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Two field strains of Haemonchus contortus resistant to rafoxanide.

Two field strains of Haemonchus contortus, one from Pretoria on the Highveld and the other from White River in the Lowveld of the Transvaal, showed pronounced resistance to rafoxanide at a dosage rate of 7.5 mg/kg live mass per os. Both of these strains originated from kikuyu pastures under irrigation. Resistance in South Africa to rafoxanide and other anthelmintics, namely, closantel, ivermectin and the benzimidazoles is reviewed. It is concluded that apart from avoiding farming practices where integrated worm control is not possible, there is at present no solution to the problem of worm resistance in the country.

Animals↗

Biochemistry of Haemonchus--a review.

Haemonchus biochemistry has been reviewed under the subheadings chemical composition (carbohydrates, lipids, proteins, amino acids, nucleic acids, hormones, inorganic elements, pigments), absorption/transport, biosynthesis (carbohydrates, lipids, proteins, amino acids, nucleic acids), catabolism/utilization (carbohydrates-glycolysis, TCA cycle, CO2 fixation, electron transport system, pentose pathways, glyoxylate cycle, strain variations in energy metabolism; lipids, proteins), biochemistry in taxonomy and miscellaneous.

Amino Acids↗

Laboratory selection of Haemonchus contortus for resistance to ivermectin.

The eighth generation of adult Haemonchus contortus, selected by subjecting infected pairs of sheep to suboptimal ivermectin treatment once per generation from parent (P; BBH isolate) through F7 (IV-A; selected isolate), required an approximate 4-fold increase in the ivermectin dose to produce 95% efficacy compared with its contemporary parent isolate. In a dose titration experiment the dose-response curve of the drug pressure-derived isolate, IV-A, was significantly (0.02 less than P less than 0.05) less steep than was the response curve of the parent, BBH, isolate. Potency estimates based upon these nonparallel dose-response curves would not remain constant over a range of efficacy levels but would decrease rapidly at efficacies greater than 95%. Passage of a closed population of the F8 generation of IV-A sequentially through pairs of sheep for an additional 11 generations (F8A-F8K) without additional drug pressure being applied produced no reversion to sensitivity to ivermectin relative to the F7 generation, thus suggesting that the selected "resistance" was stable.

Analysis of Variance↗

Changes in LC50 in an in vitro egg development assay during the patent period of Haemonchus contortus in sheep.

Using an in vitro egg development assay with thiabendazole as anthelmintic the LC50 of eggs produced by two sheep infected with either a susceptible or a resistant strain of Haemonchus contortus was followed during the patent period from 22 days to 77 days after infection. In both sheep the LC50 followed the same pattern. At the start of the patent period the average values for the susceptible and the resistant strain were, respectively, 0.05 and 0.21 micrograms ml-1 thiabendazole, after 40 to 60 days 0.09 and 0.35 micrograms ml-1, while after 77 days 0.07 and 0.24 micrograms ml-1 were recorded.

Animals↗

Protection of lambs with a purified metabolite of exsheathed third-stage Haemonchus contortus larvae.

Helminth-free lambs (approx 4 months old) were inoculated SC with a purified metabolite of exsheathed third-stage Haemonchus contortus larvae. The metabolite was obtained from in vitro cultivation and was identified as XL3FA1. After 3 periodic XL3FA1 inoculations, lambs were challenge exposed with H contortus. Multiple vaccinations with XL3FA1 did not inhibit the development of worm populations in the lambs; however, worm egg production seemed to be inhibited.

Animals↗

Effect of cool storage of faecal samples containing Haemonchus contortus eggs on the results of an in vitro egg development assay to test anthelmintic resistance.

An in vitro egg development assay was used to test the resistance of eggs from a benzimidazole susceptible and a resistant strain of Haemonchus contortus to four benzimidazoles. Storage of eggs contained in faecal samples at 4 degrees C for four, 24 or 72 hours did not appear to impair the results from the in vitro egg development assay or alter the LD50. Storage for one week, however, proved to be lethal for the eggs. The average LD50 values for thiabendazole, albendazole, mebendazole and cambendazole were 0.06, 0.07, 0.96 and 2.47 ppm for the susceptible strain. For the resistant strain the values averaged 0.26, 0.24, 1.07 and 5.11 ppm.

Animals↗

Cultivation of Haemonchus contortus (Nematoda: Trichostrongylidae) from infective larvae to the adult male and the egg-laying female.

The parasitic nematode Haemonchus contortus was cultured in vitro to the adult male and the egg-laying female. Gastric contents from 2 cannulated calves and 2 sheep as well as extracts of stomach mucosa from both hosts were added to a mixture of API-1 culture medium and Fildes' reagent (a pepsin digest of defibrinated bovine blood) adjusted to pH 6.4 for the first week and pH 6.8 thereafter. The gas phase was 85% N2:5% O2:10% CO2. Best results were obtained when API-1 culture medium plus Fildes' reagent was supplemented with ovine gastric contents. Adult males up to 10 mm were obtained, and they produced and stored sperm at about 28 days in culture. Spermatozoa were never seen in the uteri of females grown in vitro. After 36 days in culture, female worms up to 16 mm were obtained which produced and layed eggs. Some of the eggs underwent division up to the 8-cell stage but did not develop further. No live larvae were observed at a stage earlier than the fourth. Presumably, this was because no matings were observed, although large clumps of worms did occur in the culture medium and mating might have occurred. The growth of adults from young to mature phases of this stage included: for the female, oogenesis and laying of eggs; for the male, development of paired sclerotized spicules, and spermatogenesis with the production of spermatozoa.

