J. Cabaret

The role of targeted selective treatments in the development of refugia-based approaches to the control of gastrointestinal nematodes of small ruminants

Anthelmintic resistance is recognised as a major problem affecting small ruminant production worldwide and now threatens the sustainability of many of these systems. One method that has been proposed to prolong the efficacy of our current anthelmintics is the maintenance of a parasite population in refugia (unexposed to a drug) which will maintain the genes for susceptibility within the parasite population. Management strategies that employ refugia-based methods include targeted or strategically timed whole flock treatments, targeted selective treatments (TST), whereby only a proportion of the flock is treated at any one time, and the dilution of resistant with susceptible parasites. The ability to effectively target anthelmintic use relies on the identification of those animals that will most benefit from treatment. This review explains the concept of refugia, describes the role of refugia-based approaches to the management of anthelmintic resistance and reviews the markers that have been studied as indicators for TSTs as well as the implementation of refugia-based strategies. Recent results suggest that targeting anthelmintic treatment on the basis of anaemia, milk production and liveweight gain may offer a means of reducing anthelmintic usage whilst still maintaining animal performance.

Monitoring the efficacy of ivermectin and albendazole against gastro intestinal nematodes of cattle in Northern Europe

Faecal egg count reduction tests (FECRT) using ivermectin (IVM) and benzimidazole (BZ) were conducted to investigate the prevalence of anthelmintic resistance in gastro-intestinal nematodes on cattle farms in Germany, Belgium and Sweden in 2006 and 2007. Based on sufficient numbers of eggs prior to the study, between 3 and 10 farms per country were selected. 10-15 animals were randomly selected per farm and subcutaneously treated with 0.2 mg IVM/kg bodyweight (Ivomec, Merial). Faecal samples were collected individually from every animal on day 0 (treatment), day 7 (Belgium & Sweden) or 14 (Germany), and day 21 (Germany, Belgium and Sweden). Faecal egg counts (FEC) were performed at each sampling occasion to estimate the eggs per gram of faeces (EPG) and the reduction of eggs after treatment. The FECRT using IVM in 2006 revealed mean reduction of egg counts between 69-100% on day 7/14 (95% confidence interval (CI) 19-102) and 35-96% (95% CI 0-102) on day 21. Farms with a suggested problem of anthelmintic resistance have been re-visited in 2007 and except for one case all results obtained in 2006 were confirmed in 2007. Larvae obtained from faecal cultures were identified using microscopic identification keys or genus-specific real time PCR. Cooperia oncophora was the predominant species detected after treatment, but Ostertagia ostertagi was found in samples on 3 farms in Germany and 3 farms in Sweden post-treatment. In 2007 additionally a FECRT using benzimidazoles was conducted in Germany and Sweden. In Germany oral Valbazen (albendazole, 10%, Pfizer) was used at a concentration of 7.5 mg albendazole/kg bodyweight; in Sweden Valbazen Vet (albendazole, 10%, Orion Pharma) at a dose of 8 mg/kg was used. For benzimidazoles an efficacy of 100% was obtained on all tested farms in both countries. This is the first report of a multinational anthelmintic efficacy investigation in cattle in Europe. The results suggest that testing of anthelmintic efficacy should be performed more intensively due to possible insufficient efficacy of current drugs.

PCR diagnosis of benzimidazole-susceptibity or -resistance in natural populations of the small ruminant parasite, Teladorsagia circumcincta

We have developed a new molecular tool for the diagnosis of the benzimidazole (BZ)-susceptibility or resistance in Teladorsagia circumcincta a nematode parasite of small ruminants. This tool is based on the use of the PCR and allows the genotyping of resistant (rr) or susceptible (rS and SS) adult worms or larvae. By using four primers in the same reaction mixture, worms can be genotyped in regard to the mutation on the residue 200 (phenylalanine to tyrosine) of the beta-tubulin which is implicated in BZ resistance. A very high proportion of homozygous SS (Phe/Phe) individuals characterized the BZ susceptible populations, whereas a variable proportion of homozygous rr (Tyr/Tyr) individuals characterized the BZ resistant populations. A positive correlation was observed between the LD50 estimated by egg hatch assay, and the proportion of mutant homozygous individuals rr (Tyr/Tyr). Our PCR method allows the rapid genotyping of numerous worms and permits the detection of the first resistant individuals in a worm population.

Prevalence of anthelmintic resistance in gastrointestinal nematodes of dairy goats under extensive management conditions in southwestern France.

