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D Le Ray

Publications and source records attributed to D Le Ray.

At least 37 records · Page 2Linked to original sources
PubMed1999

Trypanosoma brucei spp. development in the tsetse fly: characterization of the post-mesocyclic stages in the foregut and proboscis.

Post-mesocyclic development of Trypanosoma brucei in the tsetse fly in its migration from midgut to salivary glands, was revisited by sequential microdissection, morphometry and DNA-cytofluorometry. This development started by day 6 after the infective feed, with passage of mesocyclic midgut trypomastigotes through proventriculus and upward migration along foregut and proboscis to the salivary gland ducts. Kinetics of salivary gland infection showed that colonization of the salivary glands by epimastigotes occurred only during the time-limited presence of this developmental phase in the foregut and proboscis. Post-mesocyclic trypanosomes in the foregut and proboscis were pleomorphic, with 4 morphological stages in various constant proportions and present all through from proventriculus up to the salivary gland ducts: 67% long trypomastigotes, 27% long epimastigotes, 4% long epimastigotes undergoing asymmetric cell division and 2% short epimastigotes. Measurements of DNA content demonstrated a predominant tetraploidy for 67% of these trypanosomes, the remainder consisting of the homogeneous diploid short epimastigotes and some long epimastigotes. According to the experimental data, the following sequence of trypanosome differentiation in the foregut and proboscis is proposed as the most obvious hypothesis. Incoming mesocyclic trypomastigotes (2N) from the ectoperitrophic anterior midgut start to replicate DNA to a 4N level, are arrested at this point, and differentiate into the long epimastigote (4N) which give rise, by an asymmetric cell division, to 2 unequal, diploid daughter cells: a long, probably dead-end long epimastigote and a short epimastigote. The latter is responsible for the epimastigote colonization of the salivary glands if launched at the vicinity of the gland epithelium by the asymmetric dividing epimastigote.

Animals↗
PubMed1999

Multi-centre evaluation of repeatability and reproducibility of the direct agglutination test for visceral leishmaniasis.

OBJECTIVE: To evaluate the repeatability and reproducibility of the serological direct agglutination test (DAT) for visceral leishmaniasis (VL) with aqueous antigen in a multi-centre study in VL-endemic areas in Sudan, Kenya and Nepal. METHODS: Repeatability within each centre and reproducibility between the centres' results and an external reference laboratory (Belgium) was assessed on 1596 triplicate plain blood samples collected on filter paper. RESULTS: High kappa values (range 0.86-0.97) indicated excellent DAT repeatability within the centres. The means of the titre differences between the reference laboratory and the centres in Sudan, Kenya and Nepal (2.3, 2.4 and 1.1, respectively, all significantly different from 0) showed weak reproducibility across centres. 95% of the titre differences between the reference laboratory and the respective centres were accounted for by large intervals: 0.6-9 fold titre variation for Sudan, 0.7-8 fold for Kenya and 0.26-4 fold for Nepal. CONCLUSION: High repeatability of DAT confirms its potential, but reproducibility problems remain an obstacle to its routine use in the field. Reproducibility was hindered by alteration of the antigen through temperature and shaking, especially in Kenya and Sudan, and by nonstandardization of the test reading. DAT handling procedures and antigen quality must be carefully standardized and monitored when introducing this test into routine practice.

Agglutination Tests↗
PubMed1999

Latent class analysis permits unbiased estimates of the validity of DAT for the diagnosis of visceral leishmaniasis.

BACKGROUND: Substantial uncertainty surrounds the specificity of the Direct Agglutination Test (DAT) for visceral leishmaniasis (VL) in clinical suspects, since no good gold standard exists for unequivocally identifying diseased subjects. We explored the Latent Class Analysis (LCA) modelling technique to circumvent this problem. PATIENTS AND METHODS: Data on 149 clinical suspects recruited in 1993-96 during a multicentre study in Sudan were re-examined. Clinical data, lymph node and bone marrow aspirate and DAT results were available. IFAT was performed in 1997 on stored filter paper blood of 80 individuals. Classical Validity Analysis (CVA) in a 2 x 2 contingency table with parasitology as a gold standard was compared with the parameter estimates produced by the best fitting LCA model. RESULTS: The sensitivity estimates of DAT produced by CVA (98% (89%-100%)) were almost exactly reproduced by LCA. The specificity estimates by LCA were substantially higher than those obtained in CVA. Specificity of DAT depended, however, on whether the subject was treated for VL before. In subjects without prior treatment, CVA estimated DAT specificity at 68% (56%-79%), whereas LCA estimated it at 85% (63%-100%). CONCLUSION: LCA modelling proved a useful tool, as it gave consistent estimates of test characteristics and allowed for control of confounding factors and interaction effects. Since VL is a life-threatening disease for which expensive but effective and safe treatment exists, a clinical suspect in an endemic area should be treated on the basis of a positive DAT result.

