Avirulence of viable but non-culturable Listeria monocytogenes cells demonstrated by in vitro and in vivo models

The virulence of Viable But Non-Culturable (VBNC) cells of 4 strains of Listeria monocytogenes was investigated in both a human adenocarcinoma cell line (HT-29) and a mouse model. LO 28, ATCC 19115 and CNL 895807 strains of Listeria monocytogenes became VBNC when incubated in microcosm water at 20 degrees C and Scott A strain at 4 degrees C. No culturable bacteria were detected in the VBNC state, although 104 active cells/mL were found by the Direct Viable Count (DVC) and CTC-DAPI double staining methods. A comparison of virulence in both human adenocarcinoma cell line HT-29 and the mouse model showed that culturable controls were more virulent than VBNC cells, which appeared to be avirulent regardless of the virulence methods applied. Pathogenicity was tested in each model and was lost concomitantly with culturability, whereas some cells were still metabolically active (determined by CTC and DVC). Moreover, amplification of a 388 bp fragment with Immunocapture-PCR revealed the presence of Listeria monocytogenes DNA in all mixed spleen samples after intravenous injection of VBNC cells. These results demonstrate that VBNC cells were present in the mouse spleens. The results of the study suggest that Listeria monocytogenes strains might remain in the aquatic environment for prolonged periods in the VBNC state but these cells were not pathogenic in the conditions tested. These findings demonstrate the value of VBNC studies and show the need to investigate the role of VBNC cells in environmental transmission of Listeria monocytogenes. Further studies are needed in order to investigate the virulence of VBNC cells of Listeria monocytogenes after recovery of a culturable state.     

Efficacy of Fostera PRRS modified live virus vaccine against a Canadian heterologous virulent field strain of porcine reproductive and respiratory syndrome virus

Vaccination is a useful option to control infection with porcine reproductive and respiratory syndrome virus (PRRSV), and several modified live-PRRSV vaccines have been developed. These vaccines have shown some efficacy in reducing the incidence and severity of clinical disease as well as the duration of viremia and virus shedding but have failed to provide sterilizing immunity. The efficacy of modified live-virus (MLV) vaccines is greater against a homologous strain compared with heterologous PRRSV strains. The objective of this study was to evaluate the efficacy of Fostera PRRS MLV vaccine in protecting against challenge with a heterologous field strain widely circulating in the swine herds of eastern Canada. Forty-six piglets were divided into 4 groups: nonvaccinated-nonchallenged; nonvaccinated-challenged; vaccinated-challenged; and vaccinated-nonchallenged. The animals were vaccinated at 23 d of age with Fostera PRRS and challenged 23 d later with a heterologous field strain of PRRSV (FMV12-1425619). Overall, the vaccine showed some beneficial effects in the challenged animals by reducing the severity of clinical signs and the viral load. A significant difference between nonvaccinated and vaccinated animals was detected for some parameters starting 11 to 13 d after challenge, which suggested that the cell-mediated immune response or other delayed responses could be more important than pre-existing PRRSV antibodies in vaccinated animals within the context of protection against heterologous strains.     

Use of recombinant human thyroid-stimulating hormone for thyrotropin stimulation test in healthy, hypothyroid and euthyroid sick dogs

Recombinant human thyroid-stimulating hormone (rhTSH) was evaluated for the diagnosis of canine hypothyroidism, using TSH response tests. Phase I stimulation tests were performed in 6 healthy dogs weighing over 20 kg, using 50 and then 100 microg of freshly reconstituted rhTSH administered intravenously. In phase II, the same dogs were stimulated by using 100 microg of rhTSH frozen for 3 months at -20 degrees C. Phase III stimulation tests were performed by using 50 or 100 microg of freshly reconstituted or frozen rhTSH in healthy (n = 14), euthyroid sick (n = 11) and hypothyroid dogs (n = 9). A dose of 100 microg of rhTSH was judged more appropriate for dogs weighing more than 20 kg. Biological activity of rhTSH after freezing at -20 degrees C for up to 12 weeks was maintained. When stimulated, significant (P < 0.05) increases in total thyroxine concentration were observed only in healthy and euthyroid sick dogs. Results of this study show that the rhTSH stimulation test is able to differentiate euthyroidism from hypothyroidism in dogs.     

