Chlorine dioxide inactivation of bacterial threat agents

Aims: To evaluate the efficacy of chlorine dioxide (ClO₂) against seven species of bacterial threat (BT) agents in water. Methods and Results: Two strains of Bacillus anthracis spores, Yersinia pestis, Francisella tularensis, Burkholderia pseudomallei, Burkholderia mallei and Brucella species were each inoculated into a ClO₂ solution with an initial concentration of 2·0 (spores only) and 0·25 mg l^?1 (all other bacteria) at pH 7 or 8, 5 or 25°C. At 0·25 mg l^?1 in potable water, six species were inactivated by at least three orders of magnitude within 10 min. Bacillus anthracis spores required up to 7 h at 5°C for the same inactivation with 2·0 mg l^?1 ClO₂. Conclusions: Typical ClO₂ doses used in water treatment facilities would be effective against all bacteria tested except B. anthracis spores that would require up to 7 h with the largest allowable dose of 2 mg l^?1 ClO₂. Other water treatment processes may be required in addition to ClO₂ disinfection for effective spore removal or inactivation. Significance and Impact of Study: The data obtained from this study provide valuable information for water treatment facilities and public health officials in the event that a potable water supply is contaminated with these BT agents.     

Retransformation of ts SV40 Transformants by Murine Sarcoma Virus at Non-permissive Temperature

Temperature sensitive (ts) SV40 transformed mouse fibroblasts (tsSV3T3) express their transformed phenotype in vitro when growing at 32° C but not when growing at 39° C¹. Viral mRNA is, however, apparently transcribed at 39° C, for SV40 specific T-antigen can be demonstrated and viral mRNA can be found by nucleic acid hybridization: Fusion-rescue experiments show that the transforming virus is wild type but tsSV3T3 cells cannot be re-transformed at 39° C with high multiplicity SV40. This suggests that the temperature sensitive behaviour stems from a cellular rather than a viral mutation. The question then arises of the stringency with which these ts transformants control the expression of viral transformation functions at 39° C.     

Effect of various environmental factors on the viability of Cryptosporidium parvum oocysts

Aims: To evaluate individual and combined effects of temperature (4, 18 and 25°C), pH (7 and 10), ammonia (5 and 50 mg l^?1) and exposure time (1, 2, 4 and 6 days) on the viability of Cryptosporidium parvum oocysts in water. Methods and Results: The viability of oocysts was evaluated using the fluorogenic vital dyes assay (4′,6‐diamidino‐2‐phenylindole and propidium iodide). All the factors analysed (temperature, pH, ammonia and exposure time) and their interaction were statistically significant (P < 0·005). Exposure of oocysts to pH 10 for 6 days at 25°C reduced oocyst viability from ～80% to 51%. Similarly, the exposure of C. parvum oocysts to 5 mg NH₃ l^?1 and 50 mg NH₃ l^?1 for 4 days reduced their viability from between ～80% to 41·5% and 14·8%, respectively. Conclusions: The interaction between pH, temperature and exposure time may have adverse effects on the survival of C. parvum oocysts in water. Low concentrations of ammonia, as commonly found in alga‐based wastewater systems, over a long period of time can produce high C. parvum oocyst inactivation rates. Significance and Impact of the Study: This study provides relevant data on the inactivation of C. parvum oocysts in alga‐based wastewater‐treatment systems in the northwest of Spain.     

Storage of ethyl methanesulphonate-treated barley seeds with low moisture contents

Barley seeds were treated with ethyl methanesulphonate (EMS) for 3 h at 25° C, washed with tap water for 24 h at 25° C, redried at 40° C to different moisture contents below 15% and stored at 25° C in desiccators or in sealed plastic bags. The criteria used for expressing the effect of storage were the M₁ seedling height and the frequency of chromosomal aberrations. With 14·9% seed moisture a strong increase of biological injury occurred in the course of a 2-week storage, while storage of seeds having an initial moisture content of 11·7% led to a significant increase of injury only after 6 weeks. Superdry EMS-treated seeds with 5% or less moisture can be stored at 25° C without any changes in the biological effects. A method is recommended to avoid the EMS-storage effects.     

