Natural dietary additive yellow loess as potential antibiotic replacer in Japanese eel,Anguilla japonica: Effects on growth,immune responses,serological characteristics and disease resistance against Edwardsiella tarda

The present experiment was conducted to evaluate the efficacy of an additive derived from the nature as an alternative of dietary antibiotic in Japanese eel, Anguilla japonica. Six experimental diets were formulated to contain no antibiotics or additive (yellow loess/YL) (control/CON), three graded levels of yellow loess at 5 (YL₅), 10 (YL₁₀) and 20 g/kg (YL₂₀), oxytetracycline at 5 (OTC) and amoxicillin at 10 g/kg amoxicillin (AMX) of diet. Weight gain (WG) and specific growth rate (SGR) from fish fed CON or YL₅ diets were significantly lower than those of fish fed YL₂₀ or OTC diets. Among non‐specific enzyme, lysozyme activity of fish fed YL₂₀, OTC or AMX was detected to be significantly higher than that from fish fed CON or YL₅ diets, whereas superoxide dismutase (SOD) activity of the fish fed CON was significantly lower than that for fish fed other experimental diets. Challenge test with bacteria, Edwardsiella tarda, showed improved disease resistance among the fish fed different levels of natural additive without any statistical difference from those fed antibiotics (OTC and/or AMX) supplemented diets. Therefore, these results demonstrated the potential of natural feed additive, yellow loess to replace oxytetracycline and/or amoxicillin in Japanese eel, A. japonica.     

Application of α-glucosidase activity and drug susceptibility tests to epidemiological studies on the fish pathogen <Emphasis Type="Italic">Nocardia seriolae</Emphasis>

This study determined the minimum inhibitory concentrations (MICs) for oxytetracycline hydrochloride (OTC) and erythromycin (Em), along with the α-glucosidase (α-glu) activities in 110 Nocardia seriolae strains isolated in Miyazaki and Kagoshima prefectures in 2008–2009. The strains were examined for the presence of the tet(K), tet(L), tet(M), tet(O), tet(S), erm(A), erm(B), mph(A), mef(A), and msr(D) genes. All the α-glu-positive strains (n = 15) were OTC resistant and Em sensitive, with MICs of 32–64 and <0.125 μg/ml, respectively. All the α-glu-negative strains (n = 95) were OTC sensitive, with MICs of 2–4 μg/ml, and most of them (93 of 95) were Em resistant, with MICs of >128 μg/ml. The MICs for Em in the remaining 2 α-glu-negative strains were <0.125 μg/ml. The 15 OTC-resistant strains possessed the tet(K) and/or tet(L) gene(s), and all of the 93 Em-resistant strains possessed both the mef(A) and msr(D) genes. The relationship between α-glu activity and drug sensitivity of the N. seriolae strains may explain the difference in prevalence of each phenotype. Nevertheless, the relationship should be further explored using N. seriolae isolates collected from more prefectures and farms.     

Effect of quorum quenching bacteria on growth,virulence factors and biofilm formation of Yersinia ruckeri in vitro and an in vivo evaluation of their probiotic effect in rainbow trout

Five N‐acyl homoserine lactone‐degrading bacteria (quorum quenching (QQ) strains) were selected to evaluate their impacts on growth, virulence factors and biofilm formation in Yersinia ruckeri in vitro. No difference was observed among the growth pattern of Y. ruckeri in monoculture and coculture with the QQ strains. To investigate the regulation of virulence factors by quorum sensing in Y. ruckeri, cultures were supplemented with 3oxo‐C8‐HSL. The results indicated that swimming motility and biofilm formation are positively regulated by QS (p < 0.05), whereas caseinase, phospholipase and haemolysin productions are not influenced by 3oxo‐C8‐HSL (p > 0.05). The QQs were able to decrease swimming motility and biofilm formation in Y. ruckeri. QQ bacteria were supplemented to trout feed at 10⁸ CFU/g (for 40 days). Their probiotic effect was verified by Y. ruckeri challenge either by immersion or injection in trout. All strains could significantly increase fish survival with Bacillus thuringiensis and Citrobacter gillenii showing the highest and lowest relative percentage survival (RPS) values (respectively, 85% and 38%). Besides, there was no difference between the RPS values by either immersion or injection challenge expect for B. thuringiensis. The putative involvement of the QQ capacity in the protection against Yersinia is discussed.     

