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Lactococcosis is a common bacterial fish disease caused by Lactococcus garvieae, L. petauri and L. formosensis. Although there are different PCR-based techniques to identify the etiological agent, none of these can differentiate these two bacteria without sequencing PCR-amplified fragments. In the present study, we developed a multiplex PCR assay for simultaneous detection and differentiation of L. garvieae and L. petauri. The specificity of the primers was validated against the bacterial DNA of the targeted and non-targeted bacteria. The sizes of the PCR amplicons were obtained as 204 bp for the DUF1430 domain-containing protein gene of L. garvieae, 465 bp for the Lichenan permease IIC component gene of L. petauri, and 302 bp for the teichoic acid biosynthesis protein F gene of both L. garvieae and L. petauri. The PCR amplicons were clearly separated by agarose gel electrophoresis. The multiplex PCR assay did not produce any amplification products with the DNA of the non-targeted bacteria. The multiplex PCR detection limits for L. garvieae and L. petauri were 5 and 4 CFU in pure culture and 50 and 40 CFU/g in spiked tissue samples, respectively. It takes less than 2 h from plate-cultured bacteria and 3 h from tissue samples to get results. In conclusion, the developed multiplex PCR assay is a rapid, specific, accurate, and cost-effective method for the detection and differentiation of L. garvieae and L. petauri and is suitable to be used for routine laboratory diagnosis of L. garvieae and L. petauri.

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The observation of mortality in Mediterranean mussels (Mytilus galloprovincialis) distributed in the Çanakkale Strait in recent years was influential in developing the research question for this study. In this study, the presence of bacteria (Vibrio spp.) and parasites (Marteilia spp. and Haplosporidium spp.) in mussels collected from Kumkale, Kepez, and Umurbey stations in the Çanakkale Strait was investigated seasonally. Microbiological findings, histopathology, oxidative stress enzymes and their gene expressions, lipid peroxidation, lysosomal membrane stability, and changes in haemolymph were examined. In summer samples, both the defence system and the extent of damage were higher in gill tissue. In winter samples, enzyme activities and lipid peroxidation were found to be predominantly higher in digestive gland tissues. Histological examinations and Hemacolor staining revealed the presence of protozoan cysts, and for bacterial examination, molecular analysis performed after culturing revealed the presence of 7 Vibrio species. While the total numbers of heterotrophic bacteria detected in all samples were at acceptable levels, the predominance of Vibrio spp. numbers among the total heterotrophic bacteria detected in almost all samples were noteworthy. The total hemocyte count was calculated as 5.810(4)±0.58 (cells/mm3) in winter and 7.210(4)±1.03 (cells/mm3) in summer. These factors are considered to be possible causes of mussel mortality.

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Lactococcus garvieae is the etiological agent of lactococcosis, a clinically and economically significant infectious disease affecting farmed rainbow trout. L. garvieae had been considered the only cause of lactococcosis for a long time; however, L. petauri, another species of the genus Lactococcus, has lately been linked to the same disease. The genomes and biochemical profiles of L. petauri and L. garvieae have a high degree of similarity. Traditional diagnostic tests currently available cannot distinguish between these two species. The aim of this study was to use the transcribed spacer (ITS) region between 16S rRNA and 23S rRNA as a potential useful molecular target to differentiate L. garvieae from L. petauri, saving time and money compared to genomics methods currently used as diagnostic tools for accurate discrimination between these two species. The ITS region of 82 strains was amplified and sequenced. The amplified fragments varied in size from 500 to 550 bp. Based on the sequence, seven SNPs were identified that separate L. garvieae from L. petauri. The 16S-23S rRNA ITS region has enough resolution to distinguish between closely related L. garvieae and L. petauri and it can be used as a diagnostic marker to quickly identify the pathogens in a lactococcosis outbreak.

