In vitroeffect of two commercial anti-coccidial drugs against myxospores ofKudoa septempunctatagenotype ST3 (Myxozoa, Multivalvulida)

Abstract

Kudoa septempunctata (Myxozoa: Multivalvulida) myxospores infect the trunk muscles of olive flounder (Paralichthys olivaceus). In this study, two popular commercially formulated anti-coccidial drugs (amprolium hydrochloride and toltrazuril) were serially diluted and incubated with purified mature Kudoa septempunctata myxospores. The viability of K. septempunctata spores was determined after a 2-day incubation followed by Hoechst 33342 and propidium iodide staining, and scanning electron microscopy. Amprolium hydrochloride significantly decreased spore viability (18% of control) at a concentration of 920 μg/mL, whereas toltrazuril showed almost no effect (83% of control). Viability of the control (untreated spores) was 90%. In vivo studies are required to confirm the efficacy of amprolium hydrochloride in fish infected with K. septempunctata myxospores on their growth and immune system performance.

Effect of Epigallocatechin Gallate on Viability of Kudoa septempunctata

“…It was reported several chemicals affecting the viability of K. septempunctata using approximately 100 spores with two kinds of fluorescent stains to identify the dead/live spores [11,12]. In our study, the viability of 5,000 spores was determined using one fluorescent stain.…”

“…In our study, the viability of 5,000 spores was determined using one fluorescent stain. Ahn et al [11] reported that AH had an anti-Kudoa effect at higher concentrations ≥ 1 mM and at longer treatment time (48 hr). The candidate chemicals should have anti-Kudoa effect at lower concentrations and in a shorter time since it will be used in fish farms.…”

“…Based on the differences in fluorescent stains and morphological changes of the myxozoa, several studies have been conducted to find chemotherapeutic agents that affect the viability of K. septempunctata [10][11][12]. However, the quantification and determination of accurate effective doses of chemicals based on microscopic observation can lead to inconsistency and misinterpretations, particularly due to subjective visual criteria.…”

“…The spores were purified by a modified Percoll (Sigma-Aldrich, St. Louis, Missouri, USA) density-gradient centrifugation method, as previously described [ 15 ]. The K. septempunctata spores were exposed to three kinds of chemicals including amprolium hydrochloride (AH; Dr. Ehrenstorfer, Augsburg, Germany), chlorogenic acid (CA; Carl Roth, Karlsruhe, Germany), and epigallocatechin gallate (EGCG; Sigma-Aldrich), which have been reported to be effective against the myxospores [ 11 , 12 ]. Fifty microliters of chemicals (1 and 10 mM) were added to 450 μl of phosphate-buffered saline (PBS) containing 3.2×10 5 spores, and the mixtures were incubated at room temperature (approximately 20–25°C) for 30 min.…”