Rapid assessment of the viability of Mycobacterium avium subsp. paratuberculosis cells after heating using an optimized phage amplification assay.

Thermal inactivation experiments were carried out to assess the utility of a recently optimized phage amplification assay to accurately enumerate viable Mycobacterium avium subsp. paratuberculosis cells in milk. Ultra-heat-treated (UHT) whole milk was spiked with large numbers of M. avium subsp. paratuberculosis organisms (10(6) to 10(7) CFU/ml) and dispensed in 100-μl aliquots in thin-walled 200-μl PCR tubes. A Primus 96 advanced thermal cycler (Peqlab, Erlangen, Germany) was used to achieve the following time and temperature treatments: (i) 63°C for 3, 6, and 9 min; (ii) 68°C for 20, 40, and 60 s; and (iii) 72°C for 5, 10, 15, and 25 s. After thermal stress, the number of surviving M. avium subsp. paratuberculosis cells was assessed by both phage amplification assay and culture on Herrold's egg yolk medium (HEYM). A high correlation between PFU/ml and CFU/ml counts was observed for both unheated (r(2) = 0.943) and heated (r(2) = 0.971) M. avium subsp. paratuberculosis cells. D and z values obtained using the two types of counts were not significantly different (P > 0.05). The D(68°C), mean D(63°C), and D(72°C) for four M. avium subsp. paratuberculosis strains were 81.8, 9.8, and 4.2 s, respectively, yielding a mean z value of 6.9°C. Complete inactivation of 10(6) to 10(7) CFU of M. avium subsp. paratuberculosis/ml milk was not observed for any of the time-temperature combinations studied; 5.2- to 6.6-log(10) reductions in numbers were achieved depending on the temperature and time. Nonlinear thermal inactivation kinetics were consistently observed for this bacterium. This study confirms that the optimized phage assay can be employed in place of conventional culture on HEYM to speed up the acquisition of results (48 h instead of a minimum of 6 weeks) for inactivation experiments involving M. avium subsp. paratuberculosis-spiked samples

Reperimento del virus dell'«arricciamento maculato» il del carciofo (AMCV) in Sardegna

A virus disease of «Masedu» of artichoke has been observed, in a serious form, in southern Sardinia (Italy). Affected plants are stunted with leaves heavily mottled, crinkled and laciniated. On the basis of the herbaceous hosts reactions, physical properties «in vitro», spectrophotometry, electron microscopy and serology, the virus recovered from diseased plants is identified as an isolate of AMCV, belonging to «Tombusvirus» group, serologically undistinguishable from the italian type isolate

Presenza di un «Potyvirus» sul Carciofo (<i>Cynara scolymus</i> L.) in Sardegna

A latent virus of artichoke has been isolated in Sardinia (Italy). The virus causes characteristic local lesions on Gomphrena globosa L. and Chenopodium amaranticolor Coste et Reyn., and systemic symptoms on Nicotiana benthamiana Domin. and N. clevelandii Gray. In artichoke crude sap the virus has a longevity in vitro of 20-30 hours, a dilution-end point between 10-3 and 10-4 and a thermal inactivation point between 55 and 60°C. The purification of the virus has been obtained from artichoke with two cicles of differential centrifugation followed by sucrose density gradient centrifugation. The purified suspensions had an ultraviolet light (UV) ab sorption spectrum typical of the nucleoproteins with Emax = 260-262 nm; Emin = 246 nm; and a ratio E280/E260 of 0,85 which suggests a RNA content of the virus of about 5,5%. Electron microscope observation showed that negative stained partially purified virus suspensions are composed of filamentous parti cles with a normal lenght (NL) of 730 nm and a mode of the lenght distributions of 729 nm; moreover, ultrathin sections of tissue fragments from leaves of mechanically inocu1ated Nicotiana benthamiana contained cytoplasmic inclusions of the pinwheel type. In serological tube tests, partially purified virus suspensions reacted with homologous serum (titre 1 : 1024) and a serum immune to an ALV (Artichoke Latent Virus) from Bari. These results have been substantially confirmed by serological tests with the method of «antibody coating of virus parti cles » by immune electron microscopy. I t is obvious that a latent potyvirus is widespread in artichoke growing in Sardinia. However, it cannot be exc1uded the possible occurrence in artichoke plants of a latent carlavirus too

