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.

Is the Illegal Trade of Glass Eels (Anguilla anguilla) Increasing the Spread of Disease? A Case of EVEX.

The European eel (Anguilla anguilla) is a catadromous species that inhabits the rivers of the Adriatic watershed in Croatia. It is a critically endangered fish species, according to the IUCN, due to its declining abundance in European rivers caused by overfishing and trafficking and by diseases caused by nematodes, pathogenic bacteria and viruses. An illegal parcel of glass eels was confiscated at the Zagreb Airport and was intended to re-populate Croatian rivers. Barcoding was employed to determine species affiliation, and a thorough health check was carried out. This study reports the evaluation of gross lesions, histological findings, and EVEX virus isolation and identification. Since the confiscated glass eels were of unknown origin and given the serological and genetic similarities of EVA and EVEX, we designed primers and probes for almost whole genome sequencing to elucidate the origin of glass eels based on viral phylogeny. Bayesian phylogeny showed that the isolated strain had the most common ancestor with a Danish isolate and likely evolved from the French isolate of EVEX. These findings are discussed in light of the divergence of recently isolated strains and their possible contribution to the decrease of the abundance of the European eel in European waters.

Biochemical and molecular characterization of three serologically different Vibrio harveyi strains isolated from farmed Dicentrarchus labrax from the Adriatic Sea.

Vibrio harveyi is recognized as one of the major causes of vibriosis, a disease that threatens the long-term sustainability of aquaculture. Current research shows that the Mediterranean strains of V. harveyi are serologically heterogeneous, though research comparing the traits of different strains is scarce. This study aims to describe the biochemical, physiological and genetic characteristics of three serologically different strains of V. harveyi isolated from farmed European Sea bass (Dicentrarchus labrax) from the Adriatic Sea. A total of 32 morphological and biochemical markers were examined and, the susceptibility to 13 antimicrobials tested, and then compared the results of high-throughput sequencing and in silico analyses. This study also presents the first whole genome sequences of V. harveyi isolated from European sea bass. A large number of nonsynonymous variations were detected among sequences of the three strains. The prediction analysis of resistance genes did not correspond with the in vitro antimicrobial susceptibility tests. Six virulence genes previously unrelated to virulence of vibrios were detected in all three studied strains. The results show that differences were detected at every level of comparison among the three studied strains isolated from the same fish species originating from a small geographic area.

Transmission Pathways of the VNN Introduced in Croatian Marine Aquaculture.

Due to the insufficient capacity of Croatian hatcheries, marine aquaculture depends on the importation of fry from different countries in the Mediterranean basin. Importation enables a risk of spreading pathogenic agents. Viral nervous necrosis (VNN), caused by betanodavirus is devastating for the farming of European sea bass. We described a VNN outbreak that occurred in Croatia in 2014. After the diagnosis of VNN in sea bass fry introduced from the same hatchery to five unconnected marine farms at the Adriatic Coast, we performed surveillance within one of the affected farms. It resulted in proven horizontal spreading of the virus within the farm and to feral fish around farm cages. Real-time RT-PCR tested samples showed the dependence of the virus' proliferation to the water temperature and the fish age. The highest mortality rates were noted during higher sea temperatures. Phylogenetic analysis of partial sequences of RNA1 and RNA2 supported the hypothesis that the virus was introduced to all studied farms from the same hatchery. Moreover, phylogenetic analysis of the whole genome sequences of infected farmed sea bass and thicklip mullet showed high similarity and it is unlikely that infection in Croatian sea bass farms has originated from wild reservoirs, as the first positive record in wild mullet was recorded after the disease outbreak.

First description and diagnostics of disease caused by Piscirickettsia salmonis in farmed European sea bass (Dicentrarchus labrax Linnaeus) from Croatia.

