Fibrinogen-degrading proteins from Haemonchus contortus used to vaccinate sheep.

Sixteen nonsibling sheep, approximately 12 months old, that were raised in a helminth-free environment, were used for 2 protection studies 6 months apart. Sheep were vaccinated weekly for 5 weeks by IM injection of fibrinogen-degrading proteins derived from the intestinal tract of adult Haemonchus contortus. Ten days after the last vaccination, sheep were given 2,500 infective H contortus larvae by intraruminal injection. Vaccinated sheep produced specific antibodies, and were protected from the worm challenge. Significant differences in mean fecal worm egg counts for 56 days after worm challenge, in mean numbers of H contortus worms, and female fecundity ratios at necropsy were detected in vaccinated sheep, compared with those in control sheep. These data suggest that the fibrinogen-degrading proteins have a protective role in vaccination of sheep against H contortus.     

Systemic responses to challenge infection withHaemonchus contortus in immune Merino sheep

Some systemic responses to single-dose infection with 10 000Haemonchus contortus infective larvae were examined in sheep already shown to have protective immunity against the parasite. The major haematological finding was a neutrophil leukocytosis that occurred after the infections became patent but not during the pre-patent period. There was no definitive eosinophilia and no discernible change in the erythrocyte parameters. Systemic hyperthermia was not conclusively evident during the pre-patent period. Enzyme-linked-immunosorbent assays (ELISA) were used to measure the secondary anti-helminth antibody response in serum during the pre-patent period when the establishment of patent infection is resisted. These ELISAs employed preparations from adult worms to represent the parasitic stages of the worm, preparations from infective larvae to represent the pre-parasitic stages of the worm, and exsheathing fluid, which is the soluble material obtained whenH. contortus larvae undergo ecdysis and transform from the pre-parasitic to the parasitic phase. Antibody responses to the three preparations differed qualitatively, indicating the presence of three different but perhaps overlapping sets of antigens. The three peaks in antibody against exsheathing fluid may reflect the pulses of antigen delivered to sheep as the parasite undergoes its three moults within the host.Abbreviations ELISA enzyme-linked immunosorbent assay - EDTA ethylene diamine tetraacetate     

The intensity of resistance by mature Merino ewes against Haemonchus contortus and Tichostrongylus colubriformis in single-species and combined-species infection

Three-year-old, non-lactating and non-pregnant Merino ewes, raised on pasture under a program of strategic treatment with anthelmintic and found to be extremely resistant to "trickle" infection with Haemonchus contortus, were given single-dose infections with either H. contortus or Trichostrongylus colubriformis or both species together. The purpose was to ascertain the intensity of protective immunity against the 2 parasites in sheep with immunity acquired from a presumably slight exposure to infection. To provide a criterion, some infected ewes were immunosuppressed with corticosteroid, dexamethasone. Untreated ewes were extremely resistant to challenge infection with either 15,000 or 150,000 H. contortus or 15,000 T. colubriformis. Surprisingly, when mixed infection was given, egg counts for H. contortus were significantly elevated compared with infection by that species alone. Antibody to antigens from infective larval and adult H. contortus was measured in serum by enzyme-linked-immunosorbent assay (ELISA) during the course of infection. Serum titres against larval antigens were significantly depressed when infections with either H. contortus or T. colubriformis were permitted by immunosuppression with dexamethasone, whereas those against adult antigen were depressed when infection with T. colubriformis was permitted.     

