Isolation, Purification, Characterization and Direct Conjugation of the Lipid†A-Free Lipopolysaccharide of Vibrio cholerae O139.

The lipopolysaccharide (LPS) of Vibrio cholerae O139, strain CIRS245, was isolated conventionally, and the lipid A was removed by mild acid hydrolysis (0.1 m NaOAc buffer containing 1 % SDS, pH 4.2, 95 °C, 8 h). The crude product was a complex mixture consisting mainly of constituent fragments of the O-specific polysaccharide-core (OSPc). The OSPc was only a minor component in the mixture. Two-stage purification of the crude OSPc by HPLC gave pure OSPc fragment of the LPS, as shown by NMR spectroscopy, analytical HPLC and ESI-MS. This material is the purest OSPc fragment of the LPS from Vibrio cholerae O139 reported to date. The purified OSPc was readily converted to the corresponding methyl squarate derivative and the latter was conjugated to BSA. The conjugate, when examined by ELISA, showed immunoreactivity with sera from patients in Bangladesh recovering from cholera caused by V. cholerae O139, but not O1.

Inheritance of coat colour in the cane Corso Italiano dog.

BACKGROUND: The inheritance of different coat colours in the Cane Corso Italiano dog has not been described thus far. We analysed data from 23,271 dogs and bitches using the Cane Corso Italiano Pedigree Database. We are describing for the first time the coat colour segregation ratios in Cane Corso Italiano offspring arising from crosses between parents of all possible coat colour combinations. RESULTS: Segregation ratios that do not follow a Mendelian pattern suggest that additional genes are active in the determination of coat colour. Segregation ratios of offspring produced by parental crossing (male colour A x female colour B) were compared with the ratios of offspring produced by reciprocal crossing (male colour B x female colour A) in all possible coat colour combinations. Most of the segregation ratios were the same, but some segregation ratios in reciprocal crosses differed. This result suggests that at least one gene responsible for coat colour is located on a sex chromosome. The sex ratio was analysed in the offspring of all colour groups. A ratio of 1:1 was not confirmed in 8 colour groups by the chi-square test. CONCLUSIONS: We described for the first time coat colour segregation ratios in Cane Corso Italiano dogs. Furthermore, we present the hypothesis that at least one gene responsible for coat colour is located on a sex chromosome.

Comparison of O-specific polysaccharide responses in patients following infection with Vibrio cholerae O139 versus vaccination with a bivalent (O1/O139) oral killed cholera vaccine in Bangladesh.

Cholera caused by Vibrio cholerae O139 emerged in the early 1990s and spread rapidly to 11 Asian countries before receding for unclear reasons. Protection against cholera is serogroup-specific, which is defined by the O-specific polysaccharide (OSP) component of lipopolysaccharide (LPS). V. cholerae O139 also expresses the OSP-capsule. We, therefore, assessed antibody responses targeting V. cholerae O139 OSP, LPS, capsule, and vibriocidal responses in patients in Bangladesh with cholera caused by V. cholerae O139. We compared these responses to those of age-gender-blood group-matched recipients of the bivalent oral cholera vaccine (OCV O1/O139). We found prominent OSP, LPS, and vibriocidal responses in patients, with a high correlation between these responses. OSP responses primarily targeted the terminal tetrasaccharide of OSP. Vaccinees developed OSP, LPS, and vibriocidal antibody responses, but of significantly lower magnitude and responder frequency (RF) than matched patients. We separately analyzed responses in pediatric vaccinees born after V. cholerae O139 had receded in Bangladesh. We found that OSP responses were boosted in children who had previously received a single dose of bivalent OCV 3 yr previously but not in vaccinated immunologically naïve children. Our results suggest that OSP-specific responses occur during cholera caused by V. cholerae O139 despite the presence of capsules, that vaccination with bivalent OCV is poorly immunogenic in the short term in immunologically naïve individuals, but that OSP-specific immune responses can be primed by previous exposure, although whether such responses can protect against O139 cholera is uncertain. IMPORTANCE Cholera is a severe dehydrating illness in humans caused by Vibrio cholerae serogroups O1 or O139. Protection against cholera is serogroup-specific, which is defined by the O-specific polysaccharide (OSP) of V. cholerae LPS. Yet, little is known about immunity to O139 OSP. In this study, we assessed immune responses targeting OSP in patients from an endemic region with cholera caused by V. cholerae O139. We compared these responses to those of the age-gender-blood group-matched recipients of the bivalent oral cholera vaccine. Our results suggest that OSP-specific responses occur during cholera caused by V. cholerae O139 and that the OSP responses primarily target the terminal tetrasaccharide of OSP. Our results further suggest that vaccination with the bivalent vaccine is poorly immunogenic in the short term for inducing O139-specific OSP responses in immunologically naïve individuals, but OSP-specific immune responses can be primed by previous exposure or vaccination.

Potential pathogenicity and antibiotic resistance of aquatic Vibrio isolates from freshwater in Slovakia.