Winter kill in intensively stocked channel catfish (Ictalurus punctatus): Coinfection with Aeromonas veronii,Streptococcus parauberis and Shewanella putrefaciens

Unusual persistent natural mortality occurred in a floating in‐pond raceway system intensively stocked with channel and hybrid catfish beginning in early November 2016 up until March 2017. The temperature during the period of outbreak ranged from 7.2 to 23.7°C. Gross examination of freshly dead and moribund fish revealed pale gills, slight abdominal distension and swollen inflamed vents. Comprehensive necropsy of 20 fish demonstrated vast amounts of bloody ascitic fluid in the coelomic cavity, visceral congestion, splenomegaly and pale friable livers but macroscopically normal kidneys, suggesting systemic bacterial infection. Bacterial cultures were initiated from skin, gills and major internal organs. Following incubation, a mixture of three bacterial colony phenotypes was observed on agar plates. Presumptive biochemical characterization of the isolates followed by 16S‐rRNA sequence analysis resulted in the identification of Aeromonas veronii, Streptococcus parauberis and Shewanella putrefaciens. Channel catfish juveniles were experimentally infected with the recovered isolates to fulfil Koch's postulates. Moreover, an antibiogram was used to evaluate the susceptibility of the isolates to antimicrobial drugs approved for use in aquaculture. Aquaflor was used successfully for treatment. Here, we report bacterial coinfection lead by A. veronii and the first identification of S. parauberis and S. putrefaciens from cultured catfish in North America.     

Bath efficacy of sodium hypochlorite,oxytetracycline dihydrate and chloramphenicol against bacterial black disease in fairy shrimp Branchinella thailandensis

To control black disease infecting fairy shrimp Branchinella thailandensis, the effects of concentrations and exposure time to three effective antimicrobials, which inhibited the pathogens in vitro, were evaluated. Exposure to sodium hypochlorite (NaOCl) caused a great toxicological response in the shrimp, 100% mortality was observed within 30 min–2 h at 5–20 μg mL^?1. For oxytetracycline dihydrate (OTC) and chloramphenicol (CP), short‐term exposure to four high concentrations up to 5 h and long‐term exposure (12 days) to four low concentrations were used to determine an appropriate method for bath efficacy. Long‐term exposure to low concentrations was more toxic than the short‐term. Short‐term exposure to OTC showed the highest survival rate and CP was considered more toxic. The minimum survival rate of the shrimp exposed to both antibiotics at 250 μg mL^?1 for 3 h was 83.3%. For determination of the bath efficacy, a short–term exposure (3 h) to OTC and CP was conducted using artificially infected shrimp. Administration of OTC and CP at 250 and 500 μg mL^?1 resulted in the highest survival rates of 56.7% and 46.7% respectively. This study demonstrated that bath administration with OTC could be an alternative method for the treatment of black disease in fairy shrimp cultivation.     

Oxytetracycline (OTC) uptake following bath treatment in gilthead sea bream (Sparus aurata)

The uptake and elimination profile of oxytetracycline (OTC) following a prolong bath treatment in gilthhead sea bream (Sparus aurata) were investigated in this study. The bath experiment was carried out using a OTC concentration of 50 μg/ml for 24 h at 17-18 °C water temperature. Plasma and muscle fish samples were analysed at 1, 3, 6 and 24 h during and at 1, 2, 3, 4 and 6 d following the bath. Detectable OTC levels were revealed only at the end of bath treatment (24 h) in examined tissues of gilthead sea bream, where drug concentration was measured to be as low as 0.096 and 0.047 μg/g or ml in muscle plus skin and plasma, respectively. The findings of the present study indicate that OTC bath treatment under this dosage schedule is unlikely to confront systemic bacterial infections.     

