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Kudoa septempunctata is a myxosporean parasite of Paralichthys olivaceus (olive flounder) and causes a foodborne illness that affects more than 100 cases in Japan each year. We previously reported that the consumption of raw olive flounder meat containing a high concentration of K. septempunctata spores induces transient but severe diarrhea and emesis through an unknown mechanism. Here, we demonstrate that K. septempunctata sporoplasm plays an important role in mediating the toxicity of K. septempunctata. When K. septempunctata spores were inoculated in Caco-2 human intestinal cells, K. septempunctata sporoplasms were released from spores, and they invaded the cells. Electron microscopic observations revealed that the sporoplasm invasion severely damaged the Caco-2 cells. The inoculation of K. septempunctata spores eliminated the transepithelial electrical resistance (TER) across the cell monolayer. Inhibiting the invasion of the sporoplasms prevented the observed loss in cell layer integrity, as illustrated by the rapid elimination of the TER. These results suggest that the invasion by sporoplasms severely damaged individual intestinal cells, resulting in a loss of cell monolayer integrity.

Trichothecene mycotoxins such as nivalenol and deoxynivalenol frequently contaminate foodstuffs. Recently, several trichothecene glucosides have been found in trichothecene-contaminated foods, and information about their chemistry, toxicity, and occurrence is required. In this study, a glucoside of nivalenol was isolated from nivalenol-contaminated wheat and was identified as nivalenol-3-O-β-D-glucopyranoside. Analytical methods using a multifunctional column or an immunoaffinity column have been developed for the simultaneous determination of nivalenol, nivalenol-3-O-β-D-glucopyranoside, deoxynivalenol, and deoxynivalenol-3-O-β-D-glucopyranoside in wheat. The methods were validated in a single laboratory, and recovery from wheat samples spiked at four levels ranged between 86.4 and 103.5% for the immunoaffinity column cleanup. These mycotoxins in contaminated wheat samples were quantitated by the validated method. Nivalenol-3-O-β-D-glucopyranoside was detected in the nivalenol-contaminated wheat, and the percentage of nivalenol-3-O-β-D-glucopyranoside to nivalenol ranged from 12 to 27%. This result indicates that the analytical method developed in this study is useful for obtaining data concerning the state and level of food contamination by nivalenol, deoxynivalenol, and their glucosides.

Mycotoxins are commonly present in cereal grains and are not completely destroyed during their cooking and processing. When mycotoxins contaminate staple foods, the risk for exposure becomes serious. In East Asia, including Japan, rice is consumed as a staple food, and with the increasingly Westernized lifestyle, the consumption of wheat has increased. The mycotoxins commonly associated with rice and wheat are total aflatoxin (AFL) and ochratoxin A (OTA), respectively. This study examined the retention of AFL and OTA during the cooking of rice and pasta. AFL was retained at 83%–89% the initial level after the cooking of steamed rice. In pasta noodles, more than 60% of the OTA was retained. These results show that AFL and OTA are relatively stable during the cooking process, suggesting that a major reduction in the exposure to these mycotoxins cannot be expected to occur by cooking rice and pasta. The estimated exposure assessment at the high consumer level (95th percentile) and the mycotoxin contamination level determined by taking into account these reductions in the present study should be useful for the establishment of practical regulations for mycotoxins in staple foods.

SUMMARY: Kudoa septempunctata, a myxosporean parasite, was recently identified as the causative agent of food poisoning resulting from the consumption of raw olive flounder (Paralichthys olivaceus). A single blind inter-laboratory study, involving 5 laboratories, was conducted to validate a quantitative real-time PCR assay for the detection of the parasite. We obtained relatively constant values for log rDNA copies/g from these laboratory analyses (SD ＝ 0.35-0.86), suggesting the validity of the realtime PCR method for the detection of K. septempunctata in P. olivaceus.

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SUMMARY: Kudoa septempunctata is a myxosporean parasite of Paralichthys olivaceus (olive flounder) that causes more than 50 cases of foodborne illness in Japan each year. For quantitatively assessing the presence of K. septempunctata spores in the causative fish at food poisoning outbreaks, both a direct observation method using microscopy and a quantitative real-time PCR (qRT-PCR) method are officially accepted in Japan. However, lower correlations have been often noticed between the number of spores counted using the direct observation method and the DNA amount determined using the qRT-PCR method. To elucidate the cause of this discrepancy, we observed muscle tissues of infected olive flounders with K. septempunctata by transmission electron microscopy. The images demonstrated unsynchronized development of K. septempunctata spores in plasmodia found within myofibers; in other words, the plasmodium contained not only developed spores with completed shell valves but also developing spores (sporoblasts) composed of spore-forming cells without shell valves. Furthermore, the ratio between developed spores and sporoblasts varied at different parts of muscles. The direct microscopic observation method could count developed spores, whereas the qRT-PCR method could quantify the amount of not only spores but also sporoblastic cells regardless of the cellular development and differentiation. Considering that the food toxicity caused by K. septempunctata is induced by viable spores passing through the gastric environment, the direct observation method counting only developed spores is better than the qRT-PCR method for assessing the cause of foodborne illness at the outbreak as well as the risk of human illness in monitoring surveys of aquacultured or natural-water fish.

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