Improving the aquatic organisms immune resistance with probiotics for the aquaculture sustainable development

One of the ways to strengthen the immunity of animals in aquaculture is the use of immunomodulators as a food supplement. This work presents a study on the effect of the probiotics Bacillus subtilis and Bacillus licheniformis on the immunity of crayfish and fish. It was found that the use of the probiotic “Subtilis-C” as a feed supplement strengthened the immunity of narrow-clawed crayfish: the survival rate increased almost threefold in the presence of Saprolegnia parasitica in the water and the incidence of burn-spot disease (BSD) decreased. The average cytochemical coefficient (ACC) of the lysosomal cationic protein in hemocytes decreased. It appears that crayfish consume this cytotoxic protein for immune protection. In carp fed with the probiotic, the immunity to saprolegniosis was strengthened, the body weight gain increased significantly, the amount of hemoglobin increased by 15%, and the number of erythorocytes in the blood almost doubled. An increase in metamyelocytes was noted in WBC differential count of these fish.

Copper/Carbon Core/Shell Nanoparticles: A Potential Material to Control the Fish Pathogen Saprolegnia parasitica

Copper-based fungicides have a long history of usage in agriculture and aquaculture. With the rapid development of metal-based nanoparticles, copper-based nanoparticles have attracted attention as a potential material for prevention and control of Saprolegnia parasitica. The present study investigated the effectiveness of copper/carbon core/shell nanoparticles (CCCSNs) and a commercial CCCSNs filter product (COPPERWARE®) against S. parasitica in a recirculating system. Results showed that the growth of agar plugs with mycelium was significantly suppressed after exposure to both CCCSNs powder and COPPERWARE® filters. Even the lowest concentration of CCCSNs used in our study (i.e., 100 mg/mL) exhibited significant inhibitory effects on S. parasitica. The smallest quantity of the filter product COPPERWARE® (3.75 × 3.7 × 1.2 cm, 2.58 g) used in our aquarium study also demonstrated significant inhibition compared with the control group. However, we observed leaching of copper into the water especially when larger quantities of COPPERWARE® were used. Water turbidity issues were also observed in tanks with the filter material. Besides these issues, which should be further investigated if the product is to be used on aquatic species sensitive to copper, CCCSNs has promising potential for water disinfection.

Evaluation of Potential Transfer of the Pathogen Saprolegnia parasitica between Farmed Salmonids and Wild Fish

Saprolegnia infections are among the main parasitic diseases affecting farmed salmonids. The distribution and potential transfer of Saprolegnia spp. between farms and the natural environment has been scarcely investigated. Therefore, this work aimed to study the diversity and abundance of oomycete species in salmonid farms, tributary water, and effluent water systems. Four trout farms in Italy and two Atlantic salmon farms in Scotland were considered. In Italian farms, 532 isolates of oomycetes were obtained from fish and water, at upstream, inside, and downstream the farms. In Scottish farms, 201 oomycetes isolates were obtained from water outside the farm and from fish and water inside the farming units. Isolates were identified to the species level through amplification and sequencing of the ITS rDNA region. In Italy, S. parasitica was significantly more present in farmed than in wild fish, while in water it was more frequently isolated from the wild, particularly in effluent systems, not associated with more frequent isolation of S. parasitica in wild fish downstream the farm. In Scotland, S. parasitica was the most prevalent species isolated from fish, while isolates from water were mostly Pythium spp. with few S. parasitica isolates from upstream and downstream the farms.

Efficacy of acriflavin chloride and Melaleuca alternifolia extract against Saprolegnia parasitica infection in Pterophyllum scalare

