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![Multiple virulence factors are deployed by Saprolegnia parasitica and an overview of oomycete phylogeny. (A) Schematic representation of a Saprolegnia parasitica hypha (light blue) deploying virulence factors against a fish cell (salmon color). SpHtp1 is translocated inside the host cell, and other factors are secreted to the cell surface (lectins [green circles]) or the extracellular space (proteases [red circles], CHAPs [pink triangles], toxins [HlyE, which presumably targets the host membrane, green bolts] and nucleases [purple squares]). B. An overview of oomycete phylogeny. The main genera are displayed with the plant pathogenic lineages in green, animal parasites in red, and saprophytes in blue. Some genera, such as Pythium and Aphanomyces , include both plant and animal parasitic species. The early branching Eurychasma is an obligate pathogen of marine brown algae. doi:10.1371/journal.pgen.1003590.g001](publication/240306927/figure/fig1/AS:298616964042758@1448207240505/Multiple-virulence-factors-are-deployed-by-Saprolegnia-parasitica-and-an-overview-of.png)
Multiple virulence factors are deployed by Saprolegnia parasitica and an overview of oomycete phylogeny. (A) Schematic representation of a Saprolegnia parasitica hypha (light blue) deploying virulence factors against a fish cell (salmon color). SpHtp1 is translocated inside the host cell, and other factors are secreted to the cell surface (lectins [green circles]) or the extracellular space (proteases [red circles], CHAPs [pink triangles], toxins [HlyE, which presumably targets the host membrane, green bolts] and nucleases [purple squares]). B. An overview of oomycete phylogeny. The main genera are displayed with the plant pathogenic lineages in green, animal parasites in red, and saprophytes in blue. Some genera, such as Pythium and Aphanomyces , include both plant and animal parasitic species. The early branching Eurychasma is an obligate pathogen of marine brown algae. doi:10.1371/journal.pgen.1003590.g001
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... Oomycetes represent a diverse group of eukaryotic pathogens that can infect a wide range of hosts [1][2][3] Pathogens in the Saprolegnia genus can infect and kill crustaceans and fish in freshwater, especially in aquaculture where fish are raised at high densities [4]. The most widely studied members of this genus include S. diclina, S. ferax and S. parasitica [5]. ...
Saprolegniasis is an important disease in freshwater aquaculture, and is associated with oomycete pathogens in the genus Saprolegnia . Early detection of significant levels of Saprolegnia spp. pathogens would allow informed decisions for treatment which could significantly reduce losses. This study is the first to report the development of loop-mediated isothermal amplification (LAMP) for the detection of Saprolegnia spp. and compares it with quantitative PCR (qPCR). The developed protocols targeted the internal transcribed spacer (ITS) region of ribosomal DNA and the cytochrome C oxidase subunit 1 (CoxI) gene and was shown to be specific only to Saprolegnia genus. This LAMP method can detect as low as 10 fg of S . salmonis DNA while the qPCR method has a detection limit of 2 pg of S . salmonis DNA, indicating the superior sensitivity of LAMP compared to qPCR. When applied to detect the pathogen in water samples, both methods could detect the pathogen when only one zoospore of Saprolegnia was present. We propose LAMP as a quick (about 20–60 minutes) and sensitive molecular diagnostic tool for the detection of Saprolegnia spp. suitable for on-site applications.
... Over the last decades, the production of fish, crustaceans, shellfish, and amphibians through aquaculture has become the fastest growing food sector in the world. However, the growing business of aquaculture often suffers from heavy financial losses due to the development of infections caused by microbial pathogens, particularly by different species of the genus Saprolegnia [1]. The infection caused by this genus of pathogens is known as saprolegniosis; this infection occurs in all freshwater aquatic ecosystems and significantly attacks fish and eggs, mainly when certain changes occur in the culture environment, which causes the appearance of these oomycetes [2]. ...
In this study, the essential oil (EO) from Laurelia sempervirens was analyzed by GC/MS and safrole (1) was identified as the major metabolite 1, was subjected to direct reactions on the oxygenated groups in the aromatic ring and in the side chain, and eight compounds (4 to 12) were obtained by the process. EO and compounds 4–12 were subjected to biological assays on 24 strains of the genus Saprolegnia, specifically of the species 12 S. parasitica and 12 S. australis. EO showed a significant effect against Saprolegnia strains. Compound 6 presents the highest activity against two resistant strains, with minimum inhibitory concentration (MIC) and minimum oomyceticidal concentration (MOC) values of 25 to 100 and 75 to 125 µg/mL, respectively. The results show that compound 6 exhibited superior activities compared to the commercial controls bronopol and azoxystrobin used to combat these pathogens.
