Anti-oomycetes and immunostimulatory activity of natural plant extract compounds against Saprolegnia spp.: Molecular docking and in-vitro studies.

This study aimed to investigate the effectiveness of five natural plant extract compounds Curcumin (CUR); Eugenol (EUG), Cinnamaldehyde (CIN), Stigmasterol (ST) and Morin (MOR), on two species of Saprolegnia; Saprolegnia parasitica and S. australis. Selective compounds were screened for the minimum inhibitory concentration, first for anti-oomycetes activity and then mycelium growth inhibition, spore germination inhibition and colonisation test. Nitric oxide production and myeloperoxidase activity of the compounds were tested in head kidney leukocytes of rainbow trout, Oncorhynchus mykiss to assess the immunostimulatory potential. Molecular docking of effective compounds was carried out with effector proteins of S. parasitica to investigate the target binding sites. Among all, CUR could completely inhibit zoospore production and significantly (p ≤ .05) inhibit hyphal growth at 16 mg l-1 against S. parasitica and S. australis. CIN at the concentration of 50 mg l-1 completely inhibited hyphal growth of both Saprolegnia spp., although the zoospore production of S. parasitica and S. australis was reduced at 25 mg l-1 and 10 mg l-1. In the case of EUG, significant inhibition of the hyphal growth and germination of S. parasitica zoospores was observed at 50 mg l-1. ST and MOR did not show antioomycetes activity. The molecular docking results were consistent with in vitro studies, possibly due to the binding with the vital proteins (Plasma membrane ATPase, V-type proton ATPase, TKL protein kinase, Host targeting protein 1) of S. parasitica and ultimately inhibiting their activity. CUR and CIN showed increased nitric oxide production at the highest concentration of 250 and 256 mg l-1 but the value was not significant (p ≤ .05) with control. CUR showed significantly higher peroxidase activity (p ≤ .05) at a concentration of 256 mg l-1 though values were significantly similar with concentration from 16 to 128 mg l-1. The nitric oxide and total peroxidase activity of rainbow trout leukocytes in the case of CIN showed a significant difference only at 250 mg l-1 against the control. The results conclude that CUR, CIN showed the better anti-Saprolegnia activity and could be used as phyto-additives in aquaculture. Among all, the inclusion of CUR as phyto-additives will provide additional immunostimulatory activity.

Immunity and growth improvement of rainbow trout (Oncorhynchus mykiss) fed dietary nettle (Urtica dioica) against experimental challenge with Saprolegnia parasitica.

In this study, effects of nettle (Urtica dioica) on growth, immunity, and gene expressions were examined in rainbow trout after an 8-week feeding period. A total of 264 juvenile rainbow trout (10.72 ± 0.55 g) were selected and stocked randomly in 12 aquaria. Nettle powder was added to the fish feed at three doses, 0.5,1 and 1.5% served as treatments. At the end of 8-week feeding period, the fish were exposed to Saprolegnia parasitica for 3 weeks. Results showed that all treatments fed with nettle diets exhibited significant increases in weight gain and SGR, and decreased FCR compared to the control. Feeding the fish with dietary nettle resulted in significant rises in blood indices and non-specific immunity in comparison with the control. Furthermore, fish fed 0.5% of dietary nettle showed significantly increased expressions of TNF-α, IL-1b, IL-6 and IL-8 genes following 8 weeks of feeding. A significant reduction in mortality rate was observed in the fish treated with 0.5% of nettle compared to the control following challenging with S. parasitica. Our observations indicate that the use of 0.5% nettle powder in rainbow trout diet can improve growth and immunity parameters as well as fish resistance against S. parasitica contamination.

In Vitro Activity of Essential Oils against Saprolegnia parasitica.

