Screen for anthelmintics, using larvae of Ascaris suum

A multiwell culture system was used to assay the effects of 12 known anthelmintic compounds on Ascaris suum larval development from 2nd-stage (L2; hatched from eggs) to early 3rd-stage (L3) and from in vivo-derived late L3 to early 4th-stage (L4). Larval survival, development, and motility were monitored for drug effects. Development of L2 to L3 was sensitive to thiabendazole, albendazole (ABZ), ABZ/sulfoxide, ABZ/sulfone (SO), mebendazole, L-tetramisole, D-tetramisole, piperazine, or closantel at a concentration of 0.01 microgram/ml; however, the effects of these drugs on larval development did not correlate well with known effects in vivo. The development of L3 to L4 was blocked by ABZ or mebendazole at 0.01 microgram/ml, by thiabendazole or ABZ/sulfoxide at 0.1 microgram/ml, and by ABZ/SO at 1.0 microgram/ml; however, except for ABZ/SO, most larvae were viable at these concentrations. In contrast, L-tetramisole or morantel appeared to inhibit development of L3 to L4 and to reduce survival at concentrations of greater than or equal to 1 microgram/ml; however, D-tetramisole was at least 10 times less effective. Haloxon, ivermectin, and closantel blocked development of L3 to L4 at 0.1, 1, and 10 micrograms/ml, respectively, in the absence of serum, but their activity was reduced by the presence of serum. Seemingly, in vitro development of A suum larvae was a convenient and sensitive bioassay for anthelmintic activity and could serve as a screen for anthelmintic residues in edible tissues.     

Effects of 8-epi-PGF2α on isolated bronchial smooth muscle of healthy and heaves-affected horses

8-Epi-PGF2alpha, a prostaglandin-like compound generated by oxidative stress, has been shown to be an in vitro bronchoconstrictor in airways from healthy laboratory animals and healthy humans, but it has never been studied in diseased airways. Here, the bronchoconstrictive capacity of 8-epi-PGF2alpha on isolated bronchial rings (BR) of healthy and heaves-affected horses was evaluated by comparing the maximal effect and the potency of 8-epi-PGF2alpha to those of (1) acetylcholine (ACh), (2) its stereoisomer PGF2alpha and (3) its synthetic receptor agonist, U46619. Furthermore, the potential capacity of 8-epi-PGF2alpha to enhance the cholinergic (ACh) responsiveness of bronchial smooth muscle was investigated. 8-Epi-PGF2alpha contracted BR with a rank order of efficacy of Ach > U44619 > PGF2alpha > 8-epi-PGF2alpha in both healthy and heaves-affected horses. The contractile maximal response elicited by 8-epi-PGF2alpha was significantly smaller than that elicited by the other drugs, but was significantly higher in BR from heaves-affected horses than in those sampled in healthy horses, whilst pD2 values were similar. A subthreshold concentration of 8-epi-PGF2alpha (10-7 M) did not induce in vitro cholinergic hyper-responsiveness in BR of either healthy or heaves-affected horses. In conclusion, it has been demonstrated that 8-epi-PGF2alpha is an in vitro bronchoconstrictor of minor importance in healthy horses, but whose efficacy is significantly increased in heaves-affected horses.     

Molecular mechanisms for anthelmintic resistance in strongyle nematode parasites of veterinary importance

Veterinarians rely on a relatively limited spectrum of anthelmintic agents to control nematode parasites in domestic animals. Unfortunately, anthelmintic resistance has been an emerging problem in veterinary medicine. In particular, resistance has emerged among the strongyles, a group of gastrointestinal nematodes that infect a variety of hosts that range from large herbivores to small companion animals. Over the last several decades, a great deal of research effort has been directed toward developing an understanding of the mechanisms conferring resistance against the three major groups of anthelmintics: macrocyclic lactones, benzimidazoles, and nicotinic agonists. Our understanding of anthelmintic resistance has been largely formed by determining the mechanism of action for each drug class and then evaluating drug‐resistant nematode isolates for mutations or differences in expression of target genes. More recently, drug efflux pumps have been recognized for their potential contribution to anthelmintic resistance. In this mini‐review, we summarize the evidence for mechanisms of resistance in strongyle nematodes.     

