Haemonchus Contortus Eggs Research Articles

Anthelminthic activity of acetone extract and fractions of Vernonia amygdalina against Haemonchus contortus eggs and larvae

The current control of parasitic nematodes in small ruminants relies on the use of chemical anthelminthics, but the development of resistance and the problem of drug residues require research into alternatives. Acetone extract and solvent-solvent fractions of Vernonia amygdalina Del. (Compositae) were evaluated in vitro for potential anti-parasitic effects against the eggs and larvae of Haemonchus contortus. Significant effects were obtained with the extract and fractions but differences were observed depending on the parasitic stage. The acetone extract and fractions inhibited egg hatching and inhibited larval development and killed larvae of H. contortus in a concentration-dependent manner. Best-fit 50% lethal concentration (LC(50)) values were 957.0, 76.0, 524.0, 309.0 and 224.0 μg/ml for the acetone extract, and the butanol, hexane, chloroform and 35% water in methanol fractions, respectively, when tested against nematode eggs. Best-fit LC(50) values for the larval viability test were 508.2, 485.5, 569.3, 348.9 and 196.6 μg/ml for the acetone extract, and the butanol, hexane, chloroform and 35% water in methanol fractions, respectively. The butanol fraction was most active against nematode eggs while the 35% water in methanol fraction was the most active on nematode larvae, although differences in activity between fractions were not significant (p > 0.05). Overall, these in vitro results suggest that V. amygdalina, traditionally used by small farmers in Western Africa, does possess anti-parasitic properties.

Anthelmintic properties of <i>Afzelia africana</i> sw: an in-vitro egg hatch assay

The in vitro hatch assay was used to assess the anthelmintic properties of Afzelia Africana, commonly referred to as Mahogany, against Haemonchus contortus eggs using the crude ethanol extract (CEE) of the plant back. The extract was serially diluted at a range of 5 to 0.078mg/ml using a log interval. The fecal material was cultured in test tubes containing 5 ml of each of the serial dilutions of the plant extract. Fecal materials were also exposed to similar dilutions of albendazole, (positive control) and 5 ml of water (negative control). The plant extracts showed varying degree of inhibition of egg hatch, which was significantly higher (p

In vitro screening of various forages for anthelmintic activity on Haemonchus contortus eggs

In vitro screening of various forages for anthelmintic activity on Haemonchus contortus eggs

In vitro ovicidal and larvicidal activity of Azadirachta indica extracts on Haemonchus contortus

The rapid development of anthelmintic resistance, associated with the high cost of the available anthelmintic drugs, has limited the success of gastrointestinal nematodiosis control in sheep and goats and thus created interest in studying medicinal plants as an alternative source of anthelmintics. We tested ethyl acetate and ethanol extracts of Azadirachta indica on Haemonchus contortus eggs and larvae. The extracts were evaluated at five concentrations: 0.19, 0.78, 3.12, 12.5 and 50.0 mg/ml. At 50 mg/ml, the ethyl acetate extract inhibited egg hatching by 51.31% and larval development by 68.10%. The ethanol extract was more effective, inhibiting egg hatching by 99.77% at 3.12 mg/ml and larval development by 87.11% at 50 mg/ml. These results suggest A. indica extracts may be useful in the control of gastrointestinal nematodes of small ruminants. However, in vivo studies and toxicological evaluation are still necessary.

Comparison of three different preservatives for morphological and real-time PCR analyses of Haemonchus contortus eggs

Despite the development of several recent PCR assays for the egg stages of various trichostrongyles, there have been no protocols described for preserving field samples for PCR without refrigeration. In this study, Lugol's iodine (LI), sodium azide (SA), and neutral buffered formalin (NBF) were evaluated using Haemonchus contortus eggs to determine their potential as a preservative for trichostrongyle egg samples to be processed with real-time PCR. When egg recovery, embryo development, and egg morphology were evaluated from fecal samples preserved with LI, NBF, and SA, there was equally good recovery and preservation for the first month. Preserved eggs were detectable for 1 month, but after 6 months, none could be recovered. When real-time PCR analysis was performed on eggs isolated from faeces preserved with LI and SA, there was no detectable inhibition compared to fresh, non-preserved eggs; however, NBF significantly inhibited amplification. The results from this study demonstrate that for PCR applications LI and SA are effective preservatives for H. contortus eggs, resulting in good preservation of morphology while allowing for uninhibited PCR.

