Evaluation of the effectiveness of the use of the probiotic Natupro to increase natural resistance and prevent infectious diseases of fish

The use of natural compounds can be found in many species of plants, and this holds much antimicrobial potential against fish pathogens in animal husbandry and fish farmers. In the current study, we studied the antimicrobial activity of the ethanolic extract derived from the leaves of F. villosa against the Aeromonas species: A. sobria, A. hydrophila, and A. salmonicida subsp. salmonicida, as well as Serratia liquefaciens, Yersinia ruckeri, Pseudomonas fluorescens, Shewanella putrefaciens to evaluate the possible use of this plant in preventing infections caused by these fish pathogens in aquaculture. The current study was conducted as a part of an ongoing project between five universities undertaken in the frame of a cooperation program aimed at the assessment of medicinal properties of tropical and subtropical plants. The leaves of F. villosa, cultivated under glasshouse conditions, were sampled at M.M. Gryshko National Botanic Garden (NBG), National Academy of Science of Ukraine. The Aeromonas strains used in our studies were Aeromonas hydrophila (K886), Aeromonas sobria (K825) and Aeromonas salmonicida subsp. salmonicida (St30). These microorganisms originated from the bacterial strain collection of the Department of Fish Diseases at the National Veterinary Research Institute in Puławy, Poland, and had been isolated from fish of two farmed freshwater species exhibiting clinical signs of disease: common carp (Cyprinus carpio L.) (K886 and K825) and rainbow trout (Oncorhynchus mykiss Walbaum) (St30). Bacteria Serratia liquefaciens, Yersinia ruckeri, Pseudomonas fluorescens, Shewanella putrefaciens were isolated both from apparently healthy rainbow trout (Oncorhynchus mykiss Walbaum), as well as from individuals exhibiting clinical signs of the diseases, according to the procedure developed at the Department of Fish Disease of the National Veterinary Research Institute in Poland. Antimicrobial susceptibility of the tested strains was performed by the Kirby-Bauer disc diffusion method (1966) according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI, 2014), with our some modifications. Results of the antimicrobial screening revealed, that F. villosa possessed different antibacterial properties against fish pathogens. The ethanolic extract derived from leaves of F. villosa exhibited the maximum antimicrobial activity against Shewanella putrefaciens, Aeromonas sobria, and Pseudomonas fluorescens. The percentage of increase in the diameters of inhibition zones was 122.7% for Shewanella putrefaciens, 54.9% for Aeromonas sobria, and 48.5% for Pseudomonas fluorescens, respectively. F. villosa exhibited mild antibacterial properties against Serratia liquefaciens, Yersinia ruckeri, Aeromonas hydrophila, Aeromonas salmonicida subsp. salmonicida. Thus, the Shewanella putrefaciens strain exhibited the highest sensitivity to the ethanolic extract derived from leaves of F. villosa, while Serratia liquefaciens and Yersinia ruckeri strains were resistant to this extract. These findings demonstrate that plant extracts derived from plants belonging to the Ficus genus are potential sources of botanical drugs for controlling bacterial infection in aquaculture.

The material for this report was the results of bacteriological examination of fish of various species (silver carp (Carassius gibelio); silver carp (Hypophthalmichthys nobilis); white cupid (Ctenopharyngodon idella); common carp (Cyprinus carpio); rainbow trout (Oncorhynchus mykiss), fry of sturgeon of Lena (Acipenser baeri) and sterlet (Acipenser ruthenus)) and various age groups in 2016–2018. in 7 fish farms in Belarus. As a result of research, 21 species of bacteria have been registered in introduced fish species in pond farms that are potential causative agents of bacterial diseases of introduced fish. Two types of bacteria – Shewanella putrefaciens and Aeromonas hydrophila – are invasive and harm the fish industry. Studies have shown seasonal changes in the frequency of occurrence of Sh. putrefaciens and A. hydrophila in introduced fish. During January-March, the infection of fish with A. hydrophila bacteria was not high, increasing sharply in April-May. In June-July, a decrease is observed, and in August is a sharp decrease in the infection of the internal organs and blood of fish with aeromonads. Bacteria Sh. putrefaciens were found in fish much less frequently than A. hydrophila, however, some seasonal dependence in the change in the frequency of its occurrence in introduced species is observed. The peak of occurrence is in June. The maximum values of the frequency of occurrence of pathogens of bacterial infections were noted for A. hydrophila in April (80.0%), for Sh. putrefaciens – in June (33.0%). It should be noted that the active consumption of mixed fodder and natural feed by the fish leads to the restoration of the body’s immune status after winter.

