Setting epidemiological cut-off values for Vibrio harveyi relevant to MIC data generated by a standardised microdilution method.

The lack of internationally harmonised criteria for interpreting the data generated by standardised susceptibility testing methods presents a serious obstacle for the development of prudent use of antimicrobials in aquaculture. The data required to set epidemiological cut-off values for minimum inhibitory concentrations for antibiotic agents against Vibrio harveyi was determined using a standard microdilution method that specified the use of cation-adjusted Mueller Hinton broth and incubation at 28°C for 24 to 28 h. In total, 120 observations were made in 4 independent laboratories from 109 unique isolates. The aggregated data from these laboratories were analysed by the normalised resistance method and by ECOFFinder to calculate epidemiological cut-off values. The data for chloramphenicol, meropenem and sulfamethoxazole were not considered as suitable for analysis. The data for ampicillin indicated that this species is innately resistant to this agent. No acceptable ranges for quality control strains have been set for ceftazidime and, therefore, only provisional cut-off values could be generated for this agent. The epidemiological cut-off values were, however, calculated for the other 6 agents. These values were ≤0.5 µg ml-1 for enrofloxacin, ≤1 µg ml-1 for florfenicol, oxolinic acid and oxytetracycline, ≤4 µg ml-1 for gentamicin and ≤0.5/9.5 µg ml-1 for trimethoprim/sulfamethoxazole. Evidence is presented demonstrating that the data for these 6 antimicrobial agents was of sufficient quantity and quality that they could be used by the relevant authorities to set internationally harmonised, consensus epidemiological cut-off values for V. harveyi.

Epidemiological cut-off values for Vibrio anguillarum MIC and disc diffusion data generated by standardised methods.

This work aims to generate the data needed to set epidemiological cut-off values for minimum inhibitory concentration (MIC) and disc-diffusion zone measurements of Vibrio anguillarum. A total of 261 unique isolates were tested, applying standard methods specifying incubation at 28°C for 24-28 h. Aggregated MIC distributions for a total of 247 isolates were determined in 9 laboratories for 11 agents. Data aggregations of the disc zone for the 10 agents analysed contained between 157 and 218 observations made by 4 to 7 laboratories. Acceptable ranges for quality control (QC) reference strains were available for 7 agents and the related multi-laboratory aggregated data were censored, excluding the data of a laboratory that failed to meet QC requirements. Statistical methods were applied to calculate epidemiological cut-off values. Cut-off values for MIC data were calculated for florfenicol (≤1 µg ml-1), gentamicin (≤4 µg ml-1), oxytetracycline (≤0.25 µg ml-1) and trimethoprim/sulfamethoxazole (≤0.125/2.38 µg ml-1). The cut-off values for disc zone data were calculated for enrofloxacin (≥29 mm), florfenicol (≥27 mm), gentamicin (≥19 mm), oxolinic acid (≥24 mm), oxytetracycline (≥24 mm) and trimethoprim/sulfamethoxazole (≥26 mm). MIC and disc-diffusion zone data for the other agents where not supported by QC, thus yielding only provisional cut-off values (meropenem, ceftazidime). Regardless of whether QC is available, some of the aggregated MIC distributions (enrofloxacin, oxolinic acid), disc zone (sulfamethoxazole), and MIC and disc-diffusion distributions (ampicillin, chloramphenicol) did not meet the statistical requirements. The data produced will be submitted to the Clinical Laboratory Standards Institute for their consideration in setting international consensus epidemiological cut-off values.

Characterization of a novel picornavirus isolated from moribund gilthead seabream (Sparus aurata) larvae.

Gilthead seabream represents a species of importance in Mediterranean aquaculture. The larval stage is particularly sensitive and frequently impacted in suboptimal environmental or sanitary conditions. In the present study, investigations were carried out in a seabream hatchery following an unusual mortality reaching 70% among 50-day post-hatching. Anorexia, loss of appetite and abnormal swimming behaviour were observed in absence of parasites or pathogenic bacteria. Proliferation of rod-shaped bacteria in the gut lumen was associated with focal degeneration in the intestinal mucosa. Cytopathic effects on an EK-1 cell line after 21 days of culture at 14°C and 20°C in contact with homogenized affected larvae revealed the presence of a viral agent. Molecular characterization by high-throughput sequencing showed a typical picornavirus genome organization with a polyprotein precursor of 2276 amino acids sharing 46.3% identity with that of the Eel Picornavirus-1. A specific real-time PCR confirmed the presence of the viral genome in affected larval homogenate and corresponding cell culture supernatant. We propose the name Potamipivirus daurada for this novel species within the genus Potamipivirus. The etiological role of this virus remains uncertain at this time, and future studies will be necessary to investigate its prevalence in natural and aquaculture-reared populations as well as its ability to cause diseases in gilthead seabream.

Development of a rapid assay to detect the dinoflagellate Amyloodinium ocellatum using loop-mediated isothermal amplification (LAMP).