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Background: Aeromonas hydrophila is a prevalent pathogenic bacterium in aquaculture that causes economic loss around the world. Antimicrobials are used to control and prevent the incidence of bacterial pathogens in aquaculture. However, they lead to the emergence of antimicrobial resistance strains and the accumulation of antibiotic residues in fish tissue. To address these issues, bacteriophages may be promising alternatives to many antibiotics in combating bacterial infections in aquaculture. Materials and Methods: The phage specific to A. hydrophila was isolated from domestic wastewater. The morphology of phages was analyzed using transmission electron microscopy. The genomic DNA of the Aeromonas phage T65 strain (APT65) phage was sequenced with a paired-end read length of 2 × 150 bp. The genome sequence was assembled and annotated. The tRNAs were predicted, and antimicrobial resistance and virulence genes were screened. A representation of the APT65 genome was constructed. Results: The genome of APT65 is linear double-stranded DNA with 85188 base pairs having 116 open reading frames (ORFs) and a G + C content of 39.41%. The 32 ORFs were predicted to encode proteins with known phage functions. No virulence factors, antibiotic resistance genes, or temperate lifestyle genes were found. The phage is icosahedral and measures 60 nm in diameter. Based on the whole genome sequence, APT65 belongs to Lahexavirus. Conclusions: The taxonomic analysis of the phage with a genome length of 85,188 bp revealed that it is a new species of the genus Lahexavirus. We announce the whole genome sequence of APT65, which should be named Lahexavirus APT65, as well as the absence of antimicrobial resistance and virulence factors from its genome. Based on our results, the Lahexavirus APT65 phage may have potential as a therapeutic agent to tackle antimicrobial resistance in aquaculture.

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Lactococcus garvieae Lg-per was originally isolated from rainbow trout cultured in cages located on the Turkish coast of the Black Sea in 2011. A whole genome sequence of Lg-per was performed in the present study. The complete genome of Lg-per mapped to the reference genomes of L. garvieae (GCF_000269925.1) and Lactococcus petauri (GCF_014830225.1) had a total of 1,694,407 and 1,945,297 base pairs, respectively. Lg-per had 1955 protein-coding genes and 4 rRNA, 46 tRNA and 1 tmRNA operons. The orthoANI value was 98.30% between Lg-per and L. petauri (GCF_014830225.1) and 93.1% between Lg-per and L. garvieae (GCF_000269925.1). A phylogenetic tree generated from the whole genome sequences (WGS) of several Lactococcus species found that L. petauri (GCA 002154895) was closely related to the Lg-per strain with 98% similarity. Although L. garvieae Lg-per was confirmed as L. garvieae based on phenotypical, biochemical and 16S rRNA sequence, WGS of the Lg-per strain revealed that Lg-per was L. petauri. Using a 16S rRNA-based PCR detection approach, Lg-per was misdiagnosed as L. garvieae since its 16S rRNA gene was 99.9% similar to that of L. garvieae strains. Consequently, the 16S rRNA-based PCR detection approach may not be adequate for the identification of the Lactococcus genus. This is the first study to document the presence of L. petauri in Türkiye. L. garvieae isolates should be analysed using WGS since the same issue might occur in other countries.

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Russian sturgeon (Acipenser gueldenstaedtii) is an endangered fish species and also an important resource for the sturgeon aquaculture industry in Turkiye. Recently, a fatal and persistent bacterial disease occurred in the reared sturgeon kept in a trout farm in Turkiye. The disease outbreak has been with notable external signs including petechial hemorrhages and systemic anemia. This outbreak lasted for six weeks, and cumulative mortality reached around 35.00 - 40.00%. In this study, no parasitic and viral agents were observed in the sturgeons. Citrobacter gillenii was isolated from the diseased fish and identified by biochemical and molecular methods including API 20E and 20NE and 16S rRNA gene region sequencing, respectively. As a result, C. gillenii was identified for the first time in Russian sturgeon in Turkiye. The sequence was also deposited under the Genbank with MW057770 accession number. According to the result of disc diffusion method, bacteria were sensitive to enrofloxacin, streptomycin, amoxicillin and oxytetracycline and resistant to penicillin, trimethoprim/sulfamethoxazole, florfenicol and erythromycin. Also, ampC, sul1 and floR resistance genes were detected in the isolated bacteria. The results of this study provide important information for the diagnosis and treatment of this newly emerged disease of Russian sturgeon.

