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International audience; Resistance against macrocyclic lactones such as ivermectin is widespread among parasitic gastrointestinal nematodes of small ruminants and is rapidly increasing in cattle parasites. ABC transporters of the subfamily B, the so-called P-glycoproteins (Pgps) have been frequently implicated in ivermectin resistance and are a major cause of multi-drug resistance in protozoa and helminths. The Pgp inhibitor verapamil (VPL) dramatically enhanced susceptibility of the cattle parasitic nematode Cooperia oncophora to ivermectin in vitro as measured in a larval developmental assay and a larval migration inhibition assay using third stage larvae. Moreover, VPL completely restored susceptibility to ivermectin in a resistant isolate resulting in virtually identical dose-response curves of susceptible and resistant isolates in the presence of VPL. Further characterisation of the molecular mechanisms resulting in Pgp-mediated ivermectin resistance is still hampered by the lack of molecular and biochemical information for Pgps of parasitic nematodes. Using PCR with degenerate primers, fragments of four different C. oncophora Pgps could be amplified and the Conpgp-2, previously implicated in macrocyclic lactone resistance in Haemonchus contortus, and Conpgp-3 full-length cDNAs were obtained by RACE PCR. The pgp sequences presented here contribute important data required to systematically screen resistant C. oncophora isolates for up- or down-regulation of Pgps and for the detection of single nucleotide polymorphisms in Pgps to detect selection of specific Pgp alleles by anthelmintics as early as possible.

Toll-like receptor 4 (TLR4), which recognizes lipopolysaccharide from Gram-negative bacteria, plays a major role in resistance of mice and humans to Salmonella infection. In chickens, Salmonella may establish a carrier state whereby bacteria are able to persist in the host organism without triggering clinical signs. Based on cellular morphological parameters, we developed a method, without using antibodies, to separate three cecal cell subpopulations: lymphocytes, enterocytes, and a population encompassing multiple cell types. We analyzed the mRNA expression of TLR4, interleukin-1β (IL-1β), IL-8, IL-12, and lipopolysaccharide-induced tumor necrosis factor alpha factor (LITAF) in cecal subpopulations of chicks from inbred lines resistant or susceptible to the carrier state infected with Salmonella enterica serovar Enteritidis. The results showed that resistance to the carrier state in chicks is associated with a larger percentage of lymphocytes and with higher levels of expression of TLR4 and IL-8 at homeostasis in the three cell subpopulations, as well as with a higher level of expression of LITAF in lymphocytes during the carrier state. In contrast to the early phase of infection, the carrier state is characterized by no major cell recruitment differences between infected and noninfected animals and no significant modification in terms of TLR4, IL-1β, IL-8, IL-12, and LITAF expression in all cell subpopulations measured. However, TLR4 expression increased in the lymphocytes of chicks from the susceptible line, reaching the same level as that in infected chicks from the resistant line. These observations suggest that the carrier state is characterized by a lack of immune activation and highlight the interest of working at the level of the cell population rather than that of the organ.

Efflux pump inhibitors : a progress in parasitic nematode control. Animal and human nematode infestations are controlled primarily with anthelmintics. However, their continuous administration during outbreaks represents a significant expense for livestock farms. In humans also, their high cost limits their use in poor areas where parasitic worms are most prevalent and most pathogenic. Furthermore, nematodes have developed drug resistance mechanisms, specific or not, which reduce the efficiency of treatments. Among these mechanisms, the accelerated removal of anthelmintics by efflux pumps present in cell membranes, eggshells and cuticles is a major limiting factor. This accelerated efflux is very similar to the mechanism of multidrug resistance (MDR) observed in cancer cells and protozoa. This phenomenon is all the more worrying that it applies simultaneously to several chemical families of drugs. One solution is to block the efflux pumps in parasites with inhibitors. These pumps belong to the large family of ABC transporters, which have many characteristics in common. Some have major physiological functions or protect organs from toxic agents. As much as possible, inhibitors should not have any effect on the pumps of the host and target the parasite exclusively. The diversity of these pumps is greater in nematodes than in vertebrates, and there are differences in their protein structures. Some parts of these proteins are relatively well-conserved in the animal kingdom, while other parts show little homology from one transporter to another or from one species to another. The affinity of these pumps for the substrates can vary with the mutation of a single amino acid. These differences could be used to develop inhibitors specific of nematode pumps, which could then be combined with anthelmintics., Les anthelminthiques constituent le moyen majeur de lutte contre les nématodoses animales et humaines. Administrés de façon continue en période d’infestation, ils représentent un coût considérable pour les élevages. Chez l’homme, ce coût limite leur distribution dans les zones géographiques défavorisées où pourtant les vers parasites sont les plus nombreux et les plus pathogènes. De plus, les nématodes ont développé des mécanismes de chimiorésistance, spécifiques ou non, qui réduisent l’efficacité des anthelminthiques. Parmi ces mécanismes, le rejet accéléré des molécules thérapeutiques par des pompes d’efflux est semblable au mécanisme de multi-résistance aux médicaments (MDR) des cellules cancéreuses et des protozoaires. Ces pompes sont présentes dans les membranes cellulaires, la coque des oeufs et les cuticules des nématodes. Ce rejet accéléré est d’autant plus préoccupant qu’il s’applique simultanément à plusieurs familles chimiques d’antiparasitaires. Une des solutions consiste à bloquer les pompes d’efflux des parasites à l’aide d’inhibiteurs. Ces pompes appartiennent à la grande famille des transporteurs ABC dotés de nombreuses caractéristiques communes. Certains de ces transporteurs jouent des rôles physiologiques déterminants ou protègent les organes des molécules toxiques. Les inhibiteurs doivent donc être autant que possible dépourvus d’action sur les pompes de l’hôte. La variabilité des pompes est plus importante chez les nématodes que chez les vertébrés, et il existe des différences dans leur structure protéique. Certaines parties de ces protéines sont bien conservées dans le règne animal, tandis que d’autres présentent peu d’homologie d’un transporteur à l’autre ou, pour un même transporteur, d’une espèce à l’autre. L’affinité de ces pompes pour les substrats peut varier en fonction de la mutation d’un seul acide aminé. Ces différences pourraient être mises à profit pour développer des inhibiteurs spécifiques des pompes des nématodes et les utiliser en association avec les anthelminthiques., Kerboeuf Dominique, Riou Mickaël. La maitrise des pompes d'efflux, un progrès dans la lutte contre les nématodes parasites. In: Bulletin de l'Académie Vétérinaire de France tome 164 n°3, 2011. pp. 257-264.