Development of resistance in Cronobacter sakazakii ATCC 29544 to thermal and nonthermal processes after exposure to stressing environmental conditions

Aims: The objective was to study the response of Cronobacter sakazakii ATCC 29544 cells to heat, pulsed electric fields (PEF), ultrasound under pressure (Manosonication, MS) and ultraviolet light (UV‐C) treatments after exposure to different sublethal stresses that may be encountered in food‐processing environments. Methods and Results: Cronobacter sakazakii stationary growth‐phase cells (30°C, 24 h) were exposed to acid (pH 4·5, 1 h), alkaline (pH 9·0, 1 h), osmotic (5% NaCl, 1 h), oxidative (0·5 mmol l^?1 H₂O₂, 1 h), heat (47·5°C, 1 h) and cold (4°C, 4 h) stress conditions and subjected to the subsequent challenges: heat (60°C), PEF (25 kV cm^?1, 35°C), MS (117 μm, 200 kPa, 35°C) and UV‐C light (88·55 mW cm^?2, 25°C) treatments. The inactivation kinetics of C. sakazakii by the different technologies did not change after exposure to any of the stresses. The combinations of sublethal stress and lethal treatment that were protective were: heat shock–heat, heat shock–PEF and acid pH–PEF. Conversely, the alkaline shock sensitized the cells to heat and UV‐C treatments, the osmotic shock to heat treatments and the oxidative shock to UV‐C treatments. The maximum adaptive response was observed when heat‐shocked cells were subjected to a heat treatment, increasing the time to inactivate 99·9% of the population by 1·6 times. Conclusions: Cronobacter sakazakii resistance to thermal and nonthermal preservation technologies can increase or decrease as a consequence of previous exposure to stressing conditions. Significance and Impact of the Study: The results help in understanding the physiology of the resistance of this emerging pathogen to traditional and novel preservation technologies.     

Influence of pH and sodium chloride on kinetics of thermal inactivation of the bacteriocin-like substance P34

Aims: To investigate the kinetics of thermal inactivation of the bacteriocin‐like substance P34 at different pH and sodium chloride concentration. Methods and Results: Samples of bacteriocin were treated at different time–temperature combinations in the range of 0–300 min and 90–120°C and the kinetic parameters for bacteriocin inactivation were calculated. For all treatments, the thermal inactivation reaction fitted adequately to first‐order model. D‐ and k‐values were smaller and higher, respectively, for pH 4·5 than for 6·0 or 7·0, indicating that bacteriocin P34 was less thermostable at lower pH. At 120, 115 and 100°C, the addition of sodium chloride decreased thermal stability. For other temperatures, addition of NaCl increased stability of the peptide. The presence of greater amount of the salt (50 g l^?1) resulted in a higher thermal stability of bacteriocin P34, suggesting that the reduction in water activity of the solution interfered on the stability of the peptide. Conclusions: Based on an isothermal experiment in the temperature range of 90–120°C, and by thermal death time models, bacteriocin P34 is less heat stable at low pH and has increased thermal stability in the presence of NaCl. Addition of NaCl improved the stability of the peptide P34 at high temperatures. Significance and Impact of the Study: Studies on kinetics of thermal inactivation of bacteriocins are essential to allow their proper utilization in the food industry.     

Quantitative histopathology of Oreochromis niloticus gills after copper exposure

In order to test whether histopathological changes of gills demonstrated a good dose–response relationship with water copper levels, juvenile Nile tilapia Oreochromis niloticus, of both sexes and similar mass (36·3 + 7·7 g), were kept in dechlorinated tap water (temperature 25° C, range ±1° C; pH 6·5–7·5; hardness 74·5 mg l^?1 CaCO₃) and exposed to 40 and 400 μg l^?1 of copper. Gill samples were collected after 3, 7, 14 and 21 days. Six major histopathological changes (oedema, lifting, changes in filament epithelium thickness, lamellar fusion, vasodilatation and aneurisms) and three minor ones (proliferation of the lamellar epithelium, necrosis and adjacent lamellar fusion) were found and their prevalence estimated. The extent and severity of each histopathological change were used to develop a severity gradation scale (SGS). Semi‐quantitative analysis of the histopathological changes and measurements of gill copper deposition levels revealed a good dose– and time–response relationship. Oedemas and aneurisms were significantly correlated with acute exposure periods and lamellar fusion with chronic exposure. Epithelial lifting and changes in filament epithelial thickness were seen at lower and higher metal concentrations, respectively. The data also revealed that the SGS profile of each lesion was dependent of gill copper burden.     

