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PubMed1997

Cloning and characterization of a microsomal aminopeptidase from the intestine of the nematode Haemonchus contortus.

In order to characterise the integral membrane glycoprotein H11 from the intestinal microvilli of the nematode Haemonchus contortus, cDNA libraries prepared using mRNA from adult worms from the UK and Australia were immunoscreened with anti-H11 sera. Antibodies affinity purified on the protein expressed by insert DNA (295 bp) of a positive clone from a UK library bound specifically to H11. A longer clone (948 bp) was obtained from the Australian library by hybridisation. Using a primer based on sequence common to these, a polymerase chain reaction product of 3.3 kb was generated from cDNA from UK H. contortus. The sequences from the UK and Australian nematodes were essentially identical over the 929 bp region in which both were represented. All three cloned DNAs hybridised to mRNA of about 3.5 kb. Analysis of the deduced amino acid sequence, which showed 32% identity with those of mammalian microsomal aminopeptidases, indicated that H11 has a short N-terminal cytoplasmic tail, a single transmembrane region and a long extracellular region with putative N-linked glycosylation sites and the HEXXHXW motif characteristic of microsomal aminopeptidases. Microsomal aminopeptidase activity co-purifies with H11. It is inhibited by bestatin, phenanthroline and amastatin. The recombinant protein has been expressed in active form in insect cells.

Amino Acid Sequence↗
PubMed1999

The development of vaccines against gastrointestinal nematode parasites, particularly Haemonchus contortus.

Many parasitic nematodes are developing resistance to chemical treatment, and the search is on to produce commercially viable molecular vaccines. Much progress has been made with highly protective 'hidden antigens', especially for Haemonchus contortus, and recent work with new 'natural antigens' has yielded promising results. Here, Sue Newton and Ed Munn review the most recent advances in these two main approaches to this problem.

Animals↗
PubMed1999

Vaccination against the gastrointestinal nematode, Haemonchus contortus, using a purified larval surface antigen.

Sheep were immunized with a purified antigen (Hc-sL3) expressed on the surface of L3 larvae of the gastro-intestinal parasite, Haemonchus contortus, using different adjuvant and immunization routes. In the first experiment, intradermal immunization of sheep with Hc-sL3 and QuilA did not result in reductions in faecal egg counts after subsequent challenge infection while significant reductions were obtained when aluminium hydroxide (AH) was used as the adjuvant. Significant protection with Hc-sL3 absorbed on AH was confirmed in a second experiment and this protection was maintained when dextran sulphate was added to the Hc-sL3/AH mixture while the addition of pertussis toxin abrogated the protective effect. Significant levels of protection, as determined by reductions in both faecal egg counts and worm burdens, were also obtained when the Hc-sL3/AH mixture was injected into the rectal mucosa or the Hc-sL3 antigen was deposited on the surface of the rectal mucosa with cholera toxin. No correlations with antibody levels or isotype and protection were observed.

Animals↗
PubMed2000

Ostertagia ostertagi challenge of calves vaccinated with Haemonchus placei intestinal homogenate.

The protective capacity of vaccination with Haemonchus placei whole gut homogenate against challenge with the non-blood-feeding nematode Ostertagia ostertagi was evaluated in calves. Ten helminth-free calves were randomly assigned to two groups. Group 1 received 100microg H. placei intestinal homogenate in the adjuvant 5% dextran sulfate/PBS, while Group 2 received the adjuvant alone. Injections were administered subcutaneously on Days 0 and 28. All calves were challenged with approximately 26,100 O. ostertagi larvae on Day 42. Serum antibody response and counts of nematode eggs per gram of feces (EPG) were determined throughout the study. Calves were necropsied at 5.5 weeks post-challenge for recovery of nematodes. Although significant increases were detected in both serum IgG(1) and IgG(2) of Group 1 calves (p<0.05), there was no significant difference in the total number of O. ostertagi recovered from the two groups (p>0.05). Lengths of adult nematodes were not significantly different between groups nor were the numbers of eggs present in adult females recovered from each group significantly different (p>0.05). There were also no significant differences between groups regarding fecal egg counts (p>0.05). Results suggest either: (1) the antigens targeted by the induced antibodies were not present in O. ostertagi; (2) the antigens targeted by the induced antibodies were present, but not essential to O. ostertagi survival; or (3) the antigen was present and essential, but amount of antibody ingested was insufficient to cause damage to the nematode.

