Saprolegnia Spp Research Articles

EFFICACY OF SILVER ZEOLITE ON HEMATOLOGY AND SURVIVAL RATE IN CYPRINUS CARPIO INFECTED WITH SAPROLEGNIA SPP.

This study was undertaken to evaluate the effect of silver zeolite (SZ) on hematological parameters and survival rate in common carp, Cyprinus carpio infected with Saprolegnia spp. A total of 120 fish (weight 100 g) were divided into six treated groups in duplicate (10 fish/tank) as follows: T1, T2, T3 and T4 that infected with Saprolegnia spp. (2×104 zoospore/ml) and treated with SZ at (100,200 and 300 mg/l) while T4 infected with Saprolegnia spp. and treated with copper sulfate )5g/100 L(; C+ group, fish were infected with Saprolegnia without treatment and C- served as control negative group. Following 14 days from infection with Saprolegnia spp. and treatment with SZ, considerable changes were observed in blood indices. In pre-treatment, results showed a significant increased (P≤0.05) in WBC count in all infected fish (C+, T1, T2,T3 and T4) in comparison to C- group. RBC, Hb content and PCV% registered a significantly decreased (P≤0.05) in all infected fish (C+,T1, T2,T3 and T4) relative to the control group (C-). Post treatment, WBC registered significantly decreased in treated groups (T1,T2,T3 and T4) compared to C+ in WBC count. The best survival was recorded in T3 (85%) followed by T2 and T1 (65%, and 60%) respectively. In conclusion Silver zeolite seems to be a valuable disinfectant agent against Saprolegnia spp.; at the dose of 300 mg/l.

Identification and genetic characterization of Saprolegnia parasitica, isolated from farmed and wild fish in Finland.

Oomycete infections in farmed fish are one of the most significant disease issues in salmonid aquaculture worldwide. In the present study, Saprolegnia spp. in different farmed fish species in Finland were identified, and the molecular epidemiology of especially Saprolegnia parasitica was examined. We analysed tissue samples from suspected oomycete-infected salmonids of different life stages from a number of fish farms, as well as three wild salmonids. From collected oomycete isolates, the ITS1, 5.8S and ITS2 genomic regions were amplified, analysed phylogenetically and compared with corresponding sequences deposited in GenBank. Of the sequenced isolates, 91% were identified as S. parasitica. Isolates of yolk sac fry were identified as different Saprolegnia spp. Among the isolates from rainbow trout eggs Saprolegnia diclina dominated. In order to determine potential dominating clones among the S. parasitica, isolates were analysed using Multi Locus Sequence Typing (MLST). The results showed that one main clone contained the majority of the isolates. The MLST analysis showed four main sequence types (ST1-ST4) and 13 unique STs. This suggests that the Saprolegnia infections in farmed fish in Finland are not caused by different strains originating in the farm environment. Instead, one main clone of S. parasitica is present in Finnish fish farms.

Saprolegnosis: dissemination in aquaculture and control methods

Saprolegnia spp, the causative agent of which is aquatic mold fungi, affects fish and spawn during factory hatchery and is one of the problems when growing avaculture in industrial conditions. The review describes control methods of Saprolegnia spp. Traditional methods of saprolegnia control are chemical compounds such as malachite green, purple “K” and formalin, which have carcinogenic and mutagenic effects. Safer but somewhat outdated methods are the treatment of spawn with incubation of boric acid and hydrogen peroxide, as well as water ozonization. Alternatives to these methods of control may be natural biologically active substances: thyroxine and cortisol, bark extract Drimys winteri, chitosan. Besides, various isolates of microorganisms are used as biological methods of saprolegnosis control in aquaculture, namely: bacteria of the genus Pseudomonas, Aeromonas, Bacillus subtilis, Pantoea agglomerans. The use of marine algae containing sulfated polysaccharides, including fucoidan, to control saprolegnosis has been confirmed. Fucoidan has been shown to have wide biocidal activity, but its antimicrobial properties have not been studied. In this regard, the study of the influence of the structure of fucoidan on its antimicrobial properties is a promising trend in the search for new safe means of saprolegnosis control.