International audience; We have studied the plasma membrane protein phenotype of human culture-amplified and native bone marrow mesenchymal stem cells (BM MSCs). We have found, using microarrays and flow cytometry, that cultured cells express specifically 113 transcripts and 17 proteins that were not detected in hematopoietic cells. These antigens define a lineage-homogenous cell population of mesenchymal cells, clearly distinct from the hematopoietic lineages, and distinguishable from other cultured skeletal mesenchymal cells (periosteal cells and synovial fibroblasts). Among the specific membrane proteins present on cultured MSCs, 9 allowed the isolation from BM mononuclear cells of a minute population of native MSCs. The enrichment in colony-forming units–fibroblasts was low for CD49b, CD90, and CD105, but high for CD73, CD130, CD146, CD200, and integrin alphaV/beta5. In addition, the expression of CD73, CD146, and CD200 was down-regulated in differentiated cells. The new marker CD200, because of its specificity and immunomodulatory properties, deserves further in-depth studies.

International audience; Therapeutic failure limits prophylaxis of nematode diseases and has been mainly attributed to mutations in cellular targets of anthelmintics. Besides these specific mechanisms, alterations of drug transport also occur in parasites resistant to anthelmintics and depend on both the presence of membrane pumps such as P-glycoprotein (Pgp) and on the lipid composition of membranes. We recently showed in the nematode Haemonchus contortus, using eggs as a model, that the total cholesterol (TC) concentration alters the transport of lipophilic molecules due to membrane pumps such as P-glycoprotein and the resistance to anthelmintics. The effect of TC may depend on the presence of other lipids interacting with TC. Therefore, we analysed the lipid composition and its relationship with Pgp and resistance to anthelmintics. Better correlations were found between Pgp and free cholesterol (FC) than with TC. We also showed that the relationships between lipid composition and resistance to anthelmintics or Pgp depended on the equilibrium between FC and phospholipids (PLs), mainly PLs known to be present primarily in either the external leaflets of cell membranes or the internal leaflets. The PLs phosphatidylcholine and phosphatidylethanolamine played the most significant role, but phosphatidic acid also influenced drug resistance.

International audience; Recent data have suggested that P-glycoprotein (Pgp), working as membrane efflux “pumps”, plays a major role in the transport of anthelmintic drugs in parasitic nematodes of ruminants. Flow cytometry analyses has shown that active Pgp is probably present in the external layers of Haemonchus contortus eggshells, following staining with the mouse monoclonal anti-human MDR1 antibody UIC2, which binds to Pgp in its active conformation. We evaluated the presence and distribution of this protein in the envelopes (eggshells and cuticles) of H. contortus and compared the various stages (eggs, L1–L2 larvae, L3 larvae, adult male and female worms). Electrophoresis revealed a 170-kDa band, corresponding to the molecular weight of Pgp in all stages. Indirect immunofluorescence staining with UIC2 showed Pgp to be located in the external layer of eggshells or cuticles. Transmission electron microscopy was used to localise Pgp more accurately in the three layers of the eggshells and cuticles. The conformation and biological functions of this protein, which we did not expect to find in such structures, remain to be determined.

The "multidrug resistance" (MDR) system involves the action of transmembrane P-glycoproteins (Pgp) which may be responsible for failure of chemotherapy in both invertebrates and vertebrates. We previously obtained partial reversion of anthelmintic resistance in nematodes subjected to both anthelmintics and inhibitors of this system. The results presented here are able to describe more accurately the presence of Pgp in nematodes because of the use of C219 and UIC2 monoclonal antibodies, which are used for the detection of human and mouse mdr1 gene products. These antibodies demonstrated the presence of Pgp in eggshells. Their role in these structures, which are considered to be passive barriers, remains to be determined. Flow cytometry analyses of the UIC2 staining allowed determination of the resistance of individuals, which varied within the parasite population. UIC2 demonstrated both the presence and activity of Pgp in nematodes as has previously been shown in tumour cells. Resistance seems to be due to an increase in both the number of Pgp sites and parasites with high levels of Pgp.

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

International audience; Dendritic cells (DCs) are pivotal in the development of specific T-cell responses to control pathogens, as they govern both the initiation and the polarization of adaptive immunity. To investigate the capacities of migrating DCs to respond to pathogens, we used physiologically generated lymph DCs (L-DCs). The flexible polarization of L-DCs was analysed in response to Salmonella or helminth secretions known to induce different T cell responses. Mature conventional CD1b(+) L-DCs showed a predisposition to promote pro-inflammatory (IL-6), pro-Th1 (IL-12p40) and anti-inflammatory (IL-10) responses which were amplified by Salmonella, and limited to only IL-6 induction by helminth secretions. The other major population of L-DCs did not express the CD1b molecule and displayed phenotypic features of immaturity compared to CD1b(+) L-DCs. Salmonella infection reduced the constitutive expression of TNF-alpha and IL-4 mRNA in CD1b(-) L-DCs, whereas this expression was not affected by helminth secretions. The cytokine response of T cells promoted by L-DCs was analysed in T cell subsets after co-culture with Salmonella or helminth secretion-driven CD1b(+) or CD1b(-) L-DCs. T cells preferentially expressed the IL-17 gene, and to a lesser extent the IFN-gamma and IL-10 genes, in response to Salmonella-driven CD1b(+) L-DCs, whereas a preferential IL-10, IFN-gamma and IL-17 gene expression was observed in response to Salmonella-driven CD1b(-) L-DCs. In contrast, a predominant IL-4 and IL-13 gene expression by CD4(+) and CD8(+) T cells was observed after stimulation of CD1b(+) and CD1b(-) L-DCs with helminth secretions. These results show that mature conventional CD1b(+) L-DCs maintain a flexible capacity to respond differently to pathogens, that the predisposition of CD1b(-) L-DCs to promote a Th2 response can be oriented towards other Th responses, and finally that the modulation of migrating L-DCs responses is controlled more by the pathogen encountered than the L-DC subsets.

