Geometric means provide a biased efficacy result when conducting a faecal egg count reduction test (FECRT).

The process of conducting a faecal egg count reduction test was simulated to examine whether arithmetic or geometric means offer the best estimate of efficacy in a situation where the true efficacy is known. Two components of sample variation were simulated: selecting hosts from the general population which was modelled by the negative binomial distribution (NBD), and taking an aliquot of faeces from the selected host to estimate the worm egg count by assuming a Poisson distribution of sample counts. Geometric mean counts were determined by adding a constant (C) to each count prior to log transformation, C was set at 25, 12 or 1. Ten thousand Monte Carlo simulations were run to estimate mean efficacy, the 2.5% (lower) and the 97.5% (upper) percentile based on arithmetic or geometric means. Arithmetic means best estimated efficacy for all different levels of worm aggregation. For moderate levels of aggregation and with C=1 the geometric mean substantially overestimated efficacy. The bias was reduced if C was increased to 25 but the results were no better than those based on arithmetic means. For very high levels of aggregation (over-dispersed populations) the geometric mean underestimated efficacy regardless of the size of C. It is recommended that the guidelines on anthelmintic resistance be revised to advocate the use of arithmetic means to estimate efficacy.

Efficacy of a morantel-abamectin combination for the treatment of resistant ascarids in foals.

BACKGROUND: This study was performed to investigate the efficacy of a morantel-abamectin combination for the treatment of macrocyclic lactone (ML)-resistant Parascaris spp. infections in foals. METHODS: Foals on five properties with a Parascaris faecal egg count (FEC) > 50 eggs per gram were used to estimate the FEC reduction (FECR) and efficacy of the anthelmintic combination. RESULTS & CONCLUSION: On all properties, resistance to ivermectin and abamectin was present and the Parascaris FECR in foals administered the morantel-abamectin combination was > 99%, indicating that this combination effectively controlled ML-resistant parasites.

Cloning and sequence analysis of the candidate nicotinic acetylcholine receptor alpha subunit gene tar-1 from Trichostrongylus colubriformis.

A T. colubriformis genomic library in lambda EMBL3 was screened for sequences homologous to the Caenorhabditis elegans unc-38 nicotinic acetylcholine receptor (nAChR) alpha-subunit gene. The candidate gene tar-1 (for Trichostrongylus acetylcholine receptor subunit gene 1) comprising 13704 base pairs was thus identified. BLAST comparison of the sequenced clone with GenBank, followed by comparison of translated regions in six reading frames with protein databases, identified clearly defined tracts corresponding to 12 putative exons sharing high sequence homology to other nAChR genes and able to code for sequential regions of a putative nAChR alpha-subunit protein (tar-1). Tar-1 shares sequence similarities with over 40 nAChR subunit proteins. The highest similarity (91.6%) is with unc-38, suggesting that nAChR sequences from nematodes are closely related. The sequence includes motifs typical of these molecules including adjacent cysteine residues at the ACh binding site and four transmembrane regions. The DNA sequence presents the longest genomic tract described for this organism and should prove useful as a probe source in the search for nAChR genes from this and other nematodes and for studying the molecular mechanism of resistance to levamisole, a drug which is known to act on nAChRs of worms and which is widely used for parasite control.

mle-1, a mariner-like transposable element in the nematode Trichostrongylus colubriformis.

A mariner-like element termed mle-1 was discovered in the parasitic nematode Trichostrongylus colubriformis. The mle-1 has features which support its assignment as a mariner-like transposable element. Cloned mle-1 was derived from an intron of the tar-1 gene. It comprises 893 bp, includes two 27 bp flanking perfect inverted repeats and is present at approximately 50 copies in the genome. The element contains a coding region which displays homology to transposases, with the greatest amino acid similarity to a Caenorhabditis elegans mariner-like transposase. The coding region contains two 12 bp repeats; these repeats flank an 11 bp segment which accounts for a frameshift in this region. As a candidate transposon, mle-1 provides potential for genetic manipulation of this and related organisms.

The contribution of a partial tricarboxylic acid cycle to volatile end-products in thiabendazole-resistant and susceptible Trichostrongylus colubriformis.

