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See detailPre-study and in-study validation of an ultra-high pressure LC method coupled to tandem mass spectrometry for off-line determination of oxytetracycline in nasal secretions of healthy pigs.
Bimazubute, Marcel Aimé ULg; Rozet, Eric ULg; Dizier, Isabelle ULg et al

in Journal of Chromatography. B : Analytical Technologies in the Biomedical & Life Sciences (2009), 877(23), 2349-57

In order to quantify oxytetracycline (OTC) in nasal secretions of healthy pigs after intramuscular injection of OTC at doses of 10, 20 and 40 mg/kg bodyweight, an original method based on ultra-high ... [more ▼]

In order to quantify oxytetracycline (OTC) in nasal secretions of healthy pigs after intramuscular injection of OTC at doses of 10, 20 and 40 mg/kg bodyweight, an original method based on ultra-high pressure liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) was developed and fully validated. Sample preparation consisted in protein precipitation preceded by the addition of a releasing protein reagent. Metacycline (MTC) was used as internal standard. Separation was carried out at 65 degrees C in the gradient elution mode on a short analytical column filled with Acquity BEH C(18) stationary phase. The mobile phase consisted in a mixture of water and acetonitrile containing 1 mM of oxalic acid and 0.1% (v/v) of formic acid. The triple quadrupole mass spectrometer operated in the positive electrospray ionization mode; OTC and MTC were detected using multiple reaction monitoring. The pre-study and in-study validation of this bioanalytical method was performed by applying a novel strategy based on total measurement error and accuracy profiles. The maximum risk of observing future measurements falling outside the acceptance limits during routine as well as the measurements uncertainty were also estimated. [less ▲]

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See detailLiquid chromatographic determination of enrofloxacin in nasal secretions and plasma of healthy pigs using restricted access material for on-line sample clean-up
Bimazubute, M. A.; Rozet, Eric ULg; Dizier, Isabelle ULg et al

in Journal of Chromatography. A (2008), 1189(1-2), 456-466

A new fully automated method was developed for the quantitative analysis of an antibacterial drug, enrofloxacin (ENRO), in both nasal secretions and plasma samples of healthy pigs. The method is based on ... [more ▼]

A new fully automated method was developed for the quantitative analysis of an antibacterial drug, enrofloxacin (ENRO), in both nasal secretions and plasma samples of healthy pigs. The method is based on the use of a pre-column packed with restricted access material (RAM), namely RP-18 ADS (alkyl diol silica), for on-line sample clean-up coupled to a liquid chromatographic (LC) column containing octadecyl silica. The only off-line sample preparation was the 50-fold dilution of nasal secretions and plasma samples in the washing liquid composed of 25 mM phosphate buffer of pH 7.4. A 10 μl diluted sample volume was injected directly onto the pre-column and washed for 7 min. By rotation of a switching valve, the analyte of interest was eluted in the back-flush mode with the LC mobile phase which consisted in a mixture of 25 mM phosphate buffer of pH 3.0 and acetonitrile according to a segmented gradient elution. By a new rotation of the switching valve, the pre-column and the analytical column were equilibrated for 3 min with the initial mobile phases. The flow-rate was 0.8 ml min−1 for the washing liquid and 1.5 ml min−1 for the LC mobile phase. ENRO was detected by fluorescence at excitation and emission wavelengths of 278 and 445 nm, respectively. Finally, the developed method was validated using an original strategy based on total measurement error and accuracy profiles as a decision tool. The limits of quantitation of ENRO in plasma and in nasal secretions were 30.5 and 91.6 ng/ml, respectively. The validated method was then applied successfully to the determination of ENRO in healthy pigs treated by intramuscular injection at different doses (2.5, 10 and 30 mg/kg bodyweight) for a pilot study. This method could be also used for the simultaneous analysis of ENRO and its main metabolite, ciprofloxacin (CIPRO). [less ▲]

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See detailFluctuations dans la sensibilité à l’enrofloxacine des populations nasales chez le porcelet au cours d’un traitement parentéral
Mainil, Jacques ULg; Janssen, L.; Bimazubute, M. et al

in Proceedings: 3ème Colloque International Francophone de Bactériologie Vétérinaire (2006)