Serum IgG response of Manchego lambs to infections with Haemonchus contortus and preliminary characterization of adult antigens

Manchego lambs (16-18 weeks old) were infected with 2500 infective larvae (L3) of Haemonchus contortus and challenged 2 months later with 5000 L3. The serum IgG anti-Haemonchus response was estimated by enzyme-linked immunosorbent assay (ELISA) using soluble proteins from adults and L3. Previously infected Manchego lambs failed to mount a protective immune response against challenge, at least as assessed by faecal egg counts and pre-patency periods. Primary infection did not provoke any rise in specific anti-parasite serum antibodies, whereas a weak but significant rise was observed in challenged 6.5-month-old lambs which was very similar in both infected and non-infected lambs. The serum IgG anti-parasite response was higher against larval antigens than adult soluble proteins. Preliminary characterization of adult and larval soluble proteins by electrophoresis under reducing and denaturing conditions and Western blotting showed high cross-reactivity of both extracts. Immunoblots of adult H. contortus probed with infected and challenged lambs' sera did not yield conclusive results, although some low molecular weight peptides were recognized.     

Characterization of a galectin-like activity from the parasitic nematode, Haemonchus contortus, which modulates ovine eosinophil migration in vitro

The development of eosinophilia is a characteristic feature of helminth infection, although the exact nature of the interaction between eosinophils and parasites remains to be fully defined. Previously, it has been reported that Haemonchus contortus and other nematodes produce eosinophil-specific chemoattractants. This paper describes studies aimed at isolating and identifying the factor(s) responsible. Initial studies showed that soluble extracts of infective larvae (L3) of H. contortus provoked a chemokinetic, rather than chemotactic, response in ovine bone marrow eosinophils in vitro. This activity was inhibited by lactose to a markedly greater extent than sucrose suggesting a galectin-like identity. Lactose affinity chromatography of soluble H. contortus extracts resulted in the isolation a specific bound fraction which retained biological activity. SDS-PAGE gel electrophoresis indicated a single Coomassie-stained band at between 31 and 41kDa. Subsequent, mass spectrometric analysis confirmed that the bound fraction contained a mixture of nematode galectins. The results confirm that H. contortus larvae produce several galectin-like proteins, at least one of which demonstrates eosinophil chemokinetic activity in vitro. The possibility of the parasite-derived factor mimicking the mammalian galectin-9, a known eosinophil chemokine, is discussed.     

The anthelmintic efficacy of the plant, Albizia anthelmintica, against the nematode parasites Haemonchus contortus of sheep and Heligmosomoides polygyrus of mice

Albizia anthelmintica Brong., belongs to the plant family Mimosaceae. The plant is widely used in East Africa by poor smallholder farmers and pastoralists to treat their livestock against internal parasites. The anthelmintic effects of water extracts from the bark of A. anthelmintica, obtained from three different geographic areas in Kenya and using different methods of preparation, were tested at different doses in sheep and mice infected with the nematode parasites Haemonchus contortus and Heligmosomoides polygyrus, respectively. Lambs were infected with 3000 infective larvae of H. contortus and treated with the plant preparations 28 days later, while mice were infected with 200 infective larvae of H. polygyrus and treated 18 days later. Proximate analysis established high levels of crude proteins in A. anthelmintica bark. Two sheep out of the 45 treated with the plant preparations suffered from transient bloat, which was relieved by dosing with a surfactant. Significant reductions in faecal egg counts were observed in lambs treated with A. anthelmintica in two of the three experiments undertaken, but the efficacy levels achieved were well below the 70% reduction required. Similar values of packed red cell volume and live weight gain were observed for treated and control lambs. There was no overall significant effect of treatment with A. anthelmintica on faecal egg and total worm counts in mice. A dose rate of 1000 mg/kg bodyweight of A. anthelmintica preparation resulted in death of all mice. The results show that A. anthelmintica at the doses and preparations used is not efficacious against H. contortus in sheep or against H. polygyrus in mice.     

Serum and abomasal antibody response of sheep to infections with Haemonchus contortus

Serum and abomasal IgA, IgG and IgM antibody response against adult worm, L3 and egg antigens of Haemonchus contortus was monitored by the ELISA technique after one or two infections with this nematode. Following the first infection, antibody levels in serum did not change materially. After administration of a challenge dose of infective larvae, antibodies of the three immunoglobulin classes in infected animals rose slightly, but this rise appeared later than the fall in the faecal egg counts. In contrast, in abomasal mucosa, IgA anti-larval antibody levels, which did not increase materially after the primary infection, rose rapidly after a transient inhibition when sheep were challenged. A close temporal relationship was observed between the rise in local anti-worm IgA antibodies and the self-cure reaction, but antibody levels fell rapidly after worm diminution. The local antibody response was thus considered to be related to immunity of sheep to H. contortus.     