Impact of Pseudomonas H6 surfactant on all external life cycle stages of the fish parasitic ciliate Ichthyophthirius multifiliis

A bacterial biosurfactant isolated from Pseudomonas (strain H6) has previously been shown to have a lethal effect on the oomycete Saprolegnia diclina infecting fish eggs. The present work demonstrates that the same biosurfactant has a strong in vitro antiparasitic effect on the fish pathogenic ciliate Ichthyophthirius multifiliis. Three life cycle stages (the infective theront stage, the tomont and the tomocyst containing tomites) were all susceptible to the surfactant. Theronts were the most sensitive showing 100% mortality in as low concentrations as 10 and 13 μg/ml within 30 min. Tomonts were the most resistant but were killed in concentrations of 100 μg/ml. Tomocysts, which generally are considered resistant to chemical and medical treatment, due to the surrounding protective cyst wall, were also sensitive. The surfactant, in concentrations of 10 and 13 μg/ml, penetrated the cyst wall and killed the enclosed tomites within 60 min. Rainbow trout fingerlings exposed to the biosurfactant showed no adverse immediate or late signs following several hours incubation in concentrations effective for killing the parasite. This bacterial surfactant may be further developed for application as an antiparasitic control agent in aquaculture.     

In vitro activity of chemicals and commercial products against Saprolegnia parasitica and Saprolegnia delica strains

Oomycetes of the genus Saprolegnia are responsible for severe economic losses in freshwater aquaculture. Following the ban of malachite green in food fish production, the demand for new treatments pushes towards the selection of more safe and environment‐friendly products. In the present work, in vitro activity of ten chemicals and three commercial products was tested on different strains of Saprolegnia, using malachite green as reference compound. The compounds were screened in agar and in water to assess the minimum inhibitory concentration (MIC) and the minimum lethal concentration (MLC), respectively. Two strains of Saprolegnia parasitica and one isolate of Saprolegnia delica were tested in triplicate per each concentration. Among tested chemicals, benzoic acid showed the lowest MIC (100 ppm) followed by acetic acid, iodoacetic acid and copper sulphate (250 ppm). Sodium percarbonate was not effective at any tested concentration. Among commercial products, Virkon^?S was effective in inhibiting the growth of the mycelium (MIC = MLC = 1,000 ppm). Actidrox® and Detarox® AP showed MIC = 5,000 and 1,000 ppm, respectively, while MLCs were 10‐fold lower than MICs, possibly due to a higher activity of these products in water. Similarly, a higher effectiveness in water was observed also for iodoacetic acid.     

Comprehensive antibiotic susceptibility profiling of Chilean Piscirickettsia salmonis field isolates

Antibiotics have been extensively used against infections produced by Piscirickettsia salmonis, a fish pathogen and causative agent of piscirickettsiosis and one of the major concerns for the Chilean salmon industry. Therefore, the emergence of resistant phenotypes is to be expected. With the aim of obtaining a landscape of the antimicrobial resistance of P. salmonis in Chile, the susceptibility profiles for quinolones, florfenicol and oxytetracycline (OTC) of 292 field isolates derived from main rearing areas, different hosts and collected over 5 years were assessed. The results allowed for the determination of epidemiological cut‐off values that were used to characterize the pathogen population. This work represents the first large‐scale field study addressing the antimicrobial susceptibility of P. salmonis, providing evidence of the existence of resistant types with a high incidence of resistance to quinolones. Remarkably, despite the amounts and frequency of therapies, our results disclosed that the issue of resistance to florfenicol and OTC is still in the onset.     

Development of real‐time PCR for detection and quantitation of Streptococcus parauberis

Streptococcus parauberis is an increasing threat to aquaculture of olive flounder, Paralichthys olivaceus Temminck & Schlegel, in South Korea. We developed a real‐time polymerase chain reaction (PCR) method using the TaqMan probe assay to detect and quantify S. parauberis by targeting the gyrB gene sequences, which are effective for molecular analysis of the genus Streptococcus. Our real‐time PCR assay is capable of detecting 10 fg of genomic DNA per reaction. The intra‐ and interassay coefficient of variation (CV) values ranged from 0.42–1.95%, demonstrating that the assay has good reproducibility. There was not any cross‐reactivity to Streptococcus iniae or to other streptococcal/lactococcal fish pathogens, such as S. agalactiae and Lactococcus garvieae, indicating that the assay is highly specific to S. parauberis. The results of the real‐time PCR assay corresponded well to those of conventional culture assays for S. parauberis from inoculated tissue homogenates (r = 0.957; P < 0.05). Hence, this sensitive and specific real‐time PCR is a valuable tool for diagnostic quantitation of S. parauberis in clinical samples.     