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See detailIdentification by two-dimensional electrophoresis of a new adhesin expressed by a low-passaged strain of Mycoplasma bovis
Thomas, Anne; Leprince, Pierre ULg; Dizier, Isabelle ULg et al

in Research in Microbiology (2005), 156(5-6, Jun-Jul), 713-718

A significant decrease in adherence rates of Mycoplasma bovis to bovine bronchial epithelial (BBE) cells has been observed after passage of the organism in artificial medium. Analysis of the proteins ... [more ▼]

A significant decrease in adherence rates of Mycoplasma bovis to bovine bronchial epithelial (BBE) cells has been observed after passage of the organism in artificial medium. Analysis of the proteins expressed by M. bovis isolate 2610 by two-dimensional (2-D) electrophoresis demonstrated differences between the cells harvested after the 7th and 116th passage. Three silver-stained prominent spots observed in 2-D electrophoretic separation of protein extracts of the lower-passaged cells were considerably less strongly expressed in the sample from higher-passaged cells. These spots had a molecular mass of approximately 24 kDa and an isoelectric point of about 5. The mass spectrometry analysis of these trypsin-sensitive proteins led to their identification as a unique new member of the Vsps family of membrane-associated proteins. Serum from a mouse immunized with these proteins significantly reduced adherence of M. bovis to BBE cells. This result underlines the function of this new Vsp in adherence of M. bovis to host cells. (c) 2005 Elsevier SAS. All rights reserved. [less ▲]

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See detailThe p40* adhesin pseudogene of Mycoplasma bovis
Thomas, Anne; Linden, Annick ULg; Mainil, Jacques ULg et al

in Veterinary Microbiology (2004), 104(3-4), 213-217

An analogue of the adhesin gene p40 of Mycoplasma agalactiae was found in Mycoplasma bovis. Nucleotide sequence analysis of the p40* gene in M. bovis revealed the presence of a large deletion involving a ... [more ▼]

An analogue of the adhesin gene p40 of Mycoplasma agalactiae was found in Mycoplasma bovis. Nucleotide sequence analysis of the p40* gene in M. bovis revealed the presence of a large deletion involving a frameshift that causes premature truncation of the translated protein, indicating that p40* exists as a pseudogene in M. bovis. (C) 2004 Elsevier B.V. All rights reserved. [less ▲]

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See detailConservation of the uvrC gene sequence in Mycoplasma bovis and its use in routine PCR diagnosis
Thomas, Anne; Dizier, Isabelle ULg; Linden, Annick ULg et al

in Veterinary Journal (2004), 168(1), 100-102

Mycoplasma bovis is a major cause of pneumonia and arthritis in calves, and of mastitis and genital infections in adult cows. It is responsible for high economic loss in feedlot cattle although it is ... [more ▼]

Mycoplasma bovis is a major cause of pneumonia and arthritis in calves, and of mastitis and genital infections in adult cows. It is responsible for high economic loss in feedlot cattle although it is often underestimated and is widely spread within the bovine population in enzootically infected areas. [less ▲]

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See detailAntibiotic susceptibilities of recent isolates of Mycoplasma bovis in Belgium
Thomas, Anne; Nicolas, C.; Dizier, Isabelle ULg et al

in Veterinary Record (2003), 153(14), 428-431

The susceptibilities of 40 recent Belgian field isolates of Mycoplasma bovis to 10 antimicrobial agents were assessed. Tiamulin was the most active antimicrobial agent against M bovis, with an initial ... [more ▼]

The susceptibilities of 40 recent Belgian field isolates of Mycoplasma bovis to 10 antimicrobial agents were assessed. Tiamulin was the most active antimicrobial agent against M bovis, with an initial inhibitory concentration (IIC50) of 0.06 microg/ml, but it is not licensed for the treatment of cattle. All three fluoroquinolones tested (danofloxacin, enrofloxacin and marbofloxacin) were effective against strains of M bovis, and had a minimum mycoplasmacidal concentration (MMC50) less than or equal to 1 microg/ml. Gentamicin was poorly effective, having an IIC50 of 8 microg/ml. Many strains of M bovis were resistant to tylosin, spectinomycin, lincomycin, tetracycline and oxytetracycline. [less ▲]

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See detailAdherence of Mycoplasma bovis to bovine bronchial epithelial cells
Thomas, Anne; Sachse, K.; Farnir, Frédéric ULg et al

in Microbial Pathogenesis (2003), 34(3), 141-148

Mycoplasma bovis is responsible for considerable economic losses in cattle due to pneumonia, arthritis and mastitis. As the agent was shown to be capable of adhering to neutrophils and embryonic bovine ... [more ▼]

