 ppm hydrocortisone acetate in diet) rats infected with the nematode Trichostrongylus colubriformis. Immunosuppressed rats were infected with 1500 T. colubriformis larvae, treated either orally or subcutaneously on Day 14 post-infection and necropsied 4 days after treatment. The worm counts in immunosuppressed control animals averaged 775 worms per rat. A range of benzimidazoles, levamisole hydrochloride, morantel tartrate, 22,23-dihydroavermectin B1a and alpha-milbemycin have been evaluated in the assay. The ED95 values obtained indicate that rats infected with T. colubriformis provide a highly predictive model for assaying the activity of experimental drugs in vivo prior to studies in ruminants.     

The efficacy of levamisole and ivermectin against a morantel-resistant strain of Trichostrongylus colubriformis

A controlled slaughter trial was undertaken to evaluate the efficacy of ivermectin (0.2 mg/kg) and levamisole (8 mg/kg) against a morantel-resistant field strain of Trichostrongylus colubriformis in sheep. Mean worm count reductions of greater than 99% were recorded with both anthelmintics. The apparent contradiction between the high efficacy of levamisole and the common belief that resistance to levamisole and morantel is co-inherited, is discussed.     

Effect of the morantel sustained-release bolus, used during one grazing season, on the sensitivity of Ostertagia and Cooperia to morantel tartrate in calves

Infective 3rd-stage larvae of Ostertagia and Cooperia, obtained from the feces of nonmedicated and morantel sustained-release bolus (MSRB)-treated calves, were orally administered to 2 groups of parasite-free calves. After a 42-day maturation period, a therapeutic dose of morantel tartrate was administered to half of the calves from each group. All calves were necropsied 7 days after treatment. After comparing the nematode counts for the nonmedicated and morantel-treated calves of each group, morantel tartrate was demonstrated to be equally effective against the nonmedicated-derived and MSRB-derived nematode populations. The sensitivity of Ostertagia spp and Cooperia spp to morantel tartrate, therefore, was not diminished after use of the MSRB for a single grazing season.     

South African field strains of Haemonchus contortus resistant to the levamisole/morantel group of anthelmintics

A strain of Haemonchus contortus from the Pietermaritzburg district of Natal was found to be resistant to levamisole (geometric mean efficacy 76.5%), morantel (41.9%), the benzimidazoles (oxfendazole: 33.7%) and rafoxanide (82.0%), but apparently fully susceptible to closantel and disophenol. In the case of ivermectin, a mean of 5.2% of the H. contortus was not removed at a dosage of 200 micrograms kg-1 live mass. A second strain of H. contortus, from Amsterdam in the south-eastern Transvaal, showed reduced susceptibility to levamisole (80.8%) and morantel (46.2%), the only 2 drugs tested. This is apparently the first report of resistance to the levamisole/morantel group of anthelmintics in sheep in South Africa.     

A Field Evaluation of Benzimidazole and Nonbenzimidazole Drugs in a Herd of Dairy Goats

A study was conducted to investigate the efficacy of six anthelmintics in a herd of dairy goats. Pretreatment larval cultures indicated that the goats were infected with Haemonchus contortus and Trichostrongylus colubriformis. Three separate treatment regimens were administered. In each trial, mature nonlactating goats were allocated into two treatment groups and a control group. Treatment groups received thiabendazole (TBZ) or levamisole (LEV), mebendazole (MBZ) or fenbendazole (FBZ), and morantel tartrate (MOR) or ivermectin (IVR). LEV, MOR, and IVR reduced fecal strongyle egg counts by 99% to 100% of pretreatment values. The benzimidazole (BZD) drugs changed pretreatment fecal egg counts by +2% to -32%. Results of posttreatment larval culture demonstrated the presence of H contortus larvae following the administration of BZD drugs.     

The effect of route of administration on the anthelmintic efficacy of benzimidazole anthelmintics in sheep infected with strains of Haemonchus contortus and Trichostrongylus colubriformis resistant or susceptible to thiabendazole

Observations of erratic anthelmintic activity of fenbendazole against known standardised thiabendazole-resistant strains of Haemonchus contortus and Trichostrongylus colubriformis in sheep were investigated. Fenbendazole at a dose rate of 10 mg/kg body weight was administered by oral, intra-ruminal or intra-abomasal routes, and was most effective against both resistant strains following intra-ruminal administration. In addition thiabendazole, oxibendazole, fenbendazole, parbendazole and mebendazole plus two unrelated compounds, levamisole and morantel tartrate, were used at one and a half times their suggested or recommended therapeutic dose rate against thiabendazole-resistant strains of H contortus and T colubriformis in sheep; each drug being administered by the intra-ruminal or intra-abomasal routes. Fenbendazole was more effective against both strains following intra-ruminal administration. Parbendazole was more effective against the resistant strain of T colubriformis following intra-ruminal administration. At the dose rate chosen for the other benzimidazoles used against these resistant strains, there was no difference in anthelmintic efficacy due to route of administration. Levamisole was highly effective against both resistant strains, irrespective of the route of administration. In the groups treated with morantel tartrate, the results obtained were difficult to interpret due to mortalities and a highly variable response in the surviving sheep. Fenbendazole, thiabendazole and mebendazole when used at their suggested or recommended therapeutic dose rate in sheep, were highly effective against known thiabendazole-susceptible strains of H contortus and T colubriformis following both intra-ruminal or intra-abomasal administration.     

