Why phylogenetic signal of traits is important in ecosystems: uniformity of a plant trait increases soil fauna, but only in a phylogenetically uniform vegetation.

Phylogenetically closely related plant species often share similar trait states (phylogenetic signal), but local assembly may favor dissimilar relatives and thereby decouple the diversity of a trait from the diversity of phylogenetic lineages. Associated fauna might either benefit from plant trait diversity, because it provides them complementary resources, or suffer from it due to dilution of preferred resources. We hence hypothesize that decoupling of trait and phylogenetic diversity weakens the relationship between the plant-trait diversity and the abundance and diversity of associated fauna. Studying permanent meadows, we tested for combined effects of plant phylogenetic diversity and diversity of two functional traits (specific leaf area, leaf dry matter content) on major groups of soil fauna (earthworms, mites, springtails, nematodes). We found that only in phylogenetically uniform plant communities, was uniformity in the functional traits associated with (i) high abundance in springtails, and (ii) high abundance of the sub-group that feeds more directly on plant material (in springtails and mites) or those that are more prone to disturbance (in nematodes), and (iii) high diversity in all three groups tested (springtails, earthworms, nematodes). Our results suggest that soil fauna profits from the resource concentration in local plant communities that are uniform in both functional traits and phylogenetic lineages. Soil fauna would hence benefit from co-occurrence of closely related plants that have conserved the same trait values, rather than of distantly related plants that have converged in traits. This might result in faster decomposition and a positive feedback between trait conservatism and ecosystem functioning.

The global diversity of Haemonchus contortus is shaped by human intervention and climate.

Haemonchus contortus is a haematophagous parasitic nematode of veterinary interest. We have performed a survey of its genome-wide diversity using single-worm whole genome sequencing of 223 individuals sampled from 19 isolates spanning five continents. We find an African origin for the species, together with evidence for parasites spreading during the transatlantic slave trade and colonisation of Australia. Strong selective sweeps surrounding the ß-tubulin locus, a target of benzimidazole anthelmintic drug, are identified in independent populations. These sweeps are further supported by signals of diversifying selection enriched in genes involved in response to drugs and other anthelmintic-associated biological functions. We also identify some candidate genes that may play a role in ivermectin resistance. Finally, genetic signatures of climate-driven adaptation are described, revealing a gene acting as an epigenetic regulator and components of the dauer pathway. These results begin to define genetic adaptation to climate in a parasitic nematode.

Lupin (Lupinus spp.) seeds exert anthelmintic activity associated with their alkaloid content.

The growing range of drug resistant parasitic nematode populations threatens the sustainability of ruminant farming worldwide. In this context, nutraceuticals, animal feed that provides necessary dietary requirements while ensuring parasite control, could contribute to increase farming sustainability in developed and low resource settings. In this study, we evaluated the anthelmintic potential of lupin seed extracts against the major ruminant trichostrongylids, Haemonchus contortus and Teladorsagia circumcincta. In vitro observations showed that seed extracts from commercially available lupin varieties could significantly but moderately inhibit larval migration. This anthelmintic effect was mediated by the seed alkaloid content and was potent against both fully susceptible and multidrug resistant H. contortus isolates as well as a susceptible T. circumcincta isolate. Analytical chemistry revealed a set of four lupanine and sparteine-derivatives with anthelmintic activity, and electrophysiology assays on recombinant nematode acetylcholine receptors suggested an antagonistic mode of action for lupin alkaloids. An in vivo trial in H. contortus infected lupin-fed ewes and goats failed to demonstrate any direct anthelmintic effect of crude lupin seeds but infected lupin-fed goats suffered significantly less parasite-mediated blood losses. Altogether, our findings suggest that the anthelmintic potential of lupin remains limited. However, the potent alkaloids identified could lead to the development of novel drugs or may be used in combination with current anthelmintics to improve their efficacy.

Are sainfoin or protein supplements alternatives to control small strongyle infection in horses?