Saprolegnia spp. water molds severely impact fish health in aquaculture, fish farms and hobby fish tanks colonizing mature and immature stages of fishes, as well as eggs. Considering that there are no drugs licensed for treating and/or control the organism, efficient and environmental low-impact methods to control these oomycetes in aquaculture are needed. The aim of the present report was to evaluate the in vitro sensitivity of Saprolegnia parasitica to essential oils (EOs) from Citrus aurantium L., Citrus bergamia Risso et Poiteau, Citrus limon Burm. f., Citrus paradisi Macfad, Citrussinensis Osbeck, Cinnamomum zeylanicum Blume, Cymbopogon flexuosum (Nees ex Steud.) Watson, Foeniculum vulgare Mill., Illicium verum Hook.f., Litsea cubeba (Lour.) Pers., Origanum majorana L., Origanum vulgare L., Pelargonium graveolens L'Hér., Syzygium aromaticum Merr. & L.M.Perry, and Thymus vulgaris L., by microdilution test. The most effective EOs assayed were T. vulgaris and O. vulgare, followed by C. flexuosum, L. cubeba and C. bergamia. These EOs could be of interest for controlling Saprolegnia infections. Nevertheless, further safety studies are necessary to evaluate if these products could be dispersed in tank waters, or if their use should be limited to aquaculture supplies.

Isolation of anti-Saprolegnia lignans from Magnolia officinalis and SAR evaluation of honokiol/magnolol analogs.

To control the fish fungal pathogen Saprolegnia, the effects of the petroleum ether extracts of Magnolia officinalis were evaluated by a rapeseed (Brassicanapus) microplate method in vitro. By loading on an open silica gel column and eluting with petroleum ether-ethyl acetate-methanol, honokiol (C18H18O2) and magnolol (C18H18O2) were isolated from Magnolia officinalis. Saprolegnia parasitica growth was inhibited significantly when honokiol concentration was >8.0 mg/L, and magnolol concentration was >9.0 mg/L, with EC50 values of 4.38 and 4.92 mg/L, respectively. Six honokiol and magnolol derivatives were designed, synthesized and evaluated for their anti-Saprolegnia activity. According to the results, double bond and hydroxyl played an important role in inhibiting Saprolegnia. Mechanistically, through the scanning electron microscope observation, honokiol and magnolol could cause the Saprolegnia parasitica mycelium tegumental damage including intensive wrinkles and nodular structures. Moreover, compared to traditional drugs kresoxim-methyl (LC50 = 0.66 mg/L) and azoxystrobin (LC50 = 2.71 mg/L), honokiol and magnolol showed a lower detrimental effect on zebrafish, with the LC50 values of 6.00 and 7.28 mg/L at 48 h, respectively. Overall, honokiol and magnolol were promising lead compounds for the development of commercial drugs anti-Saprolegnia.

Effect of miconazole nitrate on immunological response and its preventive efficacy in Labeo rohita fingerlings against oomycetes Saprolegnia parasitica.

This study evaluated the effect of sublethal doses of antifungal drug miconazole nitrate (MCZ) on immunological responses and its role as a prophylactic drug against S. parasitica in Labeo rohita fingerlings. Fish were fed with sublethal doses of MCZ, that is, T1-6.30 mgMCZ kgBW-1 , T2-12.61 mgMCZ kgBW-1 and T3-25.22 mgMCZ kgBW-1 , and sampling was done at different time intervals for 240 hr. Immunological parameters viz. lysozyme activity, oxygen radical production and plasma antiprotease activity showed significant enhancement (p < 0.05) in fish fed with T2 and T3 doses. Expression of immune-relevant genes such as TLR-22 and ß2-M showed significantly higher expression at 6 hr and 24 hr of sampling in both liver and head kidney. However, these genes showed a downregulation after 120 hr of sampling in both the tissues. Preventive efficacy study showed that single dose of MCZ provides protection against oomycetes up to the fourth day of infection. Significantly higher mortality was observed in control diet-fed fish as compared to fish fed with MCZ medicated diet. Thus, it can be concluded that the MCZ can act as a potent antifungal agent for preventing oomycetes infection as well as to enhance the immune response.

Chemical Characterization and Anti-Oomycete Activity of Laureliopsis philippianna Essential Oils against Saprolegnia parasitica and S. australis.

