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J Nicolet

Publications and source records attributed to J Nicolet.

At least 19 recordsLinked to original sources
PubMed1999

IS1634, a novel insertion element creating long, variable-length direct repeats which is specific for Mycoplasma mycoides subsp. mycoides small-colony type.

A new insertion sequence, IS1634, has been identified in Mycoplasma mycoides subsp. mycoides small-colony type (SC). IS1634 shows structural and functional similarities to IS1549 of Mycobacterium smegmatis and with it seems to form a new class or family of insertion sequences. IS1634 has a size of 1,872 bp, including two 13-bp terminal inverted repeats. It contains an open reading frame (ORF) encoding a product of 533 amino acids which shows similarity to the transposase of IS1549 and to a lesser extent to the transposases of IS elements of the IS4 family. IS1634 is present at about 30 copies in the genome of all 22 different field strains of M. mycoides subsp. mycoides SC tested. Characteristic of IS1634 are the long and variable-length direct repeats at the sites of insertion which were found to reach up to about 500 bp. IS1634 is specific to M. mycoides subsp. mycoides SC and is not present in any of the other members of the M. mycoides cluster. Neither was it found in other closely related Mycoplasma species of ruminants.

Cloning, Molecular↗
PubMed1999

Prevalence of beta2-toxigenic Clostridium perfringens in horses with intestinal disorders.

The incidence of a new, yet unassigned toxin type of Clostridium perfringens containing the genes for the alpha-toxin and the recently described beta2-toxin in horses with intestinal disorders is reported. The study included 18 horses suffering from typical typhlocolitis, 7 horses with atypical typhlocolitis, 16 horses with other intestinal disorders, and 58 horses without intestinal disease. In total, 20 samples of ingesta of the small and large intestines, five biopsy specimens of the intestinal wall, and 74 fecal samples were analyzed bacteriologically. C. perfringens isolates were typed for the presence of the alpha-, beta-, beta2-, and epsilon-toxin and enterotoxin genes by PCR, including a newly developed PCR for the detection of the beta2-toxin gene cpb2. beta2-Toxigenic C. perfringens was detected in samples from 13 of 25 (52%) horses with typical or atypical typhlocolitis, with a particularly high incidence in specimens of ingesta and biopsy specimens (75%), whereas only 6 of 16 specimens from horses with other intestinal diseases yielded beta2-toxigenic C. perfringens. No beta2-toxigenic C. perfringens was found in the samples from the 58 control horses, of which only one fecal sample contained C. perfringens type A. Among the samples from the 15 horses with fatal cases of typical and atypical typhlocolitis 9 (60%) were positive for beta2-toxigenic C. perfringens, whereas samples from only 4 of the 10 (40%) animals with nonfatal cases of infection were positive. We found an interesting correlation between the antibiotic-treated horses which were positive for beta2-toxigenic C. perfringens and lethal progression of the disease. No C. perfringens strains isolated in this study contained genes for the beta- and epsilon-toxins and enterotoxin. The high incidence of beta2-toxigenic C. perfringens in samples of ingesta, biopsy specimens of the intestinal wall, and feces from horses suffering or dying from typhlocolitis together with the absence of this organism in healthy horses provides strong evidence that beta2-toxigenic C. perfringens play an important role in the pathogenesis of typhlocolitis.

Animals↗
PubMed1998

Antigen heterogeneity among Mycoplasma mycoides subsp. mycoides SC isolates: discrimination of major surface proteins.

The protein and antigen profiles of 60 isolates, strains and the type strain PG1 of Mycoplasma mycoides subsp. mycoides s.c. were compared by sodium dodecyl sulphate polyacrylamide gel electrophoresis and immunoblot analysis. Analysis using contagious bovine pleuropneumonia antisera and hyperimmune rabbit sera against several representative strains revealed some differences in protein profiles and variability in antigens among strains from different geographic regions. The most common antigenic bands had the molecular masses of 110, 95, 80, 69, 62, 60, 48, 44, 39 and 38 kDa. There were differences among European strains, where a larger group coming from Italy lacked the p98 antigen, thus, with one exception, distinguishing the Italian strains from Portuguese, French and Spanish strains. African, Australian and PG1 strains showed heterogenic profiles, with quantitative differences and in a few strains some antigenic bands were absent. The group constituting African, Australian and PG1 strains was characterised by the presence of 71.5/70 kDa antigens, which were not detected in European strains. Mycoplasma mycoides subsp. mycoides s.c. membrane proteins were characterised by Triton X-114 partitioning and p110, p98, p95, p62/60 and p48 were identified as immunogenic antigens. The simultaneous presence of these five antigens was common to all the sera examined and, therefore, indicates the diagnostic potential of immunoblotting. Most immunodominant antigens are surface-exposed proteins as determined by the trypsin treatment.

