Biomedical subjects

C Saillard

Publications and source records attributed to C Saillard.

At least 19 recordsLinked to original sources

PubMed2004

Disruption of a gene predicted to encode a solute binding protein of an ABC transporter reduces transmission of Spiroplasma citri by the leafhopper Circulifer haematoceps.

Spiroplasma citri is transmitted from plant to plant by phloem-feeding leafhoppers. In an attempt to identify mechanisms involved in transmission, mutants of S. citri affected in their transmission must be available. For this purpose, transposon (Tn4001) mutagenesis was used to produce mutants which have been screened for their ability to be transmitted by the leafhopper vector Circulifer haematoceps to periwinkle plants. With one mutant (G76) which multiplied in leafhoppers as efficiently as S. citri wild-type (wt) strain GII-3, the plants showed symptoms 4 to 5 weeks later than those infected with wt GII-3. Thirty to fifty percent of plants exposed to leafhoppers injected with G76 remained symptomless, whereas for wt GII-3, all plants exposed to the transmission showed severe symptoms. This suggests that the mutant G76 was injected into plants by the leafhoppers less efficiently than wt GII-3. To check this possibility, the number of spiroplasma cells injected by a leafhopper through a Parafilm membrane into SP4 medium was determined. Thirty times less mutant G76 than wt GII-3 was transmitted through the membrane. These results suggest that mutant G76 was affected either in its capacity to penetrate the salivary glands and/or to multiply within them. In mutant G76, transposon Tn4001 was shown to be inserted into a gene encoding a putative lipoprotein (Sc76) In the ABCdb database Sc76 protein was noted as a solute binding protein of an ABC transporter of the family S1_b. Functional complementation of the G76 mutant with the Sc76 gene restored the wild phenotype, showing that Sc76 protein is involved in S. citri transmission by the leafhopper vector C. haematoceps.

ATP-Binding Cassette Transporters↗

PubMed2001

Mycoplasmas, plants, insect vectors: a matrimonial triangle.

Plant pathogenic mycoplasmas were discovered by electron microscopy, in 1967, long after the discovery and culture in 1898 of the first pathogenic mycoplasma of animal origin, Mycoplasma mycoides. Mycoplasmas are Eubacteria of the class Mollicutes, a group of organisms phylogenetically related to Gram-positive bacteria. Their more characteristic features reside in the small size of their genomes, the low guanine (G) plus cytosine (C) content of their genomic DNA and the lack of a cell wall. Plant pathogenic mycoplasmas are responsible for several hundred diseases and belong to two groups: the phytoplasmas and the spiroplasmas. The phytoplasmas (previously called MLOs, for mycoplasma like organisms) were discovered first; they are pleiomorphic, and have so far resisted in vitro cultivation. Phytoplasmas represent the largest group of plant pathogenic Mollicutes. Only three plant pathogenic spiroplasmas are known today. Spiroplasma citri, the agent of citrus stubborn was discovered and cultured in 1970 and shown to be helical and motile. S. kunkelii is the causal agent of corn stunt. S. phoeniceum, responsible for periwinkle yellows, was discovered in Syria. There are many other spiroplasmas associated with insects and ticks. Plant pathogenic mycoplasmas are restricted to the phloem sieve tubes in which circulates the photosynthetically-enriched sap, the food for many phloem-feeding insects (aphids, leafhoppers, psyllids, etc.). Interestingly, phytopathogenic mycoplasmas are very specifically transmitted by leafhoppers or psyllid species. In this paper, the most recent knowledge on phytopathogenic mycoplasmas in relation with their insect and plant habitats is presented as well as the experiments carried out to control plant mycoplasma diseases, by expression of mycoplasma-directed-antibodies in plants (plantibodies).

Animals↗

PubMed2001

[Perceived health survey of 3,800 French adolescents: methodological aspects].

An epidemiological survey was carried out by a local association network and was developed in partnership with regional and national institutions. The survey on well-being and health was conducted over an 18-month period in the French department of Charente. The purpose of the research was to test the hypothesis that "the change in context that is experienced by adolescents at the end of junior high school and at the beginning of high school leads to an increase in risky behaviours". To this end, the survey questioned 3,800 young people on a random basis, mixing those from different grades, those participating in vocational training and apprenticeships, and those from public and private schools. The accomplishment of this survey was made possible thanks to the motivation of a pre-existing strong local network organised and linked to education professionals, the technical contribution of the region's health observatory who conducted the statistical analysis of the data, and the assistance of Unit 472 of the French Institute of Health and Medical Research who provided scientific supervision and advice. The high participation rate and quality of the data (comparable with and even better than some existing studies) demonstrate that it is possible for local associations to carry out effective epidemiological surveys based on the needs keenly felt by the workers themselves, within the scope of a reasonable budget and in a short time span without sacrificing the precision demanded by the scientific method.