Flow cytometry was applied to the analysis of nematode populations. Three strains of Haemonchus contortus susceptible or resistant to anthelmintics were studied. Eggs were chosen for these analyses. Data on light-scatter emissions and native green fluorescence were collected. In addition, the size of the eggs (image analysis), the hatching rate, and the susceptibility to benzimidazoles were measured. The results showed that nematode eggs are a suitable material for multiparametric flow-cytometry analyses. Forward-scatter emission is a discriminating parameter for the egg size. The hatching rate and side-scatter emission have a significantly positive relationship. For resistant strains, the rate of resistance shows a significant regression on the native green-fluorescence pulses that might reflect the state of oxidation of associated flavin molecules.

International audience; Advances in lipid separation methods and mass spectrometry technologies allow the fine characterization of the lipidome of parasites, ranging from unicellular protists to worms, which cause threatening infections in vertebrates, including humans. Specific lipid structures or lipid metabolic pathways can inspire the development of novel antiparasitic drugs. Changes in the lipid balance in membranes of parasites can also provide clues on the dynamics of drugs and some mechanisms of drug resistance. This review highlights recent trends in parasite lipidomics, combined with functional analyses, for the discovery of novel targets and the development of novel drugs.

International audience

Abstract 1058 Redox metabolism plays an important role in self-renewal and differentiation of hematopoietic cells and it has been recently established by the Guy Sauvageau's group (Institute for Research in Immunology and Cancer, Montréal, QC) that glutathione peroxydase 3 (GPx-3) promotes competitiveness of Hoxa9-Meis1 induced leukemic stem cells in which Gpx3 overexpression is concomitant of a decrease in H2O2 level and inactivation of p38 MAPK (Herault O et al, ASH annual meeting, 2010 - submitted). Leukemic cells located in the bone marrow (BM) are interacting with a microenvironment which plays a crucial role in the development and progression of leukemia. Mesenchymal stromal cells (MSCs) constitute a population of multipotential cells giving rise to the different hematopoietic microenvironmental cells (adipocytes, osteoblasts, chondrocytes, and vascular-smooth muscle-like hematopoietic supportive stromal cells). The aim of our study was to evaluate the effects of MSC-contact on the GPx-3/H2O2/p38 MAPK axis in human leukemic cells and to assess the cell cycle changes associated with the modification of H2O2 metabolism. BM MSCs were obtained from informed and consenting patients undergoing orthopedic surgery, following a procedure approved by the local ethical committee. Nucleated cells harvested from the iliac crest were seeded (5.104 cells per cm2) in α-MEM supplemented with 10% fetal calf serum (FCS), 20 mmol/l L-glutamine, and 100 units/mL penicillin G. Cells were incubated at 37°C in 95% humidified air and 5% CO2. When fully confluent, the layer of adherent cells was trypsinized (0.25% trypsin-EDTA), and the cells were resuspended in culture medium and seeded at 103 cells per cm2 (passage 1 - P1). BM MSCs were used at P2 in all experiments. The three-lineage mesenchymal differentiation of the BM MSCs used was systematically checked by culturing cells in adipogenic, chondrogenic, and osteogenic induction media as previously described (Delorme et al, Blood 2008, 111:2631). The human KG1a leukemic cell line (FAB M0/M1 CD34+ leukemic cells) was cultured in RPMI 1640 with 20 mmoL/L L-glutamine supplemented with 10% FCS, 100 units/mL penicillin G, and 100 mg/mL streptomycin. KG1a cells were seeded at 1.5 105 cells/cm2 and cocultured with MSCs for 72 h. We have defined two distinct localizations of leukemic cells relative to MSC layer: those in supernatant (non-adherent cells) and cells adhering on the surface of MSCs. The expression of antioxydative enzymes, H2O2 level, p38 MAPK activation (T180/Y182), cell cycle, proliferation and immunophenotype of these two cell fractions were evaluated at day 0 and day 3. The expression of SOD1, SOD2, SOD3, CAT, TXN, TXN2, GLRX, GLRX2, GLRX3, GLRX5, PRDX, PRDX2, PDRX3, PRDX4, PRDX5, PRDX6, GPX1, GPX2, GPX3, GPX4, GPX5, GPX6, GPX7 and GSR antioxydative genes and CDKN1A (p21CIP1) gene was quantified by qRT-PCR (Universal ProbeLibrary, Roche). SDS-PAGE and western-blot experiments were realized to quantify GPx-3 expression and p38 MAPK activation. Flow cytometry studies were performed: (a) to quantify H2O2 level by dichlorodihydrofluorescein diacetate (DCF-DA) staining; (b) to analyze the cell cycle by staining with 7-aminoactinomycin D (7AAD), Alexa Fluor®488-conjugated anti-human Ki67 and Alexa Fluor®488-conjugated anti-phospho-histone H3 (ser10) antibodies; (c) to track the cell divisions with carboxyfluorescein diacetate N-succinimidyl ester (CFSE). Supernatant of MSCs did not modify the GPx-3/H2O2/p38 MAPK axis in KG1a cells. Conversely, when compared with cells in the supernatant of MSCs, adhering KG1a cells were characterized by the exclusive overexpression of GPX3 antioxydative gene, the induction of GPx-3 production, a major decrease in H2O2 concentration and the inactivation of p38 MAPK. These effects were concomitant of cell cycle inhibition: increase in G0 phase, decrease in S and M phase, overexpression of CDKN1A and reduced mitotic activity (CFSE). Altogether these findings suggest that the bone marrow microenvironment plays a key role in the regulation of the oxidative metabolism of leukemic cells by promoting the inhibition of the H2O2/p38 MAPK axis via the induction of GPx-3. Modulation of the GPx-3/H2O2/p38 MAPK pathway by targeting of microenvironmental interactions in leukemia may have clinical relevance and it will be important to verify if these results can be extended in vivo to other models of human leukemias. Disclosures: No relevant conflicts of interest to declare.

