A fluorescence‐based assay for in vitro screening of Saprolegnia inhibitors

The incidence of fish pathogenic oomycetes, Saprolegnia, has increased significantly in aquaculture since the ban of malachite green. For the efficient characterization of anti‐Saprolegnia therapeutics, simple accurate methods are required. However, the current screening methods are limited by time, and none of them are confirming the viability of treated spores or hyphae. In this study, a modified fluorescence‐based assay for the in vitro screening of Saprolegnia inhibitors has been developed. This method involves the use of FUN‐1 viability dye combined with calcofluor white M2R, and is based on the formation of orange‐red cylindrical intravacuolar structures (CIVS) in metabolically active spores, hyphae and biofilms. Heat‐killed and bronopol‐treated Saprolegnia spores, hyphae and biofilms exhibited diffuse bright green fluorescence which confirms complete loss of viability. For boric acid‐treated spores, no germination was observed. However, tiny CIVS were observed in 50% of treated spores which indicated reduction in their viability. Our results proved that FUN‐1 dye is an efficient tool to distinguish between live and dead Saprolegnia spores, hyphae and biofilms and to monitor the change in Saprolegnia viability during qualitative evaluation of potential anti‐Saprolegnia compounds.     

Saprolegnia diclina IIIA and S. parasitica employ different infection strategies when colonizing eggs of Atlantic salmon,Salmo salar L.

Here, we address the morphological changes of eyed eggs of Atlantic salmon, Salmo salar L. infected with Saprolegnia from a commercial hatchery and after experimental infection. Eyed eggs infected with Saprolegnia spp. from 10 Atlantic salmon females were obtained. Egg pathology was investigated by light and scanning electron microscopy. Eggs from six of ten females were infected with S. parasitica, and two females had infections with S. diclina clade IIIA; two Saprolegnia isolates remained unidentified. Light microscopy showed S. diclina infection resulted in the chorion in some areas being completely destroyed, whereas eggs infected with S. parasitica had an apparently intact chorion with hyphae growing within or beneath the chorion. The same contrasting pathology was found in experimentally infected eggs. Scanning electron microscopy revealed that S. parasitica grew on the egg surface and hyphae were found penetrating the chorion of the egg, and re‐emerging on the surface away from the infection site. The two Saprolegnia species employ different infection strategies when colonizing salmon eggs. Saprolegnia diclina infection results in chorion destruction, while S. parasitica penetrates intact chorion. We discuss the possibility these infection mechanisms representing a necrotrophic (S. diclina) vs. a facultative biotrophic strategy (S. parasitica).     

Chemical composition and in vitro antifungal activity of Sambucus ebulus and Actinidia deliciosa on the fish pathogenic fungus,Saprolegnia parasitica

The aim of the present study was to determine the chemical composition of Sambucus ebulus and Actinidia deliciosa ethanolic extracts as well as their in vitro antifungal activity on the mycelial growth of the water mold, Saprolegnia parasitica. The preliminary minimum inhibition concentration (MIC) and minimum lethal concentration (MLC) were determined by the HeMP method and finally, concentrations of each extract ranging from 1 to 10% were prepared by an agar dilution method to assess the in vitro antifungal activity, quantitatively. Both herbal extracts inhibited growth of Saprolegnia hyphae in vitro. Complete in vitro growth inhibition was found at a concentration of ≥5% for S. ebulus, whereas it was not observed even at 10% concentration of A. deliciosa. Based on gas chromatography coupled with mass spectrometry (GC/MS) analysis, the main constituents of S. ebulus include monophthalate (66.17%), fatty acids (26.47%), phytol (4.25%), and acetic acid (2.11%). Using colorimetric assays, A. deliciosa contained phenolic contents at 162 mg gallic acid (GAE)/g DW and flavonoid contents at 2.31 mg quercetin (QE)/g DW. In conclusion, the results of this study indicated that S. ebulus and A. deliciosa showed some antifungal activities against S. parasitica with formerly exhibiting stronger activity (p < 0.05).

