Dried leaves of Rosmarinus officinalis as a treatment for streptococcosis in tilapia

Dietary application of dried Rosmarinus officinalis (rosemary) leaves as a treatment for streptococcal infection was studied in tilapia, Oreochromis sp. Feeding with dried rosemary leaves significantly reduced mortality following infection with Streptococcus iniae: 44% mortality in the group fed 8% rosemary, similar to oxytetracycline treatment (43% mortality), and significantly lower than the control (65%). Dietary administration of 16% rosemary significantly reduced mortality because of Streptococcus agalactiae infection in 44 g fish (62% and 76% in 16% rosemary and control, respectively), but not in a similar experiment conducted with 5.5 g fish. The antibacterial effect of rosemary on S. iniae was studied. Activity of rosemary cultivar Israel was reduced during the winter, but there was no significant change in cultivars Oranit and Star. Storage of powdered rosemary leaves at 50 °C resulted in fourfold and eightfold higher MIC_{24 h} values after 3 and 4.5 months, respectively. Storage at ?20 °C, 4 °C and 25 °C and autoclaving (120 °C) each resulted in a twofold increase in MIC_{24 h}. Repeated exposures of S. iniae to rosemary did not affect minimal inhibitory concentration, suggesting no development of resistance to rosemary.     

Antimicrobial Activity of Selected Essential Oils on Pseudomonas Species Associated with Spoilage of Fish with Emphasis on Cinnamon Essential Oil

ABSTRACT

Eight plant essential oils (cardamom, cinnamon, clove, eucalyptus, lemongrass, lime, nutmeg and rosemary) were evaluated for their antimicrobial activity against three Pseudomonas species associated with microbial spoilage of refrigerated tilapia. In vitro test of the essential oils, using disc-diffusion method, showed various degrees of antimicrobial activity against the Pseudomonas species. Cinnamon essential oil (CEO) had the highest antimicrobial activity, followed by clove essential oil. The remaining essential oils showed consistently weak activity. The antimicrobial efficiency of CEO against the Pseudomonas species was confirmed by potential minimum inhibitory concentration and minimum bactericidal concentration values, ranging between 0.125 and 0.375 µL/mL and 0.250–0.500 µL/mL, respectively. The anti-Pseudomonas activity of CEO was also tested in fish extract model at refrigeration temperature. The essential oil reduced the Pseudomonas viable count in fish extract but to a lesser extent than when it was applied at the same concentrations in culture medium. The chemical composition of CEO showed that the oil contained 79.1% cinnamaldehyde, which was suggested to be responsible for its antimicrobial activity. These results revealed the potential of CEO as a promising natural antimicrobial agent that could efficiently contribute to the control of spoilage bacteria and retard microbial spoilage of fresh fish.     

Assessment of Aquatic Aeromonas spp. Isolates' Susceptibility to Cinnamaldehyde,Vanillin, and Crude Kigelia africana Fruit Extracts

Aeromonas spp. are primary and opportunistic fish pathogens that are responsible for severe economic losses in aquaculture, and are exacerbated by their increasing incidence of antimicrobial resistance. Phytochemicals are being explored as alternatives to conventional antimicrobial agents since they have destressing, growth‐promoting, immune‐stimulating, and antimicrobial properties. The susceptibility of 93 aquatic Aeromonas spp. and six Plesiomonas shigelloides isolates (from cultured catfish, koi carp, tilapia, and seawater) to three phytochemicals, viz.: cinnamaldehyde (10–1250 µg/mL), vanillin (5–250 µg/mL), and crude Kigelia africana fruit extracts (4–10 mg/mL ethyl acetate, dichloromethane, methanol, and hexane) were assessed using the disk diffusion assay and compared to ampicillin and tetracycline, using activity indices. No zones of inhibition were obtained with 10 µg/mL of cinnamaldehyde, or with 5–250 µg/mL of vanillin. However, varying degrees of inhibition were observed with higher concentrations of cinnamaldehyde, as well as with the K. africana methanol extract. Cinnamaldehyde (≥500 µg/mL) and the K. africana methanol extract displayed better antimicrobial activity against study isolates in comparison to vanillin and ampicillin. They appear to be promising and sustainable phytochemicals that might be useful as alternatives to the antimicrobial agents currently in use in aquaculture.     