Mechanisms of acetylcholine-induced vasorelaxation in high K+-stimulated rabbit renal arteries

To characterize the mechanisms of acetylcholine (ACh)-induced vasorelaxation in rabbit renal arteries precontracted with high K+ (100 mM), muscle tension and cytosolic free Ca2+ concentration ([Ca2+]i) were measured simultaneously in the fura-2-loaded arterial strips. In the artery with endothelium, high K+ increased both [Ca2+]i and muscle tension. Addition of ACh (10 microM) during high-K+ induced contraction significantly relaxed the muscle and induced additional increase in [Ca2+]i. In the presence of NG-nitro-L-arginine (L-NAME, 0.1 mM). ACh increased [Ca2+]i without relaxing the muscle. In the artery without endothelium, high K+ increased both [Ca2+]i and muscle tension although ACh was ineffective, suggesting that ACh acts selectively on endothelium to increase [Ca2+]i. 4-DAMP (10 nM) or atropine (0.1 microM) abolished the ACh-induced increase in [Ca2+]i and relaxation. However, pirenzepine (0.1 microM), AF-DX 116 (1 microM) and tropicamide (1 microM) were ineffective. The ACh-induced increase of [Ca2+li and vasorelaxation was significantly reduced by 3 microM gadolinium, 10 microM lanthanum or 10 microM SKF 96365. These results suggest that, in rabbit renal artery, ACh-evoked relaxation of 100 mM K+-induced contractions is mediated by the release of endothelial NO. ACh may stimulates the M3 subtype of muscarinic receptor in the endothelial cells, resulting in the opening of the nonselective cation channels followed by an increase of [Ca2+]i and stimulation of NO synthase.     

Efficacy of flubendazole against gastrointestinal and lung nematodes in pigs

Three trials were carried out on landrace pigs of various ages to assess the anthelmintic efficacy of flubendazole. The pigs were either artificially infected with Metastrongylus apri or naturally or artificially infected with the gastrointestinal nematodes Ascaris suum, Oesophagostomum dentatum or Hyostrongylus rubidus. For mass medication of young pigs and fatteners a dose regimen of 30 ppm flubendazole in the feed for 10 consecutive days was 100 per cent effective against the four nematode species. For individual medication a single dose of 5 mg/kg bodyweight administered in a small amount of feed was also 100 per cent effective. No side effects were observed.     

Caenorhabditis elegans as a model to screen plant extracts and compounds as natural anthelmintics for veterinary use

The most challenging obstacles to testing products for their anthelmintic activity are: (1) establishing a suitable nematode in vitro assay that can evaluate potential product use against a parasitic nematode of interest and (2) preparation of extracts that can be redissolved in solvents that are miscible in the test medium and are at concentrations well tolerated by the nematode system used for screening. The use of parasitic nematodes as a screening system is hindered by the difficulty of keeping them alive for long periods outside their host and by the need to keep infected animals as sources of eggs or adults when needed. This method uses the free-living soil nematode Caenorhabditis elegans as a system to screen products for their potential anthelmintic effect against small ruminant gastrointestinal nematodes, including Haemonchus contortus. This modified method uses only liquid axenic medium, instead of agar plates inoculated with Escherichia coli, and two selective sieves to obtain adult nematodes. During screening, the use of either balanced salt solution (M-9) or distilled water resulted in averages of 99.7 (± 0.73)% and 96.36 (± 2.37)% motile adults, respectively. Adult worms tolerated DMSO, ethanol, methanol, and Tween 80 at 1% and 2%, while Labrasol (a bioenhancer with low toxicity to mammals) and Tween 20 were toxic to C. elegans at 1% and were avoided as solvents. The high availability, ease of culture, and rapid proliferation of C. elegans make it a useful screening system to test plant extracts and other phytochemical compounds to investigate their potential anthelmintic activity against parasitic nematodes.     