Real-time PCR for quantifying Haemonchus contortus eggs and potential limiting factors

The purpose of this study was to evaluate the practicality of using real-time PCR for quantifying feces-derived trichostrongyle eggs. Haemonchus contortus eggs were used to evaluate fecal contaminants, time after egg embryonation, and the presence of competing and non-competing DNAs as factors that might interfere with generating reproducible results during simplex and multiplex quantitative real-time PCR (QPCR). Real-time PCR results showed linear quantifiable amplification with DNA from five to 75 eggs. However, threshold cycle (C (T)) values obtained by amplification of DNA from egg numbers between 75 and 1,000 did not differ significantly. Inhibitors of QPCR were effectively removed during DNA extraction as exemplified by the absence of any improvement in C (T) values with bovine serum albumin or phytase treatments. Changes from egg embryonation could only be detected during the first 6 h. Noncompetitive DNA did not appear to impact amplification; however, in a multiplex reaction a competing trichostrongyle such as Cooperia oncophora can hinder amplification of H. contortus DNA, when present at tenfold greater amounts. This study demonstrates the usefulness of QPCR for amplification and quantification of trichostrongyle eggs, and identifies potential limitations, which may be addressed through multiplex assays or the addition of a standard: exogenous DNA target.

Effects of legume forages on ovine gastrointestinal parasite development, migration and survival

Lambs grazing certain legumes have reduced parasite intensities compared to lambs grazing ryegrass swards. Eighteen replicates of white clover (cv. AberHerald), lucerne (cv. Luzelle), red clover (cv. Merviot) and perennial ryegrass (cv. Abersilo) were sown at equivalent field rates in 25 cm diameter PVC pots and maintained outside for 6 months. On day 0, forage in each pot was cut to 50 mm from soil level and the pots were placed in a glasshouse (at 19–25 °C and 70% humidity) in a randomised block design. Ten grams sheep faeces containing 2133 Haemonchus contortus eggs per gram were placed on the soil in each pot. Six replicates of each forage were destructively sampled on days 14, 21 and 29. Forage samples were cut at 50 mm from the soil surface and at the soil surface to give two samples per pot. The number of nematodes was determined by a modification of the Whitehead tray method. The ratio of free-living to infective-stage larvae was determined from at least 10% of the larvae. The number of H. contortus larvae kg dry matter −1 (DM) forage was calculated and the data rank transformed prior to analysis by ANOVA. There were fewer larvae on legumes compared with ryegrass on samples from forage above 50 mm ( P < 0.001) but there was no forage effect on larvae below this height. The sum of larvae present on all forage per kilogram DM showed fewer larvae on red clover compared with ryegrass on day 21 ( P < 0.05). There was an effect of day on the total number of larvae on forage ( P < 0.001) but there were no forage × day interactions. Analysis of the data according to the leaf area above 50 mm from the soil surface confirmed these results, that there were fewer larvae on legume forages than ryegrass above this height ( P < 0.01). Overall, red clover affected the development of H. contortus and all legumes affected larval migration above 50 mm compared with ryegrass but survival of larvae was similar on all forages. Further work is needed to determine if these effects of legume forages would reduce the number of parasitic larvae ingested by livestock under field conditions.

