Roles of water quality and disinfectant application on inactivation of fish pathogenic Streptococcus agalactiae with povidone iodine,quaternary ammonium compounds and glutaraldehyde

Streptococcosis is an important bacterial disease in Nile tilapia causing severe economic losses to tilapia aquaculture worldwide. The effects of water quality (low‐ [LS] and high‐level [HS] soiling, to mimic clean or dirty surface conditions and temperatures) and disinfectant application (diluted concentrations and exposure time) were characterized on the inactivation of Streptococcus agalactiae isolated from diseased tilapia. Five isolates were tested against three commercial disinfectant products with the main ingredients being povidone iodine (Anidine 100?; AD), benzalkonium chloride (Better BKC 80%?; BKC 80), and a mixture of quaternary ammonium compounds and glutaraldehyde (Chloraldehyde?; CR). CR demonstrated highest efficacy to S. agalactiae inactivation, followed by BKC 80 and AD, respectively. Higher‐level soiling, low temperature, diluted concentrations and short exposure time all decreased the disinfectant efficacy. CR and BKC 80 provided more than 5‐log inactivation at 1‐min exposure at 20°C under HS conditions, and also with ten‐fold‐diluted concentrations at 60‐min exposure time at 30°C. However, AD required 10‐min exposure to effectively remove bacteria under LS conditions at 30°C. The results could facilitate aquaculture management planning that leads to operating cost reductions and improvements in biosecurity.     

Assessing the population transmission dynamics of tilapia lake virus in farmed tilapia

A novel virus, tilapia lake virus (TiLV), has been identified as a key pathogen responsible for disease outbreak and mass mortality of farmed tilapia. We used a deterministic susceptible‐infectious‐mortality (SIM) model to derive key disease information appraised with published TiLV‐induced cumulative mortality data. The relationship between tilapia mortality and TiLV exposure dosages was described by the Hill model. Furthermore, a disease control model was proposed to determine the status of controlled TiLV infection using a parsimonious control reproduction number (R_C)‐control line criterion. Results showed that the key disease determinants of transmission rate and basic reproduction number (R₀) could be derived. The median R₀ estimate was 2.59 in a cohabitation setting with 2.6 × 10⁵ TCID50 fish^?1 TiLV. The present R_C‐control model can be employed to determine whether TiLV containment is feasible in an outbreak farm by quantifying the current level of transmission. The SIM model can then be applied to predict what additional control is required to manage R_C < 1. We offer valuable tools for aquaculture engineers and public health scientists the mechanistic‐based assessment that allows a more rigorous evaluation of different control strategies to reduce waterborne diseases in aquaculture farming systems.     

Biodegradation potential of aquaculture chemotherapeutants in marine sediments

The commercial chemotherapeutant formulations SLICE^® and AlphaMax^® [active ingredients emamectin benzoate (EB) and deltamethrin respectively] are used in fin fish aquaculture to control parasitic sea lice. In some regions, the use of these substances has drawn concern from the commercial fishing industry regarding potential adverse effects on non‐target organisms. In the present work, biodegradation of EB and deltamethrin, and their commercial formulations, was investigated over 135 days at 4 and 10°C in fresh marine sediments collected from underneath an active open net‐pen salmon farm. EB incubated as either pure substance or commercial formulation was recalcitrant at both temperatures under abiotic and biotic conditions. Deltamethrin incubated alone or as its commercial formulation degraded slowly at 10°C (t_1/2 = 330 ± 107 and 201 ± 27.1 days respectively). At 4°C, deltamethrin degradation was only significant following incubation as commercial formulation (t_1/2 = 285 ±112 days). Degradation rates of EB and deltamethrin as pure substances versus their commercial formulations were not statistically different. Depletion of deltamethrin was observed in 10°C inactive sediments indicating that transformation occurred (at least in part) via an abiotic pathway. Overall, these data provide further insight into the fate and persistence of EB from the ongoing use of SLICE^® in British Columbia's salmon aquaculture industry. AlphaMax^® is not registered in Canada but is used in other salmon farming countries to control sea lice.     