The spread of anthelmintic resistance in equine strongyle nematodes has become a major problem, advocating for the development of alternative control for strongyles. Our study consisted of both in vivo and in vitro experiments. We investigate for the first time the efficacy of a short-term consumption of tannin-rich sainfoin (Onobrychis viciifolia) or extra proteins in naturally infected horses. We used 30 horses allocated into three groups of 10 individuals that received for 18 days either (i) a tannin-rich diet with 70% DM sainfoin pellets (Sd), (ii) a protein-rich diet with 52% DM Italian rye-grass pellets and 18% DM grinded linseed expeller (Pd), or (iii) a control diet with 45% DM barley and 25% DM cereal-based pellets (Cd). The three diets were isoenergetic, covering 94% of animal energy requirements on average, and the Sd and Pd diets were isoproteic and provided extra proteins (227% of protein requirements v. 93% for the Cd diet). Pd and Cd were compared to test for benefits of receiving extra proteins, while Sd and Pd were compared to account for the effect of sainfoin secondary metabolites. There were no between-diet differences in faecal egg counts (FEC) or in worm burden evaluated from worm counts in faeces of drenched horses at the end of the experiment. However, coprocultures from the faeces collected in each group at the beginning and at the end of the experiment suggested a lower rate of strongyle larval development in the Sd group at the end of the experiment (Sd=8.1%, Pd=30.5%, Cd=22.6%). In vitro tests using sainfoin solutions evidenced the influence of sainfoin on strongyle larval development: adding 29% of sainfoin pellets to faeces reduced the strongyle egg development into infective larvae by 82% (P<0.001) and using solutions with sainfoin concentrations higher than 7.5 mg/ml reduced egg hatching by 37% (P<0.05). The short-term use of tannin-rich plants in horse diet could thus constitute a promising strategy to reduce the risk of infection by strongyles at pasture.

Risk factor analysis of equine strongyle resistance to anthelmintics.

Intestinal strongyles are the most problematic endoparasites of equids as a result of their wide distribution and the spread of resistant isolates throughout the world. While abundant literature can be found on the extent of anthelmintic resistance across continents, empirical knowledge about associated risk factors is missing. This study brought together results from anthelmintic efficacy testing and risk factor analysis to provide evidence-based guidelines in the field. It involved 688 horses from 39 French horse farms and riding schools to both estimate Faecal Egg Count Reduction (FECR) after anthelmintic treatment and to interview farm and riding school managers about their practices. Risk factors associated with reduced anthelmintic efficacy in equine strongyles were estimated across drugs using a marginal modelling approach. Results demonstrated ivermectin efficacy (96.3% ± 14.5% FECR), the inefficacy of fenbendazole (42.8% ± 33.4% FECR) and an intermediate profile for pyrantel (90.3% ± 19.6% FECR). Risk factor analysis provided support to advocate for FEC-based treatment regimens combined with individual anthelmintic dosage and the enforcement of tighter biosecurity around horse introduction. The combination of these measures resulted in a decreased risk of drug resistance (relative risk of 0.57, p = 0.02). Premises falling under this typology also relied more on their veterinarians suggesting practitionners play an important role in the sustainability of anthelmintic usage. Similarly, drug resistance risk was halved in premises with frequent pasture rotation and with stocking rate below five horses/ha (relative risk of 0.53, p < 0.01). This is the first empirical risk factor analysis for anthelmintic resistance in equids. Our findings should guide the implementation of more sustained strongyle management in the field.

Physico-chemical characteristics of topsoil for contrasted forest, agricultural, urban and industrial land uses in France.