The ability to reliably detect anthelmintic resistance is a crucial part of resistance management. If data between countries are to be compared, the same test should give the same results in each laboratory. As the egg hatch test for benzimidazole resistance is used for both research and surveys, the ability of different laboratories to obtain similar results was studied through testing of known isolates of cyathostomins, Haemonchus contortus, Ostertagia ostertagi, and Cooperia oncophora in programs supported by the EU (Cost B16 and FP6-PARASOL). Initial results showed difficulties in obtaining reproducible and similar data within and between laboratories. A series of ring tests, i.e., simultaneous and coordinated rounds of testing of nematode isolates in different laboratories was subsequently performed. By adopting identical protocols, especially the use of deionized water and making dilutions of thiabendazole in dimethyl sulfoxide in the final ring test, laboratories correctly identified both susceptible and resistant isolates. The protocols for the test and preparation of solutions of thiabendazole are described. European Commission (FOOD-CT-2005-022851)

International audience; Orientation of the immune response toward Th1, Th2, Th17 or Treg plays all important role in self-tolerance and defence against pathogens and tumors. However, this orientation has not been fully characterised in the pig and little is known about the influence of maturation stimulus Oil the capacity of dendritic cells selectively to direct different types of Th cell responses. Dendritic cell (DC) Maturation Call be induced by different agents such as inflammatory cytokines, TLR ligands and CD40L. However, the role of the latter in the maturation of pig DC has never been reported. In this study we analysed how different maturation agents influence the capacity of DC to skew the immune response. Monocyte-derived porcine DCs were matured with human CD40L-transfected L-cells, Lipopolysaccharide (LIPS) alone or LIPS in combination with Tumor necrosis factor-alpha (TNF alpha) and interferon-alpha (IFN alpha). We found that human CD40L induced DC maturation characterised by increased expression of co-stimulatory CD80/86 molecules, high production of IL-12p40 in DC and induction of IFN-gamma and t-bet mRNA in T cells, suggesting a Th I orientation. Moreover we report for the first time the appearance of CD25 after activation of porcine DC. Futhermore, DC activated with TNF + LPS + IFN showed the highest allo-stimulatory capacity of allogeneic lymphocytes and induced IL-17 mRNA in T lymphocytes, Suggesting a Th17 orientation that has never been previously reported in the pig. We also showed that immature DCs did not produce any IL-10 or IL-12 and induced both GATA-3 and IL-13 transcription in allogeneic MLR Suggesting a Th2 orientation. This study therefore underlines that the nature of the stimulus Strongly influences the capacity of DC to steer the immune response in the pig. (C) 2008 Elsevier Ltd. All rights reserved.

International audience

International audience; The chemical composition of freezing extenders plays a major role in sperm cell survival during cryopreservation. In this study we compared two extenders for freezing stallion semen: INRA82 extender (as a control) versus INRA96 (R) extender, both supplemented with egg yolk and glycerol. INRA82 contains milk, whereas INRA96 (R) is a chemically- defined extender developed for fresh semen storage at 4 degrees C or 15 degrees C. Semen from 3 stallions (7 ejaculates per stallion) was frozen in both extenders. In vitro analyses of post-thaw motility of sperm cells (computer-assisted analysis) and of membrane integrity (flow cytometry analysis) were performed. Then a fertility trial was conducted. Inseminations were conducted in a total of 84 mare cycles. INRA96 (R) extender supplemented with egg yolk and glycerol significantly improved per-cycle pregnancy rates compared with INRA82 (71% versus 40%, p < 0.01). In agreement with these fertility results, membrane integrity was better preserved in INRA96 (R) than in INRA82. In contrast, motility parameters were significantly higher in INRA82 than in INRA96 (R). Further research is needed to understand how INRA96 (R) components protect sperm cells during the cryopreservation process and highly increase their fertility potential compared with INRA82 components.

International audience; Helminthosis is one of the major constraints in the successful wool and mutton industry throughout the world. Anthelmintic Resistance (AR) is said to have been established when previously effective drug ceases to kill exposed parasitic population at the therapeutically recommended dosages. Anthelmintic resistance is almost cosmopolitan in distribution and it has been reported in almost all species of domestic animals and even in some parasites of human beings. Some of the most important species of parasites of small ruminants in which AR has been reported include: Haemonchus spp., Trichostrongylus spp. Teladorsagia spp., Cooperia spp. Nematodirus spp., and Oesophagostomum spp. All the major groups of anthelmintics have been reported for development of variable degrees of resistance in different species of gastrointestinal nematodes. This paper describes the global scenario of prevalence and methods used for detection of AR in small ruminants. Different mechanisms and contributory factors for the development of AR are discussed. Various options and alternate strategies for the control and/or delay in the onset of AR are suggested in the light of available information. (c) 2006 Elsevier Inc. All rights reserved.

Hypoosmotic swelling test (HOS) has been proposed by many authors to evaluate the functional integrity of the sperm membrane. Our approach in this experiment has consisted in exposing spermatozoa to a wide range of osmotic pressures then evaluating the reacted sperm cells by flow cytometry and finally modelling the sperm cell responses. Semen samples were diluted in skim milk or NPPC (native phosphocaseinate) extenders, and stored at 4 °C for 3 days. At D0 and D3 aliquots from each ejaculate (n = 12) were submitted to seven hypoosmotic solutions varying from 230 to 10 mOsm/kg. Sperm samples were analyzed using flow cytometry to determine two populations of spermatozoa identified by propidium iodide (PI): PI+ (including PI, red fluorescence) and PI− (excluding PI, no fluorescence). Spermatozoa PI+ were considered as spermatozoa with membrane damages. PI+ exhibited a high variation from 230 to 10 mOsm/kg which was considered as a dose–response curve. Data were modelled using Mixed procedure and probit analysis to a sigmoid curve. Each model curve characterized the profile of response of the variable PI+ to the range of osmotic pressure from 230 to 10 mOsm/kg. The estimated parameters modelling the sigmoid curves are discussed in order to evaluate the effect of extender (skim milk versus NPPC) and duration of preservation (D0 versus D3). Such modelling could help to differentiate storage method ejaculates within males or between male, contributing therefore to improve semen technology.

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

The morphogenesis and chronology of the life cycle of Nematodirus spathiger (Railliet, 1896), a parasite of ruminants, were studied in detail in an experimental host. Twenty-four worm-free rabbits were each infected per os with N. spathiger larvae and were killed at 12 h after infection (12 HAI) and every day from 1 DAI to 23 DAI. By 12 HAI , all the larvae were exsheathed and present in the small intestine. The third moult occurred between 4 DAI and 5 DAI. The last moult occurred between 13 DAI and 16 DAI. The prepatent period lasted 21-24 days. The distribution of N. spathiger along the small intestine of the rabbit was assessed. The chronology of the life cycles was compared for various Nematodirus spp from ruminants in their natural hosts and in the rabbit (N. battus, N. spathiger).