Potential of cinnamon (<Emphasis Type="Italic">Cinnamomum verum</Emphasis>) oil to control <Emphasis Type="Italic">Streptococcus iniae</Emphasis> infection in tilapia (<Emphasis Type="Italic">Oreochromis niloticus</Emphasis>)

In this study, four essential oils—cinnamon oil, leech lime oil, lemongrass oil, and turmeric oil—were examined for their antimicrobial activities against Streptococcus iniae, a bacterium that is pathogenic in fish, in which it causes streptococcosis. Cinnamon oil was the most potent antimicrobial agent among these oils, with a minimal inhibitory concentration (MIC) of 40 μg/ml. By using gas chromatography–mass spectrometry (GC–MS), it was found that the major components of cinnamon oil were cinnamaldehyde (90.24), limonene (2.42%), cinnamyl acetate (2.03%), linalool (1.16%), and α-terpineol (0.87%). Of these compounds, only cinnamaldehyde exhibited antimicrobial activity against S. iniae, with an MIC of 20 μg/ml. In an in vivo trial, no mortality was apparent in fish fed on fish diets supplemented with 0.4% (w/w) of cinnamon oil and with 0.1% (w/w) of oxytetracycline 5 days prior to infection with S. iniae. These results indicate that cinnamon oil had a protective effect on experimental S. iniae infection in tilapia, and thus has the potential to replace the antibiotics used to control this disease.     

Dietary Beta-Glucans and Nucleotides Enhance Resistance of Red-Tail Black Shark (Epalzeorhynchos bicolor, fam. Cyprinidae) to Streptococcus iniae Infection

Two experiments were conducted to examine the effect of commercially available beta‐glucan (Macrogard) and nucleotide (Aquagen) on the resistance to Streptococcus iniae infections in vaccinated or nonvaccinated juvenile red‐tail black sharks (Epalzeorhynchos bicolor, RTB) (1.4 ± 0.4 g weight total (WT), 5.6 ± 0.5 cm total length (TL)). The immunostimulants were added to a control diet formulated without any yeast source following the recommended doses of 1 g/kg feed for the beta‐glucan and 2 g/kg feed for the nucleotide. Beginning 4 d after introduction into tanks, fish were fed the experimental diets for 24 d, at 3% body weight per day, divided into two feedings. At the end of this period, fish were challenged by an intracoelomic injection of S. iniae. In the first experiment, both vaccinated and nonvaccinated fish were fed one of the two immunostimulants. In the second experiment, only vaccinated fish were fed the immunostimulants. Fish were vaccinated a week after being introduced into the system and challenged by intracoelomic injection with 1.5 × 10⁵ S. iniae colony‐forming units /fish after three additional weeks; mortality was recorded for 2 wk after the bacterial challenge. In the first experiment, the mortality of both vaccinated and nonvaccinated fish fed beta‐glucan (23 ± 7% and 82 ± 1%, respectively) or nucleotide (28 ± 6% and 86 ± 5%, respectively) was significantly lower than the mortality of the control groups (35 ± 4% and 93 ± 5%, respectively), but there was no significant difference in mortality between fish fed beta‐glucan or nucleotide. In the second experiment, the mortality of vaccinated fish fed beta‐glucan (25 ± 7%) or nucleotide (43 ± 9%) was significantly lower than that of vaccinated fish fed the control diet (69 ± 7%). In both experiments, there was no significant difference in growth rate among fish fed immunostimulants or the control diet. The results of this investigation demonstrated the efficacy of beta‐glucans and nucleotides in increasing resistance to S. iniae in RTB sharks.     