Africa↗
PubMed1998

Susceptibility of alpine ibex to conjunctivitis caused by inoculation of a sheep-strain of Mycoplasma conjunctivae.

We evaluated the susceptibility of alpine ibex (Capra ibex ibex) to mycoplasmal conjunctivitis induced by a strain of Mycoplasma conjunctivae isolated from domestic sheep by inoculation of three alpine ibexes with 1.2 x 10(6) colony forming units of M. conjunctivae in the conjunctival sac of both eyes. One more ibex was exposed to the infection by contact. Experimental animals were free of M. conjunctivae and ocular Chlamydia infection before inoculation. Conjunctivitis and serous to mucous lachrymation became apparent in all four ibexes. Clinical signs began within 2 days in inoculated animals and 22 days after the beginning of the experiment in the contact ibex. M. conjunctivae was demonstrated up to the 63th day post-inoculation by cultural and PCR-methods. After 63 days, histopathologic examination revealed nearly normal ocular tissues, and M. conjunctivae could be detected from two eyes only. No other infectious agents which might cause conjunctivitis or keratitis, including Chlamydia psittaci and Branhamella ovis, were involved. Our investigation indicates that sheep-strains of M. conjunctivae can induce conjunctivitis in alpine ibex, thus showing pathogenicity of this organism for Caprinae species other than domestic sheep and goats.

Animals↗
PubMed1998

Tularemia in a common marmoset (Callithrix jacchus) diagnosed by 16S rRNA sequencing.

We report a case of tularemia in a common marmoset (Callithrix jacchus) diagnosed by determination of the isolate's 16S ribosomal RNA (rRNA) gene sequence. Pathological examination of the animal revealed a multifocal acute necrotizing hepatitis, interstitial nephritis, splenitis, and lymphangitis of the mandibular, retropharyngeal, and cervical and mesenteric lymph nodes. Moreover, multiple foci of acute necrosis were found in the epithelium of the jejunum and the interstitium of the lung. Bacteriological investigations revealed a septicemia. The isolated infectious agent was uncommon, not routinely diagnosed in our laboratory and therefore difficult to identify by conventional tools in a reasonable time and effort. thus, we decided to perform a genetic analysis based on the 16S rRNA gene sequence. Thereby, an infection with Francisella tularensis, the causative agent of tularemia, was unambiguously diagnosed. This shows the great advantage 16S rRNA gene sequencing has as a general identification approach for unusual or rare isolates.

Animals↗
PubMed1998

Humoral and bronchial immune responses in cattle experimentally infected with Mycoplasma mycoides subsp. mycoides small colony type.

The course of immune reactions of the manifold antigens of Mycoplasma mycoides subsp. mycoides small colony type (SC) was analysed in serum and bronchial lavage of cattle experimentally infected with the African strain Afadé and the European strain L2 using Western-blots and complement fixation. Western-blot analysis of total antigens of both strains with sera from animals infected with the homologous and heterologous strain revealed the common dominant immunogenic antigens with the molecular masses of 110, 95, 85, 80, 72, 62, 48 and 39 kDa. The sequential sampling of the blood and bronchial lavages before and after contact infections allowed us to identify the antigens of 85, 80, 72, 48 and 39 kDa as particularly early immunogens. The IgA Western blots of the bronchial lavages showed distinct, early and persistent reactions to the 110, 85, 80, 72, 48 and 45 kDa proteins. These proteins were the predominant lipoproteins as determined by [14C]palmitic acid labelling. Only relatively weak reactions of the bronchial lavages were detected with IgG. In general immune responses were significantly stronger in the animals infected with the African strain Afadé, which gave positive results two weeks after contact infection. In contrast, the animals infected with the European strain L2 induced much lower reactions with a delay of three months after contact infection. In one animal strain L2 caused no sero-conversion and no infection. The results indicate a difference in virulence between the African strain Afadé and the European strain L2.

Africa↗
PubMed1998

Species identification of Mycoplasma bovis and Mycoplasma agalactiae based on the uvrC genes by PCR.