Effect of ovotransferrin and lactoferrins on Chlamydophila psittaci adhesion and invasion in HD11 chicken macrophages

The effect of ovotransferrin (ovoTF), human lactoferrin (hLF) and bovine lactoferrin (bLF) on the obligate intracellular pathogen Chlamydophila (Cp.) psittaci was evaluated using a model of Buffalo Green Monkey kidney (BGM) cells and HD11 chicken macrophages as artificial hosts. Firstly, the effect of transferrins on the infectivity of the bacteria was evaluated. Pre-incubation of Cp. psittaci with 0.5 to 5 mg/mL ovoTF prior to infecting BGM cells significantly lowered the infection rate (P < 0.05). For both lactoferrins, the infection rate could only be reduced with 5 mg/mL, albeit not significantly as compared to the infection rate created by the untreated bacteria. Secondly, transferrins were tested for their ability to influence bacterial adhesion and entry in HD11 cells. Maximal non-cytotoxic and non-bactericidal concentrations of 0.05 mg/mL ovoTF and 0.5 mg/mL hLF and bLF were used. Overall, ovoTF was more effective than human and bovine LF in inhibiting bacterial irreversible attachment and cell entry and the latter was accompanied by a dose-dependent reduction of actin recruitment at the bacterial entry site. However, once bacteria had entered HD11 cells, transferrins had apparently no effect on intracellular replication. The present findings suggest a possible role for transferrins and especially ovoTF, in preventing avian Cp. psittaci infections.     

Stand-level wind damage can be assessed using diachronic photogrammetric canopy height models

Key message

Diachronic photogrammetric canopy height models can be used to quantify at a fine scale changes in dominant height and wood volume following storms. The regular renewal of aerial surveys makes this approach appealing for monitoring forest changes.

Context

The increasing availability of aerial photographs and the development of dense matching algorithms open up new possibilities to assess the effects of storm events on forest canopies.

Aims

The objective of this research is to assess the potential of diachronic canopy height models derived from photogrammetric point clouds (PCHM) to quantify changes in dominant height and wood volume of a broadleaved forest following a major storm.

Methods

PCHMs derived from aerial photographs acquired before and after a storm event were calibrated using 25 field plots to estimate dominant height and volume using various modeling approaches. The calibrated models were combined with a reference damage maps to estimate both the within-stand damage variability, and the amount of volume impacted.

Results

Dominant height was predicted with a root mean squared error (RMSE) of 4%, and volume with RMSEs ranging from 24 to 32% according to the type of model. The volume impacted by storm was in the range of 42–76%. Overall, the maps of dominant height changes provided more details about within-stand damage variability than conventional photointerpretation do.

Conclusion

The study suggests a promising potential for exploiting PCHM in pursuit of a rapid localization and quantification of wind-throw damages, given an adapted sampling design to calibrate models.

     

Growth phenology in <Emphasis Type="Italic">Pinus halepensis</Emphasis> Mill.: apical shoot bud content and shoot elongation

Key message

The chronology of periods of organogenesis and elongation is highlighted in Pinus halepensis.The two first growth units of an annual shoot are preformed inside the bud during the previous year. The following growth units are formed during the spring or summer of the current year.

Context

Analysis of annual shoot length growth phenology is crucial to assess the impact of climate change on tree production. Little is known about the basic growth characteristics and the phenology of pines.

Aims

The present study disentangles the roles of shoot organogenesis vs elongation in the annual growth cycle of the polycyclic Aleppo pine.

Methods

Growth of young Pinus halepensis trees was monitored monthly for 1 year. At each monitoring date, the bud content and meristem dimensions of the main stem shoots apices were analyzed.

Results

The two first growth units of an annual shoot are preformed inside the bud during the previous year. The following growth units are formed during the spring or summer of the current year. The gap between a shoot organogenesis and its elongation may vary from 1 month, for the last growth unit, to half a year, for the first growth units.

Conclusion

Our results underline the importance of taking seasonal environmental conditions from both the previous and the current year into account, in order to study the plasticity of annual shoot growth and its response to climate change and variability.

     

Phenolic antioxidants and antiatherogenic effects of Marula (Sclerocarrya birrea Subsp. caffra) fruit juice in healthy humans

Antioxidant activity and composition of Israeli-grown marula ( Sclerocarrya birrea subsp. caffra) fruit juice and health-promoting aspects of juice consumption on serum lipids and lipoproteins pattern in healthy volunteers were studied. Marula juice was found to contain high vitamin C and potassium levels and low sugar concentration (267 mg dL (-1), 328 mg dL (-1), and 7.3 g dL (-1), respectively). The juice contains a significant level of phenolics (56 mg of pyrogallol equiv dL (-1)) and was found to be a potent antioxidant (382 mg of vitamin C equiv dL (-1)). The antioxidant activity was resistant to pasteurization regimens and long-term freezing and slowly decreased during refrigeration, losing up to 14% of its capacity after 4 weeks. Three-week administration of the juice as a food supplement to healthy subjects significantly reduced their serum total cholesterol (by 8%), LDL-cholesterol concentration (by 17%), and triglyceride level (by 7%), increased their serum HDL-cholesterol level (by 10%), and attenuated serum oxidative stress. Upon a 4 week "washout" period, most of these parameters returned toward baseline values. Separation of the juice soluble phenolics by HPLC produced potent antioxidant fractions, tentatively containing hydrolyzable tannins, catechins, and hydroxycinnamic acid derivatives, which could be responsible for the observed protection against atherosclerosis risk factors following marula fruit juice consumption.     

