Factors Affecting Survival of Clavibacter michiganensis subsp. sepedonicus in Water

The survival of Clavibacter michiganensis subsp. sepedonicus (Cms), the causal organism of bacterial ring rot in potato, was studied in water, to assess the risks for dissemination of Cms via surface water and infection of potato crops by irrigation. Cms was able to survive for a maximum period of 7 days in non‐sterile surface water at 10°C, a period during which Cms can be transported over long distances, but will also be strongly diluted. It is concluded that contamination of surface water with Cms can pose a threat on potato production only if aquatic host plants can multiply Cms in high densities. Survival of a fluidal and non‐mucoid strain was also studied in sterile ditch water and simulated ‘drainage water’, in sterile MilliQ water, in tap water, in physiological salt and in artificial xylem fluid. In addition, the influence of temperature and low oxygen conditions on persistence of Cms in some of these diluents was studied. A maximum survival period of 35 days was found for Cms in sterile tap water at 20°C, independent of the strain used. In the other diluents survival periods ranged between 0 and 21 days. Relatively poor survival was found in MilliQ water and artificial xylem fluid. Low temperatures of 4°C do not favour survival as it does in soil. Oxygen depletion affected survival detrimentally. Survival periods determined by agar dilution plating and a direct viable counting method, based on the use of indicators for esterase activity and membrane integrity were similar. Therefore, it was concluded that under the experimental conditions studied, Cms did not form cells in a viable but non‐culturable state.     

Challenge testing of the lactoperoxidase system in pasteurized milk

AIMS: To determine the role of lactoperoxidase (LP) in inhibiting the growth of micro-organisms in pasteurised milk. METHODS AND RESULTS: Four micro-organisms of importance in the spoilage of pasteurized milk were challenged in lactoperoxidase (LP)-enriched ultra-heat treated (UHT) milk after subsequent pasteurization. Milk samples were stored at the optimum temperatures for growth of the individual bacteria. Pasteurization was carried out at 72 degrees C/15 s and 80 degrees C/15 s to determine the effect of the LP system on the micro-organisms. An active LP system was found to greatly increase the keeping quality (KQ) of milks inoculated with Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus thermophilus and pasteurized at 72 degrees C, but had little or no effect in milks heated at 80 degrees C, presumably due to virtual inactivation of LP at 80 degrees C. However, pasteurization temperature had no effect on the KQ of milks challenged with Bacillus cereus spores. CONCLUSIONS: This study suggests that the LP system, rather than heat-shocking of spores, is responsible for the greater KQ of milk pasteurized at 72 degrees C/15 s compared with 80 degrees C/15 s. SIGNIFICANCE AND IMPACT OF THE STUDY: The study emphasizes the care required in selecting pasteurization temperatures in commercial practice and to avoid the temptation to compensate for inferior quality of raw milk by increasing pasteurization temperature.     

Storage of Resting Spores of the Gypsy Moth Fungal Pathogen, Entomophaga maimaiga

The fungal pathogen, Entomophaga maimaiga causes epizootics in populations of the important North American forest defoliator gypsy moth ( Lymantria dispar ). Increasing use of this fungus for biological control is dependent on our ability to produce and manipulate the long-lived overwintering resting spores (azygospores). E. maimaiga resting spores undergo obligate dormancy before germination so we investigated conditions required for survival during dormancy as well as the dynamics of subsequent germination. After formation in the field during summer, resting spores were stored under various moisture levels, temperatures, and with and without soil in the laboratory and field. The following spring, for samples maintained in the field, germination was greatest among resting spores stored in plastic bags containing either moistened paper towels or sterile soil. Resting spores did not require light during storage to subsequently germinate. In the laboratory, only resting spores maintained with either sterile or unsterilized soil at 4°C (but not at 20 or -20°C) germinated the following spring, but at a much lower percentage than most field treatments. To further investigate the effects of relative humidity (RH) during storage, field-collected resting spores were placed at a range of humidities at 4°C. After 9.5 months, resting spore germination was highest at 58% RH and no resting spores stored at 88 or 100% RH germinated. To evaluate the dynamics of infections initiated by resting spores after storage, gypsy moth larvae were exposed to soil containing resting spores that had been collected in the field and stored at 4°C for varying lengths of time. No differences in infection occurred among larvae exposed to fall-collected soil samples stored at 4oC over the winter, versus soil samples collected from the same location the following spring. Springcollected resting spores stored at 4°C did not go into secondary dormancy. At the time that cold storage of soil containing resting spores began in spring, infection among exposed larvae was initiated within a few days after bringing the soil to 15°C. This same pattern was also found for spring-collected resting spore-bearing soil that was assayed after cold storage for 2-7 months. However, after 31-32 months in cold storage, infections started 14-18 days after soil was brought to 15°C, indicating a delay in resting spore activity after prolonged cold storage.     