Acetate, propionate, ethanol and propanol were the predominant end-products released during incubation of a thiabendazole resistant and a susceptible strain of Trichostrongylus colubriformis. The parasites in all the incubations appeared to be deficient in reducing equivalents if the end-products arose from the classical catabolic pathway through fumarate reductase (EC 1.3.1.6). Possible alternative pathways for accounting for redox balance, including beta-oxidation, the pentose phosphate pathway and amino acid metabolism were investigated. Palmitate was oxidised aerobically. Radiolabelled tricarboxylic acid cycle intermediates, citrate and alpha-ketoglutarate, were decarboxylated to 14CO2 indicating that at least a partial tricarboxylic acid cycle to succinyl-CoA via alpha-ketoglutarate operates both anaerobically and aerobically in T. colubriformis. These data and the pattern of end-products suggest the presence of two pathways to propanol and propionate either through fumarate reduction or alpha-ketoglutarate oxidation. T. colubriformis may apportion carbon flow through these pathways to maintain a stable redox ratio. Similar calculations on previously reported data indicate that both pathways may also operate in Haemonchus contortus. Exposure of resistant T. colubriformis to thiabendazole under anaerobic conditions caused an increased accumulation of end-products, especially propanol, in the incubation medium. The alpha-ketoglutarate pathway may lower the dependence of the parasite on the fumarate reductase route which is sensitive to thiabendazole. The operation of the alpha-ketoglutarate pathway, with propanol as an end-product, may provide a mechanism for regulating redox balance in trichostrongylidae.

Ribonucleotide levels in six nematodes, a cestode and a trematode.

The levels of ribonucleotides in the nematodes Haemonchus contortus, Ostertagia circumcincta, Trichostrongylus colubriformis, Nippostrongylus brasiliensis, Ascaris suum and Stephanurus dentatus, the cestode Moniezia expansa and the trematode Fasciola hepatica were measured by high pressure liquid chromatography. The adenylate charge, ATP and total adenine nucleotide levels were in general agreement with those determined enzymatically and with published data. UDP and guanine nucleotide levels were found to be higher than those present in mammals and previously reported for helminth parasites. Cytidine nucleotide levels varied considerably between parasite species. On the other hand inosine nucleotides were present at low concentrations in all species. Adenylate and guanylate charges were similar.

Characterization of an acetylcholine receptor gene of Haemonchus contortus in relation to levamisole resistance.

The anthelminitic drug levamisole is thought to bind to nicotinic acetylcholine receptors of nematodes. It is possible that resistance to this drug is associated with either a change in binding characteristics or a reduction in the number of nicotinic acetylcholine receptors. Therefore, the molecular mechanism of levamisole resistance in the parasitic nematode Haemonchus contortus was studied by isolating and characterising cDNA clones encoding a putative ligand binding nicotinic acetylcholine receptor subunit, HCAl, of two susceptible and one levamisole resistant population. Hcal is related to unc-38, a nicotinic acetylcholine receptor subunit gene associated with levamisole resistance in Caenorhabditis elegans. Although extensive sequence analyses of hcal sequences revealed polymorphism at amino acid level, no association with levamisole resistance could be detected. Restriction fragment length polymorphism analyses confirmed that, although polymorphism was detected, no selection of a specific allele of hcal has taken place during selection for levamisole resistance in various levamisole resistant populations.

The nicotinic acetylcholine alpha-subunit gene tar-1 is located on the X chromosome but its coding sequence is not involved in levamisole resistance in an isolate of Trichostrongylus colubriformis.

The polymerase chain reaction was used to amplify fragments comprising the known reading frame of the nematode nicotinic acetylcholine alpha-subunit gene tar-1. Sequences were derived from DNA prepared from bulk collections of worms and from individual male and female Trichostrongylus colubriformis. In each case a levamisole-resistant (BCk) and a drug susceptible population were examined. Although several nucleotide transitions were detected no amino acid sequence variations were found between the isolates and between individual worms, indicating that the coding sequence of this gene is not responsible for levamisole-resistance in the isolate tested. However, an intronic allelic T/C variation at position 4955 was observed in both populations. It has been reported that levamisole-resistance in the BCk isolate of T. colubriformis is due to a sex-linked recessive gene or gene complex. A restriction fragment length polymorphism formed by the allelic variation was found and was detectable by digestion with the restriction endonuclease NlaIII. Statistical comparison of allele frequencies from individual male and female worms was consistent with sex-linkage of tar-1 (P < 0.05) but showed no correlation with levamisole resistance status. The polymorphism described will provide a useful X-chromosome marker and represents the first mapped genetic locus in this species.