Soil quality is related to soil characteristics such as fertility and contamination. The aim of this study is to assess the effect of land use on these soil characteristics and to confirm the following anthropisation gradient: (i) forest, (ii) grassland, (iii) cultivated, (iv) orchard and vineyard, (v) urban vegetable garden, and (vi) SUITMA (urban, industrial, traffic, mining and military areas). A database comprising the characteristics of 2451 soils has been constituted. In order to compare the topsoils from six contrasting land uses, a principal components analysis (PCA) was performed on nine geochemical variables (C, N, pH, POlsen, total Cd, Cu, Ni, Pb, Zn). The first axis of the PCA is interpreted as a global increase of topsoil metallic elements along the anthropisation gradient. Axis 2 reflects the variability of fertility levels. Human activity increases the pressure on soils along the proposed gradient according to six different distribution patterns. This better knowledge of topsoil quality and its dependence on current land use should therefore help to manage and preserve the soil mantle.

Exploring the limitations of pathophysiological indicators used for targeted selective treatment in sheep experimentally infected with Haemonchus contortus.

Identifying which sheep to treat as part of a Targeted Selective Treatment approach to gastro-intestinal nematode control relies entirely on the efficacy of the indicators. Indicators such as FAMACHA© (anaemia), DISCO (diarrhea) and reductions in weight gains were designed specifically to reflect those sheep experiencing symptomatic consequences of infection. Using the gastro-intestinal nematode Haemonchus contortus as a model species, this study explored the utility and sensitivity of these indicators under controlled experimental conditions on 63 adult sheep. The potential effect of sheep with different H. contortus resistance phenotypes on indicator efficacy was compared in three different phenotypes, i.e. high (Blackbelly females), medium (Blackbelly rams) and low resistance (Romane rams). The potential effect of the H. contortus isolate on indicator efficacy was also explored by using four different isolates, with varying anthelmintic resistance capacities, to infect the sheep. We limited the study to the first month of infection to evaluate the interest of these indicators as an early predictive means for controlling infection. The pathophysiological indicators FAMACHA© and DISCO do not reflect infection intensity based on Faecal Egg Counts, nor do reductions in weight gains. FAMACHA© was however a good indicator of anaemia with strong correlations to haematocrit. There was little agreement among the three indicators to identify the same animals in need of treatment and even combining them did not increase their predictive value of infection intensity or relative host damage from infection. The indicator sensitivity was influenced by the H. contortus isolate and sheep resistance phenotype in which they were tested. One isolate was poorly infective but induced high levels of anaemia (FAMACHA©) and diarrhea (DISCO) compared to the three others. The FAMACHA© and DISCO had higher values in the sheep group with a medium resistance phenotype (Blackbelly rams) indicating higher levels of damage compared to the high and low resistance phenotypes. We conclude that there is no 'one size fits all' approach to the use of indicators for Targeted Selective Treatment and the indicators should be calibrated to farm-specific conditions to increase their efficacy.

Storage of gastrointestinal nematode infective larvae for species preservation and experimental infections.

Techniques to preserve the infective third-stage larvae (L3) of gastrointestinal nematodes are of considerable interest to preserve rare species and to maintain a stable source for routine experimental infections. This study compares the relative pros and cons of the two most common techniques, cryopreservation and refrigeration by comparing how they influence consequent infection outcome parameters in terms of life-history traits and fitness as a function of time using the gastrointestinal nematode of sheep Haemonchus contortus as a study species. Establishment capacity was found to be significantly reduced in cryopreserved stocks of L3 compared to refrigerated stocks, but this was followed by significant increases in their fecundity. Refrigeration did not affect L3 stocks consequent fitness by 16 months (the maximum examined) although they did incur a significant reduction in establishment, followed once again by an augmentation in fecundity. The study highlights potential areas for bias in comparing studies using L3 larvae maintained for different periods of time under different techniques.

How does the suppression of energy supplementation affect herbage intake, performance and parasitism in lactating saddle mares?