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The present work investigated the effects of dietary incorporation of laurel (Laurus nobilis) essential oil on the zootechnical performance and digestive physiology of juvenile Black Sea salmon (Salmo labrax). In this trial, 15 fiberglass tanks (39 × 39 cm square and 33 cm high) were used. Forty-five fish (3.52 ± 0.01 g) were placed randomly per tank. Fish were fed for 90 days with the diet containing 50, 100, 200, or 400 mg kg-1 laurel (Laurus nobilis) essential oils, respectively. The work was performed in the recirculating aquaculture system (RAS) operating with freshwater. Fish were manually fed 3% level of live weight during the experiment period. Final weight (FW), weight gain (WG), feed conversion rate (FCR), and specific growth rate (SGR) were not affected by laurel essential oil supplementation. Dietary laurel essential oil (50 mg kg-1) affected positively the surface area of fish intestinal villus that required for digestion. Both villus height and villus width were affected positively in fish fed with 50 mg laurel essential oil kg-1. While incorporation with 100 mg laurel essential oil kg-1 increased the total α-amylase enzyme, 50 mg laurel kg-1 increased lipase enzyme. Moreover, 50 mg laurel essential oil kg-1 increased lactic acid bacteria (LAB) count in fish. Besides, 50 mg laurel essential oil kg-1 reduced the number of total coliform and E. coli.

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Scuticociliates are dangerous parasitic pathogens causing systemic tissue destruction and high mortality in marine fish worldwide. In this study, the first identification of Uronema marinum (Ciliophora, Scuticociliatida) from cultured turbot (Psetta maxima) larvae using mitochondrial cytochrome c oxidase 1 (cox1) gene sequence as well as species-specific primers was reported. The mean prevalence values of infected fish were calculated, and partial sequencing obtained from the mitochondrial cox1 gene region was also compared with isolates registered in the Genbank database. The sequence comparison showed 93.00% identity to U. marinum, and the parasite has also been deposited in the GenBank database. This study is the first case of U. marinum infection in Turkish marine aquaculture, contributing to the systematics and molecular epidemiology of scuticociliate in Turkey.

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Cadmium, cobalt, copper, nickel, and zinc are the most common pollutant heavy metals that can be discharged into the marine environment with different sources. Whiting (Merlangius merlangus) and mullet (Mullus barbatus) were sampled in four seasons in a year to determine Cd, Co, Cu, Ni, and Zn levels in the muscle and to determine heavy metal resistance genes (MRGs) such as copA, czc, and ncc genes in coliform bacteria isolated from the fish. In both species, zinc was the most abundant metal, while Cd and the Co levels were scarce. Co level was significantly higher in summer in mullet than that of whiting (p < 0.001). The most prevalent MRGs was determined as copA (46.2%) followed by czc (35.8%) and ncc (17.9%). Increased Co and Ni level in the muscle significantly affected the presence of ncc gene in bacteria, while the presence of ncc and copA genes was affected by Ni and Cu levels found in the fish muscle. There was a significant positive correlation between Cd level in the muscle and presence of czc and ncc gene in the bacteria (p < 0.029). When the levels of Cu, Zn, and Cd increased in the muscle of the fish, occurrence of MRGs genes was increased significantly (p < 0.0001). A strong positive correlation was found between heavy metal resistance levels in fish and the prevalence of E. coli and coliforms that harbor heavy metal resistance genes which will be a problem in aquaculture, aquatic ecosystem, and public health.