Evaluation of Anthelmintic Potential of Ethanolic Plant Extracts from Northern Cameroon Against Eggs and Infective Larvae of Haemonchus contortus

Evaluation of Anthelmintic Potential of Ethanolic Plant Extracts from Northern Cameroon Against Eggs and Infective Larvae of Haemonchus contortus

Increased resistance to anthelmintics of Haemonchus contortus eggs associated with changes in membrane fluidity of eggshells during embryonation

The embryonation of nematode eggs has been shown to increase their resistance to anthelmintics when parasites are submitted to egg hatch assays. Nevertheless, no mechanism has been suggested to explain this phenomenon. Earlier observations by other authors showed that the biochemical composition of eggshells is altered during the embryonation of eggs. The functional consequences of these changes have not been identified. We studied the changes in membrane environment (eggshells) of Haemonchus contortus eggs during the embryonation by fluidity measurements and their effects on nonspecific mechanisms of resistance to anthelmintics. We previously demonstrated that these mechanisms imply P-glycoproteins (Pgp) belonging to the multi-drug resistance (MDR) system and that the Pgp activity is very susceptible to their lipidic environment. The results obtained here show that the embryonation induced a significant and gradual increase in eggshell fluidity which was associated with an increased resistance to anthelmintics. Differences were observed between H. contortus isolates with various levels of resistance which might result from their specific biology and/or membrane composition. The membrane environment changes could act both on the solubilization of anthelmintics into the eggs and on the efflux of these lipophilic molecules by Pgp.

Ovicidal and larvicidal activity in vitro of Spigelia anthelmia Linn. extracts on Haemonchus contortus

The rapid development of anthelmintic resistance, associated with the high cost of the available anthelmintic drugs, had limited the success of gastrointestinal nematodiasis control in sheep and goats and thus awakened interest in the study of medicinal plants as alternative sources of anthelmintics. Spigelia anthelmia extracts obtained with hexane, chloroform, ethyl acetate or methanol, were tested on Haemonchus contortus eggs and larvae via egg hatch and larval development tests. The extracts were evaluated at five concentrations: 3.1, 6.2, 12.5, 25.0 and 50.0 mg ml −1. At 50.0 mg ml −1, the ethyl acetate extract inhibited 100% of the egg hatching and 81.2% of the larval development. In a similar way the methanolic extract inhibited 97.4% of the egg hatching and 84.4% of larval development. These results suggest that utilization of S. anthelmia extracts may be useful in the control of sheep and goats gastrointestinal nematodes.

Ovicidal and larvicidal activity in vitro of Spigelia anthelmia Linn. extracts on Haemonchus contortus

The rapid development of anthelmintic resistance, associated with the high cost of the available anthelmintic drugs, had limited the success of gastrointestinal nematodiasis control in sheep and goats and thus awakened interest in the study of medicinal plants as alternative sources of anthelmintics. Spigelia anthelmia extracts obtained with hexane, chloroform, ethyl acetate or methanol, were tested on Haemonchus contortus eggs and larvae via egg hatch and larval development tests. The extracts were evaluated at five concentrations: 3.1, 6.2, 12.5, 25.0 and 50.0 mg ml −1 . At 50.0 mg ml −1 , the ethyl acetate extract inhibited 100% of the egg hatching and 81.2% of the larval development. In a similar way the methanolic extract inhibited 97.4% of the egg hatching and 84.4% of larval development. These results suggest that utilization of S. anthelmia extracts may be useful in the control of sheep and goats gastrointestinal nematodes.

In vitro screening of two Nigerian medicinal plants (Vernonia amygdalina and Annona senegalensis) for anthelmintic activity.

Vernonia amygdalina and Annona senegalensis, two plants used by local/smallholder livestock farmers in Nigeria as anthelmintic were screened for in vitro anthelmintic activity using Haemonchus contortus eggs. The extract of V. amygdalina did not show any significant activity at concentrations up to 11.2mg/ml. The extract of A. senegalensis showed significant (P<0.001) reduction in egg hatch at a concentration of 7.1mg/ml. The in vitro fecal culture of eggs with A. senegalensis showed significant (P<0.001) reductions in larval recovery with increasing concentrations from 1 to 10% ((w/w) in fecal culture) when whole ground plant material was used. Although traditional veterinary healers and local livestock farmers claim both plants are effective as anthelmintics, our result indicate that only A. senegalensis showed promising anthelmintic activity especially with use of ground whole plant materials as used by some farmers.

Analysis and partial reversal of multidrug resistance to anthelmintics due to P-glycoprotein in Haemonchus contortus eggs using Lens culinaris lectin.