The DNA repair genes uvrC from Mycoplasma bovis and Mycoplasma agalactiae type strains were cloned and their nucleotide sequences were established. These sequences were used to design polymerase chain reaction (PCR) primer pairs for M. bovis and M. agalactiae. Each primer pair amplified a 1-6 kb fragment of the uvrC gene in the respective species. The specificity of the primer pairs for the two species was demonstrated through the lack of cross-amplifications in heterologous PCR reactions and in reactions using DNA from other mycoplasma species. Subsequent restriction enzyme analysis of the amplified uvrC gene segments from type and field strains of M. bovis and M. agalactiae showed that the uvrC genes are well conserved in both species but differ significantly between the two species. The diagnostic PCR assay enabled unambiguous identification of M. bovis and M. agalactiae strains isolated from geographically diverse places, even in cases where 16S rRNA gene sequence analysis was unable to discriminate between the two species.

Animals↗
PubMed1998

[Characterization of liver regeneration in the albumin-urokinase transgenic mouse].

UNLABELLED: Models of liver regeneration are essential to understand mechanisms of hepatic carcinogenesis, correct genetic diseases by gene transfer or hepatocyte transplantation. The expression in the liver of transgenic mice of a gene coding for a urokinase-type plasminogen activator (uPA mouse) induces hepatotoxicity and prolonged post-native liver regeneration from cellular clones which have inactivated the transgene. This model may have major applications but it remains necessary to characterize the liver regeneration pattern. METHODS: Histological and immunohistochemical studies of the liver of uPA and non-transgenic mice, 3, 7, 14, 21, 28, 42 and 56 days-old. Markers of cellular proliferation: 5-bromo-2'deoxyuridine (BrdU) and proliferating cell nuclear antigen (PCNA). RESULTS: Regenerative nodules were seen from day 14. These nodules then grew, became confluent and by 8 weeks constituted the entire liver mass. A semi-quantitative study of BrdU and PCNA showed a maximal labeling at day 7 (300 to 350 labeled cells/10 microscopic fields, mag 400). When the nodules appeared, 60 to 80% of the cells were labeled. The proportion of labeled cells decreased but was still greater than that observed in non transgenic mice up to day 56 (92 to 106 labeled cells vs 10 to 28, on day 28). CONCLUSIONS: In uPA mouse liver regeneration is significantly expanded, as compared to the regeneration following partial hepatectom. This study therefore has allowed to determine the best conditions for using this model.

Animals↗
PubMed1998

Genetic and serological analysis of the immunogenic 67-kDa lipoprotein of Mycoplasma sp. bovine group 7.

The gene encoding a lipoprotein of 67 kDa, named P67, was cloned from Mycoplasma sp. bovine group 7 strain PG50 and expressed in Escherichia coli K12. Analysis of the amino acid sequence derived from the DNA sequence of the P67 gene revealed a typical prokaryotic signal peptidase II membrane lipoprotein lipid attachment site and a transmembrane structure domain in the leader sequence at the amino-terminal end of the protein. Protein P67 showed 91% identical amino acid residues to the lipoprotein P72 of Mycoplasma mycoides subsp. mycoides small colony type (SC) and 53% identical amino acid residues to a peptide of an unassigned gene on the genome of Mycoplasma capricolum subsp. capricolum. Antibodies made against recombinant P67 reacted with a 67-kDa protein in all Mycoplasma sp. bovine group 7 strains tested and also, to some extent, with P72 of Mycoplasma mycoides subsp. mycoides SC. The gene encoding P67 was present in all strains of Mycoplasma sp. bovine group 7 analysed, but not in other Mycoplasma sp. of the "mycoides cluster" and not in the phylogenetically related Mycoplasma putrefaciens. PCR and restriction fragment analysis revealed that the gene of P67 is conserved in all strains of Mycoplasma sp. bovine group 7. A specific PCR reaction based on the P67 gene sequence enabled rapid identification of strains belonging to Mycoplasma sp. bovine group 7.

Amino Acid Sequence↗
PubMed1998

Detection of alpha- and epsilon-toxigenic Clostridium perfringens type D in sheep and goats using a DNA amplification technique (PCR).

Clostridium perfringens isolated from sheep and goat with enterotoxaemia at necropsy and from healthy animals at slaughter were typed using specific PCR assays for the detection of the alpha-, beta- and epsilon-toxin genes. Clostridium perfringens isolated from all 52 animals with pathological signs of enterotoxaemia showed the presence of the alpha- and epsilon-toxin genes but were devoid of the beta-toxin gene. These strains could therefore be identified as type D, characteristic for clostridial enterotoxaemia of sheep, lambs and goats. In contrast, Cl. perfringens isolated from 11 of 13 healthy animals only contained the alpha-toxin gene which is typical for type A. Two of the healthy animals contained Cl. perfringens with the alpha- and epsilon-toxin genes. However, when several individual Cl. perfringens colonies were analysed from each of these two animals, only a small percentage was found to contain the epsilon-toxin gene, whereas the majority of the colonies were of type A with the alpha-toxin gene only. This is in contrast to the findings from the diseased animals which contained practically only type D Cl. perfringens. The beta-toxin gene was not found in any Cl. perfringens isolate from goat and sheep. Comparison of the PCR data with results obtained by the classical biological toxin assay using the mouse model showed a good correlation.

