Fungal Propagules and DNA in Feces of Two Detritus-Feeding Amphipods

Aquatic shredders (leaf-eating invertebrates) preferentially ingest and digest leaves colonized by aquatic hyphomycetes (fungi). This activity destroys leaf-associated fungal biomass and detritial resources in streams. Fungal counter-adaptations may include the ability to survive passage through the invertebrate's digestive tract. When fecal pellets of Gammarus tigrinus and Hyalella azteca were incubated with sterile leaves, spores of nine (G. tigrinus) and seven (H. azteca) aquatic hyphomycete species were subsequently released from the leaves, indicating the presence of viable fungal structures in the feces. Extraction, amplification, and sequencing of DNA from feces revealed numerous fungal phylotypes, two of which could be assigned unequivocally to an aquatic hyphomycete. The estimated contributions of major fungal groups varied depending on whether 18S or ITS sequences were amplified and cloned. We conclude that a variable proportion of fungal DNA in the feces of detritivores may originate from aquatic hyphomycetes. Amplified DNA may be associated with metabolically active, dormant, or dead fungal cells.     

Separation of Fungal Propagules by Partition in Aqueous Polymer Two-Phase Systems

Conidia of Penicillium chrysogenum and Penicillium frequentans and sporangiospores of Rhizopus rhizopodiformis, Rhizomucor pusillus, and Mucor racemosus were subjected to partition in aqueous polymer two-phase systems. The partition behavior differed drastically between the conidia of the two Penicillium species and the sporangiospores of the three species of phycomycetes. This difference in partition behavior can be used for purification of fungi belonging to different taxonomical groups. P. frequentans was completely separated from M. racemosus by two extractions, whereas four extractions were needed to purify M. racemosus. This method was used on an air sample from a locality where wood fuel chips are handled. The conidia of the fungi Trichoderma viride and Rhizopus rhizopodiformis were removed completely by only two extractions.     

Inactivation of Giardia Cysts by Chlorine

Cysts of Giardia lamblia from symptomatic and asymptomatic carriers had similar resistances to chlorination.     

Chlorine Inactivation of Bacterial Bioterrorism Agents

Seven species of bacterial select agents were tested for susceptibility to free available chlorine (FAC). Under test conditions, the FAC routinely maintained in potable water would be sufficient to reduce six species by 2 orders of magnitude within 10 min. Water contaminated with spores of Bacillus anthracis spores would require further treatment.     

The Effect of Ultraviolet and Solar Radiation and Temperature on Survival of Fungal Propagules

Survival of spores, spore aggregates, sclerotia, and pycnidia of fungi was evaluated after exposure to high temperature in the dark, or exposure to ultraviolet (UV) radiation or sunlight. Under most conditions survival decreased from the most resitant Sclerotium rolfsii to Alternaria macrospora, Mycosphaerella pinodes, Aspergillus niger and Botrytis cinerea, in that order. There was no difference in survival among organisms stored in darkness at 45°C as single spores or aggregated into a mass of spores; or between spores of M. pinodes exposed before and after being released from pycnidia. Exposure to UV reduced survival much more than exposure in darkness. Longevity under UV ranged from more than 7 days for S. rolfsii to approximately 50 min for A. macrospora and 3 min for B. cinerea. Much longer survival occurred for spores of all fungi aggregated together; of spores of M. pinodes irradiated when inside rather than outside pycnidia; and in bigger, darker and older rather than in smaller, paler and younger sclerotia of S. rolfsii. Survival of sclerotia cut into slices and exposed to UV increased with thickness, irrespective of exposure to UV with the outer pigmented or innernonpigmented side. As tested with spore aggregates of A. niger, the rate of survival was linearly proportional to the number of conidia aggregated in each body. Irradiation by sunlight affected survival as described for irradiation by UV. The best protection from the detrimental effects of sun and UV radiation was provided by the infected host tissue.     