The occurrence of benzimidazole (BZ) and levamisole resistance was investigated in 18 randomly selected dairy goat herds located in southwestern France and characterized by extensive management. On each of the 18 farms, 45 adult goats were randomly allocated into three groups of 15 animals each: an untreated control group, a group that was orally administered fenbendazole (10 mg kg(-1) body weight) and a group that received orally a levamisole drench (12 mg kg(-1) body weight). Individual faecal egg counts and pooled larval cultures were done 10 days after anthelmintic treatment. Naive lambs were infected with larvae obtained from control and fenbendazole treated groups and were necropsied 35 days after infection for worm recovery. Faecal egg count reductions (FERC) were calculated for fenbendazole and levamisole and, when less than 95 per 100, were considered as indicative of anthelmintic resistance. An in vitro egg hatch test (EHT) was conducted with thiabendazole on eggs isolated from pooled faeces of fenbendazole treated goats in nine farms. Faecal egg count reductions indicated the occurrence of benzimidazole resistance in 15 out of 18 farms. Among these farms, nine had EHT values above 0.1 microg thiabendazole ml(-1) confirming the benzimidazole resistance status. Levamisole resistance was detected in two farms through FECR. Based on necropsy results, the prevalence of benzimidazole resistance was higher in Trichostrongylus colubriformis, medium in Haemonchus contortus and lower in Teladorsagia circumcincta. In nine farms the benzimidazole resistance was monospecific whereas multispecific resistance was found in the six remaining farms. A negative relationship was found between FECR for fenbendazole and the average number of anthelmintic treatments given per year on the farm. Despite extensive management including a low number of treatments, the prevalence of benzimidazole resistance was very high suggesting that the repeated and sometimes exclusive use of benzimidazole drugs, even at low frequency, is probably the main cause in developing nematode resistance in dairy goat herds. The importance of other factors such as under-dosing or buying animals already carrying resistant nematodes are discussed.

Functional reconstitution of Haemonchus contortus acetylcholine receptors in Xenopus oocytes provides mechanistic insights into levamisole resistance

BACKGROUND AND PURPOSE The cholinergic agonist levamisole is widely used to treat parasitic nematode infestations. This anthelmintic drug paralyses worms by activating a class of levamisole‐sensitive acetylcholine receptors (L‐AChRs) expressed in nematode muscle cells. However, levamisole efficacy has been compromised by the emergence of drug‐resistant parasites, especially in gastrointestinal nematodes such as Haemonchus contortus. We report here the first functional reconstitution and pharmacological characterization of H. contortus L‐AChRs in a heterologous expression system.

Relationship between helminth species diversity, intensity of infection and breeding management in dairy goats

Helminth infection, species diversity (proportion of each species in the community), species number, intensity of infection and anthelmintic resistance were investigated in 16 dairy-goat farms of south-western France. The aim of the study was to estimate which breeding management factors may affect diversity and intensity of infection. Anthelmintic resistance was investigated because this factor may modify composition of nematode communities. Farm history and breeding management were recorded by means of a questionnaire. Natural and experimental infections were compared to determine which strategy was the best to estimate diversity index and number of species from nematode community of a whole herd. A total of 17 species of helminths, among which 14 nematodes, one cestode (Moniezia spp.) and two trematodes (Paramphistomum daubneyi and Dicrocoelium lanceolatum) were recovered in the 26 necropsied culled goats. One culled goat worm burden represented conveniently species number but not species diversity (Shannon index) of parasite community harboured in the whole herd. Experimental sheep infection with larvae from pooled faeces of 15 goats was the best strategy to estimate species diversity in farms. Diversity could be predicted from the number of farms from which goats originated at constitution of the herd, the duration of goat winter withdrawal from pastures, and intensity of infection. Anthelmintic resistance was correlated with the number of farms of origin, area of permanent pastures and intensity of infection. The strategy adopted for the herd constitution was a critical step as species diversity and risk of anthelmintic resistance increased with the number of farms of origin. Intensity of infection was negatively correlated with species diversity. This suggested that more diverse communities were better controlled by the host, in agreement with the widespread statement that diversity would beget stability.

Genetic diversity of levamisole receptor subunits in parasitic nematode species and abbreviated transcripts associated with resistance.

Objectives The molecular mechanisms of levamisole (LEV) activity and expression of resistance remain largely unknown in parasitic nematodes. In contrast, genetic screens for mutants that survive exposure to LEV in the free-living nematode Caenorhabditis elegans have led to the identification of five genes (unc-38, unc-63, unc-29, lev-1 and lev-8) that encode a LEV-sensitive acetylcholine receptor (L-AChR). Loss of these genes leads to LEV resistance. In this study, orthologues of these genes were identified in three species of trichostrongylid nematodes that have a major impact on small ruminants: Haemonchus contortus, Teladorsagia circumcincta and Trichostrongylus colubriformis. Polymorphism associated with LEV resistance have been investigated by comparing transcripts of these subunits in LEV susceptible and LEV-resistant isolates of the three strongylid species. Basic methods Partial sequences were identified by PCR experiments and full-length cDNA sequences corresponding to AChR subunits in the three trichostrongylid species were obtained using 3′-rapid amplification of cDNA ends-PCR and 5′ rapid amplification of cDNA ends anchored with the spliced leader sequence, SL1. Expression of L-AChR subunits was investigated in LEV-resistant and LEV-susceptible isolates of H. contortus, T. circumcincta and T. colubriformis using reverse transcription PCR. Main results We have identified a total of 20 full-length cDNA sequences corresponding to L-AChR subunits in three parasitic trichostrongylid species of which 14 correspond to novel sequences. Genes orthologous to unc-29, unc-63, unc-38 and lev-1 were found in each trichostrongylid species, whereas no gene corresponding to lev-8 has yet been identified. We have found 11 distinct paralogous sequences corresponding to the C. elegans unc-29 gene clustered in four groups revealing an unexpected diversity of unc-29-like genes. Complete coding sequences of the L-AChR subunits in two LEV-resistant and three susceptible isolates of H. contortus, T. circumcincta and T. colubriformis were essentially unchanged, but abbreviated transcripts of the unc-63 subunit were specifically expressed in resistant isolates of all three species. Conclusion The candidate gene strategy developed in this study revealed an unexpectedly high diversity of L-AChR subunits specific to the trichostrongylid parasites that are a principal target for the drug LEV. Abbreviated variants, predicted to produce nonfunctional unc-63, were associated with LEV resistance. This study contributes significantly to a better understanding of LEV receptor constitution in parasitic nematodes and highlights the putative role of aberrant mRNA encoding L-AChR subunits in LEV resistance.