Animals↗

Influence of dietary protein on parasite establishment and pathogenesis in Finn Dorset and Scottish Blackface lambs given a single moderate infection of Haemonchus contortus.

An experiment was conducted to examine the influence of dietary protein on parasite establishment and pathogenesis of Haemonchus contortus in lambs of two breeds. Finn Dorset and Scottish Blackface. The lambs were introduced to high protein (170 g crude protein (CP) kg-1 dry matter (DM) or low protein (88 g CP kg-1 DM) diets at three months old and infected one month later with 125 larvae (kg bodyweight)-1. Blood and faecal samples were collected for analysis and bodyweights recorded weekly. The lambs were killed 20 weeks after infection. A second experiment was conducted in Blackface lambs solely to determine the influence on parasite establishment of the high and low protein diets following infection with 125 larvae kg-1. These lambs were killed five or 14 weeks after infection. Results showed that in Scottish Blackface lambs dietary protein did not significantly influence the establishment of H contortus. However, in Finn Dorset lambs dietary protein may have influenced parasite establishment since lambs on the low protein diet had a higher faecal egg output four weeks after infection and more severe clinical signs than infected lambs of the same breed on a high protein diet.

Animals↗

The possible fate of crystal harbouring larvae of Haemonchus contortus.

An examination of the occurrence of crystalline inclusions in the intestinal cells of fourth and fifth stage Haemonchus contortus revealed that a high percentage of inhibited early fourth stage larvae (L4) and developing L4 and a lower percentage of early fifth stage larvae (L5) harboured these crystals. Before the fourth moult crystals were falling into pieces, indicating that worms harbouring crystals can develop further. The presence of crystals may be associated with a retardation of development.

Animals↗

The anthelmintic efficacy of albendazole against Fasciola hepatica and benzimidazole resistant strains of Haemonchus contortus and Trichostrongylus colubriformis in sheep.

The anthelmintic efficacy of albendazole (methyl [5-(propylthio) - 1H - benzimidazole -2 -yl] carbamate) against immature and adult Fasciola hepatica and against standardised strains of benzimidazole resistant Haemonchus contortus and Trichostrongylus colubriformis was evaluated in experimentally infected sheep. A single intrarumenal treatment of dose rates of 3.8 and 7.6 mg/kg was ineffective against immature (six weeks old) F hepatica. Dose rates of 5.7 and 7.6 mg/kg reduced the number of mature (12 weeks old) F hepatica by 70 and 91 per cent respectively. Dose rates of 5.7 and 7.6 mg/kg removed 92 and 99 per cent of four-week-old, benzimidazole resistant H contortus and 89 and 99 per cent of four-week-old, benzimidazole resistant T colubriformis.

Animals↗

Investigation of Haemonchus contortus infections in sheep. Comparison of irradiated larvae and transfer factor treatment.

A comparison is made between the resistance conferred to Haemonchus contortus challenge by irradiated larval treatment and transfer factor (TF) treatment in four-month-old lambs and seven-month-old lambs. As in previous investigations the irradiated larval treatment failed to confer resistance to the four-month-old lambs challenged with 10,000 third stage larvae while similar irradiated larval treatment in seven-month-old lambs reduced the worm burdens by 40 per cent compared to controls. The TF treatment produced a 34 per cent reduction in the challenge infection in the four-month-old lambs and a 45 per cent reduction in the seven-month-old lambs compared to the control lambs. It is concluded that TF activity operates independently of immune competence.

Animals↗

Levels of benzimidazole resistance in Haemonchus contortus and Trichostrongylus colubriformis recorded from an egg hatch test procedure.

A method of using commercially available benzimidazole anthelmintics in an egg hatch test, to record levels of resistance is described. The test shows that a side-resistance is present to all the benzimidazole anthelmintics tested against both Haemonchus contortus and Trichostrongylus colubriformis after selection with thiabendazole. Additional selection on the field resistant strain of H contortus with thiabendazole showed increased levels of resistance to all the compounds tested while similar selection of T colubriformis failed to change the level of resistance after 20 generations. The level of resistance recorded by the egg hatch test in vitro is greater than that recorded for adults in vivo for the CF strain of H contortus and T colubriformis. Problems associated with reading the test are mentioned as are the different levels for the threshold of activity.

Animals↗

The dose response of several benzimidazole anthelmintics against resistant strains of Haemonchus contortus and Trichostrongylus colubriformis selected with thiabendazole.