Chlorine Inactivation of Adenovirus Type 40 and Feline Calicivirus

Ct values, the concentration of free chlorine multiplied by time of contact with virus, were determined for free-chlorine inactivation experiments carried out with chloroform-extracted (dispersed) and non-chloroform-extracted (aggregated) feline calicivirus (FCV), adenovirus type 40 (AD40), and polio virus type 1 (PV-1). Experiments were carried out with high and low pH and temperature conditions. Ct values were calculated directly from bench-scale free-chlorine inactivation experiments and from application of the efficiency factor Hom model. For each experimental condition, Ct values were higher at pH 8 than at pH 6, higher at 5°C than at 15°C, and higher for dispersed AD40 (dAD40) than for dispersed FCV (dFCV). dFCV and dAD40 were more sensitive to free chlorine than dispersed PV-1 (dPV-1). Cts for 2 log inactivation of aggregated FCV (aFCV) and aggregated PV-1 (aPV-1) were 31.0 and 2.8 orders of magnitude higher than those calculated from experiments carried out with dispersed virus. Cts for 2 log inactivation of dFCV and dAD40 in treated groundwater at 15°C were 1.2 and 13.7 times greater than in buffered-demand-free (BDF) water experiments at 5°C. Ct values listed in the U.S. Environmental Protection Agency (EPA) Guidance Manual were close to, or lower than, Ct values generated for experiments conducted with dispersed and aggregated viruses suspended in BDF water and for dispersed viruses suspended in treated groundwater. Since the state of viruses in water is most likely to be aggregated and associated with organic or inorganic matter, reevaluation of the EPA Guidance Manual Ct values is necessary, since they would not be useful for ensuring inactivation of viruses in these states. Under the tested conditions, dAD40, dFCV, aFCV, dPV-1, and aPV-1 particles would be inactivated by commonly used free chlorine concentrations (1 mg/liter) and contact times (60 to 237 min) applied for drinking water treatment in the United States.     

Chlorine Inactivation of Spores of Encephalitozoon spp.

This report is an extension of a preliminary investigation on the use of chlorine to inactivate spores of Encephalitozoon intestinalis and to investigate the effect of chlorine on two other species, E cuniculi and E. hellem, associated with human infection. The 50% tissue culture infective doses of these three species were also determined. On the basis of the results obtained, it appears that chlorination of water is an effective means of controlling spores of these organisms in the aquatic environment.     

Culture Age, Temperature, and pH Affect the Polyol and Trehalose Contents of Fungal Propagules

The growth and conidial physiology of the entomopathogenic fungi Beauveria bassiana, Metarhizium anisopliae, and Paecilomyces farinosus were studied under different conditions. The effects of culture age (up to 120 days), temperature (5 to 35(deg)C), and pH (2.9 to 11.1) were determined. Growth was optimal at pH 5 to 8 for each isolate and between 20 and 35(deg)C, depending on the isolate. The predominant polyol in conidia was mannitol, with up to 39, 134, and 61 mg g of conidia(sup-1) for B. bassiana, M. anisopliae, and P. farinosus, respectively. Conidia of M. anisopliae contained relatively small amounts of lower-molecular-weight polyols and trehalose (less than 25 mg g(sup-1) in total) in all treatments. Conidia of B. bassiana and P. farinosus contained up to 30, 32, and 25 mg of glycerol, erythritol, and trehalose, respectively, g(sup-1), depending on the treatment. Conidia of P. farinosus contained unusually high amounts of glycerol and erythritol at pH 2.9. The apparent effect of pH on gene expression is discussed in relation to the induction of a water stress response. To our knowledge, this is the first report of polyols and trehalose in fungal propagules produced over a range of temperature or pH. Some conditions and harvesting times were associated with an apparent inhibition of synthesis or accumulation of polyols and trehalose. This shows that culture age and environmental conditions affect the physiological quality of inoculum and can thereby determine its potential for biocontrol.     