     

Saprolegnia molecular phylogeny among farmed teleosts in Nova Scotia,Canada

To identify the pathogens causing saprolegniosis among farmed fish in Nova Scotia, 172 infected tissues and 23 water samples were collected from six species of teleosts: Atlantic salmon (Salmo salar), brown trout (Salmo trutta), Arctic charr (Salvelinus alpinus), brook trout (Salvelinus fontinalis), striped bass (Morone saxatilis) and rainbow trout (Oncorhynchus mykiss) at nine facilities over a 600 km range. Following laboratory culture, 132 isolates were recovered. Six species of oomycetes were identified from analysis of the internal transcribed spacer (ITS) sequence of the nrDNA: Saprolegnia parasitica, Saprolegnia ferax, Saprolegnia diclina, Saprolegnia aenigmatica, Saprolegnia torulosa, Saprolegnia sp. and Pythiopsis cymosa. Further phylogenetic analyses of the ITS and cytochrome c oxidase subunit 1 (Cox1) regions revealed four strains of Saprolegnia parasitica (named here as S1, S2, S3 and S4), of which S1 and S2 were common (37% and 42% of the isolates), and two strains of S. ferax. Among S. parasitica, S2 and S3 are more closely related to each other than to S1 based on the phylogenetic analyses and predicted RNA secondary structure of the ITS region. Sexual structures with a similar morphology were formed by S1 and S3 in vitro, but were not formed by S2.     

Colorimetric assay for the in vitro evaluation of Saprolegnia biofilm inhibitors

A quantitative and reproducible 96‐well microtiter method that is easily adaptable for the screening of Saprolegnia biofilm inhibitors is described. As opposed to other methods previously developed for the screening of Saprolegnia inhibitors on spore germination or mycelial growth, this technique is of particular significance as it investigates potential inhibitors against surface‐attached mycelial mats of Saprolegnia spp. (biofilm). In this study, we have investigated the effects of propionic acid (PPA) on reducing the viability of induced Saprolegnia biofilms using colorimetric MTS assay based on the reduction of tetrazolium salts. Viability of Saprolegnia hyphae in treated biofilms was reduced significantly following treatment with different PPA concentrations. The effect was enhanced after combining each of the tested PPA concentrations with 500 mg/L of boric acid (BA). However, the percentage of non‐viable hyphae was still higher in 200 mg L^–1 bronopol‐treated biofilms (positive control) following 6‐ and 12‐hr exposure. Similar results were observed using other recently described fluorescence‐based assays for viability.     

In Vitro Screening of Fungicidal Chemicals for Antifungal Activity against Saprolegnia

Saprolegnia is an important fish fungal pathogen that often results in significant economic losses to freshwater aquaculture. To find effective drugs to control saprolegniasis, 30 fungicidal chemicals used in agriculture were screened, in which kresoxim‐methyl and azoxystrobin, with minimum inhibitory concentration (MIC) values of 1.0 and 0.5 mg/L, respectively, showed good in vitro antifungal activities against Saprolegnia. Azoxystrobin has the most promising anti‐Saprolegnia activity with 50% effective concentration (EC₅₀) value of 0.212 mg/L against mycelial growth and minimum fungicidal concentration (MFC) value of 0.13 mg/L against spores, while EC₅₀ and MFC values to kresoxim‐methyl are 0.240 and 0.25 mg/L, respectively. Through the acute toxicity assay using goldfish, Carassius auratus, azoxystrobin exhibited wider margin of safety with a safe concentration (SC) value of 0.553 mg/L than kresoxim‐methyl with an SC value of 0.131 mg/L. These findings demonstrated that azoxystrobin has the potential for the development of therapy for the control of Saprolegnia in aquaculture. Both kresoxim‐methyl and azoxystrobin were tested with a post‐antifungal effects (PAFE) assay and the results revealed that the two chemicals had no significant effect on fungal growth inhibition after a 1‐hour exposure, indicating that the treatment needs to be carried out over an extended period.     