Antimicrobial activity of the biopolymer chitosan against Streptococcus iniae

The antimicrobial activity and mode of action of chitosan were evaluated against Streptococcus iniae, a pathogenic Gram‐positive bacterium of fish worldwide. Cell proliferation kinetics were examined following exposure to varying concentrations of chitosan. The action of chitosan on S. iniae was also investigated by measuring agglutination activity, conductivity, and extracellular and intracellular bacterial adenosine triphosphate (ATP) levels. Chitosan exhibited antibacterial activity against S. iniae at concentrations of 0.1% and above and was lethal at a concentration of 0.4% and higher. The mechanism of antibacterial activity of chitosan at the inhibitory level of bacterial growth appears to hinge upon the interaction between chitosan and the oppositely charged bacterial surface. This interplay causes agglutination, which was readily observed grossly and microscopically. After interacting with the cell surface via adsorption, an efflux of intracellular ATP was documented, which suggests that chitosan disrupts the bacterial cell causing leakage of cytosolic contents and ultimately cell death. Results suggest chitosan may be worth evaluating as a natural alternative to antibiotic against S. iniae infection of fish.     

Therapeutic and prophylactic immunization against Streptococcus iniae infection in hybrid striped bass (Morone chrysops×Morone saxatilis)

Vaccination strategies have traditionally been used as preventative or prophylactic measures against disease (prophylactic immunization) in uninfected fish. Alternatively, therapeutic or remedial measures, such as antibiotic administration, are commonly employed to treat disease in infected fish. Vaccination as a therapeutic measure (therapeutic immunization), however, has not been adequately explored in sub‐clinically infected fish. Therapeutic and prophylactic immunization with three Streptococcus iniae vaccines, formalin‐killed whole S. iniae cells (FKC vaccine), concentrated S. iniae extracellular products (greater than 2 kDa) (ECP vaccine) and a combination of killed cells and extracellular products (FKC+ECP vaccine), were tested in hybrid striped bass, Morone chrysops×Morone saxatilis, previously naturally infected with S. iniae. Fish (mean weight 10.0 g) were injected intraperitoneally (IP) or intramuscularly (IM) with one of each of the vaccines, tryptic soy broth (TSB‐control) or non‐injected (non‐injected control) to evaluate therapeutic effects (Trial 1). Survivors of the natural infection and ECP and FKC+ECP vaccine immunization and another lot of non‐injected control fish were immersion challenged with 1.47 × 10⁶ CFU of S. iniae mL^?1 at 44 days post‐immunization to evaluate vaccine efficacy (Trial 2). Hybrid striped bass (1.0 g) were also IM injected with S. iniae ECP vaccine at an aquaculture facility and immersion challenged with 1.47 × 10⁶ CFU of S. iniae mL^?1 12 weeks post‐immunization (Trial 3). The ECP and FKC+ECP vaccines, regardless of injection route, significantly (P<0.001) increased survival in asymptomatic, sub‐clinically infected fish thereby providing therapeutic merit. Hybrid bass immunized IP or IM had mean per cent survival values ranging from 78 to 96 at 44 days post‐immunization (Trial 1) and 69–97 post challenge (Trial 2). Survival of fish injected with TSB or immunized with FKC vaccine was significantly lowered and ranged from 12 to 13 by IP injection and 40 to 50 by IM injection and thus, the FKC vaccine had no therapeutic effect. The survival of hybrid striped bass IM immunized with S. iniae ECP vaccine in field Trial 3 was 91 and the RPS was 83. These results demonstrate that therapeutic immunization using S. iniae ECP and FKC+ECP vaccines can control a natural S. iniae infection. Furthermore, S. iniae ECP or FKC+ECP vaccines can also be used prophylacticly to protect hybrid striped bass against subsequent pathogen challenge.     

The Effect of Sodium Caseinate Coating Incorporated with Zataria moltiflora Essential Oil on the Quality and Shelf Life of Rainbow Trout During Refrigerated Storage

ABSTRACT

In the present study, aqueous solutions of sodium caseinate (SC) 8% (w/w) were provided in the form of four treatments—including SC without any essential oil (EO) as well as SC with 0.25, 0.5, and 1% (v/w) concentrations of Zataria moltiflora EO. Fish samples were treated with aqueous solutions and stored at 4 ± 1°C for 20 days. The control and treated fish samples were analyzed for chemical (total volatile basic nitrogen, pH, thiobarbituric acid, peroxide value), microbiological (total viable count and psychrotrophic count), and sensory analyses every 4 days. Based on results, SC edible coating had no antimicrobial effect but acted like a barrier against the fish flesh contamination. Moreover, incorporating Zataria moltiflora EO with SC edible coating reduces peroxide value and thiobarbituric acid-reactive substances values as well as the chemical and microbiological spoilage of refrigerated ?sh samples. Furthermore, sensory evaluation results showed that the presence of thyme oil improved the sensory quality of the rainbow trout.     