Fungicidal Activity of Volatiles from Selected Cruciferous Plants against Resting Propagules of Soil-borne Fungal Pathogens

The efficacy of volatiles evolved from tissues of nine cruciferous plants against resting propagules of Fusarium oxysporum var radicis f. sp. lycopersici, Sclerotium cepivorum, and Sclerotinia sclerotiorum was tested. The cruciferous plants released biocidal compounds, mainly isothiocyanates, produced during the enzymatic degradation of glucosinolates present in the plant cells. Among the plants investigated, the highest fungicidal activity and also the highest concentration of isothiocyanates were found in Brassica juncea. The resting propagules of tested fungi differed significantly in their sensitivity towards volatiles released from plant tissues.     

Inactivation of Enteric Adenovirus and Feline Calicivirus by Chlorine Dioxide

Chlorine dioxide (ClO₂) inactivation experiments were conducted with adenovirus type 40 (AD40) and feline calicivirus (FCV). Experiments were carried out in buffered, disinfectant demand-free water under high- and low-pH and -temperature conditions. Ct values (the concentration of ClO₂ multiplied by contact time with the virus) were calculated directly from bench-scale experiments and from application of the efficiency factor Hom (EFH) model. AD40 Ct ranges for 4-log inactivation (Ct_99.99%) at 5°C were >0.77 to <1.53 mg/liter × min and >0.80 to <1.59 mg/liter × min for pH 6 and 8, respectively. For 15°C AD40 experiments, >0.49 to <0.74 mg/liter × min and <0.12 mg/liter × min Ct_99.99% ranges were observed for pH 6 and 8, respectively. FCV Ct_99.99% ranges for 5°C experiments were >20.20 to <30.30 mg/liter × min and >0.68 mg/liter × min for pH 6 and 8, respectively. For 15°C FCV experiments, Ct_99.99% ranges were >4.20 to <6.72 and <0.18 mg/liter × min for pH 6 and 8, respectively. Viral inactivation was higher at pH 8 than at pH 6 and at 15°C than at 5°C. Comparison of Ct values and inactivation curves demonstrated that the EFH model described bench-scale experiment data very well. Observed bench-scale Ct_99.99% ranges and EFH model Ct_99.99% values demonstrated that FCV is more resistant to ClO₂ than AD40 for the conditions studied. U.S. Environmental Protection Agency guidance manual Ct_99.99% values are higher than Ct_99.99% values calculated from bench-scale experiments and from EFH model application.     

Development of Poliovirus Having Increased Resistance to Chlorine Inactivation

A laboratory strain of poliovirus (LSc) became progressively more resistant to chlorine inactivation during a series of repeated sublethal exposures to the halogen.     

Chlorine Dioxide Inactivation of Cryptosporidium parvum Oocysts and Bacterial Spore Indicators

Cryptosporidium parvum, which is resistant to chlorine concentrations typically used in water treatment, is recognized as a significant waterborne pathogen. Recent studies have demonstrated that chlorine dioxide is a more efficient disinfectant than free chlorine against Cryptosporidium oocysts. It is not known, however, if oocysts from different suppliers are equally sensitive to chlorine dioxide. This study used both a most-probable-number–cell culture infectivity assay and in vitro excystation to evaluate chlorine dioxide inactivation kinetics in laboratory water at pH 8 and 21°C. The two viability methods produced significantly different results (P < 0.05). Products of disinfectant concentration and contact time (Ct values) of 1,000 mg · min/liter were needed to inactivate approximately 0.5 log₁₀ and 2.0 log₁₀ units (99% inactivation) of C. parvum as measured by in vitro excystation and cell infectivity, respectively, suggesting that excystation is not an adequate viability assay. Purified oocysts originating from three different suppliers were evaluated and showed marked differences with respect to their resistance to inactivation when using chlorine dioxide. Ct values of 75, 550, and 1,000 mg · min/liter were required to achieve approximately 2.0 log₁₀ units of inactivation with oocysts from different sources. Finally, the study compared the relationship between easily measured indicators, including Bacillus subtilis (aerobic) spores and Clostridium sporogenes (anaerobic) spores, and C. parvum oocysts. The bacterial spores were found to be more sensitive to chlorine dioxide than C. parvum oocysts and therefore could not be used as direct indicators of C. parvum inactivation for this disinfectant. In conclusion, it is suggested that future studies address issues such as oocyst purification protocols and the genetic diversity of C. parvum, since these factors might affect oocyst disinfection sensitivity.     