Pharmacological characterization of muscarinic receptors in the contractions of isolated bronchi in the horse

We investigated the effects of nonselective muscarinic antagonist (atropine) and of selective muscarinic subtype 1 (M₁), 2 (M₂), 3 (M₃) antagonists (VU0255035, methoctramine, pFHHSiD, respectively) on the contractions evoked by electrical field stimulation (EFS) or by exogenous ACh in isolated horse bronchial muscle. Atropine completely inhibited neurogenic contractions in a concentration‐dependent fashion, whereas selective muscarinic antagonists induced relevant modifications only at the highest concentration tested. Experiments with selective muscarinic antagonists in combination showed that only the simultaneous blockade of M₁/M₃ or M₂/M₃ receptors was able to induce a nearly complete suppression of contractions. The contractions induced by exogenous ACh were competitively antagonized only by atropine (pA₂ = 9.01 ± 0.05). M₃ selective antagonist, up to 10⁻⁶ m , caused a moderate concentration‐dependent rightward shift of ACh curve (pA₂ = 7.96 ± 0.10). These data show that M₃ muscarinic receptors possess a central role in mediating cholinergic contraction of horse bronchi, while M₁ and M₂ receptors seem to have a cooperative role. Selective muscarinic antagonists seem unlikely to be useful against bronchoconstriction associated with airway diseases in horses. Conversely, compounds with selectivity for both M₁ and M₃ receptors could be as effective as traditional anticholinergics and induce fewer cardiac side effects.     

Muscarinic receptor subtypes in equine tracheal smooth muscle

Selective muscarinic receptor antagonists were used to identify muscarinic receptor subtypes in equine trachealis strips. The M₁ receptor antagonist pirenzepine (10^–7 mol/L to 3 × 10^–5 mol/L) and the M₃ receptor antagonist 4-diphenylacetoxy-N-methylpiperidine (4-DAMP, 10^–9 mol/L to 3 × 10^–7 mol/L³) dose dependently inhibited the contractile responses to electrical field stimulation (EFS) and exogenous acetylcholine (ACh). Schild plots yielded a pA₂ value for pirenzepine vs ACh of 6.75 ± 0.09, which is consistent with the affinity for M₂ or M₃ receptors, and a pA₂ value for 4-DAMP vs ACh of 8.47 ± 0.09, which is in agreement with the affinity for M₃ receptors. The M₂ receptor antagonist gallamine (10^–5 mol/L and 10^–4 mol/L) did not affect the response of trachealis to exogenous ACh and low-frequency EFS (0.1–2 Hz) but decreased the responses to high-frequency EFS (4–16 Hz). These results suggest that the muscarinic receptors mediating contractions induced by ACh in equine tracheal smooth muscle are of the M₃ subtype. The lack of an increase in the response to EFS following gallamine suggests that functional prejunctional inhibitory M₂ receptors are not present on the cholinergic nerves innervating equine tracheal smooth muscle.     

Influence of helminth parasite exposure and strategic application of anthelmintics on the development of immunity and growth of swine

Infection of pigs with the intestinal roundworm parasite Ascaris suum and strategic application of anthelmintic drugs during the growing phase of development were observed for specific effects on 1) development of immunity in feeder pigs and 2) growth rate during the finishing phase. Management treatments included maintenance in a parasite-free concrete environment, maintenance in a concrete environment and inoculation with 1,000 infective A. suum eggs every other day over a 52-d period, and maintenance on a dirtlot contaminated with A. suum and Trichuris suis eggs. Within each management environment, pigs were either untreated, treated with ivermectin or treated with fenbenzadole at strategic times during parasite exposure. Protective immunity, assessed by a challenge inoculation with A. suum eggs following management treatments, was not affected by ivermectin or fenbenzadole treatment during exposure, but adult worm burdens were reduced and the pattern of A. suum larval antigen serum antibody responses were different from those in control pigs not treated with drugs. Exposure to A. suum and treatment with anthelmintics during the growing phase reduced adult worm burdens following the finishing phase of growth. Rate, but not efficiency, of gain was significantly improved by anthelmintic treatment following natural exposure to parasites. Strategic treatment of pigs with anthelmintics following inoculation with A. suum eggs in a concrete management environment had no effect on rate of gain. Results suggest that natural exposure to parasites during the growing phase without therapeutic treatment causes permanent damage to growth potential.     