Experimental and modeling approaches to evaluate different aspects of the efficacy of Targeted Selective Treatment of anthelmintics against sheep parasite nematodes.

Maintaining a refugia i.e. a proportion of the population that is not exposed to selection by treatments has been suggested as an alternative to mass treatment in the management of nematode parasites of sheep. Three refugia have been identified: nematodes in untreated hosts, encysted larvae and free-living stages on pastures. Here we tested whether Targeted Selective Treatments (TST) could be relevant in controlling nematode infections and delaying anthelmintic resistance selection. We first conducted a one grazing season experiment to compare all flock treatment (the whole flock was treated monthly) and TST based on monthly excretion eggs and daily weight gain. Nematode burden was higher in TST group, but anthelmintic susceptibility of nematodes was preserved. We then used an individual-based model to evaluate the sustainability of TST on a longer period. The simulation approach indicated that TST20% and TST30% of the flock were efficient both at maintaining resistance at a low level and controlling nematode parasite infections on a mid-term period (i.e. simulation of five grazing seasons). However for an efficient TST, these percentages of treated hosts should be adapted to flock size since the number of nematode parasites exposed to selection increases in large flocks. Our results also showed a high dependence on the timing of treatment i.e. on the size of the refugia constituted by the free-living stages on the pasture.

New molecular evidence that Teladorsagia circumcincta (Nematoda: Trichostrongylidea) is a species complex

In the polymorphic Teladorsagia circumcincta (morphs circumcincta and trifurcata), a sheep and goat line (SGL) and a goat line (GL) have been previously described on the basis of the malate dehydrogenase allozyme polymorphism (MDH-2) and of the morphology of the dorsal ray. The GL were never found alone in 1 host, so the status of species was not given to these 2 lines. To investigate further whether there are other genetic markers that will delineate them, we collected T. circumcincta worms from goat and sheep at 8 farms in Touraine (west-central France). The worms were identified individually as being SGL or GL on the basis of MDH-2 polymorphism. This distinctiveness was corroborated by sequences of the β-tubulin isotype 1 gene, the second internal transcribed spacer (ITS2) of their rDNA, and the nicotinamide dehydrogenase (ND4) gene of their mDNA. The extent of the divergence in the 3 additional genetic markers was such that SGL and GL may be considered as 2 species. A third putative species was found in the SGL line based exclusively on the ND4 gene. These findings suggest that T. circumcincta is a species complex and that further investigation is required on a wider geographic scale.

Allopatric combination of Fasciola hepatica and Lymnaea truncatula is more efficient than sympatric ones

Parasites are capable of rapid evolutionary changes relative to their hosts, due to short life cycle, short generation time, and high fecundity. The direction of the evolution of parasite virulence can be studied in cross-transfer experiments, combining hosts and parasites from different localities, and comparing the outcome of established (sympatric and potentially locally adapted) and novel (allopatric) combinations of hosts and parasites. We aimed to compare the compatibility with snails hosts, the infectivity of metacercariae in rabbits and rats, and the fitness among different combinations (French-FF and Spanish-SS sympatries and allopatry-FS). The first isolate of Fasciola hepatica and its corresponding intermediate host, Lymnaea truncatula originated from Lugos northwestern Spain. The second isolate of parasite and snail was collected in the Limoges area in central France. The Spanish snails were more susceptible to their sympatric trematode than the French snails. The Spanish flukes were more infective to intermediate hosts (snails) than the French flukes, but subsequent definitive hosts (rats or rabbits) infections remained similar. The estimated fitness was low in sympatric infections and highly similar (from 4.7 to 5.3). The fitness similarity corresponds, however, to different variations in life-history traits that could represent different strategies among the host-parasite local combinations. The infection rate in snails, metacercarial productivity, metacercarial infectivity, and the estimated fitness were better for allopatric combination (FS). The susceptibility data showed a higher efficiency of flukes in the allopatric snail population than in their local snail population. However, our results were obtained after one generation and from a single isolate and it remains to be determined if all allopatric fluke-snail isolates may present a better fitness. Nevertheless our results indicate that introduction of liver fluke-infected cattle should be monitored carefully, as it could result in the introduction of more efficient parasites.