Tsuyoshi Hirata

Determination of Pyrimidine Dimers in Escherichia coli and Cryptosporidium parvum during UV Light Inactivation, Photoreactivation, and Dark Repair

ABSTRACT UV inactivation, photoreactivation, and dark repair ofEscherichia coli and Cryptosporidium parvum were investigated with the endonuclease sensitive site (ESS) assay, which can determine UV-induced pyrimidine dimers in the genomic DNA of microorganisms. In a 99.9% inactivation of E. coli, high correlation was observed between the dose of UV irradiation and the number of pyrimidine dimers induced in the DNA ofE. coli. The colony-forming ability of E. coli also correlated highly with the number of pyrimidine dimers in the DNA, indicating that the ESS assay is comparable to the method conventionally used to measure colony-forming ability. WhenE. coli were exposed to fluorescent light after a 99.9% inactivation by UV irradiation, UV-induced pyrimidine dimers in the DNA were continuously repaired and the colony-forming ability recovered gradually. When kept in darkness after the UV inactivation, however,E. coli showed neither repair of pyrimidine dimers nor recovery of colony-forming ability. When C. parvum were exposed to fluorescent light after UV inactivation, UV-induced pyrimidine dimers in the DNA were continuously repaired, while no recovery of animal infectivity was observed. When kept in darkness after UV inactivation, C. parvum also showed no recovery of infectivity in spite of the repair of pyrimidine dimers. It was suggested, therefore, that the infectivity of C. parvumwould not recover either by photoreactivation or by dark repair even after the repair of pyrimidine dimers in the genomic DNA.

Efficacy of UV irradiation in inactivating Cryptosporidium parvum oocysts.

ABSTRACT To evaluate the effectiveness of UV irradiation in inactivating Cryptosporidium parvum oocysts, the animal infectivities and excystation abilities of oocysts that had been exposed to various UV doses were determined. Infectivity decreased exponentially as the UV dose increased, and the required dose for a 2-log10 reduction in infectivity (99% inactivation) was approximately 1.0 mWs/cm2 at 20°C. However, C. parvum oocysts exhibited high resistance to UV irradiation, requiring an extremely high dose of 230 mWs/cm2 for a 2-log10 reduction in excystation, which was used to assess viability. Moreover, the excystation ability exhibited only slight decreases at UV doses below 100 mWs/cm2. Thus, UV treatment resulted in oocysts that were able to excyst but not infect. The effects of temperature and UV intensity on the UV dose requirement were also studied. The results showed that for every 10°C reduction in water temperature, the increase in the UV irradiation dose required for a 2-log10 reduction in infectivity was only 7%, and for every 10-fold increase in intensity, the dose increase was only 8%. In addition, the potential of oocysts to recover infectivity and to repair UV-induced injury (pyrimidine dimers) in DNA by photoreactivation and dark repair was investigated. There was no recovery in infectivity following treatment by fluorescent-light irradiation or storage in darkness. In contrast, UV-induced pyrimidine dimers in the DNA were apparently repaired by both photoreactivation and dark repair, as determined by endonuclease-sensitive site assay. However, the recovery rate was different in each process. Given these results, the effects of UV irradiation on C. parvum oocysts as determined by animal infectivity can conclusively be considered irreversible.

CryptosporidiumQuantitative risk assessment of in a watershed

Publisher Summary This chapter calculates the risk of the infection of Cryptosporidium via drinking water following the ILSI/RSI quantitative risk assessment framework. In addition to the source water parameters, the effect of the rainfall was also incorporated in the annual risk calculation. Monte Carlo simulations were used to calculate the annual risk of the infection of Cryptosporidium via drinking water samples, which were collected from the Sagami River and its two tributaries Koayu and Nakatsu Rivers in Kanagawa prefecture in the Kanto area of Japan. Samples were brought to the laboratory and were filtered through cellulose acetate membrane disk filters. In Sagami River, Cryptosporidium oocysts were found in 41 of the 56 samples from six sites and the geometric mean concentration was found to be 9 oocysts per 100L. In Nakatsu River, Cryptosporidium oocysts were detected in 14 of the 29 samples from three sites and the geometric mean was 9 oocysts per 100L. In Koayu River, 21 samples from two sites were examined and Cryptosporidium oocysts were detected in all the samples and the geometric mean of the samples were 320 oocysts per 100L. The chapter concludes that there was a continuous presence of Cryptosporidium in the water of Sagami River. The geometric mean concentration of oocysts in the 76 positive samples was 24 oocysts per 100L. The maximum concentration was found to be as high as 11,000 oocysts per 100L.

MICROBIAL POLLUTION OF SURFACE WATERS

ABSTRACT In order to evaluate the microbial quality of surface waters, the microbial flora of water samples collected from rivers, lakes and seasides was examined. Vibrio cholerae non 0–1, Salmonella, Staphylococcus aureus and Welchii ( Clostridium perfringens ) were selected as pathogenic microorganisms. The distribution pattern of microorganisms was much different among samples, but waters collected from the lower reaches of rivers had a similar one, indicating relatively high density levels of fecal coliforms and fecal streptococci. Water samples more than 50 % contained the pathogenic microorganisms except Welchii . A very few indicies of microorganisms had relatively high mutual relationships with only Welchii . However, it is not so difficult to find the density levels of indicator microorganisms below which pathogenic microorganisms were seldom detected. These density levels can be standard levels to ensure sanitary safety of surface waters and were around 101-102/100 ml for many indicator microorganisms, 0/100 ml for fecal coliforms by the EC method, 10/100 ml for Welchii and 103/100 ml for spore-forming bacteria.