Our previous work has shown that drug efflux pumps close to MDR1 P-glycoprotein (Pgp) can regulate anthelmintic efflux in nematodes in a way similar to that of the mutidrug resistance system (MDR) in vertebrate cancer cells. In the present study, the role of the glycosylation of Pgp was studied using a lectin specific for the alpha-mannosyl residues ( Lens culinaris agglutinin, LCA). Highly significant reversion (up to 50%) in the resistance to thiabendazole of eggs pre-treated with the lectin was obtained. Flow cytometric examinations were performed using FITC-labelled lectin. The results demonstrated that: (1) the number of Pgp sites was higher in resistant H aemonchus contortus, (2) resistance can also be associated with a decreased affinity of LCA for these sites, (3) eggs stained with LCA were also stained with specific MDR1 monoclonal antibodies. The implication of the glycosylation of Pgp in the activity and/or degradation of these pumps in eukaryotic cells is discussed.

Haemonchus contortus (Nematoda: Trichostrongylidae) is much more sensitive than Caenorhabditis elegans (Nematoda: Rhabditidae) to the ovicidal action of thiabendazole.

When eggs of the trichostrongylid nematode Haemonchus contortus were exposed to thiabendazole, the concentration required to prevent hatching in 90% of the eggs (MIC90) was found to be 0.1 microg/ml (using 1% dimethylsulfoxide [DMSO] as solvent). In contrast, eggs of the free-living rhabditid nematode Caenorhabditis elegans hatched at normal rates at a concentration 200 times higher, i.e., 20 microg/ml, and showed only a partial inhibitory effect at a concentration 1,200 times higher, i.e., 120 microg/ml (in 3% DMSO). Because solubility limitations precluded the testing of higher concentrations of thiabendazole, a more soluble derivative, 5-([1-methylethoxy]carbonylamino)-2-(4-thiazloyl)1H-++ +benzimidazolyliminoacetic acid N,N-diethylethanamine salt, was tested against C. elegans eggs. The MIC90 was found to be 400 microg/ml, and although the derivative was not tested against H. contortus eggs, this finding further suggests that C. elegans eggs have an exceptionally low degree of benzimidazole sensitivity.

Development and survival of infective larvae of nematode parasites of sheep on pasture in a cool tropical environment

The development and survival of nematode parasites of sheep were studied in a cool tropical environment of the highlands of Ethiopia on 24 plots serially contaminated with Haemonchus contortus, Longistrongylus elongata and Trichostrongylus colubriformis eggs over a period of 2 consecutive years from June 1993 to May 1995. The availability of infective larvae was monitored by monthly pasture sampling and larvae recovery. Infective larvae were recovered from the herbage collected from the plots contaminated in June, July, August, September and October of both years. The longevity of infective larvae varied between two and six weeks when eggs were deposited on pasture in June and October, respectively. During the dry and short rainy season (November through May), eggs failed to develop into L 3 stage. It is suggested that the long period (7 to 8 months) of lack of development of trichostrongylid infective larvae on the pasture can be efficiently used in a strategic treatment programme to interrupt transmission of H. contortus, L. elongata and T. colubriformis in sheep in this environment.

Inhibitory Effect of Chlorpromazine on Nematode Eggs and Larvae

Chlorpromazine inhibited the hatching of eggs of the parasitic nematode Haemonchus contortus and the free-living nematode Caenorhabditis elegans. In both species, hatching occurred at a concentration of 100 microg/ml but was almost totally blocked at 400 microg/ml. In the case of C. elegans, the effect was shown to be reversible by removal of chlorpromazine after exposure of the eggs to the drug for 1 hr. Caenorhabditis elegans larvae that hatched in a chlorpromazine concentration of 100 microg/ml were killed, but those that hatched in a concentration of 6.25 microg/ml were not. Taken together with data published by others, these observations indicate that the first-stage larva of C. elegans is less sensitive to chlorpromazine than is the adult worm.