... Acute phase response protein such as lysozyme, ceruloplasmin, myeloperoxidase, protease, anti-protease and complement component are key players of innate immunity in response to pathogen infection and some are linked with resistance to bacterial diseases . On the other hand, pathogens continuously dispose of virulence factors to subvert host defences for their benefit and thereby helping to establish infection (Banfield and Kamoun, 2013). ...
Motile Aeromonas septicaemia (MAS), caused by Aeromonas hydrophila, is one of the most significant problem among the bacterial diseases responsible for causing mortality in catfishes. The objective this study was to understand the modulation of innate immune parameters and histopathological alteration in Indian catfish Clarias magur (Hamilton, 1822), following intraperitoneal injection with A. hydrophila. At 3 to 8 h post-infection (hpi), respiratory burst activity, myeloperoxidase, protease, total anti-protease and α2 macroglobulin increased significantly (p< 0.05) indicating immune response of host against pathogen invasion at an early stage of infection. The total protein, albumin, albumin/globulin ratio and bactericidal activity were significantly decreased indicating the response of pathogen to suppress host immune response to establish infection. Serum bactericidal activity, bacterial agglutination titre and lysozyme activity increased significantly at 24 hpi. Histopathological examination of infected fish revealed the accumulation of melanomacrophage centres, congestion, hepatocyte degeneration and vacuolated hepatocytes of liver; hyaline droplet accumulation, lymphocytes infiltration and diffuse necrosis of renal tubule in kidney; unilateral fusion of secondary gill lamellae, leukocytic infiltration as well as dilated central venous sinus in gills. Knowledge regarding the immune response in magur could be useful for developing strategies for improving disease resistance against A. hydrophila infection.
... Today, aquaculture supplies an estimated 50% of all fish consumed by humans globally [1]. However, the growing business of aquaculture often suffers from heavy financial losses due to the development of infections caused by microbial pathogens-particularly by oomycetes of the genus Saprolegnia [2]. These are thought to be endemic to all fresh water habitats around the world. ...
In the present study, seven 2’,4’-dihydroxydihydrochalcone derivatives (compounds 3–9) were synthesized and their capacity as anti-Saprolegnia agents were evaluated against Saprolegnia parasitica, S. australis, S. diclina. Derivative 9 showed the best activity against the different strains, with minimum inhibitory concentration (MIC) and minimum oomyceticidal concentration (MOC) values between 100–175 μg/mL and 100–200 μg/mL, respectively, compared with bronopol and fluconazole as positive controls. In addition, compound 9 caused damage and disintegration cell membrane of all Saprolegnia strains over the action of commercial controls.
... Aquatic oomycetes (water molds) cause problems and economic losses in fish hatcheries worldwide. Oomycete infections, particularly by different species of the genus Saprolegnia [1], can also occur as a secondary infection to bacterial or viral infections [2,3]. The management of Saprolegnia sp. ...
... The descriptors considered in the gas phase were: HBD 2 , 1/Lowest Orbital Molecular Unoccupied (LUMO −1 ), 1/η, 1/ω, and Log 10 ω. However, when Hansch's analysis of statistical depuration was completed, only the descriptors LUMO −1 and Log 10 ω were able to explain reactivity differences with 99.99% of certain (p < 0.0001) (see Equation (1) and Table S1 Supplementary Material). On the other hand, descriptors considered in the solvent phase were: square hydrogen bonding donor (HBD 2 ), 1/rotatable bonding (RB −1 ), 1/Highest Occupied Molecular Orbital (HOMO −1 ), the logarithmic difference between LUMO and HOMO (Log 10 LH), the electronic chemical potential (µ), and 1/hardness (η −1 ). ...
To investigate the anti-Saprolegnia activities of chalconic compounds, nine dialkoxychalcones 2⁻10, along with their key building block 2',4'-dihydroxychalcone 1, were evaluated for their potential oomycide activities against Saprolegnia australis strains. The synthesis afforded a series of O-alkylated derivatives with typical chalcone skeletons. Compounds 4⁻10 were reported for the first time. Interestingly, analogue 8 with the new scaffold demonstrated remarkable in vitro growth-inhibitory activities against Saprolegnia strains, displaying greater anti-oomycete potency than the standard drugs used in the assay, namely fluconazole and bronopol. In contrast, a dramatic loss of activity was observed for O-alkylated derivatives 2, 3, 6, and 7. These findings have highlighted the therapeutic potential of the natural compound 1 scaffold to be exploitable as a drug lead with specific activity against various Saprolegnia strains.
