Annales de parasitologie humaine et comparée, 1990
In Meriones unguiculatus, the recovery rate of 80 inoculated larvae was low (about 20%) and irreg... more In Meriones unguiculatus, the recovery rate of 80 inoculated larvae was low (about 20%) and irregular. In the natural host Lemniscomys striatus, the recovery rate was about 50% with inoculated doses of 30, 80 or 400 L3, but slightly higher for 400 L3. This rate was constant from day 2 to month 8 post infection (p.i.). When 7-9 reinoculations were performed in one year, the recovery rate of the late inoculation was of only 14%. After subcutaneous inoculations, larvae penetrated into the peripheric lymphatic vessels from hour 6 p.i. and migrated to the lumbar and mesenteric lymph nodes; this first migratory phase was achieved 5 days p.i. Later, the larvae migrated into the digestive tract lymphatic system. Filarial localization did not depend upon the L3 dose: half were found in the caecum and anterior colon (3 cm) wall, and half were distributed in the posterior colon, mesentery and small intestine. A small number (3-5%) of the filariae were found in the pulmonary blood vessels, as a...
Techniques to preserve the infective third-stage larvae (L3) of gastrointestinal nematodes are of... more 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 f...
The control of gastro-intestinal nematodes remains largely based on anthelminthic treatments, how... more The control of gastro-intestinal nematodes remains largely based on anthelminthic treatments, however spreading of anthelmintic resistance has reduced their efficacy. The genes involved in the transition to parasitic lifestyle could constitute targets of interest to develop alternative control strategies. In the trichostrongylid nematode Haemonchus contortus, we have used a SSH (Suppressive Subtractive Hybridization) based approach to generate two distinct subtracted cDNA libraries specifically enriched in cDNA expressed during the early parasitic fourth stage larvae L4 (5 days post-infection). A total of 200 clones were subjected to dot-blot experiments and 46 clones were selected for further characterization. The 46 corresponding expressed sequence tags (EST) were found to cluster into nine contigs. The corresponding full-length cDNA was obtained for all candidates. The genes encoding potentially secreted proteins were investigated in more detail. RT-PCR experiments confirmed their specific expression or over expression from the early L4 larvae to the adult stages and search for homologs in the trichostrongylid species T. circumcincta was performed in order to investigate whether they may be novel cross-specific targets.
Acetylcholine receptors (AChRs) are pentameric ligand-gated ion channels involved in the neurotra... more Acetylcholine receptors (AChRs) are pentameric ligand-gated ion channels involved in the neurotransmission of both vertebrates and invertebrates. A number of anthelmintic compounds like levamisole and pyrantel target the AChRs of nematodes producing spastic paralysis of the worms. The muscle AChRs of nematode parasites fall into three pharmacological classes that are preferentially activated by the cholinergic agonists levamisole (L-type), nicotine (N-type) and bephenium (B-type), respectively. Despite a number of studies of the B-type AChR in parasitic species, this receptor remains to be characterized at the molecular level. Recently, we have reconstituted and functionally characterized two distinct L-AChR subtypes of the gastro-intestinal parasitic nematode Haemonchus contortus in the Xenopus laevis oocyte expression system by providing the cRNAs encoding the receptor subunits and three ancillary proteins (Boulin et al. in Br J Pharmacol 164(5):1421-1432, 2011). In the present study, the effect of the bephenium drug on Hco-L-AChR1 and Hco-L-AChR2 subtypes was examined using the two-microelectrode voltage-clamp technique. We demonstrate that bephenium selectively activates the Hco-L-AChR1 subtype made of Hco-UNC-29.1, Hco-UNC-38, Hco-UNC-63, Hco-ACR-8 subunits that is more sensitive to levamisole than acetylcholine. Removing the Hco-ACR-8 subunit produced the Hco-L-AChR2 subtype that is more sensitive to pyrantel than acetylcholine and partially activated by levamisole, but which was bephenium-insensitive indicating that the bephenium-binding site involves Hco-ACR-8. Attempts were made to modify the subunit stoichiometry of the Hco-L-AChR1 subtype by injecting five fold more cRNA of individual subunits. Increased Hco-unc-29.1 cRNA produced no functional receptor. Increasing Hco-unc-63, Hco-unc-38 or Hco-acr-8 cRNAs did not affect the pharmacological characteristics of Hco-L-AChR1 but reduced the currents elicited by acetylcholine and the other agonists. Here, we provide the first description of the molecular composition and functional characteristics of any invertebrate bephenium-sensitive receptor.