Adolescent↗
PubMed1999

Operational validation of the direct agglutination test for diagnosis of visceral leishmaniasis.

The validity of the direct agglutination test (DAT) for visceral leishmaniasis (VL) was studied with a standardized field kit on 148 clinically suspected persons and 176 healthy controls recruited between 1993 and 1994 from an endemic area in Gedaref State, Sudan. A sensitivity of 95.9% and a specificity of 99.4% were found at a 1: 8,000 cut-off titer when parasitologically confirmed cases were compared with healthy controls. While corroborating previously reported sensitivity and specificity estimates of this serodiagnostic test, this study examined the bias generated by commonly used test validation procedures. The fundamental methodologic problem in VL test validation is the absence of a reliable gold standard. Moreover, any operational guideline on DAT use has to consider the critical dependency of the predictive values of the test on VL prevalence rates. The DAT diagnostic cut-off titer depends upon many external factors, among which the prevalence of disease in the area and the case mix seem the most important.

Agglutination Tests↗
PubMed1998

Relation between variation in copy number of ribosomal RNA encoding genes and size of harbouring chromosomes in Leishmania of subgenus Viannia.

Chromosomal size polymorphism in Leishmania of subgenus Viannia has been correlated with eco-geography. The sizes of chromosomes bearing rDNA genes were determined in 69 isolates. A considerable size-variation was observed, ranging from 1100 to 1500 kb. Chromosomes of L.(V.). braziliensis, L.(V.)guyanensis and L.(V.) peruviana from northern Peru were significantly larger (200 kb) than those of L.(V.) peruviana from southern Peru. In addition, 31 out of 69 isolates presented each two different-sized homologues of the rDNA chromosome. Long range restriction mapping of three different-sized rDNA chromosomes from L.(V.)braziliensis M2903 and L.(V.)peruviana HB31 (north) and LC106 (south) each revealed three fragments delimited by PmeI restriction sites: two constant in size (the centre and one extremity of the chromosome) and one variable (the other extremity, containing a single cluster of rDNA genes). Further analysis of the M2903 rDNA chromosome allowed the localization of its 140 kb rDNA cluster at 85 kb from the telomeric end. Two arguments indicated that size-variation of the rDNA chromosome is partially due to amplification/deletion of the clustered rDNA genes: (i) size-variation of the cluster-containing fragment was proportional to the size-variation of the whole chromosome, and (ii) hybridization signal intensity of the rDNA chromosome with a small subunit rDNA probe strongly correlated with chromosomal size. Nevertheless, DNA sequences present between the rDNA cluster and the telomere might also play a role in chromosomal size polymorphism. In addition, our data suggest that rDNA gene copy number (20-40 copies cell(-1) under a diploid hypothesis) in subgenus Viannia is lower than reported previously.

Animals↗
PubMed1998

Comparison of chromosome and isoenzyme polymorphism in geographical populations of Leishmania (Viannia) peruviana.

Five chromosomes and 17 isoenzyme loci were analysed in 4 allopatric populations of Leishmania (Viannia) peruviana, and molecular distances calculated with 2 estimators, Chromosomal Size Difference Index and Jaccard Distance. Chromosome and isoenzyme data were in overall concordance: 13/30 isolates clustered similarly on the dendrograms constructed from the different estimators, and a significant correlation (P < 0.001) was observed between the molecular distances calculated from the two sets of characters. This indicates an evolutionary association between chromosomal size polymorphism and isoenzymes. Chromosomes have a faster molecular clock than isoenzymes; twice as many genotypes were identified by chromosome analysis and significant size differences (for a total of up to 500 kb for 5 chromosomes together) were observed within a given zymodeme. Chromosomes most likely represent better indicators of genetic drift than isoenzymes, as suggested by the higher correlation between both estimators of chromosomal size-polymorphism and eco-geography. Some chromosomes might present an adaptive response to environmental variation.