... Recently, Majeed et al. [38] reported that one of the serine type proteases secreted by A. invadans, is involved in degradation of rainbow trout and giant gourami's IgM. Similarly, a serine protease SPRG_14567 in oomycete Saprolegnia parasitica, has been reported to degrade the rainbow trout IgM [39,40]. Similar to the present findings, Yadav et al. [21] reported that, following A. invadans infection, antiprotease activity in susceptible L. rohita was significantly lower and it was suggested that the decline in antiprotease activity might have contributed to invasive spread of the oomycete A. invadans. ...
Infection with Aphanomyces invadans is one of the most destructive diseases of freshwater fishes. Indian major carps, the dominant cultured species in the Indian sub-continent are highly susceptible to this disease. Till date, there is no effective treatment for control of this disease and immunization can be one of the strategies to reduce disease-related losses. In the present study, inactivated germinated zoospores of A. invadans were evaluated as antigen in conjunction with and without adjuvant Montanide™ ISA 763 A VG, for assessing their efficacy in rendering protection against A. invadans infection. For the experiment, rohu Labeo rohita, (n = 160, 74 ± 12g) were divided into 4 groups (C, A, G and GA) with 40 fish in each group. The fish in groups i.e., C, A, G and GA were injected intraperitoneally with PBS, adjuvant emulsified with PBS, inactivated germinated zoospores, and inactivated germinated zoospores emulsified with adjuvant, respectively. After 21 days of immunization, the fish were given a booster dose as above. After 7 days of the booster dose, the fish were challenged with zoospores of A. invadans to determine the relative percent survival (RPS). The results revealed that all the fish in C, A and G group succumbed to infection (0% RPS), although there was delayed mortality in fish from A and G groups in comparison to the C group. However, the fish in GA group showed significantly higher (P < 0.05) protection (66.7% RPS). In addition, following booster immunization (before challenge), the antibody level in the GA group was significantly higher (P < 0.05) than the control group. In western blotting, sera from G and GA groups showed reactivity with peptides of about 54 KDa. Following challenge (on 14th day), the antibody level as well as total antiprotease activity in fish of all the groups was significantly decreased in comparison to pre-challenge, except fish in GA group not exhibiting any gross lesions. In addition, sera of surviving fish of GA group showed significant inhibition of germination of zoospores and germlings growth in comparison to other groups (P < 0.05). Further, histopathological examination of the muscle tissue revealed that, in fish of GA group without any gross lesions, there were well developed granulomas and extensive mononuclear cell infiltration restricted to the site of injection, whereas in other groups, there was extensive myonecrosis with proliferating hyphae. These preliminary findings indicate that inactivated germinated zoospores of A. invadans in combination with adjuvant could stimulate good immune responses and confer remarkable protection in rohu.
... Genes encoding peptidases are highly expressed in the mycelium of S. parasitica and the corresponding peptidases are able to degrade trout IgM (Jiang et al., 2013). It has been suggested that oomycete peptidases contribute to virulence by avoiding pathogen recognition by the fish immune system, thereby promoting successful mycelial colonization of the host tissues (Banfield and Kamoun, 2013). Alternatively, enrichment of peptidases in the mycelium may reflect a role in nutrition in environments rich in protein. ...
Several water mold species from the Saprolegnia genus infect fish, amphibians and crustaceans in natural ecosystems and aquaculture farms. Saprolegnia parasitica is one of the most severe fish pathogens. It is responsible for millions of dollars of losses to the aquaculture industry worldwide. Here, we have performed a proteomic analysis, using gel-based and solution (iTRAQ) approaches, of four defined developmental stages of S. parasitica grown in vitro, i.e., the mycelium, primary cysts, secondary cysts and germinated cysts, to gain greater insight into the types of proteins linked to the different stages. A relatively high number of kinases as well as virulence proteins, including the ricin B lectin, disintegrins and proteases were identified in the S. parasitica proteome. Many proteins associated with various biological processes were significantly enriched in different life cycle stages of S. parasitica. Compared to the mycelium, most of the proteins in the different cyst stages showed similar enrichment patterns and were mainly related to energy metabolism, signal transduction, protein synthesis and post-translational modifications. The proteins most enriched in the mycelium compared to the cyst stages were associated with amino acid metabolism, carbohydrate metabolism and mitochondrial energy production. The data presented expand our knowledge of metabolic pathways specifically linked to each developmental stage of this pathogen.
