Evaluation of the tetrodotoxin uptake ability of pufferfish Takifugu rubripes tissues according to age using an in vitro tissue slice incubation method

The tetrodotoxin (TTX) uptake ability of pufferfish Takifugu rubripes tissues and its growth-associated changes were investigated using an in vitro tissue slice incubation method. Tissue slices prepared from the liver, skin, and intestine of a non-toxic cultured adult T. rubripes (20 months old) and incubated with incubation buffer containing 25 μg/mL TTX for 1?48 h showed a time-dependent increase in the TTX content in all tissues. The TTX contents of the skin and intestine slices were comparable to or slightly higher than that of the liver slices, with a similar transition pattern, suggesting similar TTX uptake ability among the skin, intestine, and liver. The TTX uptake ability of the liver and intestine did not differ significantly between young (8 months old) and adult (20 months old) fish, but the skin slices of young fish took up approximately twice as much TTX as that of adult fish, suggesting that the TTX uptake ability of the skin is involved in the growth-dependent changes in the toxin distribution inside the body in T. rubripes. To estimate the TTX uptake pathway in each tissue, an immunohistochemical technique was used to observe temporal changes in the intra-tissue microdistribution of TTX during incubation. The findings suggested that TTX is transferred and accumulates from pancreatic exocrine cells to hepatic parenchymal cells in the liver, from connective tissues to basal cells in the skin, and from villi epithelial cells via the lamina propria to the muscle layer in the intestine

Development and Evaluation of a Rapid, Simple, and Sensitive Immunochromatographic Assay To Detect Thermostable Direct Hemolysin Produced by Vibrio parahaemolyticus in Enrichment Cultures of Stool Specimens

Thermostable direct hemolysin (TDH) is considered to be a major virulence factor in Vibrio parahaemolyticus, and most cases of V. parahaemolyticus diarrhea in humans are caused by tdh gene-positive strains. In the present study, we developed an immunochromatographic assay to detect TDH (TDH-ICA) and evaluated the utility of TDH-ICA for the diagnosis of V. parahaemolyticus diarrhea. TDH-ICA allowed the detection of 0.2 ng/ml of TDH within 10 min. Fecal homogenates were spiked with various numbers of tdh-positive V. parahaemolyticus organisms, and their enrichment cultures were tested with TDH-ICA. The results of detection of TDH in the enrichment cultures by TDH-ICA were in accord with the results of recovery of the spiked V. parahaemolyticus organisms from the enrichment cultures by plating onto thiosulfate-citrate-bile salts-sucrose agar. When enrichment cultures of 217 stool specimens from patients with diarrhea were tested with TDH-ICA, the TDH-ICA results showed 100% sensitivity and specificity compared to the results of isolation of V. parahaemolyticus from the stool specimens by a conventional bacterial culture test. Since TDH-ICA was able to detect TDH in a fecal enrichment culture within 10 min, TDH-ICA testing of a fecal enrichment culture could be completed rapidly and easily within approximately 16 h, including incubation time for the fecal enrichment culture. These results indicate that TDH-ICA is a rapid, simple, and sensitive TDH detection method and that TDH-ICA testing of a fecal enrichment culture is useful as an adjunct to facilitate the early diagnosis of V. parahaemolyticus diarrhea

Studies in the use of magnetic microspheres for immunoaffinity extraction of paralytic shellfish poisoning toxins from shellfish

toxin