Adolescent↗

PubMed2000

A nested PCR assay for the detection of Mycoplasma hyopneumoniae in tracheobronchiolar washings from pigs.

A nested polymerase chain reaction (PCR) was developed for the detection of Mycoplasma hyopneumoniae, the etiological agent of enzootic pneumonia, in tracheobronchiolar washings from live pigs. Two nested pairs of oligonucleotide primers were designed from the sequence of a specific DNA probe (I 141; accession number U02537). The primer combination was Hp1/Hp3 for the first step PCR while the nested primers (Hp4/Hp6) allowed amplification of a 706 bp fragment. All strains of M. hyopneumoniae tested in this study could be detected by the nested PCR. DNA from other bacterial species isolated from the respiratory tract of pigs or from other mycoplasmal species were not amplified. The detection limit was estimated to be 1 fg, corresponding approximately to one organism, while in the one step PCR previously described 4 x 10(2) organisms were required. The nested PCR was evaluated on 362 tracheobronchiolar lavages collected from pigs at 2, 4 and 6 months of age in eight herds chronically infected with M. hyopneumoniae. The nested PCR was compared to a blocking ELISA performed with sera collected from the same pigs at the same ages, and to an immunofluorescence test at slaughter on 65 lungs from 6-month old pigs. The comparison indicated that the nested PCR was significantly (p<0.05) more sensitive (157 positive results of 362 samples) than ELISA (118 positive results of 362 samples) for detection of M. hyopneumoniae infection. Nested PCR was also significantly more sensitive (54 positive results of 65 samples) than immunofluorescence (29 positive results of 65 samples) for detection of M. hyopneumoniae in pig lungs at slaughter. Moreover, the nested PCR was used to confirm the absence of the mollicute in a pig herd without any history of M. hyopneumoniae infection. Thus, nested PCR appears to be a useful test to assess M. hyopneumoniae infection on pig farms.

Animals↗

PubMed2000

Genetic variability of apple proliferation phytoplasmas as determined by PCR-RFLP and sequencing of a non-ribosomal fragment.

Apple proliferation phytoplasmas are considered as quarantine organisms in Europe and north America, but reliable polymerase chain reaction (PCR) primers for their identification in routine diagnosis were missing, because they show genetic variability. Therefore, 100 apple proliferation phytoplasma isolates, derived from most of the European countries where apple proliferation disease has been detected, were analysed for their genetic variability. A detailed restriction fragment length polymorphism (RFLP) analysis of a 1.5 kbp chromosomal DNA fragment amplified by PCR (PCR-RFLP) from various isolates of apple proliferation phytoplasma revealed three different subtypes named AP, AT-1 and AT-2. Sequence analysis of a 846 bp fragment of each subtype showed that the sequences differed only in the restriction sites responsible for the observed polymorphism. Thus, the apple proliferation phytoplasma subtypes are very closely related. The observed point mutations were responsible for specific amino acid changes in the putative protein PR3. No geographic prevalence of a given subtype could be observed. In a 5-year study a given subtype could be repeatedly amplified from the same tree indicating a stable maintenance of the subtype. The AP-specific primers used in this study enabled a one-step identification of all isolates suitable for routine diagnosis

DNA Primers↗

PubMed2000

PCR-RFLP and sequence analysis of a non-ribosomal fragment for genetic characterization of European stone fruit yellows phytoplasmas infecting various Prunus species.

A 927 bp non-ribosomal fragment was used to assess the genetic variability of the European stone fruit yellows (ESFY) phytoplasma infecting 14 different Prunus species. For this, 175 isolates originating from four different Mediterranean countries were tested by PCR-RFLP analysis with seven restriction enzymes. No polymorphism among the ESFY phytoplasma could be observed but 12 out of 18 restriction sites differed between the homologous fragments of ESFY and apple proliferation (AP) phytoplasmas. An 846 bp fragment of a French ESFY isolate was sequenced, it included the 3'-end of a putative nitroreductase gene, an intergenic region and a truncated open reading frame. This ESFY phytoplasma sequence showed 89.7% identity with the equivalent AP phytoplasma nucleotide sequence (83. 9% identity at the amino acid level). The G+C content of the entire sequence was extremely low (15.4%) and A+T-rich codons were highly preferred in codon usage. In this paper, we report the presence of the ESFY phytoplasma for the first time in Turkey and in five Prunus hosts never reported previously. Our results also indicate that the ESFY phytoplasma isolates affecting various Prunus species are genetically homogenous but can be distinguished from the AP phytoplasma. Therefore, they are likely to represent different taxons.