Suppression of Saprolegnia infections in rainbow trout (Oncorhynchus mykiss) eggs using protective bacteria and ultraviolet irradiation of the hatchery water

Since formalin is widely used in prevention of Saprolegnia infections in salmonid fish hatcheries, there is a need for more environmentally safe treatment methods. Therefore, we screened 360 bacterial isolates against their ability to antagonize the growth of Saprolegnia parasitica hyphae in vitro, and best strains were selected according to their antagonistic properties and colonization capability on rainbow trout egg surface. Protective bacterial cultures of Pseudomonas sp. M162, Pseudomonas sp. M174 and Janthinobacterium M169 were tested for prevention of Saprolegnia sp. infections during incubation trials of rainbow trout (Oncorhynchus mykiss) eggs with UV irradiated (400 mWs cm^?2) and non‐treated inlet water. UV irradiation of inlet water significantly decreased mortality during the incubation. Lowest mortalities were observed in protective culture treated groups incubated with UV‐irradiated inlet water. UV irradiation increased the dominance of the main bacterial colonizers and variation in the bacterial species diversity between the experimental units.     

Antifungal Activity of Rhein and Aloe‐Emodin from Rheum palmatum on Fish Pathogenic Saprolegnia sp.

Saprolegnia infections cause severe economic losses among freshwater fish farming. In this study, two known compounds, rhein and aloe‐emodin, were isolated from Rheum palmatum, and the in vitro inhibitory activity of both compounds against mycelial growth and spore germination of Saprolegnia was tested. Both rhein and aloe‐emodin were able to decrease Saprolegnia mycelial growth and spore activity in all tested concentrations after exposure for 48 h. Complete inhibition of mycelial growth was observed at 20 mg/L for rhein and at 50 mg/L for aloe‐emodin, while spore germination was 100% prevented at 16 and 40 mg/L for rhein and aloe‐emodin, respectively. Because rhein showed stronger in vitro anti‐Saprolegnia activity, it was further tested in vivo to measure the prevention and treatment efficacy on Saprolegnia infection of grass carp. Its acute activity to grass carp was also evaluated. The results revealed that exposure to rhein at 20 mg/L for 7 d could prevent 93.3% of infections by Saprolegnia in abraded grass carp, while 67.7% of infected fish could be recovered by treatment with rhein. The 48‐h median lethal concentration (48 h‐LC₅₀) to grass carp was 148.5 mg/L, which is about 7.4 times the effective dose indicating the safety for the use of rhein. This study suggests that rhein has promising anti‐Saprolegnia activity and may be an option in preventing and controlling Saprolegnia infection.     

A thicker chorion gives ova of Atlantic salmon (Salmo salar L.) the upper hand against Saprolegnia infections

Since the ban of malachite green in the fish farming industry, finding alternative ways of controlling Saprolegnia infections has become of utmost importance. Much effort has been made to elucidate the mechanisms by which Saprolegnia invades fish eggs. Little is known about the defence mechanisms of the hosts, making some eggs more prone to infection than others. One clue might lie in the composition of the eggs. As the immune system in the embryos is not developed yet, the difference in infection levels could be explained by factors influenced by the mother herself, by either transferring passive immunity, influencing the physical aspects of the eggs or both. One of the physical aspects that could be influenced by the female is the chorion, the extracellular coat surrounding the fish egg, which is in fact the first major barrier to be overcome by Saprolegnia spp. Our results suggest that a thicker chorion in eggs from Atlantic salmon gives a better protection against Saprolegnia spp. In addition to the identification of differences in sensitivity of eggs in a fish farm set‐up, we were able to confirm these results in a laboratory‐controlled challenge experiment.     

Pharmacodynamics of amoxicillin against field isolates of Streptococcus parauberis from olive flounder (Paralichthys olivaceus)

Olive flounder is the most important species for the Northeast Asian fish farming industry. However, this species is substantially affected by multiple infectious agents, including Streptococcus parauberis. Evaluation of antibiotics before their application is critical to treat infections and prevent drug resistance. Therefore, in this study, the pharmacodynamics of amoxicillin (AMX) and other antimicrobials against the planktonic‐ and biofilm‐forming bacteria were assessed. The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and Time–kill curve assay were analysed using micro‐dilution method. The minimum biofilm eradicating concentration (MBEC) was determined using the Calgary Biofilm device. The effects of temperature, pH, hardness and salinity were detected for both planktonic‐ and biofilm‐forming bacteria. The MIC of AMX ranged from 0.015 to 2 μg/ml, whereas that of cephalexin (CEP), enrofloxacin (ENR) and oxytetracycline (OTC) ranged from 0.125 to 256, 0.125 to >512 and 0.25 to >512 μg/ml respectively. No bacteria were resistant against AMX, while the percentage of resistance to CEP, OTC and ENR were 68.7%, 52.6% and 11.1% respectively. The IC₅₀ of AMX, CEP, ENR and OTC was 0.03, 0.091, 0.015 and 0.213 μg/ml respectively. The MBEC of amoxicillin against S. parauberis ranged from 0.5 to 16 μg/ml. Higher rates of bacterial growth were obtained at 30°C, pH = 8 and salinity of 7.5–10 ppt. The hardness of the media suppressed the bacterial growth. In conclusion, AMX was found to be effective against both the planktonic and the biofilm forms of the prominent fish pathogen, S. parauberis.     