Pharmacokinetics of enrofloxacin and its metabolite ciprofloxacin in healthy and Vibrio alginolyticus-infected large yellow croaker (Pseudosciaena crocea)

The present study was designed to explore pharmacokinetics of enrofloxacin and its metabolite ciprofloxacin in healthy and Vibrio alginolyticus-infected large yellow croaker (Pseudosciaena crocea) after a single 10 mg/kg oral dose. Concentrations of enrofloxacin and ciprofloxacin in serum, liver, kidney, muscle and skin of fish were determined using high-performance liquid chromatography. Pharmacokinetic parameters were analysed based on classical compartmental model analysis. The overall changes in enrofloxacin concentration–time curves in serum and tissues of diseased fish were similar to those of healthy fish. However, the peak concentration and peak time of enrofloxacin in serum and tissues were different in healthy and diseased fish. A delay of enrofloxacin peak time in serum and all tissues appeared in the diseased fish. The peak concentrations in serum and tissues of the diseased fish were lower than those of healthy fish. In healthy fish, the area under the concentration–time curve (AUC) was in the order serum >liver > kidney >muscle > skin, while AUC was serum >live > muscle >kidney > skin in the diseased fish. The peak concentrations of ciprofloxacin in the liver, serum, kidney, muscle and skin of healthy fish were 0.93 μg/g, 0.55 μg/ml, 0.36 μg/g, 0.37 μg/g and 0.12 μg/g respectively. T_max of ciprofloxacin in the corresponding tissues was 8, 24, 12, 12 and 16 h respectively. In the diseased fish, the peak concentrations of ciprofloxacin in the corresponding tissues were 0.52 μg/g, 0.52 μg/ml, 0.41 μg/g, 0.27 μg/g and 0.13 μg/g respectively. T_max in the corresponding tissues were 0.5, 8, 12, 16 and 48 h respectively. These data indicate that the health status of fish affects drug absorption and metabolism.     

Pharmacokinetic/pharmacodynamic properties and P‐glycoprotein expression in pacific white shrimp,Litopenaeus vannamei after oral administration at three dosages of oxolinic acid

The experiments explored the pharmacokinetics (PK) properties of oxolinic acid (OXA) after oral administration at three dosages (10, 30 and 80 mg/kg) via medicated feed in the shrimp. The results showed that the C_max values of 4.31, 14.93 and 16.62 mg/L and AUC_0–∞ values of 92.61, 252.30 and 364.27 mg hr^?1 L^?1 were observed at three OXA dosage groups in the haemolymph respectively. In the hepatopancreas, C_max values of 7.90, 27.23 and 60.51 mg/kg and AUC_0–∞ values of 42.01, 133.06 and 219.06 mg hr^?1 L^?1 were observed at 0.5 hr post administration respectively. In the muscle, C_max values of 1.62, 5.80 and 7.36 mg/kg and the AUC_0–∞ values of 25.64, 98.10 and 134.24 mg hr^?1 L^?1 were observed at 2 hr post administration respectively. In the gills, C_max values of 2.87, 8.08 and 12.12 mg/kg and the AUC_0–∞ values of 51.38, 118.65 and 206.48 mg hr^?1 L^?1 were observed at 4 hr post administration respectively. In addition, the in vitro MIC values of OXA at three dosages against 132 strains of Vibrio were examined and showed that the minimum inhibitory concentration (MIC) values for OXA primarily ranged from 0.15–1.25 µg/ml, including eight strains of Vibrio showing MIC values ≥5 µg/ml. The MIC₅₀ and MIC₉₀ values of 132 strains were 0.62 and 1.25 μg/ml respectively. The AUC_0–24/MIC₉₀ ratios of Vibrio were 140.4 in 30 mg/kg group. Furthermore, the P‐glycoprotein (P‐gp) expression was determined in shrimp tissues after administration to three dosage groups (10, 30 and 80 mg/kg). The results showed that P‐gp expression was up‐regulated in the hepatopancreas (5.36‐, 13.68‐ and 31.06‐fold respectively) compared with the control group.     

