Enhanced UV Inactivation of Adenoviruses under Polychromatic UV Lamps

Adenovirus is recognized as the most UV-resistant waterborne pathogen of concern to public health microbiologists. The U.S. EPA has stipulated that a UV fluence (dose) of 186 mJ cm⁻² is required for 4-log inactivation credit in water treatment. However, all adenovirus inactivation data to date published in the peer-reviewed literature have been based on UV disinfection experiments using UV irradiation at 253.7 nm produced from a conventional low-pressure UV source. The work reported here presents inactivation data for adenovirus based on polychromatic UV sources and details the significant enhancement in inactivation achieved using these polychromatic sources. When full-spectrum, medium-pressure UV lamps were used, 4-log inactivation of adenovirus type 40 is achieved at a UV fluence of less than 60 mJ cm⁻² and a surface discharge pulsed UV source required a UV fluence of less than 40 mJ cm⁻². The action spectrum for adenovirus type 2 was also developed and partially explains the improved inactivation based on enhancements at wavelengths below 230 nm. Implications for water treatment, public health, and the future of UV regulations for virus disinfection are discussed.     

Endotoxin inactivation in water by using medium-pressure UV lamps

Deionized water was spiked with various concentrations of endotoxin and exposed to UV irradiation from medium-pressure UV lamps to assess endotoxin inactivation. It was found that endotoxin inactivation was proportional to the UV dose under the conditions examined. The inactivation rate was determined to be approximately 0.55 endotoxin unit/ml per mJ/cm(2) of irradiation delivered.     