Agroecology opens up new perspectives for the design of sustainable farming systems by using the stimulation of natural processes to reduce the inputs needed for production. In horse farming systems, the challenge is to maximize the proportion of forages in the diet, and to develop alternatives to synthetic chemical drugs for controlling gastrointestinal nematodes. Lactating saddle mares, with high nutritional requirements, are commonly supplemented with concentrates at pasture, although the influence of energy supplementation on voluntary intake, performance and immune response against parasites has not yet been quantified. In a 4-month study, 16 lactating mares experimentally infected with cyathostome larvae either received a daily supplement of barley (60% of energy requirements for lactation) or were non-supplemented. The mares were rotationally grazed on permanent pastures over three vegetation cycles. All the mares met their energy requirements and maintained their body condition score higher than 3. In both treatments, they produced foals with a satisfying growth rate (cycle 1: 1293 g/day; cycle 2: 1029 g/day; cycle 3: 559 g/day) and conformation (according to measurements of height at withers and cannon bone width at 11 months). Parasite egg excretion by mares increased in both groups during the grazing season (from 150 to 2011 epg), independently of whether they were supplemented or not. This suggests that energy supplementation did not improve mare ability to regulate parasite burden. Under unlimited herbage conditions, grass dry matter intake by supplemented mares remained stable around 22.6 g DM/kg LW per day (i.e. 13.5 kg DM/al per day), whereas non-supplemented mares increased voluntary intake from 22.6 to 28.0 g DM/kg LW per day (13.5 to 17.2 kg DM/al per day) between mid-June and the end of August. Hence total digestible dry matter intake and net energy intake did not significantly differ between supplemented and non-supplemented mares during the second and third cycles. In conclusion, supplementing lactating mares at pasture should not be systematic because their adaptive capacities enable to increase herbage intake and ensure foal growth. Further research is needed to determine the herbage allowance threshold below which supplementation is required.

A genome scan for QTL affecting resistance to Haemonchus contortus in sheep.

Gastrointestinal nematodes are one of the main health issues in sheep breeding. To identify loci affecting the resistance to Haemonchus contortus, a genome scan was carried out using 1,275 Romane × Martinik Black Belly backcross lambs. The entire population was challenged with Haemonchus contortus in 2 consecutive experimental infections, and fecal egg counts (FEC) and packed cell volumes were measured. A subgroup of 332 lambs with extreme FEC was necropsied to determine the total worm burden, length of female worms, sex ratio in the worm population, abomasal pH, and serum and mucosal G immunoglobulins (IgG) responses. Pepsinogen concentration was measured in another subset of 229 lambs. For QTL detection, 160 microsatellite markers were used as well as the Illumina OvineSNP50 BeadChip that provided 42,469 SNP markers after quality control. Linkage, association, and joint linkage and association analyses were performed with the QTLMAP software. Linkage disequilibrium (LD) was estimated within each pure breed, and association analyses were carried out either considering or not the breed origin of the haplotypes. Four QTL regions on sheep chromosomes (OAR)5, 12, 13, and 21 were identified as key players among many other QTL with small to moderate effects. A QTL on OAR21 affecting pepsinogen concentration exactly matched the pepsinogen (PGA5) locus. A 10-Mbp region affecting FEC after the 1st and 2nd infections was found on OAR12. The SNP markers outperformed microsatellites in the linkage analysis. Taking advantage of the LD helped to refine the locations of the QTL mapped on OAR5 and 13.

Can efficient management of sheep gastro-intestinal nematodes be based on random treatment?