Il Risanamento degli agrumi dal viroide dell'Exocortite mediante il microinnesto

Nearly all old line citrus trees growing in Sardinia (Italy) are infected by citrus exocortis viroid (CEV). The technique of shoot-tip grafting has proved effective in recovering various citrus cultivars free of this viroid

Influenza del Potyvirus latente del carciofo «Spinoso sardo» sulla produitività delle piante in pieno campo: 2. risultati relativi al secondo anno di impianto

In order to evaluate the influence of the artichoke latent Potyvirus (ALV) on plant productivity, a comparative field trial between virus-freed by meristem-tip culture and naturally Infected «Spinoso sardo» plants has been repeated for the second year in Sardinia. On the whole the results corroborate those of the first year: the latent infection doesn't affect the earliness and the number of heads per plant; whereas it negatively influences the weight, the diamater and the stem length of the heads

Development of a specific immunomagnetic capture-PCR for rapid detection of viable <i>Mycoplasma agalactiae</i> in sheep milk samples

Aims: To develop an immunomagnetic capture (IMC) to detect viable Mycoplasma agalactiae in routine ovine milk samples. Methods and Results: Polyclonal antibodies against two M. agalactiae membrane surface proteins (P80 and P55) were covalently conjugated to magnetic beads (MBs) to form MB-Ab80 and MB-Ab55. Mycoplasma agalactiae cells were captured by a specific antigen–antibody reaction and magnetic separation. Immunomagnetic capture (IMC) was used to isolate and concentrate M. agalactiae in serial decimal dilutions and in artificially contaminated milk to facilitate subsequent detection by PCR. A 375-bp fragment of M. agalactiae was amplified using a pair of M. agalactiae-specific primers in PCR. The limit of detection of IMC-PCR method ranged from 10 to 102 CCU ml -1 when mycoplasmas were resuspended in PBS and from 102 to 103 CCU ml-1 when mycoplasmas were resuspended in uncontaminated ovine milk. This study also describes the application of IMC-PCR method to test for M. agalactiae in 516 milk samples collected from sheep with suspected contagious agalactia. Its performance was evaluated relative to culture. Conclusions: This report has demonstrated for the first time, the effective use of rapid and reliable IMC combined with PCR assay for the detection of viable M. agalactiae. Significance and Impact of the Study: The method IMC-PCR provides an alternative to conventional microbiological detection, method and it could be applied to quick detection of M. agalactiae in routine sheep milk samples.</br

Influence of different drying parameters on the composition of volatile compounds of thyme and rosemary cultivated in Sardinia

The shelf life of spices is traditionally extended by drying. Fresh herbs, due to their high water content, undergo microorganism growth and adverse biochemical reactions. On the other hand drying may result in a lot of physical and chemical alterations. Air and oven-dehydration are the main methods used to stabilize spices. During oven drying, in general, losses of volatile compounds are directly dependent on the temperature and time used. This paper deals with the effect of different drying temperatures and air fluxes on the volatiles in rosemary (Rosmarinus officinalis L.) and thyme (Thymus officinalis L.) cultivated in Sardinia. Fresh leaves were collected and soon divided in two batches, which were subjected to hydro distillation and GC-MS analysis, the first batch as fresh, the second one after drying in a laboratory pilot dryer. Three drying temperatures were used, 30, 38 and 45°C, and for each one two airflow rates were set. The fresh and dried plant material were hydro distilled for 4 hours using a Clevenger-type apparatus (Italian Official Pharmacopeias X). The oils (liquid and light yellow) were recovered directly from above the distillate without adding any solvent and stored at –20°C before analyses, which were carried out on two replicates of each sample by gas chromatography, using a flame ionization detector. The diluted samples were injected using a split/splitless automatic injector (using 2,6-dimethylphenol as internal standard). Qualitative analysis was done by GC/Mass and mass units were monitored from 10 to 450 at 70 eV. Results of the influence of the different drying conditions on volatile compounds of the two herbs will be reported