Studies on Haemonchus contortus. II. The effect of abomasal nematodes on subsequent challenge with H. contortus

Fifty-thousand infective larvae of Trichostrongylus axei dosed to 7-9-month-old Merinos protected them against a subsequent challenge with 50 000 infective larvae of Haemonchus contortus. Protection varied with the period between an initial dosage of T. axei and challenge with H. contortus as follows: After 90 days it was more than 80% effective in more than 80% of sheep; at 119 and 180 days there was still a significant reduction in the numbers of H. contortus recovered (P < 0,05 and P < 0,001, respectively). Prior infestation with either Ostertagia circumcincta or with Haemonchus placei was ineffective against challenge with H. contortus.     

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).     

Effects of the chitin synthesis inhibitor diflubenzuron on development of Ascaris suum and Haemonchus contortus

The potential of the chitin synthesis inhibitor diflubenzuron (DFB) to alter the development of the parasitic nematodes (Ascaris suum and Haemonchus contortus was investigated. DFB given orally (10 mg kg-1 per day for 30 days) to sheep inoculated with H. contortus infective larvae did not prevent the establishment of adults or affect fecal egg output. However, there was a significant (greater than 90%) decrease in the number of infective larvae recovered from fecal cultures derived from lambs harboring H. contortus adults that were treated with DFB. DFB did not affect egg hatching. Oral administration (10 mg kg-1 per day for 20 days) of DFB to swine harboring adult A. suum adults had no effect on the adult worm burden or on egg morphology, but eggs removed from worms obtained from DFB-treated swine contained less chitin than eggs removed from untreated control swine. DFB also inhibited chitin synthesis in vitro in the isolated reproductive tract of A. suum adults. These results indicate that DFB at high doses can inhibit the subsequent development of H. contortus larvae in the feces. Since H. contortus larvae lack chitin, DFB may act on these larvae by a mechanism independent of a direct effect on chitin synthesis.     

Ovicidal and larvicidal activity of the crude extracts from Phytolacca icosandra against Haemonchus contortus

The development of anthelmintic resistance has impacted on the success of conventional anthelmintics (AH) for the control of gastrointestinal nematodes in grazing/browsing sheep and goats. Medicinal plants from the traditional herbolary in Mexico may provide new candidates that can be explored as alternative sources of AHs for ruminants. This study evaluated the leaf extracts derived from Phytolacca icosandra against infective L(3) larvae and eggs from Haemonchus contortus collected from sheep. Three extracts of different polarities were obtained from the leaf plants using ethanol, n-hexane and dichloromethane as the solvents. The effectiveness of the in vitro AH activity of the plant extracts was evaluated using larval migration inhibition (LMI) and egg hatch (EHA) assays. For the LMI assays, the ethanolic extract of P. icosandra showed 55.4% inhibition of larval migration at 2mg/mL (p<0.05). The dichloromethane extract of P. icosandra showed 67.1% inhibition of migration at 3mg/mL (p<0.05) and a dose-dependent response with an LD(50) of 0.90 mg/mL. The n-hexane extract failed to show inhibition of larval migration at any concentration explored. In the EHA for the ethanol extract, the lowest concentration tested (0.15 mg/mL) resulted in inhibition of egg hatching greater than 72.6%. Therefore, the LD(50) could not be calculated for this extract. The LD(50) of the dichloromethane extract of P. icosandra was 0. 28 mg/mL. An egg hatch inhibition greater than 90% was observed with both the ethanolic and dichloromethane extracts when using a concentration of 0.90 mg/mL or higher. The n-hexane extract failed to show egg hatch inhibition at any concentration tested. The AH activity reported for P. icosandra could be attributable to the flavonoids, steroids, terpenoids, coumarins and/or saponins that were present in the ethanolic and dichloromethane extracts. A combination of more than one component may also explain the observed AH activity against the H. contortus life stages that were evaluated. In conclusion, the ethanolic and dichloromethane extracts of P. icosandra showed clear in vitro AH activity against the H. contortus eggs and the L(3) larvae. However, the hexanic extract of the plant leaves failed to show any in vitro AH activity.     