Targeted selective treatment has been proposed as a method to reduce gastro-intestinal nematode infections in ruminants and lower the selective pressure that leads to anthelmintic resistance. Since nematodes are highly aggregated within their host population, treating only the most heavily infected hosts offers an efficient and sustainable strategy to reduce infection within the flock and slow the spread of anthelmintic resistance. Although effective methods to correctly identify such hosts are available, their feasibility is often limited in the field conditions. Instead, treating part of the flock at random may prove to be a useful and practical alternative. This study examined whether such random treatment could be relevant in controlling nematode infections and delaying the selection of anthelmintic resistance compared to targeted selective treatment. Firstly, an individual based model was used to evaluate the sustainability of random treatment according to several parasitic distributions in the host population (negative binomial, uniform and normal distributions). Anthelmintic resistance was modelled based on benzimidazoles, as a monogenic trait. Anthelmintic treatment was done twice a year, week 23 and 41, corresponding to beginning of June and of October. The model was run over a five-year period corresponding to the minimum delay for anthelmintic resistance to be observed following its initial use. The model outputs show an increase in the proportion of treated hosts led to an increase in the frequency of the resistance allele for both treatment regimes. Random treatment was shown to be slightly less efficient than targeted selective treatment in controlling for the infection intensity regardless of the percentage of hosts treated. Random treatment was however more efficient than targeted selective treatment in counter-selecting for anthelmintic resistance in both the aggregated and uniformly distributed models. Secondly, a one grazing season experiment was conducted to compare a random treatment flock (20% of flock was treated at random monthly) against a mass treatment flock (the whole flock was treated monthly). Both treatment regimes produced similar pasture infectivity, similar mean infection intensity and similar final host live weight. This is the first time random treatment of a subset of hosts has been demonstrated to be a sustainable alternative to mass treatment.

In situ assessment of phytotechnologies for multicontaminated soil management.

Due to human activities, large volumes of soils are contaminated with organic pollutants such as polycyclic aromatic hydrocarbons, and very often by metallic pollutants as well. Multipolluted soils are therefore a key concern for remediation. This work presents a long-term evaluation of the fate and environmental impact of the organic and metallic contaminants of an industrially polluted soil under natural and plant-assisted conditions. A field trial was followed for four years according to six treatments in four replicates: unplanted, planted with alfalfa with or without mycorrhizal inoculation, planted with Noccaea caerulescens, naturally colonized by indigenous plants, and thermally treated soil planted with alfalfa. Leaching water volumes and composition, PAH concentrations in soil and solutions, soil fauna and microbial diversity, soil and solution toxicity using standardized bioassays, plant biomass, mycorrhizal colonization, were monitored. Results showed that plant cover alone did not affect total contaminant concentrations in soil. However, it was most efficient in improving the contamination impact on the environment and in increasing the biological diversity. Leaching water quality remained an issue because of its high toxicity shown by micro-algae testing. In this matter, prior treatment of the soil by thermal desorption proved to be the only effective treatment.

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. EXPERIMENTAL APPROACH: In the free-living nematode Caenorhabditis elegans, five AChR subunit and three ancillary protein genes are necessary in vivo and in vitro to synthesize L-AChRs. We have cloned the H. contortus orthologues of these genes and expressed them in Xenopus oocytes. We reconstituted two types of H. contortus L-AChRs with distinct pharmacologies by combining different receptor subunits. KEY RESULTS: The Hco-ACR-8 subunit plays a pivotal role in selective sensitivity to levamisole. As observed with C. elegans L-AChRs, expression of H. contortus receptors requires the ancillary proteins Hco-RIC-3, Hco-UNC-50 and Hco-UNC-74. Using this experimental system, we demonstrated that a truncated Hco-UNC-63 L-AChR subunit, which was specifically detected in a levamisole-resistant H. contortus isolate, but not in levamisole-sensitive strains, hampers the normal function of L-AChRs, when co-expressed with its full-length counterpart. CONCLUSIONS AND IMPLICATIONS: We provide the first functional evidence for a putative molecular mechanism involved in levamisole resistance in any parasitic nematode. This expression system will provide a means to analyse molecular polymorphisms associated with drug resistance at the electrophysiological level.

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.

Contrasting genetic structures of two parasitic nematodes, determined on the basis of neutral microsatellite markers and selected anthelmintic resistance markers.