Inactivation of Escherichia coli Endotoxin by Soft Hydrothermal Processing

Bacterial endotoxins, also known as lipopolysaccharides, are a fever-producing by-product of gram-negative bacteria commonly known as pyrogens. It is essential to remove endotoxins from parenteral preparations since they have multiple injurious biological activities. Because of their strong heat resistance (e.g., requiring dry-heat sterilization at 250°C for 30 min) and the formation of various supramolecular aggregates, depyrogenation is more difficult than sterilization. We report here that soft hydrothermal processing, which has many advantages in safety and cost efficiency, is sufficient to assure complete depyrogenation by the inactivation of endotoxins. The endotoxin concentration in a sample was measured by using a chromogenic limulus method with an endotoxin-specific limulus reagent. The endotoxin concentration was calculated from a standard curve obtained using a serial dilution of a standard solution. We show that endotoxins were completely inactivated by soft hydrothermal processing at 130°C for 60 min or at 140°C for 30 min in the presence of a high steam saturation ratio or with a flow system. Moreover, it is easy to remove endotoxins from water by soft hydrothermal processing similarly at 130°C for 60 min or at 140°C for 30 min, without any requirement for ultrafiltration, nonselective adsorption with a hydrophobic adsorbent, or an anion exchanger. These findings indicate that soft hydrothermal processing, applied in the presence of a high steam saturation ratio or with a flow system, can inactivate endotoxins and may be useful for the depyrogenation of parenterals, including end products and medical devices that cannot be exposed to the high temperatures of dry heat treatments.Endotoxins are lipopolysaccharides (LPS) that are derived from the cell membranes of gram-negative bacteria and are continuously released into the environment. The release of LPS occurs not only upon cell death but also during growth and division. In the pharmaceutical industry, it is essential to remove endotoxins from parenteral preparations since they have multiple injurious biological activities, including pyrogenicity, lethality, Schwartzman reactivity, adjuvant activity, and macrophage activation (2, 9, 12, 13, 25, 32). Endotoxins are very stable molecules that are capable of resisting extreme temperatures and pH values (3, 16, 17, 29, 30, 34, 38). An endotoxin monomer has a molar mass of 10 to 20 kDa and forms supramolecular aggregates in aqueous solutions (22, 39) due to its amphipathic structure, which makes depyrogenation more difficult than sterilization. Endotoxins are not efficiently inactivated with the regular heat sterilization procedures recommended by the Japanese Pharmacopoeia. These procedures are steam heat treatment at 121°C for 20 min or dry-heat treatment for at least 1 h at 180°C. It is well accepted that only dry-heat treatment is efficient in destroying endotoxins (3, 16, 29, 30) and that endotoxins can be inactivated when exposed to a temperature of 250°C for more than 30 min or 180°C for more than 3 h (14, 36). In the production of parenterals, it is necessary to both depyrogenate the final products and carry out sterilization to avoid bacterial contamination.Several studies have examined dry-heat treatment, which is a very efficient means to degrade endotoxins (6, 20, 21, 26, 41, 42). However, its application is restricted to steel and glass implements that can tolerate high temperatures of >250°C. For sterilization, dry heat treatment tends to be used only with thermostable materials that cannot be sterilized by steam heat treatment (autoclaving). Alternative depyrogenation processes include the application of activated carbon (35), oxidation (15), and acidic or alkaline reagents (27), but steam heat treatment would be an attractive option if it were sufficiently effective. However, the data on the inactivation of endotoxins by steam heat treatment are insufficient and contradictory. It has been reported that endotoxins were not efficiently inactivated by steam heat treatment at 121°C (19, 45). However, Ogawa et al. (31) recently reported that steam heat treatment was efficient in inactivating low concentrations of endotoxin, and that Escherichia coli LPS are unstable in aqueous solutions even at relatively low temperatures such as 70°C (see also reference 40). As mentioned above, these reports have shown that although studies have been carried out on the use of steam heat for depyrogenation, there is little agreement on its efficiency.The U.S. Pharmacopoeia (USP) recommends depyrogenation by dry-heat treatment at temperatures above 220°C for as long as is necessary to achieve a ≥3-log reduction in the activity of endotoxin, if the value is ≥1,000 endotoxin units (EU)/ml (11, 44). Due to the serious risks associated with endotoxins, the U.S. Food and Drug Administration (FDA) has set guidelines for medical devices and parenterals. The protocol to test for endotoxin contamination of medical devices recommends immersion of the device in endotoxin-free water for at least 1 h at room temperature, followed by testing of this extract/eluate for endotoxin. Current FDA limits are such that eluates from medical devices may not exceed 0.5 EU/ml, or 0.06 EU/ml if the device comes into contact with cerebrospinal fluid (43). The term EU describes the biological activity of endotoxins. For example, 100 pg of the standard endotoxin EC-5, 200 pg of EC-2, and 120 pg of endotoxin from E. coli O111:B4 all have an activity of 1 EU (17, 23).Steam heat treatment is comparatively easy to apply and control. If steam heat treatment could reliably inactivate endotoxins, it could be applied with sterilization, reducing labor, time, and expenditure. However, to our knowledge, few studies have addressed steam heat inactivation to determine the chemical and physical reactions that occur during the hydrothermal process, nor have any studies examined the relationship between the steam saturation ratio and the inactivation of endotoxins. Moreover, to date no study has been conducted on steam heat activation of endotoxins with reference to the chemical and physical parameters of the hydrothermal process.We have developed a groundbreaking method to thermoinactivate endotoxins by means of a soft hydrothermal process, in which the steam saturation ratio can be controlled. The steam saturation ratio is calculated as follows: steam saturation ratio (%) = [steam density (kg/m³)/saturated steam density (kg/m³)] × 100.The soft hydrothermal process lies in the part of the liquid phase of water with a high steam saturation ratio that is characterized by a higher ionic product (k_w) than that of ordinary water. The ionic product is a key parameter in promoting ionic reactions and can be related to hydrolysis. The ionic product of water is 1.0 × 10⁻¹⁴ (mol/liter)² at room temperature and increases with increasing temperature and pressure. A high ionic product favors the solubility of highly polar and ionic compounds, creating the possibility of accelerating the hydrolysis reaction process of organic compounds. Thus, water can play the role of both an acidic and an alkaline catalyst in the hydrothermal process (Fig. ​(Fig.1)1) (1, 37, 46). However, the soft hydrothermal process lies in the high-density water molecular area of the steam-gas biphasic field (Fig. ​(Fig.1)1) and is characterized by a lower dielectric constant (ɛ) than that of ordinary water. This process opens the possibility of promoting the affinity of water for nonpolar or low-polarity compounds, such as lipophilic organic compounds (46). We previously reported that most of the predominant aromatic hydrocarbons were removed from softwood bedding that had been treated by soft hydrothermal processing (24, 28).Open in a separate windowFIG. 1.Reaction field in the pressure-temperature relationship of water. The curve represents the saturated vapor pressure curve. The fields show where the pressure-temperature relationships are conducive to a variety of hydrothermal processing conditions, in which water has a large impact as a reaction medium. Because high-density water has a large dielectric constant and ionic product, it is an effective reaction medium for advancing ionic reactions, whereas water (in the form of steam) on the lower-pressure side of the saturated vapor pressure curve shows a good ability to form materials by covalent bonding. Small changes in the density of water can result in changes in the chemical affinity, which has the potential to advance a range of ionic and radical reactions.The purpose of the present study was to evaluate the thermoinactivation of endotoxins by the soft hydrothermal process, by controlling the steam saturation ratio, temperature, and time of treatment. There have been reports that endotoxins were thermoinactivated by steam heat treatment at 121°C in the presence of a nonionic surfactant and at over 135°C in its absence (4, 5, 10), but the minimum temperature for the inactivation of endotoxin remained unknown. This report provides the answer to this question.     