The clinical diagnosis of gastrointestinal strongylosis in ruminants is often difficult because of its relatively unspecific symptomatology. Moreover, as the use of prophylactic treatments is increasingly preferred over curative treatments, quantitative and accurate tests for diagnosis are required. As the gradual development of anthelmintic resistance restricts further the choice and use of molecules, laboratory diagnostic tests become useful and even often essential. Three main categories of tests are available : traditional parasitological techniques, serological assays linked to the lesions caused by the parasites, and tests for the detection of anthelmintic resistance. The principles, conditions of use and limits of interpretation of these tests are described in this brief synthesis., Le diagnostic clinique des strongyloses gastro-intestinales des ruminants est difficile dans un grand nombre de cas en raison du manque de symptomatologie très spécifique. De plus, La tendance de plus en plus grande à traiter les animaux préventivement et non plus curativement implique de disposer de moyens de diagnostic quantitatifs sensibles. Le développement progressif de phénomènes de résistance aux anthelminthiques impose des contraintes supplémentaires quant au choix et à l’utilisation des molécules. Les tests de diagnostic de laboratoire sont utiles et même souvent indispensables, dans ces démarches. Trois grandes catégories de tests sont disponibles : les tests parasitologiques classiques, les dosages sériques en relation avec les lésions occasionnées par les parasites, les tests de détection des résistances. Les principes, les conditions d’utilisation et les limites d’interprétation sont décrits dans cette courte synthèse., Kerboeuf Dominique. Apport des tests de laboratoire dans le diagnostic des strongyloses gastro-intestinales des ruminants et le choix des traitements. In: Bulletin de l'Académie Vétérinaire de France tome 157 n°4, 2004. pp. 17-23.

Brucella,the causative agent of brucellosis in animals and humans, can survive and proliferate within macrophages. Macrophages mediate mouse resistance to various pathogens through the expression of theNramp1gene. The role of this gene in the control ofBrucellainfection was investigated. When BALB/c mice (Nramp1s) and C.CB congenic mice (Nramp1r) were infected withBrucella melitensis,the number ofBrucellaorganisms per spleen was significantly larger in the C.CB mice than in the BALB/c mice during the first week postinfection (p.i.). ThisNramp1-linked susceptibility toBrucellawas temporary, since similar numbers ofBrucellawere recovered from the two strains of mice 2 weeks p.i. The effect ofNramp1expression occurred within splenocytes intracellularly infected byBrucella.However, there was no difference between in vitro replication rates ofBrucellain macrophages isolated from the two strains of mice infected in vivo or inNramp1RAW264 transfectants. In mice, infection withBrucellainduced an inflammatory response, resulting in splenomegaly and recruitment of phagocytes in the spleen, which was amplified in C.CB mice. Reverse transcription-PCR (RT-PCR), performed 5 days p.i., showed that inducible nitric oxide synthase, tumor necrosis factor alpha (TNF-α), interleukin-12 p40 (IL-12p40), gamma interferon (IFN-γ), and IL-10 mRNAs were similarly induced in spleens of the two strains. In contrast, the mRNA of KC, a C-X-C chemokine, was induced only in infected C.CB mice at this time. This pattern of mRNA expression was maintained at 14 days p.i., with IFN-γ and IL-12p40 mRNAs being more intensively induced in the infected C.CB mice, but TNF-α mRNA was no longer induced. The higher recruitment of neutrophils observed in the spleens of infected C.CB mice could explain the temporary susceptibility of C.CB mice toB. melitensisinfection. In contrast to infections withSalmonella, Leishmania,andMycobacterium,the expression of theNramp1gene appears to be of limited importance for the natural resistance of mice toBrucella.

Infestation with parasitic helminths is a common problem in human populations of third world countries and is ubiquitous in livestock and other domestic animals. The cell-membrane efflux pump, P-glycoprotein (Pgp), appears to contribute to anthelmintic resistance. Pgp have been identified from both phyla of parasitic helminths, Platyhelmintha and Nematoda, and alterations in expression levels and allele frequencies of Pgp in anthelmintic-resistant populations have been observed in nematodes. Localisation of Pgp has been studied in the free-living nematode Caenorhabditis elegans and in the sheep parasite Haemonchus contortus using specific monoclonal antibodies or lectins. Reversing agents used in human studies, such as the calcium-channel blocker verapamil (VPL), appear to have similar effects in helminths as they do in human cancer cells: the efficacy of drug treatment is increased in drug-resistant parasites when reversing agents are co-administered with the anthelmintic. The functional role of the Pgp glycosylation was also studied using a lectin specific for the alpha-mannosyl residues and showed that resistance can be associated with a decreased affinity of the lectin for Pgp sites and that up to 50% reversion in the resistance to benzimidazoles (BZ) can be obtained using this lectin. Furthermore, the current knowledge on the role of Pgp in molecular mechanisms of drug resistance in the parasitic protozoan genus Trypanosoma is discussed. In some Trypanosoma species it was shown that drug resistance was associated with reduced uptake and in other ones with increased efflux. Several trypanosome Pgp-coding sequences have been described. In contrast to earlier data, most recent observations, based on experimentally overexpressed Pgp in Trypanosoma brucei, indicate a possible involvement in the mechanism of drug resistance in this parasite.

