Impact of preheating on the behavior of Listeria monocytogenes in a broth that mimics Camembert cheese composition.

The effect of preheating on the survival of L. monocytogenes in Richard's broth, which mimics the composition of Camembert cheese composition, was examined. Experiments were carried out to reproduce contamination of cheese with environmental heat-stressed cells of L. monocytogenes surviving hot-cleaning procedures. Cells in mid-log phase were heated for 30 min at 56 degrees C before being inoculated into Richard's broth. The pHs and temperatures of Richard's broth were chosen to recreate the conditions of curd dripping (pH 5, 25 degrees C), of the beginning of cheese ripening (pH 5, 12 degrees C), and of the beginning (pH 5, 4 degrees C) and the end (pH 7, 4 degrees C) of cheese storage. Immediately after heat treatment, the viability loss was especially high for strain 306715, which exhibited only 0.6% +/- 0.2% survival, compared with 22% +/- 8.7% for strain EGD. The percentages of the surviving heated cells that were injured were 93% +/- 8% for strain 306715 and 98% +/- 3% for strain EGD. The destruction of the surviving L. monocytogenes cells was accelerated when they encountered the pH and temperature conditions of Camembert cheese during manufacturing, ripening, and cold storage (pH 5 at 25, 12, and 4 degrees C, respectively). The multiplication of the surviving heated cells was retarded under favorable growth conditions similar to those of storage by the distributor and the consumer (pH 7 at 4 and 12 degrees C, respectively).

Acid responses of Listeria monocytogenes.

The acid response and the correlated protein synthesis in Listeria monocytogenes were studied. The lowest pH value which L. monocytogenes could resist was dependent on the strain and the kind of acid used. Previous adaptation to an intermediary pH augmented bacterial resistance to a subsequent lethal acidic pH. The acid tolerance was also growth phase dependent. Organic volatile acids exerted a more deleterious effect on L. monocytogenes than inorganic acids, because weak acids infer a lower intracytoplasmic pH. The responses to acid adaptation (mildly acidic pH) and acid stress (lethal acidic pH) shared the majority of acid-induced proteins. The bacteria required more stress proteins to face severe acidic conditions. In order to obtain insights into the role these acid-induced proteins play in the mechanism of acid resistance, the proteins were analysed by two-dimensional electrophoresis and the most abundant were identified by peptide mass fingerprinting and MALDI-TOF mass spectrometry.

A proteomic approach to study the acid response in Listeria monocytogenes.

The responses of Listeria monocytogenes to acidic conditions were studied at the level of protein synthesis at a lethal acidic pH (acid stress) and an intermediary nonlethal acidic pH (acid adaptation). The radiolabeled acid-induced proteins were separated by two-dimensional (2-D) electrophoresis and analyzed by a computer-aided 2-D gel analysis system. The two acidic conditions upgraded a number of constitutive proteins and induced synthesis of a number of novel proteins. The majority of these induced proteins were common to the two pHs and the lethal acidic pH induced more proteins than the mildly acidic pH, suggesting that the responses to the two acidic conditions involve many common proteins and that additional proteins are required when the bacteria have to face more severe acidic conditions. In waiting for identification of more proteins involved in order to have a wholesome mechanistic picture of the acid response in L. monocytogenes, we present here the first results obtained from identification of the most abundant of these acid-induced proteins using peptide mass fingerprinting and matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) technique.

Microbiological hazards related to xenotransplantation of porcine organs into man.

Pigs are emerging as the most likely providers of genetically engineered organs and cells for the purpose of clinical xenotransplantation. Introduction of clinical trials has been delayed primarily by uncertainties regarding the risk of swine pathogen transmission that could harm the recipient. The concern that xenotransplantation carries the potential for a new epidemic has been highlighted by recent experiences with both bovine spongiform encephalopathy and human immunodeficiency diseases. As clinical trials have been postponed and xenotransplantation teams are working actively to gather data for an estimation of the risk, this review provides the reader with a state-of-the-art estimation of the microbiological hazards related to xenotransplantation of porcine organs to man. Particular emphasis is put on viral and retroviral hazards. Both current diagnostic tools and those under development are described, along with breeding strategies to provide donor animals that would not put the recipient or the general population at risk.