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.     

Effects of commonly used disinfectants on bacterial load,hatchability and survival of Bluefin Sea bream (Sparidentex hasta) eggs

The efficacy of four chemical reagents, iodophor, formalin, hydrogen peroxide and bronopol as fish egg surface disinfectants were evaluated in bluefin sea bream (Sparidentex hasta). Fertilized eggs were counted and subjected to a static bath dip treatment in different concentrations of the above chemicals for 4 min before being incubated at 20 ± 0.5°C for 40 h. Treatment efficacy of the different disinfectants was evaluated by assessing the bactericidal activity, egg hatch percentage and survival of larvae up to 3 days post hatch. Results showed that iodophor at medium concentrations (75 and 100 ppm) was the best of all tested disinfectants in bacterial killing ability (12% reduction in the bacterial counts), egg hatching per cent (99.8% and 99.6% respectively) and larval survival up to 3 days post hatch (50.8% and 54.8% respectively). Formalin was the second best disinfectant at levels of 100 and 150 ppm. Hydrogen peroxide gave good results compared with the control while, bronopol showed discouraging results. In conclusion, iodophor appeared to be suitable for bluefin sea bream eggs disinfection with a 4 min exposure to 75–100 ppm when applied 14–16 h after egg fertilization.     

Effects of rested-harvest using the anesthetic AQUI-S™ on channel catfish,Ictalurus punctatus,physiology and fillet quality

Trials were conducted to determine effects of AQUI-S™ sedation during harvest (rested-harvest) on physiological responses and fillet quality of channel catfish, Ictalurus punctatus. Rested-harvest is defined as application of an anesthetic immediately before harvest to reduce fish activity associated with a normal harvest. Doses of 25–35 ppm AQUI-S™ were effective for rested-harvest of catfish (loss of equilibrium in 3 to 10 min and 100% survival following recovery). Time to loss of equilibrium and time to recovery following sedation with 35 ppm AQUI-S™ increased as water temperature decreased from 30 °C to 10 °C. Catfish exposed to 25 ppm AQUI-S™, 35 ppm AQUI-S™, 100 ppm trincaine methanesulfonate, and 8 ppm metomidate had lower blood lactate, cortisol, and glucose and higher blood pH than unsedated fish exposed to a low-water stress. Rested-harvest (RH) catfish had higher muscle and blood pH, lower blood and muscle lactate, and higher muscle ATP levels than catfish exposed to a 45 min low-water stress. Rates of muscle pH decrease, ATP decrease, and lactate increase accelerated as storage temperature decreased from 15 °C to 5 °C in RH fish acclimated to summer temperatures (33 °C), conversely these rates accelerated as storage temperature increased from 5 °C to 15 °C in RH fish acclimated to winter temperatures (7 °C). Based on physiological response (higher muscle pH, lower blood lactate, delayed time to rigor), post-sedation euthanasia by CO₂ was superior to post-sedation euthanasia by AQUI-S™ overdose (150 ppm), nitrogen gas, or electrical stunning. Compared to fillets from fish exposed to simulated industry transport conditions, fillets from RH/CO₂ euthanised fish had higher pH 1 h post-slaughter, and less drip-loss and lower L^⁎ and a^⁎ color values during 7 days of iced storage. RH/CO₂ and control fillets were not different for shelf-life based on bacterial counts. Rested-harvest with AQUI-S™ followed by CO₂ euthanasia has potential to improve catfish fillet quality, but AQUI-S™ approval, development of rested-harvest strategies, and demonstration of economic benefits of rested-harvest will be required for adoption of rested-harvest to commercial catfish production.     