Objectives: The efficiency of the anthelmintics used to treat small domestic ruminants infected with nema- todes is compromised by the emergence of resistant parasites. Both specific and non-specific mechanisms of resistance exist. The non-specific mechanisms involve multiple resistance phenomena and are depend- ent on the multidrug resistance (MDR) system, which is also responsible for the development of chemo- therapy-resistant tumour cells. We showed previously that the system also exists in nematodes. Membrane 'pumps', known as P-glycoproteins (Pgp), are activated in the MDR system. The nature of the membrane, in particular the lipids, appears to condition the activity of the pumps. Thus, we studied the effects of choles- terol on drug transport activity in the nematode Haemonchus contortus. Materials and methods: We used methyl-β-cyclodextrin to carry out cholesterol depletion and cholesterol loading experiments. The resulting changes in resistance were estimated by measuring changes in drug transport (a) by means of in vitro egg hatch assays in the presence of a benzimidazole anthelmintic, thia- bendazole and (b) by measuring the transport of rhodamine 123 (R123), a specific substrate of Pgp. We used biochemical assays to estimate the cholesterol concentration in the parasites. Results: Changes in the cholesterol content induced changes in anthelmintic resistance; cholesterol depletion gave increased resistance and cholesterol loading gave decreased resistance. These changes also altered the transport of R123. Conclusion: Cholesterol depletion or cholesterol loading allow modulation of xenobiotic resistance in nematode eggs as they do in tumour cells. The effect appears to be correlated with changes in the function of membrane P-glycoproteins. The lipid environment thus influences the nematode Pgp activity.

The parasitic phase of development of both Trichostrongylus colubriformis and Trichostrongylus vitrinus, parasites of ruminants, was studied in detail in the rabbit. In T. colubriformis, the third moult appeared by 4 days after infection (DAI) and the last moult occurred between 10 and 11 DAI. In T. vitrinus, the third moult occurred between 8 and 11 DAI and the last one between 12 and 15 DAI. The prepatent period lasted 16-17 days for T. colubriformis and 20 days for T. vitrinus. The chronology of the life cycles and the distribution of the parasites along the small intestine for various Trichostrongylus spp. from lagomorphs and ruminants in the natural host or in the experimental host were compared. All of these biological parameters indicated a lower level of adaptation of T. vitrinus compared to the other species of Trichostrongylus. The results are fully compatible with the evolutionary scheme based on morphological analyses.

The morphogenesis and the chronology of the life cycle of Nematodiroides zembrae (Bernard, 1965), a parasite of Oryctolagus cuniculus from Spain, were studied in detail in its natural host. For each experiment a morphological description of the different stages of the life cycle is provided. The free-living larvae developed in eggs until infective stage. First hatching began at 10 days. Twenty-one, worm-free rabbits were each infected per os with N. zembrae larvae and killed 3 days after infection (DAI) and every day from 4 to 22 DAI. By 3 DAI all recovered larvae were exsheathed and present in the small intestine. The third moult occurred between 6 and 7 DAI. The last moult occurred between 11 and 19 DAI. The prepatent period lasted for 21-22 days. The distribution of N. zembrae along the small intestine of the rabbit is described. Significantly different distributions of the parasite along the small intestine indicated that migrations occurred during the development of N. zembrae in the rabbit. The life cycle of N. zembrae is compared with the 5 known life cycles of Nematodirus spp. in ruminants. The biological data are very similar in both groups except for the prepatent period.

Primary cultures of Sertoli cells provide an interesting model to study how signalling pathways induced by a single hormone in a single cell type evolve, depending on the developmental stage. In vivo, follicle-stimulating hormone (FSH) induces proliferation of Sertoli cells in neonate and controls the subsequent differentiation of the entire population. Molecular mechanisms underlying Sertoli cell pleiotropic responses to FSH have long been investigated. But to date, only cAMP-dependent kinase (PKA) activation has been reported to account for most FSH biological activities in male. Here, we demonstrate that FSH activates the ERK MAP kinase pathway following dual coupling of the FSH-R both to Gs and to Gi heterotrimeric proteins, in a PKA- and also Src-dependent manner. This activation is required for FSH-induced proliferation of Sertoli cells isolated 5 days after birth. Consistently, we show that the ERK-mediated FSH mitogenic effect triggers upregulation of cyclin D1. In sharp contrast, at 19 days after birth, as cells proceed through their differentiation program, the ERK pathway is dramatically inhibited by FSH treatment. Taken together, these results show that FSH can exert opposite effects on the ERK signalling cascade during the maturation process of Sertoli cells. Thus, signalling modules triggered by the FSH-R evolve dynamically throughout development of FSH natural target cells.

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International audience; This paper reports the findings of two trials into the effects of the treatment of cattle with ivermectin slow-release (SR) bolus on the larval development of the dung beetle Aphodius constans Duft. Rectal faecal samples were collected prior to treatment and every 3 and 2 weeks in a first and second trial, respectively, and up to 156 days post-administration of the SR bolus. Faecal ivermectin concentration reached a peak at 63 days post-treatment (1427 ng$\cdot$g$^{-1}$) and ivermectin was detected up to 147 days post-treatment in the first trial (7.2 ng$\cdot$g$^{-1}$). First stage larvae of A. constans were reared with control or contaminated dung and adult beetles were counted after emergence. In the first trial, the comparison of pairwise samples showed that ivermectin prevented the development of larval A. constans until day 105, while at day 135 the rate of emergence was still significantly lower than the corresponding series of control ($p < 0.05$). In the second trial, the difference between control and treated series remained significant until 143 days post-treatment, with no emergence until 128 days post-administration of SR bolus to cattle. These results show the negative effect of ivermectin on the development of larval A. constans, even at a low concentration (38.4 ng$\cdot$g$^{-1}$). The administration of ivermectin sustained-release bolus to cattle was highly effective in killing dung beetle larvae for approximately 143 days after treatment. The results were similar when dung was obtained from a single animal kept alone, or from a blending of faecal pats obtained from a group of animals kept in field conditions during the whole trial period.; Effets négatifs des résidus d'ivermectine délivrés par un bolus à diffusion lente sur la survie du coprophage Aphodius constans (Duft.) (Coleoptera: Aphodiidae). À la suite du traitement de bovins par un bolus à diffusion lente d'ivermectine (12 mg$\cdot$j$^{-1}$), deux expérimentations indépendantes ont été conduites pour en étudier les conséquences sur le développement larvaire du coléoptère coprophage Aphodius constans Duft. Des échantillons de bouse ont été prélevés avant le traitement puis régulièrement, respectivement chaque 3 et 2 semaines pour la première et la seconde expérimentation, ceci jusqu'à 156 jours après l'administration du bolus aux animaux. Les bouses utilisées ont été congelées puis stockées plusieurs mois avant leur utilisation. La concentration fécale d'ivermectine atteint un pic 63 jours après le traitement (1427 ng$\cdot$g$^{-1}$) et l'ivermectine était encore détectable dans la bouse après 147 jours (7.2 ng$\cdot$g$^{-1}$). Des larves d'A. constans ont été nourries avec de la bouse (plusieurs séries, correspondant chacune à une date après traitement) et les imagos obtenus à l'issue du développement larvaire ont été décomptés à l'émergence. Dans la première expérience (animal isolé), la mortalité des larves a été totale jusqu'à 105 jours après le traitement, tandis que 135 jours après l'administration du bolus la bouse contenait encore suffisamment d'ivermectine pour que le taux d'émergence soit significativement inférieur à celui du témoin ($p < 0.05$). Dans la seconde expérience (bouses homogénéisées provenant de plusieurs animaux traités), les différences entre séries bolus et séries témoins restaient significatives jusqu'à 143 jours après le traitement, avec une émergence nulle jusqu'à 128 jours après le traitement. Ces résultats démontrent l'effet négatif de l'ivermectine sur le développement larvaire d'Aphodius constans, même à faible concentration (38.4 ng$\cdot$g$^{-1}$). On peut considérer que l'administration d'un bolus à diffusion lente d'ivermectine a un effet négatif sur les larves de coprophages jusqu'à 143 jours après le traitement. Ces résultats sont similaires si la bouse provient d'animaux isolés ou en mélange.