Effect of initiated spores on the resistance of nongerminated resting forms of Bacillus cereus remaining in the suspension to the action of damaging agents

The elevated resistance of a Bacillus cereus spore suspension against the action of UV was found to depend on the quantity of resting forms initiated in the suspension prior to an irradiation. The resistance against UV increased 80-50 times if 60-90% of spores were initiated in the suspension as compared to that of the original resting forms. When suspensions containing 40% of non-germinated B. cereus spores were kept at 4 degrees C for 14 days, the latter became 10 and 14 times more resistant to elevated temperature (90 degrees C) and chloramine (2.5%), respectively, as compared to control intact spores. The higher resistance of non-germinated spores against the action of physical and chemical damaging agents was registered within the entire period of experiments (over three months). This phenomenon was not observed if ca. 100% of spores were initiated in a suspension. The resistance of initiated spores against the action of UV was 40 times lower than that of B. cereus resting forms.     

Eradication of Polymyxa betae by Thermal and Anaerobic Conditions and in the Presence of Compost Leachate

The abiotic conditions required for eradication of Polymyxa betae, the vector of Beet necrotic yellow vein virus in sugar beet, were investigated. Survival of resting spores of P. betae was determined under aerobic (30 min, 4 days and 21 days) and anaerobic (4 days) conditions under several temperature regimes in a water suspension and in leachate extracted from an aerobic compost heap. In water under aerobic conditions the lethal temperature was 60, 55 and 40°C for exposure times of 30 min, 4 days and 21 days, respectively. The effect of compost leachate and/or anaerobic conditions on survival of P. betae depended on temperature. After incubation for 4 days at 20°C, no significant effects of anaerobic conditions or leachate on the survival of P. betae were found. However, at 40°C for 4 days under anaerobic conditions, survival of P. betae was significantly lower than survival under aerobic conditions in water as well as in leachate. In leachate taken from an aerobic compost heap, aerobically incubated at 40°C for 4 days, survival of P. betae was significantly lower than survival in water at the same temperature. As anaerobic spots are prevalent in aerobic compost heaps, especially during the thermophilic phase, actual inactivation temperatures under composting conditions are likely to be lower than the temperatures we found for eradication in water under aerobic conditions.     

In vitro formation of resting spores by the insect pathogenic fungus Entomophaga maimaiga

Field-collected resting spores (azygospores) of the fungal pathogen of Lymantria dispar (gypsy moth), Entomophaga maimaiga, have been used to release this biological control agent in areas where this pathogen is not established. We have found that E. maimaiga can produce resting spores in vitro using Grace's insect tissue culture medium (95%) plus fetal bovine serum (5%). The majority of spores become mature between 7 and 21 days after cultures are initiated. Spore production varies by fungal isolate; of 38 isolates tested, 10 produced no resting spores while 7 produced >1000 resting spores/ml. Resting spore production was not affected when isolates were mixed. Glycerol (used for fungal storage), trehalose, and selected amino acids each inhibited resting spore formation. Fetal bovine serum was required for spore production but the presence of >5% yielded lower resting spore densities. A large surface area:volume ratio (12.5 cm(2):ml versus 4.2 cm(2):ml) was required for abundant formation of resting spores. At present, resting spores have only been produced in small volumes with a maximum of 3 x 10(4) resting spores/ml.     