Rotational grazing for control of gastrointestinal nematodes of goats in a wet tropical environment

A preliminary experiment involving contamination of pasture plots with eggs of Haemonchus contortus, Trichostrongylus colubriformis and Oesophagostomum columbianum every month for a year established that in the tropical environment of the Pacific island of Tongatapu, hatching and development of all species was rapid and continuous, with a short survival on pasture (3–7 weeks) of the resulting infective larvae. These results indicated that a rotational grazing system consisting of ten paddocks grazed in sequence for 3.5 days at a time may permit a reduction in the frequency of anthelmintic treatment of goats. In comparison with an adjacent set-stocked flock which required treatment on three occasions during the year when mean flock egg counts exceeded 2000 eggs per gram (EPG), rotationally grazed goats generally maintained mean egg counts of less than 1000 EPG. Anthelmintic treatment was only given to rotationally grazed goats individually as they kidded, and there were indications that even this precaution was unnecessary. Because of the expense of frequent anthelmintic treatment and the resulting selection of strains of anthelmintic-resistant nematodes, rotational grazing of small ruminants through fencing, tethering or herding deserves further investigation as a nematode control option in wet tropical environments.

Characterization and biological activities of anti- Brugia pahangi tubulin monoclonal antibodies

Three monoclonal antibodies (mAb) specific to β-tubulin were used to investigate the heterogeneity of tubulins from nematodes and mammals. Western blot analysis of one-dimensional SDS-PAGE showed that anti- Brugia pahangi tubulin mAb 1B6 and P3D react with epitope(s) specific to nematode β-tubulin and recognize tubulin from adults and microfilariae of B. pahangi, adult B. malayi and Dirofilaria immitis, eggs of Haemonchus contortus and adult Ascaris suum. However, the same mAb did not recognize tubulin from trophozoites of Giardia lamblia, pig brain or 3T3 mouse fibroblast cells. In two-dimensional SDS-PAGE, mAb 1B6 recognized one isoform of β-tubulin and mAb P3D recognized two β-tubulin isoforms. Limited proteolysis showed that mAb l B6 reacted with the amino-terminal fragments of β-tubulin. In contrast, mAb P3D recognized the carboxy-terminal fragments of β-tubulin. In ELISA, mAb P3D reacted with an 18 amino acid peptide corresponding to residues 430–448 of B. pahangi β-tubulin. These observations confirm that the epitope of mAb P3D is located on the extreme carboxy-terminal region. Immunogold labelling of adult B. pahangi sections with mAb P3D revealed the presence of β-tubulin isoforms in the cuticle, hypodermal layer and somatic muscle blocks of B. pahangi. Under in vitro conditions, mAb P3D caused 80% reduction in worm viability, during exposure over 48 h.

The development and mortality of the free-living stages of Haemonchus contortus in laboratory culture

Coyne M. J. and Smith G. 1992. The development and mortality of the free-living stages of Haemonchus contortus in laboratory culture. International Journal for Parasitology 22: 641–650. Haemonchus contortus eggs were cultured in intact fecal pellets at various temperatures (5–35°C) for 22 days. Temperature and relative humidity were kept constant throughout the incubation period. No larval development occurred at 5°C; peak third-stage larval recovery occurred at 20°C. Egg mortality was an agedependent phenomenon, whereas larval mortality remained constant irrespective of larval age. Development was characterized by a minimum development time followed by a transition to the next stage which occurred at a constant rate. All rates were temperature dependent. The minimum development times reported here are much less than those previously reported. Based on these results a mathematical model was used to describe the demography of the free-living stages of H. contortus at various temperatures.

A microlarval development assay for the detection of anthelmintic resistance in sheep nematodes.

A microlarval development test for the detection of anthelmintic resistance in nematodes is described. Haemonchus contortus, Teladorsagia circumcincta and Trichostrongylus colubriformis eggs were cultured to third stage larvae in the presence of Earle's balanced salt solution, yeast extract and bacteria in a total volume of 150 microliters. Good dose-response data were obtained with thiabendazole, levamisole, pyrantel tartrate and ivermectin allowing the determination of the 50 per cent lethal concentration and of resistance factors when resistant strains were available. The test was found to be accurate, sensitive, easy to carry out and applicable to the routine detection of resistance.