Structural characterisation and pharmacology of KHEYLRFamide (AF2) and KSAYMRFamide (PF3/AF8) from Haemonchus contortus.

The FMRFamide-related peptides (FaRPs), KHEYLRFamide (AF2) and KSAYMRFamide (PF3) were structurally characterised from the parasitic nematode of sheep, Haemonchus contortus (MH isolate). Both peptides were sequenced in a single gas-phase sequencing run and their structure confirmed by mass spectrometry which identified peptides of 920 Da (C-terminally amidated AF2) and 902/918 Da (C-terminally amidated non-oxidised/oxidised PF3, respectively). AF2 had inhibitory effects on H. contortus muscle and inhibited acetylcholine (ACh, 10 microM)-induced contractions, with a threshold for activity of 1 microM. PF3 induced concentration-dependent contractions of H. contortus (activity threshold, 10 nM) and enhanced ACh contractions. Compared with the MH isolate, an isolate of H. contortus which has reduced sensitivity to cholinergic drugs (Lawes isolate) was less sensitive to the effects of PF3. The concentration-response curves for the cholinergic compounds ACh and levamisole (LEV), and PF3, but not a control, KPNFIRFamide (PF4), showed a statistically similar shift. This study implicates PF3 in the modulation of cholinergic function in H. contortus.

Anthelmintic resistance: past, present and future.

Anthelmintic resistance continues to increase in geographic range, in the number of species affected and the range of drugs involved. Several aspects of resistance have emerged as important issues. They include lack of genetic reversion, presence of side resistance and lack of universality. Furthermore, resistant isolates recovered from the field may have different characteristics to those selected in pen passage. Research into anthelmintic resistance has not progressed far beyond the stage of descriptive research. Some progress has been made in developing control strategies and in diagnosing resistance, especially in the development and adoption of in-vitro tests. However, these still need improvements in their ability to detect resistance to closantel and avermectin/milbemycin anthelmintics. Less progress into understanding the basis of resistance has occurred. Research priorities include improvement of diagnostic tests and the development of molecular tests, particularly for resistance to levamisole and the avermectin/milbemycins. Resistance itself, as a selectable marker for genetic transfection in parasites, is a potential tool for investigating parasite biology.

An in vitro assay utilising parasitic larval Haemonchus contortus to detect resistance to closantel and other anthelmintics.

A new assay for detecting resistance to anthelmintics in vitro is described. The assay uses a simple culture system in which the ability of anthelmintics to kill or inhibit the migration of parasitic third and fourth stage Haemonchus contortus larvae through a 50 microns aperture mesh is assessed. The assay detects 2-10-fold resistance to closantel. Resistance to benzimidazoles, levamisole and ivermectin can also be detected.

Localization of protective antigens in Trichostrongylus colubriformis.

In order to localize protective antigens in Trichostrongylus colubriformis, adult worms were microdissected and the capacity of worm tissues to protect guinea pigs against infection examined. The results suggest that T. colubriformis protective antigens are widely distributed in the body and are not concentrated in the intestine or glandular structures.

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

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

Quid significat nomen? (What's in a name?).

For the purposes of classification and effective communication among scientists, organisms must have unequivocal names. The binomial naming system of species was devised and popularized by Linnaeus in the 18th Century. His "Botanical Latin" is an artificial language first adopted for naming plants and is now internationally accepted as a naming system for both plants and animals. Genus and species names are based on Latin and Greek words which describe characteristics of the organism, as well as words from more modern sources, such as the name of the discoverer or place of discovery. Naming follows certain rules and all of the word endings are Latinized. The history of naming parasites is interesting and the molecular age may influence naming in the future.

Levamisole resistance in Haemonchus contortus selected at different stages of infection.

Resistance to the anthelmintic levamisole is common amongst most genera of trichostrongylid nematodes of sheep, but is relatively rare in the species Haemonchus contortus. The level of resistance in at least one strain of this species varies during the life cycle. Investigation of this phenomenon, especially in parasitic stages of the parasite may reveal information on resistance mechanisms and factors affecting the selection of resistance. Compared with adult worms of the same resistant strain, immature worms are more susceptible to levamisole both in in vitro contraction assays and an in vivo efficacy experiment. To ascertain if immature parasitic worms of a susceptible strain could be selected for resistance, 3 lines of worms were selected with levamisole for 9 generations. Lines were selected with the same dose rate (2 mg kg-1) at the 4th and 24th day of infection. In addition, because levamisole is less active against immature worms a further line was selected at the 4th day with a higher dose (8 mg kg-1) but similar efficacy to the selection at day 24. In in vitro assays the line selected at day 4 with the lower dose of levamisole showed no resistance compared with an unselected line. The other 2 lines developed high levels of resistance indicating that resistance can develop after selection at either stage. In addition these data directly show that selection pressure is important in determining the rate of development of resistance.