Benzodiazepines inhibit the acetylcholine receptor-operated potassium current (IK.ACh) by different mechanisms in guinea-pig atrial myocytes

The anticholinergic effects of 7 benzodiazepines, bromazepam, camazepam, chlordiazepoxide, diazepam, lorazepam, medazepam and triazolam, were compared by examining their inhibitory effects on the acetylcholine receptor-operated potassium current (I(K).(ACh)) in guinea-pig atrial myocytes. All of these benzodiazepines (0.3-300 μM) inhibited carbachol (1 μM)-induced I(K).(ACh) in a concentration-dependent manner. The ascending order of IC(50) values for carbachol-induced I(K).(ACh) was as follows; medazepam, diazepam, camazepam, triazolam, bromazepam, lorazepam and chlordiazepoxide (>300 μM). The compounds, except for bromazepam, also inhibited I(K).(ACh) activated by an intracellular loading of 100 μM guanosine 5'-[γ-thio]triphosphate (GTPγS) in a concentration-dependent manner. The ascending order of IC(50) values for GTPγS-activated I(K).(ACh) was as follows; medazepam, diazepam, camazepam, lorazepam, triazolam chlordiazepoxide (>300 μM) and bromazepam (>300 μM). To clarify the molecular mechanism of the inhibition, IC(50) ratio, the ratio of IC(50) for GTPγS-activated I(K).(ACh) to carbachol-induced I(K).(ACh), was calculated. The IC(50) ratio for camazepam, diazepam, lorazepam, medazepam and triazolam was close to unity, while it for chlordiazepoxide could not be calculated. These compounds would act on the GTP binding protein and/or potassium channel to achieve the anticholinergic effects in atrial myocytes. In contrast, since the IC(50) ratio for bromazepam is presumably much higher than unity judging from the IC(50) values (104.0 ± 30.0 μM for carbachol-induced I(K).(ACh) and >300 μM for GTPγS-activated I(K).(ACh), it would act on the muscarinic receptor. In summary, benzodiazepines had the anticholinergic effects on atrial myocytes through inhibiting I(K).(ACh) by different molecular mechanisms.     

5-Hydroxytryptamine potentiates contraction mediated by the intramural cholinergic nerve in the longitudinal smooth muscle of the ruminant forestomach

The effects of 5-hydroxytryptamine (5-HT) on the longitudinal smooth muscle from the rumen and reticulum of the bovine forestomach were investigated. 5-HT (0.25–490 μM) caused a contraction and a relaxation of the ruminal strips while it produced only an excitatory effect on the reticular strips. These effects were not affected by tetrodotoxin, hexamethonium, atropine or morphine, but were blocked by methysergide, LSD-25 or phenoxybenzamine. 5-HT potentiated the contraction evoked by stimulation of the intramural cholinergic nerves but did not show any effect on the relaxation produced by the non-adrenergic inhibitory nerves' excitation. The 5-HT-induced potentiation was not affected by morphine, LSD-25, methysergide and hexamethonium or high concentration of nicotine. Nicotine and dimethylphenylpiperazinium also caused a transient augmentation of the nerve-mediated contraction, but these effects were abolished by the competitive ganglionic blockers. The evoked contraction was depressed in high-Mg²⁺ solution, but this depression was antagonized partly by 5-HT. The affinity of the cholinomimetics to post-synaptic muscarinic receptor was not affected by 5-HT. It is concluded that contractions or relaxations of bovine forestomach strips induced by 5-HT are mediated through activation of D-receptors in the smooth muscle, and the 5-HT-induced potentiation of the evoked contraction may be elicited through presynaptic neural effects of 5-HT on the cholinergic nerves.     

Estrous cycle-dependent differences in responsiveness to prostaglandins and contractile agents in sheep (Ovis aries) cervical smooth muscle

