Effect of dilution rate on the release of pertussis toxin and lipopolysaccharide ofBordetella pertussis

Summary The kinetics ofBordetella pertussis growth was studied in a glutamate-limited continuous culture. Growth kinetics corresponded to Monod's model. The saturation constant and maximum specific growth rate were estimated as well as the energetic parameters, theoretical yield of cells and maintenance coefficient. Release of pertussis toxin (PT) and lipopolysaccharide (LPS) were growth-associated. In addition, they showed a linear relationship between them. Growth rate affected neither outer membrane proteins nor the cell-bound LPS pattern.Nomenclature X cell concentration (g L^–1) - specific growth rate (h^–1) - _m maximum specific growth rate (h^–1) - D dilution rate (h^–1) - S concentration of growth rate-limiting nutrient (glutamate) (mmol L^–1 or g L^–1) - Ks substrate saturation constant (mol L^–1) - m_s maintenance coefficient (g g^–1 h^–1) - Y_x/s theoretical yield of cells from glutamate (g g^–1) - Y_x/s yield of cells from glutamate (g g^–1) - Y_PT/s yield of soluble PT from glutamate (mg g^–1) - Y_KDO/s yield of cell-free KDO from glutamate (g g^–1) - Y_PT/x specific yield of soluble PT (mg g^–1) - Y_KDO/x specific yield of cell-free KDO (g g^–1) - q_PT specific soluble PT production rate (mg g^–1 h^–1) - q_KDO specific cell-free KDO production rate (g g^–1 h^–1)     

Modifications of Bordetella bronchiseptica core lipopolysaccharide influence immune response without affecting protective activity

Bordetella bronchiseptica produces respiratory disease primarily in mammals including humans. Although a considerably amount of research has been generated regarding lipopolysaccharide (LPS) role during infection and stimulating innate and adaptive immune response, mechanisms involved in LPS synthesis are still unknown. In this context we searched in B. bronchiseptica genome for putative glycosyltransferases. We found possible genes codifying for enzymes involved in sugar substitution of the LPS structure. We decided to analyse BB3394 to BB3400 genes, closed to a previously described LPS biosynthetic locus in B. pertussis. Particularly, conservation of BB3394 in sequenced B. bronchiseptica genomes suggests the importance of this gene for bacteria normal physiology. Deletion of BB3394 abolished resistance to naive serum as described for other LPS mutants. When purified LPS was analyzed, differences in the LPS core structure were found. Particularly, a GalNA branched sugar substitution in the core was absent in the LPS obtained from BB3394 deletion mutant. Absence of GalNA in core LPS alters immune response in vivo but is able to induce protective response against B. bronchiseptica infection.     

Numerous transposed sequences of mitochondrial cytochrome oxidase I-II in aphids of the genus Sitobion (Hemiptera: Aphididae)

Polymerase chain reaction (PCR) products corresponding to 803 bp of the cytochrome oxidase subunits I and II region of mitochondrial DNA (mtDNA COI-II) were deduced to consist of multiple haplotypes in three Sitobion species. We investigated the molecular basis of these observations. PCR products were cloned, and six clones from one individual per species were sequenced. In each individual, one sequence was found commonly, but also two or three divergent sequences were seen. The divergent sequences were shown to be nonmitochondrial by sequencing from purified mtDNA and Southern blotting experiments. All seven nonmitochondrial clones sequenced to completion were unique. Nonmitochondrial sequences have a high proportion of unique sites, and very few characters are shared between nonmitochondrial clones to the exclusion of mtDNA. From these data, we infer that fragments of mtDNA have been transposed separately (probably into aphid chromosomes), at a frequency only known to be equalled in humans. The transposition phenomenon appears to occur infrequently or not at all in closely related genera and other aphids investigated. Patterns of nucleotide substitution in mtDNA inferred over a parsimony tree are very different from those in transposed sequences. Compared with mtDNA, nonmitochondrial sequences have less codon position bias, more even exchanges between A, G, C and T, and a higher proportion of nonsynonymous replacements. Although these data are consistent with the transposed sequences being under less constraint than mtDNA, changes in the nonmitochondrial sequences are not random: there remains significant position bias, and probable excesses of synonymous replacements and of conservative inferred amino acid replacements. We conclude that a proportion of the inferred change in the nonmitochondrial sequences occurred before transposition. We believe that Sitobion aphids (and other species exhibiting mtDNA transposition) may be important for studying the molecular evolution of mtDNA and pseudogenes. However, our data highlight the need to establish the true evolutionary relationships between sequences in comparative investigations.      

Environment and virulence factors of Vibrio cholerae strains isolated in Argentina

AIMS: To determine the presence of Vibrio cholerae in different areas of Argentina in three sample types, to determine the composition of planktonic communities in areas at which this pathogen was detected and to characterize the virulence properties and antimicrobial resistance of the recovered environmental isolates. METHODS AND RESULTS: Water and plankton samples were collected in marine, brackish and freshwater environments. Vibrio cholerae non-O1, non-O139 was isolated in 36.1% of the samples analysed. The micro-organism was detected in freshwater but not in marine or brackish samples. No relationship was found between isolation of V. cholerae and presence of any species of plankton. All the isolates presented very similar virulence profiles by PCR, lacking ctxA and tcpA El Tor and containing hlyA (98.7%), rtxA (99.0%), toxR (98.7%) and stn-sto (1.9%). Resistance to ampicillin was found in both Tucumán (21%) and Buenos Aires isolates (45%). CONCLUSIONS: We identified two geographic areas in Argentina where V. cholerae was present: freshwaters of the rivers from Tucumán and the Río de la Plata. SIGNIFICANCE AND IMPACT OF THE STUDY: The identification of V. cholerae strains in the environment, carrying both virulence factors and resistance to antimicrobial agents, highlight the need for a continuous and active surveillance of this pathogen.     