Development of a real‐time PCR assay for detection and quantification of Streptococcus iniae using the lactate permease gene

The aim of this study is the development and evaluation of a rapid and accurate quantitative PCR (qPCR)‐based protocol for detection of zoonotic pathogen Streptococcus iniae in bacterial cultures and tissues of diseased fish. For this purpose, the lactate permease‐encoding (lldY) gene was selected as a target for the design of S. iniae‐specific primers based on comparative genomic analysis using 45 sequences retrieved from NCBI genome database. Specificity and applicability of these primers were tested using 115 bacterial strains and fish tissues infected with S. iniae. Sensitivity, reproducibility and efficiency of qPCR assay were also determined. The developed qPCR assay showed 100% specificity with pure bacterial cultures or DNA extracted from S. iniae or tissues of fish infected with the bacterium. The method has high sensitivity with a detection limit of 1.12 × 10¹ amplicon copies per assay (equivalent to 2 × 10^–9 ng/µl) using bacterial DNA and of 1.44 × 10¹ gene copies in tissues of fish infected with S. iniae. In conclusion, this qPCR protocol provides an accurate and sensitive alternative for the identification of S. iniae and its detection on fish tissues that can be implemented as a routine tool in microbiological laboratories.     

Development of an ELISA to measure the humoral immune response of hybrid striped bass Morone chrysops×M. saxatilis to Streptococcus iniae

A previously developed monoclonal antibody (mAb) specific for the heavy chain of hybrid striped bass (HSB) Morone chrysops×M. saxatilis immunoglobulin was used in an assay to detect the humoral response to antigens of Streptococcus iniae. In order to validate this assay, an anti‐S. iniae antibody was produced in HSB by immunization with formalin‐killed cells of S. iniae mixed with Freund's complete adjuvant (FCA). After boosting with cells mixed with Freund's incomplete adjuvant (FIA), fish were challenged with live S. iniae by i.p. injection. This resulted in mortalities of 100%, 13% and 7% for non‐immunized, immunized and non‐challenged fish respectively. Live S. iniae were recovered only from moribund non‐immunized challenged fish, indicating that the immunization conferred protection against S. iniae. Sera were taken at 28 and 42 days post challenge and anti‐S. iniae antibody was measured by both agglutination and indirect ELISA. Titres of anti‐S. iniae antibody increased only in the immunized group as measured by agglutination and ELISA. Serum complement measured in the final bleeding was significantly higher in the immunized group. Western blotting using immune HSB serum indicated that the predominant antigen in this case was the high molecular weight polysaccharide of S. iniae.     

Growth response and acquired resistance of Nile tilapia, Oreochromis niloticus (L.) that survived Streptococcus iniae infection

This study determined the growth performance and acquired resistance of Nile tilapia, Oreochromis niloticus (L.) that survived Streptococcus iniae infection. Tilapia were challenged with three doses of S. iniae (8.8 × 10³, 8.8 × 10⁴ and 8.8 × 10⁵ CFU fish^?1 for low, medium and high challenges respectively). Groups of non‐injected and tryptic soy broth‐injected fish were maintained as controls. Significantly (P<0.05) higher mortality (45.0%) occurred in the high challenge treatment than in the low challenge treatment group (29.6%). The medium challenge group had mortality (36.3%) that did not differ significantly from the high or low treatment. Few fish died in the non‐injected and broth‐injected treatments (3.4% and 0.8% respectively). The tilapia that survived S. iniae infection used to assess growth performance were selected from survivors without gross clinical signs of disease. These fish were randomly stocked at a rate of 30 fish into each 57 L aquarium in triplicate and fed to apparent satiation for 8 weeks. No significant differences were detected in weight gain, feed intake, feed efficiency ratio or survival between S. iniae‐survived tilapia and the control treatments following the 8‐week growth performance trial. Following the 8‐week feeding study, tilapia were challenged with 1 × 10⁶ CFU fish^?1 of S. iniae to assess acquired immunity. Mean cumulative mortality was significantly higher (P<0.05) in the control treatments (41.7% for the non‐injected and 43.3% for the broth‐injected fish) than in the low, medium and high challenge treatments (7.4%, 3.3% and 8.3% respectively). Serum protein was significantly (P<0.05) elevated in the S. iniae‐survived tilapia that were subsequently challenged when compared with controls challenged for the first time. Agglutinating antibody titre was significantly higher in the fish in the medium and high challenge treatments, compared with the control fish challenged for the first time. The results suggest tilapia that survive S. iniae challenge without showing overt disease signs performed as well as non‐infected tilapia. Further, the S. iniae‐survived tilapia challenged following the 8‐week growth performance trial gained acquired resistance to homologous S. iniae challenge.     