We investigated the influence of the phase of the estrous cycle on mechanical responses elicited in sheep cervix by potassium chloride (KCl), acetylcholine chloride (ACh), prostaglandin F2 alpha (PGF2 alpha) and prostaglandin E1 (PGE1). The cervix of adult ewes (n = 48) were classified according to the presence or absence of corpora lutea (luteal or follicular phase, respectively). Muscle strips of the circular and longitudinal layers were prepared in an organ bath and coupled to an isometric force transducer. Concentration-response curves were obtained noncumulatively. KCl and ACh produced concentration-dependent contractions in all preparations in both phases of the estrous cycle. However, maximum effect, EC50 and slope values of KCl and ACh were not significantly different between muscle layers, as well as between the phases of the estrous cycle. The prostanoid, PGF2 alpha, produced a significant reduction in the amplitude of spontaneous contractions for all preparations. The depressant effect of PGF2 alpha on spontaneous contractions of circular smooth muscle was significantly greater during the follicular than the luteal phase, whilst the depressant effect of PGF2 alpha on the longitudinal layer did not differ between phases of the estrous cycle. PGE1 significantly reduced the amplitude of spontaneous contractions on circular but not on longitudinal preparations. In conclusion, we have characterized with in vitro preparations of circular and longitudinal muscle layers of ewes during the follicular and luteal phases of the estrous cycle, the parameters of the K- and ACh-induced contractions on cervix and the efficacy of PGF2 alpha and PGE1 on inhibition spontaneous contractile activity.     

Interrelationships among physicochemical properties, absorption and anthelmintic activities of 2-desoxoparaherquamide and selected analogs

The interrelationships between physicochemical properties, absorption and potency of 2-desoxoparaherquamide and five analogs, representing a new anthelmintic class, were evaluated in in vitro and in vivo assays. At pH 7.5, rates of drug absorption by the gastrointestinal nematode Haemonchus contortus and jird small intestine, parameterized by the permeability coefficient, P(e), ranged from 1.2-2.4 x 10(-4) cm/min (nematode) to 2.5-5.5 x 10(-3) cm/min (jird). In the jird intestine, absorption was pH-dependent, with P(e) at pH 7.5 being twice that at pH 4.5, reflecting the negative influence of protonation on transport of these weakly basic molecules. Each compound rapidly paralyzed H. contortus during in vitro exposure to therapeutically relevant concentrations (1-10 microm). The kinetics of drug action on motility in vivo mirrored their in vitro effects; motility concentrations were reduced in nematodes collected from jird stomach 3 h following oral drug dosing, by which time > or =50% clearance of the parasites had occurred. The nematode/medium partition coefficient K ranged from 10.1 to 16.1, consistent with the lipophilic nature of the compounds. The time required to reduce motility in vitro by 50% (t50*) and P(e) were used to determine C(n)*, the concentration of drug in the nematode at t50*, as an indicator of intrinsic potency. In the jird, the apparent potencies of the compounds were insensitive to route of administration (i.e. oral = i.v. = i.p. = i.m.) for H. contortus and two other gastrointestinal nematodes, Ostertagia ostertagi and Trichostrongylus colubriformis; topical administration, however, required three to 10-fold higher doses for equivalent efficacy.     

Target-based and whole-worm screening approaches to anthelmintic discovery

Experimental approaches for identifying new anthelmintics include target-based and whole-worm screening methods. The former involves basic research into characterising and validating new targets, mostly proteins, followed by identification of inhibitors or agonists through the use of target-based screening assays and/or in silico drug design. The latter experimental approach uses whole-worm assays to identify anthelmintic agents with unknown modes of action, or where the primary interest lies in whether analogues are able to kill (or disable) worms rather than in measuring their direct impact on their likely target. This paper focuses initially on the intestine and external layers of nematodes as potential drug targets. Specific anthelmintic agents targeting either tissue are discussed to illustrate the impact of disruption to these structures. In both cases, the activity of these agents against insects was known, and activity against nematodes was identified using whole worm screening assays. Recent literature identifying ecdysone signalling pathway receptors in nematodes is then used to provide an example of basic research into a specific target that may lead to the development of high-throughput target-based drug screening assays. Finally, the role of whole-worm screening approaches versus target-based screening is discussed briefly.     