Animals↗
PubMed2000

Interaction between Trypanosoma evansi and Haemonchus contortus infection in goats.

An experimental study on interaction between Trypanosoma evansi and Haemonchus contortus infections was conducted in 42 male Barbari goats of 6-9 months age. Parasitological observations like prepatent period, faecal egg count, worm burden and mortality were made in animals infected with H. contortus followed 1 week later with T. evansi and vice versa. These parameters were also examined in animals with single infection with each of these parasites. It was revealed that T. evansi infection in goats lowered the normal resistance to H. contortus. The prepatent period was markedly reduced to 16-18 days in cases where T. evansi preceded H. contortus as compared to 21-25 and 21-23 days in single H. contortus and in a H. contortus preceding T. evansi infection, respectively. The rate of L(3) establishment was significantly enhanced in infection where T. evansi preceded H. contortus. A higher rate of mortality and more pronounced pathological effects were observed in combined infections than in single ones.

Animals↗
PubMed2000

Uptake of albendazole and albendazole sulphoxide by Haemonchus contortus and Fasciola hepatica in sheep.

The pattern of in vivo uptake of albendazole (ABZ) and its major metabolite, ABZ-sulphoxide (ABZSO), by Haemonchus contortus and Fasciola hepatica recovered from ABZ-treated sheep, was investigated. Concentration profiles of both compounds were simultaneously measured in target tissues/fluids from the same infected sheep. In addition, the proportion of the (+) and (-) ABZSO enantiomers was determined in plasma, bile and F. hepatica recovered from treated sheep. Sheep naturally infected with H. contortus were intraruminally (i.r.) treated with ABZ (micronized suspension, 7. 5mg/kg) and the plasma concentrations of ABZSO and ABZ-sulphone (ABZSO(2)) determined in addition to the concentration of ABZ and ABZSO in H. contortus, abomasal mucosa and fluid content samples. In addition, F. hepatica artificially infected sheep were treated i.r. with the same ABZ suspension (7.5mg/kg), and samples of blood, bile, liver tissue and adult flukes were collected and analysed by HPLC to determine the concentrations of ABZ and both enantiomers of ABZSO. ABZSO and ABZSO(2) were the analytes recovered in plasma with ABZ and ABZSO present in H. contortus. ABZ was the analyte recovered at the highest concentration in H. contortus and abomasal mucosa, whereas higher concentrations of ABZSO were measured in abomasal fluid content. Only low concentrations of ABZ were detected in F. hepatica and bile, but markedly higher concentrations of ABZ were measured in liver tissue. ABZSO was the main molecule recovered in F. hepatica, plasma and bile samples collected from ABZ-treated sheep. The (+) enantiomer of ABZSO was recovered at a higher proportion in plasma (75%), bile (78%) and F. hepatica (74%) after ABZ administration to infected sheep.

Abomasum↗
PubMed2001

Experimental cross-infections of Haemonchus placei (Place, 1893) in sheep and cattle.

To examine effects on biology and morphology, Haemonchus placei infections were propagated in cattle or sheep and infective larvae were introduced into the same or opposite host. Ovine source larvae had a geometric mean (GM) prepatent period of 26.5 days regardless of host species, compared to 30.6 days for bovine source larvae regardless of host species. Similarly, ovine source H. placei had a higher GM percentage establishment versus bovine source larvae (9.6% versus 3.4%) regardless of host species. The patent daily egg count for ovine source versus bovine source H. placei was 109.1 versus 50.0 FEC regardless of host species. Mean spicule length for ovine source H. placei was 492.5 microm while bovine source measured 496.5 microm. Mean female tail length for ovine source H. placei was 586.5 and 589.5 microm for bovine source H. placei. Using synlophe morphology, all worms that were measured were identified as H. placei. Sixty-three percent of females examined for vulvar flap morphology had knob-like vulvar flaps while the remaining 37% had linguiform vulvar flaps.

Animals↗
PubMed2001

Preliminary observations on the potential of gut membrane proteins of Haemonchus contortus as candidate vaccine antigens in sheep on naturally infected pasture.