Fc receptor-mediated immunity against Bordetella pertussis.

The relevance of specific Abs for the induction of cellular effector functions against Bordetella pertussis was studied. IgG-opsonized B. pertussis was efficiently phagocytosed by human polymorphonuclear leukocytes (PMN). This process was mediated by the PMN IgG receptors, FcgammaRIIa (CD32) and FcgammaRIIIb (CD16), working synergistically. Furthermore, these FcgammaR triggered efficient PMN respiratory burst activity and mediated transfer of B. pertussis to lysosomal compartments, ultimately resulting in reduced bacterial viability. Bacteria opsonized with IgA triggered similar PMN activation via FcalphaR (CD89). Simultaneous engagement of FcalphaRI and FcgammaR by B. pertussis resulted in increased phagocytosis rates, compared with responses induced by either isotype alone. These data provide new insights into host immune mechanisms against B. pertussis and document a crucial role for Ig-FcR interactions in immunity to this human pathogen.     

A simple method for studying whole sections of rice grain

We developed a new and simple method to collect sections of a whole brown rice kernel for investigation of histological properties. A single kernel of rice was dehydrated through a graded ethanol series, transferred to xylene, and embedded in paraffin. During sectioning of the blocks using a rotary microtome, we used a special adhesive tape to collect and place the sections on slides so they remained flat. The use of the adhesive tape technique combined with autofluorescence characteristics allowed us to visualize cell walls throughout an entire longitudinal or transverse section of a whole rice kernel. We obtained scanning electron microscopy images of the sections to determine section quality.     

Initial verification of the resistance management strategy for Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae) in Australia

Shortly after the initial detection of western flower thrips (WFT), Frankiniella occidentalis (Pergande), in Australia during 1993 a resistance management strategy based on the alternation of chemical groups was implemented. This study aimed to verify this strategy by field testing α-cypermethrin against WFT with and without chemical alternation. Up to 114 times α-cypermethrin resistance (at LC50) was detected and resistance increased with and without chemical alternation; however, chemical alternation did significantly reduce numbers of thrips compared with a nonalternation strategy. Resistance has the potential to undermine the sustainable use of those chemicals to which there is no current detectable resistance. Consequently, chemicals with a high frequency and level of resistance against WFT need to be identified through monitoring and quickly eliminated from WFT chemical control recommendations.     

Effect of estrous cow serum during bovine embryo culture on blastocyst development and cryotolerance after slow freezing or vitrification

The present study investigated the effect of estrous cow serum (ECS) during culture of bovine embryos on blastocyst development and survival after cryopreservation by slow freezing or vitrification. Embryos were derived from in vitro maturation (IVM) and in vitro fertilization (IVF) of abbatoir-derived oocytes. At Day 3, embryos were cultured in three different media: Charles Ronsenkrans medium + amino acids (CR1aa; without bovine serum albumin (BSA)) + 5% estrous cow serum (CR1-ECS), CR1aa + 3 mg/mL BSA (CR1-BSA) or CR1aa + 5% ECS + 3 mg/mL BSA (CR1-ECS-BSA). At 7.5 d post-insemination (PI), blastocyst yield and quality were evaluated; blastocysts and expanded blastocysts from each media were cryopreserved by Open Pulled Straw (OPS) vitrification method or slow freezing (1.5 M ethylene glycol, EM). Total blastocyst yield did not differ among CR1-ECS, CR1-BSA and CR1-ECS-BSA (30.9, 33.1 and 32.9%, respectively, P < 0.05). Embryo survival (hatching rate) was higher in vitrified versus slow-frozen embryos (43% versus 12%, respectively, P < 0.01), and in embryos cultured in CR1-BSA (40.3%) compared with those cultured in serum-containing media (CR1-ECS, 21.5% and CR1-ECS-BSA, 19.8%; P < 0.01). In conclusion: (a) it was possible to produce in vitro bovine embryos in serum-free culture medium without affecting blastocyst yield and quality; (b) serum-free medium produced the best quality embryos (in terms of post-cryopreservation survival); and (c) vitrification yielded the highest post-cryopreservation survival rates, regardless of the presence of serum in the culture medium.     

Use of cyclodextrin as an agent to induce excretion of Bordetella pertussis antigens

Abstract This paper attempts to provide an explanation for the effect of cyclodextrin on the yield of Bordetella pertussis soluble antigens. It was demonstrated that the addition of cyclodextrin to the synthetic Stainer-Scholte liquid medium enhances the level of the intracellular form of adenylate cyclase (200 kDa) in the supernate. In addition to this effect, it has been reported that cyclodextrin also enhances the levels of two other extracellular proteins, pertussis toxin and filamentous hemagglutinin. As these antigens are structurally different, it seems that the effect of cyclodextrin is not specific. With the use of different buffer systems of well-known action on outer membrane stability it was possible to determine a relationship between the presence of cyclodextrin, destabilisation of the outer membrane and the release of proteins. It was determined that the cyclodextrin did not modify the fluidity of B. pertussis cells but produced a change of outer membrane permeability.