Amino Acid Sequence↗

PubMed2000

Use of an internal control in a nested-PCR assay for Mycoplasma hyopneumoniae detection and quantification in tracheobronchiolar washings from pigs.

We have previously reported a nested PCR assay for the detection of Mycoplasma hyopneumoniae directly in tracheobronchiolar washings from living pigs in field conditions. Here, we describe the construction and use of an internal control to monitor the presence of PCR inhibitors. A PCR modified target DNA was constructed by insertion of a small DNA fragment into the M. hyopneumoniae specific DNA target. We have demonstrated that the internal control failed to be amplified in only three tracheobronchiolar washings samples out of the 362 tested. This control molecule was inserted in a Spiroplasma citri derived plasmid vector and introduced into S. citri cells by electroporation. After a few passages we ensured that the recombinant plasmid became inserted into the genome of S. citri. PCR amplification of the DNA of this transformed S. citri strain using nested PCR primers led to amplification of a 900-bp fragment which can be discriminated from the M. hyopneumoniae PCR product 700 bp. The S. citri transformants with the integrated internal control were added to the tracheobronchiolar washings prior to PCR and used as an internal control to check the efficiency of sample processing, and to demonstrate the presence of inhibitors. Furthermore, we have been able to estimate the number of mycoplasma cells in the tracheobronchiolar washings. Quantitation was performed by comparing the PCR signal intensity of the specific M. hyopneumoniae template with known concentrations of the S. citri competitor. The titer in tracheobronchiolar washings ranged approximatively from 10(4)to 10(8)M. hyopneumoniae cells per ml of clinical specimen. Quantitative PCR can be a useful tool for monitoring the progression of M. hyopneumoniae in the disease process.

Animals↗

PubMed1998

Purification, cloning, and preliminary characterization of a Spiroplasma citri ribosomal protein with DNA binding capacity.

The rpsB-tsf-x operon of Spiroplasma citri encodes ribosomal protein S2 and elongation factor Ts, two components of the translational apparatus, and an unidentified X protein. A potential DNA-binding site (a 20-base pair (bp) inverted repeat sequence) is located at the 3' end of rpsB. Southwestern analysis of S. citri proteins, with a 30-bp double-stranded oligonucleotide probe (IRS), containing the 20-bp inverted repeat sequence and the genomic flanking sequences, detected an IRS-binding protein of 46 kDa (P46). P46 protein, which displays preferential affinity for the IRS, was purified from S. citri by a combination of affinity and gel filtration chromatographies. The native form of P46 seems to be homomultimeric as estimated by SDS-polyacrylamide gel electrophoresis analysis and gel filtration. A 3.5-kilobase pair S. citri DNA fragment comprising the P46 gene and flanking sequences was cloned and sequenced. Sequence analysis of this DNA fragment indicated that the P46 gene is located within the S10-spc operon of S. citri at the position of the gene coding for ribosomal protein L29 in the known S10-spc operons. The similarity between the N-terminal domain of P46 and the L29 ribosomal protein family and the presence of a 46-kDa IRS-binding protein in S. citri ribosomes indicated that P46 is the L29 ribosomal protein of S. citri. We suggest that P46 is a bifunctional protein with an L29 N-terminal domain and a C-terminal domain involved in IRS binding.

Bacterial Outer Membrane Proteins↗

PubMed1998

Gene organization and transcriptional analysis of the Spiroplasma citri rpsB/tsf/x operon.

The nucleotide sequence of a 6863-bp Spiroplasma citri DNA fragment comprising the spiralin gene was determined. Sequence analysis revealed eight putative ORFs that encode ribosomal protein S2, elongation factor Ts, spiralin, 6-phosphofructokinase, pyruvate kinase, and three unidentified proteins (A, B, and X). The gene organization reported here is different from that previously published. Northern blot analysis of rpsB, tsf, and x transcripts indicates that these genes are organized into a single transcriptional unit (operon). However, the detection of an additional transcript corresponding to the rpsB gene alone suggests that a transcriptional mechanism should occur in the 3' region of the rpsB gene, allowing a conditional transcription termination.

Bacterial Proteins↗

PubMed1997

Random insertion of transposon Tn4001 in the genome of Spiroplasma citri strain GII3.