In a trial lasting 11 months in South Africa, faecal egg counts and haematocrits of sheep vaccinated with gut membrane proteins of adult Haemonchus contortus were compared with unvaccinated controls grazing pasture contaminated with the parasite. Vaccination reduced egg output by >82% on average during one 4 month period of the trial and simultaneously significantly reduced the degree of anaemia and deaths due to haemonchosis. Although vaccine immunity was not sufficiently long lasting to prevent a surge in egg output which occurred after the onset of a period of irrigation, re-vaccinating the sheep at this point cleared their newly acquired infection and rapidly restored protection to approximately the level observed beforehand. It was clear that a vaccine based on parasite gut membrane proteins could offer substantial benefits in the control of natural haemonchosis.

Animals↗
PubMed2001

Testing for clinical anaemia caused by Haemonchus spp. in goats farmed under resource-poor conditions in South Africa using an eye colour chart developed for sheep.

A novel clinical assay for the assessment and subsequent treatment of Haemonchus infection in sheep to slow down the development of anthelmintic resistance--the FAMACHA system--has been developed, tested and validated in South Africa. The system is based on a colour chart with five colour categories depicting varying degrees of anaemia that are compared with the colour of the mucous membranes of the eyes of sheep. The animal is then scored from severely anaemic (pale) through anaemic to non-anaemic (red) and those animals considered in danger of succumbing to the effects of haemonchosis are treated. This method was tested in goats farmed under resource-poor conditions in South Africa. Analyses in goats performed during the summers of 1998/1999 and 1999/2000 show a test sensitivity of 76 and 85%, respectively, meaning that the system may be used to identify correctly 76-85% of those animals in need of treatment with an anthelmintic. However, the test specificity remains low at 52-55%. This means that a large proportion of those animals that would not require treatment would in fact be treated. However, when the use of the FAMACHA system is compared with conventional dosing practices where all the animals are treated, using the FAMACHA system would result in a large proportion of the animals being left untreated. The untreated animals are then able to deposit the eggs of anthelmintic-susceptible worms on the pasture, while the treated ones should pass very few ova, given an effective anthelmintic. This maintains a reservoir of susceptible larvae in refugia, and should slow down the development of anthelmintic resistance. The validation of the FAMACHA system for goats for use by resource-poor farmers, which this paper describes, may have wide application in the tropics and subtropics of sub-Saharan Africa and elsewhere.

Anemia↗
PubMed2002

Selection for resistance to macrocyclic lactones by Haemonchus contortus in sheep.

An anthelmintic-sensitive Haemonchus contortus strain was selected for moxidectin and ivermectin resistance concurrently for 22 generations. Treatment with 0.002 mg moxidectin/kg BW or 0.02 mg ivermectin/kg BW produced >99% efficacy against the susceptible parent strain passaged for 22 generations without any anthelmintic exposure. However, to obtain similar efficacy the moxidectin-selected and the ivermectin-selected strains of H. contortus required 0.05 mg moxidectin/kg BW or 0.4 mg ivermectin/kg BW. These results indicate that development of resistance to one macrocyclic lactone, simultaneously results in resistance to another macrocyclic lactone. However, rates of resistance development differ between compounds and occurs more slowly with moxidectin than with ivermectin.

Animals↗
PubMed2002

Evaluation of Duddingtonia flagrans in reducing infective larvae of Haemonchus contortus in feces of sheep.