This study aimed to evaluate the potential pathogenicity and antibiotic resistance of 31 environmental Vibrio isolates obtained from surface water in southern and eastern Slovakia. Isolates were identified as Vibrio cholerae non-O1/non-O139 and Vibrio metschnikovii by biochemical tests, MALDI biotyping, and 16S RNA gene sequencing. Analysis of the susceptibility to 13 antibacterial agents showed susceptibility of all isolates to ciprofloxacin, trimethoprim/sulfamethoxazole, chloramphenicol, gentamicin, imipenem, tetracyclin, and doxycycline. We recorded high rates of resistance to ß-lactams and streptomycin. Investigation of antibiotic resistance showed five different antibiotic profiles with resistance to antibacterials from three classes, but no multidrug resistance was observed. The investigation of the pathogenic potential of V. cholerae isolates showed that neither the cholera toxin coding gene ctxA nor the genes zot (zonula occludens toxin), ace (accessory cholera toxin), and tcpA (toxin-coregulated pilus) were present in any of 31 isolated samples. Gene ompU (outer membrane protein) was confirmed in 80% and central regulatory protein-coding gene toxR in 71% of V. cholerae isolates, respectively. A high prevalence of the hemolysin coding gene hlyA in all V. cholerae was observed. The data point toward the importance of systematic monitoring and comparative studies of potentially pathogenic vibrios in European countries.

Efficient separation of mannan-protein mixtures by ionic liquid aqueous two-phase system, comparison with lectin affinity purification.

Analysis of carbohydrates from complex biological samples often requires their isolation from proteins and other contaminants to avoid interference. An effective separation of mannan-protein mixtures by 1-butyl-3-methylimidazolium bromide/K2HPO4 ionic liquid aqueous two-phase system (IL-APTS) is reported. Extraction efficiency of bovine serum albumin (BSA) ranged from 92% to 97% while extraction efficiency of mannan reached values from 95% to about 100% depending on phase and/or model sample composition. On the contrary, lower efficiency of BSA removal (73-84%) was recorded for lectin affinity purification with concanavalin A-triazine bead cellulose (Con A-TBC); the low mannan-binding capacity was limiting factor here. The size exclusion chromatography pattern of model mannan-BSA samples after both IL-APTS and Con A-TBC treatments were consistent with the spectrophotometric component analysis. In case of biological experiment, the ionic liquid separation technique was superior in pre-purification of 2-aminobenzamide-labelled mannan from cell culture medium prior to HPLC-FLD analysis.

Longevity of Cane Corso Italiano dog breed and its relationship with hair colour.

The Cane Corso Italiano belongs among the new dog breeds that were fully recognised by Federation Cynologique Internationale (FCI) in 2007. For the first time, this study describes a median lifespan using the data of 232 dogs of the Cane Corso Italiano breed collected from kennels and individual owners from 25 countries. The median lifespan of the whole examined group is 9.29 years (IQR 6.98-11.12, IQR = Interquartile Range). This paper is the first to describe the possible relationship between median lifespan and hair colour within one breed. The longest living group is formed by black brindle coloured dogs, with a median of 10.30 years (IQR 8.33-13.00), and brindle coloured dogs, with a median of 10.13 years (IQR 7.12-11.25). The median lifespan of black brindle dogs exceeded the overall median lifespan of all dogs by 1.01 year and the median lifespan of other colour dogs by 2.21 years. Our results suggest a possible way for a prolongation of age at death of the Cane Corso Italiano breed using appropriate breeding. The median lifespan of male Cane Corso Italiano dogs is 9.25 years (IQR 6.97-11.00) and female Cane Corso Italiano dogs 9.33 years (IQR 7.00-11.31). The statistical analysis using the Independent Samples Student's t test confirmed that the lifespan of female dogs did not exceed the median lifespan of male dogs (P>0.01).

Biophysical properties of carboxymethyl derivatives of mannan and dextran.

Mannan from Candida albicans, dextran from Leuconostoc spp. and their carboxymethyl (CM)-derivatives were tested on antioxidant and thrombolytic activities. As antioxidant tests, protection of liposomes against OH radicals and reducing power assay were used. Dextran and mannan protected liposomes in dose-dependent manner. Carboxymethylation significantly increased antioxidant properties of both CM-derivatives up to concentration of 10mg/mL, higher concentrations did not change the protection of liposomes. The reducing power of CM-mannan (DS 0.92) was significantly lower (P<0.05) than underivatized mannan. No reductive activity was found for dextran and CM-dextran. All CM-derivatives demonstrated statistically significant increasing activity compared with underivatized polysaccharides. The highest thrombolytic activity was found using CM-mannan (DS 0.92). The clot lysis here amounted to 68.78 ± 6.52% compared with 0.9% NaCl control (18.3 ± 6.3%). Three-dimensional surface profiles of mannan, dextran, and their CM-derivatives were compared by atomic force microscopy.

Mannoproteins from yeast and hyphal form of Candida albicans considerably differ in mannan and protein content.

