Affinity recovery of Moloney Murine Leukaemia Virus

Lipid enveloped retroviruses such as Moloney Murine Leukaemia Virus (MoMuLV) are commonly used gene therapy vectors. Downstream processing protocols used for their purification are time consuming and a potentially generic, single step capture method for the recovery of retroviral particles is proposed that exploits streptavidin-biotin affinity chromatography. The ability of four conventional adsorbent solid phases, Fractogel, Sepharose, Magnespheres and STREAMLINE immobilised with streptavidin, to capture and recover biotinylated Moloney Murine Leukaemia Virus was studied. MoMuLV can be biotinylated whilst retaining infectivity and the biotinylated virus can be adsorbed to Streptavidin Magnespheres yielding a 2298-fold increase in titre. For optimal virus biotinylation purification using Fractogel streptavidin can yield a 1896-fold increase in cfu/mg of protein and a 1191-fold decrease in DNA/cfu. Infectious virus can be recovered from Fractogel streptavidin with a maximum recovery of 16.7%.     

Development of a microtitre-based spectrophotometric method to monitor Babesia divergens growth in vitro

Babesia divergens multiplication cycle involves erythrocyte invasion, intracellular division, and erythrocyte lysis with the simultaneous liberation of hemoglobin. We have decided to set up a spectrophotometric protocol based on hemoglobin concentration in the culture supernatants to monitor B. divergens in vitro growth. After the selection of 405 nm as the most appropriate endpoint hemoglobin wavelength in our conditions (hemoglobin concentration in the supernatant), cultures were standardized [1 x 10(9) red blood cell (RBC)/ml, 1-2.5 x 10(5) infected red blood cell (iRBC)/ml] to allow their monitoring over 3 days. The protocol was then compared to the most commonly used growth measurement methods: parasitemia counting and [(3)H]hypoxanthine incorporation. An excellent correlation was demonstrated between A(405) of the culture supernatant and parasitemia of the iRBC, whatever the RBC concentration used in the medium. This correlation was also evidenced between A(405) and [(3)H]hypoxanthine incorporation for [(3)H]hypoxanthine concentrations lower than 4 microCi/ml. Our assays also highlighted the inhibitory effect of [(3)H]hypoxanthine on B. divergens growth even when used at low concentrations (0.8 microCi/ml) and for a short incorporation duration (24 h). This effect was confirmed by both A(405) and parasitemia counting. In conclusion, A(405) measurement of B. divergens culture supernatant represents a simple, rapid, safe, and reliable way to measure the in vitro growth of this parasite. Generation times of three different B. divergens strains were then determined by the protocol described here and varied between 8 h 36 min and 13 h 8 min.     

Rapid and sensitive magnetoelastic biosensors for the detection of Salmonella typhimurium in a mixed microbial population

In this article, we report the results of an investigation into the performance of a wireless, magnetoelastic biosensor designed to selectively detect Salmonella typhimurium in a mixed microbial population. The Langmuir-Blodgett (LB) monolayer technique was employed for antibody (specific to Salmonella sp.) immobilization on rectangular shaped strip magnetoelastic sensors (2 x 0.4 x 0.015 mm). Bacterial binding to the antibody on the sensor surface changes the resonance parameters, and these changes were quantified as a shift in the sensor's resonance frequency. Response of the sensors to increasing concentrations (5 x 10(1) to 5 x 10(8) cfu/ml) of S. typhimurium in a mixture of extraneous foodborne pathogens (Escherichia coli O157:H7 and Listeria monocytogenes) was studied. A detection limit of 5 x 10(3) cfu/ml and a sensitivity of 139 Hz/decade were observed for the 2 x 0.4 x 0.015 mm sensors. Binding kinetics studies have shown that the dissociation constant (K(d)) and the binding valencies for water samples spiked with S. typhimurium was 435 cfu/ml and 2.33 respectively. The presence of extraneous microorganisms in the mixture did not produce an appreciable change in the biosensor's dose response behavior.     