Stress proteins in Listeria monocytogenes.

The proteins induced by the different stress conditions in Listeria monocytogenes were analyzed by two-dimensional (2-D) electrophoresis with the aid of a computerized 2-D gel analysis system. The stress conditions imposed were pH 4, pH 10, 0.015% sodium, dodecyl sulfate (SDS), 0.03% sodium deoxycholate and 4% ethanol. As previously seen for heat shock and cold shock, more than half of the proteins normally synthesized by Listeria cells were repressed under these stress conditions. Conversely, the synthesis of a great number of proteins was increased and novel proteins appeared upon stress. Each stress factor induced a specific set of proteins. These stress proteins were characterized by their apparent molecular mass and isoelectric point. No universal stress proteins were found to be common to all the stresses studied, while some proteins were commonly induced by two or three stress conditions. The degree of dissimilarity in stress responses was best illustrated by the induction of only two proteins common to exposure to the two detergents SDS and sodium deoxycholate. This work together with that on heat and cold shock, constitutes the basic step for the identification of stress proteins in Listeria.

A chemically defined minimal medium for the optimal culture of Listeria.

Unlike most other bacteria, many Listeria strains do not grow well in the minimal media described so far in the literature. Among the minimal media tested, a chemically defined medium modified from that of Premaratne and co-workers was found to support the best growth of Listeria spp. The promoting effect was due to the incorporation of several indispensable vitamins and growth factors.

Analysis by two-dimensional electrophoresis of proteins secreted by sheep ovarian follicles: effects of the FecB gene, follicle size and atresia.

Proteins secreted by sheep ovarian follicles at different stages of maturation (small healthy, large healthy or large atretic) and originating from ewes with different ovulation rates (homozygous carriers or noncarriers of the FecB gene) were analysed by two-dimensional electrophoresis (PAGE) followed by computerized image analysis. Follicles were incubated intact for 1 h to assess steroidogenesis, and then incubated for 24 h in the presence of [35S]methionine. Secreted proteins were then resolved by iso-electric focusing followed by migration on 10% acrylamide slab gels and fluorography. Incorporation of label into proteins was similar irrespective of genetic type or health status of the follicles (4-6%). Small follicles incorporated significantly less (P < 0.05) label. Over 100 spots were detected on the fluorographs. The presence of the FecB gene induced qualitative and quantitative changes in the follicular protein patterns. Frequency of detection of spots 116 and 129 was significantly higher (P < 0.01) in Fec+Fec+ compared with FecBFecB follicles (80% versus 8%). In addition, amounts of proteins present in spots 2 and 12 were increased in FecBFecB follicles, while those in spots 9 and 21 were decreased. Size and atresia affected protein patterns only quantitatively. Increased amounts of proteins in spots 1, 10, 38, 44 and 113 were associated with atresia (P < 0.05). Follicle enlargement was associated with increased (P < 0.05) amounts of proteins in spots 5 and 6 and decreased amounts of proteins in spot 16. Amounts of three proteins (33, 40 and 58) were related positively to oestradiol production in vitro before labelling.

Analysis of heat and cold shock proteins in Listeria by two-dimensional electrophoresis.

The proteins induced by heat and cold shock in Listeria monocytogenes (pathogenic for humans) and L. innocua (nonpathogenic) strains were analyzed by two-dimensional (2-D) electrophoresis with the help of a computerized 2-D gel analysis system. Heat (49 degrees C) and cold (4 degrees C) shock repressed roughly half the number of proteins synthesized at normal temperature (25 degrees C) and decreased the level of numerous other proteins. Conversely, the synthesis of a great number of proteins was enhanced and novel proteins appeared upon temperature stress. There were more proteins induced in the L. monocytogenes strain than in the L. innocua strain. Each stress induced a set of specific proteins. There was overlap between these sets of proteins induced by heat and cold shock. Furthermore, a number of heat or cold shock proteins were found to be induced in both Listeria species and by both heat and cold shock in both species. The induction by heat shock was more intense than that by cold shock. The most strongly induced common stress protein of Listeria had a molecular mass of 17.6 kDa and an isoelectric point of 5.1.