Electroporation of Spiroplasma citri strain GII3 with plasmid pMUT containing the Staphylococcus aureus transposon Tn4001 resulted in random insertion of Tn4001 into the spiroplasmal genome. Transformation frequencies reached 10(-8) per colony-forming unit (CFU) when 100 microg of plasmid DNA and 3 x 10(9) S. citri CFU were used. Three other strains of S. citri failed to be transformed under the same conditions. In most cases Tn4001 was randomly inserted in the genome of S. citri strain GII3, without insertion of the carrier plasmid. For most transformed spiroplasmas, Tn4001 was stably maintained in the absence of antibiotic selection for at least 80 bacterial generations, making Tn4001 a potential tool for S. citri mutagenesis.

Anti-Bacterial Agents↗

PubMed1997

Sequence analysis of Spiroplasma phoeniceum and Spiroplasma kunkelii spiralin genes and comparison with other spiralin genes.

The spiralin genes from two phytopathogenic spiroplasmas, Spiroplasma phoeniceum and Spiroplasma kunkelii, were amplified by PCR, cloned, and sequenced. Comparison of the amino acid sequences of the five spiralins analyzed to date confirm that the spiralins have a general amphiphilic character and possess a conserved lipoprotein signal peptide. It also shows that a conserved central region and an amino acid repetition, including a VTKXE consensus sequence, are present in all spiralins analyzed.

Amino Acid Sequence↗

PubMed1996

Polymerase chain reaction for Mycoplasma hyopneumoniae detection in tracheobronchiolar washings from pigs.

We have used the polymerase chain reaction (PCR) to detect Mycoplasma hyopneumoniae in tracheobronchiolar washings collected from experimentally infected piglets. On the basis of the published nucleotide sequence of M. hyopneumoniae I141 probe (accession number U02537), primers were chosen to produce an amplified fragment of 1561 bp. All the M. hyopneumoniae strains tested could be detected by the PCR test. DNA from other mycoplasmal and bacterial species currently isolated from respiratory tract of piglets gave negative result. The detection limit was estimated to be 500 fg of purified DNA corresponding to 4.10(2) organisms. The sensitivity of PCR reaction was also evaluated on microorganisms in culture, the limit sensitivity was 2.5 10(3) organisms. In the present study, a total of 143 tracheobronchiolar washings collected from experimentally infected piglets were submitted to PCR. For each tracheobronchiolar washing, PCR was performed on crude extracts treated with lysis buffer and on extracted DNA. The PCR results obtained with the two kinds of samples were compared to the immunofluorescence (IF) results. This comparison indicates a good correlation between PCR and IF test in 121/143 cases. The presence of M. hyopneumoniae is revealed in 19/143 of the washing samples only by PCR. In our hand, PCR appears to be the more sensitive test to detect M. hyopneumoniae in experimentally infected piglets.

Animals↗

PubMed1996

Serological and molecular characterization of Mesoplasma seiffertii strains isolated from hematophagous dipterans in France.

Three strains of nonhelical mollicutes previously isolated in France from two different mosquitoes and one tabanid fly were designated strains Ar 2328 (isolated from Aedes detritus), Ar 2392 (isolated from Aedes caspius), and CP 13 (isolated from Chrysops pictus). All of these strains exhibited properties of the genus Mesoplasma, a recently described genus of non-sterol-requiring mollicutes isolated from plants and insects. The results of metabolism inhibition and growth inhibition tests revealed that these strains and Mesoplasma entomophilum TAC or Mesoplasma florum L1 were not serologically related, but all three dipteran strains reacted strongly with Mesoplasma seiffertii F7T (T = type strain) antibodies. Using metabolism inhibition and growth inhibition tests, we found that the dipteran strains were related to each other and to strain F7T but were not identical. We also found that they were able to multiply and persist in the central nervous systems of suckling mice inoculated intracerebrally, a property that makes their use as biological control agents for pest dipterans inadvisable. Scanning electron microscopy revealed marked differences in the morphologies of the colonies of the different strains on SP4 solid medium. The levels of DNA-DNA homology for strains Ar 2328, Ar 2392, CP 13, and F7T were more than 70%, indicating that these strains are closely related members of the same species, M. seiffertii. In addition, one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that each strain produced about 40 protein bands. This technique also revealed differences between strains. Using the coefficient of Smeath-Jacquart, we constructed a dendrogram that allowed us to estimate of the levels of relatedness of these four strains. The results which we obtained were confirmed by two-dimensional protein electrophoresis results.

Aedes↗

PubMed1996

Analysis of putative ABC transporter genes in Mycoplasma hyopneumoniae.