The molecular mechanisms of levamisole (LEV) activity and expression of resistance remain largely... more The molecular mechanisms of levamisole (LEV) activity and expression of resistance remain largely unknown in parasitic nematodes. In contrast, genetic screens for mutants that survive exposure to LEV in the free-living nematode Caenorhabditis elegans have led to the identification of five genes (unc-38, unc-63, unc-29, lev-1 and lev-8) that encode a LEV-sensitive acetylcholine receptor (L-AChR). Loss of these genes leads to LEV resistance. In this study, orthologues of these genes were identified in three species of trichostrongylid nematodes that have a major impact on small ruminants: Haemonchus contortus, Teladorsagia circumcincta and Trichostrongylus colubriformis. Polymorphism associated with LEV resistance have been investigated by comparing transcripts of these subunits in LEV susceptible and LEV-resistant isolates of the three strongylid species. Partial sequences were identified by PCR experiments and full-length cDNA sequences corresponding to AChR subunits in the three tr...
BACKGROUND AND PURPOSE The cholinergic agonist levamisole is widely used to treat parasitic nemat... more 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 RESUL...
cDNA-AFLP (cDNA-Amplified Fragment Length Polymorphism)-based strategy has been used to identify ... more 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.
Nicotinic acetylcholine receptors (nAChRs) of parasitic nematodes are required for body movement ... more Nicotinic acetylcholine receptors (nAChRs) of parasitic nematodes are required for body movement and are targets of important "classical" anthelmintics like levamisole and pyrantel, as well as "novel" anthelmintics like tribendimidine and derquantel. Four biophysical subtypes of nAChR have been observed electrophysiologically in body muscle of the nematode parasite Oesophagostomum dentatum, but their molecular basis was not understood. Additionally, loss of one of these subtypes (G 35 pS) was found to be associated with levamisole resistance. In the present study, we identified and expressed in Xenopus oocytes, four O. dentatum nAChR subunit genes, Ode-unc-38, Ode-unc-63, Ode-unc-29 and Ode-acr-8, to explore the origin of the receptor diversity. When different combinations of subunits were injected in Xenopus oocytes, we reconstituted and characterized four pharmacologically different types of nAChRs with different sensitivities to the cholinergic anthelmintics. ...
Annales de parasitologie humaine et comparée, 1990
In Meriones unguiculatus, the recovery rate of 80 inoculated larvae was low (about 20%) and irreg... more In Meriones unguiculatus, the recovery rate of 80 inoculated larvae was low (about 20%) and irregular. In the natural host Lemniscomys striatus, the recovery rate was about 50% with inoculated doses of 30, 80 or 400 L3, but slightly higher for 400 L3. This rate was constant from day 2 to month 8 post infection (p.i.). When 7-9 reinoculations were performed in one year, the recovery rate of the late inoculation was of only 14%. After subcutaneous inoculations, larvae penetrated into the peripheric lymphatic vessels from hour 6 p.i. and migrated to the lumbar and mesenteric lymph nodes; this first migratory phase was achieved 5 days p.i. Later, the larvae migrated into the digestive tract lymphatic system. Filarial localization did not depend upon the L3 dose: half were found in the caecum and anterior colon (3 cm) wall, and half were distributed in the posterior colon, mesentery and small intestine. A small number (3-5%) of the filariae were found in the pulmonary blood vessels, as a...
Techniques to preserve the infective third-stage larvae (L3) of gastrointestinal nematodes are of... more 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 f...
The control of gastro-intestinal nematodes remains largely based on anthelminthic treatments, how... more The control of gastro-intestinal nematodes remains largely based on anthelminthic treatments, however spreading of anthelmintic resistance has reduced their efficacy. The genes involved in the transition to parasitic lifestyle could constitute targets of interest to develop alternative control strategies. In the trichostrongylid nematode Haemonchus contortus, we have used a SSH (Suppressive Subtractive Hybridization) based approach to generate two distinct subtracted cDNA libraries specifically enriched in cDNA expressed during the early parasitic fourth stage larvae L4 (5 days post-infection). A total of 200 clones were subjected to dot-blot experiments and 46 clones were selected for further characterization. The 46 corresponding expressed sequence tags (EST) were found to cluster into nine contigs. The corresponding full-length cDNA was obtained for all candidates. The genes encoding potentially secreted proteins were investigated in more detail. RT-PCR experiments confirmed their specific expression or over expression from the early L4 larvae to the adult stages and search for homologs in the trichostrongylid species T. circumcincta was performed in order to investigate whether they may be novel cross-specific targets.