Solar Disinfection of Viruses in Polyethylene Terephthalate Bottles

Solar disinfection (SODIS) of drinking water in polyethylene terephthalate (PET) bottles is a simple, efficient point-of-use technique for the inactivation of many bacterial pathogens. In contrast, the efficiency of SODIS against viruses is not well known. In this work, we studied the inactivation of bacteriophages (MS2 and ϕX174) and human viruses (echovirus 11 and adenovirus type 2) by SODIS. We conducted experiments in PET bottles exposed to (simulated) sunlight at different temperatures (15, 22, 26, and 40°C) and in water sources of diverse compositions and origins (India and Switzerland). Good inactivation of MS2 (>6-log inactivation after exposure to a total fluence of 1.34 kJ/cm²) was achieved in Swiss tap water at 22°C, while less-efficient inactivation was observed in Indian waters and for echovirus (1.5-log inactivation at the same fluence). The DNA viruses studied, ϕX174 and adenovirus, were resistant to SODIS, and the inactivation observed was equivalent to that occurring in the dark. High temperatures enhanced MS2 inactivation substantially; at 40°C, 3-log inactivation was achieved in Swiss tap water after exposure to a fluence of only 0.18 kJ/cm². Overall, our findings demonstrate that SODIS may reduce the load of single-stranded RNA (ssRNA) viruses, such as echoviruses, particularly at high temperatures and in photoreactive matrices. In contrast, complementary measures may be needed to ensure efficient inactivation during SODIS of DNA viruses resistant to oxidation.     

Development of a radio frequency heating system for sterilization of vacuum-packed fish in water

We developed equipment that quickly and uniformly heats packed whole fish in circulating tap water using radio frequency (RF) heating. Four vacuumed plastic-packed Pacific sauries in tap water were set in a radial arrangement between coaxial cylindrical electrodes in a closed vessel. For sterilization testing, Bacillus subtilis spores added in the center of the sauries were counted after treatment. For quality assurance, meat color and backbone hardness were measured after treatment. The temperature at the center of the sauries was increased up to 130 °C for 19 min using 9 kW RF heating, and up to 119 °C for 45 min using conventional heating (CH) at 120 °C. B. subtilis spores were decreased by five logarithmic orders using RF heating and by four logarithmic orders using CH. The RF-treated meat was brighter than the CH-treated meat, and the RF-treated backbone was softer than CH-treated one.     

Effects of reduction in beef surface water activity on the survival of Salmonella Typhimurium DT104 during heating

Aim: To investigate the influence of reducing beef surface water activity (a_w) on the survival of Salmonella Typhimurium DT104 during heating. Methods and Results: Beef discs were surface inoculated with S. Typhimurium DT104 and either untreated or dried to achieve surface a_w values of 0·95, 0·85 and 0·70. The samples were vacuum packed, heat‐treated at 60°C and removed at predetermined times. The inactivation curves were influenced by a_w and treatment time. Biphasic inactivation curves were observed for S. Typhimurium DT104 heat‐treated on beef samples with altered a_w values, which were characterized by an initial decline in cell numbers at commencement of heating followed by a much slower rate of inactivation during the remaining treatment period. Point estimates of the heating time required to achieve a 1 log reduction on beef surfaces with a_w of 0·99, 0·95, 0·85 and 0·70 were 0·5, 1·55, 11·25 and 17·79 min, respectively. Conclusions: A decrease in beef surface a_w can substantially enhance the survival of S. Typhimurium DT104 after heating. Significance and Impact of the Study: Caution needs to be taken using dry air as a decontamination method as this may rapidly decrease product surface and pathogen a_w values resulting in enhanced survival.     