This review aims to explore developments in the usage of multistrain probiotics in commercial freshwater‐cultured fish and to address factors that affect the efficacies of multistrain probiotics to enhance growth parameters, survival after bacterial infection and immune modulation in comparison to monostrain probiotics. From bibliographic searches in Scopus, Web of Science and PubMed, it was possible to select 81 articles that evaluated multistrain probiotics supplementation during in vivo trials in Nile tilapia (Oreochromis niloticus), rainbow trout (Oncorhynchus mykiss), common carp (Cyprinus carpio) and roho labeo (Labeo rohita). Most of the articles showed different benefits, including enhancement of fish growth performance, immune response and resistance against some pathogenic bacteria, such as Aeromonas hydrophila, Streptococcus agalactiae and Streptococcus iniae. However, only 13 journal articles included a monostrain probiotic as a control that would allow direct comparison with a probiotic bacterial mixture to determine if the mixture offered higher benefits in comparison to a monostrain probiotic and a control group without probiotic supplementation. Here we explain the main findings, including the best probiotic mixtures, and some perspectives on multistrain probiotics research and use on a commercial scale.

To investigate the response of natural resistance and immunological reactions of newborn young animals of the Volyn beef breed to the use of probiotic preparations. To conduct experimental research, three groups were formed from young Volyn meat breeds: the control group was fed only on mother's milk, the first group received a probiotic preparation with Bacillus Subtilis strain in addition to cow's milk, the second experimental group –– Lactobacillus spp. The activity of natural resistance and the body's immune response were studied on the 5th, 10th, and 30th day of the experiment. The level of nonspecific natural resistance was determined by indicators of bactericidal, lysozyme, phagocytic, and complementary activity of blood serum, and the immunological response was determined by the concentration of immunoglobulins of classes G, M, and A in the blood. It was established that starting from the 5th day of life, a difference in the nonspecific resistance of control and experimental animals was noted groups In separate age periods, probiotic supplements had a different degree of influence on the natural resistance of young animals. The greatest difference in the direction of growth in such indicators of non-specific resistance as bactericidal and complementary activity of blood serum was noted under the influence of the probiotic supplement Bacillus Subtilis (I experimental group), and indicators of lysozyme and phagocytic activity of blood serum were greater under the action of Lactobacillus spp. (II experimental group). The difference between indicators of bactericidal activity of blood under the action of Lactobacillus spp. in comparison with the control was at the age of 30 days (16.85 %, Р<0.01), and under the action of Bacillus Subtilis –– at the age of 60 days (25.49 %, Р<0.001). The level of phagocytic and complementary blood activity was the highest at the age of 60 days in the I experimental group –– by 17.17 % (Р<0.001) and 32.57 % (Р<0.001), in the II experimental group –– by 23.20 % (Р <0.001) and 36.34 % (Р<0.001), respectively. Regarding the concentration of immunoglobulins, the largest changes in their indicators were noted in the group that received Lactobacillus spp. Addition of probiotic strains of bacteria to the diet of young animals has a positive effect on the natural resistance and immune response of the body, which in the future will contribute to reducing the level of morbidity in young animals. Key words: calves, probiotics, bacterial strains, microorganisms, immunoglobulins, non-specific resistance of the organism, T-lymphocytes, phagocytic activity, bactericidal activity of blood serum.

During a 3-month period from June to the end of August 2016, ~5% mortalities were observed in a farm with rainbow trout (Oncorhynchus mykiss Walbaum) and one farm of common carp (Cyprinus carpio L.) in Bulgaria. The disease was manifested by gill ulcers/rot, asphyxiation and bloody ascites. Aeromonas hydrophila was isolated from the internal organs of all the diseased fish. Bacillus mycoides or B.pseudomycoides were recovered from the gill lesions on diseased carp and rainbow trout, respectively, with identification achieved by conventional phenotyping and by sequencing of the 16S rRNA gene. In vivo experiments confirmed that all three organisms were pathogenic to rainbow trout.

Tissue-specific Cu bioaccumulation patterns and differences in sensitivity to waterborne Cu in three freshwater fish: rainbow trout ( Oncorhynchus mykiss), common carp ( Cyprinus carpio), and gibel carp ( Carassius auratus gibelio)

Characterization of two potential probiotic strains of Levilactobacillus brevis isolated from carp (Cyprinus carpio L.) with strong immunostimulatory activity on pikeperch (Sander lucioperca L.) head kidney cells

Pathogenesis of Iridovirus: in vitro Influence on Macrophage Activity and Cytokine-Like Protein Production in Fish

Hormonal and ion regulatory response in three freshwater fish species following waterborne copper exposure

Effects of probiotics on growth, the toll-like receptor mediated immune response and susceptibility to Aeromonas salmonicida infection in rainbow trout Oncorhynchus mykiss

Respiratory control of arterial pH as temperature changes in rainbow trout Salmo gairdneri.