... These immunoglobulins immediately recognize the invading pathogens and aim to destruct them [45,46]. Banfield and Kamoun [47] have reported that degradation of IgM of rainbow trout by the proteases of S. parasitica is critical in order to cause the disease saprolegniasis. Jiang et al. [24] have also reported that at least one of the proteases produced by S. parasitica has the ability to degrade the IgM of rainbow trout, which suggests that the oomycete has the ability to manipulate and conquer the initial immune response of the fish. ...
Background
Proteases produced by many microorganisms, including oomycetes, are crucial for their growth and development. They may also play a critical role in disease manifestation. Epizootic ulcerative syndrome is one of the most destructive fish diseases known. It is caused by the oomycete Aphanomyces invadans and leads to mass mortalities of cultured and wild fish in many countries. The areas of concern are Australia, China, Japan, South and Southeast Asian countries and the USA. Extracellular proteases produced by this oomycete are believed to trigger EUS pathogenesis in fish. To address this activity, we collected the extracellular products (ECP) of A. invadans and identified the secreted proteins using SDS-PAGE and mass spectrometery. A. invadans was cultivated in liquid Glucose-Peptone-Yeats media. The culture media was ultra-filtered through 10 kDa filters and analysed using SDS-PAGE. Three prominent protein bands from the SDS gel were excised and identified by mass spectrometery. Furthermore, we assessed their proteolytic effect on casein and immunoglobulin M (IgM) of rainbow trout (Oncorhynchus mykiss) and giant gourami (Osphronemus goramy). Antiprotease activity of the fish serum was also investigated.
Results
BLASTp analysis revealed that the prominent secreted proteins were proteases, mainly of the serine and cysteine types. Proteins containing fascin-like domain and bromodomain were also identified. We could demonstrate that the secreted proteases showed proteolytic activity against the casein and the IgM of both fish species. The anti-protease activity experiment showed that the percent inhibition of the common carp serum was 94.2% while that of rainbow trout and giant gourami serum was 7.7 and 12.9%, respectively.
Conclusions
The identified proteases, especially serine proteases, could be the potential virulence factors in A. invadans and, hence, are candidates for further functional and host–pathogen interaction studies. The role of identified structural proteins in A. invadans also needs to be investigated further.
... More than 60% of known oomycetes are pathogens of plants (4) and have a devastating effect on many agriculturally important crops and ornamental plants. Members of the Saprolegniales order predominantly exhibit saprotrophic lifestyles and include the Aphanomyces animal and plant pathogens (5) as well as the fish-pathogenic Saprolegnia genus, known as "cotton molds" (6,7). The Peronosporales order consists largely of phytopathogens and includes the hemibiotrophic genus Phytophthora (the "plant destroyers"). ...
The oomycetes are a class of microscopic, filamentous eukaryotes within the stramenopiles-alveolate-Rhizaria (SAR) supergroup and include ecologically significant animal and plant pathogens. Oomycetes secrete large arsenals of effector proteins that degrade host cell components, manipulate host immune responses, and induce necrosis, enabling parasitic colonization. This study investigated the expansion and evolution of effectors in 37 oomycete species in 4 oomycete orders, including Albuginales, Peronosporales, Pythiales, and Saprolegniales species. Our results highlight the large expansions of effector protein families, including glycoside hydrolases, pectinases, and necrosis-inducing proteins, in Phytophthora species. Speciesspecific expansions, including expansions of chitinases in Aphanomyces astaci and Pythium oligandrum, were detected. Novel effectors which may be involved in suppressing animal immune responses in Ap. astaci and Py. insidiosum were also identified. Type 2 necrosis-inducing proteins with an unusual phylogenetic history were also located in a number of oomycete species. We also investigated the "RxLR" effector complement of all 37 species and, as expected, observed large expansions in Phytophthora species numbers. Our results provide in-depth sequence information on all putative RxLR effectors from all 37 species. This work represents an up-to-date in silico catalogue of the effector arsenal of the oomycetes based on the 37 genomes currently available.
... During the disease development process, hosts and pathogens are involved in a constant molecular arms race, with host deploying an array of defence mechanisms to prevent invasion and establishment of the pathogen. On the other hand, pathogens continuously develop virulence mechanisms to subvert host defences to their benefit and thereby helping to establish infection (Banfield and Kamoun, 2013). The outcome of this interaction is either adaptation and persistence of the pathogen or its clearance by the host immune response. ...
