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.     

Response of Mycobacterium avium to Ultraviolet Irradiation

The survival response of Mycobacterium avium to ultraviolet irradiation demonstrated the presence of a photoreactivating repair system.     

UV Light Inactivation of Mycobacterium avium subsp. paratuberculosis in Milk as Assessed by FASTPlaqueTB Phage Assay and Culture

UV light inactivation of Mycobacterium avium subsp. paratuberculosis in Middlebrook 7H9 broth and whole and semiskim milk was investigated using a laboratory-scale UV machine that incorporated static mixers within UV-penetrable pipes. UV treatment proved to be less effective in killing M. avium subsp. paratuberculosis suspended in milk (0.5- to 1.0-log₁₀ reduction per 1,000 mJ/ml) than that suspended in Middlebrook 7H9 broth (2.5- to 3.3-log₁₀ reduction per 1,000 mJ/ml). The FASTPlaqueTB phage assay provided more rapid enumeration of surviving M. avium subsp. paratuberculosis (within 24 h) than culture on Herrold's egg yolk medium (6 to 8 weeks). Despite the fact that plaque counts were consistently 1 to 2 log₁₀ lower than colony counts throughout the study, UV inactivation rates for M. avium subsp. paratuberculosis derived using the phage assay and culture results were not significantly different (P = 0.077).     

Electrophoretic Mobility of Mycobacterium avium Complex Organisms

The electrophoretic mobilities (EPMs) of 30 Mycobacterium avium complex organisms were measured. The EPMs of 15 clinical isolates ranged from -1.9 to -5.0 microM cm V(-1) s(-1), and the EPMs of 15 environmental isolates ranged from -1.9 to -4.6 microM cm V(-1) s(-1) at pH 7.     

Electrophoretic Mobility of Mycobacterium avium Complex Organisms

The electrophoretic mobilities (EPMs) of 30 Mycobacterium avium complex organisms were measured. The EPMs of 15 clinical isolates ranged from −1.9 to −5.0 μm cm V⁻¹ s⁻¹, and the EPMs of 15 environmental isolates ranged from −1.9 to −4.6 μm cm V⁻¹ s⁻¹ at pH 7.     

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.     

Heat Inactivation of Mycobacterium avium subsp. paratuberculosis in Milk

The effectiveness of pasteurization and the concentration of Mycobacterium avium subsp. paratuberculosis in raw milk have been identified in quantitative risk analysis as the most critical factors influencing the potential presence of viable Mycobacterium paratuberculosis in dairy products. A quantitative assessment of the lethality of pasteurization was undertaken using an industrial pasteurizer designed for research purposes with a validated Reynolds number of 62,112 and flow rates of 3,000 liters/h. M. paratuberculosis was artificially added to raw whole milk, which was then homogenized, pasteurized, and cultured, using a sensitive technique capable of detecting one organism per 10 ml of milk. Twenty batches of milk containing 10³ to 10⁴ organisms/ml were processed with combinations of three temperatures of 72, 75, and 78°C and three time intervals of 15, 20, and 25 s. Thirty 50-ml milk samples from each processed batch were cultured, and the logarithmic reduction in M. paratuberculosis organisms was determined. In 17 of the 20 runs, no viable M. paratuberculosis organisms were detected, which represented >6-log₁₀ reductions during pasteurization. These experiments were conducted with very heavily artificially contaminated milk to facilitate the measurement of the logarithmic reduction. In three of the 20 runs of milk, pasteurized at 72°C for 15 s, 75°C for 25 s, and 78°C for 15 s, a few viable organisms (0.002 to 0.004 CFU/ml) were detected. Pasteurization at all temperatures and holding times was found to be very effective in killing M. paratuberculosis, resulting in a reduction of >6 log₁₀ in 85% of runs and >4 log₁₀ in 14% of runs.     