Studies on Haemonchus contortus. X. The effect of Trichostrongylus axei in Merinos on artificial pastures

A recently established Eragrostis curvula pasture 1,1 ha in size at Hennops River in the Transvaal Highveld was contaminated from 6 October 1976 until 20 May 1977 by 30 weaned Merinos, each infested with 7 000 infective larvae of H. contortus. On 2 November 1976 3 groups of weaned Merinos and again on 10 January 1977 a further group of Merinos were infested with 40 000 infective larvae of Trichostrongylus axei. From 19 November a single group of sheep, predosed with T. axei, and a control group grazed with the seeders for 8 weeks. After 6 weeks another group of sheep dosed with T. axei grazed with them, thus ensuring a 2-week overlap. This continued until autumn, when the last groups were removed. Efficacy against challenge reached Class C (greater than 50% effective against total worm burdens of H. contortus in greater than 50% of sheep) in early summer and autumn, but only reached significant levels of either P less than 0,01 or P less than 0,05 in summer. Peak worm burdens in controls were recorded in early autumn.     

Protection against high-dose homologous infection in calves immunized with intestine or membrane extracts from Haemonchus placei

Control of Haemonchus placei, one of the most important cattle nematodes in Brazil, relies on the use of anthelmintics. However, there is a need for integrated control, which includes active immunization. The aim of this work was to assess the protection afforded to calves by immunization with adult H. placei extracts against a high-dose challenge infection, a condition frequently found in the tropics. Holstein calves aged 8-10 months were immunized four times with intestinal extracts (Group D) or with a Triton X-100-soluble fraction of adult H. placei (Group A), challenge-infected with 120,000 infective larvae and sacrificed 40 days later. Immunized animals had higher IgG titers than the controls against tested fractions after the 2nd immunization, peaking after the 4th. Sera from both immunized groups recognized bands of similar apparent mass in both antigenic preparations, some of which were similar in molecular weight to Haemonchus contortus antigens with known protective effect to sheep. Egg counts were 49% and 57% lower in Groups A and D than in controls, respectively. High levels of protection were observed in two of the four calves in Group D, as evidenced by very low worm numbers recovered at necropsy, absence of eggs in the uteri of the recovered females and reduced worm length. Group D animals also showed milder signs of anemia than the other infected animals. Results demonstrate that protection against homologous high-dose challenge can be achieved by immunizing calves with H. placei gut antigens.     

In vitro effects of four tropical plants on three life-cycle stages of the parasitic nematode, Haemonchus contortus

Alcoholic extracts of four tropical plants (Zanthoxylum zanthoxyloides, Newbouldia laevis, Morinda lucida and Carica papaya) were screened in vitro for potential anti-parasitic effects against eggs, infective larvae and adult Haemonchus contortus. Significant effects were obtained with all four plants but differences were observed depending on the parasitic stage. The effects of the four plant extracts were similar on egg hatching and were dose dependent. In contrast, no dose-response relationship was found for infective larvae and adult worms, although more potent effects were usually observed with the highest concentrations. Using a larval inhibition migration test, extracts of fagara (Z. zanthoxyloides) were found to be less active against Haemonchus infective larvae than were the other plants. N. laevis was found to be highly and rapidly effective against adult worms. Overall, these in vitro results suggest that these four plants, traditionally used by small farmers in Western Africa, do possess anti-parasitic properties. These effects remain to be confirmed through in vivo studies.     