Antifungal effects of alcoholic extract of Thymus vulgaris on Siberian sturgeon (Acipenser baerii) eggs compared with malachite green effects

The economic losses due to fungal contamination in development of sturgeon and the side effects of malachite green in the environment are very important. Therefore, it is essential to investigate and introduce suitable natural substances instead of malachite green. The aim of this study was to compare antifungal effect of Thymus vulgaris with malachite green on fungi isolated from Siberian sturgeon eggs obtained from International Sturgeon Research Institute. The samples were inoculated in culture media (SDA) and Glucose-Peptone Agar at 25°C for 5-7 days and then isolated fungi were identified. The antifungal effect of T. vulgaris extract was evaluated by disc diffusion method and MIC and MFC of fungi was measured. In this study, Saprolegnia spp ., Fusarium spp ., Rhodotorula spp ., Penicillium spp ., Trichoderma spp ., Chrysosporium spp .and sterile hyphaewere isolated and identified from fertilized eggs of A. baerii. According to the results, T. vulgaris extract had a significant effect on all isolated fungal species. The most effective sample was observed on Saprolegnia and the least effective ones were Fusarium and Trichoderma. The MIC and MFC of alcoholic extract for Saprolegnia spp ., Fusarium spp., Rhodotorula spp ., Penicillium spp ., Trichoderma spp ., Chrysosporium spp ., sterile hyphae were ≥0.75, 3, 1.5, 1.5, 0.75, 1.5, ≤0.75 mg/ml, and ≥1.5, ≥3, 3, 3, 1.5, 3 and 1.5 mg/ml, respectively. Due to high frequency of Saprolegnia spp . in A. baerii eggs, and the significant effect of alcoholic extract of T. vulgaris, it could be used as a good alternative for malachite green to reduce the frequency of fungal contamination and its economic losses during reproductive seasons of A. baerii.

INFLUENCE OF PHYTASE ENZYME ON GROWTH PERFORMANCE AND SURVIVAL RATE CHALLENGED WITH SAPROLEGNIA SPP. IN COMMON CARP

This study was aimed to investigate the effect of phytase as a feed additive on growth performance and survival rate against Saprolegnia spp.in common carp, Cyprinus carpio L. A total of 120 C. Carpio (initial mass 20.05-20.35g) were randomly stocked into six treatments in duplicate (10 fish/tank) as follows: T1, T2, and T3 were fed the basal diet supplemented with phytase at 1000, 2000, and 4000 IU/Kg diet respectively, while T4 were fed on basal diet plus β-glugan at a concentration of 8.5 g/kg as well as the control group was fed basal diet without any addition of phytase. Results showed after 60 days of feeding period, that the addition of phytase enzyme was significantly (p<0.05) affected on daily growth (DG), specific growth rate (SGR) and feed conversion ratio (FCR) in T3 and T2 followed by T4 and T1 compared to control group. At the end of the experimental period (60 days) fish were challenged with Saprolegnia spp. After 15 days of challenge test, the best survival rate was recorded in T3 (100%) followed by T2 (80%) then T1 (70%) respectively. All treatment groups were better than the positive control group which recorded 40 %. Based on these results, this study approved the potency of the phytase enzyme on growth performance and survival rate against the Saprolegniasis in common carp.

Comparative Therapeutic Effects of Natural Compounds Against Saprolegnia spp. (Oomycota) and Amyloodinium ocellatum (Dinophyceae).

The fish parasites Saprolegnia spp. (Oomycota) and Amyloodinium ocellatum (Dinophyceae) cause important losses in freshwater and marine aquaculture industry, respectively. The possible adverse effects of compounds used to control these parasites in aquaculture resulted in increased interest on the search for natural products with antiparasitic activity. In this work, eighteen plant-derived compounds (2′,4′-Dihydroxychalcone; 7-Hydroxyflavone; Artemisinin; Camphor (1R); Diallyl sulfide; Esculetin; Eucalyptol; Garlicin 80%; Harmalol hydrochloride dihydrate; Palmatine chloride; Piperine; Plumbagin; Resveratrol; Rosmarinic acid; Sclareolide; Tomatine, Umbelliferone, and Usnic Acid) have been tested in vitro. Sixteen of these were used to determine their effects on the gill cell line G1B (ATCC®CRL-2536™) and on the motility of viable dinospores of Amyloodinium ocellatum, and thirteen were screened for inhibitory activity against Saprolegnia spp. The cytotoxicity results on G1B cells determined that only two compounds (2′,4′-Dihydroxychalcone and Tomatine) exhibited dose-dependent toxic effects. The highest surveyed concentrations (0.1 and 0.01 mM) reduced cell viability by 80%. Upon lowering the compound concentration the percentage of dead cells was lower than 20%. The same two compounds revealed to be potential antiparasitics by reducing in a dose-dependent manner the motility of A. ocellatum dinospores up to 100%. With respect to Saprolegnia, a Minimum Inhibitory Concentration was found for Tomatine (0.1 mM), Piperine and Plumbagin (0.25 mM), while 2′,4′-Dihydroxychalcone considerably slowed down mycelial growth for 24 h at a concentration of 0.1 mM. Therefore, this research allowed to identify two compounds, Tomatine and 2′,4′-Dihydroxychalcone, effective against both parasites. These compounds could represent promising candidates for the treatment of amyloodiniosis and saprolegniosis in aquaculture. Nevertheless, further in vitro and in vivo tests are required in order to determine concentrations that are effective against the considered pathogens but at the same time safe for hosts, environment and consumers.