Animals↗
PubMed1998

Direct glutamate-mediated presynaptic inhibition of sensory afferents by the postsynaptic motor neurons.

An in vitro preparation of the crayfish central nervous system was used to study a negative feedback control exerted by the glutamatergic motor neurons (MNs) on to their presynaptic cholinergic sensory afferents. This negative control consists in small amplitude, slowly developing depolarizations of the primary afferents (sdPADs) strictly timed with MN bursts. They were not blocked by picrotoxin, but were sensitive to glutamate non-N-methyl-D-aspartate (NMDA) antagonists. Intracellular recordings were performed within thin branches of sensory terminals while electrical antidromic stimulation were applied to the motor nerves, or while glutamate (the MN neurotransmitter) was pressure-applied close to the recording site. Electrical motor nerve stimulations and glutamate pressure application had similar effects on to sensory terminals issued from the coxo-basipodite chordotonal organ (CBTs): like sdPADs, both stimulation-induced depolarizations were picrotoxin-resistant and were dramatically reduced by non-NMDA antagonist bath application. These results indicate that sdPADs are likely directly produced by MNs during locomotor activity. A functional scheme is proposed.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1998

The gp63 gene locus, a target for genetic characterization of Leishmania belonging to subgenus Viannia.

In the present study the gp63 gene locus was used as a target for genetic characterization of Leishmania parasites by 2 methods: (i) RFLP analysis with several restriction enzymes (gp63-RFLP), and (ii) intra-genic PCR amplification coupled with restriction analysis (PCR-RFLP). Both methods were applied to a large number of natural isolates belonging to 4 species of the subgenus Viannia, namely L. (V.) braziliensis, L. (V.) peruviana, L. (V.) guyanensis and L. (V.) lainsoni; reference stocks of subgenus Leishmania were included as outgroups. Multilocus isoenzyme typing (MLEE) was used as a reference. On the one hand gp63-RFLP evidenced an extensive polymorphism and revealed specific markers for subgenus, species and geographical populations: congruence with MLEE was demonstrated statistically. The particular interest of gp63-RFLP was illustrated by infra-specific polymorphism, because of the possible relationship with phenotype diversity. On the other hand intra-genic amplification was less resolutive than gp63-RFLP, but also allowed discrimination of the 2 subgenera (PCR alone) and all the species tested in the subgenus Viannia (PCR-RFLP). PCR-RFLP presents an important operational advantage as it allows genetic characterization of minute amounts of parasites, using Leishmania specific primers. The polymorphism revealed by gp63-RFLP and PCR-RFLP illustrates the very high genomic and genetic plasticity of gp63 genes.

Animals↗
PubMed1997

Development of a slide ELISA for canine leishmaniasis and comparison with four serological tests.

A slide ELISA for canine leishmaniasis was developed by using promastigotes of Leishmania infantum, and compared with microimmunodiffusion, immunoelectrophoresis, direct agglutination and indirect immunofluorescence assays. The sensitivity of all the tests was 100 per cent. The specificity of the direct agglutination test was 95 per cent but it was 100 per cent for the three other tests. There was also a positive correlation and a high level of concordance between the titres measured by the different tests.

Agglutination Tests↗
PubMed1997

Molecular characterization of antigen 24, a specific immunodominant antigen family from Leishmania infantum.

Leishmania infantum immunoelectrophoretic antigen 24 (AG 24), a visceral leishmaniasis associated immunodominant antigen, has been characterized with a monospecific antiserum by combining SDS-PAGE, immunoblotting, metabolic labelling, radio-immunoprecipitation and in vitro poly A+ mRNA translation. AG 24 appeared to correspond to a multi-antigen family of 6-9 members ranging from 20 to 31 kDa and proteinic by nature with no post-translational modifications. A similar banding pattern was recognized by infection sera. AG 24 was not found exposed on the cell surface.

Animals↗
PubMed1997

Neural mechanisms of reflex reversal in coxo-basipodite depressor motor neurons of the crayfish.