We studied the implication of cytochrome P450 enzymes in the in vitro metabolism of moxidectin (MXD) in homogenates of Haemonchus contortus adult stages (susceptible isolate, Weybridge, UK). After homogenisation in a phosphate buffer, 2 ml of homogenates (equivalent to 1 g of nematodes) were incubated with 5 microg [14C] MXD at 37 degrees C for 24 h. MXD and its metabolites were separated by HPLC with radiodetection on-line. Only one metabolite was detected and its production was inhibited by carbon monoxide. This result demonstrates that the cytochrome P450 system is implicated in the metabolisation of MXD in H. contortus susceptible to milbemycin. Furthermore, this metabolite did not match those previously described in vertebrates.

This study was designed to identify an extra-intestinal route of migration of Eimeria coecicola sporozoites and the types of cell harbouring the parasite during the invasion of the intestine. The presence of E. coecicola in blood, spleen and mesenteric lymph nodes of infected donor rabbits was demonstrated by immunohistology on donor organs and measurement of oocyst excretion by coccidia-free recipient rabbits injected with whole-cell suspensions prepared from donor tissues. Two types of donor lymphocyte, B (IgM+) and T (CD5+), were labelled using a two-colour immunofluorescence-labelling technique and separated with a cell-sorter (FACStar(Plus)). The presence of parasites in the sorted cells was assessed by direct examination and by using the same in vivo test after intravenous injection of IgM+ B or CD5+ T lymphocytes collected from donors at different times after inoculation. This test provided evidence that the parasites were alive and still infectious within the sorted lymphocytes. It was demonstrated that both B and T lymphocytes were infected.

Fluoroquinolones are often the treatment of choice in the cases of life-threatening salmonellosis due to multidrug-resistant strains (4, 27). Salmonella sp. strains that exhibit treatment-compromising resistance to fluoroquinolones are uncommon, but the increasing incidence of strains with decreased susceptibility is a matter of concern (12, 28). In other gram-negative bacteria, such as Escherichia coli, Neisseria gonorrhoeae, or Klebsiella pneumoniae, high-level fluoroquinolone resistance is always associated with the presence of multiple mutations in the quinolone resistance-determining regions (QRDRs) of the genes that code for the intracellular targets of these antibiotics, gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE) (2, 7, 11). For Salmonella enterica serovar Typhimurium, however, we showed in a previous study (9) that fluoroquinolone resistance is not well correlated with the presence of such mutations: highly fluoroquinolone-resistant mutants selected in vitro had no mutations in the genes that code for topoisomerase IV, and some had only one mutation in the gyrA gene, whereas E. coli isolates that exhibited the same level of resistance harbored at least two mutations in gyrA and one in parC (11). In addition to this mechanism of target modification that is strictly specific to quinolones, gram-negative bacteria can face the presence of toxic compounds, including antibiotics, by excluding them from the cell. Changes in the cell envelope, including loss of outer membrane porins or alterations of the lipopolysaccharide (LPS), can be partially responsible for decreased susceptibility to a wide range of unrelated antibiotics (5, 26). Active efflux systems that act synergistically with the outer membrane could have a high level of participation in the intrinsic and the acquired antibiotic resistance of gram-negative bacteria (16, 22). Some of the multidrug efflux pumps, which belong to several families, exhibit a low specificity and thus confer decreased susceptibility or even clinically significant resistance to several classes of antibiotics when they are overexpressed. It was recently shown that an S. enterica serovar Typhimurium mutant that overproduces the AcrAB efflux pump (initially identified in E. coli as a close homolog of the MexAB efflux system of Pseudomonas aeruginosa) was more resistant than the parent strain to a wide variety of compounds such as fusidic acid, chloramphenicol, tetracycline, norfloxacin, and penicillin G (23). The goal of the present study was to investigate if additional resistance mechanisms such as decreased cell envelope permeability or active efflux could explain the phenotype of S. enterica serovar Typhimurium mutants that were selected in vitro and that reached different levels of resistance to fluoroquinolone but that harbored not more than one gyrA mutation (9).

The effect of several modulators of the synthesis or activity of glutathione (GSH) on the susceptibility of Haemonchus contortus eggs susceptible or resistant to anthelmintics was investigated using in vitro egg-hatch assays. Diethylmaleate, d,l-buthionine-[S,R]-sulfoximine, and patulin induced an unexpected decrease in the susceptibility of resistant eggs to thiabendazole, which was chosen as a reference for resistance to benzimidazole compounds. The results demonstrate that the level of GSH or SH analog plays an important role in the toxicity of thiabendazole to nematode eggs. Comparison with changes observed in the cytotoxicity of antitumor or antiprotozoal drugs after GSH modulation suggests that in H. contortus eggs this increased thiabendazole tolerance might depend on different factors, whether associated or not, including the ability of thiabendazole to conjugate with parasitic GSH or analog, the potential toxicity of such conjugates, their cellular distribution, and their role in the expression of glutathione S-transferase activities, and, perhaps, in the regulation of apoptosis.