Efficacy of levamisole and netobimin against Haemonchus contortus in lambs in Louisiana

Efficacy of levamisole was evaluated in a suspected levamisole-resistant population of Haemonchus contortus in the Louisiana State University sheep flock. The efficacy of netobimin also was evaluated against this population of Haemonchus. In trial 1, 5 lambs naturally infected with H contortus were given 8 mg of levamisole/kg of body weight as a drench, and 5 lambs were not treated (controls). Nematode recovery after slaughter indicated 0% efficacy against H contortus. In trial 2, 30 nematode-free lambs were each given 8,300 F1 generation infective larvae of H contortus, which were derived from parent H contortus that survived 2 levamisole treatments in lambs being maintained in a nematode-free environment. Ten lambs were treated with 8 mg of levamisole/kg as a drench, 10 were treated with 20 mg of netobimin/kg as a drench, and 10 were not treated (control). Nematode recovery after slaughter revealed 62.3% and 99.8% efficacy for levamisole and netobimin, respectively, against H contortus.     

Efficiency of feeding Duddingtonia flagrans chlamydospores to grazing ewes on reducing availability of parasitic nematode larvae on pasture

Gastrointestinal nematodes are of concern in sheep production because of production and economic losses. Control of these nematodes is primarily based on the use of anthelmintic treatment and pasture management. The almost exclusive use of anthelmintic treatment has resulted in development of anthelmintic resistance which has led to the need for other parasite control options to be explored. The blood sucking abomasal parasitic nematode Haemonchus contortus causes severe losses in small ruminant production in the warm, humid sub-tropic and tropics. This study evaluated the effectiveness of a nematode trapping fungus, Duddingtonia flagrans, in reducing availability of parasitic nematode larvae, specifically H. contortus, on pasture. Chlamydospores of D. flagrans were mixed with a supplement feed which was fed daily to a group of crossbred ewes for the duration of the summer grazing season. A control group was fed the same supplement feed without chlamydospores. A reduction in infective larval numbers was observed in fecal cultures of the fungus-fed group. Herbage samples from the pasture grazed by the fungus-fed group also showed a reduction in infective larvae. There were no significant (P > 0.05) differences in overall fecal egg count, packed cell volume or animal weight between fungus-fed and control groups. Tracer animals were placed on the study pastures at the end of the study to assess pasture infectivity. Although tracer animals were only two per group, those that grazed with the fungus-fed group had substantially reduced (96.8%) nematode burdens as compared to those from the control group pasture. Results demonstrated that the fungus did have activity against nematode larvae in the feces which reduced pasture infectivity and subsequently nematode burdens in tracer animals. This study showed that D. flagrans, fed daily to grazing ewes, was an effective biological control agent in reducing a predominantly H. contortus larval population on pasture.     

Comparing the sensitivity of two in vitro assays to evaluate the anthelmintic activity of tropical tannin rich plant extracts against Haemonchus contortus

The present trial aimed at comparing the sensitivity of two in vitro methods, i.e. the larval migration inhibition assay (LMIA) and the larval exsheathment inhibition assay (LEIA), to evaluate the anthelmintic (AH) properties of tannin-rich plant extracts against Haemonchus contortus infective larvae. The two assays were applied on the same batch of H. contortus infective larvae exposed to water/acetonic extracts obtained from four tropical plants with different tannin contents: Acacia gaumeri, Brosimum alicastrum, Havardia albicans and Leucaena leucocephala. Increasing concentrations (0, 75, 150, 300, 600, 1200 μg/ml PBS) of lyophilized extracts were used in both in vitro assays. A general lineal model test was used to determine the dose-effect in the LMIA or the difference in the percentage of exsheathed larvae between the respective control and treated groups. The LMIA showed a dose-dependent AH effect for H. albicans (P<0.001) and A. gaumeri (P<0.05), but not for L. leucocephala and B. alicastrum. In contrast, the exsheathment process was significantly affected by all doses of H. albicans and A. gaumeri extracts and a significant dose-dependent effect was found for B. alicastrum and L. leucocephala. Calculation of lethal dose (LD) was possible with LEIA using B. alicastrum and L. leucocephala but not with H. albicans and A. gaumeri as the lowest tested concentration was achieving more than 50% inhibition. Calculation of LD with the LMIA results was not feasible. These results suggest that tannin-rich plant extracts are more potent inhibitors of the exsheathment of H. contortus L(3) larvae than their motility. This information underlines the difference of sensitivity between methodological procedures to evaluate the AH properties of plant extracts on the same nematode stage.     