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

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

Inheritance of levamisole and benzimidazole resistance in an isolate of Haemonchus contortus.

Reciprocal crosses between an isolate of Haemonchus contortus resistant to both benzimidazole and levamisole anthelmintics and a susceptible isolate were performed in order to determine the mode of inheritance of these resistances. F1 and F2 generations and parent isolates were assayed for susceptibility to thiabendazole and levamisole in vitro. For each drug all of the filial generations were intermediate in susceptibility between the parent isolates, and analysis indicated that resistance was inherited as an incompletely recessive character determined by more than one gene in each case. There was no evidence of maternal inheritance. Results of both the in vitro assays and in vitro selection, followed by determination of sex ratio in the survivors, as well as studies on adult worms, provided no evidence for sex-linkage. This work illustrates that in vitro assays coupled with minimal studies in sheep are useful for determining inheritance of resistance, yet use fewer experimental animals than traditional studies.

Effects of cholinergic drugs on longitudinal contraction in levamisole-susceptible and -resistant Haemonchus contortus.

A novel force transducer was used to measure the effects of cholinergic agonists on longitudinal contraction in Haemonchus contortus. Drugs were applied to whole worms or injected via a cannula in the pseudocoelomic cavity. A number of agonists, including nicotine and the anthelmintics m-aminolevamisole, levamisole and morantel, caused contractions in whole worms. Four- to 25-fold increases in concentration of the active compounds were required to cause contractions in each of two levamisole-resistant strains of H. contortus. Of the other compounds tested, bephenium had equivalent activity against susceptible and resistant strains. Anticholinesterase compounds caused contractions after a slight delay in susceptible, but not resistant worms. Numerous cholinergic agonists and other compounds did not cause contraction when applied to whole worms. One of these, acetylcholine, caused contractions in cannulated worms. Compared with the susceptible strain, five- to six-fold higher concentrations of acetylcholine were required to cause equivalent contractions in the resistant strains. Levamisole resistance in adult H. contortus is likely to be due to a change in the characteristics of the cholinergic receptor(s).

Expulsion of Trichostrongylus colubriformis by high and low responder guinea-pigs.

Guinea-pigs with genetically determined susceptibility to infection with Trichostrongylus colubriformis (or low responders) rejected both primary and secondary infections with this parasite more slowly than resistant animals (high responders). Low responders were not protected with a vaccination procedure which was highly effective in outbred animals. The relatively poor protective immune responses that develop in low responder guinea-pigs are evocative of the responses of the natural host to infection with this parasite and suggest that low responder guinea-pigs have potential for the study of T. colubriformis protective antigens and for the development of adjuvants to enhance antiparasitic effector responses in vaccinated hosts.

Binding of [3H]m-aminolevamisole to receptors in levamisole-susceptible and -resistant Haemonchus contortus.

M-aminolevamisole, a potent analogue of the commercial anthelmintic levamisole, was used to investigate ligand-binding properties of homogenates of larval and parasitic stages of the nematode parasite of sheep, Haemonchus contortus. Kinetics of the binding of [3H]m-aminolevamisole to homogenates was measured in a drug-susceptible isolate and compared with a levamisole-resistant isolate. Equilibrium binding studies and kinetic studies revealed a high affinity binding component with a KD of 3 nM. A low affinity component (KD = 2.4 microM) was also apparent in equilibrium studies. High affinity [3H]m-aminolevamisole binding was displaced in a concentration-dependent manner by levamisole analogues and cholinergic agonists. Compared with the susceptible isolate, binding in a levamisole-resistant isolate of the parasite, was quantitatively similar over a range of developmental stages and binding conditions. However, under the conditions of binding there was a reduced affinity (larger KD) and more binding sites (larger Bmax) at the low affinity site in the resistant compared with the susceptible isolate. It was concluded that the ligand was binding to acetylcholine receptor populations of the nematode and that resistance may be associated with alterations in the low affinity site of this receptor.