Soner Altun

Genotyping and antimicrobial resistance genes of Yersinia ruckeri isolates from rainbow trout farms

In this study, we compared 142 Yersinia ruckeri isolates collected between 2013 and 2016 from 6 different regions in Turkey. A total of 18 different genogroups were found, though most of the isolates clustered into the same genogroup as serotype O1. As immunization of fish with inactivated Y. ruckeri by injection, immersion, or feeding provide minimal protection against Y. ruckeri infection in Turkey, many fish producers use antimicrobials unrestrictedly, resulting in antimicrobial resistance in aquatic pathogens. Accordingly, we investigated resistance to the antimicrobials most commonly used to treat yersiniosis. More than 80% of the Y. ruckeri isolates were susceptible to sulfamethoxazole-trimethoprim (SXT), florfenicol (FFC), and tetracycline, whereas none were susceptible to sulfamethoxazole. The most commonly used antimicrobials (SXT and FFC) can be effectively administered because the resistance levels to these drugs are the lowest among those reported for agents used to control enteric red mouth disease (12.6 and 14.7%, respectively). In conclusion, to the best of our knowledge, this study is the first characterization of the antimicrobial resistance genes floR, sulI, tetC, tetD, and tetE in Y. ruckeri isolates from aquaculture. Additionally, we detected the sulII gene but not the tetA, tetB, tetM, tetS, or sulIII genes.

Molecular characterization and antimicrobial resistance profile of atypical Citrobacter gillenii and Citrobacter sp. isolated from diseased rainbow trout (Oncorhynchus mykiss)

AIMnOver the last decade, Citrobacter species have been responsible for infections in fish and many species and also new Citrobacter species have been identified. In this study, molecular identifications and the phenotypic and genotypic antimicrobial-resistance characteristics of atypical and typical Citrobacter species were determined.nnnMATERIALS AND METHODSnSeven Citrobacter isolates were investigated from rainbow trout of different lengths with signs of disease. Biochemical characteristics were determined using conventional tests and rapid test kits; moreover, molecular identifications were conducted with 16S rRNA and the gyrB housekeeping gene region. The sequencing results obtained from the gyrB gene region were deposited in the GenBank database and compared with isolates from different countries that were registered in the database. Resistances to florfenicol, sulfonamides, and tetracycline antimicrobials were determined using the broth micro dilution method, and molecular resistance genes against these antimicrobials were identified. All detected resistance genes were confirmed by sequence analyses.nnnRESULTSnIt was determined that three of the Citrobacter species with biochemical characteristics were atypical and showed oxidase-positive reactions. All the Citrobacter species were identified as Citrobacter sp. using the 16S rRNA gene; three isolates were identified as Citrobacter gillenii and four as Citrobacter sp. based on gyrB gene sequence analysis. Some isolates were found in the same group as other countries isolates in the GenBank database, while isolates with high identities were found in different genogroups. All isolates were found to be phenotypically resistant to sulfamethoxazole and susceptible to tetracycline; these isolates resistance genes included sulI, tetA, tetB, and tetD.

Infectious pancreatic necrosis virus (IPNV) serotype Sp is prevalent in Turkish rainbow trout farms

Infectious pancreatic necrosis virus (IPNV) is a common pathogen of rainbow trout (Oncorhynchus mykiss) in Turkey. We found that 455 of 1,676 sample pools tested were IPNV positive. Positive samples were found in all geographical regions where sampling was conducted. Sequence and phylogenetic analyses of VP2 from 30 isolates representing all regions showed that the viruses were highly similar in sequence and grouped within Genogroup 5 (serotype Sp-A2). No correlations between sequences, sampling sites or geographical origins were identified. Although clinical disease was evident in several farms, analyses of the amino acid sequence of VP2 showed that all virus strains harboured the P217 T221 motif, assumed to be associated with low virulence. We conclude that IPNV is prevalent in Turkish rainbow trout farms and that the viruses are very homogenous and likely to be of European origin. Frequent exchange of eggs and live fish within the farming industry may explain the homogeneity of the IPNV.