Inactivation of Baculovirus heliothis by ultraviolet irradiation,dew, and temperature

The rate and extent of inactivation of Baculovirus heliothis by artificial ultraviolet (uv) irradiation, temperature, and dew collected from foliage of cotton and sobyean plants were determined. Exposure to uv irradiation resulted in substantial inactivation of the virus. Increase in temperature from 15° to 45°C had little effect on viral activity. A significant loss in viral activity was detected as temperature was increased to 45°C with exposure to uv irradiation. Exposure to pH 9.3 cotton dew resulted in substantial loss in activity during the initial dry-down of dew. Loss of activity appeared to result from exposure to high pH and high basic ion concentration. After the dew had dried; little additional activity loss occurred unless deposited ions were resuspended in water and allowed to redry. Exposure to cotton dews at pH 7.4 or 8.8 or soybean dew (pH 7.2) produced no significant viral inactivation.     

怀山药种质资源的包埋玻璃化超低温保存与植株再生

通过对怀山药(Dioscorea opposita)种质资源的包埋玻璃化超低温保存与植株再生进行研究,结果表明:将低温下锻炼7天的怀山药试管苗带芽茎段放入预培养基中,低温下培养3天,然后在室温下用3%的海藻酸钠和0.5mol·L-1CaCl2包埋,包埋珠用MS+0.2mol·L-1蔗糖+2mol·L-1甘油+50g·L-1二甲基亚砜在0°C下装载60分钟,用30%甘油+15%乙二醇+10%二甲基亚砜+15%聚乙二醇+0.4mol·L-1蔗糖在0°C下脱水60分钟,迅速投入液氮,24小时后立即用40°C水浴快速化冻,再用MS+0.5mol·L-1蔗糖溶液洗涤2次,转入再生培养基中培养,可获得再生植株。再生植株成活率因基因型而异,铁棍山药、太谷山药、怀庆1号山药和B号山药的成活率分别为64.29%、49.21%、13.11%和39.81%。     

Leaf rosette closure in the alpine rock species Saxifraga paniculata Mill.: significance for survival of drought and heat under high irradiation

The significance of leaf rosette closure for survival of drought and heat under high irradiation on alpine rock sites was investigated in the cushion forming rosette plant, Saxifraga paniculata Mill. With decreasing water content the leaves fold over the rosette centre reducing reversibly the evaporative leaf surface area by 80%. Internal water redistribution driven by an osmotic gradient from older to younger leaves occurs. The oldest leaves dry out to promote the survival of the individual. Leaf temperatures above 45 °C (which match heat tolerance limits 45–57 °C; LT₅₀) co-occurred with low substrate water potentials (less than – 0·5 MPa) on 11·3% of summer days. Shading by leaves can be crucial to surviving high temperatures as it keeps the rosette centre up to 10 °C colder. Mutual shading prevented sustained drought-induced photoinhibition in upper leaf surfaces at relative water contents below 60%. In exposed lower leaf surfaces restoration of photosystem II took several days. Leaf temperatures above 40 °C (21·3% of summer days) induced photoinhibition in situ. Periods with sufficient water supply can be fully utilized as rehydration is fast ( < 12 h) and exposes the upper leaf surfaces that showed only minor photoinhibition. By reversible leaf rosette closure environmental extremes that otherwise could exceed tolerance are efficiently avoided.     

Heat resistance and the effects of continuous pasteurization on the inactivation of Byssochlamys fulva ascospores in clarified apple juice

Aims: To determine thermal resistance, the effect of pasteurization temperature variations (c. 2°C) in a continuous system in the number of decimal reductions (n) of a Byssochlamys strain in clarified apple juice (CAJ). Methods and Results: Thermal destruction kinetics of Byssochlamys fulva IOC 4518 in thermal death tubes were determined at 85°, 90°, 92° and 95°C by using Weibull distribution frequency model. Three processes with different heating and holding temperatures (A: 94°, 92°C; B: 95°, 93°C; C: 96°, 94°C, respectively) were performed in a continuous system. Process time was 30 s. δ (time of first decimal reduction) values were: 42·98, 8·10, 3·62 and 1·81 min. Variable n ranged from 0·16 to >4·78 for process B (equivalent to industrial). Variable n (0·95–2·66 log CFU ml^?1) were obtained in CAJ bottles processed under condition B, while process A resulted in total heat‐resistant mould (HRM) survival and process C in total HRM destruction. Conclusions: This study demonstrates that small variations in temperature during the CAJ pasteurization could result in elimination or survival of HRM due to its nonlogarithmic behaviour. Significance and Impact of the Study: This was the first study to use Weibull frequency method to model inactivation of HRM in fruit juices. Temperature variations could culminate in the presence of HRM in pasteurized juices even when low counts (<10 spores per 100 ml) were present in the raw materials.     