Pharmacokinetic–pharmacodynamic modelling for the determination of optimal dosing regimen of florfenicol in Nile tilapia (Oreochromis niloticus) at different water temperatures and antimicrobial susceptibility levels

Optimized dosing regimen is key to the effective use of antibacterials and to minimizing drug‐related side effects. The current study established a pharmacokinetic–pharmacodynamic (PK‐PD) model for the determination of optimal antibacterial dosing regimen in fish taken into consideration the temperature‐dependent PK and the pathogen‐dependent antimicrobial susceptibility, using florfenicol (FF) in Nile tilapia as an example. The calculated optimal dosages significantly varied by temperature and target MIC levels, ranging from 2.23 (MIC 1 µg/ml at 24°C) to 34.88 mg kg^?1 day^?1 (MIC 4 µg/ml at 32°C). The appropriateness of the calculated dosages was successfully verified by the in vivo studies. After 5 days of oral administration of the calculated optimal dosage at 24°C, the predicted plasma drug values were in line with the mean observed C_min(ss) while at 28 and 32°C underestimation of the C_min(ss) in a dose‐dependent manner was observed and likely due to the occurrence of non‐linear PK at high dosages. The averaged serum protein binding of FF was 19.1%. Our results demonstrated the appropriateness and clinical applicability of the developed PK‐PD approach for the determination of optimal dosing regimens at given temperatures and MICs. Saturation metabolism and PK non‐linearity of FF in tilapia warrant further study.     

In vivo and in vitro treatments against Sparicotyle chrysophrii (Monogenea: Microcotylidae) parasitizing the gills of gilthead sea bream (Sparus aurata L.)

The effect of in vitro and in vivo treatments against Sparicotyle chrysophrii, a microcotylid parasite of gilthead sea bream (Sparus aurata L.), was studied. In vitro chemical treatments were targeted to eggs, oncomiracidia and adults, and were tested both as disinfectants and therapeutics for infected animals. The compounds were: distilled water, formalin, limoseptic ®, hydrogen peroxide, chlorine, and praziquantel (PZQ). Larvae were sensitive to all the treatments, but adults were more resistant, as chlorine (60 ppm - 1 h), hydrogen peroxide (100 ppm - 30 min) and PZQ (50 ppm - 30 min) produced only 10% mortality. All adults were killed only with distilled water, limoseptic (0.1% - 5 min), formalin (300 ppm - 30 min), or hydrogen peroxide (200 ppm - 30 min). Eggs were the most resistant stage, as only 30 min in limoseptic (0.1% in distilled water) or in formalin (300 ppm) prevented hatching. PZQ was used in vivo either as a curative or preventive treatment. The highest dose tested (400 mg kg^− 1 BW; effective dose 116.3 mg kg^− 1 BW due to palatability problems leading to 45% reduction in host food intake) did not significantly decrease prevalence of infection when given for 6 consecutive days. A lower dose (200 mg kg^− 1 BW) (effective dose 158.1 mg kg^− 1 BW) was rejected to a lower degree and decreased the prevalence of infection from 90% to 40%. When a lower dose (40 mg kg^− 1 BW) was administered for longer periods (20 days), food intake was reduced slightly, but the infection did not decrease significantly. The oral intubation with PZQ (200 mg kg^− 1 BW) once a week for 4 weeks did not prevent the infection of fish by cohabitation. However, a significant reduction in the abundance of the parasite was registered. In view of the results, recommendations for fish treatment and disinfection of aquaculture facilities are discussed.     

Detection of tilapia lake virus (TiLV) infection by PCR in farmed and wild Nile tilapia (Oreochromis niloticus) from Lake Victoria