Differences between Brachiola (Nosema) algerae isolates of human and insect origin when tested using an in vitro spore germination assay and a cultured cell infection assay

Brachiola (Nosema) algerae is a microsporidian species generally believed to be an intracellular parasite of insects, especially mosquitoes. However, both mosquito and human isolates have been shown to infect mammalian cells. The present study was undertaken to determine if spores of two insect and two human isolates of B. algerae cultured at 30 degrees C and 37 degrees C differed in their ability to germinate and infect cultured green monkey kidney cells at these two temperatures. Spores from all four isolates exhibited an optimum pH of 9.5 for germination. Mercury (Hg2+) inhibited germination of all isolates equally. Germination of spores from all four isolates was significantly greater when the parasite was cultured at 30 degrees C than when cultured at 37 degrees C. However, spores from the insect isolates cultivated at 30 degrees C or 37 degrees C infected significantly fewer mammalian cells at 37 degrees C than did spores from the human isolates under the same conditions. Thus, there is no correlation between the effects of temperature on the germination and the infectivity of an isolate. In addition, while exposure of B. algerae to 37 degrees C has been reported to cause spore dysmorphism, we failed to observe any consistent ultrastructural changes that explained the greater infectivity of the human isolates at 37 degrees C.     

Survival Studies with Spores of Clostridium botulinum Type E in Pasteurized Meat of the Blue Crab Callinectes sapidus

Clostridium botulinum type E studies reported in this paper include the incidence of the organism in selected Chesapeake Bay areas, growth and toxin production in crabmeat homogenates, and the effect of pasteurization upon varying levels of spores in crabmeat. Type E spores were detected in 21 of 24 bottom mud samples taken at locations from which blue crabs were being harvested. Sterilized crabmeat homogenates inoculated with as little as five spores per 10 g became toxic after 8 days at 50 F, 2 days at 75 F, and 1 day at 85 F. Growth at 50 F and above was accompanied by gas production and a slightly sour odor. Growth and toxin production at 40 F required 55 days or longer and inocula of 10(3) spores or higher per 10 g of homogenate. At 40 F gas production was usually not apparent and no off odors could be detected. A recommended minimum pasteurization of 1 min at 185 F internal meat temperature reduced type E spore levels in inoculated packs of crabmeat from 10(8) spores per 100 g to 6 or less spores per 100 g, and the pasteurized meat remained nontoxic during 6 months of storage at 40 F.     

The influence of pre-treatment temperature on the thermal sensitivity of a mouse tumour

The response of tumours to hyperthermia was tested by giving graded heat treatments and assessing local control at 90 days. Mice were divided into three groups which were pre-treated for 3 days in ambient temperatures of 4, 21 or 35 degrees C. This enabled the mean tumour resting temperature to be varied by up to 11 degrees C, before subsequent heat treatment. For the heat treatments, the tumours were clamped in order to eliminate blood flow, resulting in uniform temperature distributions and hence more uniform thermal sensitivity. TCD50 values were used to construct Arrhenius plots. For all three pre-treatment temperatures, these plots demonstrated a factor of 1.6 increase in heating time per degree Celsius reduction in heating temperature. However, tumours kept in a 4 degrees C environment before treatment were more thermally sensitive than those kept in 21 degrees C conditions, while those in a 35 degrees C environment were more resistant. Pretreatment at 4 degrees C was equivalent to an increase of either 0.5 degree C in heating temperature or 28 per cent in heating time, compared with pre-treatment at 21 degrees C. Pre-treatment at 35 degrees C was equivalent to a reduction of either 0.6 degree C in heating temperature or 25 per cent in heating time. These data indicate that the pre-treatment tumour temperature is an important parameter, but the effect of heat treatment is more closely related to absolute heating temperature rather than to the increase in temperature above the normal resting level.     