Inactivation of Mycobacterium avium Complex by UV Irradiation

The effectiveness of two major UV technologies against a highly prevalent species of Mycobacterium avium complex was investigated. Our study indicates that M. avium is much more resistant to UV irradiation than most waterborne pathogens and that it is one of the rare microorganisms that are highly resistant to both chemical and UV disinfection in water.     

Inactivation of Giardia lamblia cysts by polychromatic UV

Aims:  Giardia lamblia is one of the most important waterborne pathogens in the world. In this study, we determined the effectiveness of a promising alternative UV technology – a polychromatic emission from a medium-pressure (MP) UV lamp – against G. lamblia cysts in phosphate buffered saline (PBS) and a filtered drinking water.
Methods and Results:  A UV collimated beam apparatus was used to expose shallow suspensions of purified G. lamblia cysts in PBS or a filtered drinking water and the UV-irradiated G. lamblia cysts were assayed in Mongolian gerbils. The inactivation of G. lamblia cysts was very rapid and reached a detection limit of >3 log₁₀ within a UV dose of 1 mJ cm⁻².
Conclusion:  The results of this study indicate that MP UV irradiation is very effective against G. lamblia cysts in both PBS and a filtered drinking water.
Significance and Impact of the Study:  It is likely that contamination of drinking water by G. lamblia cysts can be readily controlled by typical MP UV disinfection practises.     

UV Light Inactivation of Human and Plant Pathogens in Unfiltered Surface Irrigation Water

Fruit and vegetable growers continually battle plant diseases and food safety concerns. Surface water is commonly used in the production of fruits and vegetables and can harbor both human- and plant-pathogenic microorganisms that can contaminate crops when used for irrigation or other agricultural purposes. Treatment methods for surface water are currently limited, and there is a need for suitable treatment options. A liquid-processing unit that uses UV light for the decontamination of turbid juices was analyzed for its efficacy in the treatment of surface waters contaminated with bacterial or oomycete pathogens, i.e., Escherichia coli, Salmonella enterica, Listeria monocytogenes, Clavibacter michiganensis subsp. michiganensis, Pseudomonas syringae pv. tomato, and Phytophthora capsici. Five-strain cocktails of each pathogen, containing approximately 10⁸ or 10⁹ CFU/liter for bacteria or 10⁴ or 10⁵ zoospores/liter for Ph. capsici, were inoculated into aliquots of two turbid surface water irrigation sources and processed with the UV unit. Pathogens were enumerated before and after treatment. In general, as the turbidity of the water source increased, the effectiveness of the UV treatment decreased, but in all cases, 99.9% or higher inactivation was achieved. Log reductions ranged from 10.0 to 6.1 and from 5.0 to 4.2 for bacterial pathogens and Ph. capsici, respectively.     

Comparative Inactivation of Adenovirus Serotypes by UV Light Disinfection

The results of this study confirm that adenoviruses are the most resistant enteric viruses to inactivation by UV light and that adenovirus 40 appears to be the most resistant. The effect of freeze-thawing and storage in water may affect the sensitivity of some adenoviruses to inactivation by UV light.     