Tilapia lake virus disease (TiLVD) has emerged to be an important viral disease of farmed Nile tilapia (Oreochromis niloticus) having the potential to impede expansion of aquaculture production. There is a need for rapid diagnostic tools to identify infected fish to limit the spread in individual farms. We report the first detection of TiLV infection by PCR in farmed and wild Nile tilapia from Lake Victoria. There was no difference in prevalence between farmed and wild fish samples (p = .65), and of the 442 samples examined from 191 fish, 28 were positive for TiLV by PCR. In terms of tissue distribution, the head kidney (7.69%, N = 65) and spleen (10.99%, N = 191), samples had the highest prevalence (p < .0028) followed by heart samples (3.45%, N = 29). Conversely, the prevalence was low in the liver (0.71%, N = 140) and absent in brain samples (0.0%, N = 17), which have previously been shown to be target organs during acute infections. Phylogenetic analysis showed homology between our sequences and those from recent outbreaks in Israel and Thailand. Given that these findings were based on nucleic acid detection by PCR, future studies should seek to isolate the virus from fish in Lake Victoria and show its ability to cause disease and virulence in susceptible fish.     

Pre-spawning water temperature affects sperm respiration and reactivation parameters in male carps

Concentration, ability to motility, motility during the second activation (reactivation), and endogenous respiration were studied in sperm from two experimental groups of carp males. Group 1 was maintained for 7 days at 15°C (cold water (CW) group), whereas the second group was subjected to a temperature of 20°C (warm water (WW) group) before sperm sampling. Reactivation were achieved after incubation of firstly activated sperm in media with osmotic pressure adjusted up to 300 mOsm*kg⁻¹ by increasing K⁺ concentration. Statistically significant reduction of spermatozoa concentration in CW samples versus WW (from 46.0 ± 12.5 (15°C) to 59.3 ± 7 10⁹ (20°C) spermatozoa /ml) have been observed. The sperm of the CW group required a significantly longer incubation time (37 min) under isotonic conditions to achieve a maximum percentage of potent motility at repeated activation than the WW group (23 min). After activation of sperm motility, an increase of respiration rate up to maximum level has been found, this level remained the same under condition of recovering the potential to repeated activation. During the sperm movement respiration rate, in CW group (6.1 nmolO₂/min/10⁹spermatozoa) and WW (3.9 nmolO₂/min/10⁹spermatozoa), was significant higher compared to nonactivated sperm (2.4 nmolO₂/min/10⁹spermatozoa for CW and 1.1 nmolO₂ /min/10⁹spermatozoa for WW). And keeping males for 7 days at 15°C increase the respiration rate of sperm.     

Effect of chemical and physical treatments on the inactivation of Macrobrachium rosenbergii nodavirus and extra small virus

White tail disease (WTD) is found to cause immense economic losses in hatcheries, with mortalities often reaching 100% within 4 or 5 days. The pathogenic agents have been identified as Macrobrachium rosenbergii nodavirus (MrNV) associated with extra small virus (XSV), which are 27 and 15 nm in diameter respectively. The effects of some chemical disinfectants hydrogen ions (pH), heat and ultraviolet (UV) irradiation on the inactivation of MrNV and XSV were investigated. The viral inoculum exposed to UV irradiation for a period of 5 min and more was totally inactivated and failed to cause mortality in postlarvae of prawn. The viruses were totally inactivated by this high pH (8.5, 9 and 10). The viral suspension treated with sodium hypochloride, formalin, Benzalkonium chloride and Benzethonium chloride at the concentration of 200 ppm caused 100% mortality in postlarvae of prawn. Iodine was found to be effective to inactivate MrNV and XSV at the concentration of 100 ppm or more, whereas the viral suspension treated with iodine at the concentration of 50 ppm or less caused mortality in postlarvae. The infected postlarvae in treated and positive control groups showed positive by RT‐PCR for these viruses.     