The article describes cases of saprolegniosis in Pterophyllum scalare in private aquaristics and assesses the therapeutic efficacy of acriflafin chloride against Saprolegnia parasitica infection. To establish the diagnosis, the clinical signs present in sick fish, the results of mycological and microscopic examinations are taken into account. Some chemical and mycological indices of aquarium water have been studied, and also mycological studies of fish feed have been carried out. It is established that the disease of fish develops against the background of adverse changes in physical, chemical composition and microbiocenosis of aquarium water. Low water temperature, high levels of phosphates and pH, a significant level of organic pollution, compared to the norm, provoke the accumulation of opportunistic microbiota, resulting in imbalance in the parasite-host system and the development of clinical manifestations of saprolegniosis in fish. It was found that 44.4% of the studied feed samples fed to fish were contaminated with epiphytic micromycetes. Micromycetes are represented by the genera Aspergillus, Penicilium, Fusarium, Mucor, Rhizopus. Among the studied feeds, the most affected by fungi were larvae of Chironomus plumosus and dry Daphnia pulex. According to the results of our studies during outbreaks of saprolegniosis, the pH of aquarium water was 8.1 ± 0.7, the content of phosphates – 5.6 ± 1.1 mg/L, micromycetes – 18.0 ± 1.2 CFU/100 cm3. Aspergillus flavus, A. niger and Penicillium canescens were detected in the studied water samples. With saprolegniosis, the angelfish have a reduced appetite, spots, ulcers, white thin threads, and a cotton-like plaque appear on certain areas of the skin, fins, eyes, and gills. It is established that effective means for the treatment of sick fish are external use in the form of a long bath of acriflavine chloride and extract of Melaleuca alternifolia. It is also effective to increase the water temperature to 25–27 °С, to ensure the normative fish-holding density in aquariums and to exclude from the diet fish feed contaminated with micromycetes. After using the drugs for two weeks every other day, water was replaced by 20% of the aquarium volume and aerated. As a result of the treatment, gradual healing of skin lesions and recovery of 65% of fish with signs of lesions of the outer coverings were registered. Thus, the article analyzes the causes of saprolegniosis in angelfish common in private aquariums, describes the clinical signs of the disease and assesses the therapeutic efficacy of acriflavine chloride and Melaleuca alternifolia extract against Saprolegnia parasitica infection. Prospects for further research lie in search of more effective and environmentally friendly means for the treatment of saprolegniosis in aquarium fish.

Detection of fish pathogen Saprolegnia parasitica in environmental DNA samples by droplet digital PCR

Oomycetes are fungal-like microorganisms parasitic towards a large number of plant and animal species. Genera from order Saprolegniales, such as Saprolegnia and Aphanomyces, cause devastating infections of freshwater animals. Saprolegnia parasitica is a widely distributed oomycete pathogen that causes saprolegniosis, a disease responsible for significant economic losses in aquaculture, as well as declines of natural populations of fish and other freshwater organisms. Despite its negative impact, no monitoring protocol for S. parasitica has been established to date. Thus, we aimed to develop a droplet digital PCR (ddPCR) assay for the detection and quantification of S. parasitica in environmental DNA samples. Saprolegnia parasitica-specific primers were designed to target internal transcribed spacer region 2 (ITS 2), based on the alignment of ITS sequences of S. parasitica and a range of Saprolegnia spp., as well as other oomycetes. The specificity of primers was tested using genomic DNA of S. parasitica (as positive control) and DNA of non–S. parasitica oomycete isolates, as well as trout/crayfish DNA (as negative control). The primers specifically amplified a segment of the ITS region of oomycete pathogen S. parasitica, while no amplification (i.e. no positive droplets) was obtained for closely related Saprolegnia spp. (e.g. Saprolegnia sp. 1 and S. ferax) and other more distantly related oomycetes. Next, the limit of detection (LOD) of the assay was established by using serial dilutions of the S. parasitica genomic DNA. The determined sensitivity of the assay was high: LOD was 15 fg of pathogen’s genomic DNA per µL of the reaction mixture. Assay performance was further assessed with environmental DNA samples isolated from water from the trout farms and natural environments, as well as (ii) biofilm from the host surface (swab samples). Water samples were collected from 21 different locations in Croatia, while swab samples were collected from S. parasitica host/carrier species: (i) skin and eggs of the rainbow trout (Oncorhynchus mykiss Walbaum, 1792) and brown trout (Salmo trutta Linnaeus, 1758), and (ii) cuticle of signal crayfish (Pacifastacus leniusculus Dana, 1852) and narrow clawed crayfish (Pontastacus leptodactylus Eschscholtz, 1823). Samples were classified into agent levels A0 to A6, depending of the number of S. parasitica ITS copies per ng of total DNA. Saprolegnia parasitica was detected in 76 % of water samples (16/21) and the range of pathogen’s ITS copies in positive samples was between 0.02 and 14 copies/ng of total DNA (agent levels A1 to A3). Regarding the swab samples, S. parasitica load was significantly higher in diseased trout than in those with healthy appearance: 9375 vs 3.28 S. parasitica copies/ng of total swab DNA (average agent level A6 vs. A2, respectively). Despite the fact that none of the sampled crayfish had signs of infection, the pathogen was detected in all tested cuticle swabs. Swabs of P. leniusculus, a known S. parasitica host, had significantly higher S. parasitica load than swabs of P. leptodactylus, S. parasitica carrier: 390 vs 83 S. parasitica copies/ng (agent level A5 vs. A4, respectively). In conclusion, our results demonstrate the applicability of the newly developed ddPCR assay in monitoring and early detection of S. parasitica in aquaculture facilities and natural freshwater environments. This would help in a better understanding of S. parasitica ecology and its effects on the host populations.