Indagine fitosanitaria nel comprensorio di Sarroch, Villa S. Pietro e Pula (prov. di Cagliari) in relazione all'attività di uno stabilimento petrolifero

A five-year investigation (1988-92) of the phytosanitary situation in the Sarroch, Villa S. Pietro and Pula areas, belonging to the Cagliari district, where the SARAS-oil company has been operating for a long time, was carried out. Various degrees of damage caused by fungi, bacteria, viruses, insects, acari and by abiotic agents (edaphic and climatic) were detected on trees and herbaceous plants. Investigations on possible S02 damages by means of phytosanitary observations and chemical analysis, clearly showed no noxious effects of this gas on both cultivated and spontaneous plants

Profiling of the Bioactive Compounds in Flowers, Leaves and Roots of Vinca sardoa

Vinca sardoa (Stern) Pignatti (Apocynaceae) is largely distributed in Sardinia where it is considered a typical endemism. Since the plant is used in traditional folk medicine, the aim of the present work was to identify and quantify the polyphenolic metabolites, due to the well-known importance of polyphenolic compounds, as well as to evaluate antioxidant activity in different parts of the plant. The compounds were identified and determined in the methanol extracts of leaves, flowers, and roots by developing different LC-MS/MS methods. The obtained data show that leaves possess the highest amount of polyphenols, in particular quinic acid (3401 mg/100 g), chlorogenic acid (1082 mg/100 g), caffeoylquinic acid isomer 1 (190 mg/100 g), and robinin (633 mg/100 g). Likewise, antioxidant tests showed that leaves possess the main radical scavenging activities in both ABTS (49.19 ± 3.41 μg/mL, 30.88 ± 3.04 μg/mL at time zero and after 50 min, respectively) and DPPH assays (223.97 ± 30.81 μg/mL, 109.52 ± 12.89 μg/mL at time zero and after 30 min, respectively). Taking into account that leaves differed most from flowers and roots in the content of caffeoylquinic acid and chlorogenic acid, of which antioxidant properties are widely recognized, it is reasonable to assume that these two compounds are involved in the differences described. The relationship between the high polyphenolic content and interesting antioxidant activities, justifies its use in ethnobotany and may be suggest a use of this specie, after removal of the alkaloid fraction, in the pharmaceutical, phytotherapy, and cosmetic industries

Influence of different stabilizing operations and storage time on the composition of essential oil of thyme (Thymus officinalis L.) and rosemary (Rosmarinus officinalis L.)

Abstract The effect of different stabilizing techniques on the composition of essential oil of rosemary (Rosmarinus officinalis L.) and thyme (Thymus officinalis L.) during one year of storage is reported. The study was aimed to know what is the stabilizing technique to keep at the best the original essential oil composition. The fresh samples were collected and treated as follows: air-dried in a laboratory scale pilot dryer, frozen in a forced-air freezer and freeze-dried in a laboratory freeze-dryer. The fresh sample served as control. The treated samples were packaged with appropriate packaging material and stored at 20 °C or −20 °C for 12 months. All the samples were hydrodistilled every three months and the oils composition was obtained by means of gas chromatography/mass spectrometry (GC/MS). Quantification of known compounds was done with the use of an internal standard. Freezing best maintained the composition of rosemary and thyme essential oil. Appropriate packaging of air-dried and freeze-dried herbs resulted in negligible quality loss up to one year of storage. The frozen and stored thyme samples showed the best retention of thymol, the most important compound, as well as of γ-terpinene and carvacrol