Effect of lactic acid on enrofloxacin pharmacokinetics in Eriocheir sinensis (Chinese mitten crab)

As a representative acidifier, lactic acid (LA) is widely used in the diets of aquatic animals. LA is the main supporter of energy metabolism and may be associated with drug metabolism. This study was designed to explore the pharmacokinetics of enrofloxacin (ENR) in Eriocheir sinensis (Chinese mitten crab) fed diets containing 0.1%, 0.2% and 0.3% LA (designated groups LA1, LA2 and LA3 respectively). The concentrations of ENR in the haemolymph, hepatopancreas and muscle were determined by HPLC after a single oral dose of 10 mg/kg. Our results showed that LA had a significant effect on the peak ENR concentrations in all the tissues (p < 0.05) by one‐way ANOVA analysis. There was a trend that C_max (peak concentration) of ENR was elevated with LA levels increasing up to 0.3% in haemolymph, hepatopancreas, muscle and T_max (time‐point of the peak concentration of the drug), t_1/2β (elimination half‐life) and AUC_(0‐∞) of ENR were shortened. Taken together, 0.3% LA might be effective in improving ENR pharmacokinetics in E. sinensis. Furthermore, it can be speculated that the enhanced biotransformation of ENR in the hepatopancreas mediated by LA is responsible for the differences in the pharmacokinetics of ENR in E. sinensis.     

The in vitro efficacy of oxytetracycline against re‐isolated pathogenic Aeromonas hydrophila carrying the cytolytic enterotoxin gene through hybrid catfish,Clarias macrocephalus (Günther, 1864) × Clarias gariepinus (Burchell, 1822) in Thailand

Aeromonas hydrophila is a pathogen infecting farmed hybrid catfish, Clarias macrocephalus (Günther, 1864) × Clarias gariepinus (Burchell, 1822) which incurs substantial economic losses in Thailand. The study aimed at a genetic tracking of A. hydrophila infection and the in vitro assessment of the efficacy of antibiotics against its virulent strains. Five clinical strains from catfishes and Nile tilapia were employed. They were 3‐passage re‐isolated through healthy hybrid catfish and the cytolytic enterotoxin gene (AHCYTOEN) of individuals was traced. Each of the re‐isolates at a dose of ~6.67 × 10⁵ CFU/g was intraperitoneally injected into ~15 g‐healthy hybrid catfish and their pathogenicity were observed for 7 days. It was found that AHCYTOEN was carried over whereas typical signs of motile aeromonas septicaemia were found in the specimens. The bacterial strains of Nile tilapia origin did not induce mortality but those of catfish origins (80%–100% rate of mortality). The strains were susceptible to the tetracycline antibiotics, and oxytetracycline produced MIC₅₀ and MBC as low as 0.007–0.031 μg/ml and 1–8 μg/ml respectively. As oxytetracycline specifically inhibited pathogenic A. hydrophila in vitro, it is recommended that an appropriate dosage regimen of the drug should be established.     

Antiviral activity of Illicium verum Hook. f. extracts against grouper iridovirus infection

