The efficacy of Virkon-S for the control of saprolegniasis in common carp, Cyprinus carpio L

Saprolegnia parasitica cultures

S. parasitica was purchased from the Korean Collection for Type Cultures (KCTC 46452) and cultured on potato dextrose agar (PDA) at 25 °C. This strain was isolated from a farmed rainbow trout from an aquaculture farm in Wonju, Gangwondo, Korea in 2016 before deposition.

Fish and rearing conditions

In total, 100 fingerlings of common carp, Cyprinus carpio, with an average size and weight of 11.5 ± 1 cm and 17.6 ± 3 g, respectively, were obtained from Namsangju Aquaculture Farm, located in Sangju-si, Gyeongsangbuk-do, Korea. Each fish was examined for infection and acclimated in 450-L rearing tanks at 22 ± 2 °C for 10 days. During the acclimatization period, fish were fed twice daily with a proper diet according to Ellsaesser & Clem (1986).

Virkon-S

Virkon-S was purchased from Bayer Korea (Seoul, Korea) and 1 kg contained triple salt 500 g, hexametaphosphate 181 g, sodium dodecyl benzene sulphonic acid 150 g, malic acid 100 g, sulphamic acid 50 g, and sodium chloride 15 g. A 10% stock was prepared with distilled water and further dilutions were made when necessary.

Inhibition of spore germination

The inhibitory effects of Virkon-S on spore germination were tested using two methods. First, the minimum fungicidal concentration (MFC), defined as the lowest concentration of a chemical that prevents germination or the visible growth of spores, was determined as described previously (Yao et al., 2017; Hu et al., 2013). Spore suspension containing oospores and zoospores was prepared as described as Yao et al. (2017). S. parasitica was cultured on PDA at 25 °C for 12 days to induce spores. Approximately 10 ml of distilled water was added to each 87-mm diameter Petri dish and the mycelium and spores were scraped and filtered through eight layers of sterile cheesecloth. The final concentration of spores was adjusted to approximately 1 × 10⁶ spores/ml using a hemocytometer. A 10 µl sample of spore suspension was spotted on the center of a 87-mm diameter Petri dish containing 20 ml of PDA with 0, 2, 4, 10, 20, or 100 ppm Virkon-S, and incubated at 25 °C. After 72 h of incubation, the diameter of mycelial growth was measured. Inhibition of spore germination was also determined by percent spore germination as described by Király et al. (1974). PDA plates containing 0, 2, 4, 10, 20, and 100 ppm Virkon-S were prepared but the amount of total PDA was only 10 ml for each plate. This condition reduced mycelial growth and the plate was transparent enough to observe spore germination under a light microscope. Three spots of a 10-µl spore suspension were placed on each plate and incubated at 25 °C for 72 h. The percent spore germination was determined as follows: ${\displaystyle \text{Percent spore germination}=\frac{\text{No. of spores germinated}}{\text{Total no. of spores examined}}\times 100.}$

Mycelial growth inhibition on PDA plates

Inhibition of S. parasitica mycelial growth was tested on PDA plates containing different concentrations of Virkon-S as described by Hu et al. (2013). Briefly, 2 × PDA was sterilized, cooled to approximately 65 °C, and mixed with the same volume of Virkon-S to give final Virkon-S concentrations of 2, 4, 10, 20, and 100 ppm. 2 × PDA was mixed with the same volume of sterile water in control plates. Aliquots of the mixture (10 ml) were poured onto 87-mm diameter Petri dishes. A Saprolegnia-colonized PDA block of approximately 5 × 5 mm was placed on the center of the prepared plates. The plates were incubated at 25 °C for 72 h and the diameter of the mycelial growth was measured. The percentage of fungal inhibition was calculated based on the percent inhibition of radial growth (PIRG) as described by Dananjaya et al. (2017) as follows: PIRG (%) = [(R1 − R2)]/R1 × 100%, where R1 = radial growth in control and R2 = radial growth in treatment.

