Laurent Sutra

Immunogenicity in cows of Staphylococcus aureus type 5 capsular polysaccharide—ovalbumin conjugate

Six dairy cows were immunized subcutaneously with purified type 5 capsular polysaccharide (CP5) of Staphylococcus aureus or CP5-ovalbumin conjugate in Freunds incomplete adjuvant. The CP5 antibodies elicited were measured in sera and analysed with regard to isotypes by enzyme-linked immunosorbent assay. At the doses tested, the purified CP5 did not induce a humoral response in the cows. Immunization of two cows with the CP5-ovalbumin conjugate elicited a CP5 antibody response mainly of the IgG2 isotype, which culminated 4 weeks later. A second injection of conjugate, 3 months after the first one, resulted in a rapid and durable anti-CP5 response without exceeding the first antibody peak value. Intramammary infusion of purified CP5 failed to provoke an inflammatory response in the milk of the immunized cows. In contrast, a marked recruitment of cells was recorded in the milk of the sensitized cows after intramammary infusion of ovalbumin. These results demonstrate that injection of CP5-protein carrier conjugate in cows entails both antibody response against CP5 and carrier-specific recruitment of cells in milk of immunized animals.

Detection of a whitening fluorescent agent as an indicator of white paper biodegradation : a new approach to study the kinetics of cellulose hydrolysis by mixed cultures

A simple and reliable method to estimate paper degradation by cellulolytic bacteria is described. This method is based on the detection in the culture medium of a fluorescent whitening agent (FWA) added to white paper during the manufacturing process. Preliminary results using a Cellulomonas strain cultivated in a liquid medium containing FWA, indicated that this component is non-toxic at a final concentration of 0.01 per thousand (v/v) and that the fluorescence decreased during the first 24 h of incubation, i.e. during exponential growth phase, suggesting an adsorption of FWA on bacterial cells. Consequently, all experiments have been performed with a liquid medium containing FWA (0.01 per thousand v/v) and white paper (8.0 g/l) as cellulose source. Mixed bacterial populations (MBPs) were prepared from refuse samples. These MBPs, which mainly consisted of bacterial rod cells, were used as inocula and fluorescence was measured after 30 h of incubation, i.e. after the stationary phase was reached. A high linear correlation (R(2) = 0.979) was found between the percentages of degraded paper (%P) deduced from residual paper weight and the fluorescence values (F) of the culture medium and the following equation between %P and F was determined: %P = 8.71x10(-5) x F. An additional experiment using a second MBP showed a strong correlation (R(2) = 0.990) between the measured %P and the %P estimated from F values, confirming the reproducibility of the method. Moreover, the time course of paper degradation by five replicate flasks from a unique MBP was set up. Paper degradation was detected 3 to 5 days after the beginning of the stationary phase. The average degradation rate between the 7th and the 11th day of incubation was 11.4% per day. Rates of paper degradation ranged from 31 to 60% after 10 days and from 77 to 88% after 3 weeks of incubation, depending on the inoculum.

Purification of type 5 capsular polysaccharide from Straphylococcus aureus by a simple efficient method

Abstract Types 5 and 8 Straphylococcus aureus capsular polysaccharides are now considered as potential candidates for vaccination against human and animal infections. In order to get sufficient amounts of capsular polysacccharide for this use, we describe a new simple efficient purification method of type 5 capsular polysacccharide. After extraction of capsular polysaccharide from S. aureus Reynolds strain by autoclaving, the successive steps were ultrafiltration with a 30 kDa cutoff hollowfiber cartridge, treatment by sodium metaperiodate, ultrafiltration with a 100 kDa cutoff hollowfiber cartridge and high-performance size exclusion liquid chromatography. Approximatively 85% of the polysaccharide initially extracted was recovered and contaminating components represented 4.2% of the purified fraction.

Genetic Diversity of Pseudomonas syringae Pathovars and Related Species Assessed by DNA Heteroduplex Mobility Assay

Fluorescent oxidase negative pseudomonads (FONP) include Pseudomonas syringae pathovars and related species (P. amygdali, P. avellanae, P. ficuserectae, P. meliae, P. savastanoi and P. viridiflava). Using DNA-DNA hybridization, 9 genomospecies have been delineated among FONP, two of them being elevated to species level, P. tremae and P. cannabina (2). Analyses of 16S rDNA sequences have shown that Pseudomonas sensu stricto included two intrageneric clusters, designated P. aeruginosa and P. fluorescens. Five lineages were defined in P. fluorescens cluster : P. fluorescens, P. syringae, P. cichorii, P. putida and P. agarici lineages (3). Comparisons of 16S rDNA sequences of pseudomonads revealed three hypervariable (hv) regions : hv1 (positions 71–95), hv2 (positions 455–475) and hv3 (positions 998–1043) (3).

Molecular Characterisation of Spanish Pseudomonas syringae pv. phaseolicola Isolates

In this work, we focused on the molecular characterisation of Spanish Pseudomonas syringae pv. phaseolicola isolates. PCR amplification profiles of repetitive DNA were similar among the Spanish P. s. pv. phaseolicola isolates and the reference P. s. pv. phaseolicola strains, but partially different from the patterns shown by P. s. pv. glycinea. Taking these profiles into consideration, two P s. pv. phaseolicola genomic groups have been distinguished. Additionally, using a DNA heteroduplex mobility assay (HMA) performed on the internal transcribed spacer (ITS) region between 16S and 23S rRNA genes, it was possible to differentiate Group 1 isolates, which clearly present a unique HMA band, from Group 2 isolates, which show multiple HMA bands.