Characterization of lactic acid bacteria isolated from the one humped camel milk produced in Morocco

One hundred and twenty (120) strains of lactic acid bacteria (LAB) were enumerated and isolated from raw dromedary milk in Morocco using various cultured media. Strains isolated were characterized by phenotypic, physiological and biochemical properties. Results showed that high counts of LAB were found. Presumptive lactobacilli counts ranged from 2.5x10(2) to 6x10(7)cfu/ml, presumptive lactococci levels varied from 5x10(2) to 6x10(7)cfu/ml, presumptive streptococci counts varied from 4.2x10(2) to 8x10(7)cfu/ml, presumptive leuconostoc levels ranged from 5.4x10(2) to 5.4x10(7)cfu/ml. Results showed also that Lactobacillus and Lactococcus were the predominant genera with 37.5% and 25.8%, respectively. The dominated species found were Lactococcus lactis subsp. lactis (17.5%), Lactobacillus helveticus (10%), Streptococcus salivarius subsp. thermophilus (9.20%), Lactobacillus casei subsp. casei (5.80%) and Lactobacillus plantarum (5%). This is the first report on the characterization of LAB strains isolated from the one humped camel milk produced in Morocco.     

Bifidobacterium longum ATCC 15707 cell production during free- and immobilized-cell cultures in MRS-whey permeate medium

Bifidobacterium longum ATCC 15707 cell production was studied in MRS medium supplemented with whey permeate (MRS-WP) during free-cell batch fermentations and continuous immobilized-cell cultures. Very high populations were measured after 12 h batch cultures in MRS-WP medium controlled at pH 5.5 (1.7+/-0.5x10(10) cfu/ml), approximately 2-fold higher than in non-supplemented MRS. Our study showed that WP is a low-cost source of lactose and other components that can be used to increase bifidobacteria cell production in MRS medium. Continuous fermentation in MRS-WP of B. longum immobilized in gellan gum gel beads produced the highest cell concentrations in the effluent (4.9+/-0.9x10(9) cfu/ml) at a dilution rate (D) of 0.5 h(-1). However, maximal volumetric productivity (6.9+/-0.4x10(9) cfu ml(-1)h(-1)) during continuous cultures was obtained at D =2.0 h(-1), and was approximately 9.5-fold higher than during free-cell batch cultures at an optimal pH of 5.5 (7.2x10(8) cfu ml(-1)h(-1)).     

High concentration cultivation of Bifidobacterium bifidum in a submerged membrane bioreactor

In batch cultures, after 25 h, the maximum cell mass of Bifidobacterium bifidum BGN4 was 4.5 g/L, and the maximum cell count was 3.0 x 10(9) cfu/mL at pH 6.0 and 50 g/L sucrose. To increase the viable counts of bifidobacteria, cell retentive culture was applied using a submerged membrane bioreactor with suction and gas sparging. The maximum mass, count, and productivity of the cells after 36 h were 12.0 g/L, 2.2 x 10(10) cfu/mL, and 6.1 x 10(8) cfu/mL x h, respectively, at the feeding (dilution) rate of 120 mL/h (0.06 h-1) in the feeding medium. The accumulated levels of organic acids and ammonium ions at the end of the cultivation were 1.5 and 1.0 g/L, respectively. The viable counts and volumetric productivity of the cells after the cell retentive culture were 7.3- and 5.1-fold higher, respectively, than the values obtained during batch culture. These high viable counts and volumetric productivities were obtained by maintaining lower concentrations of organic acids and ammonium ions so that the growth of B. bifidum BGN4 was not inhibited. The submerged membrane bioreactor produced the highest viable counts of B. bifidum without membrane fouling and cell damage.     