Consequences of nematode infections due to Haemonchus contortus are a serious constraint for the sheep industry worldwide. Development of anthelmintic resistance and increasing concern about the impact of anthelmintic use dictate the need of alternative control. Such an alternative is using the nematode trapping fungus Duddingtonia flagrans to reduce infective larvae levels on pasture. Two trials were conducted to determine the effect of D. flagrans in reducing infective larvae (predominantly H. contortus) in feces. The first trial determined the dose effect of D. flagrans in reducing infective larvae in feces. Eighteen ewes were dewormed to remove existing infections and randomly assigned to six treatment groups: 5 x 10(4), 1 x 10(5), 2.5 x 10(5), 5 x 10(5), 1 x 10(6) or no (control) spores of D. flagrans per kg of body weight mixed in their feed for 7 days. Fecal samples were collected daily from these and from infected donor ewes. Feces from individual-treated ewes were mixed with equal amounts of donor ewe feces, theoretically approximating oral dose spore concentrations of 2.5 x 10(4), 5 x 10(4), 1.25 x 10(5), 2.5 x 10(5), 5 x 10(5) and no spores, and were cultured. Across dosages and during the 7 days of fungus feeding, percent reduction of infective larvae ranged from 76.6 to 100.0%. The second trial determined the effect of D. flagrans at the dose of 10(5) spores per kg body weight on reducing infective larvae in feces from naturally infected lambs. Twenty lambs were randomly assigned to either treatment or control groups based on fecal egg count. Treatment lambs were fed spores mixed in feed for 7 days. Feces were collected daily and cultured. During the 7 days of fungus feeding, the percent reduction of infective larvae ranged from 82.8 to 99.7%. Results of these trials demonstrated that the nematode trapping fungus D. flagrans was highly effective in reducing infective larvae in sheep feces and should be considered as a biological control agent for integrated nematode control programs.

Administration, Oral↗
PubMed2002

Excretory/secretory products of Haemonchus contortus inhibit aminopyrine accumulation by rabbit gastric glands in vitro.

Abomasal parasites inhibit gastric acid secretion and reduce the number of acid secreting parietal cells either through physical contact with gastric tissue, the release of inhibitory excretory/secretory (ES) products or by initiating the host inflammatory response. To examine the role of parasite ES products, adult Haemonchus contortus were incubated in a medium based on Hank's balanced salt solution and three incubates were tested for the presence of inhibitors of acid secretion by dispersed rabbit gastric glands in vitro, using the intracellular accumulation of 14C-aminopyrine (AP) as an indirect measure of acid secretion. Acceptably sensitive rabbit preparations (80%) for testing ES products showed increased 14C-AP accumulation with either 10(-5) or 10(-4)M histamine. Sheep glands proved unsuitable as a test system as only two of six preparations responded weakly to histamine. Inhibitory activity was demonstrated in all the three parasite incubates, although responses varied quantitatively between tests, even with the same batch of incubate. In single tests, 16% ES products significantly reduced the 14C-AP ratio (P<0.05) of 10(-4)M histamine-stimulated glands (15 of 19 tests with Batch 1 and two of six with Batch 3). Even at 1 and 5%, ES products (Batch 2) were inhibitory for 10(-5)M histamine-stimulated glands: at 1% the mean reduction was 26.0% (range 7.8-54.8%, n=10), four of which were statistically significant and at 5% was 17.6% (range -15.3-53.8%, n=17), four of which were statistically significant. ES products were overall inhibitory (-12%, P<0.05), when tested on glands exposed to increasing histamine concentrations from 10(-6)M to 10(-3)M (which increased the 14C-AP ratio (P<0.001)). Responses by individual gland preparations differed (P<0.001). The active component(s) of the parasite ES products were less than 5000 molecular weight. Ammonium chloride, tested over the range of concentrations of ammonia present in worm incubates (0.2-1 mM, final concentrations in the gland incubations of 0.005-0.1 mM), had variable, but overall inhibitory effects on 10(-5)M histamine-stimulated glands (n=3). When tested with a range of histamine concentrations, 0.01 mM NH4Cl overall reduced the response by 8.6% (P<0.05)(n=4). The similarity of responses of rabbit glands to parasite ES products and to NH4Cl suggests that ammonia may be the small molecular weight ES product of adult H. contortus, which inhibited acid secretion in vitro.

Aminopyrine↗
PubMed2002

Expression of acquired immunity to a local isolate of Haemonchus contortus by the Nigerian West African Dwarf goat.