Animals↗
PubMed1998

Detection of Mycoplasma hyopneumoniae by air sampling with a nested PCR assay.

This article describes the first successful detection of airborne Mycoplasma hyopneumoniae under experimental and field conditions with a new nested PCR assay. Air was sampled with polyethersulfone membranes (pore size, 0.2 micron) mounted in filter holders. Filters were processed by dissolution and direct extraction of DNA for PCR analysis. For the PCR, two nested pairs of oligonucleotide primers were designed by using an M. hyopneumoniae-specific DNA sequence of a repeated gene segment. A nested PCR assay was developed and used to analyze samples collected in eight pig houses where respiratory problems had been common. Air was also sampled from a mycoplasma-free herd. The nested PCR was highly specific and 10(4) times as sensitive as a one-step PCR. Under field conditions, the sampling system was able to detect airborne M. hyopneumoniae on 80% of farms where acute respiratory disease was present. No airborne M. hyopneumoniae was detected on infected farms without acute cases. The chance of successful detection was increased if air was sampled at several locations within a room and a lower air humidity.

Air Microbiology↗
PubMed1998

Detection of Tritrichomonas foetus by PCR and DNA enzyme immunoassay based on rRNA gene unit sequences.

Tritrichomonas foetus is the causative agent of bovine tritrichomonosis, a sexually transmitted disease leading to infertility and abortion. Diagnosis is hampered by putative contamination of samples with intestinal or coprophilic trichomonadid protozoa which might be mistaken for T. foetus. Therefore, we developed a PCR test optimized for applicability in routine diagnosis. Amplification is based upon primers TFR3 and TFR4 directed to the rRNA gene units of T. foetus. In order to avoid potential carryover contamination by products of previous amplification reactions, conditions were adapted to the use of the uracil DNA glycosylase system. Furthermore, documentation and interpretation of results were facilitated by including a DNA enzyme immunoassay for the detection of amplification products. Specificity was confirmed with genomic material from different related trichomonadid protozoa. The high sensitivity of the test allowed the detection of a single T. foetus organism in diagnostic culture medium or about 50 parasites per ml of preputial washing fluid. The present methods are thus proposed as (i) confirmatory tests for microscopic diagnosis following diagnostic in vitro cultivation and (ii) a direct T. foetus screening test with diagnostic samples.

Animals↗
PubMed1997

Molecular identification and epidemiology of animal mycoplasmas.

Molecular genetic techniques show a high potential for rapid and accurate identification of Mycoplasma species isolated from animals. An important field of application for such methods is the differentiation of species and/or subspecies which are phenotypically closely related, but which show significant differences in epidemiological impact. This need is particularly important for the mycoplasmas of the "mycoides group", which are phenotypically and phylogenetically very closely related. Molecular typing techniques based on 16S rRNA genes give straightforward phylogenetic answers on the species level. For more refined methods of subtyping at the subspecies level, the use of defined genes characteristic of certain Mycoplasma species or clusters is recommended. Genetic fingerprinting, especially insertion sequence typing has proved to be a valuable tool for subtyping and strain identification in particular of vaccine strains and for epidemiological investigations.

Animal Diseases↗
PubMed1997

Spontaneous and iatrogenic spreading of liver-derived cells into peripheral blood of patients with primary liver cancer.

The prognosis for patients with primary liver cancer (PLC) often depends on tumor recurrence and the development of extrahepatic metastases, particularly after liver transplantation. We have developed a sensitive test to detect both spontaneous circulation of tumor cells and the spread of liver cells due to chemoembolization and alcoholization. Reverse-transcription polymerase chain reaction was used to search for cells expressing alpha-fetoprotein (AFP) messenger RNA in the peripheral blood of 84 patients with PLC and 102 controls (55 patients with chronic hepatitis and/or cirrhosis, 10 patients with benign liver tumors or liver metastases from intestinal cancers, and 37 healthy individuals). By spiking the blood of healthy volunteers with HepG2 cells, we assessed the sensitivity limit: one HepG2 cell mixed with 10(7) leukocytes. All 102 controls tested negative. In contrast, 28 patients (33.3%) with PLC tested positive. Positivity for the test was significantly associated with portal thrombosis, tumor size, intravascular tumor emboli, serum AFP level, and extrahepatic metastases. Patients were followed up for a mean period of 39 +/- 51 weeks: the probability of developing extrahepatic metastases was significantly higher in positive than in negative patients. Eighteen negative patients with PLC were tested before, 1 hour after, and 24 hours after locoregional therapy: 9 tested positive either 1 or 24 hours after alcoholization or chemoembolization. In conclusion, we have developed a highly specific and sensitive test to detect circulating tumor cells in patients with PLC. This test is likely to be clinically useful in evaluating the risk of developing extrahepatic metastases and the possibility of iatrogenic spreading of liver-derived, possibly tumorous, cells.