Respiration profiles in monitoring the composting of by-products from the olive oil agro-industry

The composting of olive press cake (OPC) repeatedly mixed either with olive mill wastewater (OPC+OMW) or with tap water (OPC+W) was studied using the thermogradient respirometer, an apparatus that determines the respiration rates from a substrate over a wide range of different temperatures (respiratory profile). The composting processes took place over a period of five months during which nine moistenings of the OPC were performed with the respective liquids. The composting resulted in detoxification of the materials used in both treatments, as indicated by seed germination tests. However, the repeated applications of OMW resulted in recurring thermophilic phases (following each application) and in greater pH and conductivity increases in the final product, as compared to water applications. Respiration measurements performed at 35 degrees C were good indicators of the mean metabolic potential in the compost piles (the mean respiration derived from the whole respiration profile over a wide range of environmental temperatures). However, respiration measurements at higher temperatures (48.5 degrees C) were better indicators of the respiration activity occurring in situ. Following the initial thermophilic phase, the respiration potential of the composts at high temperatures (42-63 degrees C) increased drastically compared to their respiration potential at lower temperatures (17-42 degrees C) indicating the establishment of a thermophilic microflora. Subsequently, only the periodic new substrate-C applications in the form of OMW resulted in increased ratios of low temperature-to-high temperature respiration potential. These ratios decreased again following the respective thermophilic phase that each new OMW application had induced.     

Inactivation of Bacillus anthracis spores by a combination of biocides and heating under high-temperature short-time pasteurization conditions

The milk supply is considered a primary route for a bioterrorism attack with Bacillus anthracis spores because typical high-temperature short-time (HTST) pasteurization conditions cannot inactivate spores. In the event of intentional contamination, an effective method to inactivate the spores in milk under HTST processing conditions is needed. This study was undertaken to identify combinations and concentrations of biocides that can inactivate B. anthracis spores at temperatures in the HTST range in less than 1 min. Hydrogen peroxide (HP), sodium hypochlorite (SH), and peroxyacetic acid (PA) were evaluated for their efficacy in inactivating spores of strains 7702, ANR-1, and 9131 in milk at 72, 80, and 85 degrees C using a sealed capillary tube technique. Strains ANR-1 and 9131 were more resistant to all of the biocide treatments than strain 7702. Addition of 1,260 ppm SH to milk reduced the number of viable spores of each strain by 6 log CFU/ml in less than 90 and 60 s at 72 and 80 degrees C, respectively. After neutralization, 1,260 ppm SH reduced the time necessary to inactivate 6 log CFU/ml (TTI6-log) at 80 degrees C to less than 20 s. Treatment of milk with 7,000 ppm HP resulted in a similar level of inactivation in 60 s. Combined treatment with 1,260 ppm SH and 1,800 ppm HP inactivated spores of all strains in less than 20 s at 80 degrees C. Mixing 15 ppm PA with milk containing 1,260 ppm SH resulted in TTI6-log of 25 and 12 s at 72 and 80 degrees C, respectively. TTI6-log of less than 20 s were also achieved at 80 degrees C by using two combinations of biocides: 250 ppm SH, 700 ppm HP, and 150 ppm PA; and 420 ppm SH (pH 7), 1,100 ppm HP, and 15 ppm PA. These results indicated that different combinations of biocides could consistently result in 6-log reductions in the number of B. anthracis spores in less than 1 min at temperatures in the HTST range. This information could be useful for developing more effective thermal treatment strategies which could be used in HTST milk plants to process contaminated milk for disposal and decontamination, as well as for potential protective measures.     

Growth and formation of toxin by Clostridium botulinum in peeled, inoculated, vacuum-packed potatoes after a double pasteurization and storage at 25 degrees C

A process that claims to use a double pasteurization to produce vacuum-packed potatoes for storage at ambient temperature has been evaluated. After the first pasteurization, potatoes are vacuum-packed and stored at 25 degrees-35 degrees C for up to 24 h, which is intended to allow germination of bacterial spores, and are then pasteurized again. When potatoes were inoculated with spores of Clostridium botulinum and subjected to this double-pasteurization process a high proportion of spores remained viable and resulted in growth and formation of toxin within 5-9 d at 25 degrees C. To provide an appropriate reduction in the risk o survival and growth of Cl. botulinum, peeled, vacuum-packed potatoes for storage at ambient temperature should be given a heat treatment equivalent to an F(0)3 process. If they are not given such a heat treatment they should be stored at a temperature below 4 degrees C.     