Laureliopsis philippiana (Looser) R. Schodde (Monimiaceae) is a native tree widespread in the forest areas in the south of Chile and Argentina, known for its medicinal properties and excellent wood. The aim of this study was to evaluate the chemical composition of L. philippiana leaf and bark essential oils (EOs) using gas chromatography-mass spectrometry (GC-MS), and to quantify its anti-oomycete activity, specifically against Saprolegnia parasitica and S. australis. Only six components were identified in leaf EO, 96.92% of which are phenylpropanoids and 3.08% are terpenes. As for bark EO, 29 components were identified, representing 67.61% for phenylpropanoids and 32.39% for terpenes. Leaf EO was characterized mainly by safrole (96.92%) and ß-phellandrene (1.80%). Bark EO was characterized mainly by isosafrole (30.07%), safrole (24.41%), eucalyptol (13.89%), methyleugenol (7.12%), and eugenol (6.01%). Bark EO has the most promising anti-Saprolegnia activity, with a minimum inhibition concentration (MIC) value of 30.0 µg/mL against mycelia growth and a minimum fungicidal concentration (MFC) value of 50.0 µg/mL against spores; for leaf EO, the MIC and MFC values are 100 and 125 µg/mL, respectively. These findings demonstrate that bark EO has potential to be developed as a remedy for the control of Saprolegnia spp. in aquaculture.

Clotrimazole as a potent agent for treating the oomycete fish pathogen Saprolegnia parasitica through inhibition of sterol 14α-demethylase (CYP51).

A candidate CYP51 gene encoding sterol 14α-demethylase from the fish oomycete pathogen Saprolegnia parasitica (SpCYP51) was identified based on conserved CYP51 residues among CYPs in the genome. It was heterologously expressed in Escherichia coli, purified, and characterized. Lanosterol, eburicol, and obtusifoliol bound to purified SpCYP51 with similar binding affinities (Ks, 3 to 5 µM). Eight pharmaceutical and six agricultural azole antifungal agents bound tightly to SpCYP51, with posaconazole displaying the highest apparent affinity (Kd, ≤3 nM) and prothioconazole-desthio the lowest (Kd, ∼51 nM). The efficaciousness of azole antifungals as SpCYP51 inhibitors was confirmed by 50% inhibitory concentrations (IC50s) of 0.17 to 2.27 µM using CYP51 reconstitution assays. However, most azole antifungal agents were less effective at inhibiting S. parasitica, Saprolegnia diclina, and Saprolegnia ferax growth. Epoxiconazole, fluconazole, itraconazole, and posaconazole failed to inhibit Saprolegnia growth (MIC100, >256 µg ml(-1)). The remaining azoles inhibited Saprolegnia growth only at elevated concentrations (MIC100 [the lowest antifungal concentration at which growth remained completely inhibited after 72 h at 20°C], 16 to 64 µg ml(-1)) with the exception of clotrimazole, which was as potent as malachite green (MIC100, ∼1 µg ml(-1)). Sterol profiles of azole-treated Saprolegnia species confirmed that endogenous CYP51 enzymes were being inhibited with the accumulation of lanosterol in the sterol fraction. The effectiveness of clotrimazole against SpCYP51 activity (IC50, ∼1 µM) and the concentration inhibiting the growth of Saprolegnia species in vitro (MIC100, ∼1 to 2 µg ml(-1)) suggest that clotrimazole could be used against Saprolegnia infections, including as a preventative measure by pretreatment of fish eggs, and for freshwater-farmed fish as well as in leisure activities.

Novel antimicrobial activity of a dichloromethane extract obtained from red seaweed Ceramium rubrum (Hudson) (Rhodophyta: Florideophyceae) against Yersinia ruckeri and Saprolegnia parasitica, agents that cause diseases in salmonids