Inactivation of the Mycobacterium tuberculosis Antigen 85 Complex by Covalent,Allosteric Inhibitors

The rise of multidrug-resistant and totally drug-resistant tuberculosis and the association with an increasing number of HIV-positive patients developing tuberculosis emphasize the necessity to find new antitubercular targets and drugs. The antigen 85 (Ag85) complex from Mycobacterium tuberculosis plays important roles in the biosynthesis of major components of the mycobacterial cell envelope. For this reason, Ag85 has emerged as an attractive drug target. Recently, ebselen was identified as an effective inhibitor of the Ag85 complex through covalent modification of a cysteine residue proximal to the Ag85 active site and is therefore a covalent, allosteric inhibitor. To expand the understanding of this process, we have solved the x-ray crystal structures of Ag85C covalently modified with ebselen and other thiol-reactive compounds, p-chloromercuribenzoic acid and iodoacetamide, as well as the structure of a cysteine to glycine mutant. All four structures confirm that chemical modification or mutation at this particular cysteine residue leads to the disruption of the active site hydrogen-bonded network essential for Ag85 catalysis. We also describe x-ray crystal structures of Ag85C single mutants within the catalytic triad and show that a mutation of any one of these three residues promotes the same conformational change observed in the cysteine-modified forms. These results provide evidence for active site dynamics that may afford new strategies for the development of selective and potent Ag85 inhibitors.     

Assessing the Inactivation of Mycobacterium avium subsp. paratuberculosis during Composting of Livestock Carcasses

Mycobacterium avium subsp. paratuberculosis causes Johne''s disease (JD) in ruminants, with substantial economic impacts on the cattle industry. Johne''s disease is known for its long latency period, and difficulties in diagnosis are due to insensitivities of current detection methods. Eradication is challenging as M. avium subsp. paratuberculosis can survive for extended periods within the environment, resulting in new infections in naïve animals (W. Xu et al., J. Environ. Qual. 38:437-450, 2009). This study explored the use of a biosecure, static composting structure to inactivate M. avium subsp. paratuberculosis. Mycobacterium smegmatis was also assessed as a surrogate for M. avium subsp. paratuberculosis. Two structures were constructed to hold three cattle carcasses each. Naturally infected tissues and ground beef inoculated with laboratory-cultured M. avium subsp. paratuberculosis and M. smegmatis were placed in nylon and plastic bags to determine effects of temperature and compost environment on viability over 250 days. After removal, samples were cultured and growth of both organisms was assessed after 12 weeks. After 250 days, M. avium subsp. paratuberculosis was still detectable by PCR, while M. smegmatis was not detected after 67 days of composting. Furthermore, M. avium subsp. paratuberculosis remained viable in both implanted nylon and plastic bags over the composting period. As the compost never reached a homogenous thermophilic (55 to 65°C) state throughout each structure, an in vitro experiment was conducted to examine viability of M. avium subsp. paratuberculosis after exposure to 80°C for 90 days. Naturally infected lymph tissues were mixed with and without compost. After 90 days, M. avium subsp. paratuberculosis remained viable despite exposure to temperatures typically higher than that achieved in compost. In conclusion, it is unlikely composting can be used as a means of inactivating M. avium subsp. paratuberculosis associated with cattle mortalities.     

Inactivation of Airborne Viruses by Ultraviolet Irradiation

Aerosolized viruses were passed through a high-intensity ultraviolet (UV) cell. This cell consisted of a long cylindrical aluminum tube [diameter, 7 in. (17.7 cm); length, 36 in. (91.4 cm)] with a highly reflective inner surface and a longitudinally extending helical baffle system which directed airborne particles in close proximity to a centrally located UV lamp. After having been passed through the UV cell, viral aerosols were collected with an Andersen sampler, and viral concentrations were determined by plaque assay methods on tissue cultures. Inactivation rates of greater than 99.9% were obtained for Coxsackie, influenza, Sindbis, and vaccinia viruses, and slightly less for adenovirus (96.8%), when the aerosols passed through the UV cell at 100 ft³/min. At aerosol flow rates of 200 ft³/min, inactivation rates were slightly lower; 91.3 for adenovirus, 97.5 and 96.7 for Coxsackie and Sindbis, respectively, and greater than 99.9% for influenza and vaccinia viruses.     