Molecular Detection and Pathogenicity Assay of Plasmodiophora brassicae in Chicken Manure

To determine the relationship between animal excreta and the occurrence of clubroot disease of cruciferous crops caused by Plasmodiophora brassicae, chickens were fed with resting spores of the pathogen. Their faeces were collected and used to inoculate crucifers. This study proved that both fresh and composted manures could induce clubroot and the presence of the pathogen in the manure was confirmed by PCR amplification. However, composting had detrimental effects on the virulence of the resting spores in the manure. When the temperature was over 32°C, the incidence and severity of clubroot declined with the increase in the exposure time of resting spores to high temperature and the pathogenicity was completely lost when the spores were kept at 48°C for 6 h. The control measures for the clubroot disease were discussed.     

Effect of water temperature on the development, release and survival of the triactinomyxon stage of Myxobolus cerebralis in its oligochaete host.

The development of the triactinomyxon stage of Myxobolus cerebralis and release of mature spores from Tubifex tubifex were shown to be temperature dependent. In the present work, the effect of temperature over a range of 5-30 degrees C on the development and release of the triactinomyxon stages of M. cerebralis was studied. Infected T. tubifex stopped releasing triactinomyxon spores 4 days after transfer from 15 degrees C to 25 degrees C or 30 degrees C. Transmission electron microscopic examinations of the tubificids held at 25 degrees C and 30 degrees C for 3 days showed that all developmental stages degenerated and transformed to electron-dense clusters between the gut epithelial cells of T. tubifex. In contrast, tubificid worms held at 5 degrees C and 10 degrees C examined at the same time were heavily infected with many early developmental stages of triactinomyxon. At 15 degrees C, the optimal temperature for development, maturing and mature stages of the parasite were evident. Infected T. tubifex transferred from 15 degrees C to 20 degrees C stopped producing triactinomyxon spores after 15 days. However, 15 days at 20 degrees C was not sufficient to destroy all developmental stages of the parasite. When the tubificid worms were returned to 15 degrees C, the one-cell stages and the binucleate-cell stages resumed normal growth. It was also demonstrated that T. tubifex cured of infection by holding at 30 degrees C for 3 weeks and shifted to 15 degrees C could be re-infected with M. cerebralis spores. The waterborne triactinomyxon spores of M. cerebralis did not appear to be as short-lived as previously reported. More than 60% of experimentally produced waterborne triactinomyxon spores survived and maintained their infectivity for rainbow trout for 15 days at water temperatures up to 15 degrees C. In natural aquatic systems, the triactinomyxon spores may survive and keep their infectivity for periods even longer than 15 days.     

Enhancement of the thermophilic stage in cattle waste composting by addition of tofu residue

The microbial degradation and temperature rise during the composting of a cattle waste and rice straw mixture blended with tofu (soybean curd) residue was investigated using an insulated and unheated in-vessel composter (effective volume, 12 1) and a static pile with passive aeration. The addition of 11% (dry weight basis) of tofu residue shortened the time required for temperature to reach the thermophilic phase and increased the duration of the temperatures above 55 degrees C significantly, but the maximum temperature was not affected by the additive level. As shown by the change in BOD, most of the easily biodegradable matter in the tofu residue was consumed during 12 days of composting. The same results were observed in the temperature profile of the static pile with passive aeration. Tofu residue addition yielded a higher maximum temperature and a nearly two times longer duration of temperatures above 55 degrees C in almost all locations of the pile. The use of tofu residue as a co-composting material would promote thermophilic degradation throughout the entire composting mass.     

Effects of time, temperature, and honey on Nosema apis (Microsporidia: Nosematidae), a parasite of the honeybee, Apis mellifera (Hymenoptera: Apidae).

Newly emerged adult bees were fed with Nosema apis spores subjected to various treatments, and their longevity, proportions of bees infected, and spores per bee recorded. Spores lost viability after 1, 3, or 6 months in active manuka or multifloral honey, after 3 days in multifloral honey, and after 21 days in water or sugar syrup at 33 degrees C. Air-dried spores lost viability after 3 or 5 days at 40 degrees, 45 degrees, or 49 degrees C. Increasing numbers of bees became infected with increasing doses of spores, regardless of their subsequent food (active manuka honey, thyme honey, or sugar syrup). Final spore loads were similar among bees receiving the same food, regardless of dose. Bees fed with either honey had lighter infections than those fed with syrup, but this may have been due to reductions in their longevity. Bees fed with manuka honey were significantly shorter lived, whether infected or not.     