Anthelmintics and control

Anthelmintic control of Ostertagia ostertagi in cattle presents some special problems because the arrested larval stage (hypobiotic EL4) tolerates all of the older anthelmintics. The only anthelmintics on the North American market that are effective against this stage as well as the adult and developing stages are ivermectin and fenbendazole. In addition to these, the newer broad-spectrum benzimidazoles and probenzimidazoles, albendazole, febantel, netobimin and oxfendazole, are effective and available in other countries. These anthelmintics can be used for prophylaxis of Type II ostertagiasis. The older anthelmintics, levamisole, morantel tartrate, thiabendazole, coumaphos, haloxon and phenothiazine, are effective against the adults and to some extent the developing stages of O. ostertagi so that they can be used to treat Type I ostertagiasis. They can also be used to prevent incoming larvae from establishing if they are administered continuously over a long period of time. This is possible with long term in-feed administration or by the use of the morantel slow release bolus. In cooler temperature regions, where cattle are housed over the winter, this bolus is given at turnout to remove any parasites in the animals and to kill incoming larvae for 60-90 days. This can prevent the build up of significant infective larvae on pasture so that very few arrested L4 larvae accumulate in the summer/autumn, effectively preventing Type II ostertagiasis from occurring later. The use of ivermectin and the newer benzimidazoles in intermittent or slow release devices should prove highly effective in the control of Type I and II ostertagiasis, as well as of subclinical ostertagiasis. To achieve maximum economic benefit, the use of anthelmintics should be based on sound epidemiological considerations, so that stock are not rapidly reinfected after treatment.     

THE ISOLATION OF A FIELD STRAIN OF HAEMONCHUS CONTORTUS IN QUEENSLAND SHOWING MULTIPLE ANTHELMINTIC RESISTANCE

SUMMARY Following the apparent failure of levamisole to control infections of Haemonchus contortus in sheep at Lawes in south eastern Queensland, a strain of this parasite was isolated at the Animal Research Institute, Yeerongpilly. This strain was used to infect sheep at Yeerongpilly and the Merrindale Research Station, Victoria where four experiments to classify the resistance pattern of the parasite were carried out. Resistance to thiabendazole was first suspected in 1969, and these experiments confirmed that resistance to this drug was still present. They also showed that a strong degree of resistance had been developed to both levamisole and morantel tartrate. Other benzimidazole anthelmintics and also the organophosphorus compound naphthalophos were only moderately effective against the original isolate but rafoxanide, nitroxynil and phenothiazine were almost 100% effective. Other highly effective chemicals were disophenol and closantel. After passaging the strain for four generations with both levamisole and albendazole, resistance to both naphthalophos and the newer benzimidazole anthelmintics increased dramatically. This is the first report of a field strain of H. contortus exhibiting resistance to benzimidazole, non-benzimidazole and organophosphorus anthelmintics.     

A comparison of the efficacy of four different long-acting boluses to prevent infections with Dictyocaulus viviparus in calves

A field study of calves in their first grazing season tested the efficacy of four long-acting devices--a morantel sustained-release bolus, a levamisole sustained-release bolus, an oxfendazole interval bolus, and an albendazole interval bolus--against Dictyocaulus viviparus. The pasture had been previously contaminated by four calves orally inoculated with infective lungworm larvae. The calves were grazed together with four bolus-treated groups, each comprising four calves. Lungworm infection became patent in the experimentally inoculated calves between 22 and 26 days. Infection in the bolus-treated groups became patent after 54 days. The morantel bolus group excreted the most larvae, followed by the albendazole bolus group, and the levamisole bolus group. The oxfendazole bolus group excreted by far the least larvae. Eosinophil curves and ELISA titres showed that treated groups had essentially the same course of infection. The heavy infection to which the treated calves were exposed produced complete immunity in all groups. Challenge infection of 10,000 larvae at housing did not change any of the test parameters. Post-mortem examination showed only one positive calf with few worms. We concluded that when pastures are heavily infested with lungworm larvae, all boluses prevent severe clinical signs and allow build up of solid immunity, although none completely prevent excretion of larvae.     

Efficacy of morantel against nematode populations in calves exposed on a pasture stocked for two years with morantel sustained-release bolus-treated calves

Two groups of 10 parasite-free calves were maintained either for 2 weeks on a pasture grazed by nonmedicated cattle (pasture A) or for 3 weeks on a pasture grazed by morantel sustained-release bolus-treated cattle (pasture B) for the preceding 2 years. After a 4-week holding period to allow for maturation of acquired gastrointestinal nematodes, 5 calves from each group were administered a therapeutic dose (10 mg/kg of body weight) of morantel tartrate. All calves were necropsied 1 week later, and the abomasal and small intestinal nematodes were isolated, identified, and enumerated. A comparison of efficacies between nonmedicated and morantel tartrate-treated calves of each pasture demonstrated that morantel was equally effective against the gastrointestinal nematode infections, regardless of infection source (ie, pasture A vs pasture B). The overall nematode reductions due to morantel tartrate treatment of calves that grazed pastures A and B were 98% and 96%, respectively. It was concluded that the sensitivity of gastrointestinal nematodes to morantel tartrate was not diminished in calves maintained on pasture B, which had been stocked with morantel sustained-release bolus-treated calves for the preceding 2 grazing seasons.     