Cytotoxicity of Virkon-S in epithelioma papulosum cyprini (EPC) cells

The cytotoxic effect of Virkon-S was evaluated using the Ez-Cytox Cell Viability Assay Kit (Dogen-Bio Co., Ltd., Seoul, Korea) with EPC cells by following the procedures described by Park et al. (2017). EPC cells (1 × 10⁵) were grown in L-15 medium supplemented with 10% fetal bovine serum, penicillin (62.5 µg/ml), and streptomycin (100 µg/ml) in 96-well plates overnight. For treatment, the cell medium was replaced with medium (100 µl) containing 10, 100, 500, 1,000, 5,000, and 10,000 ppm of Virkon-S. Non-treated cells were used as a negative control. After 24 h of incubation, 110 µl of medium containing 10 µl of water-soluble tetrazolium solution was added to each well, and the plates were incubated for a further 4 h. The absorbance at 460 nm was measured using an enzyme-linked immunosorbent assay reader (Molecular Devices, Silicon Valley, CA, USA), and relative cell viability was calculated using cells treated with medium only as a control.

Acute toxicity in common carp

Water-only toxicity tests were carried out with cultured fingerlings of the common carp, Cyprinus carpio, using five concentrations of Virkon-S (2, 4, 10, 20, and 100 ppm) and three fish per concentration. Healthy and disease-free fish that were not previously exposed to any pollution agents or toxicants were selected based on their activity and external appearance. The fish were acclimated for 7 days at 18 °C under constant light and feeding with commercial feed. Three fish were placed in a 3-L glass flask containing different concentrations of Virkon-S and kept for 96 h in aerated water without any water changes or feeding. Dead fish were removed from the flask to prevent possible deterioration of the water quality. The percentage of fish mortality was calculated for each concentration after 24, 48, 72, and 96 h of exposure. After 96 h of exposure, all of the survivors were transferred to a 50-L tank equipped with aeration and observed for 40 days for any post-exposure effects.

Inhibition of saprolegniasis by Virkon-S

Five groups of 20 fish were used for artificial infection with S. parasitica and treatment with Virkon-S. Three groups were artificially infected and treated with 2, 4, and 10 ppm Virkon-S. Fish in the positive control group were infected with S. parasitica but not treated with Virkon-S. Fish in the negative control group were neither infected with S. parasitica nor treated with Virkon-S. Fish were kept in a 150-L glass fiber tank separated into three sections with a metal screen with six or seven fish in each section. The water temperature was fixed at 17 ± 1 °C using a room air conditioner. All fish were observed for behavioral and clinical signs of infection.

For artificial infection with S. parasitica, fish were descaled and wounded in three places on one side of the body (top of the head, center of the body, and the tail region) using a sharp scalpel. The wounded area was approximately 1 cm². Spores of S. parasitica were prepared as above and added to tanks containing the three treatment groups and the positive control group at a final concentration of 1 × 10⁵ spores/ml (Willoughby, 1994; Hatai & Hoshiai, 1994).

Ten days after the addition of spores, white cotton wool-like growth on the surface of the wounded area was observed with a light microscope and cultured on PDA at 25 °C for 3 days. The morphological and microscopic characteristics of the culture were compared with the characteristics of S. parasitica recorded from previous studies (Hatai, Willoughby & Beakes, 1990; Willoughby, Pickering & Johnson, 1984). Virkon-S solution was added to these treatment tanks at final concentrations of 2, 4, and 10 ppm to assess the treatment effect. The water in the tank was replaced with the same concentration of Virkon-S after 5 days and the cumulative mortality was recorded for 10 days. The fish were diagnosed by clinical signs and lesion healing 10 days after treatment.

Inhibition of spore germination

The inhibition of S. parasitica spore germination by Virkon-S was tested by observing the mycelial growth from a suspension of spores on PDA plates of normal thickness (20 ml media in 87-mm plates) and enumerating spore germination on PDA plates of half thickness. There was no sign of mycelial growth on plates containing 4, 10, 20, and 100 ppm Virkon-S 72 h after incubation at 25 °C (Figs. 1A–1D). Mycelial growth on a plate containing 2 ppm was 53.8% of the control plate (35 vs. 65 mm, respectively) (Fig. 1E). Therefore, MFC was defined as less than or equal to 4 ppm on PDA plates.