Antibacterial activity of oxytetracycline on microbial ecology of Nile tilapia (Oreochromis niloticus) gastrointestinal tract under laboratory condition

The antibacterial effect of oxytetracycline (OTC) on bacterial load, pathogen and possible development of antibiotic resistance in intestinal flora of apparently healthy Nile tilapia (Oreochromis niloticus) was investigated under laboratory condition. OTC, a broad spectrum approved antibiotic widely used in aquaculture, was fed to Nile tilapia through medicated diet at a rate of 2 g/ kg of feed for consecutive 7 days in treatment 1 (T₁) and 30 days in treatment 2 (T₂). Changes in physico‐chemical parameters of water were also recorded where pH and dissolved oxygen influenced bacterial load and coliform count respectively. At the start of the experiment, initial bacterial load in fish intestine was 1.61 ± 2.25 × 10¹⁰ cfu/g, which after OTC treatment decreased significantly (p < .05) to 3.06 ± 2.08 × 10⁸ cfu/g on day 7 and 3.45 ± 4.46 × 10⁷ cfu/g on day 30 when compared with the control group. Among the 10 bacterial genera identified in the intestine, 4 potential public health concern bacteria viz., Salmonella, Escherichia, Enterobacter and Staphylococcaus were predominant. There was a sharp increase in bacterial load on day 18 in T₂ that may be attributed to the development of antibiotic resistance in intestinal bacteria. Antibiotic susceptibility test for isolates against seven antibiotics: amoxycillin, ciprofloxacin, co‐trimoxazole, erythromycin, gentamicin, nitrofurantoin and tetracycline using disc diffusion method revealed significantly increased resistance of Gram‐negative rods (p < .05) that possibly caused increased frequency of OTC‐resistant microorganisms. In conclusion, short‐ and long‐term exposure to OTC treatment affected the distribution of bacterial genera including pathogens in the Nile tilapia gastrointestinal tract and concomitantly influenced their antimicrobial resistance.     

Effect of temperature on excess post-exercise oxygen consumption in juvenile southern catfish (<Emphasis Type="Italic">Silurus meridionalis</Emphasis> Chen) following exhaustive exercise

The effects of temperature on resting oxygen consumption rate (MO_2rest) and excess post-exercise oxygen consumption (EPOC) after exhaustive exercise (chasing) were measured in juvenile southern catfish (Silurus meridionalis) (8.40 ± 0.30 g, n = 40) to test whether temperature has a significant influence on MO_2rest, maximum post-exercise oxygen consumption rate (MO_2peak) and EPOC and to investigate how metabolic scope (MS: MO_2peak − MO_2rest) varies with acclimation temperature. The MO_2rest increased from 64.7 (10°C) to 160.3 mg O₂ h⁻¹ kg⁻¹ (25°C) (P < 0.05) and reached a plateau between 25 and 30°C. The post-exercise MO₂ in all temperature groups increased immediately to the peak values and then decreased slowly to a steady state that was higher than the pre-exercise MO₂. The MO_2peak did not significantly differ among the 20, 25 and 30°C groups, though these values were much higher than those of the lower temperature groups (10 and 15°C) (P < 0.05). The duration of EPOC varied from 32.9 min at 10°C to 345 min at 20°C, depending on the acclimation temperatures. The MS values of the lower temperature groups (10 and 15°C) were significantly smaller than those of the higher temperature groups (20, 25 and 30°C) (P < 0.05). The magnitude of EPOC varied ninefold among all of the temperature groups and was the largest for the 20°C temperature group (about 422.4 mg O₂ kg⁻¹). These results suggested that (1) the acclimation temperature had a significant effect on maintenance metabolism (as indicated by MO_2rest) and the post-exercise metabolic recovery process (as indicated by MO_2peak, duration and magnitude of EPOC), and (2) the change of the MS as a function of acclimation temperature in juvenile southern catfish might be related to their high degree of physiological flexibility, which allows them to adapt to changes in environmental conditions in their habitat in the Yangtze River and the Jialing River.     