Effects of UV Radiation on Photolyase and Implications with Regards to Photoreactivation following Low- and Medium-Pressure UV Disinfection

Photolyase activity following exposure to low-pressure (LP) and medium-pressure (MP) UV lamps was evaluated. MP UV irradiation resulted in a greater reduction in photolyase activity than LP UV radiation. The results suggest that oxidation of the flavin adenine dinucleotide in photolyase may have caused the decrease in activity.     

Comparative Inactivation of Enteroviruses and Adenovirus 2 by UV Light

The doses of UV irradiation necessary to inactivate selected enteric viruses on the U.S. Environmental Protection Agency Contaminant Candidate List were determined. Three-log reductions of echovirus 1, echovirus 11, coxsackievirus B3, coxsackievirus B5, poliovirus 1, and human adenovirus type 2 were effected by doses of 25, 20.5, 24.5, 27, 23, and 119 mW/cm², respectively. Human adenovirus type 2 is the most UV light-resistant enteric virus reported to date.     

Inactivation of Single-Celled Ascaris suum Eggs by Low-Pressure UV Radiation

Intact and decorticated single-celled Ascaris suum eggs were exposed to UV radiation from low-pressure, germicidal lamps at fluences (doses) ranging from 0 to 8,000 J/m² for intact eggs and from 0 to 500 J/m² for decorticated eggs. With a UV fluence of 500 J/m², 0.44- ± 0.20-log inactivation (mean ± 95% confidence interval) (63.7%) of intact eggs was observed, while a fluence of 4,000 J/m² resulted in 2.23- ± 0.49-log inactivation (99.4%). (The maximum quantifiable inactivation was 2.5 log units.) Thus, according to the methods used here, Ascaris eggs are the most UV-resistant water-related pathogen identified to date. For the range of fluences recommended for disinfecting drinking water and wastewater (200 to 2,000 J/m²), from 0- to 1.5-log inactivation can be expected, although at typical fluences (less than 1,000 J/m²), the inactivation may be less than 1 log. When the eggs were decorticated (the outer egg shell layers were removed with sodium hypochlorite, leaving only the lipoprotein ascaroside layer) before exposure to UV, 1.80- ± 0.32-log reduction (98.4%) was achieved with a fluence of 500 J/m², suggesting that the outer eggshell layers protected A. suum eggs from inactivation by UV radiation. This protection may have been due to UV absorption by proteins in the outer layers of the 3- to 4-μm-thick eggshell. Stirring alone (without UV exposure) also inactivated some of the Ascaris eggs (~20% after 75 min), which complicated determination of the inactivation caused by UV radiation alone.     

Inactivation of single-celled Ascaris suum eggs by low-pressure UV radiation

Intact and decorticated single-celled Ascaris suum eggs were exposed to UV radiation from low-pressure, germicidal lamps at fluences (doses) ranging from 0 to 8,000 J/m2 for intact eggs and from 0 to 500 J/m2 for decorticated eggs. With a UV fluence of 500 J/m2, 0.44-+/-0.20-log inactivation (mean+/-95% confidence interval) (63.7%) of intact eggs was observed, while a fluence of 4,000 J/m2 resulted in 2.23-+/-0.49-log inactivation (99.4%). (The maximum quantifiable inactivation was 2.5 log units.) Thus, according to the methods used here, Ascaris eggs are the most UV-resistant water-related pathogen identified to date. For the range of fluences recommended for disinfecting drinking water and wastewater (200 to 2,000 J/m2), from 0- to 1.5-log inactivation can be expected, although at typical fluences (less than 1,000 J/m2), the inactivation may be less than 1 log. When the eggs were decorticated (the outer egg shell layers were removed with sodium hypochlorite, leaving only the lipoprotein ascaroside layer) before exposure to UV, 1.80-+/-0.32-log reduction (98.4%) was achieved with a fluence of 500 J/m2, suggesting that the outer eggshell layers protected A. suum eggs from inactivation by UV radiation. This protection may have been due to UV absorption by proteins in the outer layers of the 3- to 4-microm-thick eggshell. Stirring alone (without UV exposure) also inactivated some of the Ascaris eggs (approximately 20% after 75 min), which complicated determination of the inactivation caused by UV radiation alone.     