Pharmacological characterization of the Haemonchus contortus GABA-gated chloride channel, Hco-UNC-49: modulation by macrocyclic lactone anthelmintics and a receptor for piperazine

Invertebrate ligand-gated chloride channels are well recognized as important targets for several insecticides and anthelmintics. Hco-UNC-49 is a GABA-gated chloride channel from the parasitic nematode Haemonchus contortus and is an orthologue to the neuromuscular receptor (Cel-UNC-49) from the free-living nematode Caenorhabditis elegans. While the receptors from the two nematodes are similar in sequence, they exhibit different sensitivities to GABA which may reflect differences in in vivo function. The aim of the current study was to further characterize the pharmacology of the Hco-UNC-49 receptor by examining its sensitivity to various insecticides and anthelmintics using two-electrode voltage clamp. Specifically, the insecticides fipronil and picrotoxin appear to inhibit the channel in a similar manner. The IC(50) of picrotoxin on the homomeric channel was 3.65 ± 0.64 μM and for the heteromeric channel was 134.56 ± 44.12 μM. On the other hand, dieldrin, a well-known insect GABA receptor blocker, had little effect on the UNC-49 channel. The anthelmintics ivermectin and moxidectin both moderately potentiated the activation of Hco-UNC-49 by GABA, while piperazine was able to directly activate both the Hco-UNC-49 homomeric and heteromeric channels with EC(50) values of 6.23 ± 0.45 mM and 5.09 ± 0.32 mM, respectively. This piperazine current was reversibly blocked by picrotoxin which demonstrates that the anthelmintic specifically targets Hco-UNC-49. These results demonstrate that Hco-UNC-49 exhibits binding sites for several molecules including piperazine and macrocyclic lactone anthelmintics. In addition, this is the first report of the heterologous expression and subsequent characterization of a receptor for piperazine.     

Prevalence of common gastrointestinal nematode parasites in scavenging pigs of different ages and sexes in eastern centre province, Burkina Faso

The range and infestation intensities of gastrointestinal parasitic nematode species depend on the type of swine production system. The present study focused mainly on nematodes of veterinary importance in scavenging pigs in Burkina Faso, and aimed at determining the prevalence of gastro-intestinal nematode parasites by means of faecal egg per gram (EPG) counts. Between November 2001 and October 2002, faecal samples from 383 pigs of different sexes and ages (< 5 months, 5-12 months and > 12 months) were collected from the rectum and examined for gastrointestinal nematodes parasites using the Mc Master method. Of the 383 pigs examined, 91% were infected by one or more parasites. Ascaris suum (40%; 100-1 400 EPG) was the most prevalent parasite followed by Strongyloides ransomi (21%; 100-4200 EPG), Oesophagostomum spp. (18%; 100-1000 EPG), Hyostrongylus rubidus (11%; 100-1 800 EPG), Globocephalus spp. (10%; 100-400 EPG) and Trichuris suis (1 %; 100-200 EPG). The prevalence was significantly higher in female pigs (n = 239) than in males. In addition, females excreted significantly (P < 0.05) more eggs in their faeces than males, except in the case of Globocephalus spp. The age of the animal had no effect on the prevalence of A. suum whereas there were significant differences in age categories concerning S. ransomi, H. rubidus, Oesophagostumum spp. and Globocephalus spp. Unexpectedly, the high prevalence of these common parasites was not accompanied by elevated EPG values, which suggests the existence of moderate infestations. The present work indicates that the common nematode infestations in pigs do not necessarily need a systematic herd anthelmintic treatment, as only a small number of worms is required to induce immunity. A further study is needed to formulate appropriate and cost-effective strategies for the control of gastro-intestinal nematode parasites in pigs in Burkina Faso.     

Slowing the spread of anthelmintic resistant nematodes of sheep and goats in the United Kingdom.

Anthelmintic resistant nematodes pose an increasing threat to animal welfare and lamb production on lowland sheep farms in the United Kingdom. Based on published information on anthelmintic resistance in nematodes and insecticide resistance in arthropods, seven recommendations are made for reducing the development and spread of anthelmintic resistant nematodes. The two most important are probably to prevent the introduction of resistant worms through the purchase of stock and to reduce the reliance on frequent anthelmintic treatments by using epidemiological principles of nematode control.     