For the first time, the neutral genetic relatedness of natural populations of Trichostrongylid nematodes was investigated in relation to polymorphism of the beta-tubulin gene, which is selected for anthelminthic treatments. The aim of the study was to assess the contribution of several evolutionary processes: migration and genetic drift by neutral genetic markers and selection by anthelminthic treatments on the presence of resistance alleles at beta-tubulin. We studied two nematode species (Teladorsagia circumcincta and Haemonchus contortus) common in temperate climatic zones; these species are characterized by contrasting life history traits. We studied 10 isolated populations of goat nematode parasites: no infected adult goat had been exchanged after the herds were established. Beta-tubulin polymorphism was similar in these two species. One and two beta-tubulin alleles from T. circumcincta and H. contortus respectively were shared by several populations. Most of the beta-tubulin alleles were 'private' alleles. No recombination between alleles was detected in BZ-resistant alleles from T. circumcincta and H. contortus. The T. circumcincta populations have not diverged much since their isolation (F(ST) <0.08), whereas H. contortus displayed marked local genetic differentiation (F(ST) ranging from 0.08 to 0.18). These findings suggest that there are severe bottlenecks in the H. contortus populations, possibly because of their reduced abundance during unfavourable periods and their high reproductive rate, which allows the species to persist even after severe population reduction. Overall, the data reported contradict the hypothesis of the origin of beta-tubulin resistance alleles in these populations from a single mutational event, but two other hypotheses (recurrent mutation generating new alleles in isolated populations and the introduction of existing alleles) emerge as equally likely.

Identification of levamisole resistance markers in the parasitic nematode Haemonchus contortus using a cDNA-AFLP approach.

cDNA-AFLP (cDNA-Amplified Fragment Length Polymorphism)-based strategy has been used to identify levamisole (LEV) resistance markers in the nematode Haemonchus contortus. Transcript profiles of adult nematodes from two LEV-resistant and two susceptible isolates were compared. Among the 17 280 transcript-derived fragments (TDFs) amplified, 26 presented a polymorphic pattern between resistant and susceptible nematodes: 11 TDFs were present in both resistant isolates and absent from both susceptible isolates whereas 15 TDFs were present in both susceptible isolates and absent from both resistant isolates. 8 TDFs specifically present in resistant isolates were cloned and sequenced. Some of these TDFs could represent novel genes, as their sequences presented no homologies in databases. Interestingly, specific expression of one candidate (HA17) in resistant nematodes from different isolates was confirmed by RT-PCR experiments. The finding that HA17 expression correlates with LEV resistance in three H. contortus isolates vs five susceptible isolates strongly suggest that we identified a new potential marker of LEV resistance. This differential approach at the transcriptome level could be of great interest for the identification of the molecular mechanism involved in this phenotype.

Substitution of benzimidazole-resistant nematodes for susceptible nematodes in grazing lambs.

Multi-drug-resistant gastrointestinal nematode parasite populations are becoming more and more prevalent. Since anthelmintic treatments are of limited effectiveness, one solution could be to replace the anthelmintic-resistant population by a susceptible population, in order to re-establish the possibility of drug-based anthelmintic control. We investigated this substitution strategy in 4 paddocks of 0.7 ha, each of which was seeded with a benzimidazole-resistant Teladorsagia circumcincta population. The proportion of benzimidazole-resistant worms in these paddocks ranged from 20% to 89%. A 2-step replacement was performed: first, the paddocks were not grazed for 6 months (from December to July), and then the grass was cut to eliminate any residual infective larvae, before contaminating each of the paddocks with 10 seeder lambs experimentally infected with a benzimidazole-susceptible strain of T. circumcincta (from July to November). At the end of the experiment, all the populations on the 4 paddocks were phenotypically benzimidazole-susceptible, but genotyping indicated that 2 populations harboured 1% and 3% resistant worms respectively. This study demonstrates that nematode replacement is feasible in temperate areas, using semi-intensive stock management, even when the initial levels of benzimidazole-resistance are very high. Further research should next assess replacing the whole community to cope with the species diversity observed under field conditions.

Relationship between testate amoeba (protist) communities and atmospheric heavy metals accumulated in Barbula indica (bryophyta) in Vietnam.