Inactivation of Cryptosporidium parvum Oocysts in Fresh Apple Cider by UV Irradiation

This study evaluated the efficacy of UV irradiation on the inactivation of Cryptosporidium parvum oocysts in fresh apple cider. Cider was inoculated with oocysts and exposed to 14.32 mJ of UV irradiation/cm². Oocyst viability was assessed with the gamma interferon gene knockout (GKO) mouse and infant BALB/cByJ mouse models. All GKO mice challenged with UV-treated cider demonstrated no morbidity or mortality, and infant BALB/c mice challenged with treated cider were negative for the presence of C. parvum. In contrast, the GKO mice challenged with non-UV-treated inoculated cider died and the parasite was detected in the ileums of all challenged infant mice. This study shows that UV irradiation can be used to inactivate C. parvum in fresh apple cider.     

Inactivation of Mycobacterium avium ssp. paratuberculosis in milk by UV treatment

Aims:  To determine the effect of UV radiation on the viability of two strains of Mycobacterium avium ssp. paratuberculosis (Map) inoculated into milk.
Methods and Results:  Mycobacterium avium ssp. paratuberculosis in a ultra heat treated milk matrix was subjected to increasing doses of UV-C radiation from 0 to 1836 mJ ml⁻¹ using a pilot-scale UV reactor (20 l capacity). Survival of Map was monitored by culture on Herrold's egg yolk medium, Middlebrook 7H10 medium and the FASTPlaque TB™ phage assay. Differences in sensitivity to UV treatment were observed between strains, however, at 1000 mJ ml⁻¹ a Map kill rate of 0·1–0·6 log₁₀ was achieved regardless of strain used or method employed to enumerate Map. Although the inactivation trend was similar on the culture and phage assay, the former gave a consistently higher viable count.
Conclusions:  The use of UV radiation alone does not represent an alternative to current pasteurization regimes for a large reduction in viable Map in milk.
Significance and Impact of the Study:  To the authors' knowledge the work here represents the first pilot-scale UV treatment process used to assess UV efficacy to inactivate Map in milk. The results are similar to those obtained with a laboratory-scale process indicating the difficulties associated with UV treatment of an opaque liquid and the recalcitrance of Map towards inimical treatments.     

Inactivation of Cryptosporidium parvum oocysts in fresh apple cider by UV irradiation

This study evaluated the efficacy of UV irradiation on the inactivation of Cryptosporidium parvum oocysts in fresh apple cider. Cider was inoculated with oocysts and exposed to 14.32 mJ of UV irradiation/cm(2). Oocyst viability was assessed with the gamma interferon gene knockout (GKO) mouse and infant BALB/cByJ mouse models. All GKO mice challenged with UV-treated cider demonstrated no morbidity or mortality, and infant BALB/c mice challenged with treated cider were negative for the presence of C. parvum. In contrast, the GKO mice challenged with non-UV-treated inoculated cider died and the parasite was detected in the ileums of all challenged infant mice. This study shows that UV irradiation can be used to inactivate C. parvum in fresh apple cider.     

Inactivation of feline calicivirus and adenovirus type 40 by UV radiation

Little information regarding the effectiveness of UV radiation on the inactivation of caliciviruses and enteric adenoviruses is available. Analysis of human calicivirus resistance to disinfectants is hampered by the lack of animal or cell culture methods that can determine the viruses' infectivity. The inactivation kinetics of enteric adenovirus type 40 (AD40), coliphage MS-2, and feline calicivirus (FCV), closely related to the human caliciviruses based on nucleic acid organization and capsid architecture, were determined after exposure to low-pressure UV radiation in buffered demand-free (BDF) water at room temperature. In addition, UV disinfection experiments were also carried out in treated groundwater with FCV and AD40. AD40 was more resistant than either FCV or coliphage MS-2 in both BDF water and groundwater. The doses of UV required to achieve 99% inactivation of AD40, coliphage MS-2, and FCV in BDF water were 109, 55, and 16 mJ/cm(2), respectively. The doses of UV required to achieve 99% inactivation of AD40, coliphage MS-2, and FCV in groundwater were slightly lower than those in BDF water. FCV was inactivated by 99% by 13 mJ/cm(2) in treated groundwater. A dose of 103 mJ/cm(2) was required for 99% inactivation of AD40 in treated groundwater. The results of this study indicate that if FCV is an adequate surrogate for human caliciviruses, then their inactivation by UV radiation is similar to those of other single-stranded RNA enteric viruses, such as poliovirus. In addition, AD40 appears to be more resistant to UV disinfection than previously reported.     