Chlorine Inactivation of Sphingomonas Cells Attached to Goethite Particles in Drinking Water

Bacteria in drinking water, attached or not attached to goethite particles, were disinfected with chlorine. No additional protection was provided to the bacteria by their attachment to particles, and the limited efficiency of inactivation by chlorine was attributed to the presence of bacterial aggregates in both types of suspension.     

Inactivation of Adenoviruses,Enteroviruses, and Murine Norovirus in Water by Free Chlorine and Monochloramine

Inactivation of infectious viruses during drinking water treatment is usually achieved with free chlorine. Many drinking water utilities in the United States now use monochloramine as a secondary disinfectant to minimize disinfectant by-product formation and biofilm growth. The inactivation of human adenoviruses 2, 40, and 41 (HAdV2, HAdV40, and HAdV41), coxsackieviruses B3 and B5 (CVB3 and CVB5), echoviruses 1 and 11 (E1 and E11), and murine norovirus (MNV) are compared in this study. Experiments were performed with 0.2 mg of free chlorine or 1 mg of monochloramine/liter at pH 7 and 8 in buffered reagent-grade water at 5°C. CT values (disinfectant concentration × time) for 2- to 4-log₁₀ (99 to 99.99%) reductions in virus titers were calculated by using the efficiency factor Hom model. The enteroviruses required the longest times for chlorine inactivation and MNV the least time. CVB5 required the longest exposure time, with CT values of 7.4 and 10 mg·min/liter (pH 7 and 8) for 4-log₁₀ inactivation. Monochloramine disinfection was most effective for E1 (CT values ranged from 8 to 18 mg·min/liter for 2- and 3-log₁₀ reductions, respectively). E11 and HAdV2 were the least susceptible to monochloramine disinfection (CT values of 1,300 and 1,600 mg-min/liter for 3-log₁₀ reductions, respectively). Monochloramine inactivation was most successful for the adenoviruses, CVB5, and E1 at pH 7. A greater variation in inactivation rates between viruses was observed during monochloramine disinfection than during chlorine disinfection. These data will be useful in drinking water risk assessment studies and disinfection system planning.Disinfection is a critical step in the drinking water treatment process to inactivate infectious viruses because primary treatment is less effective for the removal of viruses. Chlorine and monochloramine are the most widely used disinfectants in the United States (2). Free chlorine is widely used as a primary disinfectant following filtration and also as a secondary disinfectant in distribution systems. Under the Long Term 2 Enhanced Surface Water Treatment Rule (38), monochloramine can also be used as a primary disinfectant, but because it requires longer contact times to achieve the same level of disinfection as free chlorine it is primarily used as a secondary disinfectant to maintain a stable disinfectant residual in the distribution system and minimize disinfection by-product formation and biofilm growth.The efficacy of chlorine disinfection for viruses has been evaluated in numerous studies over the years. Many early studies focused on the disinfection of polioviruses by chlorine (14, 17, 26, 28, 30, 39, 40, 43). Early investigators suggested a number of variables that must be controlled in the disinfection of viruses: contact time, temperature, ionic strength, pH, chlorine concentration, and virus aggregation (29, 30). These researchers concluded that comparisons and general trends of disinfection efficacy can only be discerned for viruses when the same disinfection parameters are applied.Fewer studies have investigated the disinfection efficacy of monochloramine, but monochloramine disinfection has been found to be less effective than free chlorine for viruses. In comparative studies of chlorine and monochloramine disinfection, coxsackievirus B5, adenovirus 2, and adenovirus 41 were found to be inactivated far more readily by chlorine than monochloramine (4, 5, 32). For drinking water treatment systems where monochloramine is used as a secondary disinfectant, it is important to know its efficacy for a wide range of viruses, as infectious viruses may be introduced in the distribution system where only monochloramine is present. In addition, relatively few studies have investigated the efficacy of monochloramine as systematically as free chlorine; frequently only one concentration, pH, or temperature has been investigated. Two notable exceptions were investigations that examined monochloramine disinfection of human adenovirus 2 (HAdV2) and coxsackievirus B5 (CVB5) at multiple pH levels (21, 31).In 2005, the U.S. Environmental Protection Agency (USEPA) published its second candidate contaminant list (CCL2). The CCL2 is comprised of unregulated microbial and chemical contaminants of potential public health concern that are known or anticipated to occur in drinking water systems and includes: echovirus, coxsackievirus, adenovirus, and calicivirus (36). A number of researchers have reported the disinfection efficacy of free chlorine for representatives of the CCL2 viruses (4, 5, 7, 11, 13, 18, 20, 22, 27, 33, 34, 35), but fewer studies have investigated the disinfection efficacy of monochloramine on these viruses (4, 5, 21, 31). In addition, comparison between existing studies of chlorine or monochloramine disinfection is difficult because of differences in the viruses examined, experimental parameters investigated, and analytical methods used.The present study compared the inactivation kinetics for representative CCL2 viruses with levels of free chlorine and monochloramine recommended for drinking water disinfection. Duplicate experiments with both disinfectants were carried out in pH 7 and 8 buffered chlorine-demand-free (CDF) water at 5°C, with eight viruses chosen to represent the CCL2 virus types. Coxsackieviruses B5 and B3 (CVB5 and CVB3) and echoviruses 1 and 11 (E1 and E11) were chosen based on existing data suggesting resistance to free chlorine, disease implications, and likelihood of presence in higher numbers in natural water. Three representative human adenoviruses were studied, including both serotypes of species F HAdV (40 and 41) that cause gastroenteritis and HAdV2, a representative of respiratory HAdV that may be found in water because they are present in fecal excretions (9). Murine norovirus (MNV), phylogenetically similar to human norovirus and the only norovirus that can be propagated in cell culture, was used as a surrogate for human norovirus. Kinetic inactivation curves are presented, and CT values (disinfectant concentration × time, reported in mg·min/liter) were calculated by using the efficiency factor Hom (EFH) model (16).     