In vitro passages impact on virulence of Saprolegnia parasitica to Atlantic salmon,Salmo salar L. parr

The effect of serial in vitro subculturing on three pathogenic strains of Saprolegnia parasitica was investigated. The isolates were passed through Atlantic salmon, Salmo salar L. parr, and then re‐isolated as single spore colonies. All strains caused infection. The isolate obtained from diseased fish served as a virulent reference culture and was designated ‘AP’ (‘activated through passage’). Successive subculturing was made by obtaining an inoculum from AP to produce the 2nd subculture and then passaged to the 3rd subculture (from the 2nd), until the 15th passage was obtained. Spores used to produce storage cultures were collected at passages 5, 10 and 15. The different passages of each strain were used to artificially infect Atlantic salmon parr. Morphological characterization of growth patterns was performed to observe differences occurring due to serial in vitro subculturing. Two of the strains declined in virulence after 15 successive in vitro subcultures, whereas one did not. This study is the first to investigate attenuation of virulence in Saprolegnia and whether or not isolates of S. parasitica should be passed through the fish host prior to challenge experiments. It reveals that some strains degenerate more rapidly than others when subjected to successive in vitro subculturing on glucose–yeast extract.     

Factors influencing Saprolegnia spp. spore numbers in Norwegian salmon hatcheries

A quantitative survey of Saprolegnia spp. in the water systems of Norwegian salmon hatcheries was performed. Water samples from 14 salmon hatcheries distributed along the Norwegian coastline were collected during final incubation in the hatcheries. Samples of inlet and effluent water were analyzed to estimate Saprolegnia propagule numbers. Saprolegnia spores were found in all samples at variable abundance. Number of spores retrieved varied from 50 to 3200 L^?1 in inlet water and from 30 to >5000 L^?1 in effluent water. A significant elevation of spore levels in effluent water compared to inlet water was detected. The estimated spore levels were related to recorded managerial and environmental parameters, and the number of spores in inlet water and temperature was the factor having most influence on the spore concentration in the incubation units (effluent water). Further, the relative impact of spore concentration on hatching rates was investigated by correlation analysis. From this was found that even high spore counts did not impact significantly on hatching success.     

Reduction of in vitro growth in Flavobacterium columnare and Saprolegnia parasitica by products containing peracetic acid

Commercial products containing peracetic acid (PAA) are strong disinfectants with a wide spectrum of antimicrobial activity and have been suggested as potential therapeutic agents in aquaculture. The aim of this study was to compare the in vitro reduction of growth on two fish pathogens, Flavobacterium columnare and Saprolegnia parasitica, by seven commercial PAA‐containing products. Flavobacterium columnare was exposed to 1, 2, 4, 6, 8 and 10 mg L^?1 PAA and S. parasitica was exposed to 0.5, 1, 2, 4, 6, 8 and 10 mg L^?1 PAA in petri dishes for 24 h incubation. The reduction of growth was measured in comparison to a PAA‐free control. A reduction of the growth was observed for both pathogens with increasing PAA concentration. Hydrogen peroxide (H₂O₂) possibly has a role in the effectiveness of the products, since products with lower PAA concentrations had a higher concentration of H₂O₂. The commercial products with a low concentration of PAA and a low PAA:H₂O₂‐ratio were generally more effective against pathogens. The practical application of the products with low PAA concentration should be prioritized.     

