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A study of the mortality and fecundity of Haemonchus contortus in sheep following experimental infections.

The regulation of the fecundity and mortality of H. contortus in sheep was examined using a series of mathematical models. Six-month-old Dorset crossbred lambs were infected once only with various doses of infective H. contortus larvae (500-20,000 larvae). Parasite mortality was found to be an increasing linear function of the magnitude of the initial infection over the range of doses examined. Parasite fecundity was found to remain constant over the intensity and duration of the infection. The average fecundity for H. contortus at the time of slaughter was found to be 7037 eggs per female worm per day. There was no evidence of time-dependent changes in fecundity or density-dependent regulation of fecundity.

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Systemic immunization of sheep with surface antigens from Haemonchus contortus larvae.

Sheep immunized systemically with a surface extract from third-stage H. contortus larvae showed high serum antibody reactivity against surface antigens and whole, viable larvae. After a first infection, no significant difference was found between the mean egg counts of the vaccinates and controls although most vaccinated sheep seemed to show an increased susceptibility to infection. The local abomasal response was stimulated by giving both vaccinated and control sheep a large, abbreviated infection cured after 11 days by drenching. Thereafter, a second challenge infection was given. This immunization regime resulted in seven of the nine vaccinated sheep showing clear protection against the second challenge infection.

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In vitro activity of paraherquamide against the free-living stages of Haemonchus contortus, Trichostrongylus colubriformis and Ostertagia circumcincta.

Paraherquamide, an oxindole alkaloid recently reported to have potent nematocidal activity, was shown to have a marked inhibitory effect on the motility of the free-living larval stages of H. contortus, T. colubriformis and O. circumcincta. The effect of paraherquamide on larval motility could be distinguished from that caused by levamisole and the avermectins. After 72 h exposure, the concentration of paraherquamide required to inhibit the motility of 50% of L3 larvae present was 0.033, 0.058 and 2.7 micrograms ml-1 for O. circumcincta, T. colubriformis and H. contortus, respectively. Ivermectin (IVM)-resistant isolates of H. contortus were significantly more sensitive to the paralytic effects of paraherquamide than IVM-susceptible isolates of this species. Paraherquamide had no effect on the time for development from the egg to the L3 larval stage of H. contortus, T. colubriformis and O. circumcincta.

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Kinetics of expulsion of Haemonchus contortus from sheep and jirds after treatment with closantel.

Experiments were conducted in sheep after intramuscular treatment with closantel and in jirds after oral treatment with closantel to determine when expulsion of established H. contortus commences. Expulsion starts at about 8 h in sheep and coincides with the onset of reduced motility in worms recovered from the abomasum. In jirds, expulsion starts by 2 h after treatment. Experiments also conducted in jirds showed that infective larvae are first killed by circulating closantel 3 days after infection, when blood feeding starts, and that by 8 days 80% of larvae are lost.

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Differentiation of Haemonchus placei from H. contortus (Nematoda: Trichostrongylidae) by the ribosomal DNA second internal transcribed spacer.

There has been much debate as to whether H. placei is a separate species to H. contortus. The aim of this study is to provide molecular information to assess the species status of H. placei. Using the polymerase chain reaction, the second internal transcribed spacer (ITS-2) of ribosomal DNA was amplified and sequenced. Comparison of the 231 base pair ITS-2 sequences showed no intraspecific variation in H. contortus, among one isolate from each of the United Kingdom. Switzerland and China and 5 isolates from within Australia, or among 3 Australian isolates of H. placei. Three (1.3%) nucleotide differences were detected between the ITS-2 sequences of H. contortus and H. placei. In addition, 2 diagnostic sites for the endonucleases BfaI and FokI allowed the delineation of H. placei from H. contortus. The data presented herein support previous morphological and genetic evidence that H. placei is a separate species to H. contortus.

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Interaction between Ostertagia circumcincta and Haemonchus contortus infection in young lambs.