The efficacy of levamisole and ivermectin against a morantel-resistant strain of Trichostrongylus colubriformis

A controlled slaughter trial was undertaken to evaluate the efficacy of ivermectin (0.2 mg/kg) and levamisole (8 mg/kg) against a morantel-resistant field strain of Trichostrongylus colubriformis in sheep. Mean worm count reductions of greater than 99% were recorded with both anthelmintics. The apparent contradiction between the high efficacy of levamisole and the common belief that resistance to levamisole and morantel is co-inherited, is discussed.     

In vitro activity of various anthelmintic compounds against Haemonchus contortus larvae

Twenty-five known anthelmintic compounds were evaluated in vitro against the highly motile exsheathed non-feeding third-stage of Haemonchus contortus larvae. Activity was based on lack of motility or death of larvae after 24 h of chemical exposure. Six compounds (avermectins, closantel, levamisole, morantel, phenylhydrazone and ticarbodine) were active at a concentration of 100 μg cm^?3 or less. The most active compounds were avermectins and levamisole. When higher in vitro concentrations were used, ten compounds (bephenium, coumaphos, dichlorovos, disophenol, hygromycin b, methyridine, parbendazole, phenothiazine, pyrantel and thiabendazole) exhibited activity. Nine compounds were found to be inactive; among these were the new benzimidazoles, i.e., albendazole, fenbendazole, mebendazole and oxibendazole. Because of the inactivity of the new benzimidazoles, this in vitro system is unsuitable as a routine screening tool. Also, the system appears to favor drugs that act quickly through percuticular entry. In an initial group of 5280 untested compounds, 254 (4.8%) exhibited in vitro activity at 100 μg cm^?3 against the non-feeding larvae stage. The exogenous and in vitro cultivation techniques required for collecting, cleaning and exsheating the larvae are described.     

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.     

Overberg Research Projects. XI. First stage larval reduction test to assess anthelmintic efficacy ante mortem in sheep.

Two field trials, one with suckling Merino ewe lambs and the other with yearling Dohne Merino rams, are described. In these the anthelmintic efficacy of febantel (a benzimidazole), ivermectin, levamisole and morantel are compared, using the 1st stage larval reduction test. The mean natural log (+1 for zero values) of the post treatment larval counts of the treated groups was compared with that of the untreated controls and the percentage reduction used to assess anthelmintic efficacy. Febantel was only 87.4% effective against Teladorsagia in suckling lambs but the other anthelmintics were more than 99% effective against this genus. Efficacy against Haemonchus and Trichostrongylus ranged from 93.2%-100% for all 4 compounds. In the rams all compounds were 100% effective against Trichostrongylus, with the exception of morantel which was only 87.5% effective. None of the compounds were effective against Teladorsagia, particularly morantel, animals treated with which having more larvae than the controls. The interpretation of anthelmintic efficacy; the advantages of the first stage larval reduction test, compared with the faecal egg count reduction test; and the importance of incubating cultures at 30 degrees C for 24 h, in order to harvest first stage larvae, are discussed.     

Pharmacodynamics, pharmacokinetics and faecal persistence of morantel in cattle and goats

Morantel could not be detected (<0.05 pg/ml)in the plasma of cattle or goats followingthe oral administration of morantel tartrate at a dose rate of 10 mg/kg
bodyweight. No morantel was detected in the milk of lactating goats except in
one animal where a concentration of 0.092 pg/ml was detected at 8 h after drug
administration. Morantel was highly effective against Cooperia oncophora infec-
tions in calves treated 6, 9 or 18 days after infection; however, was highly
effective against Ostertagia ostertagi only when treated 18 days after infection.
Morantel did not affect the fecundity of adult 0.ostertagi surviving treatment 18
days after infection which had similar average numbers of eggs in their uteri
(range 13.4 f 0.73-16.8 L 0.98) as did parasites from control animals (range
12.0 k 0.70-13.6 2 0.66). Morantel could be detected at a concentration of 96 k
4.5 pgig (dry weight) in the faeces of a calf 24 h after treatment with I0 mgikg
bodyweight of morantel tartrate. The concentration of morantel in replicate
samples of this faeces exposed to natural atmosphere, but not to soil or soil
organisms, declined slowly over the following 322 days. At day 322 after the start
of the experiment 8.8 pg/g of morantel could be measured in the remaining
faecal material. Throughout the faecal degradation study the concentration of
morantel in the crusts of the replicate sample pats was lower than the
concentration in the core samples.