Inactivation of Mycobacterium avium subsp. paratuberculosis in cow's milk by means of high hydrostatic pressure at mild temperatures

Two strains of Mycobacterium avium subsp. paratuberculosis (3644/02 and ATCC 19698) were inoculated (approximately 6 log CFU/ml) into sterilized milk to evaluate inactivation by high hydrostatic pressure. Reductions of M. avium subsp. paratuberculosis increased with pressure level. Significant differences were also found between M. avium subsp. paratuberculosis strains and between the media used. Average reductions of 4 log CFU/ml after treatment with 500 MPa are comparable to those caused by thermal treatments.     

Inactivation of Mycobacterium avium subsp. paratuberculosis in Cow's Milk by Means of High Hydrostatic Pressure at Mild Temperatures

Two strains of Mycobacterium avium subsp. paratuberculosis (3644/02 and ATCC 19698) were inoculated (approximately 6 log CFU/ml) into sterilized milk to evaluate inactivation by high hydrostatic pressure. Reductions of M. avium subsp. paratuberculosis increased with pressure level. Significant differences were also found between M. avium subsp. paratuberculosis strains and between the media used. Average reductions of 4 log CFU/ml after treatment with 500 MPa are comparable to those caused by thermal treatments.     

Investigation of Spa Pools Associated with Lung Disorders Caused by Mycobacterium avium Complex in Immunocompetent Adults

Three cases of Mycobacterium avium complex-related lung disorders were associated with two poorly maintained spa pools by genotypic investigations. Inadequate disinfection of the two spas had reduced the load of environmental bacteria to less than 1 CFU/ml but allowed levels of M. avium complex of 4.3 × 10⁴ and 4.5 × 10³ CFU/ml. Persistence of the disease-associated genotype was demonstrated in one spa pool for over 5 months until repeated treatments with greater than 10 mg of chlorine per liter for 1-h intervals eliminated M. avium complex from the spa pool. A fourth case of Mycobacterium avium complex-related lung disease was associated epidemiologically but not genotypically with another spa pool that had had no maintenance undertaken. This spa pool contained low numbers of mycobacteria by smear and was culture positive for M. avium complex, and the nonmycobacterial organism count was 5.2 × 10⁶ CFU/ml. Public awareness about the proper maintenance of private (residential) spa pools must be promoted by health departments in partnership with spa pool retailers.     

Inactivation of Giardia lamblia Cysts by Ultraviolet Irradiation

Giardia lamblia cysts were found to be resistant to high doses of germicidal ultraviolet radiation.     

Proposed Definitions for Epidemiologic and Clinical Studies of Mycobacterium avium Complex Pulmonary Disease

Background

Epidemiologic and clinical studies of Mycobacterium avium complex (MAC) pulmonary disease typically use strict ATS/IDSA definitions designed for decisions about treatment. Studies based on these criteria may exclude a substantial number of patients with true disease. We reviewed patients treated for MAC pulmonary disease at an academic medical center to propose revised definitions encompass the full spectrum of MAC pulmonary disease.

Methods

We conducted a retrospective review of patients with MAC pulmonary disease treated from 1993–2006 by pulmonary or infectious disease specialists to assess whether treated patients met current ATS/IDSA microbiologic criteria and dichotomous radiologic classification as nodular/bronchiectatic (NB) or fibrocavitary (FC) disease. We propose a revised set of definitions that include categories of both probable and definite disease to include all treated patients. We further classify patients into dichotomous clinical categories as: “primary MAC” (without antecedent lung disease) or “secondary MAC” (smoking history or antecedent lung disease).

Results

Among 72 treated patients, 74% were female. Median age at diagnosis was 64 years; 41(57%) met ATS/IDSA criteria and 31 (43%) did not, most often for lack of multiple positive cultures. Dichotomous radiologic criteria were met by 48 (67%) patients (36 NB, 12 FC); the remaining 24 (33%) had both NB and FC findings or other abnormalities. Nineteen (26%) were classified as primary and 53 (74%) as secondary MAC (21 COPD, 4 bronchiectasis, 44 smoking history).

Conclusions

We propose revised definitions for epidemiologic and clinical studies of MAC pulmonary disease that describe the full spectrum of disease.     