The in vitro preparation of the fifth thoracic ganglion of the crayfish was used to investigate the mechanisms underlying the reflex reversal in a sensory-motor pathway. Sensory afferent neurons from the coxo-basipodite chordotonal organ (CBCO), which senses vertical movements of the limb, connect monosynaptically with basal limb motor neurons (MNs). In tonically active preparation, stretching the CBCO (corresponding to downward movements of the leg) stimulates the levator MNs, whereas releasing the CBCO activates the depressor (Dep) MNs. These reflexes, opposed to the imposed movement, are termed resistance reflexes. By contrast, during fictive locomotion, the reflexes are reversed and termed assistance reflexes. Intracellular recordings from all 12 Dep MNs were performed in single experiments. It allowed us to characterize three types of Dep MNs according to their response to CBCO imposed step-and-ramp movements: 8 of the 12 Dep MNs are resistance MNs that are depolarized during release of the CBCO and are connected monosynaptically to release-sensitive CBCO neurons; 1 Dep MN is an assistance MN that is depolarized during stretching of the CBCO and is connected monosynaptically to exclusively velocity-coding stretch-sensitive CBCO neurons; in our experimental conditions, 3 Dep MNs do not display any response to CBCO stimulation. Assistance reflex interneurons (ARINs), involved in polysynaptic assistance reflexes recorded from depressor MNs, are presented. During low-velocity (0.05 mm/s) stretching ramps imposed on the CBCO, ARINs display compound excitatory postsynaptic potentials (EPSPs), whereas during high-velocity (0.25 mm/s) ramps, they display a mixed excitatory and inhibitory response. Whereas a single MN generally receives monosynaptic EPSPs from three to six CBCO neurons, ARINs receive monosynaptic EPSPs from up to eight velocity-coding stretch-sensitive CBCO neurons. In addition, ARINs receive disynaptic inhibitory phasic inputs from stretch-sensitive CBCO afferents. Injection of a depolarizing current pulse into ARINs elicits a fast transient voltage-dependent depolarization. Its time to peak decreases, and its peak amplitude increases with increasing current intensity. ARINs likely are to be connected directly to Dep MNs. The synaptic delay between these nonspiking ARINs and Dep MNs is short (<2 ms) and constant. The postsynaptic EPSP amplitude increases with increasing current pulse intensity injected into ARIN. The dual sensory control (excitatory and inhibitory) makes it likely that ARIN represents a key element in reflex reversal control.

Adaptation, Physiological↗
PubMed1997

Functional analysis of the sensory motor pathway of resistance reflex in crayfish. I. Multisensory coding and motor neuron monosynaptic responses.

An in vitro preparation of the fifth thoracic ganglion of the crayfish was used to study in detail the negative feedback loop involved in the control of passive movements of the leg. Release-sensitive primary afferents of from the coxo-basipodite chordotonal organ (CBCO), a proprioceptor whose strand is released by upward movement of the leg, monosynaptically connect to depressor motor neurons (Dep MNs). Extracellular identification of sensory units from the CBCO neurogram allowed us to determine the global coding of a sine-wave movement, imposed from the most released position of the CBCO strand. Intracellular recordings from sensory terminals (CBTs) and ramp movement stimulations applied to the CBCO strand allowed us to characterize two groups of release-sensitive CBCO fibers. The first group, divided into two subgroups (phasic and phaso-tonic), is characterized by discontinuous firing patterns: phasic CBTs fired exclusively during release movements; phaso-tonic CBTs displayed both a phasic firing and a tonic discharge during the more released plateaus. The second group was continuously firing whatever the movement, with higher frequencies during the release phase of the movement stimulation. All CBTs displayed a marked sensitivity for release movements while only the phaso-tonic ones showed a clear sensitivity to maintained positions. Surprisingly, no pure tonic sensory fibers were encountered. Systematic intracellular recordings from all resistant Dep MNs, performed in high divalent cation saline, allowed us to describe two shapes of monosynaptic resistance reflex responses. A phasic response was characterized by bursts of excitatory postsynaptic potentials (EPSPs) occurring exclusively during CBCO strand release movements. A phaso-tonic response was characterized by a progressive depolarization occurring all along the release phase of the stimulation: during maintained released positions, the amplitude of the sustained depolarization was position dependent; in addition, each release movement produced a phasic burst of EPSPs in the MN. The parallel study of the Dep MN properties failed to point out any correlation between the type of reflex response recorded from the MN and the MN intrinsic properties, which would indicate that the type of MN response is entirely determined by the afferent messages it receives.

Animals↗
PubMed1997

Functional analysis of the sensory motor pathway of resistance reflex in crayfish. II. Integration Of sensory inputs in motor neurons.