Amyloodinium ocellatum is a highly pathogenic dinoflagellate parasite with global distribution that causes high mortalities in the culture of tropical and sub-tropical marine and estuarine fishes. Diagnosis typically occurs through gross examination following the onset of morbidity, at which point treatment is of limited benefit. In the present study, a new molecular diagnostic tool for the rapid detection of A. ocellatum (AO) was developed using the loop-mediated isothermal amplification method (LAMP). The AO-LAMP assay designed is highly specific using a set of four primers - two outer and two inner primers targeting six different regions on the 5' end of the Small Subunit rDNA region (SSU rDNA) of A. ocellatum. The AO-LAMP assay, optimized for 25-30 min at 62°C, amplified the DNA from A. ocellatum extracted from both water and gill tissue samples and did not amplify DNA from four closely related dinoflagellate sp ecies. The detection limit of the AO-LAMP assay was 10 fg, exceptionally higher than the conventional PCR (1 pg). In addition, the standardized AO-LAMP assay was capable of detecting single tomonts and trophonts; the assay was not affected by the presence of possible inhibitory substances present in environmental water samples or gill samples. The AO-LAMP assay developed in the present study provides a novel useful tool for the simple, rapid and sensitive detection of A. ocellatum in water and gill tissue samples, which could assist in the early detection and improved control of A. ocellatum infections in aquaculture systems.

The potential utility of the leopard pleco (Glyptoperichthys gibbiceps) as a biological control of the ciliate protozoan Ichthyophthirius multifiliis.

BACKGROUND: Infections of the ciliate protozoan Ichthyophthirius multifiliis Fouquet, 1876, can cause heavy mortalities in freshwater aquaculture systems. Following the ban of malachite green on fish for human consumption, currently available chemical therapies are only partially efficacious even when repeatedly applied. There is, therefore, an urgent necessity to identify more efficient and environmentally friendly control strategies. In the present study, the use of a substrate algae feeder leopard pleco, Glyptoperichthys gibbiceps, as a biological agent to control I. multifiliis infections on blue tilapia, Oreochromis aureus, reared in a warm-water, experimental tank-based system is explored. RESULTS: The free-swimming protomont stage of I. multifiliis demonstrated selective settlement behaviour towards biofilm-covered substrates. Following a controlled exposure, the number of I. multifiliis trophonts establishing on naive blue tilapia, O. aureus, was 4.9 ± 0.4 when reared with G. gibbiceps, significantly lower than when reared in isolation (13.3 ± 0.9; ANOVA, P < 0.001). CONCLUSION: This study demonstrates for the first time the potential of G. gibbiceps as a biological control to reduce I. multifiliis on commercially valuable fish stocks in warm-water systems. This likely originated from the grazing activity of G. gibbiceps on the biofilm layer upon which the multiplicative stage of I. multifiliis was shown preferentially to settle. Therapeutic strategies against pathogenic protozoan species like I. multifiliis could greatly benefit from incorporating, where appropriate, a biological control targeting the external multiplicative life stages of the parasite, potentially reducing the current dependence on chemical interventions.

Effects of long duration, low dose bronopol exposure on the control of Ichthyophthirius multifiliis (Ciliophora), parasitising rainbow trout (Oncorhynchus mykiss Walbaum).

Ichthyophthirius multifiliis Fouquet, 1876 infections on intensively reared fish stocks can increase rapidly, which if left unmanaged, can result in the heavy loss of stock. The present study explores the efficacy of long duration, low dose (1, 2 and 5 mg L(-1)) treatments of bronopol (marketed as Pyceze™, Novartis Ltd.) in reducing the number of trophonts establishing on juvenile Oncorhynchus mykiss held under small scale culture conditions. The effect of bronopol on the colonisation success of infective theronts was also investigated by adding 2 mg L(-1) bronopol to the water prior and during the infection process. The number of parasites surviving on fish treated this way was compared to groups of fish that only received treatment after infection had occurred. The effect of bronopol on exiting trophonts throughout their external development to the point of theront release was also assessed through the delivery of 1 mg L(-1), 2 mg L(-1) and 5 mg L(-1) bronopol for up to 27 days consecutively (days 9-36 post-infection). The trial showed that a nominal dose of 2 mg L(-1) bronopol administered prior to infection significantly reduced the number of theronts surviving in the water column at the time of the initial challenge by 35-40% (P<0.05). Similarly, doses of 2 and 5 mg L(-1) bronopol administered as the first wave of mature I. multifiliis trophonts exited fish (i.e. day 11 onwards) to develop externally, reduced the number of trophonts establishing on fish as the second cycle of infection by 52-83%. Continuous application of 2 and 5 mg L(-1) bronopol throughout the second and third cycles of I. multifiliis infection gave further reductions of between 90 and 98%. The number of trophonts on the fish in the control tanks and those treated with 1 mg L(-1) and the 2 mg L(-1) dose at the time of initial infection, by comparison, were observed to increase with successive cycles of infection. From these small scale tank trials, this study demonstrates that the strategic, long duration, low dose delivery of drugs like bronopol can significantly reduce the number of trophonts establishing on fish suggesting the potential of this drug at managing I. multifiliis infections.