Female↗
PubMed1997

Development of a sensitive nested PCR method for the specific detection of Mycoplasma mycoides subsp. mycoides SC.

A specific and sensitive test for the detection of Mycoplasma mycoides subsp. mycoides small colony type (SC), the aetiological agent of contagious bovine pleuropneumonia (CBPP) was developed using two nested PCR reactions. The PCR reactions are based on the nucleotide sequence of lipoprotein P72 of M. mycoides subsp. mycoides SC. The two specific oligonucleotide primer pairs were chosen to match those sequence segments of the P72 gene which differ most from the gene of the closely related lipoprotein P67 of Mycoplasma sp. bovine group 7 (strain PG50). The nested PCR reacted with all of the 34 different strains of M. mycoides subsp. mycoides SC analysed, and gave no amplification product with any of the closely related mycoplasmas tested, showing its high specificity. In bronchial lavage fluid experimentally contaminated with M. mycoides subsp. mycoides SC, the assay was able to detect as few as two viable cells per ml using a simple lysis procedure prior to the amplification step. With clinical samples, the sensitivity of the nested PCR was about 10(4)-10(5) higher than that of single PCR amplifications performed under the same conditions. The assay was also successfully used to detect M. mycoides subsp. mycoides SC in bronchial lavage fluid of experimentally infected cattle and proved to be more sensitive than classical culture methods.

Animals↗
PubMed1997

Identification of Clostridium chauvoei in cultures and clinical material from blackleg using PCR.

An identification system for Clostridium chauvoei, using PCR amplification of the 16S rRNA gene (rrs) with specific oligonucleotide primers and subsequent restriction digestion of the amplification product is described. The specific oligonucleotide primers were designed based on the rrs gene sequences of C. chauvoei by comparing it to the DNA sequences of the rrs genes of its most closely related species Clostridium septicum and Clostridium carnis. A subsequent restriction digestion of the 960 bp amplification product was used in order to unambiguously identify C. chauvoei. The developed identification system was evaluated on clinical material during a recent outbreak of blackleg in cattle. Thereby, C. chauvoei was identified as the etiologic agent of the outbreak either directly from clinical samples of muscle, liver, spleen and kidney or from primary cultures made with this material. A comparison of the newly developed method with standard diagnostic tools for C. chauvoei showed that it has advantages over the immunofluorescence and is, therefore, a useful option to it. Moreover, the assay is a valuable tool for the phylogenetic identification of C. chauvoei which can assist to substitute the fastidious traditional identification methods and replace laboratory animal testing currently used.

Animals↗
PubMed1997

Personalities and alimentary behaviors in obese patients.

The actual tendency in the care of obese patients is the association of dietetic information with an eating behavior therapy. Studies attempting to attribute the origin of obesity to psychiatric pathologies are contradictory. We studied whether certain eating disorders are more specific to a personality type. We studied eating disorders with the Eating Disorder Inventory (EDI) test in 281 obese women compared to 252 age-matched non-obese women. Both obese patients and non-obese volunteers were divided into four groups depending upon their personality (PERSONA test). This test defines four types of personality, based on the level of emotion (expansive or reserved) and the degree of power (dominant or consenting). According to our study, eating disorders vary between the four personality groups and were significantly higher in the facilitating group (consenting and expansive) compared to the three other obese groups. Neither promoting (expansive and dominant) nor controlling obese patients (dominant and reserved) present eating disorders. The analyzing obese patients (reserved and consenting) are reticent when it comes to consulting (18%) since they distrust others. Analyzing obese patients present an interpersonal distrust and an interoceptive awareness. The group which presents most eating disorders is that of facilitating obese patients (consenting and expansive). These present eating disorders of the compulsive types favored by interoceptive awareness, body dissatisfaction, ineffectiveness, and maturity fears. The diversity, even the absence, of eating disorders brought to evidence by our tests based upon different personality types should allow better understanding of the psychological and behavioral causes of weight gain and the means for improving compliance in the following of an obese patient.

Adult↗