Dose response lines for eight benzimidazole anthelmintics and thiophanate were determined, using standardised strains of thiabendazole selected and resistant Haemonchus contortus and Trichostrongylus colubriformis. Against H contortus, thiophanate, thiabendazole, parbendazole and oxibendazole were inactive. Mebendazole was inactive at dose rates of 6.26 and 12.5 mg/kg, although significant activity occurred at 25 mg/kg. Fenbendazole, cambendazole, oxfendazole and albendazole demonstrated significant activity at dose rates equal to or greater than the recommended therapeutic level. Thiophanate was inactive against resistant T colubriformis. The remaining compounds only showed significant activity when used at dose rates in excess of the recommended therapeutic level. These results show that a side resistance exists among the benzimidazole anthelmintics and suggests that changes in dose response lines could be expected to occur if resistant strains are selected with benzimidazoles other than thiabendazole.

Animals↗

Reversal of order of ecdysis in Haemonchus contortus (Nematoda).

Exposure of Haemonchus contortus infective larvae to carbon dioxide under specific conditions is believed to activate a succession of events that ultimately control both exsheathment and subsequent development to the fourth stage. Rarely at pH 6, but always at pH 2, worms fail to exsheath yet develop to the fourth stage surrounded by cuticles two and three. Failure to exsheath is probably a consequence of inactive enzymes in exsheathing fluid, because exposure to EDTA, which inactivates exsheathing fluid, reduced exsheathment and yielded sheathed fourth-stage worms. Although exsheathment is blocked at pH 2, CO2 can activate the receptor for exsheathment as readily at pH 2 as at pH 6. Therefore the hypothesis that an early common pathway controls both exsheathment and development is not invalidated.

Animals↗

Partial biochemical and serological characterization of exsheathing fluid and somatic extracts of infective larvae and adult females of Haemonchus contortus.

Exsheathing fluid from infective larvae of Haemonchus contortus may contain antigens which induce protective immunity upon infection in sheep. In this study, a rapid method of preparing exsheathing fluid was used to determine its biological activity, purity and antigenic complexity. Protein in exsheathing fluid ranged from 21-59.5 micrograms/mL and no carbohydrate was detected. Gel filtration showed a single broad peak which retained biological activity and polyacrylamide gel electrophoresis demonstrated a single band staining with Coomassie blue. Biological activity of exsheathing fluid was present in extracts of infective larvae but not in extracts of exsheathed larvae or in extracts of adult female H. contortus. Rabbit antiserum against exsheathing fluid proteins cross-reacted with infective larvae extracts but not with adult female worm extracts. Immunoelectrophoresis of exsheathing fluid proteins against this antiserum showed a single polydisperse precipitin line. Thus, exsheathing fluid produced by the rapid method is a pure preparation of a biologically active and antigenically simple material suitable for further studies of protective immunity in sheep to H. contortus.

Animals↗

Studies on Haemonchus contortus. IX. The effect of Trichostrongylus axei in Merinos on natural pasture.

Four groups of 6-7-month-old Merino lambs were each dosed with 40 000 infective larvae of Trichostrongylus axei on 2 November 1976 and subsequently exposed to challenge with Haemonchus contortus on natural grazing at the University of Pretoria's Experimental Farm in the eastern suburbs of Pretoria. One of these groups and one group of controls were killed every 8 weeks from the end of December 1976-June 1977. Predosing with T. axei was greater than 50% effective against 5th stage and adult H. contortus in greater than 50% of sheep for 164 days (Class C), improving to greater than 60% in greater than 60% of sheep (Class B) 220 days after dosing T. axei. The numbers of retarded 4th stage larvae (L4) of H. contortus in the undosed controls as well as in the sheep predosed with T. axei rose from a low level in summer (December) to a peak in late Autumn (June).

Animals↗

Studies on Haemonchus contortus. XI. The effect of a bovine strain of Trichostrongylus axei in Merinos on natural pastures heavily infested with H. contortus.

Sheep grazed on natural pastures heavily infested with infective larvae of Haemonchus contortus. Sixty-eight weaned Merinos were divided into 6 groups on Day 0 (23 November 1977), and on Day +14 (7 December) 79 Merinos were divided into 7 groups. There were 2 groups of undosed controls and other groups were either dosed with infective larvae of Trichostrongylus axei (bovine strain) only on Days 0 and +14, or in combination with H. contortus, or with subsequent doses of H. contortus, 28 days later. One group (Group 12) was dosed with T. axei and treated with a subcutaneous injection of di-iodonitrophenol (DNP) on Day +14. With the exception of 2 sheep, the sheep of the first 6 groups survived until slaughter in March and April 1978, while many sheep (43) of the latter 7 groups died or were killed in extremis from March-May. T. axei dosed on 23 November (Day 0) protected Group 2 by greater than 50% in greater than 50% of sheep. In the latter 7 groups the best results were achieved when DNP was combined with predosing with T. axei. The poor results were probably due either to delayed predosing with T. axei or a massive challenge in the wettest summer on record.

Animals↗