Background Enteric red mouth disease and Saprolegniasis, which are caused by the bacteria Yersinia ruckeri and the oomycete Saprolegnia parasitica, respectively, are important illnesses that affect salmonid farming. Sanitary problems in farms are addressed by the prevention of disease outbreaks or by the treatment of diseases with chemicals. Environmental and governmental restrictions, toxicity and high treatment costs limit the use of drugs. Marine organisms, such as algae, sponges and corals, have developed an antimicrobial defense strategy based on the production of bioactive metabolites. Among these organisms, seaweeds offer a particularly rich source of potential new drugs. Hence, many pharmacologically active substances have been isolated from seaweeds. In the Ceramium genus, Ceramium rubrum has been emphasized by several authors for its antimicrobial properties. Based on this background, the present study focused on the antimicrobial activity of a lipophilic extract of C. rubrum on Y. ruckeri and S. parasitica. Results The alga, collected from the Pacific coast of Chile, underwent an ethanol extraction, and the concentrated extract was partitioned between water and dichloromethane. From the dichloromethane extract, fatty acids, fatty acid esters, one hydrocarbon and phytol were identified by Gas Chromatography-Mass Spectrometry (GC/MS) analysis. The antimicrobial study showed that the whole extract was more active than the individual components, which suggests a strong synergistic effect among the components. Conclusions These results may constitute a basis for promising future applied research that could investigate the use of C. rubrum seaweed as a source of antimicrobial compounds against fish pathogens.

Evaluation of the antifungal activity of Zataria multiflora, Geranium herbarium, and Eucalyptus camaldolensis essential oils on Saprolegnia parasitica-infected rainbow trout (Oncorhynchus mykiss) eggs.

The purpose of the present study was to evaluate and assess the capability of Zataria multiflora, Geranium herbarium, and Eucalyptus camaldolensis essential oils in treating Saprolegnia parasitica-infected rainbow (Oncorhynchus mykiss) trout eggs. A total of 150 infected eggs were collected and plated on glucose-pepton agar at 24°C for 2 weeks. The antifungal assay of essential oils against S. parasitica was determined by a macrodilution broth technique. The eggs were treated with essential oils at concentrations of 1, 5, 10, 25, 50, and 100 ppm daily with three repetitions until the eyed eggs stage. Of 150 eggs examined, S. parasitica (54.3%), Saprolegnia spp. (45%), and Fusarium solani (0.7%) were isolated. The minimum inhibitory concentrations of Z. multiflora, E. camaldolensis, and G. herbarium essential oils against S. parasitica were 0.9, 2.3, and 4.8 ppm, respectively. Zataria multiflora and E. camaldolensis at concentrations of 25, 50, and 100 ppm, and G. herbarium at concentration of 100 ppm had significant differences in comparison with negative control (p<0.05). The results revealed that malachite green, followed by Z. multiflora, E. camaldolensis, and G. herbarium treated eggs had remained the most number of final eyed eggs after treatment. The highest final larvae rates belonged to malachite green, E. camaldolensis, Z. multiflora, and G. herbarium, respectively. The most hatching rates were recorded with malachite green (22%), and then Z. multiflora (11%), E. camaldolensis (7%), G. herbarium (3%), and negative control (1%). Zataria multiflora and E. camaldolensis were more effective than G. herbarium for the treatment of S. parasitica-infected rainbow trout eggs in aquaculture environment.

Treatment of microbiologically polluted aquaculture waters by a novel photochemical technique of potentially low environmental impact.

The applicability of a novel procedure for the disinfection of microbiologically polluted waters from fish-farming ponds, based on the combined action of visible light (including sunlight) and porphyrin-type photosensitising agents, has been investigated using (a) cell cultures of a Gram-positive bacterium (meticillin-resistant Staphylococcus aureus), a Gram-negative bacterium (Escherichia coli) and a fungal pathogen (Saprolegnia spp.); (b) pilot aquaculture plants involving either spontaneously or artificially Saprolegnia-infected rainbow trout (Oncorhynchus mykiss). The results obtained by using two cationic porphyrins, namely a tetra-substituted N-methyl-pyridyl-porphine (C1) and its analogue where one N-methyl group had been replaced by a N-tetradecyl chain (C14), and low intensity visible light irradiation showed an extensive (up to 6-7 log) decrease in the bacterial/fungal population after short incubation and irradiation times in the presence of micromolar photosensitiser concentrations. Moreover, C14 showed some toxic effect also in the absence of light. Extension of these studies to the pilot plants indicated that both C1 + light and C14 can prevent Saprolegnia infections or promote the cure of saprolegniasis in infected trout by treatments with submicromolar porphyrin doses. The procedure appears to be of low cost and to have a low environmental impact.