Search

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.

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.

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

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).

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.

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.

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.

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.

International audience

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.

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.

International audience

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.

Mice were multiply infected with increasing doses of Heligmosomoides polygyrus larvae. On Day 9 or on Day 30 after the beginning of infections, an anthelmintic treatment was administered and its effect on reinfections and egg production was studied. The time of treatment was found to be an important factor, since early treatment produced marked resistance to subsequent infection, whereas a similar effect was not observed for the late treatment. Most immunity appeared after a lag time of about 3 weeks following treatment. Mice placed in direct contact with high doses of larvae had massive infections, and large quantities of worm eggs were recovered in the faeces. After larval doses were no longer given, there was a decrease of the worm burden in both treated and untreated animals. Studies of egg laying showed that the increased resistance induced by the treatment was also responsible for a reduced egg production. The relationship between fertility of H. polygyrus females and the size of young parasite populations was positive, but was negative for older populations. The slope of the regression line of this relationship was steeper in treated than in untreated mice. Egg production by a female worm was depressed to a certain extent by the resistance reactions of the host, but did not decrease below a threshold value of 100 eggs per female per day.

The efficacy of the morantel sustained release bolus in controlling parasitic gastroenteritis in 153 first-season grazing cattle was assessed in three separate field trials conducted in Normandy, France. In each trial, comparisons were made on weight gain performance and parasitology data (faecal worm egg counts, herbage larval counts and- in two of the trials- worm counts from principal animals sacrificed at the end of the grazing season) when bolus treatment was given either at spring turnout or in mid-season in order to determine the optimum time for bolus administration. Cattle were allocated into three groups, each group maintained on a separate but equivalent paddock constructed from the division of a larger pasture. A morantel sustained release bolus was administered to one group of animals at the time of turnout and to a second group of animals in midsummer. The third group of animals in each trial remained nontreated. The effect of the treatment on the contamination of pasture, and parasite levels and weight gain of the principal trial animals was assessed. Similar results were observed in all three trials. Faecal worm egg counts were reduced during the first part of the grazing season in animals receiving the bolus at turnout compared with mid-season treated animals where egg counts followed a pattern similar to the controls until bolus treatment at which time counts abruptly dropped to a low level. Likewise, levels of infective larvae on pastures grazed by control and mid-season treated animals followed similar patterns, increasing to a high level in late summer, while larval levels on pastures grazed by early-season treated animals remained at low levels throughout most of the season. Serum pepsinogen levels, worm counts and weight gain reflected the results from faecal worm egg and herbage larval counts indicating that early-season treatment with the bolus provided the most efficient treatment time for controlling parasitic gastroenteritis throughout the grazing season. The overall mean weight gain advantage of the early-season bolus-treated animals over the controls was 37.2 kg (P less than 0.01) while the advantage of the mid-season treated animals over controls was 13.7 kg.

In control and Trichostrongylus colubriformis-infected rabbits histological samples were taken from the small intestine at 25-cm intervals from the pylorus. Length of villi, surface of glands of Lieberkühn, and number of epithelial cells per unit length were measured with a digitized table. Two zones were so delimited along the parasitized mucosa: in the proximal part of the gut, both shortened villi and dilated crypts were found; in the distal region of the intestine, which was also the area with the lowest density of parasites, dilatation of crypts was associated with an increase in the size of villi. In both regions, the size of enterocytes was unchanged. Correlation coefficients calculated between worm burdens and relative variation in length of villi were significant and negative. Conversely, the same coefficients were significant and positive between worm burdens and surface of crypts. The possibility of local and general actions of Trichostrongylus colubriformis on both villi and crypts is evoked to explain the changes in the two zones. The functional importance of the hypertrophy in the distal region is still to be investigated during a T. colubriformis infection.

International audience