Inactivation of Feline Calicivirus and Adenovirus Type 40 by UV Radiation

Little information regarding the effectiveness of UV radiation on the inactivation of caliciviruses and enteric adenoviruses is available. Analysis of human calicivirus resistance to disinfectants is hampered by the lack of animal or cell culture methods that can determine the viruses' infectivity. The inactivation kinetics of enteric adenovirus type 40 (AD40), coliphage MS-2, and feline calicivirus (FCV), closely related to the human caliciviruses based on nucleic acid organization and capsid architecture, were determined after exposure to low-pressure UV radiation in buffered demand-free (BDF) water at room temperature. In addition, UV disinfection experiments were also carried out in treated groundwater with FCV and AD40. AD40 was more resistant than either FCV or coliphage MS-2 in both BDF water and groundwater. The doses of UV required to achieve 99% inactivation of AD40, coliphage MS-2, and FCV in BDF water were 109, 55, and 16 mJ/cm², respectively. The doses of UV required to achieve 99% inactivation of AD40, coliphage MS-2, and FCV in groundwater were slightly lower than those in BDF water. FCV was inactivated by 99% by 13 mJ/cm² in treated groundwater. A dose of 103 mJ/cm² was required for 99% inactivation of AD40 in treated groundwater. The results of this study indicate that if FCV is an adequate surrogate for human caliciviruses, then their inactivation by UV radiation is similar to those of other single-stranded RNA enteric viruses, such as poliovirus. In addition, AD40 appears to be more resistant to UV disinfection than previously reported.     