Acetylcholine receptors (AChRs) are pentameric ligand-gated ion channels involved in the neurotra... more Acetylcholine receptors (AChRs) are pentameric ligand-gated ion channels involved in the neurotransmission of both vertebrates and invertebrates. A number of anthelmintic compounds like levamisole and pyrantel target the AChRs of nematodes producing spastic paralysis of the worms. The muscle AChRs of nematode parasites fall into three pharmacological classes that are preferentially activated by the cholinergic agonists levamisole (L-type), nicotine (N-type) and bephenium (B-type), respectively. Despite a number of studies of the B-type AChR in parasitic species, this receptor remains to be characterized at the molecular level. Recently, we have reconstituted and functionally characterized two distinct L-AChR subtypes of the gastro-intestinal parasitic nematode Haemonchus contortus in the Xenopus laevis oocyte expression system by providing the cRNAs encoding the receptor subunits and three ancillary proteins (Boulin et al. in Br J Pharmacol 164(5):1421-1432, 2011). In the present study, the effect of the bephenium drug on Hco-L-AChR1 and Hco-L-AChR2 subtypes was examined using the two-microelectrode voltage-clamp technique. We demonstrate that bephenium selectively activates the Hco-L-AChR1 subtype made of Hco-UNC-29.1, Hco-UNC-38, Hco-UNC-63, Hco-ACR-8 subunits that is more sensitive to levamisole than acetylcholine. Removing the Hco-ACR-8 subunit produced the Hco-L-AChR2 subtype that is more sensitive to pyrantel than acetylcholine and partially activated by levamisole, but which was bephenium-insensitive indicating that the bephenium-binding site involves Hco-ACR-8. Attempts were made to modify the subunit stoichiometry of the Hco-L-AChR1 subtype by injecting five fold more cRNA of individual subunits. Increased Hco-unc-29.1 cRNA produced no functional receptor. Increasing Hco-unc-63, Hco-unc-38 or Hco-acr-8 cRNAs did not affect the pharmacological characteristics of Hco-L-AChR1 but reduced the currents elicited by acetylcholine and the other agonists. Here, we provide the first description of the molecular composition and functional characteristics of any invertebrate bephenium-sensitive receptor.
The molecular mechanisms of levamisole (LEV) activity and expression of resistance remain largely... more The molecular mechanisms of levamisole (LEV) activity and expression of resistance remain largely unknown in parasitic nematodes. In contrast, genetic screens for mutants that survive exposure to LEV in the free-living nematode Caenorhabditis elegans have led to the identification of five genes (unc-38, unc-63, unc-29, lev-1 and lev-8) that encode a LEV-sensitive acetylcholine receptor (L-AChR). Loss of these genes leads to LEV resistance. In this study, orthologues of these genes were identified in three species of trichostrongylid nematodes that have a major impact on small ruminants: Haemonchus contortus, Teladorsagia circumcincta and Trichostrongylus colubriformis. Polymorphism associated with LEV resistance have been investigated by comparing transcripts of these subunits in LEV susceptible and LEV-resistant isolates of the three strongylid species. Partial sequences were identified by PCR experiments and full-length cDNA sequences corresponding to AChR subunits in the three tr...
BACKGROUND AND PURPOSE The cholinergic agonist levamisole is widely used to treat parasitic nemat... more 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 RESUL...
cDNA-AFLP (cDNA-Amplified Fragment Length Polymorphism)-based strategy has been used to identify ... more 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.
Nicotinic acetylcholine receptors (nAChRs) of parasitic nematodes are required for body movement ... more Nicotinic acetylcholine receptors (nAChRs) of parasitic nematodes are required for body movement and are targets of important "classical" anthelmintics like levamisole and pyrantel, as well as "novel" anthelmintics like tribendimidine and derquantel. Four biophysical subtypes of nAChR have been observed electrophysiologically in body muscle of the nematode parasite Oesophagostomum dentatum, but their molecular basis was not understood. Additionally, loss of one of these subtypes (G 35 pS) was found to be associated with levamisole resistance. In the present study, we identified and expressed in Xenopus oocytes, four O. dentatum nAChR subunit genes, Ode-unc-38, Ode-unc-63, Ode-unc-29 and Ode-acr-8, to explore the origin of the receptor diversity. When different combinations of subunits were injected in Xenopus oocytes, we reconstituted and characterized four pharmacologically different types of nAChRs with different sensitivities to the cholinergic anthelmintics. ...
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Papers by Jacques Cabaret