The capacity of young Nigerian West African Dwarf (WAD) goats to express good acquired immunity to their native geographic strain of Haemonchus contortus and the correlates of this responsiveness were studied in a laboratory experiment involving forty 7-8 month old kids. A primary immunising infection with 2000 L3 (equivalent to 260-450 L3/kg body weight) with or without challenge on D42 with 2000 L3 resulted in a mild chronic infection with a pre-patent period of 18-20 days and little or no reduction in worm burden between D14 and D56. In contrast, another group (D) of kids, whose immunising infection had been truncated with fenbendazole on D35 and later received similar challenge infection, developed good protection against challenge. Thus, worm burdens were largest in group E (challenge control), larger in group C (primary+challenge) and least in group D. Of the measures of infection used, namely faecal worm egg counts (FECs), circulating eosinophil (EOS) responses, packed cell volume (PCV) and body weight, FEC and EOS responses exhibited marked individual variability, but only FEC (geometric mean of transformed counts) and PCV showed strong correlation with worm burden. There was also a significant negative correlation between FEC and PCV. The size of inoculum used was well tolerated by the kids, as it induced only mild changes in PCV in some goats and no effect at all on body weights. This suggests that the WAD goat may possess a good measure of resistance to the pathogenic effects of its native strain of H. contortus. The wide individual variability in FEC and its strong relationships to worm burden and PCV are pointers to its likely genetic basis. There are, therefore, good prospects for further studies to identify H. contortus resistant genotypes among the WAD goat population.

Animals↗
PubMed2002

Estimating genetic differences in natural resistance in Rhön and Merinoland sheep following experimental Haemonchus contortus infection.

Genetic parameters of natural resistance were estimated in Rhön and Merinoland (German Merino) sheep following experimental infection with Haemonchus contortus. A total of 133 Rhön and 244 Merinoland lambs descending from 5 and 6 rams, respectively, were evaluated. Each helminth-naive lamb was orally infected with 5000 infective third-stage larvae (L(3)) of the nematode H. contortus at 12 weeks of age. Faecal egg counts (FEC) and haematocrit values were measured in all lambs at 16 and 20 weeks of age. Seventy-nine Merinoland and 29 Rhön male lambs were slaughtered immediately after the second sampling and worms were collected. Mean worm burden was calculated and the length of adults worms from an aliquot was measured.FEC of Rhön sheep was higher compared with Merinoland sheep (P<0.01). H. contortus L(3)-larvae specific antibody (IgL) level was higher in Rhön sheep (P<0.05). However, no differences in haematocrit, worm burden and IgG antibody values could be found between the breeds. Heritabilities for log FEC (+/-S.E.) were 0.0 and 0.07 (+/-0.07) for the first sample in Rhön and Merinoland sheep, respectively. Values for the second sample were higher in both breeds (Rhön 0.35+/-0.14, P<0.05; Merinoland 0.17+/-0.07, P<0.05). Corresponding heritabilities for haematocrit were higher in Merinoland (0.56+/-0.20 and 0.51+/-0.27) compared with Rhön (0.29+/-0.12 and 0.08+/-0.13). Heritabilities for worm burden were high in Rhön (0.54+/-0.2) and low in Merinoland (0.06+/-0.14 and 0.11+/-0.15). Estimated values for IgL were between 0.13 (+/-0.11) for the first sample in both breeds and 0.30 (+/-0.18) for the second sample in Rhön sheep. Corresponding heritabilities for IgG were not different from 0.0 in both breeds (P>0.05). Positive phenotypic correlations were estimated for IgG and IgL values in both breeds (P<0.01). IgG was significantly (P<0.05) and positively correlated with worm burden in male Merinoland and IgL with worm burden in male Rhön sheep.

Animals↗
PubMed2002

The effects of ivermectin and moxidectin on egg viability and larval development of ivermectin-resistant Haemonchus contortus.

The in vivo effects of ivermectin and moxidectin on egg viability and larval development of ivermectin-resistant Haemonchus contortus were examined over time after anthelmintic treatment of sheep. Twenty merino sheep, (12 months old) were allocated to five treatment groups and infected with ivermectin-resistant H. contortus. Thirty one days later, the sheep were treated with intraruminal ivermectin capsules, oral ivermectin, oral moxidectin or injectable moxidectin at the manufacturer's recommended dosages, or left untreated. At various times up to 112 days after treatment, faecal egg counts (FEC) were determined and development rates of infective larvae (L3) cultured in faeces or on agar were measured. Eggs in faecal cultures from ivermectin capsule treated sheep showed reduced L3 development percentages in comparison to faecal cultures from untreated sheep. Eggs from ivermectin capsule treated sheep, isolated from faeces, and cultured on agar showed similar L3 development to eggs from control sheep. These results demonstrate an inhibitory effect of excreted ivermectin in faeces on larval development of ivermectin-resistant H. contortus. L3 development in faecal culture from animals receiving oral ivermectin were reduced for only 3 days after treatment. Faecal egg counts and development of L3 larvae in both culture systems from moxidectin treated sheep were low, due to the high efficacy of the drug. Egg counts in moxidectin treated sheep were reduced by approximately 90% 24h after treatment, before decreasing to almost 100% at 48h, suggesting that the current quarantine recommendation of holding sheep off pasture for 24h after treatment may still lead to some subsequent pasture contamination with worm eggs.