Application of reflectance colorimeter measurements and infrared spectroscopy methods to rapid and nondestructive evaluation of carotenoids content in apricot (Prunus armeniaca L.)

The importance of carotenoid content in apricot (Prunus armeniaca L.) is recognized not only because of the color that they impart but also because of their protective activity against human diseases. Current methods to assess carotenoid content are time-consuming, expensive, and destructive. In this work, the application of rapid and nondestructive methods such as colorimeter measurements and infrared spectroscopy has been evaluated for carotenoid determination in apricot. Forty apricot genotypes covering a wide range of peel and flesh colors have been analyzed. Color measurements on the skin and flesh ( L*, a*, b*, hue, chroma, and a*/ b* ratio) as well as Fourier transform near-infrared spectroscopy (FT-NIR) on intact fruits and Fourier transform mid-infrared spectroscopy (FT-MIR) on ground flesh were correlated with the carotenoid content measured by high-performance liquid chromatography. A high variability in color values and carotenoid content was observed. Partial least squares regression analyses between beta-carotene content and provitamin A activity and color measurements showed a high fit in peel, flesh, and edible apricot portion (R(2) ranged from 0.81 to 0.91) and low prediction error. Regression equations were developed for predicting carotenoid content by using color values, which appeared as a simple, rapid, reliable, and nondestructive method. However, FT-NIR and FT-MIR models showed very low R(2) values and very high prediction errors for carotenoid content.     

Determination of proteins in refined and nonrefined oils

Five methods using aqueous/organic solvents for the separation of proteins from oils were compared. The extraction with acetone-hexane followed by amino acid analysis was found to be the most suitable method for isolation and quantification of proteins from oils. The detection limit of the method was 0.18 mg protein/kg oil, and the quantification limit was 0.6 mg protein/kg. The relative repeatability limit for samples containing 1-5 mg protein/kg sample was 27%. The protein recovery ranged between 68 and 133%. Using this method, the protein content of 14 refined and nonrefined oils was determined. In none of the refined oils were proteins detected, whereas the protein content of the unrefined oils ranged between undetectable in extra virgin olive oil to 11 mg/kg in rapeseed oil. With sodium dodecyl sulfate-polyacrylamide gel electrophoresis in combination with silver staining, many protein bands were visible in the unrefined soy, olive, peanut, and rapeseed oil samples. Proteins bands were not obtained from the refined fish oil. In the other refined oil samples, a few proteins bands could be visualized. Two protein bands with apparent molecular molecular masses of 58 and 64 kDa were always observed in these oils.     

Effect of Adding Exogenous Oxidative Enzymes on the Activity of Three Endogenous Oxidoreductases During Mixing of Wheat Flour Dough

The behavior of different exogenous enzymes (soybean lipoxygenase [SLOX], horseradish peroxidase [HPOD], catalase from bovine liver [BCAT], and glucose oxidase [GOX] from Aspergillus niger) added to dough was studied during mixing. The effect of adding these exogenous oxidoreductases on the activity of three oxidative enzymes present in wheat flour (lipoxygenase [WLOX], peroxidase [WPOD], and catalase [WCAT]) was examined. Proper assay conditions were established to differentiate between added WLOX, WPOD, and WCAT and the corresponding activities present in wheat flour. For doughs with added SLOX, an immediate loss of extractable SLOX (≈40%) was observed which remained constant during further mixing. When compared with the control dough, addition of SLOX decreased the losses in WLOX and WCAT activities, whereas WPOD activity was unaffected. With doughs supplemented by HPOD, an immediate loss of 20% in the HPOD activity was observed which did not change after 20 min of mixing. Compared with control dough, addition of HPOD did not affect the behavior of WLOX and WPOD, whereas a slight decrease in the WCAT losses was observed. Addition of BCAT to the dough did not change the behavior of WLOX and WPOD, whereas the losses in WCAT were less rapid. Half of the extractable activity of BCAT was lost at the beginning of mixing with no change during further mixing. For doughs supplemented with GOX, 25% of the GOX activity was lost in the first 5 min of mixing and an additional loss of 20% was observed after 20 min of mixing. Compared with dough without GOX, addition of GOX decreased the losses in WLOX, whereas losses in WCAT and WPOD increased. Glucose and ferulic acid were also added to doughs supplemented with GOX. Added glucose decreased the losses in GOX and WLOX and did not change the behavior of WPOD and WCAT during mixing. Addition of ferulic acid promoted a slight increase of the losses in WLOX and WCAT and almost no change for GOX and WPOD.