Simplified processing method of banana (Musa acuminata) peels possess the improvement in immunological responses of Macrobrachium rosenbergii

Musa acuminate peel extract as an immunostimulant, administrated in the diets of Macrobrachium rosenbergii, and an assessment of a simplified process to develop low‐cost feed additives are conducted in this study. The products obtained serially, during the processes of hot‐water banana peel extraction, were administrated in the diets of M. rosenbergii, including dried banana peel powder (DBP) at 8 g/kg, hot‐water‐treated banana peel (HBP) at 8 g/kg and hot‐water extract of banana peel (BPE) at 2 g/kg during the 32 days of the feeding trial. Total haemocyte count, different haemocyte count, respiratory bursts; and activity of superoxide dismutase, glutathione peroxidase, phenoloxidase and transglutaminase, as well as an accelerated haemolymph clotting time, significantly increased in M. rosenbergii fed with diets containing HBP and BPE, 32 days post‐feeding. Furthermore, the phagocytic activity and clearance efficiency of prawns against Lactococcus garvieae infection increased significantly. The respective relative survival percentages of prawns fed with HBP, and BPE containing diets after 32 days of feeding trial were 29.2% and 41.7% against L. garvieae infection for 144 hours, and 50%, and 50% against hypothermal stress for 96 hours of exposure. We may therefore conclude that HBP, obtained from a simplified procedure, without centrifugation and lyophilization, may strategically promote tolerance to hypothermal stress, and enhance immunity and resistance to L. garvieae infection.     

Protection against heterologous Streptococcus iniae isolates using a modified bacterin vaccine in Nile tilapia,Oreochromis niloticus (L.)

Streptococcus iniae is a significant pathogen impacting aquaculture production worldwide. The objectives of this study were to determine whether a developed modified S. iniae (ARS-98-60) bacterin vaccine is efficacious in Nile tilapia, Oreochromis niloticus (L.), against challenge with heterologous isolates from diverse geographical locations and to evaluate protein and antigenic variability among the isolates tested. Two groups of tilapia (approximately 5 g) were intraperitoneally (IP) vaccinated with 100 μL of the vaccine or sham vaccinated with 100 μL of sterile tryptic soy broth and held for 28 days. Fish were challenged with each isolate by IP injection of 2–3 × 10⁷ CFU per fish using calcein to mark fish prior to cohabitation for challenge. The results demonstrated significant protection against all challenge isolates, and relative percent survivals ranged from 79% to 100%. SDS–PAGE analysis of whole-cell lysate proteins from the S. iniae isolates demonstrated similar protein profiles between 10 and 31 kDa and variation in profiles between 35 and 100 kDa. Western blot analysis using antiserum from vaccinated fish (ARS-98-60) demonstrated shared immunogenic proteins among all isolates in the molecular mass range of 22–35 kDa and high molecular mass material >150 kDa. The results suggest that the developed S. iniae vaccine has broad ranging protection among isolates exhibiting different protein profiles.     