Efficacy of clove oil and ethanol against Saprolegnia sp. and usability as antifungal agents during incubation of rainbow trout Oncorhynchus mykiss (Walbaum) eggs

Inhibitory concentrations of clove oil and ethanol against growth of Saprolegnia sp. hyphae were screened by a modification of the hemp (Cannabis sativa L.) seed MicroPlate (HeMP) method and their usability as antifungal agents during incubation of rainbow trout Oncorhynchus mykiss eggs was tested. In vitro experiment showed that in continuous static exposure, clove oil at 100 mg L^?1 significantly inhibited the growth of Saprolegnia, whereas in bath exposures, clove oil at 500 mg L^?1 had no significant effect at any exposure time tested (15, 60 and 240 min), but clove oil at 10 000 mg L^?1 significantly inhibited growth at all exposure times. Clove oil and ethanol treatments had no visible effects on the onset or spread of the fungus during incubation of rainbow trout eggs. Clove oil at 1000 mg L^?1 resulted in 95–100% mortality before the eyed stage was reached. Sublethal concentrations of clove oil and ethanol had no effects on the development rate of the embryo or growth and yolk utilization efficiency after hatching. This study suggests that clove oil and ethanol may not be options in controlling aquatic fungi infestations during incubation of rainbow trout eggs.     

Use of Onion (Allium cepa) and Garlic (Allium sativum) Wastes for the Prevention of Fungal Disease (Saprolegnia parasitica) on Eggs of Rainbow Trout (Oncorhynchus mykiss)

This study was designed to prevent fungal disease (Saprolegnia parasitica) that occurs on rainbow trout eggs (Oncorhynchus mykiss) by using wastes of onion (Allium cepa) and garlic (Allium sativum) plants. For this purpose, fertilized rainbow trout eggs were exposed to garlic skin, garlic stem and onion skin aqueous methanolic extracts by bathing in concentrations of 0.4, 0.8, 1.6 and 3.2 g/L, whereas the control group was left untreated. The larvae in all groups were monitored until they become free-swimming larvae, the number of eggs died due to fungus was recorded, and the data obtained from experimental groups and control group were compared. As a result, it was determined that onion skin had no effect on the number of eggs that died due to fungus (p > .05), while garlic skin and garlic stem extracts significantly reduced the number of fungal infestations without affecting the number of live larvae or the embryological development of the eggs (p < .05). In light of these data, we conclude that garlic skin (0.4, 0.8, and 1.6 g/L) and garlic stem (0.8 and 1.6 g/L) aqueous methanolic extracts are effective at preventing Saprolegnia parasitica infestation on rainbow trout eggs and may be used in aquaculture.     

Physiological characterization of Saprolegnia parasitica isolates from brown trout

Saprolegnia parasitica has caused large mortalities in brown trout, Salmo trutta, in Spain. Several strains of Saprolegnia parasitica have been isolated from these epizootics and characterized regarding their physiological adaptation and genetic diversity. These isolates exhibit similar physiological characteristics, i.e. radial growth, zoospore mobility and germination after encystment and differ substantially from other strains of the Saprolegnia diclina-parasitica complex tested. Random amplification of polymorphic DNA polymerase chain reaction (RAPD-PCR) of total DNA revealed a genetic similarity of about 85–100% between Spanish trout isolates whereas only 20–45% similarity to other strains of the Saprolegnia diclina-parasitica complex was found. The results suggest that the Spanish isolates are closely related strains and that they might have originated from one clone.     

In vitro and in vivo antifungal activity of Satureja cuneifolia ten essential oil on Saprolegnia parasitica strains isolated from rainbow trout (Oncorhynchus mykiss,Walbaum) eggs

In the present study, the chemical composition and the antifungal properties against Saprolegnia parasitica (in vitro and in vivo) of the essential oils of thyme (Satureja cuneifolia) from Mediterranean region of Turkey were evaluated for the first time. The composition of oils was analysed using gas chromatography/mass spectrometry (GC/MS). The major constituents of oil of S. cuneifolia were cavracrol (46,84%) and cymene (16.90%). Antifungal effects of S. cuneifolia essential oil against S. parasitica strains (A1 and E1) were detected by disc diffusion and tube dilution assays. The antifungal effect of S. cuneifolia was determined to be stronger against S. parasitica E1 isolate (MIC 50 μL mL^?1, MLC 250 μL mL^?1) compared with S. parasitica A1 isolate (MIC 50 μL mL^?1, MLC 500 μL mL^?1). Following in vitro assays, effective doses of S. cuneifolia for disease control in rainbow trout eggs experimentally infected with S. parasitica were investigated. For this aim, infected eggs were treated with the essential oil (0, 5, 10, 20 and 50 ppm) during incubation period (21 days) after fertilization. Formalin (5 mL L^?1) was used as positive control. Hatching rate of eggs at the end of incubation period were calculated. The highest hatching rates were recorded in S. parasitica E1 strain at 5 and 10 ppm concentrations of S. cuneifolia and in S. parasitica A1 strain at 10 and 20 ppm (P < 0.05).     

