In this study, to evaluate whether Clostridium perfringens could be a useful fecal indicator in aquatic environments and could be employed as a potential source-tracking indicator, the distribution of C. perfringens spores and their toxin types in sewage and livestock fecal samples were analyzed. A total of 804 C. perfringens spore isolates (366 from human-related sewage and effluents, 128 from cattle, 129 from pigs, 72 from chicken, and 109 from abattoir wastewaters) were analyzed using multiplex polymerase chain reaction (PCR) to detect six C. perfringens toxin genes. On the basis of the presence of toxin genes, most of the isolates from both human sewage and livestock samples were determined as C. perfringens type A and they expressed cpa alone or cpa and C. perfringens enterotoxin (cpe) with or without cpb2. Moreover, cpe-positive C. perfringens was detected with frequencies of 29% and 32% in human sewage and effluents, respectively. However, only one isolate (from cattle feces) was cpe-positive among all the livestock samples tested. Thus, the distribution of cpe-positive C. perfringens should be considered an important source tracking indicator for human fecal pollution. Furthermore, we conclude that sewage effluents are a significant source of cpe-positive C. perfringens pollution.

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Kudoa septempunctata is the causative agent of a food-borne disease associated with the ingestion of raw olive flounder. As the current qRT-PCR method for its detection is timeconsuming, a rapid and simple method is required. Recently, a new real-time loop-mediated isothermal amplification LAMP method and an immunochromatography method, whose sensitivities are intended to be compatible with that designated in the official analytical method 10 5 spores/g olive flounder , have been developed. To validate these new methods, we performed an inter-laboratory study across seven laboratories. Both methods could not detect less than 10 4 spores/g; however, these methods were able to detect more than 10 5 spores/g in olive flounder samples. These results demonstrated that the sensitivities of these methods were compatible with the designated level in the official analytical method. We concluded that these new methods were acceptable as the screening methods for K. septempunctata.

Yeast host-vector systems have been very successful in expressing recombinant proteins. However, because there are some proteins that cannot be expressed with existing systems, there is a need for new yeast expression systems. Here we describe a new host-vector system based on the basidiomycetous yeast Cryptococcus sp. strain S-2 (S-2). Two advantages of S-2 are that it naturally produces some very useful enzymes, so it would be a good system for expressing multiple copies of some of its genes, and that, it is a nonhazardous species. The orotate phosphoribosyltransferase (OPRTase, EC 2.4.2.10) gene (URA5) was selected as a selectable marker for transformation in the new host-vector system. URA5 was isolated and introduced into a uracil auxotroph of S-2 by electroporation. To demonstrate the S-2 system, we selected one of its unique enzymes, a plastic-degrading cutinase-like enzyme (CLE). We were able to insert multiple copies of the CLE gene (CLE1) into the chromosomes in a high fraction of the targeted cells. Under optimal conditions, one transformant exhibited 3.5 times higher CLE activity than the wild type. Expression vectors, including an inducible promoter (the promoter for the xylanase or α-amylase gene), were constructed for recombinant protein production, and green fluorescent protein was expressed under the control of these promoters. The xylanase promoter was more tightly controlled. Furthermore, putting CLE1 under the control of the xylanase promoter, which is induced by xylose, increased CLE activity of the culture medium to approximately 15 times greater than that of the wild type.

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