We studied the relationships between testate amoeba communities and heavy metal (Pb, Cd, Zn, Ni, Cu, Mn, and Fe) concentrations in the moss Barbula indica sampled at 29 sites in and around the city of Hanoi (Vietnam). Our first approach was to compare the heavy metal concentrations and testate amoeba variables between the city (zone 1) and the surrounding (zone 2). Mean moss concentrations of Pb, Cd, Zn, Ni, and Cu were significantly higher and testate amoeba species richness and abundance were significantly lower in zone 1 and the abundance of eight taxa differed significantly between the two zones. We then studied the correlation between heavy metals and testate amoebae. Species richness and abundance were correlated negatively to Pb concentration. Shannon H' was negatively correlated to both Pb and Cd. The abundance of several species was negatively correlated with Pb, Cd, Zn, and Ni; however, at the community level, Pb emerged as the only significant variable in a redundancy analysis. Our results suggest that testate amoebae are sensitive to and may be good bioindicators for heavy metal pollution, especially lead. Further research is needed to understand the causal relationships underlying the observed patterns.

Estimation of abomasum strongyle nematode infections in sheep at necropsy: tentative proposals for a simplified technique.

Several necropsy techniques are available for estimating the abundance of gastro-intestinal nematodes in abomasum of ruminants. Standardization of techniques is needed to allow accurate comparisons between laboratories. Here we propose a standardized technique for estimating the abundance of worms. We intend to compare the worms' number estimations in lambs and ewes based on contents and washings, to determine the uniformity of worm counts in aliquots, and to estimate the total worm number from washings. The digesta (or "contents") and the washings of the abomasum are treated separately. The worms of each subsample are diluted with water and the total number of worms is estimated on a small volume (aliquots) of these subsamples. The use of aliquots assumes that the worms are uniformly distributed in the whole volume of each subsample. We first confirmed that the use of aliquots is appropriate in most cases. We then show that the use of the washings alone allows a faster and a suitable estimation of the total worm burden for all strongyle species of the abomasum in both ewes and lambs. The evaluation of our necropsy procedure is a first step to a standardized technique which should be improved by validation in other laboratories.

Evolution of nematode community in grazing sheep selected for resistance and susceptibility to Teladorsagia circumcincta and Trichostrongylus colubriformis: a 4-year experiment.

Field trials were undertaken to compare nematode population dynamics in two experimental flocks of rams selected for high and for low faecal egg counts (FECs) following two doses with 20000 infective larvae, 12000 Teladorsagia circumcincta (Tcirc) and 8000 Trichostrongylus colubriformis (Tcol) separated by a treatment. The selection was done by dosing 200 ram lambs (INRA 401 breed) and FECs proceeded with egg identification on days 28 and 35 post-each-infection. The 30 lambs with the lowest FECs constituted the group of permanent resistant rams (R), the 30 with the highest values the group of susceptible ones (S). Each group grazed separated pastures from April to November, this during 4 consecutive years. The contamination (number of eggs deposited) was estimated every 3 weeks on each pasture. The larval populations was measured by successive groups of tracer lambs. The infection of the permanent rams was done by slaughtering eight R and S rams on mid and end of each grazing season. Each autumn, new R and S ram lambs were selected to complete the R and S groups before turning out in spring. On third year, a third species (Haemonchus contortus, Hcon) was added to compare its behaviour with the two others. The regulation of parasite populations were studied by comparing what happens in R rams and pasture to S ones. The selected R ram lambs had a FEC of Tcirc 50% lower than S ones. This induced a similar lowered pasture contamination, a 25% decrease of infective larval population in years 2 and 3, and the worm burdens in R rams were the half of that in S rams after 2 years. The FEC of Tcol was 75% lower at the selection and induced similar difference in pasture contamination, but 65 and 96% reduction in L3 population, respectively, after 1 and 2 years. The worm burden was reduced by 99%. Adding Hcon species during the third year, in year 4, the L3 population was half in R pasture compared with S one, and R rams had 60% less worms of this species than S ones. This selection without any drench prevent outbreaks observed in young tracer lambs on S pasture. The comparison of selected resistant rams to selected susceptible ones and not to unselected controls gave more rapid information about the tendency of the regulation of the communities of nematode parasites.