Grouper iridovirus causes high mortality rates in cultured groupers, and effective treatment for grouper iridovirus infection is urgently required. Illicium verum Hook. f. is a well-known medicinal plant with a variety of biological activities. The aim of this study was to analyse the use of I. verum extracts to treat grouper iridovirus infection. The safe working concentration of each I. verum extract was identified both in vitro and in vivo as follows: I. verum aqueous extract (IVAE) ≤ 500 μg/ml; I. verum ethanol extract (IVEE) ≤ 250 μg/ml; shikimic acid (SKA) ≤ 250 μg/ml; trans-anethole (TAT) ≤ 800 μg/ml; 3,4-dihydroxybenzoic acid (DDBA) ≤ 400 μg/ml; and quercetin (QCE) ≤ 50 μg/ml. The inhibitory activity of each I. verum extract against grouper iridovirus infection was analysed using aptamer (Q2)-based fluorescent molecular probe (Q2-AFMP) and RT-qPCR. All of the I. verum extracts displayed dose-dependent antiviral activities against grouper iridovirus. Based on the achieved per cent inhibition, IVAE, IVEE, DDBA and QCE were associated with the greatest antiviral activity (all > 90%). Together, our results indicate that I. verum extracts have effective antiviral properties, making it an excellent potential source material for the development of effective treatment for grouper iridovirus infection.     

Sickeningly Sweet: L‐rhamnose stimulates Flavobacterium columnare biofilm formation and virulence

Flavobacterium columnare, the causative agent of columnaris disease, causes substantial mortality worldwide in numerous freshwater finfish species. Due to its global significance and impact on the aquaculture industry continual efforts to better understand basic mechanisms that contribute to disease are urgently needed. The current work sought to evaluate the effect of L‐rhamnose on the growth characteristics of F. columnare. While we initially did not observe any key changes during the total growth of F. columnare isolates tested when treated with L‐rhamnose, it soon became apparent that the difference lies in the ability of this carbohydrate to facilitate the formation of biofilms. The addition of different concentrations of L‐rhamnose consistently promoted the development of biofilms among different F. columnare isolates; however, it does not appear to be sufficient as a sole carbon source for biofilm growth. Our data also suggest that iron acquisition machinery is required for biofilm development. Finally, the addition of different concentrations of L‐rhamnose to F. columnare prior to a laboratory challenge increased mortality rates in channel catfish (Ictalurus punctatus) as compared to controls. These results provide further evidence that biofilm formation is an integral virulence factor in the initiation of disease in fish.     

Periphyton characteristics influence the growth and survival of Holothuria scabra early juveniles in an ocean nursery system

Floating hapas (fine mesh net enclosures) are a cost‐effective ocean nursery system to culture post‐metamorphic Holothuria scabra to release size. The growth of periphyton biofilm on hapas is a natural food source for early juveniles. This study investigated the effects of periphyton quality (i.e. chlorophyll‐a, phaeopigment, total biomass, autotrophic index or AI), water quality (nutrients, chlorophyll‐a) and environmental parameters (temperature, rainfall) on the temporal variation in the growth and survival of early juvenile (~3 mm) H. scabra reared in floating hapas. Five trials where the juveniles were reared for 60 days each in the eutrophic coastal waters of Bolinao, the Philippines were conducted during different months over 2 years. Significant differences in the growth and survival of juveniles among trials were found. Absolute growth rates (AGR) ranged widely (0.01–0.09 g/day). Trials with high AGR of juveniles (0.07–0.09 g/day) during the first 30 days of rearing had significantly higher chlorophyll‐a (chl‐a) in biofilm (15.9–27.5 mg/m²) and lower AI. Conversely, during the subsequent 30 days, trials with high AGR of juveniles (0.06–0.11 g/day) had significantly lower chl‐a and higher AI. Multivariate analyses showed that chl‐a in biofilm, AI and nutrients in the water column are good indicators of periphyton quality and juvenile growth rates in floating hapas. Further, this study validates the expansion of the feeding mode of juveniles from primarily grazing on microalgae, to feeding on detritus and heterotrophs as they grow. These results are important in optimizing ocean nursery systems.     