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OBJECTIVE: To characterize antibiotic resistance genes (ARGs) and heavy metal resistance genes (HMRGs) of Escherichia coli and Enterococcus faecium isolated from the sediment and Lactococcus garvieae isolated from fish. MATERIALS AND METHODS: The isolated bacteria were identified by sequencing 16S rRNA genes. After identification of the bacteria, tetracycline (tetA, tetB, tetD), erythromycin (ereA, ereB), sulfonamides (sulI, sulII), trimethoprim (dhfrA1), ß-lactam (blaTEM, blaCTX, ampC), florfenicol (floR), and class 1 integron (Int1) resistance gene were then determined. The presence of HMRGs, including copper (copA), mercury (mer), cadmium, zinc, cobalt (czc), and nickel, cobalt cadmium (ncc), was also analyzed by PCR. All strains were checked for the presence of ARGs and/or HMRGs on the plasmid. RESULTS: The frequency of the ß-lactam resistance gene was highest and ranged from 49.7% to 62.3%, followed by sulfonamides, tetracyclines, phenicols, and macrolide resistance genes. The cage culture fish farming practice showed significant effects on ARG frequency of bacteria isolated from the sediment, whereas it had no effect on the frequency of HMRGs. The most prevalent HMRG was determined as mercury-resistant mer gene in all bacteria. All four of the HMRGs were located on plasmids with frequency ranging from 1.20% to 32.53%. The presence of ARGs on plasmids ranged between 2.2% (Dhfr1) and 75% (AmpC, blactx, tetB), and plasmids did not contain tetD and ereB genes. CONCLUSION: The results of this study indicate that fish farming can significantly influence the antimicrobial resistance properties of bacteria isolated from sediment samples.

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Lactococcus garvieae is the causative agent of lactococcosis and has been isolated from a wide variety of animals. In the present study, 34 strains of L. garvieae isolated from fish from different sources and locations were tested for the presence or absence of the following putative virulence genes: a capsule gene cluster (CGC), hemolysins 1, 2, and 3 (hly1, -2, -3), NADH oxidase, superoxide dismutase (sod), phosphoglucomutase (pgm), adhesin Pav (adhPav), adhesin PsaA (adhPsaA), enolase (eno), LPxTG-containing surface proteins 1, 2, 3, and 4 (LPxTG-1, LPxTG-2, LPxTG-3, LPxTG-4; where LPxTG means Leu-Pro-any-Thr-Gly), adhesin clusters 1 and 2 (adhCI, adhCII), and adhesin (adh). To determine the presence of the CGC, we developed a multiplex PCR. All strains of L. garvieae had the hly1, -2, -3, NADH oxidase, pgm, adhPav, LPxTG-2, LPxTG-3, sod, eno, adhPsaA, adhCII, and adhCII genes, while only the Lg2 strain contained the CGC. The virulent Lg2 strain contained all 17 virulent genes. All Turkish, Spanish, Italian, and French strains did not contain the CGC. The multiplex PCR assay was useful for the detection of the CGC genes. In conclusion, the CGC is not the only virulent factor in L. garvieae because strains that lack the CGC are virulent to rainbow trout. Single genes also might not be responsible for the virulence of L. garvieae.

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In this study, a very sensitive and highly selective irreversible optical chemical sensor (optode) for mercury ions was described. The sensing scheme was based on the interaction of Hg (II) with a newly synthesized fluoroionophore; chloro phenyl imino propenyl aniline (CPIPA) in plasticized PVC membrane. The sensor membranes were tested for the determination of mercury ion in aqueous solutions by batch and flow-through methods. The optodes allow determination of Hg (II) in the working range of 1.0 x 10(-9)-1.0 x 10(-5) M with a detection limit of 4.3 ppb. The sensor exhibited excellent selectivity for Hg (II) with respect to several common alkali, alkaline earth and transition metal ions. The association constant of the 1:1 complex formation for Hg (II) was found to be K(a) = 1.86 x 10(5) M(-1). The CPIPA exhibited high fluorescence quantum yield, long excitation and emission wavelength and high Stokes' shift values in the solid matrix which makes it compatible with solid state optics.