Improving the Quality of Silver Carp Fish Fillets by Gamma Irradiation and Coatings Containing Rosemary Oil

The objective of this work was to study the combined effect of gamma irradiation and coatings containing 0.5% rosemary (Rosmarinus officinalis) essential oil (RO) on the chemical, microbiological, and sensorial qualities of silver carp (Hypophthalmichthys molitrix) fish fillets (SFF) during cold storage (4°C). SFF were divided into three groups: uncoated (control), coated with edible coating (without additives), and γ-irradiated (0, 1, 3, and 5 kGy) coated with coating containing 0.5% rosemary. Gamma irradiation at 1, 3, and 5 kGy with coating reduced the initial total bacterial count, psychrophilic bacteria, and lactic acid bacteria and prolonged shelf life of the samples. Coated samples irradiated at 1 kGy reduced the counts of Enterobacteriaceae, Staphylococcus aureus, and Bacillus cereus, as well as eliminating Vibrio spp. and Salmonella spp., while coated samples irradiated at 3 and 5 kGy completely eliminated these bacteria. Combined treatment showed a slight increase in thiobarbituric acid-reactive substances postirradiation during cold storage but had no effect on the total volatile basic nitrogen and trimethylamine contents, while a gradual increase in these chemical quality indices was observed during cold storage. Combined treatment had no adverse effects on the sensory properties of SFF. The increase in the bacterial inhibitory effect is caused by both rosemary oil and irradiation.     

Influence of Chitosan Nanocomposite and Rosemary (Rosmarinus officinalis L.) Extract Coating on Quality of Huso huso Fillet Inoculated with Listeria monocytogenes During Refrigerated Storage

This study aimed to evaluate the antilisterial and antioxidative effects of chitosan nanocomposites and rosemary extract coating on the fillet of Huso huso inoculated with Listeria monocytogenes during refrigerated storage (4°C). Fish fillets were subjected as control (without coating), 0.5% rosemary extract (RE), 1% chitosan (CS), and combination of chitosan and rosemary extract as chitosan nanocomposite (CS/RE). Then, samples were inoculated with L. monocytogenes. Subsequently, the chemical parameters (total volatile basic nitrogen (TVB-N), peroxide value (PV), pH, and thiobarbituric acid (TBA)) and antilisterial effects of coatings were monitored during 16 days of storage at 4°C. According to the results, CS/RE demonstrated a significant (p < 0.05) ability to inhibit the growth of L. monocytogenes from 4.14 log cfu/g to 2.23 log cfu/g at the end of the storage period, followed by CT and RET treatments, respectively, compared to the control. Even though samples coated with CS/RE had the lowest pH and TVB-N values (p < 0.05), this coating was not able to pause the protein denaturation after 8 days of storage (p > 0.05) compared to the other treatments. On the other hand, CS/RE coating retarded lipid oxidation by decreasing PV and TBA production in the samples compared to the control up to the end of refrigerated storage (p < 0.05).     

Pre‐challenge and post‐challenge haemato‐immunological changes in Oreochromis niloticus (Linnaeus, 1758) fed argan oil against Lactococcus garvieae

The present study investigated the effects of argan oil, obtained from Argania spinosa, on pre‐ and post‐challenge immuno‐haematological and biochemical responses of Nile tilapia, Oreochromis niloticus. For this purpose, the fish were fed diets containing 0, 0.5%, 1% or 2% argan oil for 45 days. Following 45 days of feeding, fish were challenged with Lactococcus garvieae and mortality was recorded for 15 days. During the pre‐challenge period, significantly higher respiratory burst activity, total white blood cell (WBC), serum lysozyme activity and myeloperoxidase activity were determined in the argan oil‐fed groups. The serum glucose and cholesterol levels decreased whilst total protein and albumin did not change in the groups fed with argan oil‐supplemented diets. After challenge with Lactococcus garvieae, the percentage survival (%) was found to be the highest in the 1% and 2% argan oil‐supplemented feeding groups. Also, there was a significant increase in weight gain, specific growth rate and feed conversion ratio in those fish fed argan oil. The results of this study indicated that after the supplementation of fish diets with argan oil, especially at 1% and 2% concentrations, the immunological, haematological and biochemical values remained similar in both the pre‐ and post‐challenge periods and the immune response against L. garvieae in Nile tilapia was modulated.     