Virulence regulation of cel‐EIIB protein mediated PTS system in Streptococcus agalactiae in Nile tilapia

Streptococcus agalactiae is a major pathogen of tilapia causing significant economic losses for the global aquatic industry yearly. To elucidate the role of cel‐EIIB protein‐mediated phosphotransferase systems (PTS) in the virulence regulation of S. agalactiae, cel‐EIIB gene deletion in a virulent strain THN0901 was achieved by homologous recombination. The cellobiose utilization of △cel‐EIIB strain was significantly decreased relative to S.a.THN0901 strain incubating in LB with 10 mg/ml cellobiose (p < 0.05). The biofilm formation ability of △cel‐EIIB strain was also significantly decreased when cultured in BHI medium (p < 0.05). Under a lower infection dose, the accumulative mortality of tilapia caused by △cel‐EIIB strain was dramatically decreased (20%), of which S.a.THN0901 strain and △cel‐EIIB::i strain were 53.33% and 50%, respectively. The competition experience using tilapia model indicated the invasion and colonization ability of △cel‐EIIB strain was significantly weaker than that of S.a.THN0901 strain (p < 0.05). Compared to △cel‐EIIB::i strain, the mRNA expression of csrS, csrR, rgfA, rgfC, bgrR and bgrS was significantly downregulated in △cel‐EIIB strain (p < 0.05). In conclusion, cel‐EIIB protein‐mediated cel‐PTS not only contributes to biofilm formation and virulence regulation, but also plays an important role in the invasion and colonization of S. agalactiae.     

Antimicrobial susceptibility of Flavobacterium psychrophilum isolates from the United Kingdom

Routine application of antimicrobials is the current treatment of choice for rainbow trout fry syndrome (RTFS) or bacterial coldwater disease (BCWD) caused by Flavobacterium psychrophilum. In this study, the antimicrobial susceptibilities of 133 F. psychrophilum isolates, 118 of which were from the UK, were evaluated by broth microdilution and disc diffusion methods following VET04‐A2 and VET03‐A guidelines of Clinical and Laboratory Standards Institute (CLSI), respectively. Isolates were categorized as wild type (fully susceptible, WT) or non‐wild type (NWT) using normalized resistance interpretation (NRI)‐determined cut‐off values (CO_WT). Broth microdilution testing showed that only 12% of UK isolates were WT to oxolinic acid (MIC CO_WT ≤ 0.25 mg/L) and 42% were WT for oxytetracycline (MIC CO_WT ≤ 0.25 mg/L). In contrast, all the isolates tested were WT (MIC CO_WT ≤ 2 mg/L) for florfenicol, the main antimicrobial for RTFS control in the UK. Disc diffusion‐based CO_WT values were ≥51 mm for 10 μg amoxicillin, ≥44 mm for 30 μg florfenicol, ≥30 mm for 2 μg oxolinic acid and ≥51 mm for 30 μg oxytetracycline. There was a high categorical agreement between the classifications of the isolates by two testing methods for florfenicol (100%), oxytetracycline (93%) and oxolinic acid (99%).     

Antiviral abilities of Curcuma kwangsiensis ingredients against grouper iridoviral infection in vitro and in vivo

As one of the most serious pathogens in mariculture, the outbreaks of grouper Iridovirus (SGIV‐Gx) caused high mortality rates in cultured groupers in Guangxi, China. Hence, effective medicines for fighting against grouper Iridovirus are urgently needed. The possible application of Curcuma kwangsiensis ingredients against SGIV‐Gx infection was evaluated in vitro and in vivo in this study. The safe working concentration of each C. kwangsiensis ingredient was identified (C. kwangsiensis ethanol ingredient, CKEE ≤5 mg/ml; curcumin ≤20 μg/ml; curdione ≤500 μg/ml; curcumenol ≤500 μg/ml; curcumol displayed no cytotoxic effects even at 2 mg/ml) in vitro and in vivo. The inhibitory activities of each C. kwangsiensis ingredient against SGIV‐Gx infection were analysed using aptamer (Q2)‐based fluorescent molecular probe (Q2‐AFMP) and RT‐qPCR. The results showed that C. kwangsiensis ingredient (CKEE, curcumin, curcumol, curdione and curcumenol) displayed antiviral activities against SGIV‐Gx infection in a dose‐dependent manner. Furthermore, according to the inhibitory percentage analysed using RT‐qPCR results, CKEE and curdione had the best antiviral activity against SGIV‐Gx above 93%. Overall, the results suggest that some C. kwangsiensis ingredients have excellent antiviral effects, making it an interesting candidate for developing effective medicines for preventing and controlling SGIV‐Gx outbreaks in mariculture.