Identification and classification of Listeria by two-dimensional protein mapping.

The cellular proteins of 29 Listeria strains belonging to different species and serotypes were analysed by two-dimensional (2-D) electrophoresis with the help of a computerized 2-D gel analysis system. The protein patterns were similar among strains within a Listeria species, but were different from one species to another. The comparative analysis of these protein maps enabled us to find specific proteins and to determine the genetic relatedness among Listeria spp. strains. The cluster analysis based on protein mapping showed a division between species and a clear separation of L. monocytogenes from other Listeria species. Inside L. monocytogenes the strains were divided into two main clusters in correlation with flagellar antigenic structures; this is in concordance with the results that have been found on the basis of multilocus enzyme electromorphs or DNA-restriction patterns. This technique enabled us to subtype the strains sharing the same serovar or the same lysovar. Because of its independence and high discriminating capacity, the 2-D protein mapping technique might provide a powerful tool for the identification and classification of Listeria strains.

Porcine retrovirus: optimal conditions for its biochemical detection.

The assay of reverse transcriptase (RT) activity was used to detect the presence of retrovirus in porcine cells. A set of optimal assay conditions was determined to design a sensitive, quantitative and reproducible RT assay for porcine systems. The template-primer poly(rA).oligo(dT) was an absolute requirement. The presence of Mn++ was indispensable, with an optimal concentration of 0.25 mM. Monocations (K+, Na+) at 50 mM greatly enhanced, but their high doses inhibited the reaction. The pH of the medium influenced very much the reaction, especially with non-purified virus samples, with which the RT activity was inhibited at pHs above 8.2. Non-ionic detergents at 1% enhanced several-fold the RT activity. It was also shown that porcine retrovirus could be spontaneously reactivated in porcine cell lines by in vitro long-term propagation and transmitted to pigs by inoculation with virus-producing cells.

Histocompatible miniature, boar model: selection of transformed cell lines of B and T lineages producing retrovirus.

A lymphoblastoid cell line (B1) was isolated in culture following a brief exposure to 5-azacytidine from peripheral-blood mononuclear cells of a boar previously injected with cells (Shimozuma) producing porcine retrovirus (Tsukuba-1) and suffering a severe non-neoplastic syndrome at autopsy. B1 cell line and 5 of its sublines were propagated for more than 100 generations, retaining doubling times comprised between 16.8 and 27.5 hr and growing readily in agarose or agar (plating efficiency: 5 to 50%). Karyotype analyses showed that 4 sublines were nearly diploid, except for cells of L14, which displayed a monosomy affecting chromosome 18 pair. Two sublines (L35 and L45) were considered as being of T-cell lineage, since MSA, antigen was observed on the surface of approximately 30% of cells. Three sublines (L23, L14 and L52) were considered of B-cell lineage, since membrane immunoglobulins were observed on the cell surface. In addition, sublines L23 and L52 were actively secreting immunoglobulin of mu isotype. Retrovirus particles were evidenced in gradient-purified preparation of 200-fold-concentrated cell culture supernatants of the B1 cell line, L14, L35 and L52 sublines, using both a reverse transcriptase activity assay and electron microscopic observation. These cell lines can be used to select for porcine retrovirus variants with transforming potential for lymphocytes of B and T lineages.

Immunochemical identification and biological characterization of cytotoxic necrotizing factor from Escherichia coli.