Identification and characterization of lactic acid bacteria isolated from rainbow trout, Oncorhynchus mykiss (Walbaum), with inhibitory activity against Lactococcus garvieae

A study was conducted to evaluate the probiotic properties of endogenous rainbow trout microbiota against pathogenic Lactococcus garvieae. A total of 335 bacterial strains were isolated from rainbow trout and screened for antagonistic activity against L. garvieae using an agar spot assay. Antagonistic strains were grouped by PCR amplification of repetitive bacterial DNA elements (rep‐PCR) and identified by 16S rRNA gene sequence analysis. The results revealed that the antagonistic strains belonged to the genera Lactobacillus, Lactococcus and Leuconostoc. Further probiotic characteristics, such as specific growth rate, doubling time, resistance to biological barriers, antibiotic resistance, hydrophobicity and production of antimicrobial substances, were also studied. These strains were able to survive low pH and high bile concentrations, showed good adherence characteristics and a broad spectrum of antibiotic resistance. The antagonistic efficacy was maintained after sterile filtration and was sensitive to proteinase K, indicating that proteinaceous extracellular inhibitory compounds were at least partially responsible for pathogen antagonism. Based on these results, these strains should be further studied to explore their probiotic effects in challenge experiments in vivo. This study shows clear evidence that the indigenous trout‐associated microbiota may provide a defensive barrier against L. garvieae.     

Dietary supplementation of garlic (Allium sativum) modulates gut microbiota and health status of tilapia (Oreochromis niloticus) against Streptococcus iniae infection

This study was conducted to characterize the causative agent of streptococcosis in tilapia (Oreochromis niloticus) and control of Streptococcus infection by means of garlic (Allium sativum) supplementation. The morphological, biochemical and polymerase chain reaction amplification confirmed 11 isolates belong to Streptococcus iniae from the infected fish eyes and tissue samples. Random screening of 12 well‐known medicinal plant parts against S. iniae revealed the garlic extract as the most effective herbal recovery against Streptococcus infection. In vivo challenge test with dietary supplementation of garlic powder significantly improved survival rates of fish against S. iniae infections, and modulate the microbial community and cytokine gene expression profiling in the intestine of the experimental tilapia. Among the two garlic supplemented treatments, 1.0 g garlic supplemented diet significantly increased (p < 0.05) the survival rates of tilapia and the gut bacterial operational transitional units abundance for Proteobacteria and Tenericutes, the phyla associated with healthy intestinal flora. The bacterial diversity index also found high with garlic supplemented diets. Significant upregulations of IL‐10 and IL‐17F gene expression in the intestinal tissue were observed with 1.0 g garlic supplemented diet where IL‐8 and IL‐1β expression levels were relatively static. The dietary supplementation of garlic, therefore, could be effective in the prevention of S. iniae infection in fish.     

Pharmacokinetics of Eugenol in Japanese Flounder,Paralichthys olivaceus

Eugenol, which has been used as an anesthetic for fish, was administered to Japanese flounder, Paralichthys olivaceus (127 ± 50.8 g), by bath treatment at concentrations of 0.025, 0.125, and 0.25 mL/L seawater for 10 min and intramuscular injection at 40 μL/fish to investigate the pharmacokinetic characteristics of eugenol in the plasma of Japanese flounder. In the bath treatment test, plasma eugenol concentration increased with increasing eugenol concentration up to 0.125 mL/L and reached steady state within 5 min. After a 10‐min bath treatment in 0.25 mL/L eugenol, plasma eugenol concentration was about 58.4 µg/mL. After transfer into running seawater, plasma eugenol concentration decreased biphasically with half‐lives of 0.0296 h (α‐phase) and 0.289 h (β‐phase). The AUC_0000→0800 was about 16.5 µg h/mL. In administration by intramuscular injection, plasma eugenol concentration increased rapidly after administration and decreased biphasically with half‐lives of 0.0329 h (α‐phase) and 8.08 h (β‐phase). The AUC_0000→0800 was about 52.5 µg h/mL. In both methods of administration, Japanese flounder with average weight of 127 g were effectively anesthetized when plasma eugenol concentrations were between 2.19 and 4.88 µg/mL.