Membrane Damage and Microbial Inactivation by Chlorine in the Absence and Presence of a Chlorine-Demanding Substrate

The relationship between cell inactivation and membrane damage was studied in two gram-positive organisms, Listeria monocytogenes and Bacillus subtilis, and two gram-negative organisms, Yersinia enterocolitica and Escherichia coli, exposed to chlorine in the absence and presence of 150 ppm of organic matter (Trypticase soy broth). L. monocytogenes and B. subtilis were more resistant to chlorine in distilled water. The addition of small amounts of organic matter to the chlorination medium drastically increased the resistance of both types of microorganisms, but this effect was more marked in Y. enterocolitica and E. coli. In addition, the survival curves for these microorganisms in the presence of organic matter had a prolonged shoulder. Sublethal injury was not detected under most experimental conditions, and only gram-positive cells treated in distilled water showed a relevant degree of injury. The exposure of bacterial cells to chlorine in distilled water caused extensive permeabilization of the cytoplasmic membrane, but the concentrations required were much higher than those needed to inactivate cells. Therefore, there was no relationship between the occurrence of membrane permeabilization and cell death. The addition of organic matter to the treatment medium stabilized the cytoplasmic membrane against permeabilization in both the gram-positive and gram-negative bacteria investigated. Exposure of E. coli cells to the outer membrane-permeabilizing agent EDTA increased their sensitivity to chlorine and caused the shoulders in the survival curves to disappear. Based on these observations, we propose that bacterial envelopes could play a role in cell inactivation by modulating the access of chlorine to the key targets within the cell.     