A previously described DNA probe specific for Mycoplasma hyopneumoniae (I-141) was fully sequenced and found to consist of 1618 bp and to contain two tandemly repeated ORFs. The deduced amino acid sequence of the two ORFs showed significant homologies with ATP-binding cassette (ABC) transporter proteins, particularly those of the eukaryotic multidrug resistance (MDR) protein family (up to 21% identity and 47% similarity). A somewhat lower homology was evident with the secretion protein HlyB of the RTX-haemolysin from Escherichia coli. The location of the two ORFs on the M. hyopneumoniae chromosome was downstream of the rrl gene encoding the 23S rRNA, but transcribed in the opposite direction. PCR amplification and subsequent chromosomal analysis by Southern blot hybridization of several M. hyopneumoniae strains showed that all field strains contained the two putative ABC transporter genes. However, some culture collection strains derived from strain J had lost these genes as the result of a 2221 bp deletion.

ATP-Binding Cassette Transporters↗

PubMed1996

Spiralin polymorphism in strains of Spiroplasma citri is not due to differences in posttranslational palmitoylation.

Spiralin is defined as the major membrane protein of the helical mollicute Spiroplasma citri. According to the S. citri strain used, spiralin shows polymorphism in its electrophoretic mobility. The spiralin gene sequences of eight S. citri strains were determined by direct sequencing of the PCR-amplified genes. All spiralins were found to be 241 amino acids long, except for the spiralin of strain Palmyre, which is 242 amino acids long. The molecular masses calculated from these sequences did not explain the differences observed in the electrophoretic mobilities. In all of the spiralins examined, the first 24 N-terminal amino acids were conserved, including a cysteine at position 24, and had the features of typical signal peptides of procaryotic lipoproteins. When S. citri strains were grown in the presence of [3H]palmitic acid, at least 10 proteins, including spiralin, became labeled. In the presence of globomycin, a lipoprotein signal peptidase inhibitor in eubacteria, apparently unprocessed spiralin could be detected. Formic acid hydrolysis of the [3H]palmitic acid-labeled spiralins of four representative S. citri strains yielded two peptide fragments for each spiralin, as expected from the gene sequence. On fragment was [3H]palmitic acid labeled, and it had almost the same electrophoretic mobility irrespective of the spiralins used. Samples of the unlabeled peptide fragments from the four representative strains had slightly different electrophoretic mobilities (delta Da approximately equal to 800 Da); however, these were much smaller than those of the whole spiralins before formic acid hydrolysis (delta Da approximately equal to 8,000 Da). These results suggest that spiralin polymorphism in S. citri is not due to differences in posttranslational modification by palmitic acid and is certainly a structural property of the whole protein or could result from an unidentified posttranslational modification of spiralin.

Amino Acid Sequence↗

PubMed1994

Differentiation of mycoplasmalike organisms (MLOs) in European fruit trees by PCR using specific primers derived from the sequence of a chromosomal fragment of the apple proliferation MLO.

A 1.8-kb chromosomal DNA fragment of the mycoplasmalike organism (MLO) associated with apple proliferation was sequenced. Three putative open reading frames were observed on this fragment. The protein encoded by open reading frame 2 shows significant homologies with bacterial nitroreductases. From the nucleotide sequence four primer pairs for PCR were chosen to specifically amplify DNA from MLOs associated with European diseases of fruit trees. Primer pairs specific for (i) Malus-affecting MLOs, (ii) Malus- and Prunus-affecting MLOs, and (iii) Malus-, Prunus-, and Pyrus-affecting MLOs were obtained. Restriction enzyme analysis of the amplification products revealed restriction fragment length polymorphisms between Malus-, Prunus, and Pyrus-affecting MLOs as well as between different isolates of the apple proliferation MLO. No amplification with either primer pair could be obtained with DNA from 12 different MLOs experimentally maintained in periwinkle.

Base Sequence↗

PubMed1993

Detection of mollicute contamination in cell cultures by 16S rDNA amplification.

A polymerase chain reaction (PCR) system was developed for the detection of mollicutes as contaminants of cell cultures. By using three oligonucleotides chosen in the 16S rDNA sequences, two sets of primers able to promote amplification of all Mycoplasma and Ureaplasma (molli1-molli2a) or all Acholeplasma (molli1-molli2b) species examined were determined. This PCR system, first applied to experimentally infected Vero cell lines, was then evaluated for the detection of mollicutes in 86 cell culture samples, comparatively to DNA staining, culture and ELISA. The results obtained by the four techniques were in agreement in 82 cases (36 positive, 46 negative). PCR allowed detection of contamination in one and two cases negative by ELISA and culture, respectively, and confirmed questionable results obtained by DNA staining. As described, PCR seems to be a very convenient tool for routine detection of cell culture contaminants.

Acholeplasma↗

PubMed1993

Spiralins.

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Amino Acid Sequence↗