Inactivation of Poliovirus 1 and F-Specific RNA Phages and Degradation of Their Genomes by UV Irradiation at 254 Nanometers

Several models (animal caliciviruses, poliovirus 1 [PV1], and F-specific RNA bacteriophages) are usually used to predict inactivation of nonculturable viruses. For the same UV fluence, viral inactivation observed in the literature varies from 0 to 5 logs according to the models and the methods (infectivity versus molecular biology). The lack of knowledge concerning the mechanisms of inactivation due to UV prevents us from selecting the best model. In this context, determining if viral genome degradation may explain the loss of infectivity under UV radiation becomes essential. Thus, four virus models (PV1 and three F-specific RNA phages: MS2, GA, and Qβ) were exposed to UV radiation from 0 to 150 mJ · cm⁻². PV1 is the least-resistant virus, while MS2 and GA phages are the most resistant, with phage Qβ having an intermediate sensitivity; respectively, 6-log, 2.3-log, 2.5-log, and 4-log decreases for 50 mJ · cm⁻². In parallel, analysis of RNA degradation demonstrated that this phenomenon depends on the fragment size for PV1 as well as for MS2. Long fragments (above 2,000 bases) for PV1 and MS2 fell rapidly to the background level (>1.3-log decrease) for 20 mJ · cm⁻² and 60 mJ · cm^{− 2}, respectively. Nevertheless, the size of the viral RNA is not the only factor affecting UV-induced RNA degradation, since viral RNA was more rapidly degraded in PV1 than in the MS2 phage with a similar size. Finally, extrapolation of inactivation and UV-induced RNA degradation kinetics highlights that genome degradation could fully explain UV-induced viral inactivation.     

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.     

Inactivation of Strains of Salmonella senftenberg by Gamma Irradiation

S ummary : Strains of Salmonella senftenberg isolated from Norwegian herring meal and strain 775W were exposed to gamma radiation from a ⁶⁰Co source. When they were irradiated in phosphate buffered saline solution, the average D₁₀ values for all the strains was 19·3 krad. On irradiating strains 56 and 775W in herring meal the D₁₀ values were 192·1 and 188·5 krad, respectively, thus indicating that the suspending medium had a great effect on the radiation resistance of the organisms. Radiation doses of the order of 0·8–1·3 Mrad are recommended for the decontamination of herring meal.     

UV light inactivation of Mycobacterium avium subsp. paratuberculosis in milk as assessed by FASTPlaqueTB phage assay and culture

UV light inactivation of Mycobacterium avium subsp. paratuberculosis in Middlebrook 7H9 broth and whole and semiskim milk was investigated using a laboratory-scale UV machine that incorporated static mixers within UV-penetrable pipes. UV treatment proved to be less effective in killing M. avium subsp. paratuberculosis suspended in milk (0.5- to 1.0-log(10) reduction per 1,000 mJ/ml) than that suspended in Middlebrook 7H9 broth (2.5- to 3.3-log(10) reduction per 1,000 mJ/ml). The FASTPlaqueTB phage assay provided more rapid enumeration of surviving M. avium subsp. paratuberculosis (within 24 h) than culture on Herrold's egg yolk medium (6 to 8 weeks). Despite the fact that plaque counts were consistently 1 to 2 log(10) lower than colony counts throughout the study, UV inactivation rates for M. avium subsp. paratuberculosis derived using the phage assay and culture results were not significantly different (P = 0.077).     

Inactivation of Lactobacillus Bacteriophage PL-1 by Microwave Irradiation

The effect of microwave irradiation on the survival of bacteriophage PL-1, which is specific for Lactobacillus casei, was studied using a commercial 2,450 MHz microwave oven. The phages were inactivated by microwave irradiation according to almost first-order reaction kinetics. The rate of phage inactivation was not affected by the difference in the continuous or intermittent irradiation, nor by the concentrations of phages used, but was affected by the volume of phage suspensions, which prevented the loss of generated heat. Microwave irradiation of phage suspensions produced a number of ghost phages with empty heads, but fragmentation of the tail was hardly noticed. The breakage of phage genome DNA was primarily caused by the heat generated by microwave irradiation, whereas the phage DNA was not affected by the same temperature achieved by heat from outside. Thus we concluded that the phage-inactivating effect of microwave irradiation was mainly attributed to a thermal microwave effect, which was much stronger than a simple thermal exposure.