The in vitro preparation of the fifth thoracic ganglion of the crayfish was used to analyze the connections supporting the monosynaptic reflex responses recorded from the depressor motor neurons (Dep MNs). Dep MNs are directly connected by the release-sensitive afferents from a proprioceptor, the coxo-basipodite chordotonal organ (CBCO), which is released by upward movements of the leg. Sine-wave movements, applied to the CBCO strand from the most released position, allowed us to stimulate the greatest part of release-sensitive CBCO fibers. Systematic intracellular recordings from all Dep MNs performed in high divalent cation saline allowed us to determine the connections between CBCO afferents and their postsynaptic Dep MNs: it highlighted the sequential activation of the different Dep MNs involved in the monosynaptic reflex. The convergence of different sensory afferents onto a given Dep MN, and the divergence of a given sensory afferent onto several Dep MNs illustrates the complexity of the sensory-motor reflex loops involved in the control of locomotion and posture. Electrophysiological experiments and simulations were performed to analyze the mechanisms by which Dep MNs integrate the large amount of sensory input that they receive. Paired intracellular recording experiments demonstrated that postsynaptic response shapes characteristic of both phasic and phaso-tonic afferents could be induced by varying the presynaptic firing frequency, whatever the postsynaptic Dep MN. Compartment model simulations were used to analyze the role of the sensory-motor synapse characteristics in the summation properties of postsynaptic MN. They demonstrated the importance of the postsynaptic compartment geometry, because large postsynaptic compartments allowed to generate greater excitatory postsynaptic potential (EPSP) summations than small ones. The results presented show that velocity information is the most effective to elicit large compound EPSPs in MNs. We therefore suggest that the negative feedback reflex is mainly based on the detection of leg movements.

Animals↗
PubMed1996

Trypanosome-binding proteins of the tsetse flies Glossina palpalis gambiensis and G. morsitans morsitans.

In this paper we describe a new, selective approach to identify protein ligand-receptor interactions between an arthropod vector and the parasite it transmits. Biotinylated vector proteins were incubated with living parasites in physiological conditions. After extensive washing, the parasites were subjected to SDS-PAGE electrophoresis and the polypeptides were electroblotted onto nitrocellulose membrane. Staining with avidin-horseradish peroxidase revealed only biotin-labeled proteins from the vector which were bound to the parasite. A multitude of tissue-specific proteins of Glossina palpalis gambiensis and G. morsitans morsitans proteins, able to bind to cultured procyclic trypanosomes of Trypanosoma brucei spp., has been demonstrated. The relevance of these interactions in relation to the developmental journey of the trypanosome in the tsetse fly is briefly discussed.

Animals↗
PubMed1995

Stimuli of differentiation regulate RNA elongation in the transcription units for the major stage-specific antigens of Trypanosoma brucei.

In Trypanosoma brucei, the mutually exclusive expression of the major surface antigens, the variant surface glycoprotein (VSG) of the bloodstream form and procyclin of the procyclic form, is due to a stage-specific accumulation of the respective mRNAs. Through the targeting of a reporter construct in the procyclin promoter region, we show that independently of any selection pressure, a relatively high level of transcription (approximately 10%) occurs from the procyclin promoter in the bloodstream form. This transcription leads to the production of detectable amounts of polyadenylated mRNAs. However, RNA elongation in the procyclin transcription unit is down-regulated at this stage. Transcription elongation in the procyclin and VSG units is inversely controlled by the combination of factors which cause the differentiation of bloodstream into procyclic forms in vitro. These factors include temperature, citrate/cis-aconitate and the incubation medium. Our results suggest that inverse regulations of primary transcription in the VSG and procyclin units are early events that underly the differentiation of the parasite.

Animals↗
PubMed1995

Putative Leishmania hybrids in the Eastern Andean valley of Huanuco, Peru.

During an outbreak of tegumentary leishmaniasis that developed in the 1990s in the Eastern Andean valley of Huanuco, Peru, the coexistence of Andean (uta) and sylvatic leishmaniases was suspected for ecological and geographical reasons, and sympatric sampling was carried out. Seven human isolates of Leishmania were characterized by multilocus enzyme electrophoresis, random amplification of polymorphic DNA and molecular karyotyping. The three methods identified 3 isolates as L. braziliensis, and 4 isolates as putative hybrids with characters of L. braziliensis and L. peruviana. Data from Huanuco are compared to previous results from other areas endemic for uta. Biological and epidemiological implications are discussed.

Animals↗