Mycoplasma bovis is responsible for considerable economic losses in cattle due to pneumonia, arthritis and mastitis. As the agent was shown to be capable of adhering to neutrophils and embryonic bovine lung (EBL) cells and invading the respiratory epithelium it is highly desirable to improve our understanding of cytadherence processes. Although several surface proteins likely to be directly involved in this initial stage of interaction between pathogen and host cells have been identified, these findings mainly referred to type strain PG45 adhering to the continuous EBL cell line. The present study provides new and complementary data about cytadherence of M. bovis based on adherence of various radiolabeled strains to a primary culture of bovine bronchial epithelial (BBE) cells using a standardized adherence assay. M. bovis was shown to adhere specifically to the primary culture of BBE cells. Inhibition of adherence was observed upon addition of monoclonal antibodies (MAbs), trypsin treatment of mycoplasmas, and competition with non-radiolabeled mycoplasma cells. Interestingly, three MAbs against proteins involved in adherence to EBL cells failed to inhibit significantly the adherence to BBE cells. On the other hand, significant reduction of adherence rates by MAbs 2A8 and 9F1 directed against epitopes of variable surface lipoproteins VspC and VspF, respectively, demonstrated the involvement of these proteins in adherence of M. bovis to primary culture of BBE cells. (C) 2003 Elsevier Science Ltd. All rights reserved. [less ▲]

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See detailLes mycoplasmes respiratoires bovins: prévalence et propriétés de cyto-adhésion
Thomas, A.; Dizier, Isabelle ULg; SACHSE, K. et al

in Annales de Médecine Vétérinaire (2003), 147(4), 267-272

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See detailMycoplasma bovis dans le complexe respiratoire bovin et propriétés de cyto-adhésion in vitro
Thomas, Anne; Dizier, Isabelle ULg; Sachse, K. et al

in Annales de Médecine Vétérinaire (2003), 147(4, AUG-SEP), 267-272

Mycoplasmas frequently infect cattle, causing especially respiratory diseases. Mycoplasma bovis is the most important pathogenic species in countries free of bovine contagious pleuropneumonia. This ... [more ▼]

Mycoplasmas frequently infect cattle, causing especially respiratory diseases. Mycoplasma bovis is the most important pathogenic species in countries free of bovine contagious pleuropneumonia. This species was frequently isolated in Belgium from cattle with respiratory disease. Furthermore, associations were often observed with pasteurellas and bovine respiratory syncytial virus. Of these M. bovis isolates, many were resistant to several antimicrobial agents which are used in cattle practice, except to fluoroquinolones. Inasmuch the high frequency of M. bovis isolation and antibiotic resistances, it is very important to understand the pathogenicity of this bacteria in order to optimize prophylactic tools. Therefore, the study of the cytadherence of M. bovis is essential since it represents the first step of the bacterial infection. According to our experimental results, PG45 is not representative of field isolates because of its low adherence rates to various cell lines. This could be explained by the high number of subcultures of this pathogenic strain underwent since its first isolation, which sharply contrasts with other isolates. M. bovis adheres specifically to bovine bronchial epithelial cells in primary culture. Proteins such as variable surface proteins C and F are involved in this step as observed by decreased adherence rates after trypsinization of mycoplasma cells or addition of monoclonal antibodies. [less ▲]

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See detailAdherence to various host cell lines of Mycoplasma bovis strains differing in pathogenic and cultural features
Thomas, Anne; Sachse, Konrad; Dizier, Isabelle ULg et al

in Veterinary Microbiology (2003), 91(2-3), 101-113

Mycoplasma bovis is known to be responsible for pneumonia and arthritis in calves, as well as mastitis in dairy cows. Despite clear evidence of its pathogenic potential, little is known about mechanisms ... [more ▼]