Development and validation of glycoprotein‐based native‐subunit vaccine for fish against Aeromonas hydrophila

Aeromonas hydrophila is known to be causative agent of an infection named as Bacterial haemorrhagic septicaemia or red pest in freshwater fish. The aim of this study was to develop and validate the glycoprotein-based fish vaccine against Aeromonas hydrophila. For this aim, after identification and characterization of A.xa0hydrophila isolates from fish farms, one A.xa0hydrophila isolate was selected as vaccine strain. Antigenic glycoproteins of this vaccine strain were determined by Western blotting and glycan detection kit. The connection types of these glycoproteins were examined by glycoprotein differentiation kit. Two glycoproteins, molecular weights of 19 and 38xa0kDa, with SNA connection type were selected for use in vaccination trials. After their purification by SNA-specific lectin and size-exclusion chromatography, protection studies with purified proteins were performed. For challenge trials, four experimental fish groups were designated: Group I (with montanide), Group II (with montanide and ginseng), Group III [with Al(OH)3 ] and Group IV [with Al(OH)3 and ginseng]. The survival ratings of fish were determined, and protection was calculated as 21.56%, 29.41%, 69.83% and 78.88% in groups I, II, III and IV, respectively. In conclusion, A.xa0hydrophila glycoproteins with Al(OH)3 and ginseng could be used as a safe and effective vaccine for fish.

Antimicrobial resistance and molecular characterization of Pantoea agglomerans isolated from rainbow trout (Oncorhynchus mykiss) fry

Aquaculture has become an important candidate as an animal protein source through its growth over the last decade. Based upon a report from the Food and Agriculture Organization of the United Nations, it is the fastest growing sector of the food industry, yet the pathogenicity of many biological agents involved in aquaculture is still unknown. In this study, we isolated Pantoe agglomerans from diseased rainbow trout on several occasions and also attempted to determine their phenotypic and genotypic characteristics, including antimicrobial resistance, of four bacterial isolates. In the present study, P. agglomerans was isolated from diseased rainbow trout as a pathogenic agent. The identification of the P. agglomerans isolates from the rainbow trout was performed through biochemical tests and 16S rRNA sequence analysis. These isolates were predominately biochemically homogeneous, although some features were different, such as seen in methyl-red, mannose and lipase activity tests. All four studied isolates were identified as 99% identical to P. agglomerans based on sequence analysis. The isolates were compared through a phylogenetic analysis with P. agglomerans sequences recovered from 16 other countries and accessed from the GenBank database. All isolates in our study were at least 98.2% similar to sequences from GenBank. Furthermore, the phenotypic antimicrobial susceptibility of the isolates in this study was analyzed through both disc diffusion and broth micro dilution minimum inhibitory concentration (MIC) tests. Although there were some differences between two phenotypic antimicrobial tests, all studied isolates were found susceptible to different antimicrobials. In addition genotypic antimicrobial resistance characteristics were assessed by the presence of antimicrobial resistance genes (ARGs), in which qnrS and sul2 were detected for the first time in P. agglomerans.

Serological and genetic characterization of Flavobacterium psychrophilum isolated from farmed salmonids in Turkey

Turkey was the largest rainbow trout producer of the European countries in 2016, and the reason for this production is mainly attributed to its egg and fry production. Flavobacteriumxa0psychrophilum cause the highest rates of mortality in the starting to feeding stages of the fish. In the present study, twenty-five F.xa0psychrophilum isolates recovered from rainbow trout, coruh trout and brook trout were analysed by RAPD-PCR, ERIC-PCR, REP-PCR and PCR-RFLP, including 16S rRNA, gyrA and gyrB gene regions and PCR-based serotyping method. The PCR-based molecular analysis showed that the isolates could not be differentiated exactly according to isolation source and geographical region. Most isolates were of type-1 and type-2, and some of them were of type-0 and type-3; in addition, one isolate showed a unique serotype. The combined analysis results showed that F.xa0psychrophilum isolates discriminated as five different genotypes and all isolates were successfully discriminated based on host.

The determination of the infectious status and prevalence of motile Aeromonas species isolated from disease cases in rainbow trout (Oncorhynchus mykiss) and aquarium fish

The aims of this study were to determine the prevalence and phylogenetic relationship of motile Aeromonas spp. that might be pathogenic species for rainbow trout in infected/mix infection cases (based upon different outbreaks on fish farms). A total of 99 motile Aeromonas isolates (and three reference strains) were analysed that were isolated from four different fish species in different sizes of fish (0.1-3,000xa0g), different months and water temperatures (6.1-21.2°C). The biochemical characteristics of the isolates were determined using conventional tests and a rapid test kit. Additionally, molecular identification was performed using the gyrB housekeeping gene region and with glycerophospholipid-cholesterol acyltransferase polymerase chain reaction (GCAT-PCR). The sequencing results obtained from the gyrB gene region were deposited in the GenBank database, and phylogenetic relationships were determined with the BioNumerics 7.6 database. Nearly half of the Aeromonas isolates that were isolated from rainbow trout showing signs of disease were determined to be possible infectious agents. Aeromonas species exhibit biochemical variability for many characters, so some Aeromonas species tested negative for GCAT-PCR despite that this test was created especially for Aeromonas identification. The phylogenetic tree based upon gyrB contained 10 different phylogroups that were based on 96% cut-off value in gyrB gene region.