Significant differences in carbohydrate composition of mannoproteins obtained from yeast and hyphal cell walls of Candida albicans (serotypes A and B) were found. Yeast mannoproteins from both serotypes consisted up to 46% of mannan while the same parts from hyphal cells contained only about 14% of mannan. Another difference was in protein content, 47-53% for yeasts, 3-4.5% for hyphae, respectively. Moreover, HPLC profiles of yeast mannoproteins were more complex compared to those of hyphal form. Subsequently, mannans were prepared from yeast and hyphal mannoproteins using cetavlon fractionation. Mannans from both yeast serotypes contained higher amounts of mannose (91.4% serotype A; 92.8% serotype B) than mannans from hyphae (66.4% serotype A; 76.3% serotype B). Unlike mannans from serotype B, mannans from serotype A contained ß-(1 → 2)-linked mannopyranosyl units in acid-stable moiety. Further, hyphal mannans were less branched than yeast mannans. The shift from yeast to hyphal form probably led to simplification of mannan structure.

Inhibition of Yeast Growth by Broadly Cross-Reactive Antisera Elicited by Heterologous Mannan-Protein Conjugate.

A new approach to obtain broadly cross-reactive antisera against important yeast pathogens by intensive hyperimmunization with polysaccharide-protein conjugates is described here. Surface mannan of Candida albicans and capsular galactoglucoxylomannan of Cryptococcus laurentii were isolated and chemically linked to human serum albumin. Antisera elicited by a 7-week vigorous immunization of rabbits with the conjugates showed effective cross-reactive growth inhibition of different representatives of Candida spp. as well as Cryptococcus spp. IgG antibodies are evidenced as the effective component of the antisera.

Immunomodulation of T-cell responses with Vibrio cholerae O135 capsular polysaccharide and its protein conjugate, novel cholera vaccine study models.

We studied T-cell immune responses to surface capsular polysaccharide (CPS) of Vibrio cholerae O135 and its protein conjugate. CPS and CPS-bovine serum albumin (BSA) activation and presentation are characterized with induced alterations in expression and upregulation of membrane antigens CD25, CD11b, CD16/32, MHCII and CD45 on blood- and spleen-derived T cells. Expression of the early activation marker CD25 revealed efficient CPS-BSA conjugate activation especially of CD4(+) CD3(+) and CD8(+) CD3(+) cells. Specific CPS-BSA-induced CD25(+) T-cell subsets in blood were observed after the first application, i.e. a 4.2-fold increase of CD4(+) CD25(+) and 7.6-fold increase of CD8(+) CD25(+) vs. preimmune levels was determined. The upregulation of surface antigens MHCII and CD45 involved in antigen presentation and cell activation of CD3(+) cells and their significant reciprocal correlation (R(2)  = 0.92) observed only with CPS-BSA conjugate suggested efficient T-cell dependency and presentation. The pattern of accelerated T-cell activation and engagement of T cells as antigen-presenting cells throughout CPS-BSA immunization contrary to CPS alone was also confirmed in CD4(+) /CD8(+) /CD3(+) splenic cells. The results revealed different T-cell antigen presentation and activation following administration of CPS and CPS-BSA conjugates, as supported also by evaluation of CD45, MHCII and CD25 expression on CD19(+) B cells.

Preparation of synthetic polyoxazoline based carrier and Vibrio cholerae O-specific polysaccharide conjugate vaccine.

Multiple chemical attachments of carbohydrate antigens to linear polymer represent promising technique for creating biologically effective conjugates. A novel conjugate consisting of detoxified lipopolysaccharide of Vibrio cholerae O135, linear polymer (polyoxazoline copolymer, serving as a matrix) and BSA (as immunogenic protein), has been prepared. The reaction conditions were optimized for obtaining high degree of conjugation. Analytical methods were evaluated to characterize conjugates obtained. Proposed chemistry is suitable for preparation of multivalent glycoconjugates in general.

Immunological efficacy of glycoconjugates derived from Vibrio cholerae O1 serotype Ogawa detoxified LPS in mice.

This study focused on changes in selected parameters of humoral and cellular immunity following vaccination of mice with unique Vibrio cholerae LPS-protein-complexed conjugates. The V. cholerae detoxified LPS (dLPS)-derived antigenic structures O-specific polysaccharide (O-SP) and de-O-acylated LPS (DeOAc-LPS) were used to prepare glycoconjugates by linking both dLPSs to glucan, the immunomodulating matrix, and then to BSA carrier. Animals were given a primary vaccination and boosted at 2-week intervals with a dose of 4.5 µg saccharide antigen. The last boost was given either subcutaneously or intraperitoneally (i.p.) to compare the boosting effect and to optimize the effective immunization route. Both conjugates (O-SP-BSA and DeOAc-LPS-BSA) induced significant levels of antigen-specific Ig isotypes, especially IgG and IgM. The i.p. booster route was more effective. A T helper 1 response was achieved only by immunization with O-SP-BSA conjugate administered i.p. Significant acceleration of phagocytic capacity and respiratory burst of neutrophils was demonstrated by both immunogenic formulations. Activation of T- and B-cell adaptive immunities was exhibited as specific changes in CD3 : CD19 and CD4 : CD8 ratios, B-cell low-affinity Fcγ II and III receptor expression and induction of CD45R antigen.