Effects of oxygen on aerobic solid-state biodegradation kinetics

Oxygen is a critical control variable for composting and other solid-state biodegradation processes. In this study we examined the effect of varying oxygen concentrations (1%, 4%, and 21% O2 (v/v)) on biodegradation kinetics under different substrate (sewage sludge and synthetic food waste), temperature (35, 45, 55, and 65 degrees C), and moisture (36-60% H2O) conditions. Three forms of a saturation or Monod-type model and one form of an exponential model were evaluated against data from extensive experiments under carefully controlled environmental conditions. The exponential model performed well at temperatures from 35 to 55 degrees C but had problems at higher temperatures. The Monod-type models yielded the best fit based on R2 values. Multiple linear regression was used to express the oxygen half-saturation coefficient as a function of temperature and moisture. For a modified one-parameter saturation model the half-saturation coefficient varied from -0.67% to 1.74% v/v O2 under the range of conditions typical of composting systems. While the positive correlation of biodegradation rate with oxygen concentration reported by previous researchers held true for temperatures below 55 degrees C, an inverse relationship was found at 65 degrees C. Although this study did not directly examine anaerobic conditions, the results under microaerophilic conditions suggest oxygen may not offer kinetic advantages for extreme thermophilic biodegradation processes.     

Occurrence of Aspergillus fumigatus during composting of sewage sludge.

Aspergillus fumigatus, a medically important fungal opportunist and respiratory allergen, was isolated from woodchips and sewage sludge used in the production of compost at the U.S. Department of Agriculture's composting research facility in Beltsville, Md. It was also regularly isolated as a dominant fungus during forced aeration composting and after 30 days in an unaerated stationary curing pile; in both cases, the fungus was found in pile zones with temperatures less than 60 degrees C. Compost stored outdoors in stationary unaerated piles from 1 to 4 months after screening out of woodchips contained easily detectable amounts of A. fumigatus in the exterior pile zones (0- to 25-cm depths). Semiquantitative studies of the airspora at the composting site revealed that A. fumigatus constituted 75% of the total viable mycoflora captured. At locations 320 m to 8 km from the compost site, the fungus constituted only 2% of the total viable mycoflora in the air. Of 21 samples of commercially available potting soil, one had levels of A. fumigatus nearly equivalent to those of 1-month-old storage compost; 15 others had lower but detectable levels.     

Occurrence of Aspergillus fumigatus during composting of sewage sludge.

Aspergillus fumigatus, a medically important fungal opportunist and respiratory allergen, was isolated from woodchips and sewage sludge used in the production of compost at the U.S. Department of Agriculture's composting research facility in Beltsville, Md. It was also regularly isolated as a dominant fungus during forced aeration composting and after 30 days in an unaerated stationary curing pile; in both cases, the fungus was found in pile zones with temperatures less than 60 degrees C. Compost stored outdoors in stationary unaerated piles from 1 to 4 months after screening out of woodchips contained easily detectable amounts of A. fumigatus in the exterior pile zones (0- to 25-cm depths). Semiquantitative studies of the airspora at the composting site revealed that A. fumigatus constituted 75% of the total viable mycoflora captured. At locations 320 m to 8 km from the compost site, the fungus constituted only 2% of the total viable mycoflora in the air. Of 21 samples of commercially available potting soil, one had levels of A. fumigatus nearly equivalent to those of 1-month-old storage compost; 15 others had lower but detectable levels.     

Thermal inactivation of Bacillus anthracis spores in cow's milk

Decimal reduction time (time to inactivate 90% of the population) (D) values of Bacillus anthracis spores in milk ranged from 3.4 to 16.7 h at 72 degrees C and from 1.6 to 3.3 s at 112 degrees C. The calculated increase of temperature needed to reduce the D value by 90% varied from 8.7 to 11.0 degrees C, and the Arrhenius activation energies ranged from 227.4 to 291.3 kJ/mol. Six-log-unit viability reductions were achieved at 120 degrees C for 16 s. These results suggest that a thermal process similar to commercial ultrahigh-temperature pasteurization could inactivate B. anthracis spores in milk.