人母乳细菌Streptococcus salivarius F286和S. parasanguinis F278对HT29细胞的细胞毒性

为了筛选到具有抗炎特性的有益菌,研究者通常将待测细菌的发酵液上清和热致死菌体与TNF-α刺激下的人类结肠腺癌细胞HT29共孵育,并测量细菌是否能够减少HT29细胞分泌的炎症因子。该测试的前提之一是待测细菌的发酵液上清或菌体不杀死或杀死<10%的HT29细胞。在前期的工作中,我们从人母乳中分离得到Streptococcus salivarius F286和S.parasanguinis F278两株菌。在研究这两株菌的抗炎能力之前,我们利用MTT法摸索不同浓度的S.salivarius F286和S.parasanguinis F278的发酵液上清和热致死菌体对HT29细胞的细胞毒性。实验表明,两株菌的发酵液上清的原液和稀释液对HT29细胞均没有细胞毒性;浓度5×10^5~7.5×10^6cfu/mL的F286热致死菌体、浓度5×10^5~2.5×10^6cfu/mL的F278热致死菌体对HT29细胞的细胞毒性低于10%,而浓度1×10^8cfu/mL的热致死F286和F278菌体分别杀死(23±5.3)%和(22±5.3)%的HT29细胞。因此,S.salivarius F286和S.parasanguinis F278的发酵液上清原液、以及浓度5×10^5~7.5×10^6cfu/mL的F286热致死菌体和5×10^5-2.5×10^6cfu/mL的F278热致死菌体可在HT29细胞模型中进行抗炎能力测试。本研究的方法可用于确定其他细菌在HT29细胞模型中进行抗炎能力测试的合理浓度范围。     

Purification of a functional gene therapy vector derived from Moloney murine leukaemia virus using membrane filtration and ceramic hydroxyapatite chromatography

The ability of membrane ultra- and diafiltration and two chromatography media, Matrex Cellufine Sulfate (Millipore) and Macro-Prep ceramic hydroxyapatite (Bio-Rad), to adsorb, elute, and purify gene therapy vectors based on Moloney murine leukaemia virus (MoMuLV) carrying the 4070A amphotropic envelope protein was studied. Membrane ultra- and diafiltration provided virus concentration up to 160-fold with an average recovery of infectious viruses of 77 +/- 14%. In batch experiments, Macro-Prep ceramic hydroxyapatite (type 2, particle size 40 microm) proved superior to Matrex Cellufine Sulfate for MoMuLV vector particle adsorption. Furthermore, functional vector particles could be eluted using phosphate buffer pH 6.8 (highest titres from >or=300 mM phosphate) from the Macro-Prep adsorbent, with higher specific titres (cfu/mg protein) than the starting material. Similar results were obtained when this ceramic hydroxyapatite was packed into a column and used in a liquid chromatography system. Recovery of transduction-competent virus was between 18 and 31% for column experiments and 32 and 46% for batch experiments.     

Investigation of a simple amperometric electrode system to rapidly quantify and detect bacteria in foods

A two electrode system mounted as a single probe was developed to measure electrochemically the rate of reduction of a redox mediator (thionine) by bacteria. The system gave a rapid (2 min) bacterial-dependent current above 2.5 x 10(5) cfu/ml with pure cultures of bacteria, but when applied to the measurement of the bacterial contamination in samples of meat and milk it was unable to detect or quantify the contamination reliably. Incubation of samples for a few hours before examination enabled the system to detect bacteria in excess of 10(6) cfu/ml.     

Colloidal gold as an electrochemical label of streptavidin-biotin interaction

A new electrochemical method to monitor biotin-streptavidin interaction, based on the use of colloidal gold as an electrochemical label, is investigated. Biotinylated albumin is adsorbed on the pretreated surface of a carbon paste electrode (CPE). This modified electrode is immersed in colloidal gold-streptavidin labelled solutions. Adsorptive voltammetry is used to monitor colloidal gold bound to streptavidin, obtaining a good reproducibility of the analytical signal (R.S.D. = 3.3%). A linear relationship between peak current and streptavidin concentration from 2.5 x 10(-9) to 2.5 x 10(-5) M is obtained when a sequential competitive assay between streptavidin and colloidal gold-labelled streptavidin is carried out. On the other hand, the adsorption of streptavidin on the electrode surface was performed, followed by the reaction with biotinylated albumin labelled with colloidal gold. In this way, a linear relationship between peak current and colloidal gold labelled biotinylated albumin concentration is achieved with a limit of detection of 7.3 x 10(9) gold particles per ml (5.29 x 10(-9) M in biotin).     

Nutrient recovery from domestic wastewater using a UASB-duckweed ponds system

The pilot-scale wastewater treatment system used in this study comprised a 40-l UASB reactor (6-h HRT) followed by three duckweed ponds in series (total HRT 15 days). During the warm season, the treatment system achieved removal values of 93%, 96% and 91% for COD, BOD and TSS, respectively. Residual values of ammonia, TKN and total phosphorus were 0.41 mg N/l, 4.4 mg N/l and 1.11 mg P/l, with removal efficiencies of 98%, 85% and 78%, respectively. The system achieved 99.998% faecal coliform removal during the warm season with final effluent containing 4 x 10(3) cfu/100 ml. During the winter, the system was efficient in removing COD, BOD and TSS but not nutrients. The system was deficient in the removal of faecal coliforms during the winter, producing effluent with 4.7 x 10(5) cfu/100 ml. During the warm season, the N removal consisted of 80% by plant uptake, 5% by sedimentation and 15% unaccounted for. A duckweed production rate of 33 t dry matter per hectare per 8 months was achieved.     