Effects of Three Anthelmintic Treatment Regimes against Fasciola and Nematodes on the Performance of Ewes and Lambs on Pasture in the Highlands of Kenya

The efficacy of strategic anthelmintic control of liver flukes (Fasciola) and gastrointestinal (GI) nematodes on the performance of ewes and lambs on pasture was assessed on a farm in the highlands of Kenya. In May 1999, 45 Corriedale ewes, aged between 2 and 3 years, were ear-tagged, weighed and allocated randomly to three equal treatment groups based on body weight. Faecal samples taken at this time revealed low levels of strongyle-type eggs per gram of faeces (EPG) and the presence of liver fluke eggs in only a few of the animals. All the animals were then set stocked for 12 months on separate paddocks in an area endemic for both Fasciola and GI nematodes. The ewes in group 1 were given a combined anthelmintic treatment against Fasciola and GI nematodes during the periods recommended for the control of Fasciola in the area (February, June and October). The ewes in group 2 were given the combined treatments 3 weeks after the onset of both the short and long rainy seasons (November and April, respectively). Those in group 3 were given separate treatments for Fasciola (February, June and October) and nematodes (3 weeks after the onset of the rainy seasons). The anthelmintic treatment against Fasciola consisted of triclabendazole at 10 mg per kg body weight, and that against nematodes was levamisole at 10 mg per kg body weight.The nematode EPG for the ewes in group 1 were higher than in groups 2 and 3 during both rainy seasons. The nematode EPG did not differ significantly between groups 2 and 3. The prevalence of Fasciola eggs (number of ewes shedding eggs in a group) in the ewes in groups 1 and 3 remained very low throughout the study period compared to those in group 2. The highest birth weights and the weight gains of lambs were recorded for the group of ewes given separate anthelmintic treatments for Fasciola and nematodes (group 3). The results of this trial indicated that, in an area like Nyandarua District, where liver flukes and GI nematodes are important constraints to sheep production, the best practice is to give separate treatments for the two groups of parasites at the recommended times.     

Cloning and structural analysis of partial acetylcholine receptor subunit genes from the parasitic nematode Teladorsagia circumcincta

Nematode nicotinic acetylcholine receptors (nAChRs) are the sites of action for the anthelmintic drug levamisole. Recent findings indicate that the molecular mechanism of levamisole resistance may involve changes in the number and/or functions of target nAChRs. Accordingly, we have used an RT-PCR approach to isolate and characterise partial cDNA clones (tca-1 and tca-2) encoding putative nAChR subunits from the economically important trichostrongyloid, Teladorsagia circumcincta. The predicted tca-1 gene product is a 248 aa fragment (TCA-1) which contains structural motifs typical of ligand-binding (alpha-) subunits, and which shows very high sequence similarities (98.8 and 97.2% amino acid identities) to the alpha-subunits encoded by tar-1 and hca-1 from Trichostrongylus colubriformis and Haemonchus contortus, respectively. Sequence analyses of partial tca-1 cDNAs from one levamisole-resistant and two susceptible populations of T. circumcincta revealed polymorphism at the predicted amino acid level, but there was no apparent association of any particular tca-1 allele with resistance. tca-2 encodes a 67 aa fragment (TCA-2) containing the TM4 transmembrane domain and carboxyl terminus of a putative nAChR structural (non-alpha) subunit. The deduced amino acid sequence of TCA-2 shows highest similarity (75% amino acid identity) to ACR-2, a structural subunit involved in forming levamisole-gated ion channels in Caenorhabditis elegans, but low similarity (43% identity) to the corresponding regions of TAR-1 and HCA-1. tca-2 is the first nAChR subunit gene of this type to be isolated from parasitic nematodes, and it provides a basis for further characterisation of structural subunits in trichostrongyloids.     

Identification of a collectin-like protein in pig serum that binds a component in perienteric fluid from Ascaris suum

A collectin-like protein (CLP) of the acute phase protein family that binds the polysaccharides mannan and alpha-1-6 dextran was isolated from the serum of pigs infected with Ascaris suum. A monoclonal antibody generated against this protein and used to characterize the CLP revealed on SDS-PAGE and western blot analysis that the protein had a molecular weight of approximately 48 kDa under reducing conditions and greater than 100 kDa under nonreducing conditions. Enzyme-linked immunosorbent assay (ELISA) showed that the CLP bound to substances in the perienteric fluid of Ascaris suum (APF). Molecular weight fractionation of APF demonstrated that CLP binds primarily to APF substances of greater than 100 kDa. Binding of CLP to APF was partially blocked by phosphatidylinositol. This is the first report of a porcine CLP and the binding of a CLP to components of the common nematode Ascaris suum.