Partial protection of lambs against Haemonchus contortus by vaccination with a fractionated preparation of the parasite

Florida Native lambs, less than 6 months of age, were successfully vaccinated against Haemonchus contortus with a high mol. wt fraction (greater than 30,000 daltons) derived from a somatic extract of H. contortus larvae (SEL) and excretions and secretions (ES) of larvae isolated during in vitro development from the infective 3rd to 4th stage. A 59% reduction in adult worm numbers was obtained in vaccinates compared to naive lambs following challenge. The protection in vaccinated lambs was similar to that seen in lambs exposed to a primary infection of H. contortus larvae which had been cleared with anthelmintic prior to the challenge infection. The unfractionated SEL/ES preparation and a low mol. wt fraction gave no significant protection against challenge infection.     

Haemonchus contortus and other trichostrongylid infections in parturient, lactating and dry ewes

From autumn (April) to spring (November), groups of pregnant and dry Merino ewes grazed pasture contaminated with infective larvae of Haemonchus contortus, Ostertagia circumcincta and Trichostrongylus colubriformis. A periparturient rise in faecal egg counts occurred in the pregnant ewes, following the maturation in early spring of arrested fourth-stage larvae of H. contortus and failure of pregnant ewes to expel the resulting adult worms. Peak egg counts were seen in September, just before lambing. In dry ewes, egg counts were slightly elevated at the time of the rise in the pregnant ewes, but fell to very low levels as adult worms developing from previously arrested larvae were expelled. Lactating ewes acquired greater burdens of O. circumcincta and T. colubriformis than did dry ewes, but were equally refractory to new infections with H. contortus. It is therefore suggested that the impairment of immunity to helminth infection seen in reproductive ewes may be more specific than was previously envisaged.     

Influence of protein supplementation during late pregnancy and lactation on the resistance of Santa Ines and Ile de France ewes to Haemonchus contortus

This experiment aimed to evaluate the effect of periparturient metabolizable protein (MP) nutrition on resistance to Haemonchus contortus in single rearing Ile de France and Santa Ines ewes. The restriction-fed iso-energetic diet was calculated to provide either 0.8 (low MP diet) or 1.3 (high MP diet) times MP, from three weeks before parturition until eight weeks into lactation. The ewes were experimentally infected with 1000 H. contortus infective larvae (L3) three times a week (Mondays, Wednesdays and Fridays), starting five weeks before the predicted date for parturition until a total of 15,000 L3 had been administered. While both breeds showed elevated fecal egg counts (FEC), these values were significantly lower for Santa Ines ewes than Ile de France ewes, but were independent of level of MP feeding. The latter also did not affect lamb weight gain and ewe body weight variation in each breed. Packed cell volume and total plasma protein for Santa Ines in all periods were significantly higher than those for Ile de France ewes (P<0.01) but were not affected by nutrition. In contrast, levels of serum IgG and IgA antibodies against somatic H. contortus infective larvae and adult antigens were similar between breeds but higher in animals that received high MP diets (P<0.05). The reduced body score of ewes at the beginning of the experiment probably influenced their high susceptibility to incoming larvae. Since, unexpectedly, MP scarcity was not achieved in this experiment, our data support the view that Santa Ines ewes are more resistant to H. contortus than Ile de France ewes.