Physical and chemical effects on viability of the Myxobolus cerebralis triactinomyxon

Abstract

Various chemical and physical methods for destroying the triactinomyxon (TAM) stage of the myxozoan parasite Myxobolus cerebralis were tested. The fluorescent stains propidium iodide and fluorescein diacetate were used as indicators of viability. Physical variables tested included freezing, drying, high temperature, sonication, and pressure of 6.2 × 10 7 Pa (9000 psi). Chemicals evaluated included chlorine bleach, povidone-iodine, and hydrogen peroxide. Freezing or drying for 1 h was effective in killing TAMs, but pressure was not. Temperatures above 75°C for at least 5 min were also effective. Sonication with a laboratory instrument cleaner for 10 to 13 min killed and ruptured TAMs, resulting in <1.9% recovery. However, among the surviving TAMs, 39 to 58% were still viable. Chlorine concentrations of 130 ppm for 10 min were also effective at temperatures ranging from ice-water to room temperature and total hardness ranging from 10 to 500 mg l -1. Lethal concentrations of hydrogen peroxide and povidone-iodine (10% solution) were quite high: 10% for 10 min, and 50% (5000 ppm active iodine) for 60 min, respectively. The stain results indicating TAM death were verified in 2 tests in which rainbow trout Oncorhynchus mykiss were exposed to TAMs that had been either frozen for 1 h or treated with 66 ppm chlorine as sodium hypochlorite for 1 min. None of the fish exposed to the treated TAMs became infected. These results should provide disinfection guidelines to prevent transfer of M. cerebralis TAMs to uninfected areas and provide information on the risks of parasite transfer under various treatment scenarios. KEY WORDS: Myxobolus · Whirling disease · Disinfection · Chlorine · UltrasoundResale or republication not permitted without written consent of the publisher

Differences in viability and reactivity of actinospores of three myxozoan species upon ageing

“…The FDA/PI double-staining method originally was developed by Mátyus et al (1983) for sperm cell viability assessment. Wagner et al (2003) managed to verify that PI-stained sporoplasms of M. cerebralis are indeed not infectious to susceptible fish any more. By this method, we observed an increase in polar filament discharge due to the pH shift (or the trace acetone from FDA dissolvent), rendering polar capsule cells non-viable due to propidium iodide influx.…”

Expression of immune-regulatory genes, arginase-2 and inducible nitric oxide synthase (iNOS), in two rainbow trout (Oncorhynchus mykiss) strains following exposure to Myxobolus cerebralis

“…For treatment, 100 µl washed TAMs were placed on one to three microscope slides. Equal amounts (50 µl) of FDA and PI were added to each slide, and a cover-slip was placed on the mixture (Wagner et al 2003). For optimal mixing, an attempt was made to dispense the staining solutions across the TAM solution.…”

Transmission Biology of the Myxozoa

“…For M. cerebralis, drying at room temperature for 15 min, freezing at -20ºC for 1 hour, temperatures above 75 ºC for 5 min and sonication (47 kHz, 130 W) for 10-13 min were effective in killing actinospores, but pressure of 6.2 x 10 7 Pa (9000 psi) was not (Wagner et al, 2003). To inactivate actinospores of M. cerebralis chemically, chlorine of 13 ppm for 10 min, hydrogen peroxide of 10% for 10 min, and povidone-iodine of 50% solution (5000 ppm active iodine) for 60 min were effective (Wagner et al, 2003). Electricity with a pulse length of 99 μsec at 3 kV induced polar filament discharge of M. cerebralis actinospores, suggesting a potential use of direct current as a means of disinfection (Wagner et al, 2002).…”

“…Sand filtration is highly effective in removing actinospores of M. cerebralis (Arndt & Wagner, 2003, Nehring et al, 2003. However, it may be applied only for large-type actinospores.…”

“…To determine the viability of actinospores, presence or absence of the sporoplasms in the spore body has been used as an indicator , Xiao & Desser, 2000b, because aged actinospores spontaneously release sporoplasms so that spores become empty. Alternatively, a vital staining technique with fluorescein diacetate (FDA) and propidium iodide (PI) can be applied (Markiw, 1992, Yokoyama et al, 1997b, Wagner et al, 2003, Kallert et al, 2005.…”