Animals↗
PubMed2002

High-level ivermectin resistance in a field isolate of Haemonchus contortus associated with a low level of resistance in the larval stage: implications for resistance detection.

The IVPro isolate of Haemonchus contortus was isolated in 1999 after significant numbers of the parasite survived an ivermectin capsule treatment of grazing sheep acquiring a natural infection at Prospect, NSW, Australia. The isolate shows high-level resistance to ivermectin (faecal egg count is unaffected by ivermectin oral treatment at 0.2mg kg(-1)). The larval LC(50), as assessed by larval development assays (LDAs), is only approximately two-fold higher than several susceptible isolates, making it difficult to detect the resistance using larval LC(50) as an indicator. However, the isolate shows the presence of a small proportion of the population (<20%) able to develop at significantly higher drug concentrations than the susceptible isolates. Hence, if the IVPro and susceptible isolates are compared at the LC(99) level, the IVPro isolate is readily identifiable as resistant. This degree of distinction at the LC(99) allows the IVPro isolate to be identified as resistant by simply observing the highest drug concentration in the development assay at which some larvae develop relative to the susceptible isolates. Assessing the development assay using this criterion allows a distinction between IVPro and the susceptible isolates equivalent to 10-fold differences in drug concentration, greatly increasing the likelihood of detecting the resistant isolate in routine resistance tests. This study highlights the need to examine this aspect of LDAs in order to detect the type of resistance displayed by IVPro H. contortus.

Animals↗
PubMed2002

Anthelmintic activity of essential oil of Ocimum gratissimum Linn. and eugenol against Haemonchus contortus.

The ovicidal activity of the essential oil of Ocimum gratissimum Linn. (Labideae) and its main component eugenol was evaluated against Haemonchus contortus, gastrointestinal parasite of small ruminants. The oil and eugenol were diluted in Tween 20 (0.5%) at five different concentrations. In the egg hatch test, H. contortus eggs were obtained from feces of goats experimentally infected. At 0.50% concentration, the essential oil and eugenol showed a maximum eclodibility inhibition. These results suggest a possible utilization of the essential oil of O. gratissimum as an aid to the control of gastrointestinal helmintosis of small ruminants.

Animals↗
PubMed2002

The effects of stage-specific selection on the development of benzimidazole resistance in Haemonchus contortus in sheep.

Resistance to the benzimidazole (BDZ) class of anthelmintics in nematodes of sheep has become a common and global phenomenon. The rate at which the selection process and development of resistance occurs is influenced by a number of factors. Of these, the effects of stage-specific exposures to anthelmintic were investigated with a BDZ-resistant strain of Haemonchus contortus (HCR) over five parasite generations. Sheep were infected at each generation with the HCR strain and were treated with thiabendazole (TBZ), either 5 days post-infection (p.i.) (larval line), 21 days p.i. (adult line), or left untreated (no selection line). Additionally eggs from each generation were exposed to TBZ (egg line). Geometric worm burdens were calculated from post-mortem worm counts, both at the start of the study, and after the final selection studies for each of the selection lines. Egg hatch assays (EHAs) were also conducted throughout the study. All data relating to worm burdens and EHAs for each generation were analysed by linear regression to produce dose titration curves and lethal dose(50) (LD(50)) values for each of the selection lines. Over the five generations, LD(50) values on dose-response were increased and worm survival occurred at higher dose rates of TBZ irrespective of the parasite stage exposed to treatment. A similar picture was seen with ED(50) values, which showed a fluctuating but generally upward trend for each of the three selection lines. In contrast, LD(50) and ED(50) values were decreased in the no selection line, indicating some degree of reversion albeit to levels still considered to be BDZ-resistant.

Aging↗