Use of Integrated Cell Culture-PCR To Evaluate the Effectiveness of Poliovirus Inactivation by Chlorine

Current standards, based on cell culture assay, indicate that poliovirus is inactivated by 0.5 mg of free chlorine per liter after 2 min; however, integrated cell culture-PCR detected viruses for up to 8 min of exposure to the same chlorine concentration, requiring 10 min for complete inactivation. Thus, the contact time for chlorine disinfection of poliovirus is up to five times greater than previously thought.     

Differential Inactivation of Fungal Spores in Water and on Seeds by Ozone and Arc Discharge Plasma

Seed sterilization is essential for preventing seed borne fungal diseases. Sterilization tools based on physical technologies have recently received much attention. However, available information is very limited in terms of efficiency, safety, and mode of action. In this study, we have examined antifungal activity of ozone and arc discharge plasma, potential tools for seed sterilization. In our results, ozone and arc discharge plasma have shown differential antifungal effects, depending on the environment associated with fungal spores (freely submerged in water or infected seeds). Ozone inactivates Fusarium fujikuroi (fungus causing rice bakanae disease) spores submerged in water more efficiently than arc discharge plasma. However, fungal spores associated with or infecting rice seeds are more effectively deactivated by arc discharge plasma. ROS generated in water by ozone may function as a powerful fungicidal factor. On the other hand, shockwave generated from arc discharge plasma may have greatly contributed to antifungal effects on fungus associated with rice seeds. In support of this notion, addition of ultrasonic wave in ozone generating water has greatly increased the efficiency of seed disinfection.     

Inactivation of Cryptosporidium parvum Oocysts and Clostridium perfringens Spores by a Mixed-Oxidant Disinfectant and by Free Chlorine

Volume 63, no. 4, p. 1600: The caption to Table 2 should read as follows: "Inactivation of Clostridium perfringens spores by 5-mg/liter doses of mixed oxidants or free chlorine in buffer at pH 7 at 25(deg)C." [This corrects the article on p. 1598 in vol. 63.].     

Inactivation Kinetics and Mechanism of a Human Norovirus Surrogate on Stainless Steel Coupons via Chlorine Dioxide Gas

Acute gastroenteritis caused by human norovirus is a significant public health issue. Fresh produce and seafood are examples of high-risk foods associated with norovirus outbreaks. Food contact surfaces also have the potential to harbor noroviruses if exposed to fecal contamination, aerosolized vomitus, or infected food handlers. Currently, there is no effective measure to decontaminate norovirus on food contact surfaces. Chlorine dioxide (ClO₂) gas is a strong oxidizer and is used as a decontaminating agent in food processing plants. The objective of this study was to determine the kinetics and mechanism of ClO₂ gas inactivation of a norovirus surrogate, murine norovirus 1 (MNV-1), on stainless steel (SS) coupons. MNV-1 was inoculated on SS coupons at the concentration of 10⁷ PFU/coupon. The samples were treated with ClO₂ gas at 1, 1.5, 2, 2.5, and 4 mg/liter for up to 5 min at 25°C and a relative humidity of 85%, and virus survival was determined by plaque assay. Treatment of the SS coupons with ClO₂ gas at 2 mg/liter for 5 min and 2.5 mg/liter for 2 min resulted in at least a 3-log reduction in MNV-1, while no infectious virus was recovered at a concentration of 4 mg/liter even within 1 min of treatment. Furthermore, it was found that the mechanism of ClO₂ gas inactivation included degradation of viral protein, disruption of viral structure, and degradation of viral genomic RNA. In conclusion, treatment with ClO₂ gas can serve as an effective method to inactivate a human norovirus surrogate on SS contact surfaces.