Mycoplasma bovis is known to be responsible for pneumonia and arthritis in calves, as well as mastitis in dairy cows. Despite clear evidence of its pathogenic potential, little is known about mechanisms of cytadherence and the molecular factors involved. The purpose of this work was to compare adherence rates of M. bovis field strains to different host cell lines and study the effects of cloning and sub-culturing M. bovis strains on their adherence properties. Eighteen metabolically labeled M. bovis strains isolated from different pathological backgrounds were examined in adherence trials using four different host cell lines, i.e. embryonic bovine lung (EBL), embryonic bovine trachea (EBTr), Madin Darby bovine kidney (MDBK) and rabbit kidney (RK) cells. Although large interstrain variations in adherence rates (3.4-19.1%) were measured they could not be correlated to the pathological background (pneumonia, arthritis or mastitis). Adherence rates to the fibroblast cell line (EBTr) were significantly lower than those to the three epithelial cell lines (EBL, MDBK and RK). The only non-pathogenic strain (221/89) exhibited lower adherence rates than three isolates from clinical mastitis. Interestingly, adherence rates were significantly reduced after in vitro passaging. In contrast, no effect of single cloning of strains on adherence was observed. There was no general correlation between expression of variable surface proteins (Vsps) as monitored by immunoblotting and adherence rates, although alterations in Vsp expression profiles were seen as a consequence of passaging. As there is probably a large number of adhesins, variable and non-variable, on the surface of M. bovis cells the issue is very complex, and the most active components have yet to be identified. [less ▲]

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See detailIsolation of Mycoplasma species from the lower respiratory tract of healthy cattle and cattle with respiratory disease in Belgium
Thomas, Anne; Ball, H.; Dizier, Isabelle ULg et al

in Veterinary Record (2002), 151(16), 472-476

Between 1997 and 2000, a total of 150 healthy cattle and 238 animals with respiratory disease were examined for six Mycoplasma species. Attempts were made to detect Mycoplasma canis, Mycoplasma dispar and ... [more ▼]

Between 1997 and 2000, a total of 150 healthy cattle and 238 animals with respiratory disease were examined for six Mycoplasma species. Attempts were made to detect Mycoplasma canis, Mycoplasma dispar and Ureaplasma diversum in calves with recurrent disease, and all three of these species were identified in calves with recurrent disease and in healthy lungs. In healthy calves, 84 per cent of bronchoalveolar lavage fluids were mycoplasma free; when cultures were positive, Mycoplasma bovirhinis was the only species isolated. Mycoplasmas were isolated from 78 per cent of animals suffering recurrent respiratory disease and from 65 per cent of acute respiratory cases. Mycoplasma bovis was isolated from bronchoalveolar lavages from 35 per cent of calves suffering recurrent respiratory disease, and from 50 per cent of acute cases, and from 20 per cent of pneumonic cases examined postmortem. M bovis was associated with other Mycoplasma species in 44 per cent of cases. M dispar was also isolated from 45.5 per cent of calves suffering recurrent respiratory disease, often in association with M bovis. M canis was identified for the first time in diseased Belgian cattle. Other mycoplasmas, including Mycoplasma arginini, Mycoplasma alkalescens and U diversum, were isolated less frequently. Associations between mycoplasmas and other pathogens were often observed. Among lungs infected with Pasteurella and/or Mannheimia species, more than 50 per cent were mixed infections with M bovis. [less ▲]

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See detailComparison of sampling procedures for isolating pulmonary mycoplasmas in cattle
Thomas, Anne; Dizier, Isabelle ULg; Trolin, A. et al

in Veterinary Research Communications (2002), 26(5), 333-339

Three sampling procedures were compared to determine the optimal technique for isolating mycoplasmas in cattle with respiratory diseases. The prevalence of mycoplasmas isolated from these animals is also ... [more ▼]

Three sampling procedures were compared to determine the optimal technique for isolating mycoplasmas in cattle with respiratory diseases. The prevalence of mycoplasmas isolated from these animals is also reported. In the first group, bronchoalveolar lavage (BAL) and nasal swab cultures were compared with the corresponding lung cultures from cattle necropsied for fatal respiratory diseases (n = 20). In a second group, nasal swabs were compared with corresponding BAL cultures in living animals with recurrent respiratory pathologies (n = 49). There was complete agreement between the paired BAL and lung cultures. In contrast, nasal cultures were not representative of the mycoplasmas present in the lower respiratory airways. The relative sensitivity and specificity of the nasal swab technique compared to BAL in living animals confirmed that the nasal swab cultures were not predictive of lower respiratory airway pathogens, such as Mycoplasma bovis. BAL is considered to be the best method for isolating M. bovis in cattle with respiratory diseases as it combines reliability and feasibility under field sampling conditions. In the present study, Mycoplasma dispar (43%) and M. bovis (29%) were mainly isolated in mixed infections. This confirms the need to search for mycoplasmas in routine examinations and to take them into account in therapeutic strategies for respiratory diseases in cattle. [less ▲]

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