Prevention of Saprolegniasis in rainbow trout (Oncorhynchus mykiss) eggs using oregano (Origanum onites) and laurel (Laurus nobilis) essential oils

The present study investigated the antifungal effects of essential oils of oregano (Origanum onites) and laurel (Laurus nobilis) on Saprolegniasis, a disease that occurs in rainbow trout eggs during the incubation period. Oregano and laurel were ground after drying, and essential oils were obtained by water distillation method using a Clevenger apparatus. The essential oils were added to potato dextrose agar (PDA) at the rates of 1–1000 ppm, and the minimum inhibitory concentration (MIC) was determined as 250 ppm whereas the minimum lethal concentration (MLC) was determined to be 500 ppm for both plants. In the in vivo trials, fertilized eggs were treated with predetermined doses either by bathing during water hardening and incubation period or only during incubation period, and death rates were monitored during embryological development. The best larvae hatching rate was determined in 500 ppm oregano and 500 ppm laurel groups treated during water hardening plus daily as 82.11% and 79.87%, respectively. According to the results, it was determined that oregano and laurel essential oils exhibited better results in all doses compared with the negative control group, and 500 ppm dose had a better effect than the positive control group treated with formalin.     

Evidence for synergism of the antimicrobial peptide piscidin 2 with antiparasitic and antioomycete drugs

Piscidins are potent, broad‐spectrum, host‐produced antimicrobial peptides (AMPs) that appear to constitute the most common AMP family in teleost fish. Here, we show that piscidin 2 has potent activity against the water mould Saprolegnia, one of the most important pathogens of freshwater fish. The minimum oomyceticidal concentration (MOC₁₀₀) of piscidin 2 against zoospores of three pathogenic isolates of Saprolegnia ranged from 12.5 to 25.0 μg mL^?1. This piscidin concentration is well within levels that have been estimated to be present in at least some fish (1–32.5 μg mL^?1). In the presence of either copper or malachite green, two drugs commonly used to treat water moulds, there was evidence for partial synergism (PSYN) with piscidin 2. There was also evidence for PSYN after exposure of the ciliate parasite Tetrahymena pyriformis to piscidin 2 plus copper. Our data provide further evidence that piscidins may be an important host defence against skin and gill pathogens and that the piscidin levels in host tissue might influence the success of drug treatments.     

Protective effects of a synbiotic against experimental Saprolegnia parasitica infection in rainbow trout (Oncorhynchus mykiss)

A combination of probiotics and prebiotics as synbiotics allows assessing their synergistic effects. This study evaluated the effects of a synbiotic supplement on growth performance, haematological parameters and resistance to Saprolegnia parasitica in rainbow trout, Oncorhynchus mykiss (Walbaum) fingerlings. Fish fed a dietary synbiotic in three levels of 0.5, 1.0 and 1.5 g kg^?1 thrice a day. The fingerlings were challenged with Saprolegnia parasitica after 60 days post feeding and their mortalities recorded up to 15 days. The fingerlings at all three experimental treatments showed significant (P < 0.05) increases in final mean weights and specific growth rates (SGR). The best feed conversion ratio (FCR), feed conversion efficiency (FCE) and maximum survival rate were also obtained by the fish fed 1.0 g synbiotic kg^?1 diet. Furthermore, supplementation with synbiotic significantly increased blood factors at all treatments. After challenges with Saprolegnia parasitica, the synbiotic‐fed groups showed significantly higher survival rates compared with the control group. These results reveal that a dietary synbiotic of 1.0 g kg^?1 fed for 60 days leads to increased growth performance and survival rate as well as improved feeding efficiency in rainbow trout fingerling, rendering them more resistant against infection by Saprolegnia parasitica.