Combined effects of spray‐drying conditions and postdrying storage time and temperature on Salmonella choleraesuis and Salmonella typhimurium survival when inoculated in liquid porcine plasma

The objective of this study was to determine the effectiveness of the spray‐drying process on the inactivation of Salmonella choleraesuis and Salmonella typhimurium spiked in liquid porcine plasma and to test the additive effect of immediate postdrying storage. Commercial spray‐dried porcine plasma was sterilized by irradiation and then reconstituted (1:9) with sterile water. Aliquots of reconstituted plasma were inoculated with either S. choleraesuis or S. typhimurium, subjected to spray‐drying at an inlet temperature of 200°C and an outlet temperature of either 71 or 80°C, and each spray‐drying temperature combinations were subjected to either 0, 30 or 60 s of residence time (RT) as a simulation of residence time typical of commercial dryers. Spray‐dried samples were stored at either 4·0 ± 3·0°C or 23·0 ± 0·3°C for 15 days. Bacterial counts of each Salmonella spp., were completed for all samples. For both Salmonella spp., spray‐drying at both outlet temperatures reduced bacterial counts about 3 logs at RT 0 s, while there was about a 5·5 log reduction at RT 60 s. Storage of all dried samples at either 4·0 ± 3·0°C or 23·0 ± 0·3°C for 15 days eliminate all detectable bacterial counts of both Salmonella spp.

Significance and Impact of the Study

Safety of raw materials from animal origin like spray‐dried porcine plasma (SDPP) may be a concern for the swine industry. Spray‐drying process and postdrying storage are good inactivation steps to reduce the bacterial load of Salmonella choleraesuis and Salmonella typhimurium. For both Salmonella spp., spray‐drying at 71°C or 80°C outlet temperatures reduced bacterial counts about 3 log at residence time (RT) 0 s, while there was about a 5.5 log reduction at RT 60 s. Storage of all dried samples at either 4.0 ± 3.0°C or 23.0 ± 0.3°C for 15 days was effective for eliminating detectable bacterial counts of both Salmonella spp.     

Effect of environmental factors on tenuazonic acid production by Alternaria alternata on soybean-based media

Aims: To determine the effects of water activity (a_W; 0·995–0·90), temperature (5, 18, 25 and 30°C), time of incubation (7–35 days) and their interactions on tenuazonic acid (TA) production on 2% soybean‐based agar by two Alternaria alternata strains isolated from soybean in Argentina. Methods and Results: TA production by two isolates of A. alternata was examined under interacting conditions of a_W, temperature and time of incubation on 2% soybean‐based agar. Maximum TA production was obtained for both strains at 0·98 a_W, but at 30 and 25°C for the strains for RC 21and RC 39, respectively. The toxin concentration varied considerably depending on a_W, temperature, incubation time and strain interactions. TA was produced over the temperature range from 5 to 30°C and a_W range from 0·92 to 0·995, however at 5 and 18°C little TA was produced at a_W below 0·94. Contour maps were developed from these data to identify areas where conditions indicate a significant risk for TA accumulation. Conclusions: The optimum and marginal conditions for TA production by A. alternata on soybean‐based agar were identified. The results indicated that TA production by A. alternata is favoured by different temperatures in different strains. Significance and Impact of the Study: Data obtained provide very useful information for predicting the possible risk factors for TA contamination of soybean as the a_W and temperature range used in this study simulate those occurring during grain ripening. The knowledge of TA production under marginal or sub‐optimal temperature and a_W conditions for growth are relevant as improper storage conditions accompanied by elevated temperature and moisture content in the grain can favour further mycotoxin production and lead to reduction in grain quality.