There was no sign of spore germination on plates of half thickness containing 4, 10, 20, or 100 ppm Virkon-S 72 h after incubation at 25 °C. A small number of spores on PDA plates containing 2 ppm germinated at a rate of 39.2% (22 out of 56). In contrast, most of the spores on control plates germinated at a rate of 96.2% (51 out of 53 spores). Therefore, the inhibition rate of spore germination was calculated as 59% at a concentration of 2 ppm [(96.2–39.2)/96. 2 × 100].

Mycelia growth inhibition

The growth of S. parasitica on PDA containing 2, 4, 10, 20, and 100 ppm Virkon-S was observed after inoculation at 25 ° C for 72 h. No growth of mycelia was observed in the plates containing 10, 20, and 100 ppm Virkon-S (Figs. 2A–2C). There was slight growth of S. parasitica on PDA plates containing 4 ppm Virkon-S (Fig. 2D). The growth inhibition rate (IR) on plates containing 4 and 2 ppm was 69.2 and 25.6%, respectively when the IR was calculated as [%IR = 100 − 100X/Y], where X = mycelia growth in sample; Y = mycelia growth in control.

Cytotoxicity of Virkon-S in cultured EPC cells

The cytotoxic effect of Virkon-S in EPC cells is shown in Fig. 3. There was no effect of Virkon-S on cell viability at 10 and 100 ppm. There was a slight decrease (97%) at 500 ppm. However, the cell viability dropped suddenly when the Virkon-S concentration was greater than 1,000 ppm. In the mycelial growth inhibition and spore germination inhibition assays, 4 ppm was enough to inhibit spore germination and the resulting mycelial growth. Even a concentration of 100 ppm did not result in any toxicity in EPC cells. Therefore, 10 ppm and lower concentrations were used in further experiments.

Acute toxicity test of Virkon-S in common carp

Acute toxicity of Virkon-S in common carp was investigated by placing fish in glass flasks containing five different concentrations of Virkon-S for up to 96 h. As shown in Table 1, all fish in the 100-ppm flask died within 24 h. Moreover, 75 and 100% cumulative mortality was observed within 24 and 46 h, respectively, in the 20-ppm flask. However, no mortality was observed in the flasks containing 10, 4, and 2 ppm Virkon-S. Furthermore, the survivors from the acute toxicity test did not show any after-exposure effects when they were kept in a culture tank for 40 days. Therefore, it was concluded that 10 ppm, which inhibited spore germination and mycelial growth, did not cause any cytotoxic effects in EPC cells, and therefore can be defined as the maximum acceptable toxicant concentration (MATC) for Virkon-S.

Induction of artificial infection and treatment with Virkon-S

Seven days after the addition of S. parasitica spores to the tanks containing artificially wounded common carp, the typical signs of saprolegniasis, including cotton shape growth of fungi and wound ulceration, appeared (Fig. 4A). The cotton wool-like mycelial growth was removed from the wound and observed with a microscope to confirm its resemblance to the inoculated S. parasitica, and placed on a PDA plate to induce mycelial growth. Both the cotton wool-like mycelial growth from the infected fish and mycelia grown on the PDA plate showed the same morphological characteristics of S. parasitica, which indicated that the disease was induced by the spores of S. parasitica that had been added to the tanks. The diseased fish were treated with Virkon-S at a final concentration of 2, 4, and 10 ppm and the results are shown in Table 2. During the 10-day treatment period, no mortality was observed among fish treated with 4 and 10 ppm. However, the cumulative mortality in the 2-ppm tank reached 50%. Furthermore, fish treated with 4 and 10 ppm showed clear recovery from the disease. As shown in Fig. 4B, all of the cotton shape fungal growth disappeared from the body of the fish. Furthermore, new scales appeared and covered the wounded area, which indicated complete recovery from the disease.