Immunoblot assay: a rapid and sensitive method for identification of salmonid fish viruses

An immunoblot assay was used to identify the viruses of infectious pancreatic necrosis, infectious hematopoietic necrosis, and viral hemorrhagic septicemia. Viral antigen in infected cell culture supernatant was adsorbed onto nitrocellulose membrane or Whatman 541 filter paper and detected by enzyme-linked immunosorbent assay techniques. The immunoblot assay took less than 4 hr to perform and required no special instrumentation. Assays using cell culture supernatant fluids showed immunoblot sensitivity was 10(5) - 10(6) PFU/ml. Assay sensitivity, determined using purified virus, is 0.85-4.0 ng of viral antigen. The immunoblot assay was used to detect and identify virus in cell culture fluids.     

Control of Lactobacillus contaminants in continuous fuel ethanol fermentations by constant or pulsed addition of penicillin G

The addition of penicillin G to combat microbial contamination in continuous fuel alcohol fermentations was performed using both continuous and pulsed addition regimes. In continuous fermentations where both Saccharomyces cerevisiae and Lactobacillus paracasei were present, the mode of addition of penicillin G determined final numbers of viable L. paracasei. When the same overall average concentration of penicillin G was added in both pulsed and continuous modes, the initial viable number of L. paracasei (8.0 x 10(9) cfu ml(-1)) decreased to a greater degree (1.02 x 10(5) cfu ml(-1) L. paracasei) when penicillin G was pulsed at 6 h frequencies at an overall average concentration of 2,475 U/l than when penicillin G was added continuously at 2,475 U/l (2.77 x 10(5) cfu ml(-1) L. paracasei). Pulsed additions over longer frequencies at 2,475 U/l were not as effective in reducing viable bacteria. Viable yeasts increased during both treatment conditions by more than 2-fold. The two addition regimes also eliminated the 40% decrease in ethanol concentration caused by the intentional bacterial infection. Although there was 3 times more bacterial death with 6 h pulsed additions compared to continuous additions of penicillin G at 2,475 U/l, there was, by that point, no practical difference in either final ethanol concentration or relative ethanol recovery.     

Elaboration of an electroporation protocol for Bacillus cereus ATCC 14579

An electro-transformation procedure was established for Bacillus cereus ATCC 14579. Using early growth-stage culture and high electric field, the ectroporation efficiency was up to 2 x 10(9) cfu microg(-1) ml(-1) with pC194 plasmid DNA. The procedure was tested with three other plasmids, of various sizes, replication mechanisms and selection markers, and the transformation efficiencies ranged between 2 x 10(6) and 1 x 10(8) cfu microg(-1) ml(-)(1). The effects of two wall-weakening agents on electroporation rates were also evaluated. The transformation rate that was reached with our procedure is 10(3) times higher than that previously obtained with members of the Bacillus genus with similar plasmids, and 10(6) times superior than that achieved with available protocols for B. cereus. The proposed method is quick, simple, efficient with small rolling circle plasmids and large theta replicating plasmids with low copy number per cell, and suitable for many genetic manipulations that are not possible without high-efficiency transformation protocols.     

Adaptation to the cell line PLC/PRF/5 of hepatitis A virus released in the cell culture medium

The propagation of hepatitis A virus (HAV), CF53 strain, released without any cytopathic effect into the PLC/PRF/5 cells supernatant, was studied in the course of six serial passages (6th to 11th). The decrease (from 5 to 1 week) of incubation time required to detect HAV, by RIA, in culture supernatant, the increase in Hepatitis A antigen (from 777 to 10,038 c.p.m./50 microliter) and infectivity titre (from 10(3.0) TCID 50/ml to 10(4.5) TCID 50/ml) were consistent with the adaptation of this virus to the cell line PLC/PRF/5.     