UV Light Inactivation of Bacterial Biothreat Agents

Seven species of bacterial biothreat agents were tested for susceptibility to UV light (254 nm). All gram-negative organisms tested required <12 mJ/cm² for a 4-log₁₀ reduction in viability (inactivation). Tailing off of the B. anthracis spore inactivation curves began close to the 2-log₁₀ inactivation point, with a fluence of approximately 40 mJ/cm², and 3-log₁₀ inactivation still was not achieved with a fluence of 120 mJ/cm².The security of our nation''s water supply is a concern for water providers and public health officials. Questions have been asked regarding the possibility of our drinking water becoming contaminated with biothreat agents and the efficacy of current disinfection practices for the reduction in viability (inactivation) of biothreat agents (5, 8, 14). The use of UV irradiation as a supplemental water disinfection practice is increasing for several reasons, among them improving control of protozoa, such as Cryptosporidium spp., and decreasing disinfection by-products created by chemical disinfectants (21). This study employed a bench-scale collimated beam test to determine the UV fluence (dose) required to inactivate seven representative bacterial biothreat agents.Seven species, two isolates each, from the Health and Human Services and U.S Dept. of Agriculture lists of select agents (http://www.selectagents.gov/resources/List%20of%20Select%20Agents%20and%20Toxins_111708.pdf) were used in this study: Bacillus anthracis Ames spores, B. anthracis 34F2 (Sterne) spores, Brucella melitensis ATCC 23456, B. melitensis IL195, Brucella suis KS528, B. suis MO562, Burkholderia mallei M9, B. mallei M13, Burkholderia pseudomallei ATCC 11688, B. pseudomallei CA650, Francisella tularensis LVS, F. tularensis NY98, Yersinia pestis A1122, and Y. pestis Harbin. B. anthracis was grown on soil extract-peptone-beef extract agar (SEA) (1) or in Schaeffer''s sporulation medium (SSM) (10) for 7 days, resulting in >99% spores as determined by phase-contrast microscopy. The cells and spores were then washed by centrifugation (8,000 × g), resuspended in ultrapure water, transferred to centrifuge tubes, treated with 50% ethanol for 1 h at room temperature, and washed five times with sterile ultrapure water before being stored in reverse-osmosis water at −70°C. F. tularensis isolates were grown on cysteine heart agar (1) and all other isolates on Trypticase soy agar with 5% sheep blood (TSA II; Becton Dickinson Microbiology Systems, Sparks, MD) for 24 h before testing. B. anthracis spores were adjusted to 10⁷ CFU/ml and other bacterial suspensions to 10⁸ CFU/ml in Butterfield buffer (3 mM KH₂PO₄, pH 7.2; Becton Dickinson Microbiology Systems), and then isolates were sonicated (40-Hz ultrasonic cleaner; VWR, Suwanee, GA) for 1 min to disperse aggregates. The suspensions were diluted 1:100 in Butterfield buffer for final test concentrations. Five milliliters of each suspension were placed into a small petri dish (50-mm diameter) along with a small sterile stir bar, and the petri dish was placed on a stir plate.UV irradiation was performed by using a collimated beam apparatus (Calgon Carbon, Pittsburgh, PA) equipped with a low-pressure lamp (254 nm) according to the standard method developed by Bolton and Linden (2). The surface of the suspension was placed 5 cm from the end of the collimating tube. The UV intensity was measured with a radiometer at 0.5-cm intervals across the test area and variability compensated for according to the UVCalc software directions (International UV association [http://www.iuva.org]). The fluences (UV doses) were determined using the UVCalc software, and the petri dishes were placed under the beam for at least five time periods to deliver a range of appropriate fluences to the organisms. Each irradiation test was conducted at room temperature (23 ± 2°C) in triplicate and in a random order of fluences. After exposure, 10-fold serial dilutions were performed, and the dilutions were spread plated in triplicate. Plates were placed in a dark incubator within 10 min of plating and incubated at the temperature appropriate for the organism, and the colonies were counted at 24 h and 48 h for B. anthracis and at 3 and 5 days for the remaining organisms. Colonies were counted, and the log₁₀ inactivation at each fluence was determined for each organism. A linear regression of the fluence response data determined the fluence required for 2-, 3-, and 4-log₁₀ inactivation.The UV fluences required for inactivation of each organism are reported in Table ​Table1.1. Little difference in UV susceptibility was seen between the gram-negative organisms. B. suis KS528 and B. melitensis ATCC 23456 required the greatest UV fluence of the gram-negative organisms for 4-log₁₀ inactivation (10.5 and 10.2 mJ/cm², respectively), while the two Y. pestis isolates required the lowest UV fluence (4.1 and 4.9 mJ/cm²) for the same 4-log₁₀ inactivation (Table ​(Table1).1). Generally, the two isolates of each species differed no more than 3 mJ/cm² in the UV fluence required for 4-log₁₀ inactivation.

TABLE 1.

UV fluence required for given log₁₀ inactivation of each organism

Inactivation of lactate dehydrogenase by UV radiation in the 300 nm wavelength region

Summary The effect of UV radiation on lactate dehydrogenase (LDH) was investigated. LDH from bovine heart was exposed to 3.6–18 kJ/m² of ultraviolet (UV) radiation in the 300 nm wavelength region. The activity of LDH was observed to decrease as a function of the dosage of UV radiation. The inactivation of LDH was independent of exposure rate at constant dosage of UV radiation. The decrease of LDH activity caused by UV 300 nm radiation was coupled to a decrease of the maximal velocity (V _max) while the Michaelis constant (K _m ) remained unchanged. The absorption spectrum of the LDH changed during exposure to UV radiation suggesting an alteration of tryptophan in the LDH molecule.On leave from Department of Ophthalmology, Ren-Ji Hospital affiliated to Shanghai No. 2 Medical University, Shanghai, People's Republic of China     

UV Inactivation of Cryptosporidium hominis as Measured in Cell Culture

The Cryptosporidium spp. UV disinfection studies conducted to date have used Cryptosporidium parvum oocysts. However, Cryptosporidium hominis predominates in human cryptosporidiosis infections, so there is a critical need to assess the efficacy of UV disinfection of C. hominis. This study utilized cell culture-based methods to demonstrate that C. hominis oocysts displayed similar levels of infectivity and had the same sensitivity to UV light as C. parvum. Therefore, the water industry can be confident about extrapolating C. parvum UV disinfection data to C. hominis oocysts.