A method for collecting eggs of Pseudocapillaria tomentosa (Nematoda: Capillariidae) from zebrafish Danio rerio and efficacy of heat and chlorine for killing the nematode's eggs

Pseudocapillaria tomentosa is a common pathogen of zebrafish (Danio rerio) in research facilities. We developed a method to collect and concentrate the nematode eggs using a modified sugar centrifugation method and documented their normal development. Embryonating stages with blastomere formation followed by elongation of the embryo prior to larva formation cumulated in developed larvae inside the eggs and hatching after 5–10 day. We then evaluated the efficacy of heat and chlorine to kill them based on a larva development assay. Eggs were exposed to 40, 50, 60 °C for 30 min and 1 h. Chlorine treatment was performed at 100, 250, 500, 1000, 3000 and 6000 ppm for 10 min. Samples exposed to 40 °C for 30 min or 1 h showed incidences of larvated eggs similar to controls. In contrast, no larvation occurred with eggs exposed to either 50 or 60 °C for 30 min or 1 h. Remarkably, in repeated assays, samples exposed to low doses of chlorine (100, 250, 500 and 1000 ppm for 10 min) showed significantly higher incidence of larvation than controls. Eggs treated with 3000 ppm for 10 min did not develop larvae, and no eggs were found after 6000 ppm treatment.     

Biomarkers of oxidative stress and antioxidant defences as indicators of different disinfectants exposure in the heart of rainbow trout (Oncorhynchus Mykiss Walbaum)

The aim of this study was to evaluate the impacts of different disinfectants' treatment using in aquaculture on the oxidative stress biomarkers such as thiobarbituric acid reactive substrates (TBARS) and carbonyl derivatives of protein oxidative modification, as well as antioxidant defences [superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and glutathione peroxidase (GPx) activity] and total antioxidant capacity (TAC) in the heart tissue of rainbow trout (Oncorhynchus mykiss). In the disinfectants exposure, fish were treated to chloramine‐T (final concentration 10 g m^?3), chloride dioxide (5 g m^?3), formalin (200 mL m^?3) and CIP disinfectant based on peracetic acid and hydrogen peroxide (16 mL m^?3) for 20 min and repeated three times every 3 days. Both chlorine dioxide and formalin treatment was indicated by a significant increase in the level of heart TBARS levels and carbonyl derivatives content and decreased SOD activity. Tissue oxidative stress biomarkers were unchanged upon chloramine‐T or CIP disinfectant exposure. Increased oxidative stress could modify antioxidant defences, principally causing increased CAT activity in the heart tissue of formalin‐ or ClO₂^?‐exposed fish. The correlation between oxidative stress biomarkers and GPx activity indicates that enzymes related to glutathione metabolism were responsible to formalin or ClO₂^?‐induced oxidative stress. Hence, TBARS, carbonyl derivatives and antioxidant defences could be used as biomarkers in evaluating the toxicity of formalin and chlorine dioxide using as disinfectants to trout.     

Determination of optimal temperature(s) in juvenile red‐spotted grouper Epinephelus akaara (Temminck & Schlegel) based on growth performance and stress responses

This study sought to determine the optimal temperature(s) for aquaculture of juvenile red‐spotted grouper Epinephelus akaara (Temminck & Schlegel) (mean initial BW: 3.1 g). Growth performance, insulin‐like growth factor 1 (IGF‐1) expression and thermal stress responses (plasma cortisol, glucose, and hepatic heat shock protein 60 expression) were evaluated at three constant temperatures (24°C, 26°C and 28°C) in a 2‐week trial. At the end of the trial, final BW was significantly higher at 26°C and 28°C than at 24°C (p < 0.05); a quadratic regression analysis of final BW showed the optimum temperature for growth was 27.5°C (p < 0.05, R² = 0.806). The highest hepatic IGF‐1 expression was observed at 26°C (p < 0.05). On the other hand, hepatic heat shock protein 60 expression was highest at 28°C (p < 0.05), suggesting thermal stress. In conclusion, temperature optima, which support excellent growth but induce minimal thermal stress, was 26°C. This fine information within a narrow temperature range is expected to give empirical information for red‐spotted grouper farmers to sustain maximal production efficiency with avoiding thermal stress and to determine the future location of production, especially in consideration of arising seawater temperatures.