During the winter of 2013 and 2016, several Croatian fish farms experienced mortalities in the fry of European sea bass, Dicentrarchus labrax. Affected fish showed abnormal swimming behaviour and reduced appetite, and death ensued several days after the onset of clinical signs of disease. Necropsy revealed pale liver, empty digestive tract, distended gall bladder, and hyperaemia and congestion of the meninges. Routine bacteriological examination tested negative, and virological examination ruled out nodavirus infection. Histological examination revealed multifocal necrosis and extensive inflammation in the brain with abundant cellular debris in the ventricles. Inflammatory cells displayed intra-cytoplasmic basophilic vacuoles leading to suspicion of Piscirickettsia salmonis infection. Fluorescent in situ hybridization using an oligonucleotide probe targeting Domain Bacterium applied to tissue sections tested positive. The pathogen was identified by 16S rRNA gene sequencing of brain material, and the sequence showed 99% similarity with P. salmonis. This result enabled the design of an oligonucleotide probe specifically targeting P. salmonis. In 2016, P. salmonis was successfully isolated on CHAB from the brain of an affected specimen and identified using 16S rRNA gene sequencing and MALDI-TOF. This study describes the first outbreak of disease caused by P. salmonis in sea bass in Croatia, while new diagnostic tools will enable further research on its epidemiology and pathogenicity.

Assessment of predominant bacteria in noble pen shell (Pinna nobilis) collected in the Eastern Adriatic Sea.

Noble pen shell (Pinna nobilis) is an endemic species and the largest known bivalve in the Mediterranean Sea. By filtering large amounts of water, they maintain a high percentage of organic matter, hence playing an important role in the marine ecosystem. The ecological community of pen shells is impressive, and there are numerous microorganisms present in its soft tissues. Since this species is highly endangered due to recently described mass mortalities throughout the Mediterranean, this study was aimed at finding out more about its microbiome. In this study, we identified the predominant bacterial populations of specimens collected at three separate locations along the Eastern Adriatic coast. The predominant bacteria were isolated and 16S rRNA sequencing was performed to identify eight different bacterial genera: Aestuariibacter sp., Aliivibrio sp., Alteromonas sp., Marinobacter sp., Pseudoalteromonas sp., Rubritalea sp., Thalassospira sp. and the Vibrio splendidus clade. The identified genera are ubiquitous in the marine environment and have previously been described as both beneficial symbionts and potential pathogens in other molluscs. There was a clear difference in the predominant bacterial populations between northern and southern sampling sites, which could be linked to water temperature. These findings indicate the need for expanded sampling over a longer time period, since more exhaustive research would provide information vital to the conservation of this critically endangered species.

Koi herpesvirus and carp edema virus threaten common carp aquaculture in Croatia.

Common carp (Cyprinus carpio) is a very important fish species for warm-water aquaculture in Croatia. All Croatian carp farms are subjected to a surveillance programme for the presence of koi herpesvirus (KHV), causing a deadly disease called koi herpesvirus disease (KHVD). However, there is no surveillance for other viral pathogens of importance like carp edema virus (CEV), a causative agent of koi sleepy disease (KSD). During regular testing within the KHVD surveillance programme, we tested samples for CEV simultaneously. The screening indicated possible outbreaks of KHVD and KSD. During 2016, KHVD broke out in an isolated area and soon thereafter a KHV eradication programme was successfully performed. However, during 2018 and 2019, two additional mortality events occurred in lakes in the southern part of Croatia during the spring. Samples from both events tested positive for CEV. An epidemiological investigation confirmed the introduction of infected carps from an infected farm to one of the lakes. To prevent the spreading of CEV into open waters, it is of utmost importance to introduce CEV testing before fish movement or to perform regular testing of all carp farms in the country to determine CEV prevalence for the purpose of implementation of control measures.

Biomonitoring of heavy metals in fish from the Danube River.

The Croatian part of the Danube River extends over 188 km and comprises 58 % of the country's overall area used for commercial freshwater fishing. To date, the heavy metal contamination of fish in the Croatian part of the Danube has not been studied. The main purpose of this study was to determine heavy metal levels in muscle tissue of sampled fish species and to analyze the measured values according to feeding habits of particular groups. Lead ranged from 0.015 µg(-1) dry weight in planktivorous to 0.039 µg(-1) dry weight in herbivorous fish, cadmium from 0.013 µg(-1) dry weight in herbivorous to 0.018 µg(-1) dry weight in piscivorous fish, mercury from 0.191 µg(-1) dry weight in omnivorous to 0.441 µg(-1) dry weight in planktivorous fish and arsenic from 0.018 µg(-1) dry weight in planktivorous to 0.039 µg(-1) dry weight in omnivorous fish. Among the analyzed metals in muscle tissue of sampled fish, only mercury exceeded the maximal level (0.5 mg kg(-1)) permitted according to the national and EU regulations determining maximum levels for certain contaminants in foodstuffs, indicating a hazard for consumers of fish from the Danube River.