Twenty-one-week-old, worm-free, pen-reared lambs were infected with either 6000 O. circumcincta L3 per week, or 3000 H. contortus L3 per week, or both (9000 L3 per week). Egg counts were monitored throughout the experiment, and worm burdens and larval establishment rates of both worm species were estimated after 4, 7, 10 and 13 weeks of infection. After 10-13 weeks of infection with H. contortus only, establishment of O. circumcincta was lower than in previously uninfected controls, demonstrating that a high level of immunity to H. contortus affords some cross-protection against O. circumcincta. Total H. contortus worm burdens and egg counts (about 2000 worms and 3000 e.p.g., respectively) in sheep infected with both worm species were less than half those observed in sheep infected with H. contortus alone (about 5000 worms and 10,000 e.p.g., respectively). Cross-protection between the two species was observed, but was probably less important than the reduction in H. contortus establishment that was caused by O. circumcincta disrupting abomasal physiology.

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The effects of closantel treatment on the ultrastructure of Haemonchus contortus.

H. contortus were recovered from sheep 0-14 h after intramuscular treatment with closantel. Ultrastructural examination revealed that mitochondria were more electron dense and contained swollen cristae compared with untreated controls. Following treatment, the basal channels in the intestine became prominent and there was vesicle formation in all organs examined. In contrast, closantel-resistant H. contortus appeared normal after drug treatment. It is likely that closantel affects membrane associated processes responsible for fluid and ion homeostasis as well as mitochondrial function. Untreated H. contortus were maintained in balanced salt solution for 12 h which caused lesions indicative of fluid imbalance, but at 23 h there were serious structural abnormalities.

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Identification and partial purification of a 26 kilodalton antigen of adult Haemonchus contortus.

Adult H. contortus soluble extracts were fractionated by means of gel filtration (S-200) and anion exchange chromatography (DEAE-Sephacel). Fractions from both analyses were checked by ELISA and western blotting (WB) with sera from lambs infected with H. contortus, monospecific heterologous sera (anti-Trichostrongylus colubriformis and anti-Teladorsagia circumcinta) and sera from naturally infected sheep with mixed trichostrongylid infections. High cross reactivity was seen between H. contortus and heterologous sera, particularly with the anti-T. colubriformis serum, when fractions from gel filtration were checked by ELISA. Individual fractions containing the highest positive/negative and positive/heterologous ratios were pooled and analysed by SDS-PAGE. One of the pools (A4) containing 2 regions around 48-55 and 25-27 kDa were strongly recognized by homologous sera in WB. Similar results were obtained with the first peak eluted in the DEAE-Sephacel chromatography with NaCl 0.1 M. The pooled fraction A4 from gel filtration was further fractionated by anion exchange chromatography and the peak obtained with the NaCl gradient contained a ca. 26 kDa antigen apparently specific for the diagnosis of H. contortus infections in lambs.

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The binding of closantel to ovine serum albumin, and homogenate fractions of Haemonchus contortus.

Closantel binds to the serum proteins of the host and affects blood sucking parasites when they ingest the blood of treated hosts. Closantel binds specifically to ovine serum albumin (K(a) of 9. 3x10(6)M(-1)) at site I, the warfarin/phenylbutazone binding site of albumin Closantel also binds to invertebrate haemocyanin and haemolymph. The strongest binding of closantel in homogenates of H. contortus is found in fractions containing soluble proteins. This binding is of low affinity and, because the site itself is not fully denaturable, it may not be proteinaceous. There is no detectable difference in binding affinity between homogenate fractions from closantel susceptible and resistant isolates of adult or larval worms suggesting that closantel resistance is not due to changes in the closantel receptor or carrier.

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The mortality and fecundity of Haemonchus contortus in parasite-naive and parasite-exposed sheep following single experimental infections.

Parasite-exposed lambs and their parasite-naive controls were experimentally infected once only with 30,000 H. contortus larvae at 3, 9, 12, and 20 weeks following termination of a moderate immunizing infection of 30,000 H. contortus larvae. Previously exposed lambs, challenged at 3 weeks, had a significant reduction in the total H. contortus worm burden as compared to parasite-naive controls. No difference in the total H. contortus worm burden was found between parasite-exposed or parasite-naive lambs challenged at 9 weeks or thereafter. Female worms were found to be significantly smaller in lambs previously exposed to the parasite as compared to those found in parasite-naive lambs. The average parasite fecundity was 4700 eggs per female worm per day. Previous exposure of the lambs to the parasite had no effect on parasite fecundity. Various mathematical models were used to examine parasite fecundity. Parasite fecundity was found to increase in the initial post-challenge period reaching a constant value approximately 58 days after challenge infection. No density-dependent constraints on fecundity were observed.

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