Evaluation of the inflammatory response to Kudoa septempunctata genotype ST3 isolated from olive flounder (Paralichthys olivaceus) in Caco-2 cells

Abstract

Kudoa septempunctata (Myxosporea, Multivalvulida) is a parasite of the trunk muscle of cultured olive flounder (Paralichthys olivaceus). We investigated whether K. septempunctata genotype ST3 spores induce cell damage and the secretion of inflammatory mediators in Caco-2 cells, which exhibit characteristics similar to human intestinal epithelial cells. Purified K. septempunctata spores were heated at 95 °C for 5 min. Lactate dehydrogenase (LDH) release was measured to determine the efficacy of denaturation. Naïve and heated spores, lipopolysaccharide (positive control) and vehicle (negative control) were added to Caco-2 cells. Cells were subjected to the cytotoxic LDH assay and western blot analysis to examine the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2. Supernatants were collected to measure nitric oxide (NO) and prostaglandin E2 (PGE2). Most spores were denaturated by heating, and the spore morphology was found to be wrinkled with shell valves and polar capsules. In addition, cytotoxicity and inflammatory mediators, such as NO, PGE2, iNOS, and COX-2, remained unchanged in Caco-2 cells following exposure to naïve and heated spores compared with the positive controls. Collectively, the findings of this study imply that spores of K. septempunctata genotype ST3 do not cause inflammation in Caco-2 cells.

Immune-triggering effect of the foodborne parasite Kudoa septempunctata through the C-type lectin Mincle in HT29 cells

“…The inflammatory responses observed in this study firmly indicate the possibility that Kudoa infection or KsAg treatment can induce inflammatory reactions in HT29 cells. In contrast, previous studies using other cell lines such as Caco-2 human intestinal and RAW264.7 macrophage-like cells did not clearly indicate the cytotoxicity of Kudoa (8,9). Meanwhile, other effort to determine the pathogenicity of K. septempunctata infection focused on the loss of cell monolayer integrity in intestinal cells after infection (7).…”

“…Several studies have debated the clinical significance of K. septempunctata and pathogenicity in in vivo test using mouse model (2)(3)(4)(5)(6). The pathogenicity of K. septempunctata was also controversial in in vitro assays using Kudoa spores (7)(8)(9). Ohnishi et al insisted that the invasion of Caco-2 human intestinal cells by Kudoa sporoplasm induced damage through the loss of cell monolayer integrity via the elimination of transepithelial electrical resistance, suggesting their role as the causative agent of food poisoning in humans (7).…”

“…Several studies have debated the clinical significance of K. septempunctata and pathogenicity in in vivo test using mouse model ( 2 - 6 ). The pathogenicity of K. septempunctata was also controversial in in vitro assays using Kudoa spores ( 7 - 9 ). Ohnishi et al .…”

“…insisted that the invasion of Caco-2 human intestinal cells by Kudoa sporoplasm induced damage through the loss of cell monolayer integrity via the elimination of transepithelial electrical resistance, suggesting their role as the causative agent of food poisoning in humans ( 7 ). Conversely, another study reported that infection of Caco-2 cells by K. septempunctata spores did not result in inflammation ( 8 ), and another report found that K. septempunctata activated TLR-2-expressing HEK293 cells and induced TNF-α production ( 9 ). As mentioned previously, the no definite conclusion can be drawn regarding the pathogenicity of K. septempunctata or the mechanism of food poisoning.…”

Kudoa ogawai (Myxosporea: Kudoidae) Infection in Cultured Olive Flounder Paralichthys olivaceus

“…Ahn et al [34] came to similar results in study using adult mice. In addition, they exhibited that K. septempunctata spores did not cause inflammation on Caco-2 cells [35]. It is therefore up for discussion whether K. septempunctata causes food poisoning or not, however a few cultured olive flounder are undeniably infected by the parasite.…”