Evaluation of Methods for Reestablishment of L-Cell Suspension Cultures Directly from Liquid Nitrogen Stored Stocks

Methods were developed and evaluated for the preservation of tissue cells grown in suspension culture and the reestablishment of suspension cultures directly from inoculum stored at -175 C. The factors investigated were processing pH, temperature of processing, freezing medium, and method of inoculation of the starter suspension cultures from the frozen stock (-175 C). Three parameters, cell viability, cell size, and growth potential in suspension culture after freezing, were used to evaluate the various factors. The results indicate that cells processed at 4 C, frozen at 1 C per min to -50 C in a medium containing 5% dimethyl sulfoxide plus 10% bovine serum at concentrations of 2 x 10(7) to 4 x 10(7) cells/ml, and stored at -175 C will reestablish suspension cultures directly from frozen seed. A 1-ml amount of frozen stock inoculated into 99 ml of medium routinely produced 2 x 10(6) to 3 x 10(6) viable cells/ml (2 x 10(8) to 3 x 10(8) total cells) in suspension culture in 4 to 5 days. Inoculum preserved by this procedure grew equally well in either serum-free or serum-containing growth medium.     

High-yield culture and purification of Chlamydiaceae bacteria

Research on intracellular bacteria of the family Chlamydiaceae, and the diseases they cause, requires large amounts of infectious elementary bodies (EB). We describe an approach that maximizes the generation of Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydia abortus, or Chlamydia pecorum EBs in several replication cycles over approximately 10 days or more in a saturated equilibrium monolayer cell culture system. Buffalo Green Monkey Kidney (BGMK) cells, Human Epidermoid Carcinoma-2 (HEp-2) cells, or mouse McCoy cells were tested. BGMK cells best supported C. pneumoniae replication when cultivated in Iscove's Modified Dulbecco's Medium. From day 1 to day 9 after inoculation, C. pneumoniae genomes per ml culture medium increased from 10(5.1) to 10(8.6) in BGMK, from 10(5.6) to 10(8.1) in HEp-2, and remained at 10(5.2) in McCoy cell cultures. Three-month pre-inoculation maintenance of BGMK cells in different culture media did not influence C. pneumoniae yields. Inoculation at multiplicities of infection (MOI) of 10 or higher and supplementation of the cell culture medium on day 7 after inoculation with 0.1% glucose enhanced C. pneumoniae EB yields in harvested cell culture medium. For purification, EBs in medium were concentrated by sedimentation, followed by low-speed centrifugation for removal of host cell nuclei, and by step-gradient centrifugation of the supernatant in a 30% RenoCal-76-50% sucrose step-gradient. Extensive sonication increased yield and infectivity of chlamydial EB. The combined method typically produced from 1000 ml infected BGMK culture medium 10 ml homogeneous, single-cell, highly infectious EB stock containing approximately 5x10(11) C. pneumoniae genomes equivalent to 4-5x10(11) inclusion forming units.     

Growth of Salmonella enterica in model mixed cultures during a two-step enrichment

Growth of Salmonella enterica was studied in model mixed cultures with Citrobacter freundii or Escherichia coli in buffered peptone water (BPW) and in Rappaport-Vassiliadis medium with soya (RVS) with modified concentrations of MgCl2 and malachite green, and at modified incubation temperatures. Selected S. enterica strains were inoculated in BPW (10(0) cfu/ml) together with selected strains of Citrobacter freundii (up to 10(8) cfu/ml) or selected strains of Escherichia coli (up to 10(8) cfu/ml), incubated overnight and then subcultured (1: 100) in RVS variants. Growth of individual bacterial species was followed by the quantitative real-time polymerase chain reaction (PCR). Optimal culture conditions during the second selective step were: MgCl2.6 H2O concentration of 29 g/l, malachite green concentration of 36 mg/1l, and the incubation temperature of 41.5 degrees C. Citr. freundii was found to be a potent competitor and E. coli was a weaker competitor. At optimal culture conditions, competition was reduced and the density of S. enterica cultures reached the level of 10(4) cfu/ml after not later than 2 h of selective enrichment. The results obtained provide a basis for the development of a short two-step enrichment to be used in rapid real-time PCR-based methods for the detection of S. enterica in food and other matrices.