The role of membrane drug-transport mechanisms in resistance to anthelmintics was examined using a flow cytometry method. This method was adapted from assays developed for the study of similar mechanisms in tumor cells. Rhodamine 123, a P-glycoprotein transport probe, associated with the reversal agent verapamil gave a significantly higher level of green fluorescence in Haemonchus contortus-resistant eggs as compared with that of susceptible eggs. In the same way, verapamil-bodipy, a new fluorescent probe for the detection of multidrug resistance in cells, showed a significantly higher degree of binding to resistant eggs. The results confirm those obtained with biological drug assays using both anthelmintics and verapamil and provide a quantitative and effective methodology for the functional study of multidrug resistance in nematodes.

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The persistent activity of moxidectin topically administered at the dose rate of 0.5 mg kg-1 bodyweight was evaluated against experimental nematode infection in 30 calves randomly allocated to six groups. Five groups were treated on days -42, -35, -28, -21 and -14. The 6th group remained untreated as a control. On Day 0, the calves were infected experimentally with 1000 Dictyocaulus viviparus and 50,000 Ostertagia ostertagi larvae and killed 3 weeks later. The formulation of moxidectin showed excellent activity against both parasites for up to 5 weeks (> 99%). Six weeks after treatment the reduction in the number of D. viviparus was still high (> 90%). No adverse reactions to moxidectin were observed in any of the animals.

There are still several obstacles to effective cryopreservation of trout spermatozoa as seen by the large variability of fertility rates obtained with frozen–thawed spermatozoa. We investigated the applicability of flow cytometry to estimate the quality of frozen–thawed trout spermatozoa. Flow cytometric analysis was performed on fresh and cryopreserved pools of sperm after fluorescent staining with rhodamine 123 to quantify mitochondrial function and propidium iodide to assess plasma membrane integrity. Several cryoprotectants that penetrate the membrane were tested: Me 2 SO, dimethylacetamide, glycerol, methanol, and propylene glycol at various concentrations in the freezing medium (0, 5, 10, and 15%). ATP content was evaluated by bioluminescence before and after freezing. The percentage of fresh spermatozoa with a functional mitochondrion and an intact membrane was 99%. After cryopreservation, the percentage of spermatozoa with damaged membrane varied between 55 and 90%, while only a few cells (between 0.5 and 17%) displayed both an undamaged membrane and an intact mitochondrion. The plasma membrane was better protected with 10% Me 2 SO, while methanol and propylene glycol showed toxic effects. The best cryoprotectant for the mitochondrion was Me 2 SO at a concentration of 5 or 10%. These results are in agreement with fertility rates generally observed. Frozen–thawed spermatozoa lost between 50 and 90% of their initial ATP content, and the origin of this loss is discussed.

New staining methods and automated instruments are now available to evaluate the sperm cell in vitro. Individual compartments of the sperm cell, such as the nucleus and the plasma and acrosomal membranes, may be investigated, as well as the cell function as shown by mitochondria activity and capacitation. Various probes are used and they can be analyzed by direct light or fluorescent microscopy or by flow cytometry. The automated instruments allow objective and accurate analysis and quantification as well as the ability to evaluate large population of cells in a shorter time, thus providing accurate evaluation of sperm quality. However, before these test can be recommended for routine clinical and investigational use, in the stallion, they need to be confirmed on a larger number of stallions and their correlation with traditional semen parameters and with stallion fertility has to be demonstrated.

Nematode infection of cattle treated in their first year at pasture with the fenbendazole slow release bolus (‘Bolus group’) was compared during the second year with that of untreated cattle. Ostertagia was the most prevalent parasite associated with Cooperia . Except for the Dictyocaulus spp. which caused clinical signs of bronchitis in the ‘Bolus’ group the infection during the second year resulted in a moderate response of the cattle whatever the group. Rises in both pepsinogen and gastrin levels were correlated with the number of Ostertagia L 3 on herbage. Damages in the abomasal mucosa were more frequent and severe in the ‘Bolus’ group where more inflammatory signs were observed in spite of a smalleer number of worms. Nevertheless, the differencesin total weight gains were not significant thanks to a compensatory effect during the second part of the grazing season in the ‘Bolus’ group. Hypotheses related to a minimum threshold of infection during the first year necessary to develop high enough protection during the second year are discussed. The pathological effects of gastrointestinal nematodes seem to vary more according to the inflammatory response than to the number of worms.

Ultrastructural changes induced in vitro by thiabendazole, levamisole, pyrantel and ivermectin in the free living larval stages of two trichostrongyles (Heligmosomoides polygyrus and Haemonchus contortus) were analysed. The observed damage for each anthelmintic is related to the known mode of action and compared to the damage commonly described in adults. The advantage of using larvae to study the effects of anthelmintics on the fine structure of nematodes rather than adults is described. Thiabendazole induced alteration of the cellular organization especially epithelial cells of the digestive tract. Changes in mitochondria were also seen. Levamisole caused contraction of muscle fibres whereas no specific lesions were observed with pyrantel. Ivermectin caused an hypertrophy of muscular groups. The interest of such a technique in research on the modes of action of anthelmintics is emphasized.

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The efficacy of orally administered moxidectin was determined against four benzimidazole-resistant nematode isolates. At the start of the trial, 30 lambs were each infected experimentally with 20,000 third stage larvae (5000 Haemonchus contortus, 7000 Teladorsagia circumcincta, 3000 Trichostrongylus colubriformis and 5000 Cooperia curticei); 28 days later they were allocated randomly to three groups of 10: one untreated group, one group treated orally with fenbendazole (5 mg/kg bodyweight) and one group treated orally with moxidectin (0.2 mg/kg). Samples of faeces were taken five and 10 days after treatment and the lambs were killed 10 days after treatment. Fenbendazole reduced the average number of nematode eggs in faeces by 95 per cent and the average number of worms by 25 to 45 per cent according to the species. The efficacy of moxidectin against these benzimidazole-resistant isolates was 100 per cent. No adverse reactions to either of the drugs were observed.

The in vivo N-dealkylation of [13C-2]-labeled aminopyrine by the L1-L2 larvae of Heligmosomoides polygyrus was demonstrated by the use of a sensitive gas chromatography-mass spectrometry method. This is the first evidence for the possible existence of a cytochrome P-450-dependent activity in helminths.