The anti-protozoal activity of bronopol on the key life-stages of Ichthyophthirius multifiliis Fouquet, 1876 (Ciliophora).

Pyceze™ (Novartis Animal Vaccines Ltd.) is licensed as a veterinary medicine to treat fungal infections in salmon, trout and their eggs. The active ingredient is bronopol, which due to its broad-spectrum activity has the potential to be an effective treatment against other important aquatic pathogens. In this study the efficacy of bronopol against Ichthyophthirius multifiliis was tested both in vitro and in vivo. In vitro trials demonstrated a 30 min exposure to 100 mg L(-1) bronopol killed 51.7% of the infective theronts. In vitro exposure of the protomonts to bronopol (0, 20, 50 and 100 mg L(-1)) for 30 min was observed to kill 0%, 76.2%, 97.2% and 100% respectively. Protomonts surviving treatment, demonstrated delayed development with the time taken from protomont until the release of theronts ranging from 28.3h for 0 mg L(-1) exposure, to 70 h for parasites in 20 and 50 mg L(-1) exposure groups. These concentrations also caused asymmetric cell division of the encysted tomonts. Exposure of encysted tomonts (min. 8 cell stage) to 100 mg L(-1) bronopol for 30 min, killed 50% within this period, with the remainder dying within the subsequent 42 h post exposure. Lower doses of bronopol were less effective in killing encysted tomonts than the higher doses (3.3% of parasites were killed in 20 mg L(-1); 10% in 50 mg L(-1)), but they still delayed theront release significantly (25.7 h for 0 mg L(-1) to 46.2h for parasites exposed to 20-50 mg L(-1)). Long, low dose (1 mg L(-1)) exposure to bronopol was also efficacious against theronts. Survival after 12h was 29% (c.f. 100% in control parasites), and <1% after 24 h exposure (c.f. 74% in control parasites). Theronts surviving these exposures demonstrated reduced infection success compared to control theronts. The findings of this study demonstrate that bronopol (Pyceze™) affects the survival of all free-living stages of I. multifiliis (protomonts, tomont and theronts), thus suggesting that bronopol may serve a useful role in the control of I. multifiliis infections.

In vitro assessment of the chemotherapeutic action of a specific hydrogen peroxide, peracetic, acetic, and peroctanoic acid-based formulation against the free-living stages of Ichthyophthirius multifiliis (Ciliophora).

Traditionally, malachite green administrated as in-bath treatment was the most effective and common strategy used in freshwater aquaculture systems to control infections of the ciliate protozoan parasite Ichthyophthirius multifiliis Fouquet, 1876. After the ban of malachite green in the USA and Europe to be used in fish for human consumption, there has been extensive research destined to find efficacious replacements. Recently, peracetic acid-based compounds have demonstrated a strong cytotoxic effect in vitro and in vivo against I. multifiliis. In the present study, we tested the efficacy of a hydrogen peroxide, peracetic, acetic and peroctanoic acid-based formulation (HPPAPA) to eliminate the free-living stages of I. multifiliis (tomonts, cysts and theronts). The results obtained showed that the administration of low doses (8, 12 or 15 mg/l) of a specific HPPAPA-based product during a short window of exposure (60 min) kills nearly all free-living stages of I. multifiliis (theronts, tomonts and cysts) within the window of treatment (∼100% mortality for all the stages; one-way ANOVA, P ≤ 0.001). Of note, even the lowest concentration of HPPAPA tested (8 mg/l) was able to disrupt normal cyst development and therefore theront release. The demonstrated in vitro efficacy of the peracetic acid-based product tested on the present study suggests its great potential to control I. multifiliis infections in commercial aquacultural systems.

Myxobolus albi n. sp. (Myxozoa) from the Gills of the Common Goby Pomatoschistus microps Krøyer (Teleostei: Gobiidae).

A recent investigation into the myxozoan fauna of common gobies, Pomatoschistus microps, from the Forth Estuary in Scotland, revealed numerous myxosporean cysts within the gill cartilage. They were composed of polysporous plasmodia containing myxobolid spores that were morphologically different from the other known species of Myxobolus and from the myxosporeans previously recorded from this host (i.e. the ceratomyxid Ellipsomyxa gobii, infecting the gall bladder, and the kudoid Kudoa camarguensis, infecting the muscle tissues). Spores were ovoid, 9.4 x 9.1 microm with a thickness of 6.6 microm, with two pyriform polar capsules, the polar filaments of which had four to five turns. Molecular analysis of the parasite's small subunit rDNA region, based upon a contiguous sequence of 1,558 base pairs, discriminated it from other myxosporean species that have been characterized so far. A comparison of the spore morphology and the molecular sequences determined for this new isolate with other myxozoans described to date, confirmed its identity as a previously unknown myxobolid supporting the proposal that this isolate be elevated to the species level as a new species within the genus Myxobolus. A phylogenetic analysis places this new myxobolid, Myxobolus albi n. sp., in a basal position of a clade containing the majority of Henneguya spp. sequenced to date and various Myxobolus spp.