Ekstrak Daun Pepaya dan Kangkung untuk Meningkatkan Daya Tetas Telur dan Kelangsungan Hidup Larva Lele (EXTRACTS OF CARICA PAPAYA AND IPOMOEA AQUATICA FOR IMPROVING EGG HATCHABILITY AND LARVAL VIABILITY OF CATFISH)

This research was aimed to investigate the potential use of leaf extract of Carica papaya and Ipomoeaaquatica lto improve egg hatchability and larval viability of catfish. Dried leaves of Carica papaya andIpomoea aquatica were macerated and extracted in water and ethanol. Eggs and larvae were tested in theaquarium size of 10 L with a a diameter of 28 cm. The extracts in concentration 600, 800 and 1.000 ppmwere tested on the egg hatchability of catfish with immersion method, and challed with Aeromonashydrophyla, Pseudomonas sp., and Saprolegnia spp. The extracts in concentration 800 and 1.000 ppm weretested on the larval viability with immersion method, and challed with pathogens. Water or ethanolextract of Carica papaya and Ipomoea aquatica can improve egg hatchability 67±8% until 90±6% andlarval viability of catfish 77±0,5 until 90±9%. Eight hundred ppm ethanol extract of Carica papaya has thebest egg hatchability and 1000 ppm can improve larval viability of catfish.

Use of Copper Sulfate to Control Egg Saprolegniasis at a Commercial Sunshine Bass Hatchery

AbstractAn obstacle to the larval production of sunshine bass (female White Bass Morone chrysops × male Striped Bass M. saxatilis) is fungal growth on the eggs caused by the water mold, Saprolegnia spp. Copper sulfate (CuSO4) is commonly used for fungus control in troughs at catfish hatcheries, but the effectiveness in other fish‐egg hatching systems is unknown. Because sunshine bass eggs begin hatching after 48 h, CuSO4 treatments (0, 10, 20, and 40 mg/L) began the afternoon of spawning with a 10‐min, aerated, static bath and were repeated the morning and afternoon of day 2; eggs were not treated after hatching began. An in vitro assay was developed for this treatment regimen that indicated maximum fungal inhibition was achieved with 20 mg/L CuSO4. In the effectiveness experiment in McDonald hatching jars (n = 3), fungus was severe in the untreated controls (27.7% survival), but very little fungus was present in treatments receiving 10 mg/L CuSO4 (31.4% survival) or higher. The control was not significantly different from the 10‐mg/L treatment. The highest survival was at 40 mg/L CuSO4 (50.5% survival); however, the survival from the 20‐mg/L CuSO4 treatment (45.8%) was not significantly different from that in the 40‐mg/L treatment and thus allows for a greater margin of safety. Fungus samples were identified as Saprolegnia ferax through genetic sequencing. Toxicity experiments on sunshine bass larvae indicated the 24‐ and 48‐h LC50 values were 5.4 and 3.9 mg/L CuSO4, respectively; the no observed effect concentrations were 0.5 and 0.25 mg/L CuSO4, respectively. This study confirms that CuSO4 can be an economical and valuable resource in hatchery management for the control of saprolegniasis on sunshine bass eggs in McDonald jars.

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.

Mitochondrial dysfunction is involved in the toxic activity of boric acid against Saprolegnia.

There has been a significant increase in the incidence of Saprolegnia infections over the past decades, especially after the banning of malachite green. Very often these infections are associated with high economic losses in salmonid farms and hatcheries. The use of boric acid to control the disease has been investigated recently both under in vitro and in vivo conditions, however its possible mode of action against fish pathogenic Saprolegnia is not known. In this study, we have explored the transformation in Saprolegnia spores/hyphae after exposure to boric acid (1 g/L) over a period 4–24 h post treatment. Using transmission electron microscopy (TEM), early changes in Saprolegnia spores were detected. Mitochondrial degeneration was the most obvious sign observed following 4 h treatment in about 20% of randomly selected spores. We also investigated the effect of the treatment on nuclear division, mitochondrial activity and function using confocal laser scanning microscopy (CLSM). Fluorescence microscopy was also used to test the effect of treatment on mitochondrial membrane potential and formation of reactive oxygen species. Additionally, the viability and proliferation of treated spores that correlated to mitochondrial enzymatic activity were tested using an MTS assay. All obtained data pointed towards changes in the mitochondrial structure, membrane potential and enzymatic activity following treatment. We have found that boric acid has no effect on the integrity of membranes of Saprolegnia spores at concentrations tested. It is therefore likely that mitochondrial dysfunction is involved in the toxic activity of boric acid against Saprolegnia spp.