The aim of this study was to identify Escherichia coli cytotoxic necrotizing factor (CNF) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and to investigate the possible dissociation of CNF from hemolysin (Hly), which is often produced by CNF-producing strains. CNF was purified from cell lysates of a CNF-producing strain by using ammonium sulfate precipitation, ion-exchange chromatography, gel filtration, and preparative nondenaturing PAGE. All eluates from successive longitudinal slices of a preparative polyacrylamide gel were tested for cytoxicity and analyzed by SDS-PAGE; CNF activity was quantitatively correlated with a protein of 115 kilodaltons (kDa). This procedure increased both cytotoxicity and lethal activity (about 300-fold). We then compared SDS-PAGE protein patterns of enriched lysates from eight field and mutant E. coli strains producing both CNF and Hly, Hly alone, or neither; the 115-kDa band was present solely in CNF-producing strains, irrespective of Hly production. A neutralizing antiserum was produced against unpurified CNF from strain BM2-1 and then extensively adsorbed with cells and extracts of a CNF-defective mutant from BM2-1. The adsorbed antiserum possessed antitoxin activity and neutralized both lethal and necrotic effects of cell lysates from all the CNF-producing strains tested. In an immunoblot of enriched extract from BM2-1, the adsorbed antiserum recognized, besides the 115-kDa protein, another protein of 59 kDa, which was present in the CNF-defective mutant from BM2-1 and was not associated with cytotoxicity. We can conclude from these findings that CNF is a protein of 115 kDa associated with both cytotoxicity and in vivo toxicity, distinct from Hly, and present in all presumed CNF-producing strains tested.

[Rapid collection of swine lymphocyte subpopulations in large quantity]. / Obtention rapide de sous-populations lymphocytaires de porc en grande quantité.

Lymphocyte subpopulations derived from different pig organs (blood, spleen, mesenteric lymph nodes) were separated by a simple, rapid and cheap panning technique, using either normal or ozone treated Petri dishes with bovine serum albumin. Slg+ lymphocytes could thus be obtained with a purity of up to 95% by adhesion onto Corning plastic dishes. The purified cells retained their proliferative activity with regard to lectins. The subpopulation including PT4 and PT8 was then separated by another panning on ozone-treated plastic dishes with a purity of 80-90%.

Increased biosynthesis and processing of fibronectin in fibroblasts from diabetic mice.

Diabetic connective tissues exhibit a deranged regulation of extracellular matrix biosynthesis. Fibronectin is shown to be increased in human dermal connective tissue by immunofluorescence, mainly at the dermoepidermal and capillary basement membranes. The rate of fibronectin biosynthesis, excretion, and incorporation in a pericellular polymeric form was investigated using genetically diabetic KK mouse skin and fibroblasts as compared to Swiss and C57BL mouse skin and fibroblasts. The rate of incorporation of [35S]methionine into proteins recovered in the culture medium or in deoxycholate and NaDodSO4 or urea extracts was investigated. The rate of incorporation in the medium and deoxycholate extracts was comparable. However, the relative rate of incorporation of the tracer in the NaDodSO4-extractable, pericellular polymeric form was increased in the diabetic KK fibroblasts both for total proteins and for fibronectin. In pulse-chase experiments, the deoxycholate-soluble and NaDodSO4-soluble fractions exhibited a precursor-product relationship. The rate of passage of fibronectin from the deoxycholate-soluble (cellular compartment) form to the NaDodSO4-soluble (pericellular polymeric) form was strongly accelerated in the diabetic fibroblast cultures. These results confirm the increased rate of synthesis of fibronectin in diabetic fibroblasts as well as its processing from the cellular compartment to the polymeric pericellular form. The increase of fibronectin in diabetic connective tissues, in the matrix as well as in the basement membranes, may play a role in the mechanism of micro- and macroangiopathies and in the perturbed permeability characteristics of the diabetic capillaries, and as a glycoprotein it may contribute to the increased periodic acid/Schiff reagent staining of diabetic capillary basement membranes.

Purification of three porcine immunoglobulin classes from the same biological source.

Porcine IgG, IgA and IgM were isolated from sow milk. They were systematically purified by a combination of gel filtration and ion exchange chromatography. The yields were 85%, 64% and 32%, respectively. These immunoglobulins were very pure, as checked by SDS-PAGE and Ouchterlony immunodiffusion. SDS-PAGE showed moreover the evidence of secretory component S in the IgA molecule and of joining piece J both in IgA and IgM molecules. The procedure can be used to purify immunoglobulin classes from other biological sources.