Summary In the present study, we investigate the possibility of spermatozoa motility activation resulted from freeze-thawing, defined by us as spontaneous sperm activation (SSA) in common carp (Cyprinus carpio L.), Eurasian perch (Perca fluviatilis L.), rainbow trout (Oncorhynchus mykiss), Northern pike (Esox lucius L.) and sterlet (Acipenser ruthenus L.) using cryopreservation methods described previously. To evaluate that, the motility percentage and velocity of frozen-thawed spermatozoa before and after dilution with activating media were used. The SSA phenomenon was found in carp and perch, but not in pike, rainbow trout and sterlet. The motility and velocity of spermatozoa for carp SSA were characterized by 16% (median value) and velocity 98 ± 2 μm s−1 (mean ± 0.95 CI), for perch 5% and 53 ± 5 μm s−1, correspondingly. Significant decrease of sperm velocity was observed, when activation media were applied for thawed sperm in carp, perch, trout and pike, while for sterlet this parameter became significantly increased. We believe that the phenomena of spontaneous activation by freeze-thawing processes are species-specific ones.

Hemolytic toxicity of Prymnesium parvum (B-type) reveals species-specific differences in freshwater fishes.

Assessment of seminal oxidative stress indicators is emerging as a valuable prognostic tool in assisted reproductive technology. We investigated levels of oxidative stress caused by reactive oxygen species (ROS) and the antioxidant enzyme defence system comprising superoxide dismutase (SOD), glutathione reductase (GR), and glutathione peroxidase (GPx) in seminal plasma (SP) of common carp Cyprinus carpio and rainbow trout Oncorhynchus mykiss over the course of the spawning season. Oxidation was determined in lipids and proteins by assessing thiobarbituric acid reactive species (TBARS) and the 2,4-dinitrophenylhydrazine carbonyl groups, respectively. Levels of SOD were assessed by the autoxidation of pyrogallol. Determination of GR and GPx was based on the rate of NADPH oxidation. We observed clear alterations in lipids and proteins over the course of the spawning season in both species. The highest levels of TBARS and CP were recorded late in the season. SOD was not significantly altered in either species. The activity of GR was higher in carp SP late in spawning compared to other times (P < 0.05), while in rainbow trout, GR significantly increased (24.13 ± 2.8 mU/mg protein) in mid-season. A significantly lower GPx activity (9.18 ± 1.32 mU/mg protein) was found in rainbow trout SP early in the spawning season, but no significant differences in GPx were observed over the course of the season in carp. These results provide further understanding of the role of fish SP antioxidants and present new data on the oxidant and antioxidant balance in SP during the spawning season that may be of value in the development of methods for artificial reproduction of teleost species.

Aeromonas salmonicida subsp. salmonicida is the etiological agent of furunculosis and a major fish health problem in salmonid aquaculture worldwide. Injection vaccination with commercial mineral oil-adjuvanted bacterin vaccines has been partly successful in preventing the disease but in Danish rainbow trout (Oncorhynchus mykiss, Walbaum) aquaculture furunculosis outbreaks still occur. In this study we tested the efficacy of experimental subunit vaccines against A. salmonicida infection in rainbow trout. We utilized in silico screening of the proteome of A. salmonicida subsp. salmonicida strain A449 and identified potential protective protein antigens that were tested by in vivo challenge trial. A total of 14 proteins were recombinantly expressed in Escherichia coli and prepared in 3 different subunit vaccine combinations to immunize 3 groups of rainbow trout by intraperitoneal (i.p.) injection. The fish were exposed to virulent A. salmonicida 7 weeks after immunization. To assess the efficacy of the subunit vaccines we evaluated the immune response in fish after immunization and challenge infection by measuring the antibody levels and monitoring the survival of fish in different groups. The survival of fish at 3 weeks after challenge infection showed that all 3 groups of fish immunized with 3 different protein combinations exhibited significantly lower mortalities (17–30%) compared to the control groups (48% and 56%). The ELISA results revealed significantly elevated antibody levels in fish against several protein antigens, which in some cases were positively correlated to the survival.