Efeito in vitro de químicos no crescimento micelial de Saprolegnia spp.

Com o aumento da produtividade na piscicultura, os peixes são expostos a altas densidades de estocagem que podem levar ao estresse e imunossupressão. Essa condição favorece a ocorrência de infecções, entre elas a saprolegniose que afeta os peixes e seus ovos, causando sérios prejuízos econômicos aos piscicultores. Dessa forma, compostos químicos eficazes e "ambientalmente amigáveis" são almejados para o controle da doença. O objetivo do presente estudo foi avaliar a suscetibilidade in vitro de 12 isolados de Saprolegnia spp., provenientes dos peixes-rei dulceaquícolas Odontesthes bonariensis e O. humensis. Para isso, testes de suscetibilidade, avaliando a inibição do crescimento micelial de Saprolegnia spp., foram realizados frente a seis químicos (cloreto de sódio, formaldeído, permanganato de potássio, iodopovidona, sal marinho e sal marinho iodado) nas concentrações seriadas de 0 a 10.000ppm. Os resultados obtidos evidenciaram que o formaldeído e o permanganato de potássio, em concentrações a partir de 10 e acima de 100ppm, respectivamente, inibiram o crescimento micelial de isolados Saprolegnia spp.. Já a iodopovidona e os compostos salinos não evidenciaram atividade antimicrobiana em nenhuma das concentrações testadas (0-10.000ppm). Embora os resultados in vitro demonstrem que formaldeído e permanganato de potássio sejam promissores no controle da saprolegniose, estudos futuros deverão avaliar a eficácia e o efeito in vivo desses compostos nos peixes-rei O. bonariensis e O. humensis.

Hematological Changes in Common Carp (Cyprinus carpio) and Rainbow Trout (Oncorhynchus mykiss), Infected with Saprolegnia spp.

The present study was conducted to investigate the physiological impairment in common carp (Cyprinus carpio) and rainbow trout (Oncorhynchus mykiss) infected with Saprolegnia spp. The following hematological parameters were studied: total erythrocyte count (TEC), total leukocyte count (TLC), hemoglobin (Hb), packed cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC). Except TLC, which significantly increased in the infected fish, all parameters decreased in response to infection. Decreases in Hb, TEC, and Hct were significant, indicating that saprolegniosis causes anemia and immunosuppression.

Dose-Confirmation of Copper Sulfate for Treating Fungus on Channel Catfish, Ictalurus punctatus, Eggs at a Commercial Hatchery

This study at a commercial hatchery was required by the U.S. Food and Drug Administration to provide independent substantiation of the results of previous laboratory dose-confirmation studies on the use of copper sulfate (CuSO4) to control fungus (Saprolegnia spp.) on the eggs of channel catfish, Ictalurus punctatus. The study compared an untreated control group of eggs to eggs treated with 10 mg/L CuSO4 in a flow-through system; mean water temperature was 23.5°C. Eggs were treated once daily until the embryos reached the eyed stage (5 treatments). Hatching was complete by day 11, and fry were counted to determine the percentage of survival in each treatment. Fungus was identified by polymerase chain reaction (PCR) as Saprolegnia spp. The mean survival in the control treatments was 4% and 40% in the CuSO4 treatments; the latter survival was significantly higher, but still lower than normal. This study confirms that 10 mg/L CuSO4 is an effective treatment to control fungus on catfish eggs when used daily until the eggs are eyed. However, continued treatment of eggs until hatching occurs may be warranted based on fungal growth rates observed after treatments were discontinued.

LACK OF SPECIFICITY OF THE MOLECULAR DIAGNOSTIC METHOD FOR IDENTIFICATION OF APHANOMYCES ASTACI

A recent PCR-test developed for identification of Aphanomyces astaci , the organism responsible for crayfish plague, provided false positives for Aphanomyces frigidophilus , Aphanomyces repetans , and some Saprolegnia spp. Real-time PCR showed that with the designed primers, A. astaci and A. frigidophilus cannot be distinguished. The results of this study show that this particular crayfish plague PCR-test ought to be improved and that molecular-based techniques need to be contrasted to histological evidences and disease history.