Pathogenicity of Saprolegnia spp. to Atlantic salmon, Salmo salar L., eggs

Live and dead Atlantic salmon eyed eggs were challenged with eight different Saprolegnia isolates, selected because of their varied origins, known morphological characteristics and growth/germination pattern. Some isolates were also tested for pathogenicity to Atlantic salmon parr. Challenge of eggs was performed by exposure to spores in suspension or by co-incubation of live eggs with infected dead eggs. The phenotypic characteristics of the isolates were evaluated in relation to their observed pathogenicity from the challenge experiment, to identify possible virulence factors leading to egg-infection by Saprolegnia. The results from the experiments confirm that live eggs are refractory to infection with Saprolegnia spores in suspension and that an infection of live eggs can only occur from an infection nucleus represented by dead eggs or debris. It was observed that strains pathogenic to salmon parr were not particularly infective towards eggs, and the isolates that gave the highest infection rates to eggs were species considered to be saprotrophs.     

Distribution patterns of saprolegniosis cutaneous lesions in wild and farmed brown trout (Salmo trutta L.) obtained using a geographic information system (GIS)

A retrospective study was conducted using 250 clinical records of brown trout (Salmo trutta L.) with saprolegniosis by Saprolegnia parasitica, which had been collected from 8 rivers and 1 fish farm in the province of León (Spain). A geographic information system (GIS) was used to obtain skin lesion distribution patterns in males and females. Lesions in wild brown trout affected 15.31 ± 13.33% of the body surface, with a mean of 12.76 ± 6.56 lesions per fish. In addition, 51.23% of wild trout presented lesions with necrosis of the skin or fins. The pattern obtained when not distinguishing between sexes indicated that saprolegniosis lesions are mainly located above the lateral line and most frequently affect the dorsal cephalic region, the adipose fin, the peduncle and the caudal fin. However, differences were observed between males and females. Farmed trout presented a lower percentage of affected body surface (2.06 ± 4.36) and a lower number of lesions with and without necrosis because they received preventive treatment for saprolegniosis.     

Morphological and physiological characteristics of Saprolegnia spp. strains pathogenic to Atlantic salmon, Salmo salar L

Seventeen strains of Saprolegnia spp. were examined for morphological and physiological characteristics, and seven were examined for their pathogenicity to Atlantic salmon, Salmo salar L. Two of the Saprolegnia strains tested caused 89 and 31% cumulative mortality in challenged salmonids and were significantly more pathogenic than the other strains tested. The positive control (Saprolegnia parasitica ATCC 90213) caused 18% mortality, but this was not significantly higher than non-pathogenic strains (0-3% cumulative mortality). All the pathogenic Saprolegnia strains and two non-pathogenic strains had secondary cysts with long, hooked hairs, a characteristic which is claimed to be typical of S. parasitica. This characteristic is apparently necessary, but does not in itself determine the ability to cause mortality in Atlantic salmon. However, all the pathogenic Saprolegnia strains in the present study showed a significantly higher initial growth rate of cysts in sterilized tap water than did non-pathogenic strains. The results of the present study suggest that initial growth rate of germinating cysts in pure water, together with the presence of long hooked hairs on the secondary cysts, may be indicators of pathogenicity of Saprolegnia strains to Atlantic salmon.     

Experimental pathogenicity in rainbow trout, Oncorhynchus mykiss (Walbaum), of two distinct morphotypes of long-spined Saprolegnia isolates obtained from wild brown trout, Salmo trutta L., and river water

Juvenile rainbow trout, Oncorhynchus mykiss (Walbaum), were experimentally infected to investigate the pathogenicity of 20 isolates of two morphotypes of long-haired Saprolegnia obtained from wild brown trout, Salmo trutta L., and river water in Spain. The trout were exposed to 2 × 10⁵ and 3 × 10⁵ L^–1 zoospores. Saprolegnia infection could not occur without 'ami-momi' treatment. Pathogenicity varied greatly among isolates as mortality ranged from 0 to 100% of the fish. There was a statistically significant difference ( P  < 0.001) between the mortality caused by morphotype I isolates and that produced by those of morphotype II. The most pathogenic isolates usually belonged to morphotype II, consisting of isolates which had secondary cysts with bundles of hooked hairs which were shorter and less numerous than those of morphotype I; the morphotype I isolates usually had low pathogenicity. Lesions were most frequently found on the fins. Cultures detected the presence of Saprolegnia in internal organs. Histopathology of the intestine suggests that Saprolegnia may reach this and other organs via the blood stream from surface lesions.     

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).     

水霉及水霉病防治的研究进展

1水霉分类地位20世纪60年代,我国学者曾将水霉(Sapro-legnia)划入藻状菌纲、水霉目、水霉科。随着研究的深入,许多研究者采用Ainsworth等真菌分类系统,摒弃旧系统中藻状菌分类说法,将水霉分在真菌界、鞭毛菌门、卵菌纲、水霉目、水霉科。但随着对卵菌纲生物的形态学、细胞学、生理学和分子生物学等方面的深入研究,结果表明卵菌生活     

Use of salt to control ichthyophthiriosis and prevent saprolegniosis in silver perch, Bidyanus bidyanus

The ciliate protozoan, Ichthyophthirius multifiliis , and the fungus, Saprolegnia parasitica , cause the diseases ichthyophthiriosis and saprolegniosis respectively. Both diseases are difficult to control and can cause high mortalities of freshwater fish, including the Australian silver perch, Bidyanus bidyanus (Mitchell). The efficacy of salt (NaCl) in controlling and preventing these diseases in silver perch was evaluated in aquaria and tanks. Low pH levels were also evaluated as a control for ichthyophthiriosis. Concentrations of 2 or 3 g L⁻¹ salt controlled infestations of I. multifiliis , and fish were free of both theronts and trophonts by day 8 at temperatures of 17.3–21.3 °C and by day 6 at 19.2–23.5 °C. Fish treated with 1 g L⁻¹ salt remained infested and all fish in a control treatment (0 g L⁻¹ salt) died. Although the mean survival rates of infested fish at pH levels of 5 or 6 were only 13.9% and 7.6%, respectively, there were no theronts or trophonts on surviving fish after 12 days. Silver perch harvested from a pond and treated with 2 or 3 g L⁻¹ salt did not become infected with S. parasitica and survival was 100%, whereas 16.6% of untreated (0 g L⁻¹ salt) fish became infected and survival was only 66.7%. A concentration of 2 g L⁻¹ NaCl is recommended for the control of ichthyophthiriosis and the prevention of saprolegniosis in silver perch held in tanks, aquaria and re-circulating aquaculture systems.     

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.     

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.     

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.     

Biocontrol of saprolegniosis in rainbow trout (Oncorhynchus mykiss Walbaum) using two bacterial isolates (LE89 and LE141) of Pseudomonas fluorescens

The probiotic activity of 15 bacterial isolates that inhibit Saprolegnia parasitica in vitro was tested for the biocontrol of saprolegniosis in rainbow trout (Oncorhynchus mykiss Walbaum), adding the bacteria to tank water for 14 days at a concentration of 10⁶ bacteria ml^?1 water. Pseudomonas fluorescens LE89 and Pseudomonas fluorescens LE141 were effective in controlling experimental infection with S. parasitica since of the fish treated with LE89, 24.5% ± 16.27% (p < 0.05) became infected, as did 42.8% ± 8.41% (p < 0.05) of those treated with LE141. Given their protective effect when administered in water, their effect was also studied when administered in feed before and after experimental infection. Both bacterial isolates survived low pH levels and the action of bile, grew in skin and intestinal mucus, were resistant to several antibiotics and survived in feed; however, neither of the two isolates prevented S. parasitica infection when administered in feed.     

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.     

In vitro Antifungal activity of essential oils from Tymbra,Origanum, Satureja species and some pure compounds on the fish pathogenic fungus,Saprolegnia parasitica

In the present study, the chemical composition and antifungal activity of the essential oils of the three Lamiaceae species, black tyme (Thymbra spicata L.), oregano (Origanum onites L.) and savory (Satureja tymbra L.) against Saprolegnia parasitica Coker strains (3) were evaluated. The composition of oils was analysed by gas chromatography/mass spectrometry (GC/MS). The major constituent of the oils was carvacrol (82.68% in Thymbra spicata, 82.24% in Origanum onites and 52.38% in Satureja tymbra). The antifungal activity of essential oils was determined by a tube dilution and disc diffusion method. All essential oils inhibited fungal strains at concentration of 100 μL mL^?1. The essential oil of O. onites and T. spicata was the most active (MIC 10 and 50 μL mL^?1, respectively). Thymol did not show inhibiting activity. The essential oils exhibited stronger inhibition than pure carvacrol. Essential oil derived from Tymbra, Origanum and Satureja species has the potential to be used as health control of rainbow trout (Oncorhyncus mykiss) against S. parasitica infection. This study describes the antifungal activity of three essential oils for developing alternative and native control methods against Saprolegniasis.     

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.     

Ectopic epithelial cell clusters in salmonid intestine are associated with inflammation

An epizootic incidence of intestinal adenocarcinomas was reported in brood fish of Atlantic salmon (Salmo salar L.) in 2009. The condition was associated with a specific diet inducing enteritis and morphological changes. Here, two field trials of fish up to slaughter size were initiated. In Trial 1, two different feed recipes were used. Feed I was predominantly based on marine ingredients, whereas plant ingredients were limited to soy protein concentrate and wheat. Feed II was lower in fishmeal and without soya protein, which was substituted with plant proteins from other sources. In Trial 2, a commercial feed (Feed III) was included. No macroscopic tumours were observed in 300 fish (Trial 1). At the end of both trials, samples from five different segments of the gastrointestinal tract of a total of 39 fish were investigated with morphological methods. Here, we show the presence of ectopic proliferating epithelial cells only occurring in inflamed intestine and predominantly in the second segment of the mid‐intestine. Presence of ectopic epithelial cells in submucosal inflammatory foci may indicate early stages in tumorigenesis, but other possibilities such as proliferative enteric disorders cannot be excluded. Together with inflammation, carcinogenesis should be a focus of investigation in future feed trials.     

一株传染性造血器官坏死病病毒的致病性研究

为了对分离于山东某虹鳟养殖场的一株传染性造血器官坏死病毒株(IHNV-Sn1203)进行致病性检测与研究,将该IHNV-Sn1203毒株进行虹鳟鱼苗人工回接感染实验。结果显示,8d内人工感染实验鱼累计死亡率高达100%。收集大批濒死的病鱼样本,制备病理组织切片;利用鲤上皮细胞(EPC)进行细胞感染实验、病毒电镜观察、空斑实验、病毒滴度检测和聚类分析。病理组织切片显示,该病毒可造成虹鳟造血器官广泛性坏死;细胞感染实验结果显示,接种24 h后EPC细胞出现葡萄串状典型细胞病变(cytopathic effect,CPE),72 h后大部分细胞崩解脱落形成网状孔洞;电镜下清晰可见弹状病毒粒子大量存在于细胞质内,其在EPC细胞上的滴度为108.36TCID50/mL,并能形成2~4 mm空斑。对病毒核蛋白氨基酸序列的聚类分析结果显示,该病毒与标准毒株RB-1和WRAC的同源性分别为97%和93%,与国内报道的zyx株具有最高的同源性(99%)。研究表明,IHNV-Sn1203毒株能够在鱼体及敏感细胞中稳定繁殖,产生典型病变,具有较高的病毒滴度,对虹鳟鱼苗有很高的感染性和致死性。     

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).     

New salmonid hosts for Tenacibaculum species: Expansion of tenacibaculosis in Chilean aquaculture

The success and sustainability of Chilean aquaculture largely depends on the control of endemic and emerging pathogens, including several species of the genus Tenacibaculum. Tenacibaculum dicentrarchi and “Tenacibaculum finnmarkense” have been detected and confirmed in Chilean Atlantic salmon (Salmo salar). However, no outbreaks of tenacibaculosis in rainbow trout (Oncorhynchus mykiss) or coho salmon (Oncorhynchus kisutch) have been reported, either in Chile or globally. The aims of this study were to determine whether the mortalities recorded for rainbow trout and coho salmon from five marine fish farms located in the Los Lagos, Aysén, and Magallanes Regions could be caused by Tenacibaculum spp. The diseased fish exhibited cutaneous haemorrhages, tail and peduncle rots, and damage on the mouth and tongue. Microbiological analysis of infected external tissues yielded 13 bacterial isolates. The isolates were identified as members of the genus Tenacibaculum through biochemical analysis (e.g. Gram-stain negative, straight rods, filamentous cells and motile by gliding), but differences existed in biochemical results, making species-level identification through biomolecular tools essential. The 16S rRNA analysis found that the majority of isolates were more closely related to “T. finnmarkense” than T. dicentrarchi, while the phylogenetic trees resulting from multilocus sequence data recovered the four main clades (clades I to IV) identified by Olsen et al. (2017, Veterinary Microbiology, 205, 39). This is the first documented occurrence of clinical tenacibaculosis in farmed rainbow trout and coho salmon globally, and it extends the known host distribution of this pathogen in Chile. Moreover, we confirm the presence of Tenacibaculum species in the Chilean Patagonia. These findings highlight the importance of establishing preventative measures to minimize the spread of this disease within the Chilean marine aquaculture industry, as well as the need for monitoring initiatives worldwide in these farmed fish species.     

鲤浮肿病毒人工感染锦鲤的试验研究

为研究鲤浮肿病毒的感染方式及在锦鲤各组织器官中的动态分布,在水温20～22℃,溶解氧7～8mg/L试验条件下,采用注射、浸泡、划伤后浸泡3种方式人工感染锦鲤,观察了试验鱼的发病和死亡情况;检测了鲤浮肿病毒在感染鱼体内各组织器官中的存在情况,探讨了主要被感染组织的病理变化。试验结果显示,采用注射、浸泡、划伤后浸泡3种方式均可导致健康锦鲤感染该病毒,病鱼出现昏睡、烂鳃、凹眼等症状,累积死亡率35%～88%。锦鲤感染该病毒后至少有3d的潜伏期,随后病毒在鳃组织中大量增殖,至7d达到高峰,病毒量314.8个/ng,之后病毒量开始逐渐下降,至12d在鳃组织中检测不出病毒。鲤浮肿病毒在肾、肝、脑、肌肉和脾中存在的时间较短且病毒量较低,在血液和肠道中未检出。结合鳃组织病理切片可知,鳃为该病毒感